Instantaneous fluorescent probe for the specific detection of H2S

Article abstract of DOI:10.1016/j.saa.2019.01.085

Novel cyanine-based fluorescent probes for the detection of H₂S were developed. The probes developed are stable under physiological conditions. The water soluble fluorescent probe 2 displayed ultrafast and specific response to H₂S di

Full text of DOI:10.1016/j.saa.2019.01.085

Accepted Manuscript
Instantaneous fluorescent probe for the specific detection of H2S
Xianfeng Lin, Xiuhong Lu, Junliang Zhou, Hang Ren, Xiaochun
Dong, Weili Zhao, Zhongjian Chen
PII:
DOI:
Reference:
S1386-1425(19)30099-X
https://doi.org/10.1016/j.saa.2019.01.085
SAA 16771
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 31 October 2018
Revised date:
Accepted
date:
11 January 2019
28 January 2019
Please cite this article as: X. Lin, X. Lu, J. Zhou, et al., Instantaneous fluorescent probe for
the specific detection of H2S, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, https://doi.org/10.1016/j.saa.2019.01.085
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Instantaneous fluorescent probe for the specific detection of H2S
Xianfeng Lin,a Xiuhong Lu,a Junliang Zhou,a Hang Ren,a Xiaochun Dong,*,a Weili
Zhao,*,a,b and Zhongjian Chen*,c
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, P.
R. China
b Key Laboratory for Special Functional Material of the Ministry of Education, Henan
University, Kaifeng, 475004, P. R. China
c Shanghai Dermatology Hospital, Shanghai, 200443, P. R. China
ABSTRACT
Novel cyanine-based fluorescent probes for the detection of H2S were developed.
The probes developed are stable under physiological conditions. The water soluble
fluorescent probe 2 displayed ultrafast and specific response to H2S displaying NIR
fluorescence of 115-fold turn-on with the detection limit of 11 nM without assistance
of organic solvent or surfactant. Cell imaging experiments indicated that probe 2 was
cell-permeable and was able to detect H2S sensitively in lysosomes. Moreover, our
probe was able to detect H2S intrinsically produced H2S through
enzymatic/non-enzymatic biosynthetic pathway from Cys/GSH. Moreover, we
applied probe 2 to detect H2S in living mice and demonstrated the fast metabolism of
H2S. Thus, probe 2 shows great promise as a reporter for H2S.
Keywords: fluorescent probe, H2S, lysosome-targeted

ACCEPTED MANUSCRIPT
1. Introduction
Recent biological reports have established the important role of H2S, and new
evidence suggests that H2S may emerge as the most important gasotransmitter which
regulates many important physiological functions in the cardiovascular, nervous,
endocrine, immune, gastrointestinal systems[1, 2]. Furthermore, H2S serves as a
scavenger for endogenous reactive oxygen species (ROS) [3]. However, abnormal
H2S production is associated with various human diseases including the symptoms of
Down syndrome [4], hypertension [5], and diabetes [6]. Physiologically, endogenous
H2S is primarily produced from cysteine or cysteine derivatives in the mitochondria or
cytosol catalyzed by cystathionine--lyase (CSE), cystathionine--synthase (CBS),
and 3-mercaptopyruvate sulphurtransferase (3MST) [7]. On the other hand,
glutathione and polysulfides can be transformed to H2S through non-enzymatic
pathway [8]. Once generated, H2S undergoes complex catabolism (with half-life of a
few minutes to be metabolized) through its interactions with reactive sulfur, oxygen,
nitrogen species (RSONs), cellular oxidants, and protein transition-metal centers [8].
H2S in an aqueous solution has a pKa of 7.0. It freely permeates plasma membranes,
and undergoes fast metabolism in the mitochondria via the sulfide oxidation pathway
[9]. In subcellular level, there is some evidence that both CBS and CSE are localized
to the cytoplasm, whereas 3MST is localized predominantly to the mitochondria [1, 7].
The complicated reactions involving H2S make the detection of H2S in biological
systems very difficult, therefore, the biologically relevant concentrations of H2S have
been debated [10-12].
The volatile, lipophilic, diffusive, and highly reactive nature (with half-life of a
few minutes to be metabolized), and complicated reactions involving H2S make the
detection of H2S in biological systems very difficult. Various H2S-mediated processes
such as reduction (reduction of azides or reduction of nitro groups), nucleophilic
addition, nucleophilic aromatic substitution (aryl nitro thiolysis), replacement of
copper, and coordinative-based approach have been applied for the probe design [7,

ACCEPTED MANUSCRIPT
13-19]. For easy use for the detection of H2S in living systems, the chemosensing
agent should meet at least the following four criteria: (1) act rapidly (within seconds)
under mild conditions; (2) be sensitive for detection under near physiological
conditions; (3) show minimal or no interference by other anions in blood serum; (4)
be functional in aqueous solutions and blood plasma [8]. Unfortunately, the existing
probes hardly satisfy all of these criteria, hampering further application of them to
tackle biological issues [20-24]. Only a few examples could detect H2S relatively fast,
but are poorly water-soluble [25-29]. Some probes are organelle-targeted (such as
mitochondria [30-35], and lysosome [36-40]). However, they frequently required long
recognition time. The lack of ideal probe for the measurement of H2S limited
advances in the field and many of its underlying molecular events remain unknown,
especially in the subcellular level.
To address the challenging responding rate and to favorite in vivo applications,
cyanine type dyes were chosen to screen H2S responsive probes and we were happy to
find that dinitrophenol ether of cyanine dye (1) was capable of fast response to H2S.
Further optimization led to the discovery of more favorable water-soluble version (2)
as shown in Chart 1.
2. Experimental Section
2.1 Materials and Instrumentation
All reagents were purchased from commercial suppliers and used without further
purification. Solvents used were purified by standard methods prior to use. All of the
intermediates were purified by silica gel chromatography using an ISCO CombiFlash
chromatography instrument. All of the final compounds were purified by preparative
HPLC (prep-HPLC) on a reversed-phase column using a Waters X-Bridge Phenyl (30
mm × 100 mm, 5 μm) or OBD RP18 (30 mm × 100 mm, 5 μm) column under acidic
conditions (A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile) or basic
1
conditions (A, 0.1% ammonia in H2O; B, acetonitrile). H NMR spectra were
recorded on a NMR spectra were obtained using Bruker AVANCE 400 MHz
spectrometer. 1H NMR chemical shifts (δ) are given in ppm (s = singlet, d = doublet, t

ACCEPTED MANUSCRIPT
= triplet, q = quartet, m = multiplet) downfield from Me4Si, determined by chloroform
13
(δ = 7.26 ppm). C NMR spectra were recorded on a Bruker AVANCE 100 MHz
spectrometer. 13C NMR chemical shifts (δ) are reported in ppm with the internal
CDCl3 at δ 77.0 as standard, respectively. All the photophysical characterization
experiments were carried out at 25 °C. Absorption spectra were collected on a Hitachi
U-2900 double beam UV-vis spectrophotometer. Fluorescence spectra were collected
on Hitachi F-2500 fluorescence spectrophotometer with slit widths to be 10 nm and
10 nm for excitement and emission respectively. Phosphate buffer saline (PBS, 10
mM, pH 7.2) was used to prepare all aqueous solutions. To test the fluorescence
responses of the probes towards reactive species, aliquots of probe stock solutions
were diluted with PBS and treated with analytes to make sure both probes and
analytes were kept at desired final concentrations. ESI-HRMS (high resolution mass
spectrometry) spectra were obtained on Agilent Technologies 6530 Accurate-Mass
Q-TOF LC-MS. LC-MS (ESI, low resolution mass spectrometry) spectra were
obtained on Waters X-Bridge Phenyl or OBD RP18 column and Waters AcQuiry
(PDA Detector, SQ Detector, ELS Detector, Sample Manager-FL, Column Manager,
Binary Solvent Manager, MassLynx).
All cell lines obtained from American Typical Cell Collection (ATCC) and the
Type Culture Collection of the Chinese Academy of Sciences were maintained in
respective growth medium which were recommended by the vendors. Cell lines
cultured in needed medium ( from Invitrogen) with 10% (vol/vol) fetal bovine serum
qualified (from Gibco, FBS, #10099-141, origin Australia) at 37 C in a humidified
incubator containing 5% CO2. Cell Counting Kit-8 (CCK8, cat. no. CK04-20,
Kumamoto, Japan) was purchased from Dojindo Molecular Tech. Lysosome tracker
green and Mito-tracker green were purchased from Thermofisher.
2.2 Preparation and Characterization of Probes
Synthesis of 4-(2’,4’-dinitrophenoxy) benzene-1,3-dicarbaldehyde
Commercially available 1-fluoro-2,4-dinitro-benzene (300 mg, 2 mmol) was
added
to
a
stirring
solution
of
commercial
available

ACCEPTED MANUSCRIPT
4-hydroxybenzene-1,3-dicarbaldehyde (372 mg, 2 mmol) and potassium carbonate
(414 mg, 3 mmol) in N,N-dimethylformamide (5 mL) at room temperature. After 1
hour, TLC indicated that the start materials consumed. The mixture was diluted with
ethyl acetate (50 mL) and washed with water (30  3 mL). The organic phase was
dried over Na2SO4, evaporated in vacuo and the residue was purified by flash column
chromatography
eluting
with
petroleum/EtOAc
(3:1)
to
afford
4-(2’,4’-dinitrophenoxy) benzene-1,3-dicarbaldehyde (537 mg, yield: 85%) as pale
yellow solid. 1H NMR (400MHz, DMSO-d6) δ = 10.33 (s, 1H), 10.10 (s, 1H), 8.97 (d,
J=2.8 Hz, 1H), 8.56 (dd, J=2.8, 9.2 Hz, 1H), 8.50 (d, J=2.2 Hz, 1H), 8.26 (dd, J=2.1,
13
8.5 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H), 7.46 (d, J=8.6 Hz, 1H). C NMR (100MHz,
DMSO-d6) δ = 192.02, 189.01, 160.17, 153.32, 143.61, 140.85, 136.84, 133.82,
132.32, 130.47, 127.48, 122.76, 122.64, 120.98.
Synthesis of probe 1
The mixture of 4-(2’,4’-dinitrophenoxy) benzene-1,3-dicarbaldehyde (95 mg,
0.30 mmol), 1,2,3,3-tetramethylindolium iodide (181 mg, 0.60 mmol) and NaOAc (74
mg, 0.90 mmol) in Ac2O (5 mL) was stirred at 80 °C for 2 hours under Ar atmosphere
and monitored by LC-MS. After completion, the reaction mixture was concentrated
by evaporation under reduced pressure. The residue was diluted with 2 mL DMF, 2
mL HOAc, and purified by preparative-HPLC (grad. 10%-90 ACN in water, 20 min)
to afford probe 1 (148 mg, 48%). 1H NMR (400MHz, Methanol-d4) δ = 9.06 (d, J=2.8
Hz, 1H), 9.00 (d, J=2.0 Hz, 1H), 8.66 (d, J=2.8 Hz, 1H), 8.65 - 8.62 (m, 2H), 8.52 (s,
1H), 8.40 (dd, J=2.3, 8.8 Hz, 1H), 7.95 - 7.88 (m, 2H), 7.86 - 7.79 (m, 2H), 7.77 -
7.66 (m, 6H), 7.41 (d, J=8.8 Hz, 1H), 4.31 (s, 3H), 4.29 (s, 3H), 1.93 (s, 6H), 1.86 (s,
13
6H). C NMR (100MHz, Methanol-d4) δ = 182.73, 182.69, 157.32, 152.50, 151.01,
150.94, 144.26, 143.89, 143.82, 141.83, 140.87, 135.25, 132.65, 132.26, 130.23,
130.00, 129.47, 129.30, 129.20, 126.36, 122.65, 122.63, 122.06, 119.17, 115.23,
114.99, 52.94, 52.91, 52.90, 35.24, 34.60, 24.88, 24.86. HRMS (ESI): calc'd for
C38H36N4O5 [M]2+ 314.13374, measured 314.13378.
Synthesis of 2,3,3-trimethyl-1-(3-sulfopropyl) indolium inner salt

ACCEPTED MANUSCRIPT
A mixture of commercially available 2,3,3-trimethylindolenine (15.9 g, 10.0
mmol) and 1,3-propanesultone (12.2 g, 10.0 mmol) in toluene (200 mL) was refluxed
overnight. After cooling, the precipitate was filtered and washed with ethyl acetate.
The product 2,3,3-trimethyl-1-(3-sulfopropyl) indolium inner salt (21.3 g, 76%) was
collected as red powder and was used in the next reaction without further purification.
Synthesis of probe 2
The mixture of 4-(2’,4’-dinitrophenoxy) benzene-1,3-dicarbaldehyde (105 mg,
0.33 mmol), 2,3,3-trimethyl-1-(3-sulfopropyl) indolium inner salt (197 mg, 0.70
mmol) and NaOAc (33 mg, 0.40 mmol) in Ac2O (5 mL) was stirred at 80 °C for 2
hours under Ar atmosphere and monitored by LC-MS. After completion, the reaction
mixture was concentrated by evaporation under reduced pressure. The residue was
diluted with 2 mL H2O, 2 mL HOAc, and purified by preparative-HPLC (grad.
1
10%90 ACN in water, 20 min) to afford probe 2 (172 mg, 62%). H NMR (400
MHz, DMSO-d6) δ = 9.45 (d, J=1.6 Hz, 1H), 9.00 (d, J=2.8 Hz, 1H), 8.72 (dd, J=1.7,
8.8 Hz, 1H), 8.65 (d, J=16.4 Hz, 1H), 8.59 (dd, J=2.8, 9.2 Hz, 1H), 8.50 - 8.43 (m,
1H), 8.37 - 8.29 (m, 1H), 8.19 - 8.05 (m, 3H), 7.91 - 7.87 (m, 2H), 7.71 - 7.62 (m, 5H),
7.50 (d, J=8.8 Hz, 1H), 5.10 - 4.93 (m, 4H), 2.78 - 2.64 (m, 4H), 2.35 - 2.19 (m, 4H),
1.89 (s, 6H), 1.74 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ = 182.70, 182.12,
156.98, 153.10, 152.12, 144.77, 144.61, 144.27, 143.49, 141.35, 141.30, 140.79,
136.33, 133.71, 133.08, 130.50, 130.21, 129.78, 129.63, 126.88, 123.66, 123.60,
122.65, 122.36, 120.31, 116.90, 116.14, 115.93, 114.77, 53.06, 52.81, 49.09, 47.58,
47.49, 46.48, 46.31, 26.18, 25.33. HRMS (ESI): calc'd for C42H42N4O11S2 [M+2H]2+
422.12185, measured [M+2H]2+ 422.12166.
2.3 Confocal Imaging Experiments
Hela or HepG2 cells were seeded at 1 × 105 cells per ml in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum, 1%
penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. 24 hours
later, cells were treated with different conditions as described in the figure legend, and

ACCEPTED MANUSCRIPT
then washed three times with 0.1 M Hank’s buffer. All confocal fluorescence images
were acquired by an LSM710 confocal microscope with a 40 × objective (Carl Zeiss).
2.4 Fluorescence Imaging in Mice
All procedures were carried out in compliance with the Guide for the Care and
Use of Laboratory Animal Resources and the National Research Council. The
bioimaging experiments on living mice utilized the Kunming mice (Sino-British
SIPPR/BK Lab. Animal Ltd, China) in vivo imaging system equipped with a sensitive
Charge Coupled Device (CCD) camera, with the excitation at 590 nm and the 680 nm
emission filter. Healthy mice (2025 g) were used, and animals had free access to
food and water. The mice were anesthetized, and the abdominal fur was removed.
Mice without any treatment was kept as a control; i) NaHS (20 mM, 0.2 mL) was
injected into the peritoneal cavity of the mouse; then the probe (50 M, 0.2 mL) was
injected into the peritoneal cavity of the mice; ii) NaHS (20 mM, 0.2 mL) was
injected into the peritoneal cavity of the mouse; 10 min later, the probe (50 M, 0.2
mL) was injected into the peritoneal cavity of the mice; iii) the probe (50 M, 0.2 mL)
was injected into the peritoneal cavity of the mouse; 10 min later, NaHS (20 mM, 0.2
mL) was injected into the peritoneal cavity of the mice . Images were taken after
incubation for different times, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min,
9 min, 10min, 20 min, 30 min, 40 min, 50 min, 60 min with the excitation filter at 590
nm and the emission filter set at 680 nm; iv) the probe (50 M, 0.2 mL) was injected
into the peritoneal cavity of the mouse.
3. Results and Discussion
3.1 Syntheses of Probes
The preparations of the probes were very straight forward (Scheme 1).
2,3,3-trimethylindolenium salt functionalized at 1 position with methyl or sulfopropyl
was reacted with 4-(2’,4’-dinitrophenoxy) benzene-1,3-dicarbaldehyde made from
1-fluoro-2,4-dinitro-benzene and 4-hydroxybenzene-1,3-dicarbaldehyde in the
presence of acetic anhydride and sodium acetate led to the desired probe 1 or 2 in
good yield (Scheme 1). The required 2,3,3-trimethylindolenium salt functionalized at

ACCEPTED MANUSCRIPT
1 position sulfopropyl as inner salt may be easily prepared by refluxing
2,3,3-trimethylindolenine with 1,3-1,3-propanesultone in toluene. The reference dye
of probe 2 (herein designated as dye 3) was also synthesized easily as shown in
Scheme S1 simply started from 4-hydroxybenzene-1,3-dicarbaldehyde. The
well-known mechanism of the reaction-based fluorescent probe capped as
dinitrophenoxy ether for the detection of H2S was that thiolation of the dinitrobenzene
portion in ipso attack to release the parent dye 3 [41].
3.2 Colorimetric and Fluorimetric Assays in Solution
The absorption spectra of probes 1 (10 μM) and 2 in PBS solution (10 mM, pH =
7.2) at 25 ºC were nearly identical with absorption maximum at 400 nm ( = 35300
M−1 cm−1 for 1, and 43200 M−1 cm−1 for 2, respectively) viewed as pale yellow color
with naked eyes (Figure 1). Both probes (1 and 2) are non-fluorescent (Φf < 0.01) due
to photo-induced electron transfer (PET) from the fluorophore to the electronic sink of
dinitrobenzene moiety [42]. Upon addition of NaHS (50 μM), two new absorption
bands for both 1 (max at 468 nm and 565 nm, respectively) and 2 (max at 476 nm and
590 nm, respectively) appeared. The color turned into gray accordingly viewed with
naked eyes. Under irradiation with UV light, red color fluorescence was clearly
generated after addition of NaHS.
The fluorescence produced by probe 1 in the presence of NaHS has the emission
maximum of 665 nm with fairly fast response and nice fluorescence enhancement
(118-fold). It was discovered that probe 2 displayed more intensive emission at
maximum of 680 nm with ultrafast response which reached to half of the saturated
intensities within 4 seconds and finished within 2 minutes while maintaining excellent
fluorescence enhancement of 115-fold (Figure 2). The fast response is extremely
important considering the quick metabolism (with half-life of a few minutes) and
volatile nature of H2S in biological systems. As far as we know, NIR fluorescent H2S
probes with such a rapid and remarkable fluorescence changes without assistance of
organic solvent or surfactant are extremely rare in the literature (Table S2). Therefore,

ACCEPTED MANUSCRIPT
the spontaneously responsive probe 2 with large Stokes shift (90 nm) was selected for
further evaluation.
To determine whether our probe 2 was selective for H2S detection, we carried
out the fluorescence measurements with 1 equiv. of the probe to react with 5 equiv. of
NaHS in PBS solution (10 mM, pH = 7.2) at 25 ºC in comparison with 100 equiv. of
various potential interferents e.g. gas transmitter (NO), various oxidants (NaClO,
H2O2, KO2), nucleophile (KSCN), various thio species (NaHSO4, Na2S2O3, Cys, Hcy,
GSH), and other common amino acids e.g. alanine (Ala), methionine (Met), lysine
(Lys), glutamine (Gln), serine (Ser), etc. The fluorescence intensities collected at 680
nm with the excitation of 590 nm of light were collected in Figure 3. To our delight,
our probe was demonstrated to specifically respond to H2S and none of other analytes
could interfere the detection of H2S. Therefore, probe 2 displayed desirable selectivity
and speed.
Our next experiment was to find the sensitivity of our probe developed.
Therefore, concentration-dependent response profile determined in PBS was provided
in Figure 4. Linearly proportional to analyte concentration was observed for probe 2
to be in the range of 110 μM. The detection limit was calculated to be around 11 nM.
For the reaction mechanism, it is well established that thiolation of the capping
moiety to release the parent dye (herein dye 3) was generally followed (Figure S1).
Our MS studies shown in Fig. S1 indicated that the desired dye 3 was released by
thiolation of probe 1 with the moiety of 2,4-dinitrothiophenol produced. Once the
capping moiety was removed, the PET effect was terminated and therefore ignited the
fluorescence of the dye [41].
3.3 Imaging Hydrogen Sulfide in Living Cells
So far, the ultrafast, sensitive and specific characteristics of probe 2 performed in
PBS buffer without assistance of organic solvent were fairly favorable for the
detection of H2S. To favorite imaging applications, toxicity studies using CCK-8
assays on the fluorescence response of probe 2 to NaHS were carried out. Various
cells used for the toxicity studies (including HCT16, HT29, A549, H1944, MCF-7,

ACCEPTED MANUSCRIPT
MDA-MB-468, MDA-MB-231, PANC1, etc) indicated that good toleration with IC50
greater than 80 M (Table S1 and Figure S2). Both the data and figures displayed in
Table S1 and Figure S2 suggested the low toxicity of our probe.
To demonstrate that probe 2 could be applied for H2S detection in live cells,
confocal fluorescent imaging experiments with probe 2 were subsequently carried out
and the results were shown in Figure S3 with Hela cells. The dim images in red
channel for intrinsic H2S suggested that the level in native live Hela cells was low
(Figure S3). Astonishingly, when external H2S was added, clear red images were
found mostly in spherical shape which was suspected to be lysosome-targeted. The
same phenomena were noticed in HepG2 cells as well (Figure S4). The observations
suggested that probe 2 was cell permeable and could detect H2S in live cells with
most probably in lysosomes.
To find solid evidence that our probe 2 could detect H2S in lysosomes,
colocalization studies were subsequently performed using commercially available
Mito-Tracker Green and Lyso-Tracker Green as standards. Both Hela and HepG2
cells were adopted for the colocalization studies. Cells were initially incubated with
known subcellular targeting dye (Lyso-Tracker Green) for 20 min and then treated
with probe 2 for 10 min. After further incubation with NaHS for 10 min, the confocal
fluorescence images were recorded and the images were illustrated in Figure 5. The
fluorescence images produced using 2 overlapped relatively well with the region
stained with Lyso-Tracker Green both in Hela (Figure 5A, 0.83) and HepG2 (Figure
5B, 0.68) cells. The overlapped parts viewed as spherical-shaped were thus expected
to be lysosome-targeted area. In stark contrast, the generated fluorescent images
overlapped poorly with Mito-Tracker Green from the merged images (Figure S5).
Since the non-fluorescent nature of probe 2 prohibited investigation of the
subcellular distribution with direct fluorescence imaging, we therefore investigated
the distribution the parent dye (3) of probe 2 instead as alternative supporting
information (Figure S6). The colocalization of parent dye 3 with Lyso-Tracker Green
in Hel cells with overlap coefficient of 0.67 (Figure S6) suggested that the dye 3 was

ACCEPTED MANUSCRIPT
most probably lysosome-targeted. The confocal fluorescence imaging experiments
strongly suggested that probe 2 was able to detect H2S elegantly inside living cells
and mostly probably in lysosomes.
In addition to supplementing cells with extraneous sources of sulphide, we put
our efforts to test whether we can detect intrinsically produced H2S through
enzymatic/non-enzymatic biosynthetic pathway. Cys as a substrate of CBS and
reduced glutathione (GSH) as a typical non-enzymatic supplier were adopted (Figure
S7). Imaging experiments were carried out with probe 2 (10 μM), as previously
described, and cells were incubated with either 100 μM Cys or GSH. After addition of
thiol species and 1 hour of incubation, further incubation with probe 2 for 10 min
elicited significant response in both cases. On the other hand, further stimulation with
PMA for ROS generation after thiol addition, subsequent incubation with probe 2
eliminated the fluorescence.
3.4 Fluorescence Imaging in Mice
To explore the potential usage of probe 2 as an in vivo imaging tool, we applied
it to H2S detection in living mice (Figure 6). Briefly, probe 2 was injected 30 s after
administrating i.p. injection of NaHS (20 mM) in mice and images were taken at
various period of time. Strong fluorescence was visualized immediately (1 min) and
the intensity declined quickly over extended time (Figure 6, upper row). In contrast,
once probe 2 was injected 10 min after administrating i.p. injection of NaHS (20 mM)
in mice, much weaker fluorescence was detected (Figure 6, middle row). When NaHS
was injected after administrating i.p. injection of probe 2, very strong intensity of
fluorescence was visualized and longer sustain of fluorescence was noted (Figure 6,
bottom row). Therefore, the results suggested that NaHS metabolized fairly rapidly in
vivo and the dye generated also metabolized. Administrating only probe 2 led to weak
fluorescence signals (Figure S8), which may indicate that even intrinsic H2S may
possibly be detected by probe 2, thus reflected the good sensitivity of our probe.
4. Conclusion

ACCEPTED MANUSCRIPT
In conclusion, a novel water soluble cyanine-based fluorescent probe for H2S
was developed. The probe developed is stable under physiological conditions. Such a
probe responded ultrafast and specifically to H2S with the detection limit of 11 nM in
PBS buffer. To the best of our knowledge, our NIR fluorescent probe with such large
Stokes shift, instantaneous and specific response to H2S in PBS buffer without
assistance of organic solvent or surfactant is unique compared to the recently reported
probes (Table S2). Moreover, our probe was lysosome-targeted and was able to detect
H2S even for the intrinsically produced H2S (both enzymatic/non-enzymatic
biosynthesized). Pleasantly, we successfully applied probe 2 to detect H2S in living
mice and demonstrated the fast metabolism of H2S. Thus, the probe shows great
promise as a reporter for H2S. We anticipate that these results would inspire and
encourage researchers to elucidate the role of H2S in biology and pharmacology.
Acknowledgements
This research was supported by National Natural Science Foundation of China
(21372063, 81601173).
References
[1] C. Szabó, Hydrogen sulphide and its therapeutic potential, Nat. Rev. Drug Discov.
6 (2007) 917-935.
[2] R. Wang, Two’s company, three’s a crowd: can H2S be the third endogenous
gaseous transmitter?, FASEB J 16 (2002) 1792-1798.
[3] B.D. Paul, S.H. Snyder, H2S signalling through protein sulfhydration and beyond,
Nat. Rev. Mol. Cell Biol. 13 (2012) 499-507.
[4] B.K. Czyzewski, D.-N. Wang, Identification and characterization of a bacterial
hydrosulphide ion channel, Nature 483 (2012) 494-497.
[5] A. Stein, S.M. Bailey, Redox biology of hydrogen sulfide: Implications for
physiology, pathophysiology, and pharmacology, Redox Biol. 1 (2013) 32-39.
[6] H. Kimura, Metabolic turnover of hydrogen sulfide, Front. Physiol. 3 (2012) 101.

ACCEPTED MANUSCRIPT
[7] V.S. Lin, W. Chen, M. Xian, C.J. Chang, Chemical probes for molecular imaging
and detection of hydrogen sulfide and reactive sulfur species in biological systems,
Chem. Soc. Rev. 44 (2015) 4596-4618.
[8] H. Peng, Y. Cheng, C. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, A
fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood,
Angew. Chem., Int. Ed. 50 (2011) 9672-9675.
[9] Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao, C. He,
Selective fluorescent probes for live-cell monitoring of sulphide, Nat. Commun. 2
(2011) 495.
[10] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for
selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc. 133 (2011)
10078-10080.
[11] V.S. Lin, A.R. Lippert, C.J. Chang, Cell-trappable fluorescent probes for
endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production,
Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 7131-7135.
[12] S.K. Bae, C.H. Heo, D.J. Choi, D. Sen, E.-H. Joe, B.R. Cho, H.M. Kim, A
ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S
production in Parkinson’s disease gene knockout astrocytes, J. Am. Chem. Soc. 135
(2013) 9915-9923.
[13] F. Yu, X. Han, L. Chen, Fluorescent probes for hydrogen sulfide detection and
bioimaging, Chem. Commun. 50 (2014) 12234-12249.
[14] V.S. Lin, C.J. Chang, Fluorescent probes for sensing and imaging biological
hydrogen sulfide, Curr. Opin. Chem. Biol. 16 (2012) 595-601.
[15] H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin, B. Wang, Thiol reactive
probes and chemosensors, Sensors 12 (2012) 15907-15946.
[16] M.H. Lee, J.S. Kim, J.L. Sessler, Small molecule-based ratiometric fluorescence
probes for cations, anions, and biomolecules, Chem. Soc. Rev. 44 (2015) 4185-4191.
[17] X. Zhou, S. Lee, Z. Xu, J. Yoon, Recent progress on the development of
chemosensors for gases, Chem. Rev. 115 (2015) 7944-8000.

ACCEPTED MANUSCRIPT
[18] N. Kumar, V. Bhalla, M. Kumar, Recent developments of fluorescent probes for
the detection of gasotransmitters (NO, CO and H2S), Coord. Chem. Rev. 257 (2013)
2335-2347.
[19] M. Strianese, C. Pellecchia, Metal complexes as fluorescent probes for sensing
biologically relevant gas molecules, Coord. Chem. Rev. 318 (2016) 16-28.
[20] B. Ke, W. Wu, W. Liu, H. Liang, D. Gong, X. Hu, M. Li, Bioluminescence probe
for detecting hydrogen sulfide in vivo, Anal. Chem. 88 (2016) 592-595.
[21] X. Jin, S. Wu, M. She, Y. Jia, L. Hao, B. Yin, L. Wang, M. Obst, Y. Shen, Y.
Zhang, J. Li, Novel fluorescein-based fluorescent probe for detecting H2S and its real
applications in blood plasma and biological imaging, Anal. Chem. 88 (2016)
11253-11260.
[22] J. Cao, R. Lopez, J.M. Thacker, J.Y. Moon, C. Jiang, S.N.S. Morris, J.H. Bauer,
P. Tao, R.P. Mason, A.R. Lippert, Chemiluminescent probes for imaging H2S in
living animals, Chem. Sci. 6 (2015) 1979-1985.
[23] Y. Qian, L. Zhang, S. Ding, X. Deng, C. He, X.E. Zheng, H.-L. Zhu, J. Zhao, A
fluorescent probe for rapid detection of hydrogen sulfide in blood plasma and brain
tissues in mice, Chem. Sci. 3 (2012) 2920-2923.
[24] K. Zhang, J. Zhang, Z. Xi, L.Y. Li, X. Gu, Q.Z. Zhang, L. Yi, A new
H2S-specific near-infrared fluorescence-enhanced probe that can visualize the H2S
level in colorectal cancer cells in mice, Chem. Sci. 8 (2017) 2776-2781.
[25] E. Karakus, M. Ucuncu, M. Emrullahoglu, Electrophilic cyanate as a recognition
motif for reactive sulfur species: selective fluorescence detection of H2S, Anal. Chem.
88 (2016) 1039-1043.
[26] Q. Fu, G. Chen, Y. Liu, Z. Cao, X. Zhao, G. Li, F. Yu, L. Chen, H. Wang, J. You,
In situ quantification and evaluation of ClO−/H2S homeostasis in inflammatory gastric
tissue by applying a rationally designed dual-response fluorescence probe featuring a
novel H+-activated mechanism, Analyst 142 (2017) 1619-1627.
[27] J. Liu, Y.-Q. Sun, J. Zhang, T. Yang, J. Cao, L. Zhang, W. Guo, A ratiometric
fluorescent probe for biological signaling molecule H2S: fast response and high
selectivity, Chem.-Eur. J. 19 (2013) 4717-4722.

ACCEPTED MANUSCRIPT
[28] Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen, Z. Guo,
A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in
mitochondria, Angew. Chem., Int. Ed. 52 (2013) 1688-1691.
[29] L. Wei, Z. Zhu, Y. Li, L. Yi, Z. Xi, A highly selective and fast-response
fluorescent probe for visualization of enzymatic H2S production in vitro and in living
cells, Chem. Commun. 51 (2015) 10463-10466.
[30] Roopa, N. Kumar, V. Bhalla, M. Kumar, Development and sensing applications
of fluorescent motifs within the mitochondrial environment, Chem. Commun. 51
(2015) 15614-15628.
[31] J. Liu, X. Guo, R. Hu, X. Liu, S. Wang, S. Li, Y. Li, G. Yang, Molecular
engineering of aqueous soluble triarylboron-compound-based two-photon fluorescent
probe for mitochondria H2S with analyte-induced finite aggregation and excellent
membrane permeability, Anal. Chem. 88 (2016) 1052-1057.
[32] Y.L. Pak, J. Li, K.C. Ko, G. Kim, J.Y. Lee, J. Yoon, Mitochondria-targeted
reaction-based fluorescent probe for hydrogen sulfide, Anal. Chem. 88 (2016)
5476-5481.
[33] N. Velusamy, A. Binoy, K.N. Bobba, D. Nedungadi, N. Mishra, S. Bhuniya, A
bioorthogonal fluorescent probe for mitochondrial hydrogen sulfide: new strategy for
cancer cell labeling, Chem. Commun. 53 (2017) 8802-8805.
[34] L. Zhou, D. Lu, Q. Wang, S. Liu, Q. Lin, H. Sun, Molecular engineering of a
mitochondrial-targeting two-photon in and near-infrared out fluorescent probe for
gaseous signal molecules H2S in deep tissue bioimaging, Biosens. Bioelectron. 91
(2017) 699-705.
[35] X. Feng, T. Zhang, J.-T. Liu, J.-Y. Miao, B.-X. Zhao, A new ratiometric
fluorescent probe for rapid, sensitive and selective detection of endogenous hydrogen
sulfide in mitochondria, Chem. Commun. 52 (2016) 3131-3134.
[36] X.J. Zou, Y.C. Ma, L.E. Guo, W.X. Liu, M.J. Liu, C.G. Zou, Y. Zhou, J.F.
Zhang, A lysosome-targeted fluorescent chemodosimeter for monitoring endogenous
and exogenous hydrogen sulfide by in vivo imaging, Chem. Commun. 50 (2014)
13833-13836.

ACCEPTED MANUSCRIPT
[37] Z. Wu, D. Liang, X. Tang, Visualizing hydrogen sulfide in mitochondria and
lysosome of living cells and in tumors of living mice with positively charged
fluorescent chemosensors, Anal. Chem. 88 (2016) 9213-9218.
[38] Y. Liu, F. Meng, L. He, K. Liu, W. Lin, A dual-site two-photon fluorescent
probe for visualizing lysosomes and tracking lysosomal hydrogen sulfide with two
different sets of fluorescence signals in the living cells and mouse liver tissues, Chem.
Commun. 52 (2016) 7016-7019.
[39] X. Zhang, H. Tan, Y. Yan, Y. Hang, F. Yu, X. Qu, J. Hua, Targetable
N-annulated perylene-based colorimetric and ratiometric near-infrared fluorescent
probes for the selective detection of hydrogen sulfide in mitochondria, lysosomes, and
serum, J. Mater. Chem. B 5 (2017) 2172-2180.
[40] T. Liu, Z. Xu, D.R. Spring, J. Cui, A lysosome-targetable fluorescent probe for
imaging hydrogen sulfide in living cells, Org. Lett. 15 (2013) 2310-2313.
[41] X. Cao, W. Lin, K. Zheng, L. He, A near-infrared fluorescent turn-on probe for
fluorescence imaging of hydrogen sulfide in living cells based on thiolysis of
dinitrophenyl ether, Chem. Commun. 48 (2012) 10529-10531.
[42] Y. Tang, D. Lee, J. Wang, G. Li, J. Yu, W. Lin, J. Yoon, Development of
fluorescent probes based on protection–deprotection of the key functional groups for
biological imaging, Chem. Soc. Rev. 44 (2015) 5003-5015.

ACCEPTED MANUSCRIPT
Chart 1. The Structures of Fluorescent Probes Developed.
Scheme 1. Syntheses of Fluorescent Probes.
Figure 1. Absorption and fluorescence spectra (ex = 590 nm) of 10 M of probes 1
(a, b) and 2 (c, d) prior to (black) and after (red) addition of NaHS (50 M) in PBS
solution (10 mM, pH = 7.2) at 25 ºC (Insets in c and d: the color change under room
light and UV irradiation viewed by naked eyes respectively).
Figure 2. Time-dependent fluorescence responses (ex = 590 nm) monitored from 4
seconds at emission maxima for probes 1 and 2 (10 M each) after addition of NaHS
(50 M) in PBS solution (10 mM, pH = 7.2) at 25 ºC.
Figure 3. Fluorescence responses of probe 2 (10 μM) to various amino acids,
oxidants (1 mM each) in comparison with NaHS (50 μM) at room temperature
monitored at 680 nm (ex = 590 nm) in PBS buffer (10 mM, pH = 7.2). Data were
recorded at 2 minutes after addition of various analytes.
Figure 4. Fluorescence intensities changes of probe 2 (10 µM) in the presence of
increasing concentrations of NaHS (0, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM) in PBS
solution (10 mM, pH = 7.2) at 25 ºC (ex/em = 590/680 nm).
Figure 5. Confocal fluorescence images of Hela (row A) and HepG2 cells (row B)
with multiple labels. Cells incubated with Lyso-Tracker Green (100 nM for A, B) for
20 min and probe 2 (10 μM) for 10 min, and then incubated with NaHS (100 μM) for
10 min. All cells were incubated at 37 °C and all cells were rinsed three times with
0.1 M Hank’s buffer before imaging (A1/B1: green channel images; A2/B2: red
channel images; A3/B3: merged images, A4/B4: Fluorescent intensity profiles of the
regions of interest in merged image).
Figure 6. In vivo imaging of H2S in living mice. Upper row: i.p. injection of probe 2
(50 M, 0.2 mL) right after administrating of NaHS (20 mM, 0.2 mL); Middle row:
i.p. injection of probe 2 (50 M, 0.2 mL) 10 min after administrating of NaHS (20
mM, 0.2 mL); Bottom row: i.p. injection of NaHS (20 mM, 0.2 mL) after

ACCEPTED MANUSCRIPT
administrating of probe 2 (50 M, 0.2 mL). Mice without injection were used as
control in all experiments.

ACCEPTED MANUSCRIPT
Highlights
 A novel water soluble cyanine-based NIR fluorescent Probe 2 for H2S.
 Responded ultrafast and specifically to H2S with the detection limit of 11 nM
in PBS buffer.
 Lysosome-targeted and capable of detecting endogenous, exogenous, and even
the
intrinsically
produced
H2S
(both
enzymatic/non-enzymatic
biosynthesized).
 Successfully applied to detect H2S in living mice and demonstrated the fast
metabolism of H2S.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7