Pergamon

PII: S0305-1978(96)00053-1

Biochemical Systernatics and Ecology, Vol. 24, No. 5, pp. 465-468,1996 Crown Copyright © 1996. Published by Elsevier Science Ltd

Printed in Great Britain 0305-1978/96 $16.00+0.00

A Cerebroside from the Marine Fungus Microsphaeropsis olivacea (Bonord.) H6hn

MICHAEL KEUSGEN,*t CHAO-MEI YU,t JONATHAN M. CURTIS,t** DON BREWERt and STEPHEN W. AYERt

"lnstitut for Pharmazeutische Biologie, U niversit~t Bonn, Nul~allee 6, D-53115 Bonn, Germany; ?Institute for Marine Biosciences, National Research Council of Canada, Halifax, B3H 3Zl Nova Scotia,

Canada; ~tSynPhar Laboratories Inc., 4290-91 A Street, Edmonton, T6E 5V2, Alberta, Canada

Key Word Index--Microsphaeropsis olivacea; marine fungus; cerebroside; NMR spectroscopy; tandem mass spectrometry.

Subject and Source Microsphaeropsis olivacea (Bonord.) Hbhn. (strain F010) was obtained from a sponge, Age~us sp., col- lected off Sombrero Key East, Florida, on 19 February 1990. The sponge was shipped to Halifax in a frozen state over dry ice, stored frozen at - 2 0 ° C for 13 weeks, and then examined for fungi. Thawed sponge was aseptically cut into thin slices (~1 x 1 cm), and one slice was placed on MYP§ agar. A colony of M. olio vacea (Bonord.) Hbhn (strain F01 O) appeared after 7 days and was transferred from the isolation plate to a 2% (w/v) malt agar slant for longer term storage at 4°C. A reference specimen of M. olivacea (Bonord.) Hbhn. (strain F010) is maintained at the Institute for Marine Biosciences (H LX1743).

Previous Work Microsphaeropsis hoohn (NRRL 15684) was shown to produce the lactone antibiotic $39163/F-I (Tscherter et aL, 1988). TAN-1496 A, C, and E, novel epi-oligothiadiketo-piperazines, as well as sirodesmin A and its deacetyl derivative, were isolated from cultures of Microsphaeropsis sp, FL-16144 (Funabashi et al., 1994).

Present Study Ten 500 ml Erlenmeyer flasks each containing 100 ml of UCII medium¶ were each inoculated with 5 ml of inoculum prepared by transferring mycelia from the storage slant to a 500 ml flask containing 100 ml of UCI medium, II and culturing for 3 days on a rotary shaker (25°C, 250 rpm). After 7 days" shaking, the ten production cultures were combined and sequentially filtered through cloth and Whatman ~;~1 filter paper. The residue (705 mg) from the ethyl acetate extract of the culture filtrate was subjected to flash chroma- tography over silica gel, using an increasing step gradient of ethyl acetate in hexane (10-50%), and then ethyl acetate in methanol (90-0%). The fraction eluting with ethyl acetate/hexane (25:75, v/v) (43 rag) was further purified by reversed-phase flash chromatography, eluting with 80% methanol/water. Final purification was achieved using Sephadex LH-20 (methanol) to give 5 mg of pure N-2"-hydroxy-3'E- octedecenoyl-1 -O-p-D-glucopyranosyl-9-methyl-4E,8E-sphingadiene (1): LSI MS: observed [M + H] + m~ z 754.5822 (calculated for C43HaoNOs: 754.5833, A= --1.1 mDa). lac NMR 8:175.5 (C-1"), 136.7 (C- 9), 134.7 (C-4"), 134.5 (C-S), 131.0 (C-4), 129.0 (C-3'), 124.9 (C-8), 104.7 (C-1"), 78.0 (C-5"), 77.9 (C-3"), 75.0 (C-2"), 74.1 (C-2"), 72.9 (C-3), 71.6 (C-4"), 69.7 (C-1), 62.7 (C-6"), 54.6 (C-2), 33.8 (C- 6), 28.8 (C-7), 40.8 (C-10), 33.5 (C-5"), 33.1 (C-16, C-16'), ~ 30.0 (C-11, C-12, C-13, C-14, C-15, C-

§Malt extract (10 g I-1), yeast extract (3 g I-1), peptone (5 g I-1), CaCO 3 (3 g I-1), agar (20 g 1-1) in deionized water.

¶Molasses (20 g I-1), dextrin (30 g I-1), fish meal (15 g I-1), Pharamedia (15 g 1-1) in deionized water.

IIGlucose (25 g I-1), Pharamedia (25 g 1-1) in deionized water. "*Corresponding author.

(Received 18 January 1996; accepted 13 March 1996)

465

466 M. KEUSGEN ETAL.

6', C-7', C-8", C-9', C-10', C-11', C-12', C-13', C-14', C-15'), 23.8 (C-17, C-17'), 16.2 (C-19), 14.5 (C- 18, C-18'). 1H NMR 5:5.83 (1H, dtd, J=15.3, 6.7, 1.2 Hz, H-4"), 5.71 (1H, dtd, J=15.5, 5.5, ~1, H-5), 5.48 (1 H, ddt, J= 15.3, 6.1,1.3 Hz, H-3"), 5.45 (1 H, ddtd, J = 15.5, 6.8 ~ 1, ~ 1 Hz, H-4), 5.14 (1 H, bt, J = 5 . 5 Hz, H-8), 4.43 (1H, rid, J=6.1 , t.1 Hz, H-2"), 4.26 (1H, d, J=7.8 Hz, H-I") , 4.13 (1H, dd, J = 10.4, 5.4 Hz, H-1 b), 4.1 2 (1 H, t, J=6.8 Hz, H-3), 3.95 (1 H, ddd, J= 6.8, 5.4, 3.5 Hz, H-2), 3.85 (1 H, dd, J = 12.0, 1.8 Hz, H-6"b), 3.69 (1 H, dd, J = 10.4, 3.5 Hz, H-1 a), 3.66 (1 H, dd, J= 12.0, 5.4 Hz, H-6"a), 3.34 (1 H, t, J=8.9 Hz, H-3"), 3.27 (2H, m, H-4", H-5"), 3.19 (1 H, dd, J=8.9, 7.8 Hz, H-2"), 2.05 (2H, m, H2-7), 2.04 (2H, m, H2-5'), 2.02 (2H, m, H2-6), 1.97 (2H, t, J=7.6 Hz, H2-10), 1.59 (3H, s, H3-19), o l . 3 0 (38H, bm, H2-11, H2-12, H2-13, H2-14, H2-15, H2-16, H2-17, H2-6", H2-7', H2-8", H2-9", H2-10", H2-11", H2-12', H2-13', H2-14', H2-15', H2-16', H2-17"), 0.89 (6H, t, J=6.9 Hz, H3-18, H3-18' ). Assignment of the 1H and 13C NMR resonances was obtained by interpretation of the results of 1H, 13C, DEPT, COSY, TOCSY, NOESY, HMBC, and HMQC NMR experiments performed with a Bruker AMX 500 spectrometer. 1H NMR spectra were obtained at 500 MHz and 13C NMR spectra at 125 MHz using CD2HOD (8 3.30) and CD30D (8 49.0) as internal standards, respectively. The 13C NMR chemical shift assignments for 1 are reported here for the first time, and are comparable to those reported for closely related cerebrosides (Wenzao et al., 1994; Sitrin et al., 1988). The 1H N MR data are in good agreement with those originally reported for I (Villas Boas eta/., 1994).

The sugar moiety of 1 was identified as a #-glucopyranose by the characteristic anomeric proton resonance at 8 4.26, and was supported by the observance of a methine carbon resonance at 5 104.7 in the 13C N MR spectrum of 1, assignable to a #-substituted anomeric carbon (Koerner et al., 1979), and a correlation between the anomeric proton and the carbon at C-5" (5 78.0) in the HMBC spectrum. H-3" couplings of 8.9 Hz with H-2" and H-4" supported the assigned trans-diaxial relationship among the glucopyranose hydrogen atoms.

The erythro (2S', 3R*) relative configuration for the long-chain sphingoid base was assigned on the basis of the 13C chemical shifts determined for C-1, C-2; and C-3, and the assigned shifts and 3JHH coupling constants for H-l , H-2, and H-3 (Wenzao et al., 1994). The E configuration of the C-4, C-5 double bond was assigned on the basis of a large coupling (15.5 Hz) between H-4 and H-5 (Silverstein et aL, 1991 ). The geometry about the C-8, C-9 double bond was also assigned as E on the basis of an NOE correlation observed between H3-19 and H2-7 in the NOESY spectrum.

The ¢-hydroxy monounsaturated fatty acid partial structure was determined by interpretation of the NMR data. The hydrogen atom at C-2' (5 4.43) shows a significant correlation to the C-1" carbon (5 175.5) in the HMBC spectrum of 1. In the COSY spectrum, a correlation between H-2' and an olefinic proton (H-3') at 5 5.48 indicated that a carbon-carbon double bond was located between C-3' and C-4'. The observation of a 1 5.3 Hz coupling between H-3' and H -4' allowed assignment of the E configuration to this double bond. The 1H and ~3C chemical shifts, and the 1H_~ H coupling constants, are very similar to those reported for the acyl moiety of related cerebrosides by Sitrin et al. (1988), where the C-2" absolute stereochemistry was determined to be R. However, on the basis of our data for 1, the chemical shifts (in CD3OD/CDCl3) assigned to the olefinic carbons (C-3', ~ 134.3; C-4", 5 128.1 ) of the (2R)-hydroxy-(3E) - hexadecenoyl moiety in the Sitrin et aL (1988) publication, which were not confirmed by 2D-NMR tech- niques, should be reversed.

The number of carbons in each of the two alkyl chains was determined by interpretation of the LSIMS tandem mass spectrometry (MS-MS) spectrum of the protonated molecular ion of 1 ([M + H] +, m/z 754.6) in the positive-ion mode (Sawabe et aL, 1994). An intense fragment ion at m/z 294 is characteristic of the protonated methyl branched sphingosine base (measured as m/z 294.2796 in the normal LSIMS spectrum; calculated for C19H35NO: 294.2797. A= -0.1 mDa), and the fatty acid must therefore be 2- hydroxy-(3E)-octadecenoic acid. A fragment ion at m/z 574 is indicative of the loss of the glucopyranose moiety ([M + H-CsH120s]+).

Chemotaxonomic Significance Cerebrosides are of interest because of their cytotoxic activity (Wenzao et al., 1 994), their synergy with the known glucan synthetase inhibitor aculeacin (Sitrin et aL, 1988), and their important biological roles as cell surface antigens and receptors (Merrill et aL, 1993; Hakomori, 1981; Ledeen and Yu, 1973; Sweeley and Siddiqui, 1977), and precursors for second messengers involved in signal transduction pathways (Bell et aL, 1993). Glucosphingolipids with a ceramide composition consisting of 9-methyl-4,8-sphingadiene linked to a 2-hydroxy-3-octadecenoic acid have previously been isolated from three species of terrestrial fungi, Pachybasium sp. (possibly a species of Tolypocladium, Sitrin et al., 1 988), Penicillium funiculosum (Kawai et aL, 1985), and Fusicoccum arnygadali (Ballio et aL, 1979), and also from three pathogenic strains of Aspergillus, the etiological agents of different lung diseases, including aspergilloma (fungus ball), and invasive aspergillosis (Villas Boas eta/., 1994). To the best of our knowledge, the isolatiQn of 1 from /t~ olivacea is the first account of the isolation of a cerebroside from a Coelomycete.

Acknowledgements--Thanks to Mr Marshall Greenwell, Mrs Margaret G. Flack, Mr Ping Seto, Mr Don Leek, and Dr John A. Walter for valuable assistance. This is NRCC publication No. 39695.

CEREBROSIDE FROM MICROSPHAEROPSIS OLIVACEA 467

O3 "1-

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b

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468 M. KEUSGENETAL.

R e f e r e n c e s Ballio, A., Casinovi, C, G., Framondino, M., Marino, G., Nota, G. and Santurbano, B. (1979) Biochem. Biophys. Acta 573, 51; Bell, R. M., Hannun, Y. A. and Merril l, A. H., Jr, (eds) (1993) Sphingolipids Part A: Functions and Breakdown Products: Advances in Lipid Research, Vol 25, Academic Press, San Diego; Funabashi, Y., Horiguchi, T., l inuma, S.daS, Tanida, S. and Harada, S. (1994) J. Antibiotics 47, 1202; Hakomori, S. Ann. Rev. Biochern. 50, 733; Kawai, G., Ikeda, Y. and Tubaki, K. (1985) Agric. Biol. Chem. 49, 2137; Koerner, T. A.W. Jr., Cary, LW. , Li, S.-C. and Li, Y.-T. (1979) J. BioL Chem. 254, 2326; Ledeen, R. W. and Yu, R. K. (1973) In Lysosomes and Storage Diseases (Hers, H. G. and Van Hoof, R., eds) pp. 105-145, Academic Press, NewYork; Merril l, A. H. Jr., Hannun, Y. A. and Bell, R. M. (1993) Adv. LipidRes. 25, 1; Sawabe, A., Mor i ta, M., Okamoto, T. and Ouchi, S. (1994) BioL Mass Spectrom. 23, 660; Silverstein, R. M., Bassler, G. C. and Morri l , T. C. (1991) In Spectrometric Identification of Organic Compounds 5th edn., p. 221, J. Wiley, NewYork; Sitrin, R. D., Chan, G., Dingerdissen, J., DeBrosse, C., Mehta, R., Roberts, G., Rottschaefer, S., Staiger, D., Valenta, J., Shader, K. a . , Stedmann, R. J. and Hoover, J. E. (1988) J. Antibiotics 41, 469; Sweeley, C. C. and Siddiqui, B. (1977) In The Glycoconjugates (Horowitz, M. I. and Pigman, W., eds) Vol I, pp. 459-540, Academic Press, NewYork; Tscherter, H., Hofmann, H., Ewald, R. and Dreyfuss, M. M. (1988) U. S. Patent 4,753,959 [ Chemical Abstracts110, 417 (22315y)]; Villas Boas, M. H. S., Egge, H., Pohlentz, G., Hartman, R. and Bergter, E. B. (1994) Chem. Phys. Lipids 70, 11; Wenzao, J., Rinehart, K. L. and Jares-Erijman, E. A. (1994) J, Org. Chem. 59,144.