Monooxygenases as Baeyer-Villigerases

This process has been employed successfully for a number of enantioselective BV reactions, including kinetic resolution of racemic ketones and desymmetrization of prochiral substrates (172-175). An example is the desymmetrization of 4-methyl-cyclohexanone, which affords the (5)-configurated 7-membered lactone with 98% ee (172,175). Of course, many ketones fail to react with acceptable levels of en-antioselectivity or are not even accepted by the enzyme. 

It has been demonstrated recently that directed evolution is ideally suited to control the enantioselectivity of partial oxidations of this kind. The results are all the more significant because no X-ray data or homology models were available at the time to serve as a possible guide (103). In a model study using whole E. coli cells containing the CHMO from Acinobacter sp. NCIMB 9871, 4-hydroxy-cyclohexanone (51) was used as the substrate. The WT leads to the preferential formation of the primary product (7 )-52 which spontaneously rearranges to the thermodynamically more stable lactone (R)-53. The ee of this desymmetrization is 

In the directed evolution study, epPCR at various mutation rates was applied, and the total number of clones screened was about 10 000. Of the positive hits identified, some were (R)-, others (iS)-selective. Eight of them were sequenced (Table I) (103). Of particular interest is mutant 1-K2-F5, characterized by a single mutation F432S, because it leads to reversal of enantioselectivity (79% ee in favor of (S)-53). 

CHMO Mutants Identified in the First Round of epPCR Showing Improved Enantioselectivity in the Air-Oxidation of 51 (Reaction Time 24 h Temperature, 23-25° C Conversion 95%) (103) 

Mutant Amino acid exchanges Favored enantiomer of 53 ee (%) 

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Cyclohexanone and Cyclopentanone Monooxygenases as Baeyer-Villigerases and Sulfoxidation Catalysts... 

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