Three- and Four-Dimensional NMR

The use of an additional 90° pulse with timing and phase cycling as given here acts as a low-pass filter, because it eliminates the high frequency component from the larger j/. This technique is also used in other complex pulse sequences as a filter. 

The two reasons for extending NMR studies beyond three dimensions are the same as those for going from one to two dimensions (1) to spread out crowded resonances and (2) to correlate resonances. 3D and 4D experiments have been carried out almost exclusively to interpret the spectra of macromolecules, principally large proteins. We return in Chapter 13 to a discussion of these applications, but here we give a brief description of some types of 3D and 4D experiments. 

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For 20 years center stage has been occupied by two-dimensional (and now three-and four-dimensional) NMR techniques. 2D NMR and its offshoots offer two distinct advantages (1) relief from overcrowding of resonance lines, as the spectral information is spread out in a plane or a cube rather than along a single frequency dimension, and (2) opportunity to correlate pairs of resonances. In the latter respect 2D NMR has features in common with various double resonance methods, but as we shall see, 2D NMR is far more efficient and versatile. Hundreds of different 2D NMR techniques have been proposed in the literature, but most of these experiments can be considered as variations on a rather small number of basic approaches. Once we develop familiarity with the basic principles, it will be relatively easy to understand most variations of the standard 2D experiments. 

Three and four-dimensional NMRs are also available. They are more complicated than one- and two-dimensional NMR, but the underlying general principles are about the same. The three- and four-dimensional NMRs are useful for the determination of stmctmes of larger proteins and protein complexes. 

Builder, S.E. and W.S. Hancock Analytical and Process Chromatography in Pharmaceutical Protein Production, Chem. Eng. Progress, 42 (August 1988). Clore, G.M, and A M. Gronenbom Structures of Larger Proteins in Solution Three- and Four-Dimensional Heteronuclear NMR Spectroscopy." Science, 1390 (June 7. 1991). 

Clore, G.M. Gronenbom, A.M. (1991). Structures of larger proteins in solution Three- and four-dimensional heteronuclear NMR spectroscopy. Science 252, 1390-1399. 

G. M. Clore and A. M. Gronenborn, Prog. NMR Spectrosc., 23, 43 (1991). Applications of Three- and Four-Dimensional Heteronudear NMR Spectroscopy to Protein Structure Determination. 

GM Clore, AM Gronenborn. Determination of structures of larger proteins in solution by three- and four-dimensional heteronuclear magnetic resonance spectroscopy. In GM Clore, AM Gronenborn, eds. NMR of Proteins. Boca Raton, FL CRC Press, 1993, pp. 1-32. 

Overlapping resonances in 7.1 J NMR have limited protein-structure elucidation to fairly small proteins. However, three- and four-dimensional melliods have been developed that enable NMR spectroscopy to be further extended to larger and larger protein structures. A third dimension can be added, for example, to spread apart a H- H Iwo-dimensional spectrum on the basis of the chemical shift of another nucleus, such as N or "C. In most three-dimensional experiments, the most effective methods for large molecules are used. Thus, CXTfsY is not often employed, but experiments like N ()F.SY-TOrSY and I Of SY-HMOr are quite effective. In some cases, the three dimensions all represent different nuclei such as These... 

A wide range of two-dimensional (2 D) experiments are now available as routine tools, and more recently three- and four-dimensional methods have been developed. In general the experiments are used to extract information from complex spectra (e.g., about which nuclei are J coupled to each other) and to measure J couplings. In this article it is only possible to give a very brief introduction to two-dimensional methodology together with a few examples of its application in H and C NMR. Further information about this important field is available in [31], [32], [99], [103], [108], [109). 

Clore GM and Gronenborn AM (1991) Application of three- and four-dimensional heteronuclear NMR spectroscopy to protein structure determination. Progress in NMR Spectroscopy 26 43. 

In 1971, the idea of 2D NMR spectroscopy was proposed by Jeener and later implemented by Aue, Bartholdi and Ernst, who published their work in 1976.47 The first experiments, carried out mostly in the liquid phase, have unambiguously proved that 2D NMR spectra provide more information about a molecule than ID NMR spectroscopy and are especially useful in determining the structure of molecules that are too complicated to work with using ID NMR. With the progress in the methodology and software improvement, three-dimensional (3D) and four-dimensional (4D) NMR experiments were gradually introduced into the laboratory practice. Such strategy, the so-called multi-dimensional (or ND) NMR spectroscopy, has found a number of spectacular applications in the structure analysis of natural products. 

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. 

The spectrum of Figure 15.1 is an example of one-dimensional NMR (the intensities of the signals not being counted as a second dimension). More sophisticated studies, in two, three and four dimensions enable a more precise localization of the position of all of the atoms in the molecule studied. This chapter only describes one-dimensional NMR (1D-NMR), for samples in solution. 

Hehnus JJ, Nadaud PS, Hofer N et al (2008) Determination of methyl [sup 13]C-[sup 15]N dipolar couplings in peptides and proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy. J Chem Phys 128 052314... 

The major components are series of homologous trimers, tetramers, and pentamers of the three acids 44-46, along with smaller quantities of dimers, hexamers, and heptamers. Furthermore, the secretion contains several isomers of each oligomer, furnishing a combinatorial library of several hundred macro-cyclic polyamines [51, 52]. Using repeated preparative HPLC fractionation, the most abundant trimeric, tetrameric and pentameric earliest-eluting compounds were isolated. One and two-dimensional H NMR spectroscopic analyses showed that these molecules were the symmetric macrocyclic lactones 48, 49, and 50 (m, n, o, p, q=7) derived from three, four or five units, respectively, of acid 46. Moreover, using preparative HPLC and NMR methods, various amide isomers, such as 53,54, and 55 (Fig. 9) were also isolated and characterized [51,52]. 

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