Repraesentins D,E and F,New Plant Growth Promoters from Lactarius repraesentaneus

Three new plant growth regulatory sesquiterpenes were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be lactarane sesquiterpenes, namely repraesentins D (1) and E (2), and a protoilludane-related sesquiterpene, namely repraesentin F (3). Repraesentin E (2) showed the strongest promotion activity, 164% at 3.6 μM, of the three compounds toward the radicle elongation of lettuce seedlings.     

New plant growth promoters,repraesentins A,B and C,from Lactarius repraesentaneus

Three new plant growth regulatory sesquiterpenoids were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be a protoilludene sesquiterpene, namely repraesentin A (1), and two related sesquiterpenes, namely repraesentins B (2) and C (3). Compounds 1-3 showed promotion activities toward the radicle elongation of lettuce seedlings by 136%, 118% and 184% at 67 ppm, respectively.     

Bioactive terpenoids from sunflower leaves cv. Peredovick

The CH(2)Cl(2) extract of dried leaves of Helianthus annuus L. cv. Peredovick(R) has yielded, in addition to the known sesquiterpene lactones annuolide E and leptocarpin, and the sesquiterpenes heliannuols A, C, D, F, G, H, I, the new bisnorsesquiterpene, annuionone E, and the new sesquiterpenes heliannuol L, helibisabonol A and helibisabonol B. Structural elucidation was based on extensive spectral (one and two-dimensional NMR experiments) and theoretical studies. The sesquiterpenes heliannuol A and helibisabonol A and the sesquiterpene lactone leptocarpin inhibited the growth of etiolated wheat coleoptiles.     

Sesquiterpenes from Cupressus macrocarpa foliage

Ten sesquiterpenes, many with unusual carbon skeletons, were identified in foliage of Cupressus macrocarpa. These are (-)-10-epi-beta-acoradiene; ent-widdra-2,4(14)-diene; (E)-iso-gamma-bisabolene, i.e., (4E)-4-(1,5-dimethylhex-5-enylidene)-1-methylcyclohexene; (-)-cumacrene, i.e., (4S)-4-[(1R,2S)-2-isopropenyl-1-methylcyclobutyl]-1-methylcyclohexene; (-)-alpha-chamipinene, i.e., (1S,6S,7S)-2,2,6,8-tetramethyltricyclo[5.3.1.01,6]undec-8-ene; and five sesquiterpenes with a 3,3,4'-trimethyl-1,1'-bi(cyclohexyl) skeleton for which the trivial name macrocarpane is proposed. The possible single-enzyme biogenesis of these sesquiterpenes is discussed.     

The influence of intact-plant and excised-leaf bioassay designs on volicitin- and jasmonic acid-induced sesquiterpene volatile release in Zea mays

Induced plant responses to insect attack include the release of volatile chemicals. These volatiles are used as host-location signals by foraging parasitoids, which are natural enemies of insect herbivores. A plant's response to herbivory can be influenced by factors present in insect oral secretions. Volicitin (N-(17-hydroxylinolenoyl)-L-glutamine), identified in beet armyworm (Spodoptera exigua) oral secretions, stimulates volatile release in corn (Zea mays L.) seedlings in a manner similar to beet armyworm herbivory. Volicitin is hypothesized to trigger release of induced volatiles, at least in part, by modulating levels of the wound hormone, jasmonic acid (JA). We compare the sesquiterpene volatile release of damaged leaves treated with aqueous buffer only or with the same buffer containing volicitin or JA. Leaves were damaged by scratching with a razor and test solutions were applied to the scratched area. The leaves were either excised from the plant or left intact shortly after this treatment. Plants were treated at three different times (designated as Evening, Midnight, and Morning) and volatiles were collected in the subsequent photoperiod. JA and volicitin treatments stimulated the release of volatile sesquiterpenes, namely beta-caryophyllene, (E)-alpha-bergamotene, and (E)-beta-farnesene. In all cases, JA stimulated significant sesquiterpene release above mechanical damage alone. Volicitin induced an increase in sesquiterpene volatiles for all excised-leaf bioassays and the Midnight intact plants. Volicitin treatments in the Evening and Morning intact plants produced more sesquiterpenes than the untreated controls, while mechanical damage alone produced an intermediate response that did not differ from either treatment group. Excised leaves produced a 2.5- to 8.0-fold greater volatile response than similarly treated intact plants. Excision also altered the ratio of JA-and volicitin-induced sesquiterpene release by preferentially increasing (E)-beta-farnesene levels relative to beta-caryophyllene. The inducibility of volatile release varied with time of treatment. On average, sesquiterpene release was highest in the Midnight excised leaves and lowest in the Morning intact plants. The duration of induced volatile release also differed between treatments. On average, JA produced a sustained release of sesquiterpenes over time, with over 20% of the combined sesquiterpenes released in the third and final volatile collection period. In contrast, less than 8% of the combined sesquiterpenes induced by volicitin were emitted during this period. The large quantitative differences between intact plants and detached leaves suggest that the results of assays using excised tissues should be cautiously interpreted when considering intact-plant models.     

Novel Zierane‐ and Guaiane‐Type Sesquiterpenes from the Root of Melicope denhamii

Two novel zierane‐type sesquiterpenes, named melicodenones A and B ( 1 and 2 , resp.), and three new guaiane‐type sesquiterpenes, named melicodenones C–E ( 3 – 5 ), were isolated from the root of Melicope denhamii (Seem. ) T. G. Hartley together with zierone ( 6 ). Their structures were established by extensive NMR‐spectroscopic analyses. Compounds 1 – 6 were tested for cytotoxicity using human colon cancer DLD‐1 cells, and melicodenone A ( 1 ) was found to exhibit moderate activity.     

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.     

Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.     

Sesquiterpenes from Achillea pannonica Scheele

From dichloromethane extracts of flowerheads of Achillea pannonica Scheele three sesquiterpenes were isolated and identified: 11,13-dehydrodesacetylmatricarin, (6E)-5-tigloxy-9-hydroxynerolidol and alpha-longipin-2-en-1-one. The structures were determined by MS, IR and NMR spectroscopic analyses, (6E)-5-Tigloxy-9-hydroxynerolidol is reported here for the first time. Additionally spathulenol, a compound of the essential oil was identified using GC-MS and GC-FTIR.     

Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.     

Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase: a single-point mutation converts aristolochene synthase into an (E)-beta-farnesene synthase

Sesquiterpene cyclases, many of which share significant structural similarity, catalyze the cyclization reactions of the universal alicyclic precursor farnesyl pyrophosphate to produce more than 300 different hydrocarbon skeletons with high regio- and stereospecificity. The molecular basis of this exquisite specificity is not well-understood, but the conformation adopted by FPP in the active site of a sesquiterpene cyclase is thought to be an important determinant of the reaction pathway. Aristolochene synthase (AS) from Penicillium roqueforti catalyzes the cyclization of farnesyl pyrophosphate to the bicyclic sesquiterpene aristolochene. The X-ray structure of AS suggested that the steric bulk of residue 92 was central in binding of FPP to the active site of AS in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused Decalin S-germacrene A. We demonstrate here that reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depended linearly on the size of the residues at position 92. ASY92A, in which Tyr92 had been replaced with Ala, produced almost 80% of alicyclic sesquiterpenes, suggesting an energetic separation of less than 0.8 kcal/mol between the cyclic and noncyclic reaction pathways. A mechanism by which FPP binds to the mutant enzymes in an extended conformation is proposed to explain the altered selectivity. The mutants also produced small amounts of additional hydrocarbons with a molecular weight of 204, namely, alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene. The production of (E)-beta-farnesene and beta-bisabolene suggested that the initial cyclization of FPP to germacrene A in AS proceeded in a stepwise fashion through farnesyl cation.     

Kelsoenethiol and dikelsoenyl ether,two unique kelsoane-type sesquiterpenes,from the Formosan soft coral Nephthea erecta

Two new kelsoane-type sesquiterpenes, namely kelsoenethiol (1) and dikelsoenyl ether (2), were obtained from the Formosan soft coral Nephthea erecta. Their structures were elucidated through extensive spectroscopic analyses, ESI orbitrap mass and quantum chemical calculations (QCC). The cytotoxicity against A-459 (human lung carcinoma), P-388 (mouse lymphocytic leukemia), and HT-29 (human colon adenocarcinoma) cancer cell lines of 1 and 2 was evaluated in vitro. Compound 1 showed cytotoxicity against P-388 and HT-29 cells with ED_50s of 1.3 and 1.8 μg/mL, respectively.     

Peroxisome Proliferator Activated Receptor-γ Agonistic Compounds from the Jellyfish-Derived Fungus Cladosporium oxysporum

In our search for peroxisome proliferator-activated receptor (PPAR) agonists, five undescribed compounds, namely two acyclic diterpenes ( 1 and 2 ; cladopsol A and cladopsol B), two sesquiterpenes ( 3 and 4 ; cladopsol C and cladopsol D), and one C21-ecdysteroid ( 5 ; cladopsol E), and 15 known compounds were isolated from the jellyfish-derived fungus – Cladosporium oxysporum. The structures of the undescribed compounds were defined using UV, NMR, HR-ESI-MS, and electronic circular dichroism (ECD) spectroscopy and a modified Mosher's method. Luciferase reporter assay and docking analysis suggested that cladopsol B may function as a PPAR-γ partial agonist with a potential antidiabetic lead which may evade the side effects of full agonists. Moreover, cladopsol B stimulated glucose uptake in HepG2 cells with an efficacy comparable to that of rosiglitazone, but with less side effect induced by lipid accumulation in 3T3-L1 cells. Therefore, cladopsol B could serve as a molecular skeleton in a study of advanced antidiabetic lead with less side effect.     

Sesquiterpene lactones from Smyrnium olusatrum.

Fruits of Smyrnium olusatrum afforded three sesquiterpene lactones, namely, 1beta,8beta-dihydroxyeudesman-3,7(11)-dien-8alpha,12-olide, 1beta,8beta-dihydroxyeudesman-4(15),7(11)-dien-8alpha,l2-olide, and 1beta,10alpha,4alpha,5beta-diepoxy-6beta-hydroxyglechoman-8alpha,12-olide. Four related known sesquiterpenes were also isolated and characterized. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses. The in vitro cytotoxicity of the extract and isolated compounds against P-388 mouse lymphoma cells will be also discussed.     

Sesquiterpenes from Warburgia ugandensis and their antimycobacterial activity

The dichloromethane extract of the stem bark of Warburgia ugandensis afforded three new coloratane sesquiterpenes, namely: 6alpha,9alpha-dihydroxy-4(13),7-coloratadien-11,12-dial (1), 4(13),7-coloratadien-12,11-olide (2), and 7beta-hydroxy-4(13),8-coloratadien-11,12-olide (3), together with nine known sesquiterpenes, i.e., cinnamolide-3beta-acetate (4), muzigadial (5), muzigadiolide (6), 11alpha-hydroxymuzigadiolide (7), cinnamolide (8), 7alpha-hydroxy-8-drimen-11,12-olide (9), ugandensolide (10), mukaadial (11), ugandensidial (12), and linoleic acid (13). Their structures were assigned on the basis of 1D and 2D-NMR spectroscopic and GC-MS analysis. The compounds were examined for their antimycobacterial activity against Mycobacterium aurum, M. fortuitum, M. phlei and M. smegmatis; and the active constituents showed MIC values ranged from 4 to 128 microg/ml compared to the antibiotic drugs ethambutol (MIC ranged from 0.5 to 8 microg/ml) and isoniazid (MIC ranged from 1 to 4 microg/ml).     

Sesquiterpenoids and diarylheptanoids from Nidus vespae and their inhibitory effects on nitric oxide production

Two coriamyrtin-type sesquiterpenes, fengfangin A (1) and tutin (2), and six diarylheptanoids, namely alnusone (3), centrolobol (4), muricarpone B (5), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-one (6), (3S)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol (7), and (3S)-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)heptan-3-ol (8), were isolated from the 95% EtOH extract of nidus vespae, the nest of Polistes species. Their structures were identified by spectroscopic methods. Compounds 1 and 8 are new products. The absolute configuration of 1 was determined by single-crystal X-ray diffraction analysis using Flack parameter. The biological tests showed that compounds 5, 6, and 8 could inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells with IC(50) values in the range of 13-17 μM, whereas the sesquiterpenes were inactive in this assay (>25 μM). In addition, the ecological significance of the presence of neurotoxic sesquiterpene lactones in nidus vespae is briefly discussed.     

Partial purification and characterization of the short-chain prenyltransferases, gernayl diphospate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir)

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa fo     

(+/−)-Alashanoid N,Two Enantiomeric Sesquiterpenes from Syringa pinnatifolia

Two enantiomeric humulane sesquiterpenes, namely (+)-alashanoid N ( 1a ) and (−)-alashanoid N ( 1b ), along with two known analogs ((2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one ( 2 ) and (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one ( 3 )), were described from the peeled stems of a folk Mongolian herbal medicine Syringa pinnatifolia. Their structures were characterized based on UV, IR, NMR, and HR-ESI-MS data analyses, and the absolute configurations were determined by data analysis of X-ray diffraction and quantum chemical calculations. (+/−)-Alashanoid N showed inhibition against NO production in RAW 264.7 macrophage cells with IC₅₀ values of 90.1 μM and 71.7 μM, and protective effect against oxygen-glucose deprivation injury to H9c2 cells at a concentration of 20 μM and 5 μM, respectively.