Enantioselective High‐Throughput Assay Showcased for the Identification of (R)‐ as well as (S)‐Selective Unspecific Peroxygenases for C−H Oxidation

Abstract

Identifying (bio)catalysts displaying high enantio/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Here, a high‐throughput, stereoselective screening assay is reported giving information on enantioselectivity, stereopreference and activity showcased for peroxygenase catalyzed hydroxylation. The assay is based on analyzing spectrophotometrically the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec‐alcohol product during the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non‐described wild‐type UPOs displaying (S)‐ as well as (R)‐stereoselectivity for the hydroxylation of representative model substrates were identified reaching up to 98% ee for the (R)‐ and 94% ee for the (S)‐enantiomer. Homology models with concomitant docking studies indicated the structural reason of the observed complementary stereopreference.