Supporting Information

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1 Supporting Information A Highly Selective Mitochondria-Targeting Fluorescent K + Sensor Xiangxing Kong, Fengyu Su, Liqiang Zhang, Jordan Yaron, Fred Lee, Zhengwei Shi, Yanqing Tian,* and Deirdre R. Meldrum* anie_ _sm_miscellaneous_information.pdf

2 List of Contents 1: Experimental section 1.1 Reagents 1.2 Instruments 1.3 Synthesis of KS6 1.4 Fluorescence quantum efficiency determination 1.5 Sensor responses to K Cell culture for imaging 1.7 Colocalization test of KS6 and MitoTracker Green FM 1.8 Monitoring the intracellular K + flux using KS6 1.9 Cytotoxicity of KS6 to cells by viable cell counts using MTT References Experimental Section 1.1 Reagents. All chemicals were purchased and used without further purification. MitroTracker Green FM was ordered from Life technology (Carlsbad, CA). 4-(6-Bromohexyloxy)-benzaldehyde (1) and TAC-CHO were synthesized according to known procedures, respectively. 1,2 1.2 Instruments. A Varian liquid-state NMR operated at 400 MHz was used for 1 H NMR spectra measurements. High resolution Mass Spectra of the intermediates and the final product were obtained by JEOL LCmate at CLAS High resolution Mass Spectrometry Lab, ASU, using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) methods. A Shimadzu UV-3600 UV-Vis-NIR spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) was used for UV- Vis absorption spectra measurements. A Shimadzu RF-5301 spectrofluorophotometer was used for fluorescence measurements. 1.3 Synthesis of KS6. Synthesis of [6-(4-formylphenoxy)hexyl]triphenylphosphonium bromide (2): 4-(6- Bromohexyloxy)-benzaldehyde (2.85g, 10 mmol) and triphenylphosphine (2.62g, 10 mmol) were added into a flask containing 30 ml of ethanol. The mixture was refluxed for 24 h. After removal of solvent by vacuum rotavapor, the remaining solid was purified by silica flash column chromatography with gradient solvent from CH 2Cl 2 to CH 2Cl 2/MeOH (v/v = 9/1) g of white solid was obtained, yield: 70%. 1 H NMR (CDCl 3, ) (ppm): 9.84 (s, 1H, CHO), (m, 6H, Ar-H), (m, 5H, Ar-H), 7.68 (m, 6H, Ar-H), 6.93 (2H, Ar-H), 4.00 (t, 2H, CH 2O), 3.91 (t, 2H; CH 2), 1.75 (m, 4H), 1.64 (m, 2H), 1.48 (m, 2H). 1

3 Synthesis of 4-[6-(triphenylphosphonium)hexyloxy]phenyl-1,3,5,7-tetramethyl borondipyrromethene bromide (3): [6-(4-formylphenoxy)hexyl]triphenylphosphonium bromide (2, 1.1 g; 2 mmol) and 2,4-dimethylpyrrole (0.52 ml; 5 mmol) were added in a dry 500 ml three-neck flask under nitrogen. 200 ml of anhydrous dichloromethane was added, followed by 2 drops of trifluoroacetic acid. After the mixture was stirred at room temperature for 12 hours, p-chloranil (0.487 g, 2 mmol) in 100 ml dichloromethane was added. After stirred for another 30 minutes, 10 ml of trimethylamine and 10 ml of BF 3OEt 2 was added in., The mixture was poured into 200 ml of water. after stirred for another 2 hours. The dark organic phase was separated using separation funnel, and washed with HCl (1.0 M), water, 10% Na 2CO 3 aqueous solution, and then water. The organic phase was removed under vacuum. The final product was purified by silica flash chromatography using CH 2Cl 2 to CH 2Cl 2/MeOH (v/v: 95:5) as eluent; 0.60 g of product was obtained. Yield: 39%. 1 H NMR (CDCl 3, ) (ppm): (m, 15H, Ph 3P + ), (dd, 2H, AR-H,), 6.96 (d, 2H, AR-H, J= 8.4 Hz), 5.95 (s, 2H, C-H); 3.97 (t, 2H, CH 2), 3.36 (m, 2H, CH 2), 2.53 (s, 6H, 2 CH 3), 1.78 (m, 2H, CH 2), 1.69 (m, 4H, 2 CH 2), 1.54 (m, 2H, CH 2), 1.41 (m, 6H, 2 CH 3). 13 C NMR (100 MHz, CDCl3) (ppm): , , , , , , , , ,130.58, , , , , , , , 67.83, 30.07, 29.91, 28.89, 25.53, 22.60, 22.55, 22.15, 21.65, 14.59, HRMS (APCI) (m/e): calculated for C 43H 45N 2OF 2PB: ; found: Synthesis of KS6. TAC-CHO (72.0 mg, 0.1 mmol) and compound 3 (76.6 mg, 0.1 mmol) were refluxed in a mixture of benzene (5 ml), acetic acid (30 L) and piperidine (36 L) overnight. The reaction mixture was cooled to room temperature, diluted with 10 ml CH 2Cl 2 and washed with water. The organic layer was dried over anhydrous Na 2SO 4, filtered and evaporated. KS6 (27 mg) was obtained by silica flash chromatography using CH 2Cl 2 to CH 2Cl 2/MeOH (v/v: 95:5) as eluent. Yield: 18.4%. KS6 was further purified by reverse-phase HPLC using water and methanol/acetic acid (0.1%) for NMR and Mass spectra characterization. 1 H NMR (CDCl 3, ) (ppm): (m, 15H, PPh 3), (m, 15H, Ar-H and vinyl-h), 5.95 (s, 1H), (m, 43H), 2.56 (s, 3H, CH 3); 2.10 (s, 6H, 2 CH 3Ph); (m, 6H, 3 CH 2); 1.54 (m, 8H, CH 2, 2 CH 3). HRMS (ESI, m/e): calculated for C 83H 98BF 2N 5O 9P + : ; found Fluorescence quantum efficiency determination. The fluorescence quantum yields ( f) of samples in solution were measured by using Rhodamine 101 in ethanol ( f=100%) as a standard excited at 540 nm and were calculated according to the following equation: Ar r As I I n n 2 s s s 2 r r where s and r are the fluorescence quantum yields of standards and the samples, respectively. A r and A s are the absorbance of the standards and the measured samples at the excitation wavelength (adjusting the concentration of both the reference and the sample to make A r and A s around 0.05), respectively. I r and I s are the integration emission intensities of the standards and samples, respectively. n r and n s are the refractive indices of the corresponding solvents of the solutions, respectively. 1.5 Sensor titration with KCl in a surfactant solution containing 2

4 A. CTAB surfactant: 10 µl of KS6 dissolved in DMSO (1 mm) was added into a solution containing 1 ml of Tris-HCl (10 mm, ph 7.4) buffer solution and 1mL of CTAB (1 mm) in water. This resulted in the final KS6 concentration of 5 µm in the solution. Different volumes of KCl (4.0 M) were added into the solution. The mixture was gently shaken for 10 seconds. Both UV-Vis spectra and fluorescence spectra were recorded to study KS6 s responses to [K + ]. KS6/metal ion complexes were excited at 540 nm and emission spectra were collected from 550 to 750 nm. The real concentration of the K + was calculated after volume correction. B. Pluronic F µl of KS6 dissolved in DMSO (1 mm) and 20 µl of Pluronic F127 (10%) in DMSO were added into a solution containing 1 ml of Tris-HCl (10 mm, ph 7.4) buffer and 1 ml of DI water. This resulted in the final KS6 concentration to be 5 µm in the buffer solution. Similar titration was performed as described with CTAB surfactant. C. Sodium dodecyl sulfate (SDS): 10 µl of KS6 dissolved in DMSO (1 mm) was added into a solution containing 1 ml of Tris-HCl (10 mm, ph 7.4) buffer and 1mL of 5mM of SDS in DI water. Titration with KCl (4.0 M) in water caused formation of white precipitate. 1.6 Cell culture for imaging. U87MG cells (American Type Culture Collection, ATCC, Manassas, VA) were cultured in Eagle s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 100 u/ml penicillin, and incubated at 37 C in 5% CO 2 atmosphere. Cells were then seeded onto 96 well plates at 10,000 cells per well, and incubated overnight at 37 C. The sensor dissolved in DMSO was added to the medium to make the sensor concentrations in a range of µm. 10 min of internalization was found to be sufficient for achieving satisfactory images. To achieve images with satisfactory signal-to-noise ratio, a sensor concentration of 2 µm was usually used for intracellular study. 1.7 Colocalization test of KS6 and MitoTracker Green FM: Cells were co-stained using the sensor KS6 in combination with MitoTracker Green FM. To co-stain the mitochondria, MitoTracker Green FM in DMSO was used. Cells were first internalized with KS6 (2 μm in cell culture medium) for 10 minutes. MitoTracker Green FM diluted in the fresh medium was then added into the wells to stain cell for 10 min at 37 C. The resulting concentration of MitoTracker Green FM in the cell medium was 50 nm. Under a Nikon C2Si Eclipse Ti confocal fluorescence microscope (Melville, NY), Sensor (KS6) was excited at 561 nm and its red emission was collected using a 605/75 nm filter set. MitoTracker Green FM was excited at 488 nm and its green emission was collected using a 515/30 nm filter set. 1.8 Monitoring the intracellular K + efflux using KS6. HeLa cells or U87MG cells were seeded onto 96 well plates with 5,000 cells and 15,000 cells per well respectively in 100 µl medium, and incubated overnight at 37 C. On the following day, the cells were internalized with KS6 (2 μm) for 10 min at 37 C. A nigericin solution was then added. Final concentrations of nigericin in the cell culture medium were 10 μm. Fluorescence in cells was visualized by Nikon C2Si Eclipse Ti confocal fluorescence microscope (Melville, NY) at 37 C. 3

5 1.9 Cytotoxicity of KS6 to cells by viable cell counts using MTT. To determine cell viability, a colorimetric MTT metabolic activity assay was used. HeLa cells ( cells/well) were cultured in a 96-well plate at 37 C, and exposed to varying concentrations of KS6 for 3, 6, and 15 hours. Cells treated with medium only served as a negative control group. After removing the supernatant of each well and washing by PBS, 15 µl of MTT solution and 100 µl of medium were then introduced. After incubation for another 4 hours, the resultant formazan crystals were dissolved in solubilization solution (100 µl) and the absorbance intensity measured by a microplate reader (BioTek Synergy H4, USA) at 570 nm. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated control cells. References 1. Jiang, S.; Liu, M.; Cui, Y. T.; Zou, D. P.; Wu, Y. J. Eur. J. Org. Chem. 2013, He, H.; Mortellaro, M. A.; Leiner, M. J. P.; Fraatz, R. J.; Tusa, J. K. J. Am. Chem. Soc. 2003, 125,

6 I/I 0 2 I/I A Time (min) B Time (min) Figure S1. Time dependent fluorescence Intensity change (at 572 nm) after mixing a solution containing KS6 (5.0 M) and KCl (0.80 M) in: A (left) Tris buffer (ph =7.4, 5 mm)/pluronic F127 (0.5%) aqueous solution; B (right): Tris buffer (ph =7.4, 5 mm)/cetrimonium bromide (500 M) aqueous solution. 5

7 Absorbance A) B) Wavelength (nm) Normalized Fluorescence Intensity em : 572 nm ex : 540 nm Wavelength (nm) Figure S2. (A) UV-Vis absorption spectra of KS6 during the titration in Tris buffer (ph = 7.4; 5.0 mm)/ctab (0.5 mm) solution from 0 mm to 0.80 M K + (left). (B) Excitation and emission spectra of KS6 in Tris buffer (ph = 7.4; 5.0 mm)/ctab (0.5 mm) solution containing 0.80 M KCl (right). 6

8 Absorbance A Wavelength (nm) 0.00 M Na M Na + B Fluorescence Intensity (a.u.) Slit: 5 nm (ex); 1.5 nm (em) Wavelength (nm) 0.00 M M M M 0.14 M 0.19 M 0.28 M 0.36 M 0.52 M 0.67 M 0.80 M Fluorescence Intensity (a.u.) C mm Na + followed by 150 mm K mm Na + KS6 only Wavelength (nm) Figure S3. (A) UV-Vis spectra and (B) fluorescence spectra of KS6 during the titration in Tris/HCl buffer (ph = 7.4; 5.0 mm)/ctab (0.5 mm) solution from 0.00 M to 0.80 M Na + ; and (C) fluorescence spectra of KS6 in Tris/HCL buffer (ph = 7.4; 5.0 mm)/ctab (0.5 mm) solution, after adding in Na + to make that the final concentration of Na + is 150 mm; and followed by adding in K + to make the final K + and Na + are 150 mm, respectively. 7

9 h 6h 15h Viability (a.u.) M 1 M 2 M 3 M KS6 concentration Figure S4. Cell viability test of HeLa cells measured using the MTT assay for KS6 at different concentrations and times. 8

10 Figure S5A. Time-dependent fluorescence images of HeLa cells in EMEM containing 10M of ionomycin and 20 mm of KCl. Figure S5B. Time-dependent fluorescence images of HeLa cells in EMEM containing 10M of ionomycin and 200 mm of KCl. 9

11 Figure S6A. Time-dependent fluorescence images of HeLa cells in EMEM containing 20 mm of KCl after stimulation with nigericin (10 M). Figure S6B. Time-dependent fluorescence images of HeLa cells in EMEM containing 200 mm of KCl after stimulation with nigericin (10 M). 10

12 Figure S7. Time-dependent fluorescence images of U87MG cells in EMEM containing 20 mm of KCl (Top) and 200 mm of KCl (Bottom) without stimulation. 11

13 Figure S8. Time-dependent fluorescence images of U87MG Cells in EMEM containing 200 mm of KCl after stimulation with ionomycin (20 M). 12

14 0 min 2 min 4 min min 10 min F/F no CCCP CCCP 10 um CCCP 20 um CCCP 40 um Time (min) Figure S9. Typical time dependent fluorescence images of U87MG cells under the stimulation of 40 M CCCP from 0 to 10 minutes. The plot at the right bottom shows the intensity ratio changes under different CCCP concentrations. F 0 is the average intensity at time of 0; F is the average intensity at various time. 13

15 0 min 2 min 4 min min 10 min F/F no CCCP CCCP 10 um CCCP 20 um CCCP 40 um Time (min) Figure S10. Typical time dependent fluorescence images of HeLa cells under the stimulation of 40 M CCCP from 0 to 10 minutes. The plot at the right bottom shows the intensity ratio changes under different CCCP concentrations. F 0 is the average intensity at time of 0; F is the average intensity at various times. 14

16 Figure S11. 1 H NMR of TAC-CHO in CDCl 3. 15

17 Figure S12. 1 H NMR of compound 3 in CDCl 3. 16

18 Figure S13. High resolution-mass spectrum (APCI ionization method) of compound 3. 17

19 Figure S14. 1 H NMR of KS6 in CDCl 3. 18

20 Figure S15. High Resolution mass spectrum (ESI ionization method) of KS6. 19