Secoiridoid glucosides from Fraxinus americana

Article abstract of DOI:10.1016/S0031-9422(00)00319-8

Investigation of the leaves of Fraxinus americana led to the isolation of five secoiridoid glucosides, demethylligstroside, (2'' R)- and (2'' S)-2''-hydroxyoleuropeins, fraxamoside and frameroside, together with 18 known compounds. Their structures were determined on the basis of spectroscopic studies and chemical evidence. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00319-8

Phytochemistry 55 (2000) 275±284
www.elsevier.com/locate/phytochem
Secoiridoid glucosides from Fraxinus americana
Yukiko Takenaka a, Takao Tanahashi a,*, Masashi Shintaku a, Takeshi Sakai a,
Naotaka Nagakura a, Parida b
aKobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
bDepartment of Pharmacy, Xinjiang Medical College, 8 Xinyi Road, Urumqi, Xinjiang 830054, People's Republic of China
Received 8 March 2000; received in revised form 25 May 2000
Abstract
Investigation of the leaves of Fraxinus americana led to the isolation of ®ve secoiridoid glucosides, demethylligstroside, (200R)-
and (200S)-200-hydroxyoleuropeins, fraxamoside and frameroside, together with 18 known compounds. Their structures were deter-
mined on the basis of spectroscopic studies and chemical evidence. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Fraxinus americana; Oleaceae; Secoiridoid glucosides; Cyclic monoterpene
1. Introduction
alcohol, p-hydroxyphenethyl alcohol, acteoside and
campneoside I, seven secoiridoid glucosides, oleoside
11-methyl ester (6), oleoside dimethyl ester (7), oleur-
opein (8), ligstroside (9), nuezhenide and (200R)- and
(200S)-200-methoxyoleuropeins (10 and 11) and seven ¯a-
vonoid glycosides.
Our previous phytochemical work on the Fraxinus
species of the family Oleaceae led to the isolation of
various new secoiridoid glucosides esteri®ed with var-
ious phenylethanoid units (Tanahashi et al., 1992,
1993a,b, 1998). In the course of our studies on the
plants of the same genus, we examined the leaves of
Fraxinus americana L., the dried barks of which have
been used as ``White Ash'' in European folk medicine
(Hansel et al., 1994) in a similar manner of those of F.
japonica in Asia. Earlier phytochemical studies of F.
americana reported the isolation of secoiridoid gluco-
sides Gl-3, 5 and 6 from the seeds (Lalonde et al., 1976),
and of acteoside, 10-hydroxyligstroside, ligstroside and
syringin from the bark (Nishibe et al., 1997). We
describe here the isolation and structural elucidation of
®ve secoiridoid glucosides (1±5).
Compound 1, C24H30O12, was isolated as a colourless
amorphous powder, [ꢀ]D -110ꢀ (MeOH). Its UV spec-
trum revealed, besides the typical absorption at 225 nm
of an iridoidic enol ether system conjugated with a car-
bonyl group, additional absorptions at 277 and 284 nm
due to a phenolic function. It showed IR bands at 3413
(OH), 1715 and 1638 (ꢀ,ꢁ-unsaturated ester), and 1518
1
(aromatic ring) cm 1. Its H NMR spectrum (Table 1)
exhibited typical signals of an oleoside (12) unit [H-3 at
ꢂ 7.38 (s), an allylic acetal proton at ꢂ 5.86 (br s), an
anomeric proton at ꢂ 4.80 (d) and an ethylidene group at
ꢂ 6.04 (1H, qd) and ꢂ 1.66 (3H, dd)] together with an
aromatic AA0BB0 spin system at ꢂ 6.71 (2H, d,
J=8.5 Hz) and ꢂ 7.05 (2H, d, J=8.5 Hz). The 13C NMR
spectroscopic signals due to the sugar moiety of 1 coin-
cided well with those ascribable to the same part of
oleoside dimethyl ester (7), indicating the presence of a
2. Results and discussion
The n-BuOH-soluble portion of the methanolic
extract of dried leaves of F. americana was fractionated
by ODS column chromatography and then puri®ed by
preparative HPLC, to a€ord ®ve new compounds 1±5
along with four phenylethanoids, 3,4-dihydroxyphenethyl
1
ꢁ-d-glucosyl moiety. These H and 13C NMR spectral
features resembled those of ligstroside (9), except for the
lack of signals due to a carbomethoxyl group. Further-
more, 1 was treated with CH2N2±Et2O to give 9. Thus,
compound 1 was designated demethylligstroside.
Compounds 2 and 3, C25H32O14, were recognized as
isomers. Their 1H NMR (Table 1) spectral features were
* Corresponding author. Tel. Fax: +81-78-441-7546.
E-mail address: tanahash@kobepharma-u.ac.jp (T. Tanahashi).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00319-8

Table 1
1H NMR spectral data for compounds 1±5 and 17 in CD3OD
H
1
2
3
4
H
5
17
1
3
5
6
5.86
7.38
4.02
2.40
2.81
6.04
1.67
br s
s
br d (9.5)
5.95 br s
7.52 s
4.00 dd
5.95 br s
7.52 s
3.99 dd
5.92 br s
7.51 s
4.05 dd
1
3
5
6
5.92 br s
7.51
5.91 br s
s
s
7.52
(9.0, 4.5)
(14.0, 9.0) 2.50 dd
(14.0, 4.5) 2.75 dd
(9.0, 4.5)
(14.0, 9.0) 2.50 dd
(14.0, 4.5) 2.93 dd
(12.0, 4.5)
(13.5, 12.0)
(13.5, 4.5)
(7.0, 1.0)
3.88 dd
2.47 dd
2.70 dd
6.10 qd
1.74 dd
(9.0, 4.5)
(14.5, 9.0)
(14.5, 4.5)
(7.0, 1.0)
(7.0, 1.0)
3.97 dd
2.47 dd
2.69 dd
6.11 qd
1.73 dd
(9.0, 4.5)
(14.0, 9.0)
(14.0, 4.5)
(7.0, 1.0)
(7.0, 1.5)
dd
m
(13.5, 9.5) 2.50 dd
2.76 dd
8
qd
dd
(7.0, 1.5)
(7.0, 1.5)
6.10 qd
1.70 dd
(7.0, 1.0)
(7.0, 1.5)
6.09 qd
1.70 dd
(7.0, 1.0)
(7.0, 1.5)
6.15 qd
1.79 dd
8
10
10
OMe
10
20
30
40
50
60
(7.0, 1.5)
±
3.72
4.81
s
3.72
4.81
s
3.73
4.66
s
OMe 3.71
s
3.72
4.81
s
4.80
d
(8.0)
d
(7.5)
d
(7.5)
d
(7.5)
10
20
30
40
50
60
4.81
d
(8.0)
d
(7.5)
9
>
;
9
>
;
9
>
;
3.34 dd
3.37 dd
2.99 dd
3.53 td
(9.5, 7.5)
(9.5, 9.0)
(9.5, 9.0)
(9.5, 2.0)
(12.0, 9.5)
(12.0, 2.0)
(12.0, 1.5)
(12.0, 9.0)
(9.0, 1.5)
3.32±3.33
3.41
m
(9.0)
3.32±3.34
3.41
m
(9.0)
>
>
>
>
=
>
=
>
=
t
t
3.30±3.41
m
3.30±3.43
m
3.30±3.43
m
>
>
>
3.32±3.33
3.32±3.33
3.69 dd
3.89 dd
m
3.32±3.34
3.32±3.34
3.69 dd
3.90 dd
m
m
m
3.68
3.87
4.08
4.20
2.81
dd
dd
dt
dt
t
(12.0, 5.0) 3.67 dd
(12.0, 1.0) 3.89 dd
(10.5, 7.0) 3.99 dd
(10.5, 7.0) 4.13 dd
(12.0, 5.5) 3.66 dd
(12.0, 2.0) 3.89 dd
(11.5, 8.0) 4.03 dd
(11.5, 4.0) 4.10 dd
(12.0, 5.5) 3.03 dd
(12.0, 2.0) 3.88 dd
(11.0, 4.5) 3.62 dd
(11.0, 8.0) 4.52 dd
(12.0, 5.0)
(12.0, 1.5)
(12.0, 5.5)
(12.0, 2.5)
100
200
400
500
100
2.51 br sextet (7.0)
2.24
m
(7.0)
(8.5)
(8.5)
4.71 dd
(8.0, 4.0)
4.72 dd
(8.0, 4.5)
4.29 dd
200
300
400
2.60 br d
2.27 brs
1.38 tt
(7.0)
(7.0)
2.50
2.50
m
m
7.04
6.71
d
d
6.82
±
d
(2.0)
6.82
±
d
(2.0)
6.71
±
d
(2.0)
(12.0, 8.0)
(12.0, 7.0)
1.54 qd
1.87
1.24 dddd (12.0, 11.5, 8.5, 7.0)
(11.5, 7.5)
1.82 br dt
1.18 m
2.12 dtd
m
500
600
(12.0, 8.0, 1.5) 2.02 dtd
(7.0)
(12.0, 7.5, 1.5)
(7.0)
0.99
2.70
d
1.07
d
700
800
6.71
7.04
d
d
(8.5)
(8.5)
6.74
6.70 dd
d
(8.0)
(8.0, 2.0)
6.74 d
6.69 dd
(8.0)
(8.0, 2.0)
6.76
6.59 dd
d
(8.0)
(8.0, 2.0)
800
900
m
2.79 ddd
4.12 dd
4.27 dd
(11.0, 6.5, 4.0)
(11.5, 6.5)
(11.5, 4.0)
4.13 dd
4.32 dd
(11.0, 4.0)
(11.0, 8.0)
OMe
OMe
±
±
3.61
3.66
s
s

Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
277
Table 2
13C NMR spectral data for 1±5, 9 and 17 in CD3OD
C
1
2
3
4
9
C
5
17
94.9
1
94.8
95.3
95.3
100.2
155.6
109.7
32.6
95.1
1
95.1
3
157.1
n.d.a
32.6
155.2
109.4
31.9
155.3
109.4
31.8
155.1
109.4
31.8
3
155.2
109.5
31.8
41.2
173.2
124.8
130.7
13.7
168.7
52.0
100.7
74.8
78.0
71.6
78.5
62.7
36.5
57.2
43.9
30.6
33.8
22.8
180.0b
48.8
66.6
180.0b
±
155.2
109.4
31.9
4
4
5
5
6
41.4
41.1
41.2
40.4
41.3
6
41.0
7
173.6
124.1
131.5
13.7
173.1
124.9
130.5
13.6
173.1
125.0
130.5
13.6
172.7
125.5
130.5
13.9
173.2
124.9
130.5
13.5
7
172.7
124.8
130.7
13.7
8
8
9
9
10
11
OMe
10
20
30
40
500
600
100
200
300
4
10
11
OMe
10
n.d.a
168.8
52.0
168.8
52.0
168.5
51.9
168.7
51.9
168.6
51.9c
100.7
74.9
±
100.9
74.9
78.0
101.0
74.8
78.0
101.0
74.8
78.0
105.0
73.0
77.8
100.9
74.7
77.9
20
30
78.0
71.7
71.5
78.4
71.5
78.5
71.5
78.5
73.0
77.5
71.4
78.4
40
500
78.6
62.9
62.8
66.8
62.7
70.5
62.7
70.5
70.7
68.7
62.7
66.9
600
100
200
300
400
5
40.5
54.9
35.2
72.7
72.6
85.1
35.4
130.1
131.0
116.3
152.8
116.3
131.0
133.8
114.6
146.4
146.2
116.3
119.0
133.8
114.6
146.4
146.2
116.3
119.0
131.0
114.6
146.8
146.7
116.5
119.3
130.7
117.1
146.2
144.9
116.4
121.3
41.1
31.2
500
600
700
800
±
34.9
21.4
175.1d
600
700
800
900
1000
OMe
OMe
47.9
65.7
±
177.3d
51.9c
52.5c
±
±
±
a
n.d. Not detected.
Not detected directly (localized through the HMBC spectrum).
Assignments may be interchangeable.
b
c,d

278
Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
similar to those of oleuropein (8), except that the pro-
tons of the phenylethanol unit in 2 and 3 appeared as an
ABX spin system instead of an ABX2 system as in 8. In
the 13C NMR spectrum of each glucoside (Table 2), the
signal of C-200 which was observed as a methylene car-
bon at ꢂ 35.4 in 8 was replaced by an oxymethine car-
bon (2: ꢂ 72.7, 3: ꢂ 72.6). These ®ndings, coupled with
the down®eld shifts of C-100, C-300, C-400, C-600 and C-800,
when compared with the corresponding signals of 8,
suggested that 2 and 3 possessed a hydroxyl group at C-
and 11), recently isolated from Jasminum ocinale var.
grandi¯orum (Tanahashi et al., 1999). In order to estab-
lish the stereochemistry at C-200 of 2-hydroxy-1-(3,4-
dihydroxyphenyl)ethanol moieties of 2 and 3, (R)- and
(S)-2-hydroxy-1-(3,4-dimethoxyphenyl)ethanols (13 and
14) were prepared from 3,4-dimethoxystyrene (15) as
illustrated in Scheme 1. Compound 15 was oxidized
with OsO4 to a€ord (Æ)-2-hydroxy-1-(3,4-dimethox-
yphenyl)ethanol, which was esteri®ed with (R)-MTPA
acid. Partial methanolysis of the resulting MTPA esters
Scheme 1. (a) (1) OsO4/Et2O-Py, (2) NaHSO3/H2O-Py, (b) (R)-MTPA, DMAP, DCC/CH2Cl2, (c) NaOMe /MeOH.
200. Moreover, there were only di€erences in the chemi-
cal shifts of the methylene protons at C-100 (2: ꢂ 3.99,
4.13; 3: ꢂ 4.03, 4.10), ascribing the structural di€erence
between 2 and 3 to the absolute con®guration at C-200.
This assumption was further supported by comparative
studies of the NMR spectra of both compounds with
those of (200R)- and (200S)-200-methoxyoleuropeins (10
13a and 14a gave 13, 13b, and 13c, and 14, 14b, and 14c,
respectively. The absolute stereochemistry of 13 and 14
was determined by comparing the chemical shifts of
proton signals of their (R)-MTPA esters, 13b and 14b
(Ohtani et al., 1991). Finally, compounds 2 and 3 were
subjected to methylation followed by methanolysis with
the respective products identi®ed as (R)- and (S)-2-
hydroxy-1-(3,4-dimethoxyphenyl)ethanols by chiral
HPLC analysis with the authentic samples 13 and 14.
Accordingly, glucosides 2 and 3 were characterized as
(200R)-200-hydroxyoleuropein and (200S)-200-hydroxyoleu-
ropein, respectively.
Compound 4, named fraxamoside, displayed a HR-
SIMS peak at m/z 537.1612 [M-H] consistent with a
molecular formula of C25H30O13, suggesting a loss of
1
H2O by comparison to those of 2 and 3. Its H and 13C
NMR (Tables 1 and 2) spectra suggested a structural
similarity to 2 and 3, that is, the oxygenated substituent
was situated at C-200 of the 2-(3,4-dihydroxyphenyl)
3
ethanol moiety of 4. Its signi®cant HMBC J correla-
tions between the oxymethine proton at ꢂ 4.29 and the
hydroxy methylene carbon (C-60) and between H2-60
and C-200 as well as correlations between H-200 and C-800
and between H2-100 and C-7, indicated that in the struc-
ture of 4, C-200 was linked with the hydroxyl group at
C-60 to form a 14 membered ether ring. The detailed
Fig. 1. Signi®cant NOESY correlations observed for 4.

Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
279
NMR spectroscopic examinations suggested an absolute
con®guration at C-200 of 4. The coupling constants
between H-5 and H2-6, between H-50 and H2-60 and
between H2-100 and H-200, together with NOESY inter-
actions between H-1 and H-10/H-6 (ꢂ 2.50) and between
H-10 and H-50, showed that 4 adopts the conformation
as shown in Fig. 1. The R-con®guration at C-200 was
deduced from the important NOEs between H-60 (ꢂ
3.03) and H-200, H-60 (ꢂ 3.88) and H-800, H2-100 and H-400
and H-200 and H-8 as observed in the NOESY spectrum.
This was further supported by the fact that H-6 (ꢂ
3.03) and H-40 (ꢂ 2.99) resonated at anomalously high
®elds due to the aromatic system in this conformation.
Consequently, the structure of fraxamoside was for-
mulated as 4.
relative con®gurations of the cyclopentane moiety in 5
was determined by NOESY experiments. The important
NOE cross-peaks observed between H3-600 and H-200, H-
300, H-500 (ꢂ 2.12) and between H-500 (ꢂ 2.12) and H-300
demonstrated that H-200 and H-300 exist on the same ꢁ
face as the methyl group (H3-600) and therefore the sub-
stituents at C-200 and C-300 have ꢀ orientations. Further
correlations between H-900 (ꢂ 4.13) and H-300, H-400ꢁ (ꢂ
1.82), and between H-400ꢀ (ꢂ 1.38) and H-800 (Fig. 2)
suggest the relative con®guration at C-800 as shown.
For the determination of the absolute con®gurations,
the cyclic monoterpene unit (16) in 5 was chemically
correlated with geniposide (18) as follows. Geniposide
(18) followed the established route to deoxyloganin,
which was subjected to enzymatic hydrolysis to give
deoxyloganin aglycone (19) (Inouye and Nishioka,
1973; Inoue et al., 1992). Subsequent oxidation of 19
with PCC gave a lactone 20. Reduction of 20 with
NaBH4 under alkaline conditions a€orded a mixture of
two 4-epimers, 21 and 22, and then the mixture was
treated with (R)-PGME in the presence of PyBOP, HBT
and TEA to give a diastereomeric mixture of (R)-
PGME amides, 23 and 24, which were separated by
preparative HPLC (Nagai and Kusumi, 1995). (S)-
PGME amides, 25 and 26 were prepared from 20 in the
same way (Scheme 2).
Compound 5 was obtained as a colourless amorphous
powder. The HR-SIMS of 5 exhibited a strong [M-H]
at m/z 601.2113, indicating a molecular formula of
C27H38O15 for 5. The UV absorption, IR bands and
NMR spectroscopic signals showed common features to
secoiridoid glucosides with an oleoside 11-methyl ester
1
(6) unit. The H NMR spectrum, moreover, displayed
signals for a secondary methyl group, three pairs of
methylene protons and four methine protons. Its 13C
NMR spectrum showed, besides the signals corre-
sponding to 6, resonances of ten carbons including two
1
carbonyls. With the aid of H-1H COSY, HMQC and
The con®guration at C-4 of 23 and 24 was determined
1
HMBC experiments, the remaining ten carbons were
evaluated as a cyclopentanoid terpene unit with the
planar structure 16. The presence of two carboxyl
groups was further con®rmed by the fact that com-
pound 5 was methylated with CH2N2-Et2O to give its
dimethylated compound 17. The hydroxyl group at C-
900 was linked to the C-7 carboxyl group on the basis of
the HMBC correlations between H2-900 and C-7. The
by detailed H NMR spedroscopic analyses. The long-
range W-coupling (J=2.0 Hz) between H-3ꢁ (ꢂ 4.33)
and H-5 as well as NOE interactions depicted in Fig. 3,
indicated a boat-like conformation of the lactone ring in
23, where H-4 must be oriented axially from the cou-
pling constant J4,5 (6.5 Hz). Accordingly, the con®g-
uration at C-4 of 23 was determined to be S. On the
other hand, the coupling constants J3ꢁ, 4 and J4, 5 (each
1
10.5 Hz) in the H NMR spectrum of 24 showed trans
diaxial relationships between H-3ꢁ and H-4 and
between H-4 and H-5, and NOE cross-peaks (Fig. 3)
suggested that 24 also possessed a lactone ring with a
boat-like form, in which the substituent at C-4 is equa-
torial and the con®guration at C-4 is R. The absolute
con®guration of C-4 of 23 and 24 was further supported
by di€erences in the chemical shifts of the correspond-
ing proton signals between 23 and 25 and between 24
and 26, respectively (Nagai and Kusumi, 1995).
Finally, compound 5 was subjected to alkaline
hydrolysis and the resulting monoterpene was converted
1
to a (R)-PGME amide, the H NMR spectral data of
which were identical to those of 23 but not to those of
24, 25 and 26, the latter two compounds of which must
show theoretically identical NMR data of (R)-PGME
amides derived from the enantiomers of 22 and 21,
respectively. Furthermore, the (R)-PGME amide
derived from 5 was identi®ed as 23 by HPLC analysis,
indicating the absolute con®gurations in the amide
compound to be 4S, 5R, 8S and 9R. Thus, the structure
Fig. 2. Signi®cant HMBC (bold arrows) and NOESY (dotted arrows)
correlations observed for the monoterpene portion in 5.

280
Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
Scheme 2. (a) PCC/CH2Cl2. (b) (1) NaBH4/0.5N NaOH, (2) Amberlite IR-120. (c) (R)- or (S)-PGMEꢁ HCl, PyBOP, HOBT, TEA/DMF.
of the new secoiridoid glucoside was represented by 5
with the absolute stereochemistry, 100S, 200R, 300S and
800S and designated as frameroside.
performed on precoated Kieselgel 60F254 plates (Merck)
and spots were visualized under UV light.
The present work gives additional examples of oleur-
opein-type secoiridoid glucosides with an O-function at
C-200. Neither interconversion of the two glucosides 2
and 3 nor transformation of 2 and 3 to 4, 10 and 11 were
observed during the separation procedures. However,
we could not completely rule out the possibility that the
isolated compounds are arti®cially formed from a sole
genuine natural product, 200-hydroxyoleuropein, as pre-
viously proposed for related compounds (Tanahashi et
al., 1999). Oleoside-type secoiridoid glucosides esteri®ed
with a cyclopentanoid monoterpene have so far been
found only in plant species of the genus Jasminum and
their cyclopentaneunitshowedtrans relationshipbetween
H-2 and H-3 in all cases. Glucoside 5 having a cyclo-
pentanoid monoterpene unit with H-2/H-3 cis relation-
ship seems to be unique for Fraxinus americana.
3.2. Plant material
The leaves of Fraxinus americana were collected in
Bole, Xinjiang, People's Republic of China. A voucher
specimen (96001) is deposited in the laboratory of Xinjiang
Medical College, Urumqi, People's Republic of China.
3.3. Isolation of glucosides
Dried leaves of F. americana (103 g) were extracted
with hot MeOH. After concentration, the extract
(17.8 g) was suspended in H2O and ®ltered through a
Celite layer. The ®ltrate and washings were combined
and extracted successively with CHCl3 and n-BuOH.
The n-BuOH extract (4.07 g) was subjected to a Wako-
gel LP-40C18 (Wako Pure Chemical Industries Ltd,
Osaka, Japan) column chromatography. Elution with
MeOH±H2O mixtures of the indicated MeOH content
gave 5 fractions. Fraction I (0±15% MeOH eluent,
318 mg) was further puri®ed by preparative HPLC
(mBondasphere 5mC18-100A, H2O±MeOH, 17:3 or 3:1),
giving 3,4-dihydroxyphenethyl alcohol (15.1 mg), p-
hydroxyphenethyl alcohol (14.8 mg), oleoside 11-methyl
ester (6) (15.1 mg) and oleoside dimethyl ester (7)
(7.1 mg) in order of elution. The following fractions
were also puri®ed by preparative HPLC (mBondasphere
5mC18-100A, H2O±MeOH, 31:19 or 3:2 or 11:9) or
preparative TLC (CHCl3±MeOH, 7:3 or n-BuOH-
AcOH-H2O, 4:1:0.5). Fr II (20% MeOH eluent, 306 mg)
yielded quercetin 3-O-(6-O-ꢀ-l-rhamnopyranosyl-ꢁ-d-
galactopyranoside) (12.4 mg), quercetin 3-O-(6-O-ꢀ-l-
rhamnopyranosyl-ꢁ-d-glucopyranoside) (20.5 mg),
acteoside (8.7 mg), 7 (16.8 mg), demethylligstroside (1)
(4.9 mg), (200R)-200-hydroxyoleuropein (2) (3.9 mg) and
3. Experimental
3.1. General
The UV spectra were recorded on a Shimadzu UV-240
spectrophotometer and the IR spectra on a Shimadzu
FTIR-8200 spectrophotometer. The optical rotations
were measured on a Jasco DIP-370 digital polarimeter.
SIMS, EIMS, HR-SIMS and HR-EIMS were obtained
with a Hitachi M-4100 mass spectrometer. Glycerol or
3-NOBA was used as the matrix for SIMS. The NMR
experiments were performed with Varian VXR-500 and
Varian Gemini-300 spectrometers, with tetramethyl
silane as internal standard. HPLC was performed using
a Waters system (510 HPLC Pump, 486 Tunable
Absorbance Detector). Thin-layer chromatography was

Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
281
Fig. 3. Signi®cant NOESY correlations observed for 23 and 24.
KBr
max
(200S)-200-hydroxyoleuropein (3) (3.5 mg); fr III (30%
MeOH eluent, 614 mg): kaempferol 3-O-(6-O-ꢀ-l-
rhamnopyranosyl-ꢁ-d-galactopyranoside) (22.8 mg),
kaempferol 3-O-(6-O-ꢀ-l-rhamnopyranosyl-ꢁ-d-gluco-
pyranoside) (25.6 mg), quercetin 3-O-(2,6-di-O-ꢀ-l-
rhamnopyranosyl-ꢁ-d-galactopyranoside) (13.0 mg),
quercetin 3-O-(2,6-di-O-ꢀ-l-rhamnopyranosyl-ꢁ-d-glu-
copyranoside) (36.6 mg), quercetin 3-O-(6-O-ꢀ-l-rham-
(3.47); IR ꢃ
cm 1: 3391, 1733, 1717, 1628, 1541,
1076, 818; 1H and 13C NMR: see Tables 1 and 2;
Signi®cant HMBC correlations: H2-6!C-7, H2-100! C-
7, C-200, C-300, H-200!C-100, C-400, C-800, OMe!C-11;
HR-SIMS Found: 555.1733 [M H] ; C25H31O14
requires 555.1715.
3.6. (200S)-200-Hydroxyoleuropein (3)
nopyranosyl-ꢁ-d-galactopyranoside)
quercetin 3-O-(6-O-ꢀ-l-rhamnopyranosyl-ꢁ-d-gluco-
(18.9
mg),
Colourless amorphous powder, [ꢀ]2D9 140ꢀ (c 0.24,
MeOH
max
pyrano side) (63.0 mg), acteoside (66.6 mg), campneo-
side I (4.7 mg) and 1 (5.4 mg); fr IV (40% MeOH eluent,
880 mg): kaempferol 3-O-(6-O-ꢀ-l±rhamnopyranosyl-ꢁ-
d-glucopyranoside) (25.6 mg), quercetin 3-O-(6-O-ꢀ-l-
rhamnopyranosyl-ꢁ-d-glucopyranoside) (63.0 mg),
acteoside (38.9 mg), oleuropein (8) (243 mg), nuezhenide
(28.3 mg), (200R)-200-methoxyoleuropein (10) (21.2 mg)
and (200S)-200-methoxyoleuropein (11) (20.7 mg); fr V
(40% MeOH eluent, 465 mg): kaempferol 7-O-(2-O-ꢀ-l-
rhamnopyranosyl-ꢁ-d-glucopyranoside) (25.5 mg), 8
(12.5 mg), ligstroside (9) (127 mg), fraxamoside (4)
(6.2 mg) and frameroside (5) (18.1 mg).
MeOH); UV l
nm (log "): 232.5 (4.20), 281.5
(3.50); IR ꢃKmaBxr cm 1: 3369, 1734, 1717, 1636, 1522, 1076,
1
818; H and 13C NMR: see Tables 1 and 2; Signi®cant
HMBC correlations: H2-6!C-7, H2-100! C-7, C-200, C-
300, H-200!C-100, C-400, C-800, OMe!C-11; HR-SIMS
Found: 555.1718 [M H] ; C25H31O14 requires 555.1715.
3.7. Fraxamoside (4)
Colourless amorphous powder, [ꢀ]2D2 137ꢀ (c 0.12,
MeOH
max
MeOH); l
nm (log "): 233 (4.14), 282 (3.45); IR
KBr
max
1
ꢃ
cm 1: 3417, 1732, 1709, 1639, 1520, 1080, 820; H
and 13C NMR: see Tables 1 and 2; Signi®cant HMBC
correlations: H2-6!C-7, H2-100!C-7, C-200, H-200!C-100,
C-300, C-400, C-800, C-60, H2 60! C 200, OMe!C 11;
HR-SIMS Found: 537.1612 [M H] ; C25H29O13 requires
537.1607.
3.4. Demethylligstroside (1)
Colourless amorphous powder, [ꢀ]2D6 110ꢀ (c 0.32,
MeOH
max
MeOH); UV l
nm (log "): 225 (4.08), 277 (3.25),
KBr
max
284 (3.19); IR ꢃ
cm 1: 3413, 1715, 1638, 1518, 1076,
822; H and 13C NMR: see Tables 1 and 2; Signi®cant
HMBC correlations: H2-6!C-7, H2-100!C-7, C-200, C-
300, H-200!C-100, C-400, C-800, OMe! C-11; HR-SIMS
Found: 509.1684 [M H] ; C24H29O12 requires 509.1660.
3.8. Frameroside (5)
1
Colourless amorphous powder, [ꢀ]2D7 134ꢀ (c 1.09,
MeOH
max
KBr
max
1
MeOH); l
nm (log "): 236 (4.03); IR ꢃ
cm :
3414, 1733, 1707, 1634, 1078, 818; H and 13C NMR:
see Tables 1 and 2; Signi®cant HMBC correlations:
H2-6!C-7, H2-900!C-7, OMe!C-11; HR-SIMS
Found: 601.2113 [M H] ; C27H37O15 requires
601.2134.
1
3.5. (200R)-200-Hydroxyoleuropein (2)
Colourless amorphous powder, [ꢀ]2D8 152ꢀ (c 0.30,
MeOH
max
MeOH); UV l
nm (log "): 232.5 (4.16), 281.5

282
Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
3.9. Methylation of 1
3.10.2. Partial methanolysis of 13a
A solution of 13a (10.7 mg, 0.017 mmol) in 0.1 M
NaOMe (1 ml) and MeOH (1 ml) was stirred for 2 h at
room temperature. After neutralization with Amberlite
IR-120 (H+ form), the reaction mixture was con-
centrated in vacuo and the residue was subjected to
prep. TLC (CHCl3-MeOH, 49:1) to give 13 (1.3 mg,
39% yield), 13b (2.0 mg, 28% yield) and 13c (2.1 mg,
A solution of 1 (0.5 mg) in MeOH was treated with
CH2N2±Et2O to give 9 (0.5 mg). Compound 9 was
identi®ed as ligstroside (1H NMR, HPLC).
3.10. Preparation of (R)- and (S)-2-hydroxy-1-(3,4-
dimethoxyphenyl)ethanols (13 and 14)
30% yield). 13: Colourless oil, [ꢀ]2D6 40ꢀ (CHCl3); H
1
3.10.1. OsO4 oxidation of 15 followed by esteri®cation
with MTPA
NMR (CDCl3): ꢂ 3.67 (1H, dd, J=11.0, 8.0 Hz, H-1),
3.75 (1H, dd, J=11.0, 3.5 Hz, H-1), 3.88, 3.90 (each 3H,
s, 5-OMe, 6-OMe), 4.78 (1H, dd, J=8.0, 3.5 Hz, H-2),
6.85 (1H, d, J=8.0 Hz, H-7), 6.91 (1H, dd, J=8.0,
2.0 Hz, H-8), 6.93 (1H, d, J=2.0 Hz, H-4). EIMS m/z
A mixture of OsO4 (0.5 g, 1.97 mmol), pyridine
(0.7 ml) and dry Et2O (15 ml) was added dropwise to
a stirred solution of 3,4-dimethoxystyrene (15) (0.3 g,
1.83 mmol) in dry Et2O (15 ml). After stirring at room
temperature for 20 h, a mixture of NaHSO3 (0.9 g,
8.65 mmol), pyridine (13 ml) and H2O (15 ml) was
added to the reaction mixture. The whole was stirred
for a further 1 h. The reaction mixture was evaporated
in vacuo and the residue was dissolved in H2O and
extracted with CHCl3. The organic layer was con-
centrated in vacuo and the resulting residue (472 mg)
was applied to a silica gel column. Elution with
MeOH±CHCl3 mixtures of increasing MeOH content
(0±10%) gave 15 (CHCl3 eluent, 36.4 mg 12 % yield)
and 2-hydroxy-1-(3,4-dimethoxyphenyl)ethanol (10%
MeOH, 310 mg 80 % yield). EIMS m/z 198 [M]+,
167, 139, 124, 108. 2-Hydroxy-1-(3,4-dimethox-
yphenyl)ethanol (50 mg) was dissolved in CH2Cl2
(2 ml), and (R)-MTPA acid (130 mg), DMAP (80 mg)
and DCC (130 mg) were added. The whole was stirred
at room temperature for 20 h, then more (R)-MTPA
acid (30 mg) and DCC (50 mg) was added and stirred.
After 5 h, the reaction mixture was poured into dil.
HCl and extracted with CHCl3. The CHCl3 layer was
dried and concentrated in vacuo. The residue was
applied to a silica gel column with CHCl3±MeOH
(19:1) as eluent to yield a mixture of 13a and 14a
(113 mg). The mixture (39.4 mg) was puri®ed by prep.
HPLC (mBondasphere 5mC18-100A, H2O-CH3CN, 3:7)
to yield 13a (10.7 mg) and 14a (23.0 mg). 13a: 1H
NMR (CDCl3): ꢂ 3.36, 3.48 (each 3H, br s, MTPA-
OMe  2), 3.66, 3.88 (each 3H, s, 5-OMe, 6-OMe),
4.49 (1H, dd, J=12.0, 8.5 Hz, H-1), 4.68 (1H, dd,
J=12.0, 3.0 Hz, H-1), 6.13 (1H, dd, J=8.5, 3.0 Hz,
H-2), 6.60±6.80 (3H, m, H-4, 7, 8), 7.20±7.50 (10H, m,
MTPA-Ph  2).* EIMS m/z 630 [M]+, 167, 139, 124,
1
198 [M]+, 167. 13b: H NMR (CDCl3): ꢂ 3.60 (3H, d,
J=0.5 Hz, MTPA-OMe), 3.73, 3.88 (each 3H, s, 5-
OMe, 6-OMe), 3.88 (1H, dd, J=12.0, 8.0 Hz, H-1), 3.94
(1H, dd, J=12.0, 4.0 Hz, H-1), 5.99 (1H, dd, J=8.0,
4.0 Hz, H-2), 6.67±6.87 (3H, m, H-4, 7, 8), 7.30±7.50
(5H, m, MTPA-Ph). EIMS m/z 414 [M]+, 189, 167, 139,
1
105. 13c: H NMR (CDCl3): ꢂ 3.55 (3H, d, J=1.0 Hz,
MTPA-OMe), 3.86, 3.88 (each 3H, s, 5-OMe, 6-OMe),
4.45 (2H, d, J=5.8 Hz, H2-1), 4.96 (1H, t, J=5.8 Hz, H-
2), 6.85 (1H, d, J=6.6 Hz, H-7), 6.92 (1H, dd, J=6.6,
2.0 Hz, H-8), 6.92 (1H, d, J=2.0 Hz, H-4), 7.37±7.53 (5H,
m, MTPA-Ph). EIMS m/z 414 [M]+, 189, 167, 139, 105.
3.10.3. Partial methanolysis of 14a
Compound 14a (10 mg, 0.016 mmol) was worked up
in the same way as described for 13, giving 13c (2.2 mg,
70 %), 14b (0.9 mg, 14 %) and 14c (1.0 mg, 15 %). 14:
Colourless oil, [ꢀ]2D6 +36ꢀ (CHCl3); H NMR (CDCl3):
1
ꢂ 3.67 (1H, dd, J=11.0, 8.0 Hz, H-1), 3.75 (1H, dd,
J=11.0, 3.5 Hz, H-1), 3.88, 3.90 (each 3H, s, 5-OMe, 6-
OMe), 4.78 (1H, dd, J=8.0, 3.5 Hz, H-2), 6.85 (1H, d,
J=8.0 Hz, H-7), 6.91 (1H, dd, J=8.0, 2.0 Hz, H-8), 6.93
(1H, d, J=2.0 Hz, H-4). EIMS m/z 198 [M]+, 167. 14b:
1H NMR (CDCl3): ꢂ 3.49 (3H, d, J=0.5 Hz, MTPA-
OMe), 3.85, 3.89 (each 3H, s, 5-OMe, 6-OMe), 3.87
(1H, dd, J=11.0, 8.0 Hz, H-1), 3.92 (1H, dd, J=11.0,
4.0 Hz, H-1), 6.03 (1H, dd, J=8.0, 4.0 Hz, H-2), 6.82±
6.97 (3H, m, H-4, 7, 8), 7.35±7.55 (5H, m, MTPA-Ph).
1
EIMS m/z 414 [M]+, 189, 167, 139, 105. 14c: H NMR
(CDCl3): ꢂ 3.54 (3H, br s, MTPA-OMe), 3.86, 3.88
(each 3H, s, 5-OMe, 6-OMe), 4.45 (2H, br d, J=6.4 Hz,
H2-1), 4.97 (1H, br t, J=5.2 Hz, H-2), 6.85 (1H, d,
J=8.0 Hz, H-7), 6.91 (1H, dd, J=8.0, 2.0 Hz, H-8), 6.92
(1H, d, J=2.0 Hz, H-4). EIMS m/z 414 [M]+, 189, 167,
139, 105.
1
108. 14a: H NMR (CDCl3): ꢂ 3.37, 3.42 (each 3H, br
s, MTPA-OMe  2), 3.77, 3.89 (each 3H, s, 5-OMe,
6-OMe), 4.48 (1H, dd, J=12.0, 7.5 Hz, H-1), 4.69
(1H, dd, J=12.0, 4.0 Hz, H-1), 5.30 (1H, dd, J=7.5,
4.0 Hz, H-2), 6.80±7.00 (3H, m, H-4, 7, 8), 7.20±7.50
(10H, m, MTPA-Ph  2).* EIMS m/z 630 [M]+, 167,
139, 124, 108. (*In order to avoid confusion, the
numbering system of the phenylethyl moiety in oleur-
opein was also used for 13a, 14a and their derivatives.
3.10.4. HPLC analysis of 13 and 14
Standard (R)- and (S)-2-hydroxy-1-(3,4-dimethoxy
phenyl)ethanols were analyzed by chiral HPLC [col-
umn, CHIRALCEL OB-H (4.6 mm i.d. Â 250 mm,
Daicel Chemical Industries Ltd); mobile phase, n-hex-

Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
283
Table 3
1H NMR spectral data for 23±26 in CDCl3
H
3
23
24
25
26
4.33
4.39
2.94
2.89
1.66
1.96
1.26
1.99
2.04
2.49
1.20
6.54
5.52
3.73
tdd
dd
ddd
m
(12.0, 3.5, 2.0)
(12.0, 10.0)
(10.0, 6.5, 3.5)
4.26
4.40
2.38
2.81
1.23
1.94
1.15
1.87
2.33
2.46
1.20
6.73
5.55
3.74
dd
dd
td
dtd
tdd
m
(11.0, 10.5)
(11.0, 3.0)
(10.5, 3.5)
4.37
4.37
2.99
2.79
1.48
1.51
1.12
1.86
1.99
2.46
1.17
6.64
5.55
3.74
br d
br d
dt
m
(7.0)
(7.0)
4.22
4.28
2.37
2.87
1.32
2.16
1.23
1.93
2.36
2.47
1.21
6.65
5.55
3.74
dd
dd
td
(11.5, 10.0)
(11.5, 4.0)
(10.0, 4.0)
4
5
6
(7.0, 6.5)
(10.5, 9.5, 7.0)
(11.5, 9.5, 6.0)
dtd
tdd
m
(11.5, 10.0, 7.0)
(11.5, 9.5, 6.5)
tdd
m
(12.0, 9.5, 6.5)
(12.0, 6.5)
m
m
7
td
m
m
td
m
(12.0, 7.0)
m
m
dtd
m
(12.5, 5.5, 1.5)
8
m
m
m
9
10
t
(10.0)
(6.5)
(7.0)
(7.0)
dd
d
(10.5, 8.0)
(7.0)
(7.0)
t
(10.5)
(6.5)
(7.5)
(7.5)
dd
d
(10.0, 8.0)
(6.5)
(7.0)
d
d
PGME-NH
PGME-CH
PGME-OMe
PGME-Ph
br d
d
br d
d
br d
d
br d
d
(7.0)
(7.0)
s
s
s
s
7.31±7.40
m
7.33±7.40
m
7.31±7.39
m
7.32±7.40
m
ane-2-propanol (22:3); ¯ow rate, 0.6 ml/min; detection,
270 nm]. Under these conditions compound 13 was
eluted at rt 22 min and 14 at rt 26 min.
3.13. Preparation of PGME amides 23, 24, 25 and 26
3.13.1. Preparation of 20 from geniposide (18)
Deoxyloganin was prepared from 18 by a modi®ca-
tion of Inouye's methods (Inouye and Nishioka, 1973;
Inoue et al., 1992). Separation of deoxyloganin tetra-
acetate from its 8-epimer was performed by preparative
HPLC (mBondasphere 5mC18-100A, H2O±MeCN, 1:1).
Deoxyloganin was hydrolysed with ꢁ-glucosidase to
deoxyloganin aglucone (19). To a solution of 19
(218 mg, 1.0 mmol) in CH2Cl2 (3 ml) was added PCC
(420 mg). After standing for 15 h at room temperature,
the reaction mixture was applied to a silica gel column,
eluted with CHCl3. Evaporation of the eluate followed
by preparative HPLC (mBondasphere 5mC18-100A,
H2O±MeCN, 1:1) gave 20 (38.7 mg, 18% yield). 1H
NMR (CDCl3): ꢂ 1.21 (3H, d, J=6.0 Hz, H3-10), 1.26,
1.42 (each 1H, m, H2-6), 1.95, 2.30 (each 1H, m, H2-7),
2.54 (2H, m, H-8, H-9), 3.16 (1H, br q, J=8.5 Hz, H-5),
3.77 (3H, s, OMe), 7.44 (1H, s, H-3). EIMS m/z 210
[M]+.
3.11. Methylation of 2 and 3 followed by methanolysis
A solution of 2 (0.7 mg, 0.0013 mmol) in MeOH was
treated with CH2N2±Et2O under ice-cooling. The
reaction mixture was evaporated in vacuo, then the
residue was dissolved in dry MeOH (1 ml) and 0.1 M
NaOMe (1 ml) and the solution was stirred for 5 h at
room temperature. After neutralization with Amberlite
IR-120 (H+ form), the reaction mixture was con-
centrated in vacuo, and the residue was distributed
between H2O and CHCl3, with the organic layer yield-
ing a residue (0.2 mg) upon evaporation. This was
shown to be identical with 13 derived from 15 (chiral
HPLC). A solution of 3 (0.7 mg, 0.0013 mmol) was
treated as described above to give a CHCl3-soluble
product (0.2 mg), which was identi®ed as 14 by chiral
HPLC analysis.
3.13.2. Preparation of (R)-PGME amides 23 and 24
from 20
3.12. Methylation of 5
To a solution of 20 (18.3 mg, 0.087 mmol) in 0.5 M
NaOH (2 ml), was added NaBH4 (10 mg, 0.26 mmol).
After standing at room temperature for 1 h, the solution
was neutralized with Amberlite IR-120, and stirred at
room temperature for 2 h. The reaction mixture was
concentrated in vacuo to give a mixture (10.5 mg) of 21
and 22. To a solution of the mixture in DMF (0.5 ml)
A solution of 5 (4.0 mg, 0.0066 mmol) in MeOH was
treated with CH2N2 and concentrated in vacuo. The
residue was subjected to preparative HPLC (H2O±
MeOH, 2:3) to give 17 (4.1 mg, 98% yield). Colourless
amorphous powder, [ꢀ]2D3-154ꢀ (c 0.21, MeOH); 1H
and 13C NMR: see Tables 1 and 2; Signi®cant NOESY
correlations: H3-600$H-200, H-400 (ꢂ 1.54)$H-100, H-800,
H2-900, H-900 (ꢂ 4.12)$1000-OMe; HR-SIMS Found:
629.2461 [M H] ; C29H41O15 requires 629.2447.
.
were added (R)-PGME HCl (d-( )-ꢀ-phenylglycine
methyl ester hydrochloride, 20 mg), PyBOP (benzotriazol-
1-yl-oxy-tris(pyrolidino)phosphonium hexa¯uoro phos-
phate, 45 mg), HBT (1-hydroxybenzotriazole, 12 mg) and

284
Y. Takenaka et al. / Phytochemistry 55 (2000) 275±284
TEA (30 mg), and the whole was stirred at room tem-
perature for 1.5 h. The reaction mixture was poured
into dil. HCl and extracted with CHCl3. The CHCl3
layer was dried and concentrated in vacuo. The residue
was puri®ed by preparative HPLC (mBondasphere
5mC18-100A, H2O±MeCN, 1:1), giving 23 (12.2 mg,
the amide compound (1.2 mg, 37%), which was identi-
®ed with 23 (1H NMR, HPLC).
Acknowledgements
Thanks are due to Dr. M. Sugiura (Kobe Pharma-
ceutical University) for 1H and 13C NMR spectra and to
Dr. K. Saiki (Kobe Pharmaceutical University) for mass
spectral measurements.
1
41%) and 24 (8.5 mg, 28%). 23: H NMR: see Table 3.
Signi®cant NOESY correlations: H-3ꢀ (ꢂ 4.39)$H-6ꢀ(ꢂ
1
1.66), H-8, H-9 $H3-10; EIMS m/z 345 [M]+. 24: H
NMR: see Table 3. Signi®cant NOESY correlations: H-
3ꢁ (ꢂ 4.26)$H-5, H-9, H3-10$H-5, H-6ꢁ (ꢂ 1.94), H-7ꢁ
(ꢂ 1.86), H-9; EIMS m/z 345 [M]+.
References
Hansel, R., Keller, K., Rimpler, H., Schneider, G. 1994. Hagers
Handbuch der Pharmazeutischen, Praxis 5, Springer Verlag, Berlin,
p. 188.
3.13.3. Preparation of (S)-PGME amides 25 and 26
from 20
Inoue, K., Ono, M., Nakajima, H., Fujie, I., Inouye, H., Fujita, T.,
1992. Radioimmunoassay of iridoid glucosides: part 1. General
methods for preparation of the haptens and the conjugates with a
protein of this series of glucosides. Heterocycles 33, 673±695.
Inouye, H., Nishioka, T., 1973. Uber die Monoterpenglucoside und
verwandte Natursto€e. XX. Uber die Struktur des Forsythids, eines
neuen Iridoidglucosides aus Forsythia viridissima. Chemical and
Pharmaceutical Bulletin 21, 497±502.
(S)-PGME was prepared from (l)-(+)-ꢀ-phenylgly-
cine and SOCl2 in MeOH according to Nagai and
Kusumi. Compound 20 (20.8 mg, 0.099 mmol) was dis-
solved in 0.5 M NaOH (0.5 ml) following which NaBH4
(10 mg, 0.26 mmol) was added, with the reaction mix-
ture treated in the same way as described above. The
reaction residue was dissolved in DMF (0.5 ml) and
Lalonde, R.T., Wong, C., Tsai, A. I.-M., 1976. Polyglucosidic meta-
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.
(S)-PGME HCl (20 mg), PyBOP (45 mg), HBT (12 mg)
and TEA (30 mg) were added, and the whole was stirred
at room temperature for 1.5 h and worked up in the
same way as described above. The resulting residue was
subjected to prep. HPLC (mBondasphere 5mC18-100A,
H2O±CH3CN, 1:1), giving 25 (12.7 mg, 37%) and 26
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determining the absolute con®guration of carboxylic acids. Tetra-
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K., Nikaido, T., 1997. Constituents of bark of Fraxinus americana.
Natural Medicines 51, 482±485.
1
(6.9 mg, 20%). 25: H NMR: see Table 3. EIMS m/z
1
345 [M]+. 26: H NMR: see Table 3. EIMS m/z 345
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-®eld
FT NMR application of Mosher's Method. The absolute con®g-
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[M]+.
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M., Fujita, T., Chen, C.-C., 1992. A secoiridoid glucoside from
Fraxinus formosana. Phytochemistry 31, 2143±2145.
3.14. Alkaline hydrolysis of 5 followed by preparation of
the (R)-PGME amide
Tanahashi, T., Watanabe, H., Itoh, A., Nagakura, N., Inoue, K., Ono,
M., Fujita, T., Morita, M., Chen, C.-C., 1993a. Five secoiridoid glu-
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Takaishi, K., Chen, C.-C., He, Z.-D., Yang, C.-R., 1993b. Isolation
of loeayunanoside from Fraxinus insularis and revision of its struc-
ture to insularoside-6000-O-b-d-glucoside. Chemical and Pharmaceu-
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A solution of 5 (5.7 mg 0.0095 mmol) in 0.5 M NaOH
(1.5 ml) was stirred for 1 h at room temperature, and
then neutralized with Amberlite IR-120 (H+ form) and
the whole was stirred for 2 h. The reaction mixture was
extracted with CHCl3 and the CHCl3 layer was con-
centrated to give a residue (2.2 mg). To a solution of the
.
residue in DMF (0.5 ml) were added (R)-PGME HCl
Tanahashi, T., Parida, Takenaka, Y., Nagakura, N., Inoue, K.,
Kuwajima, H., Chen, C.-C., 1998. Four secoiridoid glucosides from
Fraxinus insularis. Phytochemistry 49, 1333±1337.
(3 mg), PyBOP (7 mg), HBT (2 mg) and TEA (6 mg),
and the whole was stirred at room temperature for 1.5 h
and worked up in the same way as described above. The
resulting residue was subjected to preparative HPLC
(mBondasphere 5mC18-100A, H2O±CH3CN, 1:1) to give
Tanahashi, T., Sakai, T., Takenaka, Y., Nagakura, N., Chen, C.-C.,
1999. Structure elucidation of two secoiridoid glucosides from Jas-
minum ocinale L. var. grandi¯orum (L.). Chemical and Pharma-
ceutical Bulletin 47, 1582±1586.