Preparation of diastereomeric β-d-glucuronides of the bronchodilator procaterol using immobilized rabbit liver microsomal enzymes

Abstract

Liver microsomal pellets from a phenobarbital induced New Zealand white male rabbit were prepared, solubilized, and reacted with cyanogen bromide activated Sepharose beads to form an immobilized microsomal enzyme preparation. Viability of bound enzymes was determined using established methods. Racemic erythro-[3H]-procaterol hydrochloride was incubated with immobilized microsomal enzymes at room temperature in 0.1 M potassium phosphate buffer (pH 7.4) together with cofactors magnesium chloride and UDPGA for 16 h. The incubation mixture was filtered and the filtrate fractionated by a C18 solid phase extraction procedure. Two polar reaction products were isolated and characterized by TLC, HPLC-RAM (homogeneous flow-through radioactivity detection), [1H]-NMR, and Fast-Atom Bombardment mass spectrometry as diastereomeric aryl-O-glucuronides of erythro-procaterol. Based on HPLC-RAM analysis, glucuronidation of racemic procaterol proceeds with regiostereoselectivity. In addition, both diastereomers are formed in nearly equal amounts indicating lack of enantioselectivity in this reaction. Combined yield of both diastereomers was approx. 60%.