TOCSY-NOESY

Fipire 6.8 Volume chosen for a selective 3D NOESY-TOCSY spectrum of a peptide with NH region in coi, in >.2, and all protons in [Pg.354]

Different assignment strategies can be employed, depending on whether selective or nonselective pulses have been used in recording 3D spectra. A homonuclear 3D NOESY-TOCSY spectrum in which the NH/ region has been recorded is presented in schematic form in Fig. 6.9. 

The most straightforward isotope-editing method for selecting protons bound to a heteronucleus and suppressing all others is the simple acquisition of a spectrum with an indirect heteronuclear dimension (in the literature the term isotope editing is often used as a synonym for these techniques). This can be accomplished by a simple 2D HMQC or HSQC shift correlation, or a more elaborate 3D technique including an additional NOESY or TOCSY step (3D X-edited NOESY/TOCSY etc.), or even 4D experiments with a second heteronuclear shift dimension [13, 14]. 

If we consider a standard homonuclear 1H, 1H 2D experiment like NOESY, TOCSY or COSY, then all these different filter elements can be applied either before or after the mixing step, or both. Correspondingly this will lead to a selection in FI only, F2 only or both FI and F2, of the 2D spectrum. 

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. 

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). 

ID TOCSY-NOESY-TOCSY and ID NOESY-TOCSY-NOESY... 

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. 

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.

The ID ge-NOESY-TOCSY experiment is illustrated using a EPS O-polysaccharide (7) isolated from Proteus mirabilis serotype 0 57 [79]. The... 

In principle, all the combinations of homonuclear 2D spectroscopies can be performed to originate a 3D spectrum (COSY-COSY, NOESY-COSY, NOESY-TOCSY, etc.). The considerations made in this chapter for the most basic experiments can be easily extended to their combinations. The general guideline should always be that the more complex the pulse sequence is, the more the experimental sensitivity will suffer from fast nuclear relaxation. 

Selective excitation of a resolved resonance followed by homonuclear or heteronuclear Hartmann-Hahn transfer can also be advantageous in the preparation period of two-dimensional experiments. For example, two-dimensional COSY, NOESY, TOCSY, and two-dimensional /-resolved subspectra of individual spin systems can be acquired based on this principle (Homans, 1990 Sklenaf and Feigon, 1990 Nuzillard and Massiot, 1991 Gardner and Coleman, 1994). In selective two-dimensional experiments like soft COSY (Briischweiler et al., 1987 Cavanagh et al., 1987),... 

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