Carbonyl Sulfide (COS) Donor Induced Protein Persulfidation Protects against Oxidative Stress

Article abstract of DOI:10.1002/asia.201901148

The emergence of hydrogen sulfide (H₂S) as an important signalling molecule in redox biology with therapeutic potential has triggered interest in generating this molecule within cells. One strategy that has been proposed is to use carbonyl sulfide (COS) as a surrogate for hydrogen sulfide. Small molecules that generate COS have been shown to produce hydrogen sulfide in the presence of carbonic anhydrase, a widely prevalent enzyme. However, other studies have indicated that COS may have biological effects which are distinct from H₂S. Thus, it would be useful to develop tools to compare (and contrast) effects of COS and H₂S. Here we report enzyme-activated COS donors that are capable of inducing protein persulfidation, which is symptomatic of generation of hydrogen sulfide. The COS donors are also capable of mitigating stress induced by elevated reactive oxygen species. Together, our data suggests that the effects of COS parallel that of hydrogen sulfide, laying the foundation for further development of these donors as possible therapeutic agents.

Full text of DOI:10.1002/asia.201901148

DOI: 10.1002/asia.201901148
Full Paper
Carbonyl Sulfide (COS) Donor Induced Protein Persulfidation
Protects against Oxidative Stress
[
a]
[b]
[a]
[b]
Preeti Chauhan, Kavya Gupta, Govindan Ravikumar, Deepak K. Saini, and
[a]
Harinath Chakrapani*
Abstract: The emergence of hydrogen sulfide (H S) as an im-
tools to compare (and contrast) effects of COS and H S. Here
2
2
portant signalling molecule in redox biology with therapeu-
tic potential has triggered interest in generating this mole-
cule within cells. One strategy that has been proposed is to
use carbonyl sulfide (COS) as a surrogate for hydrogen sul-
fide. Small molecules that generate COS have been shown
to produce hydrogen sulfide in the presence of carbonic an-
hydrase, a widely prevalent enzyme. However, other studies
have indicated that COS may have biological effects which
we report enzyme-activated COS donors that are capable of
inducing protein persulfidation, which is symptomatic of
generation of hydrogen sulfide. The COS donors are also ca-
pable of mitigating stress induced by elevated reactive
oxygen species. Together, our data suggests that the effects
of COS parallel that of hydrogen sulfide, laying the founda-
tion for further development of these donors as possible
therapeutic agents.
are distinct from H S. Thus, it would be useful to develop
2
Introduction
have naturally assumed that any physiological effects demon-
[6b,8b]
strated by the COS donor is due to H S.
However, recent
2
Carbonyl Sulfide (COS) is a major constituent of the earth’s at-
mosphere and forms an important part of the global sulfur
cycle. COS originates from volcanoes or hot springs and this
gas has been suggested as a possible chemical component
during origin of life. COS is also a source of energy for certain
bacteria. Thiocyanate hydrolases are a class of COS synthesiz-
ing enzymes that are found in bacteria, however, its produc-
reports raised a possibility that COS may be a potential bio-
molecule with effects distinct from H S. Since certain COS
2
donors are associated with trigger-independent decomposition
[4b]
pathways, a new class of donors is necessary.
The new
donor needs to be selective, and the byproducts of activation
need to be tolerated well by cells. This would allow for system-
atically studying the effects of COS in cells and compare (and
contrast) its effects with hydrogen sulfide. Here we report the
development of enzyme activated COS donors that are highly
suited for interrogating the effects of this gas and we demon-
strate that COS donors induce persulfidation in cells that may
contribute to its observed cytoprotective effects.
[
1]
tion in mammalian cells still remains elusive. Since COS is rap-
idly hydrolyzed to hydrogen sulfide (H S) in the presence of
2
[
2]
carbonic anhydrase (CA), a widely prevalent enzyme, this gas
has been utilized as a surrogate for enhancing H S within cells.
2
[
3]
Small molecules that generate COS, known as COS donors,
[
4]
[5]
including those activatable by an enzyme, light, nucleo-
Due to the in-built catalytic efficiency of an enzyme, an
enzyme-prodrug approach was used to design new COS
[
6]
[7]
[8]
philes, click chemistry or elevated hydrogen peroxide are
in development. Some have shown potential cytoprotective ef-
fects and even endothelial proliferative effects. These studies
donors. Since the effects of H S are dependent on its concen-
2
tration as well as rate of generation, any donor must have
structural features that allow for rates to be modulated. Based
on the aforementioned criteria, NAD(P)H quinoneoxidoreduc-
tase 1 (NQO1) also referred to as DT-Diaphorase, which is pre-
dominantly a two electron reducing cytosolic enzyme used by
the cell for reduction of quinones to hydroquinones, was
[a] P. Chauhan, G. Ravikumar, Dr. H. Chakrapani
Department of Chemistry
Indian Institute of Science Education and Research Pune
Dr. Homi Bhabha Road, Pashan
Pune 411 008, Maharashtra (India)
E-mail: harinath@iiserpune.ac.in
[9]
chosen as the prodrug-metabolizing enzyme. Immunohisto-
chemical analysis of NQO1 revealed its expression in certain tis-
[b] K. Gupta, Dr. D. K. Saini
[10]
sues including epithelial cells in the colon. Increased expres-
Department of Molecular Reproduction, Development and Genetics
Indian Institute of Science
Bangalore 560012, Karnataka (India)
sion of this enzyme under oxidative stress and electrophilic
stress conditions, likely due to its role in removal of xenobiot-
[
11]
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/asia.201901148.
ics, is also reported.
NQO1 has been used for delivering
[9a,12,13]
drugs or fluorophores (for imaging).
Since H S has been
2
shown to play an important protective role in the colon as
well as under oxidative stress conditions, we designed a
NQO1-activable COS donor (Figure 1b). This compound is ex-
pected to undergo reduction to produce a hydroquinone inter-
th
This manuscript is part of a special issue celebrating the 20 anniversary of
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Scheme 1. Synthesis of NQO1 triggered COS/H
2
S donors.
Figure 1. a) Hydrolysis of COS in the presence of carbonic anhydrase (CA)
produces hydrogen sulfide (H S). The major biosynthetic pathway for H
2
2
S
generation include metabolism of cysteine. In the presence of elevated oxi-
dative species, cysteine-containing proteins are persulfidated, that is, modi-
fied to their persulfides. b) Design of a bioreductively activated COS donor.
NAD(P)H quinoneoxidoreductase 1 (NQO1), also referred to as DT-Diaphor-
ase (DT-D) reduces the quinone to a diolate intermediate, which rapidly pro-
duces a lactone. The intermediate I, which has been previously shown to be
produced by other methods rearranges by self-immolation to produce COS,
which in the presence of CA produces hydrogen sulfide. The rate of hydro-
gen sulfide production may depend on the basicity of the amine.
Scheme 2. Synthesis of NQO1 triggered COS/H
amines used in this synthesis. For structures of 7a–7c, please see SI (Fig-
ure S1).
2
S donors. Inset, aromatic
First, the H S releasing capability of the molecules in the
2
presence of NQO1 was evaluated using a methylene blue for-
[14]
mation assay. Compounds were incubated in the presence
of NQO1, NADH and CA in phosphate buffer (pH 7.4) at 378C.
At pre-determined time points, an aliquot of the reaction mix-
ture was taken, treated with the methylene blue cocktail and
further incubated to allow the formation of the complex. An
absorbance profile was measured to follow the formation of
methylene blue complex. A time dependent increase in the ab-
sorbance profile for compound 1a was observed over a period
mediate, which would lactonize to produce intermediate I. Our
lab recently reported that rates of release of COS could be
[
8a]
modulated by varying the basicity of the amine. Here, the
suggested intermediate I self-immolates to produce COS and
an amine (Figure 1b). COS is hydrolysed to H S in the presence
2
of CA.
of 4 h (Figure 2a). The kinetics of H S release was measured for
2
both the compounds under similar conditions. The rate con-
stants for compounds 1a and 1b were calculated by fitting
the initial rate data to first order kinetics. The rate constants
Results and Discussion
Since our previous data suggested that the rate of H S genera-
2
ꢀ
1
tion by the aniline-derivative was significantly higher when
compared with an aliphatic amine-based donor, we decided to
synthesize 1a, a p-anisidine-based donor and 1b, with propyla-
mine as a leaving group. Compound 6 was first synthesized in
three steps starting from compound 2 (Scheme 1). Com-
pound 6 was then reacted with thiourea for 12 h to form a thi-
ourea adduct which was further hydrolysed to give thiol. The
thiol so obtained was found to be unstable and therefore was
directly taken to the next step without further purification to
react with respective carbamates and give compound 1a in
for compound 1a and 1b were calculated to be 0.016 min
ꢀ
1
and 0.008 min respectively (See SI, Figure S2). This trend is in
accordance with our previous reports, although the magnitude
of difference between the fast and slow donor was somewhat
[8a]
diminished possibly due to the poor solubility of the donors.
Next, the decomposition of 1a in the presence of NQO1 was
monitored by HPLC analysis. Compound 1a when incubated in
buffer was eluted at a retention time of 16.6 min (See SI, Fig-
ure S5a). Upon treatment with NQO1 in phosphate buffer
(pH 7.4) containing NADH at 378C, we observed a complete
disappearance of the peak for 1a within 30 min of incubation.
This was in accordance with the previous studies, indicative of
65% and 1b in 34% yield (Scheme 2). Compound 5 was used
as a negative control in further experiments.
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phile tagged to a fluorescent reporter, to tag protein persul-
fides. The cells were then imaged under a fluorescence micro-
scope in FITC channel. A significant enhancement in the fluo-
rescence signal was observed with 1a compared to the
untreated control, indicative of the protein persulfidation in-
duced by 1a. A diminished fluorescence signal was observed
with compound 1b which could be associated to the lower
rate of H S release observed from 1b (Figure 2c). Thus, the re-
2
sults obtained suggest that the extent of persulfidation in-
duced by H S depends on the rate of its release. The observed
2
fluorescence signal for protein persulfidation was quantified
and plotted as corrected total cell fluorescence (CTCF, a.u.)
(
Figure 2d). Na S showed a diminished fluorescence signal
2
compared to compound 1a which could be due to the low ef-
fective concentration of H S obtained from the aqueous solu-
2
tion of Na S. Hence, COS donor 1a is able to permeate cells to
2
induce protein persulfidation.
Figure 2. a) Representative methylene blue formation plots determined by
spectophotometry after incubation of 1a (50 mm) in the presence of NQO1
The cytotoxicity profile of the molecules was assessed using
a standard cell viability assay. DLD-1 cells were treated with
varying concentration of compounds for 24 h following which
the cell viability was monitored. The compounds were found
to be well tolerated by the cells at 50 mm concentration (See
SI, Figure S12a,c). Human breast cancer, MCF-7 cells were next
incubated with varying concentrations of 1a and cell viability
was measured after 72 h of incubation. No significant decrease
in viable cells was seen, suggesting that the reported scaffolds
are non-toxic (See SI, Figure S9). The obtained results were fur-
ther confirmed by using LDH release assay. Here, MCF-7 cells
were treated with varying concentrations of 1a and after 24 h
measurements revealed that this compound was well tolerated
at 50 mm concentration (See SI, Figure S10). This result is in
contrast to recent reports that suggest that fast COS donors
are associated with cytotoxicity and could be related, in part,
to non-specific decomposition pathways associated with the
ꢀ
1
(
10 mgmL ), NADH (250 mm) and CA in phosphate buffer (pH 7.4). b) Repre-
sentative HPLC traces for decomposition of compound 1a, (i) 30 min (ii) 1 h
iii) 2 h. c) Representative images of persulfidation induced by NQO1 respon-
sive H S donors (1a and 1b) within human colon carcinoma (DLD-1) cells
n=3). Scale bar is 50 mm. Increase in the persulfidation signal was observed
with the H S donors, (i) ctrl represents untreated cells (ii) Na S (200 mm)
iii) 1a (10 mm) (iv) 1b (10 mm). d) Quantification of the persulfidation signal
observed by the H S donors (n=3).
(
2
(
2
2
(
2
the rapid reduction of the quinone in the presence of NQO1
enzyme; the peak at 8.3 min corresponded to the formation of
[
15]
lactone (Figure 2b). The rate of decomposition of the com-
ꢀ
1
pound was calculated to be 0.063 min which correlated well
ꢀ
1
with the rate of formation of lactone that is, 0.066 min (See
SI, Figure S5c,d). Another peak corresponding to the formation
of an intermediate was observed at 7.8 min which decom-
posed over a period of 2 h with concomitant formation of p-
anisidine at 4.8 min (See ESI, Figure S5b). The results obtained
support our hypothesis that the quinone is reduced by NQO1
to form hydroxyquinone (Figure 1b), which immediately un-
dergoes intramolecular cyclization to generate intermediate I.
The intermediate thus formed decomposes over a period of
[4b,c]
previously reported donors.
The donors reported herein were non-toxic and capable of
inducing protein persulfidation within cells. This led us to fur-
ther investigate the cytoprotective effects of these donors,
against oxidative stress induced toxicity. JCHD is a cell-permea-
ble small molecule superoxide generator; since elevated ROS is
cytotoxic, this is a good tool to study oxidative stress (See SI,
2
h to release COS which is then hydrolyzed to H S by CA.
2
Having established the in vitro release of H S from the
2
[19]
donor motifs, we next investigated the cellular aspects of con-
trolled generation of this reactive gaseous species. Protein per-
sulfidation is a protective post-translational modification asso-
Figure S11). When cells were co-treated with JCHD and 1a, a
dose-dependent increase in the cell viability was observed.
However, no increase in the cell viability was observed with
compound 1b, which is consistent with the poor capability for
this compound to induce protein persulfidation (See SI, Fig-
ure S12b,d). It appears that persulfidation by the COS donor
ciated with H S wherein, thiol of a cysteine (Cys-SH) reacts
2
with H S in the presence of oxidants to form more reactive
2
[
16]
persulfide (Cys-SSH). Due to the inherent lack of stability of
persulfides, detecting these transient species is challenging.
We used an improved tag switch technique to detect persul-
may determine the cytoprotective effects of H S.
2
To further validate the hypothesis and evaluate the cytopro-
tective effects of H S, we synthesized a H S-NSAID hybrid
[
17]
fides in cells. NQO1 enzyme is overexpressed in colon can-
cers which include human colon adenocarcinoma, DLD-1
2
2
donor and studied its effects. Mesalamine is used as first in line
[
18]
[20]
cells. Thus, in order to test the persulfidating capability of
therapy for the treatment of colitis. But, the drug is effective
the donors, DLD-1 cells were treated with H S donors (10 mm)
only against mild to moderate forms of this disease and lacks
potency to treat severe form of colitis. Wallace and co-workers
2
for 1 h to induce persulfidation. Cells were further treated with
methyl sulfonylbenzathiazole (MSBT), an aromatic thiol block-
ing agent, to block the persulfides in the form of mixed disul-
fides. This was followed by treatment with CN-BOT; a nucleo-
in 2007 showed that ATB-429, a H S-mesalamine hybrid, exhib-
2
ited marked increase in anti-inflammatory properties, thereby
increasing the effectiveness and potency of the drug in a
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[
21]
murine model of colitis. Therefore, we proceeded to investi-
gate the cytoprotective effects of these donors against xenobi-
otic induced stress in human colon carcinoma, DLD-1 cells. We
synthesized NQO1 activated COS-Mesalamine donor, NQO1-
Mes, using the above mentioned protocol in 43% yield
the cytoprotective effects of NQO1-Mes in mouse embryonic fi-
broblast, WT-MEF cells, reported previously to have low expres-
[22]
sion levels of NQO1 enzyme. The cytoprotective effects were
tested using the aforementioned protocol. WT-MEF cells were
co-treated with increasing concentrations of H S donors and
2
(
Scheme 2). This compound was found to generate H S (elec-
JCHD (15 mm) for 24 h following which the cell viability was
2
trode data, Figure 3a; methylene blue data, Figure 3b). In the
monitored. As expected, no significant increase in cell viability
presence of a known inhibitor for DT-D, diminished H S pro-
was observed with the H S donors compared to the JCHD con-
2
2
duction was observed. HPLC analysis showed complete de-
composition of this compound to produce methylester deriva-
tive of mesalamine, 9 (See SI, Figure S6). The rate constant for
hydrogen sulfide release was calculated to be 0.02 min (see
SI, Figure S3). This donor was also found to induce persulfida-
tion in cells (Figure 3c,d) and was able to protect from JCHD-
induced cytotoxicity (Figure 3 f). Mesalamine alone was not
found to be as effective possibly due to poor cell permeability.
The cell type-specificity of the donors was assessed by testing
trol (See SI, Figure S13, S14). The results indicate that the H2S
donors reported herein are selective towards activation by
NQO1 enzyme.
ꢀ
1
As COS has developed into an important delivery tool for lo-
calizing H S, it is natural to assume that the physiological ef-
2
fects observed with the reported COS donors are attributable
to H S. But recent advancements in the field have raised the
2
possibility of COS acting as a potential gaseous signalling mol-
ecule acting distinctly from H S; thus posing an important
2
question on the mechanism of action of COS within cells. We
worked towards addressing this ambiguity by synthesizing
NQO1 activated COS donors which are selective, capable of
modulating the rate of COS generation with release of poten-
tially innocuous byproducts. It is hypothesized that NQO1
along with NADH cofactor reduces the quinone moiety in com-
pound 1 to hydroquinone which undergoes immediate cyclisa-
tion to subsequently release lactone and intermediate I. The in-
termediate thus formed releases COS depending on the basici-
ty of the leaving group amine. COS released is readily hydro-
lyzed to H S in the presence of CA which is assayed by various
2
independent techniques. Therefore, the donors were found to
release H S in the presence of NQO1 and CA. Modulating the
2
release of COS/H S proved to be an important tool to delineate
2
the mechanism of action of COS within cells.
Protein persulfidation is a characteristic post translational
[23]
modification associated with H S. Protein persulfides, thus
2
assume importance in sulfur mediated signaling and therapeu-
tics. Strategies to release small molecule persulfides have been
[
24]
developed which include—spontaneous persulfide donors,
[25]
enzyme activated donors, oxidative stress induced persulfide
[19b,26]
ꢀ
[27]
release,
pH or F dependent persulfide release. These
donors provide testament to therapeutic utility of enhancing
persulfidation levels within cells. Therefore, we investigated if
the donors reported herein get activated by NQO1 present
within cells to release H S and further induce protein persulfi-
2
dation. The persulfides can be trapped and detected using im-
proved tag switch technique which is widely accepted for de-
Figure 3. a) Representative trace for H
2
S release from NQO1-Mes (50 mm) in
the presence of NQO1 (10 mgmL ), NADH (250 mm) and CA in PBS (pH 7.4,
0 mm) using sulfide selective electrode. Ctrl represents NQO1-Mes (50 mm)
alone in media containing 10% FBS. No H S was produced from the com-
ꢀ1
5
[17]
tection of persulfides in cells. Compounds 1a and 1b signifi-
cantly enhanced the persulfidation levels in DLD-1 cells which
2
pound in media. b) Methylene blue formation determined by spectopho-
tometry after incubation of NQO1-Mes(100 mm) in the presence of NQO1,
NADH and CA. Diminished signal was observed in the presence of dicoumar-
ol 8, a known inhibitor of NQO1. c) Representative images of persulfidation
induced by NQO1-Mes in human colon carcinoma (DLD-1) cells (n=3). Scale
bar is 50 mm. (i) ctrl (ii) NQO1-Mes (10 mm). d) Quantification of the persulfida-
is indicative of the release of H S within cells. However, the
2
extent of persulfidation induced by the donors was found to
be dependent on the rate of release. The result indicates that
COS produced in cells is hydrolyzed to generate H S which in-
2
duces protein persulfidation depending on the rate of release;
therefore, providing an important link towards understanding
the mechanism of COS activity. Finally, to investigate if the ob-
served differences could be translated to mediate the physio-
logical effects within cells, we tested the cytoprotective effects
of the donors against xenobiotic induced stress. Human colon
2
tion signal observed by the H S donors (n=3). e) Cell viability for NQO1-Mes
in DLD-1 cells. f) Cytoprotective effects of NQO1-Mes against JCHD (15 mm)
induced oxidative stress in DLD-1 cells over a period of 24 h. Results are ex-
pressed as Mean ꢁ SEM (n=3). [Legend: **** implies p<0.0001 vs. JCHD].
Compound 5 (50 mm) represents the negative control and Mes represents
mesalamine (50 mm). No significant increase in the cell viability was observed
during incubation of 5 and mesalamine with JCHD.
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adenocarcinoma, DLD-1 cells were treated with oxidative stress
inducing agent (JCHD) to induce cell death. When co-treated
with COS donors we observed that compound 1a showed sig-
nificant enhancement in viable cells compared to com-
pound 1b which is likely due to the difference in their persulfi-
dation capability. The results obtained were further validated
by NQO1 activated COS-NSAID hybrid donor which showed cy-
toprotective effects against oxidative stress. This is again con-
3, 7a–7c were synthesized using previously reported procedures
and were taken forward without purification.
[8a,15b]
Synthesis of 4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl 3-
methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)buta-
noate (4): To a solution of 2 (1.22 g, 4.89 mmol) and 3 (0.8 g,
4
.07 mmol) in dry DCM at 08C, EDC.HCl (1.56 g, 8.15 mmol) and
DMAP (1 g, 8.15 mmol) were added. The reaction mixture was
stirred at 08C. The progress of the reaction was monitored by TLC.
The reaction was complete after 10 min following which the reac-
tion mixture was washed with 1n HCl, organic layer was dried
over Na SO and concentrated in vacuo. The crude was purified
sistent with the behavior of COS much like H S.
2
Taken together, the effects of COS generated by this class of
donors is protein persulfidation, which is symptomatic of inter-
mediacy of hydrogen sulfide. The donors themselves as well as
their byproducts were well tolerated and the NSAID-hybrid
was able to protect cells from oxidative stress supporting the
further development of this class of compounds. These tools
lay the foundation for systematically studying the redox biol-
ogy of COS, a newly emergent bioactive gaseous molecule
with high therapeutic potential.
2
4
using column chromatography to give 4 (1.36 g, 71%) as yellow
ꢀ1
1
oily liquid. FT-IR n˜ max =1750, 1644, 1258 cm
; H NMR (400 MHz,
CDCl ): d=7.28 (d, J=8.7 Hz, 2H), 6.92 (d, J=8.6 Hz, 2H), 4.69 (s,
3
2
0
1
4
H), 3.23 (s, 2H), 2.17 (s, 3H), 1.93–1.91 (m, 6H), 1.52 (s, 6H),
13
.93(s, 9H), 0.08 (s, 6H); C NMR (100 MHz, CDCl ) d=190.9, 187.4,
3
71.4, 152.0, 149.2, 142.9, 139.1, 139.0, 138.5, 127.0, 121.2, 64.4,
7.7, 38.5, 28.9, 25.9, 18.4, 14.3, 12.6, 12.1, ꢀ5.3 ppm; HRMS (ESI-
+
TOF) for [C H O Si+H] : calcd 471.2567, found 471.2570.
27
38
5
Synthesis of 4-(hydroxymethyl)phenyl 3-methyl-3-(2,4,5-trimeth-
yl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoate (5): To a solution
of 4 (0.69 g, 1.45 mmol) in THF (5 mL), water (5 mL) and acetic acid
Conclusions
(15 mL) were added. The progress of the reaction was monitored
by TLC. Reaction was complete after 6 h of stirring. The reaction
While the geological COS production and as a consequence
exposure to this gas is not in doubt, whether COS is biosynthe-
sized remains to be determined. COS has been suggested as a
stable reservoir for hydrogen sulfide and other reports indicate
mixture was then quenched with saturated NaHCO solution and
3
the aqueous layer was extracted by DCM and dried over Na SO .
2
4
The organic layer was concentrated under vacuo. The crude was
purified by column chromatography to give 5 (0.5 g, 97%) as
distinct effects of COS when compared with H S. Since careful
ꢀ1
1
2
yellow liquid. FT-IR n˜ max =3510, 1747, 1643 cm ; H NMR (400 MHz,
generation of COS with relatively innocuous byproducts is cru-
cial to delineate such effects, the tools that we have developed
are important. Our study demonstrates that COS donors are
well tolerated by cells and induce protein persulfidation in
cells. The donors were also found to protect cells from oxida-
CDCl ): d=7.33 (d, J=8.3 Hz, 2H), 6.95 (d, J=8.4 Hz, 2H), 4.64 (s,
3
2H), 3.24 (s, 2H), 2.17 (s, 3H), 1.92–1.91 (m, 6H), 1.52 ppm (s, 6H);
13
C NMR (100 MHz, CDCl ) d=190.9, 187.4, 171.4, 152.0, 149.8,
3
1
1
42.9, 139.1, 138.6, 138.5, 128.0, 121.6, 64.7, 47.7, 38.4, 28.9, 14.4,
+
2.6, 12.1 ppm; HRMS (ESI-TOF) for [C H O H] : calcd 357.1702,
21
24 5+
found 357.1711.
tive stress, which is symptomatic of H S generation. Together,
2
Synthesis of 4-(bromomethyl)phenyl 3-methyl-3-(2,4,5-trimeth-
yl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoate (6): To a solution
of 5 (0.51 g, 1.43 mmol) in dry DCM, tribromophosphine (170 mL,
our study suggests a link between COS and persulfidation sup-
porting a significant role for H S in COS biology.
2
1
.71 mmol) was added at 08C. The reaction was stirred and the
progress was monitored by TLC. After completion of the reaction
in 1 h, the reaction mixture was quenched with saturated solution
of NaHCO3 and the aqueous layer was extracted by EtOAc. The
combined organic layer was dried over Na SO and concentrated
Experimental Section
All reactions were conducted under nitrogen atmosphere. All the
chemicals were purchased from Sigma Aldrich. The solvents were
purchased from commercial sources and used as received unless
stated otherwise. Column chromatography was performed using
silica gel-Rankem (60- 120 mesh) or silica gel Spectrochem (100–
2
4
under vacuo. Crude was purified by column chromatography and
compound was obtained as yellow liquid with 43% yield. FT-IR
ꢀ1 1
n˜ max =2924, 2856, 1747, 1648, 1601 cm ; H NMR (400 MHz, CDCl ):
3
d=7.35 (d, J=8.5 Hz, 2H), 6.95 (d, J=8.6 Hz, 2H), 4.45 (s, 2H), 3.24
2
00 mesh) as the stationary phase. Preparative high-performance
1
3
(
s, 2H), 2.17 (s, 3H) 1.93–1.92 (m, 6H), 1.52 ppm (s, 6H); C NMR
liquid chromatography (HPLC) was done using Combiflash EZ prep
UV using a KromasilꢁC-18 preparative column (250 mmꢂ21.2 mm,
(
100 MHz, CDCl ) d=190.8, 186.7, 171.2, 151.8, 150.3, 142.8, 138.6,
3
1
13
138.2, 135.4, 130.2, 121.9, 47.6, 38.4, 32.6, 28.9, 14.4, 12.7,
5
1
mm). H and C spectra were recorded on a JEOL 400 MHz (or
+
1
3
13
12.1 ppm; HRMS (ESI-TOF) for [C H BrO H] : calcd 419.0858,
00 MHz for C) or a Bruker 400 MHz (or 100 MHz for C) spec-
21 23
4+
found 419.0836.
trometer unless otherwise specified using either residual solvent
signals (CDCl dH=7.26 ppm, dC=77.2 ppm) or as an internal tet-
Synthesis of 4-((((4-methoxyphenyl)carbamoyl)thio)methyl)-
phenyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-
1-yl)butanoate (1a): To a solution of 6 (0.30 g, 0.71 mmol) in dry
THF, thiourea (0.11 g, 1.43 mmol) was added at room temperature.
The reaction was stirred overnight at room temperature. After
completion of the reaction in 12 h, the solvent was removed under
vacuum to obtain a yellow coloured solid. The crude was dissolved
in 15 mL water and 20 mL DCM under nitrogen atmosphere.
Na S O (0.54 g, 2.86 mmol) was then added to the reaction mix-
3
ramethylsilane (dH=0.00, dC=0.0). Chemical shifts (d) are report-
ed in ppm and coupling constants (J) in Hz. The following abbrevi-
ations are used: bs (broad signal), m (multiplet), s (singlet), d (dou-
blet), t (triplet) and dd (doublet of doublets). High-resolution mass
spectra were obtained from HRMS-ESI-Q-Time of Flight LC/MS. FT-
IR spectra were recorded using BRUKER-ALPHA FT-IR spectrometer.
Analytical HPLC was performed on an Agilent1260-infinity with
PhenomenexꢁC-18 reverse phase column (250 mmꢂ4.6 mm,
2
2
5
5
mm). Photometric measurements were performed using
a
ture and refluxed at 608C. After completion of the reaction in 4 h,
Thermo Scientific Varioskan microtiter plate reader. Compounds 2,
the crude was extracted using DCM. The organic layer was dried
Chem. Asian J. 2019, 00, 0 – 0
www.chemasianj.org
5
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Paper
over Na SO and concentrated under vacuo. The product obtained
was unstable and therefore was taken directly to the next step.
Crude was dissolved in dry DCM (10 mL) followed by the addition
trol with NQO1 inhibitor, 100 mm (10 mm stock in 0.1n NaOH) of
the compound 8 was added to the reaction mixture containing
NQO1-Mes in the presence of NQO1, NADH and CA. After 4 h,
100 mL aliquot was removed from each reaction vial and diluted
2
4
of 7a (0.34 g, 1.19 mmol) and Et N (470 mL, 3.37 mmol). The reac-
3
tion was stirred at room temperature. The progress of the reaction
was monitored TLC. After completion in 2 h, the reaction was
quenched by the addition of water and extracted by DCM (20 mL).
The crude was purified by preparative HPLC using kromasil C-18
column and water: acetonitrile as mobile phase to give 1a
with 100 mL of FeCl (30 mm stock in 1.2m HCl) and 100 mL of N,N-
3
dimethyl-p-pheneylenediamine sulfate (20 mm stock in 7.2m HCl).
The mixture was incubated at 378C for 30 min. After completion of
methylene blue complex formation, aliquots were transferred to a
96 well plate (250 mL/ well) and the absorbance spectra were col-
lected from 500 to 800 nm on a plate reader.
ꢀ
1
1
(
241 mg, 65%). FT-IR n˜ max =3336, 1745, 1644 cm
;
H NMR
(
8
2
400 MHz, CDCl ): d=7.33–7.26 (m, 4H), 7.01 (s, 1H), 6.90 (d, J=
3
HPLC analysis: The decomposition of compound 1a was followed
.5 Hz, 2H), 6.85 (d, J=9 Hz, 2H), 4.15 (s, 2H), 3.78 (s, 3H), 3.22 (s,
by HPLC. A stock solution of H S donors (2.5 mm) was prepared in
2
13
H), 2.17 (s, 3H), 1.92–1.91 (m, 6H), 1.51 ppm (s, 6H); C NMR
ꢀ1
DMSO. The stock of NQO1 (1 mgmL ) and NADH (10 mm) were
(
100 MHz, CDCl ) d=190.9, 187.4, 171.4, 151.9, 149.5, 142.9, 139.1,
3
prepared in phosphate buffer (pH 7.4, 10 mm). The reaction mix-
ture consisted of compound (25 mm) in buffer containing NQO1
1
1
5
38.6, 135.8, 130.0, 121.6, 114.3, 55.5, 47.7, 38.4, 33.8, 28.9, 14.4,
+
2.7, 12.1 ppm; HRMS (ESI-TOF) for [C H NO S+H] : calcd
ꢀ1
29
31
6
(10 mgmL ) and NADH (100 mm) and incubated at 378C. At prede-
22.1950, found 522.1945.
termined time points, an aliquot of the reaction mixture was re-
moved, filtered (0.22 micron filter) and injected (50 mL) in a high
performance liquid chromatography (HPLC Agilent Technologies
1260 Infinity). The mobile phase was H O/ACN. The stationary
2
phase was C-18 reverse phased column (Phenomenex, 5 mm, 4.6ꢂ
Synthesis of4-(((propylcarbamoyl)thio)methyl)phenyl 3-methyl-
-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoate
1b): Compound 1b was synthesized using procedure outlined for
a. The compound was obtained as yellow viscous liquid (110 mg,
3
(
1
3
ꢀ
1
1
4%). FT-IR n˜ max =3373, 2926, 1744, 1647 cm ; H NMR (400 MHz,
250 mm). A multistep gradient was used with a flow rate of
ꢀ1
CDCl ): d=7.28 (d, J=8.5 Hz, 2H), 6.89 (d, J=8.4 Hz, 2H), 5.52 (s,
1
1
1 mLmin starting with !0–13 min, 50:50 to 10:90!13–16 min,
3
H), 4.10 (s, 2H), 3.25–3.22 (m, 4H), 2.16 (s, 3H), 1.92–1.91 (m, 6H),
10:90 to 10:90!16–20 min, 10:90 to 50:50.
13
.52 (s, 8H), 0.90 ppm (t, J=7.4 Hz, 3H); C NMR (100 MHz, CDCl3)
A similar protocol was used for compound NQO1-Mes. The reac-
d=190.9, 187.4, 171.4, 166.4, 152.0, 149.4, 142.9, 139.1, 138.6,
36.3, 129.9, 121.5, 47.6, 43.2, 38.4, 33.5, 28.9, 22.9, 14.3, 12.7, 12.1,
tion mixture consisted of compound (50 mm) in buffer containing
ꢀ
1
1
NQO1 (10 mgmL ) and NADH (100 mm) and incubated at 378C. At
predetermined time points, an aliquot of the reaction mixture was
removed, filtered (0.22 micron filter) and injected (50 mL) in a high
performance liquid chromatography (HPLC Agilent Technologies
+
1
1.2 ppm; HRMS (ESI-TOF) for [C H NO S+H] : calcd 458.2002,
25
31
5
found 458.2002.
Synthesis of methyl 2-hydroxy-5-((((4-((3-methyl-3-(2,4,5-tri-
methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoyl)oxy)benzyl)th-
io)carbonyl)amino)benzoate (NQO1-Mes): Compound NQO1-Mes
was synthesized using procedure outlined for 1a. DMF was used
as the solvent for reaction. The compound was obtained as yellow
1
260 Infinity). The mobile phase was H O/ACN. The stationary
2
phase was C-18 reverse phased column (Phenomenex, 5 mm, 4.6ꢂ
2
1
2
50 mm). A multistep gradient was used with a flow rate of
ꢀ1
mLmin starting with !0–5 min, 40:60 to 25:75!5–10 min,
5:75 to 10:90 ! 10–15 min, 10:90 to 0:100!15–17 min 0:100 to
solid (146 mg, 43% of 2 steps). FT-IR n˜ =3330, 1745, 1681,
max
ꢀ
1
1
0:100!17–20 min, 0:100 to 40:60!20–22 min, 40: 60.
H S detection using sulfide selective electrode: Compound
2
1
7
2
2
643 cm ; H NMR (400 MHz, CDCl ): d=10.63 (s, 1H), 7.91 (s, 1H),
3
.48 (bs, 1H), 7.35 (dd, J=8.9 Hz, 2.8 Hz, 1H), 7.29 (d, J=8.6 Hz,
H), 6.89 (d, J=8.5 Hz, 3H), 4.13 (s, 2H), 3.90 (s, 3H), 3.23 (s, 2H),
.15 (s, 3H), 1.91–1.90 (m, 6H), 1.51 ppm (s, 6H); C NMR
NQO1-Mes (10 mL, 5 mm) was added to PBS pH 7.4 (925 mL) con-
taining CA (5 mL, 1% stock), NADH (50 mL, 10 mm) and NQO1
13
ꢀ
1
(
100 MHz, CDCl ) d=190.8, 187.4, 171.5, 170.0, 158.6, 151.9, 149.5,
(10 mL, 1 mgmL ) at 378C in a closed vial. The H
S produced was
2
3
1
3
42.8, 139.2, 138.6, 135.7, 130.0, 121.6, 118.1, 112.1, 52.5, 47.6, 38.4,
detected using 5 mm H S sensitive microelectrode (ISO-H2S-100)
attached to a TBR 4100 Free Radical Analyser (WPI) and shown as
picoamps current generated. A standard calibration curve was gen-
2
3.7, 28.9, 14.4, 12.7, 12.1 ppm; HRMS (ESI-TOF) for [C H NO S+
30
31
8
+
H] : calcd 566.1848, found 566.1848.
[
6b]
erated using a freshly prepared Na
lowing the manufacturer’s protocol.
S solution in PBS buffer by fol-
2
Methylene Blue method for H S detection:
Each assay de-
2
scribed here was done in triplicate in vials with closed lids, contain-
Cell Culture: Human breast carcinoma, MCF-7 cells and wild type
mouse embryonic fibroblast, WT-MEF cells were were grown in
Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal
bovine serum (FBS), 1% Penicillin Streptomycin antibiotic. Human
colon adenocarcinoma, DLD-1 cells, were grown in RPMI 1640
media containing 10% (v/v) fetal bovine serum (FBS), 1% Penicillin
Streptomycin antibiotic.
ing 746 mL of PBS, 8 mL of H S donor (5 mm stock in DMSO), 20 mL
of NADH (10 mm stock), 8 mL of NQO1 (1 mgmL ), 8 mL carbonic
anhydrase (1% stock in PBS buffer) and 10 mL Zn(OAc)2 (40 mm
2
ꢀ
1
stock in H O). The reaction mixture was incubated at 378C for 4 h.
2
1
00 mL aliquot was removed after pre-determined time points from
each reaction vial and diluted with 100 mL of FeCl (30 mm stock in
3
1
.2m HCl) and 100 mL of N,N-dimethyl-p-pheneylenediamine sulfate
(
20 mm stock in 7.2m HCl). The methylene blue mixture was fur-
Cellular persulfidation protocol: Human colon carcinoma, DLD-1
cells were seeded in a 12 well plate at a concentration of 0.1ꢂ10
cells/well in RPMI media supplemented with 10% FBS (fetal bovine
serum) and 1% antibiotic solution and incubated in an atmosphere
6
ther incubated at 378C for 30 min. After completion of methylene
blue complex formation, aliquots were transferred to a 96 well
plate (250 mL/well) and the absorbance spectra were collected
from 500 to 800 nm on a plate reader. A similar protocol was fol-
of 5% CO at 378C overnight. Next day, the cells were treated with
2
lowed to monitor the generation of H S from NQO1-Mes. Each
test compounds (10 mm) and Na S (200 mm) for 1 h. The cells were
2
2
assay was done in triplicate in vials with closed lids, containing
washed twice with sterile PBS after the treatment was over. Cells
were then fixed by incubating on ice cold methanol at ꢀ208C for
20 min and subsequently permeabilized using ice cold acetone
ꢀ208C for 5 min. The cells were then washed with PBS and treated
with 50 mm HEPES containing triton (1%) and MSBT (10 mm) for
3
74 mL of PBS, 4 mL of NQO1-Mes (10 mm stock in DMSO), 10 mL of
ꢀ1
NADH (10 mm stock), 4 mL of NQO1 (1 mgmL ), 4 mL carbonic an-
hydrase (1% stock in PBS buffer) and 4 mL Zn(OAc) (40 mm stock
2
in H O). Dicoumarol 8, is a known inhibitor of NQO1. For the con-
2
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&
Chem. Asian J. 2019, 00, 0 – 0
www.chemasianj.org
6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
overnight at room temperature. Cells were again washed with PBS
3 times) and further incubated with CN-BOT (25 mm) in PBS for 1 h
kan) to estimate the cell viability. A similar protocol was followed
(
for demonstrating the effects of H S donors against JCHD induced
2
at 378C. Finally, cells were washed 5 times with PBS and imaged
using IX83 microscope (Olympus Inc., Japan). The data that we
have presented is representative of 3 independent biological repli-
cates. Each experiment >20 fields were imaged at identical image
acquisition settings.
oxidative stress in WT-MEF cells. Cells were grown in DMEM media
supplemented with 10% FBS and 1% antibiotic solution.
Acknowledgements
Cell Viability Assay. (a) MTT assay: The cytotoxicity effect of the
H S donors was tested against various cell lines using a similar pro-
The Council for Scientific and Industrial Research (CSIR) is ac-
knowledged for research fellowships. The authors would also
like to thank Infosys Foundation for international travel grant.
2
tocol. Human colon cancer cells, DLD-1, were seeded at a concen-
4
tration of 1ꢂ10 cells/well overnight in a 96-well plate in complete
RPMI media. Cells were exposed to varying concentrations of the
test compounds prepared as a DMSO stock solution so that the
final concentration of DMSO was 0.5%. The cells were incubated Conflict of interest
for 24 h at 378C. A solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-
phenyl tetrazolium bromide (MTT) was prepared using 3.5 mg in
The authors declare no conflict of interest.
7
mL RPMI media. 100 mL of the resulting solution was added to
each well. After 4 h incubation, the media was removed carefully
and 100 mL of DMSO was added to each well. Spectrophotometric
analysis of each well using a microplate reader (Thermo Scientific
Varioskan) at 570 nm was carried out to estimate cell viability. A
similar protocol was followed for wild type mouse embryonic fibro-
blast (WT-MEF cells) and the media used was complete DMEM
media. Human breast carcinoma, MCF-7 cells were seeded at a
Keywords: carbonyl
persulfidation · prodrug
sulfide
·
hydrogen
sulfide
·
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Version of record online: && &&, 0000
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www.chemasianj.org
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ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
Preeti Chauhan, Kavya Gupta,
Govindan Ravikumar, Deepak K. Saini,
Harinath Chakrapani*
&& – &&
Carbonyl Sulfide (COS) Donor Induced
Protein Persulfidation Protects against
Oxidative Stress
NQO1 triggered release of COS induces
protein persulfidation within cells and
likely contributes to cytoprotective ef-
fects.
Chem. Asian J. 2019, 00, 0 – 0
www.chemasianj.org
9
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ