Selection, Purification, Characterisation, and Cloning of a Novel Heat-Stable Stereo-Specific Amidase fromKlebsiellaoxytoca, and Its Application in the Synthesis of Enantiomerically Pure (R)- and (S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic Acids and (S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionamide

We isolated, characterised, and cloned an enantio-specific amidase from Klebsiella oxytoca and used it to resolve ( R,S )-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide, giving ( R )-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and ( S )-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. The ( S )-amide could then be hydrolysed chemically to ( S )-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. The process can therefore be adapted to produce both ( R ) - and ( S ) - enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, or ( S )-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. The biocatalytic step is part of a combined chemical and biocatalytic route that starts from ethyl trifluoroacetoacetate. The products typically have a purity of greater than 98% and ee values of essentially 100% after isolation. The process has been used to produce 100-g amounts of the ( S )-acid, and successfully scaled up to produce 100-kg amounts of the ( R )-acid, with the biotransformation carried out at the 1500-L scale.