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NMR

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Title: NMR


1
NMR theory and experiments
2
NMR theory and experiments Analytical Chemistry
TIGP, Academia Sinica
Instructor Der-Lii M. Tzou
Place A508, IC
Hour 9001200 am June 6, 2007 (02) 2789-8524
emailtzou_at_ccvax.sinica.edu.tw
  • NMR basics and principle
  • (a) Rotation spectroscopy
  • (b) Larmor frequency
  • (c) Resonance, Fourier transfer
  • Applications of NMR to Biological Systems
  • (a) 1D NMR and chemical shifts
  • (b) J-coupling
  • (c) T1 and T2 relaxation
  • (d) NOE and 2D NOE spectroscopy
  • (d) 2D TOCSY spectroscopy
  • (e) 2D COSY spectroscopy
  • (f) 1H, 13C, 15N NMR spectroscopy and high
    resolution multi-dimensional NMR
  • (g) Other nuclear spin interactions

3
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4
With External Magnetic Field
?
?
? mi Mo ? 0
5
Number of spin states (2I1) A nucleus with
spin I can have 2I1 spin states. Each of these
states has its own spin quantum number m (
m-I,-I1,, I-1, I ). For nuclei with I1/2,
only two states are possible m1/2 and m-1/2.
Nuclear Zeeman effect
m-1/2
E-mB0(?h/2p) B0 magnetic field strength h
Plancks constant m spin quantum number ?
magnetogyric ratio
?E
0
E
m1/2
B0
6
Larmor Frequency
7
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8
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9
3D-FID (Free Induction Decay)
?????????
10
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11
Examples
Increased shielding
upfield low frequency
downfield high frequency
Increased deshielding
12
1H-1H TOCSY ?? ?? ?????? 1H-1H TOCSY (TOtal
Correlated SpectroscopY also known as HOHAHA
HOmonuclear HArtmann HAhn) is useful for dividing
the proton signals into groups or coupling
networks, especially when the multiplets overlap
or there is extensive second order coupling. A
TOCSY spectrum yields through bond correlations
via spin-spin coupling. Correlations are seen
throughout the coupling network and intensity is
not related in a simple fashion to the number of
bonds connecting the protons. Therefore a
five-bond correlation may or may not be stronger
than a three-bond correlation. TOCSY is usually
used in large molcules with many separated
coupling networks such as peptides, proteins,
oligosaccharides and polysaccharides. If an
indication of the number of bonds connecting the
protons is required, for example in order to
determine the order in which they are connected,
a COSY spectrum is preferable. The pulse sequence
used in our laboratory is the gradient enhanced
TOCSY. The spin-lock is a composite pulse and
should be applied for between 20 and 200 ms with
a pulse power sufficient to cover the spectral
width. A short spin-lock makes the TOCSY more
COSY-like in that more distant correlations will
usually be weaker than short-range ones. A long
spin-lock allows correlations over large coupling
networks. Too long a spin-lock will heat the
sample causing signal distortion and can damage
the electronics of the spectrometer. The
attenuation should be set so that the 90 pulse
with will be less than 1/(4SWH) (SWH is the
spectral width in Hz) and typically 1/(6SWH). An
attenuation of 12 dB with a 50 W amplifier
yielding a 90 pulse width of 35 µs is typical.
1H-1H spin-spin J-coupling observed by NMR
The effective magnetic field is also affected by
the orientation of neighboring nuclei. This
effect is known as spin-spin coupling which can
cause splitting of the signal for each type of
nucleus into two or more lines.
13
Returning to the example of ethylbenzene, the
methyl (CH3) group has a coupling pattern in the
form of A3X2 which to a first order approximation
looks like an AX2 multiplet. Likewise, the
methylene (CH2) group has the form A2X3 that is
equivalent to AX3. The first order approximation
works because the groups are widely separated in
the spectrum. The aromatic signals are close
together and display second order effects. The
ortho signal is a doublet AX while the meta and
para signals are triplets
14
Isolated spins (Zeeman effect, Larmor
Frequency)Electron cloud (chemical
shift)Scalar coupling (J-coupling through
chemical bonds)Dipolar interaction (NOE through
space) Spin dynamics (T1, T2 relaxations)
15
Longitudinal relaxation or spin-relaxation
relaxation T1
Longitudinal relaxation (T1) is the mechanism by
which an excited magnetization vector returns to
equilibrium (conventionally shown along the z
axis).
?

Inversion recovery curve for the methyl protons
of ethylbenzene (0.1) in CDCl3 at 400 Mhz
The inversion recovery (T1) pulse sequence yields
a signal of intensity
16
Transverse relaxation T2, spin-spin relaxation
Transverse relaxation (T2) is the mechanism by
which the excited magnetization vector
(conventionally shown in the x-y plane) decays.
This is always at least slightly faster than
longitudinal relaxation.

Spin-echo experiment for proton spectrum of
ethylbenzene (0.1) in CDCl3 at 400 MHz, the
residual water peak on the left relaxes faster
than the methyl on the right.
Exponential decay curve for the methyl protons of
ethylbenzene (0.1) in CDCl3 at 400 MHz
17
One Dimensional NMR Spectroscopy
The 1D experiment    Each 1D NMR experiment
consists of two sections preparation and
detection. During preparation the spin system is
set to a defined state. During detection the
resulting signal is recorded. In the simplest
case the preparation is a 90 degree pulse (in our
example applied along the x axis) which rotates
the equilibrium magnetization Mz onto the y axis
(My). After this pulse each spin precesses with
its own Larmor frequency around the z axis and
induces a signal in the receiver coil. The signal
decays due to T2 relaxation and is therefore
called free induction decay (FID). Usually, the
experiment is repeated several times and the data
are summed up to increase the signal to noise
ratio. After summation the data are fourier
transformed to yield the final 1D spectrum.
18
Anatomy of a 2D experiment
90x
90y
t1
t2
FID
Evolution
Preparation
Mixing Time
Detection
The construction of a 2D experiment is simple in
addition to preparation and detection which are
already known from 1D experiments the 2D
experiment has indirect evolution time t1 and a
mixing sequence. The scheme can be viewed as
Do something with the nuclei (preparation), let
them precess freely (evolution), do something
else (mixing), and detect the result (detection,
of course)
19
Anatomy of a 3D experiment
t1
t3
?m
t2
FID
MLEV
Evolution
Preparation
Mixing Time
Detection
Evolution
Mixing Time
A three dimensional NMR experiment (see picture
above) can easily be constructed from a
two-dimensional one by inserting an additional
indirect evolution time and a second mixing
period between the first mixing period and the
direct data acqusition. Each of the different
indirect time periods (t1, t2) is incremented
separately. Triple resonance experiments are the
method of choice for the sequential assignment of
larger proteins (gt 150 AA). These experiments are
called triple resonance because three different
nuclei (1H, 13C, 15N) are correlated. The
experiments are performed on doubly labeled (13C,
15N) proteins.
20
Anatomy of a 2D experiment - continued
After preparation the spins can precess freely
for a given time t1. During this time the
magnetization is labeled with the chemical shift
of the first nucleus. During the mixing time
magnetization is then transferred from the first
nucleus to a second one. Mixing sequence utilize
two mechanisms for magnetization transfer scalar
coupling (J-coupling) or dipolar interaction
(NOE). Data are acquired at the end of the
experiment (detection, often called direct
evolution time) during this time the
magnetization is labeled with the chemical shift
of the second nucleus.
21
2D COSY (COrrelated SpectroscopY)
In the COSY experiment, magnetization is
transferred by scalar coupling (J-coupling).
Protons that are more than three chemical bonds
apart give no cross signal because the 4J
coupling constants are close to 0. Therefore,
only signals of protons which are two or three
bonds apart are visible in a COSY spectrum (red
signals). The cross signals between HN and H?
protons are of special importance because the phi
torsion angle of the protein backbone can be
derived from the 3J coupling constant between
them.
22
2D TOCSY (TOtal Correlated SpectroscopY)
In the TOCSY experiment, magnetization is
dispersed over a complete spin system of an amino
acid by successive scalar coupling (J-coupling).
The TOCSY experiment correlates all protons of a
spin system. Therefore, not only the red signals
are visible (which also appear in a COSY
spectrum) but also additional signals (green)
which originate from the interaction of all
protons of a spin system that are not directly
connected via three chemical bonds. Thus a
characteristic pattern of signals results for
each amino acid from which the amino acid can be
identified. However, some amino acids have
identical spin systems and therefore identical
signal patterns. They are cysteine, aspartic
acid, phenylalanine, histidine, asparagine,
tryptophane and tyrosine ('AMX systems') on the
one hand and glutamic acid, glutamine and
methionine ('AM(PT)X systems') on the other hand.
23
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24
2D NOESY (Nuclear Overhauser Effect Spectroscopy)
The NOESY experiment is crucial for the
determination of protein structure. It uses the
dipolar interaction of spins (the nuclear
Overhauser effect, NOE) for correlation of
protons. The intensity of the NOE is in first
approximation proportional to 1/r6, with r being
the distance between the protons The correlation
between two protons depends on the distance
between them, but normally a signal is only
observed if their distance is smaller than 5 Å.
The NOESY experiment correlates all protons which
are close enough. It also correlates protons
which are distant in the amino acid sequence but
close in space due to tertiary structure. This is
the most important information for the
determination of protein structures
25
Whats happening during the pulse sequence ?
26
RF pulse I? ? I? cos ? I? sin
?
(?I?)
?, ? and ? represent permutations of the x, y and
z axes and ? is the pulse flip angle
27
Chemical shift Ix ? Ix cos (wIt)
Iy sin (wIt) Iy ? Iy
cos (wIt) Ix sin (wIt)
(wItIz)
(wItIz)
2IzSx ? 2IzSx cos ? 2Iy Sx sin ?
2IxSz ? 2IxSz cos (wIt) 2IySz sin
(wIt)
(?Ix)
(wItIz)
Iz Sz
Iy Sz
Ix
28
J-coupling Ix ? Ix cos (?JISt)
2IySz sin(?JISt) 2IySz ? 2Iy Sz
cos (?JISt) - Ix sin (?JISt)
(?JISt2IzSz)
(?JISt2IzSz)
Example Ix ? 2IySz (t
1/2JIS) 2IySz ? - Ix (t 1/2JIS)
(?JISt2IzSz)
(?JISt2IzSz)
29
COSY
90x
I
90x
S
t
b
a
c
b ? c ? ? Ixcos (wIt) Iz sin (wIt)
cos(?JISt) -2IzSy cos(wIt) 2IxSy
sin(wIt) sin(?JISt)
30
NOESY
90x
90-x
90x
t1
?m
t2
I
S
t
a
b
d
c
e
b ? c ? Ixcos (wIt) Iz sin (wIt)
cos(?JISt) 2IzSz cos(wIt) 2IxSz
sin(wIt) sin(?JISt)
c ? d ? ?
31
180x
HMQC
I
90x
90x
t
S
d
a
b
f
c
e
(?JISt2IzSz)
a ? b Ix ? 2IySz ( 1/2JIS) b
? c ? -2IySy c ? d ?
-2IySy cos(wSt) 2IySx sin(wSt) d ? e ?
-2IySz cos(wSt) 2IySx sin(wSt) e ? f
? Ix cos(wSt) 2IySx sin(wSt) HMQC Ix ?
Ix cos(wSt) ( 1/2JIS)
(90Sx)
(Dec CS)
(90Sx)
(?JISt2IzSz)
32
Anatomy of a 3D experiment
t1
t3
?m
t2
MLEV
FID
Evolution
Preparation
Mixing Time
Detection
Evolution
Mixing Time
A three dimensional NMR experiment (see picture
above) can easily be constructed from a
two-dimensional one by inserting an additional
indirect evolution time and a second mixing
period between the first mixing period and the
direct data acqusition. Each of the different
indirect time periods (t1, t2) is incremented
separately. Triple resonance experiments are the
method of choice for the sequential assignment of
larger proteins (gt 150 AA). These experiments are
called 'triple resonance' because three different
nuclei (1H, 13C, 15N) are correlated. The
experiments are performed on doubly labelled
(13C, 15N) proteins.
33
Whats kind of the structural information that we
can observe from NMR ?
34
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35
  • The distances between vicinal protons vary
    between approximately 2.15 and 2.90 A, and the
    exact values are determined by the intervening
    torsion angle. For instance, the distances
    daN(i,i) and dab(i,i) are related with ?i and ci,
    respectively.

36
1H-1H distance in Proteins
  • Notation for 1H-1H distances
  • daN(i,j) ? d(aHi, NHj)
  • dNN(i,j) ? d(NHi, NHj)
  • dbN(i,j) ? mind(bHi, NHj)
  • daa(i,j) ? d(aHi, aHj)
  • dab (i,j) ? mind(aHi, bHj)

37
Karplus relations
  • For structure determination of proteins the most
    important Karplus relations are
  • 3JNHa 6.4 cos2q 1.4cosq 1.9
  • 3Jab 9.5 cos2q 1.6cosq 1.8
  • 3JNb -4.4 cos2q 1.2cosq 0.1
  • 3JCb 8.0 cos2q 2.0cosq

38
(a) Chemical shifts and secondary structure
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40
NMR data with structural content
Chemical shifts (Ha )
a-helix
b-sheet
Random coil
down field
ltDdgt (db-sheet - drandom coil 0.76 ppm)
Coupling constants ( 3J )
For L-amino acid 3J 6.4 cos2(f 60º)
1.4cos(f 60º) 1.9 For D-amino acid 3J
6.4 cos2(f 60º) 1.4cos(f 60º) 1.9 3J gt 9
Hz, f -120º 30º 3J lt 4 Hz, f -30º 40º
41
a-13C Chemical Shift Values Categorized According
to Secondary Structural Assignmenta-d
a Experimentally measured random coil values from
Richarz and Wuthrich and from Spear and Bax are
included for comparison. Data are given in ppm. b
The compounds (DDS, TMS, or dioxane) used in
referencing the data are shown at the top of each
column. c To adjust DSS values to old dioxane
standard, substract 1.5 ppm. d To adjust DSS
values to TSP, add 0.1 ppm. e Total number of
residues observed is given in parentheses. The
data cover a grand total of 1572 amino acids.
42
Random Coil Chemical Shifts for Backbone Atoms in
Peptides and Proteinsa
a Proton and carbon shifts are relative to DDS,
nitrogen shifts are relative to NH3. Data are
given in ppm. b a-1H shifts were measured using
the hexapeptide GGXAGG in 1M urea at 25C. Wishart
and Skyes, Methods Enzymol. (1994), 239 ,363-392.

43
(b) COSY spectroscopy and its structural
connectivities
44
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45
(A) 1D 1H spectrum
(B) 2D COSY spectrum
(C) Same as (b), contour plot
46
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47
Eight areas containing different connectivities
  • a. All nonlabile, nonaromatic amino acid side
    chain protons except bH-gCH3 of Thr, dH-dH of
    Pro, and bH-bH of Ser.
  • b. aH-bCH3 of Ala and bH-gCH3 of Thr.
  • c. aH-bH of Val, Ile, Leu, Glu, Gln, Met, Pro,
    Arg, and Lys.
  • d. aH-bH of Cys, Asp, Asn, Phe, Tyr, His, and
    Trp.
  • e. aH-aH of Gly, aH-bH of Thr, dH-dH of Pro,
    aH-bH and bH-bH of Ser.
  • f. Aromatic ring protons, including the
    four-bond connectivity 2H-4H of His and side
    chain amide protons of Asn and Gln
  • g. Backone NH-aH.
  • h. dCH3eNH of Arg.

48
(A) 1D 1H spectrum
(B) 2D COSY spectrum
(C) Same as (b), contour plot
49
(c) NOE spectroscopy and its Applications to
Macromolecules
50
Nuclear Overhauser Effects (NOE)
  • The NOE phenomenon is intimately related to spin
    relaxation. Analogous to the spin relaxation T1
    and T2, the NOE varies as a function of the
    product of the Larmor frequency wo, and the
    rotational correlation time tc.
  • Considering a pair of closely spaced spins i and
    j, connected by the vector rij, and located
    either in a small or large spherical molecule. As
    a result of the collisions with the surrounding
    solvent and solute molecules, the thermal motions
    of these spheres consist of a random walk, which
    includes both translational and rotational
    movements. The relevant quantity for
    dipole-dipole relaxation and NOE is the
    rotational tumbling of the vector rij, and the
    concomitant time variation of the angle qij
    between rij and Bo.
  • If the mobility of this vector is restricted to
    the overall rotations of the molecule, rij will
    change orientation much more frequently in the
    small molecule than in the large molecule. For
    spherical particles of radius a in a solvent of
    viscosity ?, a correlation time characterizing
    the frequency range for these stochastic motions
    can be estimated as
  • tc 4??a3/3kT

51
The NOE is a consequence of modulation of the
dipole-dipole coupling between different nuclear
spins by the Brownian motion of the molecules in
solution, and the NOE intensity can be related to
the distance r between pre-irradiated and
observed spin by an equation of the general form
NOE a 1/r6 f(tc) f(tc) is a function of
the correlation time tc, which accounts for the
influence of the motional averaging process on
the observed NOE. It seems to indicate that
distance measurements with the use of NOEs
should be straightforward, provided that f(tc)
can be independently assessed. In reality, a
number of fundamental and technical obstacles
tend to render quantitative distance measurements
difficult. Thus, in all NOE experiments, and in
particular in NOESY, processes other than NOEs
may also be manifested and can lead to
falsification of apparent NOE intensities. Quite
generally, because of the low sensitivity for
observation of NOEs, the accuracy of integration
of line intensities is also limited by low S/N.
Fundamental difficulties can then also arise when
trying to correlate experimental NOE intensities
with distances, for example, because of spin
diffusion or the prevalence of intramolecular
mobility in macromolecules.
52
In a small molecules, for example, a tripeptide
or dinucleotide in aqueous solution, tc is short
relative to wo-1 (at 500 MHz, wo-1 3 x 10-10
s). In this extreme motional situation, the
frequency range covered by the rotational motion
of rij includes wo-1 and 2wo-1, which enables
dissipative transitions between different spin
states. In contrast, for macromolecules tc is
long relative to wo-1, and the frequencies of the
rotational motions are too low to allow efficient
coupling with the nuclear spin transitions.
Therefore, energy-conserving transitions of the
type aibi-biaj (cross relaxation) are favored.
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  • In the 1D experiments with relative line
    intensities I in the absence of NOEs, the line
    intensities with NOE then become
  • I 1 ?i/ 2?j (NOE factor)
  • The 1H1H NOE, which is of prime interest for
    conformational studies, is 0.5 for the extreme
    motional narrowing situation. For tc, longer than
    approximately 1 x 10-9 s it adopts a value of
    -1.0. For 13C and 31P 1H the NOE factor is
    positive over the entire tc range and becomes
    very small for long tc. For 15N 1H, the NOE
    factor is negative throughout because of the
    negative value of ?. The NOEs 1H31P, 1H13C,
    and 1H15N, are very small and relatively of
    little practical importance in macromolecules.
  • In general, mechanisms other than dipole-dipole
    coupling with the preirradiated spin contribute
    to the T1 relaxation. If T1d(j) accounts for the
    dipolar relaxation between i and j and T10 for
    all other contribution to T1 of spin i, NOE
    factor becomes
  • (?i/2?j) (T1d(j)-1/(T1d(j)-1 T10-1 )
  • Accordingly, the NOE can be partially or
    completely quenched in the presence of
    alternative, efficient relaxation pathways, for
    example, through proximity of spin i to a
    paramagnetic center.

55
NOE and structural determination
  • In principle, all hydrogen atoms of a protein
    form a single network of spins, coupled by the
    dipole-dipole interaction. Magnetization can be
    transferred from one spin to another not only
    directly but also indirectly via other spins in
    the vicinity-an effect called spin diffusion.
  • The approximation of isolated spin pairs is only
    valid for short mixing time in the NOESY
    experiment. However, the mixing time cannot be
    made arbitrarily short because the intensity of a
    NOE is proportional to the mixing time.
  • In practice, a compromise has to be made between
    the suppression of spin diffusion and sufficient
    cross-peak intensities, usually with mixing time
    in the range of 40-80 ms. Spin diffusion effects
    can also be included in the structure calculation
    by complete relaxation matrix refinement, care
    has to be taken not to bias the structure
    determination by over-interpretation of the data.

56
90x
90-x
90x
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  • Sequential distances are those between backbone
    protons or between a backbone proton and a b
    proton in residues that are nearest neighbors in
    the sequence. For simplicity, the indices i and j
    are omitted for the sequential distance for
    example,
  • daN(i,i1) ? daN
  • and dNN(i,i1) ? dNN
  • Medium-range distances are all non-sequential
    inter-residue distances between backbone protons
    or between a backbone proton and a b proton
    within a segment of five consecutive residues.
  • Long-range backbone distances are between
    backbone protons in residues that at least six
    positions apart in the sequence, that is ?i-j??
    5. All other inter-residue distances are referred
    to as long-range distances.

60
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61
Short ( lt 4.5 A) Sequential and Medium-Range
1H-1H Distances in Polypeptide Secondary
Structures
a For the turns, the first of two numbers applies
to the distance between residues 2 and 3, the
second to that between residues 3 and 4. The
range between indicated for daN(i,i3)
corresponds to the distances adopted if ?1 is
varied between -180? and 180?. b The ranges given
correspond to the distances adopted by a
b-methine proton if ?1 is varied between -180?
and 180?.
62
  • The distances between vicinal protons vary
    between approximately 2.15 and 2.90 A, and the
    exact values are determined by the intervening
    torsion angle. For instance, the distances
    daN(i,i) and dab(i,i) are related with ?i and ci,
    respectively.

63
Torsion angles for regular polypeptide
conformations
64
(c) Distance dNN and torsion angles relationship
Ramachandran plot
65
Other NMR data for structure determination
  • NOEs and scalar coupling constants are the NMR
    data that most directly provide structural
    information. Additional NMR parameters that are
    sometimes used in structure determination include
    hydrogen exchange data and chemical shifts, in
    particular 13Ca. Slow hydrogen exchange indicates
    that an amide proton is involved in a hydrogen
    bond.
  • It was recognized that the deviations of 13Ca
    (and, to some extent 13Cb) chemical shifts from
    their random coil values are correlated with the
    local backbone conformation 13Ca chemical shifts
    larger than the random coil values tend to occur
    for amino acid residues in a-helical
    conformation, whereas deviations toward smaller
    values are observed for residues in b-sheet
    conformation. Such information can be included in
    a structure calculation by restricting the local
    conformation for a residue to the a-helical or
    b-sheet region of the Ramachandran plot, although
    care should be applied because the correlation
    between chemical shift deviation and structure is
    not perfect.

66
Resonance assignment strategies for small proteins
  • Spin system identification
  • DQF-COSY and TOCSY experiments
  • Sequence-specific assignment
  • NOESY experiment
  • For protein lt 10 kDa, 2D homonuclear
    experiments may be sufficient for resolving
    overlapping NMR resonances.

DQF-COSY Double-Quantum Filter-Correlation
Spectroscopy
TOCSY Total Correlation Spectroscopy
NOESY Nuclear Overhauser Effect Spectroscopy
67
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70
Determination of Macromolecular
Structure by Multidimensional NMR Spectroscopy
71
Spin-spin coupling constants in peptides
Spin-spin coupling constants, like chemical
shifts, depend on chemical environment and are
therefore of great use in structure
determination.
72
2D HSQC (Heteronuclear single quantun
correlation) Spectroscopy
The natural abundance of 15N and 13C is very low
and their gyromagnetic ratio is markedly lower
than that of protons. Therefore, two strategies
are used for increasing the low sensitivity of
these nuclei Isotopic enrichment of these nuclei
in proteins and enhancement of the signal to
noise ratio by the use of inverse NMR experiments
in which the magnetization is tranferred from
protons to the hetero nucleus. The most important
inverse NMR experiment is the HSQC the pulse
sequence of which is shown above. It correlates
the nitrogen atom of an NHx group with the
directly attached proton. Each signal in a HSQC
spectrum represents a proton that is bound to a
nitrogen atom.
73
i-residue
(i-1)-residue
Inter-residues
(b) HN(CA)CO HN(i) N(i) Ca(i) C'(i)
C
C
C
C
N
C
C
N
H
H
O
H
O
H
Inter- (weak) and intra- (strong)
74
Ca(i-1)
(c) HNCA HN(i) N(i)
Ca(i)
Inter- (weak) and intra- (strong)
Inter-residues
75
Ca(i-1) Cb(i-1)
(e) CBCANH HN(i) N(i)
Ca(i) Cb(i)
C
C
C
C
N
N
C
C
H
H
O
H
O
H
Inter- (weak) and intra- (strong), C? and C?
Inter-residues, C? and C?
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2D NMR Spectroscopy
A two-dimensional NMR experiment involves a
series of one-dimensional experiments. Each
experiment consists of a sequence of radio
frequency pulses with delay periods in between
them. It is the timing, frequencies, and
intensities of these pulses that distinguish
different NMR experiments from one another.
During some of the delays, the nuclear spins
are allowed to freely precess (rotate) for a
determined length of time known as the evolution
time. The frequencies of the nuclei are detected
after the final pulse. By incrementing the
evolution time in successive experiments, a
two-dimensional data set is generated from a
series of one-dimensional experiments
Correlation spectroscopy is one of several types
of two-dimensional NMR spectroscopy. Other
types of two-dimensional NMR include
J-spectroscopy, exchange spectroscopy (EXSY), and
Nuclear Overhauser effect spectroscopy (NOESY).
Two-dimensional NMR spectra provide more
information about a molecule than one-dimensional
NMR spectra and are especially useful in
determining the structure of a molecule,
particularly for molecules that are too
complicated to work with using one-dimensional
NMR. The first two-dimensional experiment, COSY,
was proposed by Jean Jeener, a professor at
Université Libre de Bruxelles, in 1971. This
experiment was later implemented by Walter P.
Aue, Enrico Bartholdi and Richard R. Ernst, who
published their work in 1976
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2D NOESY spectrum of 50 mM Gramicidin in DMSO-d6
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2D NOESY spectrum of ethylbenzene
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2D NOESY spectrum of aromatic region of
12,14-ditbutylbenzogchrysene
Continuing the connectivity, we can assign H10 as
7.76 ppm H11 as 7.60 ppm and H13 as 7.86 ppm. In
the opposite direction, H7 is at 7.59 ppm , H6 at
7.55 ppm, H5 at 8.62 ppm, H4 at 8.54 ppm, H3 at
7.44 ppm, H2 at 7.34 ppm and H1 at 8.17 ppm.
Aromaric region shows connectivity and separation
into four color-coded proton groups
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2D TOCSY spectrum of ethylbenzene
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1H-1H TOCSY ?? ?? ?????? 1H-1H TOCSY (TOtal
Correlated SpectroscopY also known as HOHAHA
HOmonuclear HArtmann HAhn) is useful for dividing
the proton signals into groups or coupling
networks, especially when the multiplets overlap
or there is extensive second order coupling. A
TOCSY spectrum yields through bond correlations
via spin-spin coupling. Correlations are seen
throughout the coupling network and intensity is
not related in a simple fashion to the number of
bonds connecting the protons. Therefore a
five-bond correlation may or may not be stronger
than a three-bond correlation. TOCSY is usually
used in large molcules with many separated
coupling networks such as peptides, proteins,
oligosaccharides and polysaccharides. If an
indication of the number of bonds connecting the
protons is required, for example in order to
determine the order in which they are connected,
a COSY spectrum is preferable. The pulse sequence
used in our laboratory is the gradient enhanced
TOCSY. The spin-lock is a composite pulse and
should be applied for between 20 and 200 ms with
a pulse power sufficient to cover the spectral
width. A short spin-lock makes the TOCSY more
COSY-like in that more distant correlations will
usually be weaker than short-range ones. A long
spin-lock allows correlations over large coupling
networks. Too long a spin-lock will heat the
sample causing signal distortion and can damage
the electronics of the spectrometer. The
attenuation should be set so that the 90 pulse
with will be less than 1/(4SWH) (SWH is the
spectral width in Hz) and typically 1/(6SWH). An
attenuation of 12 dB with a 50 W amplifier
yielding a 90 pulse width of 35 µs is typical.
1H-1H COSY Spectroscopy 1H-1H COSY (COrrelated
SpectroscopY) is useful for determining which
signals arise from neighboring protons,
especially when the multiplets overlap or there
is extensive second order coupling. A COSY
spectrum yields through bond correlations via
spin-spin coupling. If a homonuclear coupling is
resolved in the 1D spectrum, a correlation will
appear in the COSY but if no splitting is
observed then no correlation is likely. Two and
three bond and sometimes four bond correlations
yield COSY signals.
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2D COSY spectrum of ethylbenzene
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2D COSY spectrum of aromatic region of
12,14-ditbutylbenzogchrysene
There are separated into four color-coded proton
groups showing connectivity. Using horizontal and
vertical lines, it is possible to separate each
group and follow its connectivity. The blue group
of four protons is connected in the order 8.62
ppm to 7.55 to 7.59 to 8.56, the green group of
four protons in the order 8.54 to 7.34 to 7.44 to
8.17 and the red group or two protons, that
correspond to H9 and 10 because they are the only
group of two protons expected to have a
three-bond coupling constant (8.9 Hz), are at
7.76 and 8.32 ppm. The yellow group of two
protons correspond to H11 and 13 because the
coupling constant is small (1.9 Hz) and
consistent with a four bond correlation.
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HMQC NMR Spectrum of of codeine
This is a 2D experiment used to correlate, or
connect, 1H and 13C peaks for directly bonded C-H
pairs.  The coordinates of each peak seen in the
contour plot are the 1H and 13C chemical shifts. 
This is helpful in making assignments by
comparing 1H and 13C spectra.     This experiment
yields the same information as the older "HETCOR"
experiment, but is more sensitive, so can be done
in less time and/or with less material.  This is
possible because in the HMQC experiment, the
signal is detected by observing protons, rather
than carbons, which is inherently more sensitive,
and the relaxation time is shorter.  This
so-called "inverse detection" experiment is
technically more difficult and is possible only
on newer model spectrometers. Contour plot of
the HMQC spectrum.  Because it is a heteronuclear
experiment, the 2 axes are different, and the
plot is not symmetrical.  Unlike a COSY spectrum,
there are no diagonal peaks.
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HMQC NMR Spectrum of of codeine
Expanded aliphatic region
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CIEAKLTDTTTES (13-mer peptide)
Fig 5-19 Peptide Den7 TOCSY spectrum at pH 5.0
and 50mM phosphate buffer 300µL?30µL D2O, and 298
K.
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CIEAKLTDTTTES (13-mer peptide)
Fig 5-17 ??Den7 DQF-COSY???, pH 5.0?50mM
phosphate buffer 300µL?30µL D2O,298K??????
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CIEAKLTDTTTES (13-mer peptide)
Fig 5-18 ??Den7 NOESY???, pH 5.0?50mM phosphate
buffer 300µL?30µL D2O,298K,mixing time?450ms??????
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1H-1H TOCSY 1H-1H TOCSY (TOtal Correlated
SpectroscopY also known as HOHAHA HOmonuclear
HArtmann HAhn) is useful for dividing the proton
signals into groups or coupling networks,
especially when the multiplets overlap or there
is extensive second order coupling. A TOCSY
spectrum yields through bond correlations via
spin-spin coupling. Correlations are seen
throughout the coupling network and intensity is
not related in a simple fashion to the number of
bonds connecting the protons. Therefore a
five-bond correlation may or may not be stronger
than a three-bond correlation. TOCSY is usually
used in large molecules with many separated
coupling networks such as peptides, proteins,
oligosaccharides and polysaccharides. If an
indication of the number of bonds connecting the
protons is required, for example in order to
determine the order in which they are connected,
a COSY spectrum is preferable. The pulse
sequence used in our laboratory is the gradient
enhanced TOCSY. The spin-lock is a composite
pulse and should be applied for between 20 and
200 ms with a pulse power sufficient to cover the
spectral width. A short spin-lock makes the TOCSY
more COSY-like in that more distant correlations
will usually be weaker than short-range ones. A
long spin-lock allows correlations over large
coupling networks. Too long a spin-lock will heat
the sample causing signal distortion and can
damage the electronics of the spectrometer. The
attenuation should be set so that the 90 pulse
with will be less than 1/(4SWH) (SWH is the
spectral width in Hz) and typically 1/(6SWH). An
attenuation of 12 dB with a 50 W amplifier
yielding a 90 pulse width of 35 µs is typical.
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Spin-spin coupling constant (J) characterizes
scalar interactions (through-bond) between nuclei
linked via a small number of covalent bonds in a
chemical structure.
If two nuclei couple with non-zero spin in the
molecule having, say, spin I1 and I2, then it is
found that the resonance of spin I1 is split into
2I21 lines of equal intensity and that of spin
I2 is similarly split into 2I11 lines. The line
separations are equal. The interaction is known
as spin-spin coupling. And J is field independent
and is customarily quoted in hertz (Hz).
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2D HSQC (Heteronuclear single quantun
correlation) Spectroscopy
The natural abundance of 15N and 13C is very low
and their gyromagnetic ratio is markedly lower
than that of protons. Therefore, two strategies
are used for increasing the low sensitivity of
these nuclei Isotopic enrichment of these nuclei
in proteins and enhancement of the signal to
noise ratio by the use of inverse NMR experiments
in which the magnetization is tranferred from
protons to the hetero nucleus. The most important
inverse NMR experiment is the HSQC the pulse
sequence of which is shown above. It correlates
the nitrogen atom of an NHx group with the
directly attached proton. Each signal in a HSQC
spectrum represents a proton that is bound to a
nitrogen atom.
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Six areas containing different connectivities
  • a. NH aromatics-NH aromatics
  • b. NH aromatics-aH dH of Pro bH of Ser and Thr
  • c. NH aromatics-aliphatic side chains
  • d. aH dH of Pro bH of Ser and Thr-aH dH of
    Pro bH of Ser and Thr
  • e. aH dH of Pro bH of Ser and Thr-aliphatic
    side chains
  • f. Aliphatic side chains-aliphatic side chains
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