Experiment TOCSY

The HOHAHA or TOCSY experiment [5.150, 5.151] has proved a popular alternative in many applications to the main homonuclear correlation experiment for sensitive nuclei, the basic COSY experiment. Both the HOHAHA and the TOCSY experiment are based on the principal of isotropic mixing but differ in the type of spinlock sequence used. Nevertheless they may be considered together and for convenience in the following discussion the expression TOCSY experiment will be used for both sequences. The TOCSY experiment uses cross polarization for the coherence 

The homonuclear correlation of direct and relayed connected nuclei is established by isotropic mixing and cross polarization. The isotropic mixing is achieved by a cw field (HOHAHA experiment) or a multipulse spinlock sequence (TOCSY experiment). 

In Check it 5.4.2.1 the ID selective COSY, ID selective relayed COSY without and with z-filter and a ID selective TOCSY spectrum are simulated for the same spin system and the results compared. As already mentioned the spinlock for isotropic mixing can be generated in different ways and this has lead to the development of improvements and elements being added to the spinlock sequence. Of these improvements the trim pulse and z-filter, adapted to the spinlock sequence [5.154], are the most popular. 

Originally it was proposed that a series of 180° pulse should be used for the spinlock sequence but this has now been superseded by more efficient sequences. Although other spinlock sequences can be implemented [5.154] the most popular sequence is MLEV-17 with z-filter [5.143] which enables lower pulse power levels to be used with less lineshape distortion. Check it 5.4.2.2 examines several spinlock sequences. 

TOCSY experiments using different spinlock sequences and configuration files, (a) A simple 180° low power pulses ch5422a.cfg, (b) a composite 180° (90°x/180y/90°x) pulse 

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The pulse sequence for the ID TOCSY experiment is shown in Fig. 7.6. The original experiment used a Gaussian pulse, but a half-Gaussian... 

Figure 7.6 (a) A ID TOCSYexjjeriment with Gaussian excitation, (b) A ID TOCSY experiment with half-Gaussian excitation and TR (trim) pulses. (Reprinted from Mag. Reson. Chem. 29, H. Kessler et al., 527, copyright (1991), with permission from John Wiley and Sons Limited, Baffins Lane, Chichester, Sussex P019 lUD, England.)... 

Nolls, P., Parella, T. Spin-edited 2D HSQC-TOCSY experiments for the measurement of homonuclear and heteronuclear coupling constants application to carbohydrates and peptides. /. Magn. Reson. 2005, 176, 15-26. 

For the complex of the inhibited catalytic domain of stromelysin-1, 2-D doubly filtered COSY and TOCSY experiments performed... 

Kay LE, Xu G-Y, Singer AU, Muhandiram DR, Forman-Kay JD. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 3H and 13C correlations in H20 samples of proteins. J Magn Reson 1993 101B 333-337. 

In Problem 45 we have irradiated the same two signals in the NOESY and TOCSY experiments. In the NOESY spectra the protons which answer (show a clearly magnitude-enhanced signal) are those which are closer to the ir-... 

NOESY and TOCSY experiments. It was noted that while the monodendrons are soluble only in CH3N02 the assemblies readily dissolve in CH2C12 on addition of NPM. 

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. 

A more sensitive alternative is to use HCCH-TOCSY experiments [63-65], which transfer magnetization through the much larger 13C-13C coupling constants. In addition, these coupling constants are almost independent of the secondary structure and very uniform... 

D or 3D HCCNH-TOCSY experiment that correlates the imino proton with H6/8, which can be independently assigned from other experiments. 

TTie TOCSY 2D NMR experiment correlates all protons of a spin system, not just those directly connected via three chemical bonds. For the protein example, the alpha proton, Ft , and all the other protons are able to transfer magnetization to the beta, gamma, delta, and epsilon protons if they are connected by a continuous chain—that is, the continuous chain of protons in the side chains of the individual amino acids making up the protein. The COSY and TOCSY experiments are used to build so-called spin systems—that is, a list of resonances of the chemical shift of the peptide main chain proton, the alpha proton(s), and all other protons from each aa side chain. Which chemical shifts correspond to which nuclei in the spin system is determined by the conventional correlation spectroscopy connectivities and the fact that different types of protons have characteristic chemical shifts. To connect the different spin systems in a sequential order, the nuclear Overhauser effect spectroscopy... 

As an example the three subspectra of a carbohydrate 1 (peracetylated triglucose) obtained with the modified pulse sequence I and with the frequencies of the selective 180° pulses adjusted to the frequencies of the three anomeric protons lA, IB and 1C are shown in fig. 3(b). One of these spectra is compared with the corresponding spectrum measured within exactly the same total measuring time with the basic single selective ID TOCSY experiment (fig. 3(a)). 

These spectra demonstrate that with the multiselective method a clean separation of the subspectra of the three independent spin systems may be achieved. They furthermore prove that - compared to the basic ID TOCSY experiment - spectra of the same quality with respect to the suppression of residual signals originating from the other spin systems and with respect to the signal-to-noise ratios can be measured. 

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. 

The ID NOESY-TOCSY experiment [39] shown in fig. 1(c) is a straighffor-ward concatenation of ID NOESY and TOCSY experiments [34] (figs 1(a), (b)). Since the NOE transfer takes place along the z axis, and thus has no phase memory, no phase correction for the second selective pulse is needed to compensate for the change of the r.f. frequency during the tnoe interval. Nevertheless, any possible phase differences between the selective and consecutive nonselective pulses must be taken into account in both steps, by adjusting the phase of soft pulses. 

In the subsequent ID ROESY-TOCSY experiment (pulse sequence of fig. 7(c)), a selective TOCSY transfer was applied from H-4c. During the... 

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