Stereoselective synthesis of vicinal diols with enzymatic cascade reactions

Alcohol dehydrogenases are of high interest for the stereoselective synthesis of building blocks with multi-chiral centres. They are implemented for single enzymatic reduction or oxidation steps or be part of synthetic enzymatic multi-step cascades for the production of industrially relevant chiral synthons. Although biotransformations with alcohol dehydrogenases are widespread, enzymes, which accept sterically demanding substrates, especially α-hydroxy ketones, are not common in nature. Therefore chemical methods for the synthesis of chiral 1,2-diols are still first choice. As a drawback, alcohol dehydrogenases require expensive nicotinamide cofactors such as NAD(H) or NADP(H) for their activity. Prices of these cofactors prevent their application in stoichiometric amounts and therefore regeneration of nicotinamide cofactors is an essential issue for biotechnological purposes. Therefore a co-substrate is required that is transformed to the respective co-product in equimolar amounts relative to the product. This co-product has to be separated from the product or must be removed in situ, which decreases atom- and process economy. In this work the carboligation of two inexpensive aldehydes catalysed by ThDP-dependent enzymes is combined with a subsequent reduction of the intermediately formed α-hydroxy ketone by alcohol dehydrogenases. Therefore, a suitable cofactor regeneration system with smart in situ co-product removal had to be developed in order to gain high eco-efficiency of the synthetic enzyme cascade. To achieve the aim, the following work packages were addressed: 1. Investigation of the substrate range of eight available alcohol dehydrogenases with focus on reduction of bulky-bulky α-hydroxy ketones; 2. Determination of the stereoselectivity of the first and the second step of the synthetic cascade; 3. Selection of the most promising enzyme(s) for the reduction of α-hydroxy ketones (which turned out to be the alcohol dehydrogenase from Ralstonia sp.) and its detailed biochemical characterisation with focus on the reduction of α-hydroxy ketones; 4. Evaluation of a suitable cofactor regeneration system (substrate-coupled and enzyme-coupled approach) for the enzymatic 2-step synthesis, where smart implementation of co-substrates (benzyl alcohol or ethanol) allows in situ removal of the co-product (benzaldehyde or acetaldehyde) from the second cascade step by reusing it in the first catalytic step (carboligation) of the cascade; 5. Reaction engineering of the synthetic multi-enzymatic cascade with assistance of mathematical modelling, to optimise the overall yield of the synthetic cascade. During this work two synthetic recycling cascade reactions using a substrate-coupled and an enzyme-coupled approach for cofactor regeneration were developed. For the substrate-coupled approach benzyl alcohol was used as co-substrate, and the co-product benzaldehyde was reused as a substrate in the carboligation step. For the enzyme-coupled approach ethanol was chosen as a co-substrate and the resulting acetaldehyde was also reused as substrate for the C–C bond formation. Whereas good conversions (> 70%), but moderate stereoselectivities (dr syn/anti 9:1) were obtained for the production of (1R,2R)-1-phenylpropane-1,2-diol in cascade reactions using the substrate-coupled approach, the enzyme-coupled approach yielded excellent conversions (> 95%) and diastereoselectivities (de > 99%). Furthermore, during this thesis the most promising alcohol dehydrogenase from Ralstonia sp. for the reduction of α-hydroxy ketones was subject of crystallisation studies for X-ray structural investigations in order to explain its high potential for conversion of especially bulky substrates.