NOESY 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.

Nuclear Overhauser enhancement spectroscopy (NOESY) experiments play a very important role in structural studies in quinolizidine derivatives. For instance, the endo-type structure of compound 28 was proven by the steric proximity of the H-3a and H-12a protons according to the NOESY cross peak, while the spatial proximity of the H-6f3 and H-8/3 protons reveals that tha A/B ring junction has a /ra t-stereochemistry. Similarly, compound 28 could be distinguished from its regioisomer 29 on the basis of the NOESY behavior of its H-13 atom <1999JST153>. 

Upper bounds b on the distance between two hydrogen atoms are derived from the corresponding NOESY cross peak volumes V according to calibration curves , V=f(b). Assuming a rigid molecule, the calibration curve is... 

Semi-automated approaches to NOESY assignment [85-87] use the chemical shifts and a model or preliminary structure to provide the user with the list of possible assignments for each cross peak. The user decides interactively about the assignment and/or temporary removal of individual NOESY cross peaks, possibly taking into account supplementary information such as line shapes or secondary structure data, and performs a structure calculation with the resulting (usually incomplete) input. In practice, several cycles of NOESY assignment and structure calculation are required to obtain a high-quality structure. 

Requirement 2. Self-consistency The peak lists must be faithful representations of the NOESY spectra, and the chemical shift positions of the NOESY cross peaks must be correctly calibrated to fit the chemical shift lists within the chemical shift tolerances. The range of allowed chemical shift variations ( tolerances ) for 1H should not exceed 0.02 ppm when working with homonuclear [1H,1H]-NOESY spectra, or 0.03 ppm when work-... 

For each NOESY cross peak, one or several initial assignments are determined based on chemical shift fitting within a user-defined tolerance range. 

Rank the initial assignments of each individual NOESY cross peak by the generalized contribution to the total peak volume and eliminate low-ranking initial assignments... 

Fig. 2.4 Three conditions that must be fulfilled by valid NOESY cross peak assignments in Candid a Agreement between chemical shifts and the...

Criterion 4. Eliminated peaks More than 80% of the NOESY peaks should have been assigned by Candid, and less than 20% of the NOESY cross peaks with exclusively long-range assignments (spanning 5 or more residues) should have been eliminated by the peak filters of Candid. 

In principle, a de novo protein structure determination requires one round of 7 Candid cycles. This is realistic for projects where an essentially complete chemical shift list is available and much effort was made to prepare a complete high-quality input of NOESY peak lists. In practice, it turned out to be more efficient to start a first round of Candid analysis without excessive work for the preparation of the input peak list, using an slightly incomplete list of safely identifiable NOESY cross peaks, and then to use the result of the first round of Candid assignment and structure determination as additional information from which to prepare an improved, more complete NOESY peak list as input for a second round of 7 Candid cycles. 

Fig. 7. The intensity of different NOESY cross-peaks in sonicated DMPC...

H/ H-ROESY (-NOESY) Cross peak.s and diagonal peaks appear in absorption. The diagonal peaks are the most intense and are best suited for phase adjustments. They should be phased for negative absorption in ROESY spectra and in NOESY spectra measured for small molecules, giving cross peaks in positive absorption in both cases. For NOESY spectra of large molecules (e.g. biomolecules), both diagonal and cross peaks should be phased to positive absorption. 

Homonuclear correlation spectroscopy (COSY) experiments (see Chapter 9) substantiate the theoretical predictions, based on molecular orbital calculation, of the pattern of spin delocalization in the 3e orbitals of low-spin Fe(III) complexes of unsymmetrically substituted tetraphenylporphyrins [46]. Furthermore, the correlations observed show that this n electron spin density distribution is differently modified by the electronic properties of a mono-orf/io-substituted derivative, depending on the distribution of the electronic effect over both sets of pyrrole rings or only over the immediately adjacent pyrrole rings [46]. No NOESY cross peaks are detectable, consistently with expectations of small NOEs for relatively small molecules and effective paramagnetic relaxation [47]. 

Fig. 8.10. Computer-simulated NOESY cross peaks between signals with different T2 values. Conditions p[ — p =40 s 1 p[ - 50 s-1 p( = 200 s I o/j = 5 s l. (A) f[" = t2m = 0.01 s (B) t["ax = T , r2max = T2J (C) t["ax = T, r2max = T. Note that the upper left cross peak has maximal intensity in case (B), the lower right cross peak has maximal intensity in case (C), whereas in case A the two intensities are equal and intermediate between the larger and the smaller of cases (B) and (C). For simplicity, fra = T[ = T( has been taken. A cos2 weighting function has been applied.

NOEs or NOESY cross peaks can be used to define distances between protons. It is well known that the three-bond scalar coupling constants 3Jjj between nuclei / and J obey a Karplus relationship of the type [39,40]... 

Fig. 2. Schematic representation of NOESY cross peaks used in peptide assignments.

This conformation would place the chromophore residue into a stacked situation within the adjacent DNA bases while replacing the counterbase of the complementary strand. To obtain more structural information, NOESY experiments were performed on the pyrene-modified nucleosides. The spectra of the modified pyrimidines Py-dU and Py-C clearly showed a significant NOESY cross peak between H-6 of the dU part or the C part, respectively and H-2 of the corresponding 2 -deoxyribose moiety. The NOESY cross peak was comparably as strong as the cross peaks between H-2 and H-l or H-3, respectively. These NMR results can only be explained with the preferred anti conformation of these nucleosides. 

During the two decades of intense interest in the 1,2-intrastrand d(G pG ) crosslink, the only anti,anti conformation proposed was HH, and only this conformation was expected, before we studied (R,S,S,R)-BipPt(d(GpG)). Remarkably, we found two N(7)-Pt-N(7) crosslink products one was an HH1 conformer, but the other was a new HH conformer (HH2) (Fig. 7). Each product had a pair of H(8) signals with a dispersion (Fig. 8) and a medium H(8)-H(8) NOESY cross-peak, features consistent with HH conformers [38] [63] [67] [68]. The very similar H(8)-H(8) distance, estimated for both in the medium-distance (2.5-3.5 A) range, is consistent with HH bases. For comparison, H(8)-H(8) distances in HT models are 4.5-5.5 A. No H(8)-H(l ) cross-peaks were observed in the 300 ms mixingtime NOESY spectrum, indicating that all of the G s are anti since an intense H(8)-H(l ) cross-peak would be observed for a syn-G. ... 

In practice, these methods offer more than just reliable interpretation of distances. Because the procedure can be used to model NOESY cross-peaks with contributions from spin diffusion, they suggest that cross-peaks from spectra with long mixing times, and thus better signal-to-noise, can be reliably used. As Nilges et al.67 point out, a cross-peak may arise entirely via indirect magnetization transfer, but may contain useful structural information. 

The coherence pathway leading to NOESY cross peaks was illustrated in Eq. 11.88, where we showed that with appropriate phase cycling we had only a term ASy (with A measuring the extent of NOE polarization transfer). Double quantum and zero quantum coherences are then generated by the pathway... 

Different methods for the study of selective solvation have been developed [118, 120] conductance and Hittorf transference measurements [119], NMR measurements (especially the effect of solvent composition on the chemical shift of a nucleus in the solute) [106-109], and optical spectra measurements like IR absorption shifts [111] or UV/Vis absorption shifts of solvatochromic dyes in binary solvent mixtures [124, 249, 371]. Recently, the preferential solvation of ionic (tetralkylammonium salts) and neutral solutes (phenol, nitroanilines) has been studied particularly successfully by H NMR spectroscopy through the analysis of the relative intensities of intermolecular H NOESY cross-peaks [372]. 

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