1 Electronic Supplementary Information Self-assembling mini cell-penetrating peptides enter by both direct translocation and glycosaminoglycan-dependent endocytosis S. A. Bode, M. Thévenin, C. Bechara, S. Sagan, S. Bregant, S. Lavielle, G. Chassaing, F. Burlina Abbreviations Boc, tert-butoxycarbonyl; BSA, bovine serum albumin; CHCA, α-cyano-4-hydroxycinnamic acid; CHO cells, Chinese hamster ovary cells; CPP, cell-penetrating peptide; DAPI, 4’,6-diamidino-2-phenylindole; DCC, dicyclohexylcarbodiimide; DLS, Dynamic light scattering, DMEM, Dulbecco's modified Eagle medium; DIEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DTT, dithiotreitol; FBS, fetal bovine serum; EDTA, ethylenediamine tetraacetic acid; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate; HCTU, O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt, 1-hydroxybenzotriazole; 1 H-PKCi, non deuterated PKCi; 2 H-PKCi, deuterated PKCi; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time- of-flight mass spectrometry; MBHA-PS, 4-methylbenzhydrylamine polystyrene; Npys, 3-nitro-2-pyridinesulphenyl; PBS, phosphate buffered saline; PKCi, protein kinase C inhibitor; rt, room temperature; SEM, standard error of the mean; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid. Reagents Coupling reagents for peptide synthesis and protected amino acids were purchased from Novabiochem (Merck Chemicals Ltd) except for (2,2-D 2 , 98%)-Boc Glycine, which was obtained from Euriso-top. Solvents for peptide synthesis and TFA were obtained from SDS. Tris-HCl, Triton X-100, trypsin inhibitor and bovine serum albumin were obtained from Sigma-Aldrich. Dulbecco’s modified Eagle medium, fetal bovine serum, trypsin-EDTA (0.05 % trypsin, 0.02 % EDTA) and Hank’s BSS were purchased from PAA. The cell counting kit (CCK8) was from Dojindo Laboratories. Streptavidin-coated magnetic beads (Dynabeads® MyOne™ Streptavidin C1 or Dynabeads® M-280 Streptavidin) were purchased from Invitrogen. The complete mini tablets of protease inhibitors were from Roche. DAPI was from Pierce. Peptide synthesis Cysteamino-Merrifield resin Merrifield resin (0.5 g, loading 1.09 mmol/g) was swollen in DMF. Cysteamine hydrochloride (0.186 g, 5.45 mmol) was added to an ice-cooled suspension of NaH 60 % in mineral oil (0.218 g, 5.45 mmol) in dry DMF (1.5 mL). The resulting mixture was stirred at rt until the generation of hydrogen gas stopped (about 20 min) and then added to the Merrifield resin in DMF. The mixture was shaken for 48 h at rt or heated at 50 °C under micro-waves (10 x 5 seconds, 60 W). The resin was washed with CH 2 Cl 2 , CH 3 OH, H 2 O and CH 2 Cl 2 and dried under vacuum. Synthesis of CPPs C4 to C18 Amounts of reagent are given in equivalents (eq.) with respect to the peptidyl-resin. CPPs were synthesised manually on the cysteamino-Merrifield resin (0.2 g, 0.218 mmol) using the Boc strategy. Boc-Arg(Tos)-OH (3 eq.) was activated by 5 min treatment with HBTU (2.9 eq.), HOBt (3 eq.) and DIEA (6 eq.) in DMF (final amino acid concentration: 0.45 M). Each coupling was performed by 5 shots of 5 s of micro-waves (50 °C, 60 W). Between each shot, the resin was cooled in liquid nitrogen. The reaction was monitored by the Kaiser test and double couplings were performed if necessary. The Boc protecting group was removed by treatment with TFA (3 x 1 min) followed by neutralisation with DIEA (20 % in DMF). Coupling of the carbon chains was performed using either the chloride derivative (3 eq.) (lauroyl chloride for C12, myristoyl chloride for C14) or the carboxylic acid derivative (3 eq.) (palmitic acid for C16, stearic acid for C18) with HBTU/DIEA activation. Reactions were performed under micro-waves (5 x 5 s, 50 °C, 60 W). Peptide cleavage from the resin was performed by treatment with HF (2 h, 0 °C) in presence of anisole (1.5 mL/g peptidyl-resin), dimethylsulfide (0.25 mL/g peptidyl-resin) and p-toluenethiol Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2012