Download Nuclear Magnetic Resonance

Nuclear Magnetic Resonance
Spectroscopy
核磁共振光譜
蘇士哲 19/Nov/2013
NMR, the Lord of spectrometers
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Resolution to atomic level.
Enable to determine structure.
Dynamic information in wide time scale.
Chemical modification is not required.
In solution condition, more close to biology.
e
Pieter Zeeman
Zeeman effect
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This splitting of spectral line into several
components in the presence of magnetic field
is called Zeeman (1902, Nobel Prize in
physics) effect.
Nuclear has Spin, too
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In 1945 the groups of both Bloch (Stanford) and Purcell (Harvard) succeeded in
detecting nuclear magnetic resonance absorption in bulk matter.
– The energy absorption was observed by irradiating the sample with
radiofrequency field and varying the strength of the magnetic field (continue
wave).
– 1952 Nobel prize in physics.
Felix Bloch
Edward Mills Purcell
Nuclear Spin
Nuclear spin is the total nuclear angular momentum quantum number (spin
number), characterized by a quantum number I. Different nuclear has different
number I, which only can be integral, half integral or zero.
The spin number I represents the magnetic quantum number mI of - I, I + 1,
….+ I. Different mI represents one energy state in magnetic field.
The number of possible orientations is give by 2I +1.
Not all nuclei have “spin”. Think about I = 0, there is only one the energy state.
12C, 16O, 32S
When I = 1/2, there are two energy states (only one energy transition).
1H, 13C, 15N, 31P
When I > 1/2, there are 2I + 1 states.
2H (I = 1), 14N (I = 1), 17O (I = 5/2)
The energy gap
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The two states have magnetic quantum numbers mI = 1/2 and mI = -1/2.
1
1
E  [( ) H] [( ) H]   H
2
2

h   H
H

2
The strength of magnetic field
 H B0

2

H = 26.7519 x 107 (T-1 S-1)
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If 300 MHz for 1H, that is ~ 7 Tesla (~ 7 x 104 Gauss). The strength of
the magnetic field of the Earth is only ~ 50 uT (0.5 Gauss)
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13C
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Super magnet to enlarge the energy gap
and 15N: 13C = 6.7283 x 107 and 15N = -2.712 x 107 ?
The location of NMR signal
High energy
Low energy
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Richard R. Ernst - The Nobel
Prize in chemistry 1991, “for his
contribution to the development of
high resolution nuclear magnetic
resonance (NMR) spectroscopy”
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Develop Fouier transform
techniques in NMR (FT-NMR)
Richard R. Ernst
Free induction decay (FID) and Fouier
transform (FT)
(A) One high energy pulse
(B) FT transform, from time domain data to
frequency domain
Jean Baptiste Fourier
1768-1830
The advantages of FT-NMR
• Fast
• Multiplex
• Informative: frequency,
intensity and line width
• Signal averaging
Modern Fourier transform NMR spectrometer
Coil and superconductor
LN2 and LHe2 tank
Typical protein proton 1D spectrum
Typical DNA proton 1D spectrum
Chemical Shift
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When an external field is applied by a magnet, the different protons in a molecule
will see different “effective” magnetic fields because of the other fields induced in
the molecule itself.
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At constant external field, the protons absorb at different frequencies depending
on local variation in the field.
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The frequency of resonance is given by:
obs (B0/2)(1)
shielding constant

shielded by electron cloud

, withdraw electron cloud
In practice, we use a reference compound to define the zero point,
such as tetramethylsilane (TMS, Si(CH3)4) or sodium 2,2-methyl-2silapentane-5-sulfonate (DSS). The protons is highly shielded.
Downfield
Less shielded
Low electron density
Higher magnetic strength
Higher frequency
Upfield
Shielded
High electron density
Lower magnetic strength
Lower frequency
Multidimensional NMR spectroscopy
1D
Why do we go beyond one dimension?
• To resolve the crowded signals in 1D spectrum by
spreading them into other dimensions.
• Homo- and hetero- nuclear NMR.
• 2D, 3D and 4D spectra.
• To elucidate the “through-bond” and “through-space”
relationships between the spins in the molecules.
Three most well-known Two-dimensional
homonuclear NMR spectra
TOCSY
Mix
NMR signal on the evolution period t1
A example of 2D COSY:
1D & 2D
2D COSY v.s. 2D TOCSY: through bond
N
2D NOESY: through space
Nuclear Overhauser Effect (NOE)
– A change in signal intensity for one type of
nucleus is caused by irradiation of another
type of nucleus that is nearby.
– Duo to the local magnetic fluctuation.
– The NOE is a dipole-dipole interaction that
depends on the distance between two
nuclei as r -6.
– The NOE can be used to measure interproton distance up to 5-6 Å.
– Most important information for structure
determination by NMR.
Ready for Heteronuclear NMR: Isotopelabeling of proteins (15N, 13C labeling)
• Grow proteins on minimal media (M9) with 15NH4Cl as
the sole nitrogen source and 13C-glucose as the sole
carbon source.
Pulse program of 2D Heteronuclear Single
Quantum Coherence
2D 1H-15N HSQC
2D 1H-13C HSQC
-13CH3
-13CH -13CH2 -
2D 1H-15N HSQC
15N
(ppm)
1H
(ppm)
Case study A
a.a. 1-240
a.a. 1-100
a.a. 101-240
Case study B
Case study C
From 2D to 3D: one block combines another block
Pair-wise backbone experiment and sequential
assignment
Assignment Strategy
Experiments for structural
determination
Three pairs of spectra for backbone assignment:
HNCA/HN(CO)CA
CBCANH/CBCA(CO)NH
HNCO/HN(CA)CO
More Spectra for side chain assignment:
HCC(CO)NH/CC(CO)NH
HBHA(CO)NH
15N HSQC-TOCSY
HCCH-TOCSY
…….
More NOESY Spectra for structural determination
15N
NOESY-HSQC
13C NOESY-HSQC
Example for Modern NMR pulse program,
CBCANH
NOESY for obtaining NOE constrains
One cross peak
represents one distance
constrain from a proton
pair.
Protein Secondary structure elements
-helix v.s. -sheet
Structure determination
1.
2.
3.
4.
Preparation of protein sample
NMR data acquisition
Resonance assignment, NOE assignments, and collection of
other geometric restraints.
Structure modeling and refinement
NMR and Dynamics
Protein-Protein complex interaction
Li et al. JMB (2004) 335:371-381
NMR in drug discovery
by line broadening &
relaxation Time
Fejzo et al.
Chemistry & Biology, 1999, 6:755-769
Fragment-based NMR screening
(SAR by NMR, Fesik)
Shuker et al.
Science, 1996, 274:1531-1534
Metabonomics
Nature review In
drug discovery
2002
Chemical exchange
1
1
 A  k , if k < 
obs
T2
T2
1
1  2
 
, if k > 
obs
T2
T2
8k
 |  A   B |
  2
1
 1 
T2
2
ka
A
kb
B
ionization States and pH
A-+H+
k1
k-1
 0 [H  ]
 
[H ]  K a
0
When   , K a  [H  ]
2
AH