Tracking isotopically labeled oxidants using boronate-based redox probes

Natalia Rios; Rafael Radi; Balaraman Kalyanaraman; Jacek Zielonka

doi:10.1074/jbc.RA120.013402

. 2020 Mar 26;295(19):6665–6676. doi: 10.1074/jbc.RA120.013402

Tracking isotopically labeled oxidants using boronate-based redox probes

Natalia Rios ^‡,^§, Rafael Radi ^‡,^§, Balaraman Kalyanaraman ^¶, Jacek Zielonka ^¶,¹

PMCID: PMC7212653 PMID: 32217693

Abstract

Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18–labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

Keywords: hydrogen peroxide, superoxide ion, reactive oxygen species (ROS), reactive nitrogen species (RNS), oxygen radicals, isotopic tracer, mass spectrometry (MS), oxygen-18, peroxymonocarbonate, peroxynitrite, reaction mechanism, redox probes, hypochlorous acid, superoxide radical anion, isotope tracing

Introduction

Reactive oxygen species (ROS),² including superoxide (O₂^˙̄/HO₂^•), hydrogen peroxide (H₂O₂), and peroxynitrite (ONOO⁻/ONOOH), have been implicated in (patho)physiological mechanisms in redox biology and medicine (1 –4). Both superoxide and H₂O₂ are relatively slow reacting and/or weak oxidants (4 –6) but in biological systems can be converted to more reactive species (see Fig. 1), including peroxynitrite (7, 8), peroxymonocarbonate (HCO₄⁻) (9, 10), or hypochlorous acid (HOCl) (11), resulting in enhanced redox signaling and/or damage to cell components (5, 12, 13). Because of the short lifetime of most ROS in biological settings, detection and quantitative analyses of those species have remained a challenge, and development of new probes for redox biology is an active area of research. Most chemical probes used for the detection of cellular oxidants lack selectivity toward a single species. For example, dichlorodihydrofluorescein (DCFH), dihydrorhodamine-123 (DHR123), and Amplex Red undergo two-electron oxidation to fluorescent dichlorofluorescein, rhodamine, and resorufin, respectively, and nitro blue tetrazolium undergoes four-electron reduction to diformazan, without incorporation of the reactive species detected into the product formed (Fig. 1). This often leads to ambiguity regarding the identity of the species detected and prevents tracking of the oxidants using isotope-labeling approach. ROS detection and their unambiguous identification in biological systems requires the use of chemical probes, which upon reaction form species-specific product(s) (14 –19). As an example, spin traps react with most radicals by the formation of a covalent bond between the probe and the radical trapped, and the product formed is typically highly specific for the trapped species. Also, the conversion of hydroethidine (HE) into 2-hydroxyethidium (2-OH-E⁺) has been used to detect O₂^˙̄ in cultured cells in vitro and in animal models in vivo (20 –27). Other products formed from the HE probe, including diethidium and 2-chloroethidium (2-Cl-E⁺), have been proposed as specific marker products of one-electron oxidants and hypochlorous acid, respectively (28, 29).

Figure 1. — In contrast to commonly used redox probes DCFH, DHR123, Amplex Red, and NBT, spin traps, boronate-based probes, and HE incorporate atoms from the oxidants into the products formed.

Oxidation of boronate-based probes into phenolic products has been utilized for the detection of H₂O₂ (30 –32). An array of boronate probes, with similar chemical reactivities and a similar mechanism of response to H₂O₂ but with different modes of detection, has been reported (33 –37). Also, fluorogenic boronate probes targeted to various subcellular compartments have been described (31, 38 –40). Triphenylphosphonium (TPP⁺)-conjugated phenylboronic acid (called MitoB) was designed for MS-based detection of mitochondrial H₂O₂ (41 –43). Resistance of boronates to heme-catalyzed oxidation makes them good candidates for the detection of oxidants in the in vivo settings. Boronate-based probes are oxidized more than a thousand times faster by HOCl and nearly a million times faster by ONOO⁻ than by H₂O₂ (k_H2O2 ∼ 1 m⁻¹s⁻¹; k_HOCl ∼ 10⁴ m⁻¹s⁻¹; k_ONOO− ∼ 10⁶ m⁻¹s⁻¹), and the reaction typically involves a minor pathway, with the formation of ONOO⁻-specific product(s) (7, 44 –48). Recently, it has been reported that peroxymonocarbonate, the product of the reaction of H₂O₂ with CO₂, reacts with coumarin boronic acid nearly 50 times faster than H₂O₂ (k_HCO4− ∼ 10² m⁻¹s⁻¹) (10).

Although the identities of the oxidation, chlorination, and nitration products of boronate probes have been established in many cases, and the reaction mechanisms have been proposed, the origin of oxygen atoms in the oxidation and nitration products of boronate probes has not been experimentally determined. Understanding the mechanisms of formation of the oxidation products is required for their rigorous use as specific ROS markers in the in vitro and in vivo settings. Also, the potential for selective monitoring of the specific oxidizing species, through use of isotopically labeled oxidant and monitoring isotopic labeling of the specific products, remains to be explored.

Here, we report on the incorporation of an oxygen atom from the biologically relevant two-electron oxidants, including H₂O₂, HCO₄⁻, HOCl, and ONOO⁻ in the oxidation and nitration products of the mitochondria-targeted phenyl boronate probe (oMitoPhB(OH)₂) (Fig. 2). In addition, we demonstrate the involvement of oxygen atoms from superoxide in the formation of the hydroxylated product, 2-OH-E⁺, during oxidation of hydroethidine by O₂^˙̄ (Fig. 3), corroborating the proposed mechanism of the conversion of HE into 2-OH-E⁺.

Figure 2. — Conversion of oMitoPhB(OH)₂ boronate probe in the phenolic product, oMitoPhOH, in the presence of nucleophilic two-electron oxidants.

Figure 3. — Conversion of HE into 2-OH-E⁺ by O₂^˙̄.

Results

We have investigated the incorporation of oxygen atoms from different biologically relevant nucleophilic oxidants (Fig. 1) capable of oxidizing boronate probes into the products formed. We chose oMitoPhB(OH)₂ as a model boronate probe (Fig. 2) because its reactivity toward H₂O₂, HOCl, and ONOO⁻ has been studied previously in detail and the products characterized (49 –51). To demonstrate the formation of ¹⁸O-labeled superoxide, we have also tracked the incorporation of the ¹⁸O atom into the hydroxylation product of hydroethidine.

Hydrogen peroxide

Upon oxidation by H₂O₂, a conversion of oMitoPhB(OH)₂ into the phenolic product (oMitoPhOH) occurs (Fig. 4). H₂O₂ oxidizes the phenylboronate substrate into a phenoxyboronate intermediate that, upon hydrolysis, yields the phenolic product and boric acid. To determine whether the phenolic oxygen atom derives from H₂O₂ or water, we performed the oxidation of oMitoPhB(OH)₂ by H₂¹⁶O₂ in H₂¹⁸O and by H₂¹⁸O₂ in H₂¹⁶O (Fig. 5). The product detected in the presence of H₂¹⁶O₂ showed the molecular mass of oMitoPh¹⁶OH (m/z = 369); in the presence of H₂¹⁸O₂, the product had a molecular mass of 371 (Fig. 5, a and b), attributed to oMitoPh¹⁸OH. Liquid chromatography with tandem MS (LC-MS/MS) analyses indicated no formation of oMitoPh¹⁸OH during the oxidation of the probe by H₂¹⁶O₂ in H₂¹⁸O, whereas it was the predominant product in the presence of H₂¹⁸O₂ (Fig. 5c). We conclude that during oxidation of boronates by H₂O₂, the oxygen atom in the phenolic product derives exclusively from H₂O₂ and not from water.

Figure 4. — Oxidation of the oMitoPhB(OH)₂ probe by H₂O₂.

Figure 5. — **Incorporation of an oxygen atom into the phenolic product during the oxidation of oMitoPhB(OH)₂ by H₂O₂.** a, chemical structures of the products. b, online mass spectra of the products. c, LC-MS/MS traces of the phenolic products containing ¹⁶O (*left panel*) or ¹⁸O (*right panel*). LC-MS/MS analyses were performed after incubation (20 min) of oMitoPhB(OH)₂ (20 μm) alone (control), with H₂¹⁶O₂ (10 mm) in H₂¹⁸O (90%), or with H₂¹⁸O₂ (10 mm) in H₂¹⁶O.

Peroxymonocarbonate

In the presence of CO₂, H₂O₂ is in equilibrium with a more potent oxidant, peroxymonocarbonate (HOOCO₂⁻) (Fig. 6a) (9, 52, 53). Formation of this species has been implicated, for example, in the enhanced hyperoxidation of cellular peroxiredoxins and protein tyrosine phosphatase 1B–mediated signaling cascade observed in the presence of bicarbonate (12, 54 –56). Recently, it was shown that the rate of oxidation of the coumarin boronate probe in the presence of H₂O₂ is increased after the addition of bicarbonate (10). Therefore, we tested if the H₂O₂-derived HCO₄⁻ incorporates the oxygen atom into oMitoPhB(OH)₂ probe.

Figure 6. — **NaHCO₃-enhanced oxidation of oMitoPhB(OH)₂ by H₂O₂ and incorporation of an oxygen atom from HCO₄⁻ into the phenolic product.** a, chemical scheme of the formation of HCO₄⁻ and acid-base equilibria involved. b, dynamics of the formation of oMitoPhOH in the absence and presence of NaHCO₃. c, relative increase in the yield of oMitoPhOH after 1-h incubation of the probe with H₂¹⁶O₂ or H₂¹⁸O₂ in the absence and presence of NaHCO₃. d and e, LC-MS/MS traces of the phenolic products containing ¹⁶O (d) or ¹⁸O (e) atoms. LC-MS/MS analyses were performed after incubation (1 h) of oMitoPhB(OH)₂ (1 μm) alone (control), with H₂¹⁶O₂ (50 μm, d), or with H₂¹⁸O₂ (50 μm, e). All solutions contained 0.1 m phosphate buffer and 0.1 mm dtpa, and the pH of the solutions was adjusted to 7.0.

First, we confirmed that the experimental conditions we used would allow us to detect increased formation of the phenolic product during the reaction of the probe with H₂O₂ upon addition of NaHCO₃. In fact, with increased concentration of NaHCO₃, the rate of product formation increased, as determined by LC-MS–based monitoring of the accumulation of oMitoPhOH over the incubation time (Fig. 6, b and c). This effect was observed for both H₂¹⁶O₂ and H₂¹⁸O₂, when monitoring the ¹⁶O- or ¹⁸O-phenolic products, respectively (Fig. 6c). The relative increase in the yield of the phenolic product in the case of oMitoPh¹⁸OH was higher than in case of oMitoPh¹⁶OH, which we attribute to the presence of small amounts of oMitoPh¹⁶OH but not oMitoPh¹⁸OH in the probe stock solution. The representative LC-MS chromatograms for both H₂¹⁶O₂ and H₂¹⁸O₂, with increased concentrations of NaHCO₃ are shown in Fig. 6, d and e. Under those conditions, HC¹⁶O₄⁻ and HC¹⁸O₂¹⁶O₂⁻ were formed, respectively. Incorporation of an ¹⁸O atom into the phenolic product indicates the involvement of the peroxyl moiety of HCO₄⁻ in the oxidation reaction. Obtained data are consistent with the addition of the deprotonated form of peroxymonocarbonate (CO₄²⁻) to the boronate moiety, with elimination of the carbonate anion and incorporation of an oxygen atom from the peroxyl part of the oxidant.

Hypochlorite

Boronates are oxidized more than a thousand times faster by HOCl than by H₂O₂ at neutral pH (44). The product of the reaction is a phenol (or alcohol), which may undergo chlorination in the presence of excess HOCl (47, 51, 57). To determine the source of the oxygen atom during the conversion of oMitoPhB(OH)₂ into oMitoPhOH, we generated H¹⁶OCl and H¹⁸OCl in situ from myeloperoxidase (MPO)-catalyzed oxidation of chloride anions by H₂¹⁶O₂ and H₂¹⁸O₂, respectively (Fig. 7a). To confirm the formation of HOCl in the investigated system, we also performed similar incubations using H₂¹⁸O₂ in the presence of the HE probe, and monitored the chlorination product, 2-Cl-E⁺ (29).

Figure 7. — **Incorporation of an oxygen atom into the phenolic product during oxidation of oMitoPhB(OH)₂ by HOCl.** a, method generating HOCl. b and c, LC-MS/MS traces of the phenolic products containing ¹⁶O (b) or ¹⁸O (c). LC-MS/MS analyses were performed after incubation (15 min) of oMitoPhB(OH)₂ (50 μm) alone (control), with H₂¹⁶O₂ (0.1 mm, b), or with H₂¹⁸O₂ (0.1 mm, c) in the presence or absence of MPO (20 nm) and KCl (0.1 m). All solutions contained 0.1 m phosphate buffer, pH 7.4. d, scheme of the conversion of HE into 2-Cl-E⁺. e, online mass spectrum of 2-Cl-E⁺ detected from reaction of HE with bolus HOCl. f, confirmation of HOCl generation using 2-Cl-E⁺ marker product. All experimental conditions were the same as described above, but the oMitoPhB(OH)₂ probe was replaced by HE (50 μm).

Incubation of oMitoPhB(OH)₂ with H₂O₂, MPO, and potassium chloride (KCl) led to a significant increase in the production of the phenolic product, confirming that HOCl was the major species responsible for oxidation under the conditions used (Fig. 7b). The omission of KCl or MPO resulted in a significantly lower yield of the product. Also, addition of small amounts of dimethyl sulfoxide (DMSO), known to rapidly scavenge HOCl (47, 58), led to a significant attenuation of the formation of the phenolic product. Formation of HOCl was further confirmed by the detection of 2-Cl-E⁺ in analogous systems, using the HE probe instead of the boronate (Fig. 7, d–f). It previously was shown that HOCl and taurine chloramine are able convert HE into 2-Cl-E⁺ (29).

Replacement of H₂¹⁶O₂ with H₂¹⁸O₂ in the incubation mixture containing oMitoPhB(OH)₂, MPO, and KCl resulted in a switch from oMitoPh¹⁶OH to oMitoPh¹⁸OH (Fig. 7c). In the case of both isotopologs, the signal was maximal in a mixture containing H₂O₂, MPO, and KCl and decreased upon the addition of DMSO. We conclude that the oxygen atom in the product of oMitoPhB(OH)₂ oxidation by HOCl derives from the oxidant.

Peroxynitrite

Similar to H₂O₂, ONOO⁻ reacts with boronates to form a corresponding phenol as the major product. The rate constant of the reaction, however, is significantly higher (∼10⁶ m⁻¹s⁻¹ for ONOO⁻ and ∼1 m⁻¹s⁻¹ for H₂O₂), and the reaction typically involves a minor pathway, leading to ONOO⁻-specific minor products (59). The high rate constant provides an opportunity to estimate the absolute flux of ONOO⁻ in cultured cells (48, 60, 61). Formation of ONOO⁻-specific products provides an opportunity to selectively monitor ONOO⁻ formation in chemical and biological systems (14). We have previously applied this approach to demonstrate the formation of ONOO⁻ during the reaction of nitroxyl with oxygen (62). In the case of oxidation of oMitoPhB(OH)₂ by ONOO⁻, the minor products include cyclo-oMitoPh and oMitoPhNO₂ (Fig. 8), formed in 10 and 0.5% yields, respectively (51). Although the mechanism of the oxidation of boronates by ONOO⁻ has been extensively studied, both experimentally and using theoretical calculations (44, 45, 47, 49), the isotope-labeling studies have not been performed. We decided to test the proposed reaction mechanism by reacting oMitoPhB(OH)₂ with ¹⁸O-labeled ONOO⁻, produced in situ from co-generated fluxes of nitric oxide (^•NO) and ¹⁸O₂^˙̄ (7, 8). ^•NO flux was generated from the decomposition of spermine NONOate, whereas ¹⁸O₂^˙̄ flux was produced during xanthine oxidase (XO)-catalyzed oxidation of hypoxanthine (HX) in the presence of ¹⁸O₂. The identity of ¹⁸O₂^˙̄ has been confirmed by using the HE probe and monitoring the incorporation of ¹⁸O atoms into the superoxide-specific product 2-OH-E⁺ (see below). Co-generation of ^•N¹⁶O and ¹⁸O₂^˙̄ leads to the formation of ¹⁶ON¹⁸O¹⁸O⁻, providing an opportunity to track different oxygen atoms from ONOO⁻ during the conversion of oMitoPhB(OH)₂ into oMitoPhOH and oMitoPhNO₂. Incubation of oMitoPhB(OH)₂ with ¹⁶ON ¹⁸O¹⁸O⁻ led to the formation of the major phenolic product, which showed the mass (m/z = 371) to be two units higher than when using ¹⁶ON¹⁶O¹⁶O⁻ (m/z = 369) (Fig. 9, a and b). LC-MS/MS traces of the phenolic products showed no formation of the oMitoPh¹⁸OH in the presence of ¹⁶ON¹⁶O¹⁶O⁻, although it was a predominant product when ¹⁶ON¹⁸O¹⁸O⁻ was generated (Fig. 9c). In addition, changing the solvent to H₂¹⁸O failed to produce oMitoPh¹⁸OH (Fig. S1). These data indicate that the formation of the phenolic product during the reaction of boronates with ONOO⁻ is associated with the incorporation of the oxygen atom from the peroxyl part of the oxidant. Among the minor, ONOO⁻-specific, products formed, cyclo-oMitoPh did not change its mass when switching from ¹⁶ON¹⁶O¹⁶O⁻ to ¹⁶ON¹⁸O¹⁸O⁻ (Fig. S1) as no oxygen atom is incorporated. Cyclo-oMitoPh was formed in maximal yields when ^•NO and O₂^˙̄ were co-generated, and its peak intensity was similar for both ¹⁶ON¹⁶O¹⁶O⁻ and ¹⁶ON¹⁸O¹⁸O⁻ (Fig. S1).

Figure 8. — Oxidation of the oMitoPhB(OH)₂ probe by ONOO⁻.

Figure 9. — **Incorporation of an oxygen atom into the phenolic and nitrated products during oxidation of oMitoPhB(OH)₂ by ONOO⁻.** a, chemical structures of the products. b, online mass spectra of the detected products. c, LC-MS/MS traces of the phenolic and nitrated products containing ¹⁶O (*left panels*) or ¹⁸O (*right panels*). LC-MS/MS analyses were performed after incubation (30 min) of oMitoPhB(OH)₂ (20 μm) alone (control) or with *in situ*–generated ON¹⁶O¹⁶O⁻ or ON¹⁸O¹⁸O⁻. ON¹⁶O¹⁶O⁻ and ON¹⁸O¹⁸O⁻ were produced by cogenerated fluxes of ^•NO (0.2 μm/min) and ¹⁶O₂^˙̄ or ¹⁸O₂^˙̄ (0.2 μm/min), respectively.

Formation of the other minor product, oMitoPhNO₂, was associated with an increase in the mass of this product by two units (m/z = 400) in the presence of ¹⁶ON¹⁸O¹⁸O⁻ as compared with the product formed by ¹⁶ON¹⁶O¹⁶O⁻ (m/z = 398) (Fig. 9, a and b). This indicates that only one oxygen atom originated from the peroxyl (¹⁸O-labeled) part of ONOO⁻. Analyses of the LC-MS/MS traces indicate that oMitoPhN¹⁶O¹⁸O was formed only when ¹⁶ON¹⁸O¹⁸O⁻ was produced (Fig. 9c), and oMitoPhN¹⁶O₂ was the product of the reaction with ¹⁶ON¹⁶O¹⁶O⁻, even when the reaction was carried out in H₂¹⁸O (Fig. S1). The data on the oxidation of oMitoPhB(OH)₂ by ONOO⁻ indicate that the oxygen atoms introduced into the products originate from the oxidant and not from the solvent. These data are consistent with the occurrence of two reaction pathways, including heterolytic and homolytic cleavage of the peroxyl bond in the adduct of ONOO⁻ to the boronate probe (Fig. 10). The major pathway, involving a heterolytic cleavage, leads to the formation of the phenolic product, with the oxygen atom incorporated from the peroxyl moiety of the oxidant, similar to the reaction with other tested oxidants, H₂O₂, HCO₄⁻, and HOCl. The minor pathway, involving the homolytic cleavage of the peroxyl bond, leads to the formation of ^•NO₂ and a phenyl-type radical, which recombine within the solvent cage to form a nitrobenzene-type product (oMitoPhNO₂) (Fig. 10). The intramolecular addition of the phenyl radical to the phenyl ring of the TPP⁺ moiety yields the cyclic product (cyclo-oMitoPh) without incorporating any atom from the oxidant.

Figure 10. — Proposed mechanism of incorporation of oxygen atoms into the oxidation and nitration products of oMitoPhB(OH)₂ from H₂O₂, HCO₄⁻, HOCl, and ONOO⁻.

Superoxide

The production of ¹⁶ON¹⁸O¹⁸O⁻ for the study of the oxidation of boronates by ONOO⁻ involved co-generation of ^•NO and ¹⁸O₂^˙̄. To confirm the formation of ¹⁸O₂^˙̄ in the incubation mixture containing HX, XO, and ¹⁸O₂, we performed the incubation in the presence of the HE probe and monitored the incorporation of ¹⁸O atoms into the 2-OH-E⁺ product. HE is the most widely used probe for the detection of O₂^˙̄ in biological systems ranging from cultured cells to animals (21, 63). In the presence of O₂^˙̄, HE is oxidized to 2-OH-E⁺, a specific marker product for O₂^˙̄ (Fig. 3) (20 –24). Derivatives of HE for site-specific detection of O₂^˙̄ have been reported (64 –66). Those probes share the same oxidative chemistry with HE (65). A multistep mechanism of the conversion of HE to 2-OH-E⁺ has been proposed that involves the oxidation of HE to the HE radical cation (HE^•+), followed by the reaction of HE^•+ with O₂^˙̄ to form 2-OH-E⁺ (21, 67). This has been supported by pulse radiolysis data, showing the formation and rapid decay of HE^•+ in the presence of pulse-generated O₂^˙̄ (68) and an increase in the yield of 2-OH-E⁺ by the addition of peroxidase in the presence of a steady flux of O₂^˙̄ (67). Here, we provide direct proof of the production of ¹⁸O₂^˙̄ in the HX/XO/¹⁸O₂ system and the incorporation of the oxygen atom from O₂^˙̄ into the product during oxidation of HE to 2-OH-E⁺.

To follow the oxygen atoms, we incubated HE with ¹⁶O₂^˙̄ or ¹⁸O₂^˙̄, produced during enzymatic oxidation of HX by XO in the presence of ¹⁶O₂ or ¹⁸O₂, and monitored the formation of 2-¹⁶OH-E⁺ and 2-¹⁸OH-E⁺ (Fig. 11a). The mass spectra of the products showed m/z values of 330 and 332 when the probe was incubated with ¹⁶O₂^˙̄ or ¹⁸O₂^˙̄, respectively (Fig. 11b). The increase in the mass of the product from ¹⁸O₂^˙̄ is consistent with incorporation of ¹⁸O into the molecule. The LC-MS/MS traces (Fig. 11c) indicate significant formation of 2-¹⁶OH-E⁺ and negligible formation of 2-¹⁸OH-E⁺ in the presence of ¹⁶O₂^˙̄ (HX/XO/¹⁶O₂). In the presence of ¹⁸O₂^˙̄ (HX/XO/¹⁸O₂), only a small peak of 2-¹⁶OH-E⁺ and an intense peak because of 2-¹⁸OH-E⁺ were observed. Furthermore, incubation of HE with ¹⁶O₂^˙̄ in a solvent containing 90% of H₂¹⁸O led to the formation of 2-¹⁶OH-E⁺ but not 2-¹⁸OH-E⁺ (not shown). These data confirm the formation of ¹⁸O₂^˙̄ in the HX/XO/¹⁸O₂ system and indicate that during the oxidation and hydroxylation of HE, the oxygen atom in the product originates from O₂^˙̄, consistent with a mechanism involving the reaction of HE^•+ with O₂^˙̄ and forming the hydroperoxyl intermediate (Fig. 12).

Figure 11. — **Incorporation of an oxygen atom into the hydroxylated product during oxidation of HE in the presence of O₂^˙̄.** a, chemical structures of the products. b, online mass spectra of the products. c, LC-MS/MS traces of 2-OH-E⁺ containing ¹⁶O (*left panel*) or ¹⁸O (*right panel*). LC-MS/MS analyses were performed after incubation (30 min) of HE (20 μm) alone (control) or with ¹⁶O₂^˙̄ or ¹⁸O₂^˙̄ (0.2 μm O₂^˙̄/min, generated from HX/XO and ¹⁶O₂ or ¹⁸O₂, respectively).

Figure 12. — Proposed mechanism of incorporation of oxygen atom from O₂^˙̄ into 2-OH-E⁺ during oxidation of the HE probe.

Discussion

Isotope tracing is a powerful technique in the study of the mechanism of chemical and enzymatic reactions as well as cellular metabolism (69 –71). Isotopically labeled oxidants have been used to identify the spin adducts of O₂^˙̄ and other oxygen-centered radicals using an EPR spin trapping technique (72). EPR spin trapping, however, is only useful for the detection of radical species and has only limited applicability to detect intracellular ROS. Oxygen tracing in other probes used for cellular oxidants has not been reported.

In this study, we have investigated the origin of the oxygen atom in the products of the reaction of mitochondria-targeted boronate probe with four biologically relevant, two-electron oxidants: hydrogen peroxide, peroxymonocarbonate, hypochlorite, and peroxynitrite. The results support the previously proposed mechanisms of the probes' oxidation and formation of the specific products and provide a solid foundation for the use of those products for identification and tracking isotopically labeled oxidants.

New insights into the selective detection of peroxynitrite

Although initially assumed to be completely selective (specific) for H₂O₂, boronate-based probes also respond to other biologically relevant nucleophilic oxidants, including HCO₄⁻, HOCl, ONOO⁻, and amino acid hydroperoxides (44, 57, 73). The main oxidation product in case of all the listed oxidants is the corresponding phenol. In the presence of excess HOCl or ONOO⁻, the phenolic product may undergo chlorination or nitration, respectively, providing an opportunity to identify the oxidant by profiling the products formed (14, 44). As an example, in the presence of HOCl, the peroxy-caged luciferin probe is converted not only to luciferin but also to chloroluciferin (47). The reaction of boronate probes with ONOO⁻ is of special interest, as this reaction typically proceeds via two pathways of the decomposition of peroxynitrite adduct to the boronate: (i) major pathway (∼85–90%) involving heterolytic cleavage of the peroxyl bond, leading to the formation the phenolic product and (ii) minor pathway (10–15%), involving a homolytic cleavage of the peroxyl bond, with the formation of phenyl-type radical and ^•NO₂, which upon recombination form nitrobenzene-type product (Fig. 10) (45). We have proposed using that product as a specific marker for ONOO⁻ (50), and with such an approach, we demonstrated the formation of ONOO⁻ during the reaction of nitroxyl with oxygen (O₂) (62). In the case of the oMitoPhB(OH)₂ probe, the nitrated product, oMitoPhNO₂, accounts for only 0.5% of ONOO⁻ consumed. The other minor product, cyclo-oMitoPh, is formed at 10% yield, via a rapid intramolecular addition of the phenyl-type radical to one of the phenyl rings of the TPP⁺ moiety (Fig. 10) (51). Both minor products have been detected in macrophages stimulated to produce ONOO⁻ (51) and can be used as specific marker products for intracellular ONOO⁻.

Detection of ONOO⁻ in cells has remained a challenge, as most methods were based on the nitrative and/or oxidative properties of ONOO⁻-derived radicals (e.g. ^•OH, ^•NO₂, CO₃^˙̄) (74). However, the same radical species can be formed in biological systems in ONOO⁻-independent reactions. For example, although nitrated tyrosine residues are commonly used as an endogenous marker of ONOO⁻, the same product is formed by ^•NO₂ from the MPO-catalyzed oxidation of nitrite by H₂O₂. Dihydrorhodamine, a fluorogenic probe used for ONOO⁻ detection, cannot distinguish the two pathways of ^•NO₂ formation either. Boronate probes, including oMitoPhB(OH)₂ provide the first chemical tool to distinguish these two nitration pathways (51). Formation of the cyclic and nitrobenzene-type products from oMitoPhB(OH)₂ occurs in the presence of ONOO⁻ but not in the presence of MPO/H₂O₂/NO₂⁻ (51). This shows that monitoring the conversion of oMitoPhB(OH)₂ into cyclo-oMitoPh and oMitoPhNO₂ products can be used to selectively detect ONOO⁻ formed in cell-free and cellular systems. Although other boronate probes may not form the cyclic product during the reaction with ONOO⁻, in most cases they produce nitrobenzene-type minor products. These products may be used to confirm the identity of the oxidant detected. For example, a new boronate probe recently was developed to detect ONOO⁻ in β-amyloid aggregates (76). The minor product(s) formed during the reaction of the probe with ONOO⁻ should be characterized and high-performance LC (HPLC)– or LC-MS–based profiling should accompany fluorescence measurements, which report the yield of the phenolic product. This product is common for various nucleophilic oxidants, as exemplified here by H₂O₂, HCO₄⁻, HOCl, and ONOO⁻. Amino acid– and protein-based hydroperoxides also oxidize boronate probes to the phenolic products (73).

Oxidation of aromatic boronates involves initial formation of phenoxyboronic acid, followed by its hydrolysis into phenolic product (Fig. 4). The results obtained in this study demonstrate that during the oxidation of boronates by H₂O₂, HCO₄⁻, HOCl, or ONOO⁻, the oxygen atom in the phenolic product derives from those oxidants, not from water. In the case of HCO₄⁻ and ONOO⁻, the oxygen atoms in these oxidants are not equivalent, and the data obtained support the mechanism involving the nucleophilic addition of CO₄²⁻ or ONOO⁻ to the boron atom, via their peroxyl moieties, followed by elimination of a carbonate or nitrite anion, respectively (Fig. 10). Also, in the case of the formation of nitrobenzene-type product, the pattern of labeling of oMitoPhNO₂ during the reaction of oMitoPhB(OH)₂ with ONOO⁻ provides insight into the mechanism of the minor pathway of the reaction. Incorporation of only one oxygen-18 atom into the nitrated product from ¹⁶ON¹⁸O¹⁸O⁻ is consistent with the initial homolytic cleavage of the peroxyl bond in the adduct, formation of phenyl-type radical and ^•NO₂, and recombination of both radicals (Fig. 10).

2-Hydroxyethidium as a specific marker for O₂^˙̄

The HPLC or LC-MS-based analysis of 2-OH-E⁺ is regarded as a “gold standard” of the detection of O₂^˙̄ in biological systems (77). However, the utility of 2-OH-E⁺ as the marker of cellular O₂^˙̄ recently has been questioned, based on the lack of increase of its amount in HepG2 cells treated with H₂O₂ or rotenone (78). However, the ability of those treatments to induce O₂^˙̄ generation in the used cell model has not been shown. Numerous reports demonstrate the utility of HE, MitoSOX Red, and hydropropidine, when coupled with HPLC-based analyses, to detect O₂^˙̄ in different cellular models, as reviewed elsewhere (20, 21, 63). In those reports, 2-OH-E⁺, 2-OH-Mito-E⁺, and 2-OH-Pr²⁺ were used as specific marker products for O₂^˙̄. Hydroxylation of ethidium-based probes remains a method of choice for the detection of O₂^˙̄ in cell-free and cellular systems (17, 22, 64 –66, 79).

The presented results demonstrate that the specificity of 2-OH-E⁺ for O₂^˙̄ derives from incorporation of an oxygen atom from this species. Together with the pulse radiolysis data on the oxidation of HE by pulse-generated O₂^˙̄ (68), the 2:1 stoichiometry of the reaction (67), and the lack of incorporation of oxygen from water, observed in this study and during oxidation of HE by Fremy's salt (80), the obtained data are consistent with the mechanism shown in Fig. 12. Initial oxidation of HE by the protonated form of O₂^˙̄ (hydroperoxyl radical, HO₂^•) produces a radical cation of HE, which rapidly reacts with O₂^˙̄ to form a hydroperoxide, containing oxygen atoms from O₂^˙̄ (Fig. 12). This product must undergo rapid transformation in aqueous solutions to 2-OH-E⁺ as no intermediates have been detected by HPLC analyses.

Concluding remarks

In summary, we demonstrated that the oxygen atoms in the oxidation and nitration products of the boronate probe, oMitoPhB(OH)₂, originate from the corresponding oxidants, H₂O₂, HCO₄⁻, HOCl, or ONOO⁻. Also, in the case the conversion of the HE probe into 2-OH-E⁺, oxygen comes from O₂^˙̄. The presented data indicate that it is possible to track isotopically labeled oxidants by monitoring the incorporation of the isotopes into the oxidation/nitration products using the boronate and hydroethidine probes. Because no incorporation of the atoms from ROS/reactive nitrogen species occurs in most other commonly used probes, including DCFH, DHR123, Amplex Red, and NBT, they cannot be used for such purposes. The results obtained also corroborate the mechanisms of the conversion of HE into 2-OH-E⁺ by O₂^˙̄ and of oxidation and nitration of boronate-based probes, proposed previously based on product analyses, EPR experiments, and density functional theory calculation. We expect that oxygen-18 labeling studies using ¹⁸O₂, H₂¹⁸O, and H₂¹⁸O₂, may also be used in cell-free or cellular systems to reveal whether metal-induced hydrolysis and/or high-valent iron-oxo species may contribute to the hydroxylation of the probes (81). The reaction of the probes with ¹⁸O-labeled oxidants also can be used to prepare isotopically labeled standards of the oxidation products. Furthermore, H₂¹⁸O₂- or H¹⁸O₂C¹⁶O₂⁻-mediated oxidation of boronates represents a convenient route for the synthesis of ¹⁸O-labeled alcohols and phenols.

Experimental procedures

Materials

Ortho-MitoPhB(OH)₂ and its oxidation and nitration products were synthesized, as described previously (49 –51). The stock solution of oMitoPhB(OH)₂ (0.1 m) was prepared in DMSO and stored at −20 °C. The HE probe was obtained from Invitrogen (Carlsbad, CA). The stock solution of HE (20 mm) was prepared in deoxygenated DMSO under argon atmosphere and stored at −80 °C. The standards of the oxidation products were synthesized, as described previously (22, 82). For experiments involving HOCl, both probes were dissolved in ethanol (EtOH) to avoid the scavenging effect of DMSO on HOCl (47). Water-¹⁸O (97% oxygen-18), H₂¹⁸O₂ (90% oxygen-18), ¹⁸O₂ (97% oxygen-18), HX, XO, superoxide dismutase (SOD), and catalase were obtained from Sigma-Aldrich. MPO was from Calbiochem.

Determination of the flux of O₂^˙̄

O₂^˙̄ was generated from the XO-catalyzed oxidation of HX in a phosphate buffer solution (25 mm, pH = 7.4) containing 0.1 mm diethylenetriamine pentaacetic acid (dtpa). The solution was continuously purged with O₂. The flux of O₂^˙̄ was determined, as described previously (7, 83, 84), by performing the incubation in the presence of ferricytochrome c (50 μm) and monitoring the rate of its reduction following an increase in absorbance at 550 nm (Δϵ = 2.1 × 10⁴ m⁻¹ cm⁻¹) (85). Superoxide dismutase completely blocked the reduction of ferricytochrome c under the conditions used.

Determination of the flux of ^•NO

^•NO was generated from the thermal decomposition of spermine NONOate in a phosphate buffer (25 mm, pH = 7.4) containing 0.1 mm dtpa. The flux of ^•NO was determined, as described previously (7, 84), by monitoring the rate of decay of spermine NONOate following a decrease in absorbance at 252 nm (ϵ = 8 × 10³ m⁻¹cm⁻¹). The release of two molecules of ^•NO per one molecule of spermine NONOate consumed was assumed in the calculations (86).

Oxidation of oMitoPhB(OH)₂ by H₂O₂

To analyze the product of oxidation of oMitoPhB(OH)₂ by H₂O₂, oMitoPhB(OH)₂ (20 μm) was incubated at room temperature with H₂O₂ (10 mm) for 20 min in a phosphate buffer (25 mm, pH = 7.4) containing 0.1 mm dtpa. When performing the reaction in water-¹⁸O, the final concentration of H₂¹⁸O was 90% (by volume).

Oxidation of oMitoPhB(OH)₂ by HCO₄⁻

Oxidation oMitoPhB(OH)₂ by HCO₄⁻ was studied by incubation of the probe with H₂O₂ in phosphate-buffered (0.1 m) aqueous solution containing dtpa (0.1 mm) in the presence of NaHCO₃ (25 and 50 mm). To maximize the involvement of HCO₄⁻ in probe oxidation, the probe concentration was lowered to 1 μm, the H₂O₂ concentration was lowered to 50 μm, and the pH was adjusted to 7.0.

Oxidation of oMitoPhB(OH)₂ by HOCl

To study oxidation of oMitoPhB(OH)₂ by HOCl, the probe (50 μm, from a stock solution in EtOH) was incubated with H₂O₂ (0.1 mm), KCl (0.1 m), and MPO (20 nm) for 15 min at 25 °C in a phosphate-buffered (0.1 m) aqueous solution. Where indicated, DMSO was added (final concentration of 0.2% v/v) to scavenge HOCl.

Oxidation of oMitoPhB(OH)₂ by ONOO⁻

To react oMitoPhB(OH)₂ with ONOO⁻, oMitoPhB(OH)₂ (20 μm) was incubated with spermine NONOate (200 μm, generating 0.2 μm/min ^•NO), HX (200 μm), and XO (0.1 milliunit/milliliter, 0.2 μm O₂^˙̄/min) in an O₂-saturated phosphate buffer (25 mm, pH = 7.4) containing 0.1 mm dtpa and 5 kilounits/milliliter catalase. The deoxygenated stock solutions of all components were mixed under argon atmosphere in a hypoxic chamber (final reaction volume: 200 μl). The incubation was started immediately after mixing by passing oxygen gas (¹⁶O₂ or ¹⁸O₂) through the solution for 10 min, followed by 20 min further incubation at room temperature. Incubation in water-¹⁸O was performed in the presence of 90% (by volume) of H₂¹⁸O.

Chlorination of HE by HOCl

The reaction of HE with HOCl and the formation of 2-Cl-E⁺ was investigated in the presence of H₂O₂, MPO, and KCl under conditions identical to those described above for the oMitoPhB(OH)₂ probe but using HE (50 μm, from a stock solution in EtOH).

Oxidation of HE by O₂^˙̄

Conversion of HE into 2-OH-E⁺ was studied by incubation of HE (20 μm) with HX (200 μm) and XO (0.1 milliunit/milliliter, 0.2 μm O₂^˙̄/min) in an oxygen-saturated phosphate buffer (25 mm, pH = 7.4) containing 0.1 mm dtpa and 5 kilounits/milliliter catalase. To better control the type of O₂ isotopolog present in the solutions, samples were first deoxygenated to remove ¹⁶O₂ and then reoxygenated using ¹⁶O₂ or ¹⁸O₂. The deoxygenated stock solutions of all components were mixed under argon atmosphere in a hypoxic chamber (final reaction volume 200 μl). The incubation was started immediately after mixing by passing O₂ gas (¹⁶O₂ or ¹⁸O₂) through the solution for 10 min, followed by 20 min further incubation at room temperature. To stop the incubation, SOD was added (final concentration: 0.1 mg/ml) and the sample was taken for LC-MS/MS analysis. The addition of SOD at the beginning of incubation resulted in complete inhibition of 2-OH-E⁺ formation. When using water-¹⁸O as a solvent, the final concentration of H₂¹⁸O was 90% (by volume).

LC-MS/MS analysis of oMitoPhB(OH)₂ oxidation products

The oxidation products of oMitoPhB(OH)₂ were analyzed using a Shimadzu Nexera2 ultra-HPLC system equipped with UV-visible absorption and LC-MS8030 MS detectors (Columbia, MD). The presence of a positive charge (because of the presence of the TPP⁺ moiety) allows a sensitive detection by MS, as reported previously for the MitoB probe (41 –43). The incubation mixture was injected into a Raptor Biphenyl column (Restek, Bellefonte, PA; 100 mm × 2.1 mm, 2.7 μm) equilibrated with a mobile phase containing 80% water, 20% MeCN, and 0.1% formic acid. The products were eluted by increasing the content of MeCN (containing 0.1% formic acid) from 20% to 60% over 5.5 min. The mobile phase flow rate was 0.5 ml/min. Detection events included continuous scanning of the spectra of the eluate, as well as detection of the specific oxidation products in an MRM mode. MRM transitions were as follows: 397 > 135 for oMitoPhB(OH)₂, 369 > 107 for oMitoPh¹⁶OH, 371 > 263 for oMitoPh¹⁸OH, 398 > 262 for oMitoPhN¹⁶O₂, 400 > 262 for oMitoPhN¹⁶O¹⁸O, and 351 > 183 for cyclo-oMitoPh. The MRM transitions of other oxidation products have been reported elsewhere (50, 51, 79).

LC-MS/MS analysis of HE oxidation products

Detection of HE oxidation products, including 2-Cl-E⁺ and 2-OH-E⁺, was performed using a Shimadzu Nexera2 ultra-HPLC system equipped with UV-visible absorption and LC-MS8030 MS detectors. The reaction mixture was injected into a Raptor Biphenyl column (Restek, Bellefonte, PA; 100 mm × 2.1 mm, 2.7 μm) equilibrated with the mobile phase containing 90% water, 10% acetonitrile (MeCN), and 0.1% formic acid. The products were eluted by increasing the content of the organic mobile phase (MeCN, 0.1% formic acid) from 10% to 65% over 4.5 min at the flow rate of 0.4 ml/min. Detection events included continuous scanning of the spectra of the eluate, as well as detection of the specific oxidation products in an MRM mode. MRM transitions for 2-Cl-E⁺, 2-¹⁶OH-E⁺, and 2-¹⁸OH-E⁺ were 348 > 320, 330 > 300, and 332 > 302, respectively. The MRM transitions of other oxidation products were as reported previously (19, 29, 75, 79).

Data availability

All data presented and discussed are contained within the manuscript or in the supporting information.

Author contributions

N. R. and J. Z. data curation; N. R. and J. Z. formal analysis; N. R., R. R., B. K., and J. Z. funding acquisition; N. R. validation; N. R. and J. Z. investigation; N. R. and J. Z. methodology; N. R., R. R., B. K., and J. Z. writing-review and editing; R. R., B. K., and J. Z. conceptualization; R. R., B. K., and J. Z. supervision; R. R. and B. K. project administration; B. K. and J. Z. resources; J. Z. writing-original draft.

Supplementary Material

Supporting Information

supp_295_19_6665__index.html^{(944B, html)}

This work was supported by NCI, National Institutes of Health Grants U01CA178960 and R01CA208648 (to B. K.) and by Espacio Interdisciplinario 2015 and CSIC Grupos 2018, Universidad de la República, Uruguay (to R. R.). This work was also supported by American Cancer Society Institutional Research Grant IRG 16-183-31 and the MCW Cancer Center (to J. Z.); by Agencia Nacional de Investigación e Innovación (ANII) and Universidad de la República fellowships (to N. R.); and by Programa de Desarrollo de Ciencias Básicas (PEDECIBA, Uruguay) (to R. R. and N. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains Fig. S1.

The abbreviations used are:

ROS: reactive oxygen species
2-Cl-E⁺: 2-chloroethidium
DCFH: dichlorodihydrofluorescein
DHR123: dihydrorhodamine-123
dtpa: diethylenetriamine pentaacetic acid
HE: hydroethidine
HPLC: high-performance LC
HX: hypoxanthine
MeCN: acetonitrile
MPO: myeloperoxidase
MRM: multiple reaction monitoring
2-OH-E⁺: 2-hydroxyethidium
oMitoPhB(OH)₂: mitochondria-targeted phenyl boronate probe
SOD: superoxide dismutase
TPP⁺: triphenylphosphonium cationic moiety
XO: xanthine oxidase

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Supporting Information

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Data Availability Statement

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PERMALINK

Tracking isotopically labeled oxidants using boronate-based redox probes

Natalia Rios

Rafael Radi

Balaraman Kalyanaraman

Jacek Zielonka

Abstract

Introduction

Figure 1.

Figure 2.

Figure 3.

Results

Hydrogen peroxide

Figure 4.

Figure 5.

Peroxymonocarbonate

Figure 6.

Hypochlorite

Figure 7.

Peroxynitrite

Figure 8.

Figure 9.

Figure 10.

Superoxide

Figure 11.

Figure 12.

Discussion

New insights into the selective detection of peroxynitrite

2-Hydroxyethidium as a specific marker for O2˙̄

Concluding remarks

Experimental procedures

Materials

Determination of the flux of O2˙̄

Determination of the flux of •NO

Oxidation of oMitoPhB(OH)2 by H2O2

Oxidation of oMitoPhB(OH)2 by HCO4−

Oxidation of oMitoPhB(OH)2 by HOCl

Oxidation of oMitoPhB(OH)2 by ONOO−

Chlorination of HE by HOCl

Oxidation of HE by O2˙̄

LC-MS/MS analysis of oMitoPhB(OH)2 oxidation products

LC-MS/MS analysis of HE oxidation products

Data availability

Author contributions

Supplementary Material

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

2-Hydroxyethidium as a specific marker for O₂^˙̄

Determination of the flux of O₂^˙̄

Determination of the flux of ^•NO

Oxidation of oMitoPhB(OH)₂ by H₂O₂

Oxidation of oMitoPhB(OH)₂ by HCO₄⁻

Oxidation of oMitoPhB(OH)₂ by HOCl

Oxidation of oMitoPhB(OH)₂ by ONOO⁻

Oxidation of HE by O₂^˙̄

LC-MS/MS analysis of oMitoPhB(OH)₂ oxidation products