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![Predicting Chemical Shift Assignments
Predicting Chemical Shift Assignments
Predict the chemical shifts of:
C
|
C – C – C – C – C – C
2
Chemical shift is determined by sum of carbon types.
From Table 3.2 in handout:
=Bs + D
∑ mAsm +SN3 +sN4 - empirical formula
S – number of directly bonded carbons
Dm – number of directly bonded carbons having M attached carbons
Np – number of carbons P bonds away
2 = B2 + [1xA23+ 1xA21 ] + [1x2] + [1x2]
2 = 15.34 + [1X16.70 +1x0] + [1x-2.69] +[1x0.25] = 29.60 ppm](https://image.slidesharecdn.com/nmr-824-10-03-240806061147-8d69348e/85/NMR-824-10-03-pptMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM-36-320.jpg)
![Predicting Chemical Shift Assignments
Predicting Chemical Shift Assignments
Predict the chemical shifts of:
C
|
C – C – C – C – C – C
2
Chemical shift is determined by sum of carbon types.
From Table 3.2 in handout:
=Bs + D
∑ mAsm +SN3 +sN4 - empirical formula
S – number of directly bonded carbons
Dm – number of directly bonded carbons having M attached carbons
Np – number of carbons P bonds away
2 = B2 + [1xA23+ 1xA21 ] + [1x2] + [1x2]
2 = 15.34 + [1X16.70 +1x0] + [1x-2.69] +[1x0.25] = 29.60 ppm](https://image.slidesharecdn.com/nmr-824-10-03-240806061147-8d69348e/75/NMR-824-10-03-pptMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM-36-2048.jpg)
NMR-824-10-03.pptMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM
- 1. Nuclear Magnetic Resonance(NMR) Nuclear Magnetic Resonance (NMR) Probe the Composition, Structure, Dynamics and Probe the Composition, Structure, Dynamics and Function of the Complete Range of Chemical Function of the Complete Range of Chemical Entities: from small organic molecules to large Entities: from small organic molecules to large molecular weight polymers and proteins. molecular weight polymers and proteins. One of the One of the MOST MOST Routinely used Analytical Routinely used Analytical Techniques Techniques
- 2. • Structural (chemical)elucidation • Natural product chemistry. • Synthetic organic chemistry. Analytical tool of choice of synthetic chemists. • Study of dynamic processes • Reaction kinetics. • Study of equilibrium (chemical or structural). • Structural (three-dimensional) studies • Proteins. • DNA. Protein/DNA complexes • Polysaccharides • Drug design • Structure Activity Relationships by NMR • Medicine - MRI Common NMR Utility Common NMR Utility
- 3. 2-phenyl-1,3-dioxep-5-ene 2-phenyl-1,3-dioxep-5-ene 13 13 C NMR spectra CNMR spectra 1 1 H NMR spectra H NMR spectra NMR NMR: “fingerprint” of the compound’s chemical structure : “fingerprint” of the compound’s chemical structure
- 4. Protein Structures fromNMR Protein Structures from NMR 2D NOESY Spectra at 900 MHz 2D NOESY Spectra at 900 MHz Lysozyme Ribbon Diagram Lysozyme Ribbon Diagram
- 5. 1937 Rabi predictsand observes nuclear magnetic resonance 1946 Bloch, Purcell first nuclear magnetic resonance of bulk sample 1953 Overhauser NOE (nuclear Overhauser effect) 1966 Ernst, Anderson Fourier transform NMR 1975 Jeener, Ernst 2D NMR 1985 Wüthrich first solution structure of a small protein (BPTI) from NOE derived distance restraints 1987 3D NMR + 13C, 15N isotope labeling of recombinant proteins (resolution) 1990 pulsed field gradients (artifact suppression) 1996/7 new long range structural parameters: - residual dipolar couplings from partial alignment in liquid crystalline media - projection angle restraints from cross-correlated relaxation TROSY (molecular weight > 100 kDa) Nobel prizes 1944 Physics Rabi (Columbia) 1952 Physics Bloch (Stanford), Purcell (Harvard) 1991 Chemistry Ernst (ETH) 2002 Chemistry Wüthrich (ETH) 2003 Medicine Lauterbur (University of Illinois in Urbana ), Mansfield (University of Nottingham) NMR History NMR History
- 6. Some Suggested NMRReferences Some Suggested NMR References “Basic One- and Two-Dimensional NMR Spectroscopy” Horst Friebolin “Modern NMR Techniques for Chemistry Research” Andrew E. Derome “NMR and Chemistry- an introduction to the fourier transform-multinuclear era” J. W. Akitt “Nuclear Magnetic Resonance Spectroscopy” R. K Harris “Protein NMR Spectroscopy: Principals and Practice” John Cavanagh, Arthur Palmer, Nicholas J. Skelton, Wayne Fairbrother “NMR of Proteins and Nucleic Acids” Kurt Wuthrich “Tables of Spectral Data for Structure Determination of Organic Compounds” Pretsch, Clerc, Seibl and Simon “Spectrometric Identification of Organic Compounds” Silverstein, Bassler and Morrill
- 7. The Basics ofNMR Hypertext based NMR course http://www.cis.rit.edu/htbooks/nmr/nmr-main.htm Educational NMR Software All kinds of NMR software http://www.york.ac.uk/depts/chem/services/nmr/edusoft.html NMR Knowledge Base A lot of useful NMR links http://www.spectroscopynow.com/ NMR Information Server News, Links, Conferences, Jobs http://www.spincore.com/nmrinfo/ Technical Tidbits Useful source for the art of shimming http://www.acornnmr.com/nmr_topics.htm BMRB (BioMagResBank) Database of NMR resonance assignments http://www.bmrb.wisc.edu/ Some NMR Web Sites Some NMR Web Sites
- 8. Basic NMR Spectrometer BasicNMR Spectrometer
- 9. Information in aNMR Spectra Information in a NMR Spectra 1) Energy E = h h is Planck constant is NMR resonance frequency 10-10 10-8 10-6 10-4 10-2 100 102 wavelength (cm) -rays x-rays UV VIS IR -wave radio Observable Observable Name Name Quantitative Quantitative Information Information Peak position Chemical shifts () (ppm) = obs –ref/ref (Hz) chemical (electronic) environment of nucleus Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei (intensity ratios) (torsion angles) Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T1 dependent Peak Shape Line width = 1/T2 molecular motion peak half-height chemical exchange uncertainty principal
- 10. Source of theNMR Signal Source of the NMR Signal From Quantum Theroy: Nuclear Spin (Think Electron Spin) NMR “active” Nuclear Spin (I) = ½: 1 H, 13 C, 15 N, 19 F, 31 P biological and chemical relevance Odd atomic mass NMR “inactive” Nuclear Spin (I) = 0: 12 C, 16 O Even atomic mass & number Quadrupole Nuclei Nuclear Spin (I) > ½: 14 N, 2 H, 10 B Even atomic mass & odd number
- 11. Zeeman Effect andNuclear Spin Quantum Number Zeeman Effect and Nuclear Spin Quantum Number I: hyperfine interaction associate with magnetization due to nuclear spin quantum transitions Zeeman effect: splitting of energy levels in magnetic field 2I +1 possible energy levels For I =1/2: m= -1/2 & 1/2 E= B magnetogyric ratio (radians/Tesla) - unique value per nucleus 1 H: 26.7519 x 107 rad T-1 s-1 Bo applied magnetic field - units:Tesla (Kg s-2 A-1 ) NMR frequency:Bo m: magnetic quantum number
- 12. NMR Spectra Terminology NMRSpectra Terminology Increasing field (Bo) Increasing frequency () Increasing Increasing energy (E, consistent with UV/IR) 1 H 13 C 2 H 600 MHz 150 MHz 92 MHz TMS CHCl3 7.27 0 ppm increasing decreasing low field high field down field up field high frequency () low frequency de-shielding high shielding Paramagnetic diamagnetic
- 13. Another Viewpoint: MagneticMoment (Nuclear Spin) Another Viewpoint: Magnetic Moment (Nuclear Spin) magnetic moment()Ih It is a vector quantity that gives the direction and magnitude (or strength) of the ‘nuclear magnet’ By convention: spin +1/2 => low energy state spin -1/2 => Analogous to current moving in a loop which induces a magnetic field (right- hand rule) quantized by Planck’s constant (h)
- 14. Bo = h/ 4 Magnetic alignment Magnetic alignment In the absence of external field, each nuclei is energetically degenerate Add a strong external field (Bo). and the nuclear magnetic moment: aligns with (low energy) against (high-energy)
- 15. NMR Sensitivity NMR Sensitivity Bo= 0 Bo > 0 E = h N / N = e E / kT Boltzmman distribution: The applied magnetic field causes an energy difference between aligned() and unaligned() nuclei The population (N) difference can be determined from The E for 1 H at 400 MHz (Bo = 9.5 T) is 3.8 x 10-5 Kcal / mol Very Small ! Very Small ! ~64 excess spins per ~64 excess spins per million in lower state million in lower state Low energy gap
- 16. NMR Sensitivity NMR Sensitivity EhBo /2 NMRsignal depends on: 1) Number of Nuclei (N) (limited to field homogeneity and filling factor) 2) Gyromagnetic ratio (in practice 3 ) 3) Inversely to temperature (T) 4) External magnetic field (Bo 2/3 , in practice, homogeneity) 5) B1 2 exciting field strength N / N = e E / kT Increase energy gap -> Increase population difference -> Increase NMR signal E ≡ Bo ≡ - Intrinsic property of nucleus can not be changed. C)3 for 13 C is 64xN)3 for 15 N is 1000x 1 H is ~ 64x as sensitive as 13 C and 1000x as sensitive as 15 N ! Consider that the natural abundance of 13 C is 1.1% and 15 N is 0.37% relative sensitivity increases to ~6,400x and ~2.7x105 x !! signal (s) 4 4 B Bo o 2 2 NB NB1 1g( g( )/T )/T
- 17. NMR Sensitivity NMR Sensitivity Increasein Magnet Strength is a Major Means to Increase Sensitivity But at a significant cost! ~$800,000 ~$2,00,000 ~$4,500,000
- 18. E = h = Bo / 2 E = h Bo / 2 NMR Frequency Range ( NMR Frequency Range (expensive radios expensive radios) ) For 1 H in normal magnets (2.35 - 18.6 T), this frequency is in the 100-800 MHz range. 10-10 10-8 10-6 10-4 10-2 100 102 wavelength (cm) -rays x-rays UV VIS IR -wave radio
- 19. = 2 o = B (radians) Precession or Larmor frequency: l angular momentum (l) Simply, the nuclei spins about its axis creating a magnetic moment Classical View of NMR ( Classical View of NMR (compared to Quantum view compared to Quantum view) ) Maxwell: Magnetic field Moving charge ≡ Bo o Apply a large external field (Bo) and will precess about Bo at its Larmor () frequency. Important: This is the same frequency obtained from the energy Important: This is the same frequency obtained from the energy transition between quantum states transition between quantum states
- 20. Bulk magnetization Bulk magnetization (M (Mo o) ) Mo y x z x y z BoBo Now consider a real sample containing numerous nuclear spins: Mo (N - N) xiyjzk Since is precessing in the xy-plane, Mo = ∑ zk – zk is quantized ( or ), Mo has a continuous number of states, bulk property.
- 21. An NMR Experiment AnNMR Experiment Mo y x z x y z Bo Bo We have a net magnetization precessing about Bo at a frequency of o with a net population difference between aligned and unaligned spins. Now What? Perturbed the spin population or perform spin gymnastics Basic principal of NMR experiments
- 22. Mo z x i B1 Transmitter coil (y) y Bo AnNMR Experiment An NMR Experiment To perturbed the spin population need the system to absorb energy. Two ways to look at the situation: (1) quantum – absorb energy equal to difference in spin states (2) classical - perturb Mo from an excited field B1
- 23. B1 off… (or off-resonance) Mo z x B1 z x Mxy yy 1 1 Right-hand rule resonant condition: frequency (1) of B1 matches Larmor frequency (o) energy is absorbed and population of and states are perturbed. An NMR Experiment An NMR Experiment And/Or: And/Or: Mo now precesses about B1 (similar to Bo) for as long as the B1 field is applied. Again, keep in mind that individual spins flipped up or down (a single quanta), but Mo can have a continuous variation.
- 24. An NMR Experiment AnNMR Experiment What Happens Next? The B1 field is turned off and Mxy continues to precess about Bo at frequency o. z x Mxy Receiver coil (x) y NMR signal o The oscillation of Mxy generates a fluctuating magnetic field which can be used to generate a current in a receiver coil to detect the NMR signal. FID – Free Induction Decay
- 25. NMR Signal Detection- FID NMR Signal Detection - FID Mxy is precessing about z-axis in the x-y plane Time (s) y y y The FID reflects the change in the magnitude of Mxy as the signal is changing relative to the receiver along the y-axis Again, it is precessing at its Larmor Frequency (o).
- 26. NMR Signal Detection- Fourier NMR Signal Detection - Fourier Transform Transform So, the NMR signal is collected in the Time - domain But, we prefer the frequency domain. Fourier Transform is a mathematical procedure that transforms time domain data into frequency domain
- 27. z x Mxy y Bo z x Mxy y o Laboratory Frame RotatingFrame Laboratory Frame vs. Rotating Frame Laboratory Frame vs. Rotating Frame To simplify analysis we convert to the rotating frame. Simply, our axis now rotates at the Larmor Freguency (o). In the absent of any other factors, Mxy will stay on the x-axis All further analysis will use the rotating frame.
- 28. Chemical Chemical Shift Shift Up to thispoint, we have been treating nuclei in general terms. Simply comparing 1 H, 13 C, 15 N etc. If all 1 H resonate at 500MHz at a field strength of 11.7T, NMR would not be very interesting Beff = Bo - Bloc --- Beff = Bo( 1 - ) is the magnetic shielding of the nucleus The chemical environment for each nuclei results in a unique local magnetic field (Bloc) for each nuclei:
- 29. Chemical Chemical Shift Shift Again, consider Maxwell’stheorem that an electric current in a loop generates a magnetic field. Effectively, the electron distribution in the chemical will cause distinct local magnetic fields that will either add to or subtract from Bo HO-CH2-CH3 Aromaticity, electronegativity and similar factors will contribute to chemical shift differences Beff = Bo( 1 - ) de-shielding high shielding Shielding – local field opposes Bo
- 30. The NMR scale( The NMR scale ( , ppm) , ppm) - ref = ppm (parts per million) ref Instead use a relative scale, and refer all signals () in the spectrum to the signal of a particular compound (ref). Bo >> Bloc -- MHz compared to Hz Comparing small changes in the context of a large number is cumbersome Tetramethyl silane (TMS) is a common reference chemical H3C Si CH3 CH3 CH3 IMPORTANT: absolute frequency is field dependent ( = Bo / 2)
- 31. The NMR scale( The NMR scale ( , ppm) , ppm) Chemical shift is a relative scale so it is independent of Bo. Same chemical shift at 100 MHz vs. 900 MHz magnet IMPORTANT: absolute frequency is field dependent ( = Bo / 2) At higher magnetic fields an NMR spectra will exhibit the same chemical shifts but with higher resolution because of the higher frequency range.
- 32. Chemical Shift Trends ChemicalShift Trends • For protons, ~ 15 ppm: 0 TMS ppm 2 10 7 5 15 Aliphatic Alcohols, protons to ketones Olefins Aromatics Amides Acids Aldehydes
- 33. Chemical Shift Trends ChemicalShift Trends • For carbon, ~ 220 ppm: ppm 50 150 100 80 210 Aliphatic CH3, CH2, CH Carbons adjacent to alcohols, ketones Olefins Aromatics, conjugated alkenes C=O of Acids, aldehydes, esters 0 TMS C=O in ketones
- 34. Predicting Chemical ShiftAssignments Predicting Chemical Shift Assignments Numerous Experimental NMR Data has been compiled and general trends identified • Examples in Handout • See also: “Tables of Spectral Data for Structure Determination of Organic Compounds” Pretsch, Clerc, Seibl and Simon “Spectrometric Identification of Organic Compounds” Silverstein, Bassler and Morrill • Spectral Databases: Aldrich/ACD Library of FT NMR Spectra Sadtler/Spectroscopy (UV/Vis, IR, MS, GC and NMR)
- 35. Predicting Chemical ShiftAssignments Predicting Chemical Shift Assignments Predict the chemical shifts of: Benzene Shift NO2 effect NH2 effect Total Change sign since table lists as downfield shift a 7.27 0.95 -0.75 7.47 ppm d 7.27 0.33 -0.75 6.85 ppm c 7.27 0.17 -0.24 7.20 ppm b 7.27 0.95 -0.63 7.59 ppm From table 3-6-1 in handout: Substituent Shift relative to benzene (ppm) ortho meta para NH2 NO2 A B C D
- 36. Predicting Chemical ShiftAssignments Predicting Chemical Shift Assignments Predict the chemical shifts of: C | C – C – C – C – C – C 2 Chemical shift is determined by sum of carbon types. From Table 3.2 in handout: =Bs + D ∑ mAsm +SN3 +sN4 - empirical formula S – number of directly bonded carbons Dm – number of directly bonded carbons having M attached carbons Np – number of carbons P bonds away 2 = B2 + [1xA23+ 1xA21 ] + [1x2] + [1x2] 2 = 15.34 + [1X16.70 +1x0] + [1x-2.69] +[1x0.25] = 29.60 ppm
- 37. Coupling Constants Coupling Constants Energylevel of a nuclei are affected by covalently-bonded neighbors spin-states 13 C 1 H 1 H 1 H one-bond three-bond I S S S I I J (Hz) Spin-States of covalently-bonded nuclei want to be aligned. The magnitude of the separation is called coupling constant (J) and has units of Hz. +J/4 -J/4 +J/4
- 38. Coupling Constants Coupling Constants IMPORTANT:Coupling constant pattern allow for the identification of bonded nuclei. Multiplets consist of 2nI + 1 lines I is the nuclear spin quantum number (usually 1/2) and n is the number of neighboring spins. The ratios between the signal intensities within multiplets are governed by the numbers of Pascals triangle. Configuration Peak Ratios A 1 AX 1:1 AX2 1:2:1 AX3 1:3:3:1 AX4 1:4:6:4:1
- 39.
- 40. NMR Relaxation NMR Relaxation Afterthe B1 field (pulse) is removed the system needs to “relax” back to equilibrium Mz = M0(1-exp(-t/T1)) T1 is the spin-lattice (or longitudinal) relaxation time constant. Think of T1 as bulk energy/magnetization exchange with the “solvent”. Please Note: General practice is to wait 5xT1 for the system to have fully relaxed.
- 41. NMR Relaxation NMR Relaxation Mx= My = M0 exp(-t/T2) T2 is the spin-spin (or transverse) relaxation time constant. In general: T1 T2 Think of T2 as the “randomization” of spins in the x,y-plane Related to line-shape Please Note: Line shape is also affected by the magnetic fields homogeneity (derived from Hisenberg uncertainty principal)
- 42. NMR Time Scale NMRTime Scale Time Scale Chem. Shift ( Coupling Const. (J) T2 relaxation Slow k << A- B k << JA- JB k << 1/ T2,A- 1/ T2,B Intermediate k = A - B k = JA- JB k = 1/ T2,A- 1/ T2,B Fast k >> A - B k >> JA- JB k >> 1/ T2,A- 1/ T2,B Range (Sec-1 ) 0 – 1000 0 –12 1 - 20 NMR time-scale refers to the chemical shift timescale.
- 43. k = (he-ho) Exchange Rates from NMR Data Exchange Rates from NMR Data k = (o 2 - e 2 )1/2 /21/2 k = o / 21/2 k = o 2 /2(he - ho) h – peak-width at half-height – peak frequency e – with exchange o – no exchange f – mole fraction – chemical shift obs = f11 + f22 f1 +f2 =1
- 44. Continuous Wave (CW)vs. Pulse/Fourier Transform Continuous Wave (CW) vs. Pulse/Fourier Transform NMR Sensitivity Issue A frequency sweep (CW) to identify resonance is very slow (1-10 min.) Step through each individual frequency. Pulsed/FT collect all frequencies at once in time domain, fast (N x 1-10 sec) Increase signal-to-noise (S/N) by collecting multiple copies of FID and averaging signal. S/N number of scans
- 45. * = tp NMR Pulse NMRPulse FT A radiofrequency pulse is a combination of a wave (cosine) of frequency o and a step function Pulse length (time, tp) The fourier transform indicates the pulse covers a range of frequencies Hisenberg Uncertainty principal again: . t ~ 1/2 Shorter pulse length – larger frequency envelope Longer pulse length – selective/smaller frequency envelope Sweep Width f ~ 1/t
- 46. NMR Pulse NMR Pulse z x Mxy y z x y Mo B1 t tp t= * tp * B1 NMR pulse length or Tip angle (tp) The length of time the B1 field is on => torque on bulk magnetization (B1) A measured quantity – instrument dependent.
- 47. NMR Pulse NMR Pulse z x Mxy y z x y Mo / 2 Some useful common pulses 90o Maximizes signal in x,y-plane where NMR signal detected z x -Mo y z x y Mo 180o 90o pulse 180o pulse Inverts the spin-population. No NMR signal detected Can generate just about any pulse width desired.
- 48. NMR Data Acquisition NMRData Acquisition Collect Digital Data ADC – analog to digital converter 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 t1 sec SR = 1 / (2 * SW) The Nyquist Theorem says that we have to sample at least twice as fast as the fastest (higher frequency) signal. Sample Rate - Correct rate, correct frequency -½ correct rate, ½ correct frequency Folded peaks! Wrong phase! SR – sampling rate
- 49. carrier 234 233 232231 230 229 228 227 226 225 224 223 ppm Quadrature detection Quadrature detection Frequency of B1 (carrier) is set to center of the spectra. • small pulse length to excite entire spectrum • minimizes folded noise How to differentiate between peaks upfield and downfield from carrier? carrier If carrier is at edge of spectra, then peaks are all positive or negative relative to carrier. But excite twice as much including noise
- 50. (B1) B F B F PH =0 PH = 90 PH = 0 PH = 90 F F S S Quadrature Quadrature detection detection Use two detectors 90o out of phase. Phase of Peaks are different.
- 51. Receiver Gain Receiver Gain TheNMR-signal received from the resonant circuit in the probehead needs to be amplified to a certain level before it can be handled by the computer. The detected NMR-signals vary over a great range due to differences in the inherent sensitivity of the nucleus and the concentration of the sample.
- 52. Data Processing –Window Functions Data Processing – Window Functions 0 0.10 0.20 0.30 0.40 0.50 t1 sec Good stuff Mostly noise The NMR signal Mxy is decaying by T2 as the FID is collected. Emphasize the signal and decrease the noise by applying a mathematical function to the FID F(t) = 1 * e - ( LB * t ) – line broadening Effectively adds LB in Hz to peak Line-widths Sensitivity Resolution
- 53. 0 0.10 0.200.30 0.40 0.50 t1 sec 0 0.10 0.20 0.30 0.40 0.50 t1 sec 0 0.10 0.20 0.30 0.40 0.50 t1 sec FT FT LB = -1.0 Hz LB = 5.0 Hz Can either increase S/N or Resolution Not Both! Increase Sensitivity Increase Resolution
- 54. NMR Data size NMRData size digital resolution (DR) as the number of Hz per point in the FID for a given spectral width. DR = SW / SI SW - spectral width (Hz) SI - data size (points) Remember: SR = 1 / (2 * SW) Also: SW = 1/2DW Dwell time DW TD A Number of Interdependent Values (calculated automatically) AQ = TD * DW= TD/2SWH Total Data Acquisition Time: Should be long enough to allow complete delay of FID Higher Digital Resolution requires longer acquisition times
- 55. 231.40 231.39 231.38231.37 231.36 231.35 231.34 231.33 231.32 231.31 231.30 231.29 231.28 231 f1 231.42 231.40 231.38 231.36 231.34 231.32 231.30 231.28 231.26 231.24 231.22 231.20 f1 ppm 0 0.20 0.40 0.60 0.80 1.00 1.2 1.4 1.6 1.8 2.0 2.2 t1 sec 8K data 8K zero-fill 8K FID 16K FID Zero Filling Zero Filling Improve digital resolution by adding zero data points at end of FID No zero-filling 8K zero-filling
- 56. MultiDimensional NMR MultiDimensional NMR 1DNMR Up to now, we have been talking about the basic or 1D NMR experiments More complex NMR experiments will use multiple “time-dimensions” to obtain data and simplify the analysis. In a 1D NMR experiment the FID acquisition time is the time domain (t1) Multidimensional NMR experiments may also observe multiple nuclei (13 C,15 N) in addition to 1 H. But usually detect 1 H.
- 57. 2D COSY (CorrelatedSpectroscopY): Correlate J-coupled NMR resonances MultiDimensional NMR MultiDimensional NMR A series of FIDs are collected where the delay between 90o pulses (t1) is incremented. t2 is the normal acquisition time.
- 58. MultiDimensional NMR MultiDimensional NMR Duringthe t1 time period, peak intensities are modulated at a frequency corresponding to the chemical shift of its coupled partner. Solid line connects diagonal peaks (normal 1D spectra). The off-diagonal or cross-peaks indicate a correlation between the two diagonal peaks – J-coupled.
- 59. Karplus Equation –Coupling Constants Karplus Equation – Coupling Constants Relates coupling constant to Torsional angle. Used to solve Structures! J = const. + 10Cos
- 60. Karplus Equation –Coupling Constants Karplus Equation – Coupling Constants For Protein Backbones
- 61. Nuclear Overhauser Effect(NOE) Nuclear Overhauser Effect (NOE) Interaction between nuclear spins mediated through empty space (5Ă) (like ordinary bar magnets). Important: Effect is Time-Averaged! Give rise to dipolar relaxation (T1 and T2) and specially to cross-relaxation and the NOE effect. the 13 C signals are enhanced by a factor 1 + = 1 + 1/2 . (1 H)/(13 C) ~ max. of 2 Perturb 1 H spin population affects 13 C spin population NOE effect
- 62. DEPT Experiment DEPT Experiment:Distortionless Enhancement by Polarization Transfer : Distortionless Enhancement by Polarization Transfer 13 C spectra is perturbed based On the number of attached 1 H Takes advantage of different patterns of polarization transfer 1 H-13 C NOE
- 63. 2D NOESY (NuclearOverhauser Effect) 2D NOESY (Nuclear Overhauser Effect) Diagonal peaks are correlated by through-space Dipole-dipole interaction. NOE is a relaxation factor that builds-up during The “mixing-time (m) The relative magnitude of the cross-peak is Related to the distance (1/r6 ) between the Protons ( 5Ă). ≥ Basis for solving a Structure!
- 64. Protein NMR Protein NMR Numberof atoms in a protein makes NMR spectra complex Resonance overlap Isotope label protein with 13 C and 15 N and spread spectra out in 3D and 4D
- 65. Protein NMR Protein NMR Howdo you assign a protein NMR spectra? A collection of “COSY”-like experiments that sequentially walk down the proteins’ backbone 3D-NMR experiments that Require 13 C and 15 N labeled Protein sample Detect couplings to NH Detect couplings to NH
- 66. Protein NMR Protein NMR Assignmentstrategy We know the primary sequence of the protein. Connect the overlapping correlation between NMR experiments
- 68. Protein NMR Protein NMR Molecular-weightProblem Higher molecular-weight –> more atoms –> more NMR resonance overlap More dramatic: NMR spectra deteriorate with increasing molecular-weight. MW increases -> correlation time increases -> T2 decreases -> line-width increases NMR lines broaden to the point of not being detected! With broad lines, correlations (J, NOE) become less-efficient
- 69. Protein NMR Protein NMR Howto Solve the Molecular-weight Problem? 1) Deuterium label the protein. • replace 1 H with 2 H and remove efficient relaxation paths • NMR resonances sharpen • problem: no hydrogens -> no NOEs -> no structure • actually get exchangeable (NH –NH) noes can augment with specific 1 H labeling 2) TROSY • line-width is field dependent