The NOE Difference Experiment

Keep the lock power just below saturation and the lock gain at approximately 20% of maximum for optimum lock stability. 

Interleave on- and off-resonance irradiation, using a block size of four to eight scans. 

A moderate line broadening ( 2 Hz, not enough to cause signal overlap) reduces spectral noise. 

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NOE-difFerence spectroscopy is particularly valuable for distinguishing stereoisomers, for it relies solely on intemuclear distances, and thus avoids any problems of ambiguity or absence associated with couplings. With smallish molecules, it is best carried out in the above 1D maimer, because 2 s are necessary for tire transmission of the NOE. The transmission process becomes more efficient with large molecules and is almost optimal for proteins. However, problems can occur with molecules of intemiediate size [3f]. A 2D version of the NOE-difference experiment exists, called NOESY. 

It is important that constant temperature be maintained throughout the nOe difference experiment. If the instrument is fitted with a constant-temperature device, then it is advisable to adjust it to a few degrees above room temperature so that it maintains a constant temperature accurately. 

The through-space distances involved in H H proximity determinations are quite small, and the effect decreases as the inverse of the sixth power of the distance, through space, between the protons. The usual observable enhancement is less than 20%. To increase the sensitivity, we use the NOE difference experiment, in which a conventional H spectrum is computer sub-... 

The experiment described above is termed selective population transfer (SPT), or more precisely in this case with proton spin inversion, selective population inversion, (SPI). It is important to note, however, that the complete inversion of spin populations is not a requirement for the SPT effect to manifest itself. Any unequal perturbation of the lines within a multiplet will suffice, so, for example, saturation of one proton line would also have altered the intensities of the carbon resonance. In heteronuclear polarisation (population) transfer experiments, it is the heterospin-coupled satellites of the parent proton resonance that must be subject to the perturbation to induce SPT. The effect is not restricted to heteronuclear systems and can appear in proton spectra when homonuclear-coupled multiplets are subject to unsymmetrical saturation. Fig. 4.20 illustrates the effect of selectively but unevenly saturating a double doublet and shows the resulting intensity distortions in the multiplet structure of its coupled partner, which are most apparent in a difference spectrum. Despite these distortions, the integrated intensity of the proton multiplet is unaffected by the presence of the SPT because of the equal positive and negative contributions (see Fig. 4.19d). Distortions of this sort have particular relevance to the NOE difference experiment described in Chapter 8. 

NOE experiments typically require significant spectrometer time because the enhancements being sought are rather small. The NOE difference experiment represents a stringent test of both short- and long-term spectrometer stability, since changes in rf phase or frequency or in magnetic field contribute to... 

In the first example, 2D NOESY spectra were used to define the stereochemistry in the synthetic cycloadduct 8.14 [7], a potential biomimetic precursor to the naturally occurring marine-sponge alkaloid Keramaphidine B, 8.15. This problem is essentially the same as that addressed for 8.7 above using the NOE difference experiment, but in this case the additional unsaturated sidechains caused extensive overlap in the proton spectrum and precluded the use of selective presaturation. Sufficient characteristic NOEs present in a 600 ms NOESY spectrum gave conclusive proof of the endo stereochemistry, as shown. Only positive NOEs were observed, consistent with a molecule of mass 436 daltons in chloroform. NOESY spectra have also been successfully applied to the structure elucidation of molecules for considerably greater mass and complexity, as illustrated by the cytotoxic macrolide cinachyrolide A, 8.16 [35], also from a marine sponge. The structure of the molecule was determined through extensive 600 MHz 2D NMR experiments, of which NOESY played... 

The through-space distances involved in H H proximity determinations are quite small, and the effect decreases as the inverse of the sixth power of the distance, through space, between the protons. The usual observable enhancement is less than 20%. To increase the sensitivity, we use the NOE difference experiment, in which a conventional H spectrum is subtracted from a specific proton-irradiated spectrum this subtraction leaves only the enhanced absorptions. Under these conditions, a measurable effect can be expected between H nuclei over a distance of up to about 4 A (0.4 nm)t for example, the distance between 1,3-diaxial protons in the cyclohexane chair form is — 2.6 A. This procedure is a powerful tool in distinguishing among isomers, all of which should be available, if feasible, for NOE difference spectra (Fig. 4.54). 

The NOE difference experiment is carried out as follows. First, we collect a regular 1-D spectrum. Next, we record the transmitter offset required to make each resonance to be irradiated on resonance. Third, we find a suitable location in or near the spectral rvindow for dummy (or control) irradiation (perhaps —5 ppm or +15 ppm) where no resonances are observed. Fourth, we prepare both FlDs using a long period of single-frequency low power RF irradiation followed by a hard 90° RF pulse, except that in the preparation of the... 

A second important consideration to bear in mind when we use the NOE difference experiment concerns competing relaxation mechanisms. If the dipolar relaxation mechanism contributes negligibly to enhancing the overall rate of relaxation for a particular spin because of other efficient and available relaxation mechanisms, then we will... 

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