NOESY-HSQC experiments

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.

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 ... 

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... 

Clearly the homonuclear and the heteronuclear experiments could be combined in the reverse order i.e. HSQC-NOESY and HSQC-TOCSY. The main advantage in these schemes relates to N-edited experiments in which the narrower amide proton spectral width is sampled during f and the full proton spectral width is collected during t. On the other hand water suppression is more effective when HSQC follows NOESY. Also the sensitivity enhancement can be incorporated into the NOESY-HSQC experiment. Eor the TOCSY-HSQC or HSQC-TOCSY it does not matter because both the TOCSY and HSQC sequences can be implemented with the sensitivity enhancement. It should be mentioned that the TOCSY type of transfer is more effective between C nuclei than between protons and therefore the HCCH-TOCSY experiment is preferred when a doubly labeled sample is available. 

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... 

No general studies have been carried out for these compounds, but there are several reports in which the stereochemistry of the final product has been elucidated by NOESY, correlation spectroscopy (COSY), or heteronuclear single quantum correlation (HSQC) experiments. For example, intensive NOESY experiments were used to establish the exact nature of each of the three cycloadducts 151a-c generated by the cycloaddition of a substituted nitrone to dimethyl (Z)-diethylenedicarboxylate <2000EJ03633>. 

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... 

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

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. 

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. 

Figure 12.12a gives a good illustration of the need for going to a third dimension to facilitate the interpretation of a crowded 2D spectrum. The NOESY spectrum of a uniformly 15N-enriched protein, staphylococcal nuclease, has so many cross peaks that interpretation is virtually impossible. However, it is possible to use, 5N chemical shifts to edit this spectrum, as indicated in Fig. 12.121) and c in a three-dimensional experiment. With the 15N enrichment, NOESY can be combined with a heteronuclear correlation experiment, in this case HMQC, but HSQC could also be used. A 3D pulse sequence can be obtained from two separate 2D experiments by deleting the detection period of one experiment and the preparation period of the other to obtain two evolution periods (q and t2) and one detection period (f3). In principle, the two 2D components can be placed in either order. For the NOESY-HMQC experiment, either order works well, but in some instances coherence transfer proceeds more efficiendy with a particular arrangement of the component experiments. We look first at the NOESY-HMQC sequence, for which a pulse sequence is given in Fig. 12.13. The three types of spins are designated I and S (as usual), both of which are H in the current example, and T, which is 15N in this case.

To obtain information about the glycosidic linkage, 1H—1H NOESY/ROESY and/or long-range 1H—13C correlated spectra, heteronuclear multiple bond correlation (HMBC) or CT (constant time)-HMBC,12 are recorded. The combined 2D HSQC(HMQC)-NOESY(ROESY) experiments could also be helpful, but have limited applications due to their low sensitivity in samples with natural abundance of 13C. 

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). 

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). 

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... 

A new element for simultaneous indirect detection of C and signals in labelled proteins was proposed by Uhrin et The CT- CHs, VT- N-HSQC sequence combines constant-time carbon evolution with variable delay nitrogen evolution. This is achieved by the variation of the position of a 90° pulse creating transverse coherence within the C constant-time period. The maximum indirect acquisition time for both nuclei is determined by constant-time period set to l/ /(C,C) = 28.6 ms. The method is best suited for detection of CH3 signals due to their slower relaxation. The proposed element was incorporated into NOESY-based experiments resulting in 3D NOESY-CH3NH and 3D HSQC-NOESY-CH3NH sequences. The experiments... 

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... 

The structures of most benzophenones have been determined by 1D ( H, l3C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) NMR experiments. The majority of benzophenone NMR spectra have been recorded in CDCI3 and CD3OD. Benzene-c4, mixtures of benzene-<4 and CDCI3 [75], or pyridine-ds have also been used [88,93]. The aforementioned solvents were used to resolve overlapping signals of studied compounds. Deuterated TFA (0.1%) has also been used to increase the rate of keto-enol interconversion in benzophenones. We now turn our attention to the structural elucidation of xanthochymol (138). 

The 3D HMQC-NOESY-HMQC (HSQC) experiment together with 4D -edited... 

NOESY-HMQC (HSQC) experiment (b) 4D i C/i W-edited and i C/ C-edited NOESY experiments. Colour notation is identical with Fig. 5.17. 

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