Trapping Reactions of the Sulfenyl and Sulfinyl Tautomers of

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Trapping Reactions of the Sulfenyl and Sulfinyl Tautomers of Sulfenic Acids Murugaeson R. Kumar and Patrick J. Farmer* Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798, United States ABSTRACT: Sulfenic acids react as both nucleophiles and electrophiles, which may be attributable to interconversion between sulfenyl and sulfinyl tautomers. We demonstrate onepot trapping of both tautomeric forms of glutathione sulfenic acid by LCMS. The sulfinyl tautomers are characterized by reaction with nucleophilic reagents such as dimedone and cyanide, giving unique products that are analogous to corresponding adducts of aldehydes. Likewise, we show that aldehyde reactive reagents such as silyl enol ethers also react with glutathione sulfenic acid to give products characteristic of both sulfenyl and sulfinyl tautomers.

S

In a typical experiment, GSH (1 mM) was reacted with H2O2 (1.2 equiv) in the presence of dimedone (5 mM) in pH 7 buffered aqueous solution. In Figure 1, the expected dimedone thioether derivative (1) is seen, as well as the hitherto unknown xathinedione (2). Compound 2 is rationalized as deriving from

ulfenic acids are initial products of biological thiol oxidations and as such act as a direct indicator of oxidative stress, which is important in the diagnosis and treatment of chronic illness.1−4 For example, oxidation of glutathione, GSH, by peroxide produces the sulfenic acid GSOH, in eq 1, as characterized by variety of a protocols, most notably by mass spectrometry;5−17 GSOH further reacts with the parent thiol to form the stable oxidized disulfide GSSG, in eq 2. Transient sulfenic acids in the biological milieu are most commonly characterized by S-alkylation with acidic carbon enolates such as dimedone, DmH, in eq 3, and subsequently characterized by fluorescence or proteomic mass spectrometry.18 GSH + H 2O2 → GSOH + H 2O

(1)

GSOH + GSH → GSSG + H 2O

(2)

GSOH + DmH → GSDm + H 2O

(3)

Sulfenic acids react as nucleophiles or electrophiles, which may be attributable to interconversion between sulfenyl and sulfinyl tautomers, Scheme 1.19,20 Recent DFT studies have Scheme 1

examined the equilibration between hydrogen thioperoxide tautomers, HSOH and H2SO, confirming the sulfenyl as the lowest energy state, but inclusion of water molecules dramatically lowers the energy of the sulfinyl tautomer.21 In this work, we report trapped products derived from glutathione (GSH) oxidation that most reasonably derive from distinct tautomers, and thus may provide a method to characterize the tautomeric equilibration in situ. © XXXX American Chemical Society

Figure 1. Selective ion chromatogram and mass spectra of compounds 3 and 4 obtained in the oxidation of GSH (1 mM) with peroxide (1.2 mM) in the presence of dimedone (5 mM). Received: November 4, 2016 Accepted: December 16, 2016 Published: December 16, 2016 A

DOI: 10.1021/acschembio.6b00980 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology the sulfinyl tautomer, by analogy to similar xanthenediones formed in reactions of aldehydes with dimedone, species 3 in Scheme 2.22,23 The Orbitrap LC/MS in the positive ion mode

Scheme 3

Scheme 2

using the gradient elution method with 0.1% formic acid− acetonitrile solvent mixture is used to identify reaction products as [M + H+] singly charged ion peaks, with expected isotope patterns for 34S and 13C abundances. Single Ion Chromatograms (SICs) are shown to confirm that the observed species are present in the reaction mixture and separated on the LC column prior to ionization. Quantification of the peak areas of the respective SICs gives a ratio of 1/2 at ca. 1000:1, suggesting a moderate free energy difference between the tautomers.24 We have no data on the rate of interconversion between the tautomers, but the observation of both suggest that it is slow. One caveat is that species 1 is formed by a single bimolecular step, while 2 derives from a series of coupling reactions. Both 1 and 2 are also observed in reactions of GSH with other oxidants such as hypochlorite and ozone. The possible presence of the sulfinyl tautomer led us to try other reactions characteristic of aldehydes. The Mukaiyama aldol reaction of aldehydes with silyl enol ethers yields 1,3 hydroxyketones,25 e.g., species 4 in Scheme 3. When GSH is oxidized in the presence of 1-trimethylsiloxycyclohexene, multiple cyclohexone adducts are observed, Figure 2. As rationalized in Scheme 3, the sulfenyl tautomer GSOH acts as a nucleophile in a Mukaiyama-like addition to the olefin, generating species 5. The sulfinyl tautomer GS(O)H undergoes an aldol-like electrophilic reaction to give species 6. A similar quantification of SICs gives the ratio of the initial products 5/6 as ca. 3:1, much lower than expected but perhaps due to the relative rates of the aldol and Mukaiyama reactivities. A second molecule of 1-trimethylsiloxycyclohexene can further undergo aldol type condensation with 5 to yield 7, Scheme 3, and likewise species 8 can be formed from 6 by a Knoevenagel-like type reaction. The selective ion chromatogram of species 8 has

Figure 2. Selective ion chromatogram and mass spectra of products 5 and 6, obtained in oxidation of GSH (1 mM) with peroxide (1.2 mM) in the presence of 1-trimethylsiloxycyclohexene (5 mM).

two peaks, suggesting it is a mixture of cis- and trans- tautomers, Figure 2. We tested the silyl enol ether trap’s ability to distinguish between tautomers generated in the aqueous peroxidation of methimazole, 9, Scheme 4. A recent report suggested sulfenyl 10 and S-oxide 11 tautomers, but not the sulfinyl 12.26 In the absence of a trap, the nominal sulfenic acid mass is seen, attributable to any tautomer. However, in the presence of the 1B

DOI: 10.1021/acschembio.6b00980 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Scheme 4

Scheme 5

trimethylsiloxycyclohexene, adducts attributable to all three tautomers are observed as shown in Figure 3; quantification of the SIC areas assigned to tautomers 10, 11, and 12 gives a relative abundance as 100:54:1, respectively.

Figure 4. Mass spectra of products glutathione adducts of cyanide obtained in oxidation of GSH (1 mM) with peroxide (1.2 mM) in the presence of varying KCN: (a, b) 5 mM and (c) 20 mM.

Scheme 6

Figure 3. Mass spectra of products methimazole sulfenic acid obtained in the oxidation of GSH (1 mM) with peroxide (1.2 mM) in the presence of 1-trimethylsiloxycyclohexene (5 mM).

Another characteristic reaction of aldehydes is the nucleophilic addition of cyanide generating cyanohydrins, eq 4.27 Indeed, the addition of KCN to the reaction mixtures during GSOH generation obtains products corresponding to the mono- and bis-cyano adducts, species 13 and 14 shown in Scheme 5, with MS data shown in Figure 4. A second addition of cyanide to the cyanohydrins is unknown, but a dicyanosulfanyl species 14 appears at high cyanide concentrations. The thiocyanide adduct, GSCN 15, is also observed, which may form by condensation of GSOH with HCN or by elimination of HCN from the dicyanide 14. At low cyanide concentrations, the ratio of 15 to 13 from their respective SIC peak areas is 100:2. RHC = O + HCN → RHC(OH)CN

(4)

the oxidative transformation of ampicillin 16 to cephalosporin 18.29 Peroxidation of 16 in the presence of 1-trimethylsiloxycyclohexene produces trapped species 21 and 22, characteristic of both sulfenyl and sulfinyl tautomers, respectively, Figure

As a proof-of-concept demonstration, we applied the aldehyde-reactive reagents to trap the sulfenic tautomers of the Morin intermediate,28 19 and 20 in Scheme 6, proposed in C

DOI: 10.1021/acschembio.6b00980 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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5; SIC quantification of the tautomers gives a ratio of 100:15. When the same reaction was carried out in the presence of

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Patrick J. Farmer: 0000-0001-9911-999X Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank K. L. Shuford and J. L.Wood for their comments and insights during the preparation of this report, which is based upon work supported by the National Science Foundation (PJF CHE-1057942), and from Baylor University Mass Spectroscopy Facility.



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Figure 5. Mass spectra of products Morin intermediate sulfenic acid obtained in oxidation of ampicillin (1 mM) with peroxide (1.2 mM) in the presence of 1-trimethylsiloxycyclohexene (5 mM).

potassium cyanide, analogous sulfenyl and sulfinyl derivatives, 23 and 24 respectively, were observed in a ratio of 100:89 calculated from their SIC plots, Figure 6.

Figure 6. Mass spectra of products Morin intermediate sulfenic acid obtained in oxidation of ampicillin (1 mM) with peroxide (1.2 mM) in the presence of KCN (5 mM).

In all reactions studied here, the sulfenyl tautomer predominates over the sulfinyl, but not to the extent that theoretical literature would predict. Especially important, in our view, is the analogous reactivity of sulfinyl tautomers and aldehydes, which may lead to new chemical strategies to identify and discriminate sulfenic acid tautomers in biological systems. D

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DOI: 10.1021/acschembio.6b00980 ACS Chem. Biol. XXXX, XXX, XXX−XXX