Light-induced hydrogen sulfide release from "caged" gem-dithiols

Article abstract of DOI:10.1021/ol401118k

"Caged" gem-dithiol derivatives that release H₂S upon light stimulation have been developed. This new class of H₂S donors was proven, by various spectroscopic methods, to generate H₂S in an aqueous/organic medium as well a

Full text of DOI:10.1021/ol401118k

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Light-Induced Hydrogen Sulfide Release
from “Caged” gem-Dithiols
Nelmi O. Devarie-Baez, Powell E. Bagdon, Bo Peng, Yu Zhao, Chung-Min Park, and
Ming Xian*
Department of Chemistry, Washington State University, Pullman, Washington 99164,
United States
mxian@wsu.edu
Received April 22, 2013
ABSTRACT
“Caged” gem-dithiol derivatives that release H2S upon light stimulation have been developed. This new class of H2S donors was proven, by
various spectroscopic methods, to generate H2S in an aqueous/organic medium as well as in cell culture.
Hydrogen sulfide (H2S) is a notorious toxic gas known
for many years to be detrimental to humans. Recently, this
molecule has been identified as a cell-signaling mediator
and constitutes a member of the gasotransmitter family,
together with its congeners nitric oxide (NO) and carbon
monoxide (CO).1 The endogenous generation of H2S has
been predominantly attributed to the enzymatic actions of
cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE),
and 3-mercaptopyruvate sulfurtransferase (3-MST or
3-MPST).2 In specific tissues, these enzymes utilized
cysteine (Cys), homocysteine (Hcy), or other cysteine deri-
vatives to produce H2S in a controllable manner. Once H2S
is produced, in addition to carrying out its biological
functions, H2S can be rapidly metabolized into two other
forms as acid-labile sulfur (FeÀS cluster) and bound-
sulfane sulfur. Both could in turn serve as in vivo H2Ssources.3
In the past decade, a number of studies have revealed
significant roles of H2S in physiology and pathology.1,2
Among many attributes given to endogenous production
of H2S and/or exogenous administration of H2S, critical
functions are especially exerted in the cardiovascular and
nervous systems and regulating inflammation.4 However,
mechanisms underlying these biological responses are still
unclear. These physiological and pathological activities
may be derived from biological chemistry occurring at
the molecular level. For example, H2S is highly reactive
toward reactive oxygen and nitrogen species including
hydrogen peroxide (H2O2),5 superoxide (O2•À),6 and per-
oxynitrite (OONOÀ),7 establishing its role as an antioxi-
dant. H2S can also react with RSNO to produce thionitrous
acid, HSNO (the smallest S-nitrosothiol), which serves as a
(4) (a) Whitffield, N. J.; Kreimier, E. L.; Verdial, F. C.; Skovgaard,
N.; Olson, K. R. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008, 294,
R1930. (b) Li, J.; Whiteman, M.; Guan, Y. Y.; Neo, K. L.; Cheng, Y.;
Lee, S. W.; Zhao, Y.; Baskar, R.; Tan, C. H.; Moore, P. K. Circulation
2008, 117, 2351. (c) Predmore, B. L.; Lefer, D. J.; Gojon, G. Antioxid.
Redox Signal. 2012, 17, 119. (d) Abe, K.; Kimura, H. J. Neurosci. 1996,
16, 1066. (e) Qu, K.; Lee, S. W.; Bian, J. S.; Low, C. M.; Wong, P. T. H.
Neurochem. Int. 2008, 52, 155. (f) Fiorucci, S.; Distrutti, E.; Cirino, G.;
Wallace, J. L. Gastroenterology 2006, 131, 259. (g) Wallace, J. L.;
Caliendo, G.; Santagada, V.; Cirino, G.; Fiorucci, S. Gastroenterology
2007, 132, 261.
(1) (a) Li, L.; Rose, P.; Moore, P. K. Annu. Rev. Pharmacol. Toxicol.
2011, 51, 169. (b) Olson, K. R.; Donald, J. A.; Dombkoski, R. A.; Perry,
S. F. Repir. Physiol. Neurobiol. 2012, 184, 117. (c) Wang, R. Physiol. Rev.
2012, 92, 791. (d) Fukuto, J. M.; Carrington, S. J.; Tantillo, D. J.;
Harrison, J. G.; Ignarro, L. J.; Freeman, B. A.; Chen, A.; Wink, D. A.
Chem. Res. Toxicol. 2012, 25, 769.
(5) Geng, B.; Yang, J.; Qi, Y.; Zhao, J.; Pang, Y.; Du, J.; Tang, C.
Biochem. Biophy. Res. Commun. 2004, 313, 362.
(6) Cheng, L.; Geng, B.; Yu, F.; Zhao, J.; Jiang, H.; Du, J.; Tang, C.
Amino Acids 2008, 34, 573.
(7) Whiteman, M.; Armstrong, J. S.; Chu, S. H.; Jia-Ling, S.; Wong,
B. S.; Cheung, N. S.; Halliwell, B.; Moore, P. L. Neurochemistry 2004,
90, 765.
(8) Filipovic, M. R.; Miljkovic, J. Lj.; Nauser, T.; Royzen, M.; Klos,
K.; Shubina, T.; Joppenol, W. H.; Lippard, S. J.; Ivanvic-Burmazovic, I.
J. Am. Chem. Soc. 2012, 134, 12016.
(2) (a) Kabil, O.; Banerjee, R. J. Biol. Chem. 2010, 285, 21903.
(b) Kimura, H. Amino Acids 2011, 41, 113. (c) Jhee, K. H.; Kruger, W. D.
Antioxid. Redox Signal. 2005, 7, 813. (d) Zhao, W.; Zhang, J.; Lu, Y.;
Wang, R. EMBO J. 2001, 20, 6008.
(3) (a) Ubuka, T. J. Chromatogr. 2002, 781, 227. (b) Ishigami, M.;
Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.; Kimura, H.
Antioxid. Redox Signal. 2009, 11, 205. (c) Shen, X.; Peter, E. A.; Bir,
S.; Wang, R.; Kevil, C. G. Free Radical Biol. Med. 2012, 52, 2276.
r
10.1021/ol401118k
XXXX American Chemical Society

cell-permeable nitrosylating agent.8 In addition, H2S can
modify protein cysteine residues to give sulfhydrated
proteins (protein-S-SH), which are believed to be a critical
pathway in regulating protein functions.9
Scheme 1. Representative Organic H2S Donors
The rapid and constant growth of the H2S-biomedical
research has led to a concomitant need of research tools.
Recent advances on H2S-fluorescent sensors and H2S
donors are perfect examples.10 In particular, H2S donors
are very attractive as many studies have highlighted the
therapeutic potentials of exogenous administration of
H2S.11 Among commonly used donors, most researchers
are using inorganic sulfide salts such as NaHS and Na2S.
However, H2S generation from these salts occurs rapidly.
It is difficult to control the timing of release, which there-
fore cannot mimic the endogenous production of H2S.12 In
some cases, the biological effects displayed by sulfide salts
may not represent physiological events induced by the
actions of H2S. Instead, it may be a systematic response
to excess amounts of H2S.13
Incontrast toinorganicsulfide salts, organic H2S donors
can exert continuous and controllable H2S release at
concentrations relative to endogenous levels. Currently,
several types of organic H2S donors have been developed
and their mechanisms of H2S production are diverse
(Scheme 1).11,14 Our group has recently disclosed two types
of controllable donors: N-(benzoylthio)benzamide-based
donors and persulfide-based donors.15 Both types are
utilizing biological thiols, such as cysteine and glutathione,
as the triggers to promote H2S generation. In addition to
the thiol-activation mechanism, we expect that a platform
capable of generating H2S upon external stimulus should
be of great interest. Such donors would enable steady and
localized concentrations of H2S at desired timing and
cellular locations. In this context, photocaged H2S donors
are potential candidates. Herein we report the design,
synthesis, and evaluation of a series of photoactivated
H2S donors.
The idea of caged-H2S donors was based on the struc-
ture of geminal-dithiols (gem-dithiols). It is known that
gem-dithiols are unstable species, particularly in aqueous
environments, and H2S can be formed as a decomposition
byproduct.16 Therefore, we envisioned gem-dithiols were
useful templates for H2S donor design. Introduction of
protecting groups on free ÀSH of gem-dithiols should lead
to stable derivatives as H2S donors. In addition, we should
be able to manipulate the deprotection strategy to achieve
controllable H2S release. As the first step to develop gem-
dithiol based donors, we decided to test photoactivation
strategy. As shown in Scheme 2, our target was compounds 1,
in which the SH groups were protected with a photosensitive
2-nitrobenzyl group. Upon light irradiation, the gem-dithiol
intermediate 1A should be produced and subsequent hydro-
lysis of 1A would liberate H2S.
Scheme 2. Design of Photoactivated H2S Donors
(9) (a) Mustafa, A. K.; Gadalla, M. M.; Sen, N.; Kim, S.; Mu, W.;
Gazi, S. K.; Barrow, R. K.; Yang, G.; Wang, R.; Snyder, S. H. Sci.
Signal. 2009, 2, ra72. (b) Krishnan, N.; Fu, C.; Pappin, D. J.; Tonks,
N. K. Sci. Signal. 2011, 4, ra86. (c) Paul, B. D.; Snyder, S. H. Nat. Rev.
Mol. Cell Bio. 2012, 13, 499.
(10) For selected reviews, see: (a) Lin, V. S.; Chang, C. J. Curr. Opin.
Chem. Biol. 2012, 16, 1. (b) Chan, J.; Dodani, S. C.; Chang, C. J. Nat.
Chem. 2012, 4, 973.
(11) (a) Kashfi, K.; Olson, K. R. Biochem. Pharmacol. 2012, 85, 689.
(b) Olson, K. R. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2011, 301,
R297. (c) Whiteman, M.; Trionnaire, Le; Chopra, M.; Fox, B.; What-
more, J. Clin. Sci. 2011, 121, 459. (d) Caliendo, G.; Cirino, G.;
Santagada, V.; Wallace, J. L. J. Med. Chem. 2010, 53, 6275.
(12) (a) DeLeon, E. R.; Stoy, G. F.; Olson, K. R. Anal. Biochem.
2012, 421, 203. (b) Calvert, J. W.; Coetzee, W. A.; Lefer, D. J. Antioxid.
Redox Signaling 2010, 12, 1203.
The synthesis of this type of donor is illustrated in
Scheme 3. Briefly, commercially available 2-nitrobenzyl
bromide 2 was treated with thioureain THFtoproduce the
thiouronium bromidesalt3. Hydrolysisof3inthepresence
of sodium metabisulfite (Na2S2O5) provided 2-nitrobenze-
nemethanethiol 4 in high yield. Finally, compound 4 was
coupled with acetone in the presenceof catalytic amount of
TiCl4 to give a model donor 1a.
With the model donor in hand, we examined its H2S
generation capability. The standard methylene blue method
was used to monitor H2S generation (the mechanistic
scheme of this method is shown in the Supporting
Information). In this study, a 200 μM solution of 1a in
pH 7.4 phosphate buffer/acetonitrile (1:1) was prepared.
The compound appeared to be stable and no H2S release
was detected. However, when the solution was subjected to
UV irradiation at 365 nm, we observed a time-dependent
H2S production. The concentrations of H2S reached a
maximum of ∼36 μM in about 7 min and dropped
afterward, presumably due to volatilization of H2S gas
(Figure 1).12a
(13) Olson, K. R. Front. Physiol. 2013, 4, 1.
(14) Zhou, Z.; von Wantoch Rekowski, M.; Coletta, C.; Szabo, C.;
Bucci, M.; Cirino, G.; Topouzis, S.; Papapetropoulos, A.; Gianns, A.
Bioorg. Med. Chem. 2012, 20, 2675.
(15) (a) Zhao, Y.; Wang, H.; Xian, M. J. Am. Chem. Soc. 2011, 133,
15. (b) Zhao, Y.; Bhushan, S.; Yang, C.; Otsuka, H.; Stein, J. D.;
Pacheco, A.; Peng, B.; Devarie-Baez, N. O.; Aguilar, H. C.; Lefer,
D. J.; Xian, M. ACS Chem. Biol. 2013dx.doi.org/10.1021/cb400090d.
(16) (a) Cairns, T. L.; Evans, G. L.; Larchar, A. W.; Mckusick, B. C.
J. Am. Chem. Soc. 1952, 74, 3982. (b) Berchtold, G. A.; Edwards, B. E.;
Campaigne, E.; Carmack, M. J. Am. Chem. Soc. 1959, 81, 3148. (c)
Voronkov, M.; Shagun, L.; Ermolyuk, L.; Timokhina, L. J. Sulfur
Chem. 2004, 25, 131.
B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 3. Synthesis of Photoactivated H2S Donors
Figure 2. Spectra of methylene blue assay. Blue line: Na2S
(50 μM). Other lines: H2S release from 1a upon irradiation at
different times.
Figure 1. Time-dependent H2S release of 1a in pH 7.4 phosphate
buffer/acetonitrile (1:1).
To confirm the signals shown in Figure 1 were indeed
from H2S, we recorded the UVÀvis spectra of methylene
blue generated from the photolysis of 1a and compared
with the spectra obtained using Na2S, a standard H2S
precursor. As shown in Figure 2, these absorbance spectra
showed identical patterns, and the levels increased when
irradiation was prolonged.
Given the potential applications of photoactivated
donors for site-specific delivery of H2S and the diverse cellular
pH values, we also studied the effects of pH on H2S
releasing activity of 1a. In mild acidic medium (pH = 5.5),
H2S level was found to be much higher than the level
generated at neutral pH (Figure 3). This result may
indicate the intermediacy of gem-dithiol, in which the
hydrolysis should be an acid-facilitated process. In con-
trast, H2S concentration dropped slightly under mild basic
pH (8.2). It should be noted that the donor did not produce
any H2S under these pH values if the irradiation of UV
light was absent.
Figure 3. H2S release of 1a (200 μM) at different pH’s.
modulate H2S release capability. As shown in Figure 4,
alkyl-substituted donors 1bÀd exhibited similar H2S
release capability as the model compound 1a. However, aryl-
substituted compounds 1e and 1f showed very low activity.
Although the methylene blue method has been widely
used to evaluate H2S donors, this method requires strong
acidic conditions and is destructive to biological samples
like cells. We expected the photoactivated donors would be
used in cell-based studies; therefore nondestructive methods
for continuously testing H2S generation in such samples
are needed. The fluorescent probes are appropriate for this
purpose. To this end, WSP-1, a H2S-specific fluorescent
probe developed by our group,17 was used to monitor
the photolysis of 1a in buffers (the structure and reac-
tion mechanism of WSP-1 is shown in the Supporting
Information). In this study, a 200 μM solution of 1a in
pH 7.4 phosphate buffer/acetonitrile (1:1) was prepared.
Having demonstrated UV light-induced H2S generation
of 1a, we turned our attention to other derivatives 1bÀf,
which were prepared using the same protocol shown in
Scheme 3. We wondered if structural modifications could
(17) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.;
Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011, 123, 10511.
Org. Lett., Vol. XX, No. XX, XXXX
C

under photolysis (aliquot taken at 9 min). The intensity
was approximately 66 folds higher than the solution with-
out light irradiation. The results proved that fluorescence
method isappropriate for the evaluation of photoactivated
H2S donors.
Finally, we wondered if these donors could be used to
selectively deliver H2S to cells and conducted a cell-based
assay to address this question. In this study, HeLa cells
were first incubated with 1d (200 μM) for 30 min, and the
mixture was then exposed to UV light (365 nm) for 15 min.
After that, cells were washed and resuspended in new
media. WSP-1 (50 μM) was applied into the system to
monitor H2S in cells. As expected, cells treated with 1d
under irradiation showed much stronger fluorescent sig-
nals than cells treated with 1d but no irradiation (Figure 6).
Figure 4. H2S release of 1aÀf. Donor concentration: 200 μM.
Under continuous irradiation.
Figure 6. H2S release of 1d in HeLa cells: (a) 1d (200 μM) and
WSP-1 (50 μM), no UV irradiation; (b) 1d (200 μM) and WSP-1
(50 μM), under UV irradiation.
In summary, a series of photoactivated H2S donors
based on the structure of gem-dithiol were prepared and
evaluated. Our evidence demonstrates the capabilities of
these compounds as specific H2S donors. These “caged”
donors could allow spatial and temporal release of H2S,
and study real time H2S activities. Screening other photo-
labile groups and examining different activation mechan-
isms to unmask gem-dithiols are ongoing in our laboratory.
Figure 5. H2S release of 1a detected by fluorescence: (a) WSP-1
only (100 μM); (b) WSP-1 (100 μM) and 1a (200 μM), in the
absence of light; (c) WSP-1 (100 μM) and 1a (200 μM), in the
presence of light.
Acknowledgment. This work is supported by an American
Chemical SocietyÀTeva USA Scholar Grant and the NIH
(R01GM088226).
The solution was subjected to UV irradiation at 365 nm,
and aliquots were withdrawn from the solution at a given
time and then detected by WSP-1. We observed a time-
dependent H2S production, similar to that observed when
using the methylene blue method. As shown in Figure 5,
fluorescence signal increased dramatically when 1a was
Supporting Information Available. Synthetic proce-
dures, spectroscopic data, and experimental procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX