Biotechnological Synthesis of the Designer Drug Metabolite 4′-Hydroxymethyl-α-pyrrolidinohexanophenone in Fission Yeast Heterologously Expressing Human Cytochrome P450 2D6-A Versatile Alternative to Multistep Chemical Synthesis

1-(4-Methylphenyl)-2-pyrrolidin-1-ylhexan-1-one (4′-methyl-α- pyrrolidinohexanophenone, MPHP) is a new designer drug that appeared on the illicit drug market. It is mainly metabolized to 4′-hydroxymethyl-α- pyrrolidinohexanophenone (HO-MPHP) followed by oxidation to the respective carboxylic acid. For studies on the quantitative involvement of human cytochrome P450 (CYP) isoenzymes in the initial hydroxylation, a reference standard of HO-MPHP was needed. Therefore, the aim of this study was to synthesize this metabolite using a biotechnological approach. MPHP·HNO3 (250 μmol) was incubated with 1 L culture of the fission yeast (Schizosaccharomyces pombe) strain CAD64 heterologously co-expressing human CYP reductase and CYP2D6. After centrifugation, the product was isolated from the incubation supernatants by solid-phase extraction. Further product cleanup was achieved by semi-preparative high-performance liquid chromatography (HPLC). After extraction of HO-MPHP from the respective eluent fractions, it was precipitated as its hydrochloric salt. The final product HO-MPHP·HCl was obtained in a yield of 138 μmol (43 mg, 55%). Its identity was confirmed by full scan gas chromatography-mass spectrometry (after trimethylsilylation), 1H-NMR, and 13C-NMR. The product purity as estimated from HPLC-ultraviolet analysis was greater than 99%. The described biotechnological approach proved to be a versatile alternative to the chemical synthesis of HO-MPHP.