Synthesis of flaccidoside II, a bidesmosidic triterpene saponin isolated from Chinese folk medicine Di Wu

Article abstract of DOI:10.1016/j.carres.2007.11.015

A total synthesis of flaccidoside II, 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-xylopyranosyloleanolic acid 28-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside, isolated from Chinese folk medicine Di Wu, has been accomplished from building

Full text of DOI:10.1016/j.carres.2007.11.015

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 462–469
Synthesis of flaccidoside II, a bidesmosidic triterpene saponin
isolated from Chinese folk medicine Di Wu
Shuihong Cheng,a Yuguo Du,a,* Feihong Bingb and Guobin Zhangb
aState Key Lab of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, PR China
bHubei College of Traditional Chinese Medicine, PR China
Received 12 October 2007; received in revised form 9 November 2007; accepted 12 November 2007
Available online 22 November 2007
Abstract—A total synthesis of flaccidoside II, 3-O-a-L-rhamnopyranosyl-(1!2)-b-D-xylopyranosyloleanolic acid 28-O-a-L-rhamno-
pyranosyl-(1!4)-b-D-glucopyranosyl-(1!6)-b-D-glucopyranoside, isolated from Chinese folk medicine Di Wu, has been accom-
plished from building blocks isopropyl 2-O-acetyl-3,4-di-O-benzoyl-1-thio-b-D-xylopyranoside, 2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl trichloroacetimidate, oleanolic acid trityl ester, ethyl 2,3-di-O-acetyl-6-O-benzoyl-1-thio-b-D-glucopyranoside
and 4-methoxyphenyl 2,3,4-tri-O-acetyl-b-D-glucopyranoside. The use of a partially protected thioglycosyl donor significantly sim-
plified the synthesis of the target saponin.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Flaccidoside II; Saponin; Di Wu; Natural product; Chinese folk medicine
1. Introduction
Di Wu is the dry rhizome of Anemone flaccida
Fr. Schmidt.6 It is distributed in the southern part of
China and is used as a folk medicine for detoxication,
expelling wind-evil and releasing wetness-evil. The
main bioactive fraction of Di Wu is proved to be
triterpenoid saponins by pharmacological studies, and
a bioactive component, named as flaccidoside II, was
isolated from the alcohol extracts of A. flaccida
Fr. Schmidt. On the basis of its chemical–physical
properties, hydrolysis reactions and spectroscopic
analyses, the chemical structure of flaccidoside II was
elucidated as 3-O-a-L-rhamnopyranosyl-(1!2)-b-D-xylo-
pyranosyloleanolic acid 28-O-a-L-rhamnopyranosyl-
(1!4)-b-D-glucopyranosyl-(1!6)-b-D-glucopyranoside.7
The structural complexity, especially having a 20-OH
branched sugar chain on C-3 of oleanolic acid, has
hindered the chemical synthesis of this type of bides-
mosidic triterpene saponin.8 In the preparation of bio-
active saponins containing a 2-OH branched sugar
chain, we have recently developed a facile method by
using partially protected glycosyl donors.9 Herein, we
report the synthesis of flaccidoside II by applying the
same methodology.
Since ancient times, traditional medicine has been used
worldwide in the treatment of many diseases and for
health care practices. Many compounds with well-
defined biological and pharmacological activities have
been isolated and structurally characterized from known
medicinal plants.1,2 Among these compounds, steroid or
triterpene saponins present a broad spectrum of biomed-
ical, food and industrial applications, which take advan-
tages of their generally nonionic surfactant and
membrane-disrupting properties. The useful commercial
applications range from their use as fish and snail poi-
sons, fire extinguishers, denatured alcohol to precursors
for cortisone and other steroid drugs and hormones.
Regarding the pharmaceutical uses, various saponins
have shown interesting anti-cancer, anti-inflammatory,
ion channel-blocking, immune-stimulating, antifungal,
antithrombotic and hypocholesterolemic properties.3–5
*
Corresponding author. Tel.: +86 10 62849126; fax: +86 10
62923563; e-mail: duyuguo@rcees.ac.cn
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.11.015

S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
463
2. Results and discussion
methanesulfonate (TMSOTf) at 0 ꢁC afforded ethyl
2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1!4)-2,3-di-
O-acetyl-6-O-benzoyl-1-thio-b-D-glucopyranoside (12)
in 92% yield. Convergently, 4-methoxyphenyl b-D-
glucopyranoside (13)12 was regioselectively silylated
with tert-butyldimethylsilyl chloride (TBSCl) in pyr-
idine, followed by acetylation with acetic anhydride in
one pot, to afford 4-methoxyphenyl 2,3,4-tri-O-acetyl-
6-O-tert-butyldimethylsilyl-b-D-glucopyranoside (14) in
good yield. BF3ÆEt2O-catalyzed removal of TBS was
carried out smoothly13 to generate the acceptor, 4-
methoxyphenyl 2,3,4-tri-O-acetyl-b-D-glucopyranoside
(15) in excellent yield, with no acetyl migration14
observed. Glycosylation of 12 and 15 in anhyd CH2Cl2
at ambient temperature using the N-iodosuccinimide
(NIS)–TMSOTf combination successfully gave trisac-
charide 16 in a yield of 86%. Treatment of 16 with ceric
ammonium nitrate (CAN) in 4:1 CH3CN–H2O,
followed by trichloroacetimidation (Cl3CCN, DBU,
CH2Cl2), furnished the trisaccharide building block 4
in 80% yield over two steps.
Saponin 1 can be disconnected into a disaccharide
donor 2, an oleanolic acid derivative 3 and a trisaccha-
ride donor 4. The sugar moieties 2 and 4 could be assem-
bled from monosaccharide building blocks 5, 6, 7 and/or
8 through standard glycosylation procedures. As shown
in Scheme 1, X, X1, X2 and X3 represent suitable leaving
groups or temporary protecting groups. The trityl ester
3 was easily prepared in quantitative yield by treating
oleanolic acid with triphenylmethyl chloride (TrCl)
and 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in
refluxing tetrahydrofuran (THF), while 2,3,4-tri-O-
benzoyl-a-L-rhamnopyranosyl trichloroacetimidate (6)
is a known compound.10
As depicted in Scheme 2, trisaccharide trichloroacet-
imidate donor 4 was obtained in a straightforward
manner. Thus, ethyl 2,3-di-O-acetyl-4,6-O-benzylidene-
1-thio-b-D-glucopyranoside (9)11 was treated with
aqueous 80% HOAc under reflux, giving ethyl 2,3-di-
O-acetyl-1-thio-b-D-glucopyranoside (10), which was
then regioselectively benzoylated with benzoyl chloride
in pyridine at 0 ꢁC obtained ethyl 2,3-di-O-acetyl-6-O-
benzoyl-1-thio-b-D-glucopyranoside (11) in 85% yield
from 9. In the 1H NMR spectrum of 11, a set of doublet
of doublet peaks at d 4.61 and 4.77 ppm corresponding
to H-6s clearly proved the selectivity. Coupling of
L-rhamnopyranosyl trichloroacetimidate 6 and 11 in
dry CH2Cl2 in the presence of trimethylsilyl trifluoro-
With compound 4 in hand, we started to formally
assemble the target saponin 1. The initial focus of this
work was the synthesis of the disaccharide–oleanolic
acid complex (Scheme 3). Thus, condensation of allyl
3,4-di-O-acetyl-b-D-xylopyranoside (17)15 and 6 in dry
CH2Cl2 with promotion of the reaction by TMSOTf
generated disaccharide 18, which was then transformed
into a disaccharide donor 19 according to our published
O
O
O
O
HO
OH
O
HO
O
O
O
HO
OH
Me
HO
O
HO
O
OH
HO
HO
OH
O
Me
HO
1
OH
OH
O
BzO
BzO
BzO
X
O
O
O
O
AcO
OTr
Me
O
AcO
O
AcO
AcO
Me
O
O
BzO
NH
BzO
BzO
AcO
OBz
BzO
HO
OCCCl3
OBz
3
4
2
OC(NH)CCl3
HO
AcO
AcO
BzO
HO
AcO
O
BzO
BzO
Me
BzO
O
O
X1
O
X3
X2
BzO
OH
AcO
AcO
OBz
5
8
6
7
Scheme 1. Retrosynthesis of Saponin 1.

464
S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
BzO
RO
HO
AcO
TMSOTf
O
O
O
Ph
O
DCM
80% HOAc
SEt
O
SEt
O
AcO
SEt
AcO
Me
BzO
O
OAc
OAc
OAc
6
10 R = H
11 R = Bz
9
BzCl, Pyr
12
BzO
I
OBz
BzO
O
II
R1O
RO
RO
TMSOTf
NIS
O
III
O
O
AcO
OMP
4
O
Me
BzO
AcO
O
AcO
1) CAN
OMP
12
AcO
OR
2) CCl3CN, DBU
OAc
BzO
OBz
13 R = R1 = H
16
TBSCl, Pyr; Ac2O
BF3-Et2O, CH2Cl2
14 R = Ac, R1 = TBS
15 R = Ac, R1 = H
Scheme 2. Synthesis of trisaccharide 4.
O
AcO
AcO
TMSOTf
DCM
TMSOTf
DCM
OR
O
AcO
AcO
O
OAll
OTr
O
6
OH
17
Me
BzO
O
O
O
3
AcO
AcO
O
BzO
OBz
1) PdCl2, HOAc, NaOAc
2) Cl3CCN, K2CO3, DCM
18 R = All
19 R = C(NH)CCl3
Me
BzO
O
20
BzO
OBz
Scheme 3. Attempted strategy for the synthesis of disaccharide saponin derivative.
procedure (PdCl2, HOAc, NaOAc; Cl3CCN, K2CO3,
CH2Cl2).16 Coupling of oleanolic ester 3 with trichloro-
acetimidate 19 was completed within 30 min in the pres-
ence of a catalytic amount of TMSOTf at low
temperature (ꢀ42 ꢁC); however, an inseparable a,b-mix-
ture of 20 was obtained. Limited efforts, owing to the
instability of trityl ester, were tried regarding solvents
(ether, toluene and acetonitrile) and catalysts (AgOTf
and HClO4–SiO2),17 but all were fruitless. We thus
turned our attention to a stepwise route.
In our previous research, we found that a thioglyco-
side having its C-2 unprotected hydroxyl group could
be a good glycosyl donor. Accordingly, orthoester 2118
was converted into 3,4-di-O-benzoyl-1,2-O-(1-methoxy-
ethylidene)-a-D-xylopyranose (22), which was then
dropped into a mixture of 2-PrSH and TMSOTf19 in
dry CH2Cl2 to afford isopropyl 2-O-acetyl-3,4-di-O-
benzoyl-1-thio-b-D-xylopyranoside (23) (Scheme 4).
Treatment of 23 with a methanolic solution containing
5% HCl (dry) gave the desired isopropyl 3,4-di-O-
PriSH
TMSOTf
O
NIS/TMSOTf
O
BzO
BzO
RO
RO
SCH(CH3)2
OTr
OR
O
3
O
O
O
23 R = Ac
24 R = H
5% HCl
MeOH
BzO
BzO
Me
OCH3
O
OH
21 R = Ac
22 R = Bz
1) NaOMe, MeOH
2) BzCl, Pyr
25
6
TMSOTf
DCM
O
O
OR
IV
I
O
O
BzO
BzO
OAc
O
O
BzO
BzO
O
4
O
I
O
O
O
O
OAc
BzO
TMSOTf
DCM
AcO
OAc
Me
BzO
O
V
Me
O
II
OBz
BzO
O
80% HOAc
70 oC
26 R = Tr
27 R = H
AcO
BzO
II
OBz
BzO
III
NaOMe
DCM-MeOH
O
Me
1
OBz
28
BzO
OBz
Scheme 4. Final assembly of target compound 1.

S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
465
benzoyl-1-thio-b-D-xylopyranoside (24) in a moderate
yield of 55% over two steps. Coupling of oleanolic ester
3 and thioglycoside 24 was carried out smoothly in dry
dichloromethane at low temperature (ꢀ42 ꢁC) under
the promotion of co-catalyst NIS/TMSOTf, providing
the desired saponin derivative 25 in 70% yield. Pure
compound 25 was glycosylated with trichloroacet-
imidate 6 under standard conditions to give 26, which
was readily converted into acid 27 in aqueous 80%
HOAc at 70 ꢁC. Ester formation between acid 27 and
trisaccharide donor 4 in dry dichloromethane under
the promotion of TMSOTf led to the fully protected
saponin derivative 28. Selective removal of the acetate
and benzoate protections using a catalytic amount of
NaOMe (2:1 CH2Cl2–MeOH) in the presence of the
C-28 ester glycosidic linkage finished the total synthesis
of flaccidoside II. The physical data obtained were
essentially identical to the natural product reported by
us6 and another group.7 The prepared samples show
good inhibition activity towards ConA-induced lympho-
cyte proliferation in a bioactivity screening,20 and the
details will be published in due course.
mixture was stirred at rt for 6 h and then concentrated
with toluene under reduced pressure. The residue was
purified by silica gel column chromatography (2:1 petro-
leum ether–EtOAc) to give compound 11 as a foamy
solid (8.83 g, 85% from 9): ½aꢁD +65 (c 1, CHCl3); H
NMR (400 MHz, CDCl3): d 1.28 (t, 3H, J 7.4 Hz,
SCH2CH3), 2.09, 2.11 (2s, 2 · 3H, 2CH3CO), 2.66–
2.78 (m, 2H, SCH2CH3), 3.68–3.69 (m, 2H, H-4, H-5),
4.55 (d, 1H, J 9.7 Hz, H-1), 4.61 (dd, 1H, J 1.3,
12.0 Hz, H-6a), 4.77 (dd, 1H, J 3.9, 12.0 Hz, H-6b),
5.01 (t, 1H, J 9.7 Hz, H-2), 5.13 (t, 1H, J 9.7 Hz, H-3),
7.47–8.10 (m, 5H, Ph). Anal. Calcd for C19H24O8S: C,
55.33; H, 5.87. Found: C, 55.04; H, 5.72.
25
1
3.3. Ethyl 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-
(1!4)-2,3-di-O-acetyl-6-O-benzoyl-1-thio-b-D-glucopy-
ranoside (12)
To a mixture of compounds 6 (1.10 g, 1.77 mmol) and
11 (610 mg, 1.48 mmol) in anhyd CH2Cl2 (10 mL) was
added TMSOTf (32 lL, 0.17 mmol) under an N2 atmo-
sphere at 0 ꢁC. The mixture was stirred under these con-
ditions for 30 min, at the end of which time TLC (2:1
petroleum ether–EtOAc) indicated that all starting
materials were consumed. The reaction mixture was
neutralized with Et3N and concentrated. Column chro-
matography (3:1 petroleum ether–EtOAc) of the residue
3. Experimental
3.1. General methods
25
gave 12 as a syrup (1.19 g, 92%): ½aꢁD +85 (c 2, CHCl3);
Optical rotations were determined at 25 ꢁC with a
Perkin–Elmer Model 241-Mc automatic polarimeter.
1H NMR, 13C NMR and 1H–1H, 1H–13C COSY spectra
were recorded with a Bruker ARX 400 spectrometer for
solutions in CDCl3 or D2O. Chemical shifts are given in
ppm downfield from internal Me4Si. Mass spectra were
measured using a MALDITOF-MS with a-cyano-4-
hydroxycinnamic acid (CCA) as matrix. Thin-layer
chromatography (TLC) was performed on silica gel
HF254 with detection by charring with 30% (v/v)
H2SO4 in MeOH or in some cases by UV detection.
Column chromatography was conducted by elution of
a column of silica gel (100–200 mesh) with EtOAc–
petroleum ether (60–90 ꢁC) as the eluent, or a column
of Bio-Gel P2 with water as the eluent. Solutions were
concentrated at <50 ꢁC under reduced pressure.
1H NMR (400 MHz, CDCl3): d 1.23 (t, 3H, J 7.4 Hz,
SCH2CH3), 1.36 (d, 3H, J 6.3 Hz, H-6II), 2.07, 2.09
(2s, 2 · 3H, 2CH3CO), 2.60–2.74 (m, 2H, SCH2CH3),
3.87–3.92 (m, 1H, H-5I), 4.07 (t, 1H, J 9.3 Hz, H-4I),
4.16–4.21 (m, 1H, H-5II), 4.61 (d, 1H, J 10.2 Hz,
H-1I), 4.64 (dd, 1H, J 12.0 Hz, H-6aI), 4.94–5.01 (m,
2H, H-3I, H-6bI), 5.21 (d, 1H, J 2.0 Hz, H-1II), 5.37
(dd, 1H, J 9.0, 10.2 Hz, H-2I), 5.52 (dd, 1H, J 2.0,
3.2 Hz, H-2II), 5.66 (t, 1H, J 10.0 Hz, H-4II), 5.75 (dd,
1H, J 3.2, 10.0 Hz, H-3II), 7.26–8.10 (m, 20H, 4Ph).
Anal. Calcd for C46H46O15S: C, 63.44; H, 5.32. Found:
C, 63.71; H, 5.20.
3.4. 4-Methoxyphenyl 2,3,4-tri-O-acetyl-b-D-glucopyr-
anoside (15)
To a solution of 13 (5.04 g, 17.6 mmol) in pyridine
(50 mL) was added TBSCl (3.2 g, 21.2 mmol) at 0 ꢁC.
The mixture was stirred at rt for 2.5 h, then Ac2O
(12 mL) was added. The mixture was stirred at rt for
another 4 h, then co-evaporated with toluene to dryness
under reduced pressure. The residue was purified by col-
umn chromatography (3:1 petroleum ether–EtOAc) to
give 14 as a solid (8.33 g, 90%). To a solution of the
above solid (8.02 g, 15.2 mmol) in dry CH2Cl2 (80 mL)
was added BF3ÆEt2O (5.4 mL, 41.8 mmol). The mixture
was stirred at rt for 40 min, at the end of which time
TLC (1:1 petroleum ether–EtOAc) indicated the
3.2. Ethyl 2,3-di-O-acetyl-6-O-benzoyl-1-thio-b-D-gluco-
pyranoside (11)
Compound 9 (10.0 g, 25.2 mmol) was dissolved in 80%
aq HOAc (100 mL). The mixture was stirred under
reflux for 5 h and then co-evaporated with the help of
toluene to dryness. The residue was subjected to column
chromatography (1:1 EtOAc–petroleum ether) to give
amorphous solid 10, which was dissolved in pyridine
(42 mL) and dropped into a mixture of benzoyl chloride
(3.1 mL, 26.3 mmol) and pyridine (10 mL) at 0 ꢁC. The

466
S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
reaction complete. The mixture was diluted with
CH2Cl2, washed with satd aq NaHCO3 and then satd
aq NaCl. The organic layer was combined, dried and
concentrated. Purification by column chromatography
(1:1 petroleum ether–EtOAc) gave 15 as a white solid
column chromatography (3:2 petroleum ether–EtOAc)
to afford a solid. To a mixture of the solid in CH2Cl2
(8 mL) were added trichloroacetonitrile (0.4 mL) and
DBU (0.04 mL). The reaction mixture was stirred at rt
for 1.5 h and then concentrated in vacuo. The residue
was purified by column chromatography (2:1 petroleum
ether–EtOAc) to give 4 as a white foamy solid (0.91 g,
25
(5.91 g, 94%): ½aꢁD ꢀ5 (c 4, CHCl3); 1H NMR
(400 MHz, CDCl3): d 2.03, 2.06, 2.07 (3s, 3 · 3H,
3CH3CO), 2.17 (br s, 1H, OH), 3.61–3.66 (m, 2H,
H-5, H-6a), 3.73–3.77 (m, 4H, H-6b, OCH3), 5.01 (d,
1H, J 7.9 Hz, H-1), 5.10 (t, 1H, J 9.4 Hz, H-4), 5.21
(dd, 1H, J 7.9, 9.4 Hz, H-2), 5.32 (t, 1H, J 9.4 Hz,
H-3), 6.81–6.94 (m, 4H, Ph). Anal. Calcd for
C19H24O10: C, 55.34; H, 5.87. Found: C, 55.08; H, 5.95.
25
80%): ½aꢁD +131 (c 1, CHCl3); 1H NMR (400 MHz,
CDCl3): d 1.29 (d, 3H, J 6.2 Hz, H-6III), 2.00, 2.02,
2.03, 2.07, 2.08 (5s, 5 · 3H, 5Ac), 3.60 (dd, 1H, J 5.2,
11.3 Hz, H-6bI), 3.87 (m, 1H, H-5II), 3.96 (dd, 1H, J
2.0, 11.3 Hz, H-6aI), 4.07 (t, 1H, J 9.2 Hz, H-4II),
4.15–4.19 (m, 2H, H-5I, H-5III), 4.63 (dd, 1H, J 4.0,
12.4 Hz, H-6bII), 4.66 (d, 1H, J 7.9 Hz, H-1II), 4.90
(dd, 1H, J 7.9, 9.2 Hz, H-2II), 4.93 (dd, 1H, J 3.6,
9.5 Hz, H-2I), 5.05–5.08 (m, 2H, H-3II, H-6aII), 5.20
(d, 1H, J 1.8 Hz, H-1III), 5.33 (t, 1H, J 9.5 Hz, H-3I),
5.51–5.56 (m, 2H, H-4I, H-2III), 5.65 (t, 1H, J 10.0 Hz,
H-4III), 5.75 (dd, 1H, J 3.2, 10.0 Hz, H-3III), 6.53 (d,
1H, J 3.6 Hz, H-1I), 7.23–8.09 (m, 20H, 4Ph), 8.69 (s,
1H, NH). Anal. Calcd for C58H58Cl3NO24: C, 55.31;
H, 4.64. Found: C, 55.53; H, 4.58.
3.5. 4-Methoxyphenyl 2,3,4-tri-O-benzoyl-a-L-rhamno-
pyranosyl-(1!4)-2,3-di-O-acetyl-6-O-benzoyl-b-D-gluco-
pyranosyl-(1!6)-2,3,4-tri-O-acetyl-b-D-glucopyranoside
(16)
To a mixture of compounds 12 (1.10 g, 1.26 mmol) and
15 (474 mg, 1.15 mmol) in anhyd CH2Cl2 (12 mL) was
added NIS (425 mg, 1.89 mmol) and TMSOTf (23 lL,
0.13 mmol) under an N2 atmosphere at 0 ꢁC. The mix-
ture was stirred under these conditions for 30 min, at
the end of which time TLC (1:1 petroleum ether–
EtOAc) indicated that all starting materials were con-
sumed. The reaction mixture was neutralized with
Et3N, then concentrated. Column chromatography
(3:2 petroleum ether–EtOAc) of the residue gave 16 as
3.7. 3,4-Di-O-benzoyl-1,2-O-methoxyethylidene-a-D-
xylopyranose (22)
To a mixture of 21 (10.02 g, 34.5 mmol) in MeOH
(80 mL) was added 1.0 M NaOMe–MeOH. The pH of
the mixture was kept at 10 at rt for 2.5 h, TLC (4:1
EtOAc–MeOH) indicated that the reaction was com-
plete. The reaction mixture was concentrated, and the
residue was purified by column chromatography
(EtOAc) to give a syrup. The syrup was dissolved in pyr-
idine (40 mL), and BzCl (8.82 mL, 75.9 mmol) in pyri-
dine (15 mL) was added dropwise to the mixture
cooled in an ice-water bath. The mixture was stirred at
rt for 4 h, and TLC (3:1 petroleum ether–EtOAc) indi-
cated that the reaction was complete. The mixture was
concentrated with toluene and purified by column chro-
matography (4:1 petroleum ether–EtOAc) to give 22 as a
25
a foamy solid (1.206 g, 86%): ½aꢁD +75 (c 2, CHCl3);
1H NMR (400 MHz, CDCl3): d 1.27 (d, 3H, J 6.2 Hz,
H-6III), 1.89, 2.01, 2.02, 2.06, 2.08 (5s, 5 · 3H, 5Ac),
3.70 (dd, 1H, J 5.3, 11.3 Hz, H-6aI), 3.75–3.78 (m, 4H,
H-5II, OCH3), 3.84–3.88 (m, 2H, H-5I, H-6bI), 4.06 (t,
1H, J 9.4 Hz, H-4II), 4.15–4.19 (m, 1H, H-5III), 4.61
(dd, 1H, J 4.0, 12.4 Hz, H-6aII), 4.67 (d, 1H, J 7.9 Hz,
H-1II), 4.90–5.01 (m, 4H, H-2I, H-6bII, H-3II, H-1I),
5.19–5.32 (m, 4H, H-1III, H-4I, H-2II, H-3I), 5.52 (t,
1H, J 2.1 Hz, H-2III), 5.68 (t, 1H, J 9.7 Hz, H-4III),
5.74 (dd, 1H, J 2.1, 9.7 Hz, H-3III), 6.88–8.09 (m, 24H,
Ph). Anal. Calcd for C63H64O25: C, 61.96; H, 5.28.
Found: C, 62.25; H, 5.16.
25
1
syrup (12.8 g, 90%): ½aꢁD +30 (c 1, CHCl3); H NMR
(400 MHz, CDCl3): d 1.79 (s, 3H, CH3), 3.32 (s, 3H,
OCH3), 3.97 (dd, 1H, J 6.0, 12.5 Hz, H-5a), 4.16 (dd,
1H, J 4.7, 12.5 Hz, H-5b), 4.41–4.43 (m, 1H, H-2),
5.21–5.22 (m, 1H, H-4), 5.69–5.72 (m, 2H, H-3, H-1),
7.41–8.09 (m, 10H, 2Ph). Anal. Calcd for C22H22O8:
C, 63.76; H, 5.35. Found: C, 63.51; H, 5.42.
3.6. 2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1!4)-
2,3-di-O-acetyl-6-O-benzoyl-b-D-glucopyranosyl-(1!6)-
2,3,4-tri-O-acetyl-b-D-glucopyranosyl trichloroacet-
imidate (4)
To a solution of 16 (1.10 g, 0.90 mmol) in 4:1 CH3CN–
H2O (v/v, 20 mL) was added CAN (1.41 g, 2.7 mmol),
and the mixture was stirred at rt for 30 min, at the end
of which time TLC (1:1 petroleum ether–EtOAc) indi-
cated that the reaction was complete. The mixture was
extracted with EtOAc, and the extract was washed with
water, dried over Na2SO4 and concentrated under
reduced pressure. The crude product was purified by
3.8. Isopropyl 3,4-di-O-benzoyl-1-thio-b-D-xylopyran-
oside (24)
To a solution of Me2CHSH (1.2 mL, 11.02 mmol) and
TMSOTf (181 lL, 1 mmol) in anhyd CH2Cl2 (25 mL)
was slowly added a solution of compound 22 (3.726 g,
9 mmol) in anhyd CH2Cl2 (10 mL) under an N2
atmosphere at 0 ꢁC. The mixture was stirred under these

S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
467
25
1
conditions for 25 min, at the end of which time TLC (2:1
petroleum ether–EtOAc) indicated that all starting
materials were consumed. The reaction mixture was
concentrated and dissolved in 1:1 CH2Cl2–MeOH
(100 mL) co-solvent containing 5% HCl. The mixture
was stirred at rt for 16 h, evaporated under reduced
pressure at rt for 5 min, and then concentrated to dry-
ness at 45 ꢁC. Column chromatography (4:1 petroleum
ether–EtOAc) of the residue gave 24 as a syrup
½aꢁD +113 (c 1, CHCl3); H NMR (400 MHz, CDCl3):
d 0.35, 0.82, 0.88, 0.90, 0.91, 1.11, 1.29, 1.31 (8s,
8 · 3H, 8CH3), 2.83 (dd, 1H, J 4.1, 13.0 Hz, H-18 of
oleanolic acid), 3.25 (dd, 1H, J 4.4, 11.6 Hz, H-3 of
oleanolic acid), 3.66 (dd, 1H, J 7.1, 11.9 Hz, H-5aI),
4.12 (t, 1H, J 7.5 Hz, H-2I), 4.40 (dd, 1H, J 4.2,
11.9 Hz, H-5bI), 4.49 (dd, 1H, J 6.2, 9.9 Hz, H-5II),
4.87 (d, 1H, J 7.5 Hz, H-1I), 5.23–5.34 (m, 2H, H-4I,
H-12 of oleanolic acid), 5.34 (d, 1H, J 1.6 Hz, H-1II),
5.57 (dd, 1H, J 1.6, 3.5 Hz, H-2II), 5.60 (t, 1H, J
10.0 Hz, H-4II), 5.70 (t, 1H, J 7.5 Hz, H-3I), 5.81 (dd,
1H, J 3.5, 10.0 Hz, H-3II), 7.21–8.04 (m, 40H, 8Ph).
Anal. Calcd for C95H100O16: C, 76.18; H, 6.73. Found:
C, 75.87; H, 6.61.
25
(2.27 g, 55% for two steps): ½aꢁD +111 (c 1, CHCl3);
1H NMR (400 MHz, CDCl3): d 2.04 (br s, 1H, OH),
1.36–1.39 (2d, 6H, J 1.2 Hz, CH(CH3)2), 3.21–3.28 (m,
1H, CH(CH3)2), 3.55 (dd, 1H, J 9.4, 11.5 Hz, H-5a),
3.77 (t, 1H, J 8.4 Hz, H-2), 4.42 (dd, 1H, J 5.1,
11.4 Hz, H-5e), 4.65 (d, 1H, H-1, J 8.8 Hz), 5.29–5.35
(m, 1H, H-4), 5.58 (t, 1H, J 8.6 Hz, H-3), 7.37–8.05
(m, 10H, 2Ph). Anal. Calcd for C22H24O6S: C, 63.44;
H, 5.81. Found: C, 63.09; H, 5.75.
3.11. 3-O-[2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl-
(1!2)-3,4-di-O-benzoyl-b-D-xylopyranosyl]oleanolic
acid (27)
3.9. 3-O-[3,4-Di-O-benzoyl-b-D-xylopyranosyl]oleanolic
acid 28-O-trityl ester (25)
Compound 26 (800 mg, 0.53 mmol) was dissolved in
80% aq acetic acid (10 mL). The mixture was stirred at
70 ꢁC for 1 h and then co-evaporated with toluene to
dryness under reduced pressure. The residue was puri-
fied by silica gel column chromatography (2:1 petroleum
ether–EtOAc) to give compound 27 as a foamy solid
To a mixture of compounds 24 (800 mg, 1.92 mmol) and
3 (1.10 g, 1.57 mmol) in anhyd CH2Cl2 (30 mL) was
added NIS (440 mg, 1.96 mmol) and TMSOTf (36 lL,
0.20 mmol) under an N2 atmosphere at ꢀ42 ꢁC. The
mixture was stirred under these conditions for 1.5 h,
quenched by Et3N, diluted with CH2Cl2 (20 mL), and
washed with aq Na2S2O3. The organic layer was dried
over Na2SO4 and concentrated. The residue was purified
by silica gel column chromatography (7:1 petroleum
ether–EtOAc) to give compound 25 as a white foamy
25
(616 mg, 92%): ½aꢁD +72 (c 1, CHCl3); 1H NMR
(400 MHz, CDCl3): d 0.35, 0.82, 0.88, 0.90, 0.91, 1.11,
1.29, 1.31 (8s, 8 · 3H, 8CH3), 2.83 (dd, 1H, J 4.1,
13.0 Hz, H-18 of oleanolic acid), 3.25 (dd, 1H, J 4.4,
11.6 Hz, H-3 of oleanolic acid), 3.66 (dd, 1H, J 6.9,
12.0 Hz, H-5aI), 4.12 (t, 1H, J 7.1 Hz, H-2I), 4.40 (dd,
1H, J 4.2, 12.0 Hz, H-5bI), 4.49 (m, 1H, H-5II), 4.87
(d, 1H, J 7.1 Hz, H-1I), 5.20–5.25 (m, 1H, H-4I), 5.29
(t, 1H, J 3.2 Hz, H-12 of oleanolic acid), 5.34 (d, 1H,
J 1.6 Hz, H-1II), 5.57 (dd, 1H, J 1.6, 3.5 Hz, H-2II),
5.60 (t, 1H, J 10.0 Hz, H-4II), 5.70 (t, 1H, J 7.1 Hz,
H-3I), 5.82 (dd, 1H, J 3.5, 10.0 Hz, H-3II), 7.21–8.04
(m, 25H, 5Ph). Anal. Calcd for C76H86O16: C, 72.71;
H, 6.90. Found: C, 72.46; H, 6.99.
25
1
solid (1.14 g, 70%): ½aꢁD +40 (c 1, CHCl3); H NMR
(400 MHz, CDCl3): d 0.33, 0.72, 0.80, 0.87, 0.89, 0.99,
1.10 (7s, 7 · 3H, 7CH3), 2.48 (br d, 1H, J 3.1 Hz,
OH), 2.84 (dd, 1H, J 4.1, 13.0 Hz, H-18), 3.20 (dd,
1H, J 4.6, 11.5 Hz, H-3 of oleanolic acid), 3.49 (dd,
1H, J 9.3, 11.6 Hz, H-5a), 3.78–3.83 (m, 1H, H-2),
4.31 (dd, 1H, J 5.2, 11.6 Hz, H-5b), 4.55 (d, 1H, J
6.9 Hz, H-1), 5.10 (t, 1H, J 3.2 Hz, H-12 of oleanolic
acid), 5.27–5.32 (m, 1H, H-4), 5.57 (t, 1H, J 8.9 Hz,
H-3), 7.20–8.01 (m, 25H, 5Ph). Anal. Calcd for
C68H78O9: C, 78.58; H, 7.56. Found: C, 78.23; H, 7.41.
3.12. 3-O-[2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl-
(1!2)-3,4-di-O-benzoyl-b-D-xylopyranosyl]oleanolic acid
28-O-[2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1!4)-
2,3-di-O-acetyl-6-O-benzoyl-b-D-glucopyranosyl-(1!6)-
2,3,4-tri-O-acetyl-b-D-glucopyranosyl] ester (28)
3.10. 3-O-[2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl-
(1!2)-3,4-di-O-benzoyl-b-D-xylopyranosyl]oleanolic acid
28-O-trityl ester (26)
To a mixture of compounds 27 (600 mg, 0.48 mmol) and
trisaccharide 4 (730 mg, 0.58 mmol) in anhyd CH2Cl2
(15 mL) was added TMSOTf (11 lL, 0.06 mmol) under
an N2 atmosphere at 0 ꢁC. The mixture was stirred
under these conditions for 30 min, quenched by Et3N,
and concentrated. The residue was purified by silica
gel column chromatography (3:2 petroleum ether–
EtOAc) to give compound 28 as a white foamy solid
To a mixture of compounds 6 (574 mg, 0.92 mmol) and
25 (800 mg, 0.77 mmol) in anhyd CH2Cl2 (12 mL) was
added TMSOTf (18 lL, 0.1 mmol) under an N2 atmo-
sphere at ꢀ42 ꢁC. The mixture was stirred under these
conditions for 40 min, quenched by Et3N, and concen-
trated. The residue was purified by silica gel column
chromatography (6:1 petroleum ether–EtOAc) to give
compound 26 as a white foamy solid (865 mg, 75%):
25
(880 mg, 78%): ½aꢁD +81 (c 2, CHCl3); 1H NMR
(400 MHz, CDCl3): d 0.76, 0.85, 0.89, 0.91, 0.95, 1.12,

468
S. Cheng et al. / Carbohydrate Research 343 (2008) 462–469
1.13, 1.27, 1.29 (9s, 9 · 3H, 9CH3), 2.00, 2.02, 2.03, 2.07,
2.08 (5s, 5 · 3H, 5Ac), 2.81 (dd, 1H, J 4.1, 13.0 Hz, H-18
of oleanolic acid), 3.10 (br s, 1H), 3.26 (dd, 1H, J 4.3,
11.3 Hz, H-3 of oleanolic acid), 3.67–3.58 (m, 2H),
3.90–3.79 (m, 3H), 4.18–4.04 (m, 3H), 4.41 (dd, 1H, J
4.2, 11.9 Hz, H-5IV), 4.54–4.45 (m, 1H, H-5III), 4.65–
4.61 (m, 2H), 5.00–4.87 (m, 4H), 5.25–5.13 (m, 4H),
5.34–5.29 (m, 3H), 5.63–5.51 (m, 4H, H-2III, H-2V,
H-4I, H-4V), 5.70–5.65 (m, 2H, H-3IV, H-4III), 5.73
(dd, 1H, J 3.2, 10.2 Hz, H-3V), 5.82 (dd, 1H, J 3.5,
10.1 Hz, H-3III), 7.23–8.20 (m, 45H, 9Ph). 13C NMR
(100 MHz, CDCl3): d 175.2, 170.16, 170.14, 169.9,
169.3, 168.8, 165.8, 165.79, 165.70, 165.48, 165.42,
165.36, 165.23, 165.06, 164.93, 142.0, 133.4, 133.36,
133.26, 133.226, 133.10, 133.00, 132.9, 132.8, 129.88,
129.85, 129.79, 129.73, 129.70, 129.61, 129.54, 129.27,
129.24, 129.22, 129.18, 129.08, 128.51, 128.42, 128.39,
128.36, 128.33, 128.24, 128.17, 128.14, 103.5, 100.1,
98.5, 97.5, 92.1, 89.5, 77.22, 74.14, 73.9, 73.8, 72.96,
72.91, 72.14, 71.87, 71.80, 71.36, 71.10, 70.52, 70.01,
69.60, 69.36, 68.79, 68.04, 67.56, 67.24, 62.31, 61.16,
55.67, 47.64, 46.70, 45.81, 41.74, 41.05, 39.31, 39.29,
39.21, 38.77, 36.77, 33.74, 32.94, 31.65, 30.54, 29.66,
28.02, 27.86, 26.01, 25.56, 23.47, 23.40, 22.87, 22.59,
21.09, 20.65, 20.63, 20.58, 20.55, 20.54, 18.14, 17.41,
17.00, 16.54, 15.50. Anal. Calcd for C132H142O39: C,
67.39; H, 6.08. Found: C, 67.71; H, 6.15. MALDI-
TOF-MS: calcd for C132H142O39: 2351 [M]+; found,
2373.8 [M+Na]+.
38.9, 26.8, 39.5, 56.0, 18.5, 18.6, 33.0, 39.8, 48.0, 36.9,
23.7, 42.0, 28.2, 23.3, 46.9, 41.6, 46.1, 30.7, 33.9, 32.4,
27.9, 17.0, 15.6, 17.4, 26.0, 23.6. MALDITOF-MS:
calcd for C59H96O25: 1204 [M]+; found, 1226.6
[M+Na]+.
Acknowledgement
This work was supported in partial by NNSF of China
(Projects 20621703 and 20572128).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.11.015.
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afford 1 as an amorphous solid (197 mg, 96%): ½aꢁD
+8.6 (c 1, MeOH); 1H NMR (400 MHz, C6D5N): d
0.87 (m, 9H, 3CH3), 1.08 (s, 3H, CH3), 1.18 (s, 3H,
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