Abstract:The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase, and further scaled up using a preparation of the two enzymes expressed in E. coli.

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Biotransformation of mutilin and pleuromutilin by microbial cultures was investigated to provide a source of 8-hydroxymutilin or 8-hydroxypleuromutilin. LC/MS analysis of culture broths showed that several strains gave M+16 products from mutilin and one culture gave an M+16 product from pleuromutilin, suggesting addition of oxygen. Biotransformation products were extracted from culture broths with ethyl acetate, dried, and purified by chromatography on silica gel. Streptomyces griseus strains SC 1754 and SC 13971 (ATCC 13273) converted mutilin to (8S)-, (7S)-, and (2S)-hydroxymutilin. Cunninghamella echinulata SC 16162 (NRRL 3655) gave (2S)-hydroxymutilin or (2R)-hydroxypleuromutilin from biotransformation of mutilin or pleuromutilin, respectively. The biotransformation of mutilin by S. griseus strain SC 1754 was scaled up in 15-, 60-, and 100-L fermentations to produce a total of 49 g of (8S)-hydroxymutilin (BMS-303786), 17 g of (7S)-hydroxymutilin (BMS-303789) and 13 g of (2S)-hydroxymutilin (BMS-303782) from 162 g of mutilin.

A new enzymatic process for the enantioselective cleavage of N-benzyloxycarbonyl (Cbz) groups from protected amino acids and related compounds has been developed. The Cbz-deprotecting enzyme was isolated from cell extracts of Sphingomonas paucimobilis SC 16113 and purified to homogeneity. The purified protein has a molecular weight of 155,000 daltons and a subunit size of 44,000 daltons.