TOCSY spin-locks

Fig. 8. ID ROESY-TOCSY. (a) H spectrum of the oligosaccharide 3 (5 mg/0.5 ml D2O). (b) ID ROESY spectrum of 3 acquired using the pulse sequence of fig. 7(a) with selective excitation of the H-lb proton. Duration of the 270° Gaussian pulse and the spin-lock pulse ( yBi/ K = 2.8 kHz) was 49.2 ms and 0.5 s, respectively. The spin-lock pulse was applied 333.3 Hz downfield from the H-lb resonance. The time used for the frequency change was 3 ms. (c) ID ROESY-TOCSY spectrum acquired using the pulse sequence of fig. 7(c) and the selective ROESY transfer from H-lb followed by a selective TOCSY transfer from H-4c. Parameters for the ROESY part were the same as in (b). A 49.2 ms Gaussian pulse was used at the beginning of the 29.07 ms TOCSY spin lock. 256 scans were accumulated. A partial structure of 3 is given in the inset. Solid and dotted lines represent TOCSY and ROESY...

Concatenation of two TOCSY steps in a ID TOCSY-TOCSY experiment [72] is a straightforward matter (fig. 10(a)). After the initial TOCSY transfer, the magnetization is returned to the 2 axis by a nonselective 90° pulse applied perpendicularly to the spin-lock axis. The carrier frequency is changed and the second 90° selective pulse applied to a different proton followed by the second TOCSY spin-lock period. 

We can resolve this ambiguity with a TOCS Y spectrum. This is just a homonuclear 2D ( H H) experiment with the TOCSY spin lock as the mixing portion of the pulse sequence. With the fragment CH1-CH2-CH3-Cq4-CH5-CH6-CH7 we expect to see HI correlated... 

If we apply the TOCSY spin lock at this point on the x axis, we will destroy the first and fourth terms (Bi field inhomogeneity) and lock the second and third terms. Because the TOCSY mixing sequence transfers coherence from in-phase to in-phase, only the third... 

The Ideal Isotropic Mixing (TOCSY Spin-Lock) Hamiltonian... 

The TOCSY spin lock can sometimes cause RF heating of the sample, especially when the mixing time gets long and the sample contains ions. Recall that RF heating can cause sample degradation— especially protein denaturation—as well as other problems. If the sample temperature is not held constant during the acquisition of... 

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... 

However, a proton TOCSY spin-locking pulse is applied before H detection, so that all of the protons that are pan of the HCaCx spin system exhibit correlations in the fs dimension. In the 3D uace at chemical shifts of Cx and Ca, all protons in the spin system of Ha can be observed. 

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]. 

Total correlation spectroscopy (TOCSY) is similar to the COSY sequence in that it allows observation of contiguous spin systems [35]. However, the TOCSY experiment additionally will allow observation of up to about six coupled spins simultaneously (contiguous spin system). The basic sequence is similar to the COSY sequence with the exception of the last pulse, which is a spin-lock pulse train. The spin lock can be thought of as a number of homonuclear spin echoes placed very close to one another. The number of spin echoes is dependent on the amount of time one wants to apply the spin lock (typically 60 msec for small molecules). This sequence is extremely useful in the identification of spin systems. The TOCSY sequence can also be coupled to a hetero-nuclear correlation experiment as described later in this chapter. 

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. 

In a ID TOCSY-NOESY experiment [39], the proton magnetization is aligned along the spin-lock axis after the initial selective TOCSY step. The... 

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. 

Doubly selective ID-TOCSY experiments have been proposed to specifically transfer in-phase magnetization from two designated spins [57, 58]. This transfer will only take place if the two spins are connected by a scalar coupling. This method is achieved by using a double-selective spin-lock after the selective excitation of transverse magnetization of a desired spin. The doubly selective spin-lock can be obtained by using cosine-modulated... 

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... 

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