A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria

Article abstract of DOI:10.1002/anie.201207701

Quick: An exogenously induced quick increase of the H₂S concentration (80 s) in MCF-7 cells can be visualized by ratiometric imaging using a new probe (CouMC) that can target mitochondria. CouMC was constructed by combining merocyanine and coumarin fluorophores. The selective nucleophilic addition of HS^- to the merocyanine derivative at neutral pH is crucial for the rapid H₂S detection. Copyright

Full text of DOI:10.1002/anie.201207701

Angewandte
Chemie
DOI: 10.1002/anie.201207701
Fluorescence Imaging
A Ratiometric Fluorescent Probe for Rapid Detection of Hydrogen
Sulfide in Mitochondria**
Yuncong Chen, Chengcheng Zhu, Zhenghao Yang, Junjie Chen, Yafeng He, Yang Jiao,
Weijiang He,* Lin Qiu, Jiajie Cen, and Zijian Guo*
Traditionally considered as a toxic gas with unpleasant smell,
hydrogen sulfide (H2S) has emerged as the third endogenous
gaseous signaling compound (gasotransmitter) after NO and
CO.[1] Endogenous H2S has been found in high concentrations
(10 to 600 mm) in the brain of bovine, rat, and human.[2] H2S
has also been recognized to mediate a wide range of
physiological processes, such as vasodilation,[3] antioxidation,
anti-apoptosis and anti-inflammation,[4] and the abnormal
H2S level was correlated to diseases such as Alzheimerꢀs
disease and Downꢀs syndrome.[5,6] The increasing interest in
endogenous H2S demand rapid, facile, and reliable detection
techniques, since the current colorimetric and electrochem-
ical assays, gas chromatography, and sulfide precipitation are
difficult to implement for in situ detection.[7] Fluorescence
imaging through staining with a fluorescent probe is now one
of the most attractive molecular imaging techniques for the
in vivo detection of biomolecules owing to its high sensitivity/
selectivity, non-invasiveness, and aptness for living cells,
tissues, and living small animals, and developing fluorescent
probes for H2S detection is now drawing much attention.
A few fluorescent turn-on probes for H2S have been
reported. Some could even be used for imaging of intra-
cellular H2S, while others were only applicable for in vitro
detection of H2S in blood samples.[8] They are chemodosim-
eters based on the specific H2S-induced reactions, such as
azide reduction,[8a–d] quencher (such as Cu2+) removal,[8f,g] and
nucleophilic reaction to achieve strong fluorescence.[8h-j] Most
of them display a response time between 20 min to two hours,
except for those that are based on the reduction of dansyl
azide and Cu2+-removal sensing mechanisms reported by
Wang and Nagano, respectively.[8b,f] These probes, however,
are not suitable for real-time imaging of quick H2S-related
biological processes, and searching for quick reactions that
are sensitive to H2S to enhance the response rate of H2S
probes has always been attractive and challenging. Of course,
improving the H2S selectivity over biological thiols is also
essential for the sensing reaction. On the other hand, these
reported turn-on probes are difficult to give quantitative
information on the H2S concentration, since molecular
emission intensity can be distinctly affected by photobleach-
ing, microenvironments, and local probe concentration.
Therefore, ratiometric probes for H2S are highly appealing
owing to their ability in quantitative tracking, because they
endow a self-calibration effect, which reduces most of the
aforementioned interferences.[9] The only ratiometric H2S
probe reported so far is Cy-N3,[10] but it responds to H2S in
20 min. Moreover, fluorescent probes that are able to provide
information on H2S in an organelle of interest are especially
appreciated, since studies have implied that H2S biology is
associated with certain organelles such as mitochondria.[11]
Herein, we report a novel ratiometric fluorescent probe,
CouMC, the function of which is based on the selective
nucleophilic addition of HSÀ to a specific merocyanine
derivative in medium of near neutral pH value (Scheme 1).
Besides the rapid and specific response to H2S, this probe can
also be applied for preferential imaging of H2S in mitochon-
dria of living cells.
Probe CouMC was constructed by connecting a coumarin
fluorophore and an indolenium block through an ethylene
group. CouMC can be considered as a hybrid fluorophore of
coumarin and merocyanine. The merocyanine moiety was
[*] Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, Prof. W. He, L. Qiu,
J. Cen, Prof. Z. Guo
State Key Laboratory of Coordination Chemistry
Coordination Chemistry Institute
School of Chemistry and Chemical Engineering, Nanjing University
22 Hankou Road, Nanjing, 210093 (P. R. China)
E-mail: heweij69@nju.edu.cn
Scheme 1. a) Reversible conversion between the merocyanine form
and the spiroform of spiropyran; b) the proposed H2S sensing
mechanism and acid-induced retrieval of CouMC.
zguo@nju.edu.cn
incorporated not only as fluorophore but also as the guest
receptor, since its indolenium C-2 atom is an effective target
for a nucleophilic analyte. Normally, simple spiropyran
undergoes a reversible conversion between the merocyanine
form and the spiroform, and displays a switch of color or
emission, since the reversible nucleophilic addition of
[**] We thank the National Basic Research Program of China (No.
2011CB935800) and National Natural Science Foundation of China
(No. 21271100, 91213305, 10979019, 21131003, and 21021062) for
financial support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201207701.
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a phenol anion to the indolenium C-2 atom alters the
conjugated system and internal charge transfer (ICT) effect
of the whole molecule (Scheme 1).[12] We envisioned that
HSÀ, the main stable form of H2S in the physiological
condition, can be added to the indolenium C-2 atom of
CouMC as a nucleophile and lead to a ratiometric sensing
behavior by eliminating merocyanine emission but retaining
coumarin emission. Since biological thiols, such as cysteine
(Cys), glutathione (GSH), and homocysteine (Hcy), possess
high pKa values (ꢀ 8.5),[13] H2S is expected to be a better
nucleophile than these biological thiols in neutral medium
owing to its lower pKa value (ca. 7.0). Therefore, CouMC was
expected to have better selectivity for H2S over these
biological thiols, which are the main interference in intra-
cellular H2S detection.
suggesting HSÀ can be detected with the naked eye when
using CouMC. The titration profile according to the absorb-
ance at 588 nm indicated that CouMC bind HSÀ with a 1:1
stoichiometry.
CouMC displays a very quick response to HSÀ. Temporal
emission tracking of CouMC (10 mm) in the presence of NaHS
(10-200 mm) suggested that the sensing reaction could be
completed within 30 s, even the total HSÀ level was as low as
10 mm (Figure S4 in the Supporting Information). If the HSÀ
level was in the range from 100 to 200 mm, the reaction could
be completed within seconds. In fact, probe CouMC is able to
detect H2S more rapidly than most of the reported H2S probes
(typically 20 min–2 h). This provides the probe an advantage
in real-time intracellular imaging, when considering the
variable nature and quick metabolism of endogenous H2S in
biological systems.
This new probe shows fine aqueous solubility, and its
fluorescence and sensing behavior to H2S were tested in
phosphate-buffered saline (PBS; 20 mm, pH 7.4) containing
2% DMSO. Free CouMC (10 mm) shows two well-resolved
emission bands centered at 510 and 652 nm (lex, 475 nm;
Figure 1a), which can be assigned as the emission band of
The ratiometric sensing selectivity of CouMC for HSÀ was
investigated in PBS buffer of pH 7.4 (Figure 2a). Only HSÀ
Figure 2. a) Emission ratio F510/F652 of CouMC (10 mm) in PBS (20 mm,
pH 7.40, 2% DMSO, v/v) in the presence of HSÀ, various anions, or
biologically relevant species. 1. Probe alone; 2. HSÀ (200 mm);
3. HCO3À; 4. ClÀ; 5. BrÀ; 6. IÀ; 7. CNÀ; 8. FÀ; 9. NO3À; 10. SO4
;
2À
11. SO32À; 12. S2O32À; 13. SCNÀ; 14. EtNH2; 15. EtOH; 16. NO; 17. S-
nitrosoglutathione; 18. CysNO; 19. ClOÀ; 20. O2À; 21. H2O2; (3–21:
1 mm). b) Emission ratio F510/F652 of CouMC (10 mm) in the same
buffer in the presence of HSÀ (200 mm), Cys (1 mm), GSH (1 mm),
Hcy (200 mm), and bovine serum albumin (BSA; 200 mm). For (b):
black bars correspond to free CouMC, or CouMC with HSÀ or CouMC
with marked biological thiols; white bars to CouMC in the presence of
both HSÀ (200 mm) and the marked biological thiols.
Figure 1. a) Fluorescence spectra of CouMC (10 mm) in PBS (20 mm,
pH 7.40, 2% DMSO, v/v) obtained upon titration with HSÀ from 0 to
200 mm. lex, 475 nm. b) Absorption spectra of CouMC (25 mm) in PBS
(20 mm, pH 7.40, 5% DMSO, v/v) obtained upon titration with HSÀ
from 0 to 100 mm. Inset in (a): photograph of CouMC solutions upon
irradiation by a UV lamp (365 nm) in the absence or presence of HSÀ.
Inset in (b): photograph of CouMC solutions in the absence or
presence of HSÀ.
coumarin and merocyanine, respectively. The distinct gap
between the two bands is over 140 nm, which makes this
probe favorable for the dual emission ratiometric imaging
owing to the minimum overlap between the two bands. Its
quantum yield was determined as 0.03 with cresyl violet as
reference.[14] Fluorescence titration of a CouMC solution with
NaHS (0–200 mm) demonstrated that its emission band at
652 nm decreased distinctly, while the band at 510 nm under-
went a noticeable increment simultaneously (Figure 1a). The
intensity ratio of the two emission bands, F510/F652, increased
from 0.17 to 21.5, and the final enhancement factor is over
120-fold. The detection limit according to the ratiometric
fluorescent sensing was determined as approximately 1 mm
(Figure S3 in the Supporting Information). The absorption
spectrum of free CouMC (25 mm) displays a strong ICT band
at 588 nm (e, 67500mÀ1 cmÀ1, Figure 1b). This band decreased
gradually upon HSÀ titration, confirming the disruption of the
ICTeffect in the whole molecule caused by HSÀ addition. The
solution turned from dark blue to very pale blue, thereby
(200 mm) induces a dramatic increment of emission ratio F510/
F652, while other anions (HCO3À, FÀ, ClÀ, BrÀ, IÀ, CNÀ,
2À
NO3À, 1 mm), inorganic reactive sulfur species (SO42À, SO3
,
,
S2O32À, SCNÀ, 1 mm), reactive oxygen species (H2O2, O2
À
ClOÀ, 1 mm), NO and NO producers (CysNO, S-nitrosoglu-
tathione, 1 mm) only trigger very minor changes. The classical
nucleophile CNÀ induces almost no change of the ratio; the
protonation of CNÀ in neutral medium might be the origin,
since HCN possess a pKa ꢁ 9.2.[15] Other nucleophiles such as
ethanol and ethylamine (1 mm) induce also no obvious
change in CouMC emission. The ratiometric HSÀ sensing
ability of CouMC in the presence of other biological thiols
was also investigated. GSH (1 mm) or Cys (1 mm) induce only
a neglectable enhancement of emission ratio F510/F652 (Fig-
ure 2b). The emission ratio enhancement induced by Hcy
(200 mm) and BSA (200 mm, a protein with exposed Cys
residues) is also very limited when compared to that induced
by HSÀ. The sensing selectivity of CouMC for H2S over Cys,
GSH, Hcy, and BSA is approximately 430-fold, 460-fold, 54-
2
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fold, and 18-fold, respectively, and its HSÀ sensing behavior in
the presence of GSH, Cys, Hcy, or BSA indicated that
CouMC is able to detect HSÀ without any distinct interfer-
ence from these biological thiols.
The color of the CouMC solution (25 mm) in the presence
of NaHS (200 mm) can be recovered to dark blue when the pH
value of the solution was adjusted to 1.0 (Figure S5a in the
Supporting Information). However, a CouMC solution with-
out HSÀ displays stable absorption spectra in the pH range
from 1.0 to 8.0. Similarly, the distinctly enhanced emission
ratio F510/F652 (21.5) of CouMC (10 mm) in PBS (1 mm, pH 7.4)
upon NaHS addition (200 mm) can be reduced back to 0.32,
when the pH value of the medium was adjusted to 2.5. If the
pH value of the medium was adjusted again to 7.4, the
emission ratio was enhanced back to approximately 15.0. The
pH-dependent recovery of emission ratio can be observed for
at least three cycles (Figure S5b in the Supporting Informa-
tion). However, the emission ratio F510/F652 of free CouMC
exhibits very minor change in the pH range from 2.5 to 8.0
(Figure S6 in the Supporting Information), thus suggesting
that the probe CouMC itself is stable in this pH range. All
these results implied that CouMC might be retrievable in
acidic condition after reacting with HSÀ. Moreover, our
attempt to separate HSÀ nucleophilic addition product
CouMC-SH was unsuccessful, and TLC analysis demon-
strated that most product has been recovered to CouMC. The
MALDI-TOF-MS spectrum of CouMC solution in the
presence of excessive NaHS at pH 7.4 showed one major
signal with m/z of 477.165 and another minor peak with m/z of
533.129, which can be assigned as [CouMC]+ and [CouMC-
SH + Na]+, respectively (Figure S7 in the Supporting Infor-
mation). This result implied a recovery of CouMC from
CouMC-SH in the MS determination procedure. Moreover,
MS determination of this solution after the pH value was
adjusted to 2.5 gave only the signal of CouMC. The 1H NMR
and 13C NMR spectra of CouMC in the presence of HSÀ
(1.2 equiv) are given in Figures S8 and S9 in the Supporting
Information. As can be seen in Figure S8, almost all proton
signals from the coumarin/merocyanine conjugated system
underwent an obvious up-field shift after HSÀ addition; this
shift is consistent with the proposed product where the
electron-withdrawing ability of indolenium N+ has disap-
Figure 3. a–c) Confocal fluorescence images of MCF-7 cells costained
by CouMC (10 mm, 30 min) and Mito marker Deep Red 633 (10 mm,
30 min). a) Pseudocolored image obtained with band path of 660–
750 nm upon excitation of CouMC at 488 nm; b) image from band
path of 665–750 nm upon excitation of Deep Red at 633 nm; c) overlay
of (a) and (b). d–k) Confocal fluorescence imaging of MCF-7 cells by
using a dual emission mode upon excitation at 488 nm. d–f) Images
of cells stained by CouMC (10 mm, 10 min); g–i) images of cells
preincubated with CouMC (10 mm) followed by incubation with NaHS
(200 mm) for 8 min. d,g) Green-channel images collected with band
path of 500–560 nm; e,h) red-channel images collected with band path
of 640–700 nm; f,i) ratiometric images obtained by mediating green
channel image with the related red channel image. j) Temporal profiles
of average green and red channel fluorescence in region of interest D
shown in (i), and the related profile of average fluorescence ratio
(Fgreen/Fred); the values on the y-axis correspond to the fluorescence
intensity of green and red channels, as well as the ratio of (Fgreen/Fred);
k) temporal profile of average fluorescence ratio of (Fgreen/Fred) in
regions of interest A, B, C, D, and E shown in (i), NaHS (200 mm) was
added at the 12th min. Scale bar in (d): 20 mm.
À
+
=
peared. In the meantime, the HS addition to C N resulted
in the formation of a chiral center at C-2, thereby changing
the singlet signals of the C-3 dimethyl groups into multiplets.
Similarly, as can be seen from the 13C NMR spectrum
(Figure S9), the conversion of sp2 to sp3 hybrids of the C-2
atom resulted in a distinct up-field shift of the lowest signal of
the indolenium C-2 atom at 182 ppm, which support the
suggested indolenium C-2 atom as HSÀ addition site.
Red (overlap coefficient 0.95), thereby implying a preferential
distribution of CouMC in mitochondria (Figure 3a–c). The
ratiometric imaging for intracellular H2S was carried out with
a dual-emission mode upon excitation at 488 nm. The
ratiometric images (Figure 3 f,i) were obtained by mediating
the green channel image (band path: 500–560 nm; Fig-
ure 3d,g) with the related red channel image (band path:
640–700 nm; Figure 3e,h) by using the software of the
microscope. MCF-7 cells stained by CouMC alone showed
very dim fluorescence in the green channel and a strong
Imaging with CouMC was investigated in MCF-7 cells
with a confocal microscope Zeiss LSM710 (Figure 3). When
the mono-emission mode imaging was used, the CouMC-
stained cells showed a bright densely punctuated pattern in
the cytoplasm, and the nonstained cells showed only a dim
background, thus indicating the fine cell membrane perme-
ability of CouMC. A colocalization assay with mitochondria
dye Mito-marker Deep Red 633 and CouMC displayed that
fluorescence of CouMC was colocalized with that of Deep
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
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Papapetropoulos, A. Pyriochou, Z. Altaany, G. Yang, A.
Marazioti, Z. Zhou, M. G. Jeschke, L. K. Branski, D. N. Hern-
don, R. Wang, C. Szabꢁ, Proc. Natl. Acad. Sci. USA 2009, 106,
21972; d) L. Li, M. Bhatia, Y. Z. Zhu, R. D. Ramnath, Z. J.
Wang, F. B. M. Anuar, M. Whiteman, M. Salto-Tellez, P. K.
Moore, FASEB J. 2005, 19, 1196.
fluorescence in the red channel. The related ratiometric
image of dim background implies a very low H2S level.
Incubation with NaHS (200 mm) at the 12th min led to
a distinct increase in green channel fluorescence, accompa-
nied by the dramatic drop of red channel fluorescence, and
a drastic enhancement of emission ratio (green/red) can be
observed in the related ratiometric image. The NaHS-induced
increase of the ratio is finished after approximately 80 s
(Figure 3k), thus indicating that the H2S level in mitochondria
can be enhanced very quickly by incubation with 200 mm
NaHS. The enhancement process was visualized and recorded
in a video (see the Supporting Information) in which NaHS
was added at the 12th second; 1 s video time equals to 1 min
of real imaging process. Figure 3j showed clearly the response
process of region of interest D upon incubation with NaHS
according to the fluorescence recorded in both green and red
channels. This preliminary imaging study suggested that
CouMC can be used for ratiometric tracking of H2S in
mitochondria, which is of great importance to clarify the
physiological roles of H2S in the organelle.[11] Moreover,
owing to the quick response rate of this probe it is possible to
effectively track the quick increase of the H2S concentration,
which is finished in 80 s.
In summary, the new probe constructed by combining
coumarin and merocyanine can be used for specific ratio-
metric sensing of H2S. It preferentially targets mitochondria
and has a quick response rate for intracellular H2S, thus
favoring the real-time intracellular H2S imaging. The visual-
ization of the increase of the mitochondrial H2S concentration
upon NaHS incubation by using CouMC staining is promising
for the practical exploration of H2S-related processes in
mitochondria. This study provides an effective rationale to
design ratiometric probes for H2S that profit from the quick
HSÀ nucleophilic addition to a merocyanine derivative in
medium of near neutral pH value. In addition, the possibility
to recover CouMC from the nucleophilic addition product in
acidic conditions (pH < 3.0) together with the quick sensing
behavior shows the potential of this reaction in the design of
H2S probes.
[3] a) G. D. Yang, L. Y. Wu, B. Jiang, W. Yang, J. S. Qi, K. Cao, Q. H.
Meng, A. K. Mustafa, W. T. Mu, S. M. Zhang, S. H. Snyder, R.
Wang, Science 2008, 322, 587; b) W. M. Zhao, J. Zhang, Y. J. Lu,
R. Wang, EMBO J. 2001, 20, 6008; c) D. J. Lefer, Proc. Natl.
Acad. Sci. USA 2007, 104, 17907.
[4] a) J. W. Calvert, S. Jha, S. Gundewar, J. W. Elrod, A. Ram-
achandran, C. B. Pattillo, C. G. Kevil, D. J. Lefer, Circ. Res. 2009,
105, 365; b) R. C. O. Zanardo, V. Brancaleone, E. Distrutti, S.
Fiorucci, G. Cirino, J. L. Wallace, FASEB J. 2006, 20, 2118.
[5] K. Eto, T. Asada, K. Arima, T. Makifuchi, H. Kimura, Biochem.
Biophys. Res. Commun. 2002, 293, 1485.
[6] P. Kamoun, M. C. Belardinelli, A. Chabli, K. Lallouchi, B.
Chadefaux-Vekemans, Am. J. Med. Genet. Part A 2003, 116, 310.
[7] a) D. M. Tsai, A. S. Kumar, J. M. Zen, Anal. Chim. Acta 2006,
556, 145; b) J. Furne, A. Saeed, M. D. Levitt, Am. J. Physiol.
2008, 295, R1479; c) D. Jimꢂnez, R. Martinez-Manez, F.
Sancenon, J. V. Ros-Lis, A. Benito, J. Soto, J. Am. Chem. Soc.
2003, 125, 9000.
[8] a) A. R. Lippert, E. J. New, C. J. Chang, J. Am. Chem. Soc. 2011,
133, 10078; b) H. Peng, Y. Cheng, C. Dai, A. L. King, B. L.
Predmore, D. J. Lefer, B. Wang, Angew. Chem. 2011, 123, 9846;
Angew. Chem. Int. Ed. 2011, 50, 9672; c) L. A. Montoya, M. D.
Pluth, Chem. Commun. 2012, 48, 4767; d) S. Chen, Z. Chen, W.
Ren, H. Ai, J. Am. Chem. Soc. 2012, 134, 9589; e) W. Xuan, C.
Sheng, Y. Cao, W. He, W. Wang, Angew. Chem. 2012, 124, 2328;
Angew. Chem. Int. Ed. 2012, 51, 2282; f) K. Sasakura, K.
Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T.
Ueno, T. Takuya, H. Kimura, T. Nagano, J. Am. Chem. Soc. 2011,
133, 18003; g) F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie,
Y. Shi, F. Cheng, X. Yao, D. Bai, Z. Zeng, Inorg. Chem. 2012, 51,
2454; h) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman,
A. R. Whorton, M. Xian, Angew. Chem. 2011, 123, 10511;
Angew. Chem. Int. Ed. 2011, 50, 10327; i) Y. Qian, J. Karpus, O.
Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J. Zhao, C. He, Nat.
Commun. 2011, 2, 495; j) Y. Qian, L. Zhang, S. Ding, X. Deng, C.
He, X. E. Zheng, H.-L. Zhu, J. Zhao, Chem. Sci. 2012, 3, 2920.
[9] K. W. Dunn, S. Mayor, J. N. Myers, F. R. Maxfield, FASEB J.
1994, 8, 573.
[10] F. Yu, P. Li, P. Song, B. Wang, J. Zhao, K. Han, Chem. Commun.
2012, 48, 2852.
Received: September 24, 2012
Revised: December 3, 2012
Published online: && &&, &&&&
[11] a) P. Nicholls, Biochem. Soc. Trans. 1975, 3, 316; b) 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, D. J.
Lefer, Proc. Natl. Acad. Sci. USA 2007, 104, 15560.
[12] a) V. I. Minkin, Chem. Rev. 2004, 104, 2751; b) G. Berkovic, V.
Krongauz, V. Weiss, Chem. Rev. 2000, 100, 1741; c) J. D. Winkler,
C. M. Bowen, V. Michelet, J. Am. Chem. Soc. 1998, 120, 3237.
[13] a) M. P. Lutolf, N. Tirelli, S. Cerritelli, L. Cavalli, J. A. Hubbell,
Bioconjugate Chem. 2001, 12, 1051; b) C. N. Salinas, K. S.
Anseth, Macromolecules 2008, 41, 6019.
[14] D. Magde, J. L. Brannon, T. L. Cremers, J. Olmsted, J. Phys.
Chem. C 1979, 83, 696.
[15] X. Lv, J. Liu, Y. Liu, Y. Zhao, Y. Q. Sun, P. Wang, W. Guo, Chem.
Commun. 2011, 47, 12843.
Keywords: fluorescence imaging · fluorescent probes ·
hydrogen sulfide · mitochondria · ratiometric probes
.
[1] a) E. Culotta, D. E. Koshland, Science 1992, 258, 1862; b) T.
Morita, M. A. Perrella, M. E. Lee, S. Kourembanas, Proc. Natl.
Acad. Sci. USA 1995, 92, 1475; c) C. Szabꢁ, Nat. Rev. Drug
Discovery 2007, 6, 917.
[2] a) J. C. Savage, D. H. Gould, J. Chromatogr. Biomed. Appl. 1990,
526, 540; b) K. Abe, H. Kimura, J. Neurosci. 1996, 16, 1066; c) A.
4
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Communications
Fluorescence Imaging
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
Quick: An exogenously induced quick
increase of the H2S concentration (80 s)
in MCF-7 cells can be visualized by
ratiometric imaging using a new probe
(CouMC) that can target mitochondria.
CouMC was constructed by combining
merocyanine and coumarin fluorophores.
The selective nucleophilic addition of HSÀ
to the merocyanine derivative at neutral
pH is crucial for the rapid H2S detection.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!