Cyclic Hydroxylamine Research Articles

Light increases resistance of thylakoid membranes to thermal inactivation.

In the region of slightly acidic pH (рН 5.7), the manganese cluster in oxygen-evolving complex of photosystem II (PSII) is more resistant to exogenous reductants. The effect of such pH on the heat inactivation efficiency of the electron transport chain (O2 evolution and 2,6-dichlorophenolindophenol reduction) in PSII membranes and thylakoid membranes was investigated. Under thylakoid membranes illumination accompanied by lumen acidification, their resistance to heat inactivation increases. In the presence of protonophores, the rate of heat inactivation increases, which seems to be associated not with the protonophore mechanism, but with structural and/or functional changes in membranes. In PSII membrane preparations, the efficiency of the oxygen evolution inhibition at pH 5.7 is also lower than at pH 6.5. The role of reactive oxygen species in thermal inactivation of photosynthetic membranes was investigated using a lipophilic cyclic hydroxylamine ESR spin probe.

Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance.

Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease. Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology. The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×1014 CP• and (9.9 ± 1.0) ×1014 DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells. The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

Modular Photochemical Flow Synthesis of Structurally Diverse Benzyne and Triazine Precursors

AbstractBenzyne and related arynes are classical high‐energy species with a rich history of widespread applications in synthesis. However, despite several synthetic routes being available to generate arynes and their precursors, none represents an ideal entry to benzyne chemistry in view of safety, scalability, and sustainability. Here we report a new photochemical flow process allowing for the generation of benzyne precursors in high yields, and throughput, with easy isolation of multigram quantities of products. This process leverages a catalyst‐free photochemical rearrangement via a photoexcited nitro arene which involves a cyclic hydroxylamine intermediate that has been fully characterized. The resulting precursors were converted to benzynes via a second photochemical flow process generating heterocyclic targets upon trapping with azide and sydnone partners. Remarkably, when reacting the benzyne precursors with secondary amines, a wide range of aryl triazines is obtained in good yields via a third photo‐flow transformation. This represents a modular approach to synthesize these species, that avoids the use of potentially explosive diazonium salts. Ultimately, three photochemical flow processes using a single high‐power LED light source (365 nm, adjustable in‐put power) are presented with manifest benefits compared to batch processing. Moreover, the functionalization of a pendent carboxyl group to form sets of biologically relevant aryl triazine‐based amides highlights further applications of these unique and industrially relevant triazine entities.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10mg/kg). At 24h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Measurement of Mitochondrial (Dys)Function in Cellular Systems Using Electron Paramagnetic Resonance (EPR): Oxygen Consumption Rate and Superoxide Production.

The oxygen consumption rate (OCR) and superoxide production are crucial when assessing mitochondrial function and/or dysfunction. EPR spectroscopy allows the measurement of both components either independently or simultaneously in a same cellular or mitochondrial preparation. OCR determination using EPR oximetry is based on the change in EPR linewidth of a paramagnetic oxygen sensing probe (a perdeuterated nitroxide) in the presence of oxygen consuming cells in a closed system. Superoxide production can be monitored by the oxidation of cyclic hydroxylamines into nitroxides. The contribution of superoxide to the nitroxide formation is deduced from experiments in the presence and in the absence of SOD and PEG-SOD as appropriate controls.

Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO−) and superoxide (HO2•/O2•−). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO−. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.

Comparative EPR Study on the Scavenging Effect of Methotrexate with the Isomers of Its Photoswitchable Derivative.

The scavenging effect of the antimetabolite dihydrofolate reductase inhibitor methotrexate (MTX) and the isomers of its photoswitchable derivate, cis- and trans-phototrexate (PHX), have been compared by ESR spectroscopy, with the application of a cyclic hydroxylamine spin probe. The results showed the most pronounced scavenging effect in the presence of trans-phototrexate (trans-PHX). At a low concentration (100 µM) cis-PHX also showed a greater scavenging effect than the parent molecule MTX. Direct antioxidant properties of the investigated molecules were measured by ABTS scavenging assay, which showed no significant difference between trans-PHX and cis-PHX, but both of the isomers of PHX showed a higher antioxidant capacity than MTX. These findings imply that trans-PHX may have more pronounced anti-inflammatory and tissue-protective effects than MTX, despite the lack of its cytotoxic, antineoplastic effect.

A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production

In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.

Hydroxylamines inhibit tyrosine oxidation and nitration: The role of their respective nitroxide radicals

In vivo, nitroxide antioxidants distribute within minutes throughout all tissues, but are reduced to their respective hydroxylamines due to the cellular reducing environment, which apparently limits their application. To distinguish their antioxidative activity from that of their respective nitroxides, the kinetics and mechanism of their inhibitory effect on the enzymatic oxidation and nitration of tyrosine have been studied. The inhibitory effect of the hydroxylamines on the oxidation and nitration of tyrosine induced by HRP/H2O2 and HRP/H2O2/nitrite was investigated by following the kinetics of the formation of their respective nitroxides, H2O2 decomposition, release of O2 and accumulation of tyrosine oxidation and nitration products. The distinction between the antioxidative activities of nitroxides and of their respective hydroxylamines is hindered due to oxidation of hydroxylamines to nitroxides, which catalytically inhibit tyrosine oxidation and nitration. The results demonstrate that (i) hydroxylamines inhibit tyrosine oxidation and nitration and their inhibitory effect increases as the reduction potential of their respective nitroxides decreases; (ii) the 6-membered ring hydroxylamines are more effective antioxidants than the 5-membered hydroxylamine derived from 3-carbamoyl proxyl and (iii) the 6-membered ring hydroxylamines are as effective antioxidants as their respective nitroxides, whereas the 3-carbamoyl proxyl is even a weaker antioxidant than its respective hydroxylamine. In general, cyclic hydroxylamines are more effective antioxidants than common antioxidants such as ascorbic and uric acids, which are depleted giving rise to secondary radicals that, might be toxic. In the case of hydroxylamines, the secondary radicals are their respective nitroxides, which are efficient catalytic antioxidants.

An EPR Study Using Cyclic Hydroxylamines To Assess The Level of Mitochondrial ROS in Superinvasive Cancer Cells.

It has been proposed that a mitochondrial switch involving a high mitochondrial superoxide production is associated with cancer metastasis. We here report an EPR analysis of ROS production using cyclic hydroxylamines in superinvasive SiHa-F3 compared with less invasive SiHa wild-type human cervix cancer cells. Using the CMH probe, no significant difference was observed in the overall level of ROS between SiHa and SiHa-F3 cells. However, using mitochondria-targeted cyclic hydroxylamine probe mitoTEMPO-H, we detected a significantly higher mitochondrial ROS content in SiHa-F3 compared with the wild-type SiHa cells. To investigate the nature of mitochondrial ROS, we overexpressed superoxide dismutase 2, a SOD isoform exclusively localized in mitochondria, in SiHa-F3 superinvasive cells. A significantly lower signal was detected in SiHa-F3 cells overexpressing SOD2 compared with SiHa-F3. Despite some limitations discussed in the paper, our EPR results suggest that mitochondrial ROS (at least partly superoxide) are produced to a larger extent in superinvasive cancer cells compared with less invasive wild-type cancer cells.

Aspartic Acid Forming α-Ketoacid-Hydroxylamine (KAHA) Ligations with (S)-4,4-Difluoro-5-oxaproline.

The α-ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. Currently, the most applied hydroxylamine is the 5-membered cyclic hydroxylamine (S)-5-oxaproline, which forms a homoserine ester as the primary ligation product. In order to access native aspartic acid residues at the ligation site, we synthesized a 4,4-difluoro version of this monomer. Upon KAHA ligation, the resulting difluoro alcohol hydrolyzes to an aspartic acid residue with little or no formation of aspartamide. We applied this monomer for the synthesis of the hormone peptides glucagon and an insulin variant, and as well for segment ligation of the peptides UbcH5a and SUMO3.

Synthesis and characterization of a 5-membered ring cyclic hydroxylamine coupled to triphenylphosphonium to detect mitochondrial superoxide by EPR spectrometry

As mitochondrial superoxide is becoming an attractive metabolic and pharmacological target, there is an important need for developing analytical tools able to detect superoxide with high sensitivity and specificity. Among EPR-based methods, it has been recently reported that cyclic hydroxylamines offer a high sensitivity to measure superoxide production. Here, we report the synthesis and evaluation of mitoCPH, in which a 5-membered ring hydroxylamine was coupled to a triphenylphosphonium moiety to allow mitochondrial accumulation. MitoCPH efficiently reacted with superoxide with a bimolecular rate constant of 1.5 × 104 M−1 s−1. We assessed the ability of this compound to detect superoxide in PBS buffer, lysates, and in paraquat-stimulated cells. We compared its performance with CMH, a nontargeted 5-membered ring hydroxylamine, and mitoTEMPO-H, a classically used 6-membered ring hydroxylamine targeted to mitochondria. MitoCPH presented a higher sensitivity for superoxide anion detection than commonly used mitoTEMPO-H, both in buffer and in cell lysates. While we have described the ability of mitoCPH to detect superoxide in different cellular media, we cannot exclude other potential contributors to the nitroxide production from this probe. Therefore, mitoCPH should be considered as a mitochondria-targeted probe and its use as selective superoxide probe should be used cautiously.

A Threonine-Forming Oxazetidine Amino Acid for the Chemical Synthesis of Proteins through KAHA Ligation.

α-Ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments through the chemoselective formation of an amide bond. Currently, the most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. In order to directly form amide-linked threonine residues at the ligation site, we prepared a new 4-membered cyclic hydroxylamine building block. This monomer was applied to the synthesis of wild-type ubiquitin-conjugating enzyme UbcH5a (146 residues) and Titin protein domain TI I27 (89 residues). Both the resulting UbcH5a and the variant with two homoserine residues showed identical activity to a recombinant variant in a ubiquitination assay.

Traceless Electrophilic Amination for the Synthesis of Unprotected Cyclic β-Amino Acids.

Electrophilic aminations involve an umpolung of a nitrogen atom, providing an alternate, distinctive synthetic strategy. The recent advent of various designed O-substituted hydroxylamines has significantly advanced this research field. An underappreciated issue is atom economy of the transformations: The necessary activating group on the oxygen atom is left in coproduced waste. Herein, we describe Rh-catalyzed electrophilic amination of substituted isoxazolidin-5-ones for the synthesis of unprotected, cyclic β-amino acids featuring either benzo-fused or spirocyclic scaffolds. Using the cyclic hydroxylamines allows for retaining both nitrogen and oxygen functionalities in the product. The traceless, redox neutral process proceeds on a gram scale with as little as 0.1 mol % catalyst loading. In contrast to related electrophilic aminations in the literature, a series of mechanistic experiments suggests a unique pathway involving spirocyclization, followed by the skeletal rearrangement. The insights provided herein shed light on a nuanced reactivity of the active species, Rh-nitrenoid generated from the activated hydroxylamine, and extend the knowledge on electrophilic aromatic substitutions.

Increased cysteine sulfonation of Complex III with destruction of heme c mediates cardiac reperfusion injury via impairing catalytic function and enhancing FADH2‐linked superoxide production

One consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which can cause mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide, which can potentially induce protein cysteine sulfonation (PrSO3H) in mitochondria. Herein we employed UV/VIS spectroscopy and LC-MS/MS analysis to study the mechanism of I/R-mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p<0.001). Visible spectroscopic and immunoblotting analysis indicated that I/R mediated destruction of heme c (c-type heme) without affecting protein level of cytochrome c (cyt c) in the mitochondria. I/R thus mediates dysfunction of cyt c via heme destruction. However, no significant impairment of complex III activity and heme c destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min ischemia (no reperfusion) in vivo despite a decreased state 3 respiration. LC-MS/MS analysis showed enhanced formation of PrSO3H, which was consistent with mitochondrial redox state following I/R (EPR measurement of cyclic hydroxylamine oxidation). Mitochondria from I/R regions had intensely increased oxidative modification with sulfonation of cysteine (at C236) of 2Fe-2S cluster of Rieske iron-sulfur protein (uqcrfs1), which would impair the main electron transfer pathway controlled by the Q-cycle mechanism of complex III. LC-MS/MS analysis also indicated that I/R intensely increased complex III PrSO3H at the subunits of hinge protein, cytochrome c1, core 1 and core 2. Carbamidomethylated C122/C125 of the cytochrome c1 via alkylating complex III was exclusively detected in the post-ischemic mitochondria by LC-MS/MS, indicating I/R mediates cleavage of thioether bonds between heme c1 and apocytochrome c1. FADH2-linked ·O2− generation by mitochondrial SCR (supercomplex hosting complex II and complex III) was induced by succinate and evaluated by EPR spin trapping with DEPMPO. FADH2-linked ·O2− generation by normal mitochondrial SCR was minimized in the presence of a functional cyt c. Whereas, FADH2-linked ·O2− generation by the post-ischemic mitochondrial SCR was intensely increased by 123% in the presence of heme-depleted cyt c, and further enhanced by 227% under the conditions of increasing pO2 in vitro (from 0.23 mM O2 to 0.8 mM O2), suggesting that physiologic conditions of hyperoxygenation during reperfusion with heme-depleted cyt c mediated overproduction of FADH2-linked ·O2− by the post-ischemic mitochondria. In conclusion, reperfusion-induced PrSO3H with heme depletion of cyt c controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction via overproducing ·O2− in the post-ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may necessitate lessening of PrSO3H and preservation of functional integrity of heme groups in mitochondria. Support or Funding Information NIH RO1HL083237 Enhancement of FADH2-linked superoxide generation by post-ischemic mitochondrial SCR (C vs B) in the presence of post-ischemic cyt c (D vs B) and hyperoxygenation (E vs D). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and Evaluation of Cyclic Acetals of Serine Hydroxylamine for Amide-Forming KAHA Ligations

The α-ketoacid–hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. The most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. While very effective, monomers that give canonical amino acid residues are in high demand. In order to preserve the stability and reactivity of cyclic hydroxylamines, but form a canonical amino acid residue upon ligation, we sought to prepare cyclic derivatives of serine hydroxylamine. An evaluation of several cyclization strategies led to cyclobutanone ketals as the leading structures. The preparation, stability, and amide-forming ligation of these serine-derived ketals are described.

Comparison of different methods for measuring the superoxide radical by EPR spectroscopy in buffer, cell lysates and cells

As superoxide anion is of keen interest in biomedical research, it is highly desirable to have a technique allowing its detection sensitively and specifically in biological media. If electron paramagnetic resonance (EPR) techniques and probes have been individually described in the literature, there is actually no comparison of these techniques in the same conditions that may help guiding researchers for selecting the most appropriate approach. The aim of the present study was to compare different EPR strategies in terms of sensitivity and specificity to detect superoxide (vs. hydroxyl radical). Three main classes of EPR probes were used, including paramagnetic superoxide scavengers (such as nitroxides TEMPOL and mitoTEMPO as well as trityl CT-03), a spin trap (DIPPMPO), and diamagnetic superoxide scavengers (such as cyclic hydroxylamines CMH and mitoTEMPO-H). We analysed the reactivity of the different probes in the presence of a constant production of superoxide or hydroxyl radical in buffers and in cell lysates. We also assessed the performances of the different probes to detect superoxide produced by RAW264.7 macrophages stimulated by phorbol 12-myristate 13-acetate. In our conditions and models, we found that nitroxides were not specific for superoxide. CT-03 was specific, but the sensitivity of detection was low. Comparatively, we found that nitrone DIPPMPO and cyclic hydroxylamine CMH were good candidates to sensitively and specifically detect superoxide in complex biological media, CMH offering the best sensitivity.

Increased Protein Cysteine Sulfonation with Heme Destruction of Mitochondrial Complex III Mediates Cardiac Reperfusion Injury

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage, which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide. The cascading reactive oxygen species can propagate and potentially induce protein cysteine sulfonation (PrCSO3H) of mitochondrial proteins. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of IR‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30 min of ischemia (coronary ligation) and 24 h of reperfusion in vivo. In the IR region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p&lt;0.001). Visible spectroscopic analysis indicated that IR mediated destruction of heme b and hemes (c+c1) in the mitochondria, thus supporting IR‐induced impairment of the enzymatic activity of complex III. However, no significant impairment of complex III activity and heme destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min coronary ligation in vivo despite a decreased state 3 respiration detected in the ischemic mitochondria. LC‐MS/MS analysis showed formation of PrCSO3H, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondria from IR regions had intensely increased oxidative modification with S‐sulfonation level on the cysteine ligand (at C236) of 2Fe‐2S cluster of Rieske iron‐sulfur protein (uqcrfs1), thus impairing the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that IR‐mediated increased PrCSO3H of hinge protein at C65 (subunit 8, uqcrh) and cytochrome c1 subunit at C140 and C220, which weakened the interaction of hinge protein with cytochrome c1 and proper complex formation between cytochrome c1 and cytochrome c. LC‐MS/MS analysis further identified 4‐HNE (4‐hydroxylnonenal) adduct of cytochrome c1 at C140, suggesting involvement of IR‐induced lipid peroxidation in heme destruction and PrCSO3H of complex III. LC‐MS/MS analysis also showed that IR‐enhancing PrCSO3H of core 1 subunit (uqcrc1 at C69, C268, C380, C410, C445, C453) and core 2 subunit (uqcrc2 at C191), which may affect the binding of subunit 9 inhibitory peptide to core 1 and core 2 subunits and reactivate the MPP (matrix processing peptidase) activity of the core 1/core 2 subunits of mammalian complex III. LC‐MS/MS analysis of ischemic mitochondria has indicated a modest reduction from the basal level of complex III PrCSO3H detected in the mitochondria of sham control heart, suggesting that physiologic conditions of hypoxia during ischemia suppressed PrCSO3H, and physiologic conditions of hyperoxygenation during reperfusion mediated enhancement of complex III PrCSO3H. In conclusion, reperfusion‐mediated heme destruction with increased PrCSO3H controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction in the post‐ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may related to the preservation of functional integrity of heme groups and protein structure within mitochondrial complex III.Support or Funding InformationNHLBI/NIH, HL083237This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electron Paramagnetic Resonance Measurements of Reactive Oxygen Species by Cyclic Hydroxylamine Spin Probes.

Oxidative stress contributes to numerous pathophysiological conditions such as development of cancer, neurodegenerative, and cardiovascular diseases. A variety of measurements of oxidative stress markers in biological systems have been developed; however, many of these methods are not specific, can produce artifacts, and do not directly detect the free radicals and reactive oxygen species (ROS) that cause oxidative stress. Electron paramagnetic resonance (EPR) is a unique tool that allows direct measurements of free radical species. Cyclic hydroxylamines are useful and convenient molecular probes that readily react with ROS to produce stable nitroxide radicals, which can be quantitatively measured by EPR. In this work, we critically review recent applications of various cyclic hydroxylamine spin probes in biology to study oxidative stress, their advantages, and the shortcomings. Recent Advances: In the past decade, a number of new cyclic hydroxylamine spin probes have been developed and their successful application for ROS measurement using EPR has been published. These new state-of-the-art methods provide improved selectivity and sensitivity for in vitro and in vivo studies. Although cyclic hydroxylamine spin probes EPR application has been previously described, there has been lack of translation of these new methods into biomedical research, limiting their widespread use. This work summarizes "best practice" in applications of cyclic hydroxylamine spin probes to assist with EPR studies of oxidative stress. Additional studies to advance hydroxylamine spin probes from the "basic science" to biomedical applications are needed and could lead to better understanding of pathological conditions associated with oxidative stress. Antioxid. Redox Signal. 28, 1433-1443.

Diruthenium Diacetate Catalysed Aerobic Oxidation of Hydroxylamines and Improved Chemoselectivity by Immobilisation to Lysozyme

AbstractA new green method for the preparation of nitrones through the aerobic oxidation of the corresponding N,N‐disubstituted hydroxylamines has been developed upon exploring the catalytic activity of a diruthenium catalyst, that is, [Ru2(OAc)4Cl]), in aqueous or alcoholic solution under mild reaction conditions (0.1 to 1 mol % catalyst, air, 50 °C) and reasonable reaction times. Notably, the catalytic activity of the dimetallic centre is retained after its binding to the small protein lysozyme. Interestingly, this new artificial metalloenzyme conferred complete chemoselectivity to the oxidation of cyclic hydroxylamines, in contrast to the diruthenium catalyst.