Benzoxepine esters as precursors of the wound-activated chemical defence of Mycena galopus

Article abstract of DOI:10.1002/ejoc.200700812

The native precursors E/Z-1a to E/Z-1e and 2a-2e of the antifungally active benzoxepines E/Z-1 and 2 have been isolated from intact fruiting bodies of Mycena galopus. These precursors turned out to be fatty acid esters of the benzoxepines (pterulones) E/Z-1 and 2. Metabolic profiling of ethyl acetate extracts from intact fruiting bodies, from the latex and from mechanically wounded fruiting bodies revealed that the known antifungally active benzoxepines E/Z-1 and the previously unknown benzoxepines 2 are only present in injured fruiting bodies. Intact fruiting bodies and the latex, however, exclusively contain the corresponding ester derivatives E/Z-1a to E/Z-1e and 2a-2e. Unlike the antifungally active benzoxepines E/Z-1 and 2, the esters E/Z-1a to E/Z-1e present in intact fruiting bodies turned out to be inactive. Upon injury the inactive esters E/Z-1a to E/Z-1e are cleaved by a putative esterase, and the bioactive benzoxepines E/Z-1 and 2 are released, suggesting a wound-activated defence mechanism of M. galopus against yeasts and parasitic fungi. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700812

FULL PAPER
DOI: 10.1002/ejoc.200700812
Benzoxepine Esters as Precursors of the Wound-Activated Chemical Defence of
Mycena galopus
Silke Peters,[a] Robert J. R. Jaeger,[a] and Peter Spiteller*[a]
Keywords: Benzoxepines / Chemical defence / Fungi / Metabolic profiling / Natural products
The native precursors E/Z-1a to E/Z-1e and 2a–2e of the anti-
fungally active benzoxepines E/Z-1 and 2 have been isolated
from intact fruiting bodies of Mycena galopus. These precur-
sors turned out to be fatty acid esters of the benzoxepines
(pterulones) E/Z-1 and 2. Metabolic profiling of ethyl acetate
extracts from intact fruiting bodies, from the latex and from
mechanically wounded fruiting bodies revealed that the
exclusively contain the corresponding ester derivatives E/Z-
1a to E/Z-1e and 2a–2e. Unlike the antifungally active benzo-
xepines E/Z-1 and 2, the esters E/Z-1a to E/Z-1e present in
intact fruiting bodies turned out to be inactive. Upon injury
the inactive esters E/Z-1a to E/Z-1e are cleaved by a putative
esterase, and the bioactive benzoxepines E/Z-1 and 2 are re-
leased, suggesting a wound-activated defence mechanism of
known antifungally active benzoxepines E/Z-1 and the pre- M. galopus against yeasts and parasitic fungi.
viously unknown benzoxepines 2 are only present in injured
fruiting bodies. Intact fruiting bodies and the latex, however,
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
esters of E/Z-1 and of the previously unknown non-chlori-
nated benzoxepine 2, respectively (Figure 1). Moreover, we
propose that the fatty acid esters of E/Z-1 serve as stable
precursors of the bioactive benzoxepine alcohols E/Z-1 and
2 in the wound-activated chemical defence of M. galopus.
Introduction
The basidiomycete Mycena galopus (Pers.: Fr.) P. Kumm.
(German name: Weißmilchender Helmling) is a small
mushroom widespread in Europe and Northern America
on mossy floors of coniferous forests.[1] The fruiting bodies
of M. galopus can easily be identified, as they contain a
characteristic white latex that is exuded if they are cut or
bruised. Recently, we isolated red pyrroloquinoline alka-
loids from the red latex of the related species Mycena san-
guinolenta and Mycena haematopus and from the red fruit-
ing bodies of Mycena rosea.[2] The broad spectrum of un-
usual natural products detected in Mycena species inspired
us to extend these investigations to M. galopus. This My-
cena species had already been screened for the presence of
bioactive compounds in 1999 by Wijnberg et al., who iden-
tified the antifungal benzoxepines (pterulones) E/Z-1 and
3.[3] 6-Hydroxypterulone (3) is structurally closely related to
pterulone, which was isolated by Anke et al. from sub-
merged cultures of the Pterula species 82168 in 1997.[4] The
antifungal activity of pterulone is based on selective inhibi-
tion of the NADH:ubiquinone oxidoreductase (complex I)
of the respiratory chain.[4b]
Figure 1. Benzoxepines from Mycena galopus.
Results and Discussion
When we reinvestigated intact fruiting bodies of M. ga-
lopus for the presence of the benzoxepines E/Z-1 and 3, we
found E/Z-1 either only in traces or not at all. A compari-
son of the metabolic profiles of intact fruiting bodies with
those of mechanically injured ones recorded by GC-MS
indicated that E/Z-1 was only present in injured fruiting
bodies. However, in the GC-MS of intact fruiting bodies a
characteristic pair of the fragment ions m/z = 205/207
(which is also typical of the trimethylsilyl ethers of the
benzoxepines E/Z-1) was recognised at later retention times
than determined for E/Z-1, pointing to the presence of de-
rivatives of E/Z-1 in the latex instead.
In this paper we describe the isolation and the structural
elucidation of E/Z-1a to E/Z-1e and 2a–2e, native constitu-
ents of the white latex, which turned out to be fatty acid
[a] Institut für Organische Chemie und Biochemie II der Tech-
nischen Universität München,
Lichtenbergstraße 4, 85747 Garching, Germany
Fax: +49-89-289-13210
E-mail: peter.spiteller@ch.tum.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
Eur. J. Org. Chem. 2008, 1187–1194
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1187

S. Peters, R. J. R. Jaeger, P. Spiteller
FULL PAPER
In order to isolate the new compounds from the fruiting moiety (Figure 2). If the molecular mass ions at m/z = 460
bodies of M. galopus, the fungal material was frozen with [M(35Cl)]+ and 462 [M(37Cl)]+ are taken into account, E-
liquid nitrogen immediately after collection and stored at 1a consists of palmitic acid esterified with the benzoxepine
–35 °C to avoid decomposition. The native latex constitu- alcohol E-1. To confirm this deduction, the benzoxepine
ents E/Z-1a to E/Z-1e and 2a–2e were extracted with ethyl ester was saponified with a methanolic solution (0.02 ) of
acetate from frozen fruiting bodies of M. galopus and sepa- potassium hydroxide. After trimethylsilylation with
rated first by preparative HPLC on RP-18 and after that MSTFA, the mixture was analysed by GC-MS. The pal-
on silica gel. In this way the esters E/Z-1a to E/Z-1e and mitic acid and E-1 present in the hydrolysate of E-1a were
2a–2e were separated from each other. A sample of frozen identified unambiguously by GC-MS comparison of the tri-
fruiting bodies (100 g) yielded E-1a (5.7 mg), Z-1a (1.8 mg), methylsilylated hydrolysate with authentic samples of tri-
E-1b (1.0 mg), Z-1b (0.5 mg), E-1d (2.5 mg), E-1e (2.0 mg), methylsilyl palmitate and the trimethylsilylated benzoxepine
Z-1e (0.7 mg), 2a (1.7 mg) and 2e (0.7 mg), while the trace alcohol E-1.
compounds E-1c, Z-1c, Z-1d, 2b, 2c, 2d and 3 were only
identified by GC-MS.
The mass spectrum of the benzoxepine E-1a exhibited
the same pair of base ions at m/z (35Cl) = 205 and m/z (37Cl)
= 207 as the trimethylsilyl derivative of E-1. In addition, E-
1a showed fragment ions at m/z = 170, 141, 131, 128 and
115 with intensities similar to those of the trimethylsilyl de-
rivative of E-1, indicating that the two compounds are likely
to contain the same benzoxepine moiety. A comparison of
the 1H and 13C NMR spectroscopic data for E-1 with those
for E-1a confirms this conclusion, since for all resonances
present in E-1 a corresponding one is present in E-1a
(Table 1).
Figure 2. Selected HMBC (Ǟ) and NOE (↔) correlations of E-1a
and 2a.
Moreover, the COSY, HSQC, HMBC and NOESY cor-
relations allowed the complete assignment of the protons
The benzoxepine ester Z-1a exhibited essentially the
and carbon atoms of the benzoxepine fragment (Table 1 same MS characteristics (EI) as E-1a. However, its reten-
and Figure 2). However, E-1a exhibited additional signals tion time on the GC column was slightly shorter than that
at δH = 0.88, 1.24–1.25, 1.28, 1.29, 1.63, 2.34 ppm, typical of E-1a. Hydrolysis of the ester Z-1a yielded Z-1 and pal-
of the presence of a saturated straight-chain fatty acid moi- mitic acid according to the GC-MS analysis.
3
ety. In the HMBC spectrum a J correlation of the signal
The non-chlorinated ester 2a differs from E/Z-1a in its
of the benzylic protons at δH = 5.05 ppm to the carbon GC-MS, exhibiting a molecular ion peak at m/z = 426
signal at δC = 173.7 ppm indicates that the fatty acid moiety [M]+ and a base ion peak at m/z = 171, indicating the ab-
is esterified with the benzylic alcohol of the benzoxepine sence of chlorine atoms. In addition, its retention time in
Table 1. NMR spectroscopic data (600 MHz, CDCl3, 298 K) for E-1a, E-1 and 2a.
#
E-1a
δC
E-1
δC
2a
δC
δH (mult., J in Hz)
δH (mult. J in Hz)
2
3
4
5
5a
6
7
8
9
72.6
4.55 (s)
72.6
74.6
4.58 (s)
136.3
124.2
130.9
130.8
133.4
127.0
129.4
120.3
159.2
119.4
136.3
124.2
130.9
130.8
133.4
127.0
129.5
120.3
159.2
119.4
143.6
131.0
128.0
130.7
132.8
128.8
128.4
120.3
159.4
117.7
6.82 (d, 11.8)
6.53 (d, 11.8)
6.47 (d, 11.5)
6.34 (d, 11.5)
7.26 (d, 1.9)
7.21 (s)
7.18 (dd, 8.2, 1.9)
6.98 (d, 8.2)
7.15 (d, 8.7)
6.98 (d, 8.7)
9a
10
6.12 (s)
5.05 (s)
5.07 (s) Hb
5.24 (s) Ha
5.04 (s)
11
65.4
65.4
65.6
1Ј
2Ј
3Ј
4Ј
173.7
34.3
24.9
173.8
34.3
25.0
2.34 (t, 7.5)
1.63 (tt, 7.5, 7.5)
1.29 (m)
2.33 (t, 7.0)
1.63 (tt, 7.0, 7.0)
29.1
29.1
5Ј–13Ј
14Ј
15Ј
16Ј
29.3–29.7
31.9
23.0
1.24–1.25 (m)
1.25 (m)
1.28 (m)
29.3 to 29.7
31.9
22.7
1.24–1.25 (m)
1.25 (m)
1.28 (m)
14.1
0.88 (t, 7.3)
14.1
0.88 (t, 7.6)
1188
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1187–1194

Benzoxepine Esters as Chemical Defence Precursors in Mycena galopus
the GC-MS was considerably shorter than that of E-1a bodies into methanol after certain periods of time to stop
(Figure 4). In the 1H NMR spectrum the signals of two the enzymatic hydrolysis. The samples were analysed by
protons of the exocyclic methylene group at δH = 5.07 and GC-MS after trimethylsilylation. After 60 min, approxi-
5.24 ppm are present in 2a instead of only one in E-1a. The mately the same amounts of free alcohols as of the corre-
protons of the exocyclic methylene group exhibit character- sponding esters were present in the extract. After 180 min,
istic NOEs to 2-H and 4-H (Figure 2 and Table 1), thus two thirds of the esters had been converted into the benzo-
allowing the unambiguous assignment of these protons in xepine alcohols E/Z-1 and 2, and after 24 h, the esters had
2a. In addition, hydrolysis of 2a yielded the free benzoxep- been cleaved quantitatively into the corresponding alcohols
ine alcohol 2 and palmitic acid as verified by GC-MS analy- (Figure 4).
sis.
The structures of E/Z-1b to E/Z-1e and of 2b–2e were
determined analogously to those of E/Z-1a and 2a, respec-
tively. In general, the GC-MS retention times of the (Z)
isomers turned out to be slightly shorter than those of the
corresponding (E) isomers.
In order to elucidate the ecological roles of the free
alcohols E/Z-1 and 2 and of the benzoxepine esters E/Z-1a
to E/Z-1e and 2a–2e, the bioactivities of the compounds
were evaluated. None of the compounds exhibited any sig-
nificant bioactivity towards bacteria such as Escherichia
coli, Bacillus brevis or Bacillus subtilis. In contrast, E-1 and
a 1:1 mixture of E/Z-1 exhibited moderate activities against
the fungus Cladosporium cucumerinum and, as already dem-
onstrated by Wijnberg,[3b] significant activities against
Figure 4. GC of trimethylsilylated methanolic extracts from intact
fruiting bodies (top), from the latex (centre) and from homogenised
fruiting bodies (bottom) of M. galopus.
yeasts such as Saccharomyces cerevisiae (Figure 3). The
non-chlorinated alcohol 2 turned out to be less active
towards S. cerevisiae than E-1 or E/Z-1 (Figure 3). The
presence of the benzoxepine moiety seems to be important
The white latex dripping from the stipes of fresh fruiting
for the bioactivity, since benzyl alcohol itself was com- bodies only consisted – according to the GC-MS analysis –
pletely inactive. Similarly, when we tested the benzoxepine of the benzoxepine esters together with some free fatty ac-
esters E/Z-1a to E/Z-1e and 2a–2e against C. cucumerinum ids. Interestingly, even after 10 h, the latex had not decom-
and S. cerevisiae, no growth inhibition occurred, demon- posed at all to give the free alcohols E/Z-1 and 2 (Figure 4).
strating that the benzoxepine esters were inactive (Figure 3). Hence, the latex itself does not contain esterases.
We therefore propose that upon wounding of the fruiting
bodies, esterases come into contact with the latex and cleave
the benzoxepine esters to release the corresponding bioac-
tive alcohols E/Z-1 and 2. It is likely that the effects of
mechanical wounding of M. galopus are comparable to
those of injury caused to the fruiting bodies by insect feed-
ing and infection with parasites. This assumption is sup-
ported by recent experiments with lima beans (Phaseolus
lunatus). Boland et al. demonstrated for this example that
continuous mechanical wounding induces defence reactions
similar to those towards insect feeding, indicating that
wounding is a crucial element of defence activation.[5]
In agreement with the proposed wound-activated chemi-
cal defence mechanism, the fruiting bodies of M. galopus
are rarely attacked by fungivores and parasitic fungi. How-
ever, M. galopus is prone to infection with the mycoparas-
itic zygomycete Spinellus fusiger, which is able to infect
many Mycena species, especially in wet and cold weather.
Figure 3. Bioactivities of the esters E-1a and E-1e, the alcohols E/
Z-1 (1:1 mixture), E-1 and 2 and of the antibiotic nourseothricin
(NTC) towards S. cerevisiae in YMG medium at a concentration
of 1.0 m.
If intact fruiting bodies of M. galopus were submerged S. fusiger indeed turned out to be insensitive to E/Z-1, thus
in methanol only a few seconds after collection, the free enabling the mycoparasite to grow on fruiting bodies of
benzoxepine alcohols E/Z-1 and 2 were not detectable at all M. galopus.
by GC-MS analysis of this methanolic extract (Figure 4).
The putative esterase of M. galopus exhibits a broad sub-
In contrast, after artificial injury of intact fruiting bodies strate specificity. Incubation experiments with homogenised
by bruising, E/Z-1 and 2 were generated. The course of the fungal material of M. galopus with selected esters showed
reaction was monitored by transferring injured fruiting that the enzymes present in M. galopus are able to cleave
Eur. J. Org. Chem. 2008, 1187–1194
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1189

S. Peters, R. J. R. Jaeger, P. Spiteller
FULL PAPER
methyl oleate and the acetylated and caprylated derivatives mentins are converted into atromentin and the feeding de-
of the benzoxepine alcohols E/Z-1 quantitatively. Even ben- terrent osmundalactone after wounding of the fruiting bod-
zyl stearate is hydrolysed to some extent to benzyl alcohol ies.[16]
and stearic acid. In addition, several other Mycena species
Similarly to M. galopus, Lactarius species also contain a
contain enzymes exhibiting an esterase activity. When we latex, which has been investigated in detail in a number of
incubated the unnatural substrate benzyl stearate with the species. For instance, upon injury of Lactarius vellereus,
homogenate of M. rosea and M. sanguinolenta, at least one fatty acid esters of the sesquiterpene velutinal that are pres-
quarter of this ester was hydrolysed after 16 h. In contrast, ent in the latex are saponified.[17] The unstable free velutinal
the yeast S. cerevisiae was inactive and did not hydrolyse is then transformed into pungent-tasting aldehydes such as
benzyl stearate at all.
velleral and isovelleral or the corresponding alcohols vel-
Plants also contain lipases, which are activated upon lerol and isovellerol. In Lactarius deliciosus and Lactarius
wounding.[6] In plants free fatty acids are usually generated deterrimus, intact fruiting bodies exclusively contain a dihy-
from lipids. The free fatty acids are subsequently degraded droazulene alcohol that is esterified with stearic acid or
by lipoxygenases into compounds such as methyl jasmon- linoleic acid. Upon injury the ester is hydrolysed, yielding
ate[6] and stimulate the production of volatiles that serve as a number of different pungent-tasting sesquiterpenes, such
signalling compounds in their chemical defence.[7] However, as lactaroviolin and delicial.[18]
plants usually do not contain fatty acid esters of secondary
metabolites as precursors for their chemical defence. In con-
Conclusion
trast, glycosides are often present in plants as inactive pre-
cursors. For instance, a number of plants hydrolyse cyano-
genic glycosides upon injury and release hydrocyanic acid.[8]
A large number of secondary metabolites is already
known from plants and higher fungi. However, the ecologi-
cal roles of many compounds from fungi are still obscure.
Well-known toxins from toadstools are the amanitins,[9]
ibotenic acid[10] or orellanine,[11] which protect the fruiting
bodies from feeding animals ranging from insects to mam-
mals. Antifungal compounds such as the strobilurins, which
have served as lead structures for the development of com-
mercially available fungicides,[12] have been found in the my-
celia of a variety of fungi including several Mycena species.
Hydroxystrobilurin D (= hydroxystrobilurin G) from
M. sanguinolenta is a benzodioxepine-type strobilurin[13]
structurally related to the benzoxepines from M. galopus.
In contrast to the examples mentioned above, which rep-
resent secondary metabolites that are present permanently
in their bioactive forms in fruiting bodies as a constitutive
chemical defence, M. galopus seems to use a wound-acti-
vated defence mechanism based on the enzymatic conver-
sion of an inactive precursor into the active agent that oc-
curs only temporarily upon activation by injury. In com-
parison with direct chemical defence mechanisms relying on
permanently present bioactive compounds, wound-acti-
vated chemical defence mechanisms have the advantage that
the intact organism is not affected negatively by its own
defence compounds.
The detection of a wound-activated chemical defence
mechanism in M. galopus shows that chemical defence
based on the cleavage of inactive fatty acid esters to active
compounds is not restricted to some species of the genus
Lactarius, but also occurs in a Mycena species. Moreover,
the unspecific esterase activity upon injury of all tested My-
cena species indicates that wound-activated chemical de-
fence mechanisms might be more widespread than assumed
previously. Secondary metabolites have often been isolated
from mushrooms under conditions such as drying or hom-
ogenisation in aqueous solution, which might alter the me-
tabolite pattern in relation to intact fruiting bodies. Thus,
the presence of wound-activated defence mechanisms and
of the corresponding inactive precursors might have been
overlooked in some other cases. Hence, consequent meta-
bolic profiling both of intact and artificially wounded spe-
cies by GC-MS or LC-MS may lead to the detection of
more wound-activated defence mechanisms in the future.
Experimental Section
General Experimental Procedures: Evaporation of the solvents was
performed under reduced pressure with a rotary evaporator. Pre-
parative HPLC separations were performed with two Waters 510
pumps equipped with an automated gradient controller 680 and
an Applied Biosystems 783A UV/Vis detector. The samples were
separated first on a Nucleodur C-18 EC, 5 µm, 10ϫ250 mm col-
umn (Macherey–Nagel) with use of the following gradient pro-
Wound-activated defence mechanisms have been ob-
served in mushrooms only in a limited number of cases: gram: from 49.97% H2O/49.97% MeOH/0.06% AcOH over 20 min
linear to 100% MeOH, then 15 min at 100% MeOH; flow rate:
6 mLmin–1; detection: UV at 230 nm. A second normal-phase sep-
aration (HPLCNP) was performed with the same HPLC instrumen-
tation and with a Nucleosil Si, 5 µm, 10ϫ250 mm column (Mach-
erey–Nagel) and use of the following isocratic separation condi-
tions: 98% n-hexane, 2% tert-butyl methyl ether; flow rate:
6 mL min–1; detection: UV at 280 nm. UV spectra were recorded
with a Varian Cary 100 Bio UV/Vis spectrometer. NMR spectra
were recorded with a Bruker DMX 250 spectrometer (1H at 250.06,
13C at 62.9 MHz), with a Bruker DMX 500 spectrometer equipped
for instance, the fruiting bodies of Aleurodiscus amorphus
contain the cyanohydrin ether aleurodisconitrile, which,
upon injury of the fruiting bodies, is converted into highly
toxic hydrocyanic acid and aleurodiscoester by an oxidative
mechanism that differs fundamentally from the hydrolysis
of cyanogenic glycosides occurring in a variety of plants.[14]
Stephanosporin is a precursor of the fungicide 2-chloro-4-
nitrophenol, which is generated in large quantities in the
rare gasteromycete Stephanospora caroticolor after injury of
the fruiting bodies.[15] In Paxillus atrotomentosus the leuco- with a TXI probe (1H at 500.11, 13C at 125.8 MHz) and a Bruker
1190 Eur. J. Org. Chem. 2008, 1187–1194
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Benzoxepine Esters as Chemical Defence Precursors in Mycena galopus
DMX 600 spectrometer equipped with a TXI cryo probe (1H at
600.13, 13C at 150.9 MHz). Chemical shifts were determined rela-
tive to the solvent CDCl3 (δH = 7.26, δC = 77.0 ppm) as internal
standard. GC-MS (EI) spectra were recorded with a Thermo Elec-
tron Trace DSQ mass spectrometer coupled with a Thermo Elec-
tron Trace GC Ultra equipped with a PTV injector. For sample
solved in water (5 mL) and extracted with AcOEt (3ϫ5 mL). The
organic phase was dried with Na2SO4, filtered and concentrated in
vacuo at 40 °C. The resulting residue was dissolved in MeOH
(5 mL), prepurified with an RP-18 cartridge and separated on an
RP-18 column by semipreparative HPLC, yielding three benzoxep-
ine-containing fractions. The first fraction at Rt = 8.9 min (1.3 mg)
consisted of 2, the second at Rt = 11.1 min (1.9 mg) consisted of
pure E-1, while the third fraction at Rt = 11.4 min (2.2 mg) con-
sisted of a 1:1 mixture of Z/E-1.
separation,
a
fused silica DB 5 ms capillary column
(15 mϫ0.25 mm, coated with a 0.25 µm layer of liquid phase) and
helium as carrier gas was used. Injection volumes were 0.2–0.5 µL
of a 1–2% (m/v) solution. For pertrimethylsilylation, N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA) was used. Temperature
program: 1 min isothermal at 50 °C, then 5 °Cmin–1 up to 300 °C,
finally 10 min isothermal at 300 °C. Retention indices Ri according
to Kováts were determined by injection of a 0.2 µL sample of a
standard mixture of saturated straight-chain alkanes (C10–C36).
HRMS (APCI) spectra were obtained with a Thermo Scientific
LTQ Orbitrap mass spectrometer. The spectrometer was operated
in the positive mode (1 spectrums–1; mass range: 50–1000) with
nominal mass resolving power of 60000 at m/z = 400 with a scan
rate of 1 Hz with automatic gain control to provide high-accuracy
mass measurements within 2 ppm deviation with polydimethylcy-
closiloxane {[(CH3)2SiO]6, m/z = 445.120025} as internal lock
mass.
Extraction and Isolation of E/Z-1a to E/Z-1e and 2a–2e: Frozen
fruiting bodies (15 g) were crushed after addition of AcOEt
(25 mL) and extracted twice with AcOEt (2ϫ25 mL) at 25 °C for
30 min. The combined extracts were then concentrated in vacuo
at 40 °C. The resulting residue was dissolved in MeOH (5 mL),
prepurified with an RP-18 cartridge and separated on an RP-18
column by preparative HPLC (UV detection at 230 nm) yielding
two benzoxepine-containing fractions. The first fraction at Rt =
26.5 min (1.1 mg) consisted of E/Z-1b, E/Z-1e, 2b and 2e, while the
second one eluting at Rt = 28.2 min (1.7 mg) consisted of E/Z-1a,
E-1d and 2a. After that, both fractions were separated by prepara-
tive HPLC (UV detection at 280 nm) on a normal-phase silica gel
column. From a sample (100 g) of frozen fruiting bodies of M. ga-
lopus, E-1b (1.0 mg), Z-1b (0.5 mg), E-1a (5.7 mg), Z-1a (1.8 mg),
E-1e (2.0 mg), Z-1e (0.7 mg), E-1d (2.5 mg), 2a (1.7 mg) and 2e
(0.7 mg) were isolated.
Mushrooms: Fruiting bodies of M. galopus (leg. et det. S. Peters and
P. Spiteller) were collected in September and October 2004, 2005
and 2006 in coniferous forests near Leutstetten, 20 km south of
Munich. Voucher samples of M. galopus are deposited at the Insti-
tut für Organische Chemie und Biochemie II der Technischen Uni-
versität München, Germany. The mushrooms were frozen with li-
quid nitrogen and stored at –35 °C after collecting.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methanol
(E-1): Colourless solid. HPLCRP18: Rt = 11.1 min. 1H NMR
(600 MHz, CDCl3, 298 K): δ = 4.55 (s, 2 H, 2-H), 4.65 (s, 2 H, 11-
H), 6.12 (s, 1 H, 10-H), 6.55 (d, J = 11.8 Hz, 1 H, 4-H), 6.83 (d, J
= 11.8 Hz, 1 H, 5-H), 6.99 (d, J = 8.2 Hz, 1 H, 9-H), 7.19 (dd, J
= 8.2, 2.0 Hz, 1 H, 8-H), 7.27 (d, J = 2.0 Hz, 1 H, 6-H) ppm.
Test Organisms: Bacillus subtilis (NCCB 1089), Bacillus brevis
(NCCB 89173), Escherichia coli XL1 blue, Cladosporium cucumeri-
num (CBS 177.54, Centraalbureau voor Schimmelcultures, Amster-
dam), Spinellus fusiger (CBS 633.80, Centraalbureau voor Schim-
melcultures, Amsterdam, and FSU857, Max-Planck-Institut für
Chemische Ökologie, Jena), Saccharomyces cerevisiae (Strain 6211,
Lehrstuhl für Biotechnologie, Technische University of München,
Garching).
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Trimethylsilyl Ether (Trimethylsilyl Derivative of E-1): GC-MS: Ri
= 2117; m/z (%) = 296 (22) [M(37Cl)]+, 294 (64) [M(35Cl)]+, 281 (3),
279 (9), 259 (13), 251 (3), 249 (11), 243 (5), 231 (3), 229 (5), 207
(34) [M(37Cl) – OSi(CH3)3]+, 205 (100) [M(35Cl) – OSi(CH3)3]+, 185
(8), 170 (44), 169 (39), 157 (4), 142 (16), 141 (63), 139 (11), 131
(16), 128 (11), 122 (12), 115 (30), 77 (9), 75 (13), 73 (48), 59 (5), 45
(11).
Metabolic Profiling: For metabolic profiling of intact fruiting bod-
ies of M. galopus, three freshly collected fruiting bodies were placed
into MeOH (5 mL) immediately after collection in the forest. In
order to monitor the hydrolytic cleavage of the benzoxepine esters,
18 freshly collected fruiting bodies of M. galopus were bruised with
a spatula. After 0, 10, 30, 60, 180 and 1440 min, three of these
fruiting bodies were placed into separate vials containing MeOH
(5 mL). In order to monitor the stability of the benzoxepine esters
in the latex, the latex of 15 freshly collected fruiting bodies of
M. galopus was dripped immediately into six separated vials. After
0, 2, 10, 60 and 600 min, respectively, MeOH (5 mL) was applied
to the vials. In the laboratory, the methanolic extract (1 mL) was
removed from each sample, and the solvent was evaporated. Each
sample was dissolved in AcOEt (200 µL) and dried with Na2SO4,
and the solution was transferred to a new vial. The solvent was
then removed in a stream of nitrogen, and each sample was tri-
methylsilylated with MSTFA (50 µL) and subjected to GC-MS
analysis.
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Trimethylsilyl Ether (Trimethylsilyl Derivative of Z-1): GC-MS: Ri
= 2105; m/z: see trimethylsilylated E-1.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Palmitate (E-1a): Colourless solid. HPLCRP18: Rt = 28.2 min.
HPLCNP: Rt = 10.2 min. 1H NMR: see Table 1. 13C NMR: see
Table 1. UV/Vis (H2O): λmax (lg ε) = 239 (4.43), 289 (4.44) nm. GC-
MS: Ri = 3622; m/z (%) = 462 (6) [M(37Cl)]+, 460 (16) [M(35Cl)]+,
224 (8) [M(37Cl) – O=C=CH(CH2)13CH3]+, 222 (23) [M(35Cl) –
O=C=CH(CH2)13CH3]+, 207 (35) [M(37Cl) – O2C(CH2)14CH3]+,
205 (100) [M(35Cl) – O2C(CH2)14CH3]+, 171 (20), 170 (29), 141
(18), 131 (4), 128 (4), 115 (7), 69 (6), 57 (5), 55 (8), 43 (9), 41 (8).
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Palmitate (Z-1a): Colourless solid. HPLCRP18: Rt = 28.2 min.
HPLCNP: Rt = 9.5 min. GC-MS: Ri = 3611; m/z: see E-1a.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Palmitoleate (E-1b): Colourless solid. HPLCRP18: Rt = 26.5 min.
Extraction and Isolation of E-1, Z-1 and 2: Frozen fruiting bodies
(40 g) were crushed after addition of AcOEt (25 mL) and extracted
twice with AcOEt (2ϫ25 mL) at 25 °C for 30 min. The combined
extracts were then concentrated in vacuo at 40 °C. The resulting
residue was heated in methanolic KOH (0.02 , 5 mL) at 70 °C for
5 h. After concentration in vacuo at 40 °C, the residue was dis-
1
HPLCNP: Rt = 10.6 min. H NMR (500 MHz, CDCl3, 298 K): δ =
0.88 (t, J = 6.3 Hz, 3 H, 16Ј-H), 1.26–1.31 (m, 16 H, 4Ј-H to 7Ј-H
and 12Ј-H to 15Ј-H), 1.63 (tt, J = 7.6, 7.6 Hz, 2 H, 3Ј-H), 2.00–
2.01 (m, 4 H, 8Ј-H, 11Ј-H), 2.34 (t, J = 7.6 Hz, 2 H, 2Ј-H), 4.55 (s,
2 H, 2-H), 5.05 (s, 2 H, 11-H), 5.33–5.35 (m, 2 H, 9Ј-H, 10Ј-H),
Eur. J. Org. Chem. 2008, 1187–1194
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1191

S. Peters, R. J. R. Jaeger, P. Spiteller
HPLCNP: Rt = 11.0 min. H NMR (600 MHz, CDCl3, 298 K): δ =
FULL PAPER
6.12 (s, 1 H, 10-H), 6.53 (d, J = 11.8 Hz, 1 H, 5-H), 6.83 (d, J =
1
11.8 Hz, 1 H, 4-H), 6.98 (d, J = 8.8 Hz, 1 H, 9-H), 7.18 (d, J = 0.89 (t, J = 6.6 Hz, 3 H, 18Ј-H), 1.29–1.30 (m, 10 H, 4Ј-H to 6Ј-H,
8.8 Hz, 1 H, 8-H), 7.26 (s, 1 H, 6-H) ppm. HRMS (APCI): calcd.
for C28H4035ClO3 459.2666, found 459.2660 [M + H]+. GC-MS: Ri
= 3609; m/z (%) = 460 (1) [M(37Cl)]+, 458 (3) [M(35Cl)]+, 224 (2)
16Ј-H, 17Ј-H), 1.33–1.37 (m, 4 H, 7Ј-H, 15Ј-H), 1.63 (tt, J = 7.4,
7.4 Hz, 2 H, 3Ј-H), 2.03–2.04 (m, 4 H, 8Ј-H, 14Ј-H), 2.34 (t, J =
7.4 Hz, 2 H, 2Ј-H), 2.77 (dd, J = 7.1, 7.1 Hz, 2 H, 11Ј-H), 4.55 (s,
[ M ( 3 7 C l ) – O = C = C H ( C 1 4 H 2 7 ) ] + , 2 2 2 ( 6 ) [ M ( 3 5 C l ) – 2 H, 2-H), 5.05 (s, 2 H, 11-H), 5.30–5.35 (m, 2 H, 10Ј-H, 12Ј-H),
O=C=CH(C14H27)]+, 207 (38) [M(37Cl) – O2C(CH2)7CHCH-
(CH2)5CH3]+, 205 (100) [M(35Cl) – O2C(CH2)7CHCH(CH2)5-
CH3]+, 171 (14), 170 (22), 141 (12), 131 (4), 128 (4), 115 (6), 69 (7),
55 (9), 43 (6), 41 (7).
5.35–5.40 (m, 2 H, 9Ј-H, 13Ј-H), 6.12 (s, 1 H, 10-H), 6.53 (d, J =
11.8 Hz, 1 H, 5-H), 6.83 (d, J = 11.8 Hz, 1 H, 4-H), 6.98 (d, J =
8.4 Hz, 1 H, 9-H), 7.18 (d, J = 8.4 Hz, 1 H, 8-H), 7.26 (s, 1 H, 6-
H) ppm. 13C NMR (151 MHz, CDCl3, 298 K): δ = 14.1 (C-18Ј),
22.6 (C-17Ј), 24.9 (C-3Ј), 25.6 (C-11Ј), 27.2 (C-8Ј and C-14Ј), 29.1–
29.7, (C-4Ј to C-7Ј and C-15Ј), 31.5 (C-16Ј), 34.3 (C-2Ј), 65.4 (C-
11), 72.6 (C-2), 119.4 (C-10), 120.3 (C-9), 124.2 (C-4), 127.0 (C-7),
127.9 (C-10Ј or C-12Ј), 128.0 (C-12Ј or C-10Ј), 129.5 (C-8), 130.0
(C-9Ј or C-13Ј), 130.2 (C-13Ј or C-9Ј), 130.8 (C-5a), 130.9 (C-5),
133.4 (C-6), 136.3 (C-3), 159.2 (C-9a), 173.7 (C-1Ј) ppm. GC-MS:
Ri = 3834; m/z (%) = 486 (0.3) [M(37Cl)]+, 484 (1) [M(35Cl)]+, 279
(12), 277 (7), 224 (1) [M(37Cl) – O=C=CH(C16H29)]+, 222 (4)
[M(35Cl) – O=C=CH(C16H29)]+, 207 (43) [M(37Cl) – O2C-
(C17H31)]+, 205 (100) [M(35Cl) – O2C(C17H31)]+, 171 (18), 170 (26),
141 (14), 131 (4), 128 (4), 115 (6), 95 (6), 81 (6), 67 (7), 55 (6).
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Palmitoleate (Z-1b): Colourless solid. HPLCRP18: Rt = 26.5 min.
1
HPLCNP: Rt = 9.8 min. H NMR (600 MHz, CDCl3, 298 K): δ =
0.88 (t, J = 7.1 Hz, 3 H, 16Ј-H), 1.24–1.33 (m, 16 H, 4Ј-H to 7Ј-H
and 12Ј-H to 15Ј-H), 1.63 (tt, J = 7.2, 7.2 Hz, 2 H, 3Ј-H), 2.33 (t,
J = 7.2 Hz, 2 H, 2Ј-H), 2.00–2.01 (m, 4 H, 8Ј-H, 11Ј-H), 4.86 (s, 2
H, 2-H), 5.04 (s, 2 H, 11-H), 5.33–5.34 (m, 2 H, 9Ј-H, 10Ј-H), 6.33–
6.34 (m, 3 H, 4-H, 5-H, 10-H), 7.01 (d, J = 8.6 Hz, 1 H, 9-H), 7.16
(dd, J = 8.6, 2.2 Hz, 1 H, 8-H), 7.18 (d, J = 2.2 Hz, 1 H, 6-H) ppm.
13C NMR (151 MHz, CDCl3, 298 K): δ = 14.1 (C-16Ј), 22.7 (C-
15Ј), 25.0 (C-3Ј), 27.2 (C-8Ј and C-11Ј) 29.1–29.8 (C-4Ј–C-7Ј and
C-12Ј–C-13Ј), 31.9 (C-14Ј) 34.3 (C-2Ј), 65.4 (C-11), 68.1 (C-2),
120.2 (C-10), 120.3 (C-9), 127.7 (C-7), 127.8 (C-4 or C-5), 128.1
(C-5 or C-4), 128.9 (C-8), 129.7 (C-9Ј or C-10Ј), 130.0 (C-10Ј or C-
9Ј), 131.0 (C-5a), 132.4 (C-6), 138.2 (C-3), 159.0 (C-9a) 173.7 (C-
1Ј) ppm. GC-MS: Ri = 3593; m/z: see E-1b.
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl Li-
noleate (Z-1e): Colourless solid. Rt = 26.5 min. HPLCNP: Rt =
10.2 min. 1H NMR (600 MHz, CDCl3, 298 K): δ = 0.88 (t, J =
7.4 Hz, 3 H, 18Ј-H), 1.29–1.37 (m, 14 H, 4Ј-H to 7Ј-H and 15Ј-H
to 17Ј-H), 1.63 (tt, J = 6.4, 6.4 Hz, 2 H, 3Ј-H), 2.00–2.05 (m, 4 H,
8Ј-H, 14Ј-H), 2.34 (t, J = 6.4 Hz, 2 H, 2Ј-H), 2.77 (dd, J = 6.9,
6.9 Hz, 2 H, 11Ј-H), 4.86 (s, 2 H, 2-H), 5.05 (s, 2 H, 11-H), 5.31–
5.35 (m, 2 H, 10Ј-H, 12Ј-H), 5.34–5.38 (m, 2 H, 9Ј-H, 13Ј-H), 6.33–
6.34 (m, 3 H, 4-H, 5-H, 10-H), 7.01 (d, J = 8.5 Hz, 1 H, 9-H), 7.17
(d, J = 8.5 Hz, 1 H, 8-H), 7.21 (s, 1 H, 6-H) ppm. GC-MS: Ri =
3815; m/z: see E-1e.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Stearate (E-1c): GC-MS: Ri = 3861; m/z (%) = 490 (14)
[M(37Cl)]+, 488 (32) [M(35Cl)]+, 279 (2), 277 (2), 224 (16) [M(37Cl) –
O=C=CH(CH2)15CH3]+, 222 (47) [M(35Cl) – O=C=CH(CH2)15
-
CH3]+, 207 (38) [M(37Cl) – O2C(CH2)15CH3]+, 205 (100) [M(35Cl) –
O2C(CH2)15CH3]+, 171 (33), 170 (46), 141 (25), 131 (10), 128 (4),
115 (10), 69 (11), 67 (10), 57 (14), 55 (14).
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methanol (2): Colour-
1
less solid. HPLCRP18: Rt = 8.9 min. H NMR (600 MHz, CDCl3,
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Stearate (Z-1c): GC-MS: Ri = 3847; m/z: see E-1c.
298 K): δ = 4.58 (s, 2 H, 2-H), 4.64 (s, 2 H, 11-H), 5.07 (s, 1 H, 10-
Hb), 5.23 (s, 1 H, 10-Ha), 6.35 (d, J = 11.6 Hz, 1 H, 5-H), 6.47 (d,
J = 11.6 Hz, 1 H, 4-H), 6.99 (d, J = 8.2 Hz, 1 H, 9-H), 7.16 (dd, J
= 8.2, 2.0 Hz, 1 H, 8-H), 7.24 (d, J = 2.0 Hz, 1 H, 6-H) ppm.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Oleate (E-1d): Colourless solid. HPLCRP18: Rt = 28.2 min.
1
HPLCNP: Rt = 10.6 min. H NMR (500 MHz, CDCl3, 298 K): δ =
0.88 (t, J = 6.7 Hz, 3 H, 18Ј-H), 1.27–1.29 (m, 20 H, 4Ј-H to 7Ј-H (2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Trimethylsilyl
and 12Ј-H to 17Ј-H), 1.64 (tt, J = 7.6, 7.6 Hz, 2 H, 3Ј-H), 1.98– Ether (Trimethylsilyl Derivative of 2): GC-MS: Ri = 1864; m/z (%)
2.03 (m, 4 H, 8Ј-H, 11Ј-H), 2.34 (t, J = 7.6 Hz, 2 H, 2Ј-H), 4.55 (s, = 260 (64) [M]+, 245 (10), 215 (13), 186 (4), 185 (5), 171 (100) [M –
2 H, 2-H), 5.05 (s, 2 H, 11-H), 5.32–5.37 (m, 2 H, 9Ј-H, 10Ј-H), OSi(CH3)3]+, 141 (13), 131 (6), 128 (19), 115 (10), 75 (6), 73 (6).
6.12 (s, 1 H, 10-H), 6.53 (d, J = 11.9 Hz, 1 H, 5-H), 6.83 (d, J =
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Palmitate (2a):
11.9 Hz, 1 H, 4-H), 6.98 (d, J = 8.0 Hz, 1 H, 9-H), 7.18 (dd, J =
Colourless solid. HPLCRP18: Rt = 28.2 min. HPLCNP: Rt = 8.4 min.
8.0, J = 2.4 Hz, 1 H, 8-H), 7.26 (d, J = 2.4 Hz, 1 H, 6-H) ppm.
1H NMR: see Table 1. 13C NMR: see Table 1. UV/Vis (H2O): λmax
13C NMR (126 MHz, CDCl3, 298 K): δ = 14.1 (C-18Ј), 22.7 (C-
(lg ε) = 236 (4.42), 282 (4.34) nm. GC-MS: Ri = 3353; m/z (%) =
17Ј), 25.0 (C-3Ј), 27.2 (C-8Ј and C-11Ј) 29.1–29.8 (C-4Ј–C-7Ј and
426 (11) [M]+, 188 (42) [M – O=C=CH(CH2)13CH3]+, 172 (78), 171
C-12Ј–C-15Ј), 31.9 (C-16Ј) 34.3 (C-2Ј), 65.4 (C-11), 72.6 (C-2),
(100) [M – O2C(CH2)14CH3]+, 159 (6), 157 (8), 141 (8), 131 (5), 128
119.4 (C-10), 120.3 (C-9), 124.2 (C-4), 127.0 (C-7), 129.4 (C-8),
(15), 115 (6), 57 (6), 55 (6), 43 (10), 41 (6).
129.7 (C-9Ј or C-10Ј), 130.0 (C-10Ј or C-9Ј), 130.8 (C-5a), 130.9 (C-
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Palmitoleate
(2b): Colourless solid. HPLCRP18: Rt = 26.5 min. HPLCNP: Rt =
8.6 min. GC-MS: Ri = 3336; m/z (%) = 424 (1) [M]+, 188 (7) [M –
O=C=CHC14H27]+, 172 (83) 171 (100) [M – O2C(CH2)7-
CHCH(CH2)5CH3]+, 157 (4), 141 (4), 131 (3), 128 (11), 115 (4), 69
(4), 55 (6), 43 (4), 41 (5).
5), 133.4 (C-6), 136.4 (C-3), 159.2 (C-9a) 173.6 (C-1Ј) ppm. HRMS
(APCI): calcd. for C30H4435ClO3 487.2979, found 487.2977 [M +
H]+. GC-MS: Ri = 3835; m/z (%) = 488 (0.3) [M(37Cl)]+, 486 (1)
[M(35Cl)]+, 279 (6), 277 (4), 224 (1) [M(37Cl) – O=C=CH-
(C16H31)]+, 222 (4) [M(35Cl) – O=C=CH(C16H31)]+, 207 (38)
[M(37Cl) – O2C(C17H33)]+, 205 (100) [M(35Cl) – O2C(C17H33)]+,
171 (12), 170 (20), 141 (10), 131 (3), 128 (3), 115 (4), 95 (3), 81 (4),
69 (5), 67 (10), 55 (7), 43 (4), 41 (6).
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Stearate (2c):
GC-MS: Ri = 3545; m/z (%) = 454 (6) [M]+, 188 (26) [M –
O2C(CH2)16CH3]+, 172 (88), 171 (100) [M – O2C(CH2)16CH3]+, 157
(8), 131 (6), 128 (14), 115 (6), 57 (86), 55 (6), 43 (12).
{(Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Oleate (Z-1d): GC-MS: Ri = 3819; m/z: see E-1d.
{(E)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl Li-
noleate (E-1e): Colourless solid. HPLCRP18: Rt = 26.5 min.
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Oleate (2d):
GC-MS: Ri = 3543; m/z (%) = 452 (1) [M]+, 281 (3), 277 (6), 188
1192
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1187–1194

Benzoxepine Esters as Chemical Defence Precursors in Mycena galopus
(6) [M – O=C=CH–(CH2)6CHCH(CH2)7CH3]+, 172 (85), 171 (100) 2132, Ri[(E) isomer] = 2146; m/z (%) = 266 (25) [M(37Cl)]+, 264
[M – O2CC17H33]+, 157 (6), 141 (9), 131 (6), 128 (17), 115 (6), 55 (65) [M(35Cl)]+, 229 (38) [M – Cl]+, 224 (15), 222 (52), 207 (9)
(9).
[M(37Cl) – CH3CO2]+, 205 (22) [M(35Cl) – CH3CO2]+, 187 (27),
170 (52), 169 (61), 157 (13), 141 (100), 139 (12), 131 (31), 128 (41),
115 (48), 43 (37).
(2,3-Dihydro-3-methylenebenzo[b]oxepin-7-yl)methyl Linoleate (2e):
Colourless solid. HPLCRP18: Rt = 26.5 min. HPLCNP: Rt = 9.0 min.
GC-MS: Ri = 3538; m/z (%) = 450 (1) [M]+, 277 (8), 188 (4) [M –
O=C=CHC16H29]+, 172 (64), 171 (100) [M – O2CC17H31]+, 157 (5),
141 (5), 128 (13), 115 (3), 67 (4), 55 (4).
{(E/Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Caprylate: Compound E/Z-1 (0.5 mg), octanoic acid (10 µL) and
(CH3)3SiCl (2 µL) were heated at 80 °C for 3 h. Then, (CH3)3SiCl
and the excess of octanoic acid were removed at 80 °C at 0.1 mbar.
Yield (GC-MS): 99%. Colourless solid. GC-MS: Ri[(Z) isomer] =
2741, Ri[(E) isomer] = 2754; m/z (%) = 350 (18) [M(37Cl)]+, 348
(48) [M(35Cl)]+, 313 (7) [M – Cl]+, 224 (31), 222 (93), 207 (39)
[M(37Cl) – C7H15CO2]+, 205 (100) [M(35Cl) – C7H15CO2]+, 187
(13), 170 (81), 169 (56), 157 (14), 141 (89), 131 (17), 128 (25), 115
(39), 57 (35), 55 (16), 43 (18), 41 (20).
1-{3-(Chloromethylene)-6-hydroxy-2,3-dihydrobenzo[b]oxepin-7-
yl}ethanone (3): GC-MS: Ri = 2180; m/z (%) = 252 (31)
[M(37Cl)]+, 250 (100) [M(35Cl)]+, 237 (4), 235 (15), 215 (38), 213
(15), 201 (6), 199 (6), 197 (8), 179 (6), 173 (16), 171 (7), 161 (29),
141 (6), 115 (18), 113 (4), 105 (13), 77 (6), 43 (12).
Hydrolysis of the Isolated Benzoxepine Esters: Pure samples
(0.5 mg) of E-1a, Z-1a and 2a were dissolved in a methanolic KOH
solution (0.02 , 2 mL) and heated to 60 °C for 1 h. The solvent
was removed at 40 °C in vacuo, and the residue was acidified with
HCl (0.1 , 1 mL) and extracted with CH2Cl2 (2ϫ1 mL). The or-
ganic phase was dried with Na2SO4, and the solvent was removed
at 40 °C in vacuo. The residue was then either trimethylsilylated
with MSTFA or methylated with CH2N2. The samples were sub-
jected to GC-MS analysis. For the identification of the fatty acids,
authentic samples of the corresponding derivatives were compared
by GC-MS with the derivatives of the compounds originating from
M. galopus.
Benzyl Stearate: Stearic acid (100 mg), benzyl alcohol (0.5 mL) and
(CH3)3SiCl (10 µL) were heated at 130 °C for 1 h. After removal of
the benzyl alcohol in vacuo, the reaction product was purified on
a silica gel column (10ϫ1 cm) with hexane/AcOEt (4:1) as eluent.
Yield: 112.5 mg (85%). Colourless solid. 1H NMR (250 MHz,
CDCl3, 298 K): δ = 0.91 (t, J = 6.5 Hz, 3 H, CH3), 1.15–1.42 (m,
28 H, 14ϫCH2), 1.67 (tt, J = 7.5, 7.0 Hz, 2 H, CH2), 2.37 (t, J =
7.5 Hz, 2 H, CH2), 5.14 (s, 2 H, CH2), 7.27–7.43 (m, 5 H, 5ϫCH)
ppm. 13C NMR (63 MHz, CDCl3, 298 K): δ = 14.1 (CH3), 22.7
(CH2), 24.9 (CH2), 29.1–29.8 (12ϫCH2), 31.9 (CH2), 34.3 (CH2),
66.0 (CH2), 128.06 (Cq), 128.08 (CH), 128.4 (CH), 136.1 (Cq), 173.5
(Cq) ppm. GC-MS: Ri = 2791; m/z (%) = 374 (1) [M]+, 283 (4) [M –
C7H7]+, 265 (5), 247 (3), 139 (3), 125 (4), 111 (6), 108 (69), 91 (100)
[C7H7]+, 85 (8), 83 (8), 81 (4), 79 (5), 77 (3), 71 (11), 69 (8), 65 (5),
57 (18), 55 (12), 43 (19).
Biological Tests: For plate diffusion assays, either E/Z-1 (100 or
500 µg), or a mixture of E/Z-1a, E/Z-1c, 2a and 2c (100 or 500 µg,
first fraction of the HPLC separation on RP-18 at Rt = 26.5 min),
or a mixture of E/Z-1b, E-1d and 2b (100 or 500 µg, second fraction
of the HPLC separation on RP-18 at Rt = 28.2 min), was dissolved
in AcOEt (50 µL) and dropped onto paper discs (Ø 6 mm, thick-
ness 0.5 mm). These discs were dried under sterile conditions and
placed on agar plates inoculated with the test organism (Bacillus
brevis, Bacillus subtilis, Escherichia coli, Cladosporium cucumerinum
and Spinellus fusiger). The plates were incubated in the case of
bacteria at 37 °C for 24 h, in the case of C. cucumerinum at 18 °C
for 48 h and in the case of S. fusiger at 18 °C for 96 h. The bioassay
against the yeast Saccharomyces cerevisiae was performed in
liquid culture according to Wijnberg.[3b] Either E-1a (0.23 mg or
0.50 µmol), E-1e (0.24 mg or 0.50 µmol), a mixture of E/Z-1b, E/
Z-1e, 2b and 2e (0.23 mg, 0.50 µmol, first fraction of the HPLC
separation on RP-18 at Rt = 26.5 min), a mixture of E/Z-1a, E-1d
and 2a (0.23 mg, 0.50 µmol, second fraction of the HPLC separa-
tion on RP-18 at Rt = 28.2 min), E/Z-1 (0.11 mg or 0.50 µmol,
1:1 mixture), E-1 (0.11 mg or 0.50 µmol) or nourseothricin (NTC,
0.5 µmol) was dissolved in CH2Cl2 (50 µL) and placed in a sterile
5 mL test tube. After evaporation of the CH2Cl2, liquid YMG me-
dium (500 µL) consisting of yeast extract (0.4%, w/v), malt extract
(1.0%, w/v) and glucose (0.4%, w/v) was added, and each test tube
was inoculated with cells (5 µL) from a freshly prepared 24 h pre-
culture of S. cerevisiae and incubated in a shaking cabinet at 30 °C.
After 18 h, the yeast cell concentration was determined by reading
the optical density at 660 nm. A pure yeast culture, grown in the
absence of test compounds, served as reference for the determi-
nation of the relative growth. The test was repeated three times
for each compound and for the control. Relative growth rates: see
Figure 3.
Esterase Activity of M. galopus: Frozen fruiting bodies (1 g) were
crushed, H2O (1 mL) and either acetylated E/Z-1, caprylated E/Z-
1, methyl oleate or benzyl stearate [100 µg dissolved in EtOH
(20 µL)] were added, and the mixture was incubated at 25 °C and
shaken vigorously. After 24 h, the mixture was extracted with
CHCl3 (2ϫ1 mL). The organic phase was dried with Na2SO4, the
solvent was removed at 40 °C in vacuo, and the residue was tri-
methylsilylated with MSTFA and subjected to GC-MS analysis.
Yields (GC-MS): 99% (starting compound: acetate of E/Z-1,
caprylate of E/Z-1, methyl oleate), 6% (starting compound: benzyl
stearate). In control experiments, in which CHCl3 (1 mL) was
added to the mixture instead of H2O, no esterase activity was de-
tectable by GC-MS.
Esterase Activity of Selected Mycena Species: Frozen fruiting bod-
ies (1 g) were crushed, H2O (1 mL) and benzyl stearate [100 µg dis-
solved in EtOH (20 µL)] were added, and the mixture was incu-
bated at 25 °C. After 16 h, the mixture was extracted with AcOEt
(2ϫ1 mL). The organic phase was dried with Na2SO4, the solvent
was removed at 40 °C in vacuo, and the residue was trimethyl-
silylated with MSTFA and subjected to GC-MS analysis. Yields of
free benzyl alcohol (GC-MS): 32% (M. sanguinolenta), 24%
(M. rosea), 6% (M. galopus), 3% (Mycena aurantiomarginata).
Supporting Information (see also the footnote on the first page of
this article): Selected NMR, UV/Vis and mass spectra of E/Z-1, E/
Z-1a to E/Z-1e, 2, 2a–2e and 3.
Acknowledgments
{(E/Z)-3-(Chloromethylene)-2,3-dihydrobenzo[b]oxepin-7-yl}methyl
Acetate: Compound E/Z-1 (0.5 mg), pyridine (1 mL) and acetic an-
hydride (100 µL) were heated at 100 °C for 2 h. The solvent and
the excess of acetic anhydride were then removed at 40 °C in vacuo.
Yield (GC-MS): 99%. Colourless solid. GC-MS: Ri[(Z) isomer] =
We are grateful to Doreen Schachtschabel and Dr. Dieter Spiteller
(Max-Planck-Institut für chemische Ökologie, Jena, Germany) for
helpful discussions and for providing us a culture of S. fusiger and
Eur. J. Org. Chem. 2008, 1187–1194
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1193

S. Peters, R. J. R. Jaeger, P. Spiteller
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Published Online: January 7, 2008
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