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.

狄尔斯-阿尔德反应是合成化学中碳-碳键的立体有择构造中最强大，应用最广泛的方法之一。尽管Diels-Alder反应在许多次生代谢产物的生物合成中很重要，但尚无天然存在的独立Diels-Alderase能够催化分子间Diels-Alder转化。在这里，我们报告黄素腺嘌呤二核苷酸依赖性酶，桑 白尔斯- Alderase（MAD​​A），从桑细胞培养，可催化分子间[4 + 2]环加成反应，从而以高效率和对映选择性产生天然异戊二烯基黄酮类查克拉霉素。密度泛函理论计算和动力学同位素效应的初步测量建立了一个协调但异步的周环途径。结构指导的诱变和对接研究表明，MaDA与二烯和亲二烯体的相互作用可催化[4 + 2]环加成反应。MaDA对二烯和亲二烯体均显示出底物混杂性，这使得能够方便地合成结构多样的天然产物。我们还报告了用于发现MaDA的基于生物合成中间探针（BIP）的目标识别策略。