2D-NMR spectroscopy Part 2

F.D. SönnichsenThursday, Oct 23 2008

The 2D - COSY

Periods in a 2D: Preparation frequency labeling acquisition

mixing

Repeat n times, each time with incrementally increased Δt1Keep 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:

90xT1=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 KernenDas HETCOR Experiment(traditionelle,ursprüngliche Weg, Protonen mit HeteroAtomen zu koppeln)Jetzt selten benutztNOTE: 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 DetectionExperiments 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 Hγ 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 Hγ 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

IpsenolNote : 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 textp. 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 lineupon saturating –changing the population of another spin in proximity

NOE = c x r -6Noes can be measured for protons that are in close proximity, generally if they are less than 5Å

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

αα

αββα

ββ

ω13 = ω24 = ωIω12 = ω34 = ωS

ω12 , S

ω24 Iω34 , 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 respectivepopulation differences are:

<Iz> = 1/2 (P1 - P3 + P2 -P4 ) and<Sz> = 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

αββα

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

2NOE

WWWWW

II

IS

++−

===ϕση

For small molecules , W2 is the dominant relaxation pathway leading to positive NOEsFor 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

Difference in z-component = population change, causes relaxation, is transfers magnetization, mixes S with I and vice versa

)()(1 SoSzIoIzdt

dIzIS −−−−= σϕ

During t1: frequency labeled at ωsDuring tmix transferred to I

During t2 detected with I (ωI) ),(),( 21 ISI

ISNOE ϖϖϕσϖϖ =

t1

y

z

90x

90x

Aphanamol-1

This NOE showed that the 5-membered ring is cis to the 7-membered ring.

5H

15CH2

• Through-bond• COSY (absolute value and

phase sensitive displays, DQF- COSY)

• TOCSY

• Through-space• NOESY• ROESY

• Through-bond• HETCOR• Long-range HETCOR or

COLOC• INADEQUATE• INDIRECT-DETECTION

(HSCQ, HMQC, HMBC)

Homonuclear Heteronuclear

Pulse Field Gradients (PFG)

NMR- the acronym soup!

(NMR – die Suppenterrine voll Abkürzungen)

Example: Lactose

• 2 sugar units• 2 separate groups of

coupled spins (termed spin systems)

• Multiple overlap of the sugar resonances, and of the resepective cross peaks

• Can we use the good separation of the anomeric protons?

Lactose - 2D TOCSY

Lactose -ROESY

• Equivalent Experiment to the NOESY

• Coupling through space

• Detects close proximity of protons

Expansions:

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.