Volatiles from the xylarialean fungus Hypoxylon invadens

Article abstract of DOI:10.3762/bjoc.14.62

The volatiles emitted by agar plate cultures of the xylarialean fungus Hypoxylon invadens were investigated by use of a closed loop stripping apparatus in combination with GC-MS. Several aromatic compounds were found that could only be identified by comparison to all possible constitutional isomers with different ring substitution patterns. For the set of identified compounds a plausible biosynthetic scheme was suggested that gives further support for the assigned structures.

Full text of DOI:10.3762/bjoc.14.62

Volatiles from the xylarialean fungus Hypoxylon invadens
Jeroen S. Dickschat*1, Tao Wang1 and Marc Stadler2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 734–746.
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.14.62
Received: 24 November 2017
Accepted: 20 March 2018
Published: 29 March 2018
Email:
Jeroen S. Dickschat* - dickschat@uni-bonn.de
Associate Editor: S. Bräse
* Corresponding author
© 2018 Dickschat 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 agar plate cultures of the xylarialean fungus Hypoxylon invadens were investigated by use of a closed loop
stripping apparatus in combination with GC–MS. Several aromatic compounds were found that could only be identified by compar-
ison to all possible constitutional isomers with different ring substitution patterns. For the set of identified compounds a plausible
biosynthetic scheme was suggested that gives further support for the assigned structures.
Introduction
The research during the past decades has shown that many fungi able interest of natural product chemists and ecologists in vola-
release a rich bouquet of volatile organic compounds [1]. Some tile secondary metabolites.
of these metabolites contribute to the pleasant aroma of edible
mushrooms, e.g., the widespread compound oct-1-en-3-ol (1) is Volatile natural products can efficiently be captured on char-
responsible for the typical odour of the button mushroom, coal filter traps by using a closed-loop stripping apparatus
Agaricus bisporus, and other delicacies from the fungal world (CLSA) [6] or on polydimethylsiloxane fibres by application of
such as the oyster mushroom, the penny bun, and shiitake [2,3]. the solid phase micro-extraction method (SPME) [7], followed
The ecological function of most fungal volatiles is unknown, by GC–MS analysis of the obtained extracts [8]. Compound
but for the alcohol 1 (Figure 1) a germination inhibitory func- identification is then usually performed by comparison of
tion has been reported [4]. For 6-pentyl-2H-pyran-2-one (2), measured mass spectra to mass spectra in electronic libraries, in
another widespread fungal volatile, a plant growth promoting addition to comparison of measured to published retention
effect and an induction of systemic resistance in plants against indices. Positive compound identification can be assumed, if the
fungi was observed [5]. The significant biological effects of mass spectrum and the retention index match reported data. The
these and other fungal volatiles recently resulted in a consider- comparison of retention indices is particularly important, if dif-
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Figure 1: Structures of the widespread fungal volatiles oct-1-en-3-ol (1) and 6-pentyl-2H-pyran-2-one (2), the stereoisomers α-muurolene (3), α-amor-
phene (4) and α-cadinene (5), and the chlorinated aromatic compounds 1-chloro-3,4-dimethoxybenzene (6) and 1,3-dichloro-4,5-dimethoxybenzene
(7) from Geniculosporium.
ferent stereoisomers need to be considered, because stereoiso- Results and Discussion
mers may have very similar mass spectra, as observed for the The volatiles released by agar plate cultures of Hypoxylon
sesquiterpenes α-muurolene (3), α-amorphene (4), and invadens MUCL 54175 were collected through the CLSA head-
α-cadinene (5). The same problem applies to the unambiguous space method and the obtained headspace extracts were
identification of regioisomers of aromatic compounds. We have analysed by GC–MS. The gas chromatogram of a representa-
recently reported on the GC–MS-based identification of the tive extract is shown in Figure 2. Several volatiles could imme-
fungal volatiles 1-chloro-3,4-dimethoxybenzene (6) and 1,3- diately be identified based on their mass spectra and retention
dichloro-4,5-dimethoxybenzene (7) from an endophytic indices (Table 1 and Figure 3), including the major compounds
Geniculosporium sp. by comparison of the natural products to benzaldehyde (8) and 2-phenylethanol (10), and the trace com-
all possible regioisomers that were obtained by chemical syn- ponents acetophenone (9), terpinen-4-ol (13), and indole (16).
thesis [9]. Here we report on the identification of the volatiles For several other compounds in the headspace extract close hits
emitted by the xylarialean fungus Hypoxylon invadens MUCL for highly substituted aromatic compounds were found in our
54175, a highly interesting pyrenomycete that was recently de- mass spectral libraries, but the mass spectra and retention
scribed as a new species [10]. This species is apparently rare indices for some of these compounds also showed some differ-
and has hitherto only been found twice in Southwestern France, ences. Furthermore, the structures of regioisomers with differ-
colonising the stromata (fruiting bodies) of another species of
the same genus, the ubiquitous Hypoxylon fragiforme. The
genus Hypoxylon was traditionally accommodated in the family
Xylariaceae, but has recently been reassigned to the Hypoxy-
laceae. This family was resurrected as a result of intensive
polyphasic studies on the biological and chemical diversity of
the ascomycete order Xylariales, which is well-known for its
diversity of bioactive secondary metabolites [11,12].
We decided to select a culture initiated from the germinating
ascospores of H. invadens among a panel of hypoxylaceous
fungi that were studied for comparison of their volatile profiles
[13]. Several aromatic compounds were detected in the head-
space extracts of Hypoxylon invadens MUCL 54175 for which
an unambiguous GC–MS-based structural assignment was only
possible by comparison to all regioisomers with different substi-
tution patterns at the benzene ring.
Figure 2: Total-ion chromatogram of the bouquet from Hypoxylon
invadens MUCL 54175 obtained by the CLSA headspace technique.
Numbers at peaks correspond to the compounds in Table 1 and
Figure 3, asterisks indicate unknown compounds. The red line shows a
500% superelevation of the chromatogram between 16.2 min and
18.3 min.
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Table 1: Volatiles identified in the bouquet of Hypoxylon invadens MUCL 54175.
Compound
I
I (Ref.)
Identificationa
Peak areab
benzaldehyde (8)
961
951 [14]
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
std
7.9%
0.2%
18.1%
2.4%
10.6%
0.1%
<0.1%
40.6%
<0.1%
4.7%
6.8%
0.1%
1.0%
acetophenone (9)
1066
1111
1152
1165
1179
1187
1240
1293
1364
1472
1059 [14]
1106 [14]
1151 [15]
2-phenylethanol (10)
2,5-dimethylphenol (11)
2-hydroxy-4-methylbenzaldehyde (12)
terpinen-4-ol (13)
1174 [14]
1290 [14]
ms, ri
std
2-methoxy-5-methylphenol (14)
3,4-dimethoxytoluene (15)
indole (16)
std
ms, ri, std
syn
2-methoxy-4-methylbenzaldehyde (17)
5-hydroxy-2-methylchroman-4-one (18)
ms
5-hydroxy-2-methyl-4H-chromen-4-one (19) 1591
1,8-dimethoxynaphthalene (20) 1657
syn
ms
aCompound identification by ms: mass spectrum identical to a library spectrum, ri: retention index identical to published data, std: comparison to an
authentic standard, syn: comparison to a compound synthesised in this work. bPeak integral (% of sum of peak integrals). Medium components, cont-
aminants such as plasticisers and unknown compounds are not listed.
Figure 3: Identified volatile organic compounds from Hypoxylon invadens MUCL 54175.
ent substitution patterns at the aromatic ring could not be of 11 and 2,5-dimethylphenol by identical retention index and
excluded, because the mass spectra and retention indices were best matching mass spectrum (Table 2).
not available for all these compounds.
The mass spectrum of the second compound 12 (Figure 4B) was
The mass spectrum of one of these compounds (11, Figure 4A) very similar to the mass spectra of several regioisomers of
suggested the structure of a dimethylphenol, but the mass spec- hydroxy-methylbenzaldehydes included in our libraries, but
tra of several regioisomers in our database proved to be nearly retention indices for the complete set of regioisomers were not
identical. The measured retention index (I = 1152) matched available from the literature. Also in this case all ten constitu-
with a published retention index for 2,5-dimethylphenol tional isomers of hydroxy-methylbenzaldehyde were commer-
(I = 1151) [15], but retention indices were not available for all cially available and a comparison of the headspace constituent
the possible molecules. Six constitutional isomers with differ- from H. invadens to all these compounds by GC–MS allowed
ent substitution patterns at the aromatic ring exist for for the unambiguous identification of 12 as 2-hydroxy-4-
dimethylphenol that were all commercially available. A direct methylbenzaldehyde (Table 3). The best MS match was ob-
comparison by GC–MS unequivocally established the identity tained for 2-hydroxy-6-methylbenzaldehyde (MS match: 904),
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Figure 4: Mass spectra of volatiles from Hypoxylon invadens MUCL 54175. Mass spectra of A) 2,5-dimethylphenol (11), B) 2-hydroxy-4-methylbenz-
aldehyde (12), C) 3,4-dimethoxytoluene (15), D) 2-methoxy-4-methylbenzaldehyde (17), E) 2-methoxy-5-methylphenol (14), F) 5-hydroxy-2-
methylchroman-4-one (18), and G) 5-hydroxy-2-methyl-4H-chromen-4-one (19).
but the mass spectrum of 2-hydroxy-4-methylbenzaldehyde pro- The main compound 15 released by H. invadens exhibited a
duced a match that was nearly as good (MS match: 902), and mass spectrum that pointed to the structure of a dimethoxy-
the structure of 2-hydroxy-6-methylbenzaldehyde could clearly toluene (Figure 4C), but again the mass spectra of various dif-
be excluded by a different retention index.
ferent regioisomers in our mass spectral libraries were too simi-
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Table 2: Retention indices of all regioisomers of dimethylphenol.
Structure Compound name
I
MS matcha
862
2,6-dimethylphenol
2,4-dimethylphenol
1108
1147
879
2,5-dimethylphenol (11)
3,5-dimethylphenol
2,3-dimethylphenol
1152
1164
1177
913
903
901
3,4-dimethylphenol
1191
892
aMS match of the mass spectrum of the natural product from H. invadens in comparison to the mass spectrum of the tabulated compound (a value of
1000 would indicate identical mass spectra, positive compound identification can usually be assumed for a value >900).
Table 3: Retention indices of all regioisomers of hydroxy-methylbenzaldehyde.
Structure
Compound name
I
MS matcha
902
2-hydroxy-3-methylbenzaldehyde
1139
2-hydroxy-5-methylbenzaldehyde
2-hydroxy-4-methylbenzaldehyde (12)
2-hydroxy-6-methylbenzaldehyde
1160
1165
1202
893
902
904
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Table 3: Retention indices of all regioisomers of hydroxy-methylbenzaldehyde. (continued)
3-hydroxy-2-methylbenzaldehyde
1374
1396
1399
1408
878
874
857
785
3-hydroxy-4-methylbenzaldehyde
3-hydroxy-5-methylbenzaldehyde
3-hydroxy-6-methylbenzaldehyde
4-hydroxy-3-methylbenzaldehyde
4-hydroxy-2-methylbenzaldehyde
1427
1452
857
857
aMS match of the mass spectrum of the natural product from H. invadens in comparison to the mass spectrum of the tabulated compound (a value of
1000 would indicate identical mass spectra, positive compound identification can usually be assumed for a value >900).
lar to distinguish between the possibilities and retention indices Notably, a common biosynthesis for all the identified aromatic
were not available for all cases. All six constitutional isomers compounds can be assumed that further strengthens their struc-
were obtained from commercial suppliers and compared to the ture elucidations (Scheme 1). Starting from 11, an oxidation
natural product, establishing the identity of 15 and 3,4- step at the 2-methyl group (red) could lead via the benzyl
dimethoxytoluene, for which an identical retention index and alcohol derivative to the corresponding aldehyde 12 that upon
the best MS match was determined (Table 4).
O-methylation (green), likely with S-adenosylmethionine,
would result in 17. The alternative oxidation of 12 by a
Baeyer–Villiger monooxygenase could result in the insertion of
an oxygen (blue) to yield the formate ester 21, followed by ester
hydrolysis to 4-methylcatechol (22). Two sequential O-methyl-
ations (green) could give rise to 15 via the hypothetical interme-
diate 2-methoxy-5-methylphenol (14). These thoughts prompted
us to search for missing biosynthetic links in the headspace
extracts of Hypoxylon invadens. Indeed, a trace compound with
a mass spectrum that could fit to the structure of 14 was ob-
served (Figure 4E). This compound exhibited the same reten-
tion index and mass spectrum as a commercially available stan-
dard of 2-methoxy-5-methylphenol. Unfortunately, not all the
constitutional isomers with different ring substitution patterns
Similarly, the mass spectrum of compound 17 hinted to the
structure of a methoxy-methylbenzaldehyde isomer
(Figure 4D), for which like for the hydroxy-methylbenzalde-
hydes ten different constitutional isomers are possible. For an
unambiguous structural assignment all ten commercially ob-
tained hydroxy-methylbenzaldehydes were converted into the
corresponding methoxy derivatives by methylation with potas-
sium carbonate and methyl iodide. The GC–MS analysis of all
the obtained methylation products unequivocally established the
identity of 17 and 2-methoxy-4-methylbenzaldehyde by the
same retention index and best matching mass spectrum
(Table 5).
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Table 4: Retention indices of all regioisomers of dimethoxytoluene.
Structure
Compound name
I
MS matcha
905
2,3-dimethoxytoluene
1176
3,4-dimethoxytoluene (15)
2,5-dimethoxytoluene
2,4-dimethoxytoluene
1240
1252
1257
937
772
742
2,6-dimethoxytoluene
3,5-dimethoxytoluene
1259
1271
779
716
aMS match of the mass spectrum of the natural product from H. invadens in comparison to the mass spectrum of the tabulated compound (a value of
1000 would indicate identical mass spectra, positive compound identification can usually be assumed for a value >900).
Table 5: Retention indices of all regioisomers of methoxy-methylbenzaldehyde.
Structure
Compound name
I
MS matcha
894
2-methoxy-3-methylbenzaldehyde
1265
3-methoxy-4-methylbenzaldehyde
3-methoxy-5-methylbenzaldehyde
1307
1313
697
670
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Beilstein J. Org. Chem. 2018, 14, 734–746.
Table 5: Retention indices of all regioisomers of methoxy-methylbenzaldehyde. (continued)
3-methoxy-6-methylbenzaldehyde
1323
1335
1335
650
889
741
2-methoxy-6-methylbenzaldehyde
3-methoxy-2-methylbenzaldehyde
2-methoxy-5-methylbenzaldehyde
1352
1364
884
934
2-methoxy-4-methylbenzaldehyde (17)
4-methoxy-3-methylbenzaldehyde
4-methoxy-2-methylbenzaldehyde
1366
1368
660
628
aMS match of the mass spectrum of the natural product from H. invadens in comparison to the mass spectrum of the tabulated compound (a value of
1000 would indicate identical mass spectra, positive compound identification can usually be assumed for a value >900).
were available from standard suppliers of fine chemicals and did, however, not constitute a major metabolite during the
thus the structure of another regioisomer cannot fully be screening of numerous other species of Hypoxylon and the
excluded for 14, but the closure of the biosynthetic gap by related Annulohypoxylon using HPLC/DAD–MS detection of
2-methoxy-5-methylphenol intriguingly favours this structural organic extracts from standardised submerged cultures [26].
assigment for 14. The alternative structure of 2-methoxy-4-
methylphenol that would be an intermediate if the methylations Another of the trace components (19) displayed a molecular ion
would proceed by a reverse order of steps was excluded for the that was reduced by 2 Da in comparison to the molecular ion of
detected compound, because the mass spectrum of 2-methoxy- 18 (compare Figure 4F and 4G), and the mass spectrum of this
4-methylphenol was included in our libraries and showed sig- compound was highly similar to a mass spectrum of 5-hydroxy-
nificant differences.
2-methyl-4H-chromen-4-one in our mass spectral library. A
synthetic standard of this compound was obtained via a known
The compound 5-hydroxy-2-methylchroman-4-one (18), one of procedure from 2,6-dihydroxyacetophenone (23) and acetyl
the major constituents in the H. invadens headspace extracts, chloride under basic conditions [27], with formation of
has been reported before from various ascomycetes, especially 3-acetyl-5-hydroxy-2-methyl-4H-chromen-4-one (24) as the
from the genus Daldinia and other endophytic fungi [16-25]. It main product (Scheme 2). The synthetic 5-hydroxy-2-methyl-
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Scheme 1: Proposed common biosynthetic pathway to volatile aromatic compounds from Hypoxylon invadens.
Scheme 2: Synthesis of 5-hydroxy-2-methyl-4H-chromen-4-one (19).
4H-chromen-4-one proved to be identical to natural 19 in terms fungus that was assigned with certainty to this genus. A few
of its gas chromatographic behaviour and mass spectrum. Com- other recent papers were dealing with volatile secondary
pound 19 has also been reported from other fungi before metabolites from endophytic isolates, but these were only tenta-
[18,21,26,28,29]. Finally, 1,8-dimethoxynaphthalene (20) was tively assigned to the genus Hypoxylon, on the basis of genera-
tentatively identified from its mass spectrum. This compound tion of internal transcribed spacer (ITS) DNA sequences and
is known from another endophytic Hypoxylon sp. that was homology comparisons [38-40]. In fact, the ITS is not regarded
isolated from the Formosan plant Litsea akoensis var. species- or even genus-specific in Xylariales and the phyloge-
chitouchiaoensis [30], and was previously reported from the nies based on this DNA locus have led to rather ambiguous
fungi Nodulisporium sp. [18,28], Sporothrix sp. [31], and results. It should be interesting to find out in the future which
Leptographium wageneri [32]. The compound comprises the other species of Hypoxylon produce the volatiles that were
bis-methylation product of 1,8-dihydroxynaphthalene, an im- detected here in the apparently rare and aberrant species
portant precursor of melanin-type pigments in fungi [33], while H. invadens. This fungus represents one of a few cases of
20 has been reported to inhibit melanin biosynthesis in fungi mycophilic Xylariales that parasitise related species [10],
[34].
whereas the vast majority of the species in this order are known
to be saprotrophs with an endophytic state in their life cycle. A
respective study embarking on the chemotaxonomic signifi-
Conclusion
The genus Hypoxylon has already been examined extensively cance of the detected volatiles, paired with the evaluation of the
for secondary metabolite production, and some of its species biological activities of these metabolites, appears very promis-
like H. pulicicidum and H. rickii are extremely creative with ing to complement our knowledge on the functional diversity of
respect to the production of non-volatile compounds [35-37]. the secondary metabolome of the Hypoxylaceae and other
The current study is the first to embark on the volatiles from a Xylariales.
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Experimental
Strain and culture conditions
119 (12), 118 (10), 105 (26), 104 (18), 91 (47), 90 (26), 89 (18),
78 (13), 77 (26).
Hypoxylon invadens MUCL 54175 was isolated from the
ascospores of the holotype specimen using the methodology de- 2-Methoxy-4-methylbenzaldehyde (17). Yield: 35 mg
scribed by Kuhnert et al. [41] and grown under identical condi- (0.23 mmol, 91%), colourless solid. TLC (cyclohexane/ethyl
tions on YMG medium as described by Pažoutová et al. [13].
acetate 10:1): Rf = 0.18; 1H NMR (400 MHz, CDCl3, TMS) δ
10.38 (s, 1H, CHO), 7.71 (d, 3JH,H = 7.8 Hz, 1H, CH), 6.82 (d,
3JH,H = 7.8 Hz, 1H, CH), 6.77 (s, 1H, CH), 3.90 (s, 3H, OCH3),
Analysis of volatiles
The volatiles released by H. invadens agar plate cultures were 2.40 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3, TMS) δ
collected using a closed-loop stripping apparatus (CLSA) [6] 189.6 (CHO), 162.0 (Cq), 147.5 (Cq), 128.7 (CH), 122.8 (Cq),
for 16 to 24 hours at room temperature and under circadian 121.8 (CH), 112.3 (CH), 55.7 (OCH3), 22.4 (CH3) ppm; MS
light-dark rhythm. The CLSA charcoal filters were extracted (EI, 70 eV) m/z (%): 150 (100) [M]+, 149 (60), 135 (11), 133
with HPLC grade CH2Cl2 (50 μL) and the obtained extracts (39), 132 (26), 118 (28), 105 (21), 104 (15), 91 (37), 90 (15), 89
were immediately analysed by GC–MS.
(13), 77 (15).
GC–MS
2-Methoxy-5-methylbenzaldehyde. Yield: 33 mg (0.22 mmol,
GC–MS analyses were performed with an Agilent 7890A GC 89%), colourless oil. TLC (cyclohexane/ethyl acetate 10:1):
and an Agilent 5975C inert mass detector (Hewlett-Packard Rf = 0.15; 1H NMR (400 MHz, CDCl3, TMS) δ 10.43 (d, 4JH,H
Company, Wilmington, USA). The GC was equipped with a = 0.7 Hz, 1H, CHO), 7.61 (d, 4JH,H = 2.4 Hz, 1H, CH), 7.34
non-polar HP5-MS fused silica capillary column (30 m, (dd, 3JH,H = 8.5 Hz, 4JH,H = 2.4 Hz, 1H, CH), 6.88 (d, 3JH,H =
0.25 mm i. d., 0.25 μm film, Agilent). Conditions were inlet 8.5 Hz, 1H, CH), 3.89 (s, 3H, OCH3), 2.30 (s, 3H, CH3) ppm;
pressure: 77.1 kPa, He 23.3 mL min−1; injection volume: 13C NMR (100 MHz, CDCl3, TMS) δ 190.1 (CHO), 160.1 (Cq),
1.5 μL; injector operation mode: splitless (60 s valve time); 136.7 (CH), 130.1 (Cq), 128.7 (CH), 124.6 (Cq), 111.7 (CH),
carrier gas: He at 1.2 mL min−1; GC program: 5 min at 50 °C, 55.8 (OCH3), 20.3 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150
then increasing with 5 °C min−1 to 320 °C; transfer line 300 °C; (100) [M]+, 149 (38), 135 (14), 133 (26), 132 (20), 121 (10),
electron energy 70 eV. Retention indices (I) were determined 118 (10), 105 (18), 104 (17), 91 (33), 90 (13), 89 (13), 77 (19).
from a homologous series of n-alkanes (C8–C38).
2-Methoxy-6-methylbenzaldehyde. Yield: 27 mg (0.18 mmol,
Preparation of methoxy-methylbenzalde-
hydes
72%), colourless solid. TLC (cyclohexane/ethyl acetate 10:1):
Rf = 0.29; 1H NMR (400 MHz, CDCl3, TMS): δ 10.64 (s,
To a solution of the hydroxy-methylbenzaldehyde (34 mg, CHO), 7.37 (dd, 3JH,H = 8.4, 7.6 Hz, 1H, CH), 6.83 (d, 3JH,H =
0.25 mmol) in anhydrous DMF (6.0 mL), K2CO3 (35 mg, 8.4 Hz, 1H, CH), 6.79 (dq, 3JH,H = 7.6 Hz, 4JH,H = 0.8 Hz, 1H,
0.25 mmol) was added and the mixture was stirred at room tem- CH), 3.89 (s, 3H, OCH3), 2.56 (s, 3H, CH3) ppm; 13C NMR
perature for 30 minutes. Then, methyl iodide (30 μL, 68 mg, (100 MHz, CDCl3, TMS) δ 192.4 (CHO), 163.3 (Cq), 142.1
0.5 mmol) was added and the reaction was stirred at room tem- (Cq), 134.6 (CH), 124.2 (CH), 123.5 (Cq), 109.2 (CH), 55.9
perature overnight. The reaction was quenched by the addition (OCH3), 21.6 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (100)
of distilled water, and the aqueous phase was extracted three [M]+, 151 (10), 149 (47), 135 (16), 134 (6), 133 (27), 132 (13),
times with ethyl acetate. The combined organic phases were 119 (5), 118 (15), 105 (13), 104 (7), 91 (30), 90 (19), 89 (12),
dried with MgSO4 and concentrated in vacuo. The residue was 79 (6), 78 (9), 77 (14).
purified by column chromatography on silica gel.
3-Methoxy-2-methylbenzaldehyde. Yield: 27 mg (0.18 mmol,
2-Methoxy-3-methylbenzaldehyde. Yield: 26 mg (0.17 mmol, 72%), pale yellow oil. TLC (cyclohexane/ethyl acetate 10:1):
69%), colourless oil. TLC (cyclohexane/ethyl acetate 10:1): Rf = 0.24; 1H NMR (400 MHz, CDCl3, TMS) δ 10.64 (d, 4JH,H
Rf = 0.18; 1H NMR (400 MHz, CDCl3, TMS) δ 10.38 (d, 4JH,H = 0.6 Hz, 1H, CHO), 7.37 (dd, 3JH,H = 8.4, 7.6 Hz, 1H, CH),
= 0.8 Hz, 1H, CHO), 7.68 (d, 3JH,H = 7.7 Hz, 1H, CH), 7.44 (d, 6.83 (d, 3JH,H = 8.4 Hz, 1H, CH), 6.80 (d, 3JH,H = 7.6 Hz, 1H,
3JH,H = 7.4 Hz, 1H, CH), 7.13 (dd, 3JH,H = 7.7, 7.4 Hz, 1H, CH), 3.89 (s, 3H, OCH3), 2.56 (s, 3H, CH3) ppm; 13C NMR
CH), 3.88 (s, 3H, OCH3), 2.34 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3, TMS) δ 192.4 (CHO), 163.4 (C), 142.1 (C),
(100 MHz, CDCl3, TMS) δ 190.4 (CHO), 161.8 (Cq), 137.7 134.6 (CH), 124.2 (CH), 123.5 (C), 109.2 (CH), 55.9 (OCH3),
(CH), 132.4 (Cq), 129.3 (Cq), 126.6 (CH), 124.5 (CH), 63.2 21.6 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (100) [M]+, 151
(OCH3), 15.6 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (100) (9), 149 (40), 135 (5), 121 (15), 120 (12), 119 (10), 107 (8), 105
[M]+, 149 (31), 135 (29), 134 (10), 133 (49), 132 (52), 121 (10), (8), 91 (39), 79 (7), 78 (7), 77 (20), 51 (5).
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3-Methoxy-4-methylbenzaldehyde. Yield: 35 mg (0.23 mmol, Rf = 0.15; 1H NMR (400 MHz, CDCl3, TMS) δ 9.84 (s, 1H,
91%), colourless solid. TLC (cyclohexane/ethyl acetate 10:1): CHO), 7.70 (dd, 3JH,H = 8.4 Hz, 4JH,H = 2.2 Hz, 1H, CH), 7.67
Rf = 0.18; 1H NMR (400 MHz, CDCl3, TMS) δ 9.92 (s, 1H, (d, 4JH,H = 1.8 Hz, 1H, CH), 6.91 (d, 3JH,H = 8.4 Hz, 1H, CH),
CHO), 7.36 (dd, 3JH,H = 7.5 Hz, 4JH,H = 1.5 Hz, 1H, CH), 7.33 3.90 (s, 3H, OCH3), 2.25 (s, 3H, CH3) ppm; 13C NMR
(d, 4JH,H = 1.4 Hz, 1H, CH), 7.29 (d, 3JH,H = 7.5 Hz, 1H, CH), (100 MHz, CDCl3, TMS) δ 191.3 (CHO), 163.0 (Cq), 131.6
3.89 (s, 3H, OCH3), 2.29 (s, 3H, CH3) ppm; 13C NMR (CH), 130.8 (CH), 129.6 (Cq), 127.8 (Cq), 109.8 (CH), 55.8
(100 MHz, CDCl3, TMS) δ 192.1 (CHO), 158.5 (Cq), 136.0 (OCH3), 16.3 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (69)
(Cq), 135.0 (Cq), 131.0 (CH), 124.6 (CH), 108.0 (CH), 55.6 [M]+, 151 (6), 149 (100), 121 (7), 106 (3), 91 (17), 89 (2), 78
(OCH3), 17.0 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (100) (3), 77 (8), 65 (2).
[M]+, 151 (9), 149 (89), 135 (4), 122 (4), 121 (22), 106 (4), 91
Synthesis of 5-hydroxy-2-methyl-4H-
chromen-4-one (19)
(25), 89 (3), 79 (3), 78 (5), 77 (13), 65 (4), 51 (3), 44 (3).
3-Methoxy-5-methylbenzaldehyde. Yield: 28 mg (0.19 mmol, According to Okombi et al. [27], potassium carbonate (1.38 g,
75%), pale yellow oil. TLC (cyclohexane/ethyl acetate 10:1): 10.0 mmol) was added to a solution of 2,6-dihydroxyacetophe-
Rf = 0.23; 1H NMR (400 MHz, CDCl3, TMS) δ 9.92 (s, 1H, none (23, 304 mg, 2.0 mmol) in acetone (10 mL), and the mix-
CHO), 7.26 (s, 1H, CH), 7.19 (br s, 1H, CH), 6.98 (m, 1H, CH), ture was stirred at room temperature for 15 minutes. Then,
3.84 (s, 3H, OCH3), 2.39 (d, 4JH,H = 0.9 Hz, 3H, CH3) ppm; acetyl chloride (157 mg, 2.0 mmol) was added and stirring was
13C NMR (100 MHz, CDCl3, TMS) δ 192.5 (CHO), 160.3 (Cq), continued under reflux for 24 hours. The reaction was cooled to
140.5 (Cq), 137.9 (Cq), 124.5 (CH), 122.3 (CH), 109.6 (CH), room temperature and hydrolysed by the addition of distilled
55.6 (OCH3), 21.3 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 water. The aqueous layer was extracted three times with diethyl
(100) [M]+, 151 (9), 149 (85), 122 (5), 121 (33), 119 (3), 106 ether. The collected organic phases were dried over MgSO4 and
(3), 105 (2), 92 (2), 91 (16), 89 (2), 79 (3), 78 (4), 77 (10), 65 concentrated in vacuo. The residue was purified by column
(3), 63 (2), 51 (2).
chromatography on silica gel to afford the title compound 19
(37 mg, 0.21 mmol, 11%) and 3-acetyl-5-hydroxy-2-methyl-
3-Methoxy-6-methylbenzaldehyde. Yield: 21 mg (0.14 mmol, 4H-chromen-4-one (24) as main product (154 mg, 0.71 mmol,
56%), colourless oil. TLC (cyclohexane/ethyl acetate 10:1): 35%). Both compounds were obtained as pale yellow solid.
Rf = 0.19; 1H NMR (400 MHz, CDCl3, TMS) δ 10.27 (s, 1H,
CHO), 7.32 (d, 4JH,H = 2.9 Hz, 1H, CH), 7.16 (d, 3JH,H = 8.4 5-Hydroxy-2-methyl-4H-chromen-4-one (19). TLC (hexane/
Hz, 1H, CH), 7.04 (dd, 3JH,H = 8.4 Hz, 4JH,H = 2.9 Hz, 1H, ethyl acetate/toluene, 3:1:1): Rf = 0.36; GC (HP-5): I = 1591;
CH), 3.84 (s, 3H, OCH3), 2.60 (s, 3H, CH3) ppm; 13C NMR 1H NMR (400 MHz, CDCl3, TMS) δ 12.54 (s, 1H, OH), 7.48
(100 MHz, CDCl3, TMS) δ 192.2 (CHO), 158.2 (Cq), 134.8 (dd, 3JH,H = 8.5, 8.3 Hz, 1H, CH), 6.84 (dd, 3JH,H = 8.5 Hz,
(Cq), 133.0 (Cq), 132.9 (CH), 120.9 (CH), 114.2 (CH), 55.6 4JH,H = 0.8 Hz, 1H, CH), 6.76 (dd, 3JH,H = 8.3 Hz, 4JH,H = 0.8
(OCH3), 18.2 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (100) Hz, 1H, CH), 6.09 (s, 1H, CH), 2.38 (s, 3H, CH3) ppm; 13C
[M]+, 151 (9), 149 (45), 135 (4), 122 (9), 121 (68), 119 (2), 107 NMR (100 MHz, CDCl3) δ 183.6 (Cq), 167.7 (Cq), 160.9 (Cq),
(7), 106 (2), 92 (2), 91 (13), 89 (3), 79 (4), 78 (6), 77 (14), 65 156.9 (Cq), 135.2 (CH), 111.3 (CH), 110.6 (Cq), 109.3 (CH),
(3), 63 (2), 51 (3).
106.9 (CH), 20.8 (CH3) ppm; IR (ATR) v: 3075 (w), 2970 (w),
2930 (w), 2850 (w), 2781 (w), 1664 (s), 1622 (s), 1596 (s),
4-Methoxy-2-methylbenzaldehyde. Yield: 32 mg (0.21 mmol, 1465 (m), 1404 (s), 1377 (s), 1305 (s), 1250 (s), 1228 (s), 1183
84%), pale yellow oil. TLC (cyclohexane/ethyl acetate 10:1): (m), 1158 (m), 1110 (m), 1061 (m), 1002 (m), 953 (s), 867 (m),
Rf = 0.18; 1H NMR (400 MHz, CDCl3, TMS) δ 10.11 (s, 1H, 838 (m), 799 (s), 745 (s), 741 (m), 681 (m), 617 (m), 587 (s)
CHO), 7.75 (d, 3JH,H = 8.6 Hz, 1H, CH), 6.84 (dd, 3JH,H = 8.5 cm−1; UV–vis (CH2Cl2): λmax (lg ε) = 325 (4.64), 253 (5.08),
Hz, 4JH,H = 2.5 Hz, 1H, CH), 6.74 (d, 4JH,H = 2.5 Hz, 1H, CH), 230 (5.30) nm; MS (EI, 70 eV) m/z (%): 176 (100) [M]+, 148
3.86 (s, 3H, OCH3), 2.65 (s, 3H, CH3) ppm; 13C NMR (32), 147 (26), 136 (22), 108 (37), 91 (10), 39 (12).
(100 MHz, CDCl3, TMS) δ 191.3 (CHO), 163.8 (Cq), 143.4
(Cq), 134.9 (CH), 128.1 (Cq), 117.1 (CH), 111.6 (CH), 55.6 3-Acetyl-5-hydroxy-2-methyl-4H-chromen-4-one (24). TLC
(OCH3), 20.0 (CH3) ppm; MS (EI, 70 eV) m/z (%): 150 (65) (hexane/ethyl acetate/toluene, 3:1:1): Rf = 0.33; GC (HP-5): I =
[M]+, 151 (6), 149 (100), 122 (3), 121 (17), 106 (2), 91 (12), 89 1800; 1H NMR (400 MHz, CDCl3, TMS) δ 12.42 (s, 1H, OH),
(2), 78 (4), 77 (9), 63 (2), 51 (3).
7.52 (dd, 3JH,H = 8.4, 8.3 Hz, 1H, CH), 6.86 (dd, 3JH,H = 8.4
Hz, 4JH,H = 0.9 Hz, 1H, CH), 6.80 (dd, 3JH,H = 8.3 Hz, 4JH,H =
4-Methoxy-3-methylbenzaldehyde. Yield: 32 mg (0.21 mmol, 0.9 Hz, 1H, CH), 2.62 (s, 3H, CH3), 2.52 (s, 3H, CH3) ppm; 13C
84%), colourless solid. TLC (cyclohexane/ethyl acetate 10:1): NMR (100 MHz, CDCl3) δ 199.3 (Cq), 181.5 (Cq), 170.4 (Cq),
744

Beilstein J. Org. Chem. 2018, 14, 734–746.
13.Pažoutová, S.; Follert, S.; Bitzer, J.; Keck, M.; Surup, F.; Šrůtka, P.;
Holuša, J.; Stadler, M. Fungal Diversity 2013, 60, 107–123.
doi:10.1007/s13225-013-0238-5
161.1 (Cq), 155.7 (Cq), 135.9 (CH), 122.3 (Cq), 112.2 (CH),
110.5 (Cq), 106.9 (CH), 32.4 (CH3), 20.2 (CH3) ppm; IR (ATR)
v: 3073 (w), 2965 (w), 2925 (w), 2851 (w), 1692 (s), 1642 (s),
1601 (s), 1503 (m), 1469 (s), 1409 (s), 1376 (m), 1348 (m),
1296 (s), 1213 (s), 1164 (m), 1129 (m), 1078 (m), 1057 (m),
1036 (m), 994 (m), 953 (m), 881 (m), 861 (m), 812 (s), 756 (s),
707 (s), 650 (m), 634 (m), 593 (m), 531 (m) cm−1; UV–vis
(CH2Cl2) λmax (lg ε): 328 (4.65), 241 (5.17), 228 (5.19) nm;
MS (EI, 70 eV) m/z (%): 218 (81) [M]+, 204 (13), 203 (100),
137 (53), 136 (17), 108 (21), 67 (26), 43 (23), 39 (13).
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Acknowledgements
This work was funded by the Deutsche Forschungs-
gemeinschaft (DI1536/9-1) and by the State of Lower Saxony
with a Ph.D. scholarship in the frame of the MINAS graduate
school (to TW). We thank Stefanie Follert and Matthias Keck
(former members of the HZI research department Microbial
Drugs) for their preliminary work on the volatiles of selected
Xylariales in preparation for the current study.
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ORCID® iDs
Jeroen S. Dickschat - https://orcid.org/0000-0002-0102-0631
Marc Stadler - https://orcid.org/0000-0002-7284-8671
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