Heteronuclear NMR experiments

CCR is easily measured by heteronuclear NMR experiments of isotopically labeled molecules. The information extracted from these experiments will significantly improve the resolution of NMR structures, especially bound conformations of weakly bound ligands, since /-couplings cannot be used in this case. The reason is the fact that the nonbound conformation significantly contributes to the averaged values of the coupling constant. 

NMR experiments. Heteronuclear NMR experiments are represented by H C HSQC (Figure 2.1) and heteronuclear multiple bond correlation HMBC) (Figure 2.2)... 

Heteronuclear NMR experiments are represented by the HSQC and HMBC spectra in Fig. FI. 4.6 and Fig. FI. 4.7. The 13C NMR spectrum (or 13C-projection) is displayed along one axis, and the H NMR spectrum (or -projection) along the other. The H-13C correlations are shown as cross-peaks in the spectrum. Contrary to homonuclear NMR experiments, there exist no diagonal and only one cross-peak for each correlation. 

Figure 15.1. (A) COSY, (B) TOCSY, (C) 1H-1T HSQC or HMQC, (D) dl- Y HMBC, for 4-oxopentanal. For clarity, only key assignments have been given as an example. Note that the double-ended arrows indicate how to interpret the spectra. In the case of COSY and TOCSY the information is represented as cross-peaks that are symmetrically oriented with respect to the central diagonal. In the single-bond correlation (HSQC/HMQC) a cross-peak represents in one dimension the carbon chemical shift and in the other dimension the proton chemical shift. Note there is no diagonal in heteronuclear NMR experiments. In the HMBC, lines are drawn vertically to connect the cross-peaks. In HMBC 2-4 bonds, H-13C correlations are often observed. Note that the 4-bond correlation is less common in NMR but has been included here as an example, and 1-bond correlation is commonly filtered from the HMBC experiment to improve detection limits for the weaker 2-4 bond correlations.

Cavanagh, J., Fairbrother, W., Palmer, A. and Skelton, N. Heteronuclear NMR Experiments from Protein NMR Spectroscopy Principles and Practice, Academic Press, San Diego, 1996. 

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). 

Figure 12 Segment of a polypeptide chain showing the magnitude of the scalar J-couplings used in heteronuclear NMR experiments.

Heteronuclear NMR experiments, which can be performed with the standard equipment of practically all modern spectrometers, require in general three separate radiofrequency (RF) channels for both spectrometer and probe head. The first two channels deliver the H (for decoupling) and "X frequencies to the sample, and the third channel is commonly tuned to D and operates the field frequency lock. In most standard probe heads, these three frequencies are delivered via two concentric coils. The inner coil with the higher Q factor is generally used for detection, the outer one only for the application of pulses and decouphng. TWo general designs are in use in normal or forward probe heads, which are optimized for direct detection of X nuclei, the inner coil is a tuneable X coil and the outer coil is normally double tuned to and the lock frequency, while in inverse probe heads which are optimized for indirect detection of "X resonances via H, this order is reversed. 

Multidimensional heteronuclear NMR experiments for determining the structure of isotopically labelled RNA were discussed in detail by Pardi. ... 

Multinuclear NMR spectroscopy experiments of various 1,3-dialkylimidazolium ILs dissolved in organic solvents have also pointed to the formation of floating aggregates through hydrogen bonds [81-84]. In particular, it has been demonstrated by heteronuclear NMR experiments on [C4CjIm]BF4 that contact ion pairs exist in the presence of small amounts of water and even in dimethyl sulfoxide (DMSO) solution [85]. 

Bermel W, Bertini 1, Felli 1C et al (2010) Exclusively heteronuclear NMR experiments to obtain structural and dynamic information on proteins. ChemPhysChem 11 689-695 Kostic M, Pochapsky SS, Pochapsky TC (2002) Rapid recycle C , N and C, C heteronuclear and homonuclear multiple quantum coherence detection for resonance... 

Crews and coworkers [68] isolated and characterized an imidazolyl-P-carboline, 64, from a marine sponge, Hyrtios reticulatus, isolated off the coast of Papua New Guinea. The structure was characterized through the usual ensemble of 2D homo- and heteronuclear NMR experiments supplemented by long-range HMBC data, the optimization of which... 

D. Jeannerat, High resolution in heteronuclear NMR experiments by opti-... 

Thus for the NOE is 2, while for it is -5. The magnetization could thus be enhanced by 200 % by adding the NOE to carbon equilibrium magnetization. For i N, the enhancement was -4, which is like a 400% increase in the signal. These enhancements have been and are routinely utilized in heteronuclear NMR experiments. 

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