Efficient Artificial Cu(II)-Diels–Alderase Based on Streptavidin for Highly Enantio- and Regioselective Diels–Alder Reactions

The enzymatic Diels–Alder reaction presents an environmentally appealing strategy for synthesizing chiral norbornene scaffolds. In this study, an artificial Diels–Alderase was constructed by incorporating a biotinylated Cu-phenanthroline cofactor (5-NH₂Phen-biotin*Cu(NO₃)₂) into streptavidin and optimizing the enzyme through genetic engineering. The S112D variant of this artificial Diels–Alderase exhibited commendable catalytic performance, facilitating highly enantio- and regioselective Diels–Alder reactions. Employing this method, we synthesized a series of norbornene pyridones with broad substrate scopes and good functional group tolerance under mild conditions, achieving high yields along with good enantio- and regioselectivities. Molecular dynamics (MD) simulations provided insights into the critical complex intermediates involved in the proposed reaction mechanism and clarified the interactions between the Diels–Alderase and its substrates, revealing the structural basis for the generation of the predominant norbornenepyridone conformation and the enhanced selectivity observed with the S112D mutant. Furthermore, quantum mechanics/molecular mechanics (QM/MM) calculations analyzed the reaction energy barriers of possible transition states, elucidating the reason for the formation of the target product from an energetic perspective.