Synthesis of (KFF)3K-N3

Azido-peptide was synthesized by manual solid-phase peptide synthesis (SPPS) using the standard Fmoc/t-Bu chemistry on a 100 µmol scale with a 3-fold molar excess of the Fmoc-protected amino acids and Rink-amide resin (TentaGel S RAM resin, amine groups loading of 240 μmol/g; this resin has a linker which yields a C-terminal amide upon TFA cleavage of the peptide). Fmoc-protected amino acids were assembled as active derivatives in a 3-fold molar excess by the use of HATU with the addition of 1-hydroxy-7-azabenzotriazole (HOAt) and collidine (1:1:2), using the DMF/NMP (1:1, v/v) solution-coupling method for 2 h. Fmoc deprotection was accomplished using 20% piperidine in DMF for 2 cycles (5 and 15 min). Removal of the protecting group (Boc from Lys) and cleavage of peptide from the resin was performed by treatment with a TFA/triisopropylsilane/m-cresol (95:2.5:2.5; v/v/v) mixture for 60 min. The obtained crude peptide was lyophilized and subsequently purified by RP-HPLC.