Reactivity of superoxide radical anion with cyclic nitrones: role of intramolecular H-bond and electrostatic effects

Limitations exist among the commonly used cyclic nitrone spin traps for biological free radical detection using electron paramagnetic resonance (EPR) spectroscopy. The design of new spin traps for biological free radical detection and identification using EPR spectroscopy has been a major challenge due to the lack of systematic and rational approaches to their design. In this work, density functional theory (DFT) calculations and stopped-flow kinetics were employed to predict the reactivity of functionalized spin traps with superoxide radical anion (O2*-). Functional groups provide versatility and can potentially improve spin-trap reactivity, adduct stability, and target specificity. The effect of functional group substitution at the C-5 position of pyrroline N-oxides on spin-trap reactivity toward O2*- was computationally rationalized at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) and PCM/mPW1K/6-31+G(d,p) levels of theory. Calculated free energies and rate constants for the reactivity of O2*- with model nitrones were found to correlate with the experimentally obtained rate constants using stopped-flow and EPR spectroscopic methods. New insights into the nucleophilic nature of O2*- addition to nitrones as well as the role of intramolecular hydrogen bonding of O2*- in facilitating this reaction are discussed. This study shows that using an N-monoalkylsubstituted amide or an ester as attached groups on the nitrone can be ideal in molecular tethering for improved spin-trapping properties and could pave the way for improved in vivo radical detection at the site of superoxide formation.     

Reaction of superoxide radical with quinone molecules

When the superoxide radical O(2)(?-) is generated on reaction of KO(2) with water in dimethyl sulfoxide, the decay of the radical is dramatically accelerated by inclusion of quinones in the reaction mix. For quinones with no or short hydrophobic tails, the radical product is a semiquinone at much lower yield, likely indicating reduction of quinone by superoxide and loss of most of the semiquinone product by disproportionation. In the presence of ubiquinone-10, a different species (I) is generated, which has the EPR spectrum of superoxide radical. However, pulsed EPR shows spin interaction with protons in fully deuterated solvent, indicating close proximity to the ubinquinone-10. We discuss the nature of species I, and possible roles in the physiological reactions through which ubisemiquinone generates superoxide by reduction of O(2) through bypass reactions in electron transfer chains.     

A stable dialkylphosphinyl radical

Dialkylphosphinyl radical 1 was synthesized as thermally stable yellow crystals and found to be monomeric both in solution and in the solid state. EPR spectrum showed that the spin density of 1 is mainly localized on the 3p orbital of the dicoordinated phosphorus atom. A distinct absorption band due to the electronic transition from nonbonding electron pair orbital to singly occupied 3p orbital on the phosphorus atom of 1 was observed at 445 nm in solution. Phosphinyl radical 1 underwent facile reaction with carbon tetrachloride, hydrogen abstraction, and a unique reaction with a persistent radical, galvinoxyl, giving a cyclic phosphaalkene and a silylether.     

Optical Absorption and Magnetic Field Effect Based Imaging of Transient Radicals

Short‐lived radicals generated in the photoexcitation of flavin adenine dinucleotide (FAD) in aqueous solution at low pH are detected with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope. Radicals can be studied under both flash photolysis and continuous irradiation conditions, providing a means of directly probing potential biological magnetoreception within sub‐cellular structures. Direct spatial imaging of magnetic field effects (MFEs) by magnetic intensity modulation (MIM) imaging is demonstrated along with transfer and inversion of the magnetic field sensitivity of the flavin semiquinone radical concentration to that of the ground state of the flavin under strongly pumped reaction cycling conditions. A low field effect (LFE) on the flavin semiquinone–adenine radical pair is resolved for the first time, with important implications for biological magnetoreception through the radical pair mechanism.     

Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium

Nitrones such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) have become the spin-traps of choice for the detection of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy. The mechanism of decomposition of the superoxide radical anion (O2(.-)) adducts of DMPO, DEPMPO and EMPO in aqueous solutions was investigated. Our findings suggest that nitric oxide (NO) was formed during the decomposition of the O2(.-) adduct as detected by EPR spin trapping using Fe(II)N-methyl-d-glucamine dithiocarbamate (MGD). Nitric oxide release was observed from the O2(.-) adduct formed from hypoxanthine-xanthine oxidase, PMA-activated human neutrophils, and DMSO solution of KO2. Nitric oxide formation was not observed from the independently generated hydroxyl radical adduct. Formation of nitric oxide was also indirectly detected as nitrite (NO2(.-)) utilizing the Griess assay. Nitrite concentration increases with increasing O2(.-) concentration at constant DMPO concentration, while NO2(.-) formation is suppressed at anaerobic conditions. Moreover, large excess of DMPO also inhibits NO2(.-) formation which can be attributed to the oxidation of DMPO to hydroxamic acid nitroxide (DMPO-X) by nitrogen dioxide (NO2), a precursor to NO2(.-). Product analysis was also conducted to further elucidate the mechanism of adduct decay using gas chromatography-mass spectrometry (GC-MS) technique.     

Simultaneous production of superoxide radical and singlet oxygen by sulphonated chloroaluminum phthalocyanine incorporated in human low-density lipoproteins: implications for photodynamic therapy

Sulfonated chloroaluminum phthalocyanines have been studied for their use in the photodynamic therapy (PDT) of tumors. Plasma low-density lipoproteins (LDL) are important carriers of phthalocyanines in the blood, but on exposure to visible light, phthalocyanine-loaded LDL undergo an oxidation process that propagates to erythrocytes. We attempted to identify the reactive species involved in LDL and erythrocyte oxidation by means of electron paramagnetic resonance (EPR) spectroscopy in the presence of 2,2,6,6-tetramethyl-4-piperidone (TEMP) and the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Irradiation of phthalocyanine-loaded LDL in the presence of DMPO resulted in the formation of a four-line EPR spectrum with relative intensity of 1:2:2:1 (a(N) = a(H) = 14.8 G), characteristic of DMPO-hydroxyl radical spin adduct. This signal was sensitive to superoxide dismutase and slightly sensitive to catalase, but a mixture of the two enzymatic activities was the most efficient in promoting a decrease in the intensity of the EPR signal. In the presence of erythrocytes, an increase in the quartet intensity for a hematocrit of 1% and 4% was observed, decreasing for higher erythrocyte concentrations. The irradiation of phthalocyanine-loaded LDL in the presence of TEMP resulted in the formation of a nitroxide radical, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl radical, intensity of which was sensitive to histidine, a singlet oxygen ((1)O(2)) quencher. Under both incubation conditions, with DMPO and TEMP, the formation of the respective EPR signals required the sensitizer (phthalocyanine), light and oxygen. Overall, the results are compatible with the simultaneous formation of superoxide anion and (1)O(2), implying that Type-I and Type-II mechanisms of photochemistry are simultaneously operative in phthalocyanine-loaded LDL. However, for a constant LDL/phthalocyanine ratio, the formation of oxygen free radicals shows a biphasic behavior with the concentration of LDL increasing and reaching a plateau, whereas the formation of (1)O(2) increases linearly with LDL concentration. Erythrocytes at high (physiological) concentrations induced a decrease in the intensity of both EPR signals. The physiological relevance of these findings in the framework of PDT is briefly discussed.     

Hydroxyl radical self-recombination reaction and absorption spectrum in water up to 350 degrees C

The rate constant for the self-recombination of hydroxyl radicals (*OH) in aqueous solution giving H2O2 product has been measured from 150 to 350 degrees C by direct measurement of the *OH radical transient optical absorption at 250 nm. The values of the rate constant are smaller than previously predicted by extrapolation to the 200-350 degrees C range and show virtually no change in this range. In combining these measurements with previous results, the non-Arrhenius behavior can be well described in terms of the Noyes equation kobs-1 = kact-1+ kdiff-1, using the diffusion-limited rate constant kdiff estimated from the Smoluchowski equation and an activated barrier rate kact nearly equal to the gas-phase high-pressure limiting rate constant for this reaction. The aqueous *OH radical spectrum between 230 and 320 nm is reported up to 350 degrees C. A weak band at 310 nm grows in at higher temperature, while the stronger band at 230 nm decreases. An isosbestic point appears near 305 nm. We assign the 230 nm band to hydrogen-bonded *OH radical, and the 310 nm band is assigned to "free" *OH. On the basis of the spectrum change relative to room temperature, over half of the *OH radicals are in the latter form at 350 degrees C.     

Transients in the oxidative and H-atom-induced degradation of 1,3,5-trithiane. Time-resolved studies in aqueous solution

The (*)OH-induced oxidation of 1,3,5-trithiacyclohexane (1) in aqueous solution was studied by means of pulse radiolysis with optical and conductivity detection. This oxidation leads, via a short-lived (*)OH radical adduct (<1 micros), to the radical cation 1(*+) showing a broad absorption with lambda(max) equal to 610 nm. A defined pathway of the decay of 1(*+) is proton elimination. It occurs with k = (2.2 +/- 0.2) x 10(4) s(-1) and yields the cyclic C-centered radical 1(-H)(*). The latter radical decays via ring opening (beta-scission) with an estimated rate constant of about 10(5) s(-1). A distinct, immediate product (formed with the same rate constant) is characterized by a narrow absorption band with lambda(max) = 310 nm and is attributed to the presence of a dithioester function. The formation of the 310 nm absorption can be suppressed in the presence of oxygen, the rationale for this being a reaction of the C-centered cyclic radical 1(-H)(*) with O(2). The disappearance of the 310 nm band (with a rate constant of 900 s(-1)) is associated with the hydrolysis of the dithioester functionality. A further aspect of this study deals with the reaction of H(*) atoms with 1 which yields a strongly absorbing, three-electron-bonded 2sigma/1sigma* radical cation [1(S therefore S)-H](+) (lambda(max) = 400 nm). Its formation is based on an addition of H(*) to one of the sulfur atoms, followed by beta-scission, intramolecular sulfur-sulfur coupling (constituting a ring contraction), and further stabilization of the S therefore S bond thus formed by protonation. [1(S therefore S)-H](+) decays with a first-order rate constant of about 10(4) s(-1). Its formation can be suppressed by the addition of oxygen which scavenges the H(*) atoms prior to their reaction with 1. Complementary time-resolved conductivity experiments have provided information on the quantification of the 1(*+) radical cation yield, the cationic longer-lived follow-up species, extinction coefficients, and kinetics concerning deprotonation processes as well as further reaction steps after hydrolysis of the transient dithioesters. The results are also discussed in the light of previous photochemical studies.     

Generation of singlet oxygen by the glyoxal-peroxynitrite system

Diacetyl, methylglyoxal, and glyoxal are α-dicarbonyl catabolites prone to nucleophilic additions of amino groups of proteins and nucleobases, thereby triggering adverse biological responses. Because of their electrophilicity, in aqueous medium, they exist in a phosphate-catalyzed dynamic equilibrium with their hydrate forms. Diacetyl and methylglyoxal can be attacked by peroxynitrite (k(2) ≈ 1.0 × 10(4) M(-1) s(-1) and k(2) ≈ 1.0 × 10(5) M(-1) s(-1), respectively), a potent biological nucleophile and oxidant, yielding the acetyl radical from the homolysis of peroxynitrosocarbonyl adducts, and acetate or formate ions, respectively. We report here that glyoxal also reacts with peroxynitrite, yielding formate ion at rates at least 1 order of magnitude greater than does methylglyoxal. A triplet EPR signal (1:2:1; a(H) = 0.78 mT) attributable to hydrated formyl radical was detected by direct flow experiments. In the presence of the spin trap 2-methyl-2-nitrosopropane, the EPR spectrum displays the di-tert-butyl nitroxide signal, another signal assignable to the spin trapping adduct with hydrogen radical (a(N) = a(H) = 1.44 mT), probably formed from formyl radical decarbonylation, and a third EPR signal assignable to the formyl radical adduct of the spin trap (a(N) = 0.71 mT and a(H) = 0.14 mT). The novelty here is the detection of singlet oxygen ((1)Δ(g)) monomol light emission at 1270 nm during the reaction, probably formed by subsequent dioxygen addition to formyl radical and a Russell reaction of nascent formylperoxyl radicals. Accordingly, the near-infrared emission increases upon raising the peroxynitrite concentration in D(2)O buffer and is suppressed upon addition of O(2) ((1)Δ(g)) quenchers (NaN(3), l-His, H(2)O). Unequivocal evidence of O(2) ((1)Δ(g)) generation was also obtained by chemical trapping of (18)O(2) ((1)Δ(g)) with anthracene-9,10-divinylsulfonate, using HPLC/MS/MS for detection of the corresponding 9,10-endoperoxide derivative. Our studies add insights into the molecular events underlying nitrosative, oxidative, and carbonyl stress in inflammatory processes and aging-associated maladies.     

DNA mediated charge transport: characterization of a DNA radical localized at an artificial nucleic acid base

DNA assemblies containing 4-methylindole incorporated as an artificial base provide a chemically well-defined system in which to explore the oxidative charge transport process in DNA. Using this artificial base, we have combined transient absorption and EPR spectroscopies as well as biochemical methods to test experimentally current mechanisms for DNA charge transport. The 4-methylindole radical cation intermediate has been identified using both EPR and transient absorption spectroscopies in oxidative flash-quench studies using a dipyridophenazine complex of ruthenium as the intercalating oxidant. The 4-methylindole radical cation intermediate is particularly amenable to study given its strong absorptivity at 600 nm and EPR signal measured at 77 K with g = 2.0065. Both transient absorption and EPR spectroscopies show that the 4-methylindole is well incorporated in the duplex; the data also indicate no evidence of guanine radicals, given the low oxidation potential of 4-methylindole relative to the nucleic acid bases. Biochemical studies further support the irreversible oxidation of the indole moiety and allow the determination of yields of irreversible product formation. The construction of these assemblies containing 4-methylindole as an artificial base is also applied in examining long-range charge transport mediated by the DNA base pair stack as a function of intervening distance and sequence. The rate of formation of the indole radical cation is >/=10(7) s(-)(1) for different assemblies with the ruthenium positioned 17-37 A away from the methylindole and with intervening A-T base pairs primarily composing the bridge. In these assemblies, methylindole radical formation at a distance is essentially coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; charge transport is not rate limiting over this distance regime. The measurements here of rates of radical cation formation establish that a model of G-hopping and AT-tunneling is not sufficient to account for DNA charge transport. Instead, these data are viewed mechanistically as charge transport through the DNA duplex primarily through hopping among well stacked domains of the helix defined by DNA sequence and dynamics.     

Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. The thermodynamics of decay and EPR spectral properties

The formation of the superoxide radical anion (O2*-) adduct of the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as detected by electron paramagnetic resonance (EPR) spectroscopy is one of the most common techniques for O2*- detection in chemical and biological systems. However, the nature of DMPO-O2H has confounded spin-trapping investigators over the years, since there has been no independently synthesized DMPO-O2H to date. A density functional theory (DFT) approach was used to predict the isotropic hyperfine coupling constants arising from the N, beta-H, and gamma-H nuclei of DMPO-O2H using explicit interactions with water molecules as well as via a bulk dielectric effect employing the polarizable continuum model (PCM). Theoretical calculation on the thermodynamics of DMPO-O2H decay shows favorable intramolecular rearrangement to form a nitrosoaldehyde and a hydroxyl radical as products, consistent with experimental observations. Some pathways for the bimolecular decomposition of DMPO-O2H and DMPO-OH have also been computed.     

High resolution electronic absorption spectra of anisole and phenoxyl radical

High resolution absorption spectra of anisole and phenoxyl radical has been recorded in the vapor phase at room temperature by flash photolysis technique, and subsequent reactions have been investigated by kinetic spectroscopy. It was possible to follow the kinetics of the radical's decay which occurred predominantly by bimolecular recombination. The concentration of the phenoxyl radical calculated from the concentration of diphenoxyl molecules formed in the reaction cell during the optical pumping. The absolute extinction coefficient of this radical was measured from the absorption band at 291 nm, and found to be 0.95 x 10(+4) mol(-1) 1 cm(-1).     

A DNA oligonucleotide-hemin complex cleaves t-butyl hydroperoxide through a homolytic mechanism

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex ( approximately 35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of approximately 250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of < or =5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from beta-scission of the primary tert-butyloxyl radical product.     

Mechanistic studies of the radical S-adenosyl-L-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-D-quinovose

DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-D-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism. This hypothesis is now confirmed by EPR spectroscopy with the detection of a TDP-D-quinovose radical intermediate having a g-value of 2.0025 with hyperfine coupling to two spin 1/2 nuclei, each with a splitting constant of 33.6 G. A significant decrease in the EPR line width is observed when the radical is generated in reactions conducted in D(2)O versus H(2)O. These results are consistent with a C3 α-hydroxyalkyl radical in which the p-orbital harboring the unpaired electron spin at C3 is periplanar with the C-H bonds at both C2 and C4.     

Coumarin 314 free radical cation: formation, properties, and reactivity toward phenolic antioxidants

We have explored the photogeneration of the coumarin 314 radical cation by using nanosecond laser excitation at wavelengths longer than 400 nm in benzene, acetonitrile, dichloromethane, and aqueous media. In addition, time-resolved absorption spectroscopy measurements allowed detection of the triplet excited state of coumarin 314 (C(314)) with a maximum absorption at 550 nm in benzene. The triplet excited state has a lifetime of 90 μs in benzene. It is readily quenched by oxygen (k(q) = 5.0 × 10(9) M(-1) s(-1)). From triplet-triplet energy transfer quenching experiments, it is shown that the energy of this triplet excited state is higher than 35 kcal/mol, in accord with the relatively large singlet oxygen quantum yield (Φ(Δ) = 0.25). However, in aqueous media, the coumarin triplet was no longer observed, and instead of that, a long-lived (160 μs in air-equilibrated solutions) free radical cation with a maximum absorbance at 370 nm was detected. The free radical cation generation, which has a quantum yield of 0.2, occurs by electron photoejection. Moreover, density functional theory (DFT) calculations indicate that at least 40% of the electronic density is placed on the nitrogen atom in aqueous media, which explains its lack of reactivity toward oxygen. On the other hand, rate constant values close to the diffusion rate limit in water (>10(9) M(-1) s(-1)) were found for the quenching of the C(314) free radical cation by phenolic antioxidants. The results have been interpreted by an electron-transfer reaction between the phenolic antioxidant and the radical cation where ion pair formation could be involved.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS. XVII. BENZANTHRONE

The photochemistry of benzanthrone (7H-benz[de]-anthracene-7-one) has been studied using electron paramagnetic resonance (EPR) in conjunction with the spin trapping technique and the direct detection of singlet molecular oxygen luminescence. Irradiation (lambda ex = 394 nm) of benzanthrone (BA) in aerated ethanol, dimethylsulfoxide or benzene resulted in the generation of superoxide (O2-.) which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The ethoxy radical was also detected in ethanol. Photolysis of BA in deaerated basic ethanol led to the formation of BA anion radical, BA-., which was detected directly by ESR. This radical anion decayed back to BA with a unimolecular rate constant of 1.5 x 10(-3) s-1. The 1O2 quantum yields (lambda ex greater than 345 nm) for BA in ethanol, 90% ethanol and basic ethanol (0.1N NaOH) were 0.89, 0.88 and 0.28 respectively relative to Rose Bengal. The lower yield of 1O2 in basic ethanol may be attributable to the reaction of oxygen with BA-. (which is generated in higher yield at alkaline pH) to give O2-.. These findings suggest that on exposure to light BA can generate active oxygen species which may be responsible for the photocontact dermatitis caused by BA in industrial workers exposed to this chemical.     

Model studies of DNA C5' radicals. Selective generation and reactivity of 2'-deoxyadenosin-5'-yl radical

The reaction of hydrated electrons (e(aq)(-)) with 8-bromo-2'-deoxyadenosine has been investigated by radiolytic methods coupled with product studies and addressed computationally by means of DFT-B3LYP calculations. Pulse radiolysis revealed that this reaction was complete in approximately 0.3 mus, and, at this time, no significant absorption was detected. The spectrum of a transient developed in 20 mus has an absorbance in the range 300-500 nm (epsilon(max) congruent with 9600 M(-1) cm(-1) at 360 nm), and it was assigned to aromatic aminyl radical 3. Computed vertical transitions (TD-UB3LYP/6-311+G) are in good agreement with the experimental observations. Radical 3 is obtained by the following reaction sequence: one-electron reductive cleavage of the C-Br bond that gives the C8 radical, a fast radical translocation from the C8 to C5' position, and an intramolecular attack of the C5' radical at the C8,N7 double bond of the adenine moiety. The rate constant for the cyclization is 1.6 x 10(5) s(-1). On the basis of the theoretical findings, the cyclization step is highly stereospecific. The rate constants for the reactions of C5' and aminyl 3 radicals with different oxidants were determined by pulse radiolysis methods. The respective rate constants for the reaction of 2'-deoxyadenosin-5'-yl radical with dioxygen, Fe(CN)(6)(3)(-), and MV(2+) in water at ambient temperature are 1.9 x 10(9), 4.2 x 10(9), and 2.2 x 10(8) M(-1) s(-1). The value for the reaction of aminyl radical 3 with Fe(CN)(6)(3-) is 8.3 x 10(8) M(-1) s(-1), whereas the reaction with dioxygen is reversible. Tailored experiments allowed the reaction mechanism to be defined in some detail. A synthetically useful radical cascade process has also been developed that allows in a one-pot procedure the conversion of 8-bromo-2'-deoxyadenosine to 5',8-cyclo-2'-deoxyadenosine in a diastereoisomeric ratio (5'R):(5'S) = 6:1 and in high yield, by reaction with hydrated electrons in the presence of K(4)Fe(CN)(6).     

A highly selective and sensitive nanoprobe for detection and imaging of the superoxide anion radical in living cells

A novel fluorescent nanoprobe with high sensitivity and selectivity for detection and imaging of the superoxide anion radical O(2)(·-) in living cells was designed and synthesized by a simple self-assembly method based on 2-chloro-1,3-dibenzothiazoline-cyclohexene (DBZTC) and Ag@SiO(2) core-shell nanoparticles.     

Reaction of the hydroxyl radical with phenol in water up to supercritical conditions

The rate constants for the reactions of phenol with the hydroxyl radical (OH*) in water have been measured from room temperature to 380 degrees C using electron pulse radiolysis and transient absorption spectroscopy. The reaction scheme designed to fit the data shows the importance of an equilibrium, giving back reactants (OH* radical and phenol) from the dihydroxycyclohexadienyl radical formed by their reaction, and the non-negligible contribution of the hydroxycyclohexadienyl radical absorption from H* atom addition. The accuracy of the reaction scheme and the reaction rate constants determined from it have been determined by the analysis of two different experiments, one under pure N2O atmosphere and the second under a mixture a N2O and O2. We report reaction rates for the H* and OH* radical addition to phenol, the formation of phenoxyl, the second-order recombination, the reaction of dihydroxycyclohexadienyl with O2, and the decay of the peroxyl adduct. Nearly all of the reaction rates deviate strongly from Arrhenius behavior.     

Interference of non-specific peroxidases in the fluorescence detection of superoxide radical by hydroethidine oxidation: a new assay for H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The present study shows that hydroethidine (HE), used for in-vivo qualitative fluorescent detection of superoxide anion, can be also oxidized by H₂O₂ via non-specific peroxidase (horseradish peroxidase and myeloperoxidase) catalysis, forming fluorescent oxidation products. These products give broad excitation/emission peaks (490–495/580–600 nm) near the excitation/emission peaks (475/580 nm) of the HE-superoxide oxidation product, and this may pose serious interference problems to the fluorescent detection of the superoxide radical. The study suggests cautionary use of the HE-superoxide anion assay mainly for detection of reactive oxygen species. A byproduct of this study was the development of a simple and sensitive HE-horseradish peroxidase assay for the in-vitro quantification of H₂O₂ in biological tissues with a sensitivity of 1 mol L^–1.