核磁共振光譜與影像導論 Introduction to NMR Spectroscopy and Imaging Lecture 05 Basic Two-Dimensional Experiments (Spring Term, 2011) (Fall Term, 2011) (Spring Term, 2014) (Spring Term, 2015) (Spring Term, 2016) (Spring Term, 2017) Department of Chemistry National Sun Yat-sen University

Basic Two-Dimensional Experiments Why multi-dimensional? Resolution Multi-quantum coherences Coherence transfer Types of multi-dimensional spectra: To Correlate To Resolve To Exchange (Diffusion)

1D is not enough for large molecules

Resolution improvement by using higher dimensions ,jasncjkhdsjkjk;dfaigvjtreitgikvcxmlfdlkvfdklvkfdkmfvkdfkvfdd.,fvdvfdmgfvdklfkfdffd 2D: ,j s k g v k d v f d n j l o g k l a b n d f j k y d f m n w s q s h a g v m d f d

Resolution improvement by using higher dimensions

Resolution improvement by using higher dimensions Hidden, complicated correlations can be revealed through cross peaks in a multi- dimensional spectrum, with which the chemical bonding, spatial closeness or dynamical processes may be indentified.

Why multi-dimensional? Resolution Multi-quantum coherences Coherence transfer Types of multi-dimensional spectra: To Correlate To Resolve To Exchange (Diffusion) structure dynamics

From 1D to 2D

From 1D to 2D t2 t1

If you look at the same data matrix in a different way…. You also get a series of FIDs.

In either direction, you see FIDs You really don’t know which is “acquisition dimension”, Do you?

A 2D Spectrum: A Single Peak FT2 t1 f2 FT1

F2 A 2D Spectrum t2 FT2 FT1 F1 t1

2D FT

Overview: Basic 2D Experiments Homonuclear experiments (proteins < 8 ~kDa): COrrelation SpectroscopY (COSY) J couplings Double Quantum Filter COSY (DQFCOSY)J couplings TOtal Correlation SpectroscopY (TOCSY) Side chains Nuclear Overhauser Effect SpectroscopY (NOESY)Distances … Heteronuclear experiments (proteins > ~8 kDa, connectivity): HETeronuclear CORrelation spectroscopy (HETCOR) Heteronuclear Single Quantum Coherence (HSQC) Heteronuclear Multiple Quantum Coherence (HMQC)

Typical 2D Lineshapes

Resolution The resolution in the second dimension is determined the same way as in 1D NMR. The resolution in the first dimension is determined by the longest evolution time in the first dimension (maximum of t1). The total evolution time in the indirect dimension is equivalent to the total acquisition time in the detection dimension.

Window and Zero-Filling t2 t1 FT2 window（pink） Zero-filling 20484096（brown） 256 FIDs F2 4096 points Increased to 4096 FIDS F2 （4096 points） F1（4096points） FT 1 Window in t1 （dashed pink）

COSY pulse sequence (top) with a presaturation pulse to suppress water signal. Also shown in the diagram are the coherence transfer pathway (middle) and phase cycling scheme (bottom).

Through Bonds: COSY F2 (ωB, ωA) (ωA, ωA) (ωA, ωB) (ωB, ωB) F1 Evolve on spin A (B) during the incremented delay t1 Partially transfer magnetization from spin A (B) to spin B (A) during mixing time Detect signal during t2

+ - - + +- -+ -+ +- First diagonal peak F1

+ - - + +- -+ -+ +- second diagonal peak

+ - - + +- -+ -+ +- second cross peak (SI) first cross peak (IS)

+- -+ -+ +- + - - + F2 Dashed arrow: former life (t1) + - - + +- -+ -+ +- coherence transfer from S to I F2 Dashed arrow: former life (t1) Solid arrow: this life (t2) coherence transfer from I to S F1

Strychnine

2D Correlation Spectroscopy (COSY) Experiment –magnetization transfer through scalar (J) coupling. No signal from correlations that are more than three bonds apart since 4J ≈ 0.

Cross-Peak Intensity and J Coupling The relationship between the cross-peak intensity and the magnitude of the J coupling is rather complicated： 1. For large J couplings (J>10 Hz), cross-peak intensity does not depend on J coupling, but is affected by relaxation rates. Weak intensity is usually from fast relaxation in the first dimension. 2. When J coupling is small and the maximum of t1 is not long enough, the cross peaks may be weak, even vanish. Increasing maximum t1 can improve (the total experimental time is increased).

A 2D dispersive peak

A 2D absorptive peak

+ - - + +- -+ -+ +-

Absorptive cross peaks Dispersive diagonal peaks Dispersive cross peaks Absorptive diagonal peaks

Absorptive cross peaks

Absorptive cross peaks

Dispersive diagonal peaks

F E D C B A

1 2 5 3 4 6 7 8 9 10 1 3 2 9 6 7 4 8 5 10

Absolute Value Display

The major drawback of COSY: cross peaks near the diagonal line may be obscured by diagonal peaks.

DQF COSY

The Advantages of DQF COSY Having the same phase for both the diagonal and cross peaks; Because the magnetization is detected in anti-phase, the multiplet structure is retained with opposite phase. In other words, the two lines of a doublet are 180° out of phase with respect to each other. While this can lead to cancellation in crowded regions of the spectrum, it also allows for the easy identification of multiplets (based on their square, or box, shape), and for measuring the size of the scalar coupling constant connecting the two spins. Another advantage of the DQF-COSY experiment is not so immediately obvious. In order for a transition to create multiple quantum levels, and to survive a multiple quantum filter, you need to have at least two spins or three spins for a 2Q or 3Q transition, respectively. Thus, singlets are drastically reduced in intensity in a DQF-COSY spectrum.

The Disadvantages of DQF COSY There are also disadvantages to the DQF-COSY relative to COSY. First, the sensitivity of DQF is about a factor of two lower than regular COSY. Second, MQ relaxation in 1Hs is very slow, so the experiments require a relatively long d1 relaxation delay. Thus, while a good COSY spectrum could be generated in about 2-4 hours of data accumulation, a good DQF-COSY requires about 16-24 hours to allow complete relaxation during d1.

COSY Family COSY COSY-45,COSY-90 R-COSY (LR-COSY) pQF COSY PE-COSY MQ COSY TOCSY zCOSY,zzCOSY, J.R.COSY …..

3JHNα = 5.9 cos2Φ - 1.3cos Φ + 2.2   3Jαβ = 9.5 cos2χ1 - 1.6cosχ1+ 1.8

J Coupling Measurement J coupling and peak intensity J coupling and linewidth J coupling and overlap

With D2O as solvent, NH2 and NH are not observed owing to exchange between D and H.

2D-NMR COSY spectrum of Rhodococcus sp. ZS402 overlaid with medium 2D-NMR COSY spectrum of Rhodococcus sp. ZS402 overlaid with medium. Signals in orange are from the sample and signals in grey are from the medium.

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