Supporting Information for

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1 Supporting Information for FRET-Based Mito-Specific Fluorescent Probe for Ratiometric Detection and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and Cy5 Xiaotong Jia, Qiangqiang Chen, Yingfang Yang, Yao Tang, Rui Wang, Yufang Xu, Xuhong Qian* State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, and Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Meilong Road 130H, Shanghai , China. Contents: 1. Materials and Instruments 2. Synthesis and Characterization of Compounds 3. Methods 3.1 Spectroscopic Materials and Methods 3.2 Generation of various ROSs 4. Cell culture and imaging 5. MTT Assay 6. Data 7. NMR, HRMS and MALDI-TOF spectra 8. Reference S1

2 1. Materials and Instruments Silica gel P60 (Qingdao) was used for column chromatography. All chemicals were purchased from TCI and J&K without further purification except especial instruction. All the organic solvents were of analytical grade. Water was purified by a Milli-Q system. All 1 H-NMR and 13 C-NMR spectra are acquired on a Bruker AV-400 spectrometer. Chemicals shifts are referenced to the residue solvent peaks and given in ppm. HRMS are acquired on a Micromass GCT spectrometer. 2. Synthesis and Characterization of Compounds Scheme S1. Synthesis of Cy3 S2

3 Scheme S2. Synthesis of Cy5 and PNCy3Cy5 Synthesis of compound 1 Phenylhydrazine (9.82 ml, 0.1 mol) and 3-methyl-2-butanone (12 ml, 0.11 mol) were dissolved in a mixture of anhydrous EtOH (30 ml) and CH3COOH (three drops). The solution was refluxed for 4 h, then allowed to cool to room temperature, and the solvent was removed under reduced pressure. To the resulting viscous residue was added CH3COOH (30 ml), and the solution was refluxed for 1 h under Ar atmosphere, then allowed to cool to room temperature, and removed most acetic acid under reduced pressure. The resulting viscous residue was washed with saturated aqueous solution of NaHCO3 to neutralize ph of the solution to 7.0. The resulting neutral mixture was extracted with CH2Cl2/H2O (1:1, v/v, 3 50 ml). The organic solution was dried with anhydrous Na2SO4 and concentrated to provide the crude product 1, which was purified by a column chromatography (silica gel, petroleum ether/ EtOAc, 40:3, v/v) to give compound 1 (12.7 g) as light yellow oil, which changed to red with time expands. Yield, 80%. 1 H NMR (400 MHz, CDCl3): δ 7.55 (d, J = 8.0 Hz, 1H), 7.26~7.29 (m, 2H), 7.20 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 1H), 2.28 (s, 3H), 1.29 (s, 6H); 13 C NMR (100 MHz, CDCl3): δ 15.18, 23.01, 53.54, , , , , , , ; GC-MS (EI): Calcd. For C11H13N [M] = ; Found, Synthesis of compound 2 S3

4 Compound 1 (4.773 g, 30 mmol) was dissolved in the mixture of ethyl iodide (24.11 ml, 300 mmol) and acetonitrile (60 ml). Under Ar, the solution was refluxed for 24 h, then allowed to cool to room temperature, and the resulting solid was collected by filtration. The solid was washed with acetone and recrystallized with EtOH to give compound 2 as yellow schistose crystal (4.246 g). Yield, 45%. 1 H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 4.0 Hz, 1H), 7.60~7.61 (m, 3H), 4.76 (q, J = 8.0 Hz, 2H), 3.16 (s, 3H), 1.62~1.66 (m, 9H). 13 C NMR (100 MHz, CDCl3): δ 13.70, 17.00, 23.10, 45.37, 54.66, , , , , , , LC-MS (ESI): Calcd. For m/z, C13H18N + I - [M-I] + = ; Found, Synthesis of compound 3 Phenylamine (18.3 ml, 200 mmol) and triethyl orthoformate (16.6 ml, 100 mmol) were refluxed for 12 h, then allowed to cool to room temperature, and the resulting solid was collected by filtration. The solid was washed with EtOH and recrystallized with EtOH to provide compound 3 as white needle-like crystal (14.7 g). Yield, 75%. 1 H NMR (400 MHz, CDCl3): δ (s, 1H), 8.33 (s, 1H), 7.38 (t, J = 8.0 Hz, 4H), 7.17 (t, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 4H). 13 C NMR (100 MHz, CDCl3): δ , , , , LC-MS (ESI): Calcd. For m/z, C13H12N2 [M+H] + = 197.1; Found, Synthesis of compound 4 Compound 2 (4.05 g, 12.8 mmol) and compound 3 (2.76 g, 14.1 mmol) were dissolved in CH3COOH (60 ml). Under Ar, the solution was refluxed for 4 h, then removed acetic acid under reduced pressure. The resulting residue was extracted with CH2Cl2/H2O (1:1, v/v, ml). The organic solution was dried with anhydrous Na2SO4 and concentrated to provide the crude product 4, which was purified by a column chromatography (silica gel, petroleum ether/ EtOAc, 2:1 and CH2Cl2/EtOAc, 3:1, v/v) to give compound 4 as light yellow scurf-like solid (1.38 g). Yield, 36%. 1 H NMR (400 MHz, DMSO-d6), δ (s, 1H), 8.70 (d, J = 12.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.46~7.59 (m, 6H), 7.29~7.37 (m, 2H), 6.27 (d, J = 16.0 Hz, 1H), 4.19 (d, J = 8.0 Hz, 2H), 1.72 (s, 6H), 1.36 (t, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 12.34, 28.10, 50.06, 91.33, , , 123.1, 126.5, , 130.3, , 141.5, 141.6, , HRMS (ESI): Calcd. For m/z, C20H23N2 + I - [M-I] + = ; Found, Synthesis of compound 5 S4

5 1-Boc-piperazine (0.558 g, 3 mmol) and anhydrous Na2CO3 (0.848 g, 8 mmol) were dissolved in the mixture of CH2Cl2 (10 ml) and deionized water (7 ml). The resulting solution was vigorously stirring at 0 o C for 20 min, then was added quickly with bromoacetyl bromide (0.568 ml, 6.6 mmol) in CH2Cl2 (10 ml) at o C. The resulting solution was added 4-Dimethylaminopyridine (DMAP) (36.7 mg, 0.3 mmol), then still vigorously stirring at 0 o C for 2 h, then stirring at room temperature for 1 h. The resulting residue was extracted with CH2Cl2/H2O (1:1, v/v, 3 20 ml). The organic solution was dried with anhydrous Na2SO4 and concentrated to provide the crude product 5, which was purified by a column chromatography (silica gel, CH2Cl2/MeOH, 60:1, v/v) to give compound 5 as white crystal (598 mg). Yield, 65%. 1 H NMR (400 MHz, CDCl3): δ 3.85 (s, 2H), 3.56 (t, J = 8.0 Hz, 2H), 3.47 (d, J = 4.0 Hz, 2H), 1.44 (s, 9H). 13 C NMR (100 MHz, CDCl3): δ 25.62, 28.35, 41.93, 46.55, 80.45, , GC-MS (EI): Calcd. For C11H19BrN2O3 [M] = ; Found, Synthesis of compound 6 Compound 1 (477 mg, 3 mmol) and compound 5 (1.38 g, 4.5 mmol) were dissolved in MeCN (15 ml). Under Ar, the solution was refluxed for 16 h. The resulting residue was concentrated under reduced pressure, then was added least CH2Cl2 to make sure the viscous residue dissolve entirely. The resulting solution was added an excess of ether to make sure all impurities were dissolved in ether, then removed most ether by dropper to make sure the resulting solid was isolated from air. The resulting mixture was added Ar to provide dry crude product 6 as light pink solid (700 mg, Yield, 50%), which was directly used for the next step without further purification. 1 H NMR (400 MHz, CDCl3): δ 7.53 (s, 4H), 6.37 (s, 2H), 3.45~3.89 (m, 8H), 2.98 (s, 3H), 1.65 (s, 6H), 1.47 (s, 9H). LC-MS (ESI): Calcd. For C22H32N3O3 + Br - [M-Br] + = ; Found, Synthesis of compound 7 Compound 4 (1.57 g, 3.76 mmol) and compound 6 (2.17 g, 4.67 mmol) were dissolved in the mixture of Ac2O (8 ml) and pyridine (8 ml). Under Ar, the solution was refluxed for 15 min, then allowed to cool to room temperature. The resulting residue was concentrated under reduced pressure, then purified by a column chromatography (silica gel, CH2Cl2/EtOAc, 1:10, v/v) to give compound 7 as red scurf-like solid (615 mg). Yield, 28%. 1 H NMR (400 MHz, CD3OD): δ 8.51 (t, J = 4.0 Hz, 1H), 7.54 (t, J = 4.0 Hz, 2H), 7.26~7.44 (m, 6H), 6.72 (d, J = 16.0 Hz, 1H), 6.60 S5

6 (d, J = 12.0 Hz, 1H), 5.28 (s, 2H), 4.24 (d, J = 8.0 Hz, 2H), 3.76 (d, J = 8.0 Hz, 4H), 3.60 (s, 2H), 3.51 (s, 2H), 1.79 (s, 6H), 1.73 (s, 6H), 1.49 (s, 9H), 1.40 (t, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, CD3OD): δ 11.68, 26.84, 27.20, 27.32, 39.31, 41.86, 44.66, 45.69, 49.20, 49.36, 80.30, , , , , , , , , , , , , , , , , , , HRMS (ESI): Calcd. For m/z, C36H47N4O3 +, ; Found, Synthesis of Cy3 To a solution of compound 7 (500 mg, 0.7 mmol) in anhydrous CH2Cl2 (10 ml) was added slowly the solution of CF3COOH in anhydrous CH2Cl2 (6 ml, 1:5, v/v) at room temperature. The mixture was stirred at room temperature for 20 h. The resulting residue was concentrated under reduced pressure, then purified by a column chromatography (silica gel, CH2Cl2/MeOH, 20:1, v/v) to give Cy3 as red scurf-like solid (300 mg). Yield, 70%. 1 H NMR (400 MHz, CD3OD): δ 8.57 (t, J = 12.0 Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.32~7.41 (m, 3H), 7.28 (t, J = 8.0 Hz, 2H), 5.27 (s, 2H), 4.25 (q, J = 8.0 Hz, 2H), 4.03 (s, 2H), 3.89 (s, 2H), 3.55 (s, 2H), 1.82 (s, 6H), 1.76 (s, 6H), 1.42 (t, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, CD3OD): δ 11.50, 26.74, 38.73, 39.21, 41.72, 42.79, 43.01, 45.27, 49.19, 49.43, , , , , , , , , , , , , , , , , , HRMS (ESI): Calcd. For m/z, C31H39N4O +, ; Found, Synthesis of compound 8 Compound 1 (1.59 g, 10 mmol) and 6-bromohexanoic acid (4.85 g, 25 mmol) were dissolved in 1,2-dichlorobenzene (10 ml). Under Ar, the solution was stirred at 110 o C for 16 h, then allowed to cool to room temperature. The resulting solid was dried under vacuum to provide the crude product (1.3 g, Yield, 37%) as brown solid, which was directly used for the next step without further purification. 1 H NMR (400 MHz, DMSO-d6): δ (s, 1H), 8.01 (d, J = 4.0 Hz, 1H), 7.86 (d, J = 4.0 Hz, 2H), 4.48 (t, J = 8.0 Hz, 2H), 2.88 (s, 3H), 2.22 (t, J = 8.0 Hz, 2H), 1.84 (t, J = 8.0 Hz, 2H), 1.54 (s, 8H), 1.44 (s, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 14.73, 22.49, 24.50, 25.86, 27.43, 33.86, 47.99, 54.64, , , , , , , , HRMS (ESI): C17H24NO2 + Br - [M-Br] + = ; Found, Synthesis of compound 9 S6

7 Compound 2 (6.3 g, 20 mmol) and Malonaldehyde dianilide hydrochloride (0.8 g, 3.1 mmol) was dissolved in CH3COOH (50 ml). Under Ar, the solution was refluxed for 4 h, then allowed to cool to room temperature. The resulting residue was concentrated under reduced pressure, then extracted with CH2Cl2/H2O (1:1, v/v, ml). The organic solution was dried with anhydrous Na2SO4 and concentrated to provide the crude product 9, which was purified by a column chromatography (silica gel, CH2Cl2/EtOAc, 1:10, v/v) to give compound 9 as black green scurf-like solid (1.678 g). Yield, 20%. 1 H NMR (400 MHz, DMSO-d6): δ (s, 1H), 8.87 (d, J = 12.0 Hz, 1H), 8.36 (t, J = 12.0 Hz, 1H), 7.62 (d, J = 4.0 Hz, 1H), 7.41~7.46 (m, 6H), 7.21~7.27 (m, 2H), 6.38~6.47 (m, 2H), 4.16 (q, J = 8.0 Hz, 2H), 1.67 (s, 6H), 1.27 (t, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 12.85, 27.58, 49.47, , , , , , , , , , , , , , HRMS (ESI): Calcd. For C22H25N2 + I - [M-I] + = ; Found, Synthesis of Cy5 Compound 9 (1.005 g, 2.2 mmol) and compound 8 (1 g, 2.8 mmol) were dissolved in the mixture of Ac2O (10 ml) and pyridine (10 ml). Under Ar, the solution was refluxed for 15 min. The resulting residue was concentrated under reduced pressure, then purified by a column chromatography (silica gel, CH2Cl2/MeOH, 30:1, v/v) to give Cy5 as blue scurf-like solid (466 mg). Yield, 42%. 1 H NMR (400 MHz, CD3OD): δ 8.25 (t, J = 16.0 Hz, 2H), 7.49 (dd, J1= 4.0 Hz, J2 = 8.0 Hz, 2H), 7.38~7.43 (m, 2H), 7.24~7.30 (m, 4H), 6.64 (t, J = 12.0 Hz, 1H), 6.30 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 2H), 4.15 (q, J = 8.0 Hz, 2H), 4.10 (t, J = 8.0 Hz, 2H), 2.25 (t, J = 4.0 Hz, 2H), 1.83 (t, J = 8.0 Hz, 2H), 1.72 (s, 12H), 1.69 (q, J = 8.0 Hz, 2H), 1.49 (t, J = 8.0 Hz, 2H), 1.38 (t, J = 4.0 Hz, 3H). 13 C NMR (100 MHz, CD3OD): δ 11.28, 25.06, 26.55, 26.65, 26.86, 35.28, 38.66, 43.52, 49.16, , , , , , , , , , , , , , , , , , , , HRMS (ESI): Calcd. For m/z, C33H40N2O2 [M+H] + = ; Found, Synthesis of compound PNCy3Cy5 Cy5 (253 mg, 0.51 mmol) and HBTU (580 mg, 1.53 mmol) were dissolved in anhydrous DMF (10 ml). To the solution was added simultaneously Cy3 (467 mg, 0.77 mmol) in anhydrous DMF (10 ml) and DIPEA (0.18 ml, 1 mmol) in anhydrous DMF (5 ml) at 0 o C under Ar atmosphere. The resulting solution was stirring for 6 h at 0 o C, then stirred overnight at room temperature. The resulting residue was S7

8 concentrated under reduced pressure, then extracted with CH2Cl2/H2O (1:1, v/v, 3 50 ml). The organic solution was dried with anhydrous Na2SO4 and concentrated to provide the crude product PNCy3Cy5, which was purified by a column chromatography (silica gel, CH2Cl2/MeOH, 100:1, v/v) to give compound PNCy3Cy5 as purple scurf-like solid (598 mg). Yield, 35%. 1 H NMR (400 MHz, CDCl3): δ 8.42 (t, J = 12.0 Hz, 1H), 7.94 (t, J = 12.0 Hz, 2H), 7.34~7.39 (m, 8H), 7.05~7.27 (m, 8H), 6.30 (s, 2H), 4.11 (q, J = 8.0 Hz, 2H), 4.03 (t, J = 8.0 Hz, 2H), 3.63~3.80 (m, 8H), 2.45 (q, J = 8.0 Hz, 2H), 1.85 (m, 2H), 1.77 (s, 6H), 1.72 (s, 5H), 1.70 (s, 11H), 1.54 (t, J = 8.0 Hz, 2H), 1.40 (q, J = 8.0 Hz, 6H). 13 C NMR (100 MHz, CDCl3): δ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 49.43, 49.40, 49.28, 49.24, 45.31, 44.86, 44.55, 44.08, 42.15, 40.93, 39.52, 39.15, 32.79, 32.53, 29.69, 28.27, 27.91, 27.86, 27.75, 27.71, 26.51, 24.86, 24.71, 12.43, P NMR (161 MHz, CDCl3): δ (sep, JPF = Hz, PF6 - ). 19 F NMR (365 MHz, DMSO-d6): δ (d, JPF = Hz, PF6 - ). HRMS (ESI): Calcd. For m/z, C64H78N6O22 2+, m/z = ; Found, Methods 3.1 Spectroscopic Materials and Methods All spectroscopic measurements were performed in 0.1 M phosphate buffer (ph 7.4, 0.2% DMF, v/v) at room temperature. Absorption spectra were recorded using a Varian Cary100 Bio UV-Visible spectrophotometer. Fluorescence spectra were recorded using a Varian Cary Eclipse scanning spectrofluorometer equipped with a Xenon flash lamp. Samples for absorption and fluorescence measurements were contained in 1 cm 1 cm quartz cuvettes (3.5 ml volume). 3.2 Generation of various ROSs 1) OONO - : Three kinds of solutions, including the mixture of hydrogen peroxide (0.7 M, 1.5 ml) and hydrochloric acid (0.6 M, 1.5 ml), solution of sodium nitrite (0.6 M, 3 ml) and solution of sodium hydroxide (1.5 M, 3 ml), were added simultaneously within 1s to make OONO - stock solution. S1 The resulting solution was stored at -20 o C. The concentration of the OONO - stock solution was determined in 0.1 M NaOH by measuring the absorbance at 302 nm with a molar extinction coefficient of 1670 M -1 cm -1. S8

9 2) ClO - : It was prepared by dilution of commercial NaClO solution in deionized water and the concentration of the ClO - stock solution was determined by measuring the absorbance at 209 nm with a molar extinction coefficient of 350 M -1 cm -1. 3) OH: It was generated in the Fenton system from ferrous ammonium sulfate and hydrogen peroxide. S2 4) H2O2: The stock H2O2 solution was purchased from Sigma-Aldrich. The concentration of the H2O2 stock solution was determined by measuring the absorbance at 240 nm with a molar extinction coefficient of 43.6 M -1 cm -1. 5) O2 - : Solid potassium superoxide was used as superoxide radical anion source. S3 6) ROO : A 10 mm stock solution of tbuooh was firstly prepared in deionized water and then added into the probe testing solutions. 7) 1 O2: NaMoO4 (20 mm) and H2O2 (20 mm) were prepared in deionized water. Equal aliquots of these two solutions were then mixed to yield 1 O2 of 10 mm. 8) H2S: A 10 mm stock solution of NaHS was firstly prepared in deionized water and then added into the probe testing solutions. 9) HSO3 - : A 10 mm stock solution of NaHSO3 was firstly prepared in deionized water and then added into the probe testing solutions. 10) SO3 2- : A 10 mm stock solution of Na2SO3 was firstly prepared in deionized water and then added into the probe testing solutions. 11) Cys: A 10 mm stock solution of Cystein was firstly prepared in deionized water and then added into the probe testing solutions. And it was stored at -20 o C. 12) Hcy: A 10 mm stock solution of Homocystein was firstly prepared in deionized water and then added into the probe testing solutions. And it was stored at -20 o C. 13) GSH: A 10 mm stock solution of Glutathione was firstly prepared in deionized water and then added into the probe testing solutions. And it was stored at -20 o C. 4. Cell Culture and Imaging RAW264.7 cells (macrophages cell line) were purchased from Stem Cell Bank, Chinese Academy of Sciences. Cells were seeded at a density of cells ml -1 for confocal imaging in DMEM Medium supplemented with 10% fetal bovine serum (FBS). Cultures were maintained at 37 under a humidified atmosphere containing 5% CO2. The cells were subcultured by scraping and seeding on 60 mm Petri-dishes according to the instructions from the manufacturer. Fluorescence imaging were acquired on Leica TCS SP8 confocal microscope. Colocalization expepriment: S9

10 (1) Mito: RAW264.7 were incubated with 10 μm PNCy3Cy5 for 30min, then 200 nm Mitotracker Green FM for 30min. Red Channel of PNCy3Cy5 was at nm with excitation at 633 nm. Green Channel of Mitotracker Green FM was at nm with excitation at 488 nm. (2) Lyso: RAW264.7 were incubated with 10 μm PNCy3Cy5 for 30min, then 200 nm Lysotracker Green DND-26 for 30min. Red Channel of PNCy3Cy5 was at nm with excitation at 633 nm. Green Channel of Lysotracker Green DND-26 was at nm with excitation at 488 nm. (3) Hoechst: RAW264.7 were incubated with 10 μm PNCy3Cy5 for 30min, then 5 μg/ml Hoechst for 30min. Red Channel of PNCy3Cy5 was at nm with excitation at 633 nm. Green Channel of 5 μg/ml Hoechst was at nm with excitation at 405 nm. 5. MTT assay MTT reduction assay was carried out to determine cell viability. RAW264.7 cells were cultured in 96-well plate at a density of cells/well and incubated for h prior to experimental treatments. Cells were exposed to different concentrations of PNCy3Cy5 or vehicle only. Cell viability was measured after incubation for 8 hours. MTT solution (5 mg/ml, 15 μl) was added to each well and continuously incubated for 4 h at 37 C. Then, MTT solution was removed and DMSO (150 μl) was added to each well to dissolve the dark blue formazan crystals. The absorbance was then measured at 570 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT). Cell viability was expressed as a percentage of MTT reduction with the untreated cells as 100%. Figure S1. Cytotoxity of PNCy3Cy5 in cultured RAW264.7 cells. S10

11 6. Data Figure S2. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with OONO -. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). Figure S3. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with ClO -. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). Figure S4. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with 1 O2. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). S11

12 Figure S5. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with H2O2. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). Figure S6. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with OH. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). Figure S7. The absorption spectra of a 2 μm solution of Cy3 (a) or Cy5 (b) after reaction with O2 -. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, 0.2% DMF, v/v). S12

13 Figure S8. The absorption and fluorescence emission spectra of PNCy3Cy5 with excitation at 530 nm. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, containing 0.2% DMF as co-solvent). Figure S9. (a) The absorption spectra of a 2 μm solution of PNCy3Cy5 after reaction with ClO -. (b) The fluorescence spectra of a 2 μm solution of PNCy3Cy5 with ClO - with excitation at 530 nm. Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, containing 0.2% DMF as co-solvent). Figure S10. Reaction-time profile of probe PNCy3Cy5 (2 μm) in the presence of 1-5 equiv OONO -. Fluorescence intensity was collected at 560 nm (with excitation at 530 nm). Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, containing 0.2% DMF as co-solvent). S13

14 Figure S11. Emission intensity ratio (F560/F660) of PNCy3Cy5 (2 μm) in the coexist of NO and OONO - or O2 - and OONO -. NO or O2 - were added with OONO - simultaneously into the solution of PNCy3Cy5 (2 μm). Data were collected at 25 o C in 0.1 M phosphate buffer (ph = 7.4, containing 0.2% DMF as co-solvent). Figure S12. Energy minimized (Gaussian 09, b3lyp module) structure of PNCy3Cy5. Calculated Ncy3-Ncy5 distance is 14.7 Å. Figure S13. Time-related real-time confocal fluorescence imaging of exogenous OONO - with PNCy3Cy5 in SIN-1 (1 mm) treated RAW264.7 cells. Green channel at nm, red channel at nm; Ratio images generated from green/red channel. S14

15 Figure S14. 1 H NMR of compound 1. Figure S C NMR of compound 1. S15

16 Figure S16. GC-MS of compound 1. S16

17 Figure S17. 1 H NMR of compound 2. Figure S C NMR of compound 2. S17

18 Figure S19. LC-MS of compound 2. S18

19 Figure S20. 1 H NMR of compound 3. Figure S C NMR of compound 3. S19

20 Figure S22. LC-MS of compound 3. Figure S23. 1 H NMR of compound 4. S20

21 Figure S C NMR of compound 4. Figure S25. HRMS of compound 4. S21

22 Figure S26. 1 H NMR of compound 5. Figure S C NMR of compound 5. S22

23 Figure S28. GC-MS of compound 5. S23

24 Figure S29. LC-MS of compound 6. Figure S30. 1 H NMR of compound 7. S24

25 Figure S C NMR of compound 7. Figure S32. HRMS of compound 7. S25

26 Figure S33. 1 H NMR of Cy3. Figure S C NMR of Cy3. S26

27 Figure S35. HRMS of Cy3. Figure S17. 1 H NMR of compound 3. Figure S36. 1 H NMR of compound 8. S27

28 Figure S C NMR of compound 8. Figure S38. HRMS of compound 8. S28

29 Figure S39. 1 H NMR of compound 9. Figure S C NMR of compound 9. S29

30 Figure S41. HRMS of compound 9. Figure S42. 1 H NMR of Cy5. S30

31 Figure S C NMR of Cy5. Figure S44. HRMS of Cy5. S31

32 Figure S45. 1 H NMR of PNCy3Cy5. Figure S C NMR of PNCy3Cy5. S32

33 Figure S F NMR of PNCy3Cy5. Figure S P NMR of PNCy3Cy5. S33

34 Figure S49. HRMS of PNCy3Cy5. S34

35 Figure S50. MALDI-TOF of PNCy3. S35

36 8. References S1. Peng, T.; Yang, D. Org. Lett. 2010, 12, S2 Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. J. Biol. Chem. 2003, 278, S3 Lippert, A. R.; New, E. J.; Chang, C. J. J. Am. Chem. Soc. 2011, 133, S36