volume 16 / number 1 / pages 1–174 - world scientific · 2019. 12. 24. · contents j. porphyrins...

MICA (P) 222/04/2011

2011 - ISSN 1088-4246

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CONTENTS

Journal of Porphyrins and PhthalocyaninesJ. Porphyrins Phthalocyanines 2012; 16: 1–174

See Masahiko Taniguchi and Jonathan S. Lindsey* pp 1–13

Combinatorial libraries of -substituted tetrapyrrole macrocycles are rich in isomers due to the location and relative orientation of the substituents. A software program (PorphyrinViLiGe) and accompanying terminology enable description of the libra-ries, their members, and the subsets of isomers located therein. The libraries can be vast: derivatization of an 8-point tetrapyrrole scaffold with 8 reagents affords > 2 million members, of which > 99% are isomers of other members of the library. The diverse macrocycles are shown in the firmament with the Andromeda galaxy (cour-tesy NASA/JPL-Caltech).

About the Cover

Articles

pp. 14–24Spectroscopic and theoretical insights on determina-tion of binding strength and molecular structure for the supramolecular complexes of a designed bisporphyrin with C60 and C70

Partha Mukherjee, Subarata Chattopadhyay and Sumanta

Bhattacharya*

The present article examines the binding affinity of the newly designed Zn2-bisporphyrin molecule, syn-1, towards C60 and C70 in toluene medium. The bisporphyrin, syn-1, serves as an effective and selective molecular tweezer for C70 as the average value of binding constants (K) for non-covalent complexes of syn-1 with C60 and C70 are estimated to be 1.65 104 and 1.05 105 M-1, respec-tively. Proton NMR studies suggest that the C70 moiety remains at the shallow part of the cleft of syn-1.

pp. 1–13Diversity, isomer composition, and design of com-binatorial libraries of tetrapyrrole macrocyclesMasahiko Taniguchi and Jonathan S. Lindsey*

Combinatorial libraries of tetrapyrrole macrocycles typically are rich in isomers. Terminology for describing isomers due to distinct patterns of peripheral substituents and an understanding of the molecular origins of isomerism should facilitate the design and creation of combinatorial libraries of diverse tetrapyrrole macrocycles.

CONTENTS

J. Porphyrins Phthalocyanines 2012; 16: 1–174

pp. 39–46Protonation-deprotonation equilibria in tetrapyr-roles. Part 2. Mono- and diprotonation of methyl pyropheophorbide a in methanolic hydrochloric

1H, 13C HSQC and 1H, 15NHMBC NMR experimentsPaavo H. Hynninen* and Markku Mesilaakso

The 1H, 13C HSQC and 1H, 15N HMBC NMR spectra recorded at 278 K verified that the CD3OH-HCl solution with [H+] 0.021 M, contained the N22-protonated monocations of methyl pyropheophorbide a, whereas the CD3OH-HCl solution with [H+] 5.0 M contained the N22, N24-protonated dications of the phorbin. No further protonations to form the trication or tetracation were observed.

pp. 55–63Correlation of photodynamic activity and singlet oxygen quantum yields in two series of hydro-phobic monocationic porphyrinsDaiana K. Deda, Christiane Pavani, Eduardo Caritá,

Maurício S. Baptista, Henrique E. Toma and Koiti Araki*

The photodynamic activity of a series of highly hydrophobic monoca-tionic porphyrin derivatives, incorporated in a polymeric formulation of marine atelocollagen/xanthan gum prepared by the coacervation method, was analyzed and compared to its photoinduced singlet oxygen quantum efficiencies.

pp. 47–54Nucleotide-driven packaging of a singlet oxygen generating porphyrin in an icosahedral virusBrian A. Cohen, Alain E. Kaloyeros and Magnus Bergkvist*

Schematic illustration showing porphyrin penetration through capsid pores and association with RNA (assembly packaging) on the interior of the capsid. Experimental results demonstrate loading of approxi-mately 250 porphyrin molecules per bacteriophage while retaining the ability to photogenerate singlet oxygen.

pp. 25–38Internal spin trapping of thiyl radical during the complexation and reduction of cobalamin with glu-tathione and dithiothrietolSomasundaram Ramasamy, Tapan K. Kundu, William Antholine,

Periakaruppan T. Manoharan* and Joseph M. Rifkind*

Reduction of cobalamine(III) by glutathione requires two molecules; the first one acts as a complexing ligand in the axial position and the second one located inside the ligand cavity causes reduction to Cbl(II), produces a thiyl radical and forms a ferromagnetically exchanged coupled species. Reduction by dithiothri-etol having two thiols is a much faster reaction.

CONTENTS


pp. 72–76Water-soluble porphyrin-based logic gatesLili Li, Peiying Cai, Yuefei Deng, Liutao Yang, Xuan He,

Lingsong Pu, Di Wu*, Jin Liu*, Haifeng Xiang and Xiangge

Zhou*

A series of simple logic gates based on a water-soluble porphyrin mo-lecule, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) is de-signed. Logic operations, including OR, NOR, INHIBIT and AND, have been built by two inputs of acid/base or metal ions (Al3+ and/or Sn4+) and two outputs of of UV-vis and fluorescence spectra. An OFF-ON switch triggered by Al3+ ion in vitro is developed based on TPPS4.

pp. 77–84Synthesis, properties and near-infrared ima-ging evaluation of glucose conjugated zinc phthalocyanine via Click reactionFeng Lv, Xujun He, Li Lu, Li Wu and Tianjun Liu*

A novel near infrared fluorescence probe, glucose conjugated zinc phthalocyanine, [2,9(10),16(17),23(24)-tetrakis((1-( -D-glucopy-ranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]zinc(II), was synthesized via Click reaction. The probe’s optical be-havior and near-infrared imaging effect in vivo were evaluated.

pp. 85–92Synthesis, circular dichroism, DNA cleavage and singlet oxygen photogeneration of 4-ami-dinophenyl porphyrinsKai Wang, Chun T. Poon, Chun Y. Choi, Wai-Kwok

Wong*, Daniel W.J. Kwong*, Fa Q. Yu, Heng Zhang

and Zao Y. Li*

5,10,15,20-tetrakis(4-amidinophenyl)porphyrin (Por 1), its Zn com-plex (Por 2) and its conjugate with tetraphenylporphyrin to form a bisporphyrin (Por 3) were prepared. Their DNA binding modes by UV-vis and CD spectra were studied. The singlet oxygen and DNA cleavage were also performed.

pp. 64–71Effect of lipophilicity on biological properties of 109Pd-porphyrin complexes: a preliminary investigationSudipta Chakraborty, Tapas Das, Haladhar D. Sarma and

Sharmila Banerjee*

The present study investigates the effect of lipophilicity of 109Pd-porphyrin complexes on their biological properties, with an aim to develop a potential agent for targeted tumor therapy with optimum tumor uptake along with adequately high tumor to background ratio. 109Pd complexes of three different porphyrin derivatives (I, II and III) with different Log P values were synthesized and their biological behaviors were evaluated in Swiss mice bearing fibrosarcoma tumors. It was found that the lipophilicity of the complexes have noticeable effect on their biological characteristics. It can be inferred from the present study that radio-metal-porphyrin complexes with appreciable tumor uptake and adequately high tumor to background ratio could be synthesized by optimization of the lipophi-licity through proper tailoring of peripheral substituents of porphyrin molecule.

N

N

N

NR'

R R

R'

R

R'

R

R'

R = OCH2COOH R' = OCH2COOH

R = OCH2COOEt R' = OCH2COOEt

R = OCH2COOH R' = H

[I]

[II]

[III]

Pd109

CONTENTS


pp. 101–113meso-tetraphenylporphy-

rins: synthesis and non-covalent interactions with a short poly(dGdC) duplexEduarda M.P. Silva, Catarina I.V. Ramos, Patrícia M.R. Pereira, Francesca Giuntini, Maria A.F. Faustino, João P.C. Tomé, Augusto C. Tomé, Artur M.S. Silva, M. Graça Santana-Marques*, M. Graça P.M.S. Neves* and José A.S. Cavaleiro

Several cationic β-vinyl-piridinium and -vinyl-quinolinium-meso-tetraphenyl-porphyrin derivatives were synthesized in a one-step process via base catalyzed al-dol-type condensation reactions. Electrospray ionization mass spectrometry (ESI-MS) and ultraviolet-visible (UV-vis) spectroscopy were used to investigate the binding mode of the synthesized cationic -vinyl-piridinium and -vinyl-quino-linium-meso-tetraphenylporphyrin derivatives with a short GC duplex oligonucle-otide. The overall results confirm previous hypothesis that the positive charge is a main contributor for the type and strength of porphyrin/duplex interactions.

pp. 114–121In vitro insulin-mimetic activity and in vivo metalloki-netic feature of oxovanadium(IV)porphyrin complexes in healthy ratsTapan K. Saha*, Yutaka Yoshikawa, Hirouki Yasui and Hiromu Sakurai

The in vitro insulin-mimetic activity and in vivo metallokinetic feature of [VO(tcpp)]Na4 are very similar to those of [VO(tpps)], however, significantly better than those of previously reported insulin-mimetic [VO(tmpyp)]-(ClO4)4

and oxovanadium(IV) sulfate. These results highlight the potential utility of suit-ably designed oxovanadium(IV)porphyrin complex [VO(tcpp)-]Na4 as a candi-date for drug with insulin-mimetic activity.

pp. 122–129Synthesis of long-wavelength chlorins by chemical

a and their in vitro cell viabilitiesJin Jun Wang*, Jia Zhu Li, Judit Jakus and Young Key Shim

A series of novel chlorophyll-a homologs with long wavelength absorption (more than 700 nm) were synthesized via introduction of electron-withdrawing group me-thylenemalononitrile moiety on the periphery of modified chlorin by Knoevenagel reaction of malononitrile with formyl group at 3-, 15-position and 131-carbonyl group on the exocyclic ring. A preliminary in vitro photodynamic anticancer effect of these new derivatives on mouse sarcoma S-180 cell line was also examined.

pp. 93–100

acid in the presence of platinum porphyrin sensitizers, air and sunlightMahdi Hajimohammadi, Hamid Mofakham, Nasser Safari* and

Anahita Mortazavi Manesh

A variety of aromatic and aliphatic aldehydes were oxidized to the corresponding carboxylic acids in the presence of platinum porphyrin, sunlight and air in aceto-nitrile solvent under mild conditions. Nitrobenzaldehydes were found to be very efficient 1O2 scavengers that quench the formation of acids from any aldehyde in the presence of free-base porphyrin sensitizers.

CONTENTS


pp. 140–148Picosecond and femtosecond optical nonlinea-rities of novel corrolesSyed Hamad, Surya P. Tewari, L. Giribabu and S. Venugopal

Rao*

Picosecond nonlinear absorption data of TPC obtained at a concentra-tion of 5 10-4 M demonstrated complex behavior with switching from reverse saturable absorption (RSA) within saturable absorption (SA) at lower peak intensities to RSA (effective 3PA) at higher peak intensities. TTC data recorded at a similar concentration exhibited saturable absorp-tion (SA) type of behavior at lower peak intensities to switching from RSA within SA at higher peak intensities.

pp. 149–153Catalytic epoxidation of cis-stilbene and naphthalene by tetra-n-butylammonium hydrogen monopersulfate in the presence of manganese(III) tetraarylporphyrins and various anionic co-catalystsZahra Solati*, Majid Hashemi, Ahmad Keshavarzi and Ezzat

Rafiee

The epoxidation of cis-stilbene by n-Bu4NHSO5 was studied in the presence of two different manganese porphyrin complexes, [MnTPFPP(Cl) and MnTPP(Cl)], and various n-Bu4NX (X = OAc, F, Cl, Br, OCN, NO3, BF4). In general direct cor-relation was found between stereoselectivity of the epoxidation reaction and the nucleophilic properties of these anionic co-catalysts. Also epoxidation of naphtha-lene was carried out in high yield and good selectivity by n-Bu4NHSO5 in the pre-sence of MnTPFPP(Cl) in association with n-Bu4NOAc or n-Bu4NF co-catalysts.

pp. 154–162Synthesis and structural characterization of a magnesium phthalocyanine(3–) anionEdwin W.Y. Wong and Daniel B. Leznoff*

The reduction of magnesium phthalocyanine (MgPc) with potas-sium graphite in 1,2-dimethoxyethane (DME) and trace O2/H2O gives [K2(DME)4]PcMg(OH) (1). Compound 1 was structurally characterized using single crystal X-ray crystallography and an absorption spectrum of 1 indicates that the Pc ligand is singly reduced and has a –3 charge. Compound 1 is the first example of a structurally characterized reduced MgPc and only the second structure of a Pc3- system.

pp. 130–139Single walled carbon nanotubes fuctionalized with nickel phthalocyanines: effects of point of substitution and nature of functionalization on the electro-oxidationof 4-chlorophenolSamson Khene and Tebello Nyokong*

Glassy carbon electrodes modified with a number of differently substitut-ed nickel phthalocyanines show improved electrocatalytic activity towards 4-chlorophenol in the presence of single walled carbon nanotubes. The best response was obtained for the octa substituted derivative containing hydroxy groups.

CONTENTS


pp. 163–174Tetra and octa(2,6-di-iso-propylphenoxy)-substitu-ted phthalocyanines: a comparative study among their photophysicochemical propertiesAhmad Tuhl, Wadzanai Chidawanayika, Hamada Mohamed

Ibrahim, Nouria Al-Awadi, Christian Litwinski, Tebello

Nyokong, Haider Behbehani, Hacene Manaa and Saad

Makhseed*

Highly non-aggregated tetra and octa(2,6-di-iso-propylphenoxy)-subs-tituted phthalocyanines (Pcs) were synthesized and their photophysico-chemical properties have been determined to evaluate the effect of both, substituents and centered transition metals.

AUTHOR INDEX (cumulative)

AAl-Awadi, Nouria 163Antholine, William 25Araki, Koiti 55

BBanerjee, Sharmila 64Baptista, Maurício S. 55Behbehani, Haider 163Bergkvist, Magnus 47Bhattacharya, Sumanta 14

CCai, Peiying 72Caritá, Eduardo 55Cavaleiro, José A.S. 101Chakraborty, Sudipta 64Chattopadhyay, Subarata 14Chidawanayika, Wadzanai 163Choi, Chun Y. 85Cohen, Brian A. 47

DDas, Tapas 64Deda, Daiana K. 55Deng, Yuefei 72

FFaustino Maria A.F. 101

GGiribabu, L. 140Giuntini, Francesca 101

HHajimohammadi, Mahdi 93Hamad, Syed 140Hashemi, Majid 149He, Xuan 72He, Xujun 77Hynninen, Paavo H. 39

JJakus, Judit 122

KKaloyeros, Alain E. 47

Keshavarzi, Ahmad 149Khene, Samson 130Kundu, Tapan K. 25Kwong, Daniel W.J. 85

LLeznoff, Daniel B. 154Li, Jia Zhu 122Li, Lili 72Li, Zao Y. 85Lindsey, Jonathan S. 1Litwinski, Christian 163Liu, Jin 72Liu, Tianjun 77Lu, li 77Lv, Feng 77

MMakhseed, Saad 163Manaa, Hacene 163Manoharan, Periakaruppan T. 25Mesilaakso, Markku 39Mofakham, Hamid 93Mohamed Ibrahim, Hamada 163Mortazavi Manesh, Anahita 93Mukherjee, Partha 14

NNeves, M. Graça P.M.S. 101Nyokong, Tebello 130, 163

PPavani, Christiane 55Pereira, Patrícia M.R. 101Poon, Chun T. 85Pu, Lingsong 72

RRafiee, Ezzat 149Ramasamy, Somasundaram 25Ramos, Catarina I.V. 101Rifkind, Joseph M. 25

SSafari, Nasser 93

Saha, Tapan K. 114

Sarma, Haladhar D. 64

Sakurai, Hiromu 114

Santana-Marques, M. Graça 101

Shim, Young Key 122

Silva, Artur M.S. 101

Silva, Eduarda M.P. 101

Solati, Zahra 149

TTaniguchi, Masahiko 1

Tewari, Surya P. 140

Toma, Henrique E. 55

Tomé, Augusto C. 101

Tomé, João P.C. 101

Tuhl, Ahmad 163

VVenugopal Rao, S. 140

WWang, Jin Jun 122

Wang, Kai 85

Wong, Edwin W.Y. 154

Wong, Wai-Kwok 85

Wu, Di 72

Wu, Li 77

XXiang, Haifeng 72

YYang, Liutao 72

Yasui, Hirouki 114

Yoshikawa, Yutaka 114

Yu, Fa Q. 85

ZZhang, Heng 85

Zhou, Xiangge 72


JPP Volume 16 - Number 1 - Pages 1–174

Aacid-base properties 39aldehyde 93aluminum(III) 72amidinophenylporphyrin 85

Bbinding mode 85

Ccarboxylic acid 93cationic porphyrins 55, 101chemical modification 122chemset 1chlorin 39, 122Click reaction 77cobalamin 25combinatorial chemistry 1conformation 39

Ddesigned bisporphyrin syn-1 14di-iso-propylphenoxy 163dithiothreitol 25DNA cleavage 85drug delivery 47

E-electron delocalization 39

electron paramagnetic resonance 25electrospray mass spectrometry 101

Ffullerenes C60 and C70 14

Gglutathione 25

HHeLa cells 55HF calculations 14

Llibrary 1lipophilicity 64logic gate 72

Mmagnesium 154mangenese porphyrin 149metallokinetics 114mass degeneracy 1methyl pyropheophorbide-a 122

Nnear infrared fluorescence probe 77NH-tautomerism 39nickel phthalocyanine 130non-covalent adducts 101nuclear magnetic resonance 39nucleotide packaging 47

Ooxovanadium(IV)porphyrin complex

114

PPDT 55p-electron delocalization 39pH 72photochemistry 163photodynamic therapy 47photooxidation 93phthalocyanine 1, 154, 163picosecond 140

platinum porphyrin 93polymeric encapsulation 55porphyrazine 1porphyrin 1, 39, 47, 64, 72prebiotic 1pyridinium salts 101pyrrole 1

Rreduction 154

Ssaccharide conjugated 77saturable absorption 140singlet oxygen 85single-walled carbon nanotube 130spectroscopic investigations 14sunlight 93

Tthyil radicals 25tin(IV) 72trageted tumor therapy 64

Vvirus 47visible absorption 122visible spectroscopy 25

Wwater 72

XX-ray 154

Zzinc phthalocyanines 77

KEYWORD INDEX (cumulative)


JPP Volume 16 - Number 1 - Pages 1–174


DOI: 10.1142/S1088424612004628

Published at http://www.worldscinet.com/jpp/

Copyright © 2012 World Scientific Publishing Company

INTRODUCTION

The isomers of substituted tetrapyrrole macrocycles have been the focus of considerable attention for many decades. In the first half of the 20th century, Fischer manually enumerated the isomers of protoporphyrin and of other tetrapyrrole macrocycles, and carried out the individual syntheses of numerous such compounds [1–3]. Systematic mathematical methods for enumerating the isomers of β-substituted tetrapyrrole macrocycles were developed in the 1970s [4–7]. The mathematical methods were chiefly applied to address questions concerning small sets of isomers of naturally occurring porphyrins, although Tapscott and Marcovich did point out that there are 5040 possible permutations (i.e. isomers) of eight

distinct entities among the eight β-pyrrolic sites of a tetrapyrrole macrocycle [7]. Study of the enumeration of isomers of substituted tetrapyrrole macrocycles subsequently became rather dormant, reflecting little interest or perhaps the appearance of a settled field, but has become relevant with the advent of combinatorial chemistry.

Combinatorial chemistry has led to practical experimental approaches for the rapid generation and screening of libraries containing vast numbers of compounds, far exceeding even those contemplated by Tapscott and Marcovich [7]. The synthesis and analysis of combinatorial libraries of tetrapyrrole macrocycles (all meso-substituted) have been pioneered by the groups of Richert [8–11], Drain [12, 13] and Boyle [14, 15]. The work to date has been comprehensively reviewed [16]. Members of such combinatorial libraries include molecular structures with distinct molecular formulas and, often, constitutional isomers thereof. By contrast,

Diversity, isomer composition, and design of combinatorial

libraries of tetrapyrrole macrocycles

Masahiko Taniguchi and Jonathan S. Lindsey*

Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA

Received 4 January 2012Accepted 25 January 2012

ABSTRACT: Combinatorial libraries of substituted tetrapyrrole macrocycles, which can now be prepared via a variety of approaches, typically are rich in isomers. Terminology for describing such isomers (due to distinct patterns of peripheral substituents) is delineated in several illustrative examples. A hierarchical relationship exists of molecular formula, condensed formula(s) of substituents, set(s) of pyrrole collocates (conveying each pair of β-pyrrolic substituents), and isomers of substituted tetrapyrrole macrocycles. Isomers with identical pyrrole collocate sets can arise owing to distinct positions or orientations of the (homo- or hetero-substituted) pyrrolic units in a macrocycle. Consideration of a handful of virtual combinatorial libraries illustrates tradeoffs of library size, chemical richness, and isomeric content. As one example, octa-derivatization of a tetrapyrrole scaffold with eight reactants A–H affords 2,099,728 members (99.7% isomers, 82,251 pyrrole collocate sets, and 6,435 condensed formulas) whereas the reversible self-condensation of four pyrroles that bear the same eight entities (AB, CD, EF, GH) affords 538 members (93.5% isomers, 35 pyrrole collocate sets, and 35 condensed formulas). Derivatization affords all combinations and permutations whereas self-condensation of substituted pyrroles carries collocational restrictions. Understanding such tradeoffs and the structural origin of isomerism are important aspects in the design of tetrapyrrole combinatorial libraries.

KEYWORDS: combinatorial chemistry, library, chemset, pyrrole, porphyrin, phthalocyanine, porphyrazine, mass degeneracy, prebiotic.

SPP full member in good standing

*Correspondence to: Jonathan S. Lindsey, email: [email protected]

Copyright © 2012 World Scientific Publishing Company J. Porphyrins Phthalocyanines 2012; 16: 2–13

2 M. TANIGUCHI AND J. S. LINDSEY

the method of Tapscott and Marcovich solely concerned permutational isomers (of β-substituted macrocycles), not products with distinct molecular formulas.

We recently developed a program (PorphyrinViLiGe)for the combinatorial generation of virtual libraries of substituted tetrapyrrole macrocycles [17]. The program employs a generative algorithm to enumerate the type and relative amounts of substituted tetrapyrrole macrocycles for each of four classes of reactions. Provisions for data mining of the virtual library enable assessment of the number of products with a chosen pattern of β- or meso-substituents. The products differ only in the nature and pattern of substituents arrayed about the perimeter of the macrocycle, and do not include free base tautomers, core modification, and compounds owing to alterations of the macrocycle skeleton.

Examination of the combinatorial libraries of tetrapyrrole macrocycles formed in the four classes of reactions revealed that the libraries were rich in isomers, with isomers often constituting >90% of the total [17]. The isomers differ only in the patterns of peripheral substituents. Such patterns play a very large role in governing the solubility, assembly, and recognition properties of the molecules. Hence, understanding the patterns that can be formed via synthetic routes and categorizing distinct patterns is central to a host of topics in tetrapyrrole chemistry. In subsequent attempts to describe the various isomers contained within such libraries, we encountered a dearth of suitable language to distinguish what appeared to us to be chemically obvious subsets of tetrapyrrole isomers. The dearth apparently is not unique to tetrapyrrole chemistry, as current terminology for the description of combinatorial libraries apparently lacks provisions for — and therefore is implicitly silent about — the presence of isomers [18, 19].

A first objective of this paper is to clearly delineate subgroups of isomers and thereby provide language for informed discussion of the members of combinatorial libraries of tetrapyrrole macrocycles. A second objective is to provide a more full articulation of the origin of isomerism as required to understand the library composition. Because such libraries typically are heavily populated with isomers, understanding the nature of the isomeric constituents is quite important for the design of suitable libraries. The paper chiefly concerns libraries of β-substituted tetrapyrrole macrocycles, which have been essentially unexplored despite their potential utility across a broad swath of the life sciences. The design of virtual libraries and delineation of the contents thereof is a valuable if not essential prelude to synthesis and screening.

RECONNAISANCE AND TERMINOLOGY

To understand the structural origins of isomerism in substituted tetrapyrrole macrocycles, consider the

13 uroporphyrins shown in Fig. 1 as a case in point. Each uroporphyrin contains four acetic acid (A) substituents and four propionic acid (P) substituents. The 13 uroporphyrins shown in Fig. 1 are isomers of each other, yet there are several subgroups that warrant distinction. In isomers I–IV, each pyrrole contains one acetic acid and one propionic acid substituent and thus is hetero-substituted. The four isomers differ only in the orientation of the pyrrole units with respect to each other in the macrocycle. Thus, isomer I has a pattern of AP-AP-AP-AP- substituents upon circumambulating the ring, whereas isomer III has a substituent pattern of AP-AP-AP-PA-.

Uroporphyrins I–IV are the only isomers available upon condensation of porphobilinogen (inset, Fig. 1) [20–22], yet in principle, nine other isomers are possible by permutation of the acetic acid and propionic acid substituents. Uroporphyrins 5 and 6, for example, each still contains four acetic acid and four propionic acid groups, but each pyrrole therein is homo-substitutedrather than hetero-substituted. In other words, each pyrrole contains two propionic acid groups or two acetic groups, but not one of each. The two propionic acid groups are positioned together on a pyrrole, as are the two acetic acid groups. Isomers 5 and 6 differ from each other owing to the position of the AA- and PP-pyrroles with respect to each other in the macrocycle: 5 has an opposite pair of AA groups whereas 6 has an adjacent pair of AA substituents.

Uroporphyrins 7–13 also each contain four acetic acid and four propionic acid groups, but in each macrocycle, two of the pyrroles are homo-substituted (with two acetic acid or two propionic acid groups) and the other two are hetero-substituted (with one acetic acid and one propionic acid group on each pyrrole). The resulting isomers stem from different orientations and different positions of the respective pyrroles in a given macrocycle.

The 13 uroporphyrin isomers are summarized in Table 1. Each uroporphyrin has the same condensed formula of substituents (A4P4). We previously used the term “substituent combinations” but henceforth abandon this term in favor of “condensed formula of substituents,” which we feel is more intuitive chemically and affords a better connection with mathematical descriptions of such distributions (e.g. Pólya’s theorem) where such descriptions have been developed [17]. We define a “set of pyrrole collocates” by the expression [st][uv][wx][yz] where each enclosed bracket defines the two β-substituents on a given pyrrole. Usage of the term collocate (Lat. collatico, “arrangement, ordering”) [23] here is analogous to that in linguistics [24]. The four brackets delineate the four pairs of substituents (due to the β-disubstituted pyrroles) in a macrocycle, but not the relative orientations or positions of the four pyrrolic units about the perimeter of the macrocycle. A more general term would be “set of quadrant collocates,” but for simplicity, we use the term pyrrole to encompass


DIVERSITY, ISOMER COMPOSITION, AND DESIGN OF COMBINATORIAL LIBRARIES 3

all tetrapyrrole macrocycles including porphyrins, phthalocyanines and porphyrazines.

Thus, pyrrole collocate set [AP][AP][AP][AP] gives four isomers (I–IV) owing to distinct orientations of the four pyrrole units; pyrrole collocate set [AA][AA][PP]-[PP] gives two isomers (5, 6) owing to distinct positions of the four pyrrole units; and pyrrole collocate set [AA][AP]-[AP][PP] gives seven isomers (7–13) owing to distinct orientations and positions of the four pyrrole units. The collection of uroporphyrins, grouped in three subsets on the basis of pyrrole collocates, is shown in Fig. 2.

The hierarchical relationship of the aforementioned terminology for describing tetrapyrrole isomers is shown

in Fig. 3. The members of a collection of porphyrin isomers all have the same molecular formula, but distinct subsets of isomers within the collection are readily delineated upon consideration of the various sets of pyrrole collocates. The condensed formula of substituents might appear to be shorthand for the molecular formula; however, coincidental mass degeneracy, when present, renders this not the case. A simple example of coincidental mass degeneracy occurs with porphyrins derived from a first pyrrole containing methyl and propyl substituents (MP), and porphyrins derived from a second pyrrole containing two ethyl substituents (EE). The M4P4-porphyrins and the E8-porphyrin would have the same molecular formula yet

N HN

NNHA

P

P

A P

A

P

A

N HN

NNHA

P

A

P P

A

A

P

N HN

NNHA

P

A

P P

A

P

A

N HN

NNHA

P

A

P A

P

P

A

I II III IV

N HN

NNHA

A

P

P P

P

A

A

N HN

NNHA

A

P

P A

A

P

P

5 6

N HN

NNHA

A

P

P P

A

P

A

N HN

NNHA

A

P

P A

P

P

A

N HN

NNHA

A

P

A P

P

P

A

N HN

NNHA

A

A

P P

P

P

A

7 8 9 10

N HN

NNHA

A

P

P P

A

A

P

N HN

NNHA

A

P

P A

P

A

P

N HN

NNHA

A

P

A P

P

A

P

11 12 13

N

A P

HH2N

porphobilinogen

Fig. 1. Thirteen uroporphyrin isomers. The isomers differ owing to the patterns of acetic acid (A) and propionic acid (P) substituents. Isomers due to N-H tautomers are ignored.



obviously distinct condensed formulas of substituents, distinct sets of pyrrole collocates thereof, and distinct molecular structures for each of the resulting porphyrin isomers. Mass degeneracy is discussed in more detail below.

COMBINATORIAL REACTIONS

The results in each of the following examples were obtained for virtual libraries generated with the program PorphyrinViLiGe [17]. Three classes of reactions are considered. For each case of pyrrole self-condensation, the reaction is considered to be reversible. Reversibility results in a hetero-substituted pyrrole entering the macrocycle in two distinct orientations, and consequently affords a greater number of products than for irreversible reaction.

Libraries of β-substituted porphyrins

Self-condensation of an AB-pyrrole. The composition of the members of a combinatorial library depends on

the nature of the synthetic route. Here we consider two routes to β-substituted porphyrins. In one reaction, the self-condensation of β-substituted pyrroles affords porphyrinogens, which upon dehydrogenation give the corresponding porphyrins (Fig. 4). Here, the accessible substituent patterns on the porphyrin are directly determined by the substituent patterns on the pyrrole reactants.

The products upon reaction of a hetero-substituted pyrrole (e.g. with A, B substituents) are shown in Table 2. Only one set of pyrrole collocates is accessible, and four isomers can be obtained. This simple case is well-known with a wide variety of examples [25], the most prominent of which is the self-condensation of porphobilinogen to give uroporphyrinogens I–IV. The number of ways that each isomer can be formed is termed the “frequency.” The ratio of the four isomers on the basis of these statistical considerations is then given by the relative frequencies.

Octa-derivatization of a porphyrin scaffold. A com-plementary route to prepare β-substituted porphyrins (or phthalocyanines) entails treatment of an octa-substituted macrocycle with one or more reactants to give the octa-derivatized product [26–34] (Fig. 5). Here, there are no constraints in principle on the collocation of the two substituents on a given pyrrole, and all possibilities

Table 1. Thirteen possible uroporphyrin isomers

Condensedformula of substituents

Pyrrole collocate sets

Porphyrins Isomer

A4P4

[AP][AP][AP][AP]

AP-AP-AP-AP- IAP-PA-AP-PA- II

AP-AP-AP-PA- III

AP-AP-PA-PA- IV

[AA][AA][PP][PP]AA-AA-PP-PP- 5AA-PP-AA-PP- 6

[AA][AP][AP][PP]

AA-AP-AP-PP- 7AA-AP-PA-PP- 8

AA-AP-PP-AP- 9

AA-AP-PP-PA- 10

AA-PA-AP-PP- 11

AA-PA-PA-PP- 12

AA-PA-PP-AP- 13

A = -CH2COOH, P = -CH2CH2COOH

Fig. 2. The thirteen uroporphyrin isomers share the same condensed substituent formula (A4P4) yet can be categorized into three subsets on the basis of pyrrole collocates

Fig. 3. Hierarchical relationship of isomeric entities with a given molecular formula

NH

R1-n R1-n

X

chemset of pyrroles

β-substituted porphyrins

N HN

NNHR1-n

R1-n R1-n

R1-n

R1-n

R1-nR1-n

R1-n

4

Fig. 4. Self-condensation of a pyrrole (or chemset [18, 19] of pyrroles) to give β-substituted porphyrins



are not only possible but expected on statistical consideration.

The use of two reactants (A, B) in the octa-derivatization process affords nine condensed formulas of substituents, 15 sets of pyrrole collocates, and 43 porphyrins as shown in Table 3. The 43 porphyrins are not all isomers of each other, however. Of the nine condensed formulas of substituents, four are represented by a single product: A8,A7B1, A1B7, and B8. A single product also implies a single corresponding pyrrole collocate set. On the other hand, 39 of the porphyrins have at least one isomeric member in the overall distribution. If there were no isomers, the number of members would equal the number of condensed formulas (nine in this case). Hence, the percentage of excess members owing to isomers is given by 100· [(number of porphyrins)–(number of condensed formulas of substituents)]/(number of porphyrins). On this basis, the percentage of excess members for the distribution in Table 3 is 100(43–9)/43 = 79%. Alternatively, the total number of members that are isomeric with other members is 39, which equates to 90% of the total. The latter method requires data mining to assess the number of condensed formulas for which there is a single member. Data mining of small libraries, as shown in Table 3, can be done by visual inspection, but can be more challenging in larger libraries. The former method is more accessible, is used here, and was employed previously [17].

Five of the nine condensed formulas of substituents each has two or three sets of pyrrole collocates. For example, the condensed formula A6B2 encompasses two sets of pyrrole collocates: [AA][AA][AA][BB] and

[AA][AA][AB][AB]. The former is composed entirely of homo-substituted pyrroles and in this particular case represents only one porphyrin. The latter case is composed of two homo-substituted pyrroles and two hetero-substituted pyrroles. The various relative positions and orientations of the four pyrroles give rise to five porphyrin isomers. Thus, porphyrin isomers with the condensed formula A6B2 are categorized in two subgroups, one member (with exclusively homo-substituted pyrroles) and a separate set of five isomers (with two homo-substituted pyrroles and two hetero-substituted pyrroles). The porphyrins with condensed formula A2B6 are subdivided in a reciprocal fashion to those with condensed formula A6B2.

The condensed formula A5B3 also encompasses two sets of pyrrole collocates ([AA][AA][AB][BB] and [AA][AB][AB][AB]), which differ in the presence of two or one homo-substituted pyrroles, respectively. The former comprises three isomers whereas the latter comprises four isomers. The seven porphyrins are all isomers yet are naturally categorized on the basis of the composition of pyrrole substitution. The porphyrins with condensed formula A3B5 are subdivided in a reciprocal fashion to those with condensed formula A5B3.

There are 13 substituted porphyrins with condensed formula A4B4; reiteration of their composition and subgroups is not necessary because the nature of this set of isomers is identical to that of the 13 uroporphyrins described above. For clarity, the 13 members categorized by three sets of pyrrole collocates are encircled in red in Table 3. One pyrrole collocate set that is accessible in this derivatization process is given by [AB][AB][AB][AB]

Table 2. Self-condensation of an AB-pyrrole to give octa-substituted porphyrins

Condensedformula of substituents

Set of pyrrole collocates

Porphyrins Frequency Yield, %

A4B4 [AB][AB][AB][AB]

AB-AB-AB-AB- 2 12.5AB-AB-AB-BA- 8 50

AB-AB-BA-BA- 4 25

AB-BA-AB-BA- 2 12.5

1 1 4 16 100

N HN

NNHXH2C

XH2C CH2X

CH2X

CH2X

CH2XXH2C

XH2C


N HN

NNH

n reactants

R1-n

R1-n R1-n

R1-n

R1-n

R1-nR1-n

R1-n

derivatization

Fig. 5. Octa-derivatization of a tetrapyrrole macrocycle, illustrated for a porphyrin



Table 3. Derivatization of eight sites with two reactants (A, B) to give octa-substituted porphyrins

Condensed formula of substituents

Pyrrole collocate sets Porphyrins Frequency Yield, %

A8 [AA][AA][AA][AA] AA-AA-AA-AA- 1 0.3906

A7B1 [AA][AA][AA][AB] AA-AA-AA-AB- 8 3.125

A6B2

[AA][AA][AA][BB] AA-AA-AA-BB- 4 1.5625

[AA][AA][AB][AB]

AA-AA-AB-AB- 8 3.125

AA-AA-AB-BA- 4 1.5625

AA-AA-BA-AB- 4 1.5625

AA-AB-AA-AB- 4 1.5625

AA-AB-AA-BA- 4 1.5625

A5B3

[AA][AA][AB][BB]

AA-AA-AB-BB- 8 3.125

AA-AA-BA-BB- 8 3.125

AA-AB-AA-BB- 8 3.125

[AA][AB][AB][AB]

AA-AB-AB-AB- 8 3.125

AA-AB-AB-BA- 8 3.125

AA-AB-BA-AB- 8 3.125

AA-BA-AB-AB- 8 3.125

A4B4

[AA][AA][BB][BB] AA-AA-BB-BB- 4 1.5625

AA-BB-AA-BB- 2 0.7813

[AA][AB][AB][BB]

AA-AB-AB-BB- 8 3.125

AA-AB-BA-BB- 8 3.125

AA-AB-BB-AB- 8 3.125

AA-AB-BB-BA- 4 1.5625

AA-BA-AB-BB- 8 3.125

AA-BA-BA-BB- 8 3.125

AA-BA-BB-AB- 4 1.5625

[AB][AB][AB][AB]

AB-AB-AB-AB- 2 0.7813

AB-AB-AB-BA- 8 3.125

AB-AB-BA-BA- 4 1.5625

AB-BA-AB-BA- 2 0.7813

A3B5

[AA][AB][BB][BB]AA-AB-BB-BB- 8 3.125

AA-BA-BB-BB- 8 3.125

AA-BB-AB-BB- 8 3.125

[AB][AB][AB][BB]

AB-AB-AB-BB- 8 3.125

AB-AB-BA-BB- 8 3.125

AB-AB-BB-BA- 8 3.125

AB-BA-AB-BB- 8 3.125

A2B6

[AA][BB][BB][BB] AA-BB-BB-BB- 4 1.5625

[AB][AB][BB][BB]

AB-AB-BB-BB- 8 3.125

AB-BA-BB-BB- 4 1.5625

AB-BB-AB-BB- 4 1.5625

AB-BB-BA-BB- 4 1.5625

AB-BB-BB-BA- 4 1.5625

A1B7 [AB][BB][BB][BB] AB-BB-BB-BB- 8 3.125

B8 [BB][BB][BB][BB] BB-BB-BB-BB- 1 0.3906

9 15 43 256 100



(encircled in blue), which in fact is the same (and only) pyrrole collocate set accessible via the pyrrole self-condensation process shown in Fig. 4.

The octa-derivatization process of Fig. 5 affords much greater diversity than that of the pyrrole self-condensation process of Fig. 4. The pyrrole self-condensation strictly constrains the accessible substituent patterns, whereas the octa-derivatization process has no such collocational restriction and affords all combinations and permutations of substitutions. Nonetheless, the products obtainable viathe pyrrole self-condensation are accessible via the octa-derivatization process, albeit in lower relative amount. The relative amounts of the porphyrins are shown in the rightmost column of Table 3.

Self-condensation of two types of pyrroles. Theself-condensation of two types of pyrroles to give octa-substituted porphyrins is shown in Table 4. The reactions differ in the nature of the collocated substituents on each of the two pyrroles. The cases range from two homo-substituted pyrroles (AA, BB) with no redundancy

(entry 1) to two hetero-substituted pyrroles (AB, CD) with no redundancy (entry 5). Two intermediate cases include one homo- and one hetero-substituted pyrrole with redundancy in one substituent (AA, AB; entry 2) or no redundancy (AA, BC; entry 3). Another intermediate case entails two hetero-substituted pyrroles with redundancy in one substituent (AB, AC; entry 4). Regardless of substituent pattern, the number of condensed formulas equals the number of pyrrole collocate sets for a given reaction. For the case of the two pyrroles, there are five condensed formulas and five pyrrole collocate sets regardless of pyrrole substitution pattern.

On the other hand, the number of porphyrins is not constant across the reaction types. The number of porphyrins is six upon use of two homo-substituted pyrroles, increases to 15 upon use of one homo-substituted pyrrole and one hetero-substituted pyrrole, and is 39 upon use of two hetero-substituted pyrroles. The number of porphyrins here does not depend on whether there is redundancy of substituents, only on whether zero, one or both pyrroles is hetero-substituted.

Table 4. Self-condensation of two pyrroles to give octa-substituted porphyrins

Entry Pyrroles Condensed formula of substituents

Pyrrole collocate sets No. of porphyrins

1 AA + BB

A8 [AA][AA][AA][AA] 1

6

A6B2 [AA][AA][AA][BB] 1

A4B4 [AA][AA][BB][BB] 2

A2B6 [AA][BB][BB][BB] 1

B8[BB][BB][BB][BB] 1

2 AA + AB

A8[AA][AA][AA][AA] 1

15

A7B1 [AA][AA][AA][AB] 1

A6B2 [AA][AA][AB][AB] 5

A5B3 [AA][AB][AB][AB] 4

A4B4 [AB][AB][AB][AB] 4

3 AA + BC

A8[AA][AA][AA][AA] 1

15

A6B1C1 [AA][AA][AA][BC] 1

A4B2C2 [AA][AA][BC][BC] 5

A2B3C3 [AA][BC][BC][BC] 4

B4C4 [BC][BC][BC][BC] 4

4 AB + AC

A4B4 [AB][AB][AB][AB] 4

39

A4B3C1 [AB][AB][AB][AC] 8

A4B2C2 [AB][AB][AC][AC] 15

A4B1C3 [AB][AC][AC][AC] 8

A4C4 [AC][AC][AC][AC] 4

5 AB + CD

A4B4[AB][AB][AB][AB] 4

39

A3B3C1D1 [AB][AB][AB][CD] 8

A2B2C2D2 [AB][AB][CD][CD] 15

A1B1C3D3 [AB][CD][CD][CD] 8

C4D4 [CD][CD][CD][CD] 4



Self-condensation of three types of pyrroles. Thenumber of condensed formulas and pyrrole collocate sets is not always identical, however. Nor does a given number of pyrrole collocate sets directly correspond to a number of porphyrins. Examples of the changing number of these three parameters are illustrated by the self-condensation of three pyrroles, as shown in Table 5.

Entry 1 provides a simple example of reaction of two homo-substituted pyrroles (AA, BB) and one hetero-substituted pyrrole (AB). Here, the number of condensed formulas (9) is fewer than that of pyrrole collocate sets (15). This deficiency is a consequence of the presence of a hetero-substituted pyrrole with substituent types (A, B) that are redundant with those of another pyrrole. The disparity between the number of condensed formulas and the number of pyrrole collocate sets was not drawn previously [17]. Interestingly, this particular case has all possible pyrrole substitution patterns for the case of two reactants (A, B). Not surprisingly, the result is identical with that upon octa-derivatization with two reactants (A, B) to give octa-substituted porphyrins (Table 3): there are 43 products, 9 condensed formulas (and hence at most 9 unimolecular masses, vide infra), and 15 pyrrole collocate sets. The condensed formulas and the 43 porphyrins from this reaction were first described by Drain and Singh [16].

The introduction of one new substituent on the hetero-substituted pyrrole (change AB to AC or CD) affords an increase in the condensed formulas but the number of pyrrole collocate sets and porphyrins remains constant (entries 2 and 3). The unchanged number of porphyrins stems from the fact that the hetero-substituted pyrrole is either present or absent in a porphyrin, and when present the change in substitution does not alter the ultimate number of products.

When one of the homo-substituted pyrroles is replaced with a hetero-substituted pyrrole, however, the number of porphyrins increases markedly. The increase

is independent of the nature of the substituents (entries 4–6) because again, the hetero-substituted pyrrole is either present or absent in a porphyrin.

When all three pyrroles are hetero-substituted, the size of the library increases further, as shown in entries 7–11. Regardless if each pyrrole has one common substituent and one distinct substituent (AB, AC, and AD, entry 9) or each pyrrole has unique substituents (AB, CD, and EF, entry 11), the number of porphyrins is identical. Such comparisons are germane in considering the reactants for the formation of combinatorial libraries. Clearly, the chemical richness of the library illustrated in entry 11 is greater than that in entry 9, because the former is composed of six types of substituents versus only four in the latter; nonetheless, the size of the two libraries is identical.

Comparison of self-condensation and derivatization. Four libraries are illustrated in Table 6. The reaction of four homo-substituted pyrroles (AA, BB, CC, DD) affords 55 porphyrins wherein the number of condensed formulas (35) is identical to the number of pyrrole collocate sets (entry 1). The equivalency is readily seen because there is no redundancy of substituent types (A, B, C, D) across the four reactants. Upon use of four hetero-substituted pyrroles (AB, AD, BC, CD) wherein each substituent type is represented twice (on each of two pyrroles), the number of porphyrins (538) increases almost by a factor of 10 vs. that of the four homo-substituted pyrroles with the same set of four substituent types (entry 2). On the other hand, the number of condensed formulas (25) is fewer than the number of pyrrole collocate sets (35) because of the substituent redundancy.

A more complex example would seem to be the use of four pyrroles where there is no redundancy of substituent among members of the reactant pool. Thus, the reaction of four such hetero-substituted pyrroles (AB, CD, EF, GH) again affords 538 porphyrins, because a given

Table 5. Self-condensation of three pyrroles to give octa-substituted porphyrins

Entry Pyrroles No. of condensed formula of substituents

No. of pyrrole collocate sets

No. of porphyrins

% of isomers

1 AA + BB + AB 9 15 43 79.1

2 AA + BB + AC 15 15 43 65.1

3 AA + BB + CD 15 15 43 65.1

4 AA + AB + BC 15 15 90 83.3

5 AA + AB + AC 15 15 90 83.3

6 AA + AB + CD 15 15 90 83.3

7 AB + AC + BC 15 15 177 91.5

8 AB + AC + BD 15 15 177 91.5

9 AB + AC +AD 15 15 177 91.5

10 AB + AC + DE 15 15 177 91.5

11 AB + CD + EF 15 15 177 91.5



hetero-substituted pyrrole is either present or absent (entry 3). Here, the number of condensed formulas (35) is identical to the number of pyrrole collocate sets.

As counterpoint, it is instructive to consider again the octa-derivatization of a tetrapyrrole macrocycle (Fig. 5), but here with eight distinct reactants (A, B, C, D, E, F, G, H) as is shown in entry 4 of Table 6. This is tantamount to the use of 82 = 64 distinct β-disubstituted pyrroles (AA, AB, AC,….FH, GH, HH). The number of porphyrins (>2 million) escalates profoundly. Each of the porphyrins accessed by the pyrrole condensation processes in entries 1–3 is present in the library created by octa-derivatization in entry 4, yet upon octa-derivatization such porphyrins are formed along with a very wide variety of other porphyrins. The very large library afforded by octa-derivatization is a consequence of unconstrained pyrrole substitution patterns vs. the constrained substitution patterns (i.e. collocation restrictions) upon condensation of a limited set of pyrrole reactants. Another comparison concerns the permutational isomers of Tapscott: there are 5040 ways of arranging eight substituents at the eight β-pyrrolic sites [7]. Such permutation is a great source of isomers contained in the library, yet the much larger size of the library (~2.1 million members) reflects the power of combinatorics: there are 6,435 distinct condensed formulas of substituents of which almost all represent ≥2 constitutional isomers. The only exceptions are the X8-porphyrins and X7Y1-porphyrins (where each of X, Y = A–H and X ≠ Y), for which there is a unique porphyrin member in each case.

In recent years, the field of combinatorial chemistry to some extent has turned away from vast libraries. Still, it warrants mention that the use of 16 distinct reactants in the derivatization process affords, in principle, 536,911,936 products [17]. Similarly, the use of 20 distinct reactants would afford 3,200,100,100 products. Representative examples of octa-substituted tetrapyrrole macrocycles amenable as scaffolds for derivatization include phthalocyanines bearing eight alcohols [31, 32] or eight ethynes [29, 30, 32, 34], and porphyrins bearing eight aryl bromides [27, 28, 33], eight carboxylic acids [26], or eight alcohols [35]. The ability to create vast

libraries with a single substrate in a one-flask reaction appears unusual and may be novel.

Meso-substituted porphyrins

The condensation of pyrrole with an aldehyde affords the corresponding meso-substituted porphyrin. The same reaction with a chemset [18, 19] of aldehydes affords a library of meso-substituted porphyrins [16]. The results upon reaction of pyrrole with three aldehydes are shown in Table 7. For meso-substituted porphyrins, the concept of a collocate set is not applicable because the four sites of substitution are each occupied by a single substituent, and in a pyrrole + aldehyde condensation the four sites are independent. By contrast, the β-substitutedporphyrins have two sites of substitution on each of four pyrrole units.

Coincidental mass degeneracies

The hierarchical relationships displayed in Fig. 3 imply that in a combinatorial library, the number of condensed formulas of substituents can exceed the number of molecular formulas. Actually, it is better said that there are cases where there are fewer molecular formulas than condensed formulas of substituents. Here, several examples are provided to illustrate the manner in which such disparities arise, all of which stem from mass degeneracies.

The reaction of the four pyrroles shown in Table 8 affords β-substituted porphyrins. Here, the number of molecular formulas is less than the number of condensed formulas of substituents owing to the presence of redundant substituents in the pyrrole chemset (see Table 6, entry 2). The number of molecular formulas depends on the chemical composition of the substituents and on their presence in the condensed formulas.

In the first example (Table 8, entry 1), the AD-pyrrole and the BC-pyrrole have the same molecular formula, which gives rise to a sizable deficiency of molecular formulas (9) versus the condensed formulas (25). In the second example (entry 2), the same four substituents (H, methyl, ethyl, propyl) are present, yet because of a

Table 6. Routes to octa-substituted tetrapyrrole macrocycles

Entry Reactants No. of condensed formula of substituents


No. of porphyrins

% of isomers

Pyrroles:

1 AA + BB + CC + DD 35 35 55 36.4

2 AB + AD + BC + CD 25 35 538 95.4

3 AB + CD + EF + GH 35 35 538 93.5

Tetrapyrrole:

4 Derivatization with eight reactants A-H

6,435 82,251 2,099,728 99.7



switch in the patterns (C = propyl not ethyl, D = ethyl not propyl), all four pyrroles now have distinct molecular formulas. Nonetheless, the combined formula of two pyrroles (AB, CD) equals that of the remaining two pyrroles (AD, BC). Accordingly, there is again a deficit of distinct molecular formulas (17) versus condensed formulas. In entry 3, all four pyrroles have distinct formulas and no combined groups of pyrroles have identical formulas, hence there are no mass degeneracies and the number of molecular formulas is equal to the number of condensed formulas.

Mass degeneracies also can occur upon derivatization (where there are no collocation restrictions), although the

origins may not always be as clear as in the two examples given in Table 8. A case is provided for octa-derivatization with four groups, which affords 165 condensed formulas and 8356 porphyrins (Table 9). Remarkably, when A, B, C, D = H, methyl, ethyl, and propyl, the number of molecular formulas is only 25. In the absence of mass degeneracy, there would be 165 molecular formulas. The deficit in molecular formulas stems the homologous nature of the A–D substituents in this case and the presence of eight groups drawn from A–D on each porphyrin. On the other hand, when there are no mass degeneracies in the four substituents, as illustrated in the second example of Table 9, the number of molecular formulas is equal to the number of condensed formulas.

The challenges caused by species of identical molecular formulas (or overlapping mass spectrometric peaks) in combinatorial libraries have been discussed previously [36, 37], but may be particularly acute in tetrapyrrole libraries. The presence of such species has obvious ramifications for characterization by mass spectrometry. The number of expected peaks upon mass spectrometry is obviously given by the number of molecular formulas. On the other hand, the incomplete information provided by molecular formula alone gives added impetus to the use of condensed formulas and sets of pyrrole collocates to identify sets of structural isomers.

OUTLOOK

Combinatorial chemistry has been a strong animating feature of the molecular sciences over the past two decades. With the important exception of the pioneering work of Drain, Richert, and Boyle in the preparation of libraries of meso-substituted porphyrins [8–15], the methodology of combinatorial chemistry has largely not penetrated the tetrapyrrole field. In this regard, tetrapyrrole combinatorial libraries represent a somewhat undeveloped domain, yet one that may be rich in potential. The issues discussed herein illustrate the variety, size, and isomeric composition of libraries that can be created. The designer of such libraries can exercise a degree of control over the number, chemical richness, and isomeric content by choice of reaction and reactant chemset. The delineation of the members of libraries is a desirable if not essential accompaniment to synthetic considerations. The PorphyrinViLiGe program (version 1.1) with terminology employed herein is freely available at www.photochemcad.com.

The libraries discussed herein are largely composed of β-substituted porphyrins, but can equally include α- or β-substituted phthalocyanines and homologues thereof. The ability to create such libraries depends on the availability of reactants, including pyrroles (for porphyrins) and diminoisoindolines or phthalonitriles (for phthalocyanines). Pyrrole chemistry often presents a number of challenges given the intrinsic reactivity of this class of compounds.

Table 7. Aldehydes (A, B, and C) + pyrrole to give meso-substituted porphyrins

meso-substituted porphyrins

N HN

NNH

R1-n

R1-n R1-n

R1-n

Condensed formula of substituents

Porphyrins Frequency Yield, %

A4 A-A-A-A- 1 1.235

A3B1 A-A-A-B- 4 4.938

A3C1 A-A-A-C- 4 4.938

A2B2 A-A-B-B- 4 4.938

A2B2 A-B-A-B- 2 2.469

A2B1C1 A-A-B-C- 8 9.877

A2B1C1 A-B-A-C- 4 4.938

A2C2 A-A-C-C- 4 4.938

A2C2 A-C-A-C- 2 2.469

A1B3 A-B-B-B- 4 4.938

A1B2C1 A-B-B-C- 8 9.877

A1B2C1 A-B-C-B- 4 4.938

A1B1C2 A-B-C-C- 8 9.877

A1B1C2 A-C-B-C- 4 4.938

A1C3 A-C-C-C- 4 4.938

B4 B-B-B-B- 1 1.235

B3C1 B-B-B-C- 4 4.938

B2C2 B-B-C-C- 4 4.938

B2C2 B-C-B-C- 2 2.469

B1C3 B-C-C-C- 4 4.938

C4 C-C-C-C- 1 1.235

15 21 81 100



In the case of porphyrins, one approach could employ the synthesis of a combinatorial library of pyrroles [38–42] with use of the resulting members in the preparation of a combinatorial library of porphyrins. Our discussion has focused on octa-derivatization of β-substituted tetrapyrrole macrocycles [26–35] as a concise route to libraries of vast size. Similarly, meso-tetraarylporphyrins bearing

two ethynes [43], two bromomethyl units [44], or two carboxylic acids [45] on each meso-aryl unit also present eight sites for derivatization.

Our own interest in combinatorial chemistry stems from studies aimed at developing a chemical model for the possible prebiogenesis of tetrapyrrole macrocycles [46, 47]. In this context, we recently described a tandem

Table 8. Mass degeneracies and molecular formulas upon self-condensation of four pyrroles

NH

A B

X


N HN

NNH

R1-n

R1-n R1-n

R1-n

R1-n

R1-nR1-n

R1-n

NH

A D

X

NH

B C

XNH

B D

X

chemset of four pyrroles

Entry Substituents upon derivatization

No. of molecularformulas

No. of condensedformula of substituents


No. of porphyrins

1 A = -H

B = -CH3

C = -CH2CH3

D = -CH2CH2CH3

9 25 35 538

2 A = -H

B = -CH3

C = -CH2CH2CH3

D = -CH2CH3

17 25 35 538

3A = -SH

B = -OCH3

C = -OCOCH3

D = -NH2

25 25 35 538

Table 9. Mass degeneracies and molecular formula upon octa-derivatization

Substituents upon derivatization

No. of molecular formulas

No. of condensed formula of substituents


No. of porphyrins

-H

-CH3

-CH2CH3

-CH2CH2CH3

25 165 715 8356

-SH

-OCH3

-OCOCH3

-NH2

165 165 715 8356



combinatorial route wherein pyrroles are created in situfrom acyclic reactants, and the pyrroles thus formed self-condense to give ultimately a collection of porphyrinogens [48, 49]. As in other areas of combinatorial science, the rapid creation of a diverse collection of molecules opens a number of opportunities that are not available viaunitary syntheses.

Acknowledgements

This work was supported by a grant from the NSF Chemistry of Life Processes Program (NSF CHE-0953010).

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DOI: 10.1142/S1088424611003811



INTRODUCTION

There is an increasing interest among the chemists in finding the cooperative effect in non-covalent interactions, which can be successfully employed for the construction of quite complex structures [1]. Several classes of porphyrins and derivatized porphyrins have been used for this purpose and in many cases, the resulting systems have been served as models for the study of photo-induced energy [2, 3] and electron transfer [4–9] process that mimic events occur-ring in natural photosynthesis. In recent past, highly effi-cient porphyrin sensitizers are reported by Campbell et al.for the construction of dye-sensitized solar cell [10]. It is already reported that fullerene and porphyrin form natu-rally assembled co-crystallates, which are characterized by an unusually short fullerene/porphyrin distance (3.0–3.5 Å) [11, 12] and that a number of fullerene/porphyrin systems form an emissive charge transfer (CT) state both

in solutions [13, 14] and in solid state [15]. These systems differ from previously studied emitting CT systems by the size of interacting electron subsystems, which are signifi-cantly larger as compared, e.g. to anthracene derivatives [16]. Recently, self-assembled supramolecular methodol-ogy using axial coordination, hydrogen bonding, crown ether-ammonium cation complexation, rotaxane forma-tion and additional studies, has been successfully utilized to construct fullerene/porphyrin assemblies [17–20]. Improved stability and control over the distance and ori-entation are achieved by utilizing multiple modes of bind-ing, such as covalent-coordinate and coordinate-hydrogen bond formation in a few instances [21]. However, the introduction of selectivity paradigm into supramolecular chemistry brings about a fundamental change in ways, means and outlook. The ultimate goal is to make a plat-form for the formation of selective and effective com-plexes. Therefore, in the pursuit of improved stability and control over the distance and orientation, molecular design of porphyrin hosts that can selectively bind with C60 or C70

is a formidable task in the field of host-guest chemistry.

Spectroscopic and theoretical insights on determination

of binding strength and molecular structure for the

supramolecular complexes of a designed bisporphyrin with

C60 and C70

Partha Mukherjeea, Subarata Chattopadhyayb and Sumanta Bhattacharya*a

a Department of Chemistry, The University of Burdwan, Golapbag, Burdwan 713 104, Indiab Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

Received 31 March 2011Accepted 23 April 2011

ABSTRACT: The present article examines the binding affinity of the newly designed Zn2-bisporphyrin molecule, syn-1, towards C60 and C70 in toluene medium. The investigation is carried out by UV-vis spectrophotometric, steady state and time-resolved fluorescence spectroscopic techniques. The bisporphyrin, syn-1, serves as an effective and selective molecular tweezer for C70 as average value of binding constants (K) for the non-covalent complexes of syn-1 with C60 and C70 are estimated to be 1.65 × 104 and 1.05 × 105 dm3 ·mol-1, respectively. Binding of C70 in the cleft of syn-1 is clearly demonstrated by the quantum chemical calculations at ab initio level of theory. Molecular electrostatic potential maps demonstrate significant redistribution of charges in these supramolecules. Proton NMR studies suggest that the C70 moiety remains at the shallow part of the cleft of syn-1.

KEYWORDS: fullerenes C60 and C70, designed bisporphyrin syn-1, spectroscopic investigations, HFcalculations.

*Correspondence to: Sumanta Bhattacharya, email: [email protected], fax: +91 342-2530452


SPECTROSCOPIC AND THEORETICAL INSIGHTS ON DETERMINATION OF BINDING STRENGTH 15

In the present study, we have adopted a two-point non-covalent bonding strategy for the construction of a highly stable fullerene/porphyrin supramolecular complex. Here, a covalently linked zinc porphyrin dimer connected through naphthalene spacer is allowed spontaneously to interact with fullerenes C60 and C70 in a toluene medium. The role played by the spacer is not just limited to structural con-cerns, but more importantly, its chemical nature influences the electronic communication between the electron accep-tor (i.e. fullerene) and photo-excited electron donor (i.e.syn-1). As a leading example for a short-spaced dyad, the

-stacked fullerene/porphyrin dyad (C60-ZnP) should be cited [22] whose electron transfer and back electron trans-fer dynamics helped to establish the presence of highly energetic charge-separated state. Similar observations stem from a parachute-shaped dyads [23], as well as C60-ZnP dyads with a variety of phenyl spacers [24]. In two of the earlier works of the authors [25, 26], a strong evi-dence for a high affinity between fullerene and designed porphyrin molecules is noticed. Thus, the search for new porphyrin receptor prompt us to design the new type of Zn2-bisporphyrin (syn-1), as designed host molecule. In our present study, in order to address the effective and selective interaction between fullerenes and the designed bisporphyrin syn-1 (Fig. 1), we have carried out a detailed UV-vis absorption spectrophotometric and steady state fluorescence measurements of the complexes of syn-1with C60 and C70 in a non-polar (i.e. toluene) medium. The spectroscopic investigations of the complexes enable us to determine the extent of binding as well as selectivity in binding constant (K) between C60/syn-1 and C70/syn-1complexes. Computational quantum chemistry methods have provided a good support in interpreting the stabil-ity difference between C60 and C70 complexes of syn-1 in terms of heat of formation ( Hf

0) and molecular electro-static potential (MEP) map calculations.

EXPERIMENTAL

Equipments

All UV-vis spectral measurements are carried out on a UV 1601 PC model spectrophotometer fitted with Peltier

controlled thermo bath. Steady state fluorescence mea-surements are recorded in a F-4500 Hitachi model spec-trofluorimeter. Theoretical calculations have been done in a Pentium IV computer using SPARTAN’06 Windows version software.

Materials

C60 and C70 are obtained from Merck, Germany and Aldrich, USA, respectively. The solvent toluene is of UV spectroscopic grade. Toluene is used as solvent to provide extra photo-stability to the samples. Syn-1 is obtained as a gift item from Prof. Hidemitsu Uno, Ehime University, Japan.

RESULTS AND DISCUSSION

UV-vis spectral studies

The ground state absorption spectrum of syn-1(Fig. 2a) displays (i) very intense Soret absorption band ( max = 403 nm) corresponding to the transition to the second excited singlet state S2, and (ii) two Q-bands at 530 and 570 nm corresponding to the vibronic sequence of the transition to the lowest excited single state S1. It should be noted at this point that although syn-1 contains two porphyrin units, noticeable splitting of the Soret absorption band may not be observed. The splitting of the Soret absorption band in case of bisporphyrin mol-ecule generally takes place due to the presence of exciton coupling that was originated from the Coulombic inter-actions between the transition dipole moments of the two porphyrin subunits [27]. Similar sort of observation is also reported by Borovkov et al. for their particular bisporphyrin-based systems [28]. Ground state electronic interactions between fullerenes and syn-1 are first evi-denced from UV-vis titration experiment. It is observed that gradual addition of a C60 solution to a toluene solution of syn-1 decreases the absorbance of Soret band and red shifted it from 403 to 406 nm (inset of Fig. 2a). However, no additional absorption peak is observed in the visible region. The former observation extends a good support in favor of the complexation between C60 and syn-1 in ground state. The latter observation indicates that the interaction is not controlled by charge transfer (CT) type transition. Isosbestic point is located at 414 nm, giving good support of 1:1 complexation between two species [29]. The stoichiometry of the C60/syn-1 complex is also confirmed by Jobs plot of continuation variation method (Fig. 2b). Similar sort of absorption spectral phenomena are also observed in C70/syn-1 system (Fig. 3a), though the Soret absorption band has been red shifted by 6 nm probably due to greater interaction between C70 and syn-1compared to C60. Isosbestic point is also observed at 412 nm (Fig. 3a). All the above phenomena indicate that syn-1 undergoes appreciable amount of interaction with both C60 and C70. Stoichiometry of the C70/syn-1 system Fig. 1. Structure of syn-1


16 P. MUKHERJEE ET AL.

is found to be 1:1 as determined from the Jobs continu-ation variation analysis (Fig. 3b). The most important observation in the present investigation is that the Soret absorption bands get affected more than the Q-absorption bands, which is already observed by Guldi et al. for their particular fullerene/porphyrin systems [30]. Therefore, the gradual decrease in the absorbance of the Soret band of syn-1 is used to determine the binding constant (K) by Benesi-Hildebrand (BH) plot [31]. Sibley et al. have also estimated K values for the complexes of C60 with aniline and its derivatives [32] using BH equation for cells with 1 cm optical path length as shown below.

[A]0[D]0/d = ([A]0/d ) + (1/K ) (1)

with d = dD0 - d (2)

Here [A]0 and [D]0 are the initial concentrations of the acceptor (i.e. fullerenes C60 and C70) and donor (syn-1)

solutions in toluene, respectively; d is the absorbance of the fullerene/syn-1 mixture at the wavelength of measu-rement (e.g. 403 nm) against the same fullerene concen-tration as reference; dD

0 is the absorbance of the syn-1solution with same molar concentration as in the mix-ture. The quantity (= c - A - D) means the correc-ted molar absorptivity of the complex, D and A being those of the syn-1 and fullerenes, respectively. K is the binding constant of the fullerene/syn-1 complex. Equation 1 is valid under 1:1 approximation for syn-1 and fullerene systems. It should be mentioned at this point that the corrected molar extinction coefficient, , is not quite that of the complex. The BH method [31] is an approximation that we have used many times and it gives decent answers. But the extinction coefficient is really a different one between the complex and free species that absorbs at the same wavelength. Experimental data for

Fig. 2. (a) (i) UV-vis absorption spectra of syn-1 (3.86 × 10-6 M) recorded against the solvent as reference and set (ii)–(viii) indica-tes UV-vis absorption spectra of syn-1 (3.86 × 10-6 M) in the presence of C60 (1.429 × 10-5 to 3.572 × 10-5 M) recorded against the pristine C60 solution as reference; (b) Jobs plot of continuous variation for C60/syn-1 system



spectrophotometric determination of binding constant for the supramolecular complexes of syn-1 with C60

and C70 are provided in Tables S1 and S2, respectively. In all the cases very good linear plots are obtained for C60 /syn-1 and C70 /syn-1 systems, as shown in Figs 4a and 4b, respectively.

Fluorescence investigations of the complexes of syn-1with C60 and C70

The photo induced behavior of the complexes of C60

(Fig. S1a) and C70 (Fig. 5a) with syn-1 are investigated by steady-state emission measurements. It is observed that the fluorescence of syn-1 upon excitation at Soret absorption band diminishes gradually during titration with C60 and C70 solutions in toluene. This indicates that there is a relaxation pathway from the excited singlet state of the porphyrin to that of the fullerene in toluene. It is already reported that charge separation can also occur from the excited singlet state of the porphyrin to

the C60 in toluene medium [33]. Competing between the energy and electron transfer processes is a univer-sal phenomenon in donor molecule-fullerene complexes [34], solvent dependent photophysical behavior is a typi-cal phenomenon of the most fullerene/porphyrin dyads studied to date [35]. Photophysical studies already prove that in conformationally flexible fullerene/porphyrin dyad, -stacking interactions facilitate the through space interactions between these two chromophores which is demonstrated by quenching of 1porphyrin* fluorescence and formation of fullerene excited states (by energy transfer) or generation of fullerene-porphyrin+ ion-pair states (by electron transfer) [36]. However, in non-polar solvent, energy transfer generally dominates (over the electron transfer process) the photophysical behavior in deactivating the photo excited chromophore 1porphyrin*

of fullerene/porphyrin dyad. Similar sort of rationale is already proved by Yin et al. for their particular cis-2,5-dipyridylpyrrolidino[3,4:1,2]C60/zinc tetraphenylpor-phyrin supramolecule [37]. In the present investigation,

Fig. 3. (a) UV-vis absorption spectra of syn-1 (5.550 × 10-6 M) recorded against the solvent as reference and set (ii)–(viii) indicates UV-vis absorption spectra of syn-1 (5.550 × 10-6 M) in the presence of C70 (3.570 × 10-6 to 4.320 × 10-5 M) recorded against the pristine C70 solution as reference; (b) Jobs plot of continuous variation for C70/syn-1 system



therefore, the quenching phenomenon can be ascribed to photo-induced energy transfer from porphyrins to fuller-enes. As we use the Soret absorption band as our source of excitation wavelength in fluorescence experiment, the 2nd excited singlet state of syn-1 is deactivated by singlet-singlet energy transfer to the fullerene. The spec-tral changes finally reach a plateau by the complexation with C70 and C60 (inset of Fig. 5a and Fig. S1b, respec-tively). Although syn-1 exhibits fluorescence quenching upon the addition of fullerenes, the quenching efficiency of C70 is higher than that of C60. The binding constants of the C60 / and C70 /syn-1 complexes have been evaluated according to the modified BH plots as shown in Figs S2 and 5b, respectively [32]. The modified BH equation is generally expressed in following form:

{F0 /(F0 - F)} = (1/A) + {(1/KA) (1/[fullerene]) (3)

In Eq. 3, F0 and F are the fluorescence intensity of syn-1without and with the fullerenes, respectively; [fullerene]

indicates the molar concentration of fullerene; A isthe constant associated with the difference in the emission quantum yield of the complexed and uncom-plexed syn-1. By plotting F0/(F0 - F) vs. 1/[fullerene], a surprisingly high binding constant (K) is obtained for C70/syn-1 system. Data for steady state fluorescence titration of for the determination of binding constant for the supramolecular complexes of syn-1 with C60

and C70 are provided in Tables S3 and S4, respecti-vely. BH plots of C60/syn-1 and C70/syn-1 complexes are demonstrated in Figs S2 and 5b, respectively. It is interesting to note that the increase in magnitude of binding constant led to increase of the fluorescence quenching efficiency. Although the details are not clear at this point, the well-defined structure of syn-1has afforded tight fixing of C70 should give rise to cor-rect host-guest orientation. The K values determined by fluorescence method agree fairly well with those obtained by spectrophotometric measurements dis-cussed in Section 3.2 (Table 1).

Binding constants

Binding constants (K) of various fullerene/syn-1complexes are summarized in Table 1. It is observed that syn-1 undergoes appreciable amount of com-plexation with both C60 and C70. The average value of binding constant of C60/syn-1 complex (K = 1.65 × 104 M-1) is found to be large compared to those of the previously reported thiacalixarene bisporphy-rin (K = 2340 M-1) [38], C60 complexes of zinc(II) resorcinarene (K = 5010 M-1) [39] and terphenyl porphyrin tetramer (K = 5800 M-1) [40]. The bind-ing of C60 to syn-1 is also found to exhibit much larger value to that reported for JAWS porphyrin (K = 1950 M-1) [41] and, somewhat surprisingly, stronger than a number of other first row transi-

tion metalloporphyrins (K = 815 M-1) [41]. Similarly, C70/syn-1 complex exhibits almost similar magnitude of binding constant with cyclic dimers of Zn-porphyrins (K = 1.1 × 105 M-1) [42]. However, both C60 and C70 dem-onstrate much lower value of K for their complexes with syn-1 compared to porphyrin hexamer (K = 1.4 × 108 M-1)[43]. However, the most interesting feature of the present investigation is that syn-1 binds C70 in preference to C60

[26] and the average KC70/C60 selectively is estimated to be ~6.5 in toluene. Although, the average value of pres-ent selectivity is found to be somewhat lower than that of bridged calix[5]arene (~10.2), [44] it exhibits higher value than those of azacalix[m]arene[n]pyridine (~1.9), [45] cyclotriveratrylenophane (~2.5) [46] and calixarene bisporphyrin (~4.3) [38]. However, the present selectiv-ity is found to be lower than those of observed for cyclic bisporphyrins (~25 to 32) [42, 47]. Practical applications on such high selectivity to C70 is also observed in the preferential precipitation of C70 over C60 with host mol-ecule like p-halohomooxacalix[3]arene [48]. The larger

Fig. 4. BH plot of (a) C60/syn-1 and (b) C70/syn-1 systems in toluene



values of the K for C70 compared to C60 with porphy-rins are commonly observed in the work of Tashiro and Aida [49] or Boyd and Reed [50]. The preference in the fullerene C70 complexation also indicates that subtle dif-ference in solvation energies between C60 and C70 is the key factor behind the variation of affinities. It is well-known that the stability of supramolecular complexes depends upon attractive interactions between host and guest, and on the solvation of the binding partners [51]. In the present investigations, since the host and guest are separately solvated in solution, the desolvation is ener-getically an uphill task and, hence, an association takes place only when the energy gain between the host and

guest interaction exceeds this unfavorable energy. From the trends in the K values of fullerene/syn-1 complexes, we may infer that the extent of solvation and desolva-tion of the binding partners might play an important role in forming weak or strong supramolecular complexes. Haino et al. [52] show that the stability of the com-plex is increased as the solubility of fullerene in solvent decreases, since less energy is required for the desolva-tion of fullerene which must necessarily precedes its complexation with a host molecule. This would be one of the reasons for the higher K values of the C70 complexes in toluene, since the solubility of C60 is higher in toluene (4.1 mg/mL) than that of C70 (2.0 mg/mL) [53].

Fig. 5. (a) Steady state fluorescence quenching of syn-1 (3.25 × 10-6 M) by C70 (from (i) 0 to (ix) 3.56 × 10-5 M) in toluene medium and in inset, plot of relative fluorescence intensity vs. [C70] for the same system at 298 K; (b) Bh fluorescence plot of C70/1 system in toluene



Computational studies

To visualize the geometry and electronic structure of the individual components as well as the fullerene/syn-1adducts, computational studies are performed at the restricted Hartree Fock (RHF)/3-21G* level, in vacuo.For this reason, the fullerene/syn-1 complexes are opti-mized on the Born-Oppenheimer potential energy sur-face, and a global minimum for each of the fullerene complexes are recognized (see Fig. 6). For the energy classification of the optimized structures, we used the following (standard) interaction energy (IE) (i.e. Hf

0)definitions. IEs have been estimated within the supramo-lecular approach IE = Ecomplex - [Efullerene + Esyn-1] (where Ecomplex, Efullerene and Esyn-1, are the energies of the fuller-ene/syn-1 complex, uncomplexed fullerene and free syn-1, respectively) in order to determine the total strength of the various interaction patterns between the fullerenes (C60 and C70) and the receptor, e.g. syn-1. In all of the structures reported here, two porphyrin units of syn-1 are arranged in such a manner so that they can form cavity for the approaching fullerene surface, facilitating a close face-to-face fullerene/porphyrin contact. It is observed that the Zn metal is pulled out of the porphyrin plane after complex formation with fullerenes. Such a square pyramidal geometry is well-known for metalloporphy-rin [54]. The center-to-center distance, i.e. the distance between the central zinc metal and the center of the C60

and C70 spheroids are computed to be 5.872 Å and 5.792 Å, respectively. Close distance between C70 and syn-1 in comparison to C60 gives clear evidence in favor of strong intermolecular interaction for C70/syn-1 supramolecule. Another interesting feature of present investigations

is that in presence of C70, the distance between Zn(1) and Zn(2) atoms of syn-1 is estimated to be 13.772 Å while the distance remains at 13.725 Å in case of C60/syn-1 supramolecular complex. Thus, cavity of syn-1 is much larger when C70 approaches to syn-1. Hf

0 for the supramolecular complexes of syn-1 with C60 and C70 are measured in present case in vacuo. Both C60/syn-1 and C70/syn-1 complexes are shown to exhibit negative Hf

0

value. The negative heat of formation values are resulted from the formation of the complexes predominate over the other opposing factors (i.e. dissociation of com-plexes). Theoretically estimated Hf

0 values (see Table 1) also support the high K value observed in case of C70/syn-1 complex. Hf

0 value is more negative in case of C70/syn-1 complex indicating that above complexation process is enthalpy favored. The trend of magnitude of

Hf0 i.e. Hf

0 (C60/syn-1) > Hf0 (C70/syn-1) can be ratio-

nalized as follows: randomness of C60 molecule is so high that this particular species failed to approach quite close to syn-1. For this reason, effective overlap between their molecular orbital may not take place. In contrast, much stronger association is predicted computationally for C70/syn-1 complex in which the aromatic moiety of syn-1molecule containing four N atoms appear to interact with the graphitic equatorial belt of C70. During complexation with syn-1, the orientation of C70 molecule towards the porphyrin is proved to be side-on rather than end-on, confirming structural deductions based on RHF/3-21G* calculations (ab initio, see Fig. 6). The van der Waals attraction is considered to be maximized in the side-on structure of the complex since the flatter equatorial regions of C70 have more contact with the porphyrin than the highly curved polar region. This can also be viewed

Fig. 6. Ab initio optimized space filling models of the C60/syn-1 and C70/syn-1 systems done by RHF/3-21G method

Table 1. Binding constants of the complexes of C60 and C70 with syn-1 at 298 K along with theoretically determined heat of formation ( Hf

0) values of C60/syn-1 and C70/syn-1 complexes by RHF/3-21G* method in vacuo

System Hf0, kJ.mol-1 K, M-1 KC70/KC60

UV-vis Fluorescence

C60/syn-1 -2.450 20,970 ± 1,050 12,140 ± 650 4.2

C70/syn-1a -6.530 88,750 ± 4,250 106,450 ± 5,300 8.8

C70/syn-1b -6.850

a End-on orientation. b Side-on orientation.



Fig. 7. Pictures of frontier highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of C60/syn-1 ((a) and (b)), C70/syn-1 (side-on) ((c) and (d)) and C70/syn-1 (end-on) ((e) and (f)) complexes done by HF/3-21G calcula-tions. Values of HOMOs and LUMOs of the fullerene/syn-1 complexes are listed in Table S1

Table 2. Comparison of five HOMOs and five LUMOs of C60/syn-1 and C70/syn-1 systems(in both side-on and end-on orientations) along with syn-1 done by RHF/3-21G* method

State Energy, eV

C60/syn-1 C70/syn-1 (end-on) C70/syn-1 (side-on) Syn-1

HOMO-4 -7.6959 -7.6885 -7.6713 -7.6762

HOMO-3 -6.7837 -6.7884 -6.7866 -6.7660

HOMO-2 -6.7729 -6.7772 -6.7772 -6.7549

HOMO-1 -6.0472 -6.0519 -6.0500 -6.0296

HOMO -6.0388 -6.0429 -6.0416 -6.0211

LUMO -0.5331 -0.7253 -0.7217 0.5476

LUMO+1 -0.5317 -0.7230 -0.7191 0.5588

LUMO+2 -0.5234 -0.5639 -0.5365 0.5882

LUMO+3 0.5303 0.0417 0.0650 0.5959

LUMO+4 0.5413 0.5257 0.5274 3.0598



in terms of higher number of 6:6 bonds present in the equatorial region of C70 compared to C60. In addition to this dispersion force, the attraction must also be subject to the subtle interplay of small effect from differences in solvation, and electrostatic interactions.

From the RHF/3-21G*-generated HOMOs and LUMOs of the supramolecular complexes of syn-1 with C60 and C70 (see Fig. 7, Table 2), the HOMO is located on the porphyrin system, while the LUMO is positioned on the fullerene spheroid. Importantly, part of the HOMO is also located on the naphthalene moiety of syn-1, sug-gesting considerable interaction between donor and acceptor moieties. It is observed that while the HOMO is mostly localized at the side-on porphyrin ring, the HOMO-1 electronic state is positioned partially at the end-on porphyrin ring. This is well understood by larger electrostatic attraction between the side-on porphyrin ring and C70 than that between the end-on porphyrin ring and C70.

NMR studies

NMR spectra of syn-1 with or without fullerenes have been measured in THF-d8. The complex of syn-1 with C70

is used for this experiment as C70 itself does not dissolve in THF and the binding constants of C70 with bispor-phyrins are larger than those of C60.

1H NMR spectra of uncomplexed syn-1 and C70/syn-1 complex are shown in Figs 8a and 8b, respectively. All the signals in 1H NMR spectra are unambiguously assigned by ROESY. Obvi-ously up-field shifts are observed for the NMR peaks of syn-1 after complexation with C70. Signals due to bridge head, inner meso, and outer meso protons of bisporphyrin syn-1 exhibit up-field shifts of 0.05, 0.07, and 0.14 ppm, respectively, upon complexation with C70. This phenom-enon suggests that the C70 moiety remain at the shallow part of the cleft of syn-1 as evidenced from the computa-tional studies.

CONCLUSION

From the foregoing discussions we can surmise the results of the following investigations as follows. (1) The

present investigations report ground state non-covalent interactions between fullerenes (C60 and C70) and a newly designed Zn2-bisporphyrin molecule, syn-1 in toluene medium. (2) Estimation of binding constants applying UV-vis and steady state fluorescence spectroscopic tech-niques reveal that syn-1 can effectively and selectively be employed as molecular tweezers for C70. (3) Quantum chemical calculations at ab initio level of theory suggest that C70 is encapsulated in side-on orientation within the cavity of syn-1. (4) Proton NMR studies clearly demon-strate the strong intermolecular interaction between C70

and syn-1. (5) The results emanating from present inves-tigations would certainly enhance the probabilities to apply syn-1 for encapsulation of nanotubes having small diameter in near future.

Acknowledgements

SB acknowledges UGC, New Delhi and DST, India for providing financial support in this work. The authors also wish to extend their gratitude to Prof. Karl M. Kadish and Prof. Francis D’Souza for their valuable comments.

Supporting information

Data for UV-vis titration of C60/syn-1 and C70/syn-1complexation processes in toluene medium, data for steady state fluorescence investigations of C60/syn-1 and C70/syn-1 complexation processes in toluene medium, steady state fluorescence spectral variation of C60/syn-1system in toluene medium and fluorescence induced curve for similar system in same solvent, BH fluores-cence plot of C60/syn-1 system in toluene are provided as Tables S1–S4 and Figs S1–S2, respectively. Tables S1–S4 and Figures S1–S2 are given as supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S1088424611004051



INTRODUCTION

Cobalamin (Cbl) dependent enzymes catalyze three important types of reactions; viz intramolecu-lar rearrangements, methylation and the reduction of

ribonucleotides to deoxyribonucleotides. Out of nearly 15 enzyme reactions that require vitamin B12, the con-version of L-methylmalonyl CoA into succinyl CoA and the formation of methionine by methylation of homo-cysteine are the only reactions occurring in mammals that are known to require coenzyme B12. The importance of regulating homocysteine is evident by the finding that mild to moderate elevations of serum homocysteine are observed in Alzheimer’s disease [1] and that there is an

Internal spin trapping of thiyl radical during the

complexation and reduction of cobalamin with

glutathione and dithiothrietol

Somasundaram Ramasamya, Tapan K. Kundub, William Antholined,

Periakaruppan T. Manoharan*b,c and Joseph M. Rifkind*a

a Molecular Dynamics Section, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard Baltimore, Maryland 21224, USAb Sophisticated Analytical Instruments Facility and Department of Chemistry, Indian Institute of Technology-Madras, Chennai 600036, Indiac School of Sciences, Indira Gandhi National Open University, New Delhi 110 068, Indiad Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

Received 27 March 2011Accepted 13 June 2011

ABSTRACT: The activation of cobalamin requires a reduction from cobalamin(III) to cobalamin(II).The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored. Glutathione first reacts with oxidized cobalamin(III). The binding of a second glutathione required for the reduction to cobalamin(II) is presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity. The reduction of cobalamin(III) by dithiothreitol, which contains two thiols, is much faster even though no stable cobalamin(III) complex is formed. The reduction, by both thiol reagents, results in the formation of thiyl radicals, some of which are released to form oxidized thiol products and some of which remain associated with the reduced cobalamin. In the reduced state the intrinsic lower affinity for the benzimidazole base, coupled with a trans effect from the initial glutathione bound to the β-axial site and a possible lowering of the pH results in an equilibrium between base-on and base-off complexes. The dissociation of the base facilitates a closer approach of the thiyl radical to the cobalamin(II) α-axial site resulting in a complex with ferromagnetic exchange coupling between the metal ion and the thiyl radical. This is a unique example of “internal spin trapping” of a thiyl radical formed during reduction. The finding that the reduction involves a peripheral site and that thiyl radicals produced during the reduction remain associated with the reduced cobalamin provide important new insights into our understanding of the formation and function of cobalamin enzymes.

KEYWORDS: cobalamin, glutathione, dithiothreitol, electron paramagnetic resonance, visiblespectroscopy, thyil radicals.

*Correspondence to: Joseph M. Rifkind, email: [email protected], tel: +1 410-558-8168, fax: +1 410-558-8397 and Periakaruppan T. Manoharan, email: [email protected], tel: +91 44-22574938, fax: +91 44-22570545


26 S. RAMASAMY ET AL.

increased risk of cardiovascular disease and stroke associ-ated with elevated levels of serum homocysteine [2–5].

Cbl (Fig. 1) contains a cobalt ion which can be in the +1, +2 or +3 valence states and an intramolecularly coor-dinated 5,6-dimethylbenzimidazole in the α-axial posi-tion. The function of the Cbl cofactor depends on the oxidation state of the cobalt and the ligand bound in the β-axial position. While Cbl is generally isolated in the +3valence state with cyanide, water or hydroxide coordi-nated in the β position, the activity of Cbl requires its reduction to the +1 or +2 valence state with the coordi-nation of a methyl group or the 5′ deoxyadenosyl group [6, 7].

Glutathione (GSH) is the most abundant non-protein thiol and is a major intracellular reducing agent pres-ent in almost all biological tissues with a concentration in the mM range. Its concentration in cells is at least an order of magnitude greater than that of other thiols such as cysteine [8]. Vitamin B12 and its derivatives have been known to form complexes with GSH [9–11]. The com-plex formed between Cbl and GSH, glutathionylcobala-min GSCbl(III), is believed to be one of the major forms of vitamin B12 found within mammalian cells [6, 7]. It is considered to be the substrate for Cbl(III)reductase [12] serving as an intermediate in the conversion of biologi-cally inactive cyanocobalamin to the active coenzyme forms, adenosylcobalamin and methylcobalamin [6]. The pathway for this process is thought to involve the reaction of cyanocobalamin with β-ligand transferase, a cytosolic enzyme that utilizes FAD, NADPH and GSH to produce GSCbl(III), which reacts with the NADH-linked microsomal cobalamin(III) reductase, that is then con-verted to adenosylcobalamin or methylcobalamin. It was, in fact, hypothesized by McCoddon [1] that GSCbl(III)

might play a role in the treatment of various neuropsychi-atric disorders associated with oxidative stress, including Alzheimer’s disease.

In vitro, GSCbl(III) can be prepared by reacting aquo-cobalamin and GSH to form a stable complex of 1:1 stoi-chiometry [10, 13–16]. The equilibrium constant for the formation of GSCbl(III) was estimated as 5 × 109 M-1.This GSCbl(III) complex was shown to be stable in the presence of air and a detailed study of the structure of this complex has been obtained by means of 1H, 13C 2D-NMR and X-ray absorption. These studies confirm that GSH is coordinated to the cobalt atom in the β-axial position viathe cysteine sulfur atom [14, 17].

The potential of low molecular weight thiols like GSH and dithiothreitol (DTT), as well as enzyme sulfhydryl groups, to form radicals when reacting with Cbl and related compounds has been well documented [18–22].

In this report we have investigated the reduction of Cbl(III)OH and cobinamide with GSH and DTT under anaerobic conditions by visible spectroscopy and elec-tron paramagnetic resonance (EPR). For GSH we have also monitored the formation of oxidized glutathione (GSSG). The reduction by GSH is shown to involve a second GSH molecule in addition to the GSH that binds to the cobalt of Cbl(III). It is found that GSH and DTT radicals remain coordinated to the cobalt ion after reduc-tion providing a model for “internal spin trapping”. The objective of this study was to identify by means of elec-tron paramagnetic resonance (EPR) and optical spectros-copy the species formed during the reduction, providing an explanation for the overall reduction process. The species formed include the base-on species, the base-offspecies as well as radicalated Cbl(II), which is a “spin trapped” exchange-coupled species.

EXPERIMENTAL

Materials

Cbl(III)OH (vitamin B12a) acetate salt, DTT, GSH, GSSG, glutathione reductase, Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and NADPH were obtained from Sigma and used without any further purification. 2-vinylpyridine was obtained from Aldrich. Cobinamide, in which the benzimidazole is chemically removed, was a gift from Dr. Vijay Sharma. All other reagents were of analytical grade and all solutions and buffers were prepared with deionized water. All solutions of Cbl’s were prepared in 100 mM sodium phosphate buffer, pH 7.3.

UV-visible studies

For UV-visible spectral measurements, 100 μMCbl(III)OH samples were prepared in 100 mM phos-phate buffer, pH 7.3 in septum sealed 10 mm quartz cells and purged with argon to remove oxygen. The reaction

Fig. 1. Molecular Structure of cobalamin(III) with the OH in the β-axial site and the benzimidazole nucleotide in the α-axialsite. Also shown the structures of GSH and DTT


INTERNAL SPIN TRAPPING OF THIYL RADICAL DURING THE COMPLEXATION 27

of Cbl(III) with GSH and DTT was initiated by adding the thiol to the anaerobic Cbl(III) solution. Spectra were run anaerobically in the 250–650 nm regions at ambi-ent temperature (~23 °C) using a Perkin Elmer Lamda 6 spectrophotometer.

Electron Paramagnetic Resonance studies

For EPR studies a 5 mM solution of Cbl(III)OH was prepared in 100 mM phosphate buffer, pH 7.3. The solu-tion was deoxygenated by bubbling with pure argon. After the reaction was completed, 30 μl samples were transferred to 4 mm quartz sample tubes under a positive argon pressure and immediately frozen by submerging the tube in liquid nitrogen. For GSH, excess thiol was added anaerobically and incubated for 3 h prior to freezing. For DTT, the samples were frozen immediately after the addition of the thiol. EPR spectroscopic measurements were carried out at X-band frequency on a Bruker EMX 6 spectrometer in the temperature range from 5 K to 77 K. The temperature was maintained using an Oxford ESR 900 continuous flow cryostat with an ITC-502 tempera-ture controller. X-band measurements were carried out at a microwave power of 2 mW and a modulation ampli-tude of 1 mT. Some measurements were also made on a Varian E-112 instrument. For Q-band measurements, a Varian E-9 system was used at the National Biomedical ESR Center, Medical College of Wisconsin. The Q-band bridge was modified with addition of a GaAs field-effect transistor signal amplifier and low-noise Gunn diode oscillator. The Q-band measurements were carried out at a microwave frequency, 35.0 GHz; power, 10 dB; modu-lation amplitude, 10 G; gain, 400; temperature, -140 °C;seven scans averaged.

GSSG assay

Griffith’s method, which was a modification of the method developed by Tietze, was followed for the deter-mination of GSSG [23, 24]. The following stock solu-tions were made up in 125 mM Na-phosphate buffer containing 6.3 mM Na-EDTA adjusted to pH 7.5 and stored at 0 °C. (1) 0.3 mM NADPH, (2) 6 mM DTNB, (3) ~50 units of glutathione reductase/mL. To 0.6 mL of the sample were added 2.1 mL of the NADPH solution and 0.3 mL of the DTNB solution giving a final volume of 3 mL in a cuvette with a 1 cm path length and equili-brated to 30 °C. To the warmed solution, 30 μl of the glutathione reductase solution was added and the absor-bance change was monitored at 412 nm until the absor-bance exceeded 2. Based on comparing the rate observed for known amounts of GSSG, a standard curve was gen-erated. To prevent any contribution from the unreacted GSH present in the sample, the GSH (for pH’s > 5.5) was derivatized by adding 2 μl of neat 2-vinylpyridine per 100 μl solution and mixing the solution vigorously for 1 min. Depending on the final pH, GSH will be fully derivatized after 20–60 min at 25 °C.

RESULTS

UV-visible spectral studies

The reaction of glutathione with Cbl(III)OH. Figure 2a shows the time dependent spectral changes produced by the reaction of a 10-fold excess of GSH with Cbl(III)OH under anaerobic conditions. A color change from red to violet is observed upon complex formation with absorp-tion peaks changing from 273, 351, 412 and 525 nm to 287.5, 331.5, 370.5, 427.5 and 534 nm. Clear isosbestic points are observed at 338, 364, 447 and 540 nm. This spectral change is attributed to the displacement of the -OH group by GSH to form GSCbl(III) complex. Under these conditions no reduction of Co(III) to Co(II) is observed. With a 50-fold excess of GSH (Fig. 2b) the formation of GSCbl(III) occurs within the mixing time of the reagents (<5 s). This initial change is followed by a slow color change from violet to brown with the absorption peaks changing to 311, 404 and 474 nm. Clear isosbestic points are now observed at 499, 388, 330 and 296 nm. This slower spectral change is attributed to the reduction of Co(III) to Co(II). Other investigators inter-ested in the reaction of GSH with cobalamin have either focused on the binding to Cbl(III) or the reduction of Cbl(III) to Cbl(II). However, by comparing both reac-tions it becomes evident that completely different con-centrations of GSH are required for both reactions.

Job plot of the reduction of Cbl(III)OH by GSH. Inorder to determine whether the GSH that reduces Cbl(III) to Cbl(II) is distinct from the initial GSH bound to Cbl(III) a continuous variation study was performed changing the relative molar ratios of GSH and Cbl(III), maintain-ing the total molar concentration the same (Job plot). The samples were then permitted to reach equilibrium by incubating at room temperature for 3 h. The resultant UV-visible Spectroscopy based Job plot (Fig. 3a) shows that at low molar ratios of GSH there is no reduction. At low GSH:Cbl ratios GSH binds to Cbl(III) resulting in the formation of GSCbl(III). This reaction is com-pleted by approximately a 1:1 ratio of Cbl(III) to GSH before any reduction takes place. Figure 3b shows the EPR based Job plot where we can directly measure the reduced Cbl(II) species, in contrast to the requirement for a fitting procedure to obtain Cbl(II) from visible spec-troscopy (Fig. 3a). The more precise EPR data indicates that only low levels of reduced species are formed at low GSH molar ratios (<1:1) when nearly all the added GSH binds to Cbl(III) without reduction. Both of these experi-ments clearly indicate the requirement for a second molecule of GSH to form the reduced species.

The reduction of Cbl(III)OH by DTT. Unlike the reduction of Cbl(III)OH by GSH, the reduction by DTT is very fast, even with a slight excess of DTT relative to Cbl(III)OH. The addition of DTT immediately resulted in a color change from red to brown with very clear spectral changes. It is to be noted that the Cbl(II) visible



spectrum obtained immediately with a 3:1 molar ratio of DTT:Cbl(III)OH is similar to that obtained with a 50-fold excess of GSH after 3 h of incubation (Fig. 2c).

Electron Paramagnetic Resonance

EPR spectrum of cobalamin(III) reduced by GSH.Because EPR requires a much higher concentration than visible spectroscopy, mass action results in significant reduction even at a 3:1 molar ratio of GSH to Cbl(III)OH (results not shown). Figure 4 shows the Q-band spectrum at 12 K of the reduced Cbl(II) obtained when Cbl(III)OH reacts with a 10-fold excess of GSH. The improved reso-lution obtained in the Q-band provides direct evidence

for two species as indicated by two broad g⊥ lines withg = 2.23 and g = 2.27, respectively, and more than 8 cobalt-59 hyperfine lines in the parallel component. The observation that 14N hyperfine splitting on the Cobalt-59 are partially resolved for some bands and completely absent for other bands indicates that one of the two spe-cies has the base dissociated from the cobalt. The EPR parameters for the two components obtained by Q-band simulation are: (1) for the base-off species: g11 = 1.992 ± 0.003, g⊥ = 2.272 ± 0.004, A11 = 105 × 10-4 cm-1, A⊥ = 3.5 ± 0.5 × 10-4 cm-1 and (2) for the base on species: g11 = 2.010 ± 0.003, g⊥ = 2.234 ± 0.0005, A11 = 102.8 × 10-4 ± 1.0 cm-1, A⊥ = 3.5 ± 0.5 × 10-4 cm-1. Figure 5 shows the EPR spectrum at 77 K when a 50-fold excess of GSH

Fig. 2. (a) Time-dependent UV-vis spectral change monitored during the reaction of 3-fold excess of GSH with 100 μM Cbl(III)OH under anaerobic condition. The time taken for the complete formation of the GSCbl(III) complex is 8–9 min. The first spectrum corresponds to that of Cbl(III)OH. After a dead time of 10 s, the accumulation of the spectra was initiated. The running time for each spectrum is 38 s. (b) Time-dependent UV-vis spectral change during the reaction of 50-fold excess of GSH with 100 μM Cbl(III)OH under anaerobic condition. The initial spectrum obtained after the 10 s dead time is the same as that of Fig. 2a at the end of the reaction with a 3:1 molar ratio of GSH and Cbl(III)OH. The subsequent reduction was followed for 5 h. (c) The UV-vis spectra of the final product formed from 100 μM Cbl(III)OH with a 50-fold excess GSH (-----) is compared with that obtained with a 3-fold excess of dithiothreitol (–––––)



is added to Cbl(III)OH and incubated for 3 h. With this large excess of GSH the base-on complex has a spectrum similar to that shown in Fig. 4 with the same A tensors, but better resolved nitrogen hyperfine lines. However, the base-off complex is completely different with changes that include: (a) sharpening of the A11 lines; (b) the shift and broadening of the g⊥ component from its earlier value of 2.272 to almost 2.39. There is also the apparent formation of a buried low intensity line with a “g” value of 2.19, (marked by a *).

EPR spectrum for cobalamin reduced by DTT and the effects of added GSH on the DTT reduced

species. The EPR spectrum at 77 K of Cbl(II) formed by the reduction of Cbl(III) with a 4-fold excess DTT (Fig. 6a) is produced rapidly and is a simple axial spectrum with poorly resolved 14N hyperfine splitting. The addition of 50-fold excess GSH to the already DTT reduced sample results in the conversion of the spectrum of DTT reduced cobalamin (Fig. 6a) into a spectrum (Fig. 6b) that is very similar to that obtained when a 50-fold excess of GSH reacts directly with Cbl(III) (Fig. 5).

Cobinamide experiments. In order to characterize the complexes formed without the base bound and particu-larly the nature of the base-off complex with the broad g⊥ at 2.39 and sharp A11 (

59Co) lines as well as the small band observed at g = 2.19 marked by a ∗ in Figs 5 and 6b we have used cobinamide (Cbi(III)), which has no benz-imidazole nucleotide. The EPR measurement of Cbi(III)

Fig. 3. (a) Job’s plot from the UV-vis spectral results for the reduction of Cbl(III)-OH with GSH. The fraction of reduced Cbl(II) for the Job’s plot was obtained by fitting the 3 h spectra with the spectra of Cbl(III), Cbl(III)-GSH and Cbl(II) taken from Figs 2a and 2b. (b) Job’s plot from the EPR spectral data for the reduction of Cbl(III)-OH when incubated with GSH for 3 h. The intensity of Cbl(II) is directly measured

Fig. 4. Q-band EPR spectrum of 5 mM Cbl(III)-OH with 10-fold excess of GSH at 12 K (solid line) and its simulation requiring two different species (dotted line)

Fig. 5. X-band EPR spectrum of 5 mM Cbl(III)-OH to which a 50-fold excess of GSH is added and measured at 77 K



reduced by DTT yields a spectrum shown in Fig. 7a simi-lar to those of Figs 5 and 6b. It is, however, puzzling to see that the Cbi(II) spectrum (Fig. 7a) includes a base-on component with 14N hyperfine splitting in addition to the base-off components.

Figure 7b shows the EPR spectrum obtained on the addition of excess GSH to already DTT reduced Cbi(III). The GSH causes (i) the obliteration of the apparent base-on species accompanied by a large increase in the intensity of the species with g⊥ = 2.39 (a broad and intense line) along with its sharp parallel hyperfine components at higher field; (ii) a substantial increase in the intensity of the g = 2.19 line.

To determine whether the 2.19 band is part of the g⊥ = 2.39 species, we carried out a computer simulation of the Co(II) base-off species with g⊥ = 2.39. The simulation (Fig. 7c, bottom) provides a very good fit with g⊥ = 2.39,g|| = 2.00 and sharp lines with A|| = 130 × 10-4 cm-1. No g = 2.19 was generated indicating that the g = 2.19 line is not part of the spectrum of this species (see Fig. 7c, middle). Also shown in Fig. 7c (top) is the simulation of a spec-trum with g⊥ = 2.19 involving a thiyl radical.

A molecule with no base bound to the cobalt, analo-gous to Cbi(II), can be generated directly from Cbl by lowering the pH to 2.0 where protonation of the axial benzimidazole results in the dissociation of the base forming a base-off Cbl(III). Under these conditions the addition of 50-fold excess GSH yields a spectrum (Fig. 7d) very similar to that obtained when GSH was added to the DTT reduced cobinamide (Fig. 7b). The pronounced g = 2.19 band in this spectrum and the Cbi(III) spectrum with GSH added (Fig. 7b) indicates that it is formed when the base is dissociated from the corrin, but does not require the cleavage of the base from the corrin. We infer that this line is also present in Figs 6b and 5 (see *), but at a much lower concentration.

Fig. 6. (a) X-band EPR spectrum at 77 K of Cbl(III)OH (5 mM) reduced by DTT (20 mM) at 77 K; (b) X-band EPR spectrum at 77 K of Cbl(III)OH (5 mM) reduced first by DTT (20 mM) to which 250 mM GSH was subsequently added

Fig. 7. (a) X-band EPR spectrum at 77 K of 965 μM cobinamide, Cbi(III), reduced by 10 mM DTT. (b) X-band EPR spectrum at 77 K of 965 μM cobinamide, Cbi(III), reduced by 10 mM DTT to which 50 mM GSH was subsequently added (see text). (c)(Middle) the X-band EPR spectrum of Cbi(III)OH reduced first by DTT and followed by GSH addition (the same as in Fig. 7b reproduced for comparison with simulated spectra); (bottom) computer simulation of the species with g⊥ = 2.39 which is attributed to the “base-weakened” species with the thiyl radical in the pocket (E′) or the spectrum where the base is replaced by a second GSH (Cbl(II)(GSH)2-E); (top) simulation of the exchange coupled species (F). (d) X-band EPR spectrum of Cbl(III) (5 m) at pH 2 reduced directly by GSH. Measurement done at 8 K at a microwave power of 0.02 mw. * and + respec-tively identify the g⊥ = 2.19 and the low intensity A|| (

59Co) of the exchange coupled species



Figure 7d is very similar to that of Fig. 7b except that many of the lines are broader. Since Fig. 7d was run at 8 K instead of 77 K, the difference can be attributed to saturation even though it was run at a lower microwave power of 0.02 mW instead of the 2 mW microwave power used in Fig. 7b. To further investigate the saturation prop-erties of the species formed, spectra were run varying the temperature and the power. The saturation produced by increasing the microwave power from 40 dB to 20 dB is shown in Fig. 8a for a sample of GSH reduced Cbi(III), and by decreasing the temperature from 77 K to 10 K,is shown in Fig. 8b for a sample of the DTT reduced Cbi(III), which was then reacted with excess GSH. A general increase in the intensity along with the broaden-ing of the whole spectrum occurs with increased power or decreased temperature. The changes are, however, not the same for all bands. Initially the intensity of the g = 2.19

line increases and then decreases indicative of satura-tion only at the highest power and lowest temperature. However, the g = 2.39 line as well as the sharper A|| linesare continuously broadened by a decrease in temperature or an increase in power indicating that these signals are saturated more readily than the g = 2.19 line. The differ-ent saturation of the g = 2.19 indicates that it is from a different species and not part of the main high intensity spectrum.

EPR saturation experiments on reduced Cbl(III)OH at pH 2. We have also performed saturation experiments on Cbl(III) at pH 2 after its reduction by (i) DTT, (ii) GSH and (iii) DTT followed by the addition of GSH. Their EPR spectral features are shown in Figs 9a, 9b and 9c, respectively. The saturation properties of DTT reduced Cbl(III) at pH 2 are substantially different from those of either GSH or DTT/GSH reduction, even though all of them give similar non-saturated signals at the lowest microwave power of 0.02 mw. For DTT without added GSH the broadening of the parallel component with 59Cohyperfine lines is a continuous process, but the g = 2.19 and g = 2.39 lines do not saturate at all until we go to the highest microwave power of 5 dB. The two reduced spe-cies with GSH respond like the Cbi experiments (Fig. 8) where the only line that is not saturated until the highest power is reached is the g = 2.19 line. This again supports the contention that the origin of this line is different from that of the others.

The formation of the g = 2.19 and g = 2.39 bands by lowering the pH of DTT reduced cobalamin. Figure 10ashows the DTT reduced cobalamin(III), which is the same as that shown in Fig. 6a. In Fig. 10b the pH of this sample was reduced to pH 2. At this low pH, the Bzm base is no longer coordinated to cobalt(II). Although the DTT can now bind in the base binding site no additional reduction takes place and, therefore, no thiyl radicals were formed. The observation of the thiyl radical coordi-nated exchange coupled species with its g⊥ = 2.19 similar to that found in Figs 7b and 7d indicates that, eventhough the signal is not detected, the thiyl radicals are present when the base is coordinated.

The formation of GSSG during the reaction of GSH with cobalamin. The reduction of cobalamin by GSH produces GS•. This thiyl radical can either remain coor-dinated with Cbl or released into solution. If the thiyl radical is released from Cbl it will rapidly undergo the reaction,

2GS• → GSSG (1)

To determine whether thiyl radicals are associated with the reduced Cbl, the formation of GSSG was deter-mined during the reaction. The results indicate that the level of GSSG formed is about half of that necessary to account for the Cbl being reduced. Thus, a significant fraction of the thiyl radical formed must be associated with the reduced Cbl.

Fig. 8. (a) EPR power saturation measurement on sample of 965 μM Cbi(III) reduced by GSH at 77 K. (b) Temperature dependent EPR spectra of 965 μM Cbi(III), first reduced by DTT and later reacted with GSH at a constant power of 40 dB (0.02 mw)



DISCUSSION

The reduction of Cbl(III) to Cbl(II) by glutathione

The GSH complex with Cbl(III), GSCbl(III), is found in mammalian cells [6, 7] and is believed to be one of the major forms of vitamin B12 serving as a substrate for Cbl(III) reductase [12]. These findings have been the impetus for extensive in vitro studies characterizing the complex. It is a very stable 1:1 complex formed by react-ing aquocobalamin with GSH [10, 13–16]. The detailed structure of this complex obtained by means of 1H, 13C2D-NMR and X-ray absorption confirm that GSH is

coordinated to the cobalt atom via the cysteine sulfur atom [14, 17]. In Fig. 2a, we show the rapid formation of the GSCbl(III) complex. This complex is stable and does not undergo reduction. However, with a larger excess of GSH there is a slow reduction of the Cbl(III) to Cbl(II), as shown in Fig. 2b. The Job plots obtained both from the visible spectra (Fig. 3a) and the EPR data (Fig. 3b) indicate that a second GSH molecule is required for Cbl reduction.

The requirement for a second GSH indicates that the tightly bound GSH is not involved in the reduction. As the X-ray absorption data [19] indicates, the first GSH is docked in the –OH position of Cbl(III)OH attached to the

(a) (b)

(c)

Fig. 9. (a) Power variation EPR measurement at 77 K of 5 mM Cbl(III) at pH 2 reduced by 20 mM DTT. (b) Power variation EPR measurement at 77 K of 5 mM Cbl(III) at pH 2 reduced by 250 mM GSH. (c) Power variation EPR measurement at 77 K of 5 mM Cbl(III) at pH 2 first reduced by 20 mM DTT with 250 mM GSH added subsequently



Co(III) atom at the β axial site where it is protected by the phytyl chains stretching out providing a “basket” kind of arrangement (see the structure of Cbl(III), Fig. 1). ThisGSH replaces the ligand (OH or water) at the β-axial site and may actually stabilize the Cbl(III) oxidation state inhibiting reduction [25]. Reduction takes place only when a second GSH binds.

Where does the second GSH involved in the reduc-tion (Fig. 2b) bind? It is unlikely that a second GSH binds directly to cobalt, with the first GSH tightly bound to the β axial site and the benzimidazole bound trans to the GSH on the other side of the corrin at the α axial site. With no available cobalt coordination site the most reasonable area that seems to permit the GSH to approach the metal center and facilitate the reduc-tion would be the void space available in the 5,6-di-methyl benzimidazole ribonucleotide ligand cavity (Fig. 1). The kinetics of the slow reduction (Fig. 2b) can be explained by the requirement for the relatively large GSH to enter this restricted cavity and achieve the proper location and orientation necessary for the trans-fer of an electron from the thiol to Co(III). The first GSH already coordinated to Cbl(III) in the β-axial site may contribute to the reduction process by lowering the reduction potential.

The general spectral features of GSH reduced cobala-min shown in the EPR spectra of Figs 4 and 5 are similar to earlier observations [26, 27]. In Fig. 5 at 77 K, with the excess of GSH required for reduction, the nitrogen super-hyperfine lines originating from the bound benzimidazole are partially resolved in one of the species. The improved resolution of these lines relative to DTT reduced cobala-min (Fig. 6a) can be explained by GSH still bound in the β axial position when cobalamin is reduced. GSH at this site will decrease the intermolecular interactions reduc-ing dipolar interactions, resulting in less broadening of the superhyperfine lines.

With GSH there is clear evidence from both X-band and Q-band EPR for an additional species even with a 10-fold excess of GSH (Fig. 4). A single species should have eight equally speced hyperfine lines that originate from the quadrupolar effect of the cobalt-59 (I = 7/2) nucleus. The presence of two species is evident by the number of hyperfine lines and the unequal splitting between these hyperfine lines. In addition nitrogen super-hyperfine splitting is observed for only some of these lines, while other lines are sharp indicating the absence of any nitrogen interactions. These two species are the base-on species with triplet superhyperfine due to 14Nand the base-off species with sharp lines due to 59Cohyperfine lines with no 14N splitting. Using the Q-band EPR spectra, it was possible to simulate both spectral components (Fig. 4) obtaining the EPR parameters (see above). The base-off species is stabilized when Co(III) is reduced to Co(II), by a trans effect on the base due to GSH still bound in the β axial site and by a reduction of the pH when excess GSH is added.

With a large excess of GSH (Fig. 5) the relative inten-sity of the base-on species is reduced and there is an increase in the base off species, with sharp A|| (

59Co) lines not broadened by nitrogen superhyperfine splitting. How-ever, at these high concentrations of added GSH the base-off complex with g⊥ = 2.272 obtained at a 10-fold excess of GSH (Fig. 4) is no longer detected. Instead there is an increase in g⊥ to 2.39 caused by the coordination of a second GSH in the α-axial site originally occupied by benzimidazole.

The thiyl radical associated with GSH reduced coba-lamin

The basis for radical involvement in Cbl chemistry goes back to the original Schrauzer [18] proposal that the reduction of Cbl by GSH involves the formation of thiyl radicals.

BzmCbl(III)(GSH)2 → GSHCbl(II)Bzm + GS• + H+ (2)

The involvement of these radicals in Cbl dependent enzymes has been extensively studied [28, 29]. In order to determine if any of the thiyl radical formed when GSH reduces Cbl stays associated with Cbl(II), we measured GSSG formed during the reaction. Interestingly, the oxidized GSSG accounted for only ~50% of the thiyl radicals formed during the reduction. The other 50% of the thiyl radicals must, therefore, be associated with reduced Cbl.

Two distinct species involving additional interactions with GSH are detected by EPR. These species are only detected in the base-off configuration, which is stabilized during the reduction by GSH (see above). The g⊥ = 2.39 line together with the sharp Co hyperfine lines are asso-ciated with the coordination of a sulfur in the α-axialposition. These lines are formed when excess GSH binds

Fig. 10. The EPR spectra describing the effect of lowering the pH on DTT reduced cobalamin: (a) cobalamin reduced by DTT at pH 7.4; (b) the pH of the DTT-reduced cobalamin was then lowered to pH 2



to reduced Cbl (complex F, see Scheme 1, vide infra).If the GSH on the α-axial site is a thiyl radical, similar EPR paramaters can be observed if the sulfur remains >2.2 Å from the Co atom. A closer approach results in the exchange coupled species (g = 2.19), which reflects the capture of a spin from the thiyl radical by Cbl(II), referred to as “internal spin trapping”.

In order to prove that the g = 2.19 band is not part of the species with g⊥ = 2.39, we simulated the spectrum of the g = 2.39 species. As shown in Fig. 7c (bottom), this simulation fits all the lines in the reduced cobinamide spectrum except for the line with g = 2.19. Confirmation that the g = 2.19 line is not part of the g⊥ = 2.39 species is also provided by differences between these two lines in saturation studies performed by increasing the power (Fig. 8a) or decreasing the temperature (Fig. 8b). These studies clearly indicate that the g = 2.39 line and the sharper A|| lines are saturated before the g = 2.19 line.

The relatively broad (15 gauss) line with a g-value of 2.19 is clearly not an isolated free radical, which should have a g value much closer to the g-value of 2.0023 for a free electron [30]. Even for sulphur based radicals, which have higher g-values, the value should be between 2.03 and 2.09 for an isolated radical [31, 32]. Very large exchange coupling or dipolar coupling between the metal center and the thiyl radical can give rise to the broad g = 2.19 isotropic line. This suggestion is, however, ruled out by the predicted very small exchange coupling constant (vide infra). Hence this large-intensity line is interpreted to represent the g⊥ of anexchange coupled species involv-ing “internal spin trapping” between base-free Cbl(II) and the coordinated thiyl radical. The g⊥ for this species should be the average between that of Cbl(II)(GSH)2

with GSH bound in the α and β axial position and that of the free thiyl radical. The ligand field for two GSH molecules bound would result in g = 2.39 (complex F, see Scheme 1, vide infra) and the estimated g⊥ ~ 2.00 for a free thiyl radical can be based on the theoretical calcula-tions and experimental data reported by van Gastel and coworkers [31]. The average results in a value of 2.195 in good agreement with the observed intense line at 2.19. It should, however, be noted that the g⊥-value of 2.00 being used for the thiyl radical is only a rough estimate. van Gastel and coworkers [31] have cautioned that the largest of the anisotropic g-parameters viz gxx, which decides the magnitude of gav or giso is very sensitive to the conforma-tion of the cysteine, the environment of sulfur and polar-ity. This is because the EPR g-values are very sensitive to the energy difference between the singly occupied almost degenerate levels and one of the lone-pair orbitals, the ΔEbeing of the order of 1000 to 4000 cm-1 [31].

The parallel component of the exchange coupled spe-cies would be in the 2.0–2.05 range taking an average of 2.0 for the gzz for the cobalt species and 2.0 to 2.10 for the thiyl radical [31, 32]. Similarly, the parallel compo-nent will have its 59Co hyperfine lines at half the A|| value observed for the g = 2.39 species i.e. ~50–60 × 10-4 cm-1

at much reduced intensities in comparison to the ampli-tude of g⊥ = 2.19. The simulated spectrum of this species with g|| = the 2.00, g⊥ = 2.19, A⊥ = 1 × 10-4 cm-1 and A|| = ~50 × 10-4 cm-1 is shown on the top of Fig. 7c. At least one or two of the predicted lines are poorly resolved as weak lines with + signs in the experimental spectrum (Fig. 7d).

The temperature dependence in the 77–20 K reveals that the Co(II)-thiyl species is ferromagnetically coupled. The intensity variation of this data was used to calculate a “J” value, i.e. Heisenberg exchange coupling, [34–36] of 2.45 ± 0.2 cm-1. In our analysis, we could not detect any effects due to dipolar coupling. Confirmation of a triplet state due to the exchange-coupled species is frequently obtained by the observation of a half-field transition. However, the intensity of the band due to this forbidden transition is proportional to the zero-field splitting (2D) and cannot be detected for the small D-value expected for our complex.

Support for the assignment of the g = 2.19 complex as a Co(II) thiyl exchange coupled species can be drawn from two important works involving ribonucleotide triphosphate reductase: (i) A protein based thiyl radical has been proposed as an intermediate in this enzyme from Lactobacillus leichmannil, which catalyzes adenosyl cobalamin-dependent reduction as well as the exchange of the 5′ hydrogens of adenosylcobalamin with solvent [37]. (ii) Modeling of this intermediate as a thiyl radical coupled to cobalamin(II) by electron–electron exchange and dipolar interaction yields a reasonable fit to both X- and Q-band spectra. The J-value calculated by Gerfen et al. [33, 37] was |Jex| > 0.45 cm-1 for the cysteine thiyl radical) detected in cobalamin(II) reacted ribonucleotide triphosphate reductase. This determination of the J-value by simulation was in the same range as the 2.45 cm-1 (see above) that we calculated from the temperature depen-dence of the intensity, which indicates that both com-plexes are similar. They also observed a minimal amount of dipolar broadening.

The reduction pathway of Cbl(III)OH by the addition of GSH is summarized in Scheme 1. The GSH adduct of Cbl(III), already established earlier by UV-vis experi-ments, is represented by A, in this scheme. The formation of this complex, GSCbl(III), requires one GSH molecule and involves the simple displacement of OH by GSH. The binding of a second GSH in the α-axial ligand cavity is represented by B. The second GSH interacts with the cobalt as represented by C resulting in the reduction of Cbl(III) to Cbl(II) forming a thiyl radical (D and D′). The thiyl radical can be released and undergoes dimerization leading to the formation of oxidized glutathione GS:SG and the base-on reduced complex D. As indicated by our analysis of the formation of GSSG, only ~50% of the rad-ical goes on to form GSSG and the remainder of the GS•

radicals formed are still associated with Cbl. The EPR parameters with the base still coordinated (the base-on complex D with g = 2.234) is not significantly affected



by having the thiyl radical (complex D′) in the pocket or if some of the excess GSH enters this pocket (complex D′′).

Under conditions where the base is still coordinated, the evidence for the formation of thiyl radicals is, there-fore, indirect. The released thiyl could not be spin-trapped using an external spin trap in this experiment, since the formation of GS:SG through the process of radical recombination as well as the internal spin trap-ping by Cbl(II) are faster than the reaction of GS• with an external spin trap. At the low temperatures used, the EPR spectrum of the thiyl in the ligand pocket is broadened and not detected (vide infra).

In the pathway with the thiyl radical released, the ini-tial low levels of a base-off complex with g⊥ = 2.272 (E) is observed (Fig. 4). This is a 5-coordinated complex with-out any ligand substituting for the displaced base in the α-axial position. The dissociation of the base in this com-plex is facilitated by a trans effect associated with GSH bound in the β-axial site and the release of protons during the reduction process that lowers the pH. The affinity of GSH for the α-axial site is much less than that of the ini-tial GSH bound to the β-axial site. However, with a large excess of GSH a second GSH binds in the α-axial site resulting in a complex with g⊥ = 2.39 (complex F).

In the pathway with the thiyl in the ligand pocket, as soon as the base is released the thiyl radical will interact with Co(II). A transient species formed as the thiyl radi-cal approaches Co(II) would have parameters similar to complex F with two thiols bound with g⊥ ~2.39. How-ever, this species is only formed transiently, because the closer approach of the thiyl radical produces an exchange coupled complex E′ with g⊥ = 2.19. This complex is pres-ent whenever base-off complexes are observed (Figs 5, 6b, 7a), but is most pronounced when the base is com-pletely removed as in cobinamide (Fig. 7b) or by lower-ing the pH and protonating the base (Fig. 7d). At high GSH levels it is also possible that the thiyl radical bound in the α-axial position (species E′) can be displaced by GSH producing complex F.

Comparison of Cbl reduction by GSH and DTT

DTT a smaller molecule with two terminal thiols reduces Cbl much more efficiently and rapidly than GSH (Fig. 2). From the kinetics and spectral differences between GSH and DTT reduced Cbl, important insights can be provided into the reduction of Cbl by thiols.

Although the EPR spectrum at 77 K obtained with DTT (Fig. 6a) is similar to that obtained for GSH (Figs 4and 5), the nitrogen superhyperfine lines with DTT are more poorly resolved than for GSH. We have attributed the better resolution for GSH (see above) to the binding of the larger GSH in the β-axial position which reduces intramolecular dipolar broadening. The broadening of the hyperfine lines for DTT can, however, also be attrib-uted to increased retention of the formed thiyl radicals in the ligand pocket.

Proof that thiyl radicals are associated with DTT reduced Cbl even when the base is bound is provided by Fig. 10b, where we show that lowering the pH of the DTT reduced base-on complex results on the formation of the exchange coupled species with g⊥ = 2.19 (complex E′). The simultaneous formation of a base-off complex with g⊥ = 2.39 (complex F) that involves the binding of two thiols instead of a g⊥ = 2.272 (complex E) also indi-cates that DTT is bound to the β-axial side of the corrin. Although, DTT binding to the β axial side without the interactions involving the glu and gly residues of GSH [22] is expected to be weaker than the binding of GSH, this linkage to the corrin can explain a greater retention of DTT thiyl radicals than the 50% retention observed for GSH. This retention can result in broadening of the nitrogen hyperfine lines observed with DTT. The initial β-axial binding also facilitates the access of the second DTT thiol to the α-axial ligand pocket, explaining the rapid reduction with DTT.

The binding of one of the DTT thiols in the β-axialposition that increases the retention of the thiyl radical in the pocket is also expected to restrain the approach of the thiyl radical to cobalt when the base is dissociated. This can result in complex F, where one of the sulfur atoms is part of a thiyl radical that does not approach Co(II) close enough to form the exchange coupled complex (E′).This is supported by the power saturation studies (Figs 9aand 9b). Thus, for the GSH reaction (Fig. 9b) the 2.39 line (complex F), which does not involve thiyl radicals, is saturated before the 2.19 line (complex E′), which involves a thiyl radical and, therefore, has a shorter relax-ation time. However, for DTT (Fig. 9a) the saturation of the 2.39 line is similar to the 2.19 line implying that the 2.39 line (complex F) also involves a thiyl radical. The resultant complex, nevertheless, has the properties of two sulfur axial ligands and has the same EPR parameters as the GSH complex F.

The dominant species when DTT reduces Cbi (Fig. 7a) is a complex with nitrogen hyperfine interactions. These nitrogen hyperfine interactions in the EPR spectrum dem-onstrate that in the absence of the benzamidazole group one of the phytyl groups containing a nitrogen such as the longer –CH2–CH2–CO–NH–CH2–CHOH–CH3 or one of the two –CH2–CH2–CO–NH2 (see Fig. 1) swing back to provide axial coordination through nitrogen, presum-ably in the α-axial site.

Support for this bonding is provided by a careful analysis of the EPR spectra obtained, which indicates a difference in the hyperfine splitting from 14N for the benzimidazole and phytyl nitrogens. The 14N hyperfine splitting is less resolved in Fig. 7a for Cbi than in Figs 5 and 6b, for the benzimidazole, attributed to a reduction in the hyperfine splitting from 22.3 G to 20.3 G that is due to weaker binding. Such differences in 14N hyperfine splittings have been observed in a study of the coordina-tion of hyperlong nitrogen ligands in a series of LCo(II)Cbi+ adducts where L represents a series of substituted



pyridines with 14N hyperfine splittings varying from 18.3 to 19.8 G [27].

These nitrogen hyperfine lines are eliminated with an excess of GSH (Fig. 7b) implying that these relatively weak interactions with the phytyl nitrogens inhibit the coordination of the DTT thiyl radicals, but not GSH or the GSH thiyl radicals (Fig. 5) with Co(II). These results are consistent with constraints on the interaction of the DTT thiyl radical imposed by binding the same molecule to the β-axial site (see above). It is only at low pH where both the benzimidazole group and any phytyl nitrogens are protonated that in the DTT spectrum the g⊥ = 2.19 is clearly observed and the g⊥ = 2.39 becomes the dominant band (Fig. 10b).

Since the g⊥ = 2.19 line (complex E′) requires that the thiyl radical interact strongly with the Co(II), complex E′ formed with DTT would have the interaction on the β-axial side further weakened. When an excess of GSH is added to DTT reduced Cbl (Figs 6b and 9c), no additional thiyl radicals are formed. However, GSH, which has a much higher affinity for the β axial Co(II) will displace DTT from the β axial side of the corrin. For the g⊥ = 2.19 complex E′, this reaction will not affect the properties of the complex. However, complex F with a constrained thiyl radical (see above) will be converted into the g⊥ = 2.19 complex E′. The conversion of complex F to com-plex E′ is seen by reduction in the F/E′ ratio from ~12 for DTT (Fig. 10b) to ~8 in Fig. 7b after an excess of GSH is added. Furthermore, the saturation experiment (Fig. 9c) indicates that after an excess of GSH is added saturation of the 2.39 line occurs at a lower power than the 2.19 line indicating that thiyl radicals are no longer associated with complex F.

Physiological relevance

The complex of GSH with cobalamin. It was first pro-posed by Wagner and Bernhauer that the Co(III) glutathi-onylcobalamin (γ-glutamylcysteinylglycinylcobalamin)could be the precursor of cobalamin coenzymes [10]. The GSCbl(III) formed in vivo can be reduced by a number of cellular reducing agents including the large excess of reduced GSH found in cells.

While the significance of the GSCbl(III) complex has been discussed in the earlier literature, our studies show that GSH is one of the few ligands that also bind to reduced Cbl(II). With GSCbl(III) the putative substrate for Cob(III)alamin reductase and the presence of high concentrations of intracellular GSH, it would be expected that the initial reduced form of cobalamin is Cbl(II)GSH. It should, therefore, be this complex which serves as an intermediate in the biosynthesis of methylcobalamin and adenosylcobalamin.

Our results clearly show that the Cbl(II)GSH complex exists as an equilibrium between a base-on and base-off complex. The binding of a relatively strong ligand like

GSH in the β-axial site is expected to weaken the benz-imidazole bound in the α-axial site through a trans effect contributing to this equilibrium. A number of studies indicate that the incorporation of the Cbl cofactor into enzymes involve the base-off configuration, which our studies show to be stabilized by GSH.

An example of this is the recent X-ray crystallographic structure determination of methylmalonyl-CoA mutase at 2.0 , which reveals that, as discovered for methylcoba-lamin dependent methionine synthase, the adenosylcoba-lamin cofactor is also bound in a dimethylbenzimidazole base-off form. The nucleotide-loop appended 5,6-dime-thylbenzimidazole serves to anchor the B12 cofactor to the enzyme, and a protein side chain, histidine imidazole, serves as cobalt’s axial base [38–41]. It has also been reported that methylcobinamide which completely lacks the lower axial nitrogen donor ligand to cobalt, is the sub-strate for acetyl-coA synthase [42].

These studies indicate that although the dimethylben-zimidazole base binds to Cbl(III), the functional role for the benzimidazole base does not seem to involve coordi-nation to the Co(II)metal center. The weakening of the bond between the base and the metal when GSH is bound to reduced Cbl may, thus, play an important role in pro-ducing the proper configuration of the corrin cofactor in biological systems.

These results support the hypothesis that lack of a lower axial nitrogen donor ligand to cobalt in the native corrinoid iron-sulfur protein from methanogenic and ace-togenic microbes enhances its propensity for reductive activation and demethylation reactions [43].

The reduction by thiols- the second thiol and com-plexes involving the thiyl radical. The major new insight in our study involve the requirement for a second thiol when GSH or other thiols reduce Cbl(III) (Fig. 3) and the finding that ~50% of the thiyl radicals formed dur-ing the reduction process remain associated with Cbl (Scheme 1). Both of these conclusions provide new insights into understanding the redox reactions which play an important role in Cbl bioactivity.

The demonstration that a peripheral site presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity is responsible for the reduction of Cbl(III) to Cbl(II) instead of coordination to one of the axial posi-tions may play a role in understanding the role of GSH and other thiols in reducing Cbl. Furthermore, such inter-actions may play a role in Cbl related enzymes.

The enzymatic activation of coenzyme B12 (5′-deoxyadenosylcobalamin) [28, 29] initiates enzymatic radical reactions thought to require the homolytic cleavage of the carbon cobalt bond producing the 5′-deoxyadenosyl radical. This radical has, however, not been directly detected, and a peripheral interaction with a thiol or other reducing agent may help explain the enzy-matic activation process. Of particular relevance to our results are the studies with ribonucleotide triphosphate



reductase [33, 37], which contains the deoxyadenosyl cobalamin cofactor. In this case the formation of an inter-mediate involving a thiyl radical coordinated to the coba-lamin has been detected [33, 37] with similar properties to the thiyl complexes found in our study (see above). While it has usually been assumed that the deoxyadeno-syl radical reacts with Cys-408 to produce the thiyl radi-cal [44], our results suggest the need to consider a direct interaction of the protein cysteine with the cobalamin that is analogous to the observed interaction of GSH, in our study producing a thiyl radical that coordinates with the Cbl.

CONCLUSION

The reduction process of Cbl with GSH is analyzed. It is found that GSH rapidly forms a stable complex with Cbl(III). The slow reduction requires a second GSH mol-ecule that does not initially bind to the cobalt, but is pre-sumably located in the dimethyl benzimidazole ligand cavity. The reduction results in the formation of thiyl radicals. About 50% of theses radicals are released from Cbl to form GSSG, but the other 50% remain associated with the reduced Cbl. In the reduced state the intrinsic lower affinity for the benzimidazole base coupled with

Scheme 1. Sequence of reduction of Cbl(III)OH with GSH



a trans effect from the initial GSH still bound in the β-axial site and a possible lowering of the pH results in an equilibrium between a base-on and base-off complex. The dissociation of the base facilitates closer approach of the thiyl radical to the metal center resulting in a com-plex with ferromagnetic exchange coupling between the metal ion in the corrin ring and the thiyl radical.

Acknowledgements

This research was supported in part by the Intramu-ral Research Program of the NIH, National Institute on Aging. PTM thanks the DST, Govt. of India for a research scheme (SP/SI/F18/00) and Ramanna Fellowship (SR/S1/RFIC-02/2006); PTM also thanks the JNCASR Bangalore for an Honorary Professorship and IGNOU, New Delhi for Sir C.V. Raman Chair Professorship.

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DOI: 10.1142/S1088424611004348



INTRODUCTION

The acid-base properties belong to fundamental aspects in the chemistry of tetrapyrroles, such as porphyrins proper, chlorins (17,18-dihydroporphyrins) and bacteriochlorins (7,8,17,18-tetrahydroporphyrins). The examination of these properties is expected to provide information on, e.g., the mechanisms of metalation and demetalation [1–7], π-electron delocalization pathway (aromaticity) [8–10], resonance energy [11],

NH-tautomerism [10, 12, 13–22], isomerization [23, 24], substitution pattern [25, 26] and stereochemistry [25, 26] of a cyclic tetrapyrrole. Furthermore, knowledge of the foregoing aspects may also be of immense importance when trying to elucidate the reaction-center mechanism of natural photosynthesis [27–29], disorders of porphyrin metabolism [30] and the mechanism of photodynamic therapy (PDT) [31–36].

The reviews by Phillips [5, 6] and by Falk and Phillips [2] are still the best sources of information on the acid-base properties of cyclic tetrapyrroles. Following the commonly used notation, the neutral form of a porphyrin or chlorin with two H-atoms at positions 21 and 23 (Scheme 1) is called “free-base porphyrin” or “free-base chlorin” (PH2). This can be deprotonated

Protonation-deprotonation equilibria in tetrapyrroles. Part 2. Mono- and diprotonation of methyl pyropheophorbide a in methanolic hydrochloric acid as verified by the 1H, 13C HSQC and 1H, 15N HMBC NMR experiments

Paavo H. Hynninen*a and Markku Mesilaaksob

a Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, A. I. Virtasen Aukio 1, P.O. Box 55, FI-00014, Finlandb Finnish Institute for Verification of the Chemical Weapons Convention, University of Helsinki, A. I. Virtasen Aukio 1, P.O. Box 55, FI-00014, Finland

Received 10 May 2011Accepted 19 June 2011

ABSTRACT: The two-dimensional 1H, 13C and 1H, 15N heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR techniques were applied to investigate the formation of N-protonated cationic species of methyl pyropheophorbide a in methanolic hydrochloric acid (CD3OH-HCl). The 1H, 13C HSQC and 1H, 15N HMBC NMR spectra, recorded at the temperature of 278 K, verified that the CD3OH-HCl solution with [H]+ = 0.021 M, contained the N22-protonated monocations of methyl pyropheophorbide a, whereas the CD3OH-HCl solution with [H]+ = 5.0 M contained the N22, N24-protonated dications of the phorbin. No further protonations to form the trication or tetracation were observed. Consequently, the two middle electronic spectra, which were previously tentatively interpreted as representing the dication and trication, were now attributed to conformational alterations originating from steric hindrance between the central hydrogens and changes in counter-ion and solvation interactions and/or conformational alterations intimately connected to NH-tautomerism. These possibilities to explain the previously observed middle electronic spectra are briefly discussed.

KEYWORDS: porphyrin, chlorin, acid-base properties, π-electron delocalization, conformation,NH-tautomerism, nuclear magnetic resonance.


*Correspondence to: Paavo H. Hynninen, email: [email protected], tel: +358 9-19150358, fax: +358 9-19150466


40 P. H. HYNNINEN AND M. MESILAAKSO

to the corresponding monoanion (PH-) and dianion (P2-). K1 and K2 denote the acid ionization constants of these deprotonations. The acid ionization constants of the four theoretically possible N-protonation steps have been denoted by Phillips with symbols K3, K4, K5 and K6.These acid-base equilibria are illustrated in Scheme 1 for an unsubstituted chlorin.

Of the six theoretically predictable ionic species, only the monoanion (PH-), the dianion (P2-), the monocation (PH3

+), and the dication (PH42+) have been reported to

be experimentally observable with certainty for fully conjugated porphyrins (for references, see Ref. 37). The existence of the porphyrin monocation as a separate species has aroused a long dispute and thus far there is no conclusive evidence for the formation of the porphyrin trication (PH5

3+) or tetracation (PH64+). In the case of

chlorins, the reported information of N-deprotonatedor N-protonated ionic species is even meager and more controversial. Apparently, though the studies of the central deprotonation-protonation equilibria in chlorins and bacteriochlorins are extremely challenging, mainly due to the functions of these compounds in Nature, such studies are extremely demanding to carry out experimentally and the results are difficult to interpret unambiguously. Owing to the general low solubility and high aggregation tendency of porphyrins, chlorins and bacteriochlorins in water, various organic solvents have to be used in the investigations of the acid-base properties of these compounds. Regarding the acid ionization constants of the tetrapyrroles, this brings about an enigma, which is hard to solve: How is the Ka value determined in water related to that determined in an organic solvent?

Methanol has rarely been used for the acid-base studies of porphyrins, even though its pKa value (ca. -2, the exact value is unknown) is quite close to that of water (pKa = -1.84) [38]. Methanol also has a high solvation power for many kinds of organic molecules owing to its high hydrogen bonding capability. In addition, it is noteworthy that the pKa value of the pyridinium ion (pKa =5.2) has been found to be precisely the same in methanol and water [39].

An ester-exchange method, that has long been used to convert chlorophyll a (1) or pheophytin a (3) to methyl pheophorbide a (5) and pyrochlorophyll a (2) or pyropheophytin a (4) to methyl pyropheophorbide a (6) (Fig. 1), involves standing of the phytyl ester in dry methanol containing sulfuric acid (5%, v/v) or saturated with hydrochloric acid, liberated from sodium chloride and dried in a standard gas generation apparatus. The procedure results in a beautiful dark-blue solution containing N-protonated form(s) of 5 or 6.Interestingly, it should be established which of the four central nitrogen atoms of 5 or 6 are protonated in these solutions.

Part 1 of this series [37] described the spectrophotometric titrations of pyropheophytin a (4) in methanolic hydrochloric acid. In addition to the neutral form, four spectrally different protonated species were observed with clear isosbestic points in the sets of spectra when the HCl concentration was gradually increased in the methanol solution of 4. As the most likely interpretation,

Scheme 1. Theoretically possible deprotonation-protonation equilibria in an unsubstituted chlorin

Fig. 1. Structures of 132(R)-chlorophyll a (1), pyrochlorophyll a (2), 132(R)-pheophytin a (3), pyropheophytin a (4), methyl 132(R)-pheophorbide a (5), methyl pyropheophorbide a (6);pyro = 132-demethoxycarbonyl; the semisystematic numbering system and nomenclature of IUPAC-IUB [40] is used in this paper


PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES 41

the four different spectra were assigned to arise from the N-protonated mono-, di-, tri- and tetracationic species. Nevertheless, this interpretation was intended to be only tentative as appears from the quotation [37]: “However, this evidence cannot be considered conclusive, and much more experimental work is necessary for the final clarification of the protonation behavior of various tetrapyrroles.” Alternative reaction possibilities, including C-protonation to the vinyl group or a methine bridge, O-protonation to the 131-oxo function followed by ketalization, electrophilic substitution at a methine bridge and aggregation, were considered but found as less likely options. The possible involvement of conformational alterations and NH-tautomerism in the spectral changes was also considered but found difficult to assess because relatively little was known about those matters in the case of chlorins at the time of the appearance of the first part [37]. In addition, at that time, an NMR instrument with a sufficiently high magnetic field was not available for us.

During the past 25 years, the NMR instruments and two-dimensional (2D) methods have experienced an enormous development, allowing for instance measurements of 2D heteronuclear 1H, 13C and 1H, 15NHSQC and HMBC NMR spectra (HSQC = heteronuclear single quantum coherence, HMBC = heteronuclear multiple bond correlation) at the natural abundances of 13C and 15N [41–50]. This development and the availability of a 500 MHz NMR instrument enabled us to focus meanwhile on investigations of NH-tautomerism in natural chlorins [21, 22]. Subsequently, it also became possible for us to revisit the problem concerning the protonation of pyropheophytin a (4, Fig. 1) or methyl pyropheophorbide a (6) in methanolic hydrochloric acid

[37]. The results from the protonation studies by NMR described in this paper led us to reinterpret the nature of the higher N-protonated species of 6.


Figure 2 shows the 1H, 15N HMBC spectrum of methyl pyropheophorbide a (6) in the CD3OH-HCl solution with [H]+ = 0.021 M. The correlation signals, arising from 10-CH, 5-CH and 20-CH methine (meso) protons and protonated 15N atoms, HN21, H+N22 and HN23, are localized at the upper part of Fig. 2 and have 15Nchemical shifts at 107, 115 and 111 ppm, respectively. The correlation signals of H+N22 and HN23 overlap each other almost completely as can be more clearly seen from Fig. 3, showing the expanded upper part of the 1H, 15N HMBC spectrum in Fig. 2. At the lower part of Fig. 2, there is one correlation signal with δ(15N) at 292 ppm. This correlation signal obviously arised from the unprotonated N24 atom. These assignments indicate that the 1H, 15N HMBC spectrum in Fig. 2 clearly belongs to the H+N22 monocation and not to the dication or trication. The origin of the low-intensity satellite signals in Fig. 2 remained unclear.

A prerequisite for the above assignments was that the assignment order of the methine (meso) proton signals had to be determined first. This was performed by recording the 1H, 13C HSQC spectrum (Fig. 4), which straightforwardly indicated that the correct order is the following: 10-CH (δH = 10.32), 5-CH (δH = 9.93) and 20-CH (δH = 9.37). This order is in agreement with our previous investigation [51] for the monocation of pyropheophytin a (4, Fig. 1). Our previous investigation

also showed that the proton derived from trifluoroacetic acid (TFA) resided at the N22 of subring B.

The 1H, 15N HMBC spectrum (Fig. 5), recorded from the NMR sample with [H]+ = 5.0 M, indicated that a higher protonated species of phorbin 6 was formed. In this spectrum, the 15N chemical shifts of N21, N23 and N22 were localized at 103, 105 and 108 ppm, respectively. These values are quite close to those obtained for the monocation. However, the 15N chemical shift of N24, δ(15N24) = 136 ppm, is distinctly different from the corresponding value of the monocation, δ(15N24) = 292 ppm. These results verify that the 1H, 15N HMBC spectrum in Fig. 5 represents the H+N22, H+N24-dicationof methyl pyropheophorbide a (6).

The 1H, 15N HSQC spectrum at 278 K of the dicationic solution of 6 exhibited two cross-signals (Fig. 6): the first cross-signal was localized at δ(1H) = 1.00 ppm, δ(15N) = 115 ppm and the second one at δ(1H) = 0.10 ppm, δ(15N)= 118 ppm. These cross-signals can be assigned to the H-N21 and H-N23 groups, respectively.

Fig. 2. 1H, 15N HMBC spectrum at 278 K of the monocation of methyl pyropheophorbide a (6) in the CD3OH-HCl solution with [H]+ = 0.021 M



Continued conduction of the HCl gas into the dicationic solution resulted in no tricationic or tetracationic species.

The foregoing results verify conclusively that only two different N-protonated species, the H+N22 monocation and the H+N22, H+N24 dication, are formed in the methanolic solution of methyl pyropheophorbide a (6)

when the HCl concentration is increased gradually up to saturation. From the results above follows that the pK values of the different protonation steps must be reestimated. The pK3 value, belonging to the formation of the monocation in the first protonation step, remains the same as reported before, pK3 = +4.14. However, because the results above clearly indicate that, from the higher protonations, only the formation of the dication in the second protonation step is possible, the new pK4 value must replace the old pK6 value (pK6 =-0.34 ± 0.04) and be around zero. This conclusion is in agreement with the titration results for chlorin e6 trimethyl ester (pK4 = 0.62) and the 71-acetalof rhodin g7 trimethyl ester (pK4 = 0.60) (P.H. Hynninen, Protonation-deprotonation equilibria in tetrapyrroles. Part 3, in preparation).

The previous paper also included the results from the spectrophotometric titration of pyro-pheophytin a (4) in the AcOH-HCl system, where only two sets of electronic spectra were observed [37]. The first set of spectra with several sharp isosbestic points (see Fig. 10 in Ref. 37) was interpreted as representing the conversion of the neutral form of 4 (Fig. 1) to its monocation and the second set of spectra (see Fig. 11 in Ref. 37), the conversion of the monocation to the tetracation of 4. In addition, the end spectrum of the titration was virtually identical with that from the titration in the MeOH-HCl system. On the basis of the NMR results of this paper, it seems clear that the second set of spectra (see Fig. 11 in Ref. 37) must be reinterpreted as representing the conversion of the monocation to the dication.

Previously, alternative reactions to the four N-protonations were sought. These included C-protonation to the vinyl group or the 20-methine bridge (meso position), O-protonation to the 131-oxo function followed by ketalization, electrophilic substitution (chlorination) to the 20-methine bridge and aggregation. Furthermore, the possible involvement of changes in conformation, NH-tautomerism, counter-ion binding and solvation were briefly discussed but could not be treated more profoundly. Nowadays, these phenomena are well-known for the crystalline dications (diacids) of many variously substituted fully-conjugated porphyrins and porphyrin isomers [52–55], but comparable information is still missing for the dications of chlorins. For the

dications of fully-conjugated porphyrins, the degrees and modes of distortion from planarity of the macrocycle to relieve conformational strain in it (saddle conformation, ruffled conformation) have been thoroughly investigated. For some dications of the porphyrins, an interesting new possibility to relieve conformational strain in the core of the macrocycle has been mentioned by Senge

Fig. 3. Expanded upper part of the 1H, 15N HMBC spectrum in Fig. 2; the expanded partial spectrum shows clearly that the N22 and N23 cross-signals overlap almost completely, whereas the N21 and N22 cross-signals appear as virtually separate signals

Fig. 4. 1H, 13C HSQC spectrum at 278 K of the monocation of methyl pyropheophorbide a (6) in the CD3OH-HCl solution with [H]+ = 0.021 M



[53, 54]: that is the pyramidalization of the N–H units by sp2 → sp3 rehybridization, which removes the inner hydrogen atoms farther away from each other, thus relieving the strain. Such a rehybridization might also be possible in the core of chlorin dications in the solution state.

When seeking a reasonable explanation for the two middle electronic spectra, formerly observed in the protonation of pyropheophytin a (4, Fig. 1) or methyl

pyropheophorbide a (6), it now seems likely that those spectra represent different conformers of the monocation of 4 or 6, because different conformers of a molecule with π-electrons may produce different electronic spectra. As major reasons for conformational changes in the monocation we see, besides moderate steric hindrance (crowding) in the center, changes in the solvation and counter-ion binding of the monocationic species, when the HCl concentration in the solution was gradually increased and the long, nonpolar phytyl chain was replaced with a methyl group in the ester exchange.

Furthermore, it is noteworthy that confor-mational changes are intimately connected to NH tautomerism in a cyclic tetrapyrrole [56, 57]. All possible aromatic and less aromatic NH tautomers of several natural chlorins were studied by the Helsinki Chlorophyll Group (under the supervision of P.H.H.) using the two-dimensional (2D), heteronuclear 1H, 13C and 1H, 15N HSQC and HMBC NMR techniques, as well as variable-temperature NMR [21, 22].

The structures of the cis and transNH-tautomers and the equilibria between them are shown in Scheme 2. The variable-temperature NMR investigations provided the activation free-energies (ΔG‡) for the NH-tautomerization of six different demetallated chlorophyll derivatives, one of which was methyl pyropheophorbide a(6). The second activation free-energy, ΔG2

‡,describing the over-all tautomeric exchange (A � A′ in Scheme 2), was found to attain the highest value, ΔG2

‡ > 18.0 kcal.mol-1,for phorbin 6. This high energy barrier was attributed to the macrocycle rigidifying effect of the isocyclic ring E in the phorbin. The rigidifying effect was considered capable of hindering conformational distortions in the macrocycle, demanded by the NH-tautomeric process. This conclusion is in line with the observation that the two lines, representing the temperature dependence of the δ(15NH) values for the H-N21 and H-N23 groups of phorbin 6, remained far away from each other in the temperature range of 210–330 K (see Fig. 4 in Ref. 22).

Consequently, the over-all NH-tautomerization seems unlikely in chlorins possessing the isocyclic ring E. However, the tautomeric conversion between an aromatic trans NH-tautomer (A/A′, in Scheme 2) and an aromatic cis NH-tautomer (C/C′, D/D′ in Scheme 2), might be feasible also for derivative 6. Such a partial tautomeric reaction would imply changes in the protonation sites, as well as changes in the counter-ion binding and solvation of the cations formed by N-protonations.

Fig. 5. 1H, 15N HMBC spectrum at 278 K of the dication of methyl pyropheophorbide a (6) in the CD3OH-HCl solution with [H]+ = 5.0 M

Fig. 6. 1H, 15N HSQC spectrum at 278 K of the dication of methyl pyropheophorbide a (6) in the CD3OH-HCl solution with [H]+ = 5.0 M



The solvation phenomena of molecules in solution are an interesting but a theoretically demanding subject still today. Applying density functional theory (DFT) and the implicit solvation method (dielectric polarizable continuum model, DPCM) or alternatively the hybrid solvation method (cluster-continuum model, CCM), Mehta and Datta [58] were able to calculate the pK3

value for the monocation of pyropheophytin a (4) in methanol. The DFT-DPCM calculation resulted in pK3 = 6.12 whereas the CCM gave pK3 = 4.70. The latter value, obtained by the cluster-continuum model, is in a better agreement with the experimentally observed value, pK3 = 4.14 [37]. The authors explain this by the presence of a strong intermolecular hydrogen bonding interaction between the solute and the solvent which is taken into account by the hybrid solvation model, CCM. The authors predict that “consideration of a large number of solvent molecules would further improve the result”. In addition, they are of the opinion that “a pure quantum mechanical treatment on a complex of a large number of atoms is still not computationally feasible, and then one would have to resort to a mixed quantum mechanical-molecular mechanical treatment” [58].

EXPERIMENTAL

NMR spectra

For the preparation of two NMR samples of methyl pyropheophorbide a (6, Fig. 1), an adequate volume of CD3OH (methanol-d3, Aldrich, 99 atom% D) solution, saturated with HCl, i.e. [HCl] = 5.0 M, was first prepared by conducting dry, O2-less HCl gas (for preparation, see Ref. 37) through 3.0 mL of CD3OH in a 5 mL volumetric flask, on an ice bath. After the conduction, the flask was closed air-tight with a Teflon stopper. On the basis of Table 1 in Ref. 37, it was calculated that 3.0 μL of the CD3OH solution with [HCl] = 5.0 M would be required to produce 0.70 mL of CD3OH-HCl solution with [H]+ = 0.021 M, assuming complete ionization of HCl. At this [H]+, complete formation of the previously postulated dication was expected. Hence, a 3.0 μL volume of the CD3OH solution with [HCl] = 5.0 M was added with a Carlsberg-type micropipette into a test tube, containing 0.70 mL of CD3OH solution of methyl pyropheophorbide a (6, 10 mg, 0.0182 mmol) to result in the first NMR sample, transferred with a Pasteur pipette into a 5 mm NMR tube,

Scheme 2. The total tautomeric NH exchange process between fully aromatic (thick black line) and less aromatic trans and cisNH-tautomers; adapted from Ref. 22



sealed air-tight. The second NMR sample was prepared by dissolving in another test tube 10 mg (0.0182 mmol) of phorbin 6 in 0.70 mL of CD3OH-HCl solution with [H]+ =5.0 M. The solution was transferred with a Pasteur pipette into a second 5 mm NMR tube, sealed air-tight.

The two-dimensional 1H, 15N HSQC and HMBC spectra were recorded at 278.0 K (4.85 °C) on a Bruker DRX 500 spectrometer and the 1H, 13C HSQC spectrum at 278.0 K on a Varian Unity 500 spectrometer, using a non-spinning sample tube, an inverse broad-band probe-head with z-gradients and the appropriate inverse (1Hdetected) pulse sequence provided by the spectrometerlibrary. The acquisition/processing parameters were as follows: ν(1H) = 500.13 MHz, ν(13C) = 125.03 MHz and ν(15N) = 50.68 MHz. The 1H and 13C chemical shifts were referenced to internal Me4S (tetramethylsilane), δ =0.0 ppm, and the 15N chemical shifts to DMF (dimethyl formamide), δ(15N) = 103.8 ppm [21, 22].

Chlorophyll a (1). Chlorophyll a (1, Fig. 1) was isolated from fresh or frozen leaves of red clover (Trifolium pratense) by the method described previously [59], adapted to large-scale preparation.

Pyrochlorophyll a [132-(demethoxycarbonyl)chlor-ophyll a] (2). Chlorophyll a was converted quantitatively into pyrochlorophyll a (2), when its deaerated pyridine solution was heated overnight at 100 °C in a sealed tube [60].

Pyropheophytin a [132-(demethoxycarbonyl)pheo-phytin a] (4). Removal of the magnesium ion from 2 with 12.5% (w/w) HCl afforded pyropheophytin a (4). The 13CNMR spectrum of 4 was identical with that previously reported [51, 61].

Methyl pyropheophorbide a [132-(demethoxycar-bonyl)pheophorbide a methyl ester] (6). The phytyl ester 4 was converted to the methyl ester 6 by ester exchange in dried MeOH containing 5% (v/v) sulfuric acid (note that a fresh preparation of concentrated H2SO4 must be used; conc. H2SO4 is hygroscopic, absorbing water from air and making an old preparation of H2SO4 unsuitable for the purpose).

Acknowledgements

We thank Dr. Harri Koskela for assisting us in the preparation of the 2D NMR spectral figures.

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DOI: 10.1142/S1088424611004324



INTRODUCTION

Porphyrins are a group of molecules that have been the subject of scientific interest for almost a century. These heterocyclic molecules play a key role in many biological phenomena ranging from oxygen transport in the blood to photosynthesis. In particular, their tendency to strongly interact with light has led to intense research using porphyrins as sensitizers in both energy applications and medicine [1–6].

One area of medicine where the light harvesting properties of porphyrins is of particular interest is photodynamic therapy (PDT). PDT aims to target and kill cancerous tissue through overproduction of reactive oxygen species (ROS) [7]. Upon light absorption, an electron in the porphyrin heterocycle is excited from its highest occupied molecular orbital (HOMO) to its lowest unoccupied molecular orbital (LUMO), with a

high conversion rate to an excited triplet state. Excited porphyrins in a triplet state can easily transfer energy to a nearby oxygen molecule resulting in excited singlet oxygen, a potent reactive oxygen species [8]. ROS facilitate the oxidation of many types of biomolecules, including nucleic acids, proteins, and lipids. Cells unable to cope with high concentrations of ROS suffer from oxidative damage, leading eventually to cell death [7].

PDT has been clinically approved for the treatment of several cancer types including skin, head, neck, and lung tumors. However, several challenges associated with effective cancer treatment remain, such as limited solubility of current sensitizers in aqueous systems, and poor localization of PDT agents to cancerous tissue, leading possibly to side-effects such as dark toxicity and increased skin photosensitivity [9]. Efforts to improve site-specific delivery have involved incorporation of PDT therapeutics in nanoscale liposome constructs combined with cancer cell targeting functionality through monoclonal antibodies, aptamers, or other ligands [4, 7, 10].

Nucleotide-driven packaging of a singlet oxygen generating

porphyrin in an icosahedral virus

Brian A. Cohen, Alain E. Kaloyeros and Magnus Bergkvist*

College of Nanoscale Science and Engineering, University at Albany, 253 Fuller Road, Albany, NY 12203, USA

Received 1 April 2011Accepted 21 June 2011

ABSTRACT: Results are reported from investigations of the interactions between MS2 bacteriophages and a cationic porphyrin with potential use in photodynamic therapy. Based on the naturally strong binding between porphyrins and nucleic acids, it is suggested that this non-enveloped capsid could act as a self-loading, nanoscale carrier of porphyrins. By applying size exclusion chromatography in conjunction with UV-vis and fluorescence spectroscopy, it is demonstrated that approximately 250 porphyrin molecules could associate and co-elute with a single capsid. Additionally, there is an observed red shift in the Soret peak of the porphyrin, indicating that the majority of the cationic porphyrin is capable of interacting with RNA on the interior of the capsid. It is also observed that removal of RNA from the interior of the MS2 capsid significantly reduces loading capacity of the porphyrin. Furthermore, MS2 bacteriophages loaded with porphyrins were shown to photogenerate singlet oxygen. These findings suggest that icosahedral viruses, such as MS2 bacteriophages, are able to function as self-packaging “nanoscale containers” and efficiently load cationic porphyrins, with potential benefits in areas such as targeted photodynamic therapy.

KEYWORDS: photodynamic therapy, virus, porphyrin, drug delivery, nucleotide packaging.

*Correspondence to: Magnus Bergkvist, email: [email protected], tel: 1 607-342-3721, fax: 1 518-437-8610


48 B. A. COHEN ET AL.

Potential issues with such strategies include cost, complexity in preparation, and poor stability of the constructs.

Viruses offer an alternative approach as a “nanoscale carrier” for targeted delivery of photodynamic therapy agents as they are capable of self-assembly and form chemically and mechanically stable constructs at physiological conditions. Viruses also present a convenient platform for chemical and/or genetic modification to incorporate desired cell-targeting moieties [11]. The combination of the aforementioned properties, such as self-assembly, stability, biocompatibility, and the multiple approaches for exterior modification make these nanocontainers interesting alternatives to polymer nanostructures [12]. Light harvesting and energy transfer using sensitizers, such as porphyrins conjugated to virus capsids, has been demonstrated with bacteriophages and plant viruses such as M13, MS2 bacteriophages and Tobacco Mosaic Virus (TMV) [13–16]. However, the incorporation of porphyrins into a virus cage for the purpose of site-specific delivery of cationic porphyrins for use in PDT has not been fully and systematically explored yet.

Research by the present investigators has focused on the study of virus capsids as potential carriers of photoactive porphyrins. This concept is just beginning to attract the interest of drug delivery researchers, but questions remain regarding the best technique for tethering the porphyrins to the capsid while retaining their photoactive properties. One approach to load the interior of these “cargo containers” would be to exploit the naturally strong interaction between nucleotides and porphine macrocycles [17]. Such a “porphyrin nucleotide-driven packaging” strategy would entail the following steps: (i) introduction of the porphyrins into the capsid; (ii) binding of the two species with sufficient affinity to enable manipulation of the “assembled” system; and (iii) achievement of a high localized concentration of molecules trapped within the capsid. One cationic porphyrin of interest is meso-tetra-(4-N,N,N,-trimethylanilinium)-porphine-tetrachloride (TMAP) (Fig. 1). TMAP has a net positive charge and is known to interact strongly with DNA [18–20]. In addition, TMAP is capable of generating ROS upon light exposure and has been applied in certain PDT treatments [21].

This report presents findings from an investigation examining the interaction between TMAP and the MS2 bacteriophage, which is suggested as a viable candidate for evaluation of the porphyrin-packaging concept. MS2 bacteriophages are well-studied, innocuous, and straightforward to produce in purified form. The capsid is arranged into an icosahedral T 3 quasi-symmetry and contains 32 pores with a diameter of approximately 1.8 nm [22]. These pores are large enough to allow molecules, such as TMAP, which is approximately 1.6 nm in size, access to the capsid interior. The MS2 capsid remains structurally stable between pH 4.5–12

and can withstand temperatures up to 68 °C [23].The MS2 capsid proteins has a pI of 3.9, and is negatively charged at neutral pH [24]. Fully assembled with its single-stranded RNA genome (3569 nucleotides), the capsid (molecular weight 3.6 × 106 Da) has an empty cavity of approximately 7 nm in diameter [25]. Upon removal of the RNA, the cavity size increases to almost 12 nm [26]. Thus, the MS2 capsid provides a hollow container with appropriate dimensions as follows: (i) pores wide enough to allow introduction of small molecules and (ii) interior volume sufficiently large to accommodate such molecules.

As mentioned earlier, the research presented here seeks to investigate the TMAP-MS2 interaction and whether the RNA contained within the virus capsid facilitates packaging of this positively charged cationic porphyrin. In the following sections, results from an investigation of the interactions between TMAP and MS2 bacteriophages are described and discussed, including an assessment of the potential for small, non-enveloped viruses to package photoactive cationic compounds, and the ability of such “nanocontainers” to photogenerate singlet oxygen.


Loading capacity and access of TMAP to MS2 interior

Figure 2 plots the absorbance from eluted size exclusion chromatography fractions as a function of elution fraction for: MS2 wild-type (MS2-wt), TMAP, and MS2-wt with TMAP (monitored at 260 nm and 412 nm respectively). As can be seen in Fig. 2, the data demonstrate that the TMAP molecule interacts and co-elutes through the column with MS2-wt. More specifically, the first appearance of MS2-wt through the column occurs in elution fraction 7 (corresponding to 1.75 mL of loaded volume). As the elution continues, the MS2-wt reaches a peak concentration in fraction 9, where 98% of the sample has eluted from the column by fraction 12.

Fig. 1. The chemical structure of TMAP


NUCLEOTIDE-DRIVEN PACKAGING OF A SINGLET OXYGEN GENERATING PORPHYRIN IN AN ICOSAHEDRAL VIRUS 49

In comparison, the molecular weight of TMAP is 989 Da, and is well below the 5 kDa cut-off value of the crosslinked dextran size-exclusion matrix. Thus, TMAP molecules enter the matrix and consequently elute in later fractions when compared with MS2. Furthermore, negligible amounts of TMAP are detected in the collected elution fractions, indicating significant interaction with the column matrix. In fact, TMAP elutes so inefficiently through the column that small amounts can be detected in the effluent even after several hundred milliliters of buffer have been passed through the column.

However, when TMAP is combined with MS2-wt (2500:1 TMAP:MS2 capsid initial ratio) and the sample applied to the column in mixed form, a direct correlation can be observed between the elution profile of MS2 (monitored at 260 nm) and TMAP (monitored at 412 nm). Based upon the total absorbance and the extinction coefficients of the individual components, the ratio of TMAP:MS2 after column elution was calculated to be approximately 250:1 (~250 TMAP molecules associated with every MS2-wt bacteriophage).

Additionally, Fig. 3 plots the absorbance from eluted size exclusion chromatography fractions as a function of elution fraction for: MS2 empty capsids (MS2-ec), and MS2-ec with TMAP (monitored at 280 nm and 412 nm respectively). The elution profile of MS2-wt with TMAP is also included for reference. Empty MS2 capsids, where RNA has been largely removed, show a similar elution profile to the MS2-wt capsids that contain RNA. The MS2-ec absorbance intensities in the eluted fractions are considerably lower than their MS2-wt analogs, even though the same molar concentration of capsids was loaded. This decrease in absorbance is attributed to the lower extinction coefficient of protein vs.that for RNA. When TMAP is combined with MS2-ec, it co-elutes to some extent with the capsid fraction. However, the TMAP:MS2-ec ratio is significantly lower than that for its MS2-wt counterpart. The calculated ratio of TMAP:MS2-ec is about 25:1, i.e. one tenth of the interaction ratio detected in the previous experiment, considering that the initial TMAP:capsid ratio was kept the same (2500:1).

Fig. 2. Elution profiles of MS2 bacteriophage (MS2-wt) and TMAP through a 5 kDa Mw cut-off size exclusion column. TMAP and MS2 were monitored at, respectively, 412 nm and 260 nm

Fig. 3. Elution profiles of MS2 empty capsids (MS2-ec) and TMAP through a 5 kDa mw cut-off size exclusion column. TMAP was monitored at 412 nm and MS2-ec at 280 nm. The elution profile of MS2-wt with TMAP is included for reference



These results reveal that there is indeed a defined interaction between the MS2 bacteriophages and the TMAP molecules, with the latter being able to bind MS2-wt at an average ratio of 250:1. Upon removal of RNA, the TMAP loading capacity is reduced by ~90%, leading to a TMAP:MS2 empty capsid ratio of approximately 25:1. This indicates that only a small percentage of the TMAP molecules associate with empty capsids, and the majority of the TMAP molecules that bind to MS2-wt enter through the ~1.8 nm pores in the capsid structure and interact with the RNA. Due to its chemical properties, TMAP by itself is retained in the column and is unable to elute efficiently.

The observation that TMAP:MS2-ec interact to some extent could be due to two potentially concurrent explanations. For one, the positively charged porphyrin molecule may be interacting with residual RNA present on the interior of the capsid, an observation that is supported by the fact that even after RNA removal, a ~98% protein purity is calculated based on the 260/280 nm ratio. Additionally, charged or polar amino acid residues that are present on the interior or exterior surfaces of the capsid structure could potentially interact with the positively charged porphyrins.

Binding interaction between MS2 and TMAP

To further investigate the nature of the interaction between TMAP and the MS2 bacteriophages, the full UV-visible Absorbance Spectra of the eluted fractions were analyzed in greater detail. In this respect, Fig. 4displays the UV-vis spectra for TMAP, TMAP with MS2-wt, TMAP with MS2-ec, TMAP with extracted MS2 RNA, and TMAP with Lambda dsDNA, after eluting through a size exclusion column (fraction 9). The TMAP and DNA samples were used as a comparative control given that the interaction between TMAP and

DNA in solution has been well documented [17–19, 27, 28]. The absorbance spectrum for TMAP in buffer without the presence of MS2 exhibiting no shift in the Soret peak (412 nm) is also included for reference in Fig. 4. Furthermore, individual absorbance spectra for MS2-wt, MS2-ec, and TMAP in buffer can be found in Fig. S1 (see Supporting information section). Upon TMAP binding to both MS2-wt, extracted MS2 RNA and DNA, there is an observed red shift in the Soret peaks of around 8–10 nm to ~420 nm. In contrast, the MS2-ec/TMAP sample shows a smaller 4–6 nm red shift and a broader Soret peak than the other samples.

Porphyrin interactions with proteins and nucleic acids have been extensively studied, and it is well-known that the interaction between TMAP and nucleotides could induce bathochromicity shifts in the porhpyrin absorbance spectra [17]. In addition, it has been previously observed that TMAP molecules tend to bind to the outside backbone of a DNA molecule and that this type of binding will cause a red-shift of the magnitude of 8–10 nm [18]. Accordingly, it is suggested that the 8–10 nm red shift in the Soret band observed in both the MS2-wt /TMAP, MS2 RNA/TMAP and DNA/TMAP spectra could be attributed to TMAP interacting with the nucleic acid backbone. Other potential interaction modes, such as intercalation between the nucleotide bases seen with other porphyrins, typically exhibit a much higher red shift, in the range of 30–40 nm [29]. In addition, the bulky R groups at the meso positions in TMAP are known to interfere with intercalation [18]. The observed red shifts provide additional evidence that TMAP is able to enter the capsid through the pores and interact with the RNA present inside the capsid. A smaller shift is observed in the MS2-ec/TMAP spectra, but this is inconclusive as to whether it could be attributed to a weak interaction of TMAP with the capsid protein or/and a small amount of RNA remaining after base hydrolysis.

Fig. 4. UV-vis absorbance spectra of MS2-wt with TMAP, MS2-ec with TMAP, MS2 RNA with TMAP, and DNA with TMAP after elution through a 5 kDa mw cut-off size-exclusion column (fraction 9). The inset shows a zoom-in of the Soret peak region of MS2 with TMAP samples — the dotted vertical line through the peak adsorption of TMAP with MS2-wt is meant to guide the eye



It is also interesting to analyze the shape of the Soret band peaks for each of the samples. The DNA/TMAP Soret band peak has a similar shape to the TMAP-only Soret band peak, although it is red shifted. The free MS2 RNA/TMAP peak on the other hand is broader, while the MS2-wt/TMAP peak has a similar appearance to the DNA/TMAP spectra with a slightly extended shoulder on the left side. Upon closer inspection it can be seen that the relative absorption of the extended shoulder vs. the main Soret peak is somewhat higher compared to the DNA/TMAP sample indicating differences in binding. This observation could signify that a fraction of the TMAP molecules associated with the MS2-wt bacteriophages are interacting with the protein capsid, or that the interactions of TMAP with packaged RNA is different from free RNA/DNA in solution. This finding further corroborates what is seen in the UV-spectra for the MS2-ec/TMAP sample. The peak shape for the latter is broader and has a smaller red shift than TMAP associated with MS2-wt, free MS2 RNA, and DNA. One possible explanation for this is that the spectra include partial contribution from TMAP associating with the capsid protein, (with little or no red-shift in the Soret band), and an additional partial contribution from TMAP binding to small amounts of RNA remaining in the capsid (giving rise to larger shifts). However additional analysis is required to elucidate this in more detail.

To this end, an investigation of peak shifts and binding characteristics of TMAP to bovine serum albumin (BSA), a generic protein with an isoelectric point of ~4.7 (thus negatively charged at neutral pH) was undertaken [30]. BSA was selected as a model protein considering that MS2 capsid proteins are also negatively charged at neutral pH (pI ~3.9). As can be observed in Fig. S2, very little TMAP co-elutes with albumin (less than 1:1 ratio). Additionally, no red shift was detected for the TMAP/BSA sample in the UV-vis spectra.

Fluorescent spectroscopy was used to further study the interaction between MS2 bacteriophages and TMAP, and normalized emission spectra of TMAP are plotted in Fig. 5. Fluorescence is extremely sensitive to the environment immediately surrounding a molecule, and hence can provide insight into how TMAP interacts with the bacteriophages. As can be seen in the emission spectra, there is a stark contrast between the peak positions of free TMAP in solution compared to when TMAP is associated with MS2-wt and free MS2 RNA. The emission peaks from the TMAP/MS2-wt and TMAP/MS2 RNA samples are red shifted approximately 8 nm, which indicates that the TMAP environment in these samples are similar. This provides additional evidence that the TMAP molecules are able to enter the pores of the capsid and associate with the RNA in the capsid interior. Interestingly, the MS2-ec

emission spectra show a smaller red shift, approximately 6 nm from that of free TMAP. This suggests that the interaction of TMAP with the empty capsids is different from that of the RNA-containing capsids, which is in agreement with the absorption data. Additionally, the peak positions of free TMAP in solution and BSA/TMAP are just about identical. A reasonable explanation is that in both of these samples, the TMAP molecule is highly solvated and there is only a weak interaction with the protein. This assumption is supported by the absorption data (Fig. S2).

MS2-TMAP singlet oxygen generation

The results from the investigation of MS2-TMAP singlet oxygen photogeneration can be found in Fig. 6. The peak output of the HeNe laser used in the experiments, (632 nm), matches the fourth absorbance Q-band of the TMAP molecule. Even though absorption is much higher at the Soret band, it is desirable to use light > 600 nm in PDT treatment because of poor depth penetration of light into tissues [31]. The data demonstrate that TMAP molecules loaded within MS2 bacteriophages are able to generate singlet oxygen when illuminated with light. Interestingly, MS2-TMAP exhibited higher fluorescence emission than an equivalent concentration of free porphyrin in solution, indicating an increase in singlet oxygen production. The increased singlet oxygen generation could potentially be caused by the inability of the porphyrin to self-stack when encapsulated inside MS2. Self-stacking is a phenomenon known to influence the optical properties of porphyrins, including absorbance peak shifting and fluorescence emission intensity [32]. It is conceivable that TMAP molecules associated with MS2 RNA could be oriented within the capsid in such a way as to prevent self-stacking from occurring, resulting in increased singlet oxygen generation. MS2 bacteriophages with no associated porphyrin exhibited background levels of fluorescence, indicating no singlet oxygen generation.

Fig. 5. Normalized fluorescence spectra of free TMAP in solution, MS2-wt with TMAP, MS2-ec with TMAP, MS2 RNA with TMAP, and BSA with TMAP after elution through a 5 kDa mw cut-off size-exclusion column (fraction 9). ex 412 nm



EXPERIMENTAL

Materials

Unless otherwise noted, all chemicals were purchased from Sigma, Ltd, MO. TMAP was purchased from Frontier Scientific Inc, UT. All solutions were prepared using 18.2 M -cm ultrapure water (Millipore, MA). MS2 wild-type bacteriophage stock was provided by Dr. David Peabody, University of New Mexico.

MS2 propagation and purification

MS2 bacteriophages were prepared and purified as reported previously with minor modifications [33]. Initially, a liquid culture (40 mL) inoculated from a single colony of E. coli C3000 (ATCC 15997) was grown for 6 h in LB media at 37 °C. Cells were pelleted by centrifugation and re-suspended in sterile MgSO4 (20 mL, 10 mM). Approximately 109 pfu MS2 were added and incubated at 37 °C for 5 min. This solution was added to LB media (1 L) supplemented with MgSO4

(10 mM) and incubated for 16 h at 37 °C.Next, chloroform (20 mL) and NaCl (120 mL, 0.5 M)

were added to lyse the cells. The cell debris was pelleted by centrifugation at 2500 RCF for 30 min at 4 °C. To

the supernatant, MgSO4 (10 mL, 1 M) and polyethylene glycol (PEG6000, 120 g) were added and then incubated at 4 °C for 1 h to precipitate the phage. The precipitated phages were collected by centrifugation at 2500 RCF for 1 h at 4 °C. The phage-containing pellet was resuspended in Tris-HCl buffer, (15 mL, pH 7.0) supplemented with MgSO4 (10 mM). This suspension was transferred to a 30 mL corex tube followed by the addition of chloroform (15 mL), vortexing and spinning at 10,000 RCF for 10 min. The upper aqueous phase containing the phages was carefully removed and the chloroform extraction was repeated to remove any residual PEG.

The phages were purified by ultracentrifugation in two steps: first, the phage suspension (8 mL) was layered on top of a CsCl step gradient (1.5 mL each of 1.4 g.mL-1,1.5 g.mL-1 and 1.7 g.mL-1) followed by centrifugation for 4 h at 38,000 RCF in a SW41 rotor (Beckman-Coulter, CA). The phages could be seen as a tight, bluish band at the interface of the 1.4 and 1.5-density steps. This band was removed by puncturing the side of the centrifuge tube and drawing out the fluid with a syringe fitted with an 18 gauge-needle. This fraction was further purified by ultracentrifugation in a CsCl equilibrium gradient (1.5 g.mL-1) at 180,000 RCF for 16 h in a SW41 rotor. Again, the phage formed a bluish band near the top of the gradient that was removed from the tube as described above. The recovered fraction was dialyzed against ultrapure H2O (2 L) for 16 h. SDS-PAGE analysis on a 15% polyacrylamide gel showed a single protein band at 13.5 kDa, corresponding to the MS2 capsid protein. Afterwards, MS2 phages were diluted to working concentrations with sodium phosphate buffer (20 mM, pH 7.0).

For preparation of RNA-free empty capsids (MS2-ec), MS2 virions were incubated in a 11.8 pH solution as reported by Hooker et al. [34]. Absorbance data (260/280 nm ratios) showed protein purity levels of ~98%, indicating the majority of the RNA was removed. Capsid concentration was determined using the Warburg–Christian equation [35]. Analysis of MS2 before and after RNA removal with dynamic light scattering (DLS), transmission electron microscopy (TEM), and tryptophan fluorescence confirmed that the phage capsids remained intact after RNA removal (Figs S3–S5).

MS2-TMAP sample preparation

Samples were prepared to contain either MS2-wt (0.075 mg.mL-1) or MS2-ec (0.05 mg.mL-1) and TMAP (50 M) in sodium phosphate buffer (20 mM, pH 7.0), resulting in a 2500:1 TMAP:MS2-wt or TMAP:MS2-ec ratio. These concentrations corresponded to the same molar concentration of empty or full capsids in each experiment. Pierce D-Salt Dextran desalting columns (crosslinked beaded dextran, molecular weight cutoff: 5 kDa, volume: 5 mL; void volume: 1.75 mL) were used for size separation experiments (Thermo Scientific, IL).

Fig. 6. Singlet oxygen production by MS2-TMAP in solution after photoexcitation for 30 or 90 sec at 632 nm with a 15 mW HeNe laser. Fluorescence was generated by the Singlet Oxygen Sensor Green reagent (excitation/emission 488/525 nm). The error bars represent the /- standard deviation of the measurements, (n 3)



Columns were first equilibrated with sodium phosphate buffer (20 mM, pH 7.0). After equilibration, 100 L of sample was added and allowed to enter the column. The controls consisted of running TMAP (50 M), MS2-wt (0.075 mg.mL-1) and MS2-ec (0.05 mg.mL-1) through columns individually. After sample was loaded onto the column, sodium phosphate (5 mL, 20 mM, pH 7.0) was added and collected in 250 L fractions.

Each eluted fraction was analyzed by UV-vis spectroscopy. Concentrations of TMAP:MS2 before and after column chromatography were calculated using the absorption coefficient for TMAP, 412 199,930 M-1.cm-1 (Fig. S6) and for MS2, 260 8.03 mL.mg-1 [36]. Eluted fractions exhibiting absorbance at wavelengths corresponding to the peak absorbances of MS2 and TMAP were pooled and the absorbance values averaged before determining the concentration of each component.

UV-vis and fluorescence spectroscopy

UV-vis and fluorescence measurements were per-formed on a Tecan Infinite M200 plate reader (Tecan Trading AG, Switzerland). For elution experiments, entire spectra were collected for each fraction and the peak absorption values for each component was monitored, (260 nm for MS2-wt, 280 nm for MS2-ec and 412 nm for TMAP). UV-visible absorbance spectra were used to investigate the nature of the MS2 bacteriophage/TMAP interaction. Additional controls consisted of lambda phage DNA (100 M) mixed with TMAP (50 M), and free MS2 RNA (extracted and purified with phenol-chloroform). For the experiment to investigate eventual peak shifts and binding characteristics of a generic protein to TMAP, bovine serum albumin was employed. BSA (0.13 mg.mL-1) was mixed with TMAP (50 M) to achieve a 2500:1 TMAP:BSA starting ratio and eluted through a size exclusion column similar as for MS2/TMAP.

Singlet oxygen assay

The ability of TMAP packaged into MS2 bacte-riophages to generate singlet oxygen was assessed with a commercially available kit, Singlet Oxygen Sensor Green, (SOSG), (Invitrogen, CA). SOSG (10 L) was added according to the manufacturers protocol to MS2, TMAP, and MS2-TMAP (50 L each taken from pooled fractions after size exclusion chromatography). Starting concentrations before addition of SOSG were MS2 (140 g/mL) and TMAP (10 M) in sodium phosphate buffer (20 mM, pH 7.0). Samples were then illuminated for either 30 or 90 sec with a 15 mW HeNe laser (peak output: 632.8 nm, CVI Melles Griot, NM), and singlet oxygen production was assayed with fluorescence, (excitation/emission 488/525 nm). Fluorescence data were normalized to emission levels of samples that had not been illuminated.

CONCLUSION

The investigation presented herein demonstrates that MS2 bacteriophage is capable of efficient “nucleotide-driven packaging” of cationic porphyrins and that MS2 bacteriophages can provide a natural nanoscale container for loading therapeutic molecules. Through size-exclusion chromatography, UV-vis and fluorescence spectroscopy, it was shown that porphyrins could enter and interact with the RNA present within the MS2 capsid. Removal of the interior RNA was observed to lead to significantly lower porphyrin interaction with the MS2 capsid. This observation indicates that this approach could potentially provide a controlled route to fine-tune the loading capacity.

Based on bathochromic shifts of the porphyrin absorption and fluorescence spectra, it is suggested that the MS2/TMAP interaction occurs primarily with the RNA in the interior of the capsid. This interaction could likely occur through binding of the positively charged porphyrin with the negatively charged RNA phosphate backbone, opposed to an intercalating base-stacking mechanism. It was also noted that a smaller amount of porphyrin molecule might have interacted with the capsid protein itself, but the exact nature of this interaction needs further investigation.

Lastly, it has been shown that the porphyrin TMAP, when packaged inside MS2 bacteriophages was capable of singlet oxygen photogeneration in solution when illuminated with light at 632 nm. These results demonstrate for the first time, ROS generation by self-packaged porphyrins contained within a nanoscale vessel. In summary, the robustness and stability of MS2 make it an attractive transport vessel for therapeutically relevant molecules such as porphyrins. However, the efficacy of the encapsulated porphyrins to generate cytotoxicity warrants additional study.

Acknowledgements

This research was funded by the College of Nanoscale Science and Engineering, University at Albany, Albany, NY, USA. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081) [37].


Figs S1–S6 are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S1088424611004336



INTRODUCTION

Photodynamic therapy (PDT) is a medical procedure, based on the combined action of a photosensitizer (dye) and light, that has been increasingly employed in the treatment of cancer and other skin diseases, such as psoriasis and vitiligo (leukoderma), with excellent results from the medical and aesthetic points of view. Among the several compounds, porphyrins and their derivatives have been extensively employed as photosensitizers. In fact, Photofrin® the first commercial phototherapeutic agent approved by FDA about 20 years ago, is based on hematoporphyrin oligomers, more exactly nine porphyrin units linked by ether, ester and carbon–carbon bonds [1–3]. Nevertheless, some limitations have been imposed, particularly by their long persistence time in the organism, leading to undesirable skin photosensibility that can last several weeks or even months [4–6]. For this reason, more effective and safe photosensitizers and formulations exhibiting well defined chemical compositions, higher

biocompatibility, preferential or specific accumulation in the tumor cells, faster elimination rate by the organism, lower toxicity and higher phototoxicity have been pursued [3, 7–10]. At the present time only Photofrin®, Levulan® Kerastick and Visudyne® have been approved by FDA for PDT treatment.

Although porphyrins and derivatives have interesting photodynamic properties, frequently their solubility in aqueous media is far too low for direct application in PDT treatment. A convenient strategy to increase the biocompatibility and dispersability in biological fluids is the micro and/or nanoencapsulation of water insoluble lipophilic compounds. Polymeric encapsulation is a very interesting choice for that purpose, because the capsules shell can be engineered using several biocompatible polymers and varying the degree of cross-linking [9, 11, 12], thus allowing the control of the permeability and the mechanical resistance. Also, polyethyleneglycol derivatives can be added to decrease the interaction of the polymeric capsule with the plasma components thus avoiding the loss of formulation by opsonization [13, 14] by phagocytary cells.

The binding properties of a series of meso-phenyl(N-methylpyridinium)porphyrins to vesicles and red cells

Correlation of photodynamic activity and singlet oxygen

quantum yields in two series of hydrophobic monocationic

porphyrins

Daiana K. Deda, Christiane Pavani, Eduardo Caritá, Maurício S. Baptista,

Henrique E. Toma and Koiti Araki*

Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes 748, Sao Paulo, 05508-000, SP, Brazil

Received 7 May 2011Accepted 2 July 2011

ABSTRACT: The photodynamic properties of eight hydrophobic monocationic methyl and ruthenium polypyridine complex derivatives of free-base and zinc(II) meso-triphenyl-monopyridylporphyrin series were evaluated and compared using HeLa cells as model. The cream-like polymeric nanocapsule formulations of marine atelocollagen/xanthan gum, prepared by the coacervation method, exhibited high phototoxicity but negligible cytotoxicity in the dark. Interestingly, the formulations of a given series presented similar photodynamic activities but the methylated free-base derivatives were significantly more phototoxic than the respective ruthenated photosensitizers, reflecting the higher photoinduced singlet oxygen quantum yields of those monocationic porphyrin dyes.

KEYWORDS: cationic porphyrins, polymeric encapsulation, PDT, HeLa cells.


*Correspondence to: Koiti Araki, email: [email protected], tel: +55 11-3091-8513, fax: +55 11-3815-5579


56 D. K. DEDA ET AL.

were shown to be correlated with their n-octanol/water partition coefficients (log(POW)). However, amphiphilic species exhibited significantly higher interaction and photodynamic activity [15–17] than predicted solely based on that parameter. More recently, the monocationic meso-triphenyl(3-N-methylpyiridinium)porphyrin, 3MMe, was shown to be a very efficient photosensitizer when encapsulated in atelocollagen polymeric nanocapsules prepared by coacervation method [18]. However, few are the reports [19, 20] on the influence of transition metal ions, coordinated to the porphyrin ring or to its periphery, on the photodynamic properties. Accordingly, a systematic study was carried out with two series (free-base and zinc(II) complex) of monocationic meso-triphenyl(pyridyl)porphyrin derivatives (Fig. 1) in order to assess the influence of the ring metallation and the coordination of a [Ru(bipy)2Cl]+ complex on the photodynamic properties of porphyrin dyes, using HeLa cells as model.


Syntheses and characterization

The monocationic porphyrins 3MPyTPP and 4MPyTPP were prepared by the reaction of stoichiometric amounts of pyrrole, benzaldehyde and 3-pyridyl or 4-pyridylcarboxaldehyde in refluxing glacial acetic acid [24]. The small amounts of meso-tetraphenylporphyrin (TPP) and disubstituted derivatives were separated by silica-gel column chromatography using a mixture of dichloromethane and ethanol (2%) as eluent. The respective zinc(II) complexes (3- and 4-ZnMPyTPP) were obtained by refluxing them with zinc acetate in a mixture of glacial acetic acid and DMF.

3MMe and 4MMe, were obtained refluxing 3MPyTPP and 4MPyTPP with 40 times excess of methyl p-toluenesulfonate in dimethylformamide (DMF), purified by neutral alumina column chromatography and precipitated as chloride salts in saturated NaCl solution to increase the biocompatibility and solubility in aqueous media. The Zn-3MMe and Zn-4MMe were prepared in a similar way.

The series of ruthenated porphyrins (3MRu, 4MRu, Zn-3MRu and Zn-4MRu) were obtained by reacting 3MPyTPP, 4MPyTPP, Zn-3MPyTPP and Zn-4MPyTPP with about 1% excess of the [Ru(bipy)2Cl(H2O)]NO3 complex, in a CH2Cl2/DMF 4:1 v/v mixture, for 60 min [17] and purified by neutral alumina column chromatography, using a mixture of CH2Cl2 and ethanol as eluent.

The methylated and ruthenated free-base porphyrin series exhibited characteristic UV-vis spectral profiles with an intense band around 420 nm (Soret band) and four less intense Q-bands at 515, 565, 596 and 650 nm (Fig. 2). A bipyridine pπ→pπ* internal transition band and a broad RuII(dπ)→bipy(pπ*) MLCT band were also observed in the ruthenated derivatives, respectively at 295 and 470 nm. A similar behavior was observed for the Zn(II) porphyrin series, but the Soret band was shifted to 428 nm and only two Q-bands were observed at 560 and 610 nm.

The two series of methylated and ruthenated amphiphilic monocationic porphyrins (Fig. 1) were characterized by 1H NMR. The β-pyrrole protons of the para-isomers were found as a broadened singlet (8H) between 8.4 and 8.9 ppm, while the inner ring protons appeared as a singlet (2H) at -2.8 ppm. The pyridyl protons were found as a pair of doublets at 8.0 and 9.7 ppm, while the phenyl group protons were

Fig. 1. Scheme showing the structure of the eight monocationic meso-triphenyl(pyridyl)porphyrin derivatives, highlighting the structure of 3MMe


CORRELATION OF PHOTODYNAMIC ACTIVITY 57

found as a double doublet and a multiplet at 7.6 and 8.1 ppm. The protons of the bipyridyl ligands appeared as very characteristic signals in the 7 to 10.1 ppm range. As expected, an intense singlet (3H) was observed at 4.6 ppm in the N-methylated derivatives. The 1H NMR signals of the [Ru(bipy)2Cl]+ derivatives of the porphyrin para-isomers were sharper and better resolved than the signals of the new meta-isomers, but the integration was always consistent with the expected number of protons. Accordingly, the presence of a [Ru(bipy)2Cl]+ group bond to the periphery of the porphyrin ring was further confirmed by cyclic voltammetry. In fact, the characteristic reversible pair of voltammetric waves at 0.9 V, assigned to the RuIII/RuII redox process, had the same intensity of the monoelectronic reversible reduction processes of the porphyrin ring at -0.9 and -1.3 V, thus confirming the presence of a ruthenium complex coordinated to the porphyrin ring. The irreversible process observed at +1.38 V was assigned to the oxidation of the porphyrin ring.

Singlet oxygen quantum yields (φΔ)

Meso-arylporphyrins are type II photosensitizers whose photodynamic activity depend on the formation of singlet oxygen by energy-transfer from electronically excited dye molecules to dioxygen [1–3, 6, 25], upon irradiation with light of suitable wavelength [2, 5, 6, 26, 27]. The photoinduced singlet oxygen quantum yields (φΔ) of all porphyrin dyes were evaluated from the slope of the 1Δg(O2) emission intensity as a function

of the laser power plot. The absorbance at 532 nm of the eight porphyrin dyes and the TPP standard in CH2Cl2

solution was adjusted to 0.02 a.u., and the power of the laser pulses varied in the 1 to 8 mJ.cm-2 range. A typical set of decay curves, used to collect the emission intensity at 1270 nm for the 4MRu and the Zn-4MRu derivatives, are shown in Fig. S1 (see Supporting information section). The phosphorescence intensities were measured 70 μs after the laser pulse and plotted as a function of the corresponding laser power. Linear correlations were found for both porphyrin series and φΔ determined from the ratio of their slopes with that found for the TPP standard, in the same experimental conditions. The free-base porphyrin derivatives exhibited significantly higher 1Δg(O2) phosphorescence than the corresponding zinc(II) porphyrin derivatives, reflecting their higher energy transfer quantum efficiencies. The φΔ values, the lifetimes (1/k) and the photodynamic efficiencies (% of cell death) for the eight porphyrin dyes are listed in Table 1.

The lifetime of 1Δg(O2) defines the maximum radius of action of this reactive species. Values in the 140–180 μsrange were measured in CH2Cl2, which are consistent with the lifetime of singlet oxygen in hydrophobic environments. In fact, it can be as high as 200 μs in organic solvents in the absence of quenchers, but decreases to 2–4 μs in aqueous solution [28]. In the cytosol, however, the lifetime decreases to about 10–50 ns, such that the perimeter of action is reduced to about 0.1 μm from the point where it is generated.

Fig. 2. Absorption spectra of (a) 3MMe and Zn-3MMe, (b) 4MMe and Zn-4MMe, (c) 3MRu and Zn-3MRu, and (d) 4MRu and Zn-4MRu in CH2Cl2. Inset: exploded view in the visible range



The free-base porphyrins, particularly the 3MMe and 4MMe derivatives, showed higher φΔ and photodynamic efficiencies than the respective zinc(II) porphyrins. Also, the ruthenium complex is contributing to diminish further the 1Δg(O2) quantum yields of the macrocyle, possibly introducing new competing relaxation pathways for the excited state species.

Toxicity and phototoxicity of the monocationic porphyrins

Coacervation is one of the most commonly used methods for the preparation of nanocapsules using natural polymeric materials such as sodium alginate and gelatin. The procedure involves two steps: (a) the o/w emulsification by mixing an organic phase containing the active substances with an aqueous polymeric phase by mechanical stirring and/or ultrasound processing; followed by (b) the coacervation process that can be induced by addition of electrolytes, decrease of temperature or dehydration agents [21, 29, 30].

The delivery efficiency of a photosensitizer [11, 31] to cells by micro/nanocapsule formulations are mainly controlled by shell properties [21, 32] such as morphology, size, mechanical strength and permeability, which generally are defined by the chemical composition, temperature, pressure and pH, that may also influence the cell viability, cytolocalization and interaction with the blood components, as described by Dash et al. [33].

The photodynamic properties of a dye depend on three major factors in addition to the pharmacokinetic properties and biodistribution: (a) their ability to interact with cells and tissues, (b) the quantum efficiency of the photosensitizer, and (c) the mechanism of the

photodynamic action. The first two are strongly dependent on the molecular structure, such that the affinity was found to be a function of the lipophilic character of the cationic dyes. However, this is abnormally enhanced in the case of amphiphilic species [16] that penetrate more deeply into the cell membrane as a consequence of the unique spatial distribution of lipophilic and hydrophilic groups in those molecules. Also, amphiphilic Zn(II) porphyrins was shown to bind preferentially to the cell membrane, improving the PDT efficiency [20]. The meso-pyridylporphyrins are type II photosensitizers, acting essentially through energy transfer to molecular oxygen and production of singlet oxygen, rather than the direct electron transfer from the excited species to biomolecules. The coordination of zinc(II) [34, 35] was shown to improve the PDT efficiency because tend to enhance the type II character of the photosensitizer. In fact, tetraruthenated porphyrins [36], particularly the zinc(II) derivative, showed to be a better type II photosensitizer than methylene blue. A similar behavior was observed for N-confused porphyrins and its Ag(III) complex [37].

The two series of monocationic porphyrins have very low solubility in aqueous media such that they can cause serious problems when injected intravenously due to precipitation and clogging of capillary veins. Unfortunately, the affinity of the dye for tumor cells and tissues is proportional to log(POW) such that high affinity usually means low solubility in biological fluids, hindering their direct use in PDT. This problem can be overcome by encapsulating them in micro and nanocapsules with appropriate surface functional groups and surface charge [11, 12].

In addition, the composition and size can be controlled to impart biocompatibility and optimal

Table 1. Fluorescence quantum yields, φfl, singlet oxygen 1Δg(O2) quantum yields, φΔ, lifetimes τ (in CHCl3 solution) and photodynamic efficiencies φPD towards HeLa cells of the eight monocationic porphyrin dyes and TPP, used as standard

Porphyrin φfl (SD) φΔ (SD) τ, μs (SD) φPD, %

TPP 0.150 0.50 183.4 (4.2)

3MMe 0.3431 (0.0086) 0.79 (0.04) 163.2 (3.1) 92.3

4MMe 0.4583 (0.0030) 0.55 (0.02) 160.2 (2.6) 92.3

3MRu 0.0210 (0.0007) 0.50 (0.02) 161.1 (4.7) 71.9

4MRu 0.0297 (0.0006) 0.51 (0.03) 162.6 (1.4) 74.9

Zn-3MMe 0.0124 (0.0003) 0.40 (0.01) 140.6 (7.5) 54.0

Zn-4MMe 0.0372 (0.0015) 0.48 (0.03) 138.9 (5.0) 54.5

Zn-3MRu 0.0148 (0.0002) 0.40 (0.03) 139.3 (2.6) 53.0

Zn-4MRu 0.0249 (0.0003) 0.36 (0.02) 144.7 (3.4) 58.8



interaction and delivery of their content to malignant cells. For this purpose, polymeric formulations were prepared by encapsulating the two series of monocationic porphyrins in marine athelocolagen/xantam gum polymeric microcapsules, that were reprocessed using a high power ultrasonic tip monitoring the size by DLS [18]. Formulations with the largest fraction of capsules in the 400–500 nm range and histograms exhibiting bimodal size distribution were obtained (see Supporting information section Fig. S2). The porphyrin concentration was 1.0 × 10-4 M in all formulations. The cytotoxicity was evaluated by incubating 1.0 × 105 HeLa cells with 1.0 mL of a 1 μM porphyrin formulation (in colorless DMEM culture media) for 6 h, in the dark.

When compared to the dark control experiments in the absence of any sensitizer, the dark toxicity of all sensitizers investigated was negligible (Fig. S3). However, the cell viability decreased dramatically when the cells, incubated with the free-base porphyrin formulations, were irradiated with the 650 nm laser, in contrast with the control (Fig. 3). Note that the 4MMe and 3MMe formulations reduced the cell viability in more than 80%, while a 50–60% decrease was found for 3MRu and 4MRu. In fact, the N-methylated derivatives consistently showed a much higher photodynamic efficiency than the N-ruthenated derivatives. The localization of the peripheral groups in the para- or meta-position had no significant influence on the photodynamic activity within experimental error, showing that the solubility and binding affinity of those photosensitizers to the HeLa cells are virtually the same for both isomers.

At this point, it is interesting to remember that the fluorescence quantum yield of 4MPyTPP in CH2Cl2 is φfl(λexc 512 nm) = 0.099, only slightly smaller than for TPP standard ((φfl(λexc 512 nm) = 0.11). The coordination of a [Ru(bipy)2Cl]+ group to the pyridyl N-atom did not change the fluorescence spectra profile (two bands

at 655 and 715 nm) indicating that the lowest excited state in 3MRu and 4MRu is localized in the porphyrin ring and not in the peripheral [Ru(bipy)2Cl(pyP)]+

complex. However, the fluorescence quantum yield decreased almost two orders of magnitude to 2.9 × 10-3

suggesting that other relaxation pathways, such as the non-radiative decay and the intersystem crossing, were favored by coordination of the ruthenium complex (Fig. S4). Interestingly, the photodynamic efficiency was reduced only about 20% suggesting that the energy transfer from the excited porphyrin to dioxygen should be much faster than the fluorescence emission and other pathways, including the non-radiative decay. However, considering that the lifetime of ruthenated porphyrins in the singlet excited state should be much smaller than that for the starting MPyTPP (~5 ns in CH2Cl2), there is not enough time for the occurrence of efficient bimolecular processes. Thus, the energy transfer to dioxygen should be taking place after intersystem crossing to the excited triplet state. In this case, the decreased of the fluorescence quantum yield can be explained by the increase of the intersystem-crossing rate induced by the coordination of the ruthenium complex. Analogous behavior was previously reported for the doubly N-confused Ag(III) complex. [37]

Unfortunately, the series of monocationic zinc(II) porphyrins have the lowest energy absorption band around 610 nm (Fig. 2) and can not be excited by the red laser (λem = 650 nm) used for the free-base series. In order to get comparable results with both series, experiments were carried out using a mercury lamp as white light source. This presents a broad emission band in the visible range that can be used to excite both, the free-base and the zinc(II) porphyrin derivatives.

Accordingly, experiments were carried out as described above for the free-base porphyrin derivatives with both series of porphyrin dyes, except for the irradiation with a white mercury lamp source (16 mW.cm-2) for an hour. About 55% decrease in cell viability was found for all zinc(II) porphyrin series (Fig. 4a), while the free-base series once again showed distinct photodynamic behavior for the methylated (90% decrease) and ruthenated (70% decrease) derivatives (Fig. 4b). These results are consistent with those described above using the red laser as light source (Fig. 3), where no significant differences were observed in the photodynamic properties of the para-and the meta-isomers. This result is also consistent with a previous report in which a convergence of the log(POW)values was shown for both mono(N-methylpyridinium)porphyrin derivatives, while increasing differences were observed as the number of N-methylpyridinium groups increased [16].

Similar results were obtained for the zinc(II) porphyrin series suggesting that the coordination of Zn(II) ion to the porphyrin ring contributed to enhance the intersystem crossing rate alike the coordination of the [Ru(bipy)2Cl]+

complex to the pyridyl N-atom. Furthermore, the energy

Fig. 3. Phototoxicity of the monocationic free-base porphyrin derivatives in polymeric nanocapsules (1 μM) towards HeLa cells. The cells (1 × 105) were incubated for 6 h and irradiated with a 650 nm laser source for 10 min



of the Zn(II) porphyrin excited triplet state should be lower than the charge transfer excited state localized in the ruthenium complex, because otherwise the photodynamic efficiency should be significantly decreased by energy transfer. In addition, electron transfer processes should not be taking place since they would decrease further the photodynamic activity.

The φΔ and photodynamic efficiency of the porphyrin derivatives are compared in Fig. 5. The highest phototoxicity observed for 3MMe can be assigned to its higher 1Δg(O2) quantum yield. Interestingly, the photodynamic activity of 4MMe was much higher than that expected based on the φΔ suggesting that the value was underestimated, probably as a consequence of aggregation even at the low concentrations used in the measurements [15]. Likewise, the lower photodynamic efficiency observed for zinc(II) porphyrin and ruthenium complex derivatives can be assigned to their much lower 1Δg(O2) quantum yields.

EXPERIMENTAL

Synthesis of the monocationic porphyrin derivatives

3MPyTPP and 4MPyTPP. The 5,10,15-triphenyl-20-(3-pyridyl)porphyrin, 3MPyTPP, and the 5,10, 15-triphenyl-20-(4-pyridyl)porphyrin, 4MPyTPP, were synthesized and characterized according to a previously described method [15, 17]. A mixture of 53 mmol of benzaldehyde and 18 mmol of 3-pyridyl carboxaldehyde (or 4-pyridyl carboxaldehyde) was reacted with 71 mmol of pyrrol in glacial acetic acid, in the dark. After 2 h, the solvent was removed in a flash evaporator and the porphyrin precipitated-out in a mixture of ethanol and N,N-dimethylformamide (9:1 v/v). The solid was filtered, washed with the same mixture of solvents, dried and purified by silica-gel column chromatograph using suitable mixtures of CH2Cl2/EtOH as eluent. Yield 11%. (3MPyTPP). UV-vis (CH2Cl2): λmax, nm (log ε)

Fig. 4. HeLa cell viability after incubation with monocationic porphyrins incorporated in polymeric nanocapsules, in the dark and after irradiation for 1 h with a 16 mW.cm-2 white mercury lamp. (a) Zn(II) and (b) free-base porphyrin derivatives

Fig. 5. Photodynamic efficiency and respective 1Δg(O2) quantum yields of the eight monocationic porphyrin derivatives encapsulated in polymeric nanocapsules, when irradiated with a 16 mW.cm-2 white mercury lamp



418 (5.68), 515 (4.32), 548 (3.95), 590 (3.83) and 650 (3.63). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 9.74 (m, 1H), 9.39 (d, 1H), 8.98 (m, 1H), 8.83 (d, 2H), 8.79 (s, 4H), 8.72 (d, 2H), 8.46 (m, 1H), 8.15 (m, 6H), 7.71 (m, 9H), -2.82 (s, 2H). (4MPyTPP). UV-vis (CH2Cl2): λmax, nm (log ε) 417 (5.70), 515 (4.28), 550 (3.92), 590 (3.80) and 650 (3.61). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 8.96 (m, 2H), 8.83 (d, 2H), 8.79 (s, 4H), 8.73 (d, 2H), 8.15 (m, 6H), 8.11 (m, 2H), 7.71 (m, 9H), -2.80 (s, 2H).

Preparation of the free-base porphyrin series3MMe and 4MMe. The monopyridylporphyrins

3MPyTPP and 4MPyTPP were refluxed with 40-fold excess of methyl p-toluenesulfonate in N,N-dimethylformamide (DMF, 4 h), concentrated in a rotary evaporator and poured into a saturated NaCl aqueous solution. The precipitate was separated by filtration, redissolved in DMF and precipitated again in saturated NaCl solution to obtain the N-methylated porphyrins as chloride salts. After repeating this process three more times, the N-methylated dyes were purified by alumina column chromatography using a mixture of CH2Cl2 and EtOH as eluent. Yield 87%. (3MMe). UV-vis (CH2Cl2):λmax, nm (log ε) 422 (5.06), 515 (3.95), 550 (3.59), 590 (3.55) and 650 (3.45). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 10.02 (s, 1H), 9.50 (d, 1H), 9.40 (d, 1H), 9.04 (s, 2H), 8.90 (m, 6H), 8.58 (t, 1H), 8.23 (m, 6H), 7.86 (m, 9H), 4.64 (s, 3H), -2.85 (s, 2H). (4MMe).UV-vis (CH2Cl2): λmax, nm (log ε) 422 (5.17), 515 (4.07), 550 (3.78), 590 (3.60) and 650 (3.58). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 9.22 (m, 2H), 8.87 (d, 2H), 8.82 (s, 4H), 8.69 (d, 2H), 8.24 (m, 2H), 8.15 (m, 6H), 7.73 (m, 9H), 4.62 (s, 3H), -2.81 (s, 2H).

3MRu and 4MRu. The ruthenated free-base porphyrin derivatives were obtained by the reaction of 3MPyTPP and 4MPyTPP with [Ru(bipy)2Cl(H2O)]NO3

prepared just before use. Typically, 85 mg of AgNO3

was dissolved in 5 mL of DI-water and added to 236 mg of [Ru(bipy)2Cl2] dissolved in 15 mL of a 2:1 DMF/ethanol mixture, stirred for 20 min at 50 °C, and filtered through a Celite layer. The filtrate was concentrated to 5 mL, mixed with an equal volume of CH2Cl2 and reacted with 60 mg of the respective porphyrins, dissolved in 10 mL of CH2Cl2/DMF 4:1 mixture. After completion of the reaction (~20 min, 50 °C), the solvent was removed in a rotary evaporator, the solid dissolved in ~5 mL of DMF and added into an aqueous lithium trifluoromethanesulphonate (LiCF3SO3) solution. The ruthenated porphyrins, 3MRu and 4MRu, were pre-cipitated (CF3SO3

- salts) and isolated as dark brown solids after filtration, washing with water and drying in a desiccator under vacuum. The compounds were finally purified by alumina column chromatography using a mixture of CH2Cl2 and ethanol as eluent. Yield ~90%. (3MRu). UV-vis (CH2Cl2): λmax, nm (log ε) 294 (4.70), 417 (5.60), 520 (4.20), 550 (4.13), 588 (3.95) and 650 (3.77). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 10.13

(s, 1H), 9.45 (s, 1H), 9.10–8.65 (10H), 8.60–8.45 (4H), 8.35(m, 3H), 8.28–7.85 (13H); 7.80–7.40 (13H), 7.05(m, 1H), -2.78 (s, 2H). (4MRu). UV-vis (CH2Cl2): λmax, nm (log ε) 293 (4.80), 417 (5.70), 520 (4.30), 552 (4.16), 588 (3.88) and 649 (3.70). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 10.19 (m, 1H), 8.93 (m, 1H), 8.80 (m, 8H), 8.59 (m, 2H), 8.59 (m, 1H), 8.48 (m, 1H), 8.32 (m, 1H), 8.25 (m, 1H), 8.12 (m, 6H), 8.12 (m, 2H), 8.02 (m, 2H), 8.01 (m, 1H), 7.80 (m, 1H), 7.68 (m, 9H), 7.62 (m, 1H), 7.35 (m, 1H), 7.10 (m, 1H), -2.80 (s, 2H).

Preparation of the zinc(II) porphyrin seriesZn(II) porphyrin derivatives. The zinc(II)

porphyrins, Zn-3MPyTPP and Zn-4MPyTPP, were obtained by refluxing 3MPyTPP and 4MPyTPP with zinc acetate in a mixture of glacial acetic acid and DMF (yield ~90%). Then, the N-methylpyridinium (Zn-3MMe and Zn-4MMe) and the ruthenium complex derivatives (Zn-3MRu and Zn-4MRu) were obtained with ~90% yield, using the procedures described above for the preparation of the respective free-base porphyrin derivatives. (Zn-3MMe). UV-vis (CH2Cl2): λmax, nm (log ε)429 (5.07), 560 (3.84) and 600 (3.40). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 10.05 (s, 1H), 9.37 (d, 1H), 9.32 (d, 1H), 8.96 (s, 2H), 8.83 (m, 6H), 8.55 (t, 1H), 8.17 (m, 6H), 7.84 (m, 9H), 4.69 (s, 3H). (Zn-4MMe).UV-vis (CH2Cl2): λmax, nm (log ε) 428 (4.90), 560 (3.90) and 610 (3.33). 1H NMR (500 MHz, DMSO, TMS): δ,ppm 9.18 (m, 2H), 8.82 (d, 2H), 8.61 (s, 4H), 8.39 (d, 2H), 8.19 (m, 2H), 8.10 (m, 6H), 7.67 (m, 9H), 4.67 (s, 3H). (Zn-3MRu). UV-vis (CH2Cl2): λmax, nm (log ε) 295 (4.88), 427 (5.52), 560 (3.97) and 600 (3.70). 1H NMR(500 MHz, DMSO, TMS): δ, ppm 9.83 (s, 1H), 9.22 (d, 1H), 9.00–8.3 (13H), 8.25–8.00 (7H), 8.00–7.70 (9H); 7.60–7.35 (14H), 6.85 (m, 1H). (Zn-4MRu). UV-vis (CH2Cl2): λmax, nm (log ε) 295 (4.76), 427 (5.45), 552 (3.02) and 603 (2.92). 1H NMR (500 MHz, DMSO, TMS): δ, ppm 9.97 (m, 1H), 8.90 (m, 1H), 8.76 (m, 8H), 8.61 (m, 1H), 8.61 (m, 2H), 8.41 (m, 1H), 8.31 (m, 1H), 8.23 (m, 1H), 8.11 (m, 2H), 8.11 (m, 6H), 8.01 (m, 1H), 8.01 (m, 2H), 7.80 (m, 1H), 7.66 (m, 3H), 7.63 (m, 9H), 7.60 (m, 1H), 7.12 (m, 1H), 6.95 (m, 1H).

Preparation of the polymeric nanocapsule formulations

The eight porphyrin derivatives were encapsulated using the coacervation method, as described previously [18]. Typically, 1.0 mL of a porphyrin derivative solution (3.0 mM in CH2Cl2) was dispersed in a mixture of isopropylmyristate, almond oil and Tween 20 (1.2% v/v) using a Turrax, and poured into an aqueous xanthan gum suspension. Finally, marine atellocollagen and sodium sulfate were added under vigorous stirring to prepare a stable cream-like formulation of polymeric microcapsules [21], with the porphyrin derivatives dissolved in the oily core. The concentration of the dye was adjusted to 1 × 10-4 M. The average size of the capsules was diminished to 200–400 nm using an ultrasonic tip (750 W,



VibraCell, from Sonics), monitoring the size distribution by dynamic light scattering in a Microtrac Inc. Nanotrac 252 equipment.

Toxicity and phototoxicity of the nanocapsule formulations

The toxicity and phototoxicity of the monocationic porphyrin dyes were evaluated using human cervical epithelioid carcinoma cells (HeLa) as model. They were cultivated in DMEM culture media, containing 10% of bovine fetal serum and 1% of peniciline/streptomicine, in a Thermo Electron Corporation HEPA class 100 incubator, at 37 °C and atmosphere with 5% of CO2.Then, 1 × 105 cells were transferred to 2.0 cm diameter culture plates with 12 wells and incubated for 15 h for cell adhesion. Then, the culture media was removed and 1.0 mL of a 100 times diluted emulsion ([porphyrin] = 1 μM)was added and incubated in the same conditions as described above.

The toxicity of the porphyrin dye formulations were evaluated by the MTT method after removal of the culture media containing the porphyrin formulation, careful washing of the wells with saline phosphate buffer (PBS) and addition of 1.0 mL of colorless DMEM culture media. The number of viable cells was estimated 14 h after by measuring the ratio of the absorbance at 550 nm found for the wells incubated with and without (control experiment, 100% viability) a porphyrin formulation, using an Infinite M200 ELISA reader (TECAN). The absorbances are proportional to the amount of formazan crystals [22] formed after incubation of the cells with a 2.0 mg.mL-1 methylthiazyldiphenyltetrazolium bromide (MTT) solution in PBS for 2 h, careful removal of the excess of the green aqueous supernatant solution and solubilization of the crystals in 1.0 mL of DMSO.

The phototoxicity was evaluated in the same way but after irradiation of the adhered cells with a 650 nm laser (LASERLine, 70 mW.cm-2, free-base series) for 10 min (periodic pulses of 1 min with 1 min intervals); or with a white light source (tungsten/halogen lamp, 16 mW.cm-2,zinc(II) porphyrin series) for 60 min. The results are the average of at least two independent experiments performed in triplicate to ensure the reproducibility of the experimental conditions.

Singlet oxygen quantum yields (φΔ)

The singlet oxygen (1Δg(O2)) quantum yields (φΔ) of the two series of monocationic porphyrin derivatives were determined from the ratio of the slopes of the phosphorescence intensity at 1270 nm vs. the laser power plots, using meso-tetraphenylporphyrin, TPP, as standard (φΔ(1O2) = 0.5) [23]. The 1Δg(O2) phosphorescence emission decay curves were registered using a time-resolved Edinburg Analytical Instruments NIR flash-photolysis instrument equipped with a Nd:YAG Continuum Surelite III pulsed laser (λ = 532 nm, 10 Hz),

a liquid nitrogen cooled (-80 °C) Hamamatsu R5509 photomultiplier and LP900 aquisition software. The emitted light was passed through a silicon filter and a monochromator before reaching the NIR-PMT. The absorbance at 532 nm of the porphyrin derivatives in chloroform solution was adjusted to about 0.02 a.u. (~1 μM), and the phosphorescence intensity measured 10 ns after the laser pulse.

CONCLUSION

Two series of monocationic porphyrin derivatives were synthesized and their photodynamic activity evaluated using HeLa cells as model, after incorporation in polymeric marine atelocollagen/xanthan gum nanocapsules. The methylated free-base porphyrin derivatives exhibited higher phototoxicity inactivating 92% of the cells after 60 min of irradiation with a white light source, but no significant differences were observed for the para and meta isomers. The activity of the ruthenated porphyrins were ~20% lower, but was diminished even further by the coordination of Zn(II) ion to the porphyrin ring. In fact, all Zn(II) porphyrin derivatives exhibited similar phototoxicity and inactivate ~50% of the HeLa cells. Those results showed a correlation with their singlet oxygen quantum yields, such as in the case of the Zn(II) porphyrin derivatives that exhibited the lowest photodynamic activities and φΔ

values. In conclusion, the meta and para isomers of the methylated free-base monocationic porphyrins exhibited the highest photodynamic efficiencies and are promising candidates as PDT photosensitizers.

Acknowledgements

We gratefully acknowledge the financial support from the Brazilian agencies, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).


Singlet oxygen fluorescence decay curves, polymeric nanocapsules size distribution histograms and toxicity in the dark and fluorescence spectra of free-base monocationic porphyrins, (Figs S1–S4) are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S1088424611004427



INTRODUCTION

The specific localization of naturally occurring hematoporphyrin and several other porphyrin derivatives in the tumor cells is well reported and has been successfully utilized in photochemotherapy (PCT) or photodynamic therapy (PDT) [1–6]. Over the last few decades, studies using a variety of porphyrin derivatives radiolabeled using a host of radionuclides have been reported and their tumor specificity has been demonstrated in animal models [7–15].

The use of radiolabeled porphyrin in targeted tumor therapy could possibly circumvent certain inherent disadvantages of PCT/PDT techniques [7–10, 15].

Recent studies have revealed that porphyrins typi-cally show preferential uptake and retention by tumor tissue, possibly via receptor-mediated endocytosis of low density lipoproteins (LDL) [6, 16, 17]. Consequently, lipophilicity of radiometalated porphyrin complexes is expected to have significant effect on their tumor uptake. Moreover, it has been well-documented that lipophilicity of a metallo-radiopharmaceutical plays an important role in determining its clearance from different organs/tissue [18, 19]. It was therefore felt pertinent to carry out a systematic study showing the effect of lipophilicity of

Effect of lipophilicity on biological properties of 109Pd-

porphyrin complexes: a preliminary investigation

Sudipta Chakrabortya, Tapas Dasa, Haladhar D. Sarmab and Sharmila Banerjee*a

aRadiopharmaceuticals Division and bRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India

Received 31 March 2011Accepted 12 July 2011

ABSTRACT: The present study is designed to investigate the effect of lipophilicity of 109Pd-porphyrin complexes on their biological properties which were evaluated in tumor-bearing animal model. The insight obtained could be utilized to develop other radiometalated porphyrin complexes with optimum tumor uptake and tumor to background ratio as potential agents for targeted tumor therapy. 109Pd was produced by thermal neutron bombardment on enriched (in 108Pd) metallic palladium target at a flux of 3 × 1013

n/cm2.s for 3 d. 109Pd complexes of three different porphyrin derivatives, namely, 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin(I), 5,10,15,20-tetrakis[3,4-bis(carboethoxymethyleneoxy)-phenyl]porphyrin(II) and 5,10,15,20-tetrakis[4-carboxymethyleneoxyphenyl]porphyrin(III), which differ in their peripheral substituents, were synthesized. The biological behavior of the complexes was studied in Swiss mice bearing fibrosarcoma tumors. 109Pd was produced with a specific activity of ~1.85 GBq/mg (50 mCi/mg) and radionuclidic purity of 100%. All the 109Pd complexes were obtained in high yield (>97%) and they exhibited satisfactory in vitro stability at room temperature. The lipophilicity of the complexes follows the order 109Pd-II >> 109Pd-III > 109Pd-I. Biodistribution studies revealed that the most lipophilic 109Pd-II complex exhibited highest initial tumor uptake but poor tumor/liver ratio, while 109Pd-III complex exhibited the best tumor/liver ratio with reasonably good tumor accumulation. Thelipophilicity of 109Pd-porphyrin complexes was found to have considerable effect on their biological characteristics and radiometal-porphyrin complexes with optimum tumor uptake and adequately high tumor to background ratio could be synthesized by optimization of the lipophilicity through proper selection of peripheral substituents.

KEYWORDS: porphyrin, 109Pd, lipophilicity, targeted tumor therapy.

*Correspondence to: Sharmila Banerjee, email: [email protected], tel: +91-22 25593910, fax: +91 22-25505151


EFFECT OF LIPOPHILICITY ON BIOLOGICAL PROPERTIES OF 109PD-PORPHYRIN COMPLEXES 65

radiometal-porphyrin complexes on their tumor uptake as well as pharmacokinetics in animal model.

In our effort to develop potential agents for targeted tumor therapy, we have synthesized a few porphyrin derivatives and radiolabeled them with different therapeutic radionuclides, either by direct complexation or through bifunctional chelators [15, 20–22]. Among the radionuclides used, 109Pd could be envisaged as a useful therapeutic radionuclide owing to its suitable nuclear decay characteristics [15, 20, 23]. Palladium-109 decays by emission of β- particles [Eβ(max) = 1.12 MeV, 100% yield] with a half-life of 13.7 h to 109mAg [T1/2 = 39.6 s] which, in turn, emits a 88 keV γ photon in 3.6% yield accompanied by emission of conversion and Auger electrons leading to the formation of stable 109Ag [24, 25]. Moreover, 109Pd could be produced in adequate quantity, specific activity and in 100% radionuclidic purity [15, 20, 23, 24]. Moreover, it is well reported that Pd-porphyrin complexes are formed by direct incorporation of the metal at the core of the porphyrin molecule where the tetrapyrrole donor array acts as the chelating moiety [26]. As a result, a direct inference can be drawn about the correlation of the lipophilicities of the porphyrins (controlled by their peripheral substituents) and the pharmacokinetic properties of the corresponding palladium complexes. Taking these factors into consideration, 109Pd was chosen as the radionuclide for the present study.

109Pd complexes of two different porphyrin derivatives, namely, 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin(I) and 5,10,15,20-tetrakis[3,4-bis(carboe-thoxy-methylenoxy)phenyl]porphyrin(II) (Fig. 1), poss-essing eight carboxylic acid and ester residues as the peripheral substituents respectively have been reported earlier [15, 20]. In the present study, an attempt is made to synthesize the 109Pd complex (Pd-III) of yet

another porphyrin derivative viz. 5,10,15,20-tetrakis-[4-carboxymethyleneoxyphenyl]-porphyrin(III) (Fig. 1), having four carboxylic acid substituents, with the pres-umption that it would exhibit an intermediate lipophilicity when compared to those exhibited by complexes Pd-I and Pd-II. The effect of the different lipophilicities of the aforementioned complexes on their biological characteristics in terms of tumor uptake and retention, clearance from blood, liver, kidney and other major organs have been systematically studied in Swiss mice bearing fibrosarcoma tumor.

RESULTS

Production of 109Pd109Pd was produced with a specific activity of ~1.85

GBq/mg (50 mCi/mg) upon irradiation of enriched palladium (98% in 108Pd) target at a thermal neutron flux of 3 × 1013 n/cm2.s for 3 days. The gamma ray spectrum of the irradiated target recorded after radiochemical processing and subsequent separation of 111Ag did not show any gamma photopeaks characteristic of 111Ag (96, 245 and 342 keV) or 110mAg (655, 678, 707, 764, 885 and 937 keV) other than the 88 keV photopeak of 109mAgformed by the β- decay of 109Pd [25]. This indicates that 109Pd was obtained with 100% radionuclidic purity. The average radiochemical yield of 109Pd after the radiochemical separation of silver radionuclides was found to be ~90%.

Radiochemical studies

The 109Pd-complexes of the porphyrin derivatives I, II and III were obtained in high yield (>97%) under the optimized reaction conditions. The percentage complextion yields and Log P values of the complexes are given in Table 1. All the complexes were found to retain their radiochemical purities to the extent of >95%after 48 h at room temperature in normal saline.

Biodistribution studies

The biodistribution patterns of the 109Pd complexes with porphyrin derivatives I [15], II [20] and III carried out in Swiss mice bearing fibrosarcoma tumors are shown in

Table 1. Complexation yields and Log P values of 109Pd-porphyrin complexes

Complex % Complexation yield Log P

109Pd-I 98.3 (0.6) -1.88 (0.10) 109Pd-II 97.5 (1.0) 0.82 (0.06)109Pd-III 98.2 (0.5) -1.29 (0.08)

The figures in the parentheses indicate standard deviations (n = 3).

N

NH

HN

NR'

R R

R'

RR'

RR'

R = OCH2COOH R' = OCH2COOH

R = OCH2COOEt R' = OCH2COOEt

R = OCH2COOH R' = H

[I]

[II]

[III]

Fig. 1. Structure of porphyrin derivatives used in the present study


66 S. CHAKRABORTY ET AL.

Tables 2–4. The complex Pd-I showed significant tumor uptake of 2.80 ± 0.57% IA/g (IA = injected activity) within 30 min post-injection (p.i.), which remained almost constant at 24 h p.i. (2.66 ± 0.14% IA/g). The initially accumulated activity cleared from the blood and other major organs within 24 h p.i. The radiolabeled porphyrin exhibited major clearance through renal pathway as 88.68 ± 4.01% of the injected activity was observed to be cleared by this route within 24 h of injection. On the other hand, the complex 109Pd-II showed significantly higher initial tumor uptake (5.28 ± 1.46% IA/g at 30 min p.i.). The observed accumulation of activity in different major organ/tissue, particularly in liver (31.88 ± 3.78% IA/g), was also considerably high at this time point. However, with the progress of time the accumulated activity in the blood and the other major organs showed gradual clearance except that in the liver (14.41 ± 2.02% IA/g at 24 h p.i.). Though the tumor uptake was also observed to reduce substantially with time, significant activity (2.77 ±0.82% IA/g) was retained in the tumor at 24 h p.i. 64.61 ± 7.03% of the injected activity was observed to be cleared via renal pathway at this time point. The complex 109Pd-III exhibited similar biodistribution pattern as that of 109Pd-I in Swiss mice bearing fibrosarcoma tumor except slightly higher initial fibrosarcoma uptake (3.55 ± 0.49% IA/g at 30 min p.i.). The retention of the complex in the tumor was also satisfactorily similar to that of the other two 109Pd complexes (2.56 ± 0.25% IA/g at 24 h p.i.). The activity accumulated in blood and other major organs was found to undergo fast clearance through renal route. 90.01 ± 5.67% of the injected activity was observed to be cleared by this route within 24 h of injection.

DISCUSSION

In the present study, we report the comparison of the tumor uptake and other pharmacokinetic properties of 109Pd complexes of three different porphyrin derivatives evaluated in Swiss mice bearing fibrosarcoma tumors. The porphyrin derivatives used in these studies have identical core but differ in their peripheral substituents (Fig. 1). While the tetrapyrrole donor array constituting the core of the porphyrin derivatives acts as the chelating moiety for complexing with palladium, the peripheral substituents are responsible for the variation of the lipophilicity of the complexes formed. Determination of octanol-water partition coefficient of the complexes revealed that their lipophilicity (Log P values) follows the order 109Pd-II >>109Pd-III > 109Pd-I (Table 1). The lipophilicities of the porphyrin complexes were found to have significant effect on their tumor uptake as well as clearance pattern from the non-target organs in Swiss mice bearing fibrosarcoma tumors. A comparison of uptake in the fibrosarcoma tumor along with the tumor to blood and tumor to liver ratios among the 109Pd-porphyrin complexes are shown in Figs 2a to 2c, respectively. It was observed that the most lipophilic complex 109Pd-II exhibited significantly higher initial tumor uptake (at 30 min p.i.) compared to the other two complexes (Fig. 2a). However, the retention of all three 109Pd-complexes in fibrosarcoma tumors was found to be comparable in the later time points (3 h p.i. onwards). From these observations it could be inferred that while the lipophilicity of the metalloporphyrin complexes plays major role in their tumor affinity, it does not have significant effect on their retention in the tumor.

Table 2. Biodistribution pattern of 109Pd-I complex in Swiss mice bearing fibrosarcoma tumors

Organ % Injected activity/g of organ/tissue

30 min 1 h 3 h 24 h

Blood 6.88 (0.74) 5.83 (0.60) 2.41 (0.78) 0.61 (0.12)

Liver 2.76 (0.06) 3.56 (1.02) 2.59 (0.01) 2.70 (0.01)

GIT 1.81 (0.47) 2.27 (0.87) 1.92 (0.26) 0.56 (0.05)

Kidneys 12.62 (2.33) 9.63 (0.01) 10.98 (4.43) 6.55 (1.87)

Stomach 1.80 (0.28) 1.61 (0.34) 3.75 (2.47) 0.07 (0.02)

Heart 3.75 (1.91) 2.40 (0.38) 1.44 (0.29) 0.25 (0.05)

Lungs 6.77 (1.75) 6.38 (2.16) 3.44 (0.62) 1.88 (0.88)

Bone 1.06 (0.15) 1.30 (0.20) 0.97 (0.48) 0.00 (0.00)

Muscle 1.60 (0.79) 1.72 (0.07) 1.69 (0.29) 0.07 (0.04)

Spleen 1.60 (0.57) 1.39 (0.28) 0.75 (0.23) 0.59 (0.13)

Tumor 2.80 (0.57) 2.58 (0.05) 2.57 (0.28) 2.66 (0.14)

Excretiona 50.37 (7.48) 48.26 (7.66) 59.95 (4.00) 88.68 (4.01)

aPercentage (%) excretion has been indirectly calculated by subtracting the activity accounted for in all the organs from total injected activity. Three animals were used for each time point. The figures in the parentheses indicate standard deviations.



A comparison of tumor to blood ratio showed that 109Pd-IIcomplex had higher initial tumor to blood ratio compared to 109Pd-I and 109Pd-III (Fig. 2b), which is mainly due to the higher initial tumor uptake of the former. On the other hand, the tumor to liver ratio of 109Pd-II was found to be significantly poorer compared to both 109Pd-I and 109Pd-III complexes throughout the time points studied (Fig. 2c). The higher liver uptake of 109Pd-II could be probably attributed to its high Log P value. Although, 109Pd-II showed significantly higher initial tumor uptake, high uptake observed in vital organs, particularly in liver would limit its applicability as a suitable agent for targeted tumor therapy.

Both the complexes 109Pd-I and 109Pd-III are hydrophilic in nature and exhibited similar biological properties in the animal model used in the present study. The porphyrin derivatives I and III differ in the number

of –OCH2–COOH groups in the peripheral phenyl rings. While I has eight such substituents, III has only four. This renders 109Pd-I relatively more hydrophilic compared to the 109Pd-III complex. A comparison of their biodistribution characteristics revealed that 109Pd-III has higher initial fibrosarcoma tumor uptake (Fig. 2a) and more favorable tumor to liver ratio (Fig. 2c), although the differences are not so significant.

The present study revealed that although the lipophilic 109Pd complex showed significantly higher initial tumor localization compared to its hydrophilic analogs, high uptake in normal organs, particularly in liver, rendered the complex unfavorable for practical application. It could therefore be inferred that, while high lipophilicity facilitates appreciable tumor accumulation of the radiometal-porphyrin complexes, optimium hydrophilicity and presence of metabolisable groups

Fig. 2. Comparison of 109Pd-porphyrin complexes in terms of their fibrosarcoma tumor uptake (a), tumor to blood (b) and tumor to liver (c) ratio



such as ethers in the periphery are beneficial for their fast clearance from the non-target organs especially, from liver, resulting in good target to non-target ratio. Moreover, optimization of the lipophilicity of the

radiometal-porphyrin complexes could be achieved through proper tailoring of peripheral substituents attached to the porphyrin ring resulting in favorable characteristics for targeted tumor therapy.

Table 3. Biodistribution pattern of 109Pd-II complex in Swiss mice bearing fibrosarcoma tumors

Organ/tissue % Injected activity/g of organ/tissue

30 min 1 h 3 h 24 h

Blood 6.83 (2.23) 5.47 (0.94) 1.73 (0.34) 0.81 (0.33)

Liver 31.88 (3.78) 20.42 (2.22) 17.55 (1.21) 14.41 (2.02)

GIT 2.29 (0.51) 1.92 (0.31) 1.64 (0.66) 0.78 (0.32)

Kidney 12.60 (0.83) 9.95 (0.35) 8.51 (0.57) 7.92 (0.77)

Stomach 1.32 (0.38) 1.34 (0.28) 0.97 (0.16) 0.24 (0.11)

Heart 3.08 (0.00) 1.75 (0.42) 0.75 (0.25) 0.18 (0.02)

Lungs 10.02 (0.72) 6.95 (0.36) 6.11 (0.51) 3.22 (0.56)

Skeleton 0.68 (0.12) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Muscle 0.59 (0.12) 0.58 (0.10) 0.57 (0.09) 0.20 (0.04)

Spleen 4.98 (1.18) 4.79 (0.22) 2.82 (0.30) 1.96 (0.44)

Tumor 5.28 (1.46) 3.50 (0.68) 2.85 (0.57) 2.77 (0.82)

Excretiona 20.73 (3.65) 44.61 (5.60) 54.49 (4.76) 64.61 (7.03)


Table 4. Biodistribution pattern of 109Pd-III complex in Swiss mice bearing fibrosarcoma tumors

Organ % Injected activity/g of organ/tissue

30 min 1 h 3 h 24 h

Blood 7.46 (0.82) 6.50 (1.11) 3.01 (0.78) 0.88 (0.12)

Liver 3.11 (0.29) 2.70 (0.39) 2.48 (0.12) 2.53 (0.35)

Intestine 2.11 (0.40) 2.02 (0.87) 1.75 (0.38) 0.69 (0.22)

Kidneys 10.69 (3.07) 8.99 (1.02) 8.00 (2.18) 5.88 (0.79)

Stomach 1.63 (0.33) 1.66 (0.30) 1.19 (1.01) 0.23 (0.11)

Heart 2.28 (0.55) 1.60 (0.22) 1.03 (0.31) 0.22 (0.05)

Lungs 3.80 (1.01) 2.86 (0.67) 1.78 (0.55) 1.68 (0.40)

Bone 0.88 (0.13) 0.95 (0.12) 0.67 (0.33) 0.03 (0.01)

Muscle 1.48 (0.66) 1.29 (0.28) 1.13 (0.23) 0.09 (0.03)

Spleen 1.58 (0.28) 1.46 (0.17) 0.91 (0.30) 0.48 (0.13)

Tumor 3.55 (0.49) 3.12 (0.81) 2.71 (0.35) 2.56 (0.25)

Excretiona 48.81 (5.23) 51.66 (8.11) 58.83 (6.01) 90.01 (5.67)




EXPERIMENTAL

Materials and methods

Isotopically enriched palladium metal (98% in 108Pd) used for the production of 109Pd was procured from M/s. Isoflex, Russia. The production and radiochemical processing of 109Pd from the enriched palladium metal target was carried out following the method reported earlier [15, 20]. 109Pd was obtained in the form of 109Pd(DMSO)2Cl2 after radiochemical processing which was subsequently used for radiolabeling. The porphyrin derivatives I, II and III used for complexation with 109Pd were synthesized and characterized following the procedure reported earlier [15, 20–22]. All other chemicals and solvents used in the experiments were of analytical grade and supplied by reputed chemical manufacturers.

Radionuclidic purity of 109Pd produced was ascertained by high resolution gamma ray spectrometry using an HPGe detector (EGG Ortec/Canberra detector, USA) coupled to a 4 K multichannel analyzer (MCA) system. A 152Eu reference source, obtained from Amersham Inc., USA, was used for energy as well as efficiency calibration of the detector. All other radioactivity measurements were carried out by using a well-type NaI(Tl) scintillation counter by keeping the baseline at 50 keV and with a window of 100 keV to utilize the 88 keV gamma photon emission of 109mAg, unless mentioned otherwise. UV-Visible spectra were recorded using JASCO V-530-UV/Visible spectrophotometer (Japan) in methanol. 1H-NMR spectra were recorded in a 300 MHz Varian VXR 300S spectrometer (USA) using d6-dimethyl sulphoxide as the solvent. Whatman 3 MM chromatography paper (UK) was used for paper chromatography (PC) studies. Fibrosarcoma cell line, used for raising the tumors in Swiss mice, was purchased from National Center for Cell Sciences, Pune, India.

Preparation and characterizationof 109Pd-porphyrin complexes

109Pd-I and 109Pd-II complexes were prepared and their complexation yields as well as radiochemical purities were determined following the reported procedure [15, 20].

The optimized protocol for the preparation of 109Pdcomplex of the porphyrin derivative III is as follows. To a solution of 1 mg ligand in 500 μL 0.1 M ammonium acetate buffer (pH ~5.5), 50 μL of 109Pd(DMSO)2Cl2

solution (containing 50 μg Pd, ~74 MBq of 109Pd) was added after the addition of 450 μL of 0.1 M ammonium acetate buffer (pH ~5.5). The reaction mixture was heated at 80 °C for 30 min without further adjustment of pH (pH 5–6). Several reaction parameters such as, ligand concentration, incubation time, reaction temperature (etc.) were varied extensively in order to arrive at the optimum protocol for obtaining maximum complexation yield. The complexation yield and radiochemical purity

of 109Pd-III was determined by employing PC technique using acetonitrile:water (1:3, v/v) as the eluting solvent [15]. The 109Pd-III complex exhibited Rf of ~0.7, while 109Pd(DMSO)2Cl2 did not show any appreciable movement from the point of application (Rf = 0-0.1).

The Pd-porphyrin Complexes were also characterized by UV-Visible and 1H-NMR spectroscopy. For recording the UV-Visible and 1H-NMR spectra corresponding inactive complexes were synthesized following the reported procedure [15, 20]. The spectroscopic data obtained for all the three Pd-porphyrin complexes are listed below.

Pd-I [15]. UV-Vis: λmax, nm (ε × 103, M-1.cm-1) 418 (11.87), 523 (0.78), 560 (0.46). 1H-NMR (DMSO-d6): δ,ppm 4.91 (16H, m, –OCH2COOH), 7.25–7.28 (4H, m, benzenoid), 8.06–8.12 (8H, m, benzenoid), 8.82 (8H, m, pyrrole).

Pd-II [20]. UV-Vis: λmax, nm (ε × 103, M-1.cm-1) 420 (18.20), 522 (0.20), 556 (0.08). 1H-NMR (DMSO-d6): δ,ppm 1.39–1.44 (24H, m, –OCH2COOCH2CH3), 4.38–4.45 (16H, m, –OCH2COOCH2CH3), 4.91 (16H, m, –OCH2COOH), 7.29 (4H, m, benzenoid), 8.09 (8H, m, benzenoid), 8.81 (8H, m, pyrrole).

Pd-III. UV-Vis: λmax, nm (ε × 103, M-1.cm-1) 418 (15.27), 516 (0.56), 554 (0.47). 1H-NMR (DMSO-d6):δ, ppm 4.85 (8H, s, –OCH2COOH), 7.16–7.35 (8H, m, benzenoid), 7.53–7.77 (8H, m, benzenoid), 8.90 (8H, m, pyrrole).

Determination of Log P values

The Log P values of 109Pd-porphyrin complexes were determined using the following protocol. 25 μL aliquots of the complexes prepared under optimized conditions were diluted to 1 mL volume using normal saline. To each of these solutions 1 mL of n-octanol was added and the mixture stirred vigorously for ~10 min. Subsequently, the mixtures were centrifuged at a speed of 3000 rpm for 5 min. Aliquots were withdrawn from both water and n-octanol layers and counted for 109Pd activity in NaI(Tl) counter. The Log P values were determined from these data and reported as an average of three independent measurements.

Stability studies

The stability of 109Pd-porphyrin complexes prepared under optimized conditions was studied at room temperature for a period of 48 h (~4 half-lives of 109Pd).Towards this, the radiochemical purities of the complexes were determined at regular time intervals by employing standard quality control techniques described earlier.

Biodistribution studies

Bio-evaluation of the 109Pd-porphyrin complexes were carried out in Swiss mice bearing fibrosarcoma tumors. This tumor was originally developed by administration of dimethylbenzidithionaphthene (DBDN) in syngenic



mice and was maintained in vivo by serial transplantation. Fibrosarcoma cells were prepared in normal saline (106

cells/mL) and 200 μL of the cell-suspension was injected subcutaneously into each Swiss mice weighing 20–25 g. The animals were observed for visibility of tumors and subsequently allowed to grow for two weeks to attain a tumor size of ~1 cm diameter. ~100 μL (~3.7 MBq) of the radiolabeled preparation was injected into each animal through the tail vein. The animals were sacrificed by cardiac puncture post-anesthesia at 30 min, 1 h, 3 h and 24 h p.i. Various organs and tumors were excised following sacrifice and the radioactivity associated with each organ/tissue was determined using a flat type NaI(Tl) counter. The percentage of injected activity accumulated in various organs/tissue and tumor was calculated from the above data. The activity excreted was indirectly determined from the difference between total injected activity and the %IA accounted for in all the organs. The uptake in blood, bone and muscles were calculated based on the assumption that 7%, 10% and 40% of the body weight of the animals are constituted by these organs, respectively [27, 28]. All the animal experiments were carried out in strict compliance with the relevant national laws relating to the conduct of animal experimentation.

CONCLUSION109Pd-complexes of three different porphyrin

derivatives namely, 5,10,15,20-tetrakis[3,4-bis(carboxy-methyleneoxy)phenyl]porphyrin(I), 5,10,15,20-tetrakis-[3,4-bis(carboethoxymethyleneoxy)phenyl]porphyrin(II) and 5,10,15,20-tetrakis[4-carboxymethyleneoxyphenyl]-porphyrin(III), which are having identical core but different peripheral substituents, were prepared in high yield (>97%) and excellent in vitro stability. The lipophilicity of the complexes were found to follow the order 109Pd-II >> 109Pd-III > 109Pd-I. Biodistribution studies carried out in Swiss mice bearing fibrosarcoma tumors revealed that the most lipophilic 109Pd-II complex had highest initial tumor uptake but poor tumor to liver ratio. On the other hand, both 109Pd-I and 109Pd-IIIcomplexes have satisfactory tumor to background ratio, with 109Pd-III exhibiting the best tumor to liver ratio. Thus the envisaged strategy of altering the lipophilicity of the radiometal-porphyrin complexes by varying the peripheral substituents of the porphyrin moiety could be utilized in designing suitable agents with the optimum lipophilicity for significant uptake in the tumor yet fast clearance from other non-target organs resulting in high tumor to background ratio.

Acknowledgements

The authors gratefully acknowledge Dr. Meera Venkatesh, Head, Radiopharmaceuticals Division and Dr. V. Venugopal, Director, Radiochemistry and Isotope

Group for their keen interest and constant encouragement. The authors acknowledge the sincere help received from the staff-members of the Animal House Facility of Bhabha Atomic Research Centre during the course of animal experimentations. The authors are grateful to Dr. S. V. Thakare and Mr. K. C. Jagadeesan for their valuable help in carrying out the irradiations of palladium targets.

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DOI: 10.1142/S1088424611004397



INTRODUCTION

Molecules can undergo changes in the ground or excited states in response to external stimuli, such as temperature, light, ions and redox potential. In most cases, these changes could be signaled by variables of photophysical properties, which are related to logic gates through the familiar Boolean logic [1, 2]. Beginning from the pioneering work by De Silva and co-workers in 1993 [3], various molecular logic gates are demonstrated by careful design of molecular structures and definition of inputs and outputs [4, 5]. Recently, much attention has been focused on the applications of ions detection, drug release/activity control, encoding of micro-objects and so on [1, 4–6]. The most interesting target is the small spaces such as the inside of a cell [7], where silicon-based analogues are not expected to get in.

With various optical properties and abundant cation and anion coordination abilities, porphyrins

are often employed to be the candidates for molecule/ion recognition [8], chemical sensing [9] and organic optoelectronic devices [10–13]. Due to the lack of sufficient solubility in water, most porphyrin-based logic gate systems are in organic solvents [14–16]. Considering the biocompatibility, the logic gate system in aqueous solution is important and challenging. In this work, a simple and water-soluble porphyrin, 5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin (TPPS4)(Scheme 1) is employed to demonstrate the logic operations in aqueous solution and in vitro. With the help of four 4-sulfonatophenyl groups, TPPS4 has a good solubility in water. In response to the inputs of acid/base or metal cations (Al3+ and Sn4+), the outputs are signaled by UV-vis absorption and fluorescent spectra.

EXPERIMENTAL

Spectroscopy

The UV-vis absorption and fluorescent spectrum are detected by UV-765 UV-vis spectrophotometer (Shanghai Precision & Scientific Instrument Co., Ltd)

Water-soluble porphyrin-based logic gates

Lili Lia†, Peiying Caib†,Yuefei Dengc, Liutao Yanga, Xuan Hea, Lingsong Pua,

Di Wu*a,d , Jin Liu*b, Haifeng Xianga and Xiangge Zhou*a,d

a Institute of Homogeneous Catalysis, College of Chemistry, Sichuan University, 610064 Chengdu, Chinab Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 610064 Chengdu, Chinac Department of Neurosurgery, The Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, 510120 Guangzhou, Chinad State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, Nanjing University, 210093 Nanjing, China

Received 25 June 2011Accepted 16 July 2011

ABSTRACT: A series of simple logic gates based on a water-soluble porphyrin molecule, 5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin (TPPS4) is designed. Logic operations, including OR, NOR, INHIBIT and AND, have been built by two inputs of acid/base or metal ions (Al3+ and/or Sn4+)and two outputs of UV-vis absorption and fluorescent spectra. An OFF–ON switch triggered by Al3+ ion in vitro is developed based on TPPS4.

KEYWORDS: logic gate, porphyrin, water, pH, aluminum(III), tin(IV).


*Correspondence to: Di Wu, email: [email protected], fax: +86 28-85412026†These authors contributed equally to this work


WATER-SOLUBLE PORPHYRIN-BASED LOGIC GATES 73

and Hitachi F-4500 fluorescent emission spectrometer, respectively. All spectroscopic experiments are carried out at room temperature.

Sample preparation

TPPS4 is purchased from Frontier Scientific Company and used without further purification. The concentration of TPPS4 is 3.0 × 10-5 M to avoid aggregation [17]. Distilled water is used as solvent for all experiments. The concentration of HCl, NaOH and metal ions (Sn4+,Al3+, Ba2+, Cd2+, Cu2+, Fe2+, Li+, Mg2+, Mn2+, Na+, Ni2+,Pb2+, Sr2+ and Zn2+) in the form of their chlorate salts is 3.0 × 10-2 M.


Effect of pH

TPPS4 is an amphophilic molecule. It has the capability of accepting or losing protons in the porphyrin core (Scheme 1), and then has the possibility of the variation of the molecular orbital symmetry [18], which would result in the changes of its UV-vis absorption and fluorescent spectrum upon the addition of H+ and OH-. In neutral aqueous solution, the UV-vis spectrum of TPPS4 shows a single Soret band (λmax = 414 nm) and four weak Q-bands (516, 552, 579 and 632 nm). The addition of H+ leads to a 20 nm red shift for the Soret band (Fig. 1A and details in Supporting information, Fig. S1A), with an isosbestic point at 420 nm. Q-bands also exhibit red shifts. Moreover, a new band around 641 nm appears. When pH decreases to 3.5 (end point of titration, CH

+ = 3.0 × 10-4 M), the molar absorption coefficient at 434 nm (ε434) is 4.0 × 104 M-1 cm-1 (Fig. S1A). However, upon adding OH-, no change is observed both in the shape and the wavelength of the absorption spectra for TPPS4 (Fig. S1B).

The pH value affects not only the absorption spectrum but also the fluorescent spectrum for TPPS4. Upon addition of H+, the emission at 646 nm is red shifted to 673 nm (Fig. S2A). On the contrary, the addition of OH-

leads to an enhancement of emission at 648 nm (Fig. S2B). Uncler both circumstances when the concentration of H+

is over 2.0 × 10-4 M (pH < 3.7, end point) and when the concentration of OH- is over 1.0 × 10-4 M (pH > 10, end

point), the fluorescent intensity of TPPS4 is almost doubled compared with the weakest case (case b in Fig. 1B).

A pH-response half-subtractor

As shown in Fig. 1A, when the input of H+ (pH < 3.5) is true (as “1”), the absorption at 434 nm (ε434) exceeds a certain threshold (defining ε434 > 4.0 × 104 M-1 cm-1 as “1”). When the input of H+ is “0”, or the two inputs of H+ and OH- are both “1” or both “0” (pH ≥ 7), the ε434

drops below the defined threshold (as “0”). Thus, the first column of the outputs in the Truth Table of half-subtractor (Table 1) is filled.

N

N N

N N

NH N

HN N

N N

N

PhSO3-

PhSO3-

PhSO3-

-O3SPh 4H

PhSO3-

PhSO3-

PhSO3-

-O3SPh

4Na+

PhSO3-

PhSO3--O3SPh

PhSO3-

2-

6Na+

OH-

H+

OH-

H+

PhSO3- = SO3

-

Scheme 1. The amphophilic nature of TPPS4.

Fig. 1. pH effects on absorption spectra (A) and fluorescent emission spectra (B) of TPPS4 (3.0 10-5 M): a: pH < 3.5 (dash), b: pH = 7.0 (solid), and c: pH > 10.0 (dot). The pH value is established with 3.0 10-2 M HCl and 3.0 10-2 M NaOH


74 L. LI ET AL.

The second column of the outputs in the Truth Table of half-subtractor is filled by simultaneously monitoring the fluorescence upon the addition of H+ and OH-. This logic function is configured by defining the emission intensity at 661 nm (I661) (I661 > 500 as “1”) as output. As shown in Fig. 1B, the emission (output) is “1” only when H+ or OH- is added alone (pH < 3.5 and pH > 10). Both gates are implemented in parallel and addressed by the same inputs, thus fulfilling the logic gate, half-subtractor [14, 19] (Table 1).

This water soluble pH-sensitive logic gate is built based on the protonation behavior of TPPS4. It shows potential applications in molecule tracking, positioning and controlling within biological systems such as tumor therapy, because cancerous tissue is reported to be relatively more acidic than healthy cells [20].

Effect of metal ions

Tin(IV) and aluminum(III) are known to play important roles in living systems. Although they are indispensable trace elements in plants and animals, excess concentration of Al3+ and Sn4+ can cause damages on organs such as brain, heart, liver and kidney. Therefore, designing molecular logic gates for monitoring Al3+ and Sn4+ in aqueous solution is of importance [21–23].

Based on absorption and fluorescent method, TPPS4 is used as sensors for detecting Al3+ and Sn4+ with a sound sensitivity and selectivity.

In UV-vis spectrum, the Soret band of TPPS4 red-shifts from 414 to 434 nm in response to the input of Sn4+

or Al3+. New Q-bands appear at 494, 644 nm when input Sn4+ (Fig. S4) and 497, 713 nm when input Al3+ (Fig. S5). When adding Sn4+ and Al3+ simultaneously, the observed shifts are similar to those when adding Al3+ alone (Fig. 2A). Notably, when adding Al3+, the red/near-infrared (NIR) signal at 713 nm penetrates into the “biological window” (650–1100 nm) [24, 25], which is significant in the photobiological imaging and treating due to a deeper penetrating depth into the skin surface and a minor background interference.

The fluorescent emission of TPPS4 is enhanced upon addition of Al3+ and/or Sn4+. The emission intensity (671 nm for Al3+ and 673 nm for Sn4+) increases around 6-fold when excited at 434 nm (Fig. S6). The emission intensity reaches the maximum when the concentrations of Al3+ and Sn4+ increases to 6.0 × 10-4 M and 18 × 10-4

M respectively. By monitoring the change of emission intensity at 672 nm, a typical titration curve is obtained.

Anti-interference studies underscore the selective response of TPPS4. There is no remarkable change in absorption spectra of Tin(IV)-porphyrin and aluminum(III)-porphyrin when other cation (Ba2+, Cd2+,Cu2+, Fe2+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sr2+, and Zn2+) is coexisted (Figs S3 and S8A,B). The fluorescent intensity at 672 nm is quenched no more than a half upon adding many other metal ions except Cu2+ (Fig. S8C,D). With careful definition of thresholds, the interference ions have little effect on the ionic sensing and logic operation.

Logic gates in response to Al3+ and Sn4+

A water soluble molecular “OR” logic gate is built with two inputs of Al3+ and Sn4+ and an outputs of the intensity on the absorption at 434 nm (ε434). As shown in Fig. 2A, when either of the two inputs (Al3+ and Sn4+)is true (as “1”), the ε434 exceeds a certain threshold (defining ε434 > 1.0 × 104 M-1 cm-1 as “1”). When both of the two inputs are false (as “0”), the ε434 drops below the defined threshold (as “0”). Similarly, when defining the appropriate outputs and thresholds, the variation of

Table 1. Truth Table for a pH-response half-subtractor

INPUT OUTPUT

X Y B D

pH > 10 pH < 3.5 ε434 I661

0 0 0 00 1 1 11 0 0 11 1 0 0

Fig. 2. Absorption spectra (A) and fluorescence emission spectra (B) of a: TPPS4 (CTPPS4 = 3.0 10-5 M) (dot) in the presence of b: Sn4+ (1.5 10-3 M) (solid); c: Al3+ (2.0 10-4 M)(dash); and d: both of Sn4+ (1.5 10-3 M) and Al3+ (2.0 10-4

M) (dash dot). Insets: Truth table for OR logic gate and AND logic gate


WATER-SOLUBLE PORPHYRIN-BASED LOGIC GATES 75

the absorption intensity at 414, 490 and 645 nm could be built as “NOR,” “OR” and “INHIBIT” logic gates, respectively (Fig. S7).

Monitoring the fluorescence at 672 nm (I672) upon addition of Al3+ and Sn4+ yields an “AND” logic gate. This logic function could be built by defining I672 (defining I672

> 4000 as “1”) as output. As shown in Fig. 2B, the outputof I672 is “1” only when both Al3+ and Sn4+ are added. If either of the two inputs (Al3+ and Sn4+) are false (as “0”), the I672 drops below the line as “0”. Therefore, TPPS4 is capable of simultaneously conveying five-channel optical signals in response to Al3+ and Sn4+.

OFF-ON switch in vitro

The in vitro OFF-ON switch is based on the fluorescent sensitivity of TPPS4 to Al3+. In vitro Al3+ sensing experiments are performed in A549 cells. The fluorescent images recorded upon excitation are shown in Fig. 3. Incubation of A549 cells with TPPS4 (30 μM) for 1 h at 25 °C gives weak red fluorescence (Fig. 3b). When Al3+ is turned “ON” by introducing during incubation, red fluorescence is turned to intensive, displaying the enhancement of Al3+

concentration inside cells (Fig. 3c). Note that the emission wavelength, around 650 nm, is inside “biological window”. Similar result is obtained in Hela cells (Fig. S9).

CONCLUSION

In summary, we build molecular logic gates based on water-soluble porphyrin TPPS4. These systems are sensitive, selective and biocompatible. Importantly, the cell membrane permeability of TPPS4 is favorable for monitoring Al3+ levels in vitro. Thus, we expect that TPPS4 will be a useful molecular tool for diverse applications in diagnosis and treatment of tumor. Further research about the application of porphyrin logic gates on the biological system is under investigation.

Acknowledgements

This project was sponsored by the Natural Science Foundation of China (Nos. 20901052 and 21072132), Sichuan Provincial Foundation (08ZQ026–041) and

Ministry of Education (NCET-10–0581). We also thank Prof. Dan Xiao in Sichuan University for fluorescent test.


UV-vis absorption, fluorescent spectra related to the article and fluorescence images of TPPS4 in Hela cells (Figs S1–S9) are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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Fig. 3. Fluorescence images of A549 cells when stained by TPPS4 solution (30 μM in PBS) at 25 °C. (a) Brightfield transmission image; (b) fluorescence image of (a); (c) fluorescence image of cells in (b) pre-treated with 150 μM AlCl3 solution for 2 h


76 L. LI ET AL.

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DOI: 10.1142/S1088424611004361



INTRODUCTION

Molecular imaging in vivo can display the biological processes at the cellular and molecular level, reveal the mechanism of physiological and pathological processes, and provide effective methods of detecting and tracking the diseases treatments. The applications of molecular imaging include the early clinical disease diagnosis, qualitative and quantitative evaluation of curative effect and so on [1, 2]. Comparing with CT, PET, MRI imaging modality, optical imaging has many advantages, such as low cost, non-ionic low-energy radiation, high sensitivity, continuous real-time monitoring, non-invasive or minimally invasive [3, 4]. Furthermore, the fact that organisms have low scattering effect and background interference in near-infrared region(NIR), increases

near-infrared image sensitivity and penetrating depth in biological tissues [5, 6]. So near-infrared fluorescence imaging can be used in deep tissues and organs for detection and imaging owning to its specific advantages [7–9].

However, imaging events in vivo by fluorescence is limited by the probes available. As optical probe several properties are required including light transmission in depth, the luminescence quantum yield, good biocompatibility as well as targeting ability, so a great scientific effort has been focused on the development of near-infrared fluorescence probe. Phthalocyanines fluorescent dyes have large visible to NIR absorption molar extinction constant, acceptable fluorescent quantum yields, and good biocompatibility [10, 11]. Upto now, several kinds of phthalocyanines compounds with emission in NIR region have been developed [12, 13]. However, their low water solubility and no cell specificity were some obstacles for biomedical applications. The combination of carbohydrate moieties with macrocycles can not only improve the substrate solubility and

Synthesis, properties and near-infrared imaging evaluation of glucose conjugated zinc phthalocyanine via Click reaction

Feng Lv†, Xujun He†, li Lu, Li Wu and Tianjun Liu*

Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, The Tianjin Key Laboratory of Biomaterial Research, Tianjin 300192, PR China

Received 8 July 2011Accepted 28 July 2011

ABSTRACT: In order to develop a novel near infrared fluorescence agent, glucose conjugated zinc phthalocyanine, [2,9(10),16(17),23(24)-tetrakis((1-(β-D-glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]zinc(II), was synthesized via Click reaction. Their chemical structures were characterized by mass spectrometry, nuclear magnetic resonance spectrum. Their light stability and fluorescence quantum yield were evaluated by UV-visible and fluorescent spectroscopic method. Optical imaging in vivo was performed with this saccharide conjugated phthalocyanine as probe on liver tumor-bearing nude mice. Near-infrared imaging effect, organ aggregation as well as distribution of probe in vivo were evaluated by in vivo fluorescence imaging technique. Results show that glucose conjugated zinc phthalocyanine has favorable water solubility, good optical stability and high emission ability in near infrared region. Imaging results demonstrate that saccharide conjugated phthalocyanine has possess obvious imaging effect in vivo, which implies its potential in cancer diagnosis as near infrared optical probe.

KEYWORDS: near infrared fluorescence probe, zinc phthalocyanines, saccharide conjugated, Click reaction.


*Correspondence to: Tianjun Liu, email: [email protected], [email protected], tel/fax: +86 22-87893236

†These authors contributed equally to this work


78 F. LV ET AL.

biocompatibility [14], but also increase tumor targeting function of imaging probes or Photodynamic Therapy (PDT) sensitizer [15, 16]. In NIR optical imaging field, glucose conjugated phthalocyanine as NIR fluorescence probe has not been reported before. In this paper, near-infrared fluorescent probe was designed to meet molecular probes function and improve imaging effects by combining phthalocyanine with saccharide.

Saccharide conjugated macrocyclic compounds usually were achieved using coupling methods such as esterification, amidation or etherification [17, 18]. Recently, saccharide decorated macrocyclic compounds including porphyrin or phthalocyanine were synthesized by Click reaction as a novel method instead of traditional synthesis method [19, 20]. Click chemistry has been exploited for the generation of neoglycoconjugates and been applied in a wide variety of research areas, including material science, polymer chemistry, and pharmaceutical sciences because of the simplicity of this reaction and the easy workup procedure for the resulting products [21]. The characteristics of tolerance of typical biological conditions, tolerance of most functional groups makes click reaction particularly ideal for bioconjugations [22, 23]. In carbohydrate chemistry, propargyl glycosides have thus been widely utilized with alkyl azides. Based on this approach, we synthesized glucose conjugated phthalocyanine [2,9(10),16(17),23(24)-tetrakis((1-(β-D-glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl) methoxyl)phthalocyaninato]zinc(II). The fluorescent imaging in vivo with glucose conjugated phthalocyanine as probe was reported with liver tumor-bearing athymic nude mice as animal model.


Glucose conjugated phthalocyanine [2,9(10),16(17), 23(24)-tetrakis((1-(β-D-glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]zinc(II) (1) was achieved according to Ref. 24 by condensation of its precursor 4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-tetra-hydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl-methoxy)phthalonitrile (9), which was achieved in a routine three step route from 4-propyne oxide phthalonitrile(4) and 1,2,3,5-tetra-O-acetyl-β-D-glycopyranosyl azide (7)through Click reaction (Scheme 1). 4-propyne oxide phthalonitrile (4) was achieved from reaction of 4-chlorophthalonitrile (2) and propiolic alcohol (3) in DMF for 24 h at 60 °C in the presence of potassium carbonate. After recrystallization from methanol, the compound (4) was obtained as a yellow-white solid in 65% yield. Hydrogen bromide in acetic acid reacted with 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (5)in CH2Cl2 at 0 °C to give 2,3,4,6-tetra-O-acetyl-β-D-glycopyranosyl bromide (6) in 80% yield. Then compound (6) reacted with NaN3 in water at 70 °C overnight with benzyltriethylammonium chloride as phase transfer catalyst to give corresponding 1,2,3,5-tetra-O-acetyl-β-D-glycopyranosyl azide (7). After

recrystallization from 95% ethanol, the compound (7)was obtained as a white solid in 89% yield. Reaction of 4-propyne oxide phthalonitrile (4) and 1,2,3,5-tetra-O-acetyl-β-D-glycopyranosyl azide (7) in the presence of anhydrous CuSO4 and sodium ascorbate in dichloro-methane/methanol at room temperature for 48 h gave compound (8) 4-((1-(1,2,3-trihydroxy-4-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)phthalonitrile in 60%yield. Compound (8) was deprotected in dry MeOH and NaOMe to give compound (9)4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)phthalonitrile. Without further purification, the deprotected 9 reacted in a mixture of DMAE and n-butanol, zinc chloride under N2 for 24 h at 100 °C. After recrystallization from water and acetone, glucose conjugated phthalocyanine [2,9(10),16(17),23(24)-tetrakis((1-(β-D-glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]zinc(II) (1) was obtained as a green solid in 62% yield.

The structures of products were characterized by NMR spectroscopy and MS. 1H NMR (DMSO-d6,300 MHz) shows typical signals of phthalcyanine and glucose. Peak at 8.437 ppm (s, 12H) is assigned as aromatic proton, broad peak at 7.07 ppm as hydroxyl protons (s, 16OH), peak from 5.562 to 5.191 (s, 8H) assigned as triazole and related sugar protons. High resolution MS recorded by Agilent 6510 confirmed the target compound. The optical characters of target compound were evaluated by UV-vis and fluorescence spetra. The absorption and fluorescence spectra of glucose conjugated zinc phthalocyanines in DMSO and in water were shown in Fig. 1. The spectrum in DMSO shows no intermolecular aggregation. Characteristic sharp bands of the zinc phthalocyanines are seen at 618 nm and at 682 nm. However, the absorption spectrum in water differs remarkably from that in DMSO. In water, the intensity of absorption peak is much lower and broader than in DMSO, which can be attributed to cofacial aggregation of phthalocyanines in water [25, 26]. Consistent with the aggregation difference in DMSO and water, the emission ability in these two solvents also show large difference [27, 28]. Glucose conjugated zinc phthalocyanine in water has weak fluorescence signal due to its aggregation, while strong fluorescence at 690 nm with excitation wavelength at 618 nm (Φf = 0.482) was detected in DMSO. However the following experiment in vivo demonstrated that the low fluorescent ability of glucose conjugated phthalocyanines in water does not limit its application as fluorescence probe in vivo.

Photo-bleaching experiment demonstrated that glucose conjugated phthalocyanine had relative high photostability. After continuous irradiation by 0.1 w/cm2/s laser with light emerge density of 60 J/cm2 at 690 nm, its absorbance spectrum was recorded every 10 min irradiation. In first 10 min, its optical density decreased less than 10%, 20 min vs. 20%, 40 min to 50%, after continuous irradiation for 70 min, it decreased 70% or so.


SYNTHESIS, PROPERTIES AND NEAR-INFRARED IMAGING EVALUATION OF GLUCOSE 79

As a NIR probe, stability of probe in physiological conditions such as salt or serum was important. The emission of probe was measured in different physiological conditions. It can be seen that emission fluorescence was greatly reduced in serum or salt solution owning to molecule aggregation. After incubation in DMSO, FCS or physiological salt solution at 37 °C for 1 h, the total fluorescence emission of probe showed a slightly increase in first 20 min and then maintained at a stable level for 40 min, which indicated that the probe was pretty stable in serum or salt solution. Considering that optical image recording costs just several minutes, the photo stability of this compound was good enough as probe.

The NIR fluorescent imaging was conducted with liver tumor-bearing athymic nude mice as animal model. Two groups of model mice, one group was injected

with 200 μL of glucose conjugated phthalocyanines at 2 × 10–4 M through the tail vein, another was injected with equal amount of saline as control. The in vivo imaging at 12 h post injections were acquired respectively using a Kodak In Vivo FX Professional Imaging System equipped with fluorescent filter sets (excitation/emission, 625/700 nm) near optimal wavelength. As shown in Fig. 2, a significant luminescence signal was observed in the body after intravenous injection of probe for 12 h, whereas no significant luminescence signal was observed in the control group. Considerable fluorescence was detected in liver and kidney at experiment group after injecting near infrared fluorescence agent. These results indicate that in vivo, glucose conjugated zinc phthalocyanines are emissive and do not undergo substantial phase transitions that drive phthalocyanines aggregation or facilitate

OH

CI CN

CN

OCN

CN2 3 4

OAcO

AcOOAc

OAc

OAc

OAcO

AcOOAc

Br

OAc

OAcO

AcOOAc

N3

OAc

5 6 7

OCN

CN

OAcO

AcOOAc

N3

OAcO

AcOAcO

OAc

OAcN

N

NO CN

CN

OHO

HOOH

OHN

N

NO CN

CN

N

N

N

N N

N

N

N

O

O

O

N NNO

H

OH

OHH

H

HO H

OH

N

NN

O

HOH

OHH

H OH

HHO

NNN

OH

HO

OHH

H

OHH

OH

Zn

4 7 8

9

1

(i)

(ii) (iii)

(iv)

(v)

(vi)

ON

NN

O

HHO

HOH

HHO

HOH

+

Scheme 1. Synthetic route of glucose conjugated zinc phthalocyanines. (i) DMF, K2CO3, 60 °C; (ii) CH2Cl2, 33% HBr/AcOH, 0 °C;(iii) CHCl3, NaN3, PTC, 70 °C; (iv) CuSO4, Na ascorbate, CH2Cl2/MeOH (4:1), rt; (v) MeOH, NaOMe, rt; (vi) DMAE, n-butanol, ZnCl2, N2, 100 °C


80 F. LV ET AL.

intermembranous fluorophore transfer to surrounding biological structures.

To further investigate the probe distribution in various organs, the animals were sacrificed immediately after intravenous injection of probe at 12 h time point, and the kidney, heart, liver, lung, spleen, muscle and tumor were harvested and subjected to the imager. Fluorescence could be detected clearly in the lung, kidney and liver, a little in the heart and muscle (Fig. 3). Comparing with the control group, the intensity of sample group was about two-fold higher. This further confirmed that the probe was accumulated in these organ after enough circulation time. Because the lung, kidney and liver are the main metabolism organs, the probe as external matter was accumulated at relative large amount in these organs.

In order to investigate the influence of glucose substituted zinc phthalocyanines to organ, histological analysis were performed. Large amount of fluorescence agent aggregated in the liver and kidney (Fig. 4), while little fluorescence was found in the other organs, which was in accordance with the result of NIR fluorescent imaging of organs. These results further revealed the distribution of fluorescence agent. Toxic effects are an important issue for probe. Histological analysis showed that the glucose conjugated phthalocyanines had no damage to the organ according to the HE staining, which proved the safety of fluorescence agent as the near infrared fluorescence probe in vivo.

In summary, a novel water-soluble glucose conjugated zinc phthalocyanines was synthesized. With mice bearing liver cancer as animal model in vivo fluorescent imaging was conducted. Near-infrared imaging effect, distribution in organs as well as its histological analysis were also assessed. The results proved that glucose conjugated phthalocyanines has obvious imaging effect

(a)

(b)

(c)

Fig. 1. (a) UV and FL spectrum of glucose conjugated phthalocyanines in different solvents (2 × 10-4 M; solid line UV: spectrum in DMSO; dot line: FL spectrum in DMSO; dash dot line: UV spectrum in H2O; short dot line: FL spectrum in H2O). (b) Photobleaching experiment recorded by UV-vis (light intensity was 0.1 w/cm2/s and light emerge density was 60 J/cm2. The photobleaching measurements was carried from 0 to 70 min). Inset: absorbance spectra data after different irradiation time. (c) Optical stability of glucose conjugated phthalocyanines incubation in DMSO, FCS or physiological salt solution at 37 °C (5 × 10-5 M, solid line: DMSO; dash line: DMSO:FCS = 1:1; dot line: DMSO:physiological saline = 1:1)

Fig. 2. Optical imaging in vivo with glucose conjugated zinc phthalocyanines as probe, mice bearing cancer as model (injected with 200 μL of 2 × 10-4 M with an exposure time of 1min (filters: excitation 625 nm, emission 700 nm)



and exhibits its potential as near infrared optical probe in the diagnosis of cancer in future.

EXPERIMENTAL

General methods and materials

The 1H NMR spectra were recorded on Varian Mercury at 300 MHz using CDCl3 or DMSO-d6 as solvent and TMS as internal reference. MS were recorded on Varin FT-ICR-MS and Agient 6510 Q-Tof instrument. The UV-vis and fluorescence spectra were recorded on a ThermoFisher scientific Varioskan TM Flash multimode microplate spectra photometer. All purchased materials were used without further purification.

Synthesis

4-propyne oxide phthalonitrile (4). 4-chlorophtha-lonitrile (1.638 g, 10 mmol) and propiolic alcohol (1.2 g,

20 mmol) were stirred for 24 h in DMF (40 mL) at 60 °C in the presence of potassium carbonate (8.3 g, 60 mmol). The reaction was then followed to complete by TLC. The reaction mixture was poured into ice-water to give a green-brown precipitate, which was extracted with dichloromethane. The organic extracts were dried over anhydrous MgSO4 and concentrated under vacuum to give a yellow-brown solid. After recrystallization from methanol, the title compound was obtained as a yellow-white solid. Yield 1.2 g (65%), mp 112.3–114.1 °C. ESI-MS: m/z (C11H6N2O) 183.0826143 (calcd. for [M + H]+ 183.0553). IR (solid): ν, cm-1 3288.3 (C≡H); 2232.9 (CN); 1593.1, 1494.3 (C=C phenyl). 1H NMR (CDCl3, 300 MHz): ΤΜ, ppm 7.762 (d, 1H, J = 8.7 Hz), 7.373 (d, 1H, J = 2.4 Hz), 7.314 (dd, 1H, J = 2.4, 8.7 Hz), 4.816 (d, 2H, J = 2.4 Hz), 2.636 (t, 1H, J = 2.4,2.7 Hz).

1,2,3,5-tetra-O-acetyl-β-D-glycopyranosyl azide (7). To a stirring solution of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (2.4 g, 6.1 mmol) in CH2Cl2 (20 mL) was added HBr/HOAc (10 mL, 33%) solution at 0 °C. The resulting solution was stirred for 7 h until 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose completely disappeared. The solution was neutralized with saturated NaHCO3

aqueous solution before being dried (MgSO4), filtered, and evaporated under vacuum to give the crude glycosyl bromide as a yellow oil. After recrystallization from ethanol, the compound was obtained as a white solid (2.0 g, 80%). 2,3,4,6-tetra-O-acetyl-β-D-glycopyranosyl bromide (0.5 g, 1.2 mmol) was dissolved in CHCl3

(20 mL) and stirred with NaN3 (0.3 g, 4.6 mmol) in water (20 mL) at 70 °C overnight in the presence of benzyltriethylammonium chloride (0.1 g, 0.4 mmol). The reaction mixture was diluted with water and extracted with CHCl3. The combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum to give an orange oil. After recrystallization from 95% ethanol, the title compound 1,2,3,5-tetra-O-acetyl-β-D-glycopyranosyl azide was obtained as a white solid. Yield 0.4 g (89%), mp 129.7–130.5 °C. ESI-MS: m/z (C14H19N3O9) 396.1013 (calcd. for [M + Na]+ 396.1014). IR (solid): ν, cm-1 2233.3 (N=N=N), 1737.8 (C=O). 1H NMR (CDCl3, 300 MHz):TM,ppm 5.247 (t, 1H, J = 9.3, 9.6 Hz), 5.133 (t, 1H, J = 9.6, 9.9 Hz), 4.982 (t, 1H, J = 8.7, 9.6 Hz), 4.661 (d, 1H, J =8.7Hz), 4.301 (dd, 1H, J = 4.8, 12.6 Hz), 4.184 (dd, 1H, J= 2.1, 12.3 Hz), 3.821–3.764 (m, 1H), 2.165 (s, 3H, CH3), 2.099 (s, 3H, CH3), 2.074 (s, 3H, CH3), 2.027 (s, 3H, CH3).

4-((1-(1,2,3-trihydroxy-4-(hydroxymethyl)-tetra-hydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)-phthalonitrile (8). A mixture of 4-propyne oxide phtha-lonitrile (4) (500 mg, 2.7 mmol), 1,2,3,5- tetra-O-acetyl-β-D-glycopyranosyl azide (7) (1100 mg, 2.9 mmol), anhydrous CuSO4 (45 mg, 0.28 mmol), and sodium ascorbate (110 mg, 0.56 mmol) in dichloromethane/methanol = 4:1 (15 mL) was stirred at room temperature for 48 h. After being filtered under vacuum, washed with water, extracted with dichloromethane, the organic extracts were dried over anhydrous MgSO4 and concentrated under vacuum. The residue was purified

(a)

(b)

Fig. 3. (a) Distribution and fluorescent intensity of dissected organs. (a) Ex vivo imaging with an exposure time of 1 min (filters: excitation 625 nm, emission 700 nm). (b) Distribution and fluorescent intensity of dissected organs. B: Quantitative analysis


82 F. LV ET AL.

Fig. 4. Fluorescent image of liver (a) and kidney (b) tissue slice recorded by confocal laser microscopy



by column chromatography (silica gel: ethyl acetate-petroleum ether, 1:1), to yield a white solid. Yield 899 mg (60%), mp 161.5–162.8 °C. ESI-MS: m/z (C25H25N5O10)578.2507 (calcd. for [M + Na]+ 578.1494). IR (solid): ν, cm-1 2233.3 (CN), 1737.8 (C=O). 1H NMR (CDCl3,300 MHz):TM, ppm 7.907 (s, 1H), 7.751 (d, 1H, J = 8.7 Hz), 7.383 (d, 1H, J = 2.4 Hz), 7.359 (dd, 1H, J = 2.7, 8.7 Hz), 5.908 (d, 1H, J = 9.3 Hz), 5.476–5.206 (m, 5H), 4.359 (dd, 1H, J = 5.1, 12.6 Hz), 4.184 (dd, 1H, J = 2.1, 12.9 Hz), 4.058–4.007 (m, 1H), 2.178 (s, 3H, CH3), 2.090 (s, 3H, CH3), 2.079 (s, 3H, CH3), 2.038 (s, 3H, CH3).

[2,9(10),16(17),23(24)-tetrakis((1-(β-D-glucopy-ranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl) phthalocyaninato]zinc(II)(I). 4-((1-(1,2,3-trihydroxy-4-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)phthalonitrile (8) (600 mg, 1.1 mmol) was suspended in dry MeOH (10 mL). NaOMe (300 μL) was added and the solution was stirred for 4–5 h at room temperature. The ion exchanger was added to neutralize the solution, then filtered off and the solvent evaporated. Without further purification, to the compound (9) 4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)phthalonitrile dissolved in a mixture of DMAE (1 mL) and n-butanol (0.5 mL), zinc chloride (70 mg, 0.5 mmol) were added. The reaction mixture was stirred under N2 for 24 h at 100 °C. After cooling, the solid was reprecipitated by adding acetone and collected after filtration. Yield 1.08 g (62%), mp > 300 °C. ESI-HRMS: m/z (C68H68N20O24Zn) 1635.3891 (calcd. for [M + Na]+

1635.3899). 1H NMR (DMSO, 300 MHz): ΤΜ, ppm 8.437 (s, 12H), 7.076 (s, 16OH), 5.562 (d, 4H, J = 9 Hz), 5.439 (s, 4H), 5.301 (s, 4H), 5.191 (s, 8H), 3.797–3.016 (m, 28H).

Optical measurement

The optical characters of target compound were evaluated by UV-vis and fluorescence spectrum. The photobleaching measurements were carried out in Multimode Microplate Spectraphotometer (VarioskanTM Flash, ThermoFisher Scientific). Dye and buffer solutions were prepared immediately prior to measuring. 200 μL2 × 10-4 M in 96 wells was used as sample and sealed by cover slips to avoid evaporation. The light intensity was 0.1 w/cm2/s and light emerge density was 60 J/cm2.The photobleaching measurements was carried from 0 to 70 min, every 10 min record one datum, with irradiation wavelength at 690 nm. Optical stability of glucose conjugated phthalocyanines incubation in DMSO, FCS or physiological salt solution at 37 °C was measured using 200 μL 5 × 10-5 M in 96 wells as sample.

In vivo imaging and distribution of glucose conjugated zinc phthalocyanines

Athymic nude mice (seven weeks old, 20–25 g) were used. All the animal experiments were performed in compliance with the Guiding Principles for the Care

and Use of Laboratory Animals, Peking Union Medical College, China. Animals can access free to food and water. Tumor-bearing mice were prepared by injecting a suspension of 1 × 106 liver tumor cells in physiological saline (100 μL) into the subcutaneous left flank. Tumors develop within a periods of 1 week. Athymic nude mice were randomly assigned to perform as follows: glucose conjugated zinc phthalocyanines, control group (n = 6 for each group). In experimental group, near infrared fluorescence agent was injected into the tail vein of the liver tumor-bearing mice at 200 μL of 2 × 10-4 M. Images were taken using a Kodak Image Station in vivoFX (filters: excitation 625 nm, emission 700 nm) with an exposure time of 1 min. At the end of the imaging, anesthetized mice were sacrificed and images of organs were made to evaluate the distribution of near infrared fluorescence agent. Fluorescence images of organ were analyzed using the Kodah Image Analysis Software. After imaging, organ tissues were immediately immersed into 4% formaldehyde in phosphate-buffered saline of pH 7.4 at 4 °C for 24 h. After fixation, the samples were embedded in paraffin and sectioned to 5-μm-thick slices. Routine staining was performed with hematoxylin-eosin.

Acknowledgements

This work is supported by the National Basic Research Program of China (No. 2006CB705703) and the Ph.D. Programs Foundation of Ministry of Education of China (Nos. 20101106120052, 20070023020) and the Natural Science Foundation of Tianjin (No. 09JCYBJC03500).


Experimental details for the synthesis, measurement method, MS data and H NMR data are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S108842461100435X



INTRODUCTION

Photodynamic therapy (PDT), with its inherent selectivity, has received increasing attention as an emerging cancer treatment modality recently. In PDT, a non-toxic photosensitizer, which can be selectively taken up by tumor cells, generates reactive oxygen species, such as singlet oxygen (1O2), upon photo-activation by visible light. The reactive oxygen species produced causes oxidative damage to the tumor tissues, resulting in cell death [1, 2].

Circular dichroism (CD) and UV-vis spectroscopy were applied to study the porphyrin-DNA interactions. Previous studies showed that the sign of the induced CD in the Soret region of the porphyrins can be used as a signature of their binding modes with DNA under low porphyrin loads (r < 0.1): a positive induced CD

signal indicates external binding whereas a negative induced CD signal indicates intercalative binding with DNA [3, 4].

Cationic porphyrins, such as 5,10,15,20-tetrakis(4-N-methylpyridinium)porphyrin (H2TMPyP), have been intensively studied as potential PDT agents for decades due to their strong binding affinity towards DNA and their water-solubility which make DNA-binding possible under physiologically relevant conditions [5, 6]. These crucial properties have been attributed to their cationic substituent which facilitates their binding interactions with the anionic DNA in aqueous media [7, 8]. But DNA-binding interactions can be achieved via complementary hydrogen bonding with DNA bases as well, particularly at the minor groove region [9, 10]. In this work, a hydrogen bonding functionality, i.e. the amidine group, was introduced onto the tetraphenylporphyrin (TPP) by synthesis. Its Zn(II) complex and its conjugate with another TPP via the −O−(CH2)3−O− linker were prepared as well (Schemes 1 and 2). The efficiency of cellular uptake of the drug is very important to play a therapeutic role, which depends on hydrophilic and lipophilic nature of molecular structure [11]. Since the previously prepared bisporphyrin with

Synthesis, circular dichroism, DNA cleavage and singlet

oxygen photogeneration of 4-amidinophenyl porphyrins

Kai Wanga,b,c, Chun T. Poonb, Chun Y. Choib, Wai-Kwok Wong*b, Daniel W.J. Kwong*b,

Fa Q. Yuc, Heng Zhangc and Zao Y. Li*a

a College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, Hubei, P.R. Chinab Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. Chinac Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, Hubei, P.R. China

Received 8 July 2011Accepted 30 July 2011

ABSTRACT: 5,10,15,20-tetrakis(4-amidinophenyl)porphyrin (Por 1), its Zn complex (Por 2) and its conjugate with a tetraphenylporphyrin to form a bisporphyrin (Por 3) were prepared. The monomeric Por 1 and Por 2 showed both intercalative and external binding with DNA whereas only external DNA binding was seen in the bisporphyrin, Por 3 by circular dichroism and UV-vis. The DNA photocleavage activities of these porphyrins followed the order: Por 1 ∼ Por 2 > Por 3, which did not correlate with their measured 1O2 production rates. It suggests 4-amidinophenylporphyrins are promising new photodynamic therapeutic agents.

KEYWORDS: amidinophenylporphyrin, singlet oxygen, DNA cleavage, binding mode.

*Correspondence to: Zaoying Li, email: [email protected], tel: +86 27-87219084, fax: +86 27-68754067; Wai-Kwok Wong, email: [email protected] and Daniel W.J. Kwong, email: [email protected], tel: +852 3411-7063, fax: +852 3411-7348


86 K. WANG ET AL.

N

NH N

HN

NC

CN

CN

CNZn(OAC)2

N

N N

N

NC

CN

CN

CNZn

N

N N

N

(a) LiN(SiMe3)2/THF(b) H2O

NHH2N

NH2

NH

HN NH2

HN

H2N

M

Por1, M = 2HPor2, M = Zn

Scheme 1

N

NH N

HN

NC

CN

CN

O OK2CO3/DMF

N

NH N

HN

O BrN

NH N

HN

NC

CN

CN

OH

N

HNN

NH

Br(CH2)3BrK2CO3/DMF

N

NH N

HN

NC

CN

CN

O Br

N

HNN

NH

HO

+

K2CO3/DMF

(a) LiN(SiMe3)2/THF(b) H2O

N

NH N

HN

O O

N

HNN

NH

NHH2N

HN

H2N

HNNH2 Por3

+

Scheme 2

hydrophilic (TMPyP) and lipophilic (TPP) structure in our lab had a good cellular uptake [12], amidine group as the hydrophilic part replaced TMPyP in the bisporphyrin structure design. Furthermore, the amidine substituent, in addition to its hydrogen-bonding capability, can also form

amidinium-carboxylate salt bridge with biomolecules bearing a pendant carboxylate/carboxylic acid. Such intermolecular amidinium-carboxylate bridges have recently been shown to facilitate photo-excited energy transfer with rates much faster than those predicted by


DNA CLEAVAGE AND SINGLET OXYGEN PHOTOGENERATION OF 4-AMIDINOPHENYL PORPHYRINS 87

the Forster mechanism [13]. Herein we reported their binding modes with DNA as studied by circular dichroism spectroscopy and UV-vis. Their DNA photocleavage activities and their relative 1O2 production rates were also investigated.


Binding mode of Por 1, Por 2 and Por 3 with DNA

The absorption titration spectra of these 4-amidi-nophenylporphyrins with calf thymus DNA (ct-DNA) was given in Fig. 1. Without DNA, λmax of the Soret bands of Por 1, Por 2 and Por 3 were 414, 423 and 422 nm, respectively. In the presence of low concentrations of ct-DNA (i.e., r = [porphyrin]/[DNA] > 0.6), the Soret bands of Por 1, Por 2 and Por 3 exhibited substantial hypochromicity (75.9%, 76.1% and 77.1%, respectively) but no significant spectral shift, which suggests an external DNA binding mode with modest effect on the π–π∗ Soret absorption. Further addition of ct-DNA to Por 1, Por 2 and Por 3 (i.e., r < 0.26) resulted in a red shift of 11, 4 and 10 nm, respectively. The substantial bathochromic shifts and hypochromicity observed suggest binding interactions between the 4-amidinophenylporphyrins and ct-DNA [14, 15]. An apparent binding constant, Kapp, can be calculated from these data using Eq. (1) as follows [16].

[ ]

| | | |[ ]

|( ) ( ) { (DNA

DNAKf b f

total

apptotal

app- -ε ε ε ε ε=

⎛

⎝⎜

⎞

⎠⎟ +

1 1

bb f- ε |)}

(1)

where εapp, εf and εb correspond to Aobsd/[porphyrin], the molar absorptivity of the free porphyrin, and the molar absorptivity of the DNA-bound porphyrin, respectively. By plotting [DNA]total/(|εapp - εf |) vs. [DNA]total, Kapp of Por 1, Por 2 and Por 3 were determined to be (1.53 0.69) × 106 M-1, (7.25 6.02) × 105 M-1 and (5.81 1.14) × 105

M-1, respectively.The induced CD spectra in the Soret region of Por 1,

Por 2 and Por 3 (at r([porphyrin]/[DNA]) = 0.05) were given in Fig. 2. From this figure, Por 1 showed two positive signals at 421 and 443 nm, and one negative signal at 431 nm. Por 2 showed a strong negative signal at 427 nm and a strong positive signal at 450 nm. These features suggest that both Por 1 and Por 2 could bind to ct-DNA via both external bounds and intercalation. Por 3showed a very weak and broad positive signal centered at 440 nm, indicating that it bond to ct-DNA rather weakly with outside binding mode in the high DNA concentration.

DNA photocleavage ability

DNA photocleavage activities of Por 1, Por 2 and Por 3were measured using plasmid DNA relaxation assay [17].

The gel images are given in Fig. 3. For Por 1 and its Zn(II) complex (Por 2), their photocleavage activities were comparable and were ca. 4-fold lower than that of H2TMPyP. At 10 μM, their DNA photocleavage activities

Fig. 1. Absorption titration spectra of the amidinophenyl-porphyrins (4.0 μM), Por 1 (a), Por 2 (b) and Por 3 (c), with increasing concentrations of ct-DNA (r = [porphyrin]/[DNA base pairs] = �, 7.14, 3.57, 1.80, 0.91, 0.62, 0.26, 0.20, 0.17, 0.11) in buffered solution (pH = 7.4, 5 mM tris-HCl, 50 mM NaCl)


88 K. WANG ET AL.

were 69% and 74%, respectively, which was ca. 4-fold higher than that of Por 3 (19%).

For Por 3, its maximum photocleavage activity of ca. 36% was seen at 75 μM. The significant DNA photocleavage activities were likely the result of a binding interaction with DNA and a more effectively oxidative attack from close range with 1O2 [18–20]. It also suggests that Por 1, Por 2 and Por 3 with hydrogen-bonding capability have relatively weaker electrostatic interaction with DNA compared with H2TMPyP.

Photogeneration of 1O2

Since the DNA photocleavage activity of a photosensitizer depends on its 1O2 yield as well as

its DNA-binding property, we measured the relative 1O2 production rates of Por 1, Por 2,Por 3 and H2TMPyP based on the 1O2-induced bleaching of 1,3-diphenylisobenzofuran (DPBF, λmax = 418 nm) [21, 22]. According to the relationship between A/A0 absorbed by DPBF and illumination time, one may indirectly obtain the 1O2 yield of those porphyrins compared with H2TMPyP. All three 4-amidinophenylporphyrins produced 1O2 more efficiently than H2TMPyP in the following order: Por 2 > Por 1 ~ Por 3 > H2TMPyP (Fig. 4).

In contrast, Zn-porphyrin 2 showed a greater yield of 1O2, as other Zn(II)-porphyrin derivatives, and this is attributed to its correspondingly high triplet quantum yield, ΦT [23]. The lack of correlation between the observed DNA photocleavage activities of these porphyrins and their relative 1O2 production rates suggests that their DNA-binding properties must be a more important factor. Presumably, the intercalative binding exhibited by H2TMPyP, Por 1 and Por 2 with DNA was crucial to their higher

DNA photocleavage activities than Por 3 which binds to DNA only externally.

EXPERIMENTAL

General

5-[p-(3-bromopropoxy)phenyl]-10,15,20-tris -phenylporphyrin, 5-(p-hydroxyphenyl)- 10,15,20- trisph-enylporphyrin and meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (H2TMPyP) were prepared according to literature procedures [24, 25]. Other chemicals were obtained from Sigma-Aldrich Company and used without further purification. Silica gel 60 (0.04–0.063 mm) for

Fig. 2. Induced CD spectra of Por 1, Por 2 and Por 3 in the tris-HCl buffer solution with the [porphyrin] = 10 μM and [ct-DNA] = 200 μM,r = 0.05

Fig. 3. Agarose gel electrophoresis images of DNA photocleavage assay of (a) Por 1, (b) Por 2, (c) Por 3 and (d) H2TMPyP at different concentrations. Photo-irradiation was conducted using a transilluminator at 455 nm for 45 min



column chromatography was obtained from Merck. Calf thymus DNA (ct-DNA) was obtained from Pharmacia Biotech Company. The concentration of ct-DNA in base-pairs was determined spectrophotometrically using the extinction coefficient ε260 = 6600 M-1.cm-1 [26].

NMR spectra were recorded on a Varian INOVA 400 NMR spectrometer. High-resolution matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra were recorded on an Autoflex Bruker MALDI-TOF mass spectrometer. FAB-mass spectra were obtained on a Finnigan TSQ710 mass spectrometer. Electronic absorption spectra in the UV-vis region were recorded with a Varian Cary 100 UV-vis spectrophotometer. Elemental analyses were performed on an instrument of VarioEL III. The IR spectra (KBr pellets) were recorded on a Nicolet Magna-FTIR 550 spectrometer. CD spectra were recorded using a JASCO J-720 spectropolarimeter. The CD spectral and UV-vis measurements were performed in 5 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl.

Synthesis routes and procedures of the 4-amidino-phenylporphyrins

The synthetic routes for the preparation of porphyrins with tris- and tetra-amidino groups are outlined in Schemes 1 and 2. The detailed procedures for the preparation of all the involved compounds, together with their characterization data, are given in the following sections.

5-(p-hydroxyphenyl)-10,15,20-tris(4-cyanophenyl)-porphyrin and 5,10,15,20-tetrakis(p-cyanophenyl)-porphyrin (H2TCNPP). Pyrrole (5.36 g, 0.08 mmol) was added dropwisely to a solution of 4-cyanobenzaldehyde (2.62 g, 0.06 mmol) and 4-hydroxybenzaldehyde (2.44 g, 0.02 mmol) in refluxing propionic acid (200 mL). The mixture was refluxed for 1 h and then

evaporated to dryness. After adding chloroform (300 mL) to the residue and mixing for 3 h, it was filtered, and chloroform layer was washed with water (300 mL × 3), which was dried by anhydrous sodium sulfate. After it had been filtered and concentrated, the residue was purified by silica gel column chromatography using dichloromethane as eluent. The first fraction was 5,10,15,20-tetrakis(p-cyanophenyl)porphyrin. Yield 220 mg (1.5%), mp > 300 °C. MS (FAB): m/z 714.6 (calcd. for [M]+

714.2); the second fraction was the title blue-violet product. Yield 665 mg (4.7%) after recrystallization from chloroform/methanol, mp > 300 °C. IR (KBr): ν, cm-1 3310.9, 2227.6, 1604.3. UV-vis (CHCl3): λmax, nm (log ε) 422 (5.62), 517 (4.24), 553 (3.90), 591 (3.73), 648 (3.68). MS (FAB): m/z 706.3 (calcd. for [M + H]+ 705.2).

5,10,15,20-tetrakis(p-cyanophenyl)-Zn-porphyrin(ZnTCNPP). The complex was

prepared by refluxing H2TCNPP (100 mg, 0.14 mmol) with an excess amount of zinc acetate in CHCl3/methanolfor 5 h and purified by column chromatography on silica gel using chloroform as eluent. Yield 103 mg (95%), mp > 300 °C. 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm 8.09–8.11 (8H, d, J = 8.0 Hz, porPhHm), 8.33–8.35 (8H, d, J = 8.0 Hz, porPhHo), 8.90 (8H, s, pyrrole H). IR (KBr): v, cm-1 2228, 1603, 996 (ZnII, OSMB). UV-vis (CHCl3):λmax, nm (log ε) 427 (5.76), 557 (4.35), 597 (3.74). HRMS (MALDI-TOF, in chloroform): m/z 776.1421 (calcd. for [M]+ 776.1409, Δm = 1.4288 ppm).

5-[p-(3-bromopropoxy)phenyl]-10,15,20-tris(4-cyanophenyl)porphyrin. In the presence of anhydrous potassium carbonate (1.0 g), 5-(p-hydroxyphenyl)-10,15,20-tris(4-cyanophenyl)porphyrin (100 mg, 0.14 mmol) and 1,3-dibromopropane (0.57 g, 2.83 mmol) in dry DMF (10 mL) was heated to 65 °C for 4 h with stirring under nitrogen. After cooling to room temperature, the reaction mixture was poured into water saturated with sodium chloride (30 mL) and then filtered. The precipitate was purified by silica gel column chromatography using chloroform as eluent. The first fraction was the title blue-violet product. Yield 82 mg (70.8%) after recrystallization from chloroform/methanol. mp > 300 °C. 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm -2.88 (2H, s, pyrrole ring NH), 2.50–2.52 (2H, m, -CH2- CH2- CH2-), 3.77–3.80 (2H, t, J = 6.4 Hz, CH2Br), 4.39–4.42 (2H, t, J = 5.6 Hz, OCH2), 7.29–7.31 (2H, m, O-PhHo), 8.06–8.10 (8H, m, porPhHm), 8.31–8.33 (6H, d, J = 7.2 Hz, porPhHo), 8.73–8.76 (8H, m, pyrrole H). UV-vis (CHCl3): λmax, nm (log ε)420 (5.65), 517 (4.22), 553 (3.85), 591 (3.70), 647 (3.66).MS (FAB): m/z 827.3 (calcd. for [M]+ 826.7).

Free CN-bisporphyrin. In the presence of anhydrous potassium carbonate (1.0 g), 5-(p-hydroxyphenyl)-10,15,20-tris(4-cyanophenyl)porphyrin (100.0 mg, 0.14 mmol) and 5-[p-(3-bromopropoxy)phenyl]-10,15,20-trisphenylporphyrin (106.5 mg, 0.14 mmol) [or 5-[p-(3-bromopropoxy)phenyl]-10,15,20-tris(4-cyanophenyl)-

Fig. 4. Normalized absorbance of DPBF (50 μM in DMSO) at 418 nm as a function of photo-irradiation time in the absence (control) and presence of 1 μM of H2TMPyP, Por 1, Por 2 and Por 3


90 K. WANG ET AL.

porphyrin (100 mg, 0.12 mmol) and 5-(p-hydroxyphenyl)-10,15,20-trisphenylporphyrin (76 mg, 0.12 mmol)] in dry DMF (15 mL) was heated to 65 °C for 5 h with stirring under nitrogen. After cooling to room temperature, the reaction mixture was poured into water saturated with sodium chloride (50 mL) and then filtered. The precipitate was purified by silica gel column chromatography using chloroform as eluent. The second fraction was the title blue-violet product. Yield 135 mg (70.4%) after recrystallization from chloroform/methanol, mp > 300 °C. 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm -2.86 (2H, s, trisphenyl pyrrole ring NH), -2.79 (2H, s, tris(cyanophenyl) pyrrole ring NH), 2.63–2.66 (2H, m, -CH2- CH2- CH2-), 4.60–4.64 (4H, m, CH2), 7.37–7.42 (4H, m, O-PhHo), 7.64–7.77 (9H, m, portrisPhHm,p), 8.31–8.33 (22H, m, porPhHo and trisCNPhHo), 8.68–8.99 (16H, m, pyrrole H). IR (KBr): v, cm-1 3311, 2227, 1604. UV-vis (CHCl3): λmax, nm (log ε) 418 (5.53), 517 (4.18), 553 (3.88), 592 (3.65), 647 (3.62). MS (FAB): m/z 1376.6 (calcd. for [M + H]+ 1375.5).

5,10,15,20-tetrakis(4-amidinophenyl)porphyrin (Por 1). A slurry of H2TCNPP (150 mg, 0.21 mmol) and LiN(SiMe3)2 (0.16 mmol/mL in THF solution, 0.32 mmol) in dry THF (20 mL) was sonicated at room temperature for 5 h [27]. The solvent was removed under vacuum. The residue was then washed with distilled water and chloroform, purified by recrystallization from methanol and acetonitrile. The resulting title compound was characterized by NMR, UV-vis, MS and elemental analysis. Yield 164 mg (91%), mp > 300 °C. Anal. calcd. for C48H38N12: C, 73.53; H, 4.85; N, 21.45. Found: C, 73.60; H, 4.79; N, 21.50. 1H NMR (400 MHz; DMSO-d6; Me4Si): δH, ppm -2.95 (2H, s, pyrrole ring NH), 8.24 (8H, s, porPhHm), 8.26 (8H, s, porPhHo), 8.87 (8H, s, pyrrole H). IR (KBr): ν, cm-1 3318, 3201, 1667, 1607, 1507, 1440, 966, 863, 799, 502. UV-vis (DMSO): λmax, nm (log ε) 421 (5.43), 515 (4.07), 551 (3.72), 589 (3.51), 645 (3.44). HRMS (MALDI-TOF, in methanol): m/z 783.3456 (calcd. for [M]+ 783.3420, Δm = 1.1489 ppm).

Zn(II)-5,10,15,20-tetrakis(4-amidinophenyl)-porphyrin (Por 2). A slurry of ZnTCNPP (50 mg, 0.06 mmol) and LiN(SiMe3)2 (0.16 mmol/mL in THF solution, 0.32 mmol) in dry THF (20 mL) was sonicated at room temperature for 5 h. The solvent was removed in vacuum. The residue was then washed with distilled water and chloroform and then purified by recrystallization from methanol and acetonitrile. Yield 51 mg (94%), mp > 300 °C. Anal. calcd. for C48H36N12Zn: C, 68.14; H, 4.26; N, 19.86. Found: C, 68.07; H, 4.31; N, 19.79. 1H NMR (400 MHz; methanol-d4; Me4Si): δH, ppm 8.10–8.12 (8H, d, J = 8.0 Hz, porPhHm), 8.28–8.30 (8H, d, J = 8.0 Hz, porPhHo), 8.82 (8H, s, pyrrole H). IR (KBr): ν, cm-1 3372, 3326, 1633, 1606, 992 (ZnII, OSMB). UV-vis (DMSO): λmax, nm (log ε) 430 (5.55), 561 (4.16), 602 (3.83). HRMS (MALDI-TOF, in methanol): m/z 845.2529 (calcd. for [M]+ 845.2550, Δm = -2.4986 ppm).

4-amidinophenylbisporphyrin (Por 3). A slurry of free CN-bisporphyrin (50 mg, 0.036 mmol) and LiN(SiMe3)2

(0.16 mmol/mL in THF solution, 0.32 mmol) in dry THF (15 mL) was sonicated at room temperature for 5 h. The solvent was removed in vacuum. The residue obtained was washed by water. Yield 50 mg (97%), mp > 300 °C.Anal. calcd. for C94H70N14O2: C, 78.96; H, 4.90; N, 13.72. Found: C, 78.82; H, 4.92; N, 13.63. 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm -2.76 (4H, s, pyrrole ring NH), 2.57–2.67 (2H, m, -CH2- CH2- CH2-), 4.50–4.63 (4H, m, CH2), 7.37–7.40 (4H, m, O-PhHo), 7.61–7.77 (15H, m, porPhHm and portrisPhHp), 8.09–8.22 (16H, m, porPhHo), 8.83–8.85 (16H, m, pyrrole H). IR (KBr): ν, cm-1 3318, 3036, 2918, 1674, 1598, 1482. UV-vis (DMSO): λmax, nm (log ε) 421 (5.77), 516 (4.44), 552 (4.14), 591 (3.88), 647 (3.89). HRMS (MALDI-TOF, in methanol): m/z 1428.5971 (calcd. for [M]+ 1428.5909, Δm = 4.2923 ppm).

Absorption titration of Por 1, Por 2 and Por 3 with DNA

The sample solutions were prepared by mixing 2 mM 4-amidinophenylporphyrin stock solution (in DMSO) and 0.169 mM ct-DNA stock solution in appropriate ratios and then diluted with Tris-HCl buffer solution to obtain the final desired concentration ratios (r = [porphyrin]/[ct-DNA]). UV-vis absorption spectra of theseporphyrin-DNA sample mixtures were recorded from 350–500 nm.

Circular dichroism (CD) measurement of Por 1, Por 2 and Por 3 with DNA

Appropriate amount of calf thymus DNA was added to a Tris-HCl buffered 10 μM porphyrin solution to obtain a porphyrin-DNA mixture with a concentration ratio r ([porphyrin]/[ct-DNA]) = 0.05. After an incubation period of 45 min, the sample mixture was scanned in the Soret region of porphyrin (i.e., 400–500 nm). Baseline correction was obtained by scanning the same buffer solution without the porphyrin. The spectral bandwidth and the cell length for the CD measurements were 1 nm and 1 cm, respectively.

DNA photocleavage assay

The DNA photocleavage activities of Por 1, Por 2, Por 3 and H2TMPyP were measured using the plasmid DNA relaxation assay. Briefly, the plasmid DNA (pBluescript, 0.5 μg), enriched with the covalently-closed circular or supercoiled conformer (Form I), and the one-phor-all plus buffer (10 mM tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, pH 7.5) was vortexed. Aliquots of the DNA were pipetted into different Eppendorf tubes. Various amounts of autoclaved water (control sample) or prophyrins (test sample) were added into the Eppendorf tubes to give a final volume of 20 μL in each sample tube.



The sample mixtures were then photo-irradiated at 400–450 nm for 45 min using a transilluminator (Vilber Lourmat) containing 4 × 15 W light tubes (Aqua Lux) with maximum emission at 435 nm. After photo-irradiation, 2 μL of the 6x sample dye solution (which contained 20% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol FF) was added to each Eppendorf tube and mixed well by centrifugation. The sample mixtures were loaded onto a 0.8% (v/v) agarose gel (Gel dimension: 13 cm × 10 cm), with 1x TBE buffer (89 mM tris-borate, 1 mM EDTA, pH 8) used as supporting electrolyte, and electrophoresized at 1.3 V.cm-1 for 3 h using a mini gel set (CBS Scientific Co., Model No. MGU-502T). After electrophoresis, the gel was stained with 0.5 μg/mL ethidium bromide solution for 30 min and then destained using deionized water for 10 min. The resulting gel image was viewed under 365 nm and captured digitally using a gel documentation system (BioRad).

The DNA photocleavage activities of Por 1, Por 2and Por 3 were measured by monitoring the conversion of supercoiled conformer (Form I) to the open-circular conformer (Form II) upon nicking by a DNA-cleavage agent.

Measurement of singlet oxygen production rate

1,3-diphenylisobenzofuran (DPBF) was used as a selective singlet oxygen (1O2) acceptor, which was bleached upon reaction with 1O2. Five sample solutions of DPBF in DMSO (50 μM) containing, respectively, no porphyrin (control sample), H2TMPyP (1 μM), Por 1(1 μM), Por 2 (1 μM) and Por 3 (1 μM) were prepared in dark. Each sample container was covered with aluminum foil with a yellow filter (with cutoff wavelength < 500 nm) on one side. The samples were then exposed to light (50 watt) through the filter. After irradiation, visible spectra of the sample solutions were measured spectrophotometrically. The normalized absorbances of DPBF at 418 nm in these samples were reported as a function of the photo-irradiation time. From this plot, the 1O2 production rates of Por 1, Por 2 and Por 3 relative to that of H2TMPyP can be determined.

CONCLUSION

The monomeric Por 1 and Por 2 showed both intercalative and external binding with DNA by the CD spectra whereas only external DNA binding was seen in the bisporphyrin, Por 3. The DNA photocleavage activities of these porphyrins followed the order: Por 1~ Por 2 > Por 3. All three 4-amidinophenylporphyrins produced 1O2 more efficiently than H2TMPyP. It suggests the porphyrins with strong hydrogen bonding capacity are promising new photo-dynamic therapeutic agents. To ascertain whether hydrogen bonding occurred between the amidine group and the DNA bases in the observed porphyrin-DNA binding interactions, 1H NMR study using oligodeoxyribonucleotides as binding substrates

is currently underway. Additionally, cellular uptake and cytotoxicity tests are also in progress.

Acknowledgements

The authors are grateful for the financial supports of National Natural Science Foundation of China (Nos. 20902071, 20672082), Hong Kong Research Grants Council (No. HKBU 202407) and Open Funds of Key Laboratory for Green Chemical Process of Ministry of Education in China (No. RGCT201003).

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DOI: 10.1142/S1088424612004483



INTRODUCTION

Numerous works have been concentrated on the oxidation of organic compounds in the last century, owing to their importance in organic synthesis and in the industry [1]. The popular conventional method for the conversion of aldehydes to their corresponding carboxylic acids involves the use of Jones’ reagent [2]. The Jones reagent however has two disadvantages; highly acidic media and the stoichiometric generation of Cr-based hazard material. Other efficient reagents have also been reported such as oxone [3], calcium hypochlorite [4] and 2-hydroperoxyhexafluoro-2-propanol [5]. Hydrogen peroxide has also been used as an oxidant under severe basic conditions [6], or in combination with other reagents such as formic acid [7], sodium chlorite [8], benzene selinic acid [9], magnesium monoperoxyphthalate (MMPP) [10] and other transition metal mediated reactions [11–17]. However, all of these methods have some limitations such as the requirement of strong acidic or basic conditions,

stoichiometric or super stoichiometric amounts of costly or hazardous oxidizing agents, or elevated temperatures. Therefore, mild, catalytic, economic and efficient alternative methods are required.

An extensive effort has also focused on metallo-porphyrins which are efficient biomimetic catalysts for hydrocarbon oxidation using molecular oxygen and various oxygen transfer reagents [18, 19]. Unfortunately, metalloporphyrins catalysts usually require use of stoi-chiometric oxidation for thermal oxidation of the substrates. Therefore, finding a catalytic system for the oxidation of hydrocarbons by O2 under mild conditions is still a challenging issue [20, 21]. A few examples of photochemical oxidations using O2 and a metalloporphyrins catalyst have been reported [22, 23]. Their reactivities are found to be dependent upon the central metal, the substituents on the porphyrin ring, and the axially coordinated ligands. Platinum porphyrins have also been investigated as oxygen sensing probes, photosensitizer, potential antitumor agents [24], molecular conductors and components of molecular devices [25]. Platinum porphyrins in high oxidation states are also known, but studies of these higher oxidation state derivatives are limited [26].

Highly efficient conversion of aldehydes to carboxylic acid

in the presence of platinum porphyrin sensitizers, air and

sunlight

Mahdi Hajimohammadi, Hamid Mofakham, Nasser Safari* and Anahita Mortazavi

Manesh

Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G.C., Evin, 19839–6313 Tehran, Iran

Received 6 June 2011Accepted 5 August 2011

ABSTRACT: A variety of aromatic and aliphatic aldehydes were oxidized to the corresponding carboxylic acids in the presence of platinum porphyrin, sunlight and air in acetonitrile solvent under mild conditions. Nitrobenzaldehydes were found to be very efficient 1O2 scavengers that quench the formation of acids from any aldehyde in the presence of free-base porphyrin sensitizers. However, nitrobenzaldehydes were converted to the corresponding acids in the presence of platinum porphyrins. The platinum porphyrins are very good and efficient catalysts for a wide range of applications in the aerobic conversion of aldehydes to acids.

KEYWORDS: photooxidation, platinum porphyrin, carboxylic acid, sunlight, aldehyde.


*Correspondence to: Nasser Safari, email: [email protected], tel: +98 21-22401765, fax: +98 21-22403041


94 M. HAJIMOHAMMADI ET AL.

Although a synthetic reaction using solar energy was reported about 50 years ago, the rate was very slow and the yield of product was poor. Sunlight has recently been applied as a sustainable light source [27]. To alleviate these problems, strong UV light (e.g. from a high pressure mercury lamp) has been used to accelerate the reaction. If solar radiation can be used to efficiently mediate the oxidation of alcohols, an environmentally benign process will be established. This concept leads back to the origin of organic photochemistry in the late 19th century [28]. In this works we first investigate the photocatalytic oxidation of aldehydes by sunlight then we describe the effects of solvent, time, metal, mechanism and the structure of sensitizers in these reactions.

EXPERIMENTAL

Chemicals and reagents

Melting points were measured on an Electrothermal 9100 apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on a BRUKER DRX-300 AVANCE spectrometer at 300.13 and 75.47 MHz. NMR spectra were obtained on solution in DMSO-d6 using TMS as internal standard. Light intensity determined with light meter [(LT lutron model YK-2500LX). The chemicals used in this work were purchased from Merck and Fluka Chemical Companies. The conversion and selectivity of products were monitored by Agilent and Finnigan TRACE gas chromatographs.

Preparation of catalyst

The meso-substituted porphyrins were synthesized from the reaction of related aldehydes and pyrrole [29]. The reaction was carried out in the boiling propionic acid with the addition of 10 vol.% acetic anhydride. The purification of porphyrins was carried out using the crystallization from different organic solvents. To introduce the platinum ion into a macrocycle molecule the benzonitrile method with divalent platinum salts was used [30]. The solvents were boiled down in vacuum, and the residue was recrystallized from the chloroform-methanol mixture.

Reaction procedures

General procedure for the synthesis of carboxylic acids. The aldehydes (0.5 mmol) and a 1 mL CH2Cl2

solution of the PtTMP (0.5 × 10-3 mmol) were dissolved in 14 mL of CH3CN in a quartz tube. Air was bubbled through the solution and the sample was irradiated using sunlight 75400 LUX (Intensity determined with light meter LT lutron model YK-2500LX) for 4–24 h. The solvent was removed under vacuum, and the residue was separated by column chromatography (silica gel,

n-hexane/EtOAc, 13:1) to give the corresponding carboxylic acid. The conversion and selectivity of products were measured by GC. The product of entry 12, Table 2 was confirmed from the melting points and 1Hand 13C NMR spectra.

Gas-chromatographic method. Analytical methods.Gas-chromatographic determinations were carried out by means of a Finnigan TRACE GC Ultra with an Innowax column (RTX-1) and H2 as the carrier gas. The flow was (1/4 mL/min). The temperature program was 50–180 °C(11 °C/min), 180–270 °C (11 °C/min).

Selected spectral data

Terephthalic acid (12). Colorless crystals; mp > 260 °C. 1H NMR (300.13 MHz, DMSO-d6): δ, ppm 8.00–8.15 (4H, m, H Ar), 13.31 (2H, br s, CO2H).13C NMR (75.47 MHz, DMSO-d6): δ, ppm 129.9, 130.4, 134.9, 139.3, 167.1.


In previous studies, efficient catalytic systems for free-base porphyrins-catalyzed aerobic oxidations of aldehydes have been developed in the presence of visible light and sunlight [31]. In continuation of our studies for aerobic oxidation of organic compounds with porphyrin and using solar energy herein, we would like to report a novel and highly efficient catalytic system for the aerobic oxidation of aldehydes using platinum porphyrins as catalyst, by which aldehydes and its substituted derivatives can be smoothly oxidized to corresponding carboxylic acids. Photo-oxygenation was performed in the oxygenated solutions of acetonitrile under sunlight in the presence of platinum porphyrins under 1 atmosphere pressure of air at room temperature (Scheme 1).

R CHO R CO2HPlatinum porphyrins (cat.)/air/sunlight

CH3CN, rt

PtTPP: R1=R2=R3=R4=R5=HPtTMP: R1=R3=R5=CH3, R2=R4=HPtTPFPP: R1=R2=R3=R4=R5=F

N

N

N

N

R5

R4

R3

R2

R1

R1

R2

R3

R4

R5

R1

R2

R3

R4

R5 R1

R5

R4

R3

R2

Pt

Scheme 1. Reaction scheme and the structure of platinum porphyrins used as catalysts


HIGHLY EFFICIENT CONVERSION OF ALDEHYDES TO CARBOXYLIC ACID 95

Initial attempts to optimize the reaction conditions for the oxidation of aldehydes to the corresponding carboxylic acids were performed by a variety of metalloporphyrins with 2-methoxybenzaldehyde as the test substrate. The yields are highest for platinum porphyrins as is indicated in Table 1. All other metals have shown negligible yields, but Pd and Ni porphyrins were slightly reactive. ZnTPP which is relatively reactive for electron-withdrawing substituted aldehydes (59% conversion for 4-bromobenzaldehyde) showed no reactivity for less reactive aldehydes such as 2-methoxybenzaldehyde. The complexes of paramagnetic metal ions of the first row transition metals have a very short triplet lifetime in solution and are not reactive [32]. In contrast, complexes of diamagnetic Pd(II) porphyrins and Pt(II) show both intense fluorescence and pho-sphorescence, with triplet lifetimes in the range of msec [33] and show highest reactivity. The catalytic activity of metalloporphyrins for photooxidation appeared to be dependent on the nature of the ligands as well. PtTMP Showed the best activity among PtTMP, PtTPP and PtTPFPP, and presented excellent catalytic performance for the oxidation of 2-methoxybenzaldehyde under mild reaction conditions. Therefore PtTMP was chosen for further investigations. When the oxidation of 2-methoxybenzaldehyde was conducted in the absence of PtTMP, the yield of 2-methoxybenzoic acid was negligible (Table 1, entry 10). However, the yield showed remarkable increase by adding 1.0 × 10-6 mol of platinum porphyrin to the reaction mixture (Table 1, 91%, entry 8).

Figure 1 shows the of formation percent of 2-methoxybenzoic acid vs. time in an oxygenated solution in acetonitrile under sunlight irradiation in the presence of PtTMP. The Soret band of PtTMP was monitored at 398.5 nm using a UV-vis method. This method showed

that sensitizer bleaching for PtTMP was complete in 8 h under our conditions. After the disappearance of the porphyrin Soret band, the formation of the oxidation products stopped, and the reaction conversion remained constant. Therefore, the end point of the reaction can be controlled by the change of color of the reaction medium (Fig. 1).

To check the generality of this method, the oxidation of a variety of aromatic and aliphatic substrates was studied under sunlight. As shown in Table 2, this catalytic system was applicable to a wide range of aromatic and aliphatic substrates. The aldehydes were converted into the corresponding carboxylic acids in high percentages within reasonable times with a high turnover number. Aromatic aldehydes possessing electron-withdrawing groups on the phenyl ring (Table 2, entries 4–9) were more reactive than those with electron-donating substrates (Table 2, entries 2, 3). Porphyrin degradation was also accelerated in the presence of less reactive substrates.

Table 2 also shows that PtTMP is more reactive than H2TMP and the patterns of reactivity for different substrates are somehow different for PtTMP and H2TMP.

To find a clue to the mechanism, the aerobic oxidation of 2-methoxybenzaldehyde in various solvents and singlet oxygen scavengers has been investigated and the results are summarized in Table 3. The engaged solvents for the oxidation reactions are dime-thylformamide, acetonitrile, dimethylsulfoxide, NaN3/Na2SO3/acetonitrile and H2O. NaN3/Na2SO3 is known as a singlet oxygen scavenger in the literature science it produces N3

- in the presence of light which N3- compete

with 1O2 [34]. Acetonitrile is a good solvent for oxidation reactions since it is relatively inert toward oxidation, and its tendency toward coordination and interaction with coordination compound is low. In addition acetonitrile is usually enough polar to dissolve the substrates, products and the catalyst.

It could be observed from Table 3 that the reaction medium played an important role in the oxidation system. It seems that acetonitrile and NaN3/Na2SO3/acetonitrile were

Table 1. Effect of various metalloporphyrins on the photooxidation of 2-methoxybenzaldehydea

Entry Catalyst Conversion into 2-methoxy-benzoic acid, %

1 ClMnTPP trace

2 ClFeTPP trace

3 ClCoTPP trace

4 ClNiTPP 6

5 ZnTPP trace

6 PdTPP 27

7 PtTPP 75

8 PtTMP 91

9 PtTPFPP 71

10 no catalyst —

a 0.5 × 10-6 mol porphyrins, 0.5 × 10-3 mol 2-methoxybenzalde-hyde, 8 h.

Fig. 1. Formation of 2-methoxybenzoic acid vs. time of irradiation in acetonitrile (Series 1); Percent of remaining PtTMP vs. time (Series 2)



Table 2. Oxidation of aldehydes to carboxylic acids in the presence of (a) PtTMP and (b) H2TMP under sunlight irradiation

Entry Aldehyde Acid Time, h Conversion, % TONc

Sunlighta Visible lightb

1

CHO CO2H

4 95 87 (72 h)31b 950

2

CHO

OMe

CO2HOMe 8 91 71 (72 h)31b 910

3

CHO

OMe

CO2H

OMe

8 81 95 (48 h)31b 810

4

CHO

Br

CO2H

Br

4 100 100 (16 h)31b 1000

5

CHO

Cl

CO2H

Cl

4 100 89 (72 h)31b 1000

6

CHO

F

CO2H

F

4 100 74 (48 h)31b 1000

7

CHO

NO2

CO2H

NO2

24 69 Trace 690

8

CHO

NO2

CO2H

NO2

24 57 Trace 570

9

CHO

NO2

CO2H

NO2 24 83 Trace 830

10O CHO O CO2H 8 100 100 (48 h)31b 1000

11O

H

O

OH8 85 96 (48 h)31b 850

(Continued)



effective in the aerobic oxidation of 2-methoxybenzalde-hyde to 2-methoxybenzoic acid, in the presence of PtTMP, but not H2TMP.

Meanwhile, according to Table 3 (entry 2, 3 and 5), it was found that the reaction was completely inhibited when we used dimethylsulfoxide, dimethylformamide

and H2O as solvents. Highly coordinated solvents such as dimethylsulfoxide inhibit the formation of an active intermediate by preventing oxygen coordination with the platinum porphyrin formed in the solution and also quench carboxylic acid formation. The reaction did not quench in the presence of NaN3/Na2SO3/acetonitrilewhich is known as a singlet oxygen scavenger (Table 3, entries 4) and this proves that, singlet oxygen generation is not the main mechanism in the oxidation of aldehydes with PtTMP sensitizers.

We found that nitrobenzaldehydes are the most effective 1O2 scavengers. They are much more effective than NaN3/Na2SO3 systems. The formations of carboxylic acids from the corresponding aldehydes

are completely quenched in the presence of nitrobenzaldehydes (Table 4). Therefore we recommend nitrobenzaldehydes as an effective 1O2 scavenger (Scheme 2).

Table 4, however, shows the photooxidation of nitrobenzaldehydes in the presence of PtTMP and H2TMP. Nitrobenzaldehydes were effectively converted to the corresponding acids in the presence of PtTMP, but not H2TMP. This observation accompanied with the observation brought in entry 6 in Table 3 rules out the 1O2 path as the only path for acid formation by PtTMP.

The production of platinum(IV)por-phyrin from Pt(II) porphyrin during the reaction period was confirmed by UV-vis study for the photooxygenation of 2-methox-ybenzaldehyde, as demonstrated in Fig. 2. Series 1, 2, 3 and 4 in Fig. 2 show the solution spectra of PtTMP in the beginning and after 5, 10 and 15 min of light irradiation, respectively. The strength of the Soret band

of PtTMP at 398.5 nm decreased and a new signal gradually increased again at 417.5 nm which was assigned to the Pt(IV) formation. Kadish and co-workers recently reported the first example for electrogeneration of a Pt(IV) porphyrin [35] and the chemical production of high valent Pt porphyrins has been recently reported

Table 3. Effect of solvent and 1O2 on the conversion of 2-methoxybenzaldehyde to 2-methoxybenzoic acid

Entry Solvent Conversion

1 acetonitrile 91a

2 dimethyl formamide tracea

3 dimethyl sulfoxide tracea

4 NaN3/Na2SO3/acetonitrile 65a

5 H2O tracea

6 NaN3/Na2SO3/acetonitrile traceb

a 0.5 × 10-6 mol PtTMP, 0.5 × 10-3 mol 2-methoxybenzaldehyde after 8 h. b0.5 × 10-6 mol H2TMP, 0.5 × 10-3 mol 2-methoxy-benzaldehyde after 8 h.

Table 4. Oxidation of nitrobenzaldehydes with free-base porphyrin and PtTMPa

Entry Aldehyde Acid Time, h

Conversion, % (PtTMP)

Conversion, % (H2TMP)

1

CHO

NO2

CO2H

NO2

24 69 trace

2

CHO

NO2

CO2H

NO2

24 57 trace

3

CHO

NO2

CO2H

NO2 24 83 trace

a 0.5 × 10-6 mol porphyrins and 0.5 × 10-3 mol aldehydes as substrates, air (1 atm), and sunlight (75400 LUX).

Entry Aldehyde Acid Time, h Conversion, % TONc

Sunlighta Visible lightb

12

CHO

CHO

CO2H

CO2H

4 100 100 (16 h)31b 2000

a 0.5 × 10-6 mol PtTMP and 0.5 × 10-3 mol aldehydes as substrates, air (1 atm) and sunlight (75400 LUX). b 1.0 × 10-6 mol H2TMPand 1.0 × 10-3 mol aldehydes as substrates, air (1 atm) and fluorescent circular lamp, six 22 W lamps (32400 LUX). c Turnover number of the PtTMP catalyst.

Table 2. (Continued)



[36]. Our results in UV-vis spectroscopy correlate with the high oxidation of platinum during the photooxidation process.

According to the above discussions, a plausible reaction mechanism for the photooxidation reaction using PtTMP as a catalyst has been proposed as shown in Scheme 3. In the presence of oxygen and sunlight thus, there may be a new photochemical pathway that involves the formation of intermediated 2 as the main oxidant. This oxidant, 2 can oxidize aldehydes to their corresponding acids and reduce itself to reform the starting catalyst [33].

Consequently, platinum porphyrin is a better catalyst than free-base porphyrin since: In 1O2 singlet pathways

Pt(II) has high triplet lifetime in the range of msec which result in better generation of 1O2. In the case of platinum another path other than 1O2 path which is formation of active Pt(IV) is involved. This path is more reactive than 1O2 since nitrobenzaldehydes are converted in good yields. In addition electron donating substituent porphyrin such as PtTMP shows highest activity. This is in accord with the mechanism proposed in Scheme 3. Light will induce electron transfer from platinum porphyrin to the O2. More electron rich porphyrin such as PtTMP is more reactive. Table 1 shows that reactivity is in the other of PtTMP > PtTPP > PtTPFPP. This trend matches with electron donating ability of the porphyrin macrocycles.

CONCLUSION

In conclusion, PtTMP is very effective at photo-oxidation for specific conversions of aldehydes to acids. PtTMP has an advantage over H2TMP for photooxidation in that its reaction time is faster and it is more reactive towards less active aldehydes in comparison with H2TMP. For example PtTMP has the ability to convert nitrobenzaldehydes which is completely inert in the case of H2TMP. However, using precious Pt metals compared to that of free-base porphyrins can be viewed as a disadvantage, however the low amount of catalyst used and its productivity where H2TMP is ineffective makes up for its use.

Acknowledgements

We gratefully acknowledge financial support from the Research Council of Shahid Beheshti University.

REFERENCES

1. a) Hollingworth GJ. In Comprehensive Organic Functional Group Transformations, Vol. 5, Katritzky AR, Meth-Cohn O, Rees CW and Pattenden G.

Fig. 2. Spectral changes, solution A: 2-methoxybenzaldehyde (0.5 mmol) and PtTMP (0.5 × 10-3 mmol) (Series 1), solution A 5 min after photoreaction (Series 2), solution A 10 min after photoreaction (Series 3) and solution A 15 min after photoreaction (Series 4)

H

PtII

hv

PtIV

O

2

R

O

H3

OH

1

PtIV

OH

OH2

PtIV

O

O

RR

O

OH5

4

H2O

PtII

6

H2O

PPtII + hv P*PtII

P*PtII + O2 P+PtII O2-+

2 O2- O2

2-+O2

O22-

H

Scheme 3. A possible mechanism for the formation of the carboxylic acids from aldehydes in the presence of PtTMP

H2TMP (cat.)/O2/ hv

CH3CN, rt

CHO

R

no reaction+

CHO

Br

PtTMP (cat.)/O2/ hv

CH3CN, rt

CHO

R

+

CHO

Br

CO2H

R

+

CO2H

BrR = NO2

Scheme 2. Oxidation of different nitrobenzaldehydes in the presence of PtTMP or H2TMP



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DOI: 10.1142/S1088424611004373



INTRODUCTION

The unique photophysical and chemical features of tetrapyrrolic macrocycles are the main reasons for the continuous interest by the scientific community in such compounds. As it is well-known their potential or successful applications are important in a range of scientific areas, such as catalysis, advanced biomimetic modeling for photosynthesis, development of new electronic materials, sensors and also medicine [1]. In the biomedical field, porphyrins and related compounds are being employed successfully for the treatment of cancer and several other diseases by photodynamic therapy (PDT) [2, 3], a technique which relies on the administration of a photosensitizing agent and subsequent

irradiation with light of appropriate wavelength [4, 5]. Recent studies demonstrated that PDT can also be very effective in the selective inactivation of microorganisms, indicating that it is a potential alternative tool for the treatment and eradication of microbial infections [6–16].

Ionic photosensitizers, especially those with an asymmetric distribution of the charges in the molecule, are very promising for medicinal use since such compounds show an increased ability to accumulate in sub-cellular compartments (enhanced membrane penetration) and to interact with the target [3]. In particular, cationic porphyrins are able to induce photoinactivation of microorganisms [8, 17], and/or to interact with DNA and also to cause its rupture upon irradiation [18]. In a previous paper, we have shown that cationic β-vinyl substituted meso-tetraphenylporphyrins are effective in the photoinactivation of herpes simplex virus type 1 (HSV-1) [19]. Such compounds were obtained by condensation of 2-formylporphyrins with 1,2- and 1,4-dimethylpyridinium salts. Further investigations

Cationic β-vinyl substituted meso-tetraphenylporphyrins:

synthesis and non-covalent interactions with a short

poly(dGdC) duplex

Eduarda M.P. Silva, Catarina I.V. Ramos, Patrícia M.R. Pereira, Francesca Giuntini,

Maria A.F. Faustino, João P.C. Tomé , Augusto C. Tomé , Artur M.S. Silva, M. Graça

Santana-Marques* , M. Graça P.M.S. Neves* and José A.S. Cavaleiro

Department of Chemistry, QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal

Received 30 July 2011Accepted 2 September 2011

ABSTRACT: Several cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenyl-porphyrin derivatives were synthesized starting from 2-formyl-meso-tetraphenylporphyrin, and the corresponding Zn(II) complex, and different N-alkyl derivatives of 2- and 4-methylpyridine and 2- and 4-methylquinoline. The new compounds were obtained in a one-step process via base catalyzed aldol-type condensation reactions. Electrospray ionization mass spectrometry (ESI-MS) and ultraviolet-visible (UV-vis) spectroscopy were used to investigate the binding mode of the synthesized cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenylporphyrin derivatives with a short GC duplex oligonucleotide. Analysis of the obtained mass spectrometry results indicates the probable occurrence of outside binding. UV-vis spectroscopy data also points to non-intercalation. The potential photosensitizing capacity of these compounds was also ascertained from preliminary photophysical studies.

KEYWORDS: cationic porphyrins, pyridinium salts, non-covalent adducts, electrospray mass spectrometry.


*Correspondence to: M. Graça Santana-Marques, email: [email protected] and M. Graça P.M.S. Neves, email: [email protected]


102 E.M.P. SILVA ET AL.

carried out in our laboratory showed that this method has a broad applicability and can be used to prepare a variety of cationic porphyrin derivatives.

Electrospray ionization mass spectrometry (ESI-MS) and tandem electrospray ionization mass spectrometry (ESI-MS/MS) have been previously used to differentiate the 2- and 4-methylpyridyl isomers of free-base and metalated cationic β-vinylpyridylporphyrins [20]. The data acquired show that of all the studied compounds, the free-base 2-methylpyridyl isomer, which was operative in the in vitro photoinactivation of Herpessimplex virus, has a different gas-phase behavior. Our previous studies have shown that local distortion of the macrocycle, due to the presence of the β-vinylpyridyl substituent, occurs for all the compounds, but that a different electron density distribution can account for the observed gas-phase behavior of this potential antivirus agent. In addition, these same techniques along with UV-vis spectroscopy, have been previously used by us to investigate the non-covalent interactions between small oligonucleotide duplexes and a group of cationic meso-(1-methylpyridinium-4-yl)porphyrins (four free-bases with one to four positive charges, and the corresponding zinc complexes) [21, 22]. The positive charges were located in the meso-positions of the macrocycle. The results obtained pointed to outside binding of the porphyrins, with the binding strength increasing with the number of positive charges.

The promising features of the β-vinyl-methylpyridinium porphyrins as virus photoinactivators, led us to synthesize several related compounds and to investigate the possibility of intercalation with a GC-motif duplex oligonucleotides using electrospray mass spectrometry.


Chemistry

Electron-poor alkyl-heterocycles (e.g. 2- and 4-alkylpyridines) undergo aldol-like condensations with carbonyl compounds in the presence of strong bases or Lewis acids, but in most cases such reactions have a limited applicability because of the harsh requirements; many chemical functions are not suited to such conditions. However, when the corresponding N-oxidesor N-alkyl derivatives are used, the condensation occurs in milder conditions, due to the increased acidity of the side-chain protons induced by the strong electron withdrawing effect exerted by the positive charge on the ring [23, 24]. Bearing this in mind, we have undertaken the synthesis of a series of β-monosubstituted cationic meso-tetraphenylporphyrin derivatives, in a one-step process, starting with 2-formyl-5,10,15,20-tetraphenyl-porphyrin (2-formyl-TPP, 1a) and the corresponding Zn(II) complex 1b and different cationic derivatives of 2- and 4-methylpyridine (Scheme 1) and 2- and 4-methylquinoline (Scheme 4).

2-formyl-5,10,15,20-tetraphenylporphyrin was obtai-ned by Vilsmeier formylation [25] of the copper complex of 5,10,15,20-tetraphenylporphyrin (TPP), according to the literature procedure [26]. The corresponding zinc complex was obtained by metalation of the free-base with zinc acetate [27]. The pyridinium and quinolinium derivatives were prepared according to literature procedures [28–30].

The condensation reaction using 2-formyl-TPP 1a with either 1,2-dimethylpyridinium iodide 2 or 1,4-dimethylpyridinium iodide 4 [29] (Scheme 1) in the

N

N

N

N

PhPh

Ph Ph

CHO

M Cl-N

N

N

M N

PhPh

Ph Ph

NMe

1a, M = 2H1b, M = Zn

3a, M = 2H3b, M = Zn

1''

2''

3''

2'

1'

3

5

6 4

+

(i), (ii)

N

N

N

N

PhPh

Ph Ph

CHO

M

Cl-

N M

R

N

N

N

PhPh

Ph Ph

N

1a, M = 2H1b, M = Zn

7, 8

1''

2''

3''

2'

1'

23

56

+

7a, R = Me, M = 2H7b, R = Me, M = Zn8a, R = (CH2)9Me, M = 2H9a, R = monosacharide unit, M = 2H

(i), (ii)

N Me

MeI-

2

+

X-

N RMe

4-6

+

Scheme 1. Reagents and conditions: (i) K2CO3, THF/MeOH; (ii) NaCl aq


CATIONIC β-VINYL SUBSTITUTED MESO-TETRAPHENYLPORPHYRINS 103

presence of K2CO3, at 30 °C, in a THF/MeOH mixture has been previously published [19]. This reaction allowed the preparation of the desired compounds 3aand 7a (M = 2H) in 60% and 65% yields, respectively. An attempt to improve these yields was performed using the Zn(II) complex 1b. The new zinc complexes 3b and 7b (Scheme 1, M = Zn) were isolated by flash chromatography on silica gel, followed by washing with brine (in order to assure the complete counter ion exchange), and by subsequent crystallization. The zinc complexes 3b and 7b (M = Zn) were obtained in 43% and 40% yields, respectively. These results show that the reactions carried out with the free-base 1a afforded better yields than those in which the corresponding zinc complex 1b was used. The position of the methyl group on the pyridine ring seems not to affect the reaction; salt 2 proved to be only slightly less reactive than 4. The synthesis of an analog of 7a (R = H) starting from 1ahas previously been reported in the literature [31–33]. However, the procedure described requires several steps, namely reduction of the carbonyl function, halogenation of the resulting hydroxymethyl group, its conversion into the corresponding triphenylphosphonium salt and then reaction with pyridine-4-carbaldehyde. This method leads to the formation of both E- and Z-isomers of the 2-(4-pyridylvinyl)-substituted porphyrins, whereas with our process only the E-isomer was formed.

In order to verify whether the presence of a bulkier alkyl substituent at the pyridine nitrogen had any effect on the course of the reaction, and to illustrate the scope of the method, we prepared salts 5 and 6 (Scheme 2). 1-Decyl-4-methylpyridinium iodide 5 was obtained in 86% yield by alkylation of 4-methylpyridine with 1-iododecane. Similarly, alkylation of 4-methylpyridine with 6-iodo-1,2:3,4-di-O-isopropylidene- -D-galactopyranose afforded salt 6 in 96% yield [34, 35].

Condensation of porphyrin 1a with 5, in the presence of K2CO3, afforded porphyrin 8a in 30% yield, while condensation of 1a with 6 gave porphyrin 9a in 53% yield (Scheme 1). The unprotected galactose derivative 10a was obtained in 66% yield by acidic hydrolysis of the isopropylidene moieties in porphyrin 9a (Scheme 3). There are several methods reported in the literature to prepare porphyrin-carbohydrate conjugates. Those conjugates are frequently used as neutral water-soluble photosensitizers since they show a better biodistribution, and are potentially more selective for cell targeting [36, 37]. In compound 10a, the simultaneous presence of a carbohydrate unit and a positive charge may render this compound as potentially useful as photosensitizer.

The mild conditions of our synthetic method show that it can be applied for the synthesis of a range of cationic vinylporphyrins bearing a diversity of groups linked to the pyridyl nitrogen. However the applicability of our synthetic method is not confined to N-alkyl-pyridinium salts. Both 1,2- and 1,4-dimethylquinolinium iodides (11and 12) [30] also underwent condensation with the Zn(II) complex of 2-formyl-TPP 1b, although under slightly different conditions (Scheme 4).

Structural elucidation

The 1H NMR spectra of the new compounds show some common features (Table 1). The resonance due to the H-3 proton is observed above 9 ppm, out of the multiplet generated by the other beta protons (8.70–8.90 ppm), except for compounds 3b (vide infra). The vinylic protons originate signals above 7 ppm for all compounds except in the cases of 13b and 14b, in which the signals of such protons fall within the multiplets generated by the meta and para protons of the meso-phenyl rings. The coupling constant values, for all compounds except

NR2

R1

NMe (CH2)9MeI I(CH2)9Me I

OO

OO

I

OMe N

OO

O O O5 6

(86%) (96%)

+

(i) (ii)

Scheme 2. Reagents and conditions: (i) 80 °C, 6 h; (ii) 120 °C, 4 h

Cl-

N

NH

HN

N

PhPh

Ph Ph

N OO

O O O

Cl-

N

NH

HN

N

PhPh

Ph Ph

N OHO

HO OHOH(i)

+

9a

+

10a

Scheme 3. Reagents and conditions: (i) TFA/H2O (9:1), rt, 30 min



for 3b, are higher than 15 Hz, confirming the transstereochemistry of the vinylic system.

For the free-bases, the singlets generated by the inner pyrrolic protons are observed, as expected, between -2.60 and -2.45 ppm. The 1H NMR spectrum of each compound bearing an N-methyl group shows a singlet between 3.55 and 4.75 ppm.

Compound 3b shows a somewhat unusual 1H NMR spectrum. The signal of H-3 , for instance, does not

appear as an isolated singlet but instead it is found within the multiplet generated by the remaining H-β,as confirmed by the HSQC spectrum. The resonances of the vinylic protons appear slightly high-field shifted when compared with the remaining compounds, with a 3J value (11.9 Hz) that does not allow to determine unequivocally the trans configuration. However, the absence of correlation between H-1 and H-2 in the NOESY spectrum clearly indicates that, in spite of the relatively small value of the 3J, the stereochemistry of the double bond is also trans.

Photostability of the compounds

The photostability of the compounds is an important parameter to evaluate since it is necessary to certify that the compounds do not undergo an extensive destruction of the tetrapyrrolic macrocycle when exposed to UV-vis light and oxygen [38]. In order to establish the rate of photodegradation of the compounds, we performed photostability studies at the same irradiation conditions used in the biological assays (white light, 50 mW/cm2)previously published for compounds 3a and 7a [19]. The obtained values were calculated by the ratio between the residual absorbance after irradiation for different periods of time and the absorbance before irradiation. The absorbances were measured at the corresponding Soret band wavelengths. Under these conditions, all compounds show a high photostability, between 98 and 100%, during at least the first 15 min of irradiation.

Singlet oxygen and fluorescence quantum yields

The ability of the porphyrin derivatives to generate singlet oxygen and its preferential location in tumor cells or microorganisms, is the reason for their importance and application in PDT. The basis for this use is dependent on

N

N

N

N

PhPh

Ph Ph

N

MN

MeI-

Cl-Me

12

1a or 1b +

Me+

+ 23

4a

5

67

8

1'

2'1''

2''

3''

14a, M = 2H, 4-yl (22%)14b, M = Zn, 4-yl (45%)

N

N

N

N

PhPh

Ph Ph

NMN

MeI-

Cl-

11

1a or 1b +Me

++

3

4a

5

67

8

1'

2'1''

2''

3''

13a, M = 2H, 2-yl (49%)13b, M = Zn, 2-yl (38%)

Me

4

(i)

(i)

Scheme 4. Reagents and conditions: (i) pyridine, 60 °C, 60 h

Table 1. Relevant common features (δ, ppm and J, Hz) in the NMR spectra of the synthesized compounds

H-3′′ H-2′ H-1′ J Me NH

3ac 9.09 (s) 7.47 (d) 7.33 (d) 15.6 4.67 -2.54

3b a 7.27 (d) 6.97 (d) 11.9 4.09 —

7ac 9.09 (s) 7.49 (d) 7.25 (d) 15.7 4.65 -2.52

7b 9.19 (s) 7.16 (d) 7.06 (d) 15.2 3.55 —

8a 9.09 (s) 7.46 (d) 7.25 (d) 16.0 — -2.53

9a 9.09 (s) 7.49 (d) 7.24 (d) 15.4 — -2.53

10a 9.16 (s) 7.66 (d) 7.41 (d) 16.0 — -2.59

13a 9.30 (s) 7.71 (AB) 7.71 (AB) 15.5 4.61 -2.49

13b 9.38 (s) b b — 4.29 —

14a 9.21 (s) 7.74 (d) 7.96 (d) 15.5 4.75 -2.51

14b 9.21 (s) b b — 4.56 —

a The signal of this proton lies within the multiplet generated by the resonances of the other H-β. b The COSY (1H/1H) spectrum showed that the signals corresponding to these protons lie within the multiplet generated by the Ph-Hmeta/paraprotons. c The structural characterization of these compounds is described in Ref. 19.



energy transfer processes [39, 40]. In this particular case, a photosensitizer in its triplet state needs to interact with the ground state triplet oxygen to produce the reactive singlet oxygen species [41]. This active molecule can oxidize important biomolecules in the cells leading to cellular damage [40, 41]. Another important process to assess, in what concerns energy transfer from the photoexcited sensitizer, is the fluorescence emission. A sensitizer might discharge the excitation energy into a radiative process and such release would allow its location and in this way the sensitizer would act as a marker. Therefore, in order to test the potential use of these new porphyrin derivatives, preliminary evaluation of the photosensitizing activity of these compounds was performed through measurement of the singlet oxygen and fluorescence quantum yields.

The capability of the new porphyrin derivatives to generate singlet oxygen was qualitatively estimated by measuring the absorption decay of 1,3-diphenyli-sobenzofuran (DPBF) at 415 nm in DMF [42–44]. The yellow DPBF reacts with 1O2 in a [4+2] cycloaddition being oxidized to colorless ortho-dibenzoylbenzene. Since DPBF has an absorption maximum at 415 nm, it is possible to follow the ability of the porphyrin to generate 1O2 by measuring the absorption decay, at this wavelength. Thus, a solution containing DPBF (50 μM)and the porphyrin derivative (0.5 μM) was irradiated over a period of 6 min using a white lamp with a cut-off filter for wavelengths < 540 nm, at a fluence rate of 50 mW/cm2.Given that the 5,10,15,20-tetraphenylporphyrin (TPP) is known to be a good photosensitizer it was used in this study as reference [45]. The results obtained, summarized in Fig. 1, show that the DPBF photodegradation is highly enhanced in the presence of the photosensitizers 3aand 9a, confirming that they are good singlet oxygen generators. The result obtained for compound 3a may

perhaps justify the significant activity exhibited by this compound that reached 99% of virus inactivation after 15 min of photoactivation [19]. Compounds 7aand 8a demonstrate behaviors comparable to TPP and compounds 13a and 14a, which contain in their structure the quinolinium moiety, do not produce singlet oxygen.

The fluorescence quantum yields (Φf) measured relatively to 5,10,15,20-tetraphenylporphyrin [Φfl (TPP) = 0.11] [46, 47] are summarized in Table 2. For all deriva-tives the introduction of cationic β-vinyl moieties led to a weakening of fluorescence emission. Moreover, the derivatives 13a and 14a do not shown any fluorescence emission. Most likely these two compounds in their excited forms can decay by other radiative or non-radiative processes [4, 5] and to entirely understand the photophysical behavior of these two compounds further studies are required.

Non-covalent interactions with d(GCGCGCGC) — mass spectrometric results

The non-covalent interactions of the cationic porphyrins 3a, 7–9a, 13a and 14a (Scheme 1 and Scheme 4) with a small DNA duplex with the GC-motif were studied by electrospray mass spectrometry (ESI-MS), with several stages of mass separation. At physiological pH the DNA backbone is mostly deprotonated, thus all the spectra were acquired in the negative ion mode using an ammonium acetate buffer. The GC motif was chosen to maximize the possible occurrence of interactions by intercalation, of the positively charged porphyrins.

In this array of porphyrins one could point out two distinct groups: (a) one group containing positional isomers: compounds 3a/7a and 13a/14a where the basic carbon skeleton remains unchanged but the N-methylpyridinium and N-methylquinolinium rings are either in an 4 or 2 position in relation to the vinyl group

Fig. 1. Comparative photooxidation of DPBF (50 μM) in DMF with or without photosensitizer (0.5 μM); irradiation with white light filtered through a cut-off filter for wavelengths < 540 nm (50 mW/cm2)



and (b) a second group where different substituents on the N-terminus are all present in position 4 in relation to the vinyl group (Scheme 1 and Scheme 4).

In general, duplex DNA binding ligands with the same binding mode display the same dissociation patterns [48, 49]. Unknown duplex DNA binding modes may be deduced by comparing the fragmentation of new duplex DNA-ligand adduct ions, with those of known binders and intercalators [50–56]. Possible ambiguities in the attribution of binding modes by this procedure, can be eliminated through the use of the same mass spectrometer and by using adduct ions with the same charge states [57, 58].

To investigate the binding mode and stability of the porphyrin-duplex adduct ions, different types of experiments were undertaken. Initially the mass spectra were obtained for all the electrosprayed oligonucleotide-porphyrin solutions, at two different cone voltages (25 and 45 V) and following that, the product ion spectra (precursor ion product ions, MS/MS or MS2) of chosen adduct ions were acquired. Finally a different type of experiments that can be considered as a pseudo MS3 (first precursor ion second precursor ion product ions)were undertaken. The designation pseudo arises from the fact that, in this type of experiments, the first precursor ion is not mass selected but fragments in the source as a result of an increase in the cone voltage.

Two types of adduct ions were observed in the mass spectra, [ds+porphyrin-(n+1)H+]n- and [ds+porphyrin-nH++CH3COO-]n- (ds = double strand, n = 3 or 4), their relative abundances varying with the porphyrin and with cone voltage.

In the mass spectra obtained at a cone voltage of 25 V, the adduct ions [ds+porphyrin-5H+]4-, [ds+porphyrin-4H++CH3COO-]4-, [ds+porphyrin-4H+]3- and [ds+porphyrin-3H++CH3COO-]3- were observed for almost all the porphyrins (see Table 3), the exceptions being discussed below.

For porphyrin 13a (N-methylquinolinium-2-yl) the adduct ion [ds+porphyrin-4H+]3- was not observed. In contrast the mass spectrum of its isomer, porphyrin 14a(N-methylquinolinium-4-yl), does not show the triply charged adduct ion [ds+porphyrin-3H++CH3COO-]3-. In the spectrum of porphyrin 9a (N-sugar-pyridinium-4-yl) (Fig. 2) the adduct [ds+porphyrin-3H++CH3COO-]3- is also absent.

The analysis of the mass spectra obtained at a cone voltage of 45 V shows in general, a decrease in the abundance of the adduct ions [ds+porphyrin-5H+]4- and [ds+porphyrin-4H++CH3COO-]4- and an increase in the abundance of the triply charged adducts in special the [ds+porphyrin-4H+]3- adduct ions. This increase in the abundance of the triply charged adducts may result from two different factors. On one hand, we had observed in our previous studies [21, 22] that duplex ions with a higher number of negative charges are less stable at higher cone voltages, the higher repulsion of the phosphate chains leading to fragmentation by unzipping of the double strand. On the other hand, the fragmentation in the source of the adduct ions [ds+porphyrin-4H++CH3COO-]4- with the formation of the [ds+porphyrin-4H+]3- and CH3COO-

ions, is a probable process. An example of this behavior is observed in the mass spectrum of the porphyrin 13a(N-methylquinolinium-2-yl). At a cone voltage of 25 V the ion [ds+porphyrin-4H+]3- is absent and the adduct ion [ds+porphyrin-4H++CH3COO-]4- is the most abundant ion, but when the cone voltage is increased to 45 V, the adduct ion [ds+porphyrin-4H++CH3COO-]4- has a very low abundance and the [ds+porphyrin-4H+]3- ion is observed.

It is interesting to note that the observed behavior with the increase in cone voltage of the isomeric pair, porphyrins 3a (N-methylpyridinium-2yl)/7a (N-methylpyridinium-4-yl) and of porphyrin 8a (N-decylpyridinium-4-yl) is similar: the [ds+porphyrin-4H++CH3COO-]4- adduct ion is not present at 45 V and the adduct [ds+porphyrin-4H+]3-

is present with higher relative abundance. Although we cannot distinguish between the positional isomers 3a and 7a it is apparent that the [ds+porphyrin-4H++CH3COO-]4- adducts for this pair and for porphyrin 8a (N-decylpyridinium-4-yl), are less strongly bound when compared with the same adduct ions of the other

Table 2. Fluorescence (Φfl) quantum yields of compounds 3a,7a, 8a, 9a, 13a and 14a in DMF

Compound 3a 7a 8a 9a 13a 14aaΦfl 0.05 0.05 0.05 0.06 0.00 0.00

a Absorbance of all samples was 0.05 at λexc = 532 nm.

Table 3. Mass to charge ratio and ionic current intensities of the observed adducts

Porph

[ds+porph-5H+]4- [ds+porph-4H++CH3COO-]4- [ds+porph-4H+]3- [ds+porph-3H++CH3COO-]3-

m/z I I m/z I I m/z I I m/z I I′

3a 1387.05 38 15 1402.55 20 0 1849.73 18 40 1870.06 12 30

7a 1387.05 28 11 1402.55 13 0 1849.73 13 23 1870.06 11 18

8a 1418.58 20 9 1434.08 12 0 1891.78 14 9 1912.11 10 0

9a 1444.07 34 10 1459.57 16 10 1925.76 20 20 1946.10 0 0

13a 1399.55 8 11 1415.05 27 15 1866.40 0 15 1886.73 16 27

14a 1399.55 15 13 1415.05 11 13 1866.40 11 25 1886.73 0 21

I — ionic current intensities at a cone voltage of 25 V; I — ionic current intensities at a cone voltage of 45 V.



three porphyrins, since their adduct ions did not totally disappear but their abundance decreases. As indicated above the adduct ions [ds+porphyrin-4H++CH3COO-]4-

can be the main source of the [ds+porphyrin-4H+]3- ions, not only for porphyrin 13a (N-methylquinolinium-2-yl), but also for porphyrins 3a and 7a. The differences in behavior between the other pair of isomers 13a and 14aare less easy to rationalize, specially the formation of the[ds+porphyrin-3H++CH3COO-]3- adduct ion at a higher cone voltage for porphyrin 14a.

The product ion spectra (precursor ion production, MS/MS or MS2) of the [ds+porphyrin-5H+]4- adduct ions were acquired by mass selecting them with the first analyzer, at the cone voltage value of 25 V for which their relative abundances were higher. The exception was porphyrin 13a (N-methylquinolinium-2-yl), due to the low relative abundance of the [ds+porphyrin-5H+]4-

precursor ion. However as, for this compound, the adduct ion [ds+porphyrin-4H++CH3COO-]4- is formed with higher relative abundance at this voltage, its MS2

spectrum was also acquired. Unzipping of the double strand, with formation of the [ss+porphyrin-3H+]2-

and [ss-2H+]2- ions (ss = single strand, monoisotopic mass = 2410.4399 Da) is the main feature in the MS2

spectra of the [ds+porphyrin-5H+]4- adduct ions. Similarly the [ds+porphyrin-4H++CH3COO-]4- adduct ion of porphyrin 13a fragments with formation of the [ss+porphyrin-2H++CH3COO-]2- and [ss-2H+]2- species.

In Fig. 3 the product ion spectrum of the [ds+porphyrin-5H+]4- adduct ion is shown for porphyrin 3a (N-methylpyridinium-2-yl).

The same type of behavior was observed by us for the [ds+porphyrin-5H+]4- adduct ions of the same duplex oligonucleotide, with the monocharged compound 5-(1-methyl- pyridynium-4-yl)-10,15,20-triphenylpor-phyrin [21]. Thus, we may conclude that the porphyrins are probably bound to the outside of the duplex structure through coulombic attractions with the phosphate ions. The formation of duplex/porphyrin/acetate ion adducts is also consistent with external binding. The acetate ion is probably electrostatically bound to the nitrogen bearing the positive charge in the substituent group.

The similarity of the MS2 spectra lead us to acquire the pseudo MS3 spectra,[ds+porphyrin-5H+]4- [ss+porphyrin-3H+]2-

product ions, for all the porphyrins, with the exception of porphyrin 13a(N-methylquinolinium-2-yl), in order to obtain further information on the [ds+porphyrin-5H+]4-

adduct ions.The first step was to increase the cone

voltage in order to generate in the source the [ss+porphyrin-3H+]2- ions, through the

unzipping of the [ds+porphyrin-5H+]4- adduct ions. Complete disappearance of the [ds+porphyrin-5H+]4-

signal, with formation of the [ss+porphyrin-3H+]2- and [ss-2H+]2- ions, was observed for porphyrins 3a, 7a and 14a for a cone voltage of 45 V. For porphyrin 9a the complete disappearance of the [ds+porphyrin-5H]4- ion was observed for a cone voltage of 50 V and for porphyrin 8a for a cone voltage of 55 V. This behavior can be observed in Fig. 4 for porphyrin 7a. At the described cone voltages, the [ss+porphyrin-3H+]2- ions were formed with relative abundances high enough to allow the acquisition of their MS2 spectra.

The [ss+porphyrin-3H+]2- ions were then mass selected and subjected to collisions with an inert gas (collision energy of 50 eV). The spectra obtained were very similar for porphyrins 3a, 7a, 8a and 14a. The signal of the [ss+porphyrin-3H+]2- ion for porphyrin 9a (N-sugar-pyridinium-4-yl) was low and it was not possible to perform the MS2 experiments.

The overall results confirm our previous hypothesis that the positive charge is the main contributor for the type and strength of porphyrin/duplex interactions [21, 22]. Nevertheless a general ranking of the binding strengths can be proposed based on the cone voltages values needed to completely unzip the [ds+porphyrin-5H+]4-

adduct ions: 3a, 7a and 14a < 9a < 8a. The observed trend points to a contribution of the more easily distorted N-decylpyridinium-4-yl (8a) and N-sugar-pyridinium-4-yl (9a) substituent groups in adduct formation.

Fig. 2. Mass spectrum of the solution of the oligonucleotide ds4GC and porphyrin 9a at a cone voltage of 25 V

Fig. 3. Product ion spectrum of the solution of the oligonucleotide ds4GC and porphyrin 3a at a cone voltage of 25 V



UV-vis experiments

In an attempt to relate the behavior of the adducts in the gas-phase to their behavior in solution, UV-vis spectrophotometric experiments were performed for all the cationic porphyrins. The alterations in the Soret absorption band of the porphyrins were analysed when the GC duplex was added. A general trend of hypochromism, although modest, was observed for all the porphyrins in 0.5 M ammonium acetate solutions. However, it was established that the ammonium acetate solutions on its own produced the same sort of hypochromic shift. Bathochromic shifts, which would be an indication of the intercalative binding to the duplex [59–62], were not observed. In the case of external or groove binding the changes in the UV-vis absorption spectra are not significant, because of the less direct contact between

-systems, when compared with intercalation [61, 63]. These data confirms that intercalation does not occur for the buffer concentrations (0.5 M) used to perform the mass spectrometric experiments.

EXPERIMENTAL

General

Melting points were measured on a Reichert Thermovar apparatus fitted with a microscope and are uncorrected. 1H and 13C solution NMR spectra were recorded on Bruker Avance 300 and 500 spectrometers at 300.13/500.13 and 75.47/126.76 MHz, respectively. DMSO-d6, CDCl3 or a mixture of CDCl3/MeOH-d4

were used as solvent and TMS as internal reference; the chemical shifts are expressed in δ (ppm) and the coupling constants (J) in Hertz (Hz). Unequivocal 1Hassignments were made with aid of 2D COSY (1H/1H),while 13C assignments were made on the basis of 2D HSQC (1H/13C) and HMBC experiments (delay for long-range J C/H couplings were optimized for 7 Hz). Mass spectra and HRMS were recorded on VG AutoSpec Q and M mass spectrometers using CHCl3 as solvent and NBA as matrix (unless otherwise stated). Elemental

analyses were performed in a Leco 999 CHN analyzer. The UV-vis spectra were recorded on an Uvikon 922 spectrophotometer using DMSO as solvent. Column chromatography was carried out using silica gel (Merck, 35–70 mesh). Analytical TLC was carried out on precoated sheets with silica gel (Merck 60, 0.2 mm thick). All the chemicals were used as supplied except 2- and 4-methylpyridine, which were distilled from KOH under an inert atmosphere and stored over KOH. Other solvents were purified or dried according to the literature procedures [64]. Both 1,2- and 1,4-dimethylpyridinium iodides (2 and 4) as

well as 1,2- and 1,4-dimethylquinolinium iodides (11 and 12) were prepared according to the literature procedures [29, 30].

Synthesis of the pyridinium salts

1-decyl-4-methylpyridinium iodide 5. 1-iododecane(1.26 g, 4.70 mmol) was added to 4-methylpyridine (0.50 mL, 5.15 mmol) in a round bottomed flask equipped with a condenser, at room temperature while stirring. The mixture was stirred at 80 °C for 6 h, then it was allowed to cool at room temperature, and Et2O was added. The sticky precipitate formed was decanted and washed several times with Et2O. The desired product was obtained as a pink oil (1.45 g, 84% yield) after purification of the crude by alumina column chromatography (eluent: CH2Cl2).

1HNMR (300 MHz, CDCl3): δ, ppm 9.17 (d, J = 6.5 Hz, 2H, H-2,6), 7.89 (d, J = 6.5 Hz, 2H, H-3,5), 4.86 (t, J = 7.4 Hz, 2H, N-CH2R), 2.69 (s, 3H, 4-CH3), 2.01 (quint, 2H, J = 7.4 Hz, N-CH2CH2R), 1.33–1.24 [m, 14H, R(CH2)7CH3], 0.87 [t, 3H, J = 6.7 Hz, R(CH2)9CH3].13C NMR (75 MHz, CDCl3): δ, ppm 159.0 (C-4), 143.9 (C-2,6), 128.8 (C-3,5), 61.5 (N-CH2R), 31.8, 31.7, 29.4, 29.3, 29.2, 29.0, 26.0, 22.6 and 22.4 (C-decyl group and 4-CH3), 14.1 (N(CH2)9CH3). ESI(+)-HRMS: m/zC16H28N, calcd. 234.2216; found 234.2211.

1-(1,2:3,4-di-O-isopropylidene-α-D-galacto-py-ranos-6-yl)-4-methylpyridinium iodide 6. 6-iodo-1,2:3, 4-di-O-isopropylidene-α-D-galactopyranose (0.20 g, 0.54 mmol) was added to 4-methylpyridine (0.50 mL, 5.15 mmol), in a round bottomed flask at room temperature while stirring. The mixture was stirred at 120 °C for 4 h, then it was allowed to cool at room temperature and light petroleum was added. The pale pink precipitate formed was decanted and washed several times with Et2O. The desired product was obtained after crystallization from CH2Cl2/petroleum ether (0.24 mg, 96% yield); mp > 300 °C. 1H NMR (300 MHz, CDCl3): δ, ppm 9.28 (d, J = 6.5 Hz, 2H, H-2,6), 7.75 (d, J = 6.5 Hz, 2H, H-3,5), 5.68 (dd, J = 2.4 and 13.6 Hz, 1H, H-6 ), 5.43 (d, J = 4.9 Hz, 1H, H-1 ), 4.73 (dd, J = 1.6 and 7.9 Hz, 1H, H-4 ), 4.67 (dd, J = 2.3 and 7.9 Hz, 1H, H-3 ), 4.62 (dd, J = 9.5 and 13.6 Hz, 1H, H-6 ), 4.36–4.32 (m, 1H,

Fig. 4. Mass spectrum of the solution of the oligonucleotide ds4GC and porphyrin 7a at a cone voltage of 45 V



H-5 ), 4.32 (dd, J = 2.3 and 4.9 Hz, 1H, H-2 ), 2.69 (s, 3H, 4-CH3), 1.42, 1.40, 1.34 and 1.25 [4s, 12H, 2 × C(CH3)2]. 13C NMR (75 MHz, CDCl3): δ, ppm 159.4 (C-4), 144.7 (C-2,6), 127.9 (C-3,5), 109.7 and 109.2 [2 × C(CH3)2], 96.1 (C-1 ), 70.6, 70.45 and 70.40 (C-2 , 3 , 4 ), 67.3 (C-5 ), 60.6 (C-6 ), 26.2, 25.9, 24.7 and 24.1 [2 × C(CH3)2], 22.4 (4-CH3). FAB(+)-MS: m/z 336 [M − I + H]+. Elemental analysis: calcd. for C18H26INO5: C, 46.66; H, 5.66; N, 3.02; found (%) C, 46.55; H, 5.84; N, 2.98.

Synthesis of the (porphyrin-2-yl)vinylpyridinium salts

Typical procedure: zinc(II) complex of 1-methyl-2-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)vinyl]pyridinium chloride 3b. 1,2-dimethylpyridinium iodide (11.5 mg, 0.05 mmol) was added to a solution of porphyrin 1b (15.7 mg, 0.02 mmol) in THF/MeOH (2:1, 4.5 mL) containing K2CO3 (20.6 mg, 0.14 mmol). The reaction mixture was stirred for 1 h at 30 °C under a N2 atmosphere. The mixture was then allowed to cool at room temperature, and the solvents were evaporated under reduced pressure. The crude was taken in CH2Cl2

and washed with water. The organic phase was dried (Na2SO4) and the solvent was evaporated. The desired product was obtained after flash chromatography on silica gel (eluent: CH2Cl2/MeOH, 95:5). The fraction containing the desired product was concentrated and washed several times with brine, the organic layer was dried (Na2SO4) and the solvent was evaporated. The porphyrin was crystallized from CH2Cl2/hexane (7.6 mg, 43% yield); some 2-formyl-TPP was recovered (4.7 mg, 30%); mp > 300 °C. 1H NMR (300 MHz, CDCl3/CD3OD): δ, ppm 8.81–8.76 (m, 5H, H-β), 8.75 (d, 1H, J = 4.8 Hz, H-β), 8.66 (d, 1H, J = 4.8 Hz, H-β), 8.36–8.14 (m, 9H, H-Ph-o and H-5), 8.03–7.73 (m, 15H, H-Ph-m,p and H-3,4,6), 7.27 (d, 1H, J = 11.9 Hz, H-2 ),6.97 (d, 1H, J = 11.9 Hz, H-1 ), 4.09 (s, 3H, N-CH3).13C NMR (75 MHz, CDCl3/CD3OD): δ, ppm 152.6 (C-1), 152.55 (C-Ph), 152.51 (C-Ph), 151.3 (C-Ph), 151.2 (C-Ph), 151.1, 144.34, 144.26 (C-5), 143.94, 143.86 (C-3), 143.0, 142.0, 140.5 (C-2 ), 139.8, 136.3 (C-2 ),134.9 and 133.6 (C-Ph-o), 133.32, 133.27, 133.1, 132.8, 132.7, 129.7; 129.0, 128.3, 128.0, 127.7, 127.4, 127.0 (C-Ph-m,p); 126.3 (C-4,6), 122.0, 120.9, 119.5 (C-1 ),46.3 (N-CH3). UV-vis (CHCl3): λmax, nm (%) 415 (100), 402 (71.1), 568 (15.3), 629 (14.4). ESI(+)-HRMS: m/zC52H36N5Zn: calcd. 794.2262; found 794.2276.

Zinc(II) complex of 1-methyl-4-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)vinyl]pyridinium chloride 7b. The synthesis was accomplished according to the procedure described for 3b. Yield 6.1 mg (40%); some 2-formyl-Zn-TPP was recovered (2.1 mg, 19%); mp > 300 °C. 1H NMR (300 MHz, CDCl3/CD3OD): δ, ppm 9.19 (s, 1H, H-3 ), 8.86–8.82 (m, 4H, H-β), 8.80 (d, 1H, J = 4.7 Hz, H-17 or 18 ), 8.70 (d, 1H, J = 4.7 Hz, H-17or 18 ), 8.28–8.25 (m, 2H, H-Ph-o), 8.22–8.17 (m, 4H, H-Ph-o), 8.00 (d, 2H, J = 7.3 Hz, H-20-Ph-o), 7.88–7.72

(m, 11H, H-Ph-m,p and H-3,5), 7.66 (t, 1H, J = 7.3 Hz, H-20-Ph-p), 7.52 (t, 2H, J = 7.3 Hz, H-20-Ph-m), 7.16 (d, 1H, J = 15.2 Hz, H-2 ), 7.06 (d, 1H, J = 15.2 Hz, H-1 ),6.88 (d, 2H, J = 6.2 Hz, H-2,6), 3.55 (s, 3H, N-CH3).13C NMR (75 MHz, CDCl3/CD3OD): δ, ppm 154.5 (C-1), 152.1 (C-Ph), 152.0, 151.9 (C-Ph), 151.5 (C-Ph), 150.9 (C-Ph), 147.9, 146.7, 144.3, 144.0 (C-3,5), 143.9, 143.6, 139.5 (C-2 ), 139.4, 135.3 and 135.2 (C-Ph-o), 133.2, 133.1, 133.0, 130.9, 132.8, 132.5 (C-3 ), 132.2, 128.8 (C-20 -Ph-p); 128.4, 128.2 (C-5 ,10 ,15 -Ph-m,p);127.6 (C-20 -Ph-m); 127.3, 127.2 (C-5 ,10 ,15 -Ph-m,p); 123.4 (C-2,6), 122.3 (C-1 ), 121.8, 120.6, 46.8 (N-CH3). UV-vis (CHCl3): λmax, nm (%) 417 (100), 491 (57.3), 633 (22.4). ESI(+)-HRMS: m/z C52H36N5Zn:calcd. 794.2262; found 794.2282.

1-decyl-4-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)vinyl]pyridinium chloride 8a. 1-decyl-4-methylpyridinium iodide (30.3 mg, 0.08 mmol) was added to a solution of porphyrin 1a (26.1 mg, 0.04 mmol) in THF/MeOH (2:1, 4.5 mL) containing K2CO3 (34.0 mg, 0.25 mmol). The reaction mixture was stirred at 60 °C,under a N2 atmosphere, for 9 h (total time). New portions of 1-decyl-4-methylpyridinium iodide were added to the reaction mixture (additions after 3, 5 and 7 h, 4 equiv. per addition). The mixture was then allowed to cool at room temperature, and the solvents were evaporated under reduced pressure. The crude was taken in CH2Cl2

and washed with water and with brine, the organic phase was dried (Na2SO4) and the solvent was evaporated. The desired product was obtained after flash chromatography on silica gel (eluent: CH2Cl2/MeOH 20/1). The fraction containing the desired product was concentrated and washed several times with brine, the organic layer was dried (Na2SO4) and the solvent was evaporated. The porphyrin was crystallized from CH2Cl2/hexane (12.1 mg, 30% yield); mp 275–278 °C. 1H NMR (500 MHz, CDCl3): δ, ppm 9.09 (s, 1H, H-3 ), 8.99 (d, 2H, J = 6.4 Hz, H-3,5), 8.85–8.77 (m, 6H, H-β), 8.23–8.18 (m, 8H, H-Ph-o), 8.03 (t, 1H, J = 7.4 Hz, 20 -H-Ph-p), 7.91–7.74 (m, 11H, H-Ph-m and 5 ,10 ,15 -H-Ph-p), 7.54 (d, 1H, J = 6.4 Hz, H-2,6), 7.46 (d, 1H, J = 16.0 Hz, H-2 ), 7.25 (d, 1H, J = 16.0 Hz, H-1 ), 4.77 (t, 2H, J = 7.5 Hz, NCH2R),2.04–2.02 (m, 2H, NCH2CH2R), 1.38–1.25 [m, 14H, R(CH2)7CH3)], 0.86 [t, 3H, J = 6.5 Hz, N(CH2)9CH3],-2.53 (s, 2H, NH). 13C NMR (126 MHz, CDCl3/CD3OD):δ, ppm 154.2 (C-1), 143.7 (C-3,5), 141.94 (C-Ph), 141.92 (C-Ph), 141.7 (C-Ph), 141.5 (C-Ph), 138.3 (C-2 ); 134.7, 134.6, 134.52, 134.50 (C-Ph-o); 129.4 (C-20 -Ph-p);128.2, 127.9, 127.7, 126.9, 126.8 (C-5 ,10 ,15 –Ph-pand C-Ph-m); 123.6 (C-3,5), 122.7 (C-1 ), 121.0, 120.63, 120.57, 119.6, 61.0 (NCH2R), 31.9 (NCH2CH2R), 31.8, 31.7, 26.1, 22.7, 22.6, 14.1 (N(CH2)9CH3). UV-vis (DMSO): λmax, nm (log ε) 431 (4.14), 523 (2.77), 579 (2.30), 599 (2.17), 666 (1.22). ESI(+)-HRMS: m/zC61H56N5: calcd. 858.4530; found 858.4562.

1-(1,2:3,4-di-O-isopropylidene-α-D-galactopyranos-6-yl)-4-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)-vinyl]



pyridinium chloride 9a. Compound 6 (34.3 mg, 0.07 mmol) was added to a solution of porphyrin 1a (24.8 mg, 0.04 mmol) in THF/MeOH (2:1, 4.5 mL) containing K2CO3 (32.7 mg, 0.24 mmol). The reaction mixture was stirred at 60 °C, under a N2 atmosphere, for 3 h (total time). New portions of 6 were added to the reaction mixture after 1 h and 2 h (2 equiv. per addition). The mixture was then allowed to cool at room temperature, and the solvents were evaporated under reduced pressure. The crude was taken in CH2Cl2 and washed with water and with brine, the organic phase was dried (Na2SO4) and the solvent was evaporated. The compound was purified by flash chromatography on silica gel (eluent: CH2Cl2/MeOH 9:1). The fraction containing compound 9a was concentrated and washed several times with brine. The organic layer was dried (Na2SO4) and the solvent was evaporated. The porphyrin was crystallized from CH2Cl2/hexane (20.5 mg, 53% yield); mp 266–268 °C. 1H NMR (300 MHz, CDCl3): δ, ppm 9.18 (d, 2H, J = 6.7 Hz, H-3,5), 9.09 (s, 1H, H-3 ), 8.86–8.77 (m, 6H, H-β), 8.25–8.19 (m, 8H, H-Ph-o), 7.96 (t, 1H, J = 7.6 Hz, 20 -H-Ph-p), 7.87–7.74 (m, 11H, H-Ph-m and 5 ,10 ,15 -H-Ph-p), 7.491 (d, 1H, J = 6.7 Hz, H-2,6), 7.489 (d, 1H, J = 15.4 Hz, H-2 ), 7.24 (d, 1H, J = 15.4 Hz, H-1 ), 5.64 (dd, 1H, J = 2.2 and 13.7 Hz, Gal-H-6), 5.50 (d, 1H, J = 4.8 Hz, Gal-H-1), 4.79 (dd, 1H, J = 1.4 and 8.0 Hz, Gal-H-4), 4.72 (dd, 1H, J = 2.3 and 8.0 Hz, Gal-H-3), 4.56 (dd, 1H, J = 9.4 and 13.7 Hz, Gal-H-6), 4.39–4.37 (m, 1H, Gal-H-5), 4.35 (dd, 1H, J = 2.3 and 4.8 Hz, Gal-H-2), 1.49, 1.44, 1.38, 1.28 [4s, 12H, 2 × C(CH3)2], –2.53 (s, 2H, NH). 13C NMR (75 MHz, CDCl3): δ, ppm 154.4 (C-1), 144.6 (C-3,5), 142.0, 141.7, 141.5, 138.5 (C-2 ), 134.8, 134.6, 134.53, 134.51, 132.9, 131.2, 129.9, 129.85, 129.77, 129.3, 128.2, 127.9, 127.6, 127.5 126.9, 126.8, 122.8 (C-2,6 and C-1 ), 121.0, 120.7, 120.6, 119.5, 109.6 [C(CH3)2], 109.3 [C(CH3)2], 96.1 (Gal-C-1), 70.7, 70.6, 70.5 (Gal-C-2,3,4), 67.6 (Gal-C-5), 60.3 (Gal-C-6), 26.3, 25.9, 24.8 and 24.1 [C(CH3)2]. UV-vis (DMSO): λmax, nm (log ε) 431 (4.89), 525 (4.11), 574 (3.86), 600 (3.86), 663 (3.72). ESI(+)-HRMS: m/z C63H54N5O5: calcd. 960.4119; found 960.4167.

1-(α,β-D-galactopyranos-6-yl)-4-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)vinyl]pyridinium chloride 10a. A solution of 9a (7.5 mg, 6.8 μmol) in TFA/H2O(9:1, 1.0 mL) was stirred for 30 min at room temperature. The reaction mixture was then partitioned between CHCl3

(10 mL) and an ice-cold aqueous Na2CO3 saturated solution (10 mL). The organic layer was washed with water, dried (Na2SO4) and the solvent was evaporated. The desired product was obtained after flash chromatography on silica gel using CH2Cl2/MeOH 9:1 as the eluent. The fraction containing the desired product was concentrated and washed several times with brine, the organic layer was dried (Na2SO4) and the solvent was evaporated. The porphyrin was crystallized from CH2Cl2/petroleum ether (4.6 mg, 66% yield); mp > 300 °C. 1H NMR (300 MHz, DMSO-d6): δ, ppm 9.16 (s, 1H, H-3 ), 8.92 (d, 2H, J = 6.3 Hz, H-3,5), 8.87–8.73 (m, 6H, H-β), 8.28–8.21 (m, 8H, H-Ph-o),

7.89–7.84 (m, 12H, H-Ph-m,p), 7.75 (d, 1H, J = 6.3 Hz, H-2,6), 7.66 (d, 1H, J = 16.3 Hz, H-2 ), 7.41 (d, 1H, J = 16.3 Hz, H-1 ), 6.73 (d, 1H, J = 6.9 Hz, Gal-H-1), δ 5.08–4.91 and 4.86–4.54 (2m, 7H, Gal-H), -2.59 (s, 2H, NH). ESI(+)−HRMS: m/z C57H46N5O5: calcd. 880.3493; found 880.3491.

Synthesis of (porphyrin-2-yl)vinylquinolinium salts

Typical procedure: 1-methyl-4-[2-(5,10,15,20-tetraph-enylporphyrin-2-yl)vinyl]quinolinium chloride 13a. 1,2-dimethylquinolinium iodide (13.7 mg, 0.05 mmol) was added to a solution of porphyrin 1a (30 mg, 0.03 mmol) in pyridine (2 mL). The resulting mixture was left overnight at 60 °C under a N2 atmosphere. After that, another portion of 1,2-dimethylquinolinium iodide (13.6 mg) was added and stirring was continued through another day. The addition was repeated again and the reaction mixture was left for an additional day. The solvent was evaporated under reduced pressure, the crude was taken in CH2Cl2 and the desired product was obtained after flash chromatography on silica gel (CH2Cl2/MeOH 9:1). The fraction containing the desired product was concentrated and washed several times with brine, the organic layer was dried (Na2SO4) and the solvent was evaporated. The porphyrin was crystallized from CH2Cl2/hexane (18.7 mg, 49% yield); 2-formyl-TPP was recovered (24.4 mg, 81%); mp 195.1–197.8 °C. 1H NMR (300 MHz, CDCl3/CD3OD): δ, ppm 9.30 (s, 1H, H-3 ), 8.87–8.76 (m, 6H, H-β), 8.59 (d, 1H, J = 8.9 Hz, H-8), 8.53 (d, 1H, J =9.0 Hz, H-5), 8.28–8.05 (m, 10H, H-3,6 and H-Ph-o),7.91–7.75 (m, 12H, H-Ph-m,p), 7.71 (AB, 2H, J = 15.5 Hz, H-1 ,2 ), 7.31 (d, 1H, J = 8.9 Hz, H-7), 7.29 (d, 1H, J= 8.3 Hz, H-4), 4.61 (s, 3H, N-CH3), 2.49 (s, 2H, NH).13C NMR (75 MHz, CDCl3/CD3OD): δ, ppm 156.0 (C-1), 143.5 (C-8 and C-2 ), 142.3 (C-Ph), 141.6 (C-Ph), 141.5 (C-Ph), 141.2 (C-Ph), 139.4 (C-4a), 136.6 (C-3), 135.9, 134.7 (C-β), 134.6 (C-β), 134.4 (C-3 ), 129.9 (C-6), 129.6 (C-β), 129.4 (C-β); 128.7, 128.3, 127.9, 127.7, 127.6, 127.5, 127.4, 126.9, 126.8, 126.7 (C-Ph-o,m,p);126.6 (C-8a), 125.8, 122.0, 121.6 (C-4), 120.7, 120.5, 120.3 (C-7), 119.1 (C-5), 117.8 (C-1 ), 40.4 (N-CH3).UV-vis (DMSO): λmax, nm (log ε) 428 (4.13), 492 (3.57), 562 (4.07), 600 (4.13), 629 (3.67).

Zinc(II) complex of 1-methyl-2-[2-(5,10,15,20-tetra-phenylporphyrin-2-yl)vinyl]quinolinium chloride 13b. The synthesis was accomplished according to the procedure described for 13a. Yield 10.0 mg (38%); mp 289–292 °C. 1H NMR (300 MHz, DMSO-d6): δ, ppm 9.38 (s, 1H, H-3 ), 8.85–8.70 (m, 6H, H-β), 8.44–7.41 (m, 24H, H-Ph and H-3 to H-8), 7.05 (d, 1H, J = 9.1 Hz), 4.29 (s, 3H, N-CH3). ESI(+)-HRMS: m/z C56H38N5Zn:calcd. 844.2413; found 844.2414.

1-methyl-4-[2-(5,10,15,20-tetraphenylporphyrin-2-yl)vinyl]quinolinium chloride 14a. The synthesis was accomplished according to the procedure described for 13a. Yield 8.4 mg (22%); 2-formyl-TPP was recovered (20.2 mg, 66%); mp 219.7–221.1 °C decomp. 1H NMR



(300 MHz, CDCl3/CD3OD): δ, ppm 10.27 (d, 1H, J = 6.2 Hz, H-3), 9.21 (s, 1H, H-3 ), 8.88–8.80 (m, 6H, H-β), 8.77 (AB, 2H, J = 4.9 Hz, H-12 ,13 ), 8.58 (d, 1H, J =8.5 Hz, H-8), 8.32–8.29 (m, 2H, H-20 -Ph-o), 8.23–8.17 (m, 6H, H-5 ,10 ,15 -Ph-o), 8.14–8.12 (m, 2H, H-5,6), 7.96 (d, 1H, J = 15.5 Hz, H-1 ), 7.98–7.90 (m, 1H, H-7), 7.88–7.72 (m, 12H, H-Ph-m,p), 7.74 (d, 1H, J = 15.5 Hz, H-2 ), 7.45 (d, 1H, J = 6.2 Hz, H-2), 4.75 (s, 3H, N-CH3), -2.51 (s, 2H, NH). 13C NMR (126 MHz, CDCl3/CD3OD): δ, ppm 153.5 (C-1), 148.7 (C-3), 142.1 (C-Ph), 141.7 (C-Ph), 141.6 (C-Ph), 141.5 (C-Ph), 140.0 (C-2 and C-2 ), 138.7 (C-4a), 135.0 (C-6), 134.7 (C-20 -Ph-o), 134.6 and 134.5 (C-5 ,10 ,15 -Ph-o), 133.1 (C-12 ,13 ), 132.4 (C-3 ), 130.2 (C-β), 129.1 (C-7), 128.1 and 127.9 (C-5 ,10 ,15 -Ph-m,p), 126.9 and 126.8 (C-20 -Ph-m,p), 126.4 (C-8a), 125.9 (C-8), 120.9, 120.7, 120.5, 119.9, 119.0 (C-1 ), 118.2 (C-5), 116.4 (C-2), 44.9 (N-CH3). UV-vis (DMSO): λmax, nm (log ε) 414 (4.42), 461 (4.24), 529 (3.69), 583 (3.47), 658 (3.12).

Zinc(II) complex of 1-methyl-4-[2-(5,10,15,20-tetraph-enylporphyrin-2-yl)vinyl]quinolinium chloride 14b. The synthesis was accomplished according to the procedure described for 13a. Yield 12.0 mg (45%); mp > 300 °C. 1H NMR (300 MHz, DMSO-d6): δ, ppm 9.39 (d, J = 6.5 Hz, 1H, H-3), 9.22 (s, 1H, H-3 ), 8.91–8.68 (m, 6H, H-β), 8.58 (d, 1H, J = 6.1 Hz, H-1 ), 8.29–7.65 (m, 25H, H-Ph, H-2 and H-5 to H-8), 7.37 (d, 1H, J = 6.5 Hz, H-2), 3.88 (s, 3H, N-CH3). ESI(+)-HRMS: m/z C56H38N5Zn: calcd. 844.2413; found 844.2428.

Photostability

In a typical experiment, 2 mL of a solution of the porphyrin (1 μM) in DMF in a glass cuvette was irradiated with white light (50 mW/cm2) at room temperature and under gentle stirring for different periods of time. The intensity of the Soret band was registered at different intervals of time by UV-visible spectroscopy, and the photostability was expressed as It/I0 (%) (It = intensity of the band at given time of irradiation, I0 = intensity of the band before irradiation).

Oxygen singlet

A solution containing DPBF (50 μM) and photosensitizer (0.5 μM) in DMF was irradiated in a glass cuvette at room temperature using a white light with a fluence rate at 50 mW/cm2. The light beam was filtered using an appropriate cut-off filter (<540 nm) and the absorption of the stirred solution was monitored at 415 nm at irradiation intervals of 10 sec for the 1st min and of 30 sec for the remaining 5 min.

Fluorescence

The fluorescence of the compounds was measured at 20 °C in 1 cm × 1 cm quartz optical cells with a computer controlled Jobin Yvon FluoroMax-3 spectrofluorometer. Fluorescence quantum yields Φfl were determined

relatively to 5,10,15,20-tetraphenylporphyrin in DMF used as the reference; Φfl (TPP) = 0.11 [47]. The samples and the reference in DMF were excited at 532 nm (OD532 nm = 0.05) and emission was monitored from 600 to 750 nm. The fluorescence quantum yield (Φfl) of porphyrins were calculated by comparison of the area below the corrected emission spectrum with that of tetraphenylporphyrin (TPP) as a fluorescence standard, exciting at = 532 nm [47].

Mass spectrometry and UV-vis experiments

The single-stranded oligonucleotide GCGCGCGC (ss4GC, monoisotopic mass = 2410.4399 Da) was purchased from Eurogentec (Liege, Belgium). The double-stranded oligonucleotide dsGCGCGCGC (ds4GC) was formed from a stock solution: 100 μM solution of the single strand in ammonium acetate 0.5 M heated to 85 °C and left to cool overnight. Seventy-five microliters of porphyrin solution in methanol (approximately 1.0 × 10-5 M) was then added to 25 μL of oligonucleotide solution. Electrospray ionization mass spectra were acquired with a Micromass Q-Tof 2 (Micromass, Manchester, UK), operating in the negative mode, equipped with a Z-spray source, an electrospray probe and a syringe pump. Source and desolvation temperatures were 80 °C and 150 °C,respectively. Capillary voltage was 2600 V. The spectra were acquired at a nominal resolution of 9000 and at cone voltages between 25 and 55 V. Nebulisation and collision gases were N2 and Ar respectively. The samples were introduced at a 10 μL.min-1 flow. MS/MS spectra were acquired by selecting the ion of interest with the quadrupole and using the hexapole as collision cell with energies from 25 to 55 eV.

UV-vis absorption spectra were obtained using an Uvikon 922 spectrophotometer (Kontron Instruments Ltd, UK) and scanning from 350 to 800 nm, a 1 cm path length cuvette was utilised in all experiments. For the UV-vis measurements, porphyrin samples were dissolved in dimethylsulfoxide/methanol and added to solutions of the double-stranded oligonucleotides in ammonium acetate 0.5 M.

CONCLUSION

New cationic porphyrins with different substituents on the N-terminus of pyridinium and quinolinium rings were efficiently prepared in moderate yields. The results obtained with the DPBF photooxidation indicated that cationic derivatives 3a and 9a are efficient singlet oxygen generators, possibly justifying their significant in vitroactivity against herpes simplex virus type 1 [19]. On the contrary, compounds 13a and 14a, which contain in their structures the quinolinium moiety, do not produce singlet oxygen. The fluorescence studies demonstrated that all the studied derivatives have shown a weak fluorescence emission revealing that these compounds most likely



decay by other radiative or non-radiative processes. Concerning the possible non-covalent interactions of the synthesized porphyrins with d(4GC), the data indicate that the porphyrins are probably bound to the outside of the duplex structure through coulombic attractions with the phosphate ions. The overall results confirm our previous hypothesis that the positive charge is a main contributor for the type and strength of porphyrin/duplex interactions. A general binding strength order is proposed (porphyrins 3a, 7a and 14a < 9a < 8a).

Acknowledgements

Thanks are due to the University of Aveiro, to the Portuguese Foundation for Science and Technology (FCT) and FEDER for funding the Organic Chemistry Research Unit and the Portuguese National NMR Network. E.M.P. Silva (Post-Doc grant SFRH/BPD/66961/2009), and C.I.V. Ramos (PhD grant SFRH/BD/30755/2006) thank FCT for their grants.

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DOI: 10.1142/S1088424612004458



INTRODUCTION

Diabetes mellitus (DM) is a group of important metabolic syndromes that results from an absolute, or relative, deficiency of insulin secretion and/or its action, and it is widely accepted as one of the leading causes of death and disability worldwide. It can affect nearly every organ system in the body and lead to blindness, end-stage renal disease, lower extremity amputations, increased risk of stroke, ischaemic heart disease, peripheral vascular disease and neuropathy. Based on its type, the treatment for DM involves either daily injections of exogenous insulin (most common for type 1, i.e. insulin-dependent DM) or oral administration of hypoglycemic

drugs, such as sulfonylureas [1], metformin [2], alpha-glucosidase inhibitors, thiazolidinediones, meglitinides and nateglinide [3, 4], and in a combination therapy for type 2 (non-insulin-dependent) DM [5]. However, this approach is not satisfactory for a large proportion of patients; hence, there have been continued efforts towards developing new hypoglycemic drugs with high potency, but little or no side-effects. It has been established that vanadium compounds exert an insulin-mimetic action in both in vitro and in vivo systems, including their ability to improve glucose homeostasis and insulin resistance in animal models of DM [6–8]. Several reports have documented vanadium therapy-induced improvements in insulin sensitivity in the liver and muscles of many type 2 diabetic human subjects [9–11].

Since the discovery of the orally active insulin mimetic oxovanadium(IV)-cysteine methyl ester complex for the treatment of streptozotocin (STZ)-induced type 1 diabetic

In vitro insulin-mimetic activity and in vivo metallokinetic

feature of oxovanadium(IV)porphyrin complexes in healthy

rats

Tapan K. Saha*a,b,Yutaka Yoshikawab, Hirouki Yasuib and Hiromu Sakuraib

aDepartment of Chemistry, Jahangirnagar University, Savar, Dhaka 1342, BangladeshbDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Misasagi, Ymashina-ku, Kyoto 607-8414, Japan

Received 7 August 2011Accepted 19 September 2011

ABSTRACT: We prepared [meso-tetrakis(4-carboxylatophenyl)porphyrinato]oxovanadium(IV)tetrasodium, ([VO(tcpp)]Na4), and investigated its in vitro insulin-mimetic activity and in vivo metallokinetic feature in healthy rats. The results were compared with those of previously proposed insulin-mimetic oxovanadium(IV)porphyrin complexes and oxovanadium(IV) sulphate. The in vitro insulin-mimetic activity and bioavailability of [VO(tcpp)]Na4 were considerably better than those of [meso-tetrakis(1-methylpyridinium-4-yl)porphyrinato]oxovanadium(IV)(4+) tetraperchlorate ([VO(tmpyp)](ClO4)4)and oxovanadium(IV) sulphate. On the other hand, [VO(tcpp)]Na4 and [meso-tetrakis(4-sulfonatophenyl)porphyrinato]oxidovanadate(IV)(4-) ([VO(tpps)]) showed very similar in vitro insulin-mimetic activity and in vivo metallokinetic feature in healthy rats. In particular, the order of in vitro insulin-mimetic activity of the complexes was determined to be: [VO(tcpp)]Na4 [VO(tpps)] > ([VO(tmpyp)](ClO4)4 >oxovanadium(IV) sulphate.

KEYWORDS: oxovanadium(IV)porphyrin complex, insulin-mimetic, metallokinetics, free fatty acid, glucose uptake.

*Correspondence to: Tapan K. Saha, email: [email protected], tel: +880 2-7791045-51 extn. 1437, fax: +880 2-7791052


IN VITRO INSULIN-MIMETIC ACTIVITY AND IN VIVO METALLOKINETIC FEATURE OF OXOVANADIUM(IV)PORPHYRIN 115

rats (STZ-rat) in 1990 [12], the therapeutic potential of oxovanadium(IV) complexes has been of great interest to us. We and other researchers have developed several types of oxovanadium(IV) complexes with different equatorial coordination spheres, such as VO(N2S2), VO(N2O2),VO(S2O2), VO(O4), VO(S4) and VO(N

4) [13–26]. These

complexes showed normoglycemic activity in type 1 and type 2 DM animals. Recently, we found that [meso-tetrakis(4-sulfonatophenyl)porphyrinato]oxovanadate(IV)(4-), [VO(tpps)], (Fig. 1), in which the equatorial coordination sphere of oxovanadium(IV) is VO(N4), is a potential insulin-mimetic oxovanadium(IV)porphyrin complex for the treatment of not only STZ-mice but also type 2 diabetic KKAy mice when the complex was introduced by oral gavage [24–26].

These important findings promoted us to develop more active oxovanadium(IV)porphyrin complex for the treatment of diabetic mellitus. In this study, we have newly synthesized and characterized [meso-tetrakis(4-carboxylatophenyl)porphyrinato]oxovanadium(IV) tetrasodium, [VO(tcpp)]Na4, complex and estimated its in vitro insulin-mimetic activity in isolated rat adipocytes treated with epinephrine and in vivometallokinetic features in the blood of healthy rats. Finally, we compare the above results with those of previously reported [VO(tpps)], [meso-tetrakis(1-methylpyridinium-4-yl)porphyrinato]oxovanadium(IV)(4+)tetraperchlorate, [VO(tmpyp)](ClO4)4, complexes and oxovanadium(IV) sulfate as positive controls [24, 27].

EXPERIMENTAL

Materials

All reagents and solvents were commercially available and of the highest grade of purity; hence, they were used without purification. Oxovanadium(IV) sulfate, VOSO4·nH2O, was obtained from Wako Pure Chemical Industries (Osaka, Japan). The ligands meso-tetrakis(4-carboxylatophenyl)porphyrin (H2tcpp),

meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (H2tmpyp), and meso-tetrakis(4-sulfonatophenyl)porphyrin (H2tpps) were purchased from Frontier Scientific Inc. (P.O. Box 31, Logan, UT, USA). VOSO4·nH2Owas standardized complexometrically with ethylenediamine-N,N,N ,N -tetraacetic acid (EDTA), determined to be the trihydrate, and used in all of the experiments. Sephadex LH-20 was obtained from Amersham Pharmacia Biotech (Tokyo, Japan). Bovine serum albumin (fraction V), (±)-epinephrinemonohydrochloride and collagenase were purchased from Sigma Chemical (St. Louis, MO, USA).

Syntheses of the oxovanadium(IV)porphyrin complexes

The oxovanadium(IV) derivative of H2tcpp was prepared according to the method used by Erdman et al. [28] with slight modification. [VO(tpps)] and [VO(tmpyp)](ClO4)4 were synthesized as described previously [24].

[VO(tcpp)]Na4. In a round-bottomed flask fitted with a ground glass joint to a reflux condenser was placed 20 mL of glacial acetic acid, 0.6 g of sodium acetate, 0.86 g (3.99 mmol) of oxovanadium(IV) sulfate (VOSO4·3H2O)and 0.2 g (0.25 mmol) of H2tcpp. The mixture was refluxed until a sample withdrawn with a pipet indicated spectrally that no more complex was being formed. The refluxing solution was then evaporated to approximately 5 mL and cooled in an ice bath. Acetone (20 mL) was added to the remaining solution. The resulting precipitate was redissolved in methanol and reprecipitated with acetone three times. The crude material was purified by gel chromatography (Sephadex LH-20; eluent: H2O).Finally, the aqueous solution was concentrated and dried under high vacuum. The composition of the complex was determined by elemental analyses, UV-visible, infra red (IR) and electron spin resonance (ESR) spectra (Table 1), and then compared with the data from relevant literature [24, 27, 28].

Physical measurements

Elemental analysis was carried out using a Perkin-Elmer 240C elemental analyzer (Wellesley, MA, USA). IR spectra of the solid samples in compressed KBr disks were measured using a Shimadzu FTIR-8100A spectrophotometer (Shimadzu, Kyoto, Japan). UV-visible absorption spectra in aqueous solvents were recorded with a Shimadzu UV-1601PC spectrophotometer equipped with an electronically thermostatic cell holder (Shimadzu); the quartz cell had a path length of 1.0 cm. Before each measurement, the base line of spectrophotometer was calibrated against solvent. ESR spectra were measured using a 1.0 mM solution of the

N

N N

N

V

OR

R

R

R

N CH3ClO4COONa SO3HR = ; ;

[VO(tcpp)]Na4 [VO(tpps)] [VO(tmpyp)](ClO4)4

Fig. 1. Structures of [VO(tcpp)]Na4, [VO(tpps)] and [VO(tmpyp)](ClO4)4


116 T.K. SAHA ET AL.

complexes in water at both room temperature (22 °C)and liquid nitrogen temperature (77 K) by an X-band ESR spectrometer (JES RE1X, JEOL, Tokyo, Japan) under the following conditions: frequency, 9.4 GHz; microwave power, 5.0 mW; modulation frequency, 100 kHz; modulation amplitude width, 0.63 mT; response, 0.03 s; scanning time, 4 min; magnetic field, 340 ± 100 mT; standards, tetracyanoquinodimethane lithium salt (TCNQ-Li) (g = 2.00252) and Mn(II) in MgO (magnetic field between the third and fourth signals due to Mn(II), 8.69 mT). The hyperfine coupling constant (A0), which was estimated as the magnetic field between the MI =-1/2 and 1/2 hyperfine components, and g0 values were obtained from the spectra at room temperature. A//, which was estimated as the mean on the basis of the spectral regions for the MI = -5/2 and -7/2 (A1) and MI = 5/2 and 7/2 (A2) hyperfine components, and g// values were obtained from the spectra at 77 K. The g⊥ and A⊥ values were obtained using the following equations:

g⊥ = 1/2 (3g0 - g//) (1)A⊥ = 1/2 (3A0 - A//) (2)

Evaluation of in vitro insulin-mimetic activity of oxovanadium(IV)porphyrin complexes

The insulin-mimetic activity of [VO(tcpp)]Na4 was evaluated by simultaneous in vitro experiments, in which both the inhibitory activity upon release of free fatty acid

[29] and the enhancement of glucose uptake ability [30] of the complex in isolated rat adipocytes treated with epinephrine were estimated and compared with those of [VO(tpps)], [VO(tmpyp)](ClO4)4 and oxovanadium(IV) sulphate as positive controls [26, 27]. Male Wistar rats (weighing 200 g) were sacrificed under anesthesia with ether. The adipose tissues were removed, chopped with scissors and digested with collagenase for 1 h at 37 °C in Krebs Ringer bicarbonate buffer, pH 7.4, containing 2% bovine serum albumin. The adipocytes, thus obtained, were then separated from undigested tissues by filtration through a nylon mesh (250 μm) and washed three times with the buffer in the absence of collagenase. The complex was dissolved in saline at various concentrations. Each complex solution (30 μL) and 10 μL of 150 mM glucose solution (final concentration: 5 mM) along with either 5 μL of H2O or 5 μL of 60 mM sodium ascorbate solution (final concentration: 1 mM) were added to 240 μL of the isolated adipocytes (1 × 106 cells.mL-1), and the resulting suspensions were incubated at 37 °C for 30 min. Finally, 15μL of 200 μM epinephrine solution (final concentration: 10 μM) was added to the suspensions, and the resulting mixture was incubated at 37 °C for 3 h. Sodium ascorbate was used to prevent the oxidation of oxovanadium(IV) species during incubation. The reaction was stopped by cooling in ice water, and the mixtures were centrifuged at 3000 rpm at 4 °C for 10 min. The free fatty acid concentration in the outer solution of the adipocytes was determined with a free fatty acid kit (NEFA C-test

Complex Solvent g-Values A-values, 10-4 cm-1

g0 g// g⊥ A0 A// A⊥

[VO(tcpp)]Na4 H2O 1.970 1.968 1.970 79 158 40

[VO(tpps)] H2O 1.967 1.938 1.982 106 183 67

[VO(tmpyp)](ClO4)4 H2O 1.982 1.966 1.990 78 160 37

Table 1. Physicochemical properties of oxovanadium(IV)porphyrin complexes

Complex Found (calcd.), %

C H N

C48H24N4O8Na4VO·4.5H2O [VO(tcpp)]Na4 56.15 (56.26) 4.20 (4.25) 5.41 (5.47)

C44H28O12S4N4VO·8H2O·2C3H7NO [VO(tpps)] 46.55 (46.21) 4.53 (4.48) 6.51 (6.55)

C44H36N8VO(ClO4)4·5H2O [VO(tmpyp)](ClO4)4 42.92 (42.86) 3.76 (3.44) 9.10 (9.46)

IR data in KBr disk and UV-visible data in H2O

Complex IR data UV-visible data

νV=O, cm-1 Solvent λ, nm (ε, 103 M-1.cm-1)

[VO(tcpp)]Na4 1007 H2O 437 (210.2) 565 (16.5) 606 (5.1)

[VO(tpps)] 1005 H2O 436 (219.5) 564 (18.8) 604 (7.3)

[VO(tmpyp)](ClO4)4 1007 H2O 439 (190.0) 563 (15.0) 603 (3.4)

ESR data in H2O



Wako, Wako Pure Chemical Industries, Osaka, Japan). The IC50 value, i.e. the 50% inhibitory concentration of the complex, was determined from the curve for the concentration-dependent inhibitory effect of the complex on free fatty acid release in isolated rat adipocytes treated with epinephrine in the absence, as well as in the presence, of sodium ascorbate. In addition, the glucose concentration in the outer solution of the adipocytes was estimated using a Fuji Dry Chem analyzer (Fuji Medical Co., Tokyo, Japan). The glucose uptake ability of the complexes was evaluated using the apparent EC50

values, which are the 50% enhancing concentration of the complex with respect to the maximal glucose uptake concentration in epinephrine-treated adipocytes.

Metallokinetic analyses of oxovanadium(IV)-porphyrin complexes by in vivo blood circulation monitoring-ESR

The metallokinetic features of the oxovanadium(IV)-porphyrin complexes in the blood of healthy rats that received either [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4,[VO(tpps)] or oxovanadium(IV) sulphate (dose: 0.5 mg of V.kg-1 body mass) were analyzed by in vivo blood circulation monitoring-ESR [31–33]. In short, the rats were anaesthetized by i.p. injection of pentobarbital and maintained at 35 °C on a Deltaphase Isothermal Pad (Model 39 DP, Braintree Scientific, MA, USA). Heparinized polyethylene tubes were cannulated into the left femoral artery and vein. The free ends of the cannula were joined with heparinized silicon tubes to form a blood circuit outside the body; this was directly connected to an ESR sample tube (a quartz 20-μL capillary tube). Blood from the femoral artery was returned to the femoral vein and recirculated after flowing through the ESR sample tube by the rat’s own heartbeat and blood pressure without depletion. [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4, [VO(tpps)] and oxovanadium(IV) sulphate in saline solutions were administered to the rats by a single i.v. injection, and the ESR spectra were recorded for every 30 s at room temperature using an X-band ESR spectrometer. Data were collected and analyzed on a Windows computer using WIN-RAD Data Analyzer (Radical Research, Tokyo, Japan). The disappearance of the ESR signal due to the oxovanadium(IV) species in the blood was plotted against time following the administration of [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4, [VO(tpps)] and oxovanadium(IV) sulphate, respectively. To determine the concentrations of the oxovanadium(IV) species, 20 μL of [VO(tcpp)]Na4,[VO(tmpyp)](ClO4)4, [VO(tpps)] or oxovanadium(IV) sulphate dissolved in the blood of untreated rats was used in ESR sample tube. The samples were freshly prepared by using fresh blood spiked with each complex and, each calibration curve was obtained by monitoring the signal intensities of the central peak due to the corresponding oxovanadium(IV) complex. Metallokinetic parameters

for oxovanadium(IV) complexes were obtained on the basis of a one-compartment model. Using the nonlinear least-squares regression program MULTI [31–33] that was rewritten with Visual Basic, the individual profiles of the determined concentrations of oxovanadium(IV) complexes in the blood of rats that were injected with [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4, [VO(tpps)] or oxovanadium(IV) sulphate were fitted to the equation [Cb = D/Vd

.exp(-ket)], where Cb is the blood concentration, D is the dose of a compound, Vd is the distribution volume, ke is the elimination rate constant, and t is the time. The area under the concentration curve (AUC), mean resistance time (MRT), total clearance (CLtot) and half life (t1/2) were calculated from the following equations: AUC = D/Vd/ke, MRT = 1/ke, CLtot = Vd·ke and t1/2 = 0.693/ke.

Statistical analysis

Experimental results are expressed as the mean values ± standard deviations (SD). Statistical analysis was performed by using analysis of variance (ANOVA) or Student’s t-test at the 5% (p < 0.05), 1% (p < 0.01), or 0.1% (p < 0.001) level of significance.


Preparation and characterizationof oxovanadium(IV)porphyrin complexes

[VO(tcpp)]Na4 was prepared according to the method used by Erdman et al. [28] with slight modifications as described earlier, and [VO(tpps)] and [VO(tmpyp)](ClO4)4 were prepared as described previously [24]. The oxovanadium(IV)porphyrin complexes were characterized by elemental analysis, visible absorption, IR and ESR spectra. The physicochemical parameters of [VO(tcpp)]Na4, along with [VO(tmpyp)](ClO4)4 and [VO(tpps)], are summarized in Table 1. The calculated and found values in the elemental analyses were very similar for all complexes (Table 1). The visible absorption spectrum of [VO(tcpp)]Na4 dissolved in H2O showed the Soret and the Q-bands at 437, 565 and 606 nm, respectively. The apparent molar absorptivity of [VO(tcpp)]Na4 was estimated to be 210.2 × 103, 16.5 × 103 and 5.1 × 103 M-1.cm-1 at 437, 565 and 606 nm, respectively. These molar absorptivity values are very similar to those of [VO(tpps)] and [VO(tmpyp)](ClO4)4, respectively (Table 1).

In the IR spectra (figure not shown), the band due to the V=O stretching vibration was found to be at 1007 cm-1

for [VO(tcpp)]Na4 (Table 1), which is very similar to the reported values at 1007 cm-1 for [VO(tpps)] [24] and [VO(tmpyp)](ClO4)4 [27], and at 1005 cm-1 for [VO(tpp)] (H2tpp = tetraphenylporphyrin) [34]. The X-band ESR spectra of [VO(tcpp)]Na4 dissolved in H2Oshowed eight-line hyperfine splitting patterns (Fig. 2a)at room temperature and an anisotropic spectrum at liquid nitrogen temperature (Fig. 2b) due to the coupling



of unpaired electron spin with the nuclear spin of 51Vnucleus (I = 7/2), indicating that only one mononuclear vanadium(IV) species is present in the examined sample solution. The estimated ESR parameters (g and A values) are listed in Table 1, and the values indicate that the oxovanadium(IV) ion has a VO(N4)equatorial coordination sphere (Fig. 1), based on the reference values of [VO(tmpyp)](ClO4)4 [27]. The small discrepancy in the ESR parameters between [VO(tpps)] and [VO(tcpp)]Na4 (Table 1) might be due to a slight aggregation of [VO(tpps)] at liquid nitrogen temperature, which was observed in the ESR spectrum [26].

In vitro insulin-mimetic activity of oxovanadium(IV)porphyrin complexes

The in vitro insulin-mimetic activity of the complexes was examined based on both the inhibition of free fatty acid release and the enhancement of glucose uptake in isolated rat adipocytes treated with epinephrine in the absence, as well as in the presence of sodium ascorbate [29, 30]. A typical example of the concentration-dependent inhibitory effect of [VO(tcpp)]Na4 on free fatty acid release in isolated rat adipocytes treated with epinephrine is illustrated in Fig. 3a. The apparent IC50 values in the absence and presence of sodium ascorbate were estimated to be 0.52 ± 0.19 mM and 0.50 ± 0.03 mM for [VO(tcpp)]Na4, 0.16 ± 0.01 mM and 0.16 ± 0.01 mM for [VO(tpps)] [26], 28.70 ± 1.22 mM and 1.22 ± 0.59 mM for [VO(tmpyp)](ClO4)4 [26], and 1.00 ± 0.34 mM and 0.34 ± 0.15 mM for oxovanadium(IV) sulfate, (Table 2) respectively. These results suggest that the sodium ascorbate had no effect on the IC50

values of [VO(tcpp)]Na4 and [VO(tpps)], and the oxidation states of these two complexes were not

changed during incubation either in the presence or in the absence of sodium ascorbate. However, the significant lower IC50 of [VO(tmpyp)](ClO4)4 and oxovanadium(IV) sulphate with sodium ascorbate strongly indicate that [VO(tmpyp)](ClO4)4 and vanadium(IV) oxide sulfate were oxidized during incubation, and the oxovanadium(IV) state sustained by sodium ascorbate is the active form that exhibits insulin-mimetic activity. As well, [VO(tcpp)]Na4 has a higher insulin-mimetic activity than that of [VO(tmpyp)](ClO4)4 or oxovanadium(IV) sulfate, whereby the order of inhibitory activity of the complexes is: [VO(tpps)] > [VO(tcpp)]Na4 > [VO(tmpyp)](ClO4)4 >oxovanadium(IV) sulfate.

[VO(tcpp)]Na4 produced a concentration-dependent increase in glucose uptake in epinephrine-treated isolated rat adipocytes in the absence of sodium ascorbate (Fig. 3b). The apparent EC50 values of were estimated to be 0.124 ± 0.016 mM for [VO(tcpp)]Na4, 0.028 ±0.006 mM for [VO(tpps)] [26], and 0.221 ± 0.001 mM for oxovanadium(IV) sulphate (Table 2). However, [VO(tmpyp)](ClO4)4 did not enhance the glucose uptake

Fig. 2. Typical ESR spectra of [VO(tcpp)]Na4 in H2O at room temperature (a) and at liquid nitrogen temperature (b)

Fig. 3. (a) A typical example of inhibitory effect of [VO(tcpp)]Na4

(0.1-0.5 mM) on free fatty acid release from isolated rat adipocytes (2.5 × 106 cells.mL-1) treated with 0.01 mM epinephrine containing 5 mM glucose in the presence (all solid bars) and absence (all dashed bars) of sodium ascorbate (1 mM). (b) Enhancing effect of [VO(tcpp)]Na4 (0.01-0.6 mM) on glucose uptake ability in isolated rat adipocytes (2.5 × 106 cells.mL-1) treated with 0.01 mM epinephrine containing 5 mM glucose in the absence (all dashed bars) of sodium ascorbate



ability under the same experimental condition [26]. Based on these results, [VO(tcpp)]Na4 is anticipated to have a higher in vitro insulin-mimetic activity than those of oxovanadium(IV) sulfate and previously reported [VO(tmpyp)](ClO4)4 [27].

Metallokinetic features of oxovanadium(IV)-porphyrin complexes in the blood of healthyrats as estimated by in vivo blood circulation monitoring-ESR

To understand the bioavailability of oxovanadium(IV)porphyrin complexes, the metallokinetic features of paramagnetic oxovanadium(IV) species in the blood of healthy rats that received [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4, [VO(tpps)] or oxovanadium(IV) sulfate in the absence of sodium ascorbate were analyzed using the blood circulation monitoring-ESR method [31–33]. Figure 4ashows a typical example for the ESR spectral change with a time-dependent decrease of its signal intensities produced by oxovanadium(IV) species during BCM-ESR in rats administered the [VO(tcpp)]Na4 complex. The central peak intensity gradually decreased with time as expected. Figure 4b shows the time courses for the determined oxovanadium(IV) species in the circulating blood of rats given oxovanadium(IV) compounds as well as the simulated curves, which represent the theoretical curves fitted to the mean data on the basis of the one-compartment model with nonlinear least-squares regression [31–33]. Detected oxovanadium(IV) species in the blood of rats given oxovanadium(IV) compounds decayed exponentially, and then the clearance curves were fitted to the individual with the one-compartment model. The in vivo oxovanadium(IV) species disappeared time-dependently in the circulating blood of rats, indicating that oxovanadium(IV) species taken up into the blood were distributed to the peripheral tissues and eliminated from the whole body [31–33]. Oxovanadium(IV) concentrations in the blood of rats given [VO(tcpp)]Na4 (t1/2 = 16.6 ± 0.3 min) complex were remained significantly higher and longer than those of [VO(tmpyp)](ClO4)4 (t1/2 = 5.4 ± 0.6 min) and oxovanadium(IV) sulfate (t1/2 = 3.6 ± 0.6 min) as shown in Fig. 4b. The metallokinetic parameters of oxovanadium(IV) species

after administration of [VO(tcpp)]Na4, [VO(tmpyp)](ClO4)4, [VO(tpps)] and oxovanadium(IV) sulfate are summarized in Table 3. The AUC and MRT values for the rats treated with [VO(tcpp)]Na4 (AUC: 3.54 ± 0.07

Table 2. Inhibitory effect (IC50-value) of oxovanadium(IV)porphyrin complexes on free fatty acid release from isolated rat adipocytes treated with epinephrine and enhancing effect (EC50-value) of those complexes on glucose uptake ability in isolated rat adipocytes treated with epinephrine

Complex In the absence ofascorbate IC50, mM

In the presence ofascorbate IC50, mM

In the absence ofascorbate EC50, mM

[VO(tcpp)]Na4 0.52 ± 0.19* 0.50 ± 0.03* 0.124 ± 0.016

[VO(tpps)] 0.16 ± 0.01* 0.16 ± 0.01* 0.028 ± 0.006

[VO(tmpyp)](ClO4)4 28.70 ± 1.22 1.22 ± 0.59 not detected

Oxovanadium(IV) sulfate 1.00 ± 0.03 0.34 ± 0.15 0.221 ± 0.001

*p < 0.001 vs. [VO(tmpyp)](ClO4)4 and oxovanadium(IV) sulfate.

Fig. 4. In vivo blood circulation monitoring-ESR feature of oxovanadium(IV) species. A rat was administered [VO(tcpp)]Na4, [VO(tpps)] or [VO(tmpyp)](ClO4)4 intravenously at a dose of 0.5 mg of V/kg body mass under anesthesia. A typical example of time-dependent blood circulation monitoring-ESR spectral change of [VO(tcpp)]Na4 (a). ESR spectra were recorded at room temperature every 30 s following administration of the complex. Time courses of oxovanadium(IV) species concentration in the blood of rats treated intravenously with [VO(tcpp)]Na4 (Δ; n = 3), [VO(tpps)] (•; n = 3), and [VO(tmpyp)](ClO4)4 ( ; n = 3) and were monitored by the blood circulation monitoring-ESR method (b). The corresponding theoretical curves (solid lines) were fitted to the mean values. Complexes were dissolved in saline



μmol.mim.mL-1; MRT: 24.0 ± 0.5 mim) and [VO(tpps)] (AUC: 3.09 ± 0.18 μmol.mim.mL-1; MRT: 20.9 ± 0.6 mim) were very similar, however, they were significantly higher than for those given [VO(tmpyp)](ClO4)4 (AUC:0.70 ± 0.10 μmol.mim.mL-1; MRT: 7.8 ± 0.8 mim) and vanadium(IV) oxide sulfate (AUC: 0.27 ± 0.04 μmol.mim.mL-1; MRT: 5.2 ± 0.8 mim). The values are related to the clearance rates of the oxovanadium(IV) species from the blood of rats. In fact, the values of CLtot and Vd in the rats treated with [VO(tcpp)]Na4 (CLtot: 2.8 ±0.1 mL.min-1.kg-1; Vd: 67 ± 1 mL.kg-1) and [VO(tpps)] (CLtot: 3.2 ± 0.2 mL.min-1.kg-1; Vd: 66 ± 4 mL.kg-1) were significantly lower than those due to [VO(tmpyp)](ClO4)4

(CLtot: 14.1 ± 2.1 mL.min-1.kg-1; Vd: 109 ± 5 mL.kg-1) and oxovanadium(IV) sulfate (CLtot: 37.0 ± 5.5 mL.min-1.kg-1; Vd: 193 ± 12 mL.kg-1). These results suggested that the bioavailability of [VO(tcpp)]Na4 was very similar to that of [VO(tpps)], however, much higher than that of [VO(tmpyp)](ClO4)4 or oxovanadium(IV) sulfate.

CONCLUSION

[VO(tcpp)]Na4 complex produced with a VO(N4)equatorial coordination sphere. The in vitro insulin-mimetic activity and in vivo metallokinetic feature of [VO(tcpp)]Na4 were very similar to those of [VO(tpps)] [26], however, significantly better than those of previously reported insulin-mimetic [VO(tmpyp)](ClO4)4 [27] and oxovanadium(IV) sulfate [27]. These results highlight the potential utility of suitably designed oxovanadium(IV)porphyrin complex [VO(tcpp)]Na4 as a candidate for drug with insulin-mimetic activity.

Acknowledgements

We acknowledge the Japan Society for the Promotion of Science (JSPS) for awarding the JSPS postdoctoral fellowship to T.K.S.

REFERENCES

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16. Sakurai H, Yasui H and Adachi Y. Expert Opin. Investig. Drugs 2003; 12: 1189–1203.

Table 3. In vivo metallokinetic parameters of oxovanadium(IV)porphyrin complexes and oxovanadium(IV) sulfate after intravenous injection to healthy rats

Metallokinetic parameters Complex

[VO(tcpp)]Na4 [VO(tpps)] [VO(tmpyp)](ClO4)4

Oxovanadium(IV)sulfate

AUC, μmol.min.mL-1 3.54 ± 0.07* 3.09 ± 0.18* 0.70 ± 0.10 0.27 ± 0.04

MRT, min 24.0 ± 0.5* 20.9 ± 0.6* 7.8 ± 0.8 5.2 ± 0.8

CLtot, mL.min-1.kg-1 2.8 ± 0.1* 3.2 ± 0.2* 14.1 ± 2.1 37.0 ± 5.5

Vd, mL.kg-1 67 ± 1* 66 ± 4* 109 ± 5 193 ± 12

ke, min-1 0.042 ± 0.002* 0.048 ± 0.001* 0.129 ± 0.013 0.191 ± 0.027

t1/2, min 16.6 ± 0.3* 14.5 ± 0.4* 5.4 ± 0.6 3.6 ± 0.6

Data are expressed as the means ± SDs for 3 or 4 rats. Rats were treated with oxovanadium(IV)porphyrin complexes such as [VO(tcpp)]Na4 (n = 3), [VO(tpps)] (n = 3), [VO(tmpyp)](ClO4)4 (n = 3) and oxovanadium(IV) sulfate (n = 4) at a dose of 0.5 mg.V.kg-1 body mass by i.v. injection under anesthesia. *Significantly different at the 1% level of ANOVA in comparison with [VO(tmpyp)](ClO4)4 or oxovanadium(IV) sulfate.



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1999; 99: 2561–2571.20. Thompson KH, Liboiron BD, Sun Y, Bellman KDD,

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27. Sakurai H, Inohara T, Adachi Y, Kawabe H, Yasui H and Takada J. Bioorg. Med. Chem. Lett. 2004; 14:1093–1096.

28. Erdman JG, Ramsey VG, Kalenda NW and Hanson WE. J. Am. Chem. Soc. 1956; 78: 5844–5847.

29. Nakai M, Watanabe H, Fujiwara C, Kakegawa H, Satoh T, Takeda J, Matushita R and Sakurai H. Biol.Pharm. Bull. 1995; 18: 719–725.

30. Adachi Y and Sakurai H. Chem. Pharm. Bull. 2004; 52: 428–433.

31. Takechi K, Tamura H, Yamaoka K and Sakurai H. Free Rad. Res. 1997; 26: 483–496.

32. Yasui H, Tamura A, Takino T and Sakurai H. J. Inorg. Biochem. 2002; 91: 327–338.

33. Sakurai H and Yasui H. J. Trace. Elem. Exp. Med.2003; 16: 269–280.

34. Tsuchimoto M, Hoshina G, Uemura R, Nakajima K, Kojima M and Ohba S. Bull. Chem. Soc. Jpn. 2000; 73: 2317–2323.


DOI: 10.1142/S1088424611004403



INTRODUCTION

Long wavelength absorbing photosensitizers (>700nm) have been described as potential candidates for achieving maximum tissue penetration in photodynamic therapy (PDT) [1–5]. Among such compounds with long-wavelength absorption, some naturally occurring bacteriochlorins have been reported as effective photosensitizers in preliminary in vitro and in vivostudies [6, 7]. However, most of these naturally occurring bacteriochlorins are extremely sensitive to oxidation, which result in rapid transformation into relative stable chlorin state which has an absorption maximum at or below 670 nm [4, 8]. Furthermore, if a laser is used to excite the bacteriochlorin in vivo, oxidation may result in the formation of a new chromophore absorbing outside the laser window, which reduces the photodynamic efficacy. In order to render PDT more generally applicable to tumor therapy, long wavelength absorbing photosensitizers,

such as stable chlorins are needed, because they should also be able to localize in relatively high concentration at the tumor site relative to normal tissues. Chlorophyll-a is a natural chlorin pigment and his degradation products stand in marked contrast to symmetric porphyrin pigments due to substantially stabilized S1 energies, a strong Qy absorption band, and unique redox reactivates. Therefore the synthesis of novel photosensitizers, possessed the basic skeleton of chlorophyll-a, have become the focus of research in photodynamic therapy [9]. For chlorophyll-a compounds, the Qy bands as a longest absorption band were strongly affected by the substitutents on the Qy axis (N21–N23, see Scheme 1) [10]. Considering that introducing strong electron-withdrawing group on the chromophore or expanding the conjugation system of the macrocycle can bring about obvious red shift of its long wavelength absorption, the modifications of methyl pyropheophorbide-a 1(MPPa), which derived from methyl pheophorbide a(MPa), the initial degradative product extracted from Spirulina pacfica alga, were carried out for synthesis of long-wavelength photosensitizers with stable chlorin structure in our studies. Here we report the synthesis

Synthesis of long-wavelength chlorins by chemical

modification for methyl pyropheophorbide-a and

their in vitro cell viabilities

Jin Jun Wang*a, Jia Zhu Lib, Judit Jakusc and Young Key Shimb

a College of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, Chinab PDT Research Institute, School of Nano Engineering, Inje University, Gimhae 621-749, Koreac Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest 1025, Hungary

Received 28 August 2011Accepted 28 September 2011

ABSTRACT: A series of novel chlorophyll-a homologs with long wavelength absorption were synthesized via modification of methyl pyropheophorbide-a used as starting material. For introducing electron-withdrawing group methylenemalononitrile moiety was established on the periphery of modified chlorin by Knoevenagel reaction of malononitrile with formyl group at 3-, 15-position and 131-carbonyl group on the exocyclic ring. All of chlorins containing the methylenemalononitrile structure show Qy-absorption at more than 700 nm. Moreover, we have examined a preliminary in vitro photodynamicanticancer effect of these new derivatives on mouse sarcoma S-180 cell line.

KEYWORDS: chlorin, methyl pyropheophorbide-a, chemical modification, visible absorption.


*Correspondence to: Jin Jun Wang, email: [email protected],fax: +86 535-6902078


SYNTHESIS OF LONG-WAVELENGTH CHLORINS BY CHEMICAL MODIFICATION 123

of chlorophyll a homologs, which possess absorption maximum around longer than 700 nm, by modification of methyl pyropheophorbide-a.


Synthesis and characterization

Malononitrile exhibits high activity and its introduction will extend greatly the Qy peak of chlorin comparing with other active methylene compounds such as dimethyl malonate and acetylacetone [11, 12], on the other hand, in our preliminary experiment, the reaction of dimethyl malonate or acetylacetone with E-ketone gave very poor yield in same condition or no react. Therefore in this study we choose malononitrile as main building block to prepare the stable long-wavelength chlorins. The reaction of starting material 1 with malononitrile in the presence of sodium ethoxide in EtOH by refluxing to give 131-dicyanomethylene chlorin 2 in 75% yield which shows Qy-absorption at 704 nm. The exocyclic carbon–carbon double bonds at 3-position of 2 was oxidized with OsO4 in dichloromethane in the presence of pyridine followed by glycol cleavage, using sodium periodate to form chlorin 3 and methyl pyropheophorbide-d 4 (MPPd)whose Qy peaks were observed at 727 and 694 nm,respectively. To expand conjugated π-system of chlorin chromophore the C3-formyl of 3 reacted continuouslywith Ph3PCH2Ph in dichloromethane in the presence of aqueous NaOH solution at room temperature for 30 min to give a C3-phenyl substituted chlorin mixture composed of pyropheophorbide 5a as cis-isomer (19%) and 5bas trans-isomer (59%), which were easily separated by chromatography and showed their Qy bands at 703 and 710 nm, respectively. The Knoevenagel reaction of 3 was performed readily in THF in the presence of TEA to give tetracyano-substituted chlorin 6 in 70% yield, whose Qy-absorption attained to 734 nm. By the same way MPP-d 4 was converted into dicyano-substituted chlorin7 (Qy = 706 nm) from which chlorin 6 was also obtained by means of refluxing in THF with malononitrile. The chlorination for MPPa 1 produced 20-chlorochlorin 9whoseπ-system was further extended through its 20-meso-position. The subsequent Knoevenagel reaction of 9 with malononitrile smoothly gave 131-dicyanomethylene-substituted chlorochlorin 10 whose Qy peak appeared at 714 nm. Although this compound also could be obtained from 2 by same chlorination with NCS, the speed and yield of the reaction were relatively low in comparison with that of MPPa 1 due to the absorb-electron effect of the dicyanomethylene group at 131-position. The Wittig reaction of MPPd 4 with benzyltriphenylphosphonium bromide generated a pair of cis-trans isomers 8a(Qy = 667 nm) and 8b (Qy = 673 nm) in 20% and 58% yields, respectively [11]. The condensations of these isomers with malononitrile were carried out under the

same condition for preparing chlorin 2 to also give 5aand 5b (Scheme 1). It is worth noting that E-ketone and C3-formyl group exhibited different activities, thereby in the reaction of malononitrile with C3-formyl group, TEA was used as a moderate catalyst, while its condensation with E-ketone need a stronger base (NaOEt).

In order to introduce methylenemalononitrile moiety at meso-position, purpurin-5 dimethyl esters 11 and 132-oxopyropheophoride-a 12, obtained from the allomerization of MPPa 1 [13], were used as reaction precursors on account of the high reactivities of their C15-carbonyl groups. The former (11) reacted with malononitrile smoothly to form expected product 13in 67% yield under the catalysis of TEA in THF. The corresponding reaction of the latter (12) did not gave designed tetracyanochlorin 15, but diacyanochlorin 14 in 52% instead. The further reaction of 14 with malononitrile using sodium ethoxide as catalyst by refluxing in EtOH also not generated 15, but rearranged into purpurin-18 16(Scheme 2).

All constructions for dicyanomethylene moiety were based on Knoevenagel reaction. Instead of forming expected product, only the condensation of C132-carbonyl of 14 with malononitrile was rearranged to known purpurin-18 ester 16, whose possible formation process was outlined in Fig. 1. we postulated that in ethanol containing sodium ethoxide the resulting malononitrile carbonion failed to attack to the carbonyl at 132-position,or carbon-carbon double bond at 131-position either due to its steric hindrance, but the smaller ethyioxide ion as catalyst attacked the carbon atom at 132-position to form Michael adduct A. Subsequent nucleophilic substitution at the carbon atom of C131-ethoxyl group brought about leaving malononitrile carbonion to rearrange to diketochlorin 12 which was readily converted into purpurin-18 eater 16 under strong alkaline condition [13].

The visible spectra of chlorins show that the introduction of dicyanomethylene moiety on the tetrapyrrole macrocycles can cause various degree of bathochromic shift in their Qy peaks (Fig. 2). Compared the differences of the longest absorption bands in their visible spectra between chlorins before and after Knoevenagel reaction, each change from C=O on the exocyclic E-ring to C=C(CN)2, such as 1→2(ΔQy = 36 nm) or 2→6 (ΔQy = 30 nm), increases their Qy wavelengths more than 30 nm. The transform from formyl group to dicyanomethylene structure at 3-position or 15-position, such as 3→6 (ΔQy = 7 nm), 4→7 (ΔQy = 12 nm) and 11→13 (ΔQy = 12 nm), bring about completelydifferent red-shift distance. Besides structure modification, these results showed that the changes of Qy wavelengths of chlorins also related to the existing substituted groups in the opposite direction of introduced dicyanomethylene along N21–N23 axis. The λmax of trans-phenyl-substituted isomer 5b was red shifted compared to those of unsubstituted vinyl compound 2 because the C3b-phenyl conjugated with the chlorin chromophore through the 3a–3b double bond. However, the corresponding Qy band of cis-isomer


124 J. J. WANG ET AL.

HN

NNH

N

O

OCH3

O

HN

NNH

N

OCH3

O NC

CN

HN

NNH

N

O

OCH3

O

HN

NNH

N

OCH3

O

NC

NC

HN

NNH

N

OCH3

O

HN

NNH

N

OCH3

O

+

12 3

5a 5b

6

(a) (b)

(d)

(c)

HN

NNH

N

O

O

OCH3

O

(d)

HN

NNH

N

O

OCH3

O

+

7

(a)

NC

CN

NC

CN

NC

NC

NC

CN

NC

CN

4

1

3132

5

10

1311718

20

132

HN

NNH

N

OCH3

O

HN

NNH

N

OCH3

O

+

8a 8bOO

(a)

HN

NNH

N

O

OCH3

O

ClHN

NNH

N

OCH3

O NC

CN9 10

(e)

(a) Cl

(e)

(c)

Scheme 1. Synthesis of dicyanomethylene bearing long-wavelength chlorins. Reagents and conditions: (a) CH2(CN)2/NaOEt/EtOH;(b) Py, OsO4/THF/NaIO4/SiO2; (c) Ph3PCH2PhCl/NaOH; (d) CH2(CN)2/CH2Cl2/N(Et)3; (e) NCS/CH2Cl2

(b)

HN

NNH

N

OCH3

O

HN

NNH

N

O

OCH3

OOO O

OCH3

HN

NNH

N

CO2CH3

OCH3

OCN

NC

HN

NNH

N

OCH3

OO CN

NC

(a)

11 12

13

14

HN

NNH

N

OCH3

O

CN

NCCNNC

HN

NNH

N

O

OCH3

O

15

1

HN

NNH

N

O

OCH3

OO O

16

+(a)

(c)

Scheme 2. Synthesis of 15-meso dicyanomethylene bearing purpurin-5 13, and attempts at synthesizing tetracyano bearing chlorin 15. Reagents and conditions: (a) LiOH/THF; (b) CH2(CN)2/CH2Cl2/N(Et)3; (c) NaOEt/EtOH



5a appeared at 703 nm, which was almost the same as that of 2 (704 nm). This difference was ascribable to that cis-isomer has less conjugation of the C3-double bond with the chlorin chromophore because of steric repulsion between the phenyl group and C2-methyl group. The Soret/Qy absorbance ratios of chlorins including 2, 5–7, 10 and 14,which are in the range of 0.88–1.16, were reduced after linking with electro-withdrawing groups at 131-position. The corresponding ratios of chlorins 3 and 13, which beared β, β-dicyanomethylene at 3- and 15-position respectively, were not much difference in comparison with that of their precursors 4 and 11. It implied that rigid dicyanomethylene structure on the E-ring, except chlorin 13, improved the absorbing intensity of Qy band more effectively than that on C3 position (Table 1).

The 1H NMR spectrum of chlorins introduced dicyanomethylene structure at 131-position, including 2, 3, 10 and 14, clearly showed the chemical shifts of all sorts of the proton signals corresponding to these of their precursor. While for the chlorins introduced dicyanomethylene structure at 3-position, after the conversion from formyl group to dicyanomethylene a 1H singlet proton signal corresponding to the proton attached to dicyanomethylene was discovered at about 8.00 ppm, at the same time the original signal for formyl group disappeared. By contrast, allmost all proton absorption signals moved to upfield except that of one hydrogen linked with central nitrogen. These regular migrations could be

explained due to introduction of β, β-dicyanomethylene to reduce ring current density and (de)shielding action.

In vitro photosensitizing efficacy

In the present study, viability of the cell using the methodology reported by Ahn et al. [14] was determined by comparison with that of MPPa 1 for new photosensitizers on mouse sarcoma S-180 cell line at 0.01, 0.05, 0.1, 0.5 and 1 μM after PDT (Fig. 3). For PDT treatment, it was observed that all compounds showed an improved effect for cell death or cell viability as the concentration of the photosensitizer increased. And all compounds obtained showed better effect than that of MPPa. Among all the dicyano- or tetracyano-substituted chlorins we obtained, compound 10 showed highest effect when compared with the others.

Table 2 shows the IC50 values of these new photosensitizers on mouse sarcoma S-180 cell line after PDT. The reference compound MPPa showed a relative low effect after PDT (IC50 > 1.0 μM), all the dicyano- or tetracyano-substituted chlorins showed lower IC50 value than that of MPPa.Compound 10 among the tested photosensitizers showed relatively high PDT effect (IC50 = 0.060 μM). Furthermore, we are aiming to explore, in greater depth, the other biological effects of these compounds for PDT.

HNN

OCH3

OO CN

CN

14

HNN

O

OCH3

O

O O

16CNNC

O C2H5

HNN

OCH3

OO CN

NC

OC2H5 OC2H5

- CH(CN)2 HNN

OCH3

OO O

A 12

Fig. 1. Possible conversion process from chlorin 14 to purpurin-18 methyl ester 16

Fig. 2. UV-vis spectra in dichloromethane of dicyano- and tetracyano-substituted chlorin 2 and 6

Table 1. Absorption properties of dicyanomethylene bearing long-wavelength chlorins

Compound Absorption λmax, nm (relative intensity)

Soret ΔSoret* Qy ΔQy*

1 414 (1.00) 0 668 (0.38) 0

2 452 (1.00) 38 704 (1.11) 36

3 393 (1.00) −21 726 (0.86) 58

5a 452 (1.00) 38 703 (1.01) 35

5b 455 (1.00) 41 710 (0.97) 42

6 422 (1.00) 8 734 (0.93) 66

7 380 (1.00) −34 706 (0.54) 38

10 456 (1.00) 42 711 (1.00) 43

13 432 (1.00) 18 700 (0.29) 32

14 407 (1.00) −7 722 (0.91) 54

*ΔSoret and ΔQy represent the change of the Soret band and Qy band between the dicyano-methylene bearing long-wavelength chlorin and their starting material MPPa 1.



In conclusion, we have developed a new approach for the preparation of some pyropheophorbide derivatives possessing Qy-absorption at more than 700 nm by constructing two or four electron-withdrawing cyano-group on the perphery of chlorin chromophore such as at 3-positon or 131-position. These long wavelength absorbing chlorins with basic skeleton of chlorophyll-a possess relative stable tetrapyrrole macrocyclic structure. Their unique optical and photochemical properties and polyfunctional groups may be valuable for the synthesis of new generation photosensitisers using in PDT. Among all the compounds we obtained, compound 10 showed relatively high PDT effect (IC50 = 0.060 μM).

EXPERIMENTAL

General

All of the reactions were monitored by thin layer chromatography (TLC) using 0.20-mm silica gel plates with or without a UV indicator (60F-254). Silica gel 60 (70–230 or 230–400 mesh, Merck) was used for flash column chromatography. The melting points (uncorrected) were measured using an Electrothermal IA9000 Series digital melting point apparatus. The electronic absorption spectra were measured using a SCINCO S-3100 UV-vis spectrophotometer. The absorption maxima (λmax) values

are given in nanometers and relative intensity. The 1HNMR spectra were obtained using a Varian spectrometer (400 MHz). The chemical shifts (δ) are given in parts per million (ppm) relative to tetramethylsilane (TMS, 0 ppm), unless otherwise indicated. Elemental analyses were carried out using a Perkin Elmer 240-C microanalyzer. Materials obtained from commercial suppliers were used without further purification. Methyl pyropheophorbide a1 was prepared according to the procedures described in the literature [15].

Synthesis

Preparation of 131-β,β-dicyanomethylene-131-deoxopyropheophorbide-a methyl ester (2). MPPa 1(120 mg, 0.219 mmol) was dissolved in 30 mL EtOH, to which malononitrile (100 mg, 1.515 mmol) and sodium ethoxide (25 mg) was added under stirring. The solution was refluxed under N2 and decrease of 1 was monitored by TLC. After disappearance of 1 the reaction mixture was poured into ice water and extracted with dichloromethane (2 × 50 mL). The combined extract was washed with water, dried over anhydrous Na2SO4. After evaporating the residue was chromatographed on silica gel (eluent: hexane/ethyl acetate, 3:1) to afford 98 mg of 2 (0.164 mmol, 75%). UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 704 (1.11), 644 (0.32), 574 (0.21), 535 (0.16), 452 (1.00), 430 (0.97), 384 (0.98), 350 (0.78). 1HNMR (400 MHz; CDCl3): δ, ppm 9.09, 8.97, 8.32 (each s, each 1H, meso-H), 7.82 (dd, J = 17.8, 12.3 Hz, 1H, 31-H), 6.20 (dd, J = 17.8, 1.5 Hz, 1H, 32-trans-H), 6.09 (dd, J = 12.3, 1.5 Hz, 1H, 32 -cis-H), 5.31 (d, J = 20.0 Hz, 1H, 132-H), 5.26 (d, J = 20.0 Hz, 1H, 132-H), 4.06–4.27 (m, 2H, 17 + 18-H), 3.58, 3.35, 3.27, 3.06 (each s, each 3H, CH3 + OCH3), 3.50 (q, J = 7.8 Hz, 2H, 81-H),2.39–2.58, 2.11–2.30 (m, 4H, 171 + 172-H), 1.70 (d, J = 7.2 Hz, 3H, 18-CH3), 1.53 (t, J = 7.8 Hz, 3H, 82-H),0.78 (br s, 1H, NH), -1.18 (br s, 1H, NH). IR (KBr): ν,cm-1 3469 (N–H), 2962, 2869 (C–H), 1737 (C=O), 1664 (C=C), 1564, 1444, 1240, 1172, 1074, 908, 796 (chlorin skeleton). Anal. calcd. for C37H36N6O2: C 74.47, H 6.08, N 14.08; found C 74.59, H 5.89, N 14.28.

Preparation of 3-formyl-131-β,β-dicyanomethylene-3-devinyl-131-deoxopyropheophorbide-a methyl ester (3) and methyl pyeopheophorbide-d (MPPd, 4). Chlorin 2 (70 mg, 0.117 mmol) was dissolved in a mixed solution of THF (20 mL) and pyridine (0.3 mL) at 0 °Cand stirred at same temperature for 30 min after adding 51 mg (0.200 mmol) osmium(VIII) oxide in THF (2 mL), and then stirred at room temperature for an additional 1 h. An excess of a solution of sodium hydrogensulfite (15 g) in a 50% mixture of methanol in water was added. The mixture was stirred violently for 20 min. After filtrating the brown osmium(VI) oxide precipitate, dichloromethane was added to the mixture. The organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed to give solid material

Fig. 3. Cell viabiliy results of photosensitizers on mouse sarcoma S-180 cell line. Compounds were tested at 0.01, 0.05, 0.1, 0.5 and 1 μM concentrations in triplicate. MPPa was used as reference compound. Mouse sarcoma S-180 cell line at 80% confluency in 96-well plates was used for in vitro PDT tests. Cells were illuminated with a lamp in a wavelength range of 640–700 nm, with a peak at 660 nm, for 20 min. Total light dose was 8.4 J. Statistical analyses were performed using unpaired Student’s t test

Table 2. IC50 results of photosensitizers on mouse sarcoma S-180 cell line

Compound 1(MPPa) 2 3 5a 5b

IC50, μM >1.0 0.559 0.394 0.194 0.123

Compound 6 7 10 13 14

IC50, μM 0.312 0.735 0.060 0.853 0.241



that was suspended in a mixture of THF (15 mL) and silica gel (2.5 g). After addition of a solution of sodium metaperiodate (1 g) in water (15 mL), the color of the solution changed from green to bronze within 30 min. After adding dichloromethane (20 mL), the mixture was filtered through cotton wool and then the resultant crude material was chromatographed on silica gel (eluent: hexane/ethyl acetate, 3:1) to give 54 mg chlorin 3 (0.090mmol, 77%) and 8 mg MPPd 4 (0.014 mmol, 12%), respectively. 3. UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 726 (0.86), 663 (0.22), 583 (0.21), 544 (0.15), 412 (0.99), 393 (1.00), 384 (0.86). 1H NMR (400 MHz; CDCl3): δ, ppm 11.36 (s, 1H, CHO), 10.06, 9.28, 8.62 (s, each 1H, meso-H), 5.61, 5.55 (each d, J = 19.8 Hz, each 1H, 132-H), 4.23–4.53, 4.13–4.20 (m, each 1H, 17, 18-H), 3.68 (q, J = 7.8 Hz, 2H, 81-H), 3.67, 3.63, 3.57, 3.15 (each s, each 3H, CH3 + OCH3), 2.49–2.69, 2.15–2.39 (m, 4H, 171 + 172-H), 1.74 (d, J = 7.4 Hz, 3H, 18-CH3), 1.58 (t, J= 7.8 Hz, 3H, 82-H), 0.80 (br s, 1H, NH), -1.50 (br s, 1H, NH). IR (KBr): ν, cm-1 3443 (N–H), 2962, 2926 (C–H), 1740, 1735 (C=O), 1654 (C=C), 1564, 1438, 1400, 1256, 1169, 1083, 940, 722 (chlorin skeleton). Anal. calcd. for C36H34N6O3: C 72.22, H 5.72, N 14.04; found C 72.40, H 5.56, N 14.19. Analytical data of MPPd 4 in line with the literature values [16].

Preparation of cis-32-phenyl-131-β,β-dicyanome-thylene-131-deoxopyropheophorbide-a methyl ester (5a) and trans-32-phenyl-131-β,β-dicyanomethylene-131-deoxopyropheophorbide-a methyl ester (5b). Formyl chlorin 3 (65 mg, 0.109 mmol) and benzyltriphenyl-phosphonium chloride (93 mg, 0.240 mmol) was dissolved in 10 mL dichloromethane and a solution of NaOH (70 mg) in H2O (3 mL) was added with stirring. The solution was stirred at room temperature under N2

and the decrease of 3 was monitored by TLC. After disappearance of 3, the reaction mixture was poured into ice water and dichloromethane. The aqueous phase was extracted with several portions of dichloromethane and the combined organic phases were washed with aqueous aq. 2% HCl, aq. 4% NaHCO3 and water and evaporated in vacuo to dryness. The residue was chromatographed on silica gel (eluent: hexane/ethyl acetate, 4:1) to give 14 mg chlorin 5a (0.021 mmol, 19%) and 43 mg chlorin 5b(0.064 mmol, 59%). 5a. UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 703 (1.01), 644 (0.34), 574 (0.23), 533 (0.19), 495 (0.16), 452 (1.00), 434 (0.94), 386 (0.88), 350 (0.72). 1H NMR (400 MHz; CDCl3): δ, ppm 9.10, 9.02, 8.27 (each s, each 1H, meso-H), 7.70 (m, 2H, Ph-H), 7.54 (d, J = 11.4 Hz, 1H, 31-H), 7.52 (m, 1H, Ph-H), 7.35 (d, J = 11.4 Hz, 1H, 32-H), 6.94–7.25 (m, 3H, Ph-H), 5.52, 5.38 (d, J = 20 Hz, each 1H, 132-H), 4.18–4.35, 4.06–4.15(m, 2H, 17 + 18-H), 3.53 (q, J = 7.8 Hz, 2H, 81-H), 3.64, 3.53, 3.00, 2.90 (each s, each 3H, CH3 + OCH3), 2.46–2.64, 2.20–2.35 (m, each 1H, 171 + 172-H), 1.75 (d, J =7.2 Hz, 3H, 18-CH3), 1.58 (t, J = 7.8 Hz, 3H, 82-H), 0.78 (br s, 1H, NH), -0.86 (br s, 1H, NH). IR (KBr): ν, cm-1

3448, 3180 (N–H), 2929, 2866 (C–H), 1741 (C=O), 1638

(C=C), 1528, 1400, 1173, 1071, 1040, 968, 728 (chlorin skeleton). Anal. calcd. for C43H40N6O2: C 76.76, H 5.99, N 12.49; found C 76.60, H 5.81, N 12.24. 5b. UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 710 (0.97), 649 (0.27), 577 (0.12), 496 (0.08), 455 (1.00), 434 (0.89), 390 (0.84), 353 (0.52). 1H NMR (400 MHz; CDCl3): δ, ppm 9.12, 9.10, 8.36 (each s, each 1H, meso-H), 8.13 (d, J =16.3 Hz, 1H, 31-H), 7.78 (d, J = 7.2 Hz, 2H, Ph-H), 7.55 (d, J = 16.3 Hz, 1H, 32-H), 7.44–7.57 (m, 3H, Ph-H), 5.54 (d, J = 21 Hz, 1H, 132-H), 5.40 (d, J = 21 Hz, 1H, 132-H), 4.24–4.40, 4.08–4.17 (m, each 1H, 17 + 18-H), 3.63, 3.34, 3.32, 3.09 (each s, each 3H, CH3 + OCH3), 3.60 (q, J = 7.8 Hz, 2H, 81-H), 2.40–2.63, 2.17–2.36 (m, each 2H, 171 + 172-H), 1.75 (d, J = 7.2 Hz, 1H, 18-CH3), 1.61 (t, J = 7.4 Hz, 3H, 82-H), 0.06 (br s, 1H, NH), -0.98 (br s, 1H, NH). IR (KBr): ν, cm-1 3449 (N–H), 2924 (C-H), 1736 (C=O), 1686 (C=C), 1560, 1459, 1341, 1086, 1040, 976, 669 (chlorin skeleton). Anal. calcd. for C43H40N6O2: C 76.76, H 5.99, N 12.49; found C 76.89, H 6.13, N 12.67.

Preparation of 32,32-dicyano-131-β,β-dicyanomethy-lene-131-deoxopyropheophorbide-a methyl ester (6).Formyl chlorin 3 (40 mg, 0.067 mmol) was dissolved in 15 mL dichloromethane containing TEA (1.5 mL), to which malononitrile (70 mg, 1.060 mmol) was added under stirring. The solution was stirred under N2 for 2 h. The reaction mixture was poured into ice water and extracted with dichloromethane (2 × 15 mL). The combined extract was washed with water, dried over anhydrous Na2SO4. The combined organic phases were evaporated to druness and the residue was chromatographed on silica gel (eluent: hexane/ethyl acetate, 3:1) to afford 20 mg 6 (0.0031 mmol, 46%). UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 734 (0.93), 590 (0.31), 545 (0.24), 422 (1.00), 353 (0.83). 1H NMR (400 MHz; CDCl3): δ, ppm 9.46, 9.12, 8.69 (each s, each 1H, meso-H), 9.25 (s, 1H, 31-H), 5.71 (d, J = 21.0 Hz, 1H, 132-H), 5.55 (d, J = 21.0 Hz, 1H, 132-H), 4.38–4.56 (m, 1H, 18-H), 4.21–4.43(m, 1H, 17-H), 3.76, 3.62, 3.48, 3.26 (each s, each 3H, CH3 + OCH3), 3.69 (q, J = 7.5 Hz, 2H, 81-H), 2.51–2.73,2.16–2.40 (m, each 2H, 171 + 172-H), 1.81 (d, J = 7.2 Hz, 3H, 18-CH3), 1.68 (t, J = 7.5Hz, 3H, 81-CH3), 0.79 (br s, 1H, NH), -1.35 (br s, 1H, NH). IR (KBr): ν, cm-1 3450 (N–H), 2978 (C–H), 1735 (C=O), 1637 (C=C), 1561, 1475, 1225, 1084, 982, 722 (chlorin skeleton). Anal. calcd. for C39H34N8O2: C 72.43, H 5.30, N 17.33; found C 72.59, H 5.17, N 17.50.

Preparation of 32,32-dicyanopyropheophorbide-amethyl ester (7). MPPd 4 (42 mg, 0.076 mmol) was dissolved in 20 mL dichloromethane, to which malononitrile (70 mg, 1.060 mmol) and TEA (1.5 mL) was added under stirring. The solution was stirred under N2 for 2 h. The reaction mixture was poured into ice water and extracted with dichloro-methane (2 × 15 mL). The combined extracts was washed with water, dried over anhydrous Na2SO4. After evaporating the solution the residue was chromatographed on silica gel (eluent: hexane/ethyl acetate, 3:1) to afford 34 mg 7 (0.057 mmol,



76%). UV-vis (CH2Cl2): λmax, nm (rel. intensity log ε) 706 (0.54), 638 (0.08), 590 (0.11), 522 (0.11), 434 (0.54), 431 (0.49), 380 (1.00). 1H NMR (400 MHz; CDCl3): δ, ppm 9.58, 9.19, 8.79 (s, each 1H, meso-H), 9.23 (s, 1H, 31-H),5.26 (d, J = 20.0 Hz, 1H, 132-H), 5.19 (d, J = 20.0 Hz, 1H, 132-H), 4.34 (q, J = 7.3 Hz, 1H, 18-H), 4.12 (d, J = 7.2 Hz, 1H, 17-H), 3.67, 3.62, 3.51, 3.28 (each s, each 3H, CH3 + OCH3), 3.70 (q, J = 7.6Hz, 2H, 81-H), 2.52–2.70,2.20–2.43 (m, each 2H, 171 + 172-H), 1.84 (d, J = 7.2 Hz, 3H, 18-CH3), 1.71 (t, J = 7.6 Hz, 3H, 81-CH3), -0.16 (br s, 1H, NH), -2.08 (br s, 1H, NH). IR (KBr): ν, cm-1 3449, 3167 (N–H), 2963 (C–H), 1740 (C=O), 1672 (C=C),1542, 1510, 1400, 1223, 1075, 1021, 980, 799 (chlorin skeleton). Anal. calcd. for C36H34N6O3: C 72.22, H 5.72, N 14.04; found C 72.06, H 5.60, N 13.91.

Preparation of 20-chloropyropheophorbide-a methyl ester (9). 150 mg of NCS was partialy added to a solution of MPPa 1 (200 mg, 0.330 mmol) in methylene chloride (50 mL), and the reaction mixture was stirred for 5 h under nitrogen in the dark. The resulting solution was then poured into 200 mL of iced water and extracted with methylene chloride (3 × 100 mL). The combined extract was washed with 10% aqueous NaHCO3, water and dried over Na2SO4 and evaporated to dryness. The residue was chromatographed on silica gel (eluent: hexane/ethyl acetate, 2:1) to afford 237 mg chlorin 9 as dark-red solid in 80% yield. mp 210–214 °C. UV-vis (CHCl3): λmax, nm (rel. intensity log ε) 676 (0.41), 620 (0.07), 550 (0.12), 518 (0.09), 416 (1.00). 1H NMR (400 MHz; CDCl3): δ,ppm 9.52, 9.55 (each s, each 1H, meso-H), 7.92 (dd, J =17.8, 11.5 Hz, 1H, 31-H), 6.27 (dd, J = 11.5, 1.5 Hz, 1H, 32-H), 6.14 (dd, J = 17.8, 1.5 Hz, 1H, 32-H), 5.24 (d, J =3.2 Hz, 2H, 132-H), 4.80 (q, J = 7.0 Hz, 1H, 18-H), 4.24 (dd, J = 9.0, 2.8 Hz, 1H, 17-H), 3.68 (q, J = 7.6 Hz, 2H, 81-CH2), 3.67, 3.60, 3.59, 3.24 (each s, each 3H, OCH3 +CH3), 2.05–2.70 (m, 4H, 171 + 172-H), 1.70 (t, J = 7.6 Hz, 3H, 82-CH3), 1.64 (d, J = 7.0 Hz, 3H, 18-CH3), 0.48 (br s, 1H, NH), −1.94 (br s, 1H, NH). Other analytical data are consistent with the literature data [17].

Preparation of 20-chloro-131-β,β-dicyanomethy-lene-131-deoxopyropheophorbide-a methyl ester (10).This compound as a red solid was obtained from compound 9 by reacting with malononitrile in the yield of 59% according to the method for preparing compound 2.UV-vis (CHCl3): λmax, nm (rel. intensity log ε) 711 (1.00), 651 (0.17), 585 (0.17), 545 (0.08), 456 (1.00), 385 (0.79). 1H NMR (400 MHz; CDCl3): δ, ppm 9.17, 9.35 (each s, each 1H, meso-H), 7.85 (dd, J = 17.8, 11.5 Hz, 1H, 31-H), 6.28 (dd, J = 11.5, 1.4 Hz, 1H, cis-32-H), 6.07 (dd, J = 17.8, 1.4 Hz, 1H, trans-32-H), 5.44 (d, J = 21.0 Hz, 1H, 132-H), 5.28 (d, J = 21.0 Hz, 1H, 132-H), 4.66 (q, J = 7.1 Hz, 1H, 18-H), 4.04 (dd, J = 9.2, 2.4 Hz, 1H, 17-H), 3.62 (q, J = 7.6 Hz, 2H, 81-H), 3.63, 3.54, 3.50, 3.19 (each s, each 3H, CH3 + OCH3), 2.53–2.61, 2.15–2.25, 1.85–1.93 (each m, 4H, 171 + 172-H), 1.64 (t, J =7.6 Hz, 3H, 82-CH3), 1.55 (d, J = 7.1 Hz, 3H, 18-CH3),-1.32 (each br s, each 1H, NH), -1.51 (each br s, each 1H,

NH). IR (KBr): ν, cm-1 3448, 3145 (N–H), 2956 (C–H), 1736 (C=O), 1665 (C=C), 1619, 1437, 1401, 1257, 1170, 1010, 976, 710, 708, 597 (chlorin skeleton). Anal. calcd. for C37H35ClN6O2: C 70.41, H 5.59, N 13.32; found C 70.26, H 5.72, N 13.25.

Preparation of 15-β,β-dicyanomethylenepurpurin-5 methyl ester (13). This compound as a red solid was obtained from compound 11 by reacting with malononitrile in the yield of 71% according to the method for preparing compound 6. UV-vis (CHCl3):λmax, nm (rel. intensity log ε) 700 (0.29), 620 (0.07), 564 (0.20), 502 (0.08), 432 (1.00), 358 (0.67) nm. 1HNMR (400 MHz; CDCl3): δ, ppm 9.59 (s, 1H, 151-H),8.45, 9.34, 9.62 (each s, each 1H, meso-H), 7.88 (dd, J = 17.7, 11.5 Hz, 1H, 31-H), 6.28 (d, J = 17.7 Hz, 1H, trans-32-H), 6.16 (d, J = 11.5 Hz, 1H, cis-32-H), 4.50 (d, J = 10.7 Hz, 1H, 17-H), 4.36 (q, J = 7.3 Hz, 1H, 18-H), 3.66 (q, J = 7.6 Hz, 2H, 81-H), 4.25, 3.37, 3.51, 3.32, 3.18 (each s, each 3H, CH3 + OCH3), 1.95–2.08, 2.38–2.41,2.58–2.69 (each m, 4H, 171 + 172-H), 1.78 (d, J = 6.6Hz, 3H, 18-CH3), 1.67 (t, J = 7.6 Hz, 3H, 82-CH3), 0.20 (br s, 1H, NH), -0.20 (br s, 1H, NH). IR (KBr): ν, cm-1

3423, 3163 (N–H), 2958 (C–H), 1740, 1735 (C=O), 1672 (C=C), 1606, 1400, 1256, 1084, 924 (chlorin skeleton). Anal. calcd. for C38H38N6O4: C 71.01, H 5.96, N 13.08; found C 71.24, H 5.79, N 13.20.

Preparation of 131-β,β-dicyanomethylene-132-oxo-131-deoxopyropheophorbide-a methyl ester (14). This compound as a red solid was obtained from compound 12 by reacting with malononitrile in the yield of 68% according to the method for preparing compound 6.UV-vis (CHCl3): λmax, nm (rel. intensity log ε) 722 (0.91), 618 (0.08), 512 (0.28), 472 (0.36), 425 (0.35), 407 (1.00). 1H NMR (400 MHz; CDCl3): δ, ppm 8.94, 9.50, 9.80 (each s, each 1H, meso-H), 8.08 (dd, J = 17.8, 11.5 Hz, 31-H), 6.36 (dd, J = 17.8, 1.0 Hz, 1H, trans-32-H), 6.30 (dd, J = 11.5, 1.0 Hz, 1H, cis-32-H), 5.11 (d, J = 8.0 Hz, 1H, 17-H), 4.63 (q, J = 7.5 Hz, 1H, 18-H), 3.75 (q, J =7.6 Hz, 2H, 81-H), 3.62, 3.51, 3.50, 3.35 (each s, each 3H, CH3 + OCH3), 1.95–2.07, 2.18–2.43, 2.67–2.80 (each m, 4H, 171 + 172-H), 1.88 (d, J = 7.3 Hz, 3H, 18-CH3), 1.70 (t, J = 7.6 Hz, 3H, 8-CH3), 0.51 (br s, 1H, NH), -1.98 (br s, 1H, NH). IR (KBr): ν, cm-1 3449 (N–H), 2989 (C–H), 1741, 1735 (C=O), 1638 (C=C), 1542, 1528, 1308, 1080, 726 (chlorin skeleton). Anal. calcd. for C37H34N6O3: C 72.77, H 5.61, N 13.76; found C 72.60, H 5.40, N 13.91.

MTT assay for in vitro photosensitizing efficacy

The photosensitizing activities of long-wavelength dicyanomethylene bearing compounds (2, 3, 5a, 5b,6, 7, 10, 13, 14) and MPPa 1 were determined in the mouse sarcoma S-180 cell line. The cells were grown in α-MEM with 10% fetal calf serum, L-glutamine, penicillin, and streptomycin. Cells were maintained in 5% CO2, 95% air, and 100% humidity. Cells were plated in 96-well plates at a density of 5 × 103 cells per well



in complete medium. After an overnight incubation at 37 °C, the photosensitizers were added at varying concentrations and incubated at 37 °C for 3 h in the dark. Prior to light treatment the cells were replaced with drug-free complete medium. Cells were then illuminated with a lamp in a wavelength range of 640–700 nm, with a peak at 660 nm, for 20 min. Total light dose was 8.4 J. After PDT, the cells were incubated for 48 h at 37 °Cin the dark. Following the 48 h incubation, 10 μL of 4.0 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) (Sigma) dissolved in PBS was added to each well. After a 4 h incubation at 37 °C, unreacted MTT and medium were removed and 100 μL DMSO was added to solubilize the formazan crystals. The 96-well plate was read on a microtiter plate reader (ELISA-reader, BioTek, Synergy HT, USA) at an absorbance of 570 nm. The results were plotted as percent survival of the corresponding dark (drug no light) control for each compound tested after subtracting medium only control absorbance. Each data point represents the mean from 3 separate experiments with 6 replicates at each dose, and the standard errors were less than 10%.

Acknowledgements

This work was supported by the Natural Science Foundation of Shandong Province of China (No. Y2008B49) and by the open project of state key laboratory breeding base of green chemistry-synthesis technology (Zhejiang University of Technology), as well as by the Framework of the Intergovernmental Sino-Hungarian Scientific and Technological Cooperation (CHN-30/04).

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DOI: 10.1142/S1088424611004439



INTRODUCTION

Single walled carbon nanotubes (SWCNTs) have remarkable electrical, chemical, mechanical and structural properties and have found applications in various disciplines, such as electrochemistry [1], material science [2] and surface science [3]. SWCNT are ideal material to use in the fabrication of electrochemical sensors, because of their electrical conductivity, large surface area, low surface fouling and ability to reduce overpotentials [4–7]. It has been demonstrated that chemical modification of electrodes with SWCNT enhances sensitivity for detection of analytes [8–10]. Metallophthalocyanine (MPc) have been used to functionalize SWCNT, covalently [11] or noncovalently (π–π interaction) [12], in order to improve electron transfer processes, in electrocatalysis. Electrooxidation of chlorophenols on glassy carbon electrode (GCE) modified with nickel based cyclam,

porphyrin and phthalocyanines has been reported [13–15]. These complexes were found to reduce fouling of electrodes to different extents [13, 16–18]. Yin et al. [19] reported on the use of cobalt phthalocyanine carbon paste electrode (CPE) and multiwalled carbon nanotubes (MWCNT) for detection of phenolic compounds and showed improved stability in the presence of carbon nanotubes. Hence in this work a series of NiPc derivatives: β-NiPc(NH2)4,β-NiPc(OH)4, α-NiPc(OH)4 and α-NiPc(OH)8 complexes (Figs 1a–1d) are adsorbed on SWCNT and employed to catalyze 4-chlorophenol, a well known pollutant [20]. The hydroxy group was chosen since hydroxyl substituted macrocycles such as porphyrins are known to be good electrocatalysts [21]. The amine group was also chosen because it can be chemically linked to SWCNT via amide bond. In addition, the electron donating nature of these substituents would make oxidation easier and hence improve their catalytic behavior. The effect of peripheral (β) vs. non-peripheral (α) substitution and the number of substituents on the behavior of the NiPc modified electrodes towards the detection of 4-chlorophenol will be explored as well the effects of chemically linking SWCNT

Single walled carbon nanotubes functionalized with nickel

phthalocyanines: effects of point of substitution and nature of

functionalization on the electro-oxidation of 4-chlorophenol

Samson Khene and Tebello Nyokong*

Department of Chemistry, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa

Received 28 July 2011Accepted 2 October 2011

ABSTRACT: In this work we report on electrochemical behavior of nickel phthalocyanine derivatives tetrasubstituted peripherally and non-peripherally with hydroxy and used to modify single walled carbon nanotubes. Nickel phthalocyanine complex octasubstituted at the peripheral positions with hydroxy groups was also used to modify single walled carbon nanotubes. Nickel phthalocyanine complex tetrasubstituted with amino groups at peripheral position was covalently and non-covalently linked to single walled carbon nanotubes. All the conjugates of nickel phthalocyanine derivatives with single walled carbon nanotubes were used for the electro oxidation of 4-chlorophenol. The nickel phthalocyanine octabsubstituted with hydroxy groups at the non-peripheral positions gave the best current response and the best resistance against electrode fouling for the oxidation of 4-chlorophenol.

KEYWORDS: nickel phthalocyanine, single-walled carbon nanotube, cyclic voltammetry, chlorophenol,electrocatalysis.


*Correspondence to: Tebello Nyokong, email: [email protected], tel: +27 46-603 8260, fax: +27 46-622 5109


SINGLE WALLED CARBON NANOTUBES FUNCTIONALIZED WITH NICKEL PHTHALOCYANINES 131

to -NiPc(NH2)4 via amide bond to form β-NiPc(NH2)4-SWCNT(linked) (5) as opposed to β-NiPc(NH2)4 adsorbed (without a chemical bond) on SWCNT (represented as β-NiPc(NH2)4-SWCNT(ads), 6). The GCE modified with the rest of the NiPc derivatives are represented as: GCE- -NiPc(NH2)4, (1), GCE-β-NiPc(OH)4 (2), GCE- -NiPc(OH)4 (3) and GCE- -NiPc(OH)8 (4) in the absence of SWCNT. When adsorbed (ads) onto SWCNT, the rest of the electrodes are represented as: GCE-β-NiPc(OH)4-SWCNT (ads) (7), GCE-α-NiPc(OH)4-SWCNT(ads) (8)and GCE- -NiPc(OH)8-SWCNT(ads) (9) (see Table 1).


The structures formed by chemical coordination of NiPc(NH2)4 to SWCNT may be represented by the dendrimer shown in Fig. 1e. This has been suggested before for the coordination of FePc(NH2)4 to SWCNT using UV-vis spectroscopy [11]. For adsorbed species, carbon nanotubes NiPc derivatives are adsorbed onto SWCNT through π–π interactions (Fig. 1f). The π–πinteraction preserves the sp2 nanotube structure and thus their electronic characteristics.

Table 1. Electrochemical parameters for electrode characterization using [Fe(CN)6]3-/[Fe(CN)6]

4- in 0.1 M KCl and the detection of 0.7 mM 4-chlorophenols in 0.1 mM NaOH. Potentials vs. Ag|AgCl

Electrodes Electrode number E/V 4-chlorophenol/V 4-chlorophenolpeak current, μA

Regeneration, %

Bare GCE none 0.09 0.40 17.9 67

GCE-SWCNT none 0.50 0.40 25.2 57

GCE-β-NiPc(NH2)4 (1) 0.13 0.41 35.4 35

GCE-β-NiPc(OH)4 (2) 0.24 0.43 18.1 81

GCE-α-NiPc(OH)4 (3) 0.25 0.36 28.6 63

GCE-α-NiPc(OH)8 (4) 0.18 0.36 29.0 69

GCE-β-NiPc(NH2)4-SWCNT(linked)

(5) 0.19 0.40 20.1 70

GCE-β-NiPc(NH2)4-SWCNT(ads)

(6) 0.14 0.36 23.9 68

GCE-β-NiPc(OH)4-SWCNT(ads)

(7) 0.13 0.38 20.1 86

GCE-α-NiPc(OH)4-SWCNT(ads)

(8) 0.14 0.36 22.9 76

GCE-β-NiPc(OH)8-SWCNT(ads)

(9) 0.27 0.37 41.0 96

Spectroscopic characterization

Figure 2 shows a Raman spectra of SWCNT (a), α-NiPc(OH)4 (b) and α-NiPc(OH)4-SWCNT(ads) (c). In order to minimize self-absorption and to obtain a good signal-to-noise ratio, KBr powder was used to dilute all complexes for recording of Raman experimental data. The Raman spectra provide an insight on the extent of functionalization of the SWCNT. The spectrum in Fig. 2a shows a characteristic ring breathing mode (RBM) at 168 cm-1, tangential vibrational mode at 1596 cm-1 (the G band) and vibrational mode due to disorder on the hexagonal lattice on the SWCNT sidewalls at 1271 cm-1

(the D band) [22]. Figure 2b shows the Raman spectra of -NiPc(OH)4 with intense bands at 756, 1075, 1340 and

1546 cm-1. A band in the region 1546 cm-1 (due to C–N–C

stretch) is known to show a remarkable sensitivity to the metal ion present and provides a specific signature for phthalocyanines [22–26]. The band at 1340 cm-1 is due to deformation of the ring. Figure 2c shows that the interaction between SWCNT and -NiPc(OH)4

causes the D band to shift from 1271 cm-1 to 1287 cm-1. This significant shift of 18 cm-1 suggests a strong interaction between SWCNT and -NiPc(OH)4. Similar Raman spectral changes were observed for all the NiPc derivatives in the presence and absence of SWCNT.

Figure 3 shows the transmission electron microscopy (TEM) image of SWCNT (a), -NiPc(OH)8–SWCNT(ads)(b), initially dispersed through ultrasonication in DMF. Figure 3(b) shows ropes of SWCNT coated with aggregates of -NiPc(OH)8 complex, thus further


132 S. KHENE AND T. NYOKONG

NiHO N

N N N

N

NNNOH

OH

HO

Ni

HO

HO

OH

OH

N

N N N

N

NNN

Ni N

N N N

N

NNN

HO

OH

OH

OH

OHHO

OH

OH

NiH2N N

N N N

N

NNNNH2

NH2

H2N

(a) (b)

(c) (d)

N

H

Ni

N N

N

NN

N

N

N

NH2

N

H

NH

O

C

N

HNi

N N

N

NN

N

N

N

N

N

H

H

O

Ni

NN

N

NN

N

N

N

N

H

OMPcMPc

MPc

N

H

Ni

NN

N

NN

N

N

N

N

H

N H

O

C

MPc

HN

NH2

H2N

H2N

NH

C CS

W

C

N

T

(e)

Fig. 1. (a) Molecular structure of β-NiPc(OH)4, (b) -NiPc(OH)4, (c) -NiPc(OH)8, (d) β-NiPc(NH2)4, (e) possible structure formed on linking β-NiPc(NH2)4 to SWCNT and (f) representation of NiPc adsorbed onto SWCNT



confirming the structural integrity of β-NiPc(OH)8–SWCNT(ads). The black spots observed on SWCNT before adsorption of NiPc are consistent with a known fact that carbon nanotubes are not 100% pure but consist of metal catalyst (which are difficult to remove) used in the synthesis of SWCNT [23].

Cyclic voltammetric characterization of adsorbed species

The [Fe(CN)6]3-/[Fe(CN)6]

4- couple is often employed to characterize the blocking ability of surfaces. Figure 4ashows cyclic voltammograms of 1 mM [Fe(CN)6]

3-/[Fe(CN)6]

4- in 0.1 M KCl on bare GCE, GCE-SWCNT, GCE-β-NiPc(OH)4-SWCNT(ads) (7) and GCE- -NiPc(OH)8-SWCNT(ads) (9) as representatives of the

rest of the electrodes. All the modified electrodes showed the [Fe(CN)6]

3-/[Fe(CN)6]4- redox probe, hence do not

block the electrode effectively. Table 1 lists the anodic to cathodic peak separations (ΔE) for the [Fe(CN)6]

3-/[Fe(CN)6]

4- redox probe on the various electrodes.The lack of blocking of [Fe(CN)6]

3-/[Fe(CN)6]4- redox

probe by MPc complexes has been reported previously for adsorbed cobalt tetra-amino phthalocyanine films on glassy carbon electrode [27]. In terms of current, GCE-β-NiPc(NH2)4-SWCNT(ads) (6), GCE-β-NiPc(OH)4-SWCNT(ads) (7) and GCE-α-NiPc(OH)4-SWCNT(ads)(8) (only 7 shown in Fig. 4a, for clarity of the figure) showed larger currents than bare GCE. GCE- β-NiPc(NH2)4-SWCNT(linked) (5) (not shown in Fig. 4a), GCE- -NiPc(OH)8-SWCNT(ads) (9) and GCE-SWCNT showed lower currents than the bare GCE,

Fig. 1. (Continued )

1600 1400 1200 1000 800 600 400 200

0.00

0.01

0.02

0.03

1600 1400 1200 1000 800 600 400 200

0.00

0.02

0.04

0.06

0.08

0.10 1600 1400 1200 1000 800 600 400 200

0.00

0.01

0.02

0.03

0.04

0.05

SWCNT (a)1596

1271 RBM

Wavenumber (cm-1)

α−NiPc(OH)4-SWCNT (ads)

1287

168

Inte

ns

ity α−NiPc(OH)

4 (b)

(c)

7561075

13401546

Fig. 2. Raman spectra of SWCNT (a), α-NiPc(OH)4 (b) and -NiPc(OH)4-SWCNT(ads) (c)



the latter two showed highly distorted voltammograms, hence showing slow kinetics and some blocking of the [Fe(CN)6]

3-/[Fe(CN)6]4- couple. The ΔE value for

the [Fe(CN)6]3-/[Fe(CN)6]

4- couple are 0.09 V for bare GCE, 0.13 V for GCE-β-NiPc(OH)4-SWCNT(ads)(7), 0.14 V for GCE-α-NiPc(OH)4-SWCNT(ads) (8),and GCE- -NiPc(NH2)4-SWCNT(ads) (6), 0.19 V for

GCE-β-NiPc(NH2)4-SWCNT (linked) (5), 0.27 V for GCE- -NiPc(OH)8-SWCNT(ads) (9) and 0.50 V for GCE-SWCNT (Table 1). Thus the largest ΔE value is for GCE-SWCNT. The ΔE values suggest that adsorbing NiPc complexes on SWCNT improves the rate of electron transfer compared to SWCNT alone. GCE- -NiPc(NH2)4-SWCNT(ads) (6), GCE- -NiPc(OH)4-SWCNT(ads) (7)

Fig. 3. TEM image of SWCNT before (a) and after (b) functionalization with NiPc(OH)8 at 200 nm resolution

Fig. 4. Cyclic voltammograms of 1 mM [Fe(CN)6]3-/[Fe(CN)6]

4- in 0.1 M KCl on (a) bare GCE, GCE- -NiPc(OH)4-SWCNT (ads) (7), GCE-α-NiPc(OH)8-SWCNT(ads) (9) and GCE-SWCNT; (b) GCE-β-NiPc(OH)4 (2), GCE-α-NiPc(OH)4 (3) and GCE-α-NiPc(OH)8 (4). Scan rate = 100 mV.s-1



and GCE-α-NiPc(OH)4-SWCNT(ads) (8) have similar ΔE values (ΔE = 0.13 or 0.14 V), suggesting similar rate of electron transfer which is faster compared to the rest of the electrodes.

The ΔE values of SWCNT modified with β-NiPc(OH)4

(7) and α-NiPc(OH)4 (8) are similar at 0.13 and 0.14, respectively, showing that the point of substitution has no effect on electrode kinetics. Comparing GCE-α-NiPc(OH)4-SWCNT(ads) (8) with GCE-α-NiPc(OH)8-SWCNT(ads) (9), with ΔE = 0.14 and 0.27, respectively, shows that the latter has about double the ΔE value of the former, suggesting that the number OH functional groups has an effect on the rate of electron transfer, when adsorbed on SWCNT. It is known that the rate of electron transfer depends on the orientation of aromatic complexes on SWCNT [28]. Thus the above results suggest that β-NiPc(OH)4 and -NiPc(OH)4 have similar orientation on SWCNT which is different from the orientation of

-NiPc(OH)8 containing a larger number of substituents. When GCE-β-NiPc(OH)4-SWCNT(ads) (7) is compared to GCE-β-NiPc(NH2)4-SWCNT(ads) (6), the ΔE values obtained for both electrodes are very close to each other, 0.13 and 0.14 V respectively. This suggests that the rate of electron transfer, for NiPcs adsorbed on SWCNT, is not affected by the electron donating ability of the NH2

group. β-NiPc(NH2)4 was then covalently linked to SWCNT via amide bond and its electrochemical behavior compared to that of -NiPc(NH2)4 adsorbed on SWCNT. The ΔE value of β-NiPc(NH2)4-SWCNT (linked) (5)is higher (ΔE = 0.19 V) compared to β-NiPc(NH2)4-SWCNT(ads) (6, ΔE = 0.14 V), suggesting that linking SWCNT to β-NiPc(NH2)4 via covalent bond reduces the rate of electron transfer.

Figure 4b shows cyclic voltammograms (in the absence of SWCNT) for GCE- -NiPc(OH)4 (2), GCE-

-NiPc(OH)4 (3) and GCE-α-NiPc(OH)8 (4) in 1 mM [Fe(CN)6]

3-/[Fe(CN)6]4- in 0.1 M KCl, with ΔE values

of 0.24, 0.25 and 0.18 V, respectively. Again, the ΔEvalues suggest that the number OH functional groups have an effect on the rate of electron transfer in the absence of SWCNT and again the point of substitution has no effect on the ΔE values as was the case in the presence of SWCNT. As already stated, in the absence of SWCNT, α-NiPc(OH)8 (4) with ΔE = 0.18, shows fast kinetics compared to α-NiPc(OH)4 (3) with ΔE = 0.25 V. The opposite was true in the presence of SWCNT as discussed above.

The effective electrode (real) area of the unmodified GCE was estimated by using the [Fe(CN)6]

3-/4- redox system and applying the Randles-Sevcik (Eq. 1) for a reversible process [29]:

Ipa = (2.69 × 105)n3/2D1/2υ1/2ACo (1)

where D and Co are the diffusion coefficient and bulk concentration of the redox probe 1 mM K3[Fe(CN)6],respectively. n is the number of electrons transferred

(n = 1), v is the scan rate and A is the effective electrode coating geometric area (= 0.079 cm2). Cyclic voltammetry experiments at different scan rates were performed with the modified electrodes containing NiPc derivatives in the absence of SWCNT (1–4) immersed in a solution of 1 mM K3[Fe(CN)6] in 0.1 M KCl. The D value for K3[Fe(CN)6] = 7.6 × 10-6 cm2.s-1 [24]. These studies could only be done for complexes in the absence of SWCNT since in the presence of SWCNT, there was no clear peak due to the adsorbed NiPc derivative.

Figure 5 shows the CV of adsorbed β-NiPc(OH)4

species as an example. The quasi-reversible peak is attributed to adsorbed NiPc derivative as observed before [30]. The surface coverage was calculated by using the charge under the adsorption peak in Fig. 5, according to Eq. 2 [31].

GMPc =Q

n AF(2)

where Q is the background corrected charge, n is the number of electrons transferred (= 1), F is Faraday’s constant and A is the real area of the bare electrode. The total surface coverage of the electrode NiPc modifier films (in the absence of SWCNT) ranged from 8.40 ×10-10 to 4.3 × 10-9 mol.cm-2. The determined values are higher than the order of magnitude of 1 × 10-10 mol.cm-2

observed for a Pc molecule lying flat, suggesting that the electrode surface is more than a monolayer [32].

Electrochemical oxidation of 4-chlorophenol

Adsorbed complexes. These studies were carried out in alkaline media where chlorophenols are deprotonated. Under alkaline conditions, NiPc derivatives readilytransform into O–Ni–O when adsorbed, however the O–Ni–O bridges did not form for NiPc adsorbed on in SWCNT. The O–Ni–O transformation has been studied and characterized elsewhere [33–35]. In the absence of SWCNT, O–Ni–O bridges formed. Covalently linked NiPc to SWCNT was able to transform into O–Ni–O

Fig. 5. Cyclic voltammogram of GCE-β-NiPc(OH)4 (ads) (2)in buffer solution of pH 9.2. Scan rate = 100 mV.s-1



bridge when cycled in 0.1 M NaOH solution (see discussed below).

Figure 6 shows the cyclic voltammograms for the oxidation of 0.7 mM of 4-chlorophenol in 0.1 mM NaOH aqueous solution on bare GCE, GCE-β-NiPc(NH2)4-SWCNT(linked) (5), GCE-β-NiPc(NH2)4-SWCNT(ads) (6) and GCE-α-NiPc(OH)4-SWCNT(ads) (8), as repre-sentatives of all electrodes. Table 1 list the peak potentials and currents for the oxidation of 4-chlorophenol on the various electrodes. The irreversible redox process, which corresponds to oxidation of 4-chlorophenol, is observed at potentials of 0.36 V for GCE- -NiPc(OH)4-SWCNT(ads) (8) and GCE-β-NiPc(NH2)4-SWCNT(ads) (6), 0.37 V for GCE- -NiPc(OH)8-SWCNT(ads) (9), 0.38 V for GCE- -NiPc(OH)4-SWCNT(ads) (7) and 0.40 V for bare GCE, GCE-SWCNT and GCE- -NiPc(NH2)4-SWCNT(linked) (5) (Table 1). Thus in terms of potential (and in the presence of SWCNT), all electrodes performed equally or better than the bare GCE. Comparing 7 and 8 shows no major difference in terms of potential for 4-chlorophenol oxidation, hence showing no effect of the point of substitution. Comparing 8 and 9 in terms of potential also shows no effects on the number of substituents on 4-chlorophenol oxidation, the same applies to 6 and 7,where the effects of the different substituents is not clear. In terms of current, GCE-α-NiPc(OH)8-SWCNT(ads) performed better compared to all other electrodes in Table 1. Even though for α-NiPc(OH)4 (8) and α-NiPc(OH)8 (9) adsorbed on SWCNT catalytic activity in terms of potential remains approximately the same, the current for 4-chlorophenol improved considerably for the latter suggesting that the number of substituents, which will affect orientation, result in improved catalytic activity in terms of current. The bare GCE and GCE-SWCNT gave the lowest current for 4-chlorophenol detection.

Peaks observed in the absence of SWCNT are also listed in Table 1. These values are 0.36 V, 0.36 V,

0.41 V and 0.43 V for GCE-α-NiPc(OH)4 (3) GCE- α-NiPc(OH)8 (4) GCE-β-NiPc(NH2)4 (1) and GCE-β-NiPc(OH)4 (2), respectively. Non-peripherally substituted derivatives showed improved potential in the absence of SWCNT compared to peripherally substituted derivatives (compare 2 and 3), contradicting results in the presence of SWCNT where there was no significant change in potential. However change of substituent from NH2 (1)to OH (2) did not affect the potential for 4-chlorophenol oxidation in the absence of SWCNT as was the case in the presence of the latter.

Table 1 suggests that the catalytic activity of β-NiPc(OH)4 towards the oxidation for 4-chlorophenol is improved in terms of potential when adsorbed on SWCNT (peak potential, Ep = 0.38 V for (7) compared to Ep = 0.43 V in the absence of SWCNT (2)). In terms of current, there is some improvement in the presence of SWCNT for -NiPc(OH)4. Similar behavior was observed also for β-NiPc(NH2)4 in terms of potential (comparing 1with Ep = 0.41 compared to 6 with Ep = 0.36 V).

It can be concluded that non-peripheral substitution improves catalytic behavior in terms of current compared to peripheral substitution in the presence (compare 7 and 8) or absence (compare 2 and 3) of SWCNT. In terms of potential, SWCNT do not improve the catalytic behavior of non-peripherally substituted derivatives, but improved that of peripherally substituted ones. This could be related to the orientation of the differently substituted derivatives onto the SWCNT.

Linking versus adsorption. In order to determine the effect of covalently linking NiPc to SWCNT, on electrocatalysis of 4-chlorophenol, β-NiPc(NH2)4 was linked to SWCNT (via amide bond) to form GCE- β-NiPc(NH2)4-SWCNT(linked) (5) and compared to GCE-β-NiPc(NH2)4(ads) (6). The GCE-β-NiPc(NH2)4-SWCNT(linked) (5) or GCE-β-NiPc(NH2)4 (ads) (6) were then cycled in 0.1 M NaOH to give the well documented peaks [34, 35] due to Ni(III)/Ni(II) (Fig. 7) following the formation of O–Ni–O. The redox potential at which Ni(III)/Ni(II) occurs for NiPc(NH2)4-SWCNT(linked) is around 0.3 V and for NiPc(NH2)4 in the absence of SWCNT was around 0.5 V (Fig. 7, insert). The shift to lower oxidation potential suggest that linking NiPc(NH2)4

to SWCNT lowers the energy required to drive the Ni(III)/Ni(II) redox process.

The E of the irreversible Ni(III)/Ni(II) redox process is 0.15 V for the linked NiPc(NH2)4-SWCNT and 0.25 V for -NiPc(NH2)4 [34]. The small Evalue for β-NiPc(NH2)4-SWCNT (linked) compared to β-NiPc(NH2)4 suggests that the rate of electron transfer for Ni(III)/Ni(II) redox process is enhanced when β-NiPc(NH2)4 is linked to SWCNTs. The above results suggest that SWCNT have an effect on the redox properties of Ni(III)/Ni(II) process, by lowering the oxidation potential where Ni(III)/Ni(II) redox process occurs with respect to adsorbed β-NiPc(NH2)4 where the process was not observed.

Fig. 6. Oxidation of 0.7 mM of 4-chlorophenol in 0.1 M NaOH aqueous solution on bare GCE, GCE-β-NiPc(NH2)4-SWCNT(linked) (5), GCE-β-NiPc(NH2)4-SWCNT (ads) (6)and GCE-α-NiPc(OH)4-SWCNT (8). Scan rate = 100 mV.s-1



GCE- -NiPc(NH2)4-SWCNT(ads) (6) gave an oxidation potential of 4-chlorophenol at 0.36 V compared to 0.40 V obtained with GCE-β-NiPc(NH2)4-SWCNT(linked) (5)and the bare GCE (Table 1). Thus, the oxidation potential obtained for 4-chlorophenol using -NiPc(NH2)4-SWCNT(ads) (6) occurs at a slightly lower values compared to GCE-β-NiPc(NH2)4-SWCNT (linked) (5), suggesting that adsorbing β-NiPc(NH2)4 on SWCNT is better than covalently linking the two together for lowering the oxidation potential of 4-chlorophenol, even though the discussion above shows better kinetics on linking. Non-covalent functionalizing of SWCNT via π–π interaction, preserves the sp2 nanotube structure and thus the electronic characteristics.

Electrode stability. Figure 8 shows cyclic voltam-mograms of 0.7 mM 4-chlorophenol in 0.1 M NaOH

solution catalyzed by GCE-α-NiPc(OH)4-SWCNT(ads). The second scan (Fig. 8(b)) shows a considerable decrease in current compared to the first scan (Fig. 8(a)). Figure 8(c) shows the scan obtained after rinsing the modified electrode with Millipore water, hence shows that there was recovery of the oxidation currents. Table 1 shows the percentage recoveries obtained for all other electrodes studied.

Of all the modified electrodes,GCE-α -NiPc(OH) 8-SWCNT(ads) (96% recovery) gave the best recovery against electrode fouling, followed by GCE-β-NiPc(OH)4-SWCNT(ads) at 86% recovery. In all cases, the % recovery considerably improves in the presence of SWCNT, showing the huge influence of SWCNT on the stability of the electrode.

EXPERIMENTAL

Material

Single walled carbon nanotube (SWCNTs, 0.7–1.2 nm in diameter and 2–20 m in length) were purchased from Sigma Aldrich. Dimethylformamide (DMF) and sodium hydroxide pellets were obtained from SAARCHEM. 4-Chlorophenol (4-CP), and ferricyanide (K3Fe(CN)6),ferrocyanide (K4Fe(CN)6) were purchased from Sigma-Aldrich. Aqueous solutions were prepared using millipore water from Milli-Q Water Systems (Millipore Corp., Bedford, MA, USA, conductivity range = 0.055–0.294 μS.cm-1). Synthesis and characterization of -NiPc(NH2)4,

-NiPc(OH)4, α-NiPc(OH)4 and α-NiPc(OH)8 have been reported before [33, 36]. The linking of SWCNT to β-NiPc(NH2)4 to form β-NiPc(NH2)4-SWCNT(linked) has been described in detail elsewhere [11]. The conjugate was characterized by various techniques including: Raman, Fourier transform infrared (FT-IR), UV-vis and X-ray diffraction spectroscopies and will not be repeated here.

Electrochemical methods

Cyclic voltammetry (CV) data were obtained withAutolab potentiostat PGSTAT 30 (Eco Chemie, Utretch, The Netherlands) driven by the General Purpose Electrochemical Systems data processing software (GPES, version 4.9, Eco Chemie), using a conventional three-electrode set up with glassy carbon electrode (GCE, area = 0.071 cm2) as a working electrode, platinum wire as counter electrode and Ag|AgCl pseudo reference electrode. The potential response of the Ag|AgCl pseudo reference electrode was less than the Ag|AgCl (3 M KCl) by 0.015 ±0.003 V. Argon was bubbled for 15 min prior to

Fig. 7. Cyclic voltammograms of α-NiPc(OH)4-SWCNT in 0.1 M NaOH, forming poly-Ni(OH)Pc(OH)4-SWCNT. Scan rate = 100 mV.s-1. Insert: Cyclic voltammogram showing the Ni(III)/Ni(II) process for GCE-β-NiPc(NH2)4 (a) and GCE-β-NiPc(NH2)4-SWCNT(linked) (b) in 0.1 M NaOH solution

Fig. 8. Cyclic voltammograms of 0.7 mM 4-chlorophenol in 0.1 M NaOH solution catalyzed by GCE-α-NiPc(OH)4-SWCNT(ads) (8) showing the first (a), second (b) and the scan(c) obtained after rinsing the modified electrode with Millipore water. Scan rate = 100 mV.s-1



the recording of cyclic voltammograms and an atmosphere of Ar was maintained throughout the CV scans.

Prior to use, the glassy carbon electrode surface was polished with alumina on a Buehler felt pad and rinsed with excess millipore water. Following modifications, the electrodes were rinsed several times with DMF to remove the catalyst used to modify the electrode. The colloidal solutions of SWCNT or SWCNT functionalized with NiPc derivatives were prepared by adding 1 mg of each to 1.5 mL DMF. The GCE was modified by the drop dry method, whereby a drop of the colloidal solution of each modifier was placed onto the electrode and allowed to dry for 10 min at 80 °C. For complexes without SWCNTs, the GCE was modified by placing a drop of DMF solutions of β-NiPc(NH2)4, β-NiPc(OH)4,α-NiPc(OH)4 or α-NiPc(OH)8 on GCE followed by drying in nitrogen atmosphere. The modified electrodes in the absence of SWCNT are represented as: GCE-SWCNT, GCE-β-NiPc(NH2)4, (1), GCE-β-NiPc(OH)4

(2), GCE- -NiPc(OH)4 (3), GCE-α-NiPc(OH)8 (4).When adsorbed onto (ads) or linked to SWCNT, the electrodes are represented as: GCE- -NiPc(NH2)4-SWCNT(linked) (5), GCE-β-NiPc(NH2)4-SWCNT(ads)(6), GCE-β-NiPc(OH)4-SWCNT(ads) (7), GCE- -NiPc(OH)4-SWCNT(ads) (8) and GCE-α-NiPc(OH)8-SWCNT(ads) (9). The numbers in brackets are used to identify the modified electrodes in order to make it easier to follow the discussion.

Equipment

Raman spectra were acquired with Bruker Vertex70-Ram II spectrometer (equipped with a 1064 nm Nd:YAG laser and a liquid nitrogen cooled germanium detector). Solid samples containing KBr were employed. FT-IR spectra (KBr pellets) were collected with Perkin-Elmer FT-IR spectrometer. Transmission electron microscope (TEM) images were recorded using JEOL JEM 1210 at 100 KV accelerating voltage

Purification of single walled carbon nanotubes and adsorption of NiPc derivatives onto SWCNT

The SWCNTs were purified by following methods described previously in literature [37–39]. Briefly, SWCNTs (100 mg) were suspended in a mixture of concentrated HNO3:H2SO4 (1:3) and stirred at 70 °C for 2 h. The SWCNTs were separated from solution and washed with distilled water. The purified SWCNTs were dried in the oven at 70 °C over night. This purification method also functionalizes the carbon nanotube with carboxylic acid functional groups.

The conjugates of NiPcs and SWCNT: β-NiPc-(NH2)4-SWCNT(ads),β-NiPc(OH)4-SWCNT(ads), β-NiPc-(OH)4-SWCNT(ads) and GCE-α-NiPc(OH)8-SWCNT(ads) were formed by mixing the corresponding complexes (20 mg) and SWCNT (1.0 mg) in DMF (1.5 mL), followed by ultrasonication for 10 min. The solid product was

separated from the solution by centrifuging and several washings with DMF (to remove excess unreacted NiPc) to give the adsorbed species.

CONCLUSION

In conclusion we have shown in this work that GCE electrode modified with -NiPc(NH2)4-SWCNT (linked) (5), -NiPc(NH2)4-SWCNT(ads)(6), β-NiPc(OH)4-SWCNT(ads) (7), α-NiPc(OH)4-SWCNT(ads) (8) and α-NiPc(OH)8-SWCNT(ads) (9)exhibit electrocatalytic activity towards the oxidation of 4-chlorophenol. We have shown that in general SWCNT improves the electrocatalytic activity of NiPc complexes, when adsorbed on SWCNT. SWCNT improves the resistance to electrode fouling by NiPc complexes. NiPcs adsorbed on SWCNT showed better response to electrocatalytic oxidation of 4-chlorophenol compared to the bare GCE and NiPc linked to SWCNT electrodes.

Acknowledgements

This work was supported by the Department of Science and Technology (DST) and National Research Foundation (NRF) of South Africa through DST/NRF South African Research Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology, and Rhodes University.

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DOI: 10.1142/S108842461200446X



INTRODUCTION

Porphyrins, phthalocyanines, and porphycenes are macromolecules with huge number of delocalized πelectrons resulting in interesting third order nonlinear optical (NLO) properties leading to extensive applications in optical limiting, all-optical switching, and optical signal processing [1–6]. Corroles are tetrapyrrolic molecules

maintaining the skeletal structure of Corrin with its three meso carbon positions and one direct pyrrole-pyrrole linkage and possessing the aromaticity of porphyrins [7–12]. Corroles generally show porphyrin type spectra, with strong absorptions in the visible range associated with very highly colored compounds. Moreover, among the interesting attributes of corroles are their photo physical properties. As well, the direct pyrrole–pyrrole linkage seems to give corroles stronger fluorescence properties than their porphyrin counterparts. These properties open up potential for using corroles in many applications, including diverse areas such as cancer diagnosis, treatment, and

Picosecond and femtosecond optical nonlinearities of novel

corroles

Syed Hamada, Surya P. Tewaria,b, L. Giribabuc and S. Venugopal Rao*b

aSchool of Physics, University of Hyderabad, Hyderabad 500046, IndiabAdvanced Centre of Research in High Energy Materials (ACRHEM), University of Hyderabad, Hyderabad 500046, IndiacInorganic & Physical Chemistry Division, Indian Institute of Chemical Technology, Hyderabad 50007, India

Received 24 August 2011Accepted 5 October 2011

ABSTRACT: We present our results from the experimental and modeling studies of picosecond (ps) and femtosecond (fs) nonlinearities of two novel corroles (a) tritolyl corrole (TTC) (b) triphenyl corrole (TPC) using the Z-scan technique. Both open and closed aperture Z-scan curves were recorded with ~2 ps/~40 fs laser pulses at a wavelength of 800 nm and nonlinear optical coefficients were extracted for both studies. Both the molecules possessed negative nonlinear refractive index (n2) as revealed by signature of the closed aperture data in both (ps and fs) time domains. Picosecond nonlinear absorption data of TPC obtained at a concentration of 5 × 10-4 M demonstrated complex behavior with switching from reverse saturable absorption (RSA) within saturable absorption (SA) at lower peak intensities to RSA at higher peak intensities. TTC data recorded at the similar concentration exhibited saturable absorption (SA) type of behavior at lower peak intensities to switching from RSA with in SA at higher peak intensities. At a concentration of 2.5 × 10-4 M, the ps open aperture data at higher peak intensities illustrated effective three-photon absorption (3PA) for both the molecules. We also report the picosecond spectral dependent Z-scan studies performed at 680 nm, 700 nm, and 740 nm. Nonlinear absorption and refraction of both the samples at these three wavelengths were studied in detail. Femtosecond nonlinear absorption data of TPC and TTC demonstrated the behavior of saturable absorption (SA) at a concentration of 1 × 10-3 M. Solvent contribution to the nonlinearity was also identified. We have also evaluated the sign and magnitude of third order nonlinearity. We discuss the nonlinear optical performance of these organic molecules.

KEYWORDS: picosecond, femtosecond, saturable absorption, effective three photon absorption.

*Correspondence to: S. Venugopal Rao, email: [email protected] and [email protected], tel: +91 040-23138811, fax: +91 040-23012800


PICOSECOND AND FEMTOSECOND OPTICAL NONLINEARITIES OF NOVEL CORROLES 141

solar cell research. Rebane et al. [13, 14] studied the NLO properties and reported two-photon absorption (2PA) cross sections and spectra of corroles within a broad spectral range of excitation wavelengths, 800–1400 nm. They also observed that the strength of 2PA peak in the Soret region strongly decreased with the electron-withdrawing ability of the side substituents. Cho et al. [15] studied corrole dimers and obtained superior values of two-photon cross-section compared to the values obtained by Rebane et al. [13, 14]. Our group has been investigating a number of porphyrins and phthalocyanines for their NLO properties in cw, nanosecond (ns), picosecond (ps) and femtosecond (fs) time domains [16–27]. The motivation for such studies is many fold. For any new molecule synthesized it is imperative to study its NLO properties with different pulse durations and at different wavelengths to identify the resonant and non-resonant contributions to the nonlinearity. A molecule might be useful for optical limiting applications when studied with ns/ps pulses while it’s utility for signal processing or all-optical switching is decided by the femtosecond NLO properties and dynamics. A molecule could be applied for broadband optical limiting or could possess interesting NLO properties at different wavelengths and input intensities. Therefore, for identification of the complete potential of any new molecule, studies at different input conditions (wavelengths, pulse duration, input intensity, surrounding matrix etc.) are indispensable. In this paper we present the results of our studies on nonlinear optical properties of triphenyl corrole (TPC) and tritolyl corrole (TTC) with Z-scan technique with ~2 ps and ~40 fs laser pulses excitation at the same 800 nm wave length. We also report here our results on the ps and fs NLO properties of two corroles [triphenyl corrole and tritolyl corrole, herewith represented as TPC and TTC, throughout this script] studied at same wavelength of 800 nm and picosecond spectral dependent studies at the wavelengths of 680 nm, 700 nm, and 740 nm with the standard Z-scan technique. The sign and magnitude of nonlinear refractive index were derived from the closed aperture Z-scan in both ps & fs domains. From the ps nonlinear absorption data, achieved through the open aperture Z-scans, we observed the two photon absorption (0.5 mM solutions) and three photon absorption (0.25 mM solutions). From the fs Z-scan data we deduced saturable absorption as the main nonlinear absorption mechanism and evaluated their third order nonlinearities. Our studies in ps domain provide sufficient evidence that these molecules possess superior nonlinear coefficients required for applications in optical switching.

EXPERIMENTAL

Synthesis of corroles

TPC and TTC were prepared according to the procedure described below [28]. For obtaining TPC

benzaldehyde (330 mg, 5 m mol) and pyrrole (670 mg, 10 mM) were dissolved in a mixture of methanol and water (400 mL, 1:1 of methanol:H2O). To this 4.25 mL of HCl was added and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was extracted with CHCl3, and the organic layer was washed with H2O and dried over anhydrous Na2SO4. To this p-chloranil (1.23 g, 5 mM) was added, and the mixture was refluxed for 1 h. The reaction mixture subjected to silica gel column chromatography and eluted with CHCl3:hexane (1:1 v/v). The solvent front running brown color band was collected and re-crystallized from CHCl3:hexane afforded pure corrole. For the synthesis of TTC P-tolylaldehyde (600 mg, 5 mM) and pyrrole (670 mg, 10 mM) were dissolved in a mixture of methanol and water (400 mL, 1:1 of methano:H2O). To this 4.25 mL of HCl was added, and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was extracted with CHCl3, and the organic layer was washed with H2O and dried over anhydrous Na2SO4. To this p-chloranil (1.23 g, 5 mM) was added, and the mixture was refluxed for 1 h. The reaction mixture subjected to silica gel column chromatography and eluted with CHCl3:hexane (1:1 v/v). The solvent front running brown color band was collected and re-crystallized from CHCl3:hexane afforded pure corrole. The spectroscopic data for these compounds is as follows. TPC: UV-vis (in CH2Cl2): λmax, nm (ε M-1.cm-1) 646 (8,600) 616 (10,200), 575 (17,100), 415 (1,12,000). TTC: UV-vis (in CH2Cl2):λmax, nm (ε M-1.cm-1) 649 (9,600) 621 (9,200), 575 (9,700), 418 (1,32,000).

Each sample was subjected to a column chromatographic purification process prior to the NLO measurements. The details of molecular structure and the absorption spectra have been detailed in Figs 1 and 2. All the experiments were performed with the samples dissolved in chloroform and placed in 1-mm glass/quartz cuvette. Picosecond pulses and fs pulses were generated by separate Ti:sapphire lasers (Coherent, Legend amplifiers) operating at a repetition rate of 1 kHz with a pulse durations ~2 ps and ~40 fs at 800 nm. The amplifiers were seeded with ~15 fs pulses from

N

NH HN

HN

RR

R

Fig. 1. Structures of corroles used (a) R = H, triphenyl corrole (TPC), molecular formula = C37H26N4; (b) R = -CH3, tritolyl corrole (TTC), molecular formula = C40H32N4


142 S. HAMAD ET AL.

the oscillator (Coherent, Micra). The input beam was spatially filtered to obtain a pure Gaussian profile in the far field. Z-scan studies [19, 24, 29] were performed by focusing the 3-mm diameter input beam using a 200 mm focal length convex lens into the sample in both psec and fsec domains. The sample was placed on a high resolution translation stage and the detector (Si photodiode, SM1PD2A, Thorlabs) output was connected to a lock-in amplifier. Both the stage and lock-in were controlled by a computer program. The picosecond studies were performed with solutions having the concentration of 0.5 mM providing ~75% (for TPC) and ~70% (for TTC) linear transmittance with

800 nm wavelength and picosecond spectral dependent studies were performed with same solution having the same concentration providing ~65% (for TPC) and ~55% (for TTC) linear transmittance at 680, 700 and 740 nm wavelengths. Picosecond studies with 0.25 mM (85% transmittance) were performed with very high peak intensities to completely understand the intensity dependent nonlinear absorption. Femtosecond studies were performed with solutions having concentration of ~1 mM providing 90% linear transmittance at the 800 nm wavelength. Closed aperture scans were performed at intensities where the contribution from the higher order nonlinear effects is negligible (the value of Δφestimated in all the cases was <π). The experiments were repeated more than once and the best data were used for obtaining the nonlinear optical coefficients from the best fits. Schematic of the experiment is shown in Fig. 3.


Picosecond NLO studies at 800 nm, 680 nm, 700 nm and 740 nm

Figures 4(a) and 4(b) illustrate the closed aperture Z-scan data recorded at 800 nm for TPC at an intensity of 33 GW/cm2 and TTC at an intensity of 40 GW/cm2, well below the peak intensities where the contribution from solvent starts to be significant. Both the samples exhibited negative nonlinearity as discovered by the peak-valley signature. The magnitudes of nonlinear refractive indices (n2) evaluated, using the standard procedure, were 1 ×10-14 cm2/W for TPC and 0.6 × 10-14 cm2/W for TTC, respectively. To determine the irradiance dependence of nonlinear absorption, we performed our measurements at different input laser energies. Figures 5(a) and 5(b) present the obtained intensity dependent Z-scan curves for TPC and TTC. In particular, we noted that the increase of laser intensity induced a switching from saturable

Fig. 2. Absorption spectra of (a) TPC and (b) TTC in chloroform. Dotted lines represent the expanded view

Fig. 3. Experimental schematic of Z-scan



absorption (SA) to reverse saturable absorption (RSA) to purely RSA behavior in the case of TPC. For the case of TTC we observed switching from saturable absorption to reverse saturable absorption. In Fig. 5(a) open circles represent the results obtained at high peak intensity of 122 GW/cm2 which clearly demonstrates pure RSA (2PA). Intriguingly, lower intensity data (open squares at 55 GW/cm2 and open stars at 93 GW/cm2, respectively) demonstrates that the nonlinear absorption transformed from SA to RSA. Figure 5(b) shows the Z-scan curves with open squares denoting the data obtained at low peak intensity of 98 GW/cm2. When the linear absorption is significant and with low peak intensity the transmission increases monotonically (SA) and the same was observed at lower peak intensities. However, for increasing intensities (open circles representing the data at 245 GW/cm2 and open stars at 170 GW/cm2, respectively) the nonlinear absorption of sample changed from SA to RSA.

To interpret and fit the data for the flip of saturable absorption around the beam waist, we combined saturable absorption coefficient and two photon absorption (TPA, β) coefficients yielding the total absorption coefficient [30, 31] as;

α α β( )II

IS

=+

+01

1I

(1)

where the first term describes the negative nonlinear absorption and the second term describes positive nonlinear absorption such as reverse saturable absorption and/or two photon absorption α0 is the linear absorption coefficient. I and IS are laser peak intensity and saturation intensity, respectively. β is the nonlinear absorption coefficient. In presence of saturable absorption only the intensity dependent nonlinear absorption is given by the equation [31–33];

α α( )II

IS

=+

01

1(2)

Using Equations 1 and 2, Is and β can be obtained from the open aperture Z-scan data by fitting it to the following equation;

dI

dz′= −α(I)I (3)

where z corresponds to sample length. If the excitation intensity I is lesser than Is, we can consider SA as a third order process and in such cases –(α0/Ιs) is the equivalent of nonlinear absorption coefficient β (negative nonlinear

Fig. 5. Open aperture Z-scan curves obtained for (a) TPC with varying input intensities like (open squares) I00 = 55 GW/cm2,(open stars) I00 = 93 GW/cm2 and (open circles) I00 = 122 GW/cm2 (b) TTC with (open squares) I00 = 98 GW/cm2, (open stars) I00 = 170 GW/cm2 and (open circles) I00 = 245 GW/cm2. The concentration used was 5 × 10-4 M. Solid line is theoretical fit

Fig. 4. Closed aperture Z-scan curves (open circles) for (a) TPC with I00 = 33 GW/cm2 and (b) TTC with I00 = 40 GW/cm2

obtained near 800 nm for a concentration of 5 × 10-4 M. Solid line is theoretical fit


144 S. HAMAD ET AL.

absorption), to a good approximation. Im [χ(3)] can be evaluated from β [33]. The observed behavior can be qualitatively explained using the energy level diagram of corroles depicted in Fig. 6. For lower peak intensities the population in the ground state is bleached initially (S0 states to S1 states). Further pumping leads to excitation into higher excited singlet states (S2 state) since the excited state absorption cross-section is significant at higher peak intensities and this is reflected in the switching of SA to RSA. This can be considered as two-step 2PA (β) and the data has been fitted accordingly [33, 34].

SA generally occurs due to depletion of the ground state population and occurs in the regions near resonance. On other hand, RSA is due to further absorption taking place from first or higher excited singlet states. This is also called excited state absorption (ESA). The condition for occurrence of ESA is that the first excited singlet state S1 should have an absorption cross-section, σex, higher than that of ground state absorption cross-section (σ0)[23]. On the other hand if σ0 is greater than σex, SA is observed. As the wavelength of excitation is shifted closer to (or away from) the wavelength of resonant absorption, σ0 increases (decreases). Consequently the figure of merit of RSA defined as the ratio σex/σ0, decreases (increases) resulting in a changeover of nonlinear absorption mechanism from RSA to SA (SA to RSA). Wavelength region over which nature of NL absorption changes between RSA and SA is very important for optical limiting applications. It is highly

desirable that optical limiter posses broad band response. Figures 7(a)–7(f) show the typical open aperture and closed aperture Z-scan graphs obtained for TPC and TTC samples in the 680–740 nm spectral range. Measured values of IS, β, n2, Im [χ(3)], Re [χ(3)] and |χ(3)| at different wavelengths of excitation are provided in Table 1. Both samples, TPC and TTC, exhibited SA from 680–740 nm, except for TTC at 700 nm with higher peak intensities, though their saturation intensity was found to be slightly different. The condition that the excitation intensity should be less than saturation intensity IS not satisfied in all cases except at 740 nm wavelength. However, at 700 nm, these two samples exhibited different behavior. While TTC showed switching to slight RSA from SA, TPC still exhibited SA. For both the samples IS was found to increase as the excitation wavelength is shifted away from the resonance.

Figures 8(a) and 8(b) shows the open aperture Z-scan data recorded for both TPC and TTC at a concentration of 0.25 mM clearly illustrating RSA kind of behavior at 800 nm recorded at peak intensity of ~400 GW/cm2. It is

Fig. 6. Energy level diagram of corroles explaining the various nonlinear absorption processes

Fig. 7. Z-scan curves (a) open aperture (open stars) and closed aperture (open circles) I00 = 57 GW/cm2 at 680 nm (b) open aperture (open stars) with I00 = 72 GW/cm2 and closed aperture (open circles) with I00 = 54 GW/cm2 at 700 nm and (c) open aperture (open stars) with I00 = 90 GW/cm2 and closed aperture (open circles) with I00 = 32 GW/cm2 at 740 nm for TPC (d) open aperture (open stars) and closed aperture (open circles) with I00 = 76 GW/cm2 at 680 nm (e) open aperture (open stars) with I00 = 108 GW/cm2 and closed aperture (open circles) with I00 = 25 GW/cm2 at 700 nm (f) open aperture (open stars) with I00 = 96 GW/cm2 and closed aperture (open circles) with I00 = 25 GW/cm2 at 740 nm for TTC. The concentration used was 5 × 10-4 M. Open aperture data has been shifted for clarity



evident from the theoretical fits [18–20] that three-photon absorption (3PA) is the dominant nonlinear absorption mechanism. The fits yielded values of γ ~ 10-21 cm3/W2.TTC had higher 3PA coefficient compared to TPC. The solvent (chloroform) contribution, though present, was quite small (less than one order of magnitude) compared to the contribution of solute. At lower peak intensities, and with the same concentration, both samples exhibited complex behavior with switching from RSA with in SA, which is as shown in Figs 9(a) and 9(b). Again, this behavior can be explained using the

energy level diagram provided in Fig. 6. At lower peak intensities SA is dominant while at moderately high peak intensities switching to RSA is observed due to presence of population in the S2 state [34]. At very high peak intensities there could be further absorption from S2

states to Sn states via two possible mechanisms depicted by (a) and (b) in Fig. 6. This can be termed as effective 3PA (two-step 3PA). Following the theory provided below [35] we fitted the data using Equation 5 or 6 to obtain and effective 3PA coefficient.

Table 1. Summary of nonlinear coefficients obtained from the present studies in picosecond domain

Wavelength Sample b × 10-11 cm/W n2 × 10-14

cm2/WIm[χ(3)] ×

10-21 m2/V2Re[χ(3)] ×

10-20 m2/V2χ(3) ×

10-21 m2/V2χ(3) ×

10-12 (e.s.u.)

800 nm TPC 2.8 (2PA) 1.0 1.95 1.11 11.31 5.3

TTC 5.4 (IS = 31 GW/cm2) 0.6 3.77 0.66 7.65 3.6

740 nm TPC -2.9 (IS = 200 GW/cm2) 1.0 -1.87 1.11 11.26 5.3

TTC -6.1 (IS = 150 GW/cm2) 2.0 -3.94 2.22 22.55 10.6

700 nm TPC 0 (IS = 28 GW/cm2) 0.8 — — — —

TTC 1.5 (IS = 8 GW/cm2) 1.0 0.92 1.11 10.14 5.25

680 nm TPC 0 (IS = 16 GW/cm2) 0.5 — — — —

TTC 0 (IS = 35 GW/cm2) 0.7 — — — —

Fig. 8. Open aperture Z-scan curves (open circles) for (a) TPC and (b) TTC obtained near 800 nm for concentration of 2.5 × 10-4 M using an intensity of 350 GW/cm2. Solid line is theoretical fit

Fig. 9. Open aperture Z-scan curves for (a) TPC with two different intensities 110 GW/cm2 (dotted line) and 82 GW/cm2

(solid line) (b) TTC with two different intensities 170 GW/cm2

(dotted line) and 110 GW/cm2 (solid line) obtained at 800 nm for concentration of 2.5 × 10-4 M


146 S. HAMAD ET AL.

Assuming a spatial and temporal Gaussian profile for laser pulses and utilizing the open aperture Z-scan theory for two photon absorption (2PA) given by Sheik-Bahae et al. [29] the equation for 2PA open aperture (OA) normalized power transmittance given by [35, 36];

T PAq z

OA ( )( , )/

21

01 20

=∞

∞

∫πln[1+ ( ,0)exp( )]0

2

-q z dτ τ

(4)

Three-photon absorption (3PA) equation is;

T PAp

OA ( )/

[ ]31

1 20

=

+

∞

∞

∫πln 1+ exp(-2 )

exp(- )

{

}

02 2 1/2

-

02

p

p d

τ

τ τ

For p0 1, and ignoring the higher order terms, we obtain;

T PAI L

z zOA

eff( )( / )( ) /3 1

2

1 3002

02 3 2= −

+γ ¢

(6)

where;

q zI L

z zp z

I L

z z

eff eff0

00

02 0

002

02

01

02

1( , )

( / ), ( , )

( / )=

+=

+

β γ ¢

I00 is the peak intensity, z is the sample position, z0 0

2= πϖ λ is the Rayleigh range: ω0 is the beam waist at the focal point (z = 0), is the laser wavelength; effective path length in the sample of length L for 2PA, 3PA is given as .

Le

Le

eff

L

eff

L

= =1 10 0- --

0

-2

0

α α

α 2α, ¢

We have evaluated the three photon cross-section (σ3)using the relation

sw

g3

2=

( )�N

(7)

where is the frequency of the laser radiation, and N is the number density. The cross-section values estimated for TPC and TTC were 25.9 × 10-76 cm6.s2/photon2 and 2.44 × 10-76 cm6.s2/photon2, respectively. These values represent one of the highest values reported in recent literature [5, 21, 22]. The values of 2P cross-sections evaluated for TPC and TTC (obtained with ps pulses) were ~103 GM. The nonlinear coefficients obtained in this study are comparable to some of the recently reported highly efficient NLO materials [5, 37]. For example, Venugopal Rao et al. [37] reported 2PA coefficients in the range of 0.05–0.13 cm/GW and

3PA coefficients in the range of 3.5–20.0 × 10-21 cm3/W2 for five different porphycenes synthesized recently. Anusha et al. [24, 38] observed 2PA with magnitude of 0.1 cm/GW and 0.275 cm/GW in alkoxy and alkyl phthalocyanines using 2 ps pulses at 800 nm. Kumar et al. [21, 22] observed 3PA with fs pulse excitation in solutions of alkyl phthalocyanines and estimated a magnitude of ~10-22 cm3/W2. Srinivas et al. [39] reported 2PA in porphyrins over a range of wavelengths in the visible spectral region with their magnitudes of ~1 cm/GW but the data was obtained with ns pulses. Venkatram et al. [23] reported large 2PA coefficients in the range of 2000 cm/GW for alkoxy phthalocyanines with 532 nm ns pulses. The coefficients are expected to be larger in the ns regime while in the ps/fs regime the nonlinearities are comparatively smaller. However, the figures of merit obtained for our molecules demand further study on the time response of these nonlinearities.

NLO studies with 800 nm, ~40 fs pulses

Open aperture scans for TPC and TTC recorded at 800 nm using ~40 fs pulses indicated SA apparent from the changes in transmittance values. The concentrations of solutions were ~1 mM. Figures 10(a) and 10(b) illustrate the open aperture data (stars) obtained with

Fig. 10. (a) Fs open aperture for TPC with I00 = 0.7 TW/cm2

(open stars) and closed aperture (open circles) with I00 = 1.0 TW/cm2 and (b) Fs open aperture for TTC with I00 = 0.7 TW/cm2 (open stars) and closed aperture with I00 = 1.0 TW/cm2

(open circles) obtained at 800 nm for a concentration of 1 × 10-3

M. Open aperture data has been shifted up for clarity

(5)



an intensity of 0.7 TW/cm2 and closed aperture data (open circles) with an intensity of 1.0 TW/cm2. The peak intensities were calculated considering the pulse duration to be ~70 fs due to broadening from the optical components (neutral density filters and lenses). An independent experiment performed for measuring the pulse duration indeed confirmed the value to be 70–75 fs. The experiment was performed using Silhouette (Coherent, USA) with optical components in the path. The data was recorded for TPC and TTC well below the intensity levels where the contribution from solvent is significant. Obtained experimental data was fitted using Equations 2 and 3. We found the best fit was obtained with transmittance equation for SA. The fs pulses possess large bandwidth (~26 nm) and in combination with linear absorption at 800 nm we can only expect saturation of the S1 state. For pumping with higher peak intensities we observed Supercontinuum generation from the solvent. Both the samples exhibited negative nonlinearity as discovered by the peak-valley signature in the fs domain also. The magnitudes of the nonlinear refractive indices (n2) evaluated, using the standard procedure, were 3.4 × 10-17 cm2/W for the TPC and 2.5 × 10-17 cm2/W for TTC. The summary of NLO coefficients is presented in Table 2. The solvent nonlinearity was positive suggesting the final values of n2 calculated for the pure solute will be higher than the calculated. Our future endeavor is to study the (1) fs NLO properties of these molecules at different wavelengths in the visible and NIR (2) excited state dynamics of both the molecules using ultrafast pump-probe techniques (3) incorporate these molecules in suitable polymer/glass matrix and later evaluate their NLO properties and dynamics.

CONCLUSION

In summary, the changeover from saturable absorption to reverse saturable absorption in both samples was investigated at the wavelength of 800 nm with 5 × 10-4 M in picosecond domain. It was found that the sample TTC demonstrated saturable absorption at low intensities, with switching from reverse saturable absorption within saturable absorption at high intensities and TPC depicted switching from RSA within SA at lower intensities to pure RSA (TPA) at higher intensities. The shift was analyzed phenomenologically in terms of an intensity-dependent saturable absorption and reverse saturable absorption. We have also investigated the

2 ps spectral dependent Z-scan studies. At 700 nm TPC exhibited predominantly SA while RSA within SA was observed in TTC. Since the structural difference between these two samples is only TPC (R = -CH3) and TTC (R = -H), observed difference in the wavelength dependence of their nonlinear absorption can be due to varying influence of this difference. We have also carried out Z-scan studies with 800 nm, ~40 fs pulses and characterized the nonlinearities in detail. From the fs open aperture data we derived that these molecules exhibit saturable absorption (SA) behavior. Both the samples exhibited negative nonlinear refractive index in both the time domains of investigation. In some of the cases we observed peak intensity being lesser than saturation intensity and we calculated nonlinear absorption coefficients. We measured the values of IS, β,n2, Im [χ(3)], Re [χ(3)] and |χ(3)| for all wavelengths.

Acknowledgements

S. Venugopal Rao acknowledges the financial support from DRDO. SH acknowledges the constant support of Ms. P.T. Anusha and Mr. P. Gopala Krishna during the entire course of experimental work. LG acknowledges the financial support from DST.

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8. D’Souza F, Chitta R, Ohkubo K, Tasior M, Subbaiyan NK, Zandler ME, Rogacki MK, Gryko DT and Fukuzumi S. J. Am. Chem. Soc. 2008; 130:14263–14272.

Table 2. Summary of nonlinear coefficients obtained from the present studies using fs pulses

Sample β(fs) × 10-13

cm/Wn2(fs) × 10-17

cm2/WIm[χ(3)] × 10-23

m2/V2Re[χ(3)] × 10-23

m2/V2χ(3) × 10-23

m2/V2χ(3) × 10-14

(e.s.u)

TPC -1.6 3.4 -1.12 3.77 3.94 1.9

TTC -3.6 2.5 -2.51 2.78 3.74 1.8


148 S. HAMAD ET AL.

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DOI: 10.1142/S1088424611004415



INTRODUCTION

Metalloporphyrins as a model for cytochrome P450

enzymes are known to mediate the oxygen atom transfer to alkenes from various oxidants such as hydrogen peroxide [1–3], periodates [4–8], iodosylarenes [9, 10], peracids [11, 12], sodium hypochlorite [13, 14], potassium monopersulfate [15, 16], tetrabutylammonium hydrogen monopersulfate [17, 18] in a catalytic way. However the search for developing and designing stable, selective and efficient oxidizing catalytic systems has been continuously increasing. The most important strategy to improve this sense is the application of co-catalysts such as imidazole, pyridine, acetate, etc. [19–29].

Cis-stibene is an interesting substrate that can be used to prove the nature of active intermediates in the metal-catalyzed oxidation reactions [30–32].

It has been shown that two different Mn-oxo species could be involved as intermediates in the epoxidation of olefins by different oxidants catalyzed

by manganese porphyrins, an Mn(V)-oxo species leading to high stereoselectivity and an Mn(IV)-oxo species leading to non-streoselectivity in the products. These complexes were characterized by UV-vis and 1H NMR spectroscopy [33–35]. It was mentioned that the involvement of these high-valent manganese-oxo species as oxidizing intermediates are strongly depended on the nature of axial ligand [7, 36, 37]. In most studies, the axial ligands used are neutral ligands, mostly imidazoles and pyridines, and there are little articles about the co-catalytic effects of anionic axial ligands on the epoxidation reactions of olefins catalyzed by manganese porphyrins [18, 20].

In this work, we have studied the effects of some different anionic co-catalysts on the yield and stereo-selectivity of the epoxidation reaction of cis-stilbeneby tetra-n-butylammonium hydrogen monoper-sulfate(n-Bu4NHSO5) in the presence of manganese(III) meso-tetrakis (pentaflurophenyl)porphyrin chloride [MnTPFPP(Cl)] or manganese(III) meso-tetraphenyl porphyrin chloride [MnTPP(Cl)]. Also MnTPFPP(Cl)/n-Bu4NHSO5 was used for the selective epoxidation of naphthalene in the presence of OAc- and F- anionic co-catalysts.

Catalytic epoxidation of cis-stilbene and naphthalene

by tetra-n-butylammonium hydrogen monopersulfate

in the presence of manganese(III) tetraarylporphyrins

and various anionic co-catalysts

Zahra Solati*a, Majid Hashemia, Ahmad Keshavarzia and Ezzat Rafieeb

aChemistry Department, Faculty of Sciences, Persian Gulf University, Bushehr 75168, IranbChemistry Department, Razi University, Kermanshah, Iran

Received 31 August 2011Accepted 16 October 2011

ABSTRACT: The epoxidation of cis-stilbene by n-Bu4NHSO5 was studied in the presence of two different manganese porphyrin complexes, [MnTPFPP(Cl) and MnTPP(Cl)], and various n-Bu4NX (X =OAc, F, Cl, Br, OCN, NO3, BF4). In general direct correlation was found between stereoselectivity of the epoxidation reaction and the nucleophilic properties of these anionic co-catalysts. Also epoxidation of naphthalene was carried out in high yield and good selectivity by n-Bu4NHSO5 in the presence of MnTPFPP(Cl) in association with n-Bu4NOAc or n-Bu4NF co-catalysts.

KEYWORDS: co-catalyst, epoxidation, catalysis, mangenese porphyrin.

*Correspondence to: Zahra Solati, email: [email protected], fax: +98 771-4541494


150 Z. SOLATI ET AL.


Table 1 shows the effects of different anionic co-catalysts on the epoxidation of cis-stilbene by n-Bu4NHSO5 catalyzed by MnTPFPP(Cl). The oxidation reaction does not proceed in the absence of a co-catalyst (entry 1). Presence of non-coordinating or very weak coordinating anions (BF4

- and NO3-) has no special

effect on the yield and stereoselectivity of the reaction (entries 2, 3). Coordinating anions (OCN-, OAc-, F-, Cl-,Br-) have great effects on the yield and stereoselectivity of the reaction (entries 4, 5, 6, 7, 8). The efficiency of the epoxidizing system considering both the yield and stereoselectivity increases in following order: F- > OAc- >OCN- > Cl- > Br-. The fluoride anion improved the yield and the stereoselectivity of the reaction more than others (the conversion is 93% and the cis:trans ratio is 22:1), OAc- as an oxygen donor ligand (conversion = 98% and cis:trans ratio = 16) is better than OCN-(conversion =30% and cis:trans ratio = 8) and Cl-(conversion = 30% and cis:trans ratio = 5) is better than Br-(conversion =25% and cis:trans ratio = 2).

Interpreting the trends in the yield and the stereoselectivity of the reaction based on the values of intrinsic basicity of these coordinating anions (pka values) is unsuccessful. But considering the trends in the hardness of these anions, there is a great correlation between the hardness of these coordinating anions and the yield and the stereoselectivity of the oxidation reaction. The most electronegative species (the hardest one), F-, gives the most cis:trans ratio while the least electronegative species (the softest one), Br-, gives the least cis:transratio. According to symbiosis principle, existence of fluoride substituents on phenyl rings of MnTPFPP(Cl) makes Mn(III) in the center of the complex harder,

then bonding between this ion and the harder donors are stronger. Perhaps it causes the oxidation reaction proceeds through Mn(V)-oxo species that leads to the greater stereoselectivity. Also, Br- may be oxidized by HSO5

- leading to lower conversion.Table 2 shows the results of oxidation of cis-stilbene with

n-Bu4NHSO5 catalyzed by MnTPP(Cl) in the presence ofdifferent anionic co-catalysts. Again, the oxidation reaction does not proceed in the absence of a co-catalyst. In the presence of BF4

- and NO3-, the MnTPP(Cl) catalyst

is inactive. Epoxidation of cis-stilbene in the presence of F- resulted in 10% conversion and cis- to trans-stilbene oxide ratio was 1.2. Epoxidation of cis-stilbene in the presence of OAc- resulted in 24% yield and cis- to trans-stilbene oxide ratio was 1. Epoxidation of cis-stilbene in the presence of OCN- proceeds with 12% yield and cis:trans ratio was 0.5. When Cl- was used as co-catalyst, the yield was 10% and cis:trans ratio was 0.1. The yield of the reaction in the presence of Br- was 9% and trans-stilbene oxide is obtained as the major product. Again the results are in accordance to hardness of these co-catalysts, but because the MnTPP(Cl) is softer than MnTPFPP(Cl), it seems that proceeding the reaction through Mn(IV)-oxo species is most probable and leads to greater amount of trans product, the conversion data are closer together and because the stability of the catalyst is lower, the conversions are generally lower.

The epoxidation of polynuclear aromatic hydrocarbons was demonstrated to be a particularly difficult process, and the oxidation to the diepoxides was only obtained with peroxyacids in very low yields [28, 38, 39]. Cavaleiro and coworkers described the formation of diepoxides from naphthalene and anthracene in high yields using hydrogen peroxide as oxidant in the presence

Table 1. Epoxidation of cis-stilbene with n-Bu4NHSO5 catalyzed by MnTPFPP(Cl) in the presence of various anionic co-catalysts in CH2Cl2

Entry Co-catalyst pka Conversion, % Cis, % Trans, % Cis/Trans Time, min

1 — — — — — 10

2 n-Bu4NBF4 0.5 — 10

3 n-Bu4NNO3 -1.3 — 10

4 n-Bu4NOCN 3.7 30 24 3 8 10

5 n-Bu4NOAc 4.76 98 80 5 16 5

6 n-Bu4NF 3.17 93 89 4 22 5

7 n-Bu4NCl -6.3 30 25 5 5 10

8 n-Bu4NBr -8.7 25 17 8 2 10

Reactions were run at least in triplicate under air at 25 2 C, and the reported values are the average of the measured values. The molar ratio for catalyst:co-catalyst:substrate:n-Bu4NHSO5

is 1:20:100:190. The solvent was removed in vacuo, the products and unreacted alkene were extracted with n-hexane. The isomer ratios were determined by 1H NMR spectroscopy. The values for pka are for the corresponding acids (HA).


CATALYTIC EPOXIDATION OF CIS-STILBENE AND NAPHTHALENE 151

Table 2. Epoxidation of cis-stilbene with n-Bu4NHSO5 catalyzed by MnTPP(Cl) in the presence various anionic co-catalysts in CH2Cl2

Entry Co-catalyst Conversion, % Cis, % Trans, % Cis/Trans Time, min

1 — — — — — 10

2 n-Bu4NBF4 — 10

3 n-Bu4NNO3 — 10

4 n-Bu4NOCN 12 4 8 0.5 10

5 n-Bu4NOAc 24 12 12 1 10

6 n-Bu4NF 10 6 4 1.2 10

7 n-Bu4NCl 10 1 9 0.1 10

8 n-Bu4NBr 9 trace 9 Only trans 10

Reactions were run at least in triplicate under air at 25 ± 2 °C, and the reported values are the average of the measured values. The molar ratio for catalyst:co-catalyst:substrate:n-Bu4NHSO5 is 1:20:100:190. The solvent was removed in vacuo, the products and unreacted alkene were extracted with n-hexane. The isomer ratios were determined by 1HNMR spectroscopy.

Table 3. Epoxidation of naphthalene with n-Bu4NHSO5 catalyzed by MnTPFPP(Cl) in the presence of n-Bu4NOAc and n-Bu4NF in CH2Cl2 at room temperature

Entry Co-catalyst Conversion, % Diepoxy, % Diepoxy selectivity, %

1 n-Bu4NOAc 97 89 92

2 n-Bu4NF 70 54 77

The molar ratio for catalyst:co-catalyst:substrate:n-Bu4NHSO5 is 1:100:100:200.The solvent was removed in vacuo, the products and unreacted naphthalene were extracted with n-hexane. Characterization of the reaction products and determination of substrate conversion (based on starting naphthalene) and product selectivity carried out by 1H NMR and GC-MS spectroscopy.

of manganese porphyrins and ammoniumacetate. They suggested the involvement of an oxo species [Mn(V)- oxo] in the reaction pathway [28, 40].

We used MnTPFPP(Cl)/n-Bu4NHSO5 oxidizing system in the presence of OAc- and F- anions to check its efficiency and its selectivity on the oxidation of naphthalene. As the results in Table 3 show it can epoxidize naphthalene to diepoxy naphthalene with high efficiency and selectivity in 1 h.

To check the probability of coordination of these anions to Mn center and involvement of Mn-oxo species in the reaction pathway, we followed the variations in the UV-vis spectra of the manganese porphyrins during the epoxidation reactions. Involvement of an Mn-oxo species in the epoxidation of alkenes by n-Bu4NHSO5 in the presence of MnTPFPP(Cl) catalyst and n-Bu4NOAc has been already reported [18].

By addition of a dichloromethane solution of n-Bu4NF to the solution of MnTPFPP(Cl) in CH2Cl2,the Soret band at 474 nm shifted to 437 nm. This blue

shift could relate to the displacement of Cl- counter ion by fluoride and stronger interaction of fluoride with metal center and perhaps to the formation of a six coordinate complex [MnTPFPP(F)2]

-. Upon addition of n-Bu4NHSO5 to this solution, this Soret band spectrum (437 nm) was subsequently transformed within 100 s into a new band at about 430 nm. The band is assigned to an Mn-oxo intermediate species [41]. In the absence of alkene substrate, this species was very stable. However, upon addition of an excess of alkene, this Soret band decayed quickly and concomitantly the Soret band at 454 nm increased (Fig. 1).

Addition of tetrabutylammonium salts of C1-, Br-,OCN- to a CH2Cl2 solution containing MnTPFPP(Cl) caused no observable change in the UV-vis spectrum. Addition of n-Bu4NHSO5 to this solution caused a slowly decrease in the Soret band at 474 nm with concomitant increase in the absorbance at 418 nm ascribed to Mn-oxo species.


152 Z. SOLATI ET AL.

EXPERIMENTAL

The free-base porphyrins TPPH2 [42], TPFPPH2

[43], were prepared and purified as reported previously. MnTPP(Cl) was synthesized using MnCl2·4H2Oaccording to the procedure of Adler et al. [44]. The synthesis of MnTPFPP(Cl) was based on the procedure given by Kadish et al. [45]. n-Bu4NOAc and n-Bu4NHSO5

was prepared by adding tetrabutylammonium hydrogen sulfate (6.5 mmol) to a solution of sodium acetate or potassium monopersulfate (5.9 mmol) in water (20 mL), the mixture was stirred for 30 min and then extracted with CH2Cl2 (40 mL) and the extract was dried over magnesium sulfate. After filtration and evaporation of the solvent, the remaining paste was washed with hexane (10 mL) and dried under vacuum [43]. Other tetrabutylammonium salts were prepared by exchanging Br- in n-Bu4NBr with other anions by a similar procedure to the above [43]. n-Bu4NF·3H2O and n-Bu4NBr were purchased from Fluka.

Stock solutions of MnPor catalysts (0.003 M) were prepared in CH2Cl2. In a typical reaction 0.001 mmol (0.3 mL) of catalyst, 0.1 mmol of alkene and x mmol (as required) of co-catalyst were dissolved in 0.5 mL of CH2Cl2. n-Bu4NHSO5 (0.2 mmol) was then added to the reaction solutions at 25 °C. The reaction solutions were stirred for the required times and the solvent of the reaction solution was removed under reduced pressure, n-hexane (2 mL) was added, the resulting mixture was filtered and n-hexane was removed under vacuum. The identification and the quantification of the products were done by 1H NMR spectroscopy (in CDCl3). In the case of naphthalene, the reaction solutions were analyzed by GC-MS spectroscopy.

CONCLUSION

The coordinating strength of an anionic co-catalyst could have a great influence on the yields and selectivities of oxidation reactions of olefins with n-Bu4NHSO5 catalyzed by manganese porphyrins. The hardest anionic axial ligand bond the metal center stronger than others, leading to higher conversion and greater stereoselectivity in the reaction. The importance of the effects of anionic co-catalysts is more pronounced when their relative activities are examined in the presence of manganese porphyrins with electron withdrawing substituents on the porphyrin periphery.

Acknowledgements

This work was supported by the Persian Gulf University Research Council.

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DOI: 10.1142/S1088424611004440



INTRODUCTION

Investigation into the spectroscopic and redox properties of metallophthalocyanines (MPcs) continue to be active areas of research [1–8]. MPc complexes can be successively reduced using chemical, electrochemical, or photochemical methods to give rise to species containing reduced Pc3-, Pc4-, Pc5-, or Pc6- ligands, which drastically affect the spectral properties of the complexes [9]. The electronic absorption spectra of these ring-reduced MPc anions have been studied extensively. Much of this work has focused on main group MPcs (M = Mg, Al, Ge, Zn) since the unvarying oxidation state of the main group metal simplifies the determination of the charge on the Pc ligand. Indeed, the first report of an absorption spectrum of a reduced MPc was of a chemically reduced MgPc [10]. The spectra of the whole series of chemically reduced MgPc anions was reported again by Clack and Yandle [9]. Magnetic circular dichroism (MCD) spectroscopy has been used in conjunction with absorption spectroscopy to gather additional information on the electronic structure of

MgPc reduced by chemical methods [11], as well as electrochemical methods [12]. However, no reduced MgPc species has ever been isolated or structurally characterized.

The anions of ZnPc have also been comprehensively studied in situ. The absorption spectra of chemically [9], electrochemically [9, 13–16], and photochemically [17] reduced ZnPc have been reported along with the MCD spectra of photochemically [16, 17] and electrochemically [14] reduced ZnPc. While MgPc and ZnPc are the most commonly investigated main group MPcs, the absorption spectra of chemically reduced AlPc [9, 18, 19] and GePc [20] species have also been reported.

In spite of the additional challenge of variable oxidation states at the metal centre, reduced transition metal Pcs have also been investigated. The absorption spectra of chemically reduced Mn, Fe, Ni [9], and Nb [21] Pcs and electrochemically reduced Fe [15, 22], Co [15, 23, 24], Ni [23], and Cu [13, 23], Pcs are available. The absorption spectra of some rare examples of electrochemically reduced actinide Pcs containing U and Th are alsoknown [25].

In spite of this body of work on the electronic absorption spectra of ring-reduced MPcs, these species are usually generated and characterized in situ and have only very rarely been isolated for structural characterization,

Synthesis and structural characterization of a magnesium

phthalocyanine(3–) anion

Edwin W.Y. Wong and Daniel B. Leznoff*

Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby BC, V5A 1S6, Canada

Received 1 October 2011Accepted 19 October 2011

ABSTRACT: The reduction of magnesium phthalocyanine (MgPc) with 2.2 equivalents of potassium graphite in 1,2-dimethoxyethane (DME) gives [K2(DME)4]PcMg(OH) (1) in 67% yield. Compound 1 was structurally characterized using single crystal X-ray crystallography and was found to be a monomeric, heterometallic complex consisting of a μ3-OH ligand that bridges a [MgIIPc3-]- anion to two potassium cations solvated by four DME molecules. An absorption spectrum of 1 confirms the Pc ligand is singly reduced and has a 3– charge. The solid-state structure of 1 does not indicate breaking of the aromaticity of the Pc ligand. Compound 1 is only the second Pc3- complex and the first reduced MgPc to be isolated and structurally characterized.

KEYWORDS: magnesium, phthalocyanine, reduction, anion, X-ray crystal structure.


*Correspondence to: Daniel B. Leznoff, email: [email protected], tel: +1 778-782-4887, fax: +1 778-782-3765


SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF A MAGNESIUM PHTHALOCYANINE(3–) ANION 155

even though structural information would be useful for interpreting the spectroscopic data [26]. This is likely due to the extreme sensitivity of these complexes, which can only be isolated under the rigorous exclusion of air and moisture [27]. Although the isolation of a series of reduced first-row transition metal and main group element Pcs was reported [27], none of these compounds were ever structurally characterized.

Indeed, only five of these elusive compounds have been isolated and structurally characterized to our knowledge. Specifically, reduced FePc complexes with the formulas K([2.2.2]cryptand)FePc and Li2(THF)(18-crown-6)2FePc (THF = tetrahydrofuran) have been structurally characterized [28]. The single crystal X-ray structures of AlIIIPc3-(anisole)2 [18] and GeIVPc4-

(pyridine)2 have also been reported [20]. We recently described the structural and electronic characterization of a reduced NbPc species, K2NbIVPc4-O·5DME (DME = 1,2-dimethoxyethane) [21]. Of the five reported structures, all feature nearly planar Pc ligands except for K2NbIVPc4-

O·5DME, which has a saddle-shaped Pc ligand [21].The addition of electrons to the aromatic

18π-electron circuit of the Pc ligand via reduction is expected to disrupt its aromaticity [20]. The effects of MPc reduction are most dramatically seen in the doubly reduced GeIVPc4-(pyridine)2 and K2NbIVPc4-O·5DME systems in the form of bond localization, which manifests itself in the form of alternating short and long bonds around the Pc ligand [20, 21]. Interestingly, in both compounds, the bond-length alternation is seen in only two of the four benzo groups situated trans to each other [20, 21]. This bonding pattern is consistent with a simple valence-bond model of the Pc ligand, where two of the four opposing benzo moieties are expected to be delocalized and the remaining two are expected to show localized bonding [20]. There is evidence of antiaromaticity in the 1H NMR spectrum of the 20π-electron GeIVPc4- system, in which resonances are shifted from their expected positions by a strong paratropic ring current [20]. Nucleus independent shift calculations also predicted the antiaromatic character of GeIVPc4- [20].

In the case of MPc3- complexes, the predicted loss of aromaticity does not manifest itself significantly in the structural data. For instance, there is little to no bond-length alternation observed in AlIIIPc3-(anisole)2 in spite of the fact that DFT calculations predict that it should be present [18]. Conversely, bond-length alternation is seen in a similar singly reduced Al(TPP)(THF)2 complex (TPP = tetraphenylporphyrin) [18].

Our previous success at isolating and structurally characterizing K2NbIVPc4-O·5DME [21] motivated us to examine the more commonly studied ring-reduced MPcs, such as MgPc in a similar fashion. Herein, we report the first isolation of a reduced MgPc system and only the second Pc3- anion to be structurally characterized, namely MgIIPc3- .


Synthesis and structure

The addition of 2.2 equivalents of potassium graphite (KC8) to a stirred mixture of MgPc in DME results in an immediate reaction to give a dark purple mixture. Filtration of the reaction mixture through a pad of Celite on a frit filter initially resulted in decomposition of the purple product into a blue-green material due to the extreme sensitivity of the purple product. After this initial decomposition, the remaining purple filtrate was collected since it retained its original colour as it passed through the frit filter. A dark purple powder of [K2(DME)4]PcMg(OH) (1) was obtained in 67% yield after removal of the solvent under reduced pressure.

Elemental analysis (C, H, N) of 1 is consistent with an empirical formula of [K2(DME)4]PcMg(OH). A mass spectrum of 1 could not be obtained because 1 decomposes immediately upon exposure to even small traces of oxygen. However, a MALDI-TOF mass spectrum of a sample of 1 exposed to air reveals that 1 decomposesto MgPc (m/z = 536.1, calcd. for C32H16N8Mg = 536.1 [M+]). As further confirmation, the absorption spectrum of the decomposition product is nearly identical with the spectrum of MgPc in DME. Extraction of the decomposition products of 1 with water yields a basic solution, which is consistent with the presence of KOH, the other expected decomposition product. A controlled oxidation of 1 with ferrocenium tetrafluoroborate in an inert atmosphere also yields MgPc and KOH.

Purple crystals suitable for single crystal X-ray diffraction were grown by layering hexanes over a DME solution of 1. At some point in the reaction, the reactants or products were inadvertently exposed to traces of oxygen and moisture, this being the source of the hydroxide ligand. Nevertheless, regardless of the details of its isolation, the structure of compound 1 is of interest due to the near absence of structurally characterized MPc3- complexes in the literature.

Compound 1 is a monomeric, heterometallic complex consisting of a μ3-OH ligand that bridges a [MgIIPc3-]-

anion and two potassium cations (Fig. 1), which are solvated by four DME molecules. The crystal structure of 1 shows that it is highly distorted in the solid state and no longer contains an axis of symmetry. The Mg centre is 5-coordinate with a distorted square-pyramidal geometry consisting of four basal isoindole N-atoms and the apical μ3-O-atom of the hydroxide ligand. The average Mg to basal N distance in 1 is 2.072(2) Å, whereas the average for MgPc (X-ray data collected at 120 K) is slightly shorter (2.048 Å) [29]. Correspondingly, the distance of the Mg centre to the PcN4 mean plane in 1 (0.595 Å) is slightly greater than in MgPc (0.557 Å) [29].

The hydroxide oxygen atom, O1, is not situated directly above Mg1 but is angled back towards the potassium cations. This is evidenced by the asymmetry observed


156 E.W.Y. WONG AND D.B. LEZNOFF

in the pairs of opposing isoindole NX–Mg1–O1 (X = 1 and 5, 3 and 7) bond angles. The N3– and N7–Mg1–O1 pair of angles are essentially the same at 106.66(11)º and 106.00(11)º, respectively, but the N1– and N5–Mg1–O1 pair of angles are quite different at 114.73(11)º and 99.06(11)º, respectively. O1 of the μ3-OH ligand adopts a distorted tetrahedral geometry. Previously, a μ3-OH was reported to bridge two potassium atoms to a transition metal atom in a bis(imino)pyridine iron dimer [30] and in a ruthenium complex [31]. The K2–O1 and K3–O1 bond lengths of 2.644(2) Å and 2.638(2) Å, respectively, are slightly shorter than previously reported K–O distances, which average 2.716(1) Å [30, 31].

The asymmetry of 1 also manifests itself in the position of the potassium cations. K2 lies 3.758 Å above the Pc-C8N8 mean plane whereas K3 lies much closer to the Pc ligand at only 2.989 Å. There appear to be significant interactions between K3 and C23, C24, N5, and N6 of the Pc ligand judging by their relatively close contacts. However, this does not appear to cause any localization of the bonds within the Pc ligand since no bond length-alternation is observed in the region of the Pc ligand near K3.

The Mg1–O1 bond distance (1.948(2) Å) is shorter than reported Mg–O distances for terminal Mg–OH bonds, which range from 2.073(3) to 2.199(3) Å [32]. However, a shorter Mg–O distance of 1.985(6) Å has been reported for a μ3-OH that bridges between Mg and two Li centres, which is more in line with the Mg1–O1 bond distance observed in 1 [33].

The nature of the Pc aromaticity after reduction is of interest, but lack of structural information on reduced MPcs [34] has limited the discussion of this topic. Therefore, the conformation of bond distances in the Pc ligand were examined closely for signs of loss of aromaticity or the presence of anti-aromaticity. The Pc ligand in 1 is distorted, with the two isoindole moieties on the same side as K3 being relatively flat whereas the isoindoles on the K2 side point downward away from K2. The dihedral angles between the C8N8 mean plane and the N5 and N7 isoindole moieties on the K3 side are 2.12º and 4.55º, respectively, while the dihedral angles between the

C8N8 mean plane and the N1 and N3 isoindole moieties are much greater at 9.36º and 12.13º, respectively. It is difficult to compare the conformation of the Pc ligand in 1 to the published structures of MgPc [29, 35] since disorder of the Mg centre in the neutral MgPc structure limits the accuracy of the metrical parameters of the Pc ligand. A survey of the high quality, (R 0.05) low temperature single crystal X-ray structures of (L)MgPc [36, 37] (where L = H2O or MeOH) suggests that the Pc ligand in MgPc can vary from distorted to nearly planar conformations, which also makes it difficult to compare the conformation of the Pc3- ligand in 1 to the published structures containing a Pc2- ligand.

To our knowledge, the only other structurally characterized Pc3- compound is AlIIIPc3-(anisole)2 (2) [18]. In contrast to 1, 2 is nearly flat and the largest dihedral angle between the C8N8 mean plane and an isoindole moiety is 3.75º. There is no strong evidence for breaking of the aromaticity of the Pc ligand in the form of bond localization in either 1 or 2, although DFT calculations on 2 predict that it should occur at the Nmeso–C bonds [18]. There is some slight bond localization in the form of bond-length alternation in the benzo moieties of the Pc ligand observed in 2, but the differences in adjacent C–C bonds range from statistically insignificant (0.003(3) Å) to 0.028(4) Å and the difference in length between short and long C–C bonds is not consistent throughout the Pc ligand.

With this in mind, the DFT geometry optimized structure (B3LYP/6–31G*) of [MgPc(OH)]2- was calculated and it predicts that there should be a pattern of alternating short and long Nmeso–C bonds in the Pc ligand with an average difference of 0.036 Å, similar to the difference of 0.037 Å calculated for 2 [18]. In 1,the experimental difference between adjacent Nmeso–Cbonds only ranges from 0.018(6) to 0.010(6) Å. This discrepancy was also observed for 2. It is interesting to contrast the structures of Pc3- ligands with those of Pc4-

ligands. In the case of the two structurally characterized Pc4- complexes [20, 21], bond-length alternation is clearly observed (vide supra).

Absorption spectroscopy of 1

The most intense absorption band for MPcs (the Q-band) arises from a ligand-based π–π* transition, which generally lies around 670 nm and does not change greatly with varying metal centres [38]. However, oxidation or reduction of the Pc ligand in a MPc complex changes the spectrum significantly, where each Pc charge state has a distinct absorption spectrum [38, 39]. The absorption spectra of reduced MgPc anions in situ have been reported previously [9, 10] and can be compared with the spectrum of 1 to determine the charge of the Pc ligand. An absorption spectrum of crystals of 1 dissolved in DME (Fig. 2) correspond well with the published spectrum of [MgIIPc3-]- in THF and, therefore, the Pc

Fig. 1. Molecular structure of [K2(DME)4]PcMg(OH) (1) with potassium-bound DME molecules removed for clarity. Thermal ellipsoids are set at 30% probability



ligand in 1 has a -3 charge [9]. The -3 charge of the Pc ligand in 1 is also consistent with its molecular formula since the sum of the charge on the remaining ions (Mg2+,2 × K+, OH-) is +3. Compound 1 is extremely unstable with respect to oxidation of the reduced Pc3- ligand and it was not possible to obtain a spectrum unobscured by the Q-band of oxidized 1, which is presumably a Pc2- species.

Insights into the reaction

The mechanism for the formation of 1 is not clear since it would be expected that the addition of 2.2 equivalents of KC8 to MgPc would result in a MgIIPc4- product rather than the MgIIPc3- product, 1, that was obtained. There are two possibilities: (a) that a Pc4- product was never formed or (b) that a Pc4- formed initially and was subsequently oxidized to the Pc3- product 1 that was isolated.

To examine this, the reduction of MgPc with KC8

was repeated in DME that was degassed using four freeze-pump-thaw cycles, thereby further minimizing any possible immediate oxidation of the products. An absorption spectrum of an aliquot of the reaction mixture was recorded and, in comparison with the spectrum of 1, the presence of an additional distinct absorption attributable to MgIIPc4- at 520 nm [9] appears to indicate that a mixture of MgIIPc4- and MgIIPc3- products are present in the reaction (Fig. 3), therefore, a Pc4- product is initially being produced. It is unclear whether 1 results from the oxidation of the initial Pc4- product or whether the Pc3- product, 1, was formed simultaneously in parallel with the Pc4- product. In the latter case, it is possible that a portion of the KC8 reacts with trace moisture present in the reaction mixture (despite our efforts to exclude moisture) to form potassium hydroxide, which is then incorporated into 1, rather than all of the KC8 acting to reduce the MgPc starting material. This is only one possibility of many and due to the inherent reactivity and extreme air and moisture sensitivity of these reduced MPcs it is difficult to determine the exact sequence of events that leads to the formation of 1.

EXPERIMENTAL

General methods and procedures

All manipulations were carried out under an atmosphere of dinitrogen in a MBraun Labmaster 130 glovebox or using standard Schlenk techniques unless otherwise stated. All glassware was rigorously dried overnight at 160 °C and cooled under vacuum before use. Potassium graphite [40] was prepared according to a literature procedure. Magnesium phthalocyanine (TCI America) was dried under vacuum overnight before being transferred into a glovebox for use and storage. 1,2-dimethoxyethane (Certified, Fisher) was distilled under a nitrogen atmosphere from a purple solution of sodium benzophenone ketyl and stored over sodium wire in a glovebox. All other reagents were obtained from commercial sources and used as received.

Electronic absorption spectra were recorded on a Varian Cary 5000 spectrophotometer in a 0.1 cm quartz cell or a 0.1 cm quartz cell equipped with a Kontes PTFE plug, HI-VAC valve for air-sensitive samples.

Elemental analyses (C, H, N) were performed by Mr. Farzad Haftbaradaran at Simon Fraser University on a Carlo Erba EA 1110 CHN elemental analyzer. Samples for elemental analyses were loaded into pre-weighed tin capsules inside a glovebox, sealed by crimping the capsules with tweezers, removed from the glovebox, weighed, and promptly loaded into the analyzer.

MALDI-TOF mass spectrometry experiments were performed on a Bruker Biflex IV equipped with a nitrogen laser. The spectra were acquired by matrix-assisted laser desorption/ionization (MALDI) using trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (dctb) as a matrix.

Synthetic procedures

Preparation of [K2(DME)4]PcMg(OH).KC8 (0.139g, 1.03 mmol) was added to a dark blue-green suspension

Fig. 2. Absorption spectrum of 1 in DME. Vertical bars rep-resent the reported absorptions of MgIIPc3- in THF with arbitrary intensities matched to that of the experimental spectrum [9](* denote absorptions due to oxidation of 1)

Fig. 3. Absorption spectrum of an aliquot of the reaction between MgPc and 2.2 equivalents of KC8 in DME. Vertical bars represent the reported absorptions of MgIIPc(3-) [9] (black) and MgIIPc(4-) [9] (orange) in THF with arbitrary intensities matched to that of the experimental spectrum



of MgPc (0.250 g, 0.466 mmol) in approximately 20 mL of DME at room temperature. An immediate colour change to dark purple was observed. The mixture was stirred overnight and filtered through Celite. Initially, the purple reaction mixture turned blue-green upon contact with the frit. The blue-green filtrate was discarded. Filtration of the reaction mixture was continued and the filtrate was collected once it retained the original dark purple color of the reaction mixture. The solvent was removed under reduced pressure and a dark purple solid was collected. Yield 0.309 g (66.9%). Anal. calcd. for C48H57K2MgN8O9:C, 58.09; H, 5.79; N, 11.29%. Found: C, 58.19; H, 5.74; N 11.21.

Purple crystals suitable for X-ray analysis were grown by layering hexanes onto a DME solution of the product in a capped one dram vial. During the recrystallization process, the atmosphere of the glovebox was contaminated with traces of oxygen and H2O (< 10 ppm) over a period of several hours on multiple occasions due to power outages. An absorption spectrum of the crystals was recorded. UV-vis (DME): λmax, nm 956, 869, 791, 663, 646, 598, 565, 428, 362, 331.

The above procedure was repeated with an additional step of degassing the DME solvent by four freeze-pump-thaw cycles prior to use. In this case, the DME was used immediately and was not stored over sodium wire. The graphite was allowed to settle to the bottom of the reaction vessel and an absorption spectrum was taken of a drop of the reaction solution diluted with degassed DME. UV-vis (DME): λmax, nm 958, 857, 794, 642, 565, 528, 427, 350, 323.

Oxidation of 1

In a glovebox, a small sample of 1 was dissolved in about 1 mL of DME resulting in a dark blue-purple solution. This solution was taken out of the glovebox and turned dark blue-green immediately upon exposure to air. The solvent was allowed to slowly evaporate to give a mixture of colorless and dark blue-green solids. A sample of the mixture was extracted with a few drops of distilled water and the water was found to be basic using pH paper. UV-vis (DME): λmax, nm 666, 637, 602, 343. MS (MALDI-TOF): m/z 536.1 (calcd. for C32N8H16Mg:536.1 [M+]).

In a glovebox, 1 (0.025 g, 0.025 mmol) was dissolved in about 7 mL of DME resulting in a dark blue-purple solution. Ferrocenium tetrafluoroborate (0.0076 g, 0.028 mmol) was added to the solution of 1 and stirred overnight resulting in a dark blue-green solution. The solution was taken out of the glovebox and the solvent was removed under reduced pressure to give a dark blue-green solid. A sample of the dark blue-green solid was extracted with a few drops of distilled water and the water was found to be basic using pH paper. UV-vis (DME): λmax, nm 667, 638, 603, 344. MS (MALDI-TOF): m/z535.9 (calcd. for C32N8H16Mg: 536.1 [M+]).

Absorption spectrum of MgPc

An absorption spectrum of MgPc in DME was recorded. UV-vis (DME): λmax, nm 668, 639, 604, 345.

Single crystal X-ray crystallographic analysis

Crystallographic data and select bond lengths and angles for 1 are listed in Tables 1, 2 and 3, respectively. Data were collected through the SCrALS (Service Crystallography at Advanced Light Source) program at the Small-Crystal Crystallography Beamline 11.3.1 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory due to the weakly diffracting nature of the crystals of 1.

Intensity data were collected at 150 K on a D8 goniostat equipped with a Bruker APEXII CCD detector at Beamline 11.3.1 at the ALS using synchrotron radiation tuned to λ = 0.77490 Å. A series of one second frames measured at 0.2º increments of ω were collected to calculate a unit cell. For data collection frames were measured for a duration of one second at 0.3º intervals of ω with a maximum 2θ value of ~60º. The data frames were collected using the program APEX2 and processed using the program SAINT routine within APEX2. The data were corrected for absorption and beam corrections based on the multi-scan technique as implemented in SADABS.

The structures were solved using direct methods (SIR92) [41] and refined by least-squares procedures

Table 1. Crystallographic data for [K2(DME)4]PcMg(OH) (1)

Empirical formula C48H57K2MgN8O9

Formula weight 992.53

Crystal system monoclinic

Space group P21/n

a, Å 12.570(3)

b, Å 20.364(5)

c, Å 20.163(5)

α, º 90

β, º 105.499(3)

γ, º 90

V, Å3 4974(2)

Z 4

T, K 150

calcd, g/cm3 1.325

μ, mm-1 0.266

Unique reflections collected 10239

Observed reflections 6373

R [I0 ≥ 3 (I0)] 0.0643

Rw [I0 ≥ 3 (I0)] 0.0761

Goodness of fit 1.0848



Table 2. Selected bond lengths (Å) for [K2(DME)4]PcMg(OH) (1)

K2–O1 2.644(2) N8–C1 1.353(4) C19–C20 1.365(6)

K3–O1 2.638(2) N8–C32 1.335(4) C20–C21 1.391(7)

Mg1–O1 1.948(2) C1–C2 1.457(4) C21–C22 1.377(6)

Mg1–N1 2.066(3) C2–C3 1.379(5) C22–C23 1.383(5)

Mg1–N3 2.078(3) C2–C7 1.404(5) C23–C24 1.453(5)

Mg1–N5 2.068(3) C3–C4 1.390(5) C25–C26 1.457(5)

Mg1–N7 2.075(3) C4–C5 1.384(6) C26–C27 1.393(4)

N1–C1 1.363(4) C5–C6 1.374(5) C26–C31 1.404(4)

N1–C8 1.381(4) C6–C7 1.400(5) C27–C28 1.371(5)

N2–C8 1.341(4) C7–C8 1.443(5) C28–C29 1.394(6)

N2–C9 1.328(5) C9–C10 1.460(5) C29–C30 1.386(5)

N3–C9 1.366(4) C10–C11 1.397(5) C30–C31 1.387(5)

N3–C16 1.373(4) C10–C15 1.389(6) C31–C32 1.465(4)

N4–C16 1.327(5) C11–C12 1.381(6) K2–O101 2.808(4)

N4–C17 1.345(5) C12–C13 1.390(7) K2–O102 2.830(3)

N5–C17 1.376(4) C13–C14 1.367(7) K2–O401 2.763(5)

N5–C24 1.370(4) C14–C15 1.399(5) K2–O402 2.819(6)

N6–C24 1.345(4) C15–C16 1.455(5) K3–O201 2.757(3)

N6–C25 1.335(4) C17–C18 1.439(5) K3–O202 2.693(3)

N7–C25 1.377(4) C18–C19 1.407(5) K3–O301 2.659(7)

N7–C32 1.367(4) C18–C23 1.402(5) K3–O302 2.967(4)

Table 3. Selected bond angles (º) for [K2(DME)4]PcMg(OH) (1)

O1–Mg1–N1 114.73(11) N8–C1–C2 122.3(3) N5–C17–C18 109.3(3)

O1–Mg1–N3 106.66(11) C1–C2–C3 133.3(3) N4–C17–C18 123.3(3)

N1–Mg1–N3 85.81(11) C1–C2–C7 105.4(3) C17–C18–C19 133.3(4)

O1–Mg1–N5 99.06(11) C3–C2–C7 121.1(3) C17–C18–C23 106.8(3)

N1–Mg1–N5 146.21(12) C2–C3–C4 118.0(3) C19–C18–C23 119.9(4)

N3–Mg1–N5 85.05(12) C3–C4–C5 120.6(3) C18–C19–C20 117.8(4)

O1–Mg1–N7 106.00(11) C4–C5–C6 122.3(3) C19–C20–C21 122.0(4)

N1–Mg1–N7 85.01(11) C5–C6–C7 117.4(3) C20–C21–C22 121.0(4)

N3–Mg1–N7 146.98(12) C2–C7–C6 120.4(3) C21–C22–C23 118.0(4)

N5–Mg1–N7 85.19(11) C2–C7–C8 107.6(3) C18–C23–C22 121.3(3)

Mg1–O1–K3 107.90(10) C6–C7–C8 131.9(3) C18–C23–C24 106.4(3)

Mg1–O1–K2 116.75(10) C7–C8–N1 108.5(3) C22–C23–C24 132.3(4)

K3–O1–K2 95.91(8) C7–C8–N2 123.2(3) C23–C24–N5 109.0(3)

Mg1–N1–C1 126.1(2) N1–C8–N2 128.2(3) C23–C24–N6 123.0(3)

Mg1–N1–C8 124.8(2) N3–C9–N2 128.3(3) N5–C24–N6 127.9(3)

C1–N1–C8 108.6(3) N3–C9–C10 108.9(3) N7–C25–N6 127.7(3)

C8–N2–C9 123.2(3) N2–C9–C10 122.7(3) N7–C25–C26 109.1(3)

Mg1–N3–C9 125.4(2) C9–C10–C11 132.9(4) N6–C25–C26 123.1(3)

(Continued )



using CRYSTALS [42]. The hydroxide hydrogen atom was located on an electron difference map and linked to its respective oxygen atom using a riding model during refinement. All other hydrogen atoms were placed in idealized geometric positions and linked to their respective carbon atoms using a riding model during refinement. The isotropic temperature factor of each hydrogen atom was initially set to 1.2 times that of the atom it is bonded to and then the temperature factors of groups of similar hydrogen atoms were linked during refinement. DME molecules were disordered and modeled accordingly (see CIF file for details). Crystal structure diagrams were generated using ORTEP-3 for Windows (v. 2.02) [43] and rendered using POV-Ray(v. 3.6.1c) [44].

Computational methods

Geometry optimizations were performed using the Gaussian 09 program (Revision B.01) [45], the B3LYP functional [46], and the 6–31G* basis set [47, 48].

CONCLUSION

The reduction of MgPc with KC8 in DME and trace moisture or oxygen generates [K2(DME)4]PcMg(OH) containing a singly reduced Pc3- ligand. The -3 charge of the Pc ligand was confirmed by absorption spectroscopy. This is only the second structurally characterized mono-reduced MPc complex reported in the literature. Unlike the structurally characterized, doubly reduced MPc complexes, which exhibit bond-length alternation within the Pc ligand [20, 21], bond-length alternation is not observed in [K2(DME)4]PcMg(OH). While both Pc3- and Pc4- complexes

have now been structurally characterized, isolating Pc5- and Pc6- complexes, which are presumably exceedingly air- and moisture-sensitive, remains a challenge for future research.

Acknowledgements

We are grateful to the Natural Sciences and Engineering Research Council of Canada for a research grant (DBL) and an Alexander Graham Bell Canada Graduate Scholarship-D (EWYW), Simon Fraser University for funding, and Compute Canada and WestGrid for providing computational resources. We thank Dr. Michael J. Katz for assistance with X-ray crystallography and Professor Tim Storr for assistance with DFT calculations.

We are grateful to Dr. Jeanette A. Krause and the SCrALS program (Lawrence Berkeley National Laboratory) for the X-ray crystallographic data collection of 1. The ALS is supported by the US Department of Energy, Office of Energy Sciences Materials Sciences Division, under contract DE-AC02-05CH11231.


Atomic coordinates of the DFT-optimized structure of 1 are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under number CCDC-851775 (1). Copies can be obtained on request, free of charge, via www.ccdc.cam.ac.uk/data_request/cif or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223-336-033 or email: [email protected]).

Table 3. (Continued)

Mg1–N3–C16 125.7(2) C9–C10–C15 106.8(3) C25–C26–C27 132.3(3)

C9–N3–C16 108.7(3) C11–C10–C15 120.3(4) C25–C26–C31 106.7(3)

C16–N4–C17 122.8(3) C10–C11–C12 117.8(5) C27–C26–C31 121.0(3)

Mg1–N5–C17 124.0(2) C11–C12–C13 121.5(4) C26–C27–C28 117.9(3)

Mg1–N5–C24 123.3(2) C12–C13–C14 121.1(4) C27–C28–C29 121.4(3)

C17–N5–C24 108.4(3) C13–C14–C15 118.0(5) C28–C29–C30 121.3(3)

C24–N6–C25 123.1(3) C14–C15–C10 121.2(4) C29–C30–C31 117.9(3)

Mg1–N7–C25 123.6(2) C14–C15–C16 132.2(4) C26–C31–C30 120.6(3)

Mg1–N7–C32 125.4(2) C10–C15–C16 106.5(3) C26–C31–C32 106.2(3)

C25–N7–C32 108.7(3) C15–C16–N3 109.0(3) C30–C31–C32 133.2(3)

C1–N8–C32 122.7(3) C15–C16–N4 122.7(3) C31–C32–N7 109.4(3)

N1–C1–N8 127.9(3) N3–C16–N4 128.2(3) C31–C32–N8 122.8(3)

N1–C1–C2 109.8(3) N5–C17–N4 127.4(3) N7–C32–N8 127.9(3)



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DOI: 10.1142/S1088424612004495



INTRODUCTION

Phthalocyanines (Pcs) and derivatives thereof show potential as photosensitizers for many applications including in non-linear optics and photodynamic therapy (PDT) [1–8]. For these applications, large triplet state yield and long triplet state lifetimes are required.

With their π-conjugated planar organic structure, Pcs interact strongly with light over most of the visible spectrum. Diversification of the phthalocyanine by substitution at the ring periphery, axial position and central metal atom could alter the solubility, electronic absorption characteristics and photosensitizing behavior of Pcs [9, 10].

MPc derivatives containing diamagnetic non-transition centrals, are photoactive, and are often employed in photosensitization [11–13]. Interest in improving the photophysical properties of the Pc ring system continues to grow rapidly. This has stirred research into examining new Pc compounds whose structures promote good photophysical behavior [14–16]. Therefore this work is focused on the synthesis and subsequent photophysicochemical characterization of novel tetra and octa di-iso-propylphenoxy substituted metal free, aluminum, zinc, gallium and indium phthalocyanines. For the di-iso-propylphenoxy tetra substituted complexes, the chlorine atom is also present on the periphery of the ring. Halogenated phthalocyanines have been reported to show improved photosensitizer activity for PDT compared to non-halogenated derivatives [17–19]. With the exception of octa-substituted zinc derivative [20, 21] to date there have been no reports on the synthesis of tetra-substituted derivatives. Ameliorating the

Tetra and octa(2,6-di-iso-propylphenoxy)-substituted

phthalocyanines: a comparative study among their

photophysicochemical properties

Ahmad Tuhla , Wadzanai Chidawanayikab, Hamada Mohamed Ibrahima, Nouria Al-

Awadia, Christian Litwinskib, Tebello Nyokongb , Haider Behbehania, Hacene Manaac

and Saad Makhseed*a

a Department of Chemistry, Kuwait University, P.O. Box 5969, Safat 13060, Kuwaitb Department of Chemistry, Rhodes University, Grahamstown 6140, South Africac Department of Physics, Kuwait University, P.O. Box 5969, Safat 13060, Kuwait

Received 10 September 2011Accepted 23 October 2011

ABSTRACT: This work reports on the synthesis of novel metal free, zinc, aluminum, gallium and indium tetra and octa (2,6-di-iso-propylphenoxy)-substituted phthalocyanine derivatives. UV-visible and 1H NMR analyses confirm that a non-planar conformation, adapted by the phenoxy substituents due to steric interaction in both derivative series, perfectly discourage cofacial aggregation. Fluorescence quantum yields vary as a function of the number of substituents on the ring periphery, while the fluorescence lifetimes display no distinct trend. Triplet quantum yields are significantly larger for the tetra 2,6-di-iso-propylphenoxy-substituted derivatives relative to their corresponding octa-substituted species. However there was no overall trend in the triplet lifetime values. For almost all of the phthalocyanine derivatives, singlet oxygen was produced with relatively good quantum yields. This study explores the possibility of fine-tuning their physicochemical properties by simple structural modification.

KEYWORDS: di-iso-propylphenoxy, phthalocyanine, photochemistry, photophysics.


∗Correspondence to: Saad Makhseed, email: saad.makhseed@ ku.edu.kw, tel: +965 24985538, fax: +965 24816482


164 A. TUHL ET AL.

photosensitizing efficiency of phthalocyanines may also be achieved by the presence of four chlorine atoms on the ring periphery (for complexes 4a–8a), due to the heavy atom effect [22, 23]. Moreover, electron-withdrawing groups (i.e. chlorine atoms) are known to increase the photo-oxidative stability, which could impart additional features for such material in PDT and non-linear optical (NLO) applications [24, 25].

The aim is thus to establish the influence of the different metal centers and substitution patterns, with respect to the photophysicochemical properties of the complexes.


Synthesis and characterization

The phthalonitrile precursors (3a and 3b) mono and di-substituted with di-iso-propylphenoxy were employed to synthesize the tetra (Scheme 1) and octa (Scheme 2)substituted metal free, aluminum, zinc, indium and

gallium phthalocyanines respectively. Phthalonitriles 3a and 3b were prepared by the well-known base catalyzed nucleophilic aromatic substitution reaction between 4,5-dichlorophthalonitrile and the anion of the sterically hindered phenol (2,6-di-iso-propylphenol) in good yield [26]. Following the metal-ion-mediated reaction procedure, the precursor 3a undergoes cyclo-tetramerisation in quinoline using the appropriate metal salt (AlCl3, GaCl3, InCl3 and Zn(OAc)2) with a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford metal-containing derivatives 5a–8a in acceptable yields as a mixture of inseparable structural isomers. Cyclization of 3a in pentanol with lithium metal (i.e.the most routinely followed procedure) to prepare the metal-free phthalocyanine derivative (4a) was avoided to protect the peripheral chlorine atoms from the possibility of being displaced by the alkyloxide anion. Therefore, the synthesis of 4a was best achieved in reasonable yield simply by urea-promoted cyclization of 3a in quinoline. 4b was synthesized using the lithium method. Due to the asymmetrical arrangement of the substituents in the periphery of the macrocycle, all 4a–8a products are

obtained as a mixture of structural isomers which are highly soluble in most organic solvents e.g. dichloromethane (DCM), CHCl3, tetrahydrofuran (THF), toluene and hexane thus facilitating their purification by column chromatography. Each of 4a–8acomplexes occurs as a mixture of structural isomers which are difficult to separate these isomers by column chromatography or high performance liquid chromatography (HPLC) technique. The octa-substituted derivatives (5b–8b) were prepared by following the same procedure used to prepare the tetra 2,6-di-iso-propylphenoxy-substituted complexes (5a–8a). The identity and purity of the prepared complexes were confirmed by 1H NMR, IR, UV-vis spectroscopies, fast atom bombardment (FAB) mass spectroscopy and elemental analysis. All the techniques employed gave further confirmation of the predicted structural configuration of the molecules as detailed in the experimental section.

Structural and self-association characte-rization using proton NMR

The efficient π–π stacking of disc-like molecules such as phthalocyanines results in aggregation, even in dilute solution, into dimer, trimer and higher oligomers. Such intrinsic behavior can considerably affect their properties (e.g. opto-electronic properties) and of course limit their uses in the intended applications such as in the

CN

Cl

Cl

CN

1

OH

2

(a)

CNCl

CNO

3a

(b)

Cl

O

N

Cl

O

N

ClO

N

Cl O

NN N

NN

M

M = H2 (4a); Zn (5a); AlCl (6a); GaCl (7a); InCl (8a)

4a-8a

+

Scheme 1. Synthetic route to tetra-substituted 2,6-di-iso-propylphenoxy phthalocyanines 4a–8a. Reagents and conditions: (a) anhydrous K2CO3, DMF, 45 °C, 24 h; (b) appropriate metal salt, quinoline, 180 °C, 12 h, inert atmosphere


TETRA AND OCTA(2,6-DI-ISO-PROPYLPHENOXY)-SUBSTITUTED PHTHALOCYANINES 165

NLO and PDT fields [9, 15, 27–30]. UV-vis [31–33] and NMR [34] spectroscopic techniques were, therefore, used to evaluate the aggregation behavior of the target complexes.

It was noted previously that the 1H NMR analyses of the disubstituted phthalonitrile 3b demonstrated that the phenyl rings bearing two iso-propyl groups are forced out of the plane of the benzene-containing dicarbonitriles due to the bulkiness exerted by the presence of two isopropyl groups [21]. This informative conformation was clearly detected by the 1H NMR spectrum which displays a doublet of doublets for the methyl groups at 298 K due to the frozen rotation about the aryl-oxygen bonds on the NMR time-scale as shown in Fig. 1. Surprisingly, the 1H NMR spectra of the mono-substituted phthalonitrile 3a at 298 K shows the same conformation adapted by the phenoxy groups in the derived phthalonitrile 3band correspondingly displays two environments for the methyl hydrogens of the iso-propyl groups. Such

findings impart that the chlorine atom next to the bulky phenoxy substituent in 3a is big enough to introduce steric hindrance which prevents rotation around the aryl ether linkage and consequently forces the phenoxy substituent out of the plane of the target molecule, where the iso-propyl groups adopt a configuration, leading to two different methyl hydrogen environments. Thus, the resulting conformational arrangement adopted by the substituents in 3a would interestingly ensure that cofacial self-association of the derived Pc cores is prohibited. Variable-temperature studies using NMR techniques showed that the coalescence of the methyl hydrogen peaks for 3b and 3a occurs at 308 K and 348 K, respectively, corresponding to a free energy of activation (ΔG*) of 65.8 and 75.8 KJ.mol-1,respectively (Figs 1 and 2 for 348 K, Table 1).

This energy barrier difference between the phthalonitriles under investigation can be attributed to the greater steric effect imposed by the chlorine atom as a result of interaction with the mono phenoxy substituent in 3a,in comparison with the effect of the second phenoxy substituent containing 3b whichprovides more freedom of movement for the conformational processes required for methyl interchange.

The intrinsic tendency of phthalocyanine molecules toward aggregation can usually result in a poor quality NMR spectrum characterized by the broadening and up field shift of proton peaks [29, 35]. Therefore, we use the NMR technique along with UV-vis spectroscopy to evaluate the self-association behavior of

CN

Cl

Cl

CN

1

OH

2

(a)

CN

CN

3b

(b)

M = H2 (4b); Zn (5b); AlCl (6b); GaCl (7b); InCl (8b)

4b-8b

O

N N

O

N

NN N

NN

MO

O

O

O

O

O

OO

+

Scheme 2. Synthetic route to octa-substituted 2,6-di-iso-propylphenoxy phthalocyanine 4b–8b. Reagents and conditions: (a) anhydrous K2CO3,DMF, 80 °C, 24 h; (b) appropriate metal salt, quinoline, 180 °C, 12 h, inert atmosphere

Fig. 1. The 1H NMR spectra of the aliphatic region (methyl groups of the di-iso-propyl units) of 4-chloro-5-(2,6-di-iso-propylphenoxy) phthalonitrile (3a) at different temperatures in DMSO-d6 (a) 298 K and (b) 348 K


166 A. TUHL ET AL.

the prepared complexes. The NMR spectra for 4a–8athe prepared complexes recorded in CDCl3 were almost identical and show complicated and overlapped resonant peaks which were difficult to assign due to the mixture of isomers characteristic of the prepared Pcs. Nonetheless, integration of the peaks correctly corresponded to the expected total number of protons for each complex, thus confirming the relative purity of the complexes which is supported by the elemental and mass spectrometry analysis. For example, the NMR spectrum of 5a consistsof two distinct aliphatic and one aromatic region: several overlapped doublet peaks at ε: 1.10–1.60 ppm belonging to the methyl protons, four septet peaks in the range of ε:3.21–4.21 ppm attributed to the methylene protons and complicated multiplet peaks at ε: 7.39–9.05 ppm related to the aromatic protons. The qualities of 1H NMR spectra of 4a–8a complexes remain unchanged even after the addition of trace amount of pyridine-D5 into the 1NMRsample solution. Although, the overlapped aromatic peaks are separated from each other due to the chemical shift induced by pyridine-D5. In addition, all octa-substituted derivatives (4b–8b) show very simple and well resolved spectra with sharp peaks in both aromatic and aliphatic regions due to their high symmetry and the total absence of aggregation. Indeed, the defined

signal patterns, including the absence of both broadening and chemical shifts of the prepared complexes, were essentially independent of the concentration and the appearance of the spectra remained unchanged over the concentration range between 1 × 10-4 and 1 × 10-2 M at room temperature [35]. Such behavior can be attributed to the position adopted by the bulky phenoxy substituents relative to the Pc core that successfully prevents efficient π–π stacking of the macrocycle units.

Ground state electronic absorption and fluorescence spectra

Figures 3 and 4 show the ground state electronic absorption spectra pertaining to the metal free, aluminum, zinc, indium and gallium tetra-di-iso-propylphenoxy (4a–8a) and octa-di-iso-propylphenoxy (4b–8b)phthalocyanines, respectively in DMF. All spectra remain monomeric up to 2 × 10-5 M (Beer–Lambert law was obeyed below these concentrations) and exhibit sharp bands typical of non-aggregated species. The presence of axial ligands associated with the aluminum, indium and gallium central metal atoms, is particularly useful in preventing aggregation. Axial ligands in this position restrict the stacking interaction of multiple MPc molecules, often reflected by broad and blue shifted absorption bands in the UV-vis spectrum [36–38].

Fig. 2. The 1H NMR spectra of the aliphatic region (methyl groups of the di-iso-propyl units) of 4,5-bis-(2,6-di-iso-propylphenoxy) phthalonitrile (3b) at different temperatures in CDCl3 (a) 273 K and (b) 308 K

Table 1. Activation energy calculations of 4-chloro-5-(2,6-di-iso-propylphenoxy)phthalonitrile (3a) in comparison with the 4,5-bis-(2,6-di-iso-propylphenoxy)phthalonitrile (3b)

Materials Tc, K Δυ, Hz ΔG*, kJ.mole

3a 348 31.8 75.8

3b 308 45.6 65.8

Fig. 3. Normalized ground state electronic absorption spectra of (i) 4a, (ii) 5a, (iii) 6a, (iv) 7a and (v) 8a in DMF

Fig. 4. Normalized ground state electronic absorption spectra of (i) 4b, (ii) 5b, (iii) 6b, (iv) 7b and (v) 8b in DMF



Complexes 5a, 6a and 8a show a slight red shift in the Q-band maxima relative to the corresponding octa-substituted complexes 5b, 6b and 8b proving that the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) energy gap is reduced upon the introduction of chlorine atoms [24, 25, 39]. As anticipated, the electronic spectra of the tetra and octa (2,6-di-iso-propylphenoxy)-substituted phthalocyanine complexes showed monomeric behavior in solution using different organic solvents (CH2Cl2,CHCl3, THF and DMF) as confirmed by the position and the appearance of intense Q-band peaks. The spectrum in less coordinating solvents such as chloroform was similar to that in DMF (Fig. 3) which is typical of non-aggregated species belonging to D4h symmetry. However, the monomeric Q-band position of 5b is blue shifted relative to that of 5a proving that the HOMO-LUMO energy gap is reduced upon the introduction of chlorine atoms. This marginal red shift, caused by chlorine atoms, is consistent with the concept that the chlorine atoms are participating in the π-electron system of the Pc complexes and act as electron donor substituents by mesomeric effects which may provoke noticeable changes in photoelectric behavior [22, 23, 40].

Similarly, a shift to longer wavelengths with an increase in central metal size is due to an increase in electron density, where the order of Q-band positions is Zn2+ < Al3+ < Ga3+ < In3+ (Table 2) for the 4a–8acomplexes. For the octa-substituted complexes the trend is the same as for the 4a–8a complexes, except 7b and 8b have the same wavelength. The notable red shift for InPc derivatives may be attributed either to distortion of the macrocycle in which the indium (i.e. the largest size among the transition metals used in this study) is out of the π-planar system creating a non-planar system or weakness of the coordination M–N bond [41], due to

a decrease in electronegativity on going from Al to In, which leads to higher π-electron density of the system and therefore the lower energy gap between HOMO and LUMO is consequently observed.

With the exception of InPc and GaPc derivatives, all the molecules showed fluorescence emission and excitation spectra typical of phthalocyanines, where the excitation is similar to absorption and both are mirror images of the fluorescence emission, Fig. 5 for 6a inDMF. The behavior of InPc and GaPc derivatives could be related to their size which may result in changes in symmetry upon excitation. A flexible σ-bond connecting the phenoxy ring to the local MPc ring may allow for photoinduced twisting which distorts the MPc molecule. Stokes shifts were typical of MPc derivatives [11].

Photophysicochemical parameters

Fluorescence quantum yields and lifetimes. Fluorescence quantum yields reflect the fraction of absorbing molecules, excited to the singlet state and subsequently deactivated via radiative means. Thus the quantum yield is a ratio of the number of photons emitted relative to the number of photons absorbed [42]. Variations in fluorescence quantum yield values have been explained by a number of prevailing conditions including temperature, molecular structure and solvent parameters (polarity, viscosity, and refractive index). In this case, considering molecules with the same number of substituents, there is a trend towards lower quantum yields with increase in size of central metal for the 4a–8aspecies i.e. Zn(5a) > Ga(7a) > In(8a) (Table 3) and similarly for the octa-di-iso-propyl-substituted species. Such behavior is expected on the grounds of spin-orbit coupling induced by the respective central metal ions. In3+ being the largest central metal ion induces a greater

Table 2. Absorption, fluorescence emission and fluorescence excitation spectral data for MPc complexes 4a–8a and 4b–8b andZnPc in DMF

MPc Q-band λmax, nm Log ε Emission λmax, nm Excitationa λmax, nm Stoke’s Shift, nm

ZnPc 669 5.37 685 669 16

4a 671, 700 5.21 707 671, 700 7

5a 680 5.35 697 677 20

6a 683 5.29 696 683 13

7a 688 5.27 702 692 10

8a 693 5.21 712 704 8

4b 667, 701 5.30 709 667, 703 6

5b 675 5.57 693 676 17

6b 679 5.48 697 679 8

7b 690 5.43 710 703 7

8b 690 5.38 713 705 8

a Excitation at 580 nm for MPc complexes.


168 A. TUHL ET AL.

effect, in terms of spin-orbit coupling, thereby increasing the likelihood of the spin-forbidden intersystem crossing (ISC) process to populate the triplet state and an attendant decrease in the spin-allowed fluorescence. Aluminum serves as an exception, due to its small size, with fluorescence quantum yields higher than the Zn metalated species (Table 3). The effect of introducing eight di-iso-propylphenoxy substituents to the Pc ring periphery is insignificant in terms of quantum yield values, relative to the tetra 2,6-di-iso-propylphenoxy-substituted derivatives (Table 3). However, 6b gives higher yields relative to the tetra 2,6-di-iso-propylphenoxy-substituted derivative, 6a. This may be in relation to the presence of the chloro-substituent groups in 6a which have a greater effect, in this case, in terms of deactivation of molecules in the excited singlet via non-radiative means such as ISC rather than by fluorescence, as a result of the heavy atom effect.

The fluorescence lifetime gives an indication of how much time the molecule spends in the excited state prior to relaxation back to the ground state. Metallophthalocyanines

decay with mono-exponential behavior with fluorescence lifetimes that range between 1–10 ns [43–45]. All phtha-locyanines, in this study, exhibit mono-exponential decay kinetic behavior typical of monomeric phthalocyanine species as shown by a typical fluorescence decay curve for 7b in Fig. 6. The data in Table 3 shows minimal variation with respect to the number of substituents; however a change in central metal is more significant. A similar effect to the fluorescence quantum yields is observed for the most part for fluorescence lifetimes, where the presence of large central metals such as Ga and In experience shortened lifetimes as a consequence of the heavy atom mediated effect. This has also been reported previously for Ga and In derivatives [15, 16]. As with fluorescence quantum yields, the aluminum derivatives 6a and 6b deviate from typical behavior to give fluorescence lifetimes higher than those of a corresponding Pc substituted with larger central metal atoms i.e. Zn2+, Ga3+ and In3+. Aluminum Pcs generally tend to exhibit elevated fluorescence yields [41, 44, 46–48] due to the small size of Al.

Triplet quantum yields and lifetimes. The spin forbidden process of intersystem crossing to populate the triplet-excited state is a result of spin-orbit coupling. Phthalocyanine species that encounter the strongest spin-orbit coupling effects are often accompanied by high triplet quantum yields (ΦT) which give a measure of the fraction of absorbing molecules that undergo intersystem crossing to the triplet excited state. The ΦT and triplet lifetimes (τT) pertaining to the molecules explored in this work are shown in Table 3. The order of variance, with respect to the octa-substituted derivatives, is consistent with the strengths of induced spin-orbit coupling in the complexes, i.e. species metalated with In3+ show the highest ΦT values with the lowest data occurring for the Zn species, although aluminum was expected to give lower values due to its size. For the 4a–8a complexes

Fig. 5. Normalized ground state electronic absorption (i), fluorescence emission (ii) and fluorescence excitation (iii) of 6a in DMF

Table 3. Photophysicochemical parameters of MPc complexes 4a–8a and 4b–8b and ZnPc in DMF

MPc ΦF ΦT ΦIC ΦΔ SΔ τF, ns (±0.04) τT, μs

ZnPc 0.1754 0.5854 0.25 0.56 [57] 0.97 3.14 330 [58]

4a 0.12 0.83 (700 nm) 0.05 0.52 0.63 3.69 17

5a 0.12 0.81 0.07 0.58 0.72 2.87 69

6a 0.14 0.85 0.01 0.25 0.30 5.52 12

7a 0.07 0.81 0.12 0.35 0.43 3.86 13

8a 0.02 0.77 0.21 0.56 0.73 2.17 19

4b 0.12 0.87 (702 nm) 0.01 0.53 0.56 5.52 10

5b 0.12 0.32 0.56 0.26 0.81 3.00 15

6b 0.24 0.40 0.64 0.36 0.90 5.88 17

7b 0.09 0.51 0.40 0.45 0.88 3.53 6

8b 0.01 0.67 0.32 0.42 0.63 2.76 13



there seems to be minimal effect with increase in size of the central metal. Interestingly the presence of eight 2,6-di-iso-propylphenoxy ring substituents gave reduced ΦTvalues suggesting that an increase in substituent groups brings about a quenching effect. Such an effect may be in relation to the “loose bolt effect” [49]. A similar effect has been reported for InPc complexes substituted with large phenoxy substituents [16]. The effect is associated with the loss of energy, through internal conversion, as a result of C–H vibrations [49]. An increase in the number of 2,6-di-iso-propylphenoxy groups, from four to eight (tetra to octa), gives rise to an increase in C–H bonds and concomitant increased rate of internal conversion and thus a more pronounced “loose bolt” effect. Worthy of note is the transient absorption spectra of 7a, as an example, shown in Fig. 7. The figure indicates the appearance of a broadened peak centered near 700 nm whereas the absorption spectrum corresponding to this complex shows a maximum at 688 nm (Table 2). Such changes in optical spectra are often a result of symmetry loss, often depicted by splitting or broadening of bands, following photoexcitation [50]. As stated above, a flexible σ-bond connecting the phenoxy ring to the local MPc ring allows for photoinduced twisting which distorts the MPc molecule and thus gives rise to the changes observed. High values of ΦT for 4a–8a derivatives could also be due to the presence of a halogen (chlorine atom).

The triplet lifetimes determined for the complexes conform to no particular trend ranging from 10–69 μs(Table 3). Such low lifetimes in DMF are common for phthalocyanines containing a large central metal such as In, Ga and Cd, corresponding to high triplet state quantum yields [15, 16, 50, and 51]. The reason for the low lifetime for the AlPc derivatives is not clear, but could be related to the substituents. Unsubstituted ZnPc has a long lifetime compared to complexes 5a and 5b,showing the effects of the substituents.

Singlet oxygen quantum yields. The generation of singlet oxygen (1O2) is a result of the transfer of energy from a phthalocyanine molecule in the triplet excited state

to ground state molecular oxygen (3Σg). The efficiency with which singlet oxygen is generated is quantified by the singlet oxygen quantum yield (ΦΔ) which varies as a function of triplet state quantum yield, triplet state lifetime, efficiency of energy transfer (SΔ), triplet state quenching effect of substituents and the triplet state energy. In essence, large triplet state quantum yields should be accompanied in turn by large singlet oxygen quantum yields as shown in Table 3, where 4a–8a derivatives generally give larger quantum yield values compared to their octa-substituted counterparts. Compounds 6a and 7a are exceptions, expressing much lower yields than expected based on their triplet quantum yields. In these complexes, the energy transfer process proves to be slightly less efficient as expressed by SΔ which is a measure of excitation energy transfer efficiency. However, values near unity, as shown in Table 3 for the octa-substituted derivatives, reflect more efficient energy transfer to produce 1O2 as opposed to MPc complexes modified by tetra-substitution with 2,6-di-iso-propylphenoxy.

EXPERIMENTAL

Materials

All solvents including dimethylformamide (DMF), dichloromethane (DCM), and tetrahydrofuran (THF) were of reagent grade and purchased from Aldrich. Any purification required was carried out according to Perrin and Amarego [52].

Thin layer chromatography (TLC) was performed using Polygram sil G/UV 254 TLC plates and visualization was carried out by ultraviolet light at 254 nm and 350 nm. Column chromatography was performed using Merck silica gel 60 of mesh size 0.040–0.063 mm.

2,6-di-iso-propylphenol (2), 4,5-dichlorophthalonitrile (1), diphenylisobenzofuran (DPBF) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from esta-blished suppliers (Sigma-Aldrich, Merck, TCI Europe). All other reagents were purchased from suppliers and used without further purification.

Fig. 6. Fluorescence decay curve of 7b (red) and instrument response function (IRF, black) in DMF

Fig. 7. Transient absorption spectrum of 7a in DMF (concentration = 1.34 × 10-5 M)


170 A. TUHL ET AL.

Instrumentation

IR spectra were recorded on a Perkin Elmer system 2000 FTIR. All absorption spectra were undertaken on a Varian Cary 5 UV-vis spectrometer and fluorescence emission and excitation spectra using a Varian Eclipse spectrofluorimeter. 1H NMR spectra were recorded using a Bruker DPX 400 or BrukerAvance II 600. Elemental Analyses were carried out using a LECO Elemental Analyzer CHNS 932. Mass analyses were obtained using a VG Autospec-Q. DSC analyses were carried out on a Shimadzu DSC-50. FAB mass spectra were obtained using a Micro-Mass Tofspec 2E spectrometer.

Fluorescence lifetimes were measured using a time correlated single photon counting (TCSPC) setup (FluoTime 200, Picoquant GmbH). The excitation source was a diode laser (LDH-P-670 with PDL 800-B, Picoquant GmbH, 670 nm, 20 MHz repetition rate, 44 ps pulse width). Fluorescence was detected under the magic angle with a peltier cooled photomultiplier tube (PMT) (PMA-C 192-N-M, Picoquant) and integrated electronics (PicoHarp 300E, Picoquant GmbH). A monochromator with a spectral width of about 4 nm was used to select the required emission wavelength band. The response function of the system, which was measured with a scattering Ludox solution (DuPont), had a full width at half-maximum (FWHM) of about 300 ps. The ratio of stop to start pulses was kept low (below 0.05) to ensure good statistics. All luminescence decay curves were measured at the maximum of the emission peak. The data was analyzed with the program FluoFit (Picoquant GmbH). The support plane approach was used to estimate the errors of the decay times [42].

A laser flash photolysis system was used for the determination of triplet absorption and decay kinetics. The excitation pulses were produced by a Quanta-Ray Nd: YAG laser (1.5 J/9 ns), pumping a Lambda Physik FL 3002 dye laser (Pyridin 1 in methanol). The analyzing beam source was from a Thermo Oriel 66902 xenon arc lamp, and a KratosLisProjekte MLIS-X3 photomultiplier tube was used as the detector. Signals were recorded with a two-channel 300 MHz digital real-time oscilloscope (Tektronix TDS 3032C); the kinetic curves were averaged over 128 laser pulses.

Photo-irradiations for singlet oxygen determinations were done using a General Electric Quartz line lamp (300 W). A 600 nm glass cut off filter (Schott) and a water filter were used to filter off ultraviolet and infrared radiations respectively. An interference filter (Intor, 700 nm with a band width of 40 nm) was additionally placed in the light path before the sample. The light intensity was measured with a POWER MAX 5100 (Molelectron detector incorporated) power meter and found to be 2.97 × 1016 photons.s-1.

Photophysicochemical parameters

Fluorescence quantum yields ( F) were determined as reported in literature [53] using ZnPc as a standard in

DMF (with F = 0.17 [54]). Similarly triplet quantum yield ( T) values were determined according to literature [55] using ZnPc in DMF as a standard ( T = 0.56 [56].

Fluorescence lifetimes were obtained by deconvolution of the decay curves, obtained by time correlated single photon counting (TCSPC), using the FluoFit Software program (PicoQuant GmbH, Germany). Triplet lifetimes ( T) were determined by exponential fitting of the kinetic curves using OriginPro 7.5 software.

Quantum yields of internal conversion ( IC) were obtained from Equation 1, which assumes that only the three intrinsic processes fluorescence, intersystem crossing and internal conversion; jointly deactivate the excited singlet state of an MPc molecule.

ΦIC = 1 - (ΦF + ΦT) (1)

The fraction of the excited triplet state quenched by ground state molecular oxygen S was calculated using Equation 2:

ST

ΔΔΦ

Φ= (2)

Singlet oxygen quantum yields

Singlet oxygen quantum yield values ( Δ) values were determined in air using the relative method with DPBF acting as a singlet oxygen chemical quencher in DMF, using Equation 3:

Φ ΦΔ = TStd abs

Std

Stdabs

R I

R I

.

.(3)

where ΦΔStd is the singlet oxygen quantum yield for the

standard ZnPc (ΦΔStd = 0.56 in DMF [57]); R and RStd are

the DPBF photobleaching rates in the presence of the respective MPc and standard respectively; Iabs and Iabs

Std

are the rates of light absorption by the MPc and standard respectively. The concentration of DPBF was lowered to ~ 3 × 10-5 M for all solutions, to avoid chain reactions [57]. DPBF degradation was monitored at ~417 nm.

Synthesis

4-chloro-5-(2,6-di-iso-propylphenoxy)phthalo-nitrile (3a). To a stirred solution of 4,5-dichloro-phthalonitrile (1, 1.97 g, 10 mmol) and 2,6-di-iso-propylphenol (2, 1.8 g, 10 mmol) in dry DMF (150 mL) finely ground anhydrous potassium carbonate (4.8 g, 35 mmol) was added. The reaction mixture was heated at 45 °C under nitrogen for 24 h. On cooling, the reaction mixture was poured into acidified water. The resulting precipitate was collected by filtration and washed with distilled water, then air-dried. The crude product was then recrystallized from methanol to give 3a. Yield 2.85 g (84%) as a white crystalline solid, mp 181 °C. Anal. calcd. for C20H19ClN2O: C, 70.90; H, 5.65; N, 8.27%. Found: C, 70.78; H, 5.58; N, 8.59. IR (KBr): ν, cm-1



2233 (CN). 1H NMR (400 MHz, CDCl3, 25 °C): δ, ppm 1.14 (d, 6H, 2-CH3), 1.22 (d, 6H, 2-CH3), 2.75 (sept, 2H, 2-CH3CHCH3 ), 6.76 (s, 1H, Pc Ar-H), 7.30 (d, 2H, Ar-H), 7.37 (t, 1H, Ar-H), 7.91 (s, 1H, Pc Ar-H). MS (EI): m/z 338 (calcd. for [M]+ 338.5998].

2,9,16,23-tetrachloro-3,10,17,24-tetra(2,6-di-iso-propylphenoxy)phthalocyanine (4a). A solution of 4-chloro-5-(2,6-di-iso-propylphenoxy)phthalonitrile (3a,1.00 g, 2.95 mmol), and urea (0.35 g, 6 mmol), in dry quinoline (10 mL) was heated at 180°C under nitrogen for 12 h. On cooling the reaction mixture was poured into 200 mL of stirred distilled water and neutralized with hydrochloric acid. The resulting solid product was collected by filtration, washed with water and methanol. The crude product was purified by column chromatography on silica (eluent DCM) to give 4a. Yield 0.28 g (28%) as a greenish-blue powder, mp > 300 °C.Anal. calcd. for C80H78Cl4N8O4: C, 70.79 H, 5.79; N, 8.26%. Found: C, 71.18; H, 5.53; N, 7.95. IR (KBr): ν, cm-1 3964 (Ar-H), 1607 (C=N). UV-vis (DMF): max,nm (log ε) 668 (5.13), 703 (5.21). 1H NMR (600 MHz, CDCl3, 25 °C): δ, ppm -0.53 (s, 2H, -NH), 1.15–1.37 (m, 48H, 16-CH3), 3.20–4.24 (m, 8H, 4-CH3CHCH3), 7.45–9.51 (m, 20H, Pc Ar-H and Ar -H). MS (FAB): m/z 1357 (calcd. for [M]+ 1356.4259).

A general procedure for synthesis of complexes 5a–8ais as follows. A mixture of 3a (1.00 g, 2.95 mmol) and excess of anhydrous metal salt (40 mg) in dry quinoline (5 mL) and few drops of DBU was stirred at 180 °C under nitrogen for 12 h. The reaction mixture was poured into stirred distilled water (200 mL) on cooling, and the solid product was collected by filtration, washed with water and methanol. The crude product was purified by column chromatography on silica using the appropriate eluent as stated for each complex below.

2,9,16,23-tetrachloro-3,10,17,24-tetra(2,6-di-iso-propylphenoxy)phthalocyaninatozinc (5a). Zincacetate used as metal salt. Eluent used: hexane-DCM (1:1) to give 5a. Yield 0.20 g (19%) as a greenish-blue powder, mp > 300 °C. Anal. calcd. for C80H76Cl4N8O4 Zn:C, 67.63; H, 5.39; N, 7.88%. Found: C, 67.37; H, 5.49; N, 7.54. IR (KBr): ν, cm-1 3962 (Ar-H), 1605 (C=N). UV-vis (DMF): max, nm (log ε) 680 (5.35). 1H NMR (600 MHz, CDCl3, 25 °C): δ, ppm 1.10–1.60 (m, 48H, 16-CH3), 3.21–4.21 (m, 8H, 4-CH3CHCH3), 7.39–9.05 (m, 20H, Pc Ar-H and Ar -H). MS (FAB): m/z 1417 (calcd. for [M]+ 1419.7903).

2,9,16,23-tetrachloro-3,10,17,24-tetra(2,6-di-iso-propylphenoxy)phthalocyaninatoaluminum chloride (6a). Aluminum chloride used as metal salt. Eluent used: ethyl acetate-THF (4:1) to give 6a. Yield 0.29 g (28%) as a green powder, mp > 300 °C. Anal. calcd. for C80H76Cl5N8O4Al: C, 67.77; H, 5.40; N, 7.90%. Found: C, 67.45; H, 5.87; N, 7.65. IR (KBr): ν, cm-1 3963 (Ar-H), 1607 (C=N). UV-vis (DMF): max, nm (log ε) 683 (5.29). 1H NMR (600 MHz, CDCl3, 25 °C): δ, ppm 1.07–1.45 (m, 48H, 16-CH3), 3.14–3.45 (m, 8H, 4-CH3CHCH3),

7.46–9.63 (m, 20H, Pc Ar-H and Ar -H). MS (FAB): m/z1417 (calcd. for [M]+ 1416.6608).

2,9,16,23-tetrachloro-3,10,17,24-tetra(2,6-di-iso-propylphenoxy)phthalocyaninatogallium chloride (7a). Gallium chloride used as metal salt. Eluent used: DCM-ethyl acetate (4:1) to give 7a. Yield 0.19 g (17.5%) as a green powder, mp > 300 °C. Anal. calcd. for C80H76Cl5N8O4Ga: C, 65.79; H, 5.25; N, 7.67%. Found: C, 65.41; H, 5.36; N, 7.39. IR (KBr): ν, cm-1 3963 (Ar-H), 1606 (C=N). UV-vis (DMF): max, nm (log ε) 688 (5.27). 1H NMR (600 MHz, CDCl3, 25 °C): δ, ppm 1.04–1.80 (m, 48H, 16-CH3), 3.21–4.25 (m, 8H, 4-CH3CHCH3),7.43–9.71 (m, 20H, Pc Ar-H and Ar -H). MS (FAB): m/z1460 (calcd. for [M]+ 1459.5859).

2,9,16,23-tetrachloro-3,10,17,24-tetra(2,6-di-iso-propylphenoxy)phthalocyaninatoindium chloride (8a). Indium chloride used as metal salt. Eluent used: hexane-DCM (1:5) to give 8a. Yield 0.25 g (22.5%) as a green powder, mp > 300 °C. Anal. calcd. for C80H76Cl5N8O4In: C, 63.82; H, 5.09; N, 7.44%. Found: C, 64.22; H, 5.27; N, 7.54. IR (KBr): ν, cm-1 3964 (Ar-H), 1605 (C=N). UV-vis (DMF): max, nm (log ε) 693 (5.21). 1H NMR (600 MHz, CDCl3, 25 °C): δ, ppm 0.94–1.46 (m, 48H, 16-CH3), 3.02–3.39 (m, 8H, 4-CH3CHCH3),7.42–9.65 (m, 20H, Pc Ar-H and Ar -H). MS (FAB): m/z1505 (calcd. for [M]+ 1504.6809).

4,5-bis-(2,6-di-iso-propylphenoxy)phthalonitrile (3b) [20]. Dry potassium carbonate (30.00g, 217 mmol) was added to a solution of 4,5-dichlorophthalonitrile (1, 10.0 g, 50.7 mmol) and 2,6-di-iso-propylphenol (2,22.25 g, 125.0 mmol) in dry dimethylformamide and were reacted according to the procedure used for 3a to give the crude compound which was purified by recrystallization from methanol to give 3b. Yield 18.3 g (75%) as a white solid, mp 161–163 °C. Anal. calcd. for C32H36N2O2: C, 79.95; H, 7.50; N, 5.83%. Found: C, 79.91; H, 7.60; N, 5.80. IR (KBr): ν, cm-1 3082, 2228, 1590, 1500, 1342, 1096, 880, 795. 1H NMR (400 MHz; CDCl3): δ, ppm 7.25 (m, 6H, Ar-H), 6.70 (s, 2H, Pc Ar-H), 2.88 (sept, 4H, J = 6.7 Hz, CH3CHCH3), 1.20 (d, 12H, J = 6.7 Hz, CH3CHCH3), 1.10 (d, 12H, J = 6.7 Hz, CH3CHCH3). MS (EI): m/z 480 (calcd. for [M]+ 480.2778).

2,3,9,10,16,17,23,24-octa(2,6-di-iso-propylphe-noxy)phthalocyaninato (4b) [20]. To a solution of 4,5-bis(2,6-di-iso-propylphenoxy)phthalonitrile (3b,0.20 g, 0.42 mmol), in refluxing anhydrous 1-pentanol (3 mL) and under nitrogen, an excess of lithium was added. The reaction mixture was refluxed for 5 h and then cooled and acetic acid added (1 mL, 0.1 M). The solvent was removed under reduced pressure to give the crude product. Purification by column chromatography on SiO2, eluting with DCM to give 4b. Yield 0.08 g (40%) as a green solid, mp > 300 °C. Anal. calcd. for C128H146N8O8: C, 79.88; H, 7.65; N, 5.82%. Found: C, 80.04; H, 7.70; N, 5.70. IR (film): ν, cm-1 3576, 2962, 1439, 1328, 1265, 1184, 1093, 1017, 877, 754. UV-vis (DMF): max, nm (log ε) 701 (5.30). 1H NMR (400 MHz,


172 A. TUHL ET AL.

CDCl3): δ, ppm 8.13 (br, 8H, Pc Ar-H), 7.50 (m, 24H, Ar-H), 3.37 (sept, 16H, J = 6.5 Hz, CH3CHCH3), 1.28 (br m, 96H, CH3CHCH3), -0.84 (s, 2H, -NH). MS (FAB): m/z 1925.71 (calcd. for [M]+ 1923.1272).

2,3,9,10,16,17,23,24-octa(2,6-di-iso-propylphe-noxy)phthalocyaninatozinc (5b) [20]. A stirred solution of 4,5-bis (2,6-di-iso-propylphenoxy)phthalonitrile (3b,0.30 g, 0.6 mmol) and zinc(II) acetate (0.1 g, 0.6 mmol) in quinoline (1 mL) was heated at 180 °C for 12 h under nitrogen. The reaction mixture was cooled to room temperature and poured into methanol (15 mL). The crude product was purified by column chromatography on SiO2, eluting with hexane-DCM (4:6) to give 5b. Yield 0.2 g (67%) as a green solid, mp > 300 °C. Anal. calcd. for C128H144N8O8Zn: C, 77.59; H, 7.32; N, 5.66%. Found: C, 77.68; H, 7.52; N, 5.52. IR (film): ν, cm-1 2961, 1612, 1456, 1462, 1413, 1353, 1269, 1186, 1095, 1050, 904, 864, 799, 777, 755, 729. UV-vis (DMF): max, nm (log ε)675 (5.57). 1H NMR (400 MHz, CDCl3): δ, ppm 8.17 (s, 8H, Pc Ar-H), 7.62 (t, 8H, J = 7.8 Hz, Ar-H), 7.5 (br s, 16H, Ar-H), 3.46 (br s, 16H, CH3CHCH3), 1.30 (br m, 96H, CH3CHCH3). MS (FAB): m/z 1988 (calcd. for [M]+

1986.4915).Complexes 6b–8b were synthesized and purified as

outlined for (5b).2,3,9,10,16,17,23,24-octa(2,6-di-iso-propylphe-

noxy)phthalocyaninatoaluminum chloride (6b). Aluminum(III) chloride used as metal salt. Eluent used: hexane-DCM-ethyl acetate (4:4:1) to give 6b. Yield 0.12 g (20%) as a green solid, mp > 300 °C. Anal. calcd. for C128H144ClN8O8Al: C, 77.48; H, 7.26; N, 5.65%]. Found: C, 77.52; H, 7.45; N, 5.80. IR (film): ν, cm-1 2962, 1621, 1463, 1414, 1354, 1330, 1272, 1185, 1093. UV-vis (DMF): max, nm (log ε) 691 (5.48). 1H NMR (400 MHz, CDCl3): δ, ppm 8.22 (s, 8H, Pc Ar-H), 7.61 (t, 8H, J = 7.8 Hz, Ar-H), 7.50 (br s, 16H, Ar-H), 3.43 (br s, 16H, CH3CHCH3), 1.27 (br m, 96H, CH3CHCH3). MS (FAB): m/z 1982 (calcd. for [M]+ 1983.5457).

2,3,9,10,16,17,23,24-octa(2,6-di-iso-propylphenoxy)phthalocyaninatogallium chloride (7b). Gallium(III) chloride used as metal salt. Eluent used: DCM to give 7b. Yield 0.04 g (40%) as a green solid, mp > 300 °C.Anal. calcd. for C128H144ClGaN8O8: C, 75.85; H, 7.11; N, 5.53%. Found: C, 75.60; H, 7.98; N, 5.78. IR (film): ν,cm-1 2963, 1610, 1452, 1400, 1265, 1185, 1090. UV-vis (DMF): max, nm (log ε) 690 (5.43). 1H NMR (400 MHz, CDCl3): δ, ppm 8.19 (s, 8H, Pc Ar-H), 7.60 (t, 8H, J = 7.6 Hz, Ar-H), 7.48 (r s, 16H, Ar-H), 3.41 (br s, 16H, CH3CHCH3), 1.33 (br m, 96H, CH3CHCH3). MS (FAB): m/z 2025 (calcd. for [M]+ 2026.2872).

2,3,9,10,16,17,23,24-octa(2,6-di-iso-propylphenoxy)phthalocyaninatoindium chloride (8b). Indium(III) chloride was used as metal salt. Eluent used: DCM to give 8b. Yield 0.03 g (38%) as a green solid, mp > 300 °C.Anal. calcd. for C128H144ClInN8O8: C, 74.17; H, 7.00; N, 5.41%. Found: C, 73.62; H, 7.36; N, 4.93. IR (film): ν,cm-1 2965, 1617, 1461, 1410, 1280, 1182, 1090. UV-vis

(DMF): max, nm (log ε) 690 (5.38). 1H NMR (400 MHz, CDCl3): δ, ppm 8.17 (s, 8H, Pc Ar-H), 7.61 (t, 8H, J = 7.6 Hz, Ar-H), 7.52 (br s, 16H, Ar-H), 3.35 (br s, 16H, CH3CHCH3), 1.28 (br m, 96H, CH3CHCH3). MS (FAB): m/z 2072 (calcd. for [M]+ 2071.3822).

CONCLUSION

The synthesis of novel metal free, zinc, aluminum, gallium and indium phthalocyanines terminated by four or eight di-iso-propylphenoxy groups has been reported. Supporting characterization techniques confirm the predicted structural configuration of these molecules. It was found that the steric factor produced by the peripheral substituents play a major role in perfectly preventing, the common aggregation behavior of Pc complexes as confirmed by 1H NMR and UV-vis spectroscopic techniques. Consequently the effects of substituents (i.e. phenoxy or chlorine atoms) or the central metals on the physicochemical properties have been investigated. Low fluorescence quantum yields were accompanied by large triplet quantum yields, particularly for the tetra-substituted species. Considering the central metal atom, an increase in the size of the central metal atom yielded a similar effect in response to the heavy atom effect. Fluorescence lifetimes fall within the range typical of phthalocyanines, with the aluminum species (6a and 6b) showing extended lifetimes in the S1 state relative to the other Pc derivatives. All phthalocyanine derivatives in this study generally produced singlet oxygen with relatively good quantum yields. The photophysicochemical properties determined thus suggest that these molecules may be considered for either PDT or optical limiting applications.

Acknowledgements

AT wishes to thank the College of Graduate Studies, Kuwait University for a Ph.D. candidacy. The financial support of ANALAB and SAF grants (Nos. GS 01/01, GS 01/05 and GS 03/01), Kuwait University, and the support by the Department of Science and Technology (DST) and National Research Foundation (NRF), South Africa through DST/NRF Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology and Rhodes University are gratefully acknowledged. WC thanks Rhodes University for funding.

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CONTENTS


Diversity, isomer composition, and design of combinatorial libraries 1of tetrapyrrole macrocycles

Masahiko Taniguchi and Jonathan S. Lindsey*

Spectroscopic and theoretical insights on determination of binding strength and molecular structure for the 14supramolecular complexes of a designed bisporphyrin with C60 and C70

Partha Mukherjee, Subarata Chattopadhyay and Sumanta Bhattacharya*

Internal spin trapping of thiyl radical during the complexation and reduction of cobalamin with 25glutathione and dithiothrietol

Somasundaram Ramasamy, Tapan K. Kundu, William Antholine, Periakaruppan T. Manoharan* and Joseph M. Rifkind*

Protonation-deprotonation equilibria in tetrapyrroles. Part 2. Mono- and diprotonation 39of methyl pyropheophorbide a in methanolic hydrochloric acid as verified by the 1H, 13C HSQC and 1H, 15N HMBC NMR experiments

Paavo H. Hynninen* and Markku Mesilaakso

Nucleotide-driven packaging of a singlet oxygen generating porphyrin in an icosahedral virus 47

Brian A. Cohen, Alain E. Kaloyeros and Magnus Bergkvist*

Correlation of photodynamic activity and singlet oxygen quantum yields in two series 55of hydrophobic monocationic porphyrins

Daiana K. Deda, Christiane Pavani, Eduardo Caritá, Maurício S. Baptista, Henrique E. Toma and Koiti Araki*

Effect of lipophilicity on biological properties of 109Pd-porphyrin complexes: a preliminary investigation 64

Sudipta Chakraborty, Tapas Das, Haladhar D. Sarma and Sharmila Banerjee*

Water-soluble porphyrin-based logic gates 72

Lili Li, Peiying Cai, Yuefei Deng, Liutao Yang, Xuan He, Lingsong Pu, Di Wu*, Jin Liu*, Haifeng Xiang and Xiangge Zhou*

Synthesis, properties and near-infrared imaging evaluation of glucose conjugated zinc phthalocyanine 77via Click reaction

Feng Lv, Xujun He, li Lu, Li Wu and Tianjun Liu*

Synthesis, circular dichroism, DNA cleavage and singlet oxygen photogeneration of 4-amidinophenyl porphyrins 85

Kai Wang, Chun T. Poon, Chun Y. Choi, Wai-Kwok Wong*, Daniel W.J. Kwong*, Fa Q. Yu, Heng Zhang and Zao Y. Li*

Highly efficient conversion of aldehydes to carboxylic acid in the presence of platinum porphyrin 93sensitizers, air and sunlight

Mahdi Hajimohammadi, Hamid Mofakham, Nasser Safari* and Anahita Mortazavi Manesh

Cationic -vinyl substituted meso-tetraphenylporphyrins: synthesis and non-covalent interactions with 101a short poly(dGdC) duplex

Eduarda M.P. Silva, Catarina I.V. Ramos, Patrícia M.R. Pereira, Francesca Giuntini, Maria A.F. Faustino, João P.C. Tomé, Augusto C. Tomé, Artur M.S. Silva, M. Graça Santana-Marques*, M. Graça P.M.S. Neves* and José A.S. Cavaleiro

In vitro insulin-mimetic activity and in vivo metallokinetic feature of oxovanadium(IV)porphyrin 114complexes in healthy rats

Tapan K. Saha*, Yutaka Yoshikawa, Hirouki Yasui and Hiromu Sakurai

Synthesis of long-wavelength chlorins by chemical modification for methyl pyropheophorbide-a and 122their in vitro cell viabilities

Jin Jun Wang*, Jia Zhu Li, Judit Jakus and Young Key Shim

Single walled carbon nanotubes fuctionalized with nickel phthalocyanines: effects of point of 130substitution and nature of functionalization on the electro-oxidation of 4-chlorophenol

Samson Khene and Tebello Nyokong*

Picosecond and femtosecond optical nonlinearities of novel corroles 140

Syed Hamad, Surya P. Tewari, L. Giribabu and S. Venugopal Rao*

Catalytic epoxidation of cis-stilbene and naphthalene by tetra-n-butylammonium hydrogen monopersulfate 149in the presence of manganese(III) tetraarylporphyrins and various anionic co-catalysts

Zahra Solati*, Majid Hashemi, Ahmad Keshavarzi and Ezzat Rafiee

Synthesis and structural characterization of a magnesium phthalocyanine(3–) anion 154

Edwin W.Y. Wong and Daniel B. Leznoff*

Tetra and octa(2,6-di-iso-propylphenoxy)-substituted phthalocyanines: a comparative study among 163their photophysicochemical properties

Ahmad Tuhl, Wadzanai Chidawanayika, Hamada Mohamed Ibrahim, Nouria Al-Awadi, Christian Litwinski, Tebello Nyokong, Haider Behbehani, Hacene Manaa and Saad Makhseed*

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volume 16 / number 1 / pages 1–174 - world scientific · 2019. 12. 24. · contents j. porphyrins...

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