Ascorbate radical anion

Spectral and Kinetic Properties of the Ascorbate Radical Anion... 

Nitrous oxide added to neutral or alkaline aqueous solutions converts the hydrated electron to OH radicals that react with ascorbate at near diffusion-controlled rates (see Table II) to give a mixture of ascorbate radical anion and OH-radical adducts ... 

Figure 1. Absorption spectrum of the ascorbate radical anion at pH 11.0...

In basic solutions ascorbate is apparently oxidized preferentially by the electron transfer process, which goes to completion in less than 2 fts after termination of the electron pulse (see Structure I). In nitrous-oxide-saturated acid solutions (pH 3.0-4.5), A and two other species which were shown to be OH-radical adducts were observed (37), thus confirming earlier observations (18,19,23, 25). The ascorbate radical anion was identified by its doublet of triplets spectrum that maintains its line position from pH 13 to 1. One OH-radical adduct (IV) shows a doublet, the lines of which start to shift below pH 3.0 it has a pK near 2.0, a decay period of about 100 fxs, and probably does not lead to formation of A". The other OH-radical adduct (II) is formed by addition of the OH radical to the C2 position its ESR parameters are = 24.4 0.0002 G and g == 2.0031 0.0002. Time growth studies suggest that this radical adduct converts to the ascorbate anion radical (III) with r 15 fxs, and accounts for 50% of the A signal intensity 40 fxS after termination of the electron pulse. The formation of the three radicals can be summarized as shown in Scheme 1. 

An ESR study (30) showed that ascorbate free radicals are formed when ascorbate and dehydroascorbic acid are mixed under anaerobic conditions. The equilibrium constant K == [AH ] /[AH2][A] is 4.85 0.38 X 10 at pH 6.4 and 25°C. Using the numerical values from Reference 30 and Equation 28, which describe the same equilibrium in terms of concentrations of the ascorbate radical anion, ascorbic acid, and ascorbate ion at any pH, the following value is obtained ... 

Ascorbic acid is a strong two-electron reducing agent that is readily oxidized in one-electron steps by metal ions and metal complexes in their higher valence states. An inner sphere mechanism for the stoichiometric oxidation of ascorbic acid by ferric ion in acid solution is illustrated by Scheme 1(8). The first step in the reaction is the formation of a monoprotonated Fe(III) complex similar to the monoprotonated ascorbate complexes listed in Table I. The intermediate monoprotonated Fe(III) complex is short-lived and rapidly undergoes an intramolecular one-electron transfer to give a deprotonated Fe(II) complex of the ascorbate radical anion, indicated by 7. This complex dissociates to the free radical anion, which may then combine with a second ferric ion to form the complex 9. Complex 9 in turn undergoes a second intramolecular electron... 

Finally it should be pointed out that there is an alternate mechanism for the two successive electron transfer processes indicated by the sequence 14-> 15 -> 16- 13 a. It is quite possible that the ascorbate radical anion dissociates from the mixed ligand complex 15, prior to reoxidation of the Cu(I) ion, and recombines with another Cu(II) chelate prior to the final electron transfer step indicated by 16 17 +... 

Almost all biological tissues contain some organic free radicals that are detectable by ESR. These radicals ( tissue radicals ) are of low reactivity in the sense that they do not react readily with molecules in the system, in particular oxygen if this is present. Thus, they tend to be radicals at the end of a radical chain. Examples are the ascorbate radical anion, melanin free radicals, and some other oxygen-insensitive species, such as some flavin semiquinones. The magnetic properties of these various radicals are sufficiently distinct that their ESR spectra can be differentiated on the basis of g value and linewidth. 

Ascorbate anion acts as a scavenger of ROS and other radicals by singleelectron transfer to yield a stable, persistent ascorbic anion-radical. Ascorbate anion reacts with most radicals at the diffusion limit and even reduces various semiquinone anions, which are usually stable and persistent, by electron transfer at a rate around 2x 10 M s at room temperature and water-soluble models for the tocopherol radical marginally more slowly (Figure 7.9). The product is a weak oxidant ascorbate radical anion oxidises semiquinone... 

In contrast, antioxidant enzymes can efficiently counteract all UV-induced ROS (Aguilera et al. 2002). These enzymes are represented by superoxide dismutase (SOD), catalase and glutathione peroxidase as well as those involved in the ascorbate-glutathione cycle, such as ascorbate peroxidase, mono-dehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase. One of the most important classes of antioxidant enzymes is the SOD family, which eliminate noxious superoxide radical anions. Different metalloforms of SOD exist (Fe, Mn, CuZn and Ni), which due to their intracellular localisation protect different cellular proteins (Lesser and Stochaj 1990). 

The oxidative behaviour of glycolaldehyde towards hexacyanoferrate(III) in alkaline media has been investigated and a mechanism proposed, which involves an intermediate alkoxide ion. Reactions of tetranitromethane with the luminol and luminol-peroxide radical anions have been shown to contribute substantially to the tetranitromethane reduction in luminol oxidation with hexacyanoferrate(III) in aerated aqueous alkali solutions. The retarding effect of crown ethers on the oxidation of triethylamine by hexacyanoferrate(III) ion has been noted. The influence of ionic strength on the rate constant of oxidation of ascorbic acid by hexacyanofer-rate(III) in acidic media has been investigated. The oxidations of CH2=CHX (where X = CN, CONH2, and C02 ) by alkaline hexacyanoferrate(III) to diols have been studied. ... 

Sugiyama H, Fujimoto K, Saito I (1995) Stereospecific 1,2-hydride shift in ribonolactone formation in the photoreaction of 2 -iododeooxyuridine. J Am Chem Soc 117 2945-2946 Sugiyama H, Fujimoto K, Saito I (1997) Preferential Cl hydrogen abstraction by a uracilyl radical in a DNA-RNA hybrid. Tetrahedron Lett 38 8057-8060 Svoboda P, Harms-Ringdahl M (1999) Protection or sensitation by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine. Radiat Res 151 605-616 Symons MCR(1990) ESR spectra for protonated thymine and cytidine radical anions their relevance to irradiated DNA. Int J Radiat Biol 58 93-96... 

Figure 9-3. The copper complex of ascorbic acid may be written in two different resonance forms. In 9.3 it is written as a cop-per(n) complex of a dianionic ligand, whereas in 9.4 it is viewed as a cop-per(i) complex of a radical anion.

Ascorbic acid inhibits light-induced yellowing for a finite time, 1 to 2 hours when irradiated with near-uv light with an intensity of 9.2 mW/cm2 (6). This limitation has been attributed in part to photooxidation of ascorbic acid. In addition to air oxidation, ascorbic acid is oxidized by photochemically produced peroxyl radicals, superoxide radical anion and singlet oxygen (27,28). If ascorbic acid is to be an effective inhibitor of light-induced yellowing it s oxidation must be slowed. 

Superoxide radical anion, hydroxyl radical, and hydrogen peroxide are known as prooxidants, whereas substances that neutralize their effects are called antioxidants. Oxidative stress occurs when the prooxidant-antioxidant balance becomes too favorable to the prooxidants. The effects of prooxidants can be neutralized by their direct reaction with small-molecule antioxidants, including glutathione, ascorbate, and tocopherols. In addition, oxidizing radicals are scavenged from a living system by several enzymes, including peroxidase, superoxide dismutase, and catalase. Oxidative lesions on DNA may be repaired by DNA repair enzymes. 

Delocalisation onto oxygen stabilizes radicals considerably. An important example is the ascorbate radical (Scheme 1.3) formed by electron-loss from the ascorbate anion, or electron-capture by dehydroascorbate. This is remarkably stable, and is characterized by an ESR doublet (1.7 G) which is quite distinctive. Because of the high sensitivity of ESR spectroscopy, and the fact that opaque samples can be used, ascorbate radical intermediates have been widely studied (Liu et al., 1988a). The most probable structure is shown in Scheme 1.3 but this is still a matter of some controversy (Liu et al., 1988a). A key factor in the formation of ascorbate radicals is that ascorbate anions... 

The first reaction describes the excitation of uranyl ions. The excited sensitizer can lose the energy A by a non-radiative process (12b), by emission (12c) or by energy transfer in monomer excitation to the triplet state (12d). Radicals are formed by reaction (12e). The detailed mechanism of step (12e) is so far unknown. Electron transfer probably occurs, with radical cation and radical anion formation these can recombine by their oppositely charged ends. The products retain their radical character. Step (12g) corresponds to propagation and step (12f) to inactivation of the excited monomer by collision with another molecule. The photosensitized initiation and polymerization of methacrylamide [69] probably proceeds according to scheme (12). Ascorbic acid and /7-carotene act as sensitizers of isoprene photoinitiation in aqueous media [70], and diacetyl (2, 3-butenedione) as sensitizer of viny-lidene chloride photopolymerization in a homogeneous medium (N--methylpyrrolidone was used as solvent) [71]. 

Both oxetanes were formed with exclusively the exo-phenyl configuration. The regio- and diastereoselectivity observed are in accord with the assumption of a PET process involving the oxidation of the ascorbic acid derivatives and the formation of the carbonyl radical anions. In these special instances 1,4-biradical and 1,4-zwitterion stabilization result in similar product regiochemistry. The relative configuration of the products favors the assumption of a PET-process. 

The chemistry of ascorbic acid free radicals is reviewed. Particular emphasis is placed on identification and charac-terization of ascorbate radicals by spectrophotometric and electron paramagnetic resonance techniques, the kinetics of formation and disappearance of ascorbate free radicals in enzymatic and nonenzymatic reactions, the effect of pH upon the spectral and kinetic properties of ascorbate anion radical, and chemical reactivity of ascorbate free radicals. 

The spectrum of A (measured 100 fts after termination of the electron pulse) is constant in the 1-10 pH range, suggesting a single species present over that pH range (23). This finding was later corroborated and explained in work (25) that demonstrated that the ascorbate radical is present in its anionic form in the 0-13 pH range, but protonates at pH < 0 (Reactions 16,-16), with pK = —0.45. Earlier reported pK values (22,23) were incorrect and arose from misinterpretation of kinetic and spectral data. 

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

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