The 100-year-old Diels–Alder reaction (DAr) is an atom economic and elegant organic chemistry transformation combining a 1,3-diene and a dienophile in a [4+2] cycloaddition leading to a set of products with several stereo centres and multiple stereoisomers. Stereoselective [4+2] cycloaddition is a challenge. Here, we describe two natural enzymes, PyrI4 and AbnU performing stereospecific intermolecular DAr on non-natural substrates. AbnU catalyses a single exo-stereoisomer by 32-fold higher than the background. PyrI4 catalyses the same stereoisomer (15-fold higher) as a major component (>50%). Structural, biochemical and fluorescence studies indicate that the dienophile enters first into the β-barrel of the enzymes followed by the 1,3-diene, yielding a stereospecific product. However, if some critical interactions are disrupted to increase the catalytic efficiency, stereoselectivity is compromised. Since it is established that natural enzymes can carry out intermolecular DAr on non-natural substrates, several hundreds of Diels-Alderases available in nature could be explored.

已有 100 年历史的 Diels-Alder 反应 (DAr) 是一种原子经济和优雅的有机化学转化，将 1,3-二烯和亲二烯体结合在 [4+2] 环加成中，产生一组具有多个立体中心的产物和多种立体异构体。立体选择性 [4+2] 环加成是一项挑战。在这里，我们描述了两种天然酶 PyrI4 和 AbnU，它们在非天然底物上进行立体特异性分子间 DAr。AbnU 催化单个外立体异构体比背景高 32 倍。PyrI4 催化相同的立体异构体（高 15 倍）作为主要成分 (>50%)。结构、生化和荧光研究表明，亲二烯体首先进入酶的 β-桶，然后是 1,3-二烯，产生立体特异性产物。然而，如果一些关键的相互作用被破坏以提高催化效率，立体选择性就会受到损害。由于已确定天然酶可以在非天然底物上进行分子间 DAr，因此可以探索自然界中存在的数百种 Diels-Alderases。