Decoding the metabolic activation of clopidogrel via an unspecific peroxygenase of Marasmius rotula

Clopidogrel ( CPG ) is an antiplatelet prodrug that requires metabolic activation in the liver by cytochrome P450 (CYP) enzymes to exert its antithrombotic effect. This activation is a two-step process: CPG is first converted to the intermediate 2-oxoclopidogrel ( 2-OxoCPG ), and then to the clopidogrel active metabolite ( CAM ). This study was focused on investigating this activation using an unspecific peroxygenase (UPO) from the fungus Marasmius rotula ( Mro UPO), which mimics CYP activity in a simpler manner. Detailed tracer studies were conducted using 2 H 2 O (D 2 O), H 2(R.P.D. Bank, n.d. 18) O 2 , and H 2(R.P.D. Bank, n.d. 18) O to follow the fate of oxygen. Results revealed that 2-OxoCPG is formed by epoxidation using an oxygen atom from H₂ 18 O₂, followed by a chemical rearrangement to stabilize the ketone. The subsequent oxidation of 2-OxoCPG to CAM is proposed to follow a Baeyer Villiger-like mechanism. This step may be initiated by the Compound 0 of Mro UPO and forms short-lived intermediates, whose structures depend on the two isomers of 2-OxoCPG (4 S ,7 S - and 4 R ,7 S -) specifically interacting with the enzyme. These intermediates are converted into an oxathiin derivative and a ring-fission product. Their subsequent hydrolysis yields sulfenic acid derivatives, which were detected indirectly by forming adducts with dimedone and which are reduced to the final active CAM isomers, CAM-H3 and CAM H4 . The study highlights the crucial role of 2-OxoCPG isomerism in the formation of intermediates that determine the subsequent biosynthetic steps for CAM formation. Our findings demonstrate that UPOs are a valuable and efficient tool for studying CYP-mediated metabolic pathways, as they offer high catalytic mimicry, yield, and ease of use.