NMR of Paramagnetic MoleculesKara L. BrenUniversity of Rochester
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren2
Resources Bertini & Luchinat, “NMR of Paramagnetic Molecules inBiological Systems,” 1986, Benjamin/Cummings: MenloPark. ISBN: 0-8053-0780-X Bertini & Luchinat, “NMR of Paramagnetic Substances,”Coord. Chem. Rev. 1996, 150, 1-296. Banci, “Nuclear and Electron Relaxation. The MagneticNucleus-unpaired Electron Coupling in Solution,” 1991, VCH:Weinheim. ISBN: 3-5272-8306-4 Bren, “NMR Analysis of Spin States and Spin Densities,” inSpin States in Biochemistry and Inorganic Chemistry:Influence on Structure and Reactivity, (Eds: Swart & Costas),2016, Wiley: Chichester. DOI: hop,Bren3
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren4
Effects of Unpaired ElectronsHorse ferricytochrome c2016PSUBioinorganicWorkshop,BrenS 1/25
Effects of Unpaired ElectronsHorse ferricytochrome cS 1/2Heme methyls2016PSUBioinorganicWorkshop,Bren6
Effects of Unpaired ElectronsCN-Fe(III) cytochrome c, D2O* heme methyls**S hromec,c,DD2O2OH 2O***FeN*NHHis2520151050-5-10 -15δ, ppmJACS 1995, 80672016PSUBioinorganicWorkshop,Bren7
Effects of Unpaired ElectronsH. thermophilus Fe(III)M61A cyt cS 1/2, 5/25 COH215 CFe25 CNNH35 CHisa,b,c,d heme methyls45 CBren group2016PSUBioinorganicWorkshop,Bren8
Effects of Unpaired ElectronsP. aeruginosa Cu(II) azurinS 1/2HisH 2CMetSNCysSCuNHHisH 2CNO6050403020100NHδ, ppmJACS 2000, 37012016PSUBioinorganicWorkshop,Bren9
Effects of Unpaired ElectronsS 1Ni(II) azurinBiochemistry 1996, 18102016PSUBioinorganicWorkshop,Bren10
Effects of Unpaired ElectronsT. thermophilus CuA domainCu(I)Cu(II) S 1/2pH 8.0, H2OpH 4.5, H2OHNHisCysNHSJACS 1996, tOSNNH11
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren12
What Can We Learn? Metal oxidation state and spin state Electron spin relaxation time (estimate) Presence of low-lying excited states Pattern and amount of electron spin delocalizationonto ligands; hyperfine coupling constants Presence of hydrogen bonds Magnetic anisotropy, magnetic axes Structural refinement is possible (In addition, 3D structure, exchange phenomena,dynamics, etc.)2016PSUBioinorganicWorkshop,Bren13
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren14
NMR FundamentalsSome nuclei have spin angular momentum and anassociated magnetic moment, µI.µIµI gN (e/2mp) IgN (1H) 5.5856947 µI (h/2π) I[I(I 1)]1/2Need nucleus with non-zero spin (I 0)Examples:1H, 13C, 15N, 19F, 31P2H, 14N,(I 1/2)(I 1)Herein we will base examples in nuclei with I 1/22016PSUBioinorganicWorkshop,Bren15
NMR FundamentalsA nucleus with spin I has states with associated MIvalues where MI -I, I 1, IWhen I 1/2, MI 1/2In the absence of a magneticfield, states with different MI aredegenerate and their magneticmoments µ orient randomly2016PSUBioinorganicWorkshop,Bren16
NMR FundamentalsApplying a magnetic field lifts this degeneracy. 1H with MI -1/2 have a higher energy and 1H with MI 1/2 have alower energy.The nuclear spins align with (MI 1/2) or opposed to (MI -1/2) theapplied magnetic field:B0The z component of themagnetic moment is shown andis MIh/2π2016PSUBioinorganicWorkshop,Bren17
NMR FundamentalsThe energies of the nuclei in the magnetic field are:E -MIµ Β0/ITransitions may be induced between MI states.The selection rule is ΔMI 1MI -1/2E0ΔΕ µB0/I hνMI 1/2B02016PSUBioinorganicWorkshop,Bren18
NMR FundamentalsThe energies of the nuclei in the magnetic field are:E -MIµ Β0/IΔΕ µB0/I hνTraditionally, the magnetogyricratio γ (T-1 s-1) is used in NMR:γ µ 2π/hI, soB0ΔE γB0/2π hν2016PSUBioinorganicWorkshop,Bren19
NMR FundamentalsΔΕ γB0/2πA transition between MI statescorresponds to a “spin flip.”At equilibrium there is a smallexcess of spins aligned with thefield:N(-1/2)N( 1/2)B0 exp [–(E-1/2 – E 1/2)/kBT]2016PSUBioinorganicWorkshop,Bren20
NMR FundamentalsWe can consider a net magnetization vector for thesample. Exciting the sample decreases the different in upand down spins, tipping this vector, after which it returns toequilibrium:MzB0MzPulse (By)zzRelaxation (T1, T2)yyxzxyxPulse width is time of pulse; adjust time to change tip angle (i.e. 90 pulse)2016PSUBioinorganicWorkshop,Bren21
NMR FundamentalsMz is undergoing precession throughout this process –think of a cone rather than just the Mz vector tipping –precession frequency is the Larmor frequency:ω0 γB0 (rad) or ν0 γB0/2π (Hz)(Larmor equation)MzB0Pulse (By)zzyyx2016PSUBioinorganicWorkshop,Brenx22
NMR FundamentalsThe observable signal is recorded in the xy plane duringrelaxation. T1 relaxation along z axis; T2 relaxation inxy plane. Precession frequency is observed.Record FID: signalin xy planezB0MzRelaxation(T1, T2)yxzyxtime2016PSUBioinorganicWorkshop,Bren23
NMR FundamentalsThe observable signal is recorded in the xy plane duringrelaxation. T1 relaxation along z axis; T2 relaxation inxy plane. Precession frequency is observed.Line widthΔν 1/(πT2)FID: time-domainsignal in xy planeFourier op,Bren24
NMR FundamentalsThe chemical shift results from small deviations from ν0Chemical shift:δ (ppm) 106 [ν(obs) - ν(ref)]/ν(ref)The chemical environment of nuclei (especiallycirculation of electrons) leads to deviations of ν(obs)giving different chemical shifts.Unpaired electrons can have very large effects onchemical shifts through different mechanisms.2016PSUBioinorganicWorkshop,Bren25
Electrons vs. NucleiQuantitySpinMagnetic momentGyromagnetic ratioTransition energy infield B0ElectronS 1/2µeγe µB ge/hhν ge µB B0I 1/2µIγI µI gI/hhν h γI B0/2πTransition betweenstatesMS 1/2MI 1/22016PSUBioinorganicWorkshop,Bren1H26
Electrons vs. NucleiTwo major differences: Electrons have a larger magnetic moment µB 658 µI(1H) Larger resonance frequency More efficient relaxation Electrons are in orbitals Delocalization Spin-orbit coupling2016PSUBioinorganicWorkshop,Bren27
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren28
Relaxation in NMRThe observable signal is recorded in the xy plane duringrelaxation. T1 relaxation along z axis; T2 relaxation inxy plane.Line widthΔν 1/(πT2)FID: time-domainsignal in xy planeFourier op,Bren29
Relaxation in NMRThe observable signal is recorded in the xy plane duringrelaxation. T1 relaxation along z axis; T2 relaxation inxy plane.Assumption here: T1 T2 (simplest case – fast motion)Relaxation must be induced by exchange of energy at theresonance frequency νEnergy provided by fluctuating magnetic field – here we’llconsider unpaired electrons as a source2016PSUBioinorganicWorkshop,Bren30
Relaxation Mechanisms Dipole-dipole Most important Through-space interaction betweenmagnetic moments and fluctuating magneticfield Modulated by molecular tumbling Quadrupolar (I 1/2), scalar Spin rotation Chemical shift anisotropy Others 2016PSUBioinorganicWorkshop,Bren31
Dipole-dipole Relaxation The relaxation of one spin (dipole) by through-spaceinteractions with other dipoles A fluctuating field is needed, and this is generated bymolecular motion Relaxation rate depends on µ12µ22, r-6, τcτc: correlation timeτc 4πa3η/3kT2016PSUBioinorganicWorkshop,Bren32
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren33
Dipole-dipole Relaxation Depends on µ12µ22, r-6, τc An unpaired electron’s µ is 658x greater than µ for 1H. Unpaired electron has a large impact on 1H relaxationFor dipole-dipole nuclear relaxationby unpaired electron:1Heµ0 2 γΙ2 ge2 µe2 S(S 1)ττscT1,2-1 R1,2 4/364πrNote dependence on S, γI, r-6, τcBut there is more to τc 2016PSUBioinorganicWorkshop,Bren34
Correlation TimeThe correlation time τc actually contains threecontributions:1. molecular tumbling time (τr), 2. electron spinrelaxation time (τs), 3. chemical exchange (τM).The overall correlation τc time is:τc-1 τr-1 τs-1 τM-1Assuming no chemical exchange:τc-1 τr-1 τs-1In a diamagnetic system, no chemical exchange:τc-1 τr-12016PSUBioinorganicWorkshop,Bren35
Correlation TimeIn a paramagnetic molecule, especially if it is not toolarge (large means long τr), τs usually dominates τcτr ranges from 10-9 s (small protein) to 10-7 s (large for NMR)τs ranges from 10-13 s to 10-8 s; but values 10-13 to 10-10 mostfeasible for high-resolution NMR.Thus τr-1 τs-1 and τs dominates τc for metalloproteins.Example: Fe(III)cytochrome c (MW 12 kDa):τc-1 τr-1 τs-1 (10-9 s)-1 (10-13 s)-1τc 10-13 s τs2016PSUBioinorganicWorkshop,Bren36
Correlation TimeIn a paramagnetic molecule, especially if it is not toolarge (large means long τr), τs usually dominates τcNote for small molecules, especially with long τs, insteadyou may have τc τrSmall Cu(II) complex, τs (10-8 s), τr (10-12 s)In this case, τs-1 τr-1 and τc τr.2016PSUBioinorganicWorkshop,Bren37
Dipole-dipole Relaxation – e-/nucleusµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3τsτc64πrτc τs1HeFor NMR of metalloproteins,variations in τs are the mostimportant factor determining linewidths. It also can be importantfor small molecules.Long τs large R1,2 smallT1,2 large Δν (broad line)2016PSUBioinorganicWorkshop,Bren38
Electron Spin Relaxation Times τsµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3ττss4πr6MetalOx stateSC.N.Linebroadening(Hz)*Fe 31/25,610-12 - 10-13Fe 35/24,5,610-9 - 10-11Fe 22410-11Fe 225,610-12 - 10-135-20Cu 21/2any(1-5) x 10-91000-5000Mn 25/24,5,610-8Mn 324,5,610-10 - 10-11τs(s)Coord. Chem. Rev. 1996, 150, p. 50100000150-1500*for 1H 5 Å from metal39
Paramagnetic Relaxation Enhancementµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3ττss4πr6 Measure distances (r) and/or refine structures by measuringeffects of a paramagnetic probe on line widths andrelaxation times (R1,2) Need to be in regime where dipolar relaxation dominates Other effects on relaxation: Contact, applicable mostly for small molecules (rapidtumbling) with large τs (slow electron relaxation) Curie, most relevant for very large molecules with high S Effects of quadrupolar nuclei2016PSUBioinorganicWorkshop,Bren40
Contact RelaxationContact:(in absence of exchangephenomena)Dipolar:(when τs-1 τr-1)R1,2 2/3 S(S 1) (A/h)2 ττsµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3ττss4πr6 Depends on S, A2, and τs Never depends on τr (why not)? May be in effect when hyperfine coupling is strong over anumber of bonds (so not extremely small r) Will be in effect when τs/τr is relatively large May be in effect for very large A values.2016PSUBioinorganicWorkshop,Bren41
What can I do with these broad peaks? Try some cool tricks, and learn what you can! Change metal or metal oxidation state (although youmay want to study the native metal in a particularoxidation state, of course). Take advantage of the situation! Fast relaxation timesmeans you can use a short acquisition time and recycletime, and get many scans in a short period of timewhile enhancing intensity of broad peaks Increase the temperature – faster tumbling (shorter τr)and faster electron relaxation (shorter τs) (and fasterexchange if present) can help significantly. Detect nuclei other than 1H – especially 13C, 15N, or 2H.Relaxation enhancement and line widths depend on γ2.2016PSUBioinorganicWorkshop,Bren42
Finding Hidden PeaksS 1/2τs 10-9 sP. aeruginosa Cu(II) azurinHisH 2CMetC/DSJ (Hα)NCysSNHACuH 2CNO60504030δ, ppmJACS 2000, 3EHis
Finding Hidden PeaksSaturation transfer experimentAz Cu(I)* Az Cu(II)intensityAz Cu(II)* Az Cu(I)Saturate Cys 1H in diamagneticCu(I), observe change in intensity inspectrum of Cu(II)ppmJACS 2000, 37012016PSUBioinorganicWorkshop,Bren44
What can I do with these broad peaks?Why does this have a 120,000Hz line width?1. It is very close to Cu(II)2. But line width can’t beexplained by dipolarrelaxation3. Contribution from contactrelaxation because of large A4. Observation consistent withefficient spin delocalizationonto Cys residueJACS 2000, 3701S 1/2τs 10-8 sHisH 2CMetC/DSJ (Hα)NCysSNHACuH 2CNOBNHC/DE800, 850 ppm120,000 Hz(!)2016PSUBioinorganicWorkshop,Bren45EHis
What can I do with these broad peaks? Try some cool tricks, and learn what you can! Change metal or metal oxidation state (although youmay want to study the native metal in a particularoxidation state, of course). Take advantage of the situation! Fast relaxation timesmeans you can use a short acquisition time and recycletime, and get many scans in a short period of timewhile enhancing intensity of broad peaks Increase the temperature – faster tumbling (shorter τr)and faster electron relaxation (shorter τs) (and fasterexchange if present) can help significantly. Detect nuclei other than 1H – especially 13C, 15N, or 2H.Relaxation enhancement and line widths depend on γ2.2016PSUBioinorganicWorkshop,Bren46
Metal SubstitutionS 1, τs 10-12 sNi(II) azurinBiochemistry 1996, 18102016PSUBioinorganicWorkshop,Bren47
What can I do with these broad peaks? Try some cool tricks, and learn what you can! Change metal or metal oxidation state (although youmay want to study the native metal in a particularoxidation state, of course). Take advantage of the situation! Fast relaxation timesmeans you can use a short acquisition time and recycletime, and get many scans in a short period of timewhile enhancing intensity of broad peaks Increase the temperature – faster tumbling (shorter τr)and faster electron relaxation (shorter τs) (and fasterexchange if present) can help significantly. Detect nuclei other than 1H – especially 13C, 15N, or 2H.Relaxation enhancement and line widths depend on γ2.2016PSUBioinorganicWorkshop,Bren48
Finding Hidden Peaks1H-15NHSQC spectra of 1 mM Hydrogenobacter thermophilus [U-15N]ferricytochrome c552 showing the effect of a decreased INEPT delay onintensity of the peak correlating the heme axial His δHN nuclei.From Encyclopedia of Inorganic Chemistry DOI: p,Bren49
What can I do with these broad peaks? Try some cool tricks, and learn what you can! Change metal or metal oxidation state (although youmay want to study the native metal in a particularoxidation state, of course). Take advantage of the situation! Fast relaxation timesmeans you can use a short acquisition time and recycletime, and get many scans in a short period of timewhile enhancing intensity of broad peaks Increase the temperature – faster tumbling (shorter τr)and faster electron relaxation (shorter τs) (and fasterexchange if present) can help significantly. Detect nuclei other than 1H – especially 13C, 15N, or 2H.Relaxation enhancement and line widths depend on γ2.2016PSUBioinorganicWorkshop,Bren50
Effect of Temperature1H26 Cresonances of heme methylgroups of Hydrogenobacterthermophilus ferricytochrome c55211 C1 C-6 CFrom Encyclopedia of Inorganic Chemistry DOI: p,Bren51
What can I do with these broad peaks? Try some cool tricks, and learn what you can! Change metal or metal oxidation state (although youmay want to study the native metal in a particularoxidation state, of course). Take advantage of the situation! Fast relaxation timesmeans you can use a short acquisition time and recycletime, and get many scans in a short period of timewhile enhancing intensity of broad peaks Increase the temperature – faster tumbling (shorter τr)and faster electron relaxation (shorter τs) (and fasterexchange if present) can help significantly. Detect nuclei other than 1H – especially 13C, 15N, or 2H.Relaxation enhancement and line widths depend on γ2.2016PSUBioinorganicWorkshop,Bren52
Detection of 13C2-D in-phase-anti-phasespectrum correlatingbackbone 13C and 15N nuclei,with detection of 13C (CONIPAP experiment). Data wereacquired on a 1.5 mM sampleof 13C,15N labeled reducedmonomeric superoxidedismutase (see ID779-) with a14.1 T Bruker Avancespectrometer equipped with acryogenically cooledprobehead optimized for 13Cdetection at 298 KProg. Nucl. Magn. Reson. Spectrosc. 2006, 48, 25.2016PSUBioinorganicWorkshop,Bren53
Detection of 13Cµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3τs64πrProg. Nucl. Magn. Reson. Spectrosc. 2006, 48, 25.2016PSUBioinorganicWorkshop,Bren54
Detection of 13Cµ0 2 γΙ2 ge2 µe2 S(S 1)R1,2 4/3τs64πrProg. Nucl. Magn. Reson. Spectrosc. 2006, 48, 25.2016PSUBioinorganicWorkshop,Bren55
Relaxation Summary Properties of metal site have a profound effect onrelaxation and thus line widths, with τs being mostimportant A number of systems show minimal line broadening –especially LS Fe(III), 4-coordinate HS Ni(II), 5- and 6coordinate HS Co(II), 5- and 6-coordinate HS Fe(II). Also –most Ln(III), (not Gd(III)), Ru(III), and many bi- andmultinuclear sites Line broadening is diminished for low γ nuclei (by γ2 factor) Relaxation enhancement provides information onmolecular and electronic structure We have ways to deal with it!2016PSUBioinorganicWorkshop,Bren56
Outline Resources Examples of effects on spectra What we can learn (why bother?) NMR fundamentals (review) Relaxation mechanisms in NMR Effects of unpaired electrons on relaxation Effects of unpaired electrons on chemical shifts2016PSUBioinorganicWorkshop,Bren57
Paramagnetic Chemical Shifts Values are not necessarily related to amount of linebroadening Can be higher or lower than diamagnetic (positive ornegative; to high frequency or low frequency) Are caused by two primary mechanisms: contact(through-bond) and dipolar (through-space) Unlike with relaxation, both contact and dipolar oftenplay a role Contain information on electronic structure, molecularstructure, spin delocalization, magnetic anisotropy2016PSUBioinorganicWorkshop,Bren58
Paramagnetic Chemical Shiftsδobs δpara δdiaδpara δcon δpcδdiais shift in isostructural diamagnetic moleculeδconis contact shift – through-bond electronnucleus interactionδpcis pseudocontact (also called dipolar) shift –through-space electron-nucleus interaction2016PSUBioinorganicWorkshop,Bren59
Contact Shiftδcon [(2πA/h)ge βe S (S 1)]/[3 kBT γI]Note dependence on: Hyperfine coupling constant (A) Spin (S (S 1)) Temperature (1/T) Value γI in denominator cancels with part of A2016PSUBioinorganicWorkshop,Bren60
Contact Shiftδcon [(2πA/
ranges from 10-9 s (small protein) to 10-7 s (large for NMR) τ s ranges from 10-13 s to 10-8 s; but values 10-13 to 10-10 most
paramagnetic salt will increase the concentration of the paramagnetic species and, thus, the magnetic susceptibility of the solution. This evaporation of solvent complicates the use and storage of paramagnetic solutions and, in some applications, requires calibration or the use of internal standards. (ii) Aqueous solutions of paramagnetic salts .
Aug 01, 2018 · 1H NMR, 19F NMR, 31P NMR experiments -about 3 5 mg 13C NMR short run experiment (0.5 -1 hr) about 20 50 mg; long run experiment about 5 10 mg 0.6-0.7 ml of NMR solvent is appropriate for the right solvent level in NMR tube. Unsuitable solvent level can lea
1985 –First Protein Structure solved by NMR 2009 –First 1 Gigahertz NMR Spectrometer (23.5 T) 2019 –High Temperature Superconducting Magnets 1.1 GHz NMR, St. Jude, Memphis TN 1.2 GHz NMR, Florence, Italy 1938 –NMR of LiCl molecular beams. Rabi (Columbia University) 1946 –NMR
NMR SOLVENTS Deuterated Solvents for NMR NMR Solvents NMR Reference Standards NMR Tubes. Cambridge Isotope Laboratories, Inc. www.isotope.com s tel: 978-749-8000 800-322-1174 (USA) fax: 978-749-2768 cilsales@isotope.com TABLE OF CONTENTS
due to the interaction of paramagnetic sites with the hydrogen nuclei in the pore space (Korringa et al., 1962) and increases when paramagnetic minerals are present (Saidian and Prasad, 2015a ). Foley et al. (1996) showed that the paramagnetic ion content, magnetic susceptibility, and the surface relaxivity
5 nuclear magnetic resonance (nmr) spectroscopy 33 5.1 the physics of nuclear spins and nmr instruments 33 5.2 continuous wave (cw) nmr spectroscopy 37 5.3 fourier-transform (ft) nmr spectroscopy 39 5.4 chemical shift in 1h nmr spectroscopy 40 5.5 spin-spin coupling in 1h nmr spectroscopy 50
14.1 An Introduction to NMR Spectroscopy A. The Basics of Nuclear Magnetic Resonance (NMR) Spectroscopy nuclei with odd atomic number have a S ½ with two spin states ( 1/2 and -1/2) 1H NMR (proton NMR): determines number and type of H atoms 13C NMR (proton
5 Experiment 8.16 - Quantitative 13C NMR Spectroscopy with Inverse Gated 1H-Decoupling 68 Experiment 8.17 - NMR Using Liquid-Crystal Solvents 68 CHAPTER 9 - HETERONUCLEAR NMR SPECROSCOPY 70 SUMMARY 70 Experiment 9.1 - 1H-Decoupled 15N NMR Spectra with DEPT 70 Experiment 9.2 - 1H-Coupled 15N NMR Spectra