Reactivity, Selectivity, and Stability in Sulfenic Acid Detection: A Comparative Study of Nucleophilic and Electrophilic Probes

Article abstract of DOI:10.1021/acs.bioconjchem.6b00181

The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes toward cysteine sulfenic acid have been thoroughly evaluated in two different settings-(i) a small molecule dipeptide based model and (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling.

Full text of DOI:10.1021/acs.bioconjchem.6b00181

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Article
Reactivity, Selectivity and Stability in Sulfenic Acid Detection:
A Comparative Study of Nucleophilic and Electropilic Probes
Vinayak Gupta, Hanumantharao Paritala, and Kate S. Carroll
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00181 • Publication Date (Web): 28 Apr 2016
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Reactivity, Selectivity and Stability in Sulfenic Acid Detection:
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A Comparative Study of Nucleophilic and Electropilic Probes
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Vinayak Gupta,a Hanumantharao Paritalaa and Kate S. Carrolla, *
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Graphical Abstract
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Abstract
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The comparative reaction efficiencies of currently used nucleophilic and electrophilic
probes towards cysteine sulfenic acid have been thoroughly evaluated in two different
settings – (i) a small molecule dipeptide based model and, (ii) a recombinant protein
model. We further evaluated the stability of corresponding thioether and sulfoxide
adducts under reducing conditions which are commonly encountered during proteomic
protocols and in cell analysis. Powered by the development of new cyclic and linear C-
nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under
competitive conditions is achieved and thus holds great promise as highly potent tools
for activity-based sulfenome profiling.
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Introduction
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Owing to the intrinsic nucleophilicity and oxidation susceptibility, cysteine
thiols (Cys-SH) are prime targets for the redox-based modulation of protein
activity. Oxidation of a protein cysteine thiol (Cys-SH) to sulfenic acid (Cys-SOH)
by reactive oxygen species (ROS) (e.g. H2O2), termed S-sulfenylation, is a
reversible post-translational modification that plays a crucial role in regulating
several protein functions.1-6 With an estimated half-life in minutes, Cys-SOH is
considered a transient species whose ultimate fate depends upon factors such as
presence of neighbouring thiol, level/duration of ROS and protein
microenvironment.5 Essentially, absence of proximal thiols capable of forming
intramolecular disulfide bond, limited solvent access and proximal hydrogen bond
acceptors contribute towards Cys-SOH stabilization. High/chronic oxidative stress
may cause –SOH to undergo further oxidation to irreversible sulfinic (Cys-SO2H)
or sulfonic acid (Cys-SO3H). Due to the abundance of biological thiols (mM
levels), an important and facile biological reaction of Cys-SOH is the disulfide
formation. The nascent disulfide may undergo thiol-disulfide exchange to
regenerate initial Cys-SH. Cys-SOH may undergo intramolecular reaction with
adjacent amide nitrogen resulting in the formation of a cyclic species known as
cyclic sulfenamide which may be reduced back to thiol via disulfide formation.
Due to the central role of Cys-SOH in reversible/irreversible pathway, it serves as
an important hub within the redox milieu.5 Indeed, over the past decade several
groups have reported the role of Cys-SOH in regulation of several proteins such
as transcription factors, kinases, phosphatases, ion channels, peroxidases and
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cysteine proteases, sirtuins, human serum albumin and many others.3,
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Moreover, aberrant Cys-SOH formation has been shown to correlate with disease
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state.12,
The aforementioned examples and many other reports establish
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protein S-sulfenylation as a global signaling mechanism akin to phosphorylation
and a potential drug target.5, 6, 15, 23-27
Fig. 1 Dual chemical reactivity of sulfenic acid.
Due to the electrophilic as well as nucleophilic character of sulfur atom in
Cys-SOH, the detection methods exploiting both have been reported (Fig. 1) 5, 10-
.
13, 19, 20, 25, 26, 28-43 Briefly, due to higher electrophilicity (than nucleophilicity) of Cys-
SOH, vast majority of probes are nucleophilic in nature and based on cyclic 1,3-
carbonyl scaffold such as dimedone (1). These nucleophilic probes reacts with
Cys-SOH to give thioether adducts. Such nucleophilic probes are extensively
employed to study qualitative and quantitative capture of Cys-SOH.1, 5, 9-11, 13-16, 18-
22, 29-32, 34-39, 42-51
Commonly used repertoire of C-nucleophilic probes consist of
commercially available 1,3-dicarbonyl-based ‘clickable’ probes such as DYn-2
(2a),15 DAz-2 (2b),10 Alk- -KE (3),46 BP1 (7),44 and under development 6-hydroxy
β
derivatives of phenalne-1,3-dione (8) (Fig. 2). Enrichment-ready/imaging-ready
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reagents such as DCP-Bio1 (2c) and DCP-Rho1 (2d) have also been made
commercially available and used extensively.30 Although these nucleophilic
probes are selective for protein sulfenic acid under aqueous physiological
conditions, they suffer from poor reaction kinetics (kobs = 0.008 M-1s-1 – 0.05 M-1s-
1) when compared to other biological reactions of Cys-SOH (such as disulfide
formation or over-oxidation to sulfinic/sulfonic acid).5 To overcome this limited
reactivity problem, we recently developed and reported a facile mass-
spectrometery-based assay and a repurposed dipeptide-based sulfenic acid
model using which we screened a library of cyclic and linear C-nucleophiles for
reactivity towards sulfenic acid under aqueous conditions. We obtained the
reaction rate constants of more than 150 C-nucleophiles and from this collection
we identified novel classes of compounds with more than 200-fold enhanced
reactivity towards Cys-SOH.25, 26
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Fig. 2 Nucleophilic and electrophilic probes reported for detecting sulfenic acid.
Sulfenic acid can also act as a nucleophile and react with electrophiles to
produce a sulfoxide adduct (Fig. 1). Given the nucleophilic properties of thiols
and several other biological species, it is generally considered that the selectivity
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towards Cys-SOH maybe difficult to achieve with electrophilic detection tools.47
Currently reported examples of electrophilic probes are commercially available
compounds such as (i) low reactive NBD-Cl (4) which also lack the
chemoselectivity and react with several biological nucleophiles including sulfenic
acid (but the resulting adducts are distinguishable due to difference in their
spectroscopic properties);5, 28, 38 (ii) arylboronates (5) which react with Cys-SOH
in a reversible manner and thus have limited utility in proteomic-based sulfenic
acid detection52 and; (iii) strained cycloalkynes (6) which were recently reported
as new, highly reactive sulfenic acid traps (kobs = 13.3 M-1s-1 – 16.7 M-1s-1) with >
100-fold higher reactivity than 1,3-dicarbonyls.40 Not surprisingly, over the years
strained cycloalkynes have been shown to react with other sulfur species such as
protein- and small molecule thiols (present in mM concentration inside the cells)
and persulfides.53-55 Moreover, the product of the reaction of Cys-SOH and
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strained cycloalkyne
6 is an activated vinylic sulfoxide which is a Michael-
acceptor and may readily react with biological thiols as well.56 As a consequence
of the promiscuous reactivity of strained cycloalkynes and the resultant sulfoxide
adduct, an additional thiol-blocking step is required to increase the selectivity for
sulfenic acid. As mentioned above, even though over the past decade several
nucleophilic and electrophilic probes for sulfenic acid detection have been
developed and commercialized, the issue of modest reactivity of such probes is
well documented.5, 26, 40 Several subsequent publications reported probes with
superior reactivity compared to dimedone-based probes at lower or higher pHs.44,
46, 47
However, such reports generally compared the reactivities of these probes
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towards Cys-SOH in specific model protein systems where the reactivity could be
biased by the protein microenvironment surrounding Cys-SOH. For this reason a
direct comparison of the reactivity of various nucleophilic/electrophilic probes
towards Cys-SOH and the stability of resulting thioether/sulfoxide adduct under
exact same conditions is needed for the better understanding of the reactivity
profile of each probe. In order to do so, we chose to utilize our peptide-based
sulfenic acid model with subsequent verification in a protein sulfenic acid model.
The peptide-based sulfenic acid model exists in the masked form as a cyclic
sulfenamide (9) which rearranges to dipeptide sulfenic acid (10) under aqueous
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conditions (Fig. 3) 26
Since this model is not influenced by a complex protein
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microenvironment, the observed reaction rates are independent of factors such
as steric hindrance and stabilization/destabilization of sulfenic acid.
Fig. 3 Nucleophiles and electrophiles tested for reactivity with sulfenic acid 10
.
Results and Discussion
Using the peptide-based model and the previously developed LC/MS
assay, we initially obtained the pseudo 1st order rate constants ([Nu/Elec] = 1
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mM, [10] = 100
ꢀ
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compounds (Fig. 3). These compounds constitute the reactive component of
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prevalent and commercially available Cys-SOH probes such as 2a-d
6 (Fig. 2). Nucleophilic cyclohexane-1,3-dione (2e, kobs = 0.4 min-1), methyl
acetoacetate (3a, kobs = 0.9 min-1) and 4-(ethylthio)cyclopentane-1,3-dione (7a
, 3, 7, 4 and
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,
kobs = 0.01 min-1) successfully reacted with dipeptide-SOH 10 to form
corresponding thioether adduct. While 2e and 3a had similar reactivity under
physiological conditions, it is noteworthy that 7a was 40-folds less reactive
compared to 2e towards sulfenic acid 10
.
Electrophilic 4-chloro-7-
nitrobenzo[c][1,2,5]oxadiazole (
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, kobs < 0.001 min-1) was extremely slow to react
and only a small amount of sulfoxide adduct could be detected even after 48 h
(ESI, Fig. S4). Conversely, while the benzoyl derivative of strained cycloalkyne
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl benzoate (6a, kobs = N.D.) was
highly reactive, the corresponding sulfoxide adduct was unstable and quickly
rearranged to disulfide (Cbz-Cys-Val-OMe)2 (ESI, Fig. S5). Next, we obtained the
pseudo 1st order rate constants of 1H-phenalene-1,3(2H)-dione (8a, kobs = 0.1
min-1), recently developed cyclic C-nucleophiles 1-benzylpiperidine-2,4-dione (11
kobs = 86.4 min-1), 1-benzyl-1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxide (12
,
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kobs = 190.5 min-1), linear C-nucleophiles ((nitromethyl)sulfonyl)benzene (13, kobs
= 7.8 min-1), and 2-(phenylsulfonyl)acetonitrile (14, kobs = 78.0 min-1) for the
reaction with dipeptide-SOH 10. Due to the opposing effect of resonance
stabilization and electron donating naphthyl group, 8a showed a reduced rate of
reaction towards sulfenic acid 10. Cyclic and linear C-nucleophiles 11-14 showed
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elevated reactivity towards 10. In brief, the reactions of various nucleophiles and
electrophiles towards dipeptide-SOH 10 clearly demonstrated the substantially
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enhanced reactivity of 11-14 compared to currently prevalent probes (Fig. 3)
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Fig. 4 Reactivity of nucleophiles and electrophiles with Gpx3-SOH.
Subsequently, we examined the reactivity of above-tested nucleophiles
and electrophiles towards a protein Cys-SOH. For these studies, we used our
well-established Cys64Ser Cys82Ser variant of the recombinant thiol peroxidase,
Gpx3.11 Incubation of nucleophiles and electrophiles at 100
with Gpx3-SOH led to the covalent modification of Gpx3-SOH. Of note, while
trapping with 2e was not quantitative, it still captured ~ 50% Gpx3-SOH (Fig. 4B)
Interestingly, even though -ketoester 3a showed a higher rate of reaction in the
ꢀM concentration
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dipeptide-SOH model, the amount of Gpx3-SOH captured was lower than 2e
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highlighting the influence of protein microenvironment on reactivity as well as the
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tendency of the thioether adduct of 3a to undergo loss of a methylketo (-COMe)
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group (Fig. 4C) 25, 57
Owing to poor reaction rate, 7a failed to capture Gpx3-SOH,
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a finding that was consistent with data obtained in the dipeptide-SOH model (Fig.
4D). In contrast to data reported using the C165A AhpC-SOH,40 the hydroxyl
version of strained cycloalkyne
6 captured only a small amount of Gpx3-SOH
(Fig. 4E). This result correlates well with the observation that the sulfoxide adduct
of 6a with dipeptide-SOH 10 quickly degrades (ESI, Fig. S5). Phenalene-1,3-
dione 8a also showed extremely poor reactivity towards Gpx3-SOH, also
consistent with results obtained in the dipeptide–SOH model (Fig. 4F). Finally,
akin to their superior reactivity towards the dipeptide-SOH, cyclic C-nucleophiles
11 and 12 quantitatively trapped Gpx3-SOH (Fig. 4G-H). Linear C-nucleophiles
13 and 14 also tagged the majority of Gpx3-SOH (Figure 4I-J). These results
gratifyingly confirmed the order of reactivity observed in our LC/MS assay using
the dipeptide-SOH model system.
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* Stable adduct formation is defined as an adduct in which no detectable cleavage has occurred within 48 h.
Table 1 Reversibility of dipeptide-S-Nu adducts under reducing conditions.
We previously showed that due to the difference in reactivity, pKa, and
carbanion stability of C-nucleophiles, the resulting thioether adducts also display
a range of stability under reducing conditions.25, 26 As cell contains millimolar
concentrations of glutathione (GSH) we tested the ability of this low-molecular
weight thiol to reduce the nascent covalent thioether bond formed between 10
and nucleophiles. Initially thioether adducts of linear C-nucleophiles were shown
to be reversible under reducing conditions – a view strengthened by the stability
of thioether adducts 15
,
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(t1/2 = 1.4 h) and 22 (t1/2 = 0.4 h) (Table 1, ESI Fig. S6 – S21 and Fig. S34 –
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cyclic C-nucleophile 11 (ESI, Fig. S22 – S25), and the stability of thioether
adduct 21 (t1/2 > 9 days) of linear C-nucleophile 14 (ESI, Fig. S30 – S33)
indicates that the cyclic/linear nature of nucleophiles is not the determining factor
with respect to the stability/instability of corresponding adducts (Table 1)
.
However, the instability of thioether adduct 19 (t1/2 > 39 h) formed by
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,
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Interestingly, enhanced reactivity of a nucleophile is also not the sole criterion for
reversibility, as illustrated by the stability of thioether adduct 20 (t1/2 > 18 days)
which results from trapping by the highly reactive cyclic C-nucleophile 12 (Table
1, ESI Fig. S26 – S29). Thioether adducts showing t1/2 < 48 h were then tested
for reversibility in the Gpx3 model. Gpx3-SOH was first labeled with nucleophiles
3a, 11 and 14. Subsequently, each Gpx3-Nu adduct was subjected to mM
glutathione (GSH), dithiothreitol (DTT) or the phosphine-based reductant, TCEP.
Correlating with the reversibility trends observed in the reduction of dipeptide-
based model, Gpx3-S-Nu adducts formed by
β-ketoester 3a and
phenylsulfonylacetonitrile 14 were almost completely reversible while the adduct
corresponding to 11 showed some reversibility under reducing conditions (ESI
Fig. S50)
.
In light of the aforementioned stability data, we subsequently explored the effect
of C-2 substitution on the reactivity of nucleophiles towards sulfenic acid. In this regard,
one key issue is that a tertiary carbanion is less stable than a secondary carbanion.
Moreover, upon reaction with sulfenic acid, a C-2 substituted nucleophile would be
‘locked’ in the keto tautomer. Such a conformational lock could potentially result in
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unique spectroscopic properties (i.e., UV/Vis or fluorescence) of the nucleophile before
and after reaction with sulfenic acid. Such difference in UV/Vis absorption or
fluorescence may be exploited to develop a ‘turn-on/turn-off switch’ based sulfenic acid
detection strategy. A representative example of a nucleophile undergoing such change
in spectroscopic properties is 2-substituted derivatives of phenalene-1,3-dione (8a).
Keto-enol tautomerism is facile for compound 8a readily and, upon reaction with Cys-
SOH, keto product 18 can easily rearrange to give enol 18’ (Fig. 5A), whereas the
thioether derivative of 2-substituted- phenalene-1,3-dione (23) would be locked in the
keto form 24 (Fig. 5B) resulting in turn-on fluorescence.58 Although new spectral
properties of the resulting thioether adduct is an attractive possibility, the effect of C-2
substitution by an electron-donating group (EDG) or electron-withdrawing group (EWG)
on nucleophile reactivity has not been studied.
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Fig. 5 Effect of C-2 substitution on the UV-vis and fluorescence properties of the
nucleophiles and corresponding thioether adducts.
To examine the issue of C-2 substitution, we studied the reactivity of dipeptide
sulfenic acid 10 with C-2 methylated (EDG) and C-2 fluorinated (EWG) derivatives of
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three nucleophiles – (a) cyclohexane-1,3-dione (2e), (b) 1H-phenalene-1,3(2H)-dione
(8a), and (c) diethylmalonate (29). The analogs were prepared following literature
reported procedures.59 The kinetic studies data are summarized in Table 2. Briefly,
while 2-methylcyclohexane-1,3-dione (25, kobs > 0.4 min-1) showed slightly higher
reactivity than 2e (kobs = 0.4 min-1), 2-fluorocyclohexane-1,3-dione (26, kobs < 0.001 min-
1) was very slow to react (Table 2A). Conversely, both 2-methyl-1H-phenalene-1,3(2H)-
dione (27, kobs = 0.002 min-1) and 2-fluoro-1H-phenalene-1,3(2H)-dione (28, kobs < 0.001
min-1) showed reduced reactivity, as compared to 8a (kobs = 0.1 min-1) (Table 2B).
Diethylmalonate (29, kobs = 0.1 min-1) exhibited similar reactivity to 8a and its C-2
substituted derivatives diethyl 2-methylmalonate (29, kobs < 0.001 min-1) and diethyl 2-
fluoromalonate (30, kobs < 0.001 min-1) were extremely slow to react as well (Table 2C).
10
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Table 2 Effect of C-2 substitution on the reactivity of nucleophiles towards dipeptide-
SOH.
The poor reactivity of C-2 fluorinated analogs 26, 28 and 31 may be attributed to
the strongly electron-withdrawing effect resulting in hydration of carbonyl group.
However, the reactivity of C-2 methylated analogs towards dipeptide-SOH 10 is more
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complex to explain. While 25 showed slightly enhanced reactivity compared to 2e the
resulting thioether adduct quickly underwent carbonyl hydration due to the electron-
withdrawing effect of -SR group (ESI Fig. S51 – S52). Similar carbonyl hydration was
observed in case of thioether adduct of 27 (ESI Fig. S53). Another consequence of
hydration is that the diketo nucleophile becomes a poorer leaving group and the
corresponding thioether adducts are stable under reducing conditions (ESI Fig. S38 –
S41). The hydration effect is not observed in case of the thioether adduct 15 of 2e or 18
of 8a because under aqueous conditions they readily undergo keto-enol tautomerism
and favor the enol form. Diethylmalonate 29 has a comparatively high pKa value of 13
and its C-2 methyl derivative 30 is expected to have an even higher pKa, which would
result in reduced reactivity towards sulfenic acid 10, as was observed. In general, C-2
substitution is detrimental for the reactivity of C-nucleophiles towards sulfenic acid and
reduced the reaction rates by 100-folds or more.
10
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Like many other critical post-translational modifications, such as phosphorylation
and methylation, protein sulfenylation is reversible with an estimated half-life occurring
on the timescale of minutes.5 In the case of sulfenic acid, its transient nature is often
attributed to its reaction with GSH and protein thiol group and these reactions compete
with chemical probes. We illustrate this issue by calculating the percent of Human
Serum Albumin sulfenic acid (HSA-SOH) and oxidized MAP Kinase Phosphatase 3
(oxMKP3) trapped by various nucleophiles in the presence of mM GSH (ESI, Fig. S54).
It is clear from the calculations that in both cases, the majority of C-nucleophiles on
which current probes are based would capture only a fraction of sulfenic acid (Table 3,
entries 2e, 3a, 7a and 8a). By contrast, the same calculations with more recently
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developed cyclic and linear C-nucleophiles predict near quantitative capture of both
HSA-SOH and oxMKP3, even at low nucleophile concentration ([Nu] = 100 ꢀM) (Table
3, entries 11-14). Collectively, these data highlight key differences among chemical
probes for detecting sulfenic acid in the face of competing biological reactions.
10
11
12
13
14
15
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Table 3 Amount of protein-SOH captured by nucleophiles in the presence of GSH at
steady state.
The impact of the slow reaction rates of currently used probes in the detection
and quantitative analysis of protein-SOH is countered by using large amount of starting
biological material (> 30 mg), higher probe concentrations (5 mM – 10 mM) and longer
incubation periods.19, 41 It is speculated that higher probe concentrations and longer
incubation times increase the rate of false positive identification.21 Thus, an ideal probe
for proteomic studies would have the following characteristics: (i) sufficiently fast rate of
reaction with protein-SOH at low probe concentrations; (ii) form stable adducts with
protein-SOH; and (iii) retain chemoselectivity for sulfenic acid. Many of the commercially
available probes do not satisfy one or more of these criterions. For example, most
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commonly used probes such as DYn-2 (2a), DAz-2 (2b), DCP-Bio1 (2c), DCP-Rho1
(2d) and many more are based on cyclohexane1,3-dione (2e) core, which has modest
reactivity towards SOH (kobs = 0.4 min-1). Factors such as the poor reactivity of
cyclopentane-1,3-dione (kobs = 0.01 min-1), strongly anionic nature and presence of a
biotin group (poor cell permeability and increased complexity for peptide identification)
make BP1 (7) less attractive for proteomic analysis of protein-SOH. On the other hand,
10
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while β-ketoester-based Alk-β
-KE (3) gives reaction rates (kobs = 0.9 min-1) similar to 2e,
the corresponding thioether adduct is susceptible to reduction as well as deacetylation.
Recently reported strained cycloalkyne BCN-Bio (6) was highly reactive towards small
molecule SOH (10), but the corresponding sulfoxide adduct was highly unstable under
the conditions of LC/MS analysis. Moreover, BCN-bio (6) showed poor reactivity
towards Gpx3-SOH and is not chemoselective for sulfenic acid.53-55 The concept of
exploiting 2-substituted-phenalene-1,3(2H)-diones (8, 27, 28) as fluorescent turn-on
probes using conformational-lock is attractive, however such compounds suffered from
poor reactivity towards dipeptide-SOH 10 (kobs < 0.001 min-1) as well as Gpx3-SOH.
In an effort to address above limitations and develop “next-generation” sulfenic
acid probes, we performed side-by-side comparisons with recently developed cyclic (11,
12) and linear C-nucleophiles (13, 14). With kobs ranging from 7.8 min-1 to 190.5 min-1,
C-nucleophiles 11-14 showed 20- to 500-fold rate enhancement, compared to 2e.
Moreover, these nucleophiles quantitatively trapped Gpx3-SOH at low concentrations
(100 ꢀM). Thioether adducts resulting from reaction of cyclic 11 and linear 14 with
sulfenic acid 10 (i.e., 19 and 22) exhibited varying stability under reducing conditions.
With a t1/2 = 39 h, thioether 19 was substantially more stable than 22 (t1/2 = 0.4 h),
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indicating that cyclic C-nucleophile 11 may be suitable for future probe development.
Thioether adducts 20 and 21 showed robust stability under reducing conditions,
suggesting that C-nucleophiles 12 and 13 are excellent candidates for “next-generation”
sulfenic acid probes.
10
11
12
13
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Fig. 6 Ratification of various nucleophiles and electrophiles on the basis of sulfenic acid
reactivity and stability/reversibility of corresponding thioether (or sulfoxide) adduct.
Conclusion
In conclusion, we have compared the reactivity of commonly used/commercially
available nucleophilic and electrophilic probes with recently developed cyclic and linear
C-nucleophiles with sulfenic acid. Using the rate constants obtained from our dipeptide-
SOH model, and comparing them with known rates of glutathionylation for protein-SOH,
our data indicate that while current probes are sufficient for qualitative analysis, they are
not capable of quantitative trapping and may not be well-suited to detect more short-
lived sulfenic acid modifications. By extension, “next-generation” probes with superior
reaction rates and sufficient stability will enable more comprehensive analysis of the
sulfenylome. Another important consideration is the effect of probe/protein structure on
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chemical reactivity. Along these lines, to obtain deep coverage of the sulfenylome, it
may be valuable to explore multiple probes with varying reactivity and distinct structural
features. Further, we have established the reversibility of the thioether adduct resulting
10
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from
β-ketoester-based Alk-β-KE (3) and other nucleophiles, such as 14, under
reducing conditions encountered during trapping or in common use during proteomic
sample preparation. Such reversibility can result in false negative results and, thus, it is
critical to evaluate the stability of thioether (or sulfoxide) adducts formed by the reaction
of nucleophilic (or electrophilic) probes (Fig. 6). Finally, we highlight the utility of
dipeptide sulfenic acid 10 as model for screening and a facile way to compare the
reactivity of nucleophiles and electrophiles with sulfenic acid. Moreover, dipeptide-SOH
10 provides a straightforward way to prepare the corresponding thioether (or sulfoxide)
adducts in order to study their stability under various conditions.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the ACS Publications website
at DOI:
Experimental procedures, 1H NMR, 13C NMR, and LC-MS data
including stability studies.
AUTHOR INFORMATION
Corresponding Author
*kcarroll@scripps.edu
Notes
The authors declare no competing financial interest.
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Acknowledgements
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We thank Dr. Brent Martin (University of Michigan) for conversations pertaining to
2-substituted-phenalene-1,3(2H)-diones. This work is supported by the National
Institutes of Health under awards No. R01 GM102187 and R01 CA174864 to
K.S.C.
References
[1] Reddie, K. G., and Carroll, K. S. (2008) Expanding the functional diversity of proteins
through cysteine oxidation, Curr Opin Chem Biol 12, 746-754.
[2] Paulsen, C. E., and Carroll, K. S. (2010) Orchestrating redox signaling networks
through regulatory cysteine switches, ACS Chem Biol 5, 47-62.
[3] Lo Conte, M., and Carroll, K. S. (2013) The redox biochemistry of protein
sulfenylation and sulfinylation, J Biol Chem 288, 26480-26488.
[4] Paulsen, C. E., and Carroll, K. S. (2013) Cysteine-mediated redox signaling:
chemistry, biology, and tools for discovery, Chem Rev 113, 4633-4679.
[5] Gupta, V., and Carroll, K. S. (2014) Sulfenic acid chemistry, detection and cellular
lifetime, Biochim Biophys Acta 1840, 847-875.
21
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Page 22 of 30
1
2
3
4
5
6
7
8
9
[6] Chouchani, E. T., Kazak, L., Jedrychowski, M. P., Lu, G. Z., Erickson, B. K., Szpyt,
J., Pierce, K. A., Laznik-Bogoslavski, D., Vetrivelan, R., Clish, C. B., et al. (2016)
Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of
UCP1, Nature 532, 112-116.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[7] Denu, J. M., and Tanner, K. G. (1998) Specific and reversible inactivation of protein
tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate
and implications for redox regulation, Biochemistry 37, 5633-5642.
[8] Carballal, S., Radi, R., Kirk, M. C., Barnes, S., Freeman, B. A., and Alvarez, B.
(2003) Sulfenic acid formation in human serum albumin by hydrogen peroxide and
peroxynitrite, Biochemistry 42, 9906-9914.
[9] Depuydt, M., Leonard, S. E., Vertommen, D., Denoncin, K., Morsomme, P., Wahni,
K., Messens, J., Carroll, K. S., and Collet, J. F. (2009) A periplasmic reducing system
protects single cysteine residues from oxidation, Science 326, 1109-1111.
[10] Leonard, S. E., Reddie, K. G., and Carroll, K. S. (2009) Mining the thiol proteome
for sulfenic acid modifications reveals new targets for oxidation in cells, ACS Chem Biol
4, 783-799.
[11] Paulsen, C. E., and Carroll, K. S. (2009) Chemical dissection of an essential redox
switch in yeast, Chem Biol 16, 217-225.
22
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Bioconjugate Chemistry
1
2
3
4
[12] Seo, Y. H., and Carroll, K. S. (2009) Profiling protein thiol oxidation in tumor cells
5
6
using sulfenic acid-specific antibodies, Proc Natl Acad Sci U S A 106, 16163-16168.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[13] Leonard, S. E., Garcia, F. J., Goodsell, D. S., and Carroll, K. S. (2011) Redox-
based probes for protein tyrosine phosphatases, Angew Chem Int Ed Engl 50, 4423-
4427.
[14] Barrett, T. J., Pattison, D. I., Leonard, S. E., Carroll, K. S., Davies, M. J., and
Hawkins, C. L. (2012) Inactivation of thiol-dependent enzymes by hypothiocyanous
acid: role of sulfenyl thiocyanate and sulfenic acid intermediates, Free Radic Biol Med
52, 1075-1085.
[15] Paulsen, C. E., Truong, T. H., Garcia, F. J., Homann, A., Gupta, V., Leonard, S. E.,
and Carroll, K. S. (2012) Peroxide-dependent sulfenylation of the EGFR catalytic site
enhances kinase activity, Nat Chem Biol 8, 57-64.
[16] Svoboda, L. K., Reddie, K. G., Zhang, L., Vesely, E. D., Williams, E. S.,
Schumacher, S. M., O'Connell, R. P., Shaw, R., Day, S. M., Anumonwo, J. M., et al.
(2012) Redox-sensitive sulfenic acid modification regulates surface expression of the
cardiovascular voltage-gated potassium channel Kv1.5, Circ Res 111, 842-853.
[17] Alili, L., Sack, M., von Montfort, C., Giri, S., Das, S., Carroll, K. S., Zanger, K., Seal,
S., and Brenneisen, P. (2013) Downregulation of tumor growth and invasion by redox-
active nanoparticles, Antioxid Redox Signal 19, 765-778.
23
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Page 24 of 30
1
2
3
4
5
6
7
8
9
[18] Kulathu, Y., Garcia, F. J., Mevissen, T. E., Busch, M., Arnaudo, N., Carroll, K. S.,
Barford, D., and Komander, D. (2013) Regulation of A20 and other OTU
deubiquitinases by reversible oxidation, Nat Commun 4, 1569.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[19] Yang, J., Gupta, V., Carroll, K. S., and Liebler, D. C. (2014) Site-specific mapping
and quantification of protein S-sulphenylation in cells, Nat Commun 5, 4776.
[20] Gould, N. S., Evans, P., Martinez-Acedo, P., Marino, S. M., Gladyshev, V. N.,
Carroll, K. S., and Ischiropoulos, H. (2015) Site-Specific Proteomic Mapping Identifies
Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein
Networks, Chem Biol 22, 965-975.
[21] Devarie-Baez, N. O., Silva Lopez, E. I., and Furdui, C. M. (2016) Biological
chemistry and functionality of protein sulfenic acids and related thiol modifications, Free
Radical Research 50, 172-194.
[22] Zito, E., Hansen, H. G., Yeo, G. S., Fujii, J., and Ron, D. (2012) Endoplasmic
reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical
scurvy in mice, Mol Cell 48, 39-51.
[23] Truong, T. H., and Carroll, K. S. (2012) Redox regulation of epidermal growth factor
receptor signaling through cysteine oxidation, Biochemistry 51, 9954-9965.
24
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Bioconjugate Chemistry
1
2
3
4
5
6
[24] Truong, T. H., and Carroll, K. S. (2013) Redox regulation of protein kinases, Crit
Rev Biochem Mol Biol 48, 332-356.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[25] Gupta, V., and Carroll, K. S. (2016) Rational design of reversible and irreversible
cysteine sulfenic acid-targeted linear C-nucleophiles, Chem Commun (Camb) 52, 3414-
3417.
[26] Gupta, V., and Carroll, K. S. (2016) Profiling the Reactivity of Cyclic C-Nucleophiles
towards Electrophilic Sulfur in Cysteine Sulfenic Acid, Chem Sci 7, 400-415.
[27] Visscher, M., Arkin, M. R., and Dansen, T. B. (2016) Covalent targeting of acquired
cysteines in cancer, Curr Opin Chem Biol 30, 61-67.
[28] Ellis, H. R., and Poole, L. B. (1997) Novel application of 7-chloro-4-nitrobenzo-2-
oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl
hydroperoxide reductase, Biochemistry 36, 15013-15018.
[29] Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. (2005)
Synthesis of chemical probes to map sulfenic acid modifications on proteins, Bioconjug
Chem 16, 1624-1628.
[30] Poole, L. B., Klomsiri, C., Knaggs, S. A., Furdui, C. M., Nelson, K. J., Thomas, M.
J., Fetrow, J. S., Daniel, L. W., and King, S. B. (2007) Fluorescent and affinity-based
25
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2
3
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tools to detect cysteine sulfenic acid formation in proteins, Bioconjug Chem 18, 2004-
5
6
2017.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[31] Reddie, K. G., Seo, Y. H., Muse Iii, W. B., Leonard, S. E., and Carroll, K. S. (2008)
A chemical approach for detecting sulfenic acid-modified proteins in living cells, Mol
Biosyst 4, 521-531.
[32] Seo, Y. H., and Carroll, K. S. (2009) Facile synthesis and biological evaluation of a
cell-permeable probe to detect redox-regulated proteins, Bioorg Med Chem Lett 19,
356-359.
[33] Leonard, S. E., and Carroll, K. S. (2011) Chemical 'omics' approaches for
understanding protein cysteine oxidation in biology, Curr Opin Chem Biol 15, 88-102.
[34] Seo, Y. H., and Carroll, K. S. (2011) Quantification of protein sulfenic acid
modifications using isotope-coded dimedone and iododimedone, Angew Chem Int Ed
Engl 50, 1342-1345.
[35] Truong, T. H., Garcia, F. J., Seo, Y. H., and Carroll, K. S. (2011) Isotope-coded
chemical reporter and acid-cleavable affinity reagents for monitoring protein sulfenic
acids, Bioorg Med Chem Lett 21, 5015-5020.
[36] Garcia, F. J., and Carroll, K. S. (2014) Redox-based probes as tools to monitor
oxidized protein tyrosine phosphatases in living cells, Eur J Med Chem 88, 28-33.
26
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Bioconjugate Chemistry
1
2
3
4
[37] Martinez-Acedo, P., Gupta, V., and Carroll, K. S. (2014) Proteomic analysis of
5
6
peptides tagged with dimedone and related probes, J Mass Spectrom 49, 257-265.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[38] Pan, J., and Carroll, K. S. (2014) Chemical biology approaches to study protein
cysteine sulfenylation, Biopolymers 101, 165-172.
[39] Brewer, T. F., Garcia, F. J., Onak, C. S., Carroll, K. S., and Chang, C. J. (2015)
Chemical approaches to discovery and study of sources and targets of hydrogen
peroxide redox signaling through NADPH oxidase proteins, Annu Rev Biochem 84, 765-
790.
[40] Poole, T. H., Reisz, J. A., Zhao, W. L., Poole, L. B., Furdui, C. M., and King, S. B.
(2014) Strained Cycloalkynes as New Protein Sulfenic Acid Traps, J Am Chem Soc
136, 6167-6170.
[41] Yang, J., Gupta, V., Tallman, K. A., Porter, N. A., Carroll, K. S., and Liebler, D. C.
(2015) Global, in situ, site-specific analysis of protein S-sulfenylation, Nat Protoc 10,
1022-1037.
[42] Garcia, F. J., and Carroll, K. S. (2016) An immunochemical approach to detect
oxidized protein tyrosine phosphatases using a selective C-nucleophile tag, Mol Biosyst.
[43] Yang, J., Carroll, K. S., and Liebler, D. C. (2016) The Expanding Landscape of the
Thiol Redox Proteome, Mol Cell Proteomics 15, 1-11.
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5
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7
8
9
[44] Qian, J., Klomsiri, C., Wright, M. W., King, S. B., Tsang, A. W., Poole, L. B., and
Furdui, C. M. (2011) Simple synthesis of 1,3-cyclopentanedione derived probes for
labeling sulfenic acid proteins, Chem Commun (Camb) 47, 9203-9205.
10
11
12
13
14
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[45] Wani, R., Qian, J., Yin, L., Bechtold, E., King, S. B., Poole, L. B., Paek, E., Tsang,
A. W., and Furdui, C. M. (2011) Isoform-specific regulation of Akt by PDGF-induced
reactive oxygen species, Proc Natl Acad Sci U S A 108, 10550-10555.
[46] Qian, J., Wani, R., Klomsiri, C., Poole, L. B., Tsang, A. W., and Furdui, C. M. (2012)
A simple and effective strategy for labeling cysteine sulfenic acid in proteins by
utilization of beta-ketoesters as cleavable probes, Chem Commun (Camb) 48, 4091-
4093.
[47] Furdui, C. M., and Poole, L. B. (2014) Chemical approaches to detect and analyze
protein sulfenic acids, Mass Spectrometry Reviews 33, 126-146.
[48] Pan, J., and Carroll, K. S. (2015) Light-Mediated Sulfenic Acid Generation from
Photocaged Cysteine Sulfoxide, Org Lett 17, 6014-6017.
[49] Waszczak, C., Akter, S., Eeckhout, D., Persiau, G., Wahni, K., Bodra, N., Van
Molle, I., De Smet, B., Vertommen, D., Gevaert, K., et al. (2014) Sulfenome mining in
Arabidopsis thaliana, Proc Natl Acad Sci U S A 111, 11545-11550.
28
ACS Paragon Plus Environment

Page 29 of 30
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
[50] Akter, S., Huang, J., Waszczak, C., Jacques, S., Gevaert, K., Van Breusegem, F.,
and Messens, J. (2015) Cysteines under ROS attack in plants: a proteomics view, J Exp
Bot 66, 2935-2944.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[51] Akter, S., Huang, J., Bodra, N., De Smet, B., Wahni, K., Rombaut, D., Pauwels, J.,
Gevaert, K., Carroll, K., Van Breusegem, F., et al. (2015) DYn-2 Based Identification of
Arabidopsis Sulfenomes, Mol Cell Proteomics 14, 1183-1200.
[52] Liu, C. T., and Benkovic, S. J. (2013) Capturing a sulfenic acid with arylboronic
acids and benzoxaborole, J Am Chem Soc 135, 14544-14547.
[53] Sun, Y., Ma, X. W., Cheng, K., Wu, B. Y., Duan, J. L., Chen, H., Bu, L. H., Zhang,
R. P., Hu, X. M., Deng, Z. X., et al. (2015) Strained Cyclooctyne as a Molecular Platform
for Construction of Multimodal Imaging Probes, Angew Chem Int Edit 54, 5981-5984.
[54] Galardon, E., and Padovani, D. (2015) Reactivity of persulfides toward strained
bicyclo[6.1.0]nonyne derivatives: relevance to chemical tagging of proteins, Bioconjug
Chem 26, 1013-1016.
[55] Tian, H., Sakmar, T. P., and Huber, T. (2016) A simple method for enhancing the
bioorthogonality of cyclooctyne reagent, Chem Commun (Camb) 52, 5451-5454.
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