Two dimensional NMR spectra

Figure 18.20 The two-dimensional NMR spectrum shown in Figure 18.17 was used to derive a number of distance constraints for different hydrogen atoms along the polypeptide chain of the C-terminal domain of a cellulase. The diagram shows 10 superimposed structures that all satisfy the distance constraints equally well. These structures are all quite similar since a large number of constraints were experimentally obtained. (Courtesy of P. Kraulis, Uppsala, from data published in P. Kraulis et ah. Biochemistry 28 7241-7257, 1989, by copyright permission of the American Chemical Society.)...

Two-dimensional NMR spectroscopy may be defined as a spectral method in which the data are collected in two different time domains acquisition of the FID tz), and a successively incremented delay (tj). The resulting FID (data matrix) is accordingly subjected to two successive sets of Fourier transformations to furnish a two-dimensional NMR spectrum in the two frequency axes. The time sequence of a typical 2D NMR experiment is given in Fig. 3.1. The major difference between one- and two-dimensional NMR methods is therefore the insertion of an evolution time, t, that is systematically incremented within a sequence of pulse cycles. Many experiments are generally performed with variable /], which is incremented by a constant Atj. The resulting signals (FIDs) from this experiment depend... 

Figure 3.3 The mechanics of obtaining a two-dimensional NMR spectrum. As the l value is varied, the magnetization vectors are caught during detection at their various positions on the x /-plane. The value of the detection time l-i is kept constant. The first set of Fourier transformations across is followed by transposition of the data, which aligns the peaks behind one another, and a second set of Fourier transformations across t then affords the 2D plot.

Figure 3.4 Schematic representation of the steps involved in obtaining a two-dimensional NMR spectrum. (A) Many FIDs are recorded with incremented values of the evolution time and stored. (B) Each of the FIDs is subjected to Fourier transformation to give a corresponding number of spectra. The data are transposed in such a manner that the spectra are arranged behind one another so that each peak is seen to undergo a sinusoidal modulation with A second series of Fourier transformations is carried out across these columns of peaks to produce the two-dimensional plot shown in (C).

Translating a two-dimensional NMR spectrum into a complete three-dimensional structure can be a laborious process. The NOE signals provide some information about the distances between individual atoms, but... 

In fact, two-dimensional solid-state NMR is far superior to one-dimensional techniques for studying structure and dynamics123. In particular, the two-dimensional exchange NMR spectrum124 of a static sample is identical with a two-time distribution function125. Thus a two-dimensional NMR spectrum, which is detected for a fixed mixing time, is an image of the state of the dynamic process under study at that time. 

Fig. 5.7. Structure analysis of a 56-amino-acid protease inhibitor peptide by 2D NMR spectroscopy. A. Onedimensional NMR spectrum of the peptide. B. A two-dimensional NMR spectrum of the same peptide. C. Set of possible structures obtained from 2D NMR spectra similar to the one shown in panel B. (Courtesy of Dr Sarah L.Heald, Miles Pharmaceutical Inc. For details see S.L.Heald et al. (1991) Biochemistry 30,10467 10477.)...

The large and fast computers associated with Fourier transform spectrometers allow for a series of precisely timed pulses and data accumulations to give a large data matrix that can be subjected to Fourier transformation in two dimensions to produce a two-dimensional nmr spectrum. 

Figure 27 provides an example of one two-dimensional NMR spectrum. It depicts a phase-sensitive two-dimensional ROESY spectrum where only negative levels are shown. It was recorded for a pure sample of standard material ginsenoside Re This figure was drawn from reference [30] and the readers are strongly encouraged to review the discussion relating to the interpretation of that spectrum as 2-D spectra are, at best, difficult to interpret despite their nice pictures appearance. 

The most important structural probe is the nuclear Overhauser effect (NOE), which provides valuable information on the structure of linear peptides. Briefly, the observation of a direct NOE between a pair of protons indicates the presence of a significant population of conformers in which the distance between these two proteins is relatively short. The overall pattern of connectivity therefore corresponds to a particular conformation. Constraints on both backbone and side chain dihedral angles are obtained from coupling contacts. An example of a two-dimensional NMR spectrum for a synthetic peptide related to residues 6-13 of human growth hormone is shown in Figure 8. 

We can readily locate the second doublet from the minor, ds isomer by examining the COSY two-dimensional NMR spectrum (see Chapter 8,... 

A two-dimensional NMR spectrum is often displayed in the form of a map. One axis, V2, contains the conventional chemical shifts. The other axis, Vi or t, may contain chemical shift or /-coupling information or both. The position of a signal on the map tells us the chemical shift of that signal and the effect on that signal due to the presence of other nuclei. 

Fig. 13.3B A representation of the two-dimensional NMR spectrum obtained by application of the COSY pulse sequence to an AX spin system.

In order to demonstrate the methodology for generating a two-dimensional NMR spectrum, the homonuclear, /-correlated experiment first proposed by Jeener will be described. This experiment has been named COSY for... 

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