Fluorogenic hydrogen sulfide (H2S) donors based on sulfenyl thiocarbonates enable H2S tracking and quantification

Article abstract of DOI:10.1039/c8sc05200j

Hydrogen sulfide (H₂S) is an important cellular signaling molecule that exhibits promising protective effects. Although a number of triggerable H₂S donors have been developed, spatiotemporal feedback from H₂S release in biological systems remains a key challenge in H₂S donor development. Herein we report the synthesis, evaluation, and application of caged sulfenyl thiocarbonates as new fluorescent H₂S donors. These molecules rely on thiol cleavage of sulfenyl thiocarbonates to release carbonyl sulfide (COS), which is quickly converted to H₂S by carbonic anhydrase (CA). This approach is a new strategy in H₂S release and does not release electrophilic byproducts common from COS-based H₂S releasing motifs. Importantly, the release of COS/H₂S is accompanied by the release of a fluorescent reporter, which enables the real-time tracking of H₂S by fluorescence spectroscopy or microscopy. Dependent on the choice of fluorophore, either one or two equivalents of H₂S can be released, thus allowing for the dynamic range of the fluorescent donors to be tuned. We demonstrate that the fluorescence response correlates directly with quantified H₂S release and also demonstrate the live-cell compatibility of these donors. Furthermore, these fluorescent donors exhibit anti-inflammatory effects in RAW 264.7 cells, indicating their potential application as new H₂S-releasing therapeutics. Taken together, sulfenyl thiocarbonates provide a new platform for H₂S donation and readily enable fluorescent tracking of H₂S delivery in complex environments.

Full text of DOI:10.1039/c8sc05200j

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Fluorogenic hydrogen sulfide (H S) donors based
2
on sulfenyl thiocarbonates enable H S tracking and
2
Cite this: DOI: 10.1039/c8sc05200j
quantification†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Yu Zhao,
Matthew M. Cerda and Michael D. Pluth
*
Hydrogen sulfide (H
Although a number of triggerable H
release in biological systems remains a key challenge in H
synthesis, evaluation, and application of caged sulfenyl thiocarbonates as new fluorescent H
These molecules rely on thiol cleavage of sulfenyl thiocarbonates to release carbonyl sulfide (COS),
which is quickly converted to H S by carbonic anhydrase (CA). This approach is a new strategy in H
release and does not release electrophilic byproducts common from COS-based H S releasing motifs.
Importantly, the release of COS/H S is accompanied by the release of a fluorescent reporter, which
enables the real-time tracking of H S by fluorescence spectroscopy or microscopy. Dependent on the
choice of fluorophore, either one or two equivalents of H S can be released, thus allowing for the
dynamic range of the fluorescent donors to be tuned. We demonstrate that the fluorescence response
correlates directly with quantified H S release and also demonstrate the live-cell compatibility of these
donors. Furthermore, these fluorescent donors exhibit anti-inflammatory effects in RAW 264.7 cells,
indicating their potential application as new H S-releasing therapeutics. Taken together, sulfenyl
S donation and readily enable fluorescent tracking of H
2
S) is an important cellular signaling molecule that exhibits promising protective effects.
S donors have been developed, spatiotemporal feedback from H
S donor development. Herein we report the
S donors.
2
2
S
2
2
2
2
S
2
2
2
2
2
Received 21st November 2018
Accepted 10th December 2018
2
DOI: 10.1039/c8sc05200j
thiocarbonates provide a new platform for H
delivery in complex environments.
2
2
S
rsc.li/chemical-science
from GYY4137, a phosphorodithioate-based H
2
S donor, is slow
Introduction
in aqueous buffer, and the low H S releasing efficiency remains
2
13
Hydrogen sulde (H S) is an important gaseous molecule that as a major concern when applying it to the living systems.
2
1
–3
plays critical roles in living systems.
primarily produced from cysteine (Cys) and homocysteine (Hcy) donors have been developed in the last decade.
by four main enzymes, including cystathionine b-synthase donors are activated by different triggers, such as enzymes,
Endogenous H S is Building from these donor scaffolds, a series of synthetic H S
2
2
14–19
These
2
0,21
22–27
28,29
30–33
(
CBS), cystathionine g-lyase (CSE), 3-mercaptopyruvate sulfur cellular thiols,
pH modulation,
and photo activation,
4
–8
transferase (3-MST), and cysteine aminotransferase (CAT).
Due to its bioregulatory and protective roles, H
as an important cellular signaling molecule, much like nitric
and the released H S exhibits promising activities in different
2
17,18
S is considered physiological and pathological processes.
To expand the library of H S donors, we recently developed
the carbonyl sulde (COS)-based platform for H S donation. In
2
2
8
–11
oxide (NO) and carbon monoxide (CO).
2
Although many H S-related biological functions have been this approach, caged-thiocarbamates are activated and the
2
discovered in the past two decades, many investigations have resultant intermediates undergo a cascade reaction to release
been limited due to the lack of controllable and rened H
delivery systems (H
S donors). Inorganic sulde salts, such as enzyme carbonic anhydrase (CA) (Fig. 1a). Expanding on this
sodium sulde (Na
S) and sodium hydrosulde (NaHS), are initial report, we, as well as others, have applied the similar
widely used in H S investigations, but they release H
S quickly caged-COS systems to include a series of triggerable COS-based
in aqueous media, thus failing to mimic the slow and well-
2
S
COS, which is quickly converted to H S by the ubiquitous
2
34
2
2
2
2
H
2
S donors which can be activated by different mechanisms,
1
2
35–37
38,39
regulated in vivo H S generation. By contrast, the H S release such as reactive oxygen species (ROS),
esterase,
and Cys, to release
S under various conditions.
Although H S release from these donors has been measured
cellular
2
2
4
0
41
42–44
45
nucleophiles, click chemistry, light,
H
4
5,46
2
Department of Chemistry and Biochemistry, Institute of Molecular Biology, Materials
Science Institute, University of Oregon, Eugene, OR 97403, USA. E-mail: pluth@
uoregon.edu
2
using different methods, the real-time tracking of donor acti-
vation and H S delivery in living systems remains a key chal-
2
†
2
Electronic supplementary information (ESI) available: H S release curves,
lenge due to the inherent limitations of current H
2
S detection
cytotoxicity data, NMR spectra. See DOI: 10.1039/c8sc05200j
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Results and discussion
To test our hypothesis that caged-sulfenyl thiocarbonates could
serve as thiol-triggered uorescent COS/H S donors, four
2
donors (FLD-1–4) were prepared by reacting uorescein or 3-O-
methyluorescein with ((benzyl)dithio)carbonyl chloride 1 or
(
(
(phenol)dithio)carbonyl chloride 2 in the presence of DIPEA
Scheme 1a). A caged thiocarbonate (TCN-1) was synthesized as
a triggerless control compound, which is expected to be stable
toward thiol activation due to the lack of a disulde functional
group (Scheme 1b).
Fig.
donors. (b) COS/H
carbonate donor motifs.
1
(a) General design of previous self-immolative COS/H
2
S
With these compounds in hand, we rst evaluated their
spectroscopic properties in PBS buffer (pH 7.4, 10 mM). As ex-
pected, FLD-1–3 and TCN-1 are not absorptive in the visible
region and are all nonuorescent because the uorescein unit is
2
S release from fluorogenic caged-sulfenyl thio-
methods. For example, the colorimetric methylene blue (MB) locked in the closed lactone form. By contrast, FLD-4 shows
assay has been widely used to measure H S levels, but requires a prominent absorbance band in the visible region (l
strongly acidic conditions, which may trigger H
acid labile sulde pools. Additionally, MB is an end-point assay rescence (l
2
max
¼
ꢁ1
ꢁ1
2
S release from 449 nm, 3 ¼ 27 300 ꢀ 2500 M cm ) with measurable uo-
¼ 514 nm, F ¼ 0.11 ꢀ 0.01) due to the free
em
which destroys experimental samples, thus making it not hydroxyl group (Table S1†). Based on the promising spectro-
feasible for in vivo H S detection. Similarly, H S-selective scopic properties, large dynamic range, and efficient release of
47
2
2
electrodes are most oen used in bulk measurements rather two equivalents of COS/H S, we chose to use FLD-1 as the model
2
47
than non-homogenized biological samples. By contrast, H2S donor for initial reactivity and selectivity evaluations.

uorescent probes have attracted much attentions due to their
To test the reactivity of FLD-1 towards Cys-induced activa-
tion, FLD-1 (10 mM) was incubated with Cys (100 mM) in PBS
high sensitivities and have been used to sense and visualize H
in biological samples. Albeit promising, H S uorescent buffer (pH 7.4, 10 mM) containing physiologically-relevant
2
probes are prone to react with reactive cellular species, such as concentrations of CA (25 mg mL ), and the uorescence
Cys or glutathione (GSH), which results in either probe intensity was measured using a uorescence spectrometer. As
consumption or false positive signals. In addition, all of the expected, Cys successfully activated FLD-1 and resulted in a 500-
2
S
48
ꢁ1
above methods consume the H S being measured. Recently, we fold uorescence turn on over 2 h, demonstrating the release of
2
reported a series of g-ketothiocarbamates (g-KetoTCM) that the uorescein upon FLD-1 activation (Fig. 2a). Fluorescein
release p-nitroaniline as a colorimetric indicator; however, formation was also conrmed by UV-vis spectroscopy under the
although the colorimetric response enables monitoring of COS/ identical conditions (Fig. S1†). A Cys-dependent uorescence
2
H S release in cuvettes, the direct tracking in live-cell environ- enhancement was observed when treating FLD-1 (10 mM) with
49
ments remains an un-met need. Taken together, a key increasing concentrations of Cys (1–20 equiv.), indicating a high
advancement compatible with live-cell and tissue experiments sensitivity of FLD-1 towards Cys. No uorescent signal was
would be the development of H S donors that deliver H S with observed in the absence of Cys, suggesting that FLD-1 is stable
2
2
a concomitant uorescence response to enable tracking of H2S in aqueous buffer, and that it is not hydrolyzed to provide false
delivery by common microscopy techniques.
Aligned with these needs, we report here the development of
a new H
bonates and apply this donor platform to access uorescent
turn-on H S donors. In this approach, cellular thiols (i.e. Cys
2
S-releasing strategy based on caged sulfenyl thiocar-
2
and GSH) activate the sulfenyl thiocarbonates through thiol-
mediated disulde reduction to release COS, which is quickly
converted to H S by CA (Fig. 1b). To the best of our knowledge,
2
the sulfenyl thiocarbonate reduction strategy provides a new
activation pathway that has not been used to trigger COS/H S
2
release from donor platforms. Unlike currently-available donors
that function through initial COS release, the sulfenyl thiocar-
bonate system does not generate reactive electrophile byprod-
ucts upon activation, which provides a signicant advance in
the eld. In addition, we leverage this new donor strategy to
access systems in which a concomitant uorescence turn-on
occurs upon donor activation, thus allowing for real-time
2
monitoring and quantication of H S release using uores-
2
Scheme 1 Synthesis of fluorescent COS/H S donors (a) and thiocar-
cence spectroscopy.
bonate control compound (b).
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2
signals (Fig. 2b). In addition, to conrm the H S delivery from
FLD-1, we treated FLD-1 (10 mM) with Cys (100 mM) in PBS
ꢁ
1
containing CA (25 mg mL ) and quantied H S release using
2
the MB assay. In this experiment, 15 mM of H S was detected
2
(75% releasing efficiency), which is consistent with our
hypothesis that 2 equivalents of COS/H S would be released
2
upon FLD-1 activation (Fig. S2†). Taken together, these experi-
ments demonstrate that FLD-1 is highly responsive to Cys
activation.
To determine whether H S release correlated directly with
2
the observed uorescence response, we measured the uores-
cent response from FLD-3 in the presence of Cys and CA and Fig. 3 (a) Time-dependent fluorescence turn on (red) and H
2
S release
(
blue) upon FLD-3 (10 mM) activation in PBS (pH 7.4, 10 mM) containing
2
quantied H S release using the MB assay. We chose to use
ꢁ
1
Cys (100 mM) and CA (25 mg mL ). No H
absence of CA (black). lex ¼ 454 nm, lem ¼ 500–650 nm, and slit width
0.3 mm. (b) Correlation between fluorescence measurement and
MB detection. The experiments were performed in triplicate. The
2
S was detected in the
FLD-3 as the model compound for these investigations because
it only contains one sulfenyl thiocarbonate moiety, and there-
fore should simplify the reaction kinetics. In comparison, FLD-1
¼
contains two sulfenyl thiocarbonate groups, and the cleavage of results are expressed as mean ꢀ SD (n ¼ 3).
one sulfenyl thiocarbamate would generate FLD-4 as a reaction
intermediate, which exhibits moderate uorescence (see Fig. 4).
Incubation of FLD-3 (10 mM) with Cys (100 mM) resulted in
a rapid uorescence response with 96% of the H S release cence turn on and COS/H S release, we next evaluated the
measured by MB. At extended time points, we observed a slight reactivity of other FLD donors towards Cys activation. Treating
decrease in measured H S, possibly due to volatilization of H
FLD-1–3 (10 mM) with Cys (100 mM) in PBS buffer (pH 7.4,
in the headspace of the closed system or adventitious oxidation 10 mM) resulted in a 120–500-fold uorescence turn on over 2 h.
of released H S. Negligible H S was detected in the absence of We attribute the faster response of FLD-2 to the more electro-
Having conrmed the efficiency of Cys-triggered uores-
2
2
2
2
S
2
2
CA, indicating that Cys-triggered H S delivery from FLD-3 philic phenyl sulfenyl thiocarbonate in comparison to the less
2
proceeds through intermediate COS formation (Fig. 3a). electrophilic benzyl sulfenyl thiocarbonate in FLD-1. FLD-4,
Importantly, the strong linear correlation between the however, provided minimal uorescence enhancement due to
2
measured uorescence and H S measured from the MB method its strong background uorescence. No uorescence response
2
detection (rst 25 min, R ¼ 0.988) demonstrates that uores- from TCN-1 (10 mM) was observed under the identical condi-
cent readouts can serve as reliable optical tools to track COS/ tions, which demonstrates the stability of the thiocarbonate
H
2
S release from FLD donors with temporal resolution (Fig. 3b). group in the presence of Cys (Fig. 4).
Moreover, this linear correlation suggests that choice of other
To further support our proposed activation mechanism in
uorophores with different brightnesses and photophysical Fig. 1b, as well as the activation of the sulfenyl thiocarbonate

properties could be used to access different dynamic ranges of group by other thiols, we incubated FLD-1 (10 mM) in PBS
H S release, thus enabling this approach to be translated to
2
different types of experimental designs.
Fig. 2 (a) Time-dependent fluorescence spectra of FLD-1 (10 mM) in Fig. 4 Fluorescence turn on of FLD-1 (gray), FLD-2 (red), FLD-3 (blue),
ꢁ1
PBS (pH 7.4, 10 mM) containing Cys (100 mM) and CA (25 mg mL ). (b) FLD-4 (black), and TCN-1 (dark yellow) (10 mM) in PBS (pH 7.4, 10 mM)
Cys-dependent (0–200 mM) fluorescence turn on of FLD-1 (10 mM) in containing Cys (100 mM). lex ¼ 490 nm for FLD-1, 2, 4, and TCN-1,
PBS. lex ¼ 490 nm, lem ¼ 500–650 nm, and slit width ¼ 0.3 mm. The
l
ex ¼ 454 nm for FLD-3, lem ¼ 500–650 nm, and slit width ¼ 0.3 mm.
experiments were performed in triplicate and results are expressed as The experiments were performed in triplicate. The results are
mean ꢀ SD (n ¼ 3).
expressed as mean ꢀ SD (n ¼ 3).
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(pH 7.4, 10 mM) with 10 equivalents of benzyl mercaptan experiments, conrming the stability of FLD-1 to common
(100 mM) for 1 h and analyzed the reaction products by HPLC RSONs (Fig. 5 bars 9–17). Because the carbonate functional
(Fig. S3†). Consistent with our proposed activation mechanism, group may be sensitive to esterase-catalyzed hydrolysis, we also
we observed FLD-1 consumption and the formation of both tested the esterase stability of the sulfenyl thiocarbonate group
benzyl disulde and uorescein, which supports the proposed by incubating FLD-1 (10 mM) with porcine liver esterase (PLE,
ꢁ
1
mechanism.
1 U mL ). Although a slight uorescence turn on was observed
In addition to Cys and BnSH, other cellular thiols and bio- aer a 2 h incubation, the observed response was much lower
logical nucleophiles were tested to determine whether they than that from thiol activation (Fig. 5 bar 18). Taken together,
resulted in donor activation. In these experiments, FLD-1 these studies demonstrate that FLD-1 is highly responsive and
(10 mM) was incubated with GSH, homocysteine (Hcy), N-acetyl selective to thiol activation and common cellular RSONs do not
cysteine (NAC), penicillamine (PEN), or bovine serum albumin trigger FLD-1 to release COS/H S.
2
(
(
2
BSA) (100 mM), in PBS buffer (pH 7.4, 10 mM) containing CA
Aer conrming the thiol-triggered COS/H S release from
2
ꢁ1
25 mg mL ) and the uorescent intensity was measured aer FLD-1 in aqueous buffer, we next investigated the H
h. As expected, FLD-1 is stable in PBS at physiological pH in capacity of FLD-1 in cellular environment. In these experiments,
the absence of thiols (Fig. 5 bar 1). Incubation of FLD-1 with HeLa cells were treated with FLD-1 (50 mM) or TCN-1 (50 mM)
Cys, NAC, GSH, and Hcy, however, led to a signicant uores- and H S release was monitored using C7-Az, a H S-responsive
cence enhancement, indicating successful donor activation and uorescent probe. Incubation of HeLa cells with C7-Az
2
S delivering
2
2
50,51
COS/H S release (Fig. 5 bars 2–5 and S4†). In addition, these (50 mM) alone resulted in a negligible uorescence response,
2
results demonstrate that the sulfenyl thiocarbonate group is indicating minimal endogenous H S (Fig. 6 top row). Treatment
2
responsive to different types of thiols. In comparison, PEN with TCN-1 also failed to provide a uorescence signal, sug-
resulted in only minimal uorescence response and BSA failed gesting that the thiocarbonate group was stable and did not
to activate the donor presumably due to the bulkiness of these decompose to generate false signals in cellular environments
two thiol species, which hindered their reactions with FLD-1 (Fig. 6 middle row). By contrast, a strong C7-Az uorescent
(Fig. 5 bars 6 and 7). Additionally, N-ethylmaleimide (NEM) signal was observed when incubating HeLa cells with FLD-1,
pretreatment of Cys samples signicantly reduced the uores- suggesting that H S release was successfully triggered by
2
cence enhancement from FLD-1, conrming the necessity of the endogenous thiols. In addition, a strong uorescence signal was
thiol-induced reduction for the donor activation (Fig. 5 bar 8). also observed from activated FLD-1 in the uorescein channel,
We also tested the response of the FLD donors in the pres- conrming the uorescence response upon donor activation
ence of other cellular reactive sulfur, oxygen, and nitrogen (Fig. 6 bottom row). Taken together, these results demonstrate
species (RSONs). FLD-1 (10 mM) was incubated with RSONs that the FLD donors not only function as efficacious H
2
S donors
), hyperchlorite in live cells, but also provide a uorescence signal that enables
), tert-butyl hydroperoxide (TBHP), observing H S release.
To further demonstrate the H
GSSG), and S-nitrosoglutathione (GSNO), and COS/H S release donors we also investigated the anti-inammatory activities of
(
(
2 2
100 mM), such as hydrogen peroxide (H O
ꢁ
ꢁ
ClO ), superoxide (O
2
2
serine (Ser), lysine (Lys), glycine (Gly), oxidized glutathione
2
S-releasing delity of the FLD
(
2
was monitored by tracking the uorescein formation. As FLD-1. We pretreated macrophage RAW 264.7 cells with FLD-1
expected, minimal uorescence was observed in these (0–25 mM) for 2 h, followed by a 24 h incubation with lipo-
ꢁ
1
polysaccharide (LPS, 0.5 mg mL ) to trigger the inammatory
Fig. 5 Fluorescence turn on of FLD-1 (10 mM) in the presence of
cellular RSONs (100 mM): (1) FLD-1 only, (2) Cys, (3) NAC, (4) GSH, (5) Fig. 6
2 2 2
Hcy, (6) PEN, (7) BSA, (8) Cys + NEM, (9) H O , (10) ClO , (11) O
H
2
S delivery from FLD-1 in HeLa cells. HeLa cells were treated
, (12) with NucRed nuclear dye and C7-Az (50 mM) in DMEM only (top row) or
TBHP, (13) Ser, (14) Lys, (15) Gly, (16) GSSG, (17) GSNO, and (18) PLE DMEM containing TCN-1 (50 mM) (middle row) or FLD-1 (50 mM)
ꢁ
ꢁ
ꢁ1
(
1 U mL ). Fluorescence intensity was measured after 2 h incubation. (bottom row) for 30 min. Cells were then washed with PBS and cell
The experiments were performed in triplicate. The results were images were taken in PBS using a fluorescent microscope. Bar scale:
expressed as mean ꢀ SD (n ¼ 3).
50 mm.
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demonstrate that FLD-1 exhibits a dose-dependent inhibition of
the LPS-induced NO formation, which is consistent with anti-
inammatory activities of H S. Taken together, caged-sulfenyl
2
thiocarbonates are promising new class of COS/H S donors
2
with high potential for accessing H
2
S-related protective activi-
ties, and the developed uorescent donors provide new
2
methods for monitoring H S release in real-time in complex
environments.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Research reported in this publication was supported by the NIH
Fig. 7 Effects of FLD-1 on LPS-induced NO accumulation. RAW (R01GM113030) and Dreyfus Foundation. NMR, uorescence
2ꢁ
2
64.7 cells were pretreated with FLD-1 (5–25 mM) or GYY4137 (25 mM)
microscopy, and MS instrumentation in the UO CAMCOR
facility is supported by the NSF (CHE-1427987, CHE-1531189,
and CHE-1625529).
ꢁ
1
for 2 h, followed by a 24 h treatment of LPS (0.5 mg mL ). Results are
expressed as mean ꢀ SD (n ¼ 4). ***P < 0.001 vs. the control group;
##
###
P < 0.01 vs. vehicle-treated group;
P < 0.001 vs. vehicle-treated
group.
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2
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generation, which can be monitored by measuring nitrite
ꢁ
3 M. R. Filipovic, J. Zivanovic, B. Alvarez and R. Banerjee,
(NO
2
) accumulation. We chose to use concentrations of FLD-1
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up to 25 mM because these concentrations did not induce
cytotoxicity (Fig. S6†).
4
O. Kabil and R. Banerjee, Antioxid. Redox Signaling, 2014, 20,
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5 S. Bruce King, Free Radical Biol. Med., 2013, 55, 1–7.
7
As expected, the pretreatment of RAW 264.7 cells with FLD-1
ꢁ
showed a dose-dependent inhibition of NO2 accumulation,
6
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8
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concentration and longer incubation time (i.e. 100–1000 mM
52
and 24 h incubation), such cytoprotection was not observed at
the 25 mM concentration used for comparison, highlighting the
efficacious H S release from FLD-1 in the cellular environment
2
(Fig. 7). To further conrm that the observed effects were due to
9
K. R. Olson, Antioxid. Redox Signaling, 2012, 17, 32–44.
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2
10 C. Szabo, Antioxid. Redox Signaling, 2012, 17, 68–80.
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ꢁ
11 L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol.,
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2
production. None of these
ꢁ
compounds attenuated NO
2
generation, conrming that the
S release
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2
2
(
1
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releases COS/H S in complex cellular environment and exhibits
2
promising anti-inammatory protections, indicating potential
applications of FLD-1 as H S-releasing therapeutics.
2
14 M. D. Pluth, T. S. Bailey, M. D. Hammers, M. D. Hartle,
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2
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1
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497–564.
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fenyl thiocarbonates as new controllable and uorescent 16 M. D. Hartle and M. D. Pluth, Chem. Soc. Rev., 2016, 45,
COS/H S donors. Thiol-triggered COS/H S release from these
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