Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene

Abstract

1-fluoro-2,4-dinitrophenyl-5-l-alanine amide has been synthesized in high yield (76%) from 1,5-difluoro-2,4-dinitrobenzene and l-Ala-NH2. This compound contains a reactive fluorine atom which can be used for the reaction with a mixture of l- and d-amino acids. The resulting diastereomers which are obtained in quantitative yield can be separated and estimated by HPLC. With the five amino acids studied (Ala, Asp, Glu, Met and Phe), l-diastereomers were eluted from the reverse-phase column before d-diastereomers. This behavior can be explained by a stronger intramolecular hydrogen bonding in the latter diastereomer. When artificial mixtures of the five amino acids containing known proportions of l- and d-isomers were derivatized with the reagent and the reaction products analyzed by HPLC, it was possible to determine the relative content of each isomer in nanomole range. © 1984 Carlsberg Laboratory.