Wakabayashi Haruto, Miyagawa Masayoshi, Koshi Yoichiro, Takaoka Yousuke, Tsukiji Shinya, Hamachi Itaru
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
Chem Asian J. 2008 Jul 7;3(7):1134-9. doi: 10.1002/asia.200800057.
A new chemical method to site-specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity-labeling-based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime-exchange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target-specific multiple modification in a protein mixture. The availability of various (photo)affinity-labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site-selective incorporation of many functional molecules into proteins.
本文描述了一种无需基因操作即可对天然蛋白质进行位点特异性修饰的新化学方法。我们的策略包括在目标蛋白表面基于亲和标记连接一个独特的反应性手柄，随后通过生物正交反应对手柄进行选择性转化，从而将多种功能探针引入蛋白质中。为了证明这种方法，我们合成了标记试剂，其包含：1）特异性靶向牛碳酸酐酶II（bCA）的苯磺酰胺配体；2）用于蛋白质标记的亲电环氧基团；3）连接配体和环氧基团的可交换腙键；4）碘苯基或乙炔手柄。通过将标记试剂与bCA孵育，反应性手柄通过环氧开环共价连接在bCA表面。在通过腙/肟交换反应去除配体（该反应可恢复酶活性）之后或之前，引入的反应性手柄均可通过铃木偶联或惠斯根环加成反应进行衍生化。该方法也适用于蛋白质混合物中的目标特异性多重修饰。各种（光）亲和标记试剂和生物正交反应的可用性应会扩展这种策略的灵活性，以便将许多功能分子位点选择性地掺入蛋白质中。
