Profiling the reactivity of cyclic C-nucleophiles towards electrophilic sulfur in cysteine sulfenic acid

Oxidation of a protein cysteine thiol to sulfenic acid, termed S-sulfenylation, is a reversible post-translational modification that plays a crucial role in regulating protein function and is correlated with disease states.


Introduction
Reactive oxygen species (ROS) are continuously generated, transformed and consumed in living organisms as a consequence of aerobic life. Due to their role in both physiology and pathology, ROS are considered scientic equivalents of "antiheroes". 1 Once generated, ROS mediates diverse arrays of reversible and irreversible modications on biomolecules such as proteins, lipids DNA and RNA. 2,3 Due to their strong nucleophilic character and low redox potential in proteins (E o , À0.27 to À0.125 V) side chain thiol(ate) of cysteines (Cys-SH) are one of the more common targets of ROS. 4 Indeed, thiolate oxidation by hydrogen peroxide (H 2 O 2 ) represents a widely studied area of redox-based post-translational protein modication. Nucleophilic attack of a protein thiolate on electrophilic H 2 O 2 releases water and results in the formation of cysteine sulfenic acid (Cys-SOH) also known as S-sulfenylation. Depending upon the protein microenvironment where the thiolate is located, the rate of oxidation by H 2 O 2 can vary substantially (1-10 8 M À1 s À1 ). This stark difference in oxidation rates is highlighted by the reaction rates of two major targets of H 2 O 2 signaling in cells, peroxiredoxin 2 (Prx2; 10 8 M À1 s À1 ) and protein tyrosine phosphatase type 1B (PTP1B; 9 M À1 s À1 ). 4,5 Reversible Cys-SOH formation plays a regulatory role among transcription factors, kinases (EGFR, JAK2, Akt2, IKK-b, RegB, PGKase, L-PYK), phosphatases (PTP1B, YopH, PTEN, Cdc25a, SHP-1 and SHP-2), ion channels, peroxidases and cysteine proteases, human serum albumin (HSA) and many other proteins. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Moreover, aberrant S-sulfenylation correlates with tumor progression and can lead to noncanonical scurvy in mice. 10,21 The aforesaid examples and many other reports demonstrate that protein Ssulfenylation constitutes a global signal mechanism, not unlike phosphorylation.
The cellular lifetime of Cys-SOH depends on numerous factors, including the level of ROS and/or duration of ROS signaling as well as the local protein environment. Essentially, the absence of proximal thiols capable of generating an intramolecular disulde is considered to be a primary stabilizing factor; limited solvent access and proximal hydrogen bond acceptors also contribute toward Cys-SOH stabilization. Cys-SOH is the rst oxidation product that results from the reaction between a cysteine thiolate and H 2 O 2 (Fig. 1A, Reaction 1). High ROS, chronic oxidative stress, and/or the lack of adjacent thiols may cause -SOH to undergo further oxidization to sulnic (-SO 2 H) or sulfonic acid (-SO 3 H) (Fig. 1A, Reactions 2 and 3). In contrast to biologically reversible Cys-SOH, these higher oxoforms are essentially irreversible (the only exception to this statement has been found to date is with Prx-SO 2 H, which can be reduced to Prx-SH by the ATP-dependent enzyme, sulredoxin 22 ). An important biological reaction of Cys-SOH is disulde bond formation. Mechanistically, the electrophilic sulfur atom of Cys-SOH reacts with the thiolate nucleophile to give the disulde with concomitant loss of water (Fig. 1A, Reaction 4). Due to the abundance of biological thiols (mM levels) including protein and low-molecular weight molecule thiols, such as glutathione (GSH), this reaction can be facile and constitutes a major pathway for disulde formation. The nascent disulde may undergo thiol-disulde exchange to give the initial thiol (Fig. 1A, Reactions 4 and 5). Cys-SOH may also undergo intramolecular reaction with adjacent amide nitrogen, which results in the formation of isothiazolidinone, also known as cyclic sulfenamide (Fig. 1A, Reaction 6). 23,24 The cyclic sulfenamide species may be reduced back to thiol via disulde formation (Fig. 1A, Reactions 7 and 5). On the basis of the reversible/irreversible reactions that Cys-SOH can undergo, this post-translational modication serves as an important hub within the redox milieu. Accordingly, an important goal to dissect regulatory redox pathways has been to develop robust, sensitive and rapid detection techniques to identify sites, conditions and the cellular lifetime of protein S-sulfenyl modications. 4,6,[25][26][27][28][29] The sulfur atom in sulfenic acid is distinguished from other cysteine redox modications by its weak nucleophilic and moderate electrophilic reactivity (due to the higher pK a leading to lower tendency to form sulfenate anion, they are better electrophiles than nucleophiles). This behavior is epitomized by the tendency of -SOH to self-condense resulting in the formation of a thiosulnate (Fig. 1B). Detection methods exploiting the electrophilic or the nucleophilic character of Cys-SOH have been reported (Fig. 1C). 4,28,30 However, the vast majority of probes capitalize on the unique electrophilic character of sulfur atom in Cys-SOH and are based on 5,5-dimethyl-1,3-cyclohexanedione (1) or dimedone scaffold. 31 Dimedone (1) and probes based on the cyclic 1,3-dicarbonyl scaffold (2) are extensively employed for qualitative and quantitative study of protein S-sulfenylation. [12][13][14][15]20 Though they are selective under aqueous physiological conditions, the above probes suffer from poor reaction kinetics when compared with other common biological reactions of Cys-SOH. 4,32 Conventional electrophilic probes are either slow and cross-react with other biological functionalities (e.g., NBD-Cl (3), Fig. 1D) 4,28 or are reversible (e.g., arylboronic acids (4), Fig. 1D). 33 Recently, however, an electrophilic ring strained alkyne, bicyclo[6.1.0]nonyne (BCN (5), Fig. 1D) was shown to react with sulfenic acid at 100-fold higher reaction rate compared to dimedone. 32 Since protein thiols and persuldes are well documented to readily react with activated alkynes such as 5, this probe has major chemoselectivity issues. [34][35][36][37][38] Thus, there is still signicant room for exploration and further improvement of chemical probes for qualitatively/quantitatively proling of cellular protein Ssulfenylation.
A signicant hurdle to study -SOH reactivity and probe development is the unstable nature of small-molecule sulfenic acid models. In principle, protein sulfenic acid model could be used, however, rates of probe reaction could be biased by the microenvironment surrounding Cys-SOH. For example, a sterically bulky probe may be very reactive, but unable to access Cys-SOH buried in an active-site pocket. Such a case also underscores the importance of developing a suite of probes to prole Cys-SOH, to maximize comprehensive detection of this modi-cation. Existing small molecule sulfenic acid models may be divided into two categories: (i) stable sulfenic acid systems that can be synthesized and stored, and (ii) small-molecule sulfenic acids generated in situ. The rst category are stabilized through hydrogen bonding (e.g. Fig. 1E, 6, 7) and/or steric factors (e.g. Fig. 1E, 8,9). Like proteins, these structures protect and stabilize the sulfenic acid through the surrounding microenvironment. 4 Ideally, however, the model should not be unduly inuenced by such factors. For this reason, we were more interested in a model wherein the sulfenic acid is generated in situ. Although such currently known reactions are highly efficient in generating small molecule sulfenic acids, these reactions either require heat and organic conditions (Fig. 1E, 11) or are kinetically slow (Fig. 1E, 13). 4,39 In the ideal case, we envisaged a cysteine-based small-molecule model that is: (i) straightforward to prepare/store, and (ii) sterically and chemically accessible (i.e., not physically hindered or excessively stabilized by electrostatic interactions). Consequently, the aim of our study was two-fold. First, we wanted to develop a facile small-molecule sulfenic acid model. Second, we wanted to use this model to screen, identify, and kinetically characterize small-molecule C-nucleophiles that react with cysteine sulfenic acid under aqueous conditions.

Synthesis and validation of a dipeptide-based sulfenic acid model
Several literature-reported persistent and transient sulfenic acid models were surveyed, but the example that caught our attention was a dipeptide-based model for its isostere, cyclic sulfenamide (Scheme 1, 14). Dipeptide 14 was originally reported by Shiau et al. at Sunesis pharmaceuticals and employed as a model of cysteine oxidation to cyclic sulfenamide in PTP1B. 40 Owing to the combination of ring strain and electronic factors, we reasoned that the sulfur of cyclic sulfenamide might also be moderately electrophilic (Scheme 1, 15). Furthermore, we were curious about the stability of the sulfenamide under aqueous conditions and wondered whether the cyclic structure could be a synthon of sorts, existing in equilibrium with the corresponding sulfenic acid (Scheme 1). The reported synthesis is low in yield but a straight-forward sequence with well-established synthetic precedent for the key oxidative cyclization step. 41 Even so, following the reported procedure, we obtained the target cyclic sulfenamide (14) in poorer and variable yield. Closer analysis of reaction products revealed the presence of precursor disulde (Cbz-Cys-Val-OMe) 2 (16) and a new compound, identied as cyclic sulnamide (17) (Scheme S1A †). To address the issue of yield and variability, we varied the ratio of bromine to pyridine and avoided the aqueous workup. With these modications in place, the cyclization step was successfully standardized at gram scale to give the dipeptide based cyclic sulfenamide product in >85% yield aer silica gel based column purication (Scheme 2).
With the dipeptide cyclic sulfenamide (14) in hand, we next evaluated its stability under aqueous conditions. In these Scheme 1 Dipeptide based cyclic sulfenamide model is hypothesized to exist in equilibrium with corresponding sulfenic acid under aqueous conditions. experiments, we observed that dipeptide cyclic sulfenamide (14) reacted over time to form cyclic sulnamide (17) and (Cbz-Cys-Val-OMe) 2 (16) (Scheme S1A †). The mechanism shown in Scheme S1B † accounts for the formation of 16 and 17 and is consistent with our proposal that cyclic sulfenamide (14) exist in equilibrium with sulfenic acid (15) under aqueous conditions. In the absence of other reactive groups, cyclic sulfenamide 14 can be reformed from 15 through attack by nitrogen. In addition, 15 can condense with itself (or cyclic sulfenamide 14) to give thiosulnate (18) as an intermediate, the eventual rearrangement of which was observed over the time (Scheme S2B †). In subsequent steps, the amide nitrogen nucleophile attacks the electrophilic sulnyl sulfur, producing cyclic sulnamide (17) and dipeptide thiolate (19). Thiolate 19 subsequently reacts with sulfenic acid 15 (or with cyclic sulfenamide 14) resulting in the formation of dipeptide disulde 16. Importantly, the dipeptide cyclic sulfenamide 14 was stable in acetonitrile over the same period of time (and longer) demonstrating that H 2 O is required for decomposition (Scheme S3 †). Further chemical evidence for the formation of sulfenic acid (15) was obtained through the addition of methyl iodide and NBD-Cl to the reaction, giving corresponding methyl and aryl sulfone respectively (Schemes S4 and S5 †).
Since formation of sulnamide 17 and disulde 16 has the potential to interfere with downstream kinetic analysis, we determined the second-order rate constant for this reaction (Scheme S2D †). In this analysis, a value of 1.2 M À1 s À1 was obtained and deemed acceptable given the anticipated rate constants for our assay (see below). Since the rate-limiting step in this rearrangement is formation of 18 and the sulfenate anion is required for facile self-condensation of sulfenic acid, we were presented with the opportunity to determine the pK a of sulfenic acid 15. Pseudo rst-order rate constants (k obs ) were obtained for the rearrangement from pH 3-9 (Scheme S6B †). The plot of k obs versus pH gave a pK a value of 7.1 for sulfenic acid 15 (Scheme S6C †). This value agrees well with small-molecule sulfenic acid pK a s, which generally range between 4 and 8 depending upon their stability. 4 The measured pK a value of 7.1 is signicant as it indicates that under our aqueous experimental conditions, sulfenic acid 15 and the corresponding sulfenate anion are present in roughly equal amounts. The existence of both species is required for facile formation of thiosulnate (18), which is clearly observed in our assay. Collectively, the aforementioned data provide strong support for the formation of sulfenic acid 15 under aqueous conditions.
Validation of the LC-MS assay for screening cyclic Cnucleophiles Having shown that sulfenic acid 15 forms under aqueous conditions, we next evaluated its ability to react with dimedone 1 to give the expected thioether adduct 20 (Scheme 3A) under pseudo rst-order conditions (i.e., $10-fold excess of C-nucleophile, Scheme S7C †). The resulting plot of k obs versus cyclic sulfenamide 14 gave a straight line, the slope of which yielded a second-order rate constant value of 11.8 M À1 s À1 , consistent with the rate constants reported for reaction between dimedone 1 and protein sulfenic acids (Scheme 3B). 4,42 Additional experiments conrmed that self-condensation of 15 (i.e., the competing background reaction shown in Scheme S1 †) was negligible under the conditions of our kinetic assay ($1 mM dimedone and #100 mM cyclic sulfenamide 14, see also Scheme S7B †).
Compared to cyclic sulfenamide 14, the sulfur of sulfenic acid 15 is considerably more electrophilic. Even so, it is formally possible that dimedone (1) could react with either sulfur center. To identify the reactive specie(s) under our aqueous assay conditions, we investigated the reaction between dimedone (1) and two additional sulfenamide models in which sulfenic acid formation was either minimized (i.e., electron-rich cyclic sulfenamide) or absent (i.e., linear sulfenamide). Cyclic sulfena- (1) to form an adduct (k obs ¼ 0.03 min À1 , Scheme S8 †); however, the observed rate was $30-fold less than the equivalent reaction with cyclic sulfenamide 14 (k obs ¼ 0.8 min À1 ). Linear sulfenamide, methyl 2-(acetamidothio)benzoate (43) failed to react with dimedone (1), as expected (Scheme S10 †). Though sulfenamides 41 and 43 are not perfect experimental models for 14 (e.g., 41 and 43 are more sterically hindered around the sulfur atom) these data are consistent with the hypothesis that sulfenic acid 15 is the reactive species. Furthermore, both 41 and 43 failed to give the diagnostic sulfoxide under aqueous conditions, which is exhibited by 14 and a hallmark of sulfenic acid formation (Schemes S9 and S11 †). In addition to the above data, we note the excellent correspondence in second order rate constants for the reaction between dimedone (1) and protein sulfenic acids or dipeptide 14. Lastly, it has been well established that dimedone does not react with the stable cyclic sulfenamide formed in the tyrosine phosphatase, PTP1B. 12,13,15,43 When taken together, these data support our proposal that sulfenic acid 15 is the major reactive species in our aqueous kinetic assay.
In subsequent studies, we characterized the pH dependence for the reaction of dimedone (1) and sulfenic acid 15. The resulting plot of pH versus k obs for formation of thioether 20 was best t to an equation with a single ionization with a pK a value of 5.4 (Scheme 3C). This value matches closely with the pK a of dimedone (1) obtained in water (pK a ¼ 5.2). 44,45 An analogous experiment was performed with a closely related C-nucleophile, 1,3-cyclopentanedione (21a). 46 In this case, the resulting pK a for 21a gave a value of 4.2, which matches closely with the reported pK a in water (pK a ¼ 4.3) 47 (Scheme S12 †). Taken together, the data from these experiments suggests that the reaction rate of sulfenic acid 15 and the aforementioned cyclic 1,3-dicarbonyl nucleophiles is inuenced by the position of the C-2 acid/base equilibrium. These ndings thus substantiate the importance of C-nucleophile pK a as an important determinant in the dimedone (1) reaction and highlight the utility of our assay to evaluate the reactivity of C-nucleophiles with sulfenic acid.

Ring size and C-nucleophile reactivity
In subsequent studies, we examined the effect of C-nucleophile ring size on reaction rate constants with sulfenic acid. To this end, we selected four commercially available nucleophiles: 1,3-cyclopentanedione (21a), 1,3-cyclohexanedione (22a), 1,3-cycloheptanedione (23) and 2,4-pentanedione (24) (Chart 1). The resulting pseudo rst-order rate constants show an increase in reactivity with increasing ring size. Due to resonance stabilization of the enolate, the pK a of the a-carbon nucleophile in 1,3dicarbonyls is relatively low (<14) (Scheme 4) and, consequently, these compounds will have varied anionic character at physiological pH. For example, the enol tautomer of 21a (pK a $ 4.3) is the dominant form under aqueous conditions at pH 7.4 and its low pK a leads to a highly stabilized enolate. Consequently, 21a has a lower tendency to react with sulfenic acid 15 (k obs ¼ 0.02 min À1 ) compared to 22a (k obs ¼ 0.4 min À1 ). As ring size increases, the pK a of the a-carbon rises and the tautomeric equilibrium shis toward the keto form. Consistent with these properties, 22a (pK a ¼ 5.23) 47 and 23 showed a respective 20-fold and 150-fold (k obs ¼ 3 min À1 ) enhancement in reaction rate constants relative to 21a. Linear 1,3-dicarbonyl 24 (pK a ¼ 8.99), 47 which favors the keto tautomer by 4 : 1, 48 displayed a 190-fold (k obs ¼ 3.8 min À1 ) rate enhancement compared to 21a. Together, the observed trend in C-nucleophile reactivity can be rationalized by two principle factors: electronics or a-carbon pK a and keto-enol tautomerism.

Cyclic C-nucleophile heteroatom incorporation
To increase the reactivity of cyclic C-nucleophiles, we next sought to destabilize the C-2 anion and shi the keto-enol equilibrium towards the keto tautomer. To this end, we pursued the substitution of one or more ring C-atoms with heteroatoms, such as nitrogen or oxygen. The most straightforward, commercially available compound was 2,4-piperidinedione (26a) and the k obs for this reaction was 11 min À1 or $15-fold faster than dimedone (1) (Chart 3). In subsequent studies, novel derivatization methods (e.g., base mediated alkylation, Ullmann-type arylation and alkyl isocyanate-based urea derivatization) were developed in order to functionalize 26a (Scheme S15 †). Urea-, arylated-and alkylated derivatives of 26a were thus prepared and evaluated for their reactivity with dipeptide sulfenic acid 15. The k obs for reaction of 26b was almost 3-fold less than 26a, suggesting that the electron-withdrawing Boc-group stabilizes the C-3 anion. On the other hand, electron-donating urea 26c (k obs ¼ 13.9 min À1 ) and N-aryl 26d (k obs ¼ 17.3 min À1 ) derivatives gave $20-fold enhancement in reactivity compared to dimedone (1). Interestingly, the k obs for 26e was 35-fold faster than dimedone (1) implying that simple alkylation is sufficient to destabilize the C-3 anion and enhance its reactivity towards sulfenic acid. N-Benzylation (26f, k obs ¼ 86.4 AE 2.2 min À1 ) augmented reactivity 100-fold compared to dimedone (1), underscoring our observation that N-alkylation with EDG leads to an increase in reaction rate constants (Chart 3). Identication of piperidine-2,4-dione 26a as a cyclic C-nucleophile with enhanced reactivity for sulfenic acid represents an important advance, since it is structurally similar to 22a, but its derivatives exhibit rate enhancements of almost two orders of magnitude relative to dimedone (1). Moreover, unlike C-4 alkylation of 22a, N-alkylation (or N-arylation) of 26a is more straightforward from a synthetic point of view. Our ndings at C-4, however, did not extend to C-5, as replacement with an N-heteroatom (26g) afforded a compound with only moderate activity (k obs ¼ 0.2 min À1 ) (Chart 3). The reduced reactivity of 26g stems from its 1,3-dicabonyl functionality (versus keto-lactams (26a-f) and thus shows similar reactivity to compounds listed in Chart 2).
Next, we studied the reactivity of cyclic C-nucleophiles containing two heteroatoms in the ring. The rst such nucleophile screened was commercially available barbituric acid (27a). Barbituric acid (27a) is based on pyrimidine heterocycle skeleton and its pK a is more than one unit lower than dimedone (1) (pK a,barbituric acid ¼ 4.01 versus pK a,dimedone ¼ 5.23). 53 Although (27a) can exist as several different tautomers, the triketo form is generally considered to be most stable. 53 Given its low pK a and greater stabilization of the anion, we anticipated that (27a) would be less reactive than dimedone (1) or 1,3-cyclohexanedione (22a). Consistent with this hypothesis, the k obs for the reaction of (27a) and sulfenic acid 15 was 0.2 min À1 (Chart 4). For subsequent studies, we prepared mono (27b) and dimethylated (27c) derivatives of 27a according to literature procedures. 54 1-Methylbarbituric acid (27b) had similar reactivity to 27a whereas 1,3-dimethylbarbituric acid (27c) displayed a slight rate enhancement ($4-fold increase over 27a). On the other hand, reaction with 2-thiobarbituric acid (27d) resulted in the formation of the expected adduct as well as side products, possibly due to the aromatization of 27d and resulting reactive thiol nucleophile. Among all barbituric acid derivatives examined in our studies, 1,3-dimethyl-2-thiobarbituric acid (27e) gave the highest k obs (2.9 min À1 ) (Chart 4). Meldrum's acid (27f), an oxygen-based heterocycle, was also evaluated for its reactivity. The expected adduct was observed, however, it rapidly decomposed owing to the aqueous instability of lactone 27f. Due to the inherent instability of such lactones, analogous nucleophiles were not pursued further. In short, due to their electron-decient heterocyclic ring, barbituric acid-based nucleophiles exhibit poor reactivity relative to dimedone (1). The slight increase in the reactivity of 27e can be attributed to resonance destabilization of the C-3 carbanion.

The effect of keto-enol tautomerism on cyclic C-nucleophile reactivity
To gain more insight into the effect of enolization on reactivity of cyclic C-nucleophiles, we selected cyclic 1,3-dicarbonyls with at least one carbonyl in conjugation with a phenyl ring, thus shiing the keto-enol tautomerism primarily towards enol form (28a-e, Chart S1 †). Owing to the added stability imparted by aromatization or extended conjugation, compounds 28a-e largely exist as 28a 0 -e 0 . Minor adduct formation was observed for each compound; however, reactions were quite slow and did not proceed to completion. To further evaluate the effect of enolization on nucleophile reactivity, several enamines and hydrazide derivatives of dimedone (1) were prepared. With enamine derivatives (29a-f) either no reaction took place or k obs was too slow to measure (Chart S2 †). Likewise, hydrazide derivatives (30a-d) showed poor reactivity and rate constants were again too slow to measure accurately (Chart S2 †). Together, these data underscore the detrimental effect of aromatic stabilization on cyclic C-nucleophile reactivity with sulfenic acid.
Next, we explored the reactivity of cyclic C-nucleophiles with tautomeric equilibria shied toward the keto form. To this end, we used the commercially available compound, dihydro-2Hthiopyran-3(4H)-one 1,1-dioxide (31a) in which one carbonyl is replaced with a sulfone. 1 H-NMR of 31a in DMSO-d 6 clearly demonstrates that the remaining carbonyl exists predominantly as the keto form (Table S1, † Entry J). k obs for reaction of 31a and sulfenic acid 15 was 2.0 min À1 , which represents a 2.5-fold increase relative to dimedone. This increase in reaction rate of 31a is attributed to the enhanced reactivity of C-2 anion owing to the loss of resonance stability compared to 1,3-dicarbonyl compounds. Following up on this result, the phenyl-conjugated derivative of 31a, isothiochroman-4-one 2,2-dioxide (31b) was prepared using a three-step literature reported procedure. 55 Like 31a, the keto form of 31b predominates (Table S1, † Entry F) and showed a rate enhancement of almost 70-fold (k obs ¼ 54.9 min À1 ), when compared to dimedone (1). Another direct followup to 31a is the class of compounds in which the sulfone is replaced with a sulfonamide moiety, as in 2-alkyl-1,2-thiazinan-5-one 1,1-dioxide (31c, d) 56 and 2-alkyl-2H-1,2-thiazin-5(6H)-one 1,1-dioxide (31e, f) 57 (prepared as described in Scheme S17 †). Both 2-isopropyl-(31c) and 2-benzyl-(31d) 1,2-thiazinan-5-one 1,1-dioxides formed the expected adduct with sulfenic acid 15 with rate constants comparable to dimedone (1) (k obs ¼ 0.6 min À1 for 31c and 0.8 min À1 for 31d). However, 31e and 31f (k obs ¼ 45 min À1 and 73.9 min À1 , respectively) were 50-and 90-fold more reactive than dimedone respectively (1) (Chart 5). It is worth noting that the only structural difference between 31c, d and 31e, f is the presence of a double bond, which is conjugated to the carbonyl. This difference leads to a substantial change in their reactivity towards sulfenic acid. Along these lines, we prepared benzo[c] [1,2]thiazine-based analogs (31g, h) to evaluate the inuence of benzene ring conjugation on sulfenic acid reactivity. 58 Both 31g and 31h readily reacted with sulfenic acid 15 to form stable thioether adducts with relatively fast rate constants (k obs ¼ 138.8 min À1 for 31g and 190.5 AE 12.7 min À1 for 31h) or 200-fold greater, compared to dimedone (1) (Chart 5). The keto forms of 31g and 31h are greatly favored and very small signals from enol tautomers were observed by 1 H-NMR (Table S1, † Entry D). In this regard, the crystal structure of 31g indicates that the heterocyclic ring adopts a half-boat conformation with the sulfone S out of the plane, thus distorting the tetrahedral geometry around the S atom. Since formation of the enol tautomer of 31g would require the ring to be planar, the non-planar heterocyclic ring forces the carbonyl to adopt the keto form. 59 Consequently, the carbanion that forms under aqueous conditions is stabilized by resonance to lesser extent and is extremely reactive. Interestingly, replacement of the sulfonamide with an amide and the carbonyl with a sulfone (i.e., converting benzo[c] [1,2]thiazine analogs to benzo[b] [1,4] thiazines) led to a signicant reduction in reactivity (k obs ¼ 4.5 min À1 for 31i and 0.9 min À1 for 31j) (Chart 5). To summarize, thiazine analogs have shown generally enhanced reactivity compared to dimedone (1) which can be attributed to the effect of two factorsdestabilization of carbanion due to reduced resonance (achieved by replacing one carbonyl with a sulfonamide) and further destabilization of carbanion due to sterics introduced by non-planar heterocyclic ring structure. The maximal cumulative effect of these factors is observed in benzo[c] [1,2]thiazine analogs 31g, h, which exhibited an increase of two orders of magnitude in reactivity compared to dimedone (1).

Reactivity of 1,3-cyclopentanedione derivatives (5-membered ring system)
To further increase the diversity of C-nucleophiles and verify the trends observed with 6-membered ring systems, we evaluated several 5-membered ring systems. As reported above, the lower pK a and complete enolization of 1,3-cyclopentanedione (21a, k obs ¼ 0.02 min À1 ) manifested as a 40-fold decrease in the observed rate constant, compared to dimedone. Our observation is in line with protein-labeling data reported by Furdui and coworkers, 46 however the effect is more pronounced in our model dipeptide sulfenic acid 15. Next, we investigated the effect of C-4 alkylation on the reactivity of 21a. 4-Benzylcyclopentane-1,3-dione (21b) did not show any rate enhancement and 4-benzylidenecyclopentane-1,3-dione (21c) exhibited a total loss of reactivity. Likewise, the C-4 aryl derivative, 4-phenylcyclopentane-1,3-dione (21d) proved unreactive. 4-(Ethylthio) cyclopentane-1,3-dione (21e) successfully reacted with sulfenic acid 15 (k obs ¼ 0.01 min À1 ), although with 2-fold decrease in reactivity relative to 21a. This observation is in contrast to protein-labeling experiments reported by Furdui et al. 46 that show a two-fold rate enhancement with 4-ethylthio substitution, compared to 21a. However, this apparent discordance with our data is readily explained by the presence of empty d-orbitals on the S atom that exerts a net electron-withdrawing effect on the 1,3-cyclopentanedione ring with concomitant stabilization of the C-2 carbanion and reduced reactivity with sulfenic acid. These contrasting data highlight the impact that protein microenvironment can have on probe reactivity and suggest that intrinsic nucleophile reactivity is best studied in smallmolecule sulfenic acid model systems (Chart 6).
Since replacement of C-4 with an N-heteroatom yielded substantial rate enhancements in 6-membered C-nucleophile ring systems, we investigated similar heteroatom substitutions in 1,3-cyclopentanedione (21a). Replacing C-4 with an Oheteroatom gave the commercially available lactone, tetronic acid (32a), which formed the expected adduct with sulfenic acid 15 (k obs ¼ 0.04 min À1 ), albeit with only a two-fold increase in reactivity compared to 21a. Next, we replaced C-4 with an Nheteroatom in the reaction of 2,4-pyrrolidinedione (32b) with sulfenic acid 15. The k obs value for this derivative was 0.8 min À1 , which represents a 40-fold increase over 21a and is also equivalent to dimedone (1). The N-alkylated nucleophile, 1-benzylpyrrolidine-2,4-dione (32c) exhibited a rate acceleration of more than 1000-fold (k obs ¼ 21.3 min À1 ) relative to 21a, representing more than a 25-fold rate enhancement compared to dimedone (1). Similarly, the N-arylated C-nucleophile, 1-phenylpyrrolidine-2,4-dione (32d) was 250-fold more reactive (k obs ¼ 5.2 min À1 ) than 21a and 5-fold more reactive than dimedone (1) (Chart 6). In this regard, we note that although 1 H-NMR analysis of (21a) in DMSO-d 6 indicates that this compound exists exclusively in the enol form, analogous spectra of 32c and 32d show a respective 10 : 3 and 1 : 1 ratio of keto to enol tautomeric forms, respectively (Table S1, † Entries K and L). These observations again suggest that shiing the tautomeric equilibrium to favor the keto form is a general mechanism to increase the reactivity of these C-nucleophiles toward sulfenic acid. Two substituting N-heteroatoms, as in 3,5-pyrazolidinedione (32e), accelerated reactivity 35-fold (k obs ¼ 0.7 min À1 ) when compared to 21a, but remained similar in reaction rate constant to dimedone (1) (Chart 6).

Evaluating C-nucleophile selectivity and thioether bond stability
Increased C-nucleophile reactivity may lead to decreased selectivity for the sulfenic acid target. Consequently, we thought it prudent to screen representative C-nucleophiles (1, 26a, 31f, 31h, 34b) for cross-reactivity with other biological functional groups (Scheme S19 †). For these studies, we utilized Fmoc (or Cbz)protected amino acids cysteine (thiol), serine (alcohol), lysine (amine), cystine (disulde) as well as sulnic acid (BnSO 2 Na) in aqueous buffer at pH 7.4. The resulting data demonstrate that the majority of nucleophiles retained their selectivity for sulfenic acid. One exception to these ndings was 2-benzyl-1,2-thiazinan-5-one 1,1-dioxide (bTD, 31f), which gave the expected Michael adduct with Fmoc-Lys-OH (Scheme S19, Fig. S13 †). Next, we evaluated the stability of the thioether bond formed between C-nucleophiles 1, 26a and 31h and 15 under reducing conditions, such as that encountered within the cytosol. For these studies, the dipeptide-nucleophile product from each reaction was puried and analyzed by NMR to establish that the correct thioether bond was formed (Scheme S20 †). Incubation of each product with millimolar concentration of dithiothreitol (DTT), glutathione (GSH) or tris(2-carboxyethyl) phosphine (TCEP) indicated that each adduct was stable for more than 12 h (Scheme S21 †). These cross-reactivity and stability studies affirm the selectivity of the C-nucleophiles for sulfenic acid and the irreversible nature of thioether adduct thus formed.

Discussion
Although numerous studies proling electrophiles as reactivity probes for thiols have been reported, 27,63-66 to our knowledge, this study represents the rst of its kind to comprehensively prole nucleophiles as reactivity probes for the related sulfur oxoform, sulfenic acid. Herein, we have conceived, synthesized and screened several classes of cyclic C-nucleophiles for their reactivity with a novel model dipeptide sulfenic acid using a newly developed, facile LC-MS assay. The observed rate constants obtained from the ts to the ensuing data enables the stratication of C-nucleophiles based on their reaction kinetics. Our approach is user-friendly and utilizes a simply prepared dipeptide that can be stored in stable form until it is needed for conversion to sulfenic acid under aqueous conditions. Thus, this work addresses a fundamental, previously unmet need for a workow that expedites the identication of compounds, which react with cysteine sulfenic acid over a broad range of time scales (10 to 2Â10 5 M À1 min À1 ).
A major goal of this study was to identify new classes of cyclic C-nucleophiles with robust reaction kinetics for future development as cellular probes of protein sulfenic acid. To this end, in the present work, we have identied several classes of cyclic C-nucleophiles with 100-to 200-fold enhanced rate of reaction compared to dimedone (1). Screening nucleophiles based on ring size showed that reactivity increases with the shi from the enol to keto forms, indicating that factors resulting in the destabilization of carbanion at C-2 positively inuence reactivity. The destabilization and reactivity of the C-2 carbanion was found to depend upon three primary factors: (i) electronic effects, as EDG substitution of the ring system enhances C-2 reactivity and vice versa; (ii) loss of resonance stability, and (iii) steric factors, which inuence the ring to achieve non-planar forms. In Chart 2, we observe the effect of EDG or EWG substitution, which cause a respective increase or decrease in reactivity of cyclic C-nucleophiles towards sulfenic acid. Nucleophiles based on the 2,4-piperidinedione (26a) scaffold had one of the carbonyls replaced with a lactam, resulting in loss of resonance stabilization and a substantially more reactive carbanion (Chart 3). In general, barbituric acid derivatives were electron-decient heterocycles and resonance stabilized, which lead to reduced reactivity towards sulfenic acid (Chart 4). The greater reactivity of thiazine nucleophiles stems from the decrease in resonance stabilization and steric factors introduced by the sulfonamide substitution (Chart 5). The maximal additive effect of these two factors was observed for benzo[c] [1,2]thiazine analogs 31g, h, which were $200-fold more reactive towards sulfenic acid compared to dimedone (1). Lastly, 5-membered cyclic nucleophiles followed same reactivity trends, but showed reduced reactivity relative to 6-membered counterparts (Chart 6). The observed enhancement in reactivity was further veried by obtaining 2 nd order rate constants for representative reactive cyclic C-nucleophiles (Chart S3 †). For example, the 2 nd order rate constant for benzyl-PRD (26f, k obs ¼ 1192 M À1 s À1 ) showed a 100-fold increase compared to dimedone (1, k obs ¼ 11.8 M À1 s À1 ). Likewise, the rate enhancement calculated from the 2 nd order rate constants of benzyl-BTD (31h, k obs ¼ 1725 M À1 s À1 ) and 1,3-indandione (34b, k obs ¼ 251 M À1 s À1 ) agreed well with the reactivity increase obtained from earlier pseudo 1 st order rate constant values (Chart S3 †).
Finally, with the re-emergence of covalent inhibition strategies, 67-71 one possible use of our cyclic C-nucleophile library is toward the development of inhibitors that target oxidized cysteine residues in therapeutically important proteins, such as kinases. With the FDA approval of afatinib 72 and ibrutinib, 73 Cys-targeting covalent inhibitors of the ErbB family of tyrosine kinases and Bruton tyrosine kinase (BTK) respectively, inhibition of receptor tyrosine kinase signaling has emerged as one of the more effective anticancer treatment strategy. Recent ndings indicate that elevated EGFR and HER2 (ErbB family) levels in cancer cells correlate with an increase in H 2 O 2 levels and global protein sulfenylation. 74,75 Moreover, we have previously reported that Cys 797 of EGFR undergoes sulfenic acid modication. 7 Because of its electrophilic nature, EGFR-Cys 797 -SOH precludes the covalent bond formation with electrophilic inhibitors like afatinib, resulting in signicant loss of overall effectiveness. However, it also presents a unique opportunity to utilize the nucleophiles library as warheads to target electrophilic EGFR-Cys 797 -SOH (Fig. 4). With nine other protein tyrosine kinases including BTK 76 (Cys 481 ) harboring a Cys residue that is structurally homologous to EGFR-Cys 797 , 68 this group of kinases may be regulated by oxidation of this key residue and susceptible to irreversible inhibition by nucleophilic redoxbased inhibitors (Fig. 5). These studies are currently underway in our laboratory and will be reported in due course.

Conclusions
We have reported a facile mass spectrometry-based assay and repurposed dipeptide-based model to screen a library of cyclic C-nucleophiles for reactivity with sulfenic acid under aqueous conditions. Observed rate constants for $100 cyclic C-nucleophiles were obtained and, from this collection, we have identi-ed novel compounds with more than 200-fold enhanced reactivity, as compared to dimedone (1). The increase in reactivity and retention of selectivity of these C-nucleophiles were validated in secondary assays, including a protein model for sulfenic acid. Together, this work represents a signicant step toward developing new chemical reporters for detecting protein S-sulfenylation with superior kinetic resolution. The enhanced rates and varied composition of the C-nucleophiles should enable more comprehensive analyses of the sulfenome and serve as the foundation for reversible or irreversible nucleophilic covalent inhibitors that target oxidized cysteine residues in therapeutically important proteins. Electrophilic covalent inhibitors inactivate their target through covalent attachment to the cysteine thiol functional group. However, the electrophilic center (e.g., acrylamide, haloacetamide, and vinyl sulfonamide) can also react with other cellular nucleophiles such as glutathione as well as the amino and imidazole groups of amino acids. (B) Nucleophilic covalent strategy as an alternative or complementary inhibition mechanism. According to this approach, active site-directed smallmolecule inhibitors containing a reactive nucleophilic center form a covalent bond with a cysteine side chain that has oxidized to sulfenic acid. Such modifications form transiently in specific proteins during H 2 O 2 -mediated signal transduction in normal cells, but form constitutively in diseases associated with chronically elevated levels of H 2 O 2 , including cancer. In the sulfenic acid oxidation state, the electron deficient sulfur exhibits enhanced electrophilic character that can be selectively targeted by certain nucleophilic compounds. Because sulfenic acid is a unique chemical moiety in biochemistry, this strategy could decrease the potential for off-target activity while retaining the advantages gained by covalent targeting.