Synthesis of Bisdesmosidic Oleanolic Acid Saponins via a Glycosylation-Deprotection Sequence under Continuous Microfluidic/Batch Conditions

Article abstract of DOI:10.1021/acs.joc.7b00841

We report the first synthesis of a series of bisdesmosidic oleanolic acid saponins using microflow reactor Comet X-01 via a continuous flow glycosylation-batch deprotection sequence. The main results of this study can be summarized as follows: (1) The microfluidic glycosylation of oleanolic acid at C-28 was achieved in quantitative yield and was applied to the synthesis of six C-28-monoglycosidic saponins. (2) The microfluidic glycosylation of oleanolic acid at C-3 was achieved in good yield without orthoester byproduct formation and was applied to the synthesis of three bisdesmosidic saponins. (3) The continuous synthesis of saponins via a microfluidic glycosylation-batch deprotection sequence was achieved in four steps involving two purifications. Thus, the continuous microfluidic glycosylation-deprotection process is expected to be suitable for the preparation of a library of bisdesmosidic oleanolic acid saponins for in vivo pharmacological studies.

Full text of DOI:10.1021/acs.joc.7b00841

Article
Synthesis of bisdesmosidic oleanolic acid saponins via a glycosylation-
deprotection sequence under continuous microfluidic/batch conditions
Naruki Konishi, Tatsuya Shirahata, Masaki Yokoyama, Tatsuya Katsumi, Yoshikazu
Ito, Nozomu Hirata, Takashi Nishino, Kazuishi Makino, Noriko Sato, Takayuki
Nagai, Hiroaki Kiyohara, Haruki Yamada, Eisuke Kaji, and Yoshinori Kobayashi
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 31 May 2017
Downloaded from http://pubs.acs.org on June 1, 2017
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Synthesis of bisdesmosidic oleanolic acid saponins via a
glycosylation-deprotection sequence under continuous
microfluidic/batch conditions
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＊
Naruki Konishia, Tatsuya Shirahataa , Masaki Yokoyamaa, Tatsuya Katsumia, Yoshikazu
Itoa, Nozomu Hirataa, Takashi Nishinoa, Kazuishi Makinoa, Noriko Satoa, Takayuki
Nagaib, Hiroaki Kiyoharab, Haruki Yamadab, Eisuke Kajia,
Yoshinori Kobayashia
a, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
b, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane,
Minato-ku, Tokyo 108-8641, Japan.
GRAPHICAL ABSTRACT
ABSTRACT
We report the first synthesis of a series of bisdesmosidic oleanolic acid saponins using
microflow reactor Comet X-01 via a continuous flow glycosylation-batch deprotection
sequence. The main results of this study can be summarized as follows: (1) The
microfluidic glycosylation of oleanolic acid at C-28 was achieved in quantitative yield
and was applied to the synthesis of six C-28-monoglycosidic saponins. (2) The
microfluidic glycosylation of oleanolic acid at C-3 was achieved in good yield without
orthoester byproduct formation, and was applied to the synthesis of three bisdesmosidic
saponins. (3) The continuous synthesis of saponins via
a
microfluidic
glycosylation-batch deprotection sequence was achieved in four steps involving two
purifications. Thus, the continuous microfluidic glycosylation-deprotection process is
expected to be suitable for the preparation of a library of bisdesmosidic oleanolic acid
saponins for in vivo pharmacological studies.
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INTRODUCTION
Naturally occurring saponins, found in plants and echinoderms, have several biological
activities including cytotoxic, antitumor, anti-inflammatory, and interfacial properties1.
Thus, they have been widely used as detergents, emulsifiers, and medicinal drugs.
During our studies on the biological effects of saponins, several onjisaponins isolated
from the rhizomes of Polygala tenuifolia Willd were found to be active as nasal vaccine
adjuvants2. However, the development of a pure saponin-based drug has been hampered
by the low yields and high costs associated with their isolation from natural sources.
Moreover, the total synthesis of saponins3 based on the regio- and stereoselective
formation of a glycosidic linkage using a variety of sugars in the presence of many
hydroxyl groups on the aglycon and sugar moieties is a challenging task. Recently, we
synthesized a series of simplified oleanolic acid saponins glycosylated at the C-28
carboxyl, which revealed that the introduction of a 4-O-cinnamoyl glucosyl ester moiety
enhanced the vaccine adjuvant activity4; nevertheless, the adjuvant activity was much
lower than that of onjisaponins.
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In order to improve the mucosal adjuvant activity and gain a deeper understanding of
the structure-activity relationship, a library of glycoforms differing in carbohydrate
composition, linkages, and position is required. In particular, we focused on the
synthesis of the simplified saponin oleanolic acid 3,28-bisdesmoside5 because some
bisdesmosidic saponins with a complex carbohydrate structure, such as QS-216, have
shown adjuvant activity7 (Figure 1). A sequential one-pot method has been reported for
the efficient synthesis of bisdesmosidic saponins5; however, in the one-pot approach
only one saponin compound is obtained. On the other hand, in a stepwise approach,
isolation of a monosaccharide glycosyl acceptor as an intermediate after the first
glycosylation followed by glycosylation with different glycosyl donors would result in
the generation of a saponin library (Figure 2). Unfortunately, this stepwise method
disturbs a continuity of reaction sequence. Moreover, glycosylation reactions usually
require extensive protecting group manipulations, and the additional
protection/deprotection steps by conventional procedures leads to lengthy and
impractical synthetic schemes (e.g., synthesis of QS-21)6. Thus, the construction of a
saponin library is a laborious and time-consuming task.
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Figure 1. Structure of onjisaponins and simplified synthetic bisdesmosidic
oleanolic acid saponins.
work:
successive
Previous
multiple
in one-batch reaction
1999)
A)
glycosylation
(Yu,
BzAra-imidate
BzGlc-imidate AcRhaGlc-imidate
COOTr
COO
BzGlc
AcGlc
HO
BzAraO
AcRha
reaction
One-Batch
work:
and
This
under continuous
microfluidic/batch
condition
process
glycosylaion
deprotection
Micromixer
B)
Micromixer
GalO
COO
COO
COOH
Glc
AcGlc
GlcO
HO
TESO
+
+
–PG
+Sugar,
BzGlc, Gal,
AcGlc-imidate
Xyl-imidate
–TES
+AcGlc,
O
Xyl
Microfluidic/batch condition
chemical library
Saponin
Figure 2. Synthesis of bisdesmosidic oleanolic acid saponins to construct saponin
chemical library
In order to solve this problem, we turned our attention to continuous flow synthesis8
including flow microreactor synthesis9. Microfluidic synthesis has various advantages
compared to conventional batch synthesis strategies: (1) strict control of temperature,
stirring, and reaction time, (2) easy scale-up, (3) safe and reproducible operations, and
(4) possibility of reactions involving short-lived reactive intermediates10.
Microflow reactors11, (e.g., Silicon microfluidic reactor, PFA reactor12, Comet X-0113)
have been widely used in carbohydrate synthesis for glycosylation reactions (Figure
3-A). However, microfluidic glycosylations have not been extensively studied for
saponin synthesis because of solubility and reactivity limitations of medium-sized
triterpenoid acceptors.
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In this study, it was expected that microfluidic glycosylation conditions would benefit
from the advantages of microflow reactors. Thus, the glycosylation step of the synthesis
of a simplified saponin was carried out for the first time in a microflow reactor (Figure
3-B). In addition, continuous glycosylation and deprotection processes for the synthesis
of saponins, such as bisdesmosides, can be efficiently performed in microfluidic
reactors, that would allow shorter synthetic routes without the need of purification steps
and make the saponin chemical library (Figure 2-B).
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In this paper, we describe the glycosylation of oleanolic acid at C-28 and C-3 in a
microfluidic system and a continuous flow glycosylation-batch deprotection process for
the synthesis of bisdesmosidic oleanolic acid saponins with different sugar moieties.
Figure 3. Applications of microflow reactor for glycosylation.
RESULTS AND DISCUSSION
Glycosylation at C-28
We initially investigated the glycosylation of oleanolic acid at C-28 in a microfluidic
system. The Schmidt conditions14 were considered for the microfluidic glycosylation,
whereas C-28 glycosylation has been reported such as Koenigs-Knorr15 glycosylation
and a phase-transfer catalyst conditions16 which are not suitable for microfluidic
systems due to clogging of the silver salts.
Substrate solubility in the reaction medium is an important requirement for successful
microfluidic reactions; thus, a key feature of this study is the solubility of oleanolic acid
derivatives in CH2Cl2, solvent used for Schmidt glycosylation. We decided to synthesize
oleanolic acid acceptor 14 with a triethylsilyl (TES) protecting group at C-3 following
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Gin’s method, which showed improved solubility in CH2Cl2 as well as different
reactivity at C-3 and C-28 allowing for orthogonal deprotection. Accordingly, acceptor
1 was prepared from commercially available oleanolic acid via 3,28-double protection
followed by treatment with silica gel to remove the 28-O-TES group. On the other
hand, selective protection of the C-3-OH with an acetyl (Ac) group was attempted by a
stepwise protection-deprotection sequence, resulting in only 32% yield of the desired
C-3 Ac-protected acceptor16.
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Next, we attempted the microfluidic β-selective glycosylation of oleanolic acid at
C-2817, after preparation of acceptor 1 and imidate donor 2a18, and the reaction
conditions were optimized by changing the following parameters: (1) the amount of
glycosyl imidate donor 2a; (2) the amount of BF3•OEt2, used as an activator (catalytic
or stoichiometric); and (3) the flow rate in the microreactor. A solution of acceptor 1 and
donor 2a in CH2Cl2 and a solution of BF3•OEt2 in CH2Cl2 were prepared and mixed
using a Comet X-01 micromixer at -40 °C (see supporting information, SI-table 1). As
shown in Table 1, in our initial attempts, the microfluidic glycosylation was carried out
at different flow rates (0.4-0.7 mL/min) and ratios of donor 2a to BF3•OEt2. The use of
1.0 eq of donor 2a slightly promoted the glycosylation except at the flow rate of 0.6
mL/min (entries 5-12), whereas the use of 2.0 eq of 2a significantly improved the
reaction efficiency to provide glycoside 3a4 (entries 13-20). In an effort to examine the
possibility of decreasing the required amount of 2a, additional conditions were tested;
however, the glycosylation did not proceed effectively when 1.5 eq of 2a were used
(entries 21-23). Moreover, a catalytic amount (0.1 eq) of BF3•OEt2 did not promote the
reaction even at the efficient flow rate of 0.6 mL/min (entry 24). Interestingly, with 2.0
eq of 2a, a lower yield was obtained at a flow rate of 0.8 mL/min as compared with
those at 0.4-0.6 mL/min (entry 25), whereas the glycosylation at the low flow rate of 0.1
mL/min proceeded in excellent yield (entry 26). These results indicate that, in the
presence of an excess amount of 2a, a sufficient residence time was required for
complete glycosylation. In contrast to the batch reaction in the presence of MS4Å19,
under microfluidic conditions the glycosylation proceeded to completion even when the
amount of BF3•OEt2 was decreased (entry 27). These results suggest that the efficient
mixing of reactants in the microflow reactor resulted in the efficient activation of 2a by
BF3•OEt2 followed by rapid coupling with 1.
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Table 1. Synthesis of glycoside 3a under microfluidic system at various reaction
condition and saponin 4a by deprotection.
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Donor 2a
X [eq]
BF3•OEt2
Y [eq]
Flow rate
Z [mL/min]
Yieldc
[%]
no reaction
Entry
1
2
0.4
0.5
0.6
0.7
0.4
0.5
0.6
0.7
0.4
0.5
0.6
0.7
0.4
0.5
0.6
0.7
0.4
0.5
0.6
0.7
0.6
0.6
0.6
0.6
0.8
0.1
-
no reaction
no reaction
no reaction
4.9
1.0
1.0
1.0
2.0
0.1
0.5
1.0
0.5
1.0
3
4
5
6
17
7
48
8
3.6
9
2.6
10
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12
13
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15
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19
20
21 a
22 a
23 a
24 a
25 a
26 a
27 b
4.5
51
4.5
quant.
quant.
quant.
quant.
76
quant.
quant.
quant.
0.9
2.0
1.5
0.1
0.5
1.0
0.1
0.5
0.5
1.0
59
66
11
2.0
2.0
75
quant.
86
a Additional experiments were performed except enties 1-20. b The reaction was performed using
a flask apparatus. c Isolated yield.
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The scope and limitation of the microfluidic glycosylation at C-28 was examined with
various sugar moieties (Table 2). Acetylated galactosyl-, xylosyl-, mannosyl-,
rhamnosyl-, glucuronyl-, and cinnamoyl-type (2b–2g, respectively) imidate donors were
prepared according to previous reports20, and the results of their microfluidic
glycosylation reactions are summarized in Table 2. Protected glycosides 3c and 3f were
obtained in moderate yields because of the low reactivity of the corresponding donors
due to the electron-withdrawing 5’-methylene or carboxyl moiety, which decreased the
nucleophilicity of the 1’-anomeric hydroxyl group (entries 2 and 3). On the other hand,
3b, 3d, 3e, and 3g were obtained in high yields (entries 1, 4, 5 and 6). In particular, the
glycosylation of 1 (1.0 eq) with 3,4-dimethoxycinnamoyl (3,4-DMC) donor 2g (1.0 eq)
in the presence of BF3•OEt2 (10.0 eq) at a flow rate of 0.1 mL/min proceeded smoothly
to produce protected cinnamoyl glucoside 3g4 in quantitative yield (entry 6), in contrast
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to the corresponding batch reaction4 (61% yield).
With protected C-28-glycosides in hand, Ac and TES deprotection of 3a, 3b, 3c, 3d, and
3e was performed by treatment with NaH (60% dispersion in mineral oil) and MeOH at
room temperature followed by acidification to pH 4 with Dowex resin to afford
saponins 4a4, 4b16a, 4c16a, 4d, and 4e16a, respectively, in excellent yields (entries 1, 2, 3,
and 4). On the other hand, glucuronyl saponin 4f21 was obtained in low yield because of
the unhydrolyzed methyl ester at the 6’ position. Other deprotection conditions, such as
alkaline hydrolysis with KOH22 and dealkylation with dodecyl methyl sulfide23, were
resulted in the undesired simultaneous cleavage of the 6’-methyl and 28-glycosyl ester
moieties in 3f (entry 5). As for compound 3g, the deprotection was conducted according
to our previous report4 (entry 6).
The structures of the C-28-glycosidic saponins were determined by 1H NMR, 13C NMR,
1H-1H COSY, HMQC, and HMBC analyses, which confirmed the glycosidic linkage at
C-28, except for mannosyl glycoside 4d and rhamnosyl glycoside 4e24. The anomeric
configuration in 4d and 4e was determined by GATE-1 decoupling experiments25 to
give α conformation (see SI-IV-2, JC-H = 173.4 Hz for 4d, JC-H = 173.0 Hz for 4e).
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Table 2. The application of glycosylation at C-28 under microfluidic system and
synthesis of various C-28-monoglycoside saponin.
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Donor
Structure
Glycoside
Saponin
Deprotection
condition
Yielda
(%)
Yielda
Entry
1
eq.
2.5
Product
Product
(%)
AcO
AcO
OAc
O
NaH (0.1 eq.), MeOH
(0.05 M),
3b
(β only)
4b
(β only)
AcO
O
CCl3
NH
quant.
quant.
r.t., 1 h
2b
O
AcO
AcO
NaH (0.1 eq.), MeOH
(0.1 M),
AcO
3c
(β only)
4c
(β only)
O
CCl3
NH
2
3
4
5
2.0
2.0
2.0
2.0
66
93
94
74
99
r.t., 1 h
2c
AcO
AcO
AcO
OAc
O
NaH (0.1 eq.), MeOH
(0.05 M),
3d
(α only)
4d
(α only)
O
CCl3
NH
quant.
quant.
27
r.t., 40 min
2d
NH
CCl3
NaH (0.1 eq.), MeOH
(0.1 M),
O
3e
(α only)
4e
(α only)
O
AcO
AcO
OAc
2e
r.t., 30 min
MeOOC
AcO
AcO
O
NaH (0.1 eq.), MeOH
(0.05 M),
3f
(β only)
4f
(β only)
AcO
O
CCl3
NH
r.t., 3.5 h
2f
1) NaH (6.4 eq.),
MeOH (0.02 M),
CH2Cl2
(0.08
M), r.t., 1.5 h
3g
4g
(β only)
6
1.0
quant.
23
2) DDQ (2.0 eq.), (β only)
H2O (0.2 M)
CH2Cl2
(0.01
M), r.t., 17 h
a Isolated yield.
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Encouraged by the results of the oleanolic acid glycosylation at C-28 in a flow
microreactor, acceptor 5 was prepared from glycoside 3a by deprotection of the TES
ether at C-3 in order to attempt the microfluidic glycosylation at C-3 (Table 3). Acidic
(AcOH-THF-H2O 5:11:326) and mildly acidic (TBAF/THF-AcOH27) conditions gave
selectively deprotected product 5 in moderate yield along with non-selectively
deprotected 4a (entries 1 and 2). Selective removal of the TES group at C-3 was not
achieved under the above conditions because of the steric hindrance of the dimethyl
groups at C-23 and C-24. On the other hand, treatment with excess ZnBr2 in aqueous
CH2Cl228 afforded acceptor 5 in excellent yield (entry 3). Thus, we were able to achieve
the microfluidic C-28 glycosylation of oleanolic acid in quantitative yield without
byproduct formation as well as efficient conditions for the subsequent selective C-3
TES deprotection, which was applied to a continuous glycosylation-deprotection
reaction.
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Table 3. Synthesis of acceptor 5
Reaction condition
COO
3a
O
OAc
HO
AcO
OAc
OAc
5
Acceptor
Entry
Reaction condition
Yielda (%)
1
2
3
AcOH-THF-H2O (5:11:3) (0.1M), 16 h
TBAF, THF (5.0 eq.) AcOH (5.0 eq.), 24 h
ZnBr2 (5.0 eq.) H2O (5.0 eq.) CH2Cl2 (0.25 M), 16 h
65
68
97
a Isolated yield.
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Continuous reaction of microfluidic C-28 glycosylation-deprotection
The advantages of the microfluidic glycosylation reactions of oleanolic acid at C-28 are
beneficial for the development of a continuous glycosylation-deprotection process by
avoiding the purification of the protected glycoside intermediate. Thus, we attempted
the continuous C-28 glycosylation-C-3 selective TES deprotection process of oleanolic
acid (Table 4). In view of the changes in physical properties required for saponin
synthesis, the continuous reaction system was designed based on safety and solubility
criteria. In the continuous system, the microfluidic glycosylation at C-28 was performed
under the previously optimized conditions, whereas the subsequent protecting group
removal required the addition of a suitable deprotecting reagent instead of the
quenching reagent triethylamine. Using small amounts of NaH (60% dispersion in
mineral oil, 1.5 or 3.0 eq) in MeOH at room temperature, the deprotection reaction did
not proceed in the continuous system (entries 1 and 2), indicating that NaH was
quenched by BF3•OEt2. On the other hand, in the batch deprotection reaction, when
excess NaH (10.0 eq) was used, only byproduct 3’a was obtained by neutralization with
Dowex resin after methanolysis (entry 3). Acidification to pH 4 with Dowex resin after
methanolysis resulted in TES deprotection to afford saponin 4a in excellent yield over
two steps (entry 4).
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49
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52
53
54
55
56
57
58
59
60
For the synthesis of glycosyl acceptor 5 in the continuous system, regioselective TES
deprotection was investigated. Treatment with TBAF/THF-AcOH afforded 5 in only
24% yield over two steps because of the use of different solvents in the two reactions
(entry 5). When 5.0 eq of ZnBr2 and H2O were used at -40 °C to room temperature,
acceptor 5 was obtained in 55% yield over two steps (entry 6). In contrast to the batch
synthesis of acceptor 5, the deprotection conditions in the continuous system were
dependent on the concentration of acceptor 5 in CH2Cl2, solvent used for the
microfluidic C-28 glycosylation. Hence, we increased the amount of ZnBr2 and H2O
(10.0 eq), which provided acceptor 5 in 78% yield over two steps (entry 7). These
results indicate that it is important to carefully select the type and concentration of the
deprotecting reagent so that the deprotection step is not affected by the glycosylation
conditions used in the microfluidic system.
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Table 4. Continuous synthesis for preparation of saponin 4a and acceptor 5 under
microfluidic glycosylation at C-28 and batch deprotection.
–
40 ºC
rate
Flow
Temp.;
1
0.03 M)
eq,
Acceptor (1.0
=
=
mm
m
1.0
1.0
φ
l
0.6
(mL/min)
&
9
Micromixer
2a
in CH2Cl2
Donor
0.06
M)
eq,
COO
O
(2.0
Batch
deprotection
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
OH
TESO
BF3•OEt
0.03
M)
eq,
2 (1.0
in CH2Cl2
–
–
TES
Ac
group
group
4a
rate
–
Flow
0.6
3'a
HO
TES
OH
OH
group
(mL/min)
5
Acceptor
Saponin
Result
yielda
Entry
Deprotection condition with batch apparatus
Product
(2 steps, %)
1
NaH (1.5 eq.), MeOH, r.t., 1 h
NaH (3.0 eq.), MeOH, r.t., 1 h
3a
3a
11
4a
5
quant.
2
quant.
40~89
95
3
NaH (4.5~10 eq.), MeOH, r.t., 40 min, neutralized to pH 7
NaH (10 eq.), MeOH, r.t., 40 min, neutralized to pH 4
TBAF,THF (5.0 eq.) , AcOH (2.5 eq.) , r.t., 2 days
4
5
24
6
ZnBr2 (5.0 eq.) H2O (5.0 eq.) , CH2Cl2 (6 mL), r.t., 30 min
ZnBr2 (10 eq.) H2O (10 eq.), CH2Cl2 (6 mL), r.t., 30 min
5
55
7
5
78
a Isolated yield.
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Glycosylation at C-3
The glycosylation of oleanolic acid at C-3 was performed under microfluidic system
(Table 5), besides trying a batch system for comparison with the microfluidic method
(see supporting information, SI-table 2). The glycosylation using acetylated 2a did not
give the protected diglycoside 7a; instead, the acyloxonium ion intermediate derived
from the acetyl imidate donor produced orthoester 8a29, which then rearranged to
acetylated oleanolic acid 9. The structure of orthoester 8a was determined by a chemical
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1
shift on the H NMR at anomeric proton compared to β glycosidic bond. The batch
glycosylation using benzoylated glucosyl imidate donor 6a30 minimize the orthoester
formation (see supporting information in detail).
As shown in Table 5, the C-3 glycosylation in a microfluidic system using the same
amount of 6a (1.5 eq) used for the batch experiment. Glycosyl acceptor 5 and donor 6a
were dissolved in CH2Cl2 and mixed with various amounts of activator in CH2Cl2 at
various temperatures using a Comet X-01 microreactor at a 0.6 mL/min flow rate. After
flowing through the reactor tube (1.0 m, i.d.: 1.0 mm), the mixture was quenched by
addition of a triethylamine solution in CH2Cl2 in a batch apparatus.
Micro-mixing of 5, benzoyl donor 6a, and 1.0 eq of BF3•OEt2, did not afford 7a at all
(entry 1), and analytical TLC showed incomplete reaction of acceptor 5. To improve the
reaction, we tested TMSOTf as an activator. Micro-mixing of 5, 6a, and a catalytic
amount of TMSOTf at different temperatures provided diglycoside 7a in only about
20% yield (entries 2-5), and orthoester 8b was observed as a byproduct in about 60%
yield. Mild reaction conditions utilizing catalytic TMSOTf in lower temperature, caused
the preferential formation of 8b29. On the other hand, a stoichiometric amount of
TMSOTf promoted the C-3 glycosylation at room temperature, and diglycoside 7a was
obtained in 83% yield without formation of orthoester 8b (entry 7), whereas at 0 °C the
yield of 7a was decreased (entry 6). These results indicate that rapid activation by an
appropriate amount of TMSOTf at a suitable temperature was critical for C-3
glycosylation. Notably, microfluidic C-3 glycosylation required a lower amount of
donor 6a and resulted in an increased yield of 7a as compared to the batch reaction.
In order to examine the versatility of this approach, the microfluidic C-3 glycosylation
was applied to different carbohydrates. Benzoylated galactosyl- and xylosyl-type (6b
and 6c, respectively) imidate donors were prepared from the corresponding
commercially available monosaccharides by literature procedures30. Under the
optimized conditions, the microfluidic glycosylation at C-3 afforded protected
diglycosides 7b and 7c in 71% and 81% yield, respectively (entries 8 and 9), which are
about 10% higher than the yields of the corresponding batch reactions.
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Next, deprotection of all acyl groups in 7a, 7b, and 7c by treatment with NaH (60%
dispersion in mineral oil) and MeOH at room temperature afforded known
bisdesmosidic saponins 10a31, 10b16a, 32, and 10c16b in 93%, 74%, and 87% yield,
respectively (Table 6).
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On the basis of these results, we envisioned the continuous synthesis of saponins by a
consecutive glycosylation-deprotection process. Moreover, suitable conditions were
found for the glycosylation at C-3 without orthoester byproduct formation. In addition,
it should be noted that the efficiency of these transformations, with trace amounts of
unreacted substrates and byproducts, allows for the easy isolation of the desired
bisdesmosidic saponins by column chromatography.
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Table 5. The study of glycosylation at C-3 under microfluidic system toward
synthesis of diglycoside 7a-7c.a
5
OBz
O
Temp.
Acceptor
0.02 M)
rate
Flow
0.6
=
1.0
φ
mm
m
eq,
&
Donor
(1.0
(mL/min)
BzO
BzO
=
R2
l
1.0
R3
9
O
O
O O
O
Micromixer
6a-6c
R1
BzO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
38
39
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41
42
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47
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50
51
52
53
54
55
56
57
58
59
60
OBz
0.03
eq,
in CH2Cl2
(1.5
M)
7a-7c
Diglycoside
=
R
=
=
7a
7b R1
7c
R1 OBz, R2
R
CH2OBz
= =
OBz, R3 CH2OBz
H,
3
NEt3 in CH2Cl2
Activator
in CH2Cl2
=
=
H,
OBz, R2
2
rate
Flow
=
=
H
R
R
H,
8a
Orthoester
1
3
0.6
(mL/min)
Entry
Donor (1.5 eq.)
Activator (eq.)
BF3・OEt2 (1.0)
TMSOTf (0.1)
TMSOTf (0.1)
TMSOTf (0.1)
TMSOTf (0.1)
TMSOTf (1.0)
TMSOTf (1.0)
Temp (°C) 7a-7c (%)
8a (%)
0
1
2
3
4
5
6
7
r.t.
-40
-20
0
0
20b
22b
24b
27b
47c
83 c
65b
54b
66b
34b
0
OBz
O
BzO
BzO
BzO
O
CCl3
NH
6a
r.t.
0
r.t.
0
BzO
BzO
OBz
O
BzO
O
d
CCl3
NH
8
9
TMSOTf (1.0)
TMSOTf (1.0)
r.t.
r.t.
71 c
81 c
-
6b
O
BzO
BzO
BzO
O
CCl3
NH
d
-
6c
a
Results under the batch conditions; See SI-Table 2 in supporting information. b The yield was based on the
7a / 8a ratio by measurement of 1H-NMR. c Isolated yield. d Corresponding orthoester was not observed.
Table 6. Synthesis of bisdesmosidic saponin 10a, 10b, 10c.
10a
10b
10c
=
=
=
R
CH2OH
3
R1 OH, R2
H,
OH, R3 CH2OH
H,
=
=
=
R
2
=
R1
H,
=
=
H
R1 OH, R2
R
3
NaH
COO
O
7a, 7b, 7c
Diglycoside
CH2Cl2
-MeOH
OH
R2
R3
r.t.
O
O
R1
HO
OH
OH
HO
OH
Bisdesmosidic
10a, 10b, 10c
saponin
Entry Diglycoside Bisdesmosidic saponin Yielda
1
2
3
7a
7b
7c
10a
10b
10c
93
74
87
a Isolated yield.
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Continuous reaction of microfluidic C-3 glycosylation-deprotection
We attempted the continuous C-3 glycosylation-acyl deprotection sequence (Table 7).
Gratifyingly, bisdesmosidic saponins 10a, 10b, and 10c were obtained in about 40%
yield over two steps by treatment with 4.5 eq of NaH (60% dispersion in mineral oil) in
MeOH at room temperature. Thus, bisdesmosidic saponins could be readily prepared
from acceptor 1 in about 30% total yield over four steps involving two purification
operations.
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23
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25
26
27
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29
30
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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49
50
51
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53
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57
58
59
60
Table 7. Continuous synthesis of bisdesmosidic saponins under microfluidic
glycosylation at C-3 and batch deprotection of acyl groups.
r.t.
mm
m
Flow rate 0.6
(mL/min)
Temp.;
5
0.0212 M)
eq,
Acceptor (1.0
=
=
1.0
1.0
φ
l
&
Micromixer
6a, 6b, 6c
Donor
0.0318
M)
eq,
(1.5
in CH2Cl2
NaH in oil
MeOH
r.t., 30 min
eq.)
(4.5
TMSOTf
0.0212
in CH Cl
M)
2 2
eq,
(1.0
Flow rate 0.6
(mL/min)
10a, 10b, 10c
Saponin
Yielda
Entry
Donor (1.5 eq.)
Product
(2 steps, %)
1
6a
6b
6c
10a
10b
10c
47
43
47
2
3
a Isolated yield.
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The assignments of bisdesmosidic acid saponins
In this work, we determined for the first time the detailed structure of synthetic
bisdesmosidic saponins 10a, 10b, and 10c, and the spectral data of their sugar moieties
are shown33 in Table 8. Because of overlapping of the sugar proton signals, the
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1
chemical structures of the synthesized saponins could not be identified by H-NMR
spectroscopy even by comparison with previous reports16a, and it was therefore
necessary to resort to TOCSY experiments34. The 1H-NMR chemical shifts,
multiplicities, and coupling constants of the sugar signals were assigned by H-1H
1
COSY and 1D TOCSY spectra; HSQC and HSQC-TOCSY experiments were used to
assign the chemical shifts and multiplicities in the 13C-NMR spectra and the
corresponding sugar proton signals (see SI-IV-3). The β-anomeric configuration of the
28-O-sugar and 3-O-sugar was demonstrated by the large J values (7.5-8.5 Hz). The
HMBC correlations between the anomeric proton (1’-H or 1’’-H) and C-28 or C-3
1
confirmed the presence of glycosidic linkages. Interestingly, the H-NMR spectra of
diglycosidic saponins showed differences in the multiplicities of the 2’-H and 3’-H
signals of the 28-O-sugar moiety, suggesting that the nature of the 3-O-sugar moiety
affected the conformation of the saponin.
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Table 8. 1H- and 13C-NMR spectral assignments for sugar moieties of bisdesmosidic
saponin 10a, 10b, 10c.
8
9
28-O- β-Glc
δH
δC
3-O- β-Glc
δH
δC
107.0
75.9
78.8
71.9
78.4
1'
2'
3'
4'
5'
6.30 (d, 8.0)
95.9
74.2
78.9
71.2
1''
2''
3''
4''
5''
4.91 (d, 8.0)
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45
46
47
48
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50
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59
60
4.18 (t, 8.5)
4.01 (t, 8.0)
4.26 (dd, 9.0, 8.5)
4.34 (dd, 9.5, 9.0)
4.22 (dd, 8.5, 8.0)
4.20 (dd, 9.0, 8.5)
3.98 (ddd, 9.0, 5.0, 2.0)
4.56 (dd, 12.0, 2.0)
4.37 (dd, 12.0, 5.0)
10a
4.00 (ddd, 9.5, 4.0, 2.0) 79.5
4.43 (dd, 12.0, 2.0)
62.2
4.38 (dd, 12.0, 5.0)
6'
6''
63.1
28-O- β-Glc δH
δC
3-O- β-Gal δH
δC
107.0
75.9
73.2
71.9
70.3
1'
2'
3'
4'
5'
6.33 (d, 8.0)
95.9
74.2
78.9
71.1
1''
2''
3''
4''
5''
4.87 (d, 8.0)
4.22 (dd, 8.5, 8.0)
4.30 (dd, 9.0, 8.5)
4.37 (dd, 9.5, 9.0)
4.46 (dd, 9.0, 8.0)
4.19 (dd, 9.0, 3.0)
4.60 (dd, 3.0, 1.0)
4.13 (td, 6.0, 1.0)
4.50 (dd, 12.0, 6.0)
4.45 (dd, 12.0, 6.0)
10b
4.04 (ddd, 9.5, 4.0, 3.0) 79.5
4.48 (dd, 12.0, 3.0)
62.2
4.41 (dd, 12.0, 4.0)
6'
6''
62.5
28-O-β-Glc δH
δC
3-O- β-Xyl δH
δC
107.8
75.6
78.7
1'
2'
3'
6.33 (d, 8.5)
95.9
74.2
78.9
1''
2''
3''
4.84 (d, 7.5)
4.21 (dd, 8.5, 8.0)
4.30 (t, 9.0)
4.03 (dd, 8.5, 7.5)
4.18 (t, 8.5)
10c
4.24 (ddd, 10.0, 8.5,
5.0)
4'
5'
6'
4.38 (dd, 9.5, 9.0)
71.1
4''
5''
71.2
67.3
4.04 (ddd, 9.5, 4.0, 2.0) 79.5
4.39 (dd, 11.0, 5.0)
3.79 (dd, 11.0, 5.0)
4.47 (dd, 12.0, 2.0)
62.2
4.41 (dd, 12.0, 4.0)
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CONCLUSION
In conclusion, we achieved the first C-28 and C-3 glycosylation of oleanolic acid in a
microfluidic system in quantitative and good yields, respectively. Six
C-28-monoglycosidic and three bisdesmosidic saponins were synthesized, and their
detailed structures were determined. The advantages of the microfluidic glycosylation
were exploited for the development of a continuous glycosylation-deprotection
sequence for saponin synthesis. This simple continuous method is based on the addition
of a deprotecting reagent, instead of a quenching reagent, after the glycosylation step.
Three bisdesmosidic saponins could be easily prepared from oleanolic acid via
continuous microfluidic glycosylation-batch deprotection in 28-31% total yield. By
reducing the number of purification steps, this synthetic approach using a microflow
reactor could readily provide a wide variety of bisdesmosidic saponins and find
application in the development of a saponin library for biological assays.
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EXPERIMENTAL SECTION
9
General Information.
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All reactions were carried out under argon atmosphere in dried glassware, unless
otherwise noted. All reagents were purchased from Tokyo Kasei Kogyo, Kanto
Chemical, Wako Pure Chemical Industries, Fluka or Aldrich companies and used
without further purification, unless otherwise noted. Dry DMF, THF, and CH2Cl2 were
purchased from Kanto Chemical Co. Pre-coated silica gel plates with a fluorescent
indicator (Merck 60 F254) were used for analytical and preparative thin layer
chromatography. Flash column chromatography was carried out with Kanto Chemical
silica gel (Kanto Chemical, silica gel 60N, spherical neutral, 0.040-0.050 mm, Cat. No.
37 563-84). Powdered and pre-dried molecular sieves 4Å were used in glycosylation.
Detection was carried out by staining with phosphomolybdic acid resulted in blue spots
1
and application of UV (254 nm). H NMR spectra were recorded on Agilent
Technologies 400-MR (400 MHz), 400-MR DD2 (400 Hz), NMR DD2 400NB (400
MHz) spectrometers. 13C NMR spectra were recorded on Agilent Technologies 400-MR
(400 MHz), 400-MR DD2 (400 Hz), NMR DD2 400NB (400 MHz) spectrometers. The
chemical shifts are expressed in ppm downfield from the internal solvent peaks for
CDCl3 (7.26 ppm, 1H-NMR), CD3OD (3.31 ppm, 1H-NMR), pyridine-d5 (8.73 ppm, 1H
NMR), CDCl3 (77.0 ppm, 13C NMR), CD3OD (49.0 ppm, 13C-NMR) or pyridine-d5
(150.0 ppm, 13C-NMR) and J values are given in Hertz. The coupling patterns are
denoted s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t
(triplet), dt (double triplet), q (quartet), m (multiplet), or br (broad). All infrared spectra
were measured on a JASCO FT/IR-460 spectrometer and absorbance bands are reported
in wavenumber (cm-1). High- and low-resolution mass spectra were measured on a
JEOL JMS-T100 LP, JMS-700 MStation and JEOL JMS-AX505 HA spectrometer.
Optical rotations were measured by using JASCO DIP-370 and P-2200 polarimeter.
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8
Olean-12-en-28-oic acid, 3-O-triethylsilyl-, 2, 3, 4, 6-Tetra-O-acetyl-β-D-
glucopyranosyl ester (3a)
9
MicroFlow. A solution of BF3•OEt2 (6.0 µL, 0.0478 mmol, 0.006 M) dissolved in
CH2Cl2 (7.5 mL) was injected to the micromixer by using the
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syringe pump at the flow rate of 0.6 mL/min. Also, a solution
COO
O
OAc
of donor 2a (94.2 mg, 0.1912 mmol, 0.025 M) and acceptor 1
(54.6 mg, 0.0956 mmol, 0.013 M) dissolved in CH2Cl2 (7.5
mL) was injected to the micromixer by the same syringe pump
TESO
AcO
OAc
OAc
3a
at the flow rate of 0.6 mL/min and mixed at –40 ˚C. After the reaction mixture was
allowed to flow at –40 ˚C for an additional 100 sec through a Teflon reactor tube (φ =
1.0 mm, l = 1.0 m), the mixture was quenched by addition of triethylamine (3.0 µL)
diluted in CH2Cl2. The mixture was concentrated under reduced pressure to give the
crude product. Purification by flash column chromatography (silica gel 20 g, hexane :
AcOEt = 6 : 1) afforded 3a (90.5 mg, 0.100 mmol, quant.) as a white foamy solid. Rf =
26
1
0.43 (hexane : AcOEt = 2 : 1); [α]D +25.1 (c 1.00, CHCl3); H-NMR (400 MHz,
CDCl3) δ : 5.58 (d, J = 8.0 Hz, 1H, 1’-H), 5.31 (dd, J = 4.8 Hz, 3.2 Hz, 1H, 12-H), 5.25
(dd, J = 10.0 Hz, 9.1Hz, 1H, 4’-H), 5.18 (dd, J = 9.1 Hz, 8.0 Hz, 1H, 2’-H), 5.13 (t, J =
9.1 Hz, 1H, 3’-H), 4.27 (dd, J = 12.5 Hz, 4.3 Hz, 1H, 6’-H), 4.04 (dd, J = 12.5 Hz, 2.2
Hz, 1H, 6’-H), 3.79 (ddd, J = 10.0 Hz, 4.3 Hz, 2.2 Hz, 1H, 5’-H), 3.19 (m, 1H, 3-H),
2.78 (m, 1H, 18-H), 2.06 (s, 3H, -OCOCH3), 2.02 (s, 3H, -OCOCH3), 2.01 (s, 6H,
-OCOCH3 x2), 1.95 (m, 1H, 11-H), 1.84 (m, 2H, 16-H), 1.60 (m, 1H, 19-H), 1.58 (m,
2H, 22-H), 1.56 (m, 1H, 15-H), 1.54 (m, 1H, 1-H), 1.53 (m, 1H, 11-H), 1.48 (m, 2H,
6-H, 9-H), 1.44 (m, 2H, 2-H), 1.40 (m, 1H, 7-H), 1.31 (m, 1H, 6-H), 1.29 (m, 1H, 21-H),
1.18 (m, 1H, 21-H), 1.15 (m, 1H, 7-H), 1.14 (m, 1H, 19-H), 1.10 (s, 3H, 27-H), 1.01 (m,
1H, 15-H), 0.93 (m, 9H, -Si(CH2CH3)3), 0.89 (m, 1H, 1-H), 0.88 (s, 6H, 23-H, 30-H),
0.87 (s, 6H, 25-H, 29-H), 0.72 (s, 3H, 24-H), 0.69 (s, 3H, 26-H), 0.65 (m, 1H, 5-H),
0.56 (m, 6H, -Si(CH2CH3)3); 13C-NMR (100 MHz, CDCl3) δ : 175.6 (C-28), 170.6
(-OCOCH3), 170.2 (-OCOCH3), 169.4 (-OCOCH3), 169.0 (-OCOCH3), 142.8 (C-13),
122.9 (C-12), 91.5 (C-1’), 79.4 (C-3), 72.8 (C-4’), 72.3 (C-5’), 69.8 (C-2’), 68.0 (C-3’),
61.5 (C-6’), 47.5 (C-5), 46.7 (C-9), 45.7 (C-17), 41.6 (C-19), 41.0 (C-14), 39.2 (C-18),
38.4 (C-8), 38.4 (C-4), 36.8 (C-1), 33.7 (C-10), 32.9 (C-21), 32.8 (C-29), 32.8 (C-7),
31.7 (C-22), 30.6 (C-20), 28.4 (C-23), 27.6 (C-15), 27.6 (C-2), 25.6 (C-27), 23.4 (C-30),
23.4 (C-16), 22.8 (C-11), 20.7 (-OCOCH3), 20.7 (-OCOCH3), 20.6 (-OCOCH3), 20.6
(-OCOCH3), 18.5 (C-6), 16.9 (C-26), 16.0 (C-24), 15.3 (C-25), 7.0 (-Si(CH2CH3)3), 5.2
(-Si(CH2CH3)3); IR (NaCl) cm-1 ν : 2950 (=C-H), 1692 (-C=O), 1075 (-C-O-); HRMS
(ESI-TOF) m/z: [M+Na]+ Calcd for C50H80O12SiNa 923.5317; Found 923.5326.
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Olean-12-en-28-oic acid, 3-O-triethylsilyl-, 2, 3, 4, 6-tetra-O-acetyl-β-D-
galactopyranosyl ester (3b) MicroFlow. A solution of BF3•OEt2 (55 µL, 0.4379 mmol,
0.03 M) dissolved in CH2Cl2 (14.6 mL) was injected to the
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micromixer by using the syringe pump at the flow rate of 0.6
COO
O
OAc
mL/min. Also, a solution of donor 2b (539 mg, 1.095 mmol,
AcO
TESO
AcO
OAc
0.075 M) and acceptor 1 (250 mg, 0.4379 mmol, 0.03 M)
3b
dissolved in CH2Cl2 (16.4 mL) was injected to the micromixer
by the same syringe pump at the flow rate of 0.6 mL/min and mixed at –40 ˚C. After the
reaction mixture was allowed to flow at –40 ˚C for an additional 100 sec through a
Teflon reactor tube (φ = 1.0 mm, l = 1.0 m), the mixture was quenched by addition of
triethylamine (0.24 mL) diluted in CH2Cl2. The mixture was concentrated under reduced
pressure to give the crude product. Purification by flash column chromatography (silica
gel 80 g, hexane : AcOEt = 5 : 1) afforded 3b (413 mg, 0.458 mmol, quant.) as a white
solid. Rf = 0.40 (hexane : AcOEt = 2 : 1); [α]D22 +44.1 (c 1.00, CHCl3); 1H-NMR (400
MHz, CDCl3) δ : 5.55 (d, J = 8.5 Hz, 1H, 1’-H), 5.40 (dd, J = 3.5 Hz, 1.0 Hz, 1H, 4’-H),
5.35 (dd, J = 10.4 Hz, 8.4 Hz, 1H, 2’-H), 5.32 (dd, J = 3.3 Hz, 3.3 Hz, 1H, 12-H), 5.07
(dd, J = 10.4 Hz, 3.5 Hz, 1H, 3’-H), 4.10 (m, 2H, 6’-H), 4.00 (ddd, J = 7.2 Hz, 5.9 Hz,
1.2 Hz, 1H, 5’-H), 3.20 (dd, J = 11.1 Hz, 4.3 Hz, 1H, 3-H), 2.80 (dd, J = 13.9 Hz, 4.0
Hz, 1H, 18-H), 2.16 (s, 3H, -OCOCH3), 2.03 (s, 3H, -OCOCH3), 2.02 (s, 3H,
-OCOCH3), 1.99 (s, 3H, -OCOCH3), 1.95 (m, 1H, 11-H), 1.86 (m, 2H, 16-H), 1.65 (m,
2H, 22-H), 1.61 (m, 1H, 19-H), 1.58 (m, 1H, 15-H), 1.54 (m, 1H, 1-H), 1.49 (m, 1H,
6-H), 1.49 (m, 2H, 9-H), 1.45 (m, 2H, 2-H), 1.40 (m, 1H, 7-H), 1.33 (m, 1H, 6-H), 1.30
(m, 1H, 21-H), 1.22 (m, 1H, 21-H), 1.18 (m, 1H, 7-H), 1.15 (m, 1H, 19-H), 1.12 (s, 3H,
27-H), 1.02 (m, 1H, 15-H), 0.95 (t, J = 7.6 Hz, 9H, -Si(CH2CH3)3), 0.91 (m, 1H, 1-H),
0.90 (s, 12H, 23-H, 25-H, 29-H, 30-H), 0.74 (s, 3H, 24-H), 0.73 (s, 3H, 26-H), 0.68 (m,
13
1H, 5-H), 0.58 (m, 6H, -Si(CH2CH3)3); C-NMR (100 MHz, CDCl3) δ : 175.6 (C-28),
170.3 (-OCOCH3), 170.2 (-OCOCH3), 169.9 (-OCOCH3), 169.1 (-OCOCH3), 142.9
(C-13), 122.8 (C-12), 92.0 (C-1’), 79.4 (C-3), 71.4 (C-5’), 70.9 (C-3’), 67.5 (C-2’), 66.8
(C-4’), 60.8 (C-6’), 55.3 (C-5), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19), 41.7 (C-14), 41.0
(C-18), 39.5 (C-1), 39.3 (C-8), 39.3 (C-4), 36.9 (C-10), 33.8 (C-21), 33.0 (C-7), 33.0
(C-29), 31.6 (C-22), 30.6 (C-20), 28.4 (C-23), 27.8 (C-15), 27.7 (C-2), 25.6 (C-27), 23.5
(C-11), 23.4 (C-16), 22.8 (C-30), 20.7 (-OCOCH3), 20.6 (-OCOCH3), 20.6 (-OCOCH3),
20.5 (-OCOCH3), 18.5 (C-6), 17.0 (C-26), 16.0 (C-24), 15.4 (C-25), 7.0 (-Si(CH2CH3)3),
5.3 (-Si(CH2CH3)3); IR (KBr) cm-1 ν : 2957 (=C-H), 1752 (-C=O), 1067 (-C-O-);
HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C50H80O12SiNa: 923.5317; Found 923.5307.
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Olean-12-en-28-oic acid, 3-O-triethylsilyl-, 2, 3, 4-tri-O-acetyl-β-D-xylopyranosyl
ester (3c) MicroFlow A solution of BF3•OEt2 (72.0 µL, 0.5705 mmol, 0.0316 M)
dissolved in CH2Cl2 (18 mL) was injected to the micromixer by
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using the syringe pump at the flow rate of 0.6 mL/min. Also, a
COO
O
OAc
solution of donor 2c (480 mg, 1.1411 mmol, 0.0634 M) and
acceptor 1 (326 mg, 0.5705 mmol, 0.0317 M) dissolved in
CH2Cl2 (18 mL) was injected to the micromixer by the same
TESO
OAc
OAc
3c
syringe pump at the flow rate of 0.6 mL/min and mixed at –40 ˚C. After the reaction
mixture was allowed to flow at –40 ˚C for an additional 100 sec through a Teflon
reactor tube (φ = 1.0 mm, l = 1.0 m), the mixture was quenched by addition of
triethylamine (79.4 µL) diluted in CH2Cl2. The mixture was concentrated under reduced
pressure to give the crude product. Purification by flash column chromatography (silica
gel 25 g, hexane : AcOEt = 6 : 1) afforded 3c (312 mg, 0.0941 mmol, 66%) as a white
foamy solid.
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Rf = 0.43 (hexane : AcOEt = 2 : 1); [α]D +28.2 (c 1.00, CHCl3); 1H-NMR (400 MHz,
CDCl3) δ : 5.63 (d, J = 6.8 Hz, 1H, 1’-H), 5.31 (t, J = 3.5 Hz, 1H, 12-H), 5.20 (t, J = 8.1
Hz, 3’-H), 5.05 (dd, J = 8.1 Hz, 6.8 Hz, 1H, 2’-H), 5.13 (m, J = 9.1 Hz, 1H, 4’-H), 4.12
(m, 1H, 5’-H), 3.49 (dd, J = 13.6 Hz, 8.4 Hz, 1H, 5’-H), 3.20 (m, 1H, 3-H), 2.84 (m,
1H, 18-H), 2.06 (s, 3H, -OCOCH3), 2.05 (s, 3H, -OCOCH3), 2.04 (s, 3H, -OCOCH3),
1.95 (m, 1H, 11-H), 1.84 (m, 2H, 16-H), 1.60 (m, 1H, 19-H), 1.58 (m, 2H, 22-H), 1.56
(m, 1H, 15-H), 1.54 (m, 1H, 1-H), 1.53 (m, 1H, 11-H), 1.48 (m, 2H, 6-H, 9-H), 1.44 (m,
2H, 2-H), 1.40 (m, 1H, 7-H), 1.31 (m, 1H, 6-H), 1.29 (m, 1H, 21-H), 1.18 (m, 1H,
21-H), 1.15 (m, 1H, 7-H), 1.14 (m, 1H, 19-H), 1.10 (s, 3H, 27-H), 1.01 (m, 1H, 15-H),
0.93 (m, 9H, -Si(CH2CH3)3), 0.89 (m, 1H, 1-H), 0.88 (s, 6H, 23-H, 30-H), 0.87 (s, 6H,
25-H, 29-H), 0.72 (s, 3H, 24-H), 0.69 (s, 3H, 26-H), 0.65 (m, 1H, 5-H), 0.56 (m, 6H,
-Si(CH2CH3)3); 13C-NMR (100 MHz, CDCl3) δ : 175.6 (C-28), 169.8 (-OCOCH3 x2),
169.0 (-OCOCH3), 143.0 (C-13), 123.0 (C-12), 91.7 (C-1’), 79.5 (C-3), 71.0 (C-3’),
69.3 (C-2’), 68.3 (C-4’), 62.5 (C-5’), 55.2 (C-5), 47.6 (C-9), 46.8 (C-17), 45.7 (C-19),
41.7 (C-14), 39.2 (C-18), 38.7 (C-8), 38.4 (C-4), 37.0 (C-1), 33.7 (C-10), 32.9 (C-21),
32.9 (C-29), 32.8 (C-7), 31.9 (C-22), 30.6 (C-20), 28.0 (C-23), 27.7 (C-15), 27.1 (C-2),
25.6 (C-27), 23.5 (C-30), 23.4 (C-16), 22.8 (C-11), 20.7 (-OCOCH3), 20.7 (-OCOCH3),
20.6 (-OCOCH3), 18.3 (C-6), 16.9 (C-26), 15.5 (C-24), 15.3 (C-25); IR (KBr) cm-1 ν :
2933 (=C-H), 1760 (-C=O); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C47H76O10SiNa
851.5074; Found 851.5078.
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Olean-12-en-28-oic acid, 3-O-triethylsilyl-, 2, 3, 4, 6-tetra-O-acetyl-α-D-
mannopyranosyl ester (3d). A solution of BF3•OEt2 (62 µL, 0.5070 mmol, 0.0035 M)
dissolved in CH2Cl2 (16.4 mL) was injected to the
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micromixer by using the syringe pump at the flow rate of
COO
0.6 mL/min. Also, a solution of donor 2d (500 mg,
OAc
OAc
O
AcO
TESO
OAc
1.0148 mmol, 0.0619 M) and acceptor 1 (289.5 mg,
3d
0.5070 mmol, 0.0309 M) dissolved in CH2Cl2 (16.4 mL)
was injected to the micromixer by the same syringe pump at the flow rate of 0.6
mL/min and mixed at –40 ˚C. After the reaction mixture was allowed to flow at –40 ˚C
for an additional 3 min through a Teflon reactor tube (φ = 1.0 mm, l = 1.0 m), the
mixture was quenched by addition of triethylamine (0.26 mL) diluted in CH2Cl2. The
mixture was concentrated under reduced pressure to give the crude product. Purification
by flash column chromatography (silica gel 40 g, hexane : AcOEt = 9 : 1) afforded 3d
(414.7 mg, 0.4601 mmol, 93 %) as a white solid. Rf = 0.40 (hexane : AcOEt = 2 : 1);
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[α]D +51.6 (c 1.00, CHCl3); H-NMR (400 MHz, CDCl3) δ : 6.06 (d, 2.0 Hz, 1H,
1’-H), 5.36 (t, J = 3.5 Hz, 1H, 12-H), 5.32 (t, J = 10.0 Hz, 1H, 4’-H), 5.26 (dd, J = 10.0
Hz, 3.2 Hz, 1H, 3’-H), 5.17 (dd, J = 3.2 Hz, 2.0 Hz, 1H, 2’-H), 4.29 (dd, J = 12.3 Hz,
5.0 Hz, 1H, 6’-H), 4.07 (dd, J = 12.3 Hz, 2.3 Hz, 1H, 6’-H), 3.99 (ddd, J = 10.0 Hz, 5.0
Hz, 2.3 Hz, 1H, 5’-H), 3.18 (dd, J = 11.0 Hz, 4.5 Hz, 1H, 3-H), 2.83 (dd, J = 13.8 Hz,
4.0 Hz, 1H, 18-H), 2.14 (s, 3H, -OCOCH3), 2.06 (s, 3H, -OCOCH3), 2.05 (s, 3H,
-OCOCH3), 1.98 (s, 3H, -OCOCH3), 1.93 (m, 2H, 11-H), 1.86 (m, 2H, 16-H), 1.62 (m,
1H, 19-H), 1.60 (m, 2H, 22-H), 1.57 (m, 1H,15-H), 1.53 (m, 1H, 1-H), 1.51 (m, 1H,
9-H), 1.50 (m, 1H, 6-H), 1.47 (m, 2H, 2-H), 1.42 (m, 2H, 7-H), 1.31 (m, 1H, 21-H),
1.31 (m, 1H, 6-H), 1.20 (m, 1H, 21-H), 1.16 (m, 1H, 19-H), 1.12 (s, 3H, 27-H), 1.08 (m,
1H, 15-H), 0.97 (t, J = 7.9 Hz, 9H, -Si(CH2CH3)3), 0.92 (s, 6H, 29-H, 30-H), 0.90 (s, 3H,
23-H), 0.90 (m, 1H, 1-H), 0.89 (s, 3H, 25-H), 0.73 (s, 3H, 24-H), 0.71 (s, 3H, 26-H),
0.68 (m, 1H, 5-H), 0.58 (m, 6H, -Si(CH2CH3)3); 13C-NMR (100 MHz, CDCl3) δ : 174.7
(C-28), 170.6 (-OCOCH3), 169.8 (-OCOCH3), 169.6 (-OCOCH3), 169.6 (-OCOCH3),
142.7 (C-13), 123.4 (C-12), 90.2 (C-1’), 79.5 (C-3), 71.0 (C-5’), 68.9 (C-3’), 68.3
(C-2’), 65.5 (C-4’), 62.2 (C-6’), 55.3 (C-5), 47.6 (C-9), 47.2 (C-17), 45.7 (C-19), 41.6
(C-14), 41.2 (C-18), 39.3 (C-8), 38.4 (C-1,C-4), 36.9 (C-10), 33.7 (C-21), 33.0 (C-7),
32.7 (C-29), 32.2 (C-22), 30.6 (C-20), 28.4 (C-23), 27.7 (C-15), 27.5 (C-2), 25.8 (C-27),
23.5 (C-30), 23.3 (C-16), 23.3 (C-11), 20.7 (-OCOCH3), 20.7 (-OCOCH3), 20.7
(-OCOCH3), 20.6 (-OCOCH3), 18.5 (C-6), 16.9 (C-26), 16.0 (C-24), 15.3 (C-25), 7.0
(-Si(CH2CH3)3), 5.3 (-Si(CH2CH3)3); IR (KBr) cm-1 ν : 2952 (=C-H), 1755 (-C=O),
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1697 (-C=C-), 1057 (-C-O-); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C50H80O12SiNa 923.5317; Found 923.5303.
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Olean-12-en-28-oic acid, 3-O-triethylsilyl-, 2, 3, 4-tri-O-acetyl-α-L-
rhamnopyranosyl ester (3e) MicroFlow. A solution of BF3•OEt2 (12.5 µL, 0.1002
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mmol, 0.0334 M) dissolved in CH2Cl2 (3.0 mL) was
OAc
OAc
OAc
O
injected to the micromixer by using the syringe pump at
COO
the flow rate of 0.6 mL/min. Also, a solution of donor 2e
TESO
(87.1 mg, 0.2004 mmol, 0.0668 M) and acceptor 1 (57.2
3e
mg, 0.1002 mmol, 0.0334 M) dissolved in CH2Cl2 (3.0
mL) was injected to the micromixer by the same syringe pump at the flow rate of 0.6
mL/min and mixed at –40 ˚C. After the reaction mixture was allowed to flow at –40 ˚C
for an additional 100 sec through a Teflon reactor tube (φ = 1.0 mm, l = 1.0 m), the
mixture was quenched by addition of triethylamine (13.9 µL) diluted in CH2Cl2. The
mixture was concentrated under reduced pressure to give the crude product. Purification
by flash column chromatography (silica gel 30 g , hexane : AcOEt = 10 : 1 → 8 : 1 →
7 : 1 → 5 : 1) afforded 3e (79.3 mg, 0.0941 mmol, 94%) as a white solid. Rf = 0.56
(hexane : AcOEt = 2 : 1); [α]D22 +16.0 (c 1.00, CHCl3); 1H-NMR (400 MHz, CDCl3)δ :
6.02 (d, J = 2.0 Hz, 1H, 1’-H), 5.32 (t, J = 3.2 Hz, 1H, 12-H), 5.27 (dd, J = 10.0 Hz, 3.3
Hz, 1H, 3’-H), 5.20 (dd, J = 3.3 Hz, 2.0 Hz, 1H, 2’-H), 5.13 (t, J = 10.0 Hz, 1H, 4’-H),
3.94 (m, 1H, 5’-H), 3.20 (m, 1H, 3-H), 2.90 (m, 1H, 18-H), 2.16 (s, 3H, -OCOCH3),
2.08 (s, 3H, -OCOCH3), 2.00 (s, 3H, -OCOCH3), 1.95 (m, 1H, 11-H), 1.84 (m, 2H,
16-H), 1.60 (m, 1H, 19-H), 1.58 (m, 2H, 22-H), 1.56 (m, 1H, 15-H), 1.54 (m, 1H, 1-H),
1.53 (m, 1H, 11-H), 1.48 (m, 2H, 6-H, 9-H), 1.44 (m, 2H, 2-H), 1.40 (m, 1H, 7-H), 1.31
(m, 1H, 6-H), 1.29 (m, 1H, 21-H), 1.20 (d, J = 6.2 Hz, 3H, 6’-CH3), 1.18 (m, 1H, 21-H),
1.15 (m, 1H, 7-H), 1.14 (m, 1H, 19-H), 1.10 (s, 3H, 27-H), 1.01 (m, 1H, 15-H), 0.93 (m,
9H, -Si(CH2CH3)3), 0.89 (m, 1H, 1-H), 0.88 (s, 6H, 23-H, 30-H), 0.87 (s, 6H, 25-H,
29-H), 0.72 (s, 3H, 24-H), 0.69 (s, 3H, 26-H), 0.65 (m, 1H, 5-H), 0.56 (m, 6H,
-Si(CH2CH3)3); 13C-NMR (100 MHz, CDCl3) δ : 174.7 (C-28), 169.9 (-OCOCH3),
169.8 (-OCOCH3), 169.8 (-OCOCH3), 143.6 (C-13), 122.9 (C-12), 90.1 (C-1’), 78.9
(C-3), 70.3 (C-4’), 69.0 (C-3’), 68.9 (C-5’), 68.7 (C-2’), 55.2 (C-5), 47.5 (C-9), 47.3
(C-17), 45.6 (C-19), 41.8 (C-14), 41.7 (C-18), 39.4 (C-8), 38.7 (C-4), 38.4 (C-1), 37.0
(C-10), 33.7 (C-21), 33.0 (C-29), 32.7 (C-7), 32.5 (C-22), 30.7 (C-20), 28.1 (C-23), 27.5
(C-15), 27.1 (C-2), 25.8 (C-27), 23.5 (C-30), 23.4 (C-16), 22.9 (C-11), 20.8 (-OCOCH3),
20.8 (-OCOCH3), 20.6 (-OCOCH3), 18.3 (C-6), 17.5 (6’-CH3) 17.0 (C-26), 15.6 (C-24),
15.3 (C-25), 7.0 (-Si(CH2CH3)3), 5.3 (-Si(CH2CH3)3); IR (KBr) cm-1 ν : 2924 (=C-H),
1761 (-C=O); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C48H78O10SiNa 865.5231;
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Found 865.5237.
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Olean-12-en-28-oic acid, 3-O-triethylsilyl-, methyl 2, 3, 4-tri-O-acetyl-β-D-
glucopyranuronosyl ester (3f) MicroFlow. A solution of BF3•OEt2 (39 µL, 0.3097
mmol, 0.03 M) dissolved in CH2Cl2 (10.3 mL) was injected
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to the micromixer by using the syringe pump at the flow rate
COO
O
OAc of 0.6 mL/min. Also, a solution of donor 2f (296.5 mg,
TESO
H
3COOC
0.6194 mmol, 0.0601 M) and acceptor 1 (176.8 mg, 0.3097
OAc
OAc
mmol, 0.03 M) dissolved in CH2Cl2 (10.3 mL) was injected
3f
to the micromixer by the same syringe pump at the flow rate of 0.6 mL/min and mixed
at –40 ˚C. After the reaction mixture was allowed to flow at –40 ˚C for an additional 3
min through a Teflon reactor tube (φ = 1.0 mm, l = 1.0 m), the mixture was quenched by
addition of triethylamine (0.17 mL) diluted in CH2Cl2. The mixture was concentrated
under reduced pressure to give the crude product. Purification by flash column
chromatography (silica gel 40 g, hexane : AcOEt = 6 : 1) afforded 3f (203.9 mg, 0.2298
mmol, 74%) as a white solid. Rf = 0.40 (hexane : AcOEt = 2 : 1); [α]D24 +30.9 (c 0.98,
CHCl3); 1H-NMR (400 MHz, CDCl3) δ : 5.63 (d, J = 8.1 Hz, 1H, 1’-H), 5.31 (dt, J = 3.7
Hz, 1H, 12-H), 5.30 (t, J = 9.5 Hz, 1H, 3’-H), 5.23 (t, J = 9.5 Hz, 1H, 4’-H), 5.20 (dd, J
= 9.5 Hz, 8.1 Hz, 1H, 2’-H), 4.12 (d, J = 9.5 Hz, 1H, 5’-H), 3.72 (s, 3H, -COOCH3),
3.20 (dd, J = 11.1 Hz, 4.4 Hz, 1H, 3-H), 2.81 (dd, J = 14.0 Hz, 4.0 Hz, 1H, 18-H), 2.03
(s, 3H, -OCOCH3), 2.02 (s, 3H, -OCOCH3), 2.02 (s, 3H, -OCOCH3), 1.96 (m, 2H,
11-H), 1.87 (m, 1H, 16-H), 1.64 (m, 2H, 22-H), 1.62 (m, 1H, 19-H), 1.60 (m, 1H, 15-H),
1.58 (m, 1H, 2-H), 1.56 (m, 1H, 1-H), 1.54 (m, 1H, 16-H), 1.52 (m, 1H, 6-H), 1.50 (m,
1H, 9-H), 1.49 (m, 1H, 2-H), 1.40 (m, 1H, 7-H), 1.35 (m, 1H, 6-H), 1.31 (m, 1H, 21-H),
1.21 (m, 1H, 21-H), 1.18 (m, 1H, 19-H), 1.12 (s, 3H, 27-H), 1.03 (m, 1H, 15-H), 0.95 (t,
J = 8.0 Hz, 9H, -Si(CH2CH3)3), 0.90 (s, 3H, 23-H), 0.90 (s, 3H, 25-H), 0.89 (m, 1H,
1-H), 0.89 (s, 3H, 29-H), 0.89 (s, 3H, 30-H), 0.74 (s, 3H, 24-H), 0.71 (s, 3H, 26-H),
0.68 (m, 1H, 5-H), 0.58 (m, 6H, -Si(CH2CH3)3); 13C-NMR (100 MHz, CDCl3) δ : 175.5
(C-28), 169.9 (-OCOCH3), 169.4 (-OCOCH3), 168.9 (-OCOCH3), 166.7 (C-6’), 142.7
(C-13), 123.0 (C-12), 91.2 (C-1’), 79.5 (C-3), 72.9 (C-5’), 72.0 (C-3’), 69.7 (C-2’), 69.3
(C-4’), 55.3 (C-5), 52.9 (-COOCH3), 47.6 (C-9), 46.8 (C-17), 45.7 (C-19), 41.7 (C-14),
41.0 (C-18), 39.3 (C-4), 39.3(C-10), 38.5 (C-1), 36.9 (C-8), 33.7 (C-21), 33.7 (C-29),
33.0 (C-7), 31.6 (C-22), 30.6 (C-20), 28.4 (C-23), 27.7 (C-2), 27.7 (C-15), 25.7 (C-27),
23.4 (C-30), 23.4 (C-16), 22.8 (C-11), 20.6 (-OCOCH3), 20.6 (-OCOCH3), 20.4
(-OCOCH3), 18.5 (C-6), 16.9 (C-26), 16.4 (C-24), 15.4 (C-25), 7.0 (-Si(CH2CH3)3), 5.3
(-Si(CH2CH3)3) IR (KBr) cm-1 ν : 2952 (=C-H), 1759 (-C=O), 1698 (-C=C-), 1068
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(-C-O-); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C49H78O12SiNa 909.5160; Found
909.5147.
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Olean-12-en-28-olic acid, 3-O-triethylsilyl-, 2’-O-acetyl-,
3’,6’-O-(4-methoxybenzyl)-, 4-O-[(E)-3, 4-dimethoxycinnamoyl]-
β-D-glucopyranosyl ester (3g) MicroFlow. A solution of BF3•OEt2 (12.0 µL, 0.0976
mmol, 0.0976 M) dissolved in CH2Cl2 (1.0 mL) was injected to
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the micromixer by using the syringe pump at the flow rate of
COO
O
O
OAc
0.1 mL/min. Also, a solution of donor 2g (7.8 mg, 9.76 µmol)
and acceptor 1 (5.6 mg, 9.76 µmol) dissolved in CH2Cl2 (7.5
mL) was injected to the micromixer by the same syringe pump
at the flow rate of 0.1 mL/min and mixed at –40 ˚C. After the
reaction mixture was allowed to flow at –40 ˚C for an
additional 18 min through a Teflon reactor tube (φ = 1.0 mm, l
TESO
PMBO
O
OPMB
MeO
MeO
3g
= 1.0 m), the mixture was quenched by addition of triethylamine (3.0 µL) diluted in
CH2Cl2. The mixture was concentrated under reduced pressure to give the crude product.
Purification by flash column chromatography (silica gel 7.5 g, hexane : AcOEt = 5 : 1)
afforded 3g (12.3 mg, 9.76 µmol, quant.) as a colorless oil. Rf = 0.29 (hexane : AcOEt =
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2 : 1); [α]D +21.1 (c 1.00, CHCl3); H-NMR (400 MHz, CDCl3) δ : 7.57 (d, J = 16.0
Hz, 1H, PhCH=CH), 7.21 - 6.73 (m, 11H, Ph-H), 6.14 (d, J = 16.0 Hz, 1H, PhCH=CH),
5.56 (d, J = 7.8 Hz, 1H, 1’-H), 5.33 (m, 1H, 12-H), 5.27 (t, J = 9.2 Hz, 1H, 4’-H), 5.22
(dd, J = 9.4 Hz, 7.8 Hz, 1H, 2’-H), 4.54 (m, 2H, -CH2Ph), 4.40 (m, 2H, -CH2Ph), 3.93 (s,
6H, -OCH3 x 2), 3.79 (t, J = 9.4 Hz, 1H, 3’-H), 3.71 (ddd, J = 9.4 Hz, 4.7 Hz, 3.7 Hz,
1H, 5’-H), 3.68 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3), 3.57 (dd, J = 11.0 Hz, 3.7 Hz, 1H,
6’-H), 3.51 (dd, J = 11.0 Hz, 4.7 Hz, 1H, 6’-H), 3.21 (m, 1H, 3-H), 2.84 (m, 1H, 18-H),
2.00 (s, 3H, -OCOCH3), 1.99 (m, 1H, 11-H), 1.87 (m, 2H, 16-H), 1.63 (m, 2H, 22-H),
1.62 (m, 1H, 19-H), 1.59 (m, 1H, 15-H), 1.58 (m, 1H, 11-H), 1.56 (m, 1H, 1-H), 1.52
(m, 1H, 6-H), 1.51 (m, 1H, 9-H), 1.47 (m, 2H, 2-H) 1.34 (m, 1H, 6-H), 1.33 (m, 2H,
21-H) 1.18 (m, 2H, 7-H), 1.15 (m, 1H, 19-H), 1.13 (s, 3H, 27-H), 1.04 (m, 1H, 15-H),
0.98 (m, 9H, -Si(CH2CH3)3), 0.91 (s, 3H, 23-H), 0.90 (m, 4H, 1-H, 30-H), 0.89 (s, 3H,
29-H), 0.88 (s, 3H, 25-H), 0.75 (s, 6H, 24-H, 26-H), 0.69 (m, 1H, 5-H), 0.59 (m, 6 H,
-Si(CH2CH3)3).; 13C-NMR (100 MHz, CDCl3) δ : 175.9 (C-28), 168.8 (-OCOCH3),
165.6 (4’-OCO-), 159.2, 159.0, 151.3, 149.2, 145.6 (PhCH=CH), 143.0 (C-13), 129.9,
129.9, 129.9, 129.7, 129.6, 129.4, 129.3, 127.1, 122.9, 122.8 (C-12), 114.8 (PhCH=CH),
113.7, 113.6, 113.6, 110.9, 109.5, 91.9 (C-1’), 79.5 (C-3’), 79.4 (C-3), 74.4 (C-5’), 73.2
(-CH2Ph), 73.1 (-CH2Ph), 71.3 (C-2’), 70.3 (C-4’), 68.7 (C-6’), 56.0 (-OCH3), 55.8
(-OCH3), 55.3 (C-5), 55.1 (-OCH3), 55.0 (-OCH3), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19),
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41.7 (C-14), 40.9 (C-18), 39.3 (C-8), 39.3 (C-4), 38.5 (C-1), 36.9 (C-10), 33.8 (C-21),
33.0 (C-29), 32.9 (C-7), 31.7 (C-22), 30.6 (C-20), 28.4 (C-23), 27.8 (C-15), 27.7 (C-2),
25.6 (C-27), 23.5 (C-30), 23.4 (C-16), 22.7 (C-11), 20.9 (-OCOCH3), 18.5 (C-6), 17.0
(C-26), 16.0 (C-24), 15.4 (C-25), 7.0 (-Si(CH2CH3)3), 5.2 (-Si(CH2CH3)3); IR (NaCl)
cm-1 ν : 2951 (=C-H), 1753 (-C=O), 1631 (-C=C-), 1514 (-C=C-), 1066 (-C-O-); HRMS
(ESI-TOF) m/z: [M+Na]+ Calcd for C71H100O14SiNa 1227.6780; Found 1227.6772.
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Olean-12-en-28-oic acid, 3-hydroxy-, β-D-galactopyranosyloxy ester (4b). To a
solution of 3b (200 mg, 0.2219 mmol) in MeOH (4.4 mL) at
ambient temperature was added NaH (0.88 mg, 0.0222 mmol,
COO
O
OH
60% disp.). The reaction mixture was stirred at ambient
temperature for 1 h, neutralized with Dowex H+ resin to pH 4
and filtered. The filtrate was concentrated under reduced
HO
HO
HO
OH
4b
pressure to give the pure saponin 4b (144.4 mg, 0.2333 mmol, quant.) as a white solid
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without further purification. Rf = 0.43 (CHCl3: MeOH = 5 : 1); [α]D +74.2 (c 0.25,
MeOH); 1H-NMR (400 MHz, pyridine-d5) δ : 6.29 (d, J = 8.2 Hz, 1H, 1’-H), 5.46 (dd, J
= 3.4 Hz, 3.4 Hz, 1H, 12-H), 4.68 (m, 1H, 3’-H), 4.65 (m, 1H, 2’-H), 4.51 (ddd, J =
10.9 Hz, 6.5 Hz, 6.5 Hz, 1H, 6’-H), 4.41 (ddd, J = 10.8 Hz, 5.4 Hz, 5.4 Hz, 1H, 6’-H),
4.25 (m, 1H, 4’-H), 4.21 (m, 1H, 5’-H), 3.45 (m, 1H, 3-H), 3.22 (dd, J = 4.2 Hz, 4.2 Hz,
1H, 18-H), 2.35 (m, 1H, 2-H), 2.06 (m, 2H, 11-H), 1.94 (m, 2H, 16-H), 1.83 (m, 2H,
2-H, 22-H), 1.78 (m, 1H, 22-H), 1.77 (m, 1H, 19-H), 1.68 (m, 1H, 9-H), 1.58 (m, 1H,
1-H), 1.54 (m, 1H, 6-H), 1.48 (m, 1H, 7-H), 1.40 (m, 1H, 6-H), 1.38 (m, 1H, 7-H), 1.35
(m, 1H, 21-H), 1.30 (m, 1H, 19-H), 1.24 (s, 6H, 23-H, 27-H), 1.16 (s, 3H, 26-H), 1.13
(m, 2H, 15-H), 1.09 (m, 1H, 21-H), 1.04 (s, 3H, 24-H), 0.99 (m, 1H, 1-H), 0.94 (s, 3H,
25-H), 0.91 (s, 3H, 29-H), 0.88 (s, 3H, 30-H), 0.85 (m, 1H, 5-H); 13C-NMR (100 MHz,
pyridine-d5) δ : 176.6 (C-28), 144.3 (C-13), 123.0 (C-12), 96.4 (C-1’), 78.2 (C-3), 77.9
(C-5’), 75.9 (C-4’), 71.6 (C-3’), 70.2 (C-2’), 62.0 (C-6’), 56.0 (C-5), 48.3 (C-9), 47.1
(C-17), 46.4 (C-19), 42.3 (C-14), 41.9 (C-18), 40.1 (C-8), 39.5 (C-4), 39.1 (C-1), 37.5
(C-10), 34.1 (C-21), 33.3 (C-7), 33.3 (C-29), 32.7 (C-22), 30.9 (C-20), 28.9 (C-23), 28.4
(C-15), 28.3 (C-2), 26.2 (C-27), 24.0 (C-30), 23.8 (C-16), 23.5 (C-11), 19.0 (C-6), 17.7
(C-26), 16.7 (C-24), 15.8 (C-25).; IR (KBr) cm-1 ν : 3435 (-O-H), 2949 (=C-H), 1736
(-C=O), 1719 (-C=C-), 1070 (-C-O-); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C36H58O8Na 641.4029; Found 641.4016.
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Olean-12-en-28-oic acid, 3-hydroxy-, β-D-xylopyranosyloxy ester (4c). To a
solution of 3c (200 mg, 0.241 mmol) in MeOH (2.4 mL) at
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ambient temperature was added NaH (0.96 mg, 0.024 mmol,
COO
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O
OH 60% disp.). The reaction mixture was stirred at ambient
HO
temperature for 1 h, neutralized with Dowex H+ resin to pH 4
OH
OH
4c
and filtered, rinsed with MeOH (20 mL). The filtrate was
concentrated under reduced pressure to give the pure 4c (141 mg, 0.239 mmol, 99%) as
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a white solid without further purification. Rf = 0.59 (CHCl3 : MeOH = 5 : 1); [α]D
1
+41.7 (c 0.31, MeOH); H-NMR (400 MHz, pyridine-d5) δ : 6.25 (d, J = 7.0 Hz, 1H,
1’-H), 5.48 (t, J = 3.3 Hz, 1H, 12-H), 4.40 (dd, J = 11.0 Hz, 4.0 Hz, 1H, 5’-H),
4.23-4.19 (m, 3H, 2’-H, 3’-H, 4’-H), 3.85 (dd, J = 11.0 Hz, 10.0 Hz, 1H, 5’-H), 3.45 (dd,
10.7 Hz, 5.7 Hz, 1H, 3-H), 3.27 (dd, J = 14.2 Hz, 3.8 Hz, 1H, 18-H), 2.32 (m, 1H,
15-H), 2.10 (m, 2H, 16-H), 2.05 (m, 1H, 22-H), 1.97 (m, 2H, 11-H), 1.85 (m, 2H, 15-H,
22-H), 1.80 (m, 1H, 19-H), 1.68 (m, 1H, 9-H), 1.54 (m, 2H, 1-H, 6-H), 1.51 (m, 2H,
7-H), 1.37 (m, 2H, 6-H, 21-H), 1.30 (m, 1H, 19-H), 1.25 (s, 3H, 27-H), 1.24 (s, 3H,
23-H), 1.18 (m, 1H, 2-H), 1.15 (m, 1H, 21-H), 1.12 (s, 3H, 26-H), 1.05 (s, 3H, 24-H),
1.00 (m, 1H, 1-H), 0.95 (s, 3H, 30-H), 0.94 (s, 6H, 25-H, 29-H), 0.86 (m, 1H, 5-H);
13C-NMR (100 MHz, pyridine-d5) δ : 176.7 (C-28), 144.3 (C-13), 123.1 (C-12), 96.4
(C-1’), 78.4 (C-2’), 78.2 (C-3), 73.8 (C-3’), 71.0 (C-4’), 67.9 (C-5’), 56.0 (C-5), 48.3
(C-9), 47.3 (C-17), 46.4 (C-19), 42.3 (C-14), 41.9 (C-18), 40.0 (C-8), 39.5 (C-4), 39.1
(C-1), 37.5 (C-10), 34.2 (C-21), 33.4 (C-7), 33.3 (C-29), 32.9 (C-22), 31.0 (C-20), 29.0
(C-23), 28.4 (C-15), 28.3 (C-2), 26.2 (C-27), 24.0 (C-11), 23.8 (C-30), 23.5 (C-16), 19.0
(C-6), 17.7 (C-26), 16.7 (C-24), 15.8 (C-25); IR (KBr) cm-1 ν : 3407 (-O-H), 2940
(=C-H), 1744 (-C=O), 1032 (-C-O-); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C35H56O7Na 611.3924; Found 611.3908.
Olean-12-en-28-oic acid, 3-hydroxy-, α-D-mannopyranosyloxy ester (4d). To a
solution of 3d (200 mg, 0.2219 mmol) stirring in dry MeOH
(4.4 mL) was added sodium hydride (0.88 mg, 0.0222
COO
OH
OH
O
HO
mmol). The mixture was stirred at ambient temperature for
40 min, neutralized with Dowex H+ resin to pH 4 and
HO
OH
4d
filtered. The filtrate was concentrated under reduced pressure to give the pure saponin
4d (150.5 mg, 0.2432 mmol, quant.) as a white solid. Rf = 0.53 (CHCl3: MeOH= 5 : 1);
[α]D22 +67.9 (c 0.24 , MeOH); 1H-NMR (400 MHz, pyridine-d5) δ : 6.89 (d, J = 1.6 Hz,
1H, 1’-H), 5.40 (t, J = 3.5 Hz, 1H, 12-H), 4.86 (dt, 9.5 Hz, 5.0 Hz, 1H, 4’-H), 4.68 (ddd,
J = 10.0 Hz, 9.5 Hz, 6.5 Hz, 1H, 3’-H), 4.65 (ddd, J = 11.5 Hz, 6.0 Hz, 2.0 Hz, 1H,
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6’-H), 4.63 (ddd, J = 10.0 Hz, 4.5 Hz, 1.6 Hz, 2’-H), 4.56 (ddd, J = 11.5 Hz, 6.5 Hz, 5.0
Hz, 1H, 6’-H), 4.51 (ddd, J = 9.5 Hz, 5.0 Hz, 2.0 Hz, 1H, 5’-H), 3.44 (ddd, J = 10.9 Hz,
5.4 Hz, 5.4 Hz, 1H, 3-H), 3.17 (dd, J = 13.8 Hz, 4.0 Hz, 1H, 18-H), 2.05 (m, 1H, 11-H),
2.02 (m, 2H, 2-H), 1.88 (m, 1H, 22-H), 1.85 (m, 2H, 16-H), 1.83 (m, 1H, 15-H), 1.78
(m, 1H, 11-H), 1.75 (m, 1H, 19-H), 1.64 (m, 1H, 9-H), 1.58 (m, 1H, 22-H), 1.53 (m, 1H,
1-H), 1.53 (m, 1H, 6-H), 1.47 (m, 1H, 7-H), 1.39 (m, 1H, 21-H), 1.34 (m, 1H, 7-H),
1.31 (m, 1H, 6-H), 1.24 (s, 3H, 23-H), 1.23 (m, 1H, 19-H), 1.21 (s, 3H, 27-H), 1.14 (m,
1H, 21-H), 1.11 (m, 1H, 15-H), 1.04 (s, 3H, 24-H), 1.00 (m, 1H, 1-H), 0.95 (s, 3H,
30-H), 0.93 (s, 3H, 26-H), 0.91 (s, 3H, 29-H), 0.86 (s, 3H, 25-H), 0.83 (m, 1H, 5-H);
13C-NMR (100 MHz, pyridine-d5) δ : 176.1 (C-28), 144.2 (C-13), 123.2 (C-12), 95.5
(C-1’), 78.5 (C-5’), 78.2 (C-3), 73.2 (C-3’), 71.4 (C-2’), 68.5 (C-4’), 62.8 (C-6’), 55.9
(C-5), 48.2 (C-9), 47.5 (C-17), 46.2 (C-19), 42.2 (C-14), 42.0 (C-18), 39.9 (C-8), 39.5
(C-4), 39.1 (C-1), 37.4 (C-10), 34.1 (C-21), 33.3 (C-7), 33.3 (C-29), 32.9 (C-22), 31.0
(C-20), 28.9 (C-23), 28.2 (C-15), 28.1 (C-2), 26.1 (C-27), 24.0 (C-30), 23.8 (C-16), 23.6
(C-11), 18.9 (C-6), 17.6 (C-26), 16.7 (C-24), 15.8 (C-25); IR (KBr) cm-1 ν: 3382 (-O-H),
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31
32
33
34
35
36
37
38
39
40
41
42
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44
45
46
47
48
49
50
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60
2933 (=C-H), 1697 (-C=O); HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C36H58O8Na
1
641.4029; Found 641.4014. GATEI (400 MHz, pyridine-d5) δ : 95.5 (C-1’), JC,H
=
173.4 Hz.
Olean-12-en-28-oic acid, 3-hydroxy-, α-L-rhamnopyranosyloxy ester (4e). To a
OH
OH
solution of 3e (200 mg, 0.237 mmol) in MeOH (2.3 mL) at
OH
O
COO
ambient temperature was added NaH (0.948 mg, 0.024
mmol, 60% disp.). The reaction mixture was stirred at
ambient temperature for 30 min, neutralized with Dowex
HO
4e
H+ resin to pH 4 and filtered, rinsed with MeOH (20 mL). The filtrate was concentrated
under reduced pressure to give the pure 4e (150 mg, 0.248 mmol, quant.) as a white
22
solid without further purification. Rf = 0.57 (CHCl3 : MeOH = 5 : 1); [α]D +25.7 (c
1.00, MeOH); 1H-NMR (400 MHz, pyridine-d5); δ : 6.83 (d, J = 1.5 Hz, 1H, 1’-H), 5.46
(t, J = 3.3 Hz, 1H, 12-H), 4.61 (dd, J = 3.3 Hz, 1.5 Hz, 1H, 2’-H), 4.54 (dd, J = 8.8 Hz,
3.3 Hz, 1H, 3’-H), 4.39 (t, J = 8.8 Hz, 1H, 4’-H), 4.39 (m, 1H, 5’-H), 3.45 (dd, 10.7 Hz,
5.7 Hz, 1H, 3-H), 3.17 (dd, J = 14.2 Hz, 3.8 Hz, 1H, 18-H), 2.05 (m, 1H, 16-H), 2.01 (m,
1H, 2-H), 1.95 (m, 2H, 11-H), 1.86 (m, 2H, 2-H, 22-H), 1.75 (m, 2H, 16-H, 19-H), 1.72
(d, J = 5.5 Hz, 3H, 6’-CH3), 1.64 (m, 1H, 9-H), 1.61 (m, 1H, 22-H), 1.57 (m, 2H, 1-H,
6-H), 1.46 (m, 1H, 7-H), 1.41 (m, 1H, 21-H), 1.36 (m, 1H, 6-H), 1.30 (m, 2H, 7-H,
19-H), 1.25 (s, 3H, 23-H), 1.22 (s, 3H, 27-H), 1.17 (m, 1H, 21-H), 1.12 (m, 2H, 15-H),
1.06 (s, 3H, 26-H),1.00 (m, 1H, 1-H), 0.92 (s, 3H, 24-H), 0.92 (s, 3H, 25-H), 0.92 (s, 3H,
29-H), 0.89 (s, 3H, 30-H), 0.87 (m, 1H, 5-H); 13C-NMR (100 MHz, pyridine-d5) δ :
29
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