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1、Introduction to Electron Spin Resonance and Spin Trapping,Michael R.GuntherWest Virginia University School of Medicine,Free Radicals and EPR,Molecules with one or more unpaired electronQuantum mechanics:unpaired electrons have spin and charge and hence magnetic momentElectronic spin can be in either
2、 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 field,The EPR experiment,Put sample into experimental magnetic field(B)Irradiate(microwave frequencies)Measure absorbance of r
3、adiation as f(B),Weil,Bolton,and Wertz,1994,“Electron Paramagnetic Resonance”,The EPR spectrometer,ElectromagnetMicrowave source and detector(typically X band,9.5 GHz)Modulation of magnetic field and phase-sensitive detectionSpectrum 1st derivative,Weil,Bolton,and Wertz,1994,“Electron Paramagnetic R
4、esonance”,The EPR spectrum,A 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/enhanced by hyperfine interactions wi
5、th nuclei with non-zero spin,The hyperfine effect,The magnetic field experienced by the unpaired electron is affected by nearby nuclei with non-zero nuclear spin,Weil,Bolton,and Wertz,1994,“Electron Paramagnetic Resonance”,New York:Wiley Interscience.,Hyperfine splitting of EPR spectra,The magnitude
6、 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 spinMagnitude of the splitting typically decreases greatl
7、y with increasing numbers of bonds between the nucleus and unpaired electron,Hyperfine splittings are additive,Direct EPR analysis of a radical,Radical cannot be diatomic Radical must be available at a detectable concentrationAt least metastableFrozen solution to greatly decrease radical decayCan gr
8、eatly complicate the spectrum due to anisotropyContinuous formation inside resonatorEnzymatic radical formationFlow experimentRadical characterized by hyperfine analysis,Direct EPR of a tyrosyl radical,Gunther,M.R.,Sturgeon,B.E.,and Mason,R.P.,Free Radic.Biol.Med.28:709-719,2000,Spin trapping:when d
9、irect EPR is not convenient or possible,Unstable free radical reacts with diamagnetic molecule(the spin trap)to form a relatively stable free radicalThe vast majority of spin traps form radical adducts through the addition of the radical to the trap to form a nitroxide radical2 major classes of trap
10、s:nitrones and nitroso compounds,Advantages of the nitrones,React with a variety of different free radicals to form nitroxide adductsRC.,RO.,RS.,in some cases RN.Adducts are often quite stableNot terribly toxic so amenable to in vivo/ex vivo spin trapping,Nitrone spin traps,DMPO,5,5-dimethylpyrrolin
11、e N-oxidePBN/4-POBN,phenyl-N-t-butylnitrone,EPR spectra from DMPO adducts,EPR spectra from 4-POBN adducts,Nitroso spin traps,Free radical adds to the nitrogen atom of a C-nitroso compound2-methyl-2-nitrosopropane,MNP3,5-dibromo-4-nitrosobenzene sulfonate,EPR spectra from methyl radical adducts of ni
12、troso traps,DMPO-trapping the tyrosyl radical,Oxidize tyrosine with HRP/H2O2,Gunther,M.R.,et al.,Biochem.J.330:1293-1299,1998.,Spin trap-derived hyperfine from MNP and MNP-d9,Each line in the EPR spectra from MNP adducts is broadened by hyperfine from the 9 equivalent protons on the spin trap,MNP-tr
13、apping the tyrosyl radical,Gunther,M.R.,et al.,Biochem.J.330:1293-1299,1998.,Why not spin trap?,Nitrone spin traps,especially DMPOAdducts 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
14、 be facilely oxidized up to the nitroxide,Why not spin trap?,Nitroso spin traps MNP and DBNBSOften acutely toxic so cant use in vivoThe C-nitroso group critical to their function is highly reactiveTend to directly add across unsaturated systems giving EPR-silent hydroxylamines that are readily oxidi
15、zed to the corresponding nitroxides,Summary,The main feature of EPR spectra that is useful for assignment to a particular free radical structure is hyperfine splittingDirect EPR spectra can provide a wealth of structural informationHighly unstable free radicals can,in many cases,be stabilized for EP
16、R 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 essential,
