O-Methylation of phenylphenalenone phytoalexins in Musa acuminata and Wachendorfia thyrsiflora

Article abstract of DOI:10.1016/j.phytochem.2009.10.019

Biosynthetic O-methylation at various sites along the backbone of inducible phenylphenalenones in Musa acuminata var. Williams (Musaceae) and Wachendorfia thyrsiflora (Haemodoraceae) was investigated using ¹³C-labelled precursors. The inducibility of O-methylated metabolites was demonstrated in both species and the origin of methoxyl group from [methyl-¹³C]l-methionine was confirmed. In addition to known phenylphenalenones, a methoxylated metabolite, 4-(4-hydroxy-3-methoxy-phenyl)-benzo[de]isochromene-1,3-dione, was detected and its structure elucidated mainly by NMR spectroscopic techniques. The experiments were used to discriminate methionine-derived and artificial methoxy groups formed during methanolic extraction. Finally, demethylation of 4′-methoxycinnamic acid and subsequent conversion to 3′,4′-methylenedioxycinnamic acid was demonstrated in M. acuminata.

Full text of DOI:10.1016/j.phytochem.2009.10.019

Phytochemistry 71 (2010) 206–213
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
O-Methylation of phenylphenalenone phytoalexins in Musa acuminata and
Wachendorfia thyrsiflora
Felipe Otálvaro a, Kusuma Jitsaeng b, Tobias Munde b, Fernando Echeverri c, Winston Quiñones c,
Bernd Schneider b,
*
a Instituto de Química, Universidad de Antioquia, Calle 67# 53-108, A.A. 1226 Medellín, Colombia
b Max-Planck-Institut für Chemische Ökologie, Beutenberg Campus, Hans Knöll Str. 8, D-07745 Jena, Germany
c Laboratorio de Química Orgánica de Productos Naturales, Edificio SIU 234-235, A.A. 1226 Medellín, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 October 2008
Received in revised form 23 October 2009
Accepted 24 October 2009
Available online 24 November 2009
Biosynthetic O-methylation at various sites along the backbone of inducible phenylphenalenones in Musa
acuminata var. ‘‘Williams” (Musaceae) and Wachendorfia thyrsiflora (Haemodoraceae) was investigated
using 13C-labelled precursors. The inducibility of O-methylated metabolites was demonstrated in both
species and the origin of methoxyl group from [methyl-13C]
L-methionine was confirmed. In addition to
known phenylphenalenones,
a
methoxylated metabolite, 4-(4-hydroxy-3-methoxy-phenyl)-
benzo[de]isochromene-1,3-dione, was detected and its structure elucidated mainly by NMR spectro-
scopic techniques. The experiments were used to discriminate methionine-derived and artificial methoxy
groups formed during methanolic extraction. Finally, demethylation of 40-methoxycinnamic acid and
subsequent conversion to 30,40-methylenedioxycinnamic acid was demonstrated in M. acuminata.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Musa acuminata
Wachendorfia thyrsiflora
Haemodoraceae
Musaceae
Elicitor
Labelling
Methylation
NMR
Phenylphenalenones
Phytoalexins
1. Introduction
fungi (Liu et al., 1999). The process of O-methylation in general is
catalyzed by S-adenosyl-L-methionine (SAM)-dependent O-meth-
Methyl transfer reactions play a crucial role in plant secondary
metabolism (Roje, 2006). O-Methylation occurs in early as well as
in downstream steps of many biosynthetic pathways, thereby con-
tributing to diversification of secondary metabolites and modulat-
ing their physico-chemical properties (Ferrer et al., 2008). As a
consequence, in comparison to the activities of non-methylated
analogues, the biological activities of O-methylated natural prod-
ucts may be altered. In the case of flavonoid and isoflavonoid deriv-
atives, for example, regio-selective methylation modulates the in
vivo activity by limiting the number of reactive hydroxyl groups,
altering the solubility properties, and ultimately determining the
extent of interaction between compounds and cell receptors (Zu-
bieta et al., 2001). Similar methylation phenomena have been ob-
served in cotton; there, methylated terpenoid phytoalexins are
synthesized by the plant in response to invasion by pathogenic
yltransferases, which have been classified according to their sub-
strate preference and molecular architecture (Ibrahim et al.,
1998; Noel et al., 2003).
Phenylphenalenones, polycyclic natural products of some
monocotyledonous plant families, occur both in (poly)phenolic
and partially or completely O-methylated form. In the Haemodor-
aceae, phenylphenalenone-type compounds mainly accumulate in
the roots but were also detected in above-ground tissue (Opitz and
Schneider, 2002). A similar distribution seems to occur in the Mus-
aceae (Luis et al., 1996; Kamo et al., 1998). In Musa, phenylphe-
nalenones have been described as phytoalexins (Luis et al., 1994;
Binks et al., 1997; Kamo et al., 1998, 2000) while they seem to
be constitutive plant defense compounds in the Haemodoraceae
or, as preliminary reported for Wachendorfia thyrsiflora (Opitz,
2002; Schüler et al., 2004), inducible and constitutive phenylphe-
nalenones co-exist in one species. Although the ecological role of
non-methylated and O-methylated phenylphenalenones remains
to be studied in detail, the two classes of compounds seem to have
different domains of activities: The accumulation in secretory
* Corresponding author. Tel.: +49 3641 571600; fax: +49 3641 571601.
E-mail address: schneider@ice.mpg.de (B. Schneider).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.10.019

F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
207
cavities (Hölscher and Schneider, 2007), which are supposed to
function as reservoirs for natural products that defend the plant
against chewing insects, support the assumption that O-permethy-
lated phenylphenalenones are involved in herbivore deterrence
rather than protection against fungal infection. In contrast, some
non-methylated phenylphenalenones are more potent antifungal
natural products than their methylated analogues (Otálvaro et al.,
2007).
Biosynthetic incorporation of [2-13C]ferulic acid into rings C and
D of musanolone F (Fig. 1, panel b, path ii) but not into rings A and
B of methoxyanigorufone (panel a, path ii) was reported in Anigo-
zanthos preissii (Schmitt et al., 2000). Therefore, it seems reason-
able that methoxyanigorufone is formed from anigorufone by O-
methylation of the hydroxyl group at C-2 (Fig. 1, panel a, path i),
and musanolone F via incorporation of ferulic acid-CoA ester dur-
ing condensation (Fig. 1, panel b, path ii).
Musa acuminata and W. thyrsiflora were selected for this study
because they belong to phytochemically related phenylphenale-
none-producing plant families, the Musaceae (banana family)
and Haemodoraceae (bloodroot family), which in part share
identical phenylphenalenone structures. Another reason for using
the two species is the availability of in vitro-cultures, which are
suitable systems for biosynthetic precursor feeding experiments.
While banana plants are economically important crop plants,
some members of the bloodroot family, especially Anigozanthos
hybrids (‘‘kangaroo paw”) are used as ornamental plants. The
genus Wachendorfia is endemic to the cape region of South Afri-
ca. W. thyrsiflora is a rich source of phenylphenalenone-type
compounds (Brand, 2005) and has been used to study biosyn-
thetic enzymes of these polycyclic natural products (Brand
et al., 2006).
The biosynthetic origin of the carbon skeleton of phenylphe-
nalenones from two phenylpropane units and C-2 of acetate or
malonate has been demonstrated to be basically the same in
Haemodoraceae (Thomas, 1973; Hölscher and Schneider,
1995a,b) and Musaceae (Kamo et al., 2000). However, many details
of the biosynthetic pathways such as the cyclization mechanism,
the origin and sequence of the oxygenation and O-methylation still
have to be established. Two different possibilities for the formation
of O-methylphenylphenalenone derivatives are illustrated in Fig. 1.
The versions indicated by the single arrows (path i) directly meth-
ylate a phenolic hydroxyl group of a pre-formed phenylphenale-
none; this process is thought to be the hypothetical final
biosynthetic step in the pathway to O-methylated phenylphenale-
nones. Alternatively, intact incorporation, as indicated by reversed
arrows (path ii), of methoxyphenylpropanoids would result in the
same methoxyphenylphenalenones to which path i leads.
The last reaction was surprising because condensation with the
diketide to form a linear diarylheptanoid requires an activated
intermediate, which is thought to be the phenylpropanoyl-CoA es-
ter. However, a specific ferulate-CoA ligase has not yet been re-
ported from plants and the feruloyl-CoA ester is considered to be
formed by caffeoyl-CoA-O-methyltransferase-catalyzed O-methyl-
ation of caffeoyl-CoA. Ferulate-CoA ligase activity would explain
the observed incorporation of ferulic acid in Anigozanthos. How-
ever, the alternative formation of musanolone F from dihydroxyan-
igorufone cannot be ruled out because caffeic acid-O-
methyltransferases have been shown to methylate the 3-position
of catechol moieties of various phenylpropanoids (Parvathi et al.,
2001; Do et al., 2007; Lin et al., 2006).
No information is available about the origin of the methoxy
group of 40-O-methylirenolone (Fig. 1, panel c). It is unclear
whether this compound is formed by direct O-methylation of iren-
olone via path i or by incorporation of 40-methoxycinnamic acid
(path ii). Taking into account that 40-methoxycinnamic acid is not
a member of the established phenylpropanoid pathway, 40-O-
methylirenolone might be formed by 40-O-methylation of ireno-
lone rather than by incorporation of 40-methoxycinnamic acid as
a unit. On the other hand, 40-methoxycinnamic acid recently was
found as a natural product in Aquilegia vulgaris (Ranunculaceae)
(Bylka, 2004) and may have been overlooked in other plants. If
present in Musa, its SCoA ester could function as a putative precur-
sor for the biosynthesis of 40-O-methylirenolone. The occurrence of
further O-methylated phenylphenalenones in various species of
the Haemodoraceae and Musaceae also raised the question
whether the methoxy groups were introduced before or after the
formation of the skeleton and prompted us to study the biosynthe-
sis of such compounds in the two families.
Fig. 1. Hypothetical formation of methoxyphenylphenalenones by O-methylation of hydroxyl groups of phenylphenalenones (i) or incorporation of intact methoxyphe-
nylpropanoids (ii) in form of their SCoA esters. More likely conversions are marked in bold (for details see text). SAM: S-adenosyl-L-methionine, SAH: S-
adenosylhomocysteine.

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F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
In this paper, we report the existence of regio-specific O-
methyl-phenylphenalenone-type natural products including
nalenones 1–4 (Fig. 2). 1H and 13C NMR spectroscopic data of
known compounds 1–3 matched those of 40-O-methylirenolone
(1) (Luis et al., 1994), (2S,3S)-(+)-2,3-dihydro-2,3-dihydroxy-4-
(40-methoxyphenyl)-phenalen-1-one (2) (Luis et al., 1994), and
musanolone F (3) (Schmitt et al., 2000). 2-(4-hydroxy-3-methoxy-
phenyl)-naphthalene-1,8-dicarboxylic anhydride (4) is a new nat-
ural product. For details of structure elucidation, Section 4.5. This
experiment showed that the O-methyl groups of compounds 1–4
a
new compound in a member of the Musaceae (M. acuminata cv.
Williams) and Haemodoraceae (W. thyrsiflora), discuss the possibil-
ity of O-methylation occurring at different steps of the phenylphe-
nalenone biosynthetic pathway, and provide evidence for the
inducibility of methoxy-phenylphenalenone biosynthesis in the
two species. In addition, O-methyl groups formed enzymatically
by methyl transfer from SAM and solvent-derived O-methyl arti-
facts are discriminated by isotopic labelling.
originated from
biosynthetic step the O-methylation occurred.
Administering [methyl-13C]
L-methionine via SAM but did not reveal at which
L-methionine to root cultures of W.
thyrsiflora and 13C NMR-guided fractionation (MeOH extraction;
semipreparative HPLC method 4; TLC; final purification by HPLC
method 1) resulted in isolation of three methoxyphenylphenale-
none derivatives 5–7. 1H and 13C NMR spectroscopic data of com-
2. Results and discussion
2.1. 13C NMR-guided isolation of methoxy-phenylphenalenones in
Musa and Wachendorfia
pounds
5
and
6
matched those of previously isolated
methoxyanigorufone (5) (Hölscher and Schneider, 1997), and 5-hy-
droxy-2-methoxy-6-oxa-benzo[def]chrysen-1-one (6) (Opitz et al.,
2002). No analytical data for compound 7 were available. Enrich-
ment of 13C was inferred from enhanced O-methyl signals in the
13C NMR and cross peaks in the HSQC spectra. In addition, 13C
labelling was detected by means of the large 1H–13C coupling con-
stant (JH–C = 144.5 Hz) of the O13CH3 resonances in the 1H NMR
spectra (Schneider et al., 2003; Schneider, 2007). The 1H NMR sig-
nals of the O-methyl groups appear as pseudo triplets, which are
composed of the central singlet of the [O12CH3]-isotopomer and a
doublet indicating the [O13CH3]-isotopomer.
The O-methylation patterns of phenylphenalenones in M.
acuminata cv. ‘‘Williams” (Musaceae) and W. thyrsiflora (Haem-
odoraceae) were previously reported to be different. W. thyrsiflora
produces phenylphenalenones bearing O-methyl groups at the tri-
cyclic nucleus but not at the lateral phenyl ring (Opitz et al., 2002).
40-Methoxyphenylphenalenones such as 40-O-methylirenolone are
typical for Musa species, which also produce phenylphenalenones
showing a 30-methoxy-40-hydroxy substitution pattern in the phe-
nyl ring (Fig. 1) (Luis et al., 1996; Kamo et al., 1998).
In order to detect methoxyphenylphenalenones in Musa selec-
tively, [methyl-13C]
L-methionine was administered to in vitro
For example, the partial 1H NMR spectrum of compound 3
(Fig. 3) from in vitro plants of M. acuminata displays the signal of
the O-methyl group. The integral ratio of the O13CH3 doublet
(a + a0) to the O12CH3 singlet (b) were used to determine the pro-
portions of 13C-labelled molecules derived from the 13C-labelled
plantlets, which simultaneously were treated with CuCl2 for elici-
tation. After 6 days, the roots of the plants were cut, frozen in li-
quid nitrogen, macerated and extracted with acetone. Extraction
with methanol was omitted in this case in order to avoid non-
enzymatic O-methylation. The crude acetone extract was submit-
ted to 1H and 13C NMR spectroscopic analysis. Greatly enhanced
precursor, [methyl-13C]
L
-methionine (99% 13C), and from unla-
-methionine. The incorporation levels for com-
13C NMR signals, in comparison to
[methyl-13C]
-methionine, appeared in the region between 55 and
a
control without
belled endogenous
L
pounds 1–7 were determined to be as follows: 1: 13%, 2: 50%, 3:
30%, 4: 33%, 5: 12%, 6: 10%, 7, 3-OCH3: natural abundance, 6-
OCH3: 12%. Isotope dilution from the 99% 13C of the precursor to
L
60 ppm, suggesting O-13CH3 groups from the labelled precursor
have been incorporated (Fig. S1). Chromatographic separation
(semipreparative HPLC method 3) of the extract gave two relevant
fractions, which showed a total of four intense 13C NMR peaks
around 55 ppm in the region of interest. Further 13C NMR-guided
isolation (TLC; HPLC method 1) resulted in methoxy-phenylphe-
Fig. 2. Structures of methoxy-phenylphenalenones isolated in this study. Com-
pounds 1–4 were isolated by 13C NMR-guided fractionation from in vitro plants of
Fig. 3. Partial 1H NMR spectrum (500 MHz, CDCl3) of musanolone F (3) from M.
acuminata displaying the 30-O-methyl signal. The integral ratio of the O13CH3
doublet (a + a0; JC–H = 144.5 Hz) to the O12CH3 singlet (b) were used to determine
the proportions of isotopomers derived from the 13C-labelled precursor,
M. acuminata after administration of [methyl-13C]
L-methionine. Compounds 5–7
were obtained from root cultures of Wachendorfia thysiflora. ꢀ, 13C incorporated
from [methyl-13C]
L
-methionine.
[methyl-13C] -methionine (99% 13C), and from unlabelled endogenous
L L-methionine.

F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
209
the specified percentages demonstrated that compounds 1–6 are
natural products of M. acuminata and W. thyrsiflora, respectively.
Interestingly, although compound possesses two O-methyl
7
groups, 3-OCH3 and 6-OCH3, only the last one was enriched with
13C (Fig. 4). Obviously, the O-methyl group in position 3 has not
been provided by [methyl-13C]
L-methionine via SAM but must be
of artificial origin, presumably due to the extraction with MeOH.
This result paralleled data previously reported by Opitz et al.
(2002), who found that extraction of Xiphidium caeruleum with
MeOH resulted, among other phenylphenalenone-type com-
pounds, in 3-methoxy-5,6-dihydroxy-7-phenyl-3H-benzo[de]iso-
chromen-1-one. This compound was not detectable after
isolation using acetone as a solvent. It was concluded, therefore,
that O-methyl groups in position 3 of 7-phenyl-benzoisochrome-
nones seem to be artifacts of the extraction procedure.
2.2. Inducibility of methoxy-phenylphenalenone biosynthesis
The biosynthesis of phenylphenalenones in Musa was shown to
be stimulated by biotic (Luis et al., 1994; Binks et al., 1997; Kamo
et al., 1998) and chemical elicitors (Luis et al., 1996). This has been
confirmed by the present experiments with M. acuminata var. Wil-
liams, which produced only minute amounts of phenylphenale-
nones when grown in vitro under sterile conditions (Fig. 5, panel
B). Treatment of in vitro plants with CuCl2 (Fig. 5, panel A) dramat-
ically increased phenylphenalenone production. Highest increase
was observed for the peak of anigorufone, a non-O-methylated
phenylphenalenone (AR, Rt 40.1 min). Additional unidentified com-
pounds were detected between 43 and 53 min. Their 1H NMR spec-
tra did not display any signal from phenolic OCH3 groups between
3.6 and 4.0 ppm. 40-O-Methylirenolone (1, Rt 42.0 min) was also
identified but it was not the major metabolite in these experi-
ments. Hence, although there was a clear CuCl2-dependent stimu-
lation of phenylphenalenone biosynthesis, specific elicitation of O-
methylated phenylphenalenones was not observed in these exper-
iments. Treating in vitro plants of M. acuminata with jasmonic acid
(JA) as an elicitor instead of CuCl2 also enhanced levels of phenyl-
phenalenones, including 40-O-methylirenolone. However, results
largely diverged even in parallel experiments and were not always
reproducible.
Fig. 5. Partial HPLC traces (HPLC method 2, UV 254 nm) of extracts from Musa
acuminata (roots of in vitro plants; A and B) and Wachendorfia thyrsiflora (cultured
roots; C and D). (A) M. acuminata treated with CuCl2; (C) Wachendorfia thyrsiflora
treated with jasmonic acid (JA); (B and D) Untreated controls. 1, 40-O-methyliren-
olone (Rt 42.0 min); 5, methoxyanigorufone (Rt 38.9 min); AR, anigorufone (Rt
40.1 min); U, unidentified compounds showing no O-methyl signals in the 1H NMR
spectrum; IS, internal standard (0.5 lg pyrene per injection; Rt 57.0 min). For
details of HPLC methods and sample preparation, see Section 4.
Anigorufone, a 1,2-oxygenated phenylphenalenone, which is a
major secondary compound in most Haemodoraceae, was detected
in W. thyrsiflora only in traces (Opitz, 2002; Brand, 2005). However,
preliminary experiments indicated an eliciting effect of JA (Opitz,
2002) and coronalone (Schüler et al., 2004) on the formation of ani-
gorufone. In addition, methoxyanigorufone (5), which was not
detectable in extracts of untreated plant roots, was formed in high
levels upon elicitation (Fig. 5, panel C, Rt 38.9 min).
In order to evaluate how the availability of L-methionine affects
the level of O-methylated phenylphenalenones, the media of in vitro
plants of M. acuminata and root cultures of W. thyrsiflora were sup-
plemented with [methyl-13C]
L
-methionine alone and with
[methyl-13C]
L-methionine together with elicitors (CuCl2, JA).
Although the 13C label was incorporated into methoxyphenylphe-
nalenone methyl groups, there was no significant increase in the le-
vel of methoxyphenylphenalenones compared to the untreated
controls and the experiments with elicitors, respectively (data not
shown).
2.3. Metabolism of [2-13C,40-O13CH3]40-methoxycinnamic acid in M.
acuminata
Fig. 4. Partial 13C NMR spectra of compound 7 isolated from W. thyrsiflora. (a) after
Labelling experiments are not only useful for NMR-guided iso-
lation of natural products (Section 2.1) but also to establish precur-
incorporation of [methyl-13C]
L
-methionine, ꢀ, signal of 13C-enriched methyl group;
s, naturally abundant 13C signal; (b) unlabelled reference. The asterisk indicates an
impurity signal.
sor – product relationships and infer biosynthetic pathways

210
F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
(Schneider, 2007). In this context, the results presented above (Sec-
tion 2.1) did not only indicate the occurrence of O-13C-methylated
natural products in extracts and fractions but, although not sur-
prising, also demonstrated that the S-methyl group of L-methio-
nine is the source of the O-methyl groups of the
methoxyphenylphenalenones 1–6 and one of the two O-methyl
groups of 7. However, O-methyl groups of phenylphenalenones
are supposed to become labelled from [methyl-13C]
L-methionine
via S-adenosylmethionine regardless of the mode of synthesis, O-
methylation of phenylphenalenones (Fig. 1, path i) or incorporation
of intact methoxyphenylpropanoids (Fig. 1, path ii). Two biosyn-
thetic origins of methoxyphenylphenalenones bearing the O-
methyl group at C-40 of the lateral phenyl ring are possible. One op-
tion is the 40-O-methylation of irenolone to 1 (path i in Fig. 1).
Alternatively, hypothesis ii, similar to the formation of musanolone
F (3) in A. preissii (Schmitt et al., 2000), suggested O-methylation of
p-coumaric acid (or the SCoA ester) followed by incorporation of
40-methoxycinnamoyl-CoA into methoxyphenylphenalenones.
To investigate the latter hypothesis, [2-13C,40-O13CH3]40-meth-
oxycinnamic acid (99% 13C) was synthesized and administered to
in vitro plants of M. acuminata. The doubly labelled precursor,
which is 13C-enriched in the side chain of the phenylpropanoid
skeleton and the O-methyl group, was used to distinguish between
intact incorporation of the precursor and demethylation before
incorporation. The crude root extract obtained after incubation
with [2-13C,40-O13CH3]40-methoxycinnamic acid did not show sig-
nals of the parent compound. Therefore it was subjected to 13C
NMR-guided fractionation. However, the 13C NMR spectra of phe-
nylphenalenone-containing n-hexane and dichloromethane frac-
tions did not exhibit the enhanced 13C NMR signals of the carbon
atoms C-5 and C-8 (128 and 132 ppm) that would be expected
from incorporation of two phenylpropanoid units into the aromatic
tricycle. Nonetheless, small amounts of two non-methylated com-
pounds, anigorufone and hydroxyanigorufone, were purified from
this feeding experiment but no label was detected by 13C NMR in
these compounds. In addition, musanolone F (3), bearing an O-
methyl group at C30, was purified and subjected to NMR and mass
spectral analysis. Again, no 13C enrichment in positions 5 and 8 of
the phenalenone skeleton and in the 30-OCH3 group was detected.
A dichloromethane-soluble fraction was obtained in the same
experiment, which surprisingly showed a strong 13C NMR signal
at d 116.6. The labelled compound was isolated and identified by
NMR and mass spectroscopic analysis as 30,40-methylenedioxycin-
namic acid. The enhanced 13C signal at d 116.6 was attributed to C-
2 (Fig. 6), indicating formation from the precursor, [2-13C,40-
O13CH3]40-methoxycinnamic acid. No label was detected in the
methylenendioxy group. This observation clearly proved loss of
the O13CH3 group of [2-13C,40-O13CH3]40-methoxycinnamic acid.
The extensive demethylation of 40-methoxycinnamic acid upon
feeding to M. acuminata may prevent intact incorporation into 40-
methoxyphenylphenalenones, even if the putative PKS-type con-
densing enzyme would accept 40-methoxycinnamoyl-CoA acid as
a starter unit. O-Demethylation, a common step in many biosyn-
thetic pathways, is frequently involved in xenobiotic metabolism
(Schuler, 1997). Hence, in the present feeding experiment, 40-O-
demethylation might detoxify exogenously applied 40-methoxy-
cinnamic acid to form p-coumaric acid, which then is transformed
to 30,40-methylenedioxycinnamic acid. This conversion requires 30-
hydroxylation, 30-O-methylation and formation of the methylene-
dioxy bridge. The last reaction could be catalyzed by a cytochrome
P450, as reported for the formation of methylenedioxy groups in
lignan biosynthesis (Ono et al., 2006). The original [2-13C]-content
of the precursor, 40-methoxycinnamic acid, of 99% was reduced to
8% in [2-13CH3]30,40-methylenedioxycinnamic acid, which indi-
cated relatively high endogenous levels of this compound in M.
acuminata.
Fig. 6. HSQC spectrum (500 MHz, MeOH-d4) of 30,40-methylenedioxycinnamic acid
obtained from in vitro-cultures of Musa acuminata upon administration of [2-13C,40-
O13CH3]40-methoxycinnamic acid. The intensity-enhanced cross signal of C-2/H-2
(dH 6.31/dC 116.7) and the satellite signals of H-2 (1JC–H = 161 Hz) in the 1H NMR
spectrum shown on top of the 2D spectrum indicate enrichment of 13C in this
position. No 13C enrichment in the methylenedioxy group (dH 6.00/dC 103.1) was
observed. The sequence of steps indicated by the triple arrows is presumably 40-O-
demethylation, 30-hydroxylation, 30-O-methylation, and finally formation of the
methylenedioxy ring.
The question why musanolone F (3) was not labelled upon incu-
bation of Musa plantlets with [2-13C,40-O13C]40-methoxycinnamic
acid cannot conclusively answered from the present experiments.
However, according to the generally accepted phenylpropanoic
pathway (Ferrer et al., 2008), 40-coumaric acid (which here may
come from 40-methoxycinnamic acid) is converted to activated es-
ters (SCoA ester, quinate, shikimate) before 30-hydroxylation and
30-O-methylation occurs. Subsequent PKS III-catalyzed condensa-
tions to form diarylheptanoids (which are intermediates in phenyl-
phenalenone biosynthesis) also require SCoA esters as substrates
(Brand et al., 2006). Hence, free ferulic acid probably is not an
intermediate in phenylphenalenone biosynthesis in Musa. Accord-
ing to such pathway considerations and previous results (Schmitt
et al., 2000), details of the musanolone F formation between Anigo-
zanthos and Musa might be different.
3. Conclusion
NMR-guided isolation of methoxyphenylphenalenones in M.
acuminata and W. thyrsiflora using [methyl-13C]
L-methionine as a
13C source for labelling O-methyl groups resulted in identification
of O-methylated phenylphenalenones of various structural types.
13C-Enrichment of O-methyl groups simultaneously indicates their
origin from [methyl-13C]
L-methionine via a S-adenosylmethionine-
dependent process. The obtained compounds include a new natu-
ral product, 2-(4-hydroxy-3-methoxyphenyl)-naphthalene-1,8-
dicarboxylic anhydride from M. acuminata and known compounds

F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
211
decorated with O-methyl groups in various positions, both in the
tricycle and the lateral phenyl ring. The O-methyl groups, in prin-
ciple can be introduced early in biosynthesis as a substituent of the
phenylpropanoid precursor or at a late step, e.g. by O-methylation
of hydroxyl groups of phenylphenalenones. Discrimination be-
tween early and late O-methylation is not trivial, especially in
cases where arguments exist for either of the two options. An
interesting case of that category is 40-O-methylirenolone (2), which
in M. acuminata could be formed by late O-methylation of the non-
methylated analogue, irenolone, or by incorporation of intact 40-
methoxycinnamic acid. Since 13C-enrichment of the O-methyl
duplicate or triplicate: CuCl2; CuCl2 + 13C-met; JA; JA + 13C-met;
13C-met; untreated control. The plants were extracted after 6 days.
W. thyrsiflora: Root cultures of W. thyrsiflora Burm. were estab-
lished from seeds (Chiltern Seeds, Ulverston, Cumbria, UK) as de-
scribed for in vitro-cultures of other Haemodoraceae (Hölscher
and Schneider, 1997). The sterile cultures were maintained in li-
quid M3 (Murashige and Skoog, 1962) medium (100 ml; pH 5.3)
in conical flasks (volume 250 ml) on a gyratory shaker (80 rpm)
at 23 °C under permanent diffuse light (4.4 l
mol mꢁ2 sꢁ1).
Administration of [methyl-13C]
L-methionine to W. thyrsiflora for
13C NMR-guided fractionation (Section 2.1): Root cultures of W.
thyrsiflora were transferred to fresh medium 4 days before an
group from labelled [methyl-13C]
L-methionine does not allow to
discriminate between early and late methoxylation, [2-13C,40-
O13CH3]40-methoxycinnamic acid as an advanced precursor was
administered to Musa. Demethylation of the labelled 40-O-methyl
group (and unexpected formation of 30,40-methylenedioxycinnam-
ic acid) suggests that 40-O-methylirenolone is not formed from a 40-
methoxycinnamoyl precursor but more likely from the 40-O-meth-
ylation of irenolone. Molecular and biochemical studies are re-
quired in Musaceae and Haemodoraceae to shed light on
methoxylation of various positions of the phenylphenalenone skel-
eton and characterize their putative O-methyltransferases. En-
hanced formation of phenylphenalenones including their O-
methylated analogues in response to elicitation with CuCl2 and jas-
monic acid, respectively, in M. acuminata and W. thyrsiflora suggest
that these natural products help defend plants against pathogens
and/or herbivores.
aqueous solution of [methyl-13C]
L
-methionine (4 mg) was added
m) to give a final concentration
M. Then JA was added in the same manner to obtain a con-
centration of 80 M in the medium. The same procedure was used
through a membrane filter (0.2
of 270
l
l
l
to study the inducibility of methoxy-phenylphenalenone biosyn-
thesis in W. thyrsiflora (Section 2.2). The following combinations
of JA and [methyl-13C] -methionine (13C-met) were performed in
L
duplicate or triplicate: JA; JA + 13C-met; 13C-met; untreated con-
trol. The plants were extracted after 6 days.
4.2. Extraction of plant material and isolation of phenylphenalenone-
type compounds and 30,40-methylenedioxycinnamic acid
After the incubation, roots of in vitro plants of M. acuminata
(Section 2.1) were cut, frozen in liquid N2, macerated and extracted
with acetone at room temperature. After evaporation (<30 °C) the
extract was dissolved in 1 ml MeOH, and phenylphenalenone-re-
lated compounds 1–4 were separated by semipreparative re-
versed-phase HPLC (HPLC method 3). Compound 1: Rt 33.3 min,
2: Rt 18.7 min, 3: Rt 24.4 min, 4: Rt 18.7 min. Further purification
of compounds 2 and 4 was achieved by preparative TLC on pre-
coated Si gel 60 F254 plates (Merck, layer thickness 0.25 mm) using
n-hexane–diethyl ether (1:2) as an eluent. HPLC method 1 was
used for final purification.
The procedure used for M. acuminata was also employed to ex-
tract cultured roots of W. thyrsiflora (Section 2.1) and isolate phe-
nylphenalenones except that MeOH was used instead of acetone
in order to also extract some hydrophilic phenylphenalenone glu-
cosides occurring in this plant. After evaporation, the MeOH ex-
tract was partitioned between n-hexane–H2O and CH2Cl2–H2O,
and compounds 5–7 were purified from the CH2Cl2 fraction using
semipreparative reversed-phase HPLC. Compound 5: Rt 30.6 min,
6: Rt 20.4 min, 7: Rt 26.0 min, HPLC method 4.
4. Experimental
4.1. Plant material and administration of elicitors and labelled
precursors
M. acuminata: Sterile in vitro plants of M. acuminata var. Wil-
liams (group AAA) were provided by Universidad Católica de Ori-
ente (Rionegro-Antioquia, Colombia). They were maintained in
MS liquid medium (Murashige and Skoog, 1962) at 23 °C under a
dark/light cycle of 12 h. Plants with both average root lengths of
5 cm and minimum heights of 5 cm were used for feeding
experiments.
Administration of [methyl-13C]
L-methionine to M. acuminata for
13C NMR-guided fractionation (Section 2.1): In a large-scale exper-
iment, 600 in vitro plants were transferred into conical flasks (vol-
ume 250 ml) containing six plants each and a total volume of 30 ml
of liquid MS medium per flask. An aqueous solution (500 ml) con-
Roots of in vitro plants from ‘‘induction” experiments with M.
acuminata (Section 2.2) were lyophilized and extracted with EtOH,
and the extracts evaporated to dryness. The residue was parti-
tioned between n-hexane–H2O, CH2Cl2–H2O and EtOAc–H2O. The
n-hexane and CH2Cl2 phases were pooled and fractionated using
semipreparative HPLC method 3. The fraction eluting with approx-
imately 60–70% MeCN in H2O was again evaporated to dryness and
a sample dissolved in MeOH to a concentration of 1 mg mlꢁ1. Then
taining of [methyl-13C]
L
-methionine (100 mg) and CuCl2 (10 mg)
was prepared. A volume of 5 ml of this solution was added through
a membrane filter (0.2 m) into each flask and then incubated at
23 °C for 6 days. Fifty plants without [methyl-13C]
-methionine
l
L
and CuCl2 treatment were used as control. The extracts were pre-
pared in identical procedures from treated and untreated plants
and equal amounts subjected to analysis. An analogous experimen-
tal setup was used for feeding [2-13C,40-O13CH3]40-methoxycin-
namic acid (30 mg, 99% 13C) to M. acuminata (Section 2.3). To
study the inducibility of methoxy-phenylphenalenone biosynthe-
sis in M. acuminata (Section 2.2), in vitro plants of M. acuminata
were transferred individually to 30 ml MS liquid medium in
250 ml Erlenmeyer flasks and maintained at 23 °C for one week be-
pyrene was added to an amount of 0.5
nal standard from a stock solution (0.1 mg pyrene per ml MeOH). A
volume of 10 l was injected to the analytical reversed-phase HPLC
lg per injection as an inter-
l
method 2. Cultured roots of W. thyrsiflora from ‘‘induction” exper-
iments (Section 2.2) were extracted with MeOH and evaporated to
dryness. A sample of the residue was dissolved in MeOH to a con-
fore the experiment started. Aqueous solutions of [methyl-13C]
methionine (0.3 ml, 1 mg mlꢁ1), CuCl2 (0.1 ml, 1 mg mlꢁ1) and JA
(0.3 ml of a 10 mM solution to obtain a concentration of 100
in the medium), respectively, were added to the cultures under
sterile conditions through a membrane filter (0.2 m) and further
cultivated as described above. The following combinations of elic-
itors and [methyl-13C] -methionine (13C-met) were performed in
L
-
centration of 2.5 mg mlꢁ1. A volume of 50
ll of this solution was
supplemented with 0.5
lg pyrene as an internal standard and ana-
lM
lyzed using HPLC method 2.
Isolation of 30,40-methylenedioxycinnamic acid and phenylphe-
nalenones (Section 2.3): 6 days after administration of [2-13C,40-
O13CH3]40-methoxycinnamic acid to M. acuminata, the roots were
extracted with acetone using the above-mentioned procedure
l
L

212
F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
and the residue partitioned between n-hexane–H2O and CH2Cl2–
H2O. The organic phases were evaporated and subjected to 13C
NMR analysis. 30,40-Methylenedioxycinnamic acid was isolated
from the CH2Cl2 fraction by HPLC method 2. Anigorufone (Rt
40.1 min), hydroxyanigorufone (Rt 24.8 min) and musanolone F
(3, Rt 25.3 min) were also purified by HPLC method 2.
30 min ? 90:10% in 5 min ? 30:70% in 5 min (total time
40 min); flow rate 3.5 ml minꢁ1; UV detection at 254 nm.
4.5. Identification of methoxy-phenylphenalenones and 30,40-
methylenedioxycinnamic acid
4.3. Synthesis of [2-13C,40-O13CH3]40-methoxycinnamic acid
Compounds 1 and 2 were identified by comparing their 1H and
13C NMR spectra with literature data. Musanolone F (3), 5-hydro-
xy-2-methoxy-6-oxa-benzo[def]chrysen-1-one (6), methoxyanigo-
rufone (5), anigorufone, and hydroxyanigorufone were identified
by comparing their retention times and 1H NMR spectra with those
of authentic references obtained in previous studies (Schmitt et al.,
2000; Opitz et al., 2002; Hölscher and Schneider, 1997).
[2-13C]p-Coumaric acid was synthesized from 4-hydroxybenz-
aldehyde and [2-13C]malonic acid (99% 13C) (Deutero GmbH,
Kastellaun, Germany) using the Knoevenagel condensation. The
product (100 mg, 0.6 mmol) was methylated using a total of 3 ml
of an ethanol-containing ethereal solution of [13C]diazomethane,
which was prepared from N-[13C]methyl-N-nitroso-p-toluenesul-
fonamide (Cambridge Isotope Laboratories) using a standard pro-
cedure. The product, [2-13C,40-O13CH3]40-methoxycinnamic acid
methyl ester (70 mg, 0.4 mmol), was refluxed with 8 ml of an aque-
ous solution of sodium hydroxide (25%) for 2 h. The solution was
acidified to pH ꢂ1 and extracted with two portions of 10 ml of
diethyl ether. Evaporation of the solvent gave [2-13C,40-
O13CH3]40-methoxycinnamic acid. 1H NMR (400 MHz, MeOH-d4):
2-(4-Hydroxy-3-methoxyphenyl)-naphthalene-1,8-dicarbox-
ylic anhydride (4) is a new natural product. 1H NMR and 1H,1H
COSY spectra showed signals of an AB spin system of H-3 and H-
4, and two AMX spin systems, one of which is due to H-5/H-6/H-
7 and the other one to H-20/H-50/H-60 of the lateral 30,40-disubsti-
tuted phenyl ring. An exchangeable broad singlet (d 8.00) indicated
a phenolic hydroxyl group. The methoxy signal at d 3.98, typical of
an O-methyl group, appears as a singlet of an [O12CH3]-isotopomer
and, due to 13C–1H coupling, a doublet (JH–C = 144.5 Hz) of the la-
belled [O13CH3]-isotopomer. The missing singlet of a ring A
methine, together with an also missing carbon (only 18 instead
of usual 19 C atoms of the phenylphenalenone skeleton were de-
tected, not counted the additional O-methyl carbon) and two car-
bonyl resonances, suggested an anhydride structure of ring A.
The downfield resonance of H-7 at d 8.63 was in agreement with
a peri position of this proton relative to a carbonyl group. A HMBC
cross signal supporting this suggestion was detected between Y-7
and the carbonyl signal at d 159.8 (C-9). The anhydride structure
was further confirmed by a weak HMBC w-correlation through
four bonds between H-3 (d 7.79) and the other carbonyl group
(C-10, d 162.1). The location of the methoxy group at C-30 was
established by a HMBC correlation of the corresponding signal (d
3.89) with the carbon at d 147.2. HREIMS obs. m/z 320.06918 (calc.
for C19H12O5, 320.06847). 1H NMR (500 MHz, acetone-d6): d 8.63
(1H, dd, J1 = 7.6 Hz; J2 = 1.3 Hz, H-7), 8.54 (1H, dd, J1 = 8.0 Hz;
J2 = 1.0 Hz, H-5), 8.48 (1H, d, J = 8.5 Hz, H-4), 8.00 (1H, s, –OH),
7.94 (1H, dd, J1 = 7.6 Hz; J2 = 7.5 Hz, H-6), 7.79 (1H, d, J = 8.5 Hz,
H-3), 7.18 (1H, d, J = 2.0 Hz, H-20), 7.01 (1H, dd, J1 = 8.0 Hz;
J2 = 2.0 Hz, H-60), 6.95 (1H, d, J = 8.0 Hz, H-50), 3.89 (3H, s, –OCH3).
13C NMR (125 MHz, acetone-d6): d 162.1 (C-9), 159.8 (C-10),
150.6 (C-10), 150.5 (C-2), 147.7 (C-40), 147.2 (C-30), 135.9 (C-5),
134.5 (C-4), 133.3 (C-7), 132.6 (C-3), 132.3 (C-4a), 132.2 (C-8a),
127.5 (C-6), 122.2 (C-60), 120.4 (C-8), 116.6 (C-1), 115.2 (C-50),
113.3 (C-20), 55.9 (–OCH3).
2
3
d 7.62 (1H, dd, JH-3–C-2 = 3.0 Hz, JH-3–H-2 = 16.0 Hz, H-3), 7.54
(2H, d, J = 8.8 Hz, H-20/60), 6.95 (2H, d, J = 8.8 Hz, H-30/H-50), 6.36
1
3
(1H, dd, JH-2–C-2 = 160.9 Hz, JH-2–H-3 = 16.0 Hz, H-2), 3.80 (3H, d,
1JH–C = 144.2, OCH3); 13C NMR (100 MHz, MeOH-d4): d 146.5 (C-
3
40), 145.8 (C-3), 130.9 (C-20/C-60, d, JC-2 /6 –C-2 = 4.5 Hz), 128.4 (C-
0
0
3
10), 116.6 (C-2), 115.4 (C-20/C-60, d, JC-3 /C-5 –OCH3 = 4.2 Hz), 55.9
(OCH3), the carbonyl signal (C-1) was not detected due to poor sig-
nal-to-noise ratio.
0
0
4.4. Spectroscopic and chromatographic methods
NMR spectroscopic analyses were performed on a Bruker AV
500 NMR spectrometer operating at 500.13 MHz (1H) and
125.75 MHz (13C) or a Bruker AV 400 NMR spectrometer operating
at 400.13 MHz (1H) and 100.75 MHz (13C) (Bruker-Biospin, Kar-
lsruhe, Germany). Chemical shifts are reported relative to tetra-
methylsilane (TMS). 13C NMR spectra of crude extracts were run
using 5 mm broadband probes. 1H NMR, 13C NMR and 2D NMR
spectra of isolated compounds were recorded using a 2.5 mm
broadband microprobe (500 MHz),
a 5 mm TXI cryoprobe
(500 MHz), or a 5 mm broadband inverse probe (400 MHz). ESI-
MS and ESI-HRMS were measured in the positive ion mode on a
Micromass Quattro II tandem quadrupole mass spectrometer
(Micromass Ltd., Manchester, UK).
Analytical reversed-phase HPLC was carried out on an Agilent
1100 chromatography system (binary pump G1312A, DAD
G1315B, autosampler G1313A). HPLC method 1: LiChrospher 100
RP-18 column (5
l
m; 250 ꢃ 5 mm); MeCN–H2O gradient (0.1%
5-Hydroxy-3,6-dimethoxy-7-phenyl-3H-benzo[de]isochromen-
1-one (7). 1H NMR (500 MHz, MeOH-d4): d 8.22 (1H, d, J = 7.1 Hz,
H-9), 7.58 (1H, s, H-4), 7.34 (5H, m, H-10–H-50), 7.32 (1H, d,
J = 7.1 Hz, H-8), 6.47 (1H, s, H-3), 3.96 (3H, s, 6-OCH3), 3.67 (3H,
s, 3-OCH3); 13C NMR (125 MHz, MeOH-d4): d 166.3 (C-1), 147.2
(C-7), 145.2 (C-5), 145.0 (6-OCH3), 144.7 (C-10), 129.8 (C-8), 128.7
(C-9), 128.0 (C-20/60), 127.8 (C-30/C-50 and C-40), 125.6 (C-9b),
122.0 (C-6a), 119.3 (C-9a), 119.0 (C-3a), 115.4 (C-4), 103.9 (C-3),
57.8 (6-OCH3), 56.4 (3-OCH3).
TFA) 5%:95% ? 65%:35% in 50 min ? 90:10% in 5 min and
90%:10% for another 5 min ? 5%:95% in 5 min (total time
65 min); flow rate 0.8 ml minꢁ1; DAD detection 200–620 nm, mon-
itoring wavelength at 254 nm. HPLC method 2: Nucleosil 100-3
C18 (3
l
m; 250 ꢃ 3 mm); MeCN–H2O gradient (0.1% TFA)
35%:65% ? 70%:30% in 60 min ? 35%:65% in 5 min (total time
65 min); flow rate 0.4 ml minꢁ1; DAD detection 200–620 nm, mon-
itoring wavelength at 254 nm.
Semipreparative reversed-phase HPLC runs were performed on
a Merck-Hitachi HPLC system composed of an L-4250 UV–Vis
detector and an L-6200A pump. HPLC method 3: LiChrospher 100
[2-13C]30,40-Methylenedioxycinnamic acid: EIMS m/z (rel. int.):
193 [M+ of 2-13C-isotopomer] (15), 192 [M+ of natural abundance
isotopomer] (100). 1H NMR (500 MHz, MeOH-d4): d 7.58 (1H, d,
J = 15.9 Hz, H-3), 7.15 (1H, d, J = 1.8 Hz, H-20), 7.07 (1H, dd, J = 8.1,
1.8 Hz, H-60), 6.84 (1H, d, J = 8.1 Hz, H-50), 6.31 (1H, d, J = 15.9 Hz,
H-2), 6.00 (2H, s, –CH2–); 13C NMR (125 MHz, MeOH-d4): d 170.5
(C-1), 150.3 (C-40), 149.3 (C-30), 146.4 (C-3), 130.4 (C-10), 125.8
(C-60), 116.7 (C-2), 109.6 (C-50), 107.6 (C-20), 103.2 (–CH2–).
RP18 column (10
l
m; 250 ꢃ 10 mm); MeCN (0.1% TFA)–H2O
(0.1% TFA) gradient 30:70% ? 90:10% in 50 min ? 30:70% in
5 min (total time 55 min); flow rate 3.5 ml minꢁ1; UV detection
at 254 nm. HPLC method 4: LiChrospher 100 RP18 column
(10
lm; 250 ꢃ 10 mm); MeCN-H2O gradient 30:70% ? 65:35% in

F. Otálvaro et al. / Phytochemistry 71 (2010) 206–213
213
Liu, J.G., Benedict, C.R., Stipanovic, R.D., Bell, A.A., 1999. Purification and
characterization of S-adenosyl-
-methionine: desoxyhemigossypol-6-O-
Acknowledgements
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We thank Colciencias, Universidad de Antioquia (Programa de
Sostenibilidad), the Max Planck Institute for Chemical Ecology for
financial support and Emily Wheeler for editorial assistance.
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Appendix A. Supplementary data
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