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NMR Spectroscopy Guangjin Hou 22-04-2009 NMR History 1H NMR spectra of water (First NMR Spectra on Water, 1946) 1H NMR spectra ethanol (First Observation of the Chemical Shift, 1951) NMR History Modern ethanol spectra 1937 1946 1953 1966 1975 1985 1987 1990 1996/7 …… Rabi predicted NMR Bloch, Purcell first NMR of bulk sample Overhauser NOE (nuclear Overhauser effect) Ernst, Anderson Fourier transform NMR Jeener, Ernst 2D NMR Wüthrich first solution structure of a small protein (BPTI) from NOE derived distance restraints 3D NMR + 13C, 15N isotope labeling of recombinant proteins (resolution) pulsed field gradients (artifact suppression) 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) NMR (Nuclear Magnetic Resonance) NMR measures the absorption of electromagnetic radiation in the radio-frequency region (~4-950 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 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! Theory of NMR Faraday’s Law = electron = neutron = proton 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 Theory of NMR l Quantum Description Nuclear Spin •Nucleus rotates about its axis (spin) •Nuclei with spin have angular momentum (p) or spin • quantized, spin quantum number I • 2I + 1 states: I, I-1, I-2, …, -I • identical energies in absence of external magnetic field When placed in a magnetic field of strength B0, the energy for different states would be different, i.g. 1H (I=1/2), there are +½, -½ states, the energy difference is (γ, gyromagnetic ratio), ΔE = h ⋅ γ ⋅ B0 Note: NMR cannot detect “inactive” Nuclear Spin (I) = 0, e.g. 12C, 16O. Fortunately, almost every element in the periodic table has an isotope with a non zero nuclear spin. NMR Signal B0 Magnetic alignment − M Bo > 0 Bo = 0 + NMR Sensitivity Strength of the NMR signal depends on the population difference between the α and β spin states, and the population (N) difference can be determined from the Boltzmman distribution and the energy separation: N− + = e −ΔE / kT N e.g. 1H at 400 MHz (Bo = 9.39 T) : ΔE = 6 x 10-5 Kcal / mol N+/N- = 1.000060 To enhance NMR signal: S ∝ γ 5/ 2 B03/ 2 NB12 g (ν ) / T 1) Number of Nuclei (N) (quantity of the sample) (limited to field homogeneity and filling factor) 2) Gyromagnetic ratio (in practice γ5/2) 3) Inversely to temperature (T) 4) External magnetic field (Bo3/2, in practice, homogeneity) 5) B12 exciting field strength (RF pulse) NMR Sensitivity To Increase Magnet Strength is a Major Means HSQC@600M HSQC@900M NMR Sensitivity To Increase Magnet Strength is a Major Means $800,000 $2,000,000 $5,000,000 NMR spectrometers Three Main Series Varian INOVA The first 40 MHz NMR spectrometer (Varian, 1953) The first 90 MHz FTNMR spectrometer (Bruker, 1967) Bruker AVANCE JEOL ECA Information in NMR Spectra chemical shift splitting linewidth Observable Name Quantitative Information Peak position Chemical shifts (δ) δ(ppm) = υobs –υref/υref (Hz) chemical (electronic) environment of nucleus Peak Splitting Coupling Constant (J) Hz peak separation (intensity ratios) neighboring nuclei (torsion angles) Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T1 dependent Peak Shape Δυ = 1/πT2 peak half-height Line width molecular motion chemical exchange uncertainty principal uncertainty in energy Development of Multi-Dimensional NMR Developing MD NMR in order to 1. Spread spectrum out – resolve peaks – remove overlap 2. Supply more information …… 1967 1971 1987 1990 …… Richard Ernst Jean Jeener Griesinger et al. Ad Bax et al. - Fourier transformations - Two dimensional NMR - COSY - Advent of three dimensional NMR - Four dimensional NMR NMR structure of Taxol (anticancer drug ) O Typical Applications of NMR O O NH O O OH O OH HO O O 1.) Structural (chemical) elucidation Natural product chemistry NMR structure of proteinSynthetic chemistry ligand complex - 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 MRI images of human brain DNA, RNA, Protein/DNA complexes Polysaccharides 4.) Drug Design Structure Activity Relationships by NMR 5) Medicine -MRI O O O 3D Structure determination by NMR Drug Discovery by NMR Structure Activity Relationships, SAR 15 N(ppm) ¾ Proposed by the Abbott Laboratories (Fesik et al, 1996)* ¾ NMR method of screening and optimization of small organic molecules that bind the protein ¾ Based on chemical shift perturbation upon substrate binding/2D Correlation Spec ¾ Good for eliminating false positives ¾ Non-overlapping ligands can potentially be linked to form a more effective (nM) compound 1 H(ppm) Dynamic investigation by NMR NMR is a convenient way to study rate of reactions or motions, provided that the lifetime of participating species is comparable to appropriate scales ps relaxation T1 ns μs CMPG T1ρ, T2 ms s min, hours … 2D exchange H/D exchange Summary on application of NMR spectroscopy NMR has extensively applied into various fields including: from gas to liquid, to solid from inorganic to organic from material to biology, to medicine from structure to dynamics form physical change to chemical reaction to biological mechanism …… Other NMR Facilities ¾ ¾ ¾ ¾ ¾ ¾ ¾ NMR imaging spectrometer NMR Teslameter 、Gaussmeter Magnetic Field Mapping system NMR Analyzer NMR-MOUSE (MObile Universal Surface Explorer) NMR water explorer NMR logging instrument e.g. Thanks for your attention! 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