P71: Preparation of chiral pharmaceutical intermediates by biocatalysis

The development of enzymatic processes for the production of chiral amine and amino acid intermediates for the synthesis of pharmaceutical compounds will be discussed. (R)-1-cyclopropylethylamine and (R)-sec-butylamine were prepared by resolution of the racemic amines with an S-specific transaminase from Bacillus megaterium (all enzymes were expressed in E. coli). S-3-hydroxyadamantylglycine, an intermediate used for synthesis of saxagliptin, was prepared from the corresponding keto acid using a modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and formate dehydrogenase from Pichia pastoris for NADH regeneration. (R)-2-Amino-3-(7-methyl-1 H-indazol-5-yl)propanoic acid, an intermediate for a CGRP antagonist, was prepared from the racemic amino acid using S-amino acid deaminase from Proteus mirabilis in combination with an R-transaminase or from the corresponding keto acid using an R-transaminase from Bacillus thuringiensis with racemic alanine as the amino donor. An efficient chemo-enzymatic route was developed for the preparation of (S)-1-cyclopropyl-2-methoxyethanamine, an intermediate for a CRF antagonist. Cyclopropylglyoxylic acid was converted to (S)-cyclopropylglycine using leucine dehydrogenase from Thermoactinomyces intermedius with NADH cofactor recycling by formate dehydrogenase from Pichia pastoris. (S)-cyclopropylglycine was isolated as the N-Boc derivative and converted to the desired amine by reduction, methylation and Boc-deprotection. (R)-5,5,5-trifluoronorvaline was prepared from the corresponding keto acid using an R-amino acid dehydrogenase and glucose dehydrogenase for cofactor recycling, then  converted without isolation to a p-chlorophenylsulfonamide amide intermediate needed for the synthetic  route.  The glutamate dehydrogenase gene was knocked out of the E. coli expression strain to eliminate background production of the S-amino acid and improve the ee of the product to >99%.