Kalia Jeet, Raines Ronald T
Department of Biochemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706-1322, USA.
Chembiochem. 2006 Sep;7(9):1375-83. doi: 10.1002/cbic.200600150.
The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.
基因组测序的成功增加了对操纵蛋白质新方法的需求。一个特别理想的目标是能够用具有正交反应性的官能团在特定位点修饰目标蛋白质。在此，我们通过利用内含肽介导的蛋白质剪接过程中形成的硫酯中间体的固有亲电性实现了这一目标。对氮亲核试剂与发色小分子硫酯反应的详细动力学分析表明，α-肼基乙酰基是在中性pH下攻击硫酯以形成稳定连接的最佳亲核试剂。以高总收率合成了一种带有α-肼基乙酰胺基和叠氮基的双功能试剂。该试剂用于攻击目标蛋白质与内含肽之间的硫酯键，从而在一步中将叠氮基附加到目标蛋白质上。叠氮基蛋白质保留了全部生物活性。此外，其叠氮基可用于通过惠斯根1,3-偶极叠氮-炔环加成进行化学修饰。因此，内含肽介导的蛋白质剪接机制提供了在几乎任何蛋白质的特定位点——C末端——安装有用官能团的方法。
