Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Reactive oxygen species (ROS) are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, superoxide radical is of special interest as the origin of several damaging species such as H 2 O 2 , hydroxyl radical, or peroxynitrite (ONOO -). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In the present manuscript, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original set up combining a stoppedflow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.3

Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPMPO, bearing mitochondria-targeting triphenylphosphonium cationic moiety or guanidinium cationic group. All the spin traps prepared have been observed to efficiently trap superoxide radical anion in cell-free system. The superoxide spin adducts exhibited similar spectral properties indicating no significant differences in the geometry of the cyclic nitroxide moieties of the spin adducts. The superoxide adduct stability was measured and observed to be highest (t1/2 = 73 min) for DIPPMPO nitrone linked to triphenylphosphonium moiety via a short carbon chain (Mito-DIPPMPO). The experimental results and DFT quantum chemical calculations indicate that the cationic property of the triphenylphosphonium group may be responsible for increased superoxide trapping efficiency and adduct stability of Mito-DIPPMPO, as compared to DIPPMPO spin trap. The studies of uptake of the synthesized traps into isolated mitochondria indicated the importance of both cationic and lipophilic properties, with the DEPMPO nitrone linked to triphenylphosphonium moiety via a long carbon chain (Mito10-DEPMPO) exhibiting the highest mitochondrial uptake. We conclude that of the synthesized traps, Mito-DIPPMPO and Mito10-DEPMPO are the best candidates as the potential mitochondria-specific spin traps for use in biologically-relevant systems.