R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethyla minosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral t agging reagent, for the determination of racemic amines and amino acids, wa s studied. The reagent reacted with P-blockers selected as representative s econdary amines to produce corresponding fluorescent diastereomers (excitat ion at 460 nm and emission at 550 nm); The yields of the derivatization rea ction were dependent on the stereostructure arround the NH group in beta -b lockers. The resulting diastereomers were completely separated with single chromatographic run using Linear gradient elutions by reversed-phase chroma tography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, cons idered to be one of the primary amines, in human urine and foodstuffs. DL-a mino acids tested equally reacted with the reagent, and the thiocarbamoyl d erivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of oppos ite diastereomers on the chromatogram. The occurence of D-amino acids (D-Al a, D-Ser, D-Asp and/or D-GIu) was identified in the samples tested. The str uctures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structu re seems to be applicable to continuous sequential analysis of peptides con taining D-amino acids. The thiocarbamoyl derivatives obtained from the reac tion with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations we re:sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoin s was not avoidable, the descrimination of D- and L-configuration was demon strated with some commercially available peptides such as beta -lipotropin and [D-Ala(2)]-deltorphin II. The Edman degaradation method using R(-)-DBD- PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separati on and the detection (UV 254 nm) conditions of the derivatives were used wi thout any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala(2)]-leucine-enkephalin and [D-Ala(2)]-leucine-enkephalin were perfe ctly identidied with the autoanalysis. Copyright (C) 2001 John Wiley & Sons , Ltd.