Generation Of HNO Research Articles

Developing Photoactive Coumarin-Caged N-Hydroxysulfonamides for Generation of Nitroxyl (HNO).

Photoactive N-hydroxysulfonamides photocaged with the (6-bromo-7-hydroxycoumarin-4-yl)methyl chromophore have been successfully synthesized, and the mechanisms of photodecomposition investigated for two of the compounds. Upon irradiation up to 97% of a diagnostic marker for (H)NO release, sulfinate was observed for the trifluoromethanesulfonamide system. In the absence of a species that reacts rapidly with (H)NO, (H)NO instead reacts with the carbocation intermediate to ultimately generate (E)-BHC-oxime and (Z)-BHC-oxime. Alternatively, the carbocation intermediate reacts with solvent water to give a diol. Deprotonation of the N(H) proton is required for HNO generation via concerted C-O/N-S bond cleavage, whereas the protonation state of the O(H) does not affect the observed photoproducts. If the N(H) is protonated, C-O bond cleavage to generate the parent N-hydroxysulfonamide will occur, and/or O-N bond cleavage to generate a sulfonamide. The undesired competing O-N bond cleavage pathway increases when the volume percentage of water in acetonitrile/water solvent mixtures is increased.

Update on Endogenous HNO Production:An Exploration of Unanswered Questions and Challenges

This review consolidates the evidence supporting endogenous HNO generation through enzymatic and non‐enzymatic pathways, emphasizing advancements in real‐time HNO sensing within living systems. Azanone (nitroxyl, HNO) is the one‐electron‐reduced congener of nitric oxide (NO•) and shares various biological actions. HNO exhibits unique properties, including positive inotropic and lusitropic effects, along with resistance to scavenging by reactive oxygen species (ROS), particularly superoxide. Research on HNO has intensified over the last two decades, focusing on its reactivity and the prospect of endogenous formation due to its potential significance. The high reactivity of HNO arises from reactions with biologically relevant species such as oxygen, NO•, and thiols, as well as self‐dimerization. Detecting and quantifying HNO has posed challenges, initially relying on nitrous oxide (N2O) detection, a byproduct of dimerization. Recent advancements, including fluorescent probes and a specific electrochemical nanomolar‐level sensor, have facilitated direct detection, enhancing understanding of HNO reactivity and formation.

Effects of Reynolds number and ammonia fraction on combustion characteristics of premixed ammonia-hydrogen-air swirling flames

Several studies have investigated the effects of equivalence ratio and blending ratio in constant power cases for ammonia-based fuels as ammonia is being investigated extensively as an alternative zero-carbon fuel in recent years. However, in many fields, such as power generation, it is common to operate practical equipment at constant Reynolds number (Re) conditions. To that extent, this study investigates the impact of Re (4000 ≤ Re ≤ 7000), equivalence ratio (0.6 ≤ Φ ≤ 1.45), and ammonia volume ratio (0.55 ≤ XNH3 ≤ 0.9) on the combustion characteristics of NH3/H2 swirling flames. To supplement the experimental work, a previously developed Chemical Reactor Network (CRN) has been used to identify the reaction pathways affecting NOx emissions through reaction analysis. Sampled gas analysis (NO, NO2, N2O, NH3) at the exhaust was reported for the first time at a wide range of equivalence ratios and XNH3 at different Re. At constant Re = 6000, NO and NO2 peaked at around Φ = 0.8 – 0.9, while N2O became substantial at Φ ≤ 0.8 and unburnt ammonia became significant at Φ ≥ 1.05. The chemiluminescence images suggested that at Re = 6000, as XNH3 or Φ increases, the NH2* region expands, and reactions occur further downstream. Furthermore, with decreasing XNH3 at Re = 6000 and Φ = 0.9, the system becomes partially abundant in H2 and H but deficient in O2, suppressing the generation of O and OH radicals, and ultimately also suppressing HNO generation. This leads to the N/NH system reactions becoming dominant and increased NO consumption. NO emissions increased steadily with increasing Re at Φ = 0.8, while at stoichiometry, NO emissions remained somewhat unchanged with changing Re. Findings from this study will aid further in the development of carbon-free zero-emissions systems.

Highly Selective Nitrite Hydrogenation to Ammonia over Iridium Nanoclusters: Competitive Adsorption Mechanism.

Wet denitrification is a promising approach to control nitrogen oxides (NOx) produced in fossil fuel combustion. Yet, the highly concentrated nitrite (NO2-) wastewater generated poses a major threat to the aqueous environment. Here, iridium nanoclusters (d = 1.63 nm) deposited on TiO2 were applied for NO2- reduction to ammonia (NRA), showing an exceptional NH4+ selectivity of 95% and a production rate of 20.51 mgN·L-1·h-1, which held significant potential for NO2- wastewater purification and ammonia resource recovery. Notably, an interesting non-first-order NO2- hydrogenation kinetics was observed, which was further confirmed to result from the competitive adsorption mechanism between H2 and NO2- over iridium. The NRA pathways on the Ir(111) surface were explored via density functional theory calculations with the NO2-* → NO* → HNO* → HNOH* → H2NOH* → NH2* → NH3* identified as the most energetically favorable pathway and the NO* → HNO* confirmed as the rate-determining step. In situ DRIFTS further experimentally verified the generation of HNO* intermediate during NO* hydrogenation on Ir(111). To verify NRA kinetics at varied NO2- concentrations or H2 pressures, a kinetic model was derived based on the Langmuir-Hinshelwood competitive adsorption mechanism. These findings provide mechanistic insights into the NRA pathways on Ir nanocatalysts, which will be beneficial for wet denitrification waste stream decontamination and valorization.

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.

A robust ratiometric photoacoustic and fluorescence dual-modal molecular probe for capturing endogenous HNO generation in tumor mice model

HNO is reported in association with a wide variety of physiological and pathological states. However, photoacoustic (PA)/fluorescence (FL) dual-modal probes with complementary advantages for detection of HNO in living systems, especially in tumors, are still lacking. Herein, the first near-infrared (NIR) ratiometric PA/FL dual-modal probe (DOP-HNO), composed of a dual ratiometric molecular skeleton caged with 2-(diphenylphosphino)-benzoate group, was designed and synthesized for capturing endogenous HNO generation in vivo. DOP-HNO was able to specifically and sensitively respond to HNO with 4-fold ratiometric fluorescence enhancement (F783/F720) and 2.6-fold ratiometric PA enhancement (PA740/PA670). Moreover, DOP-HNO successfully visualized HNO fluctuations in 4T1 cells and living mice, and the crosstalk between NO and H2S in the tumors of living mice was clearly observed through NIR dual-ratiometric PA/FL imaging, indicating the accurate detection of HNO in vivo. More importantly, using DOP-HNO, for the first time, we provided the visualization evidence of endogenous HNO generation in cancer model and further investigated its possible generation mechanisms by H2S/NO interaction. Thus, we envision that the dual-modal probe DOP-HNO will be an effective tool to study the complex relationship between HNO and cancer as well as the pathology of other HNO-related diseases.

Discovery of endogenous nitroxyl as a new redox player in Arabidopsis thaliana

Nitroxyl (HNO) is the one-electron reduced and protonated congener of nitric oxide (•NO), owning a distinct chemical profile. Based on real-time detection, we demonstrate that HNO is endogenously formed in Arabidopsis. Senescence and hypoxia induce shifts in the redox balance, triggering HNO decay or formation mediated by non-enzymatic •NO/HNO interconversion with cellular reductants. The stimuli-dependent HNO generation supports or competes with •NO signalling, depending on the local redox environment.

Theoretical investigation of [Ru(bpy)2(HAT)]2+ (HAT = 1,4,5,8,9,12-hexaazatriphenylene; bpy = 2,2′-bipyridine): Photophysics and reactions in excited state

In this article, Density Functional Theory based calculations, including dispersion corrections, PBE0(D3BJ)/Def2-TZVP(-f), were performed to elucidate the photophysics of the [Ru(bpy)2(HAT)]2+ complex in water. In addition, the thermodynamics of the charge and electron transfer excited state reactions of this complex with oxygen, nitric oxide and Guanosine-5′-monophosphate nucleotide (GMP) were investigated. The first singlet excite state, S1, strongly couples with the second and third triplet excited states (T2 and T3) giving rise to a high intersystem crossing rate of 6.26 × 1011 s−1 which is ∼106 greater than the fluorescence rate decay. The thermodynamics of the excited reactions revealed that all electron transfer reactions investigated are highly favorable, due mainly to the high stability of the triply charged radical cation 2PS•3+ species formed after the electron has been transferred. Excited state electron transfer from the GMP nucleotide to the complex is also highly favorable (ΔGsol = −92.6 kcal/mol), showing that this complex can be involved in the photooxidation of DNA, in line with experimental findings. Therefore, the calculations allow to conclude that the [Ru(bpy)2(HAT)]2+ complex can act in Photodynamic therapy through both mechanisms type I and II, through electron transfer from and to the complex and triplet–triplet energy transfer, generating ROS, RNOS and through DNA photooxidation. In addition, the work also opens a perspective of using this complex for the in-situ generation of the singlet nitroxyl (1NO−) species, which can have important applications for the generation of HNO and may have, therefore, important impact for physiological studies involving HNO.

Azanone (HNO): generation, stabilization and detection.

HNO (nitroxyl, azanone), joined the ‘biologically relevant reactive nitrogen species’ family in the 2000s. Azanone is impossible to store due to its high reactivity and inherent low stability. Consequently, its chemistry and effects are studied using donor compounds, which release this molecule in solution and in the gas phase upon stimulation. Researchers have also tried to stabilize this elusive species and its conjugate base by coordination to metal centers using several ligands, like metalloporphyrins and pincer ligands. Given HNO's high reactivity and short lifetime, several different strategies have been proposed for its detection in chemical and biological systems, such as colorimetric methods, EPR, HPLC, mass spectrometry, fluorescent probes, and electrochemical analysis. These approaches are described and critically compared. Finally, in the last ten years, several advances regarding the possibility of endogenous HNO generation were made; some of them are also revised in the present work.

A divergent mode of activation of a nitrosyl iron complex with unusual antiangiogenic activity

Nitric oxide (NO) and nitroxyl (HNO) have gained broad attention due to their roles in several physiological and pathophysiological processes. Remarkably, these sibling species can exhibit opposing effects including the promotion of angiogenic activity by NO compared to HNO, which blocks neovascularization. While many NO donors have been developed over the years, interest in HNO has led to the recent emergence of new donors. However, in both cases there is an expressive lack of iron-based compounds. Herein, we explored the novel chemical reactivity and stability of the trans-[Fe(cyclam)(NO)Cl]Cl2 (cyclam = 1,4,8,11-tetraazacyclotetradecane) complex. Interestingly, the half-life (t1/2) for NO release was 1.8 min upon light irradiation, vs 5.4 h upon thermal activation at 37 °C. Importantly, spectroscopic evidence supported the generation of HNO rather than NO induced by glutathione. Moreover, we observed significant inhibition of NO donor- or hypoxia-induced HIF-1α (hypoxia-inducible factor 1α) accumulation in breast cancer cells, as well as reduced vascular tube formation by endothelial cells pretreated with the trans-[Fe(cyclam)(NO)Cl]Cl2 complex. Together, these studies provide the first example of an iron-nitrosyl complex with anti-angiogenic activity as well as the potential dual activity of this compound as a NO/HNO releasing agent, which warrants further pharmacological investigation.

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.

Mitochondria-targeting NIR fluorescent probe for rapid, highly sensitive and selective visualization of nitroxyl in live cells, tissues and mice

Nitroxyl (HNO) has been reported to possess unique biological and pharmacological performances, and emerged as a novel therapy for congestive heart failure. Recent studies also suggest that HNO may be produced and involved in important metabolisms in mitochondria. However, due to its high reactivity and short life properties, fast, sensitive and selective observation and monitoring of HNO related dynamic changes in mitochondria still remains a great challenge. Herein, we synthesized a mitochondria-targeting near-infrared (NIR) fluorescent probe (DCMHNO) for rapid detection of HNO with remarkably high sensitivity, selectivity and photostability. DCMHNO shows fast response (about 4 min) towards HNO via 2-(diphenylphosphino)benzoyl group through the Staudinger reaction to boost the bright NIR emission (700 nm) with excellent sensitivity (detection limit of 13 nM), high pH stability and very low interference from other species. DCMHNO can selectively locate in mitochondria and visualize exogenous and endogenous HNO in live HeLa cells with high biocompatibility and photostability. The probe could also monitor the interaction between NO and H2S that gives rise to the generation of HNO in live HeLa cells. In addition, DCMHNO was further utilized in ex vivo NIR imaging of HNO in live mouse liver tissues at the depth of about 50 µm. In vivo imaging of HNO with high signal-to-noise ratio in live mice was also realized by using DCMHNO. These remarkable imaging performances could render NIR DCMNHNO as a useful tool to reveal HNO related dynamic changes in live samples.

Synthesis and photochemical studies of 2-nitrobenzyl-caged N-hydroxysulfonamides

Recently, N-hydroxysulfonamides (RSO2NHOH) caged by photolabile protecting groups have attracted significant interest as potential photoactive nitroxyl (HNO) donors. The selectivity of the desired HNO generation pathway from photocaged N-hydroxysulfonamides versus a competing pathway involving O-N bond cleavage is dependent on the specific photodeprotection mechanism of the phototrigger. We present a new class of photocaged N-hydroxysulfonamides incorporating the well-established o-nitrobenzyl photoprotecting group, including a derivative incorporating an additional carbonate linker. Photodecomposition of o-NO2Bn-ON(H)SO2CF3 and the corresponding 2-nitro-4,5-dimethoxybenzyl analog generated the desired HNO and CF3SO2- as a minor pathway, with competing photoinduced O-N bond cleavage to release CF3SO2NH2 as the major photodecomposition pathway. Photolysis of the corresponding -SO2CH3 analogs resulted in O-N bond cleavage only. The presence of the o-nitro substituent was shown to be essential for photoactivity. Photorelease of the parent HNO donor CH3SO2NHOH was observed as the major product upon irradiation of o-NO2Bn-OC(O)ON(H)SO2CH3, with the desired HNO release and O-N bond cleavage occurring as minor pathways. Photoproduct quantum yields for each species have been determined by actinometry. The effect of solvent, pH and air on the mechanism of photodecomposition was studied for o-NO2Bn-ON(H)SO2CH3. The ratio of the solvents in the solvent mixture (CH3CN and phosphate buffer, pH 7.0), the pH of the aqueous component of the buffer, and the presence of oxygen did not affect the amount of each photoproduct and the observed rate constant for O-N bond cleavage. Possible mechanisms for the various pathways are proposed.

Development of Photoactivatable Nitroxyl (HNO) Donors Incorporating the (3‐Hydroxy‐2‐naphthalenyl)methyl Phototrigger

A new family of photoactivatable HNO donors of general structure RSO2NHO‐PT [where PT represents the (3‐hydroxy‐2‐naphthalenyl)methyl (3,2‐HMN) phototrigger] has been developed, which rapidly releases HNO. Photogeneration of HNO was demonstrated using the vitamin B12 derivative aquacobalamin as a trapping agent. The amount of sulfonate RSO2– produced was essentially the same as the amount of HNO released upon photolysis, providing a convenient method to indirectly quantify HNO release. Two competing pathways were also observed; a pathway involving O–N bond cleavage leading to release of a sulfonamide, and a pathway resulting in release of the parent Nhydroxysulfonamide RSO2NHOH (for HNO donors with Me‐ and Ph‐containing leaving groups only). Up to approximately 70 % of the HNO‐generating pathway was observed with the CF3‐containing leaving group, with HNO generation favored for small percentages of aqueous buffer in the acetonitrile/pH 7.00 phosphate buffer solvent mixture. Characterization of the photoproducts obtained from steady‐state irradiation by NMR spectroscopy showed that the desired HNO‐generating pathway was less favored for HNO donors with Me‐ and Ph‐containing leaving groups compared to the CF3‐containing leaving group, suggesting that the excellent CF3‐containing leaving group promotes HNO generation.

Rapid generation of HNO induced by visible light.

We present a new method for controlled generation of HNO, based on the combination of a pH photoactuator induced by visible light with an HNO donor activated by pH increase. This method avoids the use of UV light, and in the future could be extended by using an IR photoactuator.

Amphiphilic Copolymers Capable of Concomitant Release of HNO and Small Molecule Organics.

We demonstrate concomitant release of HNO and small molecule organics from amphiphilic poly(norbornene)-based copolymers. This key function was achieved by incorporation of thermally labile oxazine units within random and block copolymer architectures. Upon thermolysis, we observed generation of HNO and release of a small molecule conjugate. Importantly, the release kinetics of HNO and a UV-active small molecule (4-nitroaniline) were found to be 1:1, signifying an ability to monitor HNO production indirectly, or to simultaneously release organic therapeutics (e.g., nonsteroidal anti-inflammatory drugs) along with HNO. To our knowledge, these are the first reported polymeric materials demonstrating HNO release from covalently attached HNO donors.

Gender specific generation of nitroxyl (HNO) from rat endothelium

Nitric oxide (NO) has long been accepted as the majority biological mediator underlying the critically important vasodilator function of the vascular endothelium yet there is growing evidence that the protonated one-electron reduction species of NO, nitroxyl (HNO), may also have biological activity. In this study we examined if there was a gender variation in the production of HNO from the endothelium of isolated segments of rat aorta. By use of recognized pharmacological inhibitors, we found that when the endothelium was stimulated by acetylcholine vascular relaxation was mediated entirely via NO in tissue from both males and females. The vasorelaxation induced by basal (non-stimulated) endothelium from females was also mediated entirely by NO. However, in contrast, the influence of basally active endothelium in males was mediated by both NO and HNO. The generation of HNO in males was dependent on endothelial nitric oxide synthase and relaxation was mediated via activation of soluble guanylate cyclase. We believe that this is the first evidence of a gender variation in the production of HNO and this may have important implications for our understanding of disparity in the development of cardiovascular disease between the sexes.

Curtailing the hydroxylaminobarbituric acid-hydantoin rearrangement to favor HNO generation.

Due to its inherent reactivity, HNO must be generated in situ through the use of donor compounds. One of the primary strategies for the development of new HNO donors has been modifying hydroxylamines with good leaving groups. A recent example of this strategy is the (hydroxylamino)barbituric acid (HABA) class of HNO donors. In this case, however, an undesired intramolecular rearrangement pathway to the corresponding hydantoin derivative competes with HNO formation, particularly in the absence of chemical traps for HNO. This competitive non-HNO-producing pathway has restricted the development of the HABA class to examples with fast HNO release profiles at physiological pH and temperature (t(1/2) < 1 min). Herein, the factors that favor the rearrangement pathway have been examined and two independent strategies that protect against rearrangement to favor HNO generation have been developed. The timecourse and stoichiometry for the in vitro conversion of these compounds to HNO (trapped as a phosphine aza-ylide) and the corresponding barbituric acid (BA) byproduct have been determined by (1)H NMR spectroscopy under physiologically relevant conditions. These results confirm the successful extension of the HABA class of pure HNO donors with half-lives at pH 7.4, 37 °C ranging from 19 to 107 min.

Six-coordinate ferric porphyrins containing bidentate N-t-butyl-N-nitrosohydroxylaminato ligands: structure, magnetism, IR spectroelectrochemisty, and reactivity.

NONOates (diazeniumdiolates) containing the [X{N2O2}](-) functional group are frequently employed as nitric oxide (NO) donors in biology, and some NONOates have been shown to bind to metalloenzymes. We report the preparation, crystal structures, detailed magnetic behavior, redox properties, and reactivities of the first isolable alkyl C-NONOate complexes of heme models, namely (OEP)Fe(η(2)-ON(t-Bu)NO) (1) and (TPP)Fe(η(2)-ON(t-Bu)NO) (2) (OEP = octaethylporphyrinato dianion, TPP = tetraphenylporphyrinato dianion). The compounds display the unusual NONOate O,O-bidentate binding mode for porphyrins, resulting in significant apical Fe displacements (+0.60 Å for 1, and +0.69 Å for 2) towards the axial ligands. Magnetic susceptibility and magnetization measurements made from 1.8-300 K at magnetic fields from 0.02 to 5 T, yielded magnetic moments of 5.976 and 5.974 Bohr magnetons for 1 and 2, respectively, clearly identifying them as high-spin (S = 5/2) ferric compounds. Variable-frequency (9.4 GHz and 34.5 GHz) EPR measurements, coupled with computer simulations, confirmed the magnetization results and yielded more precise values for the spin Hamiltonian parameters: g(avg) = 2.00 ± 0.03, |D| = 3.89 ± 0.09 cm(-1), and E/D = 0.07 ± 0.01 for both compounds, where D and E are the axial and rhombic zero-field splittings. IR spectroelectrochemistry studies reveal that the first oxidations of these compounds occur at the porphyrin macrocycles and not at the Fe-NONOate moieties. Reactions of 1 and 2 with a histidine mimic (1-methylimidazole) generate RNO and NO, both of which may bind to the metal center if sterics allow, as shown by a comparative study with the Cupferron complex (T(p-OMe)PP)Fe(η(2)-ON(Ph)NO). Protonation of 1 and 2 yields N2O as a gaseous product, presumably from the initial generation of HNO that dimerizes to the observed N2O product.

Generation of HNO and HSNO from Nitrite by Heme‐Iron‐Catalyzed Metabolism with H2S

Nitrite has been shown in the past decade to be an important source of nitric oxide that acts as a vasodilator and intrinsic signaling molecule. Numerous studies have proved that nitrite can be reduced in vivo, either non-enzymatically or enzymatically, in reactions catalyzed by xanthine oxidase, deoxyhaemoglobin, deoxymyoglobin, cytochrome c, or by thiol and metal-center-assisted processes inside the cell. The mechanism of the last process has recently been studied in detail, and has demonstrated that thiols (cysteine and gluthathione) stimulate water-soluble Fe-porphyrins to have nitrite reductase activity through an oxygen atom transfer (OAT)mechanism (Scheme 1) that leads to increased