Structure elucidation successes

The purpose of the final section of this chapter is to review the practical applications of molecular enumeration and to give the reader interested in any of these applications pointers to relevant codes and techniques. In particular, the numbers of isomers for a specific molecular series are given, popular structure elucidation codes are reviewed, computed-aided structure elucidation successes are surveyed, and the connections between structure enumeration and combinatorial library design are established. The field of molecular design with inverse quantitative structure activity relationship is also reviewed. We conclude the chapter outlining future research directions. 

In the case of being successful in calculating multiple conformations by using time- or ensemble-averaged MD restraints the solved molecular structures are presented as 3D models and can be deposited in an electronic structure database (17). Finally, it is recommended to provide an accurate explanation of the procedures used for the structure elucidation because the application of different methods (NMR, DG, MD, SA, Monte-Carlo calculations. X-ray crystallography) may result in varying conformational models which do not implicitly display the real state of a molecule. This aspect should be always kept in mind when dealing with structure determination methods. 

Capillary HPLC-MS has been reported as a confirmatory tool for the analysis of synthetic dyes [585], but has not been considered as a general means for structural information (degradant identification, structural elucidation or unequivocal confirmation) positive identification of minor components (trace component MW, degradation products and by-products, structural information, thermolabile components) or identification of degradation components (MW even at 0.01 % level, simultaneous mass and retention time data, more specific and much higher resolution than PDA). Successful application of LC-MS for additive verification purposes relies heavily and depends greatly on the quality of a MS library. Meanwhile, MB, DLI, CF-FAB, and TSP interfaces belong to history [440]. 

Limited protein stability often hampers successful structure elucidation by X-ray crystallography and/or NMR spectroscopy. Relaxation properties are usually improved at elevated temperatures, and multidimensional NMR experiments require sample lifetimes to extend over several days to weeks in order to acquire all the necessary data. In addition, the activity of contaminating proteases that are sometimes present in purified samples can be significant at the experimental temperatures. Therefore, the stability of a target protein can be a concern, in particular for expensive isotope-labeled proteins. 

SID over CID because losses of resolution due to high background pressure are avoided. SID has been successfully employed for structure elucidation of proto-nated peptides, [131] and a SID mode of operation has even been implemented with a quadrupole ion trap. [132] However, apart from the quadrupole ion trap SID requires substantial modifications of the instrumental hardware circumstances that made SID lag behind the countless applications of CID. 

In this chapter, we give an overview on how the API techniques work and which factors have an important influence on the performance. Examples are presented mostly from published work to demonstrate how LC-MS, LC-MS-MS, collision-induced dissociations (CIDs), accurate mass measurements and hydro-gen/deuterium exchange have been systematically and successfully applied in the structural elucidation of impurities, degradation products and metabolites. In addition, these also illustrate how mass spectrometry has offered a third dimension to chromatographic method development and validation. 

Remarkably, in this context, no significant sequence homology of amino acid residues could be detected. This successful structure elucidation should allow the design of non-natural specific inhibitors of this enzyme on the basis of structural information known from the bulgecins. 

Direct 3H observation should be much more direct and straightforward for rapid use in structure elucidation, preferably in a ID spectrum, and most subsequent attempts focussed on maintaining that speed and utility. Spin-echoes were applied first,34,45 utilizing the differences in transverse relaxation rates between the cross-linked polymer and relatively more mobile resin-bound molecule of interest. Wehler and Westman also had some success applying pre-saturation to the resin signals.34 While some attenuation is afforded, it is at the expense of some spectral intensity from the resin-bound molecule... 

A series of ID and 2D NMR experiments have been performed and the corresponding data will be at your disposal on your CD-ROM. If you are not familiar with these experiments you are referred to chapter 3 where they are briefly described with their field of application, their advantages and limitations. The selected NMR experiments represent to-days most popular and successful pulse sequences for structure elucidation. This selection includes ... 

To demonstrate that the proposed methods are suitable for structural elucidation of isomeric metabolites and derivatives in biological matrices, human plasma was spiked with the mixture of DL, 6-OH-DL, 3-OH-DL, /V-OH-DL, and 1-pyridine-/V-oxide-DL. The resulting sample was extracted and analyzed by LC-MS and LC-MS/MS in ESI and APCI modes as described above. HDX was successfully performed online when the extract was injected directly onto the HPLC column without drying and reconstituting the sample in a deuterated solvent. In general, there were no differences between the results obtained for the spiked plasma extract and for the mixture of the standard compounds, which indicates that the LC-MS methods with HDX described here are applicable for the analysis drug-derived material in plasma or other biological matrices. 

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