NOE DIFFERENCE SPECTRA

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CHM 512, March 24/31, 2008
NOE DIFFERENCE SPECTRA In many cases of
interpretation of NMR spectra, it would be
helpful to be able to distinguish protons by
their spatial location within a molecule. For
example, for alkenes it would be useful to
determine whether two groups are cis to each
other or whether they represent a trans isomer.
Many of these types of problems cannot be solved
by an analysis of chemical shift or by
examination of spin-spin splitting effects. A
handy method for solving these types of problems
is NOE difference spectroscopy. This technique is
based on the same phenomenon that gives rise to
the nuclear Overhauser effect in 13C-NMR, except
that it uses homonuclear, rather than a
heteronuclear, decoupling. For two nuclei to
interact via the nuclear Overhauser effect, the
two nuclei do not need to be directly bonded it
is sufficient that they be near each other
(generally within about 3 A. Nuclei that are in
close spatial proximity are capable of relaxing
one another by a dipolar mechanism. If the
magnetic moment of one nucleus, as it precesses
in the presence of an applied magnetic field,
happens to generate an oscillating field that has
the same frequency as the resonance frequency of
a nearby nucleus, the two affected nuclei will
undergo a mutual exchange of energy, and they
will relax one another. The two groups of nuclei
that interact by this dipolar process must be
very near each other the magnitude of the effect
decreases as r -6 , where r is the distance
between the nuclei. We can take advantage of this
dipolar interaction with an appropriately timed
application of a low-power decoupling pulse. If
we irradiate one group of protons, any nearby
protons that interact with it by a dipolar
mechanism will experience an enhancement in
signal intensity. The typical NOE difference
experiment consists of two separate spectra. In
the first experiment, the decoupler frequency is
tuned to match exactly the group of protons that
we wish to irradiate. The second experiment is
conducted under conditions identical to the first
experiment, except that the frequency of the
decoupler is adjusted to a value far away in the
spectrum from any peaks The two spectra are
subtracted from each other (this is done by
treating digitized data within the computer), and
the difference spectrum is plotted. The NOE
difference spectrum thus obtained would be
expected to show a negative signal for the group
of protons that had been irradiated. Positive
signals should be observed only for those nuclei
that interact with the irradiated protons by
means of a dipolar mechanism. In other words,
only those nuclei that are located within about
2.5 to 3 A of the irradiated protons will give
rise to a positive signal. All other nuclei that
are not affected by the irradiation will appear
as very weak or absent signals.
From Introduction to Spectroscopy, 3rd Ed.,
Pavia, Lampman and Kriz.
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From Introduction to Spectroscopy, 3rd Ed.,
Pavia, Lampman and Kriz.
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C7H14 NOEDIFF
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nOe and NOESY
The nuclear Overhauser enhancement (nOe) effect
provides information about the proximity in space
of protons rather than the through-bond
connectivity information provided by conventional
coupling constants. The nOe effect is manifested
as a change in the intensity (typically between
1 and 20) of a resonance in the nmr spectrum as
a result of irradiation at a second resonance.
The magnitude of the effect depends upon the
distance in space between the two nuclei and is
proportional to 1/r6, where r is the internuclear
separation. NOes are rarely seen between pairs
of protons that are separated by more than about
4.5Å, however such 'non-bonded' contacts may
provide valuable information to infer the
three-dimensional structure in large biomolecules
such as proteins or nucleic acids.
The NOESY experiment is a 2D technique in which
the spectrum appears along a diagonal and the nOe
is revealed as cross-peaks off the diagonal.
Shown below is a hypothetical NOESY spectrum for
a molecule containing four protons A-D. Protons C
and B are close, C and D are further apart and A
is distant from all the other protons.
http//www.pitt.edu/gilbertw/chem1590_web_folder/
NMR2/t9.htmldna
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C19H18O
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C19H18O - COSY
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C19H18O - NOEDIFF
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C19H18O - NOESY
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C19H18O NOESY (exp)
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C19H18O - HMQC
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C19H18O - HMBC
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C19H18O HMBC (exp)
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2D NOESY spectrum for the oligonucleotide
CGCGTTTTCGCG shown to the right (from  D. R.
Hare, B. R. Reid, Biochemistry, 1986, 25, 5341
A. Pardi, D. R. Hare, C. Wang, Proc. Natl. Acad.
Sci., 1988, 85, 8785) shows a cross-peak
connectivity map with nOe effects between the
chemical shift region 8.0 - 7.2 (the aromatic
protons and 6.1 - 5.3 ppm (the ribose protons).
The proximity of the aromatic protons of the
bases to the anomeric protons of the ribose rings
is a typical feature of the helical structure of
DNA fragments. Normally, individual protons
display more than one nOe effect, and such a
'stepping stone' approach can serve to establish
the base sequence. In this example, two exactly
vertical nOe peaks would indicate that an
individual anomeric proton was close to two
different aromatic base protons, one from its own
base, and one from an adjacent base in the
oligomer. From such information a partial or
complete base sequence can be mapped.
http//www.pitt.edu/gilbertw/chem1590_web_folde
r/NMR2/t9.htmldna
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http//www.acornnmr.com/codeine/
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COSY
NOESY
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COSY
NOESY
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