Supplementary Information

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1 4(R/S)-Guanidinylprolyl collagen peptides: On-resin synthesis, complexation with plasmid DNA and the role of peptides in enhancement of transfection Manaswini Nanda, Krishna N. Ganesh,* Chemical Biology Unit, Indian Institute of Science Education and Research, 900, NCL Innovation Park, Dr Homi Bhabha Road, Pune , India; Tel: 91 (20) ; Fax: 91 (20) ; Organic Chemistry Division, National Chemical Laboratory, Pune , India; *To whom reprint requests should be addressed; Fax: +91 (20) , Supplementary Information Contents Pag e 1 H and 13 C NMR spectra of monomer 1a S2 MS and IR spectra of 1a 1 H and 13 C NMR spectra of monomer 1b S4 MS and IR spectra of 1b General synthesis of cationic peptides Fluorescent cationic peptides UV-VIS and Fluorescence spectra of peptides (P6-P8) HPLC and MALDI-TOF profiles of synthesised peptides (P1-P8) S8- S15 CD study of peptides (P3 and P4) S16 CD thermal denaturation of peptides (P3 and P4) Cell Study Images S18- S20 Restriction enzyme assay and electrophoretic mobility shift S21 Statistical analysis of transfection S22- S23 S3 S5 S6 S7 S7 S17 S1

2 FIGURE 1: 1 H NMR of compound 1a 400 MHz, DMSO-d 6 FIGURE 2: 13 C NMR of compound 1a 100 MHz, DMSO-d 6 FIGURE 3: DEPT- 13 C NMR of compound 1a 100 MHz, DMSO-d 6 S2

3 FIGURE 4: LC-MS of compound 1a M Calc. = M Obsv.= [M+Na] + FIGURE 5: IR Spectra of compound 1a S3

4 FIGURE 6: 1 H NMR of compound 1b 400 MHz, DMSO-d 6 FIGURE 7: 13 C NMR of compound 1b 100 MHz, DMSO-d 6 FIGURE 8: DEPT- 13 C NMR of compound 1b 100 MHz, DMSO-d 6 S4

5 FIGURE 9: LC-MS of compound 1b M Calc. = M Obsv.= [M+Na] + FIGURE 10: IR Spectra of compound 1b S5

6 General Synthesis of Cationic Peptides SCHEME 1: Solid phase synthesis of cationic peptides using t-boc chemistry S6

7 Fluorescent cationic peptides (P6-P8) Peptide P6 TR-X-NH-(Pro-AmpG-Gly) 6 -NH 2 Peptide P7 TR-X-NH-(ampG-Pro-Gly) 6 -NH 2 Peptide P8 TR-X-NH-(Val-Arg-Leu-Pro-Pro-Pro) 3 -NH 2 FIGURE 11: Design of Fluorescent peptides (P6-P8). A B FIGURE 12: (A) UV-absorption spectra of TR-X-labelled peptides (B) their corresponding fluorescence emission spectra at 615 nm S7

8 HPLC and MALDI-TOF profiles of synthesised peptides FIGURE 13: HPLC of Peptide P1 (R-Am) 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.5 ml/min; λ = 220 nm FIGURE 14: MALDI-TOF of Peptide P1 (R-Am) Ac-Phe-(Pro-Amp-Gly) 6 - NH 2 M calc. = M obsv. = S8

9 FIGURE 15: HPLC of Peptide P2 (S-am) 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.5 ml/min; λ = 220 nm FIGURE 16: MALDI-TOF of Peptide P2 (S-am) Ac-Phe-(amp-Pro-Gly) 6 - NH 2 M calc. = M obsv. = S9

10 FIGURE 17: HPLC of Peptide P3 (RG) 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.5 ml/min; λ = 220 nm FIGURE 18: MALDI-TOF of Peptide P3 (RG) Ac-Phe-(Pro-AmpG-Gly) 6 - NH 2 M calc. = M obsv. = S10

11 FIGURE 19: HPLC of Peptide P4 (SG) 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.5 ml/min; λ = 220 nm FIGURE 20: MALDI-TOF of Peptide P4 (SG) Ac-Phe-(ampG-Pro-Gly) 6 - NH 2 M calc. = M obsv. = S11

12 FIGURE 21: HPLC of Peptide P5 (SAP) 30 min Gradient: 5% to 50% MeCN/H O with 0.1% TFA, flow rate = 1.5 ml/min; λ = 220 nm FIGURE 22: MALDI-TOF of Peptide P5 (SAP) Sweet Arrow Peptide (SAP) (Val-Arg-Leu-Pro-Pro- Pro) 6 M calc. = S12

13 FIGURE 23: HPLC of Peptide P6 (TR-RG) Absorbance at 220 nm Absorbance at 595 nm 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.0 ml/min; λ = 220 nm & 595 nm FIGURE 24: MALDI-TOF of Peptide P6 (TR-RG) NH 2 TR-X-NH-(Pro-AmpG-Gly) 6 - M calc. = M obsv. = (M+K) S13

14 FIGURE 25: HPLC of Peptide P7 (TR-SG) Absorbance at 220 nm Absorbance at 595 nm 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.0 ml/min; λ = 220 nm & 595 nm FIGURE 26: MALDI-TOF of Peptide P7 (TR-SG) NH 2 TR-X-NH-(ampG-Pro-Gly) 6 - M calc. = M obsv. = S14

15 FIGURE 27: HPLC of Peptide P8 (TR-SAP) Absorbance at 220 nm Absorbance at 595 nm 30 min Gradient: 5% to 50% MeCN/H 2 O with 0.1% TFA, flow rate = 1.0 ml/min; λ = 220 nm & 595 nm FIGURE 28: MALDI-TOF of Peptide P8 (TR-SAP) Texas Red-X-SAP TR-X-NH-(Val-Arg-Leu-Pro-Pro-Pro) 6 M calc. = M obsv. = S15

16 CD study of peptides FIGURE 29: Comparative CD spectra at 10 C at 0.03 mm concentration in 10 mm phosphate buffer ph 7.2 (10 mm NaCl) for peptides P2 (dark yellow), P4 (red) and P5 or SAP (blue). A B (P3) Ac-Phe-(Pro-AmpG-Gly)6-NH2 (P4) Ac-Phe-(ampG-Pro-Gly)6-NH2 FIGURE 30: CD spectra at 10 C at concentrations from 0.01 mm-0.06 mm in steps of 0.01 mm (10 mm phosphate buffer ph 7.2, 10 mm NaCl) of peptides (A) P3, λ max = nm, λ min = ~200 nm, isobestic point at nm; (B) P4, no +ve λ max, λ min = nm, isobestic point at 227 nm S16

17 CD thermal denaturation of peptides Ac-Phe-(Pro-AmpG-Gly)6-NH2 (P3) A Ac-Phe-(ampG-Pro-Gly)6-NH2 (P4) B Ac-Phe-(Pro-AmpG-Gly)6-NH2 (P3) C Ac-Phe-(ampG-Pro-Gly)6-NH2 (P4) D FIGURE 31: Peptides (A) P3 and (B) P4 (0.03 mm) Boltzman/sigmoidal fit curve for molar ellipticity at 222 nm, from variable temperature CD spectra in different solvent conditions of ph 7.2, 12.0 (10 mm phosphate buffer & borate buffer with 10 mm NaCl respectively) and ethylene glycol water mixture (3:1), corresponding first derivative curves with T m values in (C) peptide P3 and (D) peptide P4. S17

18 Cell Study Images A B C D FIGURE 32. Expession of GFP in Drosophila S2 cells in presence of peptides P3 and P4. (A) DIC image of cells (B) transfected with prmha3-gfp plasmid using Qiagen transfection kit and induced with CuSO 4 (1 mm). Improved transfection efficiency in the absence of enhancer and presence of (C) Peptide P3 and (D) Peptide P4. FIGURE 33: Confocal images of fluorescent peptides inside S2 cells. were incubated with 1µM of Peptide P6 (A) Peptide P7 (B) peptide P8 (C). S18

19 I II III P8 (SAP-F) P7 (SG-F) P6 (RG-F) FIGURE 34: Confocal fluorescence microscopy analysis of S2 cells on treatment with fluorescent peptides. (I) DAPI stained image, (II) Fluorescent image of peptide staining, (III) Superimposed DAPI stained and fluorescent image of cells. (A-C) peptide P6, (D-F) peptide P7, (G-H) peptide P8 S19

20 I II III IV P8 (SAP-F) Transfection with P7 (SG-F) P6 (RG-F) prmha3-gfp Transfection FIGURE 35: Fluorescence microscopy analysis of S2 cells on transfection with range of peptides (I) DAPI stained image, (II) Green fluorescence image for GFP expression, (III) Red fluorescence image of peptide staining, (IV) Superimposed DAPI stained and fluorescent image of cells. (A-D) pdna transfection images (E-H) transfection images with peptide P6, (I- L) transfection images with peptide P7, (M-P) transfection images with peptide P8 S20

21 Restriction enzyme assay and electrophoretic mobility shift only pdna pdna with restriction enzyme pdna + P3 (RG) pdna + P4 (SG) pdna-peptide complex Linear pdna Free pdna supercoiled Linear pdna (smaller size) FIGURE 36: Agarose gel electrophoresis after restriction enzyme (BamH1) digestion of plasmid DNA (prmha3-gfp) and its complexes with peptides P3 (RG) and P4 (SG) at 1:25 w/w ratio; (1% agarose gel, negative image, 1X-TAE buffer, 45 mins, 90 Volts); Lane 1: only pdna untreated by enzyme, Lane 2: pdna, Lane 4: pdna with P3, Lane 6: pdna with P4 treated by restriction enzyme. Lane 3 and 5 are blank. S21

22 Statistical analysis of transfection Table-1 Table-2 Fluorescent Both Effectene & Enhancer Set-1 Set-2 Set-3 Fluorescent Fluorescent pdna (control) P P P P P Fluorescent Only Effectene & No Enhancer Set-1 Set-2 Set-3 Fluorescent Fluorescent pdna (control) P P P P P Table-3 Both Effectene & Enhancer Fluorescent (%) Set-1 Set-2 Set-3 Standard Deviation Average of all Sets (%) pdna (control) P P P P P Table-4 Both Effectene & Enhancer Fluorescent (%) Set-1 Set-2 Set-3 Standard Deviation Average of all Sets (%) pdna (control) P P P P P S22