Phytochemistry 72 (2011) 871–874

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its related

anthraquinone in a Sesamum indicum hairy root culture
Toshio Furumoto ⇑, Arata Hoshikuma
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan

a r t i c l e i n f o a b s t r a c t

Article history: In order to clarify the biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its biogenetically related
Received 15 November 2010 anthraquinone, which are possible intermediates of anthrasesamones, [1–13C]glucose was administered
Received in revised form 3 March 2011 to a hairy root culture of Sesamum indicum. The labeling patterns of these quinone derivatives indicated
Accepted 11 March 2011
that the naphthoquinone ring and geranyl side-chain of geranylnaphthoquinone were respectively bio-
Available online 19 April 2011
synthesized through the shikimate and methylerythritol phosphate pathways, and that these quinone
derivatives have the same biosynthetic origin.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Sesamum indicum
Pedaliaceae
Sesame
Biosynthesis
Naphthoquinone
Anthraquinone
Anthrasesamone
Methylerythritol phosphate pathway

1. Introduction quent cyclization of the geranyl side-chain to generate the

Sesame (Sesamum indicum L., Pedaliaceae) is one of the oldest
cultivated crops, and its seeds have been utilized for millennia as
an oilseed and food material (Morris, 2002). Investigation of the
constituents of S. indicum roots, an unutilized portion of the ses-
ame plant, established that the roots contain several unusual
anthraquinones, including 2-(4-methylpent-3-en-1-yl)anthraqui-
none (MPAQ, 2) and anthrasesamones A (3), B (4), C (5), D (6)
and E (7) (Fig. 1) (Furumoto et al., 2003, 2006). Recently, a new
anthraquinone derivative, anthrasesamone F (8) (Fig. 1), was iso-
lated from black seeds, an edible and utilized portion of the sesame
plant (Kim and Park, 2008). The carbon skeleton of MPAQ (2),
which contains a C6 side-chain at C-2 in the anthraquinone ring,
is identical with that of anthrasesamones A–F (3–8), suggesting
that these anthraquinones in sesame are biosynthesized via the
same pathway. Some anthraquinones produced by plants are
formed from prenylated naphthoquinones (Herbert, 1989; Inoue
et al., 1984). Furthermore, shikonin, a well-known naphthoquinone
pigment from Lithospermum erythrorhizon, has the same C6 side-
chain as the anthraquinones in sesame (Tabata, 1996). Shikonin
is biosynthesized from 4-hydroxybenzoic acid and geranyl diphos-
phate through the formation of geranylhydroquinone and subse-
⇑ Corresponding author. Tel.: +81 87 891 3091; fax: +81 87 891 3021.
E-mail address: furumoto@ag.kagawa-u.ac.jp (T. Furumoto).
naphthoquinone ring (Tabata, 1996). On the basis of these findings,
we previously proposed that MPAQ (2) is formed from a possible
intermediate, 2-geranyl-1,4-naphthoquinone (1) (Fig. 1), and veri-
fied the existence of the key intermediates (1 and 2) of anthrases-
amone biosynthesis in an aseptic hairy root culture of S. indicum
(Furumoto et al., 2007). However, the biogenetic origin of these
quinone derivatives in sesame, in particular the origin of monoter-
penoid moiety, cannot be deduced from their chemical structures.
The monoterpenoid moiety of shikonin is known to originate from
the mevalonate pathway (Li et al., 1998). On the other hand, those
of cannabichromenic acid and tetrahydrocannabinolic acid, can-
nabinoids in Cannabis sativa, are derived from the methylerythritol
phosphate (MEP) pathway (Fellermeier et al., 2001), called the
deoxyxylulose phosphate (DXP or DOXP) pathway or the non-mev-
alonate pathway. We therefore conducted a 13C-labeled glucose
administration experiment using S. indicum hairy roots to elucidate
the biosynthetic origin of 1 and 2.
2. Results and discussion
13
D-[1– C]Glucose was administered to a two-week-old hairy
root culture of S. indicum. After two weeks of incubation with labeled
glucose, 13C-labeled geranylnaphthoquinone (1) and MPAQ (2) were
extracted and isolated as described in the Section 4. The 13C NMR
spectra of the enriched and corresponding natural abundance
compounds were measured under identical conditions.

0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.03.012
872 T. Furumoto, A. Hoshikuma / Phytochemistry 72 (2011) 871–874
O In the 13C NMR spectrum of MPAQ (2) labeled from [1–13C]glu-
1 O
O R1
R2
O R3
2 R1 = H, R2 = H, R3 = H
3 R1 = OH, R2 = H, R3 = H
4 R1 = OH, R2 = H, R3 = OH
5 R1 = OH, R2 = Cl, R3 = OH
6 R1 = OH, R2 = OH, R3 = OH
7 R1 = H, R2 = OH, R3 = OH
O the present study afforded further evidence for the formation of
HO
HO OH
8 O
Fig. 1. Structures of 2-geranyl-1,4-naphthoquinone (1), MPAQ (2) and anthrases-
amones A–F (3–8).
Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP), which are common C5 building units for geranyl diphos-
phate and various isoprenoids, are biosynthesized through a classi-
cal mevalonate pathway from acetyl-CoA. IPP and DMAPP are also
synthesized through an alternate methylerythritol phosphate
(MEP) pathway from glyceraldehyde 3-phosphate and pyruvate
(Fig. 2) (Rohmer, 2003). The labeling patterns of IPP and DMAPP
derived from [1–13C]glucose can be explained based on our knowl-
edge of the metabolism of glucose in plants (Disch et al., 1998;
Eichinger et al., 1999; Lichtenthaler et al., 1997). The label from
[1–13C]glucose is incorporated into C-2, C-4 and C-5 in IPP and
DMAPP via the mevalonate pathway, whereas it is incorporated
into C-1 and C-5 via the MEP pathway. These two pathways there-
fore can be distinguished by analysis of the labeling patterns of
metabolites enriched from [1–13C]glucose. The 13C NMR spectrum
of geranylnaphthoquinone (1) labeled from [1–13C]glucose showed
that the label was incorporated into four carbon atoms (C-10 , C-50 ,
C-90 and C-100 ) of the geranyl moiety in 1, which correspond to the
carbon atoms at C-1 and C-5 in IPP and DMAPP (Table 1 and Fig. 2).
This labeling pattern indicates that the geranyl side-chain in 1 is
biosynthesized through the MEP pathway. On the other hand, five
of the carbon signals (C-2, C-3, C-4, C-4a and C-8) of the naphtho-
quinone moiety in enriched 1 also had enhanced intensities com-
pared to natural abundance 1. Many naphthoquinone derivatives
produced by plants are biosynthesized either by the shikimate
pathway or by the polyketide pathway (Seigler, 1998). This incor-
poration pattern was in good accordance with the formation of an
intermediate such as 1,4-dihydroxy-2-naphthoate or 2-carboxy-4-
oxotetralone from o-succinylbenzoate, which is formed from
isochorismate via the shikimate pathway and 2-oxoglutarate via
the citrate cycle (Fig. 2) (Eichinger et al., 1999; Han et al., 2002;
Seigler, 1998). This indicates that the naphthoquinone ring in 1 is
biosynthesized via the shikimate pathway. Moreover, in the
biosynthesis of anthraquinones from prenylated naphthoquinones,
prenylation can occur either at C-2 or C-3 in the intermediate,
1,4-dihydroxy-2-naphthoate (Seigler, 1998). In addition to the bio-
synthetic origin of the geranyl group and naphthoquinone ring in
1, the labeling pattern observed in this experiment gave direct
evidence for geranylation at C-2 in the intermediate (Fig. 2).
cose, incorporation of the label into nine carbon atoms (C-1, C-4,
C-4a, C-5, C-8a, C-9, C-9a, C-20 and C-60 ) in 2 was observed
(Table 2). The labeling pattern of 2 was in agreement with that
of the predicted anthraquinone derivative formed by cyclization
of the geranyl side-chain in 1, as shown in Fig. 2. This suggests that
MPAQ (2) and geranylnaphthoquinone (1) are biosynthesized
through the same biogenetic route, i.e., the shikimate and MEP
pathways.
3. Concluding remarks
The incorporation of [1–13C]glucose into 2-geranyl-1,4-naph-
thoquinone (1) and MPAQ (2) indicated that these quinone deriva-
tives in S. indicum hairy roots were biosynthesized through the
shikimate and methylerythritol phosphate pathways. In addition,
MPAQ (2) from 2-geranyl-1,4-naphthoquinone (1), because the
incorporation pattern of 1 was fully consistent with that observed
in 2. The biosynthesis of several naphthoquinones and anthraqui-
nones proceeds via cyclization (C–C bond formation) between
the prenyl side-chain and aromatic ring in the prenylated quinone,
but the detailed mechanism of such a cyclization reaction is not
fully elucidated. In shikonin biosynthesis, geranylhydroquinone
300 -hydroxylase, a cytochrome P-450 monooxygenase, have been
identified as a key enzyme involved in the cyclization of geranylhy-
droquinone, in a cell suspension culture of L. erythrorhizon
(Yamamoto et al., 2000). This enzyme hydroxylates one of the
three methyl groups present in the geranyl side-chain of geranyl-
hydroquinone, a biosynthetic precursor of shikonin. This finding
therefore suggests that oxidation of the C-100 methyl group present
in the geranyl side-chain of 1 and subsequent cyclization between
the naphthoquinone ring and the oxidized side-chain to generate
the anthraquinone ring would lead to the production of 2. Anthra-
sesamones A–F (3–8) may be generated from 2 via hydroxylation
and chlorination.
4. Experimental
4.1. General experimental procedures
1
H-decoupled 13C NMR spectra of 13C-labeled and natural 13C
abundance samples were recorded with a Jeol JNM-ECA600 FT
NMR spectrometer under identical conditions (150 MHz; 25 °C;
repetition time, 2.5 s; 30° pulse angle) in CDCl3. NMR chemical
shifts were referenced to CDCl3 (dC 77.0). The relative 13C abun-
dance of individual carbon atoms was calculated by comparison
of the 13C signal integrals between the 13C-labeled and unlabeled
compounds. The values were referenced to 1.1% for the carbon
atom at C-30 in 1 and at C-2 in 2, which corresponds to C-3 in
IPP and was presumed to be unlabeled. D-[1–13C]Glucose (99
atom% 13C) was purchased from Sigma–Aldrich Co., USA. Silica
gel 60 (70–230 mesh, Nacalai Tesque, Inc., Japan) and Sephadex
LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for col-
umn chromatography (CC).
4.2. Plant material and culture method
Hairy roots of S. indicum were obtained from an established cul-
ture (Furumoto et al., 2007). The hairy roots (clone SI-37) were cul-
tured in a 100-ml conical flask containing 50 ml of B5 liquid
medium (Gamborg et al., 1968) supplemented with 2% sucrose at
25 °C on a rotary shaker at 80 rpm in the dark. [1–13C]Glucose
(0.2 g/flask) dissolved in sterile H2O was added to ten flasks con-
taining hairy roots that had been grown for two weeks (ca. 16% iso-
 T. Furumoto, A. Hoshikuma / Phytochemistry 72 (2011) 871–874 873

[1-13C]glucose

CHO
COOH CHO HOOC O O
+ HO OP
PO + CoA
OP OH S
OH
phosphoenol- erythrose glyceraldehyde pyruvate acetyl-CoA
pyruvate 4-phosphate 3-phosphate
HO COOH
O 3-deoxy- OH OH
HO 7-phospho-
OP O
OH heptulonate PO HO
OH COOH
1-deoxy-D-xylulose mevalonate
HO COOH 5-phosphate

HO
shikimate
OH OH
PO
OH OH
HOOC COOH
OH 2-C-methyl-D-erythritol
O COOH HOOC 4-phosphate
+
O

isochorismate 2-oxoglutarate
5
1
COOH 2
COOH PPO 3 4 PPO
IPP IPP
o-succinylbenzoate
O

PPO PPO

OH DMAPP DMAPP
2 COOH

1,4-dihydroxy-
2-naphthoate
OH

PPO
O 1'
10'
5'
9' geranyl diphosphate
8
8a 2
1 3' 7' 8'
4 3
5 4a
O
O 5
O 4
10a 4a 6'
10 3
2'
9
8a 9a 2
8 1 1' 3' 5'
O O
2-geranyl-1,4-naphthoquinone (1) MPAQ (2)

Fig. 2. Incorporation of [1–13C]glucose into 1 and 2. The closed circles represent carbon atoms enriched from [1–13C]glucose.

topic abundance in C-1 of the metabolized hexose). The hairy roots
were cultured under the same condition for two weeks with la-
beled glucose.
4.3. Extraction and isolation
The hairy roots and secreted metabolites were separated from
the medium by gravity filtration through filter paper. The lyophi-
lized hairy roots (2.5 g) and filter papers were sonicated for
30 min in MeOH (100 ml  4), and the MeOH soln. obtained was
concentrated to dryness under reduced pressure. The MeOH ex-
tract (1.27 g) was partitioned between CHCl3 (40 ml  3) and
H2O (80 ml) to give the CHCl3-soluble fraction (0.39 g). This frac-
tion was subjected to silica gel CC, using stepwise elution with
Me2CO–hexane containing 0.1% HOAc. The 5% Me2CO fraction
(12 mg) was purified by Sephadex LH-20 CC eluted with MeOH–
CH2Cl2 (1:1). The fraction containing 1 and 2 was further purified
by reversed-phase HPLC (column, COSMOSIL 5C18-MS,
250  10 mm, Nacalai Tesque, Inc.; detection, 254 nm) using
MeOH–HOAc (solvent, 100:0.2; flow rate, 1.5 ml/min) as the mo-
bile phase to afford 13C-labeled 1 (1.0 mg) and 2 (1.3 mg). The pur-
ity of the isolated compounds was confirmed by their NMR spectra.
874 T. Furumoto, A. Hoshikuma / Phytochemistry 72 (2011) 871–874

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a
The numbers in bold face represent significant 13
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10 183.1 1.0
10a 133.63 1.1
10 36.3 1.2
20 29.4 2.2
30 122.6 1.1
40 133.1 1.1
50 25.7 1.1
60 17.7 2.2
a 13
The numbers in bold face represent significant C incorporation from
[1–13C]glucose.