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1 Electronic upplementary Material (EI) for Chemical Communications. This journal is The Royal ociety of Chemistry 218 upporting Information A highly selective and sensitive fluorescent probe for simultaneously distinguishing and sequentially detecting hydrogen multiple thiol species in solution and in live cells Dan Qiao a, Tangliang hen a, Mengyuan Zhu b, Xiao Liang a, Lu Zhang a, Zheng Yin a, Binghe Wang b *, Luqing hang a * a. College of Pharmacy & tate Key Laboratory of Elemento-rganic Chemistry, ankai University Tianjin 371 (P.R. China) E- mail: shanglq@nankai.edu.cn b. Department of Chemistry Georgia tate University Atlanta, Georgia 333 UA wang@gsu.edu 1

2 Experimental ection 1. Apparatus 1 H-MR (4 MHz) and 13 C-MR (1 MHz) spectra were recorded on a Bruker AVACE-4 (4 MHz) (Bruker, Karlsruhe, Germany) MR spectrometer and TM as an internal standard. Electrospray ionization mass spectroscopy (EI-M) was performed on a himadzu LC/M-22 (himadzu, Kyoto, Japan). HRM were recorded on a high-resolution EI-FTICR mass spectrometer (Varian 7. T, Varian, UA). UV-vis spectra were measured by a UH-53 spectrophotometer (Hitachi). Fluorescence measurements were recorded on a F-7 spectrophotometer (Hitachi). Fluorescence images of cells were taken by a laser scanning confocal microscopy (Leica TC P8). 2. Materials Unless otherwise stated, all reagents used in this study were commercial products without further purification. Twice-distilled water was used throughout all experiments. TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel (mesh 2-3), both of which were obtained from the Qingdao cean Chemicals. Mito-Tracker Green was obtained from olarbio. 3. Detection limit The limit of detection defined by 3σ k, where σ is the standard deviation of the blank sample and k is the slope of the linear regression equation. 4. Theoretical study on the fluorescence mechanism The structure of CR was constructed using Gaussian 1, which was initially optimized using the capabilities within these aforementioned programs. Calculations were then conducted using density functional theory (DFT) with the B3LYP 2 exchange functional employing 6-31G(d) 3 level implemented via the Gaussian 9 suite of programs for the full geometry optimization and frequency calculations of the probe. Imaginary frequencies were not obtained in any of the frequency calculations. 5. Cells culture HeLa cells were cultured in DMEM (Dulbecco s modified Eagle's medium) and supplemented with 1% FB (fetal bovine serum), penicillin (1 μg/ml) and streptomycin (1 μg/ml) in an atmosphere of 5% C 2 and 95% air at 37 C. 6. MTT assay 2

3 Cell cytotoxicity was evaluated by MTT assay. Cells were cultivated in a 96-well plate until 5-7% confluence, and then incubated with different concentrations of CR (-2 μm) for 24 h. 2 μl 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ ml) was added for 4 h at 37 C. After removing the MTT, 1 μl DM was added. The absorbance intensities at 57 nm were measured using a plate reader. All experiments were repeated five times, and the data were presented as the percentage of control cells. 7. Discriminative fluorescence imaging of biothiols in live cells The cell experiment was divided into six groups. In the first group, HeLa cells were incubated with CR (5 μm) for 3 min and imaged after washing by phosphatebuffered saline (PB) for three times. For the second group, HeLa cells were pretreated with EM (.5 mm) for 3 min, subsequently incubated with CR (5 μm) for 3 min, and then washed by PB buffer for three times before imaging. In the third to sixth group, HeLa cells were pretreated with EM (.5 mm) for 3 min, subsequently incubated with ah/cys/hcy/gh (5 μm, 3 min) and probe CR (5 μm, 3 min), and then washed with PB buffer for three times. Cells were imaged by a laser scanning confocal microscopy (Leica TC P8; λex = 45 nm, λem = nm for the blue channel; λex = 561 nm, λem = nm for the red channel). 8. equential fluorescence imaging of biothiols in live cells The cell experiment was divided into four groups. In the first group, HeLa cells were pretreated with EM (.5 mm) for 3 min, subsequently incubated with CR (5 μm) for 3 min, and then imaged after washing three times by PB buffer, as the control group. In the second to fourth group, HeLa cells were pretreated with EM (.5 mm) for 3 min, subsequently incubated with Cys /Hcy/GH (5 μm) for 3 min, washed and continued to be incubated with CR (5 μm) for 3 min, and then added 5 μm ah to the culture dish, after 3 min, imaged after washing three times by PB buffer. Cells were imaged by a laser scanning confocal microscopy (Leica TC P8; λex = 45 nm, λem = nm for the blue channel; λex = 561 nm, λem = nm for the red channel). 3

4 Table 1. ummary of the optical properties of representative fluorescent probes for distinguishing biothiols with multiple sets of fluorescence signals. Multisite probe Distingui umber Emission Emission olubility Targeting References shing of wavelength/ distance/ mitochon targets emission nm nm dria for bands detection CF 3 Cys/Hcy 2 474, Ethanol/ 4.Anal. Chem., CF 3 PB 214,86,18- (4:6 187 v/v) Cl C GH, 2 517, DMF/P 5 C H 2 B (3:7.Chem.Commun. v/v) (Camb.),216, 52, Cys/Hcy 2 546, PB 6. Chem. ci.,, GH 214, 5, Anal. Chim. Cl Cys/Hcy, GH 2 47/53, 546/53 33/43 PB Acta,215, 9, Cl C C Cys, GH 2 5,56 6 CH 3 C/ PB 8. Biosens. Bioelectron.,21 7, 9, (3:7 v/v) 4

5 9.Chem. ci., F F C F F Cys, Hcy, 2 45, 5 5 PB 216, 7, C GH 1. J. Am. Chem. Cl Cys, GH 2 42, PB (1m M CTAB) oc.,214, 136, Anal. Chem., 2 Cys, 2 522,49 32 PB 215,87, H GH 66. C Cys, 2 514, DM/ 12. J. Am. Chem. 2 PB oc., 217, 139, (1:1 v/v) Cl C C GH DM/ PB 13. ensors Actuators B: Chem., 217, (1:1 v/v) 253, ensors 2 H 2, Cys/Hcy 2 465,55 85 DM/ PB Actuators B: Chem., 216,, GHp (1:4 v/v) 235, Cys 2 494, DMF/P YE 15. RC Adv., B (1:9 214,4, v/v)

6 H 3 B F F Cl Cys/Hcy, GH 2 564, CH 3 C/ HEPE (5: J. Am. Chem. oc.,212,134, v/v) H 2 H Cys, GH 2 536, CH 3 C/ PB (3:7 v/v) YE 17. J. Am. Chem. oc.,214,136, C 2 n Bu Cys/Hcy, GH 2 764,81 46 HEPE YE 18. J. Am. Chem. oc.,214, 136, C 2 n Bu 2 Cys/Hcy, GH 2 545, CH 3 C/ HEPE (3:7 v/v) 19. Anal. Chem., 216,88, Cys/Hcy 2, GH 2 55, DM/ PB (5:95 2.Biosens.Bioel ectron.,216,81, v/v) H 2 H 2, Cys/Hcy, GH 3 485,546,6 9 61/63 CH 3 C/ PB (2:8 v/v) YE 21.Chem. ci., 217,8, Cl CH Cys, 2 48, DM/ 22. Chem. Asian Hcy PB J., 217, 12, (2:8 v/v) Cl C C Cys, Hcy, 3 457,559, /3 DM/ PB 23.Angew.Chem. Int.Ed.,218,57,4 GH (6:4 v/v)

7 H 2, 2 467/ DM/ YE This work Cys/ PB 2 Hcy, GH (1:99 v/v) 7

8 H CH LAH H H + DCM H PPh 3,CBr 4 1 Br+ H K 2 C 3 DMF + H H DCM 2 3 H C,DPT,DIC DCM 4 CR 2 2 Cl + H Boc CsC 3 CH 3 C 2 Boc CF 3 CH 2 H + H Cl TEA CH 3 C H C H H + Cl Cl ZnCl 2 Ethanol H 6 cheme 1. The synthetic route of probe CR. ynthesis of 2. Compound 1 was prepared via previous method 24. Triphenylphosphine (632 mg, 2.4 mmol) and carbon tetrabromide (8 mg, 2.4 mmol) were dissolved absolutely in dichloromethane. Compound 1 (4 mg, 1.6 mmol) was slowly added. TLC was applied to monitor the reaction. After complete reaction, the solvent was evaporated under reduced pressure. The resulted residue was purified by silica gel column chromatography with PE/EA (v/v 1:1) to give a yellow oil as compound 2 (2 mg, 4%). ynthesis of 3. Resorufin (136.6 mg,.64 mmol) and potassium carbonate (132.7 mg,.96 mmol) were dissolved absolutely in dry DMF. Under the protection of nitrogen, compound 2 (2 mg,.64 mmol) was slowly added. The mixture was stirred for overnight at 55 o C. After complete reaction, 5 ml water was added into the flask and the reaction mixture was extracted with EA (1 ml 3). The organic phase was 8

9 collected and dried using Mg 4. Then the solvent was removed under reduced pressure affording the crude product, which was purified by flash chromatography column using CH 2 Cl 2 /MeH (v/v = 25:1) to afford red solid as compound 3 (21 mg, 73%). 1 H MR (4 MHz, CDCl 3 ) δ 8.47 (s, 1H), 7.7 (d, J = 8.8 Hz, 1H), (m, 4H), 7.39 (t, J = 9.6 Hz, 3H), 7.11 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 6.83 (d, J = 1.6 Hz, 2H), 6.31 (s, 1H), 5.13 (s, 2H). 13 C MR (11 MHz, CDCl 3 ) δ 186.3, 162.3, 159.3, 149.8, 149.7, 145.8, 145.6, 137.3, 136.8, 134.7, 134.6, 134.3, 131.6, 128.6, 128.3, 127.6, 121.1, 119.7, 114.2, 16.8, 11.1, 7.3. HRM: m/z [M+a] + calculated for C 24 H 16 2 a 3 2 : ; measured ynthesis of 4. Compound 3 (21 mg,.47 mmol) was dissolved absolutely in dichloromethane. 3-Mercaptopropionic acid (6.4 mg,.57 mmol) was slowly added. The reaction was monitored by TLC. After complete reaction, the solvent was evaporated under reduced pressure. The resulted residue was purified by silica gel column chromatography with CH 2 Cl 2 /MeH (v/v 1:1) to afford dark red solid as compound 4 (12 mg, 49%). 1 H MR (4 MHz, DM-d 6 ) δ (s, 1H), 7.77 (t, J = 9.6 Hz, 1H), 7.54 (dt, J = 19.2, 9.9 Hz, 5H), 7.17 (dd, J = 33.5, 9.6 Hz, 2H), 6.79 (d, J = 9.9 Hz, 1H), 6.27 (s, 1H), 5.28 (s, 2H), 2.95 (t, J = 6.8 Hz, 2H), 2.62 (t, J = 6.8 Hz, 2H). 13 C MR (11 MHz, DM-d 6 ) δ 185.8, 173., 162.7, 15.2, 145.8, 145.7, 136.9, 135.6, 135.4, 134.2, 131.8, 129.4, 128.5, 127.8, 114.8, 16.2, 11.7, 7.2, 34., HRM: m/z [M-H + ] calculated for C 22 H : ; measured ynthesis of 6. Resorcinol (1 g, 9.8 mmol), Ethyl 4-Chloroacetoacetate (14.9 g, 9.8 mmol) was dissolved absolutely in ethanol. Zinc chloride(18.5 g, 136 mmol)was added. The mixture was heated and reflux for 12 hours. After complete reaction, cooled to room temperature. The cooled reaction mixture was poured into 1 ml of.1 aqueous hydrochloride acid. Then, the solvent was evaporated under reduced pressure. The resulted residue was purified by silica gel column chromatography with CH 2 Cl 2 /MeH (v/v 1:1) to afford white solid as compound 6 (11 g, 86%). 1 H MR (4 MHz, DM-d 6 ) δ 7.65 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 6.6 Hz, 1H), 6.75 (s, 1H), 6.4 (s, 1H), 4.92 (s, 2H). 13 C MR (11 MHz, DM-d 6 ) δ 161.9, 16.6, 155.7, 151.3, 126.9, 113.5, 111.5, 19.8, 12.9, HRM: m/z [M+a] + calculated for C 1 H 7 Cla 3 : ; measured

10 ynthesis of C. Compound 5 was prepared via previous method 25. Compound 5 (237 mg,.95 mmol), Triethylamine (144.7 mg, 1.43 mmol) was dissolved absolutely acetonitrile, compound 6 (281 mg,.95 mmol) was added. The mixture was heated and reflux for 24 hours. After complete reaction, cooled to room temperature, the solvent was evaporated under reduced pressure. The resulted residue was purified by silica gel column chromatography with CH 2 Cl 2 /MeH (v/v 2:1) to afford red solid as compound C (36 mg, 75%). 1 H MR (4 MHz, DM-d 6 ) δ 1.55 (s, 1H), 8.49 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), (m, 3H), 6.32 (s, 1H), 4.17 (s, 4H), 3.74 (s, 2H), 2.76 (s, 4H), 13 C MR (11 MHz, DM-d 6 ) δ 161.6, 16.9, 155.7, 152.7, 145.8, 145.3, 145.3, 136.8, 127.3, 121.7, 113.3, 111.3, 11.7, 14.1, 12.7, 57.6, 52.9, HRM: m/z [M+a] + calculated for C 2 H 17 5 a 6 : ; measured ynthesis of CR. Compound 4 (5 mg,.11 mmol) was dissolved in anhydrous dichloromethane (5 ml) in a round-bottom flask. C (48.2 mg,.11 mmol) was added, followed by the addition of 4-(dimethylamino)-pyridinium-4-toluene sulfonate(dpt) (33.5 mg,.11 mmol) and, -Diisopropylcarbodiimide (14.4 mg,.11 mmol). The reaction mixture was stirred room temperature. The reaction was monitored by TLC, after complete reaction, diluted with dichloromethane, and washed with brine. The organic layer was dried over anhydrous a 2 4, then the solvent was evaporated under reduced pressure. The resulted residue was purified by silica gel column chromatography with CH 2 Cl 2 /MeH (v/v 15:1) to afford red solid as compound CR (6 mg, 62.5%). 1 H MR (4 MHz, CDCl 3 ) δ 8.31 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.58 (dd, J = 34.3, 8.4 Hz, 3H), (m, 3H), (m, 3H), (m, 2H), 6.47 (s, 1H), (m, 2H), 5.1 (s, 2H), 4.7 (t, J = 4.9 Hz, 4H), 3.67 (s, 2H), (m, 4H), 2.76 (t, J = 5. Hz, 4H). 13 C MR (11 MHz, CDCl 3 ) δ 186.2, 169.5, 162.4, 16.2, 154.5, 152.9, 15.4, 149.7, 145.7, 145.5, 145., 144.8, 144.7, 137.3, 135., 134.7, 134.6, 134.2, 131.6, 128.5, 128.3, 127.9, 125.6, 118., 116.5, 114.8, 114.2, 11.5, 16.7, 12.9, 11.1, 7.3, 58.6, 52.9, 49.3, 33.9, HRM: m/z [M+a] + calculated for C 42 H 32 6 a 1 2 : ; measured

11 References 1. (a) L. M. Kiefer and K. J. Kubarych, J. Phys. Chem. Lett., 216, 7, ; (b) G. Hummer, L. R. Pratt and A. E. García, J. Am. Chem. oc., 1997, 119, E. Torres and G. A. DiLabio, J. Phys. Chem. Lett., 212, 3, (a) K. Raghavachari and G. W. Trucks, J. Chem. Phys., 1989, 91, ; (b) P. J. Hay, J. Chem. Phys., 1977, 66, ; (c) A. Austin, G. A. Petersson, M. J. Frisch, F. J. Dobek, G. calmani and K. Throssell, J. Chem. Theory Comput., 212, 8, H. Lv, X. F. Yang, Y. Zhong, Y. Guo, Z. Li and H. Li, Anal. Chem., 214, 86, H. Li, W. Peng, W. Feng, Y. Wang, G. Chen,. Wang,. Li, H. Li, K. Wang and J. Zhang, Chem. Commun. (Camb.), 216, 52, J. Liu, Y.-Q. un, H. Zhang, Y. Huo, Y. hi and W. Guo, Chem. ci., 214, 5, X. Dai, Z. Y. Wang, Z. F. Du, J. Cui, J. Y. Miao and B. X. Zhao, Anal. Chim. Acta, 215, 9, Y. Li, W. Liu, P. Zhang, H. Zhang, J. Wu, J. Ge and P. Wang, Biosens. Bioelectron., 217, 9, H. Zhang, R. Liu, J. Liu, L. Li, P. Wang,. Q. Yao, Z. Xu and H. un, Chem. ci., 216, 7, J. Liu, Y. Q. un, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. ong, Y. hi and W. Guo, J. Am. Chem. oc., 214, 136, Q. Miao, Q. Li, Q. Yuan, L. Li, Z. Hai,. Liu and G. Liang, Anal. Chem., 215, 87, Y. Yue, F. Huo, P. ing, Y. Zhang, J. Chao, X. Meng and C. Yin, J. Am. Chem. oc., 217, 139, X. Li, F. Huo, Y. Yue, Y. Zhang and C. Yin, ensors Actuators B: Chem., 217, 253, Ding and G. Feng, ensors Actuators B: Chem., 216, 235, J. Liu, Y.-Q. un, H. Zhang, Y. Huo, Y. hi, H. hi and W. Guo, RC Adv., 214, 4,

12 16. L. Y. iu, Y.. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and Q. Z. Yang, J. Am. Chem. oc., 212, 134, Y. Q. un, J. Liu, H. Zhang, Y. Huo, X. Lv, Y. hi and W. Guo, J. Am. Chem. oc., 214, 136, Y. Lim, K. H. Hong, D. I. Kim, H. Kwon and H. J. Kim, J. Am. Chem. oc., 214, 136, W. Chen, H. Luo, X. Liu, J. W. Foley and X. ong, Anal. Chem., 216, 88, Q. Hu, C. Yu, X. Xia, F. Zeng and. Wu, Biosens. Bioelectron., 216, 81, L. He, X. Yang, K. Xu, X. Kong and W. Lin, Chem. ci., 217, 8, Y. Li, W. Liu, H. Zhang, M. Wang, J. Wu, J. Ge and P. Wang, Chem.Asian. J., 217, 12, G. X. Yin, T. T. iu, Y. B. Gan, T. Yu, P. Yin, H. M. Chen, Y. Y. Zhang, H. T. Li and. Z. Yao, Angew. Chem. Int. Ed., 218, 57, T. un, A. Morger, B. Castagner and J. C. Leroux, Chem. Commun. (Camb.), 215, 51, Y. Tang and G.-F. Jiang, ew J. Chem., 217, 41,

13 9. Proposed responding mechanism H 2 H H H R H H 2 2 H 2 H 2 H D H 2 CR Cys/Hcy 2 Cys/Hcy 2 H H H 2 n CH n=1 for Cys n=2 for Hcy H 2 CH n R C H C n=1 for Cys H n=2 for Hcy GH GH H H R 2 2 H HC C-GH H H 2 CH cheme 2. Proposed responding mechanism of probe CR for simultaneously distinguishing H 2 and biothiols. 13

14 Cys/Hcy 2 H 2 C H 2 H + D H H H 2 CH H 2 CH 2 CR GH 2 H 2 H H CH C-GH 2 H H H H + CH D-GH cheme 3. Proposed reaction mechanism of CR pre-treated with Cys, Hcy and GH upon continued addition of H upplementary pectra Fig. 1. The fluorescence intensity of CR (1 μm) at (A) 467 nm, (B) 588 nm, upon addition of H 2 ( 1 equiv.), Cys ( 1 equiv.), Hcy ( 1 equiv.) and GH ( 1 equiv.) and (C) 467 nm pretreated with Cys (1 equiv.), Hcy (1 equiv.), and GH (1 equiv.) upon continued addition of H 2 (-1 equiv.) in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). ex = 38 nm, em =467 nm for the first and third column. ex =56 nm, em =588 nm for the second column. Data are expressed as mean ± D of five parallel experiments. 14

15 (A1) (A2) (A3) Fl.intensity(a.u.) Fl.intensity(a.u.) Fl.intensity(a.u.) Wavelength(nm) Wavelength(nm) Wavelength(nm) Fig. 2. The fluorescence spectra of CR (1 μm) pre-treated with Cys (1 equiv.), Hcy (1 equiv.), and GH (1 equiv.) upon continued addition of H 2 (-1 equiv.) in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). ex = 38 nm, em =467nm. Upon continuing addition of increasing amount of H 2 to the mixture of CR (1 μm) and Cys (1 equiv.), a significant emission centered at 467 nm (5-fold) was generated. A similar phenomenon also occurred in the mixture of CR and Hcy (6-fold). However, the mixture of CR and GH has no obvious emission band at 467 nm (4-fold). Fig. 3. Linear correlation between the fluorescence emission intensity of CR (1 μm) and (a) H 2 ( - 4 equiv.), (b) Cys ( 4 equiv.), (c) Hcy ( 5 equiv.) and (d) GH ( - 6 equiv.) in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). 15

16 (a) (b) (c) F 467 (a.u.) 8 Blank H 2 7 Cys Hcy GH Excited at 38 nm F 588 (a.u.) 9 Blank H 8 2 Cys Hcy 7 GH Excited at 56 nm F 467 (a.u.) Blank Cys+H 2 Hcy+H 2 GH+H 2 Excited at 38 nm Time(min) Time(min) Time(min) Fig. 4. Time-dependent fluorescence spectra of CR (1 μm) in the presence of Cys (1 equiv.), Hcy (1 equiv.), GH (1 equiv.), H 2 (1 equiv.) within 3 min in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). (a) Fluorescence intensity at 467 nm, excitation at 38 nm; (b) Fluorescence intensity at 588 nm, excitation at 56 nm; (c) The fluorescence spectra of CR (1 μm) pre-treated with 1 equiv. Cys /Hcy and GH upon continued addition of H 2 (1 equiv.) within 3 min in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). Fluorescence intensity at 467 nm, excitation at 38 nm. (a).5.4 Blank H 2 (b).5.4 Blank Cys Hcy GH Absorption.3.2 Absorption (c) Blank Cys+H 2 Hcy+H 2 GH+H 2 Wavelength (nm) (d) C C+H 2 Wavelength (nm).8 Absorption.6.4 Absorption Wavelength (nm) Wavelength (nm) Fig. 5. Absorption spectra of (a) CR (1 μm) in the presence of 1 equiv. of H 2.(b) CR (1 μm ) in the presence of 1 equiv. of Cys/Hcy and GH. (c) CR (1 μm) was pretreated with 1 equiv. Cys/Hcy/GH in the presence of 1 equiv. of H 2 (d) C (2 μm) in the presence of 1 equiv. of H 2 in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). The UV-vis spectra of free CR showed a main absorption at

17 nm. Upon addition of H 2, an obvious increase was observed at 372 nm and a shoulder peak simultaneously emerged at 575 nm. These can be attributed to the products R, D and. imilarly, only an obvious shoulder peak was observed at 575 nm in the presence of Cys/Hcy and GH. After CR was pre-treated with Cys, Hcy, or GH (1 equiv.) individually, addition of H 2 resulted in a significant increase in the UV absorption at 372 nm and the simultaneous emergence of a peak at 575 nm in the Cys- and Hcypretreated solutions. However, H 2 addition only resulted in a shoulder peak at 575 nm for the GH-pretreated solution. To confirm the original attribution of those newly generated absorption peaks, the absorption spectra of control compounds C and C in the presence of H 2 were studied, and the results were consistent with the spectral performance of CR. Extinction coefficient calculations:.4 Absorption (D) y = 2983 x R 2 = C (M) Fig. 6. Linear plots of CR. The experiments were applied in 1 cm cuvette, at the indicated concentrations in DM/PB buffer (25 mm, ph 7.4, 1:99 v/v). The measurements were performed at the maximum wavelength of CR (475 nm). CR = 2983[M -1 cm -1 ] 17

18 H b H c 2 H b H c H a CR Fig 7. 1 H-MR spectra comparison: (a) CR, (b) CR-aH (c) CR-Cys, (d) CR-Hcy and (e) CR-GH. The chemical shifts for hydrogens, H a linked with resorufin; and H c linked with coumarin; and H b linked with and BD were observed during the reaction. In the CR-aH group, it was found that the chemical shifts of H a and H b displayed obvious upfield shifts compared with that of CR group, which indicated the release of resorufin and BD. In CR-Cys, CR-Hcy and CR-GH group, only upfield shift of H a was observed, but the chemical shift of H b didn t change, which indicated that the release of resorufin, and BD was not removed (cheme 2 in supporting information). 18

19 H b H c 2 H H b H c C Fig 8. 1 H-MR spectra comparison of (a) C and (b) C-aH showed that the chemical shift of H b in C-aH displayed obvious upfield shifts compared with that of C, which indicated that the BD was removed (cheme 3 in supporting information). These results are in good agreement with our proposed reaction mechanism. 1 Inten m/z 1 Inten m/z 1 Inten m/z Fig. 9. Mass spectra (EI) of CR in the presence of H 2 in aqueous solution. R m/z=(m-h + )=212., D m/z (M + H + ) = 261.2, m/z (M-H + ) =

20 1 Inten m/z 1 Inten m/z Fig. 1. Mass spectra of CR in the presence of Cys in aqueous solution. R m/z= (M-H + )=212., C m/z (M-H + ) = 422.5, C m/z (M-H + ) = Inten m/z 1 Inten m/z 2

21 Fig. 11. Mass spectra of CR in the presence of Hcy in aqueous solution. R m/z= (M-H + )=212., C m/z (M-H + ) = 422.5, H m/z (M-H + ) = Inten m/z 1 Inten m/z Fig. 12. Mass spectra of CR in the presence of GH aqueous solution. R m/z=(m- H + )=212., C-GH m/z (M-H + ) = Inten m/z 1 Inten m/z Fig. 13. Mass spectra of CR pre-treated with Cys upon continued addition of H 2. D m/z (M+H + ) = 261.2, m/z (M-H + ) =

22 1 Inten m/z 1 Inten m/z Fig. 14. Mass spectra of CR pre-treated with Hcy upon continued addition of H 2. D m/z (M + H + ) = 261.2, m/z (M-H + ) = Inten m/z Inten m/z Fig. 15. Mass spectra of CR pre-treated with GH upon continued addition of H 2. D-GH m/z (M + H + ) = , m/z (M-H + ) =

23 Fig. 16. (A) The fluorescence intensity of CR (1 μm) at (A1) 467 nm, (A2) 588 nm in the presence of (1) Blank (2) Thr, (3) Phe, (4) Asp, (5) Val, (6) His, (7) Glu, (8) Pro, (9) Met, (1) Ala, (11) Trp, (12) er, (13) 3-, (14) Br -, (15) Cl -, (16) 2-4, (17) 2-3, (18) 2 2-3, (19) Ac -, (2) 3-, (21) Ca 2+, (22) Cu 2+, (23) Mg 2+, (24) Fe 2+, (25) Fe 3+, (26) n 2+, (27) Zn 2+, (28) a +, (29) K +, (3) H 2 2, (31) Cys, (32) Hcy, (33) GH, (34) H 2 in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). Concentrations were 1 equiv. for (2) (34). (A3) CR was pre-treated with 1 equiv. (31) Cys (32) Hcy and (33) GH, upon continued addition of 1 equiv. H 2. (B). Fluorescence intensity of CR (1 μm) at (B1) 467 nm, (B2) 588 nm in the presence or absence of biothiols at different ph values ranging in DM/PB buffer (25 mm, ph 7.4, 1:99, v/v). Concentrations were 1 equiv. for Cys/Hcy, GH, H 2. (B3) CR was pretreated with 1 equiv. Cys/Hcy, GH, upon continued addition of 1 equiv. H 2. Excitation at 38 nm for (A1/B1), 56 nm for (A2/B2), and 38 nm for (A3/B3). The results showed the fluorescence intensity of free CR at two emission bands was not influenced by ph in aqueous solutions. However, at ph ranging from 7. to 8., probe CR generated significant enhancement of the fluorescence intensity in the presence of H 2, Cys/Hcy and GH. Data are expressed as mean ± D of five parallel (A) (B) LUM -3.5 ev HM -6.1eV experiments. Fig. 17. (A) ptimized structures of CR and the co-regulation of response emission by FRET and ICT mechanisms. (B) Frontier molecular orbital energy of CR at the excited state. Calculations were performed using DFT with the B3LYP exchange functional employing 6-31G(d) basis sets using Gaussian 9 programs. In the optimized 23

24 structures of CR, the calculated distance between coumarin and BD is 13 Å. Thus, the FRET donor fluorescence of coumarin can be quenched by the BD moiety through the FRET process. In addition, the electrons on both the HM and LUM efficiently distribute in the respective resorufin and BD units in CR, suggesting that CR is a typical intramolecular charge transfer (ICT)-based fluorescent probe. Fig. 18. The cell viability of living HeLa cells treated with 5, 1, or 2 μm CR for 24 hours measured by standard MTT assay. (Error bars represent the standard deviations of five trials) Fig. 19. The images of living HeLa cells pre-treated with CR (5 μm) for 3 min and subsequently co-incubated with Mito-Tracker Green (1 μm) for 3 min. (A) Red channel images of probe CR ( ex = 561 nm, em = nm); (B) Green channel images of Mito -Tracker Green ( ex = 488 nm, em = nm); (C) Merged green and red channel images; (D) The intensity profile of the RI in merged images. cale bar: 1 μm. 24

25 Fig H MR of compound 3 in CDCl f1 (ppm) Fig C MR of compound 3 in CDCl 3. 25

26 Fig. 22. HRM (EI) of compound 3 m/z [M+a] + calculated for C 24 H 16 2 a 3 2 : ; measured Fig H MR of compound 4 in DM-d 6. 26

27 f1 (ppm) Fig C MR of compound 4 in DM-d 6. Fig. 25. HRM (EI) of compound 4 m/z [M-H + ] calculated for C 22 H : ; measured

28 Fig H MR of compound 6 in DM-d f1 (ppm) Fig C MR of compound 6 in DM-d 6. 28

29 Fig. 28. HRM (EI) of compound 6 m/z [M+a] + calculated for C 1 H 7 Cla 3 : ; measured: Fig H MR of compound C in DM-d 6. 29

30 f1 (ppm) Fig C MR of compound C in DM-d 6. Fig. 31. HRM (EI) of compound C m/z [M+a] + calculated for C 2 H 17 5 a 6 : ; measured:

31 f1 (ppm) Fig H MR of compound CR in CDCl f1 (ppm) Fig C MR of compound CR in CDCl 3. 31

32 Fig. 34. HRM (EI) of compound CR m/z [M+a] + calculated for C 42 H 32 6 a 1 2 : ; measured