Wang Jing-Bo, Zhao Xue-Ru, Hu Xi-Le, Zang Yi, Li Jia, He Xiao-Peng
Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Rd., Shanghai 200237, China.
State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
ACS Appl Mater Interfaces. 2024 Dec 18;16(50):68918-68927. doi: 10.1021/acsami.4c15268. Epub 2024 Dec 9.
The sensitive detection of glycosidases in live cells is crucial to understanding their functional roles in disease progression. Here, we develop a fluorogenic labeling probe for β-galactosidase (β-Gal) based on a bright green-emitting fluorescent dye, fluorescein. Galactose was introduced to a fluoromethyl-substituted fluorescein derivative through a benzyl spacer, resulting in a quenched fluorescence due to spirocyclization of the dye. After removal of the galactosyl residue by β-Gal, an ∼210-fold enhanced green fluorescence (emission maximum at 524 nm) was detected, and the presence of other glycosidases and hydrolases did not produce false-positive signals. The probe was successfully used for imaging of the endogenous β-Gal activity in cancer and senescent cells, and the imaging results agree with the β-Gal expression level of the cells, as determined by Western blotting and polymerase chain reaction. Importantly, we demonstrated that upon hydrolysis of galactose, the fluoromethyl-substituted fluorescein derivative is covalently attached to adjacent proteins, both in solution and in live cells. This study offers a small-molecule probe for the sensitive monitoring of endogenous glycosidase activity.
活细胞中糖苷酶的灵敏检测对于理解其在疾病进展中的功能作用至关重要。在此，我们基于亮绿色荧光染料荧光素开发了一种用于β-半乳糖苷酶（β-Gal）的荧光标记探针。通过苄基间隔基将半乳糖引入氟甲基取代的荧光素衍生物中，由于染料的螺环化导致荧光猝灭。在β-Gal去除半乳糖基残基后，检测到绿色荧光增强约210倍（发射最大值在524nm），并且其他糖苷酶和水解酶的存在不会产生假阳性信号。该探针成功用于癌症和衰老细胞中内源性β-Gal活性的成像，成像结果与通过蛋白质免疫印迹和聚合酶链反应测定的细胞β-Gal表达水平一致。重要的是，我们证明在半乳糖水解时，氟甲基取代的荧光素衍生物在溶液和活细胞中均共价连接到相邻蛋白质上。本研究提供了一种用于灵敏监测内源性糖苷酶活性的小分子探针。
