Amino saxagliptin

Fig. 2 Structure drawing of saxagliptin. Binding region of DPP-IV showing saxa-gliptin (ball-stick model) interactions with key amino acid residues (stick model) from X-ray crystal structure (3BJM) (produced with Pymol)...

Reductive amination was also conducted using cell extracts from E. coli strain SC16496 expressing PDHmod and cloned FDH from Pichia pastoris. Cells from a 15-L tank had 133 u/g FDH, 65u/g PDH (phenylpyruvate assay), and 12.7 u/g PDH (assayed with keto acid 3). The extract was used for conversion of 30g 3 to 4 in close to 100% yield, and this material, after filtration for protein removal, was converted to 2 by BOC protection. Further experiments showed that the E. coli extract could be used at 2.5% w/v concentration instead of the 12.5% concentration used for batches with Pichia pastoris extract. In subsequent experiments, the substrate input was increased to 100 g/ L and the reaction was carried out at pH 8.0. Cell extracts of E. coli strain SC16496 after polyethyleneamine treatment, clarification and concentration was used to complete the reaction in 30hrs with >96% yield and >99.9% ee of product 4. PDHmod and FDH expressed in E. coli have now been used to prepare several hundred kg of BOC-protected amino acid 2 to support the development of Saxagliptin (Hanson et al., 2007). 

Reductive amination was also conducted using cell extracts from E. coli strain SC16496 expressing PDHmod and cloned FDH from Pichia pastoris. Cell extracts after polyethyleneamine treatment, clarification, and concentration were used to complete the reaction in 30 h with greater than 96% yield and more than 99.9% EE of product 7. This process has now been used to prepare several hundred kilograms of boc-protected amino acid 8 to support the development of Saxagliptin [40]. 

The synthesis of DPP-IV inhibitor Saxagliptin 5 also required (55)-5-amino-carbonyl-4,5-dihydro-lH-pyrrole-l-carboxylic acid, l-(l,l-dimethylethyl)ester 10 (Figure 16.3C). Direct chemical ammonolyses were hindered by the requirement for aggressive reaction conditions, which resulted in unacceptable levels of amide race-mization and side-product formation, while milder two-step hydrolysis-condensation protocols using coupling agents such as 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) [41] were compromised by reduced overall yields. To address this issue, a biocatalytic procedure was developed based on the Candida antartica lipase B (CALB)-mediated ammonolysis of (55)-4,5-dihydro-lH-pyrrole-l,5-dicarboxylic acid, l-(l,l-dimethylethyl)-5-ethyl ester 9 with ammonium carbamate to furnish 10 without racemization and with low levels of side-product formation. 

Hanson RL, Goldberg SL, Brzozowski DB, et al. Preparation of an amino acid intermediate for the dipeptidyl peptidase IV inhibitor, saxagliptin, using a modified phenylalanine dehydrogenase. Advanced Synthesis and Catalysis 349(8-1-9), 1369, 2007. 

Saxagliptin is a peptidyl peptidase IV inhibitor that is currently used for the treatment of type-2 diabetes [45]. Modified phenylalanine dehydrc enase (PheDH) from Thermoacti-nomyces intermedius reductively aminates 2-(3-hydroxy-l-adamanlyl)-2-oxoethanoic acid to (S)-3-hydroxyadamantylglycine, an intermediate of Saxagliptin. The engineered PheDH contains two mutations and a 12 amino acid extension of the C-terminus. 

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