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Volatiles from the hypoxylaceous fungi
Hypoxylon griseobrunneum and Hypoxylon macrocarpum
Jan Rinkel1, Alexander Babczyk1, Tao Wang1, Marc Stadler2 and Jeroen S. Dickschat*1
Full Research Paper Open Access
Address:
1Kekulé-Institut für Organische Chemie, Universität Bonn,
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany and 2Abteilung
Mikrobielle Wirkstoffe, Helmholtz-Zentrum für Infektionsforschung,
Inhoffenstraße 7, 38124 Braunschweig, Germany
Email:
Jeroen S. Dickschat* - dickschat@uni-bonn.de
* Corresponding author
Keywords:
constitutional isomerism; gas chromatography; mass spectrometry;
natural products; volatiles
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
doi:10.3762/bjoc.14.277
Received: 17 October 2018
Accepted: 22 November 2018
Published: 04 December 2018
Associate Editor: A. Kirschning
© 2018 Rinkel et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
The volatiles emitted by the ascomycetes Hypoxylon griseobrunneum and Hypoxylon macrocarpum (Hypoxylaceae, Xylariales)
were collected by use of a closed-loop stripping apparatus (CLSA) and analysed by GC–MS. The main compound class of both
species were polysubstituted benzene derivatives. Their structures could only be unambiguously determined by comparison to all
isomers with different substitution patterns. The substitution pattern of the main compound from H. griseobrunneum, the new
natural product 2,4,5-trimethylanisole, was explainable by a polyketide biosynthesis mechanism that was supported by a feeding
experiment with (methyl-2H3)methionine.
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Introduction
Fungi release a large number of different volatiles that belong to
all kinds of natural product classes [1]. Many of these com-
pounds are of interest, because they are markers for the produc-
tion of fungal toxins and thus can help to distinguish between
toxigenic and closely related non-toxigenic species. For exam-
ple, the sesquiterpene trichodiene (1, Figure 1) is the precursor
of the trichothecene family of mycotoxins [2], a class of highly
bioactive secondary metabolites that belong to the strongest
known inhibitors of protein biosynthesis in eukaryotes [3].
Similarly, the sesquiterpene aristolochene (2) is the parent
hydrocarbon of PR toxin [4,5] and has been used as a marker to
differentiate between toxin producing and non-producing Peni-
cillium roqueforti isolates [6]. On the other hand, fungal vola-
tiles are interesting, because they contribute with their aroma to
the flavour of many edible mushrooms. One of the first identi-
fied and certainly most widespread compounds is matsutake
alcohol, (R)-oct-1-en-3-ol (3), that is produced inter alia by
Tricholoma matsutake [7], a highly sought delicacy in the
Japanese cuisine, the bottom mushroom Agaricus bisporus, and
the penny bun Boletus edulis [8], as the name indicates a Euro-
pean equivalent to Matsutake in high-class cooking. Volatile
organic compounds are also important in the interaction be-
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
2975
Figure 1: Structures of fungal volatiles. Trichodiene (1), aristolochene (2), (R)-oct-1-en-3-ol (3), 3,4-dimethylpentan-4-olide (4), and 6-pentyl-2H-
pyran-2-one (5).
tween different species, e.g., between ophiostomatoid fungi and
conifer bark beetles that show different behavioural responses
to fungal volatiles [9]. Fungal volatiles can also be of impor-
tance in the interaction between plants and fungi. In some cases,
fungal volatiles seem to be involved in the plant pathogenicity
of fungi, as recently observed for 3,4-dimethylpentan-4-olide
(4), a volatile from the ash pathogen Hymenoscyphus fraxineus
that currently threatens the European ash population [10]. Both
enantiomers of this lactone were found to inhibit ash seed
germination and to cause necrotic lesions in the plant tissue. In
other cases, fungal volatiles can have beneficial effects and may
even be involved in the induction of systemic resistance in
plants, as can be assumed for 6-pentyl-2H-pyran-2-one (5) that
is produced by many fungi from the genus Trichoderma
[11,12].
Fungal volatiles can be efficiently analysed by trapping, e.g., on
charcoal filters with a closed-loop stripping apparatus (CLSA)
that was developed by Grob and Zürcher [13], followed by filter
extraction and GC–MS analysis of the obtained headspace
extracts [14]. The unambiguous compound identification
requires a good match of the recorded electron impact (EI) mass
spectrum to a database spectrum and of the retention index, a
standardised GC retention factor that is calculated from the
retention times of the analytes and of n-alkanes [15], in compar-
ison to an authentic standard or published data. A peculiar prob-
lem in the analysis of aromatic compounds with multiple sub-
stituents is that constitutional isomers with the same types of
substituents, but different substitution patterns often have very
similar mass spectra. Furthermore, some of the isomers may
also have similar retention indices, and therefore it is manda-
tory for unambiguous structure elucidation to compare analytes
that fall into this class to all the possible isomers. A similar
problem can apply to the structural assignment of compounds
with multiple stereocentres based on GC–MS data, because the
various possible diastereomers usually also produce very simi-
lar mass spectra [16], a phenomenon that is also reported for
E and Z stereoisomers and can lead to wrong structural assign-
ments, if no authentic standards are used for comparison [17].
We have recently reported on two chlorinated aromatic com-
pounds from an endophytic Geniculosporium sp. [18] and on a
series of structurally related phenols, benzaldehydes and anisole
derivatives from Hypoxylon invadens [19] that could only be
identified with certainty following this approach of extensive
compound comparisons. Members of the family Hypoxylaceae
are regarded to be extremely rich in secondary metabolites [20],
but not much is known about volatiles from these fungi [21]. In
continuation of this work, here we present the volatiles emitted
by Hypoxylon griseobrunneum MUCL 53754 and Hypoxylon
macrocarpum STMA 130423. These strains were selected,
because both species released a characteristic and strong odour,
as was already mentioned in the literature for H. macrocarpum
[22,23], but the nature of the odoriferous compounds remained
unknown. As will be shown, the bouquets of both species are
composed mainly of highly substituted aromatic compounds
whose structures were only securely identifiable by comparison
to all the possible constitutional isomers with different ring sub-
stitution patterns.
Results and Discussion
Headspace analysis
The volatiles released by agar plate cultures of H. griseobrun-
neum and H. macrocarpum were collected using a CLSA [13].
After a collection time of one day the charcoal filter traps were
removed and extracted with CH2Cl2, followed by GC–MS anal-
ysis of the obtained extracts. For both strains a large number of
compounds from different compound classes including alco-
hols, ketones, esters, terpenes and pyrazines were identified.
Besides the observed minor production of compounds from
these classes aromatic compounds dominated, but the patterns
were strain-specific.
Identification of volatiles from Hypoxylon
griseobrunneum
A representative total ion chromatogram for the volatiles re-
leased by Hypoxylon griseobrunneum is shown in Figure 2 and
the results of the analysis are compiled in Table 1. Several com-
pounds in the headspace extract were readily identified from
their mass spectra and retention indices, including the wide-
spread alcohol 2-methylbutan-1-ol (6) as one of the main com-
pounds and traces of the corresponding acetate ester 7 (Table 1
and Figure 3). Small amounts of matsutake alcohol (3) were
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
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Figure 2: Total ion chromatogram of a CLSA headspace extract from Hypoxylon griseobrunneum MUCL 53754. Peak numbers refer to compound
numbers in Table 1 and in Figure 3.
Table 1: Volatiles identified in the headspace extract from Hypoxylon griseobrunneum MUCL 53754.
compound IaI (lit.) identificationbpeak areac
2-methylbutan-1-ol (6) 723 724 [25] ms, ri, std 18.6%
methylpyrazine (9) 817 819 [25] ms, ri, std <0.1%
2-methylbutyl acetate (7) 874 875 [25] ms, ri, std <0.1%
2,5-dimethylpyrazine (10) 903 908 [25] ms, ri, std 1.5%
oct-1-en-3-ol (3) 974 974 [25] ms, ri, std <0.1%
octan-3-one (8) 982 979 [25] ms, ri, std <0.1%
trimethylpyrazine (11) 995 1000 [25] ms, ri, std 0.2%
1,8-cineole (13) 1027 1026 [25] ms, ri, std 8.5%
2-ethyl-3,6-dimethylpyrazine (12) 1074 1077 [24] ms, ri, syn <0.1%
veratrole (20) 1141 1141 [25] ms, ri, std 0.2%
3,4-dimethylanisole (23) 1141 ms, std 0.2%
1,4-dimethoxybenzene (22) 1160 1161 [25] ms, ri, std 0.2%
terpinen-4-ol (18) 1174 1174 [25] ms, ri 0.1%
3-oxo-1,8-cineole (17) 1179 1186 [25] ms, ri 0.7%
2β-hydroxy-1,8-cineole (14) 1208 1217 [26] ms, ri 0.4%
2-oxo-1,8-cineole (16) 1213 1218 [27] ms, ri <0.1%
2α-hydroxy-1,8-cineole (15) 1220 1228 [26] ms, ri <0.1%
2,4,5-trimethylanisole (24) 1225 ms, syn 54.5%
1,2,3-trimethoxybenzene (21) 1308 1309 [28] ms, ri, std <0.1%
2,5-dimethyl-p-anisaldehyde (25) 1456 ms, std 0.5%
methyl 2,5-dimethyl-p-anisate (26) 1544 ms, syn 0.4%
1,8-dimethoxynaphthalene (27) 1657 1657 [19] ms, ri 0.3%
pogostol (19) 1657 1651 [25] ms, ri 0.3%
aRetention index I on a HP5-MS column. bIdentification based on ms: identical mass spectrum, ri: identical retention index, std: comparison to a com-
mercially available standard compound, syn: comparison to a synthetic standard. cPeak area in % of total peak area. The sum is less than 100%,
because compounds originating from the medium, unidentified compounds and contaminants such as plasticisers are not mentioned.
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
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Figure 3: Volatiles from Hypoxylon griseobrunneum.
also found. This volatile is frequently accompanied by other C8
metabolites [1], which is reflected for H. griseobrunneum by
the detection of octan-3-one (8). Trace amounts of a series of
alkylated pyrazines including methylpyrazine (9), 2,5-dimethyl-
pyrazine (10), trimethylpyrazine (11) and 2-ethyl-3,6-dimethyl-
pyrazine (12) were also observed. These compounds were pre-
viously reported from the actinobacterium Corynebacterium
glutamicum in which pyrazines are biosynthetically derived
from acetoin and its higher homologs [24]. For unambiguous
structure elucidation commercially available standards of 9–11
were used, while a synthesis of 12 was performed in our earlier
study [24].
Furthermore, a group of monoterpenes and the sesquiterpene
alcohol pogostol (19) that was previously reported from other
fungi [29,30] were observed. Monoterpenes were comprised of
terpinen-4-ol (18), 1,8-cineole (13) as one of the major com-
pounds in the extracts, and small amounts of its oxidation prod-
ucts 2β-hydroxy-1,8-cineole (14), 2α-hydroxy-1,8-cineole (15),
2-oxo-1,8-cineole (16) and 3-oxo-1,8-cineole (17). The
monoterpene ether 13 has previously been reported from other
Hypoxylon spp. [31] and the responsible monoterpene synthase
has been identified [32]. Its hydroxylated derivatives 14 and 15
were found in insects feeding on leafs of Melaleuca alternifolia
that contain large amounts of 13 [26], and both alcohols 14 and
15 along with the ketones 16 and 17 were reported as metabo-
lites of 13 in human milk [27].
The mass spectrum of the main compound 24 from H. griseo-
brunneum (Figure 4A) showed several fragment ions in the low
m/z region typical for an aromatic compound, while the frag-
ment ion at m/z = 119 pointed to the loss of a methoxy group
from the molecular ion ([M − 31]+), suggesting the structure of
a trimethylanisole for 24. Six constitutional isomers of this
compound exist (Table 2). For four of these compounds the cor-
responding trimethylphenols were commercially available that
were O-methylated with methyl iodide and K2CO3 to yield
compounds 24a, 24b, 24c and 24e. The other two isomers
2,3,4-trimethylanisole (24d) and 2,4,5-trimethylanisole (24)
were obtained by ortho-methylation of 3,4-dimethylphenol (28)
via a known procedure [33], followed by HPLC purification of
the products 2,4,5-trimethylphenol (29a) and 2,3,4-trimethyl-
phenol (29b) and subsequent O-methylation (Scheme 1). Com-
parison of the GC retention index of the natural product
(I = 1225) to the retention indices of all six standards narrowed
the possible structures down to those of 2,4,5-trimethylanisole
(I = 1225) and 2,3,5-trimethylanisole (I = 1227), while all other
isomers could be ruled out. The final structural assignment of
2,4,5-trimethylanisole for 24 was based on the better matching
mass spectrum of this compound in comparison to the alterna-
tive of 24c. Compound 24 has not been reported from other
natural sources before.
The identification of 24 was further supported by a feeding ex-
periment with (methyl-2H3)methionine. While the methylation
pattern of the alternative structure 24c is difficult to understand
via a polyketide biosynthesis mechanism, the formation of the
assigned structure of 24 by a polyketide synthase (PKS) can be
easily rationalised (Scheme 2). The acetate starter unit, bound to
the acyl carrier protein (ACP) of an iterative fungal PKS, can be
elongated with malonyl-SCoA (mal-SCoA) followed by
C-methylation with S-adenosyl-L-methionine (SAM). Two
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
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Figure 4: EI mass spectra of A) 2,4,5-trimethylanisole (24), B) the coeluting mixture of 3,4-dimethylanisole (23) and veratrole (20) with major peaks
originating from 20 shown in red, C) the commercial standard of 23, D) the commercial standard of 20, E) 2,5-dimethyl-p-anisaldehyde (25),
F) methyl 2,5-dimethyl-p-anisate (26).
Table 2: Retention indices of all isomers of trimethylanisole.
structure compound nameaIb
2,4,6-trimethylanisole (24a) 1157
2,3,6-trimethylanisole (24b) 1181
2,4,5-trimethylanisole (24) 1225
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Table 2: Retention indices of all isomers of trimethylanisole. (continued)
2,3,5-trimethylanisole (24c) 1227
2,3,4-trimethylanisole (24d) 1257
3,4,5-trimethylanisole (24e) 1271
aThe natural product from H. griseobrunneum is 24, its isomers are designated 24a–e. bRetention index I on a HP5-MS column.
Scheme 1: Synthesis of trimethylanisoles 24 and 24d.
Scheme 2: Hypothetical biosynthesis of 24. ACP: acyl carrier protein, AT: acyl transferase, KR: ketoreductase, KS: ketosynthase, mal-SCoA:
malonyl-SCoA, MT: methyl transferase, SAM: S-adenosyl-L-methionine.
more rounds of elongation with mal-SCoA, the first extension
with C-methylation and action of a ketoreductase (KR), result in
a tetraketide intermediate that can be cyclised by aldol conden-
sation, followed by elimination of water to result in the aromat-
ic ring system. Thioester hydrolysis and decarboxylation
produce 29a that can be converted by SAM-dependent O-meth-
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
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Figure 5: Biosynthesis of 24. Feeding of (methyl-2H3)methionine resulted in the incorporation of labelling into up to three methyl groups of 24. The
shown ion trace chromatograms represent unlabelled 24 (black, m/z = 150), (2H3)-24 (blue, m/z = 153), (2H6)-24 (purple, m/z = 156), and (2H9)-24
(red, m/z = 159). No incorporation into the fourth methyl group was observed (no peak visible for m/z = 162). For the ion trace chromatograms of
m/z = 159 and 162 also expansions (20×) are shown.
ylation into 24. In summary, this hypothetical biosynthetic
mechanism includes three SAM-dependent methylation steps. A
feeding experiment with (methyl-2H3)methionine, the biosyn-
thetic precursor of SAM, resulted in the incorporation of
labelling into up to three methyl groups of 24, but not into the
fourth methyl group (Figure 5), which is in line with the biosyn-
thetic model of Scheme 2. Note that because of an isotope effect
the isotopomers of 24 can be separated by gas chromatography
depending on their deuterium content [34,35], which makes the
usage of (methyl-2H3)methionine superior to the usage of
13C-labelled methionine that would not have led to chromato-
graphic separation of the isotopomers. In conjunction with the
low incorporation rates obtained here, the results would have
been difficult to interpret.
Another trace compound emitted by H. griseobrunneum showed
a molecular ion at m/z = 136 and coeluted with exactly the same
retention time as a second compound with a molecular ion at
m/z = 138. In case of two coeluting compounds the individual
compounds are often enriched in the right and left peak flanks,
and their individual mass spectra can be extracted by careful
background subtraction, but this was not the case here, so only
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Table 3: Retention indices of all isomers of dimethylanisole and trimethylphenol.
structure compound nameaIb
2,6-dimethylanisole (23a) 1056
2,4-dimethylanisole (23b) 1103
2,5-dimethylanisole (23c) 1104
3,5-dimethylanisole (23d) 1114
2,3-dimethylanisole (23e) 1128
3,4-dimethylanisole (23) 1141
2,4,6-trimethylphenol (23f) 1198
2,3,6-trimethylphenol (23g) 1227
the mass spectrum of the compound mixture was obtained
(Figure 4B). The analysis of the observed fragment ions sug-
gested that the compound with the molecular ion at m/z = 136
may be one of the isomers of dimethylanisole, explaining the
fragment ion at m/z = 105 by the loss of the methoxy group
([M − 31]+), and in agreement with the 14 Da lower molecular
ion in comparison to 24. All six isomers of dimethylanisole
were commercially available and a comparison of retention
indices together with a personal inspection of the mixed mass
spectrum of Figure 4B and the mass spectrum of 3,4-dimethyl-
anisole (Figure 4C) unequivocally identified the natural prod-
uct as 3,4-dimethylanisole (23, Table 3). Furthermore, the alter-
native structure of a trimethylphenol was ruled out, because all
the isomers eluted later than 23 (Table 3). Interestingly, the
elution order of the trimethylphenols is the same as for the cor-
responding trimethylanisoles with respect to their substitution
patterns, and each trimethylphenol consistently elutes slightly
later with an increase of the retention index by ca. 30–50 points
than the trimethylanisole analogue (Table 2 and Table 3), which
is explainable by the significantly higher polarity of the phenols
compared to the anisoles. Compound 23 was recently reported
from Euphorbia golondrina [36], but was never observed as a
fungal natural product so far.
Biosynthetically, the identified compound 23 can arise by a
similar mechanism as discussed for 24, potentially as a minor
product of the same PKS, only the C-methylation step in the
second round of chain extension needs to be skipped
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Table 3: Retention indices of all isomers of dimethylanisole and trimethylphenol. (continued)
2,4,5-trimethylphenol (23h) 1262
2,3,5-trimethylphenol (23i) 1267
2,3,4-trimethylphenol (23j) 1296
3,4,5-trimethylphenol (23k) 1311
aThe natural product from H. griseobrunneum is 23, its isomers are designated 23a–k. bRetention index I on a HP5-MS column.
Table 4: Retention indices of all isomers of trimethoxybenzene.
structure compound nameaIb
1,2,3-trimethoxybenzene (21) 1308
1,2,4-trimethoxybenzene (21a) 1368
1,3,5-trimethoxybenzene (21b) 1409
aThe natural product from H. griseobrunneum is 21, its isomers are designated 21a and 21b. bRetention index I on a HP5-MS column.
(Scheme 2). However, during the feeding experiment with
(methyl-2H3)methionine the formation of 23 was suppressed,
possibly because the additional supply of methionine resulted in
a higher efficiency of the programmed methylation steps
towards 24.
The additional signals in the mixed mass spectrum (Figure 4B)
at m/z = 138, 123 and 95 that do not originate from 23 are
present with similar relative proportions as in the mass spec-
trum of veratrole (20, Figure 4D), and indeed a commercial
standard of 20 revealed the same retention index of I = 1141 as
the natural product, thus confirming the structure of veratrole
for the second of the coeluting compounds. Its isomer 1,4-
dimethoxybenzene (22) and a trimethoxybenzene 21 were also
detected. Comparison to all three commercially available
isomers of trimethoxybenzene established the identity of 21 as
1,2,3-trimethoxybenzene (Table 4). 1,8-Dimethoxynaphthalene
(27) was also found and has been reported previously from
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2983
Scheme 3: Hypothetical biosynthesis of 25 and 26.
other Hypoxylon spp. [19,37]. The corresponding compound
1,8-dihydroxynaphthalene is a known precursor of fungal
melanin pigments [38].
Two trace compounds exhibited the mass spectra shown in
Figure 4E and Figure 4F that were similar to database spectra of
2,5-dimethyl-p-anisaldehyde (25) and methyl 2,5-dimethyl-p-
anisate (26). The substitution pattern of these compounds is
well explained by polyketide biosynthesis logic (Scheme 3).
Starting from ACP-bound acetate, two non-reducing elonga-
tions with malonyl-SCoA, the first without and the second with
C-methylation, followed by another elongation with reduction
of the 3-oxo group and cyclisation yields the aromatic system of
25 and 26. Hydrolytic cleavage from the ACP and two methyla-
tions of the phenol and the carboxylic acid result in 26, while
reductive cleavage and methylation of the phenol give 25. The
aldehyde 25 was commercially available and matched the
natural product in terms of mass spectrum and retention time.
Compound 25 was transformed into the corresponding methyl
ester by treatment with iodine and potassium hydroxide in
methanol [39]. The obtained material also showed identical be-
haviour in the GC–MS analysis to natural 26. Both compounds
25 and 26 are new natural products.
Identification of volatiles from Hypoxylon
macrocarpum
The composition of the headspace extracts from H. macro-
carpum (Figure 6 and Table 5) was completely different from
the extracts of H. griseobrunneum with only the three com-
pounds 2,5-dimethylpyrazine (10), trimethylpyrazine (11) and
pogostol (19) being emitted by both species (Figure 7). The vol-
atiles benzaldehyde (32) and 2-phenylethanol (35) as two of the
main compounds, and the trace compounds 2-acetylfuran (30),
2-acetylthiazole (31), acetophenone (33), 1-phenylethanol (34),
1-phenylpropan-1,2-dione (36) and m-cresol (37) were readily
identified from their mass spectra and retention indices and by
comparison to authentic standards.
The main compounds released by H. macrocarpum were identi-
fied as 3,4-dimethoxytoluene (43) and 4-methylsalicylaldehyde
(39), while 2,5-dimethylphenol (38) and 2-methoxy-4-methyl-
benzaldehyde (40) were detected in lower amounts. All four
compounds were previously observed in the bouquet of
H. invadens and unambiguously identified by comparison to all
possible isomers with different ring substitution patterns [19].
Furthermore, comparison to all ten isomers of methoxy-methyl-
benzaldehydes described in this study allowed for the identifica-
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2984
Figure 6: Total ion chromatogram of a CLSA headspace extract from Hypoxylon macrocarpum STMA 130423. Peak numbers refer to compound
numbers in Table 5 and in Figure 7.
Table 5: Volatiles identified in headspace extract from Hypoxylon macrocarpum STMA 130423.
compound IaI (lit.) identificationbpeak areac
2,5-dimetylpyrazine (10) 903 908 [25] ms, ri, std 0.1%
2-acetylfuran (30) 906 909 [25] ms, ri, std 0.7%
benzaldehyde (32) 952 952 [25] ms, ri, std 22.8%
trimethylpyrazine (11) 995 1000 [25] ms, ri, std 0.5%
2-acetylthiazole (31) 1012 1014 [25] ms, ri, std 0.6%
1-phenylethanol (34) 1054 1057 [25] ms, ri, std 0.1%
acetophenone (33) 1059 1059 [25] ms, ri, std 0.8%
m-cresol (37) 1071 1072 [25] ms, ri 1.9%
2-phenylethanol (35) 1105 1106 [25] ms, ri, std 11.6%
2,5-dimethylphenol (38) 1154 1152 [19] ms, ri, std 3.6%
4-methylsalicylaldehyde (39) 1162 1165 [19] ms, ri, std 16.4%
1-phenylpropan-1,2-dione (36) 1171 1175 [40] ms, ri 0.1%
3,4-dimethoxytoluene (43) 1243 1240 [19] ms, ri, std 29.1%
3-methoxy-4-methylbenzaldehyde (41) 1302 1307 [19] ms, ri, std 0.2%
2-methoxy-4-methylbenzaldehyde (40) 1365 1364 [19] ms, ri, std 0.9%
3,4,5-trimethoxytoluene (44) 1405 ms, std 0.1%
2,4,5-trimethoxytoluene (45) 1436 ms, syn 0.3%
3,4-dimethoxybenzaldehyde (42) 1483 1475 [25] ms, ri, std 1.3%
2,5-dichloro-1,3-dimethoxybenzene (46) 1552 1556 [18] ms, ri, std 1.0%
pogostol (19) 1657 1651 [25] ms, ri <0.1%
aRetention index I on a HP5-MS column. bIdentification based on ms: identical mass spectrum, ri: identical retention index, std: comparison to a com-
mercially available standard compound, syn: comparison to a synthetic standard. cPeak area in % of total peak area. The sum is less than 100%,
because compounds originating from the medium, unidentified compounds and contaminants such as plasticisers are not mentioned.
tion of another trace compound from H. macrocarpum as 3-me-
thoxy-4-methylbenzaldehyde (41). The chlorinated compound
2,5-dichloro-1,3-dimethoxybenzene (46) was also rigorously
identified by comparison to all possible regioisomers that we
had synthesised in a previous study [18]. Interestingly, the sub-
stitution pattern for the compound from H. macrocarpum is dif-
ferent to an isomer from the endophyte Geniculosporium sp.
that was identified as 1,5-dichloro-2,3-dimethoxybenzene.
Compound 46 has not been described as a natural product
before. Another trace compound released by H. macrocarpum
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2985
Figure 7: Volatiles from Hypoxylon macrocarpum.
Figure 8: EI mass spectra of A) 3,4-dimethoxybenzaldehyde (42), B) 3,4,5-trimethoxytoluene (44), and C) 2,4,5-trimethoxytoluene (45).
exhibited a mass spectrum that pointed to the structure of a
dimethoxybenzaldehyde (Figure 8A). Comparison to all six
commercially available isomers (Table 6) showed the identity
of the natural product and 3,4-dimethoxybenzaldehyde (42).
Finally, two trace compounds with almost identical mass spec-
tra (Figure 8B and Figure 8C), but clear separation by gas chro-
matography, were suggested to be trimethoxytoluenes. Two
isomers, 3,4,5-trimethoxytoluene (44) and 2,4,6-trimethoxy-
toluene (44d), were commercially available. 2,3,4-Trimethoxy-
benzaldehyde (47) was reduced to 2,3,4-trimethoxytoluene
(44a) using PdCl2 and Et3SiH [41] (Scheme 4), while the other
three isomers were synthesised according to reported proce-
dures [42-44]. Comparison of all six isomers to the two natural
products (Table 7) resulted in their identification as 3,4,5-
trimethoxytoluene (44) and 2,4,5-trimethoxytoluene (45). While
44 is a relatively widespread natural product, its isomer 45 has
only once been tentatively identified by mass spectrometry in
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
2986
Table 6: Retention indices of all isomers of dimethoxybenzaldehyde.
structure compound nameaIb
2,3-dimethoxybenzaldehyde (42a) 1391
3,5-dimethoxybenzaldehyde (42b) 1445
2,5-dimethoxybenzaldehyde (42c) 1468
3,4-dimethoxybenzaldehyde (42) 1483
2,6-dimethoxybenzaldehyde (42d) 1531
2,4-dimethoxybenzaldehyde (42e) 1543
aThe natural product from H. macrocarpum is 42, its isomers are designated 42a–e. bRetention index I on a HP5-MS column.
Scheme 4: Synthesis of 2,3,4-trimethoxytoluene (44a).
plants from the genus Asarum [45], but never from fungi before.
However, it remains unclear how 45 was distinguished from 44
or other possible isomers in the earlier study.
Conclusion
Both investigated ascomycetes, Hypoxylon griseobrunneum and
Hypoxylon macrocarpum, were found to emit complex mix-
tures of volatiles, mainly composed of aromatic compounds. As
we have demonstrated, for unequivocal structural assignments
based solely on GC–MS data it is important to compare the
natural product to all possible constitutional isomers with differ-
ent ring substitution patterns, because the mass spectra of these
isomers are too similar to rely solely on MS data for compound
identification. Therefore, also the retention index of the natural
product must match the retention index of an authentic standard,
and usually the retention indices of the isomeric aromatic com-
pounds with different substitution patterns are sufficiently dif-
ferent for a confident structural assignment. Also biosynthetic
considerations can help in the structure elucidation, because
some aromatic substitution patterns are in line with a polyke-
tide biosynthesis mechanism, while other substitution patterns
may be difficult to understand. But such considerations should
be made with care and should ideally be supported, e.g.,
by feeding experiments, as we have conducted in the present
study. The main compounds of H. griseobrunneum were
2-methylbutan-1-ol, 1,8-cineol and 2,4,5-trimethylanisole,
while H. macrocarpum released a completely different bouquet
with the main compounds benzaldehyde, 2-phenylethanol,
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
2987
Table 7: Retention indices of all isomers of trimethoxytoluene.
structure compound nameaIb
2,3,4-trimethoxytoluene (44a) 1321
2,3,6-trimethoxytoluene (44b) 1397
3,4,5-trimethoxytoluene (44) 1405
2,3,5-trimethoxytoluene (44c) 1410
2,4,5-trimethoxytoluene (45) 1436
2,4,6-trimethoxytoluene (44d) 1488
aThe natural products from H. macrocarpum are 44 and 45, its isomers are designated 44a–d. bRetention index I on a HP5-MS column.
4-methylsalicylaldehyde and 3,4-dimethoxytoluene. All these
volatiles exhibit a characteristic smell and are likely main
contributors to the odour produced by the fungi, but also some
of the identified minor compounds may be important for the
fungal fragrance. Notably, fungi of the genus Hypoxylon are
interesting sources of new natural products, as exemplified by
the identification of 2,4,5-trimethylanisole, 2,5-dimethyl-p-
anisaldehyde and its corresponding methyl ester, and 2,5-
dichloro-1,3-dimethoxybenzene. Therefore, it will be of high
interest to investigate the volatiles from further Hypoxylon
species in the near future.
Experimental
Strains and culture conditions
Hypoxylon griseobrunneum was obtained from a specimen
collected in Martinique, Case Pilote, on a trail to Morne Venté
on wood and bark of a dead dicotyledon branch in a mesophilic
to xerophilic forest, on 25 August 2010 by Jacques Fournier
[46]. A voucher specimen is deposited at the herbarium of the
University of Lille, France (LIP, No MJF10120) and the cul-
ture is deposited with MUCL (Louvain-la Neuve, Belgium)
under the accession number MUCL 53754.
Hypoxylon macrocarpum was obtained from ascospores of a
specimen collected in Germany, Rhineland-Palatinate Province
in the vicinity of Forst, near the Pechsteinkopf from wood of
Fagus on 20 October 2012 by Benno and Marc Stadler [21]. A
voucher specimen is deposited in the fungarium of the
Helmholtz Centre for Infection Research (HZI, Braunschweig,
Germany) under the accession number STMA 130423.
Analysis of volatiles
The volatiles emitted by agar plate cultures of H. griseobrun-
neum and H. macrocarpum were collected through a closed-
loop stripping apparatus (CLSA) [13] for ca. 1 day at room tem-
perature and under natural light-dark rhythm. The CLSA char-
coal filter traps were extracted with CH2Cl2 (50 μL, HPLC
grade), followed by analysis of the extracts by GC–MS.
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
2988
GC–MS
GC–MS analyses were performed with a 7890A GC coupled to
a 5975C inert mass detector (Agilent, Hewlett-Packard
Company, Wilmington, USA). The GC was equipped with a
HP5-MS fused silica capillary column (30 m, 0.25 mm i. d.,
0.25 μm film, Agilent). Conditions were inlet pressure:
77.1 kPa, He 23.3 mL min−1; injection volume: 1.5 μL; injector
operation mode: splitless (60 s valve time); carrier gas: He at
1.2 mL min−1; GC program: 5 min at 50 °C, then increasing
with 5 °C min−1 to 320 °C; transfer line 300 °C; electron energy
70 eV. Retention indices (I) were determined from a homolo-
gous series of n-alkanes (C8–C38).
Synthesis of 2,4,5-trimethylphenol (29a) and
2,3,4-trimethylphenol (29b)
Diiodomethane (2.14 g, 8.0 mmol, 2 equiv) was dissolved in
dry toluene (3 mL) under an argon atmosphere and the solution
was cooled to 0 °C. To the vigorously stirred solution, Et2Zn in
toluene (5.0 mL, 1.2 M, 6.0 mmol, 1.5 equiv) was added
rapidly, followed immediately by the addition of 3,4-
dimethylphenol (500 mg, 4.0 mmol) in toluene (3 mL). The
reaction mixture was stirred at 0 °C for 5 min and then under
reflux for 1.5 h. The reaction mixture was cooled to 0 °C and
then quenched with an aqueous solution of NaHCO3
(10% w/w). The aqueous phase was extracted with diethyl ether
for three times and the combined organic layers were dried over
MgSO4. The solvent was removed under reduced pressure and
the crude product was purified by column chromatography on
silica gel (cyclohexane/ethyl acetate 5:1). The obtained product
contained 29a and 29b as a mixture which was separated by
HPLC (KNAUER Wissenschaftliche Geräte GmbH, Berlin,
Azura; DAICEL Chiralpak IA column, 5 μm, 4.6 × 250 mm;
hexane/2-propanol 95:5; retention times: 9.66 min (29b) and
10.89 min (29a)). The pure products were obtained as colour-
less liquids.
2,4,5-Trimethylphenol (29a). Yield: 14 mg (0.10 mmol, 3%).
1H NMR (500 MHz, CDCl3, 298 K) δ (ppm) 6.88 (s, 1H, CH),
6.59 (s, 1H, CH), 4.56 (br s, 1H, OH), 2.20 (s, 3H, CH3), 2.19
(s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (125 MHz,
CDCl3, 298 K) δ (ppm) 151.6 (Cq), 135.2 (Cq), 132.1 (CH),
128.4 (Cq), 120.5 (Cq), 116.3 (CH), 19.4 (CH3), 18.7 (CH3),
15.2 (CH3).
2,3,4-Trimethylphenol (29b). Yield: 11 mg (0.08 mmol, 2%).
1H NMR (500 MHz, CDCl3, 298 K) δ (ppm) 6.87 (d,
3J = 8.1 Hz, 1H, CH), 6.56 (d, 3J = 8.1 Hz, 1H, CH), 4.54 (br s,
1H, OH), 2.22 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.19 (s, 3H,
CH3); 13C NMR (125 MHz, CDCl3, 298 K) δ (ppm) 151.7 (Cq),
136.6 (Cq), 128.8 (Cq), 127.5 (CH), 122.6 (Cq), 112.0 (CH),
20.3 (CH3), 16.0 (CH3), 12.1 (CH3).
Synthesis of trimethylanisoles 24 and 24a–e
To a solution of the respective phenol derivative (23f–k,
15.0 mg, 0.11 mmol, 1 equiv) in dry DMF (2.2 mL), K2CO3
(15.2 mg, 0.11 mmol, 1 equiv) was added and the mixture was
stirred at room temperature for 30 min. Methyl iodide (31 mg,
0.22 mmol, 2 equiv) was added and the reaction mixture was
stirred at room temperature overnight. The reaction was
quenched by addition of water and the aqueous phase was
extracted three times with EtOAc. The combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography on silica gel (cyclohexane/ethyl acetate 20:1).
The pure products were obtained as pale yellow liquids.
2,4,5-Trimethylanisole (24). Yield: 5 mg (0.03 mmol, 32%).
TLC (silica, cyclohexane/ethyl acetate 20:1): Rf = 0.48;
1H NMR (500 MHz, CDCl3, 298 K) δ (ppm) 6.89 (s, 1H, CH),
6.63 (s, 1H, CH), 3.80 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.17 (s,
3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3,
298 K) δ (ppm) 155.8 (Cq), 134.6 (Cq), 132.1 (CH), 128.0 (Cq),
123.6 (Cq), 112.1 (CH), 55.7 (CH3), 20.0 (CH3), 18.8 (CH3),
15.7 (CH3).
2,4,6-Trimethylanisole (24a). Yield: 6 mg (0.04 mmol; 36%).
TLC (silica, cyclohexane/ethyl acetate 20:1): Rf = 0.31;
1H NMR (500 MHz, CDCl3, 298 K) δ (ppm) 6.82 (s, 2H,
2 × CH), 3.70 (s, 3H, CH3), 2.25 (s, 6H, 2 × CH3), 2.24 (s, 3H,
CH3); 13C NMR (125 MHz, CDCl3, 298 K) δ (ppm) 154.9 (Cq),
133.2 (Cq), 130.6 (2 × Cq), 129.5 (2 × CH), 59.9 (CH3), 20.8
(CH3), 16.1 (2 × CH3).
2,3,6-Trimethylanisole (24b). Yield: 7 mg (0.05 mmol; 42%).
TLC (silica, cyclohexane/ethyl acetate 20:1): Rf = 0.42;
1H NMR (400 MHz, CDCl3, 298 K) δ (ppm) 6.92 (d,
3J = 7.6 Hz, 1H, CH), 6.83 (d, 3J = 7.6 Hz, 1H, CH), 3.70 (s,
3H, CH3), 2.27 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.20 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm) 156.9 (Cq),
136.0 (Cq), 129.6 (Cq), 128.2 (Cq), 128.0 (CH), 125.3 (CH),
60.0 (CH3), 20.0 (CH3), 16.2 (CH3), 12.4 (CH3).
2,3,5-Trimethylanisole (24c). Yield: 8 mg (0.05 mmol; 48%).
TLC (silica, cyclohexane/ethyl acetate 20:1): Rf = 0.47;
1H NMR (400 MHz, CDCl3, 298 K) δ (ppm) 6.62 (s, 1H, CH),
6.55 (s, 1H, CH), 3.81 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.24 (s,
3H, CH3), 2.11 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3,
298 K) δ (ppm) 157.6 (Cq), 137.7 (Cq), 135.6 (Cq), 123.1 (CH),
121.9 (Cq), 109.0 (CH), 55.7 (CH3), 21.5 (CH3), 20.1 (CH3),
11.4 (CH3).
2,3,4-Trimethylanisole (24d). Yield: 5 mg (0.03 mmol, 32%).
TLC (silica, cyclohexane/ethyl acetate 20:1): Rf = 0.54;
Beilstein J. Org. Chem. 2018, 14, 2974–2990.
2989
1H NMR (400 MHz, CDCl3, 298 K) δ (ppm) 6.96 (d,
3J = 8.3 Hz, 1H, CH), 6.64 (d, 3J = 8.3 Hz, 1H, CH), 3.79 (s,
3H, CH3), 2.23 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.17 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm) 156.0 (Cq),
136.4 (Cq), 128.6 (Cq), 127.2 (CH), 125.1 (Cq), 107.8 (CH),
55.8 (CH3), 20.3 (CH3), 16.0 (CH3), 12.1 (CH3).
3,4,5-Trimethylanisole (24e). Yield: 8 mg (0.05 mmol; 48%).
TLC (silica, cyclohexane: ethyl acetate = 20:1): Rf = 0.37;
1H NMR (400 MHz, CDCl3, 298 K) δ (ppm) 6.59 (s, 2H,
2 × CH), 3.77 (s, 3H, CH3), 2.27 (s, 6H, 2 × CH3), 2.11 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm) 157.1 (Cq),
137.7 (2 × Cq), 127.2 (Cq), 113.2 (CH), 55.3 (CH3), 21.0
(2 × CH3), 14.7 (CH3).
Synthesis of methyl 2,5-dimethyl-p-anisate
(26)
Similar to a reported procedure [39], 2,5-dimethyl-p-anisalde-
hyde (25, 1 g, 6.09 mmol, 1 equiv) was dissolved in MeOH
(60 mL) and the solution was cooled to 0 °C. Solutions of KOH
(1.045 g, 15.89 mmol, 2.6 equiv, in 20 mL MeOH) and I2
(2.01 g, 7.92 mmol, 1.3 equiv, in 10 mL MeOH) were added
and the mixture was stirred for 90 min at 0 °C. The reaction was
diluted with EtOAc, washed three times with saturated aqueous
Na2S2O3 solution and subsequently with brine. The organic
layer was dried over MgSO4 and the solvent was removed
under reduced pressure. The crude product was purified via
column chromatography (cyclohexane/ethyl acetate 10:1) on
silica gel and the pure product was obtained as a colourless
solid (277 mg, 1.43 mmol, 23%). TLC (silica, cyclohexane/
ethyl acetate 3:1): Rf = 0.67. 1H NMR (500 MHz, CDCl3,
298 K) δ (ppm) 7.75 (s, 1H, CH), 6.64 (s, 1H, CH), 3.86 (s, 3H,
CH3), 3.85 (s, 3H, CH3), 2.60 (s, 3H, CH3), 2.18 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3, 298 K) δ (ppm) 167.9 (Cq), 160.6
(Cq), 140.8 (Cq), 133.4 (CH), 123.9 (Cq), 120.9 (Cq), 112.9
(CH), 55.5 (CH3), 51.6 (CH3), 22.1 (CH3), 15.8 (CH3).
Synthesis of 2,3,4-trimethoxytoluene (44a)
According to a known procedure [41], to a solution of 2,3,4-
trimethoxybenzaldehyde (47, 500 mg, 2.55 mmol, 1 equiv) in
EtOH (13 mL), SiEt3H (590 mg, 5.1 mmol, 2 equiv) was added
under an argon atmosphere. PdCl2 (45.2 mg, 0.26 mmol,
10 mol %) was added and after stirring for 1 h, the reaction was
quenched with H2O. The mixture was extracted three times with
Et2O and the combined organic layers were dried over MgSO4.
The solvent was removed under reduced pressure and the crude
product was purified via column chromatography on silica gel
(cyclohexane/ethyl acetate 10:1). The pure product was ob-
tained as a colourless liquid (267 mg, 1.47 mmol, 57%). TLC
(silica, cyclohexane/ethyl acetate 3:1): Rf = 0.50; 1H NMR
(500 MHz, C6D6, 298 K) δ (ppm) 6.73 (dq, 3J = 8.4 Hz,
4J = 0.8 Hz, 1H, CH), 6.38 (d, 3J = 8.4 Hz, 1H, CH), 3.78 (s,
3H, CH3), 3.71 (s, 3H, CH3), 3.38 (s, 3H, CH3), 2.22 (d,
4J = 0.8 Hz, 3H, CH3); 13C NMR (125 MHz, C6D6, 298 K)
δ (ppm) 152.9 (Cq), 152.8 (Cq), 143.5 (Cq), 124.7 (CH), 124.3
(Cq), 108.0 (CH), 60.6 (CH3), 60.3 (CH3), 55.8 (CH3), 15.9
(CH3).
Acknowledgements
This work was funded by the DFG (DI1536/9-1). We thank
Andreas Schneider (Bonn) for compound purification by
preparative HPLC and Jacques Fournier (Rimont, France) and
Eric Kuhnert (Leibniz University Hannover) for their previous
support in the characterisation of the H. griseobrunneum strain.
ORCID® iDs
Marc Stadler - https://orcid.org/0000-0002-7284-8671
Jeroen S. Dickschat - https://orcid.org/0000-0002-0102-0631
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