The Diels–Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels–Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels–Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels–Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP⁺/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels–Alder cycloaddition, which serves as the first example of a bifunctional Diels–Alderase that utilizes this mechanism.

第尔斯-阿尔德环加成是有机合成中最强大的方法之一，通常用于重要药物的合成。然而，严格控制狄尔斯-阿尔德反应的立体选择性具有挑战性，需要付出巨大努力通过有机催化或过渡金属策略构建具有所需手性的复杂分子。大自然已经进化出不同类型的酶来精确控制环化立体化学；然而，大多数报道的狄尔斯-阿尔德酶已被证明只能促进能量上有利的非对映选择性环加成。在这里，我们报告了 CtdP 的发现和表征，CtdP 是一类新型双功能氧化还原酶/Diels-Alderase 的成员，之前被注释为 NmrA 样转录调节因子。我们证明，CtdP 催化固有不利的环加成反应，形成具有严格 α-抗选择性的双环[2.2.2]二氮杂辛烷支架。在计算研究的指导下，我们揭示了 CtdP 催化的逆电子需求 Diels-Alder 环加成的NADP ^{+ /NADPH 依赖的氧化还原机制，这是利用该机制的双功能 Diels-Alderase 的第一个例子。}