The ID TOCSY Experiment

As mentioned in Section 7-7b, the ID version of the TOCSY experiment is especially useful for larger molecules that possess complicated and overlapping spin systems. The 2D TOCSY spectra of classes of molecules such as oligosaccharides can be very difficult to interpret. ID TOCSY experiments, however, permit the mapping of entire spin systems when the chemical shift of just one member of the system is distinct. An example is the anomeric (H-1) protons of oligosaccharides, which are situated at higher frequency (down-field) from the carbinol protons. 

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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... 

The selective excitation is referred to alternatively as semi-selective excitation , e.g., in refs [5, 6]. It is semi-selective in the sense that for the ID TOCSY experiment, the whole multiplet corresponding to a spin, rather than a component of the multiplet, is selectively excited. In this chapter, we will not make this distinction, and the term selective is used instead in this context. 

The pulse sequence for ID TOCSY is a ID modification of the original TOCSY experiment [2] introduced by Braunschweiler and Ernst. The TOCSY experiment was also referred to as HOHAHA (which stands for HOmonuclear HArtman-HAhn) by Bax and Davis [3]. The ID TOCSY experiment was proposed by Bax and co-workers [4, 5], and by Kessler et al. [6]. The essential features of the pulse sequence involve the use of selective excitation of a desired resonance, followed by a homonu-clear Hartman-Hahn (or isotropic) mixing period [2, 7]. That is, the unit -Pnonsei - in the 2D TOCSY pulse sequence is replaced by Fsei -where P stands for a pulse (or pulses), ti is the evolution period in the 2D experiment and r is a fixed delay. 

The ID TOCSY experiment is essentially the same as the 2D version, but with the parameters being those of a ID, rather than a 2D, experiment. The initial, hard 90° pulse is replaced by either a selective, soft 90° pulse or, still better, a DPFGSE sequence such as that shown in Figure 7-5 (the initial 90° pulse and the pair of 180° pulses with their attendant sets of gradients). 

There exists meanwhile a variety of frequency selective experiments still using the conventional CW irradiation as the ID NOE experiment, or upgraded with one or more selective pulses, as the ID TOCSY or the ID COSY experiment. These experiments and their many variants are probably the best choice in such cases as long as the response of a spin system to the perturbation of only one single spin or one single group of equivalent spins is of interest. If, however, and this is the most common situation, informations on several rather than only one spin-spin interaction is needed. 

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 proposed ID TOCSY-NOESY experiment is illustrated by the assignment of NOEs from anomeric protons H-lc and H-ld of the polysaccharide 1. Because the resonances of H-lc and H-ld overlapped, this assignment was not possible from a ID NOESY spectrum as shown in fig. 3(b). Although these protons differed in their chemical shifts, it was not possible to separate them by chemical-shift-selective filtration because of the very fast spin-spin relaxation of backbone protons (20-50 ms) in this polysaccharide. Instead, a ID TOCSY-NOESY experiment was performed in which the initial TOCSY transfer from an isolated resonance of H-2c was followed by a selective NOESY transfer from H-lc. The ID TOCSY-NOESY spectrum (fig. 3(c)) clearly separated NOE signals of the H-lc proton from those originating from the H-ld proton and established the linkage Ic —> 6a. 

The ID TOCSY-ROESY experiment is illustrated on the same molecule using the pulse sequence of fig. 7(d). This time the magnetization of H-4c was generated during the initial selective TOCSY transfer from H-lc (fig. 9(b), pulse sequence of fig. 7(b)). In the subsequent ID TOCSY-ROESY experiment, the ROE transfer from H-4c confirmed the expected... 

The ID TOCSY-TOCSY experiment is illustrated by the identification of two partial spin systems of a capsular polysaccharide 1, starting with two overlapping protons H-la and H-lb. A 20 ms half-Gaussian pulse was used in an exploratory ID TOCSY experiment (pulse sequence of fig. 1(b)). It has been found (fig. 11(b)) that although the corresponding H-2 protons overlapped partially, H-3a and H-3b were separated completely. In the following ID TOCSY-TOCSY experiments, using the pulse sequence of fig. 10(a), the second TOCSY transfer was initiated from protons H-3a and H-3b, respectively. The two spectra (fig. 11(c), (d)) clearly separate both partial spin system of a and b residues. 

In this chapter, the discussion will be focused on the ID TOCSY (TO-tal Correlation SpectroscopY) [2] experiment, which, together with ID NOESY, is probably the most frequently and routinely used selective ID experiment for elucidating the spin-spin coupling network, and obtaining homonuclear coupling constants. We will first review the development of this technique and the essential features of the pulse sequence. In the second section, we will discuss the practical aspects of this experiment, including the choice of the selective (shaped) pulse, the phase difference of the hard and soft pulses, and the use of the z-filter. The application of the ID TOCSY pulse sequence will be illustrated by examples in oligosaccharides, peptides and mixtures in Section 3. Finally, modifications and extensions of the basic ID TOCSY experiment and their applications will be reviewed briefly in Section 4. 

ID TOCSY and Related Techniques 4. Extensions of the basic ID TOCSY experiment... 

In the previous sections, only the basic, non-gradient ID TOCSY pulse sequence, its experimental aspects and applications were described. In the following, the more recent modifications and extensions of the basic pulse sequence and their applicability to spectral assignments and structural elucidation will be briefly reviewed. Some of these more sophisticated techniques may not be as readily implementable as the basic ID TOCSY experiments, and thus have not yet found wide applications in routine practice. 

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... 

The ID TOCSY module has been used in many pseudo-3D experiments (or alternatively referred to as ID analogues of 3D experiments in the literature) such as ID TOCSY-NOESY or ID TOCSY-ROESY experiments. The TOCSY part of these experiments are similar to that of a regular ID TOCSY where a selective excitation of a desired signal is followed by a MLEV17-type isotropic mixing. The second polarization transfer (NOESY or ROESY) step can either be non-selective [29, 59-61] or selective [62-65]. 

Since a single experiment with a fixed spin-lock period is usually performed, this experiment does not, in contrast to the H/ H decoupling experiment, establish the complete coupling network. For this purpose a series of ID TOCSY experiments, with the length of the mixing period increasing step-wise from experiment to experiment, has to be performed. 

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