NOESY and ROESY

We described the basic aspects of NOESY in Section 10.1 as an introductory example of a 2D experiment. NOESY is very widely used in measuring macro-molecular conformation, as we see in Chapter 13. However, as shown in Fig. 8.4, the H— H nuclear Overhauser enhancement 17 varies from its value of +0.5 in small molecules to a limiting value of — 1 in large polymers with very long Tc, and at intermediate values of rc the NOE may vanish. An alternative is to use the NOE measured in the rotating frame, as this quantity is always positive. By analogy to NOESY, this technique has the acronym ROESY (rotating frame Overhauser enhancement spectroscopy), 

The experimental method for obtaining ROESY is essentially the same as that for HOHAHA, application of a spin-lock pulse sequence for mixing at the end of the evolution period. HOHAHA effects can interfere with ROESY measurements but are minimized by using lower rf power and offsetting the pulse frequency to interfere with the Hartmann-Hahn condition. 

Combination of HETCOR with COSY and TOCSY provides very useful but indirect information on the proximity of particular carbon atoms. For structure elucidation, a direct method based on Jcc is desirable, but with a natural abundance of only 1.1%, the probability of having two 13C nuclei directly bonded to each other is only about 1 in 10,000. Nevertheless, with sufficient sample, it is possible to obtain the information via INADEQUATE (incredible natural 

FIGURE 10.10 (a) Pulse sequence for HETCOR, as described in text. (f ) H-13C HETCOR spectrum (300 MHz) of allylbutyl ether in CDC13. The one-dimensional H spectrum is shown at the top and the proton-decoupled 13C spectrum at the left.The 2D plot clearly indicates the H-C correlations. Spectrum courtesy of Herman J. C.Yeh (National Institutes of Health). 

The ability to manipulate spins in two-dimensional experiments and to transfer magnetization between spins has made it possible to use a sensitive nucleus (primarily H) to measure the spectral features of less sensitive nuclei, such as 13C and 15N. Several methods are commonly used, but each begins with a H pulse sequence, often resembling the one in INEPT (Section 9.7). As in INEPT, a combination of H and X pulses transfers polarization to the X spin system. In some instances further transfers are made to another spin system (Y), then back through X to H, where the signal is detected. Thus, the large polarization of the proton is used as the basis for the experiment, and the high sensitivity of H NMR is exploited for detection. Such indirect detection methods use two-, three-, and sometimes four-dimensional NMR. 

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A comparison of the methods of proton-proton NOE detection has shown that two-dimensional NOE detection such as NOESY and ROESY are better suited to the investigation of the stereochemistry of biopolymers whereas for small- to medium-sized molecules (up to 30 C atoms) NOE difference spectroscopy is less time consuming, more selective and thus more conclusive. 

The oldest and most widely used structural restraints in NMR spectroscopy are distance restraints derived from NOE experiments [1]. Transient NOE, 2D NOESY and ROESY spectra provide valuable information for interatomic distances up to 5 A that will be discussed in the following. 

NOE effects can naturally also be investigated by 2D experiments these are known as NOESY and ROESY. 

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

The use of spin-lock pulses for water suppression is illustrated with the NOESY and ROESY pulse sequences (fig. 5). Using the Cartesian product operator description [9], the effect of the NOESY pulse sequence of fig. 5(A) is readily illustrated ... 

Fig. 5. Pulse sequences of NOESY and ROESY with spin-lock purge pulses for water suppression. (A) NOESY pulse sequence. The spin-lock pulses are typically of length 0.5 ms and 2 ms, and r = 1/SW, where SW is the spectral width in the acquisition dimension. Phase cycle (pi = x,—x) 4>2 = 4 x,x,—x,—x) ...

In the spin diffusion motional regime (small molecules at low temperatures or macromolecules at all temperatures) the cross-relaxation is so efficient that it can hardly be limited to a single-step magnetization transfer. The multistep magnetization transfer is known as spin diffusion. It manifests differently in NOESY and ROESY spectra, as can be illustrated by writing eq. (29b) explicitly for the process of cross relaxation ... 

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. 

Occasionally, COSY-type artifacts appear in NOESY and ROESY spectra but these are easy to identify by their anti-phase multiplet structure. 

Today, it is possible to make complete assignments of all proton and carbon atoms in the NMR spectra of most isolated anthocyanins. These assignments are normally based on chemical shifts (8) and coupling constants (J) observed in 1-D H and l3C NMR spectra (Fig. FI.4.2), combined with correlations observed as cross-peaks in various homo- and heteronu-clear 2-D NMR experiments (see below for details on COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY). 

It should be noted that NOESY and ROESY pulse sequences also provide EXSY spectra, and therefore EXSY cross peaks may appear simultaneously in the 2D NOESY and ROESY spectra. EXSY cross peaks are always positive in both types of experiment, whereas dipolar cross peaks are negative in EXSY spectra independently of molecular weight and in NOESY spectra of small molecules. Therefore, in macromolecules the sign for NOESY and EXSY cross peaks is the same, and the two phenomena cannot be distinguished in NOESY experiments. In contrast, ROESY cross peaks have different sign from EXSY cross peaks and can be distinguished and even plotted selectively in ROESY experiments. These considerations are summarized in Table 8.3 for the reader s convenience. 

Feenstra KA, Peter C, Scheek RM, et al. A comparison of methods for calculating NMR cross-relaxation rates (NOESY and ROESY intensities) in small peptides. J Biomol NMR 2002 23 181-194. 

The structure of I19L was determined in aqueous solution at pH 6.8 by NMR [142]. Chemical shift values and the scarcity of cross-peaks in NOESY and ROESY spectra indicated that the peptide is mostly disordered in solution, although it has a helical propensity at residues 10-14 (Fig. 36). The bound conformation was investigated by means of tr-NOESY and STD experiments using a 30 1 excess of peptide relative to tubulin. The presence of intense, negative crosspeaks in the tr-NOESY spectrum proved that there is a fast binding equilibrium (Fig. 36). Unspecific binding was ruled out based on the absence of tr-NOE peaks in a control experiment where the protein was BSA instead of tubulin. The existence... 

In Chapter 3 (Section 3.16), there is a description of the nuclear Overhauser effect difference experiment, an experiment that provides information about H— H through-space proximity. Review of this section is helpful before proceeding here. The ROESY experiment, rotating-frame Overhauser effect spectroscopy, is a useful 2-D analogue of the nuclear Overhauser effect difference experiment. This experiment is useful for molecules of all sizes whereas the related experiment, NOESY (nuclear Overhauser effect spectroscopy), is not very useful with small molecules. NOESY is used primarily with biological macromolecules. Both NOESY and ROESY experiments correlate protons that are close to each other in space, typically 4.5 A or less. 

The program fits the intensity at these point values to a polynomial (up to 5th order) function and then subtracts the polynomial function from the whole dataset. This is repeated for each ID slice (row or column) of the 2D data matrix. More sophisticated methods calculate the baseline points automatically and use functions other than polynomials. For example, a program called FLATT (by Kurt Wiithrich) is very effective at removing horizontal or vertical streaks resulting from baseline curvature in rows or columns of the data matrix. Especially with NOESY and ROESY databaseline correction is essential to getting clean 2D displays and plots. 

The structures of compounds (43a,b) were established by a combination of NOE experiments and comparison with the spectra of other examples of this type of ring system <90H(3i)2l25>. A conformational study of compounds (19), (20), and (44) was carried out using NOESY and ROESY... 

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