Inhibition of kynurenine production by N,O-substituted hydroxylamine derivatives.

The inhibition of kynurenine production is considered a promising target for cancer immunotherapy. In this study, an amino acid derivative, compound 1 was discovered using a cell-based assay with our screening library. Compound 1 suppressed kynurenine production without inhibiting indoleamine 2,3-dioxygenase 1 (IDO1) activity. The activity of 1 was derived from the inhibition of IDO1 by a metabolite of 1, O-benzylhydroxylamine (OBHA, 2a). A series of N-substituted 2a derivatives that exhibit potent activity in cell-based assays may represent effective prodrugs. Therefore, we synthesized and evaluated novel N,O-substituted hydroxylamine derivatives. The structure-activity relationships revealed that N,O-substituted hydroxylamine 2c inhibits kynurenine production in a cell-based assay. We conducted an in vivo experiment with 2c, although the effectiveness of O-substituted hydroxylamine derivatives in vivo has not been previously reported. The results indicate that N,O-substituted hydroxylamine derivatives are promising IDO1 inhibitors.

Digital colloid-enhanced Raman spectroscopy by single-molecule counting.

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents1-5. Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred6. In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures7. However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations8. Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health.

Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

Hydroxylamine facilitated catalytic degradation of methylene blue in a Fenton-like system for heat-treatment modified drinking water treatment residues.

Rational treatment of drinking water treatment residues (WTR) has become an environmental and social issue due to the risk of secondary contamination. WTR has been commonly used to prepare adsorbents because of its clay-like pore structure, but then requires further treatment. In this study, a Fenton-like system of H-WTR/HA/H2O2 was constructed to degrade organic pollutants in water. Specifically, WTR was modified by heat treatment to increase its adsorption active site, and to accelerate Fe(III)/Fe(II) cycling on the catalyst surface by the addition of hydroxylamine (HA). Moreover, the effects of pH, HA and H2O2 dosage on the degradation were discussed with methylene blue (MB) as the target pollutant. The mechanism of the action of HA was analyzed and the reactive oxygen species in the reaction system were determined. Combined with the reusability and stability experiments, the removal efficiency of MB remained 65.36% after 5 cycles. Consequently, this study may provide new insights into the resource utilization of WTR.

Chemical Cyclic Amplification: Hydroxylamine Boosts the Fenton Reaction for Versatile and Scalable Biosensing.

Nucleic acid detection is undoubtedly one of the most important research fields to meet the medical needs of genetic disease diagnosis, cancer treatment, and infectious disease prevention. However, the practical detection methods based on biological amplification are complex and time-consuming and require highly trained operators. Herein, we report a simple, rapid, and sensitive method for the nucleic acid assay by fluorescence or naked eye using chemical cyclic amplification. The addition of hydroxylamine (HA) during the Fenton reaction can continuously generate hydroxyl radicals (•OH) via Fe3+/Fe2+ cycle, termed as "hydroxylamine boosts the Fenton reaction (Fenton-HA system)". Meanwhile, the reducing substances, such as terephthalic acid or o-phenylenediamine, react with •OH to generate oxidized substances that can be recognized by the naked eye or detected by fluorescence so as to realize the detection of Fe3+. The concentration of Fe3+ has a good linear relationship with fluorescence intensity in the range of 0.1 to 100 nM, and the limit of detection is calculated to be 0.03 nM (S/N = 3). Subsequently, Fe was introduced into the nucleic acid hybridization system after the Fe source was transformed into Fe3+, and the nucleic acids were indirectly determined by this method. This Fenton-HA system was used for sensing HIV-DNA and miRNA-21 to verify the validity of this method in nucleic acid detection. The detection limits were as low as 2.5 pM for HIV-DNA and 3 pM for miRNA-21. We believe that our work has unlocked an efficient signal amplification strategy, which is expected to develop a new generation of highly sensitive chemical biosensors.

Model simulation and mechanism of Fe(0/II/III) cycle activated persulfate degradation of methylparaben based on hydroxylamine enhanced nano-zero-valent iron.

The mechanism of Fe2+-activated peroxodisulfate (PDS) by hydroxylamine (HA) has been investigated, however, nano zero-valent iron-activated persulfate (nZVI/PDS) has a more optimal effect and needs further investigation. This study investigated the addition of HA to nZVI/PDS to improve Fe2+ regeneration and accelerate methylparaben (MP) degradation by Fe (0/II/III) cycle. After 60 min of reaction, the HA-enhanced nZVI/PDS (HA/nZVI/PDS) system afforded a 21% increase in MP degradation, reaching 93.26% (1 mM HA, 1 mM nZVI, and 2 mM PDS). nZVI/PDS system was a second-order reaction, but after adding HA, the reaction was more suitable for the first-order reaction. The addition of HA effectively promoted the reduction of Fe3+ to Fe2+ to improve the effect and reaction rate of PDS degradation of MP (k increased from 0.0127 min-1 to 0.0198 min-1) and broadened the reaction pH range. The results of various characterizations of nZVI before and after the reaction revealed that nZVI changed from a spherical structure to a bundle structure and was slightly oxidized. Changes in the Fe2+ and Fe3+ concentrations as well as in the pH of the reaction systems were monitored and the possible reactions of the HA/nZVI/PDS system were derived for the first time (knZVI/PDS<3.7 × 106 M-1 s-1, kFe3+/NH2O· >4.2 min-1). 12 potential compounds were investigated and MP breakdown pathways were speculated; hydroxylation was determined to be the most important pathway of degradation. And the HA/nZVI/PDS system had universal applicability.

The role of Fe dissolution in olivine-hydroxylamine-induced Fenton reaction for enhanced oxidative degradation of organic pollutant.

In this study, a dye pollutant (methyl orange, MO) was effectively oxidized in a hydroxylamine (HA)-assisted Fenton system using various Al/Si/Fe- and Fe-containing minerals. The fastest degradation kinetics of MO were observed in the olivine-HA Fenton system, whereas other Al/Si/Fe and Fe-rich minerals (magnetite and lepidocrocite) demonstrated much slower degradation kinetics. The degradation rate constants were proportional to dissolved Fe(II) quantities in mineral suspensions (R2 = 0.98), indicating the crucial role of dissolved Fe(II) quantity in HA-assisted Fenton reactions. Radical scavenging and electron spin resonance results revealed that MO was dominantly oxidized by ·HO produced in the olivine-HA Fenton system. The continuous production of aqueous Fe(II) via direct Fe(II) dissolution at a pH of 3 and further Fe dissolution from the reductive dissolution of surface Fe(III) by HA was the main driving force for efficient MO degradation. Furthermore, lowering the pH by the addition of hydroxylamine hydrochloride resulted in the effective removal of MO under various pH conditions (3-9), indicating the additional advantage of HA use in Fenton reactions. Liquid chromatography-mass spectroscopy analysis revealed that the cleavage of C-N and C-C bonds, demethylation, hydroxylation, and dehydroxylation were the main processes for MO oxidation in the olivine-HA Fenton system.

Gene expression analysis of Alcaligenes faecalis during induction of heterotrophic nitrification.

Alcaligenes faecalis is a heterotrophic nitrifying bacterium that oxidizes ammonia and generates nitrite and nitrate. When A. faecalis was cultivated in a medium containing pyruvate and ammonia as the sole carbon and nitrogen sources, respectively, high concentrations of nitrite accumulated in the medium whose carbon/nitrogen (C/N) ratio was lower than 10 during the exponential growth phase, while the accumulation was not observed in the medium whose C/N ratio was higher than 15. Comparative transcriptome analysis was performed using nitrifying and non-nitrifying cells of A. faecalis cultivated in media whose C/N ratios were 5 and 20, respectively, to evaluate the fluctuations of gene expression during induction of heterotrophic nitrification. Expression levels of genes involved in primary metabolism did not change significantly in the cells at the exponential growth phase under both conditions. We observed a significant increase in the expression levels of four gene clusters: pod cluster containing the gene encoding pyruvic oxime dioxygenase (POD), podh cluster containing the gene encoding a POD homolog (PODh), suf cluster involved in an iron-sulfur cluster biogenesis, and dnf cluster involved in a novel hydroxylamine oxidation pathway in the nitrifying cells. Our results provide valuable insight into the biochemical mechanism of heterotrophic nitrification.

Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Complexes of Formaldehyde and α-Dicarbonyls with Hydroxylamine: FTIR Matrix Isolation and Theoretical Study.

The interactions of formaldehyde (FA), glyoxal (Gly) and methylglyoxal (MGly) with hydroxylamine (HA) isolated in solid argon and nitrogen were studied using FTIR spectroscopy and ab initio methods. The spectra analysis indicates the formation of two types of hydrogen-bonded complexes between carbonyl and hydroxylamine in the studied matrices. The cyclic planar complexes are stabilized by O-H⋯O(C), and C-H⋯N interactions and the nonplanar complexes are stabilized by O-H⋯O(C) bond. Formaldehyde was found to form with hydroxylamine, the cyclic planar complex and methylglyoxal, the nonplanar one in both argon and nitrogen matrices. In turn, glyoxal forms with hydroxylamine the most stable nonplanar complex in solid argon, whereas in solid nitrogen, both types of the complex are formed.

Hydroxylamine driven advanced oxidation processes for water treatment: A review.

Hydroxylamine (HA) driven advanced oxidation processes (HAOPs) for water treatment have attracted extensive attention due to the acceleration of reactive intermediates generation and the improvement on the elimination effectiveness of target contaminants. In this review, HAOPs were categorized into three parts: (1) direct reaction of HA with oxidants (e.g., hydrogen peroxide (H2O2), peroxymonosulfate (PMS), ozone (O3), ferrate (Fe(VI)), periodate (IO4-)); (2) HA driven homogeneous Fenton/Fenton-like system (Fe(II)/peroxide/HA system, Cu(II)/O2/HA system, Cu(II)/peroxide/HA system, Ce(IV)/H2O2/HA system); (3) HA driven heterogeneous Fe/Cu-Fenton/Fenton-like system (iron-bearing material/peroxide/HA system, copper-bearing material/peroxide/HA system, bimetallic composite/peroxide/HA system). Degradation efficiency of the target pollutant, reactive intermediates, and effective pH range of various HAOPs were summarized. Further, corresponding reaction mechanism was elaborated. For the direct reaction of HA with oxidants, improvement of pollutants degradation was achieved through the generation of secondary reactive intermediates which had higher reactivity compared with the parent oxidant. For HA driven homogeneous and heterogeneous Fe/Cu-Fenton/Fenton-like system, improvement of pollutants degradation was achieved mainly via the acceleration of redox cycle of Fe(III)/Fe(II) or Cu(II)/Cu(I) and subsequent generation of reactive intermediates, which avoided the drawbacks of classical Fenton/Fenton-like system. In addition, HA driven homogeneous Fe/Cu-Fenton/Fenton-like system with heterogeneous counterpart were compared. Further, formation of oxidation products from HA in various HAOPs was summarized. Finally, the challenges and prospects in this field were discussed.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2-. Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

Hydroxylamine Complexes of Cytochrome c': Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

Design and Scalable Synthesis of N-Alkylhydroxylamine Reagents for the Direct Iron-Catalyzed Installation of Medicinally Relevant Amines*.

Secondary and tertiary alkylamines are privileged substance classes that are often found in pharmaceuticals and other biologically active small molecules. Herein, we report their direct synthesis from alkenes through an aminative difunctionalization reaction enabled by iron catalysis. A family of ten novel hydroxylamine-derived aminating reagents were designed for the installation of several medicinally relevant amine groups, such as methylamine, morpholine and piperazine, through the aminochlorination of alkenes. The method has excellent functional group tolerance and a broad scope of alkenes was converted to the corresponding products, including several drug-like molecules. Besides aminochlorination, the installation of other functionalities through aminoazidation, aminohydroxylation and even intramolecular carboamination reactions, was demonstrated, further highlighting the broad potential of these new reagents for the discovery of novel amination reactions.

Removing Formaldehyde-Induced Peptidyl Crosslinks Enables Mass Spectrometry Imaging of Peptide Hormone Distributions from Formalin-Fixed Paraffin-Embedded Tissues.

Linking molecular and chemical changes to human disease states depends on the availability of appropriate clinical samples, mostly preserved as formalin-fixed paraffin-embedded (FFPE) specimens stored in tissue banks. Mass spectrometry imaging (MSI) enables the visualization of the spatiotemporal distribution of molecules in biological samples. However, MSI is not effective for imaging FFPE tissues because of the chemical modifications of analytes, including complex crosslinking between nucleophilic moieties. Here we used an MS-compatible inorganic nucleophile, hydroxylamine hydrochloride, to chemically reverse inter- and intra-crosslinks from endogenous molecules. The analyte restoration appears specific for formaldehyde-reactive amino acids. This approach enabled the MSI-assisted localization of pancreatic peptides expressed in the alpha, beta, and gamma cells. Pancreatic islet-like distributions of islet hormones were observed in human FFPE tissues preserved for more than five years, demonstrating that samples from biobanks can effectively be investigated with MSI.

ESR Method in Monitoring of Nanoparticle Endocytosis in Cancer Cells.

Magnetic nanoparticles are extensively studied for their use in diagnostics and medical therapy. The behavior of nanoparticles after adding them to cell culture is an essential factor (i.e., whether they attach to a cell membrane or penetrate the membrane and enter into the cell). The present studies aimed to demonstrate the application of electron spin resonance (ESR) as a suitable technique for monitoring of nanoparticles entering into cells during the endocytosis process. The model nanoparticles were composed of magnetite iron (II, III) oxide core functionalized with organic unit containing nitroxide radical 4-hydroxy-TEMPO (TEMPOL). The research studies included breast cancer cells, as well as model yeast and human microvascular endothelial cells. The results confirmed that the ESR method is suitable for studying the endocytosis process of nanoparticles in the selected cells. It also allows for direct monitoring of radical cellular processes.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

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.

Enzymatic Synthesis of Ricinoleyl Hydroxamic Acid Based on Commercial Castor Oil, Cytotoxicity Properties and Application as a New Anticancer Agent.

BACKGROUND: New anticancer agents that rely on natural/healthy, not synthetic/toxic, components are very much needed. METHODS: Ricinoleyl hydroxamic acid (RHA) was synthesized from castor oil and hydroxylamine using Lipozyme TL IM as a catalyst. To optimize the conversion, the effects of the following parameters were investigated: type of organic solvent, period of reaction, amount of enzyme, the molar ratio of reactants and temperature. The highest conversion was obtained when the reaction was carried out under the following conditions: hexane as a solvent; reaction period of 48 hours; 120 mg of Lipozyme TL IM/3 mmol oil; HA-oil ratio of 19 mmol HA/3 mmol oil; and temperature of 40°C. The cytotoxicity of the synthesized RHA was assessed using human dermal fibroblasts (HDF), and its application towards fighting cancer was assessed using melanoma and glioblastoma cancer cells over a duration of 24 and 48 hours. RESULTS: RHA was successfully synthesized  and it demonstrated strong anticancer activity against glioblastoma and melanoma cells at as low as a 1 µg/mL concentration while it did not demonstrate any toxicity against HDF cells. CONCLUSION: This is the first report on the synthesis of RHA with great potential to be used as a new anticancer agent.