The microbiological transformation of some ent-beyerene diterpenoids by Gibberella fujikuroi to beyergibberellins and beyerenolides

The microbiological transformation by Gibberelia fujikuroi of ent-beyer-15-ene into the beyergibberellins A₉ and A₁₃, 7β-hydroxy- and 7β,18-dihydroxybeyerenolides, and of ent-beyer-15-en-19-ol into beyergibberellins A₄, A₇, A₉, A₁₃ and A₂₅,and 7β-hydroxy-and 7β,18-dihydroxybeyerenolides is described. In contrast, ent-beyer-15-en-18-ol gave _ent-7α, 18,19-trihydroxybeyer-15-ene, 7β,18-dihydroxybeyerenolide and _ent-7α,18-dihydroxybeyer-15-en-19-oic acid again revealing the inhibitory effect of an 18-hydroxyl group on oxidative transformations at C-6β by Gibberella fujikuroi.     

The microbiological transformation of some trachylobane diterpenoids by Gibberella fujikuroi

The microbiological transformation of ent-trachylobane, ent-7α-hydroxytrachylobane and ent-19-hydroxytrachylobane into trachylobagibberellins A₇, A₉, A₁₃, A₂₅, A₄₀ and A₄₇ by Gibberella fujikuroi is described. Whereas 7β-hydroxy- and 7β,18-dihydroxytrachylobanolides were obtained from ent-trachylobane and ent-trachyloban- 19-ol, the presence of a 7β-hydroxyl group directed metabolism exclusively into the gibberellin pathway. An 18-hydroxyl group as in ent-7α,18-dihydroxytrachylobane inhibited oxidation at C-6 affording ent-7α,18,19-trihydroxytrachylobane as the major metabolite.     

The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Steviol(ent-13-hydroxykaur-16-en-19-oic acid) is rapidly metabolised by the mutant B1-41a of Gibberellafujikuroi. The initial product is the ent- 7-α-hydroxy derivative which is then further metabolised to gibberellins A₁, A₁₈, A₁₉, A₂₀, 13-hydroxy GA₁₂, the ent-6α, 7α, 13- and ent-6β, 7α, 13 (19,6-lactone)-trihydroxykaurenoic acids, and a seco-ring B diacid. This apparently low substrate specificity of the enzymes operative beyond the block in the mutant B1-41a provides a useful model for the biosynthetic pathways to 13-hydroxylated gibberellins of higher plants and a preparative route to these plant gibberellins.     

Differential metabolism of diastereoisomeric diterpenes by Preussia minima,found as endophytic fungus in Cupressus lusitanica

The plant diastereoisomeric diterpenes ent-pimara-8(14)-15-dien-19-oic acid, obtained from Viguiera arenaria, and isopimara-8(14)-15-dien-18-oic acid, isolated from Cupressus lusitanica, were distinctly functionalized by the enzymes produced in whole cell cultures of the fungus Preussia minima, isolated from surface sterilized stems of C. lusitanica. The ent-pimaradienoic acid was transformed into the known 7β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid, and into the novel diterpenes 7-oxo-8 β-hydroxy-ent-pimara-8(14)-15-dien-19-oic and 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acids. Isopimara-8(14)-15-dien-18-oic acid was converted into novel diterpenes 11α-hydroxyisopimara-8(14)-15-dien-18-oic acid, 7β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, and 1β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, along with the known 7β-hydroxyisopimara-8(14)-15-dien-18-oic acid. All compounds were isolated and fully characterized by 1D and 2D NMR, especially ¹³C NMR. The diterpene bioproduct 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid is an isomer of sphaeropsidin C, a phytotoxin that affects cypress trees produced by Shaeropsis sapinea, one of the main phytopathogen of Cupressus. The differential metabolism of the diterpene isomers used as substrates for biotransformation was interpreted with the help of computational molecular docking calculations, considering as target enzymes those of cytochrome P450 group.     

Metabolism of Steviol and Its Derivatives by Gibberella fujikuroi

Steviol (ent-13-hydroxykaur-16-en-19-oic acid)* is metabolized by Gibberella fujikuroi in the presence of inhibitors of gibberellin biosynthesis, such as quaternary ammonium salt-type growth retardants, to afford 7β-Miydroxy- and 6β,7β-dihydroxysteviol, gibberelhns A₁, A₁₈, A₁₉, A₅₃ and 7β,13-dihydroxykaurenolide. Steviol acetate (ent-13-acetoxykaur-16-en-19-oic acid) is also metabolized to the 6β,7β-dihydroxy-derivative and to the 13-acetyl derivatives of gibberellins A₁₇ and A₂₀ and steviol methyl ester (methyl ent-13-hydroxykaur-16-en-19-oate) into the monohydroxy-, dihydroxy- and hydroxyoxo-derivatives. These results indicate a low substrate specificity of the enzymes in the fungus and provide a useful preparative methodology of several important plant gibberellins carrying the 13-hydroxyl group.     

A diterpenoid from Elaeoselinum foetidum

A new diterpene acid has been isolated from the roots of Elaeoselinum foetidum. Its structure was established as ent-7α-senecioxy-15α-hydroxy-atis-16-en-19-oic acid by ¹H NMR and ¹³C NMR spectroscopic studies of its methyl ester derivative and confirmed by correlation with a margotianin derivative.     

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.     

Microbiological conversion of 12-oxygenated and other derivatives of ent-kaur-16-en-19-oic acid by Gibberella Fujikuroi, mutant B1-41a

The metabolism of several ring C and D-functionalized ent-kaur-16-en-19-oic acids by cultures of Gibberella fujikuroi, mutant B1-41a, to the corresponding derivatives of the normal fungal gibberellins (GAs) and ent-kaurenoids is described. A range of 12α- and 12β-hydroxyGAs and ent-kaurenoids are characterized by their mass spectra and GC Kovats retention indices. The mass spectral and GC data are used to identify the 12α-hydroxy derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄ (GA₅₈), and of the 12β-hydroxy derivatives of ent-7α-hydroxy- and ent-6α, 7α-dihydroxykaurenoic acids, in seeds of Cucurbita maxima. Similarly the metabolites of GA₉, formed in seeds of Pisum sativum and cultures of G.fujikuroi, mutant B1-41a, are identified as 12α-hydroxyGA₉. ent-11β-Hydroxy- and ent-11-oxo-kaurenoic acids are metabolized by the fungus to the corresponding 11-oxygenated derivatives of the normal fungal ent-kaurenoids and some C₂₀-GAs; no 11-oxygenated C₁₉-GAs are formed. Grandiflorenic acid, 11β-hydroxygrandiflorenic acid, attractyligen and ent-15β-hydroxykaurenoic acid are metabolized to unidentified products.     

GC/MS analysis of the plant hormones in seeds of Cucurbita maxima

The GC/MS detection is reported of over 30 compounds, in extracts of the endosperm and embryos from seeds of Cucurbita maxima. The compounds which were identified from reference spectra include: cis,trans-ABA; trans,trans-ABA; dihydrophaseic acid; IAA; GA₄; GA₁₂; GA₁₃; GA₂₅; GA₃₉; GA₄₃; GA₄₉; ent-13-hydroxy-, ent-6α,7α-and ent-7α,13-dihydroxy-, and ent-6α,7α,13-trihydroxykaur-16-en-19-oic acids; ent-7α,16,17-trihydroxy- and ent-6α,7α,16,17-tetrahydroxy-kauran-19-oic acids, ent-6,7-seco-7-oxokauren-6,19-dioic acid and/or ent-6,7-secokauren-6,7,19-trioic acid, and 7β,12α-dihydroxykaurenolide. New compounds, the structures of which were deduced from GC/MS data, include: the 12α-hydroxy-derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄, and the 12β-hydroxy-derivatives of ent-7α-hydroxy- and ent-6α,7α-dihydroxykaurenoic acids.     

Beyerene derivatives and other constituents from Petunia patagonica

Two new diterpenes of the beyerene type, ent-19-hydroxy-1 7-acetoxybeyer-15-ene and ent-beyer-15-en-17-oic acid, and two previously characterized kauranoids, ent-16β-hydroxy-17-acetoxykaurane and ent-16β,1 7-dihydroxy-kaurane, as well as two known flavonoids, luteolin-7,3′,4′-trimethyl ether and luteolin-7,3′-dimethyl ether, and a triterpenoid, oleanoic acid, were obtained from a chloroform extract of Petunia patagonica. The new structures were elucidated by spectral data and chemical transformations.     

Biotransformation of 7α-hydroxy- and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi

The microbiological transformation of 7α,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7α,11β,19-trihydroxy-ent-atis-16-ene and 7α,16β,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16β,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6β-hydroxy-7-oxo-ent-atis-16-en-19-oic acid, 6β,16β,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3β,7α-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.     

Metabolic transformations of some ent-kaurenes in Gibberella fujikuroi

The conversion of ent-kaur-16-enes to gibberellic acid in Gibberella fujikuroi is blocked by A-ring modifications. Thus ent-3β-hydroxykaur-16-en-19-yl succinate gives good conversion (46%) to the 7β-hydroxy derivative.* Under the same conditions the 3β-epimer gives 7β- or 6α-hydroxylation and the former occurs for the 3-oxo analogue. The succinoyloxy function acts as a less efficient block and ent-kaur-16-en-19-yl succinate is converted to 7β-hydroxy and 6β,7β-dihydroxy derivatives along with gibberellic acid. Hydrolysis of the succinate block of the metabolities provides the 7β, 19-diol and 6β,7β, 19-triol. Of this pair only the former was effectively metabolized to gibberellic acid in G. fujikuroi.     

Gibberellin A43 and other terpenes in endosperm of Echinocystis macrocarpa

By GC-MS the following acidic constituents of the endosperm of Echinocystis macrocarpa were identified: abscisic acid and its trans,trans-isomer, 4′-dihydrophaseic acid, GA₄, GA₇, iso-GA₇, GA₂₄, GA₂₅, two isomers of GA₁₃, GA₄₃, ent-6α,7α,17-trihydroxy-16αH-kauran-19-oic acid and ent-6α,7α, 16β, 17-tetrahydroxykauran- 19-oic acid. The structures of the last three new natural products were confirmed by partial synthesis. ent-Kaurene was detected in the neutral fraction.     

Biotransformation of ent-kaur-16-ene and ent-trachylobane 7β-acetoxy derivatives by the fungus Gibberella fujikuroi (Fusarium fujikuroi)

Candol A (7β-hydroxy-ent-kaur-16-ene) (6) is efficiently transformed by Gibberella fujikuroi into the gibberellin plant hormones. In this work, the biotransformation of its acetate by this fungus has led to the formation of 7β-acetoxy-ent-kaur-16-en-19-oic acid (3), whose corresponding alcohol is a short-lived intermediate in the biosynthesis of gibberellins and seco-ring ent-kaurenoids in this fungus. Further biotransformation of this compound led to the hydroxylation of the 3β-positions to give 7β-acetoxy-3β-hydroxy-ent-kaur-16-en-19-oic acid (14), followed by a 2β- or 18-hydroxylation of this metabolite. The incubation of epicandicandiol 7β-monoacetate (7β-acetoxy-18-hydroxy-ent-kaur-16-ene) (10) produces also the 19-hydroxylation to form the 18,19 diol (20), which is oxidized to give the corresponding C-18 or C-19 acids. These results indicated that the presence of a 7β-acetoxy group does not inhibit the fungal oxidation of C-19 in 7β-acetoxy-ent-kaur-16-ene, but avoids the ring B contraction that leads to the gibberellins and the 6β-hydroxylation necessary for the formation of seco-ring B ent-kaurenoids. The biotransformation of 7β-acetoxy-ent-trachylobane (trachinol acetate) (27) only led to the formation of 7β-acetoxy-18-hydroxy-ent-trachylobane (33).     

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.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Polyoxygenated ent-kauranes and water-soluble conjugates in seed of Phaseolus coccineus

C-α and C-β, previously isolated from seed of Phaseolus coccineus, are shown respectively to be the bis-O-isopropylidene and the 16,17-mono-O-isopropylidene derivatives of ent-6α,7α,16β,17-tetrahydroxykauranoic acid. By GC-MS characterization of the products of acidic, basic and enzymatic hydrolysis, water soluble conjugates of the following compounds have been shown to occur in P. coccineus seed: GA₈, GA₁₇, GA₂₀, GA₂₈, ent-6α,7α,13-trihydroxykaurenoic acid, ent-6α,7α,17-trihydroxy-16β-kauranoic acid, ent-6α,7α,16β,17-tetrahydroxykauranoic acid, 7β,13-dihydroxykaurenolide and abscisic acid.     

ent-Kaurane diterpenes from the stem bark of Annona vepretorum (Annonaceae) and cytotoxic evaluation

This work describes a novel ent-kaurane diterpene, ent-3β-hydroxy-kaur-16-en-19-al along with five known ent-kaurane diterpenes, ent-3β,19-dihydroxy-kaur-16-eno, ent-3β-hydroxy-kaur-16-eno, ent-3β-acetoxy-kaur-16-eno, ent-3β-hydroxy-kaurenoic acid and kaurenoic acid, as well as caryophyllene oxide, humulene epoxide II, β-sitosterol, stigmasterol and campesterol from the stem bark of Annona vepretorum Mart. (Annonaceae). Cytotoxic activities towards tumor B16-F10, HepG2, K562 and HL60 and non-tumor PBMC cell lines were evaluated for ent-kaurane diterpenes. Among them, ent-3β-hydroxy-kaur-16-en-19-al was the most active compound with higher cytotoxic effect over K562 cell line (IC₅₀ of 2.49 μg/mL) and lower over B16-F10 cell line (IC₅₀ of 21.02 μg/mL).     

New alkamide and ent-kaurane diterpenoid derivatives from Senecio erechtitoides (Asteraceae)

Fractionation of a MeOH/CH₂Cl₂ (1/1) extract of the aerial parts of Senecio erechtitoides led to the isolation of six compounds including the hitherto unknown N-phenethylamide derivative named N-(p-hydroxyphenethyl)pentacosanamide (1), and a kauranoid derivative named derivative named ent-7-oxo-16α,17-dihydroxykauran-19-oic acid (2), as well as four known compounds, ent-Kaur-16-en-19-oic acid (3), ent-7β-hydroxykaur-16-en-19-oic acid (4), ent-7-oxokaur-16-en-19-oic acid (5), steppogenin 4′-O-β-d-glucoside (6). Their structures and relative configurations were elucidated on the basis of spectroscopic methods, chemical reactions, and comparison with previously known analogs. All isolates were evaluated for their antimicrobial activity and only diterpenoids were found to possess a potent inhibitor effect against the range of microorganism.     

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

Summary A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-¹⁴C into ent-kaur-16-en-19-oic acid (I), ent-7-hydroxy-kaur-16-en-19-oic acid (II), and ent-gibberell-16-en-7-al-19-oic acid (III) (gibberellin A₁₂-aldehyde). The products were identified by gas liquid chromatography and by combined gas chromatography-mass spectrometry of the methylated and trimethylsilylated fractions. The radioactivity of the compounds was established by recrystallisation to constant specific radioactivity. Gibberellin A₁₂ (IV), also detected in the system after incubation by combined gas chromatographymass spectrometry may be an artefact, derived from gibberellin A₁₂-aldehyde by a non-enzymatic conversion.With gibberellin A₁₂-aldehyde, the cell-free biosynthesis of an ent-gibberellane has been achieved for the first time.