2D NMR:

Applications
IUPAB sponsored Workshop on NMR & its
Applications in Biological Systems
November 23-30, 2009

Mamata Joshi
TIFR, Mumbai
What is 2D NMR?

When and Why?

How to apply?
 WHAT?
Stack of several 1D
spectra
Each 1D is different from
the next by a small
change in the evolution
time t1
Parameters for each
successive experiment
in the series are
constant except the
phase of the pulses
FT of the two time
domains provides a map
of spin-spin correlations
2D spectrum actual view
 Basic features of 2D spectra
HA HB
HA HB

diagonal peak (F1=F2)

H chemical shift (ppm)

crosspeak:
correlation of two
different resonances
by short interatomic
distance or through-bond
1

connection
H chemical shift (ppm)
1
 WHEN?

Overlap of signals in the 1D spectrum

No overlap, easy to assign

 WHY?
What information are you seeking? Corelation?
Characterisation/assignments of resonances in the molecule?
Orientation of different groups/segments of the molecule in
space/ w.r.t. one another (stereochemistry)?

Two main types of homonuclear 2D techniques

Scalar coupling (J) based NOE based

NOESY- Nuclear Overhauser
COSY -COrelation SpectroscopY
Effect SpectroscopY
TOCSY- TOtal Corelation ROESY- ROtating framE
SpectroscopY SpectroscopY
MQF- Multiple Quantum Filter
spectroscopy
2D-NMR sequences contain two types
of time periods:
evolution time and mixing time

evolution time: mixing time:

a second frequency magnetisation is
dimension is created by transferred from spin to
indirect detection spin via interactions
between spins
J-correlation based 2D experiments
Facilitated by electrons in
bonds separating nuclei

Interaction results in
splitting of resonance
lines in 1D spectrum or
cross peaks in 2D

Magnitude of J coupling
is dictated by torsion
angles between two
coupling nuclei given by
the Karplus equation
J = A + BCos+ CCos2
A, B & C are empirical
constants
 COSY

magnitude mode
upto three-bond coupling
network seen
ideal for small molecules with
simple coupling networks
easy processing, no phase
correction
adequate if only coupling
network has to be established
 C 3H 8O
CH3 CH2 CH2 OH

CH2 OH CH2 CH3

 C3H8O: COSY

CH3
CH2 OH
CH3 CH2 CH2 OH CH2
 DQF COSY
Phase-sensitive, pure absorption
lineshapes with +ve & -ve
components

Higher resolution

J values can be determined

Less pronounced diagonal ridge,

so easy to assign cross peaks
close to diagonal

Elimination of strong (solvent)

signals not involved in
homonuclear J-coupling
COSY Vs DQFCOSY
 TOCSY

Cross peaks generated between all members

of a coupled spin network due to
magnetisation transfer from one spin to
another even without direct coupling
Pure absorption mode spectra with positive
intensity peaks created
Ideal for assignment of spins in biomolecules
(polypeptides/nucleotides) since
characteristic peak patterns rendered for
specific spin systems.
 Other COSY-type experiments
COSYLR
Long range coupling
networks probed J-Resolve

HOMO2DJ-Resolve
renders J directly on the F1-
axis of the 2D plane with on
the F2-axis

ECOSY
simplifies complex splitting
patterns by retaining only
direct couplings & eliminating
signals due to passive
coupling
 NOE-based experiments
NOESY

Useful to identify spins

undergoing cross-relaxation

Direct dipolar couplings provide

primary means of cross-
relaxation, & so spins undergoing
this are close to one another in
space (<5A)

Intensity of peaks depends on

parameter, m, the mixing time

Indicated in the form of cross

peaks in the NOESY spectrum
 Mixing time m NOESY

For small molecules m ~ T1

For large molecules low m is

recommended to avoid spin-
diffusion effects

For medium sized molecules

(MW about 500-1000), NOE =
0 in many cases. ROESY is the
solution!

NOE 1/r6, cross-peak

intensity can be used as a
measure of distance between
the two protons
 NOESY applications
Which isomer is responsible for this spectrum?
NOESY

H4
H3

OMe
H5
 NMR of Biomolecules

NMR is the only method for

residue level structure
determination of
biomacromolecules in
aqueous solutions at near
physiological conditions

Depending on the size of the

molecule, one can go for
homunuclear/heteronuclear
2D corelation or even 3D or
4D!

Dynein light chain protein (DLC8)

89 residues, MW~10.3kD
 Structure Determination Strategy for
Polypeptides

Identify resonances for each amino acid

Put the assigned resonances in order according to
their amino acid sequence i.e. sequential assignment
(R-G-S, T-L-G-S)
Now do the sequence-specific assignment
Secondary structure determination using NOESY,
(short distances), temperature coefficients
delineation of H-bonded amide protons), coupling
constants J (torsion angles).
 COSY peak patterns of amino acids
Thr
Ala

Leu

Ser, Cys, Asp, Asn

TOCSY patterns of amino acids
TOCSY-NOESY sequential walk in polypeptides
TOCSY spectrum renders
NH- H cross peaks (J-
coupled) in fingerprint
region.

NOESY fingerprint shows

both self & sequential
NOE cross peaks
NHi-Hi (self)
NHi-H(i-1) (sequential)
 From nOes
NH-NH region is the most important followed by the NH-H & the
H-H regions
 Secondary structure elements in peptides
-helix
Clockwise spiral, NHs run upward, COs downward
Stabilised by H-bonds along the chain
Characterised by short sequential & medium-range 1H-1H distances
 -sheet
Two peptide strands running alongside each other either in the same or
opposite directions
Stabilised by H-bonds crossing between the chains
Characterised by short sequential & long-range backbone distances
 -
turns
Units of four amino Type I

acids turning back dNN(2,3)

dNN(2,4)
upon themselves dN(2,4)
Stabilised by H-bonds
between residues at
the corners Type II
dNN(2,3)
dN(2,4)
dN(2,3)
Structure Determination Strategy for nucleic acids
I (H2O) II (H2O)
Assignment of Assignment of non-
imino/amino to establish exchangeable protons
base pairing (NOESY) NOESY imino-
H2/H8/H5/H1

III (D2O)

Identification of aromatic
Sequential resonance Cytosine/Thymine Identification of sugar
Assignment NOESY H5/H6 spin systems proton spin systems
H6/H8-H1, H6/H8-H2H2 COSY/TOCSY COSY/TOCSY

IV (D2O) V

Assignment of 31P resonances & Structure Determination using

confirm/extend H3, H4, H5, H5 Torsion Constraints/Distance
assignments. Constraints
(1H,31P ) HETCOR
NOESY-
DNA H2/2 -> H1

H2/2 -> Base

H1 -> Base

H2
 ROESY-rotating frame NOESY

NOEs measured under spin-locked

conditions

Suitable for molecules with c~1

where noes are absent in NOESY

Spin exchange between dipolar coupled

spins occurs during the spin-lock period

Strength of the spin-lock needs to be

optimized to minimise TOCSY & COSY
type artifacts

Cross peaks are opposite in sign with

diagonal & chemical exchange peaks
 Heteronuclear NMR
For small molecules generally
homonuclear 2D techniques are
sufficient for structure elucidation.

However, when there is extensive

overlap even in the 2D spectrum it
helps to do heteronuclear
correlation experiments

It is a technique which helps to

determine which 1H of a molecule is
bonded to which X nucleus in the
molecule. Transfer of magnetization
takes place between nuclei of
different types. The two axis
show the chemical shift of the
respective type of nucleus. If a
transfer has taken place (due to
coupling), a signal appears at the
intersection of the two frequencies,
 Heteronuclear correlation
HSQC HMQC HMBC
one-bond 1H-X one-bond correlation two- & three-bond
correlation Shorter pulse correlation (modified
HMQC)
Longer pulse sequence & is
program, therefore therefore less Less sensitive than
sensitive to sensitive to the HMQC
calibration & tuning calibration & tuning Popular among NMR
errors errors spectroscopists in
Ideal for Ideal for small organic labs/pharma
macromolecules, molecules industries
very popular in
protein NMR
C5H8O2
5 2
34

C3
C4
C2

C5
 Heteronuclear Corelation in Proteins

Use of 13C & 15N as NMR-active nuclei

Facilitate structure determination especially of large
proteins (>100 AA)
Since both the gyromagnetic ratio as well as the natural
abundance of these is very low as compared to 1H, to
get better sensitivity, these samples are subjected to
isotopic enrichment. i.e. 12C 13
C and 14N 15
N
 15
N-1H HSQC of proteins

The 1H-15N HSQC is the most

important corelation experiment
for the protein NMR spectroscopist

The number of peaks in its

fingerprint region directly gives the
number of amides in the protein
since there is only one backbone
NH per amino acid.

It also contains signals from the

NH2 groups of the side chains of
Asn and Gln and of the aromatic
NH protons of Trp and His.
 Moral of the story!

Depending on the system you are studying, a

small organic molecule or a complex
biomolecule, a judicious choice from the vast
range of experiments available can be made
which can then make your task of structure
determination faster and simpler.
Thank you