Ratiometric Fluorescence Imaging of Cellular Glutathione

Transcription

1 Supporting Information for: Ratiometric Fluorescence Imaging of Cellular Glutathione Gun-Joong Kim, Kiwon Lee, Hyockman Kwon, and Hae-Jo Kim, * Department of Chemistry, and Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin , Korea haejkim@hufs.ac.kr Contents 1. Instruments, reagents, and preparation S2 2. Synthesis S2 3. NMR spectra S4 4. Comparison of Michael reactions of 1 and 2 S7 5. Ratiometric fluorescence response of 1 upon addition of analytes S7 6. Fluorometric determination of biothiol detection limit S8 7. Calibration curve for GSH S9 8. ph Profile S1 9. Mass spectra S11 S1

2 1. Instruments, reagents, and preparation ll fluorescence and UV-vis absorption spectra were recorded in FP 65 fluorescence spectrometer and gilent 8453 absorption spectrometer, respectively. The 1 H and 13 C NMR spectra were recorded at 3 MHz NMR spectroscopy. Mass spectra were recorded on G641 MS-spectrometer. ll experiments were carried out with commercially available reagents and solvents, and used without further purification, unless otherwise noted. Preparation for UV-vis kinetic study stock solution (1 mm) of 1 in DMS was prepared and used by dilution in aqueous DMS solution for kinetic experiments. In a typical experiment, test solutions (1 M) were prepared by placing 2 L of the probe stock solution into a test tube, adding an appropriate amount of thiols (neat liquid or solid form), and diluting the solution to 2 ml with buffered aqueous DMS (.1 M HEPES, ph 7.4). The UV-vis spectral changes were set to be monitored at 25 o C with 5 min intervals after the addition of thiols. Preparation for fluorescence study stock solution (1 mm) of 1 in DMS was prepared and used by dilution in aqueous DMS solution for in vitro and in vivo fluorescence experiments. In a typical experiment, test solutions (1 M) were prepared by placing 2 L of the probe stock solution into a test tube, adding an appropriate amount of each amino acid, and diluting the solution to 2 ml with buffered aqueous DMS (.1 M HEPES, ph 7.4). Normally, excitation was at 42 nm. Both the excitation and emission slit widths were 3 nm ⅹ 3 nm. Fluorescence spectra were monitored 1 h after addition of amino acids. Fluorescence cell imaging of HeLa cells For the detection of biothiols in live cells, HeLa cells were cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 1 units/ml penicillin, 1 g/ml streptomycin, and 1% heat-inactivated fetal bovine serum. The cells were seeded on a Ø 35mm glass-bottomed dish at a density of 1 x 1 5 cells in a culture medium overnight for live-cell imaging by confocal laser scanning microscopy (CLSM). The HeLa cells were treated with 2.5 M of probe 1 in 2 ml of serum free medium for.5 hr and washed with 3 times with prewarmed 1 x PBS before imaging by CLSM. To enhance the concentration of cellular GSH, HeLa cells were treated with -lipoic acid (LP, 5 M) for 24 h, followed by probe 1 for.5 h, and then cellular imaging experiment was performed for the live cells. In order to reduce the concentration of GSH, HeLa cells were treated with N-ethylmaleimide (NEM, 1 M) for.5 h, followed by 1 for.5 h, and the cellular images were taken under a CLSM by using three excitation channels ( ex 45 nm, 488 nm, and 555 nm). fter obtaining the live cell images, the mean fluorescence intensities at blue ( ex 45 nm) and green ( ex 488 nm) channels were measured in three different fields by ZEN imaging program. In order to reduce errors caused by background images outside of the cells, we also compared the intensity of the background image, but the level of the intensity is very low indicating that it did not seem to affect the mean fluorescence intensities. 2. Synthesis X HS H H pyrrolidine 1 (X = H) H 1-ME S H H 2 (X = H) Probe 1. To a solution of 7-(diethylamino)coumarinyl aldehyde S1 (123 mg,.5 mmol) and ketone (1.8 equiv) in 4 ml of CH 2 Cl 2 /EtH (1:1, v/v) was added 2 drops of pyrrolidine. The resulting clear red solution was stirred at rt for additional 8 hr to afford reddish precipitates, which were filtered. Further purification by recrystalization in DCM/EtC gave the desired probe 1 as red crystals (11 mg, yield 6 %). 1 H NMR (DMS-d 6, 2 MHz): (s, 1 H), 8.51(s, 1H), 8.12 (d, 3 J = 15.Hz, 1H), 8.1 (m, 1H), 7.76 (d, 3 J S2

3 = 15.Hz, 1H), 7.52 (m, 2H), 7.1 (m, 2H), 6.8(dd, 3 J = 9. Hz, 4 J = 2.4 Hz, 1H), 6.6 (d, 4 J = 9. Hz, 1H), 3.59 (q, 3 J = 7. Hz, 4H), 1.15 (t, 3 J = 7. Hz, 6H). 1 H NMR (CDCl 3, 3 MHz): δ 13.5 (s, 1H), 8.39 (d, 3 J = 15. Hz, 1H), 8.5(d, 3 J = 7.9 Hz, 1H), 7.8 (s, 1H), 7.72 (d, 3 J = 15. Hz, 1H), 7.51 (t, 3 J = 8.1 Hz, 1H), 7.36 (d, 3 J = 9. Hz, 1H), (m, 2H), 6.64 (dd, 3 J = 9. Hz, 4 J = 2.4 Hz, 1H), 6.52 (d, 4 J = 2.4 Hz, 1H), 3.5 (q, 3 J = 7. Hz, 4H), 1.28 (t, 3 J = 7. Hz, 6H). 13 C NMR (CDCl 3, 75 MHz): δ , 163.5, 16.19, 156.7, , 147.1, 14.74, , 13.25, 13.21, , 12.29, , , , 19.67, 18.94, 96.87, 45.1, 12.5 (2 carbon peaks). HRMS (FB +, m-nb): m/z obsd ([M+H] +, calcd for C 22 H 22 N 4 ). 1-ME. To a solution of 1 (1 mol, 2 mm) in DMS-d 6 was added one drop of 2-mercaptoethanol (ME). fter shaking several times, the reaction mixture was monitored in NMR and we found the reaction was complete within 1 min. 1 H NMR (DMS-d 6, 2 MHz): (s, 1 H), 7.92(m, 1H), 7.9 (s, 1H), 7.52 (m, 1H), 7.39 (d, 3 J = 8.8 Hz, 1H), 6.96 (m, 2H), 6.68 (dd, 3 J = 9. Hz, 4 J = 2.4 Hz, 1H), 6.52 (d, 4 J = 2.4 Hz, 1H), 4.76 (t, 3 J = 5.4 Hz, 1H), 4.48 (t, 3 J = 7.4 Hz, 1H), 3.78 (d, 3 J = 7.4 Hz, 2H), 3.45 (m, 6H), 2.56 (m, 2H), 1.15 (t, 3 J = 7. Hz, 6H). HRMS (FB +, m-nb): m/z obsd ([M+H] +, calcd for C 24 H 28 N 5 S). 2. To a solution of 1 (24.6 mg,.1 mmol) and acetophenone (1.8 equiv) in 1 ml CH 2 Cl 2 /EtH (4:1, v/v) was added 2 drops of pyrrolidine and the yellow reaction mixture was further stirred overnight at rt. fter evaporating all the volatiles under reduced pressure, purification by flash column chromatography on silicagel (DCM, R f =.32) afforded the desired product 2 as orange solid (yield 7 %). 1 H NMR (CDCl 3, 3 MHz): δ 8.27 (d, 3 J = 15. Hz, 1H), 8.12 (d, 3 J = 7.1 Hz, 2H), 7.8 (s, 1H), 7.68 (d, 3 J = 15. Hz, 1H), (m, 3H), 7.36 (d, 3 J = 9. Hz, 1H), 6.65 (dd, 3 J = 9. Hz, 4 J = 2.4 Hz, 1H), 6.52 (d, 4 J = 2.4 Hz, 1H), 3.5 (q, 3 J = 7. Hz, 4H), 1.28 (t, 3 J = 7. Hz, 6H). 13 C NMR (CDCl 3, 75 MHz): δ 19.8, 16.3, 156.6, 151.9, 146.3, 139.9, 138.4, , 13., 128.7, 128.5, 123., 115., 19.5, 18.9, 96.9, 45.1, 12.5 (18 carbon peaks). HRMS (FB +, m-nb): m/z obsd ([M+H] +, calcd for C 22 H 22 N 3 ). [S1] Kim, T.-K.; Lee, D.-N.; Kim, H.-J. Tetrahedron Lett. 28, 49, S3

4 3. NMR spectra H Figure S1. 1 H and 13 C NMR spectra of 1 in CDCl 3. S4

5 (t1) 15 1 Figure S2. 1 H and 13 C NMR spectra of 2 in CDCl 3. 5 S5

6 a b c H HS DMS H b' S H B H H b H H a H b H c Figure S3. Full 1 H NMR spectra of 1 upon addition of ME in DMS-d 6. () 1 (2 mm), (B) 1 + ME (1.8 equiv). c HS H Et 2N H D b' B a b 1 H DMS/D 2 S D D H b H a H b H c Figure S4. Deuterium exchange experiments with full 1 H NMR spectra of 1 upon addition of ME in DMSd 6 /D 2 (1:1, v/v). () 1 (2 mm), (B) 1 + ME in excess. S6

7 4. Comparison of Michael reactions of 1 and bs nm 485nm time/min min B.4 bs nm 485nm time/min min /nm /nm Figure S5. Time-dependent UV-vis spectral changes upon addition of 1 equiv ME to 1 μm of 1 () and 2 (B) in DMS/HEPES buffer (4:1, v/v, ph 7.4). 5. Ratiometric fluorescence response of 1 upon addition of analytes 1 F 466 nm / F 553 nm Figure S6. Ratiometric fluorescence responses of 1 (1 M) upon addition of various analytes (1 mm) in DMS/HEPES buffer (4:1, v/v, ph 7.4). If GSH is pre-treated with N-ethylmaleimide (NEM), a scavenger of GSH, the fluorescence ratio of 1 changes as small as that of 1 owing to the decreased concentration of GSH. S7

8 6. Fluorometric determination of biothiol detection limit y =.122 x R² =.998 B F 5 F 466nm /F 553nm /nm [biothiol] Figure S7. () Fluorescence responses of 1 (1 M) upon addition of various amounts of Hcy in DMS/HEPES (4:1, ph 7.4). (B) Linear plot of fluorescence intensity ratio of 1 against [Hcy] to afford 37 M Hcy as the detection limit at F/F = 3. S8

9 7. Calibration curve for GSH 15 2 B y =.989 x R² = F 6 F 466 /F F466/F nm [biothiol]/ M Figure S8. () Fluorescence responses of 1 (1 M) upon addition of various amounts of GSH in DMS/HEPES (4:1, ph 7.4). (B) Linear plot of fluorescence intensity ratio of 1 against [GSH]. lthough there exist discrepancies in the reaction conditions (emission wavelength, reaction time, and solvent) between the spectrofluorometer and the confocal laser scanning microscope, we tried to calculate the GSH concentration in the cells by the ratiometric fluorescence intensities. Based on the crude approximation, the cellular concentrations of GSH were calculated from above calibration curve of 1 against biothiol. Table S1. Cellular [GSH] calculated from the calibration curve. F Blue F Green F Blue /F Green [GSH]/ M Remarks a 1 a From above calibration curve of GSH, the concentration of the natural GSH was determined to be 1.7 mm. This value lies well within the reported cellular [GSH] of 1~1 mm:. K. Sakhi, K. M. Russnes, S. Smeland, R. Blomhoff, T. E. Gundersen, J. Chromatogr. 26, 114, 179. S9

10 8. ph Profile 1 R (F 466nm /F 553nm ) ph Figure S9. Relative fluorescence intensity ratio (R) vs ph profile of 1-Hcy ([1] = 1 M, [Hcy] = 1 mm in DMS/HEPES buffer (4:1, v/v, 8 h). S1

11 9. Mass spectra 1 H [M+H] + Figure S1. Mass spectra of 1. S11

12 S H H 1-ME Figure S11. Mass spectra of 1-ME. S12

13 2 [M+H] + Figure S12. Mass spectra of 2. S13