The nuclear Overhauser effect (NOE) itself is a manifestation of cross relaxation, stimulated by dipolar coupling. If we imagine a little magnetic field associated to each nucleus in the sample, it is easy to imagine that the nuclei will affect each other, each generating its own field and sensing the others. The prerogative for cross relaxation to take place (so for 𝜎𝐼𝑆 to be non-zero), is that the set of nuclei I and S are close to each other, as from the previous equations we have seen that 𝜎𝐼𝑆 is inversely proportional to the sixth power of the distance between the nuclei. The NOE’s dependence on the distance between sets of nuclei is what makes this technique essential in structural studies, as we will show.
1.3.3.1 NOE enhancement in the slow and fast motion regime
In the two-spin homo-nuclear system which we are considering, we can envisage the situation in which we apply a continuous selective rf pulse on a frequency corresponding to the single transition of one of the nuclei (say S), saturating the populations across energy levels 1-2 and 3-4 in Figure 1.14. In this new condition, the allowed relaxation pathways are 𝑊2𝐼𝑆 and 𝑊0𝐼𝑆, as the single quantum transitions of spin S are saturated (Figure 1.15). 𝑊2𝐼𝑆 and 𝑊0𝐼𝑆 are not single quantum transitions, so that they cannot be detected, but their effects are observable on the signals of spin I in a 1H 1D spectrum
43 | P a g e
Figure 1.15. Energy levels for a two-spin system upon saturation of spin S. Figure adapted from
55.
This results from a change in the spin I magnetisation that will manifest as a modification of the NMR signal intensity of I. This modification resulting from selective saturation of the spin S transitions is termed NOE enhancement (Figure 1.16).
Figure 1.16. Examples of the effects of cross relaxation on 1D 1H NMR spectra of spins I and S
(a) upon selective saturation of spin S. When spin S is irradiated (b), the S populations are
equalised, and we do not observe any signal (in the irradiated spectrum the situation after instantaneous saturation is depicted). If cross relaxation between S and I is taking place, the peak intensity of spin I is affected. When relaxation takes place via 𝑊2 processes, a positive
enhancement is observed (c); when relaxation takes place via 𝑊0 processes, a negative
enhancement is observed (d). Figure adapted from 54.
When spin S is irradiated, the S populations are equalised, and we do not observe any signal for that spin. At the same time, the peak intensity of spin I is affected due to cross relaxation with spin S. When cross relaxation takes place preferentially via 𝑊2 processes,
44 | P a g e a positive enhancement is observed; in contrast, if the dominant mechanism for cross relaxation involves 𝑊0 processes, a negative enhancement is observed (Figure 1.16c,d).
The NOE enhancement is indicated as 𝜂, and its sign and magnitude are proportional to the cross relaxation rate 𝜎𝐼𝑆:
𝜂 ∝ 𝜎𝐼𝑆 = 𝑊2𝐼𝑆 − 𝑊
0𝐼𝑆 Equation 1.34
The magnitude of the NOE on spin I varies according to how the NOE experiment is performed (and it can depend on other variables), but the sign of the enhancement will be dictated by which process is more favourable between 𝑊2𝐼𝑆 and 𝑊0𝐼𝑆, for the system in analysis.
We have already introduced the definition for fast motion or extreme narrowing limit, defined as 𝜔0𝜏𝑐 ≪ 1 and characterised by very short correlation times; and the slow motion or spin diffusion limit, when 𝜔0𝜏𝑐 ≫ 1 and characterised by very large correlation times.
Thus, in the extreme narrowing limit,
𝑗(2𝜔0) = 2𝜏𝑐 = 𝑗(0) Equation 1.35
and Equation 1.33 simplifies to 𝜎𝐼𝑆 = 12𝑏2𝜏
𝑐 Equation 1.36
Under this condition, the sign of 𝜎𝐼𝑆 is positive since 𝑊2𝐼𝑆 > 𝑊0𝐼𝑆, resulting in a positive NOE enhancement.
In the spin diffusion limit, 𝑗(2𝜔0) = 2
𝜔02𝜏𝑐 =
𝑗(0)
𝜔02𝜏𝑐2 Equation 1.37
and Equation 1.33 simplifies to 𝜎𝐼𝑆 = −101 𝑏2𝜏
𝑐 Equation 1.38
as 𝑗(2𝜔0) is negligible relative to 𝑗(0). Under this condition, the sign of 𝜎𝐼𝑆 is negative since 𝜏𝑐 > 0, resulting in a negative NOE enhancement.
45 | P a g e In concluding this discussion, it is important to stress that if 𝑟 is too large (usually above 5 Å), 𝑏 will tend to 0, 𝜎𝐼𝑆 will be null and there will not be NOE effect whatsoever. That is, in essence, the relevance of NOE experiments for determination of 3D structures of molecules: it is observable only for short spin-spin distances, reporting through-space connectivity associated to the 3D architecture of the molecule.
1.3.3.2 Steady state NOE
The experiment schematised in Figure 1.16, in which spin S was saturated selectively by a weak continuous wave (CW) rf irradiation, is called “steady state” NOE (ss -NOE). We define a long low power continuous wave rf as a “saturating pulse”, which ensures full saturation of the populations of spin S, and hence results in the disappearance of the peak corresponding to S, and in an enhancement in the peak corresponding to I, proportional to 𝜎𝐼𝑆.
The pulse sequence to perform this experiment is shown in Figure 1.17: in a first experiment, a continuous wave is on spin S for a time long enough to allow homogeneous saturation across the sample (long rectangle in Figure 1.17); as soon as the CW is switched off, a hard 90° pulse is given (short and tall rectangle in Figure 1.17), and acquisition follows.
Figure 1.17. Pulse sequence for steady state (SS) NOE experiments. The saturation on spin S is
obtained using a continuous wave rf irradiation, immediately followed by a hard 90° pulse and acquisition. The reference spectrum, in which the intensities are unperturbed, is a simple pulse- acquisition experiment. The difference spectrum is obtained by subtraction of the saturated spectrum from the reference spectrum. Figure adapted from 55.
Treating this with the product operator formalism, 𝑆𝑧= 0, i.e. the steady state has been reached as an effect of the CW (when this is on for long enough). This means that 𝐼𝑧 is
46 | P a g e not changing either (𝑑𝐼𝑧/𝑑𝑡 = 0). Including these two conditions, we can rewrite Equation 1.23 as it follows: 𝑑(𝐼𝑧−𝐼𝑧0) 𝑑𝑡 |𝑆𝑆 = −𝑅𝑧𝐼(𝐼𝑧,𝑆𝑆− 𝐼𝑧 0) − 𝜎 𝐼𝑆 (0 − 𝑆𝑧0) = 0 Equation 1.39 therefore: 𝐼𝑧,𝑆𝑆 = 𝜎𝐼𝑆 𝑅𝑧𝐼𝑆𝑧 0+ 𝐼 𝑧0 Equation 1.40
Hence, the enhancement 𝜂 is given by:
𝜂
𝑆𝑆=
𝐼𝑧,𝑆𝑆−𝐼𝑧,𝑟𝑒𝑓 𝐼𝑧,𝑟𝑒𝑓=
𝜎𝐼𝑆 𝑅𝑧𝐼 𝑆𝑧0 𝐼𝑧0 Equation 1.41where 𝐼𝑧,𝑟𝑒𝑓 is the magnetisation of spin I in the reference spectrum. The element to stress here is that the enhancement on spin I, when spin S is saturated to the steady state, does not only depend on 𝜎𝐼𝑆 alone, but the longitudinal relaxation of I itself, 𝑅𝑧𝐼, plays a role too.
1.3.3.3 Transient NOE
Equation 1.41 implies that the enhancement measured on spin I, in a steady state experiment saturating spin S, is not only proportional to 𝑟𝐼𝑆−6, so it can only give a qualitative measure of the distances. This is not the case for the transient NOE experiment, which relies on a shaped and selective 180° pulse on S to invert its population (rather than equalising it).
The pulse sequence to perform this experiment is shown in Figure 1.18. Again, in the first experiment, inversion of population is achieved by a shaped short pulse; a mixing time, 𝜏𝑚, is then allowed for the magnetisation to evolve, before a hard 90° pulse is given, and acquisition follows.
This means that, after the pulse (time 0), 𝐼𝑧(0) = 𝐼𝑧0 and 𝑆𝑧 (0) = −𝑆𝑧0.Dealing with a transient process means that in this case the differential equations for 𝑑𝐼𝑧/𝑑𝑡 and 𝑑𝑆𝑧/𝑑𝑡 must be solved. If we want to know what happens to spin I when the population on spin S is inverted, we must solve Equation 1.23 again:
𝑑𝐼𝑧
𝑑𝑡 = −𝑅𝑧𝐼(𝐼𝑧(𝑡) − 𝐼𝑧 0) − 𝜎
47 | P a g e where the dependence of the magnetisation of both spins with the time has been highlighted, as we are dealing with a transient phenomenon. In the initial rate approximation (in the first instance after the shaped pulse, i.e., for very short mixing times), we can re-write:
𝑑𝐼𝑧
𝑑𝑡|𝑖𝑛𝑖𝑡 = −𝑅𝑧𝐼(𝐼𝑧 0− 𝐼
𝑧0) − 𝜎𝐼𝑆 (−𝑆𝑧0− 𝑆𝑧0) = 2𝜎𝐼𝑆𝑆𝑧0 Equation 1.43
Figure 1.18. Pulse sequence for transient NOE experiments. The inversion on spin S is obtained
using a shaped 180° pulse, followed by a mixing time, 𝜏𝑚, a hard 90° pulse and acquisition. The
reference spectrum, in which the intensities are unperturbed, i s a simple pulse-acquisition experiment. Figure adapted from 55.
It is easy to integrate this equation for the length of the mixing time (that is from time 0 to 𝜏𝑚), to find that:
𝐼𝑧(𝜏𝑚) = 2𝜎𝐼𝑆𝜏𝑚𝑆𝑧0 + 𝐼
𝑧0 Equation 1.44
where the NOE enhancement is given by:
𝜂(
𝜏𝑚) =
𝐼𝑧(𝜏𝑚)−𝐼𝑧,𝑟𝑒𝑓𝐼𝑧,𝑟𝑒𝑓
=
2𝜎𝐼𝑆𝜏𝑚𝑆𝑧0
𝐼𝑧0 Equation 1.45
Unlike the steady state experiment, the behaviour of spin I does not depend on its longitudinal relaxation properties, but only on the cross relaxation rate and on the mixing time, which we can set experimentally. This make transient NOE an excellent tool for correlating NOE enhancements coming from transient NOE experiments with inter- nuclear distances between the two spins based on the inverse proportionality of 𝜎𝐼𝑆 and 𝑟𝐼𝑆6.
48 | P a g e
1.3.3.4 Truncated driven NOE (TOE)
If we apply short shaped selective pulses in a train, increasing the overall length of the irradiation period, we can measure the rate at which the steady state is reached; but in the first points of this experiment we are still in transient NOE condition. We call this approach the truncated driven NOE, abbreviated to TOE. As we will see in more detail in Chapter 2, Section 2.1, TOE is the key element for Saturation Transfer Difference NMR (STD NMR), which relies on saturation on the nuclei of a large molecule receptor (generally a protein) leading to intermolecular NOEs with the closest nuclei of the l igand. Whereas the NOE enhancement at the steady state is strongly dependent on the relaxation properties of the nuclei (in this case the ligand nuclei, which is what we observe), the rate at which the steady state is reached is affected by inter-nuclear distances. Before entering into the details concerning STD NMR, which is the core technique of the different systems studied in this thesis, in the following sub-section, we will introduce the concept of spin diffusion and give a brief overview on the wide range of NOE- or relaxation-based methods to study intermolecular interactions by NMR.