Variability of phenylpropanoid precursors in the biosynthesis of phenylphenalenones in Anigozanthos preissii

Article abstract of DOI:10.1016/S0031-9422(99)00544-0

Feeding experiments using ¹³C labelled precursors and NMR spectroscopic studies revealed general biosynthetic incorporation of phenylalanine and variable incorporation of cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid into phenylphenalenones in root cultures of Anigozanthos preissii. Evidence was obtained for parallel pathways of phenylphenalenone biosynthesis, with respect to the left phenylpropanoid unit, and a sequence involving utilisation of p-coumaric acid with late generation of an intermediate catechol moiety in the right phenylpropanoid unit. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(99)00544-0

Phytochemistry 53 (2000) 331±337
www.elsevier.com/locate/phytochem
Variability of phenylpropanoid precursors in the biosynthesis of
phenylphenalenones in Anigozanthos preissii
Bettina Schmitta, Dirk Holscherb, 1, Bernd Schneidera,*
a
È
Max-Planck-Institut fur Chemische Okologie, Tatzendpromenade 1a, D-07745 Jena, Germany
È
bInstitut fur P¯anzenbiochemie, Weinberg 3, D-06120 Halle, Germany
È
Received 24 June 1999; received in revised form 20 August 1999; accepted 4 September 1999
Dedicated to Professor H.-R. Schutte on the occasion of his 70 birthday.
Abstract
Feeding experiments using 13C labelled precursors and NMR spectroscopic studies revealed general biosynthetic incorporation
of phenylalanine and variable incorporation of cinnamic acid, p-coumaric acid, ca€eic acid and ferulic acid into
phenylphenalenones in root cultures of Anigozanthos preissii. Evidence was obtained for parallel pathways of phenylphenalenone
biosynthesis, with respect to the left phenylpropanoid unit, and a sequence involving utilisation of p-coumaric acid with late
generation of an intermediate catechol moiety in the right phenylpropanoid unit. # 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Anigozanthos preissii; Haemodoraceae; Biosynthesis; Diarylheptanoids; 13C-NMR; Phenylphenalenones; Phenylpropanoids
1. Introduction
nalenone biosynthesis was shown by feeding a labelled
precursor of that type, 1-phenyl-7-(3,4-dihydroxyphe-
nyl)-hepta-1,3-dien-5-one, carrying a catechol moiety
(Holscher & Schneider, 1995a). It has been suggested
that the catechol portion undergoes oxidation to an o-
quinone, generating an excellent dienophile capable of
involvement in a proposed Diels±Alder cycloaddition,
resulting in the phenylphenalenone ring system (Bazan,
Edwards & Weiss, 1978; Schmitt & Schneider, 1999).
However, details of the biosynthetic pathway are still
hypothetical. Until now, for example, it was uncertain
whether the second hydroxyl group of the catechol
moiety in the diarylheptanoid intermediate, originated
from ca€eic acid or, alternatively, whether it was intro-
duced by hydroxylation at a later stage of the biosyn-
thesis. In this paper, we describe in vivo feeding
experiments using labelled phenylpropanoids, having
di€erent substitution patterns in the phenyl ring, and
how these precursors are biosynthetically incorporated
into phenylphenalenones.
Phenylphenalenones are characteristic constituents
of the Haemodoraceae plant family (Cooke
&
Edwards, 1980). They accumulate in roots of Anigo-
zanthos preissii and have also been found in root cul-
tures of that species (Holscher & Schneider, 1997).
Recent biosynthetic studies demonstrated the sym-
metrical incorporation of two molecules of cinnamic
acid and p-coumaric acid into anigorufone (1)
(Holscher & Schneider, 1995a) and hydroxyanigoru-
fone (2) (Holscher & Schneider, 1995b), respectively.
The involvement of diarylheptanoids in the phenylphe-
* Corresponding author: Tel.: +49-3641-643-634; fax: +49-3641-
643-665.
E-mail address: schneider@ice.mpg.de (B. Schneider).
1 Present address: Institute of Biological Chemistry, Washington
State University, Pullman, WA 99164-6340.
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00544-0

332
B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
Fig. 1. Structure of phenylphenalenones isolated from root cultures of Anigozanthos preissii upon administration of labelled precursors: anigoru-
fone (1), hydroxyanigorufone (2), dihydroxyanigorufone (3), musanolone F (4), and methoxyanigorufone (5).
2. Results
amounts of isotopomers containing two labelled car-
2
bons in these positions (doublets, JC-6-C-7 ˆ 2:7 Hz).
Previous feeding experiments using 14C labelled
phenylalanine have established the ready incorporation
into phenylphenalenones in Haemodorum corymbosum
(Thomas, 1971) and Lachnanthes tinctoria (Edwards,
Schmitt & Weiss, 1972). Speci®c incorporation of label
from [1-13C]phenylalanine into C-7 but not C-6 of
lachnanthoside aglycone was observed in L. tinctoria
(Harmon, Edwards & Highet, 1977). In our biosyn-
thetic studies, two molecules of [1-13C]phenylalanine
were shown to be speci®cally incorporated into the
phenylphenalenones of Anigozanthos preissii. This was
indicated by enhancement of the 13C resonances of C-6
and C-7, measured directly in crude extracts obtained
from fed root cultures of A. preissii containing anigor-
ufone (1), hydroxyanigorufone (2), dihydroxyanigoru-
fone (3), musanolone F (4), and methoxyanigorufone
(5) (Holscher & Schneider, 1997) (Fig. 1). Enlarged
13C-NMR signals C-6 and C-7 in isolated 1 (d 130.2,
C-6; d 136.2, C-7) (Fig. 2), 2 (d 130.1, C-6; d 136.1, C-
7), and 5 (d 129.6, C-6; d 135.0, C-7) con®rmed this
result. The high quantity of speci®c incorporation of
13C into C-6 and C-7 was indicated by comparison of
integral ratios between non-labelled references (integral
ratio C-7:C-8:C-6 = 1:1:1) and labelled phenylphenale-
nones. In compound 1, for example, the integral ratio
C-7:C-8:C-6 is 11:1:11 (Fig. 2) corresponding to 11.2%
enhancement of both C-6 and C-7 above natural abun-
dance. This ®nding also indicated symmetric incorpor-
ation of two molecules phenylalanine into one
phenylphenalenone molecule. Due to C±C coupling,
the resonances of C-6 and C-7 appear as pseudo tri-
plets (see extensions in Fig. 2(a)), indicating the occur-
rence of singly labelled isotopomers (singlet, central
resonances of the pseudo triplets) and relatively high
Another series of feeding experiments using [2-
13C]cinnamic acid, [2-13C]p-coumaric acid, [2-13C]ca€eic
ic acid, and [2-13C]ferulic acid were carried out,
employing the same root culture line of A. preissii.
Compounds 1, 2 and 3 were isolated from the cultures
upon administration of cinnamic acid and p-coumaric
acid. 13C-NMR spectra were measured in order to ®nd
out whether incorporation into both parts or into only
one half of the phenylphenalenone molecule had
occurred. Enhanced 13C-NMR signals were expected
for either C-5 or C-8, or both of these carbon atoms.
The results are summarised in Table 1. As previously
demonstrated (Holscher & Schneider, 1995a), anigoru-
fone (1) was symmetrically labelled at C-5 (d 127.9)
and C-8 (d 132.0) following administration of [2-
13C]cinnamic acid. Symmetrical incorporation was also
shown for compounds 2 ꢀd 127.7, C-5; d 132.5, C-8)
and 3 ꢀd 127.7, C-5; d 132.5, C-8), isolated from analo-
gous feeding experiments. [2-13C]p-Coumaric acid was
symmetrically incorporated into hydroxyanigorufone
(2), as previously published (Holscher & Schneider,
1995b), and into dihydroxyanigorufone (3). Incorpor-
ation of p-coumaric acid exclusively into the right part
of 1 was a trivial ®nding. High incorporation rates of
phenylpropanoic acids into phenylphenalenones were
observed in all feeding experiments. The values, deter-
mined from EI-MS data, in this series of experiments
varied between 7.1% 13C in singly labelled (C-5 or C-
8) and 2.3% in doubly labelled molecules (both C-5
and C-8) of anigorufone 1 biosynthesised from [2-
13C]cinnamic acid and 31.7%/3.6% 13C in hydroxyani-
gorufone (2) biosynthesised from [2-13C]p-coumaric
acid. Unfortunately, mass spectral fragmentation did
not allow discrimination between incorporation into

B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
333
Fig. 2. Partial 13C-NMR spectrum of anigorufone (1) isolated from root cultures of Anigozanthos preissii upon (a) feeding [1-13C]phenylalanine.
Compared with the spectrum (b) of the non-labelled reference (integral ratio C-7:C-8:C-6 = 1:1:1), the resonances of C-6 and C-7 are enhanced
2
(integral ratio C-7:C-8:C-6 = 11:1:11) and, due to JC-C ˆ 2:7 Hz, appear as pseudo triplets (for details, see text).
C-5 and C-8. Consequently, individual incorporation
rates of doubly labelled compounds were not accessible
from our MS data. Actually this was the reason for
employing 13C-NMR spectroscopy to prove speci®c in-
corporation unambiguously. In contrast, incorporation
rates of singly labelled compounds were readily deter-
mined from the mass spectral data. 15.4% 13C were
found in anigorufone (1) biosynthesised from [2-13C]p-
coumaric acid.
Anigorufone (1), hydroxyanigorufone (2), and dihy-
droxyanigorufone (3) were also isolated upon adminis-
tration of [2-13C]ca€eic acid and [2-13C]ferulic acid.
Additionally, two phenylphenalenones bearing a meth-
oxyl group, musanolone F (4) and methoxyanigoru-
Table 1
Incorporation of [2-13C]labelled phenylpropanoids into phenylphenalenones determined by means of 13C-NMR spectroscopy of phenylphenale-
nones 1±5
[2-13C]labelled precursor
Enhancement of 13C signals
1
2
3
4
5
C-8
+
C-5
C-8
C-5
C-8
C-5
C-8
C-5
C-8
C-5
Cinnamic acid
p-Coumaric acid
Ca€eic acid
+
+
+
+
+
+
+
+
+
+
+
n.d.a
n.d
n.d
n.d
(+)
+
+
Ferulic acid
a n.d. = not determined.

334
B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
Fig. 3. Partial 13C-NMR spectra of phenylphenalenones isolated from root cultures of Anigozanthos preissii upon administration of 13C labelled
phenylpropanoids. (a) Dihydroxyanigorufone (3) biosynthesised from [2-13C]ca€eic acid, (b) musanolone F (4) biosynthesised from [2-13C]ferulic
acid.
fone (5), were isolated from these feeding experiments
and analysed by 13C-NMR. Both 4 (Luis et al., 1996)
and 5 (Luis, Fletcher, Echeverri, Abad, Kishi & Per-
ales, 1995) are known from Musa acuminata. Com-
pound 4 is described here for the ®rst time as a
constituent of a Haemodoraceae species. Compounds
1, 2 and 5, isolated upon feeding [2-13C]ca€eic acid,
did not show any incorporation of the labelled precur-
sor (Table 1). This is, again, trivial for the left part of
the phenylphenalenone molecule, because the phenyl
ring carries less substituents than the precursor. Non-
incorporation into the right part of the molecule indi-
cates that ring A and C-4 to C-6 of the phenylphenale-
nones are not formed from ca€eic acid. The 13C-NMR
spectra of dihydroxyanigorufone (3) (Fig. 3(a)) and
musanolone F (4), isolated from the same experiment,
exhibited strong enhancement of C-8. EI-MS data
indicated 5.7% 13C in singly labelled (C-5 or C-8) and
1.7% in doubly labelled molecules (both C-5 and C-8)
of compound 3, and 16.0% 13C for compound 4 (sin-
gly labelled). These ®ndings clearly indicated that the
left part of both 3 and 4 was formed from ca€eic acid.
In contrast, the right part must be derived from p-cou-
maric acid as the immediate precursor, which under-
goes condensation with a second (variably substituted)
phenylpropanoid via a central C-1 unit (Schmitt &
Schneider, 1999). Subsequent o-hydroxylation, instead
of ca€eic acid incorporation, is suggested to form the
catechol moiety involved in the pathway. In addition
to the enhanced 13C signal of C-8 in 3, slight enhance-
ment of the C-5 resonance was detected after feeding
of [2-13C]ca€eic acid. The small incorporation of caf-
feic acid into the right part of the ring system could be
observed only for 3 and not for other phenylphenale-
nones, suggesting the occurrence of an alternative sub-
pathway of minor signi®cance.
A crude extract obtained from root cultures of A.
preissii upon feeding [2-13C]ferulic acid exhibited only
a single strongly enhanced signal at d 132.5, which was
assigned to musanolone F (4). This was con®rmed
after isolation of 4 by additional NMR (Fig. 3(b)) and
MS data. Compounds 1±3 and 5 were also isolated
but enhanced signals were not detectable in the spectra
of these compounds. This result substantiates the pro-

B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
335
Fig. 4. Pathway of phenylphenalenone biosynthesis, proposed on the basis of precursor feeding experiments using 13C labelled phenylpropanoids.
Intermediates 6a, 6b, 7, and 9 are putative. Incorporation of 8 (R1, R2 = H, 1-phenyl-7-(3,4-dihydroxyphenyl)-hepta-1,3-diene-5-one) into ani-
gorufone (1) has been demonstrated previously (Holscher & Schneider, 1995a).

336
B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
posed variability of precursors forming the left part of
the phenylphenalenone molecule and the restriction of
the right-half-precursor to p-coumaric acid. Moreover,
the methoxy group at C-2 of 5 must have been formed
by hydroxylation and subsequent methylation in later
biosynthetic steps.
undergo the biosynthetic sequence proposed in Fig. 4,
since formation of catechol and o-quinone groups, and
subsequent cyclisation to phenylphenalenones, seems
to be impossible. As further demonstrated in our ex-
periments, methoxyanigorufone (5) was not formed
from ferulic acid but must be a product of anigorufone
2-O-methylation. One might also speculate that an
incompatible substitution pattern in one of the phenyl
rings of open-chain diarylheptanoids, like 6, occurring
in the Zingiberaceae and other plants could be the
reason for the lack of phenylphenalenones in these
families.
In contrast to speci®c precursor requirements in the
right part of the phenylphenalenone molecule, less
speci®c incorporation of various ring substituted
phenylpropanoic acids into the left part (C-7 to C-9
and ring D) was observed (Fig. 4). The left part is
not involved as closely as the right part in various bio-
synthetic steps and, therefore, the structural require-
ments are much less speci®c. The suggestions presented
here are in complete accordance with detailed path-
ways recently proposed for early steps of the phenyl-
phenalenone biosynthesis (Holscher, 1996; Schmitt &
Schneider, 1999). In addition, biosynthesis of phenyl-
phenalenones exhibiting diverse substitution patterns
in the phenyl ring is now explicable.
3. Discussion
The experiments described here clearly demonstrate
that in Anigozanthos preissii two phenylpropanoids are
being utilized to form the phenylphenalenone skeleton.
Equal distribution of labelling from [1-13C]phenyl-
nylalanine into C-6 and C-7 of the phenylphenalenones
provided evidence for symmetric incorporation of early
precursors into both halves of the molecule. Symmetric
incorporation of cinnamic acid and p-coumaric acid
into anigorufone (1) and hydroxyanigorufone (2),
representing the major phenylphenalenones of A. preis-
sii, has also been demonstrated. Experiments employ-
ing ca€eic and ferulic acid as precursors indicated non-
symmetric incorporation only into dihydroxyanigoru-
fone (3) and musanolone F (4), but not into 1, 2, and
methoxyanigorufone (5). This variability of incorpor-
ation was used to deduce the mechanism of phenylphe-
nalenone biosynthesis. Thus, the right portion (ring A
and C-4 to C-6) of the phenylphenalenones is predomi-
nantly formed by incorporation of p-coumaric acid
and not (or to a minor extent) ca€eic acid. By analogy
with chalcone biosynthesis (Schuz, Heller & Hahl-
brock, 1983; Schroder, 1997), p-coumaric acid seems
to serve as the preferred substrate in the ®rst step of
phenylphenalenone biosynthesis. Obviously a mechan-
ism is operating that enables discrimination between
two halves of intermediary diarylheptanoids of type 6a
and 6b (Fig. 4), which are symmetrical with respect to
the heptanoid chain.
4. Experimental
4.1. Plant material, synthesis and administration of
labelled compounds
Root cultures of Anigozanthos preissii (L.) were
initiated as previously described (Holscher & Schnei-
der, 1997) and grown in liquid MS medium (Mura-
shige & Skoog, 1962) (100 ml in 300 ml conical
Since all phenylphenalenones studied here possess an
a-ketohydroxyl (or a-ketomethoxyl) functionality in
ring A, hydroxylation at C-2' (ortho with respect to
the hydroxyl group already present in position 1') of
intermediate 7 must take place to form the catechol
moiety of intermediate 8. It was suggested that the
catechol undergoes oxidation to an o-quinone (9) pro-
viding an excellent dienophile capable of involvement
in a hypothetical Diels±Alder cycloaddition yielding
the phenylphenalenone ring system (Bazan et al., 1978;
Schmitt & Schneider, 1999). Thus, intermediary diaryl-
heptanoids (6a, 7) containing a p-hydroxyl group are
required to form a catechol moiety, as in compound 8
and, further, an o-quinone (9). Diarylheptanoids 6b
having an unsubstituted phenyl or vanillyl ring in the
right part of the molecule are expected to be formed
from cinnamic acid or ferulic acid, respectively. How-
ever, these postulated intermediates are not suitable to
¯asks) at 228C on a gyratory shaker (100 rpm)
under permanent light (4.4 mmol m
2
s
1). Three
days before administration of the precursors the
cultured roots (about 15 g) were transferred to fresh
medium. [1-13C]Phenylalanine (99% 13C† was from
Deutero GmbH, Kastellaun, Germany. [2-13C]Phenyl-
nylpropanoic acids were synthesized by an Erlenmeyer
type reaction from [2-13C]malonic acid (99% 13C†
following standard procedures. For administration to
root cultures, labelled phenylpropanoic acids (33 mmol
in each experiment) were dissolved in EtOH:H2O
7:3 (1 ml) and passed through a membrane ®lter.
[1-13C]Phenylalanine (100 mmol) was dissolved in 1 ml
H2O and administered in the same manner. The
incubation times were 1 day (cinnamic acid, coumaric
acid) or 3 days (phenylalanine, ca€eic acid and ferulic
acid).

B. Schmitt et al. / Phytochemistry 53 (2000) 331±337
337
4.2. Extraction of roots and isolation of
phenylphenalenones
Acknowledgements
The authors wish to thank Dr. N. Oldham, Jena,
for recording mass spectra and for improving the Eng-
lish of the manuscript. The Deutsche Forschungsge-
meinschaft (Bonn) is gratefully acknowledged for
providing the NMR spectrometer, and for other ®nan-
cial support. This investigation was supported by the
Fonds der Chemischen Industrie (Frankfurt/Main).
Roots were frozen in liquid N2, ground, and
extracted with MeOH at room temperature. The resi-
due obtained by evaporation (<408) of the crude
MeOH extract was fractionated by partitioning
between n-hexane±H2O, CH2Cl2±H2O, and EtOAc±
H2O. Anigorufone (1, Rt 24.1 min), methoxyanigoru-
fone (5, Rt 22.0 min), and a fraction containing hydro-
xyanigorufone (2) and musanolone F (4) ꢀRt 13.5 min)
were obtained by reversed-phase HPLC (LiChrospher
100 RP18, 250 Â 4 mm, 5 mm, 0.8 ml min 1, UV 254
nm) of the n-hexane fraction using a linear gradient
MeCN±H2O (0.1% TFA) from 45% to 75% MeCN in
30 min. Rechromatography of the fraction at Rt 13.5
min (Waters Symmetry Shield2 RP18, 250 Â 4.6 mm,
5 mm, 1.0 ml min 1, UV 254 nm, linear gradient
MeCN±H2O (0.1% TFA) from 53% to 63% MeCN in
30 min) a€orded pure 2 ꢀRt 11.8 min) and 4 ꢀRt 10.5
min). Dihydroxyanigorufone (3, Rt 15.5 min) was iso-
lated from the CH2Cl2 and EtOAc fractions by
reversed-phase HPLC (LiChrospher 100 RP18, 250 Â 4
mm, 5 mm, 0.8 ml min 1, UV 254 nm) using a linear
gradient MeCN±H2O (0.1% TFA) from 35% to 65%
MeCN in 30 min).
References
Bazan, A. C., Edwards, J. M., & Weiss, U. (1978). Synthesis of lach-
nanthocarpone [9-phenyl-2,6-dihydrophenalen-1(6)-one] by intra-
molecular Diels±Alder cyclization of
a 1,7-diarylheptanoid
orthoquinone; possible biosynthetic signi®cance of Diels±Alder
reactions. Tetrahedron, 34, 3005±3015.
Cooke, R. G., & Edwards, J. M. (1980). Naturally occurring phena-
lenones and related compounds. Progress in the Chemistry of
Organic Natural Products, 40, 153±190.
Edwards, J. M., Schmitt, R. C., & Weiss, U. (1972). Biosynthesis of
a 9-phenylphenalenone by Lachnanthes tinctoria. Phytochemistry,
11, 1717±1720.
Harmon, A. D., Edwards, J. M., & Highet, R. J. (1977). The biosyn-
thesis of 2,5,6-trihydroxy-9-phenylphenalenone by Lachnanthes
tinctoria. Incorporation of 1-13C-phenylalanine. Tetrahedron
Letters, 51, 4471±4474.
Holscher, D. (1996). Biosynthese und Strukturermittlung von
Phenylphenalenonen aus Anigozanthos preissii Endl. PhD thesis,
University of Halle (Saale), Germany.
The analytical data of compounds 1, 2, 3, and 5
ꢀ H- and 13C-NMR, MS) exactly matched those of
1
Holscher, D., & Schneider, B. (1995a). A diarylheptanoid intermedi-
ate in the biosynthesis of phenylphenalenones in Anigozanthos
authentic references (Holscher & Schneider, 1997).
Compound 4 was identi®ed by means of H- and 13C-
1
preissii.
Communications, 525±526.
Journal
of
the
Chemical
Society
Chemical
NMR, 2D heteronuclear correlation experiments
(HMQC, HMBC), and mass spectrometry. 1H-NMR:
d 3.85 (3H, s, 3'-OCH3), 6.89 (1H, dd, 8.2, 1.8 6'±H),
6.92 (1H, d, 8.2, 5'-H), 7.04 (1H, d, 1.8, 2'-H), 7.20
(1H, s, 3-H), 7.67 (1H, d, 8.2, 8-H), 7.67 (1H, dd, 8.1,
7.0, 5-H), 7.85 (1H, d, 7.0, 4-H), 8.06 (1H, d, 8.1, 6-
H), 8.40 (1H, d, 8.2, 7-H). 13C-NMR: d 56.8 (3'-
OCH3), 112.9 (3-C), 113.8 (2'-C), 115.9 (5'-C), 122.4
(6'-C), 127.8 (5-C), 130.4 (6-C), 131.2 (4-C), 132.5 (8-
C), 136.4 (7-C), 149.8 (9-C), 180.3 (1-C). EIMS (70
eV): m/z 318 [M]+. The data of 4 are in accordance
with literature values (Luis et al., 1996).
Holscher, D., & Schneider, B. (1995b). The origin of the central one-
carbon unit in the biosynthesis of phenylphenalenones in
Anigozanthos preissii. Natural Product Letters, 7, 177±182.
Holscher, D., & Schneider, B. (1997). Phenylphenalenones from root
cultures of Anigozanthos preissii. Phytochemistry, 45, 87±91.
Luis, J. G., Fletcher, W. Q., Echeverri, F., Abad, T., Kishi, M. P., &
Perales, A. (1995). New phenalenone-type phytoalexines from
Musa acuminata (Colla AAA) Grand Nain. Natural Product
Letters, 6, 23±30.
Luis, J. G., Quinones, W., Echeverri, F., Grillo, T. A., Kishi, M. P.,
&
Garcia-Garcia, F., Torres, F.,
Cardona, G. (1996).
Musanolones: four 9-phenylphenalenones from rhizomes of Musa
acuminata. Phytochemistry, 41, 753±757.
Murashige, T., & Skoog, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiologia
Plantarum, 15, 473±497.
Schmitt, B., & Schneider, B. (1999). Dihydrocinnamic acids are
involved in the biosynthesis of phenylphenalenones in
Anigozanthos preissii. Phytochemistry, 52, 45±53.
4.3. Spectroscopic methods
Bruker DRX 500 NMR spectrometer: 500.13 MHz
Schroder, J. (1997). A family of plant-speci®c polyketide synthases:
facts and predictions. Trends in Plant Science, 2, 373±378.
Schuz, R., Heller, W., & Hahlbrock, K. (1983). Substrate speci®city
of chalcone synthase from Petroselinum hortense. The Journal of
Biological Chemistry, 258, 6730±6734.
1
13
ꢀ H), 125.75 MHz ꢀ C), Me2CO-d6, TMS as int. stan-
dard. 13C-NMR spectra were run using a 2.5 mm
broadband microprobe. 1H-NMR, HMBC and
HMQC spectra were measured in a 2.5 mm inverse
detection microprobe. Mass spectra were recorded on
a Micromass MasSpec (70 eV).
Thomas, R. (1971). The biosynthesis of the plant phenalenone hae-
mocorin. Journal of the Chemical Society Chemical
Communications, 739±740.