A new 5,6-dihydro-2-pyrone derivative from Phomopsis amygdali,an endophytic fungus isolated from hazelnut (Corylus avellana)

Aims of this study were to isolate endophytes from different parts of hazelnut – Corylus avellana L. to obtain bioactive secondary metabolites and search for the presence of gene region of taxadiene synthase (Ts), a key enzyme in taxol biosynthesis, on selected fungi. Fourteen fungal species were isolated and cultured for the screening studies. The cell-free fermentation broths were extracted with chloroform. The chloroform extracts were tested for cytotoxic activity by MTT method. Based on the activity results and chemical profiles, the isolate identified as Phomopsis amygdali by internal transcribed spaces (ITS) sequence analysis using ITS1 primer was selected for further studies. After large-scale fermentation and purification studies, two major compounds, one of which turned out to be a new secondary metabolite, were isolated and characterized. Structure of the new metabolite was elucidated as (S)-4-butoxy-6-((S)-1-hydroxypentyl)-5,6-dihydro-2H-pyran-2-one by the extensive use of 1D and 2D NMR, and HR-MS, whereas the known compound was identified as (−)pestalotin. Additionally, to evaluate taxol-producing potential of the selected isolate, a PCR amplification study followed by gel electrophoresis analysis was carried out revealing no Ts gene region.     

Identification of (+)-phyllocladene, (--)-sandaracopimaradiene, and (+)-kaurene as new fungal metabolites from fusicoccin-producing Phomopsis amygdali F6

A chemical analysis of the diterpene hydrocarbons produced by fusicoccin-producing fungus Phomopsis amygdali F6 identified five phyllocladene-related tri- and tetracyclic diterpene hydrocarbons. The presence of (+)-phyllocladene, (--)-sandaracopimaradiene, (+)-isopimara-8,15-diene, and (+)-pimara-8(14),15-diene in the fungus was demonstrated by GC-MS, 1H-NMR, and [alpha]D measurements. (+)-Kaurene was also identified by GC-MS and chiral capillary GC. The possible biosynthetic relationship of these metabolites is discussed.     

Novel Fusicoccins R and S, and the fusicoccin S aglycon (phomopsiol) from Phomopsis amygdali niigata 2-A, and their seed germination-stimulating activity in the presence of abscisic acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3alpha-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3'-deacetyl-3alpha-hydroxyfusicoccin A and 3beta-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3alpha-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3alpha-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3alpha- or 3beta-hydroxy group in their diterpene moiety are briefly discussed.     

Addition reactions at the 16(17) double bond of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene

The epoxidation, the addition of hypobromous acid, and the hydroboration of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene 1 with diborane, catecholborane, and 9-BBN were investigated in order to determine the stereochemical outcome and to synthesize new 13alpha-estra-1,3,5(10)-trienes for biological and conformational investigations. It was shown that the sterically demanding reagent 9-BBN participated in a preferred beta attack (53% 16betaOH 10, 34% 17betaOH 8, 13% 16alphaOH 11). This stereochemical result is in agreement with that from another cis addition reaction, the recently described OsO4 dihydroxylation of 1 [Steroids 68 (2003) 113]. With smaller reagents such as B2H6, catecholborane, or magnesium monoperoxyphthalate, a diminished stereoselectivity was observed with only a slight excess of beta attack. The ionic trans addition of hypobromous acid gave two 17-bromo-16-alcohols with 16beta,17alpha (4, 76%) and 16alpha,17beta configuration (5, 24%) formed by trans cleavage of the 16,17alpha- and beta-bromonium ion at position 16. The same regioselective and stereoselective course was found for the cleavage of the 16alpha,17alpha- and 16beta,17beta-epoxides (3 and 2) with hydrazoic acid (3-->16betaN3,17alphaOH 7, 2-->16alphaN3,17betaOH 6). The stereochemistry of the addition reactions to 1 can be explained in terms of a twist-boat conformation involving the C ring of compound 1. From a synthetic viewpoint the synthesis of the beta-epoxide 2 from the bromohydrin 4, the cleavage of this epoxide to 16alpha-substituted-17beta-hydroxy compounds, such as 6, and hydroboration/oxidation with 9-BBN to the hitherto unknown 16beta-hydroxy compound 10 are useful procedures. The bromohydrin 5 is the first 13alpha-steroid with a 17beta-bromo substituent. X-ray analysis revealed twist-boat and 16beta-envelope conformations for rings C and D, respectively.     

Practical, asymmetric synthesis of 16-hydroxyeicosa-5(Z),8(Z), 11(Z),14(Z)-tetraenoic acid (16-HETE), an endogenous inhibitor of neutrophil activity

An asymmetric synthesis of 16-HETE, an endogenous inhibitor of neutrophil activity, was achieved in six steps from R-(-)-glycidyl benzyl ether in 28% overall yield.     

(+)-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.     

Novel Fusicoccins R and S,and the Fusicoccin S Aglycon (Phomopsiol) from Phomopsis amygdali Niigata 2-A,and Their Seed Germination-stimulating Activity in the Presence of Abscisic Acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3α-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3′-deacetyl-3α-hydroxyfusicoccin A and 3β-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3α-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3α-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3α- or 3β-hydroxy group in their diterpene moiety are briefly discussed.     

Production of 13beta-alkyl-3-methoxy-8,14-seco-1,3,5(10),9(11)-gonatetraen-14beta-ol-17-ones and 13beta-alkyl-3-methoxy-8,14-seco-1,3,5(10),9(11)-gonatetraen-17alpha-ol-14-ones by microbial enzymes.

For the purpose of producing hydroxy-keto-seco-steroids in which hydroxyl group is attached to a carbon atom having the R-configuration, numerous biochemically active microorganisms were tested without any success. The hydroxysteroid oxidoreductase enzymes of the investigated bacterial, yeast and fungal strains were suitable only for the production of 17beta-ol-14-one and 14alpha-ol-17-one derivatives. The required compounds were prepared by combinations of enzymatic reactions with chemical reduction. (i) By hydroxysteroid oxidoreductase of Saccharomyces uvarum and Saccharomyces drosophilarum, 17beta-ol-14-one and 14alpha-ol-17-one derivatives of 14,17-dione, respectively, were obtained. (ii) The above compounds were acetylated then reduced by sodium borohydride. (iii) 14beta,17beta-diol-17-acetate and 14alpha,17alpha-diol-14-acetate were dehydrogenated by dehydroxysteroid oxidoreductase of Nocardia sp. and Mycobacterum sp., respectively, in the presence of steroid esterase. The reaction mixture contained either 14beta-ol-17-one or 17alpha-ol-14-one derivatives, since oxidation by hydroxysteroid oxidoreductase was limited to the hydroxyl group attached to a carbon atom having the S-configuration.     

Synthesis of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside, an anti-inotropic cardiac glucoside

In a continuing effort to synthesize cardiac glucoside analogs with modified 17 beta-functional groups for pharmacologic testing, we used 3 beta-benzyloxy-5 beta-androst-15-en-17-one as an efficient intermediate. This report describes a preparation of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside.     

3α-Angeloyloxy-2α-hydroxycativic acid,a new diterpene from Brickellia paniculata

The investigation of Brickellia paniculata resulted in the isolation of a new diterpene of the labdane type. It was identified as 3α-angeloyloxy-     

Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.     

Cloning of Schistosoma japonicum 14-3-3 epsilon (Sj14-3-3 epsilon), a new member of the 14-3-3 family of proteins from schistosomes

A new member of the 14-3-3 protein family from Schistosoma japonicum has been identified. Phylogenetic analysis showed that this member belongs to the epsilon subfamily of the 14-3-3 proteins, and it is therefore named Sj14-3-3 epsilon. Consistent with the findings for the previously reported S. japonicum 14-3-3 protein (Sj14-3-3), Southern analysis suggested the presence of more than one gene, and/or introns or allelic polymorphism in this epsilon isoform. By RT-PCR, Sj14-3-3 epsilon was shown to be stage-specifically transcribed, being abundant in adults, present in sporocysts but absent in cercariae. Furthermore, mRNA of the epsilon isoform seemed to be much less abundant in the sporocyst stage, compared with Sj14-3-3. This suggests varying requirements of the different 14-3-3 isoforms at different stages of the life cycle.     

1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase

1-methyl-DL-Trp, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of Trp), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of Trp), each of which has a substitution at the indole nitrogen atom, were found to be the first examples of potent substrate analog competitive inhibitors (Ki 7-70 microM) with respect to the substrates D-Trp and L-Trp for rabbit small intestinal indoleamine 2,3-dioxygenase. Binding studies using optical absorption and CD spectroscopy demonstrated that these three inhibitors cause spectral changes upon binding to the native ferric, ferrous, ferrous-CO, and ferrous-NO enzymes. Such spectral effects of 1-methyl-DL-Trp on all of these enzyme derivatives were similar to those caused by L-Trp, while the sulfur and the oxygen analogs of Trp exhibit relatively small effects except for those observed for the sulfur analog with CD spectroscopy. Each of these three Trp analog inhibitors competes with L-Trp for the ferrous-CO enzyme, a model for the ferrous-O2 enzyme. The present findings demonstrate that, although substitution of a methyl group for the hydrogen atom on the indole nitrogen or of a more electron-inductive sulfur or oxygen atom for the indole nitrogen atom does not prevent the binding of the resulting Trp analog to indoleamine 2,3-dioxygenase, the free form of the indole nitrogen base is an important physical and/or electronic structural requirement for Trp to be metabolized by the enzyme. The inability of 1-methyl-Trp to serve as a substrate for the dioxygenase supports a view that singlet oxygen is not the reactive oxygen species involved in the dioxygenation of Trp by the enzyme.     

Aphaereta ceratitivora sp. n. (Hymenoptera,Braconidae), a new parasitoid of Ceratitis capitata (Wiedemann) (Diptera,Tephritidae) from the Azores

A new gregarious larval-pupal endoparasitoid of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is described and illustrated: Aphaereta ceratitivora sp. n. (Braconidae: Alysiinae: Alysiini).