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Lecture Date: February 13 th, 2008 Nuclear Magnetic Resonance 2 Slide 2 Selected Applications of NMR Structural analysis Stereochemical and conformational analysis Quantitative analysis Solid-state analysis Slide 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 Slide 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 Slide 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 Slide 6 An Example of 1D NMR Top 1H spectrum Middle Selective pulse Bottom homonuclear decoupling Slide 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 Slide 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: Slide 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 ) Slide 10 A Simple 2D NMR Spectrum Diagonal Peak Cross peak (correlation) 1 2 3 4 5 12345 F 1 (ppm) F 2 (ppm) Slide 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) Slide 12 COSY Pulse Sequences COSY-45 and double-quantum filtered COSY (the latter implements a two proton filter) Slide 13 Structural Analysis: 1 H 13 C Correlation The 1 H- 13 C HSQC analysis of clarithromycin: Slide 14 More Advanced Pulse Sequences 1 H- 13 C heteronuclear single quantum coherence (HSQC) Slide 15 Structural Analysis: Long-range 1 H 13 C Correlation The 1 H- 13 C HMBC analysis of carvedilol: Slide 16 Structural Analysis: 1 H 15 N Correlation The 1 H- 15 N long- range HMQC analysis of telithromycin: Slide 17 Determination of Relative Stereochemistry NOE difference spectroscopy Slide 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) Slide 19 Determination of Absolute Stereochemistry by Mosher-Dale Method Procedure: Derivatize a chiral alcohol with MPTA, -methoxy- - (trifluoromethyl)phenyl acetic acid Because a phenyl groups 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). Slide 20 19 F Quantitative Analysis: TFA Salt Stoichiometry Slide 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 Slide 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 Slide 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). Slide 24 An Example: Polymorphism in Carvedilol 13 C CP-TOSS spectra of the polymorphs of SKF105517 free base Amorphous forms generally give broadened spectra Slide 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 Slide 26 LC-SPE-NMR for Impurity Analysis LC separation and solid-phase extraction (SPE) concentration Slide 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 Slide 28 Magnetic Resonance Force Microscopy Rugar, D.; et al. Nature 2004, 430, 329332. 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 Slide 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