Chromatographic behaviour of opioid peptides containing beta-methylphenylalanine isomers

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed to obtain pure erythro[2S3S, 2R3R]- and threo[2S3R, 2R3S]-beta-methylphenylalanine. These amino acids were incorporated into an enkephalin, H-Tyr-D-Ala-Gly-beta-MePhe-Val-Val-Gly-NH2, and into a deltorphin C, H-Tyr-D-Ala-beta-MePhe-Asp-Val-Val-Gly-NH2, analogue, which yielded four diastereoisomers of the peptides. The diastereoisomers were separated on different columns and with different eluent systems. The sequence of elution of the peptide diastereoisomers was determined after hydrolysis of the peptides. For identification of the beta-methylphenylalanine enantiomers, enzymatic degradation and an RP-HPLC method were used, with application of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide as derivatizing reagent.