Biocatalytic Syntheses of Antiplatelet Metabolites of the Thienopyridines Clopidogrel and Prasugrel Using Fungal Peroxygenases

Abstract

Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL−1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h−1 H2O2 and 20% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays.

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

“…The ability of UPOs to generate pharmacologically relevant substrates is also of particular interest. The conversion of the antiplatelet drug clopidogrel to its active metabolite by different UPOs was also recently demonstrated (Kiebist et al, 2021).…”

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

“…The β-adrenergic blocker propranolol, the non-steroidal anti-inflammatory drug diclofenac, and the antithrombotic prodrug clopidogrel are known to be metabolized by cytochrome P450 monooxygenases in the human body. In previous studies, it was shown that wt Aae UPO is able to perform the same reactions to give the (major) human drug metabolites 5-hydroxypropranolol, 4′-hydroxydiclofenac, and 2-oxoclopidogrel, respectively ( Figure 5 ) [ 15 , 40 , 52 ].…”

“…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 ].…”

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation

“…One of the first steps in the metabolism of thienopyridines 76, clopidogrel 1q, and ticlopidine 1p was oxidation of the thiophene ring by a CYP450 monooxygenase (or UFO peroxygenase) to yield an unstable thiophene epoxide 77. This intermediate isomerized into a 2-hydroxythiophene 78 and its thiolactone tautomers 79a and 79b (Path A, Scheme 15) [159]. Further CYP450-or peroxygenase-catalyzed oxidation of tautomers 78/79a/79b led to a reactive thiolactone S-oxide 80 that hydrolyzed to a sulfenic acid 81 and was trapped using dimedone [120,[160][161][162][163][164].…”

“…The unstable S-oxide 84 was trapped by dienophiles, e.g., N-methyl-(NMM) and N-ethyl-maleimide (NEM) [119,120] to give product 86, or thiols, e.g., glutathione (GSH) to give adducts 87 [173]. The oxidation reactions (Path A and Path B) were also catalyzed by unspecific fungal peroxygenases (UFO) in the presence of ascorbic acid and hydrogen peroxide [159].…”