Hunting for the Intermolecular Diels–Alderase

ConspectusThe Diels-Alder reaction is well known as a concerted [4 + 2] cycloaddition governed by the Woodward-Hoffmann rules. Since Prof. Otto Diels and his student Kurt Alder initially reported the intermolecular [4 + 2] cycloaddition between cyclopentadiene and quinone in 1928, it has been recognized as one of the most powerful chemical transformations to build C-C bonds and construct cyclic structures. This named reaction has been widely used in synthesizing natural products and drug molecules. Driven by the synthetic importance of the Diels-Alder reaction, identifying the enzyme that stereoselectively catalyzes the Diels-Alder reaction has become an intriguing research area in natural product biosynthesis and biocatalysis. With significant progress in sequencing and bioinformatics, dozens of Diels-Alderases have been characterized in microbial natural product biosynthesis. However, few are evolutionally dedicated to catalyzing an intermolecular Diels-Alder reaction with a concerted mechanism.This Account summarizes our endeavors to hunt for the naturally occurring intermolecular Diels-Alderase from plants. Our research journey started from the biomimetic syntheses of D-A-type terpenoids and flavonoids, showing that plants use both nonenzymatic and enzymatic intermolecular [4 + 2] cycloadditions to create complex molecules. Inspired by the biomimetic syntheses, we identify an intermolecular Diels-Alderase hidden in the biosynthetic pathway of mulberry Diels-Alder-type cycloadducts using a biosynthetic intermediate probe-based target identification strategy. This enzyme, MaDA, is an