The discovery of 2,5-dialkylcyclohexan-1,3-diones as a new class of natural products.

Orchids employing sexual deceit attract males of their pollinator species through specific volatile signals that mimic female-released sex pheromones. One of these signals proved to be 2-ethyl-5-propylcyclohexan-1,3-dione (chiloglottone1), a new natural product that was shown to be most important in the relations between orchids of the genus Chiloglottis, native to Australia, and corresponding pollinator species. Systematic investigations on the mass spectrometric fragmentation pattern of 2,5-dialkylcyclohexan-1,3-diones identified key ions providing information about the structures of the substituents at positions 2 and 5. Results enabled us to identify 2-ethyl-5-pentylcyclohexan-1,3-dione (chiloglottone2) and 2-butyl-5-methylcyclohexan-1,3-dione (chiloglottone3) as new natural products that play a decisive role in the pollination syndrome of some Chiloglottis species. During field bioassays, pure synthetic samples of chiloglottone1-3 or mixtures thereof proved to be attractive to the corresponding orchid pollinators. Because of their likely biogenesis from ubiquitous fatty acid precursors, 2,5-dialkylcyclohexan-1,3-diones may represent a hitherto overlooked, widespread class of natural products.

Structure elucidation and synthesis of dioxolanes emitted by two Triatoma species (Hemiptera: Reduviidae).

Volatiles from the metasternal glands of two species of true bugs of the Triatominae subfamily, Triatoma brasiliensis and Triatoma infestans, were analyzed by SPME-GC/MS. Two sets of new natural products were found: (4S,5S)- and (4R,5R)-2,2,4-triethyl-5-methyl-1,3-dioxolane (1) (major component) and (4S*,5S*)-2,4-diethyl-2,5-dimethyl-1,3-dioxolane (2) (trace component), (2R/S,4S,5S)- as well as (2R/S,4R,5R)-4-ethyl-5-methyl-2-(1-methylethyl)-1,3-dioxolane (3) (minor component), (2R/S,4S*,5S*)-4-ethyl-5-methyl-2-(1-methylpropyl)-1,3-dioxolane (4) (trace component), and (2R/S,4S*,5S*)-4-ethyl-5-methyl-2-(2-methylpropyl)-1,3-dioxolane (5) (trace component). Syntheses of optically active 1 and 3 were carried out by reacting pure enantiomers of 2,3-pentanediol with 3-pentanone or 2-methylpropanal. The preparation of pure stereoisomers of 2,3-pentanediol involved a novel key step for the synthesis of secondary alcohols: the reduction of a carboxylic ester by means of DIBAH and in situ alkylation of the intermediate by Grignard reaction at low temperature. Starting from the pure enantiomers of methyl lactate, all four stereoisomers of 2,3-pentanediol were synthesized and transformed to the corresponding isomers of 1 and 2. Relative configurations of the natural products and enantiomeric compositions of naturally occurring 1 and 2 were determined by comparison of their mass spectra and gas chromatographic retention times (co-injection) with those of authentic reference samples.

Inhalational anesthetics stereochemistry: optical resolution of halothane, enflurane, and isoflurane.

Halothane (I), enflurane (II), and isoflurane (III), which are among the most important inhalation anesthetics, are currently administered as racemic mixtures. The pure enantiomers have not been described, and no analytical method for resolving the commercially available racemic mixtures has been reported. Complete optical resolution of (+/-)-I and (+/-)-III on per-n-pentylated alpha-cyclodextrin (Lipodex A) and of (+/-)-II and (+/-)-III on octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin capillary columns has been achieved, making rapid and convenient determination of enantiomeric ratios in samples of all three of these anesthetics possible.

Syntheses of conjugated octadecadienoic acids.

Three approaches for the synthesis of octadecadienoic acids with conjugated double bond systems are presented: synthesis of (10Z, 12Z)-octadecadienoic acid via an enyne-substructure; the use of an educt with a conjugated double bond system for the synthesis of (10E, 12E)-octadecadienoic acid; and the Suzuki cross coupling for the synthesis of (7E,9Z)-octadecadienoic acid.

Identification, structure elucidation, and synthesis of volatile compounds in the exhaust gas of food factories.

Our investigations deal with the identification and synthesis of volatile, odoriferous compounds contained in the exhaust gas of food factories and on the biodegradation of alkylpyrazines. Collection of odour emissions samples was performed with a gas sampler equipped with filter tubes containing the styrene-polymer SuperQ. After elution with solvents of different polarity, the extracts were analysed by GC/MS and chemical microreactions. Proposed structures were verified by comparison of analytical data with those of synthetic reference samples. Major components in the exhaust gas of a fat finishing factory were found to be aliphatic aldehydes, strongly dominated by hexanal. The identification of 1,2,3,3-tetramethylcyclohexene shows that for structural proof of target compounds the use of authentic reference samples is indispensable. In the exhaust gas from a chocolate factory, several carbonyl compounds and alkylated pyrazines could be identified. Biodegradation of the latter starts with hydrogenation at the nucleus.

Evolution of reproductive strategies in the sexually deceptive orchid Ophrys sphegodes: how does flower-specific variation of odor signals influence reproductive success?

The orchid Ophrys sphegodes Miller is pollinated by sexually excited males of the solitary bee Andrena nigroaenea, which are lured to the flowers by visual cues and volatile semiochemicals. In O. sphegodes, visits by pollinators are rare. Because of this low frequency of pollination, one would expect the evolution of strategies that increase the chance that males will visit more than one flower on the same plant; this would increase the number of pollination events on a plant and therefore the number of seeds produced. Using gas chromatography-mass spectrometry (GC-MS) analyses, we identified more than 100 compounds in the odor bouquets of labellum extracts from O. sphegodes; 24 compounds were found to be biologically active in male olfactory receptors based on gas chromatography with electroantennographic detection (GC-EAD). Gas chromatography (GC) analyses of odors from individual flowers showed less intraspecific variation in the odor bouquets of the biologically active compounds as compared to nonactive compounds. This can be explained by a higher selective pressure on the pollinator-attracting communication signal. Furthermore, we found a characteristic variation in the GC-EAD active esters and aldehydes among flowers of different stem positions within an inflorescence and in the n-alkanes and n-alkenes among plants from different populations. In our behavioral field tests, we showed that male bees learn the odor bouquets of individual flowers during mating attempts and recognize them in later encounters. Bees thereby avoid trying to mate with flowers they have visited previously, but do not avoid other flowers either of a different or the same plant. By varying the relative proportions of saturated esters and aldehydes between flowers of different stem positions, we demonstrated that a plant may take advantage of the learning abilities of the pollinators and influence flower visitation behavior. Sixty-seven percent of the males that visited one flower in an inflorescence returned to visit a second flower of the same inflorescence. However, geitonogamy is prevented and the likelihood of cross-fertilization is enhanced by the time required for the pollinium deposited on the pollinator to complete its bending movement, which is necessary for pollination to occur. Cross-fertilization is furthermore enhanced by the high degree of odor variation between plants. This variation minimizes learned avoidance of the flowers and increases the likelihood that a given pollinator would visit several to many different plants within a population.

Metabolism of 3-methyldiphenyl ether by Sphingomonas sp. SS31.

The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methylcatechol, and 4-methylcatechol, respectively. Phenol and 3-methylphenol were oxidized to the corresponding catechols which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.

Transformation of 3-chlorodibenzofuran by Pseudomonas sp. HH69.

The dibenzofuran-degrading bacterial strain Pseudomonas sp. HH69 showed high oxidative activity towards 3-chlorodibenzofuran (3CDF). During the co-metabolic turnover of 3CDF large amounts of 4-chlorosalicylate and temporarily small amounts of salicylate were excreted. Simultaneously a yellow colour appeared due to the excretion of two polar products. Conversion of 3CDF by a mutant, derived from Pseudomonas sp. HH69 and defective in 2,3-dihydroxybiphenyl-1,2-dioxygenase led to the formation of equal quantities of 4'-chloro-2,2',3-trihydroxybiphenyl (4'CTHBP) and 4-chloro-2,2',3-trihydroxybiphenyl (4CTHBP). Crude extracts of the wild type transformed 4'CTHBP to 4-chlorosalicylate, whilst 4CTHBP was transformed to salicylate. Hence, we propose a non-selective initial attack on both aromatic rings of 3CDF and a degradative pathway for the resulting chlorotrihydroxybiphenyls.

Cell transformation and genotoxicity induced by bis(2, 3-dichloro-1-propyl) ether.

Bis(dichloropropyl) ether isomers have been identified in a petrochemical plant effluent through a toxicity identification evaluation study in the United States. They have also been observed in the microgram per liter range along one of the largest rivers in Europe, the Elbe River. In the present investigation, the genotoxic and transforming activity of a bis(dichloropropyl) ether isomer, bis(2,3-dichloro-1-propyl) ether, was assayed in vitro. The results demonstrate that bis(2,3-dichloro-1-propyl) ether is a potent mutagen in Salmonella typhimurium strains TA 100, TA 1535, and to a lesser extent in strain TA 98, but only when tested in the presence of a metabolic activation system (S9 mix). We have also investigated the induction of micronuclei by bis(2,3-dichloro-1-propyl) ether in the metabolically competent cell line, MCL-5. A linear, dose-dependent increase in micronuclei was observed following exposure to bis(2,3-dichloro-1-propyl) ether. The DNA strand-breaking capacity of this chemical was assessed in the alkaline single-cell gel electrophoresis ("comet") assay with MCL-5 cells. Bis(2,3-dichloro-1-propyl) ether clearly induced DNA strand breaks in the 4.5-45.5 microg/ml dose range. The ether also induced malignant transformation in C3H/M2 mouse fibroblasts after metabolic activation (S9 mix). Thus, it must be suspected that bis(2, 3-dichloro-1-propyl) ether may possess a carcinogenic potential. Since the compound along with its isomers is present in considerable concentrations in surface water, their elimination is a matter of significant public concern.

Trigeminal-basal ganglia interaction: control of sensory-motor gating and positive reinforcement.

Functional interactions between the basal ganglia and the perioral area were analyzed by means of electrical brain stimulation in the rat. The first experiment showed that unilateral stimulation of the substantia nigra sensitized the contralateral perioral area for a biting reflex upon its tactile stimulation. This biting reflex consists of lip withdrawal, orienting towards and biting into the stimulus source. The same sites in the substantia nigra also produced electrical self-stimulation using bar-pressing as the operant. A positive correlation was found between threshold currents for biting and for self-stimulation. However, the current levels necessary for reinforcement were considerably higher than those to facilitate the biting reflex. In the second experiment, it was found that manipulation of the perioral area by unilateral vibrissae removal reduced the rate of electrical self-stimulation in the substantia nigra. This effect was lateralized, depended on time after vibrissae removal, and could be reversed by systemic injections of the dopamine receptor agonist apomorphine. These results, which provide evidence for a reciprocal interaction between the basal ganglia and the perioral area, are discussed with respect to mechanisms of sensory-motor gating, motivation and reinforcement.

Enantiomeric composition of the polycyclic musks HHCB and AHTN in different aquatic species

Synthetic polycyclic musk fragrances are mainly represented by the compounds HHCB (Galaxolide(TM)) and AHTN (Tonalide(TM)). Because of their volume of use and their bioaccumulation potential, there is concern with respect to their environmental safety. HHCB and AHTN are chiral compounds, and gas chromatography using modified cyclodextrins as chiral stationary phases coupled to high-resolution mass spectrometry enabled enantioselective analysis even under unfavorable matrix conditions. The gas chromatographic elution order of (4S,7RS)- and (4R,7RS)-HHCB was assigned using synthetic (4S, 7RS)-HHCB. Fish and mussels reared in a pond associated with a municipal waste water treatment plant and semipermeable membrane devices exposed in the pond were analyzed for HHCB and AHTN. The highest lipid concentrations of HHCB and AHTN were observed in mussels (Dreissena polymorpha), tench (Tinca tinca), and crucian carp (Carassius carassius). Pronounced deviations in enantiomeric composition from racemic HHCB were observed in crucian carp and from racemic AHTN in tench. Correlations between lipid levels, enrichment, and enantioselective biotransformation of HHCB or AHTN were not seen. Selective biotransformation depended on both the compound and the species involved. The present study gives the first account of the enantiomeric composition of HHCB and AHTN in aquatic species. The lactone, 1,3,4,6,7,8-hexahydro-4,6,6,7,8, 8-hexamethylcyclopenta[g]-2-benzopyran-1-one, an oxidation product of HHCB, has been identified for the first time in environmental samples. Copyright 1999 Wiley-Liss, Inc.

Catabolism of 2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas sp strain RW1.

Due to their physicochemical and toxicological properties, polychlorinated dibenzofurans are regarded as a class of compounds providing reason for serious environmental concern. While the nonhalogenated basic structure dibenzofuran is effectively mineralized by appropriate bacterial strains, its polychlorinated derivatives are not. To elucidate the ability of the strain Sphingomonas sp RW1 to metabolize some of these chlorinated derivatives, we performed turnover experiments using 2,7-dichloro- and 2,4,8-trichlorodibenzofuran. As indicated by the oxygen-uptake rates determined for these two chlorinated dibenzofurans, Sphingomonassp RW1 can catabolize these chlorinated dibenzofurans yielding small quantities of oxidation products, which we isolated and subsequently characterized employing GC/MS and (1)H- as well as (13)C-NMR spectroscopy. In the case of 2,7-dichlorodibenzofuran, two metabolites accumulated, which we identified as 6-chloro- and 7-chloro-2-methyl-4H-chromen-4-one. The single metabolite isolated from the turnover experiments performed with 2,4,8-trichlorodibenzofuran was unequivocally identified as 6,8-dichloro-2-methyl-4H-chromen-4-one.

Tracing the transformation of labelled [1-13C]phenanthrene in a soil bioreactor.

[1-(13)C]-labelled phenanthrene was incubated in a closed bioreactor to study the flux and biotransformation of polycyclic aromatic hydrocarbon (PAH) in contaminated soils on a bulk and molecular level. The degradation of extractable phenanthrene was observed by GC-MS measurements and the mineralisation was monitored by (13)CO(2) production. The transformation of the (13)C-label into non-extractable soil-bound residues was determined by carbon isotopic measurements. With these data we were able to calculate a carbon budget of the (13)C-label. Moreover, the chemical structure of non-extractable bound residues was characterised by applying selective chemical degradation reactions to cleave xenobiotic subunits from the macromolecular organic soil matrix. The obtained low molecular weight products yielded (13)C-labelled compounds which were identified using IRM (isotope ratio monitoring)-GC-MS and structurally characterised with GC-MS. Most of the (13)C-labelled products obtained by chemical degradation of non-extractable bound residues are well-known metabolites of phenanthrene. Thus, metabolites of [1-(13)C]phenanthrene formed during biodegradation appear to be reactive components which are subsequently involved in the bound residue formation. Hydrolysable amino acids of the soil residues were significantly labelled with (13)C as confirmed by IRM-GC-MS measurements. Therefore, phenanthrene-derived carbon was transformed by anabolic microbial processes into typical biologically derived compounds. These substances are likely to be incorporated into humic-like material after cell death.

Identification of a four-component sex pheromone blend in Bonagota cranaodes (Lepidoptera: Tortricidae).

Analysis of sex pheromone glands of the apple leafroller Bonagota cranaodes Meyrick by gas chromatography coupled with mass spectrometry or electroantennographic detection showed the presence of 14 structurally related acetates and alcohols of the chain length 10-18, including the main pheromone component (E,Z)-3,5-dodecadienyl acetate (E3,Z5-12Ac). Male antennae responded to the main compound, its Z,Z isomer, (E,Z) -3,5-tetradecadienyl acetate (E3,Z5-14Ac), and the monoenes (Z)-5-dodecenyl acetate (Z5-12Ac) and (Z)-9-hexadecenyl acetate (Z9-16Ac). Traps baited with a four-component blend of E3,Z5-12Ac, Z5-12Ac, E3,Z5-14Ac, and Z9-16Ac in a 100:5:5:100 ratio were significantly more attractive than the main compound alone. This improved trap lure is more suitable for monitoring population densities of B. cranaodes, and for detection of the onset of the seasonal flight period. A more complete pheromone blend is of importance also with respect to current attempts to develop mating disruption for control of this major pest of apple in Brazil.

The secretion of the ventral glands in Cladius, Priophorus and Trichiocampus sawfly larvae.

The volatile secretion from ventral glands of the larvae of Cladius pectinicornis, Priophorus morio, P. pallipes and Trichiocampus grandis was found to be principally composed of long-chain acetogenins, in majority of the esters and hydrocarbons, with more than 15 carbon atoms. The scarcity of more volatile compounds may be considered as plesiomorphic for the tribe Cladiini to which the four species belong. Further chemotaxonomic significance and chemical ecological implications of the glandular secretions are discussed. Moreover, the function of the well-developed pubescence covering the body of Cladiini larvae is discussed as a part of their defensive mechanism.

(4S,5S)-2,2,4-Triethyl-5-methyl-1,3-dioxolane: a new volatile released by a triatomine bug.

Adults of the triatomine bug Triatoma brasiliensis release 2,2,4-triethyl-5-methyl-1,3-dioxolane (1) as a mixture of the (4S,5S)- and (4R,5R)-enantiomers in a ratio of 4:1. Among the volatile acetals identified from insects so far, this is the first example resulting from an intermolecular condensation of a carbonyl moiety and a diol substructure.

Fertility signals in the bumblebee Bombus terrestris (Hymenoptera: Apidae).

In eusocial Hymenoptera, queen control over workers is probably inseparable from the mechanism of queen recognition. In primitively eusocial bumblebees (Bombus), worker reproduction is controlled not only by the presence or absence of a dominant queen but also by other dominant workers. Furthermore, it was shown that the queen dominance is maintained by pheromonal cues. We investigated whether there is a similar odor signal released by egg-laying queens and workers that may have a function as a fertility signal. We collected cuticular surface extracts from nest-searching and breeding Bombus terrestris queens and workers that were characterized by their ovarian stages. In chemical analyses, we identified 61 compounds consisting of aldehydes, alkanes, alkenes, and fatty acid esters. Nest-searching queens and all groups of breeding females differed significantly in their odor bouquets. Furthermore, workers before the competition point (time point of colony development where workers start to develop ovaries and lay eggs) differed largely from queens and all other groups of workers. Breeding queens showed a unique bouquet of chemical compounds and certain queen-specific compounds, and the differences toward workers decrease with an increasing development of the workers' ovaries, hinting the presence of a reliable fertility signal. Among the worker groups, the smallest differences were found after the competition point. Egg-laying females contained higher total amounts of chemical compounds and of relative proportions of wax-type esters and aldehydes than nest-searching queens and workers before the competition point. Therefore, these compounds may have a function as a fertility signal present in queens and workers.

Brominated indoles and phenols in marine sediment and water extracts from the north and baltic seas-concentrations and effects.

This work presents results from analytical as well as ecotoxicologic investigations of sediment and water samples from the North and Baltic Seas. A bioassay-directed procedure was used to investigate cause-effect relationships between observed effects in acute laboratory bioassays (luminescent bacteria assay with Vibrio fischeri and embryo test with Danio rerio) and analyte concentrations in extracted samples. Brominated phenols and indoles-including 4-bromophenol; 2,4-dibromophenol; 4- and 6-bromoindole; 3,4-, 4,6-, and 3,6-dibromoindole; and tribrominated compounds-were identified in partly remarkable concentrations (up to 40,000 ng g(-1) total organic carbon TOC for 4-bromophenol) in North Sea sediment samples and water samples (913 ng L(-1) 3,6-dibromoindole) from the German Bight. The toxicity of some of the identified brominated substances was low, with median effect concentration levels (EC(50)) ranging from 0.08 to 21.7 mg/L for V. fischeri and 4.3 to 46.3 mg/L for D. rerio. Comparison of the concentrations of analytes with ECs showed a toxicity contribution of brominated phenols and indoles to overall toxicity of the fraction. In the case of one water sample from the German Bight, brominated phenols and indoles accounted for the observed toxicity. Brominated phenols and indoles, which are assumed to be of biogenic origin, have rarely been discussed so far in the context of ecotoxicologic effects in marine ecosystems.

Carboxylic acids from hairpencils of male Amauris butterflies (Lep.: Danainae).

63 Carboxylic acids were identified from the male hairpencils of four species of the genus Amauris (Lep.: Danainae), namely A. echeria (Stoll), A. hecate (Butler), A. ochlea (Boisduval) and A. albimaculata Butler. Straight chain saturated as well as unsaturated carboxylic acids, some of which containing an additional oxygen function, contribute to the species-specificity of the odour bouquets. Oxygenated fatty acids form a new class of insect volatiles, 5 of the 10 ketoacids found represent new natural products. (E)-7-Oxo-11-tetradecenoic acid is the main volatile component of the hairpencils of A. echeria, the species with the highest amount of oxygenated fatty acids (70% of the extractable volatiles). 9-Hydroxyoctadecanoic acid is a major compound in both A. ochlea and A. albimaculata while in A. hecate oxygenated carboxylic acids are present in minute amounts only.

Identification of new sesquiterpenoids in cephalic secretion of cuckoo bee,Nomada lathburiana (Apoidea, Anthophoridae).

A series of new sesquiterpene ketones and norsesquiterpene ketones could be identified from the cephalic secretion of females of the cuckoo bee,Nomada lathburiana (K.). The major component proved to be 2,6,10-trimethylundeca-(5E)-2,5,9-trien-4-one. Large amounts of high-boiling-point straight-chain hydrocarbons serve as solvents for the volatile, unstable ketones.