Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

Article abstract of DOI:10.1016/j.phytochem.2012.02.020

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phe

Full text of DOI:10.1016/j.phytochem.2012.02.020

Phytochemistry 91 (2013) 165–176
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Phytochemistry
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Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora
⇑
Tobias Munde, Silke Brand, William Hidalgo, Ravi K. Maddula, Aleš Svatoš, Bernd Schneider
Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Straße 8, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 17 March 2012
The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to
which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora
were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of
recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid
and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with
13C-labelled precursors in an 18O2 atmosphere to observe incorporation of the two isotopes at defined
biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composi-
tion of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results
suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosyn-
thetic stages in the sequence O-1 ? O-2 ? O-5. In addition, the incubation of W. thyrsiflora root cultures
with 13C-labelled lachnanthocarpone established a direct biosynthetic precursor–product relationship
with 1,2,5,6-tetraoxygenated phenylphenalenones.
Dedicated to the memory of
Meinhart H. Zenk.
Keywords:
Wachendorfia thyrsiflora
Diarylheptanoids
Haemodoraceae
Isotope-induced chemical shift
Isotope effect
Isotopologues
Labelling
Nuclear magnetic resonance
Oxygen-18
Ó 2012 Elsevier Ltd. All rights reserved.
Phenylphenalenones
1. Introduction
a linear diarylheptanoid, and the carbonyl oxygen atom originated
from the hydroxyl group of the 4-coumaroyl moiety.
Phenylphenalenones are a group of polycyclic natural products
occurring in the Musaceae (banana) (Luis et al., 1994; Kamo et al.,
1998; Hölscher and Schneider, 1998; Otálvaro et al., 2007) and re-
lated plant families Strelitziaceae (Strelitzia reginae, the bird of par-
adise plant) (Hölscher and Schneider, 2000), Haemodoraceae (e.g.
Anigozanthos species, the kangaroo paw) (Cooke and Thomas,
1975; Hölscher and Schneider, 1997; Hölscher and Schneider,
1999; Opitz et al., 2002), and Pontederiaceae (e.g. Eichhornia crass-
ipes, the water hyacynth) (DellaGreca et al., 2008; Hölscher and
Schneider, 2005). Bioassay studies have demonstrated the anti-
fungal properties of (phenyl)phenalenones (Quiñones et al., 2000;
Otálvaro et al., 2007). The remarkable structural diversity of
phenylphenalenones is mainly due to their variable oxygenation
pattern. Anigorufone (3a) (Fig. 1), which occurs in most phenyl-
phenalenone-producing plants, is one of the simplest 2-hydroxy-
9-phenylphenalen-1-ones. Recently, the biosynthetic origin of the
1-keto-2-hydroxy motif of phenylphenalenones was elaborated
using 13C/18O labelling experiments and isotopologue analysis
based on nuclear magnetic resonance (NMR) spectroscopy and
high resolution electro-spray mass spectrometry (HRESIMS)
(Munde et al., 2011). It was shown that the hydroxyl group at
C-2 of 9-phenylphenalenones had been introduced at the stage of
Not only 2-hydroxy-9-phenylphenalen-1-ones such as anigo-
rufone (3a) but also compounds such as lachnanthoside aglycone
(2a) and its allophanyl glucoside (1a) (Fig. 1) with two more hydro-
xyl groups in the phenalenone nucleus occur in Wachendorfia
thyrsiflora (Fang et al., 2011), Xiphidium caeruleum (Opitz et al.,
2002) and some other Haemodoraceae (Cooke and Edwards,
1980). One of the additional oxygen atoms is located at C-6, and,
in most phenylphenalenones, the other atom is attached to C-5
but can also be found at C-4. The oxygen functionality at C-6 very
likely comes from the carbonyl thioester of 4-coumaroyl–CoA,
which in the course of phenylphenalenone biosynthesis condenses
with a diketide to form a diarylheptanoid (Brand et al., 2006).
Unlike the origin of the oxygen atom at C-6, nothing is known
about the introduction of the oxygen at C-5.
In order to probe for the origin of the oxygen functionalities
and the sequence according to which they are introduced to the
phenylphenalenone scaffold in W. thyrsiflora, two independent
approaches were used. First, the substrate specificity of WtPKS1
was investigated. The recombinant enzyme was incubated with
a-oxygenated phenylpropanoyl N-acetyl cysteamine (NAC) deriva-
tives [phenylpyruvoyl–NAC, 3-(4-hydroxyphenyl)pyruvoyl–NAC,
(R)- and (S)-phenyllactoyl–NAC), (R)- and (S)-3-(4-hydroxyphenyl)
lactoyl–NAC] as mimics of the corresponding CoA esters.
In a second approach, root cultures of W. thyrsiflora were supple-
⇑
Corresponding author. Tel.: +49 3641 571600: fax: +49 3641 571601.
E-mail address: schneider@ice.mpg.de (B. Schneider).
mented with [2-13C] -phenylalanine ([2-13C]Phe), [2-13C]4-coumaric
L
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2012.02.020

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T. Munde et al. / Phytochemistry 91 (2013) 165–176
phenylpyruvoyl–CoA, as starter units. Hypothetically, an a-oxygen-
ated 3-(4-hydroxyphenyl)propanoyl–CoA could function as a sub-
strate of WtPKS1. If the hypothetical biosynthetic pathway shown
in Fig. 2 operated in W. thyrsiflora, the a-oxygen of the phenylprop-
anoid side chain would end up at C-5 of the phenylphenalenone
skeleton.
N-Acetyl cysteamine (NAC) esters have been reported instead of
CoA esters to be the starter substrates of polyketide synthases (Cane
et al., 1991; Jacobs et al., 1991). Therefore, activated NAC esters of
(R)- and (S)-phenyllactate, (R)- and (S)-3-(4-hydroxyphenyl)lactate,
phenylpyruvate and 3-(4-hydroxyphenyl)pyruvate were synthe-
sized according to reported procedures (Gilbert et al., 1995; Pohl
et al., 1998) and, instead of CoA esters, assayed with WtPKS1. Acti-
vated phenylpropionyl- and cinnamoyl derivatives were used as
reference substrates to compare the relative activities obtained
for the incubation of WtPKS1 with NAC esters and with CoA esters
(Table 1). In a previous study, the phenylpropionyl–CoA ester was
among the best substrates in vitro and the cinnamoyl–CoA ester
was among the poorest (Brand et al., 2006). Assay conditions opti-
mized for phenylpropionyl–CoA as a starter substrate were used as
previously reported (see Experimental).
Although the relative reactivity of WtPKS1 with NAC esters was
drastically reduced compared to CoA esters, products of enzymatic
conversion were still readily detectable, demonstrating that the
NAC esters are useful starter substrates. In all experiments,
[2-14C]malonyl–CoA was used as the extender substrate. The prod-
ucts were detected in the assay by their UV absorption at 280 nm
and the radiolabel originating from [2-14C]malonyl–CoA. With (S)-
phenyllactoyl–NAC as a starter substrate, the condensation product
with two malonyl–CoA units was identified as (S)-6-[1-hydroxy-2-
(4-hydroxyphenyl)ethyl]-4-hydroxy-2H-pyran-2-one (4, Rt 28.2
min) by LCESIMS (m/z 249 [M + H]+) (see Fig. 3b). A minor, accord-
ing to its retention time, slightly more polar product was tenta-
tively assigned to benzalacetone. According to LCESIMS data and
the long retention times, the structures of enzymatic condensations
with starter substrates possessing an unsubstituted phenyl ring
were identified as (R)-6-(1-hydroxy-2-phenyl)ethyl-4-hydroxy-
2H-pyran-2-one (5) (37.7 min, m/z 231 [M-H]-), (S)-6-(1-hydroxy-
2-phenyl)ethyl-4-hydroxy-2H-pyran-2-one (6) (37.7 min, m/z 231
[M-H]-), and (E)-6-(1-hydroxy-2-phenyl)ethenyl-4-hydroxy-2H-
pyran-2-one (7) (38.8 min, m/z 231 [M + H]+) (Fig. 3).
WtPKS1 converts (S)-phenyllactoyl–NAC to the pyrone (6; rel.
activity 101%) (Fig. 4) as efficient as phenylpropionyl–NAC
(100%). In contrast, WtPKS1 was much less active (51%) with the
R-enantiomer, (R)-phenyllactoyl–NAC, to produce 5. With (S)-
3-(4-hydroxyphenyl)lactoyl–NAC, a substrate having a hydroxyl
group in 4-position of the phenyl ring, the activity of WtPKS1
was reduced to 34% compared to the non-hydroxylated substrate,
(S)-phenyllactoyl–NAC, although it was still better accepted than
cinnamoyl–NAC (24%). Phenylpyruvoyl–NAC (30%) is nearly as effi-
ciently accepted as (S)-3-(4-hydroxyphenyl)lactoyl–NAC (34%). No
product formation was observed with 3-(4-hydroxyphenyl)pyru-
voyl–NAC and (R)-3-(4-hydroxyphenyl)lactoyl–NAC.
Fig. 1. Structures of 1,2,5,6-tetraoxygenated phenylphenalenones used as target
compounds to study biosynthetic oxygenation in root cultures of W. thyrsiflora and
anigorufone; the last compound exemplifies a 2-hydroxy-9-phenylphenalen-1-one.
Because of tautomerism of 2a, the carbonyl group of 2a/2b appears at different
positions in the phenalenone tricycle. For simplicity, the position peri to the phenyl
ring is designated C-1 in the text and in structure drawings of all tautomeric
1,2,5,6-tetraoxygenated phenylphenalenones, regardless if this position is bearing a
keto or hydroxyl functionality. Thus, the numbering used in this paper is different
from that used in chemical names, which are as follows: 1a, 6-O-[(6’’-O-allophanyl-
ß-D-glucopyranosyl]-2,5-dihydroxy-7-phenylphenalen-1-one; 1b, 6-O-[(6’’-O-
allophanyl-ß-D-glucopyranosyl]-2,5-dimethoxy-7-phenylphenalen-1-one; 2a, two
tautomers of lachnanthoside aglycone, 2,5,6-trihydroxy-9-phenylphenalen-1-one
and 2,5,6-trihydroxy-7-phenylphenalen-1-one; 2b, two tri-O-methyl derivatives of
lachnanthoside aglycone, [2,5,6-(O13CH3)3]2,5,6-trimethoxy-9-phenylphenalen-1-
one and [2,5,6-(O13CH3)3]2,5,6-trimethoxy-7-phenylphenalen-1-one.
acid ([2-13C]CA), and two 13C-labelled diarylheptanoids, [6-13C]
(4E,6E)-1-(4-hydroxyphenyl)-7-phenylhepta-4,6-dien-3-one (DAH-
I), and [6-13C](4E,6E)-1-(3,4-dihydroxyphenyl)-7-phenylhepta-4,6-
dien-3-one (DAH-II), and simultaneously incubated in an 18O2
atmosphere. Phenylphenalenones isolated from these precursor
administration experiments were subjected to NMR- and HRE-
SIMS-based isotopologue analysis. The results of the enzymatic
studies and labelling experiments are discussed with respect to
the biosynthetic sequence according to which oxygen functional-
ities are introduced to the phenylphenalenone scaffold.
In addition, a 1,2,6-trioxygenated phenylphenalenone, lachnan-
thocarpone (3b), which is a potential biosynthetic precursor of
tetraoxygenated phenylphenalenones, was administered in 13C-
labelled form to probe for direct precursor–product relationships.
2. Results
2.1. a-Oxygenated phenylpropanoids are substrates of WtPKS1 in vitro
Pyrones have been formed by WtPKS1 in vitro (in addition to
benzalacetones) from phenylpropanoids, but the products in vivo
are diketides, which are converted to diarylheptanoids and, further
downstream in the biosynthetic pathway, to phenylphenalenones.
Hence, the formation of pyrones as a result of in vitro incubation
In W. thyrsiflora, WtPKS1 (Genbank™ accession number
AY727928) catalyzes the condensation of a phenylpropanoyl–CoA
with one malonyl–CoA to form a diketide. The diketide is then
condensed with another phenylpropanoyl–CoA to form
a
experiments of WtPKS1 with
a-oxygenated phenylpropanoid–
diarylheptanoid, which is further processed to form phenylphenale-
nones (Brand et al., 2006). As shown by competitive precursor
administration experiments (Schmitt and Schneider, 1999) (which
indicated reversible conversion of phenylpropionic acid and
cinnamic acid) and as discussed by Brand et al. (2006), 4-couma-
royl–CoA seems to be the starter substrate of WtPKS1 in vivo. In vitro,
however, WtPKS1 accepts various phenylpropanoyl–CoAs, including
NAC thioesters shows that some of them have the potential to
function as precursors of the diarylheptanoid/phenylphenalenone
biosynthesis. However, since the results with PKS type III enzymes
in vitro generally do not provide convincing clues for the activity of
the protein in vivo, alternative approaches were used to confirm or
disprove the outcome of the enzymatic experiments.

T. Munde et al. / Phytochemistry 91 (2013) 165–176
167
Fig. 2. Hypothetical ‘‘early hydroxylation’’ biosynthesis of 1,2,5,6-tetraoxygenated phenylphenalenones as probed by the incubation of WtPKS1 with
a-oxygenated
phenylpropanoyl thioesters and incubation of W. thyrsiflora root cultures with the corresponding 13C-labelled precursors. The hypothetical fate of oxygen atoms is coded by
color. Oxygen atoms which are suggested to be kept during phenylphenalenone biosynthesis are highlighted in red; lost oxygen atoms are displayed in bold; and oxygen
atoms incorporated from O2 are highlighted in blue. Phenylpropanoid units are marked with bold C–C bonds. d indicates the origin of the marked atom from C-2 of malonate
(Hölscher and Schneider, 1995b).
2.3. HRESIMS- and NMR-based 13C/18O isotopologue analysis
Table 1
Relative activities of WtPKS1 for cinnamoyl– and phenylpropionyl–CoA and –NAC
esters as starter substrates.
Unlike the administration of
a-oxygenated 3-(4-hydroxy-
phenyl)propanoic acids, precursor administration experiments
with radio-labelled phenylalanine and 4-coumaric acid showed
that the label in the medium decreased within 1 day to 84% for
[2,3,4,5,6-3H]Phe (1.5 mg, 0.37 kBq) and 43% for [U–14C]CA
(1.5 mg, 0.34 kBq), suggesting absorption into the root tissue. The
Starter substrate
Relative activity [%]
Cinnamoyl–CoA
Cinnamoyl–NAC
Phenylpropionyl–CoA
Phenylpropionyl–NAC
8
2
100
7
absorption of L-Phe and hydroxycinnamic acids from the medium
See experimental section for conditions.
into the cultured roots is consistent with previous studies, in which
the corresponding 13C-labelled precursors were smoothly incorpo-
rated into phenylphenalenones in W. thyrsiflora (Opitz and
Schneider, 2003; Brand et al., 2006) and root cultures of the related
species, Anigozanthos preissii (Hölscher and Schneider, 1995b;
Schmitt et al., 2000).
2.2. Early precursor administration experiments
Three 13C-labelled
a-oxygenated 3-(4-hydroxyphenyl)propa-
Recent 13C/18O labelling experiments have demonstrated the
usefulness of isotopologue analysis to infer the biosynthetic origin
of the oxygen atoms in positions 1 and 2 of simple phenylphenale-
nones (Munde et al., 2011). Thus, 13C/18O-labelled isotopologues of
compounds 1a and 2a from W. thyrsiflora were also considered to
encode useful biosynthetic information about the sequence
according to which oxygen functionalities are introduced into
phenylphenalenones.
noic acids, [2-13C](S)-3-(4-hydroxyphenyl)lactic acid, [2-13C](R)-
3-(4-hydroxyphenyl)lactic acid, and [2-13C]3-(4-hydroxyphenyl)
pyruvic acid, were synthesized and administered to root cultures
of W. thyrsiflora in order to probe for their incorporation into phe-
nylphenalenones. However, enhancement of 13C signals was ob-
served neither in the NMR spectra of the root extracts nor in
isolated lachnanthoside aglycone (2a) and its allophanyl glucoside
(1a). As shown by HPLC of aliquots taken regularly from the med-
The administration of 13C-labelled precursors to root cultures,
which were simultaneously incubated in an 18O2 atmosphere, fol-
lowed by the isolation of phenylphenalenones 1a and 2a, and
O-13CH3-methylation of the hydroxyl groups, was used to confirm
the previously reported origin of the 1-keto-2-hydroxy motif
ium, significant absorption of the a-oxygenated precursor into the
roots did not take place. Therefore, the results of these precursor
administration experiments were inconclusive, and the biosynthe-
sis of 2-oxygenated diarylheptanoids and 5-oxygenated phenyl-
phenalenones remained unclear.

168
T. Munde et al. / Phytochemistry 91 (2013) 165–176
Fig. 3. (a) Starter substrates and (b) their corresponding products formed by incubation with [2-14C]malonyl–CoA and WtPKS1. Products of two condensations are (S)-6-
[1-hydroxy-2-(4-hydroxyphenyl)ethyl]-4-hydroxy-2H-pyran-2-one (4), (R)-6-(1-hydroxy-2-phenyl)ethyl-4-hydroxy-2H-pyran-2-one (5), (S)-6-(1-hydroxy-2-phenyl)ethyl-
4-hydroxy-2H-pyran-2-one (6), and (E)-6-(1-hydroxy-2-phenyl)ethenyl-4-hydroxy-2H-pyran-2-one (7). (c) Enzymatic activity of WtPKS1 with different NAC esters, relative
to the activity with phenylpropionyl–NAC (100%). Mean values of three experiments.
(Munde et al., 2011) by NMR and MS. Whether the additional
oxygen atoms in positions 5 and 6 of 1,2,5,6-tetraoxygenated
phenylphenalenones 1a and 2a are incorporated as a unit with
18O atoms incorporated from atmospheric 18O2. The 13C NMR spec-
tra of the 2,5-di-O13CH3 derivative 1b obtained from [2-13C]Phe
and [2-13C]CA, respectively, exhibited weak but distinct
2-18O13CH3 and 5-18O13CH3 signals; these were shifted to a slightly
a-oxygenated 4-hydroxyphenylpropanoids or come from an alter-
native late hydroxylation was the subject of the double labelling
experiments.
higher field compared to the large 2-16O13CH3 1D 18O(13C) =
( d
À29 ppb) and 5-16O13CH3 signals (1
D
d
18O(13C) = À25 ppb) (Fig. 4,
[2-13C]Phe, [2-13C]CA, [6-13C]DAH-I, and [6-13C]DAH-II were
used as 13C-labelled precursors. Labels derived from [2-13C]Phe
into C-5 and C-8 (for a comment on numbering see caption of
Fig. 1) of the allophanyl glycoside 1a were reportedly incorporated
(Brand et al., 2006) and confirmed in the present investigation for
[2-13C]Phe and [2-13C]CA as precursors of phenylphenalenones in
W. thyrsiflora (data not shown). 18O-Induced 13C NMR signals of
C-2 and C-5 of 1a were not detected, indicating the low level of
18O that was incorporated. In order to enhance the sensitivity with
which 18O is detected by isotope-induced 13C NMR signals, samples
of the target compound obtained from precursor administration
experiments were methylated with 13CH2N2, thus attaching a 13C
atom to each hydroxyl group, including those groups containing
a and b). The Dd values are in the order of magnitude of a-induced
isotope shifts (Risley and van Etten, 1990). 18O-Induced 13C NMR
shifts of the same magnitude were also observed for C-2 and C-5
of compound 1b after administering [6-13C]DAH-I and
[6-13C]DAH-II (Fig. 4, c and d). These results demonstrated that
the two hydroxyl groups at C-2 and C-5 of 1a/b are derived from
atmospheric oxygen. The sequence of oxygen incorporation, how-
ever, remained unknown because the 13C NMR spectra suggested
that the occurrence of 18O in positions 2 and 5 seems to be inde-
pendent of the administered precursor (Fig. 4). The reason that
18O appeared to be attached to C-2 and C-5, regardless of the
administered precursor, is that NMR of isotopologue mixtures does
not necessarily distinguish between individual isotopologues, i.e.,

T. Munde et al. / Phytochemistry 91 (2013) 165–176
169
because heavy isotopes used for labelling did not show isotope-in-
duced shifts (e.g. 2-18O and 5-13C) or interact through spin–spin
coupling (13C-5 and 13C-8) if they were located in remote positions.
Furthermore, differences in the uptake and transportation of
precursors, pool size perturbation (Munde et al., 2011), and the
low accuracy with which small 18O13CH3 signals versus large
16O13CH3 signals in the 13C NMR spectra were integrated also made
it difficult to quantify isotopologues and understand the sequence
of 18O incorporation. Moreover, 18O-induced shifts were not ob-
served for C-1 (in 1a/b substituted with O-allophanyl-glc) and
C-6 (C = O) because O-methylation with 13CH2N2 was impossible
in these positions. Thus, NMR-based quantitative isotopologue
analysis was supplemented by mass spectrometry, especially to
determine 13C/18O-labelled isotopologues.
According to the NMR spectroscopic results, the incorporation
of 13C from labelled precursors and 18O from the gas phase
(Fig. 4) into the allophanyl glucoside 1a was low, suggesting large
proportions of unlabelled and singly labelled (18O or 13C) isotopo-
logues. This was confirmed by analysis of HRESIMS data (Table 2)
of the di-O13CH3 derivative 1b of the biosynthetically obtained
allophanyl glucoside 1a. Approximately 76% to 78% of the target
molecules accounted for the isotopologues 1bA and another
approximately 20% were natural abundance isotopologues, mainly
due to 1.1% 13C in non-enriched positions of the molecule (not
shown in Table 2). In addition to isotopologue 1bA containing
13C only in the diazomethane-derived O-methyl groups, isotopo-
logue groups B – H were identified in the case of compound 1b.
The isotopologues of group 1bB contained one additional 13C
either at C-5 or C-8 but did not incorporate 18O and therefore were
not useful for studying oxygenation. Unlike 1bB, the isotopologues
1bC, 1bE, and 1bG did not contain 13C, except in the diazomethane-
derived O13CH3 group, but incorporated one (1bC), two (1bE), or
three 18O atoms (1bG), respectively, from the gas phase, demon-
strating that the experimental system works. The value of isotopo-
logues containing only 18O for determining the sequence of
oxygenation was limited because they were not formed from the
13C-labelled precursors. The isotopologue group 1bH was also un-
able to provide new information about the sequence of oxygena-
tion because, except for the carbonyl oxygen at C-6 (which was
derived from the carbonyl oxygen of phenylpropanoic acid), 16O
atoms of the oxygen functionalities were uniformly substituted
by 18O.
Fig. 4. Partial 13C NMR spectra (125 MHz, acetone-d6, regions of O-methyl signals)
of compound 1b prepared from 1a by O-methylation using 13CH2N2 (99% 13C).
Samples of compound 1a were obtained from W. thyrsiflora root cultures incubated
in an atmosphere of 18O2 and supplemented with 13C-labelled precursors: (a)
[2-13C]Phe, (b) [2-13C]CA, (c) [6-13C]DAH-I, (d) [6-13C]DAH-II. Spectrum (e) was
recorded from unlabelled 1b (reference).
Therefore, to understand how the 18O is incorporated, only the
isotopologue groups 1bD and 1bF had to be considered; these
groups possess one 13C atom in the carbon skeleton and either
one (1bD) or two 18O atoms (1bF) in the functional groups. As pre-
viously reported (Brand et al., 2006) and confirmed here by means
it was not always evident from the NMR spectra whether the label
belongs to the same or different isotopologues. This was the case
Table 2
13C/18O Isotopologue composition of labelled 1b as determined by HRESIMS (positive ion mode) and 13C NMR (125 MHz). Compound 1a was isolated from root cultures of W.
thyrsiflora after these were incubated with 13C-labelled precursors ([2-13C]Phe, [2-13C]CA, [6-13C]DAH-I, [6-13C]DAH-II) in an atmosphere of 18O2 followed by O-methylation with
13CH2N2 to afford 1b.
Isotopologue
Isotopologue of 1b obtained from the experiment with
[2-13C]Phe
[2-13C]CA
[6-13C]DAH-I
[6-13C]DAH-II
No.
Molecular composition of
aglycone cation
m/z
m/z
Relative
abundance
(%)a
m/z
Relative
abundance
(%)a
m/z
Relative
abundance
(%)a
m/z
Relative
abundance
(%)a
[M + H]+
(calcd.)
[M + H]+
(found)
[M + H]+
(found)
[M + H]+
(found)
[M + H]+
(found)
12C 13C 1H 16O 18O
1bA 19
1bB 18
1bC 19
1bD 18
1bE 19
2
3
2
3
2
3
2
3
17
17
17
17
17
17
17
17
4
4
3
3
2
2
1
1
0
0
1
1
2
2
3
3
335.1188
336.1222
337.1231
338.1264
339.1273
340.1307
341.1316
342.1349
335.1201
336.1232
337.1254
338.1273
339.1285
340.1315
341.1345
342.1358
78.22
0.55
0.42
0.41
0.19
0.33
0.10
0.11
335.1202
-
78.46
0.00
0.07
0.29
0.51
0.28
0.81
0.00
335.1201
336.1233
337.1256
338.1275
339.1283
340.1314
341.1325
–
76.20
1.22
0.83
0.98
0.46
0.21
0.44
0.00
335.1197
336.1227
337.1252
338.1269
339.1276
-
76.27
2.06
0.26
0.42
0.66
0.00
0.82
0.00
337.1256
338.1274
339.1283
340.1316
341.1326
–
1bF
18
1bG 19
1bH 18
341.1318
–
a
Isotopologues containing the level of naturally abundant 13C were eliminated.

170
T. Munde et al. / Phytochemistry 91 (2013) 165–176
of analysis of 13C NMR and HSQC spectra (data not shown), 13C
from [2-13C]Phe appears at C-5 or C-8. The experiment with
[2-13C]CA had similar results, although the formation of [8-13C]-
isotopologues was not possible in this case and the 13C-label had
to have been located exclusively at C-5. The NMR-spectroscopic
detection of 18O in 2-OH and/or 5-OH (Fig. 4) and HRESIMS data
(Table 2) in the experiments with [2-13C]Phe and [2-13C]CA did
not clarify whether 2-OH or 5-OH comes first, i.e., the introduction
of the oxygen functionality at C-2 may occur before or after oxy-
genation at C-5.
Evidence for the sequence according to which oxygen functional-
ities are introduced into the phenylphenalenoneskeleton came from
the administration of diarylheptanoids ([6-13C]DAH-I and -II) and
18O2. The 13C labelling of C-6 in the linear seven-carbon-chain of
the diarylheptanoids determined that only isotopologues of 1a with
a single 13C atom, namely those at C-8, could be formed, which after
O-[13C]methylation using diazomethane results in the di-O13CH3
derivative 1b. Therefore, 18O incorporation and O-[13C]methylation
could result in isotopologues of the subgroups 1bB, 1bD, 1bF, and
1bH; of these, as noted above, only 1bD and 1bF were useful here.
In the experiment with [6-13C]DAH-I, isotopologues of the 1bD
group possess one 18O, which may be attached to C-2 (isotopologue
1bD-2) or C-5 (isotopologue 1bD-5). As determined by HRESIMS,
the isotopologue 1bD group was formed more efficiently (relative
abundance ꢀ 1%; Table 2) than were the other 13C/18O-labelled
isotopologues, 1bF and 1bH. Although not fully conclusive, the
abundance of 1bD may suggest that the incorporation of 18O takes
place late in the biosynthetic pathway, and it seems unlikely that
an -oxygenated phenylpropanoid (early oxidation) would be
a
used. The occurrence of isotopologue 1bF, though present in a
small quantity (0.21%) in the experiment with DAH-I, confirmed
this suggestion. Since 8-13C in 1bF comes from [6-13C]DAH-I,
which has two unlabelled oxygen atoms (16O) that end up at C-1
and C-6 of 1bF, the two ‘‘new’’ oxygen atoms at C-2 and C-5 must
be 18O. This finding rules out the possibility that an
a-oxygenated
phenylpropanoid is a precursor. Thus, one 18O atom was incorpo-
rated from the atmosphere to DAH-I and another 18O atom was
incorporated downstream in the pathway (Fig. 5). The product of
the hydroxylation of DAH-I is likely DAH-II, a compound that
was previously found to be an excellent precursor of phenylphe-
nalenones (Hölscher and Schneider, 1995a).
In addition to resulting in some singly labelled isotopologues,
the administration of [6-13C]DAH-II in an 18O atmosphere pro-
duced 1bD, the only 13C/18O labelled isotopologue (Table 2).
[6-13C]DAH-II was already oxygenated at the positions, which in
1aD and 1bD ended up in the carbonyl group (C-6) and in the oxy-
gen functionalities at C-1 and C-2, respectively. Therefore, the
simultaneous occurrence of 18O in positions 1, 5, and 6 and 13C
in C-8 was impossible. Only the isotopologue [8-13C,5-18OCH3]-1a
(1aD-5), containing 18O at C-5 as the exclusive labelled oxygen,
could be formed in the experiment with [6-13C]DAH-II.
The complete amount (0.42%) of 1bD (Table 2) generated by
derivatization with 13CH2N2 was therefore attributed to a single iso-
topologue [8-13C,5-18O13CH3,2-16O13CH3]-1 (1bD-5). The formation
Fig. 5. ‘‘Late hydroxylation’’ biosynthetic pathway of 1,2,5,6-tetraoxygenated phenylphenalenones as evident from 18O/13C-labelling experiments. The fate of oxygen atoms is
coded by color. Oxygen atoms which are suggested to retain from the phenylpropanoid precursor through the entire phenylphenalenone biosynthesis are highlighted in red;
lost oxygen atoms are displayed in bold, and oxygen atoms incorporated from O2 are highlighted in blue. Phenylpropanoid units are marked with bold C–C bonds. d indicates
the origin of the marked atom from C-2 of malonate (Hölscher and Schneider, 1995b).

T. Munde et al. / Phytochemistry 91 (2013) 165–176
171
of isotopologues 1bF and 1bH was not possible from [6-13C]DAH-II
and indeed was not observed. This result provided evidence that the
oxygen atom at C-5 of phenylphenalenones is introduced late in bio-
synthesis, probably by hydroxylation of lachnanthocarpone (Fig. 5).
In addition to compound 1a, lachnanthoside aglycone (2a) was
isolated from root cultures, which were administered with
[2-13C]Phe, [2-13C]CA, [6-13C]DAH-I and [6-13C]DAH-II, respec-
tively, and simultaneously incubated under an 18O atmosphere.
Again, as observed by 13C NMR and HRESIMS analysis of non-deriv-
atized tautomeric 2a and its O13CH3-derivatives 2b, isotope enrich-
ment was low in experiments with all precursors, and the data
obtained with [2-13C]Phe, [2-13C]CA, and [6-13C]DAH-I were not
conclusive (data not shown). However, 13C/18O-labelled tautomers
of lachnanthoside aglycone were detected after the administration
of [6-13C]DAH-II and 18O2 and were quantified after O-methylation
with 13CH2N2 as isotopologues 2bD (Table 3). The occurrence of
18O in 2bD from the experiment with [6-13C]DAH-II again
indicated the incorporation of the oxygen into position 5 late in
biosynthesis (Fig. 5). This confirmed the result, which was found
for the allophanyl glucoside 1a and, after O-methylation, for 1b.
2.4. Administration of [phenyl–13C6]lachanthocarpone
After evidence for late oxygenation at C-5 had been obtained by
13C/18O stable isotope labelling experiments, lachnanthocarpone
(3b) was considered a good candidate to function as the immediate
precursor of the 1,2,5,6-tetraoxygenated phenylphenalenone 2a.
Compound 3b has been previously reported as a constituent of W.
paniculata (Edwards, 1974). In order to probe for a precursor–
product relationship, [phenyl–13C6]lachanthocarpone (3b) (Otálvaro
et al., 2004) was administered to root cultures of W. thyrsiflora as
described in Experimental, after which compounds 1a and 2a were
isolated. The NMR spectrum of 1a (Fig. 6) showed low but significant
13C enrichment in the lateral phenyl ring. The enhancement of the
Table 3
13C/18O Isotopologue composition of the tri-O13CH3 derivatives 2b was determined by HRESIMS (positive ion mode) and 13C NMR (125 MHz). Lachnanthoside aglycone (2a) was
isolated from the root cultures of W. thyrsiflora after these were incubated with [6-13C]DAH-II in an atmosphere of 18O2 followed by O-methylation with 13CH2N2 to afford two
derivatives of 2b.
Isotopologue
Isotopologues of 2b obtained from the experiment with [6-13C]DAH-II
Tri-O13CH3 derivative 1 (1-OH, 6-C = O)
Tri-O13CH3 derivative 2 (1-C = O, 6-OH)
No.
Molecular composition of
molecular cationa
m/z [M + H]+ (calcd.) m/z [M + H]+ (found) Relative abundance (%)b m/z [M + H]+ (found) Relative abundance (%)b
12C
13C
1H
16O
18O
2bA 19
2bB 18
2bC 19
2bD 18
2bE 19
3
4
3
4
3
18
18
18
18
18
4
4
3
3
2
0
0
1
1
2
372.1204
373.1237
374.1247
375.1279
376.1288
372.1208
373.1238
374.1258
375.1278
376.1288
78.67
0.76
0.53
0.20
0.15
372.1205
373.1235
374.1255
375.1276
376.1282
78.49
0.77
0.77
0.10
0.18
Isotopologue groups 2bF (12C1813C4 H1816O218O2Na+) and 2bG (12C1913C3 H1816O218O2Na+) were not detected.
1
1
a
Molecular ions were observed as sodium adducts.
b
Isotopologues containing the level of naturally abundant 13C were eliminated.
Fig. 6. Partial 13C NMR spectra (125 MHz, acetone-d6, region of quaternary carbon signals) of 1a obtained from W. thyrsiflora fed with (a) [phenyl–13C6]lachnanthocarpone
(3b). The spectrum shows an enhanced signal for C-1’, indicating that 13C was incorporated from the labelled precursor. Spectrum (b) was recorded from unlabelled 1a
(reference). For the numbering of carbon atoms, see also the legend for Fig 1. Red dot: positions enriched with 13C, corresponding signal of [phenyl–13C6]1a shown in spectrum
(a). Black dots: positions enriched with 13C (signals not shown in 13C NMR spectra).

172
T. Munde et al. / Phytochemistry 91 (2013) 165–176
13C NMR signal of the quaternary C-1 in the spectrum (a) compared
to the corresponding signal in spectrum (b) of the non-labelled ref-
erence indicates 13C-enrichment of this particular carbon atom. The
signals of other phenyl carbon atoms of 1a (C-2’-C-6’) (not shown)
were enriched as well but are less descriptive due to overlap with
other 13C resonances, signal broadening, and magnetic inequiva-
lence (Opitz et al., 2002). In addition to 13C NMR, HRESIMS indicated
13C6-labelled isotopologues of 1a ([C2113C6H25O11N2]+, found:
559.1654, calcd: 559.1537), confirming labelling of the lateral
phenyl ring. The 13C enrichment of 2.0% is in the same order of
magnitude as determined by 13C NMR (Fig. 6). HRESIMS also estab-
lished biosynthetic 13C6-labelling of lachnanthoside aglycone (2a)
(2.0% 13C enrichment, [C1313C6H13O4]+, found: 311.1006, calcd:
311.1015) from [phenyl–13C6]lachnanthocarpone (3b) as a precur-
sor. Unfortunately the amount of 2a was insufficient for recording
a 13C NMR spectrum.
phenylpyruvoyl–CoA, although the hydroxylated pyrone was pro-
duced instead of a diarylheptanoid in vitro (Brand et al., 2006). In-
deed, a-oxygenated 4-hydroxyphenylpropanoids were accepted by
WtPKS1, which is the first report of utilization of such substrates
by PKS type III enzymes (Fig. 3). However, labelling experiments
did not confirm the origin of the oxygen at C-5 from such
substrates.
An alternative biosynthetic possibility was that not only the
oxygen at C-2 of phenylphenalenones but also the oxygen at C-5
could be introduced at the stage of diarylheptanoids, more specif-
ically to C-2 of the linear carbon chain of DAH-II. However, the
question whether the oxygen ending up at C-5 of 1a is introduced
at the stage of DAH-II or further downstream in biosynthesis, after
cyclization had yielded the phenalenone tricycle, finally was deter-
mined by administering [phenyl–13C6]lachnanthocarpone (3b) to
root cultures of W. thyrsiflora. The incorporation of 3b into lachn-
anthoside aglycone (2a) and its allophanyl glucoside 1a proved
conclusively that the hydroxylation at C-5 of the phenylphenale-
none skeleton was the final step of the biosynthesis of 1,2,5,6-tet-
raoxygenated phenylphenalenones. However, formation of
tetraoxygenated phenylphenalenones through additional routes
cannot be excluded from this experiment.
The relatively low isotopic enrichment of 1a and 2a from atmo-
spheric 18O and 13C-labelled precursors suggests the slow biosyn-
thetic flux into phenylphenalenones in W. thyrsiflora. This seems
to be a consequence of the constitutive character of the target com-
pounds. Unlike the formation of anigorufone and methoxyanigo-
rufone, the biosynthesis of compounds 1a and 2a is not inducible
in W. thyrsiflora (Schüler et al., 2004; Brand et al., 2006), and the
major proportion of these compounds is already present in the root
cultures before incubation with labelled compounds starts. In or-
der to detect low isotope enrichment with 18O, the recently re-
ported approach (Munde et al., 2011) of attaching O-13CH3
groups to the hydroxyl functionalities of phenylphenalenones by
means of 13CH2N2, followed by recording isotope-induced 13C
NMR shifts of the obtained O-13CH3 derivatives, was used. In addi-
tion, HRESIMS was employed for isotopologue analysis. The syn-
thesis of the substrates and labeled precursors has been reported
in the Experimental and of [phenyl–13C6]lachnanthocarpone by
Otálvaro et al. (2004).
3. Discussion
Recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1) ac-
cepts a wide variety of aromatic and aliphatic starter CoA esters,
including phenylpropionyl–CoA and side-chain unsaturated phe-
nylpropanoyl–CoA. This substrate specificity is consistent with the
occurrence of 2-hydroxy-9-phenylphenalen-1-one (anigorufone
3a) and its 2-O-methyl derivative (methoxyanigorufone), which in
addition to other Haemodoracae, also occur in W. thyrsiflora (Fang
et al., 2011). However, the most abundant constitutive phenylphe-
nalones of whole plants and cultured roots of W. thyrsiflora not only
contain the 1-keto-2-hydroxy motif, which is typical of simple phe-
nylphenalenones, but are also oxygenated in additional positions.
According to the occurrence of such 1,2,5,6-tetraoxygenated phe-
nylphenalenones and the availability of root cultures, W. thyrsiflora
was used to investigate the biosynthetic origin of the oxygen func-
tionalities in the tricyclic phenalenone part of phenylphenalenones.
The administration of [1-13C]phenylalanine to A. preissii root
cultures has demonstrated (Opitz and Schneider, 2003) that the
carbon atoms C-6 and C-7 (using the numbering scheme shown
in Fig. 1) of phenylphenalenones are derived from C-1 of the phe-
nylpropanoid precursors, and, although this has not been exam-
ined experimentally, it is very likely that the carbonyl in position
6 of 1,2,5,6-tetraoxygenated phenylphenalenones comes from the
carboxyl group of the precursor forming the A/B ring. Moreover,
the carbonyl oxygen atom of 9-phenylphenalenones (e.g. anigoruf-
one 3a) is derived from the hydroxyl group of 4-coumaroyl–CoA
(Munde et al., 2011) as is the oxygen at C-1 of 1,2,5,6-tetraoxygen-
ated phenylphenalenones (e.g. 1a). Unlike the oxygen atoms at C-1
and C-6, the hydroxyl groups at C-2 and C-5 of compounds 1a (and
2a) do not come from the usual phenylpropanoid precursor, 4-cou-
maroyl–CoA. The introduction of the 2-OH group of anigorufone 3a
from atmospheric oxygen at the stage of DAH-I was established
from 13C/18O-labelling experiments with root cultures of A. preissii
(Munde et al., 2011). Our results confirm that the 2-OH of 1,2,5,6-
tetraoxygenated phenylphenalenones was also inserted at the
stage of DAH-I.
4. Conclusions
The present work extends previously reported results on the
biosynthetic origin of the oxygen functionalities of phenylphenale-
nones (Fig. 7). 13C/18O labelling together with sensitivity-enhanced
NMR detection of isotope-induced chemical shift changes of
13CH2N2-derived O13CH3 groups and HRESIMS-based isotopologue
analysis have been used successfully to probe the origin of oxygen
functionalities and the biosynthetic sequence according to which
oxygen functionalities are introduced to the polyoxygenated phe-
nylphenalenone scaffold in root cultures of W. thyrsiflora.
The oxygen atoms, except those at C-6, of 1,2,5,6-tetraoxygen-
ated phenylphenalenones were incorporated from atmospheric
oxygen at different biosynthetic stages. The oxygen atoms ending
up in C-1 and C-6 are incorporated together with the 4-coumaroyl
unit (Fig. 7, panel b). Thereafter, the hydroxyl group in position 2 is
introduced at the stage of DAH-I (Fig. 5). Isotope labelling experi-
ments showed that the hydroxyl group in position 5 is introduced
late in phenylphenalenone biosynthesis, at the stage of lachnan-
thocarpone (3b). Thus, not only the 4-coumaroyl–CoA-derived
oxygen at C-1, which is introduced by cinnamate-4-hydroxylase,
but also the oxygen atoms at C-2 and C-5 of the phenalenone tricy-
cle are suggested to be the result of monooxygenase-catalyzed
reactions. From the data obtained, the ‘‘late hydroxylation’’
Two pathways, an early and a late oxygenation pathway, for
understanding the introduction of the oxygen atom at C-5 seemed
possible. According to one, this oxygen could be introduced early in
the biosynthesis as a unit with an a-oxygenated phenylpropanoid
(Fig. 2). 3-(4-Hydroxyphenyl)pyruvic acid or 3-(4-hydroxy-
phenyl)lactic acid are putative intermediates of this pathway,
and the activated esters, e.g. 3-(4-hydroxyphenyl)lactoyl–CoA,
could be substrates of the PKS type III enzyme catalyzing the
condensation with malonyl–CoA. WtPKS1 was an excellent candi-
date enzyme for this reaction because it shows broad substrate
specificity and, among other starter substrates, also accepted

T. Munde et al. / Phytochemistry 91 (2013) 165–176
173
Fig. 7. Origin of oxygen atoms in anigorufone in root cultures of A. preissii (Munde et al., 2011) and compound 1a in root cultures of W. thyrsiflora. The hydroxyl group at C-2
of 1a is incorporated at the stage of linear diarylheptanoids (DAH-I) before the hydroxyl group at C-5, which is introduced into lachnanthocarpone (3). The fate of oxygen
atoms is coded by color. Oxygen atoms which are retained from 4-coumaric acid are highlighted in red; oxygen atoms lost during phenylphenalenone biosynthesis are
displayed in bold, and oxygen atoms incorporated from 18O2 are highlighted in blue. The 4-coumaroyl unit is marked with bold C–C bonds.
pathway, as shown in Fig. 5, was concluded for 1,2,5,6-tetraoxy-
genated phenylphenalenones.
254 nm. Semipreparative and analytical HPLC was performed on
an Agilent (Palo Alto, CA, USA) series HP1100 (binary pump
G1312A; auto sampler G1313A; diode array detector G1315B,
200–700 nm). Flow rate 0.8 ml minÀ1; monitoring wavelength
254 nm.
5. Experimental
5.1. General experimental procedures
5.2. Plant material and culture conditions
1H NMR and 13C NMR of samples from biosynthetic incorpora-
tion experiments were measured on a Bruker AV 500 spectrometer
(Bruker, Rheinstetten, Germany) equipped with a 5 mm TCI Cryo-
Probe™. The Bruker pulse sequence zgpg30 (power-gated decou-
pling; 30° flip angle; relaxation delay 2 s) was used for recording
13C spectra. A Bruker DRX 500 and a Bruker AV 400 NMR spectrom-
eter were used to measure NMR spectra of synthetic phenylpropa-
noic acids and NAC diketide thioesters. Acetone-d6, MeOH-d4 and
CDCl3 were used as NMR solvents. Chemical shifts are given in d
values. Tetramethylsilane (TMS) was used as an internal standard
for referencing 1H and 13C NMR spectra.
Root cultures of W. thyrsiflora were established from seeds as
previously described for root cultures of A. preissii (Hölscher and
Schneider, 1997). Sterile cultures were grown in liquid M3
(Murashige and Skoog, 1962) medium (100 ml) in conical flasks
(volume 250 ml) on a gyratory shaker (85 rpm) at 23 °C under per-
manent diffuse light (4.4 l
mol mÀ2 sÀ1) until they reached a fresh
mass of up to 40 g. Three days before labelled precursors were
administered, the in vitro-cultured roots were transferred into
fresh medium.
The LC-MS/MS system used for HRESIMS measurements con-
sisted of an Ultimate3000 series RSLC (Dionex) system and an
LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bre-
men, Germany). UPLC was performed using an Acclaim C18 col-
5.3. Synthetic procedures and analytical data
5.3.1. Synthesis of activated ester derivatives
Phenylpropanoyl–CoA esters were prepared according to the
method described by Wang et al. (2000), using RP-18 cartridges
(LiChrolutÒ, VWR Darmstadt, Germany) in a modified purification
procedure (Beuerle and Pichersky, 2002). 3-(4-Hydroxyphenyl)
pyruvic acid and (R)- and (S)-3-(4-hydroxyphenyl)lactic acids were
prepared as described below for the 13C-labelled compounds. The
same procedure was used to prepare phenylpyruvic acid, and (R)-
and (S)-phenyl)lactic acids. To synthesize the N-acetyl cysteamine
derivatives, the corresponding acid (cinnamic acid, phenylpropionic
acid, phenylpyruvic acid, 3-(4-hydroxyphenyl)pyruvic acid, (R)- and
(S)-phenyllactic acid, (R)- and (S)-3-(4-hydroxyphenyl)lactic acid)
was dissolved in CH3CN. Dicyclohexyl-carbodiimide, N-hydroxy-
1,2,3-benzotriazole (both equimolar) and N-acetylcysteamine (in
threefold molar excess) were added and the mixture was stirred
overnight. The yield after purification by preparative HPLC was
82% for phenylpropionyl–NAC and up to 67% for R- and S-phenyl lac-
toyl–NAC’s and phenylpyruvoyl–NAC. The 4-hydroxyphenyllactic
and -pyruvic acids were protected using the tetrahydropyranyl
group (Parham and Anderson, 1948). Pyridinium 4-toluenesulfo-
nate (5 mg, 4 d, 5 ml THF) was used for deprotection. In the latter
umn (150 Â 2.1 mm, 2.2
lm, Dionex, Germany) at a constant
flow rate of 300
l
L minÀ1 using a binary solvent system: Solvent
A, H2O with 0.1% HCOOH and solvent B, MeCN with 0.1% HCOOH.
The UPLC gradient system started with 20% B and increased line-
arly to 95% B in 6 min; from there it was held for 4 min, then
brought back to the initial conditions and held for 4 more min in
order to re-equilibrate the column for the next injection. Full-scan
mass spectra were generated using 30,000 m/
and for MS2 (CID) experiments the resolution power was set to
7500 m/ m. The mass accuracy was better than 3 ppm for MS
Dm resolving power,
D
and MS2 experiments. LCESIMS for substrate incubation with
WtPKS1 were obtained using a Micromass Quattro II tandem quad-
rupole mass spectrometer (Micromass Ltd., Manchester, UK) oper-
ating in both positive and negative ion modes. Enzymatic products
were separated on a C18-RP column. Details were reported else-
where (Brand et al., 2006).
Preparative HPLC was performed on a Merck–Hitachi LiChro-
graph chromatography system (L-6200A gradient pump, L-4250
UV/Vis detector). Flow rate 3.5 ml minÀ1
; UV detection at

174
T. Munde et al. / Phytochemistry 91 (2013) 165–176
case the total yields were very low (1%) but still more than sufficient
for the enzyme assays.
chloroborane [(+)DIP-Cl], respectively. Et3N (77
ll, 0.55 mmol)
was added at –20 °C to
a
solution of [2-13C](4-hydroxy-
phenyl)pyruvic acid (100 mg, 0.55 mmol) in THF (10 ml). After
5 min, a solution of (–)DIP-Cl or (+)DIP-Cl (0.3 g, 0.94 mmol) in
THF was added dropwise to the mixture. The solution was stirred
for 8 h at room temperature and then quenched with 2 N NaOH
(1.2 ml) and H2O (0.6 ml). The combined aqueous fractions ob-
tained from extraction of the aqueous phase with t-butylmethyl
ether (2Â) and the organic phase with H20 (2Â) were acidified to
pH 1 with 2 N HCl and extracted with EtOAc. Evaporation to dry-
ness yielded [2-13C](R)-3-(4-hydroxyphenyl)lactic acid (65%) and
[2-13C](S)-3-(4-hydroxyphenyl)lactic acid (65%), respectively.
[6-13C]1-(4-Hydroxyphenyl)-7-phenylhepta-4,6-dien-1-one ([6-13C]
DAH-I) and [6-13C]1-(3,4-dihydroxyphenyl)-7-phenylhepta-4,6-
dien-1-one ([6-13C]DAH-II) were synthesized according to a previ-
ously reported procedure (Baranovsky et al., 2003; Munde et al.,
2011). The synthesis of [phenyl–13C6]lachnanthocarpone 3b (99%
13C) was reported previously (Otálvaro et al., 2004).
5.3.2. Analytical data of N-acetyl cysteamine derivatives
Cinnamoyl-N-acetylcysteamine. 1H NMR (500 MHz, MeOH-d4):
d 7.63 (1H, d, J = 15.9 Hz), 7.63 (2H, m, H-2/6), 7.41 (3H, m, H-3/
5, H-4), 6.87 (1H, d, J = 15.9 Hz, H-8), 3.39 (2H, t, J = 6.7 Hz, H-
12), 3.13 (2H, t, J = 6.7 Hz, H-11), 1.93 (3H, s, C-15).
Phenylpropionyl-N-acetylcysteamine. 1H NMR (500 MHz,
MeOH-d4): d 7.25 (2H, m, H-3/5), 7.18 (3H, m, H-2/6, H-4), 3.29
(2H, t, J = 6.7 Hz; H-12), 2.98 (2H, t, J = 6.7 Hz, H-11), 2.94 (2H, m,
H-8), 2.88 (2H, m, H-7), 1.90 (3H, s, H-15).
Phenylpyruvoyl-N-acetylcysteamine. 1H NMR (500 MHz,
CDCl3): d 7.97 (2H, dd, J = 8.0 Hz, 1.5 Hz, H-2/6), 7.60 (1H, dt,
J = 8.0 Hz, 1.5 Hz, H-4), 7.47 (2H, t, J = 8.0 Hz, H-3/5), 3.84 (0.8 H,
s, H-7, signal intensity reduced due to H/D exchange), 3.54 (2 H,
q, H-12), 3.24 (2H, t, J = 6.5 Hz, H-11), 1.98 (3H, s, H-15).
3-(4-Hydroxyphenyl)pyruvoyl-N-acetylcysteamine. 1H NMR
(500 MHz, acetone-d6): d 7.62 (2H, d, J = 8.8 Hz, H-2/6), 6.92 (2H,
d, J = 8.8 Hz, H-3/5), 3.36 (2H, dt, J = 6.7, 5.9 Hz, H-12), 3.09 (2H,
t, J = 6.7 Hz, H-11), 1.87 (3H, s, H-15). H-7 not detected due to H/
D exchange.
5.4.2. Analytical data of synthetic labelled compounds
[2-13C]3-(4-Hydroxyphenyl)pyruvic acid. 1H NMR (500 MHz,
MeOH-d4): d 7.63 (d, J = 8.8 Hz, 2H, H-2’/6’), 6.75 (d, J = 8.8 Hz,
2
(R)-Phenyllactoyl-N-acetylcysteamine. 1H NMR (500 MHz,
CDCl3): d 7.32 (2H, m, H-3/5), 7.28 (1H, m, H-4), 7.25 (2H, m, H-
2/6), 4.49 (1H, dd, J = 7.7, 4.3 Hz, H-8), 3.42 (2H, m, H-12), 3.18
(1H, dd, J = 14.2, 4.3 Hz, H-7a), 3.04 (2H, dt, J = 6.3, 1.5 Hz, H-11),
2.98 (1H, dd, J = 14.2, 7.7 Hz, H-7b) 1.96 (3H, s, H-15).
2H, H-3’/5’), 6.44 (d, JH-C = 2.5 Hz, 1H, H-3); 13C NMR (125 MHz,
MeOH-d4): d 169.1 (C-1), 158.3 (C-4’), 140.1 (C-2, signal enhanced
due to 13C enrichment), 132.4 (C-2’/6’), 128.0 (C-1’), 116.2 (C-3’/5’),
112.6 (C-3). EIMS m/z (rel. int.): 181 [M+] (16), 135 (20), 107 (100),
77 (17).
(S)-Phenyllactoyl-N-acetylcysteamine. 1H NMR (500 MHz,
CDCl3): d 7.32 (2H, m, H-3/5), 7.28 (1H, m, H-4), 7.25 (2H, m, H-
2/6), 4.49 (1H, dd, J = 7.7, 4.3 Hz, H-8), 3.42 (2H, m, H-12), 3.18
(1H, dd, J = 14.2, 4.3 Hz, H-7a), 3.04 (2H, m, H-11), 2.98 (1H, dd,
J = 14.2, 7.7 Hz, H-7b) 1.96 (3H, s, H-15).
[2-13C](R)-3-(4-Hydroxyphenyl)lactic acid. 1H NMR (500 MHz,
MeOH-d4): d 7.08 (d, J = 8.5 Hz, 2H; H-2’/6’), 6.70 (d, J = 8.5 Hz, 2H;
3
3
1
H-3’/5’), 4.26 (dd, JH-2–H-3b = 7.8 Hz, JH-2–H-3a = 4.0 Hz, JH-2–C-2
=
=
=
3
2
145.5 Hz, 1H; H-2), 3.01 (dd, JH-3a–H-2 = 4.0 Hz, JH-3a–H-3b
3
2
14.0 Hz, 1H; H-3a), 2.80 (dd, JH-3b–H-2 = 7.8 Hz, JH-3b–H-3a
(R)-4-Hydroxyphenyllactoyl-N-acetylcysteamine.
1H
NMR
14.0 Hz, 1H; H-3b); 13C NMR (125 MHz, MeOH-d4): d 174.1 (C-1),
157.1 (C-4’), 131.7 (C-2’/6’), 129.9 (C-1’), 116.1 (C-3’/5’), 74.1 (C-2,
signal enhanced due to 13C enrichment), 40.9 (C-3). EIMS m/z (rel.
int.): 183 [M+] (9), 107 (100), 77 (10).
(500 MHz, acetone-d6): d 7.24 (2H, d, J = 8.5 Hz, H-2/6), 6.92 (2H,
d, J = 8.5 Hz, H-3/5), 5.45 (1H, dd, J = 8.7, 4.4 Hz, H-8), 3.44 (2H,
dt, J = 6.8, 5.8 Hz, H-12), 3.24 (1H, dd, J = 14.2, 4.4 Hz, H-7a), 3.15
(2H, dt, J = 6.8, 2.9 Hz, H-11), 3.11 (1H, dd, J = 14.2, 8.7 Hz, H-7b),
1.86 (3H, s, H-15).
[2-13C](S)-3-(4-Hydroxyphenyl)lactic acid. 1H NMR (400 MHz,
MeOH-d4): d 7.08 (d, J = 8.5 Hz, 2H; H-2’/6’), 6.70 (d, J = 8.5 Hz,
3
3
1
(S)-4-Hydroxyphenyllactoyl-N-acetylcysteamine.
1H
NMR
2H; H-3’/5’), 4.26 (dd, JH-2–H-3b = 7.8 Hz, JH-2–H-3a = 4.4 Hz, JH-2–C-
2 = 145.5 Hz, 1H; H-2), 3.01 (dd, JH-3a–H-2 = 4.0 Hz, JH-3a–C-3b
3
2
(500 MHz, MeOH-d4): d 7.05 (2H, d, J = 8.3 Hz, H-2/6), 6.69 (2H,
d, J = 8.3 Hz, H-3/5), 4.25 (1H, dd, J = 8.7, 4.0 Hz, H-8), 3.29 (2H,
overlap, H-12), 2.98 (1H, dd, J = 14.2 Hz, 4.0 Hz, H-7a), 2.95 (2H,
dt, J = 6.7, 1.9 Hz, H-11), 2.73 (1H, dd, J = 14.2, 8.7 Hz, H-7b), 1.92
(3H, s, H-15).
=
=
3
2
14.0 Hz, 1H; H-3a), 2.80 (dd, JH-3b–H-2 = 7.8 Hz, JH-3b–H-a
14.0 Hz, 1H; H-3b); 13C NMR (100 MHz, MeOH-d4): d 174.9 (C-1),
155.8 (C-4’), 130.3 (C-2’/6’), 128.3 (C-1’), 114.7 (C-3’/5’), 71.8 (C-
2, signal enhanced due to 13C enrichment), 39.7 (C-3). EIMS m/z
(rel. int.): 183 [M+] (13), 107 (100), 77 (5).
5.4. Labelled compounds
5.5. Enzyme assay
5.4.1. Origin and synthesis of labelled compounds
[1-13C]
L
-Phenylalanine (99% 13C) and [2-13C]malonic acid (99%
Assay conditions were previously reported (Brand et al., 2006).
13C) were purchased from Deutero GmbH (Castellaun, Germany).
In brief, the phenylpropanoyl starter substrates (50
[2-14C]malonyl–CoA (80 M, 0.17 kBq nmolÀ1) and the recombi-
nant WtPKS1 protein (20 g) (EMBL/DDBJ/GenBank™ database
accession number AY727928) were added to the buffer (0.1 M
Hepes, pH 6.85; assay volume 100 l) and incubated for 1 h at
37 °C. Products were extracted with (2 Â 200 l) EtOAc. The com-
bined EtOAc extracts were evaporated, reconstituted in MeOH
(20 l), and analyzed by reversed-phase HPLC using a Supelcosil
lM),
18O2 (95%) was from EurisoTop (Saarbrücken, Germany). [2,3,4,
l
5,6-3H]
L
-Phenylalanine, [U–14C]4-coumaric acid, and [2-14C]malo-
l
nyl–CoA were from Hartmann Analytic (Braunschweig, Germany).
[2-13C]4-Coumaric acid (99% 13C) was synthesized from
4-hydroxybenzaldehyde and [2-13C]malonic acid using the Knoe-
venagel condensation. [2-13C]3-(4-Hydroxyphenyl)pyruvic acid
was synthesized under an Ar atmosphere from [2-13C]tyrosine
(99% 13C, 0.3 g, 1.6 mmol), dimethylaminopyridine (DMAP,
9.9 mg) and (CF3CO)2O TFA, (4 ml, 28.8 mmol) by refluxing for
20 h. After evaporation, the residue was reconstituted with HCl
(10 ml, 1%) and stirred for 12 h at room temperature. After 12 h
at 4 °C the precipitate was filtered, washed with cold H2O, and
dried over (P4O10) to obtain the product (0.24 g, 81%). [2-13C](R)-
and [2-13C](S)-3-(4-hydroxyphenyl)lactic acids were prepared
from [2-13C]3-(4-hydroxyphenyl)pyruvic acid using (–)-diisopino-
campheyl chloroborane [(–)DIP-Cl] and (+)-diisopinocampheyl
l
l
l
LC18 column, 250 Â 2.1 mm i.d. (Sigma–Aldrich, Taufkirchen,
Germany), solvent A: H2O containing 0.1% TFA, solvent B: MeCN,
gradient: 10% to 50% solvent B over 40 min at a flow rate of
0.25 ml minÀ1. Enzyme products were detected at 280 nm and,
after adding scintillation liquid (Ultima-Flo AP, 1 ml minÀ1, Perkin
Elmer), by radioactivity monitoring using a flow scintillation ana-
lyzer (Canberra-Packard, Dreieich, Germany). For NMR and MS
analysis, the assays were scaled up 10 to 18-fold, with the appro-
priate amount of unlabelled malonyl–CoA.

T. Munde et al. / Phytochemistry 91 (2013) 165–176
175
5.6. Administration of labelled precursors
(C-8), 141.0 (C-6), 145.3 (C-1’), 148.8 (C-7), 149.0 (C-5), 151.2
(C-2), 169.3 (C-1’’’), 171.5 (C-2’’’), 182.2 (C-1). Please note differ-
ent carbon atom numbering of 1a in the text and figures.
2,5,6-Trihydroxy-9-phenylphenalen-1-one (2a): HPLC (gradient
b): UV (MeCN–H2O) kmax 208, 278, 374 nm; 1H NMR (500 MHz,
acetone-d6): d 7.36 (s, 1H, H-3), 7.38–7.44 (m, 5H, H-2’ to H-6’),
7.54 (d, J = 8.2 Hz, 1H, H-8), 7.84 (s, 1H, H-4), 8.66 (d, J = 8.2 Hz,
1H, H-9); 13C NMR (125 MHz, acetone-d6): d 112.9 (C-3), 118.4
(C-3a), 120.7 (C-9b), 122.7 (C-4), 125.4 (C-9a), 126.9 (C-4’), 128.2
(C-3’/5’), 129.1 (C-2’/6’), 129.2 (C-9), 129.9 (C-6a), 130.6 (C-8),
139.0 (C-6), 144.2 (C-1’), 147.3 (C-7), 149.0 (C-5), 150.7 (C-2),
178.4 (C-1).
The administered amounts of labelled compounds were as
follows: [2-13C](S)-3-(4-hydroxyphenyl)lactic acid (3.0 mg),
[2-13C](R)-3-(4-hydroxyphenyl)lactic acid (3.0 mg), [2-13C]-3-(4-
hydroxyphenyl)pyruvic acid (3.0 mg), [2-13C]Phe (3.5 mg),
[2-13C]CA (2.7 mg), [6-13C]DAH-I (3.5 mg), [6-13C]DAH-II (3.5 mg),
and [phenyl–13C6]lachnanthocarpone 3b (2.2 mg). Compounds
were administered to W. thyrsiflora root cultures (100 ml medium)
as an ethanolic solution (1 ml) through a sterile membrane filter.
For incubation with 18O2, the conical flask containing the root cul-
ture was connected through a three-way cock to a vacuum pump
and an 18O2 reservoir. The normal atmosphere was removed from
the incubation flask under vacuum (72 mbar) and 18O2 filled from
the scaled reservoir. The incubation flask was also connected to a
tube containing NaOH for trapping metabolically formed CO2 from
cultured roots. The incubation time was 4-5 days except in the 18O2
incubation experiments (7 days) under continuous shaking
(85 rpm). The administration of radio-labelled [2,3,4, 5,6-3H]Phe
(1.5 mg, 0.37 kBq) and [U–14C]CA (1.5 mg, 0.34 kBq) in absorption
experiments was carried out under conditions otherwise identical
to experiments using stable isotope labelling.
[2,5-di-O13CH3]6-O-[(6’’-O-Allophanyl)-b-
D-glucopyranosyl]-
2,5-methoxy-7-phenylphenalen-1-one (1b): HPLC (gradient c):
UV (MeCN–H2O) kmax 201, 279, 374 nm; 1H NMR (500 MHz,
MeOH-d4): d 2.05 (dd, J = 7.7, 9.3 Hz, 1H, H-2’’), 2.97 (ddd,
J = 2.3, 5.6, 9.7 Hz, 1H, H-5’’), 3.03 (dd, J = 8.7, 9.7 Hz, 1H, H-4’’),
3.21 (dd, J = 8.7, 9.3 Hz, 1H, H-3’’), 3.45 (dd, J = 5.6, 11.9 Hz, 1H,
H-6’’a), 3.60 (dd, J = 2.3, 11.9 Hz, 1H, H-6’’b), 3.99 (s,
1
1JHÀC = 145.0 Hz, 3H, 2-O13CH3), 4.05 (s, JHÀC = 145.0 Hz, 3H, 5-
O13CH3), 5.11 (d, J = 7.7 Hz, 1H, H-1’’), 7.29 (s, 1H, H-3), 7.32
(br, 1H, H-5’), 7.38 (br, 2H, H-2’ and H-4’), 7.44 (br, 1H, H-6’),
7.50 (br, 1H, H-3’), 7.55 (d, J = 7.6 Hz, 1H, H-8), 7.86 (s, 1H, H-
4), 8.56 (d, J = 7.6 Hz, 1H, H-9); 13C NMR (125 MHz, MeOH-d4):
5.7. Extraction, purification and derivatization of compounds 1a and 2a
d
56.31
(2-18O13CH3, 1D
d
18O(13C)=À25 ppb),
56.34
Cultured roots were taken from the medium, shock-frozen by
liquid nitrogen, ground, and extracted with MeOH at room temper-
ature. The extract was evaporated to dryness (<40 °C), and the
residue subjected to liquid–liquid separation between n-
hexane-H2O, CH2Cl2–H2O and EtOAc–H2O. The organic phases
were subjected to preparative HPLC on a LiChrospher RP18 column
(2-16O13CH3), 57.73 (5-18O13CH3, 1D
d
18O(13C)=À29 ppb), 57.76
(5-16O13CH3), 62.8 (C-6’’), 71.5 (C-4’’), 74.6 (C-2’’), 77.8 (C-5’’),
78.2 (C-3’’), 103.0 (C-1’’), 115.6 (C-3), 121.4 (C-9b), 122.3 (C-4),
126.6 (C-3a), 128.0 (C-6a, C-2’, C-4’), 128.6 (C-9), 128.9 (C-6’),
129.5 (C-9a), 129.7 (C-3’), 130.3 (C-5’), 132.6 (C-8), 143.6 (C-
6), 145.8 (C-1’), 149.7 (C-7), 151.4 (C-5), 153.7 (C-2), 169.3 (C-
1’’’), 171.5 (C-2’’’), 179.2 (C-1). Please note different carbon atom
numbering of 1b in the text and figures.
(10
lm, 250 Â 10 mm). A linear gradient MeCN–H2O from 10% to
50% MeCN in 40 min (method a) and MeCN–H2O (0.1% TFA in
H2O), 30% to 75% MeCN in 35 min (method b) was used. Pure 1a
and 2a were obtained from the CH2Cl2 and ethyl acetate extracts
at Rt 13.0 min (method a) and Rt 16.0 min (method b), respectively.
The pure products 1a and 2a were analyzed by NMR and HRESIMS.
Compounds obtained from 12C/18O double-labelling experiments
were methylated using an ethereal solution of [13C]diazomethane,
which was prepared from N-[13C]methyl-N-nitroso-p-toluenesul-
fonamide using a standard procedure. Semipreparative HPLC on a
[2,5,6-tri-O13CH3]2,5,6-Trimethoxy-7-phenylphenalen-1-one
(2b, Rt 13.8 min): HPLC (gradient d): Rt 13.8 min, UV (MeCN–H2O)
kmax 208, 279, 373 nm; 1H NMR (500 MHz, acetone-d6): d 3.27 (s,
1JHÀC = 144.7 Hz, 3H, 6-O13CH3), 3.93 (s, JHÀC = 144.5 Hz, 3H, 2-
1
O13CH3), 4.03 (s, JHÀC = 144.6 Hz, 3H, 5-O13CH3), 7.22 (s, 1H, H-
1
3), 7.39-7.47 (br, 5H, H-2’ to H-6’), 7.56 (d, J = 7.5 Hz, 1H, H-8),
7.86 (s, 1H, H-4), 8.49 (d, J = 8.5 Hz, 1H, H-9); 13C NMR (125 MHz,
acetone-d6):
(6-18O13CH3, 1D
d
55.7 (2-16O13CH3), 57.2 (5-16O13CH3), 60.64
d
LiChrospher RP18 column (5
l
m, 250 Â 10 mm) was used to purify
18O(13C)=À28 ppb), 60.67 (6-16O13CH3), 114.1
the O-methyl derivatives 1b and 2b. Method c: linear gradient
MeCN–H2O (0.1% TFA) from 40% to 60% MeCN in 20 min and meth-
od d: linear gradient MeCN–H2O (0.1% TFA) from 40% to 75% MeCN
in 30 min were used. Pure 1b was obtained with method c at Rt
15.7 min and two O-methyl derivatives 2b with method d at Rt
13.4 and 13.8 min.
(C-3), 119.9 (C-4), 121.9 (C-9b), 125.9 (C-3a), 127.2 (C-9a), 127.4
(C-4’), 127.8 (C-3’/C-5’), 128.2 (C-9), 129.3 (C-2’/C-6’), 129.9 (C-
6a), 131.3 (C-8), 144.7 (C-1’), 146.9 (C-7), 146.6 (C-6), 151.5 (C-
5), 153.4 (C-2), 179.6 (C-1). Please note different carbon atom
numbering in the text and figures.
[2,5,6-tri-O13CH3]2,5,6-Trimethoxy-9-phenylphenalen-1-one
(2b, Rt 13.4 min): HPLC (gradient d): UV (MeCN–H2O) kmax 209,
5.8. Analytical data of biosynthetic products and their O-methyl
derivatives
277, 374 nm; 1H NMR (500 MHz, acetone-d6):
d
3.84 (s,
1JHÀC = 144.5 Hz, 3H, 2-O13CH3), 4.10 (s, JHÀC = 144.8 Hz, 3H, 6-
1
O13CH3), 4.11 (s, JHÀC = 144.7 Hz, 3H, 5-O13CH3), 7.12 (s, 1H, H-
1
6-O-[(6’’-O-Allophanyl)-b-
D
-glucopyranosyl]-2,5-dihydroxy-7-
3), 7.34 - 7.45 (br, 5H, H-2’ to H-6’), 7.56 (d, J = 8.5 Hz, 1H, H-8),
phenylphenalen-1-one (1a): HPLC (method a): UV (MeCN–H2O)
kmax 211, 279, 378, 477 nm; 1H NMR (500 MHz, MeOH-d4): d
2.36 (dd, J = 7.9, 9.0 Hz, 1H, H-2’’), 3.04 (dd, J = 9.0, 9.3 Hz, 1H,
H-4’’), 3.09 (ddd, J = 2.2, 6.5, 9.0 Hz, 1H, H-5’’), 3.19 (dd, J = 9.0,
9.3 Hz, 1H, H-3’’), 3.96 (dd, J = 2.2, 11.8 Hz, 1H, H-6’’b), 4.18 (dd,
J = 6.5, 11.8 Hz, 1H, H-6’’a), 4.67 (d, J = 7.9 Hz, 1H, H-1’’), 7.04 (s,
1H, H-3), 7.22 (br, 1H, H-5’), 7.33 (br, 2H, H-4’, H-2’’), 7.39 (br,
1H, H-6’), 7.43 (br, 1H, H-3’), 7.46 (s, 1H, H-4), 7.54 (d,
J = 7.6 Hz, 1H, H-8), 8.47 (d, J = 7.6 Hz, 1H, H-9); 13C NMR
(125 MHz, MeOH-d4): d 64.9 (C-6’’), 71.1 (C-4’’), 74.6 (C-2’’),
75.2 (C-5’’), 77.6 (C-3’’), 104.1 (C-1’’) 116.3 (C-3), 121.7 (C-9b),
124.2 (C-4), 127.3 (C-2’), 127.8 (C-6ª, C-4’) 128.3 (C-6’), 128.5
(C-3a), 128.6 (C-9), 130.0 (C-9a), 130.7 (C-3’), 131.8 (C-5’), 132.3
7.82 (s, 1H, H-4), 8.52 (d, J = 8.5 Hz, 1H, H-7); 13C NMR (125 MHz,
acetone-d6):
d
55.8 (2-16O13CH3), 57.24 (5-18O13CH3, 1D
d
18O(13C)=–28 ppb), 57.27 (5-16O13CH3), 61.9 (6-16O13CH3), 112.2
(C-3), 119.6 (C-4), 121.9 (C-6b), 126.3 (C-3a), 127.5 (C-4’), 128.4
(C-7), 128.6 (C-6a), 128.8 (C-3’/C-5’), 129.0 (C-2’/C-6’), 129.9
(C-9a), 132.2 (C-8), 144.3 (C-1’), 144.8 (C-6), 145.8 (C-5), 146.5
(C-9), 153.9 (C-2), 179.8 (C-1).
Acknowledgments
The authors thank the Deutsche Forschungsgemeinschaft for
financial support, and Emily Wheeler for editorial assistance.

176
T. Munde et al. / Phytochemistry 91 (2013) 165–176
Kamo, T., Kato, N., Hirai, N., Tsuda, M., Fujioka, D., Ohigashi, H., 1998.
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