Optimized Peptide Synthesis Protocol

All chemicals for peptide synthesis were from commercial sources and used without further purification unless otherwise stated. Solvents, including acetonitrile (CH₃CN), dimethylformamide (DMF) were purchased from Sigma Aldrich (Milano, Italy). Acetic acid, N,N-Diisopropylethylamine (DIPEA), piperidine, and TFA were also from Sigma-Aldrich. Protected amino acids and the coupling agents OxymePure^® (ethyl 2-cyano-2-(hydroxyimino)acetate, called Oxyma Pure) and N-N′ diisopropylcarbodiimide (DIC) used for peptide synthesis were from IRIS Biotech (Marktredwitz, Germany). The peptide was prepared as previously reported [27 (link),28 (link)], with some modifications. In particular as coupling and deprotection agents we used Oxyma/DIC/DIPEA and 40% piperidine in DMF to improve the final yield and to obtain purer products. The crude material was characterized by LC–MS [29 (link)] and purified by preparative RP-HPLC using a gradient from 20% solvent B (CH₃CN, 0.1% TFA) to 80% solvent B over solvent A (H₂O, 0.1% TFA) in 20 min, monitoring the eluate at 214 nm. The purified material was collected, lyophilized, and characterized for purity and identity by LC–MS using the conditions previously reported [29 (link)].