Fusicocca-3(16),10(14)-diene, and beta- and delta-araneosenes, new fusicoccin biosynthesis-related diterpene hydrocarbons from Phomopsis amygdali

Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.     

Biosynthesis of rice phytoalexins: identification of putative diterpene hydrocarbon precursors.

A procedure for the preparation of a cell-free enzyme solution from rice leaves capable of catalyzing the biosynthesis of diterpene hydrocarbons from geranylgeranyl pyrophosphate or copalyl pyrophosphate as added substrates has been developed. The rates of synthesis of a group of "pimaradiene-like" diterpene hydrocarbons are about 75-fold higher with geranylgeranyl pyrophosphate as substrate and about 8-fold higher with copalyl pyrophosphate as substrate in comparison with extracts from untreated control leaves. The maximum rate of diterpene hydrocarbon biosynthesis is seen in extracts prepared at 40 h after uv irradiation. Five diterpene hydrocarbons (compounds A-E) were present in the hydrocarbon fraction biosynthesized from [3H]geranylgeranyl pyrophosphate in large-scale incubation mixtures prepared from uv-treated rice leaves. Three of these diterpenes were identified as ent-kaur-16-ene (B), ent-sandaracopimara-8(14), 15-diene (D), and 9 beta H-pimara-7,15-diene (E) from GC retention times and GC-MS spectral characteristics in comparison with those of authentic reference compounds. Compound C has spectral characteristics analogous to those of a pimaradiene, but a specific structural assignment from the data available was not possible. Similar incubations with [3H]copalyl pyrophosphate as the substrate and enzyme prepared from uv-treated rice leaves produced ent-kaurene (B), ent-sandaracopimara-8(14),15-diene (D), and compound C, but not 9 beta H-pimara-7,15-diene (E). These results are consistent with a proposed biosynthetic scheme in which 9 beta H-pimara-7,15-diene serves as a precursor of the momilactone family, and ent-sandaracopimara-8(14),15-diene serves as a precursor of the oryzalexin family of rice phytoalexins. ent-Kaurene was the only diterpene detected in incubation mixtures containing enzyme extract from untreated rice leaves and [3H]copalyl pyrophosphate as the substrate. It is suggested that kaurene biosynthesis in rice leaves is probably associated with gibberellin biosynthesis and the regulation of vegetative growth rather than stress metabolism. The diterpene cyclization enzymes in extracts of uv-treated rice leaves show only a relatively modest inhibition by the plant growth retardants AMO-1618 and Phosfon D. No evidence was obtained for the subcellular localization of these cyclization enzymes in organellar preparations; it is tentatively concluded that the enzymes are present predominantly in the extraorganellar cytoplasm of rice leaves.     

Foliage sesquiterpenes and diterpenes of Podocarpus spicatus

Investigation of the foliage oils of 49 specimens of P. spicatus has shown marked infraspecific variation. Seven sesquiterpene hydrocarbons (δ-elemene, α-copaene, longifolene, germacrene-D, bicyclogermacrene, δ-cadinene and selina-4(14),7(11)-diene), two oxygenated sesquiterpenes (curzerenone and atractylon), ten diterpene hydrocarbons (rimuene, beyerene, sclarene, rosadiene, pimaradiene, sandaracopimaradiene, isopimara-7,15-diene, isopimara-8,15-diene, phyllocladene and kaurene), and two oxygenated diterpenes (8β-hydroxyisopimarene and ferruginol) have been isolated and identified. Of particular interest is the isolation of (+)-germacrene-D and (−)-δ-cadinene, along with the rarely reported curzerenone, atractylon, and (+)-rosadiene. This is the first report of selina-4(14),7(11)-diene in an optically active form. The occurrence of both enantiomers of sandaracopimaradiene is also of interest. The biosynthetic significance of correlations in the levels of the terpenes present is discussed.     

(+)-Menthol and its hydroxy derivatives,novel fungal monoterpenols from the fusicoccin-producing fungi,Phomopsis amygdali F6a and Niigata 2

In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (-)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.     

Sesquiterpene constituents from the liverwort Bazzania japonica

The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC-MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (-)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.     

Diterpene biosynthesis in maize seedlings in response to fungal infection

A cell-free system which catalyzes the biosynthesis of terpene hydrocarbons when supplemented with mevalonate, Mn²⁺, and ATP was prepared from the scutellum-embryonic axis region of maize seedlings. The capacity of this system for the production of terpene hydrocarbons was enhanced 50- to 100-fold when the seedlings were exposed for 48 hours to the fungus Rhizopus stolonifer prior to tissue homogenization. The fungi Aspergillus niger, Fusarium moniliforme, and Verticillium albo-atrum also elicited this biosynthetic enhancement. The terpene hydrocarbon products were separable into six fractions by argentation thin layer chromatography. Radioactivity was contributed to five of these fractions when either geranylgeranyl pyrophosphate or copalyl pyrophosphate was supplied as substrate, suggesting that polycyclic diterpenoid hydrocarbons were the main products. Large scale biosynthetic reactions led to the acquisition of about 1 milligram of terpene hydrocarbon products plus some more polar terpenoid products. Analysis of the hydrocarbon products by gas chromatography and mass spectrometry led to the separation of six distinct diterpene hydrocarbons plus a fraction with a molecular weight of about 550. Three of the diterpene hydrocarbons were identified as kaur-16-ene, kaur-15-ene (isokaurene), and pimara-8(14),15-diene. None of the terpene hydrocarbon fractions tested displayed antifungal activity in the Cladosporium cucumerinum thin layer plate assay.     

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.     

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.     

(+)-(10R)-Germacrene A synthase from goldenrod,Solidago canadensis; cDNA isolation,bacterial expression and functional analysis

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.     

Isogermacrene A, a proposed intermediate in sesquiterpene biosynthesis

In the essential oil of the liverwort Saccogyna viticulosa, collected on the island of Madeira, the new sesquiterpene hydrocarbons isogermacrene A (5) and its Cope rearrangement product iso-beta-elemene (6) were identified. 5 is proposed to act as the biogenetic precursor of several new sesquiterpenes identified in the volatiles of S. viticulosa. These include iso-alpha-humulene, alpha-gorgonene, gorgona-1,4(15), 11-triene and gorgon- 11-en-4-ol. In addition, the Cope product of zierene, isozierene, allo-aromadendra-4(15),10(14)-diene, aromadendra-4(15),10(14)-dien-1-ol and a prenylguaiane diterpene alcohol, named viticulol, were identified as new natural products.     

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.     

Biotransformation of the diterpene 15β-hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene by the fungus Fusarium fujikuroi

15β-Hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (4) was biotransformed by the fungus Fusarium fujikuroi into 3α,11β,15β-trihydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (5). The hydroxylation at C-3(α) in this diterpene reminds a similar reaction that occurs at C-13 in the biosynthesis of gibberellic acid in this fungus. The presence of the 15β-alcohol in the substrate directs the second hydroxylation at C-11(β), which had been observed in the incubation of ent-kaur-16-ene derivatives with this fungus when the C-19 hydroxylation was inhibited by the existence in the molecule of a 3α-OH or 3-oxo group. We also show that the angelate of the substrate is an undescribed natural product now identified as a component of the plant Distichoselinum tenuifolium.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Sesquiterpene hydrocarbons with trifarane backbone in the liverwort Trocholejeunea sandvicensis

The hydrocarbon fraction of the liverwort Trocholejeunea sandvicensis was investigated. Four new sesquiterpene hydrocarbons including the trifaranes (-)-trifara-9,14-diene (6) and (-)-3,7-di-epi-3,7-trifara-9,14-diene (7), as well as (+)-sandvicene (8), a sesquiterpene with uncommon skeleton, and the rearranged trifarane sesquiterpene (+)-neotrifaradiene (9) were isolated and their structure elucidated. A biogenetic pathway for the four compounds is proposed, with nerolidol as their common precursor.     

Norsesquiterpene hydrocarbon, chemical composition and antimicrobial activity of Rhaponticum carthamoides root essential oil

A detailed analysis of Rhaponticum carthamoides (Willd.) Iljin root essential oil was carried out by GC, GC-MS and GC-FTIR techniques. In total, 30 components were identified, accounting for 98.0% of total volatiles. A norsesquiterpene 13-norcypera-1(5),11(12)-diene (22.6%), followed by aplotaxene (21.2%) and cyperene (17.9%), were isolated and their structures confirmed by 1D and 2D-NMR spectra (COSY, ROESY, HSQC, HMBC and INADEQUATE). Selinene type sesquiterpenes and aliphatic hydrocarbons were among minor constituents of the essential oil. The oil exhibited antimicrobial activity against 5 of 9 strains of bacteria and yeast, when tested using broth micro-dilution method. Minimum inhibitory concentrations ranged between 32 and 256 μg/ml.     

Molecular and functional characterization of a Na(+)-K(+) transporter from the Trk family in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K(+) transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K(+) uptake is mainly dependent on the activity of Trk transporters thought to mediate H(+):K(+) symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K(+) and Na(+), and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na(+) and K(+) might result in Na(+):K(+) co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K(+) and Na(+) nutrition of the fungus (and of the plant) in nutrient-poor soils.     

Chemical study of the essential oil of Cyperus rotundus.

Minor constituents of the essential oil of Cyperus rotundus have been investigated. The three new sesquiterpene hydrocarbons (-)-isorotundene, (-)-cypera-2,4(15)-diene, (-)-norrotundene and the ketone (+)-cyperadione were isolated and their structures elucidated. The absolute configuration of (-)-rotundene was derived by chemical correlation and enantioselective gas chromatography.     

Secondary metabolites of Peucedanum tauricum fruits

From the essential oil of fruits of Peucedanum tauricum Bieb., two guaiane type sesquiterpene hydrocarbons guaia-1(10),11-diene (1) and guaia-9,11-diene (2) were identified. The structures of 1 and 2 were assigned by 1D and 2D NMR analysis. The relative configurations of the compounds were established by 2D-NOESY experiments while the absolute configurations were deduced through chemical correlations with (+)-gamma-gurjunene (9) and capillary GC analysis using modified cyclodextrins as the stationary phases. From the dichloromethane extract of the less volatile fraction of the fruits, coumarins, viz. peucedanin (3), oxypeucedanin hydrate (4) and officinalin isobutyrate (5) were isolated. Compound 5 was confirmed to be 6-carbomethoxy-7-isobutyroxycoumarin by its 1D and 2D NMR data as well as by conversion into officinalin (7) by alkaline hydrolysis. Peuruthenicin, a positional isomer of officinalin, is assigned structure 8 on spectral basis. Bergapten (6) was identified by its mass spectrum. This is the first report on the isolation of compounds 4 and 5 from P. tauricum.     

Chemical analysis and antimicrobial activity of Halimium voldii

Volatile constituents and a hexane extract of the leaves of Halimium voldii Kit Tan, Perdetzoglou & Raus, sp. nova, were analyzed by GC and GC-MS. Thirty compounds were identified in the essential oil of Halimium representing 88.7% of the oil composition. The main components were nonanal (12.8%), dodecane (10.6%), Z-caryophyllene (8.2%), gamma-muurolene (10.9%), delta-cadidene (3.5%), caryophyllene oxide (5.1%), beta-eudesmol (3.6%) and manoyl oxide (5.5%). Thymol was identified in the hexane extract as the main compound. A labdane diterpene ent-labd-7, 13 (E)-dien, 15-ol was detected by its mass spectra fragmentation pattern and its structure was determined by spectroscopic methods and its optical rotation. The essential oil and the hexane extract were assayed for their antimicrobial activity against gram (+) and gram (-) bacteria.