Spin-trapped species

The nature of the intermediates impHcated in the photooxidation of water with Ti02 has been identified in several reports using spin traps by the electron spin resonance (esr) technique under ambient conditions (53). No evidence for OH species, even at 4.2 K, was found (43), but the esr signal... 

Various oligomers formed by Diels-Alder/ene reactions are observed.333 334 For S-MA11 polymerization Sato et ci//31 used spin trapping to identify the initialing species. On the other hand, in the case of S-AN copolymerization, Ihe... 

The application of RPR in the detection and quantification of species formed by spin-trapping the products of radical-monomer reactions is described in Section 3.5.2.1, The application of time-resolved F.PR spectroscopy to study intermolecular radical-alkene reactions in solution is mentioned in Section 3.5.1. 

It is unfortunate that typical concentrations of free-radical species present in biological systems are only at the limit of e.s.r. detection sensitivity and, of course, there are major technical difficulties in studying whole animals in this manner. Therefore, the most successful e.s.r. experiments have adopted the approach of spin trapping in which very reactive and thus transient radical species are converted to long-lived adducts via reaction with a trap such as a nitrone, e.g. Equation 1.1 ... 

Despite their short half-lives, it is possible to detect free radicals in biological tissues by the addition of nonradicals such as nitrones or nitroso compounds, which act as spin traps by forming relatively stable free radicals on reaction with the endogenous radical species. Utilizing the technique of electron spin resonance (e.s.r.) spectroscopy, we have demonstrated ROM generation by human rheumatoid synovium when subjected to cycles of hypoxia/normoxia in vitro. Using 3,5-dibromo-4-nitroso-benzenesulphonate (DBNBS) as a spin trap, a... 

Albano, E., Tomasi, A., Goria-Gatti, L. and Dianzani, M.U. (1988). Spin trapping of free radical species produced during the microsomal metabolism of ethanol. Chem. Biol. Interact. 65, 223-234. 

Spin trapping EPR technique and UV-Vis spectroscopy have been used (Polyakov et al. 2001b) to determine the relative rates of reaction of carotenoids with OOH radicals formed by the Fenton reaction in organic solvents. The Fe3+ species generated via the Fenton reaction... 

Spin trapping methods were also used to show that when carotenoid-P-cyclodextrin 1 1 inclusion complex is formed (Polyakov et al. 2004), cyclodextrin does not prevent the reaction of carotenoids with Fe3+ ions but does reduce their scavenging rate toward OOH radicals. This implies that different sites of the carotenoid interact with free radicals and the Fe3+ ions. Presumably, the OOH radical attacks only the cyclohexene ring of the carotenoid. This indicates that the torus-shaped cyclodextrins, Scheme 9.6, protects the incorporated carotenoids from reactive oxygen species. Since cyclodextrins are widely used as carriers and stabilizers of dietary carotenoids, this demonstrates a mechanism for their safe delivery to the cell membrane before reaction with oxygen species occurs. 

This is known as spin trapping . Another technique, that has been used to study very short-lived radicals, is to generate them photo-lytically, from precursors, in a solid inert matrix, e.g. frozen argon. Their life is thus artificially prolonged because they are shielded from collision either with each other, or with other species that could terminate their existence. 

Spin trapping of short-lived radical species formed during... 

It should be noted that a major difficulty in the detection of nitroxyl anion is explained by the impossibility to apply ESR spectroscopy because nitroxyl is not a free radical. Moreover, the use of spin traps such as iron iV-methyl-D-glucamine dithiocarbamate (Fe-MGD) to distinguish NO and NO production by NO synthase failed because both nitrogen species reacted with this spin trap [87]. 

Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

Later on, other hydroxylamine derivatives such as 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONEH) and l-hydroxy-3-carboxy-pyrrolidine (CP-3) have been used for superoxide detection [26]. It was found that these spin traps react with both superoxide and peroxynitrite and that they might be applied for quantification of these reactive species [27]. The CP-3 radical is less predisposed to reduction by ascorbic acid and therefore is probably more suitable for superoxide detection in biological systems. 

This technique can be used to measure the production of free radicals because the unpaired electron in a free radical has magnetic resonance. However, because the radicals are unstable, owing to their high chemical reactivity, the technique of spin-trapping is used. In this technique, the generated radicals react with a suitable probe, and the EPR spectra arising from the reaction of the probe with different radical species can then be identified. 

This article concerns a simple expedient whereby short-lived reactive free radicals may be transformed into more persistent paramagnetic species, thus enabling esr techniques to be applied to systems in which the concentration of the reactive radical remains below normal detection limits. The principle is a simple one. It depends upon the addition to the reaction system of a small quantity of a diamagnetic substance (the spin-trap ) having a particularly high affinity for reactive radicals the product of this trapping reaction must be a particularly persistant free radical (the spin adduct ) whose concentration will build to readily detectable levels (>ca. 10—7—10-6 M). The general reaction is represented by equation (1). 

The tri-t-butylnitrosobenzene, TNB, is monomeric even in the solid state, but the principal advantage of this scavenger, exemplified in the mechanistic studies described in Section 3 (p. 47), is that it functions as an ambident spin trap (Terabe and Konaka, 1973). Thus, primary alkyl radicals add to form nitroxides in the normal way, but with t-alkyl radicals, addition occurs at oxygen, alkoxyaminyl radicals (ArNOR) being formed. Secondary alkyl radicals give mixtures of both species (Fig. 5). The alkoxyaminyl radicals have a lower g-value than the nitroxides (ca. 2.004 vs. 2.006) and their spectra are therefore centred at slightly higher field positions than those of the nitroxides. 

It is not uncommon to find the persistence of a spin adduct quantified in terms of half-life . This is a dangerous practice unless the experimental conditions are precisely defined, or it is known that the nitroxide decays by a unimolecular process. Decay may depend on reaction with a reducing agent present in the system, in which case the concentration of this species will influence the half-life. More commonly, decay will be second order (p. 5), in which case the time for disappearance of 50% of the spin adduct will show a profound dependence on its absolute concentration. The possibility of bimolecular association of nitroxides has been recognized for many years, but only very recently has it been suggested that this may be a complication under experimental conditions employed for spin trapping. Whilst the problem, which was encountered with the important [DMPO-HO ] system (Bullock et al., 1980), seems unlikely to be widespread, it is one which should always be borne in mind in quantitative studies. 

Two possible approaches are indicated in Schemes 4 and 5. In the first, a reactive radical R> is spin-trapped in competition with its pseudo-first order reaction with a substrate SH, which occurs at a known rate to give RH and S. The growth of both spin-adducts (ST—R ) and (ST—S ) is monitored, and simple analysis leads to the trapping rate constant kT. In the second approach, R-does not react with a substrate, but undergoes unimolecular rearrangement or fragmentation at a known rate to give a new species R. This latter procedure... 

Spin trapping of the superoxide radical anion, as well as that of hydroperoxyl and hydroxyl radicals and related species will be considered later in connection with biological chemistry (pp. 52-54). 

Spin trapping by PBN has also been employed to detect radical formation in a photo-Kolbe reaction in which acetic acid is irradiated (A > 360 nm) in the presence of platinized titanium dioxide powder (Kraeutler et al, 1978). The nitroxide observed was considered to be (PBN—Me ), but the published spectrum clearly shows the presence of a second species spectral overlap might therefore be an alternative to solvent polarity as an explanation of the discrepancy between the observed splitting parameters and those previously reported for this species. Where poor resolution obtains, it is important that... 

It was soon realized (Perkins, 1980) that such a conclusion is valid only if the spin trap is devoid of all reactivity other than the capability of reacting with X to form a persistent adduct X—ST . This ideal is not fulfilled by any of the spin traps so far used the spin trap is seldom just an innocent collector of radicals but can participate in the reaction under study in various ways. The most important of these are connected with the redox properties of spin traps and species derived from them, and the common theme to be discussed here is the... 

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