Oxidation of Ketones: A (Chemo-) Enzymatic Approach Using Oxygenases and Hydrolases

Abstract: Oxidation reactions are important in organic chemistry as well as in nature. In industry, oxidations are commonly used for the synthesis of chemicals and pharmaceuticals, however such processes have a number of limitations, they use chlorinated solvents, stoichiometric oxidation reagents, and in some cases the reagents that have risks of explosion during transportation and storage. This has called for more environment-friendly alternative technologies for oxidation reactions. Baeyer-Villiger oxidation is a reaction in which a ketone is oxidized to an ester or a cyclic ketone to a lactone by treatment with peroxyacids. Lactones constitute an important group of chemicals used in flavors, fragrances, pharmaceutical intermediates and polymer building blocks. The work presented in this thesis concerns enzymes, including Baeyer-Villiger monooxygenases (BVMOs) that catalyse the Baeyer-Villiger oxidation using molecular oxygen as an oxidant, and perhydrolytic enzymes that can be used for in situ generation of peracid for oxidation of cyclic ketones. A simple colorimetric method was developed for detection of BVMO activity and was based on the formation of a purple colored product between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. The method was shown to have potential for screening of both wild type and recombinant microbial cells as well as for quantitative measurement of BVMO activity. Further, a recombinant BVMO from a strain of Dietzia was characterized. The sequence of the enzyme suggested that it is related to Ethionamide monooxygenases. The recombinant enzyme was active in whole cells and crude lysate but lost activity on purification. The enzyme was shown to have high activity towards several linear alkenes, and was also moderately active towards cyclobutanone, phenylacetone and thioanisole. Two perhydrolytic enzymes able to produce peracids from a carboxylic ester and hydrogen peroxide were studied for oxidation of cyclohexanone to caprolactone, a chemical of immense importance. The enzymes were immobilized as cross-linked enzyme aggregates (CLEAs). The well-studied lipase B from Candida antarctica (CaLB) gave a maximal caprolactone yield of 80% with ethyl acetate as acyl donor. The perhydrolase was able to produce peracids in an aqueous medium with ethylene glycol diacetate and hydrogen peroxide, and gave caprolactone yield of 70%. In both cases the formation as acetic acid as a coproduct showed to be an important factor for the deactivation of the enzyme. The use of monooxygenases, lipases and perhydrolases for the Baeyer-Villiger reaction constitutes a greener alternative to traditional chemical processes but the problem of enzyme stability remains to be solved.