MAGNETIC RESONANCE 12/2/2011. Educational Session Short Break Educational Session Lunch
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1 //0 Educational Session Short Break.0.40 Educational Session Lunch pening & Session MAGNETIC RESNANCE «Imaging» MRI «igh field» NMR Spectroscop Liquid (high resolution) state «Low-field» TD NMR EPR Spectroscop/Imaging Solid state
2 //0 Back to basics Spin ½ no eternal magnetic field magnetic field B 0 both orientations are energ equivalent and equall possible depending on the orientation of its magnetic field a nucleus obtains a different energ Back to basics Spin ½ no eternal magnetic field magnetic field B 0 µ µ both orientations are energ equivalent and equall possible precession around the eternal field with ω 0 = γb 0
3 //0 Back to basics Man Spins no eternal magnetic field N = 0 magnetic field B 0 N > 0 ev N =N ev ev N >N ev both orientations are energ equivalent and equall possible depending on the orientation of its magnetic field a nucleus obtains a different energ Back to basics Man Spins precession magnetic field B 0 ev N >N ev Snapshot orientation defined onl in the direction // B0 depending on the orientation of its magnetic field a nucleus obtains a different energ
4 //0 Back to basics Man Spins Bulkmagnetisation ~ N z z z N N The NMR measurement Relaation time t rel Pulse time t pulse t rel t pulse t aq B off Bon Laborator frame Rotating frame B0 z M z M t pulse z' ω = γb z' B ' ' B ' ' ' B ' B0 Acquisition time t aq M B off M ω = γb 0 0 4
5 //0 The NMR measurement Relaation time t rel Pulse time t pulse t rel t pulse t aq B off Bon Laborator frame Laborator frame B0 z M z M t pulse z' ω = γb z' B ' ' B ' ' ' B ' B0 Acquisition time t aq M B off M ω = γb 0 0 NMR relaation : Bulkmagnetisation Before the 90 pulse Bulkmagnetisation After the 90 pulse z z Longitudinal ev ev N >N Transversal Longitudinal N =N N > 0 N = 0 disordered transversal ordered EQUILIBRIUM & UNPERTURBED coherent DISEQUILIBRIUM & PERTURBED 5
6 //0 NMR relaation : return to equilibrium z Longitudinal relaation M Z is the bulkmagnetisation at an time t M z t M 0 is the bulkmagnetisation at equilibrium (t = ) M z eq t z eq t ( z z ) dm t eq = R M M M z = M z pe R t dt t R = (T ) - N =N T relaation Enthalpic process ev N >N ev Echange of energ with the surroundings (lattice) NMR relaation : return to equilibrium Transversal relaation dm t, R t M, dt ordered coherent = t 0, =, M M e R t loss of order loss of coherence R = (T ) - disordered incoherent T relaation Entropic process Mutual echange of energ (spin-spin) Can be caused b chemical echange as well! 6
7 //0 Importance of T and T for spectroscop Full T relaation is essential for a quantitative response (integrations) 5*T T T R R t r = 5*T M z Since each in a molecule is characterized b a different T, one should wait a time t r equal to 5 times the longest T before equilibrium magnetisation is restored to all s. t r < 5T t r < 5T t r < 5T t r ~ 5T t r ~ 5T Importance of T and T for spectroscop Full T relaation is essential for a quantitative response (integrations) NMR, T s are generall similar and fairl short C NMR, T s are generall dissimilar and can be ver long M z M z C quat C cholesterlacetaat 7
8 //0 Importance of T and T for spectroscop The NMR signal attenuates eponentiall with time s t t/ T ( ) ~ MA cos ωat( e ) Small molecules : T ~ 0. to s Beond a time equal to times T 88% of the signal has dissipated and mostl noise is measured The value of T thus determines the time period during which the signal should be monitored T relaation determines the resonance line width s t t/ T ( ) ~ MA cos ωat( e ) FT ν ν / = πt As the value of T shortens, the resonance line will broaden Importance of T and T for spectroscop R = (T ) - scales linearl with the molecular weight Mw t>> ν (/) >> ν(/) Small molecules Bio macro molecules M w Camphor + polstrene (M w ) As the molecular Polstrene weight with increases, M w the en lines kamfer, become T PS ~ 5ms much << Tbroader, kamfer 0.5s affecting resolution and information contents 8
9 //0 Importance of T and T for spectroscop Spin echo sequence start measuring here! camphor PS The spin echo element allows to avoid phase distortions due to chemical shift evaluation in the waiting period (see later) Metabonomics Polstrene with Mr and camphor, T PS ~ 5ms << T camphor ~0.5s Sample NMR: Structure elucidation NMR Assignment Structure Restrained modelling Covalent Structure Validation against independant data Collection of conformational data 9
10 //0 D NMR Intensities C Ac C C C C NAc C C NAc PS7F ppm K. Ghesen (NMRSTR), K. Conrath (GSK) D NMR Intensit Chemical shift δ Scalar coupling C C Ac C C C C NAc C NAc PS7F ppm K. Ghesen (NMRSTR), K. Conrath (GSK) 0
11 //0 D NMR Intensit Chemical shift δ Ac C NAc PS7F NAc C ppm K. Ghesen (NMRSTR), K. Conrath (GSK) Collection of and C signals C? C Ac C C C NAc C NAc C pm Covalent Structure/Conformation K. Ghesen (NMRSTR), K. Conrath (GSK)
12 // pm Scalar coupling interaction between chemicall nearb s J δ E C D B A A A B C D E C C C C C unit unit B D C E δ - CSY : Correlating neighbouring hdrogens Individual s must be within three chemical bonds nl s from the same unit are correlated due to the glcosidic links
13 //0 Scalar coupling interaction between chemicall nearb s E C D B A J J δ A A B C D E B C C C C C unit unit D C E δ - CSY : Correlating neighbouring hdrogens Individual s must be within three chemical bonds The glcosidic links interrupt the correlation sequence nl s from the same unit are correlated due to the glcosidic links Scalar coupling interaction between chemicall nearb s E C D B A J J δ A A B C D E B C C C C C unit unit D C E δ - CSY : Correlating neighbouring hdrogens Individual s must be within three chemical bonds The glcosidic links interrupt the correlation sequence nl s from the same unit are correlated due to the glcosidic links
14 //0 R CSY C NAc R Which s belong to the same unit? 4 5 Me ppm ppm Information on carbon skeleton incomplete K. Ghesen (NMRSTR), K. Conrath (GSK) Also TCSY Scalar coupling interaction between directl bonded and C E C D B A A B C D E δ b a J C C a C b C c C d C e unit unit c d C e δ - C SQC : Single Bond Correlation Establishing C bonding pairs in the molecule No quaternar carbons C, C, C can be independantl established (mesqc) 4
15 //0 - C SQC PS7F ppm 0 0 R C NAc R 4 5 Me ppm Basic elements of the carbon skeleton of the saccharide units identified but eact linkage still unknown C C C C 4 C 5 C 6 K. Ghesen (NMRSTR), K. Conrath (GSK) - 5 N SQC: the protein NMR workhorse uman profilin (9 amino acids) N c J N A Fingerprint 5 N ppm ppm 5
16 //0 Scalar coupling interaction between nearb s and Cs A C a B C b C C c D E C d C e δ E C D B A b a J C J C J C c C A B C D E d C a C b C c C d C e e J C J C J C δ - C MBC : Multiple Bond Correlation Quaternar carbons identified Allows the immediate vicinit of the carbon skeleton to be inferred Connections across glcosidic linkages established - C MBC PS7F ppm R C NAc C R C(F)4(E) C(B)(A) C(E)(D) C(A)(B) Me ppm C C C C 4 C 5 C 6 K. Ghesen (NMRSTR), K. Conrath (GSK) 6
17 //0 Summar CSY 4 5 Me 6 Information on carbon skeleton incomplete 4 5 Me 6 - C SQC C C C C 4 C 5 C Me pm - C MBC C C C C 4 C 5 C 6 R C NAc R The NE effect: an alternative coupling through space a b NESY/RESY (ν b,ν a ) r ν I NE, = k(r, ) -6 Since I NE ~r -6, onl inter proton distances < 5Å canbe measured! (ν a,ν b ) ν NE between A and B Each cross-peak results from an inter distance shorter than 5A 7
18 //0 NESY/RESY D-Ser-6 N D-Leu-5 ppm 4 5 L-Leu-7 D-Ser-8 N N N N L-Ile-9 N D-Val-4 D-allo-Thr- N D-Gln- N N L-Leu- N (R)--hdrodecanoic acid Distribution of 7 distance restraints Alk Leu Gln Thr Val4 Leu5 Ser6 Leu7 Ser8 Ile9 Total intra inter i-->i± inter I-->>i± ppm The NESY spectrum as the basis for solution structure determination The molecular structure, defined at the level of a dense network of inter hdrogen distances, is thus encoded in the D NESY spectrum of a molecule NMR based structure determination consists in etracting the dense network of distances from the NESY spectrum and computing the corresponding D structure 8
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