Imine Synthesis by Engineered d-Amino Acid Oxidase from Porcine Kidney

Area of Science:

• Biocatalysis
• Organic Synthesis
• Enzyme Engineering

Background:

• Enzyme catalysis offers a sustainable alternative to traditional chemical synthesis.
• Developing novel biocatalysts for imine synthesis is crucial for green chemistry.
• Porcine kidney d-amino acid oxidase (pkDAO) variants were engineered for imine production.

Purpose of the Study:

• To synthesize symmetric and asymmetric imines using engineered pkDAO variants.
• To investigate the substrate scope and efficiency of the novel biocatalyst.
• To compare the catalytic performance with existing chemical catalysts.

Main Methods:

• Enzymatic oxidation of methylbenzylamine (MBA) derivatives using engineered pkDAO (Y228L/R283G and I230A/R283G).
• Trapping of reactive imine intermediates with various nucleophilic amines.
• Characterization of synthesized imines and analysis of reaction products.

Main Results:

• High yields (nearly 100%) and stoichiometric conversion (100 mM) achieved for symmetric imine synthesis using (R)-Fluoro-MBA.
• Successful synthesis of various symmetric and asymmetric imines from different (R)-MBA derivatives.
• A turnover number of 1.61 × 10^5 h⁻¹ for N-benzylidenebenzylamine synthesis, exceeding existing catalysts by over 10³-fold.
• Diastereomeric analysis of reduced products revealed a mixture of (R,R)- and (R,S)-bis(1-phenylethyl)amine.

Conclusions:

• Engineered pkDAO variants are highly effective biocatalysts for imine synthesis.
• The developed method provides a superior, environmentally friendly alternative to conventional imine synthesis.
• The high turnover number highlights the potential of enzymatic catalysis in industrial applications.