Two-Photon Small-Molecule Fluorogenic Probes for Visualizing Endogenous Nitroreductase Activities from Tumor Tissues of a Cancer Patient

Nitroreductase (NTR), a common enzymatic biomarker of hypoxia, is widely used to evaluate tumor microenvironments. To date, numerous optical probes have been reported for NTRs detection. Approaches capable of concisely guiding the probe design of NTRs suitable for deep-tissue imaging, however, are still lacking. As such, direct optical imaging of endogenous NTR activities from tumors derived from cancer patients is thus far not possible. Herein, aided by computational calculations, the authors have successfully developed a series of two-photon (TP) small-molecule fluorogenic probes capable of sensitively detecting general NTR activities from various biological samples; by optimizing the distance between the recognition moiety and the reactive site of NTRs from different sources, the authors have discovered and experimentally proven that X4 displays the best performance in both sensitivity and selectivity. Furthermore, X4 shows excellent TP excited fluorescence properties capable of directly monitoring/imaging endogenous NTR activities from live mammalian cells, growing zebrafish, and tumor-bearing mice. Finally, with an outstanding TP tissue-penetrating imaging property, X4 is used, for the first time, to successfully detect endogenous NTR activities from the liver lysates and cardia tissues of a cancer patient. The work may provide a universal strategy to design novel TP small-molecule enzymatic probes in future clinical applications.

Automated summary

This study successfully developed a series of two-photon small-molecule fluorogenic probes capable of sensitively detecting general NTR activities in various biological samples. The optimized probe, X4, displayed the best performance in terms of sensitivity and selectivity. X4 also showed excellent two-photon excited fluorescence properties and was able to directly monitor endogenous NTR activities in live cells, growing zebrafish, and tumor-bearing mice. By using its outstanding tissue-penetrating imaging property, X4 was used for the first time to detect endogenous NTR activities in liver and cardia tissues from a cancer patient.