4.1. Peptide Synthesis

Peptides were synthesized by routinely used standard solid-phase procedures in the Department of Biomolecular Chemistry (Medical University of Lodz, Poland) as described before [17]. Convenient and efficient techniques for 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids on Fmoc-Phe Wang (100–200 mesh, 0.69 mM/g, Novabiochem, La Jolla, CA, USA) or MBHA Rink-Amide peptide resin (100–200 mesh, 0.80 mM/g, Novabiochem) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as a coupling agent have been used. Crude peptides were purified by preparative reversed-phase HPLC on a Vydac C18 column (10 μm, 22 × 250 mm) equipped with a Vydac guard cartridge. For purification, a linear gradient of 0–100% acetonitrile containing 0.1% TFA over 15 min at a flow rate of 15 mL/min was used. The purity of the final peptides was verified by analytical HPLC employing a Vydac C18 column (5 μm, 4.6 × 250 mm) and the solvent system of 0.1% TFA in water (A) and 80% acetonitrile in water containing 0.1% TFA (B). A linear gradient of 0–100% solvent B over 25 min at a flow rate of 1 mL/min was used for the analysis. The absorbance was monitored at 214 nm. The final purity of synthesised peptides was >98%. Calculated values for protonated molecular ions were in agreement with those determined by FAB mass spectrometry. Table 1 showed the physicochemical data of tested peptides.