Indirect resolution of thiol enantiomers by high-performance liquid chromatography with a fluorescent chiral tagging reagent

Abstract

The fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)- 7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-PyNCS), was utilized for the resolution of thiol enantiomers as diastereomeric derivatives. The reagent reacts with thiol enantiomers in the presence of pyridine to produce the corresponding dithiocarbamate diastereomers under mild reaction conditions (50 °C for 40 min). The fluorescence properties (maximum wavelengths and intensities) of the derivatives were dependent on the solvents in the medium. Several thiols derivatized with the proposed reagent were efficiently resolved by an ODS column with water–acetonitrile containing 0.1% v/v trifluoroacetic acid as the mobile phase. The resolution (Rs) values of the thiols tested were in the range 1.05–3.33 for the diastereomers obtained with R-(–)-DBD-PyNCS. The detection limits (signal-to-noise ratio of 3) with the proposed HPLC separation and fluorescence detection were in the range 0.4–2.4 pmol. The dithiocarbamate of tiopronin resulting from the labelling reaction with R-(–)-DBD-PyNCS was fairly stable. However, the fluorophore moiety in the diastereomer produced from penicillamine transferred from the thiol functional group to the amino group (S → N conversion) with increasing reaction time. Finally, N,S-double labelled compounds were produced from the tagging reaction with excess amounts of DBD-PyNCS. These structures were elucidated from the fragments of molecular (M + H)⁺ and product ions by on-line HPLC–electrospray ionization MS–MS.