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1、Introduction to Electron Spin Resonance and Spin TrappingMichael R. GuntherWest Virginia University School of Medicine1听雨C书屋Free Radicals and EPRMolecules with one or more unpaired electronQuantum mechanics: unpaired electrons have spin and charge and hence magnetic momentElectronic spin can be in e
2、ither of two directions (formally up or down)The two spin states under normal conditions are energetically degenerateEnergetic degeneracy lost when exposed to an external magnetic field2听雨C书屋The EPR experimentPut sample into experimental magnetic field (B)Irradiate (microwave frequencies)Measure abs
3、orbance of radiation as f(B)Weil, Bolton, and Wertz, 1994, “Electron Paramagnetic Resonance”3听雨C书屋The EPR spectrometerElectromagnetMicrowave source and detector (typically X band, 9.5 GHz)Modulation of magnetic field and phase-sensitive detectionSpectrum 1st derivativeWeil, Bolton, and Wertz, 1994,
4、“Electron Paramagnetic Resonance”4听雨C书屋The EPR spectrumA 1st derivative spectrum is obtained from the unpaired electronhn = gBb0g is a characteristic of the chemical environment of the unpaired electron; for free radicals it is near 2.00; can vary widely for transition metal centersComplicated/enhan
5、ced by hyperfine interactions with nuclei with non-zero spin5听雨C书屋The hyperfine effectThe magnetic field experienced by the unpaired electron is affected by nearby nuclei with non-zero nuclear spinWeil, Bolton, and Wertz, 1994, “Electron Paramagnetic Resonance”, New York: Wiley Interscience.6听雨C书屋Hy
6、perfine splitting of EPR spectraThe magnitude of the splitting and the number of lines depend upon:The nuclear spin of the interacting nucleus# of lines = 2n(I + ) so I = gives 2 lines, etc.The nuclear gyromagnetic ratioThe magnitude of the interaction between the electronic spin and the nuclear spi
7、nMagnitude of the splitting typically decreases greatly with increasing numbers of bonds between the nucleus and unpaired electron7听雨C书屋8听雨C书屋Hyperfine splittings are additive9听雨C书屋Direct EPR analysis of a radicalRadical cannot be diatomic Radical must be available at a detectable concentrationAt le
8、ast metastableFrozen solution to greatly decrease radical decayCan greatly complicate the spectrum due to anisotropyContinuous formation inside resonatorEnzymatic radical formationFlow experimentRadical characterized by hyperfine analysis10听雨C书屋Direct EPR of a tyrosyl radicalGunther, M.R., Sturgeon,
9、 B.E., and Mason, R.P., Free Radic. Biol. Med. 28:709-719, 200011听雨C书屋Spin trapping: when direct EPR is not convenient or possibleUnstable free radical reacts with diamagnetic molecule (the spin trap) to form a relatively stable free radicalThe vast majority of spin traps form radical adducts throug
10、h the addition of the radical to the trap to form a nitroxide radical2 major classes of traps: nitrones and nitroso compounds12听雨C书屋Advantages of the nitronesReact with a variety of different free radicals to form nitroxide adductsRC., RO., RS., in some cases RN.Adducts are often quite stableNot ter
11、ribly toxic so amenable to in vivo/ex vivo spin trapping13听雨C书屋Nitrone spin trapsDMPO, 5,5-dimethylpyrroline N-oxidePBN/4-POBN, phenyl-N-t-butylnitrone14听雨C书屋EPR spectra from DMPO adducts15听雨C书屋EPR spectra from 4-POBN adducts16听雨C书屋Nitroso spin trapsFree radical adds to the nitrogen atom of a C-nitr
12、oso compound2-methyl-2-nitrosopropane, MNP3,5-dibromo-4-nitrosobenzene sulfonate17听雨C书屋EPR spectra from methyl radical adducts of nitroso traps18听雨C书屋DMPO-trapping the tyrosyl radicalOxidize tyrosine with HRP/H2O2Gunther, M.R., et al., Biochem. J. 330:1293-1299, 1998.19听雨C书屋Spin trap-derived hyperfi
13、ne from MNP and MNP-d9Each line in the EPR spectra from MNP adducts is broadened by hyperfine from the 9 equivalent protons on the spin trap20听雨C书屋MNP-trapping the tyrosyl radicalGunther, M.R., et al., Biochem. J. 330:1293-1299, 1998.21听雨C书屋Why not spin trap?Nitrone spin traps, especially DMPOAdduct
14、s can interconvert, i.e., DMPO/.OOH decays to form DMPO/.OHSubject to rare nucleophilic addition across their double bondsYields an EPR silent hydroxylamine which can be facilely oxidized up to the nitroxide22听雨C书屋Why not spin trap?Nitroso spin traps MNP and DBNBSOften acutely toxic so cant use in v
15、ivoThe C-nitroso group critical to their function is highly reactiveTend to directly add across unsaturated systems giving EPR-silent hydroxylamines that are readily oxidized to the corresponding nitroxides23听雨C书屋SummaryThe main feature of EPR spectra that is useful for assignment to a particular fr
16、ee radical structure is hyperfine splittingDirect EPR spectra can provide a wealth of structural informationHighly unstable free radicals can, in many cases, be stabilized for EPR characterization by spin trappingThe increased stability of the detected free radical comes with a loss of structural informationThe adduct may undergo chemistry between formation and detectionAdduct assignment is assisted by selective isotope labeling and EPR analysis of an independent preparation of the suspected adductThe performance of appropriate controls is essential24听雨C书屋
