2D Correlation Experiments:

HSQC, HMQC, HMBC,

BCMB/CHEM 8190
 Two Dimensional NMR Spectra

A General Scheme: other mixing and evolution

periods can be added to increase dimensions

Preparation Evolution 1 Mixing Evolution 2

(Increment t1) (observe t2)

time

Example: COSY – mixing is scalar coupling

90x 90x (mix)
d1 (recover) t1 (evolve) t2 (observe)
2D –NMR - FIDs are Transformed in t2, then in t1

t1

t1
FT

ν1

ν2
ν2
Heteronuclear Single Quantum Coherence

HSQC

1H-15N Pairs in Amide Bonds of Proteins

Provide Convenient Reporters at Each
Amino Acid Along the Backbone Just
Requires 15N Labeling
 HSQC Spectrum of H-N Amides in a Protein

Red – without PI(4)P

Blue – with PI(4)P

Phosphoinositide interactions with PH domain of FAPP1 at a bicelle surface

Heteronuclear Magnetization Transfer as Used
in the INEPT and HSQC Experiments

1H 15N

1H(I)
τ τ
90x 180y 90y
15N(S)

90x

Iz Æ -Iy Æ -2IxSz Æ -2IzSy τ = 1/4J

 Evolution and Detection in HSQC
180x 90x 180x
t2
90x 180x
t1/2 t1/2 τ τ decouple
Sz (ωS, t1) (180 decouples I)
-2IzSy -2IzSy cos(ωS t1) + 2IzSx sin(ωS t1)

Ix(π/2)+Sx(π/2)
2IySz cos(ωS t1) - 2IySx sin(ωSt1) (unobserved 2Q)

IzSz (J2τ) (chem shift refocuses)

-Ix cos(ωS t1)

Iz (ωI, t2)
-Ix cos(ωS t1)cos(ωI t2) -Iy cos(ωS t1)sin(ωI t2)
Obtaining Quadrature: Substitute a 90y 15N Pulse
and Save Separately (States)
1H(I ) τ τ
90x 180y 90y
15N(S)

90y
Iz Æ -Iy Æ -2IxSz Æ 2IzSx τ = 1/4J
180x 90x 180x
t2
90x 180x
t1/2 t1/2 τ τ decouple

Sz (ωS, t1) (180 decouples I)

2IzSx 2IzSx cos(ωS t1) - 2IzSy sin(ωS t1)
 Important Features of HSQC Spectra

• Start with 1H magnetization: gain γH / γN

A factor of 10 in sensitivity
• Detect 1H magnetization: gain (γH / γN)2
Another factor of 100
• Even though you must acquire 32-64 t1
points, far more sensitive than direct
detection.
1H-13C HSQC can be
run at Natural
Abundance

3
1
Spectral Display of HMQC Looks Just Like HSQC

1H(I)
τ τ t2
90x 180y 180x 180x
15N(S) t1/2 t1/2 τ τ decouple
90y 90x 180x

Iz Æ -Iy Æ -2IxSz Æ -2IxSx

-2IxSx = (-IxSx + IySy) + (-IxSx –IySy) = zero and 2Q coherence

180x changes sign of IySy parts, hence interchanges zero and 2Q

Evolves as chemical shift of 15N, just like HSQC

Differences in relaxation properties and lack of direct coupling

HMBC – coherence transfers can be tuned to eliminate
one-bond coupling and emphasize long-range couplings
1H(I)
τ τ t2
90x 180y 180x 180x
15N(S) t1/2 t1/2 τ τ decouple
90y 90x 180x

Choose τ = 1/4JIS1 for three-bond: Iz Æ -Iy Æ -2IxSz Æ -2IxSx

-2IxSx = (-IxSx + IySy) + (-IxSx –IySy) = zero and 2Q coherence

Choose τ = (2n+1)1/2JIS1 for one-bond: -Iy Æ Iy Æ Iz = no signal

HMBC of Carbohydrates is Useful in
Identifying Linkages
2 HMBC
HSQC
3