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
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.
Figure 3: Nonnatural L-amino-acid production by reductive amination. (FIGURE COURTESY OF KANEKA)
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
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.
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).
Figure 4: One-pot enzymatic deracemization for nonnatural L-amino acids. (FIGURE COURTESY OF KANEKA)