Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N–H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N¹-vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels–Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings.

色氨酸 (Trp) 通过其π系统和吲哚 N-H 基团在蛋白质结构、相互作用和功能的调节中发挥着关键作用。一种阻断和拯救色氨酸相互作用的通用方法将使体内各种含色氨酸蛋白质的功能获得性操作成为可能，但生成这样的平台仍然具有挑战性。在这里，我们开发了一种基因编码的N ¹ -乙烯基笼色氨酸，能够通过优化的逆电子需求狄尔斯-阿尔德反应进行快速生物正交解笼，从而允许活细胞中感兴趣的蛋白质上的色氨酸进行位点特异性激活。这种基因编码的笼状色氨酸 (Trp-CAGE) 策略的化学激活能够精确激活各种重要分子相互作用背后的目标色氨酸。我们证明了 Trp-CAGE 在各种蛋白质家族（例如过氧化氢酶-过氧化物酶和激酶）中作为翻译起始子和翻译后修饰阅读器的效用，允许以时间控制的方式调节表观遗传信号。与计算机辅助预测相结合，我们的策略为超过 28,000 种候选蛋白质在其天然细胞环境中的生物正交色氨酸激活铺平了道路。