Super-Resolution Monitoring of Mitochondrial Dynamics upon. Time-Gated Photo-Triggered Release of Nitric Oxide

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1 Supporting Information for Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide Haihong He a, Zhiwei Ye b, Yi Xiao b, *, Wei Yang b, *, Xuhong Qian a, Youjun Yang a, * (H. He and Z. Ye contributed equally to this work.) a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai, China b State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Liaoning, China, Page S2-S3 S4 S5 Content General Methods Scheme S1. The synthetic pathway of NOD550. Table S1. Crystal data and structure refinement for NOD550. Figure S1. Ortep drawings and the packing mode of NOD550 in its solid state. S6 Figure S2. EPR signal of a solution containing NOD550 (10 M) and PTIO (20 M) in aqueous phosphate buffer (50 mm, ph=7.4) with 20% DMSO upon photolysis with 365 nm light. Figure S3. Dark stability of NOD550 (10 M) without or with the presence of biological reductants. S7 S8 Figure S4. Time trace analysis of signal density during fission or fusion event marked with arrows in image a. Each color represented the fusion or fission event marked with the same color arrow in Figure 6. Figure S5. Time trace analysis of the distance between two mitochondria during fission or fusion event marked with the same color arrow in Figure 6. Figure S6. Overlay of the 1 H-NMR spectra of (A) NOD550, (B) 1 from UV-trigger decomposition of NOD550, (C) 1 from independent synthesis. S9-S12 S13 The 1 H-NMR, 13 C-NMR and HRMS characteristics of all compounds. Supporting References. S1

2 General Methods All chemicals9 and solvents were of analytical grades and used without further purification. All 1 H-NMR and 13 C-NMR spectra were collected with a Bruker AV-400 spectrometer. Chemicals shifts were referenced to the residue solvent peaks and given in unit of ppm. ESI-HRMS spectra were acquired on a TOF mass spectrometer. UV-Vis absorption spectra were collected on a SHIMADZU UV-2600 UV-vis spectrophotometer. Fluorescence emission spectra were collected on a PTI-QM4 steady-stead fluorimeter. Spectroscopic methods. UV-Vis absorption spectra were acquired over a SHIMADZU UV-2600 spectrophotometer. Fluorescence spectra were collected on a PTI-QM4 steady-state fluorimeter, equipped with a 75 W Xeon arc lamp and a model 810 type PMT. Voltage of the PMT was set to 950 V. All spectra were collected with a 1-cm quartz cuvette (3.4 ml). Molar absorptivity was calculated with the Beer-Lambert law with absorption spectra of dilute solutions of each compound (O.D. < 0.05). Fluorescence quantum yields were calculated following literature procedures. Rhodamine 6G with a fluorescence quantum yield of 0.77 in ethanol was used as the reference. 1 Synthetic scheme, procedures and characterizations. NOD550 was readily synthesized in a two-step cascade (Scheme S1). The X-ray crystal structures of NOD550 show that the oxygen atom of the nitroso group is tran to the ethyl group in the solid state (Figure S1). Yet, the NMR spectra indicates that both cis and trans conformations exist when they are in solution. Scheme S1. The synthetic pathway of NOD550. 3',6'-Bis(ethylamino)-2',7'-dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (1). 2 Rhodamine 6g (10.00 g, mmol) was dissolved in a combined solvent of ethanol and water (270 ml, v/v = 1:2),. After slowly addition of NaOH (16.70 g, mmol) into the mixture, the reaction was stirred for 4h at reflux temperature. The resulting mixture was cooled to room temperature and neutralized with 4 M HCl aqueous solution at 0 o C. After filtration, the residue was washed with water to afford a crude product, which was purified by recrystallization in ethanol and water to give compound as a red solid (8.40 g) in a 97% yield. 1 H-NMR (400 MHz, CD 3 OD, δ): 8.29 (d, J = 6.9 Hz, 1H), (q, J = 7.3 Hz, 2H), 7.35 (d, J = 6.7 Hz, 1H), 6.91 (s, 2H), 6.87 (s, 2H), (q, J = 6.8 Hz, 4H), 2.12(s, 6H), 1.34 (t, J = 6.9 Hz, 6H). N,N'-(2',7'-Dimethyl-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(N-ethy lnitrous amide) (NOD550). Compound 1 (1.00 g, 2.41 mmol) was dissolved in acetic acid (10 ml). After slowly addition of NaNO 2 (1.60 g, mmol) the solution was stirred at room temperature for 30 min. The resulting mixture was neutralized with saturated NaHCO 3 aqueous solution. After filtration the residue was washed with water to afford a crude product, which was purified by recrystallization in dichloromethane and petroleum ether to give S2

3 compound as a light yellow solid (1.10 g) in a 97% yield. 1 H-NMR (400 MHz, CDCl 3, δ): 8.11 (t, J = 7.6 Hz, 1H), (m, 2H), (t, J = 6.2 Hz, 1H), 7.22 (s, 1H), 7.19 (s, 0.3H), (m, 2+0.7H), (m, 1.3H), (m, 2.7H), 2.10 (d, 4H), 1.86 (d, 2H), (m, 2H), (m, 4H); 13 C-NMR (101 MHz, CDCl3, δ): 169.2, 169.2, 153.1, 149.3, 142.0, 139.6, 135.7, 132.0, 130.6, 130.6, 130.4, 130.4, 125.6, 125.5, 123.9, 119.5, 115.3, 115.2, 81.0, 76.7, 48.9, 41.8, 17.8, 17.4, 14.1, 11.4; HRMS (ESI) m/z: [M + Na] + calcd for C 26 H 24 N 4 O 5, ; found, S3

4 Table S1. Crystal data and structure refinement for NOD550. Identification code cd16088 Empirical formula C28 H27 Cl2 N3 O4 Formula weight Temperature 293(2) K Wavelength Å Crystal system Monoclinic Space group P 21/n Unit cell dimensions a = (13) Å a= 90. b = (3) Å b= (4). c = (2) Å g = 90. Volume (7) Å 3 Z 4 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 1128 Crystal size x x mm 3 Theta range for data collection to Index ranges -10<=h<=10, -18<=k<=24, -17<=l<=17 Reflections collected Independent reflections 4649 [R(int) = ] Completeness to theta = % Absorption correction Semi-empirical from equivalents Max. and min. transmission and Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 4649 / 12 / 354 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Extinction coefficient n/a Largest diff. peak and hole and e.å -3 S4

5 Figure S1. Ortep drawings and the packing mode of NOD550 in its solid state. S5

6 Normalized 550nm PTIO+NOD550 0min PTIO+NOD550 2min with 365nm PTIO+NOD550 10min with 365nm 1 mt Figure S2. EPR signal of a solution containing NOD550 (10 M) and PTIO (20 M) in aqueous phosphate buffer (50 mm, ph=7.4) with 20% DMSO upon photolysis with 365 nm light None 0h; 2. None 30min; 3. Resveratrol 30min; 4. Vc 30min; 5. Cystein 30min; 6. GSH 30min; 7. Tryptophan 30min. 8. After complete photolysis Figure S3. Dark stability of NOD550 (10 M) without or with the presence of biological reductants [ Ascorbic acid (10 mm), GSH (10 mm) and Cysteine (10 mm) in phosphate buffer (50 mm, ph = 7.4), Resveratrol (10 mm) and Tryptophan (10 mm) in DMSO]. The fluorescence intensity is collected at 550 nm. S6

7 To measure the signal density dynamics in Figure 6, each fusion or fission event was labeled with a same area region (smaller than the width of mitochondria). Then the mean signal density of the region was measured for each reconstructed image and then plotted vs time to provide the signal density change during fusion or fission events. The distance between mitochondria was also measured through the reconstructed PALM image sequences and plotted vs time. Figure S4. Time trace analysis of signal density during fission or fusion event marked with arrows in image a. Each color represented the fusion or fission event marked with the same color arrow in Figure 6. Figure S5. Time trace analysis of the distance between two mitochondria during fission or fusion event marked with the same color arrow in Figure 6. S7

8 Figure S6. Overlay of the 1 H-NMR spectra of (A) NOD550, (B) 1 from UV-trigger decomposition of NOD550, (C) 1 from independent synthesis. S8

9 Figure S7. The 1 H-NMR of compound 1 in CD 3 OD. S9

10 Figure S8. The 1 H-NMR of compound NOD550 in CDCl 3. S10

11 Figure S9. The 13 C-NMR of compound NOD550 in CDCl 3. S11

12 Figure S10. The HRMS of compound NOD550. S12

13 Supporting References 1. F. López Arbeloa, T. López Arbeloa, E. Gil Lage, I. López Arbeloa, F. C. De Schryver, Photochem. Photobiol. A: Chem. 1991, 56, H. Sasaki, K. Hanaoka, Y. Urano, T. Terai, T. Nagano, Bioorg. Med. Chem. 2010, 19, S13