A near-infrared fluorescent turn-on probe for fluorescence imaging of hydrogen sulfide in living cells based on thiolysis of dinitrophenyl ether

Article abstract of DOI:10.1039/c2cc34031c

We have constructed a novel NIR fluorescent turn-on hydrogen sulfide probe suitable for fluorescent imaging in living cells based on thiolysis of dinitrophenyl ether.

Full text of DOI:10.1039/c2cc34031c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 10529–10531
www.rsc.org/chemcomm
COMMUNICATION
A near-infrared fluorescent turn-on probe for fluorescence imaging of
hydrogen sulfide in living cells based on thiolysis of dinitrophenyl etherw
Xiaowei Cao, Weiying Lin,* Kaibo Zheng and Longwei He
Received 6th June 2012, Accepted 6th September 2012
DOI: 10.1039/c2cc34031c
We have constructed a novel NIR fluorescent turn-on hydrogen
sulfide probe suitable for fluorescent imaging in living cells based
on thiolysis of dinitrophenyl ether.
azides to fluorescent amines has been employed to construct
fluorescent H2S probes by Chang’s,26 Wang’s,27 and Han’s28
groups. Xian et al. introduced a turn-on strategy for fluorescent
H2S probes based on a H2S-triggered benzothiolone compounds
formation.29 Nagano’s group reported a fluorescent probe for
H2S by utilizing azamacrocyclic copper(II) ion complex chemistry
to regulate the fluorescence.30 Pluth’s group constructed fluores-
cence turn-on probes for H2S via H2S-mediated reduction of the
nitro group to amines.31
Hydrogen sulfide (H2S), traditionally considered to be a toxic
gas with the typical smell of rotten eggs, has emerged as a
member of the endogenous gaseous transmitter family of
signaling molecules including nitric oxide (NO) and carbon
monoxide (CO).1–3 The endogenous production of H2S from a
cysteine substrate or its derivatives is catalysed by several
enzymes such as cystathionine b-synthase, cystathionine
g-lyase, and 3-mercaptopyruvate sulphurtransferase4–6 in distinct
mammalian tissues. H2S is endogenously produced in mitochondria
and/or cytosol depending on the species and organs.7 H2S may
interact with downstream proteins by post-translational cysteine
sulfhydration8 and binding to heme iron centers,9 which regulates
various physiological processes including ischemia reperfusion
injury,10 vasodilation,11 apoptosis,12 angiogenesis,13 neuro-
modulation,14 inflammation,15 insulin signaling,16 and oxygen
sensing.17 Furthermore, H2S also serves as an antioxidant or
scavenger for reactive oxygen species (ROS).18 However,
abnormal levels of H2S are associated with various diseases,
like Alzheimer’s disease19 and Down syndrome.20
Herein, we present the development of NIR-H2S (Scheme 1),
a unique type of a fluorescent turn-on probe for H2S based on
dinitrophenyl ether chemistry. The dinitrophenyl group is often
used for the protection of tyrosine in peptide synthesis. The
removal of the dinitrophenyl protective group is conducted
using thiols as the thiolyting agents under basic conditions.32
H2S is a small gas molecule and has a pKa of around 6.9,33 while
the typical cellular free thiols (i.e. glutathione, cysteine) have
higher pKa values about 8.5.34 Thus, based on the marked
distinctions in terms of size and pKa values, we envisioned that,
at physiological pH, the thiolysis of the dinitrophenyl ether
reaction may be chemoselective for H2S over biologically
abundant glutathione and cysteine.
Compound NIR-H2S was readily synthesized in two steps
(Scheme 1). Condensation of the BODIPY 1 with Fisher
aldehyde 2 afforded the intermediate 3, which was then treated
with 1-fluoro-2,4-dinitrobenzene under the basic conditions to
give the target compound NIR-H2S by a nucleophilic substitu-
tion. The structures of the compounds synthesized were fully
characterized by the standard NMR and mass spectrometry.
We first evaluated the capability of NIR-H2S to detect H2S
in aqueous buffer. The titration of NaHS (a standard source
Detection of H2S in living systems has attracted great attention
recently. The current techniques for H2S detection include colori-
metric21 and electrochemical22 methods, chromatography,23 and
sulfide precipitation.24 However, these methods require the living
bio-samples to be post-mortem processed and destructed. Thus,
these methods are destructive and not suitable for monitoring
H2S in the native biological environment.
By contrast, fluorescence imaging provides an attractive
technique to study biomolecules in live cells. Recently, a limited
number of well-designed turn-on type fluorescent probes for
H2S have been constructed. For instance, He’s group developed
selective fluorescent H2S probes on the basis of H2S-induced
tandem chemical reactions.25 The H2S-mediated reduction of
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082, P. R. China.
E-mail: weiyinglin@hnu.edu.cn; Fax: +86-731-88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data, and additional spectra. See DOI:
10.1039/c2cc34031c
Scheme 1 Design and synthesis of the NIR fluorescent turn-on probe
NIR-H2S.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10529–10531 10529

View Article Online
wavelength (Fig. S8, ESIw), further substantiating the PET
signaling mechanism.
To examine the selectivity, the probe NIR-H2S (5 mM) was
treated with various biologically relevant species (e.g., the
representative anions, metal ions, reactive oxygen species,
reactive nitrogen species, reducing agents, small-molecule
thiols, and NaHS) in the aqueous buffer. As shown in
Fig. 2, addition of the representative anions (ClÀ, BrÀ, IÀ,
AcOÀ, N3À, CNÀ, CO32À, NO2À) at 1 mM, metal ions (K+,
Mg2+, Ca2+, Zn2+) at 1 mM, reactive oxygen and nitrogen
species (H2O2, OClÀ, OH, 1O2, NO), and reducing agents
ꢀ
(ascorbic acid, S2O32À, SO32À) at the biologically relevant
concentrations induced no marked fluorescence enhancement.
Notably, small-molecule thiols such as glutathione (GSH) at
10 mM and cysteine at 1 mM triggered only a small fluores-
cence enhancement (3-fold) and have nearly no interference to
H2S detection (Fig. S9, ESIw). By contrast, upon treatment
of NaHS (25 mM) with the probe, a large fluorescence signal
(18-fold fluorescence enhancement) was observed. Thus, these
data demonstrate that the probe NIR-H2S has a high selectivity
for H2S over other biological species tested including glutathione
(GSH) and cysteine at the biologically relevant concentrations,
validating the hypothesis that the H2S-triggered thiolysis of
the dinitrophenyl ether reaction is chemoselective for H2S over
other free thiols in the biological systems. The selectivity of
the probe NIR-H2S is superior or comparable to the known
fluorescent H2S probes.
Fig. 1 Fluorescence spectra of the probe NIR-H2S (5.0 mM) in the
aqueous buffer in the presence of different concentrations of NaHS
(0–40 mM). Inset: fluorescence intensity ratio (F/F0) changes at 708 nm
of the probe NIR-H2S (5.0 mM) with the amount of NaHS. Excitation
at 650 nm. Emission at 708 nm.
for hydrogen sulfide) to the probe NIR-H2S was performed
in 50 mM PBS buffer (pH 7.0) with 3 mM cetyltrimethyl-
ammonium bromide (CTAB) and 10% ethanol. The free
probe was essentially non-fluorescent in the buffer (Fig. 1).
However, introduction of NaHS elicited a significant fluores-
cence turn-on response (18-fold fluorescence enhancement) at
708 nm (inset of Fig. 1). Notably, the emission wavelength of
the probe is in the near-infrared (NIR) region, which is
favorable for fluorescent imaging studies.35 Furthermore, the
fluorescence intensities at 708 nm have an excellent linear
relationship with the concentrations of NaHS (Fig. S1, ESIw).
NIR-H2S responded rapidly to NaHS (Fig. S2, ESIw), and the
pseudo-first-order rate constant was calculated to be k =
0.006 sÀ1 (Fig. S3, ESIw). The pH effect studies suggest that the
maximal fluorescent signal was observed at around physiological
pH (Fig. S4, ESIw). The detection limit was calculated to be 5 Â
10À8 M (S/N = 3), indicating that the probe is highly sensitive and
may be suitable for studies of H2S in the living systems.
Due to the different environments between in solution and
in live cells, we decided to further investigate the suitability of
the probe to visualize H2S in living cells. First, the MTT assays
for the probe NIR-H2S and the released nitro product were
conducted, and the results showed that both compounds
with a concentration at 5 mM have only minimal cytotoxicity
after a long period (24 h) (Fig. S10, ESIw). Thus, the probe at
5 mM was selected for imaging experiments in living cells.
To shed light on the H2S-triggered fluorescence turn-on
response, we decided to characterize the thiolysis product
and carry out theoretical calculations. The ESI-MS titration
suggests the formation of compound 3 upon incubation of
NIR-H2S with NaHS (Fig. S5, ESIw), The thiolysis product 3
was further isolated and characterized by 1H NMR, 13C
NMR, MS (EI), and HRMS (EI) (see the Experimental
section, Fig. S6, ESIw). Furthermore, dinitrobenzene and the
thiolysis product 3 (the NIR dye) were examined by density
function theory (DFT) and time-dependent density function
theory (TD-DFT) calculations in the B3LYP/6-31G(d) level of
Gaussian 09 program. The large oscillator strength (0.8823) of
the S0 to S1 transition suggests that the S1 state in compound 3
is emissive (Table S1, ESIw). In addition, the LUMO of the dye
3 is significantly higher than that of dinitrobenzene, suggesting
that the photo-induced electron transfer (PET) process from
the excited dye 3 to the dinitrobenzene moiety is thermo-
dynamically favorable (Fig. S7, ESIw). Thus, NIR-H2S is
essentially nonfluorescent likely due to efficient PET. However,
after the dinitrobenzene moiety is removed by H2S, nonfluorescent
NIR-H2S is converted into fluorescent 3 to elicit a H2S-triggered
fluorescence turn-on signal. Notably, treatment of NaHS to the
probe induced no marked changes in the maximal absorption
Fig. 2 Fluorescence responses of the probe NIR-H2S (5.0 mM) to
various biologically relevant species in the aqueous buffer. Red bars
represent the addition of an excess of the representative anions, metal
ions, reactive oxygen species, reactive nitrogen species, reducing
agents, small-molecule thiols, and NaHS (1 mM for ClÀ, BrÀ, IÀ;
CH3COOÀ, NO2À, N3À,CO32À, K+, Ca2+, Mg2+ and Zn2+, 50 mM
for CNÀ, ClOÀ, ꢀOH, 1O2, and NO, 100 mM for H2O2, SO32À, and
S2O32À, 1 mM for ascorbic acid and cysteine, 10 mM for GSH, and
25 mM for NaHS). 1, probe NIR-H2S alone; 2, ClÀ; 3, BrÀ; 4, IÀ;
5, CH3COOÀ; 6, NO2À; 7, N3À; 8, CNÀ; 9, CO32À; 10, K+; 11, Ca2+
;
12, Mg2+; 13, Zn2+; 14, H2O2; 15, ClOÀ; 16, ꢀOH; 17, 1O2; 18, NO;
19, ascorbic acid; 20, SO32À; 21, S2O32À; 22, cysteine; 23, GSH; 24,
NaHS. Excitation at 650 nm. Emission at 708 nm.
c
10530 Chem. Commun., 2012, 48, 10529–10531
This journal is The Royal Society of Chemistry 2012

View Article Online
4 K. Tanizawa, J. Biochem., 2011, 149, 357.
5 D. Boehning and S. H. Snyder, Annu. Rev. Neurosci., 2003, 26, 105.
6 K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066.
7 P. Kamoun, Amino Acids, 2004, 26, 243.
8 A. K. Mustafa, M. M. Gadalla, N. Sen, S. Kim, W. Mu, S. K. Gazi,
R. K. Barrow, G. Yang, R. Wang and S. H. Snyder, Sci. Signaling,
2009, 2, ra72.
9 E. Blackstone, M. Morrison and M. B. Roth, Science, 2005,
308, 518.
10 J. W. Elrod, J. W. Calvert, J. Morrison, J. E. Doeller, D. W. Kraus,
L. Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Kimura,
C.-W. Chow and D. J. Lefer, Proc. Natl. Acad. Sci. U. S. A.,
2007, 104, 15560.
11 G. Yang, L. Wu, B. Jiang, B. Yang, J. Qi, K. Cao, Q. Meng,
A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang,
Science, 2008, 322, 587.
12 G. Yang, L. Wu and R. Wang, FASEB J., 2006, 20, 553.
13 A. Papapetropoulos, A. Pyriochou, Z. Altaany, G. Yang, A. Marazioti,
Z. Zhou, M. G. Jeschke, L. K. Branski, D. N. Herndon, R. Wang and
Fig. 3 Fluorescence response of the probe NIR-H2S with increasing
concentrations of H2S in living MCF-7 cells. (a–l) The cells were pre-treated
with the probe NIR-H2S (5 mM) and Hoechst 33342 (5 mM) for 20 min, and
then incubated with NaHS (b, c, d) 0 mM, (f, g, h) 10 mM, and (j, k, l)
100 mM for another 10 min. (a) Bright field image; (b) fluorescence
image from the red channel; (c) fluorescence image from the blue
channel (nuclear staining); (d) overlay of (b) with (c); (e) bright field
image; (f) fluorescence image from the red channel; (g) fluorescence
image from the blue channel (nuclear staining); (h) overlay of (f) with
(g); (i) bright field image; (j) fluorescence image from the red channel;
(k) fluorescence image from the blue channel (nuclear staining); (l) overlay
of (j) with (k). Scale bar = 20 mm.
C. Szabo, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 21972.
´
14 K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066.
15 L. Li, M. Bhatia, Y. Z. Zhu, Y. C. Zhu, R. D. Ramnath,
Z. J. Wang, F. B. Anuar, M. Whiteman, M. Salto-Tellez and
P. K. Moore, FASEB J., 2005, 19, 1196.
16 W. Yang, G. D. Yang, X. M. Jia, L. Y. Wu and R. Wang,
J. Physiol., 2005, 569, 519.
17 Y.-J. Peng, J. Nanduri, G. Raghuraman, D. Souvannakitti,
M. M. Gadalla and G. K. Kumar, Proc. Natl. Acad. Sci. U. S. A.,
2010, 107, 10719.
Toward this end, MCF-7 cells were pre-treated with the probe
for 20 minutes, and then incubated with varying concentra-
tions of NaHS for 10 minutes. As displayed in Fig. 3b, f and j,
the fluorescence images in the red channel became brighter as
the concentration of NaHS was increased from 0 to 100 mM.
These data establish that the probe NIR-H2S is cell membrane
permeable and can report H2S in the living cells in a dose-
dependent manner. It is worth noting that the nuclear staining
with Hoechst 33 258 (Fig. 3c, d, g, h, k and l) implies that the
cells were alive after treatment with the probe. In addition,
mitochondria staining (Fig. S11c, e, h and j, ESIw) and nuclear
staining (Fig. S11d, e, i and j, ESIw) reveal that the turn-on
signal of the probe is mainly observed in the mitochondria of
MCF-7 cells. Interestingly, this is in accordance with the
report that H2S is primarily catabolized in mitochondria by
thiosulfate reductase and sulfite oxidase.7
18 J. W. Calvert, S. Jha, S. Gundewar, W. J. Elrod, A. Ramachandran,
C. B. Pattillo, C. G. Kevil and D. J. Lefer, Circ. Res., 2009, 105, 365.
19 K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura,
Biochem. Biophys. Res. Commun., 2002, 293, 1485.
20 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and
B. Chadefaux-Vekemans, Am. J. Med. Genet., 2003, 116A, 310.
21 X. Gu, C. Liu, Y.-C. Zhu and Y.-Z. Zhu, Tetrahedron Lett., 2011,
52, 5000.
22 (a) N. S. Lawrence, R. P. Deo and J. Wang, Anal. Chim. Acta,
2004, 517, 131; (b) N. S. Lawrence, J. Davis, L. Jiang, T. G.
J. Jones, S. N. Davies and R. G. Compton, Electroanalysis, 2000,
12, 1453.
23 (a) J. Radford-Knaery and G. A. Cutter, Anal. Chem., 1993, 65, 976;
(b) C. J. Richardson, E. A. M. Magee and J. H. Cummings, Clin.
Chim. Acta, 2000, 293, 115.
24 M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii and
H. Kimura, Antioxid. Redox Signaling, 2009, 11, 205.
25 Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu,
R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011, 2, 495.
26 A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011,
133, 10078.
27 H. Peng, Y. Cheng, C. Dai, A. L. King, L. B. Predmore, D. J. Lefer
and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672.
28 F. Yu, P. Li, P. Song, B. Wang, J. Zhao and K. Han, Chem.
Commun., 2012, 48, 2852–2854.
29 (a) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman,
A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011,
50, 10327; (b) C. Liu, B. Peng, S. Li, C.-M. Park, A. R. Whorton
and M. Xian, Org. Lett., 2012, 14, 2184.
30 K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura,
T. Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 18003.
In summary, we have developed a unique NIR fluorescence
turn-on H2S probe, NIR-H2S, on the basis of dinitrophenyl
ether chemistry, a new strategy for the design of fluorescent H2S
probes. Furthermore, we have demonstrated that NIR-H2S is
suitable for fluorescent imaging in the living cells. The further
utility of the design strategy and applications of this unique
NIR fluorescent turn-on probe to investigate the biological
functions and pathological roles of H2S is underway. In
addition, we expect that the BODIPY–merocyanine conjugate
(NIR dye 3) will be useful as a NIR platform for the develop-
ment of various NIR fluorescent probes.
31 L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767.
32 (a) S. Shaltiel, Biochem. Biophys. Res. Commun., 1967, 29, 178;
(b) R. Philosof-Oppenheimer, I. Pechtz and M. Fridkin, Int. J.
Pept. Protein Res., 1995, 45, 116.
33 J. C. Mathai, A. Missner, P. Kugler, S. M. Saparov, M. L. Zeidel,
¨
J. K. Lee and P. Pohl, Proc. Natl. Acad. Sci. U. S. A., 2009,
106, 16633.
34 (a) M. P. Lutolf, N. Tirelli, S. Cerritelli, L. Cavalli and J. A. Hubbell,
Bioconjugate Chem., 2001, 12, 1051; (b) C. N. Salinas and
K. S. Anseth, Macromolecules, 2008, 41, 6019.
35 Near-Infrared Applications in Biotechnology, ed. R. Raghavachari
and M. Dekker, New York, 2001.
This work was financially supported by NSFC (20972044,
21172063), NCET (08-0175), and the Doctoral Fund of Chinese
Ministry of Education (20100161110008).
Notes and references
1 R. Wang, FASEB J., 2002, 16, 1792.
2 E. Culotta and D. E. Koshland Jr, Science, 1992, 258, 1862.
3 Y. Han, J. Qin, X. Chang, Z. Yang and J. Du, Cell. Mol. Neurobiol.,
2006, 26, 101.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10529–10531 10531