Highly Selective Molecular Recognition of Biologically Active Substances Using Liquid Phase Separation

摘要

The development of new chiral stationary phases has been very important in the a ccurate analysis of drug enantiomers and their metabolites in biological samples during drug discovery and development. New chiral stationary phases have been d eveloped using conalbumin and flavoprotein from chicken egg whites, which have b een applied to a broad range of drug enantiomers. The application and characteri zation of these two chiral columns for high-performance liquid chromatograp hy have been documented. Both specific and non-specific interactions, based on the silica gel surface and linker moiety, influenced retention and chiral separa tion of solutes. Interactions between drug enantiomers and proteins, as a pseudo chiral stationary phase, were investigated with affinity capillary electrophore sis, in order to avoid the effects of non-specific interactions. The chiral dis crimination region for ketoprofen on the flavoprotein surface was concluded to c onsist of an α-helix structure. Studies with chemically modified flavoprot ein indicated that two types of interactions at the chiral discrimination region were required for chiral separation: a π-π interaction between a tryptophan residue and the aromatic ring of ketoprofen, and an ionic interaction between th e carboxyl group of ketoprofen and an amino and carboxyl group of the protein. I n the bod y, drugs and biologically active substances having a carboxyl group have been kn own to transform various metabolites such as acyl glucuronide. The acyl adenylat e has also been noted as a chemically active intermediate of coenzyme A ligation . Both the acyl adenylate and the acyl glucuronide produced protein adducts by r eacting with nucleophilic groups such as amino groups on protein molecules. To c haracterize both active intermediates and protein adducts, analytical techniques conferring highly selective molecular recognition, such as high-performance li quid chromatography and mass spectrometry, were required.