Epicoccamide, a novel secondary metabolite from a jellyfish-derived culture of Epicoccum purpurascens.

Article abstract of DOI:10.1039/b208588g

From the inner tissue of the jellyfish Aurelia aurita a marine strain of the fungus Epicoccum purpurascens was obtained. After mass cultivation the fungus was investigated for its secondary metabolite content and found to contain the new, and most unusual tetramic acid derivative, epicoccamide (1). Epicoccamide is quite unusual since it is composed of three biosynthetically distinct subunits; glycosidic, fatty acid and tetramic acid (amino acid). The structure of the new compound was elucidated using spectroscopic methods, mainly 1D and 2D NMR, ESI-MS, and chemical degradations.

Full text of DOI:10.1039/b208588g

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Epicoccamide, a novel secondary metabolite from a jellyfish-
derived culture of Epicoccum purpurascens
Anthony D. Wright,*† Claudia Osterhage and Gabriele M. König
Institute for Pharmaceutical Biology, University of Bonn, Nußallee 6, 53115 Bonn, Germany
Received 3rd September 2002, Accepted 11th November 2002
First published as an Advance Article on the web 14th January 2003
From the inner tissue of the jellyfish Aurelia aurita a marine strain of the fungus Epicoccum purpurascens was
obtained. After mass cultivation the fungus was investigated for its secondary metabolite content and found to
contain the new, and most unusual tetramic acid derivative, epicoccamide (1). Epicoccamide is quite unusual since
it is composed of three biosynthetically distinct subunits; glycosidic, fatty acid and tetramic acid (amino acid). The
structure of the new compound was elucidated using spectroscopic methods, mainly 1D and 2D NMR, ESI-MS,
and chemical degradations.
Introduction
Results and discussion
Marine microorganisms are a new source of biologically active
metabolites with novel chemical structures.1,2 Fungi investi-
gated from the marine environment are mostly derived from
sediments,3–5 wood,6 algae,7–9 or, as in the case of Epicoccum
purpurascens, from marine animals.10–17 In the current study the
isolation of the fungus E. purpurascens from the North Sea
jellyfish Aurelia aurita is described. This fungal strain produces
a most unusual glycosylated tetramic acid containing secondary
metabolite, epicoccamide. Tetramic acid derived natural
products are interesting due to their pronounced biological
activities. Thus ascosalipyrrolidinone, a desoxy tetramic acid
derivative produced by the obligate and algae associated marine
fungus Ascochyta salicorniae, was recently patented due to its
antimicrobial activity.7 Though no obligate marine species of
the fungus Epicoccum have been described representatives of
this genus are found on beached algae and other beach wrack
material.18 Terrestrial species of the genus Epicoccum have been
studied for their natural products content with epicorazines
A and B,19 epirodin,20 and triornicin,21 all being produced by
E. nigrum (E. purpurascens),22 indicating some members of
the genus Epicoccum to have a highly developed and diverse
secondary metabolism.
After isolation, the fungus E. purpurascens was cultivated on a
solid malt soya medium containing added artificial sea salt.
Successive fractionation of the EtOAc extract of the fungus
and medium by vacuum–liquid chromatography (VLC), and
normal (Si-60) and reversed (RP-C18) phase HPLC yielded the
most unusual new natural product epicoccamide (1).
The molecular formula of epicoccamide (1) (Fig. 1) was
found to be C29H51NO9 by accurate mass measurement
(HRFAB ϩve). Of the five elements of unsaturation implied by
the molecular formula of 1, three were present as carbonyl
(enol) groups [δ 175.3 (C-1, s), 197.6 (C-3, s), 201.3, (C-7, s)] as
deduced from the 13C NMR and IR spectra of the molecule.
This deduction enabled three of the nine oxygen atoms within
the molecule to be accounted for, and also indicated the mole-
cule to be bicyclic since no other multiple bonds were present.
1
From the H and 13C NMR data it was evident that 1 was
composed of three distinct fragments; a hexose sugar, an
aliphatic chain, and a tetramic acid moiety. After all protons
† Present address: 40 Twelfth Avenue, Railway Estate, Townsville, 4810
Queensland, Australia.
Fig. 1 Structures of epicoccamide (1) and its hydrolysis products, 2 and -mannose.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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had been associated with their directly bonded carbon atoms
via a 1H–13C 2D NMR shift-correlated measurement (HMQC)
it was possible to develop the sugar and aliphatic moieties from
via C-22 at the other. From the long-range correlation observed
between the resonances for H2-22 and that of C-1Ј it was
obvious that the aliphatic chain was linked via oxygen to the
anomeric carbon C-1Ј. The electrospray (ESI) mass spectrum
(negative mode) of epicoccamide (1) contained the highest mass
signal at m/z 556, consistent with [M Ϫ H]Ϫ. Collision-induced
dissociation-tandem mass spectrometry (CID-MS/MS, neg-
ative mode) showed the 556 peak and a base peak at m/z 394
corresponding to the molecular ion less the mass of mannose.
Additionally, a m/z 126 peak was present in the ESI CID-MS/
MS (negative mode) for the molecular fragment resulting from
the α-cleavage of the tetramic acid moiety. In the ESI (ϩ) mass
spectrum m/z 580 ([M ϩ Na]ϩ) was the base peak. A CID-MS/
MS (positive mode) showed the formation of a m/z 418 base
peak which represents the sodium adduct of 1 less the mannose.
Increasing the collision energy from Ϫ39 eV to Ϫ59 eV resulted
in a loss of mannose and water from the molecule and gave a
base peak at m/z 400. Attempts to resolve the stereochemistry
of 1 at C-4 and C-8 by comparison of its CD spectra with those
of similar compounds yielded no unambiguous results.26,27
Epicoccamide (1) is quite an unusual low molecular weight
natural product since it is composed of three biosynthetically
distinct subunits; glycosidic (mannose), fatty acid (presumably
a hexa- or heptadecanoic acid derivative), and amino acid
(tetramic acid).
A number of natural products with a tetramic acid moiety
are known, e.g., ancorinoside A,26 and its magnesium salt,27
from the marine sponge Ancorina sp., both of which inhibit
star fish embryo blastulation. Aflastatin A produced by
Streptomyces sp. was found to inhibit aflatoxin biosynthesis.25
The tetramic acid glycosides aurantosides A and B from the
marine sponge Theonella sp., show cytotoxicity towards P-388
and L-1210 leukemia cells.23 From the sponge Halichondria
cylindrata cylindramide, a tetramic acid lactam, was isolated
and found to have cytotoxic effects towards B 16 melanoma
cells.24 Fischerellin A, isolated from a culture of the cyano-
bacterium Fischerella muscicola, had antifungal, and herbicidal
activities, as well as being a potent photosystem-II-inhibitor.28
As many of the reported tetramic acid derivatives discussed
above exhibited pharmacological activities, in particular
cytotoxicity, it was surprising to find that the new and unusual
natural product epicoccamide (1) had no detectable activity
in any of the applied assay systems (see Experimental
section).
1
the H–1H COSY data. Thus, crosspeaks seen between the
resonances for H-1Ј, the anomeric proton, and H-2Ј, between
the resonances for H-2Ј and H-3Ј, those for H-3Ј and H-4Ј, H-4Ј
and H-5Ј, and between the resonances for H-5Ј to those for H2-
1
6Ј, revealed the complete H–1H spin system within the sugar
moiety. The coupling constant between H-5Ј and H-4Ј (J = 9.5
Hz) showed them to have a trans-diaxial disposition, as do H-3Ј
and H-4Ј (J = 9.5 Hz). The coupling constant (J = 3.2 Hz)
between H-2Ј and H-3Ј showed H-2Ј to be equatorial, the
hexose moiety was thus concluded to be mannose. With the
mannose moiety established it was then necessary to determine
the stereochemistry of the glycosidic linkage, and if - or
-mannose were present in epicoccamide. From the magnitude
of the C–H coupling constant between C-1Ј and H-1Ј (J = 156.4
Hz) it was evident that 1 contained β-mannose since β-pyran-
oses show JCH values of around 160 Hz, and α-pyranoses values
of around 170 Hz.23 Acid hydrolysis of 1 (see Experimental
section and Fig. 1) enabled the sugar and 2 to be isolated. The
optical rotation of the sugar moiety was ϩ18.4 × 10Ϫ1 deg cm2
gϪ1, which showed epicoccamide to contain -mannose. The
mannose moiety accounted for all of the remaining oxygen
within the molecule as well as for one of the two rings within 1.
At this stage of the structural analysis 3 × CH3, 14 × CH2, 3 ×
CH groups, and one nitrogen, as deduced from the MS, 1H and
13C NMR data of 1, remained to be accounted for. Evident in
the 13C NMR spectrum of epicoccamide were four broad
resonances at δ 201.3 (C-7, s), 197.6 (C-3, s), 175.3 (C-1, s), and
101.6 (C-2, s), which were best assigned to a tetramic acid
moiety.24,25 From the 1H–1H COSY crosspeaks observed
between the resonances for H3-5 and H-4, and between the
resonances for H3-23 and H-8, and between the resonances for
H-8 and H2-9, it was evident that CH3-5 is bonded to C-4, and
that CH3-23 is bonded to C-8, which further bonded to C-9. As
the resonance for C-8 was broad in the 13C NMR spectrum it
was likely that this group was also affected by the keto–enol
1
tautomerism of the tetramic acid moiety (Fig. 2). In the H
Experimental
General
FAB (positive mode) mass spectra were recorded using a JEOL
102SX A, double focussing sector field instrument. All other
general experimental procedures were carried out as previously
described.29,30
Fig. 2 Tautomeric forms of the tetramic acid part of 1 and 2.
Isolation and taxonomy of the fungus
NMR spectrum of 1 the signal for H-8 (δ 3.80, m) was notably
deshielded, an effect which is characteristic of tetramic acid
systems.24 CH3-6 was assigned as an N–CH3 on the basis of its
The jellyfish Aurelia aurita was collected from the North Sea,
Tönning, Germany. Its inner tissue was cut into small pieces
and placed on agar plates containing isolation medium (15 g
1
1H and 13C NMR chemical shifts. Finally, long-range H–13C
L
Ϫ1 agar and 1 L sea water from the sample collecting site and
2D NMR correlations (HMBCs) observed between the reson-
ances for H3-6 and C-1 and C-4, between those associated with
H3-5 and C-3 and C-4, and between those of H3-23 and C-2, C-
7, C-8 and C-9 clearly showed C-1 and C-4 to bond to the N–
CH3, C-3 to bond to C-4, C-7 to bond to C-8 and C-2, and by
deduction C-2 to bond to both C-1 and C-3, thus completing
the tetramic acid moiety and the second ring within the mole-
cule. The remaining 14 × CH2 groups were assigned to a single
aliphatic chain connected to C-8 at one end and to the mannose
the antibiotics benzylpenicillin and streptomycin sulfate, each
250 µg LϪ1). Fungal colonies growing out of the jellyfish tissue
were transferred to the medium for sporulation (1.0 g glucose,
0.1 g yeast extract, 0.5 g peptone from meat, enzymatic digest,
15 g agar, and 1 L sea water, pH 8) in order to enable taxonomy
of the isolates. The fungal strain E. purpurascens, voucher
number N7–9, used in this study was identified by Dr S.
Draeger, Institute for Microbiology, Technical University of
Braunschweig.
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Table 1 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data for epicoccamide (1) and 1H (CD3OD, 300 MHz) and 13C NMR (CD3OD,
75.5 MHz) data for 2a
Position
δC (ppm) (1)
δH (ppm) (1)
JCH/Hz (1)
δC (ppm) (2)
δH (ppm) (2)
1
2
3
175.3 C
101.6 C
197.6 C
171.4 C
Not observed
196.5 C
4
5
6
61.8 CH
16.0 CH3
26.7 CH3
201.3 C
3.54 (br)
1.31 (d, J = 6.9 Hz)
2.91 (br s)
62.0 CH
3.68 (qm, J = 7.0 Hz)
1.37 (d, J = 7.0 Hz)
3.00 (s)
15.2 CH3
27.0 CH3
191.5 C
7
8
9
40.8 CH
35.0 CH2
30.6–30.9 CH2
27.3 CH2
30.8 CH2
70.6 CH2
3.80 (m)
1.27 (m), 1.72 (m)
1.25–1.38 (m)
1.42 (m)
40.7 CH
3.87 (q, J = 7.0 Hz)
1.27 (m), 1.74 (m)
1.25–1.38 (m)
1.42 (m)
1.55 (m)
3.58 (t, J = 6.6 Hz)
34.8 CH2
30.9–31.7 CH2
28.4 CH2
30.6 CH2
63.0 CH2
10–19
20
21
22
1.65 (m)
3.57 (ddd, J = 2.9, 6.8, 9.8 Hz)
3.98 (td, J = 6.8, 6.8, 9.8 Hz)
1.03 (d, J = 6.2 Hz)
4.54 (br s)
3.90 (br d, J = 3.2 Hz)
3.50 (dd, J = 3.2, 9.5 Hz)
3.62 (t, J = 9.5, 9.5 Hz)
3.26 (ddd, J =3.0, 5.6, 9.5 Hz)
3.77 (dd, J = 5.6, 11.8 Hz)
3.95 (dd, J = 3.0, 11.8 Hz)
23
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
18.0 CH3
101.7 CH
72.5 CH
75.3 CH
68.5 CH
78.1 CH
62.8 CH2
17.5 CH3
1.21 (d, J = 7.0 Hz)
156.4 Hz
145.9 Hz
142.0 Hz
146.2 Hz
140.5 Hz
143.5 Hz
a All assignments are based on extensive 1D and 2D NMR experiments (COSY, HMQC, HMBC).
Cultivation
LϪ1) λ/nm 218 (∆ε ϩ0.20), 232 (∆ε Ϫ0.34), 269 (∆ε ϩ0.91), 295
(∆ε Ϫ0.83); IR (film) νmax/cmϪ1 3360, 2925, 2850, 1655, 1595,
The micro-organism cultures were grown at 20 ЊC for 50 days in
4.5 L of solid malt extract soy meal agar (30 g malt extract,
Merck, 3 g peptone from soy meal, papain-digest, Merck, 7.6 g
agar, 800 mL artificial sea water and 200 mL demineralised
water, adjusted to pH 5.5). The composition of the artificial sea
water was as described by Höller et al.11
1
1480; H and 13C NMR data, see Table 1; HRFAB (ϩ) m/z
580.3379 (Ϫ8.3 mmu) [M ϩ Na]ϩ, 596.3126 (Ϫ7.5 mmu) [M ϩ
K]ϩ; ESI-MS (Ϫ) m/z 556 [M Ϫ H]Ϫ; ESI-MS (ϩ) 580 [M ϩ
Na]ϩ; CID-ESI-MS/MS (Ϫ) 556 (31), 394 (100), 126 (40);
CID-ESI-MS/MS (ϩ) 418 (28), 400 (100).
Hydrolysis of epicoccamide (1). To 8 mg of 1 2 mL 2 M HCl
(methanolic) was added and the resulting solution maintained
at 80 ЊC for 4 h. At the end of this period the residual solvent
was removed under reduced pressure and the remaining
material partitioned between H2O and CH2Cl2. The aqueous
phase contained -mannose (2.4 mg, [α]2D0/10Ϫ1 deg cm2 gϪ1
ϩ18.4 (c 0.24, H2O), lit.37 14.2), and the CH2Cl2 contained 2.
Biological activity
The activity of compounds was tested in agar diffusion assays
against the bacteria Bacillus megaterium, Escherichia coli, the
fungi Microbotryum violaceum, Eurotium repens, Fusarium
oxysporum, Mycotypha microsporum and the alga Chlorella
fusca.30 Assays with Artemia salina and Caenorrhabditis elegans
aimed at assessing cytotoxic and nematicidal activity were
also performed.31 In ELISA based enzyme assays inhibition of
tyrosine kinase p56lck and reverse transcriptase HIV-1 was
examined.32,33 Antiplasmodial activity was determined as
described by Desjardins et al.34 The activity against Mycobac-
terium tuberculosis was assessed as described by Collins and
Franzblau,35 against hemoflagellates, which cause human
sleeping sickness (Trypanosoma brucei subsp. rhodesiense), and
Chagas disease (Trypanosoma cruzi), and also the cytotoxic
activities were assessed as described by Kaminsky and Brun.36
Tetramic acid-aliphatic moiety (2). A light yellow viscous oil
(2.6 mg LϪ1); [α]2D0/10Ϫ1 deg cm2 gϪ1 Ϫ15.2 (c 0.17, CH2Cl2); UV
(EtOH) λmax/nm (ε/dm3 molϪ1 cmϪ1) 246 (9590), 285 (11250); IR
(film) νmax/cmϪ1 2924, 2853, 1714, 1652, 1610, 1457; 1H and 13
C
NMR data, see Table 1.
Acknowledgements
We thank Dr S. Draeger, Institute for Microbiology, TU
Braunschweig, for taxonomic identification of the investigated
fungal strain. We also thank Dr V. Wray and his group,
GBF Braunschweig, for recording all NMR spectra, and
Dr A. Waßmann, MS group, GBF Braunschweig, and Dr P.
Janning, Department IV, MPI for Molecular Biology,
Dortmund, for making all mass spectral measurements.
Thanks also go to Mrs L. Peters and C. Dreikorn, Institute for
Pharmaceutical Biology, TU Braunschweig, for performing
the enzyme, agar diffusion, brine shrimp and nematode assays.
We also thank Dr R. Kaminsky, Schweizerisches Tropen-
institut, Basel, for antimalarial, T. cruzi, T. brucei and cyto-
toxicity data. Financial support from the Bundesministerium
für Bildung, Forschung und Technologie (BMBF), and Bayer
AG, Leverkusen, is gratefully acknowledged.
Extraction and isolation
The solid medium and fungal mycelium were diluted with H2O,
blended using the Ultra Turrax T 25 at 8000 minϪ1, and then
extracted with 13.5 L EtOAc. The resultant EtOAc extract
(3.9 g) was purified employing a combination of chromato-
graphic techniques. First, it was passed over normal phase silica
(vacuum–liquid chromatography, VLC) using a gradient of
CH2Cl2–EtOAc–MeOH as eluent to yield 13 fractions each
of 250 mL. VLC fraction 11 (2.2 g, eluted with MeOH–EtOAc,
60 : 40) was subjected to RP-18 HPLC (Eurospher 100, 5 µm,
8 mm × 25 cm) using MeOH–H2O, 85 : 15, as eluent to yield
compound 1.
Epicoccamide (1). A white amorphous powder (2.6 mg LϪ1);
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