Synthesis Of N-Substituted-γ-Amino Acid Esters With Multiple Chiral Centers By Biocatalytic Cascade Reaction

γ-Amino acid esters and γ-lactams are very important compounds,which are widely used for the synthesis of pharmaceuticals,agrochemicals,and materials.γ-Amino acid esters are mainly obtained by chiral resolution and γ-lactams can be synthesized by chemical and enzymatic methods such as imine reductase and transaminase.In this context,it is highly desired to develop new methods for the efficient synthesis of these important compounds.In this study,we developed a novel cascade reaction involving ene reductase(ERED)and imine reductase(IRED)to synthesize single stereoisomer of γ-amino acid esters andγ-lactams with two chiral centers from α,β-unsaturated γ-ketoesters.(1)First,we studied the catalytic activity and stereoselectivity systematically of these51 EREDs toward methyl 3-oxocyclopent-1-ene-1-carboxylate 、 methyl3-oxocyclohex-1-ene-1-carboxylate 、 ethyl(E)-2-methyl-4-oxopent-2-enoate,ethyl(E)-2-ethyl-4-oxopent-2-enoate,and ethyl(E)-4-oxo-2-propylpent-2-enoate.Some enantio-complementary EREDs were found to catalyze the enantioselective reduction of these α,β-unsaturated γ-ketoesters.(2)High activity and stereoselectivity of EREDs were selected for the preparation reaction of(S/R)-methyl 3-oxocyclopentane-1-carboxylate 、(S/R)-methyl3-oxocyclohexane-1-carboxylate、(R)-ethyl 2-methyl-4-oxopentanoate、(R)-ethyl2-ethyl-4-oxopentanoate and(R)-ethyl 4-oxo-2-propylpentanoate.The saturatedγ-ketoesters were prepared at the substrate concentration of 100 mM,with 99%ee,and 80-90% isolated yields.(3)The products were used for the screening of IREDs enzyme library in the laboratory using cyclopropylamine,propargylamine,allylamine and methylamine as the amino donor.A series of IREDs were identified to catalyze the reductive amination reaction to give single stereoisomer of the corresponding products.(4)Five IREDs with high activity and stereoselectivity were selected to explore the applicability in the cascade reaction with EREDs.By selecting suitable ERED and IRED enzymes,(1S,3S),(1S,3R),(1R,3S),and(1R,3R)stereoisomers of methyl 3-(cyclopropyl amino)cyclohexane-1-carboxylate were obtained as single stereoisomer in 55-70% yield,and(3R,5R)-and(3R,5S)-1-cyclopropyl-3,5-dimethylpyrrolidin-2-one and 1-cyclopropyl-3,5-dipropylpyrrolidin-2-one were prepared with diastereomer ratio of 99:1 and60-65% yields.In conclusion,we have identified 21 ene-reductases and 13 imine reductases with high catalytic activity and stereoselectivity toward some α,β-unsaturated γ-ketoesters and the corresponding saturated γ-ketoesters,respectively.By properly selecting the ERED and IRED,we constructed a tandem reaction for the precise synthesis of single stereoisomers ofγ-amino acid esters and γ-lactams with two chiral centers,establishing a new environmentally friendly enzymatic route for the synthesis of these valuable γ-amino acid esters and γ-lactams.