NOESY elucidation

Generally, the most powerful method for stmctural elucidation of steroids is nuclear magnetic resonance (nmr) spectroscopy. There are several classical reviews on the one-dimensional (1-D) proton H-nmr spectroscopy of steroids (267). C-nmr, a technique used to observe individual carbons, is used for stmcture elucidation of steroids. In addition, C-nmr is used for biosynthesis experiments with C-enriched precursors (268). The availability of higher magnetic field instmments coupled with the arrival of 1-D and two-dimensional (2-D) techniques such as DEPT, COSY, NOESY, 2-D J-resolved, HOHAHA, etc, have provided powerful new tools for the stmctural elucidation of complex natural products including steroids (269). 

The NMR techniques discussed so far provide information about proton-proton interactions (e.g., COSY, NOESY, SECSY, 2D y-resolved), or they allow the correlation of protons with carbons or other hetero atoms (e.g., hetero COSY, COLOC, hetero /resolved). The resulting information is very useful for structure elucidation, but it does not reveal the carbon framework of the organic molecule directly. One interesting 2D NMR experiment, INADEQUATE (Incredible Natural Abundance Double Quantum Transfer Experiment), allows the entire carbon skeleton to be deduced directly via the measurement of C- C couplings. 

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>. 

The H and 13C NMR descriptions of a great number of perhydro-pyrrolo-oxazoles have appeared since the important contribution in the field by Meyers et al. They are mainly used for structural determination and to prove the stereochemistry of the substitution of these compounds. Some NOESY experiments were performed for the structural elucidation of diethyl (3R,5A,7aA)-5-methyl-3-phenylhexahydropyrrolo[2,l- ]-[l,3]oxazol-5-yl-phosphonate 181 <2004TL5175>. 

The structures of most of the compounds described have been established by FI and 13C NMR spectroscopy and in some cases the stereostructures of other compounds have been elucidated by NOESY experiments <1996TL4573, 1998J(P1)3577, 2001J(P1)2997. 

NOESY Homonuclear NOE (nuclear Overhauser) spectroscopy To elucidate structure of organic molecules To determine the spatial proximity of nuclei... 

Chemical shift correlated NMR experiments are the most valuable amongst the variety of high resolution NMR techniques designed to date. In the family of homonuclear techniques, four basic experiments are applied routinely to the structure elucidation of molecules of all sizes. The first two, COSY [1, 2] and TOCSY [3, 4], provide through bond connectivity information based on the coherent (J-couplings) transfer of polarization between spins. The other two, NOESY [5] and ROESY [6] reveal proximity of spins in space by making use of the incoherent polarization transfer (nuclear Overhauser effect, NOE). These two different polarization transfer mechanisms can be looked at as two complementary vehicles which allow us to move from one proton atom of a molecule to another proton atom this is the essence of a structure determination by the H NMR spectroscopy. 

In this chapter, the discussion will be focused on the ID TOCSY (TO-tal Correlation SpectroscopY) [2] experiment, which, together with ID NOESY, is probably the most frequently and routinely used selective ID experiment for elucidating the spin-spin coupling network, and obtaining homonuclear coupling constants. We will first review the development of this technique and the essential features of the pulse sequence. In the second section, we will discuss the practical aspects of this experiment, including the choice of the selective (shaped) pulse, the phase difference of the hard and soft pulses, and the use of the z-filter. The application of the ID TOCSY pulse sequence will be illustrated by examples in oligosaccharides, peptides and mixtures in Section 3. Finally, modifications and extensions of the basic ID TOCSY experiment and their applications will be reviewed briefly in Section 4. 

The structures of the compounds were elucidated by a combination of NMR techniques (lH-, 13C-, and 13C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. 

Nuclear magnetic resonance (NMR) has proved to be a very useful tool for structural elucidation of natural products. Recent progress in the development of two-dimensional 1H- and 13C-NMR techniques has contributed to the unambiguously assignment of proton and carbon chemical shifts, in particular in complex molecules. The more used techniques include direct correlations through homonuclear (COSY, TOCSY, ROESY, NOESY) [62-65] and heteronuclear (HMQC, HMBC) [66. 67] couplings. 

Figure 3-25 (A) Alpha-carbon plot of the structure of ribosomal protein L30 from E. coli as deduced by NMR spectroscopy and model building. (B) Combined COSY-NOESY diagram for ribosomal protein L30 used for elucidation of dm connectivities (see Fig. 3-27). The upper part of the diagram represents the fingerprint region of a COSY spectrum recorded for the protein dissolved in H20. The sequential assignments of the crosspeaks is indicated. The lower part of the diagram is part of a NOESY spectrum in H20. The dm "walks" are indicated by (->—) S11-A12 (—) H19 to L26 (-------)...

New techniques for data analysis and improvements in instrumentation have now made it possible to carry out stmctural and conformational studies of biopolymers including proteins, polysaccharides, and nucleic acids. NMR, which may be done on noncrystalline materials in solution, provides a technique complementary to X-ray diffraction, which requires crystals for analysis. One-dimensional NMR, as described to this point, can offer structural data for smaller molecules. But proteins and other biopolymers with large numbers of protons will yield a very crowded spectrum with many overlapping lines. In multidimensional NMR (2-D, 3-D, 4-D), peaks are spread out through two or more axes to improve resolution. The techniques of correlation spectroscopy (COSY), nuclear Overhausser effect spectroscopy (NOESY), and transverse relaxation-optimized spectroscopy (TROSY) depend on the observation that nonequivalent protons interact with each other. By using multiple-pulse techniques, it is possible to perturb one nucleus and observe the effect on the spin states of other nuclei. The availability of powerful computers and Fourier transform (FT) calculations makes it possible to elucidate structures of proteins up to 40,000 daltons in molecular mass and there is future promise for studies on proteins over 100,000... 

H and 13C NMR techniques have widely been used to determine the configuration of new dioxepins and dithiepins and to elucidate the constitution and conformation of new naturally occurring substances. For example, the configuration of oximes 14 was determined by H and 13C correlated spectra, correlation spectroscopy (COSY), nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear correlation (HETCOR) spectroscopy, and hetero-nuclear multiple bond correlation (E1MBC) spectroscopy <1998CCA557>. 

Notably, two isomeric products can be generated. The usual infrared (IR) and mass spectra as well as H and 13C NMR chemical shifts could not define which isomer was formed. The authors used different NMR techniques, such as 2-D heteronuclear multiple bond correlation (HMBC) experiments and phase-sensitive nuclear overhauser enhancement spectroscopy (NOESY) measurements to elucidate the product s structure. 

Complete NMR spectral assignments have been made for the trans,cis and transjrans spiroepoxides derived by epoxidation of (Z)-3-arylidenethioflavanones, their 1-oxides and 1,1-dioxides (Table 7). As a consequence of the similarity of the chemical shifts of the signals in the 13C NMR spectra of the isomer pairs, coupling constants data for 37n-2,c-8a (37hc 7.0-7.6 Hz sulfide Vhc 6.2-6.5 Hz sulfoxide Vhc 2.0-3.4 Hz sulfone) and NOESY experiments were used extensively together with ab initio MO calculations to elucidate the conformation of the various isomers <2001MRC251, 2003MRC193>. 

Although structural elucidation of lignans is not a difficult task, the similarities between the structures can create problems. In particular, the determination of stereochemistry at the chiral center requires NOE/ NOESY NMR experiments and/or X-ray analyses. The enantiomeric excesses of the known lignans (+)-lariciresinol, (-)-secoisolariciresinol and (+)-taxiresinol, isolated from Japanese yew T. cuspidata roots, were determined by chiral high-performance liquid chromatographic analyses [78] except for (+)-pinoresinol (77% enantiomeric excess), they were found to be optically pure by Kawamura et al. In an earlier study, the presence of taxiresinol in Taxus species was reported by Mujumdar et al. [69] after they had isolated it from the heartwood of T. baccata, although they did not study its stereochemistry. 

All the spectroscopic approaches applied for structural characterization of mixtures derive from methods originally developed for screening libraries for their biological activities. They include diffusion-ordered spectroscopy [15-18], relaxation-edited spectroscopy [19], isotope-filtered affinity NMR [20] and SAR-by-NMR [21]. These applications will be discussed in the last part of this chapter. As usually most of the components show very similar molecular weight, their spectroscopic parameters, such as relaxation rates or selfdiffusion coefficients, are not very different and application of these methodologies for chemical characterization is not straightforward. An exception is diffusion-edited spectroscopy, which can be a feasible way to analyze the structure of compounds within a mixture without the need of prior separation. This was the case for the analysis of a mixture of five esters (propyl acetate, butyl acetate, ethyl butyrate, isopropyl butyrate and butyl levulinate) [18]. By the combined use of diffusion-edited NMR and 2-D NMR methods such as Total Correlation Spectroscopy (TOCSY), it was possible to elucidate the structure of the components of this mixture. This strategy was called diffusion encoded spectroscopy DECODES. Another example of combination between diffusion-edited spectroscopy and traditional 2-D NMR experiment is the DOSY-NOESY experiment [22]. The use of these experiments have proven to be useful in the identification of compounds from small split and mix synthetic pools. 

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. 

A 2002 review by Reynolds and Enriquez describes the most effective pulse sequences for natural product structure elucidation.86 For natural product chemists, the review recommends HSQC over HMQC, T-ROESY (transverse rotating-frame Overhauser enhancement) in place of NOESY (nuclear Over-hauser enhancement spectroscopy) and CIGAR (constant time inverse-detected gradient accordion rescaled) or constant time HMBC over HMBC. HSQC spectra provide better line shapes than HMQC spectra, but are more demanding on spectrometer hardware. The T-ROESY or transverse ROESY provides better signal to noise for most small molecules compared with a NOESY and limits scalar coupling artefacts. In small-molecule NMR at natural abundance, the 2D HMBC or variants experiment stands out as one of the key NMR experiments for structure elucidation. HMBC spectra provide correlations over multiple bonds and, while this is desirable, it poses the problem of distinguishing between two- and three-bond correlations. 

We have seen that NOESY provides information on internuclear (principally interproton) distances. For many organic molecules (as distinguished from macromolecules such as proteins and nucleic acids) structure elucidation often involves only the establishment of the structural formula and bonding scheme. However, where ambiguities in configuration or preferred conformation remain to be settled, NOESY is often crucial for establishing stereochemistry. 

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