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Clitolactone: a banana slug antifeedant from Clitocybe flaccida

     Department of Chemistry, Humboldt State University, Arcata, California 95521

David L. Largent
Bradley L. Thompson

     Department of Biological Sciences, Humboldt State University, Arcata, California 95521

	ABSTRACT

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Clitolactone, 5-(chloromethyl)-3-methyl-2(5H)-furanone, was isolated from sporocaps of the mushroom Clitocybe flaccida. The structure was determined by HRMS, EIMS, ¹H & ¹³C NMR, 2D ¹H-¹³C COSY and ¹H-¹H COSY. This mushroom is not eaten by the banana slug Ariolimax columbianus (Gould), a mycophagist from the temperate rain forests of the Pacific Northwest. Clitolactone acts as an antifeedant because these slugs rejected 1.0 cm² pieces of lettuce treated with 25 µg of clitolactone.

Key words: Ariolimax columbianus, Basidiomycetes, chlorinated natural product, lactone, slug, sporocarp

	INTRODUCTION

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Clitocybe flaccida (Fries) Kümmer is a medium-size, buff-colored mushroom found in association with confers in western North America (Bigelow 1985). We observed that C. flaccida was not readily eaten by the banana slug Ariolimax columbianus (Gould), a natural mycophagist from the temperate rain forests of the Pacific Northwest. Clitolactone, 5-(chloromethyl)-3-methyl-2(5H)-furanone (Fig. 1A) was isolated from CH₂Cl₂ extracts of this mushroom and found to have potent slug antifeedant activity.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Structures of clitolactone (A) and lepiochlorin (B).

	MATERIALS AND METHODS

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Spectral analysis – Gas chromatography-mass spectrometry (GC-MS) of initial CH₂Cl₂ extracts was done with a Hewlett-Packard GCD Plus fitted with a 30 m x 0.25 mm cross-linked phenyl methyl silicone capillary column (HP-5MS). The gas chromatograph was programmed so the oven temperature was kept at 40 C for 4 min, then increased to a final temperature of 325 C at a rate of 30 C/min and kept at this temperature 2 min. Mass spectral fragments below m/z = 39 were not recorded. ¹H and ¹³C NMR were recorded on a Bruker QE-Plus instrument at 300 and 75 MHz, respectively, in CDCl₃. The high-resolution mass spectrum was recorded at the University of Illinois at Urbana Mass Spectrometry Laboratory.

Slug bioassay – Laboratory tests on the antifeedant activity of clitolactone were carried out using 10 freshly collected banana slugs for each concentration tested. Each slug was placed individually on a clean 20 cm by 20 cm glass plate and presented with 1.0 cm² pieces of commercial lettuce (iceberg). Slugs that did not start to eat the lettuce within 1 min were excluded from further testing. To control for repellency of the solvent, a second piece of lettuce onto which 10 µL of CH₂Cl₂ had been placed and evaporated was offered. If the slug ate the solvent control, a piece of lettuce that had 100, 50, 25 or 12.5 µg of clitolactone in 10 µL of CH₂Cl₂ (solvent evaporated as above) was offered to the slug. Antifeedant activity was rated as positive if the slug tasted the lettuce and rejected it. To ascertain whether the slug had rejected a treated sample because it no longer was feeding, another piece of untreated lettuce was offered. Only when the slug started eating the new untreated lettuce within 1 min, was antifeedant activity of the previous test recorded.

	RESULTS

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Gas Chromatography-mass spectral (GC-MS) analysis of the initial (nonconcentrated) extract of C. flaccida showed it to contain one volatile component that had an M⁺ at m/z = 146 and M⁺+2 at m/z = 148 in a 3:1 ratio indicating the presence of one chlorine atom. A high-resolution mass spectrum (HRMS) showed the molecular ion at m/z = 146.0139 (calculated for C₆H₇ClO₂, 146.0135).

The ¹³C nuclear magnetic resonance spectrum (NMR) and attached proton test (APT) spectrum of clitolactone shows 6 carbon signals and 7 hydrogens: an ester carbonyl at 173.2, a carbon-carbon double bond at 145.5 (1H) and 132.4 (quaternary), a methine (1H) at 79.0, a methylene (2H) at 43.5 and a methyl (3H) at 10.9. The molecular formula, C₆H₇ClO₂, indicates three sites of unsaturation, and because the ¹³C spectrum shows a carbonyl and a carbon-carbon double bond, the third site must be a ring. Integration of the ¹H spectrum confirms 7 hydrogens: 7. 12 (1H, t, J = 1.44 Hz), 5.11 (1H, m), 3.71 (2H, dq, J = 11.42, 6.37, 4.72 Hz), and 1.97 (3H, t, J = 1.62 Hz). With 2D ¹H-¹³C correlated spectroscopy (COSY) all distinct ¹H nuclei were assigned to the resonances of ¹³C nuclei.

The ¹H-¹H COSY shows connectivity between the ethylenic hydrogen (¹H, 7.12; ¹³C, 145.5) and the methyl group (¹H, 1.97; ¹³C, 10.9) confirming their attachment to the double bond. ¹H-¹H COSY also shows connectivity between the methine (¹H, 5.11; ¹³C, 79.0) and the chloromethyl group (¹H, 3.71; ¹³C, 43.5). The methylene (¹³C, 43.5) is a chloromethyl group with two diastereotopic H's giving a ¹H AB pattern at 3.71 (J = 11.42 Hz). This doublet of doublets is split further by the stereogenic methine (¹³C, 79.0) that couples each H of the -CH₂Cl group with a different coupling constant (J = 6.37, 4.72 Hz). The 5.11 ¹H shift of this methine is diagnostic for attachment to the oxygen of an ester group (Crews et al 1998). The last carbon at 173.2 is an ester carbonyl and because the methine at 5.11 is attached to an ester, the carbonyl must be attached to the carbon-carbon double bond.

Because the ¹H-¹H COSY shows connectivity of the methine (¹H, 5.11) to both the ethylenic H (¹H, 7.12) and the -CH₃ (¹H, 1.97) group, the location of the ethylenic H (¹H, 7.12) was deduced from its chemical shift. Due to resonance hybridization, the ethylenic hydrogen ß to the carbonyl of 2(5H)-furanone has a larger downfield shift than an H (Silverstein and Webster 1998). A close model of clitolactone is 5-(hydroxymethyl)-2(5H)-furanone, which has chemical shifts of 6.20 and 7.55 for the ethylenic Hs on C3 and C4, respectively (Poucher and Behnke 1993). Substitution by a methyl group at C3 or C4 will reduce the chemical shift of the remaining ethylenic H by 0.32 (Crews et al 1998). Because the signal of the ethylenic H of clitolactone is at 7.12, it must be on C4. These data lead unambiguously to the assignment of structure A to clitolactone.

We observed that C. flaccida was not eaten readily by the banana slug, Ariolimax columbianus (Gould), a natural mycophagist from the temperate rain forests of the Pacific Northwest. We tested clitolactone to see if it might be responsible for unpalatability of this mushroom. When 100, 50 or 25 µg of clitolactone was placed on a 1.0 cm² piece of iceberg lettuce, all 10 slugs used in the assay rejected the treated lettuce, but at 12.5 µg some slugs ate the treated lettuce. These slugs ate control pieces of lettuce including a solvent control, onto which 10 µL of CH₂Cl₂ had been placed and evaporated. GC-MS comparison of standard solutions to the original extract showed that the concentration of clitolactone in the sporocarps was 480 µg/g.

	DISCUSSION

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Clitolactone is related to lepiochlorin (Fig. 1A), a chlorinated antibiotic isolated from the mycelium of an unidentified species of a Lepiota mushroom, and differs only by the lack of a hydroxy group at the 5 position (Nair and Harvey 1979). Halogenated compounds recently have been described from the mycelium of a number of mushrooms (Swarts et al 1996, 1997, Verhagen et al 1998), but there are few cases of identification from fruiting bodies. A possible reason for this difference might be a major difference in metabolic pathways used by the mycelium and the fruiting bodies. We recently have demonstrated that, for basidiomycetes, volatile secondary metabolites in the mycelium can be totally different from those found in fruiting bodies (Wood et al 2000).

	FOOTNOTES

 
¹ Corresponding author. E-mail: wfw2{at}humboldt.edu

Accepted for publication May 28, 2003.

	LITERATURE CITED

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Bigelow HE., 1985 North American species of Clitocybe. Part II. Beihefte zur Nova Hedwigia 81:281-471

Crews P, Rodrígues J, Jaspars M., 1998 Organic structure analysis. New York: Oxford University Press. 552 p

Nair MSR, Harvey A., 1979 Structure of lepiochlorin, an antibiotic metabolite of a fungus cultivated by ants. Phytochemistry 18:326-327

Poucher CJ, Behnke J., 1993 The Aldrich library of ¹³C and ¹H NMR spectra. Milwaukee: Aldrich Chemical Co. 1148 p

Silverstein RM, Webster FX., 1998 Spectrometric identification of organic compounds. 6th ed. New York: John Wiley & Sons. 482 p

Swarts HJ, Teunisse PJM, Verhagen FJM, Field JA, Wijnberg JBPA., 1997 Chlorinated anisyl metabolites produced by basidiomycetes. Mycol Res 101:373-374

———, Verhagen FJM, Field JA, Wijnberg JBPA., 1996 Novel chlorometabolites produced by Bjerkandera species. Phytochemistry 42:1699-1701

Verhagen FJM, Swarts HJ, Wijnberg JBPA, Field JA., 1998 Organohalogen production is a ubiquitous capacity among Basidiomycetes. Chemosphere 37:2091-2104

Wood WF, Farquar GR, Largent DL., 2000 Different volatile compounds from mycelium and sporocarp of Pleurotus ostreatus. Biochem Syst Ecol 28:89-90

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