Endogenous plant nitroxyl, a new component of nitric oxide biology.

Nitroxyl (HNO), a one-electron reduced and protonated congener of nitric oxide (•NO), was recently discovered in Arabidopsis thaliana. Due to its distinct chemical properties, we believe HNO must be further studied to determine how many physiological processes it impacts.

Azobenzene-based colorimetric and fluorometric chemosensor for nitroxyl releasing.

The precise release and characterization of nitroxyl (HNO) gas signaling molecule remain a challenge due to its short lifetime to date. To solve this issue, an azobenzene-based HNO donor (Azo-D1) was proposed as a colorimetric and fluorometric chemosensor for HNO releasing, to release both HNO and an azobenzene fluorescent reporter together. Specifically, the Azo-D1 has an HNO release half-life of ∼68 min under physiological conditions. The characteristic color change from the original orange to the yellow color indicated the decomposition of the donor molecule. In addition, the stoichiometry release of HNO was qualitatively and quantitatively verified through the classical phosphine compound trap. As compared with the donor molecule by itself, the decomposed product demonstrates a maximum fluorescence emission at 424 nm, where the increase of fluorescence intensity by 6.8 times can be applied to infer the real-time concentration of HNO. Moreover, cellular imaging can also be achieved using this Azo-D1 HNO donor through photoexcitation at 405 and 488 nm, where the real-time monitoring of HNO release was achieved without consuming the HNO source. Finally, the Azo-D1 HNO donor would open a new platform in the exploration of the biochemistry and the biology of HNO.

Fluorescence Quenching Effect of a Highly Active Nitroxyl Radical on 7-amino-4-methylcoumarin and Glutathione Sensing.

Nitroxyl radical compounds, such as 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), are stable radical compounds with a variety of unique characteristics, including fluorescence quenching. In this study, we investigated the fluorescence quenching effect of nortropine N-oxyl (NNO), which is a highly active nitroxyl radical that is more active than TEMPO in oxidation catalysis. The fluorescence intensity of 7-amino-4-methylcoumarin (AMC) was quenched by NNO and TEMPO to 5% and 95% of the initial fluorescence intensity, respectively, indicating highly efficient quenching by NNO. In addition, we used this reaction to measure glutathione concentration. The quenching effect of NNO was abrogated by the chemical reaction with glutathione, resulting in restoration of AMC fluorescence. This response was observed at glutathione concentrations from 10 µM to 1 mM, and good calibration curves were obtained from 10 to 250 µM.

A Near-Infrared Fluorescent Probe for the Rapid Detection of Nitroxyl in Living Cells.

Nitroxyl (HNO) plays an important role in various physiological activities. It has the potential to be used as a treatment for certain diseases such as alcohol poisoning, acute hypertension, and atherosclerosis. However, traditional methods for detecting HNO are challenging due to its rapid polymerization and elimination into N2O. Therefore, it is crucial to establish direct and effective HNO detection methods to comprehend these physiological processes better. In this study, a new near-infrared fluorescent probe called HXM-P based on the intramolecular charge transfer (ICT) mechanism was designed and synthesized. This probe employs 2-((6-hydroxy-2,3dihydro-1 H-xanthen-4-yl)methylene)malononitrile as a fluorophore and 2-(diphenylphosphine) benzoate as a recognition group. The results showed that probe HXM-P can detect HNO with high sensitivity (1.07 × 10- 8 M). A good linear correlation was observed between the fluorescence intensities at 640 nm and the concentrations of HNO in the range of 0-80 µM (R2 = 0.997). Moreover, probe HXM-P exhibited a rapid response rate (within 15 s) toward HNO, and the fluorescent intensity reached a plateau within 5 min, making it easier to track the highly reactive and short-lived HNO in living systems. Additionally, HXM-P was successfully employed for imaging HNO in HepG2 cells.

Electrochemical Characterization of a Novel Organoelectrocatalyst, 7-Azabicyclo[2.2.1]heptan-7-ol (ABHOL), and Its Application to Electrochemical Sensors.

Electrochemical enzyme sensors are suitable for simple monitoring methods, for example, as glucose sensors for diabetic patients; however, they have several disadvantages arising from the properties of the enzyme. Therefore, non-enzymatic electrochemical sensors using functional molecules are being developed. In this paper, we report the electrochemical characterization of a new hydroxylamine compound, 7-azabicyclo[2.2.1]heptan-7-ol (ABHOL), and its application to glucose sensing. Although the cyclic voltammogram for the first cycle was unstable, it was reproducible after the second cycle, enabling electrochemical analysis of ethanol and glucose. In the first cycle, ABHOL caused complex reactions, including electrochemical oxidation and comproportionation with the generated oxoammonium ions. The electrochemical probe performance of ABHOL was more efficient than the typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and had similar efficiency to 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), which is activated by the bicyclic structure. The results demonstrated the advantages of ABHOL, which can be synthesized from inexpensive materials via simple methods.

A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Synthesis and nitroxyl (HNO) donating properties of benzoxadiazole-based Piloty's acids.

Developing functional nitroxyl (HNO) donors play a significant role in the further exploration of endogenous HNO in biochemistry and pharmacology. In this work, two novel Piloty's acids (SBD-D1 and SBD-D2) were proposed by incorporating benzoxadiazole-based fluorophores, in order to achieve the dual-function of releasing both HNO and a fluorophore in situ. Under physiological conditions, both SBD-D1 and SBD-D2 efficiently donated HNO (t1/2 = 10.96 and 8.18 min, respectively). The stoichiometric generation of HNO was determined by both Vitamin B12 and phosphine compound trap. Interestingly, due to the different substitution groups on the aromatic ring, SBD-D1 with the chlorine showed no fluorescence emission, but SBD-D2 was strongly fluorescent due to the presence of the dimethylamine group. Specifically, the fluorescent signal would decrease during the release process of HNO. Moreover, theoretical calculations were performed to understand the emission difference. A strong radiation derived from benzoxadiazole with dimethylamine group due to the large transition dipole moment (∼4.3 Debye), while the presence of intramolecular charge transfer process in the donor with chlorine group caused a small transition dipole moment (<0.1 Debye). Finally, these studies would contribute to the future design and application of novel functional HNO donors for the exploration of HNO biochemistry and pharmacology.

Photoredox Cascade Catalyst for Efficient Hydrogen Production with Biomass Photoreforming.

Biomass photoreforming is a promising method to provide both a clean energy resource in the form of hydrogen (H2 ) and valuable chemicals as the results of water reduction and biomass oxidation. To overcome the poor contact between heterogeneous photocatalysts and biomass substrates, we fabricated a new photoredox cascade catalyst by combining a homogeneous catalyst, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), and a heterogeneous dual-dye sensitized photocatalyst (DDSP) composed of two Ru(II)-polypyridine photosensitizers (RuP6 and RuCP6 ) and Pt-loaded TiO2 nanoparticles. During blue-light irradiation (λ=460±15 nm; 80 mW), the DDSP photocatalytically reduced aqueous protons to form H2 and simultaneously oxidized TEMPO• radicals to generate catalytically active TEMPO+ . It oxidized biomass substrates (water-soluble glycerol and insoluble cellulose) to regenerate TEMPO• . In the presence of N-methyl imidazole as a proton transfer mediator, the photocatalytic H2 production activities for glycerol and cellulose reforming reached 2670 and 1590 µmol H2 (gTiO2 )-1  h-1 , respectively, which were comparable to those of state-of-the-art heterogeneous photocatalysts.

Uncovering Nitrosyl Reactivity at N-Heterocyclic Carbene Center.

N-heterocyclic carbenes (NHCs) have garnered much attention due to their unique properties, such as strong σ-donating and π-accepting abilities, as well as their transition-metal-like reactivity toward small molecules. In 2015, we discovered that NHCs can react with nitric oxide (NO) gas to form radical adducts that resemble transition metal nitrosyl complexes. To elucidate the analogy between NHC and transition metal NO adducts, here we have undertaken a systematic investigation of the electron- and proton-transfer chemistry of [NHC-NO]⋅ (N-heterocyclic carbene nitric oxide radical) compounds. We have accessed a suite of compounds, comprised of [NHC-NO]+ , [NHC-NO]- , [NHC-NOH]0 , and [NHC-NHOH]+ species. In particular, [NHC-NO]- was isolated as potassium and lithium ion adducts. Most interestingly, a monomeric potassium [NHC-NO]- compound was isolated with the assistance of 18-crown-6, which is the first instance of a monomeric alkali N-oxyl compound to the best of our knowledge. Our results demonstrate that [NHC-NO]⋅ exhibits redox behavior broadly similar to metal nitrosyl complexes, which opens up more possibilities for utilizing NHCs to build on the known reactivity of metal complexes.

C-Nitrosothioformamide: A Donor Template for Dual Release of HNO and H2 S.

C-Nitrosothioformamide was demonstrated to be a donor template for dual release of HNO and COS triggered by a retro-Diels-Alder reaction. COS is an H2 S precursor in the presence of carbonic anhydrase. This process produces HNO and H2 S in a slow but steady manner. As such, the direct reaction between HNO and H2 S under this situation appears to be minor. This may provide a useful tool for studying the synergistic effects of HNO and H2 S.

Quantification of intracellular HNO delivery with capillary zone electrophoresis.

Redox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects. Coupling the inherent reactivity of thiols with highly sensitive and selective chemical analysis protocols can facilitate identification of redox signaling agents, both in solution and in cultured cells. Here, we describe use of capillary zone electrophoresis to both identify and quantify sulfinamides, which are specific markers of the reaction of thiols with nitroxyl (HNO), a putative biologically relevant reactive nitrogen species.

Nitroxyl Delivered by Angeli's Salt Causes Short-Lasting Activation Followed by Long-Lasting Deactivation of Meningeal Afferents in Models of Headache Generation.

The role of TRPA1 receptor channels in meningeal nociception underlying the generation of headaches is still unclear. Activating as well as inhibitory effects of TRPA1 agonists have been reported in animal models of headache. The aim of the present study was to clarify the effect of the TRPA1 agonist nitroxyl (HNO) delivered by Angeli's salt in two rodent models of meningeal nociception. Single fibre recordings were performed using half-skull preparations of mice (C57BL/6) in vitro. Angeli's salt solution (AS, 300 µM) caused short-lasting vigorous increases in neuronal activity of primary meningeal afferents, followed by deactivation and desensitisation. These effects were similar in TRPA1 knockout and even more pronounced in TRPA1/TRPV1 double-knockout mice in comparison to wild-type mice. The activity of spinal trigeminal neurons with afferent input from the dura mater was recorded in vivo in anesthetised rats. AS (300 µM) or the TRPA1 agonist acrolein (100 and 300 µM) was applied to the exposed dura mater. AS caused no significant changes in spontaneous activity, while the mechanically evoked activity was reduced after acrolein application. These results do not confirm the assumption that activation of trigeminal TRPA1 receptor channels triggers the generation of headaches or contributes to its aggravation. Instead, there is evidence that TRPA1 activation may have an inhibitory function in the nociceptive trigeminal system.

Oxazolidine Nitroxide Transformation in a Coordination Sphere of the Ln3+ Ions.

Upon the interaction of the hydrated lanthanide(III) salts found in acetonitrile solution with a tripodal paramagnetic compound, 4,4-dimethyl-2,2-bis(pyridin-2-yl)-1,3-oxazolidine-3-oxyl (Rad), functionalized by two pyridyl groups, three neutral, structurally characterized complexes with diamagnetic polydentate ligands-[Dy(RadH)(hbpm)Cl2], [Yb2(ipapm)2(NO3)4], and [Ce2(ipapm)2(NO3)4(EtOAc)2]-were obtained. These coordination compounds are minor uncolored crystalline products, which were formed in a reaction mixture due to the Rad transformation in a lanthanide coordination sphere, wherein the processes of its simultaneous disproportionation, hydrolysis, and condensation proceed differently than in the absence of Ln ions. The latter fact was confirmed by the formation of the structurally characterized product of the oxazolidine nitroxide transformation during its crystallization in toluene solution. Such a conversion in the presence of 4f elements ions is unique since no similar phenomenon was observed during the synthesis of the 3d-metal complexes with Rad.

Para-Substituted O-Benzyl Sulfohydroxamic Acid Derivatives as Redox-Triggered Nitroxyl (HNO) Sources.

Nitroxyl shows a unique biological profile compared to the gasotransmitters nitric oxide and hydrogen sulfide. Nitroxyl reacts with thiols as an electrophile, and this redox chemistry mediates much of its biological chemistry. This reactivity necessitates the use of donors to study nitroxyl's chemistry and biology. The preparation and evaluation of a small library of new redox-triggered nitroxyl sources is described. The condensation of sulfonyl chlorides and properly substituted O-benzyl hydroxylamines produced O-benzyl-substituted sulfohydroxamic acid derivatives with a 27-79% yield and with good purity. These compounds were designed to produce nitroxyl through a 1, 6 elimination upon oxidation or reduction via a Piloty's acid derivative. Gas chromatographic headspace analysis of nitrous oxide, the dimerization and dehydration product of nitroxyl, provides evidence for nitroxyl formation. The reduction of derivatives containing nitro and azide groups generated nitrous oxide with a 25-92% yield, providing evidence of nitroxyl formation. The oxidation of a boronate-containing derivative produced nitrous oxide with a 23% yield. These results support the proposed mechanism of nitroxyl formation upon reduction/oxidation via a 1, 6 elimination and Piloty's acid. These compounds hold promise as tools for understanding nitroxyl's role in redox biology.

Conjugate-Driven Electron Density Delocalization of Piperidine Nitroxyl Radical for Stable Aqueous Zinc Hybrid Flow Batteries.

Stable and soluble redox-active nitroxyl radicals are highly desired for high-capacity and long-life aqueous zinc hybrid flow batteries (AZHFBs). Here we report a "π-π" conjugated imidazolium and "p-π" conjugated acetylamino co-functionalized 2,2,6,6-tetramethylpiperidine-N-oxyl (MIAcNH-TEMPO) as stable catholyte for AZHFBs. The incorporation of double-conjugate substituents could delocalize the electron density of the N-O head and thus remarkably stabilize the radical and oxoammonium forms of TEMPO, avoiding the side reaction of ring-opening. Consequently, the applied MIAcNH-TEMPO/Zn AZHFB demonstrates the hardly time-dependent stability with a constant capacity retention of 99.95 % per day over 16.7 days at a high concentration catholyte of 1.5 M and high current density of 50 mA cm-2 . This proposed molecular engineering strategy based on electron density regulation of redox-active structures displays an attractive efficacy and thus represents a remarkable advance in high-performance AZHFBs.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents.

The contraction and relaxation of the heart is controlled by stimulation of the ß1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of ß1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Differences in pharmacokinetic behaviors of two lipophilic 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals, in vivo probes to assess the redox status in the brain using magnetic resonance techniques.

PURPOSE: The pharmacokinetics of 3-methoxycarbonyl- and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals (MCP and HMP, respectively), magnetic resonance probes to assess the brain redox status, were examined in healthy mouse brains. METHODS: The time course of the concentration of the radical form of the probe in the brain was examined by signal enhancements on T1 -weighted MR image after an intravenous injection. The distribution of the total probe (sum of radical and reduced forms) was investigated using brain homogenates. RESULTS: MCP distributed to the brain more than HMP. MCP exhibited biphasic decay with fast and slow components, whereas HMP exhibited monophasic decay with a similar rate constant to the slow component of MCP. Similar profiles were observed in various regions of the brain. The total probe for MCP exhibited monophasic decay at a similar rate constant to the slow component of the radical form; however, the initial content of the total probe was similar to its radical form. For HMP, decay of the total probe coincided with that of the radical form. CONCLUSION: The decay of MCP needs to consider the reduction of the probe in and its elimination from the brain, while the decay of HMP may mainly result from its elimination from the brain.

Merging N-Hydroxyphthalimide into Metal-Organic Frameworks for Highly Efficient and Environmentally Benign Aerobic Oxidation.

Two highly efficient metal-organic framework catalysts TJU-68-NHPI and TJU-68-NDHPI have been successfully synthesized through solvothermal reactions of which the frameworks are merged with N-hydroxyphthalimide (NHPI) units, resulting in the decoration of pore surfaces with highly active nitroxyl catalytic sites. When t-butyl nitrite (TBN) is used as co-catalyst, the as-synthesized MOFs are demonstrated to be highly efficient and recyclable catalysts for a novel three-phase heterogeneous oxidation of activated C-H bond of primary and secondary alcohols, and benzyl compounds under mild conditions. Based on the high efficiency and selectivity, an environmentally benign system with good sustainability, mild conditions, simple work-up procedure has been established for practical oxidation of a wide range of substrates.

Cardiovascular Therapeutic Potential of the Redox Siblings, Nitric Oxide (NO•) and Nitroxyl (HNO), in the Setting of Reactive Oxygen Species Dysregulation.

Reactive oxygen species (ROS) dysregulation is a hallmark of cardiovascular disease, characterised by an imbalance in the synthesis and removal of ROS. ROS such as superoxide (•O2-), hydrogen peroxide (H2O2), hydroxyl (OH•) and peroxynitrite (ONOO-) have a marked impact on cardiovascular function, contributing to the vascular impairment and cardiac dysfunction associated with diseases such as angina, hypertension, diabetes and heart failure. Central to the vascular dysfunction is a reduction in bioavailability and/or physiological effects of vasoprotective nitric oxide (NO•), leading to vasoconstriction, inflammation and vascular remodelling. In a cardiac context, increased ROS generation can also lead to modification of key proteins involved in cardiac contractility. Whilst playing a key role in the pathogenesis of cardiovascular disease, ROS dysregulation also limits the clinical efficacy of current therapies, such as nitrosovasodilators. As such, alternate therapies are sought. This review will discuss the impact of ROS dysregulation on the therapeutic utility of NO• and its redox sibling, nitroxyl (HNO). Both nitric oxide (NO) and nitroxyl (HNO) donors signal through soluble guanylyl cyclase (sGC). NO binds to the Fe(II) form of sGC and nitroxyl possibly to both sGC heme and thiol groups. In the vasculature, nitroxyl can also signal through voltage-dependent (Kv) and ATP-sensitive (KATP) K+ channels as well as calcitonin gene-related peptide (CGRP). In the heart, HNO directly targets critical thiols to increase myocardial contractility, an effect not seen with NO. The qualitative effects via elevation of cGMP are similar, i.e. lusitropic in the heart and inhibitory on vasoconstriction, inflammation, aggregation and vascular remodelling. Of pathophysiological significance is the fact the efficacy of NO donors is impaired by ROS, e.g. through chemical scavenging of NO, to generate reactive nitrogen oxide species (RNOS), whilst nitroxyl is apparently not.

Expansion of Substrate Scope for Nitroxyl Radical/Copper-Catalyzed Aerobic Oxidation of Primary Alcohols: A Guideline for Catalyst Selection.

Four distinctive sets of optimum nitroxyl radical/copper salt/additive catalyst combinations have been identified for accommodating the aerobic oxidation of various types of primary alcohols to their corresponding aldehydes. Interestingly, less nucleophilic catalysts exhibited higher catalytic activities for the oxidation of particular primary allylic and propargylic alcohols to give α,ß-unsaturated aldehydes that function as competent Michael acceptors. The optimum conditions identified herein were successful in the oxidation of various types of primary alcohols, including unprotected amino alcohols and divalent-sulfur-containing alcohols in good-to-high yields. Moreover, N-protected alaninol, an inefficient substrate in the nitroxyl radical/copper-catalyzed aerobic oxidation, was oxidized in good yield. On the basis of the optimization results, a guideline for catalyst selection has been established.