PUB - Publikationen an der Universität Bielefeld

The biocatalytic synthesis of nitriles and especially dinitriles were investigated within this work. Therefore, the access to dialdehydes were studied with two different approaches. The first approach was the creation of an artificial hydroformylase based on the reconstitution of a P450 monooxygenase, namely CYP119, with a Rhodium-porphyrin ligand (Rh-PO) which showed hydroformylation activity for the hydroformylation of 1-octene. After expression in minimal medium and purification high amounts of heme-free apo-CYP119 could be obtained and reconstituted with Rh-PO. The novel artificial metalloenzyme and two created mutants were tested for the hydroformylation of 1-octene and 3-butenoic acid, but no hydroformylation activity could be found. The second approach towards dialdehydes was the oxidation of dialcohols with an Anelli-type TEMPO-oxidation. For this green and mild oxidation method a solvent screening was performed, showing that nitriles are suitable solvents for this reaction type and a variety of different primary and secondary alcohols could be oxidized with high yields and selectivities by Alessa Hinzmann in her doctoral thesis. This method was then applied for the oxidation of dialcohols and after optimization of the reaction conditions five dialcohols could be oxidized with excellent selectivities of 90% and high conversions of 96% in all cases, showing the general applicability of this method. After gaining access towards dialdehydes the oxidation of dialcohols was combined with the synthesis of dialdoximes and the biocatalytic dehydration of them using aldoxime dehydratases in a three-step two-pot cascade reaction. The oxidation of the n-octan-1,8-diol and the condensation with hydroxylamine could be performed in a one-pot two-step sequential reaction and after isolation of n-octanedialdoxime with an isolated yield of 71%, the product was biocatalytically dehydrated using OxdB as whole-cell catalyst with full conversion and an isolated yield of 86% with 98% purity. The whole threestep two-pot cascade could be performed with full conversion in all steps with a high overall yield of 61%.
Furthermore, the limits of aldoxime dehydratases were explored. Therefore, the substrate scope of the aldoxime dehydratase OxdRE was expanded towards aromatic aldoximes by a semi-rational mutagenesis study. This was achieved by a systematic activity study of OxdRE and mutants for a library of 22 different substituted aldoximes, which revealed that OxdRE-Wt has high activity for ortho-substituted aromatic aldoximes and that the mutant M29G showed activity for meta-substituted aromatic aldoximes. Access to the para-substituted substrates could be generated by rational design of the mutant F306A. The applicability of OxdRE-Wt and the mutants M29G and F306A in organic synthesis was proven in preparative biotransformations in 10 mL scale with high conversions and high yields for 2-chlorobenzaldoxime, 3-phenylbenzaldoxime and 4-phenylbenzaldoxime as substrates. This study enabled the biocatalytic access to these substrates for the first time. The biocatalytic toolbox of aldoximes dehydratases could furthermore be extended by discovery of six novel aldoxime dehydratases using a 3DM database. These novel enzymes showed a divers and broad substrate scope and the novel aldoxime dehydratase OxdHR showed a remarkable high activity for the substrate n-octanaloxime. The applicability of the novel enzymes was shown by a biotransformation of n-octanaloxime with OxdHR in which 100 mM substrate could be converted with 33 mgbww/mL with full conversion after 5 h and 60% isolated yield.
As a third part of this thesis the biocatalytic synthesis of 3-phenylpropanal was investigated starting by oxidation of benzyl alcohol to benzaldehyde with an alcohol dehydrogenase, followed by an aldol condensation with acetaldehyde catalyzed by the enzyme 4-OT and a reduction of the produced 3-phenylprop-2-enal using an ene-reductase in a three step one-pot cascade in a co-factor neutral reaction. Screenings for an alcohol dehydrogenase and a suitable ene-reductase showed that the thermostable alcohol dehydrogenase BstADH and the enereductase Gox8 are suitable catalysts. However, BstADH showed activity for several intermediates of the whole cascade and Gox8 was not able to consume the produced NADH fast enough to suppress side reactions. Nevertheless, the full cascade with ADH, 4-OT and enereductase was performed and a relative conversion of 1% to the product 3-phenylpropanal and 11% relative conversion to the further by BstADH reduced side product 3-phenylpropanol was measured with no other side products.