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

2.2.3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 16 2.2.4 Application of the Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 17 probe in enzyme profiling Chapter 3 Solid-phase synthesis of peptide

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

1.4 Cysteine proteases and viny sulfone compounds 6

2.2.1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone 14

2.2.3 Synthesis of Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 16 2.2.4 Application of the Cy3-Gly-Leu-Leu-Tyr-vinyl sulfone 17

probe in enzyme profiling

Chapter 3 Solid-phase synthesis of peptide vinyl sulfone probes 20

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

Chapter 4 Combinatorial synthesis of vinyl sulfone small molecules 36

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

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

probes and papain 5.2.7 Synthesis of vinyl sulfone small molecule library 66

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 solid-

phase strategies to synthesize vinyl sulfone probes As shown in Chapter 3, our first

solid-phase strategy was based on the synthesis and immobilization of amino-vinyl sulfones onto 2-Cl-Trityl chloride resin, followed by peptide synthesis and probe generation This strategy was successful as five different phenolic-Fmoc-amino-vinyl 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 Gel-based 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-

phenolic-Fmoc-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

LIST OF TABLES

Table 2 Yield of 14, and the loading efficiency on 2-Cl-Trityl 23

-Chloride resin

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

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 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

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

LIST OF SCHEMES

Scheme 1 Synthesis of H2N-Tyr(tBu)-vinyl sulfone 15

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

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl

ABBREVIATIONS FOR AMINO ACIDS

Name Abbr Linear structure formula

Alanine ala a 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 NH-CH=N-CH=C-CH2-CH(NH2)-COOH

Isoleucine ile i 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

Serine ser s HO-CH2-CH(NH2)-COOH

Threonine thr t CH3-CH(OH)-CH(NH2)-COOH

Tryptophan trp w Ph-NH-CH=C-CH2-CH(NH2)-COOH

Tyrosine tyr y HO-p-Ph-CH2-CH(NH2)-COOH

Valine val v (CH3)2-CH-CH(NH2)-COOH

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

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,7array-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

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–small-molecule 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

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

Linker Unit

Figure 1 General structure of an activity-based probe The reactive unit is a chemical reactivity that recognizes the enzyme active site and

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 activity-based 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

Table 1 Components of activity-based probes

H N

R3

N H

H N

O

O R2

R1Peptide fragment

COOH O

O Epoxide

S O

O O Sulfonate ester

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

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

agents are widely studied Some typical examples are peptidyl chloromethyl ketones,39, 40Epoxysuccinyl Peptides41, 42 and Michael Acceptors 43-47such 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,

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

R H

R H

O H

H Gln

His+

S

-Enz

S RO

O H

H Gln

His+

S

-Enz

S RO

Site of Inhibition

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 125I-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

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

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)

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

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

O O

O

S O O

EtO P EtO OS O O

H OH

PyBOP,DIEA,

CH 3 NHOCH 3 ,

NaH

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-

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

O O

SO 3 Et

N Ph

Boc SO 3 Et

1) TFA, CH 2 Cl 2 2) Cbz-Phe-OH, EDC HOBT, DIEA 81%

N Ph

SO 3 Et

O H

Ph Cbz

1) n-Bu 4 NI, Acetone reflux 2) Ph 3 P, SO 2 Cl 2 61%

N Ph

SO 2 Cl

O H

Ph Cbz

SO 2 R

O H

Ph

R = OPh

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;

the tetrapeptide acts as a recognition unit, which mimics the substrate of the cysteine

protease; Cy3 dye is a fluorescent tag (Figure 7)

H

S O O

OH

N H N

O O

O

N I

Tag unit Peptide linker

Reactive unit

Figure 7 Structure of the Cy3-Gly-Leu-Leu-Tyr-VS proble

2.2.1 Synthesis of H 2N-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 diethylphosphonate sulfone was synthesized and used to react with Fmoc-Tyr(tBu)-H 4

4-methyl-thiophenyl-methyl-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-Wadsworth-

Emmons 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

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

SH

O P

O O

I NaH 87%

S P OEt OEt O Peracetic

acid,

1,4-dioxane

S P OEt OEt O

O O

H OH O

O HOBT,DIEA,

CH 3 NHOCH 3 , DMF

Fmoc H O O

O

N O 71%

Fmoc H H O

O

Fmoc

H

S O O

O

O O Fmoc =

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 9with 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

N I

N

OH O

O N

O

H 2 N O

Cy3

+

H2N-GLL-OCH3

EDC HOBT, DIEA DMF

O N

O

H O

20%K2CO3, MeOH

OH N

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

protecting group (tBu) on the side chain of tyrosine, the final product was obtained after

preparative HPLC purification and used for labeling experiments

H 2 N S

O O

O

OH N

O

+

EDC, HOBT DIEA DMF

H

S O O

O

N H N

O O

OH

N H N

O O

O

N I

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

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

H S O

O

OH

N H N

O O

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,

vinyl sulfone containing moiety, which was generated from a solution phase Wadsworth-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 Cy3-GLLY-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

Horner-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 N-terminal 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

SNH2

O O

Fmoc-Leu-OH (5 eq.) PyBOP (5 eq.) DIEA (6 eq) DMF

S H O

O Fmoc Piperidine

H O

O

ICH 2 CN (20 eq.) DIEA (5 eq.) NMP

O

NHZ O

S NO

O

NHZ O NC

NHZ O S

Z-LLL-VS

Figure 9 Solid-phase synthesis of vinyl sulfone compound via safety catch resin

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 side-chain 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

HOBT, DIC, DIEA

H

S

OH Fmoc

C NH

H2N O SO

OH C

NH O Peptide

Figure 10 Solid-phase synthesis of vinyl sulfone compounds via Rink amide resin and

the side chain of aspartic acid