2D-NMR spectroscopy Part 2 - 2D-NMR spectroscopy Part 2 F.D. S£¶nnichsen Thursday, Oct 23...

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Transcript of 2D-NMR spectroscopy Part 2 - 2D-NMR spectroscopy Part 2 F.D. S£¶nnichsen Thursday, Oct 23...

  • 2D-NMR spectroscopy Part 2

    F.D. Sönnichsen Thursday, Oct 23 2008

  • The 2D - COSY

    Periods in a 2D: Preparation frequency labeling acquisition

    mixing

    Repeat n times, each time with incrementally increased Δt1 Keep the individual FIDs separate.

    Directly observed = Direct dimension

    Indirect frequency determination = indirect dimension

  • Vektor-Diagramm Analyse des 2D-COSYs –die Frequenzbestimung in der indirekten Dimension

    FID 1:

    FID 2:

    FID 3:

    FID 4:

    90x T1=0

    T1=τ

    T1=2τ

    T1=3τ

    90x

    Acq

    Acq

    Acq

    Acq

    t1

    90x 90x

    t2

    n

    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?

    x

  • Sollte wie eine Sinus – Modulation aussehen

  • Fourier transform with respect to t1

  • 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

  • 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 1J-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

  • Heteronuclear Indirect Detection Experiments that detect 1H involved in coupling to an X nuclei (13C or 15N). Sensitivity is enhanced by a factor where γ

    is the gyromagnetic ratio.γ H γ X

    ⎛ ⎝⎜

    ⎞ ⎠⎟

    3/2

    Signal/Noise comparisons

    Nuclei No NOE Full NOE

    Polarization Transfer for Direct DET

    Indirect Detection

    13C +1 +3 4 32 15N +1 Š4 10 306

    γ H γ X γ H

    γ X ⎛ ⎝⎜

    ⎞ ⎠⎟

    3/2

    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!!

  • The HMBC experiment

    • We have connected neigboring protons (COSY) • We have connected protons to attached 13C with the direct 1J coupling

    (HMQC) • Can we use long range couplings and connect further protons?

    The HMBC SELECTIVELY? observes protons and 13C that are connected via small 2,3J couplings ( of the order of 10 Hz).

    MB stands for Multiple bonds

  • Comparison HMQC / HMBC

    C-H, 1J=145,

    delay = 3ms

    long range C -H, 2J or 3J ~ 10Hz,

    delay = 50ms

  • 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.

  • 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 ……

  • 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

  • 2D - INADEQUATE

    Incredible Natural Abundance Double Quantum Transfer Experiment

    Allows 13C—13C connectivities to be obtained

    1. A. Bax, Two-Dimensional Nuclear Magnetic Resonance in Liquids, Delft University Press, Delft, Holland (1982) pp. 155-174.

    2. 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. 178 - Nakanishi’s text p. 279 - Silverstein & Bassler & Morril

    Sensitivity is extremely low, because 13C 1.1% 13C—13C 0.01% Correlates double-quantum frequency of 13C—13C

    Many variations provide same information, but in different formats

    Usually the cross peaks are 2 peaks, representing AX 13C—13C doublets

    Pulse sequences are complex and involve long phase cycling to select for desired double quantum coherences

  • 13C 13C

  • 2D-TOCSY experiment

    • Total Correlation Spectroscopy • Correlates simultaneously all atoms in a

    spin system

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

  • 2D-TOCSY experiment

    B

    A

    D

    F

    A B D F

    Diagonal

    TOCSY

    Spin system : A, B, D, F

    COSY

  • TOCSY of a small peptide

  • Compare the COSY of this peptide

  • Correlation through space

    semi-quantitative measurement of of local proximity

    NOE difference spectroscopy

    t1

    t2t1

    t2

  • 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.

    η

    = I - Io

    Io

    nOe definition: 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

  • Noe-background 1 Let’s consider: 2 spins , I and S. They are not coupled ( J=0).

    αα

    αβ βα

    ββ

    ω13 = ω24 = ωI ω12 = ω34 = ωS

    ω12 , S

    ω24 I ω34 , S

    ω13 I 1

    2

    4

    3

    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:

    = 1/2 (P1 - P3 + P2 -P4 ) and = 1/2 (P1 - P2 + P3 -P4 )

    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 , WS , W2 , W0 )

    αα

    αβ βα

    ββ

    W1 , S or WS

    W1 , I or WI W1 S

    W1 I

    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

  • • It can be easily shown that

    02

    02

    2 NOE

    WWW WW

    II

    IS

    ++ −

    === ϕ ση

    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

  • 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 off- diagonal peaks, connecting the resonance frequencies of the spins which couple/ cross relax through space.

    d1 t1 tmix t2

    90 90 90

    Tmix is usually chosen to be between 500ms and 2 sec

  • 2D-NOESY: vector model

    x

    y

    z

    x

    y

    z

    x

    y

    z

    x

    y

    z

    x

    y

    z

    Different y-component

    D