Cyclohexanone oxygenase: stereochemistry, enantioselectivity, and regioselectivity of an enzyme-catalyzed Baeyer-Villiger reaction

Abstract

Cyclohexanone oxygenase, from Acinetobacter NCIB 9871, has been incubated with (2S,6S)-[2,6-2H2]-and (27?) -[ 2-2H, ]cyclohexanone. The resulting labeled c-caprolactone (2-oxepanone) samples were degraded to 1-pentanol, which was esterified by using (-)-camphanyl chloride. Analysis of the camphanates by deuterium NMR spectroscopy, using Eu(dpm)3, showed that the conversion of ketone to lactone had in each case proceeded with complete retention of configuration at the migrating carbon center. A similar degradation of (27?)-[2-2H1]cyclohexanone itself showed that reduction of [2-2H,]cyclohex-2-enone by Beauveria bassiana ATCC 7159 is also completely stereoselective. A method has been developed for assessing the enantioselectivity of enzymes toward racemic substrate mixtures. (2R)-2-[methyl-2H3]-and (2S)-2-[methyl-13C] methylcyclohexanone were synthesized and mixed in equal amounts, and the resulting mixture (a virtual racemate) was incubated with cyclohexanone oxygenase. The course of the reaction was followed by both 13C and 2H NMR spectroscopy, showing that the initial rate of oxidation of the 2S enantiomer was nearly twice that of the 2R enantiomer. (27?)-and (2S)-2-methylcyclohexanone were both converted by cyclohexanone oxygenase to 6-methyl-e-caprolactone (7-methyl-2-oxepanone).Advantages of the virtual racemate/multinuclear NMR technique over existing methodology are described.While the Baeyer-Villiger reaction3 has been a standard tool of organic chemistry for the better part of a century, only in recent years has it become clear that nature makes widespread use of this reaction in biodegradative pathways.4 Studies in several f Dedicated to Dr. Ulrich Weiss on the occasion of his 75th birthday.

Purification and Characterization of Hexahistidine-Tagged Cyclohexanone Monooxygenase Expressed in Saccharomyces cerevisiae and Escherichia coli

“…In the intact organism, cytosolic CMO catalyzes the NADPH-dependent oxidation of cyclohexanone to ⑀-caprolactone in a ring expansion reaction that occurs via a Baeyer-Villiger mechanism (2,3). This reaction is one of several degradative steps for cyclohexanol that ultimately yield diacid precursors to acetyl-CoA (4).…”

Enzymatic Baeyer–Villiger Oxidation of Benzaldehydes

“…The high preference for the formation of phenols is in disagreement with the rule derived from chemical studies that aromatic substrates with electron-withdrawing substituents mainly form benzoic acids. [8,10,16] Because the rearrangement of the tetrahedral Criegee intermediate (Scheme 1) of the enzymatic reaction is believed to be governed by the same stereoelectronic effects as in the non-enzymatic Baeyer-Villiger oxidation, [28,49,50] this suggests that interactions of the benzaldehyde substrates with amino acids or the flavin cofactor in the active site influence the selection of the migratory group in favor of the phenyl ring. The first insight into the active site topology of type I Baeyer-Villiger monooxygenases has recently come from the crystal structure determination of the uncomplexed form of phenylacetone monooxygenase.…”

ESI- and MALDI-MS Analysis of Cyclohexanone Monooxygenase from Acinetobacter NCIB 9871

“…zyme as a versatile catalyst capable of delivering nucleophilic and electrophilic oxygen to substrates from its C4a-hydroperoxyflavin active species (3)(4)(5). Investigation of the Baeyer-Villiger monooxygenases is a flourishing area fostered not only by continued interest in the vast range of metabolic reactions which these enzymes can accomplish, often with exquisite regiochemical and stereochemical control (6,7), but also by the recent cloning of several of these enzymes from different microbial genomes (8 -11).…”