β-Phenylalanine Ester Synthesis from Stable β-Keto Ester Substrate Using Engineered ω-Transaminases.

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Synthesis

Background:

• Chiral amine synthesis is crucial in pharmaceuticals.
• Amination of β-keto acids is challenging due to substrate instability.
• Transamination of β-keto esters presents a viable alternative.

Purpose of the Study:

• To investigate the use of ω-transaminases for the amination of β-keto esters.
• To identify suitable ω-transaminase variants for synthesizing chiral β-phenylalanine esters.
• To establish an alternative route for producing enantiomerically enriched β-amino acid derivatives.

Main Methods:

• Screening of an ω-transaminase library using a coloring o-xylylenediamine assay.
• Enzyme engineering and directed evolution of ω-transaminases.
• Semi-preparative synthesis for isolation of the target product.

Main Results:

• Identified ω-transaminase mutants (3FCR_4M and ATA117 11Rd) with potential for β-keto ester transamination.
• Achieved 32% conversion for the (S)-enantiomer and 13% for the (R)-enantiomer of ethyl benzoylacetate.
• Successfully isolated (S)-β-phenylalanine ethyl ester via semi-preparative synthesis.

Conclusions:

• ω-transaminases can be engineered for the efficient synthesis of chiral β-phenylalanine esters from β-keto esters.
• This biocatalytic approach offers a promising alternative to traditional chemical methods.
• The identified enzyme variants are suitable for further optimization and scale-up.