Electron Paramagnetic Resonance (22): Volume 22 - Rilegato

Rilegato

ISBN 10: 1847550614 ISBN 13: 9781847550613

Casa editrice: Royal Society of Chemistry, 2010

Riassunto

Electron Paramagnetic Resonance (EPR) highlights major developments in this area, with results being set into the context of earlier work and presented as a set of critical yet coherent overviews. The topics covered describe contrasting types of application, ranging from biological areas such as EPR studies of free-radical reactions in biology and medically-related systems, to experimental developments and applications involving EPR imaging, the use of very high fields, and time-resolved methods. Critical and up-to-the-minute reviews of advances involving the design of spin-traps, advances in spin-labelling, paramagnetic centres on solid surfaces, exchange-coupled oligomers, metalloproteins and radicals in flavoenzymes are also included. As EPR continues to find new applications in virtually all areas of modern science, including physics, chemistry, biology and materials science, this series caters not only for experts in the field, but also those wishing to gain a general overview of EPR applications in a given area.

Su questo libro

Dalla quarta di copertina

Electron Paramagnetic Resonance (EPR) covers contrasting types of application, ranging from biological areas, such as EPR studies of free-radical reactions in biology and medically-related systems, to experimental developments and applications involving EPR imaging in polymers, the design of new spin-traps, and spin-labelling approaches to the study of nanomaterials and proteins. Critical and up-to-the-minute reviews of advances involving theoretical analysis, the properties of multi-spin oligomers, identification of defects in diamond, and the use of high fields for inorganic compounds and materials are also included.

Estratto. © Ristampato con autorizzazione. Tutti i diritti riservati.

Electron Paramagnetic Resonance Volume 22

A Review of the Literature Published Between 2008 and 2009

By B. C. Gilbert, V. Chechik, D. M. Murphy

The Royal Society of Chemistry

Copyright © 2011 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84755-061-3

Contents

Preface Victor Chechik, Bruce Gilbert and Damien Murphy, v, 
Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST) Olivier Ouari, Micaël Hardy, Hakim Karoui and Paul Tordo, 1, 
EPR investigations of organic non-covalent assemblies with spin labels and spin probes Marco Lucarini and Elisabetta Mezzina, 41, 
Spin labels and spin probes for measurements of local pH and electrostatics by EPR Maxim A. Voinov and Alex I. Smirnov, 71, 
High-field EPR of bioorganic radicals Stefan Stoll, 107, 
Dynamic nuclear polarization in liquids M. Bennati, I. Tkach and M.-T. Türke, 155, 

CHAPTER 1

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 both the rate of trapping of superoxide and the t1/2 of the corresponding spin adducts were increased. Moreover, partial protection of the CD-DEPMPO-OOH adduct against bioreductant agents was observed even in blood samples. The lipophilic nitrones 18 and 19 were prepared by Han et al., and the trapping of superoxide was investigated in DMSO/water solutions.

2.3 Vectorized spin traps

In mitochondria, leakage of electrons from the respiratory chain (ETC) is an important side reaction generating superoxide radical (2 to 5% of the total amount of breathed oxygen). In healthy cells the concentration of superoxide is controlled by an appropriate pool of antioxidants, however, during mitochondria dysfunction, superoxide production may increase dramatically and worsen the cell disorders. It is now well established, that chemical probes bearing a triphenylphosphonium group can be accumulated into the mitochondrial compartment. Thus, to improve the detection of Reactive Oxygen Species (ROS) within mitochondria, various mitochondria-targeted spin traps bearing a triphenylphosphonium or a pyridinium group were synthesized (5, 25, 29, 42, 43, 45).

Mito-DEPMPO (25)18 allowed for the first time the detection of superoxide radicals generated from isolated and intact mitochondria using EPR/ ST (Scheme 3).

Mito-Spin (29) was shown to accumulate within mitochondria and its ability to reduce the concentration of oxidizing species was established. However, due to its facile oxidation to nitroxide MitoSpinox (Scheme 4), Mito-Spin is useless as spin trap to distinguish between different radicals in mitochondria.

Lipid peroxidation plays a pivotal role in several diseases associated with oxidative stress. To study the implication of ROS in lipid peroxidation processes, different EMPO derivatives (13–16, 21) and PBN derivatives (46–49, 52–64) that accumulate in lipophilic compartments were developed.

Gamliel et al. synthesized a large series of molecules (13–16, 52–64) and determined by 13C NMR their localisation within liposomal bilayers. Then, the ability of various radicals, generated by a Fenton reaction, to penetrate the lipid bilayer was determined by EPR/ST.

Hay et al. designed a series of PBN (46–49) to trap radicals at a pre- determined depth within biological membranes. Large unilamellar vesicles (LUV) were used as biological membrane models; after incorporation of the traps into the membrane, lipidyl radicals were generated by reduction of t-BuOOH by a membrane permeable CuI complex.

Durand et al. prepared an AMPO analogue (FAMPO (21)) bearing a fluorinated amphiphilic carrier conjugates. The spin trapping properties were explored as well as the cytoprotective properties against hydrogen peroxide, HNE and SIN-1 (3-morpholinosynonimine hydrochloride) in bovine aortic endothelial cells.

2.4 Miscellaneous spin traps

A series of heteroarylnitrones (83–88) designed to combine neuroprotective as well as spin trapping properties was developed. These heteroarynitrones protect cells from death induced by exposure to hydrogen peroxide. The spin trapping of oxygen-, carbon- and sulfur- centered radicals with these nitrones was performed.

N-Aryl-ketonitrone PBN like spin traps (76–82) were synthesized; their spin trapping properties were found to be limited to the trapping of carbon- and alkoxy-centered radicals.

The development of Hydrazyl PBNs (70–75) that contain in the same molecule a stable hydrazyl radical moiety and a PBN like moiety was described by Ionita. These molecules were used as conventional spin traps of short-lived radicals, particularly hydroxyl radicals, and they were also used to simultaneously generate and trap dPPh2 radicals (Scheme 5).

A dual sensor spin trap (89) was prepared by Caldwell et al. to detect and distinguish iron (III) ions from hydroxyl and methyl radicals. Typically, iron (III) reacts with the phenol unit inducing opening of the cyclopropane ring and cyclisation to yield a stable nitroxide (Scheme 6).

Benzazepine nitrones (30–39) were synthesized; they were evaluated as protectants against oxidative stress induced in rat brain mitochondria by 6-hydroxydopamine, a neurotoxin producing experimental model of Parkinson's disease. The inhibition of hydroxyl radicals, lipid peroxidation and protein carbonylation were evaluated, and all the compounds tested were more efficient than PBN. No spin trapping experiments using these nitrones were reported.

3 Applications of EPR/ST in biological systems

In the following paragraph, we mention a few recent papers using EPR/ST to characterize free radical species such as O2•- and nitric oxide (•NO) involved in physiological processes. The characterization of these species in cigarette smoke will be also emphasized.

3.1 EPR/ST of superoxide anion radical

O2•- is produced by one electron reduction of molecular oxygen during mitochondrial respiration. It constitutes the main source of various reactive oxygen species in vivo, like peroxynitrite (ONOO-), hydrogen peroxide (H2O2) and hydroxyl radical (HOd). Since the early years of EPR/ST development, it has been a challenge to detect superoxide spin adduct particularly in biological systems. Numerous spin trapping agents have been developed and the most recent reported nitrones devoted to superoxide detection are mentioned in Table 1.

Shi et al. evaluated the abilities of several nitrones to trap cell-generated superoxide induced by 1,6-benzo[a]pyrene quinone in a human epithelial cell line. Considering the superoxide spin adduct stability, among the different nitrones they used, DEPMPO and BMPO appeared as the best candidates.

During EPR/ST experiments in aqueous media, using DMPO as spin trap, spontaneous conversion of the superoxide spin adduct to the hydroxyl spin adduct is observed. In biological systems, this conversion can be mediated by endogenous reducing agent or catalyzed by glutathione peroxidase using glutathione. By using DEPMPO as spin trap, this spontaneous conversion is hardly observed when a low flux of superoxide is used.

Mojovic et al. showed that the conversion of DEPMPO-OOH to DEPMPO-OH depends on the oxygen concentration and they claimed that the conversion mechanism is independent on hypoxanthine (HX) and xanthine oxidase (XO) concentrations. However, these results must be considered with caution because, during the trapping of superoxide with DEPMPO, Tordo et al. showed that increasing XO concentration from 0.04 to 0.4 U mL-1 increased dramatically the formation of DEPMPO-OH as observed on the ESR signals (Fig 1). This observation suggests that O2•- should play a significant role in the conversion of DEPMPO-OOH to DEPMPO.

Nitric Oxide Synthases (NOS) are the enzymes responsible for nitric oxide (•NO) production using L-arginine as substrate. It has been shown that tetrahydrobiopterin (BH4) is a cofactor regulating NO production, and BH4 depletion stimulates endothelial NOS (eNOS) superoxide release causing deficient NO production. Then, O2•- released in the endothelium is thought to be responsible for oxidative stress situations that favour atherosclerosis and hypertension. Druhan et al. studied the effect of several arginine derivatives on O2•- production from eNOS under conditions of BH4 depletion. By trapping superoxide in the presence of L-arginine and endogeneous inhibitors such as asymmetric dimethylarginine and NG-monomethyl-L-arginine, more than 100% increase of eNOS-derived O2•- was evaluated.

Hardy et al. reported the synthesis of a new efficient nitrone-spin trap (Mito-DEPMPO) and the characterization of Mito-DEPMPOO-OH the corresponding superoxide spin adduct. Mito-DEPMPO-OOH was shown to be 2 to 2.5 times more persistent than DEPMPO-OOH in buffer solutions at physiological pH. Using this new nitrone, Hardy et al. detected MitoDEPMPO-OOH spin adduct obtained by trapping O2•- formed from isolated and intact mitochondria. This result constitutes the first EPR/ST characterization of mitochondrial superoxide.

It has been suggested that free radicals generated during the metabolism of acetaldehyde are responsible for initiating alcohol-induced liver injury and furthermore carcinogenic mutations and DNA damage leading to breast cancer. Aldehyde Oxidase (AO) is the major cytosolic enzyme responsible for the metabolism of endogenous aldehydes leading to the production of the corresponding carboxylic acids and reactive oxygen species such as O2•- and H2O2. Using EPR/ST with DMPO as spin trap, Kundu et al. showed that the reaction of AO with 4(dimethylamino)cinnamaldehyde (p-DMAC) in the presence of oxygen produces significant amount of O2•- and H2O2.

(Continues...)

Excerpted from Electron Paramagnetic Resonance Volume 22 by B. C. Gilbert, V. Chechik, D. M. Murphy. Copyright © 2011 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

Dettagli dell'edizione

• Editore: Royal Society of Chemistry
• Data di pubblicazione: 2010
• Lingua: Inglese
• ISBN 10: 1847550614
• ISBN 13: 9781847550613
• Rilegatura: Copertina rigida
• Numero di pagine: 194
• Redattore: Gilbert B. C., Murphy Damien M., Chechik Victor
• Contatto del produttore: non disponibile
• Persona responsabile: non disponibile