TOCSY combination with HSQC

Today, it is possible to make complete assignments of all proton and carbon atoms in the NMR spectra of most isolated anthocyanins. These assignments are normally based on chemical shifts (8) and coupling constants (J) observed in 1-D H and l3C NMR spectra (Fig. FI.4.2), combined with correlations observed as cross-peaks in various homo- and heteronu-clear 2-D NMR experiments (see below for details on COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY). 

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). 

Combinations of NOESY with HSQC (or HMQC) and TOCSY with HSQC are among the most useful edited three-dimensional experiments (D. Marion, P. C. Driscoll, L. E. Kay, P.T Wingfield, A. Bax, A. M. Gronenbom, G. M. Clore. Biochemistry 28, 6150-6156 (1989). E. R. P. Zuiderweg, S.W. Eesik Biochemistry 28, 2387-2391 (1989). Eor example the NOESY-HSQC (Pig. 14.53) begins with the NOESY sequence and is followed directly by the HSQC sequence. It is important... 

GOS) production was intensively explored. The researchers were searching for commonly available and efficient p-galactosides. Polaina and coworkers,reported efficient method for the hydrolysis of lactose and the biosynthesis of GOS by using recombinant p-galactosidase from Thermotoga maritima. The reaction mixture was veiy complex and the concentration of synthesized oligosaccharides was lower than 10%. Desired compounds were defined by means of 2D-HSQC spectra combined with selective COSY and TOCSY ID NMR experiments. 

Clearly the homonuclear and the heteronuclear experiments could be combined in the reverse order i.e. HSQC-NOESY and HSQC-TOCSY. The main advantage in these schemes relates to N-edited experiments in which the narrower amide proton spectral width is sampled during f and the full proton spectral width is collected during t. On the other hand water suppression is more effective when HSQC follows NOESY. Also the sensitivity enhancement can be incorporated into the NOESY-HSQC experiment. Eor the TOCSY-HSQC or HSQC-TOCSY it does not matter because both the TOCSY and HSQC sequences can be implemented with the sensitivity enhancement. It should be mentioned that the TOCSY type of transfer is more effective between C nuclei than between protons and therefore the HCCH-TOCSY experiment is preferred when a doubly labeled sample is available. 

The structures of the glycosylphospholipids 1 and 2 were supported by NMR spectroscopy and mass spectrometry data. The as well as P NMR spectra for compound 1 were almost identical to those for compound 2. Full structural assignment of 2 (with exception for the acyl and alkyl residues) was performed by a combination of H, COSY, ROESY, TOCSY and HSQC spectroscopy and presented in Table 1. The P NMR signals were assigned using H, P HMQC technique. The molecular masses for the GPIs 1 and 2 were confirmed by MALDI-TOF and electrospray mass spectrometry. ... 

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