Novel approaches for using dehydrogenases and ene-reductases for organic synthesis

Abstract

Oxidation of alcohols is a reaction of major interest for organic chemistry. However, the most common chemical routes developed so far involve the use of toxic or hazardous reagents or catalysts that often lack good chemoselectivity. In this respect, alcohol dehydrogenases (ADHs) represent a very valuable biocatalytic alternative, as they can operate in more sustainable conditions and with excellent selectivities. Another interesting class of oxidoreductases that catalyse the reduction of activated C¬C double bonds are the ene-reductases (ERs). Similarly to alcohol dehydrogenases, these are NAD(P)-dependent enzymes which do not have an actual chemical catalyst counterpart. For both enzyme groups one of the major hurdles en route to preparative applications is the need of nicotinamide cofactors. The most popular approach to tackle the cofactor-dependency issue is the use of regeneration systems able to catalyse the in situ recycling of NAD(P). Recently also increasing attention has been devoted to the use of NAD(P)-like molecules that potentially able to mimic the coenzymic activity of the native cofactor. This thesis is focused on the development of NAD(P)+ regeneration systems for ADH-catalysed oxidations and on evaluation of electron donors others than the NADPH to be applied for the ene-reductases catalysis. Chapter 1 gives an introduction on the potential and limitations of both ADHs-catalysed oxidations and ERs-catalysed reductions. As a major focus the cofactor dependency issue in these enzymes is being addressed: in the case of alcohol oxidations, the use of regeneration systems for the NAD(P)+ is discussed; the application of various reductants, including NAD(P)H-like structures, to promote ERs-catalysed reductions is also reviewed. In Chapter 2 a novel NAD(P)+ regeneration system based on the use of photoexcited flavins is presented. The scope of this work was to develop a catalytic system able to regenerate both NAD+ and NADP+, thereby being able to promote alcohol dehydrogenases-catalysed oxidations. Irradiation of flavins with visible light resulted in a dramatic rate acceleration of the NAD(P)H oxidation. The system was used to promote some model oxidation reactions by the horse liver and the Thermus sp.ATN1 enzymes. In Chapter 3, the further exploitation of the ADH from Thermus sp. ATN1 (TADH) in combination with the Thermus scodoductus ER to setup a catalytic sequence for the isomerisation of allylic alcohols into saturated ketones is described. The system was developed with a model substrate and proved to be suitable for the production of enantiopure ketones from the corresponding unsaturated alcohols. The cofactor regeneration is accomplished here by means of an enzyme-coupled approach. The crystal structure of TADH is reported and analysed with particular respect to the interaction with the ([Cp*Rh(bpy)(H2O)]2+) catalyst for the regeneration of NADH, in Chapter 5. A first basis towards understanding of the mechanisms leading to inactivation of the both enzyme and Rh-complex is discusses in here. Also structural features of this enzyme compared to other thermostable ADHs are highlighted. In Chapter 6 the use of ethylendiaminetetraacetic acid (EDTA) to promote ene-reductases catalysis upon illumination has been investigated and is reported together with the biochemical characterisation of two recently identified reductases. The Deinococcus radiodurans and the Ralstonia metallidurans enzymes have been characterised with respect to the substrate scope and cofactor preference. In Chapter 4 the use of synthetic nicotinamide analogs for the ene-reductases catalysis is reported. The investigated molecules proved to perform in the catalytic cycle just as good as the NADH. Additionally, a chemoselectivity advantage could be gained from the use of NADH analogs in the presence of contaminating ADHs. Overall this work dealt with the applicability of alcohol dehydrogenase and ene-reductases for biocatalytic applications. In ADHs catalysis the cofactor issue was addressed both by developing a novel regeneration system and by applying the concept of an enzyme coupled regeneration. On the other hand, in ERs catalysis the regeneration of the prosthetic group was tackled by the use of NADH or its synthetic analogs as reductants. A light-driven system was also used to promote the ER-reductions. Finally, the combination of an ER with an ADH for the isomerisation of allylic alcohols was developed which resulted in the setup of a promising new route for the production of saturated carbonyl compounds.