Male Galerucella calmariensis and Galerucella pusilla (Coleoptera: Chrysomelidae) emit an aggregation pheromone while feeding on host foliage. Isolation of the compound from collected volatiles was guided by comparisons of gas chromatograms of extracts from males and females and by gas chromatography–electroantennographic detection. The compound was identified by a combination of spectrometric methods and microchemical tests as the novel dimethylfuran lactone, 12,13-dimethyl-5,14-dioxabicyclo[9.2.1]tetradeca-1(13),11-dien-4-one. The structure was confirmed by synthesis, and the synthetic compound attracted males and females of both species in field bioassays. These beetles were previously introduced into North America as biological control agents for the invasive wetland weed, purple loosestrife Lythrum salicaria, and the pheromone could become a tool for monitoring populations. A new method is described for distinguishing the two species based on the tibial spurs of the males.

Tropical isolates of Aureobasidium pullulans previously isolated from distinct habitats in Thailand were characterized for their capacities to produce the valuable polysaccharide, pullulan. A. pullulans strain NRM2, the so-called "color variant" strain, was the best producer, yielding 25.1 g pullulan l(-1) after 7 days in sucrose medium with peptone as the nitrogen source. Pullulan from strain NRM2 was less pigmented than those from the other strains and was remarkably pure after a simple ethanol precipitation. The molecular weight of pullulan from all cultures dramatically decreased after 3 days growth, as analyzed by high performance size exclusion chromatography. Alpha-amylase with apparent activity against pullulan was expressed constitutively in sucrose-grown cultures and induced in starch-grown cultures. When the alpha-amylase inhibitor acarbose was added to the culture medium, pullulan of slightly higher molecular weight was obtained from late cultures, supporting the notion that alpha-amylase plays a role in the reduction of the molecular weight of pullulan during the production phase.

Defatted field pennycress (Thlaspi arvense L.) seedmeal was found to completely inhibit seedling germination/emergence when added to a sandy loam soil containing wheat (Triticum aestivum L.) and arugula [Eruca vesicaria (L.) Cav. subsp. sativa (Mill.) Thell.] seeds at levels of 1.0% w/w or higher. Covering the pots with Petri dishes containing the soil-seedmeal mixture decreased germination of both species at the lowest application rate (0.5% w/w), suggesting that the some of the phytotoxins were volatile. CH2Cl2, MeOH, and water extracts of the wetted seedmeal were bioassayed against wheat and sicklepod (Senna obtusifolia (L.) H. S. Irwin & Barneby) radicle elongation. Only the CH2Cl2 extract was strongly inhibitory to both species. Fractionation of the CH2Cl2 extract yielded two major phytotoxins, identified by gas chromatography–mass spectrometry and NMR as 2-propen-1-yl (allyl) isothiocyanate (AITC) and allyl thiocyanate (ATC), which constituted 80.9 and 18.8%, respectively, of the active fraction. When seeds of wheat, arugula and sicklepod were exposed to volatilized AITC and ATC, germination of all three species was completely inhibited by both compounds at concentrations of 5 ppm or less. In field studies, where seedmeal was applied at 0.50, 1.25, and 2.50 kg/m2 and tarped with black plastic mulch, all of the treatments significantly reduced dry weight of bioassay plants compared to the tarped control, with the highest seedmeal rate decreasing dry matter to less than 10% of the control 30 d after seedmeal application. Field pennycress seedmeal appears to offer excellent potential as a biofumigant for high-value horticultural crops for both conventional and organic growers.

A novel hydroxylated primary fatty amide was produced from lesquerolic acid by direct amidation with ammonia catalyzed by immobilized Pseudozyma (Candida) antarctica lipase B (Novozym 435) in organic solvent. The amides of ricinoleic acid and oleic acid were also produced for comparison. The hydroxy FA were transformed at comparable rates to that of oleic acid. The rate of amide formation was greater for the longerchain lesquerolic acid than for ricinoleic acid. All products exhibited characteristic primary-amide mass spectrum peaks with a base peak at m/z 59 and a strong ion fragment at m/z 72. Other peaks present are consistent with cleavage on either side of the hydroxyl position. The mass spectra, together with 1H and 13C NMR data, suggest that the products of lipase-catalyzed direct amidation of ricinoleic acid and lesquerolic acid are 12-hydroxy-9(Z)-octadecenamide and 14-hydroxy-11(Z)-eicosenamide, respectively.

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; 1H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by 1H NMR and 13C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by 1H NMR and 13C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.

ABSTRACT Previously, it was reported that a newly isolated microbial culture, Clavibacter sp. strain ALA2, produced trihydroxy unsaturated fatty acids, diepxoy bicyclic fatty acids, and tetrahydroxyfuranyl fatty acids (THFAs) from linoleic acid (C. T. Hou, J. Am. Oil Chem. Soc. 73:1359-1362, 1996; C. T. Hou and R. J. Forman III, J. Ind. Microbiol. Biotechnol. 24:275-276, 2000; C. T. Hou, H. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 75:1483-1487, 1998; C. T. Hou, H. W. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 78:1167-1169, 2001). In this study, we found that Clavibacter sp. strain ALA2 produced novel THFAs, including 13,16-dihydroxy-12-THFA, 15-epoxy-9( Z )-octadecenoic acid (13,16-dihydroxy-THFA), and 7,13,16-trihydroxy-12, 15-epoxy-9( Z )-octadecenoic acid (7,13,16-trihydroxy-THFA), from α-linolenic acid (9,12,15-octadecatrienoic acid). The chemical structures of these products were determined by gas chromatography-mass spectrometry and proton and 13 C nuclear magnetic resonance analyses. The optimum incubation temperature was 30°C for production of both hydroxy-THFAs. 13,16-Dihydroxy-THFA was detected after 2 days of incubation, and the concentration reached 45 mg/50 ml after 7 days of incubation; 7,13,16-trihydroxy-THFA was not detected after 2 days of incubation, but the concentration reached 9 mg/50 ml after 7 days of incubation. The total yield of both 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA was 67% (wt/wt) after 7 days of incubation at 30°C and 200 rpm. In previous studies, it was reported that Clavibacter sp. strain ALA2 oxidized the C-7, C-12, C-13, C-16, and C-17 positions of linoleic acid (n-6) into hydroxy groups. In this case, the bond between the C-16 and C-17 carbon atoms is saturated. In α-linolenic acid (n-3), however, the bond between the C-16 and C-17 carbon atoms is unsaturated. It seems that enzymes of strain ALA2 oxidized the C-12-C-13 and C-16-C-17 double bonds into dihydroxy groups first and then converted them to hydroxy-THFAs.

Oleic acid (cis-9-octadecenoic acid) was converted in excellent yield to the estolide, which was then esterified with 2,2-dimethypropan-1-ol (neopentyl alcohol), cis-9-octadence-1-ol (oleyl alcohol), and 2-propanol to generate the corresponding estolide esters. Higher-formula mass estolide esters were synthesized by reaction of the parent estolide with 1,3-propanediol, 2,2-dimethyl-1,3-propanediol, and 1,5-pentanediol to give the corresponding diesters of oleic estolide, thus doubling the molecular size of the parent estolide. Pour points and viscosities were determined in order to evaluate these products for possible industrial application.

It was previously reported that males of the crucifer flea beetle, Phyllotreta cruciferae, feeding on host foliage are attractive to both males and females in the field. Based on this evidence for an aggregation pheromone, volatiles were collected from male and female P. cruciferae feeding on cabbage (Brassica oleracea) and analyzed. For comparison, volatiles were also collected from males and females of three other flea beetle species, Aphthona flava,A. czwalinae, and A. cyparissiae, all feeding on their host, leafy spurge foliage (Euphorbia esula). Six male-specific compounds were isolated from P. cruciferae, and the same compounds plus two additional ones were isolated from males of Aphthona flava,A. czwalinae, and A. cyparissiae. The blends of compounds were relatively consistent within species, but there were characteristic differences between species. Compound structures were studied by mass spectrometry, NMR spectroscopy, UV spectroscopy, polarimetry, chiral and achiral gas chromatography, molecular modeling, and microchemical tests. Three of the compounds were identified as (+)-ar-himachalene; (+)-trans-α-himachalene; (+)-γ-cadinene. Two others were new enantiomers of himachalene hydrocarbons that were previously identified from the fir trees, Abies alba and Abies nordmanniana. Finally, there were two himachalene alcohols and one norsesquiterpene ketone that is a himachalene analog. Only (+)-ar-himachalene and (+)-γ-cadinene are previously known natural products. Electrophysiological activity was demonstrated for five of the compounds. The chemical and electrophysiological patterns are consistent with, but do not prove, a pheromonal function.

Clavibacter sp. ALA2 transformed linoleic acid into a variety of oxylipins. In previous work, three novel fatty acids were identified, (9Z)-12, 13, 17-trihydroxy-9-octadecenoic acid and two tetrahydrofuran-(di)hydroxy fatty acids. In this report, we confirm the structures of the tetrahydrofuran-(di)hydroxy fatty acids by nuclear magnetic resonance as (9Z)-12-hydroxy-13,16-epoxy-9-octadecenoic acid and (9Z)-7,12-dihydroxy-13,16-epoxy-9-octadecenoic acid. Three other products of the biotransformation were identified as novel heterobicyclic fatty acids, (9Z)-12,17;13, 17-diepoxy-9-octadecenoic acid, (9Z)-7-hydroxy-12,17;13,17-diepoxy-9-octadecenoic acid, and (9Z)-12,17;13,17-diepoxy-16-hydroxy-9-octadecenoic acid. Thus, Clavibacter ALA2 effectively oxidized linoleic acid at C-7, -12, -13, -16, and/or -17.

Allylic amination of methyl oleate with bis(N-p-toluenesulfonyl) sulfodiimide results in a mixture of methyl 11-amino-(N-p-toluenesulfonyl)-9-E-octadecenoate and methyl 8-amino-(N-p-toluenesulfonyl)-9-E-octadecenoate in 58% yield. These novel products were isolated and characterized by nuclear magnetic resonance, infrared spectroscopy, mass spectrometry, and melting point. The reaction was analyzed by highperformance liquid chromatography and thin-layer chromatography.

Several symmetrical alkenes were reacted with the selenium dioxide/tert.-butylhydroperoxide system to give three hydroxylated products each. These products were those of allylic mono- and dihydroxylation (meso andthreo dihydroxy compounds) of the double bond. Some dihydroxy products were hydrogenated to give saturated 1,4-diols. The compounds were characterized by nuclear magnetic resonance. The products have potential for application in commercial products, such as biodiesel, lubricants, greases, and cosmetics.

Several symmetrical alkenes were reacted with the selenium dioxide/tert.-butylhydroperoxide system to give three hydroxylated products each. These products were those of allylic mono- and dihydroxylation (meso andthreo dihydroxy compounds) of the double bond. Some dihydroxy products were hydrogenated to give saturated 1,4-diols. The compounds were characterized by nuclear magnetic resonance. The products have potential for application in commercial products, such as biodiesel, lubricants, greases, and cosmetics.

Stationary-phase cells of a corynebacterium (FUI-2) and a bacillus (NRRL B-14864) isolate, when grown aerobically in 1% YE medium at 25°C, converted 12-hydroxystearic acid to a major compound, 5-n-hexyl-tetrahydrofuran-2-acetic acid, and other intermediate and minor compounds (6-hydroxydodecanoic acid, 4-hydroxydecanoic acid, 4-ketodecanoic acid and γ-decanolactone). The yields of 5-n-hexyl-tetrahydrofuran-2-acetic acid, 4-hydroxydecanoic acid, and γ-decanolactone, byBacillus lentus NRRL B-14864 were 43%, 18% and 5%, respectively, after 2.5 d of incubation.

A bacterium, NRRL B-14797, isolated from composted manure, converted oleic acid exclusively to 10(R)-hydroxystearic acid in 3-day batch cultures. 9(Z)-Unsaturated fatty acids in a lipid extract from soybean soapstock were also hydrated effectively. Aerobic bioconversions by isolate B-14797 were compared with those byPseudomonas B-2994 andNocardia 5767, which produce mixtures of 10-hydroxy- and 10-ketostearic acids. The results of studies with resting cells and cell-free extracts were consistent with action of a hydratase and absence of secondary alcohol dehydrogenase in strain B-14797.

The triacylglycerols ofVernonia galamensis andCrepis alpina seed oils were characterized because these oils have high concentrations of vernolic (cis-12,13-epoxy-cis-9-octadecenoic) and crepenynic (cis-9-octadecen-12-ynoic) acids, respectively. The triacylglycerols were separated from other components of crude oils by solid-phase extraction, followed by resolution and quantitation of the individual triacylglycerols by reversed-phase high-performance liquid chromatography with an acetonitrile/methylene chloride gradient and flame-ionization detection. Isolated triacylglycerols were characterized by proton and carbon nuclear magnetic resonance and by capillary gas chromatography of their fatty acid methyl esters. The locations of the fatty acids on the glycerol moieties in the oils were obtained by lipolysis. TheVernonia galamensis oil contained 50% trivernoloyl and 21% divernoloyllinoleoyl glycerols along with 20% triacylglycerols with one vernolic and two other fatty acids. TheCrepis alpina oil contained 36% tricrepenynoyl and 33% dicrepenynoyllinoleoyl glycerols, 17% triacylglycerols with two crepenynic and one other fatty acid and 7% triacylglycerols with one crepenynic acid and two other fatty acids. Vernolic acid was found at both the 1(3)- and 2-glycerol carbons but was more abundant at the 1,(3)-position in theVernonia galamensis oil. Crepenynic acid was found at both glycerol carbon positions but was more abundant at the 2-position in theCrepis alpina oil.

Males of Carpophilus hemipterus (L.), the dried-fruit beetle, (Coleoptera: Nitidulidae) were found to emit nine all-E tetraene and one all-E triene hydrocarbons in addition to two pheromonally active tetraenes that had been reported previously. The previously known compounds are (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene (1) and (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene (2). The new tetraenes were all related to structure 1 by having one additional carbon at either one or two of the following four locations: at carbon 1 of the chain, at carbon 10 of the chain, at the 5-alkyl branch, or at the 7-alkyl branch. (Structure 2 also fits within this pattern.) The triene in C. hemipterus is (2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene. Also identified from volatile collections from the beetles were the 2Z and 4Z isomers of 1. All structures were proven by synthesis, with NMR and mass spectral data for the compounds provided. Two of the newly discovered compounds, (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, were quite active in the wind-tunnel bioassay, but others, such as (2E,4E,6E,8E)-5-ethyl-3,7-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-4,6,8-trimethyl-2,4,6,8-undecatetraene were not. Structure-activity relationships are explored among the natural compounds and additional, synthetic analogs, which were never detected from the beetles. Some of these analogs, such as (2E,4E,6E,8E)-3,5-dimethyl-7-propyl-2,4,6,8-undecatetraene, were quite active in the bioassay. The biosynthesis of the beetle-derived compounds is discussed. A single biosynthetic scheme that lacks complete enzyme specificity at four specific steps could account for the entire series of compounds found in the beetles and their relative proportions. The definition of "pheromone" is discussed in relation to these hydrocarbons.