Phosphorus fluoride exchange (PFEx), recently debuted in small molecules, represents the forefront of click chemistry. To explore PFEx’s potential in biological settings, we developed amino acids phosphoramidofluoridate tyrosine (PFY) and phosphoramidofluoridate lysine (PFK) featuring phosphoramidofluoridates and incorporated them into proteins through genetic code expansion. PFY/PFK selectively reacted with nearby His, Tyr, Lys, or Cys in proteins, both in vitro and in living cells, demonstrating that proximity enabled PFEx reactivity without external reagents. The reaction with His showed unique pH-dependent properties and created thermally sensitive linkages. Additionally, Na₂SiO₃ enhanced PFEx reactions with Tyr and Cys. PFEx, by generating defined covalent P-N/O linkages, extends the utility of phosphorus linkages in proteins, aligning with nature’s use of phosphate connectors in other biomolecules. More versatile and durable than sulfur fluoride exchange (SuFEx), PFEx in proteins expands the latent bioreactive arsenal for covalent protein engineering and will facilitate the broad application of this potent click chemistry in biological and biomedical fields.

氟化磷交换（PFEx）最近在小分子中首次亮相，代表了点击化学的前沿。为了探索 PFEx 在生物环境中的潜力，我们开发了以氟氨基磷酸酯为特征的氨基酸氟氨基磷酸酯酪氨酸 (PFY) 和氟氨基磷酸酯赖氨酸 (PFK)，并通过遗传密码扩展将它们整合到蛋白质中。在体外和活细胞中，PFY/PFK 选择性地与蛋白质中附近的 His、Tyr、Lys 或 Cys 发生反应，表明邻近性无需外部试剂即可实现 PFEx 反应。与 His 的反应显示出独特的 pH 依赖性特性并产生热敏键。此外，Na ₂ SiO ₃增强了 PFEx 与 Tyr 和 Cys 的反应。 PFEx 通过生成确定的共价 PN/O 连接，扩展了蛋白质中磷连接的效用，与其他生物分子中磷酸连接器的自然使用相一致。蛋白质中的 PFEx 比氟化硫交换 (SuFEx) 更通用、更耐用，扩大了共价蛋白质工程的潜在生物反应库，并将促进这种有效的点击化学在生物和生物医学领域的广泛应用。