Applying Biocatalysis: A Technical Forum - Pharmaceutical Technology

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Applying Biocatalysis: A Technical Forum
Scientists from DSM and Kaneka discuss various techniques in this roundtable moderated by Patricia Van Arnum.

Pharmaceutical Technology

Biocatalyst-driven Chiral Amines

By Masahiko Yamada, senior researcher, Frontier Biochemical & Medical Research Laboratories, a research division of Kaneka

Chiral amines, chiral amino acids, and chiral alcohols are key pharmaceutical intermediates. Kaneka, a producer of chiral alcohols (1-4), recently implemented a systems-biotechnology approach using protein engineering and molecular biology to introduce the chiral amine group into desired compounds.

Nonnatural L-amino acids

Figure 3: Nonnatural L-amino-acid production by reductive amination. (FIGURE COURTESY OF KANEKA)
Reductive amination. Nonnatural amino acids are increasingly becoming critical building blocks for drug manufacturers, in particular for the synthesis of protease inhibitors. Kaneka has been investigating the production of such nonnatural amino acids by the reductive amination of ketoacids (see Figure 3). The method developed by Kaneka combines an amino-acid dehydrogenase and a coenzyme recycling system in Escherichia coli. By selecting the appropriate dehydrogenases, both aliphatic and aromatic L-amino acids can be produced. Phenylalanine dehydrogenases provide a chiral aromatic amino-acid series and leucine dehydrogenases a chiral aliphatic amino-acid series.

Because the dehydrogenase requires NADH ( -nicotinamide adenine dinucleotide) as a coenzyme, the recycling of the coenzyme is critical to the reaction. Kaneka has succeeded in regenerating NADH to accelerate the reductive amination. This successful recycling is accomplished by using durable formate dehydrogenases (FDH) isolated from a specific soil-based microorganism, in which FDH survived even under high-substrate concentrations, even with electrophiles, including halogenated hydrocarbons (5).

The harmonization of the reductase library and coenzyme regeneration crafted in E. coli allows the system to produce nonnatural L-amino acids in high concentrations, sometimes as much as 100 g/L. Essentially no undesired products, except carbon dioxide from formate, are produced, and enantiomeric excesses of more than 99% are common.

Figure 4: One-pot enzymatic deracemization for nonnatural L-amino acids. (FIGURE COURTESY OF KANEKA)
One-pot enzymatic deracemization. Kaneka produces nonnatural L-amino acids from readily available racemic unprotected amino acids. Kaneka's technology uses four enzymes: a D-amino-acid oxidase and an L-amino-acid dehydrogenase for the actual reaction steps and two other enzymes, including coenzyme recycling. The mechanism is an expansion of the reductive amination technology previously described. In the oxido-reduction sequence, the oxidation of the undesired D-amino acid to the a-ketoacid or to the imino derivative takes place in concert with the reductive amination (see Figure 4). The system was scaled, giving yields of greater than 90% and enantiomeric excess of more than 99% (6, 7).


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