Trichlorinated phenols from Hypholoma elongatum

Article abstract of DOI:10.1016/S0031-9422(97)01067-4

Three trichlorinated phenols, 2,4,6-trichloro-3-methoxyphenol, 3,5,6- trichloro-2,4-dimethoxy-phenol and 3,4,6-trichloro-2,5-dimethoxyphenol, were detected as novel metabolites in the ethyl acetate extract from the culture medium of the Basidiomycete, Hyp

Full text of DOI:10.1016/S0031-9422(97)01067-4

Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 192Ð195\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S9920Ð8311"86#90956Ð3
TRICHLORINATED PHENOLS FROM HYPHOLOMA ELONGATUM
HENK J[ SWARTS\$ FRANK J[ M[ VERHAGEN\ JIM A[ FIELD and JOANNES B[ P[ A[ WIJNBERGꢀ$
Division of Industrial Microbiology\ Department of Food Science\ Wageningen Agricultural University\
P[ O[ Box 7018\ 5699 EV Wageningen\ The Netherlands^ $ Laboratory of Organic Chemistry\
Wageningen Agricultural University\ Dreijenplein 7\ 5692 HB Wageningen\ The Netherlands
"Received 18 September 0886#
Key Word Index*Hypholoma elon`atum^ Basidiomycetes^ novel metabolites^ chlorophenols[
Abstract*Three trichlorinated phenols\ 1\3\5!trichloro!2!methoxyphenol\ 2\4\5!trichloro!1\3!dimethoxy!
phenol and 2\3\5!trichloro!1\4!dimethoxyphenol\ were detected as novel metabolites in the ethyl acetate extract
from the culture medium of the Basidiomycete\ Hypholoma elon`atum "strain WIJS83!17#[ Þ 0887 Elsevier
Science Ltd[ All rights reserved
INTRODUCTION
Basidiomycetes as decomposers of forest litter rep!
resent an ecologically important group of organisms
in the environment and are known to produce a wide
variety of organohalogens ð0Ł[ Three of the most com!
monly occurring Basidiomycete genera in The Nether!
lands\ Hypholoma\ Mycena and Bjerkandera\ were
found to be dominant among the high producers of
organohalogens ð1Ł[ Among these organohalogens\
chlorinated hydroquinone methyl ethers "CHMs# and
chlorinated anisyl metabolites "CAMs# are the most
common[ The CHM\ drosophilin A "tetrachloro!3!
methoxyphenol\ 0#\ was the _rst halogenated metab!
olite identi_ed in a Basidiomycete ð2Ł and has a struc!
ture very similar to the anthropogenic penta!
chlorophenol[ Apart from drosophilin A\ several
other CHMs are known from Basidiomycetes[ To
LN medium[ Three of these compounds are novel
trichlorinated phenols which were not previously
reported as de novo metabolites\ from Basidiomycetes
or any other living organism[ The two other organo!
halogens\ drosophilin A and 2\4!dichloro!p!anisyl
alcohol\ are known as de novo metabolites from
Basidiomycetes[ Drosophilin A production by Hypho!
loma has never been demonstrated before[
date\ CHMs have been found in 09 di}erent genera
ð0\ 3Ł[ CAM production has so far been reported for 05
Basidiomycete genera ð0\4\5Ł[ Hypholoma elon`atum
"strain WIJS83!17#\ for instance\ produces almost
selectively 2\4!dichloro!p!anisyl alcohol "1# in
a
remarkably high concentration "097 mg l−0#\ when
grown on a medium with a high nitrogen "HN# content
ð5Ł[
In the present paper\ we report the results of a study
in which organohalogen production by H[ elon`atum
"strain WIJS83!17# grown on a medium with a low
nitrogen "LN# content was examined[ In contrast to
the selective production of 2\4!dichloro!p!anisyl alco!
hol by H[ elon`atum on HN medium ð5Ł\ _ve di}erent
organohalogens are produced by H[ elon`atum on
RESULTS AND DISCUSSION
The fungal strain "H[ elon`atum WIJS83!17# was
cultivated in the dark at 14> on LN medium ð6Ł[ When
the culture ~uid was completely covered by mycelium
"5 weeks#\ it was _ltered and extracted with ethyl acet!
ate ð7Ł[ The identi_cation of chlorinated compounds
in the ethyl acetate extract was performed with GC!
mass spectrometry\ and comparison of retention times
and mass spectra to data of respective reference com!
ꢀ Author to whom correspondence should be addressed[
192

193
H[ J[ SWARTS et al[
pounds[ The concentration of chlorinated compounds and:or decomposition[ Retention times and mass
was determined by GC analysis using the internal spectral data found for the two pure trichloro adducts
standard quantitation method ð8Ł[
did not match with those for the unknown trich!
In the ethyl acetate extract\ _ve chlorinated com! lorinated dimethoxyphenols produced by H[ elon!
pounds could be detected[ Two of these compounds\ `atum[ Careful GC!mass spectral analysis of the four
drosophilin A "0# and 2\4!dichloroanisyl alcohol "1#\ crude product mixtures prior to work!up\ however\
produced in concentrations of 9[6 and 39[1 mg l−0
\
revealed that the trichlorinated products derived from
respectively\ are known de novo metabolites from 1\3! ð04Ł and 1\4!dimethoxyphenol ð05Ł\ i[e[ 2\4\5!trich!
Basidiomycetes[ The production of 2\4!dichloroanisyl loro!1\3!dimethoxyphenol "3# and 2\3\5!trichloro!1\4!
alcohol by Hypholoma species is well known ð4\ 5Ł[ On dimethoxyphenol "4#\ respectively\ were identical with
the other hand\ the generation of drosophilin A\ or the two remaining unknown metabolites[ Since pure
any other CHM\ by Hypholoma has not been reported reference compounds were not available\ only the rela!
before[
tive concentration of 3 and 4 "0[4 and 1[5)\ respec!
The identity of the other three chlorinated com! tively# in the extract could be determined by means of
pounds was unknown[ The EI mass spectrum of the GC!MS analysis[ As far as we know this is the _rst
_rst unknown metabolite produced in a concentration report of the de novo biosynthesis of these compounds
of 3[9 mg l−0\ showed ðMŁ¦\ ðM¦1Ł¦\ ðM¦3Ł¦ and by a fungus or any other living organism[ Previously\
ðM¦5Ł¦ ions at m:z 115\ 117\ 129 and 121\ in a ratio of it was reported that 2\4\5!trichloro!1\3!dimethoxy!
09]8[5]2]9[2\ respectively\ suggesting a trichlorinated phenol "3# might be a degradation product of tetra!
compound with molecular formula of C6H4Cl2O1[ chloroguaiacol by Rhodococcus chlorophenolicus ð06\
These data and the prominent ion peaks "rel[ int[# at 07Ł[
m:z 102 "74#\ 100 "78#\ 074 "80#\ 072 "84#\ 038 "20# and
Hypholoma elon`atum is an ecologically important
036 "36#\ ascribed to ðM¦1ÐMeŁ¦\ ðMÐMeŁ¦\ ðM¦1Ð Basidiomycete inhabiting moss "Spa`hnum and Poy!
MeÐCOŁ¦\ ðMÐMeÐCOŁ¦\ ðM¦1ÐMeÐCOÐHClŁ¦ trichum# in wetlands[ The results presented here indi!
and ðMÐMeÐCOÐHClŁ¦\ respectively\ led to the con! cate that this fungus might be an important source
clusion that this compound is
a trichlorinated of naturally produced chlorophenols in ecosystems[
methoxyphenol ð09\ 00Ł[ Since GC retention and mass Chlorophenols are susceptible to polymerization reac!
spectral data for all the possible isomers of trich! tions by phenol oxidizing enzymes ð08Ł\ which might
lorinated 1!methoxy! ð09Ł and 3!methoxyphenol ð00Ł account for the unusually high organohalogen content
clearly di}ered from those of the unknown metabolite\ of humus recovered from wetlands ð19Ł[
the methoxyl group in the latter compound is most
likely positioned at C!2[ In order to gather additional
information about the substitution pattern of the
EXPERIMENTAL
unknown compound\ some chlorination experiments
were performed on 2!methoxyphenol[ It was found
General
0
that treatment of 2!methoxyphenol with molecular
Mps] uncorr[ H NMR "199 MHz# and 02C NMR
chlorine in glacial acetic acid a}orded a single prod! "49 MHz# CDCl2 with TMS as int[ standard[
uct\ 1\3\5!trichloro!2!methoxyphenol "2#\ with ident!
ical retention time "co!injection# and mass spectral
data as the unknown metabolite[ The position of the
Or`anism and culture conditions
three Cl substituents at C!1\ C!3 and C!5 in 2 was
Hypholoma elon`atum "strain WIJS83!17# was
deduced from its NMR spectral data ð01Ł[
obtained from the Culture Collection of Industrial
The EI mass spectra of the two other unknown Microbiology "CIMW#\ Wageningen Agricultural
metabolites were similar and both indicated the pres! University\ The Netherlands[ The fungal strain was
ence of three Cl atoms[ The appearance of ðMŁ¦ at grown on LN medium ð6Ł with the addition of NaCl
m:z 145 was consistent with a molecular formula of "9[95 g#[ Medium "09 ml# in a 099 ml serum bottle was
C7H6Cl2O2 and suggested that these compounds are sterilized at 010> for 29 min[ Medium was inoculated
trichlorinated dimethoxyphenols[ In order to establish with a plug "diameter 4 mm#\ which was taken from
their identities unequivocally\ the six possible isomers an agar medium covered with fresh mycelium of the
of dimethoxyphenol were subjected to exhaustive fungal strain[ Fungal cultures were incubated in the
chlorination[ Treatment of 2\4! and 1\5!dimethoxy! dark at 14> under air[ When the culture ~uid was
phenol with molecular chlorine in chloroform completely covered by the mycelium "5 weeks#\ the
a}orded 1\3\5!trichloro!2\4!dimethoxyphenol ð02Ł culture ~uid was harvested[
and 2\3\4!trichloro!1\5!dimethoxyphenol ð03Ł\ respec!
tively\ as pure compounds in good yield "ca 69)#[
Exhaustive chlorination of the other isomers led to
Extraction and sample preparation
rather complex product mixtures in which variable
Filtration and extraction of the culture ~uid were
amounts of the corresponding trichlorinated deriva! performed as described in Ref[ ð7Ł[ After removal of
tives were detected[ All attempts to obtain these trich! EtOAc under red[ pres[\ the resulting residue was
loro adducts in pure form resulted in demethylation redissolved in 9[14 ml of freshly dist[ EtOAc con!

Trichlorinated phenols from Hypholoma elon`atum
194
taining 43[4 mg of 3!bromoanisole as int[ standard\ ðMŁ¦ m:z 144[8352 "calcd for C7H6Cl2O2\ 144[8350#[
and then subjected to GC!MS analysis[
All attempts to obtain pure 3 from the crude reaction
mixt[ failed[ Other chlorinating agents did not pro!
duce 3[
Identi_cation and quantitation of chlorometabolites
Preparation of 2\3\5!trichloro!1\4!dimethoxyphenol
Samples were analysed on a quadrupole MS "4#[ 1\4!Dimethoxyphenol was prepd as described in
coupled to a GC equipped with a fused silica capillary Ref[ ð05Ł[ A soln of 1\4!dimethoxyphenol "9[14 g\ 0[51
column "HP!4MS\ 29 m×9[14 mm i[d[\ _lm thickness] mmol# in CH1Cl1 "04 ml# was treated with excess ben!
9[14 mm#[ Carrier gas and ~ow] He at 0[9 ml min−0
[
zyltrimethylammonium tetrachloroiodate ð11Ł[ Work!
Injector temp[ 119>^ temp[ programme] 69Ð149> at 6> up and puri_cation by CC on silica gel "petrolÐEtOAc\
min−0\ hold 14 min[ Injection vol[] 4[9 ml^ split ratio 3]0# gave 9[11 g "50)# of the corresponding dichloro
0]4[ EIMS were obtained at 69 eV[ Identi_cation of compound[ To a soln of this compound in HOAc "04
chlorinated compounds was achieved by comparison ml# was added dropwise sulfuryl chloride "79 ml# and
of Rts and MS to data of respective authentic com! the reaction mixt[ stirred at room temp[ for 2 h[
pounds[ The concns of 0Ð2 were determined by GC According to GC!MS analysis\ the crude reaction
analysis using the int[ standard quantitation method mixt[ contained ca 54) of 4[ EIMS m:z "rel int[#] 159
ð8Ł[ Because authentic compounds 3 and 4 could not "05# ðM¦3Ł¦\ 147 "32# ðM¦1Ł¦\ 145 "41# ðMŁ¦\ 134
be synthesized in pure form "see below#\ quantitation "21#\ 132 "80#\ 130 "099#\ 104 "20#\ 102 "20#\ 199 "25#\
of these compounds was not possible[ Based on the 087 "25#\ 092 "26#\ 76 "34#^ HRMS] ðMŁ¦ m:z 144[8366
total ion current\ an indication of the rel[ concn of 3 "calcd for C7H6Cl2O2\ 144[8350#[ Attempts to obtain
and 4 in the extract was obtained by GC!MS[ All pure 4 from the crude reaction mixt[ by CC failed due
measurements were done in duplicate from a duplicate to demethylation and decomposition[
set of cultures[
Acknowled`ements*This project was _nancially sup!
ported by the Technology Foundation\ Utrecht\ The
Netherlands\ under project number WLM22[2016\
entitled] {{Fungal Chlorinated Aromatic Metabolites]
Natural Priority Pollutants and Dioxin Precursors in
the Environment[|| We are grateful to Dr Juha Knuut!
Authentic compounds
Drosophilin A "0# was kindly provided by Dr J[
Knuutinen "University of Jyvaskyla\ Finland#[ 2\4!
Dichloro!p!anisyl alcohol "1# was prepd as described
in Ref[ ð4Ł[
Ã
Ã
inen "University of Jyvaskyla\ Kyllikinkatu\ Finland#
Ã
Ã
Preparation of 1\3\5!trichloro!2!methoxyphenol "2#[
A soln of 2!methoxyphenol "1[4 g\ 19[1 mmol# in
HOAc "01[4 ml# was purged with Cl1 at room temp[
for 29 min[ The reaction mixt[ was poured into H1O
"099 ml# and extracted with EtOAc "2×49 ml#[ The
combined organic layers were washed with H1O\ satd
aq[ NaHCO2 and brine\ dried over MgSO3 and concd
in vacuo[ Kugelrohr short!path distillation "009> at
09−1 mbar# of the remaining residue gave a yellow oil
"3[2 g# which was puri_ed by CC on silica gel "petrolÐ
EtOAc\ 19]0#[ Yield] 2[2 g "61)# 2\ yellow crystals\
mp 51Ð55> "lit[ ð10Ł 53>#[ 0H NMR "199 MHz\ CDCl2#]
d 2[77 "s\ 2H\ MeO!2#\ 4[89 "s\ 0H\ HO!0# 6[29 "s\ 0H\
H!4#[ 02C NMR "49 MHz\ CDCl2#] d 59[76 "q\ MeO!
2#\ 005[14 "s\ C!1 or C!5#\ 005[83 "s\ C!1 or C!5#\
019[00 "s\ C!3#\ 016[78 "d\ C!4#\ 036[58 "s\ C!0#\ 040[73
"s\ C!2#[ EIMS m:z "rel[ int[#] 121 "2# ðM¦5Ł¦\ 129
"29# ðM¦3Ł¦\ 117 "84# ðM¦1Ł¦\ 115 "099# ðMŁ¦\ 104
"17#\ 102 "74#\ 100 "78#\ 076 "18#\ 074 "80#\ 072 "84#\
038 "20#\ 036 "36#\ 010 "15#\ 008 "31#\ 73 "11#^ HRMS]
ðMŁ¦ m:z 114[8238 "calcd for C6H4Cl2O1\ 114[8244#[
Preparation of 2\4\5!trichloro!1\3!dimethoxy phenol
"3#[ 1\3!Dimethoxyphenol was prepd as described in
Ref[ ð04Ł[ A soln of 1\3!dimethoxyphenol "04 mg\ 9[0
mmol# in CHCl2 "1 ml# was purged with Cl1 at room
temp[ for 1 min[ GC!MS analysis of the crude reaction
mixt[ revealed the presence of 3 "ca 4)#[ EIMS m:z
"rel[ int[#] 159 "15# ðM¦3Ł¦\ 147 "67# ðM¦1Ł¦\ 145
"63# ðMŁ¦\ 134 "16#\ 132 "83#\ 130 "099#\ 104 "22#\ 102
"24#\ 199 "32#\ 087 "38#\ 092 "18#\ 76 "33#^ HRMS]
and Dr Alasdair H[ Neilson "Swedish Environmental
Research Institute\ Stockholm\ Sweden# for their sam!
ples of trichlorinated 1!methoxy! and 3!methoxy!
phenols[
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