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

Biomedical Chromatography2001Vol. 15(1), pp. 56–67

Toshimasa Toyo’oka, Dongri Jin, Nobue Tomoi, Tomoyuki Oe, Hidenori Hiranuma

Abstract

The usefulness of R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(−)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with β-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in β-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(−)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction 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 were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as β-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(−)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without 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-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: FITC fluorescein isothiocyanate GITC 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate MeOH methanol OPA ortho-phtalaldehyde PITC phenylisothiocyanate R(−)-DBD-PyNCS R(−)-4-(3-isothiocyanatopyrrolidin-1-yL)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran.

Abstract

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