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 AbxF, 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 AbxF and NMR complex structures of AbxF 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.