Search

H-bond complexes between 3- or 4-OH phenoxyl radicals and various H-bond accepting molecules were investigated by experimental and computational methods. The H-bond donating ability (α(2)(H)) of 2,6-di-tert-butyl-4-hydroxyphenoxyl radical (1) was determined as 0.79 ± 0.05 by measuring, using EPR spectroscopy, the variations of the hyperfine splitting constants of 1 as a function of the acceptor concentrations. A computational approach, based on DFT calculations, was employed to estimate the α(2)(H) values for OH-substituted phenoxyl radicals that were not persistent enough to be studied by EPR spectroscopy. The α(2)(H) value calculated for the 2,6-di-methyl analogue of 1 was 0.76, in good agreement with EPR experiments. The α(2)(H) values for 2-methoxy-4-hydroxy (3), 4-hydroxy (4), 4,6-di-methyl-3-hydroxy (5) and 3-hydroxy (6) phenoxyl radicals were computed as 0.77, 0.84, 0.66 and 0.71, respectively, indicating that α(2)(H) values were dependent on the presence of electron donating substituents and on the relative positions of the -OH and -O˙ groups. By correlating the α(2)(H) values for 4 and 6 with their water and gas-phase acidities, an unexpected role of water in promoting proton dissociation from these radicals was evidenced.

The design and the synthesis of a new family of hydroxy-4-thiaflavanes, in which the reactive phenolic OH is ortho to the sulfur atom of the benzofused oxathiin ring, allowed to prepare antioxidants that show rate constants for the reaction with peroxyl radicals (k(inh)), and bond dissociation energies (BDE), of the ArO-H group identical to those of α-tocopherol, the main component of vitamin E and the most effective lipophilic antioxidant known in nature. The peculiar conformation of the six-membered heterocyclic ring prevents the formation of an intramolecular hydrogen bond between the OH group and the S atom, while ensuring a good stabilization by electron donation of the phenoxyl radical formed after the reaction with peroxyl radicals. The preparation of these compounds was achieved through an inverse electron demand hetero Diels-Alder reaction of styrenes with o-thioquinones, in turn prepared from accurately designed 1,3-dihydroxy arenes. Properly arranging the substitution pattern on the aromatic ring, as in derivatives 9 and 11, allowed to reach values of k(inh) up to 4.0×10(6) M(-1) s(-1) and BDE((OH)) of 77.2 kcal mol(-1). This approach represents an innovative way to obtain highly active antioxidants without using strongly electron donating alkylamino groups which are associated with adverse toxicological profiles., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ascorbic acid (vit. C) is a cofactor whose reactivity toward peroxyl and other radical species has a key-role in its biological function. At physiological pH it is dissociated to the corresponding anion. Derivatives of ascorbic acid, like ascorbyl palmitate, are widely employed in food or in cosmetics and pharmaceuticals. While the aqueous chemistry of ascorbate has long been investigated, in non-aqueous media it is largely unexplored. In this work oxygen-uptake kinetics, EPR and computational methods were combined to study the reaction of peroxyl radicals with two lipid-soluble derivatives: ascorbyl palmitate and 5,6-isopropylidene-l-ascorbic acid in non-aqueous solvents. In acetonitrile at 303 K the undissociated AscH(2) form of the two derivatives trapped peroxyl radicals with k(inh) of (8.4 ± 1.0) × 10(4) M(-1) s(-1), with stoichiometric factor of ca. 1 and isotope effect k(H)/k(D) = 3.0 ± 0.6, while in the presence of bases the anionic AscH(-) form had k(inh) of (5.0 ± 3.3) × 10(7) M(-1) s(-1). Reactivity was also enhanced in the presence of acetic acid and the mechanism is discussed. The difference in reactivity between the AscH(2)/AscH(-) forms was paralleled by a difference in O-H bond dissociation enthalpy, which was determined by EPR equilibrations as 81.0 ± 0.4 and 72.2 ± 0.4 kcal mol(-1) respectively for AscH(2) and AscH(-) in tert-butanol at 298 K. Gas-phase calculations for the neutral/anionic forms were in good agreement yielding 80.1/69.0 kcal mol(-1) using B3LYP/6-31+g(d,p) and 79.0/67.8 kcal mol(-1) at CBS-QB3 level. EPR spectra of ascorbyl palmitate in tBuOH consisted of a doublet with HSC = 0.45 G centred at g = 2.0050 for the neutral radical AscH˙ and a doublet of triplets with HSCs of 1.85 G, 0.18 G and 0.16 G centred at g = 2.0054 for Asc˙(-) radical anion.

Hydrogenated cardanol and cardols, contained in industrial grade cardanol oil and obtained by distillation of the raw "cashew nut shell liquid" (CNSL), are easily transformed into efficient 4-thiaflavane antioxidants bearing a long alkyl chain on A ring and a catechol group on B ring.

Introduction of an octyltelluro group ortho to the phenolic moiety in 3-tert-butyl-4-hydroxyanisole (BHA) was found to significantly improve the antioxidant characteristics of the material. In contrast to BHA and the corresponding ortho-substituted octylthio- (9c) and octylseleno (9b) derivatives, the organotellurium 9a was regenerable when assayed for its capacity to inhibit azo-initiated peroxidation of linoleic acid in a chlorobenzene/water two-phase system containing N-acetylcysteine as a stoichiometric reducing agent, and peroxyl radicals were quenched more efficiently than with α-tocopherol. In the homogeneous phase, inhibition of styrene autoxidation occurred with a rate constant kinh as large as 1 × 10(7) M(-1) s(-1) but with a low (n = 0.4) stoichiometric factor. Evans-Polanij plots of log (kinh) versus BDE(O-H), which are usually linear for phenols with similar steric crowding reacting by H-atom transfer, revealed that compound 9a was more than 2 orders of magnitude more reactive than expected. Although further mechanistic investigations are needed, it seems that the ortho-arrangement of an alkyltelluro group and hydroxyl should be considered a privileged structure for phenolic antioxidants.

Addition of millimolar amounts of a weak base (pyridines) dramatically accelerates the reaction with peroxyl radicals of two biologically relevant uracil derivatives, 5-hydroxyuracil (HU) and 5-hydroxy-6-methyluracil (HMU). This is due to the formation of small amounts of the deprotonated form (pK(a) = 8.1-8.5 in water), which reacts with peroxyl radicals much faster than the parent undissociated form, via formal H-atom transfer from the OH in the 5 position.

In the presence of organic acids in organic media, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) reacts with peroxyl radicals at nearly diffusion-controlled rates by proton-coupled electron transfer from the protonated nitroxide.

DFT calculations using the B3LYP functional, medium-sized basis sets and empirical scaling of the results provide quantitative estimates of the hydrogen isotropic hyperfine splitting constants (hscs) in 2,6-di-alkyl phenoxyl radicals (1-11). Literature hscs for phenoxyl (12), 4-methylphenoxyl (13) and 4-methoxyphenoxyl (14) radicals, on the other hand, are poorly predicted by using this method. This different behaviour is explained considering that experimental hscs of 12-14 are influenced by H-bonds formed between phenoxyls and their parent phenols, usually present in large amounts in solution as radical precursors. This was confirmed experimentally by measuring the EPR spectra of 12-14 in the presence of increasing amounts of their parent phenols, and by calculating the hscs in the case of the formation of 1 : 1 and 1 : 2 complexes between these radicals and phenol. Relevance of these results to the choice of reference hscs as benchmarks for theoretical calculations and to kinetic and thermochemical determinations on unhindered phenoxyl radicals is discussed.

Remote intramolecular hydrogen bonds (HBs) in phenols and benzylammonium cations influence the dissociation enthalpies of their O-H and C-N bonds, respectively. The direction of these intramolecular HBs, para --> meta or meta --> para, determines the sign of the variation with respect to molecules lacking remote intramolecular HBs. For example, the O-H bond dissociation enthalpy of 3-methoxy-4-hydroxyphenol, 4, is about 2.5 kcal/mol lower than that of its isomer 3-hydroxy-4-methoxyphenol, 5, although group additivity rules would predict nearly identical values. In the case of 3-methoxy-4-hydroxybenzylammonium and 3-hydroxy-4-methoxybenzylammonium ions, the CBS-QB3 level calculated C-N eterolytic dissociation enthalpy is about 3.7 kcal/mol lower in the former ion. These effects are caused by the strong electron-withdrawing character of the -O(*) and -CH(2)(+) groups in the phenoxyl radical and benzyl cation, respectively, which modulates the strength of the HB. An O-H group in the para position of ArO(*) or ArCH(2)(+) becomes more acidic than in the parent molecules and hence forms stronger HBs with hydrogen bond acceptors (HBAs) in the meta position. Conversely, HBAs, such as OCH(3), in the para position become weaker HBAs in phenoxyl radicals and benzyl cations than in the parent molecules. These product thermochemistries are reflected in the transition states for, and hence in the kinetics of, hydrogen atom abstraction from phenols by free radicals (dpph(*) and ROO(*)). For example, the 298 K rate constant for the 4 + dpph(*) reaction is 22 times greater than that for the 5 + dpph(*) reaction. Fragmentation of ring-substituted benzylammonium ions, generated by ESI-MS, to form the benzyl cations reflects similar remote intramolecular HB effects.

The autoxidation of organic materials is a detrimental radical chain process often leading to their rapid deterioration unless they are protected by preventive and/or chain breaking antioxidants. The properties of the more important family of the latter ones, that one of phenols, are illustrated in this tutorial review. A short outline of diarylamine antioxidants is also given. We describe simple experimental methods employed for the determination of the two parameters more useful for estimating the inhibiting power of antioxidants, that is the kinetic rate constant for their reaction with the chain carrying peroxyl radicals, k(inh), to give persistent phenoxyl or aminyl radicals and the bond dissociation enthalpy BDE(X-H) (X = O, N) of the bond cleaved in the inhibition process. The dependence of these parameters on the number and nature of the substituents is discussed and, in the case of phenols, a simple rule allowing to predict with reasonable accuracy the BDE and k(inh) values, from their structure. The effect of solvent polarity on the antioxidant power is also described. Finally, the information on the mechanism of reaction between phenols and peroxyl radicals provided by both experiments and theoretical calculations are examined. Because of difficulties associated with the analysis of non-homogenous systems all the reported results refer to homogenous solution in which experimental data can be analysed by means of more reliable and complete treatments.

Little is known about the ED/EW character of organochalcogen substituents and their contribution to the O-H bond dissociation enthalpy (BDE) in phenolic compounds. A series of ortho- and para-(S,Se,Te)R-substituted phenols were prepared and investigated by EPR, IR, and computational methods. Substituents lowered the O-H BDE by >3 kcal/mol in the para position, while the ortho-effect was modest due to hydrogen bonding ( approximately 3 kcal/mol) to the O-H group.

The synthesis of 3-pyridinols carrying alkyltelluro, alkylseleno, and alkylthio groups is described together with a detailed kinetic, thermodynamic, and mechanistic study of their antioxidant activity. When assayed for their capacity to inhibit azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system, tellurium-containing 3-pyridinols were readily regenerable by N-acetylcysteine contained in the aqueous phase. The best inhibitors quenched peroxyl radicals more efficiently than alpha-tocopherol, and the duration of inhibition was limited only by the availability of the thiol reducing agent. In homogeneous phase, inhibition of styrene autoxidation absolute rate constants k(inh) for quenching of peroxyl radical were as large as 1 x 10(7) M(-1) s(-1), thus outperforming the best phenolic antioxidants including alpha-tocopherol. Tellurium-containing 3-pyridinols could be quantitatively regenerated in homogeneous phase by N-tert-butoxycarbonyl cysteine methyl ester, a lipid-soluble analogue of N-acetylcysteine. In the presence of an excess of the thiol, a catalytic mode of action was observed, similar to the one in the two-phase system. Overall, compounds bearing the alkyltelluro moiety ortho to the OH group were much more effective antioxidants than the corresponding para isomers. The origin of the high reactivity of these compounds was explored using pulse-radiolysis thermodynamic measurements, and a mechanism for their unusual antioxidant activity was proposed. The tellurium-containing 3-pyridinols were also found to catalyze reduction of hydrogen peroxide in the presence of thiol reducing agents, thereby acting as multifunctional (preventive and chain-breaking) catalytic antioxidants.

A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK(a) of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK(a)'s that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning one type of aryloxyl in the presence of other possible aryloxyl radicals in peptides and proteins with multiple tyrosine residues between which electrons and protons can be transferred.

The role of intramolecular hydrogen bonding (HB) on the bond-dissociation enthalpy (BDE) of the phenolic O-H and on the kinetics of H-atom transfer to peroxyl radicals (k(inh)) of several 2-alkoxyphenols was experimentally quantified by the EPR equilibration technique and by inhibited autoxidation studies. These compounds can be regarded as useful models for studying the H-atom abstraction from 2-OR phenols, such as many lignans, reduced coenzyme Q and curcumin. The effects of the various substituents on the BDE(O-H) of 2-methoxy, 2-methoxy-4-methyl, 2,4-dimethoxyphenols versus phenol were measured in benzene solution as -1.8; -3.7; -5.4 kcal mol(-1), respectively. In the case of polymethoxyphenols, significant deviations from the BDE(O-H) values predicted by the additive effects of the substituents were found. The logarithms of the k(inh) constants in cumene were inversely related to the BDE(O-H) values, obeying a linear Evans-Polanyi plot with the same slope of other substituted phenols and a y-axis intercept slightly smaller than that of 2,6-dimethyl phenols. In the cases of phenols having the 2-OR substituent included in a five-membered condensed ring (i.e, compounds 9-11), both conformational isomers in which the OH group points toward or away from the oxygen in position 2 were detected by FTIR spectroscopy and the intramolecular HB strength was thus estimated. The contribution to the BDE(O-H) of the ortho-OR substituent in 9, corrected for intramolecular HB formation, was calculated as -5.6 kcal mol(-1). The similar behaviour of cyclic and non-cyclic ortho-alkoxy derivatives clearly showed that the preferred conformation of the OMe group in ortho-methoxyphenoxyl radicals is that in which the methyl group points away from the phenoxyl oxygen, in contrast to the geometries predicted by DFT calculations.

When assayed for their capacity to inhibit azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system, tellurium-containing 3-pyridinols were readily regenerable by N-acetylcysteine contained in the aqueous phase. The best inhibitors quenched peroxyl radicals more efficiently than alpha-tocopherol, and the duration of inhibition was limited only by the availability of the thiol reducing agent. The compounds were also found to catalyze reduction of hydrogen peroxide in the presence of thiol reducing agent.

Stability constants, rates of association and dissociation, and thermodynamic and activation parameters for the formation of inclusion complexes between the radical guest, N-benzyl- tert-butyl- d 9-nitroxide and beta- or 2,6- O-dimethyl-beta-cyclodextrin (CDs), have been determined by EPR spectroscopy in water in the presence of 14 different alcohols, differing in size and lipophilicity. In all cases, it was found that addition of alcohol, depending on its structure and concentration, causes a reduction of the stability of the paramagnetic complex. Global analysis of EPR data allowed us to explain the CDs binding behavior: we discarded the formation of a ternary complex, where alcohol and radical guest are coincluded into CD cavity, while data were found more consistent with the formation of a binary complex alcohol:CD competing with the monitored complex nitroxide:CD. Both kinetic and thermodynamic analysis of the experimental results have revealed that the presence of alcohols affects to a larger extent the dissociation rather then the association of radical probe and CD and that the former process is of greater importance in determining the stability of the complex, this confirming the reliability of the competition model proposed. This competition has been used for the indirect determination of the stability constants of complexes between CD and examined alcohols. By using a similar approach, we showed EPR spectroscopy can be considered a rapid and accurate technique to investigate the CDs binding behavior toward different nonradical guest.

Brassica vegetables are attracting major attention as healthy foods because of their content of glucosinolates (GLs) that release the corresponding isothiocyanates (ITCs) upon myrosinase hydrolysis. A number of studies have so far documented the chemopreventive properties of some ITCs. On the other hand, single nutrients detached from the food itself risk being somewhat "reductive", since plants contain several classes of compounds endowed with a polyhedral mechanism of action. Our recent finding that 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) and 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC), released by the GLs purified from Japanese (Kaiware) Daikon (Raphanus sativus L.) seeds and sprouts, had selective cytotoxic/apoptotic activity on three human colon carcinoma cell lines prompted further research on the potential chemopreventive role of a standardized Kaiware Daikon extract (KDE), containing 10.5% w/w GRH and 3.8% w/w GRE, compared to its isolated components. KDE administered in combination with myrosinase at doses corresponding to 50 microM GRH-ITC plus 15 microM GRE-ITC (50 microM KDE-ITC) to three human cancer cell lines (LoVo, HCT-116 and HT-29) significantly reduced cell growth by 94-96% of control in six days (p < 0.05), outperforming pure GRH-ITC or GRE-ITC at the same dose. On the other hand, the same treatment had no significant toxicity on normal human T-lymphocytes. A 50 microM concentration of KDE-ITC had relevant apoptosis induction in all tested cancer cell lines, as confirmed by annexin V assay (e.g., 33% induction in LoVo compared to control, p < 0.05), Bax protein induction (e.g., +20% in HT-29, p < 0.05), and Bcl2 downregulation (e.g.-20% in HT-29, p < 0.05), and induced caspase-1 and PARP-1 activation in all cancer cells as shown by Western blot analysis. Unlike pure GRH or GRH-ITC, KDE also had significant chain-breaking antioxidant activity, retarding the AAPH-initiated autoxidation of methyl linoleate in SDS micelles at concentrations as low as 4.4 ppm (-50% in oxygen consumption rate), as monitored by Clark-type microelectrode oxygen-uptake kinetics, and induced very fast quenching of DPPH. radical in methanol with t(1/2) (s) = (1.47 +/- 0.25) x 10(-2)/[KDE; (g/L)], measured by stopped-flow UV-vis kinetics at 298 K. The potential chemopreventive role of KDE is discussed.

The chain-breaking antioxidant activities of two garlic-derived allyl sulfides, i.e. diallyl disulfide (1), the main component of steam-distilled garlic oil, and allyl methyl sulfide (3) were evaluated by studying the thermally initiated autoxidation of cumene or styrene in their presence. Although the rate of cumene oxidation was reduced by addition of both 1 and 3, the dependence on the concentration of the two sulfides could not be explained on the basis of the classic antioxidant mechanism as with phenolic antioxidants. The rate of oxidation of styrene, on the other hand, did not show significant changes upon addition of either 1 or 3. This unusual behaviour was explained in terms of the co-oxidant effect, consisting in the decrease of the autoxidation rate of a substrate forming tertiary peroxyl radicals (i.e. cumene) upon addition of little amounts of a second oxidizable substrate giving rise instead to secondary peroxyl radicals. The relevant rate constants for the reaction of ROO(.) with 1 and 3 were measured as 1.6 and 1.0 M(-1) s(-1), respectively, fully consistent with the H-atom abstraction from substituted sulfides. It is therefore concluded that sulfides 1 and 3 do not scavenge peroxyl radicals and therefore cannot be considered chain-breaking antioxidants.

Hydroquinones (benzene-1,4-diols) are naturally occurring chain-breaking antioxidants, whose reactions with peroxyl radicals yield 1,4-semiquinone radicals. Unlike the 1,2-semiquinone radicals derived from catechols (benzene-1,2-diols), the 1,4-semiquinone radicals do not always trap another peroxyl radical, and instead the stoichiometric factor of hydroquinones varies widely between 0 and 2 as a function of ring-substitution and reaction conditions. This variable antioxidant behavior has been attributed to the competing reaction of the 1,4-semiquinone radical with molecular oxygen. Herein we report the results of experiments and theoretical calculations focused on understanding this key reaction. Our experiments, which include detailed kinetic and mechanistic investigations by laser flash photolysis and inhibited autoxidation studies, and our theoretical calculations, which include detailed studies of the reactions of both 1,4-semiquinones and 1,2-semiquinones with O2, provide many important insights. They show that the reaction of O2 with 2,5-di-tert-butyl-1,4-semiquinone radical (used as model compound) has a rate constant of 2.4 +/- 0.9 x 10(5) M-1 s-1 in acetonitrile and as high as 2.0 +/- 0.9 x 10(6) M-1 s-1 in chlorobenzene, i.e., similar to that previously reported in water at pH approximately 7. These results, considered alongside our theoretical calculations, suggest that the reaction occurs by an unusual hydrogen atom abstraction mechanism, taking place in a two-step process consisting first of addition of O2 to the semiquinone radical and second an intramolecular H-atom transfer concerted with elimination of hydroperoxyl to yield the quinone. This reaction appears to be much more facile for 1,4-semiquinones than for their 1,2-isomers.

There is high current interest in the chemopreventive potential of Brassica vegetables (cruciferae), particularly due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isythiocyanates (ITC). Some ITCs, such as sulforaphane (SFN) from broccoli ( Brassica oleacea italica), have been found to possess anticancer activity through induction of apoptosis in selected cell lines, as well as indirect antioxidant activity through induction of phase II detoxifying enzymes. Japanese daikon ( Raphanus sativus L.) is possibly the vegetable with the highest per capita consumption within the Brassicaceae family. Thanks to a recently improved gram scale production process, it was possible to prepare sufficient amounts of the GL glucoraphasatin (GRH) as well as the corresponding ITC 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) from its sprouts. This paper reports a study on the cytotoxic and apoptotic activities of GRH-ITC compared with the oxidized counterpart 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC) on three human colon carcinoma cell lines (LoVo, HCT-116, and HT-29) together with a detailed kinetic investigation of the direct antioxidant/radical scavenging ability of GRH and GRH-ITC. Both GRH-ITC and GRE-ITC reduced cell proliferation in a dose-dependent manner and induced apoptosis in the three cancer cell lines. The compounds significantly ( p < 0.05) increased Bax and decreased Bcl2 protein expression, as well as producing caspase-9 and PARP-1 cleavage after 3 days of exposure in the three cancer cell lines. GRH-ITC treatment was shown to have no toxicity with regard to normal human lymphocytes (-15 +/- 5%) in comparison with SFN (complete growth inhibition). GRH and GRH-ITC were able to quench the 2,2-diphenyl-1-picrylhydrazyl radical, with second-order rate constants of 14.0 +/- 2.8 and 43.1 +/- 9.5 M(-1) s(-1), respectively (at 298 K in methanol), whereas the corresponding value measured here for the reference antioxidant alpha-tocopherol was 425 +/- 40 M (-1) s (-1). GRH reacted with H2O2 and tert-butyl hydroperoxide in water (pH 7.4) at 37 degrees C, with rate constants of 1.9 +/- 0.3 x 10(-2) and 9.5 +/- 0.3 x 10(-4) M(-1) s (-1) (paralleling recently developed synthetic antioxidants) being quantitatively (>97%) converted to GRE. It is demonstrated that GRH-ITC has interesting antioxidant/radical scavenging properties, associated with a selective cytotoxic/apoptotic activity toward three human colon carcinoma cell lines, and very limited toxicity on normal human T-lymphocytes.

Rotation about the Ar-S bond in ortho-(alkylthio)phenols strongly affects the bond dissociation enthalpy (BDE) and the reactivity of the OH group. Newly synthesized sulfur containing heterocycles 3 and 4, where the -SR group is almost coplanar with the phenolic ring, are characterized by unusually low BDE(O-H) values (79.6 and 79.2 kcal/mol, respectively) and by much higher reactivities toward peroxyl radicals than the ortho-methylthio derivative 1 (82.0 kcal/mol). The importance of the intramolecular hydrogen bond (IHB) in determining the BDE(O-H) was demonstrated by FT-IR experiments, which showed that in heterocycles 3 and 4 the IHB between the phenolic OH group and the S atom is much weaker than that present in 1. Since the IHB can be formed only if the -SR group adopts an out-of-plane geometry, this interaction is possible only in the methylthio derivative 1 and not in 3 and 4. The additive contribution to the phenolic BDE(O-H) of the -SR substituent therefore varies from -3.1 to +2.8 kcal/mol for the in-plane and out-of-plane conformations, respectively. These results may be relevant to understanding the role of the tyrosine-cysteine link in the active site of galactose oxidase, an important enzyme that catalyzes the two-electron aerobic oxidation of primary alcohols to aldehydes. The switching of the ortho -SR substituent between perpendicular and planar conformations may account for the catalytic efficiency of this enzyme.

We examined a possible protective effect of the nonpeptidyl low molecular weight radical scavenger IAC [bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decanedioate di-hydrochloride] on isolated human islet cells against isolation and culture oxidative stress. Islets isolated from pancreases of nondiabetic multiorgan donors by collagenase digestion were purified by density gradient centrifugation. After the isolation, islets were either exposed or not exposed for 7 days to 10 micromol/L IAC. We found that IAC markedly reduced oxidative stress and ameliorated islets function. These results suggest that the use of IAC could be an interesting pharmacological approach for the treatment of the islets before transplantation.

Oxidative stress is a putative mechanism leading to beta-cell damage in type 2 diabetes. We studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. Evidence of increased oxidative stress in diabetic islets was demonstrated by measuring nitrotyrosine concentration and by electron paramagnetic resonance. This was accompanied by reduced glucose-stimulated insulin secretion, as compared to non-diabetic islets (Stimulation Index, SI: 0.9 +/- 0.2 vs. 2.0 +/- 0.4, P<0.01), and by altered expression of insulin (approximately -60%), catalase (approximately +90%) and glutathione peroxidase (approximately +140%). When type 2 diabetic islets were pre-exposed for 24 h to the new antioxidant bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride, nitrotyrosine levels, glucose-stimulated insulin secretion (SI: 1.6+/-0.5) and gene expressions improved/normalized. These results support the concept that oxidative stress may play a role in type 2 diabetes beta-cell dysfunction; furthermore, it is proposed that therapy with antioxidants could be an interesting adjunctive pharmacological approach to the treatment of type 2 diabetes.

6-(Ethylthio)-, 6-(ethylseleno)-, and 6-(ethyltelluro)-2,2,4-trimethyl-1,2-dihydroquinoline-three heavier chalcogen analogues of ethoxyquin-were prepared by dilithiation of the corresponding 6-bromodihydroquinoline followed either by treatment with the corresponding diethyl dichalcogenide (sulfur derivative) or by insertion of selenium/tellurium into the carbon-lithium bond, oxidation to a diaryl dichalcogenide, borohydride reduction, and finally alkylation of the resulting areneselenolate/arenetellurolate. Ethoxyquin, its heavier chalcogen analogues, and the corresponding 6-PhS, 6-PhSe, and 6-PhTe derivatives were assayed for both their chain-breaking antioxidative capacity and their ability to catalyze reduction of hydrogen peroxide in the presence of a stoichiometric amount of a thiol reducing agent (thiol peroxidase activity). Ethoxyquin itself turned out to be the best inhibitor of azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system. In the absence of N-acetylcysteine as a coantioxidant in the aqueous phase, it inhibited peroxidation as efficiently as alpha-tocopherol but with a more than 2-fold longer inhibition time. In the presence of 0.25 mM coantioxidant in the aqueous phase, the inhibition time was further increased by almost a factor of 2. This is probably due to thiol-mediated regeneration of the active antioxidant across the lipid-aqueous interphase. The ethyltelluro analogue 1d of ethoxyquin was a similarly efficient quencher of peroxyl radicals compared to the parent in the two-phase system, but less regenerable. Ethoxyquin was found to inhibit azo-initiated oxidation of styrene in the homogeneous phase (chlorobenzene) almost as efficiently (kinh = (2.0 +/- 0.2) x 106 M-1 s-1) as alpha-tocopherol with a stoichiometric factor n = 2.2 +/- 0.1. At the end of the inhibition period, autoxidation was additionally retarded, probably by ethoxyquin nitroxide formed during the course of peroxidation. The N-H bond dissociation enthalpy of ethoxyquin (81.3 +/- 0.3 kcal/mol) was determined by a radical equilibration method using 2,6-dimethoxyphenol and 2,6-di-tert-butyl-4-methylphenol as equilibration partners. Among the investigated compounds, only the tellurium analogues 1d and, less efficiently, 1g had a capacity to catalyze reduction of hydrogen peroxide in the presence of thiophenol. Therefore, analogue 1d is the only antioxidant which is multifunctional (chain-breaking and preventive) in character and which can act in a truly catalytic fashion to decompose both peroxyl radicals and organic hydroperoxides in the presence of suitable thiol reducing agents.

A series of 2,3-dihydrobenzo[b]selenophene-5-ol antioxidants was prepared by subjecting suitably substituted allyl 4-methoxyphenyl selenides to microwave-induced seleno-Claisen rearrangement/intramolecular Markovnikov hydroselenation followed by boron tribromide-induced O-demethylation. The novel antioxidants were assayed for their capacity to inhibit azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system containing N-acetylcysteine as a thiol reducing agent in the aqueous phase. Antioxidant efficiency as determined by the inhibited rate of peroxidation, Rinh, increased with increasing methyl substitution (Rinh=46-26 microM/h), but none of the compounds could match alpha-tocopherol (Rinh=22 microM/h). Regenerability as determined by the inhibition time, Tinh, in the presence of the thiol regenerating agent decreased with increasing methyl substitution. Thus, under conditions where the unsubstituted compound 5a inhibited peroxidation for more than 320 min, alpha-tocopherol worked for 90 min and the trimethylated antioxidant 5g for 60 min only. Sampling of the aqueous phase at intervals during peroxidation using antioxidant 5a showed that N-acetylcysteine was continuously oxidized with time to the corresponding disulfide. In the absence of the regenerating agent, compounds 5 inhibited peroxidation for 50-60 min only. A (RO)B3LYP/LANL2DZdp//B3LYP/LANL2DZ model was used for the calculation of homolytic O-H bond dissociation enthalpies (BDE) and adiabatic ionization potentials (IP) of phenolic antioxidants 5. Both BDE (80.6-76.3 kcal/mol) and IP (163.2-156.0 kcal/mol) decrease with increasing methyl substitution. The phenoxyl radical corresponding to phenol 5g gave an intense ESR signal centered at g=2.0099. The H-O bond dissociation enthalpy of the phenol was determined by a radical equilibration method using BHA as an equilibration partner. The observed BDE (77.6+/-0.5 kcal/mol) is in reasonable agreement with calculations (76.3 kcal/mol). As judged by calculated log P values, the lipophilicity of compounds 5 increased slightly when methyl groups were introduced into the phenolic moiety (2.9>C log P<4.2). The capacity of compounds 5a (kinh=3.8x10(5) M-1 s-1) and 5g (kinh=1.5x10(6) M-1 s-1) to inhibit azo-initiated autoxidation of styrene in the homogeneous phase (chlorobenzene) was also studied. More efficient regeneration at the lipid-aqueous interphase is the most likely explanation why the intrinsically poorest antioxidant 5a can outperform its analogues as well as alpha-TOC in the two-phase system. Possible mechanisms of regeneration are discussed and evaluated.

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.

EPR spectroscopy was used for the first time to explore the binding properties of cucurbit[7]uril (CB7), a representative member of the cucurbituril family. Evidence for the formation of a complex between nitroxide radicals and the host system in an aqueous solution was provided by large changes in the nitrogen hyperfine splitting, attributed to the different polar environments experienced by the included radical. In the presence of alkali cations, the EPR spectra of benzyl tert-butyl nitroxide were characterised by new signals attributed to the radical hosted in the CB7 cavity in which one metal cation is in close contact with the nitroxidic oxygen. The formation of the coordination complex results in a substantial increase in the electron spin density on the nitrogen in inverse order with respect to the size of the cation owing to increased localisation of negative charge on the oxygen atom from bonding to the alkali cation. The EPR spectra showed selective line-broadening effects as a result of metal exchange between bulk water and the coordination complex. Analysis of the EPR linewidth variations allowed us to measure the corresponding kinetic rate constants for the first time. NMR spectroscopy showed that this behaviour is not peculiar to nitroxides but is also exhibited by the related carbonyl compounds. These data allowed us to quantify the template effect and to reach the conclusion that, in the presence of a guest having a coordinating lone pair, the formation of ternary metal-guest-CB complexes must be taken into account when discussing the complexation behaviour of cucurbituril derivatives in the presence of salts.

Sulfur-containing analogues of vitamin E (thiachromanols), either linked or not to a catechol moiety, were synthesized and their hydrogen-atom donating ability evaluated. The determination of the O--H bond dissociation enthalpy (BDE) of the alpha-tocopherol analogue 4 by the electron paramagnetic resonance (EPR) equilibration technique provided a value of 78.9 kcal mol(-1), that is, approximately 1.8 kcal mol(-1) higher than that of alpha-tocopherol. The kinetic rate constants for the reaction with peroxyl radicals (kinh), measured by inhibited autoxidation studies, showed that thiachromanols react 2.5 times slower than the corresponding tocopherols, in agreement with the higher BDE value. This behavior was explained, on the basis of crystallographic analyses and DFT calculations, in terms of a change in the molecular geometry caused by insertion of a sulfur atom into the framework of vitamin E. This behavior implies a greater deviation of the condensed ring from coplanarity with the aromatic ring, thus giving rise to a decrease in the conjugative stabilization of the phenoxyl radical and consequently to an increase in the O--H bond strength. Although less reactive than tocopherols, thiachromanols may, however, act as bimodal antioxidants as a result of the hydroperoxide decomposing ability of the sulfur atom.

A kinetic and thermodynamic investigation of phenols para-substituted with thiyl (SR), sulfinyl (SOR), and sulfonyl (SO(2)R) groups and ortho-substituted with thiyl groups is reported. The effect of the sulfur substituents on the O-H bond dissociation enthalpy values, BDE(O-H), was measured by means of the EPR radical equilibration technique and the reactivity toward peroxyl radicals, k(inh), of these phenolic antioxidants was determined by inhibited autoxidation studies. An inverse correlation between these two parameters was found. A p-SMe substituent decreased the BDE(O-H) value to a lesser extent than a p-OMe group (-3.6 vs -4.4 kcal/mol), whereas the effect of the same groups in an ortho position showed an opposite trend (-0.85 vs -0.2 kcal/mol). The latter result is explained in terms of the different strength of the intramolecular hydrogen bond between the OH proton and the sulfur or oxygen substituents in ortho derivatives. ESI-MS analysis of the products formed by reacting the sulfides with peroxyl radicals from the azoinitiator AIBN revealed the formation of a complex mixture of products, which may play an important role in determining the overall antioxidant activity of the parent compounds.

Fullerene derivatives incorporating one or two 3,5-di-tert-butyl-4-hydroxyphenyl groups were synthesized by 1,3-dipolar cycloaddition of azomethine ylides to C(60). The O-H bond dissociation enthalpies (BDEs) of these compounds were estimated by studying, by means of EPR spectroscopy, the equilibration of each of these phenols and 2,6-di-tert-butyl-4-methylphenol (BHT) with the corresponding phenoxyl radicals. The antioxidant activity of the investigated phenols was also determined by measuring the rate constants for their reaction with peroxyl radicals in controlled autoxidation experiments and compared to that recorded under identical experimental settings for [60]fullerene itself and unlinked BHT. The results indicate that linking of the BHT structure to C(60) does not substantially alter the thermochemistry and kinetics of its reaction with peroxyl radicals, but such adducts may behave as interesting bimodal radical scavengers. The inherent rate constant for trapping of peroxyl radicals by C(60) per se (k(inh)=3.1+/-1.1 x 10(2) m(-1) s(-1)) indicates that, contrary to previous reports, [60]fullerene is an extremely weak chain-breaking antioxidant.

The antioxidant activity of several phenolic acids and esters has been investigated both in organic solutions and in large unilamellar phosphatidylcholine vesicles. In solution these compounds behaved as good antioxidants, with the exception of protocatechuic acid, due to the presence of the catechol moiety. Because their antioxidant activity followed an inverse dependence on the magnitude of their O-H bond dissociation enthalpies (BDE), the key mechanism of the chain-breaking action was attributed to hydrogen atom transfer (HAT) from the phenolic OH to peroxyl radicals. In unilamellar vesicles the antioxidant activity was strongly dependent on the pH of the buffer solution. In acid media (pH 4) all of the examined phenolic acids or esters behaved as weak inhibitors of peroxidation, whereas, with increasing pH, their antioxidant activity increased substantially, becoming comparable to or even better than that of Trolox. At pH 8 they also gave rise to lag phases 2-3 times longer than that of Trolox. The increased activity being observed in proximity of the pK(a) value corresponding to the ionization of one of the catecholic hydroxyl groups, this effect has been attributed to the high antioxidant activity of the phenolate anion.

Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.

all-rac-alpha-Selenotocopherol (6c) has been synthesized in 11 steps in 6.6% total yield. Key steps include chloromethylation to approach the persubstituted aromatic 9b and cyclization of alcohol precursor 10 by radical homolytic substitution at selenium to form the selenotocopherol heterocycle. Determination of the OH bond dissociation enthalpy (BDE) of 6c by electron paramagnetic resonance (EPR) equilibration techniques gave a value of 78.1 +/- 0.3 kcal mol(-1), approximately 1 kcal mol(-1) higher than that of alpha-tocopherol. Kinetic studies performed by measuring oxygen uptake of the induced oxidation of styrene in the presence of an antioxidant showed that selenotocopherol (6c) was a slightly poorer inhibitor than alpha-tocopherol, in agreement with the BDE values. In contrast to simpler selenotocopherol analogues, 6c was not regenerable in the presence of a stoichiometric coreductant in a two-phase lipid peroxidation model.

ESR spectroscopy has been used to study the interaction of para-pentylbenzyl hydroxyalkyl nitroxide with the monolayer of water-soluble protected gold clusters having a core diameter ranging from 1.6 to 5.3 nm. The solubilization of the nitroxide probe in the more hydrophobic environment of the monolayer strongly depends on the size of the gold core. In particular, the partition equilibrium constant increases as the nanoparticle diameter decreases. These results have been attributed to the different packing of the chains in the monolayer resulting from the different radius of curvature of the investigated nanoparticles. This represents, to the best of our knowledge, the first report demonstrating that the core size of metallic nanoparticles affects the solvating properties of the protective organic monolayer.

High oxidative stress status (OSS) is known to be one of the most important factors determining cell injury and consequent organ damage in thalassaemic patients with secondary iron overload. Using an innovative hydroxylamine 'radical probe' capable of efficiently trapping majority of oxygen-radicals including superoxide we measured, by electron paramagnetic resonance (EPR) spectroscopy, OSS in peripheral blood of 38 thalassaemic patients compared with sex-/age-matched healthy controls. Thalassaemic patients showed sixfold higher EPR values of OSS than controls. Significantly higher EPR values of OSS were observed in those with a severe phenotype (thalassaemia major, transfusion-dependent) with respect to mild phenotype (sickle-cell/beta-thalassaemia, not transfusion-dependent) or thalassaemia intermedia. In patients with thalassaemia major, EPR values of OSS were positively correlated with serum ferritin and with alanine aminotransferase levels. In patients with sickle cell/beta-thalassaemia, there was no correlation between EPR value of OSS and all parameters considered. The type of chelating therapy (desferrioxamine or deferiprone) did not have an effect on EPR value of OSS. In conclusion, EPR 'radical probe' seems to be a valid innovative method to determine total OSS in patients affected by thalassaemia and might be used for evaluating new strategies of chelation, new chelators, or the efficacy of antioxidant formula.

Rocket (Eruca sativa Mill. or Eruca vesicaria L.) is widely distributed all over the world and is usually consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs). The latter phytochemicals have recently gained attention as being the precursors of isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important in the detoxification of electrophiles and protection against oxidative stress. The major GL found in rocket seeds is glucoerucin, GER (108 +/- 5 micromol g(-)(1) d.w.) that represents 95% of total GLs. The content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER possesses good direct as well as indirect antioxidant activity. GER (and its metabolite erucin, ERN) effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants of k(2) = 6.9 +/- 0.1 x 10(-)(2) M(-)(1) s(-)(1) and 4.5 +/- 0.2 x 10(-)(3) M(-)(1) s(-) , respectively, in water at 37 degrees C, thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H(2)O(2) or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 degrees C) initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant activity of GER and ERN are discussed.

The gas-phase O-H bond dissociation enthalpy, BDE, in phenol provides an essential benchmark for calibrating the O-H BDEs of other phenols, data which aids our understanding of the reactivities of phenols, such as their relevant antioxidant activities. In a recent review, the O-H BDE for phenol was presented as 90 +/- 3 kcal mol(-1) (Acc. Chem. Res. 2003, 36, 255-263). Due to the large margin of error, such a parameter cannot be used for dynamic interpretations nor can it be used as an anchor point in the development of more advanced computational models. We have reevaluated the existing experimental gas-phase data (thermolyses and ion chemistry). The large errors and variations in thermodynamic parameters associated with the gas-phase ion chemistry methods produce inconsistent results, but the thermolytic data has afforded a value of 87.0 +/- 0.5 kcal mol(-1). Next, the effect of solvent has been carefully scrutinized in four liquid-phase methods for measuring the O-H BDE in phenol: photoacoustic calorimetry, one-electron potential measurements, an electrochemical cycle, and radical equilibrium electron paramagnetic resonance (REqEPR). The enthalpic effect due to solvation, by, e.g., water, could be rigorously accounted for by means of an empirical model and the difference in hydrogen bond interactions of the solvent with phenol and the phenoxyl radical. For the REqEPR method, a second correction is required since the calibration standard, the O-H BDE in 2,4,6-tri-tert-butylphenol, had to be revised. From the gas-phase thermolysis data and three liquid-phase techniques (excluding the electrochemical cycle method), the present analysis yields a gas-phase BDE of 86.7 +/- 0.7 kcal mol(-1). The O-H BDE was also estimated by state-of-the-art computational approaches (G3, CBS-APNO, and CBS-QB3) providing a range from 86.4 to 87.7 kcal mol(-1). We therefore recommend that in the future, and until further refinement is possible, the gas-phase O-H BDE in phenol should be presented as 86.7 +/- 0.7 kcal mol(-1).

The synthesis and study of a series of 6-substituted-2,4-dimethyl-3-pyridinols having interesting antioxidant properties is reported. The general synthetic strategy leading to the compounds involved a low-temperature aryl bromide-to-alcohol conversion as the last step. 2,4-dimethyl-3-pyridinol (1a), 2,4,6-trimethyl-3-pyridinol (1b), and 2,4-dimethyl-6-(dimethylamino)-3-pyridinol (1d) were thus prepared from the corresponding 3-bromopyridine precursor. The methoxy derivative 2,4-dimethyl-6-(methoxy)-3-pyridinol (1c) was also prepared by an alternate route via a Baeyer-Villiger reaction on the substituted benzaldehyde precursor. Novel bicyclic pyridinols 2 and 3 required prior construction of the ring structure. Thus, 2 was prepared by the use of a 6-step intramolecular Friedel-Crafts strategy, and 3 required an 11-step sequence with a thermolytic intramolecular inverse-demand Diels-Alder reaction between a pyrimidine ring and an alkyne as the key step. Basicities of the pyridinols approached physiological pH with increasing electron density in the ring. Pyridinols 1a-d were found to be indefinitely stable to air oxidation while 2 and 3 decomposed upon extended exposure to the atmosphere. The reactivities of the pyridinols toward chain-carrying peroxyl radicals in homogeneous organic solution were examined by studying the kinetics of radical-initiated styrene autoxidations under controlled conditions. These experiments revealed that some of the newly synthesized pyridinols are the most effective phenolic chain-breaking antioxidants reported to date.

Characterisation of the aminoxyl (>N-O*) radical BTNO, generated from 1-hydroxybenzotriazole (HBT) by the one-electron oxidant CAN (a Ce(IV) salt), confirms BTNO as the reactive intermediate in oxidations run with the laccase/HBT system.

The antioxidant activity of the cis and trans isomers of several analogues of resveratrol and pterostilbene has been investigated, especially with regard to the effect of the stereochemistry about the olefinic double bond. The antioxidant power of these compounds was estimated by measuring the rate constants for their reactions with peroxyl radicals and, with two of them, the bond dissociation enthalpy (BDE) of the phenolic O-H bond which is cleaved in the inhibition reaction. The present data show that in homogeneous solution the various hydroxystilbenes investigated behave as mild antioxidants with the notable exceptions of the trans isomer of 4 and 6, whose activities are only slightly lower than that of alpha-tocopherol (vitamin E). The rate constants of the inhibition reaction show that the antioxidant activity of the cis-hydroxystilbene is in all the examined cases worse, by a factor ranging between 2 and 6, than that of the corresponding trans isomers. This lower reactivity depends on enthalpy factors as it can be inferred by the experimental values of the O-H bond dissociation enthalpy in the two geometric isomers of 3',5'-di-tert-butyl-4'-hydroxy-3,5-dimethoxystilbene showing that the strength of the O-H bond in the cis isomer is larger by 1.8 kcal/mol. DFT calculations provide a rationalization of this result, indicating that, although the cis geometry implies a destabilization with respect to the trans species of both phenoxyl radical and parent hydroxystilbene, the destabilization of the radical is larger because the folding of the structure strongly reduces the delocalization of the unpaired electron on the styryl group. A comparison of these results with previously reported data on the proapoptotic activity of these stilbenoids suggests that these two properties are not correlated.

The effect of water on the O-H bond dissociation enthalpy (BDE) of para-substituted phenols has been investigated by means of DFT calculations. It is shown that the experimental BDE values are fairly well-reproduced by simple B3LYP/6-31G* calculations carried out on the phenol/phenoxyl-water complexes taking into account only hydrogen-bonding (HB) interactions of water molecules with molecular sites (HB model). On the contrary, the BDE values computed with the polarizable continuum model (PCM/B3LYP/6-31G*)8 are overestimated by about 3-4 kcal/mol. Discrepancy between theory and experiment increases using the PCM method in addition to the HB model. Calculations show that, in general, the HB interaction with water molecules decreases the BDE of phenols bearing electron-releasing groups while increasing the BDE of phenols bearing electron-withdrawing substituents. This opposite effect is explained by considering the resonance structures with charge separation both in phenols and in phenoxyl radicals. With electron donors, the phenoxyl radical is preferentially stabilized by the HB acceptor interaction with two water molecules, while with electron acceptors the phenol is preferentially stabilized by the HB donor interaction with one water molecule., (Copyright 2004 American Chemical Society)

EPR spectroscopy has been used to study the interaction of para-substituted benzyl hydroxyalkyl nitroxides with the monolayer of water-soluble protected gold cluster made by a short alkyl chain and a triethylene glycol monomethyl ether unit. The inclusion of nitroxide probes in the more hydrophobic environment of the monolayer gave rise to a reduction of the value of both nitrogen and beta-proton hyperfine splittings. The spectra also showed selective line broadening attributed to modulation of the spectroscopic parameters as the result of exchange between free and complexed nitroxide. The rate constants were obtained by analyzing the EPR line shape variations as functions of nanoparticle concentration and temperature. This represents, to the best of our knowledge, the first determination of rate constants for the solubilization of organic substrates in a monolayer-protected cluster.