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

Various symmetric and asymmetric imines were synthesized using the novel amine oxidase, obtained as variants of d-amino acid oxidase (pkDAO) from porcine kidney (Y228L/R283G) and (I230A/R283G). Active primary imines produced as intermediates in the oxidation of methylbenzylamine (MBA) derivatives were trapped by aliphatic, aromatic amines and diamines as nucleophiles forming new imines. (R)-Fluoro-MBA was the best substrate for symmetric imine synthesis, providing almost stoichiometric conversion (100 mM) and achieving nearly 100% yield. Several (R)-MBA derivatives were used as substrates, and the corresponding symmetric and asymmetric imines were synthesized. The turnover number of N-benzylidenebenzylamine synthesis from benzylamine was calculated to be 1.61 × 10⁵ (number of moles of reactant consumed per mole of catalyst/h), which is more than 10³ higher than metal-, photo-, and organo-catalysts reported so far. The diastereomers of bis(1-phenylethyl)amine, the reduced products of (R)-MBA, were identified as a mixture of 84.9% (R,R)-bis(1-phenylethyl)amine and 15.1% (R,S)-bis(1-phenylethyl)amine to consider the reaction mechanism.