Esterase-sensitive trithiane-based hydrogen sulfide donors

Article abstract of DOI:10.1039/c9ob02273b

1,3,5-Trithiane functionalized with esterase-sensitive ester groups on the methylene linkers was developed as a class of enzymatic hydrolysis-based hydrogen sulfide (H₂S) donors. The amount of H₂S released from the donors was dependent on the number of ester bonds. The donors release H₂S in a controllable manner in the presence of an enzyme.

Full text of DOI:10.1039/c9ob02273b

Organic &
Biomolecular Chemistry
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Esterase-sensitive trithiane-based hydrogen sulfide
donors†
Clovis Shyaka,a Ming Xian b and Chung-Min Park
Cite this: Org. Biomol. Chem., 2019,
17, 9999
a
*
Received 20th October 2019,
Accepted 14th November 2019
DOI: 10.1039/c9ob02273b
rsc.li/obc
1,3,5-Trithiane functionalized with esterase-sensitive ester groups modulation of cellular H2S levels can have potential thera-
on the methylene linkers was developed as a class of enzymatic peutic effects.
hydrolysis-based hydrogen sulfide (H2S) donors. The amount of
Researchers typically administer exogenous H2S to modu-
H2S released from the donors was dependent on the number of late the H2S concentration in cells. Easy access to relevant com-
ester bonds. The donors release H2S in a controllable manner in mercial reagents such as NaHS and Na2S, which are H2S
the presence of an enzyme.
sources, expedites the progress in research related to the use
of this gas as a biomolecule. The sulfide salts are expected to
be rapid H2S-releasing species, based on the inherent pKa
values of H2S under physiological conditions.17,18 This
phenomenon is substantially different from the endogenous
production of H2S in cells, and it may cause excess exposure to
H2S, resulting in unavoidable side effects.19,20 Considering
this, several types of H2S-releasing synthetic molecules have
been developed and categorized according to the manner in
which they generate H2S in a cell (Scheme 1), in order to
explore the biological functions of H2S.
The most common class of H2S donors comprises the
biothiol (Cys or GSH)-activated donors such as diallyl trisulfide
(DATS), which is a natural organosulfur species21 with a series
of N-(benzoyl)-thiobenzamide derivatives.22 Hydrolysis-based
H2S donors include GYY4137,23 1,2-dithiole-3-thiones (DTTs),
and arylthioamide derivatives.24,25 Photo-induced H2S donors
include caged gem-dithiols,26 ketoprofenate-caged donors,27
and o-nitrobenzyl-caged thiocarbamates.28 Enzyme-triggered
H2S donors have also been reported.29–32 Dithiolethione (ADT)
Hydrogen sulfide (H2S) is a stinking, colorless, and toxic pollu-
tant. Despite these demerits, it is an important biological
molecule owing to its unique association with human health
and disease.3 Similar to carbon monoxide (CO) and nitric
oxide (NO), it is a gasotransmitter and hence, an important
gaseous signaling molecule in living systems.1,2 In mamma-
lian systems, biological H2S is produced by endogenous
enzymes such as cystathionine β-synthase (CBS), cystathionine
γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST),
and cysteine aminotransferase (CAT).4–8 These enzymes pri-
marily act on cysteine and homocysteine and convert them
into H2S either individually or cooperatively. This simple and
general chemical process may release H2S from the intracellu-
lar sulfane sulfur pool.9 Chemical and biochemical catabolic
reactions of both endogenous and exogenous H2S are known
to protect cells and organs against pathological damage or
damage caused due to environmental exposure.10–12 For
example, H2S can protect against myocardial ischemia reperfu-
sion (MI/R) injury caused by endogenous reactive oxygen
species (ROS) generated in the mitochondria.13,14 Additionally,
H2S has been proven to be a potent anti-inflammatory bio-
molecule in animal models.15 It relaxes vascular smooth
muscles, reduces high blood pressure, and induces vasodila-
tion of blood vessels.16 These facts strongly establish the
importance of H2S in living systems. It is also evident that
aDepartment of Chemistry, Gangneung-Wonju National University, Gangneung,
Gangwon 25457, South Korea. E-mail: parkc@gwnu.ac.kr; Fax: +82-33-640-2300;
Tel: +82-33-640-2304
bDepartment of Chemistry, Washington State University, Pullman, WA 99164, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob02273b
Scheme 1 Structures of H2S donors.
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derivatives have been conjugated with a variety of therapeutics
to enhance their biological activities.33,34 H2S production from
lactonization of thioacids was found to exhibit anti-inflamma-
tory effects.22,35–37 Recently, ROS-triggered H2S donors have
also been developed; it was demonstrated that carbonyl sulfide
(COS) generated from the donors is degraded by an enzyme
and rapidly converted to H2S.38 pH-Dependent H2S releasing
donors have also been reported,39,40 3, and are found to
exhibit cardioprotective effects against myocardial ischemia-
reperfusion (MI/R) injury.39
Scheme 3 Synthesis of trithiane-based H2S donors.
More recently, linear and cyclic polysulfides have been
studied as H2S donors and are classified as biothiol-activated
H2S donors (Fig. 1).41 Among these cyclic polysulfides (Fig. 1),
To this end, acyloxylation of a cyclic thioether by peroxy
which are composed of methylene linkers and sulfur atoms, esters in the presence of a catalytic amount of a cuprous salt
we found that 1,3,5-trithiane has a unique structure that is (CuCl) was performed according to a modified method
composed of a six-membered ring with alternating methylene (Scheme 3).43 1,3,5-Trithiane was treated with excess tert-butyl
linkers and thioether groups. The functionalization of three peroxyacetate in dry benzene to introduce an acetate group
methylene linkers of this sulfur-enriched species may lead to a into each methylene linker of the trithiane skeleton. However,
new class of H2S donors. This encouraged us to design the desired compound was not formed owing to steric hin-
trithiane-based enzyme-triggered H2S donors. Herein, we drance. Instead, we obtained trithiane bisacetate 3a as the
report the syntheses and evaluation of such trithiane-based major product. When 3a was exposed to moist air, it quickly
donors that release H2S in the presence of an esterase.
decomposed even at a low temperature and produced a distinc-
1,3,5-Trithiane, which has three sulfur atoms, could be con- tive rotten egg sulfur odor. To obtain trithiane monoacetate,
sidered as an ideal H2S donor. If it can be hydrolyzed in we changed the molar ratio of 1 and 2a. However, the com-
aqueous media, the major products would be thioformic acids pound could not be isolated due to its instability under the
(OvCHSH), which could release H2S by further hydrolysis. work-up conditions. To enhance the stability of the compound,
However, trithiane derivatives are very stable in air and water, we introduced a benzoate functionality on 1 using tert-butyl
although they can be decomposed at high temperatures.42 To peroxybenzoate. A 3.5 : 1 mixture of peroxy ester 1 and 2b was
render the compound susceptible to hydrolysis, in order to refluxed in the presence of 10 mol% CuCl in dry benzene.
trigger the release of H2S from the hydrolyzed species under Trithiane bisbenzoate 3b was obtained as the major product. A
ambient conditions, we introduced acetate functionality to reversal in the ratio of the mixture of 1 and 2b under the same
each methylene linker of 1,3,5-trithiane. The acetate functional reaction conditions afforded trithiane monobenzoate 3c.
groups may be sensitive to esterase, thereby producing an
Although we could not obtain the functionalized trithiane
unstable intermediate that may undergo further hydrolysis to donor depicted in Scheme 2, we speculated that 3b and 3c
release H2S (Scheme 2).
would be susceptible to enzymatic hydrolysis, affording frag-
ments having thiols on them—these could undergo further
hydrolysis to produce H2S.
With these compounds in hand, we examined the feasibility
of the concept of enzyme-responsive H2S release from donors
3b and 3c. The amount of H2S release was measured using an
H2S-sensitive fluorescent probe. Recently, a number of fluo-
rescent probes have been developed for H2S detection.44,45
WSP-5, a fluorescent probe,46 was selected to test the trithiane-
based H2S donors due to its fast reaction response. In these
experiments, 200 µM of each donor was incubated in 5%
DMSO in PBS buffer (pH 7.4) containing 50 µM of WSP-5. The
changes in the fluorescence emission signals at 535 nm were
detected for 4 h at the physiological temperature in the pres-
ence of porcine liver esterase (PLE) prepared in PBS. The fluo-
rescence signals were finally converted into H2S concentrations
based on the standard calibration curve prepared from a series
of NaHS standard solutions. First, we examined the capacity of
esterase to catalyze H2S release from the synthesized donors.
Fig. 2 shows that 3b and 3c slightly decomposed in aqueous
buffer and produced a negligible amount of H2S. However,
H2S release from donors 3b and 3c in the presence of PLE led
Fig. 1 Cyclic polysulfides established as H2S donors.
Scheme 2 General concept of cyclic polysulfide-based hydrogen
sulfide donors.
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Fig. 2 Time-dependent H2S-release profiles of donors 3b and 3c
detected using a fluorescent probe, WSP-5. Each donor (200 µM) was
incubated in PBS (5% DMSO) at 37 °C with 1 unit per mL of PLE. The
concentration of WSP-5 was 50 µM, and fluorescence emission was
recorded during the stated time intervals (p = 0.95, n = 3).
Fig. 3 GC-MS chromatograms of alkaline-hydrolyzed products of 3b
(A) and 3c (B).
to more intense fluorescence than when they were incubated
in aqueous buffer alone.
This indicates that esterase is responsive to the unusual
ester bond, and the donors 3b and 3c indeed release H2S by
enzyme-catalyzed hydrolysis. Although the susceptibility of the
donor to enzymatic hydrolysis was not high, they consistently
released H2S in a slow and sustainable manner. After 4 h, 3b
produced about 27.8 μM H2S, while the concentration of H2S
released from 3c was almost half that from 3b. This obser-
vation led us to investigate the H2S release mechanism, in
which the esterase-catalyzed hydrolysis should be controlled
by the number of ester bonds in the donors. The degradation
of the bonds in 3b and 3c may generate the corresponding
sulfur-containing intermediates, followed by the sustained
release of H2S in aqueous buffer.
Scheme 4 Proposed mechanism of H2S generation from the hydrolysis
of 3b (A) and 3c (B) based on GC/MS analysis.
To investigate the mechanism, we performed the alkaline
hydrolysis of 3b and 3c using lithium hydroxide, in order to
cleave the ester bonds. We expected that the hydrolysis of ester carbonothionic acid 7 (Scheme 4). More specifically, under the
bonds accelerated the decomposition of donors, resulting in hydrolysis conditions employed herein, species 4 underwent a
unstable fragments that release H2S. To identify the important rapid ring opening reaction by the abstraction of a labile acidic
species, we tried to isolate the hydrolyzed products; however, proton at the anomeric center, resulting in the formation of 7
we were not successful. Instead, we analyzed and identified (Scheme 4A, path a). This peak shows a typical M + 2 peak
the resulting hydrolyzed products by gas chromatography which is larger than usual due to the presence of a sulfur
coupled with mass spectrometry (GC-MS). Interestingly, the atom. Further hydrolysis of 7 should then afford H2S through
GC-MS chromatograms showed only a few peaks upon the fast its tautomeric equilibrium leading to carbonic acid and 2
hydrolysis of compounds 3b and 3c (Fig. 3). Three major peaks equiv. of H2S like the hydrolysis pattern of thioacetic acid.47
at 9.2, 11.3, and 11.7 min were observed upon the hydrolysis of The ring opening of 4 also took place during enzymatic hydro-
3b (Fig. 3A), while two major peaks at 9.2 and 11.7 min were lysis (Scheme 4A, path b), which resulted in the formation of
observed upon the hydrolysis of 3c; these were identical to the Na-adduct species 8 (m/z = 131, see ESI, Fig. S2†). In this
two peaks in the chromatogram of 3b (Fig. 3B).
process, thioformic acid 9 is also formed, and should be
In the two chromatograms, we could clearly observe a peak hydrolyzed rapidly to H2S due to its instability. 9 undergoes
corresponding to benzoic acid, which is the first hydrolyzed hydrolysis like thioacetic acid and gives formic acid and H2S
product. Peak 3 observed at a latter retention time (11.7 min) too.47 In the case of 3c, we can expect the ring opening of 10 to
corresponded to the typical mass spectrum pattern of benzoic take place in a similar manner to that of 4 (Scheme 4B,
acid (m/z = 122.1) (ESI, Fig. S3†). Next, we analyzed peak 1 path a), thereby yielding species 7, as confirmed by the corres-
(m/z = 94.1), which was seen in the GC-MS chromatogram of ponding GC-MS chromatogram. Hydrolyzed species 10 can
both 3b and 3c. Based on the mass fragmentation pattern of then undergo continued ring opening following enzymatic
this peak (m/z = 44.1, 61.0, 77.0, and 94.1, ESI, Fig. S1†), it was hydrolysis (Scheme 4B, path b). However, we were unable to
determined to arise from intermediate 4 and was attributed to detect 12 by GC/MS analysis, potentially due to the dominant
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Acknowledgements
This work was supported by the National Research Foundation
of Korea (NRF-2016R1D1A3B03931686 to C. M. P) and by the
research grant of the Gangneung-Wonju National University in
2017.
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