Synthesis and antitumor activities of glucan derivatives

Article abstract of DOI:10.1016/j.tet.2004.05.086

A highly efficient and practical method for the preparation of β-D-Glc-(1→6)-[β-D-Glc-(1→3)]-β-D-Glc-(1→6) -β-D-Glc-(1→6)-[β-D-Glc-(1→3)]-D-Glc-OMe was described. A dendritic nonasaccharide was also synthesized. The antitumor activities of hexasaccharide, the dendrimer, their sulfated derivatives, together with the natural glucan-protein and the corresponding polysaccharide isolated from barmy mycelium of Grifola frondosa, were preliminarily investigated based on Sarcoma-180 studies in mice tests. Our results suggest that the sulfated branching oligosaccharide and natural glycoprotein have better antitumor activities comparing to the parent sugar residue (oligosaccharide or polysaccharide).

Full text of DOI:10.1016/j.tet.2004.05.086

Tetrahedron 60 (2004) 6345–6351
Synthesis and antitumor activities of glucan derivatives
a
a
a
b
c
Yuguo Du,a Guofeng Gu, Yuxia Hua, Guohua Wei, Xinshan Ye and Guangli Yu
,
*
aResearch Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
bThe State Key Laboratory of Natural and Biomimetic Drug, School of Pharmaceutical Science, Peking University, Beijing 100083, China
cInstitute of Marine Drug and Food, Ocean University of China, Qingdao 266003, China
Received 23 February 2004; revised 12 May 2004; accepted 14 May 2004
Available online 17 June 2004
Abstract—A highly efficient and practical method for the preparation of b-D-Glc-(1!6)-[b-D-Glc-(1!3)]-b-D-Glc-(1!6)-b-D-Glc-(1!6)-
[b-D-Glc-(1!3)]-D-Glc-OMe was described. A dendritic nonasaccharide was also synthesized. The antitumor activities of hexasaccharide,
the dendrimer, their sulfated derivatives, together with the natural glucan–protein and the corresponding polysaccharide isolated from barmy
mycelium of Grifola frondosa, were preliminarily investigated based on Sarcoma-180 studies in mice tests. Our results suggest that the
sulfated branching oligosaccharide and natural glycoprotein have better antitumor activities comparing to the parent sugar residue
(oligosaccharide or polysaccharide).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
cluster compound containing three b-D-glucopyranosyl-
(1!6)-[b-D-glucopyranosyl-(1!3)]-a-D-glucopyranoside
components. Interestingly, the hexa-b-D-glucoside, b-D-
Glc-(1!6)-[b-D-Glc-(1!3)]-b-D-Glc-(1!6)-b-D-Glc-
(1!6)-[b-D-Glc-(1!3)]-D-Glc, has been well characterized
as an elicitor of plant phytoalexin accumulation.10 Our
research revealed that the sulfated hexa-b-D-glucoside may
also be a potent antitumor agent based on Sarcoma-180
model studies of mice tests.
A family of glucans containing a main chain of b-D-(1!3)-
glucopyranosyl units, and a short b-D-glucopyranosyl side
chains at O-6 have received considerable attention because
of their antitumor activities (immunomodulating action).1
Schizophyllan,2 scleroglucan,3 epiglucan4 and lentinan5 are
the most well-known members of this group of poly-
saccharides. It is known that the immunopharmacological
activities of soluble (1!3)-b-D-glucans are closely related
to the organization of the (1!3)-b-linked backbone into a
triple helix, the frequency and the complexity of side-
branching, and their molecular weight.6 However, Tsuzuki
and co-workers7 have also found that the conformation of b-
glucans, either single or triple helix, is independent on the
hematopoietic response. To investigate the structure–
activity relationship, we have synthesized a series of b-D-
glucosyl oligosaccharides to mimic the repeating units of
natural b-glucan chains.8 The mice tests revealed that our
previously synthesized b-D-glucopyranosyl oligosacchar-
ides showing weaker antitumor activities compared to the
reported natural polysaccharides. A literature survey
suggested that sulfation of the oligosaccharides could result
an increasing anti-tumor and anti-HIV activities.9 Here, we
would like to report the synthesis of sulfated methyl b-D-
glucopyranosyl-(1!6)-[b-D-glucopyranosyl-(1!3)]-b-D-
gluco pyranosyl-(1!6)-b-D-glucopyranosyl-(1!6)-[b-D-
glucopyranosyl-(1!3)]-a-D-glucopyranoside and a sulfated
2. Results and discussion
Hexa-b-D-glucopyranosides (compounds 1 and 2, Fig. 1)
have been previously prepared by Takahashi11 and
Ogawa.12 We here modified the synthesis based on our
findings of highly efficient and practical synthesis of 3,6-
branched oligosaccharides.13 Thus, phenyl 2,4-di-O-acetyl-
Keywords: Carbohydrates; Glycosylations; Antitumor agents;
Glycodendrimers; Oligosaccharides.
*
Corresponding author. Tel.: þ86-10-62914475; fax: þ86-10-62923563;
e-mail address: duyuguo@mail.rcees.ac.cn
Figure 1. Structures of hexa-b-D-glucopyranosides 1 and 2.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.05.086

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Y. Du et al. / Tetrahedron 60 (2004) 6345–6351
Scheme 1. Synthesis of hexa-b-D-glucopyranoside 1. Reaction conditions: (a) TMSOTf, CH2Cl2, 0 8C, 82%; (b) NIS, TMSOTf, 63% for 6; 86% for 10 (from
8); (c) TMSOTf, CH2Cl2, 242 8C; then TMSOTf, 0 8C, 76% (two steps); (d) 95% TFA; (e) NaOMe, MeOH, 93%; (f) SO3·Pyr, DMF.
1-thio-b-D-glucopyranoside (3)13a was condensed with
glycosyl donor 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl
trichloroacetimidate (4)14 in the presence of trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in CH2Cl2 to give
trisaccharide 5 in one pot with 82% isolated yield. Three
doublets at d 4.43 ppm (J¼7.9 Hz), 4.52 ppm (J¼8.0 Hz)
and 4.54 ppm (J¼10.0 Hz) in 1H NMR spectra of 5 clearly
indicated all three b-configuration in this trisaccharide.
Thioglycoside 5 was used as a latent glycosyl donor in the
final assembly of the target hexasaccahride. Attempt to
transfer the partially protected donor 3 into its methyl
glycoside derivative using N-iodosuccinimide (NIS) and
TMSOTf as catalysts resulted in however 6 as a major
product (63%). The formation of a isomer can be
rationalized by a SN2 reaction of methanol with
1,6-anhydrosugar intermediate formed from intermolecular
ring closure of 3 (Scheme 1).15
later, affording trisaccharide 8 in 76% yield within another
2 h. It is noteworthy that an extra amount of TMSOTf
(0.01 equiv.) was needed to complete the reaction after the
addition of 4. The treatment of 8 with 95% trifluoroacetic
acid (TFA) for 1 h gave trisaccharide acceptor 9. The
resulting crude product was co-evaporated with toluene
three times and then directly used for the next step without
further purification. Coupling of 5 and 9 in CH2Cl2 at 0 8C
under promotion of NIS and TMSOTf gave hexasaccharide
1
10 in 86% yield over two steps. H–1H COSY, TOCSY,
HMBC and HMQC spectra analyses clearly indicated 6
H-1s [dH 4.29 (H-1III), 4.49 (H-1II), 4.51 (H-1IV), 4.58
(H-1VI), 4.61 (H-1V), 4.77 (H-1I) ppm] and 6 C-1s [dC 96.4
(C-1I), 100.6 (C-1III, C-1V), 100.8 (C-1VI, C-1IV), 100.9
(C-1II) ppm], confirming the correct linkages of 10.
17
´
Standard Zemplen deacetylation of 10 furnished hexa-b-
D-glucopyranoside 1 as an amorphous solid. Sulfation of 1
with SO3·Pyr (10 equiv.) at 50 8C in N,N-dimethylforma-
mide (DMF) for 3 days, followed by conversion to the
sodium salt, removal of pyridine and purification on a
Sephadex LH-20 column, furnished a mixture of sulfated
11. The microanalysis for 11 was C 16.22%, H 1.73% and S
19.90%. This highly sulfated mixture was thus obtained in 5
steps at 38% overall yield starting from 3, and was directly
used for the following bioassay.
With 3,6-diol 6 in hand, we next applied a one-pot
sequential glycosylation to the synthesis of trisaccharide
acceptor 9. To this end, 6-O-silylated trichloroacetimidate
716 (1.1 equiv.) was regioselectively coupled with diol 6
using catalytic amount of TMSOTf (0.07 equiv.) at 242 8C
in anhydrous methylene chloride. The second donor 4
(1.5 equiv.) was added into the above mixture at 0 8C 2 h

Y. Du et al. / Tetrahedron 60 (2004) 6345–6351
6347
Scheme 2. Synthesis of nonasaccharide dendritic compound 17. Reaction conditions: (a) TMSOTf, CH2Cl2, 0 8C, 84.7%; (b) Pd(OH)2/C, H2, EtOAc–EtOH,
93.4%; (c) HOBt, DCC, DMF, rt, 57.2%; (d) NaOMe, MeOH, 91.5%; (e) SO3·Pyr, DMF.
Glycodendrimers have been prepared to give rise of new
kinds of glycoconjugate derivatives and polysaccharide
mimics.18 Some of them have shown highly improved
bioactivities compared to the monomers.19 Encouraged by
these results, we prepared a carbohydrate dendrimer based
on a combination of 3,6-branched trisaccharides as dendritic
components and noncarbohydrate units as trivalent cores
(Scheme 2). Thus, the coupling of trisaccharide imidate 128c
and 6-azido-1-hexanol under standard glycosylation con-
ditions gave 6-azidohexyl 2,3,4,6-tetra-O-benzoyl-b-D-
glucopyranosyl-(1!6)- [2,3,4,6-tetra-O-benzoyl-b-D-glu-
copyranosyl-(1!3)]-2,4-di-O-acetyl-b-D-glucopyranoside
(13), a key component for our target synthesis, in high yield.
Pd(OH)2 catalyzed hydrogenation of 13 gave amine
derivative 14, which was further condensed with triacid
1520 in the presence of HOBt and DCC in DMF to give fully
protected trimer 16 in 57.2% yield. The newly formed
amide bond was characterized by CONH peaks appearing at
d 6.04 ppm (3H, J¼5.5 Hz) in 1H NMR spectrum, and
further confirmed with a mass of 4804 (MþNa)þ of
MALDITOF-MS spectrum. Deacylation of 16 with 1 N
NaOMe afforded free nonasaccharide dendritic compound
17. Further sulfation of 17 with SO3·Pyr (10 equiv.) in DMF
as described in the preparation of 11, furnished the desired
dendrimer 18. Sodium salt of 18, after purification on LH-20
column, was directly used for the next bioassay.
Working for the same project to investigate possible
antitumor b-glucan, we have also extracted and isolated a
glucan protein (19) from barmy mycelium of Grifola
frondosa (Maitake) with a molecular weight of 95 K. After
removal of the protein (accounts for 24% of total molecular
weight), a pure polysaccharide (20) was obtained. The
structure of this polysaccharide is determined as a b-D-
glucan with the following basic repeating unit (Fig. 2) by a
NaIO4 oxidation, methylation, acetolysis and 2D NMR
spectra analysis.21
The antitumor activities of compounds 1, 11, 17, 18, 19 and
20 were preliminarily studied according to the method
described by Sasaki and co-workers.5b ICR mice weighing
about 20 g were used for the bioassay. Seven-day-old
Sarcoma-180 ascites (0.2 mL, about 5£106 cells) were
transplanted into the right groins of mice. The test samples,
dissolved in distilled water, were injected daily for 10 days
starting 24 h after tumor implantation. At the end of the 12th
day, the mice were killed, and the tumors were extirpated
and weighted. The results (Table 1), compared to lentinan
and cyclophosphamide (CTX) in the parallel test, suggest
that compound 11 and 19 may be potent antitumor agents.
Low tumor inhibition rates of 17 and 18 indicate that
structurally highly branched oligosaccharides may not be
helpful to their bioactivities. A main chain with b-(1!6)22

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Y. Du et al. / Tetrahedron 60 (2004) 6345–6351
Figure 2. Proposed structure of b-D-glucan isolated from barmy mycelium of Grifola frondosa (Maitake).
Table 1. Preliminary studies on antitumor activities of compounds 1, 11
and 17–20
4. Experimental
4.1. General methods
Sample
Dose
(mg/Kg) (g)
DBody weight Weight of tumor Inhibition rate
(%)
(g)
Optical rotations were determined at 25 8C with a Perkin–
Elmer Model 241-Mc automatic polarimeter. 1H NMR, 13
C
Control
CTX
Lentinan
1
11
11
11
17
18
19
0
30
10.4^2.3
1.42^0.45
0
NMR and 1H–1H COSY, NOESY and 1H–13C COSY
spectra were recorded with Bruker ARX 400 or 500
spectrometers in CDCl3, CD3OD or D2O. Chemical shifts
are given in ppm downfield from internal Me4Si. Mass
spectra were measured using MALDITOF-MS with CCA as
matrix or recorded with a VG PLATFORM mass spectro-
meter using the ESI technique to introduce the sample.
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 a UV detector. General
column chromatography was conducted by elution of a
column (8£200 mm, 15£300 mm, 35£400 mm) of silica gel
(100–200 mesh) with EtOAc–petroleum ether (60–90 8C)
as the eluent, while the sulfated products were purified on
Sephadex LH-20 column using water as eluent. Solutions
were concentrated at ,60 8C under reduced pressure.
8.7^1.5
12.9^2.0
9.6^1.8
12.6^3.2
11.0^1.1
10.8^1.9
6.9^1.3
9.6^1.6
12.7^1.8
10.8^1.3
0.35^0.09***
0.95^0.56**
1.15^0.41**
0.88^0.46*
0.74^0.23**
0.58^0.15***
1.11^0.60*
1.09^0.63*
0.44^0.13***
0.80^0.22**
75
33
19
38
48
59
22
23
69
43
2.0
5.0
2.5
5.0
10.0
2.0
2.0
1.0
1.0
20
t-test: *p,0.05; **p,0.01; ***p,0.001.
or b-(1!3)8b,9b linkage can be important. More details
about the action mechanism for 11 and 19 are currently
under investigation by our collaborators.
4.1.1. Methyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!6)-[2,3,4,6-tetra-O- acetyl-b-D-glucopyranosyl-
(1!3)]-2,4-di-O-acetyl-a-D-glucopyranoside (5). To a
cooled solution (0 8C) of 3 (1.1 g, 3.1 mmol) and 4 (3.2 g,
6.5 mmol) in anhydrous CH2Cl2 (20 mL) was added
TMSOTf (50 mL, 0.28 mmol). The mixture was stirred at
these conditions for 4 h and quenched with Et3N. The
solvents were evaporated in vacuo and the residue was
purified on a silica gel column (petroleum ether–EtOAc,
1:1) to give latent trisaccharide donor 5 as a syrup (2.54 g,
82%); [a]2D5¼211 (c 1, CHCl3); 1H NMR (400 MHz,
CDCl3) d 1.96, 1.97, 2.00, 2.01, 2.02, 2.03, 2.10, 2.17, 2.21,
2.23 (10 s, 10£3H, COCH3), 3.57 (ddd, 1H, J¼5.5, 11.9,
8.8 Hz), 3.60–3.66 (m, 3H), 3.78 (t, 1H, J¼8.8 Hz), 4.02
(dd, 1H, J¼5.5, 11.9 Hz), 4.11–4.17 (m, 2H), 4.43 (d, 1H,
J¼7.9 Hz, H-1II), 4.52 (d, 1H, J¼8.0 Hz, H-1III), 4.54 (d,
1H, J¼10.0 Hz, H-1I), 4.57 (dd, 1H, J¼2.0, 11.9 Hz), 4.62
3. Conclusions
A highly efficient and practical method was described for
the preparation of 3,6-branched hexa-b-D-glucopyranosyl
derivatives. A dendritic nonasaccharide was also synthe-
sized. The antitumor activities of oligosaccharide 1,
dendrimer 17, their sulfated derivatives 11 and 18, together
with a natural glucan–protein 19 and the corresponding
glucan 20, isolated from barmy mycelium of Grifola
frondosa (Maitake), were preliminarily investigated in
vivo based on Sarcoma-180 model studies. Our current
research suggests that the sulfated branching oligosacchar-
ide and natural glycoprotein have better antitumor activities
comparing to the parent sugar residue alone (oligosacchar-
ide or polysaccharide). Beside on this result, some structural
closely related glycopeptides and BSA attached glyco-
conjugates are now under preparation in our lab.

Y. Du et al. / Tetrahedron 60 (2004) 6345–6351
6349
(dd, 1H, J¼3.3, 12.5 Hz), 4.72 (dd, 1H, J¼3.3, 12.6 Hz),
4.96 (dd, 1H, J¼10.0, 10.8 Hz, H-2I), 5.00–5.05 (m, 2H),
5.11–5.21 (m, 4H), 7.25–7.50 (m, 4H, Ph). Anal. Calcd for
C44H56O25S: C, 51.97; H, 5.55. Found: C, 52.20; H, 5.48.
3.91 (dd, 1H, J¼2.5, 10.5 Hz, H-6bII), 4.01 (dd, 1H, J¼2.5,
7.5 Hz, H-6aVI), 4.03 (dd, 1H, J¼2.0, 7.5 Hz, H-6aV), 4.06–
4.12 (m, 2H, H-6bV, H-3I), 4.24 (dd, 1H, J¼4.5, 12.0 Hz,
H-6aIV), 4.29 (d, 1H, J¼8.0 Hz, H-1III), 4.30 (dd, 1H,
J¼3.0, 7.5 Hz, H-6bVI), 4.34 (dd, 1H, J¼4.0, 12.0 Hz,
H-6bIV), 4.49 (d, 1H, J¼8.0 Hz, H-1II), 4.51 (d, 1H, J¼
8.0 Hz, H-1IV), 4.58 (d, 1H, J¼8.0 Hz, H-1VI), 4.61 (d, 1H,
J¼8.0 Hz, H-1V), 4.71 (t, 1H, J¼9.5 Hz, H-4III), 4.77 (d,
1H, J¼3.5 Hz, H-1I), 4.78–4.93 (m, 7H), 4.94 (dd, 1H,
J¼8.0, 9.5 Hz, H-2IV), 5.01 (t, 1H, J¼9.0 Hz, H-4VI), 5.02
(t, 1H, J¼9.5 Hz, H-4V), 5.03 (t, 1H, J¼9.5 Hz, H-4IV), 5.08
(t, 1H, J¼9.5 Hz, H-3VI), 5.10 (t, 1H, J¼9.0 Hz, H-3V), 5.15
(t, 1H, J¼9.5 Hz, H-3II), 5.18 (t, 1H, J¼9.5 Hz, H-3IV). dC
(125 MHz, CDCl3) 20.3, 20.5, 20.6, 20.7, 20.9, 61.7, 61.8,
67.4, 68.0, 68.1, 68.3, 68.4, 68.5, 68.6, 68.7, 68.9, 71.0,
71.1, 71.2, 71.6, 71.7, 71.9, 72.5, 72.6, 72.7, 73.0, 73.2,
76.0, 78.7, 96.4 (C-1I), 100.6 (C-1III, C-1V), 100.8
(C-1VI, C-1IV), 100.9 (C-1II), 168.9, 169.0, 169.3, 169.4,
169.6, 169.7, 169.8, 170.1, 170.3, 170.4, 170.5, 170.6.
MALDITOF-MS calcd for C75H102O50: 1802 [M]þ. Found
1825 [MþNa]þ. Anal. Calcd for C75H102O50: C, 49.95; H,
5.70. Found: C, 50.21; H, 5.77.H-4H
4.1.2. Methyl 6-O-tert-butyldimethylsilyl-2,3,4-tri-O-
acetyl-b-D-glucopyranosyl- (1!6)-[2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl-(1!3)]-2,4-di-O-acetyl-a-D-
glucopyranoside (8). To a cold solution (242 8C) of 6
(1.37 g, 4.91 mmol) and 7 (2.78 g, 4.93 mmol) in anhydrous
CH2Cl2 (20 mL) was added TMSOTf (60 mL, 0.33 mmol).
The mixture was stirred at this temperature (usually 2 h)
until all starting materials were consumed according to TLC
(petroleum ether/EtOAc 1/1), and then warmed to 0 8C.
Compound 4 (2.42 g, 4.93 mmol) in dry CH2Cl2 (5 mL) was
added into the above mixture dropwise at 0 8C, followed by
the addition of extra TMSOTf (10 mL, 0.05 mmol), and the
mixture was kept at these conditions for 2 h, then quenched
with Et3N. The solvents were evaporated in vacuo and the
residue was purified by silica gel column chromatography
(petroleum ether–EtOAc, 1:1) to give trisaccharide 8 as a
1
syrup (3.77 g, 76%); [a]2D5¼þ418 (c 1, CHCl3); H NMR
(400 MHz, CDCl3) d 0.03, 0.04 (2 s, 6H, (CH3)2Si), 0.88 (s,
9H, t-Bu), 1.97, 1.98, 1.99, 2.01, 2.02, 2.03, 2.07, 2.18 (8 s,
27H, 9 CH3CO), 3.38 (s, 3H, OCH3), 3.45 (dd, 1H, J¼6.8,
10.7 Hz, H-6aIII), 3.52 (ddd, 1H, J¼2.9, 4.7, 9.9 Hz, H-5I),
3.64 (ddd, 1H, J¼2.2, 9.4, 4.6 Hz, H-5II), 3.66–3.75 (m, 2H,
H-6I), 3.88 (ddd, 1H, J¼1.8, 6.8, 9.5 Hz, H-5III), 3.93 (dd,
1H, J¼1.8, 10.7 Hz, H-6bIII), 4.04 (dd, 1H, J¼2.2, 12.4 Hz,
H-6aII), 4.11 (t, 1H, J¼9.3 Hz, H-3I), 4.34 (dd, 1H, J¼4.6,
12.4 Hz, H-6bII), 4.49 (d, 1H, J¼8.0 Hz, H-1III), 4.65 (d,
1H, J¼8.1 Hz, H-1II), 4.80 (dd, 1H, J¼9.3, 9.9 Hz, H-4I),
4.81 (d, 1H, J¼3.7 Hz, H-1I), 4.83 (dd, 1H, J¼3, 7, 9.3 Hz,
H-2I), 4.88 (dd, 1H, J¼8.1, 9.3 Hz, H-2II), 4.97 (dd, 1H,
J¼8.0, 9.5 Hz, H-2III), 5.01 (t, 1H, J¼9.5 Hz, H-4III), 5.04
(t, 1H, J¼9.4 Hz, H-4II), 5.11 (t, 1H, J¼9.4 Hz, H-3II),
5.20 (t, 1H, J¼9.5 Hz, H-3III). MALDITOF-MS calcd for
C43H66O25Si: 1010 [M]þ. Found 1033 [MþNa]þ. Anal.
Calcd for C43H66O25Si: C, 51.08; H, 6.58. Found: C, 51.27;
H, 6.52.
4.1.4. Methyl b-D-glucopyranosyl-(1!6)-[b-D-gluco-
pyranosyl-(1!3)]-b-D- glucopyranosyl-(1!6)-b-D-
glucopyranosyl-(1!6)-[b-D-glucopyranosyl-(1!3)]-a-
D-glucopyranoside (1). A solution of 10 (2.6 g, 1.44 mmol)
in ammonia–saturated MeOH (300 mL) was stirred at rt for
7 days. The solvents were evaporated, and the residue was
purified on a Sephadex LH-20 column with water as the
eluent to give 1 as an amorphous solid after lyophilization
1
(1.3 g, 90%); [a]2D5¼þ7 (c 1, H2O); H NMR (500 MHz,
D2O) d 3.28 (t, 1H, J¼9.1 Hz,), 3.30 (t, 1H, J¼9.1 Hz), 3.34
(t, 1H, J¼9.30 Hz), 3.37 (t, 1H, J¼9.5 Hz), 3.38–3.48 (m,
13H), 3.51 (t, 2H, J¼9.5 Hz), 3.58–3.93 (m 18H), 4.15–
4.23 (m, 2H, H-3I, H-3III), 4.50 (d, 2H, J¼7.9 Hz), 4.55 (d,
1H, J¼8.0 Hz), 4.69 (d, 1H, J¼8.0 Hz), 4.73 (d, 1H, J¼
8.0 Hz), 4.80 (d, 1H, J¼3.7 Hz). dC (125 MHz, CDCl3)
55.0, 60.5, 67.6, 67.7, 68.4, 68.6, 69.2, 69.3, 69.4, 70.2,
70.5, 72.6, 72.9, 73.2, 74.4, 74.6, 75.3, 75.4, 75.7, 75.8,
82.0, 84.0, 99.0, 102.5, 102.6, 102.7 (3C). ESI-MS calcd for
C37H64O31: 1004 [M]þ, found 1003 [M2H]þ.
4.1.3. Methyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!6)-[2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)]-2,4-di-O-acetyl-b-D-glucopyranosyl-(1!6)-2,3,4-
tri-O-acetyl-b-D-glucopyranosyl-(1!6)-[2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl-(1!3)]-2,4-di-O-acetyl-a-D-
glucopyranoside (10). Compound 8 (4.55 g, 4.5 mmol) was
stirred in 95% TFA (30 mL) at rt for 1 h and then evaporated
with toluene (3£50 mL) for 3 times to give the dried crude
9. To a cooled solution (0 8C) of 5 (4.576 g, 4.5 mmol) and
crude 9 (4.03 g, 4.5 mmol) in anhydrous CH2Cl2 (50 mL)
was added TMSOTf (60 mL, 0.33 mmol). The mixture was
stirred at this temperature for 2 h, and then quenched with
Et3N. The solvents were evaporated in vacuo and the
residue was purified by silica gel column chromatography
(petroleum ether–EtOAc, 1.5:1) to give hexasaccharide 10
as a syrup (6.98 g, 86%); [a]2D5¼23 (c 4, CHCl3); 1H NMR
(500 MHz, CDCl3) d 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99,
2.00, 2.01, 2.03, 2.05, 2.06, 2.13, 2.15 (14 s, 57H, 19
CH3CO), 3.35 (s, 3H, OCH3), 3.42–3.48 (m, 2H, H-5I,
H-6aI), 3.50 (dd, 1H, J¼7.0, 10.5 Hz, H-6aII), 3.54–3.60
(m, 2H, H-5II, H-5III), 3.62–3.70 (m, 3H, H-5IV, H-5V,
H-5VI), 3.79–3.88 (m, 4H, H-3III, H-6bI, H-6aIII, H-6bIII),
4.1.5. 6-Azidohexyl 2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl-(1!6)-[2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl-(1!3)]-2,4-di-O-acetyl-b-D-glucopyranoside
(13). To a solution of 2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl-(1!6)-[2,3,4,6-tetra-O-benzoyl-b-D-glucopyrano-
syl-(1!3)]-2,4-di-O-acetyl-b-D-glucopyranosyl trichloro-
acetimidate (12, 586 mg, 0.4 mmol) and 6-azido-1-
hexanol (52 mg, 0.4 mmol) in anhydrous dichloromethane
(3 mL) was added TMSOTf (10 mL, 0.06 mmol) at 0 8C
under N2 protection. The reaction mixture was stirred for
2 h, at the end of which time TLC indicated the completion
of the reaction. The mixture was neutralized with Et3N,
concentrated and purified by flash chromatography using
2:1 petroleum ether–EtOAc as the eluent to give syrupy 13
1
(476 mg, 84.7%); [a]2D0¼2101 (c 0.5, CHCl3); H NMR
(400 Hz, CDCl3) d 1.06–1.15 (m, 4H, –CH2CH2–), 1.19–
1.26 (m, 2H, –CH2CH2–), 1.45–1.52 (m, 2H, –CH2CH2–),
1.86 (s, 3H, CH3CO), 1.91 (s, 3H, CH3CO), 2.95 (m, 1H,
OCH2), 3.21 (t, 2H, CH2N3), 3.34–3.38 (m, 1H, OCH2),
3.49–3.51 (m, 1H, H-5I), 3.62 (dd, 1H, J6a,6b¼11.2 Hz,

6350
Y. Du et al. / Tetrahedron 60 (2004) 6345–6351
J6a,5¼5.7 Hz, H-6aI), 3.78–4.92 (m, 2H, H-3I, H-6bI),
4.04–4.16 (m, 3H, H-1I, H-5II, H-5III), 4.41–4.50 (m, 2H,
2H-6), 4.58–4.66 (m, 2H, 2H-6), 4.69–4.81 (m, 2H, H-2I
and H-4I), 4.89 (d, 1H, J1,2¼7.6 Hz, H-1), 4.91 (d, 1H,
J1,2¼7.8 Hz, H-1), 5.36 (dd, 1H, J2,3¼9.6 Hz, J1,2¼7.8 Hz,
H-2), 5.49 (dd, 1H, J2,3¼9.6 Hz, J1,2¼7.6 Hz, H-2), 5.60–
5.68 (m, 2H, H-4II and H-4III), 5.83–5.90 (m, 2H, H-3II and
H-3III), 7.28–8.04 (m, 40H, Ph). MALDITOF-MS calcd for
C84H79N3O26: 1545 [M]þ, found 1568 [MþNa]þ. Anal.
Calcd for C84H79N3O26: C, 65.24; H, 5.15. Found: C, 65.05;
H, 5.07.
3£2H, H-6), 4.70–4.79 (m, 3£2H, H-2I and H-4I), 4.90 (d,
3£1H, J1,2¼7.8 Hz, H-1), 4.92 (d, 3£1H, J1,2¼7.6 Hz, H-1),
5.36 (dd, 3£1H, J2,3¼9.6 Hz, J1,2¼7.8 Hz, H-2), 5.50 (dd,
3£1H, J2,3¼9.6 Hz, J1,2¼7.6 Hz, H-2), 5.60–5.68 (m,
3£2H, H-4II and H-4III), 5.84–5.91 (m, 3£2H, H-3II and
H-3III), 6.04 (br t, 3£1H, J¼5.5 Hz, NH), 7.25–8.02 (m,
3£40H, Ph). MALDITOF-MS calcd for C262H252N4O83:
4781 [M]þ, found: 4804 [MþNa]þ.
4.1.8. Free dendrimer 17. To a solution of 16 (196 mg,
0.04 mmol) in MeOH (5 mL) was added NaOMe until the
pH reached 10. The mixture was stirred at rt for 2 days,
neutralized with Amberlite IR-120 (Hþ). The solvents were
filtered, and the filtrate was concentrated to dryness under
reduced pressure. The residue was subjected to chroma-
tography on a Sephadex LH-20 column with MeOH as the
eluent to give 17 as a white solid (76 mg, 91.5%); [a]2D0¼25
(c 1, CH3OH); 1H NMR (400 Hz, CD3OD) d 1.19–1.36 (m,
3£4H, –CH2CH2–), 1.48–1.56 (m, 3£2H, –CH2CH2–),
1.58–1.66 (m, 3£2H, –CH2CH2–), 2.06–2.18 (m, 3£4H,
–CH2CH2–), 3.07 (t, 3£2H, CH2NHCO), 3.12 (dd, 3£1H,
J2,3¼9.6 Hz, J3,4¼10.2 Hz, H-3I), 3.14–3.60 (m, 3£16H),
3.71 (dd, 3£1H, J6a,6b¼11.5 Hz, J6a,5¼5.5 Hz, H-6), 3.76–
3.83 (m, 3£2H), 4.04–4.09 (m, 3£1H), 4.23 (d, 3£1H,
J1,2¼7.8 Hz, H-1), 4.31 (d, 3£1H, J1,2¼7.6 Hz, H-1), 4.47
(d, 3£1H, J1,2¼7.6 Hz, H-1); dC (100 Hz, CD3OD) 26.7,
30.2, 30.6, 31.2, 40.5 (CH2NHCO), 62.6, 62.8, 69.8, 70.0,
71.0, 71.60, 74.4, 75.1, 75.5, 76.8, 77.8, 78.0, 78.2, 94.4
(CNO2), 103.9 (C-1), 105.0 (C-1), 105.2 (C-1), 174.0
(NHCO). MALDITOF-MS Calcd for C82H144N4O53: 2032
[M]þ, found: 2055 [MþNa]þ; 2071 [MþK]þ.
4.1.6. 6-Aminohexyl 2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl-(1!6)-[2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl-(1!3)]-2,4-di-O-acetyl-b-D-glucopyranoside
(14). Compound 13 (431 mg, 0.278 mmol) was dissolved in
EtOAc–EtOH (1:1, 10 mL) containing Pd(OH)2/C (20%,
40 mg) at rt. H2 was bubbled into the mixture at the flow rate
of 100 mL/min while stirring at atmospheric pressure for
4 h. The mixture was then filtered over Celite and the filtrate
was concentrated under reduced pressure. Purification of the
residue by flash chromatography (CH2Cl2/MeOH: 5/1)
afforded 14 (394 mg, 93.4%) as a syrup; [a]2D0¼24 (c 1,
1
CHCl3); H NMR (400 Hz, CDCl3) d 1.03–1.14 (m, 4H,
–CH2CH2–), 1.17–1.24 (m, 2H, –CH2CH2–), 1.61–1.68
(m, 2H, –CH2CH2–), 1.88 (s, 3H, CH3CO), 1.89 (s, 3H,
CH3CO), 2.86–2.95 (m, 3H, CH2N3 and one proton of
OCH2), 3.34–3.37 (m, 1H, OCH2), 3.48–3.51 (m, 1H,
H-5I), 3.59 (dd, 1H, J6a,6b¼11.6 Hz, J6a,5¼5.7 Hz, H-6aI),
3.85–4.92 (m, 2H, H-3I, H-6bI), 4.06 (d, 1H, J1,2¼7.8 Hz,
H-1I), 4.07–4.16 (m, 2H, H-5II and H-5III), 4.44–4.48 (m,
2H, H-6), 4.58–4.65 (m, 2H, H-6), 4.71–4.76 (m, 2H, H-2I
and H-4I), 4.91 (d, 1H, J1,2¼7.8 Hz, H-1), 4.92 (d, 1H,
J1,2¼7.6 Hz, H-1), 5.36 (dd, 1H, J2,3¼9.6 Hz, J1,2¼7.8 Hz,
H-2), 5.50 (dd, 1H, J2,3¼9.6 Hz, J1,2¼7.6 Hz, H-2), 5.62–
5.69 (m, 2H, H-4II and H-4III), 5.85–5.91 (m, 2H, H-3II and
H-3III), 7.25–8.02 (m, 40H, Ph). MALDITOF-MS calcd for
C84H81NO26: 1519 [M]þ, found 1542 [MþNa]þ. Anal.
Calcd for C84H81NO26: C, 66.35; H, 5.37. Found: C, 66.21;
H, 5.45.
4.1.9. General procedure for sulfation of compounds 1
and 17. A mixture of oligosaccharide (1 or 17) and SO3·Pyr
(10 equiv.) in DMF was stirred at 50 8C for 3 days. Before
3 N NaOH was added, pyridine was removed in vacuo, and
the residue was purified on a Sephadex LH-20 column using
water as eluent furnished a mixture of sulfated compound
11 or 18 which were used for the next bioassay after freeze-
drying. Microanalysis data for compound 11: C 16.22%, H
1.73%, S 19.90%. Microanalysis for compound 18: C
21.01%, H 2.54%, S 17.79%.
4.1.7. Fully protected dendrimer 16. A mixture of
compound 14 (375 mg, 0.2 mmol), the triacid 15 (22 mg,
0.08 mmol) and HOBT (33 mg, 0.2 mmol) in dry DMF
(3 mL) was stirred at 0 8C for 0.5 h. Then DCC (51 mg,
0.248 mmol) was added and the reaction mixture was stirred
at 0 8C for 0.5 h, then at rt for 30 h. The mixture was filtered
and the filtrate was concentrated. The resulting crude
product was diluted in EtOAc (30 mL) and subsequently
washed successively with 5% HCl, saturated aqueous
NaHCO3 and water. The organic phase was concentrated
and purified by flash chromatography (petroleum ether–
EtOAc, 1:5) to complete 16 (219 mg, 57.2%) as a syrup;
[a]2D0¼26 (c 1, CHCl3); 1H NMR (400 Hz, CDCl3) d 1.04–
1.18 (m, 3£4H, –CH2CH2–), 1.29–1.38 (m, 3£2H,
–CH2CH2–), 1.58–1.72 (m, 3£2H, –CH2CH2–), 1.86 (s,
3£3H, CH3CO), 1.89 (s, 3£3H, CH3CO), 2.04–2.08 (m,
3£2H, CH2), 2.18–2.23 (m, 3£2H, CH2), 2.91–2.98 (m,
3£1H, OCH2), 3.06–3.13 (m, 3£2H, CH2NHCO), 3.34–
3.39 (m, 3£1H, OCH2), 3.47–3.51 (m, 3£1H, H-5I), 3.63
(dd, 3£1H, J6a,6b¼11.7 Hz, J6a,5¼6.8 Hz, H-6aI), 3.81–4.90
(m, 3£2H, H-3I and H-6bI), 4.05–4.15 (m, 3£3H, H-1I,
H-5II and H-5III), 4.42–4.49 (m, 3£2H, H-6), 4.59–4.65 (m,
Acknowledgements
This work was supported by National Basic Research
Program of China (2003CB415001) and NNSF of China
(Project 30330690, 20372081 and 30370345). The authors
would like to thank Professor Jingrong Cui of Peking
University and Dr. Hongyan Hu from General Hospital of
AP for performing the mice tests.
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