Chiral recognition mechanism studies of Tyr-Arg-Phe-Lys-NH2 tetrapeptide on crown ether-based chiral stationary phase

Abstract

Even though chiral recognition for crown-ether CSPs is generally understood, on a molecular level, exact mechanisms for the resolution are still unclear. Furthermore, short peptide analytes often contain multiple amino moieties capable of binding to the crown ether selector. In order to extend the understanding in chiral recognition mechanisms, polar organic mode separation of Tyr-Arg-Phe-Lys-NH₂ tetrapeptide llll/dddd enantiomers on S- and R-(3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 stationary phases was studied with 50-mM perchloric acid in methanol as mobile phase. Deviation from the generally acceptable 1:1 stoichiometry was supported by mass spectroscopy analysis of the formed complexes between tetrapeptide enantiomer and crown ether selectors, which revealed adducts possessing 1:1, 1:2, and 1:3 stoichiometry. Further investigation of complexation induced shifts by NMR indicated on different binding mechanisms between llll/dddd enantiomers of Tyr-Arg-Phe-Lys-NH₂ and crown ether selectors. Enantioselective proton shifts were observed in studied tetrapeptide tyrosine and phenylalanine residues exclusively for llll enantiomer upon binding with S-(3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 selector (and dddd enantiomer with R-(3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 selector), indicating that these two amino acid residues contribute to chiral recognition. The obtained results were in agreement with the LC data.