Reversed-phase high-performance liquid chromatography for the separation of peptide diastereoisomers

Diastereoisomers of peptides with biological activities often exhibit pronounced antagonistic or agonistic properties which differ greatly from each other. This fact, as well as the difficulty in obtaining many uncommon amino acids in optically pure form, underlies the importance of having at hand effective chromatographic methods for the purification and characterization of diastereoisomeric mixtures of peptides. Diastereoisomeric peptides, isomers in which one or more asymmetric centers have opposite configuration, may possess similar or different physical-chemical or biological properties. There exists a number of ways a diastereoisomeric mixture of peptides can be generated: (1) formation of a diastereoisomeric mixture may result from a peptide synthesis methodology which leads to racemization of one or more amino acid residues; (2) non-eukaryotic amino acids are often synthesized as a racemic mixture and are utilized as such for peptide synthesis; (3) commercially available D-amino acids usually contain small amounts of the L isomer; and (4) incorporation of isotopes into a peptide is usually accomplished by synthesis of a racemic modification of the amino acid followed by separation of the resulting diastereoisomeric peptides. This review will focus on the practical aspects of utilizing reversed-phase high-performance liquid chromatography (RPC) as a method for the purification and characterization of peptide hormone diastereoisomers. Since aspects of this subject were last reviewed, 1, 2 additional successful separations of peptide diastereoisomers have been reported. These recent advances will be addressed in this review, as will work from our laboratory which presents a practical approach to the separation of four closely related diastereoisomers of [ D - P en 2, D-Pe n 5 ] enkephalin (DPDPE) which differ from each other only stereochemically at a single amino acid residue.