Amphibian skin has provided a wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential. These alkaloids include batrachotoxins, which are potent activators of sodium channels, histrionicotoxins, which are noncompetitive blockers of nicotinic receptor-channels and potassium channels, pumiliotoxins/allopumiliotoxins and related homopumiliotoxins, which have myotonic and cardiotonic activity due to effects on sodium channels, epibatidine, an extremely potent and selective nicotinic agonist with potent antinociceptive activity and epiquinamide, a nicotinic agonist selective for certain receptor subtypes. Further alkaloids include decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines, lehmizidines, and a variety of tricyclic alkaloids, including spiropyrrolizidine oximes, gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines, coccinellines and coccinelline analogs. The batrachotoxins also occur in certain toxic birds and dietary beetles. 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. 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 600 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). 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. 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 conjunction in some cases with detailed NMR analysis and even synthesis for structural verification, structures of over 400 alkaloids have been delineated. Current extracts from amphibians of Central and South America and Madagascar have led to identification of nearly 50 new alkaloids, many representing new structural classes. Ants, beetles and millipedes that are dietary sources of certain classes of amphibian skin alkaloids have been identified, notably formicine ants for the pumiliotoxins, melyrid beetles for the batrachotoxins, and siphonotid millipedes for the spiropyrrolizidines. A unique pumiliotoxin 7-hydroxylase in one lineage of neotropical poison frogs stereoselectively converts a pumiliotttoxin to a more toxic allopumiliotoxin. Such alkaloids activate nociceptive sensory pathways. The major biological targets for the alkaloids appear to be both voltage-sensitive and ligand-gated ion channels, in particular sodium, calcium and nicotinic channels.
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