This article was downloaded by: [University of Calgary] On: 02 February 2015, At: 08:54 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl19 Isolation of Illudin S from the Mature Basidiocarp of Omphalotus Illudens, and Isolation of Illudins S and M from the Fermentation Broth of Omphalotus Olearis Thomas G. McCloud a , Paul A. Klueh a b , Kay C. Pearl a c , Laura K. Cartner a & Gary M. Muschik a a Chemical Synthesis and Analysis Laboratory, and KARYOL K. POOLE Fermentation Production Facility, SAIC Frederick, A Division of Science Applications International Corporation , Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201 b Washington University , St. Louis, MO c Monument Rd. , Myersville, MD, 21773 Published online: 04 Oct 2006. To cite this article: Thomas G. McCloud , Paul A. Klueh , Kay C. Pearl , Laura K. Cartner & Gary M. Muschik (1996) Isolation of Illudin S from the Mature Basidiocarp of Omphalotus Illudens, and Isolation of Illudins S and M from the Fermentation Broth of Omphalotus Olearis , Natural Product Letters, 9:2, 87-95, DOI: 10.1080/10575639608044931 To link to this article: http://dx.doi.org/10.1080/10575639608044931 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. 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Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions Downloaded by [University of Calgary] at 08:54 02 February 2015 Narurul Product Lerters 0 1996 OPA (Overseas Publisliers Associa(ioii) Volume 9, pp 87-95 Amsterdam B.V. Publishers 111 The Nethrrlands Repriiirs available directly froin [lie publisher by llarwood Acadeiiiic Publishers Photocopying perinirted by license only Printed in Malaysia ISOLATION OF ILLUDIN S FROM THE MATURE BASIPIOCARP OF OMPHALOTUS ILLUDENS, AND ISOLATION OF ILLUDINS S AND M FROM THE FERMENTATION BROTH OF OMPHALOTUS OLEARIS THOMAS G. MCCLOUD*, PAUL A. KLUEH', KAY C. PEARL", LAURA K. CARTNER, GARY M. MUSCHIK, Chemical Synthesis and Analysis Laboratory, and KARYOL K. POOLE, Fermentation Production Facility, SAIC Frederick, A Division of Science Applications International Corporation, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201 (I<eceived I0 Apt/ /9Y6) Abstract: The cytotoxic sesquiterpene illudin S was isolated from the fruiting body of the fungus Omphalotus illudens. From the fermentation broth of Omphalotus olearis, ATCC-11719, both illudin S and illudin M were isolated. Normal and reverse phase HPLC analytical methods for the title compounds were developed. Key Words: Omphalotus illudens, Omphalotus olearis, illudin M, illudin S, analytical HPLC, fermentation 'Author to whom correspondence should be addressed. 'Current Address: Washington University, St.Louis, MO '*Current Address: 2 Monument Rd., Myersville, MD 21113 INTRODUCTION The initial isolation of the cytotoxic sesquiterpenoids illudin S and M, (figure l), was accomplished from culture liquids Downloaded by [University of Calgary] at 08:54 02 February 2015 of Omphalotus illudens (formerly Clitocybe illudens),',' but isolation and quantitation from the mature basidiocarp of the species has apparently not been reported. Illudin S has been obtained from the basidiocarp of Omphalotus olivascens from California' and Lampteromyces japonicus, a related species from Japan''. The fruiting bodies of Omphalotus illudens are found throughout eastern North America, often in the fall of the year, occurring in large clumps at the base of decaying trees or stumps. The common name for 0. illudens is 'Jack-0'-Lantern' mushroom both because the basidiocarp has the orange color of a ripe pumpkin 87 88 THOMAS G. McCLOUD rr a/ from which a 'Jack-0'-Lantern' is carved, and because they sometimes exhibit bioluminescence. Although the toxicity of Omphalotus species is well known, occasional human poisonings occur when collectors confuse them with edible Chanterelles or with Laetiporus sulphureus, the 'Chicken-of-the-Woods' mushroom5*lo . 0 llludin S, R = CH20H llludin M,R = CH3 FIGURE 1 Illudin M (2',3'-Dihydro-3'~,6'a-dihydroxy-2',2',4',6'- tetramethylspiro [cyclopropane-1, 5 ' - [ SH] inden]-7 ' ( 6 H)-one) Studies using a cell-free enzyme system have demonstrated incorporation of mevalonic acid, supporting biosynthesis via the isoprenoid pathway ". Illudin S and M have shown anti-bacterial effects', and selective toxicity in the 60 human tumor cell line primary screen used by the National Cancer Institute (NCI) to detect anti-cancer activity'. The novel nature of these compounds continues to be of interest to medicinal chemists with attempts Downloaded by [University of Calgary] at 08:54 02 February 2015 currently being made to amelliorate the cytotoxicity (LD,,<10-6pg/ml to human cells in tissue culture) and improve the therapeutic index 8,9,13. In response to a request from the Developmental Therapeutics Program (DTP) of NCI for illudin M for research purposes, the Natural Products Support Group, Chemical Synthesis and Analysis Laboratory, FCRDC, undertook the investigation of several routes by which illudin M could be obtained in quantity. An examination was made of the mature fungal basidiocarp and subsequently, two species of Omphalotus growing in liquid shake culture. ISOLATION OF ILLUDINS S AND M 89 EXPERIMENTAL PART I. Isolat'ion of Illudin S from basidiocarp. Omphalotus illudens was collected (TGM) from two large clumps growing on decaying stumps on the east face of Catoctin Mountain, Frederick County, Maryland, on October 4, 1992. A voucher was deposited at the DTP Natural Products Repository in Frederick, MD, as Q67114480. The collection was quickly frozen, lyophilized, finely ground in a Wiley mill (4 mm mesh) yielding 2.63 kg, which was packed into a borosilicate glass percolator (Kontes 5848021, and steeped in dichloromethane (DCM): methanol(Me0H) (1:l). The vessel was drained and fresh solvent added repeatedly over several days, giving after solvent removal by rotary evaporation, a combined dry weight of 242 gm of crude extract. Extraction of the marc was continued with 100% MeOH and afforded an additional 86.3 gm of extract. Thin layer chromatographic analysis (tlc) (on glass backed silica 60 (EM 5729-6) developed once with chloroform: MeOH (9:1, M:Rf 0.56, S:Rf 0.24) and visualized with vanillin-sulfuric acid) demonstrated that both of these extracts contained illudin S. They were combined, resolubilized in Me0H:water (l:l), and partitioned against n-hexane. The hexane layer was withdrawn, and the aqueous layer was re-partitioned against DCM. The DCM solubles were dried in vdcuo to give 12.7 gm of illudin S enriched crude extract. This was adsorbed onto 25 gm silica (Davison Chemical, grade 22, 60-200 mesh, lot #12/89) and flash chromatographed9 on 1.27 kg of the same silica using hexane:ethyl acetate (1:l) isocratic. Ten pools were formed based on similar Downloaded by [University of Calgary] at 08:54 02 February 2015 appearance on tlc, and a spot having the same Rf as illudin S was detected in pools five through nine. No spot corresponding in Rf to illudin M was observed. Crystallization of pool 7 (245 mg) from DCM/hexane gave 139.0 mg of colorless needles. This crystalline product exhibited identical NMR, MS,& UV spectra to a standard of illudin S obtained from the NCI Repository and to data reported in the literature '*". Purity of these crystals was shown to be greater than 998 by both reverse and normal phase HPLC (method given below). Attempts to obtain additional crystalline illudin S from less-pure 90 THOMAE G McCLOUD et a/ fractions were unsuccessful, though by tlc and HPLC the compound was clearly present. A single, mature, freeze-dried basidiocarp was separated into cap, gill and stem portions, each separately solvent extracted and analyzed for illudin content by reverse-phase HPLC. The three chromatograms were similar, and indicated that illudin S was present in all parts, though in lesser concentration in the stem. No illudin M was detected in the mature fungal basidiocarp. The amount of illudin S in an average sized basidiocarp has been calculated to be -0.5mg. PART 11. Isolation of illudin S and M from fermentation broth. Cultures of Omphalotus illudens (UT 02212) and Omphalotus olearis (American Type Culture Collection (ATCC) 11719) were grown on soy-peptone-glucose-starch (SGSM) and PNSM media, shaking, and on Potato-Dextrose broth (PDB), stationary. Ethyl acetate extracts from each were assayed by tlc and HPLC (figure 2) for the production of the desired compound. In order to quantify illudin during fermentation both normal and reversed-phase HPLC analytical methods were developed. The system consisted of a Waters 600E Multi-solvent delivery system with Millennium 2010 Chromatography Manager v. 2.00 with eluate monitored by a Waters 996 photo diode array detector.