State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China.
Department of Chemistry, Tianjin University, Tianjin 300072, China.
J Am Chem Soc. 2024 Oct 2;146(39):26884-26896. doi: 10.1021/jacs.4c07958. Epub 2024 Aug 20.
Monosubstituted tetrazines are important bioorthogonal reactive tools due to their rapid ligation with -cyclooctene. However, their application is limited by the reactivity-stability paradox in biological environments. In this study, we demonstrated that steric effects are crucial in resolving this paradox through theoretical methods and developed a simple synthetic route to validate our computational findings, leading to the discovery of 1,3-azole-4-yl and 1,2-azole-3-yl monosubstituted tetrazines as superior bioorthogonal tools. These new tetrazines surpass previous tetrazines in terms of high reactivities and elevated stabilities. The most stable tetrazine exhibits a reasonable stability (71% remaining after 24 h incubation in cell culture medium) and an exceptionally high reactivity ( > 10 M s toward -cyclooctene). Due to its good stability in biological systems, a noncanonical amino acid containing such a tetrazine side chain was genetically encoded into proteins site-specifically via an expanded genetic code. The encoded protein can be efficiently labeled using cyclopropane-fused -cyclooctene dyes in living mammalian cells with an ultrafast reaction rate exceeding 10 M s, making it one of the fastest protein labeling reactions reported to date. Additionally, we showed its superiority through reactions in living mice, achieving an efficient local anchoring of proteins. These tetrazines are expected to be optimal bioorthogonal reactive tools within living systems.
单取代四嗪是一类重要的生物正交反应试剂，因其能快速与环辛烯发生点击反应而被广泛应用。然而，其在生物环境中的应用受到反应活性-稳定性悖论的限制。在本研究中，我们通过理论方法证明了空间位阻效应对解决这一悖论至关重要，并开发了一种简单的合成路线来验证我们的计算结果，发现了 1,3-唑-4-基和 1,2-唑-3-基单取代四嗪作为更优的生物正交工具。与之前的四嗪相比，这些新的四嗪具有更高的反应活性和稳定性。最稳定的四嗪具有合理的稳定性（在细胞培养基中孵育 24 小时后仍有 71%的剩余）和极高的反应活性（对环辛烯的反应速率 > 10 M s ）。由于其在生物体系中具有良好的稳定性，我们通过扩展的遗传密码将含有这种四嗪侧链的非天然氨基酸定点遗传编码到蛋白质中。该编码蛋白可在活的哺乳动物细胞中通过环丙烷融合的环辛烯染料进行高效标记，反应速率超过 10 M s ，这是迄今为止报道的最快的蛋白质标记反应之一。此外，我们还在活体小鼠中展示了其优越的反应性能，实现了蛋白质的高效局部锚定。这些四嗪有望成为活体内最佳的生物正交反应试剂。
