Hydrogen bond donating ability of meta and para hydroxy phenoxyl radicals.

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.

Optimization of the antioxidant activity of hydroxy-substituted 4-thiaflavanes: a proof-of-concept study.

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.

Kinetic and thermodynamic aspects of the chain-breaking antioxidant activity of ascorbic acid derivatives in non-aqueous media.

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.

Amphiphilic antioxidants from "cashew nut shell liquid" (CNSL) waste.

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.

Long-lasting antioxidant protection: a regenerable BHA analogue.

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.

Base-promoted reaction of 5-hydroxyuracil derivatives with peroxyl radicals.

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.

TEMPO reacts with oxygen-centered radicals under acidic conditions.

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.

Influence of "remote" intramolecular hydrogen bonds on the stabilities of phenoxyl radicals and benzyl cations.

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.

Free radical intermediates in the inhibition of the autoxidation reaction.

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.

Hydrogen hyperfine splitting constants for phenoxyl radicals by DFT methods: regression analysis unravels hydrogen bonding effects.

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.

Organochalcogen substituents in phenolic antioxidants.

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.

Catalytic chain-breaking pyridinol antioxidants.

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.

Tyrosine analogues for probing proton-coupled electron transfer processes in peptides and proteins.

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.

Hydrogen-atom transfer reactions from ortho-alkoxy-substituted phenols: an experimental approach.

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.

Catalytic chain-breaking pyridinol antioxidants.

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.

The binding behavior of cyclodextrins toward a nitroxide spin probe in the presence of different alcohols as studied by EPR.

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.

Kaiware Daikon (Raphanus sativus L.) extract: a naturally multipotent chemopreventive agent.

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.

Do garlic-derived allyl sulfides scavenge peroxyl radicals?

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.