HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC RESOLUTION OF AMINO-ACID ENANTIOMERS DERIVATIZED WITH FLUORESCENT CHIRAL EDMAN REAGENTS

The fluorescent chiral Edman 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-benzoxa- diazole [R(-)- and S(+)-DBD-PyNCS], have been utilized for the resolution of amino acid enantiomers as diastereomeric derivatives. These reagents react with amino acid enantiomers in the presence of base catalyst (triethylamine, 1-azabicyclo[2.2.2]octane and 1,8-diazabicyclo[5.4.0]-7-undecene) to produce the corresponding thiocarbamoyl-amino acid diastereomers under the mild reaction conditions of 55 degrees C for 10 min. The resulting derivatives are relatively stable, not only in alkaline reaction solution but also in acidic medium, with no measurable conversion to the thiohydantoin derivatives. The reactivities of both enantiomers of the derivatization reagents with the amino acid enantiomers (L- and D-isoleucine) are comparable. The fluorescence properties (maximal wavelengths and intensities) of the thiocarbamoyl-amino acid derivatives are dependent upon the solvents in the medium. Some amino acids labeled with the proposed reagents are efficiently resolved by an ODS column with water-acetonitrile containing 0.05% trifluoroacetic acid. The R, values are in the ranges 3.57-0.55 (13 amino acids) for the diastereomers obtained with NBD-PyNCS, and 2.57-0.68 (14 amino acids) for those with DBD-PyNCS. The R(5) values obtained from neutral and/or aromatic amino acids are larger than those of basic and acidic amino acids.