A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells

Article abstract of DOI:10.1016/j.dyepig.2014.03.039

A 2-formylbenzoate-caged methylfluorescein chemodosimeter (1) was designed and synthesized to detect hydrogen sulfide (H₂S) in aqueous solution and living cells. Chemodosimeter 1 could quantificationally detect H ₂S in a wide concent

Full text of DOI:10.1016/j.dyepig.2014.03.039

Dyes and Pigments xxx (2014) 1e5
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A highly selective fluorescent chemodosimeter for imaging hydrogen
sulfide in living cells
Caiyun Liu , Huifang Wu , Bingjun Han , Baocun Zhu ,*, Xiaoling Zhang
a
,
b
b
b
b
a,
**
a
Key Laboratory of Cluster Science of Ministry of Education, School of Chemistry, Beijing Institute of Technology, Beijing 100081, China
School of Resources and Environment, University of Jinan, Shandong Provincial Engineering Technology Research Center for Ecological Carbon Sink and
b
Capture Utilization, Jinan 250022, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 February 2014
Received in revised form
A 2-formylbenzoate-caged methylfluorescein chemodosimeter (1) was designed and synthesized to
detect hydrogen sulfide (H
cationally detect H S in a wide concentration range (2.5e1000
Chemodosimeter 1 showed excellent selectivity toward H S owing to its inherent two electrophilic re-
action sites and the dual nucleophilicity of H S. Additionally, chemodosimeter 1 exhibited the color
changes from colorless to yellow-green upon addition of H S. Thus, 1 can serve as a “naked-eye” indicator
for H S. Further, the recognition mechanism of chemodosimeter 1 for H S was confirmed using elec-
2
S) in aqueous solution and living cells. Chemodosimeter 1 could quantifi-
2
m
M) by the fluorescence turn-on method.
26 March 2014
2
Accepted 27 March 2014
Available online xxx
2
2
2
2
Keywords:
Fluorescent chemodosimeter
Methylfluorescein
trospray ionization mass spectrometry. Finally, the preliminary biological imaging experiments and low
cytotoxicity investigations demonstrated that our proposed chemodosimeter provided a promising
Hydrogen sulfide (H
-Formylbenzoate
2
S)
method for the determination of H
2
S in aqueous solution and living cells.
Ó 2014 Elsevier Ltd. All rights reserved.
2
Bioimaging
Naked-eye indicator
1. Introduction
2
H S concentrations in biological samples is needed to get useful
information about its function in organisms.
Following nitric oxide (NO) and carbon monoxide (CO),
hydrogen sulfide (H S), with rotten egg smell, is identified as the
third biologically active gas that is termed a gaseous transmitter or
a gaseous mediator [1]. H S is biosynthesized endogenously by
three enzymes: cystathionine- -synthase (CBS), cystathionine-
lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3MST) [2e
]. The production of endogenous H S has been demonstrated to
exert protective effects in pathology. For example, H S has been
In recent years, a few analytical technologies have been devel-
2
oped to detect environmental, medical and cellular H
2
S [14e16].
The major methods for the detection of H S are colorimetric and
2
2
electrochemical assays, gas chromatography, and sulfide precipi-
tation. These methods often require complicated sample process-
ing, and are limited to extracellular detection [17,18]. As such,
fluorescence-based assay could be useful in this field because of
its high sensitivity, selectivity, convenience and real time detection
[19]. Additionally, when coupled with fluorescence microscopy
techniques, small-molecule fluorescent probes offer high spatio-
temporal resolution, and are important tools used to study bio-
b
g-
5
2
2
shown to relax vascular smooth muscle, induce vasodilation of
isolated blood vessels, and reduce blood pressure [5e8]. Addi-
tionally, it has become evident that H
oxidant or a scavenger of reactive oxygen and nitrogen species in
cells [9]. The physiologically relevant concentration of H S is esti-
mated to range from nano- to millimolar levels. Once the cells
cannot maintain the level within the physiological range, H S is
2
S also performs as an anti-
2
logical signaling pathways [19]. A few fluorescent probes for H S
were reported taking using of its chemical properties of metal-
induced sulfide precipitation, the reducibility and the nucleophi-
2
2
licity [18,20,21]. However, most of probes effectively detect H
narrow concentration range. Another key challenge for selective
detection of H S in the cellular milieu is the comparatively high
concentrations of biological sulfur species such as glutathione
2
S in a
engaged in various diseases, such as arterial and pulmonary hy-
pertension, Alzheimer’s diseases, gastric mucosal injury, and liver
cirrhosis [10e13]. Obviously accurate and reliable measurement of
2
(GSH) as well as cysteine residues.
In this paper, we developed a novel highly selective fluorescent
chemodosimeter 1 for the detection of H
range (2.5e1000 M). Chemodosimeter 1 was composed of a latent
methylfluorescein fluorophore and a receptor of 2-formylbenzoate
2
S in a wide concentration
*
Corresponding author. Tel.: þ86 531 82765730; fax: þ86 531 82765969.
m
** Corresponding author. Tel./fax: þ86 10 88875298.
E-mail addresses: lcyzbc@163.com (B. Zhu), zhangxl@bit.edu.cn (X. Zhang).
http://dx.doi.org/10.1016/j.dyepig.2014.03.039
143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Liu C, et al., A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells, Dyes
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2
C. Liu et al. / Dyes and Pigments xxx (2014) 1e5
moiety [14] (Scheme 1), and it became high fluorescence upon the
spirolactone-opening reaction in the presence of H S through a
tandem nucleophilic additionecyclization reaction, accompanying
129.98, 131.58, 135.00, 135.08, 135.28, 144.17, 152.46, 158.06, 161.58,
[M þ H]þ 479.11253,
2
167.96. HRMS (ESI positive) calcd for C29
found 479.11362.
19
H O
7
with the color changes from colorless to yellow-green. Chemo-
dosimeter 1 exhibited excellent selectivity toward H
various species including GSH, which might be ascribed to its two
electrophilic reaction sites and the dual nucleophilicity of H S.
2
S over other
2.3. General procedure for analysis
2
2
Parent stock solutions (10 mM) of Na S and other biospecies
were prepared in ultrapure water. Stock solution of chemo-
2
. Materials and methods
dosimeter 1 (1 mM) was prepared in ethanol. Test solutions were
prepared by placing 25 mL probe stock solution into a test tube,
2
.1. General
adding an appropriate aliquot of each stock solution, and then
diluting the solution to 10 mL with the mixture of ethanol and
ultrapure water (1:1, v/v) containing PBS (5 mM, pH 7.0). All the
spectra were obtained in a quartz cuvette (path length ¼ 1 cm). All
All of analytical reagents in this paper were obtained from
commercial suppliers and used without further purification. Ul-
trapure water was prepared through Sartorious Arium 611DI sys-
tem and used throughout the experiment. Silica gel (200e300
mesh, Qingdao Haiyang Chemical Co.) was used for column chro-
ꢀ
measurements were made at room temperature (25 C).
2.4. Cell culture and fluorescence imaging
1
13
matography. H NMR and C NMR were recorded on a Bruker AV-
00 spectrometer with chemical shifts reported as ppm (in CDCl ),
4
3
HeLa cells (gifted from the center of cells, Peking Union Medical
College) were cultured in culture media (DMEM/F12 supplemented
using tetramethylsilane (TMS) as the internal standard. Electro-
spray ionization (ESI) mass spectra were measured with an LC-MS
2
with a UV-3101PC spectrophotometer. Fluorescence emission
spectra were carried out on a PerkineElmer Model LS-55 spectro-
photometer with excitation wavelength of 450 nm. All the fluo-
rescence spectra were uncorrected. All pH measurements were
made with a Sartorius basic pH-meter PB-10.
ꢁ
1
with 10% FBS, 50 unit/mL penicillin, and 50 mg mL of strepto-
ꢀ
010A (Shimadzu) instrument. Absorption spectra were measured
mycin) at 37 C under a humidified atmosphere containing 5% CO
2
.
4
HeLa cells were seeded in a 96-well plate at a density of 10 cells
per well in culture media. After 24 h, the cells were incubated with
ꢀ
5
m
M chemodosimeter 1 in culture media for 15 min at 37 C, and
2
then they were incubated with Na S (1 mM) for another 15 min.
Fluorescence imaging of living HeLa cells was observed under
confocal fluorescence microscope (Nikon C1eSi).
2.2. Synthesis of chemodosimeter 1
2.5. Cytotoxicity assays
According to the previous paper [22], compound 4 was prepared
from fluorescein. A mixture of compound 4 (300 mg, 0.86 mmol), 2-
carboxybenzaldehyde (193 mg, 1.3 mmol), 4-dimethylaminopyri
RAW264.7 macrophage cells were cultured in culture media
ꢀ
(DMEM) under an atmosphere of 5% CO
cells were seeded in a 96-well plate at a density of 3 ꢂ 10 cells per
2
and 95% air at 37 C. The
3
dine (DMAP, 83 mg, 0.68 mmol) and dicyclohexylcarbodiimide
ꢀ
(
DCC, 265.8 mg, 1.288 mmol) in CH
2
Cl
2
(20 mL) was stirred at 45 C
well in culture media, then 5,10, 20 and 30 M (final concentration)
m
for 4 h. After cooling to room temperature, the reaction mixture
was purified by silica column chromatography (CH Cl as eluent) to
get the pure chemodosimeter 1. mp: 120.8e140.0 C. IR (KBr)
922.592, 2850.274, 1783.832, 1764.547, 1613.163, 1575.558,
501.312, 1466.599, 1425.137, 1284.359, 1247.718, 1174.436, 1106.94,
chemodosimeter 1 were added respectively. Next, the cells were
ꢀ
2
2
ꢀ
incubated at 37 C underan atmosphere of 5% CO
2
and 95% air for 1 h.
n
:
Finally, 20 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT, 5 mg/mL) was added and cultured for another 4 h,
respectively.
2
1
ꢁ
1 1
1
049.086, 950.7343, 747.2809, 686.5342 cm
H NMR (400 MHz,
ꢁ
6
CDCl
3
)
d
(*10 ): 3.855(s, 3H), 6.784e6.801(m, 2H), 6.890(d,
3. Results and discussion
J ¼ 9.6 Hz, 1H), 7.173(t, J ¼ 6.2 Hz, 1H), 7.619e7.742(m, 5H), 7.760e
7
8
5
.824(m, 2H), 7.919(d, J ¼ 8.0 Hz, 1H), 7.963(d, J ¼ 8.0 Hz, 1H),
3.1. Characteristic spectra
13
ꢁ6
.036(d, J ¼ 7.2 Hz, 1H). C NMR (100 MHz, CDCl
5.76, 98.29, 99.05, 99.17, 100.99, 105.43, 112.08, 112.71, 124.05,
24.55, 125.26, 125.73, 125.95, 126.68, 129.20, 129.69, 129.77,
3
) d (*10 ): 29.84,
The fluorescence and absorption spectra of chemodosimeter 1
(2.5 mM) in the absence and presence of H S (Na S, a commonly
2 2
1
Scheme 1. The synthesis of chemodosimeter 1.
Please cite this article in press as: Liu C, et al., A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells, Dyes
and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.039

C. Liu et al. / Dyes and Pigments xxx (2014) 1e5
3
employed H
and ultrapure water (1:1, v/v) containing PBS (5 mM, pH 7.0). As
expected, in the fluorescence spectra, addition of Na S (1 mM) to a
2
S donor) were investigated in the mixture of ethanol
2
solution of chemodosimeter 1 resulted in a considerable fluores-
cence enhancement at around 516 nm (Fig. 1a). In addition, in the
Fig. 2. The fluorescence spectra of 1 (2.5
mM) with the addition of various concen-
tration of Na
2
S (0e1000
m
M) in the mixture of ethanol and ultrapure water (1:1, v/v)
ꢀ
containing PBS (5 mM, pH 7.0). The reaction time is 30 min under 25 C. The excitation
wavelength
l
ex ¼ 450 nm. The inset shows a good linear correlation between Na
2
S
concentration and the fluorescence intensity of 1 at 516 nm.
absorption spectra, an enhanced absorption band centered at
around 450 nm were observed in the presence of Na S (1 mM)
Fig. 1b), and simultaneously the color changes of the solution from
colorless to yellow-green were also observed. Thus, the obvious
2
(
2
color changes enable chemodosimeter 1 to detect H S by the
naked-eye (Fig. 1c).
3
.2. Determination of H
2
S
Inspiring with the above results, a high sensitivity and a wide
linear concentration-signal relationship could be expected. Next,
we further investigated the fluorescence intensity changes of che-
modosimeter 1 with various concentrations of Na
gradual increase of the fluorescence band centered at 516 nm was
observed with the increase concentrations of Na S. There was a
good linearity between the fluorescence intensity and the con-
centrations of Na S in the range of 0e1000 M with a detection
limit of 2.5 M (Fig. 2). These results demonstrated that chemo-
dosimeter 1 could detect H S quantitatively in a wide concentration
range by the fluorescence turn-on method.
2
S. As expected, a
2
2
m
m
2
2
3.3. Selectivity to H S
To check the selectivity of chemodosimeter 1 for H
2
S, various
biological species was incubated in PBS buffer, and their
Fig. 1. The fluorescence spectra (a) and absorption spectra (b) of chemodosimeter 1
Fig. 3. The fluorescence intensity of chemodosimeter 1 (2.5
mM) in the presence of
(
2.5
2
mM) in the absence and presence of Na S (1 mM) in the mixture of ethanol and
different additives (final concentration: 500 M) in the mixture of ethanol and ultra-
m
ultrapure water (1:1, v/v) containing PBS (5 mM, pH 7.0). The excitation wavelength
pure water (1:1, v/v) containing PBS (5 mM, pH 7.0). The reaction time is 30 min under
25 C. All data represent the fluorescence intensity at 516 nm, and the excitation
ꢀ
ꢀ
l
ex ¼ 450 nm, and the reaction time is 30 min under 25 C. (c) The photographs of the
solution of 1 in the absence and presence of Na
2
S.
wavelength
l
ex ¼ 450 nm.
Please cite this article in press as: Liu C, et al., A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells, Dyes
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4
C. Liu et al. / Dyes and Pigments xxx (2014) 1e5
2
Scheme 2. The proposed recognition mechanism of chemodosimeter 1 for H S.
Fig. 4. Confocal fluorescence images of living HeLa cells: (a) bright-field transmission image; (b) Fluorescence image of HeLa cells incubated with chemodosimeter 1 (5
m
M) for
ꢀ
15 min; (c) Fluorescence image of above-mentioned HeLa cells further incubated with 1 mM Na
2
S for another 15 min. Incubation was performed at 37 C under a humidified
atmosphere containing 5% CO m.
2
. Scale bar ¼ 20
m
fluorescence responses were investigated (Fig. 3). These biologi-
2
with Na S (1 mM) for 15 min (Fig. 4c). These results indicated that
cally relevant species included physiologically important metal
chemodosimeter 1 was cell-permeable and capable of visualization
of H S levels in living cells.
þ
2þ
þ
2þ
ꢁ
2ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ions (K , Ca , Na , Mg ), anions (HSO
3
, SO
COO , S ), and biothiols (GSH). In the fluorescence
spectra, it was observed small-molecule thiols GSH and other
species at 500 M have much smaller effect on fluorescence in-
4
, NO
3
, Cl , Br , I ,
2
ꢁ
ꢁ
2ꢁ
SCN , CH
3
3.6. Cytotoxicity investigations
m
tensity of chemodosimeter 1. In the same reaction conditions, the
fluorescence intensity of chemodosimeter 1 had a significant
To further estimate the cytotoxicity of chemodosimeter 1, we
carried out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assays in RAW264.7 macrophage cells with 5, 10,
enhancement in the presence of Na
2
S (500
mM). The excellent
selectivity of chemodosimeter 1 for H
2
S might be attributed to its
2
0 and 30 mM chemodosimeter 1 for 1 h, respectively. The exper-
inherent two electrophilic reaction sites and the dual nucleophi-
licity of H S (Scheme 2). The above results revealed that chemo-
dosimeter 1 with high selectivity could be used as specific indicator
for H S in aqueous solution and living cells.
iment results exhibited that the absorbances at 490 nm were
almost invariable, which were in good agreement with the control
experiment. These results indicated that our proposed chemo-
dosimeter was of low toxicity to cultured cells under the experi-
2
2
mental conditions at the concentration of 5 mM for 30 min (Fig. 5).
3.4. Recognition mechanism studies
Then, to further demonstrate the recognition mechanism of
chemodosimeter 1 for H S, the reaction of chemodosimeter 1 and
S was carried out under the same conditions as described above.
The resulted mixture of chemodosimeter 1 and H S was subjected
2
H
2
2
to the electrospray ionization mass spectral analysis, and a peak (m/
z 345) corresponding to compound 4 was obtained. Thus, a possible
mechanism was proposed as shown in Scheme 2.
3.5. Bioimaging applications
Next, to evaluate the biological imaging application of chemo-
dosimeter 1 for H
in living HeLa cells. The living HeLa cells incubated with chemo-
dosimeter 1 (5 M) showed almost no fluorescence (Fig. 4b),
2
S in living matrixes, we carried out experiments
m
implying that biological species, especially biothiols, did not cause
interference. However, strong fluorescence in the cells was
observed after the above-mentioned cells were further incubated
Fig. 5. The obtained absorbance at 490 nm.
Please cite this article in press as: Liu C, et al., A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells, Dyes
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C. Liu et al. / Dyes and Pigments xxx (2014) 1e5
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chemodosimeter 1 could be used to detect H S quantitatively in a
wide concentration range (2.5e1000 M) by the fluorescence turn-
on method. Additionally, chemodosimeter 1 showed excellent
selectivity for H S over other various species including GSH, which
might be ascribed to its two electrophilic reaction sites and the dual
nucleophilicity of H S. Preliminary fluorescence bioimaging ex-
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2
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Please cite this article in press as: Liu C, et al., A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells, Dyes
and Pigments (2014), http://dx.doi.org/10.1016/j.dyepig.2014.03.039