NMR Spectroscopy. Guangjin Hou

Transcription

1 NMR Spectroscopy Guangjin Hou

2 NMR History 1 H NMR spectra of water H NMR spectra of water (First NMR Spectra on Water, 1946) 1 H NMR spectra ethanol (First bservation of the Chemical Shift, 1951)

3 NMR History Modern ethanol spectra 1937 Rabi predicted NMR 1946 Bloch, Purcell first NMR of bulk sample 1953 verhauser NE (nuclear verhauser effect) 1966 Ernst, Anderson Fourier transform NMR 1975 Jeener, Ernst 2D NMR 1985 Wüthrich first solution structure of a small protein (BPTI) from NE derived distance restraints D NMR + 13 C, 15 N isotope labeling of recombinant proteins (resolution) 1990 pulsed field gradients (artifact suppression) 1996/7 new long range structural parameters: - residual dipolar couplings from partial alignment in liquid crystalline media - projection angle restraints from cross-correlated relaxation TRSY (molecular weight > 100 kda)

4 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. ne of the MST Routinely used Analytical Techniques!

5 Theory of NMR Faraday s 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

6 Theory of NMR Quantum Description Nuclear Spin l 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 B 0, the energy for different states would be different, i.g. 1 H (I=1/2), there are +½, -½ states, the energy difference is (γ, gyromagnetic ratio), Δ E = h γ B 0 Note: NMR cannot detect inactive Nuclear Spin (I) = 0, e.g. 12 C, 16. Fortunately, almost every element in the periodic table has an isotope with a non zero nuclear spin.

7 NMR Signal B 0 Magnetic alignment B o > 0 M B o = 0 +

8 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. 1 H at 400 MHz (B o = 9.39 T) : ΔE = 6 x 10-5 Kcal / mol N + /N - = To enhance NMR signal: 5/2 3/2 2 S γ B NB g ν T 0 1 ( )/ 1) Number of Nuclei (N) (quantity of the sample) (limited to field homogeneity and filling factor) 2) Gyromagnetic ratio (in practice γ 5/2 ) 3) Inversely to temperature (T) 4) External magnetic field (B 3/2 o, in practice, homogeneity) 5) B 2 1 exciting field strength (RF pulse)

9 NMR Sensitivity To Increase Magnet Strength is a Major Means HSQC@600M HSQC@900M

10 NMR Sensitivity To Increase Magnet Strength is a Major Means $800,000 $2,000,000 $5,000,000

11 NMR spectrometers Three Main Series Varian INVA The first 40 MHz NMR spectrometer (Varian, 1953) Bruker AVANCE The first 90 MHz FTNMR spectrometer (Bruker, 1967) JEL ECA

12 Information in NMR Spectra chemical shift splitting linewidth bservable Name Quantitative Information Peak position Chemical shifts (δ) δ(ppm) = υ obs υ ref /υ 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 curve T 1 dependent Peak Shape Line width Δυ = 1/πT 2 peak half-height molecular motion chemical exchange uncertainty principal uncertainty in energy

13 Development of Multi-Dimensional NMR Developing MD NMR in order to 1. Spread spectrum out resolve peaks remove overlap 2. Supply more information 1967 Richard Ernst - Fourier transformations 1971 Jean Jeener - Two dimensional NMR - CSY 1987 Griesinger et al. - Advent of three dimensional NMR 1990 Ad Bax et al. - Four dimensional NMR

14 NMR structure of Taxol (anticancer drug ) Typical Applications of NMR NH H 1.) Structural (chemical) elucidation Natural product chemistry Synthetic 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.) Drug Design Structure Activity Relationships by NMR 5) Medicine -MRI NMR structure of proteinligand complex H H MRI images of human brain

15 3D Structure determination by NMR

16 Drug Discovery by NMR Structure Activity Relationships, SAR Proposed by the Abbott Laboratories (Fesik et al, 1996) * NMR method of screening and optimization of small organic molecules that bind the protein Based on chemical shift perturbation upon substrate binding/2d Correlation Spec Good for eliminating false positives Non-overlapping ligands can potentially be linked to form a more effective (nm) compound 15 N(ppm) 1 H(ppm)

17 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 ns μs ms s min, hours relaxation T 1 CMPG T 1ρ, T 2 2D exchange H/D exchange

18 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

19 ther NMR Facilities NMR imaging spectrometer NMR Teslameter Gaussmeter Magnetic Field Mapping system NMR Analyzer NMR-MUSE (Mbile Universal Surface Explorer) NMR water explorer NMR logging instrument e.g.

20 The end Thanks for your attention!