ROESY spin-locks

Fig. 9. ID TOCSY-ROESY. (a) H spectrum of oligosaccharide 3 (5 mg/0.5 ml D2O). (b) ID TOCSY spectrum acquired using the pulse sequence of fig. 7(b) and a selective excitation of H-lc by a 49.2 ms 270° Gaussian pulse. Duration of the spin lock was 132.7 ms including two 2.5 ms trim pulses. 32 scans were accumulated, (c) ID TOCSY-ROESY spectrum acquired using the pulse sequence of fig. 7(d) with initial selective TOCSY transfer from H-lc and selective ROESY transfer from H-4c. Parameters for the TOCSY part were the same as in (b). A 49.2 ms 270° Gaussian pulse was used at the beginning of the ROESY transfer. A 500 ms ROESY spin-lock pulse ( yBi/2 K = 2.8 kHz) was applied 1000 Hz downfield from the H-4c resonance. The time used for the frequency change was 3 ms. 128 scans were accumulated. A partial structure of 3 is given in the inset. Solid and dotted lines represent TOCSY and ROESY transfers, respectively.

In the presence of very strong coupling, i.e., protons displaying a large coupling constant with the target, TOCSY contributions might not be completely suppressed by a non optimized T-ROESY spin lock, and therefore... 

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. 

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 8.46. The ROESY spin-lock. During this, magnetisation parallel to the B] field remains spin-locked whereas orthogonal components are driven about this (here in the xz plane) and eventually dephase through Bi field inhomogeneity.

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.

S(t, y was recorded with a. 8 homonuclear jr/2-t,-T,-t, experiment (ROESY), spin locK pulse of T = 2 ms and MAS frequency of 3 kHz. Asterisks denote spinning m sidebands. 

We may recall that the TOCSY experiment (discussed in Chapter 6) also makes use of a spin lock for mixing. The main difference between the TOCSY spin lock and the ROESY spin lock lies in the frequency of the rotating frame used for the spin lock. For the TOCSY experiment, the frequency of the spin lock is typically placed in the middle of the spectral window (normally at the frequency of the transmitter), whereas for the ROESY experiment the spin lock frequency is placed far away from the spectral window. In most... 

The pulse sequence for the ID ROESY experiment using purged half-Gaussian pulses is shown in Fig. 7.7. The purging is required to remove the dispersive components, since these are not completely eliminated by the weak spin-lock field employed in the ID ROESY experiment. 

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 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. 1. Basic pulse sequence and CP diagram for gradient-based spin-locked ID exf>eriments. A 1 (— 1) 2 gradient ratio selects N-type data (solid lines) while 1 (— 1) (—2) selects P-type data (dashed lines). When SL stands for a -filtered DIPSI-2 pulse train, a ge-lD TOeSY is performed. On the other hand, when SL stands for a T-ROESY pulse train, a GROESY experiment is performed. S stands for the gradient length.

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

The use of spin-lock pulses for water suppression is illustrated with the NOESY and ROESY pulse sequences (fig. 5). Using the Cartesian product operator description [9], the effect of the NOESY pulse sequence of fig. 5(A) is readily illustrated ... 

Fig. 5. Pulse sequences of NOESY and ROESY with spin-lock purge pulses for water suppression. (A) NOESY pulse sequence. The spin-lock pulses are typically of length 0.5 ms and 2 ms, and r = 1/SW, where SW is the spectral width in the acquisition dimension. Phase cycle (pi = x,—x) 4>2 = 4 x,x,—x,—x) ...

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... 

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... 

The 2D NOESY and the 2D ROESY experiments may also be used to measure NOE-or ROE- build-up rates. This is accomplished using a series of experiments, where the mixing period D9 or the length of the spin-lock period respectively is incremented from experiment to experiment. From build-up rates relative internuclear distances may be estimated and calculated. 

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... 

MLEV17 sequence being one of the most used sequences [23]) in such a way as to continuously refocus the chemical shift evolution of the various signals in the xy plane. Analogously to ROESY experiments, the magnetization during the spin-lock (mixing) time disappears with T p (i.e. essentially Tj, see Section 3.4). It follows that coherence transfer in the xy plane, which is built up with a sin(7T J/jt) function, also decreases with time constant p p — p[p + p p)/2 ... 

In TOCSY experiments, the problem of overheating the sample is more serious than in ROESY experiments because of the large irradiation energy required by the spin-lock pulse. Each individual component of the pulse train must have enough power to irradiate the whole spectral window of interest. Spin-lock sequences different from the MLEV17 sequence, that may alleviate the heating problem,... 

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