1 Introduction. The trapping of a short-lived free radical with a diamagnetic spin trap to ...... The photodecomposition of new free radical photoinitiators generating.
Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST) Olivier Ouari,a Micae¨l Hardy,a Hakim Karouia and Paul Tordoa 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 1960s1–3 and since then it has been widely used and its advantages and drawbacks largely debated.4–10 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. R N
R
FR
+
N
Transient Free Radical
O
FR
O EPR detectable spin adduct
Trap Scheme 1 Illustration of EPR/ST.
a
UMR 6264, Laboratoire Chimie Provence, Equipe SREP, Universite´s d’Aix-Marseille 1, 2, 3 et CNRS, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France
Electron Paramag. Reson., 2011, 22, 1–40 | 1 c
The Royal Society of Chemistry 2011
Table 1 Recently developed spin traps DEPMPO and EMPO derivatives OR5
R5O R5O O R5 O R4
R3
N
OR5
O
R5 O
β-CD =
OR5
O NH
O
OR5
R5O
O
R2 R1
O
O O
OR5 O
O R5O
O
R5O
R5O O
O OR5
R5O
R5 O
R5O
O
O
OR5
Ad =
OR5
OR5
1, 16 R1=Me, R2=CONH2, R3=H, R4=Me: CADMPO 2, 16 R1=Me, R2=CONHMe, R3=H, R4=Me: DMMCAPO 3, 16 R1=Me, R2=CONH2, R3=H, R4=Et: CAEMPO 4, 16 R1=Me, R2=CONHMe, R3=H, R4=Et: EMMCAPO 5, 36 R1=Me, R2=CO2(CH2)4P þ (Ph)3, Br , R3=H, R4=H: Mito-BMPOBr 6, 27, 50 R1=Me, R2=CO2Me, R3=H, R4=H: MeMPO 7, 27, 51R1=Me, R2=CO2i-Pr, R3=H, R4=H: iPrMPO 8, 27, 51R1=Me, R2=CO2i-Bu, R3=H, R4=H: sBuMPO 9, 27 R1=Me, R2=CO2Bn, R3=H, R4=H: BnMPO 10, 27, 52 R1=Me, R2=CO2t-Bu, R3=H, R4=H: BMPO 11, 27, 53 R1=Me, R2=CO2CH2t-Bu, R3=H, R4=H: nPtMPO 12, 27 R1=Me, R2=CO2CH2Cy, R3=H, R4=H: CMMPO 13, 41 R1=Me, R2=CO2n-C14H29, R3=H, R4=H 14, 41 R1=Me, R2=CO2n-C16H33, R3=H, R4=H 15, 41 R1=Me, R2=CO2CH2CH2Ad, R3=H, R4=H 16, 41 R1=Me, R2=CO2CH2C(H)[(CH2)9CH3](CH2)11CH3, R3=H, R4=H 17, 31 R1=Me, R2=COb-CD, R3=H, R4=H, R5=H: CD-NMPO 18, 32 R1=CONH(CH2)11CH3, R2=CH2COb-CD, R3=H, R4=H, R5=H 19, 32 R1=CH2CONH(CH2)11CH3, R2=COb-CD, R3=H, R4=H, R5=H O
20,
17
CPCOMPO
O
N O
21,
40
R1=Me, R2=Y, R3=H, R4=H: FAMPO OH
HO OH O HO
Y=
OH
OH O
OH
O
H N
O
(CH2)4
OH
N H
NH
OR
22,
18
O
P(O)(OEt)2
R=H: 4-HMDEPMPO N O
2 | Electron Paramag. Reson., 2011, 22, 1–40
(CF2)5(CF3)
Table 1 (Continued )
O O 23,
18
R=
:NHS-DEPMPO
C O N O
24,
O HN
18
O R=
C NH (CH2)3
H N
NH : Biotin-DEPMPO
O C
(CH2)4 S
25,
18, 37
26, 27,
19
28,
22
O
H C N
R= 21
+ - : Mito-DEPMPO (CH2)2 PPh3, Br
R=COb-CD, R5=Me: CD-DEPMPO R1=Me, R2=P(O)Ph2, R3=H, R4=H: DPPMDPO
O CYMPO:
N O
P O O
DMPO derivatives 29,
38
Bu Bu
O
MitoSpin Ph3P
N
Br
O
R3 R2 49
2-BENZAZEPINE nitrones
N R2 O
R1
30, R1=H, R2=Me, R3=H; 31, R1=H, R2=Me, R3=Me; 32, R1=Me, R2=Me, R3=H 33, R1=Me, R2=Me, R3=Me; 34, R1=Ph, R2=Me, R3=Me; 35, R1=H, R2=Me, Et R3=Me 36, R1=H, R2=(CH2)4, R3=H; 37, R1=H, R2=(CH2)5, R3=H; 38, R1=H, R2=(CH2)5, R3=Me 39, R1=Me, R2=(CH2)5, R3=H PBN derivatives OR4
R5O R4O O R5 O
X
N Y
O
R1 R2
O
OR4
R5O
O β-CD =
OR5
O NH
OR5
O
Z
O
O
R4 O
O
OR5
R3
O R5 O
O
R4O
O
R5O O OR5
R4 O
O
Pyr = Cl
R5O
R5O
OR5
O
N
OR4
OR5
Electron Paramag. Reson., 2011, 22, 1–40 | 3
Table 1 (Continued ) 40, 29 R1=Me, Me3CD-PBN 41, 29 R1=Me, Me2CD-PBN 42, 39 R1=Me, 43, 39 R1=Me, 45, 39 R1=Me, 46, 42 R1=Me, 47, 42 R1=Me, 48, 42 R1=Me, 49, 42 R1=Me, 50, 11 R1=Me, 51, 54 R1=Me,
41
R2=CH2OCOb-CD, R3=Me, R4=Me, R5=Me, X=H, Y=H, Z=H: R2=CH2OCOb-CD, R3=Me, R4=H, R5=Me, X=H, Y=H, Z=H: R2=Me, R3=Me, X=H, Y=Pyr, Z=H R2=Me, R3=Me, X=n-C6H13, Y=Pyr, Z=H R2=Me, R3=Me, X=n-C12H25, Y=Pyr, Z=H R2=Me, R3=Me, X=O(CH2)11CH3, Y=H, Z=O(CH2)11CH3: DIDOD R2=Me, R3=Me, X=O(CH2)3CO2H, Y=H, Z=O(CH2)3CH3: Bu-4C R2=Me, R3=Me, X=O(CH2)7CO2H, Y=H, Z=O(CH2)3CH3: Bu-8C R2=Me, R3=Me, X=O(CH2)7CO2H, Y=H, Z=O(CH2)11CH3: DOD-8C R2=P(O)(OEt)2, R3=Me, X=H, Y=H, Z=OH: 4-HOPPN R2=Me, R3=Me, X=H, Y=H, Z=NHCOCH2I
Lipophilic PBN:
R N
Ad
O 52, R=OMe; 53, R=OEt; 54, R=On-Pr; 55, R=On-Bu; 56, R=On-C5H11; 57, R=OnC6H13; 58, R=On-C7H15; 59, R=On-C8H17; 60, R=On-C10H21; 61, R=OCH2CH2Ad; 62, R=OCH2C(H)[(CH2)9CH3](CH2)11CH3; 64, R=On-C12H25.
R N
P(O)Ph2 : 65,
O R=C2H5; 69,
12
12
R=Ph; 66,
12
R=p-ClC6H4; 67,
12
R=p-ClC6H4; 68,
12
R=C3H7
R1 O2N 46
N
Hydrazyl-PBN
Z N N O
O2N R2
70, R1=H, R2=H, Z=d; 71, R1=H, R2=H, Z=H; 72, R1=H, R2=NO2, Z= d; 73, R1=H, R2=NO2, Z=H; 74, R1=NO2, R2=NO2, Z=d ; 75, R1=NO2, R2=NO2, Z=H Y Z 45
X
N-aryl-ketonitrone PBN Y
N X
CO2R1 CO2R1
O
76, R1=Me, X=H, Y=H, Z=H; 77, R1=Et, X=H, Y=H, Z=H; 78, R1=Et, X=H, Y=H, Z=N(CH3)2 79, R1=Me, X=H, Y=H, Z=CO2CH3; 80, R1=Me, X=H, Y=Me, Z=OCH3; 81, R1=Me, X=D, Y=D, Z=D 82, R1=Et, X=D, Y=D, Z=D
4 | Electron Paramag. Reson., 2011, 22, 1–40
Table 1 (Continued )
R1 W 43, 44
Heteroarylnitrones
Z
N O
R4
X Y
O
R2
N O
N
R3
O
N
83, W=N, X=C, Y=S, Z=N, R2=Ph; 84, W=S, X=N, Y=C, Z=N, R3=Ph; 85, W=C, X=N þ , Y=O, Z=N, R1=CH3, R2=O =-: FxBN; 86, W=C, X=N, Y=O, Z=N þ , R1=Ph, R4=O ; 87, W=C, X=S, Y=N, Z=N, R1=CO2Et; 88, W=C, X=N, Y=N, Z=S, R1=CH3; 89
48
O
N
Dual sensor
OH
R N O PBN
N O R = Me : DMPO; R = P(O)(OEt)2: DEPMPO; R = CO2Et: EMPO; R = CONH 2: AMPO
Scheme 2 Chemical structures of the most popular spin traps.
2.1
Influence of nitrone substituents on the spin trapping properties
A series of linear phosphorylated nitrones (50, 65–69)11,12 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 electronwithdrawing 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 Noxides to examine their influence on the spin trapping properties, especially concerning O2d– radicals in buffers.13–15 Stolze et al.16 synthesized a series of AMPO (Scheme 2) spin traps (1–4). The EPR spectra obtained during the trapping of O2d– correspond to the superimposition of the signals of many species, the estimated t1/2 for the superoxide adducts ranged from 10 to 20 min. Electron Paramag. Reson., 2011, 22, 1–40 | 5
Han et al.17 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.15 Thus, NHS-DEPMPO (23),18 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 (2226).18–20 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.21,22 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.23 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.24 When b-cyclodextrins (b-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.25 Spulber et al.26 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 b-cyclodextrin resulted in a significant increase (factor 2-3) of the lifetime of DMPO-OH and PBN-OH spin adducts. Bardelang et al.27 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 b-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, SO3d–, was trapped with DEPMPO, DPPMPO and CYMPO in the presence of glucosylated b-CD (G-b-CD).28 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 b-cyclodextrin was performed by Bardelang et al.29 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 6 | Electron Paramag. Reson., 2011, 22, 1–40
adducts were increased although they remain modest due to the very short half-lifetime of PBN-OOH. Polovyanenko et al.30 used 40 and 41 to trap glutathiyl radicals (GSd), 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 b-CD were also prepared (17–19, 26).19,31,32 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.32, 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.33 It is now well established, that chemical probes bearing a triphenylphosphonium group can be accumulated into the mitochondrial compartment.34,35 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).18,36–39 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)38 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)40,41 and PBN derivatives (46–49, 52–64)41,42 that accumulate in lipophilic compartments were developed. Gamliel et al.41 synthesized a large series of molecules (13–16, 52–64) and determined by 13C NMR their localisation within liposomal bilayers. Then,
O O
N H P(O)(OEt)2
+ P(Ph)3 Br -
N O
Mito-DEPMPO
1 mT Mitochondria
EPR detection
Scheme 3
Electron Paramag. Reson., 2011, 22, 1–40 | 7
O
Ph3P
Bu Bu N
Br
O
OH
Ph3P
Bu Bu
O
N
Br
MitoSpin
Fe3+
O
OH
Fe2++H+ O
Ph3P
Bu Bu N
Br MitoSpinox
O
OH
Ph3P
O
Br
O
Bu Bu N
OH
O
Scheme 4
the ability of various radicals, generated by a Fenton reaction, to penetrate the lipid bilayer was determined by EPR/ST. Hay et al.42 designed a series of PBN (46–49) to trap radicals at a predetermined 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 tBuOOH by a membrane permeable CuI complex. Durand et al.40 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)43,44 designed to combine neuroprotective as well as spin trapping properties was developed. These heteroarynitrones protect cells from death induced by exposure to hydrogen peroxide.43 The spin trapping of oxygen-, carbon- and sulfur- centered radicals with these nitrones was performed.44 N-Aryl-ketonitrone PBN like spin traps (76–82)45 were synthesized; their spin trapping properties were found to be limited to the trapping of carbonand alkoxy-centered radicals. The development of Hydrazyl PBNs (70–75)46,47 that contain in the same molecule a stable hydrazyl radical moiety and a PBN like moiety was described by Ionita.46,47 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.48 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 synthesized49; they were evaluated as protectants against oxidative stress induced in rat brain mitochondria by 6hydroxydopamine, a neurotoxin producing experimental model of Parkinson’s disease. The inhibition of hydroxyl radicals, lipid peroxidation and 8 | Electron Paramag. Reson., 2011, 22, 1–40
O2N
O2N N
N
N
PPh2
H N
N N
N O2N
O2N
PPh2
H N O2N
O
N
O
O2N
O
H-PPh2
Scheme 5
O
N
O
OH
O
N
O
Fe3+ Fe2++H+
N
O
N
O
O
Scheme 6
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 O2d– and nitric oxide (dNO) involved in physiological processes. The characterization of these species in cigarette smoke will be also emphasized. 3.1
EPR/ST of superoxide anion radical
d–
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.55 Electron Paramag. Reson., 2011, 22, 1–40 | 9
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.56 Mojovic´ et al. showed that the conversion of DEPMPO-OOH to DEPMPO-OH depends on the oxygen concentration57 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.58 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 O2d– should play a significant role in the conversion of DEPMPO-OOH to DEPMPO. Nitric Oxide Synthases (NOS) are the enzymes responsible for nitric oxide (dNO) 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, O2d– released in the endothelium is thought to be responsible for oxidative stress situations that favour atherosclerosis and hypertension.59 Druhan et al.60 studied the effect of several arginine derivatives on O2d– 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 XO (0.04 U/mL)
XO (0.4 U/mL)
5 min
10 min
20 min 2 mT 2 mT Fig. 1 Effect of XO concentration on the conversion of DEPMPO-OOH to DEPMPO-OH (d).
10 | Electron Paramag. Reson., 2011, 22, 1–40
NG-monomethyl-L-arginine, more than 100% increase of eNOS-derived O2d– was evaluated. Hardy et al.18,37 reported the synthesis of a new efficient nitrone-spin trap (Mito-DEPMPO) and the characterization of Mito-DEPMPO-OOH 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.18 detected MitoDEPMPO-OOH spin adduct obtained by trapping O2d– 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 acetaldehyde61 are responsible for initiating alcohol-induced liver injury and furthermore carcinogenic mutations and DNA damage leading to breast cancer.62 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 O2d– 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 O2d– and H2O2.63 Reactive oxygen species (ROS) and more particularly free radical generation by phagocytes (granulocytes, monocytes, and macrophages) constitute a line of defence against intruding microorganisms. NADPH oxidase is the enzyme responsible for the formation of O2d– from cytosolic NADPH and molecular oxygen. Lundqvist et al. used DEPMPO to detect O2d– production by human neutrophils (the most abundant granulocytes) interacting with Staphylococcus aureus and Staphylococcus epidermidis bacteria. They reported that DEPMPO is cell permeable and detect NADPH oxidase derived O2d– generated in phagosomes or human neutrophils during phagocytosis of viable Staphylococcus aureus and Staphylococcus epidermidis bacteria.64 Alzheimer disease is a neurodegenerative disease characterized by the presence of numerous amyloid plaques in the brain, it has been suggested that fibrillar deposits of the b-amyloid peptide (Ab) are responsible for the cytotoxicity by generating hydrogen peroxide causing oxidative damages via the hydroxyl radical (HOd), a highly oxidative entity. By trapping HOd after addition of exogenous Fe2 þ , Tabner et al. 65 have elaborated a methodology where the use of EPR/ST and DMPO allows characterizing the formation of hydrogen peroxide during the early stages of protein aggregation. Hydroxylamines, EPR silent diamagnetic molecules, which are easily oxidized to nitroxides, have also been used to detect O2d–. Dikalov et al. used the cell-permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Scheme 7) to detect O2d– generated in cultured human lymphoblast cell. They used three different methods of detection: superoxide dismutase (SOD)-inhibitable cytochrome c reduction-EPR/ST with EMPO and DEPMPO-oxidation of CMH monitored with EPR. They concluded that the detection of O2d– with CMH (which reacts with O2d– faster (1.2x104M 1 s 1) than the most popular nitrone spin traps) was two to five Electron Paramag. Reson., 2011, 22, 1–40 | 11
CO2CH3 H3C
CH3
H3C
CH3
N
CO2CH3 + O2
1.2x104 M-1.s-1
OH
H3C H3C
CH3 N
CH3
+
H2O2
O
Scheme 7 Superoxide dismutase-like reaction of superoxide with CMH.
times more sensitive than with cytochrome c or the spin traps EMPO and DEPMPO.66 However, during oxidative stress in vivo or in cultured cells, apart from O2d–, many other oxidative species (hydrogen peroxide, hydroxyl radical, peroxynitrite, traces of metal . . . ), can oxidize CMH to the corresponding nitroxide. Then, the identification of the oxidizing species responsible of the oxidation of CMH is not straightforward and could be a source of misinterpretations. 3.2
Trapping of nitric oxide (NO)
NO is a ubiquitous gaseous paramagnetic molecule playing an important role in many physiological processes.67 Apart the vascular smooth muscle vasorelaxation,68,69 NO has other important physiological roles, such as being a cytotoxic mediator of the immune system70 or a neurotransmitter in the central nervous system.71 Nitric oxide is stable in oxygen-free solutions but it reacts with superoxide radical to form peroxynitrite (ONOO ) at a nearly diffusion controlled rate.72 Peroxynitrite is a cytotoxic species implicated in several pathophysiological conditions like atherosclerosis73 and neurodegenerative diseases.74 In order to characterize NO production in living systems, usual EPR spin traps have been evaluated. Nitrone and nitroso compounds are not suitable because of the instability of the resulting spin adduct.75 Other methods such as trapping using cheletropic traps, exogenous iron chelates and the approach using Nitronyl Nitroxides (NNOs) as a NO scavenger were successfully developed. 3.2.1 NO cheletropic trap (NOCT). The concept of a NO cheletropic trap (NOCT) is based on the addition of NO to form a stable indolinoxyl type nitroxide with a simple and characteristic three line EPR spectrum (Scheme 8). This approach was developed in the 1990’s by Korth, Sustmann and Ingold,76,77 however, the poor solubility and thermal sensitivity of the NOCT compounds as well as the bioinstability of the resulting nitroxides limited their use to monitor NO production in cellular systems. Very recently, Lauricella et al. reported the synthesis of a cheletropic NO trap (A, Scheme 9) able to discriminate NO from NO2.78 Indeed, in t-butylbenzene and in the presence of catalytic amounts of oxygen, the trapping of NO gives rise to a persistent dialkyl nitroxide (B, aN=1.445 mT) while the trapping of NO2 generates an alkyl alkoxy nitroxide (C, aN=2.980 mT). However, the poor solubility of A in water, the need of a high concentration of trap and catalytic amounts of molecular oxygen limit the use of this NOCT to organic solvents. 3.2.2 Iron (II) chelates (Fe2 þ .(L)2). Nitric oxide can bind very easily with Fe2 þ chelates, and diethyldithiocarbamate ferrous complex (DETC)2-Fe2 þ 12 | Electron Paramag. Reson., 2011, 22, 1–40
+
NO
N
O
NOCT
Scheme 8
O
Trapping of NO with a NOCT.
OH
O A
NO
OH O N
O
NO2
O2
O2
OH
O
O
O
N
O
OH
O
O N
and/or
O
O
O
B
C
Scheme 9 Trapping of NO and NO2 with a NOCT.
NO R R1
S N
S
Fe2+ S S
R N R1
NO
R R1
S
N
S Fe2+ S S
R N R1
D : R = R1 = CH3CH2 (DETC) E : R = CH3 and R1 = CH2(CHOH)4CH2OH (MGD) Scheme 10 Structure of Fe2 þ -dithiocarbamate complexes.
(D, Scheme 10) is commonly used to trap NO produced in hydrophobic conditions79; the resulting (DETC)2-Fe2 þ @NO complex is detected as a three line EPR spectrum. The apparent rate constant (kapp) value for the reaction of NO with several (L)2-Fe2 þ complexes was shown to be around 106 M 1 s 1, and the stability of the corresponding nitrosyl Fe2 þ (NO)(L)2 was evaluated.80 Water-soluble Fe2 þ -dithiocarbamate complexes were developed and successfully used to obtain evidence of real-time NO production in septic shockmice81,82 or from purified neuronal Nitric Oxide Synthase (nNOS).74,83 (DETC)2-Fe2 þ cannot be administrated by intravenous injection (i.v.) because the aggregates formed in the blood stream cause embolism. Two novel formulations of the complex: a lipid-based carrier system stabilized by lecithin and inclusion in hydroxypropyl b-cyclodextrin that can be administrated by i.v., were developed by Charlier et al.84 Using these formulations Electron Paramag. Reson., 2011, 22, 1–40 | 13
in vitro, they showed that the sensitivity of the NO detection was increased by a factor of 4 compared to the standard spin trap agents. The trapping of NO by (MGD)2-Fe2 þ (E, Scheme 10) is not selective; Mason showed that nitrite, an oxidation product of NO, can react with (MGD)2-Fe2 þ to generate NO.85 Van Faasen and Vanin published numerous papers concerning NO trapping with iron chelates underlining the difficulty to detect NO release from non-stimulated small organs.86–89 They published the preparation of functionalized zeolite with iron-DETC complexes able to improve the detection of NO in biological systems.76 Nevertheless, dithiocarbamate complexes are widely used, even though the high quantities of added Fe2 þ and dithiocarbamate ligands can initiate unwanted reactions and a high toxicity. 3.2.3 Nitronyl Nitroxides (NNOs). NNOs are characterized by a five line EPR signal with an intensity ratio 1:2:3:2:1 due to the coupling of the single electron with two equivalent nitrogen atoms (Fig. 2); the nitrogen coupling constant is about half the value of a dialkyl nitroxide such as TEMPO. Kalyanaraman et al. were the first to suggest that nitronyl nitroxides could be a viable alternative to iron (II)-dithiocarbamate complexes90,91 to characterize nitric oxide. Indeed, 2-carboxyNNO (Fig. 2) reacts specifically with nitric oxide giving rise to an imino nitroxide (INO) (aN1=0.45 and aN2=0.90 mT) which shows a totally different EPR signal;90 the rate constant of the reaction between NNOs and NO is 104 M 1 s 1 in aqueous solutions.92 As shown by Peng et al. who reported the synthesis of 30 different NNO labelled with amino acid fragments, the possibility to modulate NNO solubility and specificity makes possible the detection of NO at different tissue sites. To improve NO rate trapping, Rosen et al. reported the first synthesis of dendrimer linked NNOs (from 2 to 8 units of NNO).93 Unfortunately the EPR spectrum of the dendrimer linked NNOs is broadened by spin exchange interactions, and the rate constant of trapping is similar to that observed with nitronyl nitroxides. Despite the easy access to various NNOs, their use for specific EPR NO detection in biological systems is not without limitation; it has been reported that NNOs can undergo fast reduction into EPR-silent diamagnetic products. Indeed, Blasig et al. reported that NNOs can react with superoxide
Fig. 2 Trapping of nitric oxide with NNO and EPR signal of the resulting iminyl nitroxide (INO).
14 | Electron Paramag. Reson., 2011, 22, 1–40
anion radical (O2d–) with a rate constant of 8.8 x105 M 1 s 1, which is more than two orders of magnitude higher than the value reported previously for reaction with NO.94 3.3
EPR/ST in cigarette smoke (CS)
Cigarette smoke is divided into mainstream (smoke inhaled) and sidestream smoke. We actually will be focusing on the mainstream smoke which is composed of particulate solid phase (tar) or tar particule matter (TPM) and the gas phase (toxic gases, free radicals . . . ). More than 4000 different chemical components are present in cigarette smoke and some of them exhibit toxic, mutagenic and carcinogenic abilities such as benzo[a]pyrene. It is now accepted that cigarette smoking is associated with numerous respiratory (emphysema) and vascular (atherosclerosis) diseases and lung cancer. Cigarette smoke produces a variety of free radicals and reactive oxygen and nitrogen species able to generate both oxidative damages and oxidative stress. Many EPR/ST studies have been devoted to analyze, characterize and determine the nature of all the free radicals produced in CS. To bring to light free radical intermediates production in gas-phase cigarette, many authors proceeded by bubbling the gas-phase smoke into an organic or a water solution containing the nitrone-spin traps PBN, DMPO, DEPMPO and iron chelates able to trap carbon-, oxygen-, or nitrogencentred radicals, respectively. The radical spin adducts were then detected and characterized by electron paramagnetic resonance (EPR) spectroscopy. The predominant gas-phase radicals were shown to be carbon-centred radicals such as acyl and alkylaminocarbonyl radicals,95,96 oxygen-centred radicals such as superoxide anion (O2d–), hydroxyl (HOd),97 alkoxyl (ROd)94 and alkylperoxyl (ROOd)98 and nitrogen-centred radicals like nitric oxide (dNO) and nitrogen dioxide (dNO2). Peroxynitrite is also formed and has to be taken into account yielding during its decomposition secondary free radicals such as HOd, nitrogen dioxide (dNO2) and carbonate anion radical (CO3d–).99 A. Valavanidis et al. using DMPO and PBN as spin trapping agents showed that aqueous cigarette tar (ACT) extracts produce reactive oxygen species especially HOd at physiological pH. They reported the presence of stable free radicals in the tar of mainstream cigarette smoke; indeed, a single broad ESR signal with a g value of 2.0035 is observed and attributed to an organic semiquinone radical (QHd) able to generate O2d– by one-electron reduction of molecular oxygen propagating the formation of several free radical species (Scheme 11).100 EPR/ST study of the mainstream gas-phase using PBN suggested the detection of carbon- and oxygen-centered radicals from the observation of nitroxide spin adducts exhibiting hyperfine coupling constants values of aN=1.40 and aHb=0.2 mT. Due to weak differences in b-hydrogen coupling constant between oxygen-centred radical spin adducts, the use of PBN does not allow efficient discrimination between superoxide, hydroxyl, alkoxyl or peroxyl radical spin adducts. The use of several nitrones exhibiting specificity for a given free radical should afford more accurate data to identify the nature of the free radical species trapped. Electron Paramag. Reson., 2011, 22, 1–40 | 15
QH + O 2 → Q + O2•– + H + 2O2•– + 2H + → H2O2 + O2 O2•– + •NO → –OONO H2O2 + Fe 2+ + H + → •OH + Fe 3+ + H2O Scheme 11 Sources of reactive oxygen species generated in ACT.
Using a sensitive spin trapping detection with DEPMPO, Culcasi et al. reported a comparative study emphasizing the respective roles of cigarette smoke- and gas phase cigarette smoke (GPCS)-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation.101 In buffer bubbled with CS they identified DEPMPO-OOH as the major detected nitroxide spin adduct. Using a computer simulation program allowing the analysis of spin adduct diastereoisomery, they showed that the DEPMPO-OH spin adduct observed in buffer solution bubbled with gas phase cigarette smoke was coming from a metal-catalyzed nucleophilic addition of water rather than a direct HOd trapping which is contradictory with previous studies suggesting HOd formation in CS. The concept of evaluating the source of HOd from the percentage contribution of each hydroxyl radical spin adduct diastereoisomer was previously reported by Nsanzumuhire et al. in 1999 using a DEPMPO phenylated derivative as spin trap.102 Nitroxides are known to be effective quenchers of excited electronic states of fluorescent moieties. Miljevic et al. reported the synthesis and the use of a profluorescent nitroxide probe (9-(1,1,3,3-tetramethylisoindolin-2-yloxyl-5ethynyl)-10-(phenylethynyl)anthracene (Scheme 12), for the detection of particle-derived ROS present in CS.103 This study has provided the first quantitative estimate of the ROS arising from sidestream cigarette smoke which is surprisingly much higher than in mainstream cigarette smoke. The fluorescence detection affords a great sensitivity to the method; however, this approach does not provide information on the nature of the species trapped by the nitroxide; only free radical species quantification is evaluated corresponding to the overall enhancement of fluorescence measured. 4 Combined liquid chromatography/EPR/Mass spectrometry (LC/EPR/ MS) approaches to identify spin adducts Approaches104 combining HPLC, EPR and MS to fully identify spin adducts generated during spin trapping processes continue to develop.105–113 These tools were first developed to allow the analysis of EPR/ST experiments giving rise to many spin adducts and resulting in composite EPR spectra very difficult to assign. MS analysis can be very useful to distinguish between different spin adducts exhibiting almost identical EPR spectra and its use to ascertain EPR assignments is increasing. Guo et al.105 used online LC/ESR/ESI-MS or MS-MS to separate and directly characterize DMPO adducts formed from the reaction of Fe2 þ with t-butyl or cumyl hydroperoxides. In each reaction they observed two oxygencentred DMPO spin adducts that were separated and unambiguously 16 | Electron Paramag. Reson., 2011, 22, 1–40
N
O
N
O R
R ROS
Ph
Ph
Non fluorescent
Fluorescent
Scheme 12 Trapping of free radical on nitroxide leading to a fluorescent alkoxyamine.
O H2O
H3 C HO
S
H2 C
S
CH3
O
O
H3C
H3 C
S
CH3
CH3SO2H
H3C
OH H3C
O2
H3COO
H3CO
CH2OH
Scheme 13 Radicals formed from reaction of HOd radicals with DMSO in water.
assigned to DMPO-OMe and DMPO-Ot-Bu or DMPO-OC(Me)2Ph respectively. This study confirmed previous reports based on 17O-labeling106 and showing that DMPO-alkylperoxyl spin adducts are not detectable in water. Qian et al.114 identified and quantified all spin trapped radicals resulting from the interaction of HOd radicals and DMSO in the presence of POBN and d9-POBN. Spin trapping experiments followed by LC/EPR and LC/MS allowed the characterization of four trapped radicals: POBN-CH3, POBNOCH3, POBN-CH2OH and POBN-CH2S(O)CH3 (Scheme 13). In the bile of rats treated with DMSO, the POBN-CH3 spin adduct was not detected, however, use of LC/MS showed the formation of a significant amount of the corresponding EPR-silent hydroxylamine. The fragmentation pathway of DEPMPO was studied by MS/MS, then DEPMPO was used to trap HOd, HOCH2d and H3Cd radicals, and the structure of the corresponding spin adducts was confirmed analyzing the fragmentation pathways obtained by tandem mass spectroscopy.107 Overall, the fragmentation pathways of the C-centred radical adducts proceed mainly via the loss of the diethoxy(oxido)phosphoranyl radical. In contrast the DEPMPO-OH adduct exclusively dissociates via the release of HOd to regenerate the DEPMPO. The same observations were made during the characterization of free radical adducts of DIPPMPO using MS and 31P NMR spin trapping.108 Yang et al. developed an LC/ESI tandem mass spectrometric method to quantify the hydroxyl radical.109 The method was applied to evaluate the HOd scavenging capacity of several phenolic acids. Morgan et al.115 studied hydroperoxides generated by hydroxyl radicals and singlet oxygen at side-chain and backbone sites on aminoacids, peptides and Electron Paramag. Reson., 2011, 22, 1–40 | 17
proteins. In conjunction with EPR/ST and LC/MS/MS, data were obtained on the sites of hydroperoxide formation. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants. Unlike many other o-6 PUFA, which tend to be unhealthy, the g-linoleic acid, GLA, (all-cis-6, 9, 12-octadecatrienoic acid) is important to maintaining good human health in a variety of ways.110 Yu et al.111 used LC/EPR and LC/MS with POBN to characterize carbon- and oxygen-centred radicals that are generated in lipoxygenase-catalysed GLA peroxidation. The results could help to understanding the biological effects of GLA. Combined EPR/ST and MS techniques were also used to investigate the nucleophilic addition of reduced glutathione on 2-methyl-2-nitrosopropane112 and the formation of a cyclic peroxide from the autoxidation of a dienolic precursor.113 5
Immuno-spin trapping
Even if Immuno-Spin Trapping (IST) technique does not mainly rely on EPR spectroscopy, we considered that a description and some comments on this technique should be included in this Chapter. IST takes advantage of the reaction specificity of nitrone spin traps with free radicals and thus DMPO is used as trapping agent in IST. Moreover, many concerns faced by EPR/ST, including radical trapping rate, spin trap distribution and spin adduct stability, apply also for IST; and because EPR/ST experiments are still challenging in biological systems, IST can be seen as a complementary technique for studying biomolecule -derived radicals in cells and tissues. 5.1
State of the art and principle of IST
Oxidative stress produces irreversible modifications of the structure and function of biomolecules such as proteins, DNA, lipids.116 These oxidative damages can end in modulation of signal transduction, mutagenesis and tissue damage. However, in different pathologies where free radicals and other ROS are implicated, the critical causative events are still unclear as well as the downstream species and sites of production of these ROS.117 As already mentioned, EPR/ST has been often used for many biological radical studies, particularly for low molecular weight free radicals but also for proteins radicals.5,7,8 Likewise, broad EPR spectra with poor resolution are obtained for trapping protein-derived radicals by spin traps such as nitroso or nitrone compounds.118,119 Identification of the protein spin adducts is still challenging,120 but these radical adducts are sometimes long-lived enough to allow the use of other techniques, such as enzymatic digestion, LC MS/MS, to gain detailed information on the nature and the site of protein radicals. Proteins are one of the major targets for oxidative damage due to their abundance, their critical role in cell functioning and their fast reaction with ROS. Number of methods has been developed to assess oxidative damage to proteins.120 In recent years, IST was developed and has appeared as an interesting tool because this technique brought the power of immunological techniques to free radical biology. This technology has shown to be reliable and has quickly evolved as shown by the large number of articles published by Mason’s group and other groups in which free radical processes in 18 | Electron Paramag. Reson., 2011, 22, 1–40
isolated biomolecules,121–140 cells,141–146 tissues147–149 and whole animal150 have been investigated. The principle of IST is based on the development of a rabbit polyclonal anti-DMPO nitrone adduct antiserum. IST takes advantage of the highly selective reaction of DMPO spin trap with free radicals. After trapping of the protein or DNA -derived radical, the subsequent oxidative transformation of the persistent nitroxide DMPO spin adduct (t1/2=min.) to a more persistent nitrone DMPO adduct (t1/2=hours-days) generates an antigen species that interacts specifically with the anti-DMPO antiserum (Scheme 14). The rabbit anti-DMPO antiboby is then detected by an antirabbit IgG conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) for ELISA or Western blot analyses. The usual protocol is based on a three step sequence: – Production of the DMPO nitrone adducts (by adding a free radical generating system to the milieu); – Separation of the nitrone adducts (by washing, precipitation or gel migration); – Immunodetection/localization of the DMPO nitrone adducts (ELISA, Western blot, immuno/histochemistry, fluorescence confocal microscopy). 5.2
Applications of IST
A few examples illustrating the range of applications of IST are described hereafter. The list is not exhaustive, the goal being to give a highlight to the reader on the large potential of the method. Recently, IST has been used to investigate the myeloperoxidase (MPO)triggered formation of DNA-centered radicals due to the production of hypochlorous acid (HOCl) in inflammatory and epithelial cells.146 Combining radiolabelled extracellular matrix (ECM), 3-NO2-Tyr detection, and IST studies, Kennett et al.148 obtained data suggesting that peroxynitrite-mediated damage to ECM occurs via a radical-mediated pathway that could contribute to ECM damage at sites of inflammation, potentially leading to rupture of atherosclerotic lesions. An approach which combines liquid chromatography, immuno-spin trapping, and offline tandem mass spectrometry for selective detection of protein residues labelled by DMPO has been developed successfully. This method allows the identification of the site of free radical production in proteins and has been applied to human haemoglobin, horse heart myoglobin, and sperm whale myoglobin.133 IST has shown also to be a sensitive method to explore the production of DNA radicals in cells (rat hepatocytes and RAW 264.7 macrophages) preloaded with Cu2 þ or Fe3 þ and treated with H2O2 or t-BOOH.142 Bonini et al.147 have studied the HOCl-mediated catalase inactivation pathways in mouse hepatocytes and they showed that IST used in conjunction with fluorescence confocal microscopy can contribute to localize protein radical formation in intact cells. Demicheli et al.131 have used a combination of techniques, including IST, to investigate the mechanism of inactivation and nitration of SOD when exposed to simultaneous fluxes of superoxide and nitric oxide radicals. The authors Electron Paramag. Reson., 2011, 22, 1–40 | 19
20 | Electron Paramag. Reson., 2011, 22, 1–40
Protein
Analysis by EPR
Protein radical
O
N
O
N
Protein
Scheme 14 Principle of IST.
EPR Spin trapping
Protein
H2O2 (oxidative decay)
O
N
Protein
O
N
Immuno Spin trapping: - Immunoassays (ELISA, Western blot, immuno / histochemistry) - Mass spectrometry (proteolytic digestion, MS / MS) - Fluorescence miscroscopy, - MRI
rabbit anti-DMPO antiserum
proposed that in addition to the action of peroxynitrite, a NO–dependent nitration mechanism can be observed in inflammatory conditions. 5.3
Concluding remarks
In recent years, IST has shown to be a reliable, sensitive, and specific approach to detecting protein-and DNA- derived radicals. IST technique brings the power of immunoassays to the field of free radical biology by combining the specificity of spin trapping and the sensitivity of the antigen/ antibody interactions. Redox processes, oxidative damages of biomolecules and their role in post-translational modifications in physiology and diseases are topics widely investigated. In combination with other techniques including LC MS/MS, immunoassays, fluorescence microscopy, and MRI, IST appears as a powerful tool to investigate biological free radical processes. 6
Kinetic aspects of spin trapping
Kinetic information, including the knowledge of rate constants of spin adduct formation (kt) and decay (kd), is a critical point for the development of new spin traps but also for the design of EPR/ST experiments in complex systems where, for instance, various competitive reactions are present and/ or quantification of the spin adduct concentrations is needed. In water solutions, the reactions of free radicals with nitrone spin traps have rate constants varying over a wide range, from the diffusion-controlled reaction with HOd (kE109 M 1 s 1) to slow reactions with O2d– (ko100 M 1 s 1). Values of trapping rate constants for other free radicals, including carbon- and sulfur-centered radicals, are usually higher than 106 M 1 s 1. 6.1
Trapping rate of superoxide radical
Determination of kt is usually performed using either competitive kinetic experiments (with a well-characterized superoxide scavenger as competitor) or by analysing the build-up of the superoxide adduct at various spin trap concentrations taking into account the competition between the trapping and the dismutation of O2d–.151–153 In the former case, the value of the rate constants for the spin trap and its competitor should be of the same order of magnitude. In the latter case, each run must be carried out using rigorously the same experimental procedure and EPR settings. Due to the weak reactivity of superoxide radical in water solution and to its implications in various side reactions, the accurate determination of the absolute trapping rate constant of superoxide radical on nitrone spin trap is not straightforward. As a consequence, there is a large scattering in the literature concerning the value of kt and the ranking of the spin traps in regard with their superoxide trapping rate (Table 2). Moreover, several groups have developed and used different systems and methods for kt determination, and that likely favours the observed discrepancy. It must also be kept in mind that kt determination in organic solvent (DMF, pyridine, DMSO) should not be compared to the values obtained in water solution for other spin traps due to the very different reactivity of superoxide radical in such solvents.154 Electron Paramag. Reson., 2011, 22, 1–40 | 21
Table 2 Reported trapping rate constants of superoxide radical by various spin traps in aqueous solution Spin trap
kt (M 1 s 1)
DMPO DEPMPO EMPO AMPO CPCMPO CDNMPO DPPMDPO CYPMPO
2.0152; 2.4153; 20.1155; 78.553; 170.0156 0.5153; 4.0152; 58.0157; 90.0158 10.9152; 74.553 25.2152 60.017 58.231 39.521, 4823 4822
In 2007, Allouch et al.159 reported a strong effect of pH on the trapping rate of superoxide/ hydroperoxyl radical (O2d– þ H þ #HOOd ; pKa=4.8) by nitrone spin traps. The authors used a kinetic approach based on a competition between the trapping and dismutation reactions of superoxide radical and they modelled the kinetic curves obtained at several pH values. Among the series of new spin traps recently described, it has been reported that Mito-DEPMPO and CD-DEPMPO exhibited improved superoxide trapping properties. The absolute rate constants for these two spin traps have not been determined yet but preliminary EPR superoxide trapping experiments have shown a 3 fold increased of kt for both molecules regarding DEPMPO.18,19 Durand et al.160 intended to correlate the reactivity of superoxide radical with PBN derivatives in organic solvents (DMF and pyridine) regarding the substituting groups present in para position of the aromatic ring. The development of spin traps with enhanced superoxide trapping feature is still a requirement for the successful application of spin trapping technique to biological systems where quantitative and qualitative data on superoxide radical are needed. An improvement in the superoxide trapping rate has been observed with various newly developed spin traps. Electrostatic interactions and/or H-bond acceptor groups have been hypothesized as possible elements responsible of this result, but more work is needed to clearly understand the origin of this effect. Moreover, the kt values obtained from different methods exhibit still a large scattering. This discrepancy makes comparison between the data obtained in different research groups very difficult, and there is a critical need for the development of a wellvalidated method that will yield reliable absolute values within a series of spin traps. 6.2
Decay of superoxide adducts
Even if the decay of superoxide spin adducts are strongly influenced by the environment (including the superoxide radical flux, the presence of ascorbate, thiols, hemoproteins, SOD, other radicals, chemical reductants or oxidants) it is highly desirable to have methods capable of providing reliable data on the intrinsic stability of the superoxide adducts, i. e. values obtained in in vitro experiments. These data could improve our understanding of factors that influence the decay of these species, and help designing better traps. 22 | Electron Paramag. Reson., 2011, 22, 1–40
Table 3 Reported half life times of superoxide radical adducts in aqueous solution Spin trap
Apparent t1/2 (in min, concentration of spin trap)
DMPO DEPMPO DEPMPO with DM-b-CD (50 mM) 4 Ph-DEPMPO 4HM-DEPMPO Mito-DEPMPO CD-DEPMPO EMPO CDMPO CPCOMPO CYPMPO DPPMDPO
0.9 (100 mM)157 18 (20 mM),161 14.3 (100 mM)157 98 (25 mM)25 14.5 (20 mM)15 21 (20 mM)20 40 (20 mM)18 40 (20 mM)19 9.9 (25 mM)162 27.5 (20 mM)31 2.4 (50 mM)31 15 (20 mM)22 8.321
The superoxide generating procedure are mostly based on the xanthine/ xanthine oxidase system or on light irradiation of riboflavin. The t1/2 are usually determined by fitting the exponential EPR kinetic curve,31,37 recorded after the production of superoxide has been stopped by adding excess of SOD or by switching off the light irradiation. Values for selected new spin traps are listed in Table 3. To get reliable measurements, the EPR signal of the superoxide adduct must be unambiguously identified and the kinetic curve recorded from a non overlapping line of this signal. Moreover, it has been observed that the use of KO2/DMSO as generating system is not recommended due to the poor reliability and accuracy of the obtained values. We also recently noticed, that the EPR settings, notably the microwave power (in a range of 10 to 30 mW, even in the absence of saturation of the EPR line), could have a strong influence on the determination of t1/2 values due to the heating of the sample during the measurement. This thermal effect is illustrated by the large difference on the half lifetime of DEPMPOOOH adduct at 22 1C and 37 1C, 15.3 and 8.7 min. respectively, all others conditions and parameters being identical.37 Some authors have reported half lifetime of superoxide adducts in DMSO or DMF, however, the determination of t1/2 in non aqueous solutions should be taken only as an indication, no comparison with values determined in water can be done due to the strong influence of the solvent on the adduct stability. The superoxide spin adducts of different new spin traps9,18,31 have half lifetimes longer than 30 min. and that corresponds to a huge improvement regarding the values characteristic of the first developed spin traps, i. e. PBN and DMPO. The origin of the enhanced stability is still not clearly understood, even though the contribution of many factors such as: anomeric effects, steric hindrance, higher oxidation potential, . . . has been suggested. With these new spin traps, the detection and characterization of superoxide radical by EPR/ST is now easily attainable in simple and well-defined systems. In complex biological systems, improvements and new strategies are needed to reduce the reactivity of the hydroperoxyl and nitroxide moieties regarding bioreductants, metallo-enzymes and free radicals. Electron Paramag. Reson., 2011, 22, 1–40 | 23
The use of cyclodextrins, as covalent or non-covalent part, has led to the most promising results. Several works have shown that the partial inclusion of the spin adduct in the cyclodextrin cavity, not only results in an enhancement of its intrinsic stability, but also provides a protection towards others species by excluding the adduct from the bulk solution.19,25 7
EPR/ST of organic, organometallic and inorganic radicals
Nowadays, most applications of EPR/ST concern the study of biological free radical processes, nevertheless, the technique continues to be used as a tool to investigate reaction mechanisms involving organic, organometallic and inorganic radicals, some recent examples are illustrated hereafter. 7.1
Organic radicals
Calicheamicin gI1 (Scheme 15) is known to display significant antitumor activity against experimental murine tumors. In the presence of thiols the ten-membered enediyne can generate a p-benzyne biradical that could initiate oxidative cleavage of double-stranded DNA. For the first time, spin trapping with phenyl t-butyl nitrone has provided spectroscopic evidence for the formation of an intermediate radical species likely the p-benzyne biradical;163,164 as confirmed by ESI MS only monoadducts of PBN were observed. Hydrazines and their hydrazide derivatives have been widely used as synthetic intermediates in industry and as therapeutic agents for various diseases. The dominant metabolic pathway normally involves their ready oxidation which leads, by the loss of nitrogen, to the generation of free radicals that can cause cellular damages. Gilbert et al. studied the oxidative decomposition of some oxalic acid arylhydrazides in aqueous base.165 Using O HO
O HO
N(H)CO2Me
N(H)CO2Me
HSCH2CH2OH MeSS
S
S RO
RO H
O
CO2Me
H
O
NH
NH PBN
HO
O N S RO
H
CO2Me
t-Bu
H Ph
HO
S RO
H
Scheme 15 Trapping with PBN of a p-benzyne biradical formed by reaction of b-mercaptoethanol with Calicheamicin gI1.
24 | Electron Paramag. Reson., 2011, 22, 1–40
Ar
H
H
O
N
N
C
CO2Et
O2
H
H2O/HO
Ar
N
N
C
Ar
CO2Et
Nitrones
N2
N
O2
C CO2Et O2
O Ar
N
N
HO
O2 Ar
N
HOO
O2
Ar
O
H
O
N
N
Ar
N
N
C
CO2Et
HCO2CO2Et
Aryl radical adducts
Scheme 16 Formation of aryl radicals during the aerobic decomposition of arylhydrazides in aqueous solution.
EPR/ST they showed that the decomposition yields aryl radicals and constitutes a mild non photochemical source of these species in aqueous solution (Scheme 16). Trifluoromethylketones were irradiated in the presence of radical initiators (H2O2, t-BuOOt-Bu) and the resulting radicals were identified by trapping with 2-methyl-2-nitrosopropane (MNP) and 2,4,6-tri-tBu-nitrosobenzene (TTNB) (Scheme 17).166 With acetone, the acetylmethyl radicals resulting from abstraction of a a-hydrogen were trapped. However, with 1,1,1-trifluoroacetone, only trifluoromethyl radicals resulting from the decomposition of the intermediate trifluoroacetylmethyl radicals were trapped. Like the methyl radical the trifluoromethyl radical adds to the nitrogen of the TTNB nitroso group and the TTNB-CF3 spin adduct is an aminoxyl radical. With trifluoromethyl alkylketones bearing a long alkyl chain, the formation of the trifluoromethyl radical was not observed. In this case, the formation of nitroxide- and anilino-type spin adducts can be accounted for, either by the trapping of the radical [RCHC(O)CF3]d or radicals resulting from transfers of the radical center within the R group (Scheme 17). The presence of phosphorus in DEPMPO and its analogs allows for the use of 31P NMR spectroscopy to investigate the detailed chemistry of radical reactions in complex reaction systems. This technique was termed ‘‘NMR spin trapping’’ by Khramtsov et al.167 and has been used by Argyropoulos et al. to detect various kinds of free radicals.168–170 EPR/ST was used to explore the mechanism of alcohol oxidation over gold catalysis.171 Reaction of secondary alcohols with supported and unsupported gold catalysts in the presence of spin traps (DMPO, PBN) led to the formation of a hydrogen spin adduct (DMPO-H, PBN-H) which was shown to result from H atom abstraction by the spin trap from Au-H intermediate. Radical intermediates resulting from two competing reactions, abstraction of hydrogen and halogen atoms, were characterized by spin trapping with DMPO during the reactions of chloroform over triphenylphosphine-protected Au nanoparticles.172 A catalytic reactor for trapping free radicals formed in gas phase catalytic reactions has been developed.173 Radical formation in the oxidation of cyclohexane over MoO3 in air was investigated. EPR spectra obtained using DMPO as spin trap displayed a large number of spin adducts Electron Paramag. Reson., 2011, 22, 1–40 | 25
26 | Electron Paramag. Reson., 2011, 22, 1–40
F3C
O
O
R
But
CF3
+
TTNB
HO
N
But
a
F3C O
But
But
CH2COCH3
CH2COCF3
O
MeCOCF3
O
N
O
H2C
CF3
+
+
+
HO
MNP or TTNB
But
CF3
O
N
TTNB
But
But
R
O
O
R CF3
CF3
nitroxide- and anilino-type spin adducts
O
CF3
MNP-CF3 or TTNB-CF3
MNP-CH2COCH3 or TTNB-CH2COCH3
O
CF3
C
O
R
or TTNB
MNP
b
Scheme 17 Spin trapping of free radical intermediates in the photolysis of trifluoroketones and free radical initiators.
O
TTNB
MeCOCH3 + HO
But
MeCOCF3 +
MNP
N
But
(DMPO-Cyclohexyl, DMPO-OOR, DMPO-OR, DMPO-OH). The observation of these species is consistent with the free radical chain mechanism oxidation of cyclohexane proposed in the literature. A review on the use of EPR techniques, including EPR/ST, to monitor working catalysts has been published.174 7.2
Organometallic radicals
The photodecomposition of new free radical photoinitiators generating silyl- and alkyl-centred radicals under light irradiation was investigated by ESR/ST and laser flash photolysis experiments175,176 (Scheme 18). The aN and aH values of the spin adduct STA4 are characteristic of the trapping of a trialkylsilyl radical (aN=1.47 mT, aH=0.60 mT for the PBNSiEt3 spin adduct). The aN and aH values of the spin adduct STA2 are identical to those of the PBN-CH2Si(CH3)3 spin adduct. Trialkyl- and triphenylsilyl radicals were trapped with PBN in a flow-through four-electrode EPR spectroelectrochemical cell, during two-electron reductions of triphenyl- or trialkylchlorosilanes followed by one-electron oxidation of the resulting Ph3Si or R3Si anions.177 Tris(trimethylsilyl)silyl radical were also trapped with PBN during irradiation of transition metal carbonyls (Mn2CO10, Re2CO10, Cp2Fe2CO4) in the presence of tris(trimethylsilyl)silane.178 Hydrotelluration of alkynes is an important synthetic methodology for stereocontrolled carbon-carbon double bond formation.179 The EPR/ST technique was used to investigate the mechanism of the reaction. In the presence of DBNBS, the formation of a nitroxide spin adduct with an unusual large nitrogen splitting (aNW2.0 mT) was attributed to the trapping of a tellurium-centred radical (Scheme 19).180 Based on spin trapping results, Cl3Ted radicals were proposed as tellurating agent during the addition of TeCl4 to an alkyne.181 The effect of Lewis base coordination on boryl radical (L-dBH2) reactivity was examined using laser flash photolysis, MO calculations and spin trapping with PBN182 (Scheme 20). The aB found in the PBN adducts were in the range 0.36 – 0.47 mT. With F (Scheme 20), the hydrogen abstraction preferentially occurs on the borane moiety as trapping of aminoalkyl radicals was not observed. For H, only Si
O
O Si Si Si A33 A A
hν
OH Ph
O Ph
O
Si(SiMe3)
+
Ph
C
Si Ph
Ph
C O
Ph
O A3
CH2
Si(SiMe3)2
PBN
A2
A1
A*
O
Si(SiMe3)2 + SiMe3 A4
PBN
STA4
STA2
aN = 1.45 mT aH = 0.26 mT
aN = 1.48 mT aH = 0.61 mT
Scheme 18 Trapping of free radicals with PBN during the photochemical decomposition of 4tris(trimethylsilyl)silyloxybenzophenone.
Electron Paramag. Reson., 2011, 22, 1–40 | 27
O
O N Br
Br
N
TeR Br
Br
RTeTeR/NaBH4/EtOH
SO3
SO3 R = n-Bu: aN = 2.16 mT, aH(2Hm) = 0.07 mT Scheme 19 Spin trapping of a tellurium-centred radical.
N
N
BH3
BH3
PH
BH3
F G N
H N
BH3 + t-BuO
BH2 + t-BuOH
O N
BH2 +
CH
N
But
O
But N
CH BH2 N
Scheme 20
phosphorus-centred radicals were trapped (aN=1.43 mT, aH=0.27 mT and aP=1.39 mT). 7.3
Inorganic radicals
Sulfur dioxide is water soluble and in aqueous solution, at neutral pH, exists primarily as sulfite (SO23 ) and (bi)sulfite (HSO3 ). Owing to its antioxidant and antimicrobial properties, (bi)sulfite is extensively used as a preservative in beverages and foods. Detoxification in vivo of (bi)sulfite is believed to occur mainly as a result of the function of sulfite oxidase that oxidizes (bi)sulfite to sulphate without any radical reaction. However, (bi)sulfite can also be oxidised to sulphate by trace transition metal ions via free radicals. The SO3d– and SO4d– radicals have been trapped in different systems using either DMPO or DEPMPO. However it has then been demonstrated183,184 that (bi)sulfite reacts with DMPO via a non radical, nucleophilic reaction, and further proposed that the radical adduct DMPO-SO3 observed in biological system is an artefact and not the result of the trapping of SO3d–. 28 | Electron Paramag. Reson., 2011, 22, 1–40
HRP-Compound I
HRP + H2O2 2
HRP-Compound I + SO3
2
HRP-Compound II + SO3
2
H2O2 + 2 SO3
2 SO3
(1)
HRP-Compound II + SO3
(2)
HRP + SO3
(3)
DMPO
2 DMPO-SO3
(4)
Scheme 21 Enzymatic oxidation of bisulfite with horseradish peroxidase/H2O2 system.
Mason et al.185 re-examine the problem by studying the enzymatic oxidation of (bi)sulfite by the horseradish peroxidase/H2O2 system (Scheme 21). To confirm the stoechiometry (2:1) in Eq. (4) (Scheme 21) they measured the yield of the radical adduct relative to the concentration of H2O2 and found a 1.95:1 stoechiometry in excellent agreement with the expected value. The authors also showed that in the presence of DMPO the uptake of oxygen correlates with the concentration of the trap in agreement with a high efficiency of DMPO trapping of SO3d– radicals. Therefore the authors concluded that the trapping of sulphur trioxide anion radical is not susceptible to artefacts arising from non-radical chemistry except when DMPO and sulfite concentrations are at non-physiological levels (W 0.1 M) and the incubations are for longer times. The trapping of sulphate radical anions (SO4d–) generated from thermal decomposition of potassium persulfate (K2S2O8) was fully investigated in DMSO using DMPO, EMPO and DIPPMPO as spin traps.186 In deoxygenated solutions, high-resolution EPR spectra of the corresponding spin adducts showed unusually rich hyperfine structure resulting from the interaction of the unpaired electron with all the magnetically active nuclei of the spin trap moiety. When the trapping was performed in DMSO/water solutions with DMPO, the DMPO-OH spin adduct was also observed and was the major adduct with water contents higher than 50 %. Interestingly, the hyperfine coupling constants of the DMPO-OH spin adduct were shown to depend significantly on the DMSO/water ratio. The study showed that the thermal generation of SO4d– in DMSO is an effective source of free radicals to test the radical scavenging properties of hydrophilic as well as hydrophobic antioxidants in the presence of conventional radical spin traps. 8 Application of EPR/ST to study the degradation of ionomer membranes used in fuel cells Proton-exchange membranes (PEMs) are used in fuel cells in order to separate the anode and cathode compartments, and to allow transport of protons from the anode to the cathode. Most of these membranes consist of perfluorinated backbones and pendant groups terminated by sulfonic acid, SO3H, and their durability is a major problem that must be solved before to consider the broad introduction of fuel cells in automotive and other applications. Work in various laboratories has demonstrated the formation of a small amount of hydrogen peroxide during the two-electron oxygen reduction at Electron Paramag. Reson., 2011, 22, 1–40 | 29
NAFION, EW 1100
3M, EW 850
AQUIVION, EW 830
(CF2CF2)mCF2CF
(CF2CF2)nCF2CF
(CF2CF2)nCF2CF
OCF2CFOCF2CF2SO3H
OCF2CF2CF2CF2SO3H
OCF2CF2SO3H
CF3 kM/M-1s-1 :
2.7 x108
0.14 x 108
0.13 x 108
Scheme 22 Reaction rates kM of fluorinated PEMs with hydroxyl radicals.
the cathode side of fuel cells. Hydrogen peroxide can then generate hydroxyl radicals that mediate the membrane degradation, and recently a large amount of work using EPR/ST approaches has been devoted to identify radical fragments in membranes used in fuel cells and in model compounds when exposed to reactive oxygen species.187–198 As an example, Danilczuk et al. have developed a competitive kinetics approach based on spin trapping to measure the kinetics of HOd attack on various perfluorinated membranes (Nafion EW 1100, 3M, EW 850 and Aquivion, EW 830) (Scheme 22). When HOd radicals reacted with CF3CF2OCF2SO3H, that mimicked the Nafion side chain, in the presence of DMPO, both carbon- and oxygen-centred were trapped thus suggesting the cleavage of the C-O bond of the Nafion side chain when exposed to HOd radicals. Thus, the increased stability of 3M, EW 850 and Aquivion compared to Nafion when exposed to HOd radicals was attributed to the absence of ether linkage in their side chain. 9
Radical formation in beer and wine studied by EPR/ST
The redox processes in beer and wine are of great importance with respect to product quality and stability and EPR/ST continues199–205, to be used to get information on free radicals involved in these redox processes. It is now well established that the shelf life of beer can be estimated from EPR/ST measurements. In the presence of PBN, the kinetics of spin adduct formation in beer oxidation exhibits an induction period if the reaction is carried out at elevated temperature and in the presence of air. This lag period lasts until the endogenous antioxidants are almost completely depleted, and its duration is used as an indicator of the flavour stability and shelf life of beer. Kocherginsky et al.170 studied the kinetics of spin adduct formation and defined a new dimensionless parameter to characterize the antioxidant pool of the beer. Frederiksen et al.171 evaluated by EPR/ST the levels of radical formation during mashing and in sweet wort. EPR/ST was used to detect and identify several free radicals in wine under oxidative conditions.173 The formation of 1-hydroxyethyl radical was unambiguously established in a red wine, thus providing the first direct evidence of the Fenton reaction in wine. The formation of sulphur trioxide radical anion arising from the sulfite added to the wine was also detected. 10
Theoretical calculations
In order to help the designing of new traps with improved properties, quantum mechanical calculations have been performed to get insights on different aspects of spin trapping processes performed with nitrones. These 30 | Electron Paramag. Reson., 2011, 22, 1–40
studies used mainly the DFT approach and were devoted to the evaluation of structural and electronic properties of different nitrones and their spin adducts. Villamena et al.152,160,206–212 reported a series of theoretical and experimental results dealing mainly with the spin trapping properties of DMPO and their comparison with those of more recently developed spin traps like DEPMPOs and EMPOs. The computed data were obtained at various levels of theory with the polarisable continuum model (PCM) to simulate the influence of the solvent. The formation of either a C-C bond (DMPO-CO2 ) or a C-O bond (DMPO-OCO ) was considered for the trapping of carbon dioxide radical anion CO2d– with DMPO206 (Scheme 23). The formation of DMPO-CO2 is exoergic by DG298K,aq=12.1 kcal/mol and the calculated values of isotropic hyperfine coupling constants while underestimated are in reasonable agreement with the experimental values. Analysis of the optimized structure of DMPO-OCO reveals two different C-O bond distances for the CO2 group (i.e., C2-O=2.14 A˚ and C6O=1.16 A˚). The C6-O bond length is close to that of carbon monoxide (1.13 A˚) and DMPO-OCO is expected to yield carbon monoxide CO and DMPO-O (Scheme 23). The pKa of DMPO-CO2H was predicted to be 7.1, in agreement with the reported pKa of 6.4 of HOCO2H. Two major types of products, a C- and an O-centred spin adduct, DMPO-CO3 and DMPO-OCO2 respectively, were predicted for the addition of the carbonate radical anion (CO3d–) to DMPO207 (Scheme 24). In agreement with the absence of spin density on the C atom of CO3d– the O-centred adduct is more preferred by DG298K,aq=-6.6 kcal/mol as compared to the C-centred adduct with DG298K,aq=93.1 kcal/mol. UV photolysis of H2O2 and DMPO in the presence of sodium carbonate (Na2CO3) or sodium bicarbonate (NaHCO3) yielded an EPR signal composed of the signal of DMPO-OH and the signal of a less persistent species (aN=1.432 mT, aHg=1.068 mT and aHg=0.137 mT) tentatively assigned to DMPO-OCO2 . Further experimental evidence is needed to definitively ascertain this assignment. To get insights on the differences observed in the half-life times of different 5,5-dimethyl-1-pyrroline N-oxide hydroperoxyl adducts, their unimolecular decay involving the homolytic cleavage of the CO-OH bond (Scheme 25) was investigated.208
2 N CO2
6 O C O
O
N O
2 N O
O 6C O
2 CO
N
O
O
Scheme 23 Spin trapping of CO2d– with DMPO.
Electron Paramag. Reson., 2011, 22, 1–40 | 31
CO3
N O
CO3
O
N
C
O
2 N
O
O
O Scheme 24 Spin trapping of CO3d– with DMPO.
OH
R N O
R
O OH
N O
O
R N
O
O
DMPO (R = Me); AMPO (R = C(O)NH2); EMPO (R = CO2Et); DEPMPO (R = P(O)(OEt2))
Scheme 25
The cleavage of the peroxyl bond is endoergic while the subsequent ringopening step leading to a nitrosoaldehyde is highly exoergic. Among AMPOOOH, EMPO-OOH and DEPMPO-OOH, the calculated overall energetics show that in the presence and absence of explicit water interactions the decomposition of DMPO-OOH is the most favourable, however, no significant differences in the energetics of decomposition were observed. Rate constants of hydroperoxyl radical addition to cyclic nitrones were investigated using various (PCM/B3LYP/6-31 þ G(d,p), PCM/mPW1K/631 þ G(d,p) . . . ) DFT methods.209 The results suggest that the transition state (TS) for HO2d addition is early on the reaction coordinate; i.e., the TS structures are closer to the reactants. The calculated second-order rate constants were in the same range (100-103M 1 s 1) than that observed experimentally, however, no correlation was found with charge densities on nitrone C2 (Scheme 25). The intramolecular H-bonding interactions presupposed to facilitate HO2d addition are unlikely to play a role in water solution. The redox properties of DMPO, AMPO, EMPO and DEPMPO and their corresponding HOOd spin adducts were investigated.210 Electron-withdrawing group substitution at C5 position results in higher EAs and IPs making these substituted nitrones more susceptible to reduction but more difficult to oxidize compared to DMPO. Among the studied spin adducts DEPMPO-OOH is the most difficult to reduce and oxidize. Liu et al.11 investigated the effect of the phosphoryl substituent on the spin trapping properties of a phosphorylated linear nitrone N-(4hydroxybenzylidene)-1-diethoxyphosphoryl-1-methylethyl-amine N-oxide (4-HOPPN) (Scheme 26). From the analysis of the optimized geometries of 4-HOPPN-OOH and PBN-OOH spin adducts the authors suggested that the stabilizing effect of the phosphoryl substituent resulted from steric protection and intramolecular nonbonding attractive interactions. 32 | Electron Paramag. Reson., 2011, 22, 1–40
O N HO
P(O)(OEt)2
4-HOPPN Scheme 26
11
Conclusion
EPR is the major method that allows specific and sensitive detection of free radicals. Its coupling with a spin trapping process (EPR/ST) constitutes the most powerful tool to detect and identify transient free radicals either in solution or in gas phase. Moreover recent advances in technology have pushed the detection limit into the region where reactive radicals at physiological concentrations can now be detected. However, EPR/ST is a demanding technique that must be used cautiously to avoid misinterpretations. Apart the never-ending problem of deciphering which signals are true adduct signals and which are artefacts, a number of problems still remain to be overcome, particularly when applying ST/EPR in free radical biology. Among these problems the most limiting are the following: – a lack of reliable data concerning the rate constants for spin trapping and the decays of spin adducts; – the perturbations that the spin traps produce on the system under study (particularly when they are toxic); – the in vivo very short half life of nitroxide spin adducts that prevent to readily examine many radical processes in humans.
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