Department of Chemistry, University of Minnesota, Minneapolis, 55455, USA.
Chembiochem. 2009 Dec 14;10(18):2934-43. doi: 10.1002/cbic.200900566.
Recently a number of nonnatural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence "CAAX", where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analogue and the "CAAX box" sequence remains. Here we report the synthesis of an alkyne-containing substrate comprised of only nine nonhydrogen atoms. This substrate was synthesized in six steps from 3-methylbut-2-en-1-ol and has been enzymatically incorporated into both proteins and peptides by using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed by using carboxypeptidase Y, leaving a single residue (the cysteine from the "CAAX box") and the prenyl analogue as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein's solubility, reactivity, or utility.
最近，人们开发了许多含有炔烃和叠氮化物的非天然异戊烯基基团，作为在以“CAAX”序列结尾的蛋白质和肽上进行点击化学的接头，其中 C 是被烷基化的半胱氨酸，A 是脂族氨基酸，X 是任何氨基酸。当修饰此类分子时，含有异戊烯基类似物和“CAAX 盒”序列的标签仍然存在。在这里，我们报告了一种仅由九个非氢原子组成的含有炔烃的底物的合成。该底物是由 3-甲基-2-丁烯-1-醇经六步合成的，并用蛋白法尼基转移酶将其酶促掺入到蛋白质和肽中。异戊烯化后，通过使用羧肽酶 Y 可以去除酶识别所需的最后三个氨基酸，留下一个残基（来自“CAAX 盒”的半胱氨酸）和异戊烯基类似物作为唯一的修饰物。我们还证明，这种小标签最小化了修饰对目标蛋白溶解度的影响。因此，这种新方法应该适用于那些大标签会阻碍修饰蛋白溶解度、反应性或实用性的应用。
