DEVELOPMENT OF A NEW CHIRAL DERIVATIZING REAGENT, (+)-2-METHYL-2-BETA-NAPHTHYL-1,3-BENZODIOXOLE-4-CARBOXYLIC ACID, FOR ENANTIOMER ANALYSIS OF AMINO-ACIDS

A new fluorescent chiral derivatizing reagent, (+)-MNB-carboxylic acid [(+)-2-methyl-2-beta-naphthyl-1,3-benzodioxole-4-carboxylic acid], was developed. This reagent was designed to have a high ability to separate enantiomeric amino acids as well as to give strong fluorescence (Ex 310 nm, Em 370 nm) for the highly sensitive HPLC analysis. It: was prepared through a CuSO4 catalyzed condensation between 3-methyl-catechol and P-acetonaphthone followed by the oxidation of the methyl group at C-4 and the optical resolution of the derived racemate with (-)-cinchonidine. The labeling of amino acids with this agent could be achieved simply: a mixture of amino acids and (+)-MNB-COCl (more than 25 fold excess) in lithium carbonate buffer (pH 9.0) was incubated at 20 similar to 22 degrees C for 30 min. Part of the reaction mixture could be directly subjected to the HPLC analysis. The D,L-separation of individual amino acids was well achieved under reverse-phase (ODS) HPLC with aqueous THF (pH 5.9). The detection limit was ca. 0.1 pmol as the injection amount (fluorescence detection, S/N = 2/1). The above data demonstrated that (+)-MNB-carboxylic acid could function as a useful agent for the highly sensitive HPLC analysis of D,L-amino acids.