Dietary derived sesquiterpenes from Phyllodesmium lizardensis

Article abstract of DOI:10.1021/np800583e

The recently discovered new aeolidean species Phyllodesmium lizardensis Burghardt, Schroedl and Waegele, 2008 was investigated concerning its secondary metabolite profile. P. lizardensis so far has only been found on Lizard Island. Analysis of P. lizarden

Full text of DOI:10.1021/np800583e

298
J. Nat. Prod. 2009, 72, 298–300
Dietary Derived Sesquiterpenes from Phyllodesmium lizardensis
Sven Affeld,† Stefan Kehraus,† Heike Wa¨gele,‡ and Gabriele M. Ko¨nig*,†
Institute for Pharmaceutical Biology, UniVersity of Bonn, Nussallee 6, D-53115 Bonn, Germany, and Department of Botany, Institute for
EVolutionary Biology and Ecology, UniVersity of Bonn, An der Immenburg 1, D-53121 Bonn, Germany
ReceiVed September 18, 2008
The recently discovered new aeolidean species Phyllodesmium lizardensis Burghardt, Schro¨dl and Wa¨gele, 2008 was
investigated concerning its secondary metabolite profile. P. lizardensis so far has only been found on Lizard Island.
Analysis of P. lizardensis led to the isolation of the new sesquiterpenes (+)-3ꢀ-hydroxy-R-muurolene (1) and (+)-3ꢀ-
acetoxy-R-muurolene (2). GC-MS analysis of the host coral, identified as Heteroxenia sp., also showed the presence of
compounds 1 and 2, whereas a sympatric Xenia species lacks these products. These results indicate that P. lizardensis
specifically sequesters these compounds from Heteroxenia sp.
Secondary metabolites as defensive mechanisms are a common
feature of shell-less opisthobranchs.1 Contrary to other groups,
like Sacoglossa, or Doridoidea, however almost nothing is known
about chemically based defensive systems in Aeolidoidea,2 since
members of this taxon usually discharge stored nematocysts
sequestered from their typical hydrozoan prey.
The genus Phyllodesmium is a group of cryptic aeolid
nudibranchs. Their members are known to feed on certain
octocorals,3 this way taking up symbiotic zooxanthellae from
the corals and storing them in their digestive gland cells, where
they are still photosynthetically active.4-6 In doing so, the slugs
are able to survive a certain time without feeding by living only
on the photosynthesis products of the zooxanthellae. However,
in contrast to other aeolidoideans, the soft coral feeding
Phyllodesmium species do not take up cnidocysts from their prey,
therefore lacking this typical defensive system.3,4 Apart from
the genus Phyllodesmium, the only aeolids that do not incorporate
cnidocysts from their prey are Phestilla lugubris, Phestilla minor,
Cuthona poritophages, and Pinufius rebusand,7-9 which feed
on hexacorals. One alternative for those species lacking cni-
docysts for their defense is the accumulation of secondary
of unsaturation. The 13C NMR spectrum contained 15 resonances
resulting from four methyl, two methylene, five sp3 methine, two
sp2 methine, and two quaternary carbons. Due to the molecular
formula and 23 protons evident from 1H NMR and DEPT135
spectra, one proton had to be present as a hydroxyl group. With
two elements of unsaturation attributed to carbon-carbon double
bonds a bicyclic structure was in agreement with the four degrees
of unsaturation. After assignment of all protons to their directly
bonded carbon atoms via a 1Hs13C HSQC experiment, it was
possible to deduce two major fragments of the molecule from the
results of a 1H-1H COSY experiment. Analysis of the COSY
spectrum of 1 gave evidence for connectivities from CH-3 to CH-5
through CH2-2, CH-1, and CH-6 (Figure 1, fragment A). Methyl
groups CH3-12 and CH3-13 both coupled to H-11, which further
coupled to H-7. H2-8 showed couplings to both H-7 and H-9, thus
completing the second spin system B (Figure 1). A 1H-13C HMBC
experiment permitted the planar structure of 1 to be further
elaborated. HMBC correlations from H3-15 to C-1, C-9, and C-10
indicated CH3-15 to be positioned at C-10 and connected COSY
fragments A and B via C-10. CH3-14 was bonded to carbon 4, as
evident by cross-peaks from H3-14 to C-3, C-4, and C-5. The
HMBC cross-peak from H3-14 to C-5 also delineated a first ring
closure between C-4 and C-5 to give a cyclohexene ring. A second
ring closure was provided by an HMBC correlation between H-6
and C-7, resulting in an R-cadinene skeleton. The missing hydroxyl
group had to be connected to C-3 because of its low-field 13C NMR
chemical shift (δC 68.5).
metabolites from their prey.
Although at least 20 described and about five undescribed
species of the genus Phyllodesmium are known, so far only two
of them have been investigated for the presence of secondary
metabolites. Coll et al. investigated P. longicirrum, which
accumulates the terpenes (+)-thunbergol and (+)-epoxythun-
bergol from the soft coral Sarcophyton trocheliophorum.10
Slattery et al. showed that P. guamensis specifically sequesters
the diterpene 11-ꢀ-acetoxypukalide from its preferred prey, the
soft coral Sinularia maxima.11 The latter diterpene has been
positively tested for feeding deterrence against reef fishes that
are potential predators for P. guamensis.
In the current study, the isolation and structure elucidation of
two new sesquiterpenes (1, 2), besides the known metabolite (+)-
R-muurolene (3) from P. lizardensis Burghardt, Schro¨dl and
Wa¨gele, 2008 are described. This species has been recorded only
from Lizard Island, Great Barrier Reef (Australia).12 GC-MS
analyses of extracts obtained from the soft coral Heteroxenia sp.
on which P. lizardensis was found regularly indicated that this coral
produces these compounds, whereas a sympatric occurring Xenia
species is lacking these metabolites.
The molecular formula of compound 1 was deduced by accurate
mass measurement (HREIMS) to be C15H24O, implying four degrees
* To whom correspondence should be addressed. Tel: +49228733747.
Fax: +49228733250. E-mail: g.koenig@uni-bonn.de.
† Institute for Pharmaceutical Biology.
‡ Zoologisches Forschungsmuseum Alexander Koenig.
10.1021/np800583e CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/27/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 2 299
Table 1. 1D and 2D NMR Spectroscopic Data for (+)-
3ꢀ-Hydroxy-R-muurolene (1)
a b
,
atom
no.
δH (mult.,
J in Hz)
a b e
, ,
δC
COSYa c HMBCa d
NOESYa c
,
,
,
1
2
35.2, CH 2.34, m
35.1, CH2 R: 1.66, m
ꢀ: 1.93, m
68.5, CH 3.93, t (3.4)
136.4, C
2R, 2ꢀ, 6
2ꢀ, 6, 15
2ꢀ, 3, 7
2R, 3
1, 3
1, 3
1, 3, 6
3
4
5
2R, 2ꢀ, 5 2, 4, 5, 14
2R, 2ꢀ, 14
129.1, CH 5.68, dd
6, 14
1, 3, 4, 6, 14 6, 7, 11, 13, 14
(1.1, 4.6)
6
7
8
9
10
11
12
13
14
15
38.2, CH 2.10, m
41.0, CH 1.47, m
25.5, CH2 1.88, m
122.6, CH 5.45, brs
136.9, C
28.1, CH 1.98, m
21.7, CH3 0.94, d (7.0) 11
16.3, CH3 0.90, d (6.6) 11
1, 5, 7
6, 8, 11
7, 9
1, 4, 5, 7
13
9
1, 5, 13
2R, 5, 8, 11, 12
9
8, 15
8, 15
1
1
Figure 1. H-1H COSY and key H-13C HMBC correlations of
7, 12, 13 8, 12, 13
5, 12, 13
11
6, 11
3, 5
compound 1.
7, 11
7, 11
3, 4, 5
1, 9, 10
21.4, CH3 1.84, brs
21.6, CH3 1.73, brs
5
9
1, 9
a MeOH-d4, 300/75.5 MHz. b Assignments are based on extensive 1D
and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to
proton resonances. d Numbers refer to carbon resonances. e Implied
multiplicities determined by DEPT.
Table 2. 1D and 2D NMR Spectroscopic Data for (+)-
3ꢀ-Acetoxy-R-muurolene (2)
1
Figure 2. H-1H NOESY correlations of compound 1.
a b
,
atom
no.
δH (mult.,
J in Hz)
a b e
, ,
COSYa c HMBCa d
NOESYa c
,
,
,
δC
A diagnostic NOE correlation between the resonances of H-1
and H-6 provided a cis-fused decalin ring system. Additional NOE
correlations between H-2R and H-7, between H-1 and H-2ꢀ,
between H-5 and H3-13, and between H-3 and both H-2R and H-2ꢀ
as well as 1H-1H coupling constants of H-3 (t, J ) 3.4 Hz; showing
an equatorial position of H-3) were indicative for the relative
configuration of 1 as 1R*, 3R*, 6S*, 7R* (Figure 2). After
derivatization with R-methoxyphenylacetic acid (MPA) the differ-
ence in the δH values of the (R)-MPA and (S)-MPA adducts
indicated C-3 possessed an R absolute configuration (see Figure
S16).13 Thus, the absolute configuration of compound 1 was
established as 1R, 3R, 6S, 7R. A comparison of the optical rotation
value of 1 (+131) with the literature value of (-)-3ꢀ-hydroxy-R-
muurolene (-147) confirmed this result.14 Thus, compound 1 is
the enantiomer of (-)-3ꢀ-hydroxy-R-muurolene, a constituent of
the essential oil of Teucrium yemense Deflers.15 We propose the
trivial name (+)-3ꢀ-hydroxy-R-muurolene for compound 1.
From accurate mass measurement, compound 2 was found to
have a molecular formula of C17H26O2. The spectroscopic data of
2 were very similar to those of 1, suggesting a related planar
structure (Tables 1 and 2). The major differences between the two
data sets were two additional resonances in the 13C NMR spectrum
of 2 (δC 21.2 and 172.8) indicative of an acetyl group. An 1H-13C
HMBC correlation from H-3 to C-16 allowed connection of this
acetyl group to C-3. The absolute configuration of compound 2
was deduced to be the same as for compound 1 because of nearly
identical optical rotation values and comparable NOESY correla-
tions. In the literature compound 2 so far has only been published
without configuration as a transformation product of khusinol.16
For compound 2 the trivial name (+)-3ꢀ-acetoxy-R-muurolene is
proposed.
1
2
35.8, CH
31.9, CH2 R: 1.76, m
2.28, m
2R, 2ꢀ, 6
2ꢀ, 6, 15
2ꢀ, 3, 7
1, 3
ꢀ: 1.94, m
5.21, t (3.3)
1, 3
3
2R, 3, 15
3
4
5
6
7
71.7, CH
132.9, C
132.0, CH 5.83, brd (4.1) 6, 14
38.1, CH
41.1, CH
2R, 2ꢀ, 5 1, 4, 14, 16 2R, 2ꢀ, 14, 17
1, 3, 14
5
6
6, 7, 11, 13, 14
1, 5, 13
2R, 5, 8, 11
2.21, m
1.50, tt
(9.8, 4.9)
1, 5, 7
6, 8
8
9
25.4, CH2 1.90, m
123.1, CH 5.47, brs
136.1, C
7, 9
8, 15
9
8, 15
10
11
12
13
14
15
16
17
28.2, CH
1.96, m
12, 13
12, 13
7, 11
7, 11, 12
3, 4, 5
9, 10
5, 7,12, 13
7, 11
5, 6, 11
3, 5
21.6, CH3 0.96, d (7.0)
16.5, CH3 0.92, d (6.6)
20.9, CH3 1.73, brs
21.5, CH3 1.70, brs
172.8, C
11
11
5
9
1, 2ꢀ, 9
21.2, CH3 2.10, s
16
a MeOH-d4, 300/75.5 MHz. b Assignments are based on extensive 1D
and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to
proton resonances. d Numbers refer to carbon resonances. e Implied
multiplicities determined by DEPT.
this xeniid synthesizes compounds 1 and 2 (Figures S12-S15).
Analysis of another sympatric occurring Xenia species did not reveal
the presence of compounds 1 and 2. This could be one reason to
explain the preference of the slugs for Heteroxenia sp. over Xenia
sp. In natural habitats, the slugs were found associated exclusively
with the former coral, although the number of Xenia colonies was
higher in the observed area around Lizard Island. Additionally, in
experiments the slugs also preferred to sit on Heteroxenia. A species
specificity association induced by chemotaxis was observed in P.
longicirrum with Sinularia maxima.10
Separate analyses of cerata and body resulted in higher quantities
of compounds in the former (data not shown). During preparation
of the living slugs, a sticky fluid was exuded in the upper half of
the cerata, which also autotomized very easily. A higher concentra-
tion of compounds in the cerata was also described for P.
longicirrum.10
The structure of (+)-R-muurolene (3) was identified by compar-
ing its NMR spectroscopic data and optical rotation with published
values.17
Compounds 1 and 2 were evaluated in antibacterial (Escherichia
coli, Bacillus megaterium), antifungal (Mycotypha microspora,
Eurotium rubrum, and Microbotryum Violaceum), and antialgal
(Chlorella fusca) assays at the 50 µg/disk level, but did not show
any activities.
Heteroxenia spp. are known to produce sesquiterpenes of the
muurolene type.17,18 Proksch and co-workers described (+)-6-
hydroxy-R-muurolene to be active against the phytopathogenic
fungus Cladosporium cucumerinum and active in the brine shrimp
GC-MS investigation of the host coral of P. lizardensis, an
unidentified species of the genus Heteroxenia, gave evidence that

300 Journal of Natural Products, 2009, Vol. 72, No. 2
Notes
lethality test.18 Thus, the uptake of secondary metabolites such as
the muurolenes and other terpenes could be an alternative defense
strategy of P. lizardensis and congeners instead of the uptake of
functional cnidocysts.
(+)-3ꢀ-Acetoxy-r-muurolene (2): colorless solid; [R]24 +122 (c
0.38 CHCl3); IR (ATR) νmax 2925, 2858, 1731, 1713, 1437, 1365, 1238
cm-1; 1H and 13C NMR data, see Table 2; ESIMS m/z 263 [M + H]+;
HREIMS m/z 262.1933 (calcd for C17H26O2, 262.1933).
Biological Assays. Compounds 1 and 2 were tested in agar diffusion
assays19 against the bacteria Bacillus megaterium and Escherichia coli,
the fungi Microbotryum Violaceum, Eurotium rubrum, and Mycotypha
microspora, and the green microalga Chlorella fusca. The pure
compounds showed no activity in these assays.
D
Experimental Section
General Experimental Procedures. All NMR spectra were recorded
in MeOH-d4 employing a Bruker Avance 300 DPX spectrometer.
Spectra were referenced to residual solvent signals with resonances at
δH/C 3.35/49.0 (MeOH-d4). IR spectra were obtained employing a
Perkin-Elmer Spectrum BX instrument. GC-MS analyses were per-
formed with a Perkin-Elmer AutoSystem XL and a TurboMass
spectrometer. HREIMS were recorded on a Finnigan MAT 95
spectrometer. ESIMS measurements were recorded employing an API
2000, Applied Biosystems/MDS Sciex. HPLC was carried out using a
Merck-Hitachi system equipped with an L-6200A pump, an L-4500A
photodiode array detector, a D-6000A interface with D-7000 HSM
software, and a Rheodyne 7725i injection system. Optical rotation was
measured on a Jasco DIP 140 polarimeter.
Animal Material. Phyllodesmium lizardensis was collected on
Lizard Island, the Great Barrier Reef, Australia, during low tide in July
2006 and stored in methanol until workup. Live specimens of the soft
corals Heteroxenia sp. and Xenia sp. were collected at the same place
and preserved in methanol. Voucher specimens of P. lizardensis have
been deposited at the Zoologische Staatssammlung, Munich, voucher
number ZSM Mol 20060654. Specimens of Heteroxenia and Xenia
are in the collection of HW.
Extraction and Isolation. After removal of the preservative MeOH,
two nudibranchs (wet wt 3.2 g) were extracted once with MeOH. The
MeOH extracts were combined and evaporated to dryness to yield 220
mg of a yellow extract. This extract was fractionated by vacuum liquid
chromatography (VLC) over Polygoprep 60-50 C18 material (Macherey-
Nagel) using gradient elution from H2O (100%) to MeOH (100%), to
yield 12 fractions. 1H NMR investigations indicated VLC fractions 10,
11, and 12 to contain pure compounds 1 (20.0 mg), 2 (4.6 mg), and 3
(73.4 mg), respectively.
GC-MS Analysis. A 1.0 mg amount of compounds 1, 2, and 3 and
2.0 mg of MeOH extracts of the soft corals were each dissolved in 1
mL of acetone. A 1 µL sample of these solutions was analyzed using
a Perkin-Elmer PE-1 column (30 m × 0.32 mm; 0.25 µm; program
rate: column temperature held at 50 °C for 5 min; 50-180 °C at 5
°C/min; flow: 2.0 mL/min; inj.: 220 °C).
Preparation of the Acid Chlorides of (R)- and (S)-MPA. Oxalyl
chloride (52 µL, 0.6 mmol) was added to a solution of the corresponding
MPA (10 mg, 0.06 mmol) and DMF (0.47 µL, 0.006 mmol) in 1 mL
of hexanes at room temperature. After 3 h, the solution was dried under
nitrogen.
Preparation of the (R)- and (S)-MPA Esters. The corresponding
MPA-Cl (10 mg, 54 µmol) was dissolved in 2 mL of CH2Cl2 and added
to a solution of compound 1 (2.5 mg, 11 µmol), Et3N (18 µL, 130
µmol), and DMAP as catalyst. After 18 h reaction time the obtained
products were dried under vacuum and further purified by HPLC using
the Merck-Hitachi system. The separation was performed with a RP18
column (Macherey-Nagel Nucleodur ISIS RP, 5 µm, 250 × 4.6 mm)
and a mobile phase (1.0 mL/min) consisting of MeCN/H2O, 80/20.
(+)-3ꢀ-Hydroxy-r-muurolene (1): colorless solid; [R]24D +131 (c
1.67 CHCl3); IR (ATR) νmax 3408, 2956, 2927, 1714, 1671, 1367, 1222
cm-1; 1H and 13C NMR data, see Table 1; ESIMS m/z 221 [M + H]+;
HREIMS m/z 220.1825 (calcd for C15H24O, 220.1827).
Acknowledgment. We thank Dr. M. Engeser and C. Sondag,
Institute for Organic Chemistry, University of Bonn, Germany, for
recording EIMS spectra. We thank Y. Grzymbowski (University of
Bonn) and K. Stemmer (University of Bochum) for help in collecting
material. K. Stemmer also identified the Heteroxenia coral. This study
was partly funded by the German Science Foundation (SPP 1127,
“Adaptive radiation-origin of biological diversity”; Wa 618/8).
Supporting Information Available: 1H and 13C NMR, 1H-1H
COSY, 1H-13C HMBC, and 1H-1H NOESY spectra of compounds 1
and 2 as well as GC-MS chromatograms. Figures with results of
modified Mosher’s method. This material is available free of charge
via the Internet at http://pubs.acs.org.
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NP800583E