High-performance liquid chromatography and nuclear magnetic resonance study of linear tetrapeptides and octapeptides containing N-methylated amino acid residues

Abstract

Chromatographic behavior of a series of N-methylated tetra and octapeptides on a reversed-phase sorbent was studied considering the information obtained on these compounds by NMR spectroscopy. The modified tetrapeptides were derived from GFFY-NH 2, GFFF-NH 2 and GFFH-NH 2 primary structures by N-methylation at various peptide bond positions. Similarly, the N-methylated octapeptides were based on TPK(Pac)T C-terminally elongated forms of GFFY and GFFF. It was found that many studied N-methylated peptides provide broad peaks as a consequence of cis/ trans isomerism of the R 1CO N(CH 3)R 2 peptide bond. The extent of the peak spreading depends on the following important factors: the nature of the surrounding amino acid residues, the location of the modified peptide bond within the peptide chain, temperature, and mobile phase flow-rate. All these aspects were critically evaluated. Nearly complete separation of the individual conformers of GF(NMe)FY-NH 2 was obtained applying fast chromatography on short column packed with 20–30 μm reversed-phase sorbent.