Aryl diazonium ions have long been used in bioconjugation due to their reactivity toward electron-rich aryl residues, such as tyrosine. However, their utility in biological systems has been restricted due to the requirement of harsh conditions for their generation in situ, as well as limited hydrolytic stability. Previous work describing a scaffold known as triazabutadiene (TBD) has shown the ability to protect aryl diazonium ions allowing for increased synthetic utility, as well as triggered release under biologically relevant conditions. Herein, we describe the synthesis and application of a novel TBD, capable of installation of a cyclooctyne on protein surfaces for later use of copper-free click reactions involving functional azides. The probe shows efficient protein labeling across a wide pH range that can be accomplished in a convenient and timely manner. Orthogonality of the cyclooctyne modification was showcased by labeling a model protein in the presence of hen egg proteins, using an azide-containing fluorophore. We further confirmed that the azobenzene modification can be cleaved using sodium dithionite treatment.

中文翻译：

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用于生物正交蛋白功能化的无铜点击启用三氮杂丁二烯

芳基重氮离子长期以来一直用于生物共轭，因为它们对富含电子的芳基残基（如酪氨酸）具有反应性。然而，由于原位生成需要苛刻的条件，它们在生物系统中的应用受到限制，以及有限的水解稳定性。先前描述称为三氮杂丁二烯 (TBD) 的支架的工作已经显示出保护芳基重氮离子的能力，从而增加了合成效用，以及在生物学相关条件下触发释放。在这里，我们描述了一种新型 TBD 的合成和应用，它能够在蛋白质表面安装环辛炔，以便以后使用涉及功能性叠氮化物的无铜点击反应。该探针在很宽的 pH 范围内显示出高效的蛋白质标记，可以方便及时地完成。通过使用含叠氮化物的荧光团在鸡蛋蛋白存在的情况下标记模型蛋白，展示了环辛炔修饰的正交性。