Department of Chemistry, Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, United States.
J Am Chem Soc. 2017 Aug 30;139(34):11670-11673. doi: 10.1021/jacs.7b05125. Epub 2017 Aug 16.
Chemoselective modification of complex biomolecules has become a cornerstone of chemical biology. Despite the exciting developments of the past two decades, the demand for new chemoselective reactions with unique abilities, and those compatible with existing chemistries for concurrent multisite-directed labeling, remains high. Here we show that 5-hydroxyindoles exhibit remarkably high reactivity toward aromatic diazonium ions and this reaction can be used to chemoselectively label proteins. We have previously genetically encoded the noncanonical amino acid 5-hydroxytryptophan in both E. coli and eukaryotes, enabling efficient site-specific incorporation of 5-hydroxyindole into virtually any protein. The 5-hydroxytryptophan residue was shown to allow rapid, chemoselective protein modification using the azo-coupling reaction, and the utility of this bioconjugation strategy was further illustrated by generating a functional antibody-fluorophore conjugate. Although the resulting azo-linkage is otherwise stable, we show that it can be efficiently cleaved upon treatment with dithionite. Our work establishes a unique chemoselective "unclickable" bioconjugation strategy to site-specifically modify proteins expressed in both bacteria and eukaryotes.
化学选择性修饰复杂生物分子已成为化学生物学的基石。尽管在过去二十年中取得了令人兴奋的发展，但对具有独特能力的新化学选择性反应的需求仍然很高，并且这些反应还需要与现有的化学方法兼容，以实现同时多部位定向标记。在这里，我们证明 5-羟基吲哚对芳族重氮离子表现出很高的反应性，并且该反应可用于选择性标记蛋白质。我们之前已经在大肠杆菌和真核生物中遗传编码了非典型氨基酸 5-羟色氨酸，从而能够有效地将 5-羟基吲哚特异性地掺入几乎任何蛋白质中。已证明 5-羟色氨酸残基可以通过偶氮偶联反应快速、选择性地进行蛋白质修饰，并且通过生成功能性抗体-荧光团缀合物进一步说明了这种生物偶联策略的实用性。尽管得到的偶氮键在其他方面是稳定的，但我们表明它可以在二硫代苏糖醇处理时有效地被切断。我们的工作建立了一种独特的化学选择性“不可点击”生物偶联策略，可用于特异性修饰在细菌和真核生物中表达的蛋白质。
