FULL PAPER DOI: 10.1002/ejoc.201200757 Epicolactone – Natural Product Isolated from the Sugarcane Endophytic Fungus Epicoccum nigrum Francisca Diana da Silva Araújo, [a] Léia Cecilia de Lima Fávaro, [b] Welington Luiz Araújo, [c] Fábio Lazzarotto de Oliveira, [a] Ricardo Aparicio, [a] and Anita Jocelyne Marsaioli* [a] Keywords: Natural products / Bioorganic chemistry / Mutagenesis / Epicoccum nigrum The endophytic fungus Epicoccum nigrum was isolated from sugarcane and the bioguided fractionation of the ethyl acet- ate extract led to the isolation of epicolactone, mellein, and 4,5-dimethylresorcinol. Characterization of epicolactone by MS, NMR and X-ray crystallography revealed a new natural Introduction Fungi are important in biotechnological processes ap- plied in the food, cosmetic, and pharmaceutical indus- tries. [1] Notwithstanding, the relevance of the predicted fungi species (1.5 million), only 5.7% have been described, and of these only a few have actually been investigated for the production of bioactive metabolites. [2] Among these, en- dophytic fungi living in association with plants are a rich source of biologically active compounds. [3] Epicoccum nigrum Link (syn. E. purpurascens Ehrenb. ex Schlecht) is an ascomycete fungus distributed worldwide, which colonizes different types of soils and host plants. [4] By 1959 about 70 species of the Epicoccum genus had been described, which were evaluated and combined into a single variable species, the E. nigrum Link. [5] However, recently Fávaro et al. [6] developed an analysis based on the poly- phasic approach, which suggested the occurrence of cryptic species within E. nigrum and that many of the sequences deposited as E. nigrum in GenBank and culture collections of microbial strains should be reclassified, including the ref- erence strain CBS 161.73. E. nigrum has been used as a biological control agent for plant pathogens [7] and produces a variety of bioactive secondary metabolites such as flavipin, [8] epicorazins A-B, [9] epirodin, [10] triornicin, [11] orevactaene, [12] epicoccamide, [13] [a] Chemistry Institute, University of Campinas, POB 6154, 13084-971, Campinas, SP, Brazil Fax: +55-19-35213023 E-mail: anita@iqm.unicamp.br Homepage: http://anita.iqm.unicamp.br/ [b] Brazilian Agricultural Research Corporation, Embrapa Agroenergia, Brasília, Distrito Federal, Brazil [c] Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201200757. Eur. J. Org. Chem. 2012, 5225–5230 © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5225 product with an unusual carbon skeleton. The production of this secondary metabolite decreased in mutants of Epi- coccum nigrum transformed by Agrobacterium tumefaciens. Additionally, these mutants produced 4-hydroxymellein. epicocconone, [14] D8646-2-6, [15] and epicoccins A–D, [16] among others. However, little is known regarding the genes related to the synthesis of the metabolites produced by Ep- icoccum nigrum. Herein we report the isolation and struc- tural elucidation of secondary metabolites from E. nigrum wild type P16, and using three E. nigrum agrotransformants P16–17, P16–47set, and P16–91, we identify genes related to the synthesis of a new natural compound produced by E. nigrum P16. Results and Discussion The culture broth from E. nigrum fermentation was ex- tracted with ethyl acetate and subjected to successive frac- tionations with open silica gel column chromatography. Further purification of the selected bioactive fractions (an- timicrobial) afforded a unique natural product, here named epicolactone (1), as white crystals. Based on HRMS (ESI) in negative mode, combined with 1 H and 13 C 2D NMR spectroscopic data, the epicolactone molecular formula is C 17 H 16 O 8 (10 degrees of unsaturation). Analysis of the 1 H, 13 C{ 1 H}, DEPT 135°, and gradient heteronuclear single quantum coherence (gHSQC) 2D NMR spectroscopic data of 1 showed the occurrence of three singlets at δ H = 6.07, 8.50, and 8.61 ppm with no correlations to carbon atoms, which are thus assigned to OH, two methyl groups, three oxymethylenes with diastereotopic hydrogen atoms, one methane group, and eleven carbon atoms nonbonded to hy- drogen atoms, three of which resonated at δ C = 176.0, 192.8, and 190.1 ppm, characteristic of carbonyl carbon atoms (Table 1). The gradient COSY (gCOSY) spectrum con- firmed the presence of three two-spin systems assigned to diastereotopic oxymethylene hydrogen atoms. The remain- ing nine hydrogen atoms showed neither scalar coupling nor homonuclear 1 H, 1 H correlations. This information