<i>N</i>-oxide-based and trioxolane-based fluorescent probes for monitoring labile iron and labile heme: an update

Iron (Fe) and heme are essential for numerous biological processes, but their dysregulation contributes to cancer, neurodegeneration, and ferroptosis. This review summarizes the development and applications of <i>N</i>-oxide chemistry-based fluorescent probes and 1,2,4-trioxolane-based probes for selective monitoring of labile Fe(II) and heme in living systems over the past decade. We describe our discovery that Fe(II) selectively deoxygenates tertiary amine <i>N</i>-oxides, enabling conversion of non-fluorescent <i>N</i>-oxide compounds into highly fluorescent tertiary amines. This approach has yielded a comprehensive palette of Fe(II)-selective probes with diverse colors and organelle-targeting capabilities. Key developments include RhoNox-4 (FerroOrange), a highly sensitive probe demonstrating >100-fold fluorescence enhancement that enabled high-throughput screening applications, and organelle-targeted probes that revealed labile Fe(II) accumulation in lysosomes and endoplasmic reticulum during ferroptosis. We also discuss H-FluNox, the first highly selective heme probe exhibiting >200-fold fluorescence increase, which successfully monitors endogenous heme dynamics and revealed simultaneous upregulation of both labile Fe(II) and heme during ferroptosis. Another approach is based on the chemical reactivity of 1,2,4-trioxolane, where the O-O bond cleavage reaction is induced by Fe(II) or heme. The 1,2,4-trioxolane motif could facilitate ferroptosis, but in contrast, the <i>N</i>-oxide-based probes function as ferroptosis inhibitors through selective depletion of labile Fe(II) involved in lipid peroxidation. We highlight the unexpected discovery that these <i>N</i>-oxide-based probes function as ferroptosis inhibitors through selective Fe(II) depletion, with activity dependent on cellular localization. These chemical tools have provided unprecedented insights into iron and heme biology, offering new opportunities for understanding oxidative stress mechanisms and developing therapeutic strategies for iron-related disorders.