Department of Chemistry, National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, Republic of China.
Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, Republic of China.
Anal Chem. 2020 Dec 1;92(23):15463-15471. doi: 10.1021/acs.analchem.0c03178. Epub 2020 Nov 12.
Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a nonfluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins that do not possess enzymatic activities. In this article, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of nonenzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our strategy, the probe binds to the protein via a specific protein-ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the activation and release of the fluorogenic reporter. In contrast, a phenyl ester linker without the difluoro substituent cannot be triggered to release the fluorogenic reporter. With this probe design, live-cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.
目前，大多数荧光探针都是为分析酶而开发的，其中需要进行键断裂或重排反应，将非荧光酶底物转化为荧光产物。然而，这种方法不能用于不具有酶活性的蛋白质。在本文中，我们表明，具有自耗损二氟苯酯连接基的荧光探针可以模拟荧光酶底物的键拆卸过程，用于快速分析非酶蛋白质。尽管许多自耗损试剂在传感器、药物传递系统和材料化学中显示出了有前途的应用，但它们都是由酶或小分子反应性分子触发的。在我们的策略中，探针通过特定的蛋白质-配体相互作用与蛋白质结合，在自耗损连接基和蛋白质的氨基酸之间引发化学反应，从而触发级联反应，导致荧光报告基团的激活和释放。相比之下，没有二氟取代基的苯酯连接基不能被触发释放荧光报告基团。通过这种探针设计，可以实现对细胞外和细胞内内源性肿瘤标志物蛋白质的高选择性和高灵敏度的活细胞成像。
