1

Structure Determina-on:
Nuclear Magne-c Resonance(NMR)

2

1Dartmouth College Department of Chemistry. h7ps://sites.dartmouth.edu/

mierkelab/nmr-facility (accessed December 19, 2015).

2Dominique Marion; An IntroducJon to Biological NMR Spectroscopy. Molecular & Cellular

Proteomics. [Online] 2013, pp 3009. h7p://www.mcponline.org/content/12/11/3006.full.pdf

+html?sid=d9388787-b9ae-4dae-b0e8-770d9d293f41 (accesed December 28, 2015).

Kwonil Kobe Ko, Cushing Academy, Senior

Project Supervisor: William R. Sponholtz, III, B.S., M.S., Ph.D.

NMR Spectroscopy
Nuclear Magne-c Resonance (NMR) Spectroscopy is an analy-cal
chemistry technique to elucidate the structures of simple and complex
organic compounds employing 1-D and 2-D NMR.
Oldest techniques

Elemental analysis

Combus-on analysis

Modern techniques
NMR(1-D and 2-D) and 3-D Shapes
GC-MS : Molecular weight
X-ray Crystallography : 3-D shapes
IR : func-onal groups (based on vibra-ons)

Classical Structure Determina'on vs Modern techniques

Degrada've vs. non-Degrada've Techniques
Large Quan''es vs. A Few Milligrams
Many Years vs. Hours
Ambiguous vs. Absolute Assignment

Top Five Greatest Discoveries in Science?

Protein crystallography involves summing the scattered X-ray waves

from a macromolecular crystal

can be studied.

NMR & X-ray crystallography

The steps in solving a protein crystal structure at high resolution are diagrammed in Figure 5-3.

Unlike X-ray diffraction, which presents a static picture

(anprotein
averagemust
in be
time
and space)
the the rate-limiting step in straightforward
First, the
crystallized.
Thisof
is often
structure of a protein, NMR has the capability of structure
measuring
certain dynamic
properties
determinations,
especially for
membraneofproteins. Then, the X-ray diffraction pattern
from the crystal must be recorded. When X-rays strike a macromolecular crystal, the atoms in
proteins over a wide range of time scales.
chemical shift (ppm)

the molecules produce scattered X-ray waves which combine to give a complex diffraction
pattern consisting of waves of different amplitudes. What is measured experimentally are the
amplitudes and positions of the scattered X-ray waves from the crystal. The structure can be
reconstructed by summing these waves, but each one must be in the correct registration with
respect to every other wave, that is, the origin of each wave must be determined so that they
sum to give some image instead of a sea of noise. This is called the phase problem. Phase
values must be assigned to all of the recorded data; this can sometimes be done computationally,
but is usually done experimentally by labeling the protein with one or more heavy atoms whose
position in the crystal can be determined independently. The phased waves are then summed
in threedata
dimensions to generate an image of the electron density
data distribution of the molecule
collection
in the
crystal. This can be done semi-automatically or byanalysis
hand on a computer graphics system.
A chemical model of part of the molecule is docked into the shape of each part of the electron
part of the electron density (as shown in Figure 5-3). This fitting provides the first picture of
13C = 22.9 ppm
the structure of the protein.
The overall model is improved by an iterative process called refinement whereby the positions of the atoms in the model are tweaked until the calculated
diffraction pattern from1Hthe
modelshift
agrees
as well as can be with the experimentally measured
chemical
(ppm)
diffraction pattern from the actual protein. There is no practical limit to the size of the protein
NMR spectrometer or protein complex whose
resonance
assignment
and
structure
can be determined
by X-ray crystallography.protein structure
1H

NMR:

purified, labeled protein

internuclear distance measurement

3 Gregory, A.; Dagmar, R.; Protein Structure and FuncJon: Chapter 5, Structure determinaJon; New Science Press Ltd, 2004; pp 168-173 For X-ray descripJon

X-ray
Crystallo
graphy:

Figure 5-4 Structure determination by NMR For protein structure determination by NMR, a labeled protein is dissolved at very high concentration and placed
in a magnetic field, which causes the spin of the hydrogen atoms to align along the field. Radio frequency pulses are then applied to the sample, perturbing the
nuclei of the atoms which when they relax back to their original state emit radio frequency radiation whose properties are determined by the environment of the
4
refinement
atom in the protein. This emitted radiation is recorded in the NMR spectrometer for pulses of differing types and durations (for simplicity, only one such record
is shown here), and compared with a reference signal to give a measure known as the chemical shift. The relative positions of the atoms in the molecule are
calculated from these data to give a series of models of the protein which can account for these data. The quality of the structure determination is measured as
the difference between the different models.
X-rays

References
Drenth, J.: Principles of Protein X-Ray Crystallography
2nd ed. (Springer-Verlag, New York, 1999).

phases

fitting

to Users of Macromolecular Models 2nd ed. (Academic

Press, New York and London, 1999).

Schmidt, A. and Lamzin, V.S.: Veni, vidi, viciatomic

electron density
maps
atomic models
the mysteries
of protein
Evans, J.N.S.: Biomolecular NMR Spectroscopy (Oxford
function. Curr.The
Opin.
Struct.
Biol.
2002,12:698703.
4 Gregory,
Figure
5-3
Structure
determination
by
X-ray
crystallography
first
step
in
structure
determination
by
X-ray
crystallography
is
the
crystallization
the descripJon
A.; D
agmar,
R.; P1995).
rotein Structure and FuncJon: Chapter 5, Structure determinaJon; New Science Press Ltd, 2004; pp 168-173 For ofX-ray
University
Press,
Oxford,

protein. The source of the X-rays is often a synchrotron and in this case the typical size for a crystal for data collection may be 0.3 0.3 0.1 mm. The crystals
Gorenstein,
N.:
Nuclear
Magnetic
Resonance
(NMR)
areMacromolecular
bombarded with X-rays
which are
scattered from the planes of the crystal lattice and are captured as a diffraction pattern on a detector such as film or an
Markley, J.L. et al.:
structure
deterTextbooks
Online):
electronic device. From this pattern, and with the(Biophysics
use of referenceor
phaseinformation
from labeled atoms in the crystal, electron density maps (shown here
mination by NMR
spectroscopy.
Methods
Biochem.
with the corresponding peptide superimposed) are
computed for different parts of the crystal. A model of the protein is constructed from the electron density
http://www.biophysics.org/btol/NMR.html
Anal. 2003, 44:89113.
maps and the diffraction pattern for the modeled protein is calculated and compared with the actual diffraction pattern. The model is then adjustedor refined
crystals (enlarged view)

diffraction
patternsunravelling
resolution

X-ray crystallography is a tool used for idenJfying the atomic and molecular structure of
a crystal by causing Rhodes,
a beam
of XMade
-rays
to
diract into many specic direcJons. Measuring the
G.: Crystallography
Crystal Clear:
A Guide
angles and intensiJes, a three-dimensional picture of the density of electrons within the
Structure Determination Chapter 5 171
crystal can be produced. And from this electron density, compounds 3-D structure can be
elucidated, including chemical bonds, chiral centers, gross connecJvity,etc. 5
5
to reduce the difference between its calculated diffraction pattern and the pattern obtained from the crystal, until the correspondence between model and reality
is as good as possible. The quality of the structure determination is measured as the percentage difference between the calculated and the actual pattern.

2004 New ScienceDefinitions

Press Ltd

phase problem: in the measurement of data from an

X-ray crystallographic experiment only the amplitude
of the wave is determined.To compute a structure, the
phase must also be known. Since it cannot be determined directly, it must be determined indirectly or by
some other experiment.

Chemwiki, Unviersity of California, Davis. h7p://chemwiki.ucdavis.edu/AnalyJcal_Chemistry/Instrumental_Analysis/DiracJon/X-

ray_Crystallography (accessed December 27, 2015).

NMR vs X-ray Crystallography

An Introduction to Biological NMR Spectroscopy

6 Dominique M
Farion;
IG. 2. Number
of structures
deposited
to the RCSB
protein
data bank
(http://www.rcsb.org/pdb/)
over the years. The number of
An IntroducJon
to Biological
NMR Spectroscopy.
Molecular
& Cellular
Proteomics.
[Online] 2013, pp 3009. h7p://www.mcponline.org/content/

structures solved by X-ray crystallography is steadily increasing

whereas
the2NMR-based
ones have hit a plateau. In the inset, the data for the
12/11/3006.full.pdf+html?sid=d9388787-b9ae-4dae-b0e8-770d9d293f41
(accesed
December
8, 2015).

early years of crystallography are displayed with an extended vertical scale for clarity. For the former method, the crystallization step remains
a major bottleneck but once suitable diffraction data are available, the structure can be obtained rather quickly. NMR is primarily hampered
by the limitation in protein size that can be studied: despite that resonance assignment and nOe interpretation have been automated, it still
requires more human input during these processes.
7

NMR spectroscopy and X-ray crystallography are the most widely used modern techniques in elds of
biology and chemistry for structure elucidaJon.
disregarded
otherwise.
The
external
magnetic
field &
B Cellular
induces
Dominique Marion;
An IntroducJon
to Biological
NMR
Spectroscopy.
Molecular
Proteomics. [Online] 2013, pp 3006. h7p://www.mcponline.org/content/12/11/3006.full.pdf
+html?sid=d9388787-b9ae-4dae-b0e8-770d9d293f41
(accesed
ecember
28, 2in
015).
currents in the electronic clouds
in Dthe
protein;
turn, these
circulating currents generate a local induced field B . As a
X-ray is highly
used when solving a protein structure because it is easy and quick to solve a protein
result, the different spins sense the vector sum of the two
fields:
structure once
suitable crystals have been obtained. 8
(accessed December 28, 2015).
B !B "B
The University Medical School of Dbrecen. h7p://www.cryst.bbk.ac.uk/pps97/assignments/projects/ambrus/html.htm
and will
thus not
same frequency.
Unlike X-ray,
which
is resonate
highly atrthe
estricted
to pChemical
rotein structure determinaJon, NMR covers a wider range of
shifts are extremely sensitive to steric and electronic effects
thus in the scase
of proteins,that
to secondary
and e
tertiary
biochemistry and
including
tructures
are not
asily crystalized. This is especially powerful in drug
structure. Unlike nOe and J-coupling, chemical shift does not
discovery, because
it a psingle
rovides
s molecular
arameters
chemical
kineJcs
by following
F .a
3.nd
Without
chemical shifts,
NMR structural
parameters the Jme-
depend on
pairwiseuinteraction
between p
well-identi
9

fied partners: its prediction
or quantitative interpretation is could not be measured and interpreted at atomic level resolution.
dependence othus
f the
data.
In fact, the magnetic field at the nucleus is generally different from the
more complex. Let us consider the chemical shifts of 1
7

ind

loc

ind

IG

applied field B0: this additional contribution (or screening)

9 The University Medical School o15
f Dbrecen. h7p://www.cryst.bbk.ac.uk/pps97/assignments/projects/ambrus/html.htm
(accessed December 28, 2015).

backbone

N in proteins: the standard chemical shift range

arises from
the interaction of the surrounding electrons with the applied field. The

Example of Impact of 2-D NMR: Strychnine

Classical structure determina-on techniques (degrada-ve) took approximately 50
years to elucidate the structure of strychnine employing the eorts of many, many
collabora-ng research groups. However, with 1H and 13C NMRs, several dierent 2-
D NMRs, and other spectroscopic data, Dr. Sponholtz was able to solve this structure
in only ten hours! The key: 2-D NMR
Isolated 1818
Elucida-on 1946
X-Ray conrma-on 1956

Strychnine

six chiral centers: 64 possible

six methylene groups;
dieren-ate pro-R and pro-S

N
O
O

Showing gross
connec-vity only
(no stereochemistry)

Collabora-on of NMR & X-ray

(a) TIC10 or ONC201

10

(b) Corrected Structure for TIC10

The patented compound, known as TIC10, was

elucidated by The Penn State group and owned by the
biotech rm OncoceuJcs.

Several insJtuJons have found TIC10 to be eecJve in
brain cancer, prostate cancer, melanoma, and sarcomas.

Thus, OncoeceuJcs has iniJated Phase I/II clinical trials of
TIC10 and was about to enter the human clinical trials.

However, When Scrippss Kim D. Janda and coworkers

synthesized the iniJal patented structure, they found it to be
biologically inacJve.

By using X-ray crystallography and NMR, Scripps
conrmed that bioacJve TIC10 has a dierent structure(b)
than the patented one.

The Scripps research group concluded that OncoceuJcs
and several insJtuJons had been working on the bioacJve
compound but had patented the inacJve structure. Thus
Scripps applied for a patent on the correct structure(b) and
licensed it exclusively to Sorrento.

10 Stu, Borman.; Tug-of-War Over Promising Cancer Drug Candidate. Drug Discovery: Structure error threatens exisJng patent and clinical trials. Chemical & Engineering News. [Online] 2014, Volume 92, Issue

21, 7. h7p://cen.acs.org/arJcles/92/i21/TugWar-Over-Promising-Cancer-Drug.html (accessed December 28, 2015).

Necessary Review
I. Basic understanding of electronega<vy dierence; i.e., must be able to predict electron
density for atoms; e.g., deshielded or shielded.
Example:

ATOMS

DESHIELDED

SHIELDED

IIIII. Basic understanding of how atoms are oriented in three-dimensions; i.e., hybridiza<on
theory.

C
sp3 Hybridized
Carbon (109.50)

sp2 Hybridized
Carbon (1200)

2pz unhybridized
orbital (one eper orbital)

2py unhybridized
orbital (one e per orbital)

sp Hybridized Carbon
(1800)

The coupling of these two topics is the key to

1 chemical shics of NMR.
understand and predict the

Obtaining NMR
3-5 mg of compound
dissolved in a suitable
solvent and transferred
to a NMR tube.

1-D Pulse NMR

Sample
Magnet

NMR tube placed into

the magne-c eld.
11

11

Magnetization
Perturbation
Response
Detection

11

Data
Fourier
Transformation
Spectrum
6

PPM

11 Dartmouth College Department of Chemistry. h7ps://sites.dartmouth.edu/mierkelab/nmr-facility (accessed December 19, 2015).

Informa-on Process
The informa-on that comes
out of the spectrometer is
called a free induc-on decay
(FID), which is in the Time
Domain. When the nuclei are
pulsed, the spins of like nuclei
group together and acer the
pulse the spins move apart or
decay.

FID

FT

The FID is transformed via a Fourier

transforma-on to yield a spectrum,
which is in the Frequency Domain.
You can determine how many
hydrogens are afached by comparing
areas under the curve (integra-on) in
the simplest ra-o. Moreover, you can
inves-gate the spligng paferns with
high resolu-on.

Spectrum

Chemists perspec-ve
Chemists look for three things: Chemical ShiJ, Intensity, and SpliLng
I): Chemical ShiJ: By looking at chemical ships of 1H and 13C, chemists can roughly predict
what group is a7ached to carbon or hydrogen. For example, if a sp2 carbon is highly
deshielded(or downeld) and thus has a chemical ship of 200-220 ppm, chemists can imagine
the presence of electron withdrawing group, such as oxygen, a7ached to that sp2 carbon.

2): Intensity: Since the signal intensity is directly proporJonal to the number of hydrogens
that give rise to the signal, chemists can see how many chemically equivalent
hydrogens(same chemical ship) are a7ached by seeing a raJo of areas under the
integrated intensity of signals in 1H NMR spectrum.

3): SpliLng : Spliqng oers informaJon of how many neighboring hydrogens exist for
a parJcular hydrogen or chemically equivalent hydrogens. Looking at spliqng pa7erns and
complex coupling constants, chemists can draw the tree diagram of complex NMR and thus
be7er understand spliqng in various cases because dierent couplings are applied
sequenJally.

1-D NMR Proton Chemical Shics (rela-ve to TMS in CDCl3)

RCOH

R 2C CH 2

Aromatics

Ethers Sulfides Sat alkanes

RO-CH RS-CH R-H
HO-CH

RCH CHR

Ar-CH R 2C CR-CH

PhO-CH
F-CH

Cl-CH

Esters
RCO 2-CH
O2N-CH
RCO 2H

RC CH

Amide RCONH

I-CH

Br-CH RCOCH
NC-CH
R 2N-CH
ROH
R 2NH

PhOH
14.0

13.0

12.0

11.0

10.0

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0 ppm
()

Dierent types of Hydrogens in NMR

Homotopic hydrogens that would result in idenJcal molecules if they
were replaced with another atom (X). 12
IdenJcal signals in H NMR

H
C

Cl

Cl

Cl

, Cl

Cl

X
C
Cl

EnanJotopic - hydrogens that would result in enanJomers if they were

replaced with another atom (X). 12
IdenJcal signals in H NMR

(R)

Cl

Cl

(S)

C
F

Cl

Diastereotopic - hydrogens that would result in diastereomers if they were

replaced with another atom (X). 12

Dierent signals in H NMR

Cl

Cl

Cl
F

C
(R)

H
X

Cl

Cl

(R)

C
(R)

Cl

, 1

12 Mater Organic Chemistry. Homotopic, EnanJotopic,

(S)

Diastereotopic. h7p://www.masterorganicchemistry.com/
2012/04/17/homotopic-enanJotopic-diastereotopic/
(accessed January 4, 2016)

1-D NMR Carbon Chemical Shic (rela-ve to TMS in CDCl3)

Aldehydes, RCH=O

R 2C=CH 2

Ketones, R 2C=O

RHC=CHR

C-F

C-Cl

C-H Saturated Alkanes

Aromatics
Heteroaromatics
Carboxylic Acids R-CO2H

RC N

C-NR 2
C-OH

C-SR

C-OR

C-Ar
C SOnR C C C

Esters R-CO2R'
C CR

Amides R-CONR2

200

150

C-Br

C-NO 2

R 2C CH 2

C-I

100

COCR

50

0 ppm()

Chemical Shics for 1-D proton and carbon NMR

Lets focus on coupling (J) constants for proton NMR
What are J values?
J(coupling constant) is a distance(Hz) between split peaks. When a proton
absorbs energy, it relaxes by giving that energy back to surrounding atoms via
the sigma bond framework. Thus, some energy is passed along to adjacent
protons. The adjacent protons provide feedback on the spectrum to the proton
that absorbs the energy. Moreover, J values are independent of the eld
strength, Bo .
H1 gives
H1
H1
H1
H2
H2
H2
energy back
(relaxation)
H1 absorbs
C
C
C
C
C
C
energy
via sigma
bonds
H2 absorbs
some of
the energy
Within the mathematics of the spectrum
H1
H2
software, we can see how many neighboring
protons absorb the energy, which gives rise to
C
C
coupling (J) constants.

What inuences the magnitude of J value?

-Distance to relaxing proton

-Angle to relaxing proton

Type

Type

J Value (Hz)

J Value (Hz)

12-15

ax-ax: 6-14
ax-eq: 0-5
eq-eq: 0-5

H
H

2-9

H
H

ortho: 6-10
H meta: 1-3
para: 0-1

0.5-3
H
H

CH

0.5-3

7-12
H
HC

CH

13-18
H

Note: circled protons a1 re NOT equivalent

0-3

Karplus Curve

H'

H
C'

H'

H
C
12

J HH' (Hz)

10

20

40

60

80

100

120

140

160

180

Spligng Pafern
13

Peak Spliqng occurs due to coupling of spins (interacJons between adjacent carbons).
Peak Spliqng is not seen for H connected to O, N. (because of hydrogen bonding)
Spliqng is based on the number of Hs on adjacent C.

13

General rules

a): If a proton has n protons a7ached to adjacent carbons, it will split into n+1 peaks.
b): Only nonequivalent protons couple.
c): If Hs are on same C and they are homotopic or enanJotopic, no spliqng will occur.
d): If Hs are on dierent Cs, but they are chemically equivalent, no spliqng will occur.
are classied by how they are split
Peaks
13

13

13 University of California, Los Angeles Department of Chemistry. Proton NMR Spectroscopy-Split the signals, not your brain! h7p://www.chem.ucla.edu/harding/ec_tutorials/tutorial37.pdf (accessed January 9, 2016).

Examples for Hydrogen chemical shics

Ethanol
Note: Only three peaks due
to equivalent protons (see
3-D model if you are not
convinced)

1.11H

Note: Deshielded protons are downeld

1.11

PPM

H 2.0
H3.57

1.11 3.57

Dimethyl Ether

3.24

3.24

3.24H

Note: All protons are equivalent

H3.24

3.24

3.24

Note: All equivalent protons are deshielded (downeld)

PPM

Propylene (1-Propene)

5.70

1.71

Note: Only three equivalent protons

H4.97

Note: sp2 more electronega-ve than sp3

carbon; thus, those afached protons are
more downeld and both are not
equivalent.

PPM
1

1.71

H
1.71

H
5.03

1.71

(E)-2-Butene

1.71

5.48

H
H

1.71

Note: Only two signals due to

equivalent protons (symmetry of
molecule)

1.71

5.48

1.71

H
1.71

PPM
1

5.03

1-Butene

PPM
1

1.06

4.97H

Note: sp2 more electronega-ve than sp3

carbon; thus, those afached protons are
more downeld and each is not equivalent.

2.00

Note: Two sets of equivalent protons.

2.00

H
H 1.06

1.06

5.70

Examples for Carbon chemical shic (x20 rules)

Ethanol
Note: Only three peaks due
to equivalent protons (see
3-D model if you are not
convinced)

16.9

Note: Deshielded protons are downeld

60

50

40

30
PPM

20

55.8

10

Ethanol
Note: Deshielded carbon is
downeld

16.9

Note: x20 rule applies very well

60

50

40

30
PPM

55.8

20

10

Dimethyl Ether

Note: both carbons are equivalent

56.1

56.1

Note: both equivalent carbons are deshielded (downeld)

60

50

40

30
PPM

20

10

Propylene (1-Propene)

H
H 132.7

Note: x20 Rule works well

H

Note: sp2 more electronega-ve than sp3

carbon; thus, those carbons are more
downeld and both are not equivalent.

140

120

100

80

PPM

60

115.9

17.2

40

20

(E) -2-Butene

Note: Only two signals due to

symmetry.
Note: sp2 more electronega-ve than sp3
carbon; thus, those equivalent carbons are
more downeld.

120

100

16.7

80

PPM

60

125.3
125.3

H
H

16.7

40

20

1-Butene

Note: sp2 more electronega-ve than sp3

H
137.3

115.1

carbon; thus, those carbons are more

downeld and each is not equivalent.

140

120

100

80
PPM

40

13.7

26.3

60

20

Predic-ng the spligng pafern of a proton signal

Example: If a proton (or group of equivalent protons) relaxes by giving o
energy to two equivalent, adjacent protons the signal will be split into a
triplet (t)
Example: If a proton (or group of equivalent protons) relaxes by
giving o energy to three equivalent, adjacent protons the signal
will be split into a quartet (q)
1.11
H

1.11H

H 2.0
H3.57

1.11 3.57

Will split into a triplet

Will split into a quartet

PPM

Example of spligng paferns for (E)-2-butene

1.71
H

1.71

5.48
H

1.71

1.71

5.48

1.71

1.71

Will split into a d

Will split into a q

1
PPM

Example of spliLng paTerns for 1-propene (can get complicated very quickly)
Mul-plet (m)
5.70

Examples of d of d of d

1.71
H

H 4.97
H

1.71

1.71

5.03

Will split into a d of d of q

Will split into a d of d of q
Will split into a d of d of q
Will split into a d of d of d

PPM