1

Biophysics
Nuclear Magnetic Resonance
1) Introduction to NMR (20, 27 of March)
2) Applications in macromolecules (27 of March)
3) Laboratory practice (17 of April)
Antxon Martinez de Ilarduya
[email protected]
ETSEIB (UPC)
Departament dEnginyeria Qumica
Master en Ingeniera Biotecnolgica 2010-2011
2
i) Introduction
- Technique for characterization of:
- Organic compounds
- Macromolecules: - Tacticity (vinyl polymers)
- Composition and sequence distribution
(copolymers)
- Conformation (biopolymers, proteins)
- Medicine: - MRI
- Evolution: - 1946 Block y Purcel :
1
H NMR
- Currently : FT-RMN
-
1
H,
13
C,
31
P,
15
N
- 2D and 3D Spectra
- Solid Samples (CP-MAS)
NUCLEAR MAGNETIC RESONANCE (NMR)
NMR
3
ii) Theory
Nuclei of atoms small magnets
E=0
H
0
Radiacin
hu = E
Observables nucleus :
a) Nuclei with rotation
- They have magnetic moment
- Can be observed by NMR
a1) Spherical charge distribution
spin I= 1/2
1
H,
13
C,
15
N,
19
F,
31
P,...
a2) Non spherical charge distribution
spin I > 1/2
2
H,
14
N,
33
S,
35
Cl, ...
(They have an electric quadrupole moment)
NMR
4
b) Nucleus without rotation
- They dont have magnetic moment
- They cant be observed by NMR
Spin I = 0
12
C,
16
O, ...
Nucleus with spin I=1/2 (
1
H,
13
C,...)
- Antiparallel
- In a magnetic field H
o
: 2 directions
- Parallel
- Small excess in parallel direction to the magnetic field
Population m= +1/2
----------------------- = 1.0000066
Population m= +1/2
NMR
5
- Condition for resonance:
Ho = Intensity of Magnetic Field
u = Radiation frequency
= Magnetogyric ratio (Depends of the type of nucleus)
2tu = H
0
H
0
(T)
u(
1
H) (MHz) u(
13
C) (MHz) u(
19
F) (MHz)
4.7 200 50.29 188.15
7.05 300 75.44 282.23
11.75 500 125.73 470.38
NMR
6
Nuclei of interest in NMR
NMR
7
1
H NMR
The chemical shift (o):
- The hydrogen nucleus (proton)
u
0
- The hydrogen atom (proton + electron)
- The shielding effect of the electron
u
0
Au
simplified scheme of the shielding effect in the hydrogen atom
NMR
8
Effect of the substituent (between the effect in the proton and the hydrogen)
Electronegativity of the substituent Unshielding
> o
Paramagnetic effect
NMR
9
Effect of the electronegativity of the substituent
e
l
e
c
t
r
o
n
e
g
a
t
i
v
i
t
y
NMR
10
Chemical shifts o of a variety of protons referred to TMS
NMR
11
Calibration of NMR spectra
- We introduce a reference (TMS)
Si
CH
3
H
3
C CH
3
CH
3

o =
u
e
u
eTMS
u
0
ppm 12 11 10 9 8 7 6 5 4 3 2 1 0
TMS
o partes por milln
NMR
12
Spin-spin Coupling
Chemical shift (o) influenced by the neighboring nuclei
A
Interest: 1) Easier assignation of signals
2) J depends on conformation (Conformational studies)
NMR
13
H A
H
x
H A
H
X
H
A
H
X
-1/2
+1/ 2
H
A
o
A
J HxHy
(ppm)
TMS
0
ACOPLAMIENTO ESPIN-ESPIN
o
A
(ppm)
TMS
0
J J
o
X
H
A
H
X
H
X
25 %
25 %
25 %
25 %
(ppm)
TMS
0
J J
o
X
o
A
J
50 %
25% 25 %
Sistema AX
Sistema AX
2
Spin-spin coupling
Hx
Ha
Hx
Hx
Ha
NMR
14
Number of signals in coupled protons
Example: diethyl ether
NMR
15
Quantitative analysis
- Integral of signals Number of protons
3
2
3
NMR
16
Problem 1: Establish the chemical structure and coupling constants of
different protons of this compound with the help of the molecular formula
and the
1
H NMR spectrum.
2
.
0
0
0
0
2
.
9
8
6
0
3
.
0
4
1
0
(ppm)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
2
.
0
0
0
0
1
2
4
7
.
6
9
1
2
4
0
.
6
3
1
2
3
3
.
5
7
1
2
2
6
.
2
5
(ppm)
4.10 4.20
3
.
0
4
1
0
3
8
5
.
6
3
3
7
8
.
3
2
3
7
1
.
2
6
(ppm)
1.20
C
4
H
8
O
2
NMR
17
Problem 2: Establish the chemical structure of this organic compound with
the help of FTIR and
1
H NMR spectra.
2 2 2 2 3 1
C
10
H
12
O
3
NMR
18
13
C NMR
Disadvantages:
- Not abundant nucleus (1%)
- Low sensibility (accumulate 200- 10000 spectra)
- It is not quantitative
Advantages:
- sharp peaks (not couplings)
- Wide spectral width (250 ppm). Better resolution.
OCH
2
CHCH
2
O
OH
CH, CH
2
1
1
2
4
3
2 3
4
NMR
19
Scheme of chemical shifts in
13
C NMR
C=O
NMR
20
Experimental Method
- Solvent without hydrogens (deuterated)
CDCl
3
; deuterated DMSO; CD
3
COCD
3
; D
2
O
- introduce TMS or derivative (one drop)
- NMR tubes of special glass
- Put the sample in the superconductive Magnet
- He y N
2
(liquid)
- Field homogenization (Shims)
NMR
21
Magnetic field direction
Irradiacin
Radiation emission
(to detector)
Pulse irradiation
(All frequencies)
I
t
FID
I
o (ppm)
Spectrum
FT
Relaxation of nuclei
(Radiation emission (FID)
(intensity vs. time)
FT NMR Spectrum
(intensity vs. frequency)
NMR
22
Pulse Diagram :
1)
1
H NMR
pulso
FID
P1 AT D1
- P1 = Pulse duration
- AT= Acquisition time
- D1= Time delay between pulse and pulse
pulso
FID
P1 AT D1
13
C
IRADIACION
CONTINUA
1
H
DESACOPLADOR
TRANSMISOR
2)
13
C NMR (with nOe and
1
H decoupled)
NMR
23
3) DEPT
Very useful multipulse sequence for the determination of the multiplicity of
different carbons (
13
C)
C, CH, CH
2
, CH
3
Depending on the 0 pulse duration it is possible to observe different spectra
NMR
24
0 135
C not observed
CH
2
down
CH, CH
3
up
0 90
CH up
C, CH
2
, CH
3
not observed
0 45
CH , CH
2
, CH
3
up
Cnot observed
13
C
DEPT 135
DEPT 90
DEPT 45
CH
2
CH
3
CH
3
CH
2
DEPT
NMR
25
Examples: Isobutanol (CH
3
)
2
CHCH
2
OH
a)
13
C
CH
3
CH
CH
2
c) DEPT 135
CH
CH
3
CH
2
CH
b) DEPT 90
NMR
26
Example: Beta-Ionona
7 8
5,6
2
1
4
1 (CH
3
)
10 3
5 (CH
3
)
CH
CH
3
CH
2
CH
CH
CH
2
CH
3
NMR
27
4) 2D NMR
Very useful for the interpretation of complicated 1D NMR spectra
- NMR signals overlapped or at very similar chemical shifts
Solution 2D NMR
Changing:
- D1
- P1, P2 Different 2D spectra
- t
1
y t
2
(COSY, HETCOR, NOESY...)
NMR
28
4.1) COSY
1
H-
1
H Homonuclear correlation
Utility
- Knowing which protons are coupled to each other
- Determination of J values
Hx
Hx
Ha
spin-spin coupling
NMR
29
Interpretation of a COSY
-Symmetric respect to the
diagonal (take one side)
-Diagonal (spectrum in one
dimension)
-Off of diagonal: Signals of
coupled protons
-Coupled protons:
1.Trace a vertical and a
horizontal line to diagonal
2. When this line meet the
diagonal trace a vertical to the
signal
H
3
C C CH
2
CH
3
O 1
2
3
Coupling signal
coupled
protons
1 2
3
NMR
30
Example 2: Ethyl Benzene
CH
2
CH
3
CH
2
CH
3
Phenyl
Coupling signal
NMR
31
Example 3: 1-propanol: CH
3
CH
2
CH
2
OH
CH
3
CH
2
OH OH
CH
2
CH
3
NMR
32
Example 4: 2-Chlorobutane: CH
3
CHClCH
2
CH
3
2 4 3 1
a b c
a
b
c
NMR
33
Problem 3: Assign the
1
H NMR signals of
the following organic compound
CH
3
CH
3
CH
3
CH
3
O
5
4
3
2
1
7
8
10
6
NMR
34
Problem 4: Assign the
1
H NMR signals of the following organic
compound (C
6
H
12
O). (FTIR absorptions at 3300 and 1640 cm
-1
)
1
H-RMN
13
C-RMN
5
10
2,5
10
10
10
10
NMR
35
FTIR absorptions at 3338 and 1642 cm
-1
NMR
36
COSY
NMR
37
4.2. HETCOR
Heteronuclear correlation
1
H-
13
C or
1
H-X
90
{
1
H}
1
H:
13
C:
90 90
t
1
- Utility:
- Correlate protons and carbons attached to each other.
- Easy assignement of
1
H y
13
C signals in complex compounds
NMR
38
Methodology:
1) Trace from
1
H peak an horizontal line to the coupling signal.
2) Then trace a vertical line (direct couplings H-C)
Example 1: Polyaspartate of ethylene glycol methyl ether
NHCHCH
2
CO
COOCH
2
CH
2
OCH
3
n
c
a b
CH
a
b
c
CH
3
CH
3
b
a
CH
c
Couplin signals
1
H-
13
C
NMR
39
Example 2: Isobutanol (CH
3
)
2
CHCH
2
OH
CH
2
CH
CH
3
1
H
13
C
NMR
40
Problem 5: Asign the
13
C NMR signals of the following organic compound
with the help of the HETCOR spectrum
CH
3
CH
3
CH
3
CH
3
O
5
4
3
2
1
7
8
10
6
NMR
41
Problem 6: Asign the
1
H y
13
C signals of the following organic compound with
the help of COSY, DEPT 135, and HETCOR spectra. FTIR (band at 1640 cm
-
1
) (C
13
H
18
O)
CDCl
3
1
H-RMN
13
C-RMN
DEPT 135
20
4
8 8
8
8 8 8
NMR
42
COSY
HETCOR
NMR
43
Other 2D experiments:
- NOE-2D (NOESY): Allows the determination of the distances of
different protons in a molecule (Conformational studies in proteins)
- INADEQUATE:
13
C-
13
C Correlacin. (Similar to COSY). Low sensibility
- COLOC: Long distance correlation
1
H-
13
C. (Assignments of C y C=O)
- J-Resolved Homonuclear and Heteronuclear: Determination of J y o.
NMR
44
Main Applications of NMR in Biopolymers
1) Determination of the chemical structure (type of
comonomers) and microstructure (comonomers sequence
distribution ).
2) Study the conformation in solution
NMR
45
1) Determination of the chemical structure (type of comonomers)
and microstructure (comonomers sequence distribution ).
NMR: Very useful tool
In most of the cases it is required to assign the signals by
2D NMR spectra:
(
1
H-
1
H COSY,
1
H-
13
C HETCOR,
1
H-
1
H NOESY y
1
H-
13
C COLOC)
NMR
46
1
.
0
0
0
0
0
.
1
8
2
2
1
.
9
7
7
9
0
.
1
9
5
5
0
.
1
9
4
3
2
.
9
7
8
7
I
n
t
e
g
r
a
l
(ppm)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Example 1: PHB (Biopolymer synthesized by bacteria)
CH CH
2
C
O
O
CH
3
O CH
2
CH
2
CH
2
C
O
x y
1
2
3 1' 2' 3'
1
3
2
1
3
2
CHCl
3
TMS
Composition:
1=K1x
0.18=K2y
x+y=100
x=91.7% y=8.3%
NMR
47
(ppm)
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
CH CH
2
C
O
O
CH
3
O CH
2
CH
2
CH
2
C
O
x y
1
2
3 1' 2' 3'
4 4'
Example 1: PHB (Biopolymer synthesized by bacteria)
1
3
2
1 3
2
CDCl
3
4
4
NMR
48
(ppm)
62.5 63.0 63.5 64.0 64.5 65.0 65.5 66.0 66.5 67.0 67.5 68.0 68.5 69.0
Automatic Deconvolution
Fit type : lorentz
No. Position (Hz/ppm) Width (Hz/ppm) Intensity Integral(rel./abs.)
1 5100.62/ 67.5860 2.36/ 0.0312 3.161070e+008 83.66/1.074359e+009
2 5084.78/ 67.3761 2.14/ 0.0284 3.243536e+007 7.81/1.002685e+008
3 4799.54/ 63.5965 2.61/ 0.0346 2.910169e+007 8.54/1.096137e+008
CH CH
2
C
O
O
CH
3
O CH
2
CH
2
CH
2
C
O
x y
Example 1: PHB (Biopolymer synthesized by bacteria)
2
CH
CH
2
A B
AA
AB
BA
AB
BA
Microstructure:
AA: 83.7 % AB+BA: 16.3 % BB: 0 %
Degree of randomness: 1/(((AA+0.5(AB+BA))/0.5(AB+BA)) + 1/(((BB+0.5(AB+BA))/0.5(AB+BA))
Degree of randomness = 1.09 (Random copolymer)
Microstructure:
NMR
49
2) Study the conformation in solution
NMR: Frequenly used for the study of the conformation of
proteins.
1
H NMR:
a) o: depends on the type of conformation (helix, |-
sheet,)
b)
3
J CH-NH: depends on the dihedral angle between
the protons.
c) Dipolar coupling (nOe effect). Used to calculate the
internuclear proton distances.
NMR
50
a) o: depends on the type of conformation (helix,
|-sheet,)
helix
|-sheet
coil
NMR
51
b)
3
J CH-NH: depends on the diedral angle of protons:
Hlix
|-Sheet
NMR
52
c) Dipolar coupling (nOe effect). Used to calculate
internuclear proton distances
Conantokin-G: Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2
NOESY:
CH
NH
MOLECULAR
DINAMICS
Helical Conformation
NMR
53
Poly(|-peptides)
N
C
CH
2
C
H
O
ROOC
H
n
R: Alkyl
-They form structures with the main chain in helix conformation
NMR
54
- Helix-coil transition (solvent effect)
NMR
55
- Helix-coil transition (effect of temperature)
NH
CH
H
H
C
C
This kind of polymers have the main chain in helix conformation that breaks with the
addition of acids or by thermal effects.
NMR
56
Bibliography
"Spectrometric Identification of Organic Compounds," R.M. Silverstein, G.C. Bassler and T.C. Morrill, John Wiley &
Sons, New York, 5th Ed. 1991.
"NMR and Chemistry; An Introduction to Nuclear Magnetic Resonance Spectroscopy," J. Akitt, Chapman and Hall,
London, 1973.
"Nuclear Magnetic Resonance for Organic Chemistry," D. W. Mathieson, Academic Press, London, 1967.
"Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry," L. M. Jackman and S. Sternhell,
Pergamon Press, Oxford, 1969.
One-dimensional and Two-dimensional NMR Spectra by Modern Pulse Techniques, K. Nakanishi, University Science
Books, California, 1990.
"NMR of Proteins and Nueclic Acids K. Wthrich, John Wiley, 1986.
"Nuclear Magnetic Resonance Spectroscopy," F. A. Bovey, Academic Press, 2nd Ed., 1988.
"Tables of Spectral Data for Structure Determination of Organic Compounds," E. Pretsch, T. Clerc, J. Seibl, W. Simon,
2nd Ed. Springer-Verlag, 1989.
"Magnetic Resonance of Biomolecules," P. F. Knowles, D. Marsh, and H. W. E. Rattle, Wiley, London, 1976.
"
13
C NMR Spectroscopy, High Resolution Methods and Applications in Organic Chemistry and Biochemistry," E.
Breitmaier and W. Voelter, Verlag Chemie, Weinheim, 1987.
NMR