Anthraquinone biosynthesis in sesame hairy roots
3
2
Administration of H-labeled MPAQ (1a).
hairy roots (about 0.3 g fresh wt.) were cultured in a
00-mL conical flask containing 50 mL of the B5 liq-
The
1′), 5.13 (1H, tqq, J = 7.2, 1.4, 1.4 Hz, H-3′), 7.59 (1H,
dd, J = 7.9, 1.8 Hz, H-3), 7.77 (0.9H, m, H-6 and H-
7), 8.11 (1H, d, J = 1.8 Hz, H-1), 8.21 (1H, d,
J = 7.9 Hz, H-4), 8.29 (0.9H, m, H-5 and H-8). EIMS
1
uid medium supplemented with 2% sucrose in the dark
at 25 °C at 70 rpm. Twenty flasks containing hairy
roots that had been grown for 14 days were washed
+
+
m/z (rel. int.): 290 (M , 19.5), 294 (M +4, 22.2).
1
with sterile H O (25 mL × 2 per flask), before the med-
Compound 2.
H NMR (CDCl ) δ : 1.86 and
3 H
2
ium was replaced with fresh B5 medium supplemented
with 0.2% sucrose and 100 mM NaCl. H-labeled 1a
1.87 (each 3H, br.s, H-5′ and H-6′), 6.37 (1H, dm,
J = 11.7 Hz, H-3′), 6.38 (1H, d, J = 11.6 Hz, H-1′),
6.62 (1H, dd, J = 11.7, 11.6 Hz, H-2′), 7.74 (1H, dd,
J = 8.0, 1.8 Hz, H-3), 7.78 (0.86H, m, H-6 and H-7),
8.24 (1H, d, J = 1.8 Hz, H-1), 8.26 (1H, d, J = 8.0 Hz,
H-4), 8.30 (0.86H, m, H-5 and H-8). EIMS m/z (rel.
2
(
10 mg, 0.5 mg/flask) dissolved in EtOH (1.2 mL) was
then fed to these flasks containing the fresh medium
and washed hairy roots. The hairy roots were cultured
2
under the same condition for four days with H-labeled
+
+
1
a. The hairy roots and secreted metabolites were sepa-
int.): 288 (M , 30.5), 292 (M +4, 47.7).
rated from the medium by gravity filtration through fil-
ter paper. The harvested hairy roots and filter paper
were freeze-dried and sonicated for 30 min in MeOH
Results and discussion
(
150 mL × 3). The MeOH solution obtained was con-
We examined whether MPAQ (1) and 2-geranyl-1,4-
naphthohydroquinone (7) serve as intermediates for
anthraquinone derivatives produced by S. indicum hairy
roots. A preliminary administration experiment using a
conventional B5 medium was unsuccessful because of
very low or no accumulation of anthraquinone deriva-
tives such as 3–5 (data not shown). In order to improve
the production of anthraquinone derivatives in the hairy
roots, several culture conditions were tested. The addi-
centrated to dryness under reduced pressure. The
MeOH extract was partitioned between CH Cl
2
2
(
100 mL × 3) and H O (100 mL) to give the CH Cl -
2 2 2
soluble fraction. This fraction was subjected to silica
gel column chromatography using stepwise elution with
acetone–hexane containing 0.1% AcOH. The 5 and
1
0% acetone fractions were combined and purified by
Sephadex LH-20 column chromatography eluted with
MeOH–CH Cl (1:1). The fractions containing the
metabolites were further purified by reversed-phase
2
2
7
)
tion of sodium chloride at 100 mM in the B5 medium
gave a sufficient amount of 2,3-epoxyanthrasesamone
B (5) together with a small amount of anthrasesamone
B (4), although anthrasesamone A (3) unfortunately did
not accumulate. Administration experiments with
HPLC (column, Cosmosil 5C -AR-II, 250 × 10 mm
i.d., Nacalai Tesque; detection, 254 nm) using MeOH–
AcOH as the mobile phase (solvent, 100:0.2; flow rate,
1
8
1
5
.5 mL/min) to afford 1 (5 mg), 2 (3 mg), 4 (0.5 mg),
(3 mg) and 6 (<0.5 mg).
2
H-labeled substrates were therefore carried out using
this improved medium. In contrast, the conventional
B5 medium was utilized as the growth medium for
reducing the endogenous production of anthraquinone
derivatives, prior to administration of the labeled
1
Compound 2.
H NMR (CDCl ) δ : 1.86 and
3 H
1
.87 (each 3H, br.s, H-5′ and H-6′), 6.37 (1H, dm,
J = 11.7 Hz, H-3′), 6.38 (1H, d, J = 11.7 Hz, H-1′),
.62 (1H, dd, J = 11.7, 11.7 Hz, H-2′), 7.73 (1H, dd,
J = 8.0, 1.8 Hz, H-3), 7.78 (0.48H, m, H-6 and H-7),
.24 (1H, d, J = 1.8 Hz, H-1), 8.25 (1H, d, J = 8.0 Hz,
H-4), 8.30 (0.48H, m, H-5 and H-8). EIMS m/z (rel.
substrates.
6
2
[5,6,7,8- H ]MPAQ (1a) was synthesized by the
4
2
Diels–Alder cycloaddition reaction of [ H ]-1,4-naph-
8
6
thoquinone and β-myrcene and subsequent aromatiza-
1
4) 2
+
+
tion of the adduct.
%
H-labeled MPAQ (1a) (>99 atom
int.): 288 (M , 3.4), 292 (M +4, 12.4).
2
H) was administered to a two-week-old hairy root
culture of S. indicum. After four days of incubation
with H-labeled MPAQ (1a), quinone derivatives were
2
2
Administration of H-labeled 2-geranyl-1,4-naphtho-
hydroquinone (7a).
The administration experiment
extracted and isolated as described in the above section.
The H NMR spectrum of (Z)-MPDEAQ (2) obtained
from this experiment showed that two aromatic proton
2
1
with H-labeled 7a was carried out by the same method
2
as that used for H-labeled 1a, except for the prepara-
tion of 7a from 6a just before the administration of the
signals at δ 7.78 (H-6 and H-7) and 8.30 (H-5 and H-
H
labeled substrate. A solution of 6a (15 mg) in Et O
8) in isolated 2 had remarkably decreased intensities
2
(
(
10 mL) was shaken with 15% sodium dithionite
(Fig. 2(A)) compared to natural abundance
2
10 mL). The Et O layer obtained was washed with a
(Fig. 2(C)) and that the content of deuterium atoms in
isolated 2 was 76% at these positions, indicating that
exogenously supplied 1a was efficiently converted to 2.
Moreover, the existence of the deuterium labels in iso-
2
mixture of saturated NaCl (10 mL) and 15% sodium
dithionite (2 mL), passed through Na SO and concen-
trated to dryness under reduced pressure. Without stor-
2
4
2
age, the reduced product (7a) was dissolved in EtOH
lated 2 was confirmed by the H NMR analysis, where
(
2.4 mL) and immediately fed to the hairy root cul-
two broad singlet signals having almost the same signal
integral were observed at the corresponding positions.
These results clearly demonstrate that MPAQ (1) is an
actual biosynthetic precursor for the formation of (Z)-
MPDEAQ (2). As expected, the hairy roots did not
convert labeled MPAQ (1a) to 2-geranyl-1,4-naphtho-
quinone (6). Contrary to our presumption, however, the
conversion of labeled MPAQ (1a) to anthrasesamone B
tures. After the final purification by reversed-phase
HPLC, 1 (1 mg), 2 (2 mg), 4 (0.5 mg) and 5 (1 mg)
and 6 (4 mg) were isolated.
1
Compound 1.
H NMR (CDCl ) δ : 1.53 (3H,
3 H
br.s, H-6′), 1.66 (3H, br.s, H-5′), 2.36 (2H, br.dt,
J = 7.2, 7.7 Hz, H-2′), 2.80 (2H, t-like, J = 7.7 Hz, H-