A red emission fluorescent probe for hydrogen sulfide and its application in living cells imaging

Article abstract of DOI:10.1016/j.tetlet.2013.03.133

Hydrogen sulfide has emerged as an important biological messenger and much attention has been paid to the design of fluorescent probes for H₂S to meet the requirement of accurate measurement of H₂S. In this work, a new red emission fluorescent probe for H₂S was developed based on the reduction reaction of azide with H₂S to amine with the fluorophore of dicyanomethylenedihydrofuran because of its long excitation and emission wavelength. The probe has a high selectivity for H₂S over competitive anions and sulfide-containing analytes. Finally, the probe was applied to sense H₂S in living cells.

Full text of DOI:10.1016/j.tetlet.2013.03.133

Tetrahedron Letters 54 (2013) 2980–2982
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A red emission fluorescent probe for hydrogen sulfide and its application
in living cells imaging
Tao Chen a,b, , Yi Zheng b,c, , Zhaochao Xu a,b, , Miao Zhao b, Yongnan Xu c, , Jingnan Cui a,
⇑
⇑
⇑
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
b Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
c The School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrogen sulfide has emerged as an important biological messenger and much attention has been paid to
the design of fluorescent probes for H2S to meet the requirement of accurate measurement of H2S. In this
work, a new red emission fluorescent probe for H2S was developed based on the reduction reaction of
azide with H2S to amine with the fluorophore of dicyanomethylenedihydrofuran because of its long exci-
tation and emission wavelength. The probe has a high selectivity for H2S over competitive anions and sul-
fide-containing analytes. Finally, the probe was applied to sense H2S in living cells.
Received 29 January 2013
Revised 21 March 2013
Accepted 28 March 2013
Available online 12 April 2013
Keywords:
Red emission
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Fluorescent probe
Hydrogen sulfide
Live cells’ imaging
Hydrogen sulfide (H2S) has emerged as an important biological
messenger, and much attention has been paid to the design of
fluorescent probes for H2S to meet the requirement of accurate
measurement of H2S, particularly in living systems.1 H2S has
recognized as the third most important gasotransmitter for regu-
lating cardiovascular, neuronal, immune, endocrine, and gastroin-
testinal systems after nitric oxide and carbon monoxide.2 H2S is
nucleophilic substitution reaction between H2S and the disulfide
moiety.11a Qian and co-workers reported a ratiometric fluorescent
probe using a similar method.11b He and co-workers used the
nucleophilic attack of H2S on the aldehyde functionality to design
an elegant fluorescent probe.12 Very recently, Guo and co-workers
reported a new fluorescent probe based on the selective nucleo-
philic addition of H2S to a specific merocyanine derivative.13
Nagano and co-workers14 and Zeng and co-workers15 used the
displacement strategy to design off–on fluorescent probes with
improved selectivity. In addition, the most applied reaction to
sense H2S is the reduction of azide with H2S to amine pioneered
by Chang16 and Wang and co-workers.17 The azide compound
usually displays weak fluorescence. After reduction to the amine
counterpart which fluoresces strongly, an off–on fluorescence
response is then obtained. This approach has been expanded to
design azide-containing fluorescent probes by altering fluoro-
phores to naphthalimide,18a resorufamine,18b NBD,18c BMF,18d
coumarin,18e,f cresyl violet,18g genetically encoded fluorescent
protein,18h pyrene,18i and phenanthroimidazole.18j However, red
emission fluorescent probes using this strategy to sense H2S are
still less common.19 Red emission can offer distinct advantages
for both in vitro and in vivo biological applications. The wave-
length range in the red region can avoid the strong interference
from UV-induced phototoxicity/autofluorescence, and corresponds
to the transparency window of a biological specimen, enabling
optimal penetration of light into various biological media. In this
regard, it is highly desirable to develop red emission fluorescent
probes for H2S to match harmless imaging and visualization of
H2S in living cells.
produced endogenously in mammalian systems from L-cysteine
in reactions catalyzed mainly by two pyridoxal-50-phosphate-
dependent enzymes, cystathionine b-synthase (CBS) and cystathi-
onine
c
-lyase (CSE).3 The endogenous levels of H2S are believed
to be related with some diseases like Alzheimer’s disease,4 Down’s
syndrome,5 diabetes,6 and liver cirrhosis.7 Thus, visualization of
the distribution and concentration of H2S in living systems would
be very important and helpful to elucidate the biological roles of
H2S. Compared with reported methods such as colorimetric,8
electrochemical analysis,9 and gas chromatography,10 small mole-
cule fluorescent probes offer high sensitivity, real-time imaging,
and high spatiotemporal resolution, and have excellent potential
to be useful tools.
The design of fluorescent probes for H2S is mainly based on
specific chemical reactions by taking advantage of the nucleophilic
or reducing properties of H2S.1 Xian and co-workers reported a
fluorescein-derived probe for selective detection of H2S through a
⇑
Corresponding authors. Tel.: +86 41 1 8437 9648; fax: +86 82 2 3277 3419.
E-mail addresses: zcxu@dicp.ac.cn (Z. Xu), ynxucn@yahoo.com.cn (Y. Xu),
jncui@dlut.edu.cn (J. Cui).
These authors contributed equally to this work.
0040-4039/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.03.133

T. Chen et al. / Tetrahedron Letters 54 (2013) 2980–2982
2981
In this Letter, we reported a new red emission fluorescent probe
1 for H2S. Dicyanomethylenedihydrofuran (DCDHF) fluorophore is
selected because of its long excitation and emission wavelength.
The azide-derived DCDHF was expected to be reduced with H2S
to give the amine-derived compound which would highly fluoresce
(Scheme 1). The amine-substituted DCDHF compound DCDHF–
NH2 was prepared independently as a control. The experimental
details of syntheses of 1 and DCDHF–NH2 were given in Supple-
mentary data.
The emission spectra and fluorescence titration experiments
with H2S were recorded in an aqueous solution (CH3CN/
HEPES = 1:2, pH 7.4) firstly (Fig. 1). The free 1 was nonfluorescent.
When H2S was added progressively from 0 to 1 equiv to the
solution of 1 (NaHS was used as a hydrogen sulfide source), a
new emission band centered at 619 nm (UF = 0.018) was observed
after 60 min and increased in intensity significantly (55-fold).
Notably, compound DCDHF–NH2 gave a same emission profile
with a little larger intensity (Fig. S1). Then the emission of 1/H2S
was believed to be attributed to the reduction of 1 to the amine
compound DCDHF–NH2. The MS analysis also confirmed that the
fluorescence emission and enhancement centered at 619 nm was
due to the formation of the amine compound (Fig. S2). In order
to get direct evidence, the product was separated and purified,
and proved to be the compound DCDHF–NH2 characterized with
1H NMR and 13C NMR (see Supplementary data). With the addition
of more than 1 equiv of H2S, the fluorescence intensity of 1/H2S did
not change but decrease to some extent (Fig. S3). Therefore, we
used 1 equiv of H2S to examine the performance of 1 in all exper-
iments. The time-dependent fluorescence responses were next
detected and the results showed that the reaction was complete
within 60 min (Fig. S4).
Figure 1. Fluorescent emission spectra of 100
different concentrations of H2S in aqueous solution (CH3CN/50 mM HEPES = 1:2, pH
7.4). Excitation at 574 nm.
l
M compound 1 in the presence of
The absorption titration of 1 with NaHS was then performed
and also indicated the reduction process of 1 to the amine deriva-
tive (Fig. 2). The absorption spectrum of 1 (100 lM) exhibited kmax
at 440 nm. On addition of 1 equiv of H2S to the solution of 1, the
absorbance at 440 nm decreased sharply to its limiting value,
while an absorption band at 564 nm developed which induced
the color change from yellow to red with an isosbestic point at
506 nm (Fig. 2). This new absorption band is in line with that of
DCDHF–NH2 but with smaller absorption.20 This may ascribe to
the incompletion of the reaction. Even so, compound 1 is still
suitable for the detection of H2S, since the background fluorescence
is so weak (1 is nonfluorescent).
Figure 2. UV–vis absorption spectra of 100 lM DCDHF–NH2 and compound 1 in
the presence of different concentrations of H2S in aqueous solution (CH3CN/
HEPES = 1:2, pH 7.4). Inset: Color change of the solution of 1 with the addition of
H2S.
The fluorescence titration of
1 with various anions was
conducted to examine the selectivity. As shown in Figure 3, the
ꢀ
addition of 10 equiv of Fꢀ, Clꢀ, Brꢀ, Iꢀ, N3ꢀ, NO2ꢀ, HCO3ꢀ, HSO3
,
background fluorescence (Fig. 4a). Then the cells were incubated
with 50 lM NaHS next and after 10 min displayed enhanced red
fluorescence (Fig. 4c). After 60 min incubation with NaHS, a higher
turn-on fluorescence response can be observed (Fig. 4d). The cyto-
toxicity of 1 was examined toward HUVEC cells by a MTT assay.
SO42ꢀ, S2O3ꢀ, S2O4ꢀ, S2O5ꢀ, CNꢀ, HPO4ꢀ, and PO4ꢀ produced a nom-
inal change in the fluorescence spectra of 1. Other tested analytes
including 10 equiv of cysteine and glutathione induced fluores-
cence but with much smaller enhancement (4- and 5-fold,
respectively). More importantly, the presence of cysteine and
glutathione do not interfere with the fluorescence response of 1
for H2S. Then probe 1 has a very high selectivity for H2S.
The results showed an IC50 value of 230
1 was of low toxicity toward cultured cell lines at the concentra-
tion of 50.0 M. These experiments indicate 1 can be used to detect
lM, demonstrating that
l
We then sought to examine whether 1 can sense H2S in living
cells. The Human Umbilical Vein Endothelial Cells (HUVEC) were
H2S in living cells.
In conclusion, we reported a dicyanomethylenedihydrofuran
derived fluorescent probe 1 for H2S based on the reduction of azide
incubated with 50
l
M 1 for 30 min and exhibited almost no
CN
CN
CN
CN
H2S
pH 7.5
NC
NC
O
O
N3
NH2
DCDHF-NH2
1
Scheme 1. The reaction of compound 1 with H2S.

2982
T. Chen et al. / Tetrahedron Letters 54 (2013) 2980–2982
Acknowledgments
We thank financial supports from the National Natural Science
Foundation of China(21276251), Ministry of Human Resources and
Social Security of PRC, the 100 talents program funded by Chinese
Academy of Sciences, and the State Key Laboratory of Fine Chemi-
cals of China (KF1105).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.
03.133.
Figure 3. Fluorescence responses of 100 lM compound 1 to various anions in
aqueous solution (CH3CN:50 mM HEPES = 1:2, pH 7.4). Excitation at 574 nm. Bars
References and notes
represent the final fluorescence intensity of 1 with 1 mM anions over the original
ꢀ
emission of free 1. (1) Free 1; (2) H2S; (3) Fꢀ; (4) Clꢀ; (5) Brꢀ; (6) Iꢀ; (7) N3 (8)
NO2ꢀ; (9) HCO3ꢀ; (10) HSO3ꢀ; (11) SO4ꢀ; (12) S2O3ꢀ; (13) S2O4ꢀ; (14) S2O5ꢀ; (15)
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with H2S to amine. With the addition of H2S to the solution of 1, an
emission centered at 619 nm was observed and increased in inten-
sity significantly. Concomitantly, the solution color changes from
yellow to red with the convenience and esthetic appeal of a color-
imetric assay. The probe has a high selectivity for H2S over compet-
itive anions and sulfide-contining analytes. Finally, the probe was
applied to sense H2S in living cells. In order to shorten the reaction
time, we are currently increasing the water-solubility of the
compound by introducing some functional groups.
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