CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping

Andy Zöllner, Maria Kristina Parr, Călin-Aurel Drăgan 1 , Stefan Dräs 1 , Nils Schlörer 3 , Frank T. Peters, Hans H. Maurer 4 , Wilhelm Schänzer 2  and Matthias Bureik 1
  • 1 PomBioTech GmbH, D-66123 Saarbrücken, Germany
  • 2 Institute of Biochemistry, German Sport University, D-50933 Cologne, Germany
  • 3 Institute of Organic Chemistry, University of Cologne, D-50939 Cologne, Germany
  • 4 Department of Experimental and Clinical Toxicology, Saarland University, D-66421 Homburg/Saar, Germany
  • 5 Institute of Forensic Medicine, Friedrich Schiller University, D-07740 Jena, Germany
  • 6 These authors contributed equally to this work.

Abstract

Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.

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