Volatiles from the fungal microbiome of the marine sponge: Callyspongia cf. flammea

Article abstract of DOI:10.1039/c7ob01837a

The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf. flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides several widespread compounds, a series of metabolites with interesting bioactivities were found, including the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and chartabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors and its absolute configuration was determined by enantioselective synthesis of a reference compound. Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential ecological function in sponge defence.

Full text of DOI:10.1039/c7ob01837a

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Volatiles from the fungal microbiome of the
marine sponge Callyspongia cf. flammea†
Cite this: DOI: 10.1039/c7ob01837a
Lena Barra,a Paul Barac,b Gabriele M. König,b Max Crüsemannb and
a
Jeroen S. Dickschat
*
The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf.
flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides
several widespread compounds, a series of metabolites with interesting bioactivities were found, including
the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the
insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and char-
tabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The
biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors
and its absolute configuration was determined by enantioselective synthesis of a reference compound.
Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential
ecological function in sponge defence.
Received 25th July 2017,
Accepted 30th August 2017
DOI: 10.1039/c7ob01837a
rsc.li/obc
the protein phosphatases 1 and 2A.9 Notably, this polyketide is
produced by the symbiotic bacterium Entotheonella and gener-
Introduction
Marine sponges (Porifera) belong to the most ancient living ated enzymatically from the pro-toxin phosphocalyculin (3)
animals that have evolved during the Cryogenian period (>635 upon tissue wounding (Fig. 1).10 The fungus Stachylidium
Myr ago), well before the Cambrian explosion.1 They are sp. 293 K04 that was isolated from the marine sponge
sessile, filter-feeding organisms, that occur in almost any Callyspongia cf. flammea produces the human leukocyte elas-
aquatic habitat from tropical reefs to the polar regions, and tase inhibitor mariline A (4) as a mixture of enantiomers.11
from the deep sea to fresh water lakes and rivers.2 They exhibit The tetrapeptide endolide A (5) containing an unusual 3-furyl-
highly variable and complex microbiomes consisting of bac- alanine subunit was isolated from the same fungus and
teria, fungi and archaea3 that can account for up to 40% of the showed affinity to the vasopressin receptor 1A.12
sponge tissue volume.4 This microbial diversity requires
In contrast to such natural products that are typically iso-
complex interactions between the symbionts and their host lated by classical extraction–isolation techniques, volatile
that are mediated by secondary metabolites with often strong metabolites are often overlooked, because these compounds
biological activities and remarkable structural architectures.5 A are easily lost during concentration steps.13 Still, knowledge
well-known example is the pyrrole-imidazole alkaloid sceptrin about these compounds is desirable, because volatiles can
(1) from the Caribbean sponge Agelas sceptrum6 that shows exhibit antibiotic properties or can be of critical importance
antimicrobial, anti-muscarinic and anti-histaminic activities, e.g. for interspecies communication, cell-to-cell signaling, or
besides a strong inhibition of cancer cell motility,7 but the control of pathogens14 which is of particularly high relevance
function of this compound in its ecological context remains in complex microbial communities as they occur in sponges.
elusive.8 Calyculin A (2) from the marine sponge Discodermia Despite these considerations studies on volatiles from sponges
calyx is a highly cytotoxic compound and a potent inhibitor of or their microbiomes are surprisingly rare and so far limited
to investigations on bacterial symbionts. Among the few
examples is a report on the North Sea sponge Halichondria
aKekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn,
Gerhard-Domagk-Straße 1, D-53121 Bonn, Germany. E-mail: dickschat@uni-bonn.de
bInstitute of Pharmaceutical Biology, University of Bonn, Nußallee 6, D-53115 Bonn,
Germany
panacea that emits a strong nauseating odour that can be
traced back to a mixture of the sulfur compounds dimethyl di-
sulfide, dimethyl trisulfide and methyl benzyl sulfide.15
Sponges of the genus Ircinia were found to release dimethyl
sulfide, methyl isocyanide and methyl isothiocyanate that may
act as chemical defense compounds.16 A series of studies on
symbiotic bacteria from Antarctic sponges demonstrated the
†Electronic supplementary information (ESI) available: Table of identified vola-
tiles, mass spectra, gas chromatograms for headspace extracts from Sporomiella
and Botrytis, NMR spectra of synthetic compounds. See DOI: 10.1039/
c7ob01837a
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identification of indoloditerpenes,21 unusual steroids, aro-
matic isocyanides,22 gliotoxins and heveadrides.23 Analysis of
the headspace extracts obtained from this fungus also revealed
a rich production of structurally diverse volatiles (Fig. 2). The
most abundant compound was identified as the sesquiterpene
alcohol (1(10)E,5E)-germacradien-11-ol (14), a known inter-
mediate in geosmin biosynthesis, found in Streptomyces
spp.,24 myxobacteria25 and liverworts.26 Other identified terpe-
noids are dauca-4(11),8-diene (13), which was previously iso-
lated from the liverwort Bazzania trilobata27 and the acyclic ter-
penoid geranylacetone (12). The acrid plant defense com-
pound protoanemonin (6) was found in minor amounts and
has previously been described from plants of the buttercup
family (Ranunculaceae) where it is enzymatically generated
from the glucoside ranunculin upon lesion of plant tissue.28
Protoanemonin was also found as a catabolite of the xeno-
Fig. 1 Secondary metabolites from sponges and sponge associated
bacteria and fungi. Structures of sceptrin (1), calyculin A (2), phosphoca-
lyculin (3), mariline A (4) and endolide A (5).
production of volatiles with activity against Burkholderia.17
Here we present the first study on volatile metabolites pro-
duced by sponge associated fungi that were previously iso-
lated18 from Callyspongia cf. flammea.
Results and discussion
The volatile metabolites released by Dichotomomyces cejpii
293 K09, Stachylidium sp. 293 K04, Botrytis sp. 293 K02,
Emericella sp. 293 K10, and Sporormiella sp. 293 K05 that were
all isolated from the marine sponge Callyspongia cf. flammea18
were collected on a charcoal filter by use of a closed-loop strip-
ping apparatus (CLSA).19 Sampling was conducted for 16–24 h
and the adsorbed volatiles were eluted with dichloromethane.
The obtained headspace extracts were analyzed by GC-MS and
compounds were identified by comparison of their mass spectra
to electronic mass spectral libraries20 and comparison of their
measured retention indices to reported data, with additional
verification by comparison to synthetic or commercially available
authentic standards in most cases. A summary of all 48 identi-
fied compounds is given in Table S1.† Two of the examined
fungi, Sporormiella and Botrytis, did not show a significant emis-
sion of volatiles (Fig. S1 and S2†). The results obtained from the
other three isolates are discussed here in detail.
Fig. 2 Volatiles from Dichotomomyces cejpii. Total ion chromatogram
of the headspace extract, structures of the identified volatiles and of tor-
quatone (18) and of C-desmethyl isotorquatone (19). Peak numbers in
the chromatogram refer to compound numbers. The absolute configur-
ations of 13, 14, 16 and 17 have not been determined, cf. main text for
Dichotomomyces cejpii 293 K09
The ascomycete D. cejpii 293 K09 has been intensively investi-
gated for its rich secondary metabolism, which resulted in the the absolute configuration of 15.
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biotic 4-chlorocatechol in Pseudomonas.29 Recently, 6 was mixture of enantiomers ((40% ee in favour of (R)-(−)-15),
shown to act as a quorum sensing inhibitor towards N-acyl- Fig. S5†). Isotorquatone is a known constituent of the essential
L-homoserine lactones (AHLs) in Pseudomonas sp. B13.30 The oils of various Eucalyptus species,35 but the absolute configu-
production of 6 by D. cejpii is interesting, as AHLs were shown ration of the plant-derived isotorquatone is unknown.
to play a key role in the interaction between sponge symbiotic
bacteria and their host.31 Additionally, protoanemonin exhi- trace compounds whose mass spectra showed a molecular ion
bits antifungal and antibacterial properties.32
at m/z = 266 and a base peak ion at m/z = 209 (Fig. S6†),
The headspace extract of D. cejpii contained two additional
Other detected metabolites included the unbranched long- suggesting the structures of desmethyl analogs of 15 with a
chain aldehydes nonanal (7), decanal (8) and dodecanal (9), missing methyl group at one of the oxygens or ring carbons.
and the benzene derivatives benzyl cyanide (10) and 3,4- For an unambiguous structural assignment all three possible
dimethoxystyrene (11) which are all frequently observed fungal derivatives were synthesized. The C-desmethyl analog 19 was
volatiles.14c A particularly interesting compound, detected as prepared from 21 via the same strategy as described for torqua-
the second most abundant volatile in the headspace extract of tone and isotorquatone, by omission of the methylation step
D. cejpii, was the highly methylated aromatic polyketide 15 to 22. Treating (rac)-15 with BBr3 yielded the O-desmethyl
along with its O-desmethyl derivatives 16 and 17. Comparison analog 16, in addition to the doubly O-demethylated com-
of the mass spectrum of 15 to data base spectra returned the pound 25, but no O-demethylation of the methoxy group in
two possible structures of torquatone (18) and isotorquatone para-position to the ketone side chain was observed.
(15). Since the mass spectra and retention indices of both com- Therefore, a published synthesis for 17 over 9 steps starting
pounds are nearly identical (Fig. S3†), a synthesis for both from 1,3,5-trimethoxybenzene was conducted (Scheme 2).33
isomers was performed to allow for an unambiguous structural Comparison of the three synthetic compounds to the natural
assignment. Two successive methylations of 1,3,5-trimethoxy- volatiles by GC-MS resulted in the identification of 16 and 17,
benzene (20) with methyl iodide gave access to 22 that was bro- but not of 19 as headspace constituents of D. cejpii. The isotor-
minated with NBS to yield 23.33 Subsequent lithiation, quatone derivative 16 (chartabomone) is known from
addition to 2-methylbutyraldehyde, and oxidation with Dess– Eucalyptus chartaboma and E. miniata,35b while 17 is a new
Martin periodinane resulted in 15 (Scheme 1). Compound 18 natural product that we wish to name dichotomone.
was prepared analogously from 23 and isovaleraldehyde.
The biosynthesis of 15 was addressed in feeding experi-
Both synthetic compounds 15 and 18 were compared to the ments with isotopically labeled (2-13C)acetate and (methyl-2H3)-
volatile emitted by D. cejpii, revealing the identity of 15 and the L-methionine. Feeding of (2-13C)acetate resulted in the incor-
natural material by GC-MS analysis (Fig. S4†). To determine poration of five labeled C2-units into 15 as indicated by the
the absolute configuration of natural 15, a stereoselective syn- resulting mass spectrum, and feeding of (methyl-2H3)-L-meth-
thesis was performed using the same synthetic route as ionine gave incorporation of labeling into six methyl groups
described above and employing enantiomerically pure (S)-2- (Fig. S7†). These findings are in line with a biosynthesis of 15
methylbutyraldehyde.34 The obtained material (S)-(+)-15 (98% ee)
and the racemate of 15 were used to determine the absolute
configuration of natural isotorquatone by HPLC on
a
homochiral stationary phase, indicating that natural 15 is a
Scheme 1 Synthesis of isotorquatone (15) and torquatone (18). (a)
s-BuLi, 40 °C, 2 h; then MeI, −78 °C to rt, 70%; (b) s-BuLi, 40 °C, 2 h;
then MeI, −78 °C to rt, 82%; (c) NBS, rt, 82%; (d) t-BuLi, −78 °C to
−10 °C; then aldehyde RCHO, −78 °C, 30 min; (e) DMP, rt, 63% for 15,
50% for 18 (2 steps). BuLi butyllithium, MeI methyl iodide, NBS
N-bromosuccinimide, DMP Dess–Martin periodinane.
Scheme 2 Synthesis of (A) C-desmethyl isotorquatone (19), (B) charta-
bomone (16), and (C) dichotomone (17). (a) NBS, 0 °C to rt, 97%; (b)
t-BuLi, −78 °C to −10 °C, then 2-methylbutyraldehyde, −78 °C, 30 min;
(c) IBX, rt, 28% (2 steps); (d) BBr3 (1.1 equiv.), −78 °C to rt, 35% of 16 and
52% of 25. IBX 2-iodoxybenzoic acid.
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by a polyketide synthase (PKS) from an acetate starter unit by
four extension steps with malonyl-CoA, with full reduction of
the 3-oxo function during the first and no reduction during
the following chain extensions, and C-methylation during the
first, third and last chain elongation (Scheme 3). Product
release by a Claisen condensation and three O-methylations
yield 15. The presence of minor amounts of 16 and 17 suggests
that the O-methyltransferase does not convert the complete
material released from the PKS. In contrast to this finding, the
polyketide synthase acts in a highly programmed fashion, as
indicated by the absence of 19 which would represent a PKS
product formed through an omitted C-methylation step.
Stachylidium sp. 293 K04
Besides mariline A (4) and endolide A (5),11,12 a variety of other
bioactive natural products has been isolated from Stachylidium
sp. 293 K04, including the marilones A–C and the stachylines
A–D.36–38 Analysis of the volatiles from Stachylidium resulted in
the identification of 14 compounds (Fig. 3). The most abun-
dant compound was 1,2,4-trimethoxybenzene (44) that is a
known constituent of the bouquet of orchid flowers39 and
Cucurbita blossoms where it acts together with indole and (E)-
cinnamaldehyde as an attractant for rootworm beetles.40 The
related compound 1,2-dimethoxybenzene (41) is only found in
traces and is also known from Aspergillus clavatus.41 The
sulphur compound benzothiazole (43), also a minor com-
ponent, has previously been reported from Trichoderma42 and
Aspergillus.41 Among the major components 1-octen-3-ol (38)
Fig. 3 Volatiles from Stachylidium sp. 293 K04. Total ion chromatogram
of the headspace extract of Stachylidium sp. 293 K04 and structures of the
identified volatiles. Asterisks indicate compounds originating from the
medium. Peak numbers in the chromatogram refer to compound numbers.
was identified that is probably the most widespread fungal
volatile. This compound is frequently accompanied by other
C8 metabolites such as 3-octanone (39).14c “Matsutake alcohol”
38 was first isolated from Tricholoma matsutake43 and its role
in chemical denfense is suggested by the triggered emission
upon wounding of fungal fruiting bodies.44
A rather unusual metabolite released by Stachylidium is
furan-3-carbaldehyde (34) whose production may be linked to
the biosynthesis of endolide A (5), a natural product that is
made up from the structurally related uncommon amino acid
3-furylalanine.45 The production of terpenes including nerolidol
(40), 2-methylisoborneol (42) and geranyl acetone (12) was also
observed. 2-Methylisoborneol is a widespread off-flavour with
a characteristic mouldy odor that is commonly found in
various bacteria24c and ascomycete fungi such as Aspergillus46
und Penicillium.47 Other identified compounds are butyl
acetate (35), 2-hydroxy-5-methylhexan-3-one (36), 4-methyl-
γ-butyrolactone (37) and the long chain aldehydes 7 and 8.
Emericella sp. 293 K02
The fungus Emericella sp. 293 K02 has not been investigated
Scheme 3 Biosynthetic model for 15 and labeling pattern after feeding
of (2-13C)acetate and (methyl-2H3)-L-methionine. AT = acyl transferase, with respect to its secondary metabolism. The headspace
C-MT = C-methyltransferase, O-MT = O-methyltransferase, DH = de-
hydratase, KR = ketoreductase, ER = enoylreductase, KS = ketosynthase,
extracts contained a large number of compounds, twelve of
which could be identified including the aldehydes 7, 8, 9 and
the higher homolog dodecanal (48), matsutake alcohol (38)
and its companion 39, and benzothiazole (43, Fig. 4). A unique
compound emitted by Emericella was 3,4-dimethylpentan-4-
CLC
= claisen condensation domain. Asterisks indicate completely
deuterated carbons and black dots indicate 13C-labeling carbons.
The indicated positions for incorporation of labelling are based on
mechanistic considerations for PKS biosynthesis.
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Table 1 Algicidal activity of fungal volatiles against Chlorella fusca (80%
inhibition)
Compound
MICa [μg mL−1
]
(rac)-15
(S)-15
16
31.3
125.0
31.3
17
18
19
>125.0
>125.0
2.0
25
15.6
1.0
<31.3
2,4-Dichlorophenolb
CuSO4·5H2Ob
a Minimal inhibitory concentration. b Positive control.
olide (47). This lactone was previously described as a phyto-
toxin that is possibly involved in the pathogenicity of the
fungus Hymenoscyphus pseudoalbidus, the causative agent of
European ash dieback. Compound testings in the laboratory
revealed a strong inhibitory activity of lactone 47 against ash
seed germination.48 H. pseudoalbidus produces mainly the
R enantiomer with 80% ee. Notably, the material emitted by
Emericella is also a 1 : 10 mixture of enantiomers, but in this
species (S)-47 is dominant (Fig. 5).
Fig. 4 Volatiles from Emericella sp. 293 K02. Total ion chromatogram
of the headspace extract of Emericella sp. and structures of the ident-
ified volatiles. Peak numbers in the chromatogram refer to compound
numbers.
Bioactivity tests
A selection of fungal volatiles identified during the course of
this study were tested for their bioactivities against bacteria
and algae. While all the tested compounds showed no or only
moderate activity against Gram-positive (Bacillus megaterium)
and Gram-negative (Escherichia coli) bacteria (Table S2†),
the racemate of 15 showed growth inhibitory effects towards
the alga Chlorella fusca (Table 1). The S enantiomer was much
less active, suggesting that the growth inhibition is mainly due
to the R enantiomer in the racemate. Notably, the isomer
torquatone (18) was inactive. The O-desmethyl derivative
chartabomone 16 revealed a similarly strong algicidal activity
as (rac)-15, while no activity was observed for the newly
identified dichotomone 17. The strongest bioactivity was
detected for the synthetic derivatives 19 and 25 (Table 1).
Conclusions
In the present study we describe the first analysis of volatiles
released by sponge-associated fungi that were previously iso-
lated from Callyspongia cf. flammea. The analyses revealed the
production of several volatiles with interesting bioactivities,
including a small family of polymethylated polyketides with
the main compound isotorquatone (15). The observed algicical
activity of racemic 15 and its O-desmethyl derivative 16 may
point to their involvement in protecting the sponge from algal
infections, but it is difficult to judge to which extent the
results from laboratory bioactivity experiments can be used to
assign a function to these compounds in a complex ecosystem
such as a sponge and its microbiome in their natural habitat.
The occurrence of compounds such as the quorum sensing
Fig. 5 Extracted ion chromatograms (m/z = 113) of (A) a pseudoracemic
mixture of synthetic (S)- and (R)-47, (B) synthetic (R)-47, (C) synthetic
(S)-47, (D) a headspace extract from Emericella, (E) headspace extract
spiked with synthetic (S)-47, and (F) headspace extract spiked with
synthetic (R)-47.
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disruptor protoanemonine (6) is also noteworthy and suggests emitted volatiles of the grown cultures were captured by use of a
a potential involvement of fungal volatiles in the intra- and CLSA and the crude headspace extracts were analyzed by GC-MS.
interspecies communication between the multiple micro-
General synthetic and analytical methods
organisms in the rich sponge microbiomes. Further research
will be required to deepen our understanding of the role of All chemicals were obtained from Acros Organics (Geel,
fungal volatiles in the complex interactions between sponges Belgium), Sigma Aldrich Chemie GmbH (Steinheim, Germay)
and their microbial symbionts.
or TCI Deutschland GmbH (Eschborn, Germany). All solvents
were purified by distillation. Whenever necessary, reactions
were carried out under inert atmosphere (Ar) using vacuum-
heated flasks and dried solvents (dried according to standard
protocols). Thin layer chromatography (TLC) was performed
on 0.20 mm silica plates (Polygram SIL G/UV254) obtained
from Macherey–Nagel (Düren, Germany). Column chromato-
graphy was performed on Merck silica gel (0.040–0.063 Mesh).
NMR spectra were recorded on Bruker AV I (400 MHz), AV III
HD Prodigy (500 MHz) and AV III HD Cryo (700 MHz) spec-
trometers, and were referenced against CDCl3 (δ = 7.26 ppm)
and C6D6 (δ = 7.16 ppm) for 1H-NMR, and CDCl3 (δ =
77.01 ppm) and C6D6 (δ = 128.06 ppm) for 13C-NMR. The
multiplicities are specified as follows: singlet (s), doublet (d),
triplet (t), quartet (q), quintet (quin), sextet (sex), septet (sept).
GC-MS analyses were carried out with an Agilent HP7890B gas
chromatograph connected to a HP5977A mass detector fitted
with a HP-5MS silica capillary column (30 m, 0.25 mm i.d.,
0.50 μm film). The GC-MS conditions were as follows: (1) inlet
pressure: 77.1 kPa, He flow 23.3 mL min−1; (2) injection
volume: 1 μL; (3) injection mode: split 50 : 1, valve time 60 s;
(4) oven temperature ramp: 5 min at 50 °C increasing at 10 °C
min−1 to 320 °C; (5) carrier gas He at 1 mL min−1; (6) transfer
line: 250 °C; (7) electron energy: 70 eV. Retention indices (I)
were determined from a homologous series of n-alkanes
(C8–C40). Optical rotary powers were recorded on a P8000
Polarimeter (Krüss). UV/Vis spectra were recorded on a Cary
100 UV/Vis spectrometer (Agilent). IR Spectra were measured
by use of an Alpha FT-IR spectrometer from Bruker.
Experimental
Fungal strains and growth conditions
The fungal strains investigated in this study (Table 2) were pre-
viously isolated from
a marine sample of the sponge
Callyspongia cf. flammea, collected on Bear Island, Sydney,
Australia as reported in ref. 18. In order to obtain the volatiles,
all strains were cultivated on biomalt salt medium, in 100 mm
× 15 mm Petri dishes, for 14 days, at room temperature.
CLSA sampling
The volatiles were collected by use of a closed-loop stripping
apparatus (CLSA).18 Sampling was conducted for 16 to 24 h at
room temperature (20 °C) and under natural day and night
light conditions. The charcoal filter was extracted with di-
chloromethane (50 μL) and the resulting headspace extracts
were directly analyzed by GC-MS. All headspace samplings
were carried out in duplicates.
GC-MS analysis of headspace extracts
The crude headspace extracts were analyzed by use of an
Agilent HP7890B gas chromatograph, fitted with a HP-5MS
silica capillary column (30 m, 0.25 mm i.d., 0.50 μm film), con-
nected to a HP5977A mass detector. The GC-MS conditions
were as follows: (1) inlet pressure: 77.1 kPa, He flow 23.3 mL
min−1; (2) injection volume: 1 μL; (3) injection mode: splitless,
valve time 60 s; (4) oven temperature ramp: 5 min at 50 °C
increasing at 5 °C min−1 to 320 °C; (5) carrier gas He at 1 mL
min−1; (6) transfer line: 250 °C; (7) electron energy: 70 eV.
Retention indices (I) were determined from a homologous
series of n-alkanes (C8–C40).
Synthetic procedures
1,3,5-Trimethoxy-2-methylbenzene (21). To a solution of
1,3,5-trimethoxybenzene (20) (5.00 g, 29.7 mmol, 1.0 equiv.) in
80 mL dry THF was slowly added a 1.4 M solution of s-butyl-
lithium (31.9 mL, 44.6 mmol, 1.5 equiv.) at room temperature
and stirred for 15 minutes. The reaction was heated to 40 °C
and stirred for 2 h. Afterwards it was cooled to −78 °C and
methyl iodide (7.40 mL, 119 mmol, 4.0 equiv.) was added
slowly. The reaction was allowed to warm to room temperature
and stirred over night. Addition of water was followed by
extraction with Et2O and the combined organic layers were
dried with MgSO4 and the solvent was removed under reduced
pressure. The crude product was purified by column chromato-
graphy on silica gel (cyclohexane/ethyl acetate 30 : 1) and 21
was isolated as a yellowish oil (3.78 g, 20.8 mmol, 70%). Rf =
0.2. GC (HP-5MS): I = 1482. 1H-NMR (500 MHz, CDCl3): δ =
6.15 (s, 2H, CH), 3.81 (s, 9H, CH3), 2.03 (s, 3H, CH3) ppm.
13C-NMR (125 MHz, CDCl3): δ = 159.1 (s, Cq), 158.9 (s, 2 × Cq),
106.9 (s, Cq), 90.7 (2 × CH), 55.8 (2 × CH3), 55.5 (s, CH3), 7.8 (s,
CH3) ppm. IR (ATR): ˜ν = 2997, 2938, 2836, 1594, 1499, 807.
Feeding experiments
Feeding experiments were performed with D. cejpii and isotopi-
cally enriched (2-13C)acetate and (methyl-2H3)-L-methionine. The
labeled compounds were directly added to the liquid agar
medium (10 mM) and D. cejpii was inoculated for 10 days. The
Table 2 Investigated strains from Callyspongia cf. flammea
Strain no.
Taxonomy
293 K02
293 K04
293 K05
293 K09
293 K10
Botrytis sp.
Stachylidium bicolor
Sporomiella sp.
Dichotomomyces cejpii
Emericella sp.
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UV/Vis (MeCN): λmax (lg ε): 271 (4.29), 241 (5.63) nm. EI-MS (70 solvent was removed under reduced pressure. The crude
eV): m/z (%) = 182 (100), 167 (21), 153 (23), 151 (25), 139 (17), product was dissolved in 5 mL dry DCM and DMP (0.36 g,
121 (22), 109 (15), 91 (14), 77 (14), 69 (14). HR-EIMS calcd for 0.84 mmol, 1.0 equiv.) was added. The reaction was stirred
+
C10H14O3 : m/z = 182.0943, found: m/z = 182.0979.
over night at room temperature, quenched with water and
1,3,5-Trimethoxy-2,4-dimethylbenzene (22). To a solution of extracted with Et2O. The crude product was purified by
21 (2.30 g, 12.6 mmol, 1.0 equiv.) in 15 mL dry THF was slowly column chromatography on silica gel (cyclohexane/ethyl
added a 1.4 M solution of s-butyllithium (13.5 mL, 19.0 mmol, acetate 20 : 1). (rac)-Isotorquatone 15 (0.15 g, 0.53 mmol, 63%)
1.5 equiv.) at room temperature and stirred for 15 minutes. was isolated as a colorless oil. Rf = 0.2. GC (HP-5MS): I = 1810.
The reaction was heated to 40 °C and stirred for 2 h. 1H-NMR (500 MHz, CDCl3): δ = 3.72 (s, 3H, CH3), 3.69 (s, 6H,
Afterwards it was cooled to −78 °C and methyl iodide 2 × CH3), 2.93–2.84 (m, 1H, CH), 2.17 (s, 6H, 2 × CH3),
(3.20 mL, 50.5 mmol, 4.0 eq.) was added slowly. The reaction 1.86–1.75 (m, 1H, CHH), 1.45–1.34 (m, 1H, CHH), 1.13 (d, 3H,
3
was allowed to warm to room temperature and stirred over 3J = 6.8 Hz, CH3), 0.94 (t, 3H, J = 7.3 Hz, CH3) ppm. 13C-NMR
night. Addition of water was followed by extraction with Et2O (125 MHz, CDCl3): δ = 209.2 (Cq), 159.0 (Cq), 153.9 (Cq),
and the combined organic layers were dried with MgSO4 and 127.2 (Cq), 121.0 (Cq), 62.5 (2 × CH3), 60.1 (CH3), 49.3 (CH),
the solvent was removed under reduced pressure. The crude 25.2 (CH2), 15.2 (CH3), 11.7 (CH3), 9.4 (CH3) ppm. IR (ATR): ˜ν =
product was purified by column chromatography on silica gel 2997, 2921, 2856, 1652. EI-MS (70 eV): m/z (%) = 280 (9),
(cyclohexane/ethyl acetate 30 : 1) and 22 was isolated as a white 225 (3), 224 (26), 223 (100), 208 (6), 193 (5), 190 (3), 179 (3),
+
crystalline solid (2.04 g, 10.4 mmol, 82%). Rf = 0.2. GC 165 (7), 91 (3). HR-EIMS calcd for C16H24O4 : m/z = 280.1675,
(HP-5MS): I = 1520. 1H-NMR (500 MHz, CDCl3): δ = 6.28 (s, 1H, found: m/z = 280.1658.
CH), 3.83 (s, 6H, 2 × CH3), 3.69 (s, 3H, CH3), 2.11 (s, 6H, 2 ×
(S)-Isotorquatone ((S)-15). Synthesis of (S)-15 was conducted
CH3) ppm. 13C-NMR (125 MHz, CDCl3): δ = 158.0 (s, Cq), 156.8 according to the procedure for (rac)-15. Enantiomerically pure
(s, 2 × Cq), 111.7 (s, 2 × Cq), 91.84 (s, CH), 60.3 (s, CH3), 55.9 (s, (S)-2-methylbutyraldehyde33 was used for the alkylation of 23.
2 × CH3), 8.7 (s, 2 × CH3) ppm. IR (ATR): ˜ν = 2985, 2938, 2840, All recorded spectral data were identical to those for (rac)-15.
1593, 1495, 803. UV/Vis (MeCN): λmax (lg ε): 281 (4.45), 235 [α]2D0 = +4.3 (c 1, EtOH).
(4.79) nm. EI-MS (70 eV): m/z (%) = 197 (12), 196 (100), 181
Analysis of the enantiomeric excess of 15. The enantiomeric
(34), 165 (31), 153 (15), 138 (10), 135 (11), 121 (11), 91 (10), 77 excess of natural 15 and synthetic (S)-15 was determined by
+
(10). HR-EIMS calcd for C11H16O3 : m/z = 196.1099, found: m/z analytical HPLC on a homochiral stationary phase (Fig. S2†)
= 196.1121.
using the following conditions: system: Fa. Knauer GmbH
(Berlin, Germany), 2 pumps P-1 HPLCplus (max. 750 bar),
1-Bromo-2,4,6-trimethoxy-3,5-dimethylbenzene (23). To
a
solution of 22 (0.20 g, 1.02 mmol, 1.0 equiv.) in 6 mL dry DCM oven T-1 with 2 integrated 6-Port valves, photodiode array
was added NBS (0.20 g, 1.12 mmol, 1.1 equiv.) and the mixture detector PDA-1 (190–1000 nm); column: Eurocel 03, 3 μm,
was stirred for 16 h at room temperature. The reaction was 4.6 mm × 250 mm; solvent: methanol/water (90/10); flow rate:
quenched with water and extracted with Et2O and the com- 1.0 mL min−1
; pressure: 209 bar, temperature: 20 °C.
bined organic layers were dried with MgSO4. The solvent was Dichotomomyces cejpii 293 K09 was cultivated on MPY medium
removed under reduced pressure and the crude product was (malt extract 20 g L−1, peptone from meat 2.5 g L−1, yeast
purified by column chromatography on silica gel (cyclohexane/ extract 2.5 g L−1, agar 15 g L−1). Cultivation was performed in
ethyl acetate 20 : 1). Product 23 was isolated as a white solid 1800 mL Fernbach flasks (20 × 250 mL per flask) for 40 days at
(0.23 g, 0.84 mmol, 82%). Rf = 0.2. GC (HP-5MS): I = 1704. room temperature with constant artificial light exposure. Each
1H-NMR (400 MHz, CDCl3): δ = 3.78 (s, 6H, 2 × CH3), 3.69 (3H, Fernbach flask was extracted with pentane (100 mL) and the
CH3), 2.23 (s, 6H, 2 × CH3) ppm. 13C-NMR (100 MHz, CDCl3): combined extracts were concentrated under reduced pressure.
δ = 157.5 (Cq), 154.5 (2 × Cq), 122.2 (2 × Cq), 108.3 (Cq), 60.5 The crude extract was purified by column chromatography on
(2 × CH3), 60.1 (CH3), 10.0 (2 × CH3) ppm. IR (ATR): ˜ν = 3000, silica gel (pentane/diethyl ether 20 : 1) and a fraction contain-
2938, 2866, 2831, 1583, 1559. UV/Vis (MeCN): λmax (lg ε): 231 ing 15 (8 mg) was isolated and directly analyzed.
(4.90) nm. EI-MS (70 eV): m/z (%) = 276 (98), 274 (100), 261
Torquatone (18). To a solution of 23 (0.05 g, 0.18 mmol, 1.0
(14), 259 (14), 233 (25), 231 (27), 216 (17), 165 (20), 137 (20), 77 equiv.) in 1 mL dry THF was slowly added a 1.7 M solution of
(16). HR-EIMS calcd for C11H15O3Br+: m/z = 274.0205, found: t-butyllithium (0.22 mL, 0.36 mmol, 2.0 equiv.) at −78 °C. The
m/z 274.0204.
reaction was warmed to −10 °C, stirred for 10 minutes and
(rac)-Isotorquatone (15). To a solution of 23 (0.23 g, again cooled to −78 °C. Aldehyde 24 (0.05 g, 0.58 mmol, 3.0
0.84 mmol, 1.0 equiv.) in 5 mL dry THF was slowly added a 1.7 equiv.) was slowly added and the reaction was stirred for
M solution of t-butyllithium (1.00 mL, 1.67 mmol, 2.0 equiv.) 30 minutes at −78 °C. The reaction was quenched with water
at −78 °C. The reaction was warmed to −10 °C, stirred for and extracted with Et2O. The combined organic layers were
10 minutes and again cooled to −78 °C. Aldehyde 2-methyl- dried with MgSO4 and the solvent was removed under reduced
butyraldehyde (0.22 g, 2.51 mmol, 3.0 equiv.) was slowly added pressure. The crude product was dissolved in 1 mL dry DCM
and the reaction was stirred for 30 minutes at −78 °C. The and DMP (0.08 g, 0.18 mmol, 1.0 equiv.) was added. The reac-
reaction was quenched with water and extracted with Et2O. tion was stirred over night at room temperature, quenched
The combined organic layers were dried with MgSO4 and the with water and extracted with Et2O. The crude product was
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purified by column chromatography on silica gel (cyclohexane/ 159.9 (Cq), 156.7 (Cq), 155.6 (Cq), 118.6 (Cq), 112.2 (Cq), 91.5
ethyl acetate 20 : 1). Torquatone 18 (0.03 g, 0.09 mmol, 50%) (CH), 62.6 (CH3), 56.0 (CH3), 55.8 (CH3), 49.2 (CH), 25.5 (CH2),
was isolated as a colorless oil. Rf = 0.2. GC (HP-5MS): I = 1824. 15.3 (CH3), 11.8 (CH3), 8.5 (CH3) ppm. IR (ATR): ˜ν = 2964,
1H-NMR (500 MHz, C6D6): δ = 3.53 (s, 6H, 2 × CH3), 3.34 (s, 2935, 2875, 2837, 1692, 1598, 805. EI-MS (70 eV): m/z (%) = 266
3H, 2 CH3), 2.73 (d, 3J = 6.8 Hz, 2H, CH2), 2.42 (tsept, 3J = (6), 209 (100), 194 (5), 165 (3), 136 (5). HR-EIMS calcd for
3
3
+
6.7 Hz, J = 6.7 Hz, 1H, CH), 2.14 (s, 6H, 2 × CH3), 0.97 (d, J = C15H23O4 : m/z = 266.1518, found: m/z = 266.1501.
6.7 Hz, 6H, 2 × CH3) ppm. 13C-NMR (125 MHz, C6D6): δ = 204.1
1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-2-methyl-
(Cq), 159.3 (Cq), 154.3 (2 × Cq), 128.6 (Cq), 121.1 (2 × Cq), 62.3 butan-1-one (16) and 1-(2,6-dihydroxy-4-methoxy-3,5-di-
(2 × CH3), 59.5 (CH3), 54.4 (CH2), 24.4 (CH), 22.7 (2 × CH3), 9.3 methylphenyl)-2-methylbutan-1-one (25). To a solution of 15
(2 × CH3) ppm. IR (ATR): ˜ν = 2955, 2868, 1696, 1581, 1452, (0.05 g, 0.16 mmol, 1.0 equiv.) in dry DCM was slowly added
1397, 1365, 1333, 1324, 1294, 1265, 1221, 1194, 1143, 1071, BBr3 (0.04 g, 0.18 mmol, 1.1 equiv.) in 0.1 mL dry DCM at
1006, 990. EI-MS (70 eV): m/z (%) = 280 (10), 223 (100), 208 (3), −78 °C. The reaction was allowed to warm to room tempera-
+
196 (3), 165 (3). HR-ESIMS calcd for C16H25O4 : m/z = ture and stirred over night. It was cooled with an ice bath,
281.1747, found: m/z = 280.1747 [M + H]+.
quenched with water and extracted with EtOAc. After drying
2-Bromo-1,3,5-trimethoxy-4-methylbenzene (24). To a solu- with MgSO4 the solvent was removed under reduced pressure
tion of 21 (0.20 g, 1.10 mmol, 1.0 equiv.) in 2 mL dry THF was and the crude product was purified by column chromato-
added NBS (0.19 g, 1.10 mmol, 1.0 equiv.) at 0 °C. The reaction graphy on silica gel (cyclohexane/ethyl acetate 15 : 1 to 5 : 1).
was warmed to room temperature and stirred for 14 h. Compounds 16 (0.015 g, 0.06 mmol, 35%) and 25 (0.021 g,
Afterwards water was added and it was extracted with Et2O. 0.08 mmol, 52%) were isolated as colorless oils. Analytical data
The combined organic layers were dried with MgSO4 and the for 16: Rf = 0.2 (15 : 1). GC (HP-5MS): I = 1855. 1H-NMR
solvent was removed under reduced pressure. The crude (500 MHz, CDCl3): δ = 12.63 (s, 1H, OH), 3.74 (s, 3H, CH3),
product was purified by column chromatography on silica gel 3.72 (m, 1H, CH), 3.69 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.13 (s,
(cyclohexane/ethyl acetate 20 : 1) and 24 was isolated as a crys- 3H, CH3), 1.82–1.74 (m, 1H, CHH), 1.48–1.38 (m, 1H, CHH),
talline white solid (0.28 g, 1.06 mmol, 97%). Rf = 0.2. GC 1.17 (d, 3J = 6.9 Hz, 3H, CH3), 0.89 (t, 3J = 7.5 Hz, 3H, CH3)
(HP-5MS): I = 1724. 1H-NMR (500 MHz, CDCl3): δ = 6.31 (s, 1H, ppm. 13C-NMR (125 MHz, CDCl3): δ = 212.2 (Cq), 163.2 (Cq),
CH), 3.88 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.77 (s, 3H, CH3), 160.7 (Cq), 158.6 (Cq), 115.7 (Cq), 115.5 (Cq), 111.6 (Cq), 62.4
2.13 (s, 3H, CH3) ppm. 13C-NMR (125 MHz, CDCl3): δ = 158.3 (CH3), 60.2 (CH3), 45.9 (CH), 27.4 (CH2), 17.2 (CH3), 12.0
(Cq), 156.7 (Cq), 155.2 (Cq), 113.8 (Cq), 98.1 (Cq), 92.8 (CH), (CH3), 9.3 (CH3), 8.9 (CH3) ppm. IR (ATR): ˜ν = 2961, 2934,
60.5 (CH3), 56.7 (CH3), 55.9 (CH3), 9.2 (CH3) ppm. IR (ATR): ˜ν = 2874, 1776, 1613, 1585, 1452, 1403, 1371, 1358, 1272, 1192,
2973, 2944, 2858, 1594, 1575, 805. EI-MS (70 eV): m/z (%) = 262 1132, 1104, 1030, 992, 922, 904, 832, 771, 709, 580, 510. EI-MS
(95), 260 (100), 217 (20), 182 (19), 181 (24), 152 (20), 151 (34), (70 eV): m/z (%) = 266 (M+, 7), 209 (100), 166 (2). HR-ESIMS
123 (20), 121 (25), 77 (18). HR-EIMS calcd for C10H13O3Br+: calcd for C15H23O4+: m/z = 267.1591, found: m/z = 267.1592
m/z = 260.0048, found: m/z = 260.0028.
[M + H]+. Analytical data for 25: Rf = 0.2 (5 : 1). GC (HP-5MS):
2-Methyl-1-(2,4,6-trimethoxy-3-methylphenyl)butan-1-one (19). I = 1928. 1H-NMR (500 MHz, CDCl3): δ = 13.22 (s, 1H, OH),
To a solution of 24 (0.10 g, 0.38 mmol, 1.0 equiv.) in 1 mL dry 5.28 (s, 1H, OH), 3.75–3.68 (m, 1H, CH), 3.69 (s, 3H, CH3), 2.13
hexane was slowly added a 1.7 M solution of t-butyllithium (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.83–1.73 (m, 1H, CHH),
(0.45 mL, 0.77 mmol, 2.0 equiv.) at −78 °C. The reaction was 1.48–1.38 (m, 1H, CHH), 1.16 (d, 3J = 6.8 Hz, 3H, CH3), 0.89
3
warmed to −10 °C, stirred for 10 minutes and again cooled to (t, J = 7.5 Hz, 3H, CH3) ppm. 13C-NMR (125 MHz, CDCl3): δ =
−78 °C. 2-Methylbutyraldehyde (0.05 g, 0.58 mmol, 1.5 equiv.) 211.4 (Cq), 161.2 (Cq), 158.9 (Cq), 158.7 (Cq), 108.7 (Cq), 108.6
was slowly added and the reaction was stirred for 30 minutes (Cq), 106.6 (Cq), 62.5 (CH3), 45.5 (CH), 27.6 (CH2), 17.4 (CH3),
at −78 °C. The reaction was quenched with water and extracted 12.1 (CH3), 8.8 (CH3), 7.7 (CH3) ppm. IR (ATR): ˜ν = 3469, 2963,
with Et2O. The combined organic layers were dried with 2933, 2874, 1601, 1453, 1410, 1373, 1288, 1223, 1171, 1142,
MgSO4 and the solvent was removed under reduced pressure. 1104, 1069, 1024, 989, 937, 901, 812, 773, 732, 689, 508. EI-MS
The crude product was dissolved in 2 mL dry DMSO and IBX (70 eV): m/z (%) = 252 (M+, 6), 195 (100), 180 (3), 152 (4).
(0.04 g, 0.15 mmol, 1.2 equiv.) was added at room temperature. HR-ESIMS calcd for C14H21O4+: m/z = 253.1434, found: m/z =
The reaction was stirred over night and quenched with sat. aq. 253.1437 [M + H]+.
NaHCO3-solution. The mixture was extracted with Et2O and
the organic layers were dried with MgSO4. Purification of the from 1,3,5-trimethoxybenzene (20) according to a literature
crude product was done by flash chromatography (cyclo- known procedure.32 GC (HP-5MS): 1885. 1H-NMR
Dichotomone (17). Compound 17 was synthesized starting
I
=
hexane/ethyl acetate 5 : 1) and 19 was isolated as a colorless oil (500 MHz, CDCl3): δ = 4.91 (s br, 1H, OH), 3.68 (s, 6H, 2 ×
(0.03 g, 0.94 mmol, 28%). Rf = 0.2. GC (HP-5MS): I = 1877. CH3), 2.95–2.87 (m, 1H, CH), 2.13 (s, 6H, 2 × CH3), 1.83–1.74
1H-NMR (500 MHz, CDCl3): δ = 6.25 (s, 1H, CH), 3.84 (s, 3H, (m, 1H, CHH), 1.43–1.33 (m, 1H, CHH), 1.12 (d, 3J = 7.1 Hz,
CH3), 3.78 (s, 3H, CH3), 3.69 (s, 3H, CH3), 2.94–2.86 (m, 1H, 3H, CH3), 0.93 (t, 3J = 7.5 Hz, 3H, CH3) ppm. 13C-NMR
CH), 2.06 (s, 3H, CH3), 1.83–1.74 (m, 1H, CHH), 1.43–1.34 (m, (125 MHz, CDCl3): δ = 209.1 (Cq), 154.4 (Cq), 154.0 (Cq), 123.5
3
3
1H, CHH), 1.11 (d, J = 7.0 Hz, 3H, CH3), 0.92 (t, J = 7.4 Hz, (Cq), 113.1 (Cq), 62.9 (2 x CH3), 49.3 (CH), 25.4 (CH2), 15.3
3H, CH3) ppm. 13C-NMR (125 MHz, CDCl3): δ = 208.8 (Cq), (CH3), 11.8 (CH3), 8.8 (CH3) ppm. IR (ATR): ˜ν = 3463, 2965,
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2936, 2874, 2838, 1682, 1584, 1457, 1406, 1376, 1295, 1227,
1186, 1126, 1102, 1063, 1022, 1001, 986, 932, 877, 844, 501.
Conflicts of interest
EI-MS (70 eV): m/z (%) = 266 (M+, 7), 209 (100), 194 (11). There are no conflicts to declare.
HR-ESIMS calcd for C15H23O4+: m/z = 267.1591, found: m/z =
267.1592.
Analysis of the enantiomeric excess of 47. The enantiomeric
Acknowledgements
excess of natural 47 and synthetic (R)- and (S)-47 47 was deter-
mined by GC-MS on a homochiral stationary phase (Fig. 5) This
work
was
funded
by
the
Deutsche
using the following conditions: system: Agilent HP7890B gas- Forschungsgemeinschaft DFG (DI1536/9-1). We thank Barbara
chromatograph connected to a HP5977A mass detector fitted Schulz (TU Braunschweig) for Chlorella fusca, Marie Reuter-
with an Agilent CycloSil-B capillary column (30 m, Schniete for contributions to the experimental work, and
0.25–0.32 mm ID, 0.25 μm film); The GC-MS conditions were Ekaterina Egereva (Pharmaceutical Biology, Bonn) for fungal
as follows: (1) inlet pressure: 77.1 kPa, He flow 23.3 mL min−1
;
strains cultivations.
(2) injection volume: 1 μL; (3) injection mode: splitless, valve
time 60 s; (4) oven temperature ramp: 5 min at 70 °C increas-
ing at 10 °C min−1 to 210 °C; (5) carrier gas He at 1 mL min−1
(6) transfer line: 250 °C; (7) electron energy: 70 eV.
;
References
1 (a) C. W. Li, J. Y. Chen and T. E. Hua, Science, 1998, 279,
879–882; (b) G. D. Love, E. Grosjean, C. Stalvies, D. A. Fike,
J. P. Grotzinger, A. S. Bradley, A. E. Kelly, M. Bhatia,
W. Meredith, C. E. Snape, S. A. Bowring, D. J. Condon and
R. E. Summons, Nature, 2009, 457, 718–721.
2 J. N. A. Hooper and R. W. M. van Soest, in System Porifera: a
guide to the classification of sponges, Academic/Plenum
Publishers, New York, NY, 2002.
Bioactivity tests against Chlorella fusca
CP medium (10 mL; yeast extract 10.0 g L−1
, D-(+)-glucose
monohydrate 10.0 g L−1, 1000 mL H2O, pH 6.2) was inoculated
with Chlorella fusca, followed by culturing for one week under
constant artificial light exposure and shaking (100 rpm) at
24 °C. The culture was diluted with sterile CP medium to a cell
count of 10 000 cells per mL distributed in 96 well plates.
Solutions of the test compounds (Table 1) in CP medium were
added with a final concentration of 125 μg mL−1, followed by a
3 (a) M. W. Taylor, R. Radax, D. Steger and M. Wagner,
Microbiol. Mol. Biol. Rev., 2007, 71, 295–347;
(b) U. Hentschel, J. Piel, S. M. Degnan and M. W. Taylor,
Nat. Rev. Microbiol., 2012, 10, 641–654; (c) T. Thomas,
L. Moitinho-Silva, M. Lurgi, J. R. Björk, C. Easson,
C. Astudillo-García, J. B. Olson, P. M. Erwin, S. López-
Legentil, H. Luter, A. Chaves-Fonnegra, R. Costa,
P. J. Schupp, L. Steindler, D. Erpenbeck, J. Gilbert,
R. Knight, G. Ackermann, J. V. Lopez, M. W. Taylor,
R. W. Thacker, J. M. Montoya, U. Hentschel and
N. S. Webster, Nat. Commun., 2016, 7, 1–12.
4 J. Vacelet, J. Microsc. Biol. Cell., 1975, 23, 271–288.
5 (a) C. A. Bewley and D. J. Faulkner, Angew. Chem., Int. Ed.,
1998, 37, 2162–2178; (b) U. R. Abdelmohsen, K. Bayer and
U. Hentschel, Nat. Prod. Rep., 2014, 31, 381–399;
(c) M. F. Mehbub, J. Lei, C. Franco and W. Zhang, Mar.
Drugs, 2014, 12, 4539–4577; (d) W. H. Gerwick and
B. S. Moore, Cell Chem. Biol., 2012, 19, 85–98;
(e) M. S. Laport, O. C. S. Santos and G. Muricy, Curr.
Pharm. Biotechnol., 2009, 10, 86–105; (f) J. Piel, Nat. Prod.
Rep., 2004, 21, 519–538.
1 : 1 dilution series (62.5 μg mL−1, 31.3 μg mL−1, 15.6 μg mL−1
,
etc.). All experiments were performed in triplicate, with 3 nega-
tive controls (no compound added) and using 2,4-dichloro-
phenol and CuSO4·5H2O as positive controls. An extra plate was
used as a blank control (no algae) to exclude light absorption
by the medium. The incubation time was 5 days at 24 °C, with
constant artificial light exposure and without shaking. OD
values were measured at 560 nm using a Sunrise Tecan micro-
plate reader plate reader. The minimal inhibitory concen-
tration was considered the lowest concentration that led to at
least 80% growth inhibition compared to the negative control.
Bioactivity tests against B. megaterium and E. coli
NP medium (10 mL; peptone from meat 7.8 g L−1, peptone
from casein 7.8 g L−1, yeast extract 2.8 g L−1
, D-(+)-glucose
monohydrate 1.0 g L−1, NaCl 5.6 g L−1, 1000 mL H2O, pH 7.5)
was inoculated with Bacillus megaterium DSM 32 or Escherichia
coli DSM 498 and the cultures were grown overnight under
constant shaking (200 rpm) at 30 °C. Agar plates were prepared
using NP medium (with 12 g L−1 agar) and the plates were
inoculated with 150 μL bacterial suspension which was evenly
distributed on the surface of the agar. With the use of 200 μL
pipette tip, three small agar plugs were taken out from each
agar plate and solutions of the test compounds (50 μL, 1 mg
mL−1) were filled into the hole from the removed agar plugs.
Ertapenem was used as a positive control. Incubation time
was 24 h at 30 °C, with constant artificial light exposure. The
inhibition zones were measured in mm (radii) and compared
to the positive control.
6 R. P. Walker, D. J. Faulkner, D. Van Engen and J. Clardy,
J. Am. Chem. Soc., 1981, 103, 6772–6773.
7 (a) V. S. Bernan, D. M. Roll, C. M. Ireland, M. Greenstein,
W. M. Maiese and D. A. Steinberg, J. Antimicrob.
Chemother., 1993, 32, 539–550; (b) R. Rosa, W. Silva,
G. Escalona de Motta, A. D. Rodriguez, J. J. Morales and
M. Ortiz, Experientia, 1992, 48, 885–887; (c) R. Mohammed,
J. Peng, M. Kelly and M. T. Hamann, J. Nat. Prod., 2006, 69,
1739–1744; (d) A. Vassas, G. Bourdy, J. J. Paillard,
J. Lavayre, M. Pais, J. C. Quirion and C. Debitus, Planta
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Med., 1996, 62, 28–30; (e) P. A. Keifer, R. E. Schwartz, 20 R. P. Adams, Identification of Essential Oil Components by
M. E. S. Koker, R. G. Hughes, D. Rittschof and
Gas Chromatography/Mass Spectrometry, Allured, Carol
K. L. Rinehart, J. Org. Chem., 1991, 56, 2965–2975;
Stream, 2009.
(f) A. D. Rodríguez, M. J. Lear and J. J. La Clair, J. Am. 21 H. Harms, V. Rempel, S. Kehraus, M. Kaiser, P. Hufendiek,
Chem. Soc., 2008, 130, 7256–7258; (g) A. Cipres,
D. P. O’Malley, K. Li, D. Finlay, P. S. Baran and K. Vuori,
ACS Chem. Biol., 2009, 5, 195–202.
C. E. Müller and G. M. König, J. Nat. Prod., 2014, 77, 673–
677.
22 H. Harms, S. Kehraus, D. Nesaei-Mosaferan, P. Hufendieck,
L. Meijer and G. M. König, Steroids, 2015, 104, 182–188.
8 T. Lindel, Alkaloids, 2017, 77, 117–219.
9 (a) Y. Kato, N. Fusetani, S. Matsunaga, K. Hashimoto, 23 H. Harms, B. Orlikova, S. Ji, D. Nesaei-Mosaferan,
S. Fujita and T. Furuya, J. Am. Chem. Soc., 1986, 108, 2780–
2781; (b) H. Ishihara, B. L. Martin, D. L. Brautigan,
G. M. König and M. Diederich, Mar. Drugs, 2015, 13, 4949–
4966.
H. Karaki, H. Ozaki, Y. Kato, N. Fusetani, S. Watabe, 24 (a) D. Gansser, F. C. Pollak and R. G. Berger, J. Nat. Prod.,
K. Hashimoto, D. Uemura and D. J. Hartshorne, Biochem.
Biophys. Res. Commun., 1989, 159, 871–877.
10 T. Wakimoto, Y. Egami, Y. Nakashima, Y. Wakimoto,
T. Mori, T. Awakawa, T. Ito, H. Kenmoku, Y. Asakawa, J. Piel
and I. Abe, Nat. Chem. Biol., 2014, 10, 648–655.
1995, 58, 1790–1793; (b) C. A. Citron, J. Gleitzmann,
G. Laurenzano, R. Pukall and J. S. Dickschat,
ChemBioChem, 2012, 13, 202–214; (c) P. Rabe, C. A. Citron
and J. S. Dickschat, ChemBioChem, 2013, 14, 2345–2354;
(d) C. A. Citron, L. Barra, J. Wink and J. S. Dickschat, Org.
Biomol. Chem., 2015, 13, 2673–2683.
11 C. Almeida, Y. Hemberger, S. M. Schmitt, S. Bouhired,
L. Natesan, S. Kehraus, K. Dimas, M. Gütschow, 25 (a) J. S. Dickschat, S. C. Wenzel, H. B. Bode, R. Müller and
G. Bringmann and G. M. König, Chem. – Eur. J., 2012, 18,
8827–8834.
12 C. Almeida, F. E. Maddah, S. Kehraus, G. Schnakenburg
and G. M. König, Org. Lett., 2016, 18, 528–531.
13 J. S. Dickschat, Nat. Prod. Rep., 2014, 31, 838–861.
14 (a) S. Schulz and J. S. Dickschat, Nat. Prod. Rep., 2007, 24,
S.
Schulz,
ChemBioChem,
2004,
5,
778–787;
(b) J. S. Dickschat, H. B. Bode, T. Mahmud, R. Müller and
S. Schulz, J. Org. Chem., 2005, 70, 5174–5182.
26 (a) M. Toyota, T. Yoshida, J. Matsunami and Y. Asakawa,
Phytochemistry, 1997, 44, 293–298; (b) D. Spiteller, A. Jux,
J. Piel and W. Boland, Phytochemistry, 2002, 61, 827–834.
814–842; (b) S. Schulz, J. S. Dickschat, B. Kunze, I. Wagner- 27 U. Warmers and W. A. König, Phytochemistry, 1999, 52, 99–104.
Döbler, R. Diestel and F. Sasse, Mar. Drugs, 2010, 8, 2976– 28 H. W. L. Ruijgrok, The distribution of ranunculin and cya-
2987; (c) J. S. Dickschat, Nat. Prod. Rep., 2017, 34, 310–328;
(d) O. Tyc, C. Song, J. S. Dickschat, M. Vos and P. Garbeva,
Trends Microbiol., 2017, 25, 280–292; (e) J. S. Dickschat,
nogenetic compounds in the Ranunculaceae, in
Comparative Phytochemistry, ed. T. Swain, Academic Press,
New York, 1966, pp. 175–186.
T. Martens, T. Brinkhoff, M. Simon and S. Schulz, Chem. 29 R. Blasco, R.-M. Wittich, M. Mallavarapu, K. N. Timmis
Biodiversity, 2005, 2, 837–865. and D. H. Pieper, J. Biol. Chem., 1995, 270, 29229–29235.
15 C. Christophersen, U. Anthoni, P. H. Nielsen, N. Jacobsen 30 R. A. B. Fazzini, M. E. Skindersoe, P. Bielecki, J. Puchalka,
and O. S. Tendal, Biochem. Syst. Ecol., 1989, 17, 459–461.
16 C. Duque, A. Bonilla, E. Bautista and S. Zea, Biochem. Syst.
Ecol., 2001, 29, 459–467.
17 (a) C. Christophersen, U. Anthoni, P. H. Nielsen,
N. Jacobsen and O. S. Tendal, Biochem. Syst. Ecol., 1989, 17,
M. Givskov and V. A. P. Martins dos Santos, Environ.
Microbiol., 2012, 15, 111–120.
31 M. W. Taylor, P. J. Schupp, H. J. Baillie, T. S. Charlton,
R. de Nys, S. Kjelleberg and P. D. Steinberg, Appl. Environ.
Microbiol., 2004, 70, 4387–4389.
459–461; (b) C. Duque, A. Bonilla, E. Bautista and S. Zea, 32 (a) N. Didry, L. Dubreuil and M. Pinkas, Phytother. Res.,
Biochem. Syst. Ecol., 2001, 29, 459–467; (c) R. Romoli,
M. C. Papaleo, D. de Pascale, M. L. Tutino, L. Michaud,
1993, 7, 21–24; (b) M. L. Martín, L. S. Román and
A. Domínguez, Planta Med., 1990, 56, 66–69.
A. L. Giudice, R. Fani and G. Bartolucci, J. Mass. Spectrom., 33 L. Pouységu, M. Marguerit, J. Gagnepain, G. Lyvinec,
2011, 46, 1051–1059; (d) M. C. Papaleo, R. Romoli, A. J. Eatherton and S. Quideau, Org. Lett., 2008, 10, 5211–5214.
G. Bartolucci, I. Maida, E. Perrin, M. Fondi, V. Orlandini, 34 L. F. Tietze, R. R. Singidi, K. M. Gericke, H. Böckemeier
A. Mengoni, G. Emiliani, M. L. Tutino, E. Parrilli, D. de and H. Laatsch, Eur. J. Org. Chem., 2007, 5875–5878.
Pascale, L. Michaud, A. L. Giudice and R. Fani, New 35 (a) C. Menut, J. M. Bessiere, A. D. Samate, J. Millogo-
Biotechnol., 2013, 30, 824–838; (e) V. Orlandini, I. Maida,
M. Fondi, E. Perrin, M. C. Papaleo, E. Bosi, D. de Pascale,
M. L. Tutino, L. Michaud, A. L. Giudice and R. Fani,
Microbiol. Res., 2014, 169, 593–601.
Rasolodimby and M. Nacro, Phytochemistry, 1999, 51, 975–
978; (b) B. F. Ireland, R. J. Goldsack, J. J. Brophy,
C. J. R. Fookes and J. R. Clarkson, J. Essent. Oil Res., 2004,
16, 89–94; (c) M. Baranska, H. Schulz, S. Reitzenstein,
U. Uhlemann, M. A. Strehle, H. Krüger, R. Quilitzsch,
W. Foley and J. Popp, Biopolymers, 2004, 78, 237–248.
36 C. Almeida, S. Kehraus, M. Prudêncio and G. M. König,
Beilstein J. Org. Chem., 2011, 7, 1636–1642.
18 U. Höller, A. D. Wright, G. F. Matthée, G. M. König,
S. Draeger, H.-J. Aust and B. Schulz, Mycol. Res., 2000, 104,
1354–1365.
19 (a) K. Grob and F. Zürcher, J. Chromatogr., 1976, 117, 285–
294; (b) J. S. Dickschat, S. C. Wenzel, H. B. Bode, R. Müller 37 C. Almeida, E. Eguereva, S. Kehraus and G. M. König,
and S. Schulz, ChemBioChem, 2004, 5, 778–787.
J. Nat. Prod., 2013, 76, 322–326.
Org. Biomol. Chem.
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38 C. Almeida, N. Part, S. Bouhired, S. Kehraus and 44 P. Spiteller, Chem. – Eur. J., 2008, 14, 9100–9110.
G. M. König, J. Nat. Prod., 2011, 74, 21–25.
39 L. Dormont, R. Delle-Vedove, J.-M. Bessière and B. Schatz,
Phytochemistry, 2014, 100, 51–59.
40 R. L. Metcalf and R. L. Lampman, Proc. Natl. Acad. Sci.
U. S. A., 1991, 88, 1869–1872.
45 F. El Maddah, S. Kehraus, M. Nazir, C. Almeida and
G. M. König, J. Nat. Prod., 2016, 79, 2838–2845.
46 B.-E. Priegnitz, U. Brandt, K. A. K. Pahirulzaman,
J. S. Dickschat and A. Fleißner, Eukaryotic Cell, 2015, 14,
602–615.
41 R. M. Seifert and A. D. King, J. Agric. Food Chem., 1982, 30, 47 (a) T. Nilsson, T. O. Larsen, L. Montanarella and
786–790.
J. O. Madsen, J. Microbiol. Methods, 1996, 25, 245–255;
(b) T. O. Larsen and J. C. Frisvad, Mycol. Res., 1995, 99,
1153–1166.
42 M. Nemcovic, L. Jakubikova, I. Viden and V. Farkas, FEMS
Microbiol. Lett., 2008, 284, 231–236.
43 S. Murahashi, Sci. Pap. Inst. Phys. Chem. Res., 1938, 34, 48 C. A. Citron, C. Junker, B. Schulz and J. S. Dickschat,
155–172.
Angew. Chem., Int. Ed., 2014, 53, 4346–4349.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.