The formation of C–N bonds—of great importance to the pharmaceutical industry—can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C–N bond formation. Here we use flavin-dependent ‘ene’-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.

C-N 键的形成（对制药行业非常重要）可以通过亲核和氮宾转移机制通过酶促方式促进。然而，目前已知天然酶和工程酶都不能产生和控制以氮为中心的自由基，而氮中心自由基是 C-N 键形成的有价值的物质。在这里，我们使用黄素依赖性“烯”还原酶和外源光氧化还原催化剂，在蛋白质活性位点内选择性地产生酰胺基。这些酶通过定向进化进行工程改造，以高水平的对映选择性催化 5-外切、6-内切、7-内切、8-内切和分子间氢氨化反应。机理研究表明，自由基引发是通过酶门控机制发生的，其中蛋白质热力学激活底物以通过光催化剂进行还原。分子动力学研究表明，酶通过非规范结合相互作用结合底物，这可以作为进一步操纵反应性的手柄。这种方法证明了这些酶在控制高能自由基中间体反应性方面的多功能性，并强调了协同催化剂策略解锁以前无法实现的酶功能的机会。