FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis

Abstract

The Diels–Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon–carbon bonds. Despite the importance of Diels–Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels–Alderase has been demonstrated to catalyse intermolecular Diels–Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels–Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.