Separation of 17 dl-Amino Acids and Chiral Sequential Analysis of Peptides by Reversed-Phase Liquid Chromatography after Labeling with R(−)-4- (3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole

✍ Scribed by Toshimasa Toyo′oka; Nobue Tomoi; Tomoyuki Oe; Taketsune Miyahara

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 232 KB
• Volume: 276
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4307

✦ Synopsis

Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(؊)-4-(3-isothiocyanatopyr-

rolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3benzoxadiazole (R(؊)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in DL- amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all DL-amino acids tested. The resulting thiohydantoin derivatives of individual DL-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of DL-amino acid. The method using the reagent was adopted to identification of DL-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions.