Spin trapping example

Spin traps are usually not practical to stabilize radicals on biomacromolecules because the reactivity is too low, presumably due to steric hindrance. Spin traps are used to stabilize physiologically relevant radicals of relatively small size such as hydroxyl, superoxide, and carbon-based radicals on organic molecules, for example, lipids. 

On the other hand, microsomes may also directly oxidize or reduce various substrates. As already mentioned, microsomal oxidation of carbon tetrachloride results in the formation of trichloromethyl free radical and the initiation of lipid peroxidation. The effect of carbon tetrachloride on microsomes has been widely studied in connection with its cytotoxic activity in humans and animals. It has been shown that CCI4 is reduced by cytochrome P-450. For example, by the use of spin-trapping technique, Albani et al. [38] demonstrated the formation of the CCI3 radical in rat liver microsomal fractions and in vivo in rats. McCay et al. [39] found that carbon tetrachloride metabolism to CC13 by rat liver accompanied by the formation of lipid dienyl and lipid peroxydienyl radicals. The incubation of carbon tetrachloride with liver cells resulted in the formation of the C02 free radical (identified as the PBN-CO2 radical spin adduct) in addition to trichoromethyl radical [40]. It was found that glutathione rather than dioxygen is needed for the formation of this additional free radical. The formation of trichloromethyl radical caused the inactivation of hepatic microsomal calcium pump [41]. 

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]. 

Early methods of superoxide detection are well known and described in many books and reviews. They include cytochrome c reduction, nitroblue tetrazolium reduction, spin trapping, etc. (see, for example, Ref. [1]). The most efficient assays are based on the ability of superoxide to reduce some compounds by one-electron transfer mechanism because such processes (Reaction (1)) proceed with high rates [2] ... 

Spin trapping has been widely used for superoxide detection in various in vitro systems [16] this method was applied for the study of microsomal reduction of nitro compounds [17], microsomal lipid peroxidation [18], xanthine-xanthine oxidase system [19], etc. As DMPO-OOH adduct quickly decomposes yielding DMPO-OH, the latter is frequently used for the measurement of superoxide formation. (Discrimination between spin trapping of superoxide and hydroxyl radicals by DMPO can be performed by the application of hydroxyl radical scavengers, see below.) For example, Mansbach et al. [20] showed that the incubation of cultured enterocytes with menadione or nitrazepam in the presence of DMPO resulted in the formation of DMPO OH signal, which supposedly originated from the reduction of DMPO OOH adduct by glutathione peroxidase. 

There is some evidence, from EPR spectroscopy and analysis of spin-trapped adducts, to suggest that OH may indeed be formed by activated neutrophils. However, caution must be exercised in interpreting such data because 02"-generated adducts may decay to form adducts that resemble those generated directly from -OH. For example, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) can react with C>2 to form DMPO-OOH, and with OH to form DMPO-OH formation of the latter adduct in phagocytosing neutrophils is taken as evidence for OH formation. However, two facts must be considered ... 

The pre-eminent advantage of C-nitroso-compounds as spin traps is that in the spin adduct the scavenged radical is directly attached to the nitroxide nitrogen. Consequently, the esr spectrum of the spin adduct is likely to reveal splittings from magnetic nuclei in the trapped radical, and these will greatly facilitate its identification. A simple example is presented in Fig. 2, which shows the spectrum of the spin adduct of the methyl radical with 2-methyl-2-nitroso-... 

The concentration of the spin trap is usually not critical, although care must be exercised in quantitative studies (next Section). When reactive radicals are being trapped in competition with attack on substrate, the scavenger concentration may have to be adjusted in order to detect substrate-derived radicals. In these experiments the variation of the scavenger concentrations can give useful information, as in the example of alcohol oxidation discussed earlier. 

A more recent example is found in the work of Schmid and Ingold (1978), who used the rate of rearrangement (17) of 5-hexenyl radicals into cyclopentylmethyl radicals (R- and R - in Scheme 5) to time the spin trapping of primary alkyl radicals. In this system, both R and R are primary alkyl, and their spin adducts with several traps therefore have virtually indistinguishable spectra. This difficulty was circumvented by labelling C-l in the hex-5-enyl radical with 13C the unrearranged radical then gives spin... 

Many of the early reports of spin-trapping experiments were focused on mechanistic investigations, and some of these feature in the early reviews (see p. 4). Unfortunately, it is in this application that inferences drawn may be most suspect. For example, the inability of the method to differentiate between radical trapping on the one hand, and a combination of nucleophile trapping with one-electron oxidation on the other, is a serious shortcoming. An early example of this was the tentative conclusion that acetoxyl radicals were spin-trapped by PBN competitively with their decarboxylation in reactions of lead tetraacetate. In view of the rapidity of the decarboxylation reaction, trapping of acetate ion and subsequent oxidation seems a likely alternative. 

The trapping of hydroxyl radicals has also been of interest in connection with electrochemistry. Bard et al. (1974) initiated electrochemical applications of spin trapping and showed, for example, that the cathodic reduction of diazonium salts in the presence of PBN gives aryl-radical spin adducts. A route... 

Examples of problems in photo-initiated spin trapping 121... 

The electrostatic terms can be reasonably well handled in solvents of high dielectric constant, but problems are raised by some solvents of widespread use in spin trapping, for example dichloromethane ( ) = 8.9), chloroform (D = 4.8) and benzene (D = 2.3), in which the electrostatic terms calculated as above for acetonitrile become -24.8, -46 and —96 kcal mol-1, respectively. Already in dichloromethane the effective standard potential of Fe(CN)6 /Fe(CN)6- is increased by 1.08 V and in benzene by an absurdly high 4.2 V ... 

The formation of the trinitromethyl adduct of PBN by photolysis of PBN and tetranitromethane (Okhlobystina et al., 1975) is an unequivocal case of inverted spin trapping. These components give an orange-red CT complex in, for example, dichloromethane when this solution is irradiated by light which only can excite the CT complex (A > 430 nm) the spin adduct (N02)3C-PBN is formed via reaction (46) (Eberson et al., 1994b). This adduct is highly persistent. When the solution is acidified by —2% trifluoroacetic acid, irradiation does not lead to spin adduct formation owing to protonation of trinitromethanide ion. 

A different electrochemical approach was applied to the cathodic reduction of sulfones in W,JV-dimethylformamide (Djeghidjegh et al., 1988), for example t-butyl phenyl sulfone, which is reduced at a more negative potential ( pc = -2.5 V) than is PBN (-2.4 V). Thus, the electrolysis of a mixture of PBN and the sulfone would possibly proceed via both true and inverted spin trapping. If a mediator of lower redox potential, such as anthracene (-2.0 V), was added and the electrolysis carried out at this potential, it was claimed that only the sulfone was reduced by anthracene - with formation of t-butyl radical and thus true spin trapping was observed. It is difficult to see how this can be reconciled with the Marcus theory, which predicts that anthracene - should react preferentially with PBN. The ratio of ET to PBN over sulfone is calculated to be 20 from equations (20) and (21), if both reactions are assumed to have the same A of 20 kcal mol-1. 

Table IS Examples of systems with a mechanistic bias towards proper spin trapping.

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