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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 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 , 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

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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

Abstract: The cytotoxic sesquiterpene illudin S was isolated from the fruiting body of the 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' 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 . Although the toxicity of Omphalotus species is well known, occasional human poisonings occur when collectors confuse them with edible 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. HPLC columns were Rainin Dynamax 8p silica, 4.6~300mm with 4.6~15mm guard column, solvent system: 95% DCM:5% MeOH isocratic @ lml/min, retention time; M: 3.6 min., S: 7.8 min. for normal phase and Waters VBondpack 101.1 C18, 3.9~300,MeOH/water 8:2, isocratic, @1.0 ml/min., retention time; M: 4.4 min., S: 3.2 min. for reverse phase. Solvents were HPLC

Downloaded by [University of Calgary] at 08:54 02 February 2015 grade and sparged with helium at 5 cc/min. The detector response was found to be linear between -0.1 and 1.0 pg/ml @ 254 nm for pure standards of illudin S and M, which were obtained from the DTP Repository. 0 I ISOLATION OF ILLUDINS S AND hl

FIGURE 2 HPLC chromatogram of the ethyl acetate extract of 0. olearis grown on SGSM. Reverse phase on C-18, method as given in text.

Both cultures produced illudin S and M, but the quantity and ratio between the two was variable and greatly affected by culture conditions, (figure 3).

MEDIA ETHYL PERCENT POTENTIAL POTENTIAL ACETATE ILLUDIN M YIELD OF YIELD OF SOLUBUS IN EtOAC ILLUDIN M ILLUDIN S I (mg/l) (mg/l)

0.ILLUneJS Downloaded by [University of Calgary] at 08:54 02 February 2015 UT 02212

, PDB 35 11.0 3.8 0.2

0. OWIS SGSM 121 6.4 11.0 4.5

ATCC PNSM 49 13.1 9.5 1.1 11719 PDB 94 4.3 4.0 0.8

FIGURE 3 Illudin S and M from 0. illudens and 0. olearis as influenced by fermentation conditions. Cultures harvested after glucose was depleted (6-15 days). Volume at harvest, 700-1000 mls. 92 THOMAS G MtCLOUD el al.

When grown shaking (250 ml in a 1L Erlenmeyer flask for 7 days) on PNSM media, which is formulated from dextrose (25 gm/l), soluble starch (10 grn/l), lexein (10 gm/l), NZ Arnine (4 gm/l), blackstrap molasses (5 gm/l), MgSO;7H,O (5 gm/l), and CaCO, (2 gm/l), 0. olearis, ATCC 11719, produced -10 mg/l of the illudins in a 9/1 ratio of M to S, (figure 3). It was observed that the ethyl acetate solubles obtained from extraction of the whole broth of this organism grown on PNSM was relatively free of contaminating materials with an Rf on silica similar to illudin M, making chromatographic purification of the desired compound straightforward. It was also confirmed by HPLC analysis that chloroform preferentially extracted illudin M, leaving illudin S in the broth'. 0. olearis seed cultures for scaled up fermentation were grown on PDB, shaking, in 4L Erlenmeyer flasks, then used to inoculate two fermentors, one of 50L (@ 8.6%v/v), the other 80L (@ 10.9%v/v) volume. The production medium was PNSM, pH 5.7, with 0.5%v/v Dow Corning type A antifoam added. A downward trend over time to pH 2.5 was observed in the 50L fermentor, which was not pH adjusted during fermentation. The pH of the 8OL fermentor was adjusted upward during fermentation to maintain a pH of 5.5. The agitation rate was 250 rpm giving a tip speed of 231 ft/min. Temperature was controlled at 25"+/- 0.5"C. Dissolved 0: was maintained at 20% by adjusting air flow. Aliquots were taken each day for HPLC quantitation. Illudin M was detected after 5 days, and reached an apparent plateau at 7 days. No significant difference in illudin production was noted between the two fermentors. Harvest of both fermentors occurred at

Downloaded by [University of Calgary] at 08:54 02 February 2015 164 hours. The combined broths were filtered to remove cellular debris. HPLC analysis of an ethyl acetate extract of the cell debris showed an insignificant amount of illudin S or M. The clarified broth was first extracted with chloroform. Solvent was removed by rotary evaporation and the extract was high vacuum dried, giving 40 gm. The crude extract was resolubilized in chloroform, adsorbed to 170 gm of Davison grade 22 silica chromatographic media, and flash chromatographed through 2,000 gm of the same silica in a 10 cm diameter x 100 cm glass column (Kontes 584800-4000) using nitrogen pressure < 5 psi. Elution was started with hexane and ISOLATION OF ILLUDINS S AND M 93

progressed in a step-gradient fashion through 10, 25, 35 and 50% ethyl acetate in hexane, followed by a strip with MeOH. Eighteen fractions (1 to 4 liters) were collected, rotary evaporated and examined by tlc. Based on similarity of appearance, six pools were made. The second of these, a yellowish solid weighing 6.8 gm, was resolubilized in hexane/ethyl acetate and, after sitting overnight at room temperature, 4.4 gm of colorless needles were obtained. Analysis by normal and reverse phase HPLC showed this material to be >99% pure illudin M. Identity was confirmed by NMR and MS comparison to an authentic sample. The fermentation broth was re-extracted exhaustively with ethyl acetate to yield 25 gm dry weight of crude material. Both tlc and HPLC analysis showed this extract contained illudin S and M. It was solubilized in ethyl acetate, adsorbed onto 80 gm of silica (Davison grade 22) and flash chromatographed on 1,000 gm of the same silica in a manner analogous to before. Illudin M was detected by tlc in pool 2 (0.372 gm) and illudin S was present in pools 5 and 6 (combined weight 2.26 gm). Each pool was resolubilized in hexane/ethyl acetate and, after sitting at. room temperature overnight, 80 mg of illudin M and 46 mg of illudin S were obtained, respectively. Decontamination of the apparatus used in the production was accomplished with strong acid and heating for 3 hours. Following this treatment no illudin S or M could be detected by HPLC in washes from the equipment, or in the spent broth.

DISCUSSION

Downloaded by [University of Calgary] at 08:54 02 February 2015 Illudin S has been isolated from the mature basidiocarp of Lairipteroniyces ~aponicusin 4x10 '% yield without illudin M being found. Likewise, only illudin S was reported from the mature hasidiocarp of Omphalotus olivascens, in similar quantity'. In this work only illudin S has been found in the mature basidiocarp of Ornphalotus illudlns at 5x10 %. This suggests that illudin S itself must be the toxic compound responsible for instances of human poisoning. The uncertain availability of the basidiocarp

diid low yield makes the fruiting body an unreliable 50urie from which to obtain illudin S in quantlty, and illudin M appears not to be present in the basidiocarp of any species so far examined. 94 THOMAS (I McCLOUD et ul

Both cultures of Omphalotus were producers of illudin S and M, but the ratio of these products was found to be variable and influenced by the composition of the media. In the work reported here, culture conditions and medium were optimized for the production of illudin M, and an isolated yield of -88 mg/L was obtained. It is probable that further improvements in yield of either product could be achieved by manipulation of fermentation conditions. The prospects for producing either illudin M or S in quantity by fermentation appears feasible.

The content of this publication do not necessarily reflect the views or polices of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Gove=rnment.

REFERENCES

1. M. Anchel, A. Hervey, W.I. Robbins, (1950) Proc. Natl. Acad. Sci., 36, 300. 2. T.C. McMorris, M. Anchel, (1963) J. Am. Chem. SOC., 85, 831. 3. K. Nakanishi, M. Ohashi, M. Tada, Y. Yamada, (1965) Tetrahedron, 21, 1231. 4. T.C. McMorris, S. Moon, G. Ungab, R.J. Kerees, (1989) J. Nat. Prod., 52, 380. 5. C.M. Christianson, (1965) Common Fleshy Fungi, Burgess Publishing Company, Minneapolis, p.32. 6. M.J. Kelner, T.C. McMorris, W.T. Beck, J.M. Zamora, R. Taetle, (1987) Cancer Res., 47, 3186. 7. T.C. McMorris, M.J. Kelner, W. Wang, L.A. Estes, M.A. Montoya, Downloaded by [University of Calgary] at 08:54 02 February 2015 and R. Taetle (1992) J. Amer Chem Soc 57, 6876-6883. 8. M.J. Kelner, T.C. McMorris, and R. Taetle, (1990) J. of the Natl Cancer Inst, 82, 1562-1565. 9. W.C. Still, M. Kahn and A. Mltra, (19781, Jol. Org. Chem. 43, 2923-2925. 10. D.R. Benjamin, (1995) : Poisons and Panaceas, W. H. Freeman and Co., New York, pp. 366-7. 11. M. Price and P. Heinstein, (1978) Lloydid, 41, 574-577. 12. A. P.W. Bradshaw and J. R. Hanson, (19821 Jol. of the Chem. SOC. Perkin Trans I, 2445-2448. ISOLATlON OF ILLUDINS S AND M 95

13. T.C. McMorris, M.J. Kelner, R.K. Chadha, J.S. Siegel, S.Moon, and M.M. Moya (1989) Tetrahedron, 45, 5433-5440. Downloaded by [University of Calgary] at 08:54 02 February 2015 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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