Design, Synthesis, and Evaluation of 2-(arylsulfonyl)oxiranes as Cell-permeable Covalent Inhibitors of Protein Tyrosine Phosphatases

Article abstract of DOI:10.1111/j.1747-0285.2012.01437.x

A structure-based design approach has been applied to develop 2-(arylsulfonyl)oxiranes as potential covalent inhibitors of protein tyrosine phosphatases. A detailed kinetic analysis of inactivation by these covalent inhibitors reveals that this class of compounds inhibits a panel of protein tyrosine phosphatases in a time- and dose-dependent manner, consistent with the covalent modification of the enzyme active site. An inactivation experiment in the presence of sodium arsenate, a known competitive inhibitor of protein tyrosine phosphatase, indicated that these inhibitors were active site bound. This finding is consistent with the mass spectrometric analysis of the covalently modified protein tyrosine phosphatase enzyme. Additional experiments indicated that these compounds remained inert toward other classes of arylphosphate-hydrolyzing enzymes, and alkaline and acid phosphatases. Cell-based experiments with human A549 lung cancer cell lines indicated that 2-(phenylsulfonyl)oxirane (1) caused an increase in intracellular pTyr levels in a dose-dependent manner thereby suggesting its cell-permeable nature. Taken together, the newly identified 2-(arylsulfonyl)oxiranyl moiety could serve as a novel chemotype for the development of activity-based probes and therapeutic agents against protein tyrosine phosphatase superfamily of enzymes.

Full text of DOI:10.1111/j.1747-0285.2012.01437.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01437.x
Chem Biol Drug Des 2012
Research Article
Design, Synthesis, and Evaluation of
2-(arylsulfonyl)oxiranes as Cell-permeable
Covalent Inhibitors of Protein Tyrosine
Phosphatases
Dibyendu Dana1, Tirtha K. Das2, Ish
Kumar3, Anibal R. Davalos1, Kevin J.
Mark4, Daryl Ramai4, Emmanuel J. Chang4,
Tanaji T. Talele5 and Sanjai Kumar1,*
Key words: covalent PTP inhibitor, irreversible PTP inhibitor, mecha-
nism-based inhibitors of PTP, protein tyrosine phosphatase inhibitor
Received 10 April 2012, revised 17 May 2012 and accepted for publica-
tion 18 June 2012
1Department of Chemistry & Biochemistry, Queens College – CUNY,
65-30 Kissena Blvd., Flushing, NY 11367, USA
Tyrosine phosphorylation of proteins is a dominant post-translational
modification that is extensively utilized by the mammalian cellular
machinery to carry out many important biological processes, such
as cell differentiation, cell proliferation, cell growth, cell survival,
vesicular trafficking, cell migration and cell–cell communication (1).
Protein tyrosine phosphatases (PTPs) are a critical class of cell sig-
naling enzymes that in conjunction with protein kinases (PKs) regu-
late the desired level of protein phosphorylation (1,2). Imbalance in
intracellular protein phosphorylation because of anomalous behavior
of PTPs has been linked to a variety of human diseases, such as
diabetes, obesity, autoimmune disease, and cancer (3–5). Despite
the significance of PTPs in cellular signaling and human diseases,
their precise physiological roles in various cellular contexts remain
largely poorly understood. Furthermore, the recent discovery that
the PTP activity is also regulated by the local redox microenviron-
ment of the cell has added another tier of complexity (6,7). To meet
these challenges, chemical approaches to investigate PTP function
has begun to emerge with many notable advantages (8,9). The pro-
gress, however, remain sluggish in this area because the majority
of potent and selective PTP inhibitors often carry multiple negative
charges which renders them poorly cell-permeable and therefore
unsuitable for cell-based studies (10). Discovery of uncharged and
selective inhibitors of PTP activities is therefore highly sought after
for their functional analysis and drug discovery efforts.
2Department of Developmental and Regenerative Biology, Mount
Sinai School of Medicine, New York, NY 10029, USA
3School of Natural Sciences, Fairleigh Dickinson University, 1000
River Road, Teaneck, NJ 07666, USA
4Department of Chemistry, York College of the City University of
New York, Jamaica, NY, 11451, USA
5Department of Pharmaceutical Sciences, College of Pharmacy and
Allied Health Professions, St. John's University, Queens, NY 11439,
USA
*Corresponding author: Sanjai Kumar, sanjai.kumar@qc.cuny.edu
A
structure-based design approach has been
applied to develop 2-(arylsulfonyl)oxiranes as
potential covalent inhibitors of protein tyrosine
phosphatases. A detailed kinetic analysis of inacti-
vation by these covalent inhibitors reveals that this
class of compounds inhibits a panel of protein tyro-
sine phosphatases in a time- and dose-dependent
manner, consistent with the covalent modification
of the enzyme active site. An inactivation experi-
ment in the presence of sodium arsenate, a known
competitive inhibitor of protein tyrosine phospha-
tase, indicated that these inhibitors were active
site bound. This finding is consistent with the mass
spectrometric analysis of the covalently modified
protein tyrosine phosphatase enzyme. Additional
experiments indicated that these compounds
remained inert toward other classes of arylphos-
phate-hydrolyzing enzymes, and alkaline and acid
phosphatases. Cell-based experiments with human
A549 lung cancer cell lines indicated that 2-(phen-
ylsulfonyl)oxirane (1) caused an increase in intra-
cellular pTyr levels in a dose-dependent manner
thereby suggesting its cell-permeable nature.
Taken together, the newly identified 2-(arylsulfo-
nyl)oxiranyl moiety could serve as a novel chemo-
type for the development of activity-based probes
and therapeutic agents against protein tyrosine
phosphatase superfamily of enzymes.
Protein tyrosine phosphatases employ a unique covalent catalytic
mechanism to carry out their biological function. This includes the
involvement of an unusually low pKa (approximately 4.2) Cys resi-
due initiating the catalytic process by attacking the electrophilic
phosphorous of pTyr substrate. Taking a mechanistic clue from this
process, novel mechanism-based inhibitory motifs, especially
uncharged, could be developed and utilized for functional elucida-
tion of PTPs in both normal and diseased physiology. Covalent
inhibitors could have a significant advantage over their non-covalent
reversible counterparts in that they have infinite binding affinities
to their targets, mainly because they covalently and permanently
inhibit their target enzymes. Additionally, the mechanism-based
1

Dana et al.
inhibitory motif could be utilized to develop selective covalent inhib-
itors of individual PTP with inclusion of a functional group that tar-
gets peripheral auxiliary pockets near the PTP active site. Such a
strategy has indeed been successful in designing potent and selec-
tive PTP inhibitory agents (11–14). Furthermore, the recent clinical
success of drugs that covalently modify their protein targets amelio-
rates earlier concerns about potential undesireable immunogenicity
of covalently modified PTP. Currently, three of the top ten selling
drugs in the United States are covalent modifiers of their cellular
targets (15). However, only a limited number of covalent and irre-
versible PTP inhibitors are reported to date, as reviewed elsewhere
(16).
entry 3D9C) (20). For this task, the Builder module of Insight II –
2005 software (Accelrys Inc., San Diego, CA, USA) was used and the
model of inhibitor (1 and 5 separately) bound to the sulfhydryl group
of Cys215 residue of hPTP1B was created. The partial charges gen-
erated for the inhibitor from the first step of calculation were now
assigned. The charges on the protein were assigned by using the
consistent valence force field provided in Insight II. While retaining
the crystallographic water molecules, a shell of water molecules in
a sphere of 15 ꢀ with the sulfhydryl group of Cys215 as the center
was created using the SOAK command. Molecular dynamics simula-
tions (50 ps) were then performed without any constraints in the
Discover standalone module with trajectories being collected every
1 ps and analyzed. A typical snapshot of the dominant conformation
was selected and energy minimized using Steepest Descent (5000
steps) followed by Conjugate Gradient (10 000 steps). The Discovery
Studio 3.1 Visualizer (Accelrys Inc., San Diego, CA, USA) was used
for viewing, analyzing, and preparing final images from the energy-
minimized structures of the inhibitory complexes.
Another important application of mechanism-based active site
directed covalent PTP inactivators is to utilize them to construct
activity-based probes for the collective analysis of PTPs in complex
proteomes. Activity-based protein profiling is an emerging area of
immense interest where protein function in native cellular contexts
can be analyzed on a global scale to understand their physiological
function (17,18).
Chemistry
Herein, we undertake a rational approach to develop 2-(arylsulfo-
nyl)oxiranes as mechanism-based inhibitors of PTPs. Molecular mod-
eling studies based on previous crystallographic data on PTP are
utilized for guidance in the inhibitor design process. Following favor-
able clues from molecular modeling studies, a focused series of
2-(sulfonyl)oxiranes (1–5) have been synthesized and kinetically
evaluated as a new class of covalent inhibitors of PTPs. Competi-
tion- and mass spectrometry-based analyses indicated that these
agents are active site directed and inhibit PTP covalently and irre-
versibly. Finally, the cell permeability of this class of inhibitors to
block intracellular PTP activities was evaluated using experiments
on human A549 lung cancer cell lines.
Isopropyl ether, anhydrous tetrahydrofuran (Acroseal), anhydrous
dichloromethane (Acroseal), and p-toluenethiol were purchased from
Acros Organics, Pittsburgh, PA, USA. Methanesulfonyl chloride,
2-bromoethanol, and triethylamine were purchased from Alfa-Aesar
Incorporated, Ward Hill, MA, USA. 1-(Vinylsulfonyl)benzene, (meth-
ylsulfonyl)ethene, 4-bromobenzenethiol, 1-chloro-4-nitrobenzene,
n-butyllithium (1.6 M in hexanes), tert-butyl hydroperoxide (5–6 M in
Decane), 30% aqueous hydrogen peroxide solution, sodium tungstate,
and potassium hydrogen sulfate were from Sigma-Aldrich Corpora-
tion, St. Louis, MO, USA. Ethyl acetate, dichloromethane, and petro-
leum ether were purchased from Thomas Scientific Inc., Swedesboro,
NJ, USA. Silica gel (230–400 mesh) for column chromatography was
purchased from Fisher Scientific Inc., Pittsburgh, PA, USA.
1
Methods and Materials
13C NMR and H spectra were recorded on a Brucker DPX 400 MHz
FT NMR with automatic sample changer. Chemical shifts (d) are
reported in parts per million (ppm) and referenced to CDCl3
(7.26 ppm for 1H and 77.0 ppm for 13C), DMSO (2.50 ppm for 1H
Molecular modeling studies
The molecular modeling studies were performed on a Dell Power-
Edge 6000 server, powered by 12 CPUs and operating on Red Hat
Enterprise Linux ES release 4. The first step of the computational
modeling was the calculation of partial charges on atoms of a
small cysteinyl peptide (CH3CONHCysCONHCH3) covalently bound to
the terminal carbon of the inhibitory compounds (1 and 5) via the
sulfhydryl group of cysteine. This was achieved in the following
way: The Builder module of the Maestro 7.5 software (Schrodinger
Inc., New York, NY, USA) was utilized for the constructions of inhib-
itor-bound modified cysteinyl peptide and the geometry was refined
by performing a semiempirical calculation. Finally, a single-point cal-
culation that yielded the partial charges on atoms was performed
in the Jaguar module employing density-functional calculation with
B3LYP functional and a 6–31G** basis set.
1
and 39.5 ppm for 13C), (CD3)2CO (2.05 ppm for H and 29.9, 206.7
1
for 13C), and CD3OD (4.87, 3.31 for H and 49.1 for 13C). Coupling
constants (J) are reported in Hertz (Hz) and multiplicities are abbre-
viated as singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
triplet of doublets (td), and multiplet (m).
The synthetic routes to acquire 2-(arylsulfonyl)oxiranes (1–4) and
2-(methylsulfonyl)oxirane (5) involved preparation of vinylsulfonyl
precursors and their subsequent oxidation using tert-butyl hydro-
peroxide and n-butyl lithium reagents (Scheme 1). Briefly, the
2-(phenylsulfonyl)oxirane (1) and 2-(methylsulfonyl)oxirane (5) were
synthesized by following a slightly modified version of an earlier
published protocol (21). This involved a one-step oxidation of 1-(vi-
nylsulfonyl)benzene and (methylsulfonyl)ethene in the presence of
tert-butyl hydroperoxide and n-butyl lithium. The 2-(4-nitro-
phenylsulfonyl)oxirane (4) was synthesized in four steps from 1-
chloro-4-nitrobenzene. In the first step, compound 4b was prepared
by a nucleophilic substitution reaction of 2-mercaptoethanol with 1-
chloro-4-nitrobenzene (4a). Subsequent oxidation of 4b yielded the
The second step of the molecular modeling involved a slight modifi-
cation of earlier published protocol (19). This methodology started
with an appropriate modification of the crystal structure of hPTP1B
enzyme covalently bound to aryl seleninic acid via a covalent seleno-
sulfide linkage with the active site cysteine sulfhydryl group (PDB
2
Chem Biol Drug Des 2012

Cell-Permeable Covalent Inhibitors of Protein Tyrosine Phosphatases
and its characterization was found to be identical to the literature
reported values (23). Yield: 70%.
2-(4-nitrophenylsulfonyl)ethanol (Compound 4c – Scheme 1). This
compound was synthesized and purified using earlier published pro-
tocol, and its characterization was found to match with the litera-
ture reported values (23). Yield: 73%.
1-nitro-4-(vinylsulfonyl)benzene (Compound 4d – Scheme 1). This
compound was synthesized by following an earlier published proto-
col with some modifications (22). 2-(4-nitrophenylsulfonyl)ethanol
(1.48 g, 6.4 mmol) was dissolved in dry tetrahydrofuran under argon
atmosphere and cooled to 0 ꢀC. Triethylamine (2.23 mL, 16 mmol)
and methanesulfonyl chloride (600 lL, 7.68 mmol) was added to
the solution and stirring was continued for 30 min. After the reac-
tion was over, the solvent was evaporated and the residue was
redissolved in dichloromethane and washed with saturated aqueous
ammonium chloride solution (2 · 20 mL). The organic layer was
Scheme 1: Synthetic strategies and chemical reagents
dried over anhydrous sodium sulfate and the resulting crude com-
pound was dried over high vacuum overnight. Purification was
achieved by flash chromatography using petroleum ether and ethyl
acetate mixture (3:2) to give the desired product as yellow powder.
employed in the acquisition of 2-(sulfonyl)oxirane compounds, 1–5:
(i) n-BuLi ⁄ tert-butyl hydroperoxide (ii) 2-mercaptoethanol ⁄ KOH ⁄ etha-
nol, reflux (iii) 30% H2O2 ⁄ Na2WO4 (iv) methanesulfonyl chlo-
ride ⁄ Et3N, dry THF, 0 ꢀC (v) 2-bromoethanol, KOH ⁄ ethanol.
1
Yield: 65%. H NMR (400 MHz, CDCl3) d ppm: 6.20 (d, J = 9.1 Hz,
1H), 6.66 (m, 2H), 8.10 (m, 2H), 8.41 (m, 2H). 13C NMR (100 MHz,
sulfonyl alcohol, 4c. A base-catalyzed elimination reaction of the
in situ generated methylsulfonate ester resulted in the formation of
compound 4d (22). In the final step, 4d was oxidized to yield 4
(21). A similar procedure was adopted for the generation of com-
pounds 2 and 3 except with the following modification: the alcohol
derivatives, 2b and 3b, were synthesized from the nucleophilic
reactions of 2-bromoethanol with their corresponding thiophenol
derivatives (23).
CDCl3) d ppm: 124.56, 129.36, 130.35, 137.35, 145.41, 150.93.
2-(4-nitrophenylsulfonyl)oxirane (Compound 4 – Scheme 1). This
compound was synthesized using the similar procedure adopted in
the synthesis of compound 1. Yield: 55%. 1H NMR (400 MHz,
((CD3)2CO) d ppm: 3.19 (dd, J = 5.3, 3.8 Hz, 1H), 3.27 (dd, J = 5.3,
2.0 Hz, 1H), 4.55 (dd, J = 3.8, 2.0 Hz, 1H), 8.11 (m, 2H), 8.42 (m,
2H). 13C NMR (100 MHz, (CD3)2CO) d ppm: 46.07, 63.67, 125.56,
131.28, 143.72, 152.42. LRMS [ESI-MS +ve] calculated for
(C8H7NO5S + Na): 252.2, observed: 252.2.
Synthesis of 2-(Phenylsulfonyl)oxirane (1)
This compound was synthesized by following a slightly modified
procedure of an earlier published protocol (21). Under strict nitrogen
environment, and at )78 ꢀC, n-butyllithium (1.4 mL of 1.6 M solu-
tion in hexane, 2.2 mmol, 1 equivalent) was added to a solution of
dry tert-butyl hydroperoxide (0.6 mL solution of 5–6 M in decane,
1.5 equivalent) in THF (30 mL). The resulting mixture was stirred for
15–20 min and brought to )10 to )20 ꢀC using ice with sodium
chloride bath. To this, a solution of phenyl vinyl sulfone (PVS) was
added (370 mg, 2.2 mmol, 1 equivalent) in dry THF and stirring was
continued at )15 ꢀC for 2.5 h. The reaction was stopped by the
addition of saturated solution of sodium sulfite (15 mL) and the
resulting mixture was extracted into ethyl acetate (3 · 100 mL).
The organic layers were combined and dried over sodium sulfate.
The final purification of product was achieved by flash chromatogra-
phy using petroleum ether and ethyl acetate (4:1) solvent mixtures.
The compound 1 was isolated as a colorless solid whose final char-
acterization was confirmed to be identical to the previously reported
literature data (24).
Synthesis of 2-(p-tolylsulfonyl)oxirane (2)
2-(p-tolylthio)ethanol (Compound 2b – Scheme 1). This compound
was synthesized by a slight modification of an earlier published
protocol (23). To a warm ethanolic solution (6 mL) of 4-methylben-
zenethiol (1 g, 8 mmol) was dissolved potassium hydroxide
(495 mg, 8.8 mmol). 2-Bromoethanol (10.4 mmol, 738 lL) was
added drop wise to this solution while stirring. The solution was
refluxed for 2 h while white mass of potassium bromide depos-
ited. The reaction mixture was cooled to room temperature, and
the reaction mixture was filtered off. The resulting filtrate con-
tained the product, as indicated by the comparison with the litera-
ture reported 1H NMR, and was used in the next step of
synthesis without any purification (25).
2-(p-tolylsulfonyl)ethanol (Compound 2c – Scheme 1). This compound
was synthesized using the same synthetic protocol as outlined in the
synthesis of 2-(4-nitrophenylsulfonyl)ethanol (4c). The chemical shifts
1
for the H and 13C NMR spectra of 2c were found to be identical to
the previously reported literature values (25). Yield: 74%.
Synthesis of 2-(4-nitrophenylsulfonyl)oxirane (4)
2-(4-nitrophenylthio)ethanol (Compound 4b – Scheme 1). This com-
pound was synthesized and purified using earlier published protocol,
1-methyl-4-(vinylsulfonyl)benzene (Compound 2d – Scheme 1). This
compound was synthesized in almost identical manner as
Chem Biol Drug Des 2012
3

Dana et al.
compound 1-nitro-4-(vinylsulfonyl)benzene (4d), except that it
required dry dichloromethane for initial dissolution of 2-(p-toly-
lsulfonyl)ethanol. Yield: 57%. 1H NMR (400 MHz, CDCl3) d ppm:
2.42 (s, 3H), 6.01 (d, J = 9.7 Hz, 1H), 6.40 (d, J = 16.3 Hz, 1H),
6.67 (dd, J = 16.3, 9.7 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.75 (d,
J = 8.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) d ppm: 21.67, 127.24,
128.0, 130.0, 136.55, 138.70, 144.74.
Enzyme kinetics procedure
The PTP enzymes (hPTP1B, vaccinia H1-related phosphatase (VHR),
YopH51* ⁄ D162, and DEP-1) and cysteine protease enzyme papain
were purchased from Enzo Life sciences, Farmingdale, NY, USA. Alka-
line phosphatase, acid phosphatase, and para-nitrophenylphosphate
(pNPP) substrate were purchased from Sigma-Aldrich Incorporated. All
enzymatic assays were performed on a temperature-controlled Lambda
25 spectrophotometer (Perkin Elmer Inc., Waltham, MA, USA). The
assays involving hPTP1B were performed at 30 ꢀC in 100 mM HEPES
buffer (pH 7) containing 1 mM freshly made dithiothreitol (DTT) solu-
tion, 1 mM Na2EDTA, adjusted to an ionic strength 50 mM using
sodium chloride. DMSO concentration in assays was maintained at
5%. At this concentration of DMSO, the enzyme activity was not
affected. A control experiment (No inhibitor) was always carried out
in parallel with PTP inactivation reaction by compounds 1–5 to
ensure that the loss of enzyme activity was not because of thermal
denaturation. Inactivation reactions for all compounds (1–5) with
hPTP1B were performed at 5 mM inhibitory concentrations.
2-(p-tolylsulfonyl)oxirane (2 – Scheme 1). This compound was syn-
thesized using the similar procedure as outlined in the synthesis of
1
2-(phenylsulfonyl)oxirane. Yield: 54%. H NMR (400 MHz, CDCl3) d
ppm: 2.47 (s, 3H), 3.11 (dd, J = 5.5, 3.6 Hz, 1H), 3.40 (dd, J = 5.5,
2.0 Hz, 1H), 4.10 (dd, J = 2.6, 2.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H),
7.83 (d, J = 7.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) d ppm: 21.79,
45.47, 63.33, 128.87, 130.11, 133.76, 145.80. LRMS [ESI-MS +ve]
calculated for (C9H10O3S + Na): 221.2, observed: 221.2.
Synthesis of 2-(4-bromophenylsulfonyl)oxirane
(3)
The following typical procedure was adopted for the hPTP1B inacti-
vation kinetics. The inactivation reaction of hPTP1B with inhibitor
was performed under pseudo-first-order condition ([I]ꢀ[E]) in a
0.5 mL-eppendorf tube maintained at 30 ꢀC in a temperature-con-
trolled bath. The reaction was initiated by the addition of inhibitor
(net 5 mM) to a buffered solution of hPTP1B (net 300 nM). At suit-
able time intervals, an aliquot of 10 lL was withdrawn and added
to an assay mixture (net 300 lL) that contained 10 mM of pNPP
substrate in buffer at 30 ꢀC. A progress curve was recorded at
405 nm, and the enzymatic activity was determined by measuring
the initial rates (usually 0–40s) of pNPP turnover. It was ensured
that the substrate consumption during the measurements of initial
rates was <2%. The initial rates thus obtained were then plotted
2-(4-bromophenyl)thioethanol (3b – Scheme 1). This compound was
synthesized using the same synthetic protocol as outlined in the
synthesis of 2-(p-tolylthio)ethanol (2b) and was used in the next
step of synthesis without any purification. 1H NMR (400 MHz,
CDCl3) d ppm: 3.25 (t, J = 6.0 Hz, 2H), 3.90 (q, 2H), 7.38 (m, 2H)
8.11 (m, 2H).
2-(4-bromophenylsulfonyl)ethanol (3c – Scheme 1). The synthesis of
this was achieved using an earlier published procedures (4c) (23).
Yield: 71%. 1H NMR (400 MHz, CDCl3) d ppm: 3.36 (m, 2H), 4.06
(m, 2H), 7.76 (m, 4H). 13C NMR (100 MHz, CDCl3) d ppm: 56.43,
58.37, 125.58, 129.58, 132.70, 132.84.
against the time of hPTP1B reaction. The data were fitted to
obs ꢂt
1-bromo-4-(vinylsulfonyl)benzene (3d
achieved in almost identical manner as was compound 1-nitro-4-(vi-
nylsulfonyl)benzene (4d). Yield: 53%. H NMR (400 MHz, CDCl3) d
ppm: 6.10 (d, J = 9.5 Hz, 1H), 6.48 (d, J = 16.4 Hz, 1H), 6.66 (dd,
J = 16.4, 9.5 Hz, 1H), 7.73 (m, 4H). 13C NMR (100 MHz, CDCl3) d
ppm: 128.53, 129.06, 129.48, 132.71, 138.07, 138.60.
–
Scheme 1). This was
vt ¼ vf ꢁ ðvf ꢁ voÞeꢁk
using Kaleidagraph software to obtain
the pseudo-first-order inactivation rate constant (kobs), where t is
the time of hPTP1B reaction, vt = initial rate at time 't' of hPTP1B
reaction, vo is the initial rate at zero time of hPTP1B reaction, vf is
the final rate after infinite time of hPTP1B reaction. In the hPTP1B
inactivation experiment involving sodium arsenate as competitive
inhibitor, identical experimental procedure described earlier was
employed with the exception that it included 5 mM of arsenate (26).
To obtain the thermodynamic inhibition constant (Ki) and first-order
inactivation rate constant (kinact) parameters for 1-mediated hPTP1B
inactivation reaction, the following simple two-step model of PTP
inactivation was assumed:
1
2-(4-bromophenylsulfonyl)oxirane (3 – Scheme 1). A similar proce-
dure as utilized in the synthesis of 1 was used. Yield: 41%. 1H
NMR (400 MHz, CDCl3) d ppm: 3.14 (dd, J = 5.6, 3.6 Hz, 1H), 3.44
(dd, J = 5.6, 2.0 Hz, 1H), 4.12 (dd, J = 3.6, 2.0 Hz, 1H), 7.79 (m,
4H). 13C NMR (100 MHz, CDCl3) d ppm: 45.53, 63.22, 130.22,
130.34, 132.85, 135.77. LRMS [ESI-MS +ve] calculated for
(C8H781BrO3S + Na): 286.9; observed: 287.1; (C8H779BrO3S + Na):
284.9, observed: 285.4.
ki
E þ I ¡ E ꢃ I k!inact E ꢁ I:
The pseudo-first-order inactivation rate constants (kobs) of hPTP1B
inactivation reaction at varying concentrations of [1] were obtained
and Kitz–Wilson analysis was performed to obtain Ki and kinact as
described before using the following equation(27):
Synthesis of 2-(methylsulfonyl)oxirane (5)
This compound was synthesized using the similar procedure as out-
lined in the synthesis of 2-(phenylsulfonyl)oxirane (1). This com-
ꢀ
ꢁ
1
1
kobs
Ki
1
1
pound was isolated as viscous oil. Yield: 26%. H NMR (400 MHz,
¼
þ
:
CDCl3) d ppm: 3.01(s, 3H), 3.15 (dd, J = 5.6, 4.0 Hz, 1H), 3.41 (dd,
J = 5.5, 1.9 Hz, 1H), 4.15 (dd, J = 4.0, 1.9 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d ppm: 39.13, 44.85, 62.01; LRMS [ESI-MS +ve]
calculated for (C3H6O3S + Na): 145.0, observed: 145.2.
kinact ½Iꢄ kinact
The time-dependent inactivation experiments involving VHR, DEP-1,
YopH, alkaline and acid phosphatase were conducted in a similar
4
Chem Biol Drug Des 2012

Cell-Permeable Covalent Inhibitors of Protein Tyrosine Phosphatases
manner as described earlier for hPTP1B, except that the following
if a suitable pTyr mimic with appropriately positioned electrophilic
element is incorporated into the inhibitor design. Consistent with
this notion, Park et al. (39) reported aryloxiranes as covalent inacti-
vators of PTPs. Although no kinetic parameters were reported in
their findings, the data suggested a very weak time-dependent inhi-
bition of PTP activity with no evidence of saturation kinetics – even
at inhibitory concentration as high as 20 mM. This perhaps indicates
that the initial binding event (and possibly the covalent modifica-
tion) to PTP is severely compromised by this class of inhibitory
agents. Any higher concentration than 20 mM in their enzymatic
assays resulted into aggregation problems, presumably due to
highly hydrophobic nature of inhibitors. In addition, based on previ-
ously reported structural data of PTP, we reasoned that the oxiranyl
group was perhaps ill-positioned for nucleophilic attack by the
active site Cys residue, therefore accounting for their poor inhibitory
activity (40,41). Interestingly and more recently, Liu et al. (42)
reported PVS as active site directed and mechanism-based inhibitor
of PTPs. More importantly, they delineated the mechanism of inacti-
vation by determining the crystal structure of PVS in complex with
YopH-PTP. This PVS-inhibited YopH-PTP structure displayed a clear
covalent bonding between the Sc of active site Cys residue and the
terminal vinylic carbon of PVS. A closer look at the inhibited com-
plex revealed that two sulfonyl oxygens of PVS interacted very
favorably with YopH-PTP active site, thereby indicating that they
played a crucial role in the binding events leading to covalent modi-
fication. For example, while three hydrogen bonds were observed
with the main-chain NH groups of Gly406, Val407, and Gly408 with
one sulfonyl oxygen, the other sulfonyl oxygen engaged in forming
two hydrogen bonds with the main-chain amides of Gly408 and
Arg409, and with the side chain of Asp356. In addition, the phenyl
group maintained van der Waals interactions with Phe229, Ile232,
Arg404, Ala405, and Val407 residues. On the basis of these obser-
vations, we hypothesized that if an inhibitory moiety such as 2-(a-
rylsulfonyl)oxirane (1) could be developed, it could provide at least
twofold advantage over the previously reported PTP inhibitors, ary-
loxiranes and PVS: (i) superior reactivity while retaining the favor-
able binding interactions of the sulfonyl group with PTP active site
residues, as observed in the complex of PVS and YopH-PTP, and (ii)
enhanced aqueous solubility.
inactivation and assay conditions were used: VHR – 50 mM sodium
succinate buffer (pH = 6.0) containing 0.1 mg ⁄ mL bovine serum
albumin (BSA), 1 mM DTT and 1 mM Na2EDTA and 150 mM sodium
chloride; DEP-1 – 100 mM HEPES buffer (pH 7.0) containing 50 mM
sodium chloride, 0.01 mg ⁄ mL BSA, 1 mM DTT and 1 mM Na2EDTA;
YopH – 100 mM HEPES buffer (pH = 7.0) containing 50 mM sodium
chloride, 1 mM DTT and 1 mM Na2EDTA; Alkaline phosphatase –
500 mM Tris–HCl buffer (pH 9.0) containing 0.5 mM magnesium
chloride; Acid phosphatase – 100 mM sodium acetate buffer (pH
5.0) containing 1 mM Na2EDTA (28–30).
Mass spectrometric analysis
The PTP inactivation reaction was initiated by addition of 1 (5 mM)
to YopH enzyme (300 nM) at 30 ꢀC in appropriate buffer condition
described earlier. Mass spectra were acquired using a Waters Mal-
diMX delayed extraction matrix-assisted laser desorption ⁄ ionization
(MALDI) time-of-flight (TOF) mass spectrometer operating in linear
mode. Samples were diluted in a 1:9 v ⁄ v ratio with a saturated
solution of recrystallized a-cyano-4-hydroxycinnamic acid (Sigma-
Aldrich Inc.) in 50% formic acid ⁄ 33% isopropyl alcohol ⁄ 17% water
(v ⁄ v ⁄ v; Fisher Scientific – all HPLC grade or higher) and prepared
for MALDI TOF mass spectrometry using the ultra-thin method
essentially as described before (31,32). External calibration was
achieved using the +1 and +4 charge states of unmodified YopH
enzyme.
Western blot analysis
Human A549 lung adenocarcinoma cells were grown using standard
technique. DMEM media supplemented with 10% Fetal Bovine
Serum and Penicillin ⁄ Streptomycin solution was used to culture
cells. Cells were grown in 75-cm2 sterile polystyrene culture flasks
to 80% confluency, trypsinized, and re-seeded in equal aliquots into
6-well plates. After 2 days and ꢅ80% confluency, media were
removed and replaced with DMSO or drug containing media. Cells
were allowed to grow another 5 h after which cells were trypsi-
nized and subjected to western analysis. For this analysis, cell
media were removed from wells and RIPA buffer, supplemented
with protease inhibitor cocktail CompleteR (Boehringer Mannheim,
Irvin, CA, USA) and phosphatase inhibitor was used to lyse cells.
Lysate was centrifuged and total protein in the cellular extract was
quantitated using Bradford Assay reagent from Bio-Rad Incorpo-
rated, Hercules, CA, USA. Ten micrograms of total protein was
loaded onto each lane and resolved by standard SDS-PAGE and
blotted onto polyvinylidene fluoride (PVDF) membranes. Western
analysis was carried out using standard techniques employing pTyr
primary antibody (Cell Signaling Technologies, Danvers, MA, USA).
To test our hypothesis, we first turned to molecular modeling stud-
ies using mammalian enzyme hPTP1B. This step was important
because the structural analysis of inhibitory complex of PVS was
performed with YopH- a PTP derived from Yersinia pestis (42).
Although YopH is considered to be a prototypical PTP, its catalytic
domain shares only 20–30% sequence identity with mammalian
PTPs (e.g. PTP1B) (43,44). Thus, the molecular models of 2-(phen-
ylsulfonyl)oxirane (1) covalently bound to hPTP1B enzyme was con-
structed, as described in Methods and materials section. Molecular
dynamics simulations were performed and the energy-minimized
structures of the most stable conformers (as determined by the
total energy criteria) were analyzed (Figure 1). The analysis of the
1-hPTP1B inhibitory complex, as shown in Figure 1, clearly indicated
that 1 interacts favorably with many key residues of hPTP1B active
site. A relatively stable H-bonding interaction between e-amino
group of Lys120 and the sulfonyl oxygen atom of 1 is observed.
This is an interesting observation because the sequence alignments
of various mammalian PTP enzymes have revealed that most PTPs
Results and Discussion
Design guided by molecular modeling studies
There are numerous precedence indicating that oxiranes are impor-
tant class of biologically active compounds (33–38). As PTPs utilize
nucleophilic chemistry involving a cysteine residue in turning over
their pTyr substrates, they could in principle be covalently inhibited
Chem Biol Drug Des 2012
5

Dana et al.
Figure 1: Stereoview images of
the energy-minimized inhibitory
complexes formed between the 2-
(phenylsulfonyl)oxirane (1) and
hPTP1B enzyme. The hydrogen-
bonding distances and interactions
are shown in red; only key active
site residues and heavy atoms are
displayed for clarity.
harbor a Lys or Arg residue at this position, indicating an important
catalytic role for this residue (45). Indeed, in a crystal structure of
insulin receptor tyrosine kinase (IRTK) in complex with hPTP1B, the
Lys120 has been shown to be involved in a salt bridge interaction
with the IRTK (46). In addition, a bi-furcated hydrogen-bonding inter-
action, with one being water-mediated, between the hydroxyl moi-
ety of 1 and the backbone amides of key P-loop residues, Arg221
and Gly220 is observed. It is also noticeable that the P- loop com-
prised of 215–221 residues is fairly stable in the inhibitory complex,
as a result of internal backbone H-bonding interaction between the
carbonyl oxygen of Ala217 and the amide nitrogen of Ile219. The
stability of the P-loop is further enhanced through a H-bond
between the OH groups of Ser216 and Tyr46 residues. Finally, an
edge-to-face aromatic interaction is evident between the phenyl
groups of 1 and Tyr46. Such aromatic–aromatic interactions are
commonly observed in stable proteins and protein ⁄ ligand complexes
and could contribute favorably to binding by as much as 1–2 kcal ⁄
mol (47,48). A favorable outcome of our molecular modeling studies
involving 1-hPTP1B complex prompted us to proceed with the syn-
thesis of compounds 1–4. Compound 5, which showed no favor-
able interactions with any of the active site PTP residues in our
molecular modeling studies, was synthesized as a negative control.
Figure 2: Chemical structures of synthesized 2-(arylsulfonyl)oxir-
anes (1–4) and 2-(methylsulfonyl)oxirane (5).
binding constant (Ki) of 0.18 € 0.01 min)1 and 7.2 € 0.1 mM,
respectively. Although the kinact value for the previously reported
PTP inactivator PVS with hPTP1B is not available for direct compari-
son, it appears that pseudo-first-order rate constant (kobs) of hPTP1B
inactivation by 1 is about 1.5-fold higher than phenyl vinylsulfonate
under identical experimental conditions (see Table 1) (42). A value
of 7.2 mM for the thermodynamic binding constant (Ki) of 1 with
hPTP1B is consistent with the binding potency of pTyr with hPTP1B
(Km = 5 mM) because 1 could be considered to be a ground state
substrate analog (49).
Kinetic analysis, structure-activity relationship,
and selectivity of PTP inhibition
Prompted by our molecular modeling studies, we synthesized a
small panel of sulfonyloxiranyl derivatives (Figure 2: compounds
1–5) and evaluated them as inhibitors of PTPs using pNPP as a
substrate. The incubation of 1 with hPTP1B resulted in a time-
dependent loss of hPTP1B activity when followed under a pseudo-
first-order kinetics conditions ([1]ꢀ[hPTP1B]). The inhibition pattern
was consistent with the covalent nature of inactivation, because
return of hPTP1B activity was not observed even after a large dilu-
tion of inactivated hPTP1B mixture (data not shown). Further investi-
gation showed that the inactivation of hPTP1B was both time- and
concentration-dependent. This data could most simply be fitted to a
scheme in which 1 initially binds reversibly to active PTP1B, and in
the following step, covalent inhibition of PTP1B occurs through
proper alignment of electrophilic center of the inhibitor 1. The
pseudo-first-order rate constant (kobs) of hPTP1B inactivation reac-
tion was determined at varying concentration of 1 and Kitz–Wilson
analysis of inactivation data was performed as described in the
Methods and materials section (Figure 3) (27,29). This analysis pro-
vided the inactivation rate constant (kinact) and the thermodynamic
As covalent inhibition of hPTP1B could also, in principle, result from
the modification of non-active site residues, a competition experi-
ment was performed to rule out this possibility. Thus, the hPTP1B
inactivation reaction was performed with 1 in the presence of arse-
nate – a known competitive inhibitor of PTP1B (26). A protection
from a time-dependent loss of hPTP1B activity in the presence of
arsenate indicated that the enzyme was partially protected from
covalent inhibition of 1, and therefore, the covalent inhibition must
be active site bound (Figure 3C). Mass spectrometry-based experi-
ments are also consistent with this observation. To demonstrate the
covalent nature of PTP inhibition by 1, Yersinia Outer Protein H
(YopH) PTP was chosen for MS analysis. As the MS analysis of 1
in complex with hPTP1B led to a poor quality MS signal, we
decided to perform this analysis with YopH-PTP. Although the exact
6
Chem Biol Drug Des 2012

Cell-Permeable Covalent Inhibitors of Protein Tyrosine Phosphatases
A
B
C
D
Figure 3: (A) A plot of time- and concentration-dependent inactivation of hPTP1B by 2-(phenylsulfonyl)oxirane (1). The experimental points are
represented by various symbols and the lines connecting them are the fitted line to obtain the kobs values at various concentration of 1. (B) Kitz–
Wilson analysis of 1-mediated inactivation reaction of hPTP1B. This analysis yielded the inactivation rate constant (kinact) and the thermodynamic
binding constant (Ki) of 0.18 € 0.01 per min and 7.2 € 0.1 mM respectively. (C). Protection of hPTP1B activity from covalent inactivation by 2-(phen-
ylsulfonyl)oxirane (1) in the absence (circle) and presence (square) of arsenate (5 mM), a known active site bound competitive inhibitor. This
experiment indicates that the inactivation of hPTP1B by 1 is active site directed. (D) Mass spectrometric analysis of the covalent modification of
YopH-PTP by 1. YopH-PTP inactivation reaction with 1 was monitored over a time course of 3 h (doubly protonated, +2 charge state shown here).
A mass shift of 180 amu is observed that is consistent with the covalent modification of PTP by 1. PTP, protein tyrosine phosphatases.
Table 1: Summary of the pseudo-first-order rate constants (kobs
)
reason of this remains unknown at this point, YopH-PTP has been
traditionally used in many instances to demonstrate a covalent nat-
ure of PTP modification by other active site bound irreversible inhib-
itors (20,29,42). A time-dependent shift in molecular weight was
observed using MALDI-TOF mass spectrometry upon incubation of
YopH PTP with 1. Before the addition of inhibitor, the molecular ion
of YopH was observed to have a molecular weight of 33 476 Da
(theoretical 33 479 Da). After 3 h, a shift of 180 (theoretical
184 Da) is observed, indicating covalent modification of the YopH
phosphatase by compound 1 (Figure 3D). These data taken together
indicate that 2-(arylsulfonyl)oxirane is an active site directed cova-
lent inhibitor of PTP.
for time-dependent inactivation of (A) hPTP1B by 5 mM 2-(sulfo-
nyl)oxiranes (1–5). (B) receptor type PTP DEP-1, dual-specific PTP
VHR, and classical-type intracellular PTP YopH, by 1 (5 mM)
(A)
Inhibitory compounds
(kobs) (per min)
1
2
3
4
5
0.068 € 0.009
0.060 € 0.008
0.067 € 0.009
0.104 € 0.022
No inhibitiona
(B)
To assess how substituents at the phenyl ring might affect enzy-
matic reactivity of hPTP1B active site, we synthesized para-substi-
tuted derivatives of 1 with the methyl, bromo, and nitro functional
groups. The compound 5 was synthesized to serve as a negative
control, based on our molecular modeling studies (data not shown).
To understand what a preferred transition state of PTP inactivation
by 2-(arylsulfonyl)oxiranes (1–4) might be, we determined the
pseudo-first-order inactivation rate constants (kobs) for substituents
bearing the methyl, bromo, and nitro groups at the phenyl ring
Enzyme
(kobs) (per min)
DEP-1
VHR
YopH
0.024 € 0.005
0.031 € 0.006
0.059 € 0.006
PTP, protein tyrosine phosphatase; VHR, vaccinia H1-related phosphatase.
aNo time-dependent inactivation of hPTP1B enzyme after 60 min of reaction
at 15 mM of 5.
Chem Biol Drug Des 2012
7

Dana et al.
(Table 1A). These studies revealed that the reactivities of 2-(p-toly-
lsulfonyl)oxirane (2) and 2-(p-bromophenylsulfonyl)oxirane (3) were
very similar to 1, suggesting that the presence of either electron-
neutral methyl or electron-withdrawing bromo-substituents at the
para-position of the phenyl ring have no significant influence on the
inhibitory activity of this class of compounds. Further, the presence
of strong electron-withdrawing, nitro group in 4 was found to be
only modestly more reactive to hPTP1B than 1. Finally, the lack of
reactivity of a non-aryl analog, 2-(methylsulfonyl)oxirane (5) with
hPTP1B indicated that an aryl moiety is required for effective hPTP
inhibition. This result, also supported by our modeling studies, was
consistent with the earlier finding that an aryl group played an
important role in binding and positioning of the catalytic groups for
the initiation of the active site chemistry (50).
line phosphatase and potato acid phosphatase (Sigma-Aldrich Inc.)
were first chosen because both, like PTPs, efficiently hydrolyze aryl
phosphates and utilize a nucleophilic residue (a serine and a histi-
dine residue, respectively) to initiate the catalytic turnover process
(54,55). Incubation of a large excess of 1 (5 mM) with alkaline
phosphatase and potato acid phosphatase did not result into any
loss of their enzymatic activities, indicating that 2-(arylsulfonyl)oxira-
ne is selective toward PTPs and perhaps the presence of a low pKa
active site Cys residue is essential for inactivation. Finally, to test
whether 1 was resistant to the presence of external nucleophile,
we carried out hPTP1B inactivation reaction in the presence of an
external nucleophile azide ion (1 mM). The hPTP1B inactivation rate
was unaffected by the presence of strong azide nucleophile,
thereby indicating that 1 remained un-reactive toward external
nucleophile such as sodium azide (data not shown). Finally, because
peptidyl epoxides are known cysteine protease inhibitors, we sought
to test whether 2-(arylsulfonyl)oxirane would inactivate a protypical
cysteine protease (56). When 1 was incubated with papain (Sigma-
Aldrich Inc.), a time-dependent loss of enzymatic activity was
observed (data not shown). Although not a desirable outcome, this
result is perhaps not too surprising because cysteine proteases also
utilize a low pKa cysteine thiolate nucleophile to initiate the turn-
over of their peptidyl substrates (57). Suitable chemical modifica-
tions of 2-(arylsulfonyl)oxiranyl moiety could be performed to
generate specific PTP inhibitors that otherwise would not be inhibi-
tory toward cysteine proteases.
In addition to classical intracellular cytosolic type PTPs (e.g. hPTP1B
and YopH), PTP enzyme superfamily has two other subclasses of
enzymes: Receptor-like PTPs (RPTPs) and Dual-specific PTPs (DSPs).
Whereas the RPTPs can only dephosphorylate pTyr residues in their
intracellular phosphatase catalytic domain, the DSPs are unusal in
that they can dephosphorylate both pSer ⁄ Thr and pTyr residues
(51,52). Although all members of PTP superfamily contain signature
active site sequence, C(X5)R(S ⁄ T), and employ a common catalytic
mechanism, they differ considerably in their cellular localization and
substrate preferences (53). As our inhibition data with hPTP1B indi-
cated that 2-(arylsulfonyl)oxiranes are active site directed, and
therefore presumably work by utilizing the conserved active site cat-
alytic machinery, we reasoned that they might also display time-
dependent inhibition pattern with RPTPs and DSPs. To test this
hypothesis, we performed kinetic experiments with a prototypical
RPTP, density-enhanced phosphatase-1 (DEP-1), and a DSP, VHR.
Indeed, a time-dependent inactivation of both DEP-1 and VHR was
observed upon incubation with 1 (Table 1B). A direct comparison of
pseudo-first-order inactivation rate constant (kobs) of 1 with hPTP1B
and VHR indicates that 1 is approximately 2-fold more selective
toward classical intracellular PTP, hPTP1B as compared to VHR. This
is in sharp contrast to previously reported 1-(vinylsulfonyl)benzene
PTP inactivator that was 10-fold more selective toward DSP, and
VHR when compared to hPTP1B (42).
Although a precise investigation of the inhibitory interactions
between the 2-(arylsulfonyl)oxiranyl species and PTP may require
crystallographic analysis, a plausible inhibitory mechanism is pro-
posed based on our current findings and earlier precedence (Fig-
ure 4) (42). This perhaps involves an initial binding step that
follows a direct nucleophilic attack of the low pKa active site Cys
residue at the sterically accessible 3-position of the aryloxiranyl
moiety, thus leading toward the formation of a non-labile carbon-
sulfur bond between the inhibitor and hPTP1B enzyme.
Cell-based studies
Intracellular signaling pathways constitute an intricate network of
phosphorylation-dependent cascades that transmit signals from the
cell surface to the nucleus to affect programs of gene expression
Next, we sought to investigate the selectivity of 2-(arylsulfonyl)oxir-
anes toward the PTP enzyme superfamily. To demonstrate this, alka-
Figure 4: Proposed mechanism
of protein tyrosine phosphatase
(PTP) inactivation by 2-(arylsulfo-
nyl)oxiranes.
8
Chem Biol Drug Des 2012

Cell-Permeable Covalent Inhibitors of Protein Tyrosine Phosphatases
Acknowledgments
The authors wish to thank Aparna Chimalamarri, Jyotsna Samarla,
Dongyue Niu, and Shanti Pabbathi for their help with organic syn-
thesis, Julio C. Padovan for support in mass spectrometry-based
experiment, Dr. Gopal Subramaniam for help with NMR experi-
ments, and Christine Tada for proofreading the final manuscript.
The funding for this research was provided by Queens College
Research Enhancement Award Committee, and Professional Staff
Congress-CUNY award number 63226-00 41.
Figure 5: Western blot analysis of pTyr level after inhibitory
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