1. Lecture Date: February 13 th, 2008 Nuclear Magnetic Resonance 2

2. Selected Applications of NMR  Structural analysis  Stereochemical and conformational analysis  Quantitative analysis  Solid-state analysis

3. NMR Experiments  NMR experiments fall into some basic categories: –Basic pulse methods  Single pulse  Selective pulse or selective decoupling  Solvent suppression –2D and multi-dimensional experiments  unravel complex spectra by separation of overlapping signals, control of “mixing” between signals (to obtain more data) –Multiple resonance (heteronuclear techniques)  Are often 2D or nD sequences –Diffusion, dynamics and relaxation experiments

4. Common Solution-state NMR Experiments for Organic Structural Analysis ExperimentAcronym Information Provided GASPE DEPT Gated-spin echo Distortionless editing by polarization transfer 13 C multiplicity (C, CH, CH 2, CH 3 ) COSY DQFCOSY correlated spectroscopy 1 H- 1 H covalent bonding, 2-4 bonds HMQCheteronuclear multiple quantum coherence 1 H- 13 C covalent bonding, 1 bond HMBCheteronuclear multiple bond correlation 1 H- 13 C covalent bonding, 2-4 bonds NOE difference, NOESY, ROESY nuclear Overhauser effect spectroscopy 1 H- 1 H proximity in space, 1.8-4.5 A

5. Pulse Sequences  Modern NMR involves flexible spectrometers that can implement pulse sequences, which are designed to extract and simplify relevant information for the spectroscopist  Designed to harness a property or properties of the nuclear spin Hamiltonians –J-coupling –Chemical shift –Quadrupolar coupling –Dipolar coupling  Or, are designed to measure a bulk effect –Relaxation –Diffusion –Chemical exchange or dynamics

6. An Example of 1D NMR Top – 1H spectrum Middle – Selective pulse Bottom – homonuclear decoupling

7. Structural Analysis – 13 C NMR and Editing 13 C spectra of cholesteryl acetate: (a) continuous 1 H decopling (b) 1 H during acquisition (no NOE) (c) GASPE (APT) (d) DEPT-135

8. The Gated Spin Echo Pulse Sequences  1D gated spin echo for 13 C editing: a mixture of pulses and delays that take advantage of J-coupling differences between C, CH, CH2, and CH 3 groups:

9. Multi-dimensional NMR  The general scheme of most 2D NMR experiments: Evolution (t 1 )Detection (t 2 ) Preparation Mixing (t m ) Experiment Time  2D NMR data has two frequency dimensions: Can include NOE or J- coupling mixing FT(t 1 )FT(t 2 )

10. A Simple 2D NMR Spectrum Diagonal Peak Cross peak (“correlation”) 1 2 3 4 5 12345 F 1 (ppm) F 2 (ppm)

11. An Example of 2D NMR – the COSY Experiment Correlations are observed between J- coupled protons! (Example is a sample of sucrose in D 2 O, analyzed with the double-quantum filtered version of COSY – “DQFCOSY”)

12. COSY Pulse Sequences  COSY-45 and double-quantum filtered COSY (the latter implements a “two proton” filter)

13. Structural Analysis: 1 H – 13 C Correlation The 1 H- 13 C HSQC analysis of clarithromycin:

14. More Advanced Pulse Sequences  1 H- 13 C heteronuclear single quantum coherence (HSQC)

15. Structural Analysis: Long-range 1 H – 13 C Correlation The 1 H- 13 C HMBC analysis of carvedilol:

16. Structural Analysis: 1 H – 15 N Correlation The 1 H- 15 N long- range HMQC analysis of telithromycin:

17. Determination of Relative Stereochemistry NOE difference spectroscopy

18. Determination of Absolute Stereochemistry Remember the ring current effect? J. A. Dale and H. S. Mosher, J. Am. Chem. Soc., 95, 512-519 (1973). C. E. Johnson and F. A. Bovey, J. Chem. Phys., 29, 1012 (1958). shielding (opposes field) deshielding (aligned with field)

19. Determination of Absolute Stereochemistry by Mosher-Dale Method  Procedure: Derivatize a chiral alcohol with MPTA,  -methoxy-  - (trifluoromethyl)phenyl acetic acid  Because a phenyl group’s deshielding effects drop off more rapidly with distance than its shielding effects, protons close to a phenyl should be more shielded!  Example: 5-nitro-2-pentanol J. A. Dale and H. S. Mosher, J. Am. Chem. Soc., 95, 512-519 (1973). A. Guarna, E. O. Occhiato, L. M. Spinetti, M. E. Vallecchi, and D. Scarpi, Tetrahedron, 51, 1775-1788 (1995).

20. 19 F Quantitative Analysis: TFA Salt Stoichiometry

21. Solid-state Nuclear Magnetic Resonance  NMR in solids, like solution-state, relies on the behavior of nuclear spin energy levels in a magnetic field. However, the interactions that affect NMR spectra act differently.  In liquids, molecules reorient and diffuse quickly, leading to narrow isotropic resonances.  In solids, the fixed orientation of individual crystallites leads to a range of resonance frequencies for anisotropic interactions. E m=+1/2 m=-1/2 No field Field = B 0  E=(  h/2  )B 0

22. Solid-state NMR: Magic-Angle Spinning  These can be averaged away over time by spinning at a root of the scaling factor:  The result of magic angle spinning (often combined with dipolar decoupling): E. R. Andrew, A. Bradbury, and R. G. Eades, Nature, 183, 1802 (1959). I. J. Lowe. Phys. Rev. Lett. 2, 285 (1959).  The following anisotropic interactions are dependent on their orientation with respect to the large magnetic field (B 0 ): –dipolar (homo- and heteronuclear) coupling –1st-order quadrupolar coupling –anisotropic chemical shift

23. Cross-Polarization  Cross-polarization is an example of a double resonance experiment –Two resonances, typically two different nuclei, are excited in a single experiment.  Cross-Polarization combined with MAS (CP-MAS): –Enhancement of signal from “sparse” spins via transfer of polarization from “abundant” spins –The “Hartmann-Hahn condition” allows for efficient energy transfer between the two spins, usually via dipolar interactions –The basic CP pulse sequence for 1 H to 13 C experiments: 1H1H 13 C CP 90  CW Decoupling E. O. Stejskal and J. D. Memory. “High Resolution NMR in the Solid State,” Oxford University Press, New York (1994). A. Pines, M. G. Gibby and J. S. Waugh. J. Chem. Phys., 59, 569 (1973).

24. An Example: Polymorphism in Carvedilol  13 C CP-TOSS spectra of the polymorphs of SKF105517 free base  Amorphous forms generally give broadened spectra

25. An Example: Polymorphism in Carvedilol  15 N SSNMR spectroscopy also shows similar effects.  Advantages: simple and easy-to-interpret spectra, valuable information about the nitrogen chemical environment  Disadvantage: much lower sensitivity

26. LC-SPE-NMR for Impurity Analysis LC separation and solid-phase extraction (SPE) concentration

27. Magnetic Resonance Imaging The basic idea: a linear magnetic field gradient imposes a linear spread of Larmor frequencies on a sample. Figure from S. W. Homans, A Dictionary of Concepts in NMR, Oxford, 1989. For more details, see P. G. Morris, NMR Imaging in Medicine and Biology, Oxford University Press, 1986. Gradient

28. Magnetic Resonance Force Microscopy Rugar, D.; et al. Nature 2004, 430, 329–332. R. Mukhopadhyay, Anal. Chem. 2005, 449A-452A.  A “combination” of AFM and EPR/NMR  Uses a nano-scale cantilever to detect spin motion induced by RF via in an magnetic field

29. Nuclear Spin Optical Rotation (NSOR) Nature 2006, 442, 1021  Measures NMR signals by detecting phase shifts induced in a laser beam as a the beam passes through a liquid  Gives excellent spatial resolution  Currently lacks sensitivity  Developed by Romalis group at Princeton