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

Abstract

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)-3,3,3trifluoro-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-2hydroxy-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.

A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide from Arthrobacter sp. S-2

“…S-2 constitutively showed hydrolysis activity toward amide 2 and hydrolyzed it S-enantioselectively. Although R-enantioselective amidase acting on amide 2 had already been purified from K. oxytoca PRS1, 5,6) , no enzymes exhibiting S-enantioselective hydrolysis activity toward amide 2 have been reported to date. Therefore, we herein purified the S-enantioselective amidase acting on amide 2 from Arthrobacter sp.…”

“…oxytoca PRS1. 5,6) For example, S-amidase exhibited its maximum activity at 40°C, and was almost inactivated after being incubated at 60°C, whereas R-amidase was reported to be heat-stable with maximum activity at 70°C. On the other hand, the specific activity of S-amidase toward amide 2 (222 U/mg) was markedly higher than that of R-amidase (2.7 U/mg).…”

“…4) Although few studies have examined the hydrolysis of trifluoromethyl-substituted aliphatic amides, Shaw developed a process for the large-scale synthesis of enantiomerically pure acid 1 by a kinetic resolution using the R-enantioselective amidase from Klebsiella oxytoca PRS1. 5,6) In our previous study, the enantioselective hydrolysis activity acting on amide 2 was screened from microbial sources, and Shinella sp. R-6 and Arthrobacter sp.…”

Hydrolysis of Amides

“…It encodes a polypeptide of 314 amino acids with clear homology to the acetamidase/formamidase family of proteins, including the stereoselective amidases from Enterobacter cloacae N-7901 [311], Thermus sp. 0-3-1 [312], and Klebsiella oxytoca PRS1 [61]. The Enterobacter and Thermus amidase genes, which were isolated by Mitsubishi researchers, encode amidases of 315 and 309 amino acids, respectively, which are 67 and 53% identical to O. anthropi LamA.…”

“…Examples are (R)-and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid (113), which are intermediates for the synthesis of several potential pharmaceuticals, including drugs for the treatment of incontinence and diabetes (see Reference [63] and primary references therein). At Lonza AG an efficient chemoenzymatic process was developed for the large-scale production of both enantiomers of this a-hydroxy acid in high optical purity [61,62,313]. The key step in this process is the amidase-catalyzed kinetic resolution of racemic 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (108,Scheme 15.20).…”