Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST)

Olivier Ouari, Micaël Hardy, Hakim Karoui and Paul Tordo

DOI: 10.1039/9781849730877-00001

1 Introduction

The trapping of a short-lived free radical with a diamagnetic spin trap to generate a persistent spin adduct which could be characterized by its EPR spectrum constitutes the well known spin trapping technique, hereafter abbreviated EPR/ST (Scheme 1).

EPR/ST was introduced in the late 1960s and since then it has been widely used and its advantages and drawbacks largely debated. In spite of the enormous progress made in four decades, EPR/ST is still faced with limitations particularly for the investigation of in vivo free radical processes. During the last five years, about 2 000 papers appeared containing references to the concept spin trapping. This important literature illustrates the wide scope of applications of EPR/ST and the continuing efforts to improve its efficiency and reliability. Some examples illustrating the range of applications of EPR/ST are described hereafter. Within the limited pages of this chapter the list could not be exhaustive, then, our goal was to give the reader the highlights on the considerable potential of the method.

2 New spin traps

Efforts continue to be devoted to the development of new spin traps especially suited to characterize free radicals involved in biological processes. A variety of substituents have been introduced around the nitronyl function of linear or cyclic nitrones to monitor their spin trapping properties, and in the last five years the synthesis and the use in EPR/ST of around hundred new nitrone spin traps (Table 1) have been described. The structures of the most popular spin traps used today and mentioned herein are shown in Scheme 2.

2.1 Influence of nitrone substituents on the spin trapping properties

A series of linear phosphorylated nitrones (50, 65–69) were synthesized and the half life time (t1/2) of their superoxide adducts was shown to range from 7 to 9 min., thus confirming that the introduction of an electron-withdrawing group on the quaternary carbon bound to the nitronyl function results in a significant improvement of the spin adduct lifetime. However, due to the limitations of linear nitrones to allow the identification of the trapped radicals the development of new spin traps has mainly concerned pyrroline N-oxide derivatives.

Various substituents have been introduced on the ring of pyrroline N-oxides to examine their influence on the spin trapping properties, especially concerning O2•- radicals in buffers. Stolze et al. synthesized a series of AMPO (Scheme 2) spin traps (1–4). The EPR spectra obtained during the trapping of O2•- correspond to the superimposition of the signals of many species, the estimated t1/2 for the superoxide adducts ranged from 10 to 20 min.

Han et al. synthesized the CPCOMPO (20), a spirolactonyl derivative of EMPO. A rate constant value of 60 M-1s-1 was measured for the trapping of superoxide, and the resulting spin adduct exhibited a t1/2 of 2.4 min.

When a substituent is introduced on the C4 of DEPMPO, in a cis position with the phosphoryl group, the half life time of the corresponding superoxide spin adduct is not significantly affected. Furthermore, the EPR pattern is simplified and the trapping reaction is almost stereospecific. Thus, NHS-DEPMPO (23), a DEPMPO analogue bearing a N-hydroxysuccinimide (NHS) active ester group on C4 was prepared. NHSDEPMPO is a very versatile building block which allows facile and straightforward synthesis of a large variety of bifunctional spin traps (22-26). Depending on the introduced substituent, the half life times of the superoxide adducts of these bifunctional spin traps were evaluated in between 21 and 40 min.. Their ability to trap oxygen-, sulfur- and carboncentered radicals was also investigated.

Other DEPMPO analogues with different phosphoryl groups on C5, 27 (CYPMPO) and 28 (DPPMPO), were prepared and tested. The spin trapping properties of CYPMPO (27) and DPPMPO (28) were compared to those of DEPMPO. Concerning the superoxide adducts, t1/2 was 15 min. for CYPMPO-OOH and 8 min. for DPPMPO-OOH. DPPMPO was used to detect superoxide radicals in activated neutrophils.

2.2 Use of cyclodextrins in EPR/ST

The ability of cyclodextrins to form inclusion complexes by noncovalent bonding with a variety of guests has become an exciting field of research. When β-cyclodextrins (β-CD) are used to encapsulate superoxide adducts of PBN, DMPO and DEPMPO, a seven-fold enhancement in adduct stability and a partial protection against glutathione peroxidase- and ascorbate anion-induced reduction was reported by Karoui et al.

Spulber et al. reported the use of cyclodextrins to encapsulate oxygenand carbon-centred radical adducts formed from DMPO, PBN and 2-methyl-2-nitroso-propane (MNP). They showed that the presence of β-cyclodextrin resulted in a significant increase (factor 23) of the lifetime of DMPO-OH and PBN-OH spin adducts.

Bardelang et al. have studied the association of a series of EMPO analogues (6–12) bearing alkyl groups which modulate the affinity of the nitrone moiety for the β-CD cavity. The influence of the association constant on the trapping properties was evaluated as well as the supramolecular protection of the superoxide adducts towards reduction.

Sulfur trioxide radical anion, SO3•-, was trapped with DEPMPO, DPPMPO and CYMPO in the presence of glucosylated β-CD (Gβ-CD). The influence of inclusion of the traps and spin adducts on the kinetics of radical trappings and spin adduct decays was investigated.

The first grafting of a nitrone spin trap with a β-cyclodextrin was performed by Bardelang et al. who prepared Me2CD-PBN (40) and Me3CDPBN (41). NMR studies showed that the nitrone moieties are included in the cyclodextrin cavity. Nevertheless, the formation of self-inclusion complexes does not prevent the spin trapping. The half life time of the superoxide spin adducts were increased although they remain modest due to the very short half-lifetime of PBN-OOH. Polovyanenko et al. used 40 and 41 to trap glutathiyl radicals (GS•), t1/2 for 40-SG and 41-SG increases by a factor of 6.8 and 5.5 respectively, compared to that of the PBN-SG adduct.

Pyrroline N-oxides covalently bound to β-CD were also prepared (17–19, 26). With CD-NMPO (17)31 and CD-DEPMPO (26),19...