Supporting Information Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR over-expressing Cells

Transcription

1 Supporting Information Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR over-expressing Cells Ning Zhao 1, Tyrslai M. Williams 1, Zehua Zhou 1, Frank R. Fronczek 1, Martha Sibrian-Vazquez 2, Seetharama D. Jois 3 and M. Graça H. Vicente 1 * 1 Louisiana State University, Department of Chemistry, Baton Rouge LA, 70803, USA 2 Portland State University, Department of Chemistry, Portland, OR, 97201, USA 3 Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe LA, 71201, USA Table of Contents 1. Spectroscopic data... S2 2. Cytotoxicity... S3 3. Cellular uptake.... S4 4. Subcellular Localization.... S5 5. HPLC traces... S6 6. SPR Analysis... S9 7. MALDI(TOF) and MALDI(TOF-TOF)... S H and 13 C NMR spectra... S16 9. HSQC... S B NMR spectra... S26 S1

2 1. Spectroscopic data Table S1. Photophysical studies of BODIPYs in CH 2Cl 2 at room temperature Compound λ abs λ em Stokes shift log ε Ф f nm nm nm Figure S1: Normalized UV-Vis (a) and fluorescence spectra (b) of BODIPYs 3-5 in CH 2Cl 2 at room temperature. S2

3 2. Cytotoxicity Figure S2. Dark toxicity of BODIPYs 5 (blue), 8 (green) and 9 (purple) in HEp2 cells using the Cell Titer Blue assay. Figure S3. Photo toxicity (~ 1.5 J/cm 2 ) of BODIPYs 5 (blue), 8 (green) and 9 (purple) in HEp2 cells using a Cell Titer Blue assay. S3

4 3. Cellular uptake. Table S2. Cellular uptake for compounds 5, 8 and 9 at 1, 2, 4, 8, 24 h based on Figure 4. Compound cellular uptake at cellular uptake cellular uptake cellular uptake cellular uptake 1 h (nm/cell) at 2 h (nm/cell) at 4 h (nm/cell) at 8 h (nm/cell) at 24 h (nm/cell) BODIPY BODIPY-peptide conjugate BODIPY-peptide conjugate S4

5 4. Subcellular Localization. Figure S4. Subcellular fluorescence of BODIPY 5 in HEp2 cells at 10 μm for 6 h. (a) Phase contrast; (b) overlay of BODIPY 5 fluorescence and phase contrast; (c) ER tracker Blue/White fluorescence; (d) overlay of BODIPY 5 fluorescence and ER Tracker; (e) BODIPY ceramide; (f) overlay of BODIPY 5 fluorescence and BODIPY ceramide; (g) MitoTracker Green fluorescence; (h) overlay of BODIPY 5 fluorescence and MitoTracker; (i) LysoSensor Green fluorescence; (j) overlay of BODIPY 5 fluorescence and LysoSensor. Scale bar: 10 μm. S5

6 5. HPLC traces Normal-phase HPLC spectra of BODIPY 5 was performed on Dionex system (organics system) including a P680 pump with a UVD 340 detector and a fraction collector III. This system is connected to a Dynamax axial compression column in the packing of irregular silica gel (Rainin 60 Å). The solvent system is: stepwise gradient 10% B and 90% A (0-5 min), 50% B and 50% A (10-20 min), 10% B and 90% A (25-30 min). Solvent A: hexanes; solvent B: ethyl acetate. Reverse-phase HPLC analysis was performed with a Waters 2485 Quaternary Gradient Module, Waters Sample Injector, and 2489 UV/Visible Detector which are controlled by Waters Empower 2 software. Separations were completed on a X-Bridge BEH300 Prep C18 (5 um, 10 x 250 mm) with a X-Bridge BEH300 Prep Guard cartridge 300 Å (5 um, 10 x 10 mm) using a linear 50% to 100% gradient of solvent B (0.1% TFA in acetonitrile) into A (0.1% TFA in water) over 25 minutes at a 4 ml/min flow rate with UV detection for peptides 220 nm, and conjugates 580 nm. Fractions of HPLC purity (> 95%) with the anticipated mass were combined and lyophilized. Figure S5: HPLC trace of BODIPY 5. S6

7 AU Minutes Figure S6. HPLC trace for peptide AU Minutes Figure S7. HPLC trace for peptide 7. S7

8 AU Minutes Figure S8. HPLC trace for conjugate AU Minutes Figure S9. HPLC trace for conjugate 9. S8

9 6. SPR Analysis Figure S10. SPR analysis for BODIPY 5 binding to EGFR. S9

10 7. Copies of MALDI(TOF) and MALDI(TOF-TOF) Figure S11. MALDI-TOF of peptide S10

11 Figure S12. MALDI-TOF of peptide S11

12 Figure S13. MALDI-TOF of conjugate S12

13 Figure S14. MALDI-TOF of conjugate S13

14 Figure S15. MALDI-TOF-TOF of conjugate 8. S14

15 Figure S16. MALDI-TOF-TOF of conjugate 9. S15

16 8. Copies of 1 H and 13 C NMR spectra Figure S17. 1 H NMR of BODIPY 2 Figure S C NMR of BODIPY 2 S16

17 Figure S19. 1 H NMR of BODIPY 3 Figure S C NMR of BODIPY 3 S17

18 Figure S21. 1 H NMR of BODIPY 4 Figure S C NMR of BODIPY 4 S18

19 Figure S23. 1 H NMR of BODIPY 5 Figure S C NMR of BODIPY 5 S19

20 Figure S25. 1 H NMR of compound 6 S20

21 Figure S26. 1 H NMR of compound 7 S21

22 Figure S27. 1 H NMR of compound 8 S22

23 Figure S28. 1 H NMR of compound 9 S23

24 9. Copies of HSQC Figure S29. HSQC NMR of 6 Figure S30. HSQC NMR of 7 S24

25 Figure S31. HSQC NMR of 8 Figure S32. HSQC NMR of 9 S25

26 10. Copies of 11 B NMR spectra Figure S B NMR of BODIPY 2 Figure S B NMR of BODIPY 3 S26

27 Figure S B NMR of BODIPY 4 Figure S B NMR of BODIPY 5 S27

28 Figure S B NMR of BODIPY-peptide conjugate 8 Figure S B NMR of BODIPY-peptide conjugate 9 S28