Alkaloids in bufonid toads (melanophryniscus): temporal and geographic determinants for two argentinian species.

Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.

The chemistry of exploding ants, Camponotus spp. (cylindricus complex).

A detailed comparative analysis of the exocrine chemistry of nine Bruneian Camponotus species in the cylindricus complex is reported. Workers of these species are known to have hypertrophied mandibular glands and release their glandular contents suicidally from the head by rupturing the intersegmental membrane of the gaster. All of the species produce mixtures of polyacetate-derived aromatics, including hydroxyacetophenones, which display pH-dependent color changes, and aliphatic hydrocarbons and alcohols. In addition, three species contained (6R)-2,6-dimethyl-(2E)-octen-1,8-dioic acid (9) or (3S)-8-hydroxycitro-nellic acid (10a), previously unreported from insects. These compounds were characterized from their spectral data, and confirmed by comparison with synthetic samples. The allomonal role of these compounds is based on numerous field observations, and their chemotaxonomic value is presented.

Evidence for biosynthesis of pseudophrynamine alkaloids by an Australian myobatrachid frog (pseudophryne) and for sequestration of dietary pumiliotoxins.

Australian myobatrachid frogs of the genus Pseudophryne have only two classes of alkaloids in skin extracts, pseudophrynamines (PSs) and pumiliotoxins (PTXs). The former are unique to such Australian frogs, while the PTXs occur worldwide in all other genera of frogs/toads that contain lipophilic alkaloids. The major alkaloid of wild-caught frogs from one population of Pseudophryne semimarmorata was PTX 267C, while PSs were only minor or trace alkaloids. Captive-raised frogs from the same parental stock had no PTXs, but had larger amounts of PSs. A PTX fed to captive-raised frogs accumulated into skin along with dihydro and hydroxy metabolites. Thus, Pseudophryne frogs appear to biosynthesize PSs, but to sequester into skin dietary PTXs. In addition, biosynthesis of PSs appears reduced when high levels of dietary PTXs have accumulated into skin. This is the first evidence indicating that certain frogs are capable of synthesizing rather than merely sequestering alkaloids. A wide range of PSs, including many with molecular weights >500, were detected using both GC-mass spectral and LC-mass spectral analysis.

Ammonia chemical ionization tandem mass spectrometry in structural determination of alkaloids. II. Tetraponerines from pseudomyrmecine ants.

Chemical ionization tandem mass spectrometry (CI-MS/MS) of alkaloids with ammonia reagent gas and collision-activated dissociation as well as EI-MS/MS were applied to the tetraponerine alkaloids in extracts from six pseudomyrmecine ants of the genus Tetraponera. The MS/MS techniques along with gas chromatography Fourier transform infrared (GC/FTIR) spectra allowed identification in two extracts of seven of the eight known tetraponerines. The EI-MS/MS fragmentations proved diagnostic for the ring system and the CI-MS/MS patterns for the C-8 or C-9 substitution, while the Bohlmann bands in FTIR spectra were diagnostic for the C-8 or C-9 configurations. An Indian ant (T. allaborans) had T-2, T-4 and T-8, while a Chinese ant (T. binghami) had T-5, T-6, T-7 and T-8. Four other ants, T. rufonigra (India), T. penzigi (Africa), T. clypeata (Africa) and T. sp. cf. emeryi (Africa), had no tetraponerines.

Structure of alkaloid 275A, a novel 1-azabicyclo[5.3.0]decane from a dendrobatid frog, Dendrobates lehmanni: synthesis of the tetrahydrodiastereomers.

The principal alkaloid 275A in skins of the Colombian poison frog Dendrobates lehmanni has been identified as the pyrrolo[1,2-a]azepane (1), the first occurrence in nature of this "izidine" system. Tetrahydro-1 proved identical to one of the four synthetic diastereomers, 2a--2d, thereby establishing that 1 has the 5Z,10E relative stereochemistry. Alkaloid 1 is often accompanied by other congeners, in particular a 5Z,10Z diastereomer 15, a dihydro analogue 16, and a ketone 17. Such izidines in frogs may arise from dietary ants, as do other classes of izidines.

Batrachotoxin alkaloids from passerine birds: a second toxic bird genus (Ifrita kowaldi) from New Guinea.

Batrachotoxins, including many congeners not previously described, were detected, and relative amounts were measured by using HPLC-mass spectrometry, in five species of New Guinean birds of the genus Pitohui as well as a species of a second toxic bird genus, Ifrita kowaldi. The alkaloids, identified in feathers and skin, were batrachotoxinin-A cis-crotonate (1), an allylically rearranged 16-acetate (2), which can form from 1 by sigmatropic rearrangement under basic conditions, batrachotoxinin-A and an isomer (3 and 3a, respectively), batrachotoxin (4), batrachotoxinin-A 3'-hydroxypentanoate (5), homobatrachotoxin (6), and mono- and dihydroxylated derivatives of homobatrachotoxin. The highest levels of batrachotoxins were generally present in the contour feathers of belly, breast, or legs in Pitohui dichrous, Pitohui kirhocephalus, and Ifrita kowaldi. Lesser amounts are found in head, back, tail, and wing feathers. Batrachotoxin (4) and homobatrachotoxin (6) were found only in feathers and not in skin. The levels of batrachotoxins varied widely for different populations of Pitohui and Ifrita, a result compatible with the hypothesis that these birds are sequestering toxins from a dietary source.

Determination of absolute stereochemistry and an alternative synthesis of homopumiliotoxin 223G: identification on chiral GC columns with the natural alkaloid.

An alternative asymmetric synthesis of (+)-(lS,9aS)-homopumiliotoxin 223G (1) was accomplished via (1R,2R, 9aS)-1-(benzyloxy)-2-hydroxy-1-methyl-3[(E)-isobutylidene]++ +quinolizidi ne (4), which was synthesized according to the intramolecular nickel(II)/chromium(II)-mediated cyclization of the N-(iodoalkenyl)aldehyde 2. Compound 4 was converted to the acetate and subjected to reduction with lithium in ammonia, whereupon deprotection of the O-benzyl group and removal of the acetoxyl group occurred in a single operation to afford (+)-homopumiliotoxin 223G. The same sequence using (+/-)-4 was applied to the synthesis of racemic 223G. Gas chromatography of a sample of racemic 223G showed no separation into enantiomers on four different cyclodextrin-based chiral GC columns. We found, however, that the O-acetates of (+/-)-223G gave a nearly baseline separation on either a beta-cyclodextrin column or a permethylated beta-cyclodextrin column. The O-acetate of synthetic (+)-223G was identical on either of these two columns, with the first eluting O-acetate from acetylated (+/-)-223G and also with the acetylated 223G present in a frog skin extract, thus allowing us to confirm unambiguously the 1S,9aS absolute configurations of natural 223G.

Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS.

Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and inactive in a high acetate producer, JM109. To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates. The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques. These distributions are next validated by predicting the NMR spectrum of glutamate. Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained with respect to the flux through the PPC anaplerotic shunt. We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109. Further, in BL21, the flux through the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt.

A stereospecific synthesis of (+/-)-5,8-disubstituted indolizidines and (+/-)-1,4-disubstituted quinolizidines found in poison frog skins.

An efficient, high-yield stereospecific route to three (+/-)-5, 8-disubstituted indolizidines, (209B (I), 209I (II), 223J (III)) and two (+/-)-1,4-disubstituted quinolizidines (207I (IV), 233A (V)), racemates of alkaloids found in the skins of neotropical and Madagascan poison frogs is reported. The structures of the natural alkaloids were thereby established by chiral GC comparison with the exception of indolizidine 209B (I) for which a natural 209B could no longer be detected.

3-methyl-4-phenylpyrrole from the ants Anochetus kempfi and Anochetus mayri.

The cephalic extracts of the ant Anochetus kempfi were found to contain 2,5-dimethyl-3-isoamylpyrazine (1) and 3-methyl-4-phenylpyrrole (2). The structures of these compounds were established from their spectral data and by comparison with synthetic samples. This is the first report of a phenylpyrrole found in an insect and only the third report of a pyrrole from ants.

Ammonia chemical ionization tandem mass spectrometry in structure determination of alkaloids. I. Pyrrolidines, piperidines, decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines and an azabicyclo[5.3.0]decane.

CI-MS/MS of alkaloids with ammonia reagent gas and collision-induced dissociation provides unique structural information for certain mono- and bi-cyclic alkaloids. This technique was applied to solve the structures of 195C, a 4,6-disubstituted quinolizidine alkaloid found in frog skin and an ant, and 275A, a novel 3,5-disubstituted azabicyclo[5.3.0]decane found in frog skin.

A gas chromatographic-mass spectral assay for the quantitative determination of oleamide in biological fluids.

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.

Alkaloid 223A: the first trisubstituted indolizidine from dendrobatid frogs.

The structure of alkaloid 223A (1), the first member of a new class of amphibian alkaloids, purified by HPLC from a skin extract of a Panamanian population of the frog Dendrobates pumilio Schmidt (Dendrobatidae) has been established as (5R,6S,8R,9S)- or (5S,6R,8S,9R)-6,8-diethyl-5-propylindolizidine, based on GC-MS, GC-FTIR, and 1H-NMR spectral studies. Three higher homologs of 223A, namely alkaloids 237L (2), 251M (3), and 267J (4), have been detected in other extracts, and tentative structures are proposed.

A 1,4-disubstituted quinolizidine from a Madagascan mantelline frog (Mantella).

A major alkaloid, 217A, in skin extracts of a mantelline frog (Mantella baroni) was isolated by HPLC and the structure defined by 1H-NMR spectral analysis as (1R,4S,10S)- or (1R,4R,10R)- 1-methyl-4-(Z)-(1-pent-2-en-4-ynyl) quinolizidine (1). Four other alkaloids-207I (2), 231A (3), 233A (4), and 235E' (5)-detected in frog skin extracts are proposed to be 1,4-disubstituted quinolizidines based on diagnostic mass and FTIR spectra.

A new secotrinervitane diterpene isolated from soldiers of the Madagascan termite species, Nasutitermes canaliculatus.

Reported herein is the X-ray crystallographic structure of a novel 10-oxygenated secotrinervitane diterpene, 3 alpha, 10 alpha-diacetoxy-7,16-secotrinervita-7,11,15(17)-triene (4), from soldiers of the endemic Madagascan termite Nasutitermes canaliculatus, which was compared with an energy-minimized structure obtained by computer molecular modeling. We also report 1H- and 13C-NMR and MS data for this new diterpene.

Primary tetradecenyl amines from the ant Monomorium floricola.

In contrast to other ants in the genus Monomorium that produce cyclic amines, extracts of Monomorium floricola contain (Z)-7-tetradecenylamine (1) and (Z)-9-tetradecenylamine (2). The structures of these compounds were established from their spectral data and by comparison with synthetic 2.

Chemistry of venom alkaloids in someSolenopsis (Diplorhoptrum) species from Puerto Rico.

A number of 15-carbon alkaloids have been identified in venom extracts of four Puerto Rican species of ants in the genusSolenopsis (Diplorhoptrum). Workers of a species from El Verde produced thecis andtrans isomers of 2-methyl-6-nonylpiperidine with the latter isomer predominating. The same compounds were identified in queens of a species from Río Grande, but in this species no alkaloids were detected in worker extracts. Workers of aDiplorhoptrum species collected on Mona Island produced primarily atrans-2-methyl-6-(Z-4-nonenyl)piperidine,3, with smaller amounts of thecis isomer, whereas the major compound found in the queens of the same species on Mona Island was (5Z,9Z)-3-hexyl-5-methylindolizidine, identical with the alkaloid produced by queens of a species collected on Cabo Rojo. Surprisingly, workers of the Cabo Rojo species produced (5Z,9Z)- and (5E,9E)-3-butyl-5-propylindolizidine (4 and5, respectively) reported earlier as the 223AB indolizidines from skins of dendrobatid frogs. The possible significance of the qualitative and quantitative differences in the venom alkaloids synthesized by queens and workers is discussed as is the possibility that ants containing such alkaloids may serve as a dietary source for the skin alkaloids used by certain frogs in chemical defense.

A new subclass of alkaloids from a dendrobatid poison frog: a structure for deoxypumiliotoxin 251H.

Deoxypumiliotoxin 251H [4], representing a new subclass of the pumiliotoxin-A class of alkaloids, has been isolated from a dendrobatid frog, Epipedobates tricolor. The structure, elucidated by nmr, gc-Ftir, and mass spectral analyses, is proposed to be an 8-methyl-6-(5-hydroxy-2-methylhexylidene)-1-azabicyclo[4.3.0]nonan e. The absolute stereochemistry and the relative configuration of the hydroxyl group are unknown.

An uptake system for dietary alkaloids in poison frogs (Dendrobatidae).

The skin of poison frogs (Dendrobatidae) contains a wide variety of alkaloids that presumably serve a defensive role. These alkaloids persist for years in captivity, but are not present in captive-raised frogs. Alkaloids fed to poison frogs (Dendrobates, Phyllobates, Epipedobates) are readily accumulated into skin, where they remain for months. The process can be selective; an ant indolizidine is accumulated, while an ant pyrrolidine is not. Frogs (Colostethus) of the same family, which do not normally contain alkaloids, do not accumulate alkaloids. Such an alkaloid uptake system provides a means of maintaining skin alkaloids and suggests that some if not all such 'dendrobatid alkaloids' may have a dietary origin.