Tetrazine-mediated inverse electron demand Diels-Alder (IEDDA) reactions are widely used ultrafast bioorthogonal reactions, and methods to control such reactions would be extremely useful. Here, we identified a high-affinity host-guest pair between synthetic macrocyclic naphthotubes and phenyltetrazine and developed a molecular-recognition strategy for controlling tetrazine reactions by host caging. Naphthotubes can recognize phenyltetrazine on various biomolecules with low-micromolar and sub-micromolar binding affinities and thus cage their IEDDA reactivity efficiently in a reversible manner. For tetrazine residues on proteins encoded by non-canonical amino acid mutagenesis, their positions could be computationally designed such that exposed residues could be reversibly caged, whereas semiburied residues that were resistant to caging retained their reactivity, thus allowing preparation of site-specific protein dual conjugates and dual protein labeling on living cells with IEDDA reactions. The reactivity of tetrazine can be further regulated in living animals. Our strategy is thus expected to expand the applications of tetrazine chemistry.

四嗪介导的逆电子需求狄尔斯-阿尔德（IEDDA）反应是广泛使用的超快生物正交反应，控制此类反应的方法将非常有用。在这里，我们确定了合成大环萘管和苯基四嗪之间的高亲和力主客体对，并开发了一种通过宿主笼蔽控制四嗪反应的分子识别策略。萘管可以以低微摩尔和亚微摩尔结合亲和力识别各种生物分子上的苯基四嗪，从而以可逆的方式有效地限制其 IEDDA 反应性。对于由非规范氨基酸诱变编码的蛋白质上的四嗪残基，可以通过计算设计它们的位置，使得暴露的残基可以可逆地笼住，而抵抗笼养的半掩埋残基保留其反应性，从而允许制备位点特异性蛋白质双通过 IEDDA 反应对活细胞进行缀合物和双重蛋白质标记。四嗪的反应性可以在活体动物中进一步调节。因此，我们的策略有望扩大四嗪化学的应用。