Homonuclear 2D methods

A protein subject to NMR analysis may have 100-200 amino acid residues, which provide a 1H NMR spectrum of many hundreds of lines. Because the amino acid sequence can be assumed to have been determined previously by non-NMR methods, the first step in the NMR study is to assign each line in the spectrum to a specific moiety (NH, ot-CH, side chain CH3, etc.) of a specific amino acid residue. Without the 2D methods that we have discussed, it would be virtually impossible to make such assignments. For relatively small proteins ( 50—100 residues) it is often possible to use conventional homonuclear 2D methods, such as COSY and HOHAHA, to define some bonding paths and to supplement these results with NOE data for residues that are very close in space as a result of secondary structural elements such as a helices. However, for proteins of moderate size such techniques are insufficient, and special methods had to be developed and now constitute the standard method of making sequential assignments. 

The methodologies based on H- H COSY and NOESY experiments are the most sensitive, as they rely on detection of high-sensitivity, abundant nuclei. COSY provides correlations among atoms that are /-coupled, while NOESY provides those among protons based on their separation in space. The drawback of using these homonuclear 2D methods is their complexity, as two coupled protons can produce many peaks in the 2D spectrum. 

Prior to the advent of 2D methods, selective spin decoupling was used extensively in both proton NMR and in heteronuclear (especially 13C) NMR to ascertain which sets of nuclei contribute to observed spin coupling. Such information is critical to assignment of resonances and to the elucidation of the structure of an unknown molecule. 2D methods now largely supply this information much more efficiently, by correlations that depend on the existence of spin coupling. The homonuclear version of one such experiment is called COSY (correlation spectroscopy), and the heteronuclear version is known by several acronyms, most commonly HETCOR (lieferonuclear correlation). 

TOCSY (Total Correlation Spectroscopy) is another important homonuclear 2D correlation experiment where correlations arise due to the presence of homonuclear scalar coupling.In the standard COSY experiment, crosspeaks appear for spins in which the scalar coupling occurs over typically two to four bonds. In the TOCSY experiment crosspeaks can appear for spins separated by many more bonds as long as they are part of a contiguous network of coupled spins. The correlations are effected by the application of a series of low-power rf pulses termed the spin-lock. The duration of the spin-lock period determines the extent to which the correlations are propagated through the spin system. The TOCSY experiment is a useful complement to the COSY methods for the elucidation of complex structures. 

Traditionally, homonuclear 2D double quantum filtered correlation spectroscopy (DQF-COSY) and total correlated spectroscopy (TOCSY) spectra are valuable in the identification of resonances of individual monosaccharide units. In the presence of small couplings, through space connectivities detected by NOESY/ROESY (nuclear Overhauser effect spectroscopy/ rotational nuclear Overhauser effect spectroscopy) experiments are also useful in completing the resonance assignment. When the H NMR spectra of complex oligosaccharides are too crowded to fully elucidate the structure by homonuclear correlation methods, it is efficient to use 2D heteronuclear correlation methods, such as heteronuclear single quantum correlation... 

The enormous simplification of crowded spectra in this manner makes this a very powerful technique. However, a point to bear in mind when considering applying these methods is that crosspeaks in the C-edited experiment originate only from C satellites because of the initial HMQC/HSQC step, and the experiment is therefore of significantly lower sensitivity than homonuclear 2D TOCSY, in which all protons may participate. The experiment will generally find use after initial stages of investigation of complex spectra... 

In Section 9.3.1, the problem of assigning the many resolved resonances in a ID MAS spectrum was mentioned, and ID spectral editing methods were introduced. In this section, we describe homonuclear 2D correlation experi-... 

It is thus not possible to obtain a phase-sensitive 45 projection of a normal homonuclear 2D J spectrum, and instead it is usual to project the absolute value or the power spectrum. This gives rise to distorted intensities and peak positions, but nevertheless is an extremely useful aid to assignment. The subject of phase-sensitive proton spectra without multiplet splittings is not however entirely closed, despite the clear message of the projection-cross-section theorem, and will be returned to later. In the interim, some of the many data acquisition and handling methods discussed above will be illustrated with some experimental spectra. 

Homonuclear 2D correlation (COSY) spectra were acquired according to the method of Nagayama et al, and Homonuclear Hartmann-Hahn experiment (HOHAHA) according to Bax. The 2D data were zero filled and weighted prior to Fourier transformation as appropriate. A sine-bell filtering function was used in both dimensions. 

In solution, dipole-dipole interactions constitute a relaxation mechanism, and the dipolar relaxation which is the basis for the well-known nuclear Overhauser effect (NOE), mostly used in the homonuclear H, H case. The 2D HOESY method between H and Li has been used to obtain structural information of many organolithium systems in solution and this field was reviewed in 1995. Li is commonly used as the relaxation is dominated by the dipole-dipole mechanism and the relaxation time is relatively long. Knowledge of the proximity of the lithium cation relative to protons in the substrate is used to derive information about the structure and aggregation of organolithium systems in solution. In a few cases quantitative investigations have been made °. An average error of the lithium position of ca 0.2 A was reported. 

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