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-NH₂) 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 (E. coli, S. aureus, and P. aeruginosa). 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.

中文翻译：

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通过近红外荧光探针在急性和慢性溃疡性结肠炎小鼠中进行氮还原酶活性的原位成像。

偶氮还原酶（AzoR）是一种必需的还原酶，与肠道疾病如溃疡性结肠炎（UC）密切相关。迄今为止，仅成功地构建了几种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。本文设计并合成了基于半菁的近红外（NIR）荧光探针（Cy-Azo）。探针的发射位于NIR区域的735 nm处，这有利于其在体内的应用。另外，Cy-Azo对AzoR活性显示出高灵敏度，荧光增强了17倍，并且对AzoR的选择性超过其他酶，离子和氨基酸。同时，₂）提出并通过HPLC，MS和理论计算验证。另外，基于低细胞毒性，Cy-Azo已成功应用于可视化两种细胞系（HCT116和HepG2细胞）和三种类型的细菌（大肠杆菌，金黄色葡萄球菌和铜绿假单胞菌）中的AzoR活性。。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，这是第一种在体内检测AzoR活性的荧光探针，可为UC的诊断和治疗提供重要信息。