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 1L batch-scale for 75h. The average space-time yield for the bioproduction of 3-OH-Ibu (125±34μmol/Ld) considerably exceeded that of 2-OH-Ibu (44±10μmol/Ld). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0±0.8μmol/gd (3-OH-Ibu) and 1.9±0.7μmol/gd (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. 44mg of 3-OH-Ibu was efficiently purified from four 1L batch cultures and its structure was clearly confirmed by one- and two-dimensional NMR.
PomBioTech GmbH, D-66123 Saarbrücken, Germany.
Ina
I
Neunzig
Anke
A
Göhring
Călin-Aurel
CA
Drăgan
Josef
J
Zapp
Frank T
FT
Peters
Hans H
HH
Maurer
Matthias
M
Bureik
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9; D006801:Humans; D007052:Ibuprofen / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D012568:Schizosaccharomyces
C109994:hydroxyibuprofen; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D007052:Ibuprofen