Versatility of cinchona-based zwitterionic chiral stationary phases: Enantiomer and diastereomer separations of non-protected oligopeptides utilizing a multi-modal chiral recognition mechanism

Abstract

Cinchona alkaloid-based zwitterionic chiral stationary phases with diverse sulfonic acid cation exchanger groups were applied for enantiomer separations of small homochiral oligopeptides (all-l/all-d Ala-n, Val-n, Phe-n) using slightly acidic polar-organic mobile phase conditions which facilitated the ionization of ion exchanger sites of the chiral selectors as well as promoting zwitterion formation of the chiral selectands. Our studies focused on retention factors and selectivity as the main chromatographic parameters illustrating selector–selectand interactions. The results confirmed that bulky, aromatic side chains such as the benzyl group of Phe are analyte-inherent structural features promoting enantiorecognition. Regarding the zwitterionic selectors, a flexible spacer between the anion exchanger-carbamoyl and the cation exchanger sites was found to enhance stereoselectivity toward bulky and aromatic peptides while a rigid cation exchanger moiety contributed favorably to enantiomer distinction of peptides with small, aliphatic side chains. The investigations included all 28 stereoisomers of Phe-2 through Phe-4 to demonstrate the applicability of zwitterionic chiral stationary phases for the separation of sequential stereoisomers. Selectivity was shown to be governed by the Cinchona alkaloid motif but heavily dependent on effective hydrogen bonding between the carbamoyl motif and the amide groups of the peptide backbone. Contrary to previous studies on enantioselectivity which found pseudo-enantiomeric behavior of quinine and quinidine-based CSPs, in peptide stereoisomer separations CSPs based on quinidine undeniably performed better.