Development of a highly selective H2S fluorescent probe and its application to evaluate CSE inhibitors

Article abstract of DOI:10.1039/c4ra09066g

In this paper, we developed a novel fluorescent probe C359 for highly selective detection of H₂S over other relevant biothiols. C359 is designed to contain a thiol-specific cleavable disulfide bond. H₂S-mediated the disulfide cleavage and subsequent intramolecular cyclization released the masked 7-hydroxyl coumarin, displaying a remarkable fluorescence enhancement. With the promising features in hand, C359 has been applied to detect the activity of CSE (one of H₂S-producing enzyme) and build up an assay for screening CSE inhibitors. We anticipated that the enzyme assay using C359 could provide a powerful methodology for screening more potent and selective enzyme inhibitors. This journal is

Full text of DOI:10.1039/c4ra09066g

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Development of a highly selective H2S fluorescent
probe and its application to evaluate CSE
inhibitors†
Cite this: RSC Adv., 2014, 4, 50097
a
a
b
c
b
*
*
Xianfeng Gu, Huikun Zhu, Suna Yang, Yi-Chun Zhu and Yi-Zhun Zhu
In this paper, we developed a novel fluorescent probe C359 for highly selective detection of H2S over other
relevant biothiols. C359 is designed to contain a thiol-specific cleavable disulfide bond. H2S-mediated the
disulfide cleavage and subsequent intramolecular cyclization released the masked 7-hydroxyl coumarin,
displaying a remarkable fluorescence enhancement. With the promising features in hand, C359 has been
applied to detect the activity of CSE (one of H2S-producing enzyme) and build up an assay for screening
CSE inhibitors. We anticipated that the enzyme assay using C359 could provide a powerful methodology
for screening more potent and selective enzyme inhibitors.
Received 22nd August 2014
Accepted 23rd September 2014
DOI: 10.1039/c4ra09066g
www.rsc.org/advances
would be valuable tools. Up to now, some inhibitors of these
enzymes have been reported, such as PAG, BCA and AOAA,
Introduction
which are the inhibitors of CSE and CBS. Methylene blue assay
is a colorimetric method for H2S detection,19 which has been
used as a common enzyme activity assay to evaluate inhibitors
of H2S-producing enzymes. However, by using this method,
complicated sample processing is oen required, and variable
results are oen yielded. To obtain more potent and selective
inhibitors, new and efficient inhibition assays are needed. In
recent years, uorescence-based assays are fast emerging in the
eld of biological molecule detection because of their high
sensitivity and convenience.20 As the studies on H2S biological
functions are proceeding, more and more uorescent probes for
H2S detection have been reported.21 However, most of these
probes are only applied to cell imaging, and rare of them used to
the research on enzyme activities, which is of great value in
understanding the role of H2S. Herein, a highly selective H2S
uorescence probe over other thiols was designed and synthe-
sized. Furthermore, an inhibition assay of CSE by using this
uorescent probe was developed.
Hydrogen sulde (H2S) has been recognized as the third
gaseous transmitter of signaling molecules in biological
systems, succeeding nitric oxide (NO) and carbon monoxide
(CO). Therefore, H2S is a topic of great interest in chemistry and
biology. In 1996, the role of H2S in human neuromodulation
was rst reported by Abe and Kimura, which had become a
research prelude for the biological signaling function of H2S.1
Since then, all aspects of the research on the biological func-
tions of H2S has been unfolded, such as cardioprotective,2–5
neuroprotective6–8 and gastroprotective effects,9 the regulation
of insulin release10 and anti-inammatory effects,11 which make
it known that H2S also plays an important, probably even pivotal
role in human and other biological systems.
In mammalian systems, the generation of H2S derives from
several enzymatic pathways. For example, both cystathionine
g-lyase (CSE, EC 4.4.1.1) and cystathionine b-synthase (CBS,
EC4.2.1.22), two pyridoxal 50-phosphate (PLP) dependent
enzymes, are able to convert cysteine into H2S within different
organs and tissues.12–17 Recently, it was reported that 3-mer-
captopyruvate sulphurtransferase (3-MST, EC2.8.1.2) and
cysteine aminotransferase (CAT cysteine lyase (CL, EC4.4.1.10))
can also catalyzed the production of H2S.18 To understanding
the biological roles of H2S, the inhibitors of these enzymes
Results and discussion
Design and synthesis of probe C359
CSE, a PLP-dependent enzyme, is able to catalyze the produc-
tion of H2S using cysteine as a substrate. Therefore, interference
from cysteine could occur in the assay of CSE activity when
using a H2S uorescent probe as an assay tool, due to the
similar reactivity of thiol-containing compounds.22 Thus, it is
highly desirable to construct a selective uorescent probe for
detecting H2S over thiols. Recently, Xian's group reported a
series of selective probes for H2S with 2-(2-pyridinyldithio)-
benzoic group.21g These probes can be triggered by H2S to
aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Rd, Shanghai, 201203, China. E-mail: xfgu@fudan.edu.cn; Fax: +86 21
51980008
bDepartment of Pharmacology, School of Pharmacy, Fudan University, 826 Zhangheng
Rd, Shanghai, 201203, China. E-mail: zhuyz@fudan.edu.cn; Fax: +86 21 51980008
cDepartments of Physiology and Pathophysiology Shanghai College of Medicine, Fudan
University, 138 Yixueyuan Rd, Shanghai, 200021, China
† Electronic supplementary information (ESI) available: NMR spectra, HPLC
spectra. See DOI: 10.1039/c4ra09066g
undergo
a
tandem nucleophilic substitution–cyclization
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reaction, and then to release the uorophores and render the
uorescence turn-on. Although these probes exhibit highly
selectivity between thiols and H2S, poor water solubility and
slow reaction rate limit their biological application. In order to
improve water solubility and uorescence turn-on rate of these
probes, they added surfactant CTAB. However, the need for a
surfactant may also limit their application in living systems.
With this consideration in mind, a selective and fast reactive
disulde containing probe (C359) was designed and synthe-
sized. C359 was readily synthesized in two steps using the
procedure shown in Scheme 1.
Scheme 2 Proposed mechanism of the reaction of C359 with H2S and
thiols.
7-Hydroxyl coumarin (C169) is a commercial available uo-
rescent dye with a broad absorption band and an emission band
centered around 350 nm (3 ¼ 1.7 ꢀ 107 Mꢁ1 cmꢁ1, in EtOH) and
450 nm (Ff ¼ 0.09), respectively. By introducing a 3-(pyridin-2-
yldisulfanyl)-propionyl group into C169, we tend to build up a
highly selective uorescent probe for H2S over other related
thiols. Notably, the introduction of this cage unit further
reduced the uorescent quantum yield to be 0.005, making
C359 a promising probe with low background interference.
Moreover, C359 exhibits fast reactivity with H2S due to its good
solubility in buffer system under experimental conditions and
the less steric effect of 3-(pyridin-2-yldisulfanyl)-propionyl
group. As shown in Scheme 2, the probe C359 could react with
H2S to afford an intermediate with a nucleophile SSH, then a
spontaneous intramolecular nucleophilic reaction would be
triggered as the electrophile carbonyl is present in a suitable
site, to release a ve membered cyclic lactone ring and the
uorescent product C169. In sharp contrast, the reaction
between thiols and C359 yielded a compound with disulde
bond, and this compound could not undergo an intramolecular
nucleophilic attack to release C169, which leads to a high
discriminative detection of H2S over thiols.
The spectroscopic response of C359 to H2S
Initially, we evaluated the optical properties of C359 toward H2S
in 200 mM Tris HCl buffer (pH ¼ 7.4) by monitoring the
changes in the absorption and uorescence spectra. In the
absence of H2S, C359 displayed a strong absorption at 280 nm
with a shoulder band around 310 nm. Introduction of H2S led to
buildup of a new band at 350 nm. Two well-dened isosbestic
points at 305 and 275 nm were noted. The absorption band at
350 nm is the characteristic feature of C169, indicating the
formation of C169 (Fig. 2a). In the uorescence spectra, free
C359 exhibited a weak emission feature centered on 450 nm.
Upon gradual addition of H2S to the solution of C359 in 200 mM
Tris HCl buffer (pH ¼ 7.4), a remarkable enhancement of
uorescence intensity at 450 nm was observed (Fig. 2b).
Notably, the uorescence intensity increased linearly with
concentrations of H2S increased from 0 to 35 mM, indicating
that C359 is a promising probe for detecting H2S at micromolar
concentration level under physiological conditions. Based on
the titration experiments, the detection limits were evaluated to
be 5.0 ꢀ 10ꢁ8 M (Fig. S3†), which are comparable to previously
reported probes.
Interference from other related analytes was then investi-
gated under the same condition. As shown in Fig. 3, no obvious
To validate the proposed mechanism of the reaction between
1
C359 and H2S, H NMR and HPLC analysis were further per-
uorescence change was observed upon addition of 50 equiv. of
formed to conrm the production of C169 in the reaction. The
partial 1H NMR spectra of C359 in the absence and presence of
H2S, and that of C169 are shown in Fig. 1. Upon addition of H2S
to the solution of C359, new peaks at 7.9, 7.5, 6.8, 6.7 and 6.2
ppm assigned to C169 were observed, suggesting the formation
of C169. In the HPLC spectra, the retention time of standard
C359 and C169 are around 5.8 min and 2.3 min respectively.
Upon addition of H2S to the solution of C359, the peak around
5.8 min decreased, while a new peak with the retention time
around 2.3 min appeared simultaneously (Fig. S1†). These
observations conrm that treatment of C359 with H2S led to
formation of C169.
Fꢁ, Clꢁ, Brꢁ, Iꢁ, SO42ꢁ, SO42ꢁ, NO3ꢁ, CO32ꢁ, SCNꢁ, S2O52ꢁ, N3
,
ꢁ
and NO2ꢁ, as well as relevant thiols and cofactor PLP. More
Scheme 1 The synthesis of C359.
Fig. 1 Partial 1H NMR spectra of C359, C359 + H2S and C169.
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Fig. 4 Utility of C359 as a H2S probe and assay for CSE activity. I0
represents the fluorescence intensity of C359 only, and Ia represents
the intensity in the presence of Cys, PLP and enzyme.
our proposal, CSE was chosen in our enzyme inhibition assay
due to the commercial availability of recombinant CSE (GST-
CSE). To assess the ability of C359 for monitoring activity of
CSE, treatment of CSE with C359 was performed rstly. As
shown in Fig. 4, signicant uorescent signal over the back-
ground was recorded when Cys was used as a substrate. In the
absence of Cys, signicantly reduced uorescence intensity was
noted. The weak uorescence response of C359 toward Cys in
the absence of CSE further conrmed the role of CSE in
production H2S. These observations indicated that C359 can be
employed to monitor CSE catalyzing Cys to produce H2S. With
this result in hand, we then assessed the capability of C359 for
the evaluation of CSE inhibitors. Two known inhibitors, BCA
and PAG, were used to test the validity of the inhibition assay. As
shown in Fig. 5, two inhibition curves of BCA and PAG against
CSE were obtained using C359 as a reporter. Interestingly, IC50
of BCA and PAG was determined to be 16.75 mM and 40.81 mM
Fig. 2 (a) Absorption spectra of C359 (5 ꢀ 10ꢁ6 M) in the presence of
different concentrations of NaHS (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 equiv.) in Tris HCl buffer (200 mM, pH ¼ 7.4). (b) Fluores-
cence spectra of C359 (lex ¼ 305 nm) in the presence of different
concentrations of NaHS (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
equiv.).
Fig. 3 Fluorescence response of C359 (450 nm) in the absence and
presence of 50 equiv. of various anions, thiols and PLP in Tris HCl
buffer (200 mM, pH ¼ 7.4). Each measuring was performed after 2 min
of mixing. I0 represents the fluorescence intensity of C359 only, and Ia
represents the intensity in the presence of various anions, PLP and
thiols.
interestingly, only addition of H2S introduced remarkable
uorescent enhancement. These results indicate the excellent
capacity of C359 for selective detection of H2S over the other
competitive anions, thiols and enzyme cofactor.
The application of C359 to evaluation of CSE inhibitors
As shown in Fig. 3, no uorescence response was observed upon
the incubation of C359 with CSE/CBS substrate (Cys) or cofactor
(PLP), enabling C359 a promising probe for testing activity of
Fig. 5 Inhibition curves for BCA and PAG against CSE. Data are pre-
H2S-producing enzymes and screening their inhibitors. To test sented as mean ꢂ SEM; n ¼ 3; *P < 0.05 versus control.
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Table 1 IC50 of CSE inhibitors
by ash chromatography (silica gel) to afford C498 2.13 g (90%).
1H NMR (400 MHz, DMSO-d6, d ppm): 8.05 (d, 2H), 7.75 (d, 2H),
7.23 (s, 1H), 7.14 (m, 2H), 6.46 (d, 2H), 5.74 (s, 1H), 3.07 (m, 8H).
13C NMR (100 MHz, DMSO-d6, d ppm): 170.5, 160.3, 154.7,
153.3, 144.5, 130.1, 119.2, 117.4, 116.3, 110.6, 34.3, 32.9. C498
(666 mg, 1.34 mmol) and 2-thiol pyridine (180 mg, 1.62 mmol)
were dissolved in ethyl acetate (20 mL). Aer the addition of 3
drops of BF3 ether solution, the reaction mixture was stirred at
room temperature for 3 days. White solid was appear, and aer
ltering, lter cake was washed by cold ethyl acetate to afford
Inhibitors
IC50 (mM)
IC50a (mM)
BCA
PAG
16.75
40.81
14.0
40.0
a
Literature data (ref. 19).
respectively, which are comparable to the literature data by
using methylene blue as a reporter (Table 1). These results
indicated that C359 is an ideal uorescent probe for monitoring
enzyme activity, which further allows the buildup of a useful
methodology to screen more potent and selective inhibitors of
H2S-producing enzyme.
1
390 mg of C359 (81%). H NMR (400 MHz, DMSO-d6, d ppm):
8.47 (d, 1H), 8.06 (d, 2H), 7.84–7.75 (m, 3H), 7.26 (m, 2H), 7.14
(d, 1H), 6.47 (d, 1H), 3.17 (m, 2H), 3.05 (m, 2H). 13C NMR (100
MHz, DMSO-d6, d ppm): 169.9, 159.6, 158.6, 153.9, 152.5, 149.5,
143.7, 137.9, 129.3, 121.4, 119.5, 118.5, 116.7, 115.6, 109.9, 33.4,
32.6. HR-MS (ESI-TOF) (m/z): C17H14N1O4S2 calcd, 360.0359;
found, 360.0356 [M + 1]+.
Conclusions
In conclusion, a uorescent probe C359 was designed and
synthesized, which show high selectivity for H2S. The reaction
between C359 and H2S triggered the disulde bond cleavage
and subsequent intramolecular cyclization, releasing 7-hydroxyl
UV and FL spectroscopic measurements
Stock solutions of probe C359 (5 ꢀ 10ꢁ3 M) and NaHS (1.5 ꢀ
10ꢁ2 M) were prepared in deionized H2O. 3 mL Tris HCl buffer
(200 mM, pH ¼ 7.4) was rstly added to a 5 mL cuvette, and then
3 mL of probe stock solutions and 0–16 mL of NaHS stock solu-
tions were added. The resulting solution was thoroughly shaken
before recording the spectra.
coumarin and resulting in
a remarkable uorescence
enhancement. Other relevant thiols introduce no observable
uorescent response. More importantly, C359 is capable of
monitoring CSE activity, which further allows the buildup of an
inhibition assay of CSE by using this uorescent probe.
CSE enzyme inhibition assay using C359
Experimental section
General method
The standard reaction was performed in the presence of 2 mg of
CSE, 6 mM PLP, 0.5 mM of L-cysteine as substrate, and 10 mM of
C359 as the probe in 100 mL Tris HCl buffer (200 mM, pH ¼ 7.4).
The concentration of inhibitors were varied from 0–5 mM, and
the assays were incubated for 75 minutes before uorescence
was measured. Data are presented as mean ꢂ SEM; n ¼ 3; *P <
0.05 versus control.
All chemical reagents and solvents for synthesis were purchased
from commercial suppliers and were used without further
purication. Sodium hydrogen sulde (NaSH) was purchased
from Sigma. cystathionine g-lyase (CSE), BCA and PAG were
purchased from Cayman Chemical. 1H NMR and 13C NMR
spectra were recorded on a Bruker AV-400 spectrometer with
chemical shis reported in ppm (in DMSO-d6) at room
temperature. The analytical HPLC was performed on Waters
600E HPLC system. Mass spectra were measured on a HP 1100
LC-MS spectrometer. UV-vis absorption spectra were recorded
on a Varian Cary 100 spectrophotometer. Fluorescence spectra
were measured with a Varian CARY Eclipse Fluorescence spec-
trophotometer. Spectral-grade solvents were used for measure-
ments of UV-vis absorption and uorescence.
Acknowledgements
We gratefully acknowledge the nancial support by the National
Science Foundation of China (no. 21001013).
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