Heteronuclear NOE

In the case of proton-proton interactions, both nuclei S and /will have the same gyromagnetic ratios, and an implication of the Equation (4) then is that there is an upper limit of 50% on the nOe obtainable, whatever the distance between nuclei S and I. This means that the observation of an nOe between two nuclei does not necessarily mean they are spatially close to one another, and nOe results must therefore be interpreted with caution. Similarly, as will be seen later, the absence of nOe between two nuclei does not necessarily mean they are far apart. In the case of heteronuclear nOe, since the gyromagnetic ratio of proton (yv) is four times the gyromagnetic ratio of carbon y,), js/ jt can be four times greater than that obtainable in homonuclear nOe. 

So far we have been concerned with homonuclear nOe effects. nOe between nuclei of different elements can also be a useful tool for structural investigations. Such heteronuclear nOe effects—for instance, between protons and carbons—can be used with advantage to locate quaternary carbon atoms. Normally, beteronuclear nOe effects are dominated by interactions between protons and directly bonded carbon atoms, and they can be recorded as either ID or 2D nOe spectra. 

Heteronuclear nOe experiments are similar to homonuclear experiments, discussed earlier. Normally, protons are irradiated and the enhancement of the heteronucleus is recorded, since if the reverse were done, the... 

In a heteronuclear nOe experiment, the first step may be identical to the homonuclear nOe experiment (i.e., involving an incoherent transfer of coherence from H to H/), while the second step could involve a coherence transfer from H to C nucleus by an INEPT sequence. These methods suffer from poor sensitivity and have therefore not been used extensively. 

If all nuclei are assigned and the spectral parameters for the conformational analysis are extracted, a conformation is calculated - usually by distance geometry (DG) or restrained molecular dynamics calculations (rMD). A test for the quality of the conformation, obtained using the experimental restraints, is its stability in a free MD run, i.e. an MD without experimental restraints. In this case, explicit solvents have to be used in the MD calculation. An indication of more than one conformation in fast equilibrium can be found if only parts of the final structure are in agreement with experimental data [3]. Relaxation data and heteronuclear NOEs can also be used to elucidate internal dynamics, but this is beyond the scope of this article. 

We have so far looked at the NOE only in a homonuclear manner, but of course there is also a heteronuclear NOE. Theory tells us that when we are dealing with C-H fragments in small molecules, the decoupling of the proton leads to an increase in the carbon signal intensity by up to almost 200% So signals of protonated carbons should be stronger than those of non-proton-ated carbons. 

Heteronuclear NOE experiments yield additional, but due to their inherent low sensitivity only sparsely exploited information on molecular structures. They are most useful to unravel structural features in the vicinity of quaternary carbons often behaving as barriers when exposed to standard routine experiments dedicated to evaluate coupling networks. Heteronuclear NOE data - in some sense complementary to the data from heteronuclear long-range couplings - are based on dipolar spin-spin interactions and strongly depend on intemuclear distances. In contrast to the sometimes similar Jch... 

Fig. 4. spectrum showing the three quaternary carbons Cl, C2 and C3 of compound 2 dissolved in CDiCN (bottom) and heteronuclear NOE spectra simultaneously acquired with pulse sequence II. Three data blocks a, b and c corresponding to three experiments with the number N of proton resonances selected for simultaneous saturation set to 1 (a), 5 (b) and 10 (c) are depicted. Five spectra with the initial saturation of protons 3, 1, 1 3" and 2 are presented. The additional five NOE difference spectra acquired in case c, with the irradiation of the sixth methyl and of four NH (NHa) proton resonances are not shown. (Continued on... 

Thus, if Pi is known, then the measurement of 77 provides the extreme narrowing conditions, the heteronuclear NOE reaches a maximum value (minimum for 71 < 0) and can be used to determine the dipole-dipole contribution to the total relaxation rate ... 

The experiment is applied for the evaluation of C T, values. T, values are usually used to optimize insensitive C experiments, i.e. to adjust the length of the preparation time in other NMR experiments. To deduce structural information it is usual to interpret the dipolar part of the longitudinal relaxation time (T, ). To separate the dipolar contribution from the contributions of other relaxation mechanisms, it is necessary to perform further experiments (gated decoupling experiments) to evaluate the heteronuclear NOE values. T °° may be exploited in a qualitative way to differentiate between carbon nuclei in less or highly mobile molecular fragments. In a more detailed analysis reliable T, values can be used to describe the overall and internal motions of molecules. 

The relative values of /, J, y/, yj are important for determining the size and the sign of heteronuclear NOE obviously, in homonuclear NOE they cancel each other and Eq. (7.7) takes the simple form... 

Analysis of the low temperature VT NMR (,9F and H) and 19F- H heteronuclear NOE spectra of 65 and 66 in conjunction with molecular modeling has revealed the presence of an equilibrium between two conformers in protic solvent systems. Interpretation of the temperature dependence of the coupling constants between H2 and H3 for 65 shows that one of these conformers (conformer C) has an unusual near-eclipsed arrangement around the H2 -C2 -C3,-H3 dihedral angle (7H2- H3-= 5.2 Hz, corresponding to the H2 -C2 -C3 -H3 torsion angle of 124° based on the MM2 calculation), and is found to be more prevalent at ambient temperatures. The... 

N and 13C labeling of peptides facilitates the study of their molecular dynamics in solution by measurements of relaxation parameters (42,43). Heteronuclear relaxation times and heteronuclear NOEs are predominantly affected by the dipole-dipole interaction of the heteronucleus with the directly attached proton. Since the intemuclear (i.e., chemical bonding) distances are known from the molecular geometry, correlation times for overall and internal motions can be determined. 

The heteronuclear NOE, for example, from to 13 C, is used to enhance the signal-to-noise ratio of 13 C peaks in the spectrum. All protons are irradiated equally and simultaneously during the relaxation delay to pump up the z magnetization of 13C above the equilibrium value of M0. Theoretically this can triple the Mz of 13C. 

Fig. 12. Heteronuclear NOE factors for (a) 15N backbone nuclei of tendamistat C45A/C73A and (b), 3Ca nuclei of the wild-type protein. Solid bars indicate the /3-strands and solid squares positions 45 and 73 in the variant. Disulfide bridges are marked by thin lines. (Reproduced from Balbach et al.64 with permission. Copyright (1998) Wiley-Liss Inc., a subsidiary of John Wiley Sons Inc.)...

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