Phantasmidine: an epibatidine congener from the ecuadorian poison frog Epipedobates anthonyi.

The skin of the Ecuadorian poison frog Epipedobates anthonyi contains the potent nicotinic agonists epibatidine (1) and N-methylepibatidine (3). In addition, a condensed tetracyclic epibatidine congener has been identified with activity at nicotinic acetylcholine receptors, but different selectivity than epibatidine. This rigid tetracycle has been named phantasmidine (4). Phantasmidine has a molecular formula of C(11)H(11)N(2)OCl, shares a chloropyridine moiety with 1, and also contains furan, pyrrolidine, and cyclobutane rings. A combination of GC-MS and GC-FTIR analysis with on-column derivatization, 1D NMR spectroscopy with selective irradiation, and spectral simulation, along with 2D NMR, were used to elucidate the structure from a total sample of approximately 20 microg of HPLC-purified 4 and its corresponding acetamide (5). After synthesis, this novel rigid agonist may serve as a selective probe for beta4-containing nicotinic receptors and potentially lead to useful pharmaceuticals.

Indolizidine 239Q and quinolizidine 275I. Major alkaloids in two Argentinian bufonid toads (Melanophryniscus).

Alkaloid profiles in skin of poison frogs/toads (Dendrobatidae, Mantellidae, Bufonidae, and Myobatrachidae) are highly dependent on diet and hence on the nature of habitat. Extracts of the two species of toads (Melanophryniscus klappenbachi and Melanophryniscus cupreuscapularis) from similar habitats in the Corrientes/Chaco Provinces of Argentina have similar profiles of alkaloids, which differ considerably in profiles from other Melanophryniscus species from Brazil, Uruguay and Argentina. Structures of two major alkaloids 239Q (1) and 275I (2) were determined by mass, FTIR, and NMR spectral analysis as 5Z,9Z-3-(1-hydroxybutyl)-5-propylindolizidine and 6Z,10E-4,6-di(pent-4-enyl) quinolizidine, respectively. A third alkaloid, 249F (3), is postulated to be a homopumiliotoxin with an unprecedented conjugated exocyclic diene moiety.

The AUUCU repeats responsible for spinocerebellar ataxia type 10 form unusual RNA hairpins.

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

A revised structure for alkaloid 235C isolated from skin extracts of mantellid (Mantella) frogs of Madagascar.

Madagascan frogs of the mantellid genus Mantella have been a rich source of alkaloids derived from dietary arthropods. Two species of frogs, inhabiting swamp forest, contain a unique set of alkaloids, previously proposed, based only on GC-MS and GC-FTIR data, to represent dehydro analogues of the homopumiliotoxins. The major alkaloid of this set, alkaloid 235C (2), now has been isolated in sufficient quantities (ca. 0.3 mg) to allow determination of the structure by NMR analysis. The structure of alkaloid 235C proved to be a 7,8-dehydro-8-desmethylpumiliotoxin. A comparison is presented between the mass, infrared, and (1)H NMR spectra of 235C (2) and a synthetic dehydrohomopumiliotoxin (1), initially proposed incorrectly as the structure for 235C.

Bioassay-guided isolation of epiquinamide, a novel quinolizidine alkaloid and nicotinic agonist from an Ecuadoran poison frog, Epipedobates tricolor.

Analytical HPLC fractionation, combined with an off-line 96-well fluorescent bioassay screen, has been developed and used for the separation and screening of a natural product extract. This method was used to guide the isolation of a novel quinolizidine alkaloid from the methanolic skin extracts of an Ecuadoran frog, Epipedobates tricolor. The structure was determined on the basis of MS, IR, and NMR analysis as (1R,10R)-1-acetamidoquinolizidine (alkaloid 196). We have named this compound epiquinamide, reflecting its origin and structure. The activity of the isolated compound was determined in five cell lines expressing various nicotinic acetylcholine receptor subtypes. The bioactivity of epiquinamide was evaluated on the basis of membrane potential fluorescence and was found to be beta2 selective. This compound represents a new structural class of nicotinic agonists and a potential lead compound for the development of new therapeutics and pharmacological probes for nicotinic receptors. The off-line screening technique was found to be very sensitive for the detection of compounds active at nicotinic receptors.

Solution structure of a cis-opened (10R)-N6-deoxyadenosine adduct of (9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex.

The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.