Todays Lecture

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Todays Lecture
10) Wed, Oct 23 Homonuclear 2D experiments a.
TOCSY Introduction to Isotropic mixing and
effective field strength b. NOESY Introduction
to the NOE c. Phase cycling
A. S. Edison University of Florida
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Before we get to TOCSY, lets review J coupling
All of the splittings are J couplings
1H NMR 1D spectra of Methyl a-D-Arabinofuranoside
in CD3CN. Collected at 11.7 T by Jim Rocca in
AMRIS.
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J coupling
Decouple 4.05 ppm
Decouple 4.93 ppm
Normal spectrum
J coupling allows you to identify atoms within 3
(sometimes more) covalent chemical bonds. This
will be even more powerful when we get to 2D NMR
experiments.
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2D COSY
As you have shown by product operators, the COSY
sequence correlates resonances that are coupled
together through J-coupling. Each time period
(t1 and t2) gives rise to a frequency axis in the
2D spectrum. Coupled spins can be identified by
cross-peaks in the spectrum.
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2D COSY gives all the J-coupling information
simultaneously
Each pair of coupled spins shows up as a
cross-peak in a 2D COSY spectrum. The diagonal
peaks correspond to the 1D spectrum.
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TOCSY OVERVIEW

• TOCSY (Total Correlation Spectroscopy) is capable
  of correlating all spins in a coupled network
  (examples shown on the next slide).
• The working end of the TOCSY pulse sequence is
  an isotropic mixing sequence that is a sequence
  of pulses that are designed to remove all
  chemical shift differences and create a strong
  coupling environment. This is called the
  Hartmann-Hahn condition. Sometimes the TOCSY
  experiment is called HOHAHA (Homonuclear
  Hartmann-Hahn).
• Several mixing sequencings have been developed.
  The most popular are MLEV-17, WALTZ-16, GARP, and
  DIPSI. These all apply a series of pulses with
  either different phases or different lengths or
  both.
• The strong coupling product operator is similar
  to the regular (weak) coupling operator but it
  includes IxSx and IySy terms.
• The net result of the TOCSY mixing sequence is
  the transfer of magnetization from I to S along
  the same axis (e.g. Iz to Sz or Ix to Sx or Iy to
  Sy). The transfer depends on the coupling
  constant but is fairly complicated because S can
  transfer to another spin, R, and so on.

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TOCSY Pulse sequence (the simplest of several
variants)
t1
The numbers are pulse lengths in degrees. The
red and black pulses are 180 degrees out of
phase. The element in the parenthesis is
repeated n-times to get the desired mixing time
(e.g. 20-80 ms)
MLEV-17 (90-y, 180x, 90-y, 90-y, 180x, 90-y)n
60x
DIPSI-2 (320, 410, 290, 285, 30, 245, 375, 265,
370)n
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Spin Systems
Any set of protons in a chain of unbroken
J-coupling interactions will give rise to sets of
TOCSY cross peaks. For example, each of the 3
sets of peaks shown above will be correlated in a
2D TOCSY spectrum.
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TOCSY spectrum of a 16 amino acid peptide
Each amino acid will produce a pattern of peaks
that represent the network of coupled spins in
that amino acid. The highlighted spin system
shown in red is a leucine. Note that there are
two leucines with very similar chemical shifts.
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NOE

• Transferring magnetization through scalar
  coupling is a coherent process. This means
  that all of the spins are doing the same thing at
  the same time.
• Relaxation is an incoherent process, because it
  is caused by random fluxuations that are not
  coordinated.
• The nuclear Overhauser effect (NOE) is in
  incoherent process in which two nuclear spins
  cross-relax. Recall that a single spin can
  relax by T1 (longitudinal or spin-latice) or T2
  (transverse or spin-spin) mechanisms. Nuclear
  spins can also cross-relax through dipole-dipole
  interactions and other mechanisms. This cross
  relaxation causes changes in one spin through
  perturbations of the other spin.
• The NOE is dependent on many factors. The major
  factors are molecular tumbling frequency and
  internuclear distance. The intensity of the NOE
  is proportional to r-6 where r is the distance
  between the 2 spins.

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Ethyl Benzene NOE
Saturation around 1.3 ppm (e.g. methyl)
Saturation around 4.5 ppm (e.g. no saturation)
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Ethyl Benzene NOE Difference spectra
The spectrum above is the difference between the
spectrum with saturation and that without.
These are expansions of the difference spectrum
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Ethyl Benzene NOE Difference spectra
Difference spectrum saturate CH3
Difference spectrum saturate CH2
Aromatic 1H no saturation
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Qualitative Description of the NOE
Two nuclear spins within about 5 Å will interact
with each other through space. This interaction
is called cross-relaxation, and it gives rise to
the nuclear Overhauser effect (NOE). Two spins
have 4 energy levels, and the transitions along
the edges correspond to transitions of one or the
other spin alone. W2 and W0 are the
cross-relaxation pathways, which depend on the
tumbling of the molecule.
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Qualitative Description of the NOE
The NOE only can measure distances up to about 5
Å, because the effect depends on r-6 where r is
the distance between the two interacting protons.
1/r6
r
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Qualitative Description of the NOE
When two nuclear spins are within 5 Å, they will
cross-relax. If one spin (S) is saturated (red
lines along the edge), the system is not in
equilibrium anymore. Magnetization will either
flow from the top to the bottom (W2 active) or
from the right to left (W0 active). The
difference in energy between bb and aa is twice
the spectrometer frequency, and molecular motions
about that frequency are required for the
transition. The difference between ab and ba is
very small, and very slow molecular motions (e.g.
proteins) will excite that transition.
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NOE vs. ROE
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NOESY pulse sequence
frec
Like all 2D sequences, t1 is the variable time to
collect frequency information in the indirect
dimension. The delay t is fixed and is the time
during which the NOE builds up. You might have
guessed that chemical exchange can also happen
during this time, and it is possible to confuse
an NOE peak with a chemical exchange peak, but
techniques have been developed to figure out
which is which. Notice that I have not indicated
the phases of the pulses. The different fs make
up a phase-cycle, and we can also adjust which
axis from which to record the signal (frec). All
2D pulse sequences have phase cycles, but I have
not indicated it until now. Next lecture we will
consider phase cycles in more detail.
A. S. Edison University of Florida
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NOESY spectrum of a 16 amino acid peptide
Each cross peak in a NOESY spectrum indicates
that the nuclei resonating at the 2 frequencies
are within 5 Å in space.
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1H-based Resonance Assignments
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1H-based Resonance Assignments
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Next Fridays Lecture
11) Fri, Oct 25 Heteronuclear 2D NMR a. HSQC b.
Gradients in NMR c. Water elimination Watergate
A. S. Edison University of Florida