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S Bruce King

Publications and source records attributed to S Bruce King.

At least 19 recordsLinked to original sources
PubMed2006

Allylic nitro compounds as nitrite donors.

Allylic nitro compounds were synthesized and evaluated as organic sources of nitrite and nitric oxide. Unactivated allylic nitro compounds do not spontaneously release nitrite and nucleophile promoted nitrite release is slow. However, 2-(nitromethyl)-cyclohex-1-ene-3-one spontaneously dissociates in buffer (pH = 7.4) to release nitrite with a kobs = 1.6 x 10(-5) s(-1). In the presence of L-cysteine, this compound rapidly yields nitrite and reacts with hemoglobin similarly to sodium nitrite. Structural modifications and the nature and amount of nucleophile modulate the rate of nitrite release. In the presence of L-cysteine and ascorbic acid, this compound forms nitric oxide. Together, these results reveal a new structural architecture for the tunable liberation of nitrite and nitric oxide from organic compounds.

Molecular Structure↗
PubMed2006

Grignard reagent-mediated conversion of an acyl nitroso-anthracene cycloadduct to a nitrone.

An intramolecular hetero-Diels-Alder cycloadduct of an acyl nitroso compound and a 9,10-dimethyl anthracene derivative was prepared as a potential nitroxyl (HNO) donor. This compound did not release HNO under any of the conditions tested. Treatment of this cycloadduct with excess MeMgCl resulted in the formation of a nitrone, whose structure was confirmed by X-ray crystallography. A mechanism where MeMgCl acts as a nucleophile, strong base, and Lewis acid possibly explains the formation of this product.

Anthracenes↗
PubMed2006

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.

Animals↗
PubMed2006

Modulation of nitrated lipid signaling by multidrug resistance protein 1 (MRP1): glutathione conjugation and MRP1-mediated efflux inhibit nitrolinoleic acid-induced, PPARgamma-dependent transcription activation.

Recent data has shown that nitrolinoleic acid (LNO(2)), an electrophilic derivative of linoleic acid, has several important bioactivities including antiinflammatory, antiplatelet, vasorelaxation, and-as a novel potent ligand of PPARgamma-transcription regulating activities. Moreover, LNO(2) is formed in abundance in vivo at levels sufficient to mediate these bioactivities. In order to investigate the role of glutathione conjugation and MRP1-mediated efflux in the regulation of PPARgamma-dependent LNO(2) signaling, regioisomers of LNO(2) were synthesized and characterized. Analysis by 1D and 2D (1)H and (13)C NMR revealed that the LNO(2) preparation consisted of four, rather than two, nitrated regioisomers in approximately equal abundance. At physiologic pH and intracellular glutathione levels, LNO(2) was rapidly and quantitatively converted to glutathione conjugates (LNO(2)-SG) via Michael addition. MRP1 mediated efficient ATP-dependent transport of LNO(2)-SG. Using a PPRE-containing reporter gene transiently transfected into MRP-poor MCF7/WT cells, we verified that the LNO(2) mixture was a potent activator of PPARgamma-dependent transcription. However, expression of MRP1 in the stably transduced MCF7 derivative, MCF7/MRP1-10, resulted in strong inhibition of LNO(2)-induced transcription activation. Taken together, these results suggest that glutathione conjugation and MRP1-mediated conjugate transport can attenuate LNO(2) bioactivity and thereby play important roles in the regulation of cellular signaling by LNO(2).

Cell Membrane↗
PubMed2006

Regio- and stereospecific syntheses and nitric oxide donor properties of (E)-9- and (E)-10-nitrooctadec-9-enoic acids.

[reaction: see text] Nitrated fatty acids act as endogenous peroxisome proliferator-activated receptor gamma (PPARgamma) ligands and nitric oxide (NO) donors. We describe the first specific preparation of the two regioisomers of nitrooleic acid, (E)-9-nitrooctadec-9-enoic acid (1) and (E)-10-nitrooctadec-9-enoic acid (2), from cis-cyclooctene and monomethyl azelate, respectively. These syntheses rely upon a Henry condensation between a nine-carbon nitro component and a nine-carbon aldehyde. Preliminary chemiluminescence NO detection studies reveal the ability of these nitrated fatty acids to release NO.

Luminescence↗
PubMed2006

Hemoglobin effects in the Saville assay.

There is a great need to establish accurate, sensitive methods for measuring the concentration of nitrosothiols. Although some progress may have been made recently, differing methodologies have lead to reports of basal levels of nitrosothiols in human plasma that differ by three orders of magnitude. The Saville assay has been widely accepted as an accurate method for measuring nitrosothiols, but one that suffers from sensitivity below that of some other methods. Recently, it has been suggested that when hemoglobin is included in reaction mixtures used for the Saville assay, the sensitivity can be increased by an order of magnitude. Here we show that, on the contrary, the presence of sufficient hemoglobin in the Saville assay decreases its sensitivity.

Azo Compounds↗
PubMed2006

Rat liver-mediated metabolism of hydroxyurea to nitric oxide.

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography-mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.

Animals↗
PubMed2005

Discriminating formation of HNO from other reactive nitrogen oxide species.

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.

Arginine↗
PubMed2005

N-Hydroxy sulfonimidamides as new nitroxyl (HNO) donors.

Chlorination and condensation of simple sulfinamides with O-benzyl and O-tert-butyl dimethyl siloxy hydroxylamine gives O-protected N-hydroxy sulfonimidamides. Deprotection of these compounds produces the corresponding sulfinamide and nitrous oxide, which provides evidence for the intermediacy of nitroxyl (HNO) and identifies these compounds as new potential HNO donors.

Cytochrome P-450 Enzyme System↗
PubMed2005

Rates of nitric oxide dissociation from hemoglobin.

Nitric oxide (NO) plays a major role in human physiology and in many pathological states. Although oxyhemoglobin is known to destroy NO activity, NO activity can, in principle, be conserved through iron nitrosylation at vacant hemes. In order for this NO activity to be delivered, the NO must dissociate from the heme. Despite its study over the past few decades, our understanding of NO dissociation from hemoglobin is incomplete. In principle, there are at least four NO dissociation rates: kR(alpha), kR(beta), kT(alpha), and kT(beta), where the subscript refers to the quaternary state and the superscript to the hemoglobin chain. In the T-state, a proportion of the proximal histidine bonds break forming pentacoordinate alpha-nitrosyl hemoglobin. In vivo, alpha-nitrosyl hemoglobin predominates over beta-nitrosyl hemoglobin. In this study we have used a fast NO trap, Fe(II)-proline-dithiocarbamate, to measure NO dissociation rates from hemoglobin. We have varied solution conditions so the rate of dissociation from pentacoordinate alpha-nitrosyl hemoglobin could be definitively measured for the first time; kT(alpha) = 4.2 +/- 1.5 x 10(-4) s(-1). We have also found that the fastest NO dissociation rate is on the order of 10(-3) s(-1) and that NO dissociation from sickle cell hemoglobin is the same as that from normal adult hemoglobin.

Electron Spin Resonance Spectroscopy↗
PubMed2005

N-hydroxyurea and acyl nitroso compounds as nitroxyl (HNO) and nitric oxide (NO) donors.

Hydroxyurea has emerged as a new therapy for sickle cell disease but a complete mechanistic description of its beneficial actions does not exist. Patients taking hydroxyurea show evidence for the in vivo conversion of hydroxyurea to nitric oxide (NO), which also has drawn interest as a sickle cell disease treatment. While the chemical oxidation of hydroxyurea produces NO or NO-related products, NO formation from the reactions of hydroxyurea and hemoglobin do not occur fast enough to account for the observed increases in patients taking hydroxyurea. Both horseradish peroxidase and catalase catalyze the rapid formation of nitric oxide and nitroxyl (HNO) from hydroxyurea. In these reactions, hydroxyurea is converted to an acyl nitroso species that hydrolyzes to form HNO. The ferric heme protein then oxidizes HNO to NO that combines with the heme iron to form a ferrous-NO complex that may act as an NO donor. In general, acyl nitroso compounds, regardless of the method of their preparation, hydrolyze to form HNO and the corresponding carboxylic acid derivative. Similarly, the incubation of blood and hydroxyurea with urease rapidly form NO-related species suggesting the initial urease-mediated hydrolysis of hydroxyurea to hydroxylamine, which then reacts quickly with hemoglobin to form these products. These studies present two NO releasing mechanisms from hydroxyurea that are kinetically competent with clinical observations.

Animals↗
PubMed2004

Nitroxyl (HNO) release from new functionalized N-hydroxyurea-derived acyl nitroso-9,10-dimethylanthracene cycloadducts.

A thermal retro-Diels-Alder decomposition of N-hydroxyurea-derived acyl nitroso compounds and 9,10-dimethylanthracene cycloadducts followed by acyl nitroso compound hydrolysis produces nitrous oxide, evidence for the formation of nitroxyl, the one-electron reduced form of nitric oxide that has drawn considerable attention for its potential roles in biological systems. EPR and NMR spectroscopy provide further evidence for nitroxyl formation and kinetic information, respectively. Such compounds may prove to be useful alternative nitroxyl donors.

Anthracenes↗
PubMed2004

Iron nitrosyl hemoglobin formation from the reaction of hydroxylamine and hemoglobin under physiological conditions.

Sickle cell disease patients receiving hydroxyurea (HU) therapy have shown increases in the production of nitric oxide (NO) metabolites, which include iron nitrosyl hemoglobin (HbNO), nitrite, and nitrate. However, the exact mechanism by which HU forms HbNO in vivo is not understood. Previous studies indicate that the reaction of oxyhemoglobin (oxyHb) or deoxyhemoglobin (deoxyHb) with HU are too slow to account for in vivo HbNO production. In this study, we show that the reaction of methemoglobin (metHb) with HU to form HbNO could potentially be fast enough to account for in vivo HbNO formation but competing reactions of either excess oxyHb or deoxyHb during the reaction reduces the likelihood that HbNO will be produced from the metHb-HU reaction. Using electron paramagnetic resonance (EPR) spectroscopy we have detected measurable amounts of HbNO and metHb during the reactions of oxyHb, deoxyHb, and metHb with excess hydroxylamine (HA). We also demonstrate HbNO and metHb formation from the reactions of excess oxyHb, deoxyHb, or metHb and HA, conditions that are more likely to mimic those in vivo. These results indicate that the reaction of hydroxylamine with hemoglobin produces HbNO and lend chemical support for a potential role for hydroxylamine in the in vivo metabolism of hydroxyurea.

Anemia, Sickle Cell↗
PubMed2004

Nitric oxide production from hydroxyurea.

Hydroxyurea is a relatively new treatment for sickle cell disease. A portion of hydroxyurea's beneficial effects may be mediated by nitric oxide, which has also drawn considerable interest as a sickle cell disease treatment. Patients taking hydroxyurea show a significant increase in iron nitrosyl hemoglobin and plasma nitrite and nitrate within 2 h of ingestion, providing evidence for the in vivo conversion of hydroxyurea to nitric oxide. Hydroxyurea reacts with hemoglobin to produce iron nitrosyl hemoglobin, nitrite, and nitrate, but these reactions do not occur fast enough to account for the observed increases in these species in patients taking hydroxyurea. This report reviews recent in vitro studies directed at better understanding the in vivo nitric oxide release from hydroxyurea in patients. Specifically, this report covers: (1) peroxidase-mediated formation of nitric oxide from hydroxyurea; (2) nitric oxide production after hydrolysis of hydroxyurea to hydroxylamine; and (3) the nitric oxide-producing structure-activity relationships of hydroxyurea. Results from these studies should provide a better understanding of the nitric oxide donor properties of hydroxyurea and guide the development of new hydroxyurea-derived nitric oxide donors as potential sickle cell disease therapies.

Anemia, Sickle Cell↗
PubMed2004

Catalase-mediated nitric oxide formation from hydroxyurea.

Hydroxyurea reduces the incidence of painful crises in patients with sickle cell disease and has recently been approved for the treatment of this condition. A number of in vitro studies show that the oxidation of hydroxyurea results in the formation of nitric oxide, which also has drawn considerable interest as a sickle cell disease therapy. While patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of catalase to catalyze the formation of nitrite and nitrate from hydroxyurea. Spectroscopic studies show that the reaction of hydroxyurea and catalase in the presence of a hydrogen peroxide generating system produces a ferrous-NO catalase complex. Trapping studies indicate the intermediacy of a nitroso species during this reaction. The proposed mechanism for this conversion includes initial hydrogen peroxide-dependent oxidation of hydroxyurea by catalase to form the nitroso species, hydrolysis of this nitroso species to produce nitroxyl, and reductive nitrosylation of the ferric heme of catalase by nitroxyl to yield the ferrous-NO catalase complex. Addition of Angeli's salt, a nitroxyl donor, to ferric catalase also produces the ferrous-NO catalase complex. Spectroscopic studies show that the ferrous-NO catalase complex releases nitric oxide as judged by the oxyhemoglobin assay and an NO specific EPR specific trap. These results demonstrate nitric oxide production from the ferric catalase oxidation of nitroxyl and identify a catalase-mediated pathway as a potential source of nitric oxide production from hydroxyurea.

Catalase↗
PubMed2003

Measurements of nitric oxide on the heme iron and beta-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation.

Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygen-linked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the beta-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an S-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or beta-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using 15N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted in vitro, we found no arterial/venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation.

Heme↗
PubMed2003

Hydroxyurea analogues as kinetic and mechanistic probes of the nitric oxide producing reactions of hydroxyurea and oxyhemoglobin.

Derivatives of N-hydroxyurea that contain an N-hydroxy group react with oxyhemoglobin to form methemoglobin and variable amounts of nitrite/nitrate. Compounds with an unsubstituted -NHOH group produce the most nitrite/nitrate, which provides evidence for nitric oxide formation. The rate of reaction of these N-hydroxyurea derivatives with oxyhemoglobin correlates well with that compound's oxidation potential. Aromatic N-hydroxyureas react 25-80-fold faster with oxyhemoglobin than with N-hydroxyurea, suggesting other N-hydroxyurea analogues may be superior nitric oxide donors. Electron paramagnetic resonance spectroscopy shows that the formation of a low-spin methemoglobin-hydroxyurea complex is critical for iron nitrosyl hemoglobin formation. These results show that iron nitrosyl hemoglobin formation from the reaction of hydroxyureas and hemoglobin requires an unsubstituted -NHOH group and that the nitrogen atom of the non-N-hydroxy group must contain at least a single hydrogen atom. These results should guide the development of new hydroxyurea-based nitric oxide donors and sickle cell disease therapies.

Electrochemistry↗