Azabicyclic vinyl sulfones for residue-specific dual protein labelling.
Azabicyclic vinyl sulfones for residue-specific dual protein labelling.
复制标题
DOI：
10.1039/c9sc00125e
复制
发表时间：
2019-04-28
期刊：
影响因子：
8.4
通讯作者：
Bernardes, Goncalo J L
中科院分区：
文献类型：
--
作者：
Gil de Montes, Enrique;Jimenez-Moreno, Ester;Bernardes, Goncalo J L
我们已经开发了[2.2.1]氮杂双环乙烯基砜试剂，其同时能够进行半胱氨酸选择性蛋白质修饰并引入用于进一步生物正交连接的手柄。相对于具有亲核侧链的其他氨基酸，该反应对于半胱氨酸是快速和选择性的，并且形成的产物在人血浆中是稳定的，并且对逆Diels-Alder降解反应具有中等抗性。模型生物素化的[2.2.1]氮杂双环乙烯基砜试剂显示在温和条件下（1-5当量）有效标记两种半胱氨酸标记的蛋白质，泛素和C2 Am。0，室温，30分钟）。所得硫醚连接的缀合物是稳定的，并保留了蛋白质的天然活性。最后，存在于官能化C2 Am蛋白上的氮杂双环部分中的亲二烯体可以通过细胞中的逆电子需求狄尔斯-阿尔德反应进行荧光标记，以允许选择性凋亡成像。直接性、位点特异性和易于制备的组合优点意味着[2.2.1]氮杂双环乙烯基砜可用于残基特异性双蛋白标记/构建策略，其仅基于[2.2.1]氮杂双环部分与半胱氨酸的连接，具有最小的天然功能扰动。
We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels-Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1-5 equiv. of reagent in NaPi pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels-Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.