Occurrence of a High Concentration of Spider Pheromones in the Ascomycete Fungus Hypoxylon truncatum

Article abstract of DOI:10.1021/np030185y

A large amount of sex pheromones of the European spider Linyphia triangularis, 3R-hydroxybutyric acid (1), its dimer 3R-(3R-hydroxybutyryloxy)butyric acid (2), and trimer 3R-[3R-(3R-hydroxybutyryloxy)-butyryloxy]butyric acid (3) were isolated from the EtOAc extract of the Japanese inedible mushroom Hypoxylon truncatum.

Full text of DOI:10.1021/np030185y

J . Nat. Prod. 2003, 66, 1613-1614
1613
Occu r r en ce of a High Con cen tr a tion of Sp id er P h er om on es in th e Ascom ycete
F u n gu s Hypoxylon tr u n ca tu m
Dang Ngoc Quang, Toshihiro Hashimoto, Masao Toyota, and Yoshinori Asakawa*
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, J apan
Received April 23, 2003
A large amount of sex pheromones of the European spider Linyphia triangularis, 3R-hydroxybutyric acid
(1), its dimer 3R-(3R-hydroxybutyryloxy)butyric acid (2), and trimer 3R-[3R-(3R-hydroxybutyryloxy)-
butyryloxy]butyric acid (3) were isolated from the EtOAc extract of the J apanese inedible mushroom
Hypoxylon truncatum.
Anderson1 isolated 3,4-dihydro-8-hydroxy-3-methyliso-
coumarin from the inedible mushroom Hypoxylon trunca-
tum. Later Uebler2 and Kern3 also reported the same
compound as a trail pheromone component of the ants
Camponotus silvicola, C. rufipes, and Lasius fuliginosus,
respectively. These results opened the avenue for research
on compounds occurring in both insects and mushrooms.
Recently, we investigated the chemical constituents of the
J apanese H. truncatum and isolated a novel perylene-
quinone, truncatone.4 Further fractionation of the ethyl
acetate extract has now resulted in the isolation of mon-
omeric 3R-hydroxybutyric acid (1), its dimer 3R-(3R-
hydroxybutyryloxy)butyric acid (2), and trimer 3R-[3R-(3R-
hydroxybutyryloxy)butyryloxy]butyric acid (3), which are
known to be sex pheromones of the spider Linyphia
F igu r e 1. Structures of compounds 1-7.
triangularis (Clerck) and related species.5
The EtOAc extract of H. truncatum was subjected to SiO2
the mushroom H. truncatum are monomeric 3R-hydroxy-
and Sephadex LH-20 column chromatography to give
butyric acid, dimeric 3R-(3R-hydroxybutyryloxy)butyric
3-hydroxybutyric acid (1) and a mixture of compounds 2
acid, and trimeric 3R-[3R-(3R-hydroxybutyryloxy)butyry-
and 3. On the basis of the IR spectral evidence to support
loxy]butyric acid and are identical to the sex pheromones
the presence of a carboxylic acid group (2400-3600 cm-1),
of the spider Linyphia triangularis and related species.5
samples of compound 1 and the mixture of 2 and 3 were
Schulz and Toft5 confirmed the presence of a very low
methylated, then purified using preparative HPLC to give
concentration (µg order per web) of these three sex phero-
compounds 4, 5, and 6 as methylated derivatives of 1, 2,
mones of the spider only by GC-MS and determined the
1
and 3, respectively. On the basis of analyses of IR, H and
absolute structures of the monomer (1) and dimer (2), but
13C NMR, and MS spectra and comparisons of the spectral
not the trimer (3) due to its very low concentration.
data with those of authentic samples and reported spectral
This is the first report of the isolation of a high
data,6,7 their stuctures were determined to be 3-hydroxy-
concentration of spider pheromones (1-3) from the fungus
butyric acid methyl ester (4), the dimer 3-(3-hydroxybu-
and very interesting from the point of view of pheromone
tyryloxy)butyric acid methyl ester (5), and the trimer 3-[3-
biogenesis in each organism.
(3-hydroxybutyryloxy)butyryloxy]butyric acid methyl ester
(6).
To determine the absolute configurations of compounds
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. IR spectra were
measured on a J ASCO FT/IR-5300 spectrophotometer. The H
1, 2, and 3, they were each converted to 3-acetoxybutyric
acid methyl ester (7), which was analyzed by GC-MS on
a chiral column. Compound 1 was methylated with trim-
ethylsilyldiazomethane and acetylated to afford compound
7. A mixture of dimer 5 and trimer 6 was saponified with
potassium hydroxide in methanol, followed by methylation
and acetylation to yield 7. Compound 7 was analyzed by
gas chromatography-mass spectroscopy (GC-MS) on a
chiral column with authentic samples (each 3R- and 3S-
acetoxybutyric acid methyl ester, which was derived from
3R- and 3S-hydroxybutyric acid, respectively) to give the
chromatograms shown in Figure 2. Consequently, the
configurations of all asymmetric carbons of 1, 2, and 3 were
established to be R. Thus, three compounds isolated from
1
and 13C NMR spectra were recorded on a Varian Unity 600
NMR spectrometer (600 MHz for 1H and 150 MHz for 13C),
using CD3OD or CD3Cl as solvent. Chemical shifts are given
relative to TMS δ 0.00 as internal standard (1H) and δ 49.0
ppm from CD3OD or 77.03 ppm as standards (13C). Mass
spectra including high-resolution mass spectra were recorded
on a J EOL J MS AX-500 spectrometer. GC-MS was carried
out on a Hewlett-Packard mass selective detector 5971 A and
a gas chromatograph 5890 Series II with chiral column â-DEX
120 (30 m × 0.25 mm, film thickness 0.25 µm). The temper-
ature programming of GC-MS analysis was performed from
a 50 °C isothermal for 3 min, then 50-230 °C at 3 °C min-1
and finally an isothermal at 230 °C for 20 min. Injection
temperature was 250 °C.
,
F u n ga l Ma ter ia l. Hypoxylon truncatum was collected in
Tokushima, J apan, in 1992 and identified by Dr. T. Hashimoto
* To whom correspondence should be addressed. Tel: +88-622-9611.
Fax: +88-655-3051. E-mail: asakawa@ph.bunri-u.ac.jp.
10.1021/np030185y CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/20/2003

1614 J ournal of Natural Products, 2003, Vol. 66, No. 12
Notes
F igu r e 2. Determination of absolute configuration of compounds 1, 2, and 3 by GC-MS on a chiral column with authentic samples.
(Tokushima Bunri University, J apan). The voucher specimen
(1992-1) was deposited at the Faculty of Pharmaceutical
Sciences, Tokushima Bunri University, J apan.
(23% in GC) (24.4 mg), and compound 6 (9.6 mg). The mixture
of compounds 5 and 6 (24.4 mg) was saponified by KOH (20
mg) in MeOH (1.5 mL), neutralized by 1 N HCl, and then
subjected repeatedly to column packing proton exchange resin
(Amberlite IR-120B/H+). The eluent was evaporated and
methylated in the same manner as mentioned above and then
acetylated with anhydride acetic (1 mL) in pyridine (1 mL) to
give 3R-acetoxybutyric acid methyl ester (7) (17.2 mg).
Extr a ction a n d Isola tion . Dried H. truncatum (173.2 g)
was extracted with EtOAc and then evaporated to give a
residue (16.6 g), which was chromatographed on SiO2 using a
CHCl3/EtOAc gradient affording three fractions [fractions 1
(667 mg), 2 (1490 mg), and 3 (880.2 mg)]. Fraction 3 contained
pure 3R-hydroxybutyric acid (1). A part of fraction 3 (1) (77.2
mg) was methylated followed by acetylation to give 3R-
acetoxybutyric acid methyl ester (7) (29.4 mg). Fraction 2 was
divided by Sephadex LH-20 column chromatography (CHCl3/
MeOH ) 1:1) into three fractions: fraction 2-1 (18.8 mg),
fraction 2-2 (947 mg), which contained 2 and 3 as methyl esters
in the ratio 2:1 by GC, and fraction 2-3 (48.1 mg). A part of
fraction 2-2 (68.8 mg) was methylated with (CH3)3SiCHN2 (1.5
mL) in MeOH (2 mL), then purified by preparative HPLC on
a Waters 5 SL-II column using hexane/EtOAc (1:3) as solvent
system and Sephadex LH-20 column chromatography to give
compound 5 (15.3 mg), a mixture of compounds 5 (76%) and 6
Refer en ces a n d Notes
(1) Anderson, J . R.; Edwards, R. L. J . Chem. Soc., Perkin Trans. 1 1983,
2185-2192.
(2) Uebler, E.; Kern, F.; Bestmann, H. J .; Boelldobler, B.; Attygalle, A.
B. Naturwissenschaften 1995, 82, 523-525.
(3) Kern, F.; Klein, R. W.; J assen, E.; Bestmann. H. J .; Attygalle, A. B.;
Schaefer, D.; Maschwits, U. J . Chem. Ecol. 1997, 23, 779-792.
(4) Hashimoto, T.; Asakawa, Y. Heterocycles 1998, 47, 1067-1110.
(5) Schulz, S.; Toft, S. Science 1993, 260, 1635-1637.
(6) Li, J .; Uzawa, J .; Doi, Y. Bull. Chem. Soc. J pn. 1997, 70, 1887-1893.
(7) Li, J .; Uzawa, J .; Doi, Y. Bull. Chem. Soc. J pn. 1998, 71, 1683-1689.
NP030185Y