title: Use of fission yeast heterologously expressing human cytochrome P450 2B6 in biotechnological synthesis of the designer drug metabolite N-(1-phenylcyclohexyl)-2-hydroxyethanamine.
abstract: Standards of drug metabolites are required for drug metabolism studies as a basis for toxicological risk assessment with respect to drug interactions and pharmacogenetic polymorphisms. They are further needed as reference compounds in analytical toxicology. However, metabolite standards are often not commercially available, particularly in the case of new designer drugs. As an alternative to often cumbersome chemical synthesis, human cytochrome P450 (CYP) isoenzymes heterologously expressed in the fission yeast Schizosaccharomyces pombe can be used for the biotechnological synthesis of drug metabolites. In the present study this concept was applied to the synthesis of N-(1-phenylcyclohexyl)-2-hydroxyethanamine (PCHEA), the common O-dealkyl metabolite of the designer drugs N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) and N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA). After adding 250 micromol PCEEA x HCl (62 mg), a 1 l culture of CAD65 (S. pombe strain co-expressing human CYP reductase and CYP2B6) was fermented over 65 h (pH 8, 30 degrees C) and centrifuged. PCHEA and remaining parent drug were isolated from the supernatant by solid-phase extraction (SPE). The eluate was evaporated to dryness and reconstituted in HPLC solvent. Aliquots were separated by semi-preparative HPLC. From the respective fraction, PCHEA was extracted by liquid-liquid extraction and precipitated as hydrochloric salt. Approximately 80% of PCEEA was converted to PCHEA. The final yield of PCHEA x HCl was 9 mg (35 micromol). Its identity was confirmed by GC-MS, (1)H NMR and (13)C NMR. The product purity, as determined by HPLC-UV, was 95%.
authors.affiliation: Department of Experimental and Clinical Toxicology, Saarland University, Building 46, D-66421 Homburg (Saar), Germany. frank.peters@uks.eu
authors.forename: Frank T
authors.initials: FT
authors.lastname: Peters
authors.forename: Calin-A
authors.initials: CA
authors.lastname: Dragan
authors.forename: Andrea E
authors.initials: AE
authors.lastname: Schwaninger
authors.forename: Christoph
authors.initials: C
authors.lastname: Sauer
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.forename: Matthias
authors.initials: M
authors.lastname: Bureik
authors.forename: Hans H
authors.initials: HH
authors.lastname: Maurer
mesh_terms: D001709:Biotechnology; D003510:Cyclohexanes / Q000378:metabolism*; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015198:Designer Drugs / Q000378:metabolism*; D005021:Ethylamines / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism*
chemical_list: D003510:Cyclohexanes; D015198:Designer Drugs; D005021:Ethylamines; C541249:N-(1-phenylcyclohexyl)-2-hydroxyethanamine; D003577:Cytochrome P-450 Enzyme System; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010622:Phencyclidine