Two-dimensional NMR methods

Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of RadiarUhus paumotensis using this approach. We find that the dominant structure is )9-sheet, with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species. 

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... 

G.E. Martin and A.S. Zektzer, Two-dimensional NMR Methods for Establishing Molecular Connectivity, VCH Publishers, New York, NY (1988). 

March, J. (1992). Advanced Organic Chemistry. Fourth Edn. John Wiley Sons New York, Chichester, Brisbane, Toronto and Singapore Martin, G. H. and Zektzer, A. S. (1988). Two-dimensional NMR Methods for Establishing Molecular Connectivity A Chemist s Guide to Experiment Selection, Performance, and Interpretation. VCH Publishers Inc. Deerfield Beach. 

Further possibilities are offered by various two-dimensional NMR methods. For example, heteronuclear solid-state correlation spectroscopy (27) is capable of correlating the spectra of abundant and dilute spins in solids, simplifying spectral assignment and permitting determination of shielding tensors. Futhermore, spin diffusion among abundant spins can be directly observed by this method. 

Note that there are two conformers, one with the two H8 atoms on the same side of the coordination plane (syn isomer) and the other with one H8 atom on each side of the coordination plane (anti isomer). Refine and save both using the MOMEC97 force field. Section 17.14 describes how to enforce planarity of a coordination compound. Two dimensional NMR methods can be used to determine which isomer dominates - as long as interconversion of the isomers is not rapid on the NMR time scale. The data used here are hypothetical and we have assumed that one isomer dominates to the exclusion of the other and that there is no interconversion, i. e., the observed NMR spectrum is that of an isomerically pure compound. 

An illustration of CP-MAS is given in Fig. 7.9. The sharp lines that are obtained permit CP-MAS to be used for structure elucidation of organic and inorganic compounds, in much the same way as liquid state spectra are used. As we see in Chapter 10, two-dimensional NMR methods are applicable to solids and form part of the analytical capability of CP-MAS and CRAMPS. One important application is in combinatorial chemistry, where molecules are synthesized on resin beads. It is feasible to obtain good H and 13C spectra from a single bead. 

The conpling constant of the AA BB portion of bisphenol A-neopentyl glycoltereph-thalate copolyesters (20) was determined in the presence of Pr(fod)3". The compositional tetrads of a 3,3 -dimethylbisphenol A-phenolphthalein copolyterephthalate (21) were resolved in the presence of En(fod)3 . The meso and racemic dyads of a poly(/l-snbstitnted- S-propiolactone) were distinguishable in the NMR in the presence of Eu(dpm)3". The H and NMR spectra of ethylene-vinyl acetate copolymers were assigned on the basis of two-dimensional NMR methods and lanthanide-induced shift data in the presence of Eu(fod)3". ... 

Molecular conformations [71,72] and associations can be determined by CP-MAS- C-NMR and two-dimensional NMR methods [73], but the primary means of determining molecular conformations has been X-ray fiber diffraction analysis coupled with molecular modeling as practiced by Chandraskeran and co-workers. Examples are found in references [74,75,76,77,78,79,80,81,82,83,84]. The technique has also been used to study polysaccharide-polysaccharide interactions [85]. Recently, powder diffraction data has been used to determine a molecular conformation [86]. Modeling alone has been used to predict possible conformations [87]. 

Although solution-state NMR has revealed much regarding the chemical structural composition of humic substances and dissolved organic matter (5), the development of more sophisticated software, pulse sequences, probes in recent years have not been exploited much in the field of soil science. Most of the novel applications have strictly relied on conventional one-dimensional spectroscopy. Two-dimensional NMR methods have become the mainstay of the chemical research field, especially for the structural elucidation of complex soluble biopolymers. The complexity of humic substances along with other inherent physical properties such as limited solubility and the presence of paramagnetics... 

Figure 6-33 The 2D INADEQUATE spectrum of menthol, with the H-decoupled spectrum. (Reproduced from G. E. Martin and A. S. Zehtzer, Two-Dimensional NMR Methods for Establishing Molecular Connectivities,...

Two-dimensional NMR methods that yield a two-dimensional frequency spectrum have not yet been attempted successfully to study metal interactions with HS, although instances of successful applications of these approaches can be found in the study of metalloproteins (Kingery et al., 2001). Mononuclear ( H) two-dimensional NMR experiments, such as total correlation spectroscopy (TOCSY), can show H- H coupling throughout the complete spin system, and exchange protons can provide information on sites to which metal attach. For example, N-containing units in HS that bind the metal can be identified since the amido protons from these structures will exchange and disappear from the spectrum. 

J. K. M. Sandersand B. K. Hunter, Modern NMR Spectroscopy. A Guide for Chemists, Oxford University Press, Oxford, 1987. The authors give a clear introduction to experimental techniques which use the most important one-and two-dimensional NMR methods. 

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