Highly selective turn-on probe for H2S with imaging applications in vitro and in vivo

Article abstract of DOI:10.1039/c7nj02869e

Hydrogen sulfide (H₂S) is the third endogenous gaseous signaling molecule, and its distribution affects several biological processes. To clarify the roles of H₂S in living systems, the exact determination of H₂S in living cells in vivo has become an important issue. Herein, we report a novel pyrene-based fluorescent probe, PyN₃, as a H₂S turn-on sensor via reduction of azide to amine, which subsequently undergoes self-immolation through intramolecular 1,6-elimination of the p-aminobenzyl moiety, releasing 1-aminopyrene and leading to the recovery of fluorescence intensity. With its high sensitivity, good selectivity, and low cytotoxicity, PyN₃ was able to recognize both exogenous and endogenously produced H₂S in biological systems. In contrast to the traditional azide-based H₂S probes, PyN₃ shows efficient and effective selectivity to H₂S at biological pH ranges. Because of these promising properties, PyN₃ is an excellent probe for visualizing endogenous H₂S in vitro and in vivo.

Full text of DOI:10.1039/c7nj02869e

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Highly selective turn-on probe for H2S with imaging applications in
vitro and in vivo
Received 00th January 20xx,
Accepted 00th January 20xx
Natesan Thirumalaivasan, a Parthiban Venkatesan a and Shu-Pao Wua,*
Hydrogen sulfide (H2S) is the third endogenous gaseous signaling molecule, and its distribution affects several biological
processes. To clarify the roles of H2S in living systems, the exact determination of H2S in living cells in vivo has become an
important issue. Herein, we report a novel pyrene-based fluorescent probe, PyN3, as a H2S turn-on sensor via reduction of
azide to amine, which subsequently undergoes self-immolation through intramolecular 1,6-elimination of the p-
aminobenzyl moiety, releasing 1-aminopyrene and leading to the recovery of fluorescence intensity. With its high
sensitivity, good selectivity, and low cytotoxicity, PyN3 was able to recognize both exogenous and endogenously produced
H2S in biological systems. In contrast to the traditional azide-based H2S probes, PyN3 shows efficient and effective
selectivity to H2S at biological pH ranges. Because of these promising properties, PyN3 is an excellent probe for visualizing
endogenous H2S in vitro and in vivo.
DOI: 10.1039/x0xx00000x
www.rsc.org/
sensitivity and selectivity.28,29 Recently, several H2S-reactive probes
have been designed by taking advantage of its strong reduction
Introduction
Hydrogen sulfide (H2S) is recognized as the third endogenous property toward azide30-42, nucleophilicity43-46 and binding affinity
with metal47,48 , as well as efficient thiolysis of dinitrophenyl ether49-
gasotransmitter in living systems, along with nitric oxide (NO) and
55. However, some drawbacks have been found with reported H2S
probes. First, probes based on nucleophilic substitution can have
interference from biothiols such as cysteine, homocysteine, and
glutathione. Second, some reported H2S probes display a long
response time (more than 20 min), which is not suitable for real-
time imaging of H2S-related biological processes. Third, only few
fluorescence probes can detect H2S in the nanomolar range.
Because biological levels of H2S vary from nanomolar to micromolar
levels, it is important to develop a sensitive fluorescent probe that
can detect a tiny change in H2S levels. Consequently, additional
works on these concerns are required.
carbon monoxide (CO).1-2 H2S production from cysteine and
homocysteine is catalyzed by the enzymes 3-mercaptopyruvate
sulfurtransferase, cystathionine-b-synthase, and cystathionine-b-
lyase (CSE).3,4 In the cardiovascular system, H2S can relax muscle
and regulate blood pressure.5 On the other hand, it is also involved
in the modulation of neuronal transmission of the nervous system
by aiding the induction of hippocampal long-term potential.6,7
Because of its reducibility and high lipid solubility at low levels, H2S
is also harmful to the human body. In addition, abnormal H2S
levels in cells is associated with various syndromes such as
8-11
12-15
16
stroke
,
Alzheimer’s disease
,
diabetes
, Down’s
syndrome17 and liver cirrhosis18-20. Consequently, it is important to
elucidate the role of H2S during disease development. Specific
Herein, we present a novel pyrene-based fluorescent probe for
H2S detection, PyN3, via azide reduction and cleavage of the
azidophenyl group. In the presence of the electron-withdrawing
group carbamate, the electron density of 1-aminopyrene
continuously declined and resulted in a significant turn-on effect on
the fluorescence intensity of PyN3. During the sensing process, the
azide group was reduced to amine by H2S, and then the carbamate
linkage was cleaved by self-immolation via intramolecular 1,6-
elimination of the p-aminobenzyl moiety, which released 1-
aminopyrene and led to the recovery of fluorescence intensity. The
formation of 1-aminopyrene during the above process has been
confirmed by ESIMS. Remarkably, PyN3 exhibited high selectivity
and sensitivity for H2S over other reactive sulfur species. After the
addition of H2S to PyN3, the strong blue emission of 1-aminopyrene
was observed. More importantly, the applicability of PyN3 was well
demonstrated using living-cell images and its distribution in live
zebrafish.
characterization and quantitation of H2S in cells and tissues remains
a great challenge.
By considering the importance of H2S, several analytical
methods have been developed for its detection in environmental
and clinical fields, such as gas chromatography21, atomic absorption
spectroscopy22
spectrometry23,24
methylene blue method26, and the monobromobimane method27
, inductively coupled plasma atomic emission
,
sulfide ion selective electrode method25
,
.
However, these methods are not appropriate for in situ analysis in
cells. The established fluorescence methodology using molecular
chemodosimeters is particularly valuable because of its low cost,
operational simplicity, and non-destructive analysis, as well as high
Results and Discussion
The synthesis of PyN3 is outlined in Scheme 1. Initially, 1-
nitropyrene was obtained from pyrene, which was further reduced
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O
NH2
within 2 hr after the addition of 15 equivalents of H2S. During H2S
titration with PyN3, a new fluorescence emission band at 453 nm
N3
HN
O
DMAP/ toluene
+
N3
Triphosgene, DCM
8000
OH
PyN3+ H2S
PyN3+ Anions+ H2S
PyN3
Scheme 1. Synthesis of PyN3.
6000
by stannous chloride to yield 1-aminopyrene. 1-Aminopyrene was
further treated with 4-(azidophenyl) methanol and triphosgene to
form PyN3. The structure of PyN3 was confirmed by 1H NMR, 13C
NMR, and mass spectra (see supporting information). PyN3 was pale
yellow, having an absorption band centered at 346 nm. It has weak
fluorescence (Ф = 0.0046) due to the presence of the electron-
withdrawing group carbamate.
4000
2000
0
-
-
4
-
-
I
-
7
c
O
O
A
l
2
C
O
P
Fig. 3 Fluorescence intensity at 455 nm of the probe PyN3 (10 µM) with
various species (15 eqiv) in a H2O–DMSO solution (v/v = 20/80). The black
bars represent single species (150 μM); the gray bars represent coexisting
species: H2S (150 μM) + other species (150 μM). The excitation wavelength
was 410 nm.
8000
PyN3
+ H2S
PyN3 with Br-
F-
H2PO4-
,
ClO4-
Hcy H2O2
NO3- NO2- OAC-
,
, , Cys
CN-
,
GSH
,
I-
H2S
,
,
,
6000
4000
2000
0
8000
,
,
,
,
OH-, OCl-, P2O74-, SCN-, S2O32-
PyN3+H2S
6000
4000
2000
0
450
500
550
Wavelength(nm)
PyN3
Fig. 1, Fluorescence changes of PyN3 (10 µM) upon the addition of various
anions (150 µM) and biothiols (Cys, GSH, Hcys, 6 mM) in H2O–DMSO (v/v =
20/80) solutions. The excitation wavelength was 410 nm.
4
5
6
7
8
9
10
pH
Fig. 4 The effect of pH on the fluorescence changes of PyN3 (10 µM) and
after the addition of H2S (150 µM) in a H2O–DMSO (v/v = 20/80, 0.2 mM
PBS) solution. The excitation wavelength was 410 nm.
8000
455nm
6000
6000
4000
2000
0
N3
0
30
60
90
120 150 180
4000
2000
0
O
H2N
H
S(µM)
2
HN
O
OH
N
N
N
DMAP/ toluene
Triphosgene, DCM
CO2
+
NH
HS
O
O
450
500
550
600
O
HN
NH2
HN
O
N
N
SH
N
Wavelength(nm)
Fig. 2 Fluorescence changes of PyN3 (10 µM) with gradual addition of H2S in
H2O–DMSO (v/v = 20/80) the excitation wavelength was 410 nm.
O
HN
O
We tested the sensing ability of the probe PyN3 toward various
amino acids and anion species, including H2S. As shown in Fig. 1,
noticeable blue fluorescence was observed only upon addition of
H2S to the PyN3 solution. Other anion species (Cys, Hcy, GSH, Br−,
N
S
N
N2S
N
H
Scheme 2. Proposed detection mechanism of PyN3.
−
−
4-
CN−, ClO4−, F−, H2O2, H2PO4−, I−, NO3 , NO2 , OAc−, OCl-, OH−, P2O7
,
appeared (Fig. 2). The fluorescence intensity reached its maximum
after the addition of 15 equivalents of H2S. In the titration curve, a
good linear range was found in the concentration of H2S (0–60 μM),
and the detection limit was found to be 158 nM (see Fig. S9 in the
supporting information), which makes the probe PyN3 sufficiently
sensitive for H2S detection in living systems. The product, 1-
aminopyrene, was confirmed by 1H NMR, 13C NMR, and MS
S2O32−, and SCN−) did not cause significant changes in fluorescence
(Fig. 1). To determine how fast the reaction of PyN3 with H2S was,
time-dependent fluorescence experiments were carried out (Fig. S8
in the supporting information). To a solution of PyN3 (10 µM), the
fluorescence emission gradually increased and reached a plateau
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spectrometry. Its quantum yield of the emission band was at Ф =
The potential application of PyN3 in monitoring H2S in living
0.59, which is 128 times higher than that of PyN3 at Ф = 0.0046 cells was further studied. Prior to cell imaging, the MTT assay using
(Scheme 2). the MCF-7 cell line was applied to estimate the cytotoxicity of PyN3.
To further evaluate the influence of other biomolecules during As shown in Fig. 5, we found more than 80% cellular viability after
H2S detection by PyN3, a competitive experiment with coexisting 24 hr incubation, which indicates low cytotoxicity of PyN3 (<25 µM).
ions was carried out. When we examined PyN3 (10 µM) with other For cell imaging, a confocal fluorescence microscope was used.
anions (15 equiv) and H2S (15 equiv). (Fig. 3), we found that there When MCF-7 cells were incubated with PyN3 (10 µM), no
was nearly no interference from other reactive sulfur species. This fluorescence was observed in cells (Fig. 6, panel a); this finding
observation indicates that other reactive sulfur species do not indicates low endogenous H2S in cells. Upon addition of Na2S (150
interfere with the reaction of PyN3 with H2S. Because of the μM) to the above cells, a strong blue emission in cells was observed
importance of pH, we also studied the emission behavior of PyN3 (Fig. 6, panel b). This observation shows that PyN3 can detect the
treated with H2S in different pH environments. As shown in Fig. 4, presence of H2S in cells. In order to visualize the endogenous H2S,
low emission was observed in the absence of H2S in the pH range of SNP (sodium nitroprusside, a NO donor) was employed to induce
4–10. With addition of H2S, the emission intensity at 453 nm the production of endogenous H2S in MCF-7 cells.56 CSE, a H2S-
increased significantly in the pH range of 7–7.5, which means that producing enzyme, is expressed in MCF-7 cells. Nitric oxide (NO) can
the probe could be used under these physiological conditions. upregulate CSE expression, stimulating the production of H2S. With
When the pH exceeded 8.0, the emission intensity dropped because incubation of SNP for 30 min, blue emission was found in MCF-7
of poor reactivity of H2S at high pH.
cells (Fig. 6, panel C). We also tested the ability for generation of
endogenous H2S in HeLa cells (Fig. 7, panel C); we observed only
weak blue emission, which indicates that less endogenous H2S was
produced in HeLa cells by SNP. In addition, higher concentration of
endogenous H2S was found in in colon cancer cells due to the
overexpression of CBS.57-59 Herein two colon cancer cells HCT-116
and HT-29 were incubated with PyN3 (10 µM) for 1.3 hr. Fig. 8
shows blue emission in HCT-116 and HT-29 and indicates that
endogenous H2S was produced in two colon cancer cells HCT-116
and HT-29.
100
80
60
40
20
0
0
5
10
15
20
25
[PyN3], µM
Fig. 5 MTT assay of MCF-7 cells in the presence of PyN3 (0–25 µM) at 37◦C
for 24 h.
Fig.7 Confocal fluorescence images of HeLa. (Left) Fluorecence; (Middle) PI
(propidium iodide, nuclear stain); and (Right) merged image. (A)The cells
were incubated with PyN3 (10 µM) alone for 15 min. (B) Cells were treated
with H2S (150 μM) for 30 min. (C) Cells were treated with SNP (50 μM) for 30
min.
Fig. 6 Confocal fluorescence images of MCF-7. (Left) Fluorecence; (Middle)
PI (propidium iodide, nuclear stain); and (Right) merged image. (A)The cells
were incubated with PyN3 (10 µM) alone for 15 min. (B) Cells were treated
with H2S (150 μM) for 30 min. (C) Cells were treated with SNP (50 μM) for 30
min.
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column chromatography (Hexane: DCM = 1: 1) to obtain PyN3 as a
light yellow solid (Scheme 1). (220 mg, 31%); 1H NMR (300 MHz,
DMSO): 10.08 (s, 1H), 8.32 (M, 9H), 7.55 (d, J = 7.8 Hz, 2H), 7.18 (d,
J = 8.1 Hz, 2H), 5.24 (s, 1H).13C NMR (75 MHz, DMSO): 155.3, 139.6,
134.1, 132.2, 131.3, 130.9, 130.4, 128.4, 127.5,126.9, 126.8, 126.8,
125.6, 125.5, 125.3, 124.8, 124.3, 123.29, 122.7, 119.6, 66.0. MS
(EI): Found [M] 392.178; HREI: calcd. for C24H16N4O2 [M]: 392.127;
found 392.127.
The reduction product 1-Aminopyrene from the reaction of PyN3
with H2S
H2S (89 mg, 15eqiv, 10% of H2O) was added to the solution of
PyN3 (30 mg) in DMSO (30 mL).The reaction mixture was stirred at
ambient temperature for 2 hr. The reaction mixture was extracted
with CH2Cl2 and cold water for three times. The crude product was
purified by column chromatography (DCM / Hexane = 1: 1) to give
the compound 1-aminopyrene as a bluish black powder. 11 mg.
(63%); 8.26 (d, J=9.0 HZ, 1H), 7.92(M, 6H), 7.71(d, J=9.0 HZ 1H),
7.37(d, J=8.4 Hz 1H), 6.34(S, 1H) ).13C NMR (75 MHz, DMSO): 144.8,
132.5, 132.2, 128.2, 127.0, 126.3, 126.2,125.6, 124.6, 123.3, 122.8,
122.6, 122.2, 121.8, 115.1, 113.5 MS (ESI): Found [M]+ 217.089;
HREI: calcd. for C16H11N [M]+: 218.096; found 218.096
Fig.8 Confocal fluorescence images of HeLa. (Left) Bright Field; (Middle)
Fluorescence; and (Right) merged image. The HCT-116 and HT-29 cells were
incubated with PyN3 (10 µM) alone for 1.3 hr.
Cytotoxicity assay
The cellular cytotoxicity of PyN3 in MCF-7 cells was evaluated by
the methyl thiazolyl tetrazolium (MTT) assay. MCF-7 cells were
grown in a 96-well cell-culture plate and several concentrations (5,
10, 15, 20, 25 mM) of PyN3 were added to the wells. Then the cells
were incubated at 37 0C under 5% CO2 for 24 h. Thereafter, each
well was incubated with 10 mL MTT (5 mg mL-1) at 37oC under 5%
CO2 for 4 h. The MTT solution was removed and the yellow
precipitates (formazan) was dissolved in 200 mL DMSO and 25 mL
Sorensen’s glycine buffer (0.1 M glycine and 0.1 M NaCl). The
absorbance at 570 nm for each well was measured by Multiskan GO
microplate reader. The viability of the cells were calculated by the
following equation:
Fig. 9 Confocal microscopy images of 3-day-old zebrafish. (a) The zebrafish
incubated with PyN3 (10 µM) for 30 min. (b) Subsequent treatment with H2S
(150 µM) for 2 hrs.
Cell viability (%) = (Mean of absorbance value of treatment group)/
(Mean of absorbance value of control group)
Cell culture and confocal fluorescence imaging of PyN3 in living
cells
To evaluate the potential of PyN3 as an imaging tool for H2S
detection in vivo, zebrafish was used a model in the study. Initially,
PyN3 was incubated with zebra fish for 30 min; no blue fluorescence
was observed (Fig. 9a). The result indicates low endogenous H2S in
MCF-7 cells were grown in DMEM media with 10% (v/v) FBS (fetal
bovine serum) and penicillin/streptomycin (100 μg/mL) at 37°C in a
5% CO2 incubator. The cells were treated with 2 µL of 10 mM PyN3
(final concentration: 10 µM) dissolved in DMSO and incubated for
30 min at 37oC. After addition of Na2S (150 µM) to the above cells, a
strong blue fluorescence in cells was observed. In addition, sodium
nitroprusside (SNP 50µM) was added to the probe-treated cells for
inducing the endogenous generation of H2S. After 30 min, the
fluorescence in cells increased significantly, indicating the
generation of endogenous H2S within the cells. The culture medium
was removed, and the treated cells were washed with 0.1 M PBS (2
mL×3) before observation. Confocal fluorescence imaging of cells
was performed with a Leica TCS SP5 X AOBS Confocal Fluorescence
Microscope (Germany), and a 63×oil-immersion objective lens was
used. The cells were excited with UV light below 410 nm and
emission was collected at 453 nm ± 10 nm.
larval zebrafish. With the incubation of H2S,
a strong blue
fluorescence in the 3-day-old zebrafish was observed (Fig. 9b).
These results show that the probe PyN3 can be utilized as a probe to
visualize H2S in zebrafish.
Experimental
Materials and instrumentation
NMR spectra were obtained from Bruker DRX-300 and
Agilent Unity INOVA-500 NMR spectrometer. UV/Vis spectra were
obtained on an Agilent 8453 UV/Vis spectrometer. Fluorescence
spectra measurements were obtained from
a Hitachi F-7000
fluorescence spectrophotometer. Fluorescent images were taken
on a Leica TCS SP5 X AOBS and Leica TCS SP5 II Confocal
Fluorescence Microscope.
Synthesis of PyN3
To a solution of dimethylaminopyridine (DMAP, 636 mg, 6.0
mmol) and triphosgene (350 mg 2.4 mmol) in dry CH2Cl2 (20 mL), a
solution of pyren-1-amine (180 mg, 2.0 mmol) in dry CH2Cl2 (10 mL)
was added dropwise in an ice bath for 1 h. Then the solution was
stirred at room temperature for 6 hours. The reaction solution was
then filtered. Thereafter, (4-azidophenyl) methanol (179 mg, 2.4
mmol) was added to the filtrate, and the mixture was stirred at
room temperature for 6 hr. The solvent was evaporated under
reduced pressure and the compound was purified by silica gel
H2S imaging in zebrafish
Animals were maintained in accordance with the guidelines of the
National Institute of Health, Taiwan, and approved by the
Institutional Animal Care and Use Committee (IACUC) of National
Chiao Tung University. Zebrafish was treated with the guidelines for
Animal Care and Use Committee of National Chiao Tung University.
Zebrafish was keep up at optimal breeding conditions at 28°C. For
mating, male and female zebrafish were maintained in one tank at
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Conclusions
With the great biological significance of H2S as an endogenous
signaling molecule, many works have focused on the development
of fluorescent probes for H2S detection to study the functions of
H2S. In this work, a novel pyrene probe, PyN3, was designed and
synthesized for highly selective H2S detection. The detection of H2S
was accomplished through significant fluorescence enhancement
resulting from H2S-triggered azide reduction. PyN3 showed a strong
turn-on blue emission in its response toward H2S and impressive
ability to detect H2S at a low detection limit (158 nM), which make
it an attractive tool for H2S study in cell imaging and live zebra fish
imaging. We believe that further application of PyN3 can serve as a
great tool to investigate the roles of H2S in living systems.
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Acknowledgements
We gratefully acknowledge the financial support of Ministry of
Science and Technology (Taiwan) for supporting this research under
the grant (MOST 105-2119-M-009-013) and National Chiao Tung
University.
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