Protection of a single-cysteine redox switch from oxidative destruction: On the functional role of sulfenyl amide formation in the redox-regulated enzyme PTP1B

Article abstract of DOI:10.1016/j.bmcl.2009.12.001

Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this enzyme against irreversible oxidative destruction. The results suggest that 'overoxidation' of the sulfenyl amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.

Full text of DOI:10.1016/j.bmcl.2009.12.001

Bioorganic & Medicinal Chemistry Letters 20 (2010) 444–447
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Bioorganic & Medicinal Chemistry Letters
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Protection of a single-cysteine redox switch from oxidative destruction:
On the functional role of sulfenyl amide formation in the redox-regulated
enzyme PTP1B
*
Santhosh Sivaramakrishnan, Andrea H. Cummings, Kent S. Gates
Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active
site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this
enzyme against irreversible oxidative destruction. The results suggest that ‘overoxidation’ of the sulfenyl
amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl
amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 7 November 2009
Revised 1 December 2009
Accepted 2 December 2009
Available online 4 December 2009
Keywords:
Protein tyrosine phosphatase
Redox regulation
Sulfenic acid
Intracellular concentrations of hydrogen peroxide (H2O2) in-
crease under conditions of oxidative stress and during some nor-
mal signal transduction processes.1–3 An important mechanism
by which cells issue temporary responses to such transitory in-
creases in H2O2 levels involves reversible oxidation of cysteine res-
idues on critical ‘sensor’ proteins.4,5 The ability of cysteine residues
to serve as reversible redox switches relies upon the unique ability
iii, Scheme 1B).10,11 Second, if ‘overoxidation’ does occur, the
resulting thiosulfinate likely could be converted cleanly back to
the native cysteine residues by reactions with biological thiols
(reaction v, Scheme 1B).14
Protein tyrosine phosphatases (PTPs) are important targets of
intracellular H2O2.15–17 These cysteine-dependent enzymes cata-
lyze the removal of phosphoryl groups from tyrosine residues on
their protein substrates.15–17 Accordingly, PTPs work in tandem
with protein tyrosine kinases to regulate critical signal transduc-
tion cascades by modulating the phosphorylation status and, in
turn, the functional properties of proteins involved in these path-
ways.15–17 The catalytic activity of some PTPs is subject to redox
of the c–sulfur atom in this amino acid to cycle easily between (at
least) two oxidation states under physiological conditions. Specifi-
cally, oxidation of a cysteine thiol by H2O2 yields a sulfenic acid
residue (reaction i, Scheme 1A) that can, over time, be returned
to the native thiol by reactions with biological thiols (reaction ii,
Scheme 1A).4–8
Protein sulfenic acid residues also have the potential to undergo
further reaction with hydrogen peroxide to generate the corre-
sponding sulfinic acid (reaction iii, Scheme 1A).4–9 This reaction
is irreversible, except in the case of some peroxiredoxins8 and,
therefore, yields an overoxidized, ‘broken’ redox switch. Alterna-
tively, in some proteins, the initially-formed sulfenic acid interme-
diate undergoes reaction with a neighboring ‘back door’ cysteine
thiol to generate a disulfide linkage (reaction ii, Scheme 1B).10–13
Rudolph and Sohn provided evidence that, at least in the context
of the phosphatase Cdc25B, disulfide formation protects the en-
zyme against irreversible overoxidation.10,11 There are at least
two possible mechanisms underlying this protection. First, the
disulfide may be relatively resistant to further oxidation (reaction
native
sulfenic
acid
sulfinic
acid
i
iii
A
B
cysteine
H2O2
H2O2
2 RSH
S
S
SH
O
OH
OH
ii
i
H2O2
SH S
SH SH
OH
v
ii
4 RSH
iv
S
S
H2O2
iii
S
S
O
thiosulfinate
disulfide
* Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754.
E-mail address: gatesk@missouri.edu (K.S. Gates).
Scheme 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.001

S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 444–447
445
regulation as part of normal cell signaling processes.15–17 For
O
CO2Et
or
example, the enzyme PTP1B, a major negative regulator of the
insulin signaling pathway, is inactivated by a burst of H2O2 that
is produced upon binding of insulin to its cell-surface receptor.18,19
Subsequent reactions with cellular thiols slowly return the enzyme
to its active form.18,19 This transient oxidative inactivation of
PTP1B serves as a ‘timing device’ that increases phosphorylation
levels on the insulin receptor and insulin receptor substrates, thus
potentiating cellular responses for a defined period of time follow-
ing insulin stimulation.18,19
O
N
O
CO2Et
S
CO2Et
O
O
N
O
N
S
2
S
O
O
1
3
Scheme 3.
For some time, it was widely assumed that redox regulation of
PTPs involved either sulfenic acid or disulfide intermediates, as
shown in Scheme 1. However, recent studies in the context of
PTP1B revealed a new mechanism for redox regulation of PTP
activity in which the initially-formed sulfenic acid undergoes reac-
tion with the neighboring amide nitrogen to yield a cyclic sulfenyl
amide (known, more formally, as an isothiazolidin-3-one, reaction
ii, Scheme 2).20–22 As required for a functional redox switch, reac-
tions with biological thiols can convert the sulfenyl amide back to
the catalytically active thiol form of the enzyme (reaction iii,
Scheme 2).20–22 Importantly, sulfenyl amide formation subse-
quently has been observed in other proteins and it has been sug-
gested that this posttranslational cysteine modification can occur
in cells.23,24
O
O
O
R
R
N
H
R'SH
R'SH
R'SH
N
N
R
H
S
SR'
S
O
S OH
O
2
R'SH
O
O
R = CH2CO2Et
R' = CH2CH2OH
R
R
N
H
N
H
SR'
SH
S
5
4
Scheme 4.
Like other cysteine-based redox switches, the sulfenyl amide
residue has the potential to undergo ‘overoxidation’ to sulfinyl
and sulfonyl derivatives (reactions iv and vii, Scheme 2). Therefore,
complete understanding of this redox switch requires consider-
ation of the reactivity of these higher oxidation states under phys-
iologically relevant conditions. In the work described here we
employed small organic molecules to model the reactivity of pro-
tein sulfinyl and sulfonyl amides that are potential ‘overoxidation’
products of the sulfenyl amide redox switch. Classic25–28 and mod-
ern29,30 studies have employed model compounds to define the
inherent reactivity of critical functional groups found at enzyme
active sites. Such studies provide insight regarding the chemical
mechanisms of enzyme catalysis and lay a foundation for under-
standing how protein microenvironments alter inherent organic
reactivities to achieve observed enzyme function.
(300 mM, pH 7) for 2 h gave the thiol derivative 5 in high isolated
yield (85%).32 Similar results were obtained when 2 was mixed
with thiol in methylene chloride containing catalytic amounts of
triethylamine as a base. These reactions presumably proceed via
the thiosulfinate, sulfenic acid, and disulfide intermediates shown
in Scheme 4.33 Consistent with this idea, HPLC analysis at early
times in the reaction between 2 (50
lM) and 2-mercaptoethanol
(500 M) in sodium phosphate buffer (50 mM, pH 7, containing
l
30% acetonitrile by volume) revealed an intermediate whose reten-
tion time and mass match that of the disulfide 4 at m/z 316 for
(M+H)+ (Fig. 1). Further, when the reaction was conducted using
only two equivalents of thiol, HPLC analysis showed the disulfide
to be a major final product alongside unreacted starting material
and 5. The thiol, 2-mercaptoethanol, is converted to the corre-
sponding disulfide in this reaction.
The present studies build upon our previous use of compound 1
to model the reactivity of the protein sulfenyl amide residue found
in PTP1B.22 For these studies, it may be important that the pKa of
the corresponding thiol (5) and, thus, the leaving group ability of
the sulfur residue in 1 resembles that of the catalytic cysteine res-
idue in PTP enzymes.22
The ‘overoxidized’ sulfenyl amide model compounds 2 and 3 for
use in the present studies were prepared by oxidation of 122 with
dimethyldioxirane (Scheme 3).31 We first examined the reactivity
of the sulfinyl amide 2 with thiol. Incubation of 2 (10 mM) with
2-mercaptoethanol (150 mM) in aqueous sodium phosphate buffer
The reaction of 2 (50 lM) with excess thiol (1 mM) in sodium
phosphate buffer (50 mM, pH 7, containing 50% acetonitrile by vol-
ume) occurs with a pseudo-first-order rate constant of 5.5 0.2 Â
10À3 sÀ1 (t1/2 ꢀ 2 min). This corresponds to an apparent second-or-
der rate constant of 5.5 0.2 MÀ1sÀ1 (Fig. 2). These results offer the
prediction that ‘overoxidation’ of the sulfenyl amide redox switch
to the sulfinyl amide in proteins is a chemically reversible event, be-
cause the sulfinyl amide can be easily returned to the native cys-
teine thiol residue via reactions with thiols (reaction v, Scheme 2).
In the absence of thiol, the sulfinyl amide 2 undergoes a rela-
tively slow reaction with water to yield the corresponding sulfinic
acid derivative 6 in 88% yield (Scheme 5, aqueous sodium phos-
phate buffer, 50 mM, pH 7, 12 h, 24ꢀC).34 Compound 6 was charac-
terized as its methyl ester derivative 7 following treatment of the
reaction mixture with excess methyl iodide.35 The hydrolysis of 2
native cysteine
sulfinyl amide
O
sulfinic acid
O
in active PTP1B
O
(50 lM) in sodium phosphate buffer (50 mM, pH 7, containing
4 RSH
H2O
NH
NH
50% acetonitrile by volume) occurs with a pseudo-first-order rate
constant of 4.5 0.2 Â 10À4 sÀ1, corresponding to a half-life of
26 min (Fig. 3). In contrast, the parent sulfenyl amide 1 is stable
under these conditions (no significant decomposition observed
over 24 h). These results forecast that protein sulfinyl amide resi-
dues can undergo chemically irreversible hydrolysis to the sulfinic
acid (reaction vi, Scheme 2); however, rate measurements in the
context of this model compound suggest that if water and physio-
logical concentrations of thiol (1–10 mM) enjoy equal access to the
sulfinyl amide, thiol-mediated recovery of enzyme activity will be
approximately 10–100 times faster than irreversible loss of activity
..
N
S
O
..
v
vi
–
SO H
2
S
i
H2O2
H2O2
iv
iii
O
O
S
O
vii
–H2O
NH
..
N
S
O
N
S
ii
O
OH
sulfenic acid
sulfenyl amide
sulfonyl amide
Scheme 2.

446
S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 444–447
0
0
2
4
6
8
10
-1
-2
-3
time (min)
Figure 2. A representative plot for the disappearance of 2 in the presence of thiol.
Compound 2 (2.5 L of a 10 mM stock in CH3CN) was added to a mixture containing
sodium phosphate buffer (50 L, 500 mM, pH 7.0), water (150 L), 2-mercap-
toethanol (50 L of a 10 mM stock) and acetonitrile (247.5 L) at 25 °C. The mixture
(final concentrations: 2, 50 M; buffer, 50 mM, pH 7.0; thiol, 1 mM; acetonitrile,
l
l
l
l
l
l
50% by volume) was vortex mixed and the disappearance of 2 (a is the peak area at
time = t and a0 is the peak area at time = 0) monitored by reverse phase HPLC at
regular time intervals as described in the legend for Figure 1. From the slope of the
plot, a pseudo-first-order rate constant of 5.5 Â 10À3 sÀ1 (t1/2 = 2 min) at 1 mM thiol
was obtained. This corresponds to an apparent second-order rate constant of
5.5 0.2 MÀ1 sÀ1
.
O
O
CO2Et
H2O
pH 7
CO2Et
N
H
N
S
OH
S
O
O
2
6
Scheme 5.
Figure 1. Reaction of 2 with 10 equiv 2-mercaptoethanol in aqueous buffer: (A)
Compound 2 alone in buffer, (B) 1 min after addition of thiol, (C) 25 min after
0.5
addition of thiol. Compound 2 (2.5
mixture containing sodium phosphate buffer (50
(275 L), 2-mercaptoethanol (25 L of a 10 mM stock in water), and acetonitrile
(147.5 L) at 25 °C (final concentrations: 2, 50 M; buffer, 50 mM, pH 7.0; thiol,
500 M; acetonitrile, 30% by volume). The disappearance of 2 was monitored at
254 nm. Aliquots (40 L) from the reaction mixture were injected onto a C-18
Varian Microsorb–MV column, 100 Å sphere size, 5 m pore size, 25 cm length,
lL of a 10 mM stock in CH3CN) was added to a
lL, 500 mM, pH 7.0), water
l
l
0
20
40
60
80
100
l
l
-0.5
l
l
l
4.6 mm i.d. eluted with a solvent system composed of water with 0.5% acetic acid
v/v (A) and acetonitrile (B), at a flow rate of 0.8 mL/min. The column was eluted
with 70:30 A/B for 4 min and then ramped to 50:50 A/B over 4 min, held at 50:50 A/
B for 7 min, and then ramped back to 70:30 A/B over the next 3 min. The peak at
10.2 min was identified as the mixed disulfide 4. The identity of 4 was confirmed by
co-injection with an authentic standard22 and by LC/MS analysis which showed that
the compound displays an m/z of 316 corresponding to that expected for the [M+H]+
ion of 4. The early peaks in the chromatogram correspond to buffer salts, 2-
mercaptoethanol, and the disulfide of 2-mercaptoethanol.
-1.5
-2.5
time (min)
Figure 3. Representative plot for the disappearance of 2 in the absence of thiol.
Compound 2 (2.5 L of a 10 mM stock in CH3CN) was incubated at 25 °C in a
solution composed of sodium phosphate buffer (50 L, 500 mM, pH 7.0), water
(200 L) and acetonitrile (247.5 L). The mixture (final concentrations: 2, 50 M;
l
l
l
l
l
due to hydrolysis. It is interesting to note that thiosulfinates may
be similarly labile to hydrolysis.36,37
buffer, 50 mM, pH 7.0; acetonitrile, 50% by volume) was vortex mixed and the
disappearance of compound 2 (a is the peak area at time = t and a0 is the peak area
at time = 0) analyzed by reverse phase HPLC as described in the legend of Figure 1.
The pseudo-first-order rate constant of 0.027 0.001 minÀ1 was obtained from the
slope of the plot. This corresponds to a half-life of 26 min for the hydrolysis of 2
under these conditions.
Finally, we examined the reactivity of the sulfonyl amide 3.38
HPLC analysis revealed that this compound is stable in aqueous so-
dium phosphate buffer (50 mM, pH 7, containing 40% acetonitrile
by volume) either in the presence or absence of the thiol, 2-
mercaptoethanol. This suggests that exhaustive oxidation of the
sulfenyl amide to the sulfonyl amide (Scheme 2, reaction vii) likely
represents chemically irreversible destruction of the sulfenyl
amide redox switch.
readily can be resolved by reactions with biological thiols to regen-
erate the native cysteine residues (Scheme 6). Thus, irreversible
oxidative destruction of these switches requires conversion to
the sulfonyl derivatives which presumably requires relatively
harsh oxidizing conditions (Scheme 6). In contrast, a sulfenic acid
redox switch has no failsafe mechanism. The sulfenic acid group
is prone to further oxidation4–9 and, in this case, conversion to
the sulfinyl oxidation state represents irreversible destruction of
the redox switch.
In conclusion, these studies offer chemical insight regarding
possible functional roles of sulfenyl amide formation in redox-
switched proteins. Sulfenyl amide formation has the potential to
protect a single-cysteine redox switch against irreversible overox-
idation in much the same way that disulfide formation protects
dithiol redox switches from oxidative destruction. In both cases,
further oxidation to the sulfinyl form yields an intermediate that

S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 444–447
447
(5 mL) was added to a dry flask under an atmosphere of nitrogen. To this was
added triethylamine (10 mol %, 14
L) and ethyl bromoacetate (1.2 equiv,
133 L) and the mixture was allowed to stir at room temperature for 2 h. The
reaction was monitored by thin layer chromatography and additional aliquots
of triethylamine (14 L) were added every 2 h until all starting material was
consumed. When the reaction was complete, the mixture was dried by rotary
evaporation, the residue taken up in water (15 mL), and the pH adjusted to 9.
This mixture was extracted with ethyl acetate (3 Â 15 mL), the combined
organic layers dried over sodium sulfate, filtered and evaporated under reduced
pressure. Purification by column chromatography (95:5, hexane:ethyl acetate)
afforded the desired compound 1 as an off-white solid (67%, 160 mg). All
spectral data for this material matched that reported previously.22 Synthesis of
THIOL
SULFENYL
SULFINYL
SULFONYL
l
O
O
O
O
l
H2O2
H2O2
H2O2
NH
..
S–
N
S
O
N
S
N
S
l
O
O
thiol
thiol
NOT thiol
recoverable
recoverable
recoverable
H2O2
H2O2
H2O2
S
S
S
S
S
S
O
SH SH
O
(via
O
ethyl 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetate 1-oxide (2). To
a stirred
sulfenic
acid)
thiol
thiol
NOT thiol
solution of 1 (20 mg, 0.085 mmol) in dry acetone (2 mL) was added a cold
recoverable
recoverable (ref 36)
recoverable (ref 36)
solution of dimethyldioxirane22 in acetone (approximately 3 mL dropwise from
a
Pasteur pipette). After about 1 min the reaction was dried by rotary
evaporation, redissolved in CDCl3, and analyzed by 1H NMR. If necessary, the
mixture was dried, redissolved in dry acetone, and an additional aliquot of cold
dimethyldioxirane sufficient to complete the reaction was added. The acetone
and any remaining DMD was then evaporated under reduced pressure to yield 2
as colorless oil (25 mg, 94%). Rf = 0.38 (3:2 hexane/ethyl acetate). 1H NMR
(CDCl3, 300 MHz) d 1.30 (3H, t, J = 7 Hz), 4.25 (2H, q, J = 7.2 Hz), 4.37 (1H, d,
J = 18 Hz), 4.80 (1H, d, J = 18 Hz), 7.81 (1H, t, J = 8 Hz), 7.87 (1H, t, J = 8 Hz), 7.97
(1H, d, J = 8 Hz); 8.07 (1H, d, J = 8 Hz); 13C-NMR (CDCl3, 75.47 MHz) d 167.64,
165.18, 145.90, 134.45, 133.25, 127.55, 126.36, 125.25, 62.03, 41.24, 14.01.
HRMS (ESI) calcd for C11H12NO4S [M+H]+ 254.0487, found 254.0493.
H2O2
H2O2
H2O2
SO3H
S
S
SH
O
OH
OH
thiol
NOT thiol
NOT thiol
recoverable
recoverable
recoverable
Scheme 6.
Acknowledgements
32. Generation of 2-(2-mercaptobenzamido)acetate (5) by reaction of compound 2
with 2-mercaptoethanol in aqueous buffer. To a stirred solution of 2 (15 mg,
We thank the National Institutes of Health for financial support
of this work (CA 83925 and CA 119131).
0.059 mmol) in acetonitrile (1.78 mL) was added
phosphate buffer (3.56 mL, 500 mM, pH 7.0), water (538
mercaptoethanol (62.2 of 14.3 M solution in water). The resulting
a
mixture of sodium
lL) and 2-
lL
a
colorless solution was stirred at 25 °C (final concentrations: 2, 10 mM;
buffer, 300 mM, pH 7.0; thiol, 150 mM; acetonitrile, 30% by volume). The
starting material disappeared in less than 5 min as indicated by the TLC. The
reaction mixture was stirred for further 2 h and acidified to pH 2 using dilute
hydrochloric acid. The aqueous solution was extracted with diethyl ether
(3 Â 5 mL), the combined organic extract washed with water, dried over
anhydrous sodium sulfate, filtered, and then evaporated to yield a colorless oil
that was purified by flash column chromatography (7:3 ethyl acetate/hexane)
to obtain 5 (11 mg, 80%). Rf = 0.61 (7:3 ethyl acetate/hexane). 1H NMR (CDCl3,
300 MHz) d 1.32 (3H, t, J = 7 Hz), 4.24 (4H, m), 4.74 (1H, s), 6.57 (1H, s, br), 7.17
(1H, m), 7.31 (2H, m), 7.53 (1H, dd, J = 7.6 Hz, 1.0 Hz); 13C NMR (CDCl3,
75.4 MHz) d 169.79, 168.45, 133.37, 132.16, 131.07, 130.94, 128.12, 125.21,
61.76, 41.83, 14.14. HRMS (ESI) calcd for C11H14NO3S [M+H]+ 240.0694, found
240.0706.
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l
l
l
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31. Synthesis of ethyl 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetate (1). A solution
of 1,2-benzisothiazol-3-one (Aldrich, 1 mmol, 150 mg) in methylene chloride
37. Nagy, P.; Ashby, M. T. Chem. Res. Toxicol. 2007, 20, 1364.
38. Synthesis of ethyl 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetate 1,1-oxide (3).
To a stirred solution of 122 (20 mg, 0.085 mmol) in dry acetone (2 mL) was
added an excess of freshly prepared dimethyldioxirane22 in acetone dropwise.
The solution was allowed to warm to room temperature and an additional
aliquot of dimethyl dioxirane equal in volume to the initial amount was added.
After 12 h of stirring at 25 °C the solution was evaporated to yield 3 as a
colorless oil (22 mg, 98%). Rf = 0.43 (3:2 hexane/ethyl acetate). 1H NMR (CDCl3,
300 MHz) d 1.30 (3H, t, J = 7 Hz), 4.27 (2H, q, J = 7.2 Hz), 4.45 (2H, s), 7.90 (3H,
m), 8.10 (1H, dd, J = 7.65 Hz, 0.9 Hz); 13C NMR (CDCl3, 75.47 MHz) d 165.85,
158.77, 137.78, 135.05, 134.47, 127.10, 125.47, 121.21, 62.27, 39.11, 14.04.
HRMS (ESI) calcd for C11H12NO5S [M+H]+ 270.0436, found 270.0446.