A reversible fluorescent probe for detecting hypochloric acid in living cells and animals: Utilizing a novel strategy for effectively modulating the fluorescence of selenide and selenoxide

Article abstract of DOI:10.1039/c3cc39269d

Based on a novel strategy for modulating the fluorescence of selenide and selenoxide, we have designed and developed a reversible fluorescent probe for hypochloric acid. And the synthesis, characterization, fluorescence properties, as well as the biological applications in living cells and animals, have all been described.

Full text of DOI:10.1039/c3cc39269d

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A reversible fluorescent probe for detecting hypochloric
acid in living cells and animals: utilizing a novel
strategy for effectively modulating the fluorescence
of selenide and selenoxide†
Cite this: Chem. Commun., 2013,
49, 2445
Received 29th December 2012,
Accepted 5th February 2013
Zhangrong Lou,ab Peng Li,a Qiang Panab and Keli Han*a
DOI: 10.1039/c3cc39269d
www.rsc.org/chemcomm
Based on a novel strategy for modulating the fluorescence of developing an effective technique to monitor the redox changes
selenide and selenoxide, we have designed and developed a between HOCl and antioxidants in living systems.
reversible fluorescent probe for hypochloric acid. And the synthesis,
Fluorescence imaging has the advantages of higher sensitivity,
characterization, fluorescence properties, as well as the biological less invasiveness, and more convenience compared with other
applications in living cells and animals, have all been described.
approaches, thereby providing more possibilities for the localization
and dynamic monitoring of cellular metabolites.11 Until now, even
Hypochloric acid (HOCl) is one of the biologically important though several fluorescent probes for HOCl12 have been reported,
reactive oxygen species (ROS) and can be produced from hydrogen few reversible fluorescent probes for visualizing the redox changes
peroxide and chloride ions catalyzed by the heme enzyme myelo- mediated by HOCl and antioxidants have been developed.13 Here
peroxidase (MPO) in living organisms.1 Although HOCl is a major we present such a reversible fluorescent probe for HOCl.
strong oxidant that functions as a microbicidal agent in living
The reversible recognition moiety for an oxidation–reduction
organisms and has a dominant role in the immune system,2 it couple relies on the activity of glutathione peroxidase (GPx), which
may also contribute to extensive oxidative stress and oxidative catalyzes the reaction between ROS and glutathione (GSH) via the
damage due to the destruction of nucleic acids, proteins, and oxidation–reduction cycles of the selenium center.14 In addition, it
lipids via both oxidation and chlorination reactions.1,3 Increasing is essential to introduce a fast excited state process to modulate
evidence shows that extensive oxidative stress and oxidative the fluorescence of selenide and selenoxide. In our previous
damage are associated with numerous diseases, such as neuro- studies,15 we have utilized photoinduced electron transfer (PET)
degeneration including Parkinson’s disease and Alzheimer’s and developed a reversible probe based on selenide and selen-
disease,4 inflammatory diseases,5 atherosclerosis6 and cancer.7 oxide successfully. In this study, we selected 1,8-naphthalimide as
Cells often have their own set of antioxidant defense systems to a signal transducer and designed a reversible fluorescent probe
maintain redox equilibrium, where intracellular thiols play (NI–Se, Fig. 1a). The existence of the PET process in NI–Se has
essential roles. For example, glutathione (GSH), which has been been denied by time-dependent density functional theory
identified as the most abundant non-protein thiol, is considered (TDDFT) calculations (ESI,† Fig. S1). However, the influences of
to be the main player in combating oxidative stress and regulating viscosity and temperature experiments demonstrated that there
the redox environment of the internal cellular compartments.8 In was an excited state configuration twist process in NI–Se, but not
addition, evidence has demonstrated that hydrogen sulfide (H2S) in NI–SeO (Fig. S2, ESI†). And it is known that the excited state
functions as an effective HOCl scavenger to protect cells from configuration twist always leads to fluorescence quenching.16
oxidative stress.9 And the complex redox biology of the cell
governs many essential biological processes and has broad impli-
cations in human health and diseases.10 In this regard, it is worth
a State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of
Chemical Physics (DICP), Chinese Academy of Sciences (CAS),
457 Zhongshan Road, Dalian 116023, P. R. China. E-mail: klhan@dicp.ac.cn;
Fax: +86-411-84675584; Tel: +86-411-84379293
b Graduate School of Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available: Additional fluorescent
Fig. 1 (a) The structures of NI–Se and NI–SeO, and the proposed mechanism of
fluorescence response to HOCl reversibly. (b) Changes in the fluorescence spectra
of NI–Se with different concentrations of NaOCl.
confocal images, fluorescence spectra assays, TDDFT calculations, details of the
synthesis, 1H NMR, 13C NMR, 77Se NMR, MS spectra of compounds discussed in
this communication. See DOI: 10.1039/c3cc39269d
c
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Chem. Commun., 2013, 49, 2445--2447 2445

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As a result, the fluorescence of NI–Se was enhanced in response to species and amino acids, except for H2S and thiols. In addition,
ROS. Here we also present the synthesis, characterization and the fluorescence titration measurement of NI–SeO showed that
fluorescence properties of this probe. And it turns out that this the fluorescent intensity decreased gradually with increasing
probe is highly sensitive, reversible and selective toward HOCl. NaHS concentrations (Fig. S8b, ESI†). Next, the kinetics experi-
Moreover, this probe is capable of visualizing HOCl and the ment of NI–Se response to NaOCl followed by H2S was
reducing repair of living RAW 264.7 cells and living mice in situ. performed (Fig. 2b). The fluorescence intensity was stable over
The spectroscopic properties of NI–Se were measured under 1 h, suggesting that the probe solution was stable to light and air
simulated physiological conditions (20 mM PBS, pH 7.40). Upon under the experimental conditions. And the emission intensity
the addition of NaOCl as a HOCl source, an enhancement in at 523 nm decreased significantly upon the addition of H2S.
fluorescence intensity was observed and the corresponding When the solution was treated again with NaOCl, the fluores-
quantum yield of NI–Se increased from 0.04 to 0.45 (Fig. S3, cence of the probe was recovered and the reversible oxidation–
ESI†). A fluorescence titration experiment demonstrated that reduction cycles can be repeated six times with the fluorescence
fluorescent intensity increased linearly with the concentrations intensity decreased by about 50% (Fig. S8c, ESI†). The redox
of NaOCl up to 15 mM (Fig. 1b, Fig. S4, ESI†). And the corre- cycles have been confirmed through HPLC analyses (Fig. S9,
sponding regression equation was F523nm = 59 772 Â [NaOCl] + ESI†). These experimental results revealed that NI–Se was
102 703 with a linear coefficient of r = 0.9941. In addition, the capable of monitoring NaOCl reversibly in the presence of H2S.
limit of detection for NaOCl was determined to be 5.86 Â 10À7 M
Subsequently, we applied NI–Se to imaging HOCl in living
under the experimental conditions (ESI,† Fig. S5). No significant cells. The mouse macrophage cell line RAW264.7, which pro-
changes in the absorption intensity were observed with the duced HOCl when stimulated by lipopolysaccharide (LPS) and
increase in NaOCl concentrations (Fig. S4, ESI†).
phorbol myristate acetate (PMA), was chosen as the bioassay
The influence of pH on NI–Se both in the absence and presence model.12j Fig. 3a shows that living RAW264.7 cells incubated
of NaOCl was examined. Fig. S6 (ESI†) shows that NI–Se is stable in with NI–Se exhibit only faint fluorescence. However, the cells
aqueous media from pH 4.0 to 12.0. Although a significant that were pretreated with LPS and then incubated with PMA
fluorescence decrease is observed over pH 9.0 in the presence of and NI–Se display green cellular fluorescence (Fig. 3b). Weaker
NaClO, NI–Se displays constant fluorescence intensity in the pH cellular fluorescence is observed when the cells were pretreated
range of 4.0–9.0, demonstrating that NI–Se would work well under with salicylhydroxamic acid (SHA), a MPO inhibitor17 (Fig. 3c).
physiological pH conditions. The emission spectra of NI–Se with Notably, co-staining with Hoechst 33342 (labels the nucleus)
the addition of various ROS were investigated to evaluate the revealed that the probe located mainly in the cytoplasm of these
selectivity of NI–Se toward NaOCl screening compared to other living RAW264.7 cells (Fig. 3d).18 And the relevant brightfield
biologically relevant species. As shown in Fig. 2a, a significant transmission images confirmed that the cells were still viable
fluorescence response is observed for NaOCl, whereas a negligible (ESI†). In addition, the MTT assay of the RAW264.7 cells with
fluorescence enhancement for other ROS. Additionally, the time probe concentrations of 10.0 mM to 100.0 mM for 24 h demon-
courses of the probe with various ROS were recorded for 30 min to strated that NI–Se has low toxicity to cultured cell lines under
check whether the fluorescence switch might turn on upon the experimental conditions at a concentration of 10.0 mM
incubation with other biological species over time (Fig. S7, ESI†). (Fig. S10, ESI†). These results indicated that NI–Se was capable
These results reveal that NI–Se is capable of responding to NaOCl of monitoring HOCl in living RAW264.7 cells.
via a turn-on fluorescence switch selectively over other ROS.
Since NI–Se is responsive to HOCl in living cells, we then
Having demonstrated that the probe can detect NaOCl selec- investigated whether it could image the reducing repair of anti-
tively and stably over pH and time, we wonder whether NI–Se can oxidants. The living RAW264.7 cells loaded with NI–Se display
respond to NaOCl reversibly in the presence of various reducing only faint fluorescence (Fig. S13a, ESI†). However, treating the
agents. As displayed in Fig. S8a (ESI†), no remarkable fluores- same NI–Se-incubated cells with PMA results in an increased
cence intensity decrease is observed upon addition of reducing cellular fluorescence intensity because of the generation of HClO
Fig. 3 Confocal fluorescence images of HOCl in living RAW264.7 cells. (a–c)
RAW264.7 cells were pretreated with 1 mg mLÀ1 LPS for 16 h. (a) Incubated with
Fig. 2 (a) Fluorescence responses of NI–Se to various analytes. Bars represent
the fluorescence intensity at 523 nm, responding to various ROS: 1, free; 2, NaOCl
(30.0 mM); 3, peroxynitrite (300.0 mM); 4, NO (150 mM); 5, tert-butylhydro-
10 mM NI–Se for 5 min. (b) Incubated with 10 mM NI–Se for 5 min and then
coincubated with 2.5 mg mLÀ1 PMA for 30 min. (c) Incubated with 10 mM SHA for
20 min and co-incubated with 2.5 mg mLÀ1 PMA for another 30 min, then
incubated with 10 mM NI–Se. (d) RAW264.7 cells loaded with 2 mM Hoechst
33342 and 10 mM NI–Se for 5 min, Overlay of the NI–Se and Hoechst 33342 sets.
NI–Se set (excitation: 488 nm, emission: 490–590 nm). Hoechst 33342 set
(excitation: 405 nm, emission: 410–460 nm). Scale bar represents 20 mm.
À
peroxide (300.0 mM); 6, cumene hydroperoxide (300.0 mM); 7, O2 (300.0 mM);
8, 1O2 (300.0 mM); 9, H2O2 (300.0 mM); 10, ꢀOH (300.0 mM). (b) Time course of
NI–Se measured by a spectrofluorometer. NI–Se was oxidized by 5 equiv. of
NaOCl for 1 h, and then treated with 20 equiv. of NaHS for 1.5 h.
c
2446 Chem. Commun., 2013, 49, 2445--2447
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This work was supported by NSFC (no. 21273234 and
21203192) and the National Basic Research Program of China
(2013CB834604). We are also grateful to Bingshuai Wang,
Songqiu Yang, Ping Song for their contribution to the discus-
sion of results. Thanks are also due to Jian Luo and Xiaohu He
for critical reading of this article.
Fig. 4 Fluorescent images of HClO production and H2S reduction in the peritoneal
cavity of the mice with NI–Se. (a) Control, neither LPS nor NI–Se was injected; (b) saline
was injected in the intraperitoneal (i.p.) cavity of mice, followed by i.p. injection of
NI–Se (100 nmol); (c) LPS (1 mg) was injected into the peritoneal cavity of the mice,
followed by i.p. injection of NI–Se (100 nmol). (d) An additional H2S (4 mmol) was
injected in parallel to (c). (e) (1)–(4) represent quantification of fluorescence emission
intensity from (a)–(d), respectively. The total number of photons from the entire body
of the groups (a)–(d) was integrated and plotted as a ratio to the control (a).
Notes and references
1 M. P. Curtis, A. J. Hicks and J. W. Neidigh, Chem. Res. Toxicol., 2011,
24, 418–428.
2 A. L. P. Chapman, M. B. Hampton, R. Senthilmohan, C. C.
Winterbourn and A. J. Kettle, J. Biol. Chem., 2002, 277, 9757–9762.
3 (a) P. Nagy and M. T. Ashby, J. Am. Chem. Soc., 2007, 129, 14082–14091;
(b) G. Drabik and J. W. Naskalski, Acta Biochim. Pol., 2001, 48, 271–275.
4 Y. W. Yap, M. Whiteman and N. S. Cheung, Cell. Signalling, 2007, 19,
219–228.
5 Y. Maruyama, B. Lindholm and P. Stenvinkel, J. Nephrol., 2004, 17,
S72–S76.
6 S. Sugiyama, K. Kugiyama, M. Aikawa, S. Nakamura, H. Ogawa and
P. Libby, Arterioscler., Thromb., Vasc. Biol., 2004, 24, 1309–1314.
7 L. C. Adam and G. Gordon, Anal. Chem., 1995, 67, 535–540.
8 C. T. Dooley, T. M. Dore, G. T. Hanson, W. C. Jackson, S. J. Remington
and R. Y. Tsien, J. Biol. Chem., 2004, 279, 22284–22293.
9 (a) B. Lee Predmore, D. Joseph Lefer and G. Gojon, Antioxid. Redox
Signaling, 2012, 17, 119–140; (b) M. Whiteman, N. S. Cheung,
Y.-Z. Zhu, S. H. Chu, J. L. Siau, B. S. Wong, J. S. Armstrong and
P. K. Moore, Biochem. Biophys. Res. Commun., 2005, 326, 794–798.
10 (a) E. W. Miller, S. X. Bian and C. J. Chang, J. Am. Chem. Soc., 2007,
129, 3458–3459; (b) Y. Koide, M. Kawaguchi, Y. Urano, K. Hanaoka,
T. Komatsu, M. Abo, T. Teraia and T. Nagano, Chem. Commun.,
2012, 48, 3091–3093; (c) B. C. Dickinson, J. Peltier, D. Stone,
D. V. Schaffer and C. J. Chang, Nat. Chem. Biol., 2011, 7, 106–112.
(Fig. S13b, ESI†). Decreased intracellular fluorescence is observed
when the stimulated RAW264.7 cells were treated with the ROS
scavenger19 Glutathione S-Transferase (GST, EC: 2.5.1.18,
5 U mLÀ1) (Fig. S13c, ESI†). The subsequent treatment of cells
with PMA resulted in an increased cellular fluorescence (Fig. S13d,
ESI†), demonstrating that the low levels of intracellular fluores-
cence in Fig. S13c (ESI†) were not due to photobleaching or dye
leakage. In summary, these results suggested that NI–Se was
capable of being developed as a fluorescence probe to image
HOCl and the reducing repair in living cells.
Finally, NI–Se was applied to visualize HOCl and the redu-
cing repair in living mice. The HClO was generated by activated 11 Z. Lou, P. Li, X. Sun, S. Yang, B. Wang and K. Han, Chem. Commun.,
2013, 49, 391–393.
12 (a) S. Kenmoku, Y. Urano, H. Kojima and T. Nagano, J. Am. Chem.
macrophages and neutrophils in a lipopolysaccharide (LPS)
model of acute inflammation.12j,20 The untreated mouse was
Soc., 2007, 129, 7313–7318; (b) Y. Koide, Y. Urano, K. Hanaoka,
set as the control (Fig. 4a), and the mouse injected with both
LPS and NI–Se (Fig. 4c) exhibited more fluorescence than that
treated with NI–Se only (Fig. 4b). However, further injection of
H2S to the mouse parallel to (c) led to a decrease in fluorescence
intensity (Fig. 4d). In addition, the fluorescent intensity from
the entire body of the mice was quantified, and it can be noted
that the mouse loaded with LPS and NI–Se exhibited higher
fluorescence than the mouse loaded with saline and NI–Se. And
lower fluorescence intensity was observed in the additional H2S
treated mouse (Fig. 4e). Taken together, NI–Se can be developed
as a fluorescent probe for imaging HOCl and the reducing
repair of antioxidants in living mice.
Mimicking the GPx centre and taking advantage of the
mechanism of the twisting process, we have designed a rever-
sible fluorescent probe for HOCl. The PET process has been
excluded by TDDFT calculations, and the twisting process in
NI–Se has been verified by the influences of viscosity and
temperature experiments. (Further studies focusing on the
T. Terai and T. Nagano, J. Am. Chem. Soc., 2011, 133, 5680–5682;
(c) S. M. Chen, J. X. Lu, C. D. Sun and H. M. Ma, Analyst, 2010, 135,
577–582; (d) G. Chen, F. Song, J. Wang, Z. Yang, S. Sun, J. Fan,
X. Qiang, X. Wang, B. Dou and X. Peng, Chem. Commun., 2012, 48,
2949–2951; (e) X. Chen, K. A. Lee, E. M. Ha, K. M. Lee, Y. Y. Seo,
H. K. Choi, H. N. Kim, M. J. Kim, C. S. Cho, S. Y. Lee, W. J. Lee and
J. Yoon, Chem. Commun., 2011, 47, 4373–4375; ( f ) X. Q. Chen,
X. C. Wang, S. J. Wang, W. Shi, K. Wang and H. M. Ma,
Chem.–Eur. J., 2008, 14, 4719–4724; (g) J. Shepherd, S. A.
Hilderbrand, P. Waterman, J. W. Heinecke, R. Weissleder and
P. Libby, Chem. Biol., 2007, 14, 1221–1231; (h) W. Y. Lin,
L. L. Long, B. B. Chen and W. Tan, Chem.–Eur. J., 2009, 15,
2305–2309; (i) L. Yuan, W. Lin, J. Song and Y. Yang, Chem. Commun.,
2011, 47, 12691–12693; ( j) L. Yuan, W. Lin, Y. Yang and H. Chen,
J. Am. Chem. Soc., 2012, 134, 1200–1211; (k) Z.-N. Sun, F.-Q. Liu,
Y. Chen, P. K. H. Tam and D. Yang, Org. Lett., 2008, 10, 2171–2174;
(l) Y. K. Yang, H. J. Cho, J. Lee, I. Shin and J. Tae, Org. Lett., 2009, 11,
859–861; (m) X. D. Lou, Y. Zhang, Q. Q. Li, J. G. Qin and Z. Li, Chem.
Commun., 2011, 47, 3189–3191.
13 B. Wang, P. Li, F. Yu, P. Song, X. Sun, S. Yang, Z. Lou and K. Han,
Chem. Commun., 2013, 49, 1014–1016.
14 (a) K. P. Bhabak and G. Mugesh, Acc. Chem. Res., 2010, 43,
1408–1419; (b) G. Mugesh, W.-W. du Mont and H. Sies, Chem.
Rev., 2001, 101, 2125–2180.
driving force of the twist are currently underway). Here we also 15 F. Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, J. Am. Chem. Soc.,
2011, 133, 11030–11033.
16 Q. Li, M. Peng, H. Li, C. Zhong, L. Zhang, X. Cheng, X. Peng,
present the synthesis, characterization and fluorescence prop-
erties of this probe. And the fluorescent probe exhibits several
Q. Wang, J. Qin and Z. Li, Org. Lett., 2012, 14, 2094–2097.
advantages in terms of selectivity, sensitivity and reversibility. 17 Y. Adachi, A. L. Kindzelskii, A. R. Petty, J. B. Huang, N. Maeda,
S. Yotsumoto, Y. Aratani, N. Ohno and H. R. Petty, J. Immunol., 2006,
176, 5033–5040.
18 M. E. Lalande, V. Ling and R. G. Miller, Proc. Natl. Acad. Sci. U. S. A.,
Importantly, combining the confocal fluorescence microscopy,
this probe has been applied to visualize the HOCl and reducing
repair of living cells and living mice in situ. Therefore, this
study highlights a promising strategy for designing fluorescent
probes, and provides a potential tool for further research in
relevant biological processes of redox biology in living systems.
1981, 78, 363–367.
19 F. Angelucci, P. Baiocco, M. Brunori, L. Gourlay, V. Morea and
A. Bellelli, Structure, 2005, 13, 1241–1246.
20 K. Kundu, S. F. Knight, N. Willett, S. Lee, W. R. Taylor and
N. Murthy, Angew. Chem., Int. Ed., 2009, 48, 299–303.
c
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