Structure determination of small molecules

This section highlights the typical applications of 13C- and 1H-detected INADEQUATE and details a selection of examples from different areas of chemistry. These include primary structure determination of small molecules, the use of partially 13C-enriched compounds in biosynthetic studies, the recent structural analysis of fullerenes and the latest development in NMR hardware that promises to advance the use of high-resolution INADEQUATE experiments for polymers. 

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. 

Several structures have been corrected and elucidated with the aid of 13C-detected INADEQUATE. For example, the aH and 13C assignment of frans-3,4, 5-trihydroxystilbene was corrected.53 The structure of a dimeric mero-terpenoid, tridentorubin, was also investigated by 13C-detected INADEQUATE,54 while the structure of A-74528, an inhibitor for 20,50-phosphodiesterase isolated from Streptomyces sp., was elucidated on the basis of several crucial carbon-carbon connectivities that could not be determined from the analysis of its HMBC spectrum.55 Additional examples of the use of 13C-detected INADEQUATE to aid structure determination of small molecules can be found in the literature.25 56-61 

The HMBC spectrum of 4 contains five cross peaks showing the correlations of H12 with carbons C2, C3, CIO, Cll and C14. Correlation of H12 with carbon at 81 ppm seen in the 1,1-ADEQUATE spectrum overlaps with an HMBC cross peak. Thus, this is a 2/CH-mediated cross peak. Also, Cll is the only carbon linked to C12. Comparison of HMBC and 1,/(-ADEQUATE spectra identified three extra carbon atoms C4, C8 and C9 that are four, five and four bonds away, respectively, from proton H12. 

These additional correlations significantly reduced the number of possible structures generated by an NMR-based structure generator used in this work. When only 1H,1H-COSY and 1H,13C-HMBC spectra were analysed, 1310 possible solutions were produced. When the 1H, 15N-HMBC and 1,1-ADEQUATE spectra were included, this number was reduced to four. 

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NMR is still the premier method for structure determination of small molecules. This is the primary use of NMR spectroscopy in pharmaceutical development. The structures of both the drug candidate and its impurities will be subject to great scrutiny as the drug moves through development. The section on structure elucidation, which includes a discussion of the uses of multinuclear NMR, will deal with this area specifically. 

Experimental distances from NOEs/ROEs of small molecules are recommended not to be classified into regions of small, medium, and large as it is often done in the structure determination of large molecules. As opposed to macromolecules, the overall correlation time Tc can be considered constant in small molecules. Thus, it is possible to measure distances in the range between 2 and 5 A with an accuracy of about 10%. Often distances between protons are almost exclusively used for the structure determination. This leads to the fact that molecules with small numbers of hydrogen atoms are more difficult to determine. 

Multidimensional and heteronuclear NMR techniques have revolutionised the use of NMR spectroscopy for the structure determination of organic molecules from small to complex. Multidimensional NMR also allows observation of forbidden multiple-quantum transitions and probing of slow dynamic processes, such as chemical exchange, cross-relaxation, transient Over-hauser effects, and spin-diffusion in solids. 

The use of lanthanide probes for structure determination in small molecules has been extensively emphasized. In practice, bond lengths and angles are taken from crystallographic data, and the rotation angles about single bonds are determined from the nmr studies. The results of small molecule studies have been reviewed elsewhere.7,8... 

Because of all of the above pitfalls, NOE is probably the most misinterpreted experiment in organic chemistry. In my experience, /-coupling measurements, both homonuclear and heteronuclear, give far more reliable information than NOE measurements in the determination of small-molecule stereochemistry. To use NOE measurements for stereochemical determinations, it is always best to do the NOESY experiment on both isomers and compare the crosspeak intensities (relative to the diagonal peak intensities) and measure distances on both isomers using an energy-minimized computer model of the structures. If the differences in distance and NOE intensity are small between the two isomers, the experiment cannot be conclusive. 

NMR spectroscopy has so far been suited for non-invasive investigation of biochemical structures, fluxes through pathways, distribution of marker-nuclei among various cellular components and enzymatic mechanisms rather than for quantitative determination of small molecules. Biochemical applications have involved NMR spectroscopy mainly for structural determination of complex molecules, e.g. [27,180], as well as inside the cells, i.e. in vivo [184,189]. In biotechnology, the potential of determining intracellular components without cell disruption is increasingly used for in vivo studies of metabolism, e.g. [15,55,88, 121,146,197,250-252,271,335], and effectors [419]. 

Makinen, M. W., Mustafi, D., and Kasa, S. (1998) ENDOR of spin labels for structure determination from small molecules to enzyme reaction intermediate, in Berliner, L. (eds.), Biological Magnetic Resonance 14, Spin Labeling Next Millennium, Plenum Press, N. Y. ppl81-249. 

Electron-diffraction studies have produced many precise structure determinations of small and medium-sized molecules, and have solved many conformational problems (Bartell, 1985). Internuclear distances can be calculated to within a few hundredths, or even thousandths, of an Angstrom unit. However, rotamer proportions are rarely determined to better than 5%, and barrier heights can often only be estimated, and are subject to uncertainties of 25% or more. 

In addition to being an alternative to X-ray diffraction for the structure determination of small proteins difficult to crystallize, NMR also offers many possibilities to study intermolecular interactions and reactions under conditions that are not suited to ciystallization. With the help of NMR spectroscopy, information can also be gained on the dynamic behavior of the systems under stucfy. Tliese aspects are important in leading us from the often merely static definition of "structure" towards a much more realistic view of biologically active molecules and their interactions. 

Among the spectroscopic methods, MS is perhaps the most useful for the identification of alkanes and cycloalkanes. A simple computer search of reference spectra of thousands of compounds greatly enhances the utility of the method. The NMR method for single compounds is very helpful in structure determination for small molecules. However, the larger the saturated hydrocarbon, the less absolute information can be obtained by this spectroscopical method. Both two-dimensional NMR and NMR can furnish decisive information as to the structure of cycloalkanes. 

ENDOR of Spin Labels for Structure Determination From Small Molecules to Enzyme Reaction Intermediates... 

For macromolecules such as proteins, the numbers of atoms that compose molecules are huge, therefore the crystal cells contain large numbers of atoms. It is not possible to apply the methods for small molecules, such as the direct method or Patterson map searching, in the structure determinations of proteins. The methods for retrieving the phases of protein crystal diffractions are molecular replacement, isomorphous replacement and anomalous scattering. In recent years, the direct method, which has been widely and successfully used in the determination of small-molecule structures, has also been applied in protein crystallography. 

Structure determination of soluble proteins of moderate size (up to 30 kDa) by multinuclear multidimensional NMR spectroscopy is now fairly standard with well-established protocols. In the chapter NMR with Multiple Receivers, an acquisition scheme employing multiple receivers is described which allows for faster structure determination in small molecules. Further, recently employed fast acquisition schemes such as Hadamard spectroscopy, projection-reconstruction techniques, and reduced dimensionality experiments are explained. 

Once the least-squares fits to Slater functions with orbital exponents 1.0 are available, fits to Slater functions with other orbital exponents can be obtained by simply multiplying the a s in the above three equations by It remains to be determined what Slater orbital exponents to use in electronic structure calculations. The two possibilities may be to use the best atom expo-nents( = 1.0 for H, for example) or to optimize exponents in each calculation. The best atom exponents might be a rather poor choice for molecular environments, and optimization of nonlinear exponents is not practical for large molecules, where the dimension of the space to be searched is very large. Acompromise is to use a set of standard exponents where the average values of exponents are optimized for a set of small molecules. The recommended STO-3G exponents are... 

Ciguatoxin. The toxin was isolated from moray eels and purified to crystals by Scheuer s group (1). Structural determination of the toxin by x-ray or NMR analyses was unsuccessful due to the unsuitability of the crystals and due to the extremely small amount of the sample. The toxin was presumed to have a molecular formula of C Hg NO from HRFAB-MS data (MH+, 1111.5570) and to have six hydroxyls, five methyls, and five double bonds in the molecule (2). The number of unsaturations (18 including the five double bonds) and the abundance of oxygen atoms in the molecule point to a polyether nature of the toxin. The toxin, or a closely related toxin if not identical, is believed to be the principal toxin in ciguatera. Ciguatoxin was separable on an alumina column into two interconvertible entities presumably differing only in polarity (J). 

Molecular sieves (zeolites) are artificially prepared aluminosilicates of alXali metals. The most common types for gas chromatography are molecular sieve 5A, a calcium aluminosilicate with an effective pore diameter of 0.5 nm, and molecular sieve 13X, a sodium aluminosilicate with an effective pore diameter of 1 nm. The molecular sieves have a tunnel-liXe pore structure with the pore size being dependent on the geometrical structure of the zeolite and the size of the cation. The pores are essentially microporous as the cross-sectional diameter of the channels is of similar dimensions to those of small molecules. This also contrilsutes to the enormous surface area of these materials. Two features primarily govern retention on molecular sieves. The size of the analyte idiich determines whether it can enter the porous... 

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