A new fluorescent probe for gasotransmitter H2S: High sensitivity, excellent selectivity, and a significant fluorescence off-on response

Article abstract of DOI:10.1039/c3cc49605h

A fluorescent off-on probe for H₂S was exploited by coupling the azide-based strategy with the excited-state intramolecular proton transfer (ESIPT) sensing mechanism, which exhibits a considerably high fluorescence enhancement (1150-fold), an e

Full text of DOI:10.1039/c3cc49605h

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A new fluorescent probe for gasotransmitter H2S:
high sensitivity, excellent selectivity, and a
significant fluorescence off–on response†
Cite this: Chem. Commun., 2014,
50, 4214
Received 19th December 2013,
Accepted 5th March 2014
Jingyu Zhang and Wei Guo*
DOI: 10.1039/c3cc49605h
www.rsc.org/chemcomm
A fluorescent off–on probe for H2S was exploited by coupling the probe. However, some of the reported H2S probes more or less fail to
azide-based strategy with the excited-state intramolecular proton meet this requirement. Secondly, considering real-time imaging of
transfer (ESIPT) sensing mechanism, which exhibits a considerably high H2S-related biological processes, the designed probe should respond
fluorescence enhancement (1150-fold), an extremely low detection fast with H2S under mild conditions. However, most of the reported
limit (0.78 nM), and a relatively fast response time (3–10 min) as well H2S probes display a delayed response time (more than 20 min).
as excellent selectivity.
Thirdly, because the biologically relevant levels of H2S vary from
nanomolar to micromolar levels,10 it is also important to develop a
sensitive fluorescent probe that could exhibit an obvious signal
change to low concentration of H2S to facilitate in-depth biological
research. Indeed, few fluorescence probes could detect H2S at a
nanomolar range so far.5f,6k Furthermore, for the improved spatio-
temporal resolution, the low background fluorescence of the probe
itself and the high luminescent efficiency upon H2S treatment are
highly desirable. Thus, further efforts to address these concerns are
highly required.
Herein, we present a simple and new fluorescent probe 1 for the
detection of H2S based on the strategy of reduction of the azido
group by H2S6 as well as the resulting ESIPT modulated fluores-
cence off–on response11 (Fig. 1). The probe exhibits considerably
high fluorescence enhancement, extremely low detection limit, and
relatively fast response time as well as excellent selectivity toward
H2S, and thus holds great potential for detecting or imaging this
important gasotransmitter in biological systems.
Hydrogen sulfide (H2S) can be endogenously produced by enzymes
such as cystathionine b-synthase, cystathionine g-lyase, and 3-mercapto-
pyruvate sulfurtransferase,1 and it plays important roles in several
pathophysiological processes, including vasodilation, angiogenesis,
regulation of cell growth, mediation of neurotransmission, inhibition
of insulin signaling, and regulation of inflammation.2 Also, studies
have shown that its deregulation is correlated with the symptoms of
Alzherimer’s disease, Down’s syndrome, diabetes, and liver cirrhosis.3
H2S has been regarded as the third gasotransmitter besides nitric oxide
(NO) and carbon monoxide (CO).4 Therefore, an efficient method for
sensitively and selectively probing H2S in biological systems is highly
required.
Among various methods, fluorescence-based assays have found
widespread application especially in biological systems due to the
high sensitivity, nondestructive detection, and high spatiotemporal
resolution. For fluorescent H2S probes, the design strategies are
commonly based on several significant characteristic properties of
H2S, namely dual nucleophilicity,5 good reducing property towards
azide,6 high binding affinity towards copper ions,7 efficient thiolysis
of dinitrophenyl ether8 as well as specific addition reaction towards
unsaturated double bonds.9 Although some advances have been
made in the field, there still exist some issues of interest and
concern. One is to attain sufficient selectivity over biologically related
species, especially biothiols such as glutathione (GSH) that is present
at levels of about 1–10 mM in cells. That is to say, these biothiols
should neither induce the fluorescent change, nor consume the
Probe 1 could be easily prepared from 2-(2-aminophenyl)benzo-
thiazole (ABT) through the Sandmeyer reaction, and its structure
1
was confirmed by H NMR, 13C NMR, and HRMS spectra (ESI†).
The probe was found to be soluble (up to 12 mM) in PBS buffer
(Fig. S1, ESI†). Thus, we first examined the reactivity of 1 (10 mM)
towards different concentrations of NaHS (a commonly employed
H2S donor) through time-dependent absorption spectroscopy in
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
China. E-mail: guow@sxu.edu.cn
† Electronic supplementary information (ESI) available: Experimental proce-
dures, supplemental spectra, and the 1H-, 13C-NMR, and MS spectrum. See
DOI: 10.1039/c3cc49605h
Fig. 1 The proposed sensing mechanism of probe 1 for H2S.
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Fig. 2 (A) Time-dependent absorption spectra of
1 (10 mM) in the
presence of 100 mM NaHS. (B) Time-dependent fluorescence intensity
changes of 1 (10 mM) at 450 nm upon addition of varied concentrations of
NaHS. Conditions: PBS buffer (10 mM, pH 7.4, 1 mM CTAB) at 25 1C.
PBS buffer [10 mM, pH 7.4, containing 1 mM CTAB12 (cetyltri-
methylammonium bromide)] at 25 1C (Fig. 2A). The free 1 showed a
main absorption at 310 nm. Upon addition of NaHS (100 mM), the
absorption of 1 at 310 nm decreased gradually within 3 min, along
with the simultaneous emergence of a new absorption at 375 nm.
In this process, a well-defined isobestic point was noted at 345 nm,
suggesting a clean chemical transformation. Based on the well-
established reduction mechanism,6 the new absorption at 375 nm
could be assigned to product ABT, which was also supported by
HRMS experiment (Fig. S2, ESI†). Furthermore, we performed the
time-dependent fluorescent spectra studies (Fig. 2B). As shown in
Fig. 2B, although 100 mM NaHS could cause the reaction to be
completed within 3 min, the low concentrations of NaHS needed a
longer reaction time of 10 min to reach the spectra saturation.
Thus, in the subsequent experiments, a time point of 10 min after
addition of NaHS was selected.
Fig. 3 Fluorescence spectra of 1 (10 mM) upon addition of HSÀ (0–100 mM
for A and 0–1 mM for C) in PBS buffer (10 mM, pH 7.4, 1 mM CTAB) and the
corresponding linear relationship between the fluorescent intensity and
HSÀ concentration (B and D). Spectra were recorded after incubation with
different concentrations of HSÀ for 10 min at 25 1C. lex = 375 nm, lem
=
450 nm. Slits: 10/10 nm.
Subsequently, we performed the fluorescence titration studies of
1 towards H2S under the same conditions. A series of spectra of the
solution of 1 with 0 to 100 mM NaSH were recorded (Fig. 3A). The
free probe 1 is almost nonfluorescent in the visible region (fluores- Fig. 4 Fluorescence intensities of 1 (10 mM) upon addition of various species in
PBS buffer (10 mM, pH 7.4, 1 mM CTAB) after 10 min at 25 1C. (1–6) K+, Na+,
cence quantum yield: F = 0.0064). Upon treatment with increasing
Ca2+, Mg2+, Zn2+, and Cu2+ (1 mM for each); (7–16) FÀ, ClÀ, BrÀ, IÀ, SO4
,
2À
concentrations of NaSH, the fluorescence intensity of 1 at 450 nm
gradually increased, and reached saturation when the amount of
HSÀ was more than 30 mM. In this case, a ca. 1150-fold fluorescence
CO32À, NO3À, AcOÀ, H2PO4À, and CNÀ (0.25 mM for each); (17) Vc-Na
2À
(0.1 mM); (18–22) SO32À, HSO3À, S2O32À, S2O42À, and S2O5 (0.1 mM for
each); (23–28) H2O2, ClOÀ, O2 , NO, ꢀOH, and 1O2 (0.1 mM for each); (29–32)
À
enhancement was observed (F = 0.4138 with quinine sulfate as a Cys (0.5 mM), Hcy (0.5 mM), GSH (1 mM), and HSÀ (0.1 mM). lex = 375 nm, lem
=
450 nm. Slits: 10/10 nm.
reference), which is in fact bigger than most of those reported in
the literature, indicative of a high signal-to-background ratio.
Moreover, a linear relationship with the HSÀ concentration from response to both biothiols (0.5 mM Cys and Hcy; 1 mM GSH, the
0 to 10 mM could be obtained (Fig. 3B). It is worth noting that the main competitive species in biological systems) as well as to reducing
probe is highly sensitive to low concentration of HSÀ (Fig. 3C conditions (0.1 mM) sodium ascorbate (Vc-Na). By contrast, only HSÀ
and D), and the detection limit for HSÀ was estimated to be elicited a dramatic increase in the fluorescence intensity of 1,
0.78 nM based on S/N = 3, which, as far as we know, is the most suggesting the high selectivity of 1 towards H2S.13 We also performed
sensitive fluorescent probe for HSÀ to date.
the competition experiments in the presence of biothiols as well as
To evaluate the specific nature of 1 for H2S, we then examined the Vc-Na. In fact, when H2S and these species coexisted, we also
fluorescence enhancement of 1 incubated with various species observed almost the same fluorescence enhancement as that only
(Fig. 4), most of which are biologically related. As expected, probe treated by H2S (Fig. 5A). Moreover, the H2S-induced fluorescence
1 is considerably inert to the common anions and cations, such as enhancement in the presence of biothiols or sodium ascorbate could
FÀ, ClÀ, BrÀ, IÀ, SO42À, CO32À, NO3À, AcOÀ, H2PO4À, CNÀ (0.25 mM be clearly observed by the naked eyes (Fig. 5B).
for each); K+, Na+, Ca2+, Mg2+, Zn2+, and Cu2+ (1 mM for each).
Next, the effect of pH on the fluorescence response of probe
Moreover, probe 1 did not exhibit any fluorescence enhancement in 1 to H2S was investigated (Fig. S3, ESI†). It was found that probe
response to reactive oxygen/sulfur/nitrogen species (ROS/RSS/RNS), 1 is stable over a wide pH range of 2–12, and displays the best
such as H2O2, ClOÀ, ꢀOH, O2À, 1O2, SO32À, HSO3À, S2O32À, S2O4
,
response for H2S in the region of 7–12. Thus, probe 1 could
2À
S2O52À, and NO (0.1 mM for each). Importantly, probe 1 showed no function properly at physiological pH.
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with the excited-state intramolecular proton transfer (ESIPT)
sensing mechanism. The probe can detect H2S with high
selectivity even in the presence of millimolar concentrations
of biothiols with a significant fluorescence of f–on response and
an extremely low detection limit. Preliminary fluorescence
imaging experiments in cells indicate its potential to probe
H2S chemistry in biological systems.
Fig. 5 (A) Fluorescence response of 1 (10 mM) to HSÀ (0.1 mM), Cys
(0.5 mM), Hcy (0.5 mM), GSH (1 mM), and Vc-Na (0.1 mM) in PBS buffer
(10 mM, pH 7.4, 1 mM CTAB) after 10 min at 25 1C. Black bar: 1 + biothiols
Notes and references
1 H. Kimura, Amino Acids, 2011, 41, 113.
2 (a) K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066; (b) O. Kabil
and R. Banerjee, J. Biol. Chem., 2010, 285, 21903.
3 (a) K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura, Biochem.
Biophys. Res. Commun., 2002, 293, 1485; (b) P. Kamoun, M.-C.
Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-Vekemans,
Am. J. Med. Genet., Part A, 2003, 116, 310; (c) S. Fiorucci, E. Antonelli,
A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah
and A. Morelli, Hepatology, 2005, 42, 539.
(or Vc-Na); Blue bar: 1 + biothiols (or Vc-Na) + HSÀ. lex = 375 nm, lem
=
450 nm. Slits: 10/10 nm. (B) The corresponding fluorescent images: (1) 1
only; (2) 1 + HSÀ; (3) 1 + Cys; (4) 1 + Cys + HSÀ; (5) 1 + Hcy; (6) 1 + Hcy +
HSÀ; (7) 1 + GSH; (8) 1 + GSH + HSÀ; (9) 1 + Vc-Na; (10) 1 + Vc-Na + HSÀ.
Encouraged by these results, we tested the capability of 1 to image
H2S in biological systems. Melanoma B16 cells incubated with 1 in
culture medium for 30 min at 37 1C showed a weak fluorescence
(Fig. 6A) due to the low H2S level in the cells. However, strong
fluorescence in the cells was observed after the cells were pre-
incubated with NaHS and further incubated with 1 (Fig. 6B), suggest-
ing that probe 1 could detect external H2S supplemented to the cell
cultures. It is well established that H2S could be biosynthesized from
Cys by the enzymes CBS and CSE.14 Also, it was reported that
melanoma cell lines express CSE.15 Thus, Cys could be regarded as
a precursor to H2S in the cell imaging assays.5c,6c,j In fact, when B16
cells were incubated with Cys for 30 min and then incubated with 1
for 30 min, we also observed the obvious fluorescence (Fig. 6C). As a
control,6c when the cells were pre-incubated in a sequence with Cys
and phorbol myristate acetate (PMA) that could decrease the H2S
level presumably by generating ROS,16 and further incubated with 1,
almost no fluorescence was observed (Fig. 6D).
4 (a) E. Lowicka and J. Beltowski, Pharmacol. Rep., 2007, 59, 4; (b) L. Li,
P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51, 169.
5 (a) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman,
A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011,
50, 10327; (b) C. Liu, B. Peng, S. Li, C.-M. Park, A. R. Whorton and
M. Xian, Org. Lett., 2012, 14, 2184; (c) Y. Qian, J. Karpus, O. Kabil,
S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao and C. He, Nat.
Commun., 2011, 2, 495; (d) Y. Qian, L. Zhang, S. Ding, X. Deng,
C. He, H.-L. Zhu and J. Zhao, Chem. Sci., 2012, 3, 2920; (e) W. Xuan,
C. Sheng, Y. Cao, W. He and W. Wang, Angew. Chem., Int. Ed., 2012,
51, 2282; ( f ) X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang,
Chem. Sci., 2013, 4, 2551; (g) Z. Xu, L. Xu, J. Zhou, Y. Xu, W. Zhu and
X. Qian, Chem. Commun., 2012, 48, 10871; (h) X. Li, S. Zhang, J. Cao,
N. Xie, T. Liu, B. Yang, Q. He and Y. Hu, Chem. Commun., 2013,
49, 8656.
6 (a) A. R. Lippert, R. J. New and C. J. Chang, J. Am. Chem. Soc., 2011,
133, 10078; (b) H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore,
D. J. Lefer and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672;
(c) S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho, Chem.
Commun., 2012, 48, 8395; (d) S. Chen, Z. Chen, W. Ren and H. Ai,
J. Am. Chem. Soc., 2012, 134, 9589; (e) Z. Wu, Z. Li, L. Yang, J. Han
and S. Han, Chem. Commun., 2012, 48, 10120; ( f ) L. A. Montoya and
M. D. Pluth, Chem. Commun., 2012, 48, 4767; (g) F. Yu, P. Li, P. Song,
B. Wang, J. Zhao and K. Han, Chem. Commun., 2012, 48, 2852;
(h) W. Sun, J. Fan, C. Hu, J. Cao, H. Zhang, X. Xiong, J. Wang,
C. Shuang, S. Sun and X. Peng, Chem. Commun., 2013, 49, 3890;
(i) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun., 2013,
49, 502; ( j) S. K. Bae, C. H. Heo, D. J. Choi, D. Swn, E.-H. Joe,
B. R. Cho and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915;
(k) H. Tian, J. Qian, H. Bai, Q. Sun, L. Zhang and W. Zhang, Anal.
Chim. Acta, 2013, 768, 136.
In summary, a reaction-type fluorescent probe 1 for detec-
tion of H2S was exploited by coupling the azide-based strategy
7 (a) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura,
T. Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 18003; (b) F. Hou, L. Huang, P. Xi, J. Cheng,
X. Zhao, G. Xie, Y. Shi, F. Cheng, X. Yao, D. Bai and Z. Zeng, Inorg.
Chem., 2012, 51, 2454; (c) X. Qu, C. Li, H. Chen, J. Mack, Z. Guo and
Z. Shen, Chem. Commun., 2013, 49, 7510.
8 (a) X.-F. Yang, L. Wang, H. Xu and M. Zhao, Anal. Chim. Acta, 2009,
631, 91; (b) X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun.,
2012, 48, 10529; (c) J. Wang, W. Lin and W. Li, Biomaterials, 2013,
34, 7429; (d) T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013,
15, 2310.
9 Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen
and Z. Guo, Angew. Chem., Int. Ed., 2013, 52, 1688.
10 Y. Han, J. Qin, X. Chang, Z. Yang and J. Du, Cell. Mol. Neurobiol.,
2006, 26, 101.
11 (a) J. Zhao, S. Ji, Y. Chen, H. Guo and P. Yang, Phys. Chem. Chem.
Phys., 2012, 14, 8803; (b) J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang,
Chem. Soc. Rev., 2011, 40, 3483.
Fig. 6 Fluorescence images of H2S in B16 cells using 1 (10 mM) at 37 1C.
(A) B16 cells incubated with 1 in the presence of CTAB (1 mM) for 30 min.
(B) B16 cells pre-incubated with NaHS (100 mM, 30 min) and further
incubated with 1 (30 min) in the presence of CTAB (1 mM). (C) B16 cells
pre-incubated with Cys (100 mM, 30 min) and further incubated with 1
(30 min) in the presence of CTAB (1 mM). (D) B16 cells pre-incubated in a
sequence with Cys (100 mM, 30 min) and PMA (1 mg mLÀ1), and further
incubated with 1 (30 min) in the presence of CTAB (1 mM). (A1–D1) The
corresponding bright-field images. Cells shown are representative images
from replicate experiments (n = 3). The mean fluorescence intensities in
(AÀD) are 456.6, 3609.4, 3980.8, and 150.8, respectively. Scale bar: 30 mm.
12 CTAB micelles were reported to be able to accelerate this type of
reaction, see H. Tian, J. Qian, H. Bai, Q. Sun, L. Zhang and
W. Zhang, Anal. Chim. Acta, 2013, 768, 136.
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13 Besides the electronic factor, the steric factor may also be
S. Singh, V. Vitvitsky and R. Banerjee, J. Biol. Chem., 2009,
284, 11601.
important for the high selectivity of 1 toward H2S over Cys or
´
GSH due to a bulky benzothiazole group adjacent to the azide 15 H. Jurkowska, W. Placha, N. Nagahara and M. Wrobel, Amino Acids,
group in 1. 2011, 41, 151.
14 (a) S. Singh, D. Padovani, R. A. Leslie, T. Chiku and R. Banerjee, 16 H. Mitsuhashi, S. Yamashita, H. Ikeuchi, T. Kuroiwa, Y. Kaneko,
J. Biol. Chem., 2009, 284, 22457; (b) T. Chiku, D. Padovani, W. Zhu,
K. Hiromura, K. Ueki and Y. Nojima, Shock, 2005, 24, 529.
This journal is ©The Royal Society of Chemistry 2014
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