SUPPORTING INFORMATION Lysine analogue of Polymyxin B as a significant opportunity for photodynamic antimicrobial chemotherapy Florent Le Guern, Tan-Sothea Ouk *, Catherine Ouk, Regis Vanderesse +, Yves Champavier, Emilie Pinault, Vincent Sol * Université de Limoges, Laboratoire de Chimie des Substances Naturelles, EA 1069, 123 Avenue Albert Thomas, 87060 Limoges Cedex, France. Université de Limoges, BISCEm, 87000 Limoges, France + Université de Lorraine, Laboratoire de Chimie Physique Macromoléculaire (LCPM), UMR 7375 CNRS, ENSIC, 1 rue Grandville, 54000 Nancy, France 1
Tables of Contents Figure S1. Synthetic route of 5Lys based on the previous work... 3 Figure S2. MS and 1 H NMR analysis of 1Lys.... 4 Figure S3. HRMS analysis of 5Lys... 5 Figure S4. 1 H NMR analysis of 5Lys... 6 Figure S5. 1 H COSY NMR analysis of 5Lys... 7 Figure S6. 1 H TOCSY NMR analysis of 5Lys... 8 Figure S7. Absorbance spectra of 5Lys and 5 in MeOH (4.3 µm)... 9 Figure S8. Absorption, fluorescence, and phosphorescence spectra... 10 Figure S9. Confocal laser scanning microscopy imaging of NHDF after incubation with 5Lys 11 Figure S10. Additional confocal laser scanning microscopy imaging of NHDF and bacteria... 13 Table S1. Fluorescence and phosphorescence properties... 10 Table S2. IC 50 (um) of the different molecules... 12 2
Figure S1. Synthetic route of 5Lys based on the previous work (Le Guern et al., 2017). a) Propionic acid, 3 h, 140 C. b) Iodomethane, anhydrous DMF, 1 h, 140 C. c) 25% TFA/H 2 O, 5 h, 100 C. d) 6-maleimidohexanoic acid, HCTU, DIEA, anhydrous DMF, 24 h, RT. e) PBS ph 6.5, 24 h, RT. 3
Figure S2. MS and 1 H NMR (in DMSO-d 6 ) analysis of 1Lys. 4
Figure S3. HRMS analysis of 5Lys 5
Figure S4. 1 H NMR (in DMSO-d 6 ) analysis of 5Lys 6
Figure S5. 1 H COSY NMR analysis of 5Lys 7
Figure S6. 1 H TOCSY NMR analysis of 5Lys 8
1 5 5Lys Absorbance (A.U.) 500 600 700 0 400 500 600 700 800 Wavelength (nm) Figure S7. Absorbance spectra of 5Lys and 5 in MeOH (4.3 µm) 9
Absorbance (a.u) 417 425 A 516 520 551 559 TMPyP 5 5Lys 584 585 637 643 500 600 700 Fluorescence intensity (a.u) 2,5x10 7 B 2,0x10 7 1,5x10 7 1,0x10 7 5,0x10 6 TMPyP 5 5Lys 300 400 500 600 700 800 Wavelength (nm) Phosphorescence intensity (a.u) 0,0016 0,0014 0,0012 0,0010 0,0008 0,0006 0,0004 0,0002 C 0,0 600 650 700 750 800 Wavelength (nm) TMPyP 5 5Lys 0,0000 1200 1220 1240 1260 1280 1300 1320 1340 Wavelength (nm) Figure S8. Absorption (A) ([TMPyP] = 13.8 µm, [5] = 16 µm, [5Lys] = 16 µm), fluorescence (B), and phosphorescence (C) spectra of TMPyP, 5 and 5Lys in D 2 O (λ exc = 425 nm, A 425 ~ 0.2). Table S1. Fluorescence and phosphorescence properties of conjugate TMPyP, 5 and 5Lys in D 2 O. a TPP as standard (Φ F (toluene) = 0.11) (Redmond and Gamlin, 1999), b TMPyP as standard (Φ (D 2 O) = 0.90) (Redmond and Gamlin, 1999), c Literature value ~ 67 µs in D 2 O (Ogilby and Foote, 1983; Wilkinson et al., 1995).(λ exc = 425 nm) compd λ F max (nm) φ F a φ τ F (ns) τ (µs) c TMPyP 708 0.06 0.90 b 6.1 69 5 717 0.07 0.79 5.2 65 5Lys 717 0.07 0.80 6.2 75 10
Figure S9. Confocal laser scanning microscopy imaging of NHDF after incubation with 5Lys. Cells were suspended in a solution of 5Lys (1 µm) for 30 min at 37 C, then washed 3 times with PBS. 11
IC 50 (um) Light NHDF Dark 1Lys - - 5 0.156-5Lys 0.156 - Table S2. IC 50 (um) of the different molecules after light irradiation (white LED, 35 J/cm²) and in the dark. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. 48h prior to experiments, 100 µl per well of cell suspension at 0.5 x 10 5 cells per ml were cultured in 96-well microplates in complete FGM culture medium (Lonza, Germany). Culture medium was removed and cells were incubated in the dark with different concentrations of PS, ranging from 5 nm to 5000 nm (in PBS 1X). After 2 hours of incubation at 37 C in the dark, cells were washed three times with PBS, and then 100 µl of fresh FGM culture medium were added in each well. One plate was exposed to a white light irradiation (4.83 mw/cm²) for 2 hours (35 J/cm²) at 37 C and then was placed at 37 C in the dark. Another plate was kept in the dark at 37 C without irradiation (dark condition). After 24 hours at 37 C, the MTT was added at a final concentration of 0.5 mg/ml in each well and cells were incubated at 37 C for 2 hours. Then, the culture medium was harvested and replaced by 100 µl of DMSO in each well in order to dissolve formazan crystals. The absorbance was measured at 595 nm using a Bio-Rad imark microplate reader. 12
Figure S10. Additional confocal laser scanning microscopy imaging of NHDF and bacteria (P. aeruginosa and S. aureus) after incubation with 5Lys. NHDF cells (5 000 cells) were seeded in a 8-well Lab-Tek TM II chamber coverglass (NUNC) in complete FBM culture medium for 48 hours at 37 C. 10 7 CFU were prepared and washed three times with PBS. 5.10 6 CFU were directly added to each well. After 30 min of incubation at 37 C, the supernatant was removed and the wells were washed once with PBS. Then, 500 µl of PS solutions (1 µm in PBS) were deposited after 30 min at 37 C in the dark, three washing steps were done, and then confocal microscopy imaging was carried out as described before, with a 40X objective lens. 13