Quantum mechanical/molecular mechanical study on the mechanism of the enzymatic Baeyer-Villiger reaction

We report a combined quantum mechanical/molecular mechanical (QM/MM) study on the mechanism of the enzymatic Baeyer-Villiger reaction catalyzed by cyclohexanone monooxygenase (CHMO). In QM/MM geometry optimizations and reaction path calculations, density functional theory (B3LYP/TZVP) is used to describe the QM region consisting of the substrate (cyclohexanone), the isoalloxazine ring of C4a-peroxyflavin, the side chain of Arg-329, and the nicotinamide ring and the adjacent ribose of NADP(+), while the remainder of the enzyme is represented by the CHARMM force field. QM/MM molecular dynamics simulations and free energy calculations at the semiempirical OM3/CHARMM level employ the same QM/MM partitioning. According to the QM/MM calculations, the enzyme-reactant complex contains an anionic deprotonated C4a-peroxyflavin that is stabilized by strong hydrogen bonds with the Arg-329 residue and the NADP(+) cofactor. The CHMO-catalyzed reaction proceeds via a Criegee intermediate having pronounced anionic character. The initial addition reaction has to overcome an energy barrier of about 9 kcal/mol. The formed Criegee intermediate occupies a shallow minimum on the QM/MM potential energy surface and can undergo fragmentation to the lactone product by surmounting a second energy barrier of about 7 kcal/mol. The transition state for the latter migration step is the highest point on the QM/MM energy profile. Gas-phase reoptimizations of the QM region lead to higher barriers and confirm the crucial role of the Arg-329 residue and the NADP(+) cofactor for the catalytic efficiency of CHMO. QM/MM calculations for the CHMO-catalyzed oxidation of 4-methylcyclohexanone reproduce and rationalize the experimentally observed (S)-enantioselectivity for this substrate, which is governed by the conformational preferences of the corresponding Criegee intermediate and the subsequent transition state for the migration step.     

Microbial Baeyer-Villiger oxidation: stereopreference and substrate acceptance of cyclohexanone monooxygenase mutants prepared by directed evolution

[reaction: see text] An array of random mutants of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was screened against a library of structurally diverse ketones for modifications in the substrate acceptance profile and stereopreference of the enzymatic Baeyer-Villiger biooxidation. While the set of mutant biocatalysts was initially evolved for the enantiocomplementary oxidation of 4-hydroxycyclohexanone, improved and/or divergent stereoselectivities were observed for several substrates. In addition, expanded substrate acceptance to facilitate biotransformation of sterically demanding ketones was found.     

Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis

Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as high-throughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.     

The steroid monooxygenase from Rhodococcus rhodochrous; a versatile biocatalyst

The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was investigated for a large range of different ketone substrates. These studies revealed that this enzyme not only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclohexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Escherichia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocarpon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher enantiomeric excess of the product lactones compared to the known BVMOs of the same enantiopreference, such as cyclopentanone monooxygenases.     

Access to optically pure 4- and 5-substituted lactones: a case of chemical-biocatalytical cooperation

Optically pure or highly enantiomerically enriched 4- and 5-substituted lactones are rather difficult to obtain. Chemical or enzymatic syntheses alone are not particularly successful. A combination of chemical catalysis and biocatalysis, however, provides a convenient route to a variety of these useful chiral compounds. In this paper we describe the synthesis of several optically pure 4- and 5-substituted lactones obtained via whole cell-catalyzed Baeyer-Villiger oxidations of highly enantiomerically enriched 3-alkyl cyclic ketones. Such chiral ketones are readily accessed by recently developed copper-catalyzed asymmetric conjugate reductions of the corresponding enones. A very high proximal regioselectivity and complete chirality transfer was obtained by employing biological Baeyer-Villiger oxidations, using recombinant E. coli strains that overexpress cyclopentanone monooxygenase (CPMO). A comparative study showed that CPMO gives superior results to those obtained with cyclohexanone monooxygenase (CHMO) catalyzed oxidations.     

Protein engineering of stereoselective baeyer-villiger monooxygenases

Baeyer-Villiger monooxygenases (BVMOs) have been used for decades as catalysts in stereoselective Baeyer-Villiger reactions, including oxidative kinetic resolution of racemic ketones and desymmetrization of prochiral substrates with high enantioselectivity. These complement catalytic BV processes based on chiral synthetic catalysts. However, as in any enzyme-catalyzed process, limitations exist due to the often observed narrow substrate scope and/or insufficient stereoselectivity. Recent protein engineering of BVMOs in the form of directed evolution and rational design have eliminated these traditional limitations, which is the subject of this Minireview. The main focus is on phenylacetone monooxygenase (PAMO); an unusually thermostable and robust BVMO, which has a very narrow substrate scope. Protein engineering of PAMO has provided a number of mutants that display relatively wide substrate scope, high stereoselectivity, and maintained thermostability.     

Blending Baeyer-Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties

The thermostable Baeyer-Villiger monooxygenase (BVMO) phenylacetone monooxygenase (PAMO) is used as a scaffold to introduce novel selectivities from other BVMOs or the metagenome by structure-inspired subdomain exchanges. This yields biocatalysts with new preferences in the oxidation of sulfides and the Baeyer-Villiger oxidation of ketones, all while maintaining most of the original thermostability.     

Towards practical biocatalytic Baeyer-Villiger reactions: applying a thermostable enzyme in the gram-scale synthesis of optically-active lactones in a two-liquid-phase system

Baeyer-Villiger monooxygenases (BVMOs) are extremely promising catalysts useful for enantioselective oxidation reactions of ketones, but organic chemists have not used them widely due to several reasons. These include instability of the enzymes in the case of in vitro and even in vivo systems, reactant/product inhibition, problems with upscaling and the necessity of using specialized equipment. The present study shows that the thermally stable phenylacetone monooxygenase (PAMO) and recently engineered mutants can be used as a practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions. For this purpose several parameters such as buffer composition, the nature of the solvent system and the co-factor regeneration system were optimized. Overall a fairly versatile and efficient catalytic system for enantioselective laboratory scale BV-oxidations of ketones was developed, which can easily be applied even by those organic chemists who are not well versed in the use of enzymes.     

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio‐, chemo‐, and enantioselectivity. However, the low stability of many Baeyer–Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate‐binding cavity explains its preference for small rather than bulky substrates. Small‐scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.     

New bioorganic reagents: evolved cyclohexanone monooxygenase--why is it more selective?

Four mutants of the cyclohexanone monooxygenase (CHMO) evolved as catalysts for Baeyer-Villiger oxidation of 4-hydroxycyclohexanone were investigated as catalysts for a variety of 4-substituted and 4,4-disubstituted cyclohexanones. Several excellent catalytic matches (mutant/substrate) were identified. The most important, however, is the finding that, in a number of cases, a mutant with a single exchange, Phe432Ser, was shown to be as robust and more selective as a catalyst than the wild-type CHMO. All biotransformations were performed on a laboratory scale, allowing full characterization of the products. The absolute configurations of two products were established. A model suggesting a possible role of the 432 serine residue in enantioselectivity control is proposed.     

Simultaneous biocatalyst production and baeyer-villiger oxidation for bioonversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase

Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce ε-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. Afed-batch process was designed to obtain high cell density for improvin production of ε-caprolactone. The fed-batch SPO process have the best results, 10.2 g/L of ε-caprolactone and 0.34 g/(L·h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.     

Baeyer–Villiger monooxygenase-catalyzed kinetic resolution of racemic α-alkyl benzyl ketones: enzymatic synthesis of α-alkyl benzylketones and α-alkyl benzylesters

The application of three BVMOs for the enantioselective oxidation of 3-phenylbutan-2-ones with different substituents in the aromatic moiety is described. By choosing the appropriate biocatalyst and substrate combination, chiral ketones and esters can be obtained with excellent enantiopurities. This methodology could also be applied to the resolution of racemic α-alkyl benzylketones with longer alkyl chains as well as with two substituted α-substituted benzylacetones. A kinetic analysis revealed that the BVMOs studied effectively convert all tested compounds showing that the enzymes are tolerant towards the substrate structure while being highly enantioselective. These properties render BVMOs as valuable biocatalysts for the preparation of compounds with high interest in organic synthesis.     

Biooxidation of bridged cycloketones using Baeyer-Villiger monooxygenases of various bacterial origin

Bridged cycloketones were synthesized and utilized as substrates to study biooxidations mediated by Baeyer-Villiger monooxygenases (BVMO) of various bacterial origin. The required enzymes were heterologously produced by recombinant overexpression systems based on Escherichia coli to enable facile recycling of the required nicotinamide cofactors during the whole-cell biotransformations. Ketone precursors of various structural demands were chosen to evaluate steric limitations and flexibility of the active site of BVMOs. By desymmetrization of the prochiral substrates, four to six stereogenic centers were generated within a single biooxidation step. The enzyme library investigated in this study allowed access to antipodal lactone products with excellent enantioselectivity in several cases. Together with a distinct substrate acceptance profile, the recently proposed classification into two groups of cycloketone converting BVMOs was supported by the biotransformation results obtained within this study.     

Hydrotalcites as catalysts for the Baeyer-Villiger oxidation of cyclic ketones with hydrogen peroxide/benzonitrile

Hydrotalcites (HTs) in variable Mg/Al ratios were used as catalysts for the Baeyer-Villiger (BV) oxidation of cyclic ketones with hydrogen peroxide. All HTs studied were found to be active in the BV oxidation of cyclohexanone, their activity increases with increasing Mg/Al ratio. The reaction, which was conducted under very mild conditions (viz. atmospheric pressure and a temperature of 70 °C), provided conversions above 70% with 100% selectivity only after 6 h. This outcome was found to require the presence of a nitrile in the reaction medium, so a mechanism involving adsorption of the nitrile and cyclohexanone onto the catalyst is proposed that is consistent with the experimental results. Based on the proposed mechanism, the presence of a surfactant should result in improved conversion and catalytic activity, as was indeed observed with sodium dodecylsulfate in the reaction medium. The best catalyst among those tested was used with other cyclic ketones and found to provide excellent conversion and selectivity results in most cases.     

What to sacrifice? Fusions of cofactor regenerating enzymes with Baeyer-Villiger monooxygenases and alcohol dehydrogenases for self-sufficient redox biocatalysis

A collection of fusion biocatalysts has been generated that can be used for self-sufficient oxygenations or ketone reductions. These biocatalysts were created by fusing a Baeyer-Villiger monooxygenase (cyclohexanone monooxygenase from Thermocrispum municipale: TmCHMO) or an alcohol dehydrogenase (alcohol dehydrogenase from Lactobacillus brevis: LbADH) with three different cofactor regeneration enzymes (formate dehydrogenase from Burkholderia stabilis: BsFDH; glucose dehydrogenase from Sulfolobus tokodaii: StGDH, and phosphite dehydrogenase from Pseudomonas stutzeri: PsPTDH). Their tolerance against various organic solvents, including a deep eutectic solvent, and their activity and selectivity with a variety of substrates have been studied. Excellent conversions and enantioselectivities were obtained, demonstrating that these engineered fusion enzymes can be used as biocatalysts for the synthesis of (chiral) valuable compounds.     

Relative Stability of the Key Enamine,Oxazolidinone, and Imine Intermediates in Some Proline-catalyzed Asymmetric Reactions

Many proline-catalyzed asymmetric addition reactions with ketones as substrates were assumed to involve a key intermediate, an enamine, produced by the condensation of proline and ketone. In this paper, the key intermediate enamines derived from L-proline and cyclohexanone （or acetone） as well as the corresponding oxazolidinone and imine tautomers have been investigated by means of density functional calculations at the B3LYP/6-311＋G^＊＊ level. The predicted order of stability for these tautomers is oxazolidinones 〉 enamines 〉 imines in gas phase and oxazolidinones 〉 imines 〉 enamines in aprotic THF solvent. This prediction explains why enamine intermediate can not be observed experimentally. The predicted energy/enthalpy difference between the formal oxazolidinone structure and the zwitterionic imine structures is very small in THF solvent, suggesting the oxazolidinone-to-imine tautomerization can be readily induced in solvent. ^13C NMR chemical shifts of the oxazolidinone and imine structures have been computed and used to explain the experimental NMR spectra observed in oxazolidinone-to-imine tautomerization induced by protic solvent.     

Enhanced shape selective catalysis of mixed cyclic ketones in aerobic Baeyer-Villiger oxidation with magnetic Cu-Fe3O4 supported mesoporous silica microspheres

Various strategies have been developed to improve the conversion for the Baeyer-Villiger oxidation. However, the catalytic effects of the Baeyer-Villiger oxidation for the mixed ketones are rarely reported, though it is also important for the natural and industrial separation processes. In this report, magnetite Cu modified Fe₃O₄ supported mesoporous silica microspheres (Cu-Fe₃O₄@mSiO₂) have been successfully synthesized by two step direct hydrothermal method (DHT). Over 99% of cyclohexanone conversion was obtained with mild air oxidation and benzaldehyde as sacrificing agent over Cu-Fe₃O₄@mSiO₂. The catalytic system also shows higher conversion rates for small molecular ketones in the mixed ketone reactants, which was attributed to the enhanced mass transfer effect and Fe-Cu composite active sites in the magnetite mesoporous silica microspheres. The catalyst could be recycled for four times with similar catalytic performance, which shows enhanced shape selectivity in aerobic Baeyer-Villiger oxidations for mixed cyclic ketones.     

Exploiting Cofactor Versatility to Convert a FAD‐Dependent Baeyer–Villiger Monooxygenase into a Ketoreductase

Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer–Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild‐type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α‐keto ester, but with poor yield and stereoselectivity. Structure‐guided, site‐directed mutagenesis drastically improved the catalytic carbonyl‐reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α‐keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild‐type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.     

Comparative Baeyer-Villiger oxidation of cyclohexanone on Fe-pillared clays and iron tetrasulfophthalocyanine covalently supported on silica

The Baeyer-Villiger oxidation of cyclohexanone to caprolactone has been investigated at room temperature over AlFe-pillared clays, using oxygen as oxidant in the presence of benzaldehyde. A nearly complete conversion is observed with a selectivity into caprolactone above 80%. The observation of an induction period in the kinetics, of high activity of the non-pillared clay, and the detection of Fe traces in the reaction medium, suggest a process involving homogenous catalysis. The reaction is indeed catalysed in homogeneous phase by a few ppm of Fe. By contrast, iron phthalocyanine covalently supported on silica appears as a true heterogeneous catalyst, giving a selectivity above 95% to caprolactone at 61% conversion.     

Emploi de l'acide monopersuccinique (PSA) dans l'eau comme agent d'oxydation dans la réaction de Baeyer-Villiger appliquée aux cétones cycliques

Baeyer-Villiger oxidation with monopersuccinic acid of cyclic ketones in water. Monopersuccinic acid (PSA) is a water-soluble peracid giving good results in the oxidation of cyclic ketones into lactones in this solvent.