Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China.
Anal Chem. 2022 May 24;94(20):7272-7277. doi: 10.1021/acs.analchem.2c00512. Epub 2022 May 12.
Nitroreductase (NTR), one of the flavin-dependent enzymes and an upregulated enzyme under tumor hypoxia, has been studied for decades. Many fluorescent probes were developed to detect NTR activity; however, these probes tend to diffuse away from their reaction site (NTR) inevitably, leading to inappropriate sample fixation, lower accuracy of NTR localization, and weaker signal-to-noise ratio. Herein, we present the design, synthesis, evaluation, and biological applications of an NTR-activatable fluorogenic and labeling probe . By integrating with quinone methide (QM) proximity-based protein labeling, the additional fluoromethyl group on serves as a potential origin of QM. Compared with conventional fluorescent probes, this new NTR probe not only offers mitochondrial localizable and fluorogenic response but also achieves permanent retention on the site of activation with an enhanced spatial resolution to improve the detection sensitivity even after cell fixation. We believe our work could offer an expandable synthetic approach to develop these permanent labeling and imaging fluorescence probes for deciphering complex biological events.
硝基还原酶（NTR）是一种黄素依赖酶，也是肿瘤缺氧下上调的酶，已经研究了几十年。已经开发出许多荧光探针来检测 NTR 活性；然而，这些探针往往不可避免地从其反应位点（NTR）扩散出去，导致不合适的样品固定、NTR 定位的准确性降低和信噪比减弱。在此，我们提出了一种 NTR 激活型荧光和标记探针的设计、合成、评价和生物学应用。通过与醌甲醚（QM）近程蛋白标记相结合，上的额外氟甲基基团可作为 QM 的潜在起源。与传统荧光探针相比，这种新型 NTR 探针不仅提供了线粒体定位和荧光响应，而且还通过增强的空间分辨率实现了在激活部位的永久性保留，从而提高了检测灵敏度，即使在细胞固定后也是如此。我们相信我们的工作可以为开发这些永久性标记和成像荧光探针提供一种可扩展的合成方法，以解析复杂的生物学事件。
