A Diazaborine Activity-Based Sensing Fluorescent Probe Reports Endogenously Produced Mitochondrial Peroxynitrite in Live Macrophages

Abstract

We present the synthesis, properties, and imaging applications of a new class of diazaborine-based probes (Peroxynitrite Probe-1, PNP-1) for selective peroxynitrite (ONOO⁻) imaging in live cells. PNP-1 features a diazaborine-based reaction motif that provides excellent discrimination between H₂O₂ and ONOO⁻, solving a persistent challenge of organoboron-based fluorescent probes for oxidative metabolite imaging. We demonstrate the utility of PNP-1 to detect endogenously produced ONOO⁻ in live RAW 264.7 macrophages by fluorescence microscopy, with probe selectivity confirmed with inhibition of NADPH oxidases and nitric oxide synthase, the requisite enzymatic machinery for ONOO⁻ production. Colocalization studies unexpectedly reveal preferential mitochondrial localization, which we show is dependent on the naphthalimide scaffold. Taken together, our results show that diazaborines are a novel motif for selective ONOO⁻ reactivity, positioning them for incorporation into other ONOO⁻-specific chemical biology tools.

Introduction

Reactive oxygen and nitrogen species (ROS/RNS) are important mediators of cellular signalling and immune responses.^[1] However, dysregulation of ROS/RNS production, maintenance, and destruction is implicated in the progression of numerous inflammatory diseases, including heart disease, stroke, diabetes, chronic respiratory diseases, and glaucoma.^[2-9] Among biological ROS/RNS, peroxynitrite (ONOO⁻) is a particularly potent and versatile species. Its high oxidative potential, nucleophilicity, and ability to nitrate biomolecules make it a double-edged sword in cellular processes, as it acts as both as a powerful tool for macrophages in pathogen destruction but can also serve as a potential cause of extensive biomolecular damage.^[10] Recent advances in ROS/RNS signalling research have underscored the important roles of ONOO⁻ in macrophage function and inflammation.^[11] However, progress in this field has been hampered by a persistent and major technological barrier: the lack of tools to monitor ONOO⁻ with high chemical selectivity. This limitation has made it challenging to distinguish between the physiological and pathological roles of ONOO⁻ in biological contexts, limiting our understanding of its complex cellular functions.

Activity-based fluorescent probes have emerged as powerful tools for tracking cellular ROS/RNS.^[12,13] These probes are designed with chemical motifs that ideally react with a single analyte, triggering a change in fluorescence that directly links the fluorescent response to the presence of the target molecule. While numerous fluorescent probes for ROS/RNS exist, significant challenges persist. The rapid production, interconversion, and varying decomposition rates of ROS/RNS necessitate a toolbox of modular motifs with high chemoselectivity for individual species to accurately profile ROS/RNS populations. This need for selectivity is particularly important for ONOO⁻ detection. Currently, the most widely used reactivity-based fluorescent probes for ONOO⁻ rely on the oxidation of arylboronic acids/esters. However, these probes suffer from a major limitation in that they also react rapidly with H₂O₂ to give an identical fluorescent product to ONOO⁻ oxidation.^[14,15] This cross-reactivity creates ambiguity in determining whether observed signals are due to H₂O₂, ONOO⁻ , or a combination of both, complicating the interpretation of cellular oxidative states.

We hypothesized that the reactivity of arylboronic acid motifs could be fine-tuned through the introduction of neighboring groups to alter their oxidative reactivity profile. This approach is supported by recent findings. Graham and colleagues demonstrated that an o-carboxyphenylboronic acid, featuring an o-carboxylate relative to its arylboronic acid motif, cyclizes to a boronolactone, dramatically slowing its H₂O₂-mediated oxidation by over five orders of magnitude compared to arylboronic acids.^[16] Complementing this work, we recently showed that cyclic diazaborines (DABs) oxidize almost 3000 times slower with H₂O₂ (k₂ ~10⁻³ M⁻¹s⁻¹) compared to acyclic o-functionalized arylboronic acids (k₂ ~10⁰ M⁻¹s⁻¹).^[17,18] Notably, DABs maintain rapid oxidation with ONOO⁻ , suggesting their potential as privileged motifs for kinetic discrimination between H₂O₂ and ONOO⁻. Very recent work has shown that derivatizing a boronic acid with a diaminonapthalene also enhances selectivity for ONOO⁻ over H₂O_2.^[19]

Here, we introduce Peroxynitrite Probe-1 (PNP-1), a novel naphthalimide-based fluorescent probe that offers a significant advancement in ONOO⁻ detection using organoboron-based detection moieties. PNP-1 combines a diazaborine (DAB) motif with a naphthalimide scaffold, resulting in high selectivity for ONOO⁻ over other ROS/RNS, including H₂O₂. PNP-1 (Φ = 0.001) exhibits a 30-fold fluorescent turn-on upon oxidation with ONOO⁻ (Φ = 0.031) and can track endogenously produced ONOO- in activated macrophages. Notably, co-localization studies reveal high subcellular localization, predominantly to the mitochondria, with further investigations indicating that the core naphthalimide scaffold is the key molecular component that drives high subcellular localization.

Results and Discussion

The synthesis of PNP-1 was accomplished in seven steps from commercially available starting materials (SI Scheme 1). With PNP-1 in hand, we characterized its in vitro response to biologically relevant ROS and RNS using steady-state fluorescence spectroscopy (Figure 2). PNP-1 (5 μM) was incubated in aqueous buffer (PBS, pH 7.4) alone or with ten equivalents (50 μM) of common biological ROS/RNS. Consistent with our hypothesis that diazaborines would enable single-species reactivity with ONOO⁻ , PNP-1 demonstrated high discrimination against all tested ROS/RNS, including H₂O₂. Liquid chromatography-mass spectrometry (LC–MS) confirms oxidation to the corresponding phenol with ONOO⁻ (SI Figure 1). This selectivity profile stands in contrast to the boronate-ester naphthalimide probe BA-1 (SI Scheme 2), which shows rapid fluorescence increases with both H₂O₂ (10-fold) and ONOO⁻ (30-fold) (SI Figure 2). These results provide support for our probe design hypothesis, highlighting the DAB motif‘s capacity to enhance ROS/RNS selectivity, particularly in discriminating between ONOO⁻ and H₂O₂.

Figure 2.

Fluorescence responses of PNP-1 (5 μM) to various reactive oxygen (ROS) and nitrogen species (RNS). Data shown are for 50 μM for all ROS and RNS over the course of 1 h. Hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (t-BuOOH), and hypochlorite (OCl⁻) were delivered from 50 mM stock solutions, diluted from 30%, 70%, and 5% aqueous solutions, respectively. Superoxide (O₂⁻) was added as solid KO₂. Hydroxyl radical (•OH) and tert-butoxy radical (•OtBu) were generated by reaction of 1 mM Fe²⁺ with 50 μM H₂O₂ or 50 μM TBHP, respectively. Nitric oxide (NO·) was added using NO· gas prepared by the action of sulfuric acid on NaNO₂, and NO⁺ was delivered using S-nitrosoglutathione (SNOG). Spectra were acquired in 20 mM HEPES, pH 7.4, and all data were obtained after incubation with the appropriate ROS at 25 °C. Collected emission was integrated between 500 and 650 nm (λ_exc = 420 nm), and bars represent fluorescent intensity at 0 (white), 15 (very light gray), 30 (light gray), 45 (gray), and 60 min (dark gray) after addition of the indicated ROS/RNS. Experiments were repeated in triplicate; error bars represent ± S.D.

To validate the ONOO- selectivity of PNP-1 in a complex cellular environment, we first carried out live cell imaging experiments with exogenously applied H₂O₂ and ONOO⁻ for both arylboronates-ester based BA-1 and diazaborine-based PNP-1. After confirming the biocompatibility of PNP-1 and BA-1 (5 μM) in RAW 264.7 macrophages (SI Figure 2) with a LIVE/DEAD assay, we compared their responses to exogenous H₂O₂ and ONOO⁻. BA-1-loaded macrophages showed a robust ~300 % increase in intracellular fluorescence upon H₂O₂ (1 mM) treatment over 60 minutes (Figure 3A, B), and a ~60% increase with SIN-1, a ONOO⁻ generator (Figure 3C). By contrast, PNP-1-loaded macrophages (Figure 3D) showed no significant change in fluorescence with H₂O₂ treatment (Figure 3E) but exhibited a ~30% increase in fluorescence intensity with SIN-1 (Figure 3F). Quantification of cellular fluorescence intensity (Figure 3G, H) reveals that DAB-based PNP-1 offers superior H₂O₂/ONOO⁻ discrimination over boronic-acid based probes in live cell imaging experiments.

Figure 3.

In cellulo response of an arylboronates-ester based probe (BA-1) and a diazaborine-based probe (PNP-1) in live RAW 264.7 macrophages to exogenously added H₂O₂ and ONOO⁻. (A) BA-1 (5 μM) loaded macrophages at t=0; (B) after addition of H₂O₂ (1 mM) at t = 60 minutes; (C) or after addition of SIN-1 (500 μM) at 240 minutes. (D) PNP-1 (5 μM) loaded macrophages at t = 0; (E) after addition of H₂O₂ (1 mM) at 60 minutes; (F) or after addition of SIN-1 (500 μM) at 240 minutes. (G) Bars represent the mean cellular fluorescence intensities of ca. 300 individually measured cells in BA-1 loaded cells across three separate experiments with ca. 100 cells/experiment; (H) Bars represent the mean cellular fluorescence intensities of ca. 300 individually measured cells in PNP-1 loaded cells across three separate experiments with ca. 100 cells/experiment for each condition. Error bars represent S.E.M. across the three samples for each condition. n.s. = not significant; * = P < 0.05; ** = P 0.001; *** = P 0.0001. Scale bar = 20 mm.

To further challenge PNP-1’s selectivity, we investigated its ability to detect endogenously produced ONOO⁻ in live macrophages stimulated with lipopolysaccharide (LPS). LPS-treated macrophages produce a complex mixture of ROS and RNS, providing a rigorous test of probe selectivity for endogenous ONOO⁻. RAW 264.7 macrophages activated with LPS (2.5 μg/mL) and loaded with PNP-1 (5 μM) showed a nearly 3-fold increase in mean fluorescence intensity compared to probe only treated cells (Figure 4A, B). Crucially, inhibition of either NADPH oxidase with VAS2870 (4 μM) or nitric oxide synthase with 1400 W (100 μM) reduced the fluorescence to baseline levels (Figure 4C-E). Because ONOO⁻ is produced in a diffusion-limited reaction of O₂⁻ and NO, these results establish that the observed fluorescence increase is dependent on both O₂⁻ and NO production, confirming PNP-1’s ability to selectively detect cell-produced ONOO⁻.

Figure 4.

Confocal fluorescence microscopy images live RAW 264.7 macrophages on collagen-coated culture plates (A–D). Cells were treated with (A) PNP-1 (5 μM) only; (B) PNP-1 (5 μM) + LPS (2.5 μg/mL); (C) NOX inhibitor VAS2870 (4 μM) and LPS (2.5 μg/mL); or (D) iNOS inhibitor 1400 W (100 μM) and LPS (2.5 μg/mL). (E) Bars represent the mean cytosolic fluorescence intensities of ca. 300 individually measured cells across three separate experiments. Error bars represent average normalized fluorescence intensity ± S.E.M. (N = 3). ** = P < 0.01; n.s. = not significant. Scale bar = 20 μm.

Intriguingly, close inspection of the subcellular distribution of PNP-1 revealed an unexpected non-uniform punctate pattern. Hypothesizing that PNP-1 was trafficking a subcellular organelle, we performed co-localization studies with MitoTracker, which revealed high mitochondrial localization of PNP-1 (Figure 5A–C). To determine whether the diazaborine or its oxidation product had different localization properties, we synthesized the product after ONOO⁻ oxidation, PNP-OH. Again, PNP-OH showed high co-localization with MitoTracker, confirming that PNP-1 does not change its subcellular localization preferences after reaction with ONOO⁻ (Figure 5D-F). ONOO⁻ is estimated to diffuse ca. 10 μm in the presence of CO₂ and ca. 2 μm in the presence of peroxiredoxin (Prx).^[20] Because mitochondria produce CO₂ during oxidative phosphorylation and express peroxiredoxin, it is reasonable to expect that PNP-1 detects ONOO⁻ generated in and/or near mitochondria.

Figure 5.

Confocal fluorescence imaging studies of live RAW 264.7 macrophages loaded with MitoTracker and either PNP-1 (A–C), PNP-OH (D–F) or BA-OH (G–I). (A) Distribution of PNP-1 in live RAW 264.7 macrophages; (B) MitoTracker; (C) overlay. (D) Live RAW 264.7 macrophages loaded with PNP-OH; (E) MitoTracker; and (F) overlay. (G) Distribution of BA-OH in live RAW 264.7 macrophages; (H) MitoTracker; (I) overlay. Values in C, F, and I are the Pearson’s coefficient showing degree of colocalization.

The mitochondrial localization is particularly noteworthy as neither compound possesses the typical cationic nature of mitochondrial targeting agents. However, a recent report noted that naphthalimide fluorophores with non-canonical targeting groups localized to the ER or lysosomes, suggesting that structural features can drive subcellular localization.^[21] To probe which structural features trigger mitochondrial localization, we first examined the effect of the sulfonyl group on subcellular distribution. We replaced the sulfonylhydrazone with a hydrazone linkage in PNP-phenyl and PNP-phenyl-OH and still observed high mitochondrial association (SI Figure S4 A-F). Next, we sought to probe the role of the hydrazone linkage with subcellular distribution. To accomplish this, we synthesized CTH, a 7-hydroxycoumarin derivative with an ortho-toluenesul-fonylhydrazone functionality (Figure S4 G-I). Co-localization studies revealed a significant decrease in mitochondrial localization compared to PNP-OH, indicating that the hydrazone was not solely responsible for subcellular localization and implicating the fluorophore scaffold in subcellular localization. To confirm the role of the naphthalimide in mitochondrial localization, we truncated the scaffold to the core naphthlimide fluorophore (BA-OH). High mitochondrial localization was observed (Pearson’s colocalization coefficient (PCC) > 0.90), indicating that the naphthalimide is the minimal molecular component needed to drive mitochondrial localization (Figure 5G-I). Follow-on studies showed that PNP-OH moderately co-localizes with lysosomes (PCC ~0.7) but to a lesser degree than with mitochondria (SI Figure S5). Moreover, a control study with both lysosomal and mitochondrial staining but without PNP-OH reveals some co-localization of the two stains (PCC ~0.25), resulting from either off-target effects of the stains or from mitophagy (SI Figure 6).

The origin of the subcellular localization is not immediately obvious. Most mitochondrial targeting probes have cationic functionality to drive association in the negatively charged mitochondrial matrix. The simplified naphthalimide core is net neutral at physiological pH. Our current hypothesis is that the planar naphthalimide architecture permits intercalation into mitochondrial DNA. No nuclear staining is seen because the compound is unable to access the nucleus. Current efforts are underway to elucidate the mechanism of the subcellular localization, as the further development of non-cationic probes that do not disrupt mitochondrial membrane potentials would be a useful complement to existing mitochondrial dyes.

Conclusions

Here we have shown that diazaborines (DABs), previously recognized for their applications in antibiotics and bioconjugation, exhibit high chemoselectivity for ONOO⁻ oxidation over other biological ROS/RNS, including over H₂O₂. When merged with a naphthalimide scaffold, the resulting probe, PNP-1, enables selective imaging of endogenously produced ONOO⁻. Inhibition studies confirm the ability of PNP-1 to detect ONOO⁻ produced in activated macrophages. The selectivity of PNP-1, particularly its discrimination between ONOO⁻ and H₂O₂, which overcomes a major limitation of existing organoboron-based probes and reveals a new strategy of ‘pendant-tuning’ to alter the selectivity of a fluorescent probe. Co-localization studies reveal a surprising and potentially valuable feature of PNP-1 to target mitochondria. Follow-on co-localization studies reveal that the mitochondrial localization is dependent on the naphthalimide scaffold, a finding that opens new possibilities in probe design and cellular targeting that complements existing known mitochondrial motifs such as triphenylphosphonium and N-alkylated pyridines.

Supplementary Material

The authors have cited additional references within the Supporting Information.^{[20,21, 22]}

Supporting information for this article is available on the WWW under https://doi.org/10.1002/cbic.202400972

Figure 1.

(Top) Diazaborine-based fluorescent probes enable kinetic high discrimination between H₂O₂ and ONOO⁻ , improving upon existing organoboron-based fluorescent probes for reactive oxygen and nitrogen species (ROS/RNS) imaging. (Bottom) Compounds studied in this work.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.