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 μmol PCEEA·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 °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·HCl was 9 mg (35 μmol). Its identity was confirmed by GC–MS, 1H NMR and 13C NMR. The product purity, as determined by HPLC-UV, was 95%.