“Newer Advances in NMR spectroscopy”

Application of NMR Techniques”

 Old CW-Instrument HR FT-NMR
( Theory-PHYSICS) (Cryo-Technology-Engineering)

FT-NMR(Application –
Chemistry/Biochemistry/Med Magnetic Resonance Imaging
icine/Material science) (Diagnosis-Medical)
 NMR Research Front (Techniques)
1946: Basic Discovery

1950-55: Chemical Shift, Coupling Constants, Over Hauser Effect, Spin Echo

1952: Nobel prize in Physics – F. Bloch and E. M. Purcell

1960-65: Spin decoupling, Cross Polarization

1966: Fourier Transform NMR (1D-NMR)

1971-76: Two Dimensional NMR (2D-NMR)

1973: Magnetic Resonance Imaging (MRI)

1985-90: Multidimensional NMR (3D-NMR and others)

1991: Nobel prize in Chemistry – R. R. Ernst

2002: Nobel prize in Chemistry – K. Wuthrich

2003: Nobel Prize in Physiology and Medicine – P. Lauterbur, P. Mansfield

NMR Research Front (Molecular Applications)

1946-66: Small molecules (< 30 atoms)

1966-75: <100 atoms, Analytical tool for

Chemists

1975-85: <1000 atoms, Biomolecules, Small

Proteins, Nucleic acids, Cellular-NMR

1985-95: <3000 atoms, MW < 15 kD

1995-till date: Large Biomolecules, MW >15 kD,

Dynamics and Molecular interactions
 NMR Technology Front
Magnetic Field Strength ( Tesla) Larmor Frequency(1H MHz)
1946-60 0.7 – 2.3 30-100

1960-70 2.3 – 4.7 100-200

1970-80 4.7 – 11.7 200-500

1980-90 11.7 – 14.1 500-600

1990-2000 14.1 – 18.8 600-800

2000-2009 21.1 – 22.3 900-950

2010 23.5 1000(Giga)

Advances in Computer Technology have contributed immensely
to spectrometer hardware
 NMR Basic Principles

2H nucleus 4He nucleus

1H nucleus
1 proton 2 protons
1 proton
1 neutron 2 neutrons
magnetic
magnetic Non-magnetic

12C nucleus 13C nucleus

6 proton 6 protons
6 neutrons 7 neutrons
Non-magnetic magnetic
 NMR Frequency table
Natural % Magnetic Magnetogyric
Isotope Spin (I)
Abundance Moment (μ) Ratio (γ) *
1H 99.9844 1/2 2.7927 26.753
2H 0.0156 1 0.8574 4,107
11B 81.17 3/2 2.6880 --
13C 1.108 1/2 0.7022 6,728
17O 0.037 5/2 -1.8930 -3,628
19F 100.0 1/2 2.6273 25,179
29Si 4.700 1/2 -0.5555 -5,319
31P 100.0 1/2 1.1305 10,840
* γ has units of 107rad T-1 sec-1
 Internal Nuclear Spin Interactions
Spin>½, 23Na, 17O ….. Spin Interactions Spin ½, 1H, 13C…..

Electric Magnetic

Quadrupolar Chemical shift Spin-spin couplings

Isotropic
chemical shift

Chemical shift Scalar, J-

Dipolar
anisotropy, CSA couplings

Isotropic quad.
shift Heteronuclear Homonuclear
1st, 2nd order quad.
Interaction, anisotropic
without Magnetic
Field

B0

Sample Spin
Energy
Level

E
 Types of NMR Spectra
Types of solution NMR High Resolution Spectra
• One-dimensional spectra(1D)
• Two-dimensional spectra(2D)
• Three-dimensional spectra(3D)
• {Homonuclear 3D,Heteronuclear double resonace 3D, Heteronuclear triple resonace 3D}

• Solid State NMR spectra (CP-MAS)

• LC-NMR {ON-FLOW,STOP-FLOW, Fraction LOOP method}
Art of NMR Spectroscopy

Intensity proportional to
population difference
 NMR Basic Equation
B0
 = ------------
2

But E = h

hB0
E = -------------
2
This is the fundamental NMR equation.
where  magnetogyric ratio or gyromagnetic
ratio.

2
 = -------------
hI
= Magnetic moment
I = Spin number
Nuclear Spin Flipping
How many spins have
parallel and anti-
parallel orientations?
N /N = e-E/kT
Assuming: B0 =9.4 T
(400 MHz magnet)
Total No.=20’00000
N =10’00001
N = 9’99999
 Field-Frequency Relationship

Energy
Level

E

200 MHz
500 MHz
4.7 T
11.4 T 900 MHz
21.1 T
 How about Sensitivity of NMR ？

IR

MS
NMR
Equilibrium Magnetization
 Resonance Absorption

The oscillating RF can be thought of as two +

rotating fields (B1) in opposite directions.
 Relaxation Process
The NMR signal (FID, free induction decay) disappears
with specific time constants (=Spin-lattice/Longitudinal
relaxation T1 and spin-spin/Transverse relaxation T2):

→ T1, (T2)

• Proximity of other nuclear spins supports relaxation →

short T1
• T1 is also dependant on molecular motion
(correlation time τc)
 Fourier Transform
z z
Bo
Field
Vector
Summation
y y

x x

Time
Domain FT
Energy
Energy

Frequency
Domain

Frequency
Time
 Pulse Sequence z

y 90o pulse y

M
B1
x x
 The NMR Information
• Chemical Shift
• Spin-Spin Coupling
• Intensity(Area) of Signal
• Relaxation(T1,T2)
• NOE
…but also dynamics and molecular
interactions:
• Dynamic NMR (effects on Lineshapes)
• Saturation transfer
• Diffusion
 Chemical Shift ()
Chemical shift position are normally expressed in ( delta )
units, which are defined as proportional difference in parts
per million ( ppm ) from an appropriate reference standard.
It is a dimensionless number and independent of field
strength.

x-TMS
x = -------------
0

where 0 is the operating frequency of the Instrument (in

MHz )
1H –NMR Spectra of Pentoxifylline

O O CH3
6 4
8
N
H3C N
7 5 1

O N N
CH3
3

CDCl3 TMS
 Chemical shift depends on :-
• Inductive effects (electron density variations,
through bonds)

• Mesomeric effects (through bonds)

• Charge effects (through space)

• Steric effects (through space)

• Anisotropy effects (electronic orbital & space)

 Spin-Spin Coupling/Spin-Spin Splitting
The number of lines (multiplicity) observed in the NMR signal for
a group of protons/carbons is not related to the number of protons in
that group, the multiplicity of lines is related to the number of
protons in neighboring groups
(n+1) rule :The simple rule is to find the multiplicity of the signal
from a group of protons, count the number of neighbors) & add 1
 Coupling Constant ( J ) ( Magnitiude of the coupling )

 The coupling constant , J in Hz is a measure of the interaction between

nuclei.

 The interaction is transmitted through the intervening electrons of it’s

own nuclear spins.

 Which is wholly independent of any external magnetic field.

Types of coupling
 Geminal coupling
 Vicinal coupling
 Long-range coupling ( W-coupling )
 Cis-Trans coupling
 Aromatic coupling
 Allylic coupling
 Virtual coupling
 Interactions between spins
Nuclei sense the presence of other nuclear spins in their
vicinity:
→ signal splitting, spin-spin coupling
→ described by the coupling constant J
• Mediated through bonds, “scalar coupling”
• Distance dependant (usually up to 3 – 4 bonds
• Dependant on parameters such as: bond order,
bond length, bond angles, orbital overlap
• Information about 3D geometry (Karplus rule)
Dihedral angle (θ):
 Homonuclear Decoupling
The chemical shift difference between signals expressed in Hz is much larger than the
coupling constant J.

  6J{Then it is the conditions for the first order spectra}

If  < 6J ( Then Non first order spectra )

Simplification of complete proton NMR spectra

1. Increased field strength

2. Spin- decoupling, Homonuclear decoupling ( Double irradiation )
3. Use of shift reagents -Eu{DPM}3, Eu{FOD}3, Eu{FHD}3

8
7 CH3
H2C
1
6 2

5 3
4
1D NOE (Nuclear Over Hauser Effect)
• Depends on relaxation between spins
• Saturation of spin A → signal intensity change for B
• Strongly dependant on distance between A and B

Difference spectrum
Proton decouple 13C NMR Spectra
(a) The convenient notation 13C –[1H].
(b) Deuterium coupling (2nI+1) splitting pattern. (with low intensity
due to high T1 time of Deuterium).
(c) NOE signal enhancement.
(d) No quantitative measurement of line intensities.
(e) No heteronuclear spin-spin couplings.
(f) So removal of coupling multiplicity makes the spectrum simpler in
appearance with sensitivity bonus.
(g) DEPT – Experiment is must

8
7 CH3
H2C
1
6 2

5 3
4
 Gated Broad Band Decoupling

(a) NOE is a slow process which has build up prior to the 13C pulse
decays only practically during the pulse and acquisition period.

(b) However coupling a fast process is established immediately after the

13
C pulse and remain through out the acquisition period.

The result is a coupled spectrum in which at least part of the NOE

enhancement retained.
Off- resonance 13C NMR Spectra

- CH3(Methyl) ------ quartet (q)

- CH2-(Methylene) ------- triplet (t)

- CH- (Methine) -------- doublet (d)

- C-(Quaternary) ------- singlet (s)

Inverse Gated Broad Band Proton Decoupling
Quantitative 13C NMR is desirable in two situations.
(1)The structural determination it is clearly useful to know whether a
signal results from more than one shift equivalent carbon.

(2) Quantitative analysis of a mixture of two or more components

requires that area of a signal be proportional to the number of carbon
atoms causing that signals.
Optimization of four parameters
1.Relaxation time, NOE, Data points and Pulse frequency amplitude

The proton broad band decoupler is Gated on only

during the 13C pulse and acquisition period and it is
Gated off during the pulse delay period.

(1)The NOE is a slow process, builds up only slightly

during the pulse and acquisition period.

(2) Decoupling, a fast process is established almost

immediately and irradiating with the 1H broad band
decoupler.

(3) So at the end result is a number of singlets whose

intensities are proportional to the carbons they
represent.
DEPT (Distortionless Enhancement by
Polarization Transfer) Experiment
Information's from DEPT-45°, 90° &135°
Type of Carbon atom DEPT EXPERIMENTAL TABLE

DEPT-45° DEPT-90° DEPT-135°

-CH3 q + Ve signal - + Ve signal

Methyl (quartet)

-CH2- t + Ve signal - - Ve signal

Methylene (triplet)

CH d + Ve signal + Ve signal + Ve signal

Methine (doublet)
s - - -
C
(Singlet)
13C proton decoupled spectra of Ethyl
Benzene with DEPT-90° & 135° in CDCl3

8
7 CH3
H2C
1
6 2

5 3
4
What is 2D NMR?
When and Why?
How?
2D-NMR Spectroscopy
 Basic features of 2D spectra

HA HB
2Å
HA HB

diagonal peak:
)
chemical shift (ppm

crosspeak:
correlation of two
different resonances
by short interatomic
1H

distance or through-bond
connection
1H chemical shift (ppm)
 WHEN?

Overlap of signals in the 1D spectrum

No overlap, easy to
assign
 2D NMR: Applications
•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
 Two main types of 2D techniques

Scalar coupling (J) based

COSY -COrelation SpectroscopY • Characterization/assignments
TOCSY- TOtal Corelation of resonances in the molecule
SpectroscopY
MQF- Multiple Quantum Filter
spectroscopy

NOE based
NOESY- Nuclear Overhauser • Orientation of different groups/
Effect SpectroscopY segments of the molecule in space
ROESY- Rotating frame nuclear or with respect to one another
Overhauser Effect SpectroscopY
 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
 HH-COSY

• magnitude mode
• only three-bond coupling
network seen
• ideal for small molecules
with simple coupling
networks
• easy processing, no phase
correction
H-H COSY Spectra of Pentoxifylline

O O CH3
6 4
8
N
H3C N
7 5 1

O N N
CH3
3
 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
J value
in homonuclear J-coupling J value
 Other COSY-type experiments
• COSYLR J-Resolve
Long range coupling
networks probed

• HOMO2DJ
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
 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 amino

acid residues of polypeptides since
characteristic peak patterns rendered for
different residues
TOCSY Spectra of Pentoxifylline

O O CH3
6 4
8
N
H3C N
7 5 1

O N N
CH3
3
 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 (<5ºA)

• Indicated in the form of cross

peaks in the NOESY spectrum
 ROESY-rotating frame NOESY

• NOE’s measured under spin-

locked conditions
• Suitable for molecules with
c~1 where NOE’s 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 minimize
TOCSY & COSY type artifacts
• Cross peaks are opposite in sign
with diagonal & chemical
exchange peaks
 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 5K-10K), 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
 Heteronuclear 2D-NMR
• For small molecules generally
HSQC spectrum of a protein
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
 Heteronuclear correlation
HSQC HMQC HMBC
• one-bond 1H-X • one-bond • two- & three-bond
correlation correlation correlation
(modified
• Longer pulse • Shorter pulse
program, HMQC)
sequence & is • Less sensitive
therefore therefore less
sensitive to than the HMQC
calibration &
sensitive to • Popular among
tuning errors calibration & NMR
tuning errors spectroscopists in
• Ideal for organic
macromolecules, • Ideal for small
labs/pharma
very popular in molecules industries
protein NMR
HSQC Spectra of Pentoxifylline

O O CH3
6 4
8
N
H3C N
7 5 1

O N N
CH3
3
HMBC Spectra of Pentoxifylline

O O CH3
6 4
8
N
H3C N
7 5 1

O N N
CH3
3
 NMR Experiment and Information
One-dimensional proton and Chemical shifts, proton
carbon spectra coupling constants

Distortionless enhancement by Identifies methines,

polarization transfer (DEPT) methylenes and methyls
spectra

COSY Identifies proton J-coupling

relationships

XHCORR (correlation of hydrogen Identifies proton – carbon

and any other nucleus),HETCOR couplings
heteronuclear multibond
correlation (HSQC)

Relayed coherence transfer Identifies long range proton-

(RELAY), total correlation proton couplings
spectroscopy (TOCSY)

Correlation by long-range coupling Identifies proton – carbon

(COLOC), HMBC long-range couplings

J-resolved Obtain proton-proton or

proton-carbon coupling
constants

NOE difference, NOE spectroscopy Identify through-space dipolar

(NOESY) couplings
 NMR of larger molecules
• Problems with overlapping signals
• Complicated multiplet patterns of signals
• Unresolved signals
• Not economic to check one signal at a time
Lysozym (129 amino acids)
 Protein NMR
1. Problems to overcome:
• Low sensitivity → High magnetic field strength, isotopic
labelling, new experiments
• Overlapping signals → 3D and 4D NMR spectra
• Large amount of information → computational methods
2. Particular problems with large molecules:
• Molecules move slowly → very broad signals
• CSA (Chemical Shift Anisotropy), fast relaxation, dipolar
couplings
3.Solutions:
• Exchange 1H for 2H (Deuterium)
• TROSY-experiment (Wüthrich, 1997)
→ Possible to study proteins up to 500 kD.
(2002: 900 kD, Wüthrich)
 Proteins
• Determine the amino acid sequence
• Determine secondary and tertiary structure
• Molecular weight: up to and exceeding 30 kD.

Advantage over X-ray crystallography

• Structure in solution (the natural environment
where proteins are active)
• Dynamic phenomena observable (such as
conformational changes)
• Interactions between molecules can be studied
(e. g., substrate binding)
 Proteins: How do we determine their structure?
• Amino acid sequence

• Non-sequential interactions

• Constraints

• Side chains

• Dynamics

• Possibly molecular interactions

 Nobel Prize in Chemistry 2002
Kurt Wüthrich (joint prize)
NMR Studies of Structure and Function of Biological Macromolecules

"for his development of nuclear magnetic resonance spectroscopy

for determining the three-dimensional structure of biological
macromolecules in solution"
 References:
1) Sanders J.K.M. and Hunter B.K. 1993, Modern NMR
Spectroscopy

(2) Robert M. Silverstein and Francis X. Webster, Spectrometric

Identification of Organic Compounds

(3) William Kemp 1994, Organic Spectroscopy

(4) Bruker-Biospin AG, Switzerland, Training course-2002 for

Avance 1D & 2D Experiments.

(5)Horst Friebolin,2002, Basic One and two dimensional NMR

Spectroscopy

(6) Jeremy N.S.Evans,Oxford University Press,1995,Biomolecular

NMR Spectroscopy.