Novel Tumor-Specific and Mitochondria-Targeted near-Infrared

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A novel tumor-specific and mitochondria-targeted near-infraredemission fluorescent probe for SO2 derivatives in living cells Jin Yang, Kun Li, Ji-Ting Hou, Ling-Ling Li, Chun-Yan Lu, Yongmei Xie, Xin Wang, and Xiaoqi Yu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00165 • Publication Date (Web): 30 Nov 2015 Downloaded from http://pubs.acs.org on December 4, 2015

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A novel tumor-specific and mitochondria-targeted near-infrared-emission fluorescent probe for SO2 derivatives in living cells

Jin Yanga, Kun Lia*,b, Ji-Ting Hou a, Ling-Ling Lia, Chun-Yan Lub, Yong-Mei Xieb, Xin Wang a, Xiao-Qi Yua* a.

Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry Sichuan

University, 29, Wangjiang Road, Chengdu, Sichuan Province,P. R. China.E-mail: [email protected]; [email protected]. b

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and

Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan Province., P. R. China.

ABSTRACT

Endogenous sulfur dioxide (SO2) is an important gaseous signal molecule, which was also regarded as one of reactive sulfur spaces (RSS) and closely related to cardiovascular diseases and many neurological disorders. However, the design and synthesis of fluorescent probes with near-infrared-emission which can detect mitochondrial SO2 and its derivatives in living cells still remain unresolved. Herein, a biotin and coumarin-benzoindole conjugate BCS-1 was presented as a ratiometric and colorimetric fluorescent probe for tracing SO2 derivatives with excellent selectivity

and

rapid

responsibility.

Notably,

it

is

the

first

mitochondria-targeted

near-infrared-emission probe that could selectively detect SO2 in tumor cells. BCS-1 could selectively enter into mitochondria of tumor cells, and the detection limit for SO2 derivatives was determined as 72 nM. Keywords: tumor-specific probe, mitochondria, near-infrared-emission, ratiometric fluorescent

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probe, sulfur dioxide

Sulfur dioxide (SO2) has been known to be an air pollutant, existing in the form of sulfite (SO32-) and bisulfite (HSO3-) in neutral solutions (3:1 M/M)[1,2]. Epidemiological studies have confirmed that not only many respiratory responses [3] but also cardiovascular diseases and many neurologicaldisorders such as brain cancer [4] are caused by SO2 and its derivatives. Therefore, it is urgent to develop analytical methods for SO2 detection; especially those could realize detection in organisms. Fluorescent probes have been regarded as an excellent detection technique because of their high selectivity and sensitivity as well as real-time imaging [5-8].

Since SO2 can be endogenously generatedin cells during oxidation of H2S or sulphur containing amino aicds, it is crucial to develop fluorescent probes which can distinguish SO2 and its derivatives from other reactive sulfur species (RSS, such as H2S, Cys, GSH) in living cells. Till this end, a number of fluorescent probes for SO2 detection have been reported. The reaction mechanisms can be probably sorted as two classes, one is the nucleophilic addition to aldehydes/ ketones [8-13], which has a limitation as it may suffer from the interference of biothiols [15, 16], the other is the nucleophilic addition to double bonds especially the unsaturated compounds addition reaction [17], which can avoid the biothiol interference [18,19]. Many reported probes have gained a low detection limit and short response time even in real-time [20]. However, to the best of our knowledge, the fluorescent probes for SO2 detection that can target a certain kind of organelle or cell are rare [21]. Mitochondria, the principle energy-producing organelle, are the main source of intracellular reactive oxygen/sulfur species (ROS/RSS) [22-26]. It is reported that endogenous SO2 can be 2


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generated by aspartate aminotransferase 2 in mitochondria [27]. Imaging SO2 derivatives in mitochondria is particularly meaningful and valuable. Meanwhile, more and more proof indicated that RSS is deeply involved in the function of tumor cell [28]. Therefore, specific imaging of mitochondria SO2 derivatives in tumor cells is crucial to understand the generation of cancer. Not long ago, we presented the conjugate of carbazole and indolium as a novel mitochondria-targeted ratiometric fluorescent probe for imaging of SO2 derivatives in living cells [29]. To continue our work, we wondered if we could develop a tumor-specific probe by introducing a targeting group. It has been reported that biotin was overexpressed in tumor cells, and can serve as useful biomarkers for the identification of them [30-32]. That is to say, biotin can be taken up preferentially in tumor cells [33-34]. To date, Kim’s group has successfully applied the biotin moiety to tumor-specific selectivity [35-40]. Additionally, it is known that many delocalized lipophilic cationic dyes, such as cyanine and rhodamine, possess an overall positive charge, which can be easily taken up and accumulate in the mitochondria of living cells [41-44]. Althgough biotin have been widely applied in cancer-targeting drugs, it has never been reported in the design of probes that may show good performance in tracking cancer process.

Herein, we presented a biotin and coumarin conjugate BCS-1 as the first tumor-specific and mitochondria-targeted ratiometric fluorescent probe for sensing of SO2 derivatives (Scheme 1). BCS-1 can realize a ratiometric and colorimetric detection of SO2 in real-time, with a low detection limit (72 nM) and NIR fluorescence character.

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Scheme 1 Synthesis of probe BCS-1.

EXPERIMENTAL General information and methods All materials were obtained from commercial suppliers and were used without further purification. All the solvents were dried according to the standard methods prior to use. All of the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies. And all reactions were monitored by TLC analyses on silica gel GF 254. Column chromatographic purifications were carried out on silica gel (HG/T2354-92). NMR spectra were measured on a F7000. The 1H NMR (400 MHz) chemical shifts were given in ppm relative to the internal reference TMS. The

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C NMR (100 MHz) chemical shifts were given using CDCl3 and

DMSO-d6as the internal standard. ESI-MS and HR-MS spectral data were recorded on a Finnigan LCQDECA and a BrukerDaltonics Bio TOF mass spectrometer, respectively. Fluorescence excitation and emission spectra were obtained using FluoroMax-4 Spectrofluoro photometer (HORIBA JobinYvon). UV-Vis absorption spectra were recorded on a Hitachi PharmaSpec

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UV-1900 UV-Visible spectrophotometer. A stock solution of BCS-1 (5 mM) was prepared in DMSO. All the stock solutions of anions and reactive sulfur were prepared with corresponding sodium salts in deionized water at a concentration of 50 mM. Test solutions were prepared by placing 5µL of the probe stock solution into a test tube, diluting the solution to 5 mL with PBS buffer (pH 7.4, 10 mM, containing 30% DMF), and adding an appropriate aliquot of each anion stock. Fluorescence spectra were measured upon the addition of anions. Synthesis of the probe BCS-1 Compound 1-8 were synthesized according to the literature, and the details could be found in supporting information (Scheme S1). Synthesis of compound 9: To a solution of compound 7 (244 mg, 0.85 mmol), compound 8 (239 mg, 0.85 mmol) in 30 mL THF– water (1:1, v/v) was added CuI (325 mg, 1.71 mmol). The mixture was stirred at 70 oC for 24 h, and monitored by TLC. After reaction, the solution was cooled down to room temperature and the precipitate was filtered to give a yellow solid (283 mg, yield: 59 %). 1H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 8.45 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 7.97 (s, 1H), 7.69 (d, J = 9.0 Hz, 1H), 6.78 (dd, J = 9.0, 2.0 Hz, 1H), 6.66 (d, J = 1.8 Hz, 1H), 6.44 (s, 1H), 6.38 (s, 1H), 4.58 (t, J = 6.2 Hz, 2H), 4.32 – 4.26 (m, 1H), 4.24 (d, J = 5.6 Hz, 2H), 4.14 – 4.09 (m, 1H), 3.92 (t, J = 6.2 Hz, 2H), 3.33 – 3.26 (m, 2H), 3.12 – 3.04 (m, 1H), 2.81 (dd, J = 12.4, 5.0 Hz, 1H), 2.56 (d, J = 12.4 Hz, 1H), 2.06 (t, J = 7.4 Hz, 2H), 1.68 – 1.16 (m, 7H), 1.02 (t, J = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) δ 187.8, 172.4, 172.4, 163.2, 161.0, 158.7, 153.9, 146.9, 145.8, 133.4, 124.0, 114.5, 111.0, 108.7, 97.5, 61.5, 59.7, 55.9, 50.2, 47.2, 45.6, 35.4, 34.5, 28.7, 28.5, 25.7, 12.2. HRMS (ESI): m/z [M+Na]+ calcd for C27H33N7NaO5S: 590.2162; found 590.2165. 5


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Synthesis of BCS-1: Compound 10 (88 mg, 0.26 mmol) and ammonium acetate (17 mg, 0.21 mmol) were dissolved in 10 mL EtOH, Then compound 9 (100 mg, 0.18 mmol) in 3 mL freshly distilled DMF were added to the mixed solution. After stirred at 60 oC for 24 h, the solution was cooled down to room temperature and the precipitate was filtered to give a bluish-green solid (105 mg, yield: 68 %).1H NMR (400 MHz, DMSO) δ 9.18 (s, 1H), 8.44 (d, J = 8.2 Hz, 1H), 8.38 (d, J = 16.2 Hz, 1H), 8.28 (d, J = 8.8 Hz, 2H), 8.20 (d, J = 5.2 Hz, 2H), 7.98 (d, J = 17.4 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.71 (t, J = 7.4 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.75 (s, 1H), 6.40 (d, J = 22.4 Hz, 2H), 4.88 (s, 2H), 4.63 (s, 2H), 4.28 (dd, J = 13.8, 5.4 Hz, 3H), 4.13 (s, 1H), 3.99 (s, 2H), 3.36 (s, 2H), 3.10 (s, 1H), 2.81 (dd, J = 12.4, 4.6 Hz, 1H), 2.72 (s, 2H), 2.58 (d, J = 12.4 Hz, 1H), 2.28 (s, 2H), 2.09 (t, J = 7.2 Hz, 2H), 2.00 (s, 5H), 1.71 – 1.18 (m, 7H), 1.07 (t, J = 6.2 Hz, 3H).13C NMR (126 MHz, ) δ 183.9, 180.5, 175.6, 171.0, 168.9, 165.6, 161.1, 157.6, 156.1, 149.8, 145.7, 140.8, 137.3, 134.0, 132.7, 131.3, 127.8, 126.0, 121.6, 116.8, 113.4, 103.2, 101.0, 88.1, 65.2, 57.2, 53.5, 50.7, 47.7, 39.8, 34.9, 28.8, 23.2, 16.7, 12.7. HRMS (ESI): m/z [M+Na]+ calcd for C45H52N8NaO7S2: 903.3298; found 903.3293 Imaging of cells Hela cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic at 37oC in a 5% CO2/95% air incubator. For fluorescence imaging, cells (4×103/well) were passed on confocal dishes and incubated for 24h. Immediately before the staining experiment, cells were washed twice with PBS (10 mM), 1 and 2 both incubated with 5 µM BCS-1 for 30 min at 37 oC. Then dish 1 incubated with 1 µM Mito Tracker Red for 10min at 37 oC; dish 2 incubated with 50 µM sulfite for 15 min at 37 oC and then dish 2 incubated with 1µM Mito Tracker Red for 10min at 37oC. Finally, wash each dish with PBS 6


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(10 mM) for 3 times, and analyzed with a confocal fluorescence microscope. The green channel in 490-520 nm was collected, the red channel in 630-670 nm was collected and the MT Red in 570-620 nm was collected. They were both excited at 488 nm. Cytotoxicity assays Hela cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic at 37 oC in a 5% CO2/95% air incubator. Immediately before the experiment, the cells well placed in a 96 - well plate, followed by addition of increasing concentrations of BCS-1. The final concentrations of the probe were kept from 0 to 20 µM. The cells were then incubated at 37 ºC in an atmosphere of 5% CO2and 95% air for 24 h, followed by MTT assays (n= 5). Untreated assay with RPMI 1640 (n = 5) was also conducted under the same conditions. Flow cytometric study The flow cytometry tests were completed with a BD Accuri C6 the λex/ λem mode (FL1 Green channel: filter 530 ±30 nm, λex: 488 nm; FL4 Red channel: filter 660 ±20 nm, λex: 640 nm. All the cells were pretreated before the injection.

RESULTS AND DISCUSSION Design and synthesis of BCS-1 To selectively detect mitochondria SO2, we introduced the benzoindoles moiety into coumarin framework, which could not only act as the mitochondria-targeted carrier, but also improve the water solubility of the probe [45-47]. To realize the tumor selectivity, biotin was firstly labeled in coumarin fluorophore via click reaction with 58% yield. Then, BCS-1 was synthesized through 7


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one simple reaction with middle yield (Scheme 1). Due to the conjugation of coumarin and benzoindol moiety, BCS-1 will exhibit a near-infrared-emission emission; while in the presence of SO2 derivatives, this conjugation will disturbed and result in hypsochromic shift of the fluorescence emission. Based on the intensity at two different emissions, a ratiometric fluorescent probe for SO2 derivatives was developed. Spectral studies We initially examined the fluorescent response of BCS-1 towards SO32-/ HSO3-in PBS buffer solution (pH7.4, 10 mM, containing 30% DMF). As shown in Fig. 1, a strong absorption at 582 nm and a moderate emission at 652 nm were observed in the absorption and fluorescence spectra, respectively. After 10 equiv. of bisulfite was added, the absorption of BCS-1 at 582 nm decreased promptly, and two new absorptions appeared at 392 nm and 456 nm, accompanied by a colour change from royal purple to bright yellow. While in fluorescence spectra, the emission peak at 652 nm rapidly decreased as a new emission peak emerged at 505 nm, and the intensity ratio changes at the two emission wavelengths (I505 nm/I652

nm)

was calculated as 290-fold (from 0.21 to 62.5). The colour

under UV-irradiation was found changing from red to blue-green (Fig. 1b).

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Fig. 1 The absorption (a) and fluorescence (b) spectra of the probe BCS-1 (5µM) upon the addition of 10 equiv. of sulfite (50 µM) in PBS buffer (pH 7.4, 10 mM, containing 30% DMF) with excitation at 467 nm. Sensing mechanism studies It was reported that HSO3- and SO32- can rapidly and quantitatively react with α, β -unsaturated compounds in aqueous solution. It is much easier to attack the double bonds in an organic solvent. We can see in Figure S1 (a), upon addition of SO32-, the absorption of BCS-1 at 582 nm decreased, along with the simultaneous emergence of a new absorption at 460 nm; after that, the absorption at 460 nm gradually decreased, and concomitantly, the two new absorptions at 387 nm and 319 nm respectively emerged. The absorption at 460 nm still existed after 90 min and we preliminarily inferred there were two additive products, one product transformed into another one. Then the UV-spectra of benzoindoles, BSC-1, BSC-1+ SO32- and diethylaminocoumarin were investigated. As shown in Figure S1 (b), the absorptions at 387 nm and 319 nm are respectively due to the diethylaminocoumarin moiety and benzoindoles moiety. That is to say, it indeed behaved an addition reaction of double bonds. In the high resolution mass spectrum (HRMS) of BCS-1 with sulfite (Figure S2), the signal of m/z 1029.2665 (calcd = 1029.2662) corresponding to [M – H + Na] + was clearly observed. On the other hand, 1H NMR spectroscopy was used to confirm the mechanism. From Sun’s work, we know that the dynamic product was the sulfite added to the coumarin ring [19]. In our experiment, this product could be confirmed by the 1HNMR (the signal peak at 8.89 ppm disappeared). However, due to the complexity of the reaction, other intermediates and products can be also existed. The other product was caused by addition of the double bonds, and it could be found form the peak at 8.89 ppm shifting to 7.93 ppm (Figure S3-5). Compared to the 1HNMR spectra after 5 min’s and after 24 h, we can confirm the existence of the 9


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two products.

Fig. 2 Frontier molecular orbital plots of BCS-1 and BCS-1-SO2. Green and red shapes are corresponding to the different phases of the molecular wave functions for HOMO and LUMO orbitals. To further investigate the reaction mechanism, theoretical calculations by the Gaussian 09 program was performed. Geometries of structures BCS-1, BCS-1-SO2 and BCS-1-SO2-B (two expected products of BCS-1 and SO32-) were fully optimized at the B3LYP/6-31G(d,p) level. The highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO) levels were computed at the same level of theory (Fig. 2, Table S1). The π electrons on the HOMO of BCS-1 are located around the whole π-conjunction structure between coumarin and benzoindole moieties; while on the HOMO of BCS-1-SO2 and BCS-1-SO2-B, the conjunction structure is destroyed, and the π electrons mostly located in benzoindole moieties. This different electron distribution could result in a blue-shift in the absorption and emission spectra. pH and anti-interference studies The pH effect on the fluorescence intensity ratio (I505

nm/I652 nm)

of BCS-1 was then

conducted. In the absence of sulphite, it showed a stable fluorescence at the range of pH 10


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3.0 to 10.0 in a PBS buffer solution (10 mM, containing 30% DMF).While after 10 equiv. of sulphite was added to the solution, I505 nm/ I652 nm has an increase from pH 4.5 to 9.0 (Fig S6).The result demonstrated that BCS-1 can be used to sense the existence of cellular sulfite and bisulfite without interference from pH effects in biological environment. Fluorescence titration experiment of BCS-1 (5 µM) with sulphite was conducted at various concentrations (0 µM to 200 µM) in PBS buffer solution (pH 7.4, 10 mM, containing 30% DMF). With the increment of sulfite concentration, the intensity at 652 nm decreased while an increase at 505 nm gradually appeared at the same time (Fig. S7a). I505 nm/

I652 nm could be amplified evidently upon addition of sulfite and reached a plateau when

the concentration is 150 µM (Fig. S7b). The sulfite concentration is linearly related to the intensity ratio (I505 nm/ I652 nm) at the range of 0-5 µM, and the detection limit for sulfite was calculated to be 72 nM (Fig. S7c).

Fig. 3 Fluorescence titration spectra (a) of BCS-1 (5 µM) upon the addition of SO32- (0, 2, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200 µM) and fluorescence ratio image (b) of

BCS-1 (5 µM) in the presence of HSO3- , SO32-, F-, Cl-, Br-, I-, S2O32-, AcO-, SO42-, N3-, NO2-, NO3-, PO43-, CO32-, S2- (10 equiv.) , GSH, Cys, Hcy (200 equiv.) at 505 and 652 nm in PBS buffer (pH 7.4, 10 mM, containing 30% DMF). Black bar: BCS-1 +various species. Red bar: BCS-1 + various species + sulfite. λex= 467 nm. 11


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To prove its good selectivity and anti-interference, we then studied the fluorescence spectra of BCS-1(5µM) in the presence of various competitive species under the same conditions (Fig.3). Addition of the species including F-,Cl-, Br-, I-, S2O32-, AcO-, SO42-, N3-, NO2-, NO3-, PO43-, CO32-, S2- (10 equiv.) did not significantly change the fluorescence of

BCS-1, and even 200 equiv. of GSH, Cys and Hcy caused no emission changes of the probe, indicating it has an excellent selectivity for SO2. When sulfite and the other competitive species existed at the same time, there is still a significant alteration in the fluorescence intensity ratio (I505

nm/

I652

nm ),

which suggested BCS-1 has a strong

anti-interference ability (Fig. 3b and Fig. S8). Imaging in living cell With the above properties, the potential application of BCS-1 for fluorescence imaging of SO2 derivatives in living cells was explored. First, we did the MTT assay for BCS-1 (0–20

µM) and the results showed that more than 95% of cells still remained alive even though 20 µM BCS-1 was internalized for 24 h (Fig.S9), suggesting it has a low cytotoxicity and could be applied to living cells. It has been reported that the biotin moiety could serve as a useful biomarker for the identification of tumour cells (Russell-Jones et al., 2004),we then investigated whether this biotin-coumarin-benzoindole framework can selectively enter tumour cells or not. BCS-1 was respectively incubated with cancer cells (HeLa) and normal cells (HEK293) for 30 min at 37 oC. As we expected, the fluorescence could be found from the red channel in cancer cells and there was no emission could be observed in normal cells, indicating BCS-1 could specifically recognize and enter tumour cells (Fig. 4).

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Fig. 4 Confocal microscopy images of HeLa and HEK293 cells treated with BCS-1 (5 µM) in PBS buffer (pH 7.4, 10 mM) for a total incubation period of 30 min at 37 oC. (a) and (d): Bright-field image; (b) and (e): the fluorescence images from the red channel from the range of 630-670 nm; (c) and (f): overlap of (a), (b) and overlap of (d), (e). λex= 488 nm. The successful imaging in living cell (in HeLa and HEK293) with BCS-1 inspires us to explore the feasibility of BCS-1 via flow cytometry, which could offer a more reliable measurement due to the observation of a very large number of cells rather than the limited cell numbers in fluorescence imaging[48]. As shown in Fig. S10, after the addition of

BCS-1, fluorescence intensity could be observed in 31.2% MCF-7 cells while the same fluorescence could only be found in 2.1% MCF-10A cells compared to theirs blank from the FL4 Red channel, and there was nearly no fluorescence change from the FL1 Green channel. The results further demonstrated that BCS-1 could dramatically target to the tumor cells.

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Fig.5 Confocal fluorescence images of HeLa cells stained with BCS-1 (5 µM) for 30 min (a-g), and then incubated with SO32- (50 µM) for 15 min (d-g). Green channel: 490–520 nm; Red channel: 630–670 nm; λex: 488 nm. Having confirmed its tumour-specific targeting ability, the imaging of SO2 derivatives in living cells was investigated. We simply incubated BCS-1 (5µM) in HeLa cells for 30 min, and observed strong fluorescence in the red channel from the range of 630–670 nm, while there was no fluorescence in the green channel from 490–520nm. After adding sulfite (50 µM) and cultured for 15 min, the fluorescencein the red channel disappeared, but a tense fluorescence emerged in the green channel simultaneously, showing that BCS-1 could rapidly react with sulfite in living cells (Fig. 5). To further identify the intracellular location of BCS-1 after entering the cells, we explored the co-localization experiment using the Mito Tracker Red FM (MT Red), which obtained from commercial suppliers. As shown in Fig. 6, the merged images of the fluorescence of green channel and red channel were both well overlaid with the fluorescence of MT Red (570–620 nm). And the Pearson’s co-localization coefficients were calculated to be 0.965 and 0.937, respectively, which proved that BCS-1 was site-specifically internalized in mitochondria of living cells.

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Fig.6 HeLa cells were stained with BCS-1 (5 µM) for 30 min, and then incubated with SO32- (50 µM) (a-e: not incubated with SO32-, g-k: for 15 min); finally, Mito Tracker Deep Red (1µM) was added to the cells and the cells were incubated for another 10 min. (a) and (g): Bright-field image; (b) and (h): the fluorescence images from the green channel; (c) and (i): the fluorescence images from the red channel; (d) and (j): the fluorescence images of Mito Tracker Deep Red; (e) and (k): overlap of (a), (c), (d) and overlap of (g), (h), (j); (f) and (l):co-localization images. Excitation both at 488 nm for the green channel (490–520 nm), the red channel (630–670 nm), and the MT Red (570–620 nm). CONCLUSION In summary, we have constructed a tumour-specific and mitochondria-targeted fluorescent probe BCS-1 by incorporating biotin into coumarin-benzoindole conjugate, which could realize colorimetric and ratiometric sensing SO2 derivatives in living cells with an excellent selectivity, rapid response (within 30s) and low detection limit (72 nm). Moreover, BCS-1 is the first ratiometric fluorescent probe for SO2 imaging with near-infrared-emission.

ASSOCIATED CONTENT Supporting Information: The Supporting Information is available free of charge. Additional experimental methods, NMR spectra, mass spectrometry, selectivity graphs and others. 15


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ACKNOWLEDGEMENT

This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603) and the National ScienceFoundation of China (nos 21232005, 21472131, 21572147 and J1103315).

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Novel Tumor-Specific and Mitochondria-Targeted near-Infrared

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