Transcript No Slide Title

Connections through space
• So far we have analyzed experiments that work because the
system has scalar couplings. COSY, HETCOR, HOMO2DJ,
MQF-COSY, and INADEQUATE tell us about the chemical
structure of the system, but nothing about the conformation
or stereochemistry (not entirely true, but bear with me here).
• We had seen at the very beginning that if we saturate a
proton in the sample, it will relax by either zero- or doublequantum processes, giving energy (enhancing) the signals
of protons dipolarly coupled to it (protons close by…). This
was the nuclear Overhauser effect (NOE):
bb (*)
W1I
(***) ab
W2IS
W1S
ba (*)
W0IS
W1S
W1I
aa (***)
• We had seen that relaxation by either W2IS or W0IS will occur
depending on the size of the molecule, actually its rate of
tumbling, or correlation time, tc.
The two-spin system case
• We had established a relationship between the different
rates or probabilities and the magnitude of the NOE. If we
limit everything to 1H-1H (gI / gS = 1), we have:
hI =
W2IS - W0IS
2W1S + W2IS + W0IS
= fI{S}
• fI{S} is the NOE enhancement of nucleus I when we saturate
nucleus S. You may also find this equation written in another
way in some textbooks. We define the numerator as the
cross-relaxation rate constant, sIS, and the denominator as
the dipolar longitudinal relaxation, rIS.
sIS = W2IS - W0IS
rIS = 2W1S + W2IS + W0IS
hI = sIS / rIS= fI{S}
• We already saw that the sign of the NOE will depend on the
tumbling rate (size) of the molecule. There were two distinct
cases, one in which w * tc << 1, or extreme narrowing limit,
and w * tc >> 1, or diffusion limit.
Correlation functions and spectral density
• We had mentioned before that the pathways for the system
had to release energy to the lattice depended on the
frequencies of different processes the system can undergo.
• In solution, this means rotation of the molecule (tc). The spins
stay aligned with the external Bo, while the molecule turns,
and this generates magnetic fields (fluctuating dipoles) at the
frequency of the rotation that allow spins to release energy:
• We need a way of analyzing the way a molecule tumbles in
solution. We define the correlation function of a system as
the average of the molecular orientation at a certain time, and
a little while (t) after that:
g(t) = R(t) * R(t + t)
• It basically (cor)relates the orientation of the molecule at two
different times. g(0) = 1, and g(t) decays exponentially as a
function of t/ tc, tc being the correlation time of the molecule.
Spectral density
• As we now know, once we have a function of time, we can
check the frequencies of the processes involved by doing an
FT. The FT of g(t) is called the spectral density function,
J(w), and since g(t) is a decaying exponential, J(w) is a
Lorentzian curve:
2 tc
J(w) =
1 + w2tc2
• Depending on tc, the sluggishness of the molecule, its
movement will be composed by frequencies slower,
comparable, or faster than wo, the Larmor frequency of the
system:
J(w)
wo
wo * tc >> 1
wo * tc ≈ 1
wo * tc << 1
log(w)
The whole enchilada
• Since the probability of a transition depends on the different
frequencies that the system has (the spectral density), the
W terms are proportional the J(w).
• Also, since we need two magnetic dipoles to have dipolar
coupling, the NOE depends on the strength of the two
dipoles involved. The strength of a dipole is proportional to
rIS-3, and the Ws will depend on rIS-6:
W0IS  gI2 gS2 rIS-6 tc / [ 1 + (wI - wS)2tc2]
W2IS  gI2 gS2 rIS-6 tc / [ 1 + (wI + wS)2tc2]
W1S  gI2 gS2 rIS-6 tc / [ 1 + wS2tc2]
W1I  gI2 gS2 rIS-6 tc / [ 1 + wI2tc2]
• The relationship is to the inverse sixth power of rIS, which
means that the NOE decays very fast as we pull the two
nuclei away from each other.
• For protons, this means that we can see things which are at
most 5 to 6 Å apart in the molecule (under ideal conditions…).
Steady-state NOE
• In small, rigid molecules the following relationships are valid:
(wI - wS) * tc<< 1
wS * tc<< 1
wS * tc << 1
(wI + wS) * tc<< 1
• Basically, they move really fast. There is a big simplification
of the probability equations (the W stuff), and we end with a
simple dependency for the NOE enhancement: hI = 0.5.
• Bummer. We had the rIS-6 dependency there and now it’s
gone. The problem is that we have only two spins, and this
basically means no ‘geometry’. This is normally the case for
1H-13C.
• Fortunately, when we look at the proton enhancements in a
molecule, we are always looking at more than one proton.
This means that if we irradiate a proton, it will be dipolarly
coupled to several protons at the same time.
b
c
a
rba
rac
Steady-state NOE (continued)
• What happens is that we have competing relaxation mechanisms for the proton we are saturating (two or more protons in
the surroundings). Now the rates of the relaxation with the
different protons becomes important (the respective Ws).
• The equations get really complicated, but if we are still in the
extreme narrowing limit, we can simplify things quite a bit. In
the end, we can establish a ‘simple’ relationship between the
NOE enhancement and the internuclear distances:
fI{S} = hmax *
rIS-6 - SX fX{S} * rIX-6
rIS-6 + SX rIX-6
• In order to estimate distances between protons in a molecule
we can saturate one nuclei and analyze the relative
enhancements of other protons. This is know as steady-state
NOE.
• We take two spectra. The first spectrum is taken without any
irradiation, and the second with. The two are subtracted, and
the difference gives us the enhancement from which we can
estimate distances...
NOE difference spectroscopy
• If our molecule has three protons, two of them at a fixed
distance (a CH2), we have:
Hb
Ha
Hb
C
Hc
Ha
Hc
_
=
hab hac
• Since we have a reference pair of protons (Ha and Hb) for
which we know the distance, we can establish a reference
NOE effect for it, and then calculate the distance between the
other protons (Ha and Hc in this example):
hab  rab-6
hac  rac-6
rac = rab * ( hab / hac ) -1/6
• The sizes of the peaks are measured by integration. Since
the effect is not symmetric, we usually do the I
S and then
the S
I enhancement and take an average value.
Transient NOE
• One of the problems of steady-state NOE is that we are
continuously giving power to the system (saturation). This
works well for small molecules, because W2 processes
(double-quantum) are dominant and we have few protons.
• However, as the size and tc increase, other processes are
more important (normal single-quantum spin-spin relaxation
and zero-quantum transitions).
• Additionally, there are more protons in the surroundings of a
larger molecule, and we have to start considering a process
called spin diffusion:
I
S
• Basically, the energy transferred from I to S then diffuses to
other nuclei in the molecule. We can see an enhancement of
a certain proton even if it is really far away from the center
we are irradiating, which would give us ambiguous results.
• Therefore, we need to control the amount of time we saturate
the system. The longer we irradiate, the more spin diffusion…
Transient NOE (continued)
• There are also some technical difficulties if we try to do this
by selective saturation. Since what we need is to see how a
system returns to equilibrium through cross-relaxation, we
can selectively invert one transition and then see how the
NOE grows with time:
bb (**)
W1I
(****) ab
W2IS
W1S
ba ()
W0IS
W1I
W1S
aa (**)
• A pulse sequence to do this could be the following:
90
180s
tm
selective inversion
• The last p / 2 pulse is usually called a read pulse, and its only
job is to convert whatever magnetization is in <z> after tm into
<xy> magnetization (detectable). All the equations are the
same, but the NOE will also depend on the mixing time, tm.
Transient NOE (…)
• If we do it for different tm values, we get NOE build-up
curves, which in the case of two isolated protons and ideal
conditions are exponentials that grow until they reach hmax.
hmax
tm
• If we also take into account the T1/T2 relaxation, the NOE
grows and then falls to zero (all the magnetization returns to
the <z> axis…):
hmax
tm
Transient NOE and NOESY
• If we had more spins and spin diffusion, the curve (for three
spins, I, S, and X) would look like this:
hmax
Diffusion to X
tm
• If we want to measure distances more or less accurately, we
have to find a compromise between the mixing time tm that
we use and the spin-diffusion we may have.
• Doing one proton at a time takes forever, so we obviously use
a 2D experiment in which we study all protons at the same
time. The sequence is called NOESY (NOE SpectroscopY),
and it’s shown below:
90
90
t1
“inversion”
90
tm
NOESY (continued)
• The first two pulses (with the t1 variable time) are basically
an inversion for all protons, in which we label everything with
chemical shifts and couplings (no re-focusing).
• What ends up in the <-z> axis evolves during the tm mixing
time, and dipolarly coupled spins will suffer NOE crossrelaxation according to all the equations we’ve seen.
• The 2D spectrum after 2D FT has chemical shifts in both f1
and f2 (t2 has all the chemical shifts also) and cross-peaks
for systems that are dipolarly coupled:
d (f1)
Hc
Hb
C
Ha
Ha
Hc
Hb
d (f2)
• The size of the cross-peak will depend on the internuclear
distances. These are measured relative to a cross-peak for
which we know a distance, using the cross-peak volumes.
NOESY (…)
• Although this pulse sequence looks like the MQF-COSY
pulse sequence, proper phase cycling removes COSY-type
cross-peaks.
• One more thing we have to consider is the sign of the crosspeaks. Thye will depend on the correlation time tc, in the
same way that the NOE enhancement does. We therefore
have positive cross-peaks for w * tc << 1 and negative crosspeaks for w * tc << 1.
• As we said earlier, there is a range of w * tc values for which
the enhancement can be zero (guess which looser had this
happening to him?). If we express the NOE enhancement as
a function of w * tc we get a sigmoid function, and we solve it
for hI = 0:
hI =
5 + w2tc2 - 4w4tc4
10 + 23w2tc2 + 4w4tc4
hI = 0 for w * tc = 1.12
• The options are to change tc (kind of hard), w (the magnet is
pretty hard to change…), or trick the system to think that we
are working at a very low field, where w * tc << 1 always.
• The experiment that does this is called ROESY, and it is
based on spin-locking, which we will see next class.
Summary
• The NOE enhancement is, among other things, proportional
to r-6, so it allows us to make determination of internuclear
distances: stereochemistry and conformation.
• Depending on the size/rigidity (tc) of the molecule, steadystate or transient NOE experiments will give more accurate
results:
• Small organic compounds: Steady-state NOE.
• Proteins, nucleic acids, saccharides: Transient NOE,
build-up curves.
• We can do everything at once using NOESY (transient NOE).
Next time
• Spin-locking - NMR at ‘lower’ fields…
• Uses in through-space correlation spectroscopy (ROESY)
and J-correlation spectroscopy (TOCSY).