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.

Peroxygenase-Catalyzed Allylic Oxidation Unlocks Telescoped Synthesis of (1S,3R)-3-Hydroxycyclohexanecarbonitrile

“…16 To further investigate the peroxygenase-catalyzed allylic hydroxylation and overoxidation, a panel of 44 UPOs from Aminoverse was screened (Figure 2A). 23 These UPOs were recently discovered and produced in Pichia pastoris. 23 Very diverse results were obtained.…”

“…Interestingly, MorUPO was also recently reported to provide racemic 1-phenylethanol from ethylbenzene. 23 Next, a panel of ADHs was screened using racemic 4, including in-house enzymes LbADH and LkADH, and the C� O-reduction kit provided by JM (Figure 3). Almost all ADHs screened achieved >90% conversion, accepting both enantiomers of 4.…”

“…On the other hand, Mor UPO showed very high levels of overoxidation under the reaction conditions and was thus chosen to be implemented in the cascade. Interestingly, Mor UPO was also recently reported to provide racemic 1-phenylethanol from ethylbenzene …”

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

“…Finally, based on variant M4, variant M5 (T125A/A129V/A247H/T244A/F243G) was obtained by mutating F to G at position 243, which further enhanced the enantiomeric excess to 84% ee ( S ) (Figure C and Table S5). To our knowledge, variant M5 represents the highest ( S )-enantioselectivity for UPOs catalyzed benzylic hydroxylation of ethylbenzene, surpassing all the reported natural UPOs or their variants [ Gdi UPO-II: 61% ee ( S ), Cab UPO-V: 20% ee ( S ), Aae UPO variant d28: 32% ee ( S ), and Abr UPO: 20% ee ( S )]. ,, …”

“…To our knowledge, variant M5 represents the highest (S)enantioselectivity for UPOs catalyzed benzylic hydroxylation of ethylbenzene, surpassing all the reported natural UPOs or their variants [GdiUPO-II: 61% ee (S), CabUPO-V: 20% ee (S), AaeUPO variant d28: 32% ee (S), and AbrUPO: 20% ee (S)]. 15,33,34 Subsequently, the two CmaUPO variants M2a and M5 were extracellularly expressed in P. pastoris X33, and the enzyme was then purified and employed for kinetic study. It was found that both variants exhibited higher catalytic efficiency (k cat /K m ) compared to that of WT CmaUPO (Table 2 and Figure S15).…”