ROESY TOCSY peaks

ROESY TOCSY peaks of opposite sign with respect to cross-relaxation ones. Very sensitive to offset. Try different carrier frequency. 

TOCSY peaks might appear in the GROESY spectrum as if truly due to cross-relaxation. This drawback can be corrected by means of a very careful calibration of the 180° pulses used for the T-ROESY spin-locking field. 

More serious are the coherence transfer cross peaks in ROESY spectra because the coherence peaks are in phase with the genuine cross-relaxation peaks and thus may modulate intensity of the genuine peaks. To emphasize the effect of coherence transfer peaks (now TOCSY peaks) we do the ROESY experiment with Tm = 300 ms and with a spin-lock field of 5 kHz (fig. 4(C)). Besides positive diagonal peaks (thick contours), several pairs... 

Figure 8.50. ROESY spectra of a tetrameric carbopeptoid 8.21 recorded with (a) a single 2.6 kHz continuous spin-lock pulse and (b) a 3.7 kHz phase-alternating Tr-ROESY spin-lock at 4.5 ppm. Spectrum (a) is dominated by TOCSY peaks which share the same phase as the diagonal, whereas these have been largely suppressed in (b), so revealing the genuine ROE peaks.

Second, undesired TOCSY peaks appear because some nuclei that are spin coupled experience similar fields during the application of the spin-lock and fulfil the Hartmann-Hahn condition. Since the TOCSY peaks are phase shifted by 180° with respect to the ROESY peaks, they can easily be recognized. However, the superposition of contributions from direct and indirect transfer results in a decrease of cross peak intensity and therefore in distances which are too long. When only lower boundaries are used as restraints in MD calculations this would lead to lower restraints and a less well-defined structure but would not induce wrong results. In addition, different internal correlation times, such as the above-mentioned different flexibility of the molecule have a smaller influence in ROESY than in NOESY spectra. 

Parhcular care has to be taken when implementing ROESY experiments. The spin-lock, which holds the spins along a defined axis perpendicular to the stahc magnetic field, can be realized in many different ways and is shU an achve field of research [18, 20]. In most spin-lock sequences the conditions for undesired TOCSY transfer are parhally fulfilled and especially cross-peaks close to the diagonal or anhdiagonal might not be accurately interpretable. Since in most cases the effechveness of the spin-lock also depends on the chemical shift offset, an offset-dependent correction has to be applied to the measured cross-peak intensities [20]. 

The easiest way to reduce the amplitude of TOCSY cross peaks in the ROESY spectra is to record a spectrum with minimal spin-lock power [23]. The other possibility is to modulate the frequency of the spin-lock field [25]. However, the most convenient way is to apply a series of 180° pulses instead of a single continuous-wave pulse during the mixing time, as is done in the T-ROESY experiment. Figure 4(D) shows the T-ROESY spectrum of cyclo(Pro-Gly) recorded with = 300 ms. Although the... 

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). 

Fig. 8.13. ROESY (A), NOESY (B) and TOCSY (C) cross peaks observed in the phenyl ring 2D pattern of the complex shown in the inset (Ln = Yb,+). Negative cross peaks are highlighted by squares, and positive cross peaks by circles [16].

Figure 4 Homonuclear 2D spectra of the 8mer peptide EWTLYWR in 90 % H2O, 10 % D2O. (a) Representation of coherence transfer pathways for COSY (soiid arrows), TOCSY (dotted arrows) and NOESY (dashed arrows) experiments, (b) Section of COSY spectrum displaying the backbone H -H -correlations, additionaiiy the side-chain H -H -correlations of R8 is visible in the upper right-hand corner. The H -H -correlation of El cannot be detected because of soivent-exchange broadening of the N-terminal amino group, (c) Section of TOCSY spectrum that displays the correlations of the backbone with all protons within the amino acid side chain, (d) Section of ROESY spectrum that displays correlations between backbone H -H intraresidual as well as to the neighboring (i-1) amino acid. The cross peaks to the (i-1) amino acid have higher intensities. Thus, a "sequential walk" is possible (arrows) that allows identification of the position of amino acids within the peptide chain. Additionally the H of El can be assigned (first arrow on the left).

Figure 5 Cross-peaks in homonuclear 2D TOCSY spectra arising due to ROESY effects. Clean TOCSY spectra were acquired with the MLEV-17 spin-lock sequence, (a) Base proton H6-to-methyl correlations in a 27-nt AT-rich DNA stem-loop structure 93 the spectrum was recorded with the 50-ms mixing sequence, (b) and (c) TOCSY spectra acquired for a 31 -nt stem-loop RNA (unpublished data), (b) H5-H6 cross-peaks in pyrimidines and a H1 -H8 cross-peak (boxed) in the syn guanine from the tetraloop UACG the spectrum was recorded with the 30-ms mixing sequence, (c) Sequential H2 -H6/H8 cross-peaks the spectrum was recorded with the 90-ms mixing sequence.

The first of these arises when the long spin-lock pulse acts in an analogous fashion to the last 90" pulse of the COSY experiment so causing coherence transfer between J-coupled spins. The resulting peaks display the usual antiphase COSY peak stmcture and tend to be weak so are of least concern. A far greater problem arises from TOCSY transfers which arise because the spin-lock period in ROESY is similar to that used in the TOCSY experiment (Section 5.7). This may, therefore, also induce coherent transfers between J-coupled spins when these experience similar rf fields, that is, when the Hartmann-Hahn matching condition is satisfied. Since the ROESY spin-lock is not modulated (i.e. not a composite pulse sequence), this match is restricted to mutually coupled spins with similar chemical shift offsets or to those with equal but opposite... 

Figure 8.47. The generation of false ROE peaks in ROESY spectra may arise from combined ROE and TOCSY mechanisms.

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