Phase TOCSY

TOCSY data are acquired in tbe pbase-sensitive mode using quadrature detection, and aU. tbe data phases are positive. Tbis increases the SNR for the matrix, and the time required for the experiment is short because very Htde, if any, phase cycling is necessary. In some cases a single scan per FID suffices, and the data can be acquired in approximately 10 min,... 

Gradient-enhanced 2D TOCSY spectrum of 10 mMof sucrose in D..,0 is shown in figure 7.26. The clean spectrum obtainable without any noise and without the nece.ssity of any phase cycling illustrates the power of this new technique in modern NMR spectroscopy. 

Haliclonacyclamine E (13) and arenosclerins A (14), B (15), and C (16) have been isolated from the marine sponge Arenosclera brasiliensis, endemic in Brazil. Crude extracts of this sponge displayed potent cytotoxic and antibiotic activities, and were subjected to fractionation by sihca-gel flash chromatography, medium pressure chromatography on a SiOH cyanopropyl-bonded column, and reversed-phase Cis column chromatography to give compounds 13-16 [18]. The structure elucidation was based on spectroscopic analysis, including HRFABMS, COSY, HSQC, HSQC-TOCSY, and HMBC NMR... 

In order to combat this, the rotating frame Overhauser effect spectroscopy (ROESY) techniques can be employed. An in-depth discussion of how this technique works is outside the remit of this book but suffice to say, in the ROESY methods (1- and 2-D), NOE data is acquired as if in a weak r.f. field rather than in a large, static magnetic field and this assures that all NOEs are present and positive, irrespective of tumbling rate and magnet size. It is possible that some TOCSY correlations can break through in ROESY spectra but these will have opposite phase to the genuine ROESY correlations and so should therefore not be a problem - unless they should overlap accidentally with them. A 2-D ROESY spectrum of the naphthalene compound is shown below (Spectrum 8.6). 

TOPHAT-shaped 90° pulses are used in other cases as the best compromise with respect to the excitation profile, the phase homogeneity and length. Depending on the type of the detected spin-spin interaction - being either scalar or dipolar coupling - each selected spin is initially perturbed only once (ID TOCSY, ID INADEQUATE, ID C/H COSY, 2D TOCSY-COSY and 2D HMBC), or for several times (ID NOE). With each of the selected spins initially perturbed only once the inherently smaller transient NOEs would be detected in the latter case, whereas with the multiple excitation of a selected spin within the NOE build-up period the stronger steady-state NOEs are more or less approximated. 

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. 

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... 

Fig. 18. The pulse sequence of a ID ge-NOESY-TOCSY experiment, tnoe is the NOE mixing time, 5 are optional delays which can be used for z-filtration [81] or for suppression of ROE effects in macromolecules (2 x (5 + Tgrad) = 0.5 x mixing time). DIPSI-2 [78] sequence was used for isotropic mixing. Phases were cycled as follows 0i = 2x, 2(—x) (j)2 = X, —x Ip = X, 2 —x), X. Rectangular PFGs, G = 6 Gauss/cm and Gi = 7 Gauss/cm, were applied along the axis for Xpad = 1 ms.

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. 

Fig. 1. The ID TOCSY pulse sequence, (a) The Bax version and (b) The Kessler version. The phase 0 for the last 90° pulse and the receiver is rotated synchronously along the four...

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... 

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