TOCSY-HSQC spectrum

Figure 12.51 shows the 2D XH, 15N HSQC spectrum of U-15N Rhodobacter capsu-latus Ferrocytochrome q in 90% H2O. The F planes of the corresponding 3D TOCSY-HSQC spectrum are indicated on the left side of the 2D spectrum. Plane 15 (lower dotted line) corresponds to a 15N chemical shift of 131.59 ppm, very close to the 15N shift of the backbone nitrogen of Ala-66 (131.66). Plane 16 (upper dotted line) corresponds to... 

A section of the strip plot (residues 15-31) taken from the 3D TOCSY-HSQC spectrum is shown in Figure 12.54. Each strip is taken from the nearest F2 (15N) plane to the backbone... 

Figire 40 Expansions from fhe 3D purged H- H- C TOCSY-HSQC spectrum of Estane 5703 showing H- H slices, at two differentiae shifts, (a) 63.2 and (b) 60.2 ppm, containing diagonal resonances from the internal butanediol units. Reprinted with permission from LeMaster, D. M. Hernandez, G. Macromolecules2000,33,3569. Copyright 2000 American Chemical Society. 

Load the spectrum of the gradient-assisted, inverse detected, 2D CH-HSQC-TOCSY experiment acquired with the echo-antiecho technique, D NMRDATA GLUCOSE 2D CH GCHICOTO 001999.RR. Check and if necessary correct its calibration in both dimensions. Set up a layout as for the basic HSQC spectrum. Compare the spectrum with the spectra of the basic HSQC and HMQC experiments. Use the same rows or columns to identify the additional TOCSY-peaks. 

Figure 7-21 HSQC spectrum (left) and HSQC-TOCSY spectrum (right) of T-2 toxin.

The heteronuclear NMR experiments discussed above highlight how much extra resonance dispersion can be gained via this approach. The power of this added dimension becomes clear if, for example, the 3H—15N HSQC experiment shown above, where each HN atom is essentially resolved, was to be combined with a TOCSY or NOESY experiment to provide a third frequency dimension. The resulting 3D 15N-HSQC-TOCSY/NOESY spectrum would contain virtually no overlap of interresidue resonances. Such experiments are indeed possible and have been the driving force in producing uniformly 15N- and/or 13C-labeled proteins. This field has been the most intensely researched area of NMR in the past 20 years, and the strategies employed to determine protein and peptide structures using heteronuclear NMR experiments are discussed in the next section (see Chepter 9.19). 

Sequence-specific a.ssignment. Each peak in the NMR spectrum (of which there will be thousands) corresponds to a particular atom (H, C, N, etc.) in the structure. These can be assigned to specific backbone or side-chain residues in the sequence using chemical shift data and correlation methods (COSY, TOCSY, HSQC, etc.). 

In 2006, Milosavljevic and co-workers64 reported a study of the complete 4H and 13C NMR assignment of a new triterpenoid saponin, leucantho-side-A (13), from Cephalaria leucantha L. In the course of determining the structure and assigning the spectra, the authors made extensive use of the normal ensemble of 2D NMR experiments in use for the characterization of natural product structures HSQC, HMBC, DQF-COSY, TOCSY, and NOESY. The authors supplemented the aforementioned list of experiments with 2D /-resolved, DINE-(Double INEPT-Edited)-HSQC, and INADEQUATE spectra. The authors made no mention of the use of the connectivity information derived from the 1,1-ADEQUATE spectrum in the assembly of the triterpene nucleus of the molecule but reported extensive tabulations of the 1,1-ADEQUATE correlations that were used to sequence and assign the saccharide resonances of the tri- and di-saccharide sub-units, 14 and 15, respectively, linked to the triterpene nucleus. 

The resulting spectrum, Cindirect/ is a symmetric matrix whose axes are defined by the indirect dimension, F1. Applying indirect covariance processing to a non-symmetric data matrix such as heteronuclear 1H-13C HSQC-TOCSY spectrum affords a symmetric homonuclear 13C-13C TOCSY spectrum analogous to that described originally by Turner.22... 

As an example, consider the complex polyether marine toxin breve-toxin-2 (7) [59]. The proton NMR spectrum of this molecule, even at 600MHz, has considerable overlap making the establishment of proton-proton connectivity information from a COSY or TOCSY spectrum difficult at best. In contrast, the IDR-HSQC-TOCSY spectrum presented in Fig. 10.19, in conjunction with an HMBC spectrum, allows the total assignment of the proton and carbon resonances of the molecule. 

Fig. 10.19. IDR-HSQC-TOCSY spectrum of the complex marine polyether toxin brevetoxin-2 (7). The data were recorded overnight using a 500 pg sample of the toxin (MW = 895) dissolved in 30 pi of d6-benzene. The data were recorded at 600 MHz using an instrument equipped with a Nalorac 1.7 mm SMIDG probe. Direct responses are inverted and identified by red contours relayed responses are plotted in black. The IDR-HSQC-TOCSY data shown allows large contiguous protonated segments of the brevetoxin-2 structure to be assembled, with ether linkages established from either long-range connectivities in the HMBC spectrum and/or a homonuclear ROESY spectrum.

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]. 

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