The 2D ROESY sequence

As stated previously, crosspeaks may arise in ROESY spectra as a result of processes that occur during the spin-lock other than cross-relaxation between spins. The principal complications that can arise are [61]  

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 

Sign of diagonal Origin of cross-peak Sign of cross-peak 

By convention the diagonal is phased to be positive and the sign of all other signals are given relative to this. 

The third complicating factor specific to ROESY is the attenuation of cross-peak intensities as a function of resonance offset from the transmitter frequency [69]. Off-resonance spins experience a spin-lock axis that is tipped out of the x-y plane (Section 3.2.1) resulting in a reduction in observable transverse signal in addition to a reduction in cross-relaxation rates. This is more of a problem for quantitative measurements, although fortunately mid-sized molecules show the weakest dependence of ROE cross-relaxation rates on offset. The so-called compensated ROESY sequence [69] eliminates these frequency-dependent losses should quantitative data be required. 

Likewise, the reverse ROE-TOCSY steps would produce a similar signal, but the conclusion would be wrong in either case. Since TOCSY transfer retains signal phase, its involvement in crosspeak generation is not obvious and can lead to drastically incorrect conclusions. Furthermore, if TOCSY and ROE transfers occur simultaneously between two spins, the ROE peak may be reduced in magnitude or even cancelled owing to opposite peak phases. The various transfer pathways that may occur during ROESY are summarised in Table 8.5. 

In parallel with the ID NOESY sequences above, the 2D ROESY experiment also has its ID equivalent (in fact, this was the original ROE experiment [69]) and gradient-selected analogues [36,79,80] all of which incorporate selective excitation of the target spin such as via the DPFGSE selection described for ID NOESY above [81]. These can be derived from ID NOESY sequences by incorporation of a suitable spin-lock in place of the 90-rm-90 segment of NOESY, and thus require no further elaboration. 

---

Figure 8.45. The 2D ROESY sequence. The mixing time, tm. is defined by the duration of the low-power spin-lock pulse.

Figure 6 Rotating frame NOE pulse sequences. The 1D experiment requires two sequences, represented by (A) and (B). (A) is the reference experiment in which a 90 non-selective pulse is applied on all the spins, followed by a spin-lock along the y-direction for a time and the state of the spin system is detected. (B) The control experiment in which a selective 180 pulse, inverts the magnetization of the spin from which the NOE is to be observed before the 90j pulse and the experiment is continued as (A). The 1D NOE spectrum is the difference between the spectra obtained with the sequence (A) and (B). (C) The 2D ROESY sequence. The times and fs are the evolution and detection periods and is the mixing time. SL refers to the low power spin-locking RF field.

The pioneering work in this field, a two-dimensional relayed-NOE experiment proposed by Wagner [7], was quickly followed by the appearance of several related NMR techniques [8-17]. Application of isotropic mixing during the J-transfer period yielded the 2D TOCSY-NOESY [11, 15] and NOESY-TOCSY [12, 14] experiments. When spin-lock sequences were applied to both J and NOE-transfers, the 2D TOCSY-ROESY and ROESY-TOCSY experiments [10, 16, 17] emerged. 

Fig. 8.2. Some of the most common 2D pulse sequences that can be employed using a proper choice of parameters to record 2D spectra of paramagnetic molecules (A) NOESY, (B) ROESY, (C) COSY, (D) ISECR COSY, (E) zero-quantum (double quantum) COSY, (F) TOCSY, (G) HMQC, (H) HSQC. Sequences (A), (B) and (F) are also used to obtain EXSY spectra. SL indicates a soft spin-lock sequence, while MLEV17 indicates a train of spin-locking hard pulses that optimizes the development of J/j coupling. In the reverse heteronuclear experiment (G) the upper and lower levels refer to H and heteronucleus, respectively. The phase cycles are not indicated. For clarity of discussion, all initial pulses can be thought to be applied along the y axis, in such a way that the coherence after the first 90° pulse is always along x. ...

The 2D ROE or ROESY experiment is an experiment to measure cross-relaxation in the rotating frame (Fig. 8.2B). After an initial 90° pulse and the variable evolution period t, a low power or soft spin-lock sequence (SL) is applied for a time during which magnetization transfer in the rotating frame occurs due to cross relaxation. Since scalar connectivities can also develop during spin lock, as... 

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. 

A 2002 review by Reynolds and Enriquez describes the most effective pulse sequences for natural product structure elucidation.86 For natural product chemists, the review recommends HSQC over HMQC, T-ROESY (transverse rotating-frame Overhauser enhancement) in place of NOESY (nuclear Over-hauser enhancement spectroscopy) and CIGAR (constant time inverse-detected gradient accordion rescaled) or constant time HMBC over HMBC. HSQC spectra provide better line shapes than HMQC spectra, but are more demanding on spectrometer hardware. The T-ROESY or transverse ROESY provides better signal to noise for most small molecules compared with a NOESY and limits scalar coupling artefacts. In small-molecule NMR at natural abundance, the 2D HMBC or variants experiment stands out as one of the key NMR experiments for structure elucidation. HMBC spectra provide correlations over multiple bonds and, while this is desirable, it poses the problem of distinguishing between two- and three-bond correlations. 

In Figures 7 and 8 the 2D proton exchange spectrum of the hydrothermally dealuminated zeolite H-Y loaded with ca. 40 H2O per unit cell (identical to the sample in Figure 6C) is shown as contour and stacked plot, respectively. A homonuclear 2D ROESY pulse sequence [5] and a spin lock pulse of 2 ms were used. The absence of cross peaks between the signals at 1.8+0.2 ppm and... 

The structures of the compounds were elucidated by a combination of NMR techniques (lH-, 13C-, and 13C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. 

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.

For the determination of the sequence and substitution positions of the different monosaccharides of a saponin, a series of proton and carbon 2D-NMR techniques can provide valuable information about the usually crowded regions of the conventional ID spectra. Thus, integrated approaches including ID or 2D H- H homonuclear NMR shift-correlation experiments (DQF- and TQF-COSY, TOCSY or HOHAHA, NOESY, ROESY or CAMELSPIN), and H-detected H-,3C heteronuclear shift correlation (HMQC, HMBC) have proved to be extremely useful. 

There is no doubt that the ROESY experiment will become the best experiment to sequence chains of sugars by NMR. Many examples of this experiment are published in the recent literatime, either ID (58, 79) or 2D (62, 73, 76, 77, 80), even though reference 77 describes a ROESY on a triterpene ... 

The structures of pseudostellarins and heterophyllins were determined by chemical degradation, 2D-NMR methods such as H- H COSY, HOHAHA, NOESY, ROESY, HMQC, and HMBC, enzymatic methods, and ESI-MS/MS analysis. The sequencing of pseudostellarins C (310) and D (311), proposed earlier was revised as shown in Fig. 39 by X-ray crystallographic and NMR analysis (205,206). 

---