chemical tools for bioconjugation : application of the

Chemical tools for bioconjugation:

Application of the thioacid-azide ligation

Inaugural-Dissertation

to obtain the academic degree

Doctor rerum naturalium (Dr. rer. nat.)

submitted by

Jamsad Mannuthodikayil

at the

The faculty of Sciences

Department of Chemistry

Date of the oral Examination: 23.01.2015

1. Referees: Professor Dr. Valentin Wittmann

2. Referees: Professor Dr. Andreas Marx

3. Referees: Professor Dr. Helmut Cölfen

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-293319

http://nbn-resolving.de/urn:nbn:de:bsz:352-0-293319

Page II

ACKNOWLEDGEMENT

I would like to express my special appreciation and thanks to my advisor Professor Dr. Valentin

Wittmann, you have been a tremendous and caring mentor for me. I would like to thank you for

encouraging my research and for allowing me to express myself in this stimulating research project.

Your advice on both inside and outside research have been priceless. I would also like to thank

Professor Dr. Andreas Marx for being the member of my graduate school thesis committee, second

referee of my thesis and for being the oral examiner. I am also thankful to Prof. Dr. Martin

Scheffner for being part of my graduate school thesis committee. I would like to express the deepest

appreciation to Prof. Dr. Helmut Cölfen for agreeing to chair the oral examination.

For the help provided in the NMR experiments, I’m thankful to Žarko Kulić, Ulrich Haunz and

Anke Friemel. I am also thankful to Dr. Inigo Göttker-Schnetmann and Dr. Peter Schmitt for their

tremendous helps on the X-ray crystallography.

I am very thankful to my Bachelor’s students Theresa Renz and Sören Radke for their hard works

on the projects. I would also like to thank my mitarbeiter students, Angelina Schwarz, Eugenia

Hoffmann, Patrick Höring, Nicolai Wagner, Patrick Anders Jennifer Knaus and Marco Frensch for

their marvelous contributions to the project. I’m also thankful to my project partner Dr. Odin Keiper

for his contributions and helps with the project.

I would like to express the deepest appreciation and gratitude to former and present members of

AG Wittmann, for their tireless efforts in fulfilling many of my everyday needs, amazing moments

in lab, and most of all for providing an excellent working atmosphere, I truly felt here home. Many

thanks goes to Daniel Wieland, for being a wonderful lab partner, putting a tremendous effort on

correcting the manuscript, having very fruitful discussions on research topics and all other helps. I

also would like to thank Dr. Meera Mohanan, Dr. Vinod Kumar, Ivan Zemskov and Oliver

Baudendistel for correcting the manuscript in a timely manner. A special thanks to Oliver

Baudendistel for writing the translation of summary. I would like to thank my big sisters of

Konstanz, Dr. Andrea Niederwieser, Ellen Batroff and Monica Boldt for their caring, affection and

all other helps. A very special thanks to Dr. Martin Dauner for giving comfortable time, assistance

for easing my initial life in Konstanz and for all other helps thereafter. I also would like to thank

Torben Seitz and Carina Immler for a memorable outdoor and night life in Konstanz.

I would like also to thank my badminton club members in Konstanz, Schlägertrupp, for giving

enjoyable moment in Konstanz. I also would like to thank members of Welcome Center, especially

to Johannes Dingler and Heike Kühmoser, for their marvelous assistance on the language

translations and helps with local bureaucracy, they are truly angels of the university. I also like to

thank international community here in Konstanz, organizing nice events and for a fantastic time.

I would further like to thank Prof. Dr. Heiko Möller, Prof. Dr. Richard R. Schmidt, Dr. Alexander

Titz, Dr. Thomas Huhn and Angelika Früh for their valuable assistance, suggestions, discussions

and the directions. I am also thankful to AG Muller, AG Titz, AG Groth and members of graduate

school chemical biology for providing a wonderful and fruitful time at here in university.

Page III

Finally I would like to thank my family and friends. Words cannot express how grateful I am to

my mother, brothers and my late father, for all of the sacrifices that you’ve made on my behalf and

supported me the entire way.

Page IV

I dedicate this work to honor Dr. Richard Dawkins and Dr. Neil deGrasse Tyson, for their marvelous

effort on building the positive public opinions towards the importance of science educations.

Page V

CONTENTS

Acknowledgement ......................................................................................................................................... II

Abbreviations ............................................................................................................................................... IX

INTRODUCTION ..................................................................................................................................... 1

General introduction ..................................................................................................................... 1

Protein conjugations and bio-orthogonal chemistry .................................................................... 4

Ketone-hydrazide/alkoxyamine condensation ...................................................................... 5

Azide-phosphine reactions ("traceless" Staudinger ligations) .............................................. 6

Azide-alkyne reactions ([3 + 2] cycloadditions, click chemistry) ........................................... 7

Strain promoted alkene/alkyne-tetrazine reactions (DAinv). ............................................... 8

Flagging the proteins ................................................................................................................... 10

Chemistry of thiocarboxylic acids ................................................................................................ 13

Thioacid azide amidation/ligation (TAL) ...................................................................................... 16

Applications of thioacid azide ligation................................................................................. 17

AIM OF THE WORK .............................................................................................................................. 22

RESULTS AND DISCUSSION .................................................................................................................. 25

Protecting groups for thiocarboxylic S-acids ............................................................................... 25

Thiocarboxylic S-esters ........................................................................................................ 25

Thiocarboxylic O-esters ....................................................................................................... 28

Safety catch approach ......................................................................................................... 38

Thioacid Azide Ligation (TAL) chemistry ...................................................................................... 44

TAL reaction with sulfonyl azides. ....................................................................................... 44

Generation of natural N-glyosidic bond using TAL chemistry ............................................. 61

TAL chemistry in presence of free sulfhydryl moiety .................................................................. 68

Influence of bases on TAL chemistry ................................................................................... 69

Further study of lissamine azide reduction in the presence of hydrogen sulfide ............... 81

SUMMARY AND OUTLOOK .................................................................................................................. 85

Summary...................................................................................................................................... 85

Outlook ........................................................................................................................................ 88

ZUSAMMENFASSUNG UND AUSBLICK ................................................................................................ 90

EXPERIMENTAL SECTION ..................................................................................................................... 92

General methods ......................................................................................................................... 92

Analytics ...................................................................................................................................... 92

Page VI

NMR Spectroscopy .............................................................................................................. 92

Mass spectrometry: ............................................................................................................. 92

CHN analysis ........................................................................................................................ 92

Analytical and preparative high performance liquid chromatography (RP-HPLC) .............. 93

Liquid chromatography mass spectrometry (LC-MS) .......................................................... 93

Preparative medium performance liquid chromatography (MPLC).................................... 93

General procedures ..................................................................................................................... 93

GP01: DCC coupling ............................................................................................................. 93

GP02: EDAC/DMAP(HOBt) coupling .................................................................................... 93

GP03: HBTU/HOBt coupling. ............................................................................................... 94

GP04: TAL with electron deficient azides ............................................................................ 94

GP05: TAL with electron rich azides .................................................................................... 94

GP06: Selective trityl thioester deprotection ...................................................................... 94

GP07 Boc and/or OtBu deprotection ................................................................................... 95

GP08: Fmoc deprotection .................................................................................................... 95

GP09: Boc, Ot-Bu, STrt, STmob global deprotection by using TFA ...................................... 95

GP10: Benzyl ester/-NHCbz de-protection. ......................................................................... 95

GP11: Acetylation of alcohols .............................................................................................. 95

Synthesis of target compounds ................................................................................................... 96

S-trityl 3-phenylpropanethioate (76) .................................................................................. 96

3-phenylpropanethioic S-acid (77) ...................................................................................... 96

S-(2-methoxypropan-2-yl) 3-phenylpropanethioate (81) ................................................... 97

4-dimethylpentan-3-yl 3-phenylpropanoate (88)106 ........................................................... 97

O-ethyl 3-phenylpropanethioate (91) 108 ............................................................................ 98

O-isopropyl 3-phenylpropanethioate (92) .......................................................................... 98

O-(2,4-dimethylpentan-3-yl) 3-phenylpropanethioate (93) ................................................ 98

N', 3-diphenylpropanehydrazide (96).................................................................................. 99

N', 3-diphenylpropanethiohydrazide (97) ........................................................................... 99

3,4-diaminobenzamide (104) 120........................................................................................ 100

Fmoc-Asn(Dbz)-Ot-Bu (107) .............................................................................................. 100

Fmoc-Val-Ala-Ot-Bu (110) ................................................................................................. 101

Fmoc-Asn(daba)-Val-Ala-Ot-Bu (112) ................................................................................ 102

Lissamine Rhodamine B derivatives (113-115) ................................................................. 102

Dansyl azide (116)129 .......................................................................................................... 104

Page VII

Boc-4-((2-aminoethyl)carbamoyl)benzenesulfonyl azide (pCSea) (119) ........................... 104

Biotin sulfonyl azide (121) ................................................................................................. 105

Fmoc-piperazyl biotin (123)............................................................................................... 106

Biotin sulfonyl azide (124) ................................................................................................. 106

Fmoc-Ala-Phe-Ot-Bu131 (130) ............................................................................................. 107

Fmoc-Gly-Phe-Ot-Bu132 (131) ............................................................................................ 108

Boc-Asp(OBn)-OH157 (139) ................................................................................................. 108

Boc-Asp(OBn)-Val-Phe-Ot-Bu (141) ................................................................................... 108

Boc-Asp(OBn)-Ala-Phe-Ot-Bu (142) ................................................................................... 109

Boc-Asp(OBn)-Gly-Phe-Ot-Bu (143) ................................................................................... 110

Boc-Asp(OBn)-Val-Ot-Bu (144) .......................................................................................... 110

Fmoc-Asp(OBn)-Val-Ot-Bu (145) ....................................................................................... 111

Boc-Asp(OBn)-Ala-Ot-Bu (146) .......................................................................................... 112

Boc-Asp(OBn)-Phe-Ot-Bu (147) ......................................................................................... 112

Boc-Asp(OBn)-Tyr(Ot-Bu)-Ot-Bu (148) .............................................................................. 113

Boc-Asp(STrt)-Ot-Bu 29 (159) ............................................................................................ 114

Cbz-Asp(STrt)-Obn (160) .................................................................................................... 114

Fmoc-Asp(STrt)-Val-Ot-Bu (161) ........................................................................................ 115

Boc-Asp(STrt)-Val-Ot-Bu (162)........................................................................................... 115

Boc-Asp(STrt)-Ala-Ot-Bu (163) .......................................................................................... 116

Boc-Asp(STrt)-Phe-Ot-Bu (164) ......................................................................................... 116

Boc-Asp(STrt)-Tyr(Ot-Bu)-Ot-Bu (165) ............................................................................... 117

Boc-Asp(STrt)-Val-Phe-Ot-Bu (166) ................................................................................... 118

Boc-Asp(STrt)-Ala-Phe-Ot-Bu (167) ................................................................................... 118

Boc-Asp(STrt)-Gly-Phe-Ot-Bu (168) ................................................................................... 119

Cbz-Asp(SH)-OBn (169) ...................................................................................................... 120

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu (170) ................................................................................. 120

Boc-Asp(SH)-Val-Phe-Ot-Bu (171) ..................................................................................... 121

TFA*NH2-Asp(SH)-Tyr(OH)-OH (172) ................................................................................. 121

Cbz-Asn(Liss)-OBn (173) .................................................................................................... 122

Boc-Asn(Liss)-Tyr(Ot-Bu)-Ot-Bu (174) ............................................................................... 122

Boc-Asn(pCSea)-Tyr(Ot-Bu)-Ot-Bu (175) ........................................................................... 123

Boc-Asn(Dn)-Tyr(Ot-Bu)- Ot-Bu (176) ................................................................................ 124

Boc-Asn(biotin1)-Val-Phe-Ot-Bu (177) .............................................................................. 125

Page VIII

Boc-Asn(biotin1)-Tyr(Ot-Bu)-Ot-Bu (not described) ......................................................... 126

Boc-Asn(Dn)-Ot-Bu (178) ................................................................................................... 126

Boc-Asn(Dn)-Val-Ot-Bu (179)............................................................................................. 127

Boc-Asn(Liss)-Val-Ot-Bu (180) ........................................................................................... 128

Boc-Asn(Liss)-Gly-Phe-Ot-Bu (181) .................................................................................... 129

Boc-Asn(biotin2)-Gly-Phe-Ot-Bu (182) .............................................................................. 129

Boc-Asn(biotin2)-Val-Ot-Bu (183) ...................................................................................... 130

Fmoc-Asn(neo-Glc)-Val-Ot-Bu (184) .................................................................................. 131

NH2-Asn(Liss)-Tyr(OH)-OH (185) ........................................................................................ 132

TFA.NH2-Asn(Dn)-Ala-OH (186) ......................................................................................... 133

TFA.NH2-Tyr(OH)-Ala-Asn(Dn)-Ser(OH)-Val-OH (188) ....................................................... 134

2-acetamido-2-deoxy-β-D-glucopyranosyl azide138 (190) ................................................. 134

2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide140 (191) .................... 135

2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide139 (192) .................... 135

N-acetylglucosamine-β-phenylpropanamide (193)........................................................... 136

Phenylpropanamido-GlcNAc(OAc)3 (194).......................................................................... 136

Phenylpropanamido-GlcNAc(OBn)3 (195) ......................................................................... 137

Boc-Asn(GlcNAc(OBn)3)-Ot-Bu (196) ................................................................................. 138

Boc-Asn(GlcNAc(OAc)3)-Ot-Bu159 (197) ............................................................................. 138

Boc-Asn(GlcNAc)-Val-Ot-Bu (198) ..................................................................................... 139

Boc-Asi-Val-Phe-Ot-Bu (199) ............................................................................................. 140

Fmoc-Cys(SH)-OMe145 (207) .............................................................................................. 140

Lissamine rhodamine B 5-sulfonnamide (209) .................................................................. 141

Oxidation products of thioacid Cbz-Asp(SH)-OBn (210-212) ............................................ 141

X-Ray crystallography ................................................................................................................ 143

Influence of bases in TAL+C chemistry (Table 7 experiments) .................................................. 143

Influence of bases in TAL-C (TAL) chemistry (Table 8 experiments) ......................................... 147

REFERENCE ........................................................................................................................................ 150

APPENDIX .......................................................................................................................................... 160

Selected spectrum ..................................................................................................................... 160

Page IX

ABBREVIATIONS

Ac acetyl

Ac2O acetic anhydride

Ala alanine

Asi aspartimide

Asn asparagine

Asp aspartic acid

Bn benzyl

Boc tert-butyloxycarbonyl

Cbz benzyloxycarbonyl

CDCl3 deuterated chloroform

CH3CN/MeCN acetonitrile

Cys cysteine

Daba 3,4-diaminobenzamide

DABCO 1,4-diazabicyclo[2.2.2]octane

DBU 1,8-diazabicyclo-[5.4.0]-undec-7-en

DBN 1,5-Diazabicyclo[4.3.0]non-5-en

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DIPEA N-ethyldiisopropylamine

DMAP 4-dimethylaminopyridine

DMF dimethylformamide

DMSO dimethylsulfoxide

DMSO-d6 hexadeuterodimethyl sulfoxide

Dn dansyl

D2O Deuterium oxide

DTBP 2,6-Di-t-butylpyridine

EA elemental analysis

EDC/EDAC N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride

Equiv equivalent

ESI-MS electrospray ionization-mass spectrometry

Et ethyl

EtOAc ethyl acetate

EtOH ethanol

Fmoc 9-fluorneylmethoxycarbonyl

GlcNAc N-Acetylglucosamine

Gly glycine

HATU N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate,

HOBt 1-hydroxybenzotriazole

Page X

HPLC high-performance-liquid-chromatography

LC-MS liquid chromatography-mass spectrometry

Liss lissamine

M molarity

m/z mass to charge ratio

Me methyl

MeOH methanol

MeOH-d4 tetradeuteromethanol

Mpe 3-Methyl-pentyl

NMR nuclear magnetic resonance spectroscopy

pCSea Boc-4-((2-aminoethyl)carbamoyl)benzenesulfonyl

PEG poly(ethylene glycol)

PG/pg protecting group

Ph phenyl

pH potentia hydrogenii (pH value)

Phe phenylalanine

ppm parts per million

RT/rt room temperature

s singlet

SPPS solid phase peptide synthesis

t time

t triplet

TAL thioacid-azide ligation

tert-Bu tertiary butyl

TFA trifluoroacetic acid

THF tetrahydrofuran

TIS triisopropylsilane

TLC thin layer chromotagrophy

Tmob S-2,4,6-trimethoxybenzyl

TMS tetramethylsilane

TMU 1,1,3,3-tetramethylurea

Trt trityl

Tyr tyrosine

UV ultraviolet

Val valine

w.r.t with respect to

Z benzyloxycarbonyl (Cbz)

INTRODUCTION

GENERAL INTRODUCTION

It is indisputable that the research interests in both biology and medicine had a quantum leap after

the arrival of the genomics in 1970. Interestingly the word genome is believed to be used by Hans

Winkler first, much long ago in 1920. He used the word genome in the context of haploid

chromosome set.1 However, the word genomics was coined first by Dr. Thomas H. Roderick, much

later in 1986, as a catchword for a yet-to-be-published journal, which is now known as Genomics.2

As we know now the term “genome” refers to the complete genetic composition of an organism,

which includes both the genes and the non-coding sequences of the deoxyribonucleic acid (DNA)

as well as the ribonucleic acid (RNA).

The so called “genomic era” has begun with the development of Maxam-Gilbert3 and Sanger4

method for sequencing the DNA. Short time later, we witnessed a big boom in the field of genomics

that started with the whole sequencing of the DNA of bacteriophage Φ-X174 (1977),5 followed by

numerous sequencing projects of eukaryotic, archaeal and prokaryotic genomes, including the

human6 genome which was made available in 2003.

Figure 1. Representation of proteome complexity: The human genome comprises approximately 20000

genes, however the proteome is estimated to encompass over a million proteins.

In spite of that, the genome sequencing does not directly define the complex architecture or the

function of an organism, but it merely represents a starting point to it. Figure 1 represents how the

human genome builds an enormous amount of distinct proteins from just around 20000 genes that

it possesses. The challenge is to unearth how the coexpression of thousands of genes can best be

examined under biological conditions, and how these patterns of expression define an organism.

The expression of genes can be analyzed by the study of proteins present in a cell or a tissue. To

define the protein-based gene expression analysis, the concept and the term, 'proteome' was coined

by Wilkins, et al.7 (1995). They defined a proteome as “the entire protein complement expressed

by a genome, or by a cell or by a tissue type”.7 Today the term proteomics contributes not only to

the systematic cataloguing, separation and study of all of the proteins produced in an organism, but

also to the study of how proteins change their structure, interact with other proteins, and ultimately,

give rise to disease or health in an organism. It is fascinating to note that, although there is only a

single unique genome of an organism, the proteome of this organism can change under different

conditions and can be dissimilar even in different tissues of that organism. In 2001, when the

Human Genome Project (HGP) completed the first maps of the human genome, Vicki Brower, a

freelance science writer, wrote in an article that “No sooner was the human genome decoded than

we found ourselves in the ‘post-genomic era’—where the name of the game is proteomics”.8

Proteins can exhibit a range of biochemical properties that are crucially dependent on the precise

spatial structure of the folded polypeptides. This erratic complexity of the proteome makes it hard

to study the structures, functions and regulations of proteins in general. Low abundance proteins

often are of utmost importance, however, it remains difficult to detect, identify and characterize

numerous proteins at their natural cellular concentrations. Very often it can be found that the

concentration of those proteins are as much as 8 orders of magnitude lower than those of high-

abundance proteins present in the same sample.9 Dissimilar to the RNA or DNA, we procured no

methods analogous to polymerase chain reaction (PCR) for amplifying the proteins, therefore, the

amount of protein present in a provided sample is the amount of protein that must be analyzed.

Additionally, in contrast to RNA or DNA, the proteins do not inherently possess well defined high-

affinity or selectivity binding partners to capitalize high-throughput methods such as microarray

techniques. This makes it difficult, laborious and expensive to devise a specific capture reagent for

each protein of interest.

Researchers are currently relying on two types of methodologies to observe the gene expression on

a large scale.7 The first one is a nucleic acid based methodology in which a probe (a DNA sequence

distinctive for that gene) that binds to its complementary mRNA sequence is exploited. However,

mRNA measurements cannot substitute protein measurements, as proteins are regulated in a variety

of ways (not all involve change in mRNA level) such as post-translational modifications,

degradation and translational regulation. A favorable alternative is the protein-based technology,

which analyzes the expression of genes by the study of proteins present in a cell/tissue. Some of

the important and relevant methods for the study of proteomics include affinity chromatography,

high-performance liquid chromatography (HPLC), mass spectrometry, sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis, and all

of these methods often are coupled with a variety of chemical tools.

To facilitate protein detection or purification, the proteomic research often requires molecular

labels that are covalently attached to the protein of interest. While multiple types of labels are

available, such as biotin, reporter enzymes, fluorophores and radioactive isotopes, their

applications are varied according to specific demands. The traditional protein labeling strategy, for

example the cysteine-maleimide conjugation, lacks its applicability for target and site-specific

protein conjugations. The most commonly used genetic method, fusing the green fluorescent

protein (GFP) in to the target, carries an extra 238 amino acids and limits detection sensitivity due

to gloomy fluorescence and photo-bleaching of the GFP. A relatively new tool recently emerged,

which is based on novel and site-specific chemistry by using modified or unnatural amino acids in

combination with chemoselective reactions. Though precise chemical modification of proteins is a

daunting challenge, this requires not only chemoselective reactions but reactions, which perform

efficiently in buffered aqueous media at or near room temperature.

PROTEIN CONJUGATIONS AND BIO-ORTHOGONAL CHEMISTRY

As the genetic tools alone cannot provide all the answers to define an organism or understand its

biomolecular dynamics and functions, scientists are keen on developing new tools that can go

beyond genetic tools. As a result, we have unearthed methods for labeling biomolecules

specifically and selectively in their native habitats. Reactions that can be carried out under

physiological conditions without interference of the biological matrix were termed as

bioorthogonal. This has then greatly improved our perception of biomolecular functions and

properties. This led to the emergence of the so called bioconjugation chemistry, which represents

the strategies to form a stable covalent linkage between two molecules, of which, at least one must

be a biomolecule. Profound studies in bioconjugation chemistry offered techniques for mild and

site-specific derivatization not only for proteins, but also for other biomolecules such as DNA,

RNA, and carbohydrates. On top, its applicability enormously helped researchers to confront the

practical challenges faced in the field of ligand discovery, disease diagnosis as well as high-

throughput screening.10, 11-13

Excellent selectivity and specificity in the protein conjugations, which is essential in the covalent

approach, typically has been achieved in a two-step manner. In the first step, a unique reporter

functionality is incorporated into a targeted biomolecule by using either genetic, chemical or

metabolic pathway manipulations. When the reporter is incorporated, an external chemical probe

can be reacted to it in a selective and a specific manner (Figure 2).10, 11

Figure 2. A representation of chemoselective labeling of biomolecules

It is fair to say that the development of bioorthogonal reactions is extremely challenging. Not many

bioorthogonal reactions are known to the date that permit covalent bond formations between the

bioorthogonal reporter of the proteins and the small-molecule probe of interest, in a satisfactory

manner. Success of these bioorthogonal reactions requires a selective covalent reaction between an

incorporated chemical functionality and the reactive probe under biological conditions. Also the

probe must not involve in any cross reactions with the functional groups occurring in biological

systems. In addition, the reactants must be kinetically, metabolically and thermodynamically stable

until the demanded reaction is completed. Besides, any of the reagents or the generated products

should preferably be nontoxic to living systems.10, 11 In spite of these challenges, researchers were

able to come across a number of chemoselective reactions that show good biocompatibility and

selectivity in different contexts.10, 11, 12

The reporter moiety placed in the target protein can be varied according to specific demands and

its influences on the system. However similar to reporter moiety for proteins, there are several

chemical functionalities known to possess the demanded qualities for biocompatible and selective

reactions. The most commonly used protein tagging approaches include chemical reporters such as

azides,14, 15, 16 ketones,17, 18, 19 alkenes 20-22 and alkynes16, 21, 23. These chemical reporters can be

selectively modified with the molecule of interest bearing functionalities such as alkynes,15

phosphines,14 hydrazines,18 tetrazines16, 20-22 or azides16, 23 (Table 1). A variety of chemical

reactions were used to react these reporter functionalities with the probes. Among these well-

known reactions such as ketone-hydrazide/alkoxyamine condensation,24 the (traceless) Staudinger

ligation of azides and phosphines25, 26, [3+2] cycloadditions of azides and alkynes27-31 and Diels-

Alder cycloadditions with inverse electron demand reactions of alkene/alkyne and tetrazines32, 33

(DAinv) are included. As there are excellent reviews10, 12 available on the topic of the bioorthogonal

reactions, including a very recent one from Lang and Chin,11 only a very brief description about

the bioorthogonal chemistry is included in this thesis.

Ketone-hydrazide/alkoxyamine condensation

Ketones and aldehydes can react with hydrazide and alkoxyamine to form hydrazone and oxime

linkages respectively (Table 1, entry 1). They are one of the first functionalities to be explored as

bioorthogonal chemical reporters. Aldehydes can react with functionalities in proteins, whereas

ketones are less reactive towards the functional groups found in proteins. Uncomplicated synthetic

accessibility to ketone derivatives and their small size allows a relatively easy incorporation into

proteins. Using expanded genetic codes, ketones can be incorporated in the form of unnatural

amino acids, to exploit the ketone−hydrazide/alkoxyamine reaction, via both residue-specific18 and

site-specific34 manner.

The reaction requires an acidic pH (4.0 to 6.5), which is difficult to obtain inside most cellular

compartments, therefore this reaction is normally suited only for in vitro or cell surface labeling.35

In addition, the reaction very often requires high concentration (millimolar range) of labeling

reagents as it possess slow36 kinetics. The second-order rate constants of the reaction normally lies

in the range of 10−4−10−3 M−1 s−1.35, 37 The use of high concentration of reagents can lead to

background reactivity and toxicity in the case of live cell imaging. To overcome these unfavorable

acidic conditions and slow kinetics, the use of aniline was exploited as a nucleophilic catalyst.38

However this reaction is classified as “biorestricted”12 chemical reaction, as it can be used only in

certain environments and under specific, often nonphysiological (acidic) conditions.

Azide-phosphine reactions ("traceless" Staudinger ligations)

Unlike ketones or aldehydes, azides are truly orthogonal to the majority of biological

functionalities.39 In addition their smaller size and kinetic stability under physiological conditions

make them good candidates for the incorporation into biomolecules, especially into proteins40 via

biosynthetic pathways.

Table 1. Bio-orthogonal reactions used for selective protein modifications.

The reaction between azide and phosphine reduces azide to primary amines, which is known as the

Staudinger reduction.41 This reaction proceeds through formation of an intermediate called

phosphazide that subsequently eliminates molecular nitrogen to yield an aza-ylide. The aza-ylide

is unstable and can hydrolyze to yield a primary amine and a phosphine oxide under aqueous

conditions.42 The azaylide hydrolyzes quickly and breaks the covalent bond between the reactants,

therefore the reaction is not a bioconjugation reaction. However, Saxon and Bertozzi showed that

an appropriately located electrophilic trap, such as an ester moiety, within the structure of the

phosphane, would direct the nucleophilic capturing of the aza-ylide to a new intramolecular

cyclization pathway.26, 43 That ultimately results in a stable covalent linkage between the two

reactants before the competing hydrolysis takes place, unlike the Staudinger reduction. This

modified Staudinger reaction is now known as Staudinger ligation,26 because it can covalently link

two molecules together (Table 1, entry 2). Shortly after, a new variant of this reaction was

described, referred to as “traceless Staudinger ligation”, where the final amide-linked product is

freed from the phosphine oxide moiety (Table 1, entry 3). Till this date there are a number of

modifications brought into this phosphane reagent to improve the quality of the reaction.

One of the main disadvantages about the Staudinger ligations in general is that it proceeds with

slow reaction kinetics (a second-order rate constant is around 10−3 M−1 s−1),42 that demands high

concentration of reagent which eventually leads to higher background signals. Other drawbacks to

note are that the azides can be reduced by endogenous thiols or other reductants. Oxidation

sensitivity of phosphines requires the use of high concentration of the reagent, however, excess of

the reagent leads to potential cross-reactivity of phosphine with disulfides.

Azide-alkyne reactions ([3 + 2] cycloadditions, click chemistry)

Alkynes can react with azides in a 1,3-dipolar [3 + 2] cycloaddition and yield a stable triazole

linkage, however, the reaction requires high temperatures and pressures to get a reasonable

conversion.27, 44 Concurrently and independently in 2002, the groups of V. V. Fokin/K. B.

Sharpless28 and M. Meldal,29 discovered that the reaction between terminal alkynes and azides can

be catalyzed by copper(I) salts and that the reaction can be performed at room temperature (Table

1, entry 4&6). In addition the copper catalyst accelerates the reaction around 6-7 orders of

magnitude faster than the uncatalyzed reactions and several orders of magnitude faster than the

Staudinger ligation.30, 45 This copper catalyzed azide alkyne reaction is now dubbed as Click

reaction, as it is one of the most popular reactions in the Click-Chemistry concept. The term was

originally coined by H. C. Kolb, M. G. Finn and K. B. Sharpless before in 2001.46

The main limitation of the click chemistry is its reliance on the toxic copper(I) catalyst. This

limitation was overcame by the group of C.R. Bertozzi, by introducing ring strain into the alkyne

(Table 1, entry 5&7), and is now dubbed as strain-promoted alkyne-azide cycloaddition

(SPAAC).30, 31 One of the drawbacks is the requirement of strained cyclooctyne derivatives, which

can be achieved via chemical synthesis, is often obtained only in very low yields. On top of that,

even with the most reactive cyclooctyne derivatives, the rate constant of SPAAC is only around

0.1-1 M−1 s−1, which is just only 2-3 magnitude better than typical Staudinger ligations.30, 42, 47

Strain promoted alkene/alkyne-tetrazine reactions (DAinv).

Alkenes, especially strained alkenes can rapidly react with tetrazines to form covalent linkages,

which is basically a form of a [4+2] Diels-Alder cycloaddition. In a classic Diels-Alder reaction,

an electron-rich diene reacts with an electron-poor dienophile. However in a Diels-Alder reaction

with inverse electron demand, an electron-rich dienophile reacts with an electron-poor diene such

as a tetrazine (Table 1, entry 8).32 The first step of the reaction between alkenes and tetrazines

involves the formation of a bridged bicyclic intermediate, which then undergoes a retro-Diels-

Alder reaction by losing molecular nitrogen to form a 4,5-dihydropyridazine product. This product

can isomerize into the 1,4-diydropyridazine in protic solvents. The dihydropyridazine can be

oxidized to pyridazine with oxidants such as elemental oxygen or amyl nitrite.

It was demonstrated that trans-cyclooctene shows the fastest reaction kinetics among the strain

promoted alkenes with a range of tetrazines. 3,6-bipyridyl-s-tetrazine reacts with trans-cyclooctene

with an extraordinary rate constant of 103−104 M−1 s−1. Though most of the tetrazines are not stable

in water, the 3,6-diaryl-s-tetrazines were identified as suitable water-stable derivatives. 20

Tetrazine-based DAinv has been used to modify and image proteins in vivo, both in bacteria and

in mammalian cells.16, 21, 48 As the DAinv is very specific and rapid, the reaction drew a lot of

attention recently. One of the drawback of DAinv chemistry is the comparatively large size of the

reactive functionalities (transcyclooctene and 3,6-diaryl-s-tetrazines) and low-yielding synthetic

accessibility. The reaction of relatively less complex norbornene and a monoaryl tetrazine, gives a

rate constant of around 2 M−1 s−1, considerably slower than the reaction between transcyclooctene

and 3,6-Diaryl-s-tetrazines, however it is still better than most strain-promoted click reactions.48, 49

Figure 3. Structure of unnatural amino acids bearing a bioorthogonal functional group as residues which can be both site and residue specifically be

reincorporated in to proteins using the biosynthetic machinery.11

FLAGGING THE PROTEINS

Intending to exploit the bioorthogonal chemistries in favor of the proteomics, methodologies were

developed to address the site-specific incorporation of designer (unnatural) amino acids into both

peptides and proteins (Figure 3). The incorporation of unnatural amino acids bearing such novel

functional groups into the proteins of interest are achieved by using methodologies such as amber

termination codon suppression mutagenesis,34, 50, 51 expressed protein ligation,52 tagging via-

substrate53 and chemical methodologies comprising solid-phase peptide synthesis54 (SPPS) and

native chemical ligation55. The chemical methodologies are very convenient to get smaller peptides

effortlessly, however synthesizing proteins, often is limited by low yields and technical challenges.

Shortly after the arrival of the concept of the expanded genetic codons, introduced by Schultz50, 56

and his competitors, incorporating unnatural amino acids into proteins became much more

attractive.11, 19, 57 To date there are over 100 unnatural amino acids that can be incorporated into

proteins by using the cellular biosynthetic machinery (Figure 3 shows some of the incorporated

amino acids bearing bio-orthogonal functionalities).11

It is interesting to note that there are two distinct ways by which the unnatural amino acids can be

incorporated into proteins, the residue-specific incorporation via selective pressure incorporation

and the site-specific incorporation via expanded genetic codes.11, 19, 57 The site-specific

incorporation of amino acids is advantageous because it provides not only the opportunity for single

molecule tracking, but also allows the three-dimensional and sequential control over the proteins,

unlike the residue-specific incorporation. The bioorthogonal group introduced during the protein

translation and the chemoselective conjugation can provide new insights into protein structure,

function and activity in cells.

Notably, almost all of the bioorthogonal reporter functionalities in the incorporated unnatural

amino acids are bearing non-polar functional moieties (Figure 3). The reason is that the available

bioorthogonal reactions applied in protein chemistry require nonpolar reporter functionalities

mostly. Some of these non-polar reporter functionalities are very large (eg: strained alkenes and

alkynes) that there might be a considerable perturbation by the nonpolar reporter to the protein if

they are not in the appropriate position to minimize it. Therefore the understanding of the three-

dimensional structure of the protein is required for identifying a suitable replacement site for the

hydrophobic reporter amino acid. Majority of the estimated protein structures are still

undiscovered, which results in an immense need for a class of hydrophilic reporter functionalities

for bioorthogonal reactions.

Considering the shape and aqueous solubility of proteins, they can be broadly classified into three

groups, the fibrous proteins, the globular proteins and the membrane proteins. Globular proteins

are one of the major, and on top, an important class of proteins influencing several functions of an

organism, unlike the other classes of proteins which mostly have structural roles. The involvement

of globular proteins in transportation, catalysis, and regulation processes, makes it very interesting

to study them in their natural environment. The globular proteins, as implied from the name, are

more or less spherical shape in nature, which is influenced by the interaction between its polar

surface amino acids and surrounding aqueous medium. Albumin is a good example of a globular

protein and a structure of bovine serum albumin is shown in Figure 4. Globular proteins are in

general, soluble in aqueous solution due to their distribution of amino acids. The hydrophobic

amino acids are arranged predominantly in the core and the hydrophilic ones at the outer sphere.

This arrangement where the hydrophobic amino acids are more likely to be buried in globular

proteins than the hydrophilic amino acids. In addition, these buried amino acids have no or

minimum accessibility to the surrounding media which makes them hard to modify with any

reporter functionality possessed by the buried residues.

Figure 4. Space fill and wireframe structural representation of albumin (BSA).58

Estimated values59 of the tendency of individual amino acids to be buried in the interior of a protein

are represented in Chart 1. The values given are based on the structures of nine proteins, in which

a total of around 2000 individual residues were studied (observed 587 of them were buried).59

Values indicate how often each amino acid was found buried, relative to the total number of

residues of this amino acid found in the proteins. By comparing the values it can be presumed that

the polar charged amino acids are less often found buried inside the proteins than the polar

uncharged, but the non-polar amino acids are found to be buried heavily in the interior of a protein.

Chart 1. Pattern of buried and unburied amino acid found in proteins.59

The change of a few atoms in one amino acid can sometimes disrupt the structure of the whole

molecule so severely, that the protein function can be lost.60 As a matter of fact, most of the

currently used bioorthogonal reactions are based on reporter functionalities that are non-polar, and

very often the introduced residues are very large in size. If such an unnatural amino acid is

incorporated into the protein surface, there is a high chance that it can disrupt the three dimensional

structure of the protein and therefore eventually alters its functions. To minimize the possibility of

protein perturbation that can be caused by non-polar designer amino acids on the protein surface,

it would be beneficial to investigate new classes of bioorthogonal reactions where we can use polar

functionalities as reporter moieties. Modifying polar amino acid residues often found on the surface

of proteins with polar reporter functionalities would have a great value.

Figure 5. Structure of designer amino acid bearing a thiocarboxylic group, and its natural analogues

aspartate and glutamate residues

Bearing that in mind we have sought of using a much overlooked but interesting functional group

which resembles carboxylic acids, the thiocarboxylic acid. Thiocarboxylic acids are very rarely

found in nature and believed to be unreactive with native functionalities of proteins themselves

under physiological conditions. Besides that, the side chains of aspartate and glutamate residues

could be easily replaced by a thiocarboxylic acid without losing the residues core polar charged

character. As the thiocarboxylic acid structurally differs from the carboxylic acid only by a

replacement of an oxygen by a sulfur atom there is only a minimal distress to a protein structure.

If an amide group (e.g. asparagine and glutamine) could be replaced by a thioacid functionality in

protiens, which would be structurally just a difference of NH2 to SH(S-). While comparing the

proposed thioacid derived aminoacids with other known bioorthogonal functionalities derived

amino acids (Figure 6), is arguably much less complex. In addition, the thioacid is a very polar and

charged group, therefore, it has a higher probability to stay on the surface of protein and be

accessible to surrounding medium and reagents. However it is required to find a unique reaction

where the thiocarboxylic acid reacts chemoselectively with the probe molecules without affecting

other biological functional groups.

CHEMISTRY OF THIOCARBOXYLIC ACIDS

Sulfur in the form of thiols plays a significant role in biology. Oxidation of two thiols into a

disulfide is one of the most distinctive property of sulfur, and these disulfidesare very ubiquitous

in nature. However, during the biosynthesis of thiamin, thioquinolobactin and cysteine, the

involvement of thioacids as “sulfide” donors has been reported.61, 62 The thioacid functionality is

not as ubiquitous as thiol group found in proteins and they cannot be even found in a fraction of

other native protein functionalities. Therefore, choosing them as bioorthogonal reporter moiety is

viable option in protein conjugations. However from the chemical perspective, thioacids are usually

ignored as a class of compounds having a very unique reactivity.

Scheme 1. Different chemical methods for synthesizing thioacids.

Different methods for preparing thiocarboxylic acids have been described in literature (Scheme 1).

Synthesis of thioacids was typically achieved by activating a carboxylic acid to an active ester

followed by nucleophilic displacement with H2S63 or some other hydrosulfide equivalents such as

sodium sulfide64 (Na2S). Thioacids can be generated from corresponding thiocarboxylic esters by

means of acidolysis or piperidine treatment. These thioesters could be synthesized easily from

active esters that were reacted with thiols.65, 66 Reaction of carboxylic acids with Lawesson’s67

reagent yields thioacids as well. The main limitation of using Lawesson’s reagent is the

epimerization of the amino acids, which can occur during the direct transformation of carboxylic

acids to the thioacids.67 In the case of the thiolysis of activated esters with H2S or NaHS, the side

reaction can become very intense and results in diacyl disulfides. In contrast to the thiolysis of

activated esters or LR treatment, acidolysis of trityl thiocarboxylic S-esters delivers thiocarboxylic

acids without a concurrent formation of diacyl disulfides and epimerisation.68 A very recent work

from Sachitanand and Hosahudya69 showed that a reaction with thioacetic acid and NaSH can

generate thioacids from the corresponding carboxylic acids, too.

Table 2. Reaction of thioacids with different functional groups.

The thiocarboxylic acids (RCOSH) reacts with various functional groups and can generate stable

covalent bonds, a few interesting reactions are shown in table 3. In the recent years, a lot of attention

was paid to the reactions of thioacids in organic chemistry, as it allows to generate amide bonds

differently compared to conventional methods (i.e. carboxylic acid activation and amine

substitution). Previous researches showed that the functionalities such as amine (-NH2),70, 71, 72, 73

azides (-N3),62, 74-81 aziridines,82, 83 dinitrofluorobenzene,84 isocyanates (-N=C=O),85 isonitriles (-

N≡C),86 nitroso (-NO) derivatives,87 sulfonamides (-SO2NR2),66 and thiocarbamates (-

OC(=S)NR2) 88 could be used for generating amide bonds on the reaction with thiocarboxylic acids.

The affinity of sulfur for metals such as ruthenium,81 silver,70 and copper71, 83 has been exploited

for derivatizing the thioacids containing amino acids/peptide, too. In addition, reactions using a

thioacid in presence of oxidizing agents has been described in literature for the synthesis of N-

acylated peptides,89 glycopeptides72, 73 and other peptide derivatives69, 90. Interesting examples of

thioacid-mediated synthesis of peptides and glycopeptides in the presence of the coupling additive

HOBt has been demonstrated recently.72, 73 Most notably is the reaction of thiocarboxylic acid with

azides which generates stable covalent (amide) bonds. Azides are found to be a bioorthogonal

functionality, therefore, the reaction with thioacid has a huge potential for biological applications.

THIOACID AZIDE AMIDATION/LIGATION (TAL)

Scheme 2. The reaction of thioacid with azide

The reaction of a thioacid with an azide was originally reported in 1980. There it was described as

a side reaction of an azide, while treating it with potassium thioacetate.91 Later in 1988, Rosen et

al.74 presented this reaction as a novel way to synthesize amide bonds by reacting azides with

thioacids (Scheme 2). However, the authors assumed that the reaction was initiated by traces of

H2S contained in the thioacids, which reduces azides to amines which reacts with thioacids to form

amides. However in 2003, a revised mechanism has been proposed for this reaction by Williams et

al.,75 where they claimed, that the thioacid directly reacts with the azide to form an amide.

Williams’s group learned that amines do not react with thioacids to amides, and excluded the

former assumption from Rosen et al., regarding the azides reduced to amines and the amine reacted

with thioacids to amides.

Table 3. Difference in reactivity of thioacids towards sulfonyl azides and azides75 in presence of the base

2,6-lutidine.

Williams and coworkers have shown that with this methodology, amide linkages can be generated

without the use of protecting groups and in aqueous solution.75 However, they observed as well, an

extreme reactivity difference between electron-rich and electron-deficient azides. The electron-

deficient azides reacted significantly faster (Table 3). In addition, the reaction with the electron-

rich azide required elevated temperatures (60-65 oC) and the reaction last up to 2-3 days. Later In

2006, they published a detailed mechanistic investigation on both TAL reactions, which is shown

in Scheme 3.92 In that article they proposed that in the primarily step of the reaction the pathway

of the electron-rich azides differs from the pathway of the electron-deficient azides.

Scheme 3. The proposed mechanism of TAL92

Highly electron-poor azides favorably react with the thioacid tautomer of thiocarboxylic S-acids

(Scheme 3A). In the first step of the reaction, thioacid and azide forms a nitrogen-sulfur bond,

resulting in a linear intermediate. In a separate step, formation of the nitrogen-carbon bond and

protonation results in the thiatriazoline intermediate. A subsequent retro-[3+2] cycloaddition ends

up in the required amide product by the elimination of nitrous sulfide. However, the electron-rich

azides (Scheme 3B) favor to react with the non-preferred thioacid tautomer, thiocarboxylic O-acid.

During the reaction, the nitrogen-sulfur and nitrogen-carbon connectivity of the thiatriazoline

intermediate is formed in a single step by anion-accelerated [3+2] cycloaddition. Subsequent

protonation and loss of nitrous sulfide via retro-[3+2] cycloaddition yields the amide product. A

further comprehensive mechanistic investigation on TAL was carried out by Yunling Gao in 2010,

where he discussed the effect of bases and solvents on the reaction mechanisms.93

Applications of thioacid azide ligation

Amides play a vital role in a large number of natural products, pharmaceuticals and bioconjugates.

In contrast to classical synthesis, where an activated ester react with amines, TAL has shown a

novel way to achieve amide bonds. Not surprisingly, there are many research groups who have

been utilizing this reaction for their advantage.76, 78, 80, 94, 95 Williams and coworkers themselves

have shown that TAL can be efficiently used for acylation of anomeric sugars azides.75 They used

thiobenzoic acid and thioacetic acid to benzoylate or acetylate the beta-1-azido group of glucose

derivatives (Table 4, entry 1-2). The reaction performed in good yields with fully unprotected

glucose derivatives, even. They also described that the reaction can be performed in a variety of

solvents from non-polar chloroform to polar water.

TAL with electron rich azides requires vigorous reaction conditions and longer reaction times

compared to electron deficient azides, even though electron rich azides are capable of generating

organic amide bonds. In the same year, 2003, Fazio and Wong81 have shown that a variant of the

TAL can be catalyzed by the addition of a transition metal catalyst, RuCl3. They described a

conversion of relatively electron-rich azides to corresponding acetamides at room temperature and

with shorter reaction time compared to Williams’s approach. They described that the reaction

works well with several azido sugar derivatives (both protected and unprotected), however, they

exclusively used thioacetic acid for the acylation and no other thioacid reagents (Table 4, entry 3).

Table 4. Acylation of anomeric 1-azido glucoside derivatives using TAL chemistry.75, 81

In the following year Schmidt et al.94 used TAL chemistry in a novel way to generate S-glycosides

and S-neoglycopeptides. S-glycosides were obtained by reacting glycosylthiomethyl azides with a

C-terminal thioacid modified amino acids and peptides. S-neoglycopeptides were obtained by

reacting glycosylthiomethyl azides with amino acids and peptide derivatives containing thioacid

derivatized aspartates and glutamates at their side chain (Scheme 4). They reported that all of the

reactions worked with a good yield, although, there is no discussion about potential side reactions

of thioacids at peptide side chains, such as the possibility of aspartimide and glutarimide formation.

Scheme 4. Synthesis of S-glycosides94

In 2005 Liskamp et al.79 used the fast reacting version of TAL to obtain a tripeptide mimic

containing a sulfonamide. A C-terminal thioacid derivatized peptide was reacted with a C-terminal

sulfonyl azide derivatized amino acid (Scheme 5). They described that the reaction was completed

within 15 minutes and resulted in a quantitative yield of the product tripeptide mimic.

Scheme 5. Synthesis of peptidyl sulfonamide 66.79

In 2009 Liu et al.76 employed the TAL reaction into the C-terminus of a protein by a chemoselective

amidation with an electron-poor organic azide carrying a biofunctional tag. They used Begley’s96

method for the production of recombinant proteins bearing a C-terminal thioacid in the model

protein ubiquitin. Then they performed C-terminus-specific PEGylation or biotinylation of

ubiquitin by using a sulfonyl azide-functionalized PEG or biotin derivative. They further described

that the amidation of the ubiquitin thioacid with an excess of PEG or biotin sulfonyl azides is a

highly specific reaction. Despite the presence of a large number of free native protein functional

groups, the reaction remains effective even with larger azide compounds.

Scheme 6. Protein bearing a C-terminal thioacid modification reacts with a sulfonyl azide to derive

biophysical probes.

A year later, in 2010, Krishnamoorthy and Begley62 attempted to detect the protein

thiocarboxylates in the bacterial proteomes by using the TAL chemistry. Protein thiocarboxylates

can naturally be found in bacterial cells, where they function as sulfide donors. They are involved

in the biosynthesis of thiamin, molybdopterin, thioquinolobactin, and cysteine. They used a

sulfonyl azide derivative of a lissamine dye, namely lissamine rhodamine B sulfonyl azide, to

detect a protein thiocarboxylate in the Pseudomonas stutzeri proteome, which is proposed to be

involved in the biosynthesis of the pyridine dithiocarboxylic acid siderophore. They have surveyed

several other bacteria for protein thiocarboxylates as well. They detected a strong labeling of a

small protein in S. coelicolor, a weakly labeled protein in S. avermitilis, and no protein

thiocarboxylates in the proteomes of B. xenovorans, Rhodococcus sp. RHA1, S. griesus, and S.

erythrea. They also explained that the reasons for not noticing a labeling in the cell-free extracts

of these organisms could be due to low reconstitution of protein thiocarboxylates in the proteome

with sulfide as the sulfur donor, or a nonexpression of thiocarboxylate-forming proteins under the

growth conditions.

Liskamp et al.78 showed a new TAL approach to decorate biologically active peptides at their side

chains with biophysical tags such as fluorescent probes, metal chelators, and small peptides

(Scheme 7A). They have denoted this thioacid sulfonyl azide ligation strategy as ‘sulfo-click’

reaction. Furthermore, they used an aminoethane sulfonyl azide derived peptide to chemo-

selectively react with a thioacid derivative of interest. They claimed that all of the reactions carried

out resulted in over 80% yield. However, they observed in the presence of free sulfhydryl moieties

that the sulfonyl azide is reduced into the corresponding amides as well. Nonetheless they argued

that the preferred coupling was faster than the concurrent reduction when an equimolar mixture of

cysteine and thioacetic acid was reacted to the sulfonyl azide (Scheme 7B).

Scheme 7. A) Chemoselective modification of a peptide side chain using biophysical probes. B) Reduction

of a sulfonyl azide in presence of thioacetic acid and cysteine.

Previously in our group (in 2007), Katja Rohmer68, 97 worked on the concept of using thioacid

sulfonyl azide ligation on the amino acid side chain to achieve the corresponding

neoglycoconjugates. She could show that the thioacid and sulfonyl azide ligation can be efficiently

used for conjugating sulfonyl azide modified carbohydrates to amino acid derivatives which

contain aspartate and glutamate thioacids. She described that the reactions performed with

excellent yields in less than 15 minutes of reaction time.

AIM OF THE WORK

In the past couple of decades, certain ligation reactions such as the “click chemistry” and strain

promoted reactions are drawing incredible attention in bioconjugation chemistry. Remarkably,

there are more and more methodologies emerging to fill the limitations left by its predecessors, and

to improve the quality of the chemical reactions under given physiological or crude conditions.

The main focus of bioorthogonal reactions lies on protein conjugation under physiological

conditions. Besides, the functional traits of the proteins must be preserved throughout the

methodology, especially for studying their biological significance. Therefore, the methodology

applied for protein tracking or purification must not cause large perturbation to the three-

dimensional structure of the protein, as this could adversely affect further investigations. Unlike

widely used unnatural functionalities for chemoselective reactions, which mostly rely on non-polar

residues, the thioacid, however is a polar and charged functional group. Therefore, a thiocarboxylic

acid (-COSH) functionality can replace the inherent protein surface moieties such as carboxylic

acids (-COOH) or an amides (-CONH2) without contributing much distress to the protein structure.

There are many reactions from thioacids reported, but its reaction with azide to amide is one of the

most interesting one. We denoted the reaction as thioacid azide ligation (TAL), which was

originally91 reported more than 30 years ago. The TAL got much attention since Williams75

reinvestigated its reaction mechanism in the year 2003. Thereafter, there have been many

researchers using this reaction because of its good chemoselectivity in different contexts, including

comparatively small peptides to larger proteins.62, 76, 78 In year 2004 Schmidt et al.,94 applied TAL

for obtaining S-neoglycopeptides by reacting glycosylthiomethyl azides to small peptide

derivatives containing aspartate and glutamate thioacids in their side chain. However, up to our

knowledge, no one else attempted to use the TAL for derivatizing peptides/proteins by using the

polar thiocarboxylic functionality in the side chain of the peptides/proteins.

Scheme 8. Proposed TAL reaction on peptides/proteins

Seeing that the thioacid functionality can be suitable for chemoselective reactions in peptides and

proteins, we sought to establish the TAL for proteins in a novel way by introducing the thioacid

functionality in amino acid side chain residues. However, it would be hard to study the potential

side reactions of TAL in large proteins, and on top of that, it would be very resourceful and

complicated to introduce the thioacid functionality in the side chain of a certain amino acid of

proteins. Therefore, we started with the idea of establishing the TAL initially on amino acids and

ω-aspartyl residues of smaller peptides.

Katja Rohmer, a former colleague, started the concept of using thioacid sulfonyl azide ligation on

the amino acid side chain to achieve corresponding neoglycoconjugates.68, 97 She showed that the

thioacid/sulfonyl azide ligation can be used efficiently for the conjugation of sulfonyl azide

modified carbohydrates to amino acid derivatives containing aspartate and glutamate thioacids.

The reactions she described performed with excellent yields and below 15 minutes of reaction time

at RT.

Scheme 9. Proposed strategy for peptide derivatization using TAL chemistry.

Part of this dissertation is meant to improve the aforesaid TAL concept by transfering the reaction

into peptides. Therefore, it is required to incorporate the thioacid functionality to the peptide side

chain, preferably suitable for the standard Fmoc-based solid phase peptide synthesis (SPPS).

Derivatization of the peptide side chain with thioacid functionality is much more difficult compared

to the C-terminal peptides or derivatization of a single amino acid. As shown in Scheme 9, we

furnished a plan for incorporating thioacids into the peptide side chain and a subsequent TAL

reactions to achieve peptide conjugates of interest. This can be divided into three main objectives:

Development of protecting groups for thiocarboxylic acids suitable for Fmoc based

SPPS.

Generation of thiocarboxylic acids in the peptide side chains from its precursors.

TAL reaction of peptide thioacids with different azides and the assessment of

potential side reactions.

However, protecting groups for thiocarboxylic acids are not a well investigated area and there are

no protecting groups available at present that are suitable for standard Fmoc-based SPPS.

Therefore, one of the objectives is to realize the concept of a protecting group for the thioacids,

suitable for ω-aspartate residues of peptides. In addition, it is required to understand all the relevant

parameters necessary for efficient TAL in peptide side chains. Furthermore, potential side reactions

of TAL that can be caused by native protein functionalities shall be unearthed. If there are any side

reactions observed, they will be characterized in order to find a solution to minimize them.

RESULTS AND DISCUSSION

PROTECTING GROUPS FOR THIOCARBOXYLIC S-ACIDS

Fmoc based SPPS (solid phase peptide synthesis) is a widely accepted chemical methodology to

obtain comparatively larger peptides. Aspartic acid, as mentioned before, is a quite abundant

residue in proteins or peptides and plays an important role in their biophysical properties. However,

it is tricky to chemically synthesize or manipulate peptides containing aspartyl residues, because

of their infamous intermolecular side reaction the “aspartimide formation”.98 Protecting groups at

the ω-aspartate carboxyl site play an important role in controlling the kinetics of the aspartimide

formation. Therefore the tert-butyl group is a widely accepted protecting group as it minimizes the

aspartimide formation. The steric hindrance of the tert-butyl moiety can effectively block

nucleophilic attack at the carboxyl carbon of the ω-aspartyl side chain, moreover, the tert-butyl

group can easily be cleaved into the corresponding carboxylic acid under acidic treatment

(acidolysis).

As previously mentioned, thiocarboxylic acids are indeed structurally and chemically similar to

carboxylic acids, so the most of their chemical reactivity is comparable, too. However protecting

groups for thiocarboxylic acids are not a well investigated area and virtually there is no protecting

group available that is compatible for Fmoc/Boc based SPPS. To realize larger peptides bearing

thiocarboxylic acids at certain ω-aspartyl residues, it is essential to investigate the applicability and

stability of different protecting groups (including those available for simple carboxylic acids),

especially towards piperidine treatment and its efficacy to convert/deprotect back to the

thiocarboxylic acid.

Thiocarboxylic S-esters

Figure 6. Protecting thiocarboxylic acids with tert-butyl, 3-Methyl-pentyl (Mpe) and 2,4,6-trimethoxybenzyl

(Tmob) groups.

Several previous investigations65, 68, 72, 99 have shown that the thiocarboxylic S-esters can be used

as a precursor for the synthesis of the thiocarboxylic acids. It only takes a single step for the

synthesis of thiocarboxylic S-esters from the corresponding carboxylic acids, and the acidolysis of

the thioesters could efficiently generate the thioacids. Inspired from this basic concept, we sought

to use the thiocarboxylic S-esters not merely as precursors, but also as a solid protecting groups for

the ω-aspartate thiocarboxylic acid in peptides. One of our colleagues, Odin Keiper, started to

investigate the possibility of using thiocarboxylic S-esters as the protecting groups for SPPS as part

of his graduation work.100 While screening several potential protecting group candidates, he

observed that the most of the thiocarboxylic S-esters are prone to nucleophilic attacks and

concurrent aspartimide formations. However, sterically much hindered tert-butyl 70 and 3-Methyl-

pentyl 71 (Mpe) were fairly capable of protecting the thioesters from both nucleophilic attack and

aspartimide formation (Figure 6). Conversely, he found that these sterically hindered tert-alkyl

groups 70&71 makes the (CO)S-C bond very strong and could not easily be cleaved to the

corresponding thioacid using the acidic treatment (acidolysis). Interestingly, his work on 2,4,6-

trimethoxybenzyl 72 (Tmob) - a sterically less hindered protecting group (Figure 6) provided a

comparable nucleophilic stability to the Mpe and tert-butyl protecting groups. On top of that, it is

easy to deprotect Tmob thioesters 72 back to thiocarboxylic acid using a simple acidolysis (60 %

TFA treatment). However, he observed the Tmob protection is not well suited for peptide side

chains as it fairly does not prevent the aspartimide formation.

Figure 7. Representation of potential protecting groups

Thiocarboxylic S-esters as protecting groups still are a prospective idea, in the case if the acidic

liability at the (CO)S-C bond for deprotection could be achieved by utilizing the advantages of the

sterically hindered tri-alkyl class of protecting groups, tert-butyl or Mpe as examples. With this

intention in mind, a replacement of an alkyl residue from the tri-alkyl moiety by an electron

donating group (+M effect) is worth to be investigated. Anticipating that the moieties such as alkyl

ethers 73 and –OAc 74 may be capable of weakening the stability of the (CO)S-C bond, we sought

to investigate the compatibility of the 2-methoxypropane 73 and 2-acetoxypropane 74 moiety as

protecting groups in a test system (Figure 7). Derivatives of 3-phenylpropanethioic S-acid101 77

was opted as the test system, since it is simple and easily traceable under UV (Scheme 10).

3.1.1.1 Synthesis of thiocarboxylic S-esters

Scheme 10. Synthesis of 3-phenylpropanethioic S-acid101

Chemical synthesis of the 3-phenylpropanethioic S-acid 77 was achieved from the commercially

available 3-phenylpropanoic acid 75 by a two-step procedure. In the first step the S-trityl 3-

phenylpropanethioate 76 was synthesized by thiotritylating the 75, by using a standard DCC (N,N-

dicyclohexylcarbodiimide) coupling procedure with a reasonable yield. The purified thioester 76

was then converted to 77 in 92 % yield by using mild acidolysis with a solution of 10% TFA in

DCM (Scheme 10).

Scheme 11. Synthesis of 2-((3-phenylpropanoyl)thio)propan-2-yl acetate and S-(2-methoxypropan-2-yl) 3-

phenylpropanethioate

Afterwards, the synthesis of 2-((3-phenylpropanoyl)thio)propan-2-yl acetate 79 was carried out by

reacting prop-1-en-2-yl acetate 78 with 77 (Scheme 11A). We observed that the reaction was

reversible. Beside that the product 79 was found to be very unstable as it hydrolyze back to the

thiocarboxylic acid 77 even under very mild acidic conditions. However, it points to the fact that -

OAc group make the (CO)S-C bond very labile, but it was too labile that it could not even be

purified for further investigations.

As next step, we synthesized S-(2-methoxypropan-2-yl) 3-phenylpropanethioate 81 from 77 by

reacting it with 2-methoxyprop-1-ene 80 (Scheme 11B). Interestingly, this reaction was irreversible

and gave quantitative yield of product compared to the acetate derivative 79. Furthermore, the 2-

methoxypropane S-thioester protection observed to be quite base stable, as it withstood an aqueous

1 M NaOH work up. However, 2-methoxypropane S-thioester protection was observed to be very

labile towards acidic medium, as the compound 81 was cleaved back to thiocarboxylic acid 77 on

silica or even in solvents like chloroform. In spite of this, it shows that the electron donating effect

of the ether and in contrast to the electron-withdrawing effect of the acyl moiety increases the

stability of the corresponding protecting group towards acidolysis. Even though we succeeded to

synthesize and purify 2-methoxypropane protected thioacid 81, the protecting group was too

reactive to put more efforts in to further investigations.

Introducing even less electron withdrawing groups like methylsulfane instead of the methoxy group

might solve the problem of acidic instability, which might yield more acid stable derivatives. As

another approach, we continued with the mostly overlooked thiocarboxylic O-esters as potential

thioacid protecting groups.

Thiocarboxylic O-esters

Figure 8. representation of the structure of carboxylic esters and thiocarboxylic O&S-esters.

Seeing that carboxylic acids are successfully protected in peptide chemistry as carboxylic esters

83, similarly the thiocarboxylic O&S-esters could be a capable protection strategy for

thiocarboxylic acids. However, Odin’s graduation work100 and the aforesaid efforts from myself

by using the thiocarboxylic S-esters 82 as protecting groups proved that the thiocarboxylic S-esters

are complicated due to their reactivity. The thiolate at the ω-aspartate residue is very prone to both

inter- and intramolecular nucleophilic attack. In addition, the strong (CO)S-C bond is more difficult

to be cleaved under acidic conditions compared to the (CO)O-C bond in the corresponding

carboxylic esters (Figure 8).

The elevated nucleophilic instability of thiocarboxylic S-esters compared to the carboxylic esters

could be explained by considering the fact that an alkylsulfanes (RS-) is a weaker base or a better

leaving group than an alkoxide (RO-). Therefore it would be worth to investigate the nucleophilic

stability of the thiocarboxylic O-esters as it replaces the thiolates to alkoxides which has the

potential to influence its nucleophilic stability. In addition, we expect that the strength of the

(CS)O-C bond of thiocarboxylic O-esters would be comparable to the (CO)O-C bond of carboxylic

esters, so that, the deprotection of thiocarboxylic O-esters to thiocarboxylic acid would not be a

major problem.

Investigating the concept of thiocarboxylic O-esters as protecting groups for thioacids were carried

out together with Theresa Renz as part of her bachelor thesis.102

3.1.2.1 Synthesis of thiocarboxylic S-esters

Scheme 12. Thionation of ester using Lawesson reagent.

Thionation -conversion of a carbonyl group into a thiocarbonyl- is a widely used method for the

preparation of organosulfur molecules. Thionation of carboxylic esters 85 to thiocarboxylic O-

esters 86 required vigorous reaction conditions, which was generally carried out using either

P4S10/HMDO103 or Lawesson’s reagent (LR)104 under the toluene reflux. We chose the Lawesson’s

reagent as it is a widely used reagent, which gives generally better yields and is comparatively easy

to handle compared to P4S10/HMDO.

In aspartic acid, it is difficult to selectively incorporate thiocarboxylic O-esters at the ω-aspartate

carboxyl position, as it carries a second carboxyl functional group. 3-phenyl propionic acid 75

bears just one carboxylic acid. So, we decided to synthesize derivatives of 75 as a model system.

We opt to study the nucleophilic stability of the thiocarboxylic O-esters via HPLC and LCMS

analysis as it allows to monitor the progress of the reaction over time. The UV detectable moiety

is advantageous, because it can easily be tracked and quantified in the HPLC and LCMS.

To synthesize a thiocarboxylic O-ester by using the LR, a prior synthesis of corresponding

carboxylic ester is required. Therefore, various types of the carboxylic O-esters, such as ethyl 3-

phenylpropanoate 90, isopropyl 3-phenylpropanoate105 87, 2,4-dimethylpentan-3-yl 3-

phenylpropanoate106 88 and tert-butyl 3-phenylpropanoate 94 were chosen, as they cover a broad

set of steric hindrance features. These derivatives allow to understand how the steric hindrance of

the thiocarboxylic O-esters could affect its reactivity and the stability. The carboxylic esters such

as the ethyl ester 90 and tert-butyl ester 94 were commercially available. The synthesis of isopropyl

3-phenylpropanoate 87 and 2,4-dimethylpentan-3-yl 3-phenylpropanoate 88 were carried out by

reacting the 3-phenylpropanoic acid with isopropanol and 2,4-dimethylpentan-3-ol respectively

using the standard DCC-DMAP coupling procedure (Scheme 13).

Scheme 13. Synthesis of isopropyl 3-phenylpropanoate105 and 2,4-dimethylpentan-3-yl 3-

phenylpropanoate106

In addition, a thiocarboxylic S-ester, S-isopropyl 3-phenylpropanethioate107 89, was synthesized to

compare its relative nucleophilic stability with corresponding thiocarboxylic O-esters and

carboxylic esters. The synthesis was achieved by coupling 75 and 2-propanethiol with the coupling

reagents DCC-DMAP, which resulted in the required product 89 with a yield of 77% (Scheme 14).

Scheme 14. Synthesis of S-isopropyl 3-phenylpropanethioate107

Employing the standard LR thionation procedure, carboxylic esters (87, 88, 90 & 94) were reacted

with LR under toluene reflux (Scheme 15). Thionation was successful for obtaining O-ethyl 3-

phenylpropanethioate108 91, O-isopropyl 3-phenylpropanethioate 92 and O-(2,4-dimethylpentan-

3-yl) 3-phenylpropanethioate 93 as pure compounds. However, the thionation of the tert-butyl 3-

phenylpropanoate 94 with LR under the refluxing toluene, showed disappearance of the starting

material and the appearance of plethora of the UV absorbing spots in TLC, none of which was

present in major amount. A thorough literature research revealed that these observations were not

made for the first time. It has been reported before that the thionation of tert-butyl esters with the

LR109 or P4S10/HMDO110 leads to decomposition. Unfortunately, there were no other synthetic

procedures available to convert 94 to 95, moreover, there was no published procedure to achieve

tert-butyl thiocarboxylic-O-esters in general. However later, Singh et al. 111 published a

transesterification methodology for synthesizing tert-butyl thiocarboxylic-O-esters.

Scheme 15. Thionation of esters.

At the time of the investigation, there was no reported procedures for synthesizing tert-butyl

thioesters, therefore, we sought to use an unconventional approach to achieve it. The idea was to

introduce less sterically hindered safety catch on carboxyl group, then thionate the carbonyl group,

followed by activating the safety catch and substituting it with tert-butanolate. Therefore, one of

the widely used safety catch, phenyl hydrazine112 was chosen for the experiment. Introduction of

phenyl hydrazine into carboxylic acid is uncomplicated, and it can be easily activated by mild

oxidation and substitute into ester with O-nucleophilic attack.113 In addition, it has been reported

that the phenyl hydrazine chemistry can be used to synthesize the tert-butyl carboxylic esters by

nucleophilic substitutiuons.114

The first step of the strategy was to synthesize a hydrazide from the corresponding carboxylic acid.

Afterwards, the hydrazide will be converted to the thiohydrazide by using the LR. Subsequently,

this phenyl hydrazine can be activated to phenyldiazen by using Cu(II) or NBS, and then substitute

with tert-butoxide to tert-butyl thiocarboxylic O-esters.

Scheme 16. Synthesis of N',3-diphenylpropanethiohydrazide

Accordingly, a derivative of the phenyl propionic acid, N',3-diphenylpropanethiohydrazide 96, was

chosen as the model system. The synthesis for 96 which started from the 3-phenyl propionic acid

75 by reacting it with phenyl hydrazine by using the standard DCC coupling procedure (Scheme

16). The resulting hydrazide 96 was then thionated with LR under toluene reflux and resulted in

quantitative yield of 97 (Scheme 16).

Scheme 17. In-situ generation of acyl diazene and the substitution

Subsequently, we went on to activate the compound 97 and try to substitute the hydrazide to the

ester with tert-butoxide. Accordingly, we performed in-situ activation and substitution of 97 by

reacting tert-butanol with a solution of copper acetate and pyridine (Scheme 17A). However, we

observed no conversion of 97 to the required O-(tert-butyl) 3-phenylpropanethioate 95. The

reaction TLC showed that the starting material was unreacted and no additional spots were

observable and no product found in LCMS. To affirm that we did not make any mistake with the

hydrazine activation procedure, we used exactly the same procedure to convert N',3-

diphenylpropanehydrazide 96 to tert-butyl 3-phenylpropanoate 94. The in-situ activation and

substitution of 96 resulted in a good yield of ester 94 (Scheme 17B). Afterwards, we abandoned

further attempts to synthesize the tert-butyl O-thioesters and focused on screening the nucleophilic

stability of other synthesized O-thioesters 91-93.

3.1.2.2 Screening the piperidine stability of thiocarboxylic S-esters

Scheme 18. Reaction of piperidine with carboxylic esters and thioesters.

Subsequently, the stability against nucleophilic attack by piperidine of the synthesized O-thioesters

91-93 were investigated. The objective was to analyze how stable the thiocarboxylic O-esters are

in a mixture of 20% piperidine in DMF, which represents the standard Fmoc cleaving conditions

for SPPS. The strategy was to analyze the reaction through HPLC, by picking and integrating the

chromatogram (UV) peaks of the target compound of the reaction mixture every hour for several

hours. The integrated area of chromatogram peak corresponding to the thiocarboxylic O-esters was

plotted against the time. Therefore we put the 0 h chromatogram peak area as the reference with

hundred percent, plotted the relative consumption of the peak over time. If the compounds were

stable, there will not be any reduction in the percentage of peak area or at least it will be very

minimal. If the compound is unstable the peak of the thiocarboxylic O-ester will gradually

disappear over time and will generate new peaks. The amount of the nucleophilic instability of the

investigated compounds can easily be compared from the plotted graphs.

Graph 1. Piperidine stability of ethyl 3-phenylpropanoate and O-ethyl 3-phenylpropanethioate.

First of all, we screened and compared the piperidine stability of the least sterically hindered

carboxylic ester ethyl 3-phenylpropanoate 90 and the corresponding thiocarboxylic O-ester 91.

Therefore, we monitored both reactions for 6 hours via HPLC and quantified the consumption of

the starting material based on the corresponding chromatogram. The results showed that the O-

ethyl 3-phenylpropanethioate108 91 is more prone to nucleophilic attack (reacted into 3-phenyl-1-

(piperidin-1-yl)propane-1-thione115 100 within 1 hour) towards piperidine treatment compared to

the analogous ester 3-phenylpropanoate (Graph 1). The results were contrary to our anticipations,

that the O-thioester might be more stable than the carboxylic ester.

Graph 2. Piperidine stability of O-(2,4-dimethylpentan-3-yl) 3-phenylpropanethioate 93 and 2,4-

dimethylpentan-3-yl 3-phenylpropanoate 88.

As the next step the piperidine stability of the sterically more hindered O-(2,4-dimethylpentan-3-

yl) 3-phenylpropanethioate 93 was compared with 2,4-dimethylpentan-3-yl 3-phenylpropanoate

88. The results showed that the nucleophilic stability of the O-thioester could be increased with the

steric hindrance of the protecting group. The results showed that only ~30% of thiocarboxylic O-

ester 93 was consumed after 24 hours of 20% piperidine treatment (Graph 2). The observation was

promising, however, Odin’s work100 on thiocarboxylic S-esters shows that even with piperidine,

stable protecting groups are prone to aspartimide formation (intermolecular nucleophilic

substitution) in peptides. So, it was important to examine whether thiocarboxylic O-esters are more

stable than thiocarboxylic S-esters. If yes, it is possible to take them into amino acids and

subsequently to the peptides.

For that reason, we evaluated and compared the piperidine stability of the analogous thiocarboxylic

O-ester 92, thiocarboxylic S-ester 89 and carboxylic ester 87, each bearing isopropyl protection.

This protectiong group was chosen due to its moderate sterical hindrance. Therefore it is easy to

analyze the variation in the reactivity of the analogues (87, 89 & 92) towards the piperidine

treatment.

Graph 3. Piperidine stability of a thiocarboxylic O-ester (92), a thiocarboxylic S-ester (89) and a carboxylic

ester (87).

Contrary to our anticipation, we found that the thiocarboxylic O-ester 92 was much less stable

compared to the thiocarboxylic S-ester 89. More than 90% of thiocarboxylic O-ester 92 was

consumed within 5 hours of piperidine treatment, while the thiocarboxylic S-ester 89 was

consumed only less than 15%, however, the carboxylic ester 87 did not react (Graph 3). This

unexpected result made us speculate that the carbonyl group of thiocarboxylic O-ester may be more

electrophilic than thiocarboxylic S-ester or the piperidine thioamides might be thermodynamically

more stable than piperidine amides. Not surprisingly, we observed that the analogous carboxylic

ester 87 was the most stable compound against piperidine treatment. There were no traces of the

corresponding piperidine amide 99116 observable. However, we concluded that the thiocarboxylic

O-ester concept is not worth for further investigation for peptides or amino acids as it shows a

much lower stability towards piperidine treatment while compared to analogous S-thioesters and

carboxylic esters.

To cut the long story short, the strategy of using O-thioesters and S-thioesters are complicated.

Experiments showed that the carbonyl carbons of the thiocarboxylic esters (both O&S) are much

more electrophilic than corresponding carboxylic esters. That makes it hard to protect the

thiocarboxylic acid as the O-thioesters or S-thioesters. Nonetheless, we were able to show the

difference in nucleophilic stability of analogous esters by introducing sulfur into the carboxylic

group.

As a matter of fact, all of the strong nucleophiles in SPPS are bearing amines as the reactive group.

Those amines can react to esters and displace thiolates or alkoxides under the basic conditions.

Therefore, it would be interesting to investigate the possibility of protecting the ω-aspartate residue

as an amide and then convert into thioacid later for SPPS.

Safety catch approach

In 1971, G. W. Kenner introduced a new strategy and a term into peptide chemistry known as the

“safety catch principle”.117 This strategy allows a linker to remain stable until it is activated for

cleavage by a chemical modification. However, nowadays, the term “safety-catch” is applied to

any moiety, which is cleaved by performing more than one reaction. The safety-catch should be

unreactive to the conditions of the pursued synthetic procedures, but can be activated by a simple

chemical transformation that permits a cleavage or substitution. The safety-catch linker strategy

was especially developed for the solid-phase-syntheses, however now it has been used for every

other synthetic chemistry. There are a number of safety catch methods that have been used, such

as sulfone-linker/Kenner’s safety-catch linker117, sulfide safety-catch linker118, hydrazide119 or 3,4-

diaminobenzamide 120 linker as examples.

Figure 9. Representation of safety catch approach for the synthesis of peptide thioacid.

The approach is to use this “safety catch” approach for protecting the carboxyl group of ω-aspartyl

residue during the polypeptide synthesis. After the complete peptide synthesis, the safety catch will

be activated and substituted by the thiolate or hydrogen sulfide (SH-) into thioesters or thioacids

respectively (Figure 5).

3.1.3.1 Phenyl hydrazine as the safety catch

Hydrazide as the protecting group for carboxyl functionality, has been exercised since around half

a century ago. 119 The strategy is using aryl hydrazines as “safety catch” in the peptide C-terminals

(in situ activation and substitution of the safety catch) has been widely used by numerous research

groups. There are a number of nucleophiles that can be introduced into carboxyl groups by using

hydrazine chemistry.121

Scheme 19. Representation of hydrazine chemistry for synthesizing thioesters/thioacids.

We sought to use the phenylhydrazine as a safety catch in the ω-aspartate residue of peptides, and

subsequently displace the corresponding hydrazides by thiolates into S-thioesters (Scheme 19).

However, it has not been previously reported to use the thiolates (or hydrogen sulfide) as

nucleophiles in this approach. Therefore the compatibility of the hydrazine chemistry with the

thiolate as nucleophile, required to be tested in a simple system before taking it into complex amino

acids or peptides. We chose here as well N',3-diphenylpropanehydrazide 96 as test system as it is

simple and has already been synthesized for the thiocarboxylic O-ester protection chemistry

(Section 3.12.1).

Scheme 20. Reaction of activated N',3-diphenylpropanehydrazide with thiol.

When the hydrazide 96 was activated by Cu(OAc)2 and pyridine, we observed quantitative

conversion (in TLC) of 96 into the presumed 3-phenyl-1-(phenyldiazenyl)propan-1-one 102.

Subsequant addition of trityl thiol resulted in complete consumption of the in-situ generated

intermediate 102 and the generation of a new major spot in TLC. Isolation of the newly generated

major spot on TLC revealed that the intermediate 102 was reduced in presence of thiol and

converted back to its precursor N',3-diphenylpropanehydrazide 96 (Scheme 20). Nonetheless, in

LCMS the product 103 could be detected but the amount was too small for an isolation or

quantification. We did not see any differences in the outcome of this reaction, when we activated

hydrazine with NBS (N-bromosuccinimide) instead of Cu(II) reagent. Here, the thiolate behaved

as the reducing agent and the in-situ generated diazene was prone to reduction. When we attempted

to do the substitution with NaSH/H2O (SH- source) instead of trityl thiol, we observed hydrolysis

product, 3-phenylpropanoic acid 75 as a major product with the precursor N',3-

diphenylpropanehydrazide 96.

So we concluded that the thiols are too strong reducing agents, so that it is not viable to displace

the reduction prone diazene with thiols. Therefore we confirmed that it is not suitable to use the

phenyl hydrazine as safety catch for the synthesis of thioesters or thioacids using thiolate or

hydrogen sulfide as nucleophiles. However, if there is a safety catch strategy where one can use

thiolate for substitution, the strategy is still worth to be investigated.

3.1.3.2 3,4-diaminobenzamide 120 as the safety catch

In 2008, Dawson et.al122 published a strategy for introducing thiolate into peptide terminal by using

safety-catch approach. In this approach the synthesis of the precursor peptide was carried out at the

amine group of 3,4-diaminobenzoyl (Dbz) 104 derived resin. In prior to the peptide release from

resin, the diaminobenzoyl moiety was converted into imidazolinone via a treatment with

p-nitrophenyl chloroformate. The imidazolinone is a mild activating group for carboxyl group, and

can be displaced with thiols into thioesters either in prior or in situ.

Scheme 21. Representation of 3,4-diaminobenzamide as safety catch in aspartyl ω-carboxyl group of a

peptide

Therefore, we sought to use this 3,4-diaminobenzamide safety catch chemistry to generate

thioesters at the carbonyl group of the ω-aspartyl residue in peptides. Seeing that this chemistry

was reported to generate thiocarboxylic S-esters from thiols,122 we intended to attach 3,4-

diaminobenzamide directly into the aspartyl ω-carboxyl group of a peptide without testing it in a

simple system (Scheme 21).

The strategy was to synthesize a simple peptide containing Asn(Dbz) residue, which can then be

converted into the corresponding peptide thioesters or thioacids. The Asn(Dbz) residue in a simple

peptide can provide information about its stability as a safety catch and the compatibility toward

peptide synthesizing procedures. Aspartyl building block, Fmoc-Asn(Dbz)-OH 105 was chosen,

because if it was compatible to these procedures, it could easily be adopted into Fmoc based SPPS

for making larger peptides.

Scheme 22. Synthesis of the aspartyl building block Fmoc-Asn(Dbz)-OH 105

To begin with this chemistry, the synthesis of the safety catch (3,4-diaminobenzamide 104) was

carried out. The 3,4-diaminobenzamide was achieved from commercially available 4-amino-2-

nitrobenzamide, according to a two-step reported120 procedure. Afterwards the synthesis of Fmoc-

Asn(Dbz)-OtBu 107 was carried out by coupling commercially available Fmoc-Asn(OH)-OtBu

106 with 3,4-diaminobenzamide. The reaction was performed via the standard HBTU/HOBt

coupling procedure and gave a good yield of the required product 107. Subsequently, tert-butyl

deprotection of Fmoc-Asn(Dbz)-OtBu 107 was performed by using 60% TFA and that resulted in

the targeted Fmoc-Asn(Dbz)-OH 105. This was then directly used for the subsequent step without

any chromatographic purifications (Scheme 22).

Scheme 23. Synthesis of dipeptide NH2-Val-Ala-OtBu 111

A simple peptide containing Asn(Dbz) was required to investigate the stability and compatibility

of the Dbz group in peptide side chain. A tripeptide Fmoc-Asn(Dbz)-Val-Ala-OtBu 112 was

chosen for that purpose, as it represents the characteristics of a peptide environment. To achieve

that, the missing dipeptide fragment NH2-Val-Ala-OtBu 111 was synthesized. The synthesis started

by coupling commercially available Fmoc-Val-OH 108 and NH2-Ala-OtBu 109 in the presence of

coupling reagent HBTU-HOBt, which gave Fmoc-Val-Ala-OtBu 110 in a quantitative yield. The

Fmoc deprotection with 20% piperidine gave the required peptide fragment NH2-Val-Ala-OtBu

111, which was then used directly for the subsequent step without any chromatographic

purifications (Scheme 23).

Scheme 24. Coupling of Fmoc-Asn(Dbz)-OH 105 and NH2-Val-Ala-OtBu 111

However, attaching the Fmoc-Asn(Dbz)-OH 105 in to NH2-Val-Ala-OtBu 111 did not work as

expected. A number of unpredicted side reactions were observed on TLC. The LCMS analysis

suggests that the amine groups from 3,4-diaminobenzamide safety catch is participating in both

inter- and intramolecular reactions resulting in large number of byproducts. If that happened during

peptide synthesis, using 3,4-diaminobenzamide as safety catch in the side chain of an aspartyl

residue of the peptide, the method would be incompatible. Nevertheless, protecting one of the

amine groups of 3,4-diaminobenzamide, and using this compound as the safety catch in the peptide

side chain could be a solution for minimizing side products.

However some of our parallel research on the derivatives of aspartic acid and aspartic thioacid in

peptides revealed that, severe amount of aspartimide formation (inter molecular reaction) can be

observed even with mildest activation at the carboxyl site of the ω-aspartyl residue. This

observation hinted that it may not be worth to investigate further on the concept of the “safety catch

approach” as it requires a mild activation in the end of the peptide synthesis, for converting to

thioesters or thioacids.

THIOACID AZIDE LIGATION (TAL) CHEMISTRY

One of the major objective of this dissertation is to study the compatibility of thiocarboxylic acids

in peptide side chain. To achieve a broad view on compatibility, reactions between a range of

dissimilar azides and peptide thioacids are required. In addition, we sought the reacting compounds

should be simple, so that, they can be easily tracked and potential side reactions can be

characterized. Besides that, the reaction should represent a close realistic environment identical to

a protein side chain labeling or conjugation. In addition, we explored the idea of generating natural

N-Glycopeptides using TAL chemistry, as it can bring the advantage of convergent123 synthesis.

To match all the requirements we synthesized a number of dissimilar azides and thioacids, which

are described below.

TAL reaction with sulfonyl azides.

As mentioned in the introduction Williams et al.75 showed that electron deficient azides, such as

sulfonyl azides, react with thiocarboxylic acids much faster than electron rich azides. Likewise,

how the electron deficiency of sulfonyl azides affects TAL reactions and potential side reactions

as well sought to investigate. Therefore, for screening of the rates, the efficiency and the potentials

of TAL in peptide side chains, a series of azides that differ in their electron withdrawing character

and their diversity of moieties, were realized. At the same time, we sought to use TAL as a

methodology to introduce widely used biophysical probes such as lissamine, biotin, dansyl and

glucose moieties.

3.2.1.1 Sulfonyl azide containing labeling reagents

3.2.1.1.1 Synthesis of lissamine rhodamine B 5-sulfonyl azide

Scheme 25. Synthesis of lissamine rhodamine B sulfonyl azide.

Lissamine Rhodamine B 113, is a widely-known protein labeling dye and the fluorescence

emission spectrum of its protein conjugates lies between those of tetramethylrhodamine and

Texas Red™. Its protein conjugates reportedly have decent chemical stability, photo-stability and

are somewhat easier to handle in general.124 In fact the synthesis of lissamine rhodamine B sulfonyl

azides were reported in literature,62, 125 however, complete characterizations and assignments of

both of its regio-isomers have not been published to date.

In order to synthesize and purify lissamine rhodamine B sulfonyl azides from the commercially

available mixture of the lissamine rhodamine B 113, we employed different synthetic methods to

the published62, 125 procedures (Scheme 25). Azidation of 113 using tetrabutylammonium azide126

gave a mixture of both azide regio-isomers with a ratio of around 1:5. Both isomers were isolated

via chromatography, from which, the position of sulfonyl azide in the major isomer, lissamine

rhodamine B 5-sulfonyl azide 114, was determined via crystallography (Figure 10). The remaining

minor isomer was then assumed as lissamine rhodamine B 3-sulfonyl azide 115 based on mass and

NMR data.

Figure 10. ORTHOP drawing (A) and, crystal data & structure refinement (B) of lissamine rhodamine B 5-

sulfonyl azide 114

X-ray Structure Determination: lissamine rhodamine B 5-sulfonyl azide 114 was crystallized

from methanol in the form of dark red needles. A well-shaped individual was chosen for X-ray data

collection and measured in single crystal x-ray diffractometer (image plate IPDS2). ORTEP

drawing of molecular structure, accurate unit cell parameters and other experimental details are

shown in Figure 10, while other crystallographic data, methods and software used are given in the

experimental part of this thesis.

3.2.1.1.2 Synthesis of other sulfonyl azides

Dansyl azide 116, a sulfonyl azide derivative of the well-known blue-green-fluorescent dye dansyl,

was already used in sulfo-click (TAL) chemistry99 and many other127, 128 contexts. The synthesis of

the dansyl azide 116 from the dansyl chloride was carried out according to the reported129

procedure. We have shown in the past that the synthesis of neoglycoamino acids can be achieved

through TAL chemistry.68 Analogously, sulfonyl azide-modified glycosides, 2-azidosulfonylethyl

2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside68 117 could be used to react with peptide

thiocarboxylic acids for the generation of neoglycopeptides. The compound 2-azidosulfonylethyl

2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside 117 was synthesized in our lab by Katja Rohmer as

part of her master thesis97 and was used here for our study.

Scheme 26. Chemical structures of dansyl azide and 2-azidosulfonylethyl 2,3,4,6-Tetra-O-acetyl-β-D-

glucopyranoside.

Scheme 27. Synthesis of sufonyl azide derived linker

The sulfonyl azide bearing Boc-protected diaminoethyl linker 119 was synthesized using a single

step procedure. A standard carbodiimide (EDAC) activated coupling reaction of the 4-

carboxybenzenesulfonazide 118 and N-Boc-ethylenediamine gave the required product 119

(Scheme 27). However, we observed large amount of by-products (such as the hydrolysis product

and diaminoethyl sulfonamides) assuming that the sulfonyl azides in general are prone to

nucleophilic attacks and that led to a huge drop in the yield of product 119. Nonetheless, we

obtained enough material of the azide 119 for further experiments, therefore, we did not try to

optimize the synthetic procedure for improving the yield.

Scheme 28. Synthesis of sulfonyl azide derived biotin.

Our desire to get an affinity tag (biotin) derived sulfonyl azide 121 was subsequently fulfilled. The

synthesis of 121 could easily be achieved from the Boc-4-((2-

aminoethyl)carbamoyl)benzenesulfonyl azide 119, via a two-step procedure (Scheme 28).

Accordingly we started with the Boc deprotection of the azide 119 by using 50% TFA to get the

corresponding amine. Subsequently, without any chromatographic purification, the obtained amine

was reacted to the biotin 120 by using standard coupling reagents HBTU/HOBt. This reaction,

however, gave as well very low yield, due to the difficulties in chromatographic purification.

Scheme 29. Alternative synthesis of biotinylated sulfonyl azide derivative 124.

To obtain pure and sufficient amounts of biotinylated sulfonyl azide for our studies, we sought to

attach the sulfonyl azide in the final step of the synthesis. Attaching sulfonyl azide in the last step

would minimize its side reactions and that would facilitate the chromatographic purification.

Accordingly, we sought to synthesize the sulfonyl azide 1-biotinyl piperazine-4-benzamido

sulfonazide 124, which has two less NH protons compared to compound 121 (assuming that the

hydrogen bonds could interfere the purifications). Synthesis of the biotinylated sulfonyl azide 124

was started by coupling biotin to 1-Boc piperazine 122 by using standard HBTU-HOBt coupling

procedure and gave good yield of Fmoc-piperazinyl biotin 123. Subsequently, Fmoc group of the

compound 123 was then deprotected via piperidine treatment and coupled into 4-

carboxybenzenesulfonazide 118 using standard DCC-DMAP coupling procedure (Scheme 29).

Even though this reaction as well gave a low yield for the required product 1-biotinyl piperazine-

4-benzamido sulfonazide 124, the isolation of 124 was easily achieved via chromatography.

To sum-up this section, we obtained several sulfonyl azides containing dissimilar, but interesting

compounds. In the case of lissamine rhodamine B sulfonyl azides, we developed a new synthetic

route to achieve it. The major regio-isomer of the lissamine azides was characterized via X-ray

crystallography and identified as the lissamine rhodamine B 5-sulfonyl azide 114 for the first time.

In addition, we have obtained the sulfonyl azide containing glycoside, the corresponding dansyl

derivative and the biotinylated compounds for studying TAL chemistry in peptides.

3.2.1.2 Synthesis of aspartyl derived peptide thiocarboxylic S-esters.

One of the interesting questions that we are studying is, how the TAL reaction can perform at the

carbonyl group of the ω-aspartyl residue in peptides. To be able to answer that question, it is

required to synthesize the peptides containing thiocarboxylic acid functionalities at the ω- aspartyl

residues of the peptides. In the past, our group has shown that trityl thiocarboxylic S-esters are

suitable precursors for generating thiocarboxylic acids in simple aspartic acid and glutamic acid

side chain.68 In contrast to the thiolysis of activated esters with H2S63 or NaHS,64 acidolysis of trityl

thiocarboxylic S-esters delivers thiocarboxylic acids without concurrent formation of diacyl

disulfides.68

Therefore, we as well selected trityl thioesters as a precursor for the thiocarboxylic acid in peptides.

As mentioned earlier trityl thiocarboxylic S-esters are easy to generate using standard carbodiimide

procedures, and can be stored for a longer period of time compared to free thiocarboxylic acids.

However, trityl thioesters are not stable enough for standard peptide coupling procedures.

Therefore the trityl thioesterification must be performed exclusively after the complete peptide

synthesis. To do a comprehensive study of TAL in peptides we synthesized a range of dipeptides

and tripeptides.

Scheme 30. Synthesis of peptide containing ω-aspartate benzyl esters for the synthesis of peptide

thioesters.

Introducing the trityl thiocarboxylic esters (-COSTrt) at the ω-aspartyl residue of a peptide, initially

required synthesis of the corresponding carboxylic acids. Synthesis of ω-aspartic acid containing

peptides can be achieved from selective deprotection of corresponding benzyl esters. For that

reason, the synthesis of the Asp(OBn) containing peptides were performed by using the standard

peptide protection (Fmoc, Boc, tBu, OBn), coupling (HBTU/HOBt) and deprotection (20%

piperidine) chemistry (Scheme 30). Accordingly, a series of dipeptides and tripeptides (141-148)

were synthesized with diverse sterical and functional properties.

To achieve the tripeptides derivatives, to begin with, the synthesis of dipeptides Fmoc-Xxx-Phe-

Ot-Bu (129-131, Xxx = Val,130 Ala131 and Gly132) were carried out. Therefore, the amino acids

Fmoc-Xxx-OH (125-127, Xxx = Gly, Ala and Val) were reacted with NH2-Phe-Ot-Bu 128 by using

standard HBTU/HOBt coupling procedure (Scheme 30). All of the reactions gave the required

products 129-131 with good yield. In the next step, Fmoc deprotection of compounds 129-131 was

achieved by 20% piperidine treatment, which resulted in the corresponding amines. Subsequently,

the coupling of freshly deprotected amines with Boc-Asp(OBn)-OH 139 was achieved by using

HBTU/HOBt procedure. All of the reactions gave the target tripeptides 141-143 with very good

yields (Scheme 30). Likewise, the synthesis of the dipeptides was achieved by coupling

(HBTU/HOBt as reagent) the commercially available amino acid derivatives 135-138 either with

Boc-Asp(OBn)-OH 139 or Fmoc-Asp(OBn)-OH 140. As expected, all of them gave quantitative

yield of the required dipeptides 144-148 (Scheme 30).

Scheme 31. Preparation of amino acid and peptide derived trityl thiocarboxylic S-esters. * Compounds 151-

157 were synthesized in situ by hydrogenation of the corresponding benzyl esters. # Synthesis of the

corresponding compound was achieved at 4 oC to reduce concurrent aspartimide formation.

The synthesized benzyl esters 141-148 were then selectively deprotected via hydrogenation (5-

10% Pd-C/H2) to get the corresponding peptide carboxylic acids at the ω-aspartate residue. All of

the hydrogenation was performed in the solvent MeOH, except in the case of peptide Fmoc-

Asp(OBn)-Val-Ot-Bu 145 where a mixture of MeOH/ethyl acetate (1:2) was used to suppress the

Fmoc cleavage. After the hydrogenation, these crude products were used directly for the

subsequent step without any chromatographic purifications, except in the case of the deprotection

of Boc-Asp(OBn)-Gly-Phe-Ot-Bu 143. As the hydrogenation of the compound 143 generated

aspartimides and its secondary reaction products, it was necessary to purify the mixture to follow

the aspartimide formation in the next step.

Afterwards, we started to synthesize the tritylated peptide thiocarboxylic S-esters. To study how

the TAL reaction behaves when it is transferred from amino acids to peptides, a couple of aspartate

derived amino acid trityl thiocarboxylic S-esters were synthesized as well. Thiotritylation of amino

acids were achieved from the commercially available compounds Boc-Asp(OH)-Ot-Bu 149 and

Cbz-Asp(OH)-OBn 150. In the case of thiotritylation of peptides, only freshly synthesized peptide

carboxylic acids were used.

Accordingly, the syntheses of the proposed trityl thiocarboxylic S-esters 159-168 were achieved

by reacting the compounds 149-158 with tritylthiol by using the DCC or EDAC coupling

procedures. Almost all of the thioesterifications gave good yields of required products (Scheme

31). However, the very aspartimide prone Boc-Asp(STrt)-Gly-Phe-Ot-Bu 168 resulted in a yield

of 21%. Even so, the obtained yield is well within the satisfactory range of such type of compounds.

The deficiency in the final yield in many of the cases here, were traced back via LCMS to the

concurrent aspartimide formations and other side reactions caused by carbodiimide activation.

Nevertheless, we managed to generate a series of peptides thiocarboxylic esters ranging from the

least aspartimide prone Asp-Val motif to the highly aspartimide prone133 Asp-Gly motif. The

synthesized tritylthioesters were stored in refrigerator and used even after years for generating

thioacids.

3.2.1.3 Synthesis of aspartyl derived peptide thiocarboxylic acids

Scheme 32. Preparation of amino acid and peptide-derived thiocarboxylic acids.

Tritylthioesters should be converted into corresponding thiocarboxylic acid before performing the

TAL reactions. To investigate how the stability and efficiency of ω-aspartyl thiocarboxylic acids

changes when it is a part of the peptide side chain, which indeed requires the corresponding

thioacids in pure form. Therefore, we generated thiocarboxylic acids, each out of an amino acid, a

dipeptide and a tripeptide tritylthioester derivative. Selective acidolysis (with 5% v/v TFA) of the

selected trityl thiocarboxylic S-esters 150, 155-156 were carried out. As expected, all of these

reactions were completed within 5 min (in TLC) at ambient temperature. The subsequent

chromatographic purifications (RP-MPLC) of the reaction mixtures gave satisfactory yields of pure

but selectively deprotected peptide thiocarboxylic acids 169-171 (Scheme 32). As next, we tried to

get a fully unprotected peptide thiocarboxylic acid in pure form. Therefore we selected Boc-

Asp(STrt)-Val-Ot-Bu 155 and vigorous acidolysis conditions were performed with a solution of

60% v/v TFA. The reaction was completed under 60 min and gave 44% yield of thiocarboxylic

acid TFA*NH2-Asp(SH)-Val-OH 172 (Scheme 33).

Scheme 33. Synthesis of fully unprotected peptide thioacids.

The decrease in the yield of tritylthioester deprotections were mainly due to the poor

chromatographic purifications. Generated thiocarboxylic acids were dragged though RP-MPLC

column, resulted in broad band of the compounds peak. However, we observed as well a small

amount of aspartimide formation and hydrolysis products via LCMS. Notably, the purified

thiocarboxylic acids generated through this procedure were quite stable for a longer period of time,

without traces of the oxidation or other side products.

3.2.1.4 TAL reaction with isolated thiocarboxylic acids

As stated before, Katja Rohmer68 has fairly investigated the TAL reactions in amino acid

derivatives (aspartic and glutamic). However, it was required to study the compatibility of the TAL

reactions in peptides by comparing it to amino acids. Therefore, we investigated the TAL reactions

in both cases, the peptide thioacids and amino thioacids, by using similar conditions. To begin with,

we performed a TAL reaction by taking two equivalents of Cbz-Asp(SH)-OBn 169 as the thioacid

component and one equivalent of lissamine rhodamine B 5-sulfonyl azide 114 as the azide

component. The ligation reaction was performed in the presence of equimolar concentration of 2,6-

lutidine with respect to the concentration of thiocarboxylic acid. The reaction completed within

few minutes and the coupling product 173 was isolated with an excellent yield (Scheme 34).

Scheme 34. TAL ligation of amino thioacid 114 with lissamine rhodamine B 5-sulfonyl azide 173.

As next, a peptide thioacid was reacted with sulfonyl azides. Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170

was selected as the peptide thioacid, given that the tyrosine residue can easily be tracked by the

UV-detector in LCMS. Two equivalents of peptide 170 were taken and reacted with the selected

sulfonyl azide derivatives of lissamine 114, 4-carboxybenzyl 116 and dansyl 119 compounds.

These selected sulfonyl azides have different electron densities and it is interesting to know, how

that would influence the speed and outcome of the ligation. An equimolar concentration of 2,6-

lutidine with respect to thiocarboxylic acid was used for the reactions and in all other TAL

mentioned in this thesis, unless otherwise specified.

We observed that the reaction of the peptide 170 with lissamine rhodamine B 5-sulfonyl azide 114

was the fastest among all investigated, completed within just 5 minutes and resulted in a

quantitative yield of product 174 (Scheme 35a). With similar reaction conditions, the peptide 170

was reacted to the 4-carboxybenzenesulfonazide derivative 116 with a slight drop in reaction speed

(15 min), though gave a quantitative yield of the required product 175 (Scheme 35b).

Scheme 35. Comparative reactivity of different sulfonyl azides with peptide thiocarboxylic acid. a) Lissamine

rhodamine B 5-sulfonyl azide; b) Boc-4-((2-aminoethyl)carbamoyl)benzenesulfonyl azide; c) Dansyl azide

However, the dansyl azide 119 reacted to the peptide thiocarboxylic acid 170 with the slowest pace

and gave the lowest yield in comparison to the two other azides (Scheme 35c). Generation of dansyl

amide128 was observed in this reaction, implying that, in the presence of base the thiocarboxylic

acid itself can reduce the sulfonyl azide into the corresponding sulfonamide. Nonetheless, the

relative yield of dansyl azide reduction to the ligation was affordable and the rate of the reduction

was much slower in comparison to concurrent TAL. In addition, the reduction products were not

observed in the cases of other fast reacting sulfonyl azides such as lissamine 114 or 4-

carboxybenzene 119 derived sulfonyl azides. Notably, the TAL reaction speed of all of these three

sulfonyl azides suggests that electron deficient residue in the aromatic ring could further contribute

into the reactivity of the aromatic sulfonyl azides. This investigation also drew our attention to the

lissamine rhodamine B 5-sulfonyl azide 114 as a potential labeling precursor in TAL chemistry, as

it reacted very fast with the thioacids in an excellent yield.

As next, the reactivity of biotinylated sulfonyl azide 121 was investigated with tripeptide thioacid,

Boc-Asp(SH)-Val-Phe-Ot-Bu 171. Here, an excess of just 1.2 equivalents of tripeptide thioacid

171 was used to react with the biotinylated sulfonyl azide 121. Reaction was completed within 15

min as expected and gave excellent yield of the product 177 (Scheme 36).

Scheme 36. Reaction between tripeptide thioacid and biotinylated sulfonyl azide

There are several published procedures in which researchers used different solvents for TAL

reactions.74-79, 81, 92, 94, 97 The coupling of the thioacid and the electron-deficient azide can be

smoothly carried out in a wide range of solvents, from non-polar chloroform to polar methanol,

DMF and even in water.75, 78, 79 However, we assumed that it is still relevant to look into the

versatility and the influence of the solvents, while taking this coupling into the ω-aspartate site of

the peptides.

Therefore, the reaction between Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170 and lissamine azide 114 was

taken as the model system. The speed and completion of the reaction, as well as potential

byproducts in chosen solvents were analyzed via LCMS. A variety of solvents were selected for

the experiments, such as DMF, DMSO, MeOH/water, MeOH/DCM and MeOH/THF. The

mixtures of methanol were used because 144 is not soluble itself in those solvents. However, we

found neither hindrance of the TAL reactions nor detectable byproduct formations in the solvents

with respect to the reaction in pure methanol. It is worth to note that even in the case of DMSO

(solvent that is capable of oxidizing thiols), the TAL reaction could be performed smoothly. It

could be the case that the selected TAL reaction occurred really fast that all the other side reactions

were left untraceable.

3.2.1.5 TAL reaction with in-situ generated thiocarboxylic acids

Seeing that the chromatographic purification of the thiocarboxylic acids resulting in a decrease in

isolated yields, we sought to generate the thiocarboxylic acids in-situ. The feasibility of using in-

situ generated thiocarboxylic acid and reacting them directly to the azides could improve the overall

yield of the coupling. Therefore, initially we attempted this approach in a simple amino acid

derivative. Accordingly, Cbz-Asp(STrt)-OBn 160 was selectively deprotected using mild

acidolysis (5% v/v TFA in DCM) and the solvent was co-evaporated just with toluene.

Subsequently, this crude thiocarboxylic acid was reacted to lissamine azide 114 using the TAL

procedure. The reaction was completed within 5 min and gave an excellent 85% overall yield of

coupling product 173 (Table 5, entry 1). The comparison of the overall yields obtained from the

purified thiocarboxylic acid (59%, Scheme 32a&34) strategy to the in-situ strategy, the latter one

is evidently far better.

As next, a reaction between the slow reactive sulfonyl azide, dansyl azide 116 and an in-situ

generated aspartyl thioacid was studied. The trityl thioester of Boc-Asp(STrt)-Ot-Bu 159 was

selectively deprotected under mild acidolysis, co-evaporated with toluene and reacted with half an

equivalent of dansyl azide. The reaction took around 2 h to complete, but gave a good yield of

required product 178 (Table 5, entry 2). Not surprisingly, we also observed (via LCMS) a small

amount of reduction of the dansyl azide to the dansyl sulfonamide. Our attempts to quantify the

reduction product failed in this case, however we had a systematic look into the reduction of azides,

which will be explained in the next section of this thesis.

Table 5. TAL reaction of in-situ generated thiocarboxylic acids with a series of azides.

Inspired by the success of these in-situ test reactions in amino acid derivatives, the applicability of

the strategy in peptides would be highly interesting. Accordingly, the thiocarboxylic acid was in-

situ generated from Boc-Asp(STrt)-Tyr(Ot-Bu)-Ot-Bu 165 and reacted with 114. Two reactions

were performed in parallel: in the first case thiocarboxylic S-ester 165 was added in excess whereas

in second case sulfonyl azide 114 was added in excess (Table 5, entry 3-4). In both cases an

acceptably good overall yield of lissamine conjugate 174 was obtained. However not surprisingly,

starting from an excess of thiocarboxylic S-ester 165 gave slightly better yields compared to the

other. This can be justified by the fact that thiocarboxylic acids are generated in-situ, and besides

they are much more labile than sulfonyl azides. However in reality, it is obligatory that an excess

of the labeling reagents is present compared to the target peptides or proteins.

Figure 11. Ligation products

To generalize the in-situ concept, thereafter, we investigated a series of TAL reactions (Table 5,

entry 5-9) starting from peptides thiocarboxylic esters containing Asp-Gly 162 and Asp-Val 168

residues. Both of the thioesters were transformed in-situ into the corresponding thiocarboxylic

acids, and then it was reacted against two equivalents of sulfonyl azides 114, 116 and 124. The less

aspartimide prone dipeptide thiocarboxylic acid, generated from Boc-Asp(STrt)-Val-Ot-Bu 162,

was reacted to the aforesaid azides (114, 116 & 124, all of the azides used excess to thioacid) with

different pace as expected. The reaction with dansyl azide 116 was completed in 2 h as expected,

and not surprisingly dansyl amide formation was detected with good overall yield of the required

dansyl conjugate 179 (Table 5, entry 5). The reaction with lissamine azide 114 was the quickest

(~5 min) and gave the required lissamine conjugate 180 with a good overall yield (Table 5, entry

6). The same reaction with biotinylated sulfonyl azide 124 took 10 min to complete and a similar

overall yield of the required biotin conjugate 183 (Table 5, entry 7).

As next, we extended our investigation into the conjugation of the more complex and aspartimide

prone tripeptide Boc-Asp(SH)-Gly-Phe-Ot-Bu. This tripeptide thioacid was in-situ generated from

Boc-Asp(STrt)-Gly-Phe-Ot-Bu 168 by mild acidolysis, and followed by co-evaporation with

MeOH below ambient temperature (to minimize aspartimide formation). The crude Boc-Asp(SH)-

Gly-Phe-Ot-Bu was then reacted into biotinylated sulfonyl azide 124, and the reaction was

completed within 10 min and gave biotin conjugate 182 with an overall yield of 58% (Table 5,

entry 8). The same peptide was then reacted to lissamine azide 114, expectedly, the reaction was

completed under 5 min, and gave lissamine conjugate 181 with a similar overall yield (Table 5,

entry 9). Notably, both of these reactions of the highly aspartimide prone Asp-Gly peptide motif

performed well within the expected range of such types reactions. Comparing the ligation reaction

of Asp-Gly with Asp-Val residual peptides, there was a 20% reduction in overall yield observable

with the Asp-Gly reaction. However, this loss in yield is presumably due to the side-reaction during

deprotection of trityl. Otherwise TAL could give better yield with lissamine azide 114 than the

biotinylated sulfonyl azide 124, as latter react faster.

As a final point by using this selective in-situ deprotection strategy, we synthesized a

neoglycopeptide 184 using TAL procedure. This was achieved by reacting in-situ generated

thiocarboxylic acid from the Fmoc-Asp(STrt)-Val-Ot-Bu 161 against 2-azidosulfonylethyl 2,3,4,6-

Tetra-O-acetyl-β- D-glucopyranoside 117. The reaction was performed in methanol, completed in

15 min and gave very good yield of required neoglycopeptide 184 (Table 5, entry 10).

These experiments undoubtedly illustrates that the in-situ TAL strategy clearly is more effective

approach than isolated thiocarboxylic acid ligation. Since all the reactions performed within the

range of satisfying yield, indicates the potentials of using TAL coupling chemistry for labeling of

peptides in a novel way. In addition we have shown that a variety of sulfonyl azides can effectively

be used here to generate amide bonds at the ω-aspartate moiety of even very aspartimide prone

peptides.

3.2.1.6 TAL reaction with fully unprotected peptide thiocarboxylic acids

From then on, we focused on studying TAL coupling with fully unprotected peptides. This will

allow us to learn about the chemo-selectivity of the ligation through our TAL approach. For that,

initially we sought to perform a reaction with a purified fully unprotected peptide thiocarboxylic

acid. Therefore, the peptide thioacid NH2-Asp(SH)-Tyr(OH)-OH 172 was selected and ligated to

the lissamine azide 114 in a mixture of 1:3 water and MeOH (with a concentration of about

300 mM). Reaction was completed within a couple of minutes as anticipated, and gave quantitative

yield of the product 185 (Scheme 37A). If we wanted to perform this reaction in proteins, there

would be a higher chance that the concentration of proteins in the medium could be very low.

Therefore we decided to reduce the concentration and see how that effects the reaction. When we

performed the reaction between 172 and 114, just by reducing the concentration of starting

materials to about 50 mM in solvent mixture of water and MeOH (1:3), not surprisingly, the

reaction took around 1.5 h to complete, nonetheless the reaction gave quantitative yield of the

anticipated product 185.

Scheme 37. Ligation with fully unprotected peptide thiocarboxylic acid and lissamine rhodamine B 5-

sulfonyl azide. A) Ligation with purified and an excess of peptide; B) Ligation with in-situ generated peptide

and an excess of sulfonyl azide.

Subsequently, we investigated the TAL reactions of the fully unprotected thiocarboxylic acid,

which was in-situ generated from Boc-Aap(STrt)-Try(Ot-Bu)-Ot-Bu 165 by using 60% v/v TFA

solution. Two equivalents of lissamine azide 114 were reacted with thiocarboxylic acid in a mixture

of water and MeOH (1:3). The reaction was completed within 5 min and gave satisfactory yield of

product 185 (Scheme 37B). If you compare both approaches (Scheme 37A&B), again shows that

the superiority of the in-situ approach on overall yield, and on top of that, one less chromatographic

purification step.

Scheme 38. Ligation with fully unprotected (in-situ) peptide thiocarboxylic acid and dansyl azide.

The slow reactive dansyl azide 116 was then investigated with a fully unprotected peptide thioacid.

In-situ generated thiocarboxylic acid from Boc-Asp(STrt)-Ala-Ot-Bu 163 was reacted with 2

equivalents of dansyl azide 116 in methanol. The reaction took around 2 h to complete as expected,

and gave satisfactory yield of the product 186 (Scheme 38).

Scheme 39. TAL reaction complex peptide thioacid with dansyl azide.

Last but not least, we sought to investigate in-situ TAL strategy in a bit more complex peptide.

Earlier in our lab O. Keiper100 synthesized a pentapeptide thiocarboxylic acid precursor using the

Tmob protecting group strategy. The acidolysis of this thioester, Boc-Tyr(Ot-Bu)-Ala-

Asp(STmob)-ψ-Thr-Val-Ot-Bu 187 can generate corresponding peptide thiocarboxylic acid H-

Tyr-Ala-Asp(SH)-Thr-Val-OH via acidolysis. Therefore we sought to use this peptide for in-situ

TAL strategy, to generate a sulfonamide bond on its ω-aspartate residue. The Boc-Tyr(Ot-Bu)-

Ala-Asp(STmob)-ψ-Thr-Val-Ot-Bu 187 was treated with 60% v/v TFA and co-evaporated with

toluene at room temperature under vacuum to minimize aspartimide formation. Subsequently the

obtained crude thioacid was reacted with two equivalents of dansyl azide 116 for 2 h. The reaction

gave the anticipated dansyl conjugate, H-Tyr-Ala-Asn(Dn)-Thr-Val-OH 188 with a good overall

yield of 58% (Scheme 39).

To sum-up this section, the aforesaid experiments clearly indicate that the TAL chemistry is indeed

very chemoselective in peptides. In addition, it can be successfully used for labeling peptides with

biophysical probes effectively and site selectively. The lissamine rhodamine B 5-sulfonyl azide

114 surfaces, a novel and fast fluorescence labeling prospect in TAL chemistry. Even though

aspartimide formation and secondary reaction products still possess a challenge to this strategy, yet

labeling aspartyl residue of peptides/proteins with this strategy is almost equivalent or one par

ahead of other available methods.

Generation of natural N-glyosidic bond using TAL chemistry

It has been estimated that N-linked protein glycosylation accommodates 90% of all glycoproteins

in mammalian cells.134 The core glycosylation commonly occurs at asparagine (Asn) residues in

the sequon Asn-Xxx-Ser/Thr, where Xxx is any amino acid except proline.135 The Fmoc-based

solid-phase peptide synthesis (SPPS) is one of the most successful chemical strategy used for the

synthesis of N-glycopeptides.136 The SPPS proceeds in a step-wise manner employing several

equivalents of building blocks. Although it is a very efficient strategy, this method requires a large

excess of precious glycosylated amino acids and protecting groups manipulations. Advantageous

might be the convergent synthesis in which, for example, an aspartic acid containing peptide is

condensed with a glycosylamine at the final stage of synthesis.123, 137 However, most known

protocols lead to the extensive formation of by-products such as aspartimides and their secondary

reaction products. In addition, orthogonal protection of aspartic acid residues is demanded which

requires additional synthetic steps.

In the previous section, we presented the applications of using TAL to obtain neoglycopeptides at

the aspartyl residue. By combining very reactive sulfonyl azide derivatives with peptides bearing

thioacids in its side chains, we have successfully synthesized a neoglycopeptides 184 (Table 5,

entry 10). However, this unnatural glycopeptides possess sulfonamide linkage, so it is interesting

to investigate whether TAL chemistry can be used for synthesizing naturally occurring β-amide

linkage. It has been reported that the TAL chemistry can be exploited to synthesize organic amide

bonds. As mentioned in the introduction (Section 1.51) Williams et al.75 showed N-glycosylation

of glucose with natural amide bond using TAL chemistry. The reaction was very slow and required

vigorous (higher temperature) condition. In the same year, Fazio et al.,81 showed that the

acetylation of anomeric azides of sugars using RuCl3 promoted TAL chemistry. This reaction was

shown to be a bit faster and can be performed at room temperature. After a year later, Schmidt et

al.94 has presented an approach to synthesize S-neoglycopeptides using the TAL chemistry. They

coupled the glycosylthiomethyl azides with peptide derivatives containing aspartate and glutamate

thioacids, which then generated equivalent glycosylthiomethyl amides. However, as far as we

know, no one has attempted to synthesize N-glycopeptides with natural β-amide linkage using the

TAL chemistry.

For that reason, we sought to exploit the TAL reaction for the synthesis of glycopeptides with

natural N-linkage, and too in a convergent manner. Before we went to investigate this in peptides

we tested how the ligation would work in a test system. 3-phenylpropanethioic acid 77 was chosen

as thioacid for this investigation. Since N-glycans are connected to the aspartyl group of proteins

via a β-GlcNAc unit, we synthesized GlcNAc derivatives bearing an anomeric β-azide group. 2-

acetamido-2-deoxy-β-D-glucopyranosyl azide138 190, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-

D-glucopyranosyl azide139 192 and 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl

azide 140 191 were synthesized to see how the protecting group influences the ligation.

Scheme 40. Synthesis of β-1-azido glycosides starting from GlcNAc

Synthesis of all of these β-1-azido glycosides 190-192 were achieved from N-acetyl glucosamine

189 through reported synthetic procedures (Scheme 40). The azidation138 of N-acetyl glucosamine

189 using DMC gave 2-acetamido-2-deoxy-β-D-glucopyranosyl azide 190 with 73% yield.

Subsequent acetylation of 190 gave 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl

azide 192 with 93% yield, while the benzylation of 190 gave 2-acetamido-3,4,6-tri-O-benzyl-2-

deoxy-β-D-glucopyranosyl azide 191 with a yield of 81% (Scheme 40).

Scheme 41. Reaction of 3-phenylpropanethioic acid with derivatives of β-1-azido glycosides

The 3-phenylpropanethioic acid 77, which was in-situ generated from corresponding thioester 76

was one by one reacted to the aforementioned β-1-azido glycosides 190-192. All of these reactions

were performed at an elevated temperature of 60 oC and kept for 2.5 days (Scheme 41). The

reactions with unprotected GlcNAc azide 190 was performed in water with equimolar w.r.t

thiocarboxylic acid amount of base NaHCO3 and gave 69% yield of glycoside 193. The reaction

of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide 192 was performed in

chloroform with 2,6-lutidine as base, in equimolar amounts compared to the thiocarboxylic acid,

and obtained 71% yield of glycoside 194. However, the reaction between in-situ generated 3-

phenylpropanethioic and 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide 191

performed in chloroform with 2,6-lutidine as base resulted in a low yield of 24% of glycoside 195.

Nevertheless, we recovered the unreacted sugar azides in all of these cases and found no secondary

reactions of these sugar azides, such as reductions, during the TAL chemistry. Therefore, based on

the reacted sugar azides, the reaction could give quantitative yield of the product.

Scheme 42. Synthesis of naturally linked N-glyco amino acids and a peptide.

After that we looked whether the TAL can be used to generate N-glycosidic amide bonds at the

aspartyl residue. Firstly tribenzylated GlcNAc azide 191 was reacted to in-situ generated

thiocarboxylic acid from Boc-Asp(STrt)-Ot-Bu 159. The reaction mixture was stirred in

chloroform at a temperature of 60-65 oC for 3 days, but gave only low yield of required glycosyl

conjugate 196 (Scheme 42A). The exact reason for the low reaction yield with tribenzylated sugar

azide 191 is unknown, but the poor solubility in chloroform might be contributing to it. The reaction

of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide 192 with the in-situ

generated Boc-Asp(SH)-Ot-Bu, gave a very good yield of required N-glycosyl amino acid141 198

(Scheme 42B).

Thereafter with much anticipations, we sought to synthesize N-glycopeptide using aforesaid TAL

approach. Aan in-situ generated peptide thiocarboxylic acid from its precursor 162 was reacted

with acetylated sugar azide 192. However, the reactions gave only low yields of the required

product, N-glycopeptide 198 (Scheme 42C). Not surprisingly, we observed extensive aspartimide

formation and its secondary products in LCMS, which obviously caused the lower yield. However,

it was not possible to isolate or quantify the amount of aspartimide formation in this TAL because

of its secondary products.

Scheme 43. Aspartimide formation during TAL conditions

Since we were unsuccessful in purifying the aspartimide generated from the aforesaid TAL

experiment, it was challenging to prove its occurrence during the ligation. Therefore we carried out

a separate reaction with a peptide thioacid, using the TAL conditions but without azide to make the

purification simple. Accordingly the in-situ generated thiocarboxylic acid from the Boc-Asp(STrt)-

Val-Phe-Ot-Bu 166 was treated with equimolar concentration of 2,6-lutidine in chloroform at 60-

65 oC for 2 days. A quick chromatographic purification of the reaction mixture gave 41% of the

aspartimide, Boc-Asi-Val-Phe-Ot-Bu 199, together with its secondary products. The secondary

products were not quantified or characterized, since their purifications were unsuccessful.

Figure 12. Proposed mechanism of aspartimide formation during TAL reaction

We assume that the aspartimide formation follows the published reaction mechanism of base

catalyzed reaction of aspartyl esters .142 Even in the presence of the base in the TAL reactions, we

anticipate that some amount thiocarboxylic acid still present in the protonated form, this would

make the hydrogensulfide moiety of thioacids act as a leaving group during any nucleophilic attack.

Therefore, we propose a mechanism for the aspartimide formation as shown in Figure 12. The

intermolecular nucleophilic attack on thiocarboxylic group at the ω-aspartyl residue and

subsequent elimination of the hydrogensulfide group yields the corresponding aspartimide. Since

the reaction takes place in presence of base, the acidic α-proton of the aspartyl residue takes part

in a double keto-enol tautomerism, which is driven by aromaticity of the subsequent pyrrole ring

of the intermediate 203. This eventually causes an epimerization of the stereochemistry at the

aspartyl residue. As a result of this, any nucleophilic attack (e.g. hydrolysis) on the aspartimide can

generate four isomeric compounds -two stereoisomers of 205 and 206 each- which make the

separation and characterization of the secondary products difficult.

Further, we attempted to use TAL chemistry to synthesize fully unprotected natural N-glycoamino

acid and N-glycopeptides. Boc-Asp(STrt)-Ot-Bu 159 and Boc-Asp(STrt)-Val-Ot-Bu 162 was

completely deprotected using 60% v/v TFA solution. After the solvent evaporation, the crude

compounds were then reacted to 2-acetamido-2-deoxy-β-D-glucopyranosyl azide 190 in water by

using NaHCO3 as base. Both reactions failed due to difficulties in chromatographic purifications.

LCMS analysis of the reaction indicated that there is some conversion to the product. As the

compounds are very polar neither flash chromatography nor HPLC were found to be effective to

isolate the compounds. Yet we were convinced that fully deprotected ligation with relatively large

peptides would resolve the trouble with purifications and provide an access to get natural

glycopeptides, however, that investigation is not part of this thesis.

Since we observed aspartimide formation during peptide TAL reactions, we sought whether the

reaction conditions can bring down to room temperature with the help of a catalyst, inspired by the

work of Fazio et al.81, in which they used RuCl3 in TAL to bring down the temperature and reaction

time. However, they worked only with thioacetic acid as thioacid, but several types of electron-

rich azides. Therefore, it was interesting to investigate whether RuCl3 or other similar catalysts can

be used to reduce the aspartimide formation during the TAL with sugar azides. Conversely, our

works on using catalysts, such as RuCl3, OsCl3, FeCl3, ZnCl2 etc. revealed that the catalyst does

more harm than good to the ligation in peptides. We observed not only extensive aspartimide

formation but also observed reduction of azides to the corresponding amines with some of the

catalysts. In addition, the chromatographic purifications were obstructed as the catalysts were

found to co-elute with the targeted compounds. Therefore extensive investigation on the concept

of catalyst promoted TAL reaction was discontinued presuming that the new side reaction -the

reduction of azide- together with an extensive aspartimide formation would heavily diminish the

advantage of the strategy.

To sum up this section, we have shown a new synthetic route to obtain glycoaminoacids and

glycopeptides with natural N-glyosidic linkage. In addition, the TAL synthetic strategy could be

used as a convergent strategy to achieve larger N-glycopeptides with natural linkage. Poor yield

duiring the synthesis of N-glycopeptide 198 leaves a space for improvement. Nevertheless, we

proposed the mechanism of aspartimide generation and isolated the corresponding by-products,

and presumably it can be used to realize an improvement in the reaction conditions for the TAL.

TAL CHEMISTRY IN PRESENCE OF FREE SULFHYDRYL MOIETY

As stated in the introduction, the sulfonyl azides have previously been used in the biological context

by many research groups and also in the TAL chemistry.62, 76, 78, 79, 143 Hypothetically, electron

deficient sulfonyl azides could react with any nucleophilic group on proteins, especially free

amines and thiols (e.g. cysteines or lysines). Nevertheless according to literatures78, 143 and our own

work with unprotected peptides evidently showed that sulfonyl azides are not affected by free

amine groups. However, it has been reported earlier that the thiol (e.g cysteines) can reduce a

sulfonyl azide into a sulfonamide.78, 143 Liskamp et al.78 described that the thiol reduction of

sulfonyl azides is very slow compared to the competitive TAL reaction. On top of everything, the

reduction of sulfonyl azide does not interfere with the protein labeling or conjugation itself, as the

reduction product (sulfonamide) does not covalently link to the proteins. In addition, the oxidized

disulfide can be reduced back into cysteine using mild reducing agents. However avoiding

reduction during TAL and gaining chemoselectivity for the ligation still has a huge value.

Scheme 44. Representation of TAL in presence of a cysteine residue.

Electron deficient lissamine rhodamine B 5-sulfonyl azide 114 is the fastest reacting azide that we

used in TAL chemistry. It is possible that the rate of the reduction as well differs with dissimilar

azides, even among different sulfonyl azides. Since we are promoting the lissamine azide 114 as a

potential site specific fluorescence reagent, it is worth to investigate how fast it can be reduced in

the presence of thiols. In addition we would like to know, whether the selection of different bases

has any influence on the thiol-reduction of sulfonyl azides. Besides, it is also interesting to

investigate the speed and the adeptness of TAL reactions in the absence of free thiol but in the

presence of different bases. Therfore the following work was carried out, together with Sören

Radke as part of his bachelor thesis.144

To study the competitive reactivity of sulfonyl azide both in the case of reduction and TAL, it is

required to perform the TAL in presence of cysteine. On the other hand, it is hard to synthesize a

peptide having both free thiol and thioacid residues. In addition, the quantification of the reaction

products and its secondary products would be really difficult, if the thiol and thioacids together are

in a single peptide. Therefore we sought to analyse the competitive reactions of sulfonyl azide with

equimolar amounts of cysteine and thioaspartic acid derivatives (Scheme 45). These individual

amino acids together will represent the scenario of a peptide/protein having both thioacid and thiol

and it is easy to quantify via LCMS/HPLC. To help an accurate quantification of reaction peaks,

we sought to introduce UV-traceable moieties in all the reactants.

Scheme 45. The reactions of sulfonyl azide with thioacid and thiol

As lissamine azide is a fluorophore, it can be easily traced via UV detector, whereas, it is not the

case with the residual thioaspartic acid and cysteine. Therefore, we decided to use already

synthesized and UV-traceable thioaspartic acid derivative, Cbz-Asp(SH)-OBn 169. Fmoc-Cys-

OMe 207 was selected as the cysteine derivative, which was synthesized by esterification of the

commercially available Fmoc-Cys-OH by using a reported procedure.145

Figure 13. Structure of the cysteine derivative, Fmoc-Cys-OMe

Influence of bases on TAL chemistry

The standard bases used for catalyzing TAL reactions, such as 2,6-Lutidine and NaHCO3, have

pKa values of 6.65 and 6.35 respectively in water.146, 147 Hence it is worth to map how the basicity

of other bases (with similar and dissimilar pKa values) influence both reduction and ligation of the

sulfonyl azides. We selected a number of widely used bases for this purpose, based on their pKa

values from 2 to 13. In addition, we carefully weighed on the non-nucleophilic nature for the

nitrogen bases as they are capable of reacting with the thioacid by themselves. Accordingly we

chose TMU, DTBP, ascorbate, acetate, bicarbonate, citrate, DABCO, DIPEA and DBN, as bases

for the screening (Table 6).

Table 6. Reported pKa values of selected bases for the screening.

Bases pKa (in H2O)

1,1,3,3-tetramethylurea (TMU) 2148

2,6-Di-t-butylpyridine (DTBP) 3.58 in 50% EtOH149

Sodium ascorbate 4.17150

Ammonium acetate 4.76151

NaHCO3 6.35146

Trisodium citrate 6.39152

2,6-Lutidine 6.65147

1,4-diazabicyclo[2.2.2]octane (DABCO) 8.82153

N,N-diisopropylethylamine (DIPEA) 10.75147

1,5-Diazabicyclo[4.3.0]non-5-en (DBN) Ca. 13154

Since the concentration of both reactants and products could change during the reaction, a reference

peak is required for an absolute quantifications of the substance peaks. The reference substance

must be inert to the reaction condition, preferably has an intense signal in UV and should give a

distinct peak in the chromatograph of LCMS/HPLC. Anthracene was selected as the reference

compound, as it can easily be tacked via LC-MS and its corresponding UV-signal (peak) is well

separated from other reaction peaks. We also anticipated that, as it does not have any reactive

groups, the anthracene will stay inert throughout the TAL chemistry.

Further, we tried to find a solvent system to perform the reaction. It is necessary to find a solvent

system in which all the reactants, bases and anthracene is soluble. Preliminary solubility studies

revealed that it is hard to dissolve all the reagents in a single solvent system, as they are very diverse

in their polarity ranging from non-polar anthracene to polar thioacid and bases. Therefore the

possibility of using mixtures of solvents were examined. We managed to get all the reagents soluble

in a mixture of MeOH, DCM and THF (1:1:1). Therefore, a stock solution of the hardly dissolving

reagents (lissamine azide 114 and anthracene) were prepared in advance in this solvent mixture,

which was used for the subsequent investigation.

In the previous section, we noticed that the ligation of lissamine azide 114 with thioacids occurs

really fast, which makes it challenging to analyze the reaction via LCMS/HPLC, as the

measurements take more time than the entire reaction time. However, we as well observed in the

previous section that the reaction indeed slows down proportionally when the reagent

concentrations in the medium were decreased. Bearing that in mind, concentration of the reagents

were reduced by a factor of ten compared to the original concentration (0.3 M), by taking

approximately 30 mM of lissamine azide 114 along with rest of the reagents (half equivalent

compared to the lissamine concentration).

After having all the substances and methods in place, a model reaction of Thioacid Azide Ligation

with Cysteine (TAL+C) was carried out. As shown in Scheme 46, one equivalent of thioacid 169

and thiol 207 were reacted to two equivalent of lissamine azide 114 in a mixture of 1:1:1

MeOH/DCM/THF with the base 2,6-lutidine. The purpose was to identify the newly generated

product peaks and optimize the LCMS gradient to get the peaks well separated for the integration

(Graph 4). We have identified five new peaks that are corresponding to, the expected ligation

product 173, reduction product 209 and oxidation product of cysteine 208 as well as three

unexpected oxidation products of the thioacid 210, 211 and 212. It was interesting to observe that,

the amount of these thioacid oxidation products could be decreased by excluding oxygen from the

medium. However, we find it hard to keep entire methodology (ligation plus LCMS analysis)

oxygen free. Therefore, the screening experiments were performed without absolutely excluding

oxygen, anticipating that the air oxidation would not affect the fast ligation (TAL performs quicker

under normal reaction concentrations). For plotting the progress of the ligation, it was required to

calculate the absolute yield of the newly generated (product) peaks and therefore the compounds

173, 209-212 were isolated on a preparative scale. However, the compound 208 was neither

isolated nor studied in this experiments. The reason is that, it was always found in very small

quantity and very close to comparatively larger reference peak of anthracene (Graph 4).

Afterwards, a standard solutions of all the substances were prepared. Known quantities of

compounds were chromatographed by using an optimised LCMS method file and UV peak

intensities were integrated. Integration of these peaks gave correlations of the quantity of

substances to their peak intensity, which was sufficient for recalculating the amount of substances

from the reaction chromatograph.

Scheme 46. Thioacid azide ligation with cysteine (TAL+C)

For the LCMS reaction profiling, the sample volume must be taken out in a regular interval of time

and the undergoing reactions must be quenched till the measurements are completed. We chose 30

minutes time interval for profiling through an optimized LCMS method (single LCMS

measurement takes up to 30 min) and quenched the reaction by further diluting the sample with

acidified solvents. Because of the high sensitivity, the MS-detector was connected to the machine

only for the identifications of peaks. Once the peaks were identified, the MS was disconnected

from LCMS and the sample volume was increased in order to lower the error range of the UV-

signal quantifications. Taken out samples were measured immediately to avoid any misleading side

reactions.

Graph 4. HPLC chromatogram of TAL+C with anthracene in presence of 2,6-lutididne.

Subsequently, we started to investigate the influence of selected bases in TAL+C chemistry. All

the experiments were carried out as oxygen free as possible by performing it under inert atmosphere

and using de-oxygenated solvents. The compounds 169, 207, 114 and anthracene together

dissolved in a solution of THF:MeOH:DCM (1:1:1) and divided into 9 portions. In each of the

portions, one of the bases mentioned in Table 6 (except NaHCO3) were added. Relative intensity

of target peaks were integrated before adding the base (which was taken as the zero time

measurement), and after adding the base, 30 minutes intervals for 1.5 h. The reactions were

analysed and we could conclude that progress of the reaction were completed after 60 minutes

measurement, therefore, the final yield of the substances were calculated from 60th minute samples.

The percentage of the relative consumptions and formations of substances were calculated by

taking anthracene peak as the reference. It was interesting to observe the influence of different

bases on the reactions, the details of the findings were described in Table 7.

208

Product (173)

Table 7. Screening the TAL+C with bases. * The yields of by-products could be influenced by presence of

dissolved oxygen; # Maximum possible yield of the substance in comparison to the thioacid.

The general observation from the experiments conducted in Table 7 was that the basicity of bases

had a significant influence on the ligations and the side reactions. Best results for the ligation was

obtained when the pKa aq. values of the bases were between 4 and 7 (Graph 5). Among these bases,

2,6-lutidine gave the best result for the ligation product (Table 7, entry 6) followed by citrate (Table

7, entry 5), then acetate (Table 7, entry 4) and ascorbate (Table 7, entry 3). Interestingly, the

ammonium acetate gave a lower reduction of sulfonyl azides followed by citrate and then only

lutidine. Although ascorbate gave relatively good yield of ligation product, we observed that it was

heavily reducing the sulfonyl azides by itself (Table 7, entry 3). Even though ascorbate gave higher

amount of the sulfonyl azide reduction, the oxidations of cysteine and thioacid stood same level

compared to the other bases. It could be that the ascorbate only reduces the sulfonyl azide but no

oxidized by-products, especially the cystine back to cysteine.

The TAL+C chemistry with bases such as TMU and DTBP (pKa aq. <4), we observed no ligation

product even after 60 minutes (Table 7, entry 1-2). Whatever the case may be, there was no

detectable consumption of lissamine azide 114 nor the cysteine 207, excluding some oxidation of

the thioacid. However when we performed the TAL+C using a base with comparatively higher

pKa, DBN (pKa aq = ca. 13), there was no ligation, but we observed severe oxidation and

decomposition of starting materials especially the thioacid (Table 7, entry 9). When we attempted

the TAL+C with DABCO and DIPEA as bases we found cysteine reduction of sulfonyl azide rather

than the demanded ligation, although, the reactions gave satisfying amount of ligation product

(Table 7, entry 7-8).

Graph 5. Comparison of product yields over 60 min with selected bases in TAL+C.

Graph 6. TAL+C chromatograph in presence of 2,6-lutidine and DTBP, at zero time and after 30 min of

ligations.

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70

pe

rce

nta

ge (

%)

time (minutes)

Yield of the ligation product over time

"Lutidine"

"Citrate"

"Acetate"

"Ascorbate"

"DIPEA"

"DABCO"

While concluding the experiments shown in Table 7, we can say that the ammonium acetate

catalyses TAL+C almost as good as 2,6-lutidine. Besides that, ammonium acetate gave 30% less

sulfonyl azide reduction, which could be interpreted as it can give a better selectivity for TAL+C

chemistry. In addition, the experiments here shown that the bases like ascorbate and citrate could

give fair amount of ligation product in the TAL+C chemistry. Therefore we believe that in the

absence of cysteine (free thiol), the bases such as acetate, ascorbate, citrate, DABCO and DIPEA

could be used for catalysing the TAL. When the results obtained from pyridine bases 2,6-lutidine

and DTBP were compared, interestingly a huge difference in the catalysis of ligation was observed.

The 2,6-lutidine gave a very fast ligation to the product, while the DTBP did not catalyze the

ligation at all (Graph 6). This result support the notion that, if the pKa value of the bases go below

4 (pKa value for thiocarboxylic acid is around 4) the ligation does not take place, as the thioacid is

required to be deprotonated.

As we learned that new bases were catalysing the ligation in TAL+C chemistry, it would be

interesting to investigate those bases in TAL (or TAL-C) chemistry as well. Therefore we selected

the two bases that performed the best (acetate and citrate) in the TAL+C chemistry together with

2,6-lutidine. In addition, an inorganic base NaHCO3 was selected for screening as it is a very

commonly used base in TAL and has a pKa value close to 2,6-lutidine. Accordingly, these four

bases were screened for their catalytic performance in the TAL-C (TAL) chemistry. As shown in

Table 8, all of the reactions with selected bases gave quantitative yield of product under 60 min of

reaction. In the case of citrate, we have observed 25% of the reduction product (sulfonamide) as

well, which could be interpreted as the citrate here itself is acting as a reducing agent. As we took

an excess of sulfonyl azide for the reaction, the citrate reduction did not influence the yield of

ligation product, which would place the citrate back on as a TAL catalyst.

Table 8. Screening bases in TAL chemistry without cysteine. # Maximum possible yield of the substance

compare to the thioacid.

When the yields of the ligation products obtained from TAL+C (Table 7 entry 6) and TAL-C (Table

8 entry 1) were compared (both reactions were catalysed by 2,6-lutidine), unsurprisingly we found

that the latter was much better (Graph 7). The loss of yield in the presence of cysteine (thiol) can

be explained as the ligation is deterred by inducing the thioacid oxidations. However, this oxidation

would be insignificant if the ligations were performed under oxygen free conditions or with a

concentrated reaction medium. Our assumption is that the use of diluted media increased the

amount of dissolved oxygen and prolongated the ligation time, which favour the oxidation of

thiocarboxylic acid over the ligations.

Graph 7. Comparing the ligation product obtained from TAL+C and TAL-C

Scheme 47. Reaction between sulfonyl azide and cysteine.

As we observed that the cysteine induces the oxidation of thioacid, it was interesting to see how

fast cysteine alone will reduce the sulfonyl azide 114 in the absence of thioacid. For that as shown

in Scheme 38, we performed a reaction just with cysteine and sulfonyl azide in the presence of

catalyst 2,6-lutidine. The reaction was monitored via LCMS for 5 h, and a graph was plotted to

monitor the amount of reactants and products over time (Graph 8). It was surprising to see that the

reduction in the absence of thioacid is much slower compared to the reaction in the presence of

thioacid. It took around 5 h to reduce just over 55% of sulfonyl azide and around same amount of

free cysteine was left unreacted. We found equivalent amounts of reduction product with respect

to the consumed sulfonyl azide and thiol.

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

pe

rce

nta

ge (

%)

time (minutes)

Yield of the ligation product with 2,6-lutidine

"TAL-C"

"TAL+C"

Graph 8. Reaction profile of cysteine reduction in the absence of thioacid

When we compared the results obtained from sulfonyl azide reduction in the presence (Table 7,

entry 6) and absence (Scheme 47, Graph 8) of thioacid, it was clear to us that the sulfonyl azide

reduction due to cysteine (thiols) could be massively catalyzed by the presence of thioacids. The

experiment which performed just with thioacid and sulfonyl azide indicated that the thioacid cannot

itself reduce the sulfonyl azide to sulfonamide (Table 8, entry 1). When we compared the reduction

product (sulfonamide) obtained from the experiments shown in Table 7, entry 6 (TAL+C) and

Scheme 47 (cysteine reduction of sulfonyl azide), revealed that the thioacid could induce the rate

of the sulfonyl reduction to almost a factor of two (Graph 9). It is not clear to us that why and how

the thioacid induces the sulfonyl azide reduction with thiol or vice versa. Finding an explanation

requires comprehensive mechanistic investigations which is beyond the scope of this thesis.

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350

pe

rce

nta

ge (

%)

time (minutes)

Reaction of thiol and sulfonyl azide

LissNH2

LissN3

Thiol

Graph 9. Reduction of sulfonyl azide in presence of cysteine with and without thioacid.

Taking excess of sulfonyl azide in TAL+C chemistry does not give much information about how

competitive the reduction over the ligation. The problem is that, both the reaction could advance

until both thioacid and thiol were consumed completely. In addition it is hard to track the reactions

of thioacid and thiol as they yield multiple products. Therefore it is interesting to investigate

TAL+C chemistry by taking an excess of thioacid and thiol compared to sulfonyl azide. Since the

sulfonyl azides react only to the ligation and the reduction product, it is easy to track their

competitiveness. Therfore with this approach, whichever reactions perform faster will give a

maximum of corresponding lissamine product. So it is much easier to quantify and compare the

reaction rates.

Into the bargain, we selected two bases (2,6-lutidine and acetate) to screen their catalytic property

by using aforesaid TAL+C approach. As shown in Table 9, in both cases, the sulfonyl azide was

completely consumed within 30 minutes of the reaction. However, an excess of the reduction

product were observed compared to the ligation product with both catalysts. This could be clearly

interpreted as the fast reduction of sulfonyl azide with thiol compared to the ligation with thioacid

in TAL+C chemistry. This finding is contradictory to the published result by Liskamp et al.78 where

they described that the preferred coupling was faster than the concurrent reduction. It could also be

that different sulfonyl azide reacts differently with free thiols and thioacids. Nonetheless, when the

two catalysts were compared to each other, interestingly, the ammonium acetate gave better

product to by-product ratio in TAL+C chemistry (Table 9).

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70

pe

rce

nta

ge (

%)

time (minutes)

Yield of the reduction product

TAL+C

Thiol

Table 9. TAL+C with an excess of thiol and thioacid. # Maximum possible yield of the substance compare to

the lissamine azide..

To sum up the results shown in this section, we have identified new bases in which the TAL

reactions will occur fast and in a quantitative yield of ligation products. In addition, sodium acetate

appeared to be a better base than standard lutidine in terms of product to byproduct ratio in the

TAL+C chemistry. Interestingly, these experiments shows that the presence of thioacid catalyzing

the reduction of sulfonyl azide in TAL+C, which is leading to reduction more than to ligation

products. Notably, we have discovered that new bases such as acetates, ascorbates and citrates

could be used for TAL chemistry to generate quantitative yield of ligation products. Last but not

least, we have observed in our case that thiol reduction of the sulfonyl azide is slightly faster than

preferred ligation.

Further study of lissamine azide reduction in the presence of hydrogen sulfide

It was reported before that dansyl azide127, 128, 155 and other azides156 could be used as a fluorescent

probe for detecting hydrogen sulfide at very low concentrations. Most of the reported reduction

takes hours to complete and would affect the real time analysis of hydrogen sulfide detection, which

is significant for biological applications. Therefore it is interesting to develop azide based

fluorescent reagents which gives a fast reduction in presence of thiol and that is fluorogenic. As we

found the lissamine azide 114 is very prone to thiol reduction, we sought to investigate its

applicability for detecting hydrogen sulfide. Primarily we sought to find how fast the reduction of

114 occur in presence of dissolved hydrogen sulfide, and subsequently whether the generated

lissamine rhodamine B 5-sulfonamide 209 has any difference in its fluorescence properties

compared to the lissamine azide 114.

Scheme 48. Reduction of lissamine rhodamine B 5-sulfonyl azide in presence of H2S.

To answer our first question, the lissamine azide 114 was reacted to NaSH/H2O in ethanol as shown

in Scheme 48. Remarkably the reduction proceeded extremely fast, completed within seconds and

gave quantitative yield of reduction product. After this encouraging result, same reaction was

performed under much diluted conditions to see whether the reduction of 114 slows down.

Astonishingly even with micro molar concentration of the reaction medium, the complete

conversion of sulfonyl azide 114 to sulfonamide 209 occurred under a minute. Amazed by this rate

of the reaction, we went further on to analyze its spectroscopic properties.

First of all for the spectroscopic data, the absorption and fluorescence spectra of lissamine

rhodamine B 5-sulfonyl azide 114 was measured (Figure 14 A&B). The compound has an

absorption maximum of 564 nm with a molar absorption coefficient of 124000 ± 3000 M-1 cm-1.

In addition, the fluorescence spectrum was measured by exciting at 505 nm wave length, which

showed an emission maximum at 587.5 nm. After that the absorption and fluorescence spectra of

lissamine rhodamine B 5-sulfonamide 209 was measured (Figure 14 C&D). We observed an

absorption maximum at 560 nm with a molar absorption coefficient of 77600 ± 10000 M-1 cm-1.

Its fluorescence spectra was measured by exciting at 505 nm and which gave an emission maximum

at 578nm.

Figure 14. A&B) Absorption and fluorescence spectrum of lissamine rhodamine B 5-sulfonyl azide and

determining its absorption coefficient; C&D) Absorption and fluorescence spectrum of lissamine rhodamine

B 5-sulfonamide and determining its absorption coefficient.

When the results were compared, there was a 40% decrease in the molar extinction coefficients

when the lissamine azide 114 was reduced to the lissamine sulfonamide 209. However, the

absorption maximum had only a small hypsochromic shift of 4 nm. Interestingly, we found a

hypsochromic shift of around 10 nm in fluorescence maximum though the shift is not sufficient

enough to use as the fluorescence prob. In spite of this, we went on further to analyze the reduction

reaction of sulfonyl azide 114 via fluorescence spectrometer to see whether there is any change in

fluorescence intensity. Suitably a reaction was performed in a cuvette by reacting lissamine azide

114 with NaSH. We measured the fluorescence spectrum of the lissamine solution, before and then

after the addition of the NaSH (Figure 15). The results showed that there is a gain in intensity after

the addition of NaSH. However the gain in intensity is only 10-20% at λmax. Therefore using

lissamine azide as a H2S sensor molecule is not viable, as the fluorescence of unreacted sulfonyl

azide itself lead to a higher background signal. That would make it harder to accurately evaluate

any titration experiments for detecting hydrogen sulfide by using available fluorescence

instrumentations.

A

B

C

D

Figure 15. Fluorescence spectra before and after the addition of NaSH in to lissamine rhodamine B 5-

sulfonyl azide

To sum-up this section, we discovered that the lissamine azide can give fast reduction with

dissolved hydrogen sulfide. Unfortunately, difference in its maximum fluorescence gain during the

reduction to sulfonamide is not within the satisfactory range. However we have measured

absorption and fluorescence spectra of both the compounds and calculated their molar absorption

coefficient for the first time.

SUMMARY AND OUTLOOK

SUMMARY

The main goal of this project was to develop a methodology that can be eventually used for

chemoselective conjugation of biomolecules. Therefore the idea was to exploit the potentials of

thioacid azide ligation (TAL) in protein or peptide side chains. To achieve the goal, the project was

divided into three main objectives: (i) introducing the thioacid functionality into peptides/proteins

(ii) thioacid azide ligation in peptides/proteins (iii) investigating and minimizing potential non-

specific reactions during TAL. As whole proteins are very complex systems to study the new

hypothesis, we chose simple amino acids and peptides for our investigations.

In the first section of this dissertation, a couple of hypotheses were tested to find a suitable

thiocarboxylic acid protecting group that is compatible with SPPS. One of the hypothesis was to

use thiocarboxylic S-ester strategy, however, previous studies showed that the tert-alkyl/aryl type

of protective groups were found to be ineffective in SPPS. The tert-alkyl type protection in

thiocarboxylic S-ester was found to be quite stable towards nucleophilic attack, but it was quite

challenging to convert them back to thioacid. On the other hand, the tert-aryl type of protection

was found to be prone to nucleophilic attack but could be easily deprotected to thioacid. Bearing

that in mind, two new protecting groups were designed by taking the steric hindrance of tert-alkyl

groups and +M effect of tert-aryl groups, i.e. 2-methoxypropane and 2-acetoxypropane. The

obtained results showed that the designed moieties does not fulfill the requirements, however the

results affirms that, the +M effect and –I effect on tert-alkyl moieties of the thiocarboxylic S-ester

can strongly influence its deprotection to thioacid. Taking that in mind, it would be worth to

investigate similar type of protecting groups varying +M effects and ±I effects at the substituting

group (Figure 16).

Figure 16. Proposed thiocarboxylic S-ester protecting groups

Afterwards, a systematic investigation of the concept of thiocarboxylic O-esters as protecting group

was carried out. The O-ethyl 3-phenylpropanethioate 91, O-isopropyl 3-phenylpropanethioate 92

and O-(2,4-dimethylpentan-3-yl) 3-phenylpropanethioate 93 were investigated for their stability

towards piperidine treatment. In contrast to our anticipation, thiocarboxylic O-esters were found to

be more prone to nucleophilic attack than the analogous thiocarboxylic S-esters. Further, safety

catch hypothesis by using phenyl hydrazine and 3,4-diaminobenzamide were attempted. The

phenyl hydrazine strategy was not compatible as the activated form of the hydrazide,

phenyldiazenyl was reduced back to hydrazide during the substitution with sulfhydryl moieties.

The 3,4-diaminobenzamide 104 strategy was reported to be compatible with sulfhydryl moieties,

however in case of ω-aspartyl residue, its free amino group was found to give side reactions.

Although protecting the free amine functionality would be beneficial to reduce its side reactions,

however, concurrent research on different protecting group strategies revealed that slightest

activation (which is required for safety catch approach) on ω-aspartyl residue could yield enormous

aspartimide formation. Therefore, further investigation on the safety catch strategy was

discontinued. Although no protecting group that matches all the requirements was found, the

obtained results are valuable for designing new and better classes of potential thioacid protecting

groups.

In the second part of this dissertation, ligation reactions of peptide thioacids with azides were

studied. For that, a variety of azide derivatives carrying biophysical probes, such as biotin 121,

124, dansyl 116, lissamine 114 and glycans 117, 110-192 were synthesized. Assignment of the

regio-isomers of lissamine rhodamine B sulfonyl azide were successfully performed by using

single crystal X-ray crystallography for the first time. Furthermore, the molar absorption coefficient

of lissamine rhodamine B 5-sulfonyl azide was determined to be 124000 ± 3000 M-1 cm-1.

Afterwards, we described the synthesis of several peptides carrying thioacids or their precursor

moieties as their ω-aspartate residue. Trityl thioesters were found to be excellent precursors for

generating thioacids on ω-aspartate residue of the peptides. The synthesized trityl thioester

derivatized peptides (159-168) includes the most aspartimide prone Asp-Gly residue to the least

aspartimide prone Asp-Val residue.

Scheme 49. Chemoselective TAL in peptide

Our results show that TAL can be successfully used for conjugating probes at the ω-aspartyl residue

of peptides. During the TAL reaction, the thioacid could react with sulfonyl azide derived lissamine

rhodamine B 5-sulfonyl azide 114 extremely fast compared to another sulfonyl azide derived

dansyl azide 116. This result shows that electron deficiency of the aromatic system can influence

the reactivity of the sulfonyl azides in TAL. In addition, the in situ generated peptide thioacids

derived from trityl thioesters can be efficiently used in TAL chemistry. This in-situ strategy could

be used for the reactions of peptide thioacids with other functionalities, such as amines (-NH2),

azides (-N3), aziridines, isocyanates (-N=C=O) and isonitriles (-N≡C). As next, chemoselective

conjugation of fully unprotected peptides with sulfonyl azides were examined. Very efficient and

chemoselective coupling between thioacid and sulfonyl azides in presence of free hydroxyl, amine

and carboxylic functionalities were observed (Scheme 49). By exploiting the reactivity of sulfonyl

azide with thioacid, a variety of peptide conjugates, which include a neoglycopeptide, were

synthesized in a very good yield. As a final point in this section, the N-glycoaminoacids and an N-

glycopeptide with natural amide linkage were obtained by using the TAL chemistry. It was

achieved by reacting derivatives of β-D-glucopyranosyl azide with the thioacid bearing amino acid

and peptide derivatives (Figure 17).

Figure 17. Structures of synthesized neoglycopeptide and N-glycopeptide using TAL chemistry.

In the final part of this dissertation, the potential side reactions of sulfonyl azides with sulfhydryl

moieties were investigated. It was shown that, in the presence of thiocarboxylic acid the reduction

rate of sulfonyl azide with thiol was increased two fold. In contrast to published results, the sulfonyl

azide reduction with thiol occurs with higher rate than concurrent ligation with thioacid. In

addition, the catalytic effectiveness of several bases was screened in TAL chemistry. We observed

that water soluble bases such as ascorbate, acetate and citrate could catalyze the TAL as good as

the standard base lutidine. Interestingly, our experiments showed that acetate could provide better

ligation to reduction product ratio than lutidine in TAL+C chemistry.

Nonetheless, it is important to note that the reduction of sulfonyl azides with sulfhydryl moieties

does not generate a covalent linkage. As it is convenient to use excess of labelling reagents rather

than peptides or proteins, the reduction of sulfonyl azide does not influence the ligation outcome.

Additionally, there are numbers of established methods for converting the oxidized cystine back to

cysteine. Therefore it can be concluded that, ligation of peptide thioacids by using sulfonyl azide

derivative could be effectively used to generate peptide conjugates even in presence of the

sulfhydryl moieties.

OUTLOOK

Figure 18. TAL chemistry in thioacid tagged proteins.

Site-specific modification of larger peptides and proteins using the TAL strategy is worth to

investigate. Therefore new methods for incorporating the thiocarboxylic acid functionality to larger

peptides and/or protein must be developed. It would also worth to investigate the possibility of

using expanded genetic code strategy for incorporating thioacid containing designer’s (unnatural)

amino acid into proteins. Incorporating structurally less complex thioacid derivatives of aspartate

and glutamate to the proteins by using amber codon suppression techniques is promising. This

technique will allow site specific tagging of proteins with minimum perturbation to their spatial

structure. Combining with TAL chemistry, the technique will allow a novel and site-specific

modification/labelling methodology of target proteins under physiological conditions (Figure 18).

Figure 19. Fluorogenic reagents used for hydrogen sulfide detection

In addition, development of novel classes of fluorogenic reagents compatible with TAL chemistry

would be beneficial. Fluorogenic reagents used for hydrogen sulfide detection could be applicable

in the TAL chemistry (Figure 19). Although, thioacids react with azides to amides unlike hydrogen

sulfide which reduce azide to amines, however, it is interesting to investigate how that affects the

fluorescence of those fluorogenic reagents.

ZUSAMMENFASSUNG UND AUSBLICK

Proteine sind die häufigsten und wichtigsten Bestandteile aller lebenden Organismen. Faszinierend

ist, dass Proteine viele unterschiedliche funktionelle und strukturelle Funktionen besitzen, jedoch

die Mehrheit der Proteine und ihre Rolle noch nicht erforscht sind. Chemoselektive Reaktionen

spielen heutzutage eine wichtige Rolle in der Forschung, da sich viele Forscher damit befassen,

neue Methoden zu entwickeln, um die Komplexität des Proteoms zu entschlüsseln.

Das Ziel dieser Arbeit war, eine Methode zu entwickeln, welche die chemoselektive Modifizierung

von Proteinen unter physiologischen Bedingungen erlaubt. Deshalb wurde in dieser Arbeit

versucht, das Potential der Thiosäure-Azide-Ligation (TAL) in Proteinen oder Proteinseitenketten

auszuschöpfen. Um unser Ziel zu erreichen, sollte der Forschungsfokus in drei große Ziele

unterteilt werden: (i) Das Einführen der Thiosäure als funktionelle Gruppe in Peptide/Proteine (ii)

Die Thiosäure-Azid-Ligation in Peptiden/Proteinen (iii) Mögliche Nebenreaktionen während der

TAL zu charakterisieren und minimieren. Da ganze Proteine sehr komplexe Systeme sind,

entschieden wir uns für Aminosäuren und kleine Peptide, um unsere Hypothesen zu testen und zu

erforschen.

Der erste Teil dieser Dissertation befasst sich mit dem seitenkettenspezifischen Einführen der

Thiosäuregruppe in Peptide. Generell müssen in der seitenkettenspezifischen

Festphasenpeptidsynthese Schutzgruppen eingeführt werden. In der Literatur sind jedoch keine

Thiosäure-Schutzgruppen beschrieben, welche seitenkettenspezifisch eingesetzt werden können.

Deshalb sollten erst einige Hypothesen getestet werden, um den Weg zu einer gut funktionierenden

Schutzgruppe zu ebnen. Dazu zogen wir Thiocarbonsäure-S-Ester, Thiocarbonsäure-O-Ester wie

auch unkonventionelle safety catch Schutzgruppen für die Thiosäuren in Erwägung. Anfangs

wurden 2-Methoxypropan- und 2-Acetoxypropanreste als Teil der Thiocarbonsäure-S-Ester-

Schutzgruppenstrategie untersucht. Anschließend wurden systematisch Thiocarbonsäure-O-

Esterals mögliche Thiosäureschutzgruppe erforscht. Im Widerspruch zu unserer Hypothese

beobachteten wir, dass die Thiocarbonsäure-O-Ester leichter als die S-Ester nukleophil angegriffen

werden. Zuletzt wird in diesem Teil der Arbeit der Versuch beschrieben, Phenylhydrazin und 3,4-

Diaminobenzamid als safety catch in ω-Position der Aspartatseitenkette eines Peptids einzusetzen.

Wie sich herausstellte, ist Phenylhydrazin Strategie nicht mit Sulfhydrylresten kompatibel und die

3,4-Diaminobenzamid-Strategie führt zu Nebenreaktionen. Obwohl die getesteten Schutzgruppen

nicht die benötigten Voraussetzungen erfüllen, können die gewonnen Erkenntnisse zum Entwerfen

neuer und besserer Thiosäure-Schutzgruppen genutzt werden.

Der zweite und gleichzeitige Hauptteil dieser Arbeit befasst sich damit, die Thiosäureligation in

kleinen Peptiden zu etablieren. Dazu wurde eine Auswahl an Azidderivaten synthetisiert, welche

biophyikalische Sonden wie Biotin, Dansyl, Lissamin oder Glykane tragen. Es wurde erstmals

erfolgreich eine Röntgenkristallstruktur von Lissamin-Rhodamin B Sulfonylazid erhalten, die es

ermöglichte, die verschiedenen Regioisomere zu charakterisieren. Darber hinaus wurde ein molarer

Absorptionskoeffizient von 124000 ± 3000 M-1cm-1 für Lissamin-Rhodamin-B-5-Sulfonylazid

ermittelt. Zudem wurde die Synthese diverser Peptide beschrieben, welche Thiosäuren oder deren

Vorläufermoleküle in ω-Position einer Aspartatseitenkette tragen. Unter diesen befanden sich

Peptide, welche die für Aspartimidbildung anfälligste Asp-Gly- und die unanfälligste Asp-Val-

Sequenz trugen. Unsere Ergebnisse zeigen, dass die TAL erfolgreich zur Konjugation von Peptiden

in der ω-aspartyl-Position eingesetzt werden kann. Während der TAL beobachteten wir, dass die

Thiosäure mit dem Lissamin-Rhodamin B 5-Sulfonylazid, verglichen mit einem anderen

Sulfonylazid, Dansylazid, wesentlich schneller reagiert. Ferner fanden wir heraus, dass aus Trityl

geschützten Peptidthioestern in situ erzeugte Peptidthiosäuren effizient in der TAL-Chemie

eingesetzt werden können. Im Anschluss wurden ungeschützte Peptide mittels TAL zu einem

Peptidkonjugat umgesetzt. Es wurde die Reaktivität eines Sulfonylazids mit einer Thiosäure

ausgenutzt, um ein Neoglycopeptid in exzellenter Ausbeute zu synthetisieren. Zuletzt wurden N-

Glycoaminosäuren und ein N-Glycopeptid mit einer natürlichen Verknüpfung dargestellt, indem

β-D-Glycopyranosylazid-Derivate in der TAL-Reaktion eingesetzt wurden.

Im letzten Teil dieser Dissertation wurden die Nebenreaktionen der Sulfonylazide in Gegenwart

von Sulfhydrylresten näher untersucht. Dabei beobachteten wir, dass sowohl in Gegenwart als auch

in Abwesenheit von Thiosäuren das Sulfonylazid vom Thiol reduziert wird. Die Reduktion wird

jedoch durch die Thiosäure katalysiert und verläuft somit schneller in Anwesenheit einer

Thiosäure. Widersprüchlich zur Literatur stellten wir fest, dass die Reduktion des Sulfonylazids

mit der Thiosöureligation konkurriert, diese dominiert. In weiteren Experimenten wurde die

katalytische Effektivität mehrerer Basen untersucht, welche 2,6-Lutidin als Standardbase in der

TAL-Chemie ablösen könnten. Dabei erwiesen sich Basen wie Ascorbat, Acetat und Citrat als

genauso gut wie Lutidin in der TAL Reaktion. Zuletzt beobachteten wir, dass Acetat ein besseres

Verhältnis von Ligation zu Reduktion als 2,6-Lutidin in der TAL Reaktion mit Sulfhydrylresten

erzielt.

EXPERIMENTAL SECTION

GENERAL METHODS

Technical solvents were distilled prior to use. Dry solvents were purchased from Fluka and Sigma-

Aldrich or dried by common methods. Reagents were purchased from Acros, Fisher Scientific,

Fluka, Glycon, Merck, MCAT and Sigma-Aldrich and used without further purification. Reactions

were performed, if necessary, with nitrogen as protecting gas using the Schlenck technique.

Analytical thin layer chromatography (TLC) was carried out on TLC silica gel 60 F254 coated

aluminum sheets (Merck) with detection by UV light ( λ = 254 nm). Additionally, following

reagents were used for visualization of spots if applicable: iodine chamber, ethanolic ninhydrin

solution (3 % w/v), anisaldehyde solution (135 mL EtOH, 5 mL H2SO4, 15 mL glacial acetic acid,

3.7 mL p-anisaldehyde), aqueous potassium permanganate (1 % w/v). After dipping into one of the

described solutions, heating was applied. Rf-values were determined under chamber saturation.

Preparative flash column chromatography (FC) was performed on silica gel Geduran 60 (40-60

µm, Merck) with solvent systems specified.

ANALYTICS

NMR Spectroscopy

NMR spectra were recorded on an Avance III 400 or 600 from Bruker at rt. 1H NMR chemical

shifts are referenced to residual protic solvent (CDCl3 δH = 7.26 ppm, D2O δH = 4.79 ppm, MeOD-

D4, DMSO-D6 δH = 2.50 ppm) or internal standard TMS (δ H = 0.00 ppm). 13C chemical shifts

are referenced to the solvent signal (CDCl3 δC = 77.1 ppm, DMSO-D6 δC = 39.5 ppm). Chemical

shifts are given as δ in ppm. For signal multiplicities the abbreviations s = singlet, br = broad singlet

d = doublet, t = triplet, q = quartet, quin = quintet and m = multiplet will be used below. When

feasible, assignments were supported by two dimensional correlation spectroscopy (COSY, HSQC,

TOCSY and HMBC).

Mass spectrometry:

ESI-IT mass spectra were recorded on an Esquire 3000 plus instrument from Bruker with electron

spray ionization or on a LC-MS instrument LCMS2020 from Shimadzu specified below. High-

resolution ESI-TOF mass spectra were recorded on a micrOTOF II instrument from Bruker.

Samples were prepared in MeOH, MeCN, THF, DMF and/or Water (approx. 1 µg/mL).

CHN analysis

Elemental analyses were performed on a vario EL instrument from Elementar.

Analytical and preparative high performance liquid chromatography (RP-HPLC)

Analytical RP HPLC was conducted on a LC-20A prominence system (pumps LC-20AT, auto

sampler SIL-20A, column oven CTO-20AC, diode array detector SPD-M2OA, controller CBM-

20A and software LC-solution) from Shimadzu. Nucleosil 100-5 C-18 PPN (4 x 250 mm, Macherey

Nagel), Nucleodur 100-5 C-18ec (4 x 250 mm, Macherey Nagel), Nucleodur 100-3 HILIC (4 x

250 mm, Macherey Nagel), Eurospher 100-10 C18 (16 x 250 mm, Knauer), Gemini 110-5 C6-

phenyl (30 x 75 mm, Phenomenex), Gemini 110-5 C6-phenyl (21.2 x 250 mm, Phenomenex) and

Luna 200-5 HILIC (21.2 x 250 mm, Phenomenex) columns were used as stationary phase. A

gradient of water with 0.1 % TFA (eluent A) in MeCN with 0.1 % TFA (eluent B) was used as

mobile phase.

Liquid chromatography mass spectrometry (LC-MS)

LC-MS measurements were performed at a LCMS2020 from Shimadzu (high pressure pumps LC-

20 AD, autosampler SIL-20AT HAT, column oven CTO-20AC, UV-Vis detector SPD-20A,

fluorescence detector RF-20A, controller CBM-20, ESI detector, software LCMS Solution, column

Nucleodur 100-3 C18ec (4 x 125 mm, Macherey Nagel). As eluent a gradient of A: H2O + 0.1 %

formic acid and B: MeCN + 0.1 % formic acid was used. Solvents were LCMS grade.

Preparative medium performance liquid chromatography (MPLC)

MPLC analyses were performed on a Reveleris I instrument from Grace. Flash cartridges

Reveleris-silica 4g, 12 g and 40g; Reveleris-reversed-phase 12g and 40g.

GENERAL PROCEDURES

GP01: DCC coupling

To a stirred suspension of carboxylic acid (in-situ generated or pure), DMAP and

triphenylmethanethiol or amine in DCM or DMF was added DCC at 4 oC. The reaction mixture

was stirred for 5 min, raised into ambient temperature and stirred for 3-6 hours. After reaction was

completed, mixture was filtered to remove DCU and filtrate was concentrated using rotary

evaporator. The resulting yellow solid was packed dry with silica and purified via flash column

chromatography.

GP02: EDAC/DMAP(HOBt) coupling

To a stirred suspension of carboxylic acid, DMAP or HOBt/DIPEA and triphenylmethanethiol or

amine in DCM/DMF was added EDAC at 4 oC. The reaction mixture was stirred for 5 min and

raised to ambient temperature and stirred for 3-6 h. After reaction was completed, solution was

diluted 10 times with organic solvent (DCM or Ethyl acetate) and washed with aqueous solution.

The organic phase was collected and dried over magnesium sulphate and concentrated using rotary

evaporator. The resulting yellow solid was packed dry with silica and purified via flash column

chromatography.

GP03: HBTU/HOBt coupling.

Carboxylic acid and HOBt were dissolved in DCM or DMF at ambient temperature. Stirring

solution was then cooled down using ice bath followed by addition of HBTU and DIPEA, stirred

for 5 min and raised to ambient temperature. After stirring for 30 min at RT, amine was added at

room temperature, and reaction mixture further stirred for 4-8 h. After reaction was completed,

solution was diluted to 10 times with organic solvent (DCM or Ethyl acetate) and extracted once

with 1 N aqueous HCl solution, saturated aqueous NaHCO3 solution, water and then brine. The

organic phase was collected and dried over magnesium sulphate and concentrated using rotary

evaporator (work-up procedure avoided with Fmoc containing compounds). The crude solid was

packed dry with silica and purified via flash column chromatography.

GP04: TAL with electron deficient azides

To a stirred solution of thiocarboxylic acid and sulfonyl azide in solvent, the base was added at

ambient temperature. The reaction mixture was stirred until the reaction was completed (TLC or

LCMS). The crude reaction mixture was purified either through Flash column chromatography

(FC) or HPLC.

GP05: TAL with electron rich azides

To a stirred solution of thiocarboxylic acid and sulfonyl azide in CHCl3 or water, was added the

base at room temperature. The reaction temperature was raised into 60 oC (reflux) and stirred until

the starting material was consumed (2-3 days). After the reaction (TLC or LCMS), the crude

mixture was purified through Flash column chromatography.

GP06: Selective trityl thioester deprotection

To a stirred solution of trityl thioester and TIS in DCM, was added TFA (5% v/v) drop-wise (very

slowly) at ambient temperature. The solution was stirred until the in situ generated yellow color

was disappeared (5 min). The solution was diluted to double volume with toluene and the solvent

was removed by either nitrogen-flux or under reduced pressure. The resulting crude product was

either used without further purifications or purified through MPLC-RP column.

GP07 Boc and/or OtBu deprotection

To a stirred solution of Boc/OtBu protected compound and TIS in DCM, was slowly added TFA

(50-60% v/v) at 4 oC. The reaction mixture was stirred at ambient temperature until the reaction

was completed (30-60 min). The solution was diluted into double volume with toluene and the

solvent was removed under reduced pressure. Afforded crude product was used for subsequent step

without further purifications.

GP08: Fmoc deprotection

Fmoc protected compound was dissolved in DMF and piperidine (20% v/v) was added. The

reaction mixture was stirred at ambient temperature until reaction was complete (5 min). Then the

solvent was removed under reduced pressure. The residual compound was co-evaporated with

toluene multiple times, dried over vacuum and used for next reaction without further purification

GP09: Boc, Ot-Bu, STrt, STmob global deprotection by using TFA

To a stirred solution of protected compound and TIS in DCM, was slowly added TFA (60% v/v)

at 4 oC. The reaction mixture was stirred at ambient temperature until the reaction was completed

(30-60 min). The solution was diluted to double volume with toluene and the solvent was removed

by either nitrogen-flux or under reduced pressure. The resulting crude product was either used

without further purification or purified through MPLC-RP column.

GP10: Benzyl ester/-NHCbz de-protection.

To a stirred solution of benzyl ester/-NHCbz protected compound in methanol (for Fmoc-protected

compounds 1:4 ethyl acetate /MeOH was used) at room temperature, was added 5-10% PdO2/C

under inert atmosphere. After, hydrogen gas was flushed through the mixture for 5 min and mixture

was stirred under hydrogen atmosphere. After reaction was finished (30 min, TLC), the mixture

was filtered through 2 cm thick celite pad to remove the catalyst Pd/charcoal, washed pad with

methanol and filtrate was evaporated under reduced pressure, then kept under vacuum for 1-2 h.

The resulting solid, carboxylic acid, was either used for next reaction without further purification

or purified with MPLC/flash-column chromatography.

GP11: Acetylation of alcohols

Compound were dissolved in 6:5 pyridine/acetic anhydride mixture and the resulting solution was

stirred overnight at room temperature. After the solvent was removed under reduced pressure, the

resulting residue was dissolved in ethyl acetate and extracted with water, saturated NaHCO3

solution and brine. Organic phase was collected, dried over MgSO4, concentrated under reduced

pressure and kept 1-2 h in vacuum. The resulting solid either used for next reaction without further

purification or purified though flash-column chromatography.

SYNTHESIS OF TARGET COMPOUNDS

S-trityl 3-phenylpropanethioate (76)

3-phenylpropanoic acid 75 (300 mg, 2 mmol), Triphenylmethanethiol (1.1 g, 4 mmol), DMAP (25

mg, 0.2 mmol) and DCC (577 mg, 3 mmol) were reacted in 10 mL of DCM for 5 h at room

temperature according to GP01. FC (0-3% ethyl acetate/petroleum ether) gave yellowish crude

product. It was then crystallized using 2% ethyl acetate/petroleum ether to get pure S-trityl-3-

phenylpropanethioate 76 (587 mg, 61%): Rf = 0.55 (ethyl acetate/petroleum ether 1:9); 1H NMR

(400 MHz, CDCl3): δ 7.31-7.13(m, 20H, aryl), 2.90- 2.81(m, 4H, Ph(CH2)2); 13C NMR (101 MHz,

CDCl3): δ 196.1 (C(O)STrt), 143.9, 140.1, 129.9, 128.6, 128.6, 127.9, 127.2, 126.5 (Carene), 70.6

(C(Ph)3), 45.2 (PhCH2CH2), 31.5 (PhCH2CH2); ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C28H25OS [M+H]+ m/z: 409.1, found 408.9.

3-phenylpropanethioic S-acid (77)

S-trityl-3-phenylpropanethioate 76 (1 g, 2.45 mmol), TIS (1.5 mL, 7.35 mmol) and TFA (10% v/v,

1.8 mL, 24.5 mmol) were reacted in 20 mL of DCM according to GP06. The organic phase was

extracted with 0.2 N NaOH and collected aqueous phase, which further acidified with 1 N HCl and

extracted with diethyl ether. Organic phase were collected, dried over MgSO4 and removed solvent

under vacuum. Afforded of 3-phenylpropanethioic S-acid 77 (374 mg, 92%) as viscous oil: Rf =

0.15-0.35 (broad, ethyl acetate/petroleum ether 1:9); 1H NMR (400 MHz, CDCl3): δ 7.37- 7.15 (m,

5H, phenyl), 4.26 (s, 1H, COSH), 3.02- 2.86 (m, 4H, Ph(CH2)2); 13C NMR (101 MHz, CDCl3): δ

196.7 (C(O)SH), 139.7, 128.7, 128.3, 126.5 (Carene), 47.2 (PhCH2CH2), 31.1 (PhCH2CH2); ESI-

MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C9H9OS [M-H]- m/z: 165.1, found 165.0.

S-(2-methoxypropan-2-yl) 3-phenylpropanethioate (81)

To a solution of 3-phenylpropanethioic S-acid 77 (358 mg, 2.2 mmol) and 2-methoxyprop-1-ene

80 (0.621 mL, 6.6 mmol) in DCM (6 mL) was added at room temperature and stirred 2 h. After the

reaction was completed, the solution was concentrated under reduced pressure and dried over

vacuum. Reaction gave S-(2-methoxypropan-2-yl) 3-phenylpropanethioate 81 (520 mg, 99%) as

colorless oil: Rf = 0.45 (ethyl acetate/petroleum ether 1:9, 2D TLC shows product decomposes in

silica); 1H NMR (400 MHz, C6D6): δ 7.12 – 7.05 (m, 2H, aryl), 7.05 – 6.99 (m, 1H, arene), 6.94

(ddd, J = 7.7, 1.6, 0.7 Hz, 2H, arene), 3.11 (s, 3H, OCH3), 2.79 (dd, J = 8.5, 6.8 Hz, 2H, PhCH2),

2.63 – 2.50 (m, 2H, CH2C(O)S), 1.68 (s, 6H, C(CH3)2OMe); 13C NMR (101 MHz, C6D6): δ 197.6

(C(O)S), 140.5, 128.7, 126.5 (Carene), 91.8 (C(CH3)2OMe), 51.6 (OCH3), 46.5 (PhCH2CH2), 31.6

(PhCH2CH2), 27.9 (C(CH3)2OMe); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C13H19O2S [M+H]+ m/z: 238.1, found 164.9; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.)

calcd for C13H18O2SNa [M+Na]+ m/z: 261.1, found 261.0.

4-dimethylpentan-3-yl 3-phenylpropanoate (88)106

3-phenylpropanoic acid 75 (2.4 g, 16.0 mmol), DMAP (0.2 g, 1.6 mmol), 2, 4-dimethylpentan-3-

ol (9.0 mL, 64.0 mmol) and DCC (3.6 g, 17.6 mmol) were reacted 50 mL of DCM according to

GP01. FC (1:19 ethyl acetate/petroleum ether) gave 86% (3.4 g) of compound 88: 1H NMR (400

MHz, CDCl3): δ = 7.36 – 7.19 (m, 5H, arene), 4.63 (t, J = 6.1, 1H, CH(iPr)2), 3.01 (t, J = 7.8, 2H,

PhCH2), 2.70 (t, J = 7.8, 2H, CH2C(O)O), 1.97 – 1.83 (m, 2H, CH(CHMe2)2), 0.86 (dd, J = 10.1,

6.9, 12H, CH(CH(CH3)2)2); 13C NMR (101 MHz, CDCl3): δ = 173.0 (C(O)O), 140.8, 128.5, 128.4,

126.3 (Carene), 82.8 (CH(iPr)2), 36.1(PhCH2CH2), 31.2 (PhCH2CH2), 29.4 (CH(CHMe2)2), 19.6,

17.3 (CH(CH(CH3)2)2).

O-ethyl 3-phenylpropanethioate (91) 108

O-ethyl 3-phenylpropanethioate 90 (0.7 g, 3.9 mmol) and Lawesson’s reagent (1.9 g, 4.7 mmol)

were refluxed in 12 mL of dry toluene overnight. FC (1:19, ethyl acetate/petroleum ether) gave

77% (0.58 g) yield of compound 91: 1H NMR (400 MHz, CDCl3): δ = 7.34 – 7.16 (m, 5H, arene),

4.50 (q, J = 7.1, 2H, OCH2Me), 3.13 – 2.99 (m, 4H, Ph(CH2)2), 1.36 (t, J = 7.1, 3H, OCH2CH3); 13C NMR (101 MHz, CDCl3): δ = 220.0 (C(S)O), 140.1, 128.2, 125.9 (Carene), 68.0 (OCH2CH3),

48.1 (PhCH2CH2), 34.4 (PhCH2CH2), 13.3 (OCH2CH3) .

O-isopropyl 3-phenylpropanethioate (92)

Isopropyl 3-phenylpropanoate 87 (0.4 g, 2.08 mmol) and Lawesson’s reagent (1.01 g, 2.5 mmol)

were refluxed in 7 mL of dry toluene overnight. FC (1:24, ethyl acetate/petroleum ether) gave 35%

(0.152 g) of compound 92: 1H NMR (400 MHz, CDCl3): δ = 7.37 – 7.21 (m, 5H, arene), 5.67 (hept,

J = 6.2, 1H, CH(Me)2), 3.14 – 3.01 (m, 4H, Ph(CH2)2), 1.35 (d, J = 6.3, 6H, CH(CH3)2); 13C NMR

(101 MHz, CDCl3): δ = 221.7 (C(S)O), 140.1, 128.2, 128.1, 125.9 (Carene), 75.0 (OCH(Me)2), 48.7

(PhCH2CH2) , 34.4 (PhCH2CH2), 20.7 (CH(CH3)2).

O-(2,4-dimethylpentan-3-yl) 3-phenylpropanethioate (93)

2, 4-dimethylpentan-3-yl 3-phenylpropanoate 88 (1.0 g, 4.03 mmol) and Lawesson’s reagent

(1.955g, 4.83 mmol) were refluxed in 13 mL of dry xylene overnight. FC (0.5% of ethyl acetate in

petroleum ether) gave 57% (0.6 g) of compound 93: 1H NMR (400 MHz, CDCl3): δ = 7.31 – 7.16

(m, 5H, arene), 5.54 (t, J = 6.1, 1H, CH(iPr)2), 3.14 – 3.03 (m, 4H, Ph(CH2)2), 2.11 – 1.96 (m, 2H,

CH(CH(Me)2)2), 0.89 (t, J = 6.5, 12H, CH(CH(CH3)2)2); 13C NMR (101 MHz, CDCl3): δ = 223.9

(C(S)O), 140.5, 128.5, 128.5, 126.3 (Carene), 90.1 (CH(iPr)2), 48.9 (PhCH2CH2), 34.6 (PhCH2CH2),

30.2 (CH(CH(Me)2)2), 19.6, 17.6 (CH(CH(CH3)2)2).

N', 3-diphenylpropanehydrazide (96)

3-phenylpropanoic acid 75 (5.0 g, 33.5 mmol), DMAP (0.4 g, 3.35 mmol), phenyl hydrazine

(2.7 mL, 40 mmol) and DCC (8.25 g, 40 mmol) were reacted in 100 mL of dry DCM according to

GP01. FC (1:2 ethyl acetate/petroleum ether) gave 75% (6.02 g) of required product 96: 1H NMR

(400 MHz, CDCl3): δ= 7.32 – 7.13 (m, 10H, arene), 6.08 (d, J = 3.7 Hz, 1H, CONHNH), 4.14 (d,

J = 7.5 Hz, 1H, CONHNH), 3.01 (t, J = 7.5 Hz, 2H, PhCH2CH2), 2.55 (t, J = 7.5 Hz, 2H,

PhCH2CH2); 13C NMR (101 MHz, CDCl3): δ= 172.3 (C=O), 147.9, 140.5, 129.3, 128.8, 128.6,

126.6, 121.3, 113.6 (Carene), 36.3 (PhCH2CH2), 31.5 (PhCH2CH2).

N', 3-diphenylpropanethiohydrazide (97)

N', 3-diphenylpropanehydrazide 96 (2.0 g, 8.33 mmol) and Lawesson’s reagent (3.7 g, 9.16 mmol)

were reacted in 25 mL of dry toluene. FC (1:2 ethyl acetate/petroleum ether) gave quantitative

yield (2.1 g) of yellowish solid, 97: HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 75 x 30 mm;

60-90% MeCN/H2O in 20 min; flow 9 mL min-1): Rt = 9 min); 1H NMR (400 MHz, CDCl3): δ 8.59

(s, 1H, CSNHNH), 7.46 (d, J = 21.6 Hz, 1H, CSNHNH), 7.32 – 7.12 (m, 7H, arene), 6.94 – 6.88

(m, 1H, arene), 6.54 (dt, J = 7.8, 1.1 Hz, 2H, arene), 3.14 (t, J = 7.1 Hz, 2H, PhCH2CH2), 3.00 –

2.95 (m, 2H, PhCH2CH2); 13C NMR (101 MHz, CDCl3): δ 201.89 (C=S), 145.70, 139.94, 129.41,

128.94, 128.75, 126.81, 122.55, 114.95 (Carene), 45.67 (PhCH2CH2), 35.22 (PhCH2CH2); ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C15H17N2S [M+H]+ m/z: 257.1, found 256.9.

3,4-diaminobenzamide (104) 120

To a stirred suspension of 4-amino-3-nitrobenzoic acid (1.0 g, 5.5 mmol), 1-hydroxybenzotriazole

(1.1 g, 8.24 mmol), and EDAC (1.5 g, 8.24 mmol) in anhydrous THF (50 mL) was added DIPEA

(1.14 mL, 8.24 mmol). The reaction mixture was stirred at ambient temperature for 10 min,

followed by addition of one portion of ammonium carbonate (1.6 g, 16.47 mmol), and the resulting

suspension was stirred at ambient temperature for 18 h. The reaction mixture was concentrated to

a light brown paste, 1:1 saturated aqueous NaHCO3/H2O (40 mL) was added, and stirring was

continued for another 2 h. The suspension was filtered through a glass frit, and the solid were dried

in a vacuum for 48 h to afford crude 4-amino-3-nitrobenzamide (913 mg) as a yellow-brown solid.

This was used in the subsequent step without further purification. A solution of the above 4-amino-

3-nitrobenzamide in ethanol (7.5 mL) and DMF (5 mL) along with 10% Pd/C (170 mg) was

hydrogenated for 24 h using hydrogen atmosphere. The reaction mixture was filtered through a pad

of Celite, and the Celite pad was washed with EtOH (3 x 5 mL). The combined filtrate was

concentrated to a dark brown solid. Trituration of the solid with 5% MeOH/Et2O (20 mL) afforded

the compound, 3,4-diaminobenzamide 104, as a brown powder (706 mg, 4.67 mmol, 85%), which

was used without further purification: Rf = 0.4 (MeOH/DCM 1:9); 1H NMR (400 MHz, DMSO-

d6): δ 7.35 (br s, 1H), 7.07 (d, J = 2.0 Hz, 1H), 6.99 (dd, J = 8.0, 2.1 Hz, 1H), 6.68 (br s, 1H), 6.46

(d, J = 8.0 Hz, 1H); 13C NMR (101 MHz, DMSO-d6): δ 168.59, 138.42, 133.52, 122.66, 117.71,

114.24, 112.65.

Fmoc-Asn(Dbz)-Ot-Bu (107)

Fmoc-Asp(OH)-Ot-Bu 106 (500 mg, 1.21 mmol), DIPEA (414 µL, 2.42 mmol), 3,4-

diaminobenzamide 104 (275 mg, 1.82 mmol), HBTU (550 mg, 1.45 mmol) and HOBt (197 mg,

1.45 mmol) were reacted in 3.5 mL of DCM according to GP03. FC (0-3% MeOH/DCM → 4%

MeOH/DCM) gave required compound 107 (445 mg, 70%) as colorless solid: Rf = 0.35 (1:9

MeOH/DCM); 1H NMR (400 MHz, MeOD-d4): δ 7.69 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 2.2 Hz,

1H), 7.59 – 7.51 (m, 3H), 7.30 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 2H), 6.76 (d, J = 8.5 Hz,

1H), 4.55 (t, J = 6.4 Hz, 1H), 4.29 (t, J = 7.3 Hz, 2H), 4.13 (t, J = 7.0 Hz, 1H), 2.89 (ddd, J = 50.6,

15.2, 6.4 Hz, 2H), 1.41 (s, 9H); 13C NMR (101 MHz, MeOD-d4): δ 172.1, 172.0, 171.5, 158.3,

147.7, 145.1, 142.4, 128.7, 128.4, 128.1, 128.0, 126.2, 123.0, 122.8, 120.9, 116.5, 83.3, 68.1, 53.2,

48.2, 39.0, 28.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C30H33N4O6

[M+H]+ m/z: 544.2, found 544.3.

Fmoc-Val-Ala-Ot-Bu (110)

Fmoc-Val-OH 108 (500 mg, 1.47 mmol), NH2-Ala-Ot-Bu 109 (320 mg, 1.76 mmol), HBTU (670

mg, 1.74 mmol), HOBt (238 mg, 1.76 mmol) and DIPEA (503 µL, 2.93 mmol) were reacted in

DCM (40 mL) according to GP03. FC (30-35 % ethyl acetate/petroleum ether) gave Fmoc-Val-

Ala-Ot-Bu 110 (671 mg, 98%) as colorless solid: Rf = 0.45 (ethyl acetate/petroleum ether 1:1); 1H

NMR (400 MHz, CDCl3): δ 7.75 (dd, J = 7.5, 1.3 Hz, 2H), 7.60 (dd, J = 7.5, 3.8 Hz, 2H), 7.38 (td,

J = 7.3, 3.0 Hz, 2H), 7.29 (tdd, J = 6.4, 3.3, 1.7 Hz, 2H), 6.83 (d, J = 7.4 Hz, 1H), 5.77 (d, J = 9.0

Hz, 1H), 4.49 (p, J = 7.5 Hz, 1H), 4.44 – 4.30 (m, 2H), 4.21 (t, J = 7.2 Hz, 1H), 4.13 (q, J = 7.0

Hz, 1H), 2.13 (td, J = 13.5, 12.7, 6.0 Hz, 1H), 1.46 (s, 9H), 1.36 (d, J = 7.2 Hz, 3H), 0.99 (dd, J =

12.3, 6.8 Hz, 6H); 13C NMR (101 MHz, CDCl3): δ 171.9, 170.9, 156.5, 144.0, 143.8, 141.3, 127.7,

127.1, 125.2, 125.2, 120.0, 119.9, 81.9, 67.1, 60.2, 48.7, 47.2, 31.6, 28.0, 19.2, 18.3, 18.0; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C27H34N2O5Na [M+Na]+ m/z: 489.2, found

489.3.

Fmoc-Asn(daba)-Val-Ala-Ot-Bu (112)

Fmoc-Asn(daba)-Ot-Bu 107 (175 mg, 0.321 mmol), TIS (330 µL, 1.60 mmol) and TFA (1.5 mL,

17 mmol) were reacted in DCM (1.5 mL) for 40 min according to GP07. Afterwards, Fmoc-Val-

Ala-Ot-Bu 110 (100 mg, 0.214 mmol) and piperidine (500 µL) were reacted in DMF (2 mL) for 5

min according to GP08. Subsequently, Fmoc-Asn(daba)-OH 105 (from first step, 0.321 mmol),

NH2-Val-Ala-Ot-Bu 111 (from second step, 0.214 mmol), HBTU (138 mg, 0.364 mmol), HOBt

(50 mg, 0.364 mmol) and DIPEA (110 µL, 0.642 mmol) were reacted in DMF (3.2 mL) according

to GP03.

Lissamine Rhodamine B derivatives (113-115)

Anhydrous lissamine rhodamine B 113 (100 mg, 0.173 mmol) and tetrabutylammonium azide (59

mg, 0.208 mmol) was dissolved in dry DCM (600 µL) at 4 oC. Solution then stirred for 3.5 h at

room temperature. FC (0-5% MeOH/DCM) gave crude mixture which was further purified via

HPLC: Eurospher 100-10 C18, 16 x 250 mm; 35-70% MeCN (with 0.1% TFA) in H2O (with 0.1%

TFA) in 25 min; flow 9 mL min-1:

Lissamine rhodamine B sulfonic acid (113): Rt = 9.5 min; 1H NMR (400 MHz, CDCl3): δ 8.67

(d, J = 1.7 Hz, 1H, benzene2), 8.09 (dd, J = 7.9, 1.6 Hz, 1H, benzene6), 7.22 (d, J = 9.5 Hz, 2H,

xanthene1,8), 7.08 (d, J = 7.8 Hz, 1H, benzene5), 6.72 (dd, J = 9.5, 2.4 Hz, 2H, xanthene2,7), 6.65

(d, J = 2.4 Hz, 2H, xanthene4,5), 3.64 – 3.45 (m, 8H, N(CH2CH3)2), 1.26 (t, J = 7.1 Hz, 12H,

N(CH2CH3)2); 13C NMR (101 MHz, CDCl3): δ 160.0, 159.8, 159.6, 159.2, 158.8, 158.0, 155.5,

147.74, 145.3, 133.5, 130.5, 129.4, 127.4, 126.4, 120.0, 117.1, 114.6, 114.2, 113.2, 111.4, 95.6

(Carene), 45.9 (N(CH2CH3)2), 12.6 (N(CH2CH3)2); ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C27H29N2O7S2 [M-H]- m/z: 557.2, found 557.2.

Lissamine rhodamine B para(5-) sulfonyl azide (114): Rt = 21.3 min; 1H NMR (400 MHz,

CDCl3): δ 8.97 (d, J = 2.0 Hz, 1H, benzene2), 8.02 (dd, J = 8.0, 2.0 Hz, 1H, benzene6), 7.32 (d, J =

8.1 Hz, 1H, benzene5), 7.20 (d, J = 9.5 Hz, 2H, xanthene1,8), 6.78 (dd, J = 9.5, 2.5 Hz, 2H,

xanthene2,7), 6.67 (d, J = 2.5 Hz, 2H, xanthene4,5), 3.54 (qd, J = 7.3, 3.4 Hz, 8H, N(CH2CH3)2),

1.28 (t, J = 7.1 Hz, 12H, N(CH2CH3)2); 13C NMR (101 MHz, CDCl3): δ 157.9, 157.8, 155.6, 149.4,

139.9, 136.3, 133.2, 130.5, 128.3, 127.3, 114.2, 113.7, 95.9 (Carene), 46.0 (N(CH2CH3)2), 12.7

(N(CH2CH3)2); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C27H30N5O6S2

[M+H]+ m/z: 584.2, found 584.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for

C27H28N5O6S2 [M-H]- m/z: 582.2, found 582.4.

Lissamine rhodamine B ortho sulfonyl azide (115): Rt = 16.87 min; 1H NMR (400 MHz, CDCl3):

δ 8.85 (d, J = 1.5 Hz, 1H, benzene2), 8.47 (dd, J = 7.8, 1.6 Hz, 1H, benzene6), 7.39 (d, J = 7.8 Hz,

1H, benzene5), 7.00 (d, J = 9.4 Hz, 2H, xanthene1,8), 6.82 (dd, J = 9.5, 2.4 Hz, 2H, xanthene2,7),

6.78 (d, J = 2.4 Hz, 2H, xanthene4,5), 3.61 (qd, J = 7.3, 2.0 Hz, 8H, N(CH2CH3)2), 1.32 (t, J = 7.8

Hz, 12H, N(CH2CH3)2); 13C NMR (101 MHz, CDCl3): δ 160.4, 160.1, 157.7, 155.8, 153.3, 150.4,

137.8, 133.0, 131.6, 131.4, 128.3, 117.5, 114.6, 114.2, 113.9, 96.4 (Carene), 46.2 (N(CH2CH3)2),

12.6 (N(CH2CH3)2); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C27H30N5O6S2

[M+H]+ m/z: 584.2, found 584.2.

Dansyl azide (116)129

Dansyl chloride (3 g, 11.12 mmol) was dissolved in 45 mL of ethanol (EtOH) at 45 oC and added

drop wise into a stirred solution of sodium azide (1.5 g, 22.22 mmol) in 20 mL of a mixed solvent

(H2O/EtOH, 3:2). Then the reaction mixture was stirred at ambient temperature for 3 h. The

organic solvent was evaporated in vacuum, and the aqueous solution was extracted by DCM. The

combined organic layers was washed with brine and then dried over MgSO4. Solvent evaporation

gave the crude product, which was purified by flash chromatography: eluent 20-50% ethyl

acetate/petroleum ether, which gives crude product (2.94 g) as a light yellow viscous oil. The crude

material was further purified by crystallization in MeOH, yield 2.05 g (7.45 mmol, 67%) of

yellowish-brown crystals, Dansyl Azide 116: Rf = 0.55 (1:1 ethyl acetate/petroleum ether); 1H

NMR (400 MHz, CDCl3): δ 8.67 (dt, J = 8.5, 1.1 Hz, 1H), 8.34 (dd, J = 7.4, 1.3 Hz, 1H), 8.21 (dt,

J = 8.7, 1.0 Hz, 1H), 7.64 (dd, J = 8.7, 7.6 Hz, 1H), 7.58 (dd, J = 8.5, 7.4 Hz, 1H), 7.24 (dd, J =

7.6, 0.9 Hz, 1H), 2.90 (s, 6H); 13C NMR (101 MHz, CDCl3): δ 152.3, 133.8, 132.8, 130.2, 130.2,

129.8, 129.4, 123.1, 118.9, 116.0, 45.5; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd

for C12H13N4O2S [M+H]+ m/z: 277.1, found 277.1.

Boc-4-((2-aminoethyl)carbamoyl)benzenesulfonyl azide (pCSea) (119)

4-Carboxybenzenesulfonazide 118 (1.72 g, 7.6 mmol), N-Boc-ethylenediamine (1.6 mL, 9.12

mmol), EDAC (1.3 g, 8.36 mmol) and DMAP (92 mg, 0.76 mmol) were reacted in DCM (50 mL)

according to GP02. FC (10-25% ethyl acetate/petroleum ether) gave crude white solid, which then

recrystallized in 1:7 ethyl acetate/petroleum ether afforded Boc-4-((2-

aminoethyl)carbamoyl)benzenesulfonyl azide 119 (880 mg, 31%) as colorless needles: Rf = 0.5

(ethyl acetate/petroleum ether 1:1); 1H NMR (400 MHz, CDCl3): δ 8.09 – 8.04 (m, 2H, benzene3,5),

8.02 – 7.98 (m, 2H, benzene2,6), 7.87 (s, 1H, CONHCN2), 5.12 (t, J = 6.4 Hz, 1H, NHBoc), 3.63 –

3.50 (m, 2H, CH2CH2NHBoc), 3.43 (q, J = 5.5 Hz, 2H, CH2CH2NHBoc), 1.42 (s, 9H,

C(O)O(CH3)3); 13C NMR (101 MHz, CDCl3): δ 165.5 (CONHCN2), 158.4 (NHC(O)OC(Me)3),

140.7, 140.1, 128.5, 127.8 (Carene), 80.6 (NHC(O)OC(Me)3), 43.4 (CH2CH2NHBoc), 39.8

(CH2CH2NHBoc), 28.4 (NHC(O)OC(CH3)3); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.)

calcd for C14H20N5O5S [M+H]+ m/z: 370.1, found 370.1; ESI-MS (H2O/MeCN (with 0.1% formic

acid), pos.) calcd for C14H19N5O5SNa [M+Na]+ m/z: 392.1, found 392.1.

Biotin sulfonyl azide (121)

Boc-4-((2-aminoethyl)carbamoyl)benzenesulfonyl azide 119 (665 mg, 1.75 mmol), TIS (1.08 mL,

5.25 mmol) and TFA (5 mL) were reacted in DCM (5 mL) for 30 min at room temperature

according to GP07. Subsequently, 4-((2-aminoethyl)carbamoyl)benzenesulfonyl azide (from first

step, 1.75 mmol), biotin (470 mg, 1.93 mmol), HBTU (835 mg, 2.1 mmol), HOBt (283 mg, 2.1

mmol) and DIPEA (900 µL, 5.25 mmol) were reacted in DMF (6 mL) for 4 h according to GP03.

MPLC (Grace-Reveleris-silica 12 g; 0-10-15% MeOH/DCM in 16 min; flow 30 mL min-1: Rt = 8

min) gave Biotin sulfonyl azide 121 (300 mg, 35%) as white solid: Rf = 0.4 (MeOH/DCM 1:9); 1H

NMR (400 MHz, MeOD-d4): δ 8.09 (br s, 4H, arene), 4.47 (dd, J = 7.9, 4.8 Hz, 1H, biotin6a), 4.24

(dd, J = 7.9, 4.4 Hz, 1H, biotin3a), 3.53 (t, J = 7.0, 5.2 Hz, 2H, CH2CH2NHCO), 3.44 (t, J = 6.6,

4.8 Hz, 2H, CONHCH2CH2), 3.11 (ddd, J = 8.8, 5.9, 4.4 Hz, 1H, biotin4), 2.89 (dd, J = 12.8, 5.0

Hz, 1H, biotin6), 2.68 (d, J = 12.7 Hz, 1H, biotin6), 2.22 (td, J = 7.3, 1.9 Hz, 2H, NHCOCH2), 1.74

– 1.47 (m, 4H, CH2CH2), 1.40 (p, J = 7.1 Hz, 2H, CH2CH2CH2CH2); 13C NMR (101 MHz, MeOD-

d4): δ 176.6 (NHC(O)(CH2)4), 168.3 (ArC(O)NH, 166.1 (NHC(O)NH), 142.1, 141.7, 129.9, 128.8

(Carene), 63.3 (biotin-C3a), 61.6 (biotin-C6a), 56.9 (biotin-C4), 41.2, 41.0, 39.8, 36.8, 29.7, 29.5, 26.8

(CH2); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C19H26N7O5S2 [M+H]+ m/z:

496.1, found 496.1; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C19H25N7O5S2Na[M+Na]+ m/z: 518.1, found 518.0.

Fmoc-piperazyl biotin (123)

Biotin 120 (1.66 g, 6.78 mmol), Fmoc-piperazine (1.8 g, 5.22 mmol), HOBt (915 mg, 6.78 mmol),

HBTU (2.7 g, 6.78 mmol) and DIPEA (3.6 mL, 20.88 mmol) were reacted in DMF (30 mL)

according to GP03. MPLC (Grace-Reveleris-silica 40 g; 0-20% MeOH/DCM in 16 min; flow 35

mL min-1: Rt = 9 min) gave white solid, Fmoc-piperazyl biotin 123 (2.4 g, 86%): Rf = 0.35

(MeOH/DCM 1:9); 1H NMR (400 MHz, DMSO-d6) δ 7.93 – 7.83 (m, 2H, Fmoc4,5), 7.63 (dd, J =

7.5, 1.2 Hz, 2H, Fmoc1,8), 7.42 (t, J = 7.4 Hz, 2H, Fmoc3,6), 7.34 (td, J = 7.5, 1.2 Hz, 2H, Fmoc2,7),

6.43 (s, 1H, NHC(O)NH), 6.36 (s, 1H, NHC(O)NH), 4.41 (d, J = 6.4 Hz, 2H, Fmoc10), 4.34 – 4.24

(m, 2H, biotine3a,6a), 4.14 (ddd, J = 7.9, 4.4, 1.5 Hz, 1H, Fmoc9), 3.40 – 3.23 (m, 8H, piperazine-

CH2), 3.11 (ddd, J = 8.6, 6.2, 4.3 Hz, 1H, biotine4), 2.83 (dd, J = 12.5, 5.1 Hz, 1H, biotin6), 2.59

(d, J = 12.4 Hz, 1H, biotin6) 2.30 (t, J = 7.5 Hz, 2H, NC(O)CH2), 1.69 – 1.27 (m, 6H, (CH2)3); 13C

NMR (101 MHz, DMSO-d6) δ 170.8, 162.8, 162.8 (C=O), 154.4, 143.8, 140.8, 127.7, 127.2, 125.0,

120.1 (Carene), 66.6 (Fmoc10), 61.1 (biotin-C3a), 59.3 (biotin-C6a), 55.5 (biotin-C4), 46.81 (biotin-

C6), 44.5, 43.6, 43.2 (piperazine-CH2), 32.1, 28.3, 28.1, 24.8 (biotin-CH2); ESI-MS (H2O/MeCN

(with 0.1% formic acid), pos.) calcd for C26H24N5O5S [M+H]+ m/z: 535.2, found 535.3.

Biotin sulfonyl azide (124)

Fmoc-piperazyl biotin 123 (963 mg, 1.8 mmol) and piperidine (20% v/v, 2 mL) were reacted in

DMF (8 mL) for 5 min according to GP08. Subsequently, 4-Carboxybenzenesulfonazide (830 mg,

3.64 mmol), piperazyl biotin (from first step, 1.8 mmol), DCC (900 mg, 4.36 mmol) and DMAP

(44 mg, 0.36 mmol) were reacted in DMF (20 mL) for 8 h according to GP01. MPLC (Grace):

Reveleris-C18(RP) 40 g (40-100% MeCN/H2O in 16 min; flow 40 mL min-1): Rt = 8 min; Afforded

title compound, Biotin sulfonyl azide 124, 115 mg (0..22 mmol, 12%) as white solid; 1H NMR

(400 MHz, MeOD-d4): δ 8.23 – 8.00 (m, 2H, benzene3,5), 7.83 – 7.69 (m, 2H, benzene2,6), 4.50 (dd,

J = 7.8, 4.8 Hz, 1H, biotin6a), 4.31 (dd, J = 8.0, 4.4 Hz, 1H, biotin3a), 3.94 – 3.36 (m, 8H, piperazine-

CH2), 3.21 (dt, J = 9.7, 5.3 Hz, 1H, biotin4), 2.93 (dd, J = 12.8, 4.9 Hz, 1H, biotin6), 2.71 (d, J =

12.8 Hz, 1H, biotin6), 2.56 – 2.37 (m, 2H, NC(O)CH2), 1.85 – 1.40 (m, 6H, (CH2)3); 13C NMR

(101 MHz, MeOD-d4): δ 174.2, 170.2, 166.0 (C=O), 143.0, 140.9, 129.7, 129.1 (Carene), 63.3

(biotin-C3a), 61.6 (biotin-C6a), 57.0 (biotin-C4), 46.4 (double, piperazine-CH2), 42.9 (quart,

piperazine-CH2), 41.1 (biotin-C6), 33.6, 29.8, 29.5, 26.2 (biotin-CH2); ESI-MS (H2O/MeCN (with

0.1% formic acid), pos.) calcd. for C21H27N7O5S2Na [M+Na]+ m/z: 544.1, found 544.0; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C21H28N7O5S2 [M+H]+ m/z: 522.2, found

522.0.

Fmoc-Ala-Phe-Ot-Bu131 (130)

Fmoc-Ala-OH 126 (2.63 g, 8.44 mmol), HCl*NH2-Phe-Ot-Bu 128 (2.6 g, 10.13 mmol), HBTU

(4.02 g, 10.13 mmol), HOBt (1.37 g, 10.13 mmol) and DIPEA (4.33 mL, 25.32 mmol) were reacted

in DMF (40 mL) according to GP03. MPLC (Grace-Reveleris-silica 40 g; 15-60% ethyl

acetate/petroleum ether in 20 min; flow 35 mL min-1: Rt = 10 min) gave Fmoc-Ala-Phe-Ot-Bu 130

(3.5 g, 81%) as colorless solid: Rf = 0.45 (ethyl acetate/petroleum ether 1:1); 1H NMR (400 MHz,

CDCl3): δ 7.77 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (td, J =

7.5, 1.2 Hz, 2H), 7.27 – 7.11 (m, 5H), 6.46 (d, J = 7.8 Hz, 1H), 5.41 (d, J = 7.7 Hz, 1H), 4.74 (dt,

J = 7.9, 6.0 Hz, 1H), 4.48 – 4.17 (m, 4H), 3.10 (dd, J = 6.1, 4.3 Hz, 2H), 1.41 (s, 12H); 13C NMR

(101 MHz, CDCl3): δ 171.7, 170.3, 155.9, 143.9, 141.4, 136.1, 129.6, 128.5, 127.8, 127.2, 127.1,

125.2, 125.2, 120.1, 82.6, 67.2, 53.7, 50.5, 47.2, 38.1, 28.1, 18.9; ESI-MS (H2O/MeCN (with 0.1%

formic acid), pos.) calcd for C31H35N2O5 [M+H]+ m/z: 515.3, found 515.3.

Fmoc-Gly-Phe-Ot-Bu132 (131)

Fmoc-Gly-OH 127 (5 g, 16.81 mmol), HCl*NH2-Phe-Ot-Bu 128 (5.18 g, 20.18 mmol), HBTU (8

g, 20.18 mmol), HOBt (2.7 g, 20.18 mmol) and DIPEA (8.7 mL, 50.43 mmol) were reacted in

DMF (85 mL) according to GP03. MPLC (Grace-Reveleris-silica 40 g; 20-60% ethyl

acetate/petroleum ether in 20 min; flow 35 mL min-1: Rt = 10 min) gave Fmoc-Gly-Phe-Ot-Bu (7.1

g, 84%) as colorless solid,: Rf = 0.3 (ethyl acetate/petroleum ether 1:1); 1H NMR (400 MHz,

CDCl3): δ 7.77 (d, J = 7.5 Hz, 2H), 7.60 (t, J = 7.1 Hz, 2H), 7.40 (td, J = 7.6, 1.2 Hz, 2H), 7.31

(tdd, J = 7.5, 3.4, 1.2 Hz, 2H), 7.28 – 7.18 (m, 3H), 7.16 – 7.12 (m, 2H), 6.49 (d, J = 7.8 Hz, 1H),

5.51 (t, J = 5.6 Hz, 1H), 4.78 (dt, J = 7.8, 6.0 Hz, 1H), 4.49 – 4.33 (m, 2H), 4.29 – 4.16 (m, 1H),

3.96 – 3.81 (m, 2H), 3.10 (d, J = 6.0 Hz, 2H), 1.41 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 170.4,

168.4, 156.6, 143.9, 141.4, 136.0, 129.6, 128.5, 127.8, 127.2, 127.2, 127.2, 125.2, 120.1, 82.7,

67.4, 53.7, 47.2, 44.5, 38.2, 28.1.

Boc-Asp(OBn)-OH157 (139)

Followed published procedure.158 NH2-Asp(OBn)-OH (8 g, 35.86 mmol), 1:1 THF/water (150

mL), (Boc)2O (9 mL, 39.45 mmol)) and NaOH ( 3.15 g, 78.89 mmol). Reaction afforded Boc-

Asp(OBn)-OH 139 (11 g, 95%) as white solid; 1H NMR (400 MHz, CDCl3): δ 9.85 (s, 1H), 7.43

– 7.30 (m, 5H), 5.58 (d, J = 8.6 Hz, 1H), 5.21 – 5.06 (m, 2H), 4.65 (dt, J = 9.2, 4.7 Hz, 1H), 3.16

– 2.80 (m, 2H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 175.6, 171.0, 155.6, 135.3, 128.6,

128.4, 128.3, 127.1, 80.5, 67.0, 49.8, 36.6, 28.3.

Boc-Asp(OBn)-Val-Phe-Ot-Bu (141)

Fmoc-Val-Phe-Ot-Bu 129 (6.55 g, 12.8 mmol) and piperidine (20% v/v, 12 mL) were reacted in

DMF (48 mL) for 5 min according to GP08. Subsequently, Boc-Asp(OBn)-OH 139 (4.68 g, 14.5

mmol), NH2-Ala-Phe-Ot-Bu 132 (from first step, 18.8 mmol), HBTU (5.5 g, 14.5 mmol), HOBt

(1.96 mg, 14.5 mmol) and DIPEA (6.16 mL, 36.2 mmol) were reacted in DMF (45 mL) according

to GP03. FC (20% ethyl acetate/petroleum ether → 30% ethyl acetate/petroleum ether) gave Boc-

Asp(OBn)-Val-Phe-Ot-Bu 141 (2 g, 93%) as colorless solid: Rf = 0.5 (ethyl acetate/petroleum ether

1:1); 1H NMR (400 MHz, CDCl3): δ 7.41 – 7.10 (m, 11H, arene, NH), 6.94 (d, J = 8.6 Hz, 1H,

NH), 6.45 – 6.31 (m, 1H, NH), 5.66 (d, J = 7.6 Hz, 1H, NH), 5.10 (s, 2H, OCH2Ph), 4.72 (dd, J =

14.2, 6.3 Hz, 1H, Cα-Asp), 4.53-4.39 (m, 1H, Cα-Phe), 4.22 (dd, J = 8.7, 5.9 Hz, 1H, Cα-Val),

3.05 (d, J = 6.3 Hz, 2H, Cβ-Phe), 2.98 (dd, J = 17.1, 5.2 Hz, 1H, Cβ-Asp), 2.68 (dd, J = 17.1, 6.2

Hz, 1H, Cβ-Asp), 2.23 – 2.10 (m, 1H, Cβ-Val), 1.44 (s, 9H, OtBu), 1.37 (s, 9H, OtBu), 0.88 (d, J

= 6.8 Hz, 3H, Cγ-Val), 0.85 (d, J = 6.8 Hz, 3H, Cγ-Val); ESI-MS (H2O/MeCN (with 0.1% formic

acid), pos.) calcd for C34H48N3O8 [M+H]+ m/z: 626.3, found 626.4.

Boc-Asp(OBn)-Ala-Phe-Ot-Bu (142)

Fmoc-Ala-Phe-Ot-Bu 130 (1.86 g, 3.61 mmol) and piperidine (20% v/v, 4 mL) were reacted in

DMF (16 mL) for 5 min according to GP08. Subsequently, Boc-Asp(OBn)-OH 139 (1.4 g, 4.34

mmol), NH2-Ala-Phe-Ot-Bu 133 (from first step, 3.61 mmol), HBTU (1.64 g, 4.34 mmol), HOBt

(586 mg, 4.34 mmol) and DIPEA (1.86 mL, 10.84 mmol) were reacted in DCM (18 mL) according

to GP03. FC (20% ethyl acetate/petroleum ether → 50% ethyl acetate/petroleum ether) gave Boc-

Asp(OBn)-Ala-Phe-Ot-Bu 142 (2 g, 93%) as colorless solid: Rf = 0.4 (ethyl acetate/petroleum ether

1:1); 1H NMR (400 MHz, CDCl3): δ 7.42 – 7.10 (m, 10H, arene), 6.92 (d, J = 7.5 Hz, 1H, NH),

6.48 (d, J = 7.8 Hz, 1H, NH), 5.60 (d, J = 8.8 Hz, 1H, NH), 5.12 (s, 2H, OCH2Ph), 4.75 – 4.66 (m,

1H, Cα-Asp), 4.52-4.41 (d, 1H, Cα-Phe), 4.40 (p, J = 7.1 Hz, 1H, Cα-Ala), 3.15 – 3.02 (m, 2H,

Cβ-Phe), 2.98 (dd, J = 17.1, 5.0 Hz, 1H, Cβ-Asp), 2.75 (dd, J = 17.1, 6.2 Hz, 1H, Cβ-Asp), 1.46

(s, 9H, OtBu), 1.40 (s, 9H, OtBu), 1.33 (d, J = 7.1 Hz, 3H, Cβ-Ala); 13C NMR (101 MHz, CDCl3):

δ 171.2, 170.4, 155.5 (C=O), 136.2, 135.5, 129.6, 128.7, 128.5, 128.4, 127.1 (Carene), 82.5

(OC(CH3)3), 67.1 (OCH2Ph), 53.8, 49.2 (Cα-Phe, Cα-Asp, Cα-Val), 38.0 (Cβ-Phe, Cβ-Asp), 28.4,

28.1 (OC(CH3)3), 18.0 (Cβ-Ala); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C32H44N3O8 [M+H]+ m/z: 598.3, found 598.4.

Boc-Asp(OBn)-Gly-Phe-Ot-Bu (143)

Fmoc-Gly-Phe-Ot-Bu 131 (5 g, 9.99 mmol) and piperidine (20% v/v, 10 mL) were reacted in DMF

(40 mL) for 5 min according to GP08. Subsequently, Boc-Asp(OBn)-OH 139 (5.47 g, 17 mmol),

NH2-Ala-Phe-Ot-Bu 134 (from first step, 9.99 mmol), HBTU (7.15 g, 18 mmol), HOBt (2.43 g,

17.98 mmol) and DIPEA (8.7 mL, 51 mmol) were reacted in DMF (60 mL) for 6 h according to

GP02. All reaction and evaporation carried out under 30 oC to avoid aspartimide formation. MPLC

(Grace): Reveleris-silica 40 g; 10-100% ethyl acetate/petroleum ether in 20 min; flow 35 mL min-

1: Rt = 12 min) gave Boc-Asp(OBn)-Gly-Phe-Ot-Bu 143 (4.27 g, 73%) as colorless solid: Rf = 0.65

(ethyl acetate/petroleum ether 4:1); 1H NMR (400 MHz, CDCl3): δ 7.39 – 7.14 (m, 10H, arene),

7.10 (d, J = 6.1 Hz, 1H, NH), 6.71 (d, J = 7.8 Hz, 1H, NH), 5.60 (d, J = 8.9 Hz, 1H, NH), 5.15 –

4.99 (m, 2H, OCH2Ph), 4.74 (dt, J = 7.9, 6.3 Hz, 1H, Cα-Asp), 4.61-4.52 (m, 1H, Cα-Phe), 3.94

(m, 2H, Cα-Gly), 3.17 – 2.72 (m, 4H, Cβ-Phe, Cβ-Asp), 1.46 (s, 9H, OtBu), 1.39 (s, 9H, OtBu); 13C NMR (101 MHz, CDCl3): δ 171.9, 171.4, 170.6, 168.2, 155.5 (C=O), 136.3, 135.4, 129.6,

128.7, 128.5, 128.5, 128.3, 127.1 (Carene), 82.5, 80.9 (OC(CH3)3), 67.1 (OCH2Ph), 53.9 (Cα-Phe),

50.9 (Cα-Asp), 43.3 (Cα-Gly), 38.1, 36.4 (Cβ-Phe, Cβ-Asp), 28.4, 28.1 (OC(CH3)3); ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C31H42N3O8 [M+H]+ m/z: 584.3, found 584.3.

Boc-Asp(OBn)-Val-Ot-Bu (144)

Boc-Asp(OBn)-OH 139 (5 g, 15.46 mmol), NH2-Val-Ot-Bu 135 (3.9 g, 18.55 mmol), HBTU (7 g,

18.55mmol), HOBt (2.5 g, 18.55 mmol) and DIPEA (8 mL, 46.38 mmol) were reacted in DCM

(50 mL) according to GP03. FC (25-35% ethyl acetate/petroleum ether) gave Boc-Asp(OBn)-Val-

Ot-Bu 144 (9.7, quant) as white solid: Rf = 0.3 (petroleum ether/ethyl acetate 3:1); 1H NMR (400

MHz, CDCl3): δ 7.42 – 7.30 (m, 5H, phenyl), 6.98 (d, J = 8.6 Hz, 1H, NH), 5.72 (d, J = 8.6 Hz,

1H, NH), 5.22 – 5.07 (m, 2H, OCH2Ph), 4.60-4.50 (m, 1H, Cα-Asp), 4.38 (dd, J = 8.8, 4.6 Hz, 1H,

Cα-Val), 3.04 (dd, J = 17.1, 4.6 Hz, 1H, Cβ-Asp), 2.74 (dd, J = 17.1, 6.3 Hz, 1H, Cβ-Asp), 2.15

(pd, J = 6.9, 4.5 Hz, 1H, Cβ-Val), 1.46 (s, 18H, OtBu, OtBu), 0.93 (d, J = 6.9 Hz, 3H, Cγ-Val),

0.91 (d, J = 6.9 Hz, 3H, Cγ-Val); 13C NMR (101 MHz, CDCl3): δ 171.9, 170.5, 170.4, 155.5 (C=O),

135.5, 128.6, 128.3, 128.3 (Carene), 81.9, 80.4 (OC(CH3)3), 66.9 (OCH2Ph), 57.8 (Cα-Val), 50.7

(Cα-Asp), 36.1 (Cβ-Asp), 31.4 (Cβ-Val), 28.3, 28.1 (OC(CH3)3), 18.9, 17.5 (Cγ-Val); ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C25H39N2O7 [M+H]+ m/z: 479.3, found 479.3;

ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C25H37N2O7 [M-H]- m/z: 477.3,

found 477.5.

Fmoc-Asp(OBn)-Val-Ot-Bu (145)

Fmoc-Asp(OBn)-OH 140 (1 g, 2.25 mmol), HCl*NH2-Val-OtBu 135 (393 g, 1.87 mmol), HBTU

(851 mg, 2.25 mmol), HOBt (303 mg, 2.25 mmol) and DIPEA (977 µL, 5.61 mmol) were reacted

in DCM (11 mL) for 8 h according to GP03. FC (30% ethyl acetate/petroleum ether) gave Fmoc-

Asp(OBn)-Val-OtBu 145 (1.07 g, 95%) as colorless solid: Rf = 0.4 (20% ethyl acetate/petroleum

ether); 1H NMR (400 MHz, CDCl3): δ 7.76 (dd, J = 7.7, 2.3 Hz, 2H, Fmoc4,5), 7.59 (d, J = 7.5 Hz,

2H, Fmoc1,8), 7.48 – 7.27 (m, 9H, Fmoc3,6, Fmoc2,7, phenyl), 7.01 (d, J = 8.7 Hz, 1H, NH), 6.04 (d,

J = 8.4 Hz, 1H, NH), 5.25 – 5.10 (m, 2H, OCH2Ph), 4.67 (q, J = 7.1, 6.2 Hz, 1H, Cα-Asp), 4.50 –

4.33 (m, 3H, Fmoc10, Fmoc9), 4.23 (t, J = 7.0 Hz, 1H, Cα-Val), 3.09 (dd, J = 17.3, 4.3 Hz, 1H, Cβ-

Asp), 2.77 (dd, J = 17.2, 6.7 Hz, 1H, Cβ-Asp), 2.18 (pd, J = 6.9, 4.6 Hz, 1H, Cβ-Val), 1.46 (s, 9H,

OtBu), 0.92 (dd, J = 11.1, 6.9 Hz, 6H, Cγ-Val); 13C NMR (101 MHz, CDCl3): δ 172.0, 170.4,

170.2, 156.1 (C=O), 143.8, 143.8, 141.4, 135.4, 128.7, 128.5, 128.4, 127.8, 127.2, 125.1, 120.1,

120.1 (Carene), 82.1 (OC(CH3)3), 67.5 (Fmoc10), 67.1 (OCH2Ph), 57.9 (Cα-Val), 51.1 (Cα-Asp),

47.2 (Fmoc9), 36.5 (Cβ-Asp), 31.3 (Cβ-Val), 28.1 (OC(CH3)3), 19.0, 17.6 (Cγ-Val); ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C35H41N2O7 [M+H]+ m/z: 601.3, found

601.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C35H40N2O7Na [M+Na]+

m/z: 623.3, found 623.2.

Boc-Asp(OBn)-Ala-Ot-Bu (146)

Boc-Asp(OBn)-OH 139 (5 g, 15.46 mmol), HCl*NH2-Ala-Ot-Bu 136 (3.37 g, 18.55 mmol), HBTU

(7 g, 18.55mmol), HOBt (2.5 g, 18.55 mmol) and DIPEA (8 mL, 46.38 mmol) were reacted in

DCM (50 mL) for 30 min according to GP03. FC (15-25% ethyl acetate/petroleum ether) gave

Boc-Asp(OBn)-Ala-Ot-Bu 146 (6.6 g, quant) as white solid: Rf = 0.35 (ethyl acetate/petroleum

ether 1:3); 1H NMR (400 MHz, CDCl3): δ 7.41 – 7.29 (m, 5H, phenyl), 6.99 (br s, 1H, NH), 5.64

(d, J = 8.8 Hz, 1H, NH), 5.20 – 5.07 (m, 2H, OCH2Ph), 4.58-4.48 (m, 1H, Cα-Asp), 4.40 (p, J =

7.2 Hz, 1H, Cα-Ala), 3.07 (dd, J = 17.1, 4.4 Hz, 1H, Cβ-Asp), 2.72 (dd, J = 17.2, 6.1 Hz, 1H, Cβ-

Asp), 1.45 (s, 18H, OtBu, OtBu), 1.34 (d, J = 7.1 Hz, 3H, Cβ-Ala); 13C NMR (101 MHz, CDCl3):

δ 171.7, 170.1 (C=O), 135.5, 128.7, 128.50, 128.4 (Carene), 82.1 (OC(CH3)3), 67.0 (OCH2Ph), 50.6

(Cα-Asp), 49.1 (Cα-Ala), 36.4 (Cβ-Asp), 28.4, 28.1 (OC(CH3)3), 18.6 (Cβ-Ala); ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C23H34N2O7Na [M+Na]+ m/z: 473.2, found

473.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C23H35N2O7 [M+H]+ m/z:

451.2, found 451.2.

Boc-Asp(OBn)-Phe-Ot-Bu (147)

Boc-Asp(OBn)-OH 139 (6 g, 18.56 mmol), HCl*NH2-Phe-Ot-Bu 137 (5.26 g, 20.41 mmol),

EDAC (3.9 g, 20.41 mmol), HOBt (2.76 g, 20.41 mmol) and DIPEA (9.53 mL, 55.66 mmol) were

reacted in DCM (50 mL) for 3o min according to GP02. FC (10-20% ethyl acetate/petroleum ether)

gave Boc-Asp(OBn)-Phe-Ot-Bu 147 (9.6 g, quant) as white solid: Rf = 0.45 (ethyl

acetate/petroleum ether 1:3); 1H NMR (400 MHz, CDCl3): δ 7.40 – 7.12 (m, 10H, arene), 6.97 (d,

J = 7.7 Hz, 1H, NH), 5.67 (d, J = 8.9 Hz, 1H, NH), 5.16 – 5.09 (m, 2H, OCH2Ph), 4.74 – 4.63 (m,

1H, Cα-Asp), 4.55 (dt, J = 9.6, 5.0 Hz, 1H, Cα-Phe), 3.13 – 2.96 (m, 3H, Cβ-Phe, Cβ-Asp), 2.72

(dd, J = 17.1, 5.9 Hz, 1H, Cβ-Asp), 1.44 (s, 9H, OtBu), 1.38 (s, 9H, OtBu); 13C NMR (101 MHz,

CDCl3): δ 170.2, 170.1, 170.0, 155.5 (C=O), 136.2, 135.5, 129.7, 128.7, 128.4, 128.3, 127.0

(Carene), 82.3, 80.4 (OC(CH3)3), 66.9 (OCH2Ph), 54.0 (Cα-Phe), 50.6 (Cα-Asp) 38.1 (Cβ-Phe), 36.1

(Cβ-Asp), 28.3, 28.0 (OC(CH3)3); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C29H38N2O7Na [M+Na]+ m/z: 549.3, found 549.3; ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C29H39N2O7 [M+H]+ m/z: 527.3, found 527.3.

Boc-Asp(OBn)-Tyr(Ot-Bu)-Ot-Bu (148)

Boc-Asp(OBn)-OH 139 (5.88 g, 18.19 mmol), HCl*NH2-Tyr(Ot-Bu)-Ot-Bu 138 (5 g, 15.15

mmol), HBTU (7.2 g, 18.19 mmol), HOBt (2.46 g, 18.19 mmol) and DIPEA (7.8 mL, 45.5 mmol)

were reacted in DMF (54 mL) according to GP03. FC (15% ethyl acetate/petroleum ether → 30%

ethyl acetate/petroleum ether) gave Boc-Asp(OBn)-Tyr(Ot-Bu)-Ot-Bu 148 (9.01 g, quant.) as

colorless solid: Rf = 0.6 (ethyl acetate/petroleum ether 1:3); 1H NMR (400 MHz, CDCl3): δ 7.42 –

7.28 (m, 5H, phenyl), 7.12 – 7.04 (m, 2H, benzene3,5), 6.95 (d, J = 7.9 Hz, 1H, NH), 6.93 – 6.89

(m, 2H, benzene2,6), 5.61 (d, J = 8.8 Hz, 1H, NH), 5.18 – 5.04 (m, 2H, OCH2Ph), 4.72 – 4.60 (m,

1H, Cα-Asp), 4.58 – 4.46 (m, 1H, Cα-Tyr), 3.02 (pd, J = 13.6, 5.4 Hz, 3H, Cβ-Tyr, Cβ-Asp), 2.71

(dd, J = 17.2, 6.1 Hz, 1H, Cβ-Asp), 1.44 (s, 9H, OtBu), 1.35 (s, 9H, OtBu), 1.32 (s, 9H, OtBu); 13C

NMR (101 MHz, CDCl3): δ 170.2, 170.1, 154.4 (C=O), 135.6, 131.1, 130.1, 128.7, 128.5, 128.4,

124.2 (Carene), 82.3, 78.4 (OC(CH3)3), 67.0 (OCH2Ph), 54.2 (Cα-Phe), 50.7 (Cα-Asp), 37.7 (Cβ-

Tyr), 36.3 (Cβ-Asp), 29.0, 28.4, 28.1 (OC(CH3)3); ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C33H46N2O8Na [M+Na]+ m/z: 621.3, found 621.3; ESI-MS (H2O/MeCN (with 0.1%

formic acid), pos.) calcd for C33H47N2O8 [M+H]+ m/z: 599.3, found 599.3.

Boc-Asp(STrt)-Ot-Bu 29 (159)

Boc-Asp(OH)-Ot-Bu 149 (2.82 g, 9.75 mmol), DCC (3.02 g, 14.63 mmol), DMAP (120 mg, 0.975

mmol) and tritylthiol (5.39 g, 19.5 mmol) was reacted in 15 mL of DCM according to GP01. FC

(petroleum ether/ethyl acetate, 19:1 petroleum ether/ethyl acetate 9:1) gave Boc-Asp(STrt)-Ot-

Bu 159 (4.86 g, 91%) as pale yellowish solid. Rf = 0.45 (petroleum ether/ethyl acetate, 4:1); 1H

NMR (400 MHz, CDCl3) δ 7.32 – 7.17 (m, 15H, Trt), 5.30 (d, J = 8.6 Hz, 1H, Boc-NH), 4.40 –

4.24 (m, 1H, Cα-Asp), 3.16 (dd, J = 16.5, 4.8 Hz, 1 H, Cβ-Asp), 3.03 (dd, J = 16.3, 4.6 Hz, 1 H,

Cβ-Asp), 1.44 (s, 9H, OtBu), 1.38 (s, 9H, OtBu); 13C NMR (101 MHz, CDCl3) δ 195.0 (C(O)STrt),

169.7 (C(O)Ot-Bu), 155.5 (C(O)NH), 143.7, 129.9, 128.0, 127.3 (Carene), 82.4

(CHC(O)OC(CH3)3), 79.9 (NHC(O)OC(CH3)3), 71.1 ((C(O)S-C(Ph3)), 51.2 (Cα-Asp), 45.0 (Cβ-

Asp), 28.5, 28.0 (C(O)OC(CH3)3); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C32H38NO5S [M+H]+ m/z: 548.2, found 547.3.

Cbz-Asp(STrt)-Obn (160)

Cbz-Asp(OH)-OBn 150 (3g, 8.39 mmol), TrtSH (4.36 g, 16.78 mmol), DCC (2.6 g, 12.59 mmol)

and DMAP (103 mg, 0.84 mmol) were reacted in DCM (30 mL) according to GP01. FC (0-15%

ethyl acetate/petroleum ether gave Cbz-Asp(STrt)-OBn 160 (5 g, 96%) as pale yellow solid: Rf =

0.4 (ethyl acetate/petroleum ether 1:4); 1H NMR (400 MHz, CDCl3): δ 7.36 – 7.15 (m, 25H, arene),

5.50 (d, J = 8.4 Hz, 1H, NH), 5.17 – 5.00 (m, 4H, OCH2Ph, OCH2Ph), 4.56 (dt, J = 8.9, 4.7 Hz,

1H, Cα-Asp), 3.22 (dd, J = 16.1, 5.0 Hz, 1H, Cβ-Asp), 3.08 (dd, J = 16.2, 4.7 Hz, 1H, Cβ-Asp); 13C NMR (101 MHz, CDCl3): δ 197.3, 170.3 (C=O), 143.5, 135.3, 129.9, 128.7, 128.7, 128.5,

128.3, 128.3, 128.1, 128.0, 127.4 (Carene), 67.7, 67.2 (OCH2Ph), 51.2 (Cα-Asp), 44.9 (Cβ-Asp);

ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H33NO5SNa [M+Na]+ m/z: 638.2,

found 638.2. ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H34NO5S [M+H]+

m/z: 616.2, found 616.2.

Fmoc-Asp(STrt)-Val-Ot-Bu (161)

Fmoc-Asp(OBn)-Val-OtBu 145 (800 mg, 1.33 mmol) and 5-10% PdO2/C (200 mg) were reacted

in 1:2 MeOH/ethyl acetate (6.6 mL) under H2 for 30 min according to GP10. Subsequently, Fmoc-

Asp(OH)-Val-OtBu 151 (product from 1st step, 1.33 mmol), TrtSH (735 mg, 2.66 mmol), DCC

(329 g, 1.6 mmol) and DMAP (16.3 mg, 0.13 mmol) were reacted in DCM (5 mL) according to

GP01. FC (5 ethyl acetate/petroleum ether → 20% ethyl acetate/petroleum ether) gave Fmoc-

Asp(STrt)-Val-OtBu 161 (685 mg, 67%) as pale yellow solid: Rf = 0.4 (ethyl acetate/petroleum

ether 1:4); 1H NMR (400 MHz, CDCl3): δ 7.75 (dd, J = 7.6, 3.1 Hz, 2H), 7.64 (t, J = 8.2 Hz, 2H),

7.44 – 7.31 (m, 2H), 7.30 – 7.15 (m, 17H), 6.59 (d, J = 9.9 Hz, 1H), 6.17 (d, J = 8.6 Hz, 1H), 4.65

– 4.52 (m, 2H), 4.48 (dd, J = 8.6, 4.3 Hz, 1H), 4.31 – 4.20 (m, 2H), 2.81 (ddd, J = 176.6, 16.4, 4.2

Hz, 2H), 2.16 (qd, J = 6.8, 4.3 Hz, 1H), 1.50 (s, 9H), 1.00 – 0.83 (m, 6H); 13C NMR (101 MHz,

CDCl3): δ 198.2, 170.0, 156.0, 143.8, 143.7, 141.3, 130.0, 127.7, 127.0, 125.2, 120.0, 82.4, 70.1,

67.5, 57.9, 57.6, 47.2, 37.3, 31.7, 19.1, 17.7; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.)

calcd. for C47H48N2O6SNa [M+Na]+ m/z: 791.3, found 791.3; ESI-MS (H2O/MeCN (with 0.1%

formic acid), neg.) calcd. for C47H47N2O6S[M-H]- m/z: 767.3, found 767.1.

Boc-Asp(STrt)-Val-Ot-Bu (162)

Boc-Asp(OBn)-Val-Ot-Bu 144 (7.32 g, 15.34 mmol) and 5-10% PdO2/C (2.3 g) were reacted in

MeOH (50 mL) under H2 for 30 min according to GP10. Subsequently, Boc-Asp(OH)-Val-Ot-Bu

152 (product from 1st step, 15.34 mmol), TrtSH (8.47 g, 30.68 mmol), DCC (3.8 g, 18.4 mmol)

and DMAP (187 mg, 1.53 mmol) were reacted in DCM (40 mL) according to GP01. FC (10-20%

ethyl acetate/petroleum ether) gave Boc-Asp(STrt)-Val-Ot-Bu 162 (8.1 g, 72%) as pale yellow

solid: Rf = 0.4 (ethyl acetate/petroleum ether 1:3); 1H NMR (400 MHz, CDCl3): δ 7.30 – 7.19 (m,

15H), 6.89 (d, J = 8.7 Hz, 1H), 5.55 (d, J = 8.4 Hz, 1H), 4.48 – 4.24 (m, 2H), 3.04 (ddd, J = 171.8,

16.3, 4.2 Hz, 2H), 2.17 – 2.06 (m, 1H), 1.63 (d, J = 14.9 Hz, 2H), 1.46 (s, 9H), 1.44 (s, 9H), 0.86

(dd, J = 11.1, 6.9 Hz, 6H); 13C NMR (101 MHz, CDCl3): δ 170.6, 170.2, 143.6, 129.9, 128.0, 127.3,

82.0, 57.8, 31.5, 28.4, 28.2, 19.0, 17.7; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd

for C37H47N2O6S [M+H]+ m/z: 647.3, found 647.3; ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C37H46N2O6SNa [M+Na]+ m/z: 669.3, found 669.3.

Boc-Asp(STrt)-Ala-Ot-Bu (163)

Boc-Asp(OBn)-Ala-Ot-Bu 146 (7.18 g, 15.46 mmol) and 5-10% PdO2/C (1.93 g) were reacted in

MeOH (50 mL) under H2 according to GP10. Subsequently, Boc-Asp(OH)-Ala-Ot-Bu 153

(product from 1st step, 15.46mmol), TrtSH (8.5 g, 30.92 mmol), DCC (4.78 g, 23.19 mmol) and

DMAP (190 mg, 1.55 mmol) were reacted in DCM (60 mL) for 6 hours at ambient temperature

according to GP01. FC (10-20% ethyl acetate/petroleum ether) gave Boc-Asp(STrt)-Ala-Ot-Bu

163 (4.68 g, 49%) as pale yellow solid: Rf = 0.45 (ethyl acetate/petroleum ether 1:3); 1H NMR (400

MHz, CDCl3): δ 7.26 – 7.06 (m, 15H), 6.77 (s, 1H), 5.40 (d, J = 8.6 Hz, 1H), 4.33 (s, 1H), 4.27 (p,

J = 7.2 Hz, 1H), 3.00 (dd, J = 189.7, 16.1 Hz, 2H), 1.38 (s, 9H), 1.36 (s, 9H), 1.22 (d, J = 7.2 Hz,

3H); 13C NMR (101 MHz, CDCl3): δ 196.8, 171.7, 169.8, 143.6, 129.9, 128.0, 127.3, 82.0, 71.2,


for C35H43N2O6S [M+H]+ m/z: 619.3, found 619.3; ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C35H42N2O6SNa [M+H]+ m/z: 641.3, found 641.3.

Boc-Asp(STrt)-Phe-Ot-Bu (164)

Boc-Asp(OBn)-Phe-Ot-Bu 147 (6.03 g, 11.45 mmol) and 5-10% PdO2/C (1.71 g) were reacted in

MeOH (35 mL) under H2 according to GP10. Subsequently, Boc-Asp(OH)-Phe-Ot-Bu 154

(product from 1st step, 11.45 mmol), TrtSH (6.33 g, 22.9 mmol), DCC (3.07 g, 14.89 mmol) and

DMAP (139 mg, 1.14 mmol) were reacted in DCM (60 mL) according to GP01. FC (10% ethyl

acetate/petroleum ether → 25-30% ethyl acetate/petroleum ether) gave Boc-Asp(STrt)-Phe-Ot-Bu

164 (4.86 g, 60%) as pale yellow solid: Rf = 0.4 (ethyl acetate/petroleum ether 1:3); 1H NMR (400

MHz, CDCl3): δ 7.30 – 7.09 (m, 20H), 6.80 (d, J = 7.7 Hz, 1H), 5.38 (d, J = 9.5 Hz, 1H), 4.67 –

4.57 (m, 1H), 4.41 (s, 1H), 3.35 – 2.75 (m, 4H), 1.41 (s, 9H), 1.36 (s, 9H); ESI-MS (H2O/MeCN

(with 0.1% formic acid), pos.) calcd for C41H46N2O6SNa [M+Na]+ m/z: 717.3, found 717.3; ESI-

MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C41H47N2O6S [M+H]+ m/z: 695.3, found

695.3.

Boc-Asp(STrt)-Tyr(Ot-Bu)-Ot-Bu (165)

Boc-Asp(OBn)-Tyr(Ot-Bu)-Ot-Bu 148 (9 g, 15 mmol) and 5-10% PdO2/C (2.25 g) were reacted

in MeOH (45 mL) under H2 according to GP10: 1H NMR (400 MHz, CDCl3): δ 7.06 (d, J = 8.0

Hz, 2H), 6.89 (d, J = 8.0 Hz, 2H), 5.62 (s, 1H), 4.65 (q, J = 6.7 Hz, 1H), 4.52 (s, 1H), 3.10 – 2.64

(m, 4H), 1.44 (s, 9H), 1.34 (s, 9H), 1.31 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 170.5, 170.3,

154.4, 131.1, 130.1, 124.2, 82.6, 78.6, 54.3, 37.7, 36.0, 29.0, 28.4, 28.0.

After, Boc-Asp(OH)-Tyr(Ot-Bu)-Ot-Bu 155 (product from 1st step, 15 mmol), TrtSH (7.6 g, 27.5

mmol), DCC (3.16 g, 16.5 mmol) and DMAP (168 mg, 1.38 mmol) were reacted in DCM (50 mL)

according to GP02. FC (10% ethyl acetate/petroleum ether → 30% ethyl acetate/petroleum ether)

gave Boc-Asp(STrt)-Try(Ot-Bu)-Ot-Bu 165 (9.35 g, 81%) as pale yellow solid: Rf = 0.35 (ethyl

acetate/petroleum ether 1:4); 1H NMR (400 MHz, CDCl3): δ 7.34 – 7.17 (m, 15H), 7.06 – 6.99 (m,

2H), 6.90 – 6.85 (m, 2H), 6.85 – 6.80 (m, 1H), 5.39 (d, J = 8.8 Hz, 1H), 4.60 (td, J = 7.0, 5.3 Hz,

1H), 4.51 – 4.31 (m, 1H), 3.38 – 2.78 (m, 4H), 1.43 (s, 9H), 1.34 (s, 9H), 1.33 (s, 9H); 13C NMR

(101 MHz, CDCl3): δ 196.6, 170.1, 169.8, 154.3, 143.6, 130.1, 129.9, 127.9, 127.3, 124.1, 82.3,


for C45H54N2O7S [M+Na]+ m/z: 789.4, found 789.4; ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C45H55N2O7S [M+H]+ m/z: 767.4, found 767.4.

Boc-Asp(STrt)-Val-Phe-Ot-Bu (166)

Boc-Asp(OBn)-Val-Phe-Ot-Bu 141 (5.87 g, 9.38 mmol) and 5-10% PdO2/C (1.4 g) reacted in

MeOH (60 mL) under H2 according to GP10. Subsequently, Boc-Asp(OH)-Val-Phe-Ot-Bu 156

(product from 1st step, 9.38 mmol), TrtSH (5.2 g, 18.76 mmol), DCC (2.9 mg, 14.07 mmol) and


acetate/petroleum ether → 35% ethyl acetate/petroleum ether) gave Boc-Asp(STrt)-Val-Phe-Ot-

Bu 166 (3.43 g, 46%) as pale yellow solid: Rf = 0.27 (ethyl acetate/petroleum ether 1:3); 1H NMR

(400 MHz, CDCl3): δ 7.30 – 7.17 (m, 15H), 7.16 – 7.09 (m, 2H), 6.85 (d, J = 8.1 Hz, 1H), 6.30 (d,

J = 7.4 Hz, 1H), 5.50 (d, J = 7.3 Hz, 1H), 4.71 (dt, J = 7.8, 6.3 Hz, 1H), 4.33 (dd, J = 13.1, 5.4 Hz,

1H), 4.19 (dd, J = 8.5, 6.1 Hz, 1H), 3.22 (dd, J = 16.3, 5.8 Hz, 1H), 3.03 (d, J = 6.3 Hz, 2H), 2.16

– 2.06 (m, 1H), 1.44 (s, 9H), 1.39 (s, 9H), 0.87 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H); 13C

NMR (101 MHz, CDCl3): δ 196.5, 170.4, 170.4, 170.2, 155.7, 143.6, 136.2, 129.9, 129.6, 128.6,

128.0, 127.3, 127.1, 82.4, 80.8, 77.4, 71.1, 58.7, 53.7, 52.1, 43.5, 38.2, 30.8, 28.4, 28.1, 19.3, 17.8,

14.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C46H56N3O7S [M+H]+ m/z:

794.4, found 794.5.

Boc-Asp(STrt)-Ala-Phe-Ot-Bu (167)

Boc-Asp(OBn)-Ala-Phe-Ot-Bu 142 (1.95 g, 3.26 mmol) and 5-10% PdO2/C (500 mg) reacted in

MeOH (13 mL) under H2 according to GP10. Subsequently, Boc-Asp(OH)-Ala-Phe-Ot-Bu 157

(product from 1st step, 3.26 mmol), TrtSH (1.8 g, 6.25 mmol), EDAC (658 mg, 4.24 mmol) and


acetate/petroleum ether → 35-50% ethyl acetate/petroleum ether) gave Boc-Asp(STrt)-Ala-Phe-

Ot-Bu 167 (1.62 g, 65%) as pale yellow solid: Rf = 0.5 (ethyl acetate/petroleum ether 1:1); 1H NMR

(400 MHz, CDCl3): δ 7.34 – 7.17 (m, 18H), 7.12 (dd, J = 6.9, 1.7 Hz, 2H), 6.70 (d, J = 7.5 Hz,

1H), 6.46 (d, J = 7.7 Hz, 1H), 5.44 (d, J = 8.6 Hz, 1H), 4.68 (dt, J = 7.7, 6.2 Hz, 1H), 4.41 – 4.25

(m, 2H), 3.32 – 2.74 (m, 4H), 1.44 (s, 9H), 1.41 (s, 9H), 1.26 (d, J = 7.0 Hz, 3H); 13C NMR (101

MHz, CDCl3): δ 200.1, 171.2, 170.4, 170.2, 143.6, 136.2, 129.9, 129.6, 128.5, 128.0, 127.4, 127.1,

82.5, 80.8, 71.2, 53.8, 51.7, 49.2, 38.0, 28.4, 28.1, 17.9; ESI-MS (H2O/MeCN (with 0.1% formic

acid), pos.) calcd for C44H51N3O7SNa [M+Na]+ m/z: 788.3, found 788.3; ESI-MS (H2O/MeCN

(with 0.1% formic acid), pos.) calcd for C44H52N3O7S [M+H]+ m/z: 766.3, found 766.4.

Boc-Asp(STrt)-Gly-Phe-Ot-Bu (168)

Boc-Asp(OBn)-Gly-Phe-Ot-Bu 143 (4 g, 6.85 mmol) and 5-10 % Pd-C (1.03 g) were reacted in

MeOH (25 mL) for 30 min according to GP10. MPLC (Grace-Reveleris-silica 40 g; 0-15%

MeOH/DCM in 16 min; flow 35 mL min-1: Rt = 12 min) gave Boc-Asp(OH)-Gly-Phe-Ot-Bu 158

(2.85 g, 84%) as colorless viscous solid. ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd

for C24H34N3O8 [M-H]- m/z: 492.2, found 492.4.

Subsequently, Boc-Asp(OH)-Gly-Phe-Ot-Bu 158 (1.74 g, 3.52 mmol), TrtSH (6.96 g, 25.166

mmol), EDAC (1.27 g, 8.18 mmol) and DMAP (77 mg, 0.629 mmol) were reacted in DMF (9 mL)

overnight at 4 oC according to GP02. All purification steps carried out under 30 oC to reduce

aspartimide formation. MPLC (Grace-Reveleris-silica 40 g; 10-100% ethyl acetate/petroleum ether

in 16 min; flow 35 mL min-1: Rt = 13 min) gave Boc-Asp(STrt)-Gly-Phe-Ot-Bu 168 (646 mg, 24%)

as pale yellow solid: Rf = 0.7 (ethyl acetate/petroleum ether 4:1); 1H NMR (400 MHz, CDCl3): δ

7.35 – 7.17 (m, 20H), 6.70 (t, J = 5.6 Hz, 1H), 6.48 (d, J = 7.8 Hz, 1H), 5.28 (d, J = 8.2 Hz, 1H),

4.66 (dt, J = 7.8, 6.4 Hz, 1H), 4.37 (dt, J = 8.0, 5.3 Hz, 1H), 3.96 – 3.70 (m, 2H), 3.34 – 2.86 (m,

4H), 1.45 (s, 9H), 1.38 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 170.7, 170.5, 168.0, 155.5, 143.5,

136.2, 129.9, 129.5, 128.5, 128.0, 127.4, 82.4, 71.3, 53.9, 51.7, 43.3, 38.0, 28.5, 28.1; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C43H50N3O7S [M+H]+ m/z: 752.3, found

752.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C43H48N3O7S [M-H]- m/z:

750.3, found 750.5.

Cbz-Asp(SH)-OBn (169)

Cbz-Asp(S-Trt)-OBn 150 (1 g, 1.62 mmol), TIS (1 mL, 4.88 mmol) and TFA (1.25 mL, 15.26

mmol) were reacted in DCM (23 mL) according to GP06. MPLC (Grace-Reveleris-C18(RP) 12 g;

5-5-60-90-100-100 MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 3+5+15+5 min; flow 25

mL-1 min: Rt = 13 min) gave Cbz-Asp(S-Trt)-OBn 169 (390 mg, 65%) as white foam; 1H NMR

(400 MHz, CDCl3) δ 7.41 – 7.27 (m, 10H), 5.69 (d, J = 8.2 Hz, 1H), 5.18 (s, 2H), 5.12 (s, 2H),

4.66 – 4.50 (m, 2H), 3.44 – 3.16 (m, 2H);13C NMR (101 MHz, CDCl3): δ 195.3, 170.1, 156.0,

136.2, 135.1, 128.8, 128.7, 128.5, 128.4, 128.2, 68.0, 67.4, 50.8, 47.3; ESI-MS (H2O/MeCN (with

0.1% formic acid), pos.) calcd. for C19H20NO5S [M+H]+ m/z: 374.1,found 373.9; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C19H19NO5SNa [M+Na]+ m/z: 396.1,found

395.9.

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu (170)

Boc-Asp(STrt)-Tyr(Ot-Bu)-Ot-Bu 155 (2 g, 2.6 mmol), TIS (1.6 mL, 7.82 mmol) and TFA (5%

v/v, 2 mL, 26 mmol) were reacted in DCM (40 mL) according to GP06. MPLC (Grace-Reveleris-

C18(RP) 12 g; 35-80% MeCN/H2O in 11 min; flow 25 mL min-1: Rt = 8 min) gave Boc-Asp(SH)-

Tyr(Ot-Bu)-Ot-Bu 170 (737 mg, 54%) as white foam. 1H NMR (400 MHz, CDCl3): δ 7.13 – 7.02

(m, 2H), 6.97 – 6.88 (m, 2H), 6.85 (d, J = 7.6 Hz, 1H), 5.40 (d, J = 9.0 Hz, 1H), 4.64 (dt, J = 7.4,

6.0 Hz, 1H), 4.49 (s, 1H), 3.46 – 2.93 (m, 4H), 1.44 (s, 9H), 1.36 (s, 9H), 1.33 (s, 9H); 13C NMR

(101 MHz, CDCl3): δ 197.6, 170.1, 169.6, 154.1, 131.0, 130.2, 124.3, 82.5, 78.5, 54.1, 51.2, 46.9,

37.6, 29.0, 28.4, 28.1; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C26H39N2O7S

[M-H]- m/z: 523.3, found 523.5.

Boc-Asp(SH)-Val-Phe-Ot-Bu (171)

Boc-Asp(STrt)-Val-Phe-Ot-Bu 156 (240 mg, 0.302 mmol), TIS (186 µL, 0.907 mmol) and TFA

(5% v/v, 231 µL, 3.022 mmol) were reacted in DCM (4.5 mL). MPLC (Grace-Reveleris-C18(RP)

4 g; 20-80% MeCN/H2O in 11 min; flow 15 mL min-1: Rt = 9 min) gave Boc-Asp(SH)-Val-Phe-

Ot-Bu 171 (80 mg, 48%) as colorless solid: ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.)

calcd for C27H40N3O7S [M-H]- m/z: 550.3, found 550.2.

TFA*NH2-Asp(SH)-Tyr(OH)-OH (172)

Boc-Asp(STrt)-Tyr(Ot-Bu)-Ot-Bu 155 (500 mg, 0.75 mmol), TIS (1.6 mL, 7.82 mmol) and TFA

(60% v/v, 4 mL) were reacted in DCM (1.6 ML) for 1 h according to GP09. MPLC (Grace-

Reveleris-C18(RP) 4 g; 5-100% MeCN/H2O in 12 min; flow 16 mL min-1: Rt = 8 min) gave

TFA*NH2-Asp(SH)-Tyr(OH)-OH 172 (135 mg, 44%) as white foam; 1H NMR (400 MHz,

DMSO-d6): δ 9.23 (br s, 2H), 8.75 (d, J = 7.9 Hz, 1H), 8.24 (br s, 3H), 7.04 (d, J = 8.5 Hz, 2H),

6.73 – 6.61 (m, 2H), 4.31 (ddd, J = 9.6, 7.8, 4.5 Hz, 1H), 3.98 (dd, J = 10.3, 3.1 Hz, 1H), 2.92 (ddd,

J = 25.8, 15.5, 3.8 Hz, 2H), 2.78 – 2.53 (m, 2H); 13C NMR (101 MHz, DMSO): δ 209.7, 172.6,

168.9, 156.0, 130.1, 127.5, 115.1, 54.4, 51.0, 49.1, 35.7; ESI-MS (H2O/MeCN (with 0.1% formic

acid), neg.) calcd for C13H15N2O5S [M-H]- m/z: 311.1, found 311.3.

Cbz-Asn(Liss)-OBn (173)

Cbz-Asp(STrt)-OBn 150 (25 mg, 0.04 mmol), TIS (25 µL) and TFA (5% v/v, 31 µL, 04 mmol)

were reacted in DCM (600 µL) for 5 min according to GP06. Subsequently, Cbz-Asp(SH)-OBn

169 (from first step, 0.04 mmol), lissamine azide 114 (11.5 mg, 0.02 mmol) and 2,6-lutidine (4.4

µL, 0.04 mmol) were reacted in 1:1 DCM/MeOH (120 µL) for 5 min according to GP04. HPLC

(Phenomenex-Gemini 110-5 C6-phenyl, 21.2 x 250 mm; 65-80% MeCN (with 0.1% TFA) in H2O

(with 0.1% TFA) in 18 min; flow 8 mL min-1: Rt = 12.5 min) gave red foam, Cbz-Asn(Liss)-OBn

(16.7 mg, 85 %, TFA adduct); 1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 2.0 Hz, 1H), 8.07

(dd, J = 8.0, 2.0 Hz, 1H), 7.41 (d, J = 12.6 Hz, 1H), 7.30 (qd, J = 7.7, 7.0, 4.5 Hz, 10H), 7.04 –

6.83 (m, 6H), 5.23 – 4.86 (m, 4H), 4.51 (dd, J = 7.7, 5.8 Hz, 1H), 3.62 (q, J = 7.9 Hz, 8H), 3.38 (s,

2H), 2.90 (dd, J = 16.6, 5.9 Hz, 1H), 2.72 (dd, J = 16.5, 7.8 Hz, 1H), 1.20 (q, J = 8.1, 7.1 Hz, 12H); 13C NMR (101 MHz, DMSO-d6) δ 170.9, 157.2, 155.9, 155.1, 147.8, 136.9, 135.9, 132.7, 130.5,

128.5, 128.4, 128.1, 127.8, 127.7, 126.6, 113.6, 113.5, 95.5, 66.3, 65.7, 45.3, 12.5; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C46H49N4O11S2 [M+H]+ m/z:897.3, found

897.2.

Boc-Asn(Liss)-Tyr(Ot-Bu)-Ot-Bu (174)

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170 (18 mg, 0.034 mmol), lissamine azide 114 (10 mg, 0.0171

mmol) and 2,6-lutidine (4 µL, 0.034 mmol) were reacted in MeOH (200 µL) for 5 min according

to GP04. HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 21.2 x 250 mm; 80% MeCN (with 0.1%

TFA) in H2O (with 0.1% TFA) in 12 min; flow 9 mL min-1: Rt = 11 min) gave title compound,

Boc-Asn(Liss)-Tyr(Ot-Bu)-Ot-Bu 174, 19.2 mg (98%, TFA salt) as red foam; 1H NMR (600 MHz,

DMSO-d6): δ 12.39 (s, 1H), 8.50 (d, J = 1.9 Hz, 1H), 8.10 (dd, J = 8.0, 2.0 Hz, 1H), 8.05 (d, J =

7.2 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.15 – 6.92 (m, 10H), 6.86 (d, J = 8.4 Hz, 2H), 4.28 (dq, J =

26.6, 8.0, 7.2 Hz, 2H), 3.63 (dh, J = 12.6, 7.4 Hz, 8H), 2.87 (qd, J = 13.8, 7.1 Hz, 2H), 2.72 – 2.55

(m, 2H), 1.36 (s, 9H), 1.26 (s, 9H), 1.25 (s, 9H), 1.20 (t, J = 7.1 Hz, 12H); 13C NMR (151 MHz,

DMSO-d6): δ 171.0, 170.3, 168.8, 158.2, 157.1, 155.1, 153.6, 148.0, 140.4, 134.4, 132.6, 131.6,

130.7, 129.8, 127.9, 126.4, 123.5, 113.7, 113.6, 113.4, 95.5, 80.8, 78.3, 77.7, 54.3, 49.8, 45.3, 37.5,

36.2, 28.5, 28.2, 27.5, 12.5; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for

C53H70N5O13S2 [M+H]+ m/z: 1048.4, found 1048.4; ESI-MS (H2O/MeCN (with 0.1% formic acid),

pos.) calcd for C53H68N5O13S2 [M-H]- m/z: 1046.4, found 1046.6.

Boc-Asn(pCSea)-Tyr(Ot-Bu)-Ot-Bu (175)

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170 (63 mg, 0.122 mmol), Boc-4-((2-

aminoethyl)carbamoyl)benzenesulfonyl azide 119 (30.6 mg, 0.084 mmol) and 2,6-lutidine (13.5

µL, 0.122 mmol) were reacted in MeOH (365 µL) for 15 min according to GP04. FC (0-7%

MeOH/DCM) gave Boc-Asn(pCSea)-Tyr(Ot-Bu)-Ot-Bu 175 (70 mg, quant.) as colorless viscous

solid: Rf = 0.3 (MeOH/DCM 1:19); 1H NMR (400 MHz, CDCl3): δ 8.05 – 7.67 (m, 4H), 7.65 –

7.47 (m, 1H), 7.18 (s, 1H), 6.98 (d, J = 8.0 Hz, 2H), 6.91 – 6.77 (m, 2H), 5.94 (s, 1H), 5.46 (d, J =

12.2 Hz, 1H), 4.52 (q, J = 6.9 Hz, 2H), 3.58 – 3.24 (m, 4H), 3.08 – 2.61 (m, 4H), 1.39 (s, 8H), 1.37

(s, 9H), 1.30 (s, 9H), 1.28 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 170.9, 170.1, 166.7, 157.4,

155.8, 154.2, 138.6, 130.9, 130.2, 128.1, 127.8, 124.2, 121.2, 82.6, 80.8, 80.0, 78.6, 54.3, 54.1,

51.1, 41.6, 40.0, 37.3, 28.9, 28.5, 28.3, 28.0; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.)

calcd for C40H60N5O12S [M+H]+ m/z: 834.4, found 834.4; ESI-MS (H2O/MeCN (with 0.1% formic

acid), neg.) calcd for C40H58N5O12S [M-H]- m/z: 832.4, found 832.6.

Boc-Asn(Dn)-Tyr(Ot-Bu)- Ot-Bu (176)

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170 (40 mg, 0.076 mmol), dansyl azide 116 (10.3 mg, 0.037

mmol) and 2,6-lutidine (8.4 µL, 0.076 mmol) were reacted in MeOH (450 µL) for 2 h at room

temperature according to GP04. HPLC (Eurospher 100-10 C18, 16 x 250 mm; 55-65-85% MeCN

(with 0.1% TFA) in H2O (with 0.1% TFA) in 4+16 min; flow 9 mL min-1: Rt = 14 min) gave Boc-

Asn(Dn)-Tyr(Ot-Bu)- Ot-Bu*TFA 176 (24.5 mg, 79% ) as pale yellow foam; 1H NMR (400 MHz,

DMSO-d6): δ 12.40 (s, 1H), 8.53 (dt, J = 8.6, 1.1 Hz, 1H), 8.29 (dd, J = 7.3, 1.3 Hz, 1H), 8.22 (d,

J = 8.7 Hz, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.66 (ddd, J = 16.6, 8.7, 7.5 Hz, 2H), 7.28 (dd, J = 7.7,

0.9 Hz, 1H), 7.08 – 7.01 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 6.86 – 6.79 (m, 2H), 4.17 (dq, J = 19.8,

7.4 Hz, 2H), 2.84 (s, 6H), 2.81 (d, J = 6.8 Hz, 2H), 2.50 (p, J = 1.9 Hz, 2H), 1.34 – 1.16 (m, 27H); 13C NMR (101 MHz, DMSO-d6): δ 177.0, 168.3, 153.6, 151.4, 134.3, 131.5, 130.8, 130.7, 129.7,

128.9, 128.8, 128.4, 123.5, 123.4, 118.2, 115.3, 80.6, 78.1, 77.6, 54.2, 45.1, 36.1, 28.5, 28.1, 27.4;

ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H53N4O9S [M+H]+ m/z: 741.4,

found 741.4.

Boc-Asn(biotin1)-Val-Phe-Ot-Bu (177)

Boc-Asp(SH)-Val-Phe-Ot-Bu 171 (40 mg, 0.0725 mmol), biotin1 azide (30 mg, 0.061 mmol) and

2,6-lutidine (10.3 µL, 0.092 mmol) were reacted in MeOH (300 µL) for 15 min at room temperature

according to GP04. HPLC (Eurospher 100-10 C18, 16 x 250 mm; 40-70% MeCN (with 0.1%

TFA) in H2O (with 0.1% TFA) in 20 min; flow 9 mL min-1: Rt = 15.8 min) gave Boc-Asn(biotin1)-

Val-Phe-Ot-Bu 1 177 (46 mg, 76%) as white foam; 1H NMR (400 MHz, DMSO-d6): δ 12.20 (s,

1H), 8.72 (t, J = 5.5 Hz, 1H), 8.36 (d, J = 7.1 Hz, 1H), 8.07 – 7.95 (m, 4H), 7.91 (t, J = 5.6 Hz,

1H), 7.37 (d, J = 9.0 Hz, 1H), 7.30 – 7.15 (m, 5H), 6.41 (s, 2H), 4.36 – 4.26 (m, 2H), 4.24 – 4.15

(m, 2H), 4.10 (dd, J = 7.7, 4.4 Hz, 1H), 3.32 (q, J = 6.2 Hz, 2H), 3.22 (q, J = 6.6, 6.2 Hz, 2H), 3.06

(ddd, J = 8.3, 6.1, 4.2 Hz, 1H), 2.91 (dd, J = 7.6, 2.1 Hz, 2H), 2.84 – 2.52 (m, 4H), 2.07 (t, J = 7.4

Hz, 2H), 1.91 (h, J = 6.6 Hz, 1H), 1.66 – 1.38 (m, 4H), 1.38 – 1.19 (m, 20H), 0.86 – 0.66 (m, 6H); 13C NMR (101 MHz, DMSO-d6): δ 172.4, 170.5, 170.4, 170.3, 168.9, 165.1, 162.7, 155.3, 141.3,

139.0, 137.0, 129.1, 128.1, 127.9, 127.5, 126.5, 80.5, 78.5, 61.0, 59.2, 56.7, 55.4, 54.2, 50.1, 38.1,


acid), pos.) calcd for C46H67N8O12S2 [M+H]+ m/z: 987.4, found 987.4.

Boc-Asn(biotin1)-Tyr(Ot-Bu)-Ot-Bu (not described)

Boc-Asp(SH)-Tyr(Ot-Bu)-Ot-Bu 170 (62 mg, 0.118 mmol), biotin1 azide 121 (30.4 mg, 0.061

mmol) and 2,6-lutidine (10.2 µL, 0.092 mmol) were reacted in MeOH (300 µL) for 15 min

according to GP04. MPLC (Grace-Reveleris-silica 4 g; 0-5-12% MeOH/CHCl3 in 14 min; flow 15

mL min-1: Rt = 7 min) gave Boc-Asn(biotin1)-Tyr(Ot-Bu)-Ot-Bu (52 mg, 88%) as white solid: Rf

= 0.4 (CHCl3/MeOH 1:9); 1H NMR (400 MHz, MeOD-d4): δ 7.93 – 7.84 (m, 2H), 7.79 – 7.74 (m,

2H), 6.91 (dd, J = 8.8, 2.6 Hz, 2H), 6.76 – 6.65 (m, 2H), 4.34 – 4.10 (m, 3H), 4.04 (dd, J = 7.9, 4.4

Hz, 1H), 3.32 (t, J = 5.8 Hz, 2H), 3.25 (qd, J = 8.2, 7.2, 5.0 Hz, 2H), 2.90 (ddd, J = 8.8, 5.8, 4.4

Hz, 1H), 2.79 – 2.32 (m, 6H), 2.02 (td, J = 7.2, 1.9 Hz, 2H), 1.54 – 1.29 (m, 4H), 1.27 – 1.14 (m,

20H), 1.12 (s, 9H); 13C NMR (101 MHz, MeOD-d4): δ 176.7, 176.6, 173.1, 171.7, 168.7, 166.1,

157.5, 155.4, 144.5, 139.7, 132.9, 131.1, 129.1, 128.8, 125.2, 83.2, 81.0, 79.5, 63.3, 61.6, 56.9,

55.8, 52.2, 41.1, 41.0, 39.9, 37.9, 36.8, 29.7, 29.5, 29.2, 28.7, 28.2, 26.8; ESI-MS (H2O/MeCN

(with 0.1% formic acid), pos.) calcd for C45H66N7O12S2 [M+H]+ m/z: 960.4, found 960.4; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C45H65N7O12S2Na[M+Na]+ m/z: 982.4, found

982.6.

Boc-Asn(Dn)-Ot-Bu (178)

Boc-Asp(STrt)-Ot-Bu 159 (150 mg, 0.274 mmol), TIS (169 µL, 0.822 mmol) and TFA (5% v/v,

210 µL, 2.741 mmol) were reacted in DCM (4.5 mL) for 5 min according to GP06. Subsequently,

Boc-Asp(SH)-Ot-Bu (from first step, 0.274 mmol), dansyl azide 116 (38 mg, 0.137 mmol) and 2,6-

lutidine (32 µL, 0.274 mmol) were reacted in MeOH (500 µL) for 2 h at room temperature

according to GP04. FC (15-25% ethyl acetate/petroleum ether) gave Boc-Asn(Dn)-Ot-Bu 178

(53.5 mg, 75%) as pale yellow solid. Rf = 0.55 (petroleum ether/EtOAc, 1:1); 1H NMR (400 MHz,

CDCl3): 9.5 (br s, 1 H, SO2NH), = 8.58 (d, J = 8.5 Hz, 1 H, dansyl), 8.47 (dd, J = 7.4, 1.3 Hz, 1

H, dansyl), 8.25–8.19 (m, 1 H, dansyl), 7.55 (ddd, J = 8.7, 7.5, 6.3 Hz, 2 H, dansyl), 7.17 (dd, J =

7.7, 0.9 Hz, 1 H, dansyl), 5.49 (d, J = 8.3 Hz, 1 H, Boc-NH), 4.30 (dt, J = 8.7, 4.6 Hz, 1 H, H-),

2.87 (s, 6 H, N(CH3)2), 2.85–2.71 (m, 2 H, CH2), 1.41 (s, 9 H, C(CH3)3), 1.07 (s, 9 H, C(CH3)3); 13C NMR (101 MHz, CDCl3): = 169.4 (C(O)Ot-Bu), 168.8 (C(O)NHSO2), 152.3 (C(O)NH),

133.6, 132.4, 131.9, 130.0, 129.7, 128.9, 128.1, 123.4, 118.4, 115.4 (Carene), 82.6

(CHC(O)OC(CH3)3), 80.5 ((NHC(O)OC(CH3)3), 50.2 (C-), 45.6 (N(CH3)), 39.2 (C-), 28.4, 27.6

(C(CH3)3); ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C25H36N3O7S [M+H]+

m/z: 522.2, found 522.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for

C25H34N3O7S [M-H]- m/z: 520.2, found 520.3.

Boc-Asn(Dn)-Val-Ot-Bu (179)

Boc-Asp(STrt)-Val-Ot-Bu 162 (248 mg, 0.384 mmol), TIS (240 µL, 1.16 mmol) and TFA (5%

v/v, 266 µL, 3.84 mmol) were reacted in DCM (5 mL) for 5 min according to GP06. Subsequently,

Boc-Asp(SH)-Ot-Bu (from first step, 0.384 mmol), dansyl azide 116 (212 mg, 0.769 mmol) and

2,6-lutidine (42 µL, 0.385 mmol) were reacted in MeOH (3 mL) for 2 h at room temperature

according to GP04. FC (0-3% MeOH/DCM) gave Boc-Asn(Dn)-Val-Ot-Bu 179 (173.2 mg, 73%)

as pale yellow viscous solid: Rf = 0.35 (MeOH/DCM 1:9); 1H NMR (400 MHz, CDCl3): δ 8.48 (d,

J = 8.6 Hz, 1H), 8.36 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.37 – 7.20 (m, 1H), 7.10 (d, J

= 7.5 Hz, 1H), 5.99 (br s, 1H), 4.55 (br s, 1H), 4.38 – 4.14 (m, 1H), 3.49 – 3.31 (m, 1H), 2.84 (s,

6H), 2.72 (td, J = 19.1, 17.4, 6.9 Hz, 2H), 2.09 – 1.82 (br m, 1H), 1.50 – 1.25 (m, 18H), 0.84 – 0.52

(br m, 6H); 13C NMR (101 MHz, CDCl3): δ 171.6, 170.6, 155.9, 151.6, 129.9, 129.8, 128.2, 123.2,

119.7, 115.1, 82.1, 80.5, 58.1, 51.3, 45.5, 31.1, 28.4, 28.1, 18.8, 17.6; ESI-MS (H2O/MeCN (with

0.1% formic acid), pos.) calcd for C30H45N4O8S [M+H]+ m/z: 621.3, found 621.3; ESI-MS

(H2O/MeCN (with 0.1% formic acid), neg.) calcd for C30H43N4O8S [M-H]- m/z: 619.3, found

619.4.

Boc-Asn(Liss)-Val-Ot-Bu (180)

Boc-Asp(STrt)-Val-Ot-Bu 162 (13 mg, 0.02 mmol), TIS (12.5 µL, 0.06 mmol) and TFA (5 % v/v,

15.3 µL, 0.2 mmol) reacted in dry DCM (300 µL) for 5 min according to GP06. In the subsequent

step, Boc-Asp(SH)-Val-Ot-Bu (from first step, 0.02 mmol), lissamine azide 114 (23.3 mg, 0.04

mmol) and 2,6-lutidine (2.2 µL, 0.02 mmol) were reacted in 1:1 Chloroform/MeOH (120 µL) for

5 min according to GP04. HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 21.2 x 250 mm; 75%

MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 13 min; flow 9 mL min-1): Rt = 12.5 min)

gave title compound, Boc-Asn(Liss)-Val-Ot-Bu 73% (14.7 mg, TFA adduct) as red foam; 1H NMR

(600 MHz, DMSO-d6): δ 12.37 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 8.0, 2.1 Hz, 1H),

7.76 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.03 (ddd, J = 18.9, 9.6,

2.4 Hz, 2H), 6.94 (dt, J = 4.8, 1.9 Hz, 5H), 4.33 (td, J = 8.7, 4.4 Hz, 1H), 4.03 (dd, J = 8.3, 5.5 Hz,

1H), 3.69 – 3.59 (m, 8H), 2.75 – 2.57 (m, 2H), 2.01 (dq, J = 13.1, 6.7 Hz, 1H), 1.40 (d, J = 2.9 Hz,

9H), 1.37 (s, 9H), 1.20 (t, J = 7.1 Hz, 12H), 0.85 (d, J = 6.9 Hz, 6H); 13C NMR (151 MHz, DMSO-

d6) δ 171.2, 170.4, 168.8, 158.0, 157.1, 157.1, 156.9, 155.3, 155.1, 155.0, 147.9, 140.3, 134.4,

132.6, 130.6, 127.9, 126.4, 113.7, 113.6, 113.4, 95.4, 80.8, 78.3, 57.7, 49.8, 45.3, 37.2, 30.2, 28.2,

27.7, 27.6, 18.9, 18.8, 17.8, 12.5; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for


neg.) calcd for C45H60N5O12S2 [M-H]- m/z: 926.4, found 926.6.

Boc-Asn(Liss)-Gly-Phe-Ot-Bu (181)

Boc-Asp(STrt)-Gly-Phe-Ot-Bu 160 (15 mg, 0.02 mmol), TIS (12.5 µL, 0.06 mmol) and TFA (5 %

v/v, 15.3 µL, 0.2 mmol) reacted in dry DCM (300 µL) for 5 min according to GP06. In the

subsequent step, Boc-Asp(SH)-Gly-Phe-OtBu (from first step, 0.02 mmol), lissamine azide 114

(23.3 mg, 0.04 mmol) and 2,6-lutidine (2.2 µL, 0.02 mmol) were reacted in 1:1 DCM/MeOH (120

µL) for 5 min according to GP04. HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 30 x 75 mm; 60-

60-100% MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 15+8 min; flow 8 mL min-1): Rt =

15 min) gave title compound, Boc-Asn(Liss)-Gly-Phe-Ot-Bu 181 54% (12 mg, TFA adduct) as red

foam; 1H NMR (400 MHz, DMSO-d6): δ 12.35 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 7.8,

2.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 1H), 7.35 – 7.10 (m, 6H), 7.13 – 6.88 (m, 6H), 4.46 – 4.14 (m,

2H), 3.74 – 3.49 (m, 10H), 2.99 – 2.83 (m, 2H), 2.81 – 2.54 (m, 2H), 1.38 (s, 9H), 1.29 (s, 9H),

1.21 (t, J = 7.0 Hz, 12H); 13C NMR (101 MHz, DMSO-d6) δ 170.3, 170.0, 169.0, 168.5, 157.1,

155.0, 147.9, 137.0, 134.3, 132.6, 129.2, 128.2, 126.5, 113.4, 95.4, 80.7, 78.4, 54.1, 50.1, 45.3,

41.9, 37.1, 28.2, 27.5, 12.5; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd. for


neg.) calcd. for C51H63N6O13S2 [M-H]- m/z: 1031.4, found 1031.1.

Boc-Asn(biotin2)-Gly-Phe-Ot-Bu (182)

Boc-Asp(STrt)-Gly-Phe-OtBu 168 (15 mg, 0.02 mmol), TIS (12.5 µL, 0.06 mmol) and TFA (5 %

v/v, 15.3 µL, 0.2 mmol) reacted in dry DCM (300 µL) for 5 min according to GP06. In the

subsequent step, Boc-Asp(SH)-Gly-Phe-OtBu (from first step, 0.02 mmol), biotin azide 124 (20.8

mg, 0.04 mmol) and 2,6-lutidine (2.2 µL, 0.02 mmol) were reacted in 1:1 DCM/MeOH (120 µL)

for 15 min according to GP04. HPLC (Phenomenex-Kinetex 100-5 C18, 21.2 x 250 mm; 40-60%

MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 17 min; flow 8 mL min-1): Rt = 15.5 min)

gave title compound, Boc-Asn(biotin2)-Gly-Phe-Ot-Bu 182 58% (11.7 mg) as white foam; 1H

NMR (400 MHz, DMSO-d6): δ 12.23 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 8.00 – 7.95 (m, 2H), 7.94

(d, J = 5.8 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.32 – 7.18 (m, 5H), 7.13 (d, J = 7.7 Hz, 1H), 6.42

(br s, 2H), 4.38 – 4.29 (m, 2H), 4.25 – 4.21 (br m, 1H), 4.16 – 4.09 (br m, 1H), 3.77 – 3.36 (br m,

10H), 3.36 – 3.18 (br m, 2H), 3.10 (q, J = 6.6, 5.5 Hz, 1H), 3.00 – 2.52 (m, 4H), 2.43 – 2.18 (m,

2H), 1.52 (br m, 4H), 1.42 – 1.18 (m, 20H); 13C NMR (101 MHz, DMSO-d6): δ 172.3, 171.0, 170.8,

170.3, 168.9, 168.4, 167.6, 162.7, 155.1, 140.1, 137.7, 137.0, 129.2, 128.2, 127.7, 127.6, 126.5,

80.7, 78.4, 61.0, 59.2, 55.4, 54.1, 41.8, 37.6, 37.1, 32.1, 28.3, 28.2, 28.1, 27.5, 24.8; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C45H63N8O12S2 [M+H]+ m/z: 971.4, found

971.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd. for C45H61N8O12S2 [M-H]- m/z:

969.4, found 969.2.

Boc-Asn(biotin2)-Val-Ot-Bu (183)

Boc-Asp(STrt)-Val-Ot-Bu 162 (13 mg, 0.02 mmol), TIS (12.5 µL, 0.06 mmol) and TFA (5 % v/v,

15.3 µL, 0.2 mmol) reacted in dry DCM (300 µL) for 5 min according to GP06. In the subsequent

step, Boc-Asp(SH)-Gly-Phe-OtBu (from first step, 0.02 mmol), biotin azide 124 (20.8 mg, 0.04

mmol) and 2,6-lutidine (2.2 µL, 0.02 mmol) were reacted in 1:1 DCM/MeOH (120 µL) for 15 min

according to GP04. HPLC (Phenomenex-Kinetex 100-5 C18, 21.2 x 250 mm; 40-60% MeCN (with

0.1% TFA) in H2O (with 0.1% TFA) in 18 min; flow 8 mL min-1): Rt = 16.5 min) gave title

compound, Boc-Asn(biotin2)-Val-Ot-Bu 183 75% (12.7 mg) as white foam; 1H NMR (400 MHz,

DMSO-d6) δ 12.21 (s, 1H), 8.05 – 7.88 (m, 2H), 7.66 (t, J = 10.3 Hz, 3H), 7.14 (d, J = 8.0 Hz, 1H),

6.42 (s, 2H), 4.36 – 4.20 (m, 2H), 4.15 – 4.07 (m, 1H), 4.00 (dd, J = 8.3, 5.5 Hz, 1H), 3.56 (t, J =

38.2 Hz, 8H), 3.35 – 3.19 (m, 1H), 3.14 – 3.03 (m, 1H), 2.82 (dd, J = 12.5, 5.1 Hz, 1H), 2.71 – 2.52

(m, 2H), 2.40 – 2.20 (m, 2H), 2.05 – 1.91 (m, 1H), 1.72 – 1.44 (m, 4H), 1.37 (d, J = 14.2 Hz, 20H),

0.81 (dd, J = 11.9, 6.8 Hz, 6H); 13C NMR (101 MHz, DMSO-d6) δ 171.1, 170.8, 170.3, 168.7,

167.6, 162.7, 155.2, 140.1, 127.7, 127.6, 80.8, 78.3, 61.0, 59.2, 57.6, 55.4, 37.2, 32.1, 30.1, 28.3,

28.1, 28.1, 27.6, 24.8, 18.8, 17.7; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd. for


neg.) calcd. for C39H58N7O11S2 [M-H]- m/z: 864.4, found 864.3.

Fmoc-Asn(neo-Glc)-Val-Ot-Bu (184)

Fmoc-Asp(STrt)-Val-Ot-Bu 161 (35 mg, 0.046 mmol), TIS (30 µL, 0.146 mmol) and TFA (5%

v/v, 35 µL, 0.455 mmol) were recated in DCM (700 mL) for 5 min according to GP06.

Subsequently, Fmoc-Asp(SH)-Val-Ot-Bu (from first step, 0.046 mmol), peracetylated sulfonyl

azide-modified glucose 117 (10.5 mg, 0.21 mmol) and 2,6-lutidine (5.3 µL, 0.046 mmol) were

reacted in MeOH (500 µL) for 15 min according to GP04. HPLC (Eurospher 100-10 C18, 16 x

250 mm; 60-95% MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 20 min; flow 9 mL min-1:

Rt = 11.0 min) gave Fmoc-Asn(neo-Glc)-Val-Ot-Bu 184 (17 mg, 83%) as colorless solid; 1H NMR

(400 MHz, MeOD-d4): δ 7.79 (d, J = 7.5 Hz, 2H), 7.67 (dd, J = 7.4, 3.0 Hz, 2H), 7.39 (t, J = 7.4

Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 5.21 (t, J = 9.5 Hz, 1H), 5.00 (t, J = 9.7 Hz, 1H), 4.90 (t, J = 8.8

Hz, 1H), 4.87 – 4.83 (br m, 1H), 4.65 (d, J = 8.0 Hz, 1H), 4.54 (t, J = 6.4 Hz, 1H), 4.41 – 4.32 (m,

2H), 4.26 – 4.20 (m, 3H), 4.15 – 3.99 (m, 2H), 3.83 – 3.57 (m, 2H), 2.80 (qd, J = 15.4, 6.4 Hz, 2H),

2.11 (dq, J = 13.4, 6.8 Hz, 1H), 2.03 (d, J = 1.7 Hz, 3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.89 (s, 3H),

1.46 (d, J = 4.5 Hz, 9H), 0.94 (dd, J = 6.7, 1.8 Hz, 6H); 13C NMR (101 MHz, MeOD-d4): δ 172.5,

172.4, 172.0, 171.7, 171.6, 171.3, 145.3, 145.2, 142.6, 128.9, 128.3, 126.4, 126.4, 121.0, 102.1,

83.0, 74.2, 73.0, 72.6, 69.8, 65.3, 63.1, 60.0, 48.4, 31.9, 28.4, 20.8, 20.7, 20.6, 20.5, 19.6, 18.5;

ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C44H57N3O18SNa [M+Na]+ m/z:

970.3, found 970.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C44H56N3O18S

[M-H]- m/z: 946.3, found 946.4.

NH2-Asn(Liss)-Tyr(OH)-OH (185)

TFA*NH2-Asp(SH)-Tyr(OH)-OH 172 (11 mg, 0.026 mmol), lissamine azide 114 (10.3 mg, 0.0176

mmol) and 2,6-lutidine (3 µL, 0.026 mmol) were reacted in 3:1 MeOH/H2O (100 µL) for 5 min

according to GP04. HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 21.2 x 250 mm; 40% MeCN

(with 0.1% TFA) in H2O (with 0.1% TFA) in 15 min; flow 9 mL min-1: Rt = 12.5 min) gave

TFA*NH2-Asn(Liss)-Tyr(OH)-OH 185 (14.4 mg, 97%, TFA salt ) as red foam; 1H NMR (600

MHz, DMSO-d6): δ 9.25 (s, 1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.05 (d, J = 59.4 Hz, 4H), 7.06 – 7.00

(m, 4H), 6.98 – 6.92 (m, 4H), 6.66 (d, J = 8.3 Hz, 2H), 4.31 (s, 1H), 3.96 (s, 1H), 3.64 (tq, J = 12.6,

6.9, 5.5 Hz, 8H), 2.99 – 2.68 (m, 2H), 2.54 – 2.50 (m, 2H), 1.20 (td, J = 7.1, 2.9 Hz, 12H); 13C

NMR (151 MHz, DMSO): δ 172.6, 169.3, 168.9, 157.1, 156.1, 155.0, 132.8, 130.1, 127.3, 115.1,

113.5, 104.6, 95.4, 54.5, 45.3, 35.7, 12.6; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.)

calcd for C40H46N5O11S2 [M+H]+ m/z: 836.3, found 836.3; ESI-MS (H2O/MeCN (with 0.1% formic

acid), neg.) calcd for C40H44N5O11S2 [M-H]- m/z: 834.3, found 834.5.

TFA.NH2-Asn(Dn)-Ala-OH (186)

Boc-Asp(STrt)-Ala-Ot-Bu 163 (250 mg, 0.404 mmol), TIS (831 µL, 4.043 mmol) and TFA (60%

v/v, 1.8 mL) were reacted in DCM (300 µL) for 40 min according to GP09. Subsequently, Boc-

Asp(SH)-Ot-Bu 116 (from first step, 0.404 mmol), dansyl azide (223 mg, 0.808 mmol) and 2,6-

lutidine (45 µL, 0.405 mmol) were reacted in MeOH (2 mL) for 2 h at room temperature according

to GP04. Solvent was removed under reduced pressure and then diluted with 10 mL of DCM. The

organic phase were extracted with water, to remove nonpolar compounds, and aqueous phase was

lyophilized. The afforded yellow powder was further purified through HPLC (Eurospher 100-10

C18, 16 x 250 mm; 2-25% MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 20 min; flow 9

mL min-1: Rt = 7.1 min) gave TFA.NH2-Asn(Dn)-Ala-OH (91.5 mg, 41%) as pale yellow foam; 1H NMR (400 MHz, DMSO-d6): δ 8.63 (d, J = 7.1 Hz, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.33 (dd, J

= 7.3, 1.2 Hz, 1H), 8.23 (d, J = 8.6 Hz, 1H), 8.08 (s, 3H), 7.68 (dt, J = 13.9, 7.9 Hz, 2H), 7.29 (d,

J = 7.6 Hz, 1H), 4.16 (p, J = 7.2 Hz, 1H), 3.97 (d, J = 9.0 Hz, 1H), 2.84 (s, 6H), 2.82 – 2.65 (m,

2H), 1.22 (d, J = 7.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): δ 173.3, 167.9, 167.3, 151.6,

134.1, 130.9, 128.9, 128.9, 128.6, 123.5, 118.0, 115.3, 47.8, 47.5, 45.0, 36.7, 16.8; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C19H25N4O6S [M+H]+ m/z: 437.1, found

437.1; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C19H23N4O6S [M-H]- m/z:

435.1, found 435.3.

TFA.NH2-Tyr(OH)-Ala-Asn(Dn)-Ser(OH)-Val-OH (188)

Boc-Tyr(Ot-Bu)-Ala-Asp(STmob)-ψ-Thr-Val-Ot-Bu 187 (17.5 mg, 0.0172 mmol), TIS (67 µL,

0.3445 mmol) and TFA (60% v/v, 1.3 mL) were reacted in DCM (600 µL) for 40 min according

to GP09. Subsequently, TFA.NH2-Tyr(OH)-Ala-Asp(SH)-Ser(OH)-Val-OH (from first step,

0.0172 mmol), dansyl azide 116 (18 mg, 0.0648 mmol) and 2,6-lutidine (2 µL, 0.0172 mmol) were

reacted in MeOH (150 µL) for 2 h at room temperature according to GP04. HPLC (Eurospher 100-

10 C18, 16 x 250 mm; 20-70% MeCN (with 0.1% TFA) in H2O (with 0.1% TFA) in 20 min; flow

9 mL min-1: Rt = 9.7 min) gave TFA.NH2-Tyr(OH)-Ala-Asn(Dn)-Ser(OH)-Val-OH 188 (9mg,

58%) as pale yellow powder; 1H NMR (400 MHz, MeOD-d4): δ 8.55 (d, J = 8.5 Hz, 1H), 8.30 (d,

J = 8.8 Hz, 1H), 8.26 (dd, J = 7.4, 1.2 Hz, 1H), 7.62 (m, 2H), 7.42 (dd, J = 8.6, 7.4 Hz, 1H), 7.32

(d, J = 7.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 4.61 – 4.54 (m, 1H), 4.29

(m, 2H), 4.22 (q, J = 7.1 Hz, 1H), 4.10 (dd, J = 6.5, 4.6 Hz, 1H), 4.04 (m, 1H), 3.24 – 2.94 (m, 2H),

2.91 (s, 6H), 2.84 (dd, J = 8.1, 5.9 Hz, 2H), 2.16 (dq, J = 13.1, 6.7 Hz, 1H), 1.25 (d, J = 7.2 Hz,

3H), 1.08 (d, J = 6.4 Hz, 3H), 0.94 (dd, J = 6.9, 2.1 Hz, 6H); 13C NMR (101 MHz, MeOD-d4): δ

172.3, 170.8, 170.1, 158.3, 152.5, 132.7, 132.1, 131.7, 130.8, 129.6, 126.3, 124.4, 120.2, 117.0,

116.8, 108.5, 68.4, 59.9, 59.2, 56.0, 51.0, 50.8, 45.9, 37.9, 37.5, 31.6, 19.8, 19.5, 18.4, 17.5; ESI-

MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C37H50N7O11S [M+H]+ m/z: 800.9, found

800.9; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C37H48N7O11S [M-H]- m/z:

798.9, found 799.1.

2-acetamido-2-deoxy-β-D-glucopyranosyl azide138 (190)

Followed slightly published138 procedure by slight modification: DMC (4.61 g, 27.24 mmol) was

added to a mixture of N-acetyl glucosamine 189 (2 g, 9.08 mmol), NaN3(4.38 g, 67.5 mmol, 2.5

M) and 2,6-lutidine (6.35 ml, 54.48 mmol) in water (27 ml), and the reaction was stirred for 48 h

at 0 oC. After concentration of reaction mixture, added CHCl3/MeOH (3:1 v/v) and the undissolved

solid was removed by filtration. The filtrate was concentrated under reduced pressure and the crude

product was purified through FC (20% MeOH/CHCl3) gave white solid, 2-acetamido-2-deoxy-β-

D-glucopyranosyl azide 190 (1.4 g, 63%): Rf = 0.5 (DCM/MeOH 3:1); 1H NMR (400 MHz, D2O):

δ 4.65 (d, J = 9.3 Hz, 1H), 3.83 (dd, J = 12.4, 2.2 Hz, 1H), 3.69 – 3.57 (m, 2H), 3.50 – 3.34 (m,

3H), 1.95 (s, 3H); 13C NMR (101 MHz, D2O): δ 175.5, 89.3, 78.5, 74.3, 70.1, 61.2, 55.7, 22.7.

2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide140 (191)

2-acetamido-2-deoxy-β-D-glucopyranosyl azide 190 (5g, 20.3 mmol) was dissolved in dry DMF,

then added to a suspension of NaH (60% in min oil, 3.2 g, 80 mmol) in DMF (10 mL) at 4 oC.

Benzyl bromide was introduced dropwise after 30 min further stirring at 4 oC and mixture stirred

for additional 16 h at room temperature. After crushed ice was added, leading to precipitation of

white solid. The precipitate was filtered and washed with water and cold ether afforded titled

compound, 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide 191, 81% (8.53 g,

16.51 mmol) as white solid: Rf = 0.38 (MeOH/DCM 1:19); 1H NMR (400 MHz, CDCl3): δ 7.40 –

7.15 (m, 15H), 5.39 (d, J = 8.0 Hz, 1H), 4.96 (d, J = 8.9 Hz, 1H), 4.87 – 4.75 (m, 2H), 4.66 – 4.53

(m, 4H), 3.99 (dd, J = 10.0, 8.3 Hz, 1H), 3.78 – 3.65 (m, 3H), 3.62 (dt, J = 9.6, 3.1 Hz, 1H), 3.43

(dt, J = 10.0, 8.4 Hz, 1H), 1.85 (s, 3H); 13C NMR (101 MHz, CDCl3): δ 170.6, 138.3, 138.1, 138.0,

128.7, 128.6, 128.5, 128.2, 128.1, 128.0, 128.0, 127.8, 88.0, 80.5, 78.4, 75.0, 74.9, 73.7, 68.6, 56.6,

23.6; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C29H32N4O5Na [M+Na]+ m/z:

539.2, found 539.2.

2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide139 (192)

2-acetamido-2-deoxy-β-D-glucopyranosyl azide 190 (1.3 g, 5.3 mmol), acetic anhydride (5 mL)

and Pyridine (6 mL) were reacted according to GP11. Evaporation of solvent gave 2-acetamido-

3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide 192 (1.84 g, 93%): Rf = 0.3 (MeOH/DCM

1:19); 1H NMR (400 MHz, CDCl3): δ 5.70 (d, J = 8.9 Hz, 1H), 5.24 (dd, J = 10.6, 9.3 Hz, 1H),

5.10 (t, J = 9.7 Hz, 1H), 4.76 (d, J = 9.2 Hz, 1H), 4.30 – 4.14 (m, 2H), 3.91 (dt, J = 10.6, 9.1 Hz,

1H), 3.79 (ddd, J = 10.0, 4.8, 2.4 Hz, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H); 13C

NMR (101 MHz, CDCl3): δ 171.1, 170.8, 170.6, 169.4, 88.6, 74.1, 72.3, 68.1, 62.0, 54.3, 23.4,

20.9, 20.8, 20.7; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C14H21N4O8


C14H19N4O8 [M-H]- m/z: 371.1, found 371.3.

N-acetylglucosamine-β-phenylpropanamide (193)

S-trityl-3-phenylpropanethioate (1 g, 2.45 mmol), TIS (1.52 mL, 7.35 mmol) and TFA (10% v/v,

1.88 mL, 24.5 mmol) were reacted in DCM (18 mL) for 5 min according to GP06. 3-

phenylpropanethioic-S-acid 77 (from first step, 2.45 mmol), 2-acetamido-2-deoxy-β-D-

glucopyranosyl azide 190 (270 mg, 1.09 mmol) and NaHCO3 (210 mg, 2.45 mmol) were reacted

in H2O (8 mL) for 2.5 days at 60 oC according to GP05. FC (0-20% MeOH/DCM) gave white

solid, N-acetylglucosamine-β-phenylpropanamide 193 (260 mg, 69%): Rf = 0.4 (DCM/MeOH 4:1); 1H NMR (400 MHz, MeOD-d4): δ 7.31 – 7.14 (m, 5H), 4.98 (d, J = 9.7 Hz, 1H), 3.86 (dd, J = 12.0,

1.6 Hz, 1H), 3.80 – 3.65 (m, 2H), 3.53 – 3.43 (m, 1H), 3.37 – 3.34 (m, 2H), 2.90 (td, J = 7.6, 4.7

Hz, 2H), 2.51 (t, J = 7.8 Hz, 2H), 1.91 (s, 3H); 13C NMR (101 MHz, MeOD-d4): δ 175.8, 174.4,

142.1, 129.5, 129.3, 127.2, 80.5, 79.7, 76.3, 71.9, 62.7, 56.2, 38.9, 32.4, 22.9; ESI-MS (H2O/MeCN

(with 0.1% formic acid), pos.) calcd for C17H25N2O6 [M+H]+ m/z: 353.2, found 353.2; ESI-MS

(H2O/MeCN (with 0.1% formic acid), neg.) calcd for C17H23N2O6 [M-H]- m/z: 351.2, found 351.4.

Phenylpropanamido-GlcNAc(OAc)3 (194)

3-phenylpropanetritylthio-S-ester 76 (1 g, 2.45 mmol), TIS (1.52 mL, 7.35 mmol) and TFA (10%

v/v, 1.8 mL, 24.5 mmol) were reacted in DCM (20 mL) for 5 min according to GP06. Subsequently,

3-phenylpropanethioic S-acid 77 (from first step, 2.45 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-

deoxy-β-D-glucopyranosyl azide 192 (456 mg, 1.23 mmol) and 2,6-lutidine (272 µL, 2.45 mmol)

were reacted in CHCl3 (8 mL) for 2.5 days at 60 oC according to GP05. FC (25% ethyl

acetate/petroleum ether → 0-10%er MeOH/DCM) gave white solid, phenylpropanamido-

GlcNAc(OAc)3 193 (416 mg, 71%): Rf = 0.4 (MeOH/DCM 1:9); 1H NMR (400 MHz, CDCl3): δ

7.30 – 7.24 (m, 2H), 7.21 – 7.15 (m, 3H), 6.96 (d, J = 8.3 Hz, 1H), 5.89 (d, J = 8.0 Hz, 1H), 5.12

(t, J = 9.6 Hz, 1H), 5.08 – 4.99 (m, 2H), 4.30 (dd, J = 12.5, 4.4 Hz, 1H), 4.15 – 4.02 (m, 2H), 3.75

(ddd, J = 10.0, 4.3, 2.2 Hz, 1H), 2.98 – 2.86 (m, 2H), 2.61 – 2.36 (m, 2H), 2.09 (s, 3H), 2.06 (s,

3H), 2.04 (s, 3H), 1.86 (s, 3H); 13C NMR (101 MHz, CDCl3): δ 172.8, 172.1, 172.0, 170.7, 169.2,

140.5, 128.5, 128.3, 126.2, 80.5, 73.5, 73.0, 67.6, 61.7, 53.5, 38.1, 30.9, 23.1, 20.8, 20.7, 20.6; ESI-

MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C23H31N2O9 [M+H]+ m/z: 479.2, found

479.2; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C23H30N2O9Na [M+Na]+ m/z:

501.2, found 501.2.

Phenylpropanamido-GlcNAc(OBn)3 (195)

3-phenylpropanetritylthio-S-ester 76 (1 g, 2.45 mmol), TIS (1.52 mL, 7.35 mmol) and TFA (10%

v/v, 1.8 mL, 24.5 mmol) were reacted in DCM (20 mL) for 5 min according to GP06. Subsequently,

3-phenylpropanethioic S-acid 77 (from first step, 2.45 mmol), 2-acetamido-3,4,6-tri-O-benzyl-2-


were reacted in CHCl3 (8 mL) for 2.5 days at 60 oC according to GP05. FC (20% ethyl

acetate/petroleum ether → 50% ethyl acetate/petroleum ether) gave white solid,

phenylpropanamido-GlcNAc(OBn)3 195 (183 mg, 24%): Rf = 0.3 (MeOH/DCM 1:19); 1H NMR

(400 MHz, CDCl3): δ 7.43 – 7.12 (m, 20H), 7.05 (d, J = 8.3 Hz, 1H), 4.88 – 4.75 (m, 4H), 4.70 –

4.55 (m, 3H), 4.49 (d, J = 12.1 Hz, 1H), 3.91 – 3.71 (m, 4H), 3.50 (dt, J = 9.9, 2.6 Hz, 1H), 3.43

(dd, J = 10.6, 8.7 Hz, 1H), 2.87 (t, J = 7.9 Hz, 2H), 2.53 – 2.33 (m, 2H), 1.62 (s, 3H); 13C NMR

fakno(101 MHz, CDCl3): δ 173.0, 172.1, 140.8, 138.1, 138.0, 137.9, 129.1, 129.1, 128.6, 128.6,

128.5, 128.4, 128.2, 128.1, 127.9, 126.3, 80.7, 80.6, 78.6, 76.7, 75.2, 74.4, 73.8, 68.3, 53.6, 38.3,

31.1, 23.2: ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H42N2O6Na [M+Na]+

m/z: 645.3, found 645.5.

Boc-Asn(GlcNAc(OBn)3)-Ot-Bu (196)

Boc-Asp(STrt)-Ot-Bu 159 (700 mg, 1.14 mmol), TIS (703 µL, 3.41 mmol) and TFA (5% v/v, 870

µL, 11.38 mmol) were reacted in DCM (8 mL) for 5 min according to GP06. Subsequently, Boc-

Asp(SH)-Ot-Bu (from first step, 1.138 mmol), 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-

glucopyranosyl azide 191 (294 mg, 0.57 mmol) and 2,6-lutidine (125 µL, 0.14 mmol) were reacted

in CHCl3 (500 µL for 2.5 days at 60 oC according to GP05. FC (50% ethyl acetate/petroleum ether

→ DCM) gave Boc-Asn(GlcNAc(OBn)3)-Ot-Bu 196 (114 mg, 26%) as white solid: Rf = 0.55

(MeOH/DCM 1:19); 1H NMR (400 MHz, CDCl3): δ 7.43 – 7.28 (m, 12H), 7.23 – 7.14 (m, 3H),

5.60 (d, J = 9.1 Hz, 1H), 5.09 (d, J = 7.5 Hz, 1H), 4.90 – 4.74 (m, 3H), 4.65 (dd, J = 12.0, 10.4 Hz,

2H), 4.59 (d, J = 10.7 Hz, 1H), 4.49 (d, J = 12.1 Hz, 1H), 4.44 – 4.36 (m, 1H), 3.89 (td, J = 10.0,

7.5 Hz, 1H), 3.84 – 3.68 (m, 3H), 3.51 (d, J = 9.6 Hz, 2H), 2.67 (ddd, J = 52.2, 16.4, 4.7 Hz, 2H),

1.76 (s, 3H), 1.43 (s, 9H), 1.42 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 172.3, 171.0, 170.5, 155.7,

138.1, 137.9, 129.0, 128.8, 128.6, 128.5, 128.1, 128.2, 127.8, 81.8, 80.9, 80.5, 79.7, 78.5, 76.6,


acid), pos.) calcd for C42H55N3O10 [M+H]+ m/z: 762.4, found 762.4; ESI-MS (H2O/MeCN (with

0.1% formic acid), neg.) calcd for C27H42N3O13 [M-H]- m/z: 760.4, found 760.6.

Boc-Asn(GlcNAc(OAc)3)-Ot-Bu159 (197)

Boc-Asp(STrt)-Ot-Bu 159 (161 mg, 0.295 mmol), TIS (182 µL, 1.05 mmol) and TFA (5% v/v,

225 µL, 2.95 mmol) were reacted in DCM (4.5 mL) for 5 min according to GP06. In subsequent

step GP05: Boc-Asp(SH)-OtBu (from first step, 0.295 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-


were reacted in CHCl3 (500 µL) for 2.5 days at 60 oC according to GP05. FC (0-2% MeOH/DCM)

gave Boc-Asn(GlcNAc(OAc)3)-Ot-Bu 197 (73.2 mg, 83%) as white solid: Rf = 0.35 (MeOH/DCM

1:19); 1H NMR (400 MHz, CDCl3): δ 7.20 (d, J = 8.5 Hz, 1H), 6.43 (d, J = 8.5 Hz, 1H), 5.61 (d, J

= 8.9 Hz, 1H), 5.16 – 5.10 (m, 1H), 5.10 – 5.03 (m, 2H), 4.39 (dd, J = 8.7, 4.5 Hz, 1H), 4.25 (dd,

J = 12.5, 4.3 Hz, 1H), 4.11 (q, J = 9.7 Hz, 1H), 4.03 (dd, J = 12.5, 2.2 Hz, 1H), 3.79 – 3.71 (m,

1H), 2.69 (ddd, J = 52.8, 16.3, 4.8 Hz, 2H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.94 (s, 3H),

1.39 (s, 18H); 13C NMR (101 MHz, CDCl3): δ 172.1, 171.7, 171.1, 170.7, 170.4, 169.3, 155.6,

81.8, 79.8, 79.7, 73.4, 73.0, 67.9, 61.8, 53.2, 50.6, 38.0, 28.3, 28.5, 27.9, 27.8, 23.1, 20.7, 20.7,

20.6; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C27H44N3O13 [M+H]+ m/z:

618.3, found 618.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), neg.) calcd for C27H42N3O13

[M-H]- m/z: 616.3, found 616.5.

Boc-Asn(GlcNAc)-Val-Ot-Bu (198)

Boc-Asp(STrt)-Val-Ot-Bu 162 (225 mg, 0.3478 mmol), TIS (214 µL, 1.05 mmol) and TFA (5%

v/v, 250 µL, 3.5 mmol) were reacted in DCM (4.5 mL) for 5 min according to GP06. Subsequently,

Boc-Asp(SH)-Val-Ot-Bu (from first step, 0.3478 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-

β-D-glucopyranosyl azide 192 (63 mg, 0.1692 mmol) and 2,6-lutidine (40 µL, 0.385 mmol) were

reacted in CHCl3 (500 µL) for 2 days at 60 oC according to GP05. FC (0-3% MeOH/DCM) gave,

Boc-Asn(GlcNAc)-Val-Ot-Bu 198 (45 mg, 37%) as white solid: Rf = 0.25 (MeOH/DCM 1:19); 1H

NMR (400 MHz, CDCl3): δ 7.31 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 6.24 – 6.09 (m, 2H),

5.08 – 4.99 (m, 2H), 4.51 – 4.35 (m, 1H), 4.26 – 4.22 (m, 1H), 4.21 – 4.18 (m, 1H), 4.14 – 4.04

(m, 1H), 4.00 (dd, J = 12.5, 2.3 Hz, 1H), 3.75 – 3.63 (m, 1H), 2.58 (ddd, J = 63.1, 16.1, 4.8 Hz,

2H), 2.11 – 2.04 (m, 1H), 2.01 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H), 1.94 (s, 3H), 1.37 (d, J = 4.9 Hz,

18H), 0.85 (dd, J = 7.0, 5.6 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 172.7, 172.5, 172.4, 171.7,

170.9, 170.7, 170.4, 169.3, 155.9, 81.7, 80.2, 73.5, 72.8, 67.8, 61.8, 57.8, 53.2, 50.9, 37.0, 31.2,

28.3, 28.0, 23.1, 20.7; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C32H53N4O14


C32H52N4O14Na [M+Na]+ m/z: 739.3, found 739.4.

Boc-Asi-Val-Phe-Ot-Bu (199)

To a solution of Boc-Asp(STrt)-Val-Phe-Ot-Bu 166 (45 mg 0.0567 mmol) and TIS (39 µL, 0.189

mmol) in 1 mL DCM, were added slowly 48 µL (5% v/v, 0.69 mmol) of TFA at ambient

temperature. Reaction mixture was stirred for 5 min, after solvent was removed via nitrogen-flux

and dried in vacuum for 40 min. The crude thioacid was dissolved in 1 mL of CHCl3, followed by

addition of 2,6-lutidine (28 µL, 0.25 mmol). Then the reaction mixture was stirred for 2 days at 60 oC. After the solvent was removed under reduced pressure, packed dry in FC: eluent 20-25% ethyl

acetate/petroleum ether. The title compound, Boc-Asi-Val-Phe-Ot-Bu 199 yield 12 mg (0.0232

mmol, 41%) as colorless solid: Rf = 0.55 (ethyl acetate/petroleum ether 1:1); 1H NMR (400 MHz,

CDCl3): δ 7.30 – 7.15 (m, 5H), 6.97 (d, J = 8.0 Hz, 1H), 5.27 (d, J = 6.7 Hz, 1H), 4.71 (td, J = 7.7,

6.4 Hz, 1H), 4.26 (d, J = 10.6 Hz, 1H), 4.17 (dt, J = 9.8, 6.4 Hz, 1H), 3.12 – 2.96 (m, 3H), 2.81 –

2.68 (m, 2H), 1.43 (s, 9H), 1.36 (s, 9H), 0.94 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H); 13C

NMR (101 MHz, CDCl3): δ 176.3, 174.2, 170.5, 167.8, 155.3, 136.7, 129.4, 128.5, 126.9, 82.1,

81.4, 62.5, 53.9, 50.0, 38.2, 35.7, 28.4, 28.1, 26.9, 20.5, 19.4.

Fmoc-Cys(SH)-OMe145 (207)

Fmoc-Cys(SH)-OH (0.5 g, 1.38 mmol) were dissolved in 10 mL of MeOH. Then 6 drops of 12 N

HCl were added. The mixture was left stirring overnight at room temperature. A white solid was

obtained, and dissolved in acetone. The solvents were evaporated under reduced pressure to obtain

the required product Fmoc-Cys(SH)-OMe 207 (0.47 g, 97%) as white solid; 1H NMR (400 MHz,

CDCl3): δ 7.77 (dt, J = 7.6, 1.0 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.41 (tq, J = 7.4, 1.0 Hz, 2H),

7.33 (tdd, J = 7.5, 2.0, 1.2 Hz, 2H), 5.66 (d, J = 7.7 Hz, 1H), 4.78 – 4.60 (m, 1H), 4.44 (d, J = 7.4

Hz, 2H), 4.24 (t, J = 6.8 Hz, 1H), 3.81 (s, 3H), 3.14 – 2.88 (m, 2H), 1.36 (t, J = 8.9 Hz, 1H); ESI-

MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd. for C19H19NO5SNa [M+Na]+ m/z: 380.1,

found 380.1.

Lissamine rhodamine B 5-sulfonnamide (209)

Fmoc-Cys(SH)-OH (18.6 mg, 0.051 mmol), lissamine azide 114 (15 mg, 0.025 mmol) and 2,6-

lutidine (5.7 µL, 0.051 mmol) were reacted in methanol (300 µL) for 4h at room temperature.

HPLC (Phenomenex-Gemini 110-5 C6-phenyl, 30 x 75 mm; 65-70-100% MeCN in H2O in 8+9

min; flow 10 mL min-1: Rt = 12 min) gave lissamine rhodamine B 5-sulfonamide 209 (7 mg, 49%); 1H NMR (600 MHz, DMSO-d6): δ 8.47 (s, 1H), 7.96 (dd, J = 7.9, 2.0 Hz, 1H), 7.66 (s, 2H), 7.44

(d, J = 7.9 Hz, 1H), 7.04 (dd, J = 9.6, 2.4 Hz, 2H), 7.00 – 6.86 (m, 4H), 3.63 (dt, J = 11.1, 7.0 Hz,

8H), 1.20 (t, J = 7.0 Hz, 12H); 13C NMR (151 MHz, DMSO-d6): δ 157.7, 157.2, 155.1, 147.8,

145.1, 132.7, 132.6, 130.6, 125.9, 125.2, 113.8, 113.6, 95.5, 45.4, 12.6; ESI-MS (H2O/MeCN (with

0.1% formic acid), pos.) calcd for C27H32N3O6S2 [M+H]+ m/z: 558.2, found 557.95; ESI-MS

(H2O/MeCN (with 0.1% formic acid), pos.) calcd for C27H30N3O6S2 [M-H]- m/z: 556.2, found

556.1.

Oxidation products of thioacid Cbz-Asp(SH)-OBn (210-212)

(Cbz-Asp(S-)-OBn)2 (210): 1H NMR (400 MHz, CDCl3): δ 7.81 – 7.72 (m, 4H), 7.65 – 7.53 (m,

4H), 7.45 – 7.35 (m, 4H), 7.35 – 7.14 (m, 8H), 5.71 (d, J = 8.1 Hz, 2H), 4.68 (d, J = 6.6 Hz, 2H),

4.48 – 4.35 (m, 4H), 4.22 (t, J = 7.0 Hz, 2H), 3.62 – 3.42 (m, 2H), 3.19 (s, 4H); 13C NMR (101

MHz, CDCl3): δ 170.9, 143.8, 141.5, 127.9, 127.2, 125.2, 120.2, 67.4, 53.4, 53.0, 47.3, 41.3; ESI-

MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H36N2O10S2Na [M+Na]+ m/z:767.2,

found 767.1.

(Cbz-Asp(SO-)-OBn)2 (211): 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.6 Hz, 4H), 7.64 – 7.52

(m, 4H), 7.39 (t, J = 7.5 Hz, 4H), 7.35 – 7.19 (m, 8H), 5.75 (d, J = 8.0 Hz, 2H), 4.74 (q, J = 6.0 Hz,

2H), 4.41 (d, J = 7.2 Hz, 4H), 4.22 (t, J = 7.0 Hz, 2H), 3.59 – 3.43 (m, 2H), 3.43 – 2.71 (m, 4H); 13C NMR (101 MHz, CDCl3): δ 170.7, 155.8, 143.8, 141.5, 127.9, 127.2, 125.3, 120.1, 67.4, 53.3,

53.0, 47.3; ESI-MS (H2O/MeCN (with 0.1% formic acid), pos.) calcd for C38H36N2O12S2Na

[M+Na]+ m/z:799.2, found 799.3.

(Cbz-Asp(SO2-)-OBn)2 (212): 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.5 Hz, 4H), 7.59 (dd,

J = 7.9, 3.2 Hz, 4H), 7.39 (t, J = 7.4 Hz, 4H), 7.34 – 7.24 (m, 8H), 5.70 (d, J = 7.4 Hz, 2H), 4.68

(d, J = 6.7 Hz, 2H), 4.45 – 4.33 (m, 4H), 4.22 (t, J = 7.0 Hz, 2H), 3.52 (q, J = 9.5, 8.4 Hz, 2H),

3.43 – 3.05 (m, 4H); 13C NMR (101 MHz, CDCl3): δ 143.8, 141.5, 127.9, 127.2, 125.3, 120.2, 77.5,


for C38H36N2O14S2Na [M+Na]+ m/z:831.2, found 830.9.

X-RAY CRYSTALLOGRAPHY

Crystals of lissamine rhodamine B 5-sulfonyl azide 114 suitable for single crystal X-Ray

diffraction analysis were grown after layering a solution of the compound (4 mg) in methanol (0.2

mL) with pentane (1 mL) in an NMR at room temperature. X-Ray diffraction analysis of was

performed at 100 K on a STOE IPDS-II diffractometer equipped with a graphite-monochromated

radiation source (Mo Kα radiation, λ = 0.71073 Å) and an image plate detection system. The

Crystal was mounted on a fine glass fiber with silicon grease. The selection, integration and

averaging procedure of the measured reflex intensities, the determination of the unit cell

dimensions and a least-squares fit of the 2θ values as well as data reduction, LP-correction and

space group determination were performed using the X-Area software package delivered with the

diffractometer.160 A semiempirical absorption correction was performed. The structure was solved

by the Patterson method (SHELXS-97), completed with difference fourier syntheses, and refined

with full-matrix least-square using SHELXL-97 minimizing ω(F02-Fc2)2.161 Weighted R factor

(wR2) and the goodness of fit GooF (GoF, S) are based on F2. All non-hydrogen atoms were refined

with anisotropic displacement parameters. All hydrogen atoms were treated in a riding model.

Graphical output (Ortep plots) were created using ORTEP-3 V2.02 for Windows XP.162

INFLUENCE OF BASES IN TAL+C CHEMISTRY (TABLE 7 EXPERIMENTS)

General procedure for the base screening in TAL+C chemistry: Two equivalent of thioacid 169,

two equivalent of thiol 207, one equivalent of lissamine azide 114, two equivalent of base and one

equivalent of anthracene 208 (as internal standard) were reacted in a solution of THF:MeOH:DCM

(1:1:1). Relative intensity of target peaks were integrated before adding the base (which was taken

as the zero time measurement) after 30 minutes and 60 minutes. The percentage of the relative

consumption and formation of substance was calculated by taking anthracene peak as the reference.

Graph 10. TAL+C chemistry with base 2,6-lutidine

Graph 11. TAL+C chemistry with base ascorbate

Graph 12. TAL+C chemistry with base citrate

Graph 13. TAL+C chemistry with base acetate

Graph 14. TAL+C chemistry with base DIPEA

Graph 15. TAL+C chemistry with base DABCO

INFLUENCE OF BASES IN TAL-C (TAL) CHEMISTRY (TABLE 8 EXPERIMENTS)

General procedure for the base screening in TAL-C (TAL) chemistry: Two equivalent of thioacid

169, one equivalent of lissamine azide 114, two equivalent of base and one equivalent of anthracene

208 (as internal standard) were reacted in a solution of THF:MeOH:DCM (1:1:1). Relative

intensity of target peaks were integrated before adding the base (which was taken as the zero time

measurement) after 30 minutes and 60 minutes. The percentage of the relative consumptions and

formations of substances were calculated by taking anthracene peak as the reference.

Graph 16. TAL-C chemistry with base 2,6-lutidine

Graph 17. TAL-C chemistry with base citrate

Graph 18. TAL-C chemistry with base acetate

Graph 19. Graph 20. TAL-C chemistry with base acetate

REFERENCE

1. Lederberg, J.; McCray, A. T., 'Ome sweet 'omics - A genealogical treasury of words. Scientist 2001, 15 (7), 8-8.

2. Kuska, B.; Roderick, T. H., Beer, Bethesda, and biology: How "genomics" came into being. J Natl Cancer I 1998, 90 (2), 93-93.

3. Maxam, A. M.; Gilbert, W., A new method for sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (2), 560-4.

4. Sanger, F.; Nicklen, S.; Coulson, A. R., DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (12), 5463-7.

5. Sanger, F.; Air, G. M.; Barrell, B. G.; Brown, N. L.; Coulson, A. R.; Fiddes, J. C.; Hutchison, C. A.; Slocombe, P. M.; Smith, M., Nucleotide-Sequence of Bacteriophage Phichi174 DNA. Nature 1977, 265 (5596), 687-695.

6. Pennisi, E., Human genome. Reaching their goal early, sequencing labs celebrate. Science 2003, 300 (5618), 409.

7. Wilkins, M. R.; Sanchez, J. C.; Gooley, A. A.; Appel, R. D.; Humphery-Smith, I.; Hochstrasser, D. F.; Williams, K. L., Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol. Genet. Eng. Rev. 1996, 13, 19-50.

8. Brower, V., Proteomics: biology in the post-genomic era - Companies all over the world rush to lead the way in the new post-genomics race. Embo Rep. 2001, 2 (7), 558-560.

9. Aebersold, R.; Cravatt, B. F., Proteomics - advances, applications and the challenges that remain. Trends Biotechnol. 2002, 20 (12), S1-S2.

10. Sletten, E. M.; Bertozzi, C. R., Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew. Chem., Int. Ed. 2009, 48 (38), 6974-6998; Lim, R. K. V.; Lin, Q., Bioorthogonal chemistry: recent progress and future directions. Chem. Commun. 2010, 46 (10), 1589-1600.

11. Lang, K.; Chin, J. W., Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins. Chem. Rev. 2014, 114 (9), 4764-4806.

12. Prescher, J. A.; Bertozzi, C. R., Chemistry in living systems. Nat. Chem. Biol. 2005, 1 (1), 13-21. 13. Hinner, M. J.; Johnsson, K., How to obtain labeled proteins and what to do with them. Curr. Opin.

Biotechnol. 2010, 21 (6), 766-776; Jing, C. R.; Cornish, V. W., Chemical Tags for Labeling Proteins Inside Living Cells. Acc. Chem. Res. 2011, 44 (9), 784-792.

14. Tsao, M. L.; Tian, F.; Schultz, P. G., Selective staudinger modification of proteins containing p-azidophenylalanine. Chembiochem 2005, 6 (12), 2147-2149; Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S., Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode N(epsilon)-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification. Chem. Biol. 2008, 15 (11), 1187-1197.

15. Link, A. J.; Tirrell, D. A., Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J. Am. Chem. Soc 2003, 125 (37), 11164-11165.

16. Plass, T.; Milles, S.; Koehler, C.; Szymanski, J.; Mueller, R.; Wiessler, M.; Schultz, C.; Lemke, E. A., Amino acids for Diels-Alder reactions in living cells. Angew. Chem., Int. Ed. 2012, 51 (17), 4166-70.

17. Tang, Y.; Wang, P.; Van Deventer, J. A.; Link, A. J.; Tirrell, D. A., Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase. Chembiochem 2009, 10 (13), 2188-2190; Wang, L.; Zhang, Z.; Brock, A.; Schultz, P. G., Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (1), 56-61.

18. Datta, D.; Wang, P.; Carrico, I. S.; Mayo, S. L.; Tirrell, D. A., A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins. J. Am. Chem. Soc 2002, 124 (20), 5652-5653.

19. Ngo, J. T.; Tirrell, D. A., Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis. Acc. Chem. Res. 2011, 44 (9), 677-685.

20. Blackman, M. L.; Royzen, M.; Fox, J. M., Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J. Am. Chem. Soc 2008, 130 (41), 13518-9.

21. Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman, M. L.; Fox, J. M.; Chin, J. W., Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. J. Am. Chem. Soc 2012, 134 (25), 10317-20.

22. Liu, D. S.; Tangpeerachaikul, A.; Selvaraj, R.; Taylor, M. T.; Fox, J. M.; Ting, A. Y., Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J. Am. Chem. Soc 2012, 134 (2), 792-5.

23. Deiters, A.; Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, J. C.; Schultz, P. G., Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J. Am. Chem. Soc 2003, 125 (39), 11782-11783.

24. Jencks, W. P., Studies on the Mechanism of Oxime and Semicarbazone Formation. J. Am. Chem. Soc 1959, 81 (2), 475-481.

25. Saxon, E.; Armstrong, J. I.; Bertozzi, C. R., A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2000, 2 (14), 2141-2143.

26. Saxon, E.; Bertozzi, C. R., Cell surface engineering by a modified Staudinger reaction. Science 2000, 287 (5460), 2007-2010.

27. Huisgen, R., 1,3-Dipolar Cycloadditions. Past and Future. Angew. Chem., Int. Ed. 1963, 2 (10), 565-598.

28. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B., A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem., Int. Ed. 2002, 41 (14), 2596.

29. Tornoe, C. W.; Christensen, C.; Meldal, M., Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67 (9), 3057-3064.

30. Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R., A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 2006, 1 (10), 644-648.

31. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R., A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc 2004, 126 (46), 15046-7.

32. Boger, D. L., Diels-Alder reactions of heterocyclic aza dienes. Scope and applications. Chem. Rev. 1986, 86 (5), 781-793.

33. Thalhammer, F.; Wallfahrer, U.; Sauer, J., Reaktivität einfacher offenkettiger und cyclischer dienophile bei Diels-Alder-reaktionen mit inversem elektronenbedarf. Tetrahedron Lett. 1990, 31 (47), 6851-6854; Balcar, J.; Chrisam, G.; Huber, F. X.; Sauer, J., Reaktivität von stickstoff-heterocyclen genenüber cyclooctin als dienophil. Tetrahedron Lett. 1983, 24 (14), 1481-1484.

34. Chin, J. W.; Cropp, T. A.; Anderson, J. C.; Mukherji, M.; Zhang, Z. W.; Schultz, P. G., An expanded eukaryotic genetic code. Science 2003, 301 (5635), 964-967.

35. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R., Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997, 276 (5315), 1125-1128.

36. Nauman, D. A.; Bertozzi, C. R., Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting. Biochim. Biophys. Acta, Bioenerg. 2001, 1568 (2), 147-154.

37. Rideout, D., Self-Assembling Cytotoxins. Science 1986, 233 (4763), 561-563.

38. Dirksen, A.; Hackeng, T. M.; Dawson, P. E., Nucleophilic catalysis of oxime ligation. Angew. Chem., Int. Ed. 2006, 45 (45), 7581-7584.

39. Griffin, R. J., The medicinal chemistry of the azido group. Prog. Med. Chem. 1994, 31, 121-232. 40. Beatty, K. E.; Fisk, J. D.; Smart, B. P.; Lu, Y. Y.; Szychowski, J.; Hangauer, M. J.; Baskin, J. M.;

Bertozzi, C. R.; Tirrell, D. A., Live-cell imaging of cellular proteins by a strain-promoted azide-alkyne cycloaddition. Chembiochem 2010, 11 (15), 2092-5.

41. Staudinger, H.; Meyer, J., Über neue organische Phosphorverbindungen III. Phosphinmethylenderivate und Phosphinimine. Helv. Chim. Acta 1919, 2 (1), 635-646.

42. Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R., Mechanistic Investigation of the Staudinger Ligation. J. Am. Chem. Soc. 2005, 127 (8), 2686-2695.

43. Kohn, M.; Breinbauer, R., The Staudinger ligation - A gift to chemical biology'. Angew. Chem., Int. Ed. 2004, 43 (24), 3106-3116.

44. Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V., Copper(I)-Catalyzed Synthesis of Azoles. DFT Study Predicts Unprecedented Reactivity and Intermediates. J. Am. Chem. Soc. 2005, 127 (1), 210-216.

45. Schmidt, M. J.; Summerer, D., A Need for Speed: Genetic Encoding of Rapid Cycloaddition Chemistries for Protein Labelling in Living Cells. Chembiochem 2012, 13 (11), 1553-1557.

46. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click chemistry: diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 2001, 40 (11), 2004-2021.

47. Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R., Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry. J. Am. Chem. Soc. 2008, 130 (34), 11486-11493; Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R., Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (43), 16793-16797.

48. Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C. J.; Deiters, A.; Chin, J. W., Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 2012, 4 (4), 298-304.

49. Han, H.-S.; Devaraj, N. K.; Lee, J.; Hilderbrand, S. A.; Weissleder, R.; Bawendi, M. G., Development of a Bioorthogonal and Highly Efficient Conjugation Method for Quantum Dots Using Tetrazine−Norbornene Cycloaddition. J. Am. Chem. Soc. 2010, ASAP.

50. Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G., Expanding the genetic code of Escherichia coli. Science 2001, 292 (5516), 498-500.

51. Xie, J. M.; Schultz, P. G., Adding amino acids to the genetic repertoire. Curr. Opin. Chem. Biol. 2005, 9 (6), 548-554.

52. Schnolzer, M.; Kent, S. B. H., Constructing Proteins by Dovetailing Unprotected Synthetic Peptides - Backbone-Engineered Hiv Protease. Science 1992, 256 (5054), 221-225; Muir, T. W.; Sondhi, D.; Cole, P. A., Expressed protein ligation: A general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 6705-6710; Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W., Insertion of a synthetic peptide into a recombinant protein framework: A protein biosensor. J. Am. Chem. Soc 1999, 121 (5), 1100-1101; Arnold, U.; Hinderaker, M. P.; Nilsson, B. L.; Huck, B. R.; Gellman, S. H.; Raines, R. T., Protein prosthesis: A semisynthetic enzyme with a beta-peptide reverse turn. J. Am. Chem. Soc 2002, 124 (29), 8522-8523; Pellois, J. P.; Muir, T. W., Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can't. Curr. Opin. Chem. Biol. 2006, 10 (5), 487-491.

53. Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C., Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (36), 13132-13137; Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K., A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 2003, 21 (1), 86-89; Kho, Y.; Kim, S. C.; Jiang, C.; Barma, D.; Kwon, S. W.;

Cheng, J. K.; Jaunbergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.; Zhao, Y. M., A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (34), 12479-12484; Tai, H. C.; Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C., Parallel identification of O-GlcNAc-modified proteins from cell lysates. J. Am. Chem. Soc 2004, 126 (34), 10500-10501; Chen, I.; Howarth, M.; Lin, W. Y.; Ting, A. Y., Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2005, 2 (2), 99-104; Lin, C. W.; Ting, A. Y., Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc 2006, 128 (14), 4542-4543.

54. Merrifield, R. B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc 1963, 85 (14), 2149-2154.

55. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B., Synthesis of proteins by native chemical ligation. Science 1994, 266 (5186), 776-779.

56. Noren, C. J.; Anthonycahill, S. J.; Griffith, M. C.; Schultz, P. G., A General-Method for Site-Specific Incorporation of Unnatural Amino-Acids into Proteins. Science 1989, 244 (4901), 182-188.

57. Johnson, J. A.; Lu, Y. Y.; Van Deventer, J. A.; Tirrell, D. A., Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 2010, 14 (6), 774-780; Liu, C. C.; Schultz, P. G., Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 2010, 79, 413-444; Davis, L.; Chin, J. W., Designer proteins: applications of genetic code expansion in cell biology. Nat. Rev. Mol. Cell Biol. 2012, 13 (3), 168-182.

58. Bujacz, A., Structures of bovine, equine and leporine serum albumin. Acta Crystallogr, Sect.D. 2012, 68, 1278-1289.

59. Simpson, R. J., Proteins and Proteomics: A Laboratory Manual. Lab Manual ed.; Cold Spring Harbor Laboratory Press: 2002; p 926.

60. Alberts, B.; Johnson, A.; Lewis, J., Molecular biology of the cell. 4th ed.; Garland Science: New York, 2002; p 1548.

61. Krishnamoorthy, K.; Begley, T. P., Protein Thiocarboxylate-Dependent Methionine Biosynthesis in Wolinella succinogenes. J. Am. Chem. Soc 2011, 133 (2), 379-386; Jurgenson, C. T.; Burns, K. E.; Begley, T. P.; Ealick, S. E., Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis. Biochemistry-Us 2008, 47 (39), 10354-10364.

62. Krishnamoorthy, K.; Begley, T. P., Reagent for the Detection of Protein Thiocarboxylates in the Bacterial Proteome: Lissamine Rhodamine B Sulfonyl Azide. J. Am. Chem. Soc 2010, 132 (33), 11608-11612.

63. Goldstein, A. S.; Gelb, M. H., An alternate preparation of thioester resin linkers for solid-phase synthesis of peptide C-terminal thioacids. Tetrahedron Lett. 2000, 41 (16), 2797-2800.

64. Merkx, R.; van Haren, M. J.; Rijkers, D. T. S.; Liskamp, R. M. J., Resin-Bound Sulfonyl Azides: Efficient Loading and Activation Strategy for the Preparation of the N-Acyl Sulfonamide Linker. J. Org. Chem. 2007, 72 (12), 4574-4577.

65. Vetter, S., Direct Synthesis of DI-and Trimethoxybenzyl Thiols from the Corresponding Alcohol. Synth. Commun. 1998, 28 (17), 3219-3223.

66. Crich, D.; Sana, K.; Guo, S., Amino Acid and Peptide Synthesis and Functionalization by the Reaction of Thioacids with 2,4-Dinitrobenzenesulfonamides. Org. Lett. 2007, 9 (22), 4423-4426.

67. Rao, Y.; Li, X. C.; Nagorny, P.; Hayashida, J.; Danishefsky, S. J., A simple method for the conversion of carboxylic acids into thioacids with Lawesson's reagent. Tetrahedron Lett. 2009, 50 (48), 6684-6686.

68. Rohmer, K.; Keiper, O.; Mannuthodikayil, J.; Wittmann, V., Preparation of Thioacid-Containing Amino Acids and Peptides and their Application in Ligation Reactions. In Peptides 2010: Tales of

Peptides (Proc. 31st European Peptide Symposium), Lebl, M.; Meldal, M.; Jensen, K. J.; Høeg-Jensen, T., Eds. European Peptide Society: 2010; pp 218-219.

69. Mali, S. M.; Gopi, H. N., Thioacetic Acid/NaSH-Mediated Synthesis of N-Protected Amino Thioacids and Their Utility in Peptide Synthesis. J. Org. Chem. 2014, 79 (6), 2377-2383.

70. Blake, J., Peptide Segment Coupling in Aqueous Medium: Silver Ion Activation of the Thiolcarboxyl Group. Int. J. Pept. Protein Res. 1981, 17 (2), 273-274; Blake, J.; Li, C. H., New segment-coupling method for peptide synthesis in aqueous solution: application to synthesis of human [Gly17]-beta-endorphin. Proc. Natl. Acad. Sci. U.S.A. 1981, 78 (7), 4055-4058.

71. Mali, S. M.; Jadhav, S. V.; Gopi, H. N., Copper(II) mediated facile and ultra fast peptide synthesis in methanol. Chem. Commun. 2012, 48 (56), 7085-7087; Mali, S. M.; Bhaisare, R. D.; Gopi, H. N., Thioacids Mediated Selective and Mild N-Acylation of Amines. J. Org. Chem. 2013, 78 (11), 5550-5555; Joseph, R.; Dyer, F. B.; Garner, P., Rapid formation of N-Glycopeptides via Cu(II)-promoted glycosylative ligation. Org Lett 2013, 15 (4), 732-5.

72. Wang, P.; Li, X.; Zhu, J.; Chen, J.; Yuan, Y.; Wu, X.; Danishefsky, S. J., Encouraging Progress in the ω-Aspartylation of Complex Oligosaccharides as a General Route to β-N-Linked Glycopolypeptides. J. Am. Chem. Soc 2011, 133 (5), 1597-1602.

73. Wang, P.; Danishefsky, S. J., Promising General Solution to the Problem of Ligating Peptides and Glycopeptides. J. Am. Chem. Soc 2010, 132 (47), 17045-17051.

74. Rosen, T.; Lico, I. M.; Chu, D. T. W., A Convenient and Highly Chemoselective Method for the Reductive Acetylation of Azides. J. Org. Chem. 1988, 53 (7), 1580-1582.

75. Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J., The Reaction of Thio Acids with Azides: A New Mechanism and New Synthetic Applications. J. Am. Chem. Soc. 2003, 125 (26), 7754-7755.

76. Zhang, X.; Li, F.; Lu, X. W.; Liu, C. F., Protein C-terminal modification through thioacid/azide amidation. Bioconjugate chemistry 2009, 20 (2), 197-200.

77. Barlett, K. N.; Kolakowski, R. V.; Katukojvala, S.; Williams, L. J., Thio Acid/Azide Amidation: An Improved Route to N-Acyl Sulfonamides. Org. Lett. 2006, 8 (5), 823-826.

78. Rijkers, D. T. S.; Merk, R.; Yim, C. B.; Brouwer, A. J.; Liskamp, R. M., Sulfo-click for ligation as well as for site-specific conjugation with peptides, fluorophores, and metal chelators. J. Pept. Sci. 2010, 16 (1), 1-5.

79. Merkx, R.; Brouwer, A. J.; Rijkers, D. T. S.; Liskamp, R. M. J., Highly Efficient Coupling of β-Substituted Aminoethane Sulfonyl Azides with Thio Acids, toward a New Chemical Ligation Reaction. Org. Lett. 2005, 7 (6), 1125-1128.

80. Yim, C. B.; Dijkgraaf, I.; Merkx, R.; Versluis, C.; Eek, A.; Mulder, G. E.; Rijkers, D. T.; Boerman, O. C.; Liskamp, R. M., Synthesis of DOTA-conjugated multimeric [Tyr3]octreotide peptides via a combination of Cu(I)-catalyzed "click" cycloaddition and thio acid/sulfonyl azide "sulfo-click" amidation and their in vivo evaluation. J. Med. Chem. 2010, 53 (10), 3944-53.

81. Fazio, F.; Wong, C. H., RuCl3-promoted amide formation from azides and thioacids. Tetrahedron Lett. 2003, 44 (51), 9083-9085.

82. Assem, N.; Natarajan, A.; Yudin, A. K., Chemoselective Peptidomimetic Ligation Using Thioacid Peptides and Aziridine Templates. J. Am. Chem. Soc 2010, 132 (32), 10986-10987.

83. Dyer, F. B.; Park, C. M.; Joseph, R.; Garner, P., Aziridine-Mediated Ligation and Site-Specific Modification of Unprotected Peptides. J. Am. Chem. Soc 2011, 133 (50), 20033-20035.

84. Crich, D.; Sharma, I., Epimerization-Free Block Synthesis of Peptides from Thioacids and Amines with the Sanger and Mukaiyama Reagents. Angew. Chem., Int. Ed. 2009, 48 (13), 2355-2358.

85. Crich, D.; Sasaki, K., Reaction of Thioacids with Isocyanates and Isothiocyanates: A Convenient Amide Ligation Process. Org. Lett. 2009, 11 (15), 3514-3517.

86. Rao, Y.; Li, X. C.; Danishefsky, S. J., Thio FCMA Intermediates as Strong Acyl Donors: A General Solution to the Formation of Complex Amide Bonds. J. Am. Chem. Soc 2009, 131 (36), 12924-+.

87. Pan, J.; Devarie-Baez, N. O.; Xian, M., Facile Amide Formation via S-Nitrosothioacids. Org. Lett. 2011, 13 (5), 1092-1094.

88. Chen, W. T.; Shao, J. A.; Hu, M.; Yu, W. W.; Giulianotti, M. A.; Houghten, R. A.; Yu, Y. P., A traceless approach to amide and peptide construction from thioacids and dithiocarbamate-terminal amines. Chem. Sci. 2013, 4 (3), 970-976.

89. Liu, R. H.; Orgel, L. E., Oxidative acylation using thioacids. Nature 1997, 389 (6646), 52-54. 90. Maurel, M. C.; Orgel, L. E., Oligomerization of alpha-thioglutamic acid. Orig. Life Evol. Biosph.

2000, 30 (5), 423-430. 91. Hosein, H. G.; Just, G., A simple synthesis of 2,2-disubstituted tetrahydrothiophenes.

Tetrahedron Lett. 1980, 21 (22), 2119-2122. 92. Kolakowski, R. V.; Shangguan, N.; Sauers, R. R.; Williams, L. J., Mechanism of thio acid/azide

amidation. J. Am. Chem. Soc. 2006, 128 (17), 5695-702. 93. Gao, Y. L., A new specific mechanism for thioacid/azide amidation: electronic and solvent effects.

Cent. Eur. J. Chem. 2010, 8 (2), 308-319. 94. Zhu, X.; Pachamuthu, K.; Schmidt, R. R., Synthesis of novel S-neoglycopeptides from

glycosylthiomethyl derivatives. Org. Lett. 2004, 6 (7), 1083-5. 95. Merkx, R.; Brouwer, A. J.; Rijkers, D. T.; Liskamp, R. M., Highly efficient coupling of beta-

substituted aminoethane sulfonyl azides with thio acids, toward a new chemical ligation reaction. Org. Lett. 2005, 7 (6), 1125-8.

96. Kinsland, C.; Taylor, S. V.; Kelleher, N. L.; McLafferty, F. W.; Begley, T. P., Overexpression of recombinant proteins with a C-terminal thiocarboxylate: Implications for protein semisynthesis and thiamin biosynthesis. Protein Sci. 1998, 7 (8), 1839-1842.

97. Rohmer, K. Anwendung der Thiosäure/Sulfonylazid-Ligation zur Darstellung von Neoglycokonjugaten. Masterarbeit, Universität Konstanz,, 2007.

98. Subirós-Funosas, R.; El-Faham, A.; Albericio, F., Aspartimide formation in peptide chemistry: occurrence, prevention strategies and the role of N-hydroxylamines. Tetrahedron 2011, 67 (45), 8595-8606.

99. Namelikonda, N. K.; Manetsch, R., Sulfo-click reaction via in situ generated thioacids and its application in kinetic target-guided synthesis. Chem. Commun. 2012, 48 (10), 1526-1528.

100. Keiper, O. Anwendung der Thiosäure/Sulfonylazid Ligation zur Darstellung von Neoglycokonjugaten. University of Konstanz, Konstanz, 2013.

101. Kuwajima, I.; Mori, A.; Kato, M., Conversion of carbon-sulfur linkages into carbon-silicon ones via reductive silylation. Preparation of silyl enol ethers of acyltrimethylsilanes. Bull. Chem. Soc. Jpn. 1980, 53 (9), 2634-8.

102. Renz, T. Synthesis and stability of thiocarboxylic acid esters. Bachelor thesis, Universität Konstanz, Konstanz, 2011.

103. Schmidt, U.; Luttringhaus, A.; Trefzger, H., Uber Trithione .12. Trithione Aus Beta-Ketoestern. Liebigs Ann. Chem. 1960, 631 (1), 129-138.

104. Pedersen, B. S.; Lawesson, S. O., Studies on Organophosphorus Compounds .28. Syntheses of 3h-1,2-Dithiole-3-Thiones and 4h-1,3,2,-Oxazaphosphorine Derivatives from the Dimer of Para-Methoxyphenylthionophosphine Sulfide and Derivatives of 3-Oxo Carboxylic-Acids. Tetrahedron 1979, 35 (20), 2433-2437.

105. Cepanec, I.; Zivkovic, A.; Bartolincic, A.; Mikuldas, H.; Litvic, M.; Merkas, S., Potassium phosphate / benzyltriethylammonium chloride as efficient catalytic system for transesterification. Croat. Chem. Acta 2008, 81 (3), 519-523; Kopecky, D. J.; Rychnovsky, S. D., Improved procedure for the reductive acetylation of acyclic esters and a new synthesis of ethers. J. Org. Chem. 2000, 65 (1), 191-198.

106. Saitoh, K.; Shiina, I.; Mukaiyama, T., O,O'-Di-2-pyridyl thiocarbonate as an efficient reagent for the preparation of carboxylic esters from highly hindered alcohols. Chem. Lett. 1998, (7), 679-680.

107. Magens, S.; Plietker, B., Fe-Catalyzed Thioesterification of Carboxylic Esters. Chem. - Eur. J. 2011, 17 (32), 8807-8809, S8807/1-S8807/40.

108. Charette, A. B.; Grenon, M., Mild Method for the Conversion of Amides to Thioamides. J. Org. Chem. 2003, 68 (14), 5792-5794.

109. Baxter, S. L.; Bradshaw, J. S., New Conversion of Esters to Ethers and Its Application to the Preparation of Furano-18-Crown-6. J. Org. Chem. 1981, 46 (4), 831-832.

110. Curphey, T. J., Thionation with the reagent combination of phosphorus pentasulfide and hexamethyldisiloxane. J. Org. Chem. 2002, 67 (18), 6461-73.

111. Devi, N. S.; Singh, S. J.; Singh, O. M., An efficient transesterification of beta-oxodithioesters catalyzed by stannous chloride under solvent-free conditions. Tetrahedron Lett. 2013, 54 (11), 1432-1435.

112. Woo, Y. H.; Mitchell, A. R.; Camarero, J. A., The use of aryl hydrazide linkers for the solid phase synthesis of chemically modified peptides. Int. J. Pept. Res. Ther. 2007, 13 (1-2), 181-190.

113. Millington, C. R.; Quarrell, R.; Lowe, G., Aryl hydrazides as linkers for solid phase synthesis which are cleavable under mild oxidative conditions. Tetrahedron Lett. 1998, 39 (39), 7201-7204.

114. Yamaguchi, J.; Aoyagi, T.; Fujikura, R.; Suyama, T., An oxidative transformation of N '-phenylhydrazide to t-butyl ester using a copper(II) halide-lithium t-butoxide system. Chem. Lett. 2001, (5), 466-467.

115. Lee, V. J.; Curran, W. V.; Fields, T. F.; Learn, K., 1,2,3-Thiadiazoles .1. Synthesis of Sodium (or Potassium) 1,2,3-Thiadiazole-4-Thiolates Via Thiocarbazonate Esters and N-Acylthiohydrazonate Esters. J. Heterocycl. Chem. 1988, 25 (6), 1873-1891.

116. Wang, W. B.; Roskamp, E. J., Tin(Ii) Amides - New Reagents for the Conversion of Esters to Amides. J. Org. Chem. 1992, 57 (23), 6101-6103.

117. Kenner, G. W.; McDermott, J. R.; Sheppard, R. C., The safety catch principle in solid phase peptide synthesis. Chem. Commun. 1971, (12), 636-637.

118. Wade, W. S.; Yang, F.; Sowin, T. J., Application of base cleavable safety catch linkers to solid phase library production. J. Comb. Chem. 2000, 2 (3), 266-75.

119. Cheung, H. T.; Blout, E. R., The hydrazide as a carboxylie-protecting group in peptide synthesis. J. Org. Chem. 1965, 30 (1), 315-6; Wieland, T.; Lewalter, J.; Birr, C., Subsequent activation of carboxylic derivatives by oxidation or elimination of water; application for cyclizing peptides. Liebigs. Ann. Chem. 1970, 740, 31-47.

120. Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.; Lee, A. C.; Blevitt, J.; Neff, D. K.; Huang, L.; Crawford, S.; Pandit, C. R.; Karlsson, L.; Breitenbucher, J. G., Checkpoint Kinase Inhibitors: SAR and Radioprotective Properties of a Series of 2-Arylbenzimidazoles. J. Med. Chem. 2004, 48 (6), 1873-1885.

121. Camarero, J. A.; Kimura, R. H.; Woo, Y. H.; Shekhtman, A.; Cantor, J., Biosynthesis of a fully functional cyclotide inside living bacterial cells. Chembiochem 2007, 8 (12), 1363-6.

122. Blanco-Canosa, J. B.; Dawson, P. E., An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem., Int. Ed. 2008, 47 (36), 6851-5.

123. Kaneshiro, C. M.; Michael, K., A convergent synthesis of N-glycopeptides. Angew. Chem., Int. Ed. 2006, 45 (7), 1077-81; Cohen-Anisfeld, S. T.; Lansbury, P. T. J., A Practical, Convergent Method for Glycopeptide Synthesis. J. Am. Chem. Soc. 1993, 115, 10531-10537; Anisfeld, S. T.; Lansbury, P. T. J., A Convergent Approach to the Chemical Synthesis of Asparagine-Linked Glycopeptides. J. Org. Chem. 1990, 55 (21), 5560-5562.

124. Hermanson, G. T., Chapter 9 - Fluorescent Probes. In Bioconjugate Techniques (Second Edition), Hermanson, G. T., Ed. Academic Press: New York, 2008; pp 396-497.

125. Heindl, D.; Seidel, C.; Thuer, W. Oligophosphoramidates, WO2008141799 A1, PCT/EP2008/004022. 2008.

126. Hendrickson, J. B.; Wolf, W. A., Direct introduction of the diazo function in organic synthesis. J. Org. Chem. 1968, 33 (9), 3610-3618.

127. Wang, K.; Peng, H.; Ni, N.; Dai, C.; Wang, B., 2,6-Dansyl Azide as a Fluorescent Probe for Hydrogen Sulfide. J. Fluoresc. 2014, 24 (1), 1-5.

128. Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B., A Fluorescent Probe for Fast and Quantitative Detection of Hydrogen Sulfide in Blood. Angew. Chem., Int. Ed. 2011, 50 (41), 9672-9675.

129. Takadate, A.; Tahara, K. E. N.; Fujino, H.; Goya, S., Fluorescent Labeling of Olefines with 5-Dimethylaminonaphthalene-1-sulfonyl Azide. Yakugaku Zasshi 1986, 106 (1), 36-40.

130. Comely, A. C.; Gibson, S. E.; Hales, N. J.; Peplow, M. A., Transition metal complexes as linkers for solid phase synthesis: chromium carbonyl complexes as linkers for arenes. J. Chem. Soc., Perkin Trans. 1 2001, (20), 2526-2531.

131. Maulucci, N.; Chini, M. G.; Di Micco, S.; Izzo, I.; Cafaro, E.; Russo, A.; Gallinari, P.; Paolini, C.; Nardi, M. C.; Casapullo, A.; Riccio, R.; Bifulco, G.; De Riccardis, F., Molecular Insights into Azumamide E Histone Deacetylases Inhibitory Activity. J. Am. Chem. Soc. 2007, 129 (10), 3007-3012.

132. Coin, I.; Beerbaum, M.; Schmieder, P.; Bienert, M.; Beyermann, M., Solid-Phase Synthesis of a Cyclodepsipeptide: Cotransin. Org. Lett. 2008, 10 (17), 3857-3860.

133. Lauer, J. L.; Fields, C. G.; Fields, G. B., Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Lett. Pept. Sci. 1995, 1 (4), 197-205.

134. Apweiler, R.; Hermjakob, H.; Sharon, N., On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta 1999, 1473 (1), 4-8.

135. Marshall, R. D., The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins. Biochem. Soc. Symp. 1974, (40), 17-26.

136. Kunz, H.; Schult, M.; Ernst, B.; Hart, G. W.; Sinaý, P., Glycopeptide Synthesis in Solution and on the Solid Phase. In Carbohydrates in Chemistry and Biology, Wiley-VCH Verlag GmbH: 2008; pp 267-304.

137. Michael, K., Convergent N-Glycopeptide Synthesis. In Acs Sym Ser, American Chemical Society: 2007; Vol. 960, pp 328-353.

138. Tanaka, T.; Nagai, H.; Noguchi, M.; Kobayashi, A.; Shoda, S., One-step conversion of unprotected sugars to beta-glycosyl azides using 2-chloroimidazolinium salt in aqueous solution. Chem. Commun. 2009, (23), 3378-3379.

139. Kunz, H., Synthesis of Glycopeptides, Partial Structures of Biological Recognition Components [New Synthetic Methods (67)]. Angew. Chem., Int. Ed. 1987, 26 (4), 294-308.

140. Kerekgyarto, J.; Agoston, K.; Batta, G.; Kamerling, J. P.; Vliegenthart, J. F. G., Synthesis of fully and partially benzylated glycosyl azides via thioalkyl glycosides as precursors for the preparation of N-glycopeptides. Tetrahedron Lett. 1998, 39 (39), 7189-7192.

141. Ito, Y.; Gerz, M.; Nakahara, Y., Amino acid fluoride for glycopeptide synthesis. Tetrahedron Lett. 2000, 41 (7), 1039-1042.

142. Tam, J. P.; Riemen, M. W.; Merrifield, R. B., Mechanisms of aspartimide formation: the effects of protecting groups, acid, base, temperature and time. Pept. Res. 1988, 1 (1), 6-18.

143. Gattner, M. J.; Ehrlich, M.; Vrabel, M., Sulfonyl azide-mediated norbornene aziridination for orthogonal peptide and protein labeling. Chem. Commun. 2014, 50 (83), 12568-71.

144. Radke, S. Mechanistic investigation of the Thioacid-Azide-Ligation in presence of thiol and the influence of the bases. Bachelor thesis, Universität Konstanz, Konstanz, 2014.

145. Diaz-Rodriguez, V.; Mullen, D. G.; Ganusova, E.; Becker, J. M.; Distefano, M. D., Synthesis of Peptides Containing C-Terminal Methyl Esters Using Trityl Side-Chain Anchoring: Application to the Synthesis of a-Factor and a-Factor Analogs. Org. Lett. 2012, 14 (22), 5648-5651.

146. Perrin, D. D. I., Dissociation constants of inorganic acids and bases in aqueous solution. Butterworths: London,, 1969; p v, 133-236 p.

147. Perrin, D. D. I., Dissociation constants of organic bases in aqueous solution. Butterworths: London,, 1965; p vii, 473, xlii p.

148. Lüttringhaus, A.; Dirksen, H. W., Tetramethylharnstoff als Lösungsmittel und Reaktionspartner. Angew. Chem. 1963, 75 (22), 1059-1068.

149. Kostikov, R. R., 2,6-Di-t-butylpyridine. In Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons, Ltd: 2001.

150. O'Neil, M. J., The Merck index : an encyclopedia of chemicals, drugs, and biologicals. 14th ed.; Merck: Whitehouse Station, N.J., 2006.

151. Serjeant, E. P.; Dempsey, B., Ionisation constants of organic acids in aqueous solution. Pergamon Press: Oxford ; New York, 1979; p xi, 989 p.

152. Acton, Q. A., Phosphoric Acids—Advances in Research and Application: 2013 Edition: ScholarlyBrief. ScholarlyEditions: 2013.

153. Crampton, M. R.; Robotham, I. A., Acidities of some substituted ammonium ions in dimethyl sulfoxide. J. Chem. Res. (S) 1997, (1), 22-23.

154. Sandstroom, A.; Chattopadhyaya, J., Enhancement of molecular ion intensity of nucleic acids in negative ion fast-atom bombardment mass spectroscopy. J. Chem. Soc., Chem. Commun. 1987, (11), 862-864.

155. Singha, S.; Kim, D.; Rao, A. S.; Wang, T.; Kim, K. H.; Lee, K. H.; Kim, K. T.; Ahn, K. H., Two-photon probes based on arylsulfonyl azides: Fluorescence detection and imaging of biothiols. Dyes Pigm. 2013, 99 (2), 308-315.

156. Santos-Figueroa, L. E.; de laTorre, C.; El Sayed, S.; Sancenon, F.; Martinez-Manez, R.; Costero, A. M.; Gil, S.; Parra, M., A Chemosensor Bearing Sulfonyl Azide Moieties for Selective Chromo-Fluorogenic Hydrogen Sulfide Recognition in Aqueous Media and in Living Cells. Eur. J. Org. Chem. 2014, 2014 (9), 1848-1854; Lippert, A. R.; New, E. J.; Chang, C. J., Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells. J. Am. Chem. Soc 2011, 133 (26), 10078-10080; Chen, B. F.; Lv, C.; Tang, X. J., Chemoselective reduction-based fluorescence probe for detection of hydrogen sulfide in living cells. Anal. Bioanal. Chem. 2012, 404 (6-7), 1919-1923; Chen, S.; Chen, Z. J.; Ren, W.; Ai, H. W., Reaction-Based Genetically Encoded Fluorescent Hydrogen Sulfide Sensors. J. Am. Chem. Soc 2012, 134 (23), 9589-9592.

157. Sowie, E. S.; Herzog, H.; Hoffmann, P.; Klauke, E.; Ugi, I., Synthese und Verwendung von tert.-Butyloxycarbonylfluorid. und anderen Fluorkohlensäureestern zur Darstellung säurelabiler Urethan-Derivate von Aminosäuren. Justus Liebigs Ann. Chem. 1968, 716 (1), 175-185.

158. Kerr, M. S.; Read de Alaniz, J.; Rovis, T., An Efficient Synthesis of Achiral and Chiral 1,2,4-Triazolium Salts: Bench Stable Precursors for N-Heterocyclic Carbenes. J. Org. Chem. 2005, 70 (14), 5725-5728.

159. Ito, Y.; Gerz, M.; Nakahara, Y., Amino acid fluoride for glycopeptide synthesis. Tetrahedron Lett. 2000, 41 (7), 1039-1042.

160. X-RED version 1.31 Stoe Data Reduction Program: Darmstadt, Germany, 2005. 161. Sheldrick, G. M. SHELXS-97 Program for Crystal Structure Analysis and Refinement: University of

Göttingen, Germany, 1997. 162. Faruggia, L. J. ORTEP-3 V2.02 for Windows XP: University of Glasgow, Scotland, 2008.

APPENDIX

SELECTED SPECTRUM

Illustration 1. 13C NMR spectrum (101 MHz, CDCl3) of 114

Illustration 2. 1H NMR spectrum (400 MHz, CDCl3) of 114



Illustration 7. 1H NMR spectrum (400 MHz, MeOD-d4) of 124

Illustration 8. 13C NMR spectrum (101 MHz, MeOD-d4) of 124



Illustration 13. 1H NMR spectrum (400 MHz, DMSO-d6) of 174

Illustration 14. 13C NMR spectrum (101 MHz, DMSO-d6) of 174



Illustration 25. 13C NMR spectrum (151 MHz, DMSO-d6) of 209

chemical tools for bioconjugation : application of the

Documents