Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells

Abstract

Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPA UPO). 4-OH-CPA UPO-induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPA UPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

“…Recently, Scheibner et al. used Mro UPO for hydroxylation of the cytostatic drug cyclophosphamide ( 34 ) (CPA), to produce HDM 4‐hydroxycyclophosphamide ( 35 ) (4‐OH‐CPA) in 32 % yield (>97.6 % purity) [158] . Cgl UPO has been studied for the oxyfunctionalisation of testosterone, producing 90 % of the 4,5‐epoxide product and 10 % HDM 16α‐hydroxytestosterone, both in high diastereoselectivities (>98 %) [159] .…”

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions

“…Mro UPO was also found to be the superior UPO among four including Aae UPO, Cci UPO, and Cg lUPO for the oxygenation of cyclophosphamide 66 ( Scheme 14 ). 97 …”

Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases

“…Essential for this type of reaction is the heme cofactor that is coordinated proximally by the thiol of a cysteine residue. Due to their P450-like catalytic mechanism and reaction spectrum, UPOs can be used for the conversion of pharmaceuticals and drugs, e.g., of volixibat ( N -dealkylation) [ 10 ], cyclophosphamide (aliphatic hydroxylation) [ 11 ], propranolol (aromatic hydroxylation) [ 12 ], testosterone (epoxidation) [ 13 ], corticosteroids (side-chain removal by C-C scission) [ 14 ] and clopidogrel (sulfoxidation and epoxidation) [ 15 ].…”