Redox-active metals dominate the essential transition metals, as well as those used in therapeutic metal complexes, and therefore cellular redox status is key to determining the sub-cellular fate of such species. Hypoxia, the depletion of oxygen below 5%, is associated with many diseases and conditions including cancer, heart attack and stroke. Hypoxia is known to be induced by metals such as cobalt, and a number of hypoxia-activated prodrugs have been designed based on redox couples such as Pt(IV)/Pt(II) and Co(III)/Co(II).1 There is therefore much interest in monitoring hypoxia in living cells.
One common method to detect hypoxia involves fluorescent sensors bearing a nitroaromatic group, which can be reduced to the corresponding amine in the presence of the nitroreductase enzyme.2 The nitro group acts as a fluorescent quencher, which is alleviated upon reduction, leading to a fluorescence turn-on. We have previously shown that nitro-bearing coumarins exhibit this fluorogenic behaviour upon reduction. However, these probes lack selectivity, being reduced in both hypoxic and normoxic cells.3 The aim of this project is to determine how to prepare analogues with a higher reduction potential, to enable greater hypoxia selectivity.
Using DFT, we calculated the LUMO energy of 30 coumarin analogues and showed that the energy increased with the addition of electron-donating groups. The five analogues with highest LUMO energy, predicted to have the highest reduction potentials, were then synthesised and studied. All five probes showed higher reduction potentials than the parent compound. The probes with highest reduction potential were used to understand the reduction window of the hypoxia environment. The live-cell microscopic imaging of the probes reports a significant enhancement in the fluorescent signal when the probes were incubated under 2% O2 and could be used to study the behaviour of metal complexes in hypoxic conditions.