Isotope Edited Experiments

Schubert et al. proposed two triple-resonance experiments for selection of amide resonances of residues sequential to proline in HSQC spectra. 

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Isotope edited experiments 3D HCCH-COSY 3D HCCH-TOCSY ID spin-state selective HMQC and HSQC-based 3D MUSIC CBCANH 3D MUSIC CBCA(CO)NH 2D Pro-HSQC... 

In the isotope edited/ filtered spectra of a protein-ligand complex, the species actually observed is generally the complex itself. This is an important difference from transferred NOE or saturation difference techniques, where the existence of an equilibrium between free and bound species - and a certain rate of exchange between them - is essential (Chapts. 13 and 16). The general conditions for isotope filtering/editing are therefore identical to those required for standard protein NMR sample concentrations are usually limited by availability and solubility of the components to the order of 1 mM. Considerably lower concentrations will reduce the sensitivity of the experiments to unacceptable levels,... 

When combining isotope filtering/editing with coherence transfer steps to multidimensional experiments, then further size restrictions apply. For example, isotope edited / filtered H TOCSY or COSY experiments are generally limited to systems of <10 kDa, because of their sensitivity to T2 relaxation. In larger systems, heteronuclear correlation spectroscopy can be used for the correspondingly labeled component, while structural information about both the labeled and unlabeled moiety can be extracted from isotope edi-ted/filtered NOESY spectra, respectively. 

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]. 

With another immunophilin, FK binding protein (FKBP), experiments were performed using isotope editing of the [U-13C]-labeled inhibitor ascomycin (bound to unlabeled FKBP) [34], as well as by isotope filtering with unlabeled ascomycin derivatives (bound to labeled FKBP) [35],... 

Sarkar et al. [32] report the use of MAS for single-bead NMR . A simple molecule on Wang resin 1 (Fig. 3) was fully 13C-labeled. A 13C-filtered H NMR spectrum was then recorded for a single bead. The isotope editing eliminates the interferences from solvents and contaminants, allowing the signal to be observed. The only information obtained is the resonance for the methyl protons directly attached to the labeled carbon, making this experiment mainly a novelty. 

Another possibility is to use half-edited/ half-filtered 2D experiments to detect NOEs that specifically involve interactions between protons attached to or and those that are not. This approach is used, for example, to detect intermolecular NOEs between a labeled protein and an unlabeled ligand. Examples of isotope editing/filtering are given in section 3.2.4. 

A between the free radical and Isotope-edited EPR experiments employing or H-... 

The formation of ethanolamine by the decarboxylation of serine has been established with certainty by isotopic labeling experiments. The experimental evidence will not be presented here and the reader is referred to the first edition of this work for references. 

Generally the sensitivity of the isotope filtered/edited version of an NMR experiment will be comparable to that of the corresponding standard experiment. However, some reduction in signal intensity will occur caused by the additional pulses (due to pulse imperfections and Bi inhomogeneity) and delays (due to relaxation) of the filter elements. These losses can become significant in the case of large molecular weight complexes. 

All the spectroscopic approaches applied for structural characterization of mixtures derive from methods originally developed for screening libraries for their biological activities. They include diffusion-ordered spectroscopy [15-18], relaxation-edited spectroscopy [19], isotope-filtered affinity NMR [20] and SAR-by-NMR [21]. These applications will be discussed in the last part of this chapter. As usually most of the components show very similar molecular weight, their spectroscopic parameters, such as relaxation rates or selfdiffusion coefficients, are not very different and application of these methodologies for chemical characterization is not straightforward. An exception is diffusion-edited spectroscopy, which can be a feasible way to analyze the structure of compounds within a mixture without the need of prior separation. This was the case for the analysis of a mixture of five esters (propyl acetate, butyl acetate, ethyl butyrate, isopropyl butyrate and butyl levulinate) [18]. By the combined use of diffusion-edited NMR and 2-D NMR methods such as Total Correlation Spectroscopy (TOCSY), it was possible to elucidate the structure of the components of this mixture. This strategy was called diffusion encoded spectroscopy DECODES. Another example of combination between diffusion-edited spectroscopy and traditional 2-D NMR experiment is the DOSY-NOESY experiment [22]. The use of these experiments have proven to be useful in the identification of compounds from small split and mix synthetic pools. 

Uniform and labeling provides improved signal dispersion and editing by multidimensional experiments or filtering by isotope or spin-state as well as various routes for coherence transfer and ways to measure scalar and dipolar couplings. Solvent suppression in heteronudear correlation experiments is also much easier than in homonuclear correlation experiments. 

Isotopic Effects. Recent Developments in Theory and Experiment", edited by E. Buncel and C.C. Lee (Elsevier, Amsterdam, 1984), p. 161. 

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