DEVELOPMENT OF OPTICALLY-ACTIVE FLUORESCENT EDMAN-TYPE REAGENTS

The optically active fluorescent 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-dimethyl-aminosulfonyl)-2,1,3-benzoxadiazole [(R)-(-)- and (S)-(+)-DBD-PyNCS] were synthesized and characterized by their physico-chemical properties. High optical purities of all four enantiomers were identified by high-performance liquid chromatography (HPLC) with a chiral stationary phase (CSP) column. Good stability in both solid and liquid forms is one of the excellent properties of these novel Edman-type reagents. The applicability of these reagents to the resolution of mirror-image enantiomers of amines, including amino acids and some drugs (beta-adrenergic blocking agents), by reversed-phase HPLC was demonstrated. The proposed reagents read quantitatively with primary and secondary amino functional groups under mild conditions (55 degrees C for 10 min) in the presence of triethylamine (TEA) to produce the corresponding fluorescent thiourea derivatives. The maximum excitation/emission wavelengths of the resulting diastereomers in acetonitrile-water (1 + 1) are around 490/530 nm for NBD-PyNCS derivatives and around 460/550 nm for DBD-PyNCS derivatives. The fluorescence wavelengths are independent of the amino compounds. The diastereomers that were derived from the amines were efficiently resolved on an achiral ODS column with water-acetonitrile containing 0.05% trifluoroacetic acid (TFA). The R(S) values were in the range 0.83-4.12 for the diastereomers obtained with NBD-PyNCS and 1.35-6.06 for those with DBD-PyNCS. The diastereomer corresponding to the R configuration of the amines elute faster than those of the S configuration with the (R)-(-)-enantiomer of the Edman-type reagents used as the derivatization reagent. As expected, the opposite elution order is obtained with the use of the (S)-(+)-enantiomers.