Superoxide Adduct Research Articles

Porous Salts as Platforms for Heterogeneous Catalysis.

The preparation of a new class of reactive porous solids, prepared via straightforward salt metathesis reactions, is described here. Reaction of the dimethylammonium salt of a magnesium-based porous coordination cage with the chloride salt of [CrII Cl(Me4 cyclam)]+ affords a porous solid with concomitant removal of dimethylammonium chloride. The salt consists of the ions combined in the expected ratio based on their charge as confirmed by UV-vis and X-ray photoelectron spectroscopies, ion chromatography (IC), and inductively coupled plasma mass spectrometry (ICP-MS). The porous salt boasts a Brunauer-Emmett-Teller (BET) surface area of 213m2 g-1 . Single crystal X-ray diffraction reveals the chromium(II) cations in the structure reside in the interstitial space between porous cages. Importantly, the chromium(II) centers, previously shown to react with O2 to afford reactive chromium(III)-superoxide adducts, are still accessible in the solid state as confirmed by UV-vis spectroscopy. The site-isolated reactive centers have competence toward hydrogen atom abstraction chemistry and display significantly increased stability and reactivity as compared to dissolved ions.

Molecular insights into the role of O2 in reversed C2H6/C2H4 separation on metal–organic frameworks

Previous experimental studies have shown that C2H4-selective metal–organic frameworks (MOFs) can be reversed to C2H6-selective MOFs by introducing an oxygen molecule (O2) into the MOF. The O2 functionalization strategy provides a new insight into one-step C2H4 purification; however, the molecular-level understanding of the reversed separation mechanism remains theoretically and experimentally unclear, which limits further material refinement. In this study, we explored the influence of O2 on the C2H6/C2H4 selectivity of a Cr-BTC MOF via density functional theory (DFT) calculations and grand canonical Monte Carlo simulations within the newly DFT-derived force fields. Results suggest that O2 was antiferromagnetically coupled to the open Cr site, with an end-on pattern forming the superoxo site. The presence of superoxide adducts decreased the dipole moment of the open-metal site by 5.95 debye. Furthermore, the altered gas selectivity could be attributed to the superoxo site blocking the donation and back-donation interactions between Cr and C2H4. This removes the highly polarized C2H4-selective site, while enhancing the pore channel environmental inertness for preferential C2H6 adsorption. To assess the performance of the reversed separation strategy under humid air conditions, ab initio molecular dynamics with metadynamics were used to determine the water stabilities of Cr-BTC and oxidized conjugates. We revealed that O2 introduction weakened the metal-linker bond and reduced the free-energy barrier for the initial framework decomposition process, which may hinder the application of gas separation in further practical environments. These findings provide theoretical support for oxidized MOFs with reversed separation performance and facilitate intentional post-synthetic MOF modifications for paraffin-selective adsorbent design, a critical need for energy applications and the petroleum industry.

Synthesis and Characterization of Manganese Dithiocarbamate Complexes: New Evidence of Dioxygen Activation.

(1) Background: Metal dithiocarbamate compounds have long been the subject of research due to their ease of formation, excellent properties and potential applications. However, manganese complexes with dithiocarbamates, to our knowledge, have never been used for medical imaging applications. With the aim of developing a new class of mononuclear manganese(II)-based agents for molecular imaging applications, we performed a specific investigation into the synthesis of mononuclear bis-substituted Mn(II) complexes with dithiocarbamate ligands. (2) Methods: Synthesis in either open or inert atmosphere at different Mn(II) to diethyldithiocarbamate molar ratios were performed and the products characterized by IR, EA, ESI-MS and XRD analysis. (3) Results: We found that only under oxygen-free atmospheric conditions the Mn(II) complex MnL2, where L = diethyldithiocarbamate ligand, is obtained, which was further observed to react with dioxygen in the solid state to form the intermediate superoxo Mn(III) complex [MnL2(η2-O2)]. The existence of the superoxo complex was revealed by mass spectroscopy, and this species was interpreted as an intermediate step in the reaction that led the bis-substituted Mn(II) complex, MnL2, to transform into the tris-substituted Mn(III) complex, MnL3. A similar result was found with the ligand L’ (= bis(N-ethoxyethyl)dithiocarbamate). (4) Conclusions: We found that in open atmosphere and in aqueous solution, only manganese(III) diethyldithiocarbamate complexes can be prepared. We report here a new example of a small-molecule Mn(II) complex that efficiently activates dioxygen in the solid state through the formation of an intermediate superoxide adduct.

Spectroelectrochemistry of Non-Aromatic Explosives in Acetonitrile and the Influence of Superoxide

The spectroelectrochemistry of non-aromatic explosive compounds, namely two nitramines (RDX and HMX), a nitrate ester (PETN), and a plastic explosive composite (Semtex 1A) in acetonitrile (AN) is reported. The redox behavior of these compounds is studied using cyclic voltammetry while monitoring changes in UV absorbance to identify transient intermediates and product species. In search of a low-cost solution-phase detection technique for explosives, we compare the spectroelectrochemistry of these compounds in the presence and absence of naturally dissolved oxygen (2 mM in AN), where the superoxide radical is co-electrogenerated during analyte reduction. Free superoxide yields a prominent UV signal in this medium (λmax = 250 nm) that the explosives’ nitro or methylene groups attenuate stoichiometrically. Overlapping UV spectra of multiple species are deconvoluted using a new strategy, spectral regional baselining, for time- and potential-resolved spectroelectrochemical (SEC) analysis. SEC data for nitrite (NO2 -), nitrate (NO3 -), and nitromethane (CH3NO2) were also collected to support proposed reaction mechanisms for the transformation of explosives that include superoxide adducts and dimerized azoxy reduced intermediates. The findings expand the understanding of high-explosive solution-phase chemistry and offer a route to novel signal transduction for (electro-)chemical sensors for energetic materials.Figure 1. Spectroelectrochemistry of PETN in aerated acetonitrile with regions denoted for differential baseline analysis. (a) 3-D projected cyclic voltammogram (black) with UV absorbance from relevant wavelength (red, unscaled) as a function of time and potential; (b) full spectra (time domain) during cyclic voltammogram. Figure 1

Modeling Tryptophan/Indoleamine 2,3-Dioxygenase with Heme Superoxide Mimics: Is Ferryl the Key Intermediate?

Tryptophan oxidation in biology has been recently implicated in a vast array of paramount pathogenic conditions in humans, including multiple sclerosis, rheumatoid arthritis, type-I diabetes, and cancer. This 2,3-dioxygenative cleavage of the indole ring of tryptophan with dioxygen is mediated by two heme enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), during its conversion to N-formylkynurenine in the first and rate-limiting step of kynurenine pathway. Despite the pivotal significance of this enzymatic transformation, a vivid viewpoint of the precise mechanistic events is far from complete. A heme superoxide adduct is thought to be the active oxidant in both TDO and IDO, which, following O-O bond cleavage, presumably generates a key ferryl (FeIV=O) reaction intermediate. This study, for the first time in model chemistry, demonstrates the potential of synthetic heme superoxide adducts to mimic the bioinorganic chemistry of indole dioxygenation by TDO and IDO, challenging the widely accepted categorization of these metal adducts as weak oxidants. Herein, an electronically divergent series of ferric heme superoxo oxidants mediates the facile conversion of an array of indole substrates into their corresponding 2,3-dioxygenated products, while shedding light on an unequivocally occurring, putative ferryl intermediate. The oxygenated indole products have been isolated in ∼31% yield, and characterized by LC-MS, 1H and 13C NMR, and FT-IR methodologies, as well as by 18O2(g) labeling experiments. Distinctly, the most electron-deficient superoxo adduct is observed to react the fastest, specifically with the most electron-rich indole substrate, underscoring the cruciality of electrophilicity of the heme superoxide moiety in facilitating the initial indole activation step. Comprehensive understanding of such mechanistic subtleties will benefit future attempts in the rational design of salient therapeutic agents, including next generation anticancer drug targets with amplified effectivity.

Nickel(II) Complexes with Electron-Rich, Sterically Hindered PNP Pincer Ligands Enable Uncommon Modes of Ligand Dearomatization

We report the reactivity and characterization of hydride, methyl, and bromo NiII complexes with a new class of electron-rich and sterically hindered PNP pincer ligands, Me4PNPR (R = iPr, tBu), in which a classical metal–ligand cooperative mode of reactivity via CH2 arm deprotonation is blocked by methylation. This enables new, uncommon modes of PNP ligand dearomatization that involve reactivity in the para position of the pyridine ring. In particular, the reduction of [(Me4PNPiPr)NiIIMe]B(ArF)4 with KC8 leads to the formation of a new C–C bond via dimerization of two complexes through the para position. This reactivity stands in sharp contrast to the previously reported bromo or chloro complexes, where stable NiI halogen moieties are formed. Computational analysis showed a greater propensity for ligand-centered radical formation for the presumed intermediate one-electron-reduced species. UV-induced homolysis of the NiII–Me bond in [(Me4PNPiPr)NiIIMe]B(ArF)4 leads to the formation of a Me radical detected by radical traps and NiI intermediates that can be trapped in the presence of halide ions to give previously characterized, stable NiI halogen complexes. In addition, treatment of the bromo complexes [(Me4PNPR)NiIIBr]BPh4 with a powerful hydride source, LiBEt3H, leads to the reduction of the pyridine ring and the formation of NiII complexes with an anionic amide donor reduced pincer ligand, although aromatic NiII hydride complexes could also be obtained with a weaker hydride source. We have observed that steric bulk at the phosphine donors controls the reactivity of the resulting NiIIH complexes. While t-Bu-substituted [(Me4PNPtBu)NiIIH]Y (Y = BPh4, B(ArF)4) does not react with O2, the less sterically hindered iPr-substituted [(Me4PNPiPr)NiIIH]Y reacts instantaneously to give an unstable superoxide adduct that can be observed by EPR.

Mechanism of Catalytic O2 Reduction by Iron Tetraphenylporphyrin.

The catalytic reduction of O2 to H2O is important for energy transduction in both synthetic and natural systems. Herein, we report a kinetic and thermochemical study of the oxygen reduction reaction (ORR) catalyzed by iron tetraphenylporphyrin (Fe(TPP)) in N, N'-dimethylformamide using decamethylferrocene as a soluble reductant and para-toluenesulfonic acid ( pTsOH) as the proton source. This work identifies and characterizes catalytic intermediates and their thermochemistry, providing a detailed mechanistic understanding of the system. Specifically, reduction of the ferric porphyrin, [FeIII(TPP)]+, forms the ferrous porphyrin, FeII(TPP), which binds O2 reversibly to form the ferric-superoxide porphyrin complex, FeIII(TPP)(O2•-). The temperature dependence of both the electron transfer and O2 binding equilibrium constants has been determined. Kinetic studies over a range of concentrations and temperatures show that the catalyst resting state changes during the course of each catalytic run, necessitating the use of global kinetic modeling to extract rate constants and kinetic barriers. The rate-determining step in oxygen reduction is the protonation of FeIII(TPP)(O2•-) by pTsOH, which proceeds with a substantial kinetic barrier. Computational studies indicate that this barrier for proton transfer arises from an unfavorable preassociation of the proton donor with the superoxide adduct and a transition state that requires significant desolvation of the proton donor. Together, these results are the first example of oxygen reduction by iron tetraphenylporphyrin where the pre-equilibria among ferric, ferrous, and ferric-superoxide intermediates have been quantified under catalytic conditions. This work gives a generalizable model for the mechanism of iron porphyrin-catalyzed ORR and provides an unusually complete mechanistic study of an ORR reaction. More broadly, this study also highlights the kinetic challenges for proton transfer to catalytic intermediates in organic media.

Multiconfigurational and DFT analyses of the electromeric formulation and UV–vis absorption spectra of the superoxide adduct of ferrous superoxide reductase

The putative initial adduct of ferrous superoxide reductase (SOR) with superoxide has been alternatively formulated as ferric-peroxo or ferrous-superoxo. The ~600-nm UV–vis absorption band proposed to be assigned to this adduct (either as sole intermediate in the SOR catalytic cycle, or as one of the two intermediates) has recently been interpreted as due to a ligand-to-metal charge transfer, involving thiolate and superoxide in a ferrous complex, contrary to an alternative assignment as a predominantly cysteine thiolate-to-ferric charge transfer in a ferric-peroxo electromer. In an attempt to clarify the electromeric formulation of this adduct, we report a computational study using a multiconfigurational complete active space self-consistent field (MC-CASSCF) wave function approach as well as modelling the UV–vis absorption spectra with time-dependent density functional theory (TD-DFT). The MC-CASSCF calculations disclose a weak interaction between iron and the dioxygenic ligand and a dominant configuration with an essentially ferrous-superoxo character. The computed UV–vis absorption spectra reveal a marked dependence on the choice of density functional – both in terms of location of bands and in terms of orbital contributors. For the main band in the visible region, besides the recently reported thiolate-to-superoxide charge transfer, a more salient, and less functional-dependent, feature is a thiolate-to-ferric iron charge transfer, consistent with a ferric-peroxo electromer. By contrast, the computed UV–vis spectra of a ferric-hydroperoxo SOR model match distinctly better (and with no qualitative dependence on the DFT methodology) the 600-nm band as due to a mainly thiolate-to-ferric character – supporting the assignment of the SOR “600-nm intermediate” as a S=5/2 ferric-hydroperoxo species.

Co(salophen)-Catalyzed Aerobic Oxidation of p-Hydroquinone: Mechanism and Implications for Aerobic Oxidation Catalysis.

Macrocyclic metal complexes and p-benzoquinones are commonly used as co-catalytic redox mediators in aerobic oxidation reactions. In an effort to gain insight into the mechanism and energetic efficiency of these reactions, we investigated Co(salophen)-catalyzed aerobic oxidation of p-hydroquinone. Kinetic and spectroscopic data suggest that the catalyst resting-state consists of an equilibrium between a Co(II)(salophen) complex, a Co(III)-superoxide adduct, and a hydrogen-bonded adduct between the hydroquinone and the Co(III)-O2 species. The kinetic data, together with density functional theory computational results, reveal that the turnover-limiting step involves proton-coupled electron transfer from a semi-hydroquinone species and a Co(III)-hydroperoxide intermediate. Additional experimental and computational data suggest that a coordinated H2O2 intermediate oxidizes a second equivalent of hydroquinone. Collectively, the results show how Co(salophen) and p-hydroquinone operate synergistically to mediate O2 reduction and generate the reactive p-benzoquinone co-catalyst.

Oxidative stress and DNA damages induced by 1-nitropyrene in human lung fibroblasts: New insights into the mechanisms of genotoxicity and EPR-spin trapping direct monitoring of free radicals at subcellular levels

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous contaminants released in the atmosphere from the incomplete combustion of organic matter and pesticide intermediates nitration. Many studies have established that 1-nitropyrene (1-NP), one the most abundant nitro-PAH pollutant in diesel exhaust, is associated with increased risk of cancer and inflammatory diseases via possible metabolic pathways, including ring oxidation, nitroreduction or conjugation reactions. In addition, genotoxicity tests (e.g. comet assay and Ames test), demonstrated that 1-NP is a potent mutagen and genotoxic agent. However, the mechanisms underlying the promotion of DNA damages remain to be fully elucidated in relation with the progression of oxidative stress due to the stimulation of cellular free radical sources. Methods: On human lung fibroblasts exposed to environmentally relevant doses of 1-NP (1-10 microM) for 24–48 h, we performed: (i) cytokinesis-blocked micronucleus (CBMN) assay in association with immunofluorescence staining of centromere protein A in micronuclei to distinguish between induction of structural or numerical chromosome changes; (ii) EPR experiments using a set of spin trapping agents (i.e. the phosphorylated nitrone DEPMPO (Culcasi et al., 2012), the new stereoselective superoxide probe Me4CyDEPMPO (Gosset et al., 2011)) and mitochondrial-targeted nitrones; and (iii) the evaluation of oxidative stress from protein carbonyls, lipid peroxidation and antioxidant depletion. Major results: One advantage of the CBMN assay is its ability to elucidate the genotoxic mechanism of agents with clastogenic or aneugenic modes of action. The data provide the first evidence for a superoxide-related aneugenic mode of action at low 1-NP doses and a dominant clastogenic mechanism as the 1-NP doses and exposure time increased. This two-steps mechanism differs from that induced by CeO2-nanoparticles, an oxidant present in diesel exhausts (Benameur et al., 2014). From the EPR detection of superoxide and hydroxyl radical spin adducts, two free radical cellular sources activated by 1-NP exposure were hypothetized, i.e. NADPH oxidase and mitochondria, this latter being likely involved in oxidative stress damages development. References Culcasi, et al., 2012. Chem. Biol. Interact.. Gosset, et al., 2011. Bioorg. Med. Chem.. Benameur, et al., 2014. Nanotoxicology.

Diagnostic Features of EPR Spectra of Superoxide Intermediates on Catalytic Surfaces and Molecular Interpretation of Their g and A Tensors

The use of electron paramagnetic resonance spectroscopy to study the superoxide intermediates, generated by end-on and side-on adsorption of the naturally abundant and 17O-enriched dioxygen on catalytic surfaces is discussed. Basic mechanisms of O2 − radical formation via a cationic redox mechanism, an anionic redox mechanism, and an electroprotic mechanism are illustrated with selected oxide-based systems of catalytic relevance. Representative experimental spectra of various complexities are analyzed and their diagnostic features have been identified and interpreted. The molecular nature of the g and A tensors of the electrostatic and covalent superoxide adducts is discussed in detail within the classic and density functional theory based approaches.

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.

Mitochondria-targeted spin traps: synthesis, superoxide spin trapping, and mitochondrial uptake.

Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPPMPO, bearing a mitochondria-targeting triphenylphosphonium cationic moiety or guanidinium cationic group. All of the spin traps prepared have been observed to efficiently trap superoxide radical anions in a cell-free system. The superoxide spin adducts exhibited similar spectral properties, indicating no significant differences in the geometry of the cyclic nitroxide moieties of the spin adducts. The superoxide adduct stability was measured and observed to be highest (t1/2 = 73 min) for DIPPMPO nitrone linked to triphenylphosphonium moiety via a short carbon chain (Mito-DIPPMPO). The experimental results and DFT quantum chemical calculations indicate that the cationic property of the triphenylphosphonium group may be responsible for increased superoxide trapping efficiency and adduct stability of Mito-DIPPMPO, as compared to the DIPPMPO spin trap. The studies of uptake of the synthesized traps into isolated mitochondria indicated the importance of both cationic and lipophilic properties, with the DEPMPO nitrone linked to the triphenylphosphonium moiety via a long carbon chain (Mito10-DEPMPO) exhibiting the highest mitochondrial uptake. We conclude that, of the synthesized traps, Mito-DIPPMPO and Mito10-DEPMPO are the best candidates for potential mitochondria-specific spin traps for use in biologically relevant systems.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Synthesis and Spin-Trapping Properties of a Trifluoromethyl Analogue of DMPO: 5-Methyl-5-trifluoromethyl-1-pyrrolineN-Oxide (5-TFDMPO)

The 5-diethoxyphosphonyl-5-methyl-1-pyrroline N-oxide superoxide spin adduct (DEPMPO-OOH) is much more persistent (about 15 times) than the 5,5-dimethyl-1-pyrroline N-oxide superoxide spin adduct (DMPO-OOH). The diethoxyphosphonyl group is bulkier than the methyl group and its electron-withdrawing effect is much stronger. These two factors could play a role in explaining the different half-lifetimes of DMPO-OOH and DEPMPO-OOH. The trifluoromethyl and the diethoxyphosphonyl groups show similar electron-withdrawing effects but have different sizes. We have thus synthesized and studied 5-methyl-5-trifluoromethyl-1-pyrroline N-oxide (5-TFDMPO), a new trifluoromethyl analogue of DMPO, to compare its spin-trapping performance with those of DMPO and DEPMPO. 5-TFDMPO was prepared in a five-step sequence by means of the Zn/AcOH reductive cyclization of 5,5,5-trifluoro-4-methyl-4-nitropentanal, and the geometry of the molecule was estimated by using DFT calculations. The spin-trapping properties were investigated both in toluene and in aqueous buffer solutions for oxygen-, sulfur-, and carbon-centered radicals. All the spin adducts exhibit slightly different fluorine hyperfine coupling constants, thereby suggesting a hindered rotation of the trifluoromethyl group, which was confirmed by variable-temperature EPR studies and DFT calculations. In phosphate buffer at pH 7.4, the half-life of 5-TFDMPOOOH is about three times shorter than for DEPMPO-OOH and five times longer than for DMPO-OOH. Our results suggest that the stabilization of the superoxide adducts comes from a delicate balance between steric, electronic, and hydrogen-bonding effects that involve the β group, the hydroperoxyl moiety, and the nitroxide.

Investigation of the Hydroxylation Mechanism of Noncoupled Copper Oxygenases by Ab Initio Molecular Dynamics Simulations

In Nature, the family of copper monooxygenases comprised of peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM) is known to perform dioxygen-dependent hydroxylation of aliphatic C-H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C-H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen-abstraction step promoted by the cupric-superoxide adduct. The hydrogen-abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long-range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO−). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.

Sivelestat Attenuates Myocardial Reperfusion Injury during Brief Low Flow Postischemic Infusion

The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.

Solution behavior of iron(iii) and iron(ii) porphyrins in DMSO and reaction with superoxide. Effect of neighboring positive charge on thermodynamics, kinetics and nature of iron-(su)peroxo product

The solution behavior of iron(III) and iron(II) complexes of 5(4),10(4),15(4),20(4)-tetra-tert-butyl-5,10,15,20-tetraphenylporphyrin (H(2)tBuTPP) and the reaction with superoxide (KO(2)) in DMSO have been studied in detail. Applying temperature and pressure dependent NMR studies, the thermodynamics of the low-spin/high-spin equilibrium between bis- and mono-DMSO Fe(II) forms have been quantified (K(DMSO) = 0.082 ± 0.002 at 298.2 K, ΔH° = +36 ± 1 kJ mol(-1), ΔS° = +101 ± 4 J K(-1) mol(-1), ΔV° = +16 ± 2 cm(3) mol(-1)). This is a key activation step for substitution and inner-sphere electron transfer. The superoxide binding constant to the iron(II) form of the studied porphyrin complex was found to be (9 ± 0.5) × 10(3) M(-1), and does not change significantly in the presence of the externally added crown ether in DMSO (11 ± 4) × 10(3) M(-1). The rate constants for the superoxide binding (k(on) = (1.30 ± 0.01) × 10(5) M(-1) s(-1)) and release (k(off) = 11.6 ± 0.7 s(-1)) are not affected by the presence of the external crown ether in solution. The resulting iron(II)-superoxide adduct has been characterized (mass spectrometry, EPR, high-pressure UV/Vis spectroscopy) and upon controlled addition of a proton source it regenerates the starting iron(II) complex. Based on DFT calculations, the reaction product without neighboring positive charge has iron(II)-superoxo character in both high-spin side-on and low-spin end-on forms. The results are compared to those obtained for the analogous complex with covalently attached crown ether, and more general conclusions regarding the spin-state equilibrium of iron(II) porphyrins, their reaction with superoxide and the electronic structure of the product species are drawn.

Synthesis and characterization of 5-hydroxymethyl-5-methyl-pyrroline N-oxide and its derivatives

Synthesis and spin trapping behavior of three novel DMPO derivatives, namely 5-hydroxymethyl-5-methyl-pyrroline N-oxide (HMMPO), 5-(2-furanyl)-oxymethyl-5-methyl-pyrroline N-oxide (FMMPO), and 5-(2-pyranyl)-oxymethyl-5-methyl-pyrroline N-oxide (PMMPO) towards different oxygen- and carbon-centered radicals are described. The stabilizing effect of a series of cyclodextrins on the superoxide adducts was tested.