Spin trapping of superoxide by diester-nitrones

Abstract

The nitrone N-[(1-oxidopyridin-1-ium-4-yl)-methylidene]-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPyON) was synthesized and used as a spin trapping agent. The kinetic aspects of the superoxide detection by this new spin trap and by two other diester-nitrones, i.e. 2,2-diethoxycarbonyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPO) and N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN), were examined by determining the rate constants for the trapping reaction and for the spin adduct decay at pH 7.2. Comparing the results obtained to those given by analogous monoester-nitrones showed that both the spin trapping and the adduct decay reactions were faster in the presence of a second ester group in the cyclic nitrone series, while the superoxide trapping capacities of linear diester-nitrones were found to be dramatically weak. It follows from this study that DEPO and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) are superior when it comes to superoxide detection. Below 0.005 mol dm(-3), DEPO is to date the only nitrone capable of clearly detecting superoxide, while EMPO should be preferred at higher spin trap concentration.

Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

“…Titration of the EPR spectra of the RSSR with GSH Figure 5 demonstrates the typical changes of the EPR spectra of the biradicals R 1 SSR 1 and R 2 SSR 2 upon the addition of thiols. The decrease of biradical spectral components and the simultaneous increase of the monoradical ones is consistent with the splitting of the disulfide bond of the biradical and the formation of two monoradicals with conventional triplet EPR spectra according to reaction [1]. A double integration of the EPR spectra of the biradicals obtained at different concentrations of GSH indicates the preservation of the integral intensity of the EPR signal, indicating that there was negligible reduction of the nitroxide by GSH under these conditions.…”

“…The principal advantage of the biradical disulfide reagents over monoradical labels is the large EPR spectral changes that accompany the reaction [1] even with low-molecular-weight thiols (46) such as GSH (Fig. 5).…”

“…This "static" EPR approach, however, while being simple and reliable for in vitro systems, cannot be used in vivo because vital thiols are consumed in the reaction with the label. The biradical R 2 SSR 2 reacts with thiols significantly slower, with the bimolecular rate constant for the reaction [1] with GSH being equal to 0.26 M −1 s −1 (47) at pH 7.0 (cf., with k 1 ≈ 5 × 10 3 M −1 s −1 for the R 1 SSR 1 (48)). This complicates the use of the static EPR approach using the R 2 SSR 2 reagent due to a possible incomplete reaction with the thiols and, therefore, requires a longer incubation time.…”

Synthesis of Azulenylsilane Nitrones as Diagnostic Tools for Superoxide Detection

“…The rate constant for the superoxide trapping reaction of EMPO was reported three times higher than BMPO at pH 7.2 which indicates that EMPO is superior to BMPO. 243 At the same pH, the rate constant of BMPO is fairly equal to DEPMPO, but at pH 7.4, the rate constant of DEPMPO is twice as much as BMPO 259 suggesting DEPMPO is twice as efficient as BMPO at trapping superoxide. One of the problems of using DEPMPO is that it decomposes on heating which makes its preparation in high purity very difficult.…”

“…To improve their efficiency, efforts were made by elaborating phosphorylated analogues and replacing methyl group with ester moieties. 242,243 However, no better However, the DMPO-OOH adduct was found to be unstable and spontaneously decomposes into the DMPO-hydroxyl adduct (DMPO-OH) and a non radical species. The first ESR evidence of superoxide generation, in ischemia/reperfusion of isolated rat myocardium, has been detected by using the DEPMPO spin trap.…”