High-performance liquid chromatographic separation of dl-amino acids derivatized with chiral variants of Sanger's reagent

Abstract

Substitution of one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene by l-alanine amide yields N 2-(5-fluoro-2,4-dinitrophenyl)- l-alanine amide (FDNP-Ala-NH 2, “Marfey's reagent”). This reagent (or analogues, termed FDNP reagents, in which Ala is replaced with other chiral α-amino acids (AAs), oligopeptides or amino components) might be considered as a chiral variant of “Sanger's reagent” (1-fluoro-2,4-dinitrobenzene). The remaining fluorine atom in FDNP reagents can be substituted by chiral AAs to furnish diastereomeric derivatives which are separable by high-performance liquid chromatography (HPLC). A number of chiral variants of Sanger's reagent have been synthesized with the general structures (a) FDNP-Val-NHR (R = H, tert.-butyl, chiral aralkyl, phenyl, p-nitrophenyl), (b) FDNP-Val-OR (R = H, CH 3, tert.-butyl), (c) FDNP-(Ala) n-NH 2, n = 1,2, and (d) FDNP-NHR (R = aralkyl and hydroxyalkyl), and their ability to resolve certain dl-AAS as diastereomers in reversed-phase HPLC was investigated. Reagents with the structures (a), (b) and (c), but not (d), made possible the separation of dl-AAs as diastereomers. From the results obtained and from observations of Corey-Pauling-Koltun (CPK) space-filling molecular models of the diastereomers, it is concluded that the AA side-chains and the carboxy and carboxamide substituents in the reagents have an influence on the retention times of diastereomers and that the differences in free energy as a result of the formation of an intramolecular hydrogen bridge in the l-l and non-formation in the d-l diastereomers (first letter refers to the configuration of the amino acid to be analysed) are in particular responsible for large differences in the retention times of certain diastereomers in HPLC.