A BODIPY fluorescent probe with selective response for hypochlorous acid and its application in cell imaging

Sensors and Actuators B 182 (2013) 1–6

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A BODIPY fluorescent probe with selective response for hypochlorous acid and its application in cell imaging Lizhi Gai a , John Mack b , Hui Liu a , Zheng Xu a , Hua Lu a,∗ , Zhifang Li a,∗ a b

Key Laboratory of Organosilicon Chemistry and Material Technology, Ministry of Education, Hangzhou Normal University, Hangzhou, 310012, PR China Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa

a r t i c l e

i n f o

Article history: Received 15 January 2013 Received in revised form 24 February 2013 Accepted 25 February 2013 Available online xxx Keywords: Hypochlorous acid BODIPY TD-DFT Fluorescent probe Cell image

a b s t r a c t A boron-dipyrromethene (BODIPY) derivative with a hydroxymethyl group at the meso-position, 1, was synthesized, which exhibits a highly specific and rapid ‘turn-off’ response for HOCl during confocal fluorescence microscopy experiments under physiological conditions and in live cells. The formation of an electron-withdrawing formyl substituent upon oxidation results in highly efficient nonradiative decay of the S1 state and hence in a drastic decrease in fluorescence emission intensity. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hypochlorous acid (OCl− /HOCl) is one of the most biologically significant reactive oxygen species (ROS), since it mediates a wide variety of biological processes in living systems and plays a critical role in the immune system [1]. Biologically, it is believed to be produced from hydrogen peroxide and chloride ions in activated neutrophils under the catalysis of a heme-containing enzyme, myeloperoxidase (MPO), which is localized mainly in leukocytes, such as neutrophils, macrophages, and monocytes [2]. Endogenous hypochlorous acid is essential to life and has many important antibacterial properties [3]. However, deficient or excessive levels of endogenous hypochlorous acid are harmful to the human health [4]. Therefore, there has been a strong research focus on the development of highly selective sensing and recognition of hypochlorous acid. Recently, there has been a particularly strong focus on fluorescent chemosensor-based detection, usually based on the strong oxidation properties of hypochlorous acid. For example, the oxidation reactions of p-methoxyphenol to benzoquinone [5], dibenzoylhydrazine to dibenzoyldiimide [6], hydroxamic acid to the acyl nitroso group [7], oxim- and dinitrophenylhydrazones to aldehydes [8], and thiols to sulfonate derivatives [9] and palkoxyanilines [10] have been reported as the basis for the design of HOCl-selective fluorescent probes. However, only a few fluorescent probes have been developed for selective imaging of OCl− /HOCl

∗ Corresponding authors. Tel.: +86 571 28867825; fax: +86 571 28865135. E-mail addresses: [email protected] (H. Lu), [email protected] (Z. Li). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.106

in live cells [5b,7,8b,11]. Enhanced sensitivity and reactivity are required for biological imaging applications. Herein, the synthesis of a boron-dipyrromethene (BODIPY) dye, 1, with a hydroxymethyl group at the meso-position is reported. The conversion of the hydroxymethyl group into a formyl group due to an oxidation reaction in the presence of HOCl was expected to serve as a sensing trigger that would substantially modify the optical properties of the BODIPY ␲-system. The BODIPY fluorophore was selected for study due to its well known desirable properties such as high molar absorption coefficients, high fluorescence quantum yields, excellent photostability, and versatile chemistry [12,13]. In recent years, these dyes have been widely employed in the detection of various kinds of ions, small molecules and viscosity [14]. However, only three examples of monitoring HOCl with a BODIPY have previously been reported [8b,11g,11f]. In this study, the BODIPY dye, 1, exhibits a highly selective, rapid and sensitive fluorescence response for HOCl. Confocal fluorescence microscopy experiments demonstrate that 1 can successfully be used to monitor HOCl in living cells. The quenching of the fluorescence emission intensity upon oxidation with HOCl, can be readily explained based on theoretical calculations. 2. Experimental 2.1. Materials and instrumentation All reagents were obtained from commercial suppliers and used without further purification unless otherwise indicated. All air and moisture-sensitive reactions were carried out under a nitrogen

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atmosphere. Dichloromethane and THF were distilled over calcium hydride and sodium, respectively. Triethylamine was obtained by simple distillation. Hypochlorous acid was prepared by distillation from a 5% commercial sodium hypochlorite solution and stored at 4 ◦ C, for periods of less than one week, as a 10 mM solution. The pH was adjusted to 11 by adding sodium hydroxide. Before use, sodium hypochlorite was assayed using a molar absorptivity of 391 M−1 cm−1 at 292 nm [15]. Deionized distilled water was used throughout. NMR spectra were recorded on a Bruker DRX400 spectrometer and referenced to the residual proton signals of the solvent. Mass spectra were measured with a Bruker Daltonics AutoflexIITM MALDI–TOF spectrometer. 2.2. Synthesis 2.2.1. Synthesis of 8-acetoxymethyl-1,3,5,7-tetramethyl BODIPY (2) 2,4-Dimethyl pyrrole (100 mg, 1.05 mmol) was dissolved in dry CH2 Cl2 (50 mL). Acetoxyacetyl chloride (0.07 mL, 0.65 mmol) was added to the solution, which was then stirred at 40 ◦ C under argon. After 2 h, the reaction mixture was cooled to room temperature and treated with triethylamine (0.5 mL) for 15 min. BF3 ·Et2 O (0.6 mL) was added and the solution was stirred for another 30 min, then washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated at reduced pressure. The crude product was purified by silica-gel flash column chromatography (elution with 50% EtOAc/hexane) to yield the target compound as orange-green crystals (120 mg, 71.4% yield). 1 H NMR (400 MHz, CDCl3 ) ı 6.08 (s, 2H), 5.30 (s, 2H), 2.53 (s, 6H), 2.36 (s, 6H), 2.13 (s, 3H); MALDI-TOF: calcd [C16 H19 BF2 N2 O2 ]+ m/z = 320.14, found m/z = 320.08 [M]+ , 301.05 [M−F]+ . 2.2.2. Synthesis of 8-Hydroxymethyl-1,3,5,7-tetramethyl BODIPY (1) 2 (100 mg, 0.31 mmol) was dissolved in dry 25 mL THF under argon. LiOH·H2 O (65 mg, 1.5 mmol), dissolved in 25 mL of water, was added to the solution. The reaction mixture was stirred for 4 h at room temperature under argon and was then extracted with ethylacetate (3 × 50 mL). The organic layers were washed with saturated aqueous NH4 Cl solution and brine, dried over MgSO4 , filtered, and evaporated under reduced pressure. The crude product was loaded onto a silica gel flash column and eluted with 50% EtOAc/hexane to yield 1 as an orange powder (55 mg, 64% yield). UV–vis (DMSO-HEPES buffer), max (ε) = 511 nm (81,000 dm3 mol−1 cm−1 ); 1 H NMR (400 MHz, CDCl3 ) ı 6.08 (s, 2H), 4.91 (s, 2H), 2.51 (s, 6H), 2.26 (s, 6H); HRMS-ESI: m/z: calcd for [C14 H17 N2 OBF2 Na]+ : 301.1294 [M+ Na]+ , found: 301.1298.

Scheme 1. Synthetic procedures for the preparation of 1.

2.5. Cell culture and confocal imaging MCF-7 cells were maintained by following the protocols provided by the American Type Culture Collection. Cells were seeded at a density of 1 × 106 cells mL−1 for confocal imaging in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS), NaHCO3 (2 g/L), and 1% antibiotics (penicillin/streptomycin, 100 U/ml). Cultures were maintained at 37 ◦ C under a humidified atmosphere containing 5% CO2 . Confocal fluorescence imaging studies were performed on a LSM 710 confocal laser-scanning microscope (Carl Zeiss Co., Ltd.). Cell imaging was carried out after washing the cells three times with PBS. The excitation wavelength was 488 nm, and emission intensity was measured in the 490–650 nm region. 2.6. Preparation of solutions Solutions of a series of ions (1 mM for I− , F− , SCN− , IO4 − , CH3 COO− , H2 PO4 − , ClO4 − , SO4 2− , Cr2 O7 2− , S2 O8 2− , H2 O2 , Cu2+ , Fe3+ and 1 mM, 10 mM for NaOCl) and a blank solution were prepared in deionized water. A stock solution of 1 (1 mM) was prepared in DMSO, which was diluted to 10 ␮M with HEPES buffer solution (1:99, v/v, pH 7.2) prior to the spectral measurements. During the titration experiments, a 2 mL solution of 1 was poured into a quartz optical cell with a 1 cm optical path length and the concentrated ion and blank solutions were added using a micro-pipette. Spectral data were recorded immediately after each addition. For all measurements, the excitation and emission slit widths were set at 5 nm.

2.3. Spectroscopic measurements 2.7. Theoretical calculations UV–vis absorption spectra were recorded on a Shimadzu 3000 spectrophotometer. Fluorescence spectra were measured on a Hitachi F-2700 FL spectrophotometer equipped with a xenon arc lamp. Absorption and emission measurements were carried out in 1 cm × 1 cm quartz cuvettes. The fluorescence lifetimes of the sample were measured on Horiba Jobin Yvon Fluorolog-3 spectrofluorimeter. For all measurements, the temperature was kept constant at (298 ± 2) K. 2.4. Generation of reactive oxygen species (ROS) Nitric oxide (NO• ) was generated from SNP (sodium nitroferricyanide (III) dihydrate). • O2 − was generated using xanthine and xanthine oxidase. Xanthine oxidase was added first. Xanthine was then added once the xanthine oxidase had fully dissolved. The mixture was then stirred at 25 ◦ C for 30 min.

Geometry optimization and TD-DFT calculations were carried out using the B3LYP functional of the G03W software package [16] with 6-31G(d) basis sets. 3. Results and discussion 1 was synthesized in two steps in 46% overall yield via the condensation reaction of acetoxyacetyl chloride and 2 equiv. of 2,4-dimethyl pyrrole under reflux in dichloromethane followed by treatment at room temperature of the reaction mixture with triethylamine and BF3 ·Et2 O (Scheme 1). The meso-acetoxymethyl BODIPY derivative was readily hydrolyzed to form the corresponding meso-hydroxymethyl BODIPY, 1, under basic conditions [17]. The main emission band of BODIPY 1 lies at 525 nm in DMSOHEPES buffer (1: 99, v/v, pH 7.2) solution (Fig. 1). There is a Stokes

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Fig. 1. Left: the fluorescence response of 1 (10 ␮M) in DMSO-HEPES buffer (50 mM HEPES, 1: 99, v/v, pH 7.2) after the addition of 10 equiv. of an anion or oxidative cation. ex = 480 nm. Right: solutions of 1 (100 ␮M) in the presence of different ions (excited at 365 nm using a UV lamp). From left to right: top: I− , NaOCl, F− , SCN− , IO4 − , CH3 COO− , H2 PO4 − ; bottom: ClO4 − , SO4 2− , Cr2 O7 2− , S2 O8 2− , H2 O2 , Cu2+ , Fe3+ .

shift of 14 nm relative to the corresponding absorption band, which is associated with the main S0 → S1 transition of the BODIPY chromophore. A shoulder of intensity associated with a vibrational band is also observed at around 560 nm (Fig. 1). Upon addition of NaOCl, the fluorescence intensity disappeared. In contrast, the fluorescence intensity of 1 was found to be almost unaffected by the addition of I− , F− , SCN− , IO4 − , CH3 COO− , H2 PO4 − , ClO4 − , SO4 2− , Cr2 O7 2− , S2 O8 2− , H2 O2 , Cu2+ , and Fe3+ and a blank solution. The quenching of the fluorescence intensity in the presence of NaOCl can be assigned to the conversion of the hydroxymethyl group at the meso-position into a formyl group due to an oxidation reaction. The formation of the formyl group was confirmed by the mass spectrometry of 1 in the presence of NaOCl (Fig. 2). The peak observed at 275.1174 m/z is consistent with the value calculated for the parent peak of a formyl-substituted BODIPY. The 1 H NMR spectroscopy of 1 in CDCl3 was also analyzed (Fig. 2). After the addition of NaOCl, the proton signals of the methylene group at 4.91 ppm completely disappeared, and a new singlet signal was observed at 10.59 ppm. This is consistent with the oxidation of the hydroxymethyl group to form a formyl substituent. Since the pKa of OCl− /HOCl equilibrium is 7.6, it is HOCl rather than OCl− that is being detected, as has also

Fig. 2. HR − MS spectrum of 1 + NaOCl (top) and the partial 1 H NMR (400 MHz) spectra of a) 1, b) 1 + NaOCl (bottom).

been the case with the probes reported previously by other groups [9,10]. The sensitivity of 1 to hypochlorous acid was investigated by fluorescence spectroscopy. Upon addition of NaOCl at different concentrations in a DMSO-HEPES buffer (1: 99, v/v, pH 7.2) solution (Fig. 3), the fluorescence intensity of 1 decreased gradually with the intensity maximum remaining virtually unchanged. The observed fluorescence intensity was proportional to the concentration of NaOCl within the 1 to 30 ␮M range. The interaction of 1 with hypochlorite was completed within a few seconds. This demonstrates that 1 can potentially be used for real-time and real-space analyses of hypochlorite in cells and organisms. The changes in the optical properties of 1, which occur upon oxidation with HOCl, can be readily explained based on theoretical calculations. When the hydroxymethyl group is converted into a formyl, the inductive effect of the substituent stabilizes both the HOMO and the LUMO. The HOMO of 1 has a nodal plane at the meso-carbon position, while there is a significant MO coefficient in the context of the LUMO (Fig. 4). The LUMO mixes with a low-lying MO associated with the formyl group (LUMO + 1) resulting in a stabilizing mesomeric interaction between the BODIPY ␲-system and the formyl group. There is a narrowing of the HOMO–LUMO band gap and an increased dipole moment in the context of the S1 state due to the electron withdrawing properties of the formyl group. The lowest lying absorption band of the hydroxymethyl-substituted BODIPY, 1, is predicted by TD-DFT to be associated primarily with the HOMO → LUMO transition (419 nm, f = 0.48; 64% HOMO → LUMO, 7% HOMO − 1 → LUMO). In the presence of the formyl substituent, a one-electron transition into the LUMO + 1 is also predicted to play a role (467 nm, f = 0.35; 64% HOMO → LUMO, 4% HOMO → LUMO+ 1, 3% HOMO − 1 → LUMO). As has been reported previously in the context of cyano-substituted BODIPYs, the electron withdrawing properties of the meso-substituent result in a quenching of the fluorescence intensity, since electron density is drawn out of the ␲-conjugation system of the fluorophore [18]. Despite the rigid structure of the BODIPY fluorophore, the fluorescence intensity can also be further quenched in polar solvents by nonradiative decay via conical intersections between the ground state and what can be viewed as being an intramolecular charge transfer (ICT) state derived from the S1 state of the fluorophore [19,20]. In the context of 1, this quenching may be further enhanced by steric interactions between the formyl group and the methyl substituents at the 1- and 7-positions, since it has been demonstrated that the ICT character of non-emissive S1 states is enhanced when there is a twisting of the bond linking the donor and acceptor moieties [20].

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Fig. 3. Left: fluorescence spectra of 1 (10 ␮M) in DMSO-HEPES buffer (50 mM HEPES, 1: 99, v/v, pH 7.2) after the addition of NaOCl. The excitation wavelength was 480 nm. The inset shows a Stern–Volmer plot of I0 /I vs [NaOCl], where I is the quenched fluorescence, I0 is the unquenched fluorescence and [NaOCl] the hypochlorite concentration. The Stern–Volmer constant (ksv ) is 4.45 × 104 M−1 from the slope of the line, the quenching rate constant (kq = 7.05 × 1012 M−1 s−1 ), is determined from the equation kq = ksv / o , where  o is the lifetime of the fluorophore. Right: the reaction–time profile of probe 1 (10 ␮M) in the presence of 10 equiv. of NaOCl in DMSO-HEPES buffer (50 mM HEPES, 1: 99, v/v, pH 7.2).

Fig. 4. Left: the nodal patterns of the HOMO and LUMO of 1 (top) and its formyl substituted analog (bottom) at an isosurface value of 0.05 a.u. Right: the relative energies of the frontier ␲-MOs.

The sensing properties of 1 in the presence of various reactive oxygen species (ROS) were investigated to assess the selectivity of the response. No significant change in fluorescence intensity was observed upon addition of H2 O2 , • O2 − , NO• (Fig. 5). Fluorescent quenching was observed only upon reaction with NaOCl. Therefore, 1 exhibits excellent selectivity toward HOCl when the various

ROS that are likely to be present in abiotic systems are taken into consideration. The practical applicability of 1 as a probe for hypochlorous acid in living cells was also evaluated. Upon incubation with 20 ␮M of 1 for 30 min at 37 ◦ C, the cells displayed a strong intracellular fluorescence image. When further treated with NaOCl for 30 min in

Fig. 5. The emission color changes (left) and fluorescence intensity changes (right) of 1 (100 ␮M) in DMSO-HEPES buffer (50 mM HEPES, 1: 99, v/v, pH 7.2) in the presence of the following ROS (from left to right) none, HOCl, H2 O2 , • O2 − , NO• . 50 mM NaOCl and 20 mM H2 O2 were added prior to the HOCl and H2 O2 measurements. Prior to the • O2 − measurement, 20 mM KO2 was added and the mixture was stirred at 37 ◦ C for 30 min. 20 mM sodium nitroferricyanide(III) was added and the mixture was stirred at 25 ◦ C for 30 min prior to the NO• measurement.

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Fig. 6. Confocal fluorescence images of HOCl in MCF-7 cells. a) MCF-7 cells loaded with 20 ␮M probe for 10 min at 37 ◦ C. b) probe-loaded cells incubated with 60 ␮M NaOCl for 30 min at 37 ◦ C. c) and d) represent the bright field images of a) and b) respectively. Scale bars: 20 ␮m.

the culture medium, and washed with phosphate buffered saline to remove extracellular HOCl, the bright green fluorescence image disappeared in these cells (Fig. 6). The fluorescence and bright-field images revealed that the fluorescence signals were localized in the perinuclear area of the cytosol, indicative of a subcellular distribution due to the high cell membrane permeability of 1 [21]. These results demonstrate that the use of 1 for monitoring HOCl in living cells merits further investigation.

4. Conclusion In summary, we have demonstrated that a borondipyrromethene (BODIPY) derivative, 1, bearing a hydroxymethyl group at the meso-position can act as a fluorescence turn-off chemosensor for hypochlorite in aqueous media with high sensitivity, a rapid response time, and high selectivity in the presence of other ions and oxidative molecules. The quenching of the fluorescence intensity after oxidation with HOCl can be attributed to the influence of the mesomeric interaction between the formyl group that is formed at the meso-position and the LUMO of the fluorophore ␲-system. An increase in the ICT character of the S1 state probably results in the formation of conical intersections with the ground state resulting in efficient nonradiatiave decay in a manner similar to that reported previously for analogous molecules [20]. Moreover, confocal fluorescence microscopy experiments have established the utility of 1 for monitoring the presence of hypochlorous acid in living cells, thus demonstrating its potential applicability for studying the effect of hypochlorous acid in biological systems.

Acknowledgment This research was financially supported by NSFC (nos. 21101049 and 20903032). References [1] (a) A. Gomes, E. Fernandes, J.L. Lima, Fluorescence probes used for detection of reactive oxygen species, Journal of Biochemical and Biophysical Methods 65 (2005) 45–80; (b) X.H. Li, G.X. Zhang, H.M. Ma, D.Q. Zhang, J. Li, D.B. Zhu, 4,5-Dimethylthio-4 [2-(9-anthryloxy)ethylthio]tetrathiafulvalene, a highly selective and sensitive chemiluminescence probe for singlet oxygen, Journal of the American Chemical Society 126 (2004) 11543–11548. [2] (a) J.M. Pullar, M. Vissers, C. Winterbourn, Living with a killer: the effects of hypochlorous acid on mammalian cells, IUBMB Life 50 (2000) 259–266; (b) Y.W. Yap, M. Whiteman, N.S. Cheung, Chlorinative stress: an under appreciated mediator of neurodegeneration? Cellular Signalling 19 (2007) 219–228. [3] Y. Yan, S.H. Wang, Z.W. Liu, H.Y. Wang, D.J. Huang, CdSe-ZnS quantum dots for selective and sensitive detection and quantification of hypochlorite, Analytical chemistry 82 (2010) 9775–9781. [4] (a) E.A. Podrez, H.M. Abu Soud, S.L. Hazen, Myeloperoxidase-generated oxidants and atherosclerosis, Free Radical Biology and Medicine 28 (2000) 1717–1725; (b) D.I. Pattison, M.J. Davies, Evidence for rapid inter-and intramolecular chlorine transfer reactions of histamine and carnosine chloramines: implications for the prevention of hypochlorous- acid-mediated damage, Biochemistry 45 (2006) 8152–8162; (c) S. Sugiyama, Y. Okada, G.K. Sukhova, R. Virmani, J.W. Heinecke, P. Libby, Macrophagemy eloperoxidase regulation by granulocyte macrophage colonystimulating factor in human atherosclerosis and implications in acute coronary syndromes, American Journal of Pathology 158 (2001) 879–891; (d) L.J. Hazell, L. Arnold, D. Flowers, G. Waeg, E. Malle, R. Stocker, Presence of hypochlorite-modified proteins in human atherosclerotic lesions, Journal of Clinical Investigation 97 (1996) 1535–1544; (e) S.M. Wu, S.V. Pizzo, Alpha(2)-macroglobulin from rheumatoid arthritis synovial fluid: functional analysis defines a role for oxidation in

6

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

L. Gai et al. / Sensors and Actuators B 182 (2013) 1–6 inflammation, Archives of Biochemistry and Biophysics 391 (2001) 119–126; (f) H.E. Jasin, Oxidative modification of inflammatory synovial fluid immunoglobulin G, Inflammation 17 (1993) 167–181. (a) W.J. Zhang, C. Guo, L.H. Liu, J.G. Qin, C.L. Yang, Naked-eye visible and fluorometric dual-signaling chemodosimeter for hypochlorous acid based on water-soluble p-methoxyphenol derivative, Organic & Biomolecular Chemistry 9 (2011) 5560–5563; (b) Z.N. Sun, F.Q. Liu, Y. Chen, P.K.H. Tam, D. Yang, A highly specific BODIPYbased fluorescent probe for the detection of hypochlorous acid, Organic Letters 10 (2008) 2171–2174; (c) K. Cui, D.Q. Zhang, G.X. Zhang, D.B. Zhu, A highly selective naked-eye probe for hypochlorite with the p-methoxyphenol-substituted aniline compound, Tetrahedron Letters 51 (2010) 6052–6055; (d) W.J. Zhang, C.G. Li, J.G. Qin, C.L. Yang, Water-soluble poly(p-phenylene) incorporating methoxyphenol units: highly sensitive and selective chemodosimeters for hypochlorite, Polymer 53 (2012) 2356–2360. X.Q. Chen, X.C. Wang, S.J. Wang, W. Shi, K. Wang, H.M. Ma, A highly selective and sensitive fluorescence probe for the hypochlorite anion, Chemistry-A European Journal 14 (2008) 4719–4724. X.Q. 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, J. Yoon, A specific and sensitive method for detection of hypochlorous acid for the imaging of microbe-induced HOCl production, Chemical Communications 47 (2011) 4373–4375. (a) J. Hwang, M.G. Choi, J. Bae, S.K. Chang, Signaling of hypochlorous acid by selective deprotection of dithiolane, Organic & Biomolecular Chemistry 9 (2011) 7011–7015; (b) Y.K. Yang, H.J. Cho, J. Lee, I. Shin, J. Tae, A rhodamine-hydroxamic acid-based fluorescent probe for hypochlorous acid and its applications to biological imagings, Organic Letters 11 (2009) 859–861; (c) J. Shi, Q. Li, X. Zhang, M. Peng, J. Qin, Z. Li, Simple triphenylamine-based luminophore as a hypochlorite chemosensor, Sensors and Actuators B: Chemical 145 (2010) 583–587. S. Kenmoku, Y. Urano, H. Kojima, T. Nagano, Development of a highly specific rhodamine-based fluorescence probe for hypochlorous acid and its application to real-time imaging of phagocytosis, Journal of the American Chemical Society 129 (2007) 7313–7318. J. Shepherd, S.A. Hilderbrand, P. Waterman, J.W. Heinecke, R. Weissleder, P. Libby, A fluorescent probe for the detection of myeloperoxidase activity in atherosclerosis-associated macrophages, Chemistry & Biology 14 (2007) 1221–1231. (a) S.M. Chen, J.X. Lu, C.D. Sun, H.M. Ma, A highly specific ferrocene-based fluorescent probe for hypochlorous acid and its application to cell imaging, Analyst 135 (2010) 577–582; (b) X.Q. 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, J. Yoon, A specific and sensitive method for detection of hypochlorous acid for the imaging of microbe-induced HOCl production, Chemical Communications 47 (2011) 4373–4375; (c) X.H. Cheng, H.Z. Jia, T. Long, J. Feng, J.G. Qin, Z. Li, A “turn-on” fluorescent probe for hypochlorous acid: convenient synthesis, good sensing performance, and a new design strategy by the removal of C N isomerization, Chemical Communications 47 (2011) 11978–11980; (d) L. Yuan, W.Y. Lin, Y.N. Xie, B. Chen, J.Z. Song, Fluorescent detection of hypochlorous acid from turn-On to FRET-based ratiometry by a HOCl-mediated cyclization reaction, Chemistry-A European Journal 18 (2012) 2700–2706; (e) X.Q. Zhan, J.H. Yan, J.H. Su, Y.C. Wang, J. He, S.Y. Wang, H. Zheng, J.G. Xu, Thiospirolactone as a recognition site: Rhodamine B-based fluorescent probe for imaging hypochlorous acid generated in human neutrophil cells, Sensors and Actuators B: Chemical 150 (2010) 774–780; (f) Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, Development of a Sirhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging, Journal of the American Chemical Society 133 (2011) 5680–5682; (g) T. Kim, S. Park, Y. Choi, Y. Kim, A BODIPY-based probe for the selective detection of hypochlorous acid in living cells, Chemistry – An Asian Journal 6 (2011) 1358–1361; (h) S.R. Liu, S.P. Wu, Hypochlorous acid turn-on fluorescent probe based on oxidation of diphenyl selenide, Organic Letters 15 (2013) 878–881. (a) A. Treibs, F.H. Kreuzer, Difluorboryl-komplexe von di-und tripyrrylmethenen, Justus Liebigs Annalen der Chemie 718 (1968) 208–223; (b) Y.H. Yu, A.B. Descalzo, Z. Shen, H. Röhr, Q. Liu, Y.W. Wang, M. Spieles, Y.Z. Li, K. Rurack, X.Z. You, Mono- and di(dimethylamino)styryl-substituted borondipyrromethene and borondiindomethene dyes with intense nearinfrared fluorescence, Chemistry – An Asian Journal 1–2 (2006) 176–187. (a) H. Lu, Z.L. Xue, J. Mack, Z. Shen, X.Z. You, N. Kobayashi, Specific Cu2+ -induced J-aggregation and Hg2+ -induced fluorescence enhancement based on BODIPY, Chemical Communications 46 (2010) 3565–3567; (b) Z. Shen, H. Röhr, K. Rurack, H. Uno, M. Spieles, B. Schulz, G. Reck, N. Ono, Boron-diindomethene (BDI) dyes and their tetrahydrobicyclo precursors—en route to a new class of highly emissive fluorophores for the red spectral range, Chemistry-A European Journal 10 (2004) 4853–4871;

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

(c) H. Lu, L. Xiong, H. Liu, M. Yu, Z. Shen, F. Li, X. You, A highly selective and sensitive fluorescent turn-on sensor for Hg2+ and its application in live cell imaging, Organic & Biomolecular Chemistry 7 (2009) 2554–2558; (d) H. Lu, Q.H. Wang, L.Z. Gai, Z.F. Li, Y. Deng, X.Q. Xiao, G.Q. Lai, Z. Shen, Tuning the solid-state luminescence of BODIPY derivatives with bulky arylsilyl groups: synthesis and spectroscopic properties, Chemistry-A European Journal 18 (2012) 7852–7861; (e) L.Z. Gai, H. Lu, B. Zou, G.Q. Lai, Z. Shen, Z.F. Li, Synthesis and spectroscopic properties of bodipy dimers with effective solid-state emission, RSC Advances 2 (2012) 8840–8846. (a) N. Boens, V. Leen, W. Dehaen, Fluorescent indicators based on BODIPY, Chemical Society Reviews 41 (2012) 1130–1172; (b) M.K. Kuimova, G. Yahioglu, J.A. Levitt, K. Suhling, Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging, Journal of the American Chemical Society 130 (2008) 6672–6673. J.R. Kanofsky, Singlet oxygen production from the reactions of alkylperoxy radicals. Evidence from 1268-nm chemiluminescence, Journal of Organic Chemistry 51 (1986) 3386–3388. Gaussian 03 (RevisionC.02), M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery, Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. AlLaham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M. W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian, Inc., Wallingford CT, 2004. (a) K. Krumova, G. Cosa, Bodipy dyes with tunable redox potentials and functional groups for further tethering: preparation, electrochemical, and spectroscopic characterization, Journal of the American Chemical Society 132 (2010) 17560–17569; (b) P. Oleynik, Y. Ishihara, G. Cosa, Design and synthesis of a BODIPY-␣tocopherol adduct for use as an off/on fluorescent antioxidant indicator, Journal of the American Chemical Society 129 (2007) 1842–1843; (c) M.T. Whited, N.M. Patel, S.T. Roberts, K. Allen, P.I. Djurovich, S.E. Bradforth, M.E. Thompson, Symmetry-breaking intramolecular charge transfer in the excited state of meso-linked BODIPY dyads, Chemical Communications 48 (2012) 284–286. F. López Arbeloa, J. Bac¸uelos Prieto, V. Martínez Martínez, T. Arbeloa López, I. López Arbeloa, Intramolecular charge transfer in pyrromethene laser dyes: photophysical behaviour of PM650, ChemPhysChem 5 (2004) 1762–1771. G.I.I. Jones, S. Kumar, O. Klueva, D. Pacheco, Photoinduced electron transfer for pyrromethene dyes, The Journal of Physical Chemistry A 107 (2003) 8429–8434. Z.R. Grabowski, K. Rotkiewicz, W. Rettig, Structural changes accompanying intramolecular electron transfer: focus on twisted intramolecular chargetransfer states and structures, Chemical Review 103 (2003) 3899–4031. J. Cao, C.C. Zhao, X.Z. Wang, Y.F. Zhang, W.H. Zhu, Target-triggered deprotonation of 6-hydroxyindole-based BODIPY: specially switch on NIR fluorescence upon selectively binding to Zn2+ , Chemical Communications 48 (2012) 9897–9899.

Biographies Lizhi Gai received his B.Sc. from Qufu Normal University in 2010. His research interests concern the design of fluorescent probes. John Mack received his Ph.D. from the University of Western Ontario in 1994. He is currently a senior researcher at Rhodes University, South Africa. His research interests are mainly focused on porphyrinoid and, BODIPY dyes, MCD spectroscopy and TD-DFT calculations. Hui Liu received her B.Sc. from Liaocheng University in 2012. Her research interests concern the design of organosilicon luminescent materials. Zheng Xu received her Ph.D. in 2007 from Zhejiang University. Her research interests are mainly in the areas of reaction mechanism studies involved organosilicon compounds. Hua Lu received his Ph.D. in 2010 from Nanjing University. His research interests are mainly in the areas of dyes, photochemistry, TD-DFT Calculations and organosilicon functional materials. Zhifang Li received his Ph.D. in 2003 from Zhejiang University. His research interests are mainly in the areas of silylenes and polysilanes.