Biosynthesis of Sesterterpenes,Head‐to‐Tail Triterpenes,and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head‐to‐tail triterpenes, and sesquarterpenes. We have identified an (all‐E)‐isoprenyl diphosphate synthase (E‐IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl‐β‐curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E‐IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone‐7, respectively.     

Diterpene Synthase-Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers

The diterpene synthase clerodienyl diphosphate synthase 1 (PvCPS1) from the crop plant switchgrass (Panicum virgatum) stereoselectively converts (E,E,E)-geranylgeranyl diphosphate (GGPP) into the clerodane natural product, cis-trans-clerodienyl diphosphate (CLPP, 1 ). Structure-guided point mutations of PvCPS1 redirected product stereoselectivity toward the formation of a rare cis-clerodane diastereomer, cis-cis-CLPP ( 2 ). Additionally, an alternative cis-clerodane diastereomer, (5S,8S,9R,10R)-13Z-CLPP ( 3 ), was produced when treating PvCPS1 and select variants thereof with the cis-prenyl substrate (Z,Z,Z)-nerylneryl diphosphate (NNPP). These results support the hypothesis that substrate configuration and minor active-site alterations impact precatalysis substrate folding in the stereoselective biosynthesis of clerodane diterpenoid scaffolds, and can be employed to provide enzymatic access to a broader range of bioactive clerodane natural products.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Insight into Isoprenoid Biosynthesis by Functional Analysis of Isoprenyl Diphosphate Synthases from Mycobacterium vanbaalenii and Mycobacterium tuberculosis

Comprehensive functional analyses of E-isoprenyl diphosphate synthases (E-IDSs) from nonpathogenic Mycobacterium vanbaalenii have been performed. Mv0992 and Mv1577 represent a nonaprenyl diphosphate (E-C₄₅) synthase and a geranylgeranyl diphosphate (E-C₂₀) synthase, respectively. Although Mv3536 was identified as an E-C₂₀ synthase using a single enzyme, co-incubation of Mv3536 and Z-IDSs (Mv4662 and Mv3822) strongly suggested it releases an intermediate geranyl diphosphate (E-C₁₀) during a continuous condensation reaction. Mv0992 and Mv3536 functions differed from those of the previously reported pathogenic Mycobacterium tuberculosis homologues Rv0562 and Rv2173, respectively. Re-analysis of Rv0562 and Rv2173 demonstrated that their functions were similar to those of Mv0992 and Mv3536 (Rv0562: E-C₄₅ synthase; Rv2173: E-C_10–15 synthase). The newly proposed functions of Rv0562 and Rv2173 would be in the biosynthesis of menaquinone and glycosyl carrier lipids essential for growth. Furthermore, a reduced allylic diphosphate could be used as the Z-IDS of the Mv3822 substrate, thereby introducing a potentially novel pathway of cyclic sesquarterpene biosynthesis.     

Sesquiterpene Synthases Cop4 and Cop6 from Coprinus cinereus: Catalytic Promiscuity and Cyclization of Farnesyl Pyrophosphate Geometric Isomers

Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of a fungal genome survey. This study investigates the reaction mechanism and catalytic fidelity of the two enzymes. Cyclization of all‐trans‐FPP ((E,E)‐FPP) was compared to the cyclization of the cis–trans isomer of FPP ((Z,E)‐FPP) as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases, which are capable of isomerizing the C2? C3 π bond of all‐trans‐FPP. Cop6 is a “high‐fidelity” α‐cuprenene synthase that retains its fidelity under various conditions tested. Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)‐FPP into multiple products, including (?)‐germacrene D and cubebol. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Cyclization of (Z,E)‐FPP by Cop4 and Cop6 yields products that are very different from those obtained with (E,E)‐FPP. Conversion of (E,E)‐FPP proceeds via a (6R)‐β‐bisabolyl carbocation in the case of Cop6 and an (E,E)‐germacradienyl carbocation in the case of Cop4. However, (Z,E)‐FPP is cyclized via a (6S)‐β‐bisabolene carbocation by both enzymes. Structural modeling suggests that differences in the active site and the loop that covers the active site of the two enzymes might explain their different catalytic fidelities.     

Chemical protection of pheromones containing an internal conjugated diene system from isomerization and oxidation

Conjugated diene systems are common in natural products, including pheromones. The systems are sensitive to heat, light, and oxygen, among other things. They can be protected by antioxidants and UV absorbers, which slow downcis-trans isomerization and oxidation. Three sex pheromones (one as an analog) containingZ,E, E,Z, andE,E units were studied: (Z,E)-9,11-C₁₄OAc, (E,Z)-7,9-C₁₂OAc, and (E,E)-10,12-C₁₆OAc. The UV absorber 2-hydroxy-4-methoxybenzophenone and the antioxidants BHT and BHA were found to be effective in solution. The protective effect of the UV absorber against photoisomerization on paper carriers was not as good as that in solution. Preliminary studies on the utilization of formulations containing these compounds and (Z,E)-9,11-C₁₄OAc in the mass trapping of Egyptian cotton leafworm male in cotton fields showed the new combinations to be as good as a previously used formulation with UOP 688, a compound which is unpleasant to handle.     

An artificial substrate for undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26

In order to investigate the reactivity of the homologs of homoallylic substrates, we examined the enzymatic reactions of E- or Z-3-methylhex-3-enyl diphosphate with farnesyl diphosphate using the undecaprenyl diphosphate synthase (UPS) of Micrococcus luteus B-P 26. E-3-methylhex-3-enyl diphosphate reacted with farnesyl diphosphate to give a chiral (4S)-(2Z,6E,10E,14E)-4-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. However, the Z-isomer is not accepted as a substrate for the bacterial UPS at all.     

Olefinic acetates, Δ-9,11–14: OAc and Δ-7,9–12: OAc used as sex pheromone components in three geometrid moths,Idaea aversata,I. straminata,andI. biselata (Geometridae,Lepidoptera)

Pheromone compounds so far identified from most geometrid moths consist of all-Z diene, triene, or tetraene hydrocarbons with chain lengths of C₁₇ to C₂₁, and their monoepoxide derivatives biosynthesized from linoleic and linolenic acids. The present study reports the occurrence of olefinic acetates as sex pheromones in three species of Geometridae. (Z,Z)-9,11-tetradecadienyl acetate and (Z,Z)-7,9-dodecadienyl acetate found in female gland extracts ofIdaea aversata elicited significant responses from conspecific male antennae in gas chromatography with electroantennographic detection (GCEAD). In extracts ofI. straminata, (Z,Z)-7,9-dodecadienyl acetate, (E,Z)-7,9-dodecadienyl acetate, and (Z,Z)-7,9-dodecadienyl acetate were found, and the synthetic compounds elicited strong responses from conspecific male antennae. In the third species,I. biselata, only (Z,Z)-7,9-dodecadienyl acetate was found in the female extracts, and this compound elicited a strong EAD response from the conspecific male antenna. The identities of the pheromone components inI. aversata andI. straminata were further confirmed according to their characteristic ions after GC-MS analyses. Single sensillum recordings fromI. aversata showed two types of pheromone-detecting sensilla present on the male antenna. One type contained two receptor neurons, one of which was specifically tuned to (Z,Z)-9,11-tetradecadienyl acetate, the other to (Z,E)-9,11-tetradecadienyl acetate. A second type contained one neuron responding to (Z,Z)-7,9-dodecadienyl acetate. The two types were clearly different also with respect to external morphology, the former being considerably longer and having a larger base diameter. Also inI. straminata two physiological types of sensilla could be distinguished. One type contained two neurons, one of which responded to (Z,Z)-7,9-dodecadienyl acetate, the other to (Z,E)-9,11-tetradecadienyl acetate. The second type contained one neuron, responding to (Z,Z)-7,9-dodecadienyl acetate. No correlation between external morphology and physiological response of the investigated sensilla was observed inI. straminata. In field tests, a two-component blend containing (Z,Z)-9,11-tetradecadienyl acetate and (Z,Z)-7,9-dodecadienyl acetate in a ratio of 10:1 was attractive to males ofI. aversata. This two-component blend was also attractive to males ofI. straminata, but in a ratio of 1:1. High numbers of maleI. biselata were caught in traps baited with (Z,Z)-7,9-dodecadienyl acetate alone. The incorporation of deuterium labels into pheromone components after topical application of deuterium-labeled palmitic acid confirmed that the pheromone components ofI. aversata could be synthesized from this precursor, as has been previously observed for acetate pheromone components of many other moth species. Our results suggest that an evolutionary reversal back to the production of palmitic acid-derived pheromone components has occurred within the geometrid subfamily Sterrhinae.     

Structure,Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase

We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a “catalytic ” subunit (SaHepPPS‐2) having two “DDXXD” motifs and a “regulatory” subunit (SaHepPPS‐1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (K_i～200 nm ) were N‐alkyl analogues of zoledronate containing ～C₆ alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially “rescued” by the addition of menaquinone‐7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.     

Novel Components of the Sex Pheromones Produced by Emerald Moths: Identification,Synthesis, and Field Evaluation

The subfamily Geometrinae (Lepidoptera: Geometridae) includes many species called emerald moths. Based on our recent finding of novel polyenyl compounds, including a double bond at the 12-position from two geometrine species, Hemithea tritonaria and Thalassodes immissaria intaminata, (6Z,9Z,12Z)-6,9,12-trienes and (3Z,6Z,9Z,12Z)-3,6,9,12-tetraenes with a C₁₇–C₂₀ straight chain were synthesized and analyzed by GC-MS. The 6,9,12-trienes, which were prepared by a double Wittig reaction between two alkanals and an ylide derived from (Z)-1,6-diiodo-3-hexene, characteristically produced fragment ions at m/z 79, 150, and M–98. The 3,6,9,12-tetraenes, which were prepared by a coupling between (Z)-3-alkenal and an ylide derived from (3Z,6Z)-1-iodo-3,6-nonadiene, showed fragment ions at m/z 79, 148, and M–96. These diagnostic ions were useful to distinguish these compounds from other known polyenyl pheromones, such as 4,6,9- and 6,9,11-trienes and 1,3,6,9-tetraenes. With reference to the GC-MS data, pheromone extracts of other species in Geometrinae inhabiting the Iriomote Islands were analyzed, and the 6,9,12-trienes were identified in the pheromone gland extracts of Pamphlebia rubrolimbraria rubrolimbraria and Maxates versicauda microptera. Furthermore, a field evaluation of the synthetic polyenes in a mixed forest of Tokyo revealed the following new male attractants for emerald moths: Idiochlora ussuriaria by a C₁₇ 6,9,12-triene and Jodis lactearia by a C₂₀ 3,6,9,12-tetraene, indicating the characteristic chemical structures of Geometrinae pheromones. On the other hand, through reexamination of the pheromone extract of H. tritonaria, (3E,6E)-α-farnesene was identified as an electrophysiologically active component in addition to the C₁₇ 6,9,12-triene. The binary mixture attracted more males than the single component lure baited with the triene in the Iriomote Islands.     

Sex pheromone of European grapevine moth (Lobesia botrana) its chemical transformations in sunlight and heat

Photo- and radical isomerization of (E, Z)-7,9-dodecadien-1-yl acetate (DDA) leads to an equilibrium mixture of all four possible geometric isomers of 7,9-DDA in the ratio ofE,E, 69–76%;Z,E, 11–13%;E, Z, 12–15%; andZ, Z, 1–3%. Iodine catalysis of the isomerization takes place even in dark at room temperature and is probably a radical reaction.     

Structural Analysis of the Minor Cerebrosides from a Glass Sponge Aulosaccus sp.

The minor cerebrosides from a Far‐Eastern glass sponge Aulosaccus sp. were analyzed as constituents of some multi‐component RP‐HPLC fractions. The structures of eighteen new and one known cerebrosides were elucidated on the basis of NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. These β‐D‐glucopyranosyl‐(1→1)‐ceramides contain sphingoid bases N‐acylated with straight‐chain (2R)‐2‐hydroxy fatty acids, namely, (2S,3S,4R,11Z)‐2‐aminoeicos‐11‐ene‐1,3,4‐triol, acylated with 15E‐22:1, 16Z‐21:1, 15Z‐21:1, 15Z‐20:1, 15E‐20:1, 19:0, 18:0 acids, (2S,3S,4R)‐2‐amino‐13‐methyltetradecane‐1,3,4‐triol—with 19Z‐26:1, 16Z‐23:1, 23:0, 22:0 acids, (2S,3S,4R)‐2‐amino‐14‐methylpentadecane‐1,3,4‐triol—with 16Z‐23:1, 16E‐23:1, 15Z‐22:1, 22:0 acids, (2S,3S,4R)‐2‐amino‐14‐methylhexadecane‐1,3,4‐triol, linked to 16Z‐23:1, 15Z‐22:1 acids, (2S,3S,4R)‐2‐amino‐9‐methylhexadecane‐1,3,4‐triol—to 16Z‐23:1 acid, and (2S,3S,4R)‐2‐aminohexadecane‐1,3,4‐triol, attached to 15Z‐22:1 acid. The 13‐methyl and 9‐methyl‐branched trihydroxy sphingoid base backbones (C₁₅ and C₁₇, respectively) have not been found previously in sphingolipids. The ceramide parts, containing other backbones, present new variants of N‐acylation of the marine sphingoid bases with the 2‐hydroxy fatty acids. The combination of the instrumental and chemical methods used in this study improved the efficiency of the structural analysis of such complex cerebroside mixtures that gave more detailed information on glycosphingolipid metabolism of the organism.     

Sex pheromone ofStenoma cecropia Meyrick (Lepidoptera: Elachistidae)

(Z,E)-9,11-tetradecadienal (Z9,E11–14: Ald; 11%), (Z,E)-9,11,13-tetradecatrienal (Z9,E11, 13–14: Ald; 67%), (Z,E)-9,11-tetradecadienyl acetate (Z9,E11–14: Ac, 5.5%), and (Z,E)-9,11,13-tetradecatrienyl acetate (Z9,E11,13–14: Ac; 16.5%) were identified in the extracts of female pheromone glands ofStenoma cecropia (Lepidoptera: Elachistidae). From electroantennograms and single sensillum recordings, receptors toZ9,E11,13–14:Ald andZ9,E11–14: Ald were found on male antenna. Behavioral data were obtained from olfactometric tests in the laboratory and field trapping experiments in Colombia. It appeared that a blend ofZ9,E11,13–14:Ald (83%) andZ9,E11–14:Ald (17%) was attractive to males. These aldehydes are assumed to be components of the sex pheromone ofS. cecropia, whereas the acetates found in gland extracts might be precursors of the pheromone.     

Long-chain alkenes of the haptophytes Isochrysis galbana and Emiliania huxleyi

The major alkenes of the haptophytes Isochrysis galbana (strain CCAP 927/14) and Emiliania huxleyi (strains CCAP 920/2 and VAN 556) have been identified by nuclear magnetic resonance spectroscopy and by mass spectrometric analysis of their dimethyl disulfide adducts. The dominant alkene in I. galbana is (22Z)-1,22-hentriacontadiene, with 1,24-hentriacontadiene and 1,24-tritriacontadiene present in much lower abundance; (22Z)-1,22-hentriacontadiene also occurs in E. huxleyi (strain CCAP 920/2), together with (2Z,22Z)-2,22-hentriacontadiene (the major hydrocarbon) and (3Z,22Z)-3,22-hentriacontadiene. Minor abundances of 2,24-hentriacontadiene and 2,24-tritriacontadiene are also present in this strain. In contrast, the dominant alkene in E. huxleyi (strain VAN 556) is (15E,22E)-1,16,23-heptatriacontatriene with the related alkatriene 1,15,22-octatriacontatriene also present and (22Z)-1,22-hentriacontadiene occurring as a minor component. From structural relationships (15E,22E)-1,15,22-heptatriacontatriene is proposed to derive from the same biosynthetic pathway as that of the characteristic C₃₇ alkenones which occur in both E. huxleyi and I. galbana. The C₃₁ and C₃₃ dienes likely derive from chain extension and decarboxylation of (Z)-9-octadecenoic acid or (Z)-7-hexadecenoic acid, using a pathway analogous to that elucidated previously in the chlorophyte Botryococcus braunii. Therefore, long-chain dienes and trienes, which can co-occur in haptophytes, may have distinct biosynthetic pathways. Dedicated to the memory of Dr. Gareth Rieley, 1969–1998.     

Production and release of (Z,E)-9,12-tetradecadienal by sex pheromone glands of females ofPlodia interpunctella (lepidoptera: pyralidae)

Extracts of sex pheromone glands obtained from females ofPloida interpunctella contained detectable amounts of (Z,E,)-9,12-tetradecadien-1-ol acetate (Z9,E12–14:Ac) and (Z,E.)-9,12-tetradecadien-1-ol (Z9,E12–14:OH) 4 hr prior to the first scotophase after adult emergence. The amount of pheromone increased during the first 4 hr of the scotophase and then declined to low levels during the subsequent photophase. Decapitation of females immediately after emergence, prior to expansion of the wings, inhibited production of pheromone during the subsequent 48 hr. Injection of extracts of the heads of 1-day-old females ofP. interpunctella of partially purified extracts of the cephalic ganglia of females of the corn earworm moth into decapitated females stimulated production of bothZ9,E12–14:Ac andZ9,E12–14:OH as well as production of (Z,E)-9,12-tetradecadienal (Z9,E12–14:Al). This aldehyde was subsequently identified from extracts of pheromone glands obtained from naturally calling females as well as from volatiles emitted by calling females. Studies on the terminal steps in biosynthesis of the pheromone showed thatZ9,E12–14:OH was produced from the corresponding acetate and thatZ9,E12–14:Al was produced from the alcohol via the action of an oxidase(s).     

Epoxidation of a conjugated linoleic acid isomer

Epoxidation of methyl (9Z, 11E)‐octadecadienoate ( 1 ) with various epoxidizing agents viz. m‐chloroperoxybenzoic acid, dimethyl dioxirane, methyltrioxorhenium/hydrogen peroxide, potassium peroxomonosulfate (Oxone, 2KHSO₅ · KHSO₄ · K₂SO₄)/tetrahydrothiopyran‐4‐one, and Novozyme 435/hydrogen peroxide is described. The reactions furnished the corresponding mono‐epoxy [methyl (11, 12E)‐epoxy‐(9Z)‐octadecenoate ( 2 ) and methyl (9, 10Z)‐epoxy‐(11E)‐octadecenoate ( 3 )] and a mixture of diastereomers of syn‐ and anti‐diepoxy‐stearate [methyl (9, 10Z;11, 12E)‐diepoxystearate ( 4a‐4d )], which were identified by a combination of spectroscopic and spectrometric analyses.     

Sex attractant for three species of the genusOncocnemis: O. chandleri (Grt.),O. cibalis (Grt.), andO. mackiei (B. & Benj.) (Lepidoptera: Noctuidae)

Oncocnemis chandleri, O. cibalis, andO. mackiei were attracted to chemically baited traps in the field. In all three cases, (5E,7Z)-dodecadienyl acetate was a key component for attraction. Attraction ofO. chandleri to traps baited with the (5E,7Z)-dodecadienyl acetate was inhibited by addition of (Z)-7-dodecenyl acetate.O. cibalis required both (5E,7Z)-dodecadienyl acetate and (Z)-7-dodecenyl acetate for attraction. Electroantennogram responses for the three species are also reported.     

Sex pheromone components of three species ofSemiothisa (Geometridae), (Z,Z,Z)-3,6,9-heptadecatriene and two monoepoxydiene analogs

Adult males ofSemiothisa signaria dispuncta (Walker) were attracted to field traps baited with (Z,Z,Z)-3,6,9-octadecatriene and (Z,Z)-6,9-cis-3,4-epoxy-octadecadiene. However, analyses of sex pheromone gland extracts of females of this species by GC-MS and by GC in combination with an electroantennograph detector (GC-EAD) showed the pheromone to be comprised of a mixture of the next lower homologs: (Z,Z,Z)-3,6,9-heptadecatriene and (Z,Z)-6,9-cis-3,4-epoxy-heptadecadiene. Blends of these two C₁₇ compounds were subsequently found to be more attractive to males in the field than the corresponding C₁₈ mixtures. Sex pheromones of two otherSemiothisa species were also found to contain C₁₇ components. (Z,Z,Z)-3,6,9-Heptadecatriene, detected by GC-EAD analysis of a female abdominal tip extract ofS. bicolorata (Fabricius), attracted conspecific males, and this attraction was significantly reduced by additions of (Z,Z)-6,9-cis-3,4-epoxyheptadecadiene, the major pheromone component ofS. signaria dispuncta, to the lure. (Z,Z)-3,9-cis-6,7-Epoxy-heptadecadiene was detected by GC-EAD analysis as the primary male antennal stimulatory component present in abdominal tip extracts ofS. ulsterata (Pearsall), and males of this species were attracted to traps baited with this epoxide. Each of these three C₁₇ compounds constitute previously unknown lepidopteran sex pheromone components. Blends of (Z,Z, Z)-3,6,9-heptadecatriene and (Z,Z)-3,9-cis-6,7-epoxyheptadecadiene attracted males of a fourth species,S. delectata Hulst, but no females of this species were obtained to permit analysis of its sex pheromone. The occurrence of (Z,Z,Z)-3,6,9-heptadecatriene inS. neptaria (Guenee) females was indicated by GC-MS analysis of an abdominal tip extract; however, no males were attracted to any of the fielded mixtures containing this hydrocarbon.     

Syntheses of pure (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z,11E)-9,11-hexadecadienals-possible candidate pheromones

The title compounds were prepared by six different routes, and recommendations are given for the more convenient procedures in laboratory-scale syntheses. Modifications in the literature preparations of the 9E,11E and 9E,11Z isomers are described. Baseline separation of a prepared mixture of all four isomers of the (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z, 11E)-9,11-hexadecadienals was achieved using GC methods with standard capillary columns. [¹³C]NMR spectroscopy of the alkene carbon atoms clearly differentiates between theZ,Z, E,E and eitherE,Z orZ,E isomers of the precursor dienols and thus of the dienals.     

Unusual Shorter-Chain C35 and C36 Alkenones from Commercially Grown Isochrysis sp. Microalgae

Unusual shorter-chain alkenones (C₃₅ and C₃₆) were the dominant alkenones (rather than C₃₇ and C₃₈) isolated from commercially grown Tisochrysis lutea microalgae. Structures of the alkenones were determined by analyzing the alkenones and their products following a butenolysis reaction by comprehensive two-dimensional gas chromatography coupled to a high-resolution time-of-flight mass spectrometer (GC × GC-TOF HRMS). The major alkenones were identified as (15E,22E)-heptatriaconta-15,22-dien-2-one and (16E,23E)-octatriaconta-16,23-dien-3-one, similar to one previous finding of abundant shorter-chain alkenones in a culture of Emiliania huxleyi CCMP2758, yet distinguishing them from C₃₅ and C₃₆ alkenones extracted from Black Sea sediments with different double bond positions. Our data suggest a possible shared trigger (e.g. nutrient stress) that may occur during cultivation resulting in shorter-chain alkenones from both the aforementioned E. huxleyi and T. lutea. Moreover, for T. lutea, short-chain alkenone biosynthesis can be accompanied by lower alkenone content but higher amounts of valuable docosahexaenoic acid (DHA). These results, therefore, extend beyond alkenones and are important to established and emerging algal oil-based industries.