NOESY spectra

Figure Bl.13.6. The basic elements of a NOESY spectrum. (Reproduced by penuission of Wiley from Williamson M P 1996 Encyclopedia of Nuclear Magnetic Resonance ed D M Grant and R K Harris (Chichester Wiley) pp 3262-71).

A related experiment TOCSY (Total Correlation Spectroscopy) gives similar information and is relatively more sensitive than the REIAY. On the other hand, intensity of cross peak in a NOESY spectrum with a short mixing time is a measure of internuclear distance (less than 4A). It depends on the correlation time and varies as . It is positive for small molecules with short correlation time (o r <<1) and is negative for macromolecules with long correlation time (wr >>l) and goes through zero for molecules with 1 Relaxation effects should be taken into consideration for quantitative interpretation of NOE intensities, however. 

Figure 5.43 A typical NOESY spectrum the off-diagonal cross-peaks represent the nOe interactions between various nuclei.

Figure 5.44 (a) Phase-sensitive absorption-mode NOESY spectrum of bovine phos-... 

The NOESY spectrum and H-NMR chemical shift assignments of 7-hydroxyfrullanolide are shown. Interpret the NOESY spectrum. What conclusions can you draw about the stereochemistry at C-6 and C-10 ... 

The NOESY spectrum of buxatenone shows four cross-peaks, A-D. Cross-peak B represents the dipolar coupling between the most upfield C-19 cyclopropyl proton (8 0.68) with the most downfield olefinic proton (8 6.72). This could be possible only when the double bond is located either between C-1 and C-2 or between C-11 and C-12. The possibility of placing a double bond between C-11 and C-12 can be excluded on the basis of chemical shift considerations, since conjuga-... 

The NOESY spectrum of 7-hydroxyfrullanolide reveals the spatial proximities between the various protons. Cross-peak D arises from the gemi-nal coupling between the exomethylenic geminal protons (8 5.71 and 6.06). Dipolar interaction between the 06 proton (8 4.97) and the allylic methyl protons (8 1.64) is inferred from cross-peak C. This interaction is possible only when the C-6 proton is a-oriented. The C-1/3 and 02/8 protons (8 1.31 and 1.45, respectively) exhibit cross-peaks... 

Figure 6.3 Schematic representation of the resolution advantages of 3D NMR spectroscopy, (a) Both pairs of protons have the same resonance frequency, (b) Due to the same resonance frequency, both pairs exhibit overlapping crosspeaks in the 2D NOESY spectrum, (c) When the frequency of the carbon atoms is plotted as the third dimension, the problem of overlapping is solved, since their resonance frequencies are different. The NOESY cross-peaks are thus distributed in different planes.

In the case of a COSY-NOESY spectrum having an unequal mixing, let us consider two nuclei (say, A and B) that are spatially close but belong to different coupling networks, and nuclei B and C, which have scalar coupling with each other but are spatially distant. The only transfers allowed in this situation are ... 

Figure 6.12 A 3D heteronuclear HMQC-NOESY spectrum of a tripeptide. The (o,-axis represents N chemical shifts, whereas <1)2- and (Uj-axes exhibit proton chemical shifts. The assignment pathways are indicated in the top spectrum for reference purposes, not as part of the 3D experiment. (Reprinted from J. Mag. Reson. 78, S. W. Fesik and E. R. P. Zuiderweg,. 588, copyright (1988), with permission from Academic Press, Inc.)...

The methyl protons correlate with C5 and with the carbon C4 consistent with the neighboring C4-C5 connection. The NOESY spectrum shows a nOe correlation between the methoxylic protons and the methyl protons. 

The structure of this compoimd 63a was determined by H and C NMR spectroscopy. The H NMR spectrum shows singlets at 1.45 ppm for the methyl protons, and at 4.17 and 6.30 ppm for the hydroxylic and ethylenic protons. The addition regioselectivity in the formation of 63a was established by H- C HMBC 2D-NMR, which shows the C5-C4-C3-Me linkages. The ethylenic proton correlates only with three carbon atoms Ce, C5, and C3. The methyl protons correlate with C3 and with the ethylenic carbon C4, consistent with the neighboring C3-C4 connection. The NOESY spectrum shows... 

Now let us look at the NOESY spectrum (b) just as in COSY, we can identify a diagonal and a series of associated off-diagonal cross peaks. Thus the interpretation of the results is analogous to the method we have already learned for COSY. However, the cross peaks are not due to spin-spin coupling but to NOE effects between the protons concerned. However, if we look more closely we can see one big difference between the diagonal peaks, which look like irregular circles, and the cross peaks, which look just like all the peaks in the COSY spectrum. 

Fig. 25 2D spectra of compound 3. Top COSY (200 MHz, CDC13, measurement time 15 min) below NOESY spectrum (200 MHz, CDC13, measurement time 40 min)... 

The 2D NOESY spectrum of the Schiff base being a derivative of histidine ethyl ester and 3-hydroxypyridin-4-carboxaldehyde has shown that in THF-d8 solution the E conformation of the molecule is preferred.22... 

Figure 3.25 The NOESY spectrum of CuNiSOD. (Reprinted with permission from Figure 45 of Ming, L.-J., in Que, L., ed. Physical Methods in Bioinorganic Chemistry Spectroscopy and Magnetism, University Science Books, Sausalito, CA, 2000, pp. 375 -64. Copyright 2000, University Science Books.)...

The intensity of an NOE, given by the volume V of the corresponding cross peak in a NOESY spectrum [11, 13, 14] is related to the distance r between the two interacting spins by... 

The quantification of an NOE amounts to determining the volume of the corresponding cross peak in the NOESY spectrum. Since the linewidths can vary appreciably for different resonances, cross-peak volumes should in principle be determined by integration over the peak area rather than by measuring peak heights. However, one should also keep in mind that, according to Eq. (1), the relative error of the distance estimate is only one sixth of the relative error of the volume determination. Furthermore, Eq. (1) involves factors that have their origin in the complex internal dynamics of the macromolecule and are beyond practical reach such that even a very accurate measurement of peak volumes will not yield equally accurate conformational constraints. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

Fig. 3.3 A contour plot of a 750 MHz two-dimensional NOESY spectrum of the mating pheromone Er-22 adapted from Ref. [6]. B Cross section along to, displaying strong artifacts at multiples of A=340 Hz corresponding to a time variation of...

As expected, the performance of this filter over the whole 13C shift range surpasses that of a conventional single or even double filter. The authors have successfully applied this technique to a 2D NOESY spectrum with 13C-filtering in both dimensions, acquired on the 26 kDA complex between a [U-13C,15N]-labeled DNA-binding protein domain and a 15-mer DNA ligand (unlabeled) [24]. 

More recent applications comprise, for example, the identification of the binding site of 18 kDa human cardiac troponin C for the drug bepridil [36]. For this study, the unlabeled ligands were bound to selectively [13CH3-Met, Phe-d8]-labeled protein (Fig. 17.8). First, the 13CH3-Met signals of troponin C were easily identified from 13C-HSQC spectra. In a 2D NOESY spectrum with 13C-editing in one dimension, intermolecular NOEs could... 

Fig. 17.8 Intermolecular NOEs between [13CH3-Met, Phe-ds]-labeled cardiac troponin C and the drug bepridil (left panels, drug protein 1.5 1 right panels, 3.5 1). A, D methyl region of the H spectrum B, E HSQC spectra showing the 13CH3-Met signals of the protein C, F section from the NOESY spectrum with 13C-editing in one dimen-...

Fig. 17.10 F1.F2 13C,, 5N-filtered NOESY spectrum of a complex of an unlabeled HIV peptide with an [U-13C, 15N]-labeled antibody fragment (left). The chemical shifts of the peptide amide...

Fig. 17.11 Region from a 13C-edited NOESY spectrum of an HIV peptide 13C,15N- labeled at positions Glyl2, Prol3 and Gly 14. The characteristic NOEs for a cis configuration of the Gly-Pro peptide bond are indicated (left). Similarly, the ex-...

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