Highly selective red-emitting H2S fluorescent probe with a large Stokes shift

Article abstract of DOI:10.1016/j.dyepig.2014.09.035

The O-2,4-dinitrobenzensulfonate of 1,3-bis(bispyridin-2ylimino)isoindolin-4-ol has been developed as a novel red-emitting fluorescent probe for the detection of H₂S. The dinitrobenzenesulfonate moiety in the probe both prohibits the excited st

Full text of DOI:10.1016/j.dyepig.2014.09.035

Dyes and Pigments 113 (2015) 596e601
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Dyes and Pigments
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Highly selective red-emitting H2S fluorescent probe with a large
Stokes shift
, c, *
Wenqiang Chen a, Song Chen a, Bingjiang Zhou b, Hongbo Wang a, Xiangzhi Song a
,
,
**
Hongyan Zhang b
a College of Chemistry & Chemical Engineering, Central South University, 932 Lushan South Road, Changsha, Hunan Province, 410083, PR China
b Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing,
100190, PR China
c State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, Liaoning Province, 116024, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The O-2,4-dinitrobenzensulfonate of 1,3-bis(bispyridin-2ylimino)isoindolin-4-ol has been developed as a
novel red-emitting fluorescent probe for the detection of H2S. The dinitrobenzenesulfonate moiety in the
probe both prohibits the excited state intramolecular proton transfer process and produces a photo-
induced electron transfer process, which renders the probe non-fluorescent in the absence of the analyte.
In the presence of H2S a specific H2S-mediated cleavage reaction converts the probe into 1,3-
bis(bispyridin-2-ylimino)isoindolin-4-ol which exhibits a strong red fluorescence with a large Stokes
shift (218 nm) via an excited state intramolecular proton transfer process upon excitation. It's noteworthy
that this new probe shows good selectivity and sensitivity to H2S over glutathione, cysteine and ho-
mocysteine. Moreover successful detection and imaging of intracellular H2S in living cells was achieved.
To our knowledge this is the first application of this type of fluorescent probe for intracellular H2S
detection.
Received 15 June 2014
Received in revised form
23 September 2014
Accepted 24 September 2014
Available online 2 October 2014
Keywords:
Red-emitting
Large Stokes shift
ESIPT
Fluorescent probe
Hydrogen sulfide
Cell imaging
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
quantitative detection of H2S in living systems is tremendously
meaningful.
For several centuries, hydrogen sulfide (H2S) was conventionally
considered as a toxic gas and environmental hazard affecting the
nervous, respiratory, and cardiovascular system of mammals [1].
Resembling CO and NO, H2S has more recently emerged as the third
endogenous gasotransmitter and cellular signaling molecule
involved in many physiological activities, i.e., regulating the car-
diovascular, neuronal, immune, endocrine and gastrointestinal
systems [2e5]. Furthermore, H2S serves as a scavenger for endog-
enous reactive oxygen species (ROS) [6]. Due to its important roles
in human health, abnormal levels of H2S can lead to serious dis-
eases such as Alzheimer's disease [7] and Down's syndrome [8].
Therefore, interest in the role and detection of H2S has increased
Owing to its operational simplicity, potential high sensitivity
and selectivity, fluorescent sensing as a powerful technique has
been frequently used to detect and image H2S in vivo [9]. Many
fluorescent H2S probes have been developed in the past several
years [10e14]. Generally, 2,4-dinitrobenzensulfonyl unit is used as
a recognition group to construct fluorescent probes for the detec-
tion of biothiols (Cys, GSH, Hcy) [15e17]. Since H2S is more reactive
than biothiols, it can also cleave the 2,4-dinitrobenzensulfonyl
moiety and previous DNBS-based fluorescent probes didn't show
selectivity between H2S and biothiols [18]. Therefore, to date and to
our knowledge, there is no DNBS-based fluorescent probe reported
to detect and image intracellular H2S in living cells due to the
interference of biothiols.
during the last decade. As
a consequence, qualitative and
It is well known that fluorescent dyes with emission in the red
and NIR regions and large Stokes shift are favorable for fluorescence
imaging in cells because: 1) photons with longer wavelength tend
to reduce environmentally-induced light scattering, be less sus-
ceptible to the experiment noise from endogenous chromophores,
and usually cause less light-induced organelle damage [19]; 2) the
large Stokes shift can highly improve the sensitivity of fluorescence
* Corresponding author. State Key Laboratory of Fine Chemicals, Dalian University
of Technology, Dalian, Liaoning Province, 116024, PR China. Tel./fax: þ86 731
88836954.
** Corresponding author.
E-mail addresses: xzsong@csu.edu.cn (X. Song), zhanghongyan@mail.ipc.ac.cn
(H. Zhang).
http://dx.doi.org/10.1016/j.dyepig.2014.09.035
0143-7208/© 2014 Elsevier Ltd. All rights reserved.

W. Chen et al. / Dyes and Pigments 113 (2015) 596e601
597
microscopy wherein emission photons can be detected against the
background from excitation photons [21]. Recently, Thompson et al.
reported a class of ESIPT (excited state intramolecular proton
transfer) fluorescent dyes involving the 1,3-bis(imino)isoindolediol
motif (BPI), having an emission in red region (about 600 nm), high
quantum yields (up to 45%) and large Stokes shift (>165 nm) [20].
Inspired by these traits of the BPI dyes, our group synthesized a
new BPI dye, 1,3-bis(bispyridin-2ylimino)isoindolin-4-ol (3)
(shown in Scheme 1), and deliberately studied its photophysical
properties. In aqueous solution, reference dye 3 emits bright red
lights (lmax ¼ 585 nm) with a 218 nm Stokes shift (shown in Fig. 1)
and is photostable. Considering these advantages, we envision that
reference dye 3 might be an excellent candidate upon which fluo-
rescent probes are based.
In this work, we reported a DNBS-based red-emitting fluores-
cent probe, BPI-DNBS (shown in Scheme 1), for the selective and
sensitive detection of H2S. This probe is non-fluorescent in the
absence of H2S. However, the probe rapidly displays a remarkable
fluorescence enhancement (lmax ¼ 585 nm) upon the addition of
H2S. Particularly, biothiols including Cys, Hcy and GSH only induce
weak fluorescence signal. To the best of our knowledge, this is the
first DNBS-based fluorescent probe to distinguish H2S from bio-
thiols. The capability of BPI-DNBS to visualize intracellular H2S in
living A594 cells has been demonstrated.
Fig. 1. The absorption (-) and emission spectra (:) of reference dye 3 and BPI-DNBS
in HEPES-DMSO solution (20 mM, v/v ¼ 4:1, pH ¼ 7.0). Black dotted lines: dye 3; red
solid lines: BPI-DNBS. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
C1si inverted microscope. TLC analysis was performed on silica gel
plates and column chromatography was conducted over silica gel
(mesh 200e300), both of which were obtained from Qingdao
Ocean Chemicals. The solutions of various testing species were
prepared from NaClO4$H2O, Na2SO4, NaF, KI, NaCl, NaBr, NaN3,
NaNO3, Na2S2O3$5H2O, Na2SO3, NaNO2, CH3COONa, Na2CO3, NaBF4,
KSCN, Hcy, GSH, Cys, H2O2, NaClO in twice-distilled water. Density
functional theory (DFT) calculations with the B3LYP exchange
functional employing 6-31G basis sets using a suite of Gaussian 09
programs were performed.
2. Experimental
2.1. Chemicals and apparatus
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purification. All
solvents were purified by standard methods. Twice-distilled water
was used throughout all experiments. NMR spectra were recorded
on a BRUKER 400 spectrometer, using TMS as an internal standard.
FT-IR spectra were recorded on a Thermo Scientific ET6700 FT-IR
spectrophotometer. All accurate mass spectrometric experiments
were performed on a micrOTOF-Q II mass spectrometer (Bruker
Daltonik, Germany). UVeVis absorption spectra were measured
using a Shimadzu UV-2450 spectrophotometer. Emission spectra
were recorded at room temperature using a HITACHI F4600 fluo-
rescence spectrophotometer with both the excitation and emission
slit widths set at 5.0 nm. Cell imaging was performed with a Nikon
2.2. Synthesis
The synthetic route of BPI-DNBS was shown in Scheme 1. 3-
Hydroxypathalonitrile was condensed with 2-aminopyridine to
give reference dye 3, which subsequently reacted with 2,4-
dinitrobenzenesulfonyl chloride to afford the fluorescent probe.
2.2.1. Synthesis of compound 2
3-Hydroxypthalonitrile was synthesized according to the liter-
ature method [22]. To a 50 mL round-bottom flask were added 3-
Scheme 1. Synthesis of BPI-DNBS and the approach to detecting H2S.

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W. Chen et al. / Dyes and Pigments 113 (2015) 596e601
nitropthalonitrile (2.0 g, 11.6 mmol), K2CO3 (1.8 g, 12.7 mmol) and
NaNO2 (0.8 g, 11.6 mmol) in DMSO (30 mL), and the reaction
mixture was stirred under reflux for 30 min. After cooling to room
temperature, the reaction mixture was diluted with water (90 mL)
and subsequently acidified with 2 M HCl to pH ¼ 3 to produce a
precipitate. Then, the solid was collected by filtration and washed
successively with water and methanol. The pure product was ob-
tained by recrystallization in acetic acid as a brown crystal (0.8 g,
48%). Mp: 263e265 ꢀC.
cells were washed with HEPES buffer solution. A549 cells were then
incubated with a solution of BPI-DNBS (5.0 mM in HEPES buffer
containing 1% DMSO) at 37 ꢀC for 30 min. After washing 3 times
with HEPES buffer, the pretreated A549 cells were then incubated
with NaHS (200.0 mM) for another 30 min. Fluorescence imaging
was performed with excitation wavelength at 405 nm using Cy3
channel. For a control experiment, A549 cells were incubated with
the solution of BPI-DNBS (5.0 m C
M) in the culture medium at 37 ꢀ
for 30 min, and the fluorescence imaging was carried out after
washing the cells with HEPES buffer 3 times.
2.2.2. Synthesis of reference dye 3
To a 25 mL round-bottom flask were added compound 2 (288 mg,
2 mmol), 2-aminopyridine (385 mg, 4.1 mmol) and CaCl2 (46 mg,
0.41 mmol) in n-BuOH (6 mL). The reaction mixture was refluxed
under an argon atmosphere for 5 days with stirring. After cooling to
room temperature, the precipitate was collected by filtration and
washed with water (3 ꢁ 20 mL). The obtained solid was further
purified by silica gel column chromatography (dichloromethane as
eluent) to yield the pure reference dye 3 as an orange yellow powder
3. Results and discussion
3.1. Spectroscopic evaluation of reference dye 3 and BPI-DNBS
The photophysical properties of reference dye 3 and BPI-DNBS
were investigated in HEPES-DMSO solution (20 mM, v/v ¼ 4:1,
pH ¼ 7.0). As shown in Fig. 1, reference dye 3 absorbs at 367 nm and
emits at 585 nm. The extremely large Stokes shift (218 nm) was
ascribed to the effective ESIPT process from the hydroxyl proton to
the imine nitrogen. The fluorescent quantum yield of reference dye
3 was 3.8% in HEPES-DMSO solution (coumarin 1 in EtOH with
Фf ¼ 0.73 as a reference) [23]. However, BPI-DNBS displays an
absorption band centered at 396 nm with a faint yellow color. In
BPI-DNBS, the ESIPT process was inhibited by the DNBS protection,
which can strongly quench the fluorescence. Moreover, the density
function theory calculations showed that the LUMO energy level
(ꢂ0.084 eV) of reference dye 3 is higher than that of dinitrobenzene
(ꢂ0.134 eV) moiety (Fig. 2). Thus, the photo-induced electron
transfer (PET) process from the excited reference dye 3 to the
dinitrobenzene moiety is thermodynamically favorable, which also
leads to the effective fluorescence quenching. Due to the inhibition
of ESIPT and the effective PET process, BPI-DNBS is essentially non-
fluorescent (not detectable).
(68 mg,12%). Mp: 200e202 ꢀC. 1H NMR (400 MHz, CDCl3)
d 13.70 (s,
1H), 8.62 (m, 2H), 7.78 (t, J ¼ 7.9 Hz, 2H), 7.60 (s,1H), 7.52 (d, J ¼ 7.3Hz,
2H), 7.38 (d, J ¼ 8.0 Hz, 1H), 7.19e7.09 (m, 3H). 13C NMR (101 MHz,
CDCl3) d 160.10, 159.34, 155.81, 155.59, 153.70,147.98, 147.76,138.12,
138.08, 135.66, 133.58, 123.43, 122.31, 120.50, 120.27, 118.98, 118.29,
114.52. IR (KBr) nmax cmꢂ1: 3446, 1631, 1580, 1550, 1459, 1431, 1309,
1226, 1151, 1057, 790. HRMS (EI) Calcd. for C18H14N5O ([M þ H]þ)：
316.1198; Found: 316.1197.
2.2.3. Synthesis of BPI-DNBS
A
solution of reference dye 3 (32 mg, 0.1 mmol), 2,4-
dinitrobenzenesulfonyl chloride (53 mg, 0.2 mmol) and triethyl-
amine (26 mg, 0.24 mmol) in dichloromethane (5 mL) was stirred at
room temperature for 2 h. After the removal of solvent under
reduced pressure, the resulting residue was further purified by
column chromatography (silica gel, dichloromethane as eluent) to
give the target compound, BPI-DNBS, as a light yellow solid (16 mg,
3.2. Sensing response of BPI-DNBS to H2S
30%). Mp: 184e187 ꢀC. 1H NMR (400 MHz, CDCl3)
d 13.83 (s, 1H),
8.57e8.52 (m, 2H), 8.49 (d, J ¼ 9.9 Hz, 2H), 8.37 (d, J ¼ 8.6 Hz, 1H),
8.17 (s, 1H), 7.80 (t, J ¼ 7.7 Hz, 1H), 7.73 (t, J ¼ 7.9 Hz, 2H), 7.55 (d,
J ¼ 8.1 Hz, 1H), 7.50 (s, 1H), 7.18e7.10 (m, 2H), 7.04 (d, J ¼ 7.9 Hz,1H).
First, we investigated the UVevis absorption spectral changes of
BPI-DNBS in response to H2S. The addition of H2S with an
increasing amount to the solution of BPI-DNBS (5.0 mM) gradually
13C NMR (101 MHz, CDCl3)
d 159.6, 150.3, 148.8, 148.0, 147.7, 144.2,
elicited a blue shift to 367 nm in its absorption spectra, which is
138.3, 138.1, 135.1, 133.7, 133.2, 127.1, 126.8, 126.1, 123.4, 122.6, 120.8,
120.7, 120.0. IR (KBr) nmax cmꢂ1: 3453, 3034, 1630, 1579, 1558, 1458,
1391, 1357, 1262, 1200, 1171, 1097, 1047, 962, 800, 732. HRMS (EI)
Calcd. for C24H16N7O7S ([M þ H]þ): 546.0832; Found: 546.0817.
2.3. Absorption and fluorescence spectroscopy
The stock solution of the probe, BPI-DNBS, was prepared at
1 mM in DMSO. The test solution of BPI-DNBS (5.0 mM) in 2 mL of
20 mM HEPES buffer (20 mM, pH ¼ 7.0) was prepared by placing
0.01 mL stock solution of BPI-DNBS and 0.39 mL DMSO in 1.59 mL
HEPES buffer. The resulting solution was shaken well and incubated
with 0.01 mL appropriate testing species for 60 min at 37 ꢀC before
recording the spectra. For all measurements of fluorescence
spectra, excitation wavelength was set at 368 nm and the scan
speed is 1200 nm minꢂ1
.
2.4. Cell culture and fluorescence confocal imaging
A549 was gifted from the center of cells, Peking Union Medical
College. Cells were grown in McCoy'5A, supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin, incubated under
5% CO2 at 37 ꢀC. Cells were seeded on confocal dish for imaging 24 h
prior to conducting the experiments. Before the experiments, the
Fig. 2. Frontier orbital diagrams of reference dye 3 (the dye scaffold) and dinitro-
benzene (the PET switch) for the probe BPI-DNBS. Orbital energies were calculated by
using Gaussian 09 program in B3LYP/6-31G level.

W. Chen et al. / Dyes and Pigments 113 (2015) 596e601
599
Fig. 3. A) Fluorescence spectra of BPI-DNBS (lex ¼ 368 nm, 5.0
intensity at 585 nm of BPI-DNBS (5.0 M) as a function of NaHS concentration; b) fluorescence images of BPI-DNBS in the absence (left) and presence of NaHS (right). B) Fluo-
rescence intensity ratio (F/F0) at 585 nm of BPI-DNBS (5.0
M) as a function of NaHS concentration in HEPES-DMSO solution (20 mM HEPES, v/v ¼ 4:1, pH ¼ 7.0).
m
M) upon the addition of NaHS (0.0e20.0
m
M) in HEPES-DMSO solution (v/v ¼ 4:1, pH ¼ 7.0). Inset: a) fluorescence
m
m
quite similar to the absorption spectra of reference dye 3 (Fig. S2).
The emission spectra of BPI-DNBS toward H2S in HEPES-DMSO
solution (20 mM, v/v ¼ 4:1, pH ¼ 7.0) are displayed in Fig. 3. The
solution of BPI-DNBS rapidly exhibits a strong red fluorescence
enhancement (up to 55-fold) upon the treatment of H2S. The
fluorescence spectrum of the reaction product is identical to that of
reference dye 3. The UVevis and fluorescence studies clearly indi-
cated that H2S could effectively cleave 2,4-dinitrobenzensulfonate
unit in BPI-DNBS, generating reference dye 3 which displays a
strong red fluorescence upon excitation (shown in Scheme 1). In-
spection of Fig. 3 shows that the fluorescence intensity at 585 nm
exhibits negligible fluorescence in response to the representative
anions (AcOꢂ, BF4ꢂ, Brꢂ, Clꢂ, ClOꢂ4 , CO23ꢂ, Fꢂ, Iꢂ, N3ꢂ, NOꢂ2 , NOꢂ3 , SO42ꢂ
SCNꢂ), reactive oxygen (ClOꢂ, H2O2), and reducing anions (S2O25ꢂ
,
,
S2O23ꢂ, SO23ꢂ). It's noteworthy that biothiols (Cys, Hcy and GSH) only
produce a weak fluorescence signal even at a high concentration
(200.0 mM for Cys, Hcy and GSH). The results demonstrate that BPI-
DNBS can be used to detect H2S with good selectivity over biothiols,
which makes it capable of detecting H2S in living cells. This selectivity
might be attributed to the differences of molecular size and pKa be-
tween H2S and biothiols. First, due to the steric effect by the adjacent
pyridinyl group in BPI-DNBS, H2S can effect the thiolysis of 2,4-
dinitrobenzensulfonate more effectively than biothiols owing to its
smaller size. Second, the pKaofH2S is6.9 in aqueoussolutionwhereas
it is approximately around 8.5 for biothiols. Thus, at physiological pH
value, H2S mainly exists in the more reactive form, HSꢂ, and can
readily react with the probe [24,25]. Ongoing studies are being per-
formed in our laboratory to prove this postulation.
increased with increasing the H2S concentrations (0e15
mM) with a
good linearity, R ¼ 0.9983. Specifically, the addition of 1.0
m
M H2S
could result in a fluorescence enhancement more than 3 folds
(Fig. S3). The detection limit was calculated to be 1.5 ꢁ 10ꢂ8 mol Lꢂ1
under experimental conditions (S/N ¼ 3). Thus, it can be concluded
that BPI-DNBS is highly sensitive to H2S.
3.3. Selectivity studies
3.4. Effect of pH on fluorescence detection of H2S
To evaluate the selectivity of this probe, we then measured the
fluorescence response of BPI-DNBS to typical anions and other bio-
logically relevant species. As shown in Fig. 4, the fluorescent probe
In order to confirm the practical applicability of BPI-DNBS, we
further investigate the influence of pH on the fluorescence of this
probe. It can be seen in Fig. 5 that BPI-DNBS shows little
Fig. 4. Fluorescence intensity ratio (F/F0) at 585 nm of the probe BPI-DNBS (5.0
response to various anions, reactive oxygen species, reducing agents, biothiols and
NaHS (20.0
M for AcOꢂ, BFꢂ4 , Brꢂ, Clꢂ, ClO4ꢂ, CO32ꢂ, Fꢂ, Iꢂ, N3ꢂ, NO2ꢂ, NOꢂ3 , SO42ꢂ, SCNꢂ
S2O25ꢂ, S2O23ꢂ and SO32ꢂ; 50.0 M for ClOꢂ and H2O2; 200.0
M for GSH, Hcy and Cys;
20.0 M for NaHS).
mM) in
Fig. 5. pH effect on the fluorescence intensity at 585 nm of BPI-DNBS (5.0
mM) in the
m
,
absence (black line) and presence (red line) of NaHS (20.0 M) in H2O-DMSO solution
m
m
m
(v/v ¼ 4:1). (For interpretation of the references to color in this figure legend, the
m
reader is referred to the web version of this article.)

600
W. Chen et al. / Dyes and Pigments 113 (2015) 596e601
Fig. 6. Confocal fluorescence contrast images of living A594 cells incubated with 5.0 m mM NaHS for 30 min
M BPI-DNBS for 30 min at 37 ꢀC (top row) and further treated with 200.0
at 37 ꢀC (bottom row). (a, d) Fluorescence images; (b, e) bright field images; (c, f) fluorescence and bright field overlay images. Scale bar ¼ 10
mm.
fluorescence within a wide pH range (1e10), indicating that this
fluorescent probe is stable between pH 1e10. When treated with 5
equivalents of H2S, BPI-DNBS displays a strong fluorescence be-
tween pH 6e8. This result suggested that BPI-DNBS could work
effectively under physiological condition.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.09.035.
References
3.5. Cell imaging
[1] Beauchamp RO, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA, Leber P. A critical
review of the literature on hydrogen sulfide toxicity. CRC Crit Rev Toxicol
1984;13:25e97.
[2] Villa Bianca RE, Sorrentino R, Mirone V, Cirino G. Hydrogen sulfide and erectile
function: a novel therapeutic target. Nat Rev Urol 2011;8:286e9.
[3] Miller TW, Isenberg JS, Roberts DD. Molecular regulation of tumor angio-
genesis and perfusion via redox signaling. Chem Rev 2009;109:3099e124.
[4] Szabo C. Gaseotransmitters: new frontiers for translational science. Sci Transl
Med 2010;2:54e8.
[5] Wang R. The gasotransmitter role of hydrogen sulfide. Antioxid Redox Signal
2003;5:493e501.
[6] Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, et al.
Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res
2009;105:365e74.
Finally, we applied BPI-DNBS to capture H2S in living A594 cells
(human lung cancer cells), as shown in Fig. 6. When A594 cells were
incubated with 5.0
m
M BPI-DNBS for 30 min at 37 ꢀC and then
washed twice with PBS buffer, only weak fluorescence was
observed due to the weak response to biothiols. However, intense
red fluorescence in cells emerged when similar treated cells were
further incubated with NaHS (200.0 m
M) for 30 min at 37 ꢀC. This
result indicated that BPI-DNBS was permeable to cell membrane
and able to response to endogenous H2S in living cells.
[7] Eto K, Asada T, Arima K, Makifuchi T, Kimura H. Brain hydrogen sulfide is
severely decreased in Alzheimer's disease. Biochem Biophys Res Commun
2002;293:1485e8.
[8] Kamoun P, Belardinelli MC, Chabli A, Lallouchi K, Chadefaux-Vekemans B.
Endogenous hydrogen sulfide overproduction in down syndrome. Am J Med
Genet A 2003;116:310e1.
[9] Kim JS, Quang DT. Calixarene-derived fluorescent probes. Chem Rev
2007;107:3780e99.
[10] Peng H, Cheng Y, Dai C, King AL, Predmore BL, Lefer DJ, et al. A fluorescent
probe for fast and quantitative detection of hydrogen sulfide in blood. Angew
Chem Int Ed 2011;50:9672e5.
[11] Lippert AR, New EJ, Chang CJ. Reaction-based fluorescent probes for selective
imaging of hydrogen sulfide in living cells. J Am Chem Soc 2011;133:10078e80.
[12] Sasakura K, Hanaoka K, Shibuya N, Mikami N, Kimura Y, Komatsu T, et al.
Development of a highly selective fluorescence probe for hydrogen sulfide.
J Am Chem Soc 2011;133:18003e5.
4. Conclusions
In summary, we have successfully developed a novel red-
emitting H2S fluorescence probe employing a 2,4-dinitrosulfonyl
moiety as the sensing group (lmaxem ¼ 585 nm). This probe dis-
plays a large Stokes shift (218 nm) in response to H2S that is
beneficial for signal detection in fluorescence microscopy. It's
noteworthy that this DNBS-based probe is highly sensitive and
selective toward H2S against biothiols and other relevant ions.
Importantly, BPI-DNBS displayed good cell permeability and was
successfully applied to visualize intracellular H2S in living cells.
[13] Liu C, Pan J, Li S, Zhao Y, Wu LY, Berkman CE, et al. Capture and visualization of
hydrogen sulfide by a fluorescent probe. Angew Chem Int Ed 2011;123:10511e3.
[14] Chen Y, Zhu C, Yang Z, Chen J, He Y, Jiao Y, et al. A ratiometric fluorescent
probe for rapid detection of hydrogen sulfide in mitochondria. Angew Chem
Int Ed 2013;125:1732e5.
[15] Maeda H, Matsuno H, Ushida M, Katayama K, Saeki K, Itoh N. 2,4-
Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman's
reagent in thiol-quantification enzyme assays. Angew Chem Int Ed 2005;44:
2922e5.
Acknowledgments
The research was supported by the Fundamental Research
Funds for the Central Universities, the State Key Laboratory of Fine
Chemicals (KF1202) and the Hunan Nonferrous Fund (No.
YSZN2013CL02). We thank Modern Analysis and Testing Center of
CSU for NMR spectral data of all the compounds.

W. Chen et al. / Dyes and Pigments 113 (2015) 596e601
601
[16] Bouffard J, Kim Y, Swager TM, Weissleder R, Hilderbrand SA. A highly selective
fluorescent probe for thiol bioimaging. Org Lett 2008;10:37e40.
[17] Shao J, Guo H, Ji S, Zhao J. Styryl-BODIPY based red-emitting fluorescent
OFFeON molecular probe for specific detection of cysteine. Biosens Bio-
electron 2011;26:3012e7.
[18] Yang XF, Wang L, Xu H, Zhao M. A fluorescein-based fluorogenic and chro-
mogenic chemodosimeter for the sensitive detection of sulfide anion in
aqueous solution. Anal Chim Acta 2009;631:61e5.
[19] Yuan L, Lin W, Yang Y, Chen H. A unique class of near-infrared functional
fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF
switching: rational design, synthesis, optical properties, theoretical calcula-
tions, and applications for fluorescence imaging in living animals. J Am Chem
Soc 2012;134:1200e11.
[21] Hou P, Chen S, Wang H, Wang J, Voitchovskya K, Song X. An aqueous red
emitting fluorescent fluoride sensing probe exhibiting a large Stokes shift and
its application in cell imaging. Chem Commun 2014;50:320e2.
[22] Ranta J, Kumpulainen T, Lemmetyinen H, Efimov A. Synthesis and charac-
terization of monoisomeric 1,8,15,22-substituted (A3B and A2B2) phthalo-
cyanines and phthalocyanine-fullerene dyads. J Org Chem 2010;75:5178e94.
[23] Jones G, Jackson WR, Choi C. Solvent effects on emission yield and lifetime for
coumarin laser dyes. Requirements for a rotatory decay mechanism. J Phys
Chem 1985;89:294e300.
[24] Mathai JC, Missner A, Kügler P, Saparov SM, Zeidel ML, Lee JK, et al. No
facilitator required for membrane transport of hydrogen sulfide. Proc Natl
Acad Sci U S A 2009;106:16633e8.
[25] Salinas CN, Anseth KS. Mixed mode thiol acrylate photopolymerizations for
the synthesis of PEG peptide hydrogels. Macromolecules 2008;41:6019e26.
[20] Hanson K, Patel N, Whited MT, Djurovich PI, Thompson ME. Substituted 1,3-
bis(imino)isoindole diols:
a new class of proton transfer dyes. Org Lett
2011;13:1598e601.