Hydrogen Sulfide Mediated Tandem Reaction of Selenenyl Sulfides and Its Application in Fluorescent Probe Development

Article abstract of DOI:10.1021/acs.orglett.9b02844

A unique reaction between H₂S and a selenenyl sulfide containing benzoate ester template was discovered. This reaction could be specifically triggered by H₂S and lead to ester bond cleavage. The reaction was not affected by the presence of thiols such as glutathione and cysteine. With this reaction, a series of fluorescent probes were synthesized and evaluated. The probes exhibited high sensitivity/selectivity for H₂S in both buffers and cells.

Full text of DOI:10.1021/acs.orglett.9b02844

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Hydrogen Sulfide Mediated Tandem Reaction of Selenenyl Sulfides
and Its Application in Fluorescent Probe Development
Yingying Wang,† Chun-tao Yang,†,‡ Shi Xu,† Wei Chen,*,† and Ming Xian*,†
†Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
‡Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, Guangdong 510436, China
S
* Supporting Information
ABSTRACT: A unique reaction between H2S and a selenenyl sulfide containing benzoate ester template was discovered. This
reaction could be specifically triggered by H2S and lead to ester bond cleavage. The reaction was not affected by the presence of
thiols such as glutathione and cysteine. With this reaction, a series of fluorescent probes were synthesized and evaluated. The
probes exhibited high sensitivity/selectivity for H2S in both buffers and cells.
Scheme 1. Design Concept of SeSP Probes
ydrogen sulfide (H2S) is a critical gasotransmitter in
H
biological systems.1,2 Current studies have proven that
H2S exists in different tissues and regulates cellular redox status
and various physiological functions involved in human disease
and health.3−7 However, the exact mechanisms of its action
remain elusive mainly due to the lack of reliable and
noninvasive methods for H2S detection in biological samples.
Traditional detection methods, such as colorimetric measure-
ments, electrochemical assays, and gas chromatography
methods, often require complicated post-mortem processing,
which could lead to the destruction of samples.8−11 Thus, they
cannot be applied in real-time and noninvasive detection.
Alternatively, fluorescent probes have obvious advantages due
to their great temporal and spatial sampling capability. This
topic has been well studied in recent years, and a large number
of such probes have been invented.12−15 In general, these
probes are reaction-based sensors, meaning their optical
changes are based on specific H2S reactions. As such, exploring
novel and more specific reactions of H2S is important for the
development of the next generation of H2S probes. Our group
reported in 2011 the first nucleophilic reaction-based design
strategy for H2S probes (WSP).16,17 These probes utilize a
unique tandem reaction: H2S reacts with pyridyl disulfide to
form a persulfide intermediate, which then undergoes an
intramolecular cyclization to release the fluorophore. This is
specific for H2S, not for other biothiols (Scheme 1a). While the
selectivity of WSP for H2S is excellent, the probes can be
consumed by thiols (RSH), which could lead to decreased
sensitivity when high concentrations of RSH are presented. To
address this problem, in 2015, we developed diselenide-based
probes SePs (Scheme 1b).18 While a SeP can react with RSH,
the resulting Se−S intermediate still retains high reactivity
toward H2S, which eventually leads to H2S-mediated
cyclization and fluorophore release. This design overcomes
the problem of WSP. However, it is not flexible enough to tune
their reactivity toward H2S as two identical fluorophore
moieties are needed in SeP. Moreover, this diselenide template
often shows poor solubility in aqueous systems. To further
optimize these nucleophilic reaction-based probes, we
envisioned a design using the selenenyl sulfide (Se−S)
template,19 inspired by the intermediate structure shown in
Received: August 10, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02844
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1b. We reasoned that this template (shown as SeSP in
Scheme 1c) should have reactivity similar to that of SeP
toward H2S, but it would allow easier modification and tuning
of the structures of the probes. Herein, we report the study of
the H2S-mediated tandem reaction with the selenenyl sulfide
template. We also prepared and evaluated a series of
fluorescent probes based on this reaction.
Scheme 3. Synthetic Route and Structures of SeSP1−9
As shown in Scheme 1c, the probe template is based on the
intermediate of SeP probes’ reaction with RSH. Some studies
have revealed that nucleophilic attack by RSH at the Se atom is
thermodynamically favorable and kinetically faster.20 More-
over, in this template there should be a strong Se---O
interaction with the CO carbonyl group which would
facilitate the attack of −SH at the Se atom rather than at the S
atom.21 Therefore, the selenenyl sulfide linkage of the probe is
expected to be highly reactive to both H2S and cellular thiols
(Cys and GSH). With H2S, the −Se−S− linkage should be
converted to −Se−SH rapidly. The following cyclization
should produce benzothiaselenol-3-one and release the
fluorophore to turn on fluorescence. With thiols (RSH), the
original −Se−S− linkage will be converted into another −Se−
S− linkage in an equilibrium process, and this should not turn
on fluorescence. Nor would it consume the probe. Only when
H2S is present will the equilibrium be broken to release the
fluorophore. Overall, the SeP probes are expected to be specific
for H2S and will not be affected by the presence of biothiols.
With this idea in mind, we first prepared a model substrate 1
and tested its reaction with H2S and cysteine. The reaction
between 1 and H2S (Scheme 2) was fast. The expected
these molecules showed nonfluorescence in buffers due to
esterification of two −OH groups of fluorescein. However,
upon treatment with 50 μM H2S (using Na2S as the
equivalent) for 30 min, SeSP1−3 gave significant fluorescence
increases while SeSP4−9 showed much smaller fluorescence
enhancement (Figure 1). These results indicated that S-aryl
Scheme 2. Model Reactions of the −Se−S− Template
Figure 1. Fluorescence intensity of SeSP (10 μM) with 50 μM Na2S.
Reaction time: 30 min, in PBS (50 mM, pH 7.4).
substituents made the −Se−S− linkage more reactive to H2S,
while substrates with S-alkyl substituents (SeSP4−9) seem to
react with H2S slowly.
products 2 and phenol were produced in very good yields. The
reaction was equally effective when H2S and cysteine coexisted.
Even when 1 was pretreated with cysteine for 1 h and then
treated with H2S, the same products were obtained. These
results demonstrated that the selenenyl sulfide template indeed
could effectively capture H2S and the presence of biothiols will
not cause any problems.
To validate the application of this reaction template in the
design of H2S fluorescent probes, we next synthesized a series
of possible probes (Scheme 3). These compounds employed
fluorescein as the common fluorophore due to its excellent
fluorescence property. It is also known that the fluorescence of
fluorescein can be quenched upon acylation on hydroxy
groups. Different R groups were used in the probe structures to
explore their effects on the reactivity/selectivity of the −Se−
S− linkage to H2S. Their synthesis was straightforward. Briefly,
a known starting material 322 was treated with various thiols to
yield −Se−S−-containing benzoic acids 4a−4i. Subsequent
esterification with fluorescein gave the desired probes SeSP1−
9. Their characterization data can be found in the Supporting
Information.
It should be noted that we tested the effects of cetrimonium
bromide (CTAB) in the responses of the probes in the studies
shown in Figure 1. As a cationic surfactant, CTAB is normally
considered not biologically friendly as it could disrupt the cell
membrane. In our previous works, we found that the use of a
small amount of CTAB could dramatically increase the probes’
reactivity (e.g., turn-on rates) and sensitivity (e.g., enhanced
fluorescence intensity) in aqueous systems.17 The effects of
CTAB can be attributed to two reasons: (1) with CTAB the
solubility of the probe in aqueous buffers is increased, and (2)
as a cationic surfactant CTAB may absorb sulfide anion. This
would facilitate the reaction between sulfide anion and the
probe.
As SeSP1−3 showed better performance than other probes,
these three probes were evaluated. As shown in Figure 2, time-
dependent responses demonstrated they were fast turn-on
probes for H2S because the maximum signals could be reached
in 5−15 min. They were also found to be very sensitive as their
fluorescence turn-on folds were at 500−700 for SeSP2 and
SeSP3 or even 1800 for SeSP1.
We next tested the selectivity of SeSP1−3 for H2S. These
With these compounds in hand, we then studied their
fluorescence responses to H2S in different conditions. All of
probes were treated separately with some representative sulfur
2−
species such as GSH, Cys, homocysteine (Hcy), SO32−, SO4
,
B
DOI: 10.1021/acs.orglett.9b02844
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Organic Letters
Letter
As SeSP1 was identified as the best in this series, we then
tested its detection limit for H2S. It was tested with different
concentrations of H2S (0−60 μM) (Figure 4). The
Figure 2. (a) Time-dependent fluorescence responses of SeSP (10
μM) to Na2S (50 μM) with 100 μM CTAB at 25 °C. (b) Turn-on
fold of SeSP (10 μM) to Na2S (50 μM) with 100 μM CTAB at 25 °C.
and S2O32−. We also tested the responses of SeSP1−3 to
several reactive oxygen species, reactive nitrogen species, and
selected amino acids: alanine, lysine, arginine, proline, serine,
hypochlorite (ClO−), superoxide (O2 ), hydrogen peroxide
−
Figure 4. Fluorescence emission spectra of SeSP1 (10 μM) with
different concentrations of Na2S (0, 1, 4, 8, 12, 15, 20, 40, 60 μM for
curves 1−9. Reaction time: 30 min, room temperature in PBS (50
mM, pH 7.4, 100 μM CTAB).
(H2O2), peroxynitrite (ONOO−), nitroxyl (HNO), and nitric
oxide (NO). As shown in Figure 3, these molecules did not
fluorescence intensities were linearly related to the concen-
trations in the range of 1−20 μM (Figure S1). The detection
limit of SeSP1 was found to be 6.7 nM, indicating SeSP1 was
suitable for detecting H2S in biological systems. We also tested
pH effects on the sensor and SeSP1 worked well in normal
biological pH (7−9) (Figure S2).
To demonstrate the application of SeSP1, it was used in H2S
imaging in HeLa cells. Briefly, freshly cultured cells were
incubated with SeSP1 (10 μM) for 45 min. Cells were then
washed with PBS to remove excess SeSP1. No significant
fluorescence in cells was observed (Figure 5). However, after
Figure 3. Fluorescence intensity of SeSP1−3 (10 μM) to different
sulfur molecules: (1) probes alone; (2) 50 μM Na2S; (3) 0.2 mM
Na2SO3; (4) 0.2 mM Na2S2O3; (5) 0.2 mM Na2SO4; (6) 0.2 mM
Cys; (7) 1 mM GSH; (8) 0.1 mM Hcy; (9) 0.1 mM Ala; (10) 0.1
mM Arg; (11) 0.1 mM Lys; (12) 0.1 mM Pro; (13) 0.1 mM Ser; (14)
−
25 μM ClO−; (15) 25 μM O2 ; (16) 0.25 mM H2O2; (17) 0.1 mM
ONOO−; (18) 0.1 mM Anglie’s salt (HNO donor); (19) 0.1 mM V-
PYRRO/NO (NO donor); (20) 0.1 mM Hcy + 50 μM Na2S; (21) 1
mM GSH + 50 μM Na2S; (22) 0.2 mM Cys + 50 μM Na2S; (23) 1
mM GSH + 0.2 mM Cys + 0.1 mM Hcy +50 μM Na2S. Reaction
time: 30 min, rt, in PBS (50 mM, pH 7.4, 100 μM CTAB).
Figure 5. Fluorescence images of H2S in HeLa cells. Cells were
treated with SeSP1 (45 min), washed, and subjected to different
treatments: (a) control (no H2S donor was added); (b) 50 μM H2S
donor (60 min); (c) 100 μM H2S donor (60 min).
cause significant fluorescence increase. When H2S coexisted
with high concentrations of biothiols (Cys, GSH, Hcy),
SeSP1−3 still elicited strong fluorescence. These results
demonstrated that SeSP were not only highly selective for
H2S but also very resistant to probe consumption by biothiols.
While SeSP1−3 showed excellent properties, the results
shown in Figures 2 and 3 indicated that SeSP1 was better than
the other two. This superiority may be attributed to the
nitrogen atom in pyridine deprotonates thiol to form a six-
membered ring intermediate and accelerate the reaction.23
(Scheme 4).
treatment with a H2S donor (NSHD-124) for 60 min, we
observed obvious fluorescence and the intensity was correlated
to the concentration of donor applied. Moreover, cell viability
assay showed SeSP1 was almost noncytotoxic (Figure S3).
These data suggest SeSP1 is cell permeable and can be used
for visualizing H2S fluctuation in cells. Of note, CTAB was not
needed for cell imaging.
Finally, we wondered if SeSP1 could measure endogenous
H2S concentration changes. We treated HeLa cells with either
L-Cys (an H2S biosynthestic substrate) or 2-amino-4-pentynoic
acid (PAG, a CSE inhibitor). The fluorescence intensity
changes in cells loaded SeSP1 were measured by a plate reader
and referred to control (detailed protocols were presented in
the Supporting Information). As shown in Figure 6, L-Cys
treated cells showed enhanced fluorescence, while PAG-
pretreated cells showed decreased fluorescence. These experi-
ment results suggested SeSP1 is suitable for monitoring
endogenous H2S changes.
Scheme 4. Proposed Mechanism and Intermediates for
SeSP1
In summary, we reported here a unique reaction between
H2S and the selenenyl sulfide template. This reaction was
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02844.
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AUTHOR INFORMATION
Corresponding Authors
■
*Tel: 509-335-6073. E-mail: w.chen@wsu.edu.
*Tel: 509-335-6073. E-mail: mxian@wsu.edu.
ORCID
Ming Xian: 0000-0002-7902-2987
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by NIH (R21DA046386) and NSF
(CHE1738305).
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DOI: 10.1021/acs.orglett.9b02844
Org. Lett. XXXX, XXX, XXX−XXX