Microbial models of mammalian metabolism: conversion of warfarin to 4'-hydroxywarfarin using Cunninghamella bainieri

Warfarin, an anticoagulant and "metabolic probe" for cytochrome P-450 isozyme multiplicity, is metabolized to 4'-hydroxywarfarin, a principle mammalian metabolite, using the fungus Cunninghamella bainieri (UI-3065). The metabolite was isolated from cell suspension cultures and characterized by analytical (TLC, HPLC, GC-MS) and spectral (HRMS, EI-MS, PMR) comparisons with authentic 4'-hydroxywarfarin. The mechanism of aromatic hydroxylation was examined in C. bainieri using 4'-deuterowarfarin. The absence of a primary isotope effect (KH/KD = 1.13), migration and retention of deuterium in the phenolic product [80% migration and retention (M&R)], and inhibition of the hydroxylation by carbon monoxide (93% inhibition in a 50:50 CO:O2 atmosphere) are consistent with a cytochrome P-450-mediated hydroxylation involving the classic NIH shift (arene oxide) pathway.