The NOESY experiment

The likelihood of signal cancelation due to EXSY artifacts is rather remote. If such nulling is believed to be occurring, the same remedies as those given for COSY artifacts are applicable. In addition to their aforementioned effects on NOE s, decreases in temperature reduce the EXSY cross peaks of small molecules by decreasing exchange rates. 

The successful observation of NOESY correlations can depend critically on the choice of two parameters the mixing (t ) and repetition times, both of which depend on the spread of H in a molecule. Recommended RT s are about 2T for small to intermediate-sized molecules. They should be set to 1.2-1.8 s for small molecules (MW 400-450), 0.9-1.2 s for intermediate-sized molecules (MW 500-750), and, more conservatively, to 2-3 s when the T s are unknown. The choice of mixing times also is important, because if is too short, NOE enhancements do not have a chance to develop to detectable intensities, and NOESY cross peaks are not observed. On the other hand, if is too long, the NOE enhancements disappear because of relaxation, and, again, cross peaks are absent. Compromise mixing times should be about the average T value and can be set to 0.3-0.6 s for MW 400-750 and 1-2 s for very small molecules. 

Another reason for the careful selection of RT is that NOESY spectra are susceptible to artifacts from pulsing too rapidly. Problems also occur for molecules (MW 750-2,000) in the crossover region in which NOESY cross-peak intensities approach zero for even 

EXSY experiments are also sometimes performed with ROESY sequences (see Section 8.4). 

NOESY experiments deal with dipolar interactions between nuclei. Successful experiments are easily planned for nuclei with large magnetic moments like protons, or for heteronuclei when the dipole-dipole interaction is very strong. 

The basic pulse sequence for the NOESY experiment is just that illustrated 

Optimal homonuclear NOESY mixing times f pl for various T (p l) values (ms) of the two spins I and J, calculated using Eq. (8.2). Cross peaks intensities (%) for a o7a value of — 1 s 1 are shown in parentheses [9] 

Calculated maximal intensities of NOESY cross peaks (%)a for various values of rr and R[m (= R m). For each tr and R[u value two NOE s are calculated for interproton distances of 1.8 and 3.0 A and for nuclear Larmor frequencies vp of 200 and 600 MHz 

A great many advanced techniques can be applied to complex molecules. We have introduced only a few of the most important ones here. As computers become faster and more powerful, as chemists evolve their understanding of what different pulse sequences can achieve, and as scientists write more sophisticated computer programs to control those pulse sequences and treat data, it will become possible to apply NMR spectroscopy to increasingly complex systems. 

The NMR detection probe that is used for most heteronuclear experiments (such as the HETCOR experiment) is designed so that the receiver coils for the less-sensitive nucleus (the insensitive nucleus) are located closer to the sample than the receiver coils for the more sensitive (generally the H) nucleus. This design is aimed at maximizing the signal that is detected from the insensitive nucleus. As was described in Chapter 6, owing to a combination of low natural abundance and a low magnetogyric ratio, a C nucleus is about 6000 times more difficult to detect than a H nucleus. A nucleus is also similarly more difficult to detect than a H nucleus. 

The difficulty with this probe design is that the initial pulse and the detection occur in the insensitive channel, while the evolution period is detected in the channel. The resolution that is possible, however, is much lower in the channel where the evolution of spins is detected. In the case of a carbon-hydrogen correlation (a HETCOR), this means that the greatest resolution will be seen in the spectrum (in which every peak is a singlet), and the lowest resolution will be observed in the 

The nuclear Overhauser effect was described in Chapter 6, Sections 6.5 and 6.6. A two-dimensional NMR experiment that takes advantage of the nuclear Overhauser effect is nuclear Overhauser effect spectroscopy, or NOESY. Any H nuclei that may interact with one another through a dipolar relaxation process will appear as cross peaks in a NOESY spectrum. This type of interaction 

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The nuclear Qverhauser effect was described in Chapter 4, Sections 4.5 and 4.6 (pp. 184-189). A two-dimensional NMR experiment that takes advantage of the nuclear Overhauser effect is nuclear Qverhauser effect spectroscopy, or NOESY. Any nuclei that may interact with one another through a dipolar relaxation process will appear as cross peaks in a NOESY spectrum. This type of interaction includes nuclei that are directly coupled to one another, but it also includes nuclei that are not directly coupled but are located near to one another through space. The result is a two-dimensional spectrum that looks very much like a COSY spectrum but includes, besides many of the expected COSY cross peaks, additional cross peaks that arise from interactions of nuclei that interact through space. In practice, the nuclei must be within 5 A of each other for this spatial interaction to be observed. 

NOESY spectroscopy has become especially useful in the study of large molecules, such as proteins and polynucleotides. Very large molecules tend to tumble more slowly in solution, which means that nuclear Overhauser effect interactions have more time to develop. Small molecules tumble more quickly in solution the nuclei move past one another too quickly to allow a significant development of dipolar interactions. The result is that NOESY cross peaks may be too weak to be observed. 

Because the cross peaks in NOESY spectra arise from spatial interactions, this type of spectroscopy is particularly well suited to the study of configurations and conformations of molecules. The example of acetanilide demonstrates the capabilities of the NOESY experiment. The structural formula is shown, with the proton NMR chemical shifts of the relevant protons indicated. 

614 Nuclear Magnetic Resonance Spectroscopy Part Five Advanced NMR Techniques 

The problem to be solved is to decide which of two possible conformations is the more important for this molecule. The two conformations are shown, with circles drawn around the protons that are close to each other spatially and would be expected to show nuclear Overhauser interactions. 

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Chemical shift anisotropy evidenced the helical structure of the complexes. NOESY experiment also suggested the presence of a stable secondary structure. On the basis of the NOESY experiments, the... 

Several basic 2D schemes have been applied in these experiments, based on (1) the use of the SQ evolutions during t and f2, in analogy to the NOESY experiment, (2) the MQ-SQ experiment, in which the evolutions of MQ coherences during t are correlated with the CTs of recoupled spins to enhance the resolution in analogy to MQMAS, and (3) the DQCT-SQ protocol similar to that used in DQMAS NMR of spin-1/2 nuclei. 

NOESY) experiment must be used. See Section 3.4.10. The NOESY experiment transfers magnetization through space, and, therefore, it will show cross peaks to all protons that are close in space (within 4-5 A of each other) regardless of whether they are in the same spin system or not. In a folded protein, aa residues are inherently close in space, so assignments can be made by comparing the peaks in the NOESY experiment with other spin systems determined by COSY and TOCSY. 

The NOESY experiment has also been very useful for revealing the presence of rotational conformers of dimeric flavonoids and flavone C-glycosides (Figure 2.3). Strong exchange crosspeaks between equivalent protons of each conformer revealed the rotational equilibriums. This NOE phenomenon was first noted by Hatano et al. in two conformers of procyanidin dimers. The volume of the NOESY crosspeaks is related to the distance... 

In contrast to the 1D experiment, where steady-state NOEs may be obtained, only the less intense transient NOE.s are measured in the NOESY experiment. ROEs can only be obtained as transient effects in both the ID and the 2D experiment. Furthermore the intensities of the NOESY and ROESY cross peaks depend upon the molecular size as well as the length of the mixing period. In the case of large molecules, e.g. polypeptides, rather short mixing times are usually chosen to avoid spin diffusion. 

The ROESY used to be a bit difficult to set up because the low power spin-lock RF had to come from a different source than the hard pulse RF. Now rapid solid-state power switching is so routine that all H RF comes from the same source, with no variation of phase or frequency. ROESY tends to replace the NOESY experiment for NOE measurements, especially for small molecules where T2 is relatively long. Because the NOE builds up about twice as fast in the x -y plane as it does on the z axis, ROESY mixing times are set to about half of what would be the NOESY mixing time. There is one additional parameter to set up the power level ( Z i field strength ) of the spin lock pulse. This is typically... 

Because of all of the above pitfalls, NOE is probably the most misinterpreted experiment in organic chemistry. In my experience, /-coupling measurements, both homonuclear and heteronuclear, give far more reliable information than NOE measurements in the determination of small-molecule stereochemistry. To use NOE measurements for stereochemical determinations, it is always best to do the NOESY experiment on both isomers and compare the crosspeak intensities (relative to the diagonal peak intensities) and measure distances on both isomers using an energy-minimized computer model of the structures. If the differences in distance and NOE intensity are small between the two isomers, the experiment cannot be conclusive. 

The spectra were acquired with 2048 t2 complex data points and 256 ri increments in the phase sensitive mode with quadrature detection using the method described by States et al. (25). Water resonance was supressed during the 1.5s relaxation period used in the NOESY, DQF-COSY and TOCSY experiments and the mixing period of the NOESY experiments by irradiating continuously at its resonance frequency. The amide exchange experiments were carried out by... 

While nOe experiments can be carried out using selective excitation of individual signals, one at a time, to determine the identity of protons proximal to the selected peak, the NOESY experiment enables the determination of nOe information between all spins in one experiment. Cross peaks in a NOESY experiment indicate which protons are close to one another. Typically nOe crosspeaks can be observed for protons less than 5 A apart in the molecule. Fig. 7 shows the NOESY spectrum of dutasteride with several key correlations highlighted. The results of this experiment enable complete stereochemical assignment of all the protons in the molecule. 

The duration of the fixed time depends on the relaxation time T, the rate of chemical exchange, and the rate of NOE buildup. In the case of the NOESY experiment, valuable information can be ascertained about the distance between various protons within a molecule. Figure 6-29 illustrates the NOESY spectrum for a complex heterocycle. As with COSY, the ID spectrum is found along the diagonal, and off-diagonal or cross peaks occur when two protons are close to each other. Thus, methyl group I shows an expected cross peak with the adjacent alkenic proton a (upper left). Additional cross peaks of methyl I indicate its closeness to the methinyl proton f and the acetal methyl n. The ester methyl e is close to the other acetal methyl m. The NOESY experiment can provide both structural and conformational information. In practice, cross peaks become unobservable when the proton-proton distance exceeds about 5 A. 

Second, in small molecules, the NOE builds up slowly and attains a theoretical maximum of only 50%, as noted earlier in the ID context. (See Section 5-4 and Appendix 5.) Because a single proton may be relaxed by several neighboring protons, the actual maximum normally is much less than 50%. (Of course, the same problem exists in the ID NOE experiment.) Moreover, as the molecular size increases and behavior departs from the extreme narrowing limit, the maximum NOE decreases to zero and becomes negative. Thus, particularly for medium-sized molecules, the NOESY experiment may fail. For larger molecules, whose relaxation is dominated by the Wq term, not only is the maximum NOE —100% rather than +50%, but also the NOE buildup occurs more rapidly. The NOESY experiment thus has been of particular utility in the analysis of the structure and conformation of large molecules such as proteins and polynucleotides. 

For the NOESY experiment, the following pulse sequence may be used Gl-90°-fi-90 -Tm, G2-90°-f2(acquire). The first PFG (GI) destroys residual transverse... 

The NOESY experiment provides information about the proximity of protons and hence is used primarily for distinguishing structures that have clear stereochemical differences. For larger molecules, the ROESY experiment may offer some advantages because of its lower tendency to exhibit spin diffusion. The related EXSY experiment is used only when chemical exchange is being investigated. 

With the completion of the two-dimensional structural elucidation of the unknown compound, questions arise concerning its three-dimensional shape [i.e., the relative orientations of substituents (e.g., Hm or methyl 24) at various carbons). For a molecule with MW = 466, the NOESY experiment can provide a wealth of such stereochemical information. The NOESY spectrum of the 15-mg sample of the compound is shown in Figure 7-22. The data from these contour plots are summarized in Table 8-5 and illustrated, in part, in structure 8-6. 

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