Anal Chem. 2019 Aug 20;91(16):10901-10907. doi: 10.1021/acs.analchem.9b02857. Epub 2019 Aug 9.
Azoreductase (AzoR) is an essential reductive enzyme which is closely associated with the intestinal disease such as ulcerative colitis (UC). To date, only a few fluorescent probes for detecting AzoR activity in bacteria or cells have been constructed successfully. It is still challenging to design fluorescent probes for in situ monitoring AzoR in vivo. In this paper, a near-infrared (NIR) fluorescent probe (Cy-Azo) based on hemicyanine is designed and synthesized. The emission of the probe is located at 735 nm in the NIR region, which is favorable for its application in vivo. In addition, Cy-Azo shows high sensitivity to AzoR activity with 17-fold fluorescence enhancement and is particularly selective to AzoR over other enzymes, ions, and amino acids. Meanwhile, a possible response mechanism (the azo group in Cy-Azo is reduced by AzoR and cleaved resulting in the production of Cy-NH) was proposed and verified by HPLC, MS, and theory calculation. In addition, based on low cell cytotoxicity, Cy-Azo is successfully applied in visualizing the activity of AzoR in two cell lines (HCT116 and HepG2 cells) and three types of bacteria (, , and ). In particular, due to its NIR emission, the probe can monitor AzoR activity in acute and chronic UC mice models. To our knowledge this is the first fluorescent probe for detecting AzoR activity in vivo, which can provide much important information for the diagnosis and treatment of UC.
偶氮还原酶（AzoR）是一种重要的还原酶，与溃疡性结肠炎（UC）等肠道疾病密切相关。迄今为止，仅成功构建了少数用于检测细菌或细胞中 AzoR 活性的荧光探针。设计用于体内原位监测 AzoR 的荧光探针仍然具有挑战性。本文设计并合成了一种基于半花菁的近红外（NIR）荧光探针（Cy-Azo）。探针的发射位于 NIR 区域的 735nm 处，有利于其在体内的应用。此外，Cy-Azo 对 AzoR 活性具有高灵敏度，荧光增强 17 倍，对 AzoR 的选择性特别高，超过其他酶、离子和氨基酸。同时，通过 HPLC、MS 和理论计算提出并验证了可能的响应机制（Cy-Azo 中的偶氮基团被 AzoR 还原并裂解，导致生成 Cy-NH）。此外，基于低细胞毒性，Cy-Azo 成功应用于两种细胞系（HCT116 和 HepG2 细胞）和三种类型的细菌（、和）中 AzoR 活性的可视化。特别是，由于其 NIR 发射，该探针可以监测急性和慢性 UC 小鼠模型中的 AzoR 活性。据我们所知，这是第一个用于体内检测 AzoR 活性的荧光探针，可为 UC 的诊断和治疗提供重要信息。
