ISOLATION OF ILLUDIN S FROM THE MATURE BASIDIOCARP OF OMPHALOTUS ILLUDENS, AND
ISOLATION OF ILLUDIN 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,* and Karyol K. Poole$
*Chemical Synthesis and Analysis Laboratory and $Fermentation Production Facility,
SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, MD

Presented at the American Society of Pharmacognosy - 37th Annual Meeting - July 27-31, 1996, Santa Cruz, CA

ABSTRACT

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 of Omphalotus illudens, which was found to contain only illudin S, and subsequently, two species of Omphalotus growing in liquid shake culture, both of which produced illuden M and S. Normal and reverse phase chromatographic methods where developed that allowed for rapid quantitation of the desired compounds from fermentation broth. Fermentation conditions where optimized for the production of illudin M by O. olearis and 4.4 gm of the desired compound was isolated.

Illudin

PART 1: Isolation of illudin M from mature basidiocarp

   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 O. illudens is 'Jack-O'-Lantern' mushroom both because the basidiocarp has the orange color of a ripe pumpkin from which a 'Jack-O'-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' mushroom.5, 10

   Omphalotus illudens was collected (TGM) on Catoctin Mountain, Frederick County, MD, on October 4, 1992. These where quickly frozen, lyophilized, finely ground in a Wiley mill (4mm mesh) yielding 2.63 kg, which was extracted exhaustively with dichloromethane (DCM): methanol (MeOH)(1:1). A combined dry weight of 242 gm of crude extract was obtained. 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 onece 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 where combined, resolubilized in MeOH:water (1:1), and partitioned against n-hexane. Thge hexane layer was withdrawn, and the aqueous layer waas re-partitioned against DCM. The DCM solubles where dried in vacuo 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 (1:1) isocratic. Ten pools where formed based on similar appearance on tlc. No spot corresponding in Rf to illudin M was observed. Crystallization of pool 7 (245 mg) from DCM/hexane gave 139.0mg of colorless needles. This crystalline product exhibited identical NMR, MS, & UV spectra to a standard of illudin S obtained form the DTP Repository and to data reported in the literature.3, 12 Purity of these crystals was shown to be greater than 99% by both reverse and normal phase HPLC.

   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 ammount of illudin S in an avarage sized basidiocarp has been calculated to be ~0.5 mg.

*Most mycologist consider O. illudens and O. olearis to be synonymous, however, both names are still used. Separation into   two species was formerly based on the larger spore size of O. olearis.


PART II. 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 Multisolvent delivery system with Millenium 2010 Chromatography Manager v. 2.00 with eluate monitored by a Waters 996 photo diode array detector. HPLC columns were Rainin Dynamax 8 silica, 4.6 x 300 mm with 4.6 x 15 mm guard column, solvent system: 95% DCM: 5% MeOH isocratic @ 1 ml/min, retention time; M:4.4 min, S: 7.8 min for normal phase and Waters Bondpack 10 C18, 3.9 x 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 grade and sparged with helium at 5 cc/min. The detector response was found to be linear between ~0.1 and 1.0 g/ml @ 254 nm for pure standards of illudin S and M, which were obtained from the DTP Repository.

   Both cultures were found to produce illudin S and M, but the quantityand ratio between the two was variable and greatly affected by culture conditions (figure 3).

hplc

FIGURE 2. C-18 reverse phase HPLC quantitation of illudin S and M in ethyl acetate extract of O. olearis fermented on SGSM (top) and PNSM (bottom)

table

Large Scale Fermentation

   When grown shaking on PNSM media, [dextrose (25 gm/l), soluble starch (10 gm/l), lexein (10 gm/l), NZ Amine (4 gm/l), blackstrap molasses (5 gm/l), MgSO4*7H2O (5 gm/l), and CaCO3 (2 gm/l)], O. olearis, ATCC 11719, produced ~10 mg/L of the illudins in a 9/1 ratio of M to S, (figure 3). Extract of the whole broth of this organism grown on PNSM waas relatively free of contaminating materials with an HPLC analysis that chloroform preferentially extracted illudin M, leaving illudin S in the broth.1

   O. olearis seed cultures for scaled up fermentation were grown on PDB, shaking, in 4 L Erlenmeyer flasks, then used to inoculate two fermentors, one of 50 L (@ 8.6% v/v), the other 80 L (@ 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 50 L fermentor, which was not pH adjusted during fermentation. The pH of the 80 L fermentor was adjusted upward during fermentation to maintain a pH of 5.5. The agitation rate was 250 rpm giving a tip speed of 237 ft/min. Temperature was controlled at 25% o.5%C. Dissolved O 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 164 hours. The combined broths were filtered to remove cellular debris. HPLC analysis of an ethyl acetate extract of cell debris showed an insignificant amount of illudin S or M.


Purification of illudin M and S

   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 x100 cm glass column (Kontes 584800-4000) using nitrogen pressure <5 psi. Elution was started with hexane and 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. This extract was flash chromatographed on silica in a manner analogous to before. Illudin M was detcted 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.


DISCUSSION

   Illudin S has been isolated from the mature basidiocarp of Lampteromyces japonicus in 4 x 10-4% yield without illudin M being found.3 Likewise, only illudin S was reported from the mature basidiocarp of Omphalotus olivascens, in similar quantity.4 In this work only illudin S has been in the mature basidiocarp of Omphalotus illudens at 5x 10-3%. This suggests that illudin S itself must be the toxic compound responsible for instances of human poisoning. The uncertain availability of the basidiocarp and low yield makes the fruiting body an unreliable source from which to obtain illudin S in quantity, and illudin M appears not to be present in the basidiocarp of any species so far examined.

   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.


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 Publiching 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, R. Taetle (1992) J. Am. Chem. Soc., 57, 6876-6883.
  8. M.J. Kelner, T.C. McMorris, R. Taetle (1990) J. Natl. Cancer Inst., 82, 1562-1565.
  9. W.C. Still, M. Kahn, A. Mitra (1978) J. Org. Chem. 43, 2923-2925.
  10. D.R. Benjamin (1995) Mushrooms: Poisons and Panaceas, W.H. Freeman and Co., New York, pp. 366-367.
  11. M. Price, P. Heinstein (1978) Lloydia, 41, 574-577.
  12. A.P.W. Bradshaw, J.R. Hanson (1982) J. Chem. Soc. Perkin Trans. I, 2445-2448.
  13. T.C. McMorris, M.J. Kelner, R.K. Chadha, J.S. Siegel, S. Moon, M.M. Moya (1989) Tetrahedron, 45, 5433-5440.




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