Determination of d-amino acids. I. Hydrolysis of DNP-l-amino acid methyl esters with carboxypeptidase-Y

Abstract

l- and d-amino acids were treated with fluoroniditrobenzene to form DNP-derivatives. The latter were esterified with methanol in the presence of dry HCl gas to the corresponding methyl esters. Treatment of the ester mixture with carboxypeptidase-Y resulted in the hydrolysis of only l-esters. All d-esters and some l-esters were unaffected. The produced DNP-l-amino acids could be separated from DNP-d-amino acid methyl esters by high performance liquid chromatography allowing quantitation of the d-isomers. When a test mixture consisting of Ala, Asp, Glu, Met and Phe, present in different proportions as l- and d-isomers, was subjected to the above procedure, all d-amino acids could be quantitated. The method allows detection of d-amino acids in the nanomole range. © 1984 Carlsberg Laboratory.