Crosspeaks

Figure 5.27 Schematic diagram of a COSY spectrum illustrating how intrinsically different cross-peaks are from diagonal peaks. The circles represent the cross-peaks in an AB coupled system. The diagonal peaks have dispersion shape, and the crosspeaks have an absorption shape though they alternate in sense. The diagonal peaks are centered at d 8,i) and (8 , 8 ) the cross-peaks appear at (8., 8 ) and (8 , 8 ).

One problem associated with COSY spectra is the dispersive character of the diagonal peaks, which can obliterate the cross-peaks lying near the diagonal. Moreover, if the multiplets are resolved incompletely in the crosspeaks, then because of their alternating phases an overlap can weaken their intensity or even cause them to disappear. In double-quantum filtered COSY spectra, both the diagonal and the cross-peaks possess antiphase character, so they can be phased simultaneously to produce pure 2D absorption line... 

Figure 5.47 Two-dimensional exchange spectrum of N,Af-dimethylacetamide and its generation, (a) The first set of spectra results from the first series of Fourier transformations with respect to The modulation of signals as a function of t is observed, (b) The second set of spectra is obtained by the second series of Fourier transformations. The unmodulated signals appear on the diagonal at (v, v ), (vx, Vx), and (v, v ), whereas the modulations due to exchange show up as crosspeaks on either side of the diagonal at (vx, Vx) and (vx, Vx). (c) A contour plot representation of (b). (Reprinted from Science 232, A. Bax, et ai, 960, copyright (1986), with permission from Science-AAAS, c/o Direct Partners Int., P.O. Box 599, 1200 AN Hilversum, The Netherlands)...

The HOHAHA spectrum (100 ms) of podophyllotoxin is presented. The HOHAHA, or TOCSY (total correlation spectroscopy), spectrum (100 ms) shows coupling interactions of all protons within a spin network, irrespective of whether they are directly coupled to one another or not. As in COSY spectra, peaks on the diagonal are ignored as they arise due to magnetization that is not modulated by coupling interactions. Podophyllotoxin has only one large spin system, extending from the C-1 proton to the C4 and 015 protons. Identify all homonuclear correlations of protons within this spin system based on the crosspeaks in the spectrum. 

The HOHAHA spectrum (100 ms) of vasicinone is shown. Interpret the spectrum, and determine the different spin systems from the crosspeaks in the spectrum. 

The one-bond hetero-COSYspectrum of 7-hydroxyfrullanoIide exhibits interactions for all nine protonated carbons. The most downfield crosspeaks, K and L, represent one-bond heteronuclear correlations of the two vinylic exomethylenic protons resonating at 8 5.71 and 6.06 with the C-13 carbon (8 120.5). The C-6a proton, which resonates downtield at 8 4.97 due to the directly bonded oxygen atom, displays correlation with the carbon resonating at 8 80.9 (cross-peak D). Cross-peaks G and M represent h interactions of the C-1 methylene protons (8 1.33 and 1.31, respectively) with C-1 (8 38.1). Similarly, cross-peaks E and F display heteronuclear interactions of the C-8 methylenic protons (8 1.48 and 1.72) with C-8 (8 30.7), while cross-peak C couplings of C-3 methylene protons at 8 1.97 and 1.99 with C-3 (8 32.5). Couplings between the C-1 methylene protons and C-1 (8 38.1) can be inferred from cross-peak A, though in this case both the C-1 a and protons resonate very close to each other (i.e., 8 1.31 and 1.33). Cross-peak C is due to C-9 methylene, while cross-peak I represents the C-15 methyl. The heteronuclear interactions between the most upheld C-2 methy-... 

The most downfield cross-peaks, V-Y, are due to heteronuclear couplings of the aromadc or vinylic protons and carbons. For instance, cross-peak Y represents heteronuclear interaction between the C-1 vinylic proton (8 5.56) and a carbon resonating at 8 134.0 (C-1). The downfield cross-peaks, V and W, are due to the heteronuclear correlations of the ortho and meta protons (8 7.34 and 7.71) in the aromatic moiety with the carbons resonating at 8 128.3 and 126.9, respectively. The remaining cross-peak X is due to the one-bond correlation of the C-4 aromatic proton (8 7.42) with the C-4 carbon appearing at 8 131.4. The cross-peak U displays direct H/ C connectivity between the carbon at 8 77.9 (C-6) and C-6 methine proton (8 4.70). The crosspeak T is due to the one-bond heteronuclear correlation of carbon... 

The HMQC spectrum of podophyllotoxin shows heteronuclear crosspeaks for all 13 protonated carbons. Each cross-peak represents a one-bond correlation between the C nucleus and the attached proton. It also allows us to identify the pairs of geminally coupled protons, since both protons display cross-peaks with the same carbon. For instance, peaks A and B represent the one-bond correlations between protons at 8 4.10 and 4.50 with the carbon at 8 71.0 and thus represent a methylene group (C-15). Cross-peak D is due to the heteronuclear correlation between the C-4 proton at 8 4.70 and the carbon at 8 72.0, assignable to the oxygen-bearing benzylic C-4. Heteronuclear shift correlations between the aromatic protons and carbons are easily distinguishable as cross-peaks J-L, while I represents C/H interactions between the methylenedioxy protons (8 5.90) and the carbon at 8 101.5. The C-NMR and H-NMR chemical shift assignments based on the HMQC cross-peaks are summarized on the structure. 

Cross-peaks A and B represent interactions of the C-4 proton (H ) resonating at 8 2.20 with the C-3 and C-5 carbons (8 72.2 and 43.7, respectively). Cross-peak C corresponds to the coupling between the C-4 proton (8 2.70) (H ) with the C-2 iminic carbon (8 160.4). Crosspeaks D, E, and F represent long-range correlations between the C-5a... 

The ROESY spectrum of podophyllotoxin exhibits a number of crosspeaks (A-D) representing interactions between dipolarly coupled (space coupling) hydrogens, which can be helpful to determine the stereochemistry at different asymmetric centers. For example, based on the assumption that the C-1 proton (8 4.53) is /3-oriented, we can trace out the stereochemistry of other asymmetric centers. Cross-peak B represents dipolar coupling between the C-1 proton (8 4.53) and the C-2 proton (8 2.8), thereby confirming that the C-2 proton is also... 

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.

So a simplified 3D spectrum is obtained having eight peaks, i.e., one crosspeak, four cross-diagonal peaks (two at V] = V2 and two at V2 = vj), three diagonal peaks, and no back-transfer peaks (Fig. 6.5b). 

Considering all potential experimental and systematic errors of NOE/ROE crosspeak intensities, it is remarkable how robust the derived distance restraints still are. The reason Ues in the dependence of the cross-relaxation rate even if a cross-peak intensity is determined wrongly by a factor 2, the resulting distance restraint is only affected by the factor 1.12, which usually lies within the error range of distance restraints used in structure calculations. It should be further noted that the quaUty of a resulting structure is not so much determined by the... 

Our preferred experiment of this type is the so called DEPT-edited HSQC which is both relatively artefact-free and sensitive. It also has one other major advantage up its sleeve. This experiment is not an absolute value technique like most of the others, but it allows for discrimination between different types of carbons. Methyl and methine carbons give crosspeaks that are phased opposite to the methylene carbons and so the results are best plotted on a colour plotter which can portray this clearly by plotting positive and negative cross peaks in different colors. 

Fig. 9 TrNOESY and QUIET-trNOESY spectra of the peptide DRPVPY in the presence of the antibody SA-3. A Regions of a trNOESY spectrum (r = 200 ms) showing Val-4 HN-Pro-3,-5 H8 cross-peaks and Pro-3,-5 H5-H5 cross-peaks. B Corresponding regions of a QUIET-trNOESY spectrum (r = 200 ms) with inversion of 0.75 ppm wide bands centered at 4.15 ppm and 8.1 ppm within the intersection of the quiet bands (quiet window), the Val HN-Pro-5 U8 cross-peaks are still present, while the Val HN-Pro-3 B.82 crosspeak is absent, indicating cancelation due to spin diffusion. Reproduced with permission from [125]. 2002 American Chemical Society...

Kessler et al. developed the Difference and Sums of Traces within Cosy Spectra (DISCO) technique for the extraction of couplings from the crosspeaks of two-dimensional (COSY) spectra. This post-processing technique combined selected cross-sections of these peaks in order to extract the active coupling , i.e., /ab from the cross-peak of A and B. After such combination, the active coupling was the separation of the in-phase and anti-phase DISCO peaks. DISCO requires accurate scaling of the constituent spectra and resolved cross-peaks. An alternative and simpler method for the accurate measurement of vicinal couplings from COSY spectra has been presented by Kim and Prestergard. ... 

Fig. 4. Bottom the Double-Band-Filtered COSY spectrum obtained by selection through DANTE-Z of the H region (prior to the evolution interval) and by the selection through SPlN-PlNGING [11] of the amide region (before the acquisition interval) of toxin 7. Top the corresponding region of a standard COSY spectrum. Note, in the bottom diagram, the considerable increase in spectral resolution as well as the occurrence of additional crosspeaks (indicated with asterisks). Experiments were performed at 360 MHz (Bruker AMX360) in H2O at 318 K. The 50 W class C amplifier of the proton channel was used as transmitter.

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