DETERMINATION OF D-AMINO-ACID AND L-AMINO-ACID RESIDUES IN PEPTIDES WITH FLUORESCENT CHIRAL TAGGING REAGENTS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Derivatization of amino acid enantiomers with fluorescent chiral Edman-type reagents, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [R(-)- and S(+)-NBD-PyNCS] and 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)- and S(+)-DBD-PyNCS], yields corresponding diastereomers separable by reversed-phase HPLC on normal achiral columns. The resolved diastereomers were detected fluorometrically at 530 nm with excitation at 490 nm for the NBD-PyNCS derivatives and at 560 nm with excitation at 450 nm for those derived from DBD-PyNCS reagents. This HPLC-derivatization method was used for evaluation of stereochemical purity for some synthetic commercial peptides. The enantioanalysis was reliable down to 0.05 % racemization of the amino acid residues and a quantity of 100 mu g peptide sample was usually enough for the analysis. Two acid hydrolysis methods, i.e. the standard procedure with constant-boiling hydrochloric acid (HCl) and a rapid vapor-phase procedure with HCl-trifluoroacetic acid (TFA) mixture, were compared. The later was found to be unsuitable owing to increased racemization of the amino acid residues during the hydrolysis. Judging from the results obtained for proline and leucine residues, most of the tested peptides including biologically active peptides, such as neurotensin, [D-Ala(2), D-Leu(5)]-enkepharin and morphine tolerance peptide, possessed stereochemical purities higher than 98 %. Influence of structural features of the peptides on the racemization of the amino acid residues was found to be significant.