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.     

Hydrolysis of acyloxy nitroso compounds yields nitroxyl (HNO)

Nitroxyl (HNO/NO(-)), the reduced form of nitric oxide, has gained attention based on its separate chemistry and biology from nitric oxide. The inherent reactivity of HNO requires new and mechanistically unique donors for the detailed study of HNO chemistry and biology. Oxidation of cyclohexanone oxime with lead tetraacetate yields 1-nitrosocyclohexyl acetate, whereas oxidation of oximes in the presence of excess carboxylic acid gives various acyloxy nitroso compounds. These bright blue compounds exist as monomers as indicated by their infrared, proton, and carbon NMR spectra, and X-ray crystallographic analysis reveals the nitroso groups possess a "nitroxyl-like" bent configuration. Hydrolysis of these compounds produces nitrous oxide, the dimerization and dehydration product of HNO, and provides evidence for the intermediacy of HNO. Both thiols and oxidative metal complexes inhibit nitrous oxide formation. Hydrolysis of these compounds in the presence of ferric heme complexes forms ferrous nitrosyl complexes providing further evidence for the intermediacy of HNO. Kinetic analysis shows that the rate of hydrolysis depends on pH and the structure of the acyl group of the acyloxy nitroso compound. These compounds relax pre-constricted rat aortic rings similar to known HNO donors. Together, these results identify acyloxy nitroso compounds as a new class of HNO donors.     

Development of N-substituted hydroxylamines as efficient nitroxyl (HNO) donors

Due to its inherent reactivity, nitroxyl (HNO), must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N-substituted hydroxylamines with carbon-based leaving groups have been synthesized, and their structures confirmed by X-ray crystallography. These compounds generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. A barbituric acid and a pyrazolone derivative have been developed as efficient HNO donors with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min, respectively.     

Dual mechanisms of HNO generation by a nitroxyl prodrug of the diazeniumdiolate (NONOate) class

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)═NO(-)Na(+) (1, IPA/NO) with BrCH(2)OAc produced a stable derivative of structure iPrHN-N(O)═NO-CH(2)OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH(3)-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.     

Reactions of acyl nitroso compounds with amines: Production of nitroxyl (HNO) with the preparation of amides

Oxidation of hydroxamic acids in the presence of amines generates nitrous oxide (N₂O) and the corresponding amide. The identification of N₂O suggests the intermediacy of nitroxyl (HNO). Retro-Diels Alder dissociation of cyclopentadiene-acyl nitroso compound cycloadducts releases N₂O with amide formation.     

HNO trapping and assisted decomposition of nitroxyl donors by ferric hemes

The role of nitric oxide (NO) as a signalling molecule in biological systems has been thoroughly studied in the last decades. More recently, there has been an increasing interest in the one-electron reduction product of NO, namely nitroxyl (HNO/NO⁻). Some studies suggest that nitroxyl can be produced by nitric oxide synthases under certain conditions, and that distinct pharmacological effects are observed for NO and nitroxyl donors. HNO is capable of react with heme proteins, thiols, molecular oxygen, NO and HNO itself. However, only recently the different reactivity patterns are being thoroughly understood. Heme model compounds offer the opportunity to study the reaction kinetics without the complexity arising from ligand interactions with the protein matrix. In this study we analyzed the reaction between the commonly used nitroxyl donors sodium trioxodinitrate and toluene sulfohydroxamic acid, with the ferric model compounds microperoxidase-11 (MP11) and the cationic metalloporphyrin [Fe^IIITEPyP]⁵⁺ (Tetrakis N-ethylpyridinium-2yl porphyne). Our results show that there are two alternative modes of reactivity for nitroxyl donors towards heme in aqueous solutions. The first one comprises the heme assisted decomposition of the donor, enhancing its decomposition rate more than 100-fold. In the second, the donor produces HNO which subsequently reacts with the porphyrin. The observed rate constants (of about 10⁵ M⁻¹ s⁻¹) are consistent with the estimated data for the HNO reaction with heme proteins, and may be controlled by the leaving water ligand. This rate constant probably represents an upper limit for the bimolecular rate constant of HNO towards these proteins.     

A theoretical study of the excited states of the nitroxyl radical (HNO) via the equations of motion method

The excited states of the HNO radical have been studied using the equations of motion method. These calculations confirm the presence of a low-lying ³A″ state at 5485 cm^−I, which lies between the ^IA′ ground state and ^IA″ excited state.     

Theoretical study of hydrogen bonding interaction in nitroxyl (HNO) dimer: interrelationship of the two N-H...O blue-shifting hydrogen bonds

The hydrogen bonding interactions of the HNO dimer have been investigated using ab initio molecular orbital and density functional theory (DFT) with the 6-311++G(2d,2p) basis set. The natural bond orbital (NBO) analysis and atom in molecules (AIM) theory were applied to understand the nature of the interactions. The interrelationship between one N-H...O hydrogen bond and the other N-H...O hydrogen bond has been established by performing partial optimizations. The dimer is stabilized by the N-H...O hydrogen bonding interactions, which lead to the contractions of N-H bonds as well as the characteristic blue-shifts of the stretching vibrational frequencies nu(N-H). The NBO analysis shows that both rehybridization and electron density redistribution contribute to the large blue-shifts of the N-H stretching frequencies. A quantitative correlations of the intermolecular distance H...O (r(H...O)) with the parameters: rho at bond critical points (BCPs), s-characters of N atoms in N-H bonds, electron densities in the sigma*(N-H), the blue-shift degrees of nu(N-H) are presented. The relationship between the difference of rho (|Deltarho|) for the one hydrogen bond compared with the other one and the difference of interaction energy (DeltaE) are also illustrated. It indicates that for r(H...O) ranging from 2.05 to 2.3528 A, with increasing r(H...O), there is the descending tendency for one rho(H...O) and the ascending tendency for the other rho(H...O). r(H...O) ranging from 2.3528 to 2.85 A, there are descending tendencies for the two rho(H...O) with increasing r(H...O). On the potential energy surface of the dimer, the smaller the difference between one rho(H...O) and the other rho(H...O) is, the more stable the structure is. As r(H...O) increases, the blue-shift degrees of nu(N-H) decrease. The cooperative descending tendencies in s-characters of two N atoms with increasing r(H...O) contribute to the decreases in blue-shift degrees of nu(N-H). Ranging from 2.05 to 2.55 A, the increase of the electron density in one sigma*(N-H) with elongating r(H...O) weakens the blue-shift degrees of nu(N-H), simultaneously, the decrease of the electron density in the other sigma*(N-H) with elongating r(H...O) strengthens the blue-shift degrees of nu(N-H). Ranging from 2.55 to 2.85 A, the cooperative ascending tendencies of the electron densities in two sigma*(N-H) with increasing r(H...O) contribute to the decreases in blue-shift degrees of nu(N-H).     

Spectral analysis of naringenin deprotonation in aqueous ethanol solutions

The deprotonation of 5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one (naringenin) was studied in aqueous solutions of ethanol and 0.1 mol L^?1 sodium perchlorate at 25°C. The chemical species that contributed to deprotonation were evaluated together with their pure spectral characteristics and concentration profiles by some chemometric methods. The deprotonation constants assigned by pK ₁, pK ₂, and pK ₃ were determined by multivariate curve analysis of spectral data at different pcH values. The pure spectral analysis concordant with the theoretical prediction of deprotonation constants indicates that the acidity of hydroxyl groups in naringenin decreases in the order: 7-OH, 4′-OH, 5-OH. The effects of the solvent on deprotonation were analysed in terms of the linear solvation energy relationships using the model of Kamlet, Abboud, and Taft (KAT). Multiple linear regressions were aimed towards correlating the deprotonation constants with the microscopic parameters containing hydrogen-bond acidity (α), dipolarity/polarisability (π*), and hydrogen-bond basicity (β). The most significant parameter was found to be the hydrogen-bond acidity of binary mixtures.     

Adsorption of Cr(VI) from aqueous solutions by HNO3-purified and chemically activated pyrolytic tire char

Pyrolytic tire char adsorbents either demineralized by nitric acid (purified char, PC) or activated with KOH-calcination (activated char, AC) were used for Cr(VI) removal from aquatic solutions and studied by adsorption kinetics, isotherms, and thermodynamics. Adsorbent’s physicochemical characteristics were studied by several techniques such as X-ray diffraction, porosimetry, scanning electron microscopy, elemental analysis, and Boehm titration. For PC, acid treatment leads partially to a mesoporous structure while for AC, KOH activation creates also a microporosity enhancing the specific surface area at 443 m²g^?1. Cr(VI) adsorption onto both adsorbents followed better second-order kinetics and Langmuir isotherm models and it was exothermic (ΔH < 0) and spontaneous (ΔG < 0). The maximum Cr(VI) adsorption capacity for AC and PC was 114 and 79.47 mg g^?1, respectively, at pH = 4. The present work reveals that AC and PC can be efficient sorbents for the removal of heavy metal ions, contributing both positively to wastewater treatment and waste tire pyrolysis plants.     

Infrared signatures of HNO3 and NO3(-) at a model aqueous surface. A theoretical study

The infrared signatures of nitric acid HNO3 and its conjugate anion NO3(-) at the surface of an aqueous layer are derived from electronic structure calculations at the HF/SBK+* level of theory on the HNO3 x (H2O)3 --> NO3(-) x H3O(+) x (H2O)2 model reaction system embedded in clusters comprising 33, 40, 45, and 50 classical, polarizable waters, mimicking various degrees of solvation [Bianco, R.; Wang, S.; Hynes, J. T. J. Phys. Chem. A 2007, 111, 11033]. The molecular level character of the various bands is discussed, and the solvation patterns are described in terms of hydrogen bonding and resulting polarization of the species' intramolecular bonds. Connection is made with assorted experimental results, including surface-sensitive Sum Frequency Generation spectroscopy of aqueous nitric acid solutions, infrared spectroscopy of amorphous thin films of nitric acid monohydrate (NAM) and dihydrate (NAD), and infrared and Raman spectroscopic results for bulk aqueous solutions of nitric acid and nitrate salts.     

The deprotonation and protonation equilibria of a hypericin derivative in aqueous solution

Summary A hypericin derivative ,'-appended at the methyl groups with two polyethylene glycol moieties (about 23 units long) and capped with acetyl groups was synthesized starting from emodin. This derivative proved to be soluble in water and was investigated by means of spectrophotometric titrations and electrophoresis experiments. Deprotonation at thebay-region hydroxyl group was observed atpK _a=1.6. This was followed by a second deprotonation step of aperi-hydroxyl group at apK _a value of 9.4. This derivative could be protonated at the carbonyl group characterized by apK _a value of –5.7. FrompK _a determinations in water-ethanol mixtures the correspondingpK _a values of hypericin itself determined in such mixtures were extrapolated to the aqueous phase. This resulted in estimatedpK _a values of 1.8, 9.2, and –6.

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Deprotonierungs- und Protonierungsgleichgewichte eines Hypericinderivates in wäßriger Lösung

Zusammenfassung Ein an den Methylgruppen von Hypericin mit endständig acetylierten Polyethylenglykolketten (ungefähr 23 Einheiten lang) ,'-disubstituiertes Derivat wurde ausgehend von Emodin synthetisiert. Dieses Derivat ist wasserlöslich, und es wurde mit Hilfe von spektrophotometrischen Titrationen und Elektrophorese untersucht. Die Deprotonierung derbay-Hydroxylgruppe erfolgt beipK _a=1.6. Diese wird von einem zweiten Deprotonierungsschritt an einerperi-Hydroxylgruppe bei einempK _a-Wert von 9.4 gefolgt. Dieses Derivat konnte an der Carbonylgruppe protoniert werden, was durch einenpK _a-Wert von –5.7 charakterisiert ist. Ausgehend vonpK _a-Messungen in Wasser-Ethanol-Mischungen wurden die entsprechendenpK _a-Werte auch für das Hypericin selbst in wäßriger Phase extrapoliert. Dies führte zu geschätztenpK _a-Werten von 1.8, 9.2 und –6.

     

Reductive chlorination of bis(4-tert-butylphenyl)amine-N-oxyl nitroxyl radical

Unlike cyclic aliphatic nitroxyls, whose oxidation with halogens gives oxoammonium salts, bis(4-tert-butylphenyl)amine-N-oxyl (1) treated with chlorine undergoes reductive chlorination to the corresponding di- and trichlorodiphenylamines. Chlorine partially dealkylates the compounds obtained. Plausible mechanisms for these reactions were suggested.     

A computational study of the intramolecular deprotonation of a carbon acid in aqueous solution

Proton transfer reactions are the rate-limiting steps in many biological and synthetic chemical processes, often requiring complex cofactors or catalysts to overcome the generally unfavourable thermodynamic process of carbanion intermediate formation. It has been suggested that quantum tunnelling processes enhance the kinetics of some of these reactions, which when coupled to protein motions may be an important consideration for enzyme catalysis. To obtain a better fundamental and quantitative understanding of these proton transfer mechanisms, a computational analysis of the intramolecular proton transfer from a carbon acid in the small molecule, 4-nitropentanoic acid, in aqueous solution is presented. Potential-energy surfaces from gas-phase, implicit and QM/MM (quantum mechanical/molecular mechanical) explicit solvation quantum chemistry models are compared, and the potential of mean force, for the full reaction coordinate, using umbrella-sampling molecular dynamics is analysed. Semi-classical multidimensional tunnelling corrections are also used to estimate the quantum tunnelling contributions and to understand the origin of the primary deuterium kinetic isotope effects (KIEs). The computational results are found to be in excellent agreement with the KIEs and the energetics obtained experimentally.     

Reactions of bis(trifluoromethyl)nitroxyl with tris(pentafluorophenyl)-phosphine arsine and stibine

Tris(pentafluorophenyl)phosphine is oxidised by bis(trifluoromethyl)nitroxyl to form tris(pentafluorophenyl)phosphine oxide and perfluoro(2,4-dimethyl-3-oxa-2,4-diazapentane). With the corresponding arsine and stibine, addition products are obtained, namely tris(pentafluorophenyl)-arsenicdi[bis(trifluoromethyl)nitroxide] and tris(pentafluorophenyl)-antimonydi[bis(trifluoromethyl)nitroxide] respectively. Some reactions of the new derivatives are described.     

Aminium cation radical of glycylglycine and its deprotonation to aminyl radical in aqueous solution

The photochemical reaction between glycylglycine and triplet 4-carboxybenzophenone has been investigated using time-resolved chemically induced dynamic nuclear polarization (CIDNP). It is shown that the mechanism of the peptide reaction with triplet excited carboxybenzophenone is electron transfer from the amino group of the peptide, leading to the formation of an aminium cation radical that deprotonates to a neutral aminyl radical. Simulation of the CIDNP kinetics leads to an estimation of the paramagnetic relaxation time for the alpha-protons at the N-terminus at 20 to 40 mus with the best-fit value of 25 mus.     

Colorimetric recognition of Cu(II) by (2-dimethylaminoethyl)amino appended anthracene-9,10-diones in aqueous solutions: deprotonation of aryl amine NH responsible for colour changes

Chromogenic receptors 2 and 3 undergo distinct colour changes from magenta to blue on gradual addition of Cu(II) and can be used as colorimetric probes for spectrophotometric and visual analysis of Cu(II) in the presence of biological metal ions Na(I), K(I), Mg(II), Ca(II), Fe(II), Zn(II) etc. in aqueous solution (methanol-water 1 : 1 v/v). On addition of Cu(II), both 2 and 3 exhibit a bathochromic shift of Delta lambda(max) = 114 nm for 2 and Delta lambda(max)= 150 and 265 nm for receptor 3. The protonation constants and formation constants of Cu(II) complexes of receptors 2 and 3 (at pH 7) and the effect of pH on formation of these complexes has been determined by the combination of UV-vis-pH titrations of receptors 2 and 3 and their Cu(II) complexes. These results and the emergence of only one peak at 610 nm for 2 and two distinct absorption peaks at 715 and 800 nm for 3 on addition of Cu(II) unambiguously point to mono- and di-deprotonation for 2 and 3, respectively.     

Synthesis and deprotonation of 2-(halophenyl)pyridines and 1-(halophenyl)isoquinolines

The ability of the 2-pyridyl and 1-isoquinolyl groups to direct metalation was studied in the benzene series. For this purpose, 2-(halophenyl)pyridines and 1-(halophenyl)isoquinolines were prepared. Interestingly, nucleophilic addition reactions on the azine ring were not observed under kinetic control using butyllithium, and the substrates were cleanly deprotonated on the benzene ring: the azine ring acidifies the adjacent hydrogen H2' (N-H2' interaction through space and/or inductive electron-withdrawing effect) and probably favors the approach of butyllithium (chelation). Under thermodynamic conditions using lithium dialkylamides, the presence of the azine group makes the lithio derivative at C2' more stable (chelation and/or inductive electron-withdrawing effect). This was evidenced in two ways: (1) syntheses of 2-halophenyllithiums (F, Cl, Br) substituted at C6 by a 2-pyridyl or 1-isoquinolyl group without elimination of lithium halide and (2) iodine migration from C2' to C4' when treating 2-(3-halo-2-iodophenyl)pyridines or 1-(3-fluoro-2-iodophenyl)isoquinoline with LTMP. Comparisons between the 2-pyridyl and fluoro units showed the latter was the stronger directing group for deprotonation.     

Synthesis and deprotonation of 2-(pyridyl)phenols and 2-(pyridyl)anilines

2-(2- and 3-Pyridyl)anilines (1, 2), 2,2-dimethyl-N-[2-(2- and 3-pyridyl)phenyl]propanamides (3, 4), and 2-, 3- and 4-(2-methoxyphenyl)pyridines (7-9) are readily synthesized using cross-coupling reactions. Whereas the amines 1, 2 undergo side reactions, the corresponding amides 3, 4 are deprotonated with lithium 2,2,6,6-tetramethylpiperidide (LTMP): the compound 3 at C6' under in situ quenching, and the compound 4 at C4'. When the ether 7 is subjected to the same reagent, lithiation occurs at C6'.     

Decomposition of sodium hypochlorite in an aqueous solution in the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) nitroxyl radical as a catalyst

Nitroxyl radical TEMPO is found to catalyze the decomposition of sodium hypochlorite in an aqueous-alkali medium. The mechanism of NaOCl decomposition to form ClO· radical is proposed which involves protonated TEMPO, oxoammonium salt TEMPO⁺, and hydroxyl radical.