Endophytic fungi reside in the intercellular spaces of higher plants, and a single plant species typically serves as the host for several fungal species. The secondary metabolism of endophytic fungi is largely unexplored, but these fungi should be a fruitful source of biologically active secondary metabolites because of the magnitude of the resourcesconservatively 1.5 × 10 6 speciessand their intimate association and coevolution with other organisms. 1,2 As part of a program to explore the biosynthetic potential of endophytic fungi, we investigated the Florida torreya (Torreya taxifolia), an endangered species closely related to the taxol-producing Pacific yew (Taxus brevifolia). 3,4 We identified the endophyte Pestalotiopsis microspora as the likely cause of the Florida torreya's decline and characterized several phytotoxic and antifungal secondary metabolites produced by P. microspora. 5 We now wish to report the isolation and structure determination of torreyanic acid (1), another metabolite from P. microspora. Torreyanic acid (1) is an unusual dimeric quinone with selective cytotoxicity against human cancer cell lines.P. microspora was cultured in potato dextrose broth, and its organic extract (375 mg/L) was obtained by sequential whole culture extractions with ethyl acetate. The organic extract exhibited a zone of inhibition against Bacillus subtilis in an agar diffusion assay. Bioassayguided fractionation using silica gel (95:5, 90:10 CH 2 Cl 2 -MeOH) and flash reverse-phase chromatography (C-18, 50% MeOH-H 2 O to 100% MeOH) afforded active crystalline torreyanic acid (50 mg/L), which could be separated from contaminating oils by careful MeOH trituration.Torreyanic acid, [R] D +92.3°(c 0.11, MeOH) and mp 160°C dec, was structurally analyzed by one-and twodimensional NMR spectroscopy. The 1 H NMR spectrum displays a large methylene envelope at δ 1.20 and two methyl triplets at δ 0.86, suggesting two long aliphatic chains. The molecular formula C 38 H 44 O 12 derived from HRFABMS is consistent with the 38 carbon resonances observed in the 13 C NMR spectrum. With six carbonyls and eight olefinic carbons ( 13 C NMR), compound 1 must contain seven rings. The intense absorption at 1690 cm -1 and the shoulder at 1700 cm -1 in the IR spectrum indicated both unsaturated carbonyl(s) and a ketone. In addition, the molecular formula and DEPT spectrum indicated two free hydroxyls.

Immunosuppressive drugs are used today to prevent allograft rejection in transplant patients, and in the future they could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes.1 Currently approved immunosuppressive agents such as cyclosporin A and FK506 possess some undesirable side effects, and the search for better immunosuppressive agents continues.2 We have been investigating endophytic fungi-the fungi that live in the intercellular spaces inside living plants-as a source of novel compounds. This search has emphasized fungi from plants with demonstrated biological activity, and in screening the fungi from the perennial twining vine Tripterygium wilfordii, we discovered two novel immunosuppressive compounds that form the basis of this report. T. wilfordii, which has been used in Chinese traditional medicine for over two thousand years, is familiar to chemists as the source of cytotoxic diterpene lactones such as trip-diolide3 and insecticidal alkaloids4 56such as wilfordine. Recently, several groups5,6 have reported the isolation of immunosuppressive triterpenes from T. wilfordii. An endophytic fungus from T. wilfordii, identified as Fusarium subglutinans, produces the immunosuppressive but noncytotoxic diterpene pyrones subglutinol A (1) and B(2).F. subglutinans was cultured in modified M-l-D medium, and immunosuppressive activity, as judged by the mixed lymphocyte reaction, appeared in the ethyl acetate extract of the culture medium. The extract was chromatographed on silica gel using CH2Cl2-MeOH mixtures. The active fractions were subjected to reverse phase HPLC on a C-18 column with a solvent gradient of 75% MeOH-H20 to 100% MeOH to give two active compounds, subglutinol A (1) and B (2) (3:1). Compounds 1 (mp 185 °C dec; [

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Hydrogenation of the C(4') exocyclic olefin of the pacidamycins has been shown to produce a series of semisynthetic compounds, the dihydropacidamycins, with antimicrobial activity similar to that of the natural products. Elucidation of stereochemistry in the pacidamycins has been completed through a campaign of natural product degradation experiments in combination with the total synthesis of the lowest-molecular weight dihydropacidamycin, dihydropacidamycin D. The stereochemical identities of the tryptophan and two alanine residues contained in pacidamycin D have been shown to be of the natural (S) configuration, and the unique 3-methylamino-2-aminobutyric acid contained in this series of antibiotics has been shown to be of the (2S,3S) configuration. Finally, the stereochemistry obtained by hydrogenation of the C(4')-C(5') exocyclic olefin has been shown to be (R) at the C(4') nucleoside site.

The filamentous fungus, P. microspora, has an endophytic-pathologic relationship with T. taxifolia. The fungus resides in the inner bark of symptomless trees, and physiological or environmental factors could trigger its pathological activity. P. microspora produces the phytotoxins pestalopyrone, hydroxypestalopyrone, and pestaloside which give rise to the disease. Pestaloside, which also has antifungal activity, could reduce competition from other fungal endophytes within the host.

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Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.

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