Production and NMR analysis of the human ibuprofen metabolite 3-hydroxyibuprofen

The anti-inflammatory drug ibuprofen (Ibu) is metabolized in the human liver to a number of metabolites including 1-hydroxyibuprofen (1-OH-Ibu), 2-OH-Ibu, and 3-OH-Ibu, respectively. The only human CYP known to produce relevant amounts of 3-OH-Ibu is CYP2C9 and as genetic polymorphisms of CYP2C9 influence the metabolization of numerous drugs, the availability of reference standards for CYP2C9-specific metabolites is of considerable interest. The aim of this study was to develop a biological production process for 3-OH-Ibu and to affirm its NMR characteristics. The recombinant fission yeast strain CAD68 coexpressing human CYP2C9 and CPR was used for the whole-cell biotransformation of Ibu to 3-OH-Ibu in 1 L batch-scale for 75 h. The average space-time yield for the bioproduction of 3-OH-Ibu (125 +/- 34 mu mol/L d) considerably exceeded that of 2-OH-Ibu (44 +/- 10 mu mol/L d). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0 +/- 0.8 mu mol/g d (3-OH-Ibu) and 1.9 +/- 0.7 mu mol/g d (2-OH-Ibu). The metabolite was prepurified on preparative TLC-plates, isolated by HPLC fractionation, and characterized by LC-MS and NMR. As expected, differential fragmentation patterns of 2-OH-Ibu and 3-OH-Ibu were detected in ESI-LC-MS analysis. 44 mg of 3-OH-Ibu was efficiently purified from four 1 L batch cultures and its structure was clearly confirmed by one-and two-dimensional NMR. (C) 2012 Elsevier B. V. All rights reserved.