A wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential, has been provided by amphibian skin. These alkaloids include batrachotoxins, which are potent activators of sodium channels, histrionicotoxins, which are noncompetitive blockers of nicotinic receptor-channels, pumiliotoxins/allopumiliotoxins/homopumiliotoxins and related congeners, some of which have myotonic and cardiotonic activity due to effects on sodium channels and epibatidine, an extremely potent and selective nicotinic agonist with potent antinociceptive activity. Further alkaloids include decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines, lehmizidines, and a variety of tricyclic alkaloids, including spiropyrrolizidine oximes, gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines and coccinellines. Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (vapor-phase IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful separation and spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin, arthropods, and other sources. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR, MS and hopefully, NMR data. These techniques, along with development of microchemical reactions including hydrogenation, acylation, butylboronation of cis-diols and N-methylation on GC analysis with formaldehyde and formic acid, have been responsible for the detailed characterization of over 800 alkaloids, representing some 26 structural classes in frog skin extracts. HPLC-MS allows study of all alkaloids, even those of high molecular weight or polarity that do not GC, but gives 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 (identification of functional groups like OH, carbonyl, double and triple bonds, etc.), to providing valuable stereochemical insights (cis- or trans-ring junctions, Bohlmann band analysis to indicate orientation of hydrogens on carbons adjacent to nitrogen, etc.). A new method in FTIR, applied during the last year by our group and still being developed by us, named methyl counting, allows a detailed CH integration in the IR spectrum, greatly enhancing our ability in the process of structure elucidation. Chiral GC analysis has established with synthetic samples the absolute stereochemistry of many alkaloids. GC-MS and GC-FTIR, in conjunction in some cases with detailed NMR analysis and even synthesis for structural verification, have delineated structures of over 400 alkaloids. NMR analysis with microprobe has now been applied to a few alkaloid samples of only 10 ug. Current extracts from amphibians and arthropods of Central and South America and Madagascar have led to identification of about 100 new alkaloids, some representing new structural classes, including N-methyldecahydroquinolines, dialkylamines and dehydroizidines. Certain melyrid beetles were found to contain batrachotoxins and appear likely to be the dietary source of batrachotoxins found in poison dart frogs and certain birds. The mites, ants, beetles and millipedes that are dietary sources of many classes of amphibian skin alkaloids have been identified, notably oribatid mites for pumiliotoxins and many izidines with branched carbon skeletons, myrmicine ants for other izidines with linear carbon skeletons, beetles for the tricyclic coccinelline alkaloids and siphonotid millipedes for the spiropyrrolizidines. Further novel alkaloids from ants have been structurally defined. The sequestration of ryanodine from plants (Spigelia) by larvae of the spider moth (Eudulophasia) has been discovered. The use of HPLC-UV-MS to re-examine an old extract of a Melanophryniscus toad skin has revealed at least 5 bufadienolides and 3 cardenolides. This result is consistent with the original bioassay, the inhibition by that extract of ouabain-binding to a Na/K-ATP-ase enzyme, essential in heart muscle function. Here, negative-ion MS and use of deuterated water in the HPLC solvent were useful in assigning partial structures. One of the pumiliotoxins, namely PTX 251D, had enantioselective contact toxicity for mosquitoes and fire ants. The major biological targets for the amphibian alkaloids appear to be both voltage-sensitive and ligand-gated ion channels, in particular sodium, calcium and nicotinic channels. Certain pumiliotoxins were found to activate nociceptive sensory pathways, presumably through interaction with sodium channels.

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Lloyd, John R; Jayasekara, P Suresh; Jacobson, Kenneth A (2016) Characterization of Polyamidoamino (PAMAM) Dendrimers Using In-Line Reversed Phase LC Electrospray Ionization Mass Spectrometry. Anal Methods 8:263-269
Sionov, Edward; Chang, Yun C; Garraffo, H Martin et al. (2009) Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob Agents Chemother 53:2804-15
Wijdeven, Marloes A; Wijtmans, Roel; van den Berg, Rutger J F et al. (2008) N,N-acetals as N-acyliminium ion precursors: synthesis and absolute stereochemistry of epiquinamide. Org Lett 10:4001-3
Nelson, Angela; Garraffo, H Martin; Spande, Thomas F et al. (2008) Facile synthesis of two diastereomeric indolizidines corresponding to the postulated structure of alkaloid 5,9E-259B from a Bufonid toad (Melanophryniscus). Beilstein J Org Chem 4:6
Garraffo, H (2008) John William Daly, 1933-2008. Cell Mol Neurobiol :
Daly, John W; Garraffo, H Martin; Spande, Thomas F et al. (2008) Individual and geographic variation of skin alkaloids in three species of Madagascan poison frogs (Mantella). J Chem Ecol 34:252-79
Toyooka, Naoki; Zhou, Dejun; Nemoto, Hideo et al. (2007) Flexible synthetic routes to poison-frog alkaloids of the 5,8-disubstituted indolizidine-class I: synthesis of common lactam chiral building blocks and application to the synthesis of (-)-203A, (-)-205A, and (-)-219F. Beilstein J Org Chem 3:29
Saporito, Ralph A; Donnelly, Maureen A; Garraffo, H Martin et al. (2006) Geographic and seasonal variation in alkaloid-based chemical defenses of Dendrobates pumilio from Bocas del Toro, Panama. J Chem Ecol 32:795-814
Weldon, Paul J; Kramer, Matthew; Gordon, Scott et al. (2006) A common pumiliotoxin from poison frogs exhibits enantioselective toxicity against mosquitoes. Proc Natl Acad Sci U S A 103:17818-21
Fitch, Richard W; Kaneko, Yumika; Klaperski, Paul et al. (2005) Halogenated and isosteric cytisine derivatives with increased affinity and functional activity at nicotinic acetylcholine receptors. Bioorg Med Chem Lett 15:1221-4

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