Nitric Oxide Reacts Very Fast with Hydrogen Sulfide, Alcohols, and Thiols to Produce HNO: Revised Rate Constants.

The chemical reactivity of NO and its role in several biological processes seem well established. Despite this, the chemical reduction of •NO toward HNO has been historically discarded, mainly because of the negative reduction potential of NO. However, this value and its implications are nowadays under revision. The last reported redox potential, E'(NO,H+/HNO), at micromolar and picomolar concentrations of •NO and HNO, respectively, is between -0.3 and 0 V at pH 7.4. This potential implies that the one-electron-reduction process for NO is feasible under biological conditions and could be promoted by well-known biological reductants with reduction potentials of around -0.3 to -0.5 V. Moreover, the biologically compatible chemical reduction of •NO (nonenzymatic), like direct routes to HNO by alkylamines, aromatic and pseudoaromatic alcohols, thiols, and hydrogen sulfide, has been extensively explored by our group during the past decade. The aim of this work is to use a kinetic modeling approach to analyze electrochemical HNO measurements and to report for the first-time direct reaction rate constants between •NO and moderate reducing agents, producing HNO. These values are between 5 and 30 times higher than the previously reported keff values. On the other hand, we also showed that reaction through successive attack by two NO molecules to biologically compatible compounds could produce HNO. After over 3 decades of intense research, the •NO chemistry is still there, ready to be discovered.

The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine.

Azanone (HNO, nitroxyl) is a highly reactive molecule that, in the past few years, has drawn significant interest because of its pharmacological properties. However, the understanding of how, when, and where endogenous HNO is produced remains a matter of discussion. In this study, we examined the ability of myoglobin to produce HNO via the peroxidation of hydroxylamine with H2O2 using both experimental and computational approaches. The production of HNO was confirmed using an azanone selective electrochemical method and by the detection of N2O using FTIR. The catalytic capacity of myoglobin was characterized by the determination of the turnover number. The reaction kinetics of the hydroxylamine peroxidation were studied by both electrochemical and UV-vis methods. Further evidence about the reaction mechanism was obtained by EPR spectroscopy. Additionally, quantum mechanical/molecular mechanics experiments were performed to calculate the energy barrier for HNO production and to gain insight into the reaction mechanism. Our results confirm that myoglobin produces HNO via the peroxidation of hydroxylamine with a great catalytic capacity. In addition, our mechanistic study allows us to state that the Mb ferryl state is the most likely intermediate that reacts with hydroxylamine, yielding important evidence for endogenous HNO generation.

Synthesis, structural elucidation and antiradical activity of a copper (II) naringenin complex.

Coupling the extraction and derivatization of flavonoids to the Citrus processing industry is attractive from both the environmental and economic points of view. In the present work, the flavonoid naringin, obtained by "green" extraction with a water:ethanol mixture from waste grapefruit industry, was hydrolyzed to obtain naringenin. This flavonoid was used to synthesize the complex trans-di(aqua) bis(7-hydroxy-2-(4-hydroxyphenyl)-4-oxo-5-chromanolato) copper (II). This compound was characterized by spectroscopic techniques (UV/Vis, IR, Raman, NMR and EPR), and by thermal analysis (TG and DSC). Then, a monocrystal of the complex obtained by dissolution and recrystallization in DMF was analyzed by single crystal X-ray diffraction. This is the first report of the crystal structure of a Citrus flavonoid complex. Additionally, its antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined and compared with that for naringenin, demonstrating that coordination to copper enhances the antiradicalar activity of naringenin. According to the Mulliken population analysis conducted, by copper favors the delocalization and stabilization of the produced radical, since it acts as an electronic density acceptor.

HNO Is Produced by the Reaction of NO with Thiols.

Azanone (nitroxyl, HNO) is a highly reactive compound whose biological role is still a matter of debate. One possible route for its formation is NO reduction by biological reductants. These reactions have been historically discarded due to the negative redox potential for the NO,H+/HNO couple. However, the NO to HNO conversion mediated by vitamins C, E, and aromatic alcohols has been recently shown to be feasible from a chemical standpoint. Based on these precedents, we decided to study the reaction of NO with thiols as potential sources of HNO. Using two complementary approaches, trapping by a Mn porphyrin and an HNO electrochemical sensor, we found that under anaerobic conditions aliphatic and aromatic thiols (as well as selenols) are able to convert NO to HNO, albeit at different rates. Further mechanistic analysis using ab initio methods shows that the reaction between NO and the thiol produces a free radical adduct RSNOH•, which reacts with a second NO molecule to produce HNO and a nitrosothiol. The nitrosothiol intermediate reacts further with RSH to produce a second molecule of HNO and RSSR, as previously reported.

Nitric oxide is reduced to HNO by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to HNO in biological media?

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical.

Reactions of HNO with metal porphyrins: underscoring the biological relevance of HNO.

Azanone ((1)HNO, nitroxyl) shows interesting yet poorly understood chemical and biological effects. HNO has some overlapping properties with nitric oxide (NO), sharing its biological reactivity toward heme proteins, thiols, and oxygen. Despite this similarity, HNO and NO show significantly different pharmacological effects. The high reactivity of HNO means that studies must rely on the use of donor molecules such as trioxodinitrate (Angeli's salt). It has been suggested that azanone could be an intermediate in several reactions and that it may be an enzymatically produced signaling molecule. The inherent difficulty in detecting its presence unequivocally prevents evidence from yielding definite answers. On the other hand, metalloporphyrins are widely used as chemical models of heme proteins, providing us with invaluable tools for the study of the coordination chemistry of small molecules, like NO, CO, and O2. Studies with transition metal porphyrins have shown diverse mechanistic, kinetic, structural, and reactive aspects related to the formation of nitrosyl complexes. Porphyrins are also widely used in technical applications, especially when coupled to a surface, where they can be used as electrochemical gas sensors. Given their versatility, they have not escaped their role as key players in chemical studies involving HNO. This Account presents the research performed during the last 10 years in our group concerning azanone reactions with iron, manganese, and cobalt porphyrins. We begin by describing their HNO trapping capabilities, which result in formation of the corresponding nitrosyl complexes. Kinetic and mechanistic studies of these reactions show two alternative operating mechanisms: reaction of the metal center with HNO or with the donor. Moreover, we have also shown that azanone can be stabilized by coordination to iron porphyrins using electron-attracting substituents attached to the porphyrin ring, which balance the negatively charged NO¯. Second, we describe an electrochemical HNO sensing device based on the covalent attachment of a cobalt porphyrin to gold. A surface effect affects the redox potentials and allows discrimination between HNO and NO. The reaction with the former is fast, efficient, and selective, lacking spurious signals due to the presence of reactive nitrogen and oxygen species. The sensor is both biologically compatible and highly sensitive (nanomolar). This time-resolved detection allows kinetic analysis of reactions producing HNO. The sensor thus offers excellent opportunities to be used in experiments looking for HNO. As examples, we present studies concerning (a) HNO donation capabilities of new HNO donors as assessed by the sensor, (b) HNO detection as an intermediate in O atom abstraction to nitrite by phosphines, and (c) NO to HNO interconversion mediated by alcohols and thiols. Finally, we briefly discuss the key experiments required to demonstrate endogenous HNO formation to be done in the near future, involving the in vivo use of the HNO sensing device.

Discussing endogenous NO(•)/HNO interconversion aided by phenolic drugs and vitamins.

The reduction of NO(•) to HNO/NO(-) under biologically compatible conditions has always been thought as unlikely, mostly because of the negative reduction potential: E°(NO(•),H(+)/HNO) = -0.55 V vs NHE at physiological pH. Nonetheless, during the past decade, several works hinted at the possible NO-to-HNO conversion mediated by moderate biological reductants. Very recently, we have shown that the reaction of NO(•) with ascorbate and aromatic alcohols occurs through a proton-coupled nucleophilic attack (PCNA) of the alcohol to NO(•), yielding an intermediate RO-N(H)O(•) species, which further decomposes to release HNO. For the present work, we decided to inspect whether other common biological aromatic alcohols obtained from foods, such as Vitamin E, or used as over-the-counter drugs, like aspirin, are able to undergo the reaction. The positive results suggest that the conversion of NO to HNO could occur far more commonly than previously expected. Taking these as the starting point, we set to review our and other groups' previous reports on the possible NO-to-HNO conversion mediated by biological compounds including phenolic drugs and vitamins, as well as several thiol-bearing compounds. Analysis of revised data prompted us to ask ourselves the following key questions: What are the most likely physio/pathological conditions for NO(•)-to-HNO conversion to take place? Which effects usually attributed to NO(•) are indeed mediated by HNO? These inquiries are discussed in the context of 2 decades of NO and HNO research.

N-nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms.

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light-induced phase advances of the clock is mediated through a neuronal nitric oxide synthase-guanilyl cyclase pathway. We have employed a novel nitric oxide-donor, N-nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub-saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase-advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N-nitrosomelatonin had no effect on light-induced phase delays at circadian time 14. The photic-enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N-nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6-h advance in the light:dark cycle (but not resynchronization to a 6-h delay). Here, we demonstrate the chronobiotic properties of N-nitrosomelatonin, emphasizing the importance of nitric oxide-mediated transduction for circadian phase advances.

Redox potential determines the reaction mechanism of HNO donors with Mn and Fe porphyrins: defining the better traps.

Azanone ((1)HNO, nitroxyl) is a highly reactive molecule with interesting chemical and biological properties. Like nitric oxide (NO), its main biologically related targets are oxygen, thiols, and metalloproteins, particularly heme proteins. As HNO dimerizes with a rate constant between 10(6) and 10(7) M(-1) s(-1), reactive studies are performed using donors, which are compounds that spontaneously release HNO in solution. In the present work, we studied the reaction mechanism and kinetics of two azanone donors Angelís Salt and toluene sulfohydroxamic acid (TSHA) with eight different Mn porphyrins as trapping agents. These porphyrins differ in their total peripheral charge (positively or negatively charged) and in their Mn(III)/Mn(II) reduction potential, showing for each case positive (oxidizing) and negative (reducing) values. Our results show that the reduction potential determines the azanone donor reaction mechanism. While oxidizing porphyrins accelerate decomposition of the donor, reducing porphyrins react with free HNO. Our results also shed light into the donor decomposition mechanism using ab initio methods and provide a thorough analysis of which MnP are the best candidates for azanone trapping and quantification experiments.

Nitrite reduction mediated by heme models. Routes to NO and HNO?

The water-soluble ferriheme model Fe(III)(TPPS) mediates oxygen atom transfer from inorganic nitrite to a water-soluble phosphine (tppts), dimethyl sulfide, and the biological thiols cysteine (CysSH) and glutathione (GSH). The products with the latter reductant are the respective sulfenic acids CysS(O)H and GS(O)H, although these reactive intermediates are rapidly trapped by reaction with excess thiol. The nitrosyl complex Fe(II)(TPPS)(NO) is the dominant iron species while excess substrate is present. However, in slightly acidic media (pH ≈ 6), the system does not terminate at this very stable ferrous nitrosyl. Instead, it displays a matrix of redox transformations linking spontaneous regeneration of Fe(III)(TPPS) to the formation of both N2O and NO. Electrochemical sensor and trapping experiments demonstrate that HNO (nitroxyl) is formed, at least when tppts is the reductant. HNO is the likely predecessor of the N2O. A key pathway to NO formation is nitrite reduction by Fe(II)(TPPS), and the kinetics of this iron-mediated transformation are described. Given that inorganic nitrite has protective roles during ischemia/reperfusion (I/R) injury to organs, attributed in part to NO formation, and that HNO may also reduce net damage from I/R, the present studies are relevant to potential mechanisms of such nitrite protection.

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.

3-Methyl-5-methyl-sulfanyl-1,3,4-thia-diazole-2(3H)-thione.

The title compound, C(4)H(6)N(2)S(3), has two very similar mol-ecules per asymmetric unit. The nine non-H atoms in each mol-ecule are coplanar, both having comparable r.m.s. deviations of 0.002 Å. The main inter-est in the rather simple structure resides in a survey of very weak (in some cases, borderline) non-bonding inter-actions of various kinds, viz. S⋯S, C-H⋯π, π-π [centroid-centroid distance = 3.8958 (13) Å] and C-S⋯π [3.7271 (11) Å], which act as the major driving force for the arrangement of mol-ecules in the structure. The role of long, though highly directional, S⋯S contacts (d > 3.60 Å), and their relevance to the stability of the structure is discussed.

Bis(acetato-κ(2)O,O')(4,4'-dimethyl-2,2'-bipyridine-κ(2)N,N')-zinc.

The mol-ecular structure of the title compound, [Zn(CH(3)COO)(2)(C(12)H(12)N(2))], consists of isolated mol-ecules bis-ected by a twofold rotation axis which goes through the Zn(II) cation and halves the organic base through the central C-C bond. The Zn(II) ion is coordinated by two N atoms from one mol-ecule of the aromatic base and four O atoms from two bidentate, symmetry-related acetate anions, which coordinate asym-metrically [Zn-O distances of 2.058 (2) and 2.362 (3) Å], while the two Zn-N bond distances are equal as imposed by symmetry [2.079 (2) Å]. The crystal structure is supported by a number of weak C-H⋯O inter-actions and C-H⋯π contacts, with no π-π inter-actions present, mainly hindered by the substituent methyl groups and the relative mol-ecular orientation. The result is a three-dimensional structure in which each mol-ecule is linked to eight different neighbors.

N-butylpyridinium undecachlorocarbadodecaborate and comparison with similar compounds.

The title compound, C(9)H(14)N(+)·CHB(11)Cl(11)(-), was obtained in the course of our continuing studies of the low-melting salts of closo- and nido-carborane cage anions with alkylpyridinium and dialkylimidazolium cations. The title compound is the first example of a pyridinium salt of a perchlorinated carborane anion. The structure consists of one N-butylpyridinium cation counterbalanced by one perchlorinated carborane cage anion per asymmetric unit. By changing the counter-ion, different packings are observed, and to try to understand this the new structure is compared with five similar compounds.

Chloro-bis-(naphthalen-1-yl)phosphane.

In the title compound, C(20)H(14)ClP, the dihedral angle between the naphthyl rings is 81.77 (6)°. The crystal packing suggests weak π-π stacking inter-actions between the naphthyl rings in adjacent units [minimum ring centroid separation 3.7625 (13) Å].

Updating NO•/HNO interconversion under physiological conditions: A biological implication overview.

Azanone (HNO/NO-), also called nitroxyl, is a highly reactive compound whose biological role is still a matter of debate. A key issue that remains to be clarified regarding HNO and its biological activity is that of its endogenous formation. Given the overlap of the molecular targets and reactivity of nitric oxide (NO•) and HNO, its chemical biology was perceived to be similar to that of NO• as a biological signaling agent. However, despite their closely related reactivity, NO• and HNO's biochemical pathways are quite different. Moreover, the reduction of nitric oxide to azanone is possible but necessarily coupled to other reactions, which drive the reaction forward, overcoming the unfavorable thermodynamic barrier. The mechanism of this NO•/HNO interplay and its downstream effects in different contexts were studied recently, showing that more than fifteen moderate reducing agents react with NO• producing HNO. Particularly, it is known that the reaction between nitric oxide and hydrogen sulfide (H2S) produces HNO. However, this rate constant was not reported yet. In this work, firstly the NO•/H2S effective rate constant was measured as a function of the pH. Then, the implications of these chemical (non-enzymatic), biologically compatible, routes to endogenous HNO formation was discussed. There is no doubt that HNO could be (is?) a new endogenously produced messenger that mediates specific physiological responses, many of which were attributed yet to direct NO• effects.

A surface effect allows HNO/NO discrimination by a cobalt porphyrin bound to gold.

Nitroxyl (HNO) is a small short-lived molecule for which it has been suggested that it could be produced, under certain cofactors conditions, by nitric oxide (NO) synthases. Biologically relevant targets of HNO are heme proteins, thiols, molecular oxygen, NO, and HNO itself. Given the overlap of the targets and reactivity between NO and HNO, it is very difficult to discriminate their physiopathological role conclusively, and accurate discrimination between them still remains critical for interpretation of the ongoing research in this field. The high reactivity and stability of cobalt(II) porphyrins toward NO and the easy and efficient way of covalently joining porphyrins to electrodes through S-Au bonds prompted us to test cobalt(II) 5,10,15,20-tetrakis[3-(p-acetylthiopropoxy)phenyl]porphyrin [Co(P)], as a possible candidate for the electrochemical discrimination of both species. For this purpose, first, we studied the reaction between NO, NO donors, and commonly used HNO donors, with Co(II)(P) and Co(III)(P). Second, we covalently attached Co(II)(P) to gold electrodes and characterized its redox and structural properties by electrochemical techniques as well as scanning tunneling microscopy, X-ray photoelectron spectroscopy, and solid-state density functional theory calculations. Finally, we studied electrochemically the NO and HNO donor reactions with the electrode-bound Co(P). Our results show that Co(P) is positioned over the gold surface in a lying-down configuration, and a surface effect is observed that decreases the Co(III)(P) (but not Co(III)(P)NO(-)) redox potential by 0.4 V. Using this information and when the potential is fixed to values that oxidize Co(III)(P)NO(-) (0.8 V vs SCE), HNO can be detected by amperometric techniques. Under these conditions, Co(P) is able to discriminate between HNO and NO donors, reacting with the former in a fast, efficient, and selective manner with concomitant formation of the Co(III)(P)NO(-) complex, while it is inert or reacts very slowly with NO donors.

Synthesis and crystallographic, spectroscopic and computational characterization of 3,3',4,4'-substituted biphenyls: effects of OR substituents on the intra-ring torsion angle.

Presented here are the synthesis, characterization and study (using single crystal X-ray diffraction, Raman scattering, quantum mechanics calculations) of the structures of a series of biphenyls substituted in positions 3, 3', 4 and 4' with a variety of R (R = methyl, acetyl, hexyl) groups connected to the biphenyl core through oxygen atoms. The molecular conformation, particularly the torsion angle between aromatic rings has been extensively studied both in the solid as well as in the liquid state. The results show that the compounds appearing as rigorously planar in the solid present instead a twisted conformation in the melt. The solid versus melt issue strongly suggests that the reasons for planarity are to be found in the packing restraints. A `rule of thumb' is suggested for the design of biphenyls with different molecular conformations, based on the selection of the OR substituent.

Two rhodamine 6G derivative compounds: a structural and fluorescence single-crystal study.

The synthesis, characterization, structural analysis and fluorescence properties of two rhodamine 6G derivatives are described, namely a propargylamine derivative, 3',6'-bis(ethylamino)-2',7'-dimethyl-2-(methylcyanide)spiro[isoindole-1,9'-xanthen]-3(2H)-one (I), and a γ-aminobutyric acid (GABA) derivative, 3',6'-bis(ethylamino)-2',7'-dimethyl-3-oxospiro[isoindole-1,9'-xanthen]-2(3H)-yl)butyricacid (II). Both structures are compared with four similar ones from the Cambridge Structural Database (CSD), and the interactions involved in the stabilization are analyzed using the atoms in molecules (AIM) theory. Finally, a single-crystal in-situ reaction study is presented, carried out by fluorescence methods, which enabled the `opening' of the spirolactam ring in the solid phase.

Br...Br and van der Waals interactions along a homologous series: crystal packing of 1,2-dibromo-4,5-dialkoxybenzenes.

The crystalline structures of four homologues of the 1,2-dibromo-4,5-dialkoxybenzene series [Br2C6H2(OCnH2n + 1)2 for n = 2, 12, 14 and 18] have been solved by means of single-crystal crystallography. Comparison along the series, including the previously reported n = 10 and n = 16 derivatives, shows a clear metric trend (b and c essentially fixed along the series and a growing linearly with n), in spite of some subtle differences in space groups and/or packing modes. A uniform packing pattern for the aliphatic chains has been found for the n = 12 to 18 homologues, which slightly differs from that of the n = 10 derivative. The crystalline structures of all the higher homologues (n = 10-18) seem to arise from van der Waals interchain interactions and, to a lesser extent, type II Br...Br interactions. The dominant role of interchain interactions provides direct structural support for the usual interpretation of melting point trends like that found along this series. Atoms in Molecules (AIM) analysis allows a comparison of the relative magnitude of the interchain and Br...Br interactions, an analysis validated by the measured melting enthalpies.