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.     

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.     

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.     

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).     

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.     

The microbiological transformation of some ent-3β-hydroxykaur-16-enes by Gibberella fujikuroi

The preparation of ent-3β-hydroxykaur-16-ene from linearol and of ent -3β,18-dihydroxykaur-16-ene from foliol is described. The microbiological transformation of these and of foliol by Gibberella fujikuroi has been studied. A 3α-hydroxyl group appears to exert an inhibitory effect on transformations involving oxidation at C-19.     

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.     

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.     

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.     

Chemical composition of Xylopia nitida: Diterpenes and alkaloids

From the roots of Xylopia nitida, were obtained a new natural diterpene, the ent-kaur-16-en-18,19-diol and a new aporphine alkaloid, the 5,6,6a,7-tetrahydro-1-methoxy-(6aS)-4H-benzo[de][1,3]benzodioxolo [5,6-g]quinoline. Some known compounds were also isolated, ent-trachylobane, ent-trachyloban-18,19-diol, ent-trachyloban-18-oic acid, ent-trachyloban-19-oic acid, (−)-xylopine, 1-O-ethyl-β-D-glucopyranose and a mixture of β-sitosterol and stigmasterol. This is the first phytochemical study about X. nitida. In this paper chemotaxonomic significance of these compounds is discussed.     

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.     

Three ent-trachylobane diterpenes from the leaf exudates of Psiadia punctulata

Three ent-trachylobane diterpenes have been isolated from the leaf exudates of Psiadia punctulata and characterised as 6α,17,19-ent-trachylobantriol; 2α,18,19-ent-trachylobantriol; and 2β,6α,18,19-ent-trachylobantetraol. The structures were determined on the basis of spectroscopic evidence.     

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).     

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.     

Structures of the Metabolites from Steviol Methyl Ester by Gibberella fujikuroi

Steviol methyl ester (methyl ent-13-hydroxykaur-16-en-19-oate)* was converted into five new metabolites together with a known compound, methyl ent-7α,13-dihydroxykaur-16-en-19-oate, by Gibberella fujikuroi in the presence of a plant growth retardant. The structures of these new metabolites were elucidated to be methyl ent-7β,13-dihydroxykaur-16-en-19-oate, methyl ent-11α,13-dihydroxykaur-16-en-19-oate, methyl ent-7β,11α,13-trihydroxykaur-16-en-19-oate, methyl ent-11α, 13,15β-trihydroxykaur-16-en-19-oate and methyl ent-13,15β-dihydroxy-11-oxokaur-16-en-19-oate mainly by spectroscopic analyses.     

Four new diterpenes from Sideritis serrata

Four new diterpenes have been isolated from Sideritis serata: lagascol (4, ent-8,5-friedopimar-5-ene-15S,16-diol), tobarrol (8, ent-15-beyerene-12α,17-diol), benuol (12, ent-15-beyerene-7α,17-diol) and serradiol (18, ent-16R-atis-13-ene-16,17-diol). The previously known diterpenes lagascatriol (1, ent-8,5-friedopimar-5-ene-11β,15S,16-triol), jativatriol (2, ent-15-beyerene-1β,12α,17-triol), conchitriol (3, ent-15-beyerene-7α,12α,17-triol) and sideritol (17, ent-16R-atis-13-ene-1β,16,17-triol) have also been obtained from the same source.     

New diterpenes from Sideritis sicula

Two new diterpenes have been isolated from Sideritis sicula: sideripol, ent-18-acetoxy-7α-hydroxykaur-15-ene and epoxysideritriol, ent-15β,16β-epoxykauran-7α,17,18-triol. The previously known diterpene eubol, ent-7α-acetoxykaur-16-en-15β,18 diol has also been obtained from the same source.     

The microbiological transformation of epicandicandiol,ent-7α,18-dihydroxykaur-16-ene,by Gibberella fujikuroi

Incubation of ent-7α,18-dihydroxykaur-16-ene with Gibberella fujikuroi affords ent-7α,18,19-trihydroxykaur-16-ene and ent-7α,18-dihydroxykaur-16-en-19-oic acid. There was no transformation into 7,18-dihydroxykaurenolide.     

Three Novel Gibberellins Produced by Gibberella fujikuroi

Three novel gibberellins, GA₅₄ (ent-1α, 3α, 10-trihydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone), GA₅₅ (ent-1α, 3α, 10, 13-tetrahydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone) and GA₅₆ (ent-2β, 3α, 10, 13-tetrahydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone) were shown to occur in the culture broth of Gibberella fujikuroi. Their structures were determined mainly by mass spectrometrical comparison of the derivatives with those of authentic compounds prepared from known gibberellins.     

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.