美国普林斯顿大学Hyster Todd K.团队报道了光酶中C-N键形成的独特机制。相关研究成果发表在2024年10月8日出版的《自然》。

由于氮杂环在小分子药物和农用化学品中的普遍存在，C-N键的形成是现代化学合成不可或缺的一部分。烯烃与未活化烯烃的加氢胺化是构建这些键的原子经济策略。然而，在制备完全取代的碳立体中心时，这些反应很难使其不对称。

该文中，研究人员报道了一种通过Baeyer-Villiger单加氧酶进行光酶烯烃氢胺化，制备2,2-二取代吡咯烷的方法。五轮蛋白质工程提供了一个突变体，提供了优异的产品产量和立体选择性。与依赖于胺或烯烃氧化形成C-N键的相关光化学氢胺化不同，该项工作利用了还原产生的苄基自由基和氮孤对的空间相互作用。

这种反键相互作用降低了自由基的氧化电位，使电子能够转移到黄素辅因子。实验表明，酶微环境对于实现小分子催化中没有平行的新型C-N键形成机制至关重要。研究人员进行分子动力学模拟以研究酶活性位点中的底物，进一步支持了这一假设。该项工作是非天然生物催化中一种新兴机制的罕见例子，在这种机制中，酶可以获得其单个成分所不具备的机制。

该研究展示了利用蛋白质工程增强新兴机制的潜力，为化学合成中尚未解决的挑战提供独特的机制解决方案。

附：英文原文

Title: Emergence of a distinct mechanism of C–N bond formation in photoenzymes

Author: Raps, Felix C., Rivas-Souchet, Ariadna, Jones, Chey M., Hyster, Todd K.

Issue&Volume: 2024-10-08

Abstract: C–N bond formation is integral to modern chemical synthesis due to the ubiquity of nitrogen heterocycles in small-molecule pharmaceuticals and agrochemicals. Alkene hydroamination with unactivated alkenes is an atom economical strategy for constructing these bonds. However, these reactions are challenging to render asymmetric when preparing fully substituted carbon stereocenters. Here, we report a photoenzymatic alkene hydroamination to prepare 2,2-disubstituted pyrrolidines by a Baeyer-Villiger Monooxygenase. Five rounds of protein engineering afforded a mutant, providing excellent product yield and stereoselectivity. Unlike related photochemical hydroaminations, which rely on the oxidation of the amine or alkene for C–N bond formation, this work exploits a through-space interaction of a reductively generated benzylic radical and the nitrogen lone pair. This antibonding interaction lowers the oxidation potential of the radical, enabling electron transfer to the flavin cofactor. Experiments indicate that the enzyme microenvironment is essential in enabling a novel C–N bond formation mechanism with no parallel in small molecule catalysis. Molecular dynamics simulations were performed to investigate the substrate in the enzyme active site which further support this hypothesis. This work is a rare example of an emerging mechanism in non-natural biocatalysis, where an enzyme has access to a mechanism that its individual components do not. Our study showcases the potential of enhancing emergent mechanisms using protein engineering to provide unique mechanistic solutions to unanswered challenges in chemical synthesis.

DOI: 10.1038/s41586-024-08138-w

Source: https://www.nature.com/articles/s41586-024-08138-w