The radical cation of N,N-diethyl-para-phenylendiamine: A possible indicator of oxidative stress in biological samples

The alkoxy and peroxy radicals formed in the degradation of hydroperoxides brought about by transition metal ions in acidic media can convert substrates with suitably low oxidation potentials, such as N,N-diethyl-para-phenylendiamine, to the corresponding radical cations. The possibility that these reactions are used in the evaluation of oxidative stress in human beings is discussed.     

Simultaneous Determination of Hydrogen Peroxide and Organic Hydroperoxides in Water

The kinetics of the reactions of H 2 O 2 and of methyl, ethyl, tert -butyl, and cumene hydroperoxides with I m were investigated in the presence and absence of molybdate as catalyst. These results were utilized to develop an analytical method for the simultaneous determination of H 2 O 2 and organic hydroperoxides in aqueous solutions. The total amount of H 2 O 2 and organic hydroperoxides can be determined by the spectrophotometric measurement of $ {\rm I}_3^ - $ formed quantitatively during 30 min of heating at 60°C. Catalase selectively decomposes H 2 O 2 in solutions containing organic hydroperoxides. The total amount of the latter can therefore be determined iodometrically after H 2 O 2 decomposition. In the oxidation of leuco crystal violet to crystal violet by H 2 O 2 and organic hydroperoxides, horseradish peroxidase exerts similar activities in the reactions involving methyl and ethyl hydroperoxides and H 2 O 2 , but its activity is much lower with tert -butyl and cumene hydroperoxide. It was observed that acetate buffer is unsuitable for pH adjustment in this type of hydroperoxide determination in consequence of the slow oxidation of the dye in the blank solution.     

Photoinduced Oxidation of Sterically Hindered Amines in Acetonitrile Solutions and Titania Suspensions (An EPR Study)

The reactions of sterically hindered amines (SHA) were investigated in acetonitrile solutions and TiO₂ suspensions upon exposure to monochromatic radiation, λ = 365 nm, by means of in situ EPR spectroscopy. The formation of singlet oxygen, as one of the possible oxidation agents for SHA, in these systems is affected significantly by solvent used and the experimental conditions. Experiments in homogeneous media evidenced alternative pathways for the SHA oxidation with a variety reactive oxygen species involved. In anhydrous acetonitrile solutions containing KO₂, the SHA oxidation was negligible not only in the dark but also on continuous exposure. However, the presence of water, even at low concentrations, led to the transformation of O₂^?? to singlet oxygen and hydrogen peroxide, which served as a source of hydroxyl radicals. These species participated in oxidation of SHA resulting in the generation of nitroxide radicals. To investigate the influence of different competitive reactions of SHA with other ROS formed upon TiO₂ photoexcitation, a series of experiments using different additives (e.g. KO₂, H₂O₂, NaN₃, dimethylsulfoxide, methanol as organic cosolvents) under air or argon were performed. The detailed analysis of paramagnetic intermediates formed upon the irradiation of the studied systems was accomplished using EPR spin trapping technique.     

Intramolecular chain reaction of artemisinin oxidation

The intramolecular chain oxidation of artemisinin was analyzed using the parabolic model. The competition of the mono- and bimolecular peroxy radicals formed from artemisinin was considered. Artemisinin is predominantly oxidized via the intramolecular chain mechanism to form polyatomic hydroperoxides. This results in the situation when, under aerobic conditions, artemisinin is transformed from the monofunctional into polyfunctional initiator with several hydroperoxide groups. The enthalpy was calculated, and the activation energies and rate constants of the intramolecular reactions of the artemisinin peroxy radicals, as well as those of their bimolecular reactions with C-H, S-H, and O-H bonds of biological substrates and their analogs, were calculated in the framework of the parabolic model. A new kinetic scheme for artemisinin oxidation was proposed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 267–275, February, 2008.     

Reactions of alkoxy and peroxy radicals formed upon the decomposition of hydroxyperoxide groups in the artemisinin derivatives

The enthalpies of intramolecular reactions of alkoxy and peroxy radicals formed from polyatomic artemisinin hydroperoxides and of their bimolecular reactions with C—H, S—H, and O—H bonds of biological substrates were calculated. The activation energies and rate constants of these reactions were calculated using the intersecting parabolas method. The decomposition of artemisinin hydroperoxides can initiate the cascade of intramolecular oxidation reactions involving radicals R·, RO·, HO·, HO₂·, and RO₂·. The main sequences of transformation of these radicals were established. The oxidative destruction of the artemisinin peroxy derivatives generates radicals RO₂·, HO·, and HO₂· in an amount of 4.5 radicals per peroxide derivative molecule on the average. The kinetic scheme of oxidative transformations of the hydroperoxide with four OOH groups and radicals formed from it was constructed using this radical as an example.     

A Closer Look at Dark Toxicity of the Photosensitizer TMPyP in Bacteria

Photodynamic inactivation of bacteria (PIB ) is based on photosensitizers which absorb light and generate reactive oxygen species (ROS ), killing cells via oxidation. PIB is evaluated by comparing viability with and without irradiation, where reduction of viability in the presence of the photosensitizer without irradiation is considered as dark toxicity. This effect is controversially discussed for photosensitizers like TMP yP (5,10,15,20‐Tetrakis(1‐methyl‐4‐pyridinio)porphyrin tetra(p‐toluensulfonate). TMP yP shows a high absorption coefficient for blue light and a high yield of ROS production, especially singlet oxygen. Escherichia coli and Bacillus atrophaeus were incubated with TMP yP and irradiated with different light sources at low radiant exposures (μW per cm²), reflecting laboratory conditions of dark toxicity evaluation. Inactivation of E. coli occurs for blue light, while no effect was detectable for wavelengths >450 nm. Being more susceptible toward PIB , growth of B. atrophaeus is even reduced for light with emission >450 nm. Decreasing the light intensities to nW per cm² for B. atrophaeus , application of TMP yP still caused bacterial killing. Toxic effects of TMP yP disappeared after addition of histidine, quenching residual ROS . Our experiments demonstrate that the evaluation of dark toxicity of a powerful photosensitizer like TMP yP requires low light intensities and if necessary additional application of substances quenching any residual ROS .     

Composition and Stability of Peroxide Products of Liquid-Phase Cyclohexanol Oxidation

The kinetics of accumulation of peroxide compounds during noncatalytic cyclohexanol oxidation at 130°C is studied. The oxidation of cyclohexanol at the CH bond in the 1-position yields hydrogen peroxide and 1-hydroxycyclohexyl hydroperoxide along with peroxides resulting from the oxidation of the CH bonds of the alcohol in the 2-, 3-, and 4-positions, namely, cis- and trans-2-, -3- and -4-hydroxycyclohexyl hydroperoxides. Product accumulation curves and the variation of the proportions of cis- and trans-2-, -3-, and -4-hydroxycyclohexyl hydroperoxides as a function of cyclohexanol conversion indicate that these hydroperoxides have different stabilities during oxidation. The highest stability is inherent in cis-2-, trans-2-, and cis-3-hydroxycyclohexyl hydroperoxides, while cis-4-, trans-4-, and trans-3-hydroxycyclohexyl hydroperoxides are characterized by the lowest stability. This fact is explained by the stabilization of the first three isomers by an intramolecular hydrogen bond, which prevents their decomposition involving substrate molecules.__________Translated from Kinetika i Kataliz, Vol. 46, No. 3, 2005, pp. 366–369.Original Russian Text Copyright © 2005 by Puchkov, Buneeva, Perkel’.Based on a report at the XI International Conference on the Chemistry of Organic and Organoelement Peroxides (Moscow, June 24–26, 2003)     

Synthesis and Characterization of Near‐Infrared Absorbing Water Soluble Squaraines and Study of their Photodynamic Effects in DLA Live Cells

Here, we report the synthesis, photophysical properties and photodynamic effects in DLA live cells of three water soluble squaraine dyes, viz. bisbenzothiazolium squaraine dyes SQMI and SQDI with iodine in one and both benzothiazolium units, respectively, and an unsymmetrical squaraine dye ASQI containing iodinated benzothiazolium and aniline substituents. The diiodinated SQDI showed an anomalous trend in both fluorescence and triplet quantum yields over the monoiodinated SQMI, with SQDI showing higher fluorescence and lower triplet quantum yields compared to SQMI. Nanosecond laser flash photolysis of SQDI and SQMI indicated the formation of triplet excited states with quantum yield of 0.19 and 0.26, respectively. On photoirradiation, both the SQDI and SQMI generate singlet oxygen and it was observed that both dyes undergoing oxidation reactions with the singlet oxygen generated. ASQI which exhibited a lower triplet quantum yield of 0.06 was, however, stable and did not react with the singlet oxygen generated. In vitro cytotoxicity studies of these dyes in DLA live cells were performed using Trypan blue dye exclusion method and it reflect an order of cytotoxicity of SQDI>SQMI>ASQI. Intracellular generation of the ROS was confirmed by dichlorofluorescein assay after the in vitro PDT.     

Die photosensibilisierte Oxydation einwertiger Phenole zu Chinonen

The photosensitized oxidation of methyl substituted phenols with free para-positions starts with an electrophilic attack of position 4 by singlet oxygen. This follows from the course of the reaction using phenols with methyl groups in different positions. The influence of the solvent shows that the hydroperoxide formation from the primary oxygen adduct proceeds via an inter-molecular hydrogen shift. The solvent is the hydrogen donator, whereas the phenoxy radicals resulting from the oxidation of the phenol by the excited sensitizer are the hydrogen acceptors. Finally, the quinones are formed from the hydroperoxides by elimination of water.     

Findings pertaining to polyethylene photooxidation

The kinetics and mechanisms of photolysis of the hydroperoxides formed on thermal oxidation of polyethylene have been investigated. The difficulties to explain the results by the generally accepted decomposition mechanism based on homolytic splitting of the hydroperoxy bond are discussed. New mechanisms of hydroperoxide photolysis are proposed. The intermolecular reactions considered account for the main products found experimentally, ketones and trans-vinylene groups. They are also in agreement with the kinetics of product formation. The intramolecular photolysis reactions envisaged seem to involve a small amount only of the hydroperoxides present in polyethylene after thermal oxidation. However, they affect directly the mechanical properties of the polymer because they involve main-chain scission.     

DYE-SENSITIZED PHOTOOXIDATION OF 1,4-POLYDIENES

Abstract— The reaction of singlet oxygen with polydiene polymers produces hydroperoxides by the typical 'ene' type reaction. The observed chain scission process cannot be explained by the photodecom-position of hydroperoxide formed by visible light, because these hydroperoxides do not absorb light in this repion. Spectroscopic and EPR studies of the dye-solvent systems show the formation of reactive free radicals. which are probably responsible for the abstraction of hydrogen from the polymer molecules. The next step is the well known free radical oxidation mechanism which is responsible for the chain scission reactions.     

HRGC separation of hydroperoxides formed during the photosensitized oxidation of (R)—(+)-Limonene

(R)—(+)-Limonene was photooxidized in the presence of Rose Bengal as catalyst. After TLC isolation, the hydroperoxides formed were separated directly by HRGC and analyzed by MS (El; Cl). Each hydroperoxide isomer was then isolated by HPLC for structure determination which after reduction of the HOO group with sodium borohydride was performed by ¹H-NMR and ¹³C-NMR. Six hydroperoxide isomers formed by oxidation of the endocyclic double bond were identified. The compounds eluted from the HRGC column in the following order (proportions are given in brackets) I (40.1%) (1S, 4R)-p-mentha-2, 8-diene 1-hydroperoxide; II (5.8%) (1R, 4R)-p-mentha-2, 8-diene 1-hydroperoxide; III (20.6%) (2R, 4R)-p-mentha-[1(7), 8]-diene 2-hydroperoxide; IV (8.5%) (2R, 4R)-p-mentha-6, 8-diene 2-hydroperoxide; V (4%) (2S, 4R)-p-mentha-6, 8-diene 2-hydroperoxide; and VI (21.0%) (2S, 4R)-p-mentha-[1(7), 8]-diene 2-hydroperoxide. Direct HRGC separation of the limonene hydroperoxides offers, inter alia, the possibility of determining their flavor qualities by HRGC/effluent sniffing.     

Pathways of Liquid-Phase Oxidation of Cyclohexanol

The kinetics of product accumulation in uncatalyzed oxidation of cyclohexanol at 403 K was studied. Along with the compounds originating from oxidation of cyclohexanol at position 1 (cyclohexanone, hydrogen peroxide, 1-hydroxycyclohexyl hydroperoxide), products formed by oxidation of C-H bonds at positions 2-4 were detected: 2-, 3-, and 4-hydroxycyclohexyl hydroperoxides (cis and trans isomers), 1,2-, 1,3-, and 1,4-dihydroxycyclohexanes (cis and trans isomers), 2- and 4-hydroxycyclohexanones, and 2-cyclohexenone.     

Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA‐Expressing Prostate Cancer Cells

Reactive oxygen species (ROS) forming enzymes are of significant interest as anticancer agents due to their potent cytotoxicity. A key challenge in their clinical translation is attaining site‐specific delivery and minimizing biodistribution to healthy tissues. Here, complexes composed of the ROS enzyme glucose oxidase (GOX), poly‐l ‐lysine‐grafted‐polyethylene glycol (PLL‐g‐PEG), and anti‐prostate specific membrane antigen (anti‐PSMA) monoclonal antibody are synthesized for localized delivery and uptake in prostate cancer cells. Formation of anti‐PSMA‐PLL‐g‐PEG/GOX results in nanoscale complexes ≈30 nm in diameter with a ζ‐potential of 6 mV. The anti‐PSMA‐PLL‐g‐PEG/GOX complexes show significant cytotoxicity (≈60% reduction in cell viability) against PSMA‐expressing LNCaP cells compared to unmodified GOX. Importantly, cytotoxicity in LNCaP cells occurrs concurrently with anti‐PSMA‐PLL‐g‐PEG/GOX uptake and increases in intracellular generation of ROS. These results demonstrate that cytotoxicity of ROS inducing enzymes can be enhanced by intracellular delivery compared to equivalent concentrations of free enzyme, providing a novel means for cancer therapy.     

Dual Effect of Organomercury Compounds on Peroxide Oxidation of Oleic Acid

Oxidation of oleic acid with atmospheric oxygen in the presence of HgCl₂ and various organo- mercury compounds (methylmercury iodide, isopropylmercury bromide, n-hexylmercury bromide, phenylmercury bromide, diphenylmercury, p-tolylmercury bromide, bis-p-tolylmercury) was studied. Mercury compounds exert a dual effect on accumulation of oleic acid hydroperoxide in the temperature range 20-90°C. Below 50°C, the concentration of the hydroperoxides formed in the presence of mercury compounds is lower, and at higher temperatures, higher than in the experiments performed without mercury compounds. Comparison of the concentrations of oleic acid hydroperoxides with those of their transformation products, carbonyl compounds, determined spectrophotometrically, shows that actually organomercury compounds and HgCl₂ accelerate peroxide oxidation at all the studied temperatures. Decreased accumulation of peroxides below 50°C is apparently due to the fact that the rate of their reaction with organomercury compounds is higher than the rate of their formation.     

New protocol for oxidation in water: micellar oxidation systems composed of novel amphiphilic hydroperoxides

A series of novel amphiphilic hydroperoxides, alpha-alkoxyalkyl hydroperoxides (alpha-AHPs) and their related hydroperoxides, were designed and prepared with the intention of developing new oxidizing agents bearing a micelle-forming character in water. After their fundamental physical and interfacial properties were elucidated, both oxidation of benzyl sulfide and epoxidation of geraniol promoted by these hydroperoxides were investigated in detail under various conditions. Effective oxidation ofbenzyl sulfide to the corresponding sulfoxide was achieved in aqueous micellar systems composed of alpha-AHPs la-d and a catalytic amount of MoO2(acac)2 at 30 degrees C. Up to 100% conversion was observed under the optimum conditions. In the case of epoxidation of geraniol in water, the corresponding 2,3-epoxide was selectively formed in good yields. Because the conversion of each substrate in the micellar systems was higher than that in nonmicellar media, the solubilization of substrates into micelles was certainly effective in promoting oxidations of insoluble substrates in aqueous media. Micellar oxidation systems composed of novel amphiphilic hydroperoxides afford a new protocol in order to derive safer conditions under which these reactions may be carried out.     

Formation of radicals in the reactions of tetralin, 2-cyanopropane,and ethylbenzene hydroperoxides with styrene

The formation of free radicals in the reactions of structurally different hydroperoxides with styrene is investigated. The free-radical chain oxidation of styrene initiated by hydroperoxides has been studied volumetrically by measuring O₂ consumption during the reaction. The bimolecular rate constants of radical initiation in the reactions of styrene with tetralin, 2-cyanopropane, and ethylbenzene hydroperoxides are 1.5 × 10^?8, 2.6 × 10^?7, and 6.5 × 10^?9 l mol^?1 s^?1) (323 K), respectively. The reactivity of a hydroperoxide increases with increasing electron-acceptor properties of the substituent in its molecule.     

Photohemolysis Sensitized by the Furocoumarin Derivative Alloimperatorin and its Hydroperoxide Photooxidation Product

The dark and photosensitized effects of alloimperatorin methyl ether 1 (hereafter simply alloimperatorin) and its photooxygenation product alloimperatorin hydroperoxide 2 were investigated on human erythrocytes. The results reveal that the furocoumarin 1 photosensitizes efficiently the hemolysis of erythrocytes. The rate of photohemolysis increases on raising the temperature of the postirradiated incubation from 4°C to 37°C. Thermal activation of the photohemolysis and inhibition by 2,6‐di‐tert‐butyl‐p‐cresol (BHT) suggest that the furocoumarin 1 photosensitizes lipid peroxidation, increasing permeability in the erythrocyte membrane. The hydroperoxide 2 induces dark and photosensitized hemolysis more efficiently than the furocoumarin 1. The rate of hemolysis induced by 2 increases with the incubation temperature and decreases in the presence of tert‐butanol and BHT. The hydroperoxide 2 photosensitizes the formation of lipid peroxidation products as shown by the reaction with thiobarbituric acid. This process is diminished by BHT. Our data imply that the photohemolysis sensitized by the furocoumarin 1 is caused by the in situ‐formed photooxygenation product 2. Such hydroperoxides are potent hemolytic agents in the dark and especially on photosensitization.     

Formation of oligomeric alkenylperoxides during the oxidation of unsaturated fatty acids: an electrospray ionization tandem mass spectrometry study

This study reports the identification of oligomeric alkenylperoxides by electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (ESI‐MS²), during the oxidation of oleic, linoleic and linolenic acids with Fenton's (Fe²⁺/H₂O₂) and Fe²⁺/O₂ systems. The reactions were followed by ferrous oxidation‐xylenol orange method together with GC‐MS and GC‐FID, allowing to observe that both oxidation systems are different in terms of hydroperoxide evolution, probably due to the presence of different intermediate reactive species: perferryl ion and OH^· radical responsible for the decomposition of lipid hydroperoxides and formation of new compounds. The analysis of ESI‐MS in the negative mode, obtained after oxidation of each fatty acid, confirmed the presence of the monomeric oxidation products together with other compounds at high mass region above m/z 550. These new ions were attributed to oligomeric structures, identified by the fragmentation pathways observed in the tandem mass spectra. Copyright © 2012 John Wiley & Sons, Ltd.     

Solvation Accounts for the Counterintuitive Nucleophilicity Ordering of Peroxide Anions

The nucleophilic reactivities (N , s _N) of peroxide anions (generated from aromatic and aliphatic peroxy acids or alkyl hydroperoxides) were investigated by following the kinetics of their reactions with a series of benzhydrylium ions (Ar₂CH⁺) in alkaline aqueous solutions at 20 °C. The second‐order rate constants revealed that deprotonated peroxy acids (RCO₃⁻), although they are the considerably weaker Brønsted bases, react much faster than anions of aliphatic hydroperoxides (ROO⁻). Substitution of the rate constants of their reactions with benzhydrylium ions into the linear free energy relationship lg k =s _N(N +E ) furnished nucleophilicity parameters (N , s _N) of peroxide anions, which were successfully applied to predict the rates of Weitz–Scheffer epoxidations. DFT calculations with inclusion of solvent effects by means of the Integral Equation Formalism version of the Polarizable Continuum Model were performed to rationalize the observed reactivities.