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.