Supplemental Information. A Visible and Near-Infrared, Dual-Channel. Fluorescence-On Probe for Selectively. Tracking Mitochondrial Glutathione

Transcription

1 Chem, Volume 4 Supplemental Information A Visible and Near-Infrared, Dual-Channel Fluorescence-On Probe for Selectively Tracking Mitochondrial Glutathione Zhiqiang Xu, Xiaoting Huang, Xie Han, Di Wu, Bibo Zhang, Ying Tan, Meijiao Cao, Sheng Hua Liu, Jun Yin, and Juyoung Yoon

2 Tables of contents 1. Synthesis of probe CyP-SNp UV-Vis absorption spectra of probe CyP-SNp with various amino acids Fluorescence spectra of probe CyP-SNp with various amino acids The MALDI TOF mass spectrometry of probe CyP-SNp with GSH Fluorescence changes of probe CyP-SNp with GSH in the presence of various amino acids The UV titration absorption spectra of probe CyP-SNp with GSH The titration fluorescence spectra of probe CyP-SNp with GSH The time-dependent fluorescence spectra of probe CyP-SNp with GSH The time-dependent fluorescence spectra of probe CyP-SNp with Cys The time-dependent fluorescence spectra of probe CyP-SNp with Hcy The fluorescence spectra of probe CyP-SNp in serum Confocal images of probe CyP-SNp towards GSH Confocal images of probe CyP-SNp towards exogenous GSH in mitochondria Confocal images of probe CyP-SNp in green channel and red channel towards the exogenous GSH in mitochondria Confocal images by staining separately cells with probe CyP-SNp and MitoTracker Red The UV-Vis absorption of probe CyP-SNp in aggregation state The MALDI-TOF mass spectrometry The 1 H NMR spectra of Np-GSH, Np-Cys and Np-Hcy The HPLC data of the reactions of probe CyP-SNp with GSH, Cys and Hcy The spectra of NMR and MS...34

3 1. Synthesis of probe CyP-SNp Scheme S1. Synthetic route. The synthetic route of probe CyP-SNp. Synthesis of CyP-SNp. To a solution of CyP (200 mg, 0.28mmol) in anhydrous THF (20 ml) under an argon atmosphere was added the pyridine (2 ml). After the reaction mixture was stirred for 5 min, a solution of Np-SO 2 Cl (394 mg, 1.12 mmol) in THF (20 ml) was added dropwise at 0. After heating at reflux for 16 h, the solvent was removed under reduced pressure. The residue was purified by silica column (dichloromethane/methanol: 70:1 to obtain probe CyP-SNp (113 mg, 0.11 mmol) as dark grey solid in 39% yield. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm): 9.24 (d, J= 8 Hz, 1H, Ar-H), 8.77 (t, J= 8 Hz, 2H, Ar-H), 8.50 (d, J= 8 Hz, 1H, Ar-H), 8.03 (t, J= 8 Hz, 1H, Ar-H), 7.59 (t, J= 12 Hz, 2H, Ar-H), (m, 2H, Ar-H), 7.18 (d, J= 4 Hz, 4H, Ar-H), 7.00 (d, J= 8 Hz, 2H, CH 2 =CH 2 ), 5.95 (d, J= 16 Hz, 2H, CH 2 =CH 2 ), 4.22 (t, J= 8 Hz, 2H, CH 2 ), 3.98 (t, J= 8 Hz, 4H, CH 2 ), 3.59 (t, J= 8 Hz, 4H, CH 2 ), 3.44 (t, J= 8 Hz, 4H, CH 2 ), 2.49 (t, J= 8 Hz, 4H, CH 2 ), (m, 6H, CH 2 ), 1.73 (t, J= 8 Hz, 2H, CH 2 ), 1.45 (t, J= 8 Hz, 2H, CH 2 ), 1.38 (s, 12H, CH 3 ), 1.03 (t, J= 8 Hz, 6H, CH 3 ), 0.95 (t, J= 8 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ (ppm): 170.1, 168.7, 163.2, 162.6, 142.4, 141.5, 140.2, 137.7, 132.1, 131.4, 130.6, 129.6, 129.4, 129.0, 128.6, 127.7, 127.2, 127.1, , 123.2, 121.8, 110.2, 99.8, 53.2, 48.2, 47.5, 45.7, 40.6, 30.1, 28.4, 25.3, 21.3, 20.5, 20.2, 13.6, MALDI-TOF MS m/z = [M-I - ] + ; calculated exact mass =

4 2. UV-Vis absorption spectra of probe CyP-SNp with various amino acids Figure S1. UV-Vis absorption spectra of probe CyP-SNp with various amino acids. The UV-vis absorption spectra (A) and the color changes (B) of probe CyP-SNp (10 μm) with various amino acids (100 μm) in HEPES buffer (0.02 M, ph= 7.4) containing 10% DMSO.

5 3. Fluorescence spectra of probe CyP-SNp with various amino acids Figure S2. Fluorescence spectra of probe CyP-SNp with various amino acids in visible channel and near-infrared channel. The fluorescence spectra (A, C) and fluorescence intensity (B, D) of probe CyP-SNp (10 μm) with various amino acids (100 μm) in HEPES buffer (0.02 M, ph = 7.4) containing 10% DMSO in visible channel (A, B) and near-infrared channel (C, D). Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

6 4. The MALDI-TOF mass spectrometry of probe CyP-SNp with GSH Figure S3. The MALDI-TOF mass spectrometry of probe CyP-SNp with GSH. The MALDI-TOF spectral data were recorded after the treatment of probe CyP-SNp and GSH on a Bruker ultraflextreme MALDI-TOF-TOF mass spectrometer, DHB as matrix.

7 5. Fluorescence changes of probe CyP-SNp with GSH in the presence of various amino acids Figure S4. Fluorescence changes of probe CyP-SNp with GSH in the presence of various amino acids in visible channel and near-infrared channel. The fluorescence response of probe CyP-SNp (10 μm) towards GSH (100 μm) in the absence and presence of various amino acids (100 μm) in HEPES buffer (0.02 M, ph = 7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (B). Black bars are probe with other amino acids; red bars are probe with GSH (100 μm) in the present of other amino acids. Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

8 6. The UV titration absorption spectra of probe CyP-SNp with GSH Figure S5. The UV titration absorption spectra of probe CyP-SNp with GSH. The absorption spectra of probe CyP-SNp (10 μm) in the presence of GSH (0 100 μm) in HEPES buffer (0.02 M, ph= 7.4) containing 10% DMSO.

9 7. The titration fluorescence spectra of probe CyP-SNp with GSH Figure S6. The titration fluorescence spectra of probe CyP-SNp with GSH in visible channel and near-infrared channel. The titration fluorescent spectra of probe CyP-SNp (10 μm) upon addition of GSH (0 100 μm) in HEPES (0.02 M, ph=7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (C). The linear relationship between the concentration of GSH and fluorescence intensity changes at 495 nm (B) and at 795 nm (D). Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

10 Figure S7. Normalized fluorescence titration responses of probe CyP-SNp to changing GSH concentrations in visible channel and near-infrared channel. Normalized fluorescence responses of probe CyP-SNp (1 μm) to changing GSH concentrations (0 2 μm) in HEPES (0.02 M, ph=7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (B). The linear relationships between (F-F min )/(F max -F min ) and log[gsh] were observed, where F, F min and F max are fluorescence intensity at 495 nm (A) and 795 nm (B) with GSH, the initial fluorescence intensity without GSH and the maximum fluorescent intensity with GSH, respectively. Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/10 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 8/20 nm. (Visible channel: detection limit = M; Near-infrared channel: detection limit = M)

11 8. The time-dependent fluorescence spectra of probe CyP-SNp with GSH Figure S8. The time-dependent fluorescence spectra of probe CyP-SNp with GSH in visible channel and near-infrared channel. The time-dependent fluorescence spectra of probe CyP-SNp (10 μm) with GSH (100 μm) in HEPES (0.02 M, ph = 7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (B) (Record the spectra per 5 min). Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

12 9. The time-dependent fluorescence spectra of probe CyP-SNp with Cys Figure S9. The time-dependent fluorescence spectra of probe CyP-SNp with Cys in visible channel and near-infrared channel. The time-dependent fluorescence spectra of probe CyP-SNp (10 μm) with Cys (100 μm) in HEPES (0.02 M, ph = 7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (B) (Record the spectra per 5 min). Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

13 10. The time-dependent fluorescence spectra of probe CyP-SNp with Hcy Figure S10. The time-dependent fluorescence spectra of probe CyP-SNp with Hcy in visible channel and near-infrared channel. The time-dependent fluorescence spectra of probe CyP-SNp (10 μm) with Hcy (100 μm) in HEPES (0.02 M, ph = 7.4) containing 10% DMSO in visible channel (A) and near-infrared channel (B) (Record the spectra per 5 min). Visible channel: λ ex = 370 nm, λ em = 495 nm, slit: 10/3 nm; Near-infrared channel: λ ex = 700 nm, λ em = 795 nm, slit: 10/10 nm.

14 11. The fluorescence spectra of probe CyP-SNp in serum Figure S11. The fluorescence spectra of probe CyP-SNp in serum. The fluorescence of probe CyP-SNp (10 µm) in serum (0, 10, 30, 50, 100%) in visible channel (A) and near-infrared channel (B). Visible channel: λ ex = 370 nm, λ em = 442 nm; Slit: 10/10 nm; Near-infrared channel: λ ex = 700 nm, λ em = 810 nm; Slit: 10/10 nm. Each spectrum was recorded at 180 min after the addition of GSH.

15 12. Confocal images of probe CyP-SNp towards GSH Figure S12. Confocal images of CyP-SNp towards GSH in green channel and red channel. Fluorescence images of HeLa cells pretreated with NMM (1 mm) for 30 min, and then treated with GSH (100 μm) for 30 min and incubated with probe CyP-SNp (10 μm) for another 30 min. (A) Fluorescence images of HeLa cells in green channel, λ ex = 405 nm, λ em = nm; (B) Fluorescence images of HeLa cells in red channel, λ ex = 635 nm, λ em = nm; (C) Merged image of images in green channel and red channel; (D) Intensity correlation plot of fluorescence signals in green channel and red channel (Pearson s correlation coefficient 0.90 and overlap coefficient 0.91); (A - C) Scale bar, 20 μm.

16 13. Confocal images of probe CyP-SNp towards exogenous GSH in mitochondria Figure S13. Confocal images of probe CyP-SNp towards exogenous GSH in mitochondria with MitoTracker Red and MitoTracker Green. HeLa cells pretreated with NMM (1 mm) for 30 min, incubated with CyP-SNp (10 μm) for 30 min, and then treated with GSH (100 μm) and MitoTracker Red (20 nm) (A - C) or MitoTracker Green (200 nm) (E - G) for another 30 min, respectively. (A) Fluorescence image in green channel, λ ex = 405 nm, λ em = nm; (B) Fluorescence image in MitoTracker Red channel, λ ex = 559 nm, λ em = nm; (C) Merged image of images in green channel and MitoTracker Red channel; (D) Intensity correlation plot of fluorescence signals in green channel and MitoTracker Red channel (Pearson s correlation coefficient 0.86 and overlap coefficient 0.88); (E) Fluorescence image in red channel, λ ex = 635 nm, λ em = nm; (F) Fluorescence image in MitoTracker Green channel, λ ex = 488 nm, λ em = nm; (G) Merged image of images in red channel and MitoTracker Green channel; (H) Intensity correlation plot of fluorescence signals in red channel and MitoTracker Green channel (Pearson s correlation coefficient 0.86 and overlap coefficient 0.87); (A - C, E - G) Scale bar, 20 μm.

17 14. Confocal images of probe CyP-SNp in green channel and red channel towards the exogenous GSH in mitochondria Figure S14. Confocal images of probe CyP-SNp in green channel and red channel towards the exogenous GSH in mitochondria with MitoTracker Red. HeLa cells pretreated with NMM (1 mm) for 30 min, incubated with probe CyP-SNp (10 μm) for 30 min, and then treated with GSH (100 μm) and MitoTracker Red (20 nm) for another 30 min, respectively. (A) Fluorescence image in green channel, λ ex = 488 nm, λ em = nm; (B) Fluorescence image in MitoTracker Red channel, λ ex = 559 nm, λ em = nm; (C) Fluorescence image in red channel (The red fluorescence was changed to blue for discrimination), λ ex = 635 nm, λ em = nm; (D) Merged image of images in green channel and MitoTracker Red channel; (E) Merged image of images in MitoTracker Red channel and red channel; (F) Merged image of images in green channel, MitoTracker Red channel and red channel; (G) Intensity correlation plots of fluorescence signals in green channel and MitoTracker Red channel (Pearson s correlation coefficients 0.79 and overlap coefficients 0.84); (H) Intensity correlation plots of fluorescence signals in MitoTracker Red channel and red channel (Pearson s correlation coefficients 0.94 and overlap coefficients 0.95); (A - F) Scale bar, 20 μm.

18 15. Confocal images by staining separately cells with probe CyP-SNp and MitoTracker Red Figure S15. Confocal images of probe CyP-SNp in green channel and red channel towards the endogenous GSH in mitochondria with MitoTracker Red. (A, B, C) HeLa cells incubated with probe MitoTracker Red (20 nm) for 30 min; (D, E, F) HeLa cells incubated with probe CyP-SNp (3 μm) for 30 min; (G, H, I) HeLa cells incubated with probe CyP-SNp (3 μm) for 15 min, followed by MitoTracker Red (20 nm) for 15 min; (J, K, L) Merged images. (A, D, G) Fluorescence images in green channel; λ ex = 488 nm, λ em = nm; (B, E, H) Fluorescence images in MitoTracker Red channel; λ ex = 559 nm, λ em = nm; (C, F, I) Fluorescence images in red channel (The red fluorescence was modified graphically to the color blue for graphical discernment), λ ex = 635 nm, λ em = nm.

19 16. The UV-Vis absorption of probe CyP-SNp in aggregation state Figure S16. UV-Vis absorption of probe CyP-SNp. Time-dependent UV Vis spectral changes of probe CyP-SNp (10 μm) in HEPES (0.02 M, ph 7.4) containing 10% DMSO from 0 to 90 min (Record the spectra per 3 min). Figure S17. UV-Vis absorption of probe CyP-SNp. UV-Vis absorption spectra of probe CyP-SNp (10 μm) in HEPES (0.02 M, ph 7.4) containing 10% DMSO upon the addition of surfactant CTAB (10 mm) at different time (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57 and 60 min) (Record the spectra per 3 min).

20 Figure S18. UV-Vis absorption of probe CyP-SNp. UV-Vis absorption spectra of probe CyP-SNp (10 μm) in different solvents.

21 17. The MALDI-TOF mass spectrometry Figure S19. The MALDI-TOF mass spectrometry. The MALDI-TOF mass spectrometry of mixing probe CyP-SNp with GSH for 3 h (A) and amplified spectra (B, C, D, E).

22 Figure S20. The MALDI-TOF mass spectrometry. The MALDI-TOF mass spectrometry of mixing probe CyP-SNp with Cys for 5-8 h (A, B) and amplified spectra (C, D, E, F).

23 Figure S21. The MALDI-TOF mass spectrometry. The MALDI-TOF mass spectrometry of mixing probe CyP-SNp with Hcy for 5-8 h (A, B) and amplified spectra (C, D, E, F).

24 18. The 1 H NMR spectra of Np-GSH, Np-Cys and Np-Hcy Figure S22. 1 H NMR spectrum. 1 H NMR spectrum of Np-GSH in CD 3 OD.

25 Figure S23. 1 H NMR spectrum. 1 H NMR spectrum of Np-Cys in CD 3 OD.

26 Figure S24. 1 H NMR spectrum. 1 H NMR spectrum of Np-Hcy in CD 3 OD.

27 19. The HPLC data of the reactions of probe CyP-SNp with GSH, Cys and Hcy Figure S25. HPLC data. High performance liquid chromatography (HPLC) in different systems. (A) Probe Cyp-SNp (10 μm), monitored at 700 nm; (B) Product CyP (10 μm), monitored at 700 nm; (C) Product Np-GSH (10 μm), monitored at 390 nm; The reaction of probe Cyp-SNp (10 μm) with GSH (100 μm) for 3 h, monitored at 390 nm (D) and 700 nm (E); The reaction of probe Cyp-SNp (10 μm) with GSH (1000 μm) for 3 h, monitored at 390 nm (F) and 700 nm (G), respectively.

28 Figure S26. HPLC data. High performance liquid chromatography (HPLC) in different systems. (A) Probe Cyp-SNp (10 μm), monitored at 700 nm; (B) Product CyP (10 μm), monitored at 700 nm; (C) Product Np-Cys (10 μm), monitored at 390 nm; The reaction of probe Cyp-SNp (10 μm) with Cys (100 μm) for 3 h, monitored at 390 nm (D) and 700 nm (E); The reaction of probe Cyp-SNp (10 μm) incubated with Cys (1000 μm) for 3 h, monitored at 390 nm (F) and 700 nm (G), respectively.

29 Figure S27. HPLC data. High performance liquid chromatography (HPLC) in different systems. (A) Probe Cyp-SNp (10 μm), monitored at 700 nm; (B) Product CyP (10 μm), monitored at 700 nm; (C) Product Np-Hcy (10 μm), monitored at 390 nm; The reaction of probe Cyp-SNp (10 μm) with Hcy (100 μm) for 3 h, monitored at 390 nm (D) and 700 nm (E); The reaction of probe Cyp-SNp (10 μm) with Hcy (1000 μm) for 3 h, monitored at 390 nm (F) and 700 nm (G), respectively.

30 Figure S28. Calibration curve. The calibration curve of CyP (A), Np-GSH (B), Np-Cys (C) and Np-Hcy (D) by HPLC.

31 Table S1. HPLC data. Reactions of probe CyP-SNp with biothiols and calculated yields Probe Amino acids Products Area Output (uv*sec) (μm) Yield (%) GSH (100 μm) Np-GSH CyP Cys (100 μm) Np-Cys 584 <0.1 <1 CyP Np-Hcy 479 <0.1 <1 Hcy (100 μm) CyP-SNp (10 CyP μm) GSH (1000 Np-GSH μm) CyP Cys (1000 μm) Np-Cys CyP Hcy (1000 μm) Np-Hcy CyP Note: Probe Cyp-SNp (10 μm) was treated with biothiols (100 μm or 1000 μm) for 3 h. Products were quantified using a High performance liquid chromatography equipped with a UV detector and calculated yields according to the calibration curve and areas of corresponding products.

32 Table S2. HPLC parameters. Gradient program for HPLC Instrument Waters1525 liquid chromatography Column SunFire C18 column, 5 μm, 4.6 x 250 mm, 25 C Detector Waters 2489 UV/Visible Detector Flow rate 1 ml/min Injection volume 20 µl Absorbance 390 nm or 700 nm Solution A: 0.05% trifluoroacetate in H 2 O Solution B: 0.05% trifluoroacetate in acetonitrile Gradient program gradient elution: 1-8 min, 5-95%B; min, 95-5% B; Isocratic elution: 0-1 min, 5%B; 8-11 min, 95%B.

33 Table S3. Data of Calibration Curves. Equations of Calibration Curves of compounds CyP, Np-GSH, Np-Cys, Np-Hcy, and linear range standard calibration curves Correlation coefficient(r 2 ) Linear range (μm) CyP y= x Np-GSH y= x Np-Cys y= x Np-Hcy y= x

34 20. The spectra of NMR and MS Figure S29. 1 H NMR Spectrum of probe CyP-SNp.

35 Figure S C NMR Spectrum of probe CyP-SNp.

36 Figure S31. MALDI-TOF mass spectra of probe CyP-SNp.