Germacrenes from fresh costus roots

Four germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots (Saussurea lappa). The structures of (+)-germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of these compounds yields (-)-beta-elemene, (-)-elema-1,3,11(13)-trien-12-ol, (-)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien-12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the germacrenes yields selinene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are artefacts.     

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.     

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.     

Biosynthesis of costunolide,dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory

Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.     

Isolation and characterization of two germacrene A synthase cDNA clones from chicory

Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K(m) value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.     

8-Hydroxypegolettiolide,a sesquiterpene lactone with a new carbon skeleton and further constituents from Pegolettia senegalensis

The investigation of Pegolettia senegalensis afforded several new sesquiterpene lactones, eight cis-6,12-germacra-trans,trans-1(10),4,11-trienolides, five cis-6,12-eudesmanolides, two elemanolides, 8,14-cyclogermacra-1(10),4,7(11)-trien-6,12-olide with a new carbon skeleton, three germacra-1(10),4,11(13)-trien-12-oic acids with ester residues at C-8, 18-hydroxygeranyl nerol, 1,3-dihydroxyoctadecane and a mixture of esters of 3,4-dihydroxy-dihydrocinnamyl alcohol. The structures were elucidated by high field ¹HNMR spectroscopy and some chemical transformations. The C-10 configuration of 6,12-cis-eudesmanolides from Calostephane divaricata and Inula crithmoides most likely has to be corrected. The chemotaxonomic situation of the genus Pegolettia and biogenetic considerations are discussed briefly.     

Anti-tumor promoting diterpenes from the stem bark of Thuja standishii (Cupressaceae).

Three new labdane-type diterpenoids, labda-8(17),13-dien-15,12R-olid-19-oic acid (1), 12S-hydroxylabda-8(17),13(16),14-trien-19-oic acid (2) and 13-ethoxylabda-8(17),11,14-trien-19-oic acid (3), along with known diterpenoids, trans-communic acid (4), totarol (5), 12-methoxyabieta-8,11,13-trien-11-ol (6), and 7 alpha,8 alpha-epoxy-6 alpha-hydroxyabieta-9(11),13-dien-12-one (7) were isolated from the stem bark of Thuja standishii. The structures of 1--3 were established by spectroscopic methods and chemical conversion. These compounds together with standishinal (8), 12-hydroxy-6,7-seco-abieta-8,11,13-trien-6,7-dial (9) and 6 alpha-hydroxysugiol (10) were tested for their inhibitory effects on Epstein--Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compound 10 showed strong inhibitory effect on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA), and compounds 2 and 6 showed moderate inhibitory effects on EBV-EA induction. In addition, 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11) was found to exhibit the anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.     

The germacranolides of Viguiera buddleiaeformis structures of budlein-A and -B

The structure of budlein-A, the main sesquiterpene lactone of Viguiera buddleiaeformis was established as the 8 angeloyl ester of 1 keto, 8-β, 14-dihydroxy germacra-2,4,11 (13)-trien-3, (10 β) oxido-6 α, 12-olide. Its structure and stereochemistry was determined by chemical and spectroscopic means. Budlein-B, found in the same plant as a minor constituent, is 8 α, 15-dihydroxygermacra-1 (10), 4, 11 (13)-trien-6 α, 12-olide.     

Ent-kauren-19-oic acid derivatives from the stem bark of Croton pseudopulchellus Pax

Two new ent-kauren-19-oic acid derivatives, ent-14S*-hydroxykaur-16-en-19-oic acid and ent-14S*,17-dihydroxykaur-15-en-19-oic acid together with eleven known compounds ent-kaur-16-en-19-oic acid, ent-kaur-16-en-19-al, ent-12β-hydroxykaur-16-en-19-oic acid, ent-12β-acetoxykaur-16-en-19-oic acid, 8R,13R-epoxylabd-14-ene, eudesm-4(15)-ene-1β,6α-diol, (?)-7-epivaleran-4-one, germacra-4(15), 5E,10(14)-trien-9β-ol, acetyl aleuritolic acid, β-amyrin, and stigmasterol were isolated from the stem bark of Croton pseudopulchellus (Euphorbiaceae). Structures were determined using spectroscopic techniques. Ent-14S*-hydroxykaur-16-en-19-oic acid, ent-kaur-16-en-19-oic acid, ent-12β-hydroxykaur-16-en-19-oic acid, ent-12β-acetoxykaur-16-en-19-oic acid and 8R,13R-epoxylabd-14-ene were tested for their effects on Semliki Forest virus replication and for cytotoxicity against human liver tumour cells (Huh-7 strain) but were found to be inactive. Ent-kaur-16-en-19-oic acid, the major constituent, showed weak activity against the Plasmodium falciparum (CQS) D10 strain.     

石菖蒲的化学成分研究

运用色谱法从石菖蒲根茎提取物中分离得到18个化合物,经波谱学分析鉴定为:(7S,8R)-4,9’-di-hydroxyl-3,3’-dimethoxyl-7,8-dihydrobenzofuran-1’-propylneolignan(1),(7S,8R)-4,9’-dihydroxyl-3,3’-dimethoxyl-7,8-dihydrobenzofuran-1’-propylneoligan-9-O-β-D-glucopyranoside(2),7’-hydroxylariciresinol-9-acetate(3),5-羟基-3,7,4’-三甲氧基黄酮(4),野漆树苷(5),紫云英苷(6),松属素-3-O-芸香糖苷(7),山奈酚-3-O-芸香糖苷(8),德钦红景天苷(9),isoschaftoside(10),5-羟甲基糠醛(11),反式桂皮酸(12),3,7-dihydroxy-11,15,23-trioxo-lanost-8,16-dien-26-oicacid(13),3,7-dihydroxy-11,15,23-trioxo-lanost-8,16-dien-26-oic acid methyl ester(14),环阿屯醇(15),胡萝卜苷(16),羽扇豆醇(17),(22E,24R)-ergosta-5,7,22-trien-3β-ol(18)。除化合物4、11和16外,其余15个化合物均为首次从该植物中分离得到。     

Synthesis and Microbial Transformation of Ent-12β-hydroxykaur-16-ene

A study was made of the microbial transformation of cis-abienol, a main precursor of the tobacco aroma principle, by an unidentified soil bacterium JTS-162. Four new compounds were isolated from the culture broth as transformation products. They were elucidated to be (12Z)-labda-8(17),12,14-triene, (12Z)-labda-8(17),12,14-trien-18-ol, (12Z)-labda-8(17),12,14-trien-18-al and 4,18,19-tri-nor-3,4-seco-5-oxo-(12Z)-labda-8(17),12,14-trien-3-oic acid by spectrometry and syntheses. Based on experiments with a degradation sequence, the biotransformation pathway of cis-abienol by the bacterium was proposed.     

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.     

Volatile components from European liverworts Marsupella emarginata,M. aquatica and M. alpina

The hydrodistillation products of the liverworts Marsupella emarginata, M. aquatica and M. alpina were investigated by spectroscopic methods. A number of new compounds could be isolated by preparative gas chromatography (GC) and identified by spectroscopic techniques including GC-mass spectrometry, NMR and chemical correlations in conjunction with enantioselective GC. From M. emarginata, in addition to many known compounds, the sesquiterpene hydrocarbon (-)-7-epi-eremophila-1(10),8,11-triene (1) and the sesquiterpene derivatives (-)-4-epi-marsupellol (2), (-)-marsupellol acetate (18), (-)-4-epi-marsupellol acetate (4), (+)-5-hydroxymarsupellol acetate (5) and (-)-9-acetoxygymnomitr-8(12)-ene (24) could be identified. In M. aquatica the sesquiterpene hydrocarbons (-)-myltayl-8(12)-ene (7), ent-(+)-amorpha-4,11-diene (8), (-)-amorpha-4,7(11)-diene (9), the sesquiterpene alcohol (+)-9-hydroxyselina-4,11-diene (10) and (-)-2-acetoxyamorpha-4,7(11)-diene (11) were identified. In M. alpina (-)-trans-selina-4(15),11-dien-5-ol (12), (+)-8,9-epoxyselina-4,11-diene (13) and (+)-cis-selina-4(15),11-dien-5-ol (14) were found as new natural products.     

CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis.     

Antifungal highly oxygenated guaianolides and other constituents from Ajania fruticulosa.

Three highly oxygenated guaianolides were isolated from the aerial parts of Ajania fruticulosa along with 17 known phytochemicals including a triterpene (alpha-amyrin), two plant sterols (beta-sitosterol, daucosterol), four flavonoids (axillarin, centaureidin, santin and 5,7,4'-trihydroxy-3,3'-dimethoxyflavone), and ten sesquiterpenes [1alpha-hydroperoxy-4beta,8alpha,10alpha,13-tetrahydroxyguaia-2-en-12,6alpha-olide, 1alpha-hydroperoxy-4alpha,10alpha-dihydroxyguaia-9alpha-angeloyloxyguaia-2,11(13)-dien-12,6alpha-olide, 3beta,4alpha-dihydroxyguaia-11(13),10(14)-dien-12,6alpha-olide, 1alpha,4alpha,10alpha-trihydroxy-9alpha-angeloyloxyguaia-2,11(13)-dien-12,6alpha-olide, 1beta,2beta-epoxy-3beta,4alpha,10alpha-trihydroxy-guaia-11(13)-en-12,6alpha-olide and 2-oxo-8alpha-hydroxyguaia-1(10),3,11(13)-trien-12,6alpha-olide, ketoplenolide B, alantolactone, 9beta-hydroxyeudesma-4,11(13)-dien-12-oic acid and 9beta-acetoxyeudesma-4,11(13)-dien-12-oic acid]. The structures of the three guaianolides were elucidated by a combination of spectroscopic methods (EIMS, HREIMS, COSY, HMQC, HMBC and NOESY) as 1beta,2beta-epoxy-3beta,4alpha,8beta,10alpha-tetrahydroxyguaia-11(13)-en-12,6alpha-olide (1), 1beta,2beta-epoxy-3beta,4alpha,9alpha,10alpha-tetrahydroxyguaia-11(13)-en-12,6alpha-olide (2) and 1beta,2beta-epoxy-10alpha-hydroperoxy-3beta,4alpha,8beta-trihydroxyguaia-11(13)-en-12,6alpha-olide (3), respectively. Antifungal bioassay of all isolates showed that guaianolides 1, 2, 3, and 1beta,2beta-epoxy-3beta,4alpha,10alpha-trihydroxyguaia-11(13)-en-12,6alpha-olide were inhibitory to the growth of Candida albicans with MICs being 20, 20, 20, and 40 microg/ml, respectively.     

思茅松松香中的一个新二萜

从思茅松（Pinus kesiya var.langbianensis）的松香中分离得到一个新奇的松香烷二萜化合物——思茅松素,经现代波谱分析将其化学结构确定为13（14）-烯-8,12-环氧-1-松香酸（1）,同时分离得到5个已知化合物,分别为abiet-8,11,13-trien-15-hydroxy-18-oic acid（2）;pimarol（3a）;iso-pimarol（3b）;abiet-trien-18．oic acid（4）;15．hydroxy abietic acid（5）。     

Triterpenes in Ganoderma lucidum

Four new oxygenated triterpenes, isolated from the mycelia of the fungus Ganoderma lucidum, were determined to be lanosta-7,9(11),24-trien-3α, 15α-dihydroxy-26-oic acid, lanosta-7,9(11),24-trien-3β, 15α-dihydroxy-26-oic-acid, lanosta-7,9(11),24-trien-3β,22β-diacetoxy-15α-hydroxy-26-oic acid and lanosta-7,9(11),24-trien-15α,22β-diacetoxy-3β-hydroxy-26-oic acid by spectroscopic methods.     

Diterpenoids and a sesquiterpene lactone from viguiera ladibractate

Eight known diterpene acids, ent-12-oxokaur-9(11),16-dien-19-oic acid, ent-12β-hydroxykaur-9(11),16-dien-19-oic acid, ent-isokaur-15(16)-en-17,19-dioic acid, ent-15α,16-epoxy-17-hydroxykaura-19-oic acid, ent-kaura-17,19-dioic acid, ent-kaur-16-en-19-oic acid, grandifloric acid, angeloyloxygrandifloric acid, as well as a new sesquiterpene lactone, ladibranolide, were isolated from Viguiera ladibractate. The stereochemistry of the sesquiterpene lactone was established by NOE experiments.     

Two New Triterpenoids from the Roots of Sanguisorba officinalis L.

Two new triterpenoids, octanordammar- 1,11,13(17)-trien- 17-ol-3,16-dione (1) and lup- 12-en- 15α,19β-diol-3,11-dioxo-28-oic acid (4), as well as 13 known compounds were isolated from the roots of Sanguisorba officinalis L. (Rosaceae). Their structures were determined using spectroscopic methods.     

Antialgal ent-labdane diterpenes from Ruppia maritima

Seven ent-labdane diterpenes have been isolated from Ruppia maritima. The structures 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-al; 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-ol acetate; methyl 15,16-epoxy-12-oxo-ent-labda-8(17),13(16),14-trien-19-oate; 15,16-epoxy-ent-labd-8(17),13E-dien-15-ol and 13-oxo-15,16-bis-nor-ent-labd-8(17)-ene have been assigned to the five new compounds by spectroscopic means and chemical correlations. The phytotoxicity of the diterpenes has been assessed using the alga Selenastrum capricornutum as organism test.