Nuclear Magnetic Resonance Lecture Date: February 13th, 2008 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 Common Solution-state NMR Experiments for Organic Structural Analysis Information Provided Experiment Acronym GASPE Gated-spin echo DEPT Distortionless editing by polarization transfer COSY correlated spectroscopy HMQC heteronuclear multiple quantum coherence 1H-13C covalent bonding, bond HMBC heteronuclear multiple bond correlation 1H-13C NOE difference, NOESY, ROESY nuclear Overhauser effect spectroscopy 13C multiplicity (C, CH, CH2, CH3) 1H-1H covalent bonding, 2-4 bonds covalent bonding, 2-4 bonds 1H-1H proximity in space, 1.8-4.5 A 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 Basic Pulse Sequences  A single pulse and acquire An Example of 1D NMR Top – 1H spectrum Middle – Selective pulse Bottom – homonuclear decoupling Multi-dimensional NMR  The general scheme of 2D and multi-dimensional NMR: Can include NOE or Jcoupling mixing Preparation Evolution (t1) Mixing (tm) Detection (t2) Experiment Time  2D NMR data has two frequency dimensions: FT(t1) FT(t2) A Simple 2D NMR Spectrum Cross peak (“correlation”) F1 (ppm) Diagonal Peak 5 F2 (ppm) An Example of 2D NMR – the COSY Experiment Correlations are observed between J-coupled protons! (Example is a sample of sucrose in D2O) Applications of NMR  Structural analysis  Quantitative analysis  Stereochemical and conformational analysis  Solid-state analysis Structural Analysis – 13C NMR and Editing 13C spectra of cholesteryl acetate: (a) continuous 1H decopling (b) 1H during acquisition (no NOE) (c) GASPE (APT) (d) DEPT-135 Structural Analysis: 1H –13C Correlation The 1H-13C HSQC analysis of clarithromycin: Structural Analysis: Long-range 1H –13C Correlation The 1H-13C HMBC analysis of carvedilol: Structural Analysis: 1H –15N Correlation The 1H-15N longrange HMQC analysis of telithromycin: Determination of Relative Stereochemistry NOE difference spectroscopy Determination of Absolute Stereochemistry Che mical Shie lding around the Be nze ne Ring 12 Absolute Isotropic Shielding (ppm) Remember the ring current effect? 10 Above Ring In Ring Plane -2 0.0 2.0 4.0 6.0 8.0 Dis t ance fr om Rin g Ce nte r ( A) Che mical Shie lding around the Be nze ne Ring (Expande d Vie w ) Absolute Isotropic Shielding (ppm) 0.8 0.6 0.4 shielding (opposes field) Abov e Ring In Ring Plane 0.2 -0.2 -0.4 -0.6 deshielding (aligned with field) -0.8 -1 4.0 5.0 6.0 7.0 8.0 Dis tance f ro m Ring C e nt e r (A) 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) 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 NO2 3.51q 7.4-7.5m H 3CO NO 4.45t 3.55q H3CO 2.02m Ph F3C 1.69m O O (S)-MPTA-Cl => (R)-MPTA ester Ph 1.26d CH3 10 F 3C 11 H 5.15m (R)-alcohol |5J H9,F10| = 1.2 Hz | J H11,H5| = 6.2 Hz |3J H2,H3| = 6.9 Hz |4J H2,H4| = Hz 7.4-7.5m 1.35d 11 H3C O 1.83m 1.62m 10 O (S)-MPTA-Cl => (R)-MPTA ester 4.34dt H 5.15m |5J H9,F10| = 1.1 Hz |3J H11,H5| = 6.3 Hz (S)-alcohol | J H2,H3| = 6.8 Hz |4J H2,H4| = 2.2 Hz 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) 19F Quantitative Analysis: TFA Salt Stoichiometry 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 No field Field = B0 m=-1/2 E=(h/2)B0 E m=+1/2  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 Solid-state NMR: Magic-Angle Spinning  The following anisotropic interactions are dependent on their orientation with respect to the large magnetic field (B0): – 1st-order quadrupolar coupling – P2 cos   cos2    dipolar (homo- and heteronuclear) coupling – broadening  anisotropic chemical shift  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) 10 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 1H to 13C experiments: 90 CP CW Decoupling 1H 13C CP 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) An Example: Polymorphism in Carvedilol  13C CP-TOSS spectra of the polymorphs of SKF105517 free base O O H 3C 10 NH 11 12 OH 15 13 O 16 14 17 19 18 20 NH 21 22 26 23 25 24  Amorphous forms generally give broadened spectra 11 An Example: Polymorphism in Carvedilol  15N SSNMR spectroscopy also shows similar effects O O H 3C 10 NH 11 12 OH 15 13 O 16 14 17 19 18 20 NH 21 22 26 23 25 24  Advantages: simple and easy-to-interpret spectra, valuable information about the nitrogen chemical environment  Disadvantage: much lower sensitivity Magnetic Resonance Imaging • The basic idea: a linear magnetic field gradient imposes a linear spread of Larmor frequencies on a sample    B0 Gradient    B0 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 12 Magnetic Resonance Force Microscopy  A “combination” of AFM and EPR (and hopefully NMR)  Uses a nano-scale cantilever to detect spin motion induced by RF via in an magnetic field Rugar, D.; et al Nature 2004, 430, 329–332 R Mukhopadhyay, Anal Chem 2005, 449A-452A Nuclear Spin Optical Rotation (NSOR)  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 Nature 2006, 442, 1021 13 Homework 19-10 19-15 Optional Homework Also, please answer one of these based on the article you chose to read: MRI: Describe the basic action of a field gradient on a sample Also describe how the spin-warp imaging method obtains a 2D image, and why it is similar to conventional 2D NMR Solid-state NMR: What effect(s) in solid-state NMR spectra allow for the analysis of hydrogen-bonding? NMR-MOUSE: List the differences and similarities between unilateral lowfield NMR and traditional high-field NMR instrumentation Why are T2 measurements so analytically useful with this technique? MRFM: Briefly describe the current AFM-derived devices used to detect electron spins What advances need to be made to take the technique forward to nuclear spins? 14 ... CP-TOSS spectra of the polymorphs of SKF105517 free base O O H 3C 10 NH 11 12 OH 15 13 O 16 14 17 19 18 20 NH 21 22 26 23 25 24  Amorphous forms generally give broadened spectra 11 An Example: Polymorphism...  15N SSNMR spectroscopy also shows similar effects O O H 3C 10 NH 11 12 OH 15 13 O 16 14 17 19 18 20 NH 21 22 26 23 25 24  Advantages: simple and easy-to-interpret spectra, valuable information... 5-nitro -2- pentanol NO2 3.51q 7.4-7.5m H 3CO NO 4.45t 3.55q H3CO 2. 02m Ph F3C 1.69m O O (S)-MPTA-Cl => (R)-MPTA ester Ph 1 .26 d CH3 10 F 3C 11 H 5.15m (R)-alcohol |5J H9,F10| = 1 .2 Hz | J H11,H5| = 6.2