Protein Modification at Tyrosine with Iminoxyl Radicals
Protein Modification at Tyrosine with Iminoxyl Radicals
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DOI：
10.1021/jacs.1c09066
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发表时间：
2021-12-01
影响因子：
15
通讯作者：
Kanai, Motomu
中科院分区：
文献类型：
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作者：
Maruyama, Katsuya;Ishiyama, Takashi;Kanai, Motomu
蛋白质翻译后修饰(PTM)是一种可逆控制蛋白质功能的生物学机制。针对特定典型氨基酸的合成蛋白质修饰(SPM)可以模拟PTMS。然而，蛋白质中疏水氨基酸残基的可逆SPM尤其有限。在这里，我们报告了一种酪氨酸(Tyr)选择性扫描电子显微镜，它利用了持久的亚胺氧自由基，这些自由基很容易通过单电子氧化从空间受阻的肟类化合物中产生。亚胺氧基与酪氨酸的反应活性取决于酮类的空间和电子需求，异丙基甲基哌啶肟1f形成稳定的加合物，而叔丁基甲基哌啶肟1o的反应是可逆的。1f和1o之间的可逆性差异是由于亚胺氧基的稳定性，这部分是由O-H亚胺基团的键离解能决定的。用1f和1o进行的Tyr选择性修饰是在生理相关的温和条件下进行的。具体地说，稳定的1f酪氨酸修饰向蛋白质引入了功能小分子，包括偶氮苯光开关。此外，用10o的SPM掩盖关键的Tyr残基，以及随后用硫醇处理引发的去结合，使蛋白质功能的按需控制成为可能。我们应用这种可逆的酪氨酸修饰，在修饰/去结合时改变了单抗的酶活性和与抗原的结合亲和力。通过酪氨酸选择性和可逆的共价键形成的蛋白质功能的按需开/关将在生物研究和治疗中提供独特的机会。
Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here, we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single-electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologically relevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins. Moreover, masking critical Tyr residues by SPM with 1o, and subsequent deconjugation triggered by the treatment with a thiol, enabled on-demand control of protein functions. We applied this reversible Tyr modification with 1o to alter an enzymatic activity and the binding affinity of a monoclonal antibody with an antigen upon modification/deconjugation. The on-demand ON/OFF switch of protein functions through Tyr-selective and reversible covalent-bond formation will provide unique opportunities in biological research and therapeutics.