Phenylalanine-independent biosynthesis of
1,3,5,8-tetrahydroxyxanthone
A retrobiosynthetic NMR study with root cultures of
Swertia chirata
Chang-Zeng Wang
1
, Ulrich H. Maier
1
, Michael Keil
2
, Meinhart H. Zenk
1
, Adelbert Bacher
3
,
Felix Rohdich
3
and Wolfgang Eisenreich
3
1
Biozentrum-Pharmazie, Universita
¨
t Halle, Halle/Saale, Germany;
2
Boehringer Ingelheim Pharma KG, Ingelheim, Germany;
3
Lehrstuhl fu
¨
r Organische Chemie und Biochemie, Technische Universita
¨
tMu

in [1]). Using this approach, we previously showed that the
biosynthetic pathways of the tannic acid precursor, gallic
acid (8), and the bitter compound, amarogentin (9),
produced in Swertia chirata (Gentianaceae) branch off the
shikimate pathway at a level before phenylpyruvate (6)
(Fig. 1) [2,3].
We now exploit the retrobiosynthetic method to analyse
the biosynthesis of a xanthone derivative. Xanthones are
formed in at least 30 families of higher plants (e.g.
Gentianaceae and Guttiferae) [4,5]. 1,3,5,8-Tetrahydroxy-
xanthone (13, Fig. 2) is found in considerable amounts in
the roots of S. chirata. A root culture of the latter plant has
been used successfully in stable isotope incorporation
experiments aimed at analysing the biosynthesis of
amarogentin [3], and therefore this root culture appeared
to be well suited for the present study.
Early studies on the biosynthesis of xanthones suggested
that the aromatic ring A (Fig. 2) is assembled via a
polyketide-type pathway, whereas rings B and C are derived
from a C
6
–C
1
benzoic acid moiety in a similar way to
flavonoid biosynthesis [6]. More specifically, xanthones
were proposed to be biosynthesized from hydroxybenzoyl-
CoA and three molecules of malonyl-CoA (10)[7].An
enzyme catalysing the condensation of 3-hydroxybenzoyl-
CoA (11) and malonyl-CoA (10) to a benzophenone
intermediate (12, Fig. 2A) has been isolated from Hyperi-

benzoyl-CoA, is formed from an early shikimate pathway
intermediate (at a level before phenylpyruvate) and not
from phenylalanine (7) via cinnamic acid or benzoic acid.
Experimental procedures
Materials
[1-
13
C]Glucose and [U-
13
C
6
]glucose were from Omicron
(South Bend, IN, USA). [7-
13
C]Benzoic acid, [1-
13
C]
bromoacetic acid and
L
-[U-
13
C
9
]phenylalanine were from
Correspondence to W. Eisenreich, Lehrstuhl fu
¨
r Organische Chemie
und Biochemie, Technische Universita
¨
tMu

C]bromoacetic
acid [18,19].
Incorporation experiments
Root cultures of S. chirata were grown with supplements of
[carboxy-
13
C]shikimate, [carboxy-
13
C]benzoate, [ring-
13
C
6
]-
cinnamic acid, [1-
13
C]glucose or a mixture of [U-
13
C
6
]glu-
cose and unlabelled glucose, as described previously [3].
Briefly, the cultures were grown in medium containing
glucose instead of sucrose as carbon source without
significant loss of viability or xanthone productivity. In the
first experiment, the cultures were supplemented with a
mixture of [1-
13
C]glucose and unlabelled glucose proffered
at a ratio of 1 : 2.3 (w/w). Although this experiment could
also have been performed with the labelled glucose as the

]shikimate, [carboxy-
13
C]benzoate or
[ring-
13
C
6
]cinnamic acid were proffered at concentrations of
0.5 m
M
, respectively, in medium containing 30 g glucose per
litre. The cultures were incubated for 21 days.
Isolation of 1,3,5,8-tetrahydroxyxanthone
Plant material (fresh weight, 50 g) was pulverized under
liquid nitrogen. The cold slurry was transferred to a flask and
extracted three times with 200 mL methanol under a
nitrogen atmosphere for 15 min. The slurry was filtered.
The solution was concentrated to dryness under reduced
pressure. The residue (500 mg) was applied to a column of
silica gel (Silica Gel 60, 220–440 mesh, 20 · 1.8 cm; Merck,
Darmstadt, Germany), which was developed with a mixture
of chloroform and methanol (30 : 1, v/v). Fractions were
combined and concentrated to dryness under reduced
pressure. The residue was crystallised from methanol (yield,
30 mg).
Fig. 2. Polyketide-type biosynthesis of 1,3,5,8-tetrahydroxyxanthone
(13) with 3-hydroxybenzoyl-CoA (11) as starter unit.
Fig. 1. Shikimate pathway as the source of phenylalanine and other
plant metabolites. Equivalent positions originally derived from phos-
phoenolpyruvate (1) and erythrose 4-phosphate (2)areindicatedbyred

pulse program INAD using a 135 ° read pulse (11.5 ls).
Assessment of isotopomer composition
The relative abundance of
13
C at specific positions of a given
metabolite was calculated from the signal intensities in 1D
13
C NMR spectra (Fig. 3). Specifically, the signal integrals
were determined for each
13
C-NMR signal of a metabolite
from the labelling experiment and of the same compound at
natural
13
C abundance [22]. The ratios of the signal integrals
of the biolabelled compound and of the compound at
natural abundance were then calculated for each respective
carbon atom. Absolute
13
C abundances for certain carbon
atoms (i.e. for carbon atoms with at least one attached
hydrogen atom displaying a
1
H-NMR signal in a non-
crowded region of the spectrum) were then determined from
the
13
C coupling satellites in the
1
H-NMR spectra. As an

C
6
]Glucose [1-
13
C]Glucose
[carboxy-
13
C]-
Shikimate
d
13
C d
1
H J
HH
J
CC
a
J
CH
a
%
13
C
b
%
13
C
13
C

Determined from spectra of
13
C-enriched samples. Coupling partners are given in parentheses.
b
Absolute
13
C abundance.
c
Calculated as
the fraction of
13
C coupled satellite pairs in the total signal intensity for a given carbon. Carbons coupled to the respective index carbon are
indicated in parentheses.
d
Determined from
13
C satellites in the
1
H-NMR signal of the index atom.
2952 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
words, 4.6% of 13 isolated from the experiment with
[U-
13
C
6
]glucose contained
13
C at position 6. The relative
13
C

]-13 (sample from
the experiment with [U-
13
C
6
]glucose) accounted for  20%
in the overall signal intensities for C-8a (Fig. 5 and %
13
C
13
C in Table 1). On the basis of the overall
13
C
abundance of C-8a (4.9%), the molar contribution of
[4b,8a-
13
C
2
]-13 was calculated as 1.0 mol% (see Fig. 7A).
Results
As a prerequisite for the interpretation of labelling data by
NMR spectrometry, unequivocal assignments of all signals
are required. Although
13
C NMR signal assignments of
1,3,5,8-tetrahydroxyxanthone (13) were published on the
basis of chemical-shift arguments [23], we independently
assigned all signals by 2D carbon-carbon correlation
experiments (INADEQUATE) (Fig. 6). This double-quan-
tum filtered experiment reveals scalar couplings between

6
]glucose derived precursors
(see below). Ten pairs of carbon signals detected in the
spectrum provided a solid basis for the assignments
summarized in Table 1.
Some of the 1D
13
C-NMR signals of 1,3,5,8-tetra-
hydroxyxanthone from the experiment with [U-
13
C
6
]glucose
are displayed in Fig. 5. The signals show satellites attributed
to
13
C
13
C coupling involving one or two adjacent
13
C
atoms, which indicate that metabolic precursors carrying
two or more adjacent
13
C atoms have been incorporated
into the biosynthetic product.
Under the experimental conditions used, the proffered
[U-
13
C

C
13
Ccou-
plings are indicated.
Fig. 6. Part of an INAEDQUATE spectrum of 1,3,5,8-tetrahydroxy-
xanthone from the experiment with [U-
13
C
6
]glucose.
Ó FEBS 2003 Biosynthesis of 1,3,5,8-tetrahydroxyxanthone (Eur. J. Biochem. 270) 2953
produced with natural
13
C abundance from the natural
abundance glucose proffered in large excess.
Anabolic (biosynthetic) processes extract labelled and
unlabelled molecules at random from the intermediary
metabolite pools for utilization as building blocks. Natural
products biosynthesized under the experimental conditions
are therefore mosaics of labelled and unlabelled building
blocks. Hence, they represent complex mixtures comprising
a variety of
13
C-labelled isotopomers which can be present
at relatively high abundance compared with their occur-
rence in natural abundance material. A systematic decon-
volution of the multiplets in the
13
C-NMR spectrum gives
the molar fraction of each isotopomer that can be detected

carbon atom; the accompanying numbers indicate the
mol% excess of the respective [
13
C
1
]-isotopomer above the
natural
13
C abundance of 1.1%. A total of three single-
labelled and 11 multiply
13
C-labelled isotopomers showed
increased abundance compared with unlabelled material.
The symmetric labelling pattern of ring A in the
experiment with [U-
13
C
6
]glucose implies that the biosyn-
thetic pathway must involve a c
2
symmetric moiety which is
free to rotate before giving rise to ring A. This result is in full
accordance with the known polyketide-type pathway of
xanthone biosynthesis [7–9] via the benzophenone inter-
mediate 12 [8,9] comprising a c
2
symmetric trihydroxy-
phenyl moiety (Fig. 2).
A much simpler pattern of isotopomers was observed in

alanine from the experiments with [U-
13
C
6
]glucose and
[1-
13
C]glucose were determined as described above and are
shown in Fig. 7D,E, respectively. The structural formulas
have been oriented to match the labelling patterns of the
amino acid with those of ring C of the xanthone
derivative. In each experiment, it is immediately obvious
that the labelling pattern of the aromatic ring in phenyl-
alanine is closely similar to that of ring C in the xanthone
Fig. 8. Reconstruction of the labelling patterns of phosphoenolpyruvate
(1), erythrose 4-phosphate (2) and shikimate (3) from the observed
labelling patterns of phenylalanine (7). (A) From the experiment with
[1-
13
C]glucose. (B) From the experiment with [U-
13
C
6
]glucose. The
colours indicate equivalent positions biosynthetically derived from
phosphoenolpyruvate (in red), erythrose 4-phosphate (in green) or the
fragment of phosphoenolpyruvate after decarboxylation (in blue)
(Fig. 1).
Fig. 7. Labelling patterns of 1,3,5,8-tetrahydroxyxanthone (A–C) and
phenylalanine (D and E). (A), (D) From the experiment with

C abundances. (C) From the experiment with [carb-
oxy-
13
C]shikimate; the filled triangle indicates the carbon atom that
acquired significant
13
C label; the number indicates the
13
C abundance.
2954 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
derivative. It is also obvious that the similarity of the
labelling does not extend further to include the b carbon of
the tyrosine side chain and ring B of the tricyclic
compound. As discussed in more detail below, the
comparison between the isotopomer patterns shows con-
clusively that the specific precursor of 1,3,5,8-tetra-
hydroxyxanthone is a shikimate pathway intermediate
before the level of phenylpyruvate.
In line with that conclusion, the carboxylic group of
shikimate is incorporated into the xanthone derivative as
shown by the experiment with [carboxy-
13
C]shikimate.
1,3,5,8-[9-
13
C
1
]Tetrahydroxyxanthone was found with an
abundance of 14.3 mol% (Fig. 7C). No excess
13

similar to those found with other plants [1,2]. The side chain
reflects the labelling pattern of phosphoenolpyruvate from
which it is biosynthetically obtained via the shikimate
pathway of aromatic amino-acid biosynthesis (Fig. 1). The
aromatic ring of phenylalanine reflects the labelling patterns
of C2–C3 of phosphoenolpyruvate and of C1–C4 of
erythrose 4-phosphate. Owing to the symmetry of the ring,
the ortho and meta carbon atoms become pairwise homo-
topic in the experiment with [1-
13
C]glucose. As a conse-
quence, only an averaged value of 5.4%
13
C can be obtained
for the ortho ring carbon atoms (reflecting C3 of phos-
phoenolpyruvate and C4 of erythrose 4-phosphate, respect-
ively). However, as the
13
C abundance for C3 of
phosphoenolpyruvate can be gleaned from the b-carbon
atom in the side chain (i.e. 5.8%
13
C), the enrichment for C4
of erythrose 4-phosphate can be determined as 5.0%
13
C
from the average value. The deduced labelling patterns of
the intermediary metabolites can then be used to reconstruct
the labelling patterns of shikimate (3) in each respective
experiment (Fig. 8A,B).

detected patterns. Therefore, the phenylalanine hypothesis
must be abandoned.
Hence, 1,3,5,8-tetrahydroxyxanthone biosynthesized in
S. chirata joins the growing list of secondary plant meta-
bolites that are derived from an early shikimate derivative as
opposed to a pathway via phenylalanine and cinnamate. A
hypothetical mechanism for the conversion of shikimic acid
(3) into 3-hydroxybenzoate (15) is shown in Fig. 11 [3]. We
propose that the vinylogous elimination of phosphate from
shikimic acid 3-phosphate (16) affords the dihydroxy-diene
intermediate 17 which appears to be well suited for a
subsequent dehydration yielding the aromatic ring system of
3-hydroxybenzoate (15). The CoA ester of 15 could then
provide the starter unit for the downstream steps of the
polyketide-type biosynthesis of 13.
In line with this interpretation, the experiment with
[carboxy-
13
C
1
]shikimate shows that the carboxylic group,
which is lost in the formation of phenylalanine and tyrosine
(Fig. 1) is in fact incorporated into ring B of 1,3,5,8-
tetrahydroxyxanthone (Figs 7C and 11). Phenylalanine,
tyrosine and metabolites downstream from the amino acids
would not have been advantageous to that labelling pattern.
By comparison with the [
13
C]glucose incorporation
studies, the data structure in the experiment with [

]glucose. (A) Prediction via
phenylalanine (7), benzoic acid (14) and 3-hydroxybenzoic acid (15). (B) Prediction via an early shikimate pathway intermediate, such as shikimate
(3); bold lines connect
13
C-labelled carbon atoms that are transferred from the same molecule of [U-
13
C
6
]glucose; filled dots represent
13
C
1
isotopomers with
13
C enrichment significantly above the natural abundance contributions; numbers indicate
13
C enrichments in mol%; the
isotopomer composition of phenylalanine was determined experimentally (see Fig. 7D); the isotopomer composition of shikimate (3)was
reconstructed from that of phenylalanine (7) (see Fig. 8B). (C) Experimentally determined. For other details, see legend of Fig. 8.
2956 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
13 predicted to be synthesized by a route via phenylalanine
(7) could not be detected (for example [8a,8-
13
C
2
]-13 and
[4b,5,6-
13
C
3

Fonds der Chemischen Industrie and the Hans-Fischer-Gesellschaft.
The expert help of Angelika Werner and Fritz Wendling with the
preparation of the manuscript is gratefully acknowledged.
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