Cysteine-activated hydrogen sulfide (H2S) delivery through caged carbonyl sulfide (COS) donor motifs

Article abstract of DOI:10.1039/c8cc02428f

Hydrogen sulfide (H₂S) is an important biomolecule, and controllable H₂S donors are needed to investigate H₂S biological functions. Here we utilize cysteine-mediated addition/cyclization chemistry to unmask an acrylate-functionalized thiocarbamate and release carbonyl sulfide (COS), which is quickly converted to H₂S by carbonic anhydrase (CA).

Full text of DOI:10.1039/c8cc02428f

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Cysteine-Activated Hydrogen Sulfide (H2S) Delivery through Caged
Carbonyl Sulfide (COS) Donor Motifs
Yu Zhao,† Andrea K. Steiger,† and Michael D. Pluth*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Hydrogen sulfide (H2S) is an important biomolecule, and release COS, which is quickly converted to H2S by the
controllable H2S donors are needed to investigate H2S ubiquitous enzyme carbonic anhydrase (CA). We have
biological functions. Here we utilize cysteine-mediated demonstrated that caged-thiocarbamates and thiocarbonates
addition/cyclization chemistry to unmask an acrylate- can serve as promising COS donors and can be activated to
functionalized caged thiocarbamate and release carbonyl release COS through a self-immolative cascade reaction.5 One
sulfide (COS), which is quickly converted to H2S by carbonic important advantage of this strategy is that COS-releasing
anhydrase (CA).
scaffolds can be designed to deliver H2S under well-defined
conditions. For example, H2S delivery from these caged-COS
Hydrogen sulfide (H2S) has joined the gasotransmitter donors can be modulated by judicious trigger selection, and
family since its first recognition as an endogenous the rate of release can be manipulated through modification of
neuromodulator in 1996.1 Four main enzymes, including the donor structure.5 Following our initial report, we, as well
cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), as others, have expended this strategy to include donors
are responsible for endogenous H2S production, converting activated by different triggers, such as reactive oxygen species
cysteine (Cys) and homocysteine (Hcy) to H2S.2 Significant (ROS),5-6 esterases,7 nucleophiles,8 click chemistry,9 and light10
efforts have been contributed to develop H2S releasing agents (Figure 1).
(H2S donors) because the regulation of H2S levels has been
Y
found to mediate a wide variety of physiological processes,
S
Ar
X
Z
including anti-inflammation, oxidative stress reduction, and
vasorelaxation.3 Although sulfide salts, such as sodium
hydrosulfide (NaHS) and sodium sulfide (Na2S), have been
widely used in the field, they are far from ideal donors because
they release H2S spontaneously, resulting in a concentrated
bolus of sulfide that oxidizes rapidly and does not mimic well-
regulated endogenous H2S production. The use of these
inorganic sulfide sources has even led to contradictory
results,3c demonstrating the need for improved sources of H2S.
These limitations suggest that controllable H2S donors, which
are stable, only release H2S upon activation by certain stimuli,
and have slower and controllable kinetics of sulfide release,
are key research tools for H2S investigations.
N
O
O
X = O or S
Y = S or O
Z = O, S or NH
H
B
O
O
S
S
N
O
O
NHR
R
O
O
R
O
R
S
X
O
N
F
N
B
X = NH or O
O
O
O
S
O
O
NHR
Ar
N
S
NO2
H
Figure 1. Examples of currently-available COS-based H2S donors that
are activated by different triggering stimuli.
Aligned with this need, our group recently reported the use
of caged-carbonyl sulfide (COS) molecules as new H2S donors.4
Unlike other known H2S donors, which directly release H2S as
the activation product, COS-based donors are activated to
Cellular nucleophiles play crucial roles in biological
systems. Among these, thiol species, such as Cys and reduced
glutathione (GSH), attract the most attention due to their
cellular abundance and potent reactivity. Cys and GSH have
been widely used to trigger biologically active molecules and
prodrugs to release caged compounds, including sulfur dioxide
(SO2),11 nitroxyl (HNO),12 and anti-cancer drugs.13 Importantly,
thiol activation strategies have been adopted in H2S donor
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development and several thiol labile H2S donors exhibit
promising protections in animal models with some of them
currently in clinical trials.3b, 3c, 3e Motivated by these findings,
we report here the first example of a Cys-activated COS/H2S
donor through functionalization of a thiocarbamate with a Cys-
reactive acrylate moiety. We envision that such thiocarbamate
compounds will expand the current COS-based H2S donor
family and serve as promising research tools for H2S studies.
The reactions of Cys with acrylates in the preparation of
substituted 1,4-thiazepines have been known for decades.14
The initial attack by Cys on the acrylate generates a thioether,
which then undergoes an intramolecular cyclization to yield
1,4-thiazepines. This cyclization strategy has been leveraged by
Strongin,15 as well as others,16 to design a series of acrylate-
based fluorescent probes for Cys detection. Similarly, the
Berreau group has recently used a similar approach to develop
a class of Cys-responsive CO donors.17 Building from these
approaches, we adopt the Cys-acrylate reaction as a triggering
mechanism to access new COS/H2S donors in which an aryl
acrylate-functionalized thiocarbamate is activated through a
Cys-mediated addition/cyclization sequence. The resultant
phenolic intermediate then undergoes a 1,6-elimination to
release COS, which is quickly converted to H2S by CA (Scheme
1).
S
O
OH
OH
PhNCS
O
O
NHPh
DBU, THF
oC, 4 h, 35%
O
0
O
OA-CysTCM-1
O
O
PhNCO
TEA, THF
65 oC, 5 h
47%
O
O
NHPh
O
O
OA-CysCM-1
S
OH
PhNCS
O
NHPh
DBU, THF
0 - r.t.
4 h, 75%
OA-TCM-1
Scheme 2. Synthesis of OA-CysTCM-1, OA-CysCM-1, and OA-TCM-1.
To evaluate Cys-activated H2S release from the donor
motif, we used the methylene blue (MB) assay to monitor H2S
release from OA-CysTCM-1 (50 µM) in the presence of Cys (0 –
500 µM) in PBS buffer (pH 7.4, 10 mM) containing cellularly-
relevant concentrations of CA (25 µg/mL). The MB assay was
chosen to measure H2S production since it has been widely
used to detect H2S from previously developed Cys-activated
H2S donors. In the absence of Cys, OA-CysTCM-1 was stable in
aqueous buffer and did not release COS/H2S spontaneously. By
contrast, the addition of Cys led to a dose-dependent COS/H2S
release from OA-CysTCM-1 (Figure 2). These results
demonstrate that OA-CysTCM-1 can be activated by Cys and
the resultant COS is quickly converted to H2S in the presence
of CA.
S
S
O
O
NHR
O
Cys
O
NHR
O
S
O
NH2
O
S
NH
CO2H
CO2H
S
RNH2
CA
NHR
O
H2S
COS
O
O
Scheme 1. General design of Cys-triggered COS/H2S release from
caged-thiocarbamate donors.
To test our hypothesis that acrylate-functionalized
thiocarbamates could serve as Cys-triggered COS/H2S donors,
we prepared O-alkyl cysteine-sensitive thiocarbamate (OA-
CysTCM-1) with an aryl acrylate trigger and an aniline payload
by reacting 4-(hydroxymethyl)phenyl acrylate and phenyl
isothiocyanate. Upon activation, OA-CysTCM-1 releases COS,
which is quickly hydrolyzed to H2S by CA. In addition to OA-
CysTCM-1, we also prepared the corresponding carbamate
18
(
OA-CysCM-1) and triggerless thiocarbamate (OA-TCM-1)
Figure 2. COS/H2S Release from OA-CysTCM-1 (50 µM) in the presence
of 0 µM (black), 50 µM (red), 250 µM (blue), and 500 µM (green) Cys.
control compounds. OA-CysCM-1, obtained from the reaction
between 4-(hydroxymethyl)phenyl acrylate and phenyl The experiments were performed in triplicate and results are
expressed as mean ± SD (n = 3).
isocyanate, is expected to undergo the same Cys activation but
would release CO2 instead of COS. OA-TCM-1, on the other
To further demonstrate that the observed H2S release is
hand, maintains the thiocarbamate scaffold but lacks the
due to Cys activation via the proposed mechanism, we
acrylate trigger, and thus is not expected to react with Cys or
pretreated Cys with N-ethylmaleimide (NEM), a Cys scavenger,
decompose to otherwise release COS (Scheme 2).
for 20 min, followed by the addition of OA-CysTCM-1. When
compared to the regular activation conditions (Figure 3, bar 1),
NEM pretreatment significantly diminished H2S release from
OA-CysTCM-1, confirming that Cys was required for donor
2 | Chem Commun, 2018, 00, 1-3
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Cys - CA, (4) Cys + AAA (10 µM), (5) Hcy, (6) NAC, (7) GSH (5.0 mM), (8)
Ser, (9) Lys, (10) GSSG, and (11) PLE (1 U/mL). Cys (500 µM) effects on
OA-CysCM-1 (12), and OA-TCM-1 (13) toward COS/H2S release. H2S
concentration was measured after 3-h incubation. The experiments
were performed in triplicate and the results were expressed as mean ±
SD (n = 3).
activation (Figure 3, bar 2). No H2S release was observed in the
absence of CA, and, similarly, pretreatment of CA with
acetazolamide (AAA), a CA inhibitor, also failed to provide H2S,
confirming that H2S release from OA-CysTCM-1 proceeds
through a COS-dependent pathway (Figure 3, bars 3 and 4).
In addition to Cys, other biologically relevant nucleophiles,
such as GSH, oxidized glutathione (GSSG), Hcy, N-
acetylcysteine (NAC), serine (Ser), and lysine (Lys), were
evaluated towards donor activation. As expected, none of
these species triggered OA-CysTCM-1, and no COS/H2S release
was observed due to the lack of the addition/cyclization
activation sequence (Figure 3, bars 5-10). Considering the high
abundance of GSH in cellular environment, we also evaluated
Cys-triggered COS/H2S release from OA-CysTCM-1 in the
presence of GSH. In these experiments, OA-CysTCM-1 (50 µM)
was co-incubated with Cys (500 µM) and GSH (0 – 1000 µM)
and COS/H2S release was monitored by MB assay (Figure S2). A
decrease of H2S release was observed as the GSH
concentration increased, indicating a potential GSH-induced
donor consumption. Although the effects of GSH were not
significant in aqueous buffer, it should be taken into
consideration when applying donors in biological systems.
Since the acrylate trigger may be prone to esterase-catalyzed
hydrolysis, we also incubated OA-CysTCM-1 with porcine liver
esterase (PLE) to determine whether common esterases could
generate COS/H2S release. Although we did observe H2S
release, it was significantly less efficient than Cys activation
(Figure 3, bar 11). As expected, treatment of control
compounds OA-CysCM-1 and OA-TCM-1 with Cys in the
presence of CA failed to generate H2S, demonstrating that
both the aryl-acrylate trigger and the caged COS-containing
thiocarbamate scaffold are crucial for COS release from this
scaffold (Figure 3, bars 12 and 13). Taken together, these
selectivity studies demonstrate that OA-CysTCM-1 is highly
sensitive towards Cys activation to release COS/H2S and inert
to activation by other biomolecules, such as GSH, GSSG, Hcy,
NAC, Ser, and Lys.
We next sought to confirm that OA-CysTCM-1 would
release COS/H2S upon reaction with Cys in
a cellular
environment. We incubated bEnd.3 cells with OA-CysTCM-1 in
the presence of Cys and visualized H2S-release using SF7-AM, a
cell-trappable H2S-responsive fluorescent probe.19 In the
absence of OA-CysTCM-1, negligible SF7-AM fluorescence was
observed, suggesting a minimum amount of endogenous H2S
present in bEnd.3 cells. By contrast, addition of OA-CysTCM-1
resulted in a significant increase in SF7-AM fluorescence,
confirming that OA-CysTCM-1 can be activated by Cys to
release H2S in a cellular environment (Figure 4). These results
demonstrate that OA-CysTCM-1 is a potent COS/H2S donor
and Cys-triggered H2S delivery can be visualized in complex
biological systems, indicating applications of OA-CysTCM-1 as
a potential H2S-related therapeutic or research tool.
Figure 4. H2S Release from OA-CysTCM-1 in bEnd.3 cells. Top: DIC (left)
and GFP (right) channels with cysteine (250 µM) and SF7-AM (5 µM).
Bottom: DIC (left) and GFP (right) channels with cysteine (250 µM),
OA-CysTCM-1 (100 µM), and SF7-AM (5 µM). Scale bar represents 100
µM.
In summary, we prepared and evaluated OA-CysTCM-1 as a
Cys-triggered COS/H2S donor. Our studies demonstrate that
OA-CysTCM-1 is stable in aqueous media and does not release
COS/H2S until being activated by Cys. Importantly, H2S delivery
from OA-CysTCM-1 is observed in a cellular environment,
indicating OA-CysTCM-1 can be used as a new efficacious Cys
labile COS/H2S donor in complex biological systems. Taken
together, our investigations demonstrate that H2S delivery
from OA-CysTCM-1 can be controlled and regulated through a
COS-dependent pathway, making OA-CysTCM-1
a new
member of COS-based H2S donor family with potential
applications in the study of both H2S and COS chemical biology,
especially when used in combination with available Cys-
activated H2S donors. Further applications of this as well as
other COS-releasing constructs are currently ongoing in our
laboratory.
Figure 3. COS/H2S Release from OA-CysTCM-1 (50 µM) in the presence
of cellular nucleophiles (500 µM): (1) Cys, (2) Cys + NEM (10 mM), (3)
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This work was supported in part by the NIH (MDP;
R01GM113030), Dreyfus Foundation, and NSF/GRFP (AKS;
DGE-1309047). NMR spectroscopy and fluorescence
microscopy instrumentation in the CAMCOR facility is
supported by the NSF (CHE-1427987, CHE-1531189).
Huang, B. Li, F. Zeng and S. Wu, Anal. Chem., 2018, 90, 1014-
1020; (c) J. F. Wang, B. Li, W. Y. Zhao, X. F. Zhang, X. Luo, M. E.
Corkins, S. L. Cole, C. Wang, Y. Xiao, X. M. Bi, Y. Pang, C. A.
McElroy, A. J. Bird and Y. Z. Dong, Acs Sensors, 2016, 1, 882-
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Guan, S. Zhao and X. Yu, Talanta, 2018, 181, 118-124.
17. T. Soboleva, H. J. Esquer, A. D. Benninghoff and L. M. Berreau,
J. Am. Chem. Soc., 2017, 139, 9435-9438.
Conflicts of interest
18. A. K. Steiger, Y. Zhao, W. J. Choi, A. Crammond, M. R. Tillotson
and M. D. Pluth, Biochem. Pharmacol., 2018, 149, 124-130.
19. V. S. Lin, A. R. Lippert and C. J. Chang, Proc. Natl. Acad. Sci. U S
A, 2013, 110, 7131-7135.
There are no conflicts to declare
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Cysteine-activated acrylate-functionalized caged thiocarbamate
provides access to triggered COS/H2S donors
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