Oxygen-dependent fluctuation of labile Fe(ii) was visualized by a new N-oxide-based fluorescent probe for Fe(ii) ion.

Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe(2+)-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe(2+)-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe(2+), compared with the first generation N-oxide based Fe(2+) probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe(2+) but also the endogenous Fe(2+) levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.

Fluctuation of labile Fe(ii) at Golgi apparatus was specifically detected by a new fluorescent probe.

High-throughput methods for monitoring subcellular labile Fe­(II) are important for conducting studies on iron homeostasis and for the discovery of potential drug candidates for the treatment of iron deficiency or overload. Herein, a highly sensitive and robust fluorescent probe for the detection of intracellular labile Fe­(II) is described. The probe was designed through the rational optimization of the reactivity and responsiveness for an Fe­(II)-induced fluorogenic reaction based on deoxygenation of an N-oxide, which was developed in-house. The probe is ready to use for a 96-well-plate-based high-content imaging of labile Fe­(II) in living cells. Using this simple method, we were able to conduct high-throughput screening of a chemical library containing 3399 compounds. The compound lomofungin was identified as a potential drug candidate for the intracellular enhancement of labile Fe­(II) via a novel mechanism in which the ferritin protein was downregulated.

Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.