An enzymatic dual-oxa Diels–Alder reaction constructs the oxygen-bridged tricyclic acetal unit of (–)-anthrabenzoxocinone

The hetero-Diels–Alder (HDA) reaction is a key method for synthesizing six-membered heterocyclic rings in natural products and bioactive compounds. Despite its importance in synthetic chemistry, naturally occurring enzymatic HDA reactions are rare and limited to a single heteroatom. Here we report Abx₍₋₎F, a bifunctional vicinal oxygen chelate (VOC)-like protein that catalyses dehydration and dual-oxa Diels–Alder reactions to stereoselectively form the oxygen-bridged tricyclic acetal of (–)-anthrabenzoxocinone ((−)-ABX). Isotope assays and density functional theory calculations reveal a dehydration-coordinated, concerted HDA mechanism. The crystal structure of Abx₍₋₎F and NMR complex structures of Abx₍₋₎F with its substrate analogue and (−)-ABX define the reaction’s structural basis. Mutational analysis identifies Asp17 as a general base that mediates dehydration, forming an o-quinone methide intermediate for stereoselective dual-oxa HDA. This work establishes the molecular and structural basis of a polyheteroatomic Diels–Alderase, paving the way for designing polyheteroatomic DA enzymatic tools. (Figure presented.)