Facile synthesis of combinatorial vinyl sulfone libraries and their applications in large scale proteomics

FACILE SYNTHESIS OF COMBINATORIAL VINYL SULFONE LIBRARIES AND THEIR APPLICATIONS IN LARGE SCALE PROTEOMICS WANG GANG NATIONAL UNIVERSITY OF SINGAPORE 2004 FACILE SYNTHESIS OF COMBINATORIAL VINYL SULFONE LIBRARIES AND THEIR APPLICATIONS IN LARGE SCALE PROTEOMICS WANG GANG A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I would like to express my greatest gratitude to my supervisor Assistant Professor Yao Shao Qin for his patient guidance, stimulating ideas and invaluable advice throughout my study. I benefited a lot from his instructions and demonstrations. I would also like to express my appreciation to my group members, Dr. Zhu Qing, Dr Li Dongbo, Elaine Chan, Ming-Lee Liau, Resmi and Rajavel from the chemistry lab, Y. J. Chen, Grace, Hu Yi and other people from the DBS lab, for their help and encouragement during my research. I appreciate the support of the research laboratory staff Mdm Han Yanhui and Ms Peggy Ler from the NMR laboratory, Mdm Wong Lai Kwan and Mdm Lai Hui Ngee from the MS lab. I can always receive help from them when I was facing technical problems. I am also grateful to the National University of Singapore, for providing me research scholarship. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF SCHEMES xii ABBREVIATIONS xiii PUBLICATIONS xvii Chapter 1 Introduction 1 1.1 Proteomics 1 1.2 Activity-based proteomics 1 1.3 Activity-based probes 3 1.4 Cysteine proteases and viny sulfone compounds 6 1.5 Positional Scanning Library 9 1.6 Aim of our project 11 Chapter 2 Solution phase synthesis of a vinyl sulfone probe 12 2.1 Introduction 12 2.2 Results and discussion 13 2.2.1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone 14 2.2.2 Synthesis of Cy3-Gly-Leu-Leu-OH 15 ii 2.2.3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 2.2.4 Application of the Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 16 17 probe in enzyme profiling 2.3 Conclusion 18 Chapter 3 Solid-phase synthesis of peptide vinyl sulfone probes 20 3.1 Introduction 20 3.2 Results and discussion 22 3.2.1 Solid-phase synthesis of peptide vinyl sulfone probe 22 via 2-Cl-Trityl-Chloride resin 3.2.1.1 Synthesis and immobilization of 14 onto 2-Cl-Trityl resin 23 3.2.1.2 Synthesis of vinyl sulfone probe Cy3-GLLY-VS on 25 2-Cl-Trityl resin 3.2.1.3 SDS-PAGE results for Cy3-GLLY-VS probe 18 26 3.2.2 Solid-phase synthesis of peptide vinyl sulfone probes via 27 Rink-amide resin 3.2.2.1 Synthesis of peptide vinyl sulfone H2N-CLFL-VS 28 3.2.2.2 Combinatorial synthesis of vinyl sulfone probes with 30 P1 variation 3.2.2.3 Labelling papain with 20 vinyl sulfone probes 33 3.3 34 Conclusion Chapter 4 Combinatorial synthesis of vinyl sulfone small molecules 36 iii 4.1 Introduction 36 4.2 Results and discussion 38 4.2.1 Overall scheme 38 4.2.2 Synthesis of Rink resin bound sulfide phosphonate 31 39 4.2.3 Synthesis of Rink resin bound sulfone phosphonate 32 40 4.2.4 Solid-phase Horner-Wadsworth-Emmons reaction 40 4.2.5 Determination of the racemization of solid-phase 42 Horner-Wadsworth-Emmons reaction products 4.2.6 Synthesis of a 30-member vinyl sulfone small molecule library 44 4.3 Conclusion 48 Chapter 5 Experimental 50 5.1 General information 50 5.2 Experimental procedures 50 5.2.1 Solution phase synthesis of vinyl sulfone probe 50 5.2.2 Solid-phase synthesis of peptide vinyl sulfone probe via 57 Trityl-chloride resin 5.2.3 Determination of resin loading efficiency by Fmoc analysis 51 5.2.4. Activity based protein labeling in SDS-PAGE experiments 59 using probe 18 5.2.5 Combinatorial synthesis of peptide vinyl sulfone probe 59 via Rink-amide resin 5.2.6 SDS-PAGE experiments with combinatorial vinyl sulfone 66 iv probes and papain 5.2.7 Synthesis of vinyl sulfone small molecule library 66 5.2.8 Qualitative ninhydrin test 77 Chapter 6 References 78 Chapter 7 Appendices 87 7.1 Fmoc-Tyr(tBu)-H (4) 87 7.2 Fmoc-Tyr(tBu)-vinyl sulfone (5) 88 7.3 H2N-Tyr(tBu)-vinyl sulfone (6) 89 7.4 Cy3-Gly-Leu-Leu-OH (10) 90 7.5 ESI-MS for Cy3-GLLY-VS (12) 91 7.6 4-Hydroxy-thiophenyl-methyl-diethylphosphonate sulfone (13) 92 7.7 Fmoc-Asp(tBu)-vinyl sulfone (14a) 93 7.8 Fmoc-Leu-vinyl sulfone (14b) 94 7.9 Fmoc-Lys(Boc)-vinyl sulfone (14c) 96 7.10 Fmoc-Phe-vinyl sulfone (14d) 98 7.11 Fmoc-Tyr(tBu)-vinyl sulfone (14e) 99 7.11 Fmoc-Asn(Trt)-H (21b) 100 7.12 Fmoc-Asp(tBu)-H(21c) 102 7.13 Fmoc-Cys(Trt)-H (21d) 103 7.14 Fmoc-Gln(Trt)-H (21e) 105 7.15 Fmoc-Glu(tBu)-H (21f) 107 v 7.16 Fmoc-His(Trt)-H (21h) 108 7.17 Fmoc-Ile-H (21i) 110 7.18 Fmoc-Leu-H (21j) 111 7.19 Fmoc-Lys(Boc)-H (21k) 113 7.20 Fmoc-Met-H (21l) 115 7.21 Fmoc-Phe-H (21m) 117 7.22 Fmoc-Ser(tBu)-H (21n) 119 7.23 Fmoc-Thr(tBu)-H (21o) 121 7.24 Fmoc-Trp(Boc)-H (21p) 123 7.25 Fmoc-Val-H (21r) 125 7.26 4-[(Diethoxyphosphoryl)-thiomethyl]-benzoic acid (30a) 126 7.27 11-[(Diethoxyphosphoryl)-thiomethyl]-undecanoic acid (30b) 127 7.28 2-[(Diethoxyphosphoryl)-thiomethyl]-nicotinic acid (30c) 128 7.29 Anisoyl-Leu-vinyl-sulfonyl-undecanamide (37.1) 129 7.30 Isonicotinoyl-Leu-vinyl-sulfonyl-undecanamide (37.2) 130 7.31 Isonicotinoyl-Asp-vinyl-sulfonyl-undecanamide (37.4) 131 7.32 Isonicotinoyl-Tyr-vinyl-sulfonyl-undecanamide (37.6) 133 7.33 Anisoyl-Lys-vinyl-sulfonyl-undecanamide (37.9) 134 7.34 Isonicotinoyl-Leu-vinyl-sulfonyl-benzamide (37.12) 136 7.35 Isonicotinoyl-Asp-vinyl-sulfonyl-benzamide (37.14) 138 vi SUMMARY Activity-based proteomics plays an important role in profiling those proteins with enzymatic activities. The design and synthesis of chemical probes for enzymes are essential to the success of this strategy. Vinyl sulfone compounds have been shown to be extremely useful as activity-based inhibitors or probes for cysteine proteases. We aim to expand the application of vinyl sulfone compounds in large scale proteomics by designing new synthetic strategies and applying them to the generation of libraries of vinyl sulfone probes and small molecule inhibitors. In Chapter 2, we synthesized a fluorescent-tagged probe Cy3-GLLY-VS based on a solution phase synthesis strategy. This probe was proved to be effective in selectively labeling cysteine protease in the presence of other proteases in a microarray experiment. The synthesis, although successful, is very inefficient. Thus, we designed new solidphase strategies to synthesize vinyl sulfone probes. As shown in Chapter 3, our first solid-phase strategy was based on the synthesis and immobilization of phenolic-Fmocamino-vinyl sulfones onto 2-Cl-Trityl chloride resin, followed by peptide synthesis and probe generation. This strategy was successful as five different phenolic-Fmoc-aminovinyl sulfones were immobilized onto 2-Cl-Trityl chloride resin with high loading efficiency, and one vinyl sulfone probe was successful synthesized and tested in a Gelbased experiment. Our second strategy was more suitable for the generation of positional scanning library of vinyl sulfone probes with P1 variation. By taking advantage of the successful implementation of both solid phase oxidation and Horner-Wadsworth- vii Emmons reaction, we successfully synthesized a library of vinyl sulfone probes. Preliminary test with papain showed the probes were effective in enzyme profiling. In Chapter 4, we discussed the successful synthesis of a 30-member vinyl sulfone small molecule library. Three points of diversity (P1, P2 and P1′) within the vinyl sulfone scaffold were introduced. Potentially large libraries of vinyl sulfone small molecules could be synthesized this way and used to identify specific small molecule inhibitors for disease related cysteine protease. In Chapter 5, all the details of the experiments as well as the characterization of products by NMR and MS are described. Selected NMR and MS spectra and listed in the Appendices. viii LIST OF TABLES Table 1 Components of activity-based probes 5 Table 2 Yield of 14, and the loading efficiency on 2-Cl-Trityl 23 -Chloride resin. Table 3 Yield of Fmoc-AA-H 31 Table 4 ESI-MS data for 20 vinyl sulfone probes with P1 variation 32 Table 5 Horner-Wadsworth-Emmons reaction from 32a to 33a 42 under different conditions Table 6 ESI-MS and HPLC data of vinyl sulfone small molecules 46 ix LIST OF FIGURES Figure 1 General structure of an activity-based probe 3 Figure 2 Strategy for activity-based protein profiling 5 Figure 3a Interaction between substrate and enzyme active site 8 Figure 3b Interaction between peptide vinyl sulfone and enzyme active site. 8 Figure 3c Mechanism of peptide vinyl sulfone inhibiting cysteine protease 8 Figure 4 Positional scanning library in the generation of affinity 10 fingerprint of peptide epoxide inhibitors Figure 5 Synthesis of Boc-Leu-vinyl sulfone 12 Figure 6 Synthesis of vinyl sulfonate esters and vinyl sulfonamides 13 Figure 7 Structure of the Cy3-Gly-Leu-Leu-Tyr-VS proble 14 Figure 8 Activity-based protein profiling using probe 12 in a 18 microarray-based experiment Figure 9 Solid-phase synthesis of vinyl sulfone compound via 20 safety catch resin Figure 10 Solid-phase synthesis of vinyl sulfone compounds via 21 Rink amide resin and the side chain of aspartic acid Figure 11 Immobilization of 14 onto Wang resin under Mitsunobu 23 reaction condition Figure 12 Immobilization of phenolic alcohol onto trichloroacetimidate 25 activated Wang resin x Figure 13 Activity-based protein profiling using probe 7 27 Figure 14 HPLC spectrum of peptide vinyl sulfone H2N-CLFL-VS 29 Figure 15 Intramolecular cyclization of Fmoc-Arg(pbf)-CHO 30 Figure 16 SDS-PAGE result for labelling papain with 20 vinyl 34 sulfone probes Figure 17 APC-3328, a potential lead compound for osteoporosis 37 Figure 18 A vinyl sulfone small molecule binding to the active site 38 of a cysteine protease. Figure 19 HPLC spectra for diastereomeric and enantiomeric dipeptides 44 vinyl sulfones Figure 20 Piperidine adduct of vinyl sulfone small molecule 45 xi LIST OF SCHEMES Scheme 1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone 15 Scheme 2 Synthesis of Cy3-Gly-Leu-Leu-OH 16 Scheme 3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 17 Scheme 4 Solid-phase synthesis of vinyl sulfone probes via 22 2-Cl-Trityl -chloride resin Scheme 5 Solid-phase synthesis of Cy3-GLLY-VS probe via 26 2-Cl-Trityl -chloride resin Scheme 6 Solid-phase synthesis of vinyl sulfone compounds via 28 Rink amide resin Scheme 7 Synthesis of peptide vinyl sulfone H2N-CLFL-VS 29 Scheme 8 Combinatorial synthesis of vinyl sulfone probes with 32 P1 variation Scheme 9 Synthesis of a 30-member vinyl sulfone small molecule library 39 Scheme 10 Generation of diastereomeric and enantiomeric dipeptide 43 vinyl sulfones xii ABBREVIATIONS AA Amino acid BF3.Et2O Boron trifluoride ether complex Boc t-Butoxycarbonyl br Broad Bu4NI Tetrabutylammonium iodide tBu tert-Butyl Cy3 Cyanine dye3 δ Chemical shift Da Dalton DBU 1,8-Diazobicyclo[5.4.0]undec-7-ene DCC N,N’-Dicyclohexylcarbodiimide DCM Dichloromethane dd Doublet of doublet DEAD Diethyl azodicarboxylate DIC N,N’-diisopropylcarbodiimide DIEA N,N’-diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethylsulfoxide DTT Dithiothreitol EA Ethyl acetate xiii EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl EDTA Ethylenediaminetetracetic acid ESI Electron Spray Ionization Et Ethyl Fmoc 9-Fluorenylmethoxycarbonyl HATU O-(7-azabenzotrizol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate HOBT N-Hydroxybenzotriazole HPLC High Performance Liquid Chromatography Hz Hertz KHMDS Potassium bis(trimethylsilyl)amide LAH Lithium aluminum hydride LDA Lithium diisopropyl amide LHMDS Lithium bis(trimethylsilyl)amide m Multiplet m-CPBA m-Chloroperbenzoic acid MS Mass spectrometry NaH Sodium hydride NHS N-Hydroxysuccinimide NMR N-Methylpyrrolidinone PPh3 Triphenylphosphine q Quartet r.t. Room temperature xiv s Singlet SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis t Triplet TBTU O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetraborofluorate TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography Tris Trishydroxymethylamino methane uv Ultraviolet VS Vinyl sulfone Z Benzyloxycarbonyl or Cbz xv ABBREVIATIONS FOR AMINO ACIDS Name Alanine Abbr. ala a Linear structure formula CH3-CH(NH2)-COOH Arginine arg r HN=C(NH2)-NH-(CH2)3-CH(NH2)-COOH Asparagine asn n H2N-CO-CH2-CH(NH2)-COOH Aspartic acid asp d HOOC-CH2-CH(NH2)-COOH Cysteine cys c HS-CH2-CH(NH2)-COOH Glutamine gln q H2N-CO-(CH2)2-CH(NH2)-COOH Glutamic acid glu e HOOC-(CH2)2-CH(NH2)-COOH Glycine gly g NH2-CH2-COOH Histidine his h Isoleucine ile i NH-CH=N-CH=C-CH2-CH(NH2)-COOH |__________| CH3-CH2-CH(CH3)-CH(NH2)-COOH Leucine leu l (CH3)2-CH-CH2-CH(NH2)-COOH Lysine lys k H2N-(CH2)4-CH(NH2)-COOH Methionine met m CH3-S-(CH2)2-CH(NH2)-COOH Phenylalanine phe f Ph-CH2-CH(NH2)-COOH Proline pro p Serine ser s NH-(CH2)3-CH-COOH |_________| HO-CH2-CH(NH2)-COOH Threonine thr t CH3-CH(OH)-CH(NH2)-COOH Tryptophan trp w Tyrosine tyr y Ph-NH-CH=C-CH2-CH(NH2)-COOH |_______| HO-p-Ph-CH2-CH(NH2)-COOH Valine val v (CH3)2-CH-CH(NH2)-COOH xvi PUBLICATIONS Wang, G., Yao, S.Q. “Combinatorial synthesis of a small-molecule library based on the vinyl sulfone scaffold”, Org. Lett. 2003; 5(23); 4437-4440 Wang, G., Uttamchandani, M., Chen, Y.J.G, Yao, S.Q. “Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases”, Org. Lett. 2003; 5(5); 737-740 Hu, Y.; Wang, G., Chen, G.Y.J., Fu, X.; Yao, S.Q. “Proteome analysis of Saccharomyces Cerevisiae under metal stress by 2-D differential gel electrophoresis (DIGE)”, Electrophoresis 2003, 24(9), 1458-1470. Chen, G.Y.J., Uttamchandani, M., Zhu, Q., Wang, G., Yao, S.Q. “Developing a novel strategy for detection of enzymatic activities on a protein array”, Chembiochem, 2003; No 4, 336-339. xvii Chapter 1 Introduction 1.1 Proteomics Proteomics aims to study the function of all expressed proteins in a given organism through the global analysis of protein expression and protein function.1 The correlation of proteins with certain cellular functions or diseases can bring enormous benefits in medicine and human health.2 With the accomplishment of the Human Genome Project (HGP) that provides the “blueprint” of gene products, the opportunity of enquiring into protein properties and activities in cellular context has been created.3 To accelerate the proteomic study, different methods and technologies have been applied and developed.4 Gel-based proteomics,5, 6 mass spectrometry-based proteomics,7 array-based proteomics8, 9 et al. are the most important approaches. These strategies enable the global quantification of protein expression and/or the global characterizations of protein activities at variable degree of efficiency and fidelity.3 1.2 Activity-based Proteomics Our strategy for the functional analysis of proteins is based on the activity-based protein profiling. Conventional proteomics strategy for the separation, quantification, and identification of proteins relies heavily on two-dimensional gel electrophoresis coupled with protein staining and mass-spectrometry analysis (2DE-MS).10 This method suffers from an inherent lack of resolving power of two-dimensional gel electrophoresis, several important classes of proteins, including membrane-associated and low-abundance proteins, are difficult to be analyzed by this technique.10 Recent proteomics approach 1 using isotope-coded affinity tag combined with mass spectrometry has enhanced the sensitivity and accuracy in measuring protein expression level,11 but all these methods have some intrinsic drawbacks since they use the relative abundance of proteins to directly correlat with cellular function, which is a potential risk in proteomics studies. Activity-based proteomics provides a complementary chemical approach to profile dynamics in protein activities in complex proteomes.12 By a combination of techniques such as two-dimensional gel electrophoresis, mass spectrometry and microarray, we are able to use chemically reactive probes to profile and identify proteins in a proteome complex by virtue of their activities.13, 14 These chemical probes can be designed to react with proteins sharing a similar enzymatic activity, or to target a wide range of proteins which are mechanically distinct. Currently most chemical probes are designed to target specific classes of enzymes, such as serine hydrolases,15, 16 cysteine proteases,17-19 phosphotases,20 kinases et al..21 These enzymes play critical roles in modulating a variety of biological processes,22-24 they function through the fine control of their catalytic activities. Numerous post-translational events, protein–protein and protein–smallmolecule interactions can regulate enzyme activities.25 Activity-based proteomics may reveal more insights into how enzymes function in a particular biological event by studying enzyme activities directly, which are more closely related to their cellular functions.26, 27 An example of activity-based proteomics is shown in the profiling of protein tyrosine phosphatases (PTPs) in the whole proteome using PTPs specific chemical probes.28 PTPs are involved in the regulation of many aspects of cellular activity including proliferation, metabolism, migration, and survival. Except for the large number and complexity of PTPs in cell signaling, the activities of many PTPs are tightly 2 regulated by post-translational mechanisms, which restrict the use of standard genomics and proteomics methods for functional characterization of these enzymes. To facilitate the functional analysis of PTPs, two activity-based probes that consist of bromobenzylphosphonate as a PTP-specific trapping device were synthesized. These probes are active site-directed irreversible inhibitors of PTPs, and they are extremely specific toward PTPs while remaining inert to other proteins. These probes can be used to profile PTPs on the basis of changes in their activity and could consequently facilitate the profiling of PTPs activities in complex proteomes and the elucidation of PTPs cellular function. To broaden the scope and impact of activity-based proteomics, one crucial element is the design and use of activity-based chemical probes for diverse enzymes or proteins. Activity-based probes usually react with active enzymes or proteins through a covalent bond.29 The successful generation of proteomics-compatible probes for additional enzyme and protein classes will probably require the synthesis of more structurally diverse libraries of candidate probes.12 1.3 Activity-based probes The general structure of an activity-based probe is shown in Figure 1, which consists of three units: a reactive unit, a linker unit and a tag unit.30 Tag Unit Linker Unit Reactive Unit Figure 1 General structure of an activity-based probe The reactive unit is a chemical reactivity that recognizes the enzyme active site and covalently modifies it. Reactive units are mostly derived from enzyme inhibitors, they are 3 usually electrophilic chemical groups since most enzymes contain nucleophilic groups within their active site.31 As shown in Table 1, activity-based probes with vinyl sulfone32 or epoxide19 as reactive unit can selectively target cysteine proteases, while sulfonate ester-containing probes can target different classes of enzymes such as thiolases, aldehyde dehydrogenases, epoxide hydrolase.14 This class of probes are called activitybased probes because they only target active enzymes which utilize enzyme catalytic mechanism. If the reactive unit modifies the enzyme through an affinity interaction, the probe is called an affinity-based probe.29 By incorporating these key scaffolds into our probes, we can generate diverse probes which could target different classes of enzymes. The linker unit is a bridge between the reactive unit and the tag unit. It could be a peptide fragment, an alkyl chain or others. Peptide fragments are often used to improve the selectivity and potency of the probe toward certain class of enzymes.33 The tag unit is used to facilitate the detection of proteins upon labeling by the probe. A biotin tag enables the detection of labeled proteins through its antibody, as well as the purification of the labeled protein via streptavidin-agarose beads.16 A fluorescent tag such as Cy3 dye can offer a much higher sensitivity than the biotin tag, using this kind of tags, quantitative assessment of separated proteins and potential high-throughput applications become possible.26 4 Table 1 Components of activity-based probes Tag Unit Linker Unit O HN O H N NH R1 R2 N H Reactive Unit H N O R3 Peptide fragment S Biotin tag O Epoxide O N N COOH O O O S O Alkyl linker Vinyl sulfone I Cy3 Fluorescent tag I125 O O HO O2N O O Polyethene linker O O S O Sulfonate ester Isotope tag Figure 2 shows a general approach to activity-based enzyme profiling. In a complex proteome containing different enzymes, the fluorescent activity-based probe will only selectively label a particular class of active enzymes. The labeled enzymes can be separated by SDS-PAGE and visualized by fluorescent imaging, which could be further characterized by mass spectrometry. Figure 2 Strategy for activity-based protein profiling 5 1.4 Cysteine proteases and viny sulfone compounds Currently the proteins that we are interested in are cysteine proteases. Cysteine proteases are an important class of enzymes involved in the hydrolysis of peptide amide bonds. They play vital roles in numerous physiological processes such as arthritis, osteoporosis, Alzheimer’s disease, cancer cell invasion, and apoptosis.34-36 According to their tertiary structures, they are classified as the papain, calpain, cathepsin, caspase and other families.37 The structural differences among the cysteine proteases are useful for the design of specific inhibitors or probes.22 Over the last few decades, many research groups have developed chemical approaches capable of generating diverse small-molecule inhibitors that target different classes of cysteine proteases with various degrees of efficacy fidelity.37 Most of these enzyme inhibitors are active site directed. According to the type of interaction between inhibitors and enzymes, they are further divided into reversible and irreversible inhibitors.22 Reversible inhibitors usually involve a non-covalent interaction between enzyme and inhibitor, although there are some exceptions, such as peptide aldehydes, which interact with enzymes through hydrolytically labile covalent bond 38 . Irreversible inhibitors interact with enzymes through a tight covalent bond which are compatible with proteomics techniques such as gel electrophoresis. By attaching fluorophore or biotin molecule to the inhibitors, these molecules can be used to probe various proteases either in vitro or in vivo. Many irreversible inhibitors of cysteine proteases have been designed. These inhibitors interact with the active thiol of cysteine protease through alkylating, acylating, phosphonylating, or sulfonylating functional groups. 20 Inhibitors employing alkylating 6 agents are widely studied. Some typical examples are peptidyl chloromethyl ketones,39, 40 Epoxysuccinyl Peptides41, 42 and Michael Acceptors 43-47 such as vinyl sulfones. Peptidyl chloromethyl ketones are the first active site-directed irreversible inhibitors reported for serine and cysteine proteases. These inhibitors have high reactivity toward enzymes but lack selectivity. Epoxysuccinyl peptide, such as E-64, is a potent and specific irreversible inhibitor of cysteine proteases. One advantage of epoxysuccinyl peptide inhibitors is their stability under physiological conditions toward simple thiols, and it does not inhibit serine proteases, aspartic proteases, or metalloproteases.41. Although, E-64 and its derivatives have limited selectivity toward different cysteine proteases, and not all cysteine proteases can be inhibited by them, they are still a useful tool for profiling cysteine proteases.42 Inhibitors containing different types of Michael acceptors are also potent and specific toward cysteine proteases.37 They work by irreversibly inactivating the catalytic cysteine residue within the active site of the enzyme.34 Many of these inhibitors, including α,β-unsaturated ketones, acrylamides, vinyl sulfones, et al., have been successfully synthesized, and some have been tested in clinical experiments.48 Peptidyl vinyl sulfones, in particular, have been shown to be extremely useful as activity-based probes for high-throughput profiling of cysteine proteases, largely due to their negligible cross-reactivity toward other classes of enzymes.34, 49 Under physiological conditions, the electrophilic, vinyl sulfone moiety of the inhibitor is not reactive toward most nucleophilic elements present in a biological species (i.e., amine and thiol groups in a protein), which is a potential advantage for in vivo studies. However, upon binding to the active site of an enzyme having a catalytic cysteine residue, i.e., a cysteine protease, 7 the vinyl sulfone would react, at a highly specific rate, with the thiol in the cysteine residue, which results in the formation of a covalent enzyme-inhibitor adduct, leading to subsequent irreversible inactivation of the enzyme (Figure 3).34 Vinyl sulfones can be manipulated on both the P and P’ side of the molecule, allowing for greater selectivity and reactivity toward target enzymes. Peptide vinyl sulfones could also be used as irreversible, active site-directed inhibitors of the proteasome. S3 O S1 P3 O H N H N N H R (a) NH P2 O S4 P3 Site of Hydrolysis O H N N H R N H P4 S P2 O S1' P1 O H N P1' O S2 O R N H P4 (b) O P1 H N R O Site of Inhibition Gln H O R Gln S O (c) H His H O O R R S S + O H His+ O His SEnz S- S Enz Enz Figure 3 (a) Interaction between substrate and enzyme active site, site labeled with S designate binding pocket of the enzyme, site labeled with P designate side chain of peptide substrate. (b) Interaction between peptide vinyl sulfone and enzyme active site. (c) Mechanism of peptide vinyl sulfone inhibiting cysteine protease 8 1.5 Positional Scanning Library Selective inhibitors and probes are of considerable use for the deconvolution of the protease or proteosome’s role in a wide range of biological processes.50-52 The design of potent and selective inhibitors is largely dependent on the determination of enzyme substrate specificity.53 To study the substrate specificities of enzymes, positional scanning libraries are extremely powerful in the generation of binding data of each substrate position.54, 55 A positional scanning library is usually a peptide-based small molecule library, where each amino acid residue of the peptide would occupy the binding pocket of the enzyme, mimicking the enzyme-substrate interaction.56 By varying one of the substrate residues with different amino acids, while keeping other positions constant, this library of compounds would react with the enzyme at different rate and extent. The resulting data can be used to generate an affinity fingerprint of these small molecules, which provides a rapid visual readout of enzyme active site topology.57 Bogyo and co-workers have successfully synthesized P2, P3 and P4 peptide epoxide positional scanning libraries and studied their specificities toward cysteine proteases.58 As shown in Figure 4, a P2 positional scanning library of peptide epoxide inhibitors was generated by varying P2 position with 20 different amino acids, while P3 and P4 positions were fixed with a mixture of 20 amino acids. This P2 diverse peptide epoxide library was screened against a cysteine protease, Cathepsin K, by first incubating the epoxide inhibitors with the enzyme, followed by labeling with the 125 I-DCG-04 probe. After gel separation and phosphoimaging, the percent competition values of these inhibitors toward Cathepsin K were generated by comparing the inhibitor-treated samples with the control untreated sample. Peptide epoxide inhibitors having different amino 9 acids at P2 position would give varying percent competition values. As was clearly seen from the gel, different amino acids at the P2 position led to varying degree of labeling intensity. After screening against multiple enzymes and subsequent computational processes, an affinity fingerprint of the P2 diverse peptide epoxide library toward different cysteine proteases was generated, which was shown in a color format. P3 P4 125 I-DCG-04 Probe P2 Cathepsin K Different enzymes Data readout and computation process Affinity fingerprint Figure 4 Positional scanning library in the generation of affinity fingerprint of peptide epoxide inhibitors. (Adopted from Reference 52) 10 1.6 Aim of our project We aim to develop a facile and efficient solid-phase strategy to combinatorially synthesize vinyl sulfone probes for the study of cysteine proteases. This library of probes would have a P1 variation and serves as a positional scanning library for the generation of affinity data for the S1 binding pocket which is known as a crucial position for the substrate recognition by enzymes. We are also interested in developing a strategy to generate a vinyl sulfone small molecule library which allows the introduction of three points of diversities at the nonprime positions (P1 and P2), as well as the prime position (P1′). Potentially large library of small molecules containing the important vinyl sulfone pharmacophore could be synthesized and used for the identification of potent and specific small molecule inhibitor for cysteine proteases. 11 Chapter 2 Solution phase synthesis of a vinyl sulfone probe 2.1 Introduction There are two approaches for the solution phase synthesis of vinyl sulfone compounds. A versatile scheme to synthesize vinyl sulfone compounds was first examined by Palmer et al.34 and further developed by Bogyo et al..59 As shown in Figure 5, Boc-Leucinal was prepared from Boc-Leu-OH in a two-step procedure. This aldehyde was further reacted with methyl-thiomethyl diethylphosphonate to give a Boc-Leu-vinyl sulfone in a Horner-Wadsworth-Emmons reaction. H N O PyBOP,DIEA, CH3NHOCH3, CH2Cl2 O OH O H N O O O N LAH, Et2O O O S EtO P EtO O O NaH H N O O H N O O H O O S O Figure 5 Synthesis of Boc-Leu-vinyl sulfone Another approach to synthesize vinyl sulfone compound was developed by Roush et al..34 In their pursuit of developing a potent and selective inhibitor for cruzain, they synthesized tens of vinyl sulfone compounds and studied the interaction of these inhibitors with the S1, S2, S1′ S2′ binding site of curzain. The vinyl sulfonate ester and vinyl sulfonamide were synthesized from vinyl sulfonyl chloride and an appropriate amine or phenol. The key intermediate, vinyl sulfonyl chloride, was prepared from a general method reported by Gennari.60 As shown in Figure 6, N-Boc-L- 12 homophenylalanal was reacted with triethyl α-phosphorylmethanesulfonate to give ethyl vinyl sulfonate in a Horner-Wadsworth-Emmons reaction. Deprotection with TFA in CH2Cl2 provided the corresponding amine, which was further coupled with Z-Phe-OH, giving the dipeptide ethyl vinyl sulfonate in 81% overall yield. Treatment of the dipeptide ethyl vinyl sulfonate with n-Bu4NI in refluxing acetone gave the corresponding tetrabutylammonium sulfonate, which was converted to the sulfonyl chloride through a Widlanski’s procedure. The sulfonyl chloride was reacted with an amine or phenol to give the corresponding vinyl sulfonate ester or vinyl sulfonamide. SO3Et O P O O Ph Ph 1) TFA, CH2Cl2 2) Cbz-Phe-OH, EDC H Boc N H O SO3Et Boc N H BuLi, THF 59% HOBT, DIEA 81% Ph Ph H Cbz N 1) n-Bu4NI, Acetone reflux O N H SO3Et O 2) Ph3P, SO2Cl2 N H SO2Cl Ph 61% Ph Ph PhNH2 (84%) or PhOH, DBU (52%) H Cbz N H Cbz N O N H SO2R R = NHPh or R = OPh Ph Figure 6 Synthesis of vinyl sulfonate esters and vinyl sulfonamides 2.2 Results and discussion Our target compound Cy3-Gly-Leu-Leu-Tyr-VS, a cysteine protease probe, contains three units as described in Chapter 1. Vinyl sulfone moiety acts as a reactive unit, which could form a covalent bond with the thiol residue in the active site of cysteine protease; 13 the tetrapeptide acts as a recognition unit, which mimics the substrate of the cysteine protease; Cy3 dye is a fluorescent tag (Figure 7) N N O N H I H N O O N H H N O O S O OH . Tag unit Peptide linker Reactive unit Figure 7 Structure of the Cy3-Gly-Leu-Leu-Tyr-VS proble 2.2.1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone The vinyl sulfone moiety having tyrosine at P1 position was synthesized using the strategy described by Bogyo et al. (Scheme 1). Fmoc-Tyr(tBu)-OH was converted to the corresponding Weinreb amides, Fmoc-Tyr(tBu)-N(CH3)OCH3 3, followed by reduction with lithium aluminum hydride to give Fmoc-Tyr(tBu)-H 4. 4-methyl-thiophenyl-methyldiethylphosphonate sulfone was synthesized and used to react with Fmoc-Tyr(tBu)-H 4 to generate Fmoc-Tyr(tBu)-vinyl sulfone 5 in a Horner-Wadsworth-Emmons reaction using NaH as a base. It had been previously shown that having a phenyl or phenolic group (instead of a methyl group) next to the vinyl sulfone could enhance the potency of the inhibitor toward its targeting enzyme in many cases.61 The Horner-WadsworthEmmons reaction product, Fmoc-Tyr(tBu)-vinyl sulfone 5, was obtained in good yield and determined to have an E configuration as the major product from the NMR spectra. This was further confirmed in chapter 2 by synthesizing five different Fmoc-AA-vinyl sulfones and examining the coupling constant of the allelic protons in NMR spectra. 14 After removing the Fmoc protecting group by a 20% piperidine/DMF solution, the final product H2N-Tyr(tBu)-vinyl sulfone 6 was obtained after flash column purification. Fmoc SH O H N OH Fmoc = O O O P O O I O NaH HOBT,DIEA, CH3NHOCH3, DMF 87% S OEt P O OEt 1 Fmoc O H N Peracetic acid, 1,4-dioxane 71% O O N O 3 LAH O O S OEt P O OEt Fmoc + 2 H N 65% O H 4 O Fmoc NaH 43% H N O S O O 5 20% Piperidine in DMF H2N 89% O S O O 6 Scheme 1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone 2.2.2 Synthesis of Cy3-Gly-Leu-Leu-OH Cy3-Gly-Leu-Leu-OH 10 was synthesized from a coupling reaction between the Cy3 dye 8 and the tripeptide methyl ester, H2N-GLL-OCH3 7, followed by hydrolysis of 15 the Cy3-tripeptide-methyl ester 9 (Scheme 2). The intermediate Cy3 dye 8 was synthesized from a procedure described by Korbel et al.62 and H2N-GLL-OCH3 7 was prepared from a typical solution phase peptide synthesis.63 After hydrolysis of 9 with 20% K2CO3 in methanol solution the desired product Cy3-Gly-Leu-Leu-OH 10 was obtained after purification by preparative HPLC. It was found that the light sensitive Cy3 fluorescent dye was stable under the base hydrolysis condition. O H2N N H 7 H N O + O N N O O OH I H2N-GLL-OCH3 8 Cy3 EDC HOBT, DIEA DMF O H Cy3 N N H H N O O O 9 20%K2CO3, MeOH H Cy3 N O N H 10 H N O OH O Cy3-GLL-OH Scheme 2 Synthesis of Cy3-Gly-Leu-Leu-OH 2.2.3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone The Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone probe 12 was readily synthesized from H2N-Tyr(tBu)-vinyl sulfone 6 and Cy3-Gly-Leu-Leu-OH 10 (Scheme 3). After a coupling reaction and the subsequent TFA deprotection step to remove the acid labile 16 protecting group (tBu) on the side chain of tyrosine, the final product was obtained after preparative HPLC purification and used for labeling experiments. Cy3 O H N N H O H N O S O H2N + OH O O EDC, HOBT DIEA DMF 10 Cy3 N H H N 6 O N H O O S O H N O O 11 50% TFA/CH2Cl2 N N O N H I H N O O N H H N O O S O OH 12 Cy3-GLLY-VS Scheme 3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 2.2.4 Application of the Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone probe in enzyme profiling In a microarray experiment performed by Grace Y. J. Chen et al. in our lab,49 the Cy3-GLLY-VS 12 probe was shown to selectively label the cysteine proteases immobilized on a microarray slide. Cy3-GLLY-VS probe was prepared as a 2 µM mixture using 0.5 µl of 200 µM stock Cy3-GLLY-VS solution in 49 µl of Tris buffer (50 mM, pH 8), and 0.5 µl BSA (1% w/v) added as a blocking agent to prevent non-specific binding. 50 µl of this freshly prepared mixture was applied to the glass slide and 17 incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS with tween (0.2% v/v) for 15 min on a shaker. The slides were then washed with distilled water, air-dried and scanned using an ArrayWorxTM microarray scanner (Applied Precision, USA), under 548/595 nm. As was shown in Figure 8, only lane 4 and 5, where cysteine proteases were immobilized, gave fluorescent signals; other lanes having other classes of enzymes immobilized showed no signals, which indicates only cysteine proteases are selectively labeled. This is consistent with the Gel-based labeling experiment (Ref. 49 and Chapter 3). Potentially, this kind of activity-based probes could be applied to high-throughput detection and identification of enzymes in a protein microarray. 1 2 3 4 5 - - - + + N N O N H I H N O O N H H N O 6 7 8 9 10 11 12 - - - - - - - O S O OH 12 Cy3-GLLY-VS Figure 8 Activity-based protein profiling using probe 12 in a microarray-based experiment. Enzymes on the microarray slide: 1. Type I-S Alkaline phosphatase; 2. Type VIII Alkaline Phosphatase; 3. Type IV Alkaline Phosphatase; 4. Chymopapain; 5. Papain; 6. α-Chymotrypsin; 7. β-Chymotrypsin; 8. γ-Chymotrypsin; 9. Proteinase K; 10. Subtilism; 11. Lysozyme; 12. Lipase 2.3 Conclusion In conclusion, we have successfully synthesized a vinyl sulfone probe using a solution phase synthesis strategy. The generation of this activity-based probe features the solution phase synthesis of a tripeptide methyl ester using Boc protected amino acids, followed by coupling with the free carboxylic acid of Cy3 fluorescent dye. This fragment, after deprotection of the methyl ester, will be further coupled with an N-terminal free, 18 vinyl sulfone containing moiety, which was generated from a solution phase HornerWadsworth-Emmons. Although the overall yield is low due to the long synthetic route, this method provides a useful tool for the small scale generation of a vinyl sulfone containing, activity-based probe for cysteine proteases. We also proved the probe Cy3GLLY-VS was useful in selectively labeling cysteine proteases in a microarray experiment. The bulky Cy3 fluorescent tag, which was added as a labeling reporter for this activity-based probe, seems to have no adverse effect on the enzyme labeling. Accordingly, we may be able to use this kind of probes to directly monitor the enzyme activities in different crude cell lysates. Generating diverse vinyl sulfone probes for proteomics applications will be the center for our next endeavor. 19 Chapter 3 Solid-phase synthesis of peptide vinyl sulfone probes 3.1 Introduction Vinyl sulfone compounds have been prepared by methods based on conventional solution-phase synthesis, but the whole process, as shown in the previous chapter, is inefficient and time-consuming. Recently, a number of solid-phase strategies have been developed. By taking advantage of Kenner’s safety catch strategy, Overkleeft et al. first synthesized the Nterminal peptide fragment of the vinyl sulfone on a solid support, followed by nucleophilic cleavage/ligation using a desired vinyl sulfone-containing, C-terminal amino acid (H2N-Leu-vinyl sulfone). Deprotection of the resulting product followed by HPLC purification gave the final peptide vinyl sulfone in 20-40% yield (Figure 9).64 Fmoc-Leu-OH (5 eq.) NH2 S O O H N S O O PyBOP (5 eq.) DIEA (6 eq) DMF Piperidine O N H Fmoc DMF O S H N O NC Z-Leu-OH H N S O O PyBOP ( 5 eq.) DIEA ( 6 eq.) DMF H2N O S O NHZ N H ICH2CN (20 eq.) DIEA (5 eq.) O NMP S O N O O N H NHZ O 5 eq. DIEA ( 7eq.) THF O NH2 O O S O O H N O N H NHZ Z-LLL-VS Figure 9 Solid-phase synthesis of vinyl sulfone compound via safety catch resin 20 This method is intrinsically inefficient and low yielding, due to the generation of a fully protected peptide product following the cleavage/ligation step. Consequently, this makes it difficult to synthesize vinyl sulfone compounds having longer peptide chains. Alternatively, Nazif and Bogyo reported a solid-phase method for generating positional-scanning combinatorial libraries of peptide vinyl sulfones (Figure 10).57 By attaching a vinyl sulfone-containing aspartic acid onto a Rink amide resin via its sidechain carboxylic acid, they were able to generate P2-P4 positional-scanning tetrapeptidic vinyl sulfone libraries while holding the P1 position constant. This strategy is limited only to the synthesis of peptide vinyl sulfones having carboxyl side chains at the P1 position (e.g. Asp and Glu), thus, they were unable to generate P1 positional scanning library to study the S1 bind pocket of cysteine proteases H Fmoc N O S O HOOC NH2 H Fmoc N OH HOBT, DIC, DIEA O O S O Piperidine/DMF C NH OH Rink resin H2N O O S O H Peptide N O S O Peptide Synthesis C NH OH O C NH OH Figure 10 Solid-phase synthesis of vinyl sulfone compounds via Rink amide resin and the side chain of aspartic acid 21 3.2 Results and discussion 3.2.1 Solid-phase synthesis of peptide vinyl sulfone probe via 2-Cl-Trityl-Chloride resin We developed a facile solid-phase strategy that may be used for the preparation of vinyl sulfone compounds having any amino acid at the P1 position. By anchoring the vinyl sulfone-derivatized P1 amino acid residue onto 2-Cl-Trityl resin via the phenolic alcohol moiety of the vinyl sulfone (Scheme 4), we can generate peptide vinyl sulfone inhibitors or probes from any peptide sequence, either individually or combinatorially. Fmoc H N OEt Cl P O OEt 13 O Cl O O S HO Fmoc H R NaH, THF R O S O H N Pyridine, Cs2CO3, THF Loading = 0.43-0.52 59-59% R = side chain of amino acid 15 OH 14 21 Cl O O S O R N H Fmoc Cl Peptide Synthesis O O S O Peptide NH2 16 1) Cy3, HOBT, DIC Cy3-Peptide-VS probe 2) Resin Cleavage Scheme 4 Solid-phase synthesis of vinyl sulfone probes via 2-Cl-Trityl -chloride resin This strategy took advantage of peptide vinyl sulfones containing a phenol group adjacent to the vinyl sulfone moiety. By modification of the P1 amino acid with a phenolic vinyl sulfone, followed by loading the resulting product 14 onto a suitable solid 22 support via the phenolic alcohol, any peptide vinyl sulfones may be potentially synthesized with high efficiency using conventional solid-phase peptide synthesis. We choose Fmoc protected amino acids as the starting material, because Fmoc chemistry is the preferred method for solid-phase peptide synthesis. 3.2.1.1 Synthesis and immobilization of 14 onto 2-Cl-Trityl resin Subsequent steps were carried out with five randomly chosen amino acids. Fmocamino aldehyde 21 were prepared65, 66 and reacted with 4-hydroxy-thiophenyl-methyldiethylphosponate 13, to give phenolic-Fmoc-amino-vinyl sulfones 14 in a HornerWadsworth-Emmons condensation reaction. The reaction was carried out smoothly in the presence of NaH at room temperature, generating the trans isomer as the predominantly major product with acceptable yield (59-78%, Table 2). All the NMR spectra showed the coupling constant for the vinylic proton is around 15 Hz, which corresponds to coupling constant of trans isomers. Table 2 Yield of 14, and the loading efficiency on 2-Cl-Trityl-Chloride resin. Compound Yield (%) 14a Fmoc-Asp(tBu)-VS 59 loading efficiency (mmol/g) 14 → 16 0.48 14b Fmoc-Leu-VS 74 0.44 14c Fmoc-Lys(Boc)-VS 78 0.43 14d Fmoc-Phe-VS 67 0.45 14e Fmoc-Tyr(tBu)-VS 72 0.52 23 A number of loading strategies were tested to immobilize 14 onto solid support via the phenolic alcohol. Mitsunobu reaction is a valuable tool for the formation of ether bond under mild condition.67 Alcohols or phenols could be immobilized onto resin through etherification with the resin-bound benzyl alcohol in the presence of coupling reagent such as triphenylphosphine (PPh3) and diethyl azodicarboxylate (DEAD).68 Different alcohols were successful loaded and reported,69-71 while the results from immobilization of phenol derivatives varied, with substitution level ranging from 0.020.5 mmol/g.72, 73 We tried different reaction conditions, such as changing the order of the reagent addition, varying the n-fold excess of the reagents, using different solvent such as dichloromethane, N-methylmorpholine, and tetrahydrofuran, but the results were not good, with substitution level ranging from 0.1-0.2 mmol/g. The poor nucleophilicity of the phenolic group of 14 caused by the electron-withdrawing sulfone group may attribute to the low substitution level.72 PPh3 OH O-PPh3 C2H5OOC-N=N-COOC2H5 Wang Resin 14 Fmoc H N R O S O OH O O S O H N Fmoc R Figure 11 Immobilization of 14 onto Wang resin under Mitsunobu reaction condition Trichloroacetimidate activated wang resin was also reported for attaching alcohol, thiol and carboxylic acid onto the resin under the catalysis of Lewis acids such as BF3.Et2O (Figure 12).74, 75 We applied this strategy to the immobilization of 14 via the 24 phenolic alcohol, but the results were unsatisfactory, with loading level below 0.1 mmol/g. CCl3CN OH CCl3 O DBU, CH2Cl2 NH Wang Resin Trichloroacetimidate activated wang resin 14 Fmoc H N R O S O BF3.Et2O OH O O S O H N Fmoc R Figure 12 Immobilization of 14 onto trichloroacetimidate activated Wang resin Finally 2-Cl-Trityl resin gave a good result for the immobilization of phenolic-NFmoc-amino-vinyl sulfones 14. Usually pyridine was used as a solvent as well as a base for the attachment of alcohols and phenols to trityl chloride resin,76 but in our case, due to the low nucleophilicity of the phenolic group, only using cesium carbonate together with pyridine as a mild base, the phenolic alcohol on 14 could be loaded onto this solid support with high efficiency. Different phenolic-N-Fmoc-amino-vinyl sulfones 14 were loaded onto the resin, with loading level ranging from 0.43-0.52 mmol/g (Table2). The loading of the resin was determined by a modified method of UV quantification of the Fmoc-piperidine adduct.77 3.2.1.2 Synthesis of vinyl sulfone probe Cy3-GLLY-VS on 2-Cl-Trityl resin A vinyl sulfone probe 18 was synthesized using the Fmoc-Tyr(tBu)-vinyl sulfone 14e attached 2-Cl-Trityl resin 16e (Scheme 5). The Cy3 dye was conveniently coupled onto the solid support at the end of peptide synthesis, using the standard 25 DIC/HOBT/DIEA strategy. Upon cleavage of the resin, the resulting probe 18 was precipitated with cold ether and purified by preparative HPLC. HO Cl 20% Piperidine/DMF O S O O N H Fmoc Fmoc-AA-OH, TBTU, HOBT 16e HO Cl O O S O O N H O H N O N H H N Fmoc 1) 20% Piperidine/DMF 2) Cy3, DIC, HOTB, DIEA 3) TFA Cleavage 17 N N O N H I H N O O N H H N O O S O OH OH 18 Cy3-GLLY-VS Scheme 5 Solid-phase synthesis of Cy3-GLLY-VS probe via 2-Cl-Trityl -chloride resin 3.2.1.3 SDS-PAGE results for Cy3-GLLY-VS probe 18 The probe 18 was subsequently tested for selective labeling of cysteine proteases on the basis of their enzymatic activity (Figure 13). A panel of 12 enzymes was used, of which two were cysteine proteases (lane 4 and 5). Only cysteine proteases were selectively labeled, indicating the specific nature of the probe. To further confirm that the fluorescence labeling of the cysteine proteases by the probe is due to their enzymatic activity, the enzymes were first inactivated by heat and then treated with the probe followed by SDS-PAGE analysis. No labeling of the cysteine proteases was observed, indicating that the enzymatic activity is a prerequisite for the labeling reaction to occur. 26 Protein #: 1 2 3 4 5 6 7 8 9 10 11 12 Figure 13 Activity-based protein profiling using probe 18 in a gel-based experiment. Lane: 1. Type I-S Alkaline Phosphatase, from bovine intestinal; 2. Type VIII Alkaline Phosphatase, from rabbit intestine; 3. Type IV Alkaline Phosphatase, from porcine intestinal mucosa; 4. Chymopapain, from papaya latex; 5. Papain, from papaya latex; 6. α-Chymotrypsin; 7. β-Chymotrypsin; 8. γ-Chymotrypsin; 9. Proteinase K, from tritirachium album; 10. Subtilisin, from bacillus licheniformis; 11. Lysozyme, from chicken egg white; 12. Lipase, from candida rugosa. 3.2.2 Solid-phase synthesis of peptide vinyl sulfone probes via Rink-amide resin Another solid-phase synthesis strategy was also developed (Scheme 6). After solution phase synthesis of sulfide phosphonate 19, it was immobilized onto Rink amide resin via the carboxylic acid at the terminal. By employing solid-phase oxidation and Horner-Wadsworth-Emmons reaction, we are able to generate the vinyl sulfone skeleton on the solid support (resin 22) with efficiency and good quality. Details of the solid-phase oxidation and Horner-Wadsworth-Emmons reaction will be discussed in chapter 4. This strategy is convenient and advantageous in generating combinatorial library of peptide vinyl sulfone inhibitors or vinyl sulfone probes. 27 Rink resin HOOC SH OEt I + DBU P O OEt HOOC S MeOH NH2 OEt P O OEt DIC, HOBT,DIEA 19 O H N C S OEt P O OEt O H N C m-CPBA Fmoc O LHMDS, THF 32a N(CH3)OCH3 Fmoc OH DCC, HOBT R OEt P O OEt CH2Cl2 31a H N O O S O H N N R O LAH Fmoc THF 20 O H N H R 21 TFA Cleavage O H N C O S O Peptide synthesis R NH-Fmoc 22 Peptide vinyl sulfone (Cysteine protease inhibitor) 1) Cy3, HATU/DIEA Vinyl sulfone probes 2) TFA Cleavage Scheme 6 Solid-phase synthesis of vinyl sulfone compounds via Rink amide resin 3.2.2.1 Synthesis of peptide vinyl sulfone H2N-CLFL-VS Scheme 7 showed the synthesis of peptide vinyl sulfone H2N-CLFL-VS 25 starting from resin 22j which was prepared from Fmoc-Leu-H 21j and Rink sulfone phosphonate resin 32a after a solid-phase Horner-Wadsworth-Emmons reaction. We noted that the subsequent peptide synthesis using 20% piperidine/DMF solution as Fmoc deprotection reagent was not successful, possibly due to the formation of a piperidine adduct via the addition of piperidine to the double bond of vinyl sulfone compound. This problem was circumvented by using 2% DBU/DMF solution as Fmoc deprotection reagent. Further discussions could be found in chapter 4. After the completion of peptide synthesis on resin, the product was cleaved from the resin using reagent R (90% TFA, 5% thioanisole, 2.5% anisole and 2.5% 1,2-ethanedithiol),57 which can prevent the side chain of cysteine 28 from being attacked by electrophiles or forming disulfide bond. After cleavage, the peptide vinyl sulfone was precipitated from cold ether and directly analyzed by analytical HPLC. O H N C O S O O H N C 2% DBU/DMF O S O NH-Fmoc NH2 22j 1) Fmoc-Phe-OH, HOBT,DIC,DIEA O H N C O S O 2) 2% DBU/DMF 1) Fmoc-Cys(Trt)-OH, HOBT,DIC,DIEA Leu-Phe NH2 2) 2% DBU/DMF 23 O H N C O S O Reagent R Leu-Phe-Leu-Cys(Trt) H2N-CLFL-VS NH2 24 25 Scheme 7 Synthesis of peptide vinyl sulfone H2N-CLFL-VS The HPLC spectrum (Figure 14) showed the crude peptide vinyl sulfone H2NCLFL-VS 25 had a good purity. The major peak was confirmed by MS to be the desired product. 2487Channel 1 (214.00 nm) 3.00 H2N-CLFL-VS AU 2.00 1.00 0.00 0.00 5.00 10.00 15.0 0 20.00 25.00 30.00 35.00 Minutes Figure 14 HPLC spectrum of peptide vinyl sulfone H2N-CLFL-VS 29 3.2.2.2 Combinatorial synthesis of vinyl sulfone probes with P1 variation. Following the successful development of new solid phase strategies for making vinyl sulfone compounds, we started to synthesize a positional scanning library of vinyl sulfone probes with P1 variation, which was introduced from 20 different Fmoc amino aldehydes 21. 18 natural Fmoc-amino acids and 2 unnatural amino aicds (FmocOrn(Boc)-OH and Fmoc-(D)-Phe-OH) were used to make the aldehydes. Fmoc-Met-OH and Fmoc-Cys(Trt)-OH were not chosen because they are thiol containing amino acids which may complicate the synthesis and storage due to thiol oxidation and disulfide formation. Most Fmoc-amino-aldehydes were synthesized in good yield (52-90%; Table 3) and high purities, except for arginine, which formed an intramolecular ring after the aldehyde formation.78 (Figure 15) So for arginine, after LAH reduction, we can’t find the proton signal for the desired aldehyde H N O O O NH N H H N O H O OH N H N Pbf NH N Pbf H Figure 15 Intramolecular cyclization of Fmoc-Arg(pbf)-CHO 30 Table 3 Yield of Fmoc-AA-H Compound yield (%) Compound yield (%) 21a Fmoc-Ala 81 21k Fmoc-Lys(Boc) 87 21b Fmoc-Asn(Trt) 78 21l Fmoc-Met 80 21c Fmoc-Asp(tBu) 86 21m Fmoc-Phe 90 21d Fmoc-Cys(Trt) 89 21n Fmoc-Ser(tBu) 80 21e Fmoc-Gln(Trt) 76 21o Fmoc-Thr(tBu) 87 21f Fmoc-Glu(tBu) 52 21p Fmoc-Trp(Boc) 64 21g Fmoc-Gly 89 21q Fmoc-Tyr(tBu) 85 21h Fmoc-His(Trt) 67 21r Fmoc-Val 52 21i Fmoc-Ile 79 21s Fmoc-(D)-Phe 82 21j Fmoc-Leu 81 21t Fmoc-Orn(Boc) 75 After solid-phase Horner-Wadsworth-Emmons reactions, 20 different vinyl sulfone containing resins 22 (a-t) were obtained. These resins were subsequently used for peptide syntheses using the DBU cleavage strategy (Scheme 8). Because we are interested in the study of enzyme substrate specificities toward P1 position residues, we chose Fmoc-LeuOH as the P2 and P3 amino acids of the probes instead of a mixture of 20 natural amino acids which was a general practice for the peptide based positional scanning library. After the synthesis tripeptide on resin, Cy3 was coupled with the free amine at the terminal of growing peptide chain 26 to give resin 27. After cleavage with TFA solution, the crude mixture was concentrated and filtered for preparative HPLC separation. 31 O H N C O S O P1 Peptide synthesis NH-Fmoc O H N C O S O DBU cleavage 22 (a-t) P1 HN Leu-Leu NH2 26 O H N C Cy3, HATU, DIEA O S O P1 TFA/CH2Cl2 HN Leu-Leu Cy3 27 Cy3 N H H N P1 O O 28 (a-t) N H O S O CONH2 P1 = 20 Different amino acids Scheme 8 Combinatorial synthesis of vinyl sulfone probes with P1 variation Ether precipitation was not employed for the purification of peptide vinyl sulfone probes because the Cy3 containing probes are quite soluble in ether solution. All 20 probes were separated by preparative HPLC and confirmed by MS (Table 4). The purity of the probes after HPLC separation ranges from 80-95%. Table 4 ESI-MS data for 20 vinyl sulfone probes with P1 variation Compound (P1 amino acid) (M-I)+ calc. Found 28a Cy3-LLA-VS (Ala) 905.5 905.5 28b Cy3-LLR-VS (Arg) 990.6 990.4 28c Cy3-LLN-VS (Asn) 948.5 948.4 28d Cy3-LLD-VS (Asp ) 949.5 949.5 28e Cy3-LLG-VS (Gly) 891.5 891.5 32 28f Cy3-LLQ-VS (Gln) 962.5 962.5 28g Cy3-LLE-VS (Glu) 963.5 963.5 28h Cy3-LLH-VS (His) 971.5 971.5 28i Cy3-LLI-VS (Ile) 947.5 947.5 28j Cy3-LLL-VS (Leu) 947.5 947.5 28k Cy3-LLK-VS (Lys) 962.5 962.6 28l Cy3-LLF-VS (Phe) 981.5 981.5 28m Cy3-LLP-VS (Pro) 931.5 931.5 28n Cy3-LLS-VS (Ser) 921.5 921.4 28o Cy3-LLT-VS (Thr) 935.5 935.5 28p Cy3-LLW-VS (Trp) 1020.5 1020.5 28q Cy3-LLY-VS (Tyr) 997.5 997.6 28r Cy3-LLV-VS (Val) 933.5 933.6 28s Cy3-LL-Orn-VS (Orn) 948.5 948.5 28t Cy3-LL-(D)-F-VS (D-Phe) 981.5 981.5 3.2.2.3 Labeling papain with 20 vinyl sulfone probes This library of vinyl sulfone probes with P1 variation was tested with a cysteine protease, papain. The probes were prepared as a 5 mM stock solution in DMF. The final concentration of these probes in the reaction solution was 10 uM. From the gel we can clearly see the labeling of papain with these probes (Figure 16). The intensity of the labeling varied with probes having different P1 amino acids. Papain was reported to be good at cleaving peptides at Arg and Lys residues at P1 position4. We observed strong 33 labeling with probes having lys and Arg at P1 position. Since the S2 subsite prefers hydrophobic site chains such as Phe.22 We also observed strong labeling with other amino acids at P1 position, such as Leu, Val and Thr. Further experiments with other cysteine proteases are still in progress. Probes Leu Ile His Glu Gln Asp Asn Arg Ala Gly (P1 amino acids) Probes D-Phe Orn Val Tyr Trp Thr Ser Pro Phe Lys Figure 16 SDS-PAGE result for labelling papain with 20 vinyl sulfone probes 3.3 Conclusion In conclusion, we have successfully developed two solid-phase strategies that allowed the facile synthesis of peptide vinyl sulfone inhibitors and probes. The first strategy is based on the immobilization of Phenolic-Fmoc-AA-vinyl sulfone onto 2Chloro-Trityl chloride resin, followed by solid phase peptide synthesis and cleavage to generate the cysteine protease inhibitor; or after coupling with a fluorescent tag and cleavage, to generate the cysteine protease probe. This strategy makes the generation of vinyl sulfone inhibitors or probes with P1 variation possible. Using this strategy, five different Phenolic-Fmoc-AA-vinyl sulfones were efficiently immobilized onto the resin, and a Cy3-GLLY-VS probe was successful synthesized and tested with a panel of 34 enzymes in a gel-based experiment, only cysteine proteases were selectively labeled, which confirmed the applicability of this strategy. The second strategy employs the solidphase oxidation reaction to convert the Rink sulfide phosphonate resin to the Rink sulfone phosphonate resin, followed by solid phase Horner-Wadsworth-Emmons reaction with Fmoc-amino aldehydes to generate the vinyl sulfone skeleton on the solid support. This method furnishes the easy introduction of P1 variation, although at the loss of certain degree of purity. Using this method, a combinatorial library of vinyl sulfone probes with 20 different amino acids at P1 position was synthesized. Preliminary experiment with papain showed this library of probes was able to label cysteine protease at variable degree of intensities, which may be derived from the enzyme substrate specificity toward different amino acid residues at the P1 position. This library of probes could potentially be used to profile diverse cysteine proteases as well as crude cell lysates, which may reveal more insight into the P1 substrate specificity of cysteine proteases. 35 Chapter 4 Combinatorial synthesis of vinyl sulfone small molecules 4.1 Introduction Protease inhibitors are not only effective biological tools in proteomics studies, but could also be developed into lead compounds by improving their potency and selectivity in binding to disease related enzymes.79-82 To be a drug candidate, however, it requires those inhibitors have good membrane permeability, good bioavailability, low molecular weight.83 Peptide-based inhibitors are usually not suitable as drug candidate due to their poor bioavailability and instability in cells.84-86 Small molecules having minimal peptide characters while still selective toward proteases would be more promising as potential lead compounds.87,88 Several cysteine protease inhibitors such as peptidyl α-substituted ketone,53, 89, 90 Michael acceptor inhibitors91 and α-keto amide inhibitors92 were identified from compounds resulting from combinatorial library syntheses. Structure or mechanismbased design provided another approach. Peptide vinyl sulfone derivatives34, 93, peptidyl nitriles,94, 95 and non-peptidic small molecules96-99 have been developed for papain-family cysteine proteases. Peptidyl α-hydroxamate derivatives100, 101 and some peptidomimetics102 were designed for calpain-family cysteine proteases. Some other small molecule inhibitors were also developed for caspase103, 104 and picornaviral cysteine proteases105. Vinyl sulfone compounds have been widely studied for their inhibition toward cysteine protease.22, 37 Some of them were found to be promising as drug candidate.106-108 APC-3328, a vinyl sulfone containing peptidomimetic inhibitor for treating osteoporosis, 36 has a ksec. = 5.7 x 106 M-1 s-1 towards cathepsin K. It was observed that there was a close interaction between APC-3328 and cathepsin S3, S2, S1, S1′ binding pocket.106 HN O H N N O N H O S O Figure 17 APC-3328, a potential lead compound for osteoporosis Current synthesis strategies are not suitable for the combinatorial generation of vinyl sulfone small molecule library because they are either based on inefficient solution phase synthesis strategy,32, 35 or solid-phase strategies which are unable to introduce diversities at positions such as P1 and P1′.57, 64 Here we developed a strategy for solid-phase synthesis of vinyl sulfone small molecules, which allows an easy introduction of diversity at not only the P positions (e.g., P1 and P2) but also the P1′ position of the inhibitor. As proof-of-concept experiments, we have successfully applied this strategy to the synthesis of a 30-member small-molecule-based (MW < 500) vinyl sulfone library that installs three, five, and two different variables at P1′, P1, and P2 positions of the inhibitor, respectively. The interaction between small molecules and cysteine protease is shown in Figure 18. 37 Site of inhibition P2 S P1 ' O O S2 O Protease binding pocket P1 SH S1 S1' Figure 18 A vinyl sulfone inhibitor binding to the active site of a cysteine protease. 4.2 Results and discussion 4.2.1 Overall scheme Our strategy takes advantage of the successful implementation of both m-CPBA oxidation of sulfide 30 and its subsequent Horner-Wadsworth-Emmons reaction on solid support with high yield and purity, thus making it possible to install three points of diversity (i.e., P1′, P1, and P2) within the vinyl sulfone scaffold. As shown in Scheme 9, steps in our synthesis include (a) solution-phase nucleophilic reaction between a commercially available thiol-containing acid, 29, and diethyl iodomethyl phosphonate to generate the sulfide phosphonate 30; (b) loading of 30 onto Rink amide resin to give 31; (c) solid-phase oxidation of 31 to give sulfone 32; (d) solid-phase Horner-WadsworthEmmons reaction with an amino acid-derived, Fmoc-protected aldehyde 21 to give 33; and (e) deprotection of Fmoc group under optimized conditions followed by (f) acylation and (g) TFA cleavage to release the desired vinyl sulfone 37 in solution. 38 P1 ' HO SH O I a P + O O O Cs2CO3 or DBU O O P1 ' P O SH HO NH2 S HO O O HOBT, DIC, DIEA n=10 b 30 N SH O HO 29 c O FmocNH O O P1 ' S NH m-CPBA O P O O NH CH2Cl2 31 O P1 NH LHMDS, O P O O O P1 DBU/DMF P1 ' HN O S O HOBT/DIC/DIEA NH2 HN P1 TFA cleavage O P1' H2N O S O 36 a OMe O b P1 NH NH P2 N = O 35 O S O OH OH P2 34 33 P1 ' 21 P1 P1 = Leu, Asp, Gln, Lys, Tyr P1 NH-Fmoc O H 32 O S O ' P1 ' O S O P2 O O 37 Scheme 9 Synthesis of a 30-member vinyl sulfone small molecule library 4.2.2 Synthesis of Rink resin bound sulfide phosphonate 31 Starting from three mercaptoacids (29a-c), 30a-c were prepared through the nucleophilic reaction with diethyl iodomethyl phosphonate in the presence of a suitable base (i.e., DBU or NaH for 29a and 29c, Cs2CO3 for 29b, respectively) in good yields (60-85%). When aliphatic, 11-mercaptoundecanoic acid 29b was used with bases such as DBU, NaH, or NaOEt, the major product isolated upon workup was the disulfide, rather 39 than the desired product 30b. This problem was overcome by using Cs2CO3 as the base in the reaction, producing the desired product in high yield (85%). The sulfide phosphonates 30a-c were conveniently loaded onto the Rink amide resin using the standard DIC/HOBT/DIEA coupling procedures to afford the resin-bound sulfide phosphonates 31a-c with high substitution levels (0.65-0.7 mmol/g). Ninhydrin test after the coupling reaction showed the resin had no color change which indicated that no free amine was on the resin and the coupling was complete. 4.2.3 Synthesis of Rink resin bound sulfone phosphonate 32 Oxidation of 31a-c was accomplished by treatments of the resin with m-CPBA (5 equiv) for 1-1.5 h, to give 32a-c in nearly quantitative yields (95-100%). Shorter oxidation times gave rise to the formation of partially oxidized products, the sulfoxide phosphonates. The best condition was found to be 1 to 1.5 hrs oxidation time with 3 equiv. m-CPBA. 4.2.4 Solid-phase Horner-Wadsworth-Emmons reaction The resin-bound sulfone phosphonates 32a-c were used as the precursors in the Horner-Wadsworth-Emmons reaction, which would generate the critical vinyl sulfone scaffold and at the same time introduce the P1 diversity in the library. HornerWadsworth-Emmons reaction is a valuable carbon-carbon bond-forming reaction that has been successfully applied in the solid-phase synthesis of olefins and some peptidyl Michael acceptors.91, 110-117 Most published Horner-Wadsworth-Emmons protocols 40 require the presence of an excess of a strong base such as NaH and LHMDS to complete the reaction. They are therefore not suitable for our synthesis because the presence of an excess of a strong base may (1) cause the Fmoc group in the aldehyde to be cleaved off prematurely, (2) lead to severe epimerization at the aldehyde chiral center, and (3) lead to potential epimerization in the vinyl sulfone product 34. Consequently, it is necessary to optimize the Horner-Wadsworth-Emmons conditions in our strategy to minimize epimerization and increase the conversion rate. Five different bases (e.g., DBU, LHMDS, KHMDS, LDA, NaH) were tried with different reaction times and temperatures. Leucine was used as the P1 amino acid; LHMDS and LDA were 1 M and 2 M solutions in THF respectively. Only LHMDS under the optimized reaction condition produced the best result, giving rise to quantitative conversion from 32 to 33 (100% conversion) and nearly quantitative yield (>95% including both epimers). The degree of epimerization in 33 under these conditions was determined to be 95% DBU 90min/25 oC 8 [...]... S O Figure 5 Synthesis of Boc-Leu -vinyl sulfone Another approach to synthesize vinyl sulfone compound was developed by Roush et al 34 In their pursuit of developing a potent and selective inhibitor for cruzain, they synthesized tens of vinyl sulfone compounds and studied the interaction of these inhibitors with the S1, S2, S1′ S2′ binding site of curzain The vinyl sulfonate ester and vinyl sulfonamide... inhibitors Figure 5 Synthesis of Boc-Leu -vinyl sulfone 12 Figure 6 Synthesis of vinyl sulfonate esters and vinyl sulfonamides 13 Figure 7 Structure of the Cy3-Gly-Leu-Leu-Tyr-VS proble 14 Figure 8 Activity-based protein profiling using probe 12 in a 18 microarray-based experiment Figure 9 Solid-phase synthesis of vinyl sulfone compound via 20 safety catch resin Figure 10 Solid-phase synthesis of vinyl. .. 45 xi LIST OF SCHEMES Scheme 1 Synthesis of H2N-Tyr(tBu) -vinyl sulfone 15 Scheme 2 Synthesis of Cy3-Gly-Leu-Leu-OH 16 Scheme 3 Synthesis of Cy3-Gly-Leu-Leu-Tyr -vinyl sulfone 17 Scheme 4 Solid-phase synthesis of vinyl sulfone probes via 22 2-Cl-Trityl -chloride resin Scheme 5 Solid-phase synthesis of Cy3-GLLY-VS probe via 26 2-Cl-Trityl -chloride resin Scheme 6 Solid-phase synthesis of vinyl sulfone compounds... of vinyl sulfone compounds via 28 Rink amide resin Scheme 7 Synthesis of peptide vinyl sulfone H2N-CLFL-VS 29 Scheme 8 Combinatorial synthesis of vinyl sulfone probes with 32 P1 variation Scheme 9 Synthesis of a 30-member vinyl sulfone small molecule library 39 Scheme 10 Generation of diastereomeric and enantiomeric dipeptide 43 vinyl sulfones xii ABBREVIATIONS AA Amino acid BF3.Et2O Boron trifluoride... structure of an activity-based probe 3 Figure 2 Strategy for activity-based protein profiling 5 Figure 3a Interaction between substrate and enzyme active site 8 Figure 3b Interaction between peptide vinyl sulfone and enzyme active site 8 Figure 3c Mechanism of peptide vinyl sulfone inhibiting cysteine protease 8 Figure 4 Positional scanning library in the generation of affinity 10 fingerprint of peptide... designate binding pocket of the enzyme, site labeled with P designate side chain of peptide substrate (b) Interaction between peptide vinyl sulfone and enzyme active site (c) Mechanism of peptide vinyl sulfone inhibiting cysteine protease 8 1.5 Positional Scanning Library Selective inhibitors and probes are of considerable use for the deconvolution of the protease or proteosome’s role in a wide range of biological... Probe P2 Cathepsin K Different enzymes Data readout and computation process Affinity fingerprint Figure 4 Positional scanning library in the generation of affinity fingerprint of peptide epoxide inhibitors (Adopted from Reference 52) 10 1.6 Aim of our project We aim to develop a facile and efficient solid-phase strategy to combinatorially synthesize vinyl sulfone probes for the study of cysteine proteases... example of activity-based proteomics is shown in the profiling of protein tyrosine phosphatases (PTPs) in the whole proteome using PTPs specific chemical probes.28 PTPs are involved in the regulation of many aspects of cellular activity including proliferation, metabolism, migration, and survival Except for the large number and complexity of PTPs in cell signaling, the activities of many PTPs are tightly... cyclization of Fmoc-Arg(pbf)-CHO 30 Figure 16 SDS-PAGE result for labelling papain with 20 vinyl 34 sulfone probes Figure 17 APC-3328, a potential lead compound for osteoporosis 37 Figure 18 A vinyl sulfone small molecule binding to the active site 38 of a cysteine protease Figure 19 HPLC spectra for diastereomeric and enantiomeric dipeptides 44 vinyl sulfones Figure 20 Piperidine adduct of vinyl sulfone. .. active site of the enzyme.34 Many of these inhibitors, including α,β-unsaturated ketones, acrylamides, vinyl sulfones, et al., have been successfully synthesized, and some have been tested in clinical experiments.48 Peptidyl vinyl sulfones, in particular, have been shown to be extremely useful as activity-based probes for high-throughput profiling of cysteine proteases, largely due to their negligible .. .FACILE SYNTHESIS OF COMBINATORIAL VINYL SULFONE LIBRARIES AND THEIR APPLICATIONS IN LARGE SCALE PROTEOMICS WANG GANG A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY... 3.2.2.2 Combinatorial synthesis of vinyl sulfone probes with 30 P1 variation 3.2.2.3 Labelling papain with 20 vinyl sulfone probes 33 3.3 34 Conclusion Chapter Combinatorial synthesis of vinyl sulfone. .. peptide vinyl sulfone and enzyme active site Figure 3c Mechanism of peptide vinyl sulfone inhibiting cysteine protease Figure Positional scanning library in the generation of affinity 10 fingerprint