Supporting Information for: Tunable Heptamethine-Azo Dye Conjugate as an IR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione over Cysteine and Homocysteine Soo-Yeon Lim, 1 Keum-Hee Hong, 1 Dae Il Kim, 2 Hyockman Kwon, 2 and Hae-Jo Kim 1 * 1 Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea 2 Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea haejkim@hufs.ac.kr Contents 1. Instruments, reagents, and preparation S2 2. Synthesis S2 3. MR spectra S4 4. Mass spectra S9 5. Titration graph S15 6. MR spectral change of MitoGP upon addition of Cys S16 7. Fluorescence kinetics of MitoGP upon addition of Cys and Hcy S17 8. ph Profile S18 9. Fluorescence kinetics of MitoGP and its analogues upon addition of biothiol S18 10. Cellular images of MitoGP S19 11. UV-vis and fluorescence spectra of control compounds S20 12. MR and mass spectra of control compounds S21 S1
1. Instruments, reagents, and preparation All experiments were carried out from commercially available reagents and solvents, and used without further purification, unless otherwise noted. Chromatography was carried out on silica gel 60 (230-400 mesh ASTM; Merck). Thin layer chromatography (TLC) was carried out using Merck 60 F 254 alumina plates with a thickness of 0.25 mm. 1 H MR and 13 C MR spectra were recorded using Bruker 400 or Varian 400. Mass spectra were obtained using MARDI-TF mass spectroscopy All fluorescence and UV-Vis absorption spectra were recorded in FP 6500 fluorescence spectrometer and HP 8453 absorption spectrometer, respectively. Preparation for UV-vis and fluorescent study A stock solution (10 mm) of MitoGP in DMS was prepared and used for UV-vis and fluorescence measurement by dilution with HEPES buffer (0.1 M, ph 7.4). In a typical experiment, test solutions (10 M) of MitoGP 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 HEPES buffer. ormally, fluorescence excitation wavelength was set at 600 nm. Both the excitation and emission slit widths were 10 nm ⅹ 10 nm. Fluorescence spectra were monitored 3 h after addition of amino acids unless otherwise stated. Fluorescence 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 100 units/ml penicillin, 100 mg/ml streptomycin, and 10 % heat-inactivated fetal bovine serum. The cells were seeded on a Ø 35 mm glass-bottomed dish at a density of 1.0 x 10 5 cells in a culture medium and incubated overnight for live-cell imaging by confocal laser scanning microscopy (CLSM). The HeLa cells were washed twice with phosphate-buffered saline (PBS) before incubation of probes. The cells were treated with 1 or 2 M MitoGP in 1 x PBS for 0.5 h and then washed with twice with pre-warmed 1 x PBS before imaging by CLSM. In order to reduce the concentration of mitochondrial GSH, HeLa cells were pretreated with 3-hydroxy-4-pentenoate (3-HP) or 3-oxo-4-pentenoate (3-P) for 5 or 10 min respectively, followed by MitoGP for 0.5 h, and washed with 3 times with pre-warmed 1 x PBS before imaging by CLSM by using two excitation channels ( ex 488 nm, 555 nm). MitoGP was pretreated with -lipoic acid (LPA, 1.8 mm, 24 h) in order to increase GSH in the live cells. After the live cell imaging, the mean fluorescence intensities at a red channel ( ex 555 nm) were measured with at least 20 HeLa cells in three different fields by ZE 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 Synthesis of 1 Compound 1 was prepared according the modified literature method [1]. 1-Hydroxycarbonylethyl-2,3,3-trimethylbenzoindoleniuum bromide (3.6 g, 11.5 mmol) and 2-chloro-1-formyl-3-(hydroxymethylene)- chclohex-1-ene (1.0 g, 5.75 mmol) were dissolved in 100 ml of n-butanol and benzene (10:1) in a flask equipped with a Dean-Stark trap. The mixture was refluxed for 6 h to give dark green solution while the water formed was collected in the trap. After the reaction was complete, the solvent was removed under reduced S2
pressure. The crude product was purified by column chromatography on silicagel using DCM and methanol (10:1, v/v) as eluent, to give compound 1 as a green solid (2.5 g) in 54 %yield. 1 H MR (CDCl 3, 400 MHz): 8.35 (d, 3 J = 14 Hz, 2H), 7.40-7.21(m, 8H), 6.38(d, 3 J = 14 Hz, 2H), 4.62 (t, 3 J = 6.4 Hz, 4H), 4.02 (t, 3 J = 6.8 Hz, 4H), 2.95 (t, 3 J = 6.4 Hz, 4H), 2.77(t, 3 J = 6.0Hz, 4H), 1.81( q, 3 J = 6.0 Hz, 2H), 1.70 (s, 12H), 1.50 (q, 3 J = 6.8 Hz, 4H), 1.30(q, 3 J = 7.2 Hz, 4H), 0.85 (t, 3 J = 7.2 Hz, 6H). 13 C MR (CDCl 3, 100 MHz): 172.29, 170.91, 150.55, 144.35, 141.84, 140.87, 128.83, 128.22, 125.42, 122.22, 111.29, 102.13, 65.17, 49.30, 40.83, 32.28, 30.42, 28.16, 28.11, 26.66, 18.99, 13.63. HRMS (MALDI +, DHB): [M] + calcd. for C 44 H 56 Cl 2 4, 711.3926; found, 711.3923. [1] Achilefu, S. et al. Synthesis and Evaluation of Polyhydroxylated ear-infrared Carbocyanine Molecular Probes, rg. Lett., 2004, 6, 2067-2070. 2 2 H 2 1.a 2, HCl 0 o C/1h 2. Phenol, ah 0 o C/1h H 2 Synthesis of 2 Diluted HCl (5 ml of conc. HCl, 58 mmol) was added slowly to p-nitroaniline (1 g, 7.24 mmol) in water at 0 o C and the mixture was stirred for 30 min. A solution of a 2 (750 mg, 10.86 mmol) in water is dropped into the slurry mixture. The resulting clear mixture was added dropwise to a solution of phenolate (680 mg of phenol, 1.16 g of ah ) for 1 h. The mixture was neutralized and the precipitate was filtered and washed with water to give compound 2 as an orange solid (0.90 g) in 51.7 % yield. 1 H MR (DMS-d 6, 400 MHz): 10.61 (s, 1H), 8.42 (d, 3 J = 8.8 Hz, 2H), 8.02 (d, 3 J = 8.8 Hz, 2H), 7.91 (d, 3 J = 8.8 Hz, 2H), 7.00 (d, 3 J = 8.8 Hz, 2H) 13 C MR (DMS-d 6, 400 MHz): 162.82, 156.02, 148.24, 145.87, 126.29, 125.52, 123.47, 116.71. HRMS (MALDI +, DHB): [M+H] + calcd. for C 12 H 9 3 3, 244.0717; found, 244.0715. S3
3. MR spectra 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 13.628 18.988 20.747 26.664 28.160 30.419 32.279 40.826 49.304 65.165 76.827 77.146 77.464 102.128 111.285 122.224 125.416 128.226 128.834 140.872 141.841 144.351 150.551 170.909 172.287 200 150 100 50 0 Fig. S1. 1 H and 13 C MR spectra of compound 1 in CDCl 3. S4
2 H 10.0 5.0 39.391 39.600 39.809 40.017 40.226 40.435 40.644 116.717 123.476 125.518 126.295 145.870 148.248 156.027 162.821 200 150 100 50 Fig. S2. 1 H and 13 C MR spectra of compound 2 in DMS-d 6 S5
2 n Bu 2 C C 2 n Bu 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 13.639 19.002 21.115 24.626 27.932 30.425 32.255 40.615 48.945 65.167 76.759 77.077 77.395 101.451 111.097 115.432 122.029 123.122 123.368 124.728 125.236 126.160 128.746 140.786 141.380 141.808 147.687 148.578 155.612 162.649 162.667 170.905 171.851 200 150 100 50 Fig. S3. 1 H and 13 C MR spectra of MitoGP in CDCl 3. S6
2 H H Fig. S4. 1 H and 13 C MR spectra of compound 3 in CD 3 D. S7
2 H H Fig. S5. 1 H and 13 C MR spectra of compound 4 in CDCl 3. S8
4. Mass spectra Fig. S6. Mass spectra of compound 1. HRMS (MALDI +, DHB): m/z obsd 711.3923 ([M] +, cald 711.3926 for C 44 H 56 Cl 2 4 ) Fig. S7. Mass spectra of compound 2. HRMS (MALDI +, DHB): m/z obsd 244.0715 ([M] +, cald 244.0717 for C 12 H 9 3 3 ) S9
2 n Bu 2 C C 2 n Bu Fig. S8. Mass spectra of MitoGP. HRMS (MALDI +, DHB): m/z obsd 918.4806 ([M] +, cald 918.4802 for C 56 H 64 5 7 ). S10
2 C 2 H 3 C2H Fig. S9. Mass spectra of compound 3. HRMS (FAB +, m-ba): [M] + calcd. for C 48 H 48 5 7, 806.3554; found, 806.3550. S11
2 (CH 2 ) 2 HC CH(CH2)2 4 Fig. S10. Mass spectra of compound 4. HRMS (FAB +, m-ba): [M] + calcd. for C 60 H 72 9 7, 1030.5555; found, 1030.5530. S12
H H H H 2 H S n Bu n Bu Fig. S11. Mass spectra of MitoGP-GSH. MS (FAB +, m-ba): m/z obsd 982 ([M] +, cald 982.4994 for C 54 H 72 5 10 S + ). SH H H Fig. S12. Mass spectra of compound MitoGP-Cys, where 1:1 ratio of MitoGP (10 mm)/cys (10 mm) was incubated in MeH. MS (FAB +, m-ba): m/z obsd 796 ([M] +, cald 796.4354 for C 47 H 62 3 6 S). S13
n Bu n Bu S H n Bu n Bu [M + ] 1471 Fig. S13. Mass spectra of (MitoGP) 2 -Cys, where 2:1 ratio of MitoGP (10 mm)/cys (5 mm) was incubated in MeH. MS (FAB +, m-ba): m/z obsd 1471 ([M] +, cald 1470.8437 for C 91 H 116 Br 5 10 S + ). S14
5. Titration graph of MitoGP upon addition of GSH 75 75 A 20 mm B Flu (a.u.) 50 25 0 mm F 810 50 25 F 810 6 4 2 0 y = 0.438x + 0.2851 R² = 0.9949 0 4 8 12 [GSH]/ M 0 670 730 790 850 nm 0 0 5 10 15 20 [GSH]/m Fig. S14. (A) The fluorescence changes of MitoGP (10 M, ex 600 nm) upon the addition of GSH in HEPES buffer (0.10 M, ph 7.4). (B) Its titration graph of fluorescence intensity at 810 nm vs [GSH]; (inset) linear plot of MitoGP against GSH. S15
6. MR spectral change of MitoGP upon addition of Cys 2 C n 2 Bu H f C 2 n Bu SH H a Cys H C 2H H e S C 2H C 2 n Bu H b C 2 n Bu C 2 n Bu C 2 n Bu H d H c H E C 2 n Bu C 2 n Bu D H c H d C H e H f B H a H b A 9.0 8.0 Fig. S15. Partial 1 H MR spectra of MitoGP (10 mm) upon addition of Cys in CD 3 D. (A) MitoGP. (B) MitoGP + 0.5 equiv Cys, 30 min. (C) MitoGP + 1.2 equiv Cys, 30 min, (D) 1-Cys. (E) 1--BocCys. 7.0 6.0 The initial thioether underwent a subsequent intramolecular amination reaction in the presence of 0.5 equiv Cys and the resulting free thiol group reacted with another MitoGP to produce a stable 2:1 complex between MitoGP and Cys (Fig. S10 B). Further addition of Cys did not induce any other spectral changes. However, we expect that the real reaction takes place in a manner of 1:1 ratio in highly diluted condition such as in a fluorescence experiment, whose stoichiometry was proven in the Job s plot. S16
7. Fluorescence kinetics of MitoGP upon addition of Cys and Hcy 9 9 (A) (B) Flu (a.u.) 6 3 6 3 Cys 0 660 720 /nm 780 840 Hcy F 747 Flu (a.u.) 0 660 720 780 840 /nm 9 (C) 6 3 0 0 30 60 90 Time/min Fig. S16. Time-dependent fluorescence spectra of MitoGP (10 M) upon the addition with (A) Hcy (10 mm) and (B) Cys (10 mm) in HEPES buffer (0.10 M, ph 7.4). (C) Fluorescence kinetics for Cys and Hcy. S17
8. ph Profile 80 MitoGP MitoGP+GSH 60 F 810 40 20 0 3 5 7 9 11 13 Fig. S17. The fluorescence response of MitoGP (10 M,) in the presence and absence of GSH (10 mm) under different phs. ph 9. Fluorescence kinetics of MitoGP and its analogues upon addition of biothiol 9 6 F/F 0 MitoGP 3 4 3 0 0 400 800 1200 time/min Fig. S18. Fluorescence kinetics of MitoGP and its analogues in the presence of GSH. (A) MitoGP with ester functionality, (B) 3 with acid, (C) 4 with amide. S18
10. Cellular Images of MitoGP Fig. S19. Confocal laser scanning microscopic images of MitoGP and rhodamine 123 in live HeLa cells. (A) Images with the spectral detection setup at the excitation wavelength, 488 nm, (B) images with the spectral detection setup at the excitation wavelength, 555 nm, (C) merged images. A B C Fig. S20. Confocal laser scanning microscopic images of MitoGP (2.0 M, 30 min) with 3-oxo-4-pentenoate (3-P) in HeLa cells ( ex 555 nm). (A) MitoGP only, (B) MitoGP after pretreatment with 3-P (0.5 mm, 10 min), (C) with 3-P (1.0 mm, 10 min). The live cells showed to lose adhesion ability on the slide glass by the treatment of 3-P. S19
11. UV-vis and fluorescence spectra of control compounds H H H S n Bu n Bu MitoGP-EA n Bu n Bu MitoGP-ME 1.2 A(MitoGP EA) F(MitoGP EA) A(MitoGP ME) F(MitoGP ME) orm. A or F 0.8 0.4 0 500 700 900 nm Fig. S21. ormalized UV-vis and fluorescence spectra of control compounds with mercaptoethanol (MitoGP- ME) and ethanol amine (MitoGP-EA). S20
12. MR and mass spectra of control compounds H S n Bu n Bu Fig. S22. 1 H MR (10 mm in methanol-d 4 ) and mass spectra of MitoGP-ME. MS (MALDI +, DHB): m/z obsd. 753.4863 ([M] +, calcd. 753.4298 for C 46 H 61 2 5 S). H H n Bu n Bu Fig. S23. 1 H MR (10 mm in methanol-d 4 ) and mass spectra of MitoGP-EA. MS (MALDI +, DHB): m/z obsd. 736.3744 ([M] +, calcd. 736.4687 for C 46 H 62 3 5 ). S21