Reductive aminations phenylpyruvate

Scheme 2.14 Reductive amination of phenylpyruvic acid derivatives under biphasic conditions.

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). 

L-Phe can be prepared via the enantioselective hydrolysis of N-acetyl-D,L-Phe and microbial reductive amination of phenylpyruvate (see Fig. 8.14) [87]. The stoichiometric yields of these processes were high but the precursors required 3-4 synthetic steps from the basic starting materials in most cases. The phenyl-ammonia lyase route, in contrast [89], provided L-Phe in only two steps from the basic chemicals benzaldehyde and acetic anhydride [90]. The enzymatic step... 

The biosynthetic pathway from SA into L-Phe [69, 70] is shown in Fig. 8.15. The synthesis of chorismate (CHA), the common intermediate in the biosynthesis of the aromatic amino acids, requires an extra equivalent of PEP, which limits the yield of L-Phe from glucose to 0.30 mol mol-1 if PEP is not conserved [91]. The further transformation of CHA into phenylpyruvic acid (PPY) suffers from inhibition by L-Phe and is also subject to transcriptional control [69, 92]. The final step is a reductive amination of PPY into L-Phe with consumption of l-G1u. 

Two reactions for the production of L-phenylalanine that can be performed particularly well in an enzyme membrane reactor (EMR) are shown in reaction 5 and 6. The recentiy discovered enzyme phenylalanine dehydix enase plays an important role. As can be seen, the reactions are coenzyme dependent and the producticm of L-phenylalanine is by reductive amination of phenylpyruvic add. Electrons can be transported from formic add to phenylpyruvic add so that two substrates have to be used formic add and an a-keto add phenylpyruvic add (reacticm 5). Also electrons can be transported from an a-hydroxy add to form phenylpyruvic add which can be aminated so that only one substrate has to be used a-hydroxy acid phenyllactic add (reaction 6). 

For reductive amination, basically no thermodynamic limitation exists for the leucine/ketoleucine reaction at pH 11.0, JCq equals 9xl012[2S1, for phenylalanine/ phenylpyruvate at pH 7.95 a Keq of 2.5xl07 has been reported1181, thus, the maximum degree of conversion is very close to 100%. Coupling of the reductive amination reaction with cofactor regeneration via the FDH/formate reaction, which is irreversible, further helps to pull the equilibrium towards the amino acid product. 

As shown in Table 7, phenylpyruvate analogs substituted at the phenyl ring are relatively good substrates. The enzyme utilizes the compounds substituted at the 3-position of pyruvic acid with a longer or a bulkier group, although the relative rate of the reductive amination reaction is low. 

Starting from 2-oxo acids, various optically pure (S)-amino acids can be quantitatively synthesized by using PheDII and formate dehydrogenase. Tabic 8 shows the yields of the (S)-amino acids thus synthesized. The reductive amination products derived from (3/ /,S )-3-methy 1-2-ox ovaierate and (37 /5)-3-methyl-2-oxo-3-phenylpyruvate have been identified as diastereomeric mixtures of (S)-isoleucine and (S)-alloisoleucine, and (2S,3f / S)-2-amino-3-methyl-3-phenyl-propionic acid, respectively. The product from 2-oxo-5-phcnylvalerate solidified as the reaction proceeded. 

T. intermedins IFO14230 (ATCC 33205) was first identihed as a source of PDH by Ohshima et al." The enzyme was purified and characterized" and then cloned and expressed in E. coli by the same workers. The enzyme was reported to be moderately specihc for deamination of phenylalanine and to carry out the amination of some keto acids at a much lower rate than amination of phenylpyruvate." In our screening, the enzyme was the most effective amino acid dehydrogenase identified for the reductive amination of the keto acid acetal. 

Figure. 2. Enzymatic synthesis of L-phenylalanine by reductive amination starting from phenylpyruvate (using EMR technology).

L-PheDH/FDH Reductive amination of phenylpyruvate L-Phenylalanine 456 1500 L-PheDH 150 FDH 600000... 

In one of the first examples, the reductive amination of phenylpyruvate catalyzed by a NADH-dependent L-phenylalanine dehydrogenase (PheDH) was coupled with the in situ generation of the substrate from acetamidocinnamic add (ACA) by a suitable acylase, thus avoiding both substrate inhibition and instability (Scheme 11.12a) [20]. An intracellular acylase was selected from a Brevibacterium strain and employed in this one-pot process at ACA concentrations up to 0.3 M with quantitative conversions into the desired product L-phenylalanine. 

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