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