Alkylamine-Substituted Perthiocarbamates: Dual Precursors to Hydropersulfide and Carbonyl Sulfide with Cardioprotective Actions

Article abstract of DOI:10.1021/jacs.9b12180

The recent discovery of hydropersulfides (RSSH) in mammalian systems suggests their potential roles in cell signaling. However, the exploration of RSSH biological significance is challenging due to their instability under physiological conditions. Herein, we report the preparation, RSSH-releasing properties, and cytoprotective nature of alkylamine-substituted perthiocarbamates. Triggered by a base-sensitive, self-immolative moiety, these precursors show efficient RSSH release and also demonstrate the ability to generate carbonyl sulfide (COS) in the presence of thiols. Using this dually reactive alkylamine-substituted perthiocarbamate platform, the generation of both RSSH and COS is tunable with respect to half-life, pH, and availability of thiols. Importantly, these precursors exhibit cytoprotective effects against hydrogen peroxide-mediated toxicity in H9c2 cells and cardioprotective effects against myocardial ischemic/reperfusion injury, indicating their potential application as new RSSH- and/or COS-releasing therapeutics.

Full text of DOI:10.1021/jacs.9b12180

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Article
Alkylamine-Substituted Perthiocarbamates: Dual Precursors to
Hydropersulfide and Carbonyl Sulfide with Cardioprotective Actions
Vinayak Shahaji Khodade, Blaze M. Pharoah, Nazareno Paolocci, and John P. Toscano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12180 • Publication Date (Web): 14 Feb 2020
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Alkylamine-Substituted Perthiocarbamates: Dual Precursors to
Hydropersulfide and Carbonyl Sulfide with Cardioprotective Actions
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Vinayak S. Khodade,† Blaze M. Pharoah,† Nazareno Paolocci,‡,§ and John P. Toscano*,†
†Department of Chemistry, Johns Hopkins University, Baltimore, Maryland
‡Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland
§Department of Biomedical Sciences, University of Padova, Padova, Italy
Supporting Information Placeholder
ABSTRACT: The recent discovery of hydropersulfides (RSSH) in mammalian systems suggests their potential roles in cell
signaling. However, the exploration of RSSH biological significance is challenging due to their instability under physiological
conditions. Herein, we report the preparation, RSSH-releasing properties, and cytoprotective nature of alkylamine-
substituted perthiocarbamates. Triggered by a base-sensitive, self-immolative moiety, these precursors show efficient RSSH
release, andalsodemonstrate the abilitytogenerate carbonyl sulfide (COS) inthe presence of thiols. Using this dually reactive
alkylamine-substituted perthiocarbamate platform, the generation of both RSSH and COS is tunable with respect to half-life,
pH, and availability of thiols. Importantly, these precursors exhibit cytoprotective effects against hydrogen peroxide-
mediated toxicity in H9c2 cells and cardioprotective effects against myocardial ischemic/reperfusion injury, indicating their
potential application as new RSSH- and/or COS-releasing therapeutics.
8-9, 18-22
signaling intermediates.5-6,
Recent reports have
INTRODUCTION
demonstrated that RSSH are efficient H-atom transfer
agents toward alkyl, alkoxyl, peroxyl, and thiyl radicals,
confirming their promise as potent antioxidants.23-24 Unlike
thiols, RSSH can also undergo transsulfuration reactions
because of their electrophilic properties in the neutral
state.18, 25 The sulfane sulfur in RSSH can be reversibly
transferred to other free thiols such as glutathione (GSH) or
cysteine (Cys-SH) to form GSSH or Cys-SSH, respectively.
Furthermore, studies have suggested RSSH involvement in
the detoxification of environmental electrophiles.26-28 Yet,
despite the increasing evidence of the role of RSSH in redox
signaling, the biological functions of RSSH remain elusive.
This deficiency is partly due to the instability of RSSH under
physiological conditions.
The discovery of H2S as an endogenously produced
signaling molecule has stimulated interest in H2S-derived
species as possible biological mediators. H2S signaling is
proposed to occur via post-translational modification of
protein cysteine residues (RSH) to form hydropersulfides
(RSSH),1-4 and recent reports indicate that much of the
biological effects attributed to H2S could instead be due to
RSSH and polysulfides.5-7 Several reports have shown that
small molecule hydropersulfides such as cysteine
hydropersulfide (Cys-SSH) and glutathione hydropersulfide
(GSSH) are ubiquitous and highly prevalent in mammalian
5, 8-9
cells, tissue, and plasma.1,
Furthermore, numerous
enzymes and proteins have been reported to have RSSH
modifications at many cysteine residues.10-14 Recently,
Akaike and co-workers have shown that Cys-SSH is
biosynthesized and attached to tRNA by the cysteinyl tRNA
synthetases (CARS), and subsequently is translationally
incorporated into proteins.15 The prevalent nature of RSSH
in cells suggests that they could have important biological
functions.
Small molecule donors of reactive sulfur species are
essential tools that can be used to elucidate their biological
chemistry. To this end, several RSSH donors have been
reported (Figure 1). For example, precursors containing an
activated disulfide bond have been developed to rearrange
spontaneously at physiological pH thereby producing
RSSH.29 We recently reported a novel class of S-substituted-
thioisothioureas as efficient RSSH precursors.30 Wang and
co-workers have developed esterase-sensitive RSSH
prodrugs and demonstrated their cardioprotective effects.31
Similarly, Xianandco-workershave reported fluoride/acid-
activated RSSH donors.32 Recently, H2O2-triggered self-
immolative RSSH donors have been developed that exhibit
RSSH display distinct chemistry and this may be important
for their biological utility. For example, RSSH are superior
nucleophiles, and more potent reductants than their
corresponding thiols because of the presence of unshared
electron pairs on the sulfur atom adjacent to the
nucleophilic sulfur atom.16-18 RSSH andrelated species have
been proposed to behave as potent antioxidants and redox
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cytoprotective effects against oxidative stress.33-34 These
in Scheme 1, Path B. Under biological conditions, the RSSH
released from Path A may react further with thiols to
produce H2S. Similarly, COS generated from Path B would
be converted to H2S by CA.
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findings highlight the therapeutic potential of small
molecule RSSH donors against oxidative stress-related
diseases. Although chemical tools for RSSH generation have
emerged, no convenient methodology for the controlled and
extended release of RSSH over long time periods is
currently available.
Scheme 1. Design of Hydropesulfide/Carbonyl Sulfide
Precursors
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RESULTS AND DISCUSSION
To synthesize alkylamine-substituted perthiocarbamates,
N-acetyl-cysteine methyl ester was treated with
chlorocarbonylsulfenyl chloride to obtain the S-
perthiocarbonyl chloride 3, which was immediately reacted
with tert-butyl methyl(2-(methylamino)ethyl)carbamate in
the presence of triethylamine to obtain 4 in 69% overall
yield (Scheme 2). The tert-butoxycarbonyl (Boc) protecting
group was removed by treatment with trifluoroacetic acid
to obtain precursor 1a in 95% yield.
Figure 1. Selected small molecule RSSH and COS/H2S donors
Activation of prodrugs via intramolecular cyclization-
elimination has been a widely used strategy for drug
delivery.35-37 In this approach, active drug release is
dependent upon a predictable intramolecular cyclization-
elimination reaction. We envisioned RSSH release using
such a strategy with the sulfhydryl group of RSSH protected
in the form of perthiocarbamate 1 (Scheme 1) and a
terminal non-nucleophilic quaternary ammonium salt. As
shown in Scheme 1 (Path A), neutralization of the
quaternary ammonium salt under physiological conditions
forms an active amine nucleophile that can then undergo an
intramolecular cyclization torelease RSSH and a cyclic urea,
presumably a biologically innocuous byproduct. Varying
the substituent on the trigger nitrogen and changing the
length of the methylene spacer should allow the rate of
cyclization to be tuned, thereby varying RSSH release rates.
We also reasoned that the alkyl substituent on the
perthiocarbamate nitrogen would improve the aqueous
stability of these precursors.
Scheme 2. Synthesis of Hydropesulfide Precursor 1a
With 1a in hand, we first examined RSSH generation using
ultra-performance liquid chromatography-mass spec-
trometry (UPLC-MS). We used b-(4-hydroxyphenyl)ethyl
iodoacetamide (HPE-IAM) as an RSSH trap. HPE-IAM was
chosen because it is a soft electrophile and has been widely
used to estimate RSSH yields from biological samples.15, 48
Incubation of 1a with HPE-IAM (50 equiv.) in ammonium
bicarbonate buffer (pH 7.4, 50 mM) shows RSS-HPE-AM 5
formation (SI, Figure S1; Scheme 3, eq. 1), demonstrating
the release of RSSH. However, dialkyltrisulfide 6 formation
is also observed as a major product (Scheme 3, eq. 2),
suggesting that precursor 1a is a competitive trap for the
initially released RSSH. As expected, the byproduct 1,3-
dimethyl-2-imidazolidinone (2a) is observed in 52% yield
under these conditions, confirming that RSSH release
Recently, Pluth and co-workers have reported caged
sulfenyl thiocarbonates (Figure 1) that release carbonyl
sulfide (COS) in the presence of biological thiols.38 Under
physiological condition, COS is rapidly hydrolyzed to H2S by
the ubiquitous enzyme, carbonic anhydrase (CA).39 The
detection of COS in human tissues suggests that it may also
have regulatory roles in biology,40 however, our
understanding of these roles remains limited. To advance
future investigations intothe biologicalroles of COS, a series
of COS donors that are activated by different triggers have
been developed.41-47 We reasoned that perthiocarbamates
1 may also produce COS in the presence of thiols as shown
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occurs via intramolecular-cyclization reaction. To verify
that the control compound 8 does not release RSSH via
intermolecular reactionsin the presence ofamines or thiols.
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7
RSSH generation, 1a was independently incubated with N-
ethyl maleimide (NEM, 50 equiv.) in pH 7.4 buffer and
UPLC-MS analysis shows RSS-NEM adduct formation (SI,
Figure S5a). In addition, we observe improved yield of the
byproduct 2a (74%) and decreased level of trisulfide in the
presence of NEM (SI, Figure S5b), presumably due to its
better RSSH-trapping efficiency vs. HPE-IAM.
Scheme 4. Reaction of 8 with n-BuNH2 and N-acetyl
cysteine
8
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Scheme 3. Proposed Mechanism of RSSH and Trisulfide
Formation from the Precursor 1a
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To minimize the reaction of released RSSH with its
precursor, we synthesized donor 7a (Figure 2), equipped
with an inhibiting dimethyl substituent alpha to the
disulfide. RSSH generation from 7a was examined with
HPE-IAM trapping and shows RSS-HPE-AM 9 formation
with no evidence of dialkyltrisulfide generation (SI, Figure
S10).
Next, we monitored the kinetics of RSSH release from 7a by
HPE-IAM trapping in pH 7.4 phosphate buffer at 37 °C using
HPLC. An increase in peak intensity at 14.1 min attributed to
RSS-HPE-AM 9 is observed (Figure 3b). To quantify RSSH, we
independently synthesized 9 (SI, Scheme S2). HPLC analysis
shows 89% formation of 9 from 7a with a first-order rate
constant of k = 0.505 min-1 (t1/2= 1.4 min, Table 1). In addition,
87% of byproduct 2a formation, analyzed using UPLC-MS, is
also observed (SI, Figure S10). We also analogously measured
the kinetics of 9 (k = 0.58 ± 0.02 min-1; t1/2 = 1.2 min) and 2a
(0.57 ± 0.02 min-1; t1/2 = 1.2 min) formation from 7a using
UPLC-MS and observe similar rate constants, indicating that
RSSH trapping with HPE-IAM is rapid under these conditions
(SI, Figure S42). We also examined the effect of pH on the
kinetics of RSSH release. As expected, the rate of RSSH release
from 7a decreases at pH 6.0 (k = 0.031 min-1; t1/2 = 22.2 min)
and increases at pH 8.0 (k = 2.58 min-1; t1/2 = 0.27 min).
Figure 2. Structures of RSSH precursors 7a-d with synthetic
yields
To support the proposed mechanism for RSSH release, we
examined RSSH release from a control compound 8
(Scheme 4) lacking the terminal amine group. No RSSH
release is observed from 8 under similar conditions (SI,
Figure S25), confirming that the terminal amine is required
for precursor activation. We also tested the ability 8 to
release RSSH via intermolecular reactions with amines
(Scheme 4, eq. 1). However, incubation of 8 with a model
amine, n-butylamine (5 equiv.), shows no reactivity, at least
over 2 h (SI, Figure S27), suggesting that 8 is stable under
these conditions and does not release RSSH via
intermolecular reaction. Additionally, we tested RSSH
release from 8 in the presence of N-acetyl cysteine (NAC) in
pH 7.4 ammonium bicarbonate buffer. We anticipated that
if thiol attacks the perthiocarbamate carbonyl group of 8,
we should observe RSSH and/or RSSH derived polysulfides,
and the NAC-thiocarbamate byproduct (Scheme 4, eq. 2).
However, UPLC-MS analysis shows no evidence of these
products (SI, Figure S29). Instead, mixed disulfide (R1SSR3)
formation is observed, presumably formed by the thiol
attack on the internal sulfur of the compound 8 (Scheme 4,
eq. 3). Furthermore, mixed disulfide R1SSR3 undergoes
disulfide exchange reaction with NAC to produce N-acetyl
cystine (R3SSR3) and N-acetyl-penicillamine methyl ester
(R1SH) (Scheme 4, eq. 4). Together, these results indicate
To tune the kinetics of RSSH release, precursor 7b with a
terminal free amine was synthesized. HPLC analysis shows
an increase in half-life (16.7 min, Table 1) at pH 7.4. Similar
to precursor 7a, we also observed a pH effect on RSSH
release for 7b (t1/2 = 280 min at pH 6.0; t1/2 = 5.1 min at pH
8.0). Precursor 7c, equipped with three methylene spacers,
was synthesized to measure its effect on RSSH release. We
anticipated that inclusion of a longer spacer compared with
that in precursor 7a would reduce the rate of the
intramolecular-cyclization reaction and therefore RSSH
release. Asexpected, the half-life of7c increasesto 118 min,
still with 90% RSSH release. In addition, 88% of the
expected byproduct 1,3-dimethyltetrahydropyrimidin-
2(1H)-one (2c) is also observed (SI, Figure S16).
Analogously, we also observe significantly slower RSSH
release (t1/2 = 484 min) from 7d, equipped with both a
terminal free amine and three methylene spacers. Taken
together, these results demonstrate the ability of the
perthiocarbamate platform to release RSSH efficiently with
tunable rates and over long time frames.
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SCHEME 5. RSSH and COS Generation from 7a-d in the
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Presence of Thiol
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Figure 3. (a) Reaction scheme showing RSSH generation from
7a in the presence of HPE-IAM. (b) HPLC analysis of RSSH
generation from 7a (100 µM) in the presence of HPE-IAM (5
mM) incubated in pH 7.4 phosphate buffer (100 mM) with
DTPA (100 µM) at 37 ˚C. An aliquot of the reaction mixture was
withdrawn at the specified time and quenched with 1% formic
acid. Asterisks indicate the presence of impurities in the
commercial HPE-IAM sample. (c) Kinetics of RSS-HPE-AM 9
generation. Data represent the average ± SD (n = 3). The curve
is the calculated best fit to a single-exponential function (k =
0.505 ± 0.019 min−1; t1/2 = 1.4 ± 0.1 min).
When 7a is incubated with NAC in pH 7.4 buffer, a new peak
at 5.67 min with m/z = 238.0556 [M+H]+ corresponding to
RSSH (expected m/z = 238.0566) is observed (Figure 4 and
SI, Figure S48). Furthermore, we also observe symmetrical
dialkyl polysulfide (R1SSnSR1, n = 1-4) formation (Figure 4,
cyan highlight), presumably formed by the decomposition
of RSSH through disproportionation (Scheme 5, eq. 5) and
RSSH-polysulfide exchange reactions (Scheme 5, eq. 6). In
addition, unsymmetrical dialkyl polysulfide (R1SSnSR3, n =
1-3) (Figure 4, pink highlight), likely produced by the NAC
reaction with symmetrical dialkyl polysulfides, are also
observed. The presence of an observable MS peak for RSSH
under these conditions is likely due to its equilibrium with
polysulfidesand itsrelative stability asa sterically hindered
persulfide. Notably, 87% of byproduct 2ais observedunder
these conditions (Table 2), suggesting that the efficiency of
RSSH generation for short-lived precursor 7a is unaffected
by thiol.
Table 1. Hydropersulfide Yields and Half-lives for
Precursors 7a-d
Hydropersulfide t1/2 (min)
Precursor R2
n
Yield (%)a
89 ± 3
7a
7b
7c
7d
CH3
H
CH3
H
1
1
2
2
1.4 ± 0.1
16.7 ± 0.3
118 ± 4
94 ± 1
90 ± 1
82 ± 1
484 ± 10
aRSSH precursors (100 µM) were incubated in the presence of
HPE-IAM (5 mM) in pH 7.4 phosphate buffer containing DTPA at
37 °C. Reported data represent averages ± SD (n = 3).
We also examined RSSH generation from 7a in the absence
of a trapping agent. As shown in SI, Figure S54, we again
observe a peak at 5.67 min corresponding to RSSH as well
The ability of these precursors to release RSSH was also
examined in the presence of thiols, likely to be present in
significant concentrations under physiological conditions.
Because thiols can also readily react with HPE-IAM, we
measured RSSH release in its absence. We anticipated that
if thiol reaction with the precursor (Scheme 5, eq. 2)
competes with RSSH release (Scheme 5, eq. 1), we should
observe reduced yields of RSSH and cyclic ureas 2a-d, and
increased formation of thiocarbamate-derived COS and
unsymmetrical disulfide 10 (R1SSR3). Since RSSH is an
unstable species under aqueous conditions, the cyclic-urea
yields were measured as an indication of RSSH yield.
as polysulfides (R1SSnSR1,
n = 1-4) and N-acetyl-
penicillamine methyl ester, indicating that RSSH undergoes
disproportionation reactions and its presence is likely due
to equilibrium reactions with polysulfides. In contrast,
UPLC-MS analysis of RSSH release from precursor 1a in the
absence of trap shows no evidence of an MS-observable
RSSH peak (SI, Figure S62), consistent with the relatively
unstable nature of primary alkyl persulfides.
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Table 2. Yields of 2a-d from Precursors 7a-d in the
Presence of N-acetyl Cysteine
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4
Precursor
Byproduct %a
7a
7b
7c
7d
87±1
58±0.7 19.2±0.5 5.9±0.4
aRSSH precursors (100 µM) were incubated in the presence of NAC
(500 µM) in pH 7.4 ammonium bicarbonate buffer containing
DTPA (100 µM) at 37 °C. Reported data represent averages ± SD
(n = 3).
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9
We also examined thiocarbamate 11a formation, another
anticipated product of precursor 7a reaction with NAC
(Scheme 5, eq. 2). UPLC-MS analysis showed no evidence of
thiocarbamate formation, suggesting that if formed, it
rapidly decomposes under aqueous conditions to release
COS (Scheme 5, eq. 4). We monitored COS production using
membrane inlet mass spectrometry (MIMS), a technique
used to detect hydrophobic gases dissolved in aqueous
solution using semi-permeable membrane that allows gases
and not the liquid phase to enter a mass spectrometer.49
When precursor 7a is examined in the absence of NAC, a
very small increase in the m/z = 60 signal attributed to COS
(Figure 5a) is observed, likely arising from released RSSH
reaction with precursor 7a producing thiocarbamate 11a,
which subsequently decomposes to give COS (Scheme 5, eq.
3 and 4). Only a small additional increase in COS signal
(Figure 5b) is observed in the presence of NAC, suggesting
that 7a rapidly releases RSSH (Scheme 5, eq. 1) and thus is
less available to react with NAC (Scheme 5, eq. 2). Taken
together, these results again demonstrate that 7a mainly
produces RSSH, even in the presence of thiols.
Figure 5. COS measurement using MIMS generated from 7a-d
(50 µM) either (a) without NAC or (b) with NAC (0.25 mM, 5
equiv.) in pH 7.4 phosphate buffer saline (10 mM) with DTPA
(100 µM) at 37 °C.
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Next, precursor 7b decomposition was examined in the
presence of NAC. UPLC-MS analysis shows decreased
byproduct 2b yield (58%), and reduced levels of R1SSnSR1
and R1SSnSR3 (SI, Figure S70). Consistent with this
observation, we also observe increased production of COS
(Figure 5b). These results suggest that with decreasing
RSSH release rate, precursor reaction with thiol becomes
more competitive. Furthermore, we observe mainly
unsymmetrical disulfide 10 (SI, Figure S84 and S90) and
COS (Figure 5b), and reduced yields of 2c and 2d (Table 2)
from precursors 7c and 7d, respectively, in the presence of
NAC. These results indicate that donors 7c and 7d produce
mainly COS in the presence of thiol. In addition to COS
formation, thiocarbamates 11b-d can also potentially
undergo an intramolecular cyclization to produce cyclic
ureas 2b-d and H2S. However, we observe reduced yields
of 2b-d during 7b-d decomposition in the presence of NAC,
indicating that this cyclization reaction is not a major
contributor. Furthermore, the predicted pKa of the
thiocarbamate sulfhydryl group is ca. 5.5,50 indicating that
it will be predominantly present in anionic form at pH 7.4,
thus disfavoring intramolecular cyclization to release H2S.
We also examined the control compound 8 for COS release
under similar conditions. Relatively slow COS release
compared with that from 7a-d is found (Figure 5b). This
result suggests that in the cases of precursors 7a-d, in
addition to the thiol-mediated COS release pathway
(Scheme 5, eq. 2), RSSH reaction with the precursor to
produce the thiocarbamate intermediate (Scheme 5, eq. 3)
presumably contributes to the observed enhanced rate of
COS release.
Although partial COS production is observed from longer-
lived precursors 7b-7d in the presence of thiols, the COS
generation pathway might be disfavored under certain
conditions. For example, patients with cardiovascular
disease often have reduced levels of glutathione.51-52 This
result implies that under reduced thiol levels, these
precursors may favor the RSSH generation pathway.
Furthermore, during myocardial ischemia injury, the local
pH changes to mildly acidic,53-54 and under these conditions,
thiol reaction with the RSSH precursor may be diminished.
Based on these conditions, we examined COS release from
7b-d with NAC in pH 6.0 buffer at 37 °C. As expected, we
observe diminished levels of COS (SI, Figure S106). Under
Figure 4. UPLC-MS chromatograms of RSSH generation from
7a (100 μM) in the presence of NAC (500 µM) incubated in pH
7.4 ammonium bicarbonate (50 mM) with the metal chelator
DTPA (100 μM) at 37 °C. Aliquots taken at various times were
quenched with 1% formic acid, and analyzed by UPLC-MS. A
peak at 5.67 min attributed to RSSH is observed under these
conditions. RSSH-derived symmetrical dialkyl polysulfide,
labeled as S3 to S6 (R1SSnSR1, n =1-4, cyan highlight), and
unsymmetrical dialkyl polysulfides labeled as ’S3 to ’S5
(R1SSnSR3, n =1-3, pink highlight) formation isevident. A peak
at 3.62 min attributed to the byproduct 2a is also observed.
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the same conditions, we still observe RSSH release from 7b,
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albeit at a slower rate (Figure S96).
We also compared the reactivity of NAC vs. GSH with
precursor 7b at pH 7.4. We anticipate that if GSH reaction
with the precursor is slower than NAC, we should observe
increased yield ofRSSH and cyclic urea 2b (Scheme 5, eq. 1),
and reduced levels of thiocarbamate-derived COS and
unsymmetrical disulfide 10 (R1SSR3) (Scheme 5, eq. 2).
When 7b is incubated with NAC, UPLC-MS analysis shows
58% of byproduct 2b formation. In the presence of GSH, we
observe a small decrease in 2b yield (42%), suggesting that
GSH reaction with 7b is slightly faster than NAC. Consistent
with this observation, we also observe increased levels of
unsymmetrical disulfide (R1SSR3) and reduced levels of
RSSH-derived symmetrical dialkyl polysulfides (R1SSnSR1, n
=1 and2) and unsymmetrical dialkyl polysulfides (R1SSnSR3,
n =1 and 2) (SI, Figure S97) in the case of GSH reaction with
7b. Additionally, we observe slightly higher/faster COS
release from 7b in the presence of GSH compared with NAC,
analyzed by MIMS (SI, Figure 107). Together, these results
indicate that precursor 7b reaction with GSH (pKa 8.83) is
slightly faster than with NAC (pKa 9.52), likely due to the
higher concentration of the corresponding thiolate at
neutral pH.46, 55
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Figure 6. Results from H9c2 cardiac myoblasts pretreated
with the RSSH precursor 7b at (50, 100 and 150 µM) and the
byproduct 1-methylimidazolidin-2-one (2b) at 150 µM for 2 h
followed by exposure to H2O2 (200 µM) for 2 h. (a)
Quantification of viability was carried out using Cell Counting
Kit-8 (CCK-8). Results are expressed as the mean ± SEM (n = 5
for each treatment group) with three independent
experiments. (b) Quantification of cytotoxicity was carried out
using Sytox Green nucleic acid stain. Results are expressed as
the mean ± SEM (n = 5 for each treatment group) with five
independent experiments. # P < 0.05, * P < 0.01, ** P < 0.001 for
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comparisons with the H2O2 treatment group.
Group
comparisons are determined by a one-way analysis of variance
(ANOVA) with Dunnett’s correction post-hoc test using
GraphPad Prism 8.
Several studies have speculated that intracellular RSSH and
related species protect cells from oxidative stress.5-6, 8-9, 18-20,
To build on our results from these in vitro studies, we also
tested 7b in isolated-perfused (ex vivo) mouse hearts. The
Langendorff model of myocardial ischemia-reperfusion is a
widely used technique whereby the ionotropic and
chronotropic effects of a drug can be studied directly
without confounding neural/hormonal influences and
minimizes changes in coronary vascular tone.59-60
Following 20 min of global ischemia, 7b was infused for the
first 7 min of reperfusion at a concentration of 100 µM.
Reperfusion is continued for a total duration of 90 min
before the heart is infused with triphenyltetrazolium
chloride (TTC), a stain for determining cellular viability
within a given tissue.61 Figure 7a shows coronal sections of
murine hearts stained with TTC. After 20 min global
ischemia (I/R), Krebs-Henseleit (KH) perfused hearts show
42% infarct size (Figure 7b). This loss in viable myocardial
tissue was significantly attenuated in 7b-perfused infarcted
hearts (16% infarct size). These data demonstrate that
RSSH and/or COS can provide protection when given at
reperfusion in hearts subjected to I/R injury. Although
more work remains to be done to determine the mechanism
by which RSSH conditions the tissue to deal with the stress
of reperfusion and/or compensates for the damage
incurred during ischemia, these data combined with in-vitro
cellular studies imply that the alkylamine-substituted
perthiocarbamates reported here can reduce the extent of
myocardial ischemia-reperfusion injury and may be
pharmacologically useful.
23
We sought to determine whether our alkylamine-
substituted perthiocarbamates exert protective effects
against oxidative stress and myocardial ischemia-
reperfusion (I/R) injury. The medium-lived precursor 7b
(t1/2 = 16.7 min) was chosen for the studies. First, we
measured the cytotoxicity of 7b on H9c2 myoblasts using
the nucleic acid stain, Sytox, a probe for compromised cell
membrane integrity.56 Both precursor 7b and its byproduct
2b show no toxicity toward H9c2 cells after 24 h of
exposure at varied concentrations (0-150 µM) (SI, Figure
S110). We then measured the cytoprotective effects of 7b
against oxidative stress in H9c2 cells. H2O2 (200 µM) was
given as a pro-oxidant source, and drastically reduced cell
viability was observed using CCK-8 staining (Figure 6a).57-58
However, pretreating myoblasts with precursor 7b for 2 h
resulted in a dose-dependent attenuation of H2O2-induced
toxicity (Figure 6a).
Under similar conditions, the
byproduct 2a shows no protective effect against H2O2-
mediated toxicity, suggesting that the protection is due to
RSSH and/or COS. Next, the cytoprotective effect of 7b was
independently evaluated using the Sytox assay, due to the
potential background reduction of CCK-8 by reactive sulfur
species leading to artifactual viability measurements.26-27
As shown in Figure 6b, 7b consistently shows protective
effects against H2O2-mediated toxicity. Under similar
conditions, the COS precursor 8 also shows protective
effects against H2O2-mediated toxicity, but to a lesser extent
compared with 7b (SI, Figure S111). Altogether these
results suggest that 7b is not cytotoxic to cardiac-derived
tissue, can be taken up by the cells, and confers protection
against oxidative stress.
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ACKNOWLEDGMENTS
We gratefully acknowledge the National Science Foundation
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8
(CHE-1900285 to J.P.T.), the National Institutes of Health (T32
GM080189 for support of B.M.P.; R01 HL136918 and R01
HL063030 to N.P.), and Johns Hopkins University (Magic-That-
Matters Fund to N.P.) for generous support for this research.
We also thank Dr. Stephen Chelko (Johns Hopkins University)
for providing access to a cellculture facility and Drs. Jon Fukuto
and Joseph Lin (Sonoma State University) for advice on the cell
viability studies.
9
Figure 7. Cardioprotective effects of 7b postconditioning in
the isolated-perfused murine heart. (a) Representative images
of coronal slices of the heart following TTC staining. (b)
Comparison of the volume of infarcted tissue following
ischemia-reperfusion and when the heart is conditioned with
precursor 7b (100 µM) at the onset of reperfusion. Results are
expressed as the mean ± SEM (n = 4 for each treatment group)
with four independent experiments. ** P < 0.001 for
comparisons with the IR group. Group comparisons are
determined by a one-way analysis of variance (ANOVA) with
Dunnett’s correction post-hoc test using GraphPad Prism 8.
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CONCLUSIONS
In summary, we have prepared alkylamine-substituted
perthiocarbamates as a new, versatile, and readily
modifiable platform for controllable RSSH release. These
precursors show efficient RSSH release with half-lives
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Synthetic procedures, kinetics of RSSH release, analysis of
RSSH and COS generation in the presence of thiol, cytotoxicity,
cytoprotective and cardioprotective effects of RSSH/COS
precursors, detailed HRMS, 1H, and 13C NMR data (PDF)
AUTHOR INFORMATION
(12) Dóka, É.; Pader, I.; Bíró, A.; Johansson, K.; Cheng, Q.;
Ballagó, K.; Prigge, J. R.; Pastor-Flores, D.; Dick, T. P.; Schmidt, E.
E.; Arnér, E. S. J.; Nagy, P. A novel persulfide detection method
reveals protein persulfide- and polysulfide-reducing functions
of thioredoxin and glutathione systems. Sci. Adv. 2016, 2,
e1500968.
Corresponding Author
*jtoscano@jhu.edu
Notes
The authors declare no competing financial interests.
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