Queensland University of Technology

A „CLICK APPROACH‟ TO THE GENERATION OF

NOVEL PROFLUORESCENT NITROXIDES

SUBMITTED BY

Jason Christopher Morris

Bachelor of Science (Honours, Chemistry)

A thesis submitted for the degree of Masters of Applied Science

Discipline of Chemistry

Queensland University of Technology

February 2011

i

Abstract

This project focused on the first application of the copper catalyzed azide alkyne

cycloaddition reaction for the generation of novel profluorescent systems. Through

this approach four novel profluorescent nitroxides were prepared both rapidly and in

good yield from coumarin and nitroxide CuAAC coupling partners. Specifically, 7-

hydroxy, 7-diethylamino, 6-bromo and unsubstituted coumarin analogues bearing an

azide group in the 3-position were prepared and conjugatively joined to an alkyne

isoindoline nitroxide previously reported by our group.

To explore the impact of the nitroxide moiety on the fluorescence of these systems,

methoxyamine analogues of the corresponding nitroxide analogues were prepared.

Spectrophotometric analysis of these methoxyamine analogues revealed that the

aromatic systems possessed high quantum yields. However, the quantum yield

efficiency was found to be dependent on the presence of electron donating

substituents in the 7-position of the coumarin motif, which enhanced the charge-

transfer character of the system.

Furthermore, spectrophotometric analysis of nitroxide analogues demonstrated that

the triazole effectively mediated fluorophore-nitroxide communication, as evidenced

by the low quantum yield values of the nitroxide analogues.

These results suggest that this technique can be used to conjugatively join any azide

bearing fluorescent system with the key alkyne isoindoline coupling partner allowing

for the rapid generation of diverse profluorescent systems.

ii

Table of Contents

Abstract ......................................................................................................................... i

List of Figures .............................................................................................................. v

List of Tables ............................................................................................................... vi

List of Schemes ........................................................................................................... vi

Abbreviations ............................................................................................................. vii

Declaration ................................................................................................................ viii

Acknowledgements ..................................................................................................... ix

1 Chapter 1. Introduction ......................................................................................... 2

1.1 Nitroxide Free Radicals ................................................................................. 2

1.1.1 Nitroxide stability ................................................................................... 3

1.1.2 Isoindoline nitroxides ............................................................................. 5

1.1.3 Profluorescent nitroxides ........................................................................ 7

1.2 Coumarins .................................................................................................... 11

1.3 The „Click‟ Approach .................................................................................. 12

1.4 Potential Applications of Profluorescent Probes ......................................... 14

1.5 Project Outline ............................................................................................. 15

2 Chapter 2. Results and Discussion ...................................................................... 17

2.1 Synthesis of Alkyne Isoindoline CuAAC Coupling Partner ....................... 20

2.1.1 Synthesis of N-benzylphthalimide (25) ................................................ 20

2.1.2 Synthesis of 2-benzyl-1,1,3,3-tetramethylisoindoline (26) .................. 21

2.1.3 Synthesis of 5-bromo-1,1,3,3-tetramethylisoindoline (27) .................. 22

2.1.4 Synthesis of 5-iodo-1,1,3,3-tetramethylisoindoline (28) ..................... 24

2.1.5 Synthesis of 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (29) ........ 25

2.1.6 Synthesis of 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-

tetramethylisoindolin-2-yloxyl (30) .................................................................... 26

2.1.7 Synthesis of 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (21) ... 28

2.2 Synthesis of 3-Azido Coumarin CuAAC Coupling Partners ...................... 29

2.2.1 Coumarin synthesis .............................................................................. 29

2.2.2 Synthesis of 3-azido coumarin analogues ............................................ 32

2.3 Synthesis of Profluorescent Nitroxides ....................................................... 34

2.4 Synthesis of Methoxyamine Analogues ...................................................... 38

iii

2.5 Spectrophotometric Analysis of Profluorescent Nitroxides ........................ 39

3 Chapter 3. Conclusions and Future Work ........................................................... 48

3.1 Conclusions ................................................................................................. 48

3.2 Future Work ................................................................................................ 51

4 Chapter 4. Experimental ..................................................................................... 53

4.1 General Procedures ...................................................................................... 53

4.2 Synthesis ...................................................................................................... 54

4.2.1 N-Benzylphthalimide (25) .................................................................... 54

4.2.2 2-Benzyl-1,1,3,3-tetramethylisoindoline (26) ...................................... 55

4.2.3 5-Bromo-1,1,3,3-tetramethylisoindoline (27) ...................................... 56

4.2.4 5-Iodo-1,1,3,3-tetramethylisoindoline (28) .......................................... 57

4.2.5 5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (29) ............................. 58

4.2.6 5-(3-Hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl

(30) .............................................................................................................. 58

4.2.7 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (21) ....................... 59

4.2.8 3-Acetamido-7-acetoxy-2H-chromen-2-one (35) ................................ 60

4.2.9 3-Acetamido-2H-chromen-2-one (36) ................................................. 60

4.2.10 3-Acetamido-6-bromo-2H-chromen-2-one (37) .................................. 61

4.2.11 3-Nitro-7-diethylamino-2H-chromen-2-one (58)................................. 62

4.2.12 3-Amino-7-diethylamino-2H-chromen-2-one (59) .............................. 62

4.2.13 3-Azido-7-hydroxy-2H-chromen-2-one (39) ....................................... 63

4.2.14 3-Azido-2H-chromen-2-one (22) ......................................................... 64

4.2.15 3-Azido-6-bromo-2H-chromen-2-one (40) .......................................... 64

4.2.16 3-Azido-7-diethylamino-2H-chromen-2-one (41) ............................... 65

4.2.17 General procedure 1 - CuAAC reactions ............................................. 66

4.2.17.1 7-Hydroxy-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (42) ................................................... 66

4.2.17.2 3-(4-(1,1,3,3-Tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-

yl)-2H-chromen-2-one (23) ............................................................................ 67

4.2.17.3 6-Bromo-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (43) ............................................................. 67

4.2.17.4 7-(Diethylamino)-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-

1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (44) ............................................. 68

4.2.18 General procedure 2 - Fenton reactions ............................................... 69

iv

4.2.18.1 7-Hydroxy-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (45) ................................ 69

4.2.18.2 3-(4-(2-Methoxy-1,1,3,3-tetramethylisoindolin-5-yl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (46) ............................................................. 70

4.2.18.3 6-Bromo-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (47) ................................ 71

4.2.18.4 7-(Diethylamino)-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-

2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48) ............................. 71

4.3 X - Ray Crystallographic Analysis of 3-(4-(1,1,3,3-Tetramethylisoindolin-

2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (23) ..................................... 72

4.4 Spectrophotometric Analysis of Profluorescent Nitroxides ........................ 73

4.4.1 Molar extinction coefficient measurements ......................................... 73

4.4.2 Quantum yield measurements .............................................................. 75

4.4.3 Absorbance and emission measurements of 7-hydroxy analogues (42

and 45) with pH .................................................................................................. 77

Appendices ................................................................................................................. 78

References .................................................................................................................. 95

v

List of Figures

Figure 1.1. Stable bis(tert-alkyl) nitroxides ................................................................ 5

Figure 1.2. Structure and numbering of 1,1,3,3-tetraalkylisoindolin-2-yloxyls ......... 5

Figure 1.3. Jablonski diagram of electronic transitions leading to fluorescence ........ 8

Figure 1.4. Electron exchange mechanism for Dn → D1 and D1 → D0 transitions ..... 9

Figure 1.5. Profluorescent probes linked through cleavable frameworks ................. 10

Figure 1.6. Profluorescent nitroxides linked through non cleavable carbon

frameworks ................................................................................................................. 10

Figure 1.7. Coumarin structure and numbering ........................................................ 11

Figure 1.8. Coumarin push - pull structural feature .................................................. 12

Figure 2.1. Representative 1H NMR spectrum of 23 ................................................ 36

Figure 2.2. (a) ORTEP depiction of the crystal structure of 3-(4-(1,1,3,3-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (23). (b)

An alternate view of 23 illustrating the bow-like distortion from planarity. ............. 37

Figure 2.3. Absorbance spectra of nitroxide (23 and 42 - 44) and methoxyamine (45

- 48) analogues ........................................................................................................... 39

Figure 2.4. Parent 7-hydroxy and 7-diethylamino coumarin structures 60 and 61 ... 40

Figure 2.5. Fluorescence spectra of (a) unsubstituted 23 and 46, (b) 6-bromo 43 and

47, (c) 7-hydroxy 42 and 45 (d) 7-diethylamino 44 and 48 nitroxide and

methoxyamine analogues ........................................................................................... 41

Figure 2.6. Absorbance spectra of 48 in cyclohexane, THF and DMSO .................. 43

Figure 2.7. Emission spectra of 48 in cyclohexane, THF and DMSO following

excitation at 402, 410 and 416 nm respectively ......................................................... 43

Figure 2.8. Emission spectra of the 7-hydroxy methoxyamine analogue 45 with

increasing pH following excitation at 340 nm ........................................................... 44

Figure 2.9. Absorbance spectra of the 7-hydroxy methoxyamine analogue 45 with

increasing pH ............................................................................................................. 44

Figure 2.10. Emission spectra of the 7-hydroxy methoxyamine analogue 45 with

increasing pH following excitation at 400 nm ........................................................... 45

Figure 2.11. Absorbance spectra of the 7-hydroxy methoxyamine analogue 45 at

neutral pH ................................................................................................................... 45

Figure 2.12. Emission spectra of the 7-hydroxy methoxyamine analogue 45 at

neutral pH after neutralization of pH 9-14 solutions with HCl (340 nm excitation) . 46

Figure 2.13. Absorbance spectra of the 7-hydroxy nitroxide analogue 42 at neutral

pH ............................................................................................................................... 46

Figure 4.1. Absorbance of nitroxide analogues with increasing concentration ........ 74

Figure 4.2. Absorbance of methoxyamine analogues with increasing concentration74

Figure 4.3. Integrated fluorescence intensities of anthracene and nitroxide analogues

23, 42 and 43 with increasing absorbance ................................................................. 75

Figure 4.4. Integrated fluorescence intensities of perylene and nitroxide analogue 44

with increasing absorbance ........................................................................................ 76

vi

Figure 4.5. Integrated fluorescence intensities of anthracene and methoxyamine

analogues 45 - 47 with increasing absorbance ........................................................... 76

Figure 4.6. Integrated fluorescence intensities of perylene and methoxyamine

analogue 48 with increasing absorbance .................................................................... 77

List of Tables

Table 2.1. Reaction conditions used in attempts to optimize the synthesis of 38 ..... 31

Table 2.2. Molar extinction coefficients for nitroxide and methoxyamine analogues

.................................................................................................................................... 40

Table 2.3. Quantum yield values for nitroxide and methoxyamine analogues ......... 42

Table 3.1. Conditions to assess specificity of profluorescent probes ........................ 52

List of Schemes

Scheme 1.1. Delocalization of the unpaired electron of a nitroxide ............................ 3

Scheme 1.2. Bimolecular degradation of a phenyl substituted nitroxide .................... 4

Scheme 1.3. β-Hydrogen induced disproportionation of nitroxides ............................ 4

Scheme 1.4. Photoexcitation induced α cleavage and recombination of TMIO ......... 6

Scheme 1.5. Photodegradation of TEMPO .................................................................. 7

Scheme 1.6. CuAAC reaction to generate the 1,4 regioisomer ................................. 13

Scheme 1.7. CuAAC reaction of alkyne nitroxide 21 and coumarin azide 22 .......... 14

Scheme 2.1. Proposed synthetic route to the alkyne isoindoline nitroxide analogue 21

.................................................................................................................................... 17

Scheme 2.2. Proposed synthetic route to 3-azido coumarin analogues 22 and 39 - 41

.................................................................................................................................... 18

Scheme 2.3. Proposed synthetic route to profluorescent nitroxides 23 and 42 - 44 .. 19

Scheme 2.4. Proposed synthetic route to methoxyamine analogues 45 - 48 ............. 19

Scheme 2.5. Synthetic route to N-benzylphthalimide 25 ........................................... 20

Scheme 2.6. Key intermediates in the formation of N-benzylphthalimide 25 .......... 20

Scheme 2.7. Synthetic route to 2-benzyl-1,1,3,3-tetramethylisoine 26 ..................... 21

Scheme 2.8. Synthetic route to 5-bromo-1,1,3,3-tetramethylisoindoline 27 ............. 22

Scheme 2.9. Key intermediates in the formation of 5-bromo-1,1,3,3-

tetramethylisoindoline 27 ........................................................................................... 23

Scheme 2.10. Synthetic route to 5-iodo-1,1,3,3-tetramethylisoindoline 28 .............. 24

Scheme 2.11. Key intermediates in the formation of 5-iodo-1,1,3,3-

tetramethylisoindoline 28 ........................................................................................... 24

Scheme 2.12. Synthetic route to 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 29 . 25

Scheme 2.13. Generation of bis-acetylenes through Glaser coupling ....................... 26

vii

Scheme 2.14. Proposed catalytic cycle for the Sonogashira reaction incorporating a

copper co-catalyst and amine base ............................................................................. 27

Scheme 2.15. Proposed catalytic cycle for the copper-free Sonogashira reaction .... 27

Scheme 2.16. Synthetic route to 5-(3-hydroxy-3methyl)butynyl-1,1,3,3-

tetramethylisoindolin-2-yloxyl 21 .............................................................................. 28

Scheme 2.17. Proposed synthetic route to 3-acetamido coumarin analogues 35 - 3830

Scheme 2.18. Knoevenagel condensation reaction conditions for the synthesis of 58

.................................................................................................................................... 31

Scheme 2.19. Synthetic route to 3-azido coumarin analogues 22, 39 and 40 ........... 32

Scheme 2.20. Reaction conditions for the reduction of the 3-nitro coumarin analogue

58 ................................................................................................................................ 33

Scheme 2.21. Synthetic route to profluorescent nitroxide analogues 23 and 42 - 44 34

Scheme 2.22. Proposed catalytic cycle for the CuAAC reaction .............................. 35

Scheme 2.23. Synthetic route to methoxyamine analogues 45 - 48 .......................... 38

Scheme 2.24. Phenolate induced scission of coumarin lactone................................. 47

Abbreviations

CuAAC copper catalyzed azide alkyne cycloaddition

DABCO 4-diazabicyclo[2.2.2]octane

DCM dichloromethane

DMSO dimethyl sulfoxide

DTBN di-tert-butyl nitroxide

EI

electron impact

EPR electron paramagnetic resonance

EPS exopolysaccharides

Equiv equivalent

ESI

electrospray ionization

EtOAc ethyl acetate

EtOH ethanol

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

IR infrared

ISC intersystem crossing

mCPBA 3-chloroperoxybenzoic acid

MS mass spectrometry

NMR nuclear magnetic resonance

PROXYL 2,2,5,5-tetramethylpyrrolidin-1-yloxyl

TEMPO 2,2,6,6-tetramethylpiperidin-1-yloxyl

THF tetrahydrofuran

TMIO 1,1,3,3-tetramethylisoindoline-2-yloxyl

UV ultraviolet

viii

Declaration

This work has not previously been submitted for a degree or diploma in any

university. To the best of my knowledge and belief, this dissertation contains no

material previously published or written by another person except where due

reference is made in the dissertation itself.

Jason C. Morris

ix

Acknowledgements

I would like to take this opportunity to sincerely thank both QUT and my supervisors

Dr. Kathryn Fairfull-Smith and Prof. Steven Bottle for financial support through the

QUT Masters Scholarship. To Kathryn and Steven, your support and guidance

throughout this year was appreciated, in particular I would like to thank you for

making group meetings an enjoyable experience. I would also like to thank Dr. John

McMurtrie for performing X ray crystallographic analysis.

To my family, I would like to thank you for supporting me throughout the year, your

support made this endeavor possible. Marie, I lack the words to adequately describe

just how important your support and guidance this year been. Know that I will be

forever grateful for the help you have given to me.

2

1 Chapter 1. Introduction

1.1 Nitroxide Free Radicals

Free radicals are defined as any chemical species containing one or more unpaired

electrons and have long been considered to be highly reactive transient species.

Whilst this is true of a large number of free radical species, certain classes of long-

lived radical species exist, which through stabilization of the unpaired electron have

made possible the isolation and application of free radical species to a variety of

fields.1 Possibly the most important class of stable radical species is

thermodynamically stable, kinetically persistent nitroxide free radicals.

Derivatives of nitrogen oxide, nitroxides are composed of a disubstituted nitrogen

atom attached to a univalent oxygen atom as the third substituent. The unpaired

electron is delocalized between the nitrogen and oxygen atoms forming a stable three

electron π system with a bond energy midway between the N–O single bond and the

N=O double bond.

Central to the importance of nitroxides is the persistence of the unpaired electron

giving rise to paramagnetic properties unique to this class of molecules.1 The positive

magnetic susceptibility arising from the spin of the unpaired electron (S = ½) has

been a driving force in the application of nitroxides as spin labels and probes which

have been used to monitor radical related processes through electron paramagnetic

resonance spectroscopy (EPR).

More recently there has been a resurgence of interest in nitroxides due to their ability

to quench excited singlet, triplet and excimeric states responsible for

fluorescence.2,3,4,5,6,7,8,9,10

This technique, first applied to the intermolecular processes

of di-tert-butyl nitroxide (DTBN) and aromatic hydrocarbons,11

has since been

applied to the intramolecular processes of otherwise fluorescent molecules covalently

tethered to stable nitroxide moieties.12,13

In contrast to the abundance of reactive radical species possessing inherently short

lifetimes, the existence of a thermodynamically stable, kinetically persistent radical

species which can be isolated and stored in pure form has ensured nitroxides rise to

prominence.

3

1.1.1 Nitroxide stability

The inherent stability of nitroxides arises due to the delocalization of the unpaired

electron between the nitrogen and oxygen atoms (Scheme 1.1). Delocalization of the

unpaired electron between the two hetero atoms generates significant delocalization

stabilization (~ 130 kJ/mol) granting nitroxides a survival advantage over other

radical species.1

Scheme 1.1. Delocalization of the unpaired electron of a nitroxide

An important consequence of the stabilization of the radical centre is an aversion to

the formation of stable adducts with other oxygen centered radical species.

Importantly this means that nitroxides do not undergo dimerization to any extent.

Although nitroxides can undergo a variety of reactions with reactive oxygen species,

this is not through direct interaction with the oxygen centered radical and therefore

dimerization does not occur as the loss in delocalization stabilization of two nitroxide

molecules (2 × ~ 130 kJ/mol) for the formation of a weak peroxide bond (~ 140

kJ/mol) is thermodynamically disfavored.14,15

Whilst the delocalization stabilization of nitroxides provides a thermodynamically

stable radical species, the persistence of the nitroxide moiety is dependent upon the

unpaired electron remaining delocalized between the two hetero atoms. The

persistence of nitroxides is therefore dependent on the absence of degradation

pathways, most notably arising from the substituents present on the carbon atom α to

the nitroxide moiety.1

In the case of tert-butyl phenyl nitroxide 1 (Scheme 1.2), delocalization of spin

density of the unpaired electron into the aromatic ring increases the thermodynamic

stability of the system, however, generation of a highly reactive carbon centered

radical facilitates bimolecular degradation of the nitroxide moiety to the

corresponding amine 4 and nitrone 5 species.1

4

Scheme 1.2. Bimolecular degradation of a phenyl substituted nitroxide

The most prolific degradation pathway affecting nitroxides arises due to the presence

of methyl (CH3–), methylene (RCH2–) or methine (R2CH–) hydrogen atoms adjacent

to the nitroxide moiety. The presence of β-hydrogens induces disproportionation of

the nitroxide to the corresponding hydroxylamine and nitrone species (Scheme 1.3).1

Scheme 1.3. β-Hydrogen induced disproportionation of nitroxides

Bis(tert-alkyl) nitroxides such as the non-cyclic di-tert-butylnitroxide (DTBN) 6, as

well as analogues of the five membered pyrrolidine system 2,2,5,5-

tetramethylpyrrolidin-1-yloxyl (PROXYL) 7, the six membered piperidine system

2,2,6,6-tetramethylpiperidin-1-yloxyl (TEMPO) 8 and the fused aromatic isoindoline

system 1,1,3,3-tetramethylisoindoline-2-yloxyl (TMIO) 9 (Figure 1.1) have emerged

as the dominant nitroxide species due to the absence of the aforementioned

degradation pathways.

5

Figure 1.1. Stable bis(tert-alkyl) nitroxides

Notably, the majority of reported literature pertaining to nitroxides has focused on

the aliphatic derivatives DTBN (6), PROXYL (7) and TEMPO (8), an occurrence

which could be rationalized by the commercial availability of these compounds.

Despite receiving less attention, TMIO (9) has been shown to possess a considerably

more robust carbon framework than its commercially available counterparts.

1.1.2 Isoindoline nitroxides

Isoindoline nitroxides have evolved from the first tetraethyl analogue reported by

Rozantsev et.al16,17

to incorporate all 1,1,3,3-tetrasubstituted isoindolin-2-yloxyls as

well as their aromatic substituted analogues (Figure 1.2).

Figure 1.2. Structure and numbering of 1,1,3,3-tetraalkylisoindolin-2-yloxyls

The first tetramethyl analogue 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) (9)

was published by Griffiths et.al18

in 1983. The reduced steric bulk of the methyl

groups surrounding the nitroxide moiety led to an increase in reaction rates with

carbon, sulfur and phosphorous centered radicals to near diffusion controlled rates

(~ 107 - 10

9 M

-1s

-1), increasing the effectiveness of isoindoline nitroxides as

scavengers of reactive radical species.19,20,21

6

Since this time numerous studies have been devoted to determining the structural

stability unique to the isoindoline nitroxide system. Structural analysis performed by

Busfield et.al22

included the x-ray crystal structure of TMIO as well as the solid state

13C NMR spectrum for an alkoxyamine derivative. These analyses established the

planarity of the fused aromatic system, for which the remarkable thermal and

chemical stability of isoindolines nitroxides has been attributed.22

Furthermore, the photodegradation study of Bottle et.al23

established the stability of

TMIO to ring opening reactions. The resistance of isoindoline nitroxides to ring

opening reactions was attributed to the rigidity of the fused aromatic framework

hindering α cleavage of the nitroxide moiety. Furthermore, it was expected that

following any photolytic α cleavage, the rigidity of the fused aromatic system 10

would favor recombination to reform the nitroxide moiety (Scheme 1.4).

Scheme 1.4. Photoexcitation induced α cleavage and recombination of TMIO

Underlying isoindoline nitroxides propensity for recombination following α

cleavage, the rigidity of the fused aromatic system restricts the freedom of movement

of the benzylic carbon radical keeping the radical in close proximity to the

corresponding nitrone 10. The constrained proximity of the radical and nitrone

moieties allows for rapid recombination to reform the nitroxide moiety 9.

Lacking the more rigid carbon framework of isoindoline nitroxides, TEMPO and to

some extent PROXYL derivatives are more susceptible to degradation through ring

opening reactions. Furthermore, recombination following α cleavage is less prevalent

as both lack the restriction of bond movement that keeps the radical in close

proximity to the resulting nitrone moiety (Scheme 1.5).24

7

Scheme 1.5. Photodegradation of TEMPO

In addition to providing structural stability the incorporation of the aromatic group

grants isoindoline nitroxides an analytical advantage over the aliphatic alternatives

PROXYL and TEMPO. The intrinsic UV chromophore enables detection of radical

adducts by standard HPLC systems. Furthermore, the presence as well as proximity

of the aromatic group to the nitroxide moiety has also proven beneficial to the

application of isoindoline nitroxides as profluorescent probes.

1.1.3 Profluorescent nitroxides

The presence of a nitroxide group has been shown to exert a profound impact on

fluorescence emission due to the paramagnetic nature of the unpaired

electron.25,26,27,28,29,30,31

In the normal process of fluorescence, a fluorescent molecule

(fluorophore) in the singlet ground state is excited to a vibrational energy level of an

excited singlet state through absorption of a photon (Figure 1.3).32

The excited

fluorophore can then dissipate some of this energy through vibrational relaxation and

internal conversion until the lowest vibrational level of the excited state is reached.

The excited fluorophore can then return to the ground state via a number of

pathways. Where the energy difference between excited and ground states is small,

energy loss through internal conversion to the ground state can occur. Where there is

significant energy difference between excited and ground states, energy loss can

8

occur by the emission of a photon (fluorescence) or by the classically spin forbidden

process of intersystem crossing (ISC). The process of ISC derives from a change in

the spin angular momentum of the excited electron which results in the formation of

a triplet excited state. Subsequent energy loss to the ground state from the excited

triplet state can occur through the emission of a photon (phosphorescence), however

due to the inherent longevity of the triplet state, energy loss through chemical and

physical interactions with the surrounding environment most often prevails.

Figure 1.3. Jablonski diagram of electronic transitions leading to fluorescence

Paramagnetic species such as nitroxides have been shown to enhance the prevalence

of fluorophore ISC resulting in a reduction of observed fluorescence.33

This process

utilizes the unpaired nitroxide electron, which changes the multiplicity of the

electronic states, such that doublet states (D0 and Dn) are formed from the singlet

ground state (S0) and the lowest singlet excited state (S1) respectively, whilst the

excited triplet state (T1) becomes the lowest excited doublet state (Figure 1.4).

Consequently, the previously „spin forbidden‟ transitions (S1 → T1 and T1 → S0)

become spin allowed processes.34

9

Figure 1.4. Electron exchange mechanism for Dn → D1 and D1 → D0 transitions

As the ISC of profluorescent systems is inherently dependent on the presence of the

radical species, formation of a diamagnetic species (either by a change in the redox

state or radical trapping) prevents the change of spin angular momentum of the

excited state electron and subsequent formation of a triplet excited state.

Accordingly, the system is returned to a singlet excited state and fluorescence is

restored. Systems such as these can be considered „profluorescent‟ as they possess

natural suppressed fluorescence which is restored upon formation of a diamagnetic

species.

Profluorescent systems utilizing intramolecular ISC of nitroxide bearing

fluorophores was first proposed by Stryer,35

however it was not until Blough et.al36

combined TEMPO with a series of naphthalene based fluorophores that the potential

of these systems was realized. Whilst intermolecular ISC relied on the chance

collision of radical and fluorophore molecules, the advantage of the intramolecular

system was the formation of a permanent „collision complex‟ capable of greatly

enhanced quenching of expected fluorescence emissions.

Early applications of profluorescent nitroxides employed commercially available

nitroxides and fluorophores attached through relatively labile linkages such as

esters37,38,39

(15), amides40,41,42

(16) and sulfonamides43,44,45,46

(17) (Figure 1.5).

Potentially labile linkages such as these present the opportunity for separation of the

fluorophore and nitroxide components and subsequent loss of the fluorescence

10

quenching mechanism. In cases such as these, the fluorescence detected could arise

from the untethered fluorophore rather than a radical or redox related product.

Figure 1.5. Profluorescent probes linked through cleavable frameworks

This problem is particularly apparent in biological systems, where potentially labile

linkages such as these may be susceptible to enzymatic cleavage from enzymes such

as esterases. Nitroxide bearing fluorophores linked through non-cleavable carbon

frameworks present a more desirable goal for profluorescent probes.

Towards this our group has previously reported isoindoline nitroxides bearing

stillbene47

and azaphenalene48

fluorophores as well as molecules with nitroxides

incorporated into the fluorescent cores of phenanthrene (18),49

naphthalene (19),48

diphenylanthracene (20)50

and fluorescein51

(Figure 1.6).

Figure 1.6. Profluorescent nitroxides linked through non cleavable carbon frameworks

Central to our approach has been the utilization of robust, non cleavable carbon

frameworks which position the nitroxide in close proximity to the aromatic system.

11

As the ability of the nitroxide to suppress fluorescence is inherently dependent on the

proximity of nitroxide and fluorophore components, a key area of design of probe

molecules has been the conjugation of fluorophore and isoindoline nitroxide

components. This approach has enabled the nitroxide moiety to be positioned within

two bond lengths of the aromatic system, generating highly sensitive profluorescent

probes.

Expanding upon this work the prospect of incorporating a coumarin fluorophore

(which has yet to be explored within an isoindoline nitroxide profluorescent system)

attached through robust carbon framework was envisaged.

1.2 Coumarins

Coumarin (2H-chromen-2-one) and its various substituted analogues have received

considerable attention in recent years owing to their application as components of

fluorescence probes, sensors and switches.52

The small size, biocompatibility and

synthetic utility of the coumarin structural motif (Figure 1.7) have been driving

forces in the resurgence of interest in coumarins as powerful components of

fluorescent systems.52

Figure 1.7. Coumarin structure and numbering

Though small in size, the extended π-conjugation of the benzopyrone system

furnishes coumarins with desirable spectrophotometric properties.52

Furthermore,

substituent manipulation in the 3- or 7- position has been shown to drastically alter

the spectrophotometric properties of coumarins.53,54

Of particular interest is the

incorporation of electron donating substituents in the 7- position of the coumarin

structure (Figure 1.8) to generate a “push-pull” structural feature.

12

Figure 1.8. Coumarin push - pull structural feature

In this case, the extended π-conjugation connects the electron-withdrawing carbonyl

moiety with the electron-donating substituent of position 7 to generate a charge-

transfer complex.52

The specific charge-transfer transition energy is dependent on the

nature of both the electron-donating and electron-withdrawing substituents which are

governed by the ionization potential and electron affinity respectively. Whilst the

coumarin carbonyl is an intrinsic moiety of the coumarin motif, the nature of the

electron-donating substituent can be optimized toward increasing ionization potential

to enhance the charge-transfer character of the fluorescent system. Although an exact

prediction of spectrophotometric properties from molecular structure remains an

underdeveloped art, the synthetic utility of coumarins makes optimization of the

coumarin structure and associated spectrophotometric properties a facile process.52

Furthermore, as the chemistry surrounding the synthesis of 3-azido coumarin

analogues is well established,55

a novel method for linking fluorophore and nitroxide

components through the copper catalyzed azide alkyne cycloaddition „click‟ reaction

was proposed.

1.3 The ‘Click’ Approach

The concept of „click chemistry‟ was proposed by Sharpless56

in 2001 to describe a

series of synthetically valuable bond forming reactions which were selective, high

yielding and wide in scope. Whilst the term „click chemistry‟ applies to a broad

variety of reactions such as the Diels-Alder reaction and numerous nucleophilic

substitution and addition reactions, the premiere example of „click chemistry‟ is

undoubtedly the copper catalyzed azide alkyne cycloaddition reaction

(CuAAC)57,58,59

both in terms of adherence to the „click‟ criteria as well as the

volume of publications pertaining to it.

13

Central to the importance of the CuAAC reaction is the use of a Cu (I) catalytic

species for the in situ generation of cuprous acetylides. The cuprous acetylide

underlies the value of the CuAAC reaction by enhancing reaction rates 1000 fold

compared with the copper free approach, whilst ensuring the 1,4 regioisomer as the

exclusive product (Scheme 1.6).

Scheme 1.6. CuAAC reaction to generate the 1,4 regioisomer

Furthermore, the reaction is typically carried out in a mixture of water and an alcohol

co-solvent enabling the desired product to precipitate from the reaction mixture in

high purity.56

The solvent mixture can be easily optimized to a given substrate with

short chain alcohols (such as methanol or ethanol) used in the case of polar starting

materials or longer, branched chain alcohols (such as tert-butanol) used to solublize

non-polar substrates. In many cases purification via column chromatography can be

avoided making this technique ideally suited to the rapid development of molecular

libraries.

The stability of the resulting triazole to reactive conditions such as oxidation,

reduction and hydrolysis has seen the CuAAC reaction extend beyond synthetic and

medicinal chemistry to surface and polymer chemistry60,61

as well as applications in a

bioconjugation context.62,63

A recent application of this reaction has been the

incorporation of nitroxide spin labels onto adenosine and other biomolecules such as

amino acids and carbohydrates.64,65

Despite the inherent benefits the CuAAC

reaction provides, no example of CuAAC chemistry in the generation of

profluorescent nitroxide has been reported.

As our research group has previously reported an alkyne isoindoline derivative (5-

ethynyl-1,1,3,3-tetramethylisoindolin-2yloxyl) 21,66

the prospect of utilizing the

CuAAC reaction to conjugatively join this alkyne with an azide bearing coumarin

fluorophore 22 coupling partner for profluorescent nitroxide applications was of

interest (Scheme 1.7).

14

Scheme 1.7. CuAAC reaction of alkyne nitroxide 21 and coumarin azide 22

1.4 Potential Applications of Profluorescent Probes

A potential application of the CuAAC based profluorescent probes could be in the

monitoring of bacterial biofilm dispersal events, which has recently been linked to

conditions of oxidative and nitrosative stress experienced by the biofilm.

Oxidative stress is caused by the exposure to reactive oxygen species (ROS) such as

superoxide (O2-), hydrogen peroxide (H2O2) and the extremely reactive hydroxyl

radical (˙OH) whilst nitrosative stress is caused by the exposure to reactive nitrogen

species (RNS) such as nitric oxide (NO˙), peroxynitrite (ONOO-), nitrous acid

(HNO2) and nitrogen trioxide (N2O3).67

Of increasing interest is the role of NO˙ in moderating dispersal events. A common

signaling molecule in eukaryotic and multicellular prokaryotic biology, NO˙

underpins a number of biological processes including apoptosis, differentiation and

cell proliferation.67,68,69

Within the biofilm NO˙ is produced as an intermediate of the

denitrification enzyme pathway nitrite reductase, which catalyses the four step

sequential reduction of nitrate to nitrogen gas under anaerobic conditions.67,70

Recently Barraud et.al67

demonstrated that low, sub lethal concentrations of NO˙

could be used to both impede the formation and induce dispersal of Pseudomonas

aeruginosa biofilms. Furthermore, it was found that a p. aeruginosa mutant lacking

the enzyme nitrite reductase did not disperse, whilst a p. aeruginosa mutant with

15

increased expression of the nitrite reductase enzyme exhibited greatly enhanced

dispersal under these conditions.67

The link between NO˙ production and moderation of biofilm dispersal is evident,

however much is still unknown of the underlying mechanisms responsible for

dispersal.67

Under anaerobic conditions radical related processes appear to govern

dispersal of the biofilm, however the extent and nature of this process remains

unknown. Molecular based approaches to monitor these processes are needed to

further develop this area of research.

Profluorescent nitroxides71

provide a facile yet extremely sensitive means for

monitoring radical related processes and may provide insight into the mechanisms

underlying biofilm dispersal.

1.5 Project Outline

The objectives of this work focus on the first application of a „click approach‟ to the

preparation profluorescent nitroxides with potential application in monitoring

processes involving free radicals. The versatility of the CuAAC reaction provides the

potential to construct structurally diverse profluorescent systems from azide and

alkyne building blocks which could be independently optimized for a given

application.

The key to our approach is the use of the alkyne isoindoline nitroxide (21) CuAAC

coupling partner previously reported by our group.71

This approach retains the

triazole-isoindoline nitroxide portion of CuAAC products whilst allowing for

manipulation of the fluorophore to address the spectrophotometric requirements of

potential profluorescent systems. Specifically, in this project a library of coumarin

fluorophores possessing both electron-withdrawing and electron-donating

functionality will be explored to enhance the charge-transfer character of coumarin

fluorophores which has been shown to be a major determinant of fluorescence

emission capacities. In order to explore the impact of the nitroxide moiety on the

fluorescence suppression of click products, methoxyamine derivatives of the

profluorescent probes will also be prepared.

16

This library of profluorescent nitroxides as well as their corresponding

methoxyamine analogues will be subjected to spectrophotometric analysis to assess

the applicability of the prepared profluorescent systems. In conjunction with the

measurement of absorbance maxima and molar extinction coefficients, these

analyses will incorporate the measurement of quantum yields of fluorescence for

paramagnetic profluorescent systems as well as their diamagnetic methoxyamine

analogues.

Fundamental to the potential application of a „click‟ based approach to

profluorescent nitroxides will be effective nitroxide-fluorophore communication.

Comparison of the quantum yields of paramagnetic and diamagnetic analogues will

be used to provide a measure of fluorescence suppression exhibited by the nitroxide

moiety.

17

2 Chapter 2. Results and Discussion

To support our „click approach‟, the synthesis of both 3-azido coumarin and alkyne

nitroxide CuAAC coupling partners was proposed to proceed according to literature

procedures.

Bottle et.al72

have previously reported a convenient route to the synthesis of the

alkyne isoindoline nitroxide analogue 21 (Scheme 2.1).

(a) benzylamine, acetic acid, reflux (b) 1. MeMgI, Et2O; 2. Toluene, 110 °C (c) AlCl3, Br2, 0 °C (d) n-

BuLi, THF, I2 (e) mCPBA, DCM, 0°C (f) 2-methyl-3-butyn-2-ol Pd(OAc)2, DABCO,75°C (g)

toluene, KOH

Scheme 2.1. Proposed synthetic route to the alkyne isoindoline nitroxide analogue 21

This synthetic approach involves the initial formation of N-benzylphthalimide 25

through the acid catalyzed condensation of commercially available phthalic

anhydride 24 with benzylamine, followed by exhaustive methylation under Grignard

18

reaction conditions to generate the desired tetramethyl analogue 26. Consecutive

bromination (27) and iodination (28) of the isoindoline aromatic ring then facilitates

the incorporation of the alkyne moiety (30) through Sonogashira coupling following

formation of the nitroxide species 29. Subsequent deprotection of the

hydroxyisopropyl protecting group under basic conditions then generates the

terminal alkyne functionality (21) required for CuAAC chemistry.

The synthesis of 3-azido coumarin CuAAC coupling partners (22 and 39 - 41) was

proposed to proceed in a similar manner to that previously reported by Wang et.al.73

This approach utilizes the condensation of appropriately substituted 2-

hydroxybenzaldehydes 31 - 34 with N-acetylglycine to afford the corresponding 3-

acetamido coumarin analogues 35 - 38. Subsequent reduction of the 3-acetamido

coumarin analogues under acidic aqueous conditions to generate the corresponding

3-amino coumarin derivatives would then facilitate the formation of 3-azido

coumarin analogues (22 and 39 - 41) through standard diazo transfer conditions

(Scheme 2.2).

(a) N-acetylglycine, NaOAc, Ac2O, 120 °C (b) 1. HCl, EtOH, reflux; 2. NaNO2, NaN3

Scheme 2.2. Proposed synthetic route to 3-azido coumarin analogues 22 and 39 - 41

Synthesis of both alkyne (21) and azide (22 and 39 – 41) CuAAC coupling partners

would then enable their application toward the generation of profluorescent probes

through use of the CuAAC reaction. Standard CuAAC reaction conditions

incorporating copper (II) sulfate (5 mol %) and sodium ascorbate (10 mol %) in an

ethanol/water solvent system are proposed for our synthesis of nitroxide analogues

23 and 42 - 44, as the versatility of this reaction often negates the need for

optimization of reaction conditions (Scheme 2.3).

19

Scheme 2.3. Proposed synthetic route to profluorescent nitroxides 23 and 42 - 44

For additional characterization and in order to explore the impact of the nitroxide

moiety on the fluorescence suppression of CuAAC products, methoxyamine

derivatives of the profluorescent probes will also be prepared. Fenton chemistry

provides a convenient route to the generation of methoxyamine analogues (45 - 48)

from the corresponding nitroxide analogues (23 and 42 - 44). Through this approach,

the in situ generation of hydroxyl radicals rapidly reacts with DMSO to generate

carbon centered radicals which are trapped by the nitroxide moiety (Scheme 2.4).

Scheme 2.4. Proposed synthetic route to methoxyamine analogues 45 - 48

20

2.1 Synthesis of Alkyne Isoindoline CuAAC Coupling Partner

2.1.1 Synthesis of N-benzylphthalimide (25)

The first stage of the synthetic approach involved the synthesis of N-

benzylphthalimide 25 through the acid catalyzed condensation of commercially

available phthalic anhydride 24 and benzylamine (Scheme 2.5).

Scheme 2.5. Synthetic route to N-benzylphthalimide 25

The convenience of this approach is attributed to the facile nature of the reaction

which proceeded to completion in one hour under refluxing conditions. Furthermore,

purification of the desired N-benzylphthalimide 25 was achieved rapidly in excellent

yield (92 %) by precipitation with cold water followed by recrystallization from

ethanol.

The reaction begins with nucleophilic attack by benzylamine on the carboxyl group

to generate the corresponding ring-opened adduct 49 (Scheme 2.6). Refluxing

conditions then promote the acid catalyzed ring-cyclization to generate the

hemiaminal species 50 which undergoes dehydration to generate the desired N-

benzylphthalimide 25.

Scheme 2.6. Key intermediates in the formation of N-benzylphthalimide 25

21

Characterization by 1H NMR spectroscopy, ESI

+ HRMS and melting point analysis

confirmed the identity of N-benzylphthalimide 25. Characteristic CH2 (4.9 ppm) and

aromatic peaks (7.9 and 7.3 ppm) corresponding to the benzylic protons were

observed in the 1H NMR spectrum of N-benzylphthalimide 25 which were consistent

with literature values.74

Furthermore, the [MH+] parent peak for 25 was observed at

238.1025 m/z (calc. 238.0863) in the ESI mass spectrum, whilst a comparison of the

observed melting point with literature values further validated formation of the

desired product 25 (115 - 116 °C, lit.74

112 - 115 °C).

2.1.2 Synthesis of 2-benzyl-1,1,3,3-tetramethylisoindoline (26)

The desired tetramethyl isoindoline analogue 2-benzyl-1,1,3,3-tetramethylisoindoline

26 was prepared from N-benzylphthalimide 25 through an exhaustive Grignard

reaction (Scheme 2.7). The reaction proceeded smoothly on a large scale (100 g),

albeit in low yield 23 % (following recrystallization) which is consistent with the

literature yield of 37 % (prior to recrystallization).75

Scheme 2.7. Synthetic route to 2-benzyl-1,1,3,3-tetramethylisoine 26

The Grignard reagent was generated in situ under an inert atmosphere of argon

through the drop-wise addition of methyl iodide to a mixture of magnesium in

diethyl ether. Once the Grignard reagent (MeMgI) was formed, subsequent

concentration of the reaction mixture was performed through distillation followed by

the drop-wise addition of N-benzylphthalimide 25 solubilized in anhydrous toluene.

Diethyl ether has previously been implicated in the stabilization of mono-, di-, and

tri- methylated intermediates preventing exhaustive methylation.75

The complete

removal of diethyl ether through distillation was therefore essential to facilitate

22

exhaustive methylation of N-benzylphthalimide 25. When the reaction had proceeded

to completion, purification of the desired Grignard product 26 was achieved by

passing a solution of 26 solubilized in n-hexanes through a basic alumina column

followed by recrystallization from methanol.

Characterization of the desired Grignard product 26 was achieved through 1H NMR

spectroscopy, ESI+ HRMS and melting point analysis. Characteristic methyl peaks

integrating for 12 protons were observed for the tetramethyl analogue 26 in the 1H

NMR spectrum at 1.3 ppm and were consistent with literature values.75

Furthermore,

the [MH+] parent peak for 26 was observed at 266.1931 m/z (calc. 266.1903) in the

ESI mass spectrum, whilst a comparison of the observed melting point with literature

values further validated the formation of the desired product 26 (62 - 63 °C, lit.

75 63 -

64 °C).

2.1.3 Synthesis of 5-bromo-1,1,3,3-tetramethylisoindoline (27)

The next stage of the synthetic approach was the one-pot oxidative debenzylation

and bromination of the isoindoline aromatic ring to afford 5-bromo-1,1,3,3-

tetramethylisoindoline 27 in excellent yield (92 %) (Scheme 2.8).

Scheme 2.8. Synthetic route to 5-bromo-1,1,3,3-tetramethylisoindoline 27

The initial action of the generated catalytic species (AlCl3Br2) results in the

formation of the brominated species 51 followed by bromination of the isoindoline

aromatic ring through electrophilic aromatic substitution (Scheme 2.9). The use of an

excess (3.6 equiv.) of anhydrous aluminium chloride and an approximately

stoichiometric volume of bromine results in the highly selective monobromination of

23

the isoindoline aromatic ring. Subsequent loss of the benzylic group proceeds

through the formation of an imminum ion which is hydrolysed upon work up to

afford benzaldehyde and the desired secondary amine 27. The presence of a small

excess of bromine remaining in solution results in the formation of the bromoamine

species 53 which is converted to the free amine by reduction with hydrogen peroxide

to afford the desired 5-bromo-1,1,3,3-tetramethylisoindoline 27.

Scheme 2.9. Key intermediates in the formation of 5-bromo-1,1,3,3-tetramethylisoindoline 27

Characterization of the brominated analogue 27 was achieved through 1H NMR

spectroscopy and ESI+ HRMS. The

1H NMR spectrum was particularly valuable for

confirming successful debenzylation of the 2-benzyl-1,1,3,3-tetramethylisoindoline

26 precursor. The loss of the characteristic CH2 (4.0 ppm) and aromatic hydrogen

signals (7.2 and 7.3 ppm) corresponding to the benzylic protons of the precursor 26

coupled with the appearance of a broad singlet peak corresponding to the generated

secondary amine (1.87 ppm) confirmed successful debenzylation of 2-benzyl-

1,1,3,3-tetramethylisoindoline 26. Furthermore, the loss of one isoindoline aromatic

peak supported monobromination of the aromatic ring. As the isoindoline skeleton

possesses a plane of symmetry monobromination is only possible in either the 4- or

5- positions of the isoindoline structure. The observed coupling patterns were

indicative of bromination in the 5- position and are consistent with literature values.76

24

Further confirmation of successful bromination was provided through ESI+ HRMS

analysis. Parent [MH+] peaks were observed for 27 at 254.0548 m/z and 256.0528

m/z in the ESI mass spectrum which corresponded to 79

Br and 81

Br isotopes of 27

respectively (calc. 254.0544 and 256.0524). Both [MH+] parent peaks for 27 were

found to be within 1.5 ppm of the calculated masses confirming formation of the

desired product 27.

2.1.4 Synthesis of 5-iodo-1,1,3,3-tetramethylisoindoline (28)

In previous studies our research group has shown that the brominated analogue 27

exhibits low reactivity to palladium catalysis.77

To address this problem, the more

reactive 5-iodo-1,1,3,3-tetramethylisoindoline 28 was synthesized from the

brominated analogue 27 in good yield (73 %) through standard lithiation techniques

(Scheme 2.10).

Scheme 2.10. Synthetic route to 5-iodo-1,1,3,3-tetramethylisoindoline 28

The reaction was performed at low temperature (- 78 °C) due to the inherent

instability of the lithiated species 54 (Scheme 2.11). An excess (3 equiv.) of iodine

was then added to quench the lithiated species 54 affording the N-iodoamine species

55. Hydrogen peroxide was then used to reduce the resulting N-iodoamine species 55

to the desired secondary amine 5-iodo-1,1,3,3-tetramethylisoindoline 28.

Scheme 2.11. Key intermediates in the formation of 5-iodo-1,1,3,3-tetramethylisoindoline 28

25

A modest downfield shift of aromatic proton peaks was observed in 1H NMR

spectrum of 5-iodo-1,1,3,3-tetramethylisoindoline 28 with respect to the brominated

precursor 27 which can be rationalized by the reduced electron withdrawing

character of the iodo substituted analogue 28. Furthermore, the [MH+] parent peak

for 28 was observed at 302.0402 m/z (calc. 302.0400) in the ESI mass spectrum. The

loss of the characteristic bromine isotopic distribution further supported the

formation of the iodo analogue 28.

2.1.5 Synthesis of 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (29)

Formation of the desired nitroxide species, 5-iodo-1,1,3,3-tetramethylisoindolin-2-

yloxyl 29, was achieved rapidly (30 minutes) and in good yield (77 %) through the

oxidation of 5-iodo-1,1,3,3-tetramethylisoindoline 28 with 3-chloroperoxybenzoic

acid (mCPBA) (Scheme 2.12).

Scheme 2.12. Synthetic route to 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 29

Our research group has previously utilized a H2O2-tungstate oxidation approach for

the generation of the nitroxide moiety.50

Whilst conversion to the nitroxide moiety

proceeds in good yield, the H2O2-tungstate approach suffers from extended reaction

times (3 days) compared with the mCPBA approach which proceeded to completion

in just 30 minutes.

The reaction proceeds through the displacement of the weak mCPBA peroxide bond

by the isoindoline secondary amine generating the thermodynamically favored

hydroxylamine species. Once formed, the hydroxylamine is further oxidized to the

desired nitroxide moiety through reaction with molecular oxygen.

26

The presence of the nitroxide moiety and the paramagnetic broadening effect it

exhibits prevented characterization by NMR spectroscopy. Alternatively, melting

point, EI+ HRMS and IR spectroscopy confirmed the identity of 5-iodo-1,1,3,3-

tetramethylisoindolin-2-yloxyl 29. The measured melting point was consistent with

reported literature values for 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 29 ( 131 -

133 °C, lit.78

132 - 134 °C). However, repeated attempts to confirm the identity of

the desired product through ESI+ HRMS failed to generate the desired [MH

+] parent

peak. Believing this to be attributable to known difficulties associated with the

ionization of nitroxides through ESI,79

EI+ HRMS was employed which afforded the

desired [M+] parent peak at 316.0913 m/z (calc. 316.0913) in the EI spectrum and

confirmation of the desired 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 29.

Furthermore, a characteristic IR absorption band at 1386 cm-1

further validated the

presence of the nitroxide moiety.80

2.1.6 Synthesis of 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-

tetramethylisoindolin-2-yloxyl (30)

Our research group has previously reported the synthesis of the alkyne isoindoline

derivative 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 30

prepared under Sonogashira reaction conditions.77,78

Initial attempts to synthesize 30

through traditional Sonogashira reaction conditions, incorporating a PdCl2(PPh3)2

catalyst, copper co-catalyst and amine base suffered from a high degree of Glaser

coupling (Scheme 2.13) restricting yields of the desired Sonogashira product 30 to 10

%.

Scheme 2.13. Generation of bis-acetylenes through Glaser coupling

To prevent this unwanted side reaction a copper free approach was implemented.

Under traditional Sonogashira reaction conditions, coordination of the Cu (I) co-

27

catalyst to the alkyne is proposed to weaken the terminal C-H bond to facilitate its

deprotonation and subsequent formation of the cuprous alkyne (Scheme 2.14).

Scheme 2.14. Proposed catalytic cycle for the Sonogashira reaction incorporating a copper co-catalyst

and amine base

In the copper free approach, coordination to palladium is proposed to fulfill this role

however the coordinative ability of the palladium catalyst to the alkyne is

appreciably lower than Cu (I) coordination (Scheme 2.15). Consequently, reaction

times are inherently longer for the copper free approach due to the limited life time

of the palladium - alkyne π - complex.

Scheme 2.15. Proposed catalytic cycle for the copper-free Sonogashira reaction

28

To overcome this problem three equivalents of base (4-diazabicyclo[2.2.2]octane)

were used to enhance base and palladium-alkyne π-complex interactions. The

reaction proceeded to completion in 16 hours affording 5-(3-hydroxy-3-

methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 30 in good yield (73 %).

The presence of the nitroxide moiety and the paramagnetic broadening effect it

exhibits again prevented characterization by NMR spectroscopy. Alternatively, ESI+

HRMS and IR spectroscopy confirmed the identity of 5-(3-hydroxy-3-

methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 30. ESI+ HRMS afforded the

desired [MNa+] parent peak at 295.1543 m/z (calc. 295.1543) and confirmation of

the desired product 30. Characteristic IR absorption bands at 2132 and 1430 cm-1

further validated the presence of the alkyne (C≡C) and nitroxide (N-O˙) moieties

respectively.

2.1.7 Synthesis of 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (21)

5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 was prepared in good yield (84

%) by refluxing 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl 30 in toluene with solid KOH (Scheme 2.16).

Scheme 2.16. Synthetic route to 5-(3-hydroxy-3methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl 21

Vigorous refluxing and stirring of the reaction mixture was required to generate a

fine dispersion of KOH to enhance the available surface area. Subsequent

deprotonation of the hydroxyisopropyl protecting group to eliminate acetone then

facilitated the formation of 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 21.

29

The presence of the nitroxide moiety and the paramagnetic broadening effect it

exhibits again prevented characterization through NMR spectroscopy. Alternatively,

melting point, ESI+ HRMS and IR spectroscopy confirmed the identity of 5-ethynyl-

1,1,3,3-tetramethylisoindolin-2-yloxyl 21. The measured melting point was

consistent with literature values for 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl

21 (126 - 127 °C, lit.78

126 - 128 °C). The ESI mass spectrum of 5-ethynyl-1,1,3,3-

tetramethylisoindolin-2-yloxyl 21 showed a characteristic M - 58 m/z peak with

respect to the precursor 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-

tetramethylisoindolin-2-yloxyl 30, which is consistent with a loss of the isopropanol

protecting group. Characteristic IR absorption bands were again observed at 2093

and 1425 cm-1

corresponding to alkyne (C≡C) and nitroxide (N-O˙) moieties of 5-

ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 respectively.

2.2 Synthesis of 3-Azido Coumarin CuAAC Coupling Partners

2.2.1 Coumarin synthesis

Substitution at the 3- and 7- positions of the coumarin ring is known to modify

coumarin fluorescence properties. As such, a library of coumarin fluorophores

possessing both electron-donating and electron-withdrawing substituents were

envisaged to provide interesting fluorescence properties.

Our initial synthetic approach focused on the preparation of coumarin analogues

through the Perkin condensation of an appropriately substituted 2-

hydroxybenzaldehyde 31 - 34 with N-acetylglycine in the presence of acetic

anhydride (Scheme 2.17).

30

Scheme 2.17. Proposed synthetic route to 3-acetamido coumarin analogues 35 - 38

Under these conditions coumarin derivatives 35 - 37 were prepared in moderate to

good yield (27 - 68 %), with purification simplified through precipitation with cold

water.

Characterization of the substituted coumarin analogues 35 - 37 was achieved with

desired [MH+] parent peaks observed in the ESI spectra. Furthermore, characteristic

allylic peaks at ~ 8.6 ppm (corresponding to the coumarin H - 4 proton) were

observed in the 1H NMR spectra supporting coumarin formation as well as

characteristic acetamide peaks at ~ 9.7 ppm and ~ 2.2 ppm corresponding to amine

and acetyl group hydrogen atoms respectively. Melting point analysis further

validated these assignments with measured melting points consistent with reported

literature values.

Interestingly, the diethylamino analogue 38 could not be prepared under these

conditions, presumably electron donating groups para to the aldehyde moiety

disfavors coumarin formation. This rationalization does not preclude the phenolic

coumarin derivative 35 which proceeded in moderate yield, as 4-phenol acetylation

of the coumarin precursor 31 prior to coumarin formation weakens the electron

donating character of this substituent.

Attempts to optimize the reaction conditions used to generate 38 was attempted

(Table 2.1) however in each case utilizing acetic anhydride as the solvent, 2-phenol

acetylated starting material was isolated as the dominant species. When alternate

31

solvents were employed the reaction failed to proceed and the starting material 34

could be recovered in a quantitative yield.

Table 2.1. Reaction conditions used in attempts to optimize the synthesis of 38

Entry Solvent Sodium

Acetate

(Equiv.)

N-acetylglycine

(Equiv.)

Temp

(°C)

Time

(Hours)

1 Acetic anhydride 3 1.1 120 4

2 Acetic anhydride 5 1.1 120 4

3 Acetic anhydride 3 1.5 120 4

4 Acetic anhydride 5 1.5 120 6

5 Acetic anhydride 3 1.1 135 8

6 Acetonitrile 3 1.1 80 24

7 THF 3 1.1 60 24

As Perkin reaction conditions had failed to generate the desired coumarin species 38,

we instead turned our attention to Knoevenagel condensation reaction conditions

(Scheme 2.18).

Scheme 2.18. Knoevenagel condensation reaction conditions for the synthesis of 58

This reaction proceeds in a similar manner to the Perkin reaction, with deprotonation

of the ethyl nitroacetate α-hydrogen facilitating nucleophilic attack of benzaldehyde.

Subsequent transesterification with the ortho hydroxy group then generated the

desired coumarin species 58 in good yield (73 %).

32

Characterization of the diethylamine substituted coumarin analogue 58 was achieved

with the desired [MH+] parent peak observed at 263.1030 m/z (calc. 263.1026) in the

ESI mass spectrum. Furthermore, the characteristic allylic peak at 8.8 ppm

(corresponding to the coumarin H - 4 proton) was again observed in the 1H NMR

spectrum confirming the formation of the coumarin species.

2.2.2 Synthesis of 3-azido coumarin analogues

Coumarin azides 22, 39 and 40 were synthesized using literature procedures from

their corresponding coumarin acetamides 35 - 37. Coumarin acetamides 35 - 37 were

refluxed in aqueous acid to generate the corresponding deacetylated 3-amino

coumarin derivatives followed by the in situ generation of 3-diazonium salts by

treatment with sodium nitrite. Sodium azide was then used to afford the desired

coumarin azides 22, 39 and 40 in moderate to good yield (30 - 90 %) (Scheme 2.19).

Scheme 2.19. Synthetic route to 3-azido coumarin analogues 22, 39 and 40

In the case of 3-nitro-7-diethylamino-2H-chromen-2-one 58, reduction of the nitro

group with tin(II) chloride in an acidic aqueous environment afforded the desired 3-

amino-7-diethylamino-2H-chromen-2-one 59 in moderate yield (65 %) (Scheme

2.20).

33

Scheme 2.20. Reaction conditions for the reduction of the 3-nitro coumarin analogue 58

The appearance of a peak at 3.9 ppm in the 1H NMR spectrum which integrated for

two protons was attributed to the formation of the amine, confirming successful

reduction of the nitro group. Subsequent diazo transfer to prepare the desired 3-

azido-7-diethylamino-2H-chromen-2-one 41 proceeded in moderate yield (64 %)

under similar reaction conditions to that previously mentioned.

The 3-azido-7-diethylamino coumarin analogue 41 was found to be highly unstable,

necessitating its immediate use as a CuAAC coupling partner following precipitation

from the reaction mixture. Photodegradation of 41 was observed as a distinct color

change from green to black within 15 minutes of exposure to light.

Although the 1H NMR spectrum confirmed the loss of the peak corresponding to the

primary amine of the precursor 59 it could not be used to confirm the presence of the

azide moiety (41). Repeated attempts to confirm the identity of the desired product

41 through ESI+ HRMS failed to generate the desired [MH

+] parent peak. This was

attributed to the instability of the 3-azido-7-diethylamino coumarin analogue 41 and

a failure to withstand ionization conditions. As the instability of 41 necessitated its

immediate use as a CuAAC coupling partner alternate ionization sources were not

pursued as degradation during transit was expected. Accordingly, IR spectroscopy

was used to confirm the presence of the azide moiety with a characteristic IR

absorption band at 2114 cm-1

observed corresponding to the N≡N triple bond of the

azide moiety.

Following preparation of both alkyne (21) and azide (22 and 39 - 41) coupling

partners we were able to turn our attention toward their application in the CuAAC

reaction.

34

2.3 Synthesis of Profluorescent Nitroxides

Standard CuAAC reaction conditions employing a copper (II) sulfate and sodium

ascorbate catalytic system were utilized for the formation of desired profluorescent

nitroxide systems 23 and 42 - 44. Treatment of an ethanol/water solution of coumarin

azides (22 and 39 - 41) and alkyne (21) with copper (II) sulfate (5 mol %) and

sodium ascorbate (10 mol %) afforded the desired profluorescent systems 23 and 42 -

44 in good to excellent yield (60 - 90 %) (Scheme 2.21).

Scheme 2.21. Synthetic route to profluorescent nitroxide analogues 23 and 42 - 44

Underlying the efficiency of the CuAAC reaction, the in situ generation of cuprous

acetylides facilitates the regioselective synthesis of 1,4-disubstituted 1,2,3-triazole

products with reaction rates 1000 fold greater than that of the copper free approach.

The coupling is proposed to proceed through the stepwise Cu (I) catalyzed dipolar

cycloaddition of coumarin azides (22 and 39 - 41) and terminal alkyne (21) coupling

partners (Scheme 2.22).

35

Scheme 2.22. Proposed catalytic cycle for the CuAAC reaction

Coordination of the terminal alkyne to Cu (I) forming the Cu (I)-alkyne π-complex

(a) weakens the alkyne C-H bond facilitating its deprotonation and subsequent

formation of the cuprous acetylide (b). Complexation of the copper acetylide with the

coumarin azide follows (c) activating the terminal nitrogen atom toward nucleophilic

attack from the cuprous acetylide. The resulting metallocycle (d) then collapses into

the 1,4-disubstituted 1,2,3-triazole (e) via transannular association of the N-1 lone

pair of electrons to C-5.

Notably, purification to isolate CuAAC products (23 and 42 - 44) was extremely

facile, with desired compounds precipitating directly from reaction mixtures. An

ethanol/water 1:1 ratio was found to be optimal for precipitation of the desired

compounds from reaction mixtures, whilst ensuring the solubility of coumarin azides

(22 and 39 - 41) and alkyne (21) coupling partners.

Due to the paramagnetic broadening effect exhibited by the nitroxide moiety,

analysis of CuAAC products 23 and 42 - 44 by 1H NMR spectroscopy was limited.

Complete broadening of the signals corresponding to the hydrogen atoms of the

isoindoline portion of CuAAC products was apparent, whilst a diminished

broadening effect of the triazole proton (~ 9 ppm) was observed. Hydrogen signals

36

corresponding to the coumarin portions of CuAAC products were visible with

broadening of their expected multiplicities to singlets likely to be attributable to

minor impacts from both intra-molecular and inter-molecular paramagnetic

broadening (Figure 2.1).

Figure 2.1. Representative 1H NMR spectrum of 23

In the absence of complete analysis by NMR spectroscopy, analytical HPLC

revealed the purity of CuAAC products 23 and 42 - 44 to be greater than 95 %,

whilst characterization of the obtained CuAAC products was provided by ESI+

HRMS and IR spectroscopy. Desired [MH+] and [MNa

+] parent peaks for CuAAC

products 23 and 42 - 44 were observed through ESI+ HRMS whilst in each case

analysis by IR spectroscopy revealed characteristic absorption bands at ~ 1420 and

1595 cm-1

corresponding to nitroxide (N-O˙) and allylic coumarin (C=C-COO)

moieties respectively. Furthermore, the X-ray crystal structure of the unsubstituted

nitroxide analogue 23 was obtained, further confirming the structure of the

unsubstituted nitroxide analogue 23 (Figure 2.2).

37

Figure 2.2. (a) ORTEP depiction of the crystal structure of 3-(4-(1,1,3,3-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (23). (b) An alternate view of 23 illustrating the

bow-like distortion from planarity.

The crystal structure of 23 as illustrated in Figure 2.2 (a) reveals the N(4)O(3) bond

length, which is 1.2788(13) Å is consistent with typical bond lengths for isoindoline

nitroxides.22,81

The dihedral angle between the mean plane of the atoms of the

isoindoline moiety and the plane of the triazole ring is 28°. The plane of the

coumarin moiety is rotated in the reverse direction making a dihedral angle of 29°

with the plane of the triazole. These dihedral angles are a result of torsional twisting

around the bonds connecting the isoindoline (N(3)-C(11)-C(12)-C(13)) = -26) and

coumarin (N(2)-N(1)-C(9)-C(8) = 23°) moieties to the central triazole. As a result the

molecule has a bow-like molecular structure as illustrated in Figure 2.2 (b).

For additional characterization and in order to explore the impact of the nitroxide

moiety on the fluorescence suppression of CuAAC products, methoxyamine

derivatives of the profluorescent probes were required.

(a)

(a)

(b)

38

2.4 Synthesis of Methoxyamine Analogues

Methoxyamine analogues 45 - 48 were prepared from their corresponding nitroxides

analogues 23 and 42 - 44 by reaction with methyl radicals, which were generated

using Fenton chemistry from DMSO, ferrous ions and hydrogen peroxide (Scheme

2.23).

Scheme 2.23. Synthetic route to methoxyamine analogues 45 - 48

The in situ generation of highly reactive hydroxyl radicals react with DMSO to give

methyl radicals which rapidly react with nitroxides 23 and 42 - 44 to afford the

desired methoxyamine analogues 45 - 48 in good to excellent yield (78 - 90 %).

Formation of methoxyamine analogues 45 - 48 eliminated the paramagnetic radical

species and consequently the fluorescence quenching mechanism, allowing for

maximum observable fluorescence to be measured. Furthermore, the loss of the

paramagnetic species allowed complete NMR analysis to be obtained which further

validated the identity of the profluorescent systems 23 and 42 - 44. In addition to

peaks attributable to hydrogen atoms of the coumarin portion of methoxyamine

analogues which were consistent with the nitroxide analogues 23 and 42 - 44,

analysis by 1H NMR spectroscopy revealed the presence of characteristic methyl

peaks at ~ 3.5 ppm corresponding to the newly formed methoxyamines, coupled with

the emergence of aromatic and methyl peaks (~ 7 - 8 and 1.2 ppm respectively)

corresponding to the hydrogen atoms of the isoindoline portion of methoxyamine

analogues 45 - 48.

39

2.5 Spectrophotometric Analysis of Profluorescent Nitroxides

Spectrophotometric analysis of nitroxides 23 and 42 - 44 and methoxyamines 45 - 48

was performed to assess the potential applicability of these probes as tools to monitor

processes involving free radicals. To find application in a biological context, such as

for monitoring bacterial biofilm dispersal, probes must possess sufficient absorbance

at biologically convenient wavelengths, in order to minimize the effect of

autofluorescence.82

Furthermore, a significant difference in the intensity of

fluorescence emission between the paramagnetic nitroxide analogues (23 and 42 -

44) and their corresponding diamagnetic methoxyamine analogues (45 - 48) is

required for generating sensitive probes for the detection of radical species.

The absorbance spectra of nitroxide analogues (23 and 42 - 44) closely resembled

that of their corresponding methoxyamine analogues (45 - 48) in THF (Figure 2.3).

Figure 2.3. Absorbance spectra of nitroxide (23 and 42 - 44) and methoxyamine (45 - 48) analogues

Bathochromic shifts were observed in the absorption spectra of 7-hydroxy (42 and

45, λmax = 354 nm) and 7-diethylamine (44 and 48, λmax = 413 nm) substituted

coumarins compared with the unsubstituted (23 and 46, λmax = 338 nm) and 6-bromo

coumarins (43 and 47, λmax = 345 nm). Comparable bathochromic shifts have

previously been observed for coumarins possessing electron donating functionality

and are consistent with an increase in charge-transfer character.73,83,84

0

0.05

0.1

0.15

0.2

0.25

500450400350300

Ab

sorb

an

ce (

a.u

)

Wavelength (nm)

42

23

43

44

45

46

47

48

40

Molar extinction coefficients for each nitroxide (23 and 42 - 44) and methoxyamine

analogue (45 - 48) were obtained. Molar extinction coefficients were calculated from

absorbance measurements taken at five different concentrations in THF to ensure

linearity of results. These analyses demonstrated that molar extinction coefficients

for both nitroxide (23 and 42 - 44) and methoxyamine analogues (45 - 48) were

relatively uniform ranging between 15969 and 20057 M-1

cm-1

in THF (Table 2.2).

Table 2.2. Molar extinction coefficients for nitroxide and methoxyamine analogues

Entry Compound

Extinction

coefficient

(M-1

cm-1

)

1 42 19 000a

2 23 17 200 a

3 43 16 900 a

4 44 17 500b

5 45 20 000 a

6 46 17 400 a

7 47 16 000 a

8 48 20 100 b

a (λ = 340nm),

b (λ= 375nm)

Furthermore, coumarin conjugation through the triazole ring did not cause a

significant shift in absorption maxima from parent coumarin structures 60 and 61

which showed absorbance maxima at 325 nm85

and 373 nm86

respectively (Figure

2.4).

Figure 2.4. Parent 7-hydroxy and 7-diethylamino coumarin structures 60 and 61

41

The quantum yields of fluorescence were obtained for nitroxide (23, 42 and 43) and

methoxyamine analogues (45 - 47) at an excitation wavelength of 340 nm, whilst an

excitation wavelength of 375 nm was used for the 7-diethylamino analogues (44 and

48) due to the observed bathochromic shift in absorption spectra. To ensure linearity

of results, integrated fluorescence emissions were measured for five concentrations

of nitroxide (23 and 42 - 44) and methoxyamine analogues (45 - 48) in THF

corresponding to approximately 0.02, 0.04, 0.06, 0.08 and 0.10 absorbance units. The

upper limit of concentrations corresponding to 0.10 absorbance units was used to

minimize the effect of intermolecular quenching which could otherwise alter the

observed fluorescence emissions. A comparison of the fluorescence emission of

nitroxide (23 and 42 - 44) and methoxyamine analogues (45 - 48) revealed modest

fluorescence suppression arising from the presence of the nitroxide moiety (Figure

2.5).

Figure 2.5. Fluorescence spectra of (a) unsubstituted 23 and 46, (b) 6-bromo 43 and 47, (c) 7-hydroxy

42 and 45 (d) 7-diethylamino 44 and 48 nitroxide and methoxyamine analogues

However the low quantum yields of the unsubstituted and 6-bromo substituted

methoxyamine analogues (0.001 and 0.005 M-1

cm-1

respectively) provided

insufficient sensitivity to find application as fluorescent probes.

0

100

200

300

400

500

600

700

800

900

35

0

40

0

45

0

50

0

55

0

60

0

I flu

ore

scen

ce(a

.u)

35

0

40

0

45

0

50

0

55

0

60

0

Nitroxide

35

0

40

0

45

0

50

0

55

0

60

0

Methoxyamine

40

0

45

0

50

0

55

0

60

0

Wavelength (nm)

(a) (b) (c) (d)

42

Substitution of the coumarin ring with electron-donating substituents is known to

exert a profound impact on fluorescence emissions due to an associated increase in

charge transfer character.73,83,85

Accordingly, the measured quantum yields of 7-

hydroxy and 7-diethylamine substituted methoxyamine analogues (45 and 48

respectively) were substantially greater than that observed for the unsubstituted 46

and 6-bromo substituted 47 methoxyamine analogues (Table 2.3). Furthermore,

significant fluorescence suppression due to the presence of the nitroxide moiety was

observed for 7-hydroxy 42 and 7-diethylamine 44 substituted nitroxide analogues

with measured quantum yields of 0.02 and 0.2 M-1

cm-1

respectively.

Table 2.3. Quantum yield values for nitroxide and methoxyamine analogues

Entry Compound Quantum Yield

(M-1

cm-1

)

1 42 0.020a

2 23 0.001a

3 43 0.005a

4 44 0.200b

5 45 0.550a

6 46 0.010a

7 47 0.015a

8 48 0.950a

a (λexcitation = 340nm),

b (λexcitation= 375nm)

The measured Stokes shifts for 7-hydroxy (42 and 45) and 7-diethylamino (44 and

48) analogues in THF were 76 nm and 54 nm respectively and were found to be

strongly influenced by solvent polarity. Bathochromic shifts in both absorbance

(Figure 2.6) and emission spectra (Figure 2.7) of 48 were observed with increasing

solvent polarity (cyclohexane > THF > DMSO) due to increasing charge-transfer

stabilization.

43

Figure 2.6. Absorbance spectra of 48 in cyclohexane, THF and DMSO

Interestingly, despite the observed bathochromic shift in absorbance spectra with a

concomitant increase in absorbance intensity, the fluorescence intensities of 48 for

each solution were relatively uniform when excited at their absorbance maxima

(cyclohexane λexcitation= 402 nm, THF λexcitation= 410 nm, DMSO λexcitation= 416 nm)

(Figure 2.7).

Figure 2.7. Emission spectra of 48 in cyclohexane, THF and DMSO following excitation at 402, 410

and 416 nm respectively

Furthermore, both the emission and absorbance of 7-hydroxy substituted analogues

42 and 45 were found to be strongly influenced by pH. Under basic conditions

phenol deprotonation of 45 to afford the corresponding phenolate caused a

0

0.02

0.04

0.06

0.08

0.1

0.12

600550500450400350300

Ab

sorb

an

ce (

a.u

)

Wavelength (nm)

Cyclohexane

THF

DMSO

0

100

200

300

400

500

600

700

800

408 458 508 558 608 658

I Flu

ore

scen

ce(a

.u)

Wavelength (nm)

Cyclohexane

THF

DMSO

44

bathochromic shift in fluorescence emission from 430 nm to 486 nm with a

concomitant reduction in fluorescence intensity (Figure 2.8).

Figure 2.8. Emission spectra of the 7-hydroxy methoxyamine analogue 45 with increasing pH

following excitation at 340 nm

A bathochromic shift in the absorbance maxima of the 7-hydroxy substituted

methoxyamine analogue 45 from 354 nm to 426 nm was also observed under basic

conditions, corresponding to formation of the phenolate species (Figure 2.9).

Figure 2.9. Absorbance spectra of the 7-hydroxy methoxyamine analogue 45 with increasing pH

The bathochromic shift in absorbance which occurs upon formation of the phenolate

species accounts for the reduction in observed fluorescence under basic conditions.

As an excitation wavelength of 340 nm (corresponding to the approximate absorption

maxima of the 7-hydroxy analogues in THF) was used, the bathochromic shift in

0

100

200

300

400

500

600

350 400 450 500 550 600

IF

luore

scen

ce (a

.u)

Wavelength (nm)

pH1234567891011121314

0

0.1

0.2

0.3

0.4

0.5

0.6

500450400350300

Ab

sorb

an

ce (

a.u

)

Wavelength (nm)

pH1234567891011121314

45

absorbance with increasing pH resulted in a diminished absorbance at the excitation

wavelength and a corresponding reduction in the intensity of fluorescence. When an

excitation wavelength of 400 nm was applied an inverse trend in fluorescence was

observed for 45 (Figure 2.10). Solutions corresponding to pH 6 - 14 exhibited

increased fluorescence intensity at 486 nm due to formation of the phenolate moiety,

whilst solutions corresponding to pH 1 - 5 exhibited low fluorescence intensity.

Figure 2.10. Emission spectra of the 7-hydroxy methoxyamine analogue 45 with increasing pH

following excitation at 400 nm

This process was shown to be reversible as the neutralization of basic conditions with

HCl regenerated the phenol moiety 45 causing an associated hypsochromic shift in

absorbance from 426 nm to 354 nm (Figure 2.11).

Figure 2.11. Absorbance spectra of the 7-hydroxy methoxyamine analogue 45 at neutral pH

0

100

200

300

400

500

600

700

800

900

405 455 505 555 605 655

I Flu

ore

scen

ce (a

.u)

Wavelength (nm)

pH1234567891011121314

0

0.05

0.1

0.15

0.2

0.25

0.3

500450400350300

Ab

sorb

an

ce (

a.u

)

Wavelength (nm)

Neutralized

from pH

9

10

11

12

13

14

46

Furthermore, a hypsochromic shift in emission from 486 nm to 430 nm following

neutralization was also observed with a concomitant increase in emission intensity

(Figure 2.12).

Figure 2.12. Emission spectra of the 7-hydroxy methoxyamine analogue 45 at neutral pH after

neutralization of pH 9-14 solutions with HCl (340 nm excitation)

Similar bathochromic trends in absorbance were observed for the corresponding

nitroxide analogue 42 (Figure 2.13) however an associated trend in fluorescence

emission was difficult to detect due to the low baseline fluorescence of the

paramagnetic analogue 42.

Figure 2.13. Absorbance spectra of the 7-hydroxy nitroxide analogue 42 at neutral pH

0

50

100

150

200

250

300

350

350 400 450 500 550 600

I F

luore

scen

ce(a

.u)

Wavelength (nm)

Neutralized

from pH

9

10

11

12

13

14

0

0.1

0.2

0.3

0.4

0.5

0.6

500450400350300

Ab

sorb

an

ce

(a.u

)

Wavelength (nm)

pH1

2

3

4

5

6

7

8

9

10

11

12

13

14

47

Accordingly, these analyses suggest that the applicability of the phenolic nitroxide

probe 42 is diminished as the lability of the phenolic hydrogen and the associated

bathochromic shifts in both absorbance and emission spectra provide a degree of

uncertainty with respect to the observed emissions. As both phenol 42 and phenolate

62 species are present in equilibrium (from pH 6 - 14) an exact measure of

fluorescence emission which could be extrapolated to the quantification of radical

species present in the system being monitored is unreliable. Furthermore, formation

of the phenolate moiety 62 provides the possibility for scission of the coumarin

lactone and subsequent irreversible loss of fluorescence emission (Scheme 2.24).

Scheme 2.24. Phenolate induced scission of coumarin lactone

48

3 Chapter 3. Conclusions and Future Work

3.1 Conclusions

This project has focused on the first application of the CuAAC reaction for the

preparation of novel profluorescent systems as potential tools to monitor processes

involving free radicals. One such application may be in the area of bacterial biofilm

dispersal, which has recently been shown to be governed by the presence of free

radical signaling molecules.

The benefits of the CuAAC approach toward the generation of profluorescent

systems are inherently related to the ease of synthesis and purification of the desired

probes as well as the potential to incorporate structural diversity as a means to

modulate the sensitivity, wavelengths of absorbance and emission, as well as

biological compatibility of profluorescent probes.

Facilitating this approach, synthesis of the alkyne isoindoline analogue 21 proceeded

smoothly according to literature procedures. The low overall yield for this synthetic

approach (6.4 % over seven steps) was attributable to the low yield of the Grignard

reaction (22 %). Although this yield is consistent with literature values, it presents a

major limitation to this synthetic approach necessitating the large scale synthesis of

the precursor N-benzylphthalimide 25. Conveniently, the synthesis of N-

benzylphthalimide 25 from readily available phthalic anhydride 24 and benzylamine

proceeded rapidly and in excellent yield, providing a degree of compensation for the

low yield of the Grignard reaction. Subsequent oxidative debenzylation and

bromination of the isoindoline aromatic ring proceeded smoothly and in good yield

according to literature procedures.

Notably, oxidation of 5-iodo-1,1,3,3-tetramethylisoindoline with mCPBA provided a

convenient synthetic route to the synthesis of 5-iodo-1,1,3,3-tetramethylisoindolin-2-

yloxyl 29. Whilst the preceding H2O2-tungstate oxidation approach employed by our

group suffered from long reaction times (3 days), the mCPBA oxidation approach

proceeded to completion in just 30 minutes in comparable yield to that obtained

through H2O2-tungstate oxidation.

49

Fundamental to the „click‟ approach was the successful incorporation of the alkyne

functionality through Sonogashira coupling to the isoindoline system. As the

brominated analogue 27 was known to possess limited reactivity to palladium

catalysis the iodinated analogue 28 was prepared through standard lithiation

techniques. Furthermore, as our group had previously demonstrated that traditional

Sonogashira reaction conditions are not applicable to this system due to the

associated high degree of Glaser coupling, a copper-free approach was implemented.

Under these conditions no discernable impact from Glaser coupling was evident and

the reaction proceeded in good yield. Subsequent deprotection of the isopropanol

group then afforded the terminal alkyne functionality required for use in the CuAAC

reaction.

Synthesis of 3-azido coumarin CuAAC coupling partners (22 and 39 - 10) was

achieved through the condensation of appropriately substituted 2-

hydroxybenzaldehydes with N-acetylglycine to initially afford the corresponding 3-

acetamido coumarin analogues. Deacetylation, followed by the in situ generation of

corresponding 3-diazonium salts upon treatment with sodium nitrite in aqueous acid,

followed by the addition of sodium azide gave the desired 3-azido coumarin

analogues 22, 39 and 40. Repeated attempts to generate the 3-acetamido-7-

diethylamine substituted coumarin analogue 38 using this methodology failed to

yield the desired product. This was attributed to the electron donating diethylamine

group para to the aldehyde moiety disfavoring coumarin formation. Accordingly, an

alternate approach was implemented which successfully prepared the desired 7-

diethylamino coumarin species through Knoevenagel condensation conditions. This

approach provided a convenient synthetic route to the preparation of coumarin

analogues from deactivated 2-hydroxybenzaldehyde systems.

Following the synthesis of the alkyne isoindoline 21 and 3-azido coumarin analogues

(22 and 39 - 41), the CuAAC approach enabled the facile preparation of four novel

profluorescent nitroxides (23 and 42 - 44) through standard CuAAC reaction

conditions in high yield. The corresponding methoxyamine analogues 45 - 48 were

then prepared by reaction of nitroxide analogues 22 and 39 - 41 with methyl radicals

generated through Fenton chemistry, in high yield.

50

Spectrophotometric analysis of nitroxide (23 and 42 - 44) and methoxyamine

analogues (45 - 48) revealed that the triazole provided effective nitroxide-

fluorophore communication, as evidenced by the observed fluorescence suppression

of nitroxide analogues (23 and 42 - 44) compared with the corresponding

methoxyamine analogues (45 - 48).

Furthermore, spectrophotometric analysis of the 7-hydroxy and 7-diethylamino

methoxyamine analogues 45 and 48 respectively, revealed that high fluorescence

emissions were obtainable with the use of coumarin fluorophores possessing

electron-donating substituents in the 7 position (as evidenced by the high quantum

yields measured). Furthermore, increasing solvent polarity was shown to stabilize the

charge-transfer complex generating associated bathochromic shifts in both

absorbance and emission spectra of the 7-diethylamino substituted methoxyamine

analogue 48 compared with the unsubsustituted methoxyamine analogue 46.

Although the incorporation of electron-donating functionality was shown to be

essential for the production of high fluorescence emissions, the applicability of the

prepared 7-hydroxy substituted analogues 42 and 45 appears diminished due to the

observed pH susceptibility arising from the labile phenolic hydrogen. Analysis of

both absorbance and emission profiles of the 7-hydroxy substituted analogues 41 and

45 under basic conditions revealed that bathochromic shifts in absorbance and

emission occurred with a concomitant reduction of fluorescence emission. Whilst

this process was shown to be reversible, as neutralization of basic conditions caused

associated hypsochromic shifts corresponding to phenolate protonation, the

persistence of the phenolate moiety offers the possibility for scission of the coumarin

lactone and subsequent irreversible loss of fluorescence emission.

The culmination of this work has identified the 7-diethylamine substituted nitroxide

analogue 44 as a potential probe for monitoring bacterial biofilm dispersal. The

corresponding methoxyamine analogue 48 was shown to possess a high quantum

yield whilst the low quantum yield of the nitroxide analogue revealed significant

fluorescence suppression arising from the nitroxide moiety. These characteristics

position this novel profluorescent nitroxide as a candidate for potential application as

a molecular probe to monitor bacterial biofilm dispersal.

51

3.2 Future Work

Whilst this project has demonstrated that a „click‟ approach to the generation of

novel profluorescent nitroxides can furnish desirable spectrophotometric properties,

further elaboration of our molecular library could lead to the discovery of novel

profluorescent probes with enhanced fluorescence properties. Specifically, we

demonstrated that substitution of the 7- position of the coumarin ring with electron

donating groups enhanced the corresponding charge-transfer complex leading to

improved quantum yields of diamagnetic methoxyamine analogues. We further

demonstrated that Knoevenagel condensation reaction conditions provide a

convenient synthetic route to the synthesis of coumarin analogues possessing

substitution in the 7- position with electron-donating groups. These reaction

conditions could be applied to the preparation of novel coumarin based probes

possessing substitution in the 7- position with electron-donating functionality to

further optimize the coumarin structure toward enhanced fluorescence emissions.

Alternatively, as we have established that the triazole mediates effective nitroxide-

fluorophore communication to facilitate effective fluorescence suppression of

fluorescent probes, the CuAAC approach could be applied to the incorporation of

additional fluorescent molecules, provided azide analogues could be prepared. As an

extension of the work performed in this thesis, specifically the bromination and

subsequent iodination of the aromatic ring, iodo analogues of the polyaromatic

fluorophores such as perylene, anthracene and 9,10-diphenylanthracene could be

prepared. This would allow for subsequent formation of their corresponding azido

analogues and facilitate their application to the click approach.

As we have identified the 7-diethylamino coumarin analogue as a potential probe for

monitoring bacterial biofilm dispersal, a major avenue for future work will be to first

assess this compound as a tool for detecting ROS and RON both in the presence and

absence of radical scavengers. These results will then be independently compared

with commercially available fluorescent probes (Table 3.1).

52

Table 3.1. Conditions to assess specificity of profluorescent probes

RONS Scavenger Fluorescent dye

Nitric oxide

(NO˙)

PTIO

Deoxyhemoglogin

DAFFM-DA

Nitrite

(NO2-)

Cu + ascorbic acid

Nitrate

(NO3-)

Nitrate reductase

Superoxide

(O2-˙)

Superoxide

dismutase

Hydroethidium

Hydrogen

peroxide (H2O2)

Catalase Dichlorofluorescein

Peroxynitrite

(ONOO-)

MnTBAP Dihydrorhodamine

Furthermore, the fluorescent probe uptake in planktonic and biofilm cells will be

measured exclusively. Confocal microscopy will be used to visualize profluorescent

probe uptake at various stages of biofim development. These analyses are currently

being undertaken through collaborations with the Environmental Biotechnology

Cooperative Research Centre at the University of New South Wales and will

establish the applicability of the prepared 7-diethylamino coumarin analogue toward

monitoring bacterial biofilm dispersal.

53

4 Chapter 4. Experimental

4.1 General Procedures

All starting materials and reagents were purchased from Sigma Aldrich. All reactions

were monitored by Merck Silica Gel 60 F254 TLC and visualized with UV light.

Silica gel column chromatography was performed using silica gel 60 Å (230 - 400

mesh). 1H NMR spectra were run at 400 MHz and

13C NMR spectra at 100 MHz.

Chemical shifts () for 1H and

13C NMR spectra run in CDCl3 are reported in ppm

relative to the solvent residual peak: proton ( = 7.28 ppm) and carbon (= 77.2

ppm). Chemical shifts for 1H and

13C NMR run in DMSO-d6 are reported in ppm

relative to residual solvent proton ( = 2.50 ppm) and carbon (= 39.5 ppm) signals,

respectively. Multiplicity is indicated as follows: s (singlet); d (doublet); t (triplet); m

(multiplet); dd (doublet of doublet); ddd (doublet of doublet of doublet); br s (broad

singlet). Coupling constants are reported in Hertz (Hz). Mass spectra were recorded

using electrospray and electron impact (where specified) as the ionization technique

in positive ion mode. All MS analysis samples were prepared as solutions in

methanol.

Infrared spectra were recorded as neat samples using a Nicolet 870 Nexus Fourier

Transform infrared spectrometer equipped with a DTGS TEC detector and an

Attenuated Total Reflectance (ATR) accessory (Nicolet Instrument Corp., Madison,

WI) using a Smart Endurance single reflection ATR accessory equipped with a

composite diamond IRE with a 0.75 mm2 sampling surface and a ZnSe focussing

element. An Optical Path Difference (OPD) velocity of 0.6329 cm s-1

and a gain of 8

were used. Spectra were collected in the spectral range 4000-525 cm-1

with a

minimum of 16 scans, and 4 cm-1

resolution.

Analytical HPLC was performed on a Hewlett Packard 1100 series HPLC, using an

Agilent prep-C18 scalar column (10 μm, 4.6 × 150 mm) at a flow rate of 1 mL/min.

All UV/Vis spectra were recorded on a single beam Varian Cary 50 UV-Vis

spectrophotometer. Fluorescence measurements were performed on a Varian Cary

54

Eclipse fluorescence spectrophotometer equipped with a standard multicell Peltier

thermostatted sample holder.

Melting points were measured on a Gallenkamp Variable Temperature Apparatus by

the capillary method and are uncorrected.

4.2 Synthesis

4.2.1 N-Benzylphthalimide (25)

A solution of phthalic anhydride 24 (2.0 g, 14 mmol) and benzylamine (2.2 g, 20

mmol) in glacial acetic acid (10 mL) was brought to reflux and maintained with

stirring for 1 hour. The hot reaction mixture was then poured into ice / H2O (50 mL)

yielding a white precipitate which was collected by vacuum filtration and washed

with H2O (50 mL). The precipitate was then recrystallized from ethanol to afford 25

as a white crystalline solid (3.1 g, 92 % yield) (Rf = 0.53, EtOAc : n-hexanes, 1 : 1).

M.p. 115 - 116 °C, lit.74

112 - 115 °C . 1H NMR (400 MHz, CDCl3): (ppm) = 7.86

(dd, J = 5.6, 3.2 Hz, 2H, 2×Harom), 7.73 (dd, J = 5.6, 3.2 Hz, 2H, 2×Harom), 7.45 (d, J

= 7.2 Hz, 2H, 2×Harom), 7.34 (m, 3H, Harom), 4.87 (s, 2H, CH2). MS (ESI): m/z (%) =

238 (10) [MH+], 260 (10) [MNa

+]. HRMS: calculated for C15H12NO2 [MH

+]

238.0863; found 238.1025. HRMS: calculated for C15H11NO2Na [MNa+] 260.0687;

found 260.0844. These data agree with those previously reported by Cheng et.al.87

55

4.2.2 2-Benzyl-1,1,3,3-tetramethylisoindoline (26)

Pre-dried magnesium (120 g, 11.7 equiv.) and three small crystals of I2 were placed

in a round bottom flask (3 L) fitted with a still head, two dropping funnels,

thermometer, mechanical stirrer and two twin helix condensers connected in series

above the still head to which ice cold water was delivered with a peristaltic pump. A

positive pressure of argon was applied, with subsequent evacuation of the system

under vacuum and a positive pressure of argon reapplied.

Addition of anhydrous diethyl ether (400 mL) to the vessel was followed by the

drop-wise addition of methyl iodide (155 mL, 5.9 equiv.) via one of the dropping

funnels. The other dropping funnel was maintained with a constant supply of

anhydrous diethyl ether which was added periodically in order to keep a constant rate

of reaction. Once addition of the methyl iodide was complete, the mixture was stirred

until all activity had subsided, with subsequent concentration of the Grignard

solution by distillation until the interior temperature reached 80 °C.

Upon cooling the reaction mixture to 64 °C, a solution of N-benzylphthalimide 25

(100 g, 0.421 mol, 1.0 equiv.) in dry toluene (800 mL) was added via both dropping

funnels at such a rate as to maintain a constant temperature. Following this addition,

diethyl ether was further removed through distillation until a reaction temperature of

110 °C was reached. The reaction mixture was refluxed for 3 hours and then further

concentrated by distillation.

Once cooled, the mixture was diluted with n-hexanes (1.5 L), mixed thoroughly and

exposed to the atmosphere. The resulting purple slurry was filtered through celite

under vacuum, and the filtrate bubbled with air over night. This filtrate was

subsequently passed through a column of basic alumina, and the solvent was

removed under reduced pressure to give a golden oil which crystallized under

vacuum (24.2 g, 22 % yield) (Rf = 0.71, n-hexanes). M.p. 62 - 63 °C, lit.75

63 - 64

56

°C. 1H NMR (400 MHz, CDCl3): (ppm) = 7.50 (d, J = 7.2 Hz, 2H, 2×Harom), 7.28

(m, 5H, 5×Harom), 7.16 (dd, J = 5.6, 2.4 Hz, 2H, 2×Harom), 4.02 (s, 2H, CH2), 1.33 (s,

12H, 4×CH3). MS (ESI): m/z (%) = 266 (95) [MH+]. HRMS: calculated for C19H24N

[MH+] 266.1903; found 266.1931. These data agree with those previously reported

by Griffiths et.al.75

4.2.3 5-Bromo-1,1,3,3-tetramethylisoindoline (27)

A solution of 2-benzyl-1,1,3,3-tetramethylisoindoline 26 (5.0 g, 19 mmol, 1.0 equiv.)

in DCM (60 mL) was cooled in an ice bath to 0 °C and placed under an atmosphere

of argon. A solution of liquid Br2 (2.2 mL, 43 mmol, 2.3 equiv.) in DCM (40 mL)

was then added followed by anhydrous AlCl3 (9.0 g, 68 mmol, 3.6 equiv.). The

reaction was maintained with stirring for one hour then poured onto ice (~ 150 mL)

and stirred for 15 minutes. The resulting solution was basified (pH 14) with 10M

NaOH and extracted with DCM (3 × 100 mL). The combined organic phases were

washed with H2O (50 mL) and dried over anhydrous sodium sulfate. The solvent was

removed in vacuo affording a yellow residue. The residue was then dissolved in

methanol (~ 30 mL) and NaHCO3 (~ 200 mg) added. To this solution was added

aqueous H2O2 (30%) until no further effervescence could be detected. 2M H2SO4 (75

mL) was then added (caution: effervescent) and the solution then washed with DCM

(3 × 100 mL). The combined organic phases were then back extracted with 2M

H2SO4 (3 × 100 mL). The combined acidic aqueous phases were then cooled in an

ice bath, basified (pH 14) with 10M NaOH and extracted with DCM (5 × 100 mL).

The combined organic phases were then washed with H2O (100 mL) and dried over

anhydrous sodium sulfate. The solvent was removed in vacuo to afford 27 as a low

melting white solid (4.4 g, 92 % yield). 1H NMR (400 MHz, CDCl3): (ppm) = 7.37

(dd, J = 8.0, 1.6 Hz, 1H, Harom), 7.25 (d, J = 1.6 Hz, 1H, Harom), 7.00 (d, J = 8.0 Hz,

1H, Harom), 1.87 (s, 1H, NH), 1.45 (d, J = 3.6 Hz, 12H, 4×CH3); MS (ESI): m/z (%)

= 254/256 (95) [MH+]. HRMS: calculated for C12H17

79BrN [MH

+] 254.0544; found

57

254.0548. HRMS: calculated for C12H1781

BrN [MH+] 256.0524; found 256.0528.

These data agree with those previously reported by Bottle et.al.66

4.2.4 5-Iodo-1,1,3,3-tetramethylisoindoline (28)

A solution of 5-bromo-1,1,3,3-tetramethylisoindoline 27 (9.0 g, 35 mmol, 1.0 equiv.)

in anhydrous THF (100 mL) was cooled to - 78 °C (dry ice / acetone). n-BuLi (1.6 M

in n-hexanes, 60 mL, 96 mmol, 2.7 equiv.) was then added (dropwise) and the

resulting mixture stirred for 15 minutes. A solution of I2 (27.0 g, 107 mmol, 3.0

equiv.) in anhydrous THF (225 mL) was then added (dropwise) and the reaction

allowed to return to room temperature. The reaction mixture was then poured into ice

/ H2O (~ 500 mL) and basified (pH 14) with 5M NaOH. The resulting solution was

then extracted with DCM (3 × 500 mL) and the combined organic phases washed

with water and dried over anhydrous sodium sulfate. The solvent was removed in

vacuo affording a clear residue which was then dissolved in methanol (~ 200 mL)

and NaHCO3 (~ 500 mg) added. To this solution was added aqueous H2O2 (30 %) (~

100 mL) followed by 2M H2SO4 (500 mL) (caution: effervescent). The resulting

solution was washed with DCM (3 × 500 mL) and the combined organic phases back

extracted with 2M H2SO4 (3 × 500 mL). The combined acidic aqueous phases were

then basified (pH 14) with 10M NaOH and extracted with DCM (5 × 500 mL). The

combined organic phases were then washed with H2O (200 mL) and dried over

anhydrous sodium sulfate. The solvent was removed in vacuo to afford 28 as a low

melting white solid (7.8 g, 73 % yield). 1H NMR (400 MHz, CDCl3): (ppm) = 7.58

(dd, J = 8.0, 1.6 Hz, 1H, Harom), 7.46 (d, J = 1.2 Hz, 1H, Harom), 1.76 (s, 1H, NH),

1.45 (s, 12H, 4×CH3). MS (ESI): m/z (%) = 302 (95) [MH+]. HRMS: calculated for

C12H17IN [MH+] 302.0400; found 302.0402. These data agree with those previously

reported by Bottle et.al.66

58

4.2.5 5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (29)

5-Iodo-1,1,3,3-tetramethylisoindoline 28 (4.0 g, 13 mmol, 1.0 equiv.) was dissolved

in DCM (160 mL) and cooled to 0 °C in an ice bath. To this solution was added 3-

chloroperoxybenzoic acid (mCPBA) (77%, 3.9 g, 17 mmol, 1.3 equiv.) and stirred

for 20 minutes at 0 °C. The reaction mixture was then allowed to return to room

temperature and H2O (200 mL) added. The organic phase was then washed with 2M

NaOH (3 × 100 mL) then brine (100 mL) and dried over anhydrous sodium sulfate.

The solvent was then removed in vacuo followed by recrystallization from ethanol to

afford 29 as an orange crystalline solid (3.2 g, 77 % yield) (Rf = 0.62, EtOAc : n-

hexanes, 1 : 1). M.p. 131 - 133 °C, lit.78

132 - 134 °C. IR (ATR) νmax 1386 (N-O˙),

1471 and 1432 (aryl C–C), 2976 and 2924 (alkyl CH3), 3039 (aryl CH) cm−1

. MS

(EI): m/z (%) = 316 (100) [M+]. HRMS: calculated for C12H15INO [M

+]316.0913;

found 316.0913. These data agree with those previously reported by Bottle et.al.66

4.2.6 5-(3-Hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl

(30)

A solution of 5-iodo-1,1,3,3-tetramethylisoindoline 29 (3.0 g, 9.5 mmol, 1.0 equiv.),

4-diazabicyclo[2.2.2]octane (DABCO) (3.2 g, 28.5 mmol, 3.0 equiv.) and palladium

(II) acetate (55 mg, 0.3 mmol, 0.03 equiv.) in acetonitrile (75 mL) was heated to 75

°C . 2-methyl-3-butyn-2-ol (4.5 mL, 4.0 g, 47 mmol, 5.0 equiv.) was then added and

the reaction maintained with stirring for 16 hours. The solvent was removed in vacuo

and the crude reaction mixture purified via silica gel chromatography to afford 30 as

59

a low melting brown solid (1.9 g, 73 % yield) (Rf = 0.50, EtOAc : n-hexanes, 1 : 1).

IR (ATR) νmax 1430 (N-O˙), 1492 and 1465 (aryl C–C), 2162 (C≡C), 2979 and 2932

(alkyl CH3), 3042 (aryl CH) cm−1

. MS (ESI): m/z (%) = 295 (20) [MNa

+]. HRMS:

calculated for C17H22NO2Na [MNa+] 295.1543; found 295.1543. These data agree

with those previously reported by Bottle et.al.66

4.2.7 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (21)

To a solution of 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl 30 ( 1.5 g, 6 mmol, 1.0 equiv.) in anhydrous toluene (300 mL) was added

solid KOH (2.4 g, 43 mmol, 7.8 equiv.). The reaction was brought to reflux and

maintained with stirring for 1 hour. The reaction was then allowed to return to room

temperature, washed with H2O (3 × 200 mL), brine (200 mL) and dried over

anhydrous sodium sulfate. The solvent was removed in vacuo and the crude reaction

mixture purified by silica gel column chromatography then recrystallized from

ethanol to afford 21 as an orange crystalline solid (1.0 g, 84 % yield) (Rf = 0.62,

EtOAc : n-hexanes, 1 : 1). M.p. 126 - 127 °C, lit.78

126 - 128 °C. IR (ATR) νmax 1425

(N-O˙), 1483 and 1462 (aryl C–C), 2093 (C≡C), 2979 and 2928 (alkyl CH3), 3190

(≡C-H) cm−1

. MS (ESI): m/z (%) = 237 (15) [MNa+]. HRMS: calculated for

C14H16NONa [MNa+] 237.1124; found 237.1136. These data agree with those

previously reported by Bottle et.al.66

60

4.2.8 3-Acetamido-7-acetoxy-2H-chromen-2-one (35)

A mixture of 2,4-dihydroxybenzaldehyde 31 (5.0 g, 36 mmol, 1.0 equiv.), N-

acetylglycine (4.7 g, 40 mmol, 1.1 equiv.) and anhydrous sodium acetate (14.9 g, 182

mmol, 5.0 equiv.) was made up in acetic anhydride (50 mL). The reaction was heated

to 120°C and maintained with stirring for 4 hours then cooled in an ice bath. Cold

H2O (200 mL) was then added and the resulting precipitate collected by vacuum

filtration, washed with H2O (100 mL) and recrystallized from ethanol to afford 35 as

a white crystalline solid (2.6 g, 27 % yield) (Rf = 0.30, EtOAc : n-hexanes, 1 : 1).

M.p. 231 - 232 °C, lit.88

234 - 236 °C. 1H NMR (400 MHz, DMSO-d6): (ppm) =

9.77 (s, 1H, NH), 8.62 (s, 1H, C=CH), 7.75 (d, J = 8.4 Hz, 1H, Harom), 7.27 (d, J =

2.0 Hz, 1H, Harom), 7.13 (dd, J = 8.4, 2.0 Hz, 1H, Harom), 2.30 (s, 3H, CH3), 2.16 (s,

3H, CH3). MS (ESI): m/z (%) = 284 (70) [MNa+]. HRMS: calculated for

C13H11NO5Na [MNa+] 284.0529; found 284.0531. These data agree with those

previously reported by Wang et.al.73

4.2.9 3-Acetamido-2H-chromen-2-one (36)

A mixture of 2-hydroxybenzaldehyde 32 (5.1 g, 41 mmol, 1.0 equiv.), N-

acetylglycine (5.3 g, 45 mmol, 1.1 equiv.) and anhydrous sodium acetate (17.0 g, 208

mmol, 5.0 equiv.) was made up in acetic anhydride (50 mL). The reaction was heated

to 120°C and maintained with stirring for 4 hours then cooled in an ice bath. Cold

H2O (200 mL) was then added and the resulting precipitate collected by vacuum

filtration, washed with H2O (100 mL) and recrystallized from ethanol to afford 36 as

a white crystalline solid (4.3 g, 52 % yield) (Rf = 0.40, EtOAc : n-hexanes, 1 : 1).

61

M.p. 193 - 195 °C, lit.88

195 - 196 °C. 1H NMR (400 MHz, DMSO-d6): (ppm) =

9.77 (s, 1H, NH), 8.62 (s, 1H, C=CH), 7.70 (dd, J = 7.6, 1.6 Hz, 1H, Harom), 7.50

(ddd, J = 8.4, 7.6, 1.2 Hz, 1H, Harom), 7.39 (d, J = 8.0 Hz, 1H, Harom), 7.33 (ddd, J =

8.4, 7.6, 1.2 Hz,1H, Harom), 2.17 (s, 3H, CH3). MS (ESI): m/z (%) = 226 (95) [MNa+].

HRMS: calculated for C11H9NO3Na [MNa+] 226.0475; found 226.0473. These data

agree with those previously reported by Kudale et.al.88

4.2.10 3-Acetamido-6-bromo-2H-chromen-2-one (37)

A mixture of 5-bromo-2-hydroxybenzaldehyde 33 (5.2 g, 26 mmol, 1.0 equiv.), N-

acetylglycine (3.3 g, 28 mmol, 1.1 equiv.) and anhydrous sodium acetate (10.5 g, 128

mmol, 5.0 equiv.) was made up in acetic anhydride (50 mL). The reaction was heated

to 120 °C and maintained with stirring for 4 hours then cooled in an ice bath. Cold

H2O (200 mL) was then added and the resulting precipitate collected by vacuum

filtration, washed with H2O (100 mL) and recrystallized from ethanol to afford 37 as

a white crystalline solid (4.93 g, 68 % yield) (Rf = 0.43, EtOAc : n-hexanes, 1 : 1).

M.p. 262 - 264 °C, lit.88

262 - 263 °C. 1H NMR (400 MHz, DMSO-d6): (ppm) =

9.84 (s, 1H, NH), 8.58 (s, 1H, C=CH), 7.80 (d, J = 2.0 Hz, 1H, Harom), 7.63 (dd, J =

8.8, 2.4 Hz, 1H, Harom), 7.35 (d, J = 8.8 Hz, 1H, Harom), 2.17 (s, 3H, CH3). MS (ESI):

m/z (%) = 303/305 (40) [MNa+]. HRMS: calculated for C11H8

79BrNO3Na [MNa

+]

303.9585; found 303.9576. HRMS: calculated for C11H881

BrNO3Na [MNa+]

305.9565; found 305.9557. These data agree with those previously reported by

Kudale et.al.88

62

4.2.11 3-Nitro-7-diethylamino-2H-chromen-2-one (58)

A solution of 4-diethylamino-2-hydroxybenzaldehyde 34 (0.63 g, 3.26 mmol, 1.0

equiv.), ethyl nitroacetate (0.40 mL, 3.62 mmol, 1.1 equiv.), piperidine (0.05 mL)

and acetic acid (0.1 mL) was made up in tert-butanol (10 mL) and refluxed for 24

hours. The reaction mixture was then allowed to return to room temperature followed

by the addition of water (30 mL). The resulting precipitate was collected by vacuum

filtration, washed with H2O (30 mL) and dried in vacuo to afford 58 as a red solid

(0.62 g, 73 % yield) (Rf = 0.37, EtOAc : n-hexanes, 1 : 1). M.p. 194 - 195 °C, lit.73

193 - 195 °C. 1H NMR (400 MHz, CDCl3): (ppm) = 8.75 (s, 1H, C=CH), 7.46 (d, J

= 9.2 Hz, 1H, Harom), 6.73 (dd, J = 9.2, 2.4 Hz, 1H, Harom), 6.51 (d, J = 2.4 Hz, 1H,

Harom), 3.52 (q, J = 7.2 Hz, 4H, 2×CH2), 1.29 (t, J = 7.2 Hz, 6H, 2×CH3). MS (ESI):

m/z (%) = 263 (45) [MH+], 285 (65) [MNa

+]. HRMS: calculated for C13H15N2O4

[MH+] 263.1026; found 263.1030. HRMS: calculated for C13H14N2O4Na [MNa

+]

285.0846; found 285.0855. These data agree with those previously reported by Wang

et.al.73

4.2.12 3-Amino-7-diethylamino-2H-chromen-2-one (59)

To a suspension of stannous chloride dihydrate (1.60 g, 7.12 mmol, 7.5 equiv.) in

37.4% HCl (5 mL) was added 3-nitro-7-diethylamino-2H-chromen-2-one 58 (0.25 g,

0.95 mmol, 1.0 equiv.) in portions over a period of 30 minutes. The reaction was

maintained with stirring for 4 hours then poured onto ice (~ 30 mL) and made

alkaline using sodium hydroxide solution (5 M). The resulting suspension was then

63

extracted with DCM (3 × 50 mL) and washed with H2O (30 mL). The combined

organic phases were then dried over anhydrous sodium sulfate and the solvent

removed in vacuo which upon triturating with n-hexanes afforded 59 as a dark red

solid (145 mg, 65 % yield) (Rf = 0.48, EtOAc : n-hexanes, 1 : 1). M.p. 83 - 85 °C,

lit.73

85 - 87 °C. 1H NMR (400 MHz, CDCl3): (ppm) = 7.13 (d, J = 8.8 Hz 1H,

Harom), 6.72 (s, 1H, C=CH), 6.59 (dd, J = 8.8, 2.4 Hz, 1H, Harom), 6.55 (d, J = 2.0 Hz,

1H, Harom), 3.88 (s, 2H, NH2), 3.39 (q, J = 7.2 Hz, 4H, 2×CH2), 1.20 (t, J = 7.2 Hz,

6H, 2×CH3). MS (ESI): m/z (%) = 233 (100) [MH+]. HRMS: calculated for

C13H17N2O2 [MH+] 233.1285; found 233.1290. These data agree with those

previously reported by Wang et.al.73

4.2.13 3-Azido-7-hydroxy-2H-chromen-2-one (39)

A mixture of 3-acetamido-7-acetoxy-2H-chromen-2-one 35 (102 mg, 0.39 mmol, 1.0

equiv.) was made up in conc. HCl and ethanol (2 : 1, v / v, 10 mL) and refluxed for

one hour with stirring. The reaction mixture was then diluted with cold water (20

mL) and placed in an ice bath for 15 minutes. Sodium nitrite (90 mg, 1.30 mmol, 3.3

equiv.) was added in portions and the reaction maintained for 15 minutes. Sodium

azide (112 mg, 1.72 mmol, 4.4 equiv.) was then added in portions and the reaction

maintained with stirring for a further 30 minutes. The resulting precipitate was

collected by vacuum filtration, washed with H2O (20 mL) and dried in vacuo to

afford 39 as a brown solid (71 mg, 90 % yield) (Rf = 0.49, EtOAc : n-hexanes, 1 : 1).

1H NMR (400 MHz, DMSO-d6): (ppm) = 10.53 (s, 1H, OH), 7.60 (s, 1H, C=CH),

7.48 (d, J = 8.8 Hz, 1H, Harom), 6.81 (dd, J = 8.8, 2.4 Hz, 1H, Harom), 6.76 (d, J = 2.0

Hz, 1H, Harom). MS (ESI): m/z (%) = 226 (20) [MNa+]. HRMS: calculated for

C9H5N3O3Na [MNa+] 226.0223; found 226.0220. These data agree with those

previously reported by Wang et.al.73

64

4.2.14 3-Azido-2H-chromen-2-one (22)

A mixture of 3-acetamido-2H-chromen-2-one 36 (116 mg, 0.57 mmol, 1.0 equiv.)

was made up in conc. HCl and ethanol (2 : 1, v / v, 10 mL) and refluxed for one hour

with stirring. The reaction mixture was then diluted with cold water (20 mL) and

placed in an ice bath for 15 minutes. Sodium nitrite (105 mg, 1.52 mmol, 2.7 equiv.)

was added in portions and the reaction maintained for 15 minutes. Sodium azide (132

mg, 2.03 mmol, 3.6 equiv.) was then added in portions and the reaction maintained

with stirring for a further 30 minutes. The resulting precipitate was collected by

vacuum filtration, washed with H2O (10 mL) and dried in vacuo to afford 22 as a

brown solid (92 mg, 87 % yield) (Rf = 0.63, EtOAc : n-hexanes, 1 : 1). 1H NMR

(400 MHz, DMSO-d6): (ppm) = 7.68 (s, 1H, C=CH), 7.66 (dd, J = 7.6, 1.6 Hz, 1H,

Harom), 7.55 (ddd, J = 8.4, 7.6, 1.6 Hz, 1H, Harom), 7.44 (d, J = 8.4 Hz, 1H, Harom),

7.36 (ddd, J = 8.4, 7.6, 1.2 Hz, 1H, Harom). MS (ESI): m/z (%) = 210 (45) [MNa+].

HRMS: calculated for C9H5N3O2Na [MNa+] 210.0274; found 210.0272. These data

agree with those previously reported by Wang et.al.73

4.2.15 3-Azido-6-bromo-2H-chromen-2-one (40)

A mixture of 3-acetamido-6-bromo-2H-chromen-2-one 37 (0.53 g, 1.90 mmol, 1.0

equiv.) in conc. HCl and ethanol (2 : 1, v / v, 10 mL) was refluxed for two hours with

stirring. The reaction mixture was then diluted with cold water (5 mL) and placed in

an ice bath for 15 minutes. Sodium nitrite (390 mg, 5.65 mmol, 3.0 equiv.) was

added in portions over a period of 10 minutes and the reaction maintained for a

65

further 5 minutes. Sodium azide (490 mg, 7.54 mmol, 4.0 equiv.) was then added in

portions and the reaction maintained with stirring for one hour at 0°C then 24 hours

at room temperature. The resulting precipitate was collected by vacuum filtration,

washed with H2O (20 mL) and purified by silica gel chromatography to afford 40 as

a brown solid (152 mg, 30 % yield) (Rf = 0.68, EtOAc : n-hexanes, 1 : 1). 1H NMR

(400 MHz, DMSO-d6): (ppm) = 7.90 (d, J = 2.4 Hz, 1H, Harom), 7.68 (dd, J = 8.8,

2.4 Hz, 1H, Harom), 7.59 (s, 1H, C=CH), 7.41 (d, J = 8.8 Hz, 1H, Harom). MS (ES):

m/z (%) = 264/266 (30) [M+]. HRMS: calculated for C9H4

79BrN3O2 [M

+] 264.9487;

found 264.9481. HRMS: calculated for C9H481

BrN3O2 [M+] 266.9461; found

266.9464. These data agree with those previously reported by Wang et.al.73

4.2.16 3-Azido-7-diethylamino-2H-chromen-2-one (41)

3-Amino-7-diethylamino-2H-chromen-2-one 59 (100 mg, 0.43 mmol, 1.0 equiv) was

dissolved slowly in HCl aq. (17.2%, 4 mL) at room temperature then cooled in an ice

bath. Sodium nitrite (33 mg, 0.43 mmol, 1.1 equiv.) was then added in portions and

the reaction maintained with stirring for one hour. Potassium acetate (2 g) in water (5

mL) was then added to adjust the pH of the resulting solution to 4. Sodium azide (60

mg, 0.92 mmol, 2.1 equiv.) was then added in portions and the mixture stirred for a

further 5 hours. The resulting precipitate was collected by vacuum filtration, washed

with cold water (20 mL) and dried in vacuo to afford 41 as a green solid (71 mg, 64

% yield) (Rf = 0.51, EtOAc : n-hexanes, 2 : 1). IR (ATR) νmax 805 (=C-H), 1130 (C-

O), 1446 and 1469 (aryl C-C), 1599 (C=C-COO), 1703 (C=O), 2114 (N≡N), 2931

and 2973 cm-1

(alkyl CH3). 1H NMR (400 MHz, CDCl3): (ppm) = 7.21 (d, J = 8.8

Hz, 1H, Harom), 7.12 (s, 1H, C=CH), 6.60 (dd, J = 10.0, 2.8 Hz, 1H, Harom), 6.52 (d, J

= 2.4 Hz, 1H, Harom), 3.43 (q, J = 7.2 Hz, 4H, 2×CH2), 1.22 ( t, J = 7.2 Hz, 6H,

2×CH3). These data agree with those previously reported by Wang et.al.73

66

4.2.17 General procedure 1 - CuAAC reactions

A mixture of 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 (1 equiv.) and 3-

azido-coumarin (22 and 39 - 41) (1.1 equiv.) was made up in ethanol and water (1 :

1, v / v, 20 ml). To this was added copper (II) sulfate pentahydrate (5 mol %) and

sodium ascorbate (10 mol %). The reaction was maintained with stirring for 16 hours

at room temperature in the absence of light and the resulting precipitate collected by

vacuum filtration and washed with water.

4.2.17.1 7-Hydroxy-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (42)

The title compound was prepared from 39 (50 mg, 0.25 mmol) according to general

procedure 1 and purified via silica gel chromatography to afford 42 as an orange

solid (94 mg, 90 % yield) (Rf = 0.19, EtOAc : n-hexanes, 1 : 1). M.p. 249 - 250 °C.

IR (ATR) νmax 853 (=C-H), 1118 (C-O), 1231 (O-H), 1416 (N-O˙), 1460 and 1488

(aryl C-C), 1606 (C=C-COO), 1713 (C=O), 2930 and 2977 (alkyl CH3), 3150 (O-H)

cm-1

. 1H NMR (400 MHz, DMSO-d6): (ppm) = 9.01 (s, 1H, Htriazole), 8.69 (s, 1H,

C=CH), 7.79 (s, 1H, Harom), 6.91 (m, 2H, 2×Harom). MS (ESI): m/z (%) = 440 (45)

[MNa+]. HRMS: calculated for C23H21N4O4Na [MNa

+] 440.1455; found 440.1459.

The purity of 42 was confirmed to be > 95 % using Analytical HPLC.

67

4.2.17.2 3-(4-(1,1,3,3-Tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-

chromen-2-one (23)

The title compound was prepared from 22 (47 mg, 0.25 mmol) according to general

procedure 1 and purified by silica gel chromatography to afford 23 as a brown solid

(86 mg, 86 % yield) (Rf = 0.22 EtOAc : n-hexanes, 1 : 1). M.p. 200 - 201 °C. IR

(ATR) νmax 801 (=C-H), 1120 (C-O), 1432 (N-O˙), 1463 and 1484 (aryl C-C), 1610

(C=C-COO), 1733 (C=O), 2931 and 2977 cm-1

(alkyl CH3). 1H NMR (400 MHz,

DMSO-d6): (ppm) = 9.07 (s, 1H, Htriazole), 8.82 (s, 1H, C=CH), 7.98 (s, 1H, Harom),

7.75 (s, 1H, Harom), 7.58 (s, 1H, Harom), 7.49 (s, 1H, Harom). MS (ESI): m/z (%) = 424

(100) [MNa+]. HRMS: calculated for C23H21N4O3Na [MNa

+] 424.1506; found

424.1508. The purity of 23 was confirmed to be > 95 % using Analytical HPLC.

4.2.17.3 6-Bromo-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-

1-yl)-2H-chromen-2-one (43)

The title compound was prepared from 40 (40 mg, 0.15 mmol) according to general

procedure 1 and purified by silica gel chromatography to afford 43 as an orange solid

(61 mg, 84 % yield) (Rf = 0.33, EtOAc : n-hexanes, 1 : 1). M.p. 246 - 248 °C. IR

(ATR) νmax 659 (C-Br), 815 (=C-H), 1119 (C-O), 1418 (N-O˙), 1462 and 1481 (aryl

C-C), 1602 (C=C-COO), 1730 (C=O), 2927 and 2978 cm-1

(alkyl CH3). 1H NMR

(400 MHz, DMSO-d6): (ppm) = 9.11 (s, 1H, Htriazole), 8.77 (s, 1H, C=CH), 8.24 (s,

1H, Harom), 7.89 (s, 1H, Harom), 7.52 (s, 1H, Harom). MS (ESI): m/z (%) = 480/482 (25)

[MH+], 502/504 (25) [MNa

+]. HRMS: calculated for C23H21N4O3

79Br [MH

+]

68

480.0797; found 480.0782. HRMS: calculated for C23H21N4O381

Br [MH+] 482.0777;

found 482.0774. HRMS: calculated for C23H20N4O379

BrNa [MNa+] 502.0616; found

502.0604. HRMS: calculated for C23H20N4O381

BrNa [MNa+] 504.0596; found

504.0624. The purity of 43 was confirmed to be > 95 % using Analytical HPLC.

4.2.17.4 7-(Diethylamino)-3-(4-(1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (44)

The title compound was prepared from 41 (110 mg, 0.43 mmol) according to general

procedure 1 and purified by silica gel chromatography to afford 44 as a yellow solid

(122 mg, 60 % yield) (Rf = 0.20, EtOAc : n-hexanes, 1 : 1). M.p. 238 - 239 °C. IR

(ATR) νmax 809 (=C-H), 1131 (C-O), 1421 (N-O˙), 1430 and 1439 (aryl C-C), 1595

(C=C-COO), 1716 (C=O), 2929 and 2978 cm-1

(alkyl CH3). 1H NMR (400 MHz,

DMSO-d6): (ppm) = 8.96 (s, 1H, Htriazole), 8.55 (s, 1H, C=CH), 7.66 (s, 1H, Harom),

6.85 (s, 1H, Harom), 6.71 (s, 1H, Harom), 3.49 (s, 4H, 2×CH2), 1.16 (s, 6H, 2×CH3).

MS (ESI): m/z (%) = 473 (65) [MH+], 495 (100) [MNa

+]. HRMS: calculated for

C27H31N5O3 [MH+] 473.2421; found 473.2433. HRMS: calculated for C27H30N5O3Na

[MNa+] 495.2241; found 495.2255. The purity of 44 was confirmed to be > 95 %

using Analytical HPLC.

69

4.2.18 General procedure 2 - Fenton reactions

To a solution of nitroxide analogue (23 and 42 - 44) (1.0 equiv) in DMSO (3 mL)

was added FeSO4.7H2O (2.0 equiv.) and H2O2 (50 µL). The reaction was maintained

with stirring for 30 minutes at room temperature under an atmosphere of argon.

4.2.18.1 7-Hydroxy-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (45)

The title compound was prepared from 42 (24 mg, 0.06 mmol) according to general

procedure 2 and purified by silica gel chromatography to afford 45 as a yellow solid

(22 mg, 90 % yield) (Rf = 0.38, EtOAc : n-hexanes, 1 : 1). M.p. 270 - 272 °C. IR

(ATR) νmax 801 (=C-H), 1049 (N-O-C), 1170 (C-O), 1237 (O-H), 1445 and 1461

(aryl C-C), 1605 (C=C-COO), 1702 (C=O), 2930 and 2977 (alkyl CH3), 3150 cm-1

(O-H). 1H NMR (400 MHz, DMSO-d6): (ppm) = 9.01 (s, 1H, Htriazole), 8.66 (s, 1H,

C=CH), 7.85 (dd, J = 8.0, 1.6 Hz, 1H, Harom), 7.80 (m, 2H, 2×Harom), 7.31 (d, J = 8.0

Hz, 1H, Harom), 6.92 (dd, J = 8.4, 2.0 Hz, 1H, Harom), 6.87 (s, 1H, Harom), 3.73 (s, 3H,

OCH3), 1.42 (s, 12H, 4×CH3). 13

C NMR (400 MHz, DMSO-d6): δ (ppm) = 156.78,

155.24, 147.17, 146.01, 145.30, 137.02, 131.49, 129.82, 125.15, 122.65, 122.43,

70

119.59, 119.18, 114.89, 110.74, 102.69, 67.20, 67.07, 65.52. MS (ESI): m/z (%) =

433 (100) [MH+], 455 (53) [MNa

+]. HRMS: calculated for C24H25N4O4 [MH

+]

433.1870; found 433.1874. HRMS: calculated for C24H24N4O4Na [MNa+] 455.1695;

found 455.1690.

4.2.18.2 3-(4-(2-Methoxy-1,1,3,3-tetramethylisoindolin-5-yl)-1H-1,2,3-triazol-1-

yl)-2H-chromen-2-one (46)

The title compound was prepared from 23 (21 mg, 0.05 mmol) according to general

procedure 2 and purified by silica gel chromatography to afford 46 as a yellow solid

(18 mg, 82 % yield) (Rf = 0.57, EtOAc : n-hexanes, 1 : 1). M.p. 170 - 172 °C. IR

(ATR) νmax 810 (=C-H), 1047 (N-O-C), 1167 (C-O), 1464 and 1488 (aryl C-C), 1607

(C=C-COO), 1726 (C=O), 2930 and 2972 cm-1

(alkyl CH3). 1H NMR (400 MHz,

DMSO-d6): (ppm) =9.10 (s, 1H, Htriazole), 8.80 (s, 1H, C=CH), 7.97 (dd, J = 7.6, 0.8

Hz, 1H, Harom), 7.87 (dd, J = 8.0, 1.6 Hz, 1H, Harom), 7.82 (s, 1H, Harom), 7.75 (m, 1H,

Harom), 7.57 (d, J = 8.0 Hz, 1H, Harom), 7.48 (t, J = 7.2, 1H, Harom), 7.32 (d, J = 8.0

Hz, 1H, Harom), 3.73 (s, 3H, OCH3), 1.42 (s, 12H, 4×CH3). 13

C NMR (400 MHz,

DMSO-d6): δ (ppm) = 155.85, 152.49, 146.86, 145.61, 144.94, 134.99, 132.96,

129.57, 129.19, 125.37, 124.74, 123.14, 122.19, 121.89, 118.79, 118.23, 116.31,

66.72, 66.60, 65.04. MS (ESI): m/z (%) = 417 (70) [MH+]. HRMS: calculated for

C24H25N4O3 [MH+] 417.1921; found 417.1917.

71

4.2.18.3 6-Bromo-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (47)

The title compound was prepared from 43 (26 mg, 0.05 mmol) according to general

procedure 2 and purified by silica gel chromatography to afford 47 as a white solid

(21 mg, 78 % yield) (Rf = 0.65, EtOAc : n-hexanes, 1 : 1). M.p. 254 - 256 °C. IR

(ATR) νmax 664 (C-Br), 809 (=C-H), 1070 (N-O-C), 1167 (C-O), 1449 and 1469

(aryl C-C), 1709 (C=O), 2927 and 2978 cm-1

(alkyl CH3). 1H NMR (400 MHz,

DMSO-d6): (ppm) = 9.12 (s, 1H, Htriazole), 8.75 (s, 1H, C=CH), 8.23 (d, J = 2.4 Hz,

1H, Harom), 7.87 (m, 3H, 3×Harom), 7.56 (d, J = 8.8 Hz, 1H, Harom), 7.32 (d, J = 8.0

Hz, 1H, Harom), 3.74 (s, 3H, OCH3), 1.42 (s, 12H, 4×CH3). 13

C NMR (400 MHz,

DMSO-d6): δ (ppm) = 155.87, 151.98, 147.42, 146.10, 145.48, 135.58, 133.54,

131.85, 129.55, 125.24, 124.52, 122.68, 122.26, 120.74, 119.30, 119.05, 117.35,

67.20, 67.08, 65.52. MS (ESI): m/z (%) = 495/497 (100) [MH+]. HRMS: calculated

for C24H24N4O379

Br [MH+] 495.1032; found 495.1028. HRMS: calculated for

C24H24N4O381

Br [MH+] 497.1011; found 497.1009.

4.2.18.4 7-(Diethylamino)-3-(4-(2-methoxy-1,1,3,3-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48)

The title compound was prepared from 44 (20 mg, 0.04 mmol) according to general

procedure 2 and purified by silica gel chromatography to afford 48 as a yellow solid

(17 mg, 84 % yield) (Rf = 0.57, EtOAc : n-hexanes, 1 : 1). M.p. 228 - 229 °C. IR

72

(ATR) νmax 808 (=C-H), 1053 (N-O-C), 1191 (C-O), 1437 and 1458 (aryl C-C), 1606

(C=C-COO), 1729 (C=O), 2926 and 2983 cm-1

(alkyl CH3). 1H NMR (400 MHz,

CDCl3): (ppm) = 8.83 (s, 1H, Htriazole), 8.49 (s, 1H, C=CH), 7.79 (dd, J = 7.6, 1.2

Hz, 1H, Harom), 7.70 (s, 1H, Harom), 7.47 (d, J = 8.0 Hz, 1H, Harom), 7.20 (d, J = 8.0

Hz, 1H, Harom),6.71 (dd, J = 8.8, 2.4Hz), 6.60 (d, J = 2.0 Hz, 1H, Harom), 3.82 (s, 3H,

OCH3), 3.49 (q, J = 7.2 Hz, 4H, 2×CH2), 1.50 (m, 12H, 4×CH3), 1.28 (t, J = 7.2 Hz,

6H, 2×CH3). 13

C NMR (400 MHz, CDCl3): δ (ppm) = 157.01, 155.80, 151.57,

147.81, 145.97, 145.43, 134.46, 130.04, 129.55, 125.07, 121.98, 120.16, 119.09,

116.94, 110.11, 107.16, 97.03, 45.02, 12.44. MS (ESI): m/z (%) = 488 (10) [MH+],

510 (15) [MNa+]. HRMS: calculated for C28H34N5O3 [MH

+] 488.2656; found

488.2645. HRMS: calculated for C28H33N5O3Na [MNa+] 510.2476; found 510.2476.

4.3 X - Ray Crystallographic Analysis of 3-(4-(1,1,3,3-

Tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-

chromen-2-one (23)

Data were collected at 173(2) K under the software control of CrysAlis CCD [Oxford

Diffraction, CrysAlis CCD and CrysAlis RED. Oxford Diffraction Ltd, Abingdon,

Oxfordshire, England (2007).] on an Oxford Diffraction Gemini Ultra diffractometer

using Mo-Kα radiation generated from a sealed tube. Data reduction was performed

using CrysAlis RED. [Repeat - Oxford Diffraction, CrysAlis CCD and CrysAlis

RED. Oxford Diffraction Ltd, Abingdon, Oxfordshire, England (2007).] A multi-

scan empirical absorption correction was applied using spherical harmonics,

implemented in the SCALE3 ABSPACK scaling algorithm, within CrysAlis RED

[Repeat - Oxford Diffraction, CrysAlis CCD and CrysAlis RED. Oxford Diffraction

Ltd, Abingdon, Oxfordshire, England (2007).] and subsequent computations were

carried out using the WinGX-32 graphical user interface89

. The structure was solved

by direct methods using SIR9790

and refined with SHELXL-97.91

Full occupancy

non hydrogen atoms were refined with anisotropic thermal parameters. CH

hydrogen atoms were included in idealised positions and a riding model was used for

their refinement. The refinement residuals are defined as R1 = ||Fo| - |Fc||/|Fo| for

73

Fo > 2(Fo) and wR2 = {[w(Fo2 - Fc

2)2]/[w(Fc

2)2]}

1/2 where w = 1/[2

(Fo2) +

(0.25P)2 + 0P], P = (Fo

2 + 2Fc

2)/3.

Crystal data: 23; Formula C23H21N4O3, M = 401.44, triclinic, P1̄ , a = 5.8079(2) Å, b

= 12.9919(11) Å, c = 14.7368(9) Å, α = 112.916(7), = 99.112(4), = 95.001(5),

V = 997.66(13) Å3, Dc = 1.336 g cm

-3, Z = 2, crystal size 0.30 0.26 0.12 mm,

yellow prism, temperature 173(2) K, (Mo-K) = 0.71073, T(multi-scan)min,max =

0.998, 1, 2max = 57.52, hkl range -7 to 7, -14 to 17, -19 to 19, N = 6999, Nind = 4461

(Rmerge = 0.018), Nobs = 2942 (I > 2(I)), Nvar = 275, residuals R1(F, 2) = 0.036,

wR2(F2, all) = 0.062, GoF(all) = 1.032, min,max = -0.187, 0.224 e Å

-3.

4.4 Spectrophotometric Analysis of Profluorescent Nitroxides

4.4.1 Molar extinction coefficient measurements

Molar extinction coefficients were calculated as the gradient (m) of absorbance

measurements taken from five solutions of nitroxide (23 and 42 - 44) (Figure 4.1)

and methoxyamine (45 - 48) (Figure 4.2) solutions in THF.

7-Hydroxy nitroxide analogue (42); stock solution of 0.024 mM (1 mg/100 mL).

Diluted to give solutions of 5.3 µM, 4.0 µM, 3.0 µM, 2.0 µM, 1.1 µM. m = 1900

Unsubstituted nitroxide analogue (23); stock solution of 0.025 mM (1 mg/100 mL).

Diluted to give solutions of 6.5 µM, 5.0 µM, 3.5 µM, 2.5 µM, 1.0 µM. m = 17224

6-Bromo nitroxide analogue (43); stock solution of 0.021 mM (1 mg/100 mL).

Diluted to give solutions of 5.2 µM, 4.2 µM, 3.0 µM, 1.9 µM, 1.1 µM. m = 16861

7-Diethylamino nitroxide analogue (44); stock solution of 0.021 mM (1 mg/100 mL).

Diluted to give solutions of 5.3 µM, 4.2 µM, 3.0 µM, 1.9 µM, 1.1 µM. m = 17519

74

Figure 4.1. Absorbance of nitroxide analogues with increasing concentration

7-Hydroxy methoxyamine analogue (45); stock solution of 0.023 mM (1 mg/100

mL). Diluted to give solutions of 4.6 µM, 3.8 µM, 2.9 µM, 1.9 µM, 1.1 µM. m =

19972

Unsubstituted methoxyamine analogue (46); stock solution of 0.024 mM (1 mg/100

mL). Diluted to give solutions of 5.0 µM, 4.2 µM, 3.1 µM, 2.0 µM, 1.1 µM. m

=17400

6-Bromo nitroxide methoxyamine (47); stock solution of 0.020 mM (1 mg/100 mL).

Diluted to give solutions of 5.0 µM, 4.0 µM, 2.9 µM, 1.8 µM, 1.1 µM. m = 15969

7-Diethylamino methoxyamine analogue (48); stock solution of 0.020 mM (1

mg/100 mL). Diluted to give solutions of 5.1 µM, 3.7 µM, 2.6 µM, 1.5 µM, 0.8 µM.

m = 20057

Figure 4.2. Absorbance of methoxyamine analogues with increasing concentration

0

0.02

0.04

0.06

0.08

0.1

0.12

0 1 2 3 4 5 6 7

Ab

sorb

an

ce (

a.u

)

Concentration [mol/L (x 10-6)]

42

23

43

44

0

0.02

0.04

0.06

0.08

0.1

0.12

0 1 2 3 4 5 6

Ab

sorb

an

ce (

a.u

)

Concentration [mol/L (x 10-6)]

45

46

47

48

75

4.4.2 Quantum yield measurements

Quantum yield efficiencies of fluorescence for compounds 23 and 42 - 48 were

obtained from measurements at five different concentrations in THF using the

following equation:

ФF sample = ФF std × (Absstd/Abssample) × (Σ[Fsample]/ Σ[Fstd]) × (n2

sample/n2

std)

where Abs and F denote the absorbance and fluorescence intensity, respectively,

Σ[F] denotes the peak area of the fluorescence spectra (calculated by summation

of the fluorescence intensity) and n denotes the refractive index of the solvent.

Anthracene (ФF = 0.36 in cyclohexane) and perylene (ФF = 0.94 in cyclohexane)

were used as standards.

7-Hydroxy nitroxide analogue (42); stock solution of 0.024 mM (1 mg/100 mL).

Diluted to give solutions of 5.3 µM, 4.0 µM, 3.0 µM, 2.0 µM, 1.1 µM. ФF = 0.020

Unsubstituted nitroxide analogue (23); stock solution of 0.025 mM (1 mg/100 mL).

Diluted to give solutions of 6.5 µM, 5.0 µM, 3.5 µM, 2.5 µM, 1.0 µM. ФF = 0.001

6-Bromo nitroxide analogue (43); stock solution of 0.021 mM (1 mg/100 mL).

Diluted to give solutions of 5.2 µM, 4.2 µM, 3.0 µM, 1.9 µM, 1.1 µM. ФF = 0.005

7-Diethylamino nitroxide analogue (44); stock solution of 0.021 mM (1 mg/100 mL).

Diluted to give solutions of 5.3 µM, 4.2 µM, 3.0 µM, 1.9 µM, 1.1 µM. ФF = 0.200

Figure 4.3. Integrated fluorescence intensities of anthracene and nitroxide analogues 23, 42 and 43

with increasing absorbance

0

5000

10000

15000

20000

0 0.02 0.04 0.06 0.08 0.1 0.12Inte

gra

ted

Flu

ore

scen

ce

Inte

nsi

ty (

a.u

)

Absorbance (a.u)

Anthracene

42

23

43

76

Figure 4.4. Integrated fluorescence intensities of perylene and nitroxide analogue 44 with increasing

absorbance

7-Hydroxy methoxyamine analogue (45); stock solution of 0.023 mM (1 mg/100

mL). Diluted to give solutions of 4.6 µM, 3.8 µM, 2.9 µM, 1.9 µM, 1.1 µM. ФF =

0.550

Unsubstituted methoxyamine analogue (46); stock solution of 0.024 mM (1 mg/100

mL). Diluted to give solutions of 5.0 µM, 4.2 µM, 3.1 µM, 2.0 µM, 1.1 µM. ФF =

0.010

6-Bromo nitroxide methoxyamine (47); stock solution of 0.020 mM (1 mg/100 mL).

Diluted to give solutions of 5.0 µM, 4.0 µM, 2.9 µM, 1.8 µM, 1.1 µM. ФF = 0.015

7-Diethylamino methoxyamine analogue (48); stock solution of 0.020 mM (1

mg/100 mL). Diluted to give solutions of 5.1 µM, 3.7 µM, 2.6 µM, 1.5 µM, 0.8 µM.

ФF = 0.950

Figure 4.5. Integrated fluorescence intensities of anthracene and methoxyamine analogues 45 - 47

with increasing absorbance

0

10000

20000

30000

40000

50000

0 0.02 0.04 0.06 0.08 0.1

Inte

gra

ted

Flu

ore

scen

ce

Inte

nsi

ty (

a,u

)

Absorbance (a.u)

Perylene

44

0

5000

10000

15000

20000

25000

30000

35000

0 0.02 0.04 0.06 0.08 0.1 0.12

Inte

gra

ted

Flu

ore

scen

ce

Inte

nsi

ty (

a.u

)

Absorbance (a.u)

Anthracene

45

46

47

77

Figure 4.6. Integrated fluorescence intensities of perylene and methoxyamine analogue 48 with

increasing absorbance

4.4.3 Absorbance and emission measurements of 7-hydroxy analogues (42 and

45) with pH

Solutions of 7-hydroxy analogues (42 and 45) from pH 1 - 14 were prepared by

adding 300 µL of 7-hydroxy analogues (42 and 45) in THF to 2.7 mL of solutions

corresponding to pH 1 - 14.

Neutralization of solutions corresponding to pH 9 - 14 was achieved by combining

solutions pH 1 and pH 14, pH 2 and pH 13, pH 3 and pH 12, pH 4 and pH 11, pH 5

and pH 10, pH 6 and pH 9. This approach ensured a consistency in concentration of

the 7-hydroxy analogues.

7-Hydroxy nitroxide analogue (42); stock solution of 0.024 mM (1 mg/100 mL).

7-Hydroxy methoxyamine analogue (45); stock solution of 0.023 mM (1 mg/100

mL).

10 M HCl was diluted 100-fold to give a solution of pH 1. An aliquot of the solution

corresponding to pH 1 was diluted 10-fold to give a solution of pH 2 followed by

successive dilutions to pH 6. Deionized water was used for the solution

corresponding to pH 7. 10 M NaOH was diluted 100-fold to give a solution of pH 14.

An aliquot of the solution corresponding to pH 14 was diluted 10-fold to give a

solution of pH 13 followed by successive dilutions to pH 8.

0

10000

20000

30000

40000

50000

60000

0 0.02 0.04 0.06 0.08 0.1 0.12

Inte

gra

ted

Flu

ore

scen

ce

Inte

nsi

ty (

a.u

)

Absorbance (a.u)

Perylene

48

78

Appendices

Appendix 1. 1H NMR spectrum of N-benzylphthalimide (25) .................................. 80

Appendix 2. 1H NMR spectrum of 2-benzyl-1,1,3,3-tetramethylisoindoline (26)..... 80

Appendix 3. 1H NMR spectrum of 5-bromo-1,1,3,3-tetramethylisoindoline (27) ..... 81

Appendix 4. 1HNMR spectrum of 5-iodo-1,1,3,3-tetramethylisoindoline (28) ......... 81

Appendix 5. 1H NMR spectrum of 3-acetamido-7-acetoxy-2H-chromen-2-one

(35) ............................................................................................................... 82

Appendix 6. 1H NMR spectrum of 3-acetamido-2H-chromen-2-one (36) ................ 82

Appendix 7. 1H NMR spectrum of 3-acetamido-6-bromo-2H-chromen-2-one (37) . 83

Appendix 8. 1H NMR spectrum of 3-nitro-7-diethylamino-2H-chromen-2-one

(58) ............................................................................................................... 83

Appendix 9. 1H NMR spectrum of 3-amino-7-diethylamino-2H-chromen-2-one

(59) ................................................................................................................... 84

Appendix 10. 1H NMR spectrum of 3-azido-7-hydroxy-2H-chromen-2-one (39) ... 84

Appendix 11. 1H NMR spectrum of 3-azido-2H-chromen-2-one (22) ..................... 85

Appendix 12. 1H NMR spectrum of 3-azido-6-bromo-2H-chromen-2-one (40) ..... 85

Appendix 13. 1H NMR spectrum of 3-azido-7-diethylamino-2H-chromen-2-one

(41) ............................................................................................................ 86

Appendix 14. 1H NMR spectrum of 7-hydroxy-3-(4-(1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (42) ........... 86

Appendix 15. HPLC trace of 7-hydroxy-3-(4-(1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (42) ................................................. 87

Appendix 16. 1H NMR spectrum of 3-(4-(1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (23) ................................................. 87

Appendix 17. HPLC trace of 3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (23) ..................................................................... 88

Appendix 18. 1H NMR spectrum of 6-bromo-3-(4-(1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (43) ........... 88

Appendix 19. HPLC trace of 6-bromo-3-(4-(1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (43) ................................................. 89

Appendix 20. 1H NMR spectrum of 7-(diethylamino)-3-(4-(1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (44) ........... 89

Appendix 21. HPLC trace of 7-(diethylamino)-3-(4-(1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (44) ........... 90

Appendix 22. 1H NMR spectrum of 7-hydroxy-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (45) ........... 90

Appendix 23. 13

C NMR spectrum of 7-hydroxy-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (45) ........... 91

Appendix 24. 1H NMR spectrum of 3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (46) ........... 91

Appendix 25. 13

C NMR spectrum of 3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (46) ........... 92

79

Appendix 26. 1H NMR spectrum of 6-bromo-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (47) .......... 92

Appendix 27. 13

C NMR spectrum of 6-bromo-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (47) .......... 93

Appendix 28. 1H NMR spectrum of 7-(diethylamino)-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48) .......... 93

Appendix 29. 13

C NMR spectrum of 7-(diethylamino)-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48) .......... 94

80

Appendix 1. 1H NMR spectrum of N-benzylphthalimide (25)

Appendix 2. 1H NMR spectrum of 2-benzyl-1,1,3,3-tetramethylisoindoline (26)

81

Appendix 3. 1H NMR spectrum of 5-bromo-1,1,3,3-tetramethylisoindoline (27)

Appendix 4. 1HNMR spectrum of 5-iodo-1,1,3,3-tetramethylisoindoline (28)

82

Appendix 5. 1H NMR spectrum of 3-acetamido-7-acetoxy-2H-chromen-2-one (35)

Appendix 6. 1H NMR spectrum of 3-acetamido-2H-chromen-2-one (36)

83

Appendix 7. 1H NMR spectrum of 3-acetamido-6-bromo-2H-chromen-2-one (37)

Appendix 8. 1H NMR spectrum of 3-nitro-7-diethylamino-2H-chromen-2-one (58)

84

Appendix 9. 1H NMR spectrum of 3-amino-7-diethylamino-2H-chromen-2-one (59)

Appendix 10. 1H NMR spectrum of 3-azido-7-hydroxy-2H-chromen-2-one (39)

85

Appendix 11. 1H NMR spectrum of 3-azido-2H-chromen-2-one (22)

Appendix 12. 1H NMR spectrum of 3-azido-6-bromo-2H-chromen-2-one (40)

86

Appendix 13. 1H NMR spectrum of 3-azido-7-diethylamino-2H-chromen-2-one (41)

Appendix 14. 1H NMR spectrum of 7-hydroxy-3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (42)

87

Appendix 15. HPLC trace of 7-hydroxy-3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (42)

Appendix 16. 1H NMR spectrum of 3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (23)

88

Appendix 17. HPLC trace of 3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-

2H-chromen-2-one (23)

Appendix 18. 1H NMR spectrum of 6-bromo-3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (43)

89

Appendix 19. HPLC trace of 6-bromo-3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-1,2,3-

triazol-1-yl)-2H-chromen-2-one (43)

Appendix 20. 1H NMR spectrum of 7-(diethylamino)-3-(4-(1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (44)

90

Appendix 21. HPLC trace of 7-(diethylamino)-3-(4-(1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (44)

Appendix 22. 1H NMR spectrum of 7-hydroxy-3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (45)

91

Appendix 23. 13

C NMR spectrum of 7-hydroxy-3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (45)

Appendix 24. 1H NMR spectrum of 3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-yloxyl)-1H-

1,2,3-triazol-1-yl)-2H-chromen-2-one (46)

92

Appendix 25. 13

C NMR spectrum of 3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-yloxyl)-

1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (46)

Appendix 26. 1H NMR spectrum of 6-bromo-3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (47)

93

Appendix 27. 13

C NMR spectrum of 6-bromo-3-(4-(2-methoxy-1,1,3,3,-tetramethylisoindolin-2-

yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (47)

Appendix 28. 1H NMR spectrum of 7-(diethylamino)-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48)

94

Appendix 29. 13

C NMR spectrum of 7-(diethylamino)-3-(4-(2-methoxy-1,1,3,3,-

tetramethylisoindolin-2-yloxyl)-1H-1,2,3-triazol-1-yl)-2H-chromen-2-one (48)

95

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