Isotope Filtered Experiments

Isotope filtered experiments 2D WURST-based C-filtered NOESY... 

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,... 

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. 

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. 

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],... 

The idea of back transformation of a three-dimensional NMR experiment involving heteronuclear 3H/X/Y out-and-back coherence transfer can in principle be carried to the extreme by fixing the mixing time in both indirect domains. Even if one-dimensional experiments of this kind fall short of providing any information on heteronuclear chemical shifts, they may still serve to obtain isotope-filtered 3H NMR spectra. A potential application of this technique is the detection of appropriately labelled metabolites in metabolism studies, and a one dimensional variant of the double INEPT 111/X/Y sequence has in fact been applied to pharmacokinetics studies of doubly 13C, 15N labelled metabolites.46 Even if the pulse scheme relied exclusively on phase-cycling for coherence selection, a suppression of matrix signals by a factor of 104 proved feasible, and it is easily conceivable that the performance can still be improved by the application of pulsed field gradients. 

Lee, W., Revington, M. J., Arrowsmith, C. and Kay, L. E. (1994). A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts... 

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. 

After the Chernobyl accident the Academy of Sciences and KGB of Ukraine tested the household filters with fibroid sorbents and ten thousands filters were produced in the Institute of Nuclear Physics of Uzbekistan and given to Ukraine through "Isotope" Corp. (USSR). The experience of using the filters for purification of drinking water from radionuclides in Chernobyl region is described in the paper. 

The single filter elements (as shown in Figs. 17.4a-c) are often called X-halffilters since each of them acts only in one dimension of a 2D experiment, to be distinguished from Xfilters that select (or suppress) 1H-X pairs in both dimensions of a 2D experiment [17, 20, 21]. Of course, X-half filters can be employed twice in a 2D experiment, to yield isotope selection in both dimensions (see Sect. 17.3.3). 

Selecting the C-bound protons before performing a homonuclear two-dimensional experiment enables to measure small heteronuclear coupling constants [16]. Such an experiment with a sample of natural isotopic abundance was first published by Otting and Wuthrich in 1990, where the half-filter element with spin-lock purge pulse was used to select the C-bound protons in a small protein in aqueous solution [6]. Later applications illustrated the usefulness of the same half-filter element with smaller molecules [17, 18]. 

In some applications like newborn screening and filter paper blood spots, the internal standard that is labeled cannot be mixed with blood. It can only be present in the extraction solvents. Therefore, only the extracted metabolites can be quantitatively measured. I have denoted a term called pseudo-isotope dilution to account for the differences between traditional isotope dilution and the technique commonly used in newborn screening by MS/MS. A special analysis is capable using this technique, however, in terms of an extraction efficiency experiment. With isotope-labeled standards you can perform an experiment whereby a traditional isotope-dilution technique (internal standard added to liquid blood and spotted) is compared to pseudo-isotope dilution techniques (internal standard is added to the extraction matrix). The ratio of the results of these two analysis (pseudo/traditional) is the extraction efficiency. 

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. 

These filters are usually employed to suppress strong and undesirable resonances, e.g. parent signals of isotopically diluted spin systems. In a typical experiment the undesirable magnetization is inverted and allowed to relax until the equilibrium state is reached. At this point the inverted magnetization is not observable and the basic experiment may be started. In the simplest case a jump and return inversion pulse or binomial pulses can provide the necessary selective inversion. A simple example of application of a T relaxation filter is the J/-BIRD (BI linear Rotation Decoupling) HMQC... 

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