Biocatalytical production of (5S)-hydroxy-2-hexanone.

Biocatalytical approaches have been investigated in order to improve accessibility of the bifunctional chiral building block (5S)-hydroxy-2-hexanone ((S)-2). As a result, a new synthetic route starting from 2,5-hexanedione (1) was developed for (S)-2, which is produced with high enantioselectivity (ee >99%). Since (S)-2 can be reduced further to furnish (2S,5S)-hexanediol ((2S,5S)-3), chemoselectivity is a major issue. Among the tested biocatalysts the whole-cell system S. cerevisiae L13 surpasses the bacterial dehydrogenase ADH-T in terms of chemoselectivity. The use of whole-cells of S. cerevisiae L13 affords (S)-2 from prochiral 1 with 85% yield, which is 21% more than the value obtained with ADH-T. This is due to the different reaction rates of monoreduction (1-->2) and consecutive reduction (2-->3) of the respective biocatalysts. In order to optimise the performance of the whole-cell-bioreduction 1 2 with S. cerevisiae, the system was studied in detail, revealing interactions between cell-physiology and xenobiotic substrate and by-products, respectively. This study compares the whole-cell biocatalytic route with the enzymatic route to enantiopure (S)-2 and investigates factors determining performance and outcome of the bioreductions.

Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.

Benzaldehyde lyase-catalyzed enantioselective carboligation of aromatic aldehydes with mono- and dimethoxy acetaldehyde.

[reaction: see text] Benzaldehyde lyase from the Pseudomonas fluorescens catalyzes the reaction of aromatic aldehydes with methoxy and dimethoxy acetaldehyde and furnishes (R)-2-hydroxy-3-methoxy-1-arylpropan-1-one and (R)-2-hydroxy-3,3-dimethoxy-1-arylpropan-1-one in high yields and enantiomeric excess via acyloin linkage. Aromatic aldehydes and benzoins are converted into enamine-carbanion-like intermediates prior to carboligation.

A short olefin metathesis-based route to enantiomerically pure arylated dihydropyrans and alpha, beta-unsaturated delta-valero lactones.

The synthesis of arylated dihydropyrans and unsaturated lactones starting from enantiomerically pure alpha-hydroxy ketones (prepared by an enzyme-catalyzed benzoin condensation) is described. The key steps are a highly diastereoselective addition of vinyl metal compounds under chelate control and a ruthenium-catalyzed ring-closing olefin metathesis reaction. Elucidation of the relative configuration of the final products was achieved by NOE experiments.

Development of a donor-acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation.

Highly enantioenriched mixed benzoins are obtained selectively through a biocatalytical cross-coupling reaction of aromatic aldehydes using ThDP-dependent enzymes.