High-performance liquid chromatography of isopeptides

High-performance liquid chromatographic (HPLC) methods are described for the structural analysis of clavicepamines, their analogues and branched-chain polypeptides, the analysis in synthetic stages of isopeptides (analysis of half-protected derivatives and purity control of active esters) and the differentiation between alpha- and iso-peptides (such as alpha- and gamma-glutamyl peptides). Pre-column derivatization was used to label the free amino groups for the structural investigation. Dansylated and hydrolysed isopeptides were analysed by HPLC methods based on isocratic separation of alpha-, epsilon- and bis-Dns-lysines using Hypersil, ODS-Hypersil and Partisil PAC columns. For the analysis of peptide active esters, an RP-HPLC method was developed, with methanol-acetonitrile-water mobile phases containing an acidic buffer. Peptides containing alpha-glutamyl residues were analysed for gamma-isomer content in the same system.