Biotechnological synthesis of the designer drug metabolite 4'-hydroxymethyl-alpha-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-alpha-pyrrolidinohexanophenone, MPHP) is a new designer drug that appeared on the illicit drug market. It is mainly metabolized to 4'-hydroxymethyl-alpha-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.HNO(3) (250 micromol) 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 micromol (43 mg, 55%). Its identity was confirmed by full scan gas chromatography-mass spectrometry (after trimethylsilylation), (1)H-NMR, and (13)C-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.
Peters
Frank T
FT
Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421 Homburg (Saar), Germany.
Dragan
Calin-Aurel
CA
Kauffels
Anne
A
Schwaninger
Andrea E
AE
Zapp
Josef
J
Bureik
Matthias
M
Maurer
Hans H
HH
D001711:Biotransformation; D002090:Butyrophenones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D015198:Designer Drugs / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011759:Pyrrolidines / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
C541457:1-(4-methylphenyl)-2-pyrrolidin-1-ylhexan-1-one; C522834:4'-hydroxymethyl-alpha-pyrrolidinobutyrophenone; D002090:Butyrophenones; D015198:Designer Drugs; D011759:Pyrrolidines; D011994:Recombinant Proteins; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase