Biotransformation of umbelliferone by Panax ginseng root cultures

Article abstract of DOI:10.1016/S0040-4039(02)01138-3

Panax ginseng root cultures biotransformed umbelliferone (1) to its 7-O-β-D-glucopyranoside (2), 7-O-β-D-glucopyranosyl (1→6) β-D-glucopyranoside (3), 7-O-β-D-xylopyranosyl (1→6) β-D-glucopyranoside (4) and 7-O-α-L-rhamnopyranosyl (1→2) β-D-glucopyranosid

Full text of DOI:10.1016/S0040-4039(02)01138-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5633–5635
Biotransformation of umbelliferone by Panax ginseng
root cultures
a
a
b
b
a,
Wei Li, Kazuo Koike, Yoshihisa Asada, Takafumi Yoshikawa and Tamotsu Nikaido *
a
Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-city, Chiba 274-8510, Japan
b
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Received 20 May 2002; accepted 7 June 2002
Abstract—Panax ginseng root cultures biotransformed umbelliferone (1) to its 7-O-b-
osyl (1“6) b- -glucopyranoside (3), 7-O-b- -xylopyranosyl (1“6) b- -glucopyranoside (4) and 7-O-a-
b- -glucopyranoside (5). The roots showed high glycosylation ability to the 7-hydroxycoumarin molecule. The glycosylation was
D
-glucopyranoside (2), 7-O-b-
D-glucopyran-
-rhamnopyranosyl (1“2)
D
D
D
L
D
catalyzed by glycosyltransferase rather than glycosidase, as was demonstrated by administration of inhibitors. © 2002 Elsevier
Science Ltd. All rights reserved.
A great number of biologically active natural products
exist as glycosides. Chemical synthesis of glycosides is
sometimes limited by the unstable structure of the
glycosyl acceptor or donor. An alternative route to
obtaining glycoconjugates would be by using enzymatic
synthesis, such as glycosyltransferase (or glycosidase) as
administered to hairy roots (ca. 12 g, Fr. Wt.) cultured
for 3 weeks in B5K liquid medium (120 ml per flask)
and then cultured for 1 week. The cultures were sepa-
rated into roots and medium by filtration through a
filter paper. The roots were extracted with 200 ml
methanol under supersonic, 1 h for three times at room
temperature. The methanol extract and the medium
were then analyzed by HPLC. Four biotransformation
products were found in the methanol extracts of roots,
but none from the medium. Further separation of the
extract gave a main product 2 (40.0 mg, biotransforma-
tion yield, 40.1%) and three minor products 3 (10.2 mg,
1
biocatalysts. But the high cost of purified enzyme or
glycosyl-donor, e.g. UDP-sugars, led us to turn our
interest to biological in vivo synthesis of the glycosides
by plant whole cells or organ cultures. Coumarin is a
kind of very important secondary metabolite of the
plant kingdom and has been reported to have various
2
pharmaceutical and biological activities. The low water
6
.8%), 4 (4.1 mg, 2.9%) and 5 (1.9 mg, 1.3%). 2 was
-glucopyranoside
solubility of most coumarins and the lack of their
natural glycosides led us to investigate an approach to
bio-synthesize the coumarin glycosides by plant tissue
cultures. Herein, we report the biotranformation of
umbelliferone (1), a simple 7-hydroxycoumarin wide-
spread in the plant kingdom, by Panax ginseng root
cultures.
confirmed as umbelliferone 7-O-b-
by its [h] , FAB-MS and NMR spectra. The structures
D
5
D
of new compounds 3–5 were further determined by
6
spectral and chemical analysis.
Product 3 was obtained as a colorless powder, [h]D
−63.3 (c 0.67, MeOH, 24°C). Negative high-resolution
FAB-MS analysis of 3 provided a molecular formula of
The P. ginseng root cultures were established as
−
3
C H O [(M−H) ] (found 485.1305, calcd 485.1296),
described in a previous paper. The root cultures were
21 25 13
4
suggesting that 3 is larger than 2 by a hexose unit. Acid
hydrolysis of 3 by 1 M hydrochloric acid in dioxane/
subcultured on Murashige and Skoog’s basal medium
containing 5 mg/l IBA and 0.1 mg/l kinetin (B5K
medium) at 25°C in the dark at 140 rpm on a rotary
shaker at 4 weeks intervals. 50 mg of umbelliferone
water (1:1) gave only
D-glucose as component sugars,
which was identified by HPLC analysis following its
(
Nihon kaisei. Co.) dissolved in ethanol (2 ml) were
conversion to the 1-[(S)-N-acetyl-a-methylbenzyl-
7
amino]-1-deoxy-alditol acetate derivatives. The b-
anomeric configurations for glucose were determined
Keywords: biotransformation; umbelliferone; Panax ginseng root cul-
tures.
3
from their large J
coupling constants (7–8 Hz).
H1,H2
*
Corresponding author. Tel.: +81-47-4721391; fax: +81-47-4721404;
The position of the sugar moiety attached to the agly-
e-mail: nikaido@phar.toho-u.ac.jp
con and the interglycosidic linkage were confirmed by
0
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01138-3

5
634
W. Li et al. / Tetrahedron Letters 43 (2002) 5633–5635
observing the correlations between H-1 (l 5.03) of the
inner glucose and C-7 (l 160.7) of aglycone, H-1 (l
were assigned as shown in Table 1, indicating that
component sugars in 5 are one b-glucopyranoses and
one a- or b-rhamnopyranose. Two sugar units were
4
.36) of the terminal glucose and C-6 (l 69.0) of the
inner glucose in the HMBC spectrum. Based upon all
further identified as D-glucose and L-rhamnose by
7 13
of the above evidence, the structure of 3 was elucidated
HPLC analysis of the acid hydrolysate. The C NMR
data of rhamnose were compared with those of methyl-
as umbelliferone 7-O-b-
D
-glucopyranosyl (1“6) b-D-
glucopyranoside.
a-
L
and methyl-b-
L
rhamnopyranosides. They agreed
- rhamnopyranoside. The
well with those of methyl-a-
L
Product 4 was obtained as a colorless powder, [h]D
66.0 (c 0.24, MeOH, 24°C). Negative high-resolution
interglycosidic linkage was determined unambiguously
by the HMBC spectrum. Namely, the correlation
between H-1 (l 5.28) of rhamnose and C-2 (l 77.8) of
glucose indicated that rhamnose was linked to C-2 of
glucose. Based upon all the above evidence, the struc-
−
FAB-MS analysis of 4 provided a molecular formula of
−
C H O [(M−H) ] (found: 455.1180; calcd 455.1189),
20
23 12
which is larger by a pentose unit than 2. Acid hydroly-
sis of 3 gave
D
-glucopyranose and
D
-xylopyranose in a
ture of 5 was elucidated as umbelliferone 7-O-a-
L-
ratio of 1:1, which were identified by HPLC analysis
following their conversion to the 1-[(S)-N-acetyl-a-
methylbenzylamino]-1-deoxy-alditol acetate deriva-
rhamnopyranosyl(1“2) b- -glucopyranoside.
D
The biotransformation pathway of umbelliferone (1) by
P. ginseng root cultures is considered as shown in Fig.
1. P. ginseng root cultures do not contain coumarins as
metabolites. To investigate the enzyme responsible for
this glycosylation, glucosyltransferase inhibitors [hex-
adecylpyridinium chloride (final conc. 4.2 mM), hex-
adecyltrimethyl ammonium bromide (4.1 mM) and
sodium dodecyl sulfate (17.3 mM)], and b-glucosidase
inhibitors [imidazole (4.6 mM), benzimidazole (6.5
mM) and 2-aminopyridine (16.0 mM)], were adminis-
tered to P. ginseng root cultures that were pre-cultured
for 2 weeks, respectively. After 3 days, umbelliferone
(each 30 mg) was administered to the root cultures, and
the root cultures were further cultured for 1 week, then
harvested and extracted with 20 ml methanol, respec-
7
tives. The b-anomeric configurations for the glucose
3
and xylose were determined from their large J
H1,H2
coupling constants (7–8 Hz). In the HMBC spectrum,
correlations of H-1 (l 4.31) of the xylose with C-6 (l
6
9.0) of the glucose gave the linkage of the sugar units
to be b- -xylopyranosyl(1“6) b- -glucopyranose.
Thus, the structure of 4 was established as umbellifer-
one 7-O-b- -xylopyranosyl(1“6) b- -glucopyranoside.
D
D
D
D
Product 5 was obtained as a colorless powder, [h]D
76.2 (c 0.13, MeOH, 24°C). Negative high resolution-
FAB-MS of 5 gave the molecular formula as C H O
−
2
1
25 12
−
[
(M−H) ] (found: 469.1360; calcd 469.1346). On the
1 1
basis of the H– H COSY spectrum, all proton signals
1
13
Table 1. H (500 MHz) and C NMR (125 MHz) data for the biotransformation products 3–5 in methanol-d at 30°C
4
Position
3
4
5
l 1H (mult, J in Hz)
l 13
C
l 1H (mult, J in Hz)
l 13
C
l 1H (mult, J in Hz)
l 13
C
2
3
4
5
6
7
8
9
161.9
112.9
144.3
129.2
114.0
160.7
103.8
155.4
114.0
161.9
112.9
144.3
129.2
114.0
160.8
103.8
155.4
114.0
161.8
113.0
144.2
129.1
113.7
160.5
103.4
155.3
114.0
6.28 (d, 9.6)
7.89 (d, 9.6)
7.57 (d, 8.5)
7.12 (dd, 8.5, 1.8)
6.28 (d, 9.6)
7.89 (d, 9.6)
7.57 (d, 8.8)
7.11 (dd, 8.8, 2.3)
6.28 (d, 9.6)
7.89 (d, 9.6)
7.56 (d, 8.8)
7.03 (dd, 8.8, 2.3)
7.16 (d, 1.8)
7.15 (d, 2.3)
7.04 (d, 2.3)
1
0
Glc
Glc
Glc
1%
2%
3%
4%
5%
6%
5.03 (d, 7.8)
100.4
73.5
76.5
70.2
76.1
69.0
4.99 (d, 7.3)
100.6
73.4
76.5
70.2
76.1
69.0
5.16 (d, 7.8)
98.8
77.8
77.7
70.1
76.9
61.1
a
a
3.49
3.48
3.48
3.69 (dd, 9.2, 7.8)
3.61 (dd, 9.6, 9.2)
3.39 (dd, 9.6, 9.6)
3.51 (ddd, 9.6, 5.9, 1.8)
3.89 (dd, 11.9, 1.8)
3.68 (dd, 11.9, 5.9)
Rham
3.49a
a
3.40 (dd, 8.7, 8.7)
3.82 (m)
4.16 (dd, 12.0, 2.5)
3.37 (dd, 8.7, 8.7)
3.75 (ddd, 8.7, 6.5, 1.8)
4.10 (dd, 11.4, 1.8)
3.79 (dd, 11.4, 6.5)
Xyl
3
.83 (dd, 12.0, 5.5)
Glc
1%%
2%%
3%%
4%%
5%%
4.36 (d, 7.3)
103.8
73.9
76.8
70.4
76.8
4.31 (d, 7.3)
104.4
73.7
76.5
69.8
65.6
5.28 (d, 1.8)
101.2
70.9
70.9
72.6
68.6
a
3.26 (dd, 8.7, 7.3)
3.33 (dd, 8.7, 8.7)
3.35 (dd, 8.7, 8.7)
3.24 (ddd, 8.7, 6.2, 2.3)
3.26
3.26
3.93 (dd, 1.8, 1.8)
3.56 (dd, 9.6, 1.8)
3.40 (ddd, 9.6, 9.6)
3.93 (dq, 9.6, 6.4)
a
3.56 (ddd, 10.2, 8.7, 5.5)
3.14 (dd, 11.5, 10.2)
3.87 (dd, 11.5, 5.5)
6
%%
3.86 (dd, 11.9, 2.3)
61.5
1.29 (dd, 6.4)
16.8
3
.66 (dd, 11.9, 6.2)
a
Overlapped signals.

W. Li et al. / Tetrahedron Letters 43 (2002) 5633–5635
5635
HO
O
O
1
OH
OH
O
HO
HO
O
O
HO
HO
O
O
O
OH
HO
O
OH
O
O
O
HO
OH
3
2
OH
O
O
HO
HO
O
O
O
HO
HO
O
OH
HO
O
O
O
O
O
HO
O
HO
OH
4
HO
OH
5
Figure 1. Biotransformation of umbelliferone (1) by P. ginseng root cultures.
8
tively. Further HPLC analysis of main biotransformed
MS (negative) m/z: Found: 485.1305; Calcd for C H O
22 25 13
−
1
product 2 showed that glucosyltransferase inhibitors
hexadecylpyridinium chloride, hexadecyltrimethyl
[(M−H) ]: 485.1296. H NMR (500 MHz, methanol-d4)
13
(
and C NMR (125 MHz, methanol-d ): see Table 1.
4
ammonium bromide and sodium dodecyl sulfate) inhib-
ited the glycosylation completely, on the contrary to the
fact that the glucosidase inhibitors showed almost no
effect on the glycosylation to umbelliferone. Thus, the
glycosylation was most likely catalyzed by the glycosyl-
tranferase. The glycosyltransferase is now under further
investigation.
Umbelliferone 7-O-b-
D
-xylopyranosyl(1“6) b-
D
D-glucopy-
ranoside (4): Powder, [h] −66.0 (c 0.24, MeOH, 24°C). IR
−
1
w cm : 3446, 1702, 1619. FAB-MS (negative) m/z: 455
−
[
(M−H) ]. HR-FAB-MS (negative) m/z: Found: 455.1180;
−
1
Calcd for C H O [(M−H) ]: 455.1189. H NMR (500
20
23 12
13
MHz, methanol-d ) and C NMR (125 MHz, methanol-
4
d4): see Table 1. Umbelliferone 7-O-b-
osyl(1“2) b- -glucopyranoside (5): Powder, [h] −76.2 (c
.13, MeOH, 24°C). IR w cm : 3432, 1706, 1619. FAB-
D-rhamnopyran-
D
D
−
1
0
Acknowledgements
−
MS (negative) m/z: 469 [(M−H) ]. HR-FAB-MS (nega-
tive) m/z: Found: 469.1360; Calcd for C H O
25 12
21
−
1
[
(M−H) ]: 469.1346. H NMR (500 MHz, methanol-d4)
The work was supported by the Sasakawa Scientific
Research Grant from The Japan Science Society.
13
and C NMR (125 MHz, methanol-d ): see Table 1.
4
7
. (a) Oshina, R.; Kumanotani, J. Chem. Lett. 1981, 943; (b)
Oshina, R.; Yamauchi, Y.; Kumanotani, J. Carbohydr.
Res. 1982, 107, 169; (c) HPLC conditions for 1-[(S)-N-ace-
tyl-a-methylbenzylamino]-1-deoxy-alditol acetate deriva-
tives of the monosaccharides: Column, Senshu Pak Pegasil
References
1
2
. Koeller, K. M.; Wong, C.-H. Chem. Rev. 2000, 100, 4465.
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Prod. 1984, 47, 70.
ODS, 4.6×150 mm; Solvent: 33% CH CN; flow rate, 0.8
3
ml/min; detection, UV 230 nm. The derivatives of
cose, -xylose and -rhamnose were detected as follows: tR
min) 38.30 (derivative of -glucose), 26.50 (derivative of
-xylose) and 40.24 (derivative of -rhamnose).
D-glu-
D
L
3
(
D
D
L
4
5
. Murashige, T.; Skoog, F. Physiol. Plant 1962, 15, 473.
. Cussans, N. J.; Huckerby, T. N. Tetrahedron 1975, 31,
8
. HPLC condition for analysis of main product 2. Detector:
SPD-M10 Avp Diode Array (Shimadzu); 280 nm.
Column: Shenshu Pak, Pegasil ODS II (4.6×250 mm).
2
719.
. Data for new compounds. Umbelliferone 7-O-b-
glucopyranosyl(1“6) b- -glucopyranoside (3): Powder,
h]D −63.3 (c 0.67, MeOH, 24°C). IR w cm : 3430, 1702,
6
D-
Solvent: MeOH/H O (0.06% TFA), Linear gradient; 0%
D
2
−
1
ꢀ
100% MeOH in 45 min, then 100% MeOH at flow rate
[
−
1
617. FAB-MS (negative) m/z: 485 [(M−H) ]. HR-FAB-
0.7 ml/min. Column temperature: 40°C. t : 26.0 min.
R