Synthesis of penetratin
Penetratin (RQIKIWFQNRRMKWKK-amide, molecular weight 2245.75 g/mol) was synthesized manually on TentaGel R RAM (Rapp Polymere, Tübingen, Germany), a low crosslinked polystyrene PEG copolymer resin (the substitution was 0.2 mmol/g) by the solid-phase method of Merrifield with standard Fmoc chemistry. Amino acids for peptide synthesis were purchased from Iris Biotech (Marktredwitz, Germany). Amino acid side chains were protected as follows: Trt for Gln and Asn,t -butoxycarbonyl (Boc) for Trp and Lys, and Pbf for Arg. A typical coupling reaction contained 3 equivalents of Fmoc-amino acid, 3 equivalents of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and 6 equivalents of diisopropylethylamine (DIPEA), and was allowed to proceed with mixing in dimethylformamide (DMF) for at least 2.5 hours. Completion of the coupling was assessed by Kaiser test. The Fmoc group was removed by treatment with 2% piperidine, 2% 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF, followed by six washes with DMF-MeOH-CH2Cl2. Completion of deprotection was assessed by Kaiser test.