Chapter 5 無機物理方法（核磁共振部分） The Physical Methods in Inorganic Chemistry (Fall Term, 2004) (Fall Term, 2005) Department of Chemistry National Sun Yat-sen University Chapter 5

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)

From 1D to 2D, 3D… 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?

F2 A 2D Spectrum t2 FT2 FT1 F1 t1

2D FT

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.

Acquisition and data processing: F2 F1

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).

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).

Recollecting Chapter 2

Four basic lineshapes Anti-phase absorptive FT Real part In-phase dispersive FT Real part Anti-phase dispersive FT Real part FT Real part In-phase absorptive

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 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

1 2 5 3 4 6 7 8 9 10

1 2 5 3 4 6 7 8 9 10

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.

ε δ γ β α

TOCSY region of amide protons from residues Lysine 29 and 48

TOCSY of BPTI at different pressures

TOCSY of BPTI at different pressures

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

Basic Heteronuclear 2D Experiments Introduction Heteronuclear correlation (HETCOR) and its variants (COLOC, HMBC etc) Heteronuclear multiple-quantum coherence (HMQC) Heteronuclear single-quantum coherence (HSQC)

Introduction The availability of other (“hetero”) nuclei than proton for multi-dimensional NMR spectroscopy is extremely useful, particularly, for macromolecules (of synthetic or biological origin) because the connectivity between protons and heteronuclear spins can be established in addition to the homonuclear multi-dimensional spectra. Like homonuclear 2D spectroscopy, the central task in heteronulcear 2D experiments is to select proper coherence pathways so that the desired interaction can be elucidated. Heteronuclear 2D experiments can be more diverse because, e.g., the data acquisition can be carried out on heteronuclei (C-13, N-15 etc) or on protons (inverse detection).

Heteronuclear correlation (HETCOR) and its variants

During t1, the carbons that are coupled to protons are selected so that only those carbons are “visible” in the detection period. The choice of two fixed delays Δ1 and Δ2 is determined by smallest J-coupling we want to observe, i.e., when long- range coupled carbons (in addition to directly bonded carbons) are also to be observed: Δ1=1/2Jmin, Δ2=1/3Jmin.

Enhanced Heteronuclear Correlation S(δI, δS) ωI ωS

COrrelation via LOng-range Coupling(COLOC) The choice of two fixed delays Δ1 and Δ2 is determined by smallest J-coupling we want to observe, i.e., when long- range coupled carbons (in addition to directly bonded carbons) are also to be observed: Δ1=1/2Jmin, Δ2=1/3Jmin.

Heteronuclear Multi-Bond Correlation (HMBC)

Inverse Detection

HSQC

Points a-d (INEPT)

Points e-g (Reversed INEPT)

Phase Cycling

HMQC

Difference of HMQC and HSQC(I)

Difference of HMQC and HSQC(II)

2D J Spectroscopy 2D methods that show coupling multiplets versus chemical shift. There is both a homonuclear and a heteronuclear version. Both methods are useful for resolving overlapping multiplets. They are also used to measure coupling constants. These sequences allow both J coupling and chemical shift evolution but re-focus the chemical shifts like a spin echo sequence (90 - - 180 -- acq).

t1: J; t2: J+CS t1: JIS, t2: JIS+CSS

Summary HETCOR, COLOC HMBC HSQC/HMQC J spectrosc. HSQC is now a “platform” for many more complicated pulse sequences.