电子自旋共振 经典讲座

1、Free Radicals and EPR Molecules with one or more unpaired electron Quantum mechanics: unpaired electrons have spin and charge and hence magnetic moment Electronic spin can be in either of two directions (formally up or down) The two spin states under normal conditions are energetically degenerate En

2、ergetic degeneracy lost when exposed to an external magnetic fieldThe EPR experiment Put sample into experimental magnetic field (B) Irradiate (microwave frequencies) Measure absorbance of radiation as f(B)Weil, Bolton, and Wertz, 1994, “Electron Paramagnetic ResonanceThe EPR spectrometer Electromag

3、net Microwave source and detector (typically X band, 9.5 GHz) Modulation of magnetic field and phase-sensitive detection Spectrum 1st derivativeWeil, Bolton, and Wertz, 1994, “Electron Paramagnetic ResonanceThe EPR spectrum A 1st derivative spectrum is obtained from the unpaired electron hn = gBb0 g

4、 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 centers Complicated/enhanced by hyperfine interactions with nuclei with non-zero spinThe hyperfine effect The magnetic field experienced by the unpaired

5、electron is affected by nearby nuclei with non-zero nuclear spinWeil, Bolton, and Wertz, 1994, “Electron Paramagnetic Resonance, New York: Wiley Interscience.Hyperfine splitting of EPR spectra The magnitude of the splitting and the number of lines depend upon: The nuclear spin of the interacting nuc

6、leus # of lines = 2n(I + ) so I = gives 2 lines, etc. The nuclear gyromagnetic ratio The magnitude of the interaction between the electronic spin and the nuclear spin Magnitude of the splitting typically decreases greatly with increasing numbers of bonds between the nucleus and unpaired electron10 G

7、aussNo hyperfine1 I=1/2 nucleus (1H)1 I=1 nucleus (14N)2 identical I=1/2 nuclei1 I=5/2 nucleus (17O)Hyperfine splittings are additive1 N, = 14.9 Ga1N, = 14.9 G, 1H, = 2.5 Gaa1N, = 14.9 G, 1H, = 14.9 GaaDirect EPR analysis of a radical Radical cannot be diatomic Radical must be available at a detecta

8、ble concentration At least metastable Frozen solution to greatly decrease radical decay Can greatly complicate the spectrum due to anisotropy Continuous formation inside resonator Enzymatic radical formation Flow experiment Radical characterized by hyperfine analysisDirect EPR of a tyrosyl radicalGu

9、nther, M.R., Sturgeon, B.E., and Mason, R.P., Free Radic. Biol. Med. 28:709-719, 2000HHHHO.CH2HCCORNHRSpin trapping: when direct EPR is not convenient or possible Unstable free radical reacts with diamagnetic molecule (the spin trap) to form a relatively stable free radical The vast majority of spin

10、 traps form radical adducts through the addition of the radical to the trap to form a nitroxide radical 2 major classes of traps: nitrones and nitroso compoundsAdvantages of the nitrones React with a variety of different free radicals to form nitroxide adducts RC., RO., RS., in some cases RN. Adduct

11、s are often quite stable Not terribly toxic so amenable to in vivo/ex vivo spin trappingNitrone spin traps DMPO, 5,5-dimethylpyrroline N-oxide PBN/4-POBN, phenyl-N-t-butylnitroneNH3CH3CHONH3CH3CHRO.R.CHNC(CH3)3OCHNC(CH3)3RO.R.EPR spectra from DMPO adductsEPR spectra from 4-POBN adducts2 0 G a u s sN

12、CHNO.C(CH3)3CRONCHNO.C(CH3)3O13CRNitroso spin traps Free radical adds to the nitrogen atom of a C-nitroso compound 2-methyl-2-nitrosopropane, MNP 3,5-dibromo-4-nitrosobenzene sulfonateNOC(CH3)3NC(CH3)3.ORR.NOBrBrSO3-NBrBrSO3-R.OR.EPR spectra from methyl radical adducts of nitroso trapsMNP/ CH3, = 17

13、.2 G; = 14.2 G (3H) .aNaHDBNBS/ CH3, = 14.3 G; = 13.3 G (3H).aNaH40 GaussDMPO-trapping the tyrosyl radicalNatural isotope abundance17O-labeled tyrosine Oxidize tyrosine with HRP/H2O2NCH3CH3HOCH2RO.Gunther, M.R., et al., Biochem. J. 330:1293-1299, 1998.Spin trap-derived hyperfine from MNP and MNP-d9E

14、ach line in the EPR spectra from MNP adducts is broadened by hyperfine from the 9 equivalent protons on the spin trapNRO.CCH3CH3CH3NRO.CCD3CD3CD3MNP-trapping the tyrosyl radical.OHHHHCHHCHNH2CO2-MNP/ tyrosine.MNP9/ tyrosined.NO.OHHCH2R(CH3)3C.OHHHHCHHCHNH2CO2-Gunther, M.R., et al., Biochem. J. 330:1

15、293-1299, 1998.Why not spin trap? Nitrone spin traps, especially DMPO Adducts can interconvert, i.e., DMPO/.OOH decays to form DMPO/.OH Subject to rare nucleophilic addition across their double bonds Yields an EPR silent hydroxylamine which can be facilely oxidized up to the nitroxideNOH3CH3CHNOH3CH

16、3CHORHROHMen+- H+- e-NH3CH3CHORO.Why not spin trap? Nitroso spin traps MNP and DBNBS Often acutely toxic so cant use in vivo The C-nitroso group critical to their function is highly reactive Tend to directly add across unsaturated systems giving EPR-silent hydroxylamines that are readily oxidized to

17、 the corresponding nitroxidesSummary The main feature of EPR spectra that is useful for assignment to a particular free radical structure is hyperfine splitting Direct EPR spectra can provide a wealth of structural information Highly unstable free radicals can, in many cases, be stabilized for EPR characterization by spin trapping The increased stability of the detected free radical comes with a loss of structural