Biocatalytic Disproportionation of Enones

The asymmetric bioreduction of activated alkenes catalysed by flavoproteins from the old-yellow-enzyme family generates (up to) two stereocenters and is thus an important tool for the chemo-enzymatic synthesis of chiral carbonyl compounds, nitro-compounds and a variety of carboxylic acid derivatives. In order to recycle the flavin- cofactor, traditional methodology relies on a second nicotinamide cofactor as a hydride-shuttle, which in turn requires a second (glucose-, formate- or phosphite-) dehydrogenase enzyme for its regeneration. In order to reduce the complexity of these systems, the flavin-cofactor will be recycled internally by the flavoprotein by making use of the flavoprotein-catalysed disproportionation of enones, which was generally regarded as a `promiscuous` catalytic activity. The project has the following main aims: (i) Detailed investigation of common structural features `dismutases` and of ideal co-substrates enhances the fundamental knowledge of the enzyme-catalysed disproportionation of enones and will significantly broaden the understanding of the catalytic capabilities and the natural role of flavoproteins from the old-yellow-enzyme family. (ii) By making use of this enzymatic side-reaction, the need for a second nicotinamide-dependent recycling system for the asymmetric bioreduction of activated alkenes will be eliminated, which overall generates a significantly simplified system for the regeneration of flavin-cofactors. The latter will allow to use organic solvent systems, which enables to transform hydrolytically labile substrates, such as acid anhydrides, and to tune the stereoselectivity of these biotransformations by `solvent-engineering`. (iii) The optimised system will be employed to significantly extend the substrate range of old-yellow-enzymes by transforming novel types of activated alkenes, which are important building blocks or end-products for pharma- and flavor & fragrance-applications. The main emphasis lies on a or ß-amino acids (from the corresponding a,ß- dehydro-precursors), O-protected acyloins (from the corresponding a-substituted keto-enolethers), a- or ß- substituted aldehydes (from the corresponding cinnamaldehyde precursors), and ß-O-substituted methacrylate derivatives.

The enzymatic hydrogen-transfer from a H-donor onto an unsaturated substrate bearing a C=C-bond represents an elegant method for the synthesis of bioactive compounds. This strategy is especially relevant for the pharma-industry and finds application in the synthesis of so-called GABA-analogs, which are used as transmitter-molecules for nerve-signals. GABA-analogs are widely used for the treatment of chronic neuropathic pain disorders and they can be synthesized by employing ene-reductases. In a similar fashion, an aldehyde used as aroma of the lily-of-the-valley can be obtained. This product is widely used in the fragrance industry. In general, the regeneration of the used (oxidized) cofactor is mediated via an indirect fashion: the oxidized flavin-cofactor within the ene-reductase is in turn reduced by a nicotinamide-cofactor, which requires a second (helper) enzyme for its regeneration. In order to simplify this process and make it more efficient, the direct hydrogen-transfer mediated by ene-reductases was investigated, which requires only a single enzyme. In a detailed study, suitable hydrogen-donor molecules were tested, which revealed several promising candidates. Among them is the so-called 'coffee-furanone', a cheap aroma compound, which is widely used in the food- and soft-drink industry as aroma additive. Special emphasis was laid on the synthesis of compounds, which occur as mirror-image isomers. The latter could be obtained by employing enzymes, which exhibit a stereo-complementary behaviour. In an alternative approach, this was possible by attaching protecting groups of various size on the substrate, which forced its binding in the active site pocket in a flipped mode. A general problem of the direct enzymatic hydrogen-transfer is the formation of a phenolic by-product, which inhibits the activity of the ene-reductase. In order to circumvent this reversible effect, several protocols for its removal were investigated: (i) The addition of adsorptive polymer resins facilitated the selective adsorption of the inhibiting by-products, which allowed to enhance the conversions significantly. (ii) Alternatively, the phenols could be enzymatically oxidized in a cascade-reaction, which cancelled their inhibiting properties. A great advantage of the direct hydrogen-transfer is its independence from nicotinamide, which due to its high polarity can only be employed in aqueous solution. As a consequence, the bioreduction could be performed for the first time in organic co-solvents, which allows the transformation of water-sensitive compounds. At the end of this project, the application of these various basic technologies could be successfully demonstrated on a bioactive pharma-ingredient (Pregabalin).