2D-NMR spectroscopy Part 2. F.D. Sönnichsen Thursday, Oct

Transcription

1 2D-NMR spectroscopy Part 2 F.D. Sönnichsen Thursday, Oct

2 The 2D - COSY Periods in a 2D: Preparation frequency labeling acquisition Indirect frequency determination = indirect dimension mixing Directly observed = Direct dimension Repeat n times, each time with incrementally increased Δt1 Keep the individual FIDs separate.

3 Vektor-Diagramm Analyse des 2D-COSYs die Frequenzbestimung in der indirekten Dimension 90x 90x t 1 t 2 n FID 1: x 90x T1=0 T1=τ 90x Acq FID 2: Acq FID 3: T1=2τ Acq FID 4: T1=3τ Acq Eine Komponente der Magnetisierung wird durch den zweiten Puls in Z-Richtung weitergedreht, während die zweite Komponente in der transversalen Ebene bleibt. Die Amplitude ist jedoch verringert, sie ist Sinus-moduliert. Was passiert wenn wir das Experiment wiederholen, jedesmal mit einer systematisch verlängerten Delay-Zeit?

4 Sollte wie eine Sinus Modulation aussehen

5 Fourier transform with respect to t 1

6 Correlating different types of nuclei The HMQC experiment Uses the direct coupling of attached nuclei to provide 2D correlation spectrum. Very sensitive. The value used for J is typically 145 Hz. Note: the peak splitting due to the coupling is suppressed via decoupling during t2, so that we see singuletts in the spectra

7 Korrelation von unterschiedlichen Kernen Das HETCOR Experiment (traditionelle,ursprüngliche Weg, Protonen mit HeteroAtomen zu koppeln) Jetzt selten benutzt NOTE: Detektion des Kohlenstoffes Das HMQC Experiment (Heteronuclear multiple quantum coherences) Benutzt die direkte 1 J-Kopplung der gebundenen Atome. Detektiert das Proton. Sehr empfindlich. Der benutzte Wert für J ist typischerweise145 Hz. Note: der Entkoppler Decoupler während der Detektionszeit führt zur Aufnahme von Singuletts,ungespaltete Signale

8 Heteronuclear Indirect Detection Experiments that detect 1 H involved in coupling to an X nuclei ( 13 C or 15 N). Sensitivity 3/2 is enhanced by a factor γ H where γ is the gyromagnetic ratio. γ X Signal/Noise comparisons γ H γ X γ H γ X 3/2 Polarization Transfer for Direct DET Nuclei No NOE Full NOE 13 C N +1 Š Indirect Detection The much larger sensitivity have lead to the almost exclusive use of indirectly detected heteronuclear experiments. Note, that these however do not provide information on quarternary Carbon nuclei!!

9 The HMBC experiment MB stands for Multiple bonds We have connected neigboring protons (COSY) We have connected protons to attached 13 C with the direct 1 J coupling (HMQC) Can we use long range couplings and connect further protons? The HMBC SELECTIVELY? observes protons and 13 C that are connected via small 2,3 J couplings ( of the order of 10 Hz).

10 Comparison HMQC / HMBC C-H, 1 J=145, delay = 3ms long range C -H, 2 J or 3 J ~ 10Hz, delay = 50ms

11 Ipsenol Note : the arrows indicate artifacts, i.e. direct couplings which are not completely removed. These are 1J couplings, and can be identified by their large splitting. Since we don t decouple during t2, the 1J couplings constant splits the signal into a doublett, with a separation of ~145Hz due to the 1J-coupling.

12 More on 2D: The principle of 2D NMR can be extended to 3D, 4D and even higher experiments, i.e. even more frequencies can be simultaneously detected. What is the caveat? A 1D spectrum takes minutes, a 2D spectrum hours, a 3D days, a 4D

13 More on 2D: Many more 2D experiments can be created, that correlate the same or further nuclei with desired specificity Most of these experiments correlate nuclei using coupling constants, i.e. the correlate through bonds. Some also offer the possibility to correlate nuclei through space

14 2D - INADEQUATE Allows 13 C 13 C connectivities to be obtained Incredible Natural Abundance Double Quantum Transfer Experiment 1. A. Bax, Two-Dimensional Nuclear Magnetic Resonance in Liquids, Delft University Press, Delft, Holland (1982) pp D. L. Turner, J. Magn. Reson., 53, 259 (1983) 3. D. L. Turner, J. Magn. Reson., 49, 175 (1982) 4. A. Bax and T. H. Mareci, J. Magn. Reson., 53, 360 (1983) p Nakanishi s text p Silverstein & Bassler & Morril Sensitivity is extremely low, because 13 C 1.1% 13 C 13 C 0.01% Correlates double-quantum frequency of 13 C 13 C Many variations provide same information, but in different formats Usually the cross peaks are 2 peaks, representing AX 13 C 13 C doublets Pulse sequences are complex and involve long phase cycling to select for desired double quantum coherences

15 13C 13C

16 2D-TOCSY experiment Total Correlation Spectroscopy Correlates simultaneously all atoms in a spin system

17 Same data as COSY (geminal and vicinal couplings), plus RELAY information

18 2D-TOCSY experiment Spin system : A, B, D, F F Diagonal TOCSY COSY D B A A B D F

19 TOCSY of a small peptide

20 Compare the COSY of this peptide

21 t1 t2 Correlation through space NOE difference spectroscopy semi-quantitative measurement of of local proximity t1 t2

22 The nuclear Overhauser enhancement In contrast to all previous experiments, in which we connected spins through bonds, the nuclear Overhauser effects enables us to connect nuclei through space. This effect is fundamentally different as exchange between the nuclei does not involve scalar coupling. Instead, the direct magnetic coupling (no electrons in between) termed dipolar coupling is involved, which usually does not have an observable effect in solution (in contrast to solid state!). The noe can be correlated with internuclear distances and molecular motion. noe definition: η = I - Io Io The noe is defined as the normalized intensity change of a resonance line upon saturating changing the population of another spin in proximity NOE = c x r -6 Noes can be measured for protons that are in close proximity, generally if they are less than 5Å

23 Noe-background 1 Let s consider: 2 spins, I and S. They are not coupled ( J=0). ω 34, S βα 4 ω 13 I ββ αα ω 24 I αβ ω 12, S ω 13 = ω 24 = ω I ω 12 = ω 34 = ω S The intensity of the magnetization/ the transition for I and S is given by the relative populations of these four levels. For I and S, the respective population differences are: <Iz> = 1/2 (P 1 -P 3 + P 2 -P 4 ) <Sz> = 1/2 (P 1 -P 2 + P 3 -P 4 ) and The irradiation with resonance frequency of S, leads to the equalization of the Populations of the levels for S:P1 = P2, and P3 = P4. At the same time, relaxation will oppose the non-equilibrium situation, leading to relaxation of the spins proportional to the following transition probabilities (W I, W S, W 2, W 0 ) W 1 S βα W 1 I ββ αα W 1, I or W I αβ W 1, S or W S In addition, if the spins are close enough, the additional two relaxation mechansims (blue arrows) also exist. These are doublequantum or zero quantum transistion, involving the spin flip of both spins simultaneously. Relaxation via these transistion leads to populationchanges, and thus the sensitivty enhancement

24 It can be easily shown that NOE= η = σ IS ϕ I = W2 W0 2W + W + W I 2 0 For small molecules, W2 is the dominant relaxation pathway leading to positive NOEs For larger molecules, W0 is dominant, and one obtains negative NOEs. (intensity reduction) Most importantly, the NOE can be quantified and correlated with a distance, since

25 NOE-experiments 1D- steady-state noe experiment: constant irradiation 1D difference spectrum 2D-NOESY experiment: The 2D-NOESY experiment is the most useful experiments, as all noe effect between spins can be measured simultaneously. The noe is generated by simultaneous, temporary population changes, which is called transient noe, not a selective inversion or steady-state irradiation. The noe gives rise to magnetization transfer and offdiagonal peaks, connecting the resonance frequencies of the spins which couple/ cross relax through space d1 t1 tmix t2 Tmix is usually chosen to be between 500ms and 2 sec

26 z 2D-NOESY: vector model z z 90x t 1 y y y x x x Different y-component z z z 90x y y y x Difference in z-component = population change, causes relaxation, is transfers magnetization, mixes S with I and vice versa diz dt = ϕ 1( Iz Io ) σ IS ( Sz So ) x During tmix transferred to I During t1: frequency labeled at ωs IS During t2 detected with I (ωi) NOE ( ϖ 1, ϖ 2 ) = ( ϖ S, ϖ I ) I σ ϕ

27 Aphanamol-1 This NOE showed that the 5-membered ring is cis to the 7-membered ring. 15CH 2 5H

28 Homonuclear Through-bond COSY (absolute value and phase sensitive displays, DQF- COSY) TOCSY Through-space NOESY ROESY Heteronuclear Through-bond HETCOR Long-range HETCOR or COLOC INADEQUATE INDIRECT-DETECTION (HSCQ, HMQC, HMBC) Pulse Field Gradients (PFG) NMR- the acronym soup! (NMR die Suppenterrine voll Abkürzungen)

29 Example: Lactose

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31 2 sugar units 2 separate groups of coupled spins (termed spin systems) Multiple overlap of the sugar resonances, and of the resepective cross peaks Canweusethegood separation of the anomeric protons?

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33 Lactose - 2D TOCSY

34 Lactose -ROESY Equivalent Experiment to the NOESY Coupling through space Detects close proximity of protons Expansions:

35 References J. B. Lambert & E. P. Mazzola, Nuclear Magnetic Resonance Spectroscopy, Pearson Prentice Hall, 2004 RM Silverstein, FX Webster & DJ Kiemle, Spectrometric Identification of Organic compounds Wiley 2005.