Trapping Reactions of the Sulfenyl and Sulfinyl Tautomers of Sulfenic Acids

Article abstract of DOI:10.1021/acschembio.6b00980

Sulfenic acids react as both nucleophiles and electrophiles, which may be attributable to interconversion between sulfenyl and sulfinyl tautomers. We demonstrate one-pot trapping of both tautomeric forms of glutathione sulfenic acid by LCMS. The sulfinyl

Full text of DOI:10.1021/acschembio.6b00980

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pubs.acs.org/acschemicalbiology
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 one-
pot 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.
ulfenic acids are initial products of biological thiol
oxidations and as such act as a direct indicator of oxidative
In a typical experiment, GSH (1 mM) was reacted with H2O2
S
(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
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 + H2O2 → GSOH + H2O
(1)
GSOH + GSH → GSSG + H2O
(2)
GSOH + DmH → GSDm + H2O
(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.
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
© XXXX American Chemical Society
A
DOI: 10.1021/acschembio.6b00980
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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 Chromato-
grams (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 1-
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DOI: 10.1021/acschembio.6b00980
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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 concen-
trations. 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
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
(4)
the oxidative transformation of ampicillin 16 to cephalosporin
18.29 Peroxidation of 16 in the presence of 1-trimethylsilox-
ycyclohexene produces trapped species 21 and 22, character-
istic of both sulfenyl and sulfinyl tautomers, respectively, Figure
C
DOI: 10.1021/acschembio.6b00980
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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
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Patrick_Farmer@Baylor.edu.
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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