Valentina Venturi


In this PhD thesis the first application of acetylacetoin synthase (AAS), by B. licheniformis DSM 13, as a biocatalyst for the stereoselective formation of C-C bonds is described. AAS, a thiamine diphosphate (ThDP) dependent enzyme, catalyzes the condensation of dialkyl or alkyl-aryl-1,2-diketones into the corresponding α-hydroxy-β-diketones with the elimination of a carboxylic acid. The reactions were carried out using a single α-diketone as donor and acceptor (homo-coupling) or two different α-diketones (cross-coupling). The AAS enzymatic reaction of a new C-C bond formation is highly chemo-, regio- and enantioselective. The α-hydroxy-β-diketones obtained from the reactions of homo- and cross-coupling were reduced with acetylacetoin reductase (AAR), a dehydrogenase obtained from the same bacterium. The combined use of AAS and AAR allowed the preparation of a new range of optically pure α-alkyl-α,β-dihydroxyketones starting from commercial α-diketones. The stereochemistry of the enantiopure syn-α-alkyl-α,β-dihydroxyketones was assigned on the basis of NOE experiments, while their absolute configuration was determined transforming one of these compounds in the natural product (+)-citreodiol. The absolute configuration of α-alkyl-α, β-dihydroxyketones confirmed the S-stereospecificity of the AAR-reduction and R-stereospecificity of AAS homo and cross-coupling reactions. On the basis of the AAS activity, an alternative synthetic biomimetic route, reminiscent the ThDP-dependent enzymes activity, was studied. Both thiamine hydrochloride and its simplified analogue, thiazolium salt, act as pre-catalysts coupled with an appropriate basis and are able to activate α-diketones such as acyl-anion equivalents that can be transferred to enable ketonic acceptors as α-diketones and α-ketoesters. These carboligation reactions have been optimized in catalytic conditions using polyethylene glycol (PEG400), an eco-friendly reaction medium, that made easier the reaction workup allowing, in addition, the catalyst recycling. A further synthetic application of AAS was the chemo-enzymatic synthesis of the natural aroma of green tea. The chirality of this compound, closely related to its organoleptic properties, is actually studied in our laboratories. The versatility of AAS as biocatalyst for C-C bond forming reactions and the raised interest by its particular applications in organic synthesis promped us to purify the enzyme, with the ultimate goal of identifying the gene encoding for AAS in the genome of B. licheniformis DSM 13.

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ISSN: 1974-918X