A novel long-wavelength off-on fluorescence probe for nitroreductase analysis and hypoxia imaging

Hypoxia is one of the most important features of various diseases including solid tumors. It is of significant clinical importance for hypoxia analysis in tumors, and the analysis of hypoxia can be moved away to the indirect detection of nitroreductase. Various fluorescence methods have been developed for the analysis of nitroreductase and tumor hypoxia due to their simplicity, non-invasiveness and excellent spatial-temporal resolution. Here, we synthesized a multifunctional fluorophore, which possesses larger π-conjugation structure, leading to the red-shift of absorption and fluorescence emission to long-wavelength region. This fluorophore (RCOH) can be further used to prepare the off-on hypoxia probe (RCO-NTR) by introducing the electron withdrawing group of nitrobenzyl, leading to the quenching of fluorescence emission through an electron transfer process. After reaction with NTR accompanied by NADH as an electron donor, the nitro group in the RCO-NTR probe can be reduced and then the RCOH fluorophore can be released through rearrangement and elimination, leading to the enhancement of fluorescence signal. Thus, the prepared RCO-NTR probe can be used for nitroreductase analysis, which can be further used for hypoxia imaging at both cellular and animal levels.