Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin and other sources. Because of restrictions on collecting and/or destruction of natural habitat, further material often cannot be obtained. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR and MS data. These techniques, along with development of microchemical reactions (deuterium exchange, hydrogenation, acylation, butylboronation of cis-diols, reductive N-methylation on GC analysis with formaldehyde, and other microreactions) have been responsible for the detailed characterization of over 500 alkaloids, representing more than 20 structural classes in frog skin extracts. HPLC is the most general separation tool, allowing study of all alkaloids, even those of high molecular weight or polarity that do not GC, but giving only limited structural insights because of lack of extensive fragmentation with either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), the two methods mostly used for HPLC-MS analysis. GC-MS analysis using electron impact ionization (EIMS) provides rich, diagnostic patterns of fragmentation, while chemical ionization (CIMS) provides molecular weight and, with deuterated ammonia, the number of exchangeable OH and NH groups. Such pioneering spectroscopic research has been extended to developing and applying tandem mass spectrometry in the collision-activated CIMS mode, demonstrating and elucidating fragmentations different from and complementary to conventional EIMS. This is so because, in the collision-activated CIMSn mode the protonated molecular ion fragments releasing neutral molecules only, to produce stable cations, while the radical molecular ion of EIMS fragments to also produce radical cations, and thus the spectra produced are totally different, based on different processes. Rules of fragmentation and applications of these methods to the study of alkaloids have been and are being developed in our Section. The analytical potential of vapor-phase GC-FTIR (Fourier transform IR) has allowed extension from traditional uses of IR (to identify functional groups like OH, carbonyl, double and triple bonds, etc.), to the obtaining of stereochemical insights (cis- or trans-ring junctions, use of Bohlmann band analysis information as to orientation of hydrogens on carbons adjacent to nitrogen, etc.). In this regard, pioneering studies (only a limited number of these instruments are in use in the world) on the significance of certain IR bands and application of GC-FTIR to the structure determination of alkaloids have been developed in our Section in the last few years, thus renewing interest in IR spectroscopy, one of the oldest techniques available. Such spectroscopic and microchemical techniques on analysis of complex mixtures, in conjunction with detailed NMR analysis, when it was feasible to isolate submilligram quantities of an alkaloid, have led to identification of over 300 alkaloids from frog skin extracts, with partial characterization of over 100 additional ones, many still representing new structural classes. In several cases, synthesis either in our Section or in collaboration, has been required to confirm structures and provide sufficient material for biological investigation. Analyses of extracts of small arthropods, have identified some 30 of these alkaloids, providing evidence for ants, beetles and millipedes as dietary sources.
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