1. Nuclear Magnetic Resonance (NMR) Introduction : Nuclear Magnetic Resonance (NMR) measures the absorption of electromagnetic radiation in the radio-frequency region (~4-900 MHz) - nuclei (instead of outer electrons) are involved in absorption process - sample needs to be placed in magnetic field to cause different energy states NMR was first experimentally observed by Bloch and Purcell in 1946 (received Nobel Prize in 1952) and quickly became commercially available and widely used. Probe the Composition, Structure, Dynamics and Function of the Complete Range of Chemical Entities: from small organic molecules to large molecular weight polymers and proteins. NMR is routinely and widely used as the preferred technique to rapidly elucidate the chemical structure of most organic compounds. One of the MOST Routinely used Analytical Techniques

2. Typical Applications of NMR: 1.) Structural (chemical) elucidation ‚ Natural product chemistry ‚ Synthetic organic chemistry - analytical tool of choice of synthetic chemists - used in conjunction with MS and IR 2.) Study of dynamic processes ‚ reaction kinetics ‚ study of equilibrium (chemical or structural) 3.) Structural (three-dimensional) studies ‚ Proteins, Protein-ligand complexes ‚ DNA, RNA, Protein/DNA complexes ‚ Polysaccharides 4.) Metabolomics 5.) Drug Design ‚ Structure Activity Relationships by NMR 6.) Medicine -MRI MRI images of the Human Brain NMR Structure of MMP-13 complexed to a ligand Taxol (natural product)

3. 1937 Rabi predicts and 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 1984Nicholson NMR metabolomics 1985 Wüthrich first solution structure of a small protein (BPTI) from NOE derived distance restraints 1987 3D NMR + 13 C, 15 N isotope labeling of recombinant proteins (resolution) 1990 pulsed field gradients (artifact suppression) 1996/7 residual dipolar couplings (RDC) from partial alignment in liquid crystalline media TROSY (molecular weight > 100 kDa) 2000sDynamic nuclear polarisation (DNP) to enhance NMR sensitivity 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

4. First NMR Spectra on Water Bloch, F.; Hansen, W. W.; Packard, M. The nuclear induction experiment. Physical Review (1946), 70 474-85. 1 H NMR spectra of water

5. NMR History First Observation of the Chemical Shift 1 H NMR spectra ethanol Modern ethanol spectra Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. 19: p. 507.

6. Bloch Equations Net Magnetization (M) placed in a magnetic Field (B) will precess: and relax (R) and relax individual components:

7. Course Goals We will NOT Cover a Detailed Analysis of NMR Theory We will NOT Derive the Bloch Equations We will NOT Discuss, in detail, a Quantum Mechanical Description of NMR We will NOT use Product Operator Formulism to Describe NMR Experiments If Interested, Dr. Harbison will Cover These Topics in a Separate Special Topics Course We Will Discuss Practical Aspects of Using an NMR We Will Take a Conceptual Approach to Understanding NMR A Focus of the Course will be the Application of NMR to Solving the Structure of Organic Molecules We Will Discuss the Basic Operations of an NMR Spectrometer. We Will Discuss the Proper Approaches to the Set-up, Process and Analysis of NMR Experiments We Will Use NMR to Solve the Structure of Unknowns The Goal is to Not Treat an NMR Spectrometer as a Black Box, but to be a Competent Experimentalist

8. Course Goals N-(2-Fluoroethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)methyl)-1H-pyrrolo[3,2- b]pyridine-3-carboxamide (9) MS (ES + ) m/z: 344. 1 H NMR (300 MHz, DMSO-d 6 ): δ 2.24 (s, 3H), 3.67 (d, J = 5.46 Hz, 1H), 3.76 (d, J = 5.46 Hz, 1H), 3.93 (s, 3H), 4.50 (t, J = 4.99 Hz, 1H), 4.66 (t, J = 4.99 Hz, 1H), 5.69 (s, 2H), 7.26 (dd, J = 8.29, 4.71 Hz, 1H), 7.94 (d, J = 8.52 Hz, 1H), 8.28 (s, 1H), 8.41 (s, 1H), 8.49 (d, J = 4.57 Hz, 1H), 8.95 (s, J = 5.89, 5.89 Hz, 1H). HRMS (M + H) calcd for C 17 H 18 FN 5 O 2, 344.1517; found, 344.15242. To Be Able to Go From This: To This: And Understand How to Read and Interpret the NMR Spectral Data

9. Course Goals To Be Able to Go From This: To This:

10. 2-phenyl-1,3-dioxep-5-ene 13 C NMR spectra 1 H NMR spectra Each NMR Observable Nuclei Yields a Peak in the Spectra “fingerprint” of the structure

11. Information in a NMR Spectra Observable Name QuantitativeInformation 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 curveT 1 dependent Peak Shape Line width  = 1/  T 2 molecular motion peak half-height chemical exchange uncertainty principal uncertainty in energy

12. A Basic Concept in ElectroMagnetic Theory Magnets Attract and Align

13. A Basic Concept in ElectroMagnetic Theory A Direct Application to NMR A moving perpendicular external magnetic field will induce an electric current in a closed loop An electric current in a closed loop will create a perpendicular magnetic field

14. For a single loop of wire, the magnetic field, B through the center of the loop is:  o – permeability of free space (4  x 10 -7 T · m / A) R – radius of the wire loop I – current A Basic Concept in ElectroMagnetic Theory

15. Faraday’s Law of Induction If the magnetic flux (  B ) through an area bounded by a closed conducting loop changes with time, a current and an emf are produced in the loop. This process is called induction. The induced emf is: A Basic Concept in ElectroMagnetic Theory Simple AC generator

16. Lenz’s Law An induced current has a direction such that the magnetic field of the current opposes the change in the magnetic flux that produces the current. The induced emf has the same direction as the induced current A Basic Concept in ElectroMagnetic Theory Direction of current follows motion of magnet

17. Quantum Description Nuclear Spin (think electron spin) Nucleus rotates about its axis (spin) Nuclei with spin have angular momentum (p) or spin 1) total magnitude 1) quantized, spin quantum number I 2) 2 I + 1 states: I, I-1, I-2, …, -I I=1/2:-1/2, 1/2 3) identical energies in absence of external magnetic field l Theory of NMR

18. NMR Periodic Table NMR “active” Nuclear Spin (I) = ½: 1 H, 13 C, 15 N, 19 F, 31 P biological and chemical relevance Odd atomic mass I = +½ & -½ 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 I = +1, 0 & -1

19. Magnetic Moment (  ) spinning charged nucleus creates a magnetic field Similar to magnetic field created by electric current flowing in a coil Magnetic moment “Right Hand Rule” determines the direction of the magnetic field around a current- carrying wire and vice-versa

20. Gyromagnetic ratio (  ) related to the relative sensitive of the NMR signal magnetic moment (  ) is created along axis of the nuclear spin where: p – angular momentum  – gyromagnetic ratio (different value for each type of nucleus) magnetic moment is quantized (m) m = I, I-1, I-2, …, -I for common nuclei of interest: m = +½ & -½

21. Gyromagnetic ratio (  ) magnetic moment is quantized (m) m = I, I-1, I-2, …, -I for common nuclei of interest: m = +½ & -½ Isotope Net Spin  / MHz T -1 Abundance / % 1H1H1/2 42.5899.98 2H2H1 6.540.015 3H3H1/245.410.0 31 P1/2 17.25100.0 23 Na3/2 11.27100.0 14 N1 3.0899.63 15 N1/24.310.37 13 C1/2 10.711.108 19 F 1/2 40.08100.0

22. BoBo =  h / 4  Magnetic alignment In the absence of external field, each nuclei is energetically degenerate Add a strong external field (B o ) and the nuclear magnetic moment: aligns with (low energy) against (high-energy)

23. Spins Orientation in a Magnetic Field (Energy Levels) Magnetic moment are no longer equivalent Magnetic moments are oriented in 2 I + 1 directions in magnetic field  Vector length is:  Angle (  ) given by:  Energy given by: where, B o – magnetic Field  – magnetic moment h – Planck’s constant For I = 1/2

24. Spins Orientation in a Magnetic Field (Energy Levels) The energy levels are more complicated for I >1/2

25. Spins Orientation in a Magnetic Field (Energy Levels) Magnetic moments are oriented in one of two directions in magnetic field (for I =1/2) Difference in energy between the two states is given by:  E =  h B o / 2  where: B o – external magnetic field h – Planck’s constant  – gyromagnetic ratio

26. Frequency of absorption: =  B o / 2  Spins Orientation in a Magnetic Field (Energy Levels) Transition from the low energy to high energy spin state occurs through an absorption of a photon of radio-frequency (RF) energy RF

27. NMR Signal (sensitivity) The applied magnetic field causes an energy difference between the aligned (  ) and unaligned (  ) nuclei NMR signal results from the transition of spins from the  to  state Strength of the signal depends on the population difference between the  and  spin states The population (N) difference can be determined from the Boltzmann distribution and the energy separation between the  and  spin states: N  / N  = e  E / kT B o = 0 B o > 0  E = h   Low energy gap

28. NMR Signal (sensitivity) Since:  E = h  E = h and =  B o / 2  then: The  E for 1 H at 400 MHz (B o = 9.39 T) is 6 x 10 -5 Kcal / mol      Very Small ! ~60 excess spins per million in lower state N  /N  = e (  hB o /2  kT) N  / N  = e  E / kT

29. NMR Sensitivity  E  h  B o /  2  NMR signal (s) 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 (B o 2/3, in practice, homogeneity) 5) B 1 2 exciting field strength (RF pulse) N  / N  = e  E / kT Increase energy gap -> Increase population difference -> Increase NMR signal EE ≡ BoBo ≡  %  4 B o 2 NB 1 g(  )/T s %  4 B o 2 NB 1 g(  )/T

30.  - Intrinsic property of nucleus can not be changed.    C ) 3 for 13 C is 64x    N ) 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 ~2.7x10 5 x !! NMR Sensitivity Relative sensitivity of 1 H, 13 C, 15 N and other nuclei NMR spectra depend on  Gyromagnetic ratio (  )  Natural abundance of the isotope 1 H NMR spectra of caffeine 8 scans ~12 secs 13 C NMR spectra of caffeine 8 scans ~12 secs 13 C NMR spectra of caffeine 10,000 scans ~4.2 hours

31. NMR Sensitivity Increase in Magnet Strength is a Major Means to Increase Sensitivity

32. NMR Sensitivity But at a significant cost! ~$800,000 ~$2,00,000~$4,500,000

33. NMR Sensitivity An Increase in concentration is a very common approach to increase sensitivity 30 mg 1.2 mg http://web.uvic.ca/~pmarrs/chem363/nmr%20files/363%20nmr%20signal%20to%20noise.pdf

34. NMR Sensitivity But, this can lead to concentration dependent changes in the NMR spectra (chemical shift & line-shape) resulting from compound property changes Beilstein J. Org. Chem. 2010, 6, No. 3. Dimerization as concentration increases from 0.4 to 50 mM Beilstein J. Org. Chem. 2010, 6, 960–965. H-bond and multimer formation as concentration increases from 1 to 30 mM

35. NMR Sensitivity An Increase in the number of scans (NS) or signal-averaging is the most common approach to increase sensitivity (signal-to-noise (S/N) S/N ≈ NS 1/2

36. NMR Sensitivity But, it takes significantly longer to acquire the spectrum as the number of scans increase: Experimental Time = Number of Scans x Acquisition Time 8 scans ~12 secs 10,000 scans ~4.2 hours

37. Dalton Trans.,2014, 43, 3601-3611 NMR Sensitivity Lowering the temperature is usually not an effective means of increasing sensitivity because of chemical shift changes and peak broadening Significant peak broadening Significant chemical shift changes

38. Classical Description Theory of NMR Spinning particle precesses around an applied magnetic field A Spinning Gyroscope in a Gravity Field

39. Classical Description Angular velocity of this motion is given by:  o =  B o Larmor where the frequency of precession or Larmor frequency is: =  B o /2  Same as quantum mechanical description

40. Classical Description Net Magnetization  Nuclei either align with or against external magnetic field along the z-axis.  Since more nuclei align with field, net magnetization (M o, M Z ) exists parallel to external magnetic field.  Net Magnetization along +Z, since higher population aligned with B o.  Magnetization in X,Y plane (M X,M Y ) averages to zero. MoMo z x yy x z BoBo BoBo

41. Net Magnetization in a Magnetic Field MoMo y x z x y z BoBo BoBo B o > 0  E = h   BoBo Classic View: - Nuclei either align with or against external magnetic field along the z-axis. - Since more nuclei align with field, net magnetization (M o ) exists parallel to external magnetic field Quantum Description: - Nuclei either populate low energy ( , aligned with field) or high energy ( , aligned against field) - Net population in  energy level. - Absorption of radio- frequency promotes nuclear spins from   .

42. An NMR Experiment MoMo y x z x y z BoBo BoBo We have a net magnetization precessing about B o 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

43. The Basic 1D NMR Experiment Experimental details will effect the NMR spectra and the corresponding interpretation

44. B 1 off… (or off-resonance) MoMo z x B1B1 z x M xy yy 11 11 Right-hand rule resonant condition: frequency (  1 ) of B 1 matches Larmor frequency (  o ) energy is absorbed and population of  and  states are perturbed. An NMR Experiment And/Or: And/Or: M o now precesses about B 1 (similar to B o ) for as long as the B 1 field is applied. Again, keep in mind that individual spins flipped up or down (a single quanta), but M o can have a continuous variation.

45. RF pulse B 1 field perpendicular to B 0 M xy MzMz Classical Description Observe NMR Signal  Need to perturb system from equilibrium.  B 1 field (radio frequency pulse) with  B o /2  frequency  Net magnetization (M o ) now precesses about B o and B 1  M X and M Y are non-zero  M x and M Y rotate at Larmor frequency  System absorbs energy with transitions between aligned and unaligned states  Precession about B 1 stops when B 1 is turned off

46. Classical Description Rotating Frame  To simplify the vector description, the X,Y axis rotates about the Z axis at the Larmor frequency (X’,Y’)  B 1 is stationary in the rotating frame M xy MzMz +

47. Classical Description NMR Pulse  Applying the B 1 field for a specified duration (Pulse length or width)  Net Magnetization precesses about B 1 a defined angle (90 o, 180 o, etc) B 1 off… (or off-resonance) MoMo z x B1B1 z x M xy yy 11 11  1 =  B 1 90 o pulse

48. Classic View: - Apply a radio-frequency (RF) pulse a long the y-axis - RF pulse viewed as a second field (B 1 ), that the net magnetization (M o ) will precess about with an angular velocity of  1 - - precession stops when B 1 turned off Quantum Description: - enough RF energy has been absorbed, such that the population in  /  are now equal - No net magnetization along the z-axis B 1 off… (or off-resonance) MoMo z x B1B1 z x M xy yy 11 11  1 =  B 1 90 o pulse B o > 0  E = h   Please Note: A whole variety of pulse widths are possible, not quantized dealing with bulk magnetization Absorption of RF Energy or NMR RF Pulse

49. 10 o 20 o 35 o 60 o 90 o A whole variety of pulse widths are possible, not quantized dealing with bulk magnetization Signal intensity decreases significantly when not at optimal 90 o pulse length

50. An NMR Experiment What Happens Next? The B 1 field is turned off and M xy continues to precess about B o at frequency  o. z x M xy Receiver coil (x) y  NMR signal oo FID – Free Induction Decay yyy Mxy is precessing about z-axis in the x-y plane Time (s)

51. =  B o /2  RF pulse along Y Detect signal along X X y Classical Description Observe NMR Signal  Remember: a moving magnetic field perpendicular to a coil will induce a current in the coil.  The induced current monitors the nuclear precession in the X,Y plane

52. NMR Signal Detection - FID The FID reflects the change in the magnitude of M xy as the signal is changing relative to the receiver along the y-axis Again, the signal is precessing about B o at its Larmor Frequency (  o ). RF pulse along Y Detect signal along X

53. NMR Signal Detection - FID The appearance of the FID depends on how the frequency of the signal differs from the Larmor Frequency Time Domain Frequency Domain Larmor Frequency

54. NMR Signal Detection - Fourier 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

55. NMR Signal Detection - Fourier Transform After the NMR Signal is Generated and the B1 Field is Removed, the Net Magnetization Will Relax Back to Equilibrium Aligned Along the Z-axis T 2 relaxation Two types of relaxation processes, one in the x,y plane and one along the z-axis Peak shape also affected by magnetic field homogeneity or shimming

56. NMR Relaxation a)No spontaneous reemission of photons to relax down to ground state Probability too low  cube of the frequency b)NMR signal relaxes back to ground state through two distinct processes

57. NMR Relaxation c) Spin-Lattice or Longitudinal Relaxation (T 1 ) i. transfer of energy to the lattice or solvent material ii. coupling of nuclei magnetic field with magnetic fields created by the ensemble of vibrational and rotational motion of the lattice or solvent. iii. results in a minimal temperature increase in sample M z = M 0 (1-exp(-t/T 1 )) Recycle Delay: General practice is to wait 5xT 1 for the system to have fully relaxed.

58. NMR Relaxation c) Spin-Lattice or Longitudinal Relaxation (T 1 ) i. Commonly measure T 1 with an inversion recovery pulse sequence 5xT 1 fully relaxed

59. NMR Relaxation d) spin-spin or transverse relaxation (T 2 ) i. exchange of energy between excited nucleus and low energy state nucleus ii. randomization of spins or magnetic moment in x,y-plane M x = M y = M 0 exp(-t/T 2 )

60. NMR Relaxation d) spin-spin or transverse relaxation (T 2 ) iii. related to NMR peak line-width ( derived from Heisenberg uncertainty principal) Please Note: Line shape is also affected by the magnetic fields homogeneity

61. NMR Relaxation d) spin-spin or transverse relaxation (T 2 ) iii. Usually measured with a Carr-Purcell-Meiboom-Gill (CPMG) Pulse sequence

62. NMR Relaxation d) Another way to look at T 1 and T 2 i. T 2 is the time it takes a stove to heat a house and T 1 is the time it takes for the heat to be lost to the surrounding environment ii. If the house is insulated, T 1 will be larger than T 2

63. NMR Relaxation d) One more way to look at T 1 and T 2 i. Relaxation of 13 C occurs through two distinct interactions ii. Magnetic fields produced by the 1 H magnetic dipoles are felt by the 13 C spin These magnetic fields are modulated by random Brownian motions (rotational, translational) iii. Relaxation between 13 C and 1 H in same molecule - intramolecular relaxation (T 2 ) iv. Relaxation between 13 C and 1 H from solvent- intermolecular relaxation (T 1 )