A FRET-ICT dual-quenching fluorescent probe with large off-on response for H2S: Synthesis, spectra and bioimaging

Article abstract of DOI:10.1039/c5cc01184a

A FRET-ICT dual-quenching probe with large off-on fluorescent response upon H₂S treatment is reported. The probe can be used for bioimaging of endogenous H₂S in living cells. This journal is

Full text of DOI:10.1039/c5cc01184a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Zhang, L. Wei,
C. Wei, J. Zhang, R. Wang, Z. Xi and L. Yi, Chem. Commun., 2015, DOI: 10.1039/C5CC01184A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

View Article Online
DOI: 10.1039/C5CC01184A
ChemComm
Page 1 ofJ5ournal Name
Dynamic Article Links ►
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
A FRET-ICT dual-quenching fluorescent probe with large off-on
response for H2S: synthesis, spectra and bioimaging
Changyu Zhang,‡a Lv Wei,‡b,c Chao Wei,b,c Jie Zhang,a Runyu Wang,b,c Zhen Xi*,b,c and Long Yi*,a,c
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
respectively. As a result, the fluorescence should be heavily
50 quenched through FRET-ICT dual-quenching effects.12
Consequently, the fluorescent turn-on fold of the probe after H2S
treatment can be significantly improved. To this end, we rational
designed and synthesized a coumarin-based fluorescent H2S
probe 1 was with significant off-on response by using FRET-ICT
55 dual-quenching effects (Scheme 1). The probe 1 is highly
sensitive and selective towards H2S in buffer, which can be used
for bioimaging of endogenous H2S in living cells.
A
FRET-ICT dual-quenching probe with large off-on
fluorescent response upon H2S treatment was reported. The
probe can be used for bioimaging of endogenous H2S in living
cells.
10 Hydrogen sulfide (H2S) is an important endogenous signalling
molecule with multi-functions.1-3 In vivo, H2S can be enzymatic
generated in many organs (heart, liver, kidney, brain, ileum,
uterus, etc.) and tissues (connective tissues, adipose tissues, etc.).2
Studies have shown that the endogenous concentration of H2S is
15 correlated with numerous diseases, including the symptoms of
Alzheimer’s disease, Down syndrome, diabetes and liver
cirrhosis.4 Despite H2S has been recognized to be linked to
numerous physiological and pathological processes, many of its
underlying molecular events remain unknown. Therefore, it
20 presents significant research value to develop efficient methods
for detection of H2S in living biological systems.
Traditionally, the major methods for H2S detection are
colorimetry, electrochemical assay, gas chromatography and
sulfide precipitation.5 However, these methods are destructive
25 and require tedious preparation sequence. Recent work indicated
that fluorescence-based methods are highly desirable and
sensitive for in-situ and real-time visualization of H2S in living
biological systems.6-11 These probes are majorly based on the
specific H2S-induced reactions, including reduction-based
30 probes,6-8 metal sulfide precipitation-based probes,9 and
nucleophilic-based probes.10 Though the great success of these
fluorescent H2S probes,6-11 we still need to develop advance
probes with highly sensitivity, highly selectivity and fast-
response properties for real-time detection of low concentration
35 H2S in biological samples. Recently, we reported a new
fluorescent thiol probe with more than 400-fold turn-on response
based a dual-quenching strategy.12 In this work, we intend to
develop highly sensitive fluorescent H2S probes based on the
dual-quenching strategy for the first time.
60
Scheme 1. Chemical structures of fluorescent probes 1-3 and their
reaction with H2S.
1 and 2 were synthesized by conveniently coupling reactions
65 and were well characterized by H NMR, 13C NMR and HR-MS
1
(see ESI). As shown in Scheme 1, the coumarin fluorophore in 1
was dual-quenched by the FRET-ICT effects, while fluorophores
in both 2 and 3 are quenched by single effect of ICT or FRET,
respectively. We examined the emission spectra of probes 1-3 in
70 PBS buffer (10 mM, pH 7.4). As shown in Fig. 1A, 1 showed
significantly weak background fluorescence in PBS buffer, while
2 and 3 showed relative strong fluorescence under similar test
condition. These results are consistent with our design that the
40
We discovered a FRET-based fluorescent H2S probe based on
selective thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amine
by H2S to give a turn-on response.13 Lin et al. reported a
fluorescent probe for H2S based on thiolysis of DNP (dinitro-
phenyl) ether.10c To obtain highly sensitive fluorescent probes for
coumarin fluorescence in
1 is heavily quenched through
45 H2S, we intend to install both NBD amine and DNP ether as
quenching groups into one fluorophore. DNP and NBD can
quench the fluorescence via intramolecular charge transfer
(ICT)10k and fluorescence resonance energy transfer (FRET),13
75 combined usage of FRET-ICT dual-quenching effects.
After reacting with 100 µM H2S at room temperature overnight,
a significant fluorescent off-on response was observed for 1 (Fig.
1B, Fig. S1), and the increase of maximal fluorescent intensity at
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

View Article Online
DOI: 10.1039/C5CC01184A
ChemComm
Page 2 of 5
455 nm is more than 2000-fold for 1. To our knowledge, this 45 H2S from 0 to 100 µM (Fig. 3B). Specifically, the addition of
large off-on response is the biggest among H2S probes in the
literature.6-11 The off-on response of 2 and 3 upon H2S treatment
(Fig. S2) was much smaller than that of 1 under similar condition.
Therefore, we can draw the conclusion that the fluorescence turn-
on fold upon reacting with H2S for 1 is indeed greatly increased
via dual-quenching effect, which agrees well with our initial
hypothesis.
even 2 µM H2S into 1 could lead to more than 10-fold fluorescent
increase. The detection limit was determined to be 0.28 µM (Fig.
S5). These results clearly indicated that 1 has shown excellent
sensitivity due to installation of the dual-quenching groups.
5
50
Fig. 3 Fluorescence response of 1 with different concentrations of H2S
(A, inset) in PBS buffer and linear relationship (r = 0.9966) between
fluorescence intensity and H2S concentration (B).
10
15
55
In order to evaluate the specific nature of 1 for H2S, it was
incubated with various biological-related species in PBS buffer
and the fluorescent increase at 455 nm was measured accordingly
(Fig. 4). Among all the tested molecules, only biothiols showed
noticeable fluorescence response. The fluorescence increase of
60 biothiols (Cys and GSH), however, is limited and far below that
of H2S. As evidenced by these results, probe 1 is selective to H2S
for further biological applications. In addition, we also
investigated the fluorescence response of probe 1 to H2S under
different pH values (Fig. S5). Results indicated that the probe can
65 function over a wide range of pH from 6.0 to 8.5.
Fig. 1 (A) The fluorescence spectra of probes 1-3 (1 µM) upon excitation
at 405 nm in PBS buffer. Slits: 10/5 nm. (B) Fluorescence response of 1
(1 µM) toward H2S (100 µM) overnight. Slits: 2.5/5 nm.
We further examined the absorption spectra of probes in PBS
buffer (Fig. S3). After reacting with H2S, 1 and 2 displayed a
decrease absorbance at 350 nm and an increase absorbance at 405
nm, respectively. H2S-mediated cleavage of the electron-
20 withdrawing DNP group releases oxygen donor at pH 7.4,
increasing the push-pull character of the dye and resulting in
large bathochromic shifts in the absorption and the ICT effect.
The decrease absorbance at 510 nm (Fig. S3A) is due to the
cleavage of NBD moiety from coumarin fluorophore.13
25
Encouraged by these preliminary results, we moved forward to
study the reaction kinetics of the probes 1 and 2. Both probes
exhibited relative fast fluorescent increase upon H2S treatment
and showed comparable reaction rate (Fig. S4). The probe 3
showed faster reaction rate toward H2S than that of 1 and 2,13 and
30 the reaction kinetics of the dual-quenching probe 1 was mainly
decided by the property of the slow reaction site DNP. A control
experiment with only 1 or 2 was incubated in PBS buffer for the
stability test. The results indicated that 2 was prone to slow
hydrolysis at pH 7.4 (Fig. 2), while the fluorescence increase due
35 to hydrolysis in 1 can be almost negligible, further indicating the
advance of the dual-quenching probe 1.
Fig. 4 Fluorescent intensity at 455 nm for 1 (1 µM) toward different
70 species (1 mM) in PBS buffer. All reactions were performed in PBS (pH
7.4) at 37 ºC for 30 min.
To demonstrate the biological applicability of probe 1, we
examined whether 1 can be used to detect intracellular H2S in
75 living cells. HEK293A and HeLa cells were treated with probe 1
(2 µM) for 30 min and then with different concentrations of Na2S
(50 and 200 µM) for another 30 min. Bright-field microscopy
images show that the two cell types both retained good
morphology after incubation with 1. The fluorescent microscopy
80 results (Fig. S6, S7) clearly indicated that probe 1 can be used for
imaging exogenous H2S in living cells in a concentration-
dependent fashion. The cytotoxicity of the probe 1 was further
evaluated using HEK293 by MTT assay (Fig. S9). The probe 1
did not show significant cytotoxicity at 0.5-2 µM range, implying
Fig. 2 Time-course experiments of 1 µM probes 1 and 2 in PBS buffer.
40
To gain detailed information about the sensitivity of 1, the
fluorescence intensity change was closely monitored by addition
of various concentrations of H2S into the probe (Fig. 3). The
fluorescence intensity was linearly related to the concentration of 85 that the probe is suitable for bioimaging of H2S in living cells. To
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

View Article Online
DOI: 10.1039/C5CC01184A
Page 3 of 5
ChemComm
c Collaborative Innovation Center of Chemical Science and Engineering,
Nankai University, Tianjin, 300071, China.
50 † Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
test whether 1 could detect endogenous production of H2S, cells
were treated with Cys and then with 1. As shown in Fig. 5,
HEK293A cells with 1 displayed a fairly weak fluorescence
under confocal fluorescence microscope (Fig. 5A). After using
Cys to induce endogenous H2S production,10f significantly
fluorescent enhancement can be observed (Fig. 5B), implying that
1 can be used for bioimaging of endogenous H2S in HEK293A
cells. Above results suggest that 1 is cell-permeable and can react
with intracellular H2S efficiently.
5
‡ The authors pay equal contributions to this work.
55 1
a) C. Szabó, Nat. Rev. Drug Discovery, 2007, 6, 917-935; b) L. Li, P.
Rose, P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51, 169-
187.
2
a) B. Predmore, D. Lefer, G. Gojon, Antioxid. Redox Signaling, 2012,
17, 119-140; b) M. Whiteman, P. K. Moore, J. Cell. Mol. Med., 2009,
13, 488-507; c) H. Kimura, Exp. Physiol., 2011, 96, 833-835.
a) G. D. Yang, L. Y. Wu, B. Jiang, W. Yang, J. S. Qi, K. Cao, Q. H.
Meng, A. K. Mustafa, W. T. Mu, S. M. Zhang, S. H. Snyder, R.
Wang, Science, 2008, 322, 587-590; b) L. F. Hu, M. Lu, Z. Y. Wu, P.
T. Wong, J. S. Bian, Mol. Pharmacol., 2009, 75, 27-34; c) K. H.
Kulkarni, E. M. Monjok, R. Zeyssig, G. Kouamou, O. N. Bongmba,
C. A. Opere, Y. F. Njie, S. E. Ohia, Neurochem. Res., 2009, 34, 400-
406; d) L. Li, M. Bhatia, P. K. Moore, Curr. Opin. Pharmacol., 2006,
6, 125-129; (e) H. Kimura, Amino Acids, 2011, 41, 113-121.
S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G.
Rizzo, E. Distrutti, V. Shah, A. Morelli, Hepatology, 2005, 42, 539-
548.
10
60
3
65
4
70
5
a) D. Jiménez, R. Martinez-Manez, F. Sancenon, J. V. Ros-Lis, A.
Benito, J. Soto, J. Am. Chem. Soc., 2003, 125, 9000-9001; b) D. G.
Searcy, M. A. Peterson, Anal. Biochem., 2004, 324, 269-275; c) N. S.
Lawrence, J. Davis, L. Jiang, T. G. J. Jones, S. N. Davies, R. G.
Compton, Electroanalysis, 2000, 18, 1453-1460; d) J. Radford-
Knoery, G. A. Cutter, Anal. Chem., 1993, 65, 976-982.
75
Fig. 5 Confocal microscopy images of intracellular H2S detection in
living HEK293A cells using 1. HEK293 cells were incubated with (A) 1
6
a) A. R. Lippert, E. J. New, C. J. Chang, J. Am. Chem. Soc., 2011, 133,
10078-10080; b) H. J. Peng, Y. F. Cheng, C. F. Dai, A. L. King, B. L.
Predmore, D. J. Lefer, B. H. Wang, Angew. Chem. Int. Ed., 2011, 50,
9672-9657; c) L. A. Montoya, M. D. Pluth, Chem. Commun., 2012,
48, 4767-4769; d) S. Chen, Z. J. Chen, W. Ren, H. W. Ai, J. Am.
Chem. Soc., 2012, 134, 9589-9592; e) S. K. Das, C. S. Lim, S. Y.
Yang, J. H. Han, B. R. Cho, Chem. Commun., 2012, 48, 8395-8397;
f) B. F. Chen, W. Li, C. Lv, M. M. Zhao, H. W. Jin, H. F. Jin, J. B.
Du, L. R. Zhang, X. J. Tang, Analyst, 2013, 138, 946-951; g) W. Sun,
J. L. Fan, C. Hu, J. F. Cao, H. Zhang, X. Q. Xiong, J. Y. Wang, S.
Cui, S. G. Sun, X. J. Peng, Chem. Commun., 2013, 49, 3890-3892; h)
V. S. Lin, A. R. Lippert, C. J. Chang, Proc. Natl. Acad. Sci. U.S.A.,
2013, 110, 7131-7135; i) G. D. Zhou, H. L. Wang, H. L. Ma, X. Q.
Chen, Tetrahedron, 2013, 69, 867-870.
15 (2 µM) for 30 min; (B) L-cysteine (200 uM) for 30 min and then 1 (2 µM)
for 30 min. Emission was collected at blue channel (425-475 nm) with
405 nm excitation. Scale bar, 50 µm.
80
In summary, we report the first fluorescent H2S probe based on
20 FRET-ICT dual-quenching effects. Preliminary tests indicated
that probe 1 has potential to image endogenous H2S in living cells.
The fluorescent enhancement of the probe upon H2S treatment
can reach more than 2000 folds, which is the biggest among
probes in the literature. Compared with other DNP-based H2S
25 probes,10 the probe 1 has large off-on response for H2S and better
stability toward hydrolysis. However, the FRET-based probes by
Yuan10n and our group11a exhibited ratiometric behaviors for H2S,
which are more accurate in bioimaging than that of the turn-on
probe. The development of fast-response and ratiometric H2S
30 probes based on the dual-quenching strategy is certainly deserved
to perform. We believe that this design strategy could be
employed as a general method for preparation of other highly
sensitive and selective H2S probes in future.
85
90
7
For recent reviews: a) V. S. Lin, W. Chen, M. Xian, C. J. Chang,
Chem. Soc. Rev., 2015, DOI: 10.1039/C4CS00298A; b) F. Yu, X.
Han, L. Chen, Chem. Commun., 2014, 50, 12234-12249.
95 8 a) F. B. Yu, P. Li, P. Song, B. S. Wang, J. Z. Zhao, K. L. Han, Chem.
Commun., 2012, 48, 2852-2854; b) Q. Q. Wan, Y. C. Song, Z. Li, X.
H. Gao, H. M. Ma, Chem. Commun., 2013, 49, 502-504; c) S. K. Bae,
C. H. Heo, D. J. Choi, D. Sen, E. H. Joe, B. R. Cho, H. M. Kim, J.
Am. Chem. Soc., 2013, 135, 9915-9923; d) L. Zhang, S. Li, M. Hong,
35 This work was supported by the MOST (2010CB126102), NSFC
(21332004, 21402007), the Fundamental Research Funds for the
Central Universities (YS1401). We are very thankful for the
comments and suggestions by anonymous reviewers.
100
Y. Xu, S. Wang, Y. Liu, Y. Qian, J. Zhao, Org. Biomol. Chem., 2014,
12, 5115-5125; e) B. Chen, P. Wang, Q. Jin, X. Tang, Org. Biomol.
Chem., 2014, 12, 5629-5633.
a) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T.
Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, J. Am. Chem.
Soc., 2011, 133, 18003-18005; b) J. L. Long, L. P. Wang, D. Xie, Y.
W. Zhan, J. Luminescence, 2013, 139, 40-46.
9
105
110
115
40 Notes and references
10 a) C. R. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R.
Whorton, M. Xian, Angew. Chem. Int. Ed., 2011, 50, 10327-10329;
b) J. Liu, Y. Q. Sun, J. Y. Zhang, T. Yang, J. B. Cao, L. S. Zhang, W.
Guo, Chem. Eur. J., 2013, 19, 4717-4722; c) X. W. Cao, W. Y. Lin,
K. B. Zheng, L. W. He, Chem. Commun., 2012, 48, 10529-10531; d)
Y. C. Chen, C. C. Zhu, Z. H. Yang, J. J. Chen, Y. F. He, Y. Jiao, W.
J. He, L. Qiu, J. J. Cen, Z. J. Guo, Angew. Chem. Int. Ed., 2013, 52,
1688-1691; e) Z. Xu, L. Xu, J. Zhou, Y. F. Xu, W. P. Zhu, X. H.
Qian, Chem. Commun., 2012, 48, 10871-10873; f) Y. Qian, J.
a Beijing Key Laboratory of Bioprocess and College of Chemical
Engineering, Beijing University of Chemical Technology, Beijing 100029,
China. E-mail: yilong@mail.buct.edu.cn
b State Key Laboratory of Elemento-Organic Chemistry and Department
45 of Chemical Biology, National Pesticide Engineering Research Center
(Tianjin), Nankai University, Tianjin, 300071, China. Tel: 86 22
23504782; E-mail: zhenxi@nankai.edu.cn
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

View Article Online
DOI: 10.1039/C5CC01184A
ChemComm
Page 4 of 5
Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J. Zhao, C.
He, Nat. Commun., 2011, 2, 495; g) W. Sun, J. L. Fan, C. Hu, J. F.
Cao, S. G. Sun, F. L. Song, J. Y. Wang, X. Peng, J. Org. Lett., 2013,
15, 4022-4025; h) X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv, B.
Tang, Chem. Sci., 2013, 4, 2551-2556; i) Y. Qian, L. Zhang, S. T.
Ding, X. Deng, C. He, X. E. Zheng, H. L. Zhu, J. Zhao, Chem. Sci.,
2012, 3, 2920-2923; j) L. Yuan, Q.-P. Zuo, Sensor Actuat. B-Chem.,
2014, 196, 151-155; k) T. Liu, Z. Xu, D. R. Spring, J. Cui, Org. Lett.,
2013, 15, 2310-2313; l) L. Yuan, Q.-P. Zuo, Chem. Asian J., 2014, 9,
1544-1549; m) Z. Huang, S. Ding, D. Yu, F. Huang, G. Feng, Chem.
Commun., 2014, 50, 9185-9187; n) L. Yuan, F. Jin, Z. Zeng, C. Liu,
S. Luo, J. Wu, Chem. Sci., 2015, DOI: 10.1039/C4SC03883E.
11 a) L. Wei, L. Yi, F. B. Song, C. Wei, B. F. Wang, Z. Xi, Sci. Rep.,
2014, 4, 4521; b) C. Wei, R. Wang, L. Wei, L. Cheng, Z. Li, Z. Xi
and L. Yi, Chem. Asian J., 2014, 9, 3586-3592.
12 H. Zhang, C. Zhang, R. Liu, L. Yi and H. Sun, Chem. Commun.,
2015, 51, 2029-2032.
13 C. Wei, L. Wei, Z. Xi, L. Yi, Tetrahedron Lett., 2013, 54, 6937-6939.
5
15
10
20
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

View Article Online
DOI: 10.1039/C5CC01184A
Page 5 of 5
ChemComm
Coverpicture
A FRET-ICT dual-quenching fluorescent probe with
significant off-on response toward H2S is rational design
and prepared for imaging of H2S in living cells.
5
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5