Glucosyloxybenzyl 2-isobutylmalates from the tubers of Gymnadenia conopsea

Article abstract of DOI:10.1021/np0581115

Seven new glucosyloxybenzyl 2-isobutylmalates, gymnosides I-VII (1-7), were isolated from the tubers of Gymnadenia conopsea. The structures of 1-7 were determined on the basis of chemical and physicochemical evidence.

Full text of DOI:10.1021/np0581115

J. Nat. Prod. 2006, 69, 881-886
881
Glucosyloxybenzyl 2-Isobutylmalates from the Tubers of Gymnadenia conopsea1
Toshio Morikawa, Haihui Xie, Hisashi Matsuda, and Masayuki Yoshikawa*
Kyoto Pharmaceutical UniVersity, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
ReceiVed October 12, 2005
Seven new glucosyloxybenzyl 2-isobutylmalates, gymnosides I-VII (1-7), were isolated from the tubers of Gymnadenia
conopsea. The structures of 1-7 were determined on the basis of chemical and physicochemical evidence.
Gymnadenia conopsea R. Br., an Orchidaceae perennial herb,
is widely distributed in the northern parts of China such as Hebei,
Liaoning, and Gansu Provinces. The tubers of this herb have been
used in traditional Chinese medicine for the treatment of asthma,
neurasthenia, and chronic hepatitis.2 Recently, we found that the
MeOH extract of this natural medicine showed antiallergic activity
in the passive cutaneous anaphylaxis (PCA) reactions in mice.3 In
the course of our characterization studies on bioactive constituents
from Chinese natural medicines,1,4-10 we previously reported the
structures of three dihydrophenanthrenes, gymconopins A-C, and
a dihydrostilbene, gymconopin D, as the less polar constituents from
the tubers of G. conoposa, together with 10 known constituents
and their inhibitory activities on antigen-induced degranulation in
RBL-2H3 cells.3 As a continuing study of this natural medicine,
we additionally isolated seven new glycosyloxybenzyl 2-isobutyl-
malates named gymnosides I-VII (1-7). This paper deals with
the isolation and elucidation of absolute configuration of the
glycosides 1-7.
Chart 1
Results and Discussion
The tubers of G. conoposa were extracted with MeOH under
reflux. The MeOH extract was subjected to Diaion HP-20 column
chromatography to give H2O-, MeOH-, and acetone-eluted fractions.
The MeOH-eluted fraction was subjected to ordinary and reversed-
phase silica gel column chromatography and finally HPLC to
furnish gymnosides I (1, 0.0024% from the natural medicine), II
(2, 0.0009%), III (3, 0.017%), IV (4, 0.0006%), V (5, 0.0014%),
VI (6, 0.0021%), and VII (7, 0.0025%), together with dactylorhin
A11 (8, 0.12%) and militarine11 (9, 0.072%). Gymnoside I (1) was
obtained as a white powder and exhibited a negative specific
rotation ([R]24 -28.0 in MeOH). In the UV spectrum of 1,
D
absorption maxima were observed at 223 (log ꢀ 4.10) and 256 (3.20)
nm. The IR spectrum of 1 showed absorption bands at 1736, 1710,
1615, 1590, 1514, and 1233 cm-1 assignable to ester carbonyl and
carboxyl functions and an aromatic ring in addition to strong
absorption bands at 3410 and 1075 cm-1 suggestive of a glycoside
moiety. In the positive- and negative-ion FABMS of 1, quasimo-
lecular ions were observed at m/z 481 [M + Na]+ and 457 [M -
H]-, and HRFABMS analysis revealed the molecular formula of 1
to be C21H30O11. The acid hydrolysis of 1 with 1.0 M HCl liberated
D-glucose, which was identified by HPLC analysis using an optical
1
rotation detector.1,4,6-8,10 The H (pyridine-d5, Table 1) and 13C
NMR (Table 2) spectra of 1, which were assigned by various NMR
experiments,12 showed signals assignable to two methyls [δ 0.95,
1.03 (3H each, both d, J ) 6.7 Hz, H3-7, 8)], three methylenes [δ
1.90, 1.93 (1H each, both dd, J ) 6.1, 14.0 Hz, H2-5), 3.11, 3.37
(1H each, both d, J ) 15.9 Hz, H2-3), 5.36, 5.39 (1H each, both d,
J ) 12.2 Hz, H2-7′)], a methine [δ 2.12 (1H, m, H-6)], and an
A2B2 type aromatic pattern [δ 7.32, 7.47 (2H each, both d, J ) 8.5
Hz, H-3′,5′, 2′,6′)], together with a â-glucopyranosyl moiety [δ 5.61
* To whom correspondence should be addressed. Tel: +81-75-595-4633.
Fax: +81-75-595-4768. E-mail: shoyaku@mb.kyoto-phu.ac.jp.
10.1021/np0581115 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/19/2006

882 Journal of Natural Products, 2006, Vol. 69, No. 6
Morikawa et al.
Figure 1.
Table 1. 1H NMR (500 MHz, pyridine-d5) Data of Gymnosides
I (1) and II (2)
Table 2. 13C NMR (125 MHz, pyridine-d5) Data of
Gymnosides I (1) and II (2)
1
2
C
1
2
H
δ (J Hz)
HMBC
C-1,4,5
δ (J Hz)
HMBC
C-1,4,5
1
2
3
4
5
6
7
8
176.0 (s)
76.2 (s)
46.1 (t)
173.8 (s)
48.5 (t)
24.4 (d)
23.8 (q)a
24.7 (q)a
130.0 (s)
130.4 (d)
116.8 (d)
158.5 (s)
66.9 (t)
178.5 (s)
75.9 (s)
45.9 (t)
170.9 (s)
48.6 (t)
24.7 (d)
23.0 (q)a
24.8 (q)a
3
5
3.11 (d, 15.9)
3.37 (d, 15.9)
3.10 (d, 15.0)
3.40 (d, 15.0)
1.90 (dd, 6.1,14.0) C-1,3,7,8 1.94 (dd, 6.1,14.0) C-1,3,7,8
1.93 (dd, 6.1,14.0)
2.12 (m)
2.05 (dd, 6.1,14.0)
6
C-2,5,7,8 2.21 (m)
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′
C-2
C-5,8
C-5,7
7
0.95 (d, 6.7)a
1.03 (d, 6.7)a
7.47 (d, 8.5)
7.32 (d, 8.5)
5.36 (d, 12.2)
5.39 (d, 12.2)
1.05 (d, 6.7)a
1.08 (d, 6.7)a
8
1′
2′,6′
3′,5′
4′
7′
1′′
2′′,6′′
3′′,5′′
2′,6′
3′,5′
7′
2′′,6′′
3′′,5′′
7′′
7.35 (d, 8.9)
7.27 (d, 8.9)
5.13 (d, 12.2)
5.18 (d, 12.2)
C-4′′,7′′
C-1′′
C-4,2′′,6′′
130.4 (s)
130.2 (d)
116.8 (d)
158.4 (s)
66.1 (t)
4′′
7′′
4′-O-Glc-1 5.61 (d, 7.6)
C-4′
2
3
4
5
6
4.31 (dd, 7.6,8.9)
4′-O-Glc-1
2
3
4
5
6
102.0 (d)
74.9 (d)
78.5 (d)
71.2 (d)
78.8 (d)
62.3 (t)
4.37 (m)
4.36 (m)
4.10 (m)
4.40 (dd, 5.2,11.9)
4.52 (dd, 2.1,12.2)
4′′-O-Glc-1
5.60 (d, 7.6)
4.31 (dd, 7.6,9.5)
4.37 (m)
4.36 (m)
4.11 (m)
4.41 (dd, 5.2,12.2)
4.54 (dd, 2.5,12.2)
C-4′′
2
3
4
5
6
4′′-O-Glc-1
102.0 (d)
74.9 (d)
78.5 (d)
71.2 (d)
78.9 (d)
62.3 (t)
2
3
4
5
6
a May be interchanged within the same column.
a May be interchanged within the same column.
(1H, d, J ) 7.6 Hz, H-4′-O-Glc-1)]. The enzymatic hydrolysis of
1 with â-glucosidase gave (2R)-hydroxy-2-(2-methylpropyl)bu-
tanedioic acid11 [1b ) (2R)-2-isobutylmalic acid], which was
deduced to be formed via the p-hydroxybenzyl ester (1a). Com-
pound 1b was also obtained by the enzymatic hydrolysis of 1 with
tannase, which is known to hydrolyze the ester bond of aromatic
compounds such as tannins, flavonoids, and chromones.13,14 In the
HMBC experiment of 1, long-range correlations were observed
between the following proton and carbon pairs: H-3 and C-1, 4,
5; H2-5 and C-1, 3, 7, 8; H-6 and C-2, 5, 7, 8; H3-7 and C-5, 8;
H3-8 and C-5, 7; H-2′,6′ and C-4′, 7′; H-3′,5′ and C-1′; H2-7′ and
C-1, 1′, 2′,6′; H-4′-O-Glc-1 and C-4′ (Table 1, Figure S1). Thus
the positions of the â-glucopyranosyl and p-hydroxybenzyl alcohol
moieties in 1 were clarified. On the basis of those findings, the
structure of gymnoside I was determined to be 1-(4-â-D-glucopy-
ranosyloxybenzyl)-(2R)-2-isobutylmalate (1). Gymnoside II (2) was
also isolated as a white powder with negative specific rotation
([R]24D -21.8 in MeOH). The UV spectrum of 2 showed absorption
maxima at 223 (log ꢀ 4.10) and 257 (3.31) nm. The IR spectrum
of 2 showed absorption bands at 3432, 1734, 1719, 1617, 1592,
1514, 1235, and 1076 cm-1 ascribable to hydroxyl, ester carbonyl,
carboxyl, and ether functions and an aromatic ring. The molecular
formula C21H30O11 of 2, which was the same as 1, was determined
by the quasimolecular ion peaks in positive- and negative-ion
FABMS and by HRFABMS. The acid hydrolysis of 2 with 1.0 M
HCl liberated D-glucose, which was identified by HPLC analysis
using an optical rotation detector.1,4,6-8,10 The proton and carbon
1
signals in the H (pyridine-d5, Table 1) and 13C NMR (Table 2)
spectra12 of 2 were very similar to those of 1 {two methyls [δ 1.05,
1.08 (3H each, both d, J ) 6.7 Hz, H3-7, 8)], three methylenes [δ
1.94, 2.05 (1H each, both dd, J ) 6.1, 14.0 Hz, H2-5), 3.10, 3.40
(1H each, both d, J ) 15.0 Hz, H2-3), 5.13, 5.18 (1H each, both d,
J ) 12.2 Hz, H2-7′′)], a methine [δ 2.21 (1H, m, H-6)], and an
A2B2 type aromatic pattern [δ 7.27, 7.35 (2H each, both d, J ) 8.9
Hz, H-3′′,5′′, 2′′,6′′)], together with a â-glucopyranosyl moiety [δ
5.60 (1H, d, J ) 7.6 Hz, H-4′′-O-Glc-1)]}. The enzymatic
hydrolysis of 2 with â-glucosidase or tannase gave 1b via 2a (vide
ante). The positions of the â-D-glucopyranosyl and p-hydroxybenzyl
alcohol moieties in 2 were confirmed by the HMBC experiment,
which showed long-range correlations between the following proton
and carbon pairs: H-3 and C-1, 4, 5; H2-5 and C-1, 3, 7, 8; H-6
and C-2, 5, 7, 8; H3-7 and C-5, 8; H3-8 and C-5, 7; H-2′′,6′′ and
C-4′′, 7′′; H-3′′,5′′ and C-1′′; H2-7′′ and C-4, 2′′,6′′; H-4′′-O-Glc-1
and C-4′′ (Table 1, Figure S1). Consequently, the structure of
gymnoside II was determined as 4-(4-â-D-glucopyranosyloxyben-
zyl)-(2R)-2-isobutylmalate (2).
Gymnoside III (3) was isolated as a white powder with negative
specific rotation ([R]22 -47.6 in MeOH). The positive- and
D
negative-ion FABMS of 3 showed quasimolecular ions at m/z 953
[M + Na]+ and m/z 929 [M - H]-. The molecular formula

Glucosyloxybenzyl 2-Isobutylmalates from Gymnadenia
Journal of Natural Products, 2006, Vol. 69, No. 6 883
Table 3. 1H NMR (500 MHz, pyridine-d5) Data of Gymnosides III-VII (3-7)
3
4
5
6
7
H
δ (J Hz)
HMBC
C-1,4,5
δ (J Hz)
HMBC
δ (J Hz)
HMBC
δ (J Hz)
HMBC
δ (J Hz)
HMBC
C-1,4,5
3
5
3.37 (d, 17.7)
3.56 (d, 17.7)
1.94 (m)
3.30 (d, 17.7)
3.46 (d, 17.7)
C-1,4,5 3.31 (d, 17.7)
3.52 (d, 17.7)
C-1,3,7,8 1.90 (2H, m)
C-1,4,5 3.39 (d, 17.5)
3.48 (d, 17.5)
C-1,3,7,8 1.94 (m)
2.01 (m)
C-1,4,5 3.38 (d, 17.4)
3.54 (d, 17.4)
C-1,3,7,8 1.96 (m)
2.06 (m)
C-1,3,7,8 1.87 (m)
2.03 (m)
C-1,3,7,8
2.02 (m)
6
7
8
2′,6′
3′,5′
7′
2.02 (m)
C-2,5,7,8 2.03 (m)
C-2,5,7,8 2.00 (m)
C-2,5,7,8 2.01 (m)
C-2,5,7,8 2.05 (m)
C-2,5,7,8
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′
0.87 (d, 6.1)a
0.91 (d, 6.1)a
7.43 (d, 8.9)
7.38 (d, 8.9)
5.27 (d, 11.9)
5.33 (d, 11.9)
7.45 (d, 8.9)
7.37 (d, 8.9)
5.13 (d, 11.9)
5.25 (d, 11.9)
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′
0.85 (d, 6.7)a
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′ 5.33 (d, 12.2)
5.39 (d, 12.2)
0.86 (d, 6.7)a
0.91 (d, 6.7)a
7.45 (d, 8.9)
7.39 (d, 8.9)
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′ 5.28 (d, 11.9)
5.35 (d, 11.9)
0.86 (d, 5.8)a
0.93 (d, 5.8)a
7.44 (d, 7.9)
7.36 (d, 7.9)
C-5,8
C-5,7
C-4′,7′
C-1′
C-1,2′,6′ 5.28 (d, 12.2)
5.33 (d, 12.2)
0.89 (d, 6.4)a
0.93 (d, 6.4)a
7.44 (d, 8.9)
7.40 (d, 8.9)
0.93 (d, 6.7)a
7.40 (d, 8.9)
7.38 (d, 8.9)
5.30 (d, 11.9)
5.35 (d, 11.9)
7.46 (d, 8.9)
7.38 (d, 8.9)
2′′,6′′
3′′,5′′
7′′
C-4′′,7′′
C-1′′
C-4′′,7′′ 7.47 (d, 8.9)
C-4′′,7′′ 7.41 (d, 7.9)
C-4′′,7′′ 7.48 (d, 8.9)
C-4′′,7′′
C-1′′
C-4,2′′,6′′
C-1′′
7.39 (d, 8.9)
C-1′′
7.36 (d, 7.9)
C-1′′
7.41 (d, 8.9)
C-4,2′′,6′′ 5.14 (d, 11.9)
C-4,2′′,6′′ 5.22 (d, 12.2)
C-4,2′′,6′′ 5.12 (d, 11.9)
C-4,2′′,6′′ 5.15 (d, 12.2)
5.22 (d, 11.9)
5.34 (d, 12.2)
5.26 (d, 11.9)
5.21 (d, 12.2)
2-O-Glc-1 5.47 (d, 7.6)
C-2
5.62 (d, 7.6)
4.16 (m)
6.05 (dd, 9.2,9.5) Cin.-9
4.48 (dd, 9.5,9.5)
3.89 (m)
C-2
5.69 (d, 7.9)
4.11 (m)
4.38 (m)
5.80 (dd, 9.5,9.8) Cin.-9
3.90 (m)
4.08 (dd, 5.2,12.2)
4.15 (dd, 2.0,12.2)
C-2
5.55 (d, 7.6)
C-2
5.56 (d, 7.6)
4.15 (d, 7.6,9.2)
5.98 (dd, 9.2,9.5) Cin.-9
4.23 (dd, 9.5,9.8)
3.89 (m)
4.71 (dd, 5.2,12.2) -OCOCH3
4.80 (dd, 1.8,12.2)
C-2
2
3
4
5
6
4.03 (dd, 7.6,8.9)
4.19 (dd, 8.9,9.2)
4.08 (dd, 8.2,9.2)
3.83 (m)
4.72 (dd, 5.5,11.9) -OCOCH3 4.38 (2H, m)
4.78 (dd, 1.8,11.9)
4.09 (dd, 7.6,7.9)
4.24 (dd, 7.6,8.8)
4.20 (dd, 7.9,8.8)
3.91 (m)
4.94 (2H, m)
Cin.-9
6-Ac
Cin.-2,6
3,5
4
1.94 (s)
-OCOCH3
2.00 (s)
7.47 (dd, 2.1,7.9) Cin.-4,7 7.56 (dd, 2.4,7.9) Cin.-4,7 7.49 (dd, 2.4,7.9) Cin.-4,7 7.50 (dd, 2.4,7.9) Cin.-4,7
7.32 (dd, 7.9,7.9) Cin.-1 7.36 (dd, 7.9,7.9) Cin.-1 7.36 (dd, 7.9,7.9) Cin.-1 7.33 (dd, 7.9,7.9) Cin.-1
7.32 (tt, 2.1,7.9) Cin.-2,6 7.36 (tt, 2.4,7.9) Cin.-2,6 7.36 (tt, 2.4,7.9) Cin.-2,6 7.33 (tt, 2.4,7.9) Cin.-2,6
-OCOCH3
7
8
7.89 (d, 15.9)
6.69 (d, 15.9)
5.69 (d, 7.6)
4.33 (m)
4.39 (m)
4.38 (m)
Cin.-9
Cin.-1,9 6.72 (d, 16.2)
C-4′
7.91 (d, 16.2)
Cin.-9
Cin.-1,9 6.70 (d, 15.9)
C-4′
7.90 (d, 15.9)
Cin.-9
Cin.-1,9 6.71 (d, 15.9)
C-4′
7.91 (d, 15.9)
Cin.-9
Cin.-1,9
C-4′
4′-O-Glc-1 5.67 (d, 7.6)
C-4′
5.64 (d, 7.9)
4.32 (dd, 7.9,7.9)
4.39 (m)
4.36 (m)
4.13 (m)
4.41 (dd, 4.8,12.2)
4.55 (dd, 2.1,12.2)
5.63 (d, 7.9)
4.32 (dd, 7.9,7.9)
4.39 (m)
4.36 (m)
4.13 (m)
5.65 (d, 7.6)
4.32 (dd, 7.6,9.1)
4.39 (m)
4.37 (m)
4.12 (m)
4.41 (dd, 4.9,11.9)
4.55 (dd, 2.1,11.9)
5.62 (d, 7.6)
4.32 (dd, 7.6,9.1)
4.39 (m)
4.37 (m)
4.12 (m)
5.70 (d, 7.6)
4.33 (dd, 7.6,8.9)
4.39 (m)
4.38 (m)
4.18 (m)
4.41 (dd, 5.0,12.1)
4.56 (dd, 2.1,12.1)
5.66 (d, 7.6)
4.33 (dd, 7.6,8.9)
4.39 (m)
4.38 (m)
4.18 (m)
2
3
4
5
6
4.32 (dd, 7.6,8.5)
4.40 (m)
4.37 (m)
4.12 (m)
4.40 (dd, 5.2,11.9)
4.55 (dd, 1.8,11.9)
4.16 (m)
4.40 (dd, 5.5,12.0)
4.56 (dd, 2.1,12.0)
5.68 (d, 7.6)
4.33 (m)
4.39 (m)
4.38 (m)
4.16 (m)
4.40 (dd, 5.5,12.0)
4.54 (dd, 2.1,12.0)
4′′-O-Glc-1 5.62 (d, 7.6)
C-4′′
C-4′′
C-4′′
C-4′′
C-4′′
2
3
4
5
6
4.32 (dd, 7.6,8.5)
4.40 (m)
4.37 (m)
4.12 (m)
4.41 (dd, 5.2,12.2)
4.53 (dd, 1.8,12.2)
4.41 (dd, 4.8,12.2)
4.55 (dd, 2.1,12.2)
4.41 (dd, 4.9,11.9)
4.53 (dd, 2.1,11.9)
4.41 (dd, 5.0,12.1)
4.54 (dd, 2.1,12.1)
a May be interchanged within the same column.
C42H58O23 of 3 was determined by HRFABMS. In the UV spectrum
of 3, absorption maxima were observed at 224 (log ꢀ 4.36) and
271 (3.28) nm. The IR spectrum of 3 showed absorption bands at
1736, 1613, 1592, 1514, and 1287 cm-1, assignable to an ester
carbonyl function and aromatic ring, and strong absorption bands
at 3432 and 1075 cm-1 suggestive of a glycoside moiety. Acid
hydrolysis of 3 with 1.0 M HCl liberated D-glucose, which was
identified by HPLC analysis using an optical rotation detector.1,4,6-8,10
The 1H (pyridine-d5, Table 3) and 13C NMR (Table 4) spectra12 of
3 showed signals assignable to two methyls [δ 0.87, 0.91 (3H each,
both d, J ) 6.1 Hz, H3-7, 8)], four methylenes [δ 1.94, 2.02 (1H
each, both m, H2-5), 3.37, 3.56 (1H each, both d, J ) 17.7 Hz,
H2-3), 5.13, 5.25 (1H each, both d, J ) 11.9 Hz, H2-7′′), 5.27,
5.33 (1H each, both d, J ) 11.9 Hz, H2-7′)], a methine [δ 2.02
(1H, m, H-6)], an acetyl group [δ 1.94 (3H, s)], and two A2B2
type aromatic spin systems [δ 7.37, 7.45 (2H each, both d, J ) 8.9
Hz, H-3′′,5′′, 2′′,6′′), 7.38, 7.43 (2H each, both d, J ) 8.9 Hz,
H-3′,5′, 2′,6′)], together with three â-glucopyranosyl signals [δ 5.47
(1H, d, J ) 7.6 Hz, H-2-O-Glc-1), 5.62 (1H, d, J ) 7.6 Hz, H-4′′-
O-Glc-1), 5.67 (1H, d, J ) 7.6 Hz, H-4′-O-Glc-1)]. In the HMBC
experiment of 3, long-range correlations were observed between
the following proton and carbon pairs: H-3 and C-1, 4, 5; H2-5
and C-1, 3, 7, 8; H-6 and C-2, 5, 7, 8; H3-7 and C-5, 8; H3-8 and
C-5, 7; H-2′,6′ and C-4′, 7′; H-3′,5′ and C-1′; H2-7′ and C-1, 2′,6′;
H-2′′,6′′ and C-4′′, 7′′; H-3′′,5′′ and C-1′′; H-7′′ and C-4, 2′′,6′′;
H-4′-O-Glc-1 and C-4′; H-2-O-Glc-1 and C-2; H-2-O-Glc-6 and
-OCOCH3; H-4′′-O-Glc-1 and C-4′′ (Table 3, Figure S1). The
enzymatic hydrolysis of 3 with â-glucosidase or tannase gave the
same product (3a), whose 1H NMR (pyridine-d5, Table 5) spectrum
showed signals assignable to a 2-isobutylmalic acid moiety {two
methyls [δ 1.02, 1.08 (3H each, both d, J ) 6.4 Hz, H3-7, 8)], two
methylenes [δ 2.07 (2H, br d, J ) 6 Hz, H2-5), 3.62, 3.69 (1H
each, both d, J ) 17.4 Hz, H2-3)], and a methine [δ 2.30 (1H, m,
H-6)]} and a â-glucopyranosyl [δ 6.01 (1H, d, J ) 7.6 Hz, H-2-
O-Glc-1)] together with an acetyl group [δ 2.00 (3H, s)]. Com-
parison of the 1H and 13C NMR spectra of 3a with those of
dactyrohin C11 (4b, vide infra) showed an acetylation shift around
the 6-position of the 2-O-glucosyl part in 3a. Acetylation of 3a
with Ac2O and pyridine gave the tetra-O-acetyl derivative (3b),
which was also obtained by the similar acetylation of 4b. The
absolute configuration at C-2 in 3 is thus R.Gymnosides IV (4), V
(5), and VI (6) were also isolated as white powders with negative
specific rotations (4, [R]25D -20.2; 5, [R]27D -11.6; 6, [R]27D -45.1
all in MeOH), respectively. In the positive- and negative-ion
FABMS of 4-6, the same quasimolecular ions were observed at
m/z 1041 [M + Na]+ and m/z 1017 [M - H]-, and the molecular
formula C49H62O23 was determined by HRFABMS measurements.
The acid hydrolysis of 4-6 liberated D-glucose.1,4,6-8,10 The proton
1
and carbon signals in the H (pyridine-d5, Table 3) and 13C NMR
(Table 4) spectra12 of 4 showed signals assignable to a 1,4-bis(p-
hydroxybenzyl)-2-isobutylmalate moiety {two methyls [δ 0.85, 0.93
(3H each, both d, J ) 6.7 Hz, H3-7, 8)], four methylenes [δ 1.87,
2.03 (1H each, both m, H2-5), 3.30, 3.46 (1H each, both d, J )
17.7 Hz, H2-3), 5.14, 5.22 (1H each, both d, J ) 11.9 Hz, H2-7′′),
5.30, 5.35 (1H each, both d, J ) 11.9 Hz, H2-7′)], a methine [δ
2.03 (1H, m, H-6)], and two A2B2 type aromatic spin systems [δ
7.38, 7.40 (2H each, both d, J ) 8.9 Hz, H-3′,5′, 2′,6′), 7.38, 7.46
(2H each, both d, J ) 8.9 Hz, H-3′′,5′′, 2′′,6′′)]} and three
â-glucopyranosyl signals [δ 5.62 (1H, d, J ) 7.6 Hz, H-2-O-Glc-
1), 5.68 (1H, d, J ) 7.6 Hz, H-4′′-O-Glc-1), 5.69 (1H, d, J ) 7.6

884 Journal of Natural Products, 2006, Vol. 69, No. 6
Morikawa et al.
Table 4. 13C NMR (125 MHz, pyridine-d5) Data of
Gymnosides III-VII (3-7)
methine [δ 2.30 (1H, m, H-6)]} and a â-glucopyranosyl [δ 5.98
(1H, d, J ) 7.6 Hz, H-2-O-Glc-1)] together with a trans-cinnamoyl
ester moiety [δ 6.73, 7.89 (1H each, both d, J ) 16.2 Hz), 7.31
(2H, dd, J ) 7.9, 7.9 Hz), 7.31 (1H, tt, J ) 2.4, 7.9 Hz), 7.48 (2H,
dd, J ) 2.4, 7.9 Hz)]. Enzymatic hydrolysis of 4 with tannase gave
trans-cinnamic acid15 and 4b. The connectivity of the trans-
cinnamoyl ester moiety in 4 was clarified by an HMBC experiment,
in which a long-range correlation was observed between H-3 of
the 2-O-glucosyl unit [δ 6.05 (1H, dd, J ) 9.2, 9.5 Hz)] and the
trans-cinnamoyl ester carbonyl carbon (δC 166.9), as shown in
Table 3 and Figure S1. Consequently, the structure of 4 was
determined as shown.
C
3
4
5
6
7
1
2
3
173.1 (s)
80.9 (s)
42.2 (t)
173.1 (s)
80.9 (s)
42.7 (t)
173.3 (s)
80.7 (s)
42.8 (t)
173.2 (s)
80.9 (s)
42.4 (t)
172.7 (s)
81.3 (s)
42.0 (t)
170.8 (s)
46.6 (t)
4
5
170.9 (s)
46.9 (t)
170.9 (s)
47.6 (t)
171.1 (s)
47.8 (t)
170.9 (s)
47.2 (t)
6
7
8
1′
2′,6′
3′,5′
4′
7′
1′′
2′′,6′′
3′′,5′′
4′′
7′′
24.1 (d)
23.9 (q)a
24.5 (q)a
129.5 (s)
130.7 (d)
117.0 (d)
158.7 (s)
67.3 (t)
129.7 (s)
130.6 (d)
117.0 (d)
158.7 (s)
66.5 (t)
24.1 (d)
24.0 (q)a
24.6 (q)a
129.5 (s)
130.7 (d)
117.0 (d)
158.7 (s)
67.4 (t)
129.7 (s)
130.6 (d)
117.0 (d)
158.7 (s)
66.6 (t)
24.0 (d)
23.9 (q)a
24.6 (q)a
129.5 (s)
130.6 (d)
117.0 (d)
158.7 (s)
67.3 (t)
129.8 (s)
130.6 (d)
117.0 (d)
158.6 (s)
66.7 (t)
24.1 (d)
23.9 (q)a
24.5 (q)a
129.5 (s)
130.7 (d)
117.0 (d)
158.7 (s)
67.3 (t)
129.7 (s)
130.6 (d)
117.0 (d)
158.7 (s)
66.5 (t)
24.1 (d)
23.9 (q)a
24.5 (q)a
129.5 (s)
130.7 (d)
117.0 (d)
158.7 (s)
67.3 (t)
129.7 (s)
130.6 (d)
117.0 (d)
158.7 (s)
66.5 (t)
99.8 (d)
73.2 (d)
79.4 (d)
69.2 (d)
74.7 (d)
63.9 (t)
20.8 (q)
170.6 (s)
135.0 (s)
128.5 (d)
129.2 (d)
130.5 (d)
144.8 (d)
119.3 (d)
166.8 (s)
102.1 (d)
74.9 (d)
78.5 (d)
71.3 (d)
78.8 (d)
62.3 (t)
1
The proton and carbon signals in the H (pyridine-d5, Table 3)
and 13C NMR (Table 4) spectra12 of 5 and 6 were very similar to
those of 4. Enzymatic hydrolysis of 5 and 6 with tannase gave 4b
together with trans-cinnamic acid. Treatment of 5 and 6 with â-
glucosidase afforded 5a and 6a, respectively. Comparison of the
1
2-O-Glc-1
2
3
4
5
6
100.0 (d)
75.4 (d)
78.4 (d)
70.9 (d)
74.9 (d)
64.3 (t)
100.0 (d)
73.5 (d)
80.0 (d)
69.0 (d)
78.0 (d)
61.9 (t)
100.1 (d)
75.8 (d)
76.0 (d)
72.5 (d)
75.9 (d)
62.0 (t)
100.1 (d)
75.5 (d)
78.4 (d)
70.9 (d)
75.0 (d)
64.4 (t)
proton signals in the H NMR spectrum (pyridine-d5, Table 5) of
5a and 6a with those of 4b showed acylation shifts around the 4-
and 6-positions of the 2-O-glucosyl part, respectively. On the basis
of this evidence and the long-range correlations in the HMBC
experiment for 5 and 6, the structures of 5 and 6 were determined
as shown.
Gymnoside VII (7), [R]19D -12.0 (in MeOH), had the molecular
formula C51H64O24 as shown by HRFABMS. Treatment of 7 with
â-glucosidase gave 7a, while treatment of 7 with tannase furnished
3a and trans-cinnamic acid. Comparison of the 1H NMR (pyridine-
d5, Table 5) spectrum of 7a with those of 3a showed an acylation
shift around the 3-position of the 2-O-glucosyl unit in 7a. The
position of the trans-cinnamoyl ester moiety of 7 was elucidated
on the basis of an HMBC experiment as shown in Table 3 and Fi-
gure S1. Consequently, the structure of 7 was constructed as shown.
6-Ac
20.7 (q)
170.8 (s)
Cin.-1
Cin.-2,6
Cin.-3,5
Cin.-4
Cin.-7
Cin.-8
Cin.-9
4′-O-Glc-1
2
3
4
5
6
135.0 (s)
128.4 (d)
129.2 (d)
130.7 (d)
144.6 (d)
119.5 (d)
166.9 (s)
102.1 (d)
74.9 (d)
78.5 (d)
71.3 (d)
78.8 (d)
62.3 (t)
134.9 (s)
128.6 (d)
129.3 (d)
130.7 (d)
145.2 (d)
118.9 (d)
166.6 (s)
102.1 (d)
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
134.8 (s)
128.6 (d)
129.3 (d)
130.7 (d)
145.0 (d)
118.8 (d)
167.0 (s)
102.1 (d)
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
102.1 (d)
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
Experimental Section
4′′-O-Glc-1 102.1 (d)
102.0 (d)
74.9 (d)
78.5 (d)
71.2 (d)
78.8 (d)
62.3 (t)
102.0 (d)
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
102.1 (d)
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
102.1 (d)
75.0 (d)
78.5 (d)
71.2 (d)
78.8 (d)
62.3 (t)
General Experimental Procedures. The following instruments were
used to obtain physical data: optical rotations, Horiba SEPA-300 digital
polarimeter (l ) 5 cm); UV spectra, Shimadzu UV-1600 spectrometer;
IR spectra, Shimadzu FTIR-8100 spectrometer; 1H NMR spectra, JEOL
JNM-LA500 (500 MHz) spectrometer; 13C NMR spectra, JEOL JNM-
LA500 (125 MHz) spectrometer with TMS as an internal standard;
FABMS and HRFABMS, JEOL JMS-SX 102A mass spectrometer;
HPLC detector, Shimadzu RID-6A refractive index and SPD-10A UV-
vis detectors. An HPLC column and YMC-Pack ODS-A (250 × 4.6
mm i.d. and 250 × 20 mm i.d.) columns were used for analytical and
preparative purposes, respectively.
2
3
4
5
6
74.9 (d)
78.4 (d)
71.2 (d)
78.8 (d)
62.3 (t)
a May be interchanged within the same column.
Hz, H-4′-O-Glc-1)], together with a trans-cinnamoyl ester moiety
[δ 6.69, 7.89 (1H each, both d, J ) 15.9 Hz), 7.32 (2H, dd, J )
7.9, 7.9 Hz), 7.32 (1H, tt, J ) 2.1, 7.9 Hz), 7.47 (2H, dd, J ) 2.1,
7.9 Hz)]. The enzymatic hydrolysis of 4 with â-glucosidase gave
4a, whose 1H (pyridine-d5, Table 5) spectrum showed signals
assignable to a 2-isobutylmalic acid moiety {two methyls [δ 1.00,
1.14 (3H each, both d, J ) 6.4 Hz, H3-7, 8)], two methylenes [δ
2.15 (2H, br d, J ) 6 Hz, H2-5), 3.63 (2H, br s, H2-3)], and a
The following experimental conditions were used for chromatogra-
phy: normal-phase silica gel column chromatography, silica gel BW-
200 (Fuji Silysia Chemical, Ltd., Aichi, Japan, 150-350 mesh);
reversed-phase silica gel column chromatography, Diaion HP-20
Table 5. 1H NMR (500 MHz, pyridine-d5) Data of 3a-7a
3a
4a
5a
δ (J Hz)
6a
7a
δ (J Hz)
H
δ (J Hz)
δ (J Hz)
δ (J Hz)
3
3.62 (d, 17.4)
3.69 (d, 17.4)
2.07 (2H, br d, 6)
2.30 (m)
3.63 (2H, br s)
3.63 (2H, br s)
3.63 (d, 17.4)
3.75 (d, 17.4)
2.10 (2H, br d, 6)
2.31 (m)
3.65 (2H, br s)
5
6
7
8
2.15 (2H, br d, 6)
2.30 (m)
2.12 (2H, br d, 6)
2.30 (m)
2.08 (2H, br s)
2.29 (m)
1.02 (d, 6.4)a
1.08 (d, 6.4)a
6.01 (d, 7.6)
1.00 (d, 6.4)a
1.14 (d, 6.4)a
5.98 (d, 7.6)
1.03 (d, 6.4)a
1.11 (d, 6.4)a
5.99 (d, 7.9)
1.02 (d, 6.4)a
1.09 (d, 6.4)a
6.04 (d, 7.9)
4.22 (m)
1.01 (d, 6.4)a
1.10 (d, 6.4)a
6.02 (d, 7.6)
2-O-Glc-1
2
3
4
5
6
4.12 (dd, 7.6,8.9)
4.23 (dd, 8.9,9.2)
4.16 (dd, 8.2,9.2)
4.02 (m)
4.76 (dd, 5.2,11.3)
4.92 (dd, 1.8,11.3)
2.00 (s)
4.26 (m)
4.23 (m)
4.25 (dd, 7.6,9.2)
6.05 (dd, 9.2,9.5)
4.26 (dd, 9.5,9.8)
4.08 (m)
4.74 (dd, 5.5,11.6)
4.92 (dd, 2.1,11.6)
2.03 (s)
6.08 (dd, 8.9,9.5)
4.53 (dd, 9.2,9.5)
3.92 (m)
4.37 (dd, 4.6,11.6)
4.50 (dd, 1.5,11.6)
4.42 (dd, 9.2,9.2)
5.87 (dd, 9.2,9.8)
4.02 (m)
4.23 (dd, 5.8,11.2)
4.27 (dd, 2.2,11.2)
4.29 (dd, 8.2,8.2)
4.22 (m)
4.09 (m)
4.90 (dd, 5.5,11.6)
5.12 (dd, 1.8,11.6)
6-Ac
Cin.-2,6
3,5
4
7
8
7.48 (dd, 2.4,7.9)
7.31 (dd, 7.9,7.9)
7.31 (tt, 2.4,7.9)
7.89 (d, 16.2)
7.49 (dd, 2.1,7.9)
7.33 (dd, 7.9,7.9)
7.33 (tt, 2.1,7.9)
7.84 (d, 16.2)
7.55 (dd, 2.4,7.9)
7.33 (dd, 7.9,7.9)
7.33 (tt, 2.4,7.9)
7.88 (d, 15.9)
7.50 (dd, 2.4,7.9)
7.32 (dd, 7.9,7.9)
7.32 (tt, 2.4,7.9)
7.91 (d, 15.9)
6.74 (d, 15.9)
6.73 (d, 16.2)
6.61 (d, 16.2)
6.77 (d, 15.9)
a May be interchanged within the same column.

Glucosyloxybenzyl 2-Isobutylmalates from Gymnadenia
Journal of Natural Products, 2006, Vol. 69, No. 6 885
Figure 2.
(Nippon Rensui Co., Tokyo, Japan), Chromatorex ODS DM1020T (Fuji
Silysia Chemical, Ltd., Aichi, Japan, 100-200 mesh); TLC, precoated
TLC plates with silica gel 60F254 (Merck, 0.25 mm) (normal-phase)
and silica gel RP-18 F254S (Merck, 0.25 mm) (reversed-phase); reversed-
phase HPTLC, precoated TLC plates with silica gel RP-18 WF254S
(Merck, 0.25 mm); detection was achieved by spraying with 1% Ce-
(SO4)2-10% aqueous H2SO4, followed by heating.
Gymnoside II (2): white powder, [R]24 -21.8 (c 0.98, MeOH);
D
UV (MeOH) λmax (log ꢀ) 223 (4.10), 257 (3.31); IR (KBr) νmax 3432,
2957, 1734, 1719, 1617, 1592, 1514, 1235, 1076 cm-1; 1H NMR data,
see Table 1; 13C NMR data, see Table 2; positive-ion FABMS m/z 481
[M + Na]+; negative-ion FABMS m/z 457 [M - H]-; HRFABMS
m/z 481.1691 (calcd for C21H30O11Na [M + Na]+, 481.1686).
Gymnoside III (3): white powder, [R]22 -47.6 (c 2.19, MeOH);
D
Plant Material. Reported before.3
UV (MeOH) λmax (log ꢀ) 224 (4.36), 271 (3.28); IR (KBr) νmax 3432,
1
2957, 1736, 1613, 1592, 1514, 1287, 1075 cm-1; H NMR data, see
Extraction and Isolation. The dried tubers of G. conopsea (12.0
kg) were powdered and extracted three times with MeOH under reflux
for 3 h. Evaporation of the solvent under reduced pressure provided a
MeOH extract (930 g, 7.8% from the dried tubers). The MeOH extract
(307 g) was subjected to Diaion HP-20 column chromatography [3.0
kg, H2O-MeOH-acetone] to give H2O-, MeOH-, and acetone-eluted
fractions (251, 51, and 5 g, respectively). Normal-phase silica gel
column chromatography [1.4 kg, n-hexane-EtOAc (5:1-1:1, v/v) to
CHCl3-MeOH-H2O (10:3:1-7:3:1, lower layer 6:4:1)-MeOH] of the
MeOH-eluted fraction (45.5 g) gave 10 fractions [1 (1.82 g), 2 (1.03
g), 3 (0.40 g), 4 (0.95 g), 5 (1.35 g), 6 (3.34 g), 7 (6.59 g), 8 (21.36 g),
9 (6.79 g), and 10 (1.87 g)]. From fractions 3-5, phenanthrenes and
stilbenes were isolated.3 Fraction 6 (3.34 g) was subjected to reversed-
phase silica gel column chromatography [100 g, MeOH-H2O (20:80-
30:70-50:50, v/v) to MeOH] to give six fractions [6-1 (920 mg), 6-2
(384 mg), 6-3 (352 mg), 6-4 (772 mg), 6-5 (280 mg), and 6-6 (632
mg)]. Fraction 6-4 (772 mg) was further purified by HPLC [YMC-
Pack ODS-A (20 × 250 mm), MeOH-H2O (40:60, v/v)] to give
gymnosides I (1, 85 mg, 0.0024%) and II (2, 33 mg, 0.0009%). Fraction
7 (6.59 g) was subjected to reversed-phase silica gel column chroma-
tography [210 g, MeOH-H2O (10:90-30:70-50:50-70:30, v/v) to
MeOH] to give seven fractions [7-1 (1070 mg), 7-2 (370 mg), 7-3 (3260
mg), 7-4 (190 mg), 7-5 (380 mg), 7-6 (840 mg), and 7-7 (480 mg)].
Fraction 7-3 (710 mg) was subjected to HPLC [MeOH-H2O (50:50,
v/v)] to give militarine (9, 557 mg, 0.072%). Fraction 7-6 (0.84 g)
was further purified by HPLC [MeOH-H2O (65:35, v/v)] to give
gymnosides IV (4, 23 mg, 0.0006%), V (5, 47 mg, 0.0014%), VI (6,
14 mg, 0.0004%), and VII (7, 89 mg, 0.0025%). Fraction 8 (21.36 g)
was subjected to reversed-phase silica gel column chromatography [630
g, MeOH-H2O (10:90-20:80-30:70-60:40-50:50-70:30, v/v) to
MeOH] to give nine fractions [8-1 (4.24 g), 8-2 (3.26 g), 8-3 (4.17 g),
8-4 (4.71 g), 8-5 (1.05 g), 8-6 (0.29 g), 8-7 (0.44 g), 8-8 (0.13 g), and
8-9 (3.07 g)]. Fraction 8-3 (0.27 g) was subjected to HPLC [MeOH-
H2O (45:55, v/v)] to give dactylorhin A (8, 229 mg, 0.11%). Fraction
8-5 (1.05 g) was subjected to HPLC [MeOH-H2O (50:50, v/v)] to
give gymnoside III (3, 595 mg, 0.017%) and 8 (78 mg, 0.0022%).
Fraction 8-8 (0.13 g) was further purified by HPLC [MeOH-H2O (65:
35, v/v)] to give 6 (62 mg, 0.0017%). Dactylorhin A (8) and militarine
(9) were identified by comparison of the physical data ([R]D, UV, IR,
1H and 13C NMR, MS] with reported values.11
Table 3; 13C NMR data, see Table 4; positive-ion FABMS m/z 953 [M
+ Na]+; negative-ion FABMS m/z 929 [M - H]-; HRFABMS m/z
953.3258 (calcd for C42H58O23Na [M + Na]+, 953.3267).
Gymnoside IV (4): white powder, [R]25 -20.2 (c 1.32, MeOH);
D
UV (MeOH) λmax (log ꢀ) 223 (4.57), 278 (4.37); IR (KBr) νmax 3432,
1
2928, 1734, 1636, 1613, 1514, 1233, 1075 cm-1; H NMR data, see
Table 3; 13C NMR data, see Table 4; positive-ion FABMS m/z 1041
[M + Na]+; negative-ion FABMS m/z 1017 [M - H]-; HRFABMS
m/z 1041.3574 (calcd for C49H62O23Na [M + Na]+, 1041.3580).
Gymnoside V (5): white powder, [R]27 -11.6 (c 1.61, MeOH);
D
UV (MeOH) λmax (log ꢀ) 223 (4.56), 278 (4.36); IR (KBr) νmax 3432,
2955, 1736, 1638, 1613, 1514, 1458, 1232, 1075 cm-1; 1H NMR data,
see Table 3; 13C NMR data, see Table 4; positive-ion FABMS m/z
1041 [M + Na]+; negative-ion FABMS m/z 1017 [M - H]-;
HRFABMS m/z 1041.3582 (calcd for C49H62O23Na [M + Na]+,
1041.3580).
Gymnoside VI (6): white powder, [R]25 -45.1 (c 2.74, MeOH);
D
UV (MeOH) λmax (log ꢀ) 223 (4.51), 278 (4.29); IR (KBr) νmax 3432,
1
2957, 1736, 1638, 1514, 1451, 1233, 1075 cm-1; H NMR data, see
Table 3; 13C NMR data, see Table 4; positive-ion FABMS m/z 1041
[M + Na]+; negative-ion FABMS m/z 1017 [M - H]-; HRFABMS
m/z 1041.3574 (calcd for C49H62O23Na [M + Na]+, 1041.3580).
Gymnoside VII (7): white powder, [R]19 -12.0 (c 0.78, MeOH);
D
UV (MeOH) λmax (log ꢀ) 223 (4.49), 278 (4.28); IR (KBr) νmax 3432,
1
2926, 1736, 1638, 1613, 1514, 1235, 1075 cm-1; H NMR data, see
Table 3; 13C NMR data, see Table 4; positive-ion FABMS m/z 1083
[M + Na]+; negative-ion FABMS m/z 1059 [M - H]-; HRFABMS
m/z 1083.3676 (calcd for C51H64O24Na [M + Na]+, 1083.3685).
Acid Hydrolysis of 1-7. A solution of 1-7 (each 1.5 mg) in 1 M
HCl (2.0 mL) was heated under reflux for 3 h. After cooling, the
reaction mixture was extracted with EtOAc. The aqueous layer was
subjected to HPLC analysis under the following conditions, respec-
tively: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250
mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation
[Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase,
CH3CN-H2O (75:25, v/v); flow rate 0.8 mL/min]. Identification of
D-glucose in the aqueous layer was carried out by comparison of its
retention time and specific rotation with those of an authentic sample,
tR: 12.3 min (D-glucose, positive specific rotation).
Gymnoside I (1): white powder, [R]24D -28.0 (c 3.52, MeOH); UV
(MeOH) λmax (log ꢀ) 223 (4.10), 256 (3.20); IR (KBr) νmax 3410, 2957,
Enzymatic Hydrolysis of 1-7 with â-Glucosidase. A solution of
1 or 2 (5.5 mg each, both 0.012 mmol) in H2O (2.0 mL) was treated
with â-glucosidase (5.0 mg, from Almond, Oriental Yeast Co., Ltd.,
Tokyo, Japan), and the solution was stirred at 37 °C for 24 h. After
EtOH was added, the solvent was removed under reduced pressure and
the residue was purified by HPLC [MeOH-1% aqueous HOAc (45:
1
1736, 1710, 1615, 1590, 1514, 1233, 1075 cm-1; H NMR data, see
Table 1; 13C NMR data, see Table 2; positive-ion FABMS m/z 481 [M
+ Na]+; negative-ion FABMS m/z 457 [M - H]-; HRFABMS m/z
481.1694 (calcd for C21H30O11Na [M + Na]+, 481.1686).

886 Journal of Natural Products, 2006, Vol. 69, No. 6
Morikawa et al.
55, v/v)] to furnish (2R)-hydroxy-2-(2-methylpropyl)butanedioic acid11
(1b, 1.8 mg, 79% from 1; 1.7 mg, 71% from 2), respectively. Through
a similar procedure, a solution of 3 (7.5 mg, 0.008 mmol), 4 (7.0 mg,
0.007 mmol), 5 (7.0 mg, 0.007 mmol), 6 (9.4 mg, 0.009 mmol), or 7
(8.8 mg, 0.008 mmol) in H2O (2.0 mL) was treated with â-glucosidase
(5.0 mg), and the solution was stirred at 37 °C for 24 h. Workup as
above gave a residue, which was purified by HPLC [MeOH-1%
aqueous HOAc (45:55, v/v)] to give 3a (1.8 mg, 57% from 3), 4a (2.6
mg, 79% from 4), 5a (2.3 mg, 70% from 5), 6a (2.7 mg, 61% from 6),
and 7a (2.4 mg, 55% from 7), respectively.
and trans-cinnamic acid (0.7 mg, 80% from 4, 0.7 mg, 70% from 5, or
1.0 mg, 72% from 6 or 1.0 mg, 73% from 7).
Acetylation of 3a and 4b. A solution of 3a (2.3 mg, 0.006 mmol)
in pyridine (1.0 mL) was treated with Ac2O (0.8 mL), and the mixture
was stirred at room temperature for 12 h. The reaction mixture was
poured into ice-water and extracted with EtOAc. The EtOAc extract
was successively washed with 5% HCl, saturated aqueous NaHCO3,
and brine, then dried over MgSO4 powder and filtered. Removal of
the solvent under reduced pressure furnished a residue, which was
purified by HPLC [MeOH-1% aqueous HOAc (60:40, v/v)] to give
3b (2.5 mg, 83%). Through a similar procedure, 3b (2.7 mg, 87%)
was also prepared from 4b (2.1 mg, 0.006 mmol).
Compound 3a: white powder, [R]15 -17.8 (c 0.11, MeOH); UV
D
(MeOH) λmax (log ꢀ) 256 (4.14); IR (KBr) νmax 3423, 1736, 1718, 1619,
1
1509, 1458, 1248, 1090 cm-1; H NMR data, see Table 5; 13C NMR
Compound 3b: white powder, [R]23 -6.7 (c 0.14, MeOH); IR
D
(125 MHz, pyridine-d5) δC 177.6 (C-1), 81.4 (C-2), 44.2 (C-3), 173.8
(C-4), 49.4 (C-5), 24.6 (C-6), 24.2 (C-7), 24.8 (C-8), 100.4 (Glc-1),
76.0 (Glc-2), 79.2 (Glc-3), 71.0 (Glc-4), 75.1 (Glc-5), 64.5 (Glc-6),
20.8 (-OCOCH3), 171.0 (-OCOCH3); positive-ion FABMS m/z 417
[M + Na]+; HRFABMS m/z 417.1367 (calcd for C16H26O11Na [M +
Na]+, 417.1373).
(KBr) νmax 2957, 1748, 1368, 1235, 1038 cm-1; H NMR (500 MHz,
1
pyridine-d5) δ 0.91, 1.00 (3H each, both d, J ) 6.4 Hz, H3-7, 8), 1.97
(2H, m, H2-5), 1.99 (1H, m, H-6), 1.91, 2.03, 2.04, 2.04 (3H each, all
s, -OCOCH3), 3.22, 3.60 (1H each, both d, J ) 17.4 Hz, H2-3), 4.23
(1H, m, H-5′), 4.93 (1H, dd, J ) 8.2, 9.2 Hz, H-2′), [4.38 (1H, dd, J
) 2.0, 12.2 Hz), 4.53 (1H, dd, J ) 5.2, 12.2 Hz), H2-6′], 5.58 (1H, dd,
J ) 9.2, 9.8 Hz, H-4′), 6.00 (1H, dd, J ) 9.8, 9.8 Hz, H-3′), 6.06 (1H,
d, J ) 7.6 Hz, H-1′); 13C NMR (125 MHz, pyridine-d5) δC 177.6 (C-
1), 80.4 (C-2), 45.4 (C-3), 173.7 (C-4), 48.6 (C-5), 24.4 (C-6), 23.7
(C-7), 24.7 (C-8), 94.4 (Glc-1), 77.8 (Glc-2), 72.6 (Glc-3), 69.3 (Glc-
4), 73.6 (Glc-5), 62.4 (Glc-6), 20.4, 20.4, 20.5, 20.5 (-OCOCH3), 170.0,
170.0, 170.4, 170.5 (-OCOCH3); positive-ion FABMS m/z 543 [M +
Na]+; HRFABMS m/z 543.1682 (calcd for C22H32O14Na [M + Na]+,
543.1690).
Compound 4a: white powder, [R]24 -22.6 (c 0.20, MeOH); UV
D
(MeOH) λmax (log ꢀ) 218 (4.35), 276 (4.28); IR (KBr) νmax 3423, 2962,
1
1719, 1636, 1509, 1456, 1076 cm-1; H NMR data, see Table 5; 13C
NMR (125 MHz, pyridine-d5) δC 177.6 (C-1), 80.7 (C-2), 44.2 (C-3),
173.8 (C-4), 48.8 (C-5), 24.6 (C-6), 24.4 (C-7), 24.8 (C-8), 100.5 (Glc-
1), 73.8 (Glc-2), 80.7 (Glc-3), 68.8 (Glc-4), 78.2 (Glc-5), 62.0 (Glc-
6), 135.1 (Cin.-1), 128.4 (Cin.-2,6), 129.2 (Cin.-3,5), 130.6 (Cin.-4),
144.5 (Cin.-7), 119.7 (Cin.-8), 167.2 (Cin.-9); positive-ion FABMS m/z
505 [M + Na]+; HRFABMS m/z 505.1692 (calcd for C23H30O11Na
[M + Na]+, 505.1686).
Compound 5a: white powder, [R]23 -22.4 (c 0.15, MeOH); UV
Acknowledgment. Part of this work was supported by the 21st COE
Program, Academic Frontier Project, and a Grant-in Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
D
(MeOH) λmax (log ꢀ) 217 (4.42), 278 (4.35); IR (KBr) νmax 3432, 2962,
1
1718, 1638, 1509, 1455, 1078 cm-1; H NMR data, see Table 5; 13C
NMR (125 MHz, pyridine-d5) δC 177.7 (C-1), 81.3 (C-2), 44.3 (C-3),
173.9 (C-4), 49.0 (C-5), 24.6 (C-6), 24.3 (C-7), 24.8 (C-8), 100.4 (Glc-
1), 76.1 (Glc-2), 76.8 (Glc-3), 72.4 (Glc-4), 76.2 (Glc-5), 62.1 (Glc-
6), 134.9 (Cin.-1), 128.6 (Cin.-2,6), 129.2 (Cin.-3,5), 130.7 (Cin.-4),
145.1 (Cin.-7), 119.0 (Cin.-8), 166.5 (Cin.-9); positive-ion FABMS m/z
505 [M + Na]+; HRFABMS m/z 505.1692 (calcd for C23H30O11Na
[M + Na]+, 505.1686).
Supporting Information Available: H-H COSY, H-H HO-
HAHA, and HMBC correlations of gemnosides I-VII (1-7) (Figure
S1). This information is available free of charge via the Internet at
http://pubs.acs.org.
Compound 6a: white powder, [R]27 -24.8 (c 0.11, MeOH); UV
D
References and Notes
(MeOH) λmax (log ꢀ) 217 (4.20), 277 (4.30); IR (KBr) νmax 3432, 2962,
1
1719, 1638, 1509, 1458, 1078 cm-1; H NMR data, see Table 5; 13C
(1) This paper is number 16 in our series Bioactive Constituents from
Chinese Natural Medicines. For paper number 15, see: Xie, H.;
Wang, T.; Matsuda, H.; Morikawa, T.; Yoshikawa, M.; Tani, T.
Chem. Pharm. Bull. 2005, 53, 1416-1422.
(2) Dictionary of Traditional Chinese Medicines; Shanghai Scientific and
Technologic Press, Ed.; Shogakkan: Tokyo, 1985; pp 1185-1186.
(3) Matsuda, H.; Morikawa, T.; Xie, H.; Yoshikawa, M. Planta Med.
2004, 70, 847-855.
(4) Matsuda, H.; Morikawa, T.; Tao, J.; Ueda, K.; Yoshikawa, M. Chem.
Pharm. Bull. 2002, 50, 208-215.
(5) Morikawa, T.; Matsuda, H.; Toguchida, I.; Ueda, K.; Yoshikawa,
M. J. Nat. Prod. 2002, 65, 1468-1474.
(6) Tao, J.; Morikawa, T.; Toguchida, I.; Ando, S.; Matsuda, H.;
Yoshikawa, M. Bioorg. Med. Chem. 2002, 10, 4005-4012.
(7) Morikawa, T.; Tao, J.; Ando, S.; Matsuda, H.; Yoshikawa, M. J.
Nat. Prod. 2003, 66, 638-645.
(8) Tao, J.; Morikawa, T.; Ando, S.; Matsuda, H.; Yoshikawa, M. Chem.
Pharm. Bull. 2003, 51, 654-662.
NMR (125 MHz, pyridine-d5) δC 177.6 (C-1), 81.4 (C-2), 44.2 (C-3),
173.9 (C-4), 49.3 (C-5), 24.6 (C-6), 24.2 (C-7), 24.8 (C-8), 100.5 (Glc-
1), 76.0 (Glc-2), 79.3 (Glc-3), 71.1 (Glc-4), 75.2 (Glc-5), 64.7 (Glc-
6), 135.0 (Cin.-1), 128.6 (Cin.-2,6), 129.2 (Cin.-3,5), 130.5 (Cin.-4),
144.8 (Cin.-7), 119.1 (Cin.-8), 167.1 (Cin.-9); positive-ion FABMS m/z
505 [M + Na]+; HRFABMS m/z 505.1682 (calcd for C23H30O11Na
[M + Na]+, 505.1686).
Compound 7a: white powder, [R]27 -21.5 (c 0.12, MeOH); UV
D
(MeOH) λmax (log ꢀ) 217 (4.21), 276 (4.31); IR (KBr) νmax 3432, 2957,
1731, 1638, 1509, 1451, 1246, 1086 cm-1; 1H NMR data, see Table 5;
13C NMR (125 MHz, pyridine-d5) δC 177.4 (C-1), 81.8 (C-2), 44.2
(C-3), 173.7 (C-4), 49.2 (C-5), 24.7 (C-6), 24.3 (C-7), 24.7 (C-8), 100.3
(Glc-1), 73.9 (Glc-2), 80.3 (Glc-3), 69.4 (Glc-4), 74.9 (Glc-5), 64.2
(Glc-6), 20.8 (-OCOCH3), 170.1 (-OCOCH3), 135.0 (Cin.-1), 128.5
(Cin.-2,6), 129.2 (Cin.-3,5), 130.5 (Cin.-4), 144.7 (Cin.-7), 119.5 (Cin.-
8), 166.8 (Cin.-9); positive-ion FABMS m/z 547 [M + Na]+; HR-
FABMS m/z 547.1796 (calcd for C25H32O12Na [M + Na]+, 547.1791).
(9) Sun, B.; Morikawa, T.; Matsuda, H.; Tewtrakul, S.; Wu, L. J.; Harima,
S.; Yoshikawa, M. J. Nat. Prod. 2004, 67, 1464-1469.
(10) Morikawa, T.; Sun, B.; Matsuda, H.; Wu, L. J.; Harima, S.;
Yoshikawa, M. Chem. Pharm. Bull. 2004, 52, 1194-1199.
(11) Kizu, H.; Kaneko, E.; Tomimori, T. Chem. Pharm. Bull. 1999, 47,
1618-1625.
Enzymatic Hydrolysis of 1-7 with Tannase. A solution of 1 (5.7
mg, 0.012 mmol) or 2 (5.1 mg, 0.011 mmol) in H2O (2.0 mL) was
treated with tannase (3.5 mg, from Aspergillus oryzae, Wako Pure
Chemical Ind., Ltd., Osaka, Japan), and the solution was stirred at 37
°C for 24 h. After EtOH was added, the solvent was removed under
reduced pressure and the residue was purified by HPLC [MeOH-1%
aqueous HOAc (45:55, v/v)] to furnish 1b11 (2.0 mg, 85% from 1, 1.6
mg, 76% from 2). Through a similar procedure, a solution of 3 (10.1
mg, 0.011 mmol), 4 (6.1 mg, 0.006 mmol), 5 (7.0 mg, 0.007 mmol),
6 (9.8 mg, 0.010 mmol), or 7 (10.0 mg, 0.009 mmol) in H2O (2.0 mL)
was treated with tannase (5.0 mg), and the solution was stirred at 37
°C for 24 h. Workup as above gave a residue, which was purified by
HPLC [MeOH-1% aqueous HOAc (45:55 or 50:50 v/v)] to furnish
3a (2.8 mg, 66% from 3 or 2.4 mg, 65% from 7), dactylirhin C (4b,
1.5 mg, 72% from 4, 1.6 mg, 66% from 5, or 2.4 mg, 71% from 6),
(12) The 1H and 13C NMR spectra of 1-7 and their derivatives were
assigned with the aid of DEPT, DQF COSY, HMQC, HMBC, and
1H-1H and 13C-1H HOHAHA experiments.
(13) Yoshida, T.; Tanaka, K.; Chen, X.-M.; Okuda, T. Chem. Pharm. Bull.
1989, 37, 920-924.
(14) Ito, H.; Kasajima, N.; Tokuda, H.; Nishino, H.; Yoshida, T. J. Nat.
Prod. 2004, 67, 411-415.
(15) trans-Cinnamic acid was identified by comparison of the physical
data (UV, IR, 1H and 13C NMR, MS) with a commercially available
sample.
NP0581115