Volatiles from the hypoxylaceous fungi Hypoxylon griseobrunneum and Hypoxylon macrocarpum

Article abstract of DOI:10.3762/bjoc.14.277

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.

Full text of DOI:10.3762/bjoc.14.277

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