Overriding Traditional Electronic Effects in Biocatalytic Baeyer–Villiger Reactions by Directed Evolution

Abstract

Controlling the regioselectivity of Baeyer-Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer-Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

“…Mutations to the bulkier amino acids either significantly improved the regioselectivity for 'normal' lactone production from 2-methylcyclohexanone or allowed access to more of the 'abnormal' lactone from 2-methylcyclopentanone. This was similarly observed in directed evolution studies by Reetz and co-workers [33], where W492Y in TmCHMO did result in reduced activity, but at the gain of significantly improved regioselectivity for abnormal lactone products.…”

“…Interestingly, directed evolution studies with TmCHMO targeting the same amino acids in iterative saturation mutagenesis (ISM), found no hits with W492 in the reversal of enantioselectivity during the desymmetrization of the prochiral ketone 4-methylcyclohexanone [32], but W492Y/F mutations did significantly change the regioselelctiviey with 1-phenyl-2-propanone (phenylacetone) and 1-phenyl-2-butanone [33]. In silico analysis through substrate docking, molecular dynamics (MD) and quantum mechanical (QM/MM) simulations with density function theory (DFT) of the Criegee intermediate, however, do show the corresponding Trp in related BVMOs to contact the substrate or intermediate, rationalizing its involvement in changes in selectivity/specificity [30,[33][34][35]. In PAMO, the same changes in regioselectivity observed with W501A was also observed in Y502A, of which the side chain would not directly contact the substrate or reaction intermediates [28].…”

Unleashing the Synthetic Power of Plant Oxygenases: From Mechanism to Application

“…There are also Baeyer-Villiger-like oxidations of plant metabolites with unknown biosynthetic routes, such as the degradation of (+)-camphor in Salvia officinalis (sage; Croteau et al, 1984). The BVMOs have been used in numerous chemo-enzymatic applications, and their selectivity has also been successfully engineered (van Beek et al, 2012;Li et al, 2018a;Woo et al, 2018).…”

Transition‐Metal‐Free Deconstructive Lactamization of Piperidines

“…[24] According to the prevailing mechanism of the BV,the Criegee intermediate (CI)f avors the migration of the group that can best stabilize the partial positive charge. [25] Therefore,t he migrating atom should be C4, and not C2 from the carbonyl group (Scheme 4). Computational analysis at the wB97XD/6-311 + G(d,p) level of theory [26] revealed the existence of two stable Criegee intermediate conformers (CI1 and CI2)prone to form N-carboxyanhydride intermediates.B ecause of the presence of an intramolecular hydrogen bond (with an energy E(2) = 2.24 kcal mol À1 ), Criegee intermediate CI1 (in which the C2-C3 bond and OÀOb ond are antiperiplanar) is 4.36 kcal mol À1 more stable than CI2.F rom the optimization and the characterization of the TS1 and TS2 transition state structures for the reactions CI1!F and CI2!E,respectively, we found that the former transformation occurs at much lower energetic cost (DG°(TS1) = 20.22 kcal mol À1 )t han the latter (DG°(TS2) = 28.35 kcal mol À1 ).…”