Three-dimensional NMR pulse sequences

More recently, three-dimensional (3D) pulse sequences with DOSY have been presented where a diffusion coordinate is added to the conventional 2D map. As in the conventional 2D spectra, these experiments reduce the probability of signal overlap by spreading the NMR frequency of the same species over a 2D plane, and distribute the diffusion coefficient. 

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. 

Basic NMR Experiments—A Practical Course by S. Braun et al,41 describes the operation of an NMR spectrometer and, as its title implies, gives guidance, with specific experimental parameters, for carrying out a variety of NMR procedures—from measuring the width of a 90° pulse to complex pulse sequences in two- and three-dimensional NMR. 

ID, one-dimensional 2D, two-dimensional 3D, three-dimensional AMP, adenosine monophosphate CNDO, complete neglect of differential overlap COSY, correlation spectroscopy CPMG, Carr-Purcell-Meiboom-Gill NMR pulse sequence CT, constant time dAMP, deoxyadenosine monophosphate DFT, density functional... 

Three dimensional NMR spectroscopy and improved pulse sequences... 

An interesting alternative to three dimensional NMR techniques is to suppress one of the evolution times while retaining the basic 3D pulse sequence. The spectral resolution is no longer increased, which is usually not a problem with smaller molecules, but the extra information is still available. The gs-HMQC-TOCSY experiment represents one such experiment. The combination of the HMQC method with the TOCSY sequence leads, in principle, to a 3D technique. However, if the tj evolution period of the TOCSY part is omitted, a 2D sequence is obtained which provides a 13C edited TOCSY spectrum. Starting from each HMQC cross-signal, one finds in the same row additional signals which are caused by a TOCSY transfer. When the structure elucidation is difficult, this experiment can fruitfully complement the HMBC experiment. 

NOESY NMR spectroscopy is a homonuclear two-dimensional experiment that identifies proton nuclei that are close to each other in space. If one has already identified proton resonances in one-dimensional NMR spectroscopy or by other methods, it is then possible to determine three dimensional structure through NOESY. For instance, it is possible to determine how large molecules such as proteins fold themselves in three-dimensional space using the NOESY technique. The solution structures thus determined can be compared with solid-state information on the same protein obtained from X-ray crystallographic studies. The pulse sequence for a simple NOESY experiment is shown in Figure 3.23 as adapted from Figure 8.12 of reference 19. 

The selection of the pulse sequence to be used out of the hundreds that have been published depends on the information desired. NMR spectroscopy cannot only be used to determine high-quality three-dimensional structures, but can provide information about the global fold, interactions with other molecules or just the identification of the secondary... 

So-called multidimensional NMR techniques can provide important information about macromolecular conformation. In these cases, the sequence of a protein is aheady known, and establishing covalent connectivity between atoms is not the goal. Rather, one seeks through-space information that can reveal the solution conformation of a protein or other macromolecule. Two-or three-dimensional techniques use pulses of radiation at different nuclear frequencies, and the response of the spin system is then recorded as a free-induction decay (FID). Techniques like COSY and NOESY allow one to deduce the structure of proteins with molecular weights less than 20,000-25,000. 

In 2D NMR spectroscopy, a two-dimensional data set is acquired as a function of two time variables tx and t2 as shown schematically in Figure 14.4 [1, 7]. Figure 14.4a shows the general case while the three pulse sequence of Figure 14.4b represents a typical example. 

The projection-reconstruction approach is a technique unrelated to covariance processing which can provide data typically inaccessible to the natural product chemist. For example, 13C-15N correlation spectra were obtained for vitamin B12 at natural abundance.104 Compared with a conventional three-dimensional 13C-15N correlation experiment, the projection-reconstruction method provides a sensitivity enhancement of two orders of magnitude. The final 13C-15N spectrum was reconstructed from data obtained from ll l5N and H- C correlations acquired using a time-shared evolution pulse sequence that allowed all the information to be obtained in one experiment.104 Martin and co-workers also demonstrated the ability to generate 13C-15N correlation spectra using unsymmetrical indirect covariance NMR with vinblastine as an example.105 In the latter case, 13C-15N correlation spectra were obtained from - C HSQC data and H-1sN HMBC data that were acquired separately. Both methods provide access to correlations that would be inaccessible for most natural products at natural abundance. 

The ability to manipulate spins in two-dimensional experiments and to transfer magnetization between spins has made it possible to use a sensitive nucleus (primarily H) to measure the spectral features of less sensitive nuclei, such as 13C and 15N. Several methods are commonly used, but each begins with a H pulse sequence, often resembling the one in INEPT (Section 9.7). As in INEPT, a combination of H and X pulses transfers polarization to the X spin system. In some instances further transfers are made to another spin system (Y), then back through X to H, where the signal is detected. Thus, the large polarization of the proton is used as the basis for the experiment, and the high sensitivity of H NMR is exploited for detection. Such indirect detection methods use two-, three-, and sometimes four-dimensional NMR. 

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