Silvia M. Glueck

Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives

The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the β-carbon atom of styrenes forming (E)-cinnamic acids.

The flavoprotein-catalyzed reduction of aliphatic nitro-compounds represents a biocatalytic equivalent to the Nef-reaction

The bioreduction of aliphatic sec-nitro compounds catalyzed by purified flavoproteins from the old-yellow-enzyme family unexpectedly furnished the corresponding carbonyl compounds instead of the expected amines and thus represents a biocatalytic equivalent to the Nef-reaction. The pathway was shown to proceed via initial reduction of the nitro-group to yield the nitroso-derivative, which spontaneously tautomerized to yield the more stable oxime, which was enzymatically reduced in a second step to furnish a hydrolytically unstable imine-species, which spontaneously hydrolyzed to finally give a carbonyl compound and ammonia.

Regioselective Enzymatic β-Carboxylation of para-Hydroxy- styrene Derivatives Catalyzed by Phenolic Acid Decarboxylases

Abstract We report on a ‘green’ method for the utilization of carbon dioxide as C1 unit for the regioselective synthesis of (E)‐cinnamic acids via regioselective enzymatic carboxylation of para‐hydroxystyrenes. Phenolic acid decarboxylases from bacterial sources catalyzed the β‐carboxylation of para‐hydroxystyrene derivatives with excellent regio‐ and (E/Z)‐stereoselectivity by exclusively acting at the β‐carbon atom of the C=C side chain to furnish the corresponding (E)‐cinnamic acid derivatives in up to 40% conversion at the expense of bicarbonate as carbon dioxide source. Studies on the substrate scope of this strategy are presented and a catalytic mechanism is proposed based on molecular modelling studies supported by mutagenesis of amino acid residues in the active site. WILEY-VCH

Oxidative Enzymatic Alkene Cleavage : Indications for a Nonclassical Enzyme Mechanism

An enzyme preparation of Trametes hirsuta cleaves alkenes following neither the classical dioxygenase mechanism nor via a monooxygenase mechanism. A catalytic cycle for an alternative enzymatic alkene cleavage was proposed, whereby two oxygen atoms derived from two different oxygen molecules are incorporated into the product(s).

Enzymatic Racemization of Amines Catalyzed by Enantiocomplementary ω‐Transaminases

A strategy for the biocatalytic racemization of primary α-chiral amines was developed by employing a pair of stereocomplementary PLP-dependent ω-transaminases. The interconversion of amine enantiomers proceeded through reversible transamination by a prochiral ketone intermediate, either catalyzed by a pair of stereocomplementary ω-transaminases or by a single enzyme possessing low stereoselectivity. To tune the system, the type and concentration of a nonchiral amino acceptor proved to be crucial. Finally, racemization could be achieved by the cross-transamination of two different amines without a requirement for an external amino acceptor. Several synthetically and industrially important amines could be enzymatically racemized under mild reaction conditions.

A highly efficient ADH‐coupled NADH‐recycling system for the asymmetric bioreduction of carbon‐carbon double bonds using enoate reductases

The asymmetric bioreduction of activated alkenes catalyzed by flavin-dependent enoate reductases from the OYE-family represents a powerful method for the production of optically active compounds. For its preparative-scale application, efficient and economic NADH-recycling is crucial. A novel enzyme-coupled NADH-recycling system is proposed based on the concurrent oxidation of a sacrificial sec-alcohol catalyzed by an alcohol dehydrogenase (ADH-A). Due to the highly favorable position of the equilibrium of ene-reduction versus alcohol-oxidation, the cosubstrate is only required in slight excess.

The substrate tolerance of alcohol oxidases

Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities.

Amination of ω‐Functionalized Aliphatic Primary Alcohols by a Biocatalytic Oxidation–Transamination Cascade

Amination of non‐activated aliphatic fatty alcohols to the corresponding primary amines was achieved through a five‐enzyme cascade reaction by coupling a long‐chain alcohol oxidase from Aspergillus fumigatus (LCAO_Af) with a ω‐transaminase from Chromobacterium violaceum (ω‐TA_Cv). The alcohol was oxidized at the expense of molecular oxygen to yield the corresponding aldehyde, which was subsequently aminated by the PLP‐dependent ω‐TA to yield the final primary amine product. The overall cascade was optimized with respect to pH, O2 pressure, substrate concentration, decomposition of H2O2 (derived from alcohol oxidation), NADH regeneration, and biocatalyst ratio. The substrate scope of this concept was investigated under optimized conditions by using terminally functionalized C4–C11 fatty primary alcohols bearing halogen, alkyne, amino, hydroxy, thiol, and nitrile groups.

Advances in biocatalytic synthesis. Enzyme-triggered asymmetric cascade reactions*

Organic compounds can be transformed through enzyme-triggered domino (or cascade) reactions via several (inseparable) consecutive steps in an asymmetric fashion to yield nonracemic products. Despite the fact that these sequences often involve the occurrence of highly reactive unstable intermediates, the overall efficiency of these processes can be high, provided that the reaction rates of the individual steps match each other in order to minimize side reactions.