Fluorescent Probe HKSOX-1 for Imaging and Detection of ...
Supporting Information
Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo Jun Jacob Hu,†,‡ Nai-Kei Wong,†,‡ Sen Ye,† Xingmiao Chen,ζ Ming-Yang Lu,† Angela Qian Zhao,† Yuhan Guo,§Alvin Chun-Hang Ma,§Anskar Yu-Hung Leung,§Jiangang Shen,ζ and Dan Yang*,† †
Morningside Laboratory for Chemical Biology and Department of Chemistry, School of Chinese Medicine, and §Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China ζ
*To whom correspondence should be addressed. E-mail: [email protected] ‡
These authors contributed equally to this work. Contents
Figure S1. Absorption and emission spectra of HKSOX-1 before and after treatment of superoxide
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Figure S2. Time course of fluorescence change in detecting superoxide with HKSOX-1
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Figure S3. Stability of HKSOX-1 toward pH changes
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Figure S4. Two-photon confocal imaging of superoxide with HKSOX-1r in RAW264.7 cells
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Figure S5. Confocal imaging of superoxide with HKSOX-1r in Hela cells
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Figure S6. Confocal imaging of superoxide induced by FCCP, antimycin A and oligomycin A with
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HKSOX-1r in RAW264.7 cells Figure S7. Performance of HKSOX-1r in FACS analysis
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Figure S8. Confocal imaging of mitochondrial superoxide with HKSOX-1m in THP-1 cells
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Figure S9. Confocal imaging of superoxide induced by zymosan in RAW264.7 cells
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Figure S10. Confocal imaging of superoxide induced by fMLP in RAW264.7 cells
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Figure S11. Confocal imaging of superoxide induced by cytoD in RAW264.7 cells
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Figure S12. Cytotoxicity of HKSOX-1, HKSOX-1r and HKSOX-1m
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Figure S13. Imaging of endogenous superoxide in intact live zebrafish embryos
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Figures S14–S36. NMR spectra
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Table S1. Fluorescence intensity of HKSOX-1 toward various analytes
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General Methods
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References
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1
Figure S1. Absorption and emission spectra of HKSOX-1 before and after treatment of superoxide. The probe (10 M) was dissolved in 0.1 M phosphate buffer at pH 7.4 (containing 0.1% DMF). Then, the probe solution was treated with 4 equiv O2 and the fluorescence intensity was monitored at the emission wavelength of 534 nm with an excitation of 509 nm.
Figure S2. Time course of fluorescence change in detecting superoxide with HKSOX-1. The probe (10 μM) was dissolved in 0.1 M phosphate buffer at pH 7.4 (containing 0.1% DMF). Then, the probe solution was treated with 10 equiv O2 and the fluorescence intensity was monitored at the emission wavelength of 534 nm with an excitation of 509 nm.
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Figure S3. Stability of HKSOX-1 toward pH changes. Fluorescence intensity of HKSOX-1 (10 μM) in 0.1 M potassium phosphate buffer (containing 0.1% DMF) at various pH (2–8.8) at 25 °C was recorded after 30 min. As a positive control, the fluorescence response of HKSOX-1 (10 μM) toward O2 (40 μM) generated by enzymatic reaction of xanthine (X) and xanthine oxidase (XO) was measured at 534 nm with an excitation at 509 nm.
Figure S4. Two-photon confocal imaging of O2 with HKSOX-1r. (left) Untreated RAW264.7 mouse macrophages incubated with probe alone (2 μM) for 30 min. (right) Cells co-incubated with probe and antimycin A (5 μM) for 30 min. Upper: fluorescence images; lower: bright field images. Scale bar 10 μm.
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Figure S5. Confocal imaging (single photosection) of O2 with HKSOX-1r in Hela cells. Cells were co-incubated with probe (2 μM), with or without antimycin A (5 μM) and increasing doses of mito-TEMPO (50, 100 and 300 μM) for 30 min. Scale bar 10 μm.
Figure S6. Confocal imaging of superoxide induced by FCCP, antimycin A and oligomycin A with HKSOX-1r in RAW264.7 cells. Representative images of RAW264.7 mouse macrophages co-incubated with HKSOX-1r (2 μM) and mitochondrial respiratory inhibitor FCCP (5 μM), antimycin A (5 μM) or oligomycin A (5 μM) for 30 min, before confocal imaging at high magnification (a; 189×) or lower magnification (b; 63×). Scale bar = 10 μm.
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Figure S7. Performance of HKSOX-1r in FACS analysis. RAW264.7 cells were co-incubated with HKSOX-1r (4 M) in the absence or presence of (a) antimycin A (0.05, 0.1, 0.5, 1, 5, and 10 M) or (b) PMA (0.1, 0.2, 0.5, and 1 g/mL) for 30 min. Results are representative of at least 3 independent experiments. The NOX inhibitor DPI (500 nM) was added to block superoxide production. Representative dot plots (left) and histograms (right) of RAW264.7 cell response, as reported by HKSOX-1r.
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Figure S8. Confocal imaging of mitochondrial superoxide with HKSOX-1m in differentiated human THP-1 cells. (left) Untreated THP-1 cells incubated with probe alone (10 μM) for 30 min. (right) Cells co-incubated with probe and antimycin A (1 μM) for 30 min. Upper: fluorescence images; lower: bright field images. Scale bar 10 μm. Single-photosection images were acquired on a Zeiss LSM 510 Meta confocal microscope, by using the following settings: Ex 514 nm (1% laser intensity), Em 527–559 nm (band-pass).
Figure S9. Confocal imaging of superoxide induced by zymosan in RAW264.7 cells. RAW264.7 cells were first preloaded with HKSOX-1r (2 M) for 30 min, and briefly challenged with zymosan (100 g/mL; 30 min) before confocal imaging. Local fluorescence maxima co-localized with intense superoxide production induced by phagocytosed zyamosan fragments but not non-internalized fragments. Scale bar 10 m.
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Figure S10. Confocal imaging of superoxide induced by fMLP in RAW264.7 cells. RAW264.7 cells were co-incubated with HKSOX-1r (2 M) and a dose gradient of fMLP (0.5 to 10 M) for 30 min, before confocal imaging at high magnification (189) (below). Relative mean fluorescence levels of cells in the same groups imaged in lower magnification (63) were quantified (above). Data are mean s.e.m., n = 4675 cells; , p 0.001 versus untreated cells. Scale bar 10 m.
Figure S11. Confocal imaging of superoxide induced by cytoD in RAW264.7 cells. RAW264.7 cells were co-incubated with HKSOX-1r (2 M) and a dose gradient of cytoD (0.1 to 10 M) for 30 min, before confocal imaging at high magnification (189) (below). Relative mean fluorescence levels of cells in the same groups imaged in lower magnification (63) were quantified (above). Data are mean s.e.m., n = 3457 cells; , p 0.001 versus untreated cells. Scale bar 10 m. 7
Figure S12. Cytotoxicity of HKSOX-1 and HKSOX-1r in RAW264.7 cells, and HKSOX-1m in THP-1 cells. (Left) HKSOX-1; (middle) HKSOX-1r; (right) HKSOX-1m. RAW264.7 and THP-1 cells were allowed to incubate with increasing concentrations of the probes overnight. The probes showed negligible or no cytotoxicity after 24-h incubation. Data represent mean s.e.m. for Cell-Titer Glo assays performed in triplicates.
Figure S13. Epifluorescence imaging of endogenous superoxide in intact live zebrafish embryos with HKSOX-1r. At 72 hpf, live zebrafish embryos were first preloaded with HKSOX-1r (10 M) for 20 min, and briefly challenged with PMA (200 ng/mL) in the absence or presence of DPI (100 nM; NOX inhibitor) for 15 min, before imaging at low magnification (4). Scale bar represents 250 m.
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Figure S14. 1H NMR spectrum of compound HKSOX-1.
Figure S15. 13C NMR spectrum of compound HKSOX-1. 9
Figure S16. 19F NMR spectrum of compound HKSOX-1.
Figure S17. 1H NMR spectrum of compound HKSOX-1r.
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Figure S18. 13C NMR spectrum of compound HKSOX-1r.
Figure S19. 19F NMR spectrum of compound HKSOX-1r.
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Figure S20. 1H NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
Figure S21. 13C NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
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Figure S22. 31P NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
Figure S23. 1H NMR spectrum of compound 3.
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Figure S24. 13C NMR spectrum of compound 3.
Figure S25. 31P NMR spectrum of compound 3.
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Figure S26. 1H NMR spectrum of compound 1.
Figure S27. 13C NMR spectrum of compound 1.
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Figure S28. 19F NMR spectrum of compound 1.
Figure S29. 1H NMR spectrum of compound 2.
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Figure S30. 13C NMR spectrum of compound 2.
Figure S31. 19F NMR spectrum of compound 2.
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Figure S32. 31P NMR spectrum of compound 2.
Figure S33. 1H NMR spectrum of compound HKSOX-1m.
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Figure S34. 13C NMR spectrum of compound HKSOX-1m.
Figure S35. 19F NMR spectrum of compound HKSOX-1m.
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Figure S36. 31P NMR spectrum of compound HKSOX-1m.
Table S1. Fluorescence Intensity of HKSOX-1 toward various analytes.
Entry Blank (probe only; 10 M) H2O2 (100 M) NO (100 M) 1O (100 M) 2 ROO (100 M) TBHP (100 M) OH (100 M) ONOO− (100 M) HOCl (100 M)
F.I. (au)
Entry
F.I. (au)
7.7 61.8 53.6 75.1 56.6 53.2 55.2 64.3 50.3
Fe2+
(100 M) ascorbic acid (AA; 100 M) 1,4-hydroquinone (HQ; 100 M) glutathione (GSH; 5 mM) esterase (0.4 U/mL)
60.6 61.6 62.6 76.3 54.5 5029 72.2 71.1 117.7
O2 (40 M) O2 (40 M) TEMPOL (40 μM) O2 (40 M) FeTMPyP (40 μM) O2 (40 M) SOD (40 U/mL)
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General Methods
Synthetic routes of probes HKSOX-1, HKSOX-1r and HKSOX-1m
HKSOX-1 To a solution of 5-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (220 mg, 0.49 mmol) in anhydrous pyridine (5 mL) and dry DCM (5 mL) at 78 °C was added triflic anhydride (Tf2O; 246 μL, 1.47 mmol) dropwise under argon atmosphere. The resulting mixture was stirred at −78 °C for 10 min and then at room temperature for another 10 min. Then, the reaction was quenched with saturated NaHCO3 aqueous solution at room temperature. The mixture was diluted with ethyl acetate (50 mL) and washed with 1 N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound HKSOX-1 was isolated as a white sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:9) as an eluent. Yield 143 mg (41%). 1H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.06 (s, 1H), 7.03 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 168.5, 167.6, 155.7, 151.9 (d, 1JC,F = 251.8 Hz), 145.5 (d, 1JC,F = 260.8 Hz), 138.3, 138.0 (dd, 2JC,F = 10.1 Hz, 4 JC,F = 2.5 Hz), 135.8, 128.6 (dd, 2JC,F = 18.3 Hz, 2JC,F = 13.1 Hz), 128.4, 127.1, 125.4, 121.6 (d, 3 JC,F = 7.4 Hz), 120.0 (q, 1JC,F = 319.9 Hz), 111.2 (dd, 2JC,F = 21.8 Hz, 4JC,F = 3.5 Hz), 79.9; 19F NMR (376 MHz, CDCl3) δ 74.8 (s, 6F), 130.0 (s, 2F), 143.0 (s, 2F); LRMS (EI, 20 eV) m/z (%) 711.9 (M+; 7), 149.0 (100); HRMS (EI): calcd for C23H6O11F10S2 (M+): 711.9192, found: 711.9200. HKSOX-1r
To a solution of HKSOX-1 (71 mg, 0.10 mmol) in anhydrous SOCl2 (2.5 mL) at room temperature was added anhydrous DMF (77 L, 0.001 mmol) under argon atmosphere. The resulting mixture was stirred under reflux for 1 h and then allowed to cool down to room temperature. The mixture was concentrated in vacuo to afford a crude residue. The crude acid chloride thus obtained was dissolved in dry THF (5 mL), and added with K2CO3 (35 mg, 0.25 mmol) and dimethyl iminodiacetate (24 mg, 0.15 mmol) under argon atmosphere. The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with ethyl acetate (25 mL) and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound HKSOX-1r was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:Hexane (v:v = 1:4) as an eluent. Yield 62 mg (72%). 1H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H), 7.90 (dd, J = 7.9, 1.3 Hz, 21
1H), 7.29 (d, J = 7.9 Hz, 1H), 6.67 (d, J = 1.6 Hz, 1H), 6.66 (d, J = 1.6 Hz, 1H), 4.35 (s, 2H), 4.17 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 169.7, 169.2, 169.0, 166.6, 152.3, 151.0 (d, 1JC,F = 255.0 Hz), 144.6 (d, 1JC,F = 263.8 Hz), 138.5, 136.5 (dd, 2JC,F = 10.3 Hz, 4 JC,F = 2.4 Hz), 135.4, 127.9 (dd, 2JC,F = 17.6 Hz, 2JC,F = 12.9 Hz), 125.2, 125.1, 124.3, 119.7 (d, 3 JC,F = 6.7 Hz), 118.6 (q, 1JC,F = 321.0 Hz), 109.3 (dd, 2JC,F = 21.4 Hz, 4JC,F = 3.6 Hz), 78.6, 53.1, 52.7, 51.8, 47.8; 19F NMR (376 MHz, CDCl3) δ 72.7 (t, J = 5.8 Hz, 6F), 126.0 (m, 2F), 138.5 (m, 2F); LRMS (EI, 20 eV) m/z (%) 855 (M+; 2), 695 (100); HRMS (EI): calcd for C29H15O14N1F10S2 (M+): 854.9774, found: 854.9784. HKSOX-1m
A solution of 1,4-dibromobutane (1.18 mL, 10.0 mmol) and triphenylphosphine (2.62 g, 10.0 mmol) in dry toluene (20.0 mL) was heated to reflux under argon atmosphere for 12 h. Then, the reaction was cooled down to room temperature, followed by filtration to give a white solid, which was subsequently washed with ethyl ether three times and dried in air to give (4-bromobutyl)triphenylphosphonium bromide [7333-63-3] (3.58 g; 75%) as a white sticky solid. 1H NMR (400 MHz, CDCl ) δ 7.76–7.56 (m, 15H), 3.77–3.61 (m, 2H), 3.38 (t, J = 6.1 Hz, 2H), 3 2.20–2.05 (m, 2H), 1.74–1.62 (m, 2H); 13C NMR (100 MHz, CDCl3) δ134.7 (d, 4JC,P = 2.5 Hz), 133.2 (d, 2JC,P = 9.4 Hz), 130.1 (d, 3JC,P = 12.8 Hz), 117.6 (d, 1JC,P = 86.0 Hz), 33.23, 31.6 (d, 2JC,P = 16.9 Hz), 21.2 (d, 1JC,P = 51.3 Hz), 20.4 (d, 3JC,P = 3.0 Hz); 31P NMR (162 MHz, CDCl3) δ 24.3; LRMS (ESI) m/z (%) 399.1 (M+; 100), 397.1 (M+; 98).
To a mixture of piperazine (516 mg, 6.0 mmol) and K2CO3 (524 mg, 4.0 mmol) in acetonitrile (50 mL) was added (4-bromobutyl)triphenylphosphonium bromide (956 mg, 2.0 mmol) in acetonitrile (20 mL) slowly at room temperature under argon atmosphere. Then, the resulting mixture was heated to reflux for 12 h. The mixture was allowed to cool to room temperature, diluted with ethyl acetate, and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give crude compound 3 (510 mg; 53%) as a white sticky solid. 1H NMR (400 MHz, CDCl ) δ 7.58–7.36 (m, 15H), 3.47–3.31 (m, 2H), 2.56–2.44 (m, 3H), 2.34 (s, 3 1H), 2.21–1.85 (m, 6H), 1.59–1.46 (m, 2H), 1.45–1.31 (m, 2H); 13C NMR (100 MHz, CDCl3) δ134.3 (d, 4JC,P = 2.5 Hz), 132.8 (d, 2JC,P = 10.1 Hz), 129.7 (d, 3JC,P = 12.3 Hz), 117.3 (d, 1JC,P = 85.9 Hz), 56.2, 53.3, 45.1, 25.3 (d, 2JC,P = 16.2 Hz), 21.1 (d, 1JC,P = 50.5 Hz), 19.3 (d, 3JC,P = 3.2 Hz); 31P NMR (162 MHz, CDCl3) δ 24.3; LRMS (ESI) m/z (%) 403.3 (M+; 20), 360.5 (100); HRMS (ESI): calcd for C26H32N2P (M+): 403.2303, found: 403.2302.
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(1) The mixture of trimellitic anhydride (88 mg, 0.46 mmol), 2,4-difluororesorcinol (134 mg, 0.92 mmol) in MeSO3H (3 mL) was heated to 120 °C under argon atmosphere for 2 h. Then, the resulting mixture was allowed to cool to room temperature and poured into cold water. A red precipitate was collected by vacuum filtration, washed by water, and dried in air to afford 5(6)-carboxy-2’,4’,5’,7’-tetrafluorofluorescein as a red solid. (2) To a solution of 5(6)-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (198 mg, 0.443 mmol) and DIPEA (0.366 mL, 2.22 mmol) in dry DCM (2 mL) was added chloromethyl methyl ether (0.168 mL, 2.22 mmol) dropwise at room temperature under argon atmosphere. The resulting mixture was stirred at room temperature for 12 h. Then the reaction mixture was diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The methoxymethyl protected product was isolated by flash chromatography on silica gel, by using MeOH: DCM (1: 99) as an eluent. (3) To a solution of the methoxymethyl protected product (180 mg, 0.31 mmol) in THF (6 mL) was added NaOH (124 mg, 3.10 mmol) in water (2 mL) dropwise at room temperature. The resulting mixture was stirred at room temperature for 1 h, diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 1 (165 mg; 80%) was isolated as a white sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:24) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 0.5H), 8.42 (d, J = 8.1 Hz, 0.5H), 8.39 (d, J = 8.0 Hz, 0.5H), 8.15 (d, J = 8.0 Hz, 0.5H), 7.88 (s, 0.5H), 7.29 (d, J = 8.1 Hz, 0.5H), 6.35 (t, J = 8.6 Hz, 2H), 5.23 (s, 4H), 3.60 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 169.1, 167.2, 156.0, 153.5, 151.8, 151.0, 146.7, 144.2, 137.2, 136.3, 132.4, 129.6, 128.0, 126.0, 125.6, 124.2, 113.6, 113.5, 108.4, 108.2, 99.0, 80.5, 57.4; 19F NMR (376.5 MHz, CDCl3) δ 130.6 (m, 2F), 145.3 (d, J = 6.2 Hz, 2F); LRMS (EI, 20 eV) m/z (%) 536.4 (M+; 72), 337.3 (100); HRMS (EI): calcd for C25H16F4O9 (M+): 536.0730, found: 536.0756.
To a solution of compound 1 (42 mg, 0.0789 mmol) in dry DCM (5 mL) was added EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; 29 mg, 0.118 mmol) at room temperature under argon atmosphere. After 15 min, a solution of compound 3 (46 mg, 0.0953 mmol) in dry DCM (2 mL) was added. The resulting solution was stirred at room temperature for 12 h, diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 2 (64 mg; 81%) 23
was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOH:DCM (v:v = 3:7) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 7.8 Hz, 0.5H), 7.99 (s, 0.5H), 7.92–7.54 (m, 16H), 7.21 (d, J = 7.9 Hz, 0.5H), 7.18 (s, 0.5H), 6.36 (d, J = 10.3 Hz, 2H), 5.19 (s, 4H), 4.03–3.81 (m, 2H), 3.78–3.68 (m, 1H), 3.68–3.62 (m, 1H), 3.56 (s, 6H), 3.46–3.36 (m, 1H), 3.30–3.16 (m, 1H), 2.74–2.32 (m, 6H), 2.07–1.79 (m, 2H), 1.73–1.57 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.8, 167.5, 153.4, 152.3, 152.2, 150.9, 146.5, 144.0, 143.2, 138.7, 134.9, 134.7, 133.7, 133.6, 130.4, 130.3, 129.4, 125.9, 125.6, 124.2, 122.6, 118.9, 118.0, 113.8, 108.3, 98.9, 80.1, 57.3, 56.5, 52.8, 26.6 (d, 2JC,P = 17.0 Hz), 22.0 (d, 1JC,P = 50.3 Hz), 20.2; 19F NMR (376.5 MHz, CDCl3) δ 130.5 (m, 2F), 145.6 (d, J = 15.4 Hz, 2F); 31P NMR (162 MHz, CDCl3) δ 24.6; LRMS (ESI) m/z (%) 921.3 (M+; 100), 877.3 (39); HRMS (ESI): calcd for C51H46F4N2O8P (M+): 921.2922, found: 921.2953.
(1) To a solution of compound 2 (64 mg, 0.0639 mmol) in anhydrous 1,4-dioxane (1 mL) was added 4 M HCl in 1,4-dioxane (1 mL) dropwise at room temperature for 30 min, followed by concentration in vacuo. (2) The crude product was dissolved in a mixture of dry DCM (2 mL) and anhydrous pyridine (2 mL), and allowed to cool down to 78 °C. Then, Tf2O (54 mg, 0.192 mmol) was added dropwise at 78 °C under argon atmosphere. The resulting mixture was stirred at –78 °C for 10 min and then at room temperature for another 10 min. The reaction mixture was diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. (3) Amberlite IRA-400 (Cl) was stirred in brine for 30 min, filtered and dried in air. To the crude product in MeOH (10 mL) at room temperature was added the pretreated Amberlite IRA-400 (Cl), and stirred for 30 min, followed by filtration. The filtrate was concentrated in vacuo. The target compound HKSOX-1m (58 mg; 78%) was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOH:DCM (v:v = 3:7) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.9 Hz, 0.5H), 8.07 (s, 0.5H), 7.85–7.66 (m, 16H), 7.29 (d, J = 7.6 Hz, 1H), 6.67 (dd, J = 9.0, 1.8 Hz, 2H), 3.75 (s, 1H), 3.68 (s, 1H), 3.50–3.37 (m, 3H), 3.32 (s, 1H), 2.60–2.44 (m, 6H), 1.88–1.77 (m, 2H), 1.73–1.62 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.48, 167.25, 166.84, 166.73, 152.10, 151.34, 149.57, 145.73, 143.75, 143.14, 139.51, 136.47, 135.38, 135.22, 135.20, 133.52, 133.41, 130.62, 130.50, 130.24, 126.64, 125.10, 124.98, 124.10, 123.29, 122.62, 120.10, 119.89, 119.83, 119.75, 119.68, 118.54, 118.50, 117.68, 117.65, 116.91, 109.45, 109.28, 109.08, 78.49, 56.39, 52.68, 52.46, 26.47 (d, 2JC,P = 16.6 Hz), 21.74 (d, 1JC,P = 51.3 Hz), 20.16 (d, 3JC,P = 3.1 Hz); 19F NMR (376.5 MHz, CDCl3) δ 73.1, 126.4, 126.6, 138.9, 139.3; 31P NMR (162 MHz, CDCl ) δ 24.1; LRMS (ESI) m/z (%) 1097 (M+; 100), 965 (11); HRMS (ESI): 3 calcd for C49H36F10N2O10PS2 (M+): 1097.1389, found: 1097.1401.
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Product analysis for the reaction of HKSOX-1 with KO2
Solid KO2 (7.1 mg, 0.1 mmol, 10 equiv) was added to a solution of HKSOX-1 (7.1 mg, 0.01 mmol) in 0.1 M potassium phosphate buffer pH 7.4 (0.1% DMF; 20 mL) at room temperature. The solution was stirred at room temperature for 10 min. The resulting mixture was diluted with ethyl acetate (10 mL) and washed with 1 N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 5-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (4.1 mg; 92%) was isolated as a red sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:9; with 1% AcOH) as an eluent. 1H NMR (400 MHz, CD OD) δ 8.65 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 3 6.41 (d, J = 1.7 Hz, 1H), 6.39 (d, J = 1.7 Hz, 1H). Compound characterization Xanthine, xanthine oxidase, SNP (sodium nitroferricyanide(III) dihydrate), 2,2’-azobis(2-amidinopropane)dihydrochloride, tert-butyl hydroperoxide solution, hydrogen peroxide solution, L-ascorbic acid, hydroquinone, glutathione (GSH) and esterase were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade and were purchased from Acros or Sigma-Aldrich. Potassium phosphate buffer was prepared by mixing aqueous solution of KHPO4 (1 M, 1.98 mL) and KH2PO4 (1 M, 8.02 mL) (final pH 7.4) followed by dilution with deionized water to 100 mL in a volumetric flask. NMR spectra were recorded in deuteriochloroform unless otherwise stated, with tetramethylsilane (TMS) as internal reference at ambient temperature, mainly on a Bruker Avance DPX 300 Fourier Transform Spectrometer operating at 300 MHz for 1H and at 75 MHz for 13C and Bruker Avance DPX 400 Fourier Transform Spectrometer operating at 400 MHz for 1H, 100 MHz for 13C, 376 MHz for 19F and 162 MHz for 31P. Mass spectra were recorded with a Thermo Scientific DFS High Resolution Magnetic Sector mass spectrometer for both low resolution and high resolution mass analysis Fluorometric analysis All fluorescence measurements were carried out at room temperature on a Hitachi F-7000 fluorescence spectrophotometer. The probe testing solutions were excited at 509 nm with the excitation and emission slit widths set at 2.5 nm. The emission spectrum was scanned from 520 to 700 nm at 1200 nm/min and the photomultiplier voltage was set at 700 V. The probe was dissolved in DMF to make a 10 mM stock solution, which was diluted to the required concentration of testing solution for measurement. Aliquots of analyte solutions were slowly added to probe testing solution (5 mL) with vigorous stirring at room temperature in the dark. The volume changes after addition of analyte solutions were less than 1%. The fluorescence intensities of the testing solutions were recorded after 30 min. Preparation of analyte solutions 25
ROO: Alkylperoxyl radical was generated from 2,2’-azobis(2-amidinopropane) dihydrochloride (10 mM), which was added into the testing solutions directly. 1O2: Singlet oxygen was generated from 3,3'-(naphthalene-1,4-diyl)dipropionic acid (10 mM). H2O2: H2O2 solution (10 mM) was added directly. TBHP: tert-Butyl hydroperoxide solution (10 mM) was added into the testing solutions directly. NO: Nitric oxide was generated from SNP (sodium nitroferricyanide(III) dihydrate) (10 mM). O2: Superoxide was generated from xanthine/xanthine oxidase system. Xanthine oxidase (0.01 U/mL) was added before addition of xanthine (30 mM). HOCl: NaOCl solution (10 mM) was added directly. OH: Hydroxyl radical was generated by Fenton reaction. To generate OH, ferrous chloride was added in the presence of 10 equiv of H2O2. The concentration of OH was equal to the Fe(II) concentration (10 mM). ONOO: Peroxynitrite solution was synthesized according to literature report.1 Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added within 1–2 s to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The resulting solution was split into small aliquots and stored at lower than −18 C. The aliquots were thawed immediately before use, and the concentration of peroxynitrite was determined by measuring the absorption of the solution at 302 nm. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH is 1670 M−1 cm−1 at 302 nm. Fe2+: Freshly prepared ferrous chloride solution (10 mM) was added directly. Vitamin C: Freshly prepared Vitamin C solution (10 mM) was added directly. HQ: Freshly prepared 1,4-dihydroquinone solution (10 mM) was added directly. GSH: Glutathione was added directly (5 mM). Esterase: Freshly prepared stock solution of esterase (40 U/mL) was added directly. Kinetic analysis of the reaction between HKSOX-1 and superoxide The rate constant for the reaction between HKSOX-1 and superoxide was calculated from competition experiments according to literature report.2 The rate constant between HKSOX-1 and superoxide was estimated according to the following equation:
V0 k SOD [SOD] 1 V k HKSOX-1[HKSOX - 1] The ratio between V and V0 corresponds to the rate of the fluorescent product formation in the presence and absence of superoxide scavenger SOD, respectively, where the values of V and V0 can be calculated from variations in fluorescence intensity of the fluorescent product over time. In this experiment, bovine Cu,Zn-SOD was used to dismutate superoxide. The rate constant used for the reaction between Cu,Zn-SOD and superoxide was 3 × 109 M-1 s-1. The rate constant between the superoxide and HKSOX-1 was estimated to be ca 2.0 × 105 M-1 s-1. Cell culture RAW264.7 cells, a mouse monocytic macrophage line, were obtained from ATCC (American Type Culture Collection), and maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum) and 1% penicillin/streptomycin. For regular confocal imaging, RAW264.7 cells at 80% confluence were harvested by scraping, washed with fresh medium and spun down (500 rpm in Eppendorf microfuge) for cell counting. Cells were then seeded into a 35-mm confocal culture dish (Mat-Tek) at a density of about 2×104 26
cells/mL in 2-mL seeding volume. BV-2 mouse microglia were obtained as a gift from Department of Pediatrics, University of Hong Kong, and maintained in the same manner as RAW264.7 cells. HCT116 human colon carcinoma cells were obtained from ATCC, and maintained in McCoy’s medium with serum and appropriate antibiotics. THP-1 cells, a human monocytic macrophage line, was obtained from ATCC, and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 55 M 2-mercaptoethanol. To achieve adherence, THP-1 cells were differentiated with a very low dose PMA (5 ng/mL) for 3 days before drug treatment. Cell imaging For overnight O2 induction, RAW264.7 cells were incubated in fresh medium in the presence of LPS (500 ng/mL) and IFN-γ (50 ng/mL) for 14 h. NADPH oxidase inhibitors were loaded into the medium throughout overnight treatment and probe incubation (30 min), whereas O2 scavenger FeTMPyP was added only during probe incubation. For acute O2 induction, mitochondrial respiratory inhibitors were added at specified doses to HBSS (supplemented with 0.6 mM L-arginine, and 0.01% chloramphenicol) and co-incubated with our fluorescent probe until imaging. Cells were typically incubated for 30 min at 37 C with 5% CO2. For co-staining experiments involving commercial organelle probes (MitoTracker Red and Hoechst 33342), the dyes were dissolved in HBSS together with our probe and loaded into the cells in confocal culture dish. During imaging, the dish was mounted onto a live cell imaging module (Axiovision). Single-photosection images were acquired on a Zeiss LSM 510 Meta confocal microscope, (unless otherwise specified) by using the following regular settings: Ex 514 nm (18% laser intensity), Em 527–559 nm (band-pass). For confocal imaging in two-photon mode, images were acquired with the following settings: Ex 730 nm (4% laser intensity), Em 527–559 nm (band-pass). Cytotoxicity assay To assess potential toxicity of our probes (HKSOX-1, HKSOX-1r and HKSOX-1m), RAW264.7 and THP-1 cells were seeded at 2105 cells/mL and 1106 cells/mL respectively in 100 L per well in a 96-well microplate (Corning), in DMEM or RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were allowed to attach for overnight. Probe stock solutions of different concentrations in DMF were diluted in fresh medium (HKSOX-1: 200 nM to 10 M; HKSOX-1r: 500 nM to 20 M; HKSOX-1m: 500 nM to 50 M) and added to the cells (100 L per well) in triplicates. After 24-h probe incubation, cells were loaded with 50-L Cell-Titer Glo reagent (Promega) and subjected to gentle shaking for cell lysis at room temperature (10 min). The microplate was mounted onto a DTX multimode plate reader (Molecular Devices) for luminescence detection, by using cellular ATP contents as a measure of cell viability. Data was collected for three separate serial dilutions and averaged. The cells viability was calculated according to the following equation: Cell viability (%) = 100 Awith probe / Acontrol. 96-well microplate fluorometric measurement RAW264.7 mouse macrophages were plated in 96-well microplates (Costar 3603; Corning), at a density of 5105 cells/mL (100 L/well in triplicates) one day before assay. The medium used was DMEM (Dulbecco’s modified Eagle medium; high glucose) supplemented with 10% 27
heat-inactivated FBS (fetal bovine serum) and 1% penicillin/streptomycin. For fluorometric measurement, all reagents and probe solutions were freshly dissolved in HBSS mix (HBSS supplemented with 0.6 mM L-arginine and 0.01% chloramphenicol). Before drug addition, cells were washed with PBS, and preloaded with 2 M HKSOX-1r in HBSS mix (50 L/well) for 30 min. Then, 50 L of 2 M HKSOX-1r together with 2 working doses of mitochondrial respiratory inhibitors (oligomycin A, FCCP, antimycin A) in HBSS mix was added, making up 100 L of probe and drug at their expected concentrations (1 working dose). At the end of 30-min co-incubation, drug gradients were removed by aspiration. Cells were briefly washed with PBS, and re-loaded with probe-free HBSS mix (100 L/well) before fluorescence reading on a DTX880 multimode detector (Beckman Coulter) with the following settings: excitation at 485 nm and emission at 535 nm, with an integration time of 400 ms. Data are presented, without subtracting background fluorescence, as mean s.e.m. for fluorometric measurement with replicates (n = 4) in at least three independent experiments. Flow cytometry analysis of superoxide production RAW264.7 cells were seeded into a 150-mm culture dish (Corning), cultured overnight for attachment and recovery, and grown to a required cell density (70-80% confluence or about 1.5107 cells). For harvest, cells were scraped off gently and collected into a clean 50-mL Falcon tube. Cells were spun down (500 rpm, room temperature, 3 min). After discarding the supernatant, 5 mL of warm HBSS was added gently to re-suspend the cell pellet. Then, cells were spun down (500 rpm, room temperature, 2 min). After this wash step, 2 mL of HBSS mix (HBSS supplemented with 0.6 mM L-arginine and 0.01% chloramphenicol) to re-suspend the cell pellet into single cells, followed by addition of another 6 mL HBSS mix. For superoxide detection, cells were co-incubated with 4 M probe HKSOX-1r in the absence or presence of antimycin A at different concentrations (0.05, 0.1, 0.5, 1, 5 and 10 M) in HBSS mix at 37 C for 30 min. The antioxidant NAC (10 mM) was added as a scavenger along with 1 M antimycin A for test of selectivity. Alternatively, cells were co-incubated with 4 M probe HKSOX-1r in the absence or presence of PMA at different concentrations (100, 200, 500, 1000 ng/mL) in HBSS mix at 37 C for 30 min. DPI (500 nM) was added to inhibit NOX-dependent superoxide production. Finally, superoxide levels were evaluated by measuring fluorescence intensity in FITC channel by using a flow cytometer (BD LSR Fortessa Analyzer). Zebrafish culture and imaging Zebrafish were maintained at 28 °C as described previously.3 Wild-type zebrafish were obtained from local fish farm. Embryos were obtained by natural spawning and were maintained in E3 zebrafish water at 28.5 °C and staged according to Kimmel et al.4 The study was conducted according to regulations by the Committee of the Use of Laboratory and Research Animals at The University of Hong Kong. At 72 hpf, zebrafish embryos were first preloaded with HKSOX-1r (10 M) for 20 min, and briefly challenged with PMA (200 ng/mL) or antimycin A (500 nM) for 15 min for O2 induction. DPI (100 nM) was optionally added to block off the effects of PMA. Confocal images were acquired with a Zeiss LSM 510 confocal microscope, by using the following settings: Ex 514 nm (18% laser intensity), Em 527–559 nm (band-pass). Other images by conventional epifluorescence microscopy were taken under Olympus IX70 (Olympus Corporation) and 10/0.3 NA objective in 3% methylcellulose, with Olympus DP71 (Olympus 28
Corporation) and Olympus DP-BSW basic Software. Bright-field images were taken under Nikon SMZ800 (Nikon Hong Kong Ltd) and P-Plan 1 objective in 3% methylcellulose with Nikon Digital Sight DS-Fi1 (Nikon Hong Kong Ltd.).
References
(1) Reed, J. W.; Ho, H. H.; Jolly, W. L. J. Am. Chem. Soc. 1974, 96, 1248–1249. (2) H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vasquez-Vivar, B. Kalyanaraman, Free Radic. Biol. Med. 2003, 34, 1359–1368. (3) Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. The University of Oregon Press, Eugene, Oregon, 1993. (4) Kimmel, D. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Dev. Dyn. 1995, 203, 253–310.
29
Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo Jun Jacob Hu,†,‡ Nai-Kei Wong,†,‡ Sen Ye,† Xingmiao Chen,ζ Ming-Yang Lu,† Angela Qian Zhao,† Yuhan Guo,§Alvin Chun-Hang Ma,§Anskar Yu-Hung Leung,§Jiangang Shen,ζ and Dan Yang*,† †
Morningside Laboratory for Chemical Biology and Department of Chemistry, School of Chinese Medicine, and §Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China ζ
*To whom correspondence should be addressed. E-mail: [email protected] ‡
These authors contributed equally to this work. Contents
Figure S1. Absorption and emission spectra of HKSOX-1 before and after treatment of superoxide
2
Figure S2. Time course of fluorescence change in detecting superoxide with HKSOX-1
2
Figure S3. Stability of HKSOX-1 toward pH changes
3
Figure S4. Two-photon confocal imaging of superoxide with HKSOX-1r in RAW264.7 cells
3
Figure S5. Confocal imaging of superoxide with HKSOX-1r in Hela cells
4
Figure S6. Confocal imaging of superoxide induced by FCCP, antimycin A and oligomycin A with
4
HKSOX-1r in RAW264.7 cells Figure S7. Performance of HKSOX-1r in FACS analysis
5
Figure S8. Confocal imaging of mitochondrial superoxide with HKSOX-1m in THP-1 cells
6
Figure S9. Confocal imaging of superoxide induced by zymosan in RAW264.7 cells
6
Figure S10. Confocal imaging of superoxide induced by fMLP in RAW264.7 cells
7
Figure S11. Confocal imaging of superoxide induced by cytoD in RAW264.7 cells
7
Figure S12. Cytotoxicity of HKSOX-1, HKSOX-1r and HKSOX-1m
8
Figure S13. Imaging of endogenous superoxide in intact live zebrafish embryos
8
Figures S14–S36. NMR spectra
9
Table S1. Fluorescence intensity of HKSOX-1 toward various analytes
20
General Methods
21
References
29
1
Figure S1. Absorption and emission spectra of HKSOX-1 before and after treatment of superoxide. The probe (10 M) was dissolved in 0.1 M phosphate buffer at pH 7.4 (containing 0.1% DMF). Then, the probe solution was treated with 4 equiv O2 and the fluorescence intensity was monitored at the emission wavelength of 534 nm with an excitation of 509 nm.
Figure S2. Time course of fluorescence change in detecting superoxide with HKSOX-1. The probe (10 μM) was dissolved in 0.1 M phosphate buffer at pH 7.4 (containing 0.1% DMF). Then, the probe solution was treated with 10 equiv O2 and the fluorescence intensity was monitored at the emission wavelength of 534 nm with an excitation of 509 nm.
2
Figure S3. Stability of HKSOX-1 toward pH changes. Fluorescence intensity of HKSOX-1 (10 μM) in 0.1 M potassium phosphate buffer (containing 0.1% DMF) at various pH (2–8.8) at 25 °C was recorded after 30 min. As a positive control, the fluorescence response of HKSOX-1 (10 μM) toward O2 (40 μM) generated by enzymatic reaction of xanthine (X) and xanthine oxidase (XO) was measured at 534 nm with an excitation at 509 nm.
Figure S4. Two-photon confocal imaging of O2 with HKSOX-1r. (left) Untreated RAW264.7 mouse macrophages incubated with probe alone (2 μM) for 30 min. (right) Cells co-incubated with probe and antimycin A (5 μM) for 30 min. Upper: fluorescence images; lower: bright field images. Scale bar 10 μm.
3
Figure S5. Confocal imaging (single photosection) of O2 with HKSOX-1r in Hela cells. Cells were co-incubated with probe (2 μM), with or without antimycin A (5 μM) and increasing doses of mito-TEMPO (50, 100 and 300 μM) for 30 min. Scale bar 10 μm.
Figure S6. Confocal imaging of superoxide induced by FCCP, antimycin A and oligomycin A with HKSOX-1r in RAW264.7 cells. Representative images of RAW264.7 mouse macrophages co-incubated with HKSOX-1r (2 μM) and mitochondrial respiratory inhibitor FCCP (5 μM), antimycin A (5 μM) or oligomycin A (5 μM) for 30 min, before confocal imaging at high magnification (a; 189×) or lower magnification (b; 63×). Scale bar = 10 μm.
4
Figure S7. Performance of HKSOX-1r in FACS analysis. RAW264.7 cells were co-incubated with HKSOX-1r (4 M) in the absence or presence of (a) antimycin A (0.05, 0.1, 0.5, 1, 5, and 10 M) or (b) PMA (0.1, 0.2, 0.5, and 1 g/mL) for 30 min. Results are representative of at least 3 independent experiments. The NOX inhibitor DPI (500 nM) was added to block superoxide production. Representative dot plots (left) and histograms (right) of RAW264.7 cell response, as reported by HKSOX-1r.
5
Figure S8. Confocal imaging of mitochondrial superoxide with HKSOX-1m in differentiated human THP-1 cells. (left) Untreated THP-1 cells incubated with probe alone (10 μM) for 30 min. (right) Cells co-incubated with probe and antimycin A (1 μM) for 30 min. Upper: fluorescence images; lower: bright field images. Scale bar 10 μm. Single-photosection images were acquired on a Zeiss LSM 510 Meta confocal microscope, by using the following settings: Ex 514 nm (1% laser intensity), Em 527–559 nm (band-pass).
Figure S9. Confocal imaging of superoxide induced by zymosan in RAW264.7 cells. RAW264.7 cells were first preloaded with HKSOX-1r (2 M) for 30 min, and briefly challenged with zymosan (100 g/mL; 30 min) before confocal imaging. Local fluorescence maxima co-localized with intense superoxide production induced by phagocytosed zyamosan fragments but not non-internalized fragments. Scale bar 10 m.
6
Figure S10. Confocal imaging of superoxide induced by fMLP in RAW264.7 cells. RAW264.7 cells were co-incubated with HKSOX-1r (2 M) and a dose gradient of fMLP (0.5 to 10 M) for 30 min, before confocal imaging at high magnification (189) (below). Relative mean fluorescence levels of cells in the same groups imaged in lower magnification (63) were quantified (above). Data are mean s.e.m., n = 4675 cells; , p 0.001 versus untreated cells. Scale bar 10 m.
Figure S11. Confocal imaging of superoxide induced by cytoD in RAW264.7 cells. RAW264.7 cells were co-incubated with HKSOX-1r (2 M) and a dose gradient of cytoD (0.1 to 10 M) for 30 min, before confocal imaging at high magnification (189) (below). Relative mean fluorescence levels of cells in the same groups imaged in lower magnification (63) were quantified (above). Data are mean s.e.m., n = 3457 cells; , p 0.001 versus untreated cells. Scale bar 10 m. 7
Figure S12. Cytotoxicity of HKSOX-1 and HKSOX-1r in RAW264.7 cells, and HKSOX-1m in THP-1 cells. (Left) HKSOX-1; (middle) HKSOX-1r; (right) HKSOX-1m. RAW264.7 and THP-1 cells were allowed to incubate with increasing concentrations of the probes overnight. The probes showed negligible or no cytotoxicity after 24-h incubation. Data represent mean s.e.m. for Cell-Titer Glo assays performed in triplicates.
Figure S13. Epifluorescence imaging of endogenous superoxide in intact live zebrafish embryos with HKSOX-1r. At 72 hpf, live zebrafish embryos were first preloaded with HKSOX-1r (10 M) for 20 min, and briefly challenged with PMA (200 ng/mL) in the absence or presence of DPI (100 nM; NOX inhibitor) for 15 min, before imaging at low magnification (4). Scale bar represents 250 m.
8
Figure S14. 1H NMR spectrum of compound HKSOX-1.
Figure S15. 13C NMR spectrum of compound HKSOX-1. 9
Figure S16. 19F NMR spectrum of compound HKSOX-1.
Figure S17. 1H NMR spectrum of compound HKSOX-1r.
10
Figure S18. 13C NMR spectrum of compound HKSOX-1r.
Figure S19. 19F NMR spectrum of compound HKSOX-1r.
11
Figure S20. 1H NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
Figure S21. 13C NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
12
Figure S22. 31P NMR spectrum of compound (4-bromobutyl)triphenylphosphonium bromide.
Figure S23. 1H NMR spectrum of compound 3.
13
Figure S24. 13C NMR spectrum of compound 3.
Figure S25. 31P NMR spectrum of compound 3.
14
Figure S26. 1H NMR spectrum of compound 1.
Figure S27. 13C NMR spectrum of compound 1.
15
Figure S28. 19F NMR spectrum of compound 1.
Figure S29. 1H NMR spectrum of compound 2.
16
Figure S30. 13C NMR spectrum of compound 2.
Figure S31. 19F NMR spectrum of compound 2.
17
Figure S32. 31P NMR spectrum of compound 2.
Figure S33. 1H NMR spectrum of compound HKSOX-1m.
18
Figure S34. 13C NMR spectrum of compound HKSOX-1m.
Figure S35. 19F NMR spectrum of compound HKSOX-1m.
19
Figure S36. 31P NMR spectrum of compound HKSOX-1m.
Table S1. Fluorescence Intensity of HKSOX-1 toward various analytes.
Entry Blank (probe only; 10 M) H2O2 (100 M) NO (100 M) 1O (100 M) 2 ROO (100 M) TBHP (100 M) OH (100 M) ONOO− (100 M) HOCl (100 M)
F.I. (au)
Entry
F.I. (au)
7.7 61.8 53.6 75.1 56.6 53.2 55.2 64.3 50.3
Fe2+
(100 M) ascorbic acid (AA; 100 M) 1,4-hydroquinone (HQ; 100 M) glutathione (GSH; 5 mM) esterase (0.4 U/mL)
60.6 61.6 62.6 76.3 54.5 5029 72.2 71.1 117.7
O2 (40 M) O2 (40 M) TEMPOL (40 μM) O2 (40 M) FeTMPyP (40 μM) O2 (40 M) SOD (40 U/mL)
20
General Methods
Synthetic routes of probes HKSOX-1, HKSOX-1r and HKSOX-1m
HKSOX-1 To a solution of 5-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (220 mg, 0.49 mmol) in anhydrous pyridine (5 mL) and dry DCM (5 mL) at 78 °C was added triflic anhydride (Tf2O; 246 μL, 1.47 mmol) dropwise under argon atmosphere. The resulting mixture was stirred at −78 °C for 10 min and then at room temperature for another 10 min. Then, the reaction was quenched with saturated NaHCO3 aqueous solution at room temperature. The mixture was diluted with ethyl acetate (50 mL) and washed with 1 N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound HKSOX-1 was isolated as a white sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:9) as an eluent. Yield 143 mg (41%). 1H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.06 (s, 1H), 7.03 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 168.5, 167.6, 155.7, 151.9 (d, 1JC,F = 251.8 Hz), 145.5 (d, 1JC,F = 260.8 Hz), 138.3, 138.0 (dd, 2JC,F = 10.1 Hz, 4 JC,F = 2.5 Hz), 135.8, 128.6 (dd, 2JC,F = 18.3 Hz, 2JC,F = 13.1 Hz), 128.4, 127.1, 125.4, 121.6 (d, 3 JC,F = 7.4 Hz), 120.0 (q, 1JC,F = 319.9 Hz), 111.2 (dd, 2JC,F = 21.8 Hz, 4JC,F = 3.5 Hz), 79.9; 19F NMR (376 MHz, CDCl3) δ 74.8 (s, 6F), 130.0 (s, 2F), 143.0 (s, 2F); LRMS (EI, 20 eV) m/z (%) 711.9 (M+; 7), 149.0 (100); HRMS (EI): calcd for C23H6O11F10S2 (M+): 711.9192, found: 711.9200. HKSOX-1r
To a solution of HKSOX-1 (71 mg, 0.10 mmol) in anhydrous SOCl2 (2.5 mL) at room temperature was added anhydrous DMF (77 L, 0.001 mmol) under argon atmosphere. The resulting mixture was stirred under reflux for 1 h and then allowed to cool down to room temperature. The mixture was concentrated in vacuo to afford a crude residue. The crude acid chloride thus obtained was dissolved in dry THF (5 mL), and added with K2CO3 (35 mg, 0.25 mmol) and dimethyl iminodiacetate (24 mg, 0.15 mmol) under argon atmosphere. The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with ethyl acetate (25 mL) and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound HKSOX-1r was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:Hexane (v:v = 1:4) as an eluent. Yield 62 mg (72%). 1H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H), 7.90 (dd, J = 7.9, 1.3 Hz, 21
1H), 7.29 (d, J = 7.9 Hz, 1H), 6.67 (d, J = 1.6 Hz, 1H), 6.66 (d, J = 1.6 Hz, 1H), 4.35 (s, 2H), 4.17 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 169.7, 169.2, 169.0, 166.6, 152.3, 151.0 (d, 1JC,F = 255.0 Hz), 144.6 (d, 1JC,F = 263.8 Hz), 138.5, 136.5 (dd, 2JC,F = 10.3 Hz, 4 JC,F = 2.4 Hz), 135.4, 127.9 (dd, 2JC,F = 17.6 Hz, 2JC,F = 12.9 Hz), 125.2, 125.1, 124.3, 119.7 (d, 3 JC,F = 6.7 Hz), 118.6 (q, 1JC,F = 321.0 Hz), 109.3 (dd, 2JC,F = 21.4 Hz, 4JC,F = 3.6 Hz), 78.6, 53.1, 52.7, 51.8, 47.8; 19F NMR (376 MHz, CDCl3) δ 72.7 (t, J = 5.8 Hz, 6F), 126.0 (m, 2F), 138.5 (m, 2F); LRMS (EI, 20 eV) m/z (%) 855 (M+; 2), 695 (100); HRMS (EI): calcd for C29H15O14N1F10S2 (M+): 854.9774, found: 854.9784. HKSOX-1m
A solution of 1,4-dibromobutane (1.18 mL, 10.0 mmol) and triphenylphosphine (2.62 g, 10.0 mmol) in dry toluene (20.0 mL) was heated to reflux under argon atmosphere for 12 h. Then, the reaction was cooled down to room temperature, followed by filtration to give a white solid, which was subsequently washed with ethyl ether three times and dried in air to give (4-bromobutyl)triphenylphosphonium bromide [7333-63-3] (3.58 g; 75%) as a white sticky solid. 1H NMR (400 MHz, CDCl ) δ 7.76–7.56 (m, 15H), 3.77–3.61 (m, 2H), 3.38 (t, J = 6.1 Hz, 2H), 3 2.20–2.05 (m, 2H), 1.74–1.62 (m, 2H); 13C NMR (100 MHz, CDCl3) δ134.7 (d, 4JC,P = 2.5 Hz), 133.2 (d, 2JC,P = 9.4 Hz), 130.1 (d, 3JC,P = 12.8 Hz), 117.6 (d, 1JC,P = 86.0 Hz), 33.23, 31.6 (d, 2JC,P = 16.9 Hz), 21.2 (d, 1JC,P = 51.3 Hz), 20.4 (d, 3JC,P = 3.0 Hz); 31P NMR (162 MHz, CDCl3) δ 24.3; LRMS (ESI) m/z (%) 399.1 (M+; 100), 397.1 (M+; 98).
To a mixture of piperazine (516 mg, 6.0 mmol) and K2CO3 (524 mg, 4.0 mmol) in acetonitrile (50 mL) was added (4-bromobutyl)triphenylphosphonium bromide (956 mg, 2.0 mmol) in acetonitrile (20 mL) slowly at room temperature under argon atmosphere. Then, the resulting mixture was heated to reflux for 12 h. The mixture was allowed to cool to room temperature, diluted with ethyl acetate, and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give crude compound 3 (510 mg; 53%) as a white sticky solid. 1H NMR (400 MHz, CDCl ) δ 7.58–7.36 (m, 15H), 3.47–3.31 (m, 2H), 2.56–2.44 (m, 3H), 2.34 (s, 3 1H), 2.21–1.85 (m, 6H), 1.59–1.46 (m, 2H), 1.45–1.31 (m, 2H); 13C NMR (100 MHz, CDCl3) δ134.3 (d, 4JC,P = 2.5 Hz), 132.8 (d, 2JC,P = 10.1 Hz), 129.7 (d, 3JC,P = 12.3 Hz), 117.3 (d, 1JC,P = 85.9 Hz), 56.2, 53.3, 45.1, 25.3 (d, 2JC,P = 16.2 Hz), 21.1 (d, 1JC,P = 50.5 Hz), 19.3 (d, 3JC,P = 3.2 Hz); 31P NMR (162 MHz, CDCl3) δ 24.3; LRMS (ESI) m/z (%) 403.3 (M+; 20), 360.5 (100); HRMS (ESI): calcd for C26H32N2P (M+): 403.2303, found: 403.2302.
22
(1) The mixture of trimellitic anhydride (88 mg, 0.46 mmol), 2,4-difluororesorcinol (134 mg, 0.92 mmol) in MeSO3H (3 mL) was heated to 120 °C under argon atmosphere for 2 h. Then, the resulting mixture was allowed to cool to room temperature and poured into cold water. A red precipitate was collected by vacuum filtration, washed by water, and dried in air to afford 5(6)-carboxy-2’,4’,5’,7’-tetrafluorofluorescein as a red solid. (2) To a solution of 5(6)-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (198 mg, 0.443 mmol) and DIPEA (0.366 mL, 2.22 mmol) in dry DCM (2 mL) was added chloromethyl methyl ether (0.168 mL, 2.22 mmol) dropwise at room temperature under argon atmosphere. The resulting mixture was stirred at room temperature for 12 h. Then the reaction mixture was diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The methoxymethyl protected product was isolated by flash chromatography on silica gel, by using MeOH: DCM (1: 99) as an eluent. (3) To a solution of the methoxymethyl protected product (180 mg, 0.31 mmol) in THF (6 mL) was added NaOH (124 mg, 3.10 mmol) in water (2 mL) dropwise at room temperature. The resulting mixture was stirred at room temperature for 1 h, diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 1 (165 mg; 80%) was isolated as a white sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:24) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 0.5H), 8.42 (d, J = 8.1 Hz, 0.5H), 8.39 (d, J = 8.0 Hz, 0.5H), 8.15 (d, J = 8.0 Hz, 0.5H), 7.88 (s, 0.5H), 7.29 (d, J = 8.1 Hz, 0.5H), 6.35 (t, J = 8.6 Hz, 2H), 5.23 (s, 4H), 3.60 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 169.1, 167.2, 156.0, 153.5, 151.8, 151.0, 146.7, 144.2, 137.2, 136.3, 132.4, 129.6, 128.0, 126.0, 125.6, 124.2, 113.6, 113.5, 108.4, 108.2, 99.0, 80.5, 57.4; 19F NMR (376.5 MHz, CDCl3) δ 130.6 (m, 2F), 145.3 (d, J = 6.2 Hz, 2F); LRMS (EI, 20 eV) m/z (%) 536.4 (M+; 72), 337.3 (100); HRMS (EI): calcd for C25H16F4O9 (M+): 536.0730, found: 536.0756.
To a solution of compound 1 (42 mg, 0.0789 mmol) in dry DCM (5 mL) was added EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; 29 mg, 0.118 mmol) at room temperature under argon atmosphere. After 15 min, a solution of compound 3 (46 mg, 0.0953 mmol) in dry DCM (2 mL) was added. The resulting solution was stirred at room temperature for 12 h, diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 2 (64 mg; 81%) 23
was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOH:DCM (v:v = 3:7) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 7.8 Hz, 0.5H), 7.99 (s, 0.5H), 7.92–7.54 (m, 16H), 7.21 (d, J = 7.9 Hz, 0.5H), 7.18 (s, 0.5H), 6.36 (d, J = 10.3 Hz, 2H), 5.19 (s, 4H), 4.03–3.81 (m, 2H), 3.78–3.68 (m, 1H), 3.68–3.62 (m, 1H), 3.56 (s, 6H), 3.46–3.36 (m, 1H), 3.30–3.16 (m, 1H), 2.74–2.32 (m, 6H), 2.07–1.79 (m, 2H), 1.73–1.57 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.8, 167.5, 153.4, 152.3, 152.2, 150.9, 146.5, 144.0, 143.2, 138.7, 134.9, 134.7, 133.7, 133.6, 130.4, 130.3, 129.4, 125.9, 125.6, 124.2, 122.6, 118.9, 118.0, 113.8, 108.3, 98.9, 80.1, 57.3, 56.5, 52.8, 26.6 (d, 2JC,P = 17.0 Hz), 22.0 (d, 1JC,P = 50.3 Hz), 20.2; 19F NMR (376.5 MHz, CDCl3) δ 130.5 (m, 2F), 145.6 (d, J = 15.4 Hz, 2F); 31P NMR (162 MHz, CDCl3) δ 24.6; LRMS (ESI) m/z (%) 921.3 (M+; 100), 877.3 (39); HRMS (ESI): calcd for C51H46F4N2O8P (M+): 921.2922, found: 921.2953.
(1) To a solution of compound 2 (64 mg, 0.0639 mmol) in anhydrous 1,4-dioxane (1 mL) was added 4 M HCl in 1,4-dioxane (1 mL) dropwise at room temperature for 30 min, followed by concentration in vacuo. (2) The crude product was dissolved in a mixture of dry DCM (2 mL) and anhydrous pyridine (2 mL), and allowed to cool down to 78 °C. Then, Tf2O (54 mg, 0.192 mmol) was added dropwise at 78 °C under argon atmosphere. The resulting mixture was stirred at –78 °C for 10 min and then at room temperature for another 10 min. The reaction mixture was diluted with ethyl acetate, and washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. (3) Amberlite IRA-400 (Cl) was stirred in brine for 30 min, filtered and dried in air. To the crude product in MeOH (10 mL) at room temperature was added the pretreated Amberlite IRA-400 (Cl), and stirred for 30 min, followed by filtration. The filtrate was concentrated in vacuo. The target compound HKSOX-1m (58 mg; 78%) was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOH:DCM (v:v = 3:7) as an eluent. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.9 Hz, 0.5H), 8.07 (s, 0.5H), 7.85–7.66 (m, 16H), 7.29 (d, J = 7.6 Hz, 1H), 6.67 (dd, J = 9.0, 1.8 Hz, 2H), 3.75 (s, 1H), 3.68 (s, 1H), 3.50–3.37 (m, 3H), 3.32 (s, 1H), 2.60–2.44 (m, 6H), 1.88–1.77 (m, 2H), 1.73–1.62 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.48, 167.25, 166.84, 166.73, 152.10, 151.34, 149.57, 145.73, 143.75, 143.14, 139.51, 136.47, 135.38, 135.22, 135.20, 133.52, 133.41, 130.62, 130.50, 130.24, 126.64, 125.10, 124.98, 124.10, 123.29, 122.62, 120.10, 119.89, 119.83, 119.75, 119.68, 118.54, 118.50, 117.68, 117.65, 116.91, 109.45, 109.28, 109.08, 78.49, 56.39, 52.68, 52.46, 26.47 (d, 2JC,P = 16.6 Hz), 21.74 (d, 1JC,P = 51.3 Hz), 20.16 (d, 3JC,P = 3.1 Hz); 19F NMR (376.5 MHz, CDCl3) δ 73.1, 126.4, 126.6, 138.9, 139.3; 31P NMR (162 MHz, CDCl ) δ 24.1; LRMS (ESI) m/z (%) 1097 (M+; 100), 965 (11); HRMS (ESI): 3 calcd for C49H36F10N2O10PS2 (M+): 1097.1389, found: 1097.1401.
24
Product analysis for the reaction of HKSOX-1 with KO2
Solid KO2 (7.1 mg, 0.1 mmol, 10 equiv) was added to a solution of HKSOX-1 (7.1 mg, 0.01 mmol) in 0.1 M potassium phosphate buffer pH 7.4 (0.1% DMF; 20 mL) at room temperature. The solution was stirred at room temperature for 10 min. The resulting mixture was diluted with ethyl acetate (10 mL) and washed with 1 N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The target compound 5-carboxy-2’,4’,5’,7’-tetrafluorofluorescein (4.1 mg; 92%) was isolated as a red sticky solid by flash chromatography on silica gel, by using MeOH:DCM (v:v = 1:9; with 1% AcOH) as an eluent. 1H NMR (400 MHz, CD OD) δ 8.65 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 3 6.41 (d, J = 1.7 Hz, 1H), 6.39 (d, J = 1.7 Hz, 1H). Compound characterization Xanthine, xanthine oxidase, SNP (sodium nitroferricyanide(III) dihydrate), 2,2’-azobis(2-amidinopropane)dihydrochloride, tert-butyl hydroperoxide solution, hydrogen peroxide solution, L-ascorbic acid, hydroquinone, glutathione (GSH) and esterase were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade and were purchased from Acros or Sigma-Aldrich. Potassium phosphate buffer was prepared by mixing aqueous solution of KHPO4 (1 M, 1.98 mL) and KH2PO4 (1 M, 8.02 mL) (final pH 7.4) followed by dilution with deionized water to 100 mL in a volumetric flask. NMR spectra were recorded in deuteriochloroform unless otherwise stated, with tetramethylsilane (TMS) as internal reference at ambient temperature, mainly on a Bruker Avance DPX 300 Fourier Transform Spectrometer operating at 300 MHz for 1H and at 75 MHz for 13C and Bruker Avance DPX 400 Fourier Transform Spectrometer operating at 400 MHz for 1H, 100 MHz for 13C, 376 MHz for 19F and 162 MHz for 31P. Mass spectra were recorded with a Thermo Scientific DFS High Resolution Magnetic Sector mass spectrometer for both low resolution and high resolution mass analysis Fluorometric analysis All fluorescence measurements were carried out at room temperature on a Hitachi F-7000 fluorescence spectrophotometer. The probe testing solutions were excited at 509 nm with the excitation and emission slit widths set at 2.5 nm. The emission spectrum was scanned from 520 to 700 nm at 1200 nm/min and the photomultiplier voltage was set at 700 V. The probe was dissolved in DMF to make a 10 mM stock solution, which was diluted to the required concentration of testing solution for measurement. Aliquots of analyte solutions were slowly added to probe testing solution (5 mL) with vigorous stirring at room temperature in the dark. The volume changes after addition of analyte solutions were less than 1%. The fluorescence intensities of the testing solutions were recorded after 30 min. Preparation of analyte solutions 25
ROO: Alkylperoxyl radical was generated from 2,2’-azobis(2-amidinopropane) dihydrochloride (10 mM), which was added into the testing solutions directly. 1O2: Singlet oxygen was generated from 3,3'-(naphthalene-1,4-diyl)dipropionic acid (10 mM). H2O2: H2O2 solution (10 mM) was added directly. TBHP: tert-Butyl hydroperoxide solution (10 mM) was added into the testing solutions directly. NO: Nitric oxide was generated from SNP (sodium nitroferricyanide(III) dihydrate) (10 mM). O2: Superoxide was generated from xanthine/xanthine oxidase system. Xanthine oxidase (0.01 U/mL) was added before addition of xanthine (30 mM). HOCl: NaOCl solution (10 mM) was added directly. OH: Hydroxyl radical was generated by Fenton reaction. To generate OH, ferrous chloride was added in the presence of 10 equiv of H2O2. The concentration of OH was equal to the Fe(II) concentration (10 mM). ONOO: Peroxynitrite solution was synthesized according to literature report.1 Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added within 1–2 s to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The resulting solution was split into small aliquots and stored at lower than −18 C. The aliquots were thawed immediately before use, and the concentration of peroxynitrite was determined by measuring the absorption of the solution at 302 nm. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH is 1670 M−1 cm−1 at 302 nm. Fe2+: Freshly prepared ferrous chloride solution (10 mM) was added directly. Vitamin C: Freshly prepared Vitamin C solution (10 mM) was added directly. HQ: Freshly prepared 1,4-dihydroquinone solution (10 mM) was added directly. GSH: Glutathione was added directly (5 mM). Esterase: Freshly prepared stock solution of esterase (40 U/mL) was added directly. Kinetic analysis of the reaction between HKSOX-1 and superoxide The rate constant for the reaction between HKSOX-1 and superoxide was calculated from competition experiments according to literature report.2 The rate constant between HKSOX-1 and superoxide was estimated according to the following equation:
V0 k SOD [SOD] 1 V k HKSOX-1[HKSOX - 1] The ratio between V and V0 corresponds to the rate of the fluorescent product formation in the presence and absence of superoxide scavenger SOD, respectively, where the values of V and V0 can be calculated from variations in fluorescence intensity of the fluorescent product over time. In this experiment, bovine Cu,Zn-SOD was used to dismutate superoxide. The rate constant used for the reaction between Cu,Zn-SOD and superoxide was 3 × 109 M-1 s-1. The rate constant between the superoxide and HKSOX-1 was estimated to be ca 2.0 × 105 M-1 s-1. Cell culture RAW264.7 cells, a mouse monocytic macrophage line, were obtained from ATCC (American Type Culture Collection), and maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum) and 1% penicillin/streptomycin. For regular confocal imaging, RAW264.7 cells at 80% confluence were harvested by scraping, washed with fresh medium and spun down (500 rpm in Eppendorf microfuge) for cell counting. Cells were then seeded into a 35-mm confocal culture dish (Mat-Tek) at a density of about 2×104 26
cells/mL in 2-mL seeding volume. BV-2 mouse microglia were obtained as a gift from Department of Pediatrics, University of Hong Kong, and maintained in the same manner as RAW264.7 cells. HCT116 human colon carcinoma cells were obtained from ATCC, and maintained in McCoy’s medium with serum and appropriate antibiotics. THP-1 cells, a human monocytic macrophage line, was obtained from ATCC, and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 55 M 2-mercaptoethanol. To achieve adherence, THP-1 cells were differentiated with a very low dose PMA (5 ng/mL) for 3 days before drug treatment. Cell imaging For overnight O2 induction, RAW264.7 cells were incubated in fresh medium in the presence of LPS (500 ng/mL) and IFN-γ (50 ng/mL) for 14 h. NADPH oxidase inhibitors were loaded into the medium throughout overnight treatment and probe incubation (30 min), whereas O2 scavenger FeTMPyP was added only during probe incubation. For acute O2 induction, mitochondrial respiratory inhibitors were added at specified doses to HBSS (supplemented with 0.6 mM L-arginine, and 0.01% chloramphenicol) and co-incubated with our fluorescent probe until imaging. Cells were typically incubated for 30 min at 37 C with 5% CO2. For co-staining experiments involving commercial organelle probes (MitoTracker Red and Hoechst 33342), the dyes were dissolved in HBSS together with our probe and loaded into the cells in confocal culture dish. During imaging, the dish was mounted onto a live cell imaging module (Axiovision). Single-photosection images were acquired on a Zeiss LSM 510 Meta confocal microscope, (unless otherwise specified) by using the following regular settings: Ex 514 nm (18% laser intensity), Em 527–559 nm (band-pass). For confocal imaging in two-photon mode, images were acquired with the following settings: Ex 730 nm (4% laser intensity), Em 527–559 nm (band-pass). Cytotoxicity assay To assess potential toxicity of our probes (HKSOX-1, HKSOX-1r and HKSOX-1m), RAW264.7 and THP-1 cells were seeded at 2105 cells/mL and 1106 cells/mL respectively in 100 L per well in a 96-well microplate (Corning), in DMEM or RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were allowed to attach for overnight. Probe stock solutions of different concentrations in DMF were diluted in fresh medium (HKSOX-1: 200 nM to 10 M; HKSOX-1r: 500 nM to 20 M; HKSOX-1m: 500 nM to 50 M) and added to the cells (100 L per well) in triplicates. After 24-h probe incubation, cells were loaded with 50-L Cell-Titer Glo reagent (Promega) and subjected to gentle shaking for cell lysis at room temperature (10 min). The microplate was mounted onto a DTX multimode plate reader (Molecular Devices) for luminescence detection, by using cellular ATP contents as a measure of cell viability. Data was collected for three separate serial dilutions and averaged. The cells viability was calculated according to the following equation: Cell viability (%) = 100 Awith probe / Acontrol. 96-well microplate fluorometric measurement RAW264.7 mouse macrophages were plated in 96-well microplates (Costar 3603; Corning), at a density of 5105 cells/mL (100 L/well in triplicates) one day before assay. The medium used was DMEM (Dulbecco’s modified Eagle medium; high glucose) supplemented with 10% 27
heat-inactivated FBS (fetal bovine serum) and 1% penicillin/streptomycin. For fluorometric measurement, all reagents and probe solutions were freshly dissolved in HBSS mix (HBSS supplemented with 0.6 mM L-arginine and 0.01% chloramphenicol). Before drug addition, cells were washed with PBS, and preloaded with 2 M HKSOX-1r in HBSS mix (50 L/well) for 30 min. Then, 50 L of 2 M HKSOX-1r together with 2 working doses of mitochondrial respiratory inhibitors (oligomycin A, FCCP, antimycin A) in HBSS mix was added, making up 100 L of probe and drug at their expected concentrations (1 working dose). At the end of 30-min co-incubation, drug gradients were removed by aspiration. Cells were briefly washed with PBS, and re-loaded with probe-free HBSS mix (100 L/well) before fluorescence reading on a DTX880 multimode detector (Beckman Coulter) with the following settings: excitation at 485 nm and emission at 535 nm, with an integration time of 400 ms. Data are presented, without subtracting background fluorescence, as mean s.e.m. for fluorometric measurement with replicates (n = 4) in at least three independent experiments. Flow cytometry analysis of superoxide production RAW264.7 cells were seeded into a 150-mm culture dish (Corning), cultured overnight for attachment and recovery, and grown to a required cell density (70-80% confluence or about 1.5107 cells). For harvest, cells were scraped off gently and collected into a clean 50-mL Falcon tube. Cells were spun down (500 rpm, room temperature, 3 min). After discarding the supernatant, 5 mL of warm HBSS was added gently to re-suspend the cell pellet. Then, cells were spun down (500 rpm, room temperature, 2 min). After this wash step, 2 mL of HBSS mix (HBSS supplemented with 0.6 mM L-arginine and 0.01% chloramphenicol) to re-suspend the cell pellet into single cells, followed by addition of another 6 mL HBSS mix. For superoxide detection, cells were co-incubated with 4 M probe HKSOX-1r in the absence or presence of antimycin A at different concentrations (0.05, 0.1, 0.5, 1, 5 and 10 M) in HBSS mix at 37 C for 30 min. The antioxidant NAC (10 mM) was added as a scavenger along with 1 M antimycin A for test of selectivity. Alternatively, cells were co-incubated with 4 M probe HKSOX-1r in the absence or presence of PMA at different concentrations (100, 200, 500, 1000 ng/mL) in HBSS mix at 37 C for 30 min. DPI (500 nM) was added to inhibit NOX-dependent superoxide production. Finally, superoxide levels were evaluated by measuring fluorescence intensity in FITC channel by using a flow cytometer (BD LSR Fortessa Analyzer). Zebrafish culture and imaging Zebrafish were maintained at 28 °C as described previously.3 Wild-type zebrafish were obtained from local fish farm. Embryos were obtained by natural spawning and were maintained in E3 zebrafish water at 28.5 °C and staged according to Kimmel et al.4 The study was conducted according to regulations by the Committee of the Use of Laboratory and Research Animals at The University of Hong Kong. At 72 hpf, zebrafish embryos were first preloaded with HKSOX-1r (10 M) for 20 min, and briefly challenged with PMA (200 ng/mL) or antimycin A (500 nM) for 15 min for O2 induction. DPI (100 nM) was optionally added to block off the effects of PMA. Confocal images were acquired with a Zeiss LSM 510 confocal microscope, by using the following settings: Ex 514 nm (18% laser intensity), Em 527–559 nm (band-pass). Other images by conventional epifluorescence microscopy were taken under Olympus IX70 (Olympus Corporation) and 10/0.3 NA objective in 3% methylcellulose, with Olympus DP71 (Olympus 28
Corporation) and Olympus DP-BSW basic Software. Bright-field images were taken under Nikon SMZ800 (Nikon Hong Kong Ltd) and P-Plan 1 objective in 3% methylcellulose with Nikon Digital Sight DS-Fi1 (Nikon Hong Kong Ltd.).
References
(1) Reed, J. W.; Ho, H. H.; Jolly, W. L. J. Am. Chem. Soc. 1974, 96, 1248–1249. (2) H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vasquez-Vivar, B. Kalyanaraman, Free Radic. Biol. Med. 2003, 34, 1359–1368. (3) Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. The University of Oregon Press, Eugene, Oregon, 1993. (4) Kimmel, D. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Dev. Dyn. 1995, 203, 253–310.
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