Laboratory of Organic Chemistry, ETH Zurich , Vladimir-Prelog-Weg 3, CH-8093 Zurich, Switzerland.
Scientific Center for Optical and Electron Microscopy, ETH Zurich , Otto-Stern-Weg 3, CH-8093 Zurich, Switzerland.
J Am Chem Soc. 2017 Sep 20;139(37):13200-13207. doi: 10.1021/jacs.7b07748. Epub 2017 Sep 5.
A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron-poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is nonemissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich, and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules. Single-molecule localization microscopy was used to reconstruct a super-resolved image of esterase activity. These single-molecule images of enzymatic activity changed significantly upon treatment of the cells with inhibitors of human carboxylesterase I and II, both in terms of total number of signals and intracellular distribution. This proof-of-principle study introduces a sensing mechanism for single-molecule detection of enzymatic activity that could be applied to many other biologically relevant targets.
开发了一种双重激活的荧光探针，用于以单分子分辨率感应酯酶活性。在没有酶的预激活的情况下，基于重氮茚酮的探针具有电子贫乏的核心，并且在照射下经历沃尔夫重排，得到非发光的扩环呫吨核。如果探针被羧酸酯酶预先激活，那么三环核就会变得富电子，光诱导的沃尔夫重排产生高发光的罗丹明染料。活细胞和溶液研究证实了探针的选择性，并表明光激活的染料不会从激活的原始位置扩散出去，因为中间的碳烯与周围的大分子形成共价键。单分子定位显微镜用于重建酯酶活性的超分辨图像。用人类羧酸酯酶 I 和 II 的抑制剂处理细胞后，酶活性的单分子图像在信号总数和细胞内分布方面都发生了显著变化。这项原理验证研究为单分子检测酶活性引入了一种传感机制，该机制可应用于许多其他与生物学相关的靶标。
