Simon C. Willies

Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution.

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.

Directed Evolution of the Enzyme Monoamine Oxidase (MAO‐N): Highly Efficient Chemo‐enzymatic Deracemisation of the Alkaloid (±)‐Crispine A

We have previously reported a general method for the deracemisation of racemic chiral amines (primary, secondary and tertiary) by using variants of the enzyme monoamine oxidase N (MAO-N) from Aspergillus niger. This deracemisation process employs a combination of an enantioselective enzymatic oxidation of the amine to afford the corresponding imine or iminium ion, together with a non-selective chemical reduction of the imine or iminium ion back to the racemic starting material (Scheme 1). The use of an (S)-selective MAO-N

Enzymatic desymmetrising redox reactions for the asymmetric synthesis of biaryl atropisomers.

Atropisomeric biaryls carrying ortho-hydroxymethyl and formyl groups were made enantioselectively by desymmetrisation of dialdehyde or diol substrates. The oxidation of the symmetrical diol substrates was achieved using a variant of galactose oxidase (GOase), and the reduction of the dialdehydes using a panel of ketoreductases. Either M or P enantiomers of the products could be formed, with absolute configurations assigned by time-dependent DFT calculations of circular dichroism spectra. The differing selectivities observed with different biaryl structures offer an insight into the detailed structure of the active site of the GOase enzyme.

Deracemisation of benzylisoquinoline alkaloids employing monoamine oxidase variants

Chemo-enzymatic deracemisation was applied to obtain the (S)-enantiomer of 1-benzylisoquinolines from the racemate in high isolated yield (up to 85%) and excellent optical purity (ee > 97%). The one-pot deracemisation protocol encompassed enantioselective oxidation by a monoamine oxidase (MAO-N) and concomitant reduction of the resulting iminium species by ammonia-borane. The challenge was the oxidation at the sterically demanding chiral centre. Recently developed variants of MAO-N, featuring an enlarged active-site pocket, turned out to be suitable biocatalysts for these substrates. In contrast to previous MAO-N variants, which preferentially converted the (S)-enantiomer, the MAO-N variant D11 used in the present study was found to oxidise all tested benzylisoquinoline substrates with (R)-enantiopreference. The structural determinants of enantioselectivity were investigated by means of protein–ligand docking simulations. The applicability of the deracemisation system was demonstrated on preparative scale (150 mg) for three benzylisoquinoline alkaloids (natural as well as non-natural), including the hypotensive and antispasmodic agent (S)-reticuline.

A stereospecific solid-phase screening assay for colonies expressing both (R)- and (S)-selective ω-aminotransferases

A novel solid-phase screening assay was developed for colonies expressing both (R)- and (S)-selective ω-aminotransferases. This high-throughput assay can be used to screen rapidly large variant libraries with enhanced substrate selectivity and enantioselectivities.