Detection Of HNO Research Articles

Time-Resolved Electrochemical Quantification of Azanone (HNO) at Low Nanomolar Level

Azanone (HNO, nitroxyl) is a highly reactive and short-lived compound with intriguing and highly relevant properties. It has been proposed to be a reaction intermediate in several chemical reactions and an in vivo, endogenously produced key metabolite and/or signaling molecule. In addition, its donors have important pharmacological properties. Therefore, given its relevance and elusive nature (it reacts with itself very quickly), the development of reliable analytical methods for quantitative HNO detection is in high demand for the advancement of future research in this area. During the past few years, several methods were developed that rely on chemical reactions followed by mass spectrometry, high-performance liquid chromatography, UV-vis, or fluorescence-trapping-based methodologies. In this work, our recently developed HNO-sensing electrode, based on the covalent attachment of cobalt(II) 5,10,15,20-tetrakis[3-(p-acetylthiopropoxy)phenyl] porphyrin [Co(P)] to a gold electrode, has been thoroughly characterized in terms of sensibility, accuracy, time-resolved detection, and compatibility with complex biologically compatible media. Our results show that the Co(P) electrode: (i) allows time-resolved detection and kinetic analysis of the electrode response (the underlying HNO-producing reactions can be characterized) (ii) is able to selectively detect and reliably quantify HNO in the 1-1000 nM range, and (iii) has good biological media compatibility (including cell culture), displaying a lack of spurious signals due to the presence of O2, NO, and other reactive nitrogen and oxygen species. In summary, the Co(P) electrode is to our knowledge the best prospect for use in studies investigating HNO-related chemical and biological reactions.

A Reductant-Resistant and Metal-Free Fluorescent Probe for Nitroxyl Applicable to Living Cells

Nitroxyl (HNO) is a one-electron reduced and protonated derivative of nitric oxide (NO) and has characteristic biological and pharmacological effects distinct from those of NO. However, studies of its biosynthesis and activities are restricted by the lack of versatile HNO detection methods applicable to living cells. Here, we report the first metal-free and reductant-resistant HNO imaging probe available for use in living cells, P-Rhod. It consists of a rhodol derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent rhodol derivative. P-Rhod showed high selectivity for HNO in the presence of various biologically relevant reductants, such as glutathione and ascorbate, in comparison with previous HNO probes. We show that P-Rhod can detect not only HNO enzymatically generated in the horseradish peroxidase-hydroxylamine system in vitro but also intracellular HNO release from Angeli's salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.

Quantitative Detection of Nitroxyl Upon Trapping with Glutathione and Labeling with a Specific Fluorogenic Reagent

Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH2) were also detected. This suggests that GS(O)NH2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

Detection of Nitric Oxide and Nitroxyl with Benzoresorufin-Based Fluorescent Sensors

A new family of benzoresorufin-based copper complexes for fluorescence detection of NO and HNO is reported. The copper complexes, CuBRNO1-3, elicit 1.5-4.8-fold emission enhancement in response to NO and HNO. The three sensors differ in the nature of the metal-binding site. The photophysical properties of these sensors are investigated with assistance from density functional theory calculations. The fluorescence turn-on observed upon reaction with HNO is an unexpected result that is discussed in detail. The utility of the new sensors for detecting HNO and NO in HeLa cells and RAW 264.7 macrophages is demonstrated.

Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides

Nitric oxide, •NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO−, one-electron reduced analog of •NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative •NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with •NO and HNO with similar rate constants of about 104 M− 1s− 1 but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative •NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, •NO and HNO. The sensitivity of this approach for the detection of the rates of •NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative •NO/HNO detection might become a valuable tool in nitric oxide-related studies.

Reduced glutathione-resisting 19F NMR sensors for detecting HNO

The 19F NMR probes for the HNO detection are reported. We synthesized the probe molecules with the paramagnetic Cu(II) complex and fluorine atoms using a cubic silsesquioxane. By using the magnetism changes of the Cu(II) to Cu(I) in the complex by the reduction with HNO, the 19F NMR signal intensities of the probe increased. Noteworthily, our probes have superior resistance to reduced glutathione which is the major intracellular molecule to maintain the reductive environment and the competitor in the reduction of Cu(II) against HNO.

Rapid and Selective Nitroxyl (HNO) Trapping by Phosphines: Kinetics and New Aqueous Ligations for HNO Detection and Quantitation

Recent studies distinguish the biological and pharmacological effects of nitroxyl (HNO) from its oxidized/deprotonated product nitric oxide (·NO), but the lack of HNO detection methods limits the understanding its in vivo mechanisms and the identification of endogenous sources. We previously demonstrated that reaction of HNO with triarylphosphines provides aza-ylides and HNO-derived amides, which may serve as stable HNO biomarkers. We now report a kinetic analysis for the trapping of HNO by phosphines, ligations of enzyme-generated HNO, and compatibility studies illustrating the selectivity of phosphines for HNO over other physiologically relevant nitrogen oxides. Quantification of HNO using phosphines is demonstrated using an HPLC-based assay and ligations of phosphine carbamates generate HNO-derived ureas. These results further demonstrate the potential of phosphine probes for reliable biological detection and quantification of HNO.

Nitroxyl (HNO): A Novel Redox Signaling Molecule

Nitroxyl (HNO), the one electron reduced and protonated congener of nitric oxide, is emerging as a novel nitrogen oxide with distinct chemistry and biological actions as compared with its redox sibling. The "thiophilic" nature of HNO underlies many of its unique properties, and attention has been focused on its regulation of cellular function and therapeutic potential, particularly in the treatment of cardiovascular disease. The present Forum issue summarizes the intriguing chemistry and biology of HNO and highlights its impact in the cardiovascular and central nervous systems. Recent advances in the development of new HNO donors and their potential use as tools to study HNO signaling and therapeutic agents are discussed. Evidence is also provided for a role of HNO as a putative, endogenous regulator of vascular function. However, as highlighted in this Forum issue, the development of sensitive methods for HNO detection in a biological system is needed to conclusively prove its in vivo generation. As research expands in this area, it is likely that new targets and pharmacological applications of HNO will be discovered.

Visualization of Nitroxyl in Living Cells by a Chelated Copper(II) Coumarin Complex

The coumarin-based probe Cu(II)-COT1 was successfully developed for the detection of HNO on the basis of the reduction reaction. In addition, highly selective "turn on" type fluorogenic behavior upon the addition of Angeli's salt (Na(2)N(2)O(3)) was also applied to bioimaging in A375 cells.

Discriminating Detection of NO and HNO using Encapsulated Nitronyl Nitroxides

Discriminating Detection of NO and HNO using Encapsulated Nitronyl Nitroxides

Reductive Phosphine-Mediated Ligation of Nitroxyl (HNO)

Nitroxyl (HNO) demonstrates a unique chemical and biological profile compared to nitric oxide (NO). Phosphorus NMR studies reveal that HNO reacts with triarylphosphines to give the corresponding phosphine oxide and aza-ylide. In the presence of a properly situated electrophilic ester, the aza-ylide undergoes a Staudinger ligation to yield an amide with the nitrogen atom being derived from HNO. These results define new HNO reactivity and provide the basis of new HNO detection methods.

The shy Angeli and his elusive creature: the HNO route to vasodilation

what we now call Angeli's salt (AS) was discovered more than a century ago by the Italian chemist Angelo Angeli (Fig. 1) . He found that “salts of nitroxylaminic acid are readily resolved into the corresponding nitrites and the unsaturated residue nitroxyl” (HNO), proposing their use as suppliers of HNO (3). However, by analogy with the genial but very reclusive personality of the discoverer (17), the physiological properties of HNO didn't draw too much attention until early 1990s. In their seminal work, Fukuto and colleagues (19) reported that HNO elicits vasorelaxation in both the rabbit aorta and bovine intrapulmonary artery by a soluble guanylate cyclase (sGC)- dependent pathway. Later, it was evident that the thiol-donating agent l-cysteine can discriminate the vasodilatative profile of HNO from that of nitric oxide (NO·) or nitrosothiols: AS/HNO action can be blocked by this agent, whereas the NO· effect is potentiated (36). In combination with the suggestion that NO· is the only species able to directly activate sGC (9), these data led to the conclusion that HNO-induced vasorelaxation is due to its intracellular conversion to NO· with subsequent activation of sGC. For several years, this idea has relegated HNO to a mere precursor of the presence and biological activity of NO·, de facto negating to HNO its own right to exist.

Xerogel Optical Sensor Films for Quantitative Detection of Nitroxyl

Xerogel sensing films were synthesized via sol-gel chemistry were used to fabricate optical nitroxyl (HNO) sensors [corrected] Selective detection of HNO in solution was achieved by monitoring the rates of manganese(III) meso-tetrakis(4-sulfonatophenyl) porphyrinate (MnIIITPPS) reductive nitrosylation in the anaerobic interior of aminoalkoxysilane-derived xerogel films. Nitroxyl permeability in sensor films deposited in round-bottom 96-well plates was enhanced via incorporation of trimethoxysilyl-terminated poly(amidoamine-organosilicon) dendrimers in the xerogel network. The selectivity of MnIIITPPS for HNO, the overall sensitivity, and the working dynamic range of the resulting sensors were characterized. The HNO-sensing microtiter plates were used to quantify pH-dependent HNO generation by the recently described HNO-donor sodium 1-(isopropylamino)diazene-1-ium-1,2-diolate (IPA/NO), and compare HNO production efficiency between IPA/NO and Angeli's salt, a traditional HNO-donor.

Using chemical ionization mass spectrometry for detection of HNO 3, HCl, and ClONO 2 in the atmosphere

Ion chemistry schemes involving three different reagent ions are described for measuring nitric acid (HNO 3), hydrochloric acid (HCl), and chlorine nitrate (ClONO 2) in the upper troposphere and lower stratosphere using chemical ionization mass spectrometry (CIMS). These schemes were evaluated in the laboratory for sensitivity and selectivity of the neutral species. The use of one scheme, based on the SF 5 − reagent ion, is described for airborne measurements. Initial atmospheric measurements show that HNO 3, HCl, and ClONO 2 can be measured using SF 5 − with high precision and accuracy.

EPR Detection of HNO2•-in the Radiolysis of Aqueous Nitrite and Quantum Chemical Calculation of Its Stability and Hyperfine Parameters

Experimental and theoretical evidence is presented to support a prior suggestion [Lymar et al. J. Phys. Chem. A 2002, 106, 7245] that radiolytically generated hydrogen atoms attack at the nitrogen, rather than the oxygen, of nitrite ions in aqueous solution. Time-resolved electron spin resonance detection was used to unambiguously identify the HNO2 ?- radicals formed. At pH 9 the radicals live about 10 ?s, and have quite broad (0.6 G) lines. The observed hyperfine splitting at nitrogen was aN 19.6 G, with each of the three nitrogen lines further split by the small hydrogen coupling, aH 4.5 G. The g factor for the radical is 2.0053. Although this is the first observation of this radical in fluid solution, the ESR parameters are consistent with previous observations in the solid phase. The identity of the radical was also confirmed by quantum chemical calculation of the ESR parameters, including the g factor. It was necessary to take into account vibrational modulation of the computed hyperfine parameters when comparing theory to experiment because of the large-amplitude motion of the hydrogen atom in the pyramidal radical. The yield of HNO2 ?- radicals was estimated at 70% of the available H atoms by a kineticmore » method. Computed thermodynamic parameters confirm that, in the gas phase, both HNO2 ?- and HONO?- are stable relative to the asymptotes H + NO2- and OH- + NO, with HNO2 ?- 40 kJ mol-1 above HONO?- but protected from rearrangement by a large barrier. In solution, calculations indicate that while HNO2 ?- is still bound with respect to dissociation into H + NO2-, it is now only 3.2 kJ mol-1 above HONO?- which, in turn, lies about 73 kJ mol-1 above OH- + NO, and dissociation is driven by the strong solvation of the hydroxide ion.« less

Reactions of SiF 5− with atmospheric trace gases: ion chemistry for chemical ionization detection of HNO 3 in the troposphere

The reactions of SiF 5 − with a series of trace atmospheric species were studied using the flowing afterglow technique at 293 K. SiF 5 − reacts at the collision rate by fluoride transfer with HI and SO 3. SiF 5 − reacts rapidly with HNO 3 to primarily form a strongly bound cluster ion. SiF 5 − also forms more weakly bound cluster ions with HCO 2H, HCl, CH 3CO 2H, and H 2O. SiF 5 − was found to be unreactive with HBr, N 2O 5, HO 2NO 2, NO 2, ClONO 2, O 3, SO 2, CH 3NO 3, CH 3C(O)O 2NO 2, CO 2, H 2O, and NH 3. These results demonstrate that SiF 5 − is an excellent reagent ion for the selective detection of HNO 3 in ambient air with a chemical ionization mass spectrometer.