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1 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 , Republic of Korea 2 Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin , 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 S Cellular images of MitoGP S UV-vis and fluorescence spectra of control compounds S MR and mass spectra of control compounds S21 S1

2 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 ( 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

3 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), (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): , , , , , , , , , , , , 65.17, 49.30, 40.83, 32.28, 30.42, 28.16, 28.11, 26.66, 18.99, HRMS (MALDI +, DHB): [M] + calcd. for C 44 H 56 Cl 2 4, ; found, [1] Achilefu, S. et al. Synthesis and Evaluation of Polyhydroxylated ear-infrared Carbocyanine Molecular Probes, rg. Lett., 2004, 6, 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, 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): (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): , , , , , , , HRMS (MALDI +, DHB): [M+H] + calcd. for C 12 H 9 3 3, ; found, S3

4 3. MR spectra Fig. S1. 1 H and 13 C MR spectra of compound 1 in CDCl 3. S4

5 2 H Fig. S2. 1 H and 13 C MR spectra of compound 2 in DMS-d 6 S5

6 2 n Bu 2 C C 2 n Bu Fig. S3. 1 H and 13 C MR spectra of MitoGP in CDCl 3. S6

7 2 H H Fig. S4. 1 H and 13 C MR spectra of compound 3 in CD 3 D. S7

8 2 H H Fig. S5. 1 H and 13 C MR spectra of compound 4 in CDCl 3. S8

9 4. Mass spectra Fig. S6. Mass spectra of compound 1. HRMS (MALDI +, DHB): m/z obsd ([M] +, cald for C 44 H 56 Cl 2 4 ) Fig. S7. Mass spectra of compound 2. HRMS (MALDI +, DHB): m/z obsd ([M] +, cald for C 12 H ) S9

10 2 n Bu 2 C C 2 n Bu Fig. S8. Mass spectra of MitoGP. HRMS (MALDI +, DHB): m/z obsd ([M] +, cald for C 56 H ). S10

11 2 C 2 H 3 C2H Fig. S9. Mass spectra of compound 3. HRMS (FAB +, m-ba): [M] + calcd. for C 48 H , ; found, S11

12 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 , ; found, S12

13 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 for C 54 H 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 for C 47 H S). S13

14 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 for C 91 H 116 Br 5 10 S + ). S14

15 5. Titration graph of MitoGP upon addition of GSH A 20 mm B Flu (a.u.) mm F F y = 0.438x R² = [GSH]/ M nm [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

16 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 Fig. S15. Partial 1 H MR spectra of MitoGP (10 mm) upon addition of Cys in CD 3 D. (A) MitoGP. (B) MitoGP equiv Cys, 30 min. (C) MitoGP equiv Cys, 30 min, (D) 1-Cys. (E) 1--BocCys 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

17 7. Fluorescence kinetics of MitoGP upon addition of Cys and Hcy 9 9 (A) (B) Flu (a.u.) Cys /nm Hcy F 747 Flu (a.u.) /nm 9 (C) 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

18 8. ph Profile 80 MitoGP MitoGP+GSH 60 F 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 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

19 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

20 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 nm Fig. S21. ormalized UV-vis and fluorescence spectra of control compounds with mercaptoethanol (MitoGP- ME) and ethanol amine (MitoGP-EA). S20

21 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 ([M] +, calcd for C 46 H 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 ([M] +, calcd for C 46 H ). S21