Mandelic Amide Research Articles

Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids: Cascade reactions employing a hydroxynitrile lyase, nitrile-converting enzymes and an amidase

Enantiomerically pure 2-hydroxycarboxylic acids are valuable synthetic building blocks. This review summarises the development of bienzymatic cascades to convert aldehydes, via hydrocyanation and subsequent hydration or hydrolysis, into the corresponding (S)-2-hydroxycarboxylic amides and acids. The biocatalysts comprised an (S)-specific hydroxynitrile lyase combined with a nonselective nitrile hydratase or nitrilase. The key to success was preventing racemisation of the intermediate (S)-2-hydroxynitrile while adequately protecting the nitrile-converting enzyme, either in a cross-linked enzyme aggregate (CLEA) or in resting cells.Two biocatalyst systems for the synthesis of (S)-mandelic acid were developed: a combined crosslinked enzyme aggregate (combi-CLEA) of an (S)-hydroxynitrile lyase and a nitrilase, as well as a whole-cell Escherichia coli biocatalyst expressing both enzymes. The nitrilase formed large amounts of mandelic amide, which was remedied by including an amidase in the combi-CLEA as well as by using nitrilase variants obtained by directed mutagenesis in the whole-cell biocatalyst. Eventually, excellent results – >95% conversion of benzaldehyde into (S)-mandelic acid with near-quantitative enantiomeric purity – were obtained with both biocatalyst systems.Directed mutagenesis of the nitrilase provided an amide-selective whole-cell biocatalyst, which produced (S)-mandelic amide in near-stoichiometric yields. (S)-2-hydroxylalkanoic carboxamides were synthesised in the presence of CLEAs of hydroxynitrile lyase and nitrile hydratase.

The combi-CLEA approach: enzymatic cascade synthesis of enantiomerically pure (S)-mandelic acid

Enantiomerically pure (S)-mandelic acid was synthesised from benzaldehyde by sequential hydrocyanation and hydrolysis in a bienzymatic cascade at starting concentrations up to 0.25M. A cross-linked enzyme aggregate (CLEA) composed of the (S)-selective oxynitrilase from Manihot esculenta and the non-selective nitrilase from Pseudomonas fluorescens EBC 191 was employed as the biocatalyst. The nitrilase produces approx. equal amounts of (S)-mandelic acid and (S)-mandelic amide from (S)-mandelonitrile under standard conditions, but we surprisingly found that high (up to 0.5M) concentrations of HCN induced a marked drift towards amide production. By including the amidase from Rhodococcus erythopolis in the CLEA we obtained (S)-mandelic acid as the sole product in 90% yield and >99% enantiomeric purity.

Construction of Recombinant Escherichia coli Catalysts which Simultaneously Express an (S)‐Oxynitrilase and Different Nitrilase Variants for the Synthesis of (S)‐Mandelic Acid and (S)‐Mandelic Amide from Benzaldehyde and Cyanide

AbstractRecombinant Escherichia coli strains were constructed which simultaneously expressed the genes encoding the (S)‐oxynitrilase from cassava (Manihot esculenta) together with the wild‐type or a mutant variant of the arylacetonitrilase from Pseudomonas fluorescens EBC191 in a single organism under the control of a rhamnose‐inducible promoter. The whole cell catalysts obtained converted benzaldehyde and potassium cyanide in aqueous media at pH 5.2 mainly to (S)‐mandelic acid and/or (S)‐mandelic amide and synthesized only low amounts of the corresponding (R)‐enantiomers. The conversion of benzaldehyde and potassium cyanide (KCN) by a whole‐cell catalyst simultaneously expressing the (S)‐oxynitrilase and the wild‐type nitrilase resulted in a ratio of (S)‐mandelic acid to (S)‐mandelic amide of about 4:3. This could be explained by the strong nitrile hydratase activity of the wild‐type nitrilase with (S)‐mandelonitrile as substrate. The relative proportion of (S)‐mandelic amide formed in this system was significantly increased by coexpressing the (S)‐oxynitrilase with a carboxy‐terminally truncated variant of the nitrilase. This whole‐cell catalyst converted benzaldehyde and KCN to mandelic amide and mandelic acid in a ratio of about 9:1. The ee of the (S)‐mandelic amide formed was calculated to be >95%.

Synthesis of enantiomerically pure ( S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity

Benzaldehyde was converted into enantiomerically pure ( S)-mandelic acid by sequential HCN addition and hydrolysis in the presence of a cross-linked enzyme aggregate composed of the ( S)-selective oxynitrilase from Manihot esculenta and the non-selective recombinant nitrilase from Pseudomonas fluorescens EBC 191. Surprisingly, ( S)-mandelic amide was formed in large amounts. It was shown, in separate experiments, that the nitrilase hydrolyses ( S)-mandelonitrile into an approx. equimolar mixture of acid and amide, whereas with the ( R)-enantiomer only 10% of amide was formed.