Electrophilic Oxidation Research Articles

Covalent Adduction of Human Serum Albumin by 4-Hydroxy-2-Nonenal: Kinetic Analysis of Competing Alkylation Reactions

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.

Electronic Structure and Reactivity of Isomeric Oxo-Mn(V) Porphyrins: Effects of Spin-State Crossing and pKa Modulation

The reactivity of the isomeric oxo-Mn(V)-2-tetra-N-methylpyridyl (2-TMPyP) and oxo-Mn(V)-4-tetra-N-methylpyridyl (4-TMPyP) porphyrins has been investigated by a combined experimental and theoretical approach based on density functional theory. The unusual higher reactivity of the more electron-rich 4-TMPyP species appears to be related to both the higher basicity of its oxo ligand, compared to that of the 2-TMPyP isomer, and the smaller low-spin-high-spin promotion energy of 4-TMPyP, compared to that of 2-TMPyP, because of the stabilization of the A2u orbital in the latter isomer. Therefore, in a two-state energy profile involving crossing of the initial singlet and final quintet potential energy surfaces, the 4-TMPyP isomer should be kinetically favored. The calculated differences in the singlet-quintet gaps for the 2-TMPyP and 4-TMPyP systems compare well with the measured differences in the activation energies for two isomeric porphyrins. Both effects, proton affinity and electron-promotion energy, contribute to reduce the reactivity of the more electrophilic oxidant when electron-withdrawing groups are closer to the active site, contrary to the usual expectations based on simple chemical reactivity correlations. These theoretical results are in accord with new experimental data showing O=Mn(V)-O-H pK(a)s of 7.5 and 8.6 for the isomeric 2-TMPyP and 4-TMPyP systems, respectively.

Reactivities of Mononuclear Non-Heme Iron Intermediates Including Evidence that Iron(III)−Hydroperoxo Species Is a Sluggish Oxidant

There is an intriguing, current controversy on the involvement of iron(III)-hydroperoxo species as a "second electrophilic oxidant" in oxygenation reactions by heme and non-heme iron enzymes and their model compounds. In the present work, we have performed reactivity studies of the iron-hydroperoxo species in nucleophilic and electrophilic reactions, with in situ-generated mononuclear non-heme iron(III)-hydroperoxo complexes that have been well characterized with various spectroscopic techniques. The intermediates did not show any reactivities in the nucleophilic (e.g., aldehyde deformylation) and electrophilic (e.g., oxidation of sulfide and olefin) reactions. These results demonstrate that non-heme iron(III)-hydroperoxo species are sluggish oxidants and that the oxidizing power of the intermediates cannot compete with that of high-valent iron(IV)-oxo complexes. We have also reported reactivities of mononuclear non-heme iron(III)-peroxo and iron(IV)-oxo complexes in the aldehyde deformylation and the oxidation of sulfides, respectively.

Induction of the permeability transition and cytochrome c release by 15-deoxy-Δ12,14-prostaglandin J2 in mitochondria

The electrophilic lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is known to allow adaptation to oxidative stress in cells at low concentrations and apoptosis at high levels. The mechanisms leading to adaptation involve the covalent modification of regulatory proteins, such as Keap1, and augmentation of antioxidant defences in the cell. The targets leading to apoptosis are less well defined, but mitochondria have been indirectly implicated in the mechanisms of cell death mediated by electrophilic lipids. To determine the potential of electrophilic cyclopentenones to induce pro-apoptotic effects in the mitochondrion, we used isolated liver mitochondria and demonstrated that 15d-PGJ2 promotes Ca2+-induced mitochondrial swelling and cytochrome c release. The mechanisms involved are consistent with direct modification of protein thiols in the mitochondrion, rather than secondary formation of reactive oxygen species or lipid peroxidation. Using proteomic analysis in combination with biotinylated 15d-PGJ2, we were able to identify 17 potential targets of the electrophile-responsive proteome in isolated liver mitochondria. Taken together, these results suggest that electrophilic lipid oxidation products can target a sub-proteome in mitochondria, and this in turn results in the transduction of the electrophilic stimulus to the cell through cytochrome c release.

Induction of mitochondrial ROS production by electrophilic lipids: a new pathway of redox signaling?

reactive oxygen species (ROS), such as superoxide or hydrogen peroxide, are usually thought of as damaging agents that nonspecifically disrupt proteins, lipids, and nucleic acids ([1][1]). However, there is now considerable evidence that ROS also act as signaling agents in a range of biological

ｖｉｃ‐ジスルホキシド結合をもつ化合物の合成と性質

vic-Disulfoxides have been recognized as very unstable intermediates in the electrophilic oxidation of disulfides. In 1999, we succeeded in the isolation and structure determination of tetrathiolane 2, 3-dioxide 11, which was the first isolable compound having an-S (O) -S (O) -moiety. After this study, we could prepare 1, 2, 4-trithiolane 1, 2-dioxides 12-14, 7, 8-dithiabicyclo [4.2.1] nona2, 4-diene 7, 8-dioxide 15, and dithiirane 1, 2-dioxide 16. The-S (O) -S (O) -moieties of 11-16 reside in a 5-or 3-membered ring, which should contribute to the stability of vic-disulfoxide bonds. Here we report the syntheses of vic-disulfoxides isolable under ambient conditions and their chemical and physical properties.

Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Delta12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.

Reactions of the Peroxo Intermediate of Soluble Methane Monooxygenase Hydroxylase with Ethers

Soluble methane monooxygenase (sMMO) isolated from Methylococcus capsulatus (Bath) utilizes a carboxylate-bridged diiron center and dioxygen to catalyze the conversion of methane to methanol. Previous studies revealed that a di(mu-oxo)diiron(IV) intermediate termed Q is responsible for the catalytic activity with hydrocarbons. In addition, the peroxodiiron(III) intermediate (H(peroxo)) that precedes Q formation in the catalytic cycle has been demonstrated to react with propylene, but its reactivity has not been extensively investigated. Given the burgeoning interest in the existence of multiple oxidants in metalloenzymes, a more exhaustive study of the reactivity of H(peroxo) was undertaken. The kinetics of single turnover reactions of the two intermediates with ethyl vinyl ether and diethyl ether were monitored by single- and double-mixing stopped-flow optical spectroscopy. For both substrates, the rate constants for reaction with H(peroxo) are greater than those for Q. An analytical model for explaining the transient kinetics is described and used successfully to fit the observed data. Activation parameters were determined through temperature-dependent studies, and the kinetic isotope effects for the reactions with diethyl ether were measured. The rate constants indicate that H(peroxo) is a more electrophilic oxidant than Q. We propose that H(peroxo) reacts via two-electron transfer mechanisms, and that Q reacts by single-electron transfer steps.

An Aerobic, Organocatalytic, and Chemoselective Method for Baeyer–Villiger Oxidation

Highly chemoselective Baeyer–Villiger oxidations can be performed in the presence of other reactive functionalities such as alcohols, olefins, and sulfides, which would undergo electrophilic oxidation under conventional conditions (see scheme). [DMRFlEt]+[ClO4]− (depicted blue) is a new class of flavin compound that catalyzes aerobic Baeyer–Villiger oxidations in the presence of Zn dust as the electron source.

Use of the Antioxidant BHT in Asymmetric Membrane Tablet Coatings to Stabilize the Core to the Acid Catalyzed Peroxide Oxidation of a Thioether Drug

The application of a controlled-release asymmetric membrane (AM) coating containing cellulose acetate and polyethylene glycol 3350 (PEG3350) to a stable osmotic tablet core resulted in the oxidative degradation of active ingredient located in the core. It was hypothesized that the production of hydroperoxides from PEG3350 in the coating was responsible for the electrophilic oxidation of drug to the sulfoxide degradation product. The type and solubility of carboxylic acid excipient used to formulate the drug release profile of the osmotic tablet significantly influenced the amount of oxidation. By adding the antioxidant butylated hydroxytoluene (BHT) to the coating, oxidation was significantly inhibited in tablets placed on accelerated stability. Of three additives that were used to prevent oxidation [BHT, ferrous sulfate, and ethylenediaminetetraacetic acid (EDTA)], BHT was shown to be the most effective at preventing sulfoxide formation. The BHT was also shown to be more effective in the coating rather than in the core due to its location closer to the source of the oxidizing species, PEG3350, in the coating.

Human Heme Oxygenase Oxidation of 5- and 15-Phenylhemes

Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.

Hydroperoxoferric heme intermediate as a second electrophilic oxidant in cytochrome P450-catalyzed reactions.

Experimental evidence supporting the catalytic activity of the peroxoferric and hydroperoxoferric cytochrome P450 intermediates as alternative oxidants to the compound I (ferryl) state in the oxygenation of organic substrates is reviewed. The peroxoferric P450 state is proposed to function as a nucleophile in the lyase step of the P450-aromatase reaction. Several systems are reviewed in which the hydroperoxoferric P450 intermediate likely functions as a second electrophilic oxidant, the "two-oxidants" model. These include alkene epoxidation, sulfoxidation, and hydroxylation of methyl groups on cyclopropane rings. The key use of the P450 mutants from different sources in which the conserved threonine in the distal substrate binding pocket is replaced with alanine, in order to minimize the formation of the compound I intermediate and unmask the reactivity of the hydroperoxoferric state, is emphasized. These data are discussed in the context of the "two-states" model, which proposes that the compound I P450 intermediate has both high- and low-spin states with different reactivities. A complicated reaction profile emerges for the wide range of P450 reactions involving up to three reactive intermediates, of which the most reactive, the compound I P450 state, has two spin states with different reactivities.

Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products.

The molecular mechanisms through which oxidized lipids and their electrophilic decomposition products mediate redox cell signalling is not well understood and may involve direct modification of signal-transduction proteins or the secondary production of reactive oxygen or nitrogen species in the cell. Critical in the adaptation of cells to oxidative stress, including exposure to subtoxic concentrations of oxidized lipids, is the transcriptional regulation of antioxidant enzymes, many of which are controlled by antioxidant-responsive elements (AREs), also known as electrophile-responsive elements. The central regulator of the ARE response is the transcription factor Nrf2 (NF-E2-related factor 2), which on stimulation dissociates from its cytoplasmic inhibitor Keap1, translocates to the nucleus and transactivates ARE-dependent genes. We hypothesized that electrophilic lipids are capable of activating ARE through thiol modification of Keap1 and we have tested this concept in an intact cell system using induction of glutathione synthesis by the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-prostaglandin J2. On exposure to 15-deoxy-Delta12,14-prostaglandin J2, the dissociation of Nrf2 from Keap1 occurred and this was dependent on the modification of thiols in Keap1. This mechanism appears to encompass other electrophilic lipids, since 15-A(2t)-isoprostane and the lipid aldehyde 4-hydroxynonenal were also shown to modify Keap1 and activate ARE. We propose that activation of ARE through this mechanism will have a major impact on inflammatory situations such as atherosclerosis, in which both enzymic as well as non-enzymic formation of electrophilic lipid oxidation products are increased.

Dioxirane Oxidation of Sulfur‐Containing Organic Compounds

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of 3,7‐Dihydroimidazo[1,2a]pyrazine‐3‐ones and Their Chemiluminescent Properties.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Direct and Enantioselective Organocatalytic α‐Oxidation of Aldehydes.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes.

Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.

Diastereoselective 14beta-hydroxylation of baccatin III derivatives.

14beta-Hydroxybaccatin III, a compound with limited availability by natural sources, is the starting material for the synthesis of the second-generation anticancer taxoid ortataxel. The 7-tert-butoxycarbonyl (1a) and 7-triethylsilyl (1b) derivatives of 14beta-hydroxybaccatin III 1,14-carbonate were synthesized from 10-deacetylbaccatin III (3). The crucial steps were (a) the C(14)beta hydroxylation of the corresponding 13-oxobaccatin III derivatives by oxaziridine-mediated electrophilic oxidation and (b) the reduction of the C(13) carbonyl group with sodium or alkylammonium borohydrides. This protocol provides a practical way for the semisynthesis of ortataxel from 10-deacetylbaccatin III, a compound readily available from various yews.

Synthesis of 3,7-dihydroimidazo[1,2 a]pyrazine-3-ones and their chemiluminescent properties

A series of 3,7-dihydroimidazo[1,2 a]pyrazine-3-ones were prepared from 2-amino-3,5-dibromopyrazine. The concise synthesis of coelenterazine ( 5j ), in three steps, 48% overall yield and >99% purity exemplifies the strategy. Further, the synthetic approach facilitated the regiospecific incorporation of carboxyalkyl linkers on the 3,7-dihydroimidazo[1,2 a]pyrazine-3-one nucleus that are required for bioconjugation. Peroxymonocarbonate, an electrophilic oxidant, was used to initiate ‘pseudo-flash’ chemiluminescence from this class of molecules.

Electronic and steric effects on the oxygenation of organic sulfides and sulfoxides with oxo(salen)chromium(V) complexes.

The kinetics of oxygenation of several para-substituted phenyl methyl sulfides and sulfoxides with a series of 5-substituted and sterically hindered oxo(salen)chromium(V) complexes have been studied by a spectrophotometric technique. Though the reaction of sulfides follows simple second-order kinetics, sulfoxides bind strongly with the metal center of the oxidant and the oxygen atom is transferred from the oxidant-sulfoxide adduct to the substrate. The reduction potentials, E(red), of eight Cr(V) complexes correlate well with the Hammett sigma constants, and the reactivity of the metal complexes is in accordance with the E(red) values. The metal complexes carrying bulky tert-butyl groups entail steric effects. Organic sulfides follow a simple electrophilic oxidation mechanism, and the nonligated sulfoxides undergo electrophilic oxidation to sulfones using the oxidant-sulfoxide adduct as the oxidant. Sulfoxides catalyze the Cr(V)-salen complexes' oxygenation of organic sulfides, and the catalytic activity of sulfoxides is comparable to pyridine N-oxide and triphenylphosphine oxide. The rate constants obtained for the oxidation of sulfides and sulfoxides clearly indicate the operation of a pronounced electronic and steric effect in the oxygenation reaction with oxo(salen)chromium(V) complexes.