Thiol-activated gem-dithiols: A new class of controllable hydrogen sulfide donors

Article abstract of DOI:10.1021/ol502088m

A class of novel thiol-activated H₂S donors has been developed on the basis of the gem-dithiol template. These donors release H₂S in the presence of cysteine or GSH in aqueous solutions as well as in cellular environments.

Full text of DOI:10.1021/ol502088m

Letter
pubs.acs.org/OrgLett
Open Access on 08/20/2015
Thiol-Activated gem-Dithiols: A New Class of Controllable Hydrogen
Sulfide Donors
Yu Zhao, Jianming Kang, Chung-Min Park, Powell E. Bagdon, Bo Peng, and Ming Xian*
Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
S
* Supporting Information
ABSTRACT: A class of novel thiol-activated H2S donors has
been developed on the basis of the gem-dithiol template. These
donors release H2S in the presence of cysteine or GSH in
aqueous solutions as well as in cellular environments.
ydrogen sulfide (H2S) has been recently recognized as a
new member of the family of gasotransmitters, along with
rapidly form in NaHS solution.27 All of these problems may
lead to disparate results when using sulfide salts as H2S
precursors. Considering these drawbacks, researchers have
started to use organic molecules as H2S donors. Several types of
synthetic H2S donors have been developed and used in studies.
Representative donors include GYY4137, dithiolethiones, N-
mercapto-based molecules, perthiol-based molecules, geminal-
dithiol (gem-dithiol) species, and thioamino acids (Figure 1).
These compounds release H2S under different conditions, and
their H2S-related biological actions have been explored.28−35
H
nitric oxide (NO), carbon monoxide (CO), and dioxygen
(O2).1−5 Biosynthesis of H2S has been attributed to at least
three enzymes: cystathionine β-synthase (CBS), cystathionine
γ-lyase (CSE), and 3-mercaptopyruvate sulfur-transferase
(MPST).6−8 These enzymes convert cysteine or cysteine
derivatives to H2S in different tissues and organs. It is believed
that H2S-induced biological actions, such as anti-inflammation,
vasodilation, and cardioprotection, are related to some specific
reactions of H2S in living systems.9−12 For example, H2S can
interact with methemoglobin to form sulfhemoglobin, and this
reaction might act as a metabolic sink for H2S.13 H2S has been
reported to cause protein S-sulfhydration to form protein-S-SH,
while how this reaction occurs is still under debate.14−16
Nevertheless, this process is potentially significant as it provides
a possible route by which H2S can alter functions of a wide
range of cellular proteins and enzymes.17,18 As a potential
reducing agent, H2S can rapidly scavenge reactive oxygen
species, such as hydrogen peroxide, superoxide, and peroxyni-
trite. These reactions account for H2S’s protective functions in
cardiovascular systems.19−22 In addition, the reaction between
H2S and nitrosothiols could result in the formation of
thionitrous acid (HSNO), the smallest S-nitrosothiol. HSNO
possibly serves as a cell-permeable nitrosylating agent.23 All of
these findings suggest that regulation of endogenous H2S
formation and exogenous H2S administration may have
therapeutic benefits.
In this field, H2S releasing agents (also known as H2S
donors) are important tools.24,25 Currently, sulfide salts (i.e.,
sodium sulfide Na2S and sodium hydrogen sulfide NaHS) are
still the most often used H2S donors in this field. Although
these salts have the advantage of boosting H2S concentration
fast, the uncontrollable H2S release makes them not ideal to
mimic slow and controllable H2S release in living systems. In
addition, H2S can quickly escape from solution due to
volatilization under laboratory conditions.26 The effective
residence time of sulfide salts in testing samples, therefore, is
very short. It should also be noted that commercially available
sulfide salts, especially NaHS, always contain a significant
amount of impurities. Recent studies revealed that polysulfides
Figure 1. Representative synthetic H2S donors.
It should be noted that although a number of H2S donors
have been reported, donors with controllable H2S release
capability are still very limited and under high demand. The
research in our group focuses on the development of
controllable H2S donors. We have reported two types of
thiol-activated donors based on N-mercapto and perthiol
templates (Figure 1).31,33 These molecules do not release
H2S without the interaction with thiols (i.e., cysteine and
glutathione). Recently, we also reported a series of gem-dithiol-
based H2S donors (GDDs), which were activated by UV
irradiation.34 Based on these results, we envisioned that gem-
dithiols are valuable templates for the design of H2S donors and
decided to explore new donors using this structure. Herein, we
reported a new class of thiol-activated gem-dithiol-based H2S
Received: July 16, 2014
© XXXX American Chemical Society
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donors (TAGDDs). H2S release from these TAGDDs can be
triggered by cellular thiols.
It is known that gem-dithiols (1) are unstable species in
aqueous environments and the decomposition of 1 should lead
to H2S release.36,37 In the development of TAGDDs, an acetyl
group was selected to stabilize 1. More importantly, this
protecting group would be selectively removed in the presence
of thiols to retrieve 1, therefore achieving controllable H2S
release (Figure 2).
Figure 2. Design of TAGDDs.
Figure 4. H2S release from TAGDD-1 in the presence of cysteine.
With this idea in mind, a series of TAGDDs were synthesized
from the substituted benzaldehydes (Figure 3). Briefly,
drop after peak time probably due to volatilization. In our
following studies we decided to use 500 μM cysteine to trigger
H2S release from TAGDDs.
In addition to cysteine, GSH’s capability in promoting H2S
release from TAGDDs was also evaluated. As shown in Figure
5, GSH (500 μM) successfully triggered H2S release, but at a
Figure 3. Chemical synthesis of TAGDDs.
benzaldehyde derivatives 2 reacted with boron tribromide
(BBr3) to form the corresponding dibromide intermediates 3.
Then compounds 3 were treated with potassium thioacetate to
provide the desired TAGDDs. In this study, eight TAGDDs
were synthesized in yields of 51−75%.
Unlike hydrolysis-based H2S donors (i.e., GYY4137 and
Na2S/NaHS), TAGDDs were stable in aqueous solutions. They
did not release H2S upon hydrolysis. Cellular nucleophiles, such
as lysine and serine, did not trigger H2S release, either.
However, a time-dependent H2S generation was observed in
the presence of cysteine, indicating thiols were essential to
trigger H2S release. In order to systematically compare H2S
generation capability of these donors we studied the effects of
donor concentrations, cysteine concentrations, solvent systems,
as well as reaction time/temperatures. H2S release was
monitored at room temperature for 2 h. The standard
methylene blue (MB) method was used to measure H2S
generation. Eventually the optimized conditions were found to
be 100 μM donors in PBS buffer (pH 7.4, 50 mM) containing
10% THF. Varied cysteine concentrations caused different H2S
release profiles (Figure 4). Taking TAGDD-1 as the example, a
maximum of 93 μM of H2S (peak H2S concentration) at 25
min (peak time) was detected from 100 μM of the donor in the
presence of 1000 μM cysteine. H2S concentrations started to
Figure 5. H2S release from TAGDD-1 in the presence of cysteine and
GSH.
relatively lower level. This is presumably due to increased steric
hindrance of GSH, therefore leading to a slower reaction to
liberate H2S. Homocysteine showed similar effects as GSH
(data shown in Figure S1, Supporting Information).
H2S-releasing profiles of all 8 TAGDDs were determined
under the optimized conditions. Briefly, a solution of donor
(100 μM) and cysteine (500 μM) was prepared in a mixed PBS
(pH 7.4, 50 mM)/THF (9:1, v:v) solvent. Reaction aliquots
(1.0 mL) were taken to MB cocktail (0.5 mL) at different
reaction times. After 15 min, UV absorbance at 670 nm was
measured. H2S concentrations were calculated by using a
standard curve generated by Na2S. Each donor was tested three
times, and their average results are summarized in Table 1. The
results showed that peak times of TAGDDs ranged from 29 to
38 min with peak H2S concentrations of 25.3−94.3 μM. The
B
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a
intracellular H2S release. Before conducting experiments to test
this hypothesis, we evaluated cytotoxicity of a representative
donor, TAGDD-1, in HeLa cells. A cell counter kit (CCK-8)
assay was used to detect cell viability (Figure 7). The results
Table 1. H2S Release from TAGDDs
CysSH
GSH
Tpeak
(min)
[H2S]peak
(μM)
Tpeak
(min)
[H2S]peak
(μM)
TAGDDs
R
1
2
3
4
5
6
7
8
H
30
37
38
34
37
34
29
35
69.5
94.0
25.3
35.8
27.2
94.3
70.6
36.1
40
34.6
24.6
10.1
16.8
N/A
52.2
24.8
17.6
4-CH3
4-Br
43
48
4-Cl
53
4-CF3
3-OH
3-CH3
3-NO2
N/A
49
39
47
a
Data were reported as the average value of three measurements.
profiles in the presence of GSH were also measured. In general,
GSH led to slower and much decreased H2S release from these
donors. These results demonstrated that TAGDDs are potent
H2S donors and structure modifications could regulate H2S
release ability.
The mechanism of H2S release is proposed as follows (Figure
6): the reaction is initiated by a reversible thiol exchange
Figure 7. Effects of TAGDD-1 on cell viability. HeLa cells were
treated with different concentrations of TAGDD-1 (5−25 μM) for 1
h. The cell counter kit (CCK)-8 assay was performed to measure cell
viability. Data were shown as the mean SD (n = 4).
showed that 1 h exposure of HeLa cells to TAGDD-1 at varied
concentrations (5−25 μM) did not decrease cell viability,
indicating that TAGDDs do not induce cytotoxicity to HeLa
cells at doses used (cytotoxicity data of other TAGDDs are
shown in Figure S3, Supporting Information).
Experiments were then conducted to test whether TAGDDs
could release H2S in cells. As shown in Figure 8, HeLa cells
Figure 6. Proposed mechanism for H2S release from TAGDD-1.
between TAGDD-1 and cysteine to generate S-acetyl cysteine
(4) and gem-dithiol (5). Compound 4 should undergo a fast S-
to-N acyl transfer to form N-acetylcysteine (6) and drive the
equilibrium.31 Meanwhile, 5 should release H2S spontaneously
in aqueous solution to yield benzaldehyde (7). To prove the
mechanism, we analyzed the reaction between TAGDD-1 and
cysteine (5 equiv) by HPLC equipped with a UV detector.
Indeed, the formation of benzaldehyde 7 and 2-phenyl-
thiazolidine-4-carboxylic acid, a product from benzaldehyde
and cysteine, was observed in high yields by using authentic
samples (see the Supporting Information). The mechanism,
when activated by GSH, should be similar to the mechanism
when activated by cysteine. However, GSH, compared with
cysteine, is more bulky. In addition, there is no S-to-N acyl
transfer in GSH-involved reactions. Therefore, the initial
equilibrium might be slow. The consumption of the final
product benzaldehyde by cysteine is also expected to be faster
than GSH due to the formation of 2-phenylthiazolidine-4-
carboxylic acid. Because of these reasons, it is expectable the
reactions between TAGDDs and GSH are slower and less
effective. The GSH experiments proved our hypothesis.
Figure 8. H2S production from TAGDD-1 in HeLa cells. Cells were
incubated with vehicle (left) and TAGDD-1 (25 μM) (right) for 30
min. After removal of excess TAGDD-1, a H2S fluorescent probe
(WSP-4) was added. Images were taken after 30 min.
were incubated with TAGDD-1 (25 μM) for 30 min. Then
cells were washed by PBS twice to remove extracellular
TAGDD-1. A selective H2S fluorescent probe, WSP-4,42 was
then applied to detect H2S generation. As expected, donor-
treated cells exhibited significantly enhanced fluorescent signals
compared to vehicle-treated group, demonstrating that
TAGDDs can release H2S in cells.
In conclusion, a series of thiol-activated H2S donors have
been developed on the basis of gem-dithiol structures. These
donors are stable in aqueous solutions. However, a time-
dependent H2S generation was observed in the presence of
thiols. In addition, H2S release of TAGDDs in cells was also
proved. Further development of these donors and evaluation of
their H2S-related biological activities are currently ongoing in
our laboratory.
Considering significant amounts of free cysteine and GSH in
living systems,38−41 we envisioned that TAGDDs could achieve
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ASSOCIATED CONTENT
* Supporting Information
Detailed synthetic procedures, characteristic data, and exper-
imental procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: mxian@wsu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by an American Chemical Society Teva
USA Scholar Grant and the NIH (R01HL116571).
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