Light-Mediated Sulfenic Acid Generation from Photocaged Cysteine Sulfoxide

Article abstract of DOI:10.1021/acs.orglett.5b02981

S-Sulfenylation is a post-translational modification with a crucial role in regulating protein function. However, its analysis has remained challenging due to the lack of facile sulfenic acid models. We report the first photocaged cysteine sulfenic acid w

Full text of DOI:10.1021/acs.orglett.5b02981

Letter
pubs.acs.org/OrgLett
Light-Mediated Sulfenic Acid Generation from Photocaged Cysteine
Sulfoxide
Jia Pan and Kate S. Carroll*
The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: S-Sulfenylation is a post-translational modification
with a crucial role in regulating protein function. However, its
analysis has remained challenging due to the lack of facile sulfenic
acid models. We report the first photocaged cysteine sulfenic acid
with efficient photodeprotection and demonstrate its utility by
generating sulfenic acid in a thiol peroxidase after illumination in
vitro. These caged sulfoxides should be promising for site-specific
incorporation of Cys sulfenic acid in living cells via genetic code
expansion.
he amino acid cysteine plays an essential role in redox signal
transduction owing to its susceptibility to oxidation by
T
various reactive oxygen species (ROS).1 Among possible
oxidative post-translational modifications of cysteine (Cys), S-
sulfenylation has become the subject of growing attention in
recent years.2 During redox signaling or under conditions of
oxidative stress, a reactive Cys thiol (−SH) can be oxidized to
sulfenic acid (−SOH) by ROS, namely hydrogen peroxide
(H2O2), and this process can be reversed by biological
reductants, such as the enzyme, thioredoxin3 (Trx), or the
tripeptide glutathione4 (GSH) (Scheme 1). To date, regulatory
Figure 1. Examples of small-molecule sulfenic acids stabilized via an
intramolecular hydrogen bond (1) or steric effects (2).
istics or reactivity of Cys sulfenic acid, and cannot be stored for an
extended period of time. In proteins, the stability of Cys sulfenic
acid is determined by the surrounding microenvironment, and
the absence of vicinal thiols14 and presence of basic residues15 are
often cited as key features. Protein sulfenic acid formation in vitro
and in cells is most often achieved by incubation with exogenous
oxidants like H2O2, organo hydroperoxides,2b or elevating
endogenous ROS production via treatment with growth factor
or insulin.1b Nevertheless, uncontrolled oxidation of reactive Cys
residue(s) stemming from such methods often makes it difficult
to study sulfenylation of specific proteins at defined sites within
redox signaling pathways.
Caged compounds are precursors of biologically active
molecules that have been rendered inactive by installation of a
photolabile protecting group (PPG) onto the essential
functionality.16 After illumination, the PG is cleaved and the
caged biomolecule is released irreversibly, thus revealing the
active species. Photocaged Cys has been site-specifically
incorporated to study thiol function and targeted covalent
labeling in small molecules,17 peptides,18 and proteins.19
Photocaged selenocysteine has also been reported.20 Despite
Scheme 1. Biologically Relevant Cysteine Oxoforms
Cys sulfenic acid modifications have been identified in many
signaling proteins such as tyrosine phosphatases,5 kinases,6
transcription factors,7 proteases,a deubiquitinases,9 and ion
channels.10 Aberrant protein sulfenylation has also been
correlated with human pathologies, including cancer11 and
cardiovascular disease.10
While protein sulfenylation is an important post-translational
modification, the analysis of this Cys oxoform has remained
challenging due to the lack of facile sulfenic acid model systems.
The challenge is significant given that sulfenic acids are often
unstable, transient species that can rapidly react to form
thiosulfinate and disulfide species.2b Some progress has been
made in defining sulfenic acid stabilization and properties in
small-molecule models such as 112 and 213 (Figure 1); however,
such compounds still suffer from complex syntheses, poor
aqueous solubility, do not adequately recapitulate the character-
Received: October 14, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02981
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
these encouraging advances and the benefits of photocontrol,
methods to incorporate defined Cys “oxoforms”, such as sulfenic
acid, have not yet been described. Herein, we report the first
photocaged Cys sulfenic acid analogues and establish conditions
for efficient photodeprotection. We demonstrate the utility of
this approach by generating Cys sulfenic acid in a thiol
peroxidase, following illumination in vitro. Overall, these
photocaged cysteine sulfenic acid analogues should have
considerable utility for the site-specific incorporation of Cys
sulfenic acid within small molecules, proteins, and eventually,
living cells via genetic code expansion.
Next, we tested whether we could observe the formation of
sulfenic acid from model caged sulfoxide 3b. Illumination of 3b
(λmax = 350 nm, ε350 nm = 18590 M−1 cm−1, ε365 nm = 16500 M−1
cm−1, Φchem = 0.13) with UV light at 365 nm (0.35 W/cm2) led
to complete consumption of 3b and formation of sulfinic acid 10
(45% yield) and disulfide 11 (39% yield) as the major products
(Scheme 4A). Formation of these products can be explained due
Scheme 4. (A) Uncaging Reaction of Caged Sulfoxide 3b
under UV. (B) LC Traces of the Uncaging Reaction of 3b in 4
h
In the current investigation, we have explored a novel strategy
based on a photolabile (o-nitrobenzyl, ONB or 4,5-dimethoxy-2-
nitrobenzyl, DMNB) group to protect Cys sulfoxide (CysO)
analogs (Scheme 2). In this approach, we reasoned that PG
Scheme 2. Proposed Mechanism for Formation of Sulfenic
Acid from Caged Sulfoxide Species via Illumination
to the disproportionation reaction of sulfenic acid to sulfinic acid
10 and a thiol, which then reacts with sulfenic acid to give
disulfide 11.21 By measuring the consumption of 3b, the pseudo-
first-order rate constant (kobs) for photocleavage was determined
to be 0.17 min−1 (Scheme 4B and Figures S2 and S3).
To verify formation of the expected sulfenic acid product, we
exploited the dual electrophilic and nucleophilic reactivity of this
sulfur oxoform.1f,2b When N-ethylmaleimide (NEM) was added
to the reaction, the anticipated sulfoxide−NEM adduct 12 was
formed (36% yield, Scheme 5A).22 Correspondingly, when
cleavage would proceed via benzoisoxazole 4, which would
rearrange to give α-hydroxyl sulfoxide 5, and conclude with
elimination of 6 to give sulfenic acid 7. We first focused our
attention on the chemical synthesis of o-nitrobenzyl (ONB) and
4,5-dimethoxy-2nitrobenzyl (DMNB) CysO and Cys analogues,
such that photocleavage and reactivity of CysO and its reduced
counterpart could be effectively compared (Scheme 3). In short,
Scheme 3. Synthesis of Caged Sulfoxides 3a and 3b
Scheme 5. Uncaging and Chemical Trapping of (A) Caged
Sulfoxide 3b and (B) Reduced analogue 8b
photocaged Cys compounds (8a, 8b) were generated from N-
acetylcysteine benzylamide 9 following literature procedures.17
Subsequently, oxidation of photocaged Cys analogues to the
corresponding sulfoxides (3a, 3b) was achieved using H2O2 in
the presence of acetic acid in excellent yields (≥90%). In the
absence of illumination, all compounds were stable in aqueous
media at neutral pH values, as expected (Figure S1).
carbon-based nucleophiles like piperidine-2,4-dione (PD)23
were included to trap the nacent sulfenic acid, the expected
sulfide adduct 13 was observed (80% yield, Scheme 5A). Control
experiments, performed under the same reaction conditions,
clearly demonstrate that caged Cys analogue 8b formed the
appropriate sulfide adduct 14 with NEM (42% yield) and did not
react with the PD nucleophile (Scheme 5B).24 These data
B
DOI: 10.1021/acs.orglett.5b02981
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
establish formation of sulfenic acid from the caged sulfoxide
starting material, as anticipated by our strategy.
Figure 2A) would be a substrate for the MetO repair enzyme
(MsrA).28 As shown in Figure 2B, MetO was efficiently reduced
To determine whether our approach could be applied to
proteins, we next examined photolytic generation of Cys sulfenic
acid from a DMNB-protected sulfoxide precursor in the thiol
peroxidase, Gpx3 (Cys64Ser Cys82Ser Gpx3 was used to avoid
multiple alkylation events). Caged Gpx3 Cys32 sulfoxide, termed
Cys32O, was prepared by alkylation of the catalytic Cys32 of
Gpx3 with dimethoxy-o-nitrobenzyl bromide (DMNB-Br),
followed by oxidation with H2O2. During the oxidation step,
we observed that methionines (Met) in Gpx3 were converted to
Met sulfoxide (MetO) (expanded intact ESI-MS spectra
presented in Figure S4).25 As shown in Scheme 6A, illumination
Scheme 6. Photochemical Generation of Sulfenic Acid in a
Protein. (A) ESI-MS Spectrum of Intact Gpx3. (B) Extracted
Ion Intensities of Gpx3 Cys32O Consumption (Black Dots)
and Gpx3-PD Formation (Gray Dots)
Figure 2. (A) Unprotected analogue of 3a. (B) MsrA activity assay with
sulfoxide 15 (gray dots) and MetO (black dots) as putative substrates.
by MsrA, while 15 remained intact. These data demonstrate that
photocaged CysO is not a substrate for MsrA, which bodes well
for the intracellular stability and application of caged Cys
sulfoxide toward genetic encoding of Cys sulfenic acid in future
work.
In summary, we have designed, synthesized, and evaluated
first-in-class photocaged cysteine sulfenic acid analogues. From
practical and conceptual standpoints, this work represents an
important advance for the thiol-based redox regulation and
chemical biology communities due to the potential for site-
specific incorporation of an oxidative cysteine post-translational
modification. From a chemistry perspective, caged sulfenic acids
should find broad application owing to their facile preparation
and storage within a large range of scaffolds, including peptides
and proteins. Evolution of the requisite orthogonal synthetase/
tRNA pair for site-specific incorporation of Cys sulfenic acid
within living cells via genetic code expansion is currently
underway and will be reported in due course.
of Gpx3 Cys32O−DMNB at 365 nm followed by intact ESI-MS
demonstrates the full conversion of the DMNB-caged sulfoxide
to Gpx3 Cys32 sulfenic acid with nearly quantitative trapping of
this intermediate by the PD nucleophile. Control photo-
uncaging experiments, in which Gpx3 Cys32 was alkylated
with DMNB-Br but not oxidized with H2O2, did not lead to the
formation of PD-tagged Gpx3, as expected (Figure S3). A time−
course study was also carried out to show controllable release of
Gpx3 sulfenic acid via consumption of caged Gpx3 Cys32O (kobs
= 0.013 min−1) and robust formation of the anticipated Gpx3-PD
adduct (kobs = 0.011 min−1, Scheme 6B). Although removal of
additional Cys residues and Met oxidation may limit the utility of
our approach in this context, these experiments serve as an
important proof-of-principle that CysO-DMNB can be effi-
ciently uncaged in a protein model to afford Cys sulfenic acid.
Many applications of this approach can be envisioned, and we
are particularly interested in the ability to genetically encode the
site-specific incorporation26,27 of a photocaged Cys sulfenic acid
residue into a target protein within cells. Evolution of the
requisite orthogonal synthetase/tRNA pair to incorporate the
aforementioned unnatural amino acid (UAA) will be reported in
due course. At this stage, however, given the structural similarity
with MetO, we deemed it essential to test whether caged CysO
15 (analogue of 3a with free amino and carboxylate groups,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.5b02981.
■
S
Experimental procedures and spectroscopic data (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: kcarroll@scripps.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM102187 and CA174864 to K.S.C.). We also thank Dr.
Prakash Palde (The Scripps Research Institute) for his support
on this project.
REFERENCES
■
(1) (a) Reddie, K. G.; Carroll, K. S. Curr. Opin. Chem. Biol. 2008, 12,
746. (b) Paulsen, C. E.; Carroll, K. S. ACS Chem. Biol. 2010, 5, 47.
(c) Miki, H.; Funato, Y. J. Biochem. 2012, 151, 255. (d) Jacob, C.;
C
DOI: 10.1021/acs.orglett.5b02981
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Battaglia, E.; Burkholz, T.; Peng, D.; Bagrel, D.; Montenarh, M. Chem.
Res. Toxicol. 2012, 25, 588. (e) Chung, H. S.; Wang, S. B.; Venkatraman,
V.; Murray, C. I.; Van Eyk, J. E. Circ. Res. 2013, 112, 382. (f) Paulsen, C.
E.; Carroll, K. S. Chem. Rev. 2013, 113, 4633.
(20) Rakauskaite, R.; Urbanaviciute, G.; Ruksenaite, A.; Liutkeviciute,
Z.; Juskenas, R.; Masevicius, V.; Klimasauskas, S. Chem. Commun. 2015,
51, 8245.
(21) (a) Davis, F. A.; Jenkins, R. H.; Rizvi, S. Q. A.; Yocklovich, S. G. J.
Org. Chem. 1981, 46, 3467. (b) Nagy, P.; Ashby, T. Chem. Res. Toxicol.
2007, 20, 1364. (c) Nagy, P.; Winterbourn, C. C. Adv. Mol. Toxicology
2010, 4, 183. (d) Acharjee, S. R.; Bhattacharjee, S. K. IOSR J. Appl. Chem.
2013, 3, 40.
(22) Another major product was sulfide adduct 14 in 33% yield.
(23) Gupta, V.; Carroll, K. S. Chem. Sci. 2015, DOI: 10.1039/
C5SC02569A.
(24) The uncaging of 8b was less effective than 3b, and almost half of
8b remained unreacted after 3 h. In the reaction of 8b with PD the major
product was reduced cysteine analogue 9.
(2) (a) Lo Conte, M.; Carroll, K. S. J. Biol. Chem. 2013, 288, 26480.
(b) Gupta, V.; Carroll, K. S. Biochim. Biophys. Acta, Gen. Subj. 2013,
1840, 847. (c) Pan, J.; Carroll, K. S. Biopolymers 2014, 101, 165.
(3) (a) Claiborne, A.; Miller, H.; Parsonage, D.; Ross, R. P. FASEB J.
1993, 7, 1483. (b) Crane, E. J., 3rd; Parsonage, D.; Poole, L. B.;
Claiborne, A. Biochemistry 1995, 34, 14114. (c) Kim, H. Y.; Kim, J. R.
Biochem. Biophys. Res. Commun. 2008, 371, 490. (d) Depuydt, M.;
Leonard, S. E.; Vertommen, D.; Denoncin, K.; Morsomme, P.; Wahni,
K.; Messens, J.; Carroll, K. S.; Collet, J. F. Science 2009, 326, 1109.
(4) (a) Mieyal, J. J.; Gallogly, M. M.; Qanungo, S.; Sabens, E. A.;
Shelton, M. D. Antioxid. Redox Signaling 2008, 10, 1941. (b) Townsend,
D. M. Mol. Interventions 2007, 7, 313.
(5) (a) Denu, J. M.; Tanner, K. G. Biochemistry 1998, 37, 5633.
(b) Salmeen, A.; Andersen, J. N.; Myers, M. P.; Meng, T. C.; Hinks, J. A.;
Tonks, N. K.; Barford, D. Nature 2003, 423, 769. (c) Michalek, R. D.;
Nelson, K. J.; Holbrook, B. C.; Yi, J. S.; Stridiron, D.; Daniel, L. W.;
Fetrow, J. S.; King, S. B.; Poole, S. B.; Grayson, J. M. J. Immunol. 2007,
179, 6456.
(25) Seven Met in Gpx3 are oxidized to Met sulfoxide.
(26) Lemke, E. A.; Summerer, D.; Geierstanger, B. H.; Brittain, S. M.;
Schultz, P. G. Nat. Chem. Biol. 2007, 3, 769.
(27) (a) Wu, N.; Deiters, A.; Cropp, T. A.; King, D.; Schultz, P. G. J.
Am. Chem. Soc. 2004, 126, 14306. (b) Deiters, A.; Groff, D.; Ryu, Y.; Xie,
J.; Schultz, P. G. Angew. Chem., Int. Ed. 2006, 45, 2728.
(28) (a) Lowther, W. T.; Brot, N.; Weissbach, H.; Matthews, B. W.
Biochemistry 2000, 39, 13307. (b) Weissbach, H.; Etienne, F.; Hoshi, T.;
Heinemann, S. H.; Lowther, W. T.; Matthews, B.; John, G. S.; Nathan,
C.; Brot, N. Arch. Biochem. Biophys. 2002, 397, 172. (c) Boschi-Muller,
S.; Gand, A.; Branlant, G. Arch. Biochem. Biophys. 2008, 474, 266.
(6) (a) Paulsen, C. E.; Truong, T. H.; Garcia, F. J.; Homann, A.; Gupta,
V.; Leonard, S. E.; Carroll, K. S. Nat. Chem. Biol. 2011, 8, 57. (b) Wani,
R.; Qian, J.; Yin, L.; Bechtold, E.; King, S. B.; Poole, L. B.; Paek, E.;
Tsang, A. W.; Furdui, C. M. Proc. Natl. Acad. Sci. U. S. A. 2011, 108,
10550.
(7) (a) Lee, C.; Lee, S. M.; Mukhopadhyay, P.; Kim, S. J.; Lee, S. C.;
Ahn, W. S.; Yu, M. H.; Storz, G.; Ryu, S. E. Nat. Struct. Mol. Biol. 2004,
11, 1179. (b) Reynaert, N. L.; van der Vliet, A.; Guala, A. S.; McGovern,
T.; Hristova, M.; Pantano, C.; Heintz, N. H.; Heim, J.; Ho, Y. S.;
Matthews, D. E.; Wouters, E. F.; Janssen-Heininger, Y. M. Proc. Natl.
Acad. Sci. U. S. A. 2006, 103, 13086. (c) Marinho, H. S.; Real, C.; Cyrne,
L.; Soares, H.; Antunes, F. Redox Biol. 2014, 2, 535.
(8) (a) See ref 3a. (b) Percival, M. D.; Ouellet, M.; Campagnolo, C.;
Claveau, D.; Li, C. Biochemistry 1999, 38, 13574.
(9) Kulathu, Y.; Garcia, F. J.; Mevissen, T. E.; Busch, M.; Arnaudo, N.;
Carroll, K. S.; Barford, D.; Komander, D. Nat. Commun. 2013, 4, 1569.
(10) 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.; Carroll, K. S.; Martens, J. R. Circ. Res. 2012, 111, 842.
(11) Seo, Y. H.; Carroll, K. S. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
16163.
(12) Fries, K. Ber. Dtsch. Chem. Ges. 1912, 45, 2965.
(13) (a) Goto, K.; Tokitoh, N.; Okazaki, R. Angew. Chem., Int. Ed. Engl.
1995, 34, 1124. (b) Saiki, T.; Goto, K.; Tokitoh, N.; Okazaki, R. J. Org.
Chem. 1996, 61, 2924. (c) Goto, K.; Holler, M.; Okazaki, R. J. Am. Chem.
Soc. 1997, 119, 1460.
(14) (a) Allison, W. S. Acc. Chem. Res. 1976, 9, 293. (b) Miller, H.;
Claiborne, A. J. Biol. Chem. 1991, 266, 19342. (c) Sohn, J.; Rudolph, J.
Biochemistry 2003, 42, 10060. (d) Buhrman, G.; Parker, B.; Sohn, J.;
Rudolph, J.; Mattos, C. Biochemistry 2005, 44, 5307.
(15) Salsbury, F. R.; Knutson, S. T.; Poole, L. B.; Fetrow, J. S. Protein
Sci. 2008, 17, 299.
(16) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.
(17) Smith, A. B., III; Savinov, S. N.; Manjappara, U. V.; Chaiken, I. M.
Org. Lett. 2002, 4, 4041.
(18) (a) Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B.
Org. Lett. 2002, 4, 2865. (b) Kotzur, N.; Briand, B.; Beyermann, M.;
Hagen, V. J. Am. Chem. Soc. 2009, 131, 16927. (c) Kotzur, N.; Briand, B.;
Beyermann, M.; Hagen, V. Chem. Commun. 2009, 14, 3255.
(19) (a) Ghosh, M.; Song, X.; Mouneimne, G.; Sidani, M.; Lawrence,
D. S.; Condeelis, J. S. Science 2004, 304, 743. (b) Chang, C. Y.; Niblack,
B.; Walker, B.; Bayley, H. Chem. Biol. 1995, 2, 391. (c) Chang, C. Y.;
Fernandez, T.; Panchal, R.; Bayley, H. J. Am. Chem. Soc. 1998, 120, 7661.
(d) Arabaci, G.; Guo, X. C.; Beebe, K. D.; Coggeshall, K. M.; Pei, D. J.
Am. Chem. Soc. 1999, 121, 5085.
D
DOI: 10.1021/acs.orglett.5b02981
Org. Lett. XXXX, XXX, XXX−XXX