Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method

Abstract

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5- l-leucinamide ( l-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a “meso” configuration such as diaminopimelic acid (A 2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A 2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the dl-FDLA method, and the resulting elution behavior was summarized as follows: (1) the ll- and meso-isomers were eluted prior to the dd-isomer except for one case; (2) the ll- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the l- and d-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH 3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.