Hypoxia, the depletion of oxygen levels below 5% within biological systems due to the dysregulation of vascular supply, is associated with many diseases and health problems including cancer, heart attack and stroke. There is therefore much interest in monitoring hypoxia in living cells. The nitroreductase enzyme, overexpressed in reducing environments, participates in essential redox processes, drug activation, and cellular responses to hypoxia.1 Tracking nitroreductase accumulation enables researchers to detect cellular environment and processes.
Fluorescent sensors are considered valuable tools in biochemical imaging for their simple operation, low cost, high analyte sensitivity and selectivity, and low toxicity.2 Nitro-containing fluorescent sensors are reduced to the corresponding amine in the presence of the nitroreductase enzyme as representative of hypoxia event.3 The key of nitroreductase sensing is the fluorescent quenching properties of nitro group is alleviated upon reduction, leading to a fluorescence turn-on. One factor that has hampered development of hypoxia probes is poor selectivity for hypoxic over normoxic cells.4
This project investigates the use of nitrocoumarins as hypoxia probes. We wanted to understand the fluorogenic behaviour of nitrocoumarins upon reduction, and to further probe the interaction of these compounds with the nitroreductase enzyme.
DFT calculations were performed to explain the change in the optical signal upon the reduction of 7-(diethylamino)-coumarin probe bearing a nitrophenyl group at the 3-position. We were able to show that the nitrophenyl ring is perpendicular to the coumarin, leading to fluorescence quenching: upon reduction to the aniline, planarity and fluorescence are restored. We confirmed these findings by synthesizing a range of coumarin analogues bearing different substituents, enabling us to propose the design of other responsive sensors. We then performed docking studies to understand the interaction with our coumarin probes with the nitroreductase enzyme and correlated our findings with their subcellular behaviour.