Recently, a collaborative research team comprising Professor Qu Xudong's group from the Zhangjiang Advanced Research Institute and the School of Life Sciences and Technology at Shanghai Jiao Tong University, alongside Professor Mehdi Mobli's group from The University of Queensland, Australia, and Professor Zhao Yilei's group from the School of Life Sciences and Technology at Shanghai J the teams of Zhao Yilei and Kong Xudong from the School of Life Sciences and Technology at Shanghai Jiao Tong University. Their paper, titled ‘An enzymatic dual-oxa Diels-Alder reaction constructs the oxygen-bridged tricyclic acetal unit of (-)-anthrabenzoxocinone’, was published in Nature Chemistry. This study has for the first time elucidated the catalytic mechanism of a novel dual-oxa Diels-Alder (HDA) enzyme, responsible for forming the chiral oxygen-bridged tricyclic acetal unit in the type II polyketide natural product (-)-anthrabenzoxocinone ((-)ABX). Postdoctoral researcher Yan Xiaoli from the School of Life Sciences and Technology and Dr Jia Xinying from the University of Queensland are the co-first authors. Professor Qu Xudong, Professor Mehdi Mobli, and Professor Zhao Yilei are the co-corresponding authors. Associate Professor Kong Xudong (tenured track), along with students Ji Shunjia and Zhang Mengjie, are co-authors.

Heterocyclic Diels-Alder reactions (HDA) represent one of the most efficient methods for constructing six-membered heterocyclic compounds, finding extensive applications in medicinal chemistry, materials science, and natural product synthesis. Among these, oxygenated HDA reactions (involving oxygen atoms in dienes or diene-like structures) are the most prevalent type, enabling efficient construction of oxygen-containing heterocyclic skeletons. However, despite the discovery of numerous enzymes catalysing single oxygen-containing HDA reactions in nature (e.g., LepI, EupfF, Tsn11), no double oxygen-containing DA reaction (involving simultaneous [4+2] cycloaddition of two oxygen atoms) has ever been reported. Its catalytic mechanism and enzymatic basis remain entirely unknown.

Figure 1. Reaction types of oxo-HDA and representative natural products containing tricyclic ketal oxygen-bridge structural units.

The Xu Dong Qu research group has long been dedicated to the biosynthesis of molecular skeletons. Previous work elucidated the formation mechanism of the tetracyclic aromatic nucleus in (-)ABX (Proc Natl Acad Sci USA 2024, 121, e2321722121), yet the biosynthetic pathway for its key structural unit—the tricyclic ketal oxygen bridge—remained unresolved. Notably, this tricyclic ketoxime bridge structure is widely present in numerous biologically active natural products (Figure 1b), where its unique rigid conformation decisively influences the molecule's pharmacological activity. Therefore, elucidating its biosynthetic mechanism holds significant implications for natural product chemistry and innovative drug discovery.

In this project, the collaborative research team, through systematic in vivo and in vitro experiments, has for the first time demonstrated that the VOC superfamily protein Abx(-)F functions as a dual-catalytic DA enzyme: it first catalyzes a dehydration reaction, converting compound 2 into the specific enantiomerically pure o-quinone methylene (o-QM) intermediate (Z, Ra)-3; Subsequently, it stereoselectively catalyses an intramolecular dioxy-DA reaction on (Z, Ra)-3, precisely constructing a chiral tricyclic ketal oxygen bridge structure. The research team further employed a comprehensive approach combining X-ray crystallography, nuclear magnetic resonance (successfully resolving the structures of Abx(-)F and its substrate complexes), and computational chemistry. This revealed a novel catalytic mechanism termed ‘dehydration-coordination synergistic HDA’ (Figure 2), providing a paradigm shift for understanding the biosynthesis of such complex structures.

Figure 2. Catalytic mechanism of Abx(-)F.

The scientific significance of this research manifests across multiple dimensions:

(1) Novel enzymological discovery: Identification of the first-ever dioxy-HDA enzyme not only fills a gap in research on polyheteroatom Diels-Alder enzymes but also suggests the potential existence of an entirely new family of polyheteroatom HDA enzymes in nature, significantly broadening our understanding of enzyme-catalysed HDA reactions;

(2) Breakthrough in natural product synthesis: It provides the first enzymatic template for understanding the ubiquitous ketal oxygen bridge structure, resolving a long-standing critical scientific question in this field;

(3) Novel insights into polyketide chemistry: The ketal oxygen bridge structure in (-)-ABX represents the ninth skeletal type discovered among nearly ten thousand Type II polyketides, and constitutes the second chiral skeleton identified by the research group following their recent discovery (J Am Chem Soc 2025, 147, 5596–5601), establishing a crucial foundation for rational design of complex aromatic polyketones.

This research not only deepens understanding of the chemical diversity of the HDA reaction but also provides innovative tools for directed biosynthesis and enzyme engineering of natural products. The associated techniques hold promise for green manufacturing of high-value oxygen-containing heterocyclic compounds.

This research received funding from the National Key R&D Programme of China, the National Natural Science Foundation of China, and the Shanghai Academic/Technical Research Leading Talent Programme.

Paper link: https://doi.org/10.1038/s41557-025-01804-0