NOESY-HSQC

This problem cannot be solved by the usual NMR experiments (COSY, NOESY, HSQC, HMBC,...). It turns out that the selective HOESY experiment (Figure 9, bottom) applied to proton H5 provides an unambiguous... 

Further confirmation of the structure of the AHLs often follows from the determination of their proton and carbon-13 NMR spectra. A large number of AT-acyl, AT-(3-oxoacyl) and AT-(3-hydroxyacyl)-L-HSL derivatives have been prepared and their NMR data reported [14-16]. Also a detailed study by Lao et al. [49] on the complete assignments of the 13C NMR resonances of AT-acyl and N-(3-oxoacyl)-L-HSL derivatives has been published. These assignments were made by comparison with values for AT-butanoyl-L-HSL (C4-HSL), whose structure was comprehensively established by a combination of 1-D and 13C spectra and 2-D COSY,NOESY, HSQC and HMBC experiments. The assignments... 

In many cases, the analytical tasks are simply to detect and quantify a specific known analyte. Examples include the detection and quantification of commonly used buffer components (e.g., Tris, acetate, citrate, MES, propylene glycol, etc.). These simple tasks can readily be accomplished by using a standard one-dimensional NMR method. In other situations, the analytical tasks may involve identifying unknown compounds. This type of task usually requires homonuclear and heteronuclear two-dimensional NMR experiments, such as COSY, TOCSY, NOESY, HSQC, HMBC, etc. The identification of unknown molecules may also require additional information from other analytical methods, such as mass spectrometry, UV-Vis spectroscopy, and IR spectroscopy.14... 

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

S.6 Two- and Three-Dimensional HSQC-TOCSY, HSQC-NOESY, HSQC-ROESY, HMQC-NOESY, HMQC-ROESY... 

Furthermore, the allohimachalane (see Section 13.11.9.1) <1999T14623> as well as boletunones A and B, highly functionalized sesquiterpenes from the fruit body of the mushroom Boletus calopus <20040L823>, have been characterized by 2D-NMR (heteronuclear single quantum correlation (HSQC), HMBC, and 111-COSY). The structure of a drimen-ll,12-acetonide, isolated from Maya s herb, was deduced by means of 111 and 13C NMR, distortionless enhancement by polarization transfer (DEPT), COSY, NOESY, HSQC, and HMBC analyses <2005MRC339>. 

The elucidation of the primary structure of small organic molecules by tracing their carbon skeletons was, traditionally, the main focus of INADEQUATE experiments. It is not the first NMR experiment to be considered for such a task typically a standard set of NMR spectra, that is COSY, TOCSY, NOESY, HSQC and HMBC, are performed and analysed first. If ambiguities remain after inspection of standard spectra, the tracing of carbon-carbon connectivities is embarked on. Nevertheless, an example is presented below where carbon-carbon connectivities are included at an earlier stage in order to reduce the number of computer-generated structures compatible with the experimental data. 

In a later study, a high resolution structure of the complete ascomycin-FKBP complex was calculated by heteronuclear 3D and 4D NMR by Meadows et al. (120). Elniformly labeled [ N]FKBP and [ C, N]FKBP were prepared and incubated with unlabeled ascomycin to form the complexes. Three-dimensional NOESY-HSQC spectra, resolved according to shifts, were used to obtain the NH-NH NOEs within FKBP. CH-NH NOEs were de-rivedfrom a4D [ C, H, N/H]-NOESY spectrum of the doubly labeled material in HgO... 

Concatenation of the 3H—15N HSQC (or HMQC) sequence with a JH—JH NOESY gives rise to the 3D 15N-edited NOESY-HSQC (or 3D NOESY-HMQC) experiment.66-68 Here, two of the frequency dimensions represent the amide JH and 15N chemical shifts, while the third dimension provides information about the chemical shift of protons with which each amide proton is dipolar coupled (i.e., separated by <5.5 A). The spectrum is routinely viewed as narrow 2D (JH—JH) strips taken at the 15N chemical shift of each crosspeak in the JH—15N HSQC spectrum (see Figure 14). 

As exemplified in Figure 14, the increase in resolution compared to a simple 2D NOESY is dramatic, due in part to the lack of a straightforward correlation between 15N chemical shift and the secondary structure in which a residue is located (in contrast to the case of HN, Ha, and Ca chemical shifts). An analogous combination of TOCSY and HMQC/HSQC yields 3D TOCSY-IIMQC/I ISQC,6 7" where the third dimension as described above shows the chemical shifts of protons to which the amide protons would exhibit correlations in a conventional TOCSY (i.e., those protons in the same spin system). Thus, when satisfactory NOESY-HSQC and TOCSY-HSQC spectra are obtained, a semiclassical route to resonance assignment... 

Figure 14 Comparison of 2D NOESY and 3D 15N-edited NOESY-HSQC spectra of a 41-residue peptide toxin from the Australian funnel-web spider Hadronyche infensa. A strip from the 2D NOESY spectrum is shown on the far left and it illustrates overlapping NOE correlations from three different amide protons (those of Trp13, Lys17, and Gly33). Fortunately, the 15N nuclei for these three amide groups have unique chemical shifts and hence they appear on different 2D planes in the 3D NOESY-HSQC experiment. Strips from these three planes are shown on the right, and they demonstrate that all of the NOE correlations are perfectly resolved in the 3D experiment.

Note that analogous experiments, such as the 13C-edited HSQC—NOESY,72 can be performed on 13C-labeled proteins. For labeled proteins, this latter experiment provides the largest number of conformational restraints for protein structure calculations (see Section 9.09.5.2). The 15N-edited NOESY-HSQC only provides distance information for protons that are close in space to amide protons (since magnetization originates and/or terminates... 

This techniqne can be easily extended into three or higher dimensions. For example, the three-dimensional NOESY-HSQC experiment records the following path of magnetization ... 

The resolntion that is gained by the additional freqnency dimension (N in this case) is illnstrated in Figure 3.6. The two-dimensional NOESY spectrnm of a 50 residne a-helical protein is shown in the left part of this fignre. In this experiment, the magnetization is transferred from proton H to proton Hy by dipolar coupling. The first freqnency dimension corresponds to the chemical shift of H and the second frequency dimension corresponds to the chemical shift of Hy. As with the NOESY-HSQC described previously, the intensity of the peak is related to the distance between H and Hy. Note the large number of unresolved overlapping peaks in the... 

Kazimierczuk and co-workers apphed their semi-automatic CLEAN procedure to suppress artifacts in a randomly sampled N-labeled NOESY-HSQC spectrum of ubiquitin [60]. It was demonstrated that the process does not systematically influence relative peak amplitudes, and is therefore applicable to NOESY spectra. Similar conclusions were later drawn by Stanek and Kozmihski [85], and by Werner-AUen and co-workers [84], who compared their reconstructions with conventionally sampled three-dimensional spectra of the same spectral resolution. The algorithm proposed by Kazimierczuk and co-workers was later also applied to higher-dimensional experiments [80]. 

Fig. 7 Comparison of alternative processing on a 3D N-NOESY-HSQC spectrum of human translation initiation factor eIF4e. (a) Uniformly sampled reference. The time domain data were acquired as 6,400 hyper-complex points sampled in the two indirect dimensions [128 (Hjndir) x 50 ( N)]. The spectra were measured on a 700-MHz spectrometer with sweep widths of 9765 Hz and 2270 Hz, respectively. The t ,ax hence were 0.013 and 0.022 s each for the indirect proton and nitrogen dimensions, respectively, representing nearly an optimal situation for the nitrogen dimension, but not for the indirect proton dimension. Data were transformed with the standard KFT procedure after cosine apodization and doubling the time domain by zero filling, (b) Reducing the number of complex points to 42 (32%) (bl) and 13 (10%) (b2) in the indirect proton dimension, cosine apodization, and zero filling result in low resolution spectra in the indirect...

Gutmanas A, Jarvoll P, Orekhov VY, Billeter M (2002) Three-way decomposition of a complete 3D 15N-NOESY-HSQC. J Biomol NMR 24 191-201... 

Muhandham DR et al (1993) A gradient 13C NOESY-HSQC experiment for recording NOESY spectra of 13C-labeled proteins dissolved in H20. J Magn Reson B 102(3) 317-321... 

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