Mycologia
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Mycologia, 95(5), 2003, pp. 807-808.
© 2003 by The Mycological Society of America

Indole and 3-chloroindole: The source of the disagreeable odor of Hygrophorus paupertinus


William F. Wood 1
Joshua Smith
Kjirsten Wayman

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

David L. Largent

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

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 DISCUSSION
 LITERATURE CITED
 

The odor emanating from sporocarps of Hygrophorus paupertinus is disagreeable and fecal-like. Solid phase microextraction (SPME) and analysis by gas chromatography-mass spectrometry (GC-MS) showed 1-octen-3-ol, indole and 3-chloroindole were responsible for the odor. This is the first case in which 3-chloroindole has been identified from a terrestrial organism.

Key words: 3-chloroindole, Hygrophorus paupertinus, indole, 1-octen-3-ol, odor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 DISCUSSION
 LITERATURE CITED
 
Hygrophorus paupertinus Sm. & Hesl. (Agaricales, Hygrophoraceae) is described as having a "exceedingly strong, penetrating, and disagreeable" odor (Hesler and Smith 1963Citation, Largent 1985Citation). To us the odor was distinctly fecal-like. Because this odor is not one we commonly have encountered in mushrooms, its source was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
DISCUSSION
 LITERATURE CITED
 
Fresh specimens of H. paupertinus were collected at Big Lagoon, Humboldt County, California, and tightly wrapped in a waxed-paper bag. A voucher specimen, DLL 5665, was deposited in the Humboldt State University Fungal Herbarium. Within a few hours of collection, volatiles escaping from the sporocarp were collected with a Supelco solid-phase microextraction (SPME) apparatus, equipped with a 100 µm polydimethylsiloxane fiber assembly. The absorbent tip of the collection apparatus was placed between the gills of the sporocarp for 15 min to trap odoriferous compounds emanating from the mushroom; the compounds then were analyzed immediately by gas chromatography-mass spectrometry (GC-MS).

GC-MS analysis using the SPME apparatus was performed in the splitless mode (0.5 min), using 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 that 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 for 2 min. Mass spectral fragments below m/z = 39 were not recorded. The relative amount of each component was determined as the percent of the total ion current (TIC). Indole and 1-octen-3-ol were purchased from Aldrich Chemical.

Identification 3-chloroindole was done by comparison of the GC retention times of the six isomers of this compound. For these analyses, the sporocarps (1.5 g) were extracted with 2 mL of dichloromethane. GC-MS analyses of this extract and the six authentic samples of isomeric chloroindole were modified so that the oven temperature was kept at 100 C for 1 min and increased to a final temperature of 150 C at a rate of 2 C/min. The 4-, 5-, 6- and 7-monochloro isomers of indole were purchased from Acros Organics. 3-Chloroindole was prepared from indole and sulfuryl chloride (Brennan et al 1986Citation, Mazzara and Borgo 1905aCitation, bCitation), and 2-chloroindole was prepared from oxindole and phosphorous oxychloride (Powers 1966Citation).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 DISCUSSION
LITERATURE CITED
 
GC-MS analysis of the SPME experiment showed three volatile compounds are responsible for the odor of H. paupertinus: 1-octen-3-ol (1, 6%), indole (2, 11%) and 3-chloroindole (3, 83%) (Fig. 1). 1-Octen-3-ol and indole initially were identified by comparison of mass spectra in the NIST 1998 computerized mass spectral library and confirmed by GC-MS comparison with samples of these compounds using the SPME apparatus. 3-chloroindole was identified using a dichloromethane extract of the sporocarp. The six isomers of chloroindole (2-, 3-, 4-, 5-, 6- and 7-chloroindole) have identical mass spectra, so identification of 3-chloroindole in H. paupertinus was done by comparison of gas chromatographic retention times of these compounds. The mass spectra of these compounds are: 1-octen-3-ol [m/z = 85 (8), 81 (5), 72 (17), 68 (5), 58 (7), 57 (100), 55 (10), 43 (19), 41 (12), and 39 (6)], indole [m/z = 118 (9), 117 (M+, 100), 116 (8), 91 (3), 90 (35), 89 (26), 63 (8), 62 (4), 58 (9) and 39 (3)], and 3-chloroindole [m/z = 153 (M+ + 2, 32), 152 (11), 151 (M+, 100), 124 (14), 123 (8), 116 (23), 89 (56), 75 (16), 63 (19) and 62 (14)].



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  		FIG. 1. Odoriferous compounds (1–3) from the sporocarp of H. paupertinus and skatol (4)

 
This is the first identification of 3-chloroindole from a terrestrial organism. It previously has been identified from the marine acorn worm, Ptychodera flava (Higa et al 1980Citation, Higa and Scheuer 1975Citation). 3-Chloroindole is highly odoriferous, has the same molecular shape and size as 3-methylindole (4, skatol) and to us has a very similar odor. Indole and 3-methylindole are found in human feces and are reported to have intense fecal odors at high concentrations that become pleasant in very dilute solutions (Poucher 1974Citation). To us, the overpowering scents of indole and 3-chloroindole masked the mushroom odor of 1-octen-3-ol.

Indole and 3-methylindole previously have been identified in other mushroom species. Indole was reported as a major component of Tricholoma bufonium, T. inamoenum, T. lascivum, T. sulphureum and Lepiota bucknallii. (Hilber 1968Citation) and a minor constituent of T. inamoenum (Watson et al 1986Citation), Morchella conica (Audouin et al 1989Citation), Coprinus picaceus (Laatsch and Matthies 1992Citation), T. sulfureum (Rapior et al 1996Citation), Boletus calopus (Rapior et al 1997Citation) and Gyrophragmium dunalii (Rapior et al 2000Citation). Laatsch and Matthies (1992)Citation report 3-methylindole as the major constituent of C. picaceus, and Hilber (1968)Citation reported it to be present in T. inamoenum, T. sulphureum and T. lascivuma.


   FOOTNOTES
 
1 Corresponding author, Email: wfw2{at}humboldt.edu Back

Accepted for publication February 28, 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 DISCUSSION
 LITERATURE CITED
 
Audouin P, Vidal JP, Richard H., 1989 Volatile compounds in the aroma of some wild edible mushrooms: morel (Morchella conica), wood blewitt (Lepista nuda), clouded agaric (Clitocybe nebularis), and false chanterelle (Hygrophoropsis aurantiaca). Sciences des Aliments 9:185-193

Brennan MR, Erickson KL, Szmalc FS, Tanse MJ, Thornton JM., 1986 The preparation and spectral characterization of 2-haloindoles, 3-haloindoles, and 2,3-dihaloindoles. Heterocycles 24:2879-2885

Hesler LR, Smith AH., 1963 North American Species of Hygrophorus. Knoxville: U. of Tenn. Press, 416 p

Higa T, Fujiyama T, Scheuer PJ., 1980 Halogenated phenol and indole constituents of acorn worms. Comp Biochem Physiol 65B:525-530

Higa T, Scheuer PJ., 1975 3-Chloroindole, principal odorous constituent of the hemichordate Ptychodera flava laysanica. Naturwiss 62:395-396

Hilber O., 1968 Indole as the main component of odor of some Tricholoma species and Lepiota buckmallii. Zeitschrift fuer Pilzkunde 34:153-158

Largent DL., 1985 The Agaricales (Gilled Fungi) of California 5: Hygrophoraceae. Eureka, California. Mad River Press Inc. 208 p

Laatsch H, Matthies L., 1992 The characteristic odor of Coprinus picaceus: a rapid enrichment procedure for apolar, volatile indoles. Mycologia 84:264-266

Mazzara G, Borgo A., 1905a The action of sulfuryl chloride on indole. Gazz Chim Ital 35:320-326

Mazzara G, Borgo A., 1905b The action of sulfuryl chloride on monochloro- and dichloroindole. Gazz Chim Ital 35:563-569

Poucher WA., 1974 Perfumes, cosmetics and soaps. 7th ed. London: Chapman and Hall. 381 p

Powers JC., 1966 Chloroindoles. J Org Chem 31:2627-2631[Medline]

Rapior S, Cavalie S, Croze P, Andary C, Pelissier Y, Bessiere J-M., 1996 Volatile components of ten frozen mushrooms (Basidiomycetes). J Essential Oil Res 8:63-66

Rapior S, Marion C, Pelissier Y, Bessiere J-M., 1997 Volatile composition of fourteen species of fresh wild mushrooms (Boletales). J Essential Oil Res 9:231-234

Rapior S, Mauruc M-J, Guinberteau J, Masson C-L, Bessiere J-M., 2000 Volatile composition of Gyrophragmium dunalii. Mycologia 92:1043-1046

Watson RL, Largent DL, Wood WF., 1986 The "Coal Tar" Odor of Tricholoma inamoenum. Mycologia 78:965-966





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