Selective Synthesis of the Human Drug Metabolite 5′-Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase

Abstract

Propranolol is a widely used beta-blocker that is metabolized by human liver P450 monooxygenases into equipotent hydroxylated human drug metabolites (HDMs). It is paramount for the pharmaceutical industry to evaluate the toxicity and activity of these metabolites, but unfortunately, their synthesis has hitherto involved the use of severe conditions, with poor reaction yields and unwanted byproducts. Unspecific peroxygenases (UPOs) catalyze the selective oxyfunctionalization of C−H bonds, and they are of particular interest in synthetic organic chemistry. Here, we describe the engineering of UPO from Agrocybe aegerita for the efficient synthesis of 5′-hydroxypropranolol (5′-OHP). We employed a structure-guided evolution approach combined with computational analysis, with the aim of avoiding unwanted phenoxyl radical coupling without having to dope the reaction with radical scavengers. The evolved biocatalyst showed a catalytic efficiency enhanced by 2 orders of magnitude and 99% regioselectivity for the synthesis of 5′-OHP. When the UPO mutant was combined with an H 2 O 2 in situ generation system using methanol as sacrificial electron donor, total turnover numbers of up to 264 000 were achieved, offering a cost-effective and readily scalable method to rapidly prepare 5′-OHP.

Photoenzymatic Hydroxylation of Ethylbenzene Catalyzed by Unspecific Peroxygenase: Origin of Enzyme Inactivation and the Impact of Light Intensity and Temperature

“…For the selective oxyfunctionalization of C−H− and C=C‐bonds, Unspecific Peroxygenases (UPOs, E.C. 1.11.2.1) are promising enzymes catalyzing a variety of oxidation reactions like selective hydroxylations, which can be used for example for the synthesis of human drug metabolites . Particularly, the UPO from Agrocybe aegerita ( Aae UPO) available from recombinant fermentation in Pichia pastoris is an attractive catalyst.…”

Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2O2‐Dependent Biocatalytic Oxidation Reactions

“…The proposed H 2 O 2 ‐generation system enabled excellent catalytic performance of the peroxygenase used. Both product concentrations and r Aae UPO‐turnover numbers were at least as high as for previous methods using more complicated H 2 O 2 generation systems …”

Formiat‐Oxidase (FOx) aus Aspergillus oryzae: ein Katalysator für verschiedene H2O2‐abhängige biokatalytische Oxidationen

“…Zunächst untersuchten wir weitere Peroxygenase‐katalysierte Hydroxylierungen und Epoxidierungen. In allen Beispielen ermöglichte das vorgeschlagene System exzellente Katalysatorwechselzahlen (TONs), die wenigstens im Bereich der Werte von zuvor berichteten, allerdings komplexeren Systeme lagen . Die Hydroxylierung von Ethylbenzol gelang im semipräparativen Maßstab und erbrachte 434 mg des enantiomerenreinen Produktes (( R )‐1‐Phenylethanol, >99 % ee ; siehe Hintergrundinformationen für weitere Details).…”