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

Abstract

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 N-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 N-oxides, enabling conversion of non-fluorescent N-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 N-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 N-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.

Biological Activity of Fe and Heme

Iron (Fe) is the most abundant transition metal in the human body and plays crucial roles in oxygen delivery, enzyme activity, nucleotide synthesis, and energy production.^(1,2) These processes depend on the high redox activity of Fe ions, which readily interconvert between Fe(II) and Fe(III) states, and achieve higher oxidation states cooperatively with oxygen. Labile iron species—protein-free or weakly protein-bound iron—exist in living cells and act as transient mediators of cellular iron dynamics (Fig. 1). The predominant redox state of labile iron species is Fe(II) rather than Fe(III), owing to higher water solubility, the reductive cellular environment, and the presence of transporters and chaperone proteins that recognize Fe(II) as their substrate.^(3,4) Fe(II) is well-established as a mediator of oxidative stress through the Fenton reaction, where Fe(II) reacts with hydrogen peroxide to generate highly reactive oxygen species such as hydroxyl radicals.^(5,6) Fe(II)-mediated lipid peroxidation is widely recognized as a hallmark of ferroptosis, an iron-dependent form of programmed cell death.^(7,8) Consequently, iron overaccumulation often causes severe diseases, including cancer, neurodegenerative disorders, and hepatitis. Selective monitoring Fe(II) in cells and living organisms is crucial for understanding the roles of labile iron species in both physiological and pathological conditions.

Fig. 1.

Schematic representation of cellular metabolism and homeostasis of iron and heme.

Heme, a complex of Fe(II) and protoporphyrin IX, is utilized across diverse organisms and species.^(9,10) The biological roles and cellular distribution of heme parallel those of Fe ions. Most heme exists as tightly bound hemoproteins, rendering it unavailable for heme-mediated cell signaling and oxidative stress (Fig. 1). Similar to labile iron species, protein-free or weakly protein-bound heme exists in living cells and is termed “labile heme”, which participates in various physiological processes including transcriptional regulation and circadian rhythms. Several diseases—hemolysis, malaria, sickle cell disease, and porphyria—are associated with labile heme accumulation and oxidative stress.^(11–15) However, the intracellular dynamics of labile Fe(II) species and heme remain poorly understood, primarily due to the lack of chemical tools for selective monitoring of labile Fe(II) and heme levels in living cells. Prior to our development of the first Fe(II)-selective fluorescent probe,⁽¹⁶⁾ RhoNox-1, in 2013 (Fig. 2), no turn-on fluorescent probes could recognize Fe(II) in a redox-state-selective manner.^(17–19) Our strategy for Fe(II) detection is based on a chemical reaction mediated by Fe(II), which we termed “N-oxide chemistry”.⁽²⁰⁾ We discovered that Fe(II) can selectively deoxygenate tertiary amine N-oxides to regenerate the original tertiary amines. By incorporating N-oxide into a tertiary amine structure within the π-conjugation system of a fluorophore, fluorescence is quenched. When the N-oxide reacts with Fe(II), deoxygenation occurs to produce the parent fluorophore, resulting in fluorescence turn-on (Fig. 2A–C). This design strategy is universally applicable to fluorophores containing tertiary amines in their π-conjugation structure. Several N-oxide-based probes have been developed by other groups and comprehensively reviewed elsewhere.⁽²¹⁾ Another chemical strategy to construct fluorescent probes for labile Fe(II) and heme is 1,2,4-trioxolane-based compounds, containing a cyclic O–O bond that is broken by single-electron reduction from Fe(II) or labile heme.^(18,22,23) Using the trioxolane strategy, several small-molecule-based fluorescent probes for Fe(II) and labile heme have been reported. These two chemical strategies for Fe(II) and labile heme detection have similarities and differences regarding their reaction properties. Several N-oxide-based probes exhibit inhibitory effects on ferroptosis,⁽²⁴⁾ while the 1,2,4-trioxolane compounds act as ferroptosis inducers, highlighting the differences in biological activity and chemistry between N-oxides and 1,2,4-trioxolanes.⁽²⁵⁾ Here, we focus on the probes based on the two leading chemistries for chemical biology of Fe(II) and labile heme and their applications over the past decade.

Fig. 2.

(A) Detection mechanism of N-oxide-based Fe(II)-selective fluorescent probe, RhoNox-1. (B) Fluorescence spectral change of RhoNox-1 (2 ‍μM) upon addition of FeSO₄ (20 ‍μM) in HEPES buffer (50 ‍mM, pH ‍7.4). (C) Fluorescence intensity of RhoNox-1 after incubation with metal ions (20 ‍μM) for 1 ‍h. (D) Fe(II)-selective fluorescent probes with a fluorophore other than rhodamine.

N-oxide-based Probes for Fe(II) Imaging and their Applications: Multi-color Imaging of Labile Iron in Living Cells

We demonstrated that N-oxide chemistry functions as an Fe(II)-responsive unit for various fluorophores beyond rhodamine (RhoNox-1), including coumarin (CoNox-1), rhodol (FluNox-1), and silicon-rhodamine (SiRhoNox-1), establishing a comprehensive color palette of Fe(II)-selective fluorescent probes (Fig. 2D).⁽²⁶⁾ Leveraging this feature, we developed organelle-targeted probes with distinct colors: green mitochondria-targeted probe (Mito-FluNox),⁽²⁷⁾ orange lysosome-targeted probe (Lyso-RhoNox),⁽²⁸⁾ and deep-red endoplasmic reticulum (ER)-targeted probe (ER-SiRhoNox)⁽²⁶⁾ (Fig. 2D and 3A). These three probes exhibited comparable responsiveness, enabling multi-color imaging of labile Fe(II) in specific organelles within living cells.⁽²⁹⁾ Fe(II)-treated cells stained with a cocktail of Mito-FluNox, Lyso-RhoNox, and ER-SiRhoNox showed increased fluorescence signals with characteristic staining patterns of their target organelles. This multicolor imaging approach was applied to monitor labile Fe(II) level changes during ferroptosis. Since Fe(II) facilitates radical chain reactions that induce lipid peroxidation and damage during ferroptosis, we investigated which organelles are responsible for Fe(II)-mediated lipid damage. HT1080 cells treated with erastin (a conventional ferroptosis inducer) and stained with organelle-targeted probes showed significant fluorescence increases in lysosomes and ER (Fig. 3B), which were suppressed by deferoxamine (DFO). While mitochondrial fluorescence signals appeared to increase, this was not suppressed by DFO treatment and was attributed to probe overaccumulation in mitochondria. We identified that labile Fe(II) is upregulated in ER and lysosomes but not in mitochondria during ferroptosis. This observation is consistent with recent findings from the Stockwell⁽³⁰⁾ and Yamada⁽³¹⁾ groups showing that lipid peroxidation initiates in lysosomes and ER during early-stage ferroptosis.

Fig. 3.

(A) Organelle-targeted fluorescent probes for Fe(II). Mito-FluNox: mitochondria, Lyso-RhoNox: lysosomes. (B) Multicolor imaging of labile Fe(II) at the early stage (5 ‍h) of ferroptosis induced by erastin (10 ‍μM). The probe concentrations: Lyso-RhoNox (1 ‍μM), Mito-FluNox (1 ‍μM), and ER-SiRhoNox (5 ‍μM). Scale bars indicate 25 ‍μm.

RhoNox-4: A Highly Sensitive Fe(II) Fluorescent Probe (FerroOrange)

The prototype probe RhoNox-1 had limited sensitivity, showing only ~30-fold fluorescence increase after 1-h incubation with 10 equivalents of Fe(II).⁽¹⁶⁾ Since intracellular labile Fe(II) concentrations are maintained at low levels (~1 ‍μM) to prevent uncontrolled oxidative damage, more sensitive probes are required to monitor endogenous labile Fe(II) fluctuations. Sensitivity depends on the turn-on ratio (fluorescence intensity ratio of off/on states) and response kinetics. We improved both parameters by screening N-oxide domain structures. Based on experimental knowledge that cyclic amine N-oxides exhibit higher Fe(II) responsiveness than linear alkyl amine N-oxides, we synthesized rhodamine N-oxide compounds with cyclic amine N-oxide structures (Fig. 4A) and screened their fluorescence responses.⁽³²⁾ All cyclic amine N-oxide probes showed enhanced responses regarding off/on rates and reaction kinetics. RhoNox-4, RhoNox-7, and RhoNox-8 demonstrated particularly high responses with >100-fold fluorescence increases within 15 ‍min (Fig. 4B). However, only RhoNox-4 functioned effectively in live-cell imaging, showing significantly better contrast and fluorescence signals in Fe(II)-treated cells compared to RhoNox-1 (Fig. 4C). The newly synthesized probes, except for RhoNox-4, were unable to permeate the cell membrane, resulting in undetectable fluorescence signals in the live-cell imaging application. The enhanced selectivity of RhoNox-4 enabled high-throughput screening (HTS) for monitoring subcellular labile iron levels. We applied this HTS to a chemical library containing 3,399 compounds to discover drug candidates that modulate subcellular iron status. The HTS detected both upregulators and downregulators of subcellular labile iron. Through repeated screening, lomofungin was identified as a novel labile iron upregulator that downregulates ferritin (an iron storage protein), resulting in labile iron release. However, the detailed mechanism of ferritin downregulation remains unknown. RhoNox-4 is the most sensitive and user-friendly fluorescent probe for labile Fe(II) among rhodamine-based probes and is commercially available as FerroOrange.

Fig. 4.

(A) Chemical structures of the RhoNox series with cyclic amine N-oxide. (B) Fluorescence response of RhoNox-1, RhoNox-4, RhoNox-7, and RhoNox-8 (2 ‍μM, each) upon addition of FeSO₄ (20 ‍μM) in HEPES buffer (50 ‍mM, pH ‍7.4). (C) Representative fluorescence microscopic images of HepG2 cells using RhoNox-1 (5 ‍μM) or RhoNox-4 (1 ‍μM) after incubation with or without ferrous ammonium sulfate. Scale bars indicate 25 ‍μm.

N-Oxide-based Probe for Labile Heme and Applications

The intracellular dynamics of labile heme are less understood than those of labile Fe(II). Live-cell imaging of labile heme was initially achieved using fluorescent protein (FP)-based sensors.^(33,34) Although FP-based sensors revealed that exchangeable labile heme exists and functions as a signaling molecule in mammalian cells,^(33,34) their requirement for genetic manipulation limits accessibility. We hypothesized that our N-oxide-based strategy could serve as a heme-specific chemical trigger, since the metal center of labile heme is Fe(II). N-oxide deoxygenation is analogous to heterolytic O–O bond cleavage of peracids, a biomimetic peroxidase reaction reported by Watanabe and Groves.⁽³⁵⁾ Based on their findings, we designed H-FluNox⁽³⁶⁾ (Fig. 5A) as a heme-selective fluorescent probe by incorporating a difluoromethylene unit proximal to the N-oxide, which introduces electron-withdrawing properties and facilitates heme-mediated heterolytic N–O bond cleavage. H-FluNox showed >200-fold fluorescence increase in response to 1 ‍μM heme in the presence of glutathione, while exhibiting only a few-fold increase with Fe(II), indicating high selectivity for labile heme over Fe(II) (Fig. 5B and C). Ac-H-FluNox, an acetylated cell-membrane-permeable analog, was applied to live-cell imaging. Heme biosynthesis upregulation by 5-aminolevulinic acid (5-ALA) supplementation and downregulation by succinylacetone (SA) resulted in significant fluorescence increases and decreases, respectively (Fig. 5D). The probe successfully monitored labile heme levels in response to nitric oxide (NO) treatment, showing fluorescence response to heme release from endogenous hemoproteins upon NO treatment (Fig. 5E). This represents the first detection of heme alterations induced by indirect stimulation using a small-molecule fluorescent probe. Since H-FluNox is highly sensitive and selective to labile heme but insensitive to Fe(II), Ac-H-FluNox was applied to monitor labile heme levels during ferroptosis. Using SiRhoNox-1 [a deep-red Fe(II) fluorescent probe with non-overlapping emission] combined with Ac-H-FluNox, we simultaneously monitored labile Fe(II) and heme levels during ferroptosis. Both labile Fe(II) and heme were upregulated in early-stage erastin-induced ferroptosis (Fig. 5F). Although the mechanism underlying labile heme upregulation remains unclear, we found that inhibiting endogenous heme biosynthesis did not rescue cells from ferroptosis. The probe successfully revealed heme-exporting properties of ATP-binding cassette transporter G2 (ABCG2), the potential heme-storage ability of guanine quadruplexes, and the potential role of feline leukemia virus subgroup C cellular receptor 1 (FLVCR1).

Fig. 5.

(A) Chemical structure and detection mechanism of an N-oxide-based heme-selective fluorescent probe, H-FluNox. (B) Fluorescence response of H-FluNox (0.2 ‍μM) upon addition of FeSO₄ (10 ‍μM, gray) or heme (as hemin, 1 ‍μM) in the presence of glutathione (100 ‍μM) in HEPES buffer (50 ‍mM, pH ‍7.4). (C) Fluorescence intensity of H-FluNox after incubation with the indicated metal ions (10 ‍μM) or heme (1 ‍μM). (D) Fluorescence images of HeLa cells stained with Ac-H-FluNox (10 ‍μM) (acetylated analogue of H-FluNox for improved cell permeability) after incubation with 5-aminolevulinic acid (5-ALA) in the presence or absence of succinylacetone (SA) for 24 ‍h. (E) Fluorescence images of HeLa cells stained with Ac-H-FluNox (10 ‍μM) after incubation with NOC-5 (100 ‍μM, NO donor) for 20 ‍min. (F) Fluorescence images of HT1080 cells stained with SiRhoNox-1 (5 ‍μM) and Ac-H-FluNox (10 ‍μM) after incubation with erastin (10 ‍μM) with or without deferoxamine (DFO, 100 ‍μM). Scale bars indicate 25 ‍μm.

1,2,4-Trioxolane-based Probes for Fe(II) and Labile Heme

Renslo and colleagues pioneered the development of the first 1,2,4-trioxolane-based Fe(II) fluorescent probe⁽³⁷⁾ employing an Fe(II)-cleavable linker,⁽³⁸⁾ which has been comprehensively reviewed previously.^(18,23) Subsequently, the application of this Fe(II)-cleavable 1,2,4-trioxolane linker was extended to Förster resonance energy transfer (FRET)-based fluorescent probes.⁽³⁹⁾ In recent investigations, Renslo’s group reported the development of FIPC-1 (Fig. 6A),⁽⁴⁰⁾ a chemical probe designed for interactome analysis of the endoperoxide-based ferroptosis inducer FINO2.⁽²⁵⁾ Their mechanistic studies demonstrated that Fe(II)-mediated O–O bond cleavage generates a secondary carbon radical, which was successfully applied to proteomics analysis through reaction with the radical intermediate of FIPC-1 during Fe(II)-mediated ferroptosis. The 1,2,4-trioxolane analog FINO3 exhibited comparable ferroptotic activity to FINO2, with EC₅₀ values of 3.2 ‍μM and 2.7 ‍μM against HT1080 cells, respectively. FIPC-1, a FINO2 derivative containing an alkyne moiety as a chemical handle, was employed for comprehensive interactome analysis. This approach identified protein disulfide isomerase (P4HB) and 5'-nucleotidase domain-containing protein 2 (NT5DC2) as molecular targets of FIPC-1-mediated ferroptosis. While P4HB inhibitors are known ferroptosis inducers, the specific role of NT5DC2 in ferroptotic cell death remains to be elucidated.⁽⁴⁰⁾

Fig. 6.

(A) Structure and mechanism of FIPC-1. (B) Structure and mechanism of ¹⁸F-TRX. (C) Left: Representative PET imaging of mice treated with ferrous ammonium citrate (FAC), deferoxamine (DFO), or deferiprone (DFP, iron chelator). Right: Relative ¹⁸F-signals in each organ. Adapted with permission from Ref 41: ACS Cent Sci 2019; 5(4): 727–736. Copyright 2019 American Chemical Society. (D) Structure and reaction mechanism of IG1-FM. (E) Structure and detection mechanism of HNG-1. (F). Representative fluorescence images (upper) of the liver isolated from different mice (λ_ex = 465 ‍nm, λ_em = green channel) and two-photon (TP, bottom) confocal microscopy images of the corresponding liver tissues (λ_ex = 780 ‍nm, λ_em = 500–600 ‍nm): − −, control; + −, only phenylhydrazine (PHZ); − +, only HNG; + +, PHZ and HNG combined. Adapted with permission from Ref 22: J Am Chem Soc 2020; 142(5): 2129–2133. Copyright 2020 American Chemical Society.

The radical-generating properties of endoperoxide probes have been leveraged for positron emission tomography (PET) imaging applications, exploiting ferrous ion-dependent accumulation through covalent bond formation with biomolecules.⁽⁴¹⁾ Utilizing this principle, Renslo and Evans developed ¹⁸F-TRX (Fig. 6B), a novel PET imaging probe for labile ferrous ion detection, and demonstrated its efficacy in murine PET imaging studies. The probe architecture consists of an artefenomel scaffold conjugated to an ¹⁸F-PET tracer. Artefenomel contains an adamantyl 1,2,4-trioxolane unit that undergoes ferrous ion-mediated activation to generate carbon-centered radicals, analogous to the mechanism observed with the chemoproteomics probe FIPC-1. The accumulation of ¹⁸F-TRX reflects tissue-specific labile ferrous ion concentrations, as the short-lived radical intermediates are trapped by proximal proteins within approximately 1 ‍mm. Application of ¹⁸F-TRX to in vivo PET imaging (Fig. 6C) revealed elevated labile ferrous iron levels in tumor tissues compared to surrounding normal tissues in xenograft mouse models.

Chang and colleagues exploited the 1,2,4-trioxolane motif, which undergoes Fe(II)-mediated O–O bond cleavage followed by retro-Michael reaction, to develop IG1-FM (Fig. 6D), a tandem activity-based probe for Fe(II) detection.⁽⁴²⁾ This probe generates an electrophilic quinone intermediate upon Fe(II) interaction, forming covalent bonds with biomolecules including proteins, and exhibits fluorescence enhancement in response to Fe(II). The extended cellular retention and labile iron-dependent accumulation of the fluorescent product represent key advantages of IG1-FM. Imaging studies utilizing IG1-FM elucidated the direct contribution of ferritin degradation to intracellular labile Fe(II) homeostasis. Furthermore, these investigations revealed that the KEAP1/NRF2 signaling pathway regulates labile iron levels in cancer cells and determines cellular vulnerability to iron chelation therapy.

Zhang and co-wokers developed HNG, the first small-molecule fluorescent probe for labile heme detection.⁽²²⁾ The probe design exploited the reactivity of the 1,2,4-trioxolane motif as a reactive trigger for Fe(II), following the principles established by Renslo’s group. HNG comprises a 4-amino-1,8-naphthalimide fluorophore conjugated to an adamantyl-1,2,4-trioxolane unit via carbamoylation at the 4-position, which initially quenches fluorescence (Fig. 6E). Labile heme-induced cleavage of the 1,2,4-trioxolane unit, followed by β-elimination, restores fluorescence signaling, generating a turn-on response. Treatment with HNG (2 ‍μM) resulted in a 13-fold fluorescence intensity increase following reaction with equimolar heme after 1-h incubation. The probe exhibited minimal cross-reactivity with ferrous ions (20 ‍μM, approximately 2-fold increase), although potential interference from labile ferrous ions cannot be excluded given their higher physiological concentrations (~1 ‍μM) compared to labile heme (~20 nM). Live-cell imaging applications of HNG successfully detected labile heme upregulation and downregulation induced by 5-ALA and SA, respectively. Additionally, HNG detected labile heme release in a phenylhydrazine (PHZ)-induced hemolysis model in murine hepatic tissues (Fig. 6F).

N-oxide-based Probes as Ferroptosis Inhibitors

Ferroptosis is an iron-dependent programmed cell death first reported by Stockwell et al.^(7,8) This cell death is characterized by accumulation of damaged lipid molecules in cell membranes through Fenton-type reactions between Fe(II) and lipid peroxides (Fig. 7A).⁽⁷⁾ Fe(II) is essential for ferroptosis progression, as demonstrated by our group and others showing Fe(II) upregulation in early-stage ferroptosis. While many ferroptosis inducers have been reported, inhibitors are limited in their modes of action. Conventional ferroptosis inhibitors are classified into two groups (Fig. 7A): radical-trapping agents (RTAs) and iron chelators. RTAs, including ferrostatin-1, often comprise electron-rich aromatics that cause hepatotoxicity.⁽⁴³⁾ DFO, a first-generation clinically used iron chelator, exhibits ferroptosis-inhibitory activity but requires high-dose treatment due to poor oral bioavailability from high hydrophilicity. Other clinical iron chelators, including deferiprone (DFP) and deferasirox (DFS), also require high-dose treatment due to their binding stoichiometry (3:1 for DFP:Fe and 2:1 for DFS:Fe).^(44,45) Novel ferroptosis inhibitors based on new mechanisms are urgently needed.

Fig. 7.

(A) Schematic representation of the mechanism of ferroptosis and its inducers and inhibitors. (B) Cell viability of HT1080 cells treated with erastin (10 ‍μM) in the presence of the indicated fluorescent probes. Error bars indicate ± SEM (n = 4) *p<0.01, **p<0.001.

We focused on N-oxide reactivity, which undergoes deoxygenation upon reaction with Fe(II) ions. This reaction reduces N-oxide to tertiary amines while oxidizing Fe(II) to higher oxidation states. Since Fenton-like reactions require Fe(II) for radical initiation, our N-oxide probes can selectively react with Fe(II),⁽²⁰⁾ enabling selective depletion of Fe(II) involved in lipid damage during ferroptosis.

X-ray absorption near-edge structure (XANES) spectrometry revealed that Fe(II) is oxidized to Fe(III) after reaction with N-oxide compounds. We assayed N-oxide-based fluorescent probes for ferroptosis inhibitory activity, demonstrating that some probes are effective while others are not for inhibiting erastin-induced ferroptosis. RhoNox-1, RhoNox-4, SiRhoNox-1, and Lyso-RhoNox inhibited ferroptosis at 3.2–12.8 ‍μM concentrations, while H-FluNox and Mito-FluNox showed no inhibitory activity (Fig. 7B). This indicates that intracellular probe localization is critical for inhibitory effects, where labile Fe(II) in lysosomes, ER, and Golgi is responsible for lipid peroxidation and ferroptotic lethality. The probes are also effective against other ferroptosis inducers, including RSL-3, FIN56, and FINO2^(25,46,47) (Fig. 7A), while other programmed cell death types were unaffected. The unique inhibitory mechanism of N-oxide-based probes against ferroptosis relies on their selectivity for labile Fe(II) in a cellular localization-dependent manner. These probes are powerful tools for understanding the origins of labile Fe(II) involved in ferroptosis.

Conclusion

Our group has developed N-oxide chemistry-based fluorescent probes for monitoring labile Fe(II) and heme in living cells over the past decade. These probes have contributed to diverse biological studies aimed at understanding labile Fe(II) functions and roles during pathological processes, including age-related macular degeneration, iron-induced tumorigenesis, asbestos-induced mesothelioma, ovarian endometriosis, renal tubulointerstitial injury, and ferroptosis. The highly sensitive Fe(II) fluorescent probe RhoNox-4 was successfully applied to HTS of chemical libraries to identify novel drug candidates regulating iron homeostasis. RhoNox-4 is now commercially available as FerroOrange and widely used in iron biology research, particularly in ferroptosis studies. The heme-selective fluorescent probe H-FluNox exhibits the highest sensitivity and selectivity among reported small-molecule fluorescent probes. Fluorescence imaging with these chemical tools provides powerful insights into the biological and pathological functions of iron and heme, which are highly relevant to oxidative stress. The 1,2,4-trioxolane-based strategy developed by Renslo et al. represents a robust approach for Fe(II) and labile heme detection. This design exploits Fe(II)-mediated endoperoxide cleavage through retro-Michael elimination and carbon radical formation, yielding fluorogenic, ratiometric, PET, and proteomics probes for Fe(II) detection. Users should note that both of N-oxide-based and 1,2,4-endoperoxide-based fluorescent probes exhibit irreversible fluorescence responses (off-to-on). Additionally, we discovered that N-oxide-based fluorescent probes can function as ferroptosis inhibitors. The use of probes for ferroptosis inhibition was first reported by Ikeda et al.,⁽⁴⁸⁾ who utilized the mitochondria-targeted probe Mito-FluNox (commercially available as Mito-FerroGreen) to alleviate doxorubicin-induced ferroptotic damage in cardiomyocytes. Our group elucidated the detailed inhibitory mechanism from a chemical perspective, where probes selectively deplete Fe(II) involved in Fenton-type radical reactions through selective oxidation of labile Fe(II). Inhibitory activity strongly depends on cellular probe localization, suggesting that specific organelles—lysosomes, ER, and Golgi—are foci of Fe(II)-mediated lipid damage in ferroptosis. The 1,2,4-trioxolane motif induces ferroptosis through Fe(II)-mediated radical generation, whereas N-oxide-based probes inhibit ferroptosis. This suggests these strategies target distinct subcellular labile iron pools; however, the speciation of cellular labile iron and potential differentiation between these pools requires further investigation. Further development of chemical tools enabling visualization of labile iron and heme in tissues, in vivo animal models, and super-resolution microscopy will contribute to expanding and deepening oxidative stress research in the future.

Conflict of Interest

No potential conflicts of interest were disclosed.