TOCSY transfer

Figure 7.9 A ID HMQC-TOCSYpulse sequence. The sequence involves a TOCSY transfer and BIRD presaturation. (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.)...

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]. 

Another ingenious modification of the HNN-COSY scheme involves the replacement of the homonuclear 15N-COSY transfer by a 15N-TOCSY transfer [37]. As the homonuclear TOCSY transfer is twice as fast as the COSY transfer, a significant sensitivity increase is achieved. The application is, however, limited to cases where the 15N donor and acceptor resonances are at similar frequencies, so that the power of the 15N-TOCSY radio frequency pulses needed is not too strong. A very interesting application of this scheme was presented for the sensitive detection of very small (0.14 Hz, Tab. 9.1) h4/NiNi couplings in N1-H1 06=C6-N1 H-bonds of guanosine tetrads. 

By applying the NOESY step first, this experiment allows us to jump from one spin system to another or to overcome a bottleneck in TOCSY transfer caused by an occurrence of a small coupling constant in the chain of J-connectivities. Both these features are illustrated in ID NOESY-TOCSY spectra of the type VI group B Streptococcus capsular polysaccharide (1). 

By reversing the order of polarization transfer steps, NOE contacts of signals hidden under the bulk of other resonances can be established in a ID TOCSY-NOESY [39] experiment (fig. 1(d)). The necessary requirement is that an efficient TOCSY transfer can be made to a particular proton from... 

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 NOESY and TOCSY polarization transfers can also be arranged so that two NOESY steps are interrupted by one TOCSY transfer. This is useful for situations when a proton which is intended as a starting point for a ID TOCSY-NOESY experiment cannot be selectively excited, nevertheless it has a NOE contact to an isolated proton. The ID NOESY-TOCSY-NOESY sequence [72] (fig. 4(b)) is obtained by appending another NOESY step to the ID NOESY-TOCSY pulse sequence of fig. 1(c). The last NOESY step can be either selective or nonselective depending whether a selective 180° pulse is applied after the nonselective 90° pulse at the end of the TOCSY transfer. 

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

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...

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... 

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. 

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. 

Mixed phase lineshapes, typical for the multistep RELAY spectra, sometimes make the identification of individual signals difficult. At the same time, for extensive networks of coupled spins, the TOCSY transfer proved... 

Replacement of the TOCSY transfer in a ID COSY-TOCSY experiment by the NOESY step yields a ID COSY-NOESY sequence [38] (fig. 13(c)). The experiment is illustrated by the determination of NOEs from the H-7 proton of a terminal 3,6-dideoxy-4-C-(l-hydroxyethyl)-D-xylohexose (6)... 

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