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Biocatalytic Baeyer–Villiger Reactions: Uncovering the Source of Regioselectivity at Each Evolutionary Stage of a Mutant with Scrutiny of Fleeting Chiral Intermediates

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Reetz,  Manfred T.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences;
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Citation

Dong, Y., Li, T., Zhang, S., Sanchis, J., Yin, H., Ren, J., et al. (2022). Biocatalytic Baeyer–Villiger Reactions: Uncovering the Source of Regioselectivity at Each Evolutionary Stage of a Mutant with Scrutiny of Fleeting Chiral Intermediates. ACS Catalysis, 12(6), 3669-3680. doi:10.1021/acscatal.2c00415.


Cite as: http://hdl.handle.net/21.11116/0000-000A-6E32-3
Abstract
In this study, we report the discovery of unexpected mechanistic intricacies of Baeyer–Villiger monooxygenases (BVMOs) and provide insights that promise to help in extending their applications in synthetic organic chemistry and biotechnology. The basic mechanism of BVMOs as catalysts in the oxidation of unsymmetrical ketones R1–(C═O)–R2 is well known, which involves the intermediacy of short-lived Criegee intermediates. The tendency of R1 or R2 to migrate preferentially in the breakdown of the Criegee intermediate follows the traditional requirement of an antiperiplanar conformation with maximum stabilization of the incipient positive charge. The challenge of inverting the regioselectivity of group migration with the formation of abnormal products was recently met by the semi-rational directed evolution of TmCHMO with the generation of a quadruple mutant. Although a reasonable model explaining the mutational effect was suggested, the theoretical analysis did not include the calculation of both enantiomeric forms of the fleeting chiral Criegee intermediate in transition states and focused only on the wild-type enzyme and the quadruple mutant. The present investigation utilizes complete mutational deconvolution with the experimental construction of a fitness-pathway landscape comprising 4! = 24 upward climbs. We were confronted by the discovery that the absolute configuration of the Criegee intermediate switches from (R) to (S), depending upon the stage of the evolutionary process. On the basis of X-ray structural data, the physical basis of this phenomenon was illuminated by quantum chemical analyses performed on the enzymes at all evolutionary steps of a selected pathway. The hitherto unexplored role of fleeting chiral intermediates in the mechanism of other enzyme types deserves increased attention.