Dorina Clay

Formal Asymmetric Biocatalytic Reductive Amination

Asymmetric methods to prepare optically active a-chiral primary amines are highly demanded in asymmetric synthesis owing to the biological/pharmacological activity of many amines. Various techniques have been reported, such as asymmetric 1,2-addition to imines and asymmetric amination of a,a-disubstituted aldehydes, transformation of allylic alcohols into amines, (dynamic) kinetic resolution, and cyclic deracemization employing racemic amines as substrates. Asymmetric reductive amination of ketones has been investigated with transition-metal catalysts and organocatalysts, as well as via sulfinyl imine intermediates. Although tremendous progress in organo/metal catalysis has been achieved for the asymmetric reductive amination of ketones to access a-chiral amines, improved protocols are still required that are simple, green, and economically viable and that lead to high enantiomeric excesses. Biocatalytic reductive amination or transamination is well established for accessing a-amino acids from the corresponding a-keto carboxylic acids. However, the situation is different for primary amines that are not adjacent to a carbonic acid moiety. w-Transaminases have recently received attention for the preparation of such a-chiral unprotected amines. w-Transaminases are employed mainly in one way, namely for the kinetic resolution of racemic chiral amines; only a few reports deal with asymmetric synthesis by starting from a prochiral ketone, probably due to problems in shifting the equilibrium to the product side, as well as due to the moderate stereoselectivity of the employed w-transaminases. These asymmetric synthetic processes usually require at least stoichiometric amounts of an amine donor (for example, alanine). The latter leads to a side product (pyruvate), which has to be removed during the transformation by using, for instance, pyruvate decarboxylase or lactate dehydrogenase. Additionally, limitations due to inhibition by the product amine and by pyruvate have been reported. An ideal process would use ammonium as the amine donor, together with a cheap reducing agent (for example, formate, hydrogen, or glucose; see Scheme 1). Even

Synthesis of Optically Active Amines Employing Recombinant ω-Transaminases in E. coli Cells

Various recombinant ω‐transaminases, overexpressed in E. coli cells and employed as whole‐cell catalysts, are tested for the synthesis of enantiomerically pure amines from the corresponding prochiral ketones. Optically pure (S)‐amines are obtained by formal reductive amination, consuming just ammonia and a cheap reducing agent (formate) with up to 99 % ee and 97 % yield. The other enantiomer was accessible by employing the same ω‐transaminases in a kinetic resolution starting from racemic amines. A ω‐transaminase derived from an Arthrobacter species displayed the highest stereoselectivity for all substrates tested, both for the kinetic resolution of rac‐amines and for the amination of ketones.

Deracemization of Mexiletine Biocatalyzed by ω-Transaminases

(S)- as well as (R)-mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral orally effective antiarrhythmic agent, was prepared by deracemization starting from the commercially available racemic amine using omega-transaminases in up to >99% ee and conversion with 97% isolated yield by a one-pot two-step procedure. The absolute configuration could be easily switched to the other enantiomer, just by switching the order of the applied transaminases. The cosubstrate pyruvate needed in the first oxidative step was recycled by using an amino acid oxidase.

Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds.

Highlights ► Activated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds bearing electron-withdrawing groups are efficiently reduced by flavoproteins from the OYE family. ► The application of ene-reductases for the pharma- and perfumery industry has been demonstrated. ► Access to both stereoisomeric products is feasible by choice of stereo-complementary enzymes or via proper substrate engineering.

Chemoenzymatic Asymmetric Synthesis of Pregabalin Precursors via Asymmetric Bioreduction of β-Cyanoacrylate Esters Using Ene-Reductases

The asymmetric bioreduction of a library of β-cyanoacrylate esters using ene-reductases was studied with the aim to provide a biocatalytic route to precursors for GABA analogues, such as pregabalin. The stereochemical outcome could be controlled by substrate-engineering through size-variation of the ester moiety and by employing stereochemically pure (E)- or (Z)-isomers, which allowed to access both enantiomers of each product in up to quantitative conversion in enantiomerically pure form. In addition, stereoselectivities and conversions could be improved by mutant variants of OPR1, and the utility of the system was demonstrated by preparative-scale applications.

Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases.

Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation (“dismutation”) of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co‐product phenol could be circumvented via in situ co‐product removal by scavenging the phenol using the polymeric adsorbent MP‐carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a “coupled‐substrate” approach via nicotinamide‐free hydrogen transfer with >90% conversion and complete stereoselectivity. Biotechnol. Bioeng. 2013;110: 3085–3092.

Reductive dehalogenation of β-haloacrylic ester derivatives mediated by ene-reductases

The enzymatic bioreduction of β-halo-α,β-unsaturated carboxylic esters proceeded via sequential enzymatic CC reduction—β-elimination to afford saturated carboxylic esters. This novel biodegradation pathway combines the reductive activity of ene-reductases with the spontaneous β-elimination of hydrohalous acid from the unstable (saturated) intermediates. Both enantiomers of methyl 2-chloro-, 2-bromo- and 2-iodopropionate were obtained in good to excellent enantiopurity via enzyme-based stereocontrol using various members of the ‘Old Yellow Enzyme’ family of flavoproteins. Overall, this pathway resembles a reductive dehalogenation of β-halogenated acrylic esters.

Nitrile as Activating Group in the Asymmetric Bioreduction of β-Cyanoacrylic Acids Catalyzed by Ene-Reductases.

Asymmetric bioreduction of an (E)-β-cyano-2,4-dienoic acid derivative by ene-reductases allowed a shortened access to a precursor of pregabalin [(S)-3-(aminomethyl)-5-methylhexanoic acid] possessing the desired configuration in up to 94% conversion and >99% ee. Deuterium labelling studies showed that the nitrile moiety was the preferred activating/anchor group in the active site of the enzyme over the carboxylic acid or the corresponding methyl ester.

NAD(P)H‐Independent Asymmetric CC Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co‐substrates as the Hydrogen Donor

To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems.

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β‐diketones by 6‐oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β‐diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily. Biotechnol. Bioeng. 2011;108: 2815–2822.