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)... S10 8. 1 H and 13 C NMR spectra... S16 9. HSQC... S24 10. 11 B NMR spectra... S26 S1
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 3 572 613 nm 41 4.68 0.19 4 608 627 nm 19 4.54 0.013 5 575 616 nm 41 4.73 0.16 Figure S1: Normalized UV-Vis (a) and fluorescence spectra (b) of BODIPYs 3-5 in CH 2Cl 2 at room temperature. S2
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
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 5 0.00028 0.00063 0.00073 0.00090 0.0016 BODIPY-peptide conjugate 8 0.0254 0.0344 0.0398 0.0424 0.0431 BODIPY-peptide conjugate 9 0.016 0.0224 0.0287 0.036 0.0485 S4
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
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
2.50 13.954 2.00 1.50 AU 1.00 0.50 14.626 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes Figure S6. HPLC trace for peptide 6. 2.00 12.003 1.50 AU 1.00 0.50 0.00 3.537 4.004 4.667 6.617 14.556 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes Figure S7. HPLC trace for peptide 7. S7
0.060 0.050 11.006 0.040 AU 0.030 0.020 0.010 24.681 0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes Figure S8. HPLC trace for conjugate 8. 0.40 0.35 11.986 0.30 0.25 AU 0.20 0.15 0.10 0.05 0.00 15.625 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes Figure S9. HPLC trace for conjugate 9. S8
6. SPR Analysis Figure S10. SPR analysis for BODIPY 5 binding to EGFR. S9
7. Copies of MALDI(TOF) and MALDI(TOF-TOF) 888.542 888.542 Figure S11. MALDI-TOF of peptide 6. 889.551 890.559 S10
Figure S12. MALDI-TOF of peptide 7. 1799.972 1800.976 1801.9811 1801.981 1800.976 S11
1493.726 1491.717 1492.730 1493.726 Figure S13. MALDI-TOF of conjugate 8. 1494.739 1495.735 1496.731 1497.745 S12
2405.028 2404.006 Figure S14. MALDI-TOF of conjugate 9. 2406.018 2407.018 2408.006 1801.9811 2409.029 1801.981 1800.976 2407.018 2410.054 S13
Figure S15. MALDI-TOF-TOF of conjugate 8. S14
Figure S16. MALDI-TOF-TOF of conjugate 9. S15
8. Copies of 1 H and 13 C NMR spectra Figure S17. 1 H NMR of BODIPY 2 Figure S18. 13 C NMR of BODIPY 2 S16
Figure S19. 1 H NMR of BODIPY 3 Figure S20. 13 C NMR of BODIPY 3 S17
Figure S21. 1 H NMR of BODIPY 4 Figure S22. 13 C NMR of BODIPY 4 S18
Figure S23. 1 H NMR of BODIPY 5 Figure S24. 13 C NMR of BODIPY 5 S19
Figure S25. 1 H NMR of compound 6 S20
Figure S26. 1 H NMR of compound 7 S21
Figure S27. 1 H NMR of compound 8 S22
Figure S28. 1 H NMR of compound 9 S23
9. Copies of HSQC Figure S29. HSQC NMR of 6 Figure S30. HSQC NMR of 7 S24
Figure S31. HSQC NMR of 8 Figure S32. HSQC NMR of 9 S25
10. Copies of 11 B NMR spectra Figure S33. 11 B NMR of BODIPY 2 Figure S34. 11 B NMR of BODIPY 3 S26
Figure S35. 11 B NMR of BODIPY 4 Figure S36. 11 B NMR of BODIPY 5 S27
Figure S37. 11 B NMR of BODIPY-peptide conjugate 8 Figure S38. 11 B NMR of BODIPY-peptide conjugate 9 S28