Peptide synthesis and purification

Peptides were synthesized by CEM Discover microwave synthesizer using Fmoc chemistry at 100-μmol scales. The Fmoc protecting group was removed by piperidine/dimethylformamide solution (20/80 v/v); at each coupling step reactants were added with the amino acid:HBTU:DIEA:resin ratio of 5:4.9:10:1. Products were cleaved from the H-Rink Amide-ChemMatrix (PCAS, 0.53 mmol g⁻¹ loading) in a cleavage cocktail solution (trifluoroacetic acid (TFA)/triisopropyl silane/deionized water, 95/2.5/2.5 v/v) for 2 h and the remaining solution was vapourized with N₂ gas. Peptide was precipitated with cold diethyl ether (Aldrich) and dried in vacuum. After dissolving the peptides in DI H2O, purification proceeded by preparative reverse-phase high-performance liquid chromatography (Waters prep 150 LC System) using preparative C4 column (XBridge BEH300 Prep C4 5 um) and a linear gradient of buffer A (99.9% H2O and 0.1 % TFA) and buffer B (90% acetonitrile, 9.9% H2O and 0.1 % TFA). Molecular mass of the peptide was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker Ultraflex III). Products had over 95 % purity.