Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

Radical SAM enzymes involved in the biosynthesis of purine-based natural products

The radical S-adenosyl-l-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5'-deoxyadenosyl radical, which is produced by the reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F(420). This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins

The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field.     

Radical mechanisms in adenosylmethionine- and adenosylcobalamin-dependent enzymatic reactions

A class of enzymatic reactions of S-adenosylmethionine (AdoMet) has recently been recognized, in which AdoMet plays a novel role by initiating free radical formation through the intermediate formation of 5'-deoxyadenosine-5'-yl, the 5'-deoxyadenosyl radical. The reactions are in this way related to adenosylcobalamin-dependent processes, which also depend on the formation of the 5'-deoxyadenosyl radical as an intermediate. The mechanisms by which the 5'-deoxyadenosyl radical is generated by the AdoMet- and adenosylcobalamin-dependent enzymes are very different. However, the functions of the 5'-deoxyadenosyl radical are similar in that in all cases it abstracts hydrogen from a substrate to form 5'-deoxyadenosine and a substrate-derived free radical. In this paper, the role of the 5'-deoxyadenosyl radical in the reaction of the adenosylcobalamin-dependent reactions will be compared with its role in the AdoMet-dependent reaction of lysine 2,3-aminomutase. The mechanism by which AdoMet is cleaved to the 5'-deoxyadenosyl radical at enzymatic sites will also be discussed.     

Recent advances in the stereoselective synthesis of isoprostanes and neuroprostanes

This review delineates several reported methods for the synthesis of isoprostanes and neuroprostanes with particular emphasis on the stereocontrolled construction of a suitably functionalized cyclopentane core. The alpha- and omega-side chains of these PG-like molecules are typically assembled by Wittig-type olefination reactions, standard transformations in the PG synthesis. The synthetic strategies include free radical cyclizations, a palladium-promoted coupling of three different components, an intramolecular cyclopropanation reaction-ring-opening sequence, a [2+2] photocycloaddition-ring-opening metathesis approach, and an intramolecular cross-coupling reaction of an alkyl iodide and a tethered alkenylsiloxane.     

Gold compounds for catalysis and metal-mediated transformations in biological systems

One of the challenges of modern inorganic chemistry is translating the potential of metal catalysts to living systems to achieve controlled non-natural transformations. This field poses numerous issues associated with the metal compounds biocompatibility, stability, and reactivity in complex aqueous environment. Moreover, it should be noted that although referring to ‘metal catalysis’, turnover has not yet been fully demonstrated in most of the examples within living systems. Nevertheless, transition metal catalysts offer an opportunity of modulating bioprocesses through reactions that are complementary to enzymes. In this context, gold complexes, both coordination and organometallic, have emerged as promising tools for bio-orthogonal transformations, endowed with excellent reactivity and selectivity, compatibility within aqueous reaction medium, fast kinetics of ligand exchange reactions, and mild reaction conditions. Thus, a number of examples of gold-templated reactions in a biologically relevant context will be presented and discussed here in relation to their potential applications in biological and medicinal chemistry.     

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals.

Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.     

Characterization of the half and overall reactions catalyzed by L-lysine:2-oxoglutarate 6-aminotransferase

Significant differences were found in the reaction rate, and the substrate and reaction specificities between the half reactions and the overall reactions catalyzed by L-lysine: 2-oxoglutarate 6-aminotransferase. The half reactions between an amino donor and the enzyme-bound pyridoxal 5'-phosphate, and also between an amino acceptor and the bound pyridoxamine 5'-phosphate followed first order reaction kinetics. The extrapolated first order rate constants and dissociation constants of the substrates were determined for the half reactions: lysine, 0.87 min-1 and 5.5 mM; glutamate, 1.1 min-1 and 10.5 mM; alanine, 0.66 min-1 and 6.6 mM; 6-aminohexanoate, 0.43 min-1 and 13.3 mM; and 2-oxoglutarate, 0.33 min-1 and 2.5 mM. As compared with the values reported for the overall reactions [Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, 4102-4109], the reactivity of the inherent substrates was lower by over 4 orders in the half reaction than that in the overall reaction, and the reactivity of alanine with the bound pyridoxal 5'-phosphate was reduced to 10% of that in the overall reaction. The substrate specificity in the half reaction was much lower than that in the overall reaction, which was re-examined in a reaction system containing the same concentration of the enzyme as that for the half reactions. Lysine 6-aminotransferase catalyzes the transfer of only the terminal amino group of lysine to 2-oxoglutarate in the overall reaction. However, in the half reaction, the 2-amino group as well as the terminal one was transferred to the bound pyridoxal 5'-phosphate. The ratio of reactivity of the 2-amino group to that of the 6-amino group was considerably influenced by the pH of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective aldol addition reactions of Fischer carbene complexes via electronic tuning of the metal center for enolate reactivity

The aldol reactions of tetracarbonyl(phosphine)methyl(methoxy)methylene chromium complexes and pentacarbonylmethyl (dialkylamino)methylene chromium complexes with aldehydes and ketones were examined. The reactions of the phosphine complexes give only aldol condensation products, but the desired aldol addition products can be isolated from the reactions of amino carbene complexes. This was attributed to the greater reactivity of the enolates of amino carbene complexes which is supported by a determination of the thermodynamic acidity of the dimethylamino complex 13 (pK_a=20.4). The aldol reactions of amino complexes with -chiral aldehydes occur with very high facial selectivities rivaling the best methods that have been developed for facial selectivity in the aldol reaction. The aldol reactions of amino complexes can be considered as direct synthons for amides since amide functions can be obtained in the oxidative cleavage of the aldol adducts of these complexes. As illustrative of the versatility of carbene complexes, it is also demonstrated in a photo-induced carbon-homologative demetallation, that in combination with the aldol addition reaction the unique reactions of carbene complexes provide powerful and novel overall transformations.     

Scavenging of chlorpromazine cation radical by ascorbic acid or glutathione

The report presented here demonstrates that scavenging of chlorpromazine cation radical (an absorption maximum = 530 nm) by ascorbic acid or glutathione can be kinetically and stoichiometrically analyzed at pH 1.5 but not at pH 3.0 and 6.0 using a conventional absorption spectrophotometer. The cation radical decays spontaneously about 10 and 200 times faster at pH 3.0 and 6.0, respectively, than at pH 1.5.At pH 1.5, ascorbic acid scavenges the cation radical faster than glutathione does, and the following different scavenging mechanisms are postulated from the above kinetic and stoichiometric analysis. The reaction of the cation radical with ascorbic acid is second order. The ascorbic acid free radical, which decays mainly by dismutation, is generated by the bimolecular reaction. In the case of glutathione, on the other hand, about 70% of the scavenged cation radical disappears through free radical chain reactions that glutathione thiol anion and glutathione free radical probably initiate. The remaining (about 30%) disappears by conjugation with glutathione. It may be due to relative nonreactivity of ascorbic acid free radical that free radical chain reactions, found commonly in radical chemistry, do not occur in the scavenging reaction by ascorbic acid.Based on the above results, the physiological scavenging mechanisms of the cation radical by the two reducing substances are discussed briefly.     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Oxygen radical chemistry of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) are readily susceptible to autoxidation. A chain oxidation of PUFA is initiated by hydrogen abstraction from allylic or bis-allylic positions leading to oxygenation and subsequent formation of peroxyl radicals. In media of low hydrogen-donating capacity the peroxyl radical is free to react further by competitive pathways resulting in cyclic peroxides, double bond isomerization and formation of dimers and oligomers. In the presence of good hydrogen donators, such as alpha-tocopherol or PUFA themselves, the peroxyl radical abstracts hydrogen to furnish PUFA hydroperoxides. Given the proper conditions or catalysts, the hydroperoxides are prone to further transformations by free radical routes. Homolytic cleavage of the hydroperoxy group can afford either a peroxyl radical or an alkoxyl radical. The products of peroxyl radicals are identical to those obtained during autoxidation of PUFA; that is, it makes no difference whether the peroxyl radical is generated in the process of autoxidation or from a performed hydroperoxide. Of particular interest is the intramolecular rearrangement of peroxyl radicals to furnish cyclic peroxides and prostaglandin-like bicyclo endoperoxides. Other principal peroxyl radical reactions are the beta-scission of O2, intermolecular addition and self-combination. Alkoxyl radicals of PUFA, contrary to popular belief, do not significantly abstract hydrogens, but rather are channeled into epoxide formation through intramolecular rearrangement. Other significant reactions of PUFA alkoxyl radicals are beta-scission of the fatty chain and possibly the formation of ether-linked dimers and oligomers. Although homolytic reactions of PUFA hydroperoxides have received the most attention, hydroperoxides are also susceptible to heterolytic transformations, such as nucleophilic displacement and acid-catalyzed rearrangement.     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4-5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyI-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H₂O₂ adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H₂O₂-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of the phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.     

Controlling the reactivity of radical intermediates by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Adenosylcobalamin or coenzyme B(12)-dependent enzymes are members of the still relatively small group of radical enzymes and catalyse 1,2-rearrangement reactions. A member of this family is methylmalonyl-CoA mutase, which catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA and, unlike the others, is present in both bacteria and animals. Enzymes that catalyse some of the most chemically challenging reactions are the ones that tend to deploy radical chemistry. The use of radical intermediates in an active site lined with amino acid side chains that threaten to extinguish the reaction by presenting alternative groups for abstraction poses the conundrum of how the enzymes control their reactivity. In this review, insights into this issue that have emerged from kinetic, mutagenesis and structural studies are described for methylmalonyl-CoA mutase.     

Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine.

The reaction of oxyhaemoglobin and acetylphenylhydrazine, which results in haemoglobin denaturation and precipitation, was found to be influenced by H202 and superoxide (O2-.) generated during the reaction. By analysing the different haemoglobin oxidation products, it was found that by influencing the rate at which oxyhaemoglobin was oxidized, H2O2 accelerated the overall haemoglobin breakdown, and O2-. inhibited it. By adding GSH (reduced glutathione) or ascorbate, it was possible to slow down the rates of both oxyhaemoglobin oxidation and O2-. production, and the overall rate of haemoglobin breakdown. These results are compatible with a mechanism involving production of the acetylphenylhydrazyl free radical, and with GSH, ascorbate and O2-. acting as radical scavengers and preventing its further reactions. The reaction produced choleglobin, as well as acetylphenyldiazine and methaemoglobin, which combined to form a haemichrome. The haemichrome was less stable and precipitated first. It was also less stable than the haemichrome formed by direct reaction of acetylphenyldiazine with methaemoglobin, and it is proposed that this is because the methaemoglobin produced from oxyhaemoglobin and acetylphenylhydrazine was modified by the free radicals and H2O2 produced in the reaction.     

Regioselectivity in the homolytic cleavage of S-adenosylmethionine

The observed regioselectivity of the homolytic cleavage of S-adenosylmethionine (SAM) by the radical SAM enzymes is modeled by free radical displacement reactions at sulfoxide centers. These displacements are also regioselective, in direct consequence of the reaction mechanism. The selectivity in the radical SAM reactions is explained by the geometry of the free radical displacement mechanism, required by the chemical reaction and arranged in the active site by the radical SAM proteins.     

Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

The detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody sensitivity and specificity greatly expands the utility of the spin trapping technique, which is no longer dependent on the quantum mechanical electron spin resonance (ESR). The specificity of the reactions of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Now the development of an immunoassay for the nitrone adducts of protein radicals brings the power of immunological techniques to bear on free radical biology. Polyclonal antibodies have now been developed that bind to protein adducts of the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In initial studies, anti-DMPO was used to detect DMPO protein adducts produced on myoglobin and hemoglobin resulting from self-peroxidation by H2O2. These investigations demonstrated that myoglobin forms the predominant detectable protein radical in rat heart supernatant, and hemoglobin radicals form inside red blood cells. In time, all of the immunological techniques based on antibody-nitrone binding should become available for free radical detection in a wide variety of biological systems.