Dual-Reactable Fluorescent Probes for Highly Selective and Sensitive Detection of Biological H2S

Article abstract of DOI:10.1002/asia.201600262

Hydrogen sulfide (H₂S) is an important endogenous signaling molecule with a variety of biological functions. Development of fluorescent probes for highly selective and sensitive detection of H₂S is necessary. We show here that dual-reactable fluorescent H₂S probes could react with higher selectivity than single-reactable probes. One of the dual-reactable probes gives more than 4000-fold turn-on response when reacting with H₂S, the largest response among fluorescent H₂S probes reported thus far. In addition, the probe could be used for high-throughput enzymatic assays and for the detection of Cys-induced H₂S in cells and in zebrafish. These dual-reactable probes hold potential for highly selective and sensitive detection of H₂S in biological systems. Two heads are better than one: Fluorescent probes with two types of reactive heads (R¹ and R²) for H₂S can lead to a higher selectivity than that of single-reactable probes (See Figure). The probe was used for highly selective and sensitive detection of biological H₂S.

Full text of DOI:10.1002/asia.201600262

DOI: 10.1002/asia.201600262
Communication
Fluorescent Probes
Dual-Reactable Fluorescent Probes for Highly Selective and
Sensitive Detection of Biological H2S
Chao Wei,[b] Runyu Wang,[b] Changyu Zhang,[a] Guoce Xu,[c] Yanyan Li,[b] Qiang-Zhe Zhang,[c]
Lu-Yuan Li,[c] Long Yi,*[a] and Zhen Xi*[b]
been fully elucidated.[5] To understand the physiological roles
Abstract: Hydrogen sulfide (H2S) is an important endoge-
of H2S in relation to its biological concentration, it is also desir-
nous signaling molecule with a variety of biological func-
able to develop probes for the accurate measurement of en-
tions. Development of fluorescent probes for highly selec-
dogenous H2S levels in vivo.[6]
tive and sensitive detection of H2S is necessary. We show
Fluorescence-based methods have recently emerged as
here that dual-reactable fluorescent H2S probes could
a useful approach for H2S detection in biological systems.[7–17]
react with higher selectivity than single-reactable probes.
An ideal intracellular H2S sensor should have a large dynamic
One of the dual-reactable probes gives more than 4000-
range for the selective detection of H2S over higher intracellu-
fold turn-on response when reacting with H2S, the largest
lar concentrations of biothiols (such as millimolar concentra-
response among fluorescent H2S probes reported thus far.
tions of reduced glutathione, GSH). The reactivity of the probe
In addition, the probe could be used for high-throughput
toward H2S is also essential for real-time detection. Although
enzymatic assays and for the detection of Cys-induced
great advances have been made in the development of fluo-
H2S in cells and in zebrafish. These dual-reactable probes
rescent H2S probes, probes with higher sensitivity and higher
hold potential for highly selective and sensitive detection
selectivity for the detection of H2S levels in biological samples
of H2S in biological systems.
still need to be developed. We previously proposed a dual-
quenching probe with the largest off-on response (2000-fold)
reported based on FRET-ICT dual-quenching effects,[17] but the
Hydrogen sulfide (H2S) is an important gaseous signaling mole-
cule with a multitude of physiological and pathological func-
tions in various tissues.[1] H2S can be enzymatically produced in
vivo by three distinctive pathways including cystathionine b-
synthase (CBS), cystathionine g-lyase (CSE), and 3-mercaptopyr-
uvate sulfurtransferase (3MPST) coupled with cysteine amino-
transferase (CAT).[2] Previous studies indicate that H2S is both
protective[3] and toxic[4] to cells depending on its concentra-
tions. Due to the complexity of sulfur resources in vivo, the
exact concentration and source of endogenous H2S have not
probe exhibited a medium selectivity for H2S over biothiols
present at millimolar concentration. Herein, we report dual-re-
actable H2S probes (Figure 1) with higher selectivity and sensi-
tivity than single-reactable probes and the dual-quenching
probe.[17] Moreover, probe 2 could be used to develop a high-
throughput enzymatic assay and to image Cys-induced endog-
enous H2S.
A dual-quenching probe can lead to a large fluorescent
turn-on response to give a low detection limit (the 3s/k
value).[18] To design a H2S probe with higher selectivity, herein
we used two types of reactable groups on one fluorophore
(Figure 1a). The reactable group is also the quenching group.
A competitor partly reacts with one reactable group while an-
other reactable group can still quench the fluorescence. As
a result, the maximum turn-on fluorescence will only be ob-
tained with a competitor that can completely undergo the two
different reaction events (here, nucleophilic and redox reac-
tions). Such a dual-reactable strategy should increase the
probe’s selective response between H2S and biothiols.
To this end, we employed FRET-ICT dual-quenching effects
and nucleophilic–redox dual-reactable groups to prepare the
two probes 1 and 2 (Figure 1b). Both NBD amine and aromatic
azide were installed into one fluorophore as reactable groups
for H2S, while the fluorescence of the designed compound was
quenched by NBD and azide through FRET and ICT effects, re-
spectively.
[a] C. Zhang, Prof. Dr. L. Yi
Beijing Key Laboratory of Bioprocess
Beijing University of Chemical Technology (BUCT)
15 Beisanhuan East Road, Chaoyang District, Beijing 100029 (P. R. China)
E-mail: yilong@mail.buct.edu.cn
[b] C. Wei, R. Wang, Y. Li, Prof. Dr. Z. Xi
Department of Chemical Biology
State Key Laboratory of Elemento-Organic Chemistry
National Engineering Research Center of Pesticide (Tianjin)
Collaborative Innovation Center of Chemical Science and Engineering (Tian-
jin)
Nankai University
Weijin Road 94, Tianjin 300071 (P. R. China)
E-mail: zhenxi@nankai.edu.cn
[c] G. Xu, Q.-Z. Zhang, Prof. Dr. L.-Y. Li
State Key Laboratory of Medicinal Chemical Biology
Nankai University
Weijin Road 94, Tianjin 300071 (P. R. China)
The synthesis of 1 was achieved by a coupling reaction of
3[14] and NBD-Cl (see the Supporting Information). Probe 2 was
Supporting information for this article can be found under http://
dx.doi.org/10.1002/asia.201600262.
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coumarin 10 by a Sandmeyer reaction. Probe 2 was obtained
by a couping reaction of 10 and NBD amine 12.[17] Probes
1
1 and 2 were well characterized by H NMR and 13C NMR spec-
troscopies and high-resolution mass spectrometry (HRMS) (see
the Supporting Information).
Emission spectra of the probes 1 and 2 were examined in
the absence or presence of H2S in phosphate-buffered saline
(PBS, pH 7.4) (Figure 3 and Figures S1, S2). As expected, the
coumarin emission was nearly completely quenched for both
probes due to the FRET-ICT dual-quenching effects (quantum
yield F for 2, 0.0016). After reacting with H2S, the probe
showed a significant turn-on fluorescent response, with an off–
on response of up to ca. 3150-fold for 1 and 4010-fold for 2
(F, 0.6063). Such a fluorescent turn-on response could be
clearly visualized by the naked eye (Figure 3a). To our knowl-
edge, the observed increase in fluorescence emission for 2 is
the largest among the fluorescent probes reported so far for
H2S.[7–17]
To determine the detection limit for 1 and 2, the fluores-
cence intensity change was closely monitored by addition of
various concentrations of H2S to a solution of the probe (Fig-
ure 3b and Figure S1). For both probes, the fluorescence inten-
sity was linearly related to the concentration of H2S from 0 to
100 mm, indicating a large dynamic range for the detection of
H2S. The detection limit for 1 or 2 was calculated to be 27 and
24 nm, respectively, by using the 3s/k method.[18] The results
implied that probes 1 and 2 are highly sensitive toward H2S in
buffer solution. In addition, the probe 2 could function over
a wide range of pH from 6.0 to 8.5 (Figure S2).
Figure 1. The design strategy for a dual-reactable-dual quenching H2S fluo-
rescent probe (a) and structures of probes 1 and 2 used in this work (b).
FRET, fluorescence resonance energy transfer; ICT, intramolecular charge
transfer; NBD, 7-nitro-1,2,3-benzoxadiazole.
prepared as shown in Figure 2. Treatment of p-amino salicylic
acid with methanol led to the formation of 4, which under-
went selective acetylation on the 4-amino group to provide
5.[19] Reduction of 5 by LiAlH4 yielded 6, which was further oxi-
dized to afford the corresponding aldehyde 7.[20] Coumarin 8
was formed by a Knoevenagel condensation of aldehyde 7
with diethyl malonate in the presence of piperidine. Hydrolysis
of 8 yielded free amine 9, which was transformed into azido-
The absorption spectra of the probes were further examined
(Figure S3). The coumarin absorbance for 1 and 2 exhibited
a decrease at 330–350 nm and an increase at 350–380 nm
after treatment with H2S, due to the reduction of azide to
amine. The NBD absorbance for 1 and 2 displayed a decrease
at around 480 nm and an increased absorbance at 530 nm, re-
Figure 2. Synthesis of probe 2 and its reaction with H2S. Conditions: (a) Conc. H2SO4, MeOH, reflux, 12 h; (b) Ac2O, CH2Cl2, 0.5 h; (c) LiAlH4, dry THF, 2 h; (d) pyr-
idinium dichromate (PDC), dry THF, 2 h; (e) diethyl malonate, piperidine, EtOH, reflux, 4 h; (f) 4m NaOH, reflux, 0.5 h; then conc. H2SO4, pH 2.0; (g) 20% H2SO4,
NaNO2, 0–58C, 0.5 h; then NaN3, 08C to room temperature, 1 h; (h) HATU, DIPEA, CH2Cl2, 12 (1 equiv), overnight. Yields of isolated product are given are given
below the arrow.
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Figure 4. The dual-reactable probe 2 is highly selective toward H2S over bio-
thiols. (a) Experiments were performed in PBS (pH 7.4) for 30 min with exci-
tation at 400 nm and relative emission at 460 nm. Columns 1–18 correspond
to: 2 only (1 mm); 2 and species of SO4 (100 mm), S2O3 (100 mm), SO3
(100 mm), H2O2 (100 mm), ClOÀ (100 mm), NO2À (100 mm), Zn2+ (100 mm), Fe3+
Figure 3. The dual-reactable probe 2 is highly sensitive toward H2S with
4010-fold turn-on response and 24 nm detection limit. (a) Time-dependent
fluorescent spectra of 2 (1 mm) in the presence of H2S (250 mm) in PBS
(pH 7.4) with excitation at 400 nm. (b) The linear relationship of relative fluo-
rescence intensity at 460 nm of 2 (1 mm) treated with different concentra-
tions of H2S (0, 10, 20, 30, 40, 50, 75 and 100 mm) for 30 min in PBS.
2À
2À
2À
(100 mm), Cys (1 mm), Hcy (1 mm), GSH (5 mm), trolox (100 mm), ascorbic acid
2+
C
(100 mm), NO (100 mm), KO2 (100 mm), OH from H2O2 and Fe (100 mm) or
H2S (100 mm), respectively. The selectivity of 2 toward H2S is 174-fold or 228-
fold over 1 mm Cys or 5 mm GSH, respectively. (b) Fluorescence intensity at
460 nm of 2 (1 mm) incubated with various analytes (SO32À, 100 mm; Cys and
Hcy, 1 mm; GSH, 5 mm) in the presence of H2S (100 mm).
spectively, due to the production of nitrobenzofurazan
thiol.[17,21] The absorption spectra of single-reactable probes
(with only azide or NBD amine as the reactive group) showed
a response similar to that of the one-reactive site from the
dual-reactable probes. The NBD-based probe showed a faster
reaction rate toward H2S than the azide-based probes (Fig-
ure S3), and the reaction kinetics of the dual-quenching probes
were mainly determined by the property of the relative slow
azide-based reaction site.[17] These results showed that H2S
could cleave the NBD moiety and reduce the azide group of
the probe 2 to give 11, which was further supported by the
HRMS results (see the Supporting Information).
change was measured accordingly (Figure 4 and Figue S4). The
tested species included biothiols (GSH, 5 mm; Cys, 1 mm; Hcy,
1 mm), trolox, ascorbic acid, reactive oxygen species (H2O2,
À
KO2, ClO , OH), reactive sulfur species (SO42À, S2O32À, SO32À),
C
anions (NO2 ) and cations (Zn2+, Mg2+). For both probes, the
À
fluorescence intensity enhancement for any tested analyte in
PBS (pH 7.4) was very small except for H2S. Thus, both 1 and 2
are highly selective toward H2S over other biologically relevant
species. Probe 2 was further tested for its selectivity over bio-
thiols at millimolar concentration, due to its larger off–on re-
sponse and lower detection limit compared to those of
probe 1. The tests indicated that probe 2 is 228-fold more se-
lective toward 100 mm H2S over 5 mm reduced GSH and more
than 1000-fold more selective toward 100 mm H2S over 100
The dual-reactable probes were incubated in PBS buffer for
thermo- and photo-stability tests (Figure S5). The results indi-
cated that the fluorescence increase of the dual-reactable
probe 2 was almost negligible even under UV light for 1 h,
while the single-reactable azide-based probes showed to some
extent a fluorescent increase. These results further indicated
the advantages of the dual-reactable probe.
2À
mm SO3 (Figure 4). Therefore, probe 2 has a higher selectivity
A major challenge for H2S detection in biological systems is
to develop a selective probe that exhibits a distinctive re-
sponse to H2S over biothiols present at millimolar concentra-
tion. Probes 1 and 2 were incubated with various biologically
related species in PBS for 30 min, and the maximal emission
than that of the single-reactable probes or the dual-quenching
probe.[17]
The high selectivity and sensitivity of probe 2 encouraged us
to apply it for the detection of biological H2S. Herein, probe 2
was employed to monitor the cystathionine b-synthase (CBS)
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Figure 5. Use of probe 2 in a high-throughput enzymatic assay. (a) Schematic of the high-throughput fluorometric CBS assay. (b) Time-dependent experiments
were carried out in the assay buffer (200 mm Tris-HCl, pH 8.0, 5 mm pyridoxal 5’-phosphate (PLP), 10 mm GSH, 0.5 mgmLÀ1 BSA, 10% DMSO) at 378C. The re-
action solution contained 10 mmL-Cys, 2 mg CBS enzyme, and 10 mm probe 2. Emission spectra were recorded for each time point with 370 nm excitation.
(c) The high-throughput experiments were carried out in the assay buffer (volume 200 mL) in a 96-well plate at 378C for 1 h. The reaction solution contained
10 mmL-Cys, 2 mg CBS enzyme, 10 mm probe 2 and no inhibitor or 1 mm inhibitor. The normalized fluorescence intensity at 450 nm was monitored upon exci-
tation at 370 nm for each sample. The structures of tested molecules are shown in Figure S5. One of the most potent inhibitors, H3, is sparfloxacin. (d) CBS ac-
tivity curve in the presence of different concentrations of sparfloxacin (see inset for its structure); the solid line represents the best fit to give an IC50 of
133.7 mm.
activity with l-Cys as substrate in a high-throughput assay (Fig-
ure 5a). The truncated version of human CBS (D414–551) was
expressed and purified (see the Supporting Information for de-
tails).[22] The enzymatic reaction can be visualized by monitor-
ing the fluorescence increase of probe 2 (Figure 5b). The CBS
activity can be inhibited by an analogue of Cys (propargylgly-
cine, PPG),[22–23] and therefore we selected amino acids and
their analogues, Cys/imidazole derivatives, and quinolones
drugs (Figure S5) for screening new potent CBS inhibitors. The
high-throughput enzymatic reactions were performed in a 96-
well plate, and the fluorescence of each well was recorded
(Figure 5c and Figure S6). Strong inhibition of CBS should
reduce the enzymatically produced H2S and further lead to
a lower fluorescent off-on response from probe 2. The results
showed that aminooxyacetic acid (a known CBS inhibitor),[23]
nadifloxacin, and sparfloxacin were the most efficient inhibi-
tors. Further tests indicated that the half maximal inhibitory
concentration (IC50) for nadifloxacin and sparfloxacin was
269.2 mm and 133.7 mm, respectively. The tested compounds
with medium inhibition potency included Cys derivatives, qui-
nolones, methacrylic acid, and hydroxylamine. Our preliminary
studies successfully showed the application of probe 2 for
high-throughput screening of CBS inhibitors in vitro.
plying that the probes are suitable for H2S detection in living
cells at this concentration. Herein we employed probe 2 for
imaging of Cys-induced H2S in vivo (Figure 6 and Figures S8–
S10). When the cells were first incubated with 2 and then
treated with Na2S (200 mm) for 30 min, a significant fluores-
cence increase was observed in the cells, further indicating
that 2 is cell-permeable and can be used for imaging intracel-
lular H2S in living cells. Next, cells were treated with l-Cys fol-
lowed by incubation with 2, and a strong fluorescence was ob-
served in the Cys-stimulated HEK293 cells (Figure 6a). As our
study revealed sparfloxacin to be a potent CBS inhibitor, bio-
imaging of its inhibitory effect was further tested in l-Cys-
stimulated HEK293 cells, considering that H2S can be produced
from l-Cys in HEK293 cells via the CBS pathway.[3b,22] The re-
sults showed the suppression of l-Cys-induced H2S production
by sparfloxacin (Figure 6a, 6b), implying that sparfloxacin
could inhibit endogenous H2S biosynthesis.
Finally, we examined the suitability of probe 2 for visualizing
endogenous H2S in living zebrafish (Figure 6c). Three-day-old
zebrafish showed no fluorescence. However, when the zebra-
fish was incubated with probe 2 (1 mm), a moderate fluores-
cence in the fish was observed, implying the existence of en-
dogenous H2S in the zebrafish. Moreover, exposure of the fish
to d-Cys (100 mm) and probe 2 (1 mm) led to a significant fluo-
rescence increase, implying that probe 2 could be used for
tracing the d-Cys-induced endogenous H2S in zebrafish.
The cytotoxicity of both probes 1 and 2 was evaluated by
MTT assay using HEK293 cells (Figure S7).[11i] Both probes did
not show any obvious cytotoxicity at 2 mm concentration, im-
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In summary, we have obtained the first set of dual-reactable
fluorescent H2S probes, which showed a higher selectivity than
that of a dual-quenching fluorescent H2S probe,[17] and single-
reactable H2S probes including thiolysis of the NBD amine or
reduction of the aromatic azide.[14] The detection limit for the
dual-reactable probes 1 and 2 was below 30 nm, implying that
both probes are also highly sensitive. Furthermore, probe 2
could be successfully used to develop a high-throughput enzy-
matic assay and to image Cys-induced endogenous H2S in
vivo, indicating that the dual-reactable probe could be a prom-
ising tool for applications in H2S biology. Our results further il-
lustrate that using such dual-reactable (including multi-reacta-
ble) strategy is likely to be general for preparation of highly se-
lective and highly sensitive H2S probes for various biological
applications.
Acknowledgements
This work was supported by the MOST (2010CB126102), NSFC
(21332004, 21402007, 81272356), the MOE Science and Tech-
nology Development Center(20130031120040), Tianjin Munici-
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Keywords: dual-quenching
imaging · hydrogen sulfide · imaging agents
· dual-reactable · fluorescent
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Manuscript received: February 29, 2016
Accepted Article published: March 9, 2016
Final Article published: April 1, 2016
Chem. Asian J. 2016, 11, 1376 – 1381
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1381
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim