Site-Specific Modification of Proteins through N-Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction
Site-Specific Modification of Proteins through N-Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction
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DOI：
10.1021/acs.bioconjchem.9b00515
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发表时间：
2019-09-01
影响因子：
4.7
通讯作者：
Hayashi, Takashi
中科院分区：
文献类型：
--
作者：
Inoue, Nozomu;Onoda, Akira;Hayashi, Takashi
肽和蛋白质的位点特异性修饰是将人工功能引入蛋白质表面的重要方法。最近，我们发现新的生物缀合试剂，6-（叠氮甲基）-2-吡啶甲醛（6AMPC）衍生物，允许特定的N-末端修饰，并提高随后的生物缀合在螯合辅助CuAAC反应的反应速率。生物活性肽和蛋白质的N-末端特异性叠氮标记在温和的反应条件下与6AMPC衍生物（血管紧张素I：90%，核糖核酸酶A：90%）发生。与叠氮化物标记的蛋白质的CuAAC反应的动力学分析表明，在铜螯合吡啶部分的存在下促进连接。重要的是，将给电子甲氧基引入吡啶部分进一步加速了CuAAC连接。我们证明，这种方法使位点特异性共轭的各种功能分子，如荧光团，生物素和聚乙二醇。
Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.