A universal fluorogenic switch for Fe(ii) ion based on N-oxide chemistry permits the visualization of intracellular redox equilibrium shift towards labile iron in hypoxic tumor cells

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe²⁺. In this study, a universal fluorogenic switching system is established, which is responsive to Fe²⁺ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe²⁺ to generate various fluorescent probes of Fe²⁺-CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe²⁺ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe²⁺ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe²⁺ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.