Discrimination of d/l-mamino acid in peptide sequence based on fluorescent chiral derivatization by reversed-phase liquid chromatography

A fluorescent chiral derivatization reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3benzoxadiazole (R(-)-DBD-PyNCS), was used for identification of d/l-amino acid in peptide sequence. The reagent reacted with N-terminal amino functional group in the peptide under the conditions of 65 °C for 60 min in 25% pyridine. The resulting thiocarbamoyl derivative of peptide was converted to thiohydantoin via thiazolinone at 55 °C for 1 min in trifluoroacetic acid (TFA). The maximum excitation and emission wavelengths of the derivative were around 460 and 550 nm, respectively. The racemization during the reactions of cleavage and cyclization was low judging from the chromatogram of the reaction solution. The thiohydantoin derivatives of N-terminal amino acid liberated from the peptides labelled were separated by reversed-phase chromatography with acetonitrile/phosphate buffer (pH 6.8) as the eluent. The separation of the thiohydantoins yielded from neutral and aromatic amino acids was good enough for the identification of d/l-amino acid. However, those from aspartic acid, glutamic acid, histidine, serine, proline and cystine were impossible or insufficient for resolution by the solvent composition. The peaks corresponding to d-amino acids were eluted faster than those from l-amino acids using R(-)-DBDPyNCS. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [d-Ala2]-leucine-enkephalin. l-Tyr in first residue, d-Ala in second residue and Gly in third residue of the peptide were completely identified with the recommended method.