State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
Org Biomol Chem. 2021 Apr 26;19(15):3469-3478. doi: 10.1039/d1ob00356a.
Hypoxia is a pathological hallmark of solid tumors. Detection of hypoxia is therefore of great interest for tumor diagnosis and treatment. As a well-established biomarker of hypoxia, nitroreductase (NTR) has been widely exploited in the development of hypoxia-responsive fluorescent probes on the basis of its enzymatic activity to reduce nitroaryl groups. However, studies on the relationship between the nitroaryl structure and the probe performance for optimal probe design are still rare. Here we report a comparative investigation of nitroaryl groups and identification of the optimal nitroaryl structure for developing new fluorescent probes with extremely high efficiency in the detection of NTR and the imaging of hypoxic tumor cells. Specifically, we synthesized a series of resorufin-based fluorescent probes containing different nitroaryl groups, compared their fluorescence responses to NTR, and identified 2-nitro-N-methyl-imidazolyl as the optimal nitroaryl group that is much more efficient than the most widely used 4-nitrophenyl for NTR detection. The structure-performance relationship was then studied by theoretical molecular docking, revealing the unique features of 2-nitro-N-methyl-imidazolyl in binding and reaction with NTR. We further incorporated the 2-nitro-N-methyl-imidazolyl group into a near-infrared (NIR) hemicyanine fluorophore and developed a NIR fluorescent probe NFP-7 for the detection of NTR and hypoxic tumor cells. NFP-7 exhibits a strong fluorescence increase toward NTR in vitro with an ultrafast (within 40 seconds to fluorescence maximum) and ultrasensitive (0.2 ng mL-1 detection limit) response. NFP-7 has also been demonstrated for imaging the degree of hypoxia in live tumor cells and, more importantly, in a murine tumor model. Our study provides important insights into hypoxia probe development and new tools for hypoxia imaging.
缺氧是实体瘤的一种病理标志。因此，检测缺氧对于肿瘤的诊断和治疗具有重要意义。硝基还原酶（NTR）作为缺氧的一种既定生物标志物，由于其酶活性可以还原硝基芳基基团，已被广泛用于开发缺氧响应荧光探针。然而，关于硝基芳基结构与探针性能之间的关系的研究，对于最佳探针设计仍然很少。在这里，我们报告了对硝基芳基基团的比较研究，并确定了开发新的荧光探针的最佳硝基芳基结构，这些探针在检测 NTR 和缺氧肿瘤细胞成像方面具有极高的效率。具体来说，我们合成了一系列含有不同硝基芳基基团的基于 Resorufin 的荧光探针，比较了它们对 NTR 的荧光响应，并确定 2-硝基-N-甲基-咪唑基为最佳硝基芳基基团，其效率比最广泛使用的 4-硝基苯基高得多，用于 NTR 检测。然后通过理论分子对接研究了结构-性能关系，揭示了 2-硝基-N-甲基-咪唑基与 NTR 结合和反应的独特特征。我们进一步将 2-硝基-N-甲基-咪唑基基团引入近红外（NIR）半花菁荧光团中，并开发了用于检测 NTR 和缺氧肿瘤细胞的 NIR 荧光探针 NFP-7。NFP-7 在体外对 NTR 表现出强烈的荧光增加，具有超快（40 秒内达到荧光最大值）和超灵敏（检测限为 0.2ng/mL）的响应。NFP-7 还已被证明可用于活肿瘤细胞中缺氧程度的成像，更重要的是，可用于在鼠肿瘤模型中成像。我们的研究为缺氧探针的开发提供了重要的见解，并为缺氧成像提供了新的工具。
