A new kinetic method for quantification phenoxyl free radicals

In this work, a new kinetic method was proposed for quantification phenoxyl radicals generated in enzyme reaction. Instead of direct detecting the spectral signals of phenoxyl radicals, a molecular probe, the reduced form of nicotinamide adenine dinucleotide (NADH), was employed to indicate the formation of phenoxyl free radicals. It was found that the reactions of NADH and phenoxyl radicals are very fast, but can be followed by using stopped-flow fast scanning spectrophotometric technique. The initial rate of accelerated-oxidation of NADH represents the reactivity of phenoxyl free radical, which is proportional in a certain range to the initial concentration of the parent chlorophenols of the radicals. With this method, the phenoxyl radicals generated in oxidation reaction of chlorophenols (2-CP; 4-CP; 2,4-DCP; 2,4,6-TCP and 2,3,4,6-Tetra-CP) with hydrogen peroxide, catalyzed by horseradish peroxidase, were investigated. The method is highly sensitive. Phenoxyl radicals generated from as low as 1 × 10⁻⁸ M 2,4-DCP, for example, can be readily detected with the proposed method. The results show that the reactivity of various phenoxyl radicals are in the following order: 2,4-DCP > 4-CP > 2-CP > 2,4,6-TCP > 2,3,4,6-Tetra-CP. A mechanism is proposed to explain the possible pathway of the probe reaction. The feasibility of this method was assessed by the determination of enzymatic generation of phenoxyl radicals in lake water samples.     

UV-induced free radicals in oriented DNA.

Abstract— Samples of oriented DNA containing 30% water were UV-irradiated at 77 K and investigated by electron paramagnetic resonance (EPR). The EPR spectra recorded in directions parallel and perpendicular to the DNA fibre direction showed that at low UV doses the induced free radicals are very similar to those induced by γ-irradiation at the same temperature. The γ-induced free radicals have previously been analysed and found to consist mainly of anionic free radicals on thymine and cationic free radicals on guanine. At higher UV doses or by suitable annealing of the samples given a low UV dose, significant amounts of hydrogen-addition radicals on thymine were observed. The quantum yield of free radical formation for irradiation at 300nm ± 10nm was estimated to 10^--⁴. We also made a quantitative determination of the UV-induced free radicals inside an optically effective volume of the sample. The following free radical induction frequencies at 77 K were estimated: γ-rays: 2 × 10^--¹² free radicals per rad per dalton and UV (300nm): 6 × 10^--¹² free radicals per J/m² per dalton.     

Identification of free radicals trapped in solid methacrylated resins

Photopolymerization of methacrylated dental resins at room temperature leads to the appearance of long‐life free radicals because of system vitrification. These free radicals were observed by electronic paramagnetic resonance (EPR), and their characterization was undertaken by reference to the reactional mechanism, from the comparison with the model EPR signal and from theoretical simulation. Overlapping of two EPR signals assigned to two different kinds of radicals because of methacrylate end groups accounted for all the experimental and theoretical results. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1691–1699, 2003     

Investigation of organosilicon radicals : II. Methylsilyl radicals

CNDO/2 calculations for a series of Me_xH₃?_xSi (where x = 0,1,2,3) free radicals confirm the suggestion, based on electron spin resonance measurements and chemical evidence, that these radicals are not planar. The deviation from planarity decreases with increasing number of methyl groups, but the angle between the carbonsilicon bond and the plane of an assumed planar configuration is the same for methylsilyl, dimethylsilyl, and trimethylsilyl radicals.     

Organic free radicals in superheated water studied by muon spin spectroscopy

There is a pressing need to identify and monitor reaction intermediates in water at high temperatures and pressures, but conventional techniques have limited capability for studying transient free radicals under such challenging conditions. Apparatus has now been developed to permit muon avoided-level crossing spectroscopy (muLCR) of organic free radicals in superheated water. The combination of muLCR with transverse-field muon spin rotation (TF-muSR) provides the means to identify and characterize free radicals via their nuclear hyperfine coupling constants. Because the radicals are derived from the addition of muonium (Mu = mu+ e-) to unsaturated compounds, the ensuing muoniated free radicals correspond to conventional organic free radicals but with a muon spin label substituted for one of the protons. Muon spin spectroscopy is the only technique presently being used to characterize transient free radicals under hydrothermal conditions in an unambiguous manner, free from interference from other reaction intermediates. This paper demonstrates how muoniated radicals can be used to monitor the species present in hydrothermal systems, and examples are presented from two classes of reaction: dehydration of alcohols and enolization of ketones. Spectra are displayed and hyperfine constants reported for muoniated forms of the following free radicals in superheated water (typically 350 degrees C at 250 bar): 2-propyl, 2-methyl-2-propyl (tert-butyl), and 2-hydroxy-2-propyl. The latter radical is the product of muonium addition to both the keto and the enol forms of acetone, but different isotopomers are produced according to which reaction channel is dominant. This should prove invaluable in future studies of the role of enols in combustion.     

Free radicals in the reactions of organic cations

By using the ESR and CIDNP methods the formation of free radicals in the reaction of nucleophilic and electrophilic substitution is proved. The role of free radicals in the mechanism of these reactions is discussed.     

Radical reactions in the coordination field of transition metals. VII—H-transfer between phenolic and aryl-aminic H-donors and their radicals

An ESR method for studying the mechanism of H-transfer reactions between H-donors of different reactivity (A₁H, A₂H…) and their free radicals (A₁; A₂^.…) in non-polar solvents at ambient temperature is presented. The new technique is based on a pulsed initiation of various secondary phenoxy or nitroxy radicals in binary mixtures of hindered phenols, unhindered phenols, partially hindered thiobisphenols and diphenylamine, employing a high concentration of free RO₂^. and coordinated (Co^III)RO₂^. tert-butyl peroxy radicals generated in the redox-reaction of Co(acac)₂ with tert-butyl hydroperoxide. The consecutive H-transfer reactions proceed to equilibrium until the most stable radicals are formed. In this way criteria are obtained for ranking the compared free and coordinated phenoxy radicals according to their relative stabilities. The secondarily generated phenoxy radicals from unhindered phenols after coordination to Co^III are stabilized and cannot take part in further H-transfer reactions.     

Verdazyl radicals with perfluorophenyl rings

New formazans, tetrazines, and verdazyl radicals containing pentafluorophenyl residues were synthesized on the basis of the reaction of perfluorophenylhydrazones with benzenediazonium salts. The stabilities of the free radicals obtained considerably exceed the stability of the diphenylpicrylhydrazyl radical.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 266–269, February, 1978.     

Polymerization in nonuniform latex particles: Distribution of free radicals

Previous kinetic studies in emulsion polymerization have almost always involved an assumption of uniform distribution of free radicals in the latex particle. Such an assumption is not likely to reflect reality in many systems that employ water-soluble initiators because the hydrophilic end-group of the oligomeric free radical will preferentially stay in the surface layer of the particle. This constrained end-group location would result in nonuniform distribution of free radicals in the polymerizing latex particles. A Monte Carlo simulation of the growth of a single polymer chain within the latex particle supports this hypothesis. Such a nonuniform distribution of free radicals in the latex particle is expected to have an influence on reaction kinetics and product properties. The mechanism for transport of free radicals out of polymerizing latex particles is reexamined based on the proposed concept, and a modified expression for the desorption rate constant is presented.     

The study of rotational mobility of stable nitroxyl radicals in polyvinylacetate

Analyses of the rotational diffusion characters of free and bonded nitroxyl radicals in polyvinylacetate were carried out. The radical rotational character essentially depends on the molecular sizes of the radicals. The movement of the “small” radical is matched by the arbitrary jump tumbling model. The rotation of the “large” radical (both probe and label) occurs by the Brownian rotational diffusion mechanism. The correlation times in the slow-motion region are calculated by taking into account the radical rotation mechanism. Comparison of the τ_c values for the free and bonded radicals with those obtained by the NMR technique shows that movements of the spin probes and labels depend not only on the short polymer segments but on other factors also.     

A Tr Esr study of the quenching of photoexcited dioxouranium (VI) salts by stable nitroxyl free radicals

TR ESR spectroscopy was applied to the study of the quenching of excited dioxouranium (VI) (uranyl) nitrate and sulfate by stable nitroxyl radicals of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) family. Photoexcitation of uranyl in solutions of alcohols of moderate viscosity (η = 3-10 cP) in the presence of TEMPO leads to CIDEP signals of TEMPO due to a radical triplet pair mechanism (RTPM). Polarized nitroxyls were also observed in solutions of polyelectrolyte sodium poly(styrenesulfonate), NaPSS, in the presence of the nitroxyl with a positively charged trimethylammonium group. Photolysis of uranyl salts in solutions of alcohols leads to the generation of free radicals of alcohols. No CIDEP of these radicals was observed, distinguishing U₂ ^2+* from its organic analog, the triplet benzophenone. The probable reason for the lack of polarization in uranyl photoreduction reactions is the difficult access of free radicals to the U atom of the solvated radical UO₂₊ (V); this atom bears the unpaired electron. The role of polyelectrolytes in the enhancement of the quenching of excited states is discussed. Results are in agreement with the statement that photoexcited uranyl has a triplet multiplicity.     

Formation of free radicals during machining and fracture of polymers

Electron paramagnetic resonance measurements of the number of free radicals formed during cutting and grinding of polymers are described. It was found in the semicrystalline polymers studied that below the glassy transition temperature that the number of free radicals formed is about 2 × 10¹³/cm² of surface formed. It is proposed that this number results from the crack progressing selectively through the glassy regions about more ordered regions in the polymers.     

ESR study of free radicals in some new hydrazones of terpenoids class

The effect of gamma-irradiation on some new hydrazones of terpenoids using electron spin resonance (ESR) is reported. Gamma-irradiation of three derivates of hydrazone and of compounds resulting from the condensation of these derivates with terpenoids produces stable free radicals at room temperature. The analysis of the ESR parameters (g-factors and hyperfine coupling) and the simulation performed lead us to conclude that free radicals are of form R–N–NH₂ (arising from hydrazine derivates) and R–N–N=R (arising from condensation compounds). The thermal stability of formed radicals is discussed and the activation energy involved in the process of recombination of free radicals is calculated.     

On the interactions of free radicals with gold nanoparticles

Electron paramagnetic resonance (EPR) spectroscopy was used to study the interactions between stable free radicals and gold nanoparticles. The nitroxyl free radicals used were TEMPO, TEMPAMINE, and TEMPONE. Two sizes of Au particles, 15 and 2.5 nm in diameter, were synthesized to investigate the interactions with the metallic particles. We find that the EPR signal is reduced upon adsorption of the radicals onto the 15 nm Au particle surface. Despite the strong adsorption of TEMPAMINE on the particles, the signal intensity recovers upon the introduction of a high concentration of ethanolamine to the solution. The signal reduction was proportional to the concentration of Au particles, and the signal totally disappeared at high concentrations of Au particles. Possible explanations of the signal reduction are discussed in this Article. We propose that the reduction in signal intensity arises from exchange interactions between the unpaired electrons of the adsorbed radicals and conduction-band electrons of the metallic particles. In addition, in the presence of oxygen, the adsorbed TEMPAMINE radicals are catalytically oxidized to the carbonyl derivative, TEMPONE. A mechanism for this unexpected catalytic reaction is proposed.     

Perfluorophenyl-containing verdazyl radicals

New formazans, tetrazines, and verdazyl radicals containing perfluorophenyl substituents were obtained on the basis of perfluorophenylhydrazides. The stabilities of mono-, bis-, and tris(perfluorophenyl)verdazyl radicals and their leuco compounds are compared qualitatively. The stabilities of the free radicals obtained considerably exceed the stability of the diphenylpicrylhydrazyl (DPPH) radical.Chlorohydrazone IIa was previously obtained [3] by chlorination of hydrazide Ia with phosphorus pentachloride.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 8, pp. 1130–1133, August, 1980.     

Fe~(2+)协同热活化过一硫酸氢钾盐诱导自由基脱除NO的研究

在气液撞击流反应器中,研究了Fe~(2+)协同热活化过一硫酸氢钾盐诱导自由基脱除模拟烟气中的NO。考察了主要工艺参数(溶液温度、Fe~(2+)浓度、过一硫酸氢钾盐浓度、溶液pH值、NO入口浓度)对NO脱除效率的影响。分析检测了反应产物和自由基。基于不同系统的对比研究、反应产物检测和活性自由基的捕获,揭示了NO脱除过程的机制和反应路径。结果表明,提高溶液温度、Fe~(2+)浓度和过一硫酸氢钾盐浓度均提高了NO的脱除效率,而提高溶液pH值和NO入口浓度均降低了NO的脱除效率。Fe~(2+)和热对活化过一硫酸氢钾盐产生自由基有显著的协同效应。自由基氧化是NO脱除的主要路径,而过一硫酸氢钾盐直接氧化是次要的脱除路径。Fe~(2+)和热的协同活化体系具有比其他体系高得多的NO脱除率。     

Gamma-radiolysis of some crown ethers and their analogues. Electron spin resonance specra of free radicals

The method of low temperature ESR spectroscopy was used to study the free radicals generated by -irradiation of crown ethers: 12-crown-4 /1/; 15-crown-5 /2/; dicyclohexane-24-crown-8 /3/; and their analogues: tetrahydrofurane /4/ and 1,4-dioxane /5/. ESR spectra of radicals 4 and 5 taken at 77 K represent a simple singlet; ESR spectra of radicals generated from 1, 2 and 3 have a complex, multiplet structure. The kinetics of thermal decay of free radicals 4 in dependence on temperature starting from 103 K was investigated. The radicals 4 decay very fast at 253 K.     

EPR study of persistent free radicals in cross-linked EPDM rubbers

Cross-linking of ethylene propylene diene monomer (EPDM) rubbers containing different amounts of dicyclopentadiene (DCPD), 5-ethylidene-2-norbornene (ENB) and 5-vinyl-2-norbornene (VNB) dienes was examined by EPR spectroscopy. The cross-linking was initiated by thermal decomposition of dicumyl peroxide at 440 K. The concentration of free radicals increased towards the end of the cross-linking process before reaching a maximum and decaying to zero. This is explained by the spatial confinement of the radicals in the cross-linked rubber, which leads to increased life time and, hence, higher radical concentration at a time when most peroxide has decomposed. The EPR spectra showed the presence of two components: a well-resolved spectrum overlapping a broad line. Both components are assigned to allyl radicals possessing different mobility. The more mobile component is assigned to allyl radicals along the EPDM chains, whereas the immobilised allyl radicals are formed in the cross-links. The stability of the allyl radicals decreases in the order DCPD > ENB > VNB. EPDMs containing two dienes show more persistent radicals than their single-diene counterparts. The most persistent radicals are observed for highly cross-linked (e.g., 28% ENB) or mixed diene EPDMs (e.g., 2.2% DCPD-4.4% ENB); the EPR spectra of free radicals in these systems can be observed for several hours.     

Nitroxides scavenge myeloperoxidase-catalyzed thiyl radicals in model systems and in cells

Nitroxide radicals possess important antioxidant activity in live tissues because of their ability to scavenge reactive radicals. Despite the fact that, in cells, damaging free radicals are primarily quenched by glutathione (GSH) with subsequent formation of harmful glutathionyl radical (GS(*)), interactions of nitroxide radicals with GS(*) and thiols have not been studied in detail. In addition, intracellular metabolic pathways leading to the formation of secondary amines from nitroxides are unknown. Here we report that GS(*) radicals react efficiently and irreversibly with nitroxides to produce secondary amines. We developed a sensitive method for the detection of GS(*) based on their specific interaction with Ac-Tempo, a nonfluorescent conjugate of fluorogenic acridine with paramagnetic nitroxide Tempo, and used it to characterize interactions between nitroxide and thiyl radicals generated through phenoxyl radical recycling by peroxidase. During reaction of Ac-Tempo with GS(*), Tempo EPR signals decayed and acridine fluorescence concurrently increased. DMPO and PBN, spin traps for GS(*), inhibited this interaction. Using combined HPLC and mass spectrometry, we determined that 90% of the Ac-Tempo was converted into fluorescent acridine (Ac)-piperidine; GSH was primarily oxidized into sulfonic acid. In myeloperoxidase-rich HL-60 cells, Ac-piperidine fluorescence was observed upon stimulation of GS(*) generation by H(2)O(2) and phenol. Development of fluorescence was prevented by preincubation of cells with the thiol-blocking reagent N-ethylmaleimide as well as with peroxidase inhibitiors. Furthermore, Ac-Tempo preserved intracellular GSH and protected cells from phenol/GS(*) toxicity, suggesting a new mechanism for the free-radical scavenging activity of nitroxides in live cells.     

Light- or oxidant-induced generation of free radicals in hemoproteins

Upon reactions with oxidants, hemoproteins retain their oxidizing equivalents not only in the heme iron, but also in the porphyrin or amino acid residues. HRP has a protein structure that stabilizes the porphyrin π-cation radical, which can easily be formed through various reactions. CCP reacts with H₂O₂ to form free radicals in amino acid residues as a catalytic intermediate species, which is stable in the absence of added electron donor. Ferric myoglobin apparently behaves as CCP, but its amino acid free radicals are unstable. These properties seem to be reflected on photooxidation of these hemoproteins.