Bioorthogonal reactions are selective transformations that are not affected by any biological functional group and are widely used for chemical modification of biomolecules. Recently, we reported that vinylboronic acids (VBAs) gave exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction with tetrazines bearing a boron-coordinating pyridyl moiety compared to tetrazines lacking such a substituent. In this integrated experimental and theoretical study, we show how the reaction rate of the VBA-tetrazine ligation can be accelerated by shifting the equilibrium from boronic acid to the boronate anion in the reaction mixture. Quantum chemical activation strain analyses reveal that this rate enhancement is a direct consequence of the excellent electron-donating capability of the boronate anion in which the π HOMO is pushed to a higher energy due to the net negative potential of this species. We have explored the second-order rate constants of several tetrazines containing potential VBA-coordinating hydroxyl substituents. We observed an increase in rate constants of several orders of magnitude compared to the tetrazines lacking a hydroxyl substituent. Furthermore, we find the hydroxyl-substituted tetrazines to be more selective toward VBAs than toward the commonly used bioorthogonal reactant norbornene, and more stable in aqueous environment than the previously studied tetrazines containing a pyridyl substituent.

中文翻译：

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高度稳定和选择性的四嗪，用于与乙烯基硼酸的配位辅助生物正交连接。

生物正交反应是不受任何生物官能团影响的选择性转化，被广泛用于生物分子的化学修饰。最近，我们报道了乙烯基硼酸（VBA）与带有硼配位吡啶基部分的四嗪相比，缺乏缺乏这种取代基的四嗪，在生物正交逆电子需求的Diels-Alder（iEDDA）反应中给出了异常高的反应速率。在这项综合的实验和理论研究中，我们显示了如何通过将平衡从硼酸转移到反应混合物中的硼酸根阴离子来加速VBA-四嗪连接的反应速率。量子化学活化应变分析表明，这种速率提高是硼酸根阴离子出色的供电子能力的直接结果，其中由于该物种的净负电势，πHOMO被推至更高的能量。我们已经探索了几个潜在的VBA配位羟基取代基的四嗪的二阶速率常数。与缺乏羟基取代基的四嗪相比，我们观察到速率常数增加了几个数量级。此外，我们发现羟基取代的四嗪对VBA的选择性比对常用的生物正交反应物降冰片烯的选择性高，并且在水性环境中比先前研究的含吡啶基取代基的四嗪更稳定。