Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst.

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol-4-(1-hydroxyethyl)-2-methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent-inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p-substituents while tolerating o-methoxy substituents.

DNA-Accelerated Catalysis of Carbene-Transfer Reactions by a DNA/Cationic Iron Porphyrin Hybrid.

A novel DNA-based hybrid catalyst comprised of salmon testes DNA and an iron(III) complex of a cationic meso-tetrakis(N-alkylpyridyl)porphyrin was developed. When the N-methyl substituents were placed at the ortho position with respect to the porphyrin ring, high reactivity in catalytic carbene-transfer reactions was observed under mild conditions, as demonstrated in the catalytic enantioselective cyclopropanation of styrene derivatives with ethyl diazoacetate (EDA) as the carbene precursor. A remarkable feature of this catalytic system is the large DNA-induced rate acceleration observed in this reaction and the related dimerization of EDA. It is proposed that high effective molarity of all components of the reaction in or near the DNA is one of the key contributors to this unique reactivity. This study demonstrates that the concept of DNA-based asymmetric catalysis can be expanded into the realm of organometallic chemistry.

Exploring the biocatalytic scope of a bacterial flavin-containing monooxygenase.

A bacterial flavin-containing monooxygenase (FMO), fused to phosphite dehydrogenase, has been used to explore its biocatalytic potential. The bifunctional biocatalyst could be expressed in high amounts in Escherichia coli and was able to oxidize indole and indole derivatives into a variety of indigo compounds. The monooxygenase also performs the sulfoxidation of a wide range of prochiral sulfides, showing moderate to good enantioselectivities in forming chiral sulfoxides.

Synthesis of chiral 3-alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger monooxygenase.

Baeyer-Villiger monooxygenases have been tested in the oxidation of racemic benzofused ketones. When employing a single mutant of phenylacetone monooxygenase (M446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with high yields and optical purities through regioselective dynamic kinetic resolution processes.

BVMO-catalysed dynamic kinetic resolution of racemic benzyl ketones in the presence of anion exchange resins.

4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB was employed in the presence of a weak anion exchange resin to perform dynamic kinetic resolutions of racemic benzyl ketones with high conversions and good optical purities. Different parameters that affect to the efficiency of the enzymatic Baeyer-Villiger oxidation and racemisation were analyzed in order to optimize the activity and selectivity of the biocatalytic system.

Biocatalysed concurrent production of enantioenriched compounds through parallel interconnected kinetic asymmetric transformations.

Parallel interconnected kinetic asymmetric transformations were performed in order to obtain enantioenriched derivatives starting from a set of racemic or prochiral compounds. Thus, in a one-pot reaction using two redox biocatalysts (a BVMO and an ADH) and a catalytic amount of cofactor that acts as a mediator, enantioenriched ketones, sulfoxides, and sec-alcohols were concurrently obtained in a strict parallel way, minimising the quantity of reagents employed. By selecting the appropriate biocatalysts, this methodology represents a potential tool for performing stereodivergent transformations.

DNA-based hybrid catalysis.

In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed.

Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids.

Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel-Crafts alkylation reactions and up to 83% ee for the product was achieved.

Improvement of the biocatalytic properties of one phenylacetone monooxygenase mutant in hydrophilic organic solvents.

The presence of different hydrophilic organic solvents or a water soluble polymer such as PEG 4000 led to an enhancement in the enzymatic activity of the M446G mutant of phenylacetone monooxygenase when it is employed in enantioselective sulfoxidations and Baeyer-Villiger reactions. By solvent engineering new substrates were found to be effectively converted by this Baeyer-Villiger monooxygenase. The use of 5% methanol together with the weak anion exchange resin Lewatit MP62 also allows the dynamic kinetic resolution of a set of racemic benzylketones. By this approach (S)-benzylesters could be obtained with high yields and optical purities.