Synthesis of Calocybe indica var. APK2 polysaccharide repeating unit

Article abstract of DOI:10.1016/j.carres.2014.04.004

The first total synthesis of p-methoxyphenyl α-l-fucopyranosyl- (1→6)-α-d-galactopyranosyl-(1→4)-β-d-glucopyranosyl- (1→6)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (2) was achieved starting from five monosaccharide building blocks. This structure represents the repeating unit of the polysaccharide isolated from edible mushroom Calocybe indica var. APK2, and was synthesized in high overall yield via a convergent '3+2' glycosylation strategy.

Full text of DOI:10.1016/j.carres.2014.04.004

Carbohydrate Research 391 (2014) 43–47
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of Calocybe indica var. APK2 polysaccharide repeating unit
Lei Zhang a, Xiangming Zhu a,b,
⇑
a College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China
b Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of p-methoxyphenyl
glucopyranosyl-(1?6)-b-D-glucopyranosyl-(1?6)-b-D-glucopyranoside (2) was achieved starting from
five monosaccharide building blocks. This structure represents the repeating unit of the polysaccharide
isolated from edible mushroom Calocybe indica var. APK2, and was synthesized in high overall yield
via a convergent ‘3+2’ glycosylation strategy.
a
-
L
-fucopyranosyl-(1?6)-
a-D-galactopyranosyl-(1?4)-b-D-
Received 21 February 2014
Received in revised form 1 April 2014
Accepted 2 April 2014
Available online 12 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Mushroom polysaccharide
Repeating unit
Synthesis
Glycosidation
1. Introduction
2. Results and discussion
A water-soluble polysaccharide was isolated recently from a hot
aqueous extract of fruit bodies of edible mushroom Calocybe indica
var. APK2.1 The primary structure of the repeating unit of this poly-
The target pentasaccharide is relatively simple in structure, but
it contains two a-glycosidic linkages that are considered to be syn-
thetic challenges in carbohydrate chemistry.3 Our retrosynthetic
analysis revealed a convergent [3+2] approach to be attractive
(Scheme 1). Target structure 2 may be obtained from fully pro-
saccharide was identified as ?3)-[ -Fucp-(1?6)]-
a
-
L
a-D-Galp-
(1?4)-b- -Glcp-(1?6)-b- -Glcp-(1?6)-b-D-Glcp-(1?, that is, the
D
D
pentasaccharide structure 1 depicted in Figure 1. The subsequent
biological assay showed that this polysaccharide could inhibit
effectively human cervical cancer HeLa cell growth with IC50 being
tected pentasaccharide 3 through a three-step deprotection
sequence, including hydrolysis of the isopropylidene group,
hydrogenolysis, and cleavage of the ester groups. In turn, pentasac-
charide 3 will be assembled from trisaccharide donor 4 and disac-
1
100
l
g/mL, whereas no significant growth inhibition was found up to
l
g/mL against normal cells.1 Furthermore, this mushroom-
charide acceptor 5. Trisaccharide 4 can be derived from
derived polysaccharide exhibited strong immunostimulatory
activity by stimulating proliferation of splenocytes, thymocytes
and bone marrow cells;1 it may thus find use in immunotherapy
as well.
In view of the interesting anticancer and immunostimulatory
activities, investigations toward the synthesis of the pentasaccha-
ride repeating unit of the polysaccharide that may prove useful
in biological studies were initiated. We wish to report here a con-
vergent synthesis of the para-methoxyphenyl (MP) glycoside of
pentasaccharide 2 as part of our research program to identify
potent immunostimulants.2 The reducing end MP group could be
readily removed on demand, and would also facilitate the
purification and characterization of the final product.
thiofucoside 6,4 MP galactoside 7,5 and thioglucoside 8.6 The ben-
zyl protecting group at the 2-O-position of 6 and 7 will favor the
formation of the corresponding
a-glycosidic linkages by virtue of
kinetic anomeric effect. This effect was well elaborated in a recent
commentary article.7 Disaccharide 5 can be accessed from thiog-
lucoside 98 and MP glucoside 10.9 The 2-hydroxyl groups of 8
and 9 are protected with benzoyl and acetyl groups, respectively,
with a view to make use of neighboring group participation10 to
ensure the formation of the corresponding b-glycosidic bonds.
All the monosaccharide building blocks 6–10 that are needed
for the synthesis of pentasaccharide 2 are known compounds and
can be readily accessed following literature procedures. The
assembly of the pentasaccharide started with the coupling of donor
6 and acceptor 7. Thiofucoside 6 and galactoside 7 were dissolved
in CH2Cl2, and the mixture was treated with N-iodosuccinimide
(NIS)11 and trimethylsilyl triflate (TMSOTf) at À15 °C to produce
the desired disaccharide 11 in 85% yield (Scheme 2). Structural
assignments of 11 were made on the basis of NMR spectroscopy
⇑
Corresponding author. Tel.: +353 17162386; fax: +353 17162501.
E-mail address: Xiangming.Zhu@ucd.ie (X. Zhu).
http://dx.doi.org/10.1016/j.carres.2014.04.004
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

44
L. Zhang, X. Zhu / Carbohydrate Research 391 (2014) 43–47
acceptor 8 was then performed under normal Schmidt conditions12
OH
OH
O
to synthesize trisaccharide donor 4, but unfortunately, a significant
amount of byproduct formed by transfer of the phenylthio group
from 8 to 12 was isolated from the reaction. Aglycon transfer of
thioglycosides has been reported numerous times in the past
decades.13 When a glycosyl acceptor possessing a thioglycoside
aglycon is reacted with a glycosyl donor, the sulfide group can be
transferred from the acceptor to the donor. This is relatively com-
mon side reaction in glycosidation reactions involving thioglyco-
side as acceptor. We attributed this side reaction to the poor
nucleophilicity of the 4-OH group of acceptor 8, which rendered
glycosylation of anomeric thio group possible. To avoid the side
reaction, the inverse procedure14 that might boost the nucleophi-
licity of acceptor was applied to the glycosidation reaction. Hence,
acceptor 8 was premixed with catalytic amounts of TMSOTf in
CH2Cl2, and imidate 12 was then added slowly to the resulting
solution. As expected, this time the desired trisaccharide 4 was
produced smoothly as indicated by TLC and was isolated in 84%
yield. The anomeric stereochemistry of the galactose residue in 4
was confirmed by the chemical shift of the anomeric proton
and the coupling constant 3JH1–H2 value: 4.94 ppm and
3JH1–H2 = 3.5 Hz. The 13C chemical shift of the anomeric carbon of
OH
O
O
HO
HO
OH
HO
O
O
O
O
HO
HO
O
OH
HO
O
HO
OH
OR
HO
1
2
R = H
R = MP
OH
Figure 1. Structures of target pentasaccharide 1 and its MP glycoside 2.
O
O
2
O
OBn
O
O
BnO
BnO
OBz
BnO
O
O
O
O
BzO
AcO
AcO
O
BzO
O
3
AcO
AcO
OMP
AcO
O
OAc
[3+2]
O
the galactosidic bond was also in the expected region for
(97.6 ppm).
a anomer
O
OBn
O
OH
O
O
BnO
AcO
AcO
Meanwhile, synthesis of the disaccharide fragment 5 was also
carried out as shown in Scheme 2. Glycosylation of pre-prepared
acceptor 10 with thioglycoside donor 9 under the action of
NIS/TMSOTf smoothly generated the desired disaccharide 13 in
very high yield (86%), which was then subjected to standard desi-
lylation conditions (TBAF/HOAc/THF) to give 5 in 85% yield.
Having trisaccharide donor 4 and disaccharide acceptor 5 in
hand, attention was then focused on their coupling to assemble
the target molecule. Fortunately, activation of 4 with NIS/TMSOTf
in the presence of 5 led successfully to the desired pentasaccharide
3 in 86% yield (Scheme 3). Clearly, the complete b-stereoselectivity
in this glycosidation resulted from the assistance of the participat-
ing benzoyl group at the C-2 position of glucose moiety in donor 4.
Final deprotection of 3 to convert into the target molecule 2 was
achieved in three steps, as shown in Scheme 3: firstly, hydrolysis
of the isopropylidene group with 80% HOAc followed by acetyla-
tion; secondly, debenzylation by catalytic hydrogenation in the
presence of Pd(OH)2/C; finally, cleavage of the ester groups under
Zemplén conditions. The target molecule 2 was produced in 76%
yield over three steps. It was purified by size exclusion chromatog-
raphy on Bio-Gel P2 (eluant: H2O), and characterized by NMR and
HR-ESIMS.
O
+
O
AcO
AcO
OBz
BnO
OMP
AcO
BnO
O
5
OAc
O
SPh
BzO
4
OBz
OTBS
O
AcO
AcO
SPh
OBz
O
SEt
OBn
O
OAc
9
HO
BzO
O
SPh
O
OBz
6
OH
8
OH
O
BnO
BnO
AcO
AcO
O
OMP
OMP
OAc
10
OBn
7
Scheme 1. Retrosynthetic analysis of pentasaccharide 2.
O
O
O
O
O
O
a
OBn
O
c
OBn
O
6
7
+
+
O
BnO
BnO
O
BnO
BnO
X
OBz
O
BnO
BnO
Y
O
In summary, the first total synthesis of the repeating pentasac-
charide unit of a polysaccharide isolated from edible mushroom C.
indica var. APK2 was achieved in this report. An efficient [3+2]
SPh
BzO
11 X = OMP; Y = H
4
b
OBz
12 X = H; Y = OC(NH)CCl3
OR
O
a
AcO
O
9
10
AcO
O
AcO
AcO
OMP
O
AcO
O
OAc
O
13 R = TBS
d
OBn
5
R = H
O
BnO
BnO
a
O
4
5
+
Scheme 2. Synthesis of trisaccharide 4 and disaccharide 5. Reagents and condi-
tions: (a) NIS, TMSOTf, CH2Cl2, À15 °C, 85% for 11, 86% for 13; (b) (i) CAN, CH3CN,
H2O; (ii) CCl3CN, DBU, CH2Cl2, 90% (two steps); (c) 8, TMSOTf, CH2Cl2, inverse
procedure, À15 °C, 84%; and (d) TBAF, HOAc, THF, 85%.
OBz
BnO
O
O
O
O
BzO
AcO
O
BzO
AcO
O
AcO
AcO
3
OMP
AcO
OAc
and exact mass spectral data. The
a configuration of the newly
b,c,d
formed glycosidic bond was readily determined by the coupling
constant JH1–H2 value, which is about 3.6 Hz. Subsequently, 11
3
2
was converted into the corresponding trichloroacetimidate 12 in
two steps by deprotection with ceric ammonium nitrate (CAN) fol-
lowed by imidation with CCl3CN. The coupling of imidate 12 with
Scheme 3. Synthesis of target molecule 2. Reagents and conditions: (a) NIS,
TMSOTf, CH2Cl2, À25 °C, 86%; (b) (i) 80% HOAc; (ii) Ac2O, Py; (c) Pd(OH)2/C, EtOAc,
MeOH; and (d) NaOMe, MeOH, 76% (three steps).

L. Zhang, X. Zhu / Carbohydrate Research 391 (2014) 43–47
45
convergent strategy was applied to the synthesis, and all the glyco-
sidic bonds were introduced in highly stereoselective mode. In
view of the important bioactivities of the mushroom polysaccha-
ride, especially the immunostimulatory activity, the synthesized
pentasaccharide is of great interest for studying its biological prop-
erties. Work is in progress to test the immunological bioactivity of
the synthesized sample.
reaction mixture was diluted with EtOAc and H2O. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
in vacuo to give a residue, which was purified by flash column
chromatography (petroleum ether/EtOAc, 2:1) to give a yellowish
amorphous solid. To
1.37 mmol) in CH2Cl2 (10 mL) were added trichloroacetonitrile
(0.41 mL, 4.10 mmol) and DBU (41 L) at 0 °C. After stirring for
a solution of the above solid (1.00 g,
l
3 h, the reaction mixture was concentrated, and the residue was
purified by flash column chromatography (petroleum ether/EtOAc,
3. Experimental
3:1) to give 12 (1.07 g, 90%) as an amorphous white solid: [
a]D À46
(c 1.1, CHCl3); 1H NMR (600 MHz, CDCl3) d 8.58 (s, 1H), 7.40–7.29
(m, 20H), 6.61 (d, J = 3.4 Hz, 1H), 5.15 (d, J = 11.5 Hz, 1H), 4.82 (d,
J = 6.8 Hz, 1H), 4.80 (br s, 3H), 4.77 (d, J = 12.3 Hz, 1H), 4.74 (d,
J = 11.8 Hz, 1H), 4.71 (d, J = 12.4 Hz, 1H), 4.63 (d, J = 11.5 Hz, 1H),
4.31 (dd, J = 3.4, 10.0 Hz, 1H), 4.28–4.24 (m, 2H), 4.16 (br s, 1H),
4.08 (dd, J = 2.6, 10.1 Hz, 1H), 4.00 (dd, J = 2.4, 6.6 Hz, 1H), 3.95
(dd, J = 2.4, 5.5 Hz, 1H), 3.81 (dd, J = 7.9, 10.5 Hz, 1H), 3.61 (dd,
J = 5.8, 10.5 Hz, 1H), 3.53 (dd, J = 3.4, 7.7 Hz, 1H), 1.44 (s, 3H),
1.38 (s, 3H), 1.21 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) d
161.3, 138.7, 138.5, 138.4, 138.3, 128.4, 128.3, 128.2, 127.8,
127.6, 127.5, 127.4, 108.8, 97.5, 95.3, 91.5, 78.0, 76.0, 75.8, 75.7,
73.1, 73.0, 65.8, 63.4, 28.1, 26.3, 16.2; ESI-HRMS calcd for C45H50-
Cl3NNaO10 [M+Na]+ 892.2535, found 892.2539.
3.1. General methods
Reactions were performed in oven-dried glassware. Solvents
were evaporated under reduced pressure while maintaining the
water bath temperature below 50 °C. All reactions were monitored
by thin-layer chromatography (TLC) using silica gel 60 F254 and the
compounds visualized by UV or by treatment with 8% H2SO4 in
methanol followed by heating. Flash chromatography was per-
formed with the appropriate solvent system using 100–200 mesh
silica. Optical rotations were measured at 20 °C with a Perkin–El-
mer 241-MC polarimeter (1 dm cell). 1H NMR spectra were ob-
tained on a 400 or 600 MHz and reported in parts per million (d)
relative to the response of the solvent or to tetramethylsilane
(0.00 ppm). Coupling constants (J) are reported in Hertz (Hz). 13C
NMR spectra were recorded at 100 or 150 MHz and reported in d
relative to the response of the solvent. Yields refer to chromato-
graphically pure compounds and are calculated based on reagents
consumed.
3.4. Phenyl 2-O-benzyl-3,4-O-isopropylidene-
fucopyranosyl-(1?6)-2,3,4-tri-O-benzyl- -galactopyranosyl-
(1?4)-2,3,6-tri-O-benzoyl-1-thio-b- -glucopyranoside (4)
a-L-
a-D
D
A
suspension of
8 (168 mg, 0.29 mmol), TMSOTf (5.7 lL,
44
lmol), and 4 Å molecular sieves in CH2Cl2 (5 mL) was stirred
3.2. p-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-a-L-
at À15 °C for 20 min under nitrogen, and a solution of 12
(275 mg, 0.32 mmol) in CH2Cl2 (3 mL) was added dropwise to the
suspension. The resulting mixture was stirred until TLC indicated
complete consumption of acceptor 8, then quenched with Et3N
fucopyranosyl-(1?6)-2,3,4-tri-O-benzyl-b-D-galactopyranoside
(11)
To a mixture of 6 (0.76 g, 2.25 mmol) and 7 (1.44 g, 1.88 mmol)
in CH2Cl2 (14 mL) were added NIS (0.84 g, 3.75 mmol) and TMSOTf
(60
residue, which was purified by flash column chromatography
(petroleum ether/EtOAc, 2:1) to yield the title compound
(312 mg, 84%) as an amorphous white solid: [
À6.8 (c 0.9,
lL), filtered. The filtrate was concentrated in vacuo to give a
(40
at this temperature for 1.5 h, the reaction mixture was quenched
with Et3N (50 L) and diluted with CH2Cl2 (20 mL). The mixture
lL, 0.22 mmol) under N2 atmosphere at À15 °C. After stirring
4
a]
D
l
CHCl3); 1H NMR (600 MHz, CDCl3) d 8.04 (d, J = 7.1 Hz, 2H), 7.94
(d, J = 7.1 Hz, 2H), 7.91 (d, J = 7.1 Hz, 2H), 7.67 (t, J = 7.8 Hz, 1H),
7.47–7.02 (m, 33H), 5.85 (t, J = 9.3 Hz, 1H), 5.36 (t, J = 9.7 Hz, 1H),
5.02 (dd, J = 1.9, 12.2 Hz, 1H), 5.00 (d, J = 3.8 Hz, 1H), 4.92 (t,
J = 10.9 Hz, 2H), 4.96 (d, J = 3.5 Hz, 1H), 4.74 (s, 2H), 4.54 (s, 2H),
4.51 (dd, J = 6.0, 12.1 Hz, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.24–4.21
(m, 2H), 4.11 (dd, J = 6.0, 7.2 Hz, 1H), 4.07 (t, J = 9.6 Hz, 1H), 3.97
(d, J = 12.6 Hz, 1H), 3.94–3.92 (m, 2H), 3.91 (dd, J = 2.1, 6.5 Hz,
1H), 3.88–3.85 (m, 2H), 3.80 (dd, J = 3.5, 10.2 Hz, 1H), 3.60 (dd,
J = 7.2, 10.2 Hz, 1H), 3.48 (dd, J = 3.5, 7.7 Hz, 1H), 3.45 (dd, J = 6.5,
10.2 Hz, 1H), 1.36 (s, 3H), 1.28 (s, 3H), 1.15 (d, J = 6.6 Hz, 3H); 13C
NMR (150 MHz, CDCl3) d 166.0, 165.5, 165.3, 138.6, 138.5, 138.4,
138.2, 133.2, 133.0, 132.8, 132.2, 129.9, 129.8, 129.6, 129.3,
128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.5,
127.4, 108.8, 100.0, 97.6, 85.8, 78.8, 77.5, 76.0, 75.9, 75.8, 75.6,
75.1, 74.9, 74.5, 73.1, 73.0, 71.8, 70.8, 70.4, 66.8, 64.0, 63.2, 28.1,
26.3, 16.2; ESI-HRMS calcd for C76H76NaO17S [M+Na]+ 1315.4865,
found 1315.4869.
was then washed with aqueous Na2S2O3 (1 M, 10 mL), the organic
layer was dried over Na2SO4 and concentrated in vacuo to give a
residue, which was purified by flash column chromatography
(petroleum ether/EtOAc, 3:1) to give 11 (1.34 g, 85%) as a syrup:
[
a]
D
À36.6 (c 0.8, CHCl3); 1H NMR (600 MHz, CDCl3) d 7.37–7.21
(m, 19H), 7.22 (m, 1H), 7.05 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.9 Hz,
2H), 5.08 (d, J = 11.7 Hz, 1H), 5.01 (d, J = 10.8 Hz, 1H), 4.85 (d,
J = 10.8 Hz, 1H), 4.82 (d, J = 7.7 Hz, 1H), 4.76 (d, J = 11.8 Hz, 1H),
4.74 (d, J = 3.6 Hz, 1H), 4.69 (t, J = 11.8 Hz, 2H), 4.61 (d,
J = 12.9 Hz, 2H), 4.22 (dd, J = 5.5, 7.7 Hz, 1H), 4.08 (dd, J = 7.8,
9.6 Hz, 1H), 3.90 (d, J = 2.6 Hz, 1H), 3.88 (dd, J = 1.8, 6.6 Hz, 1H),
3.86 (dd, J = 2.4, 5.5 Hz, 1H), 3.70 (s, 3H), 3.68 (d, J = 2.1 Hz, 1H),
3.66 (s, 1H), 3.65 (d, J = 5.7 Hz, 1H), 3.58 (dd, J = 2.7, 9.8 Hz, 1H),
3.45 (dd, J = 3.3, 7.7 Hz, 1H), 1.37 (s, 3H), 1.32 (s, 3H), 1.13 (d,
J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) d 155.4, 151.7, 138.6,
138.5, 138.4, 138.3, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6,
127.5, 119.2, 114.4, 108.6, 103.6, 97.9, 82.2, 79.3, 76.2, 76.0, 75.7,
75.5, 74.4, 73.7, 73.2, 72.2, 67.2, 63.3, 55.6, 28.1, 26.3, 16.2; ESI-
HRMS calcd for C50H56NaO11 [M+Na]+ 855.3829, found 855.3823.
3.5. p-Methoxyphenyl 2,3,4-tri-O-acetyl-6-O-tert-
butyldimethylsilyl-b-
D-glucopyranosyl-(1?6)-2,3,4-tri-O-
3.3. 2-O-Benzyl-3,4-O-isopropylidene-
2,3,4-tri-O-benzyl-a-D-galactopyranosyl trichloroacetimidate
a
-
L
-fucopyranosyl-(1?6)-
acetyl-b- -glucopyranoside (13)
D
(12)
To
2.20 mmol) in CH2Cl2 (20 mL) were added NIS (1.04 g, 4.40 mmol)
and TMSOTf (39.8
L, 0.26 mmol) under N2 atmosphere at À15 °C.
The mixture was stirred at this temperature for 1 h, then quenched
with Et3N (50 L), diluted with CH2Cl2 (20 mL), and washed with
a solution of 9 (1.18 g, 2.31 mmol) and 10 (0.91 g,
To a solution of 11 (1.27 g, 1.53 mmol) in CH3CN (20 mL) and
H2O (5 mL) was added ammonium cerium nitrate (1.36 g,
2.48 mmol) at room temperature. After stirring for 1.5 h, the
l
l

46
L. Zhang, X. Zhu / Carbohydrate Research 391 (2014) 43–47
aqueous Na2S2O3 (1 M, 12 mL). The organic layer was dried over
Na2SO4 and concentrated to give a residue, which was purified
by flash column chromatography (petroleum ether/EtOAc, 3:2) to
4.92 (t, J = 7.6 Hz, 2H), 4.87 (t, J = 8.4 Hz, 1H), 4.82 (t, J = 10.8 Hz,
1H), 4.78 (dd, J = 3.6, 8.4 Hz, 2H), 4.75 (d, J = 4.7 Hz, 2H), 4.57 (s,
3H), 4.47 (d, J = 11.4 Hz, 1H), 4.42 (d, J = 7.9 Hz, 1H), 4.28 (d,
J = 12.4 Hz, 1H), 4.25 (dd, J = 7.4, 9.4 Hz, 1H), 4.22 (d, J = 9.0 Hz,
1H), 4.14 (t, J = 6.6 Hz, 1H), 4.01 (d, J = 12.4 Hz, 1H), 3.97 (s, 1H),
3.94–3.92 (m, 2H), 3.91–3.87 (m, 2H), 3.85 (d, J = 3.6 Hz,
1H), 3.82 (dd, J = 3.6, 9.6 Hz, 1H), 3.78 (s, 3H), 3.72 (d, J = 11.4 Hz,
1H), 3.65–3.61 (m, 3H), 3.58 (t, J = 7.2 Hz, 1H), 3.50 (dd, J = 3.3,
7.7 Hz, 1H), 3.46 (dd, J = 6.3, 10.1 Hz, 1H), 3.39 (dd, J = 6.5,
11.4 Hz, 1H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H),
1.96 (s, 3H), 1.91 (s, 3H), 1.38 (s, 3H), 1.31 (s, 3H), 1.16 (d,
J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) d 177.7, 170.3, 170.1,
169.6, 169.5, 169.4, 166.0, 165.5, 165.2, 155.7, 151.1, 138.6,
138.5, 138.3, 133.3, 133.1, 133.0, 129.8, 129.7, 129.4, 128.6,
128.4, 128.3, 128.2, 128.1, 127.9, 127.7, 127.4, 127.3, 118.4,
114.7, 108.8, 101.2, 100.2, 100.1, 100.0, 97.4, 78.8, 76.2, 76.0,
75.8, 75.7, 75.1, 74.9, 74.4, 74.2, 73.7, 73.2, 73.1, 72.9, 72.8, 72.0,
71.8, 70.2, 69.1, 68.7, 68.5, 67.7, 66.6, 63.7, 63.2, 55.6, 29.6, 28.1,
26.3, 20.7, 20.6, 20.5, 16.1; ESI-HRMS calcd for C101H110NaO35
[M+Na]+ 1905.6842, found 1905.6899.
give the disaccharide 13 (1.54 g, 86%) as a white foam: [a] +2.1
D
(c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3) d 6.91 (d, J = 4.5 Hz,
2H), 6.85 (d, J = 9.1 Hz, 2H), 5.23 (t, J = 7.8 Hz, 1H), 5.17 (dd,
J = 7.8, 9.6 Hz, 1H), 5.12 (t, J = 9.5 Hz, 1H), 5.02 (t, J = 9.5 Hz, 1H),
4.94–4.13 (m, 3H), 4.55 (d, J = 7.9 Hz, 1H), 3.85 (dd, J = 1.8,
11.3 Hz, 1H), 3.80 (dd, J = 1.8, 9.6 Hz, 1H), 3.76 (s, 3H), 3.67–3.63
(m, 3H), 3.43–3.41 (m, 1H), 2.03 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H),
1.98 (s, 3H), 1.97 (s, 3H), 1.84 (s, 3H), 0.85 (br s, 9H), 0.02 (s, 3H),
0.01 (s, 3H); 13C NMR (150 MHz, CDCl3) d 170.3, 170.2, 169.5,
169.4, 169.3, 169.2, 155.7, 151.1, 129.0, 128.2, 118.1, 114.8,
100.3, 99.9, 74.6, 73.8, 73.2, 72.7, 71.3, 71.2, 68.8, 68.6, 67.7,
62.1, 55.6, 25.8, 20.7, 20.6, 20.5, 18.3, À5.3; ESI-HRMS calcd for C37-
H54NaO18Si [M+Na]+ 837.3145, found 837.3149.
3.6. p-Methoxyphenyl 2,3,4-tri-O-acetyl-b-D-glucopyranosyl-
(1?6)-2,3,4-tri-O-acetyl-b- -glucopyranoside (5)
D
Disaccharide 13 (3.09 g, 3.80 mmol) was dissolved in dry THF
(40 mL) and cooled to 0 °C under N2. HOAc (1.87 mL) was added
dropwise followed by addition of a solution of TBAF in THF
(1.0 M, 4.54 mL, 4.54 mmol). The cooling bath was removed and
the reaction was allowed to warm to room temperature and stirred
overnight. Brine was then added and the mixture was extracted
several times with EtOAc. The combined organic phases were dried
over Na2SO4 and concentrated to give a residue, which was purified
by flash column chromatography (petroleum ether/EtOAc, 2:1) to
3.8. p-Methoxyphenyl
galactopyranosyl-(1?4)-b-
glucopyranosyl-(1?6)-b- -glucopyranoside (2)
a
-
L
-fucopyranosyl-(1?6)-
a-D-
D-glucopyranosyl-(1?6)-b-D-
D
A solution of pentasaccharide 3 (420 mg, 0.22 mmol) in 80%
AcOH (20 mL) was stirred at 80 °C for 5 h and then concentrated
in vacuo, and the traces of HOAc and water were removed by
coevaporation several times with toluene. The residue was treated
directly with Ac2O (2 mL) and pyridine (4 mL), and stirred at room
temperature for 5 h. The mixture was then concentrated, diluted
with EtOAc, washed successively with 5% HCl, saturated aqueous
NaHCO3 and brine, dried over Na2SO4, and concentrated to give a
white foam, which was directly used in the next step without puri-
fication. The foam was dissolved in MeOH/EtOAc (15 mL, 1:1 v/v).
Pd(OH)2/C (20%, 60 mg) was added and the suspension was stirred
under H2 atmosphere for 8 h, after which time the mixture was fil-
tered, and the filtrate was concentrated in vacuo to afford the
crude intermediate, the partially protected pentasaccharide. The
crude material was then dissolved in MeOH (20 mL) and treated
with a 1 M solution of NaOMe in MeOH until PH of the solution
reached 9–10. The mixture was stirred at room temperature for
10 h and then neutralized with Amberlite IR-120 resin. The mix-
ture was filtrated and concentrated, and the resulting residue
was purified by size exclusion chromatography on a Bio-Gel P2 col-
umn (15 mm  820 mm) using H2O as eluent. After lyophilization
with water, the target pentasaccharide 2 (134 mg, 76% over three
give the title compound 5 (2.26 g, 85%) as a syrup: [
a
]
À6.3 (c
D
1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d 6.95 (d, J = 6.0 Hz, 2H),
6.89 (d, J = 6.0 Hz, 2H), 5.28 (t, J = 6.3 Hz, 1H), 5.21 (dd, J = 6.3,
12.5 Hz, 2H), 5.03 (dd, J = 5.2, 11.6 Hz, 2H), 4.98 (d, J = 5.2 Hz,
2H), 4.64 (d, J = 5.3 Hz, 1H), 4.14 (dd, J = 4.7, 9.4 Hz, 1H), 3.91 (dd,
J = 1.6, 7.4 Hz, 1H), 3.81 (s, 3H), 3.76–3.73 (m, 2H), 3.58 (dd,
J = 3.2, 4.4 Hz, 1H), 3.45–3.43 (m, 1H), 2.54 (br s, 1H), 2.08–2.06
(m, 9H), 2.04 (s, 3H), 2.03 (s, 3H), 1.92 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 170.5, 170.1, 169.9, 169.3, 169.2, 155.5,
150.7, 118.6, 114.4, 100.1, 98.8, 73.6, 72.6, 71.8, 71.1, 70.3, 69.6,
69.0, 68.2, 65.3, 61.7, 55.4, 25.7, 20.9, 20.8, 20.6, 20.5, 18.2, 14.1;
ESI-HRMS calcd for
723.2198.
C
31H40O18Na [M+Na]+ 723.2212, found
3.7. p-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-
fucopyranosyl-(1?6)-2,3,4-tri-O-benzyl- -galactopyranosyl-
(1?4)-2,3,6-tri-O-benzoyl-b- -glucopyranosyl-(1?6)-2,3,4-tri-O
-acetyl-b- -glucopyranosyl-(1?6)-2,3,4-tri-O-acetyl-b-
glucopyranoside (3)
a-L-
a-D
D
D
D-
steps) was obtained as an amorphous solid: [
a
]
À142 (c 0.5,
D
H2O); H NMR (600 MHz, D2O) d 7.17 (d, J = 8.9 Hz, 2H), 7.01 (d,
J = 8.9 Hz, 2H), 5.07 (d, J = 4.6 Hz, 1H), 4.97 (d, J = 3.3 Hz, 1H),
4.50 (d, J = 7.8 Hz, 2H), 4.44 (d, J = 7.9 Hz, 1H), 4.20 (t, J = 10.8 Hz,
2H), 4.13 (t, J = 1.7 Hz, 2H), 3.98 (s, 1H), 3.93–3.90 (m, 4H), 3.84–
3.79 (m, 10H), 3.69 (dd, J = 2.2, 10.0 Hz, 1H), 3.65 (dd, J = 1.2,
6.0 Hz, 1H), 3.62 (d, J = 9.6 Hz, 1H), 3.58 (d, J = 7.9 Hz, 3H), 3.55
(d, J = 9.6 Hz, 2H), 3.50 (d, J = 4.2 Hz, 2H), 3.34 (d, J = 8.4 Hz, 1H),
3.31 (d, J = 9.1 Hz, 1H), 1.24 (d, J = 6.4 Hz, 3H); 13C NMR
(150 MHz, D2O) d 154.8, 150.8, 118.3, 115.1, 103.2, 102.8, 100.9,
99.3, 98.9, 83.1, 81.0, 79.3, 76.2, 75.5, 75.3, 74.5, 74.3, 73.7, 73.1,
72.9, 72.8, 72.6, 71.8, 70.7, 69.5, 68.6, 68.2, 67.4, 66.8, 60.1, 55.8,
15.3; ESI-HRMS calcd for C37H58O26Na [M+Na]+ 941.3264, found:
941.3083.
Donor
4
(243 mg, 0.19 mmol) and acceptor
5
(120 mg,
0.17 mmol) were dissolved in CH2Cl2 (5 mL) and cooled to À25 °C
under N2 atmosphere. NIS (77 mg, 0.34 mmol) was added to this
solution, followed by addition of TMSOTf (3.1
reaction mixture was stirred at this temperature for 1 h, then
quenched with Et3N (20 L), diluted with CH2Cl2 (10 mL), and
washed with aqueous Na2S2O3 (1 M, 5 mL). The organic layer was
dried over Na2SO4 and concentrated to give a residue, which was
purified by flash column chromatography (petroleum ether/EtOAc,
3:2) to give the pentasaccharide 3 (277 mg, 86%) as a white foam:
lL, 17 lmol). The
l
[a
]
À14.9 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3) d 8.07 (d,
D
J = 7.4 Hz, 2H), 7.95 (dd, J = 3.9, 7.4 Hz, 4H), 7.63–7.25 (m, 23H),
7.21 (d, J = 7.2 Hz, 2H), 7.17 (d, J = 7.4 Hz, 2H), 7.07 (d, J = 5.3 Hz,
2H), 6.95 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 5.88 (t,
J = 9.4 Hz, 1H), 5.41 (dd, J = 7.8, 9.6 Hz, 1H), 5.25 (t, J = 9.5 Hz,
1H), 5.19 (t, J = 7.9 Hz, 1H), 5.10 (d, J = 3.4 Hz, 1H), 5.06 (t,
J = 9.4 Hz, 1H), 5.00 (d, J = 11.0 Hz, 1H), 4.94 (d, J = 11.6 Hz, 1H),
Acknowledgements
This work was supported by the Natural Science Foundation of
Zhejiang Province (R4110195), the Educational Commission of

L. Zhang, X. Zhu / Carbohydrate Research 391 (2014) 43–47
47
O’Reilly, V.; Dunne, M. R.; Dere, R. T.; Zeng, S. G.; O’Brien, C.; Amu, S.; Fallon, P.
G.; Exley, M. A.; O’Farrelly, C.; Zhu, X.; Doherty, D. G. Clin. Immunol. 2011, 140,
196–207.
Zhejiang Province (Y201328123) and the Foundation of State Key
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.04
.004.
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