Synthesis of polyanionic glycopolymers for the facile assembly of glycosyl arrays

Article abstract of DOI:10.1016/j.tet.2005.03.102

Polyanionic glycopolymers were synthesized aiming at establishing a simple process for assembling glycosyl arrays. The synthetic glycopolymers carry the key carbohydrate epitopes of α-d-galactobioside (Gb₂), β-lactoside, and α-d-mannopyranoside

Full text of DOI:10.1016/j.tet.2005.03.102

Tetrahedron 61 (2005) 5895–5905
Synthesis of polyanionic glycopolymers for the facile assembly
of glycosyl arrays
a
a
b
b
Hirotaka Uzawa,a, Hiroki Ito, Masayuki Izumi, Hideo Tokuhisa, Kazuhiro Taguchi
*
and Norihiko Minouraa
aResearch Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST),
Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Japan
bNanoarchitectonics Research Center, National Institute of Advanced Industrial Science and Technology (AIST),
Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Japan
Received 3 March 2005; accepted 25 March 2005
Available online 10 May 2005
Abstract—Polyanionic glycopolymers were synthesized aiming at establishing a simple process for assembling glycosyl arrays. The
synthetic glycopolymers carry the key carbohydrate epitopes of a-D-galactobioside (Gb2), b-lactoside, and a-D-mannopyranoside, each of
which serves as a ligand of bacterial toxins and adhesion proteins. The Gb2 epitope, prepared from penta-O-acetyl-D-galactopyranose, was
coupled with poly(ethylene-alt-maleic anhydride) in a polymer reaction to afford a Gb2-embedded glycopolymer having also carboxylate
(COOK) polyanions at the side chain. The polyanionic glycopolymer was then applied to a preparation of sugar-coated gold electrodes,
which involves an alternating layer-by-layer adsorption based on electrostatic interactions. The presence of the Gb2-coat on the surface was
evidenced by Fourier transform infrared reflection absorption spectroscopy. The Gb2-coated glyco-chip was stable in 10 mM HEPES buffer
containing 150 mM NaCl aq. Other glycopolymers carrying the b-lactoside and a-D-mannopyranoside epitopes were applied to the same
assembling process. The derived glycosyl arrays will be useful for detecting Shiga toxins, other pathogenic toxins and viruses when applied
as glyco-chips for surface plasmon resonance or quartz crystal microbalance technique.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
formation of the self-assembled monolayers between the
thiolated compounds and gold surfaces.4
The cell-surface oligosaccharides bound to glycoproteins
and glycolipids are associated with various biological roles
on cell surfaces like cell-adhesion, signal transduction and
regulation as well as bacterial and viral infections.1
Therefore, much attention is paid to the species-specific
interactions involved in carbohydrate–protein and carbo-
hydrate–carbohydrate bindings. These interactions are
analyzed with artificial models assisted by different
techniques of quartz crystal microbalance (QCM), surface
plasmon resonance (SPR), Au-nanoparticles, and other
biological assays.2 For all of these analyses, the fabrication
of glycosyl arrays, chips, or nanoparticles is of crucial
significance.3 For such purpose, simple and convenient
immobilization of carbohydrates to these sensor substrates
is indispensable. Conventional immobilization processes
apply the natural affinity of biotin and streptavidin and the
Recently, Houseman et al. proposed a method for sugar
immobilization, which is based on the Diels–Alder coupling
reaction on gold surface between benzoquinone and
glycosyl cyclopentadiene.3a Wong et al. proposed 1,3-
dipolar cycloaddition between azido sugars and alkynes.3b,e
Park et al. attached sugars onto substrate surfaces by Michel
addition between maleimides and thiols.3f In these studies,
monomeric carbohydrate molecules are routinely utilized.
However, with an aim to establish a simple process for
assembling glycosyl arrays, the utility of glycopolymers has
not yet been fully explored,5 albeit glycomaterials consist-
ing of the glycopolymers can be generally expected to have
a multivalent and cluster effect.6 This effect can potentially
enhance binding interactions with the receptor proteins. We
describe herein the synthesis and convenient use of
polyanionic glycopolymers for a facile immobilization of
carbohydrates to Au-substrates. In this paper, an alternating
layer-by-layer adsorption technique was applied, which is
clearly distinct from the previous immobilization methods.
Keywords: Glycopolymers; Glycosyl arrays; Carbohydrate epitopes; Layer-
by-layer; Polyanions.
*
Corresponding author. Tel.: C81 29 861 4431; fax: C81 29 861 4680;
e-mail: h.uzawa@aist.go.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.102

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H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
As part of our ongoing project to assemble a library of various
sulfo sugars,7 we are interested in the artificial glycopolymers
carrying Gb3 and Gb2 in clusters, which are able to adsorb the
pathogenic Escherichia coli O-157 bacteria and their
produced Shiga toxins.8 In our preceding study, we applied
the Gb3-glycopolymers8 as well as alkyl Gb2-monolayers9 in
our QCM sensors targeting the Shiga toxins.
been accomplished in the literature and found to show
notable activity to block the Shiga toxins-host cell
infections.8 Most of the synthetic studies have focused on
the molecular assembly to construct the glycosyl clusters as
glycopolymers, glycodendrimers and starfish models.10
Moreover, substituted polyacrylate-based neoglycoconju-
gates are reported as versatile chemical tools for biochemi-
cal and medicinal applications.11 In the present approach,
we synthesized novel polyanionic glycopolymers to fabri-
cate glycosyl arrays based on the alternating layer-by-layer
adsorption method.
In the present study, we synthesized various polyanionic
glycopolymers involving galactobioside (Gb2) and other
biologically important sugar epitopes for the purpose of
simple, rapid and practical immobilization. These glyco-
polymers can be applicable to the fabrication of glycosyl
arrays and glyco-chips. Our approach has an intriguing feature
in that the polymer has two different functional groups,
carbohydrates like Gb2 as a toxin–ligand and an anionic
species like COOK. The anions distributed at the sugar-
embedded polymer chain can tether the cationic counterpart
surfaces coated with, for instance, quaternary ammonium ions
through electrostatic interactions as depicted in Figure 1.
For the aimed glycopolymers, the key intermediate 5 was
synthesized from the known 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl bromide 1 in a manner similar to our
previously reported way (Scheme 1).12 10-Bromodecyl
2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside 2 carrying a
hydrophobic spacer at the reducing terminal was derived
from commercially available 1 in 72% yield. The anomeric
configuration in 2 was confirmed by 1H and 13C NMR
spectroscopies. The doublet signal of H-1 with JZ8.0 Hz
appears at d 4.45 ppm in its 1H NMR spectrum, showing the
b-coupling. The peak at d 101.2 ppm (C-1) in 13C NMR also
supports this result.
Compound 2 was then converted to 10-azidodecyl 2,3,6-tri-
O-benzyl-b-D-galactopyranoside 3 possessing a reaction
point at O-4 in the following way: (1) azide substitution of
the terminal bromide by treatment of NaN3 at 80 8C, (2) de-
O-acetylation with NaOMe-MeOH, (3) 3,4-O-isopropylide-
nation by treatment with acetone and 2,2-dimethoxypropane
in the presence of camphorsulfonic acid (CSA), (4) 2,6-di-
O-benzylation with BnBr and NaH in DMF, (5) de-O-
isopropylidenation by treatment of CSA, and (6) selective
3-O-benzylation using Bu2SnO, BnBr and Bu4NBr (56% in
6 steps).
Figure 1. Schematic figure of alternating layer-by-layer membranes onto
the substrate surface.
2. Results and discussion
Stereoselective glycosylation of 3 and 2,3,4,6-tetra-O-
benzyl-a-D-galactopyranosyl chloride13 was carried out in
the presence of AgClO4 as activator in diethylether to obtain
a1-4 globobioside 4 and b1-4 disaccharide (a/bZca. 10:1),
which was isolated by silica gel column chromatography,
respectively. The 13C NMR signal at d 100.4 ppm (C-10) of
2.1. Synthesis of Gb2-embedded polyanionic glycopolymers
for the detection of E. coli O-157 Shiga toxins
Synthesis of the Gb3-carrying glycopolymers has already
Scheme 1. Synthesis of a key intermediate Gb2 derivative with an amino group at the terminal. Reagents and conditions: (a) 10-bromo-1-decanol, Ag2CO3,
CH2Cl2, MS4A (72%); (b) NaN3, DMF, 80 8C (99%); (c) NaOMe, MeOH; (d) 2,2-dimethoxypropane, acetone, camphorsulfonic acid (CSA) (73%, two steps);
(e) BnBr, NaH (quant.); (f) MeOH, CSA (84%); (g) Bu2SnO, toluene, reflux, then BnBr, Bu4NBr (0.5 equiv), 93%; (h) 2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl chloride, AgClO4, Et2O, MS4A, 0 8C–rt, 22 h (a, 67%; b, 7%); (i) Pd(OH)2, H2, MeOH–AcOEt (2:5, v/v) (85%).

H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
5897
disaccharide 4 shows to be the a-linkage of the newly
formed glycosidic bond. COSY and HMQC analyses also
confirmed the a1-4 linkage. The FAB-MS spectrum also
supports the disaccharide ([MCNa]C 1176.8). Compound 4
was then subjected to catalytic hydrogenolysis with H2 and
Pd(OH)2 to give the fully deprotected 5 carrying an amino
group at the terminal (for 5; d 4.97 ppm (d, JZ2.9 Hz, H-10)
and d 4.26 ppm (d, JZ7.3 Hz, H-1)), which gave similar
results to the data reported for Gala1-4Gal octadecyl
glycoside.9
This method is a reliable and sensitive analysis to determine
total sugar (Gb2) amounts after complete hydrolysis of 6. In
a similar approach shown in Scheme 2, various different
Gb2-containing anionic polymers depending on the feed
ratio of the key module segment 5 were synthesized, and
each sugar content was determined as summarized in
Table 1. These polymers can be useful for the fabrication of
sensor chips according to varying sugar density.
Table 1. Reaction conditions for preparing polyanionic glycopolymer 6
with various Gb2 contents
Coupling of 2.0 mol equiv of the key monomeric epitope 5
(0.02 mmol) with a reactive polymer, poly(ethylene-alt-
maleic anhydride) (MwZ100,000–500,000) (0.01 mmol) at
70 8C was performed to produce the Gb2-embedded anionic
polymer 6 containing 43% of the Gb2 sugar moiety after
being dialyzed (Mw 2000 cut-off) in water for 3 days, further
purified with a gel-permeation chromatography and lyo-
philized (Scheme 2). In its IR spectrum, two characteristic
signals appeared at 1643 and 1566 cmK1, showing the
presence of amide (NHCO) groups in the polymer.
c
Monomer 5
(mol equiv)a
Reaction Temperature
time (h)
Sugar content
of 6 (%)b
Mw
(8C)
0.1
1.0
2.0
2.0
35
60
45
32
rt
rt
rt
70
3
4.0!105
5.0!105
6.7!105
7.8!105
13
25
43
a mol equiv to poly(ethylene-alt-maleic anhydride).
b Determined by the H2SO4-phenol method.
c Determined by the static light scattering method.
A particularly notable point is that the terminal amino group
selectively reacted with an anhydride moiety in the polymer,
almost alternately, to generate an amide group and a free
carboxylic acid in a ratio of 1:1. Thus, sugar contents do not
exceed the 50% value theoretically. The carboxylate anions
(COOK) can effectively serve as the binding sites with the
counterpart cationic surfaces or the cationic polymers
through electrostatic interactions. The characteristic poly-
mer 6 carrying both Gb2 carbohydrate and carboxylate
anions is advantageous for the immobilization onto sensor
surfaces rather than random or block polymers, because the
polyanionic glycopolymer 6 distributes carboxylate anions
alternately to give almost homogeneous clusters in the
polymer chain, while random or block polymerization gave
heterogeneous clusters on which carboxylate anions tend to
be irregularly distributed in the polymers.
The structure of 6 was further confirmed by an alternative
synthesis shown in Scheme 3. Another key carbohydrate
intermediate 9 was obtained from commercially available 7
in 6 steps (67% overall yield). Then, compound 9 was
reacted with poly(ethylene-alt-maleic anhydride) to pro-
duce the fully protected Gb2-embedded polymer 10. In the
1H NMR spectrum, broad signals appeared at near d
7.4 ppm in DMF-d7, indicating the presence of the amide
groups. Deprotection of 10 was achieved in DMF containing
excess amount of 1 M NaOH aq to give polyanionic
1
glycopolymer 6. In the H NMR spectrum, none of the Ac
groups was observed. The H NMR of 6 in Scheme 3 is
1
well-consistent with the data for 6 synthesized in Scheme 2.
This strategy completely eliminates the possibility of the
ester formation by a side reaction between hydroxyl groups
like O-6 in the Gb2 moiety and the active anhydride in the
polymer.
To determine the precise sugar content for the Gb2 moiety in
polymer 6, the phenol-H2SO4 method14 was carried out.
When using another polymer, poly(isobutylene-alt-maleic
Scheme 2. Synthesis of Gb2-embedded anionic polymer 6 in the polymer reaction.

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H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
Scheme 3. Synthesis of another key module Gb2 derivative 9 and polyanionic glycopolymer 6. Reagents and conditions: (a) Ac2O, DMAP, pyridine (98%);
(b) hydrazine–AcOH (93%); (c) Cl3CCN, DBU (90%; a only); (d) 10-bromo-1-decanol, trimethylsilyltriflate, 0 8C (90%); (e) NaN3, DMF, 80 8C (99%);
(f) Pd(OH)2, H2, MeOH (91%); (g) poly(ethylene-alt-maleic anhydride), DMAP, DMF, then H2O; (h) 1 M NaOH aq, DMF.
anhydride) (MwZ60,000), the polymer reaction similar to 6
hardly proceeded. This large difference probably is ascribed
to the steric hindrance for the isobutyl group neighboring on
the reaction site in the polymer chain.
2.3. Alternating layer-by-layer formation of sugar-
embedded polyanionic glycopolymer 6 and its
confirmation by the FTIR-RAS
The alternating layer-by-layer adsorption technique has
provided a convenient way to fabricate thin-layer films
onto solid surfaces or polymer-support,16 and is applied
to the study of molecular imprinting and chemical
filters.17 We applied this technique to prepare the
glycosyl surfaces.
2.2. Synthesis of other sugar-embedded anionic
polymers
One advantage of glycosyl arrays is the potential of
concurrent analysis for various viruses, bacteria and
other carbohydrate receptor proteins. To this end,
several sugar-embedded polymers are required. As
model compounds, we newly synthesized two poly-
anionic polymers carrying b-lactosyl and a-D-mannosyl
residues, respectively. These sugar epitopes ubiquitously
exist on the cell surfaces.
Mercaptopropionic acid was adsorbed onto a gold surface
by a self-assembled monolayer technique18 to fabricate the
surface A in Scheme 5. On this surface A, quaternary
ammonium cationic polymer, [poly(diallyldimethylammo-
nium chloride); Mw 400,000–500,000] was adsorbed in
10 mM HEPES buffer (pH 7.4) for 10 min to obtain surface
B. Finally, Gb2-embedded polyanionic polymer 6 was
adsorbed by electrostatic interactions to form a three-layer
structure (surface C) as illustrated in Scheme 5.
In this section, we chose the p-nitrophenyl (pNP) group at
the aglycon because the pNP group is readily linked to the
polymer chains, after transformation to a p-aminophenyl
(pAP) group by chemical reduction.8,15
The surface C was analyzed by Fourier transform infrared
reflection absorption spectroscopy (FTIR-RAS). Two well-
Commercially available pNP lactoside 11 was reduced
in the presence of Pd under H2 atmosphere to obtain
pAP lactoside 12, quantitatively. Compound 12 was
then reacted with poly(ethylene-alt-maleic anhydride) in
a similar manner to polymer 6 to give 13 carrying
lactoside residues. In a similar approach, the mannose-
embedded anionic polymer 16 was produced from the
corresponding pNP a-D-mannoside 14 in Scheme 4.
This characterization was carried out as stated above,
summarized in Table 2.
defined adsorptions were observed at 1650 and 1570 cmK1
,
showing amide groups (Fig. 2). Apparently, this result
supports the presence of the Gb2-sugar epitope on the
surface C.
Subsequently, we examined the stability of the Gb2-
containing polyanionic glycopolymer 6 on the surface C.
The FTIR-RAS analysis shows that the corresponding
amide peaks are nearly completely retained even when

H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
5899
Scheme 4. Synthesis of other sugar-embedded polymers. Reagents and conditions: (a) Pd(OH)2, H2, H2O (99% for 12, 99% for 15); (b) poly(ethylene-alt-
maleic anhydride), DMF.
Table 2. Reaction conditions for preparing polyanionic glycopolymer 13 and 16.
Monomer (mol equiv)a
Reaction time (h)
Temperature 8C
Sugar content (%)b
Mw
c
Polymer
13
16
12 (2.0)
15 (3.6)
50
22
rt
rt
40
37
7.2!105
6.0!105
a mol equiv to poly(ethylene-alt-maleic anhydride).
b Determined by the H2SO4-phenol method.
c Determined by the static light scattering method.
Scheme 5. Formation of layer-by-layer adsorption. Reagents and conditions: (a) 3-mercaptopropionic acid in ethanol; (b) poly(diallyldimethylammonium
chloride) in 10 mM HEPES buffer (pH 7.4); (c) 6 in the same condition as (b).
washed with 50 mM HCl aq and also exposed to 10 mM
HEPES buffer containing 150 mM NaCl aq for at least 24 h
(data are not shown), and are stable during the SPR
measurements. Those results show that the Gb2-coated
surface C can be usefully applied for toxin detection.
3. Conclusion
In conclusion, we have synthesized novel polyanionic
glycopolymers for the facile fabrication of glycosyl arrays
by applying an alternating layer-by-layer method. The
present polyanionic glycopolymers are useful to assemble
glycosyl arrays or glycosyl chips, for instance, targeting
E. coli O-157 Shiga toxins. This approach will be a highly
practical and promising way to detect toxins, viruses,
bacteria and other receptor proteins with the SPR and a
quartz crystal microbalance (QCM) method, and the details
will be reported in due course.
Other surfaces coated with polymers 13 and 16 were also
prepared in a similar manner to 6. More detailed
information for surface characterization by the FTIR-RAS,
the X-ray photoelectron spectroscopy (XPS) analysis and
thickness of each layer by ellipsometry are under study and
will be reported in a separate paper.

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H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
measurements were made using a Digilab FTS-7000
spectrometer equipped with a Harrick Scientific reflection
accessory and a liquid-N2-cooled MCT detector.
4.1.1. Preparation of layer-by-layer substrates by the
alternating adsorption. Gold substrates were cleanly
washed with a freshly prepared ‘piranha solution’,
H2SO4–H2O2 (3/1, v/v) to completely remove any organic
adsorption, and they were rinsed with milli-Q water several
times. The gold substrates were then immersed into
ethanolic solution containing mercaptopropionic acid
(1 mM). After 16 h, the substrates were rinsed extensively
with absolute ethanol and dried under nitrogen atmosphere
to give surface A in Scheme 5. On this surface A, quaternary
ammonium cationic polymer, [poly(diallyldimethylammo-
nium chloride); Mw 400,000–500,000] (100 mg/mL) was
adsorbed in 10 mM HEPES buffer (pH 7.4) for 10 min to
obtain surface B. Finally, Gb2-embedded polyanionic
polymer 6 (100 mg/mL) was adsorbed in 10 mM HEPES
buffer (pH 7.4) for 10 min to form a three-layer structure
(surface C).
Figure 2. FTIR-RAS spectrum of surface C in Scheme 5.
4. Experimental
4.1.2. FTIR-RAS measurements. FTIR-RAS measure-
ments were carried out using refractor top plate under
nitrogen atmosphere.
4.1. General methods
Globobiose was purchased from Toronto Research Chemi-
cals (Canada). pNP b-lactoside and pNP a-D-mannopyrano-
side was purchased from Sigma. 2,3,4,6-Tetra-O-acetyl-a-
D-galactopyranosyl bromide, poly(ethylene-alt-maleic
anhydride) (MwZ100,000–500,000) and poly(diallyldi-
methylammonium chloride) (Mw 400,000–500,000) were
purchased from Aldrich. Reactions were monitored by thin-
layer chromatography (TLC) on Silica Gel 60 F254
(E. Merck), which were visualized by UV light and by
spraying with 20% H2SO4 in EtOH followed by charring at
180 8C. Column chromatography was performed on Silica
Gel 60 N (Cica Reagent). NAPe-10 Column (Sephadex G-
25) was purchased from Amersham Pharmacia Biotech,
Ultrafree-MC filter from Millipore and they were used for a
gel permeation chromatography. Optical rotations were
measured with JASCO DIP-1000 digital polarimeter at
4.1.3. 10-Bromodecyl 2,3,4,6-tetra-O-acetyl-b-D-galacto-
pyranoside 2. A solution of 10-bromo-1-decanol (123 mg,
0.52 mmol) and Ag2CO3 (137.9 mg, 0.5 mmol) in dry
˚
CH2Cl2 (2 mL) containing freshly activated MS-4 A
(200 mg) was stirred for 1 h at 25 8C under N2. To the
reaction mixture, 2,3,4,6-tetra-O-acetyl-a-D-galactopyrano-
syl bromide 1 (206 mg, 0.50 mmol) in dry CH2Cl2 (3 mL)
was slowly added during 40 min. The reaction mixture was
stirred at 25 8C for 20 h and then filtered through a Celite
pad. The organic layer was washed with satd aq NaHCO3,
and water, respectively. The organic layer was dried
(MgSO4), filtered and concentrated. Column chromato-
graphy of the product on silica gel with 4:6 EtOAc–hexane
afforded compound 2 (209 mg, 72%). [a]D K9.98 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 5.39 (dd, H-4, JZ
3.2, 0.8 Hz), 5.20 (dd, H-2, JZ8.0, 10.4 Hz), 5.02 (dd, H-3,
JZ3.2, 10.4 Hz), 4.45 (d, H-1, JZ8.0 Hz), 4.19 (dd, H-6,
JZ6.4, 11.2 Hz), 4.13 (dd, H-60, JZ7.2, 11.2 Hz), 3.91–
3.85 (m, H-5 and –(CH2)–, 2H), 3.49–3.44 (m, –(CH2)–,
1H), 3.41 (t, –CH2–Br, JZ6.8 Hz, 2H), 2.149 (Ac), 2.051
(Ac), 2.049 (Ac), 1.986 (Ac), 1.88–1.81 (m, –(CH2)–, 2H),
1.62–1.24 (m, –(CH2)–, 14H); 13C NMR (100 MHz,
CDCl3): d 170.3, 170.2, 170.1, 169.2, 101.2, 70.8, 70.4,
70.1, 68.8, 67.0, 61.2, 33.9, 32.7, 29.30, 29.27, 29.22, 29.1,
28.6, 28.0, 25.6, 20.64, 20.55, 20.47; IR (liquid film): 2931,
2857, 1749, 1648, 1434, 1369, 1223, 1175, 1138, 1079,
1057, 957, 901, 840, 737, 645, 597 cmK1; HRMS calcd for
C24H39BrO10Na [MCNa]C 589.1625, found 589.1635.
ambient temperature, using a 10-cm micro cell. 1H and 13
C
NMR spectra were recorded on a Varian INOVA 400 or a
JEOL LA-600 spectrometer for solutions in CDCl3, D2O or
DMF-d7. Chemical shifts are given in ppm and referenced to
internal tert-butyl alcohol (dH 1.23 in D2O or dC 31.2 in
D2O), TMS (dH 0.00 in CDCl3 and DMF-d7), or CDCl3 (dC
77.7 in CDCl3). All data are assumed to be first order with
apparent doublet and triplets reported as d and t,
respectively. Resonances that appear broad are designated
b. A digital resolution is ca. 0.4 Hz. FAB mass spectra
(FAB-MS) were recorded using a JEOL DX 303 mass
spectrometer, and high-resolution mass spectra (HR-MS)
were recorded using a Hitachi M 80 mass spectrometer or
Mariner Biospectroscopy Workstation ESI-TOF MS.
Elemental analyses were performed with a Carlo Elba
EA-1108 or Perkin-Elmer EA-2400 instrument. Average
molecular weights (Mw) of each glycopolymer were
determined by static light scattering using Zetasizer Nano
ZS instrument (Malvern Instruments Ltd, Worcestershire,
UK), and poly[ethylene-alt-maleic acid] was used as the
standard polymer (Mw 300,000). Fourier transform
infrared reflection absorption spectroscopy (FTIR-RAS)
4.1.4. 10-Azidodecyl 2,3,6-tri-O-benzyl-b-D-galacto-
pyranoside 3.
4.1.4.1. Synthesis of 10-azidodecyl 2,3,4,6-tetra-O-
acetyl-b-D-galactopyranoside. 10-Bromodecyl glycoside
2 (1.46 g, 2.58 mmol) and NaN3 (300 mg, 4.61 mmol) was
dissolved in DMF (20 mL). The reaction mixture was stirred
at 80 8C for 4 h. The organic layer was diluted with EtOAc,
washed with brine solution, dried (MgSO4), filtered and

H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
5901
concentrated. Column chromatography of the product on
silica gel with 4:6 EtOAc–hexane gave 10-azidodecyl
2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside (1.35 g,
JZ7.2 Hz, 2H), 1.34 and 1.32 (s, 2!Me), 1.69–1.24
(m, –(CH2)–, 16H); 13C NMR (100 MHz, CDCl3): d 138.3,
138.2, 128.3, 128.1, 127.5, 127.4, 109.8 (Me2C), 102.9
(C-1), 79.6, 79.0, 73.8, 73.5, 72.2, 69.8, 69.5, 51.4 (–CH2–
N3), 29.6, 29.4, 29.32, 29.30, 29.1, 28.7, 27.7, 26.6, 26.3,
26.0; IR (liquid film): 3031, 2986, 2931, 2857, 2095, 1497,
1455, 1371, 1243, 1219, 1163, 1097, 1080, 1045, 1029, 871,
806, 736, 697 cmK1; HRMS calcd for C33H47N3O6Na [MC
Na]C 604.3363, found 604.3350.
1
99%). [a]D K12.98 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): d 5.39 (d, H-4, JZ3.2 Hz), 5.20 (dd, H-2, JZ8.0,
10.4 Hz), 5.02 (dd, H-3, JZ3.2, 10.4 Hz), 4.45 (d, H-1, JZ
8.0 Hz), 4.19 (dd, H-6, JZ6.4, 11.2 Hz), 4.13 (dd, H-60, JZ
7.2, 11.2 Hz), 3.91–3.85 (m, H-5 and –(CH2)–, 2H), 3.49–
3.44 (m, –(CH2)–, 1H), 3.26 (t, –CH2–N3, JZ6.8 Hz, 2H),
2.147, 2.051, 2.049 and 1.986 (4!Ac), 1.64–1.24 (m,
–(CH2)–, 16H). 13C NMR (100 MHz, CDCl3): d 170.3,
170.2, 170.1, 169.2, 101.2, 70.8, 70.4, 70.1, 68.8, 67.0, 61.2,
51.3, 29.3, 29.2, 29.1, 29.0, 28.7, 26.6, 25.7, 20.62, 20.55,
20.47; IR (liquid film): 2932, 2858, 2098, 1756, 1456, 1435,
1370, 1224, 1175, 1135, 1080, 1058, 957, 902, 737,
597 cmK1. Anal. Calcd for C24H39O10N3: C, 54.43; H,
7.42; N, 7.93. Found C, 54.33; H, 7.54; N, 7.90.
4.1.4.4. Synthesis of 10-azidodecyl 2,6-di-O-benzyl-b-
D-galactopyranoside. A mixture of the above di-O-benzyl
compound (508 mg, 0.84 mmol) and camphorsulfonic acid
(139 mg, 0.6 mmol) in dry MeOH (10 mL) was stirred at
25 8C for 2 h. The reaction mixture was then neutralized
with Et3N. The reaction mixture was then diluted with
EtOAc. The organic layer was washed with water, dried
(MgSO4), filtered and concentrated. Column chromato-
graphy on silica gel with EtOAc–hexane (4:6) gave 10-
azidodecyl 2,6-di-O-benzyl-b-D-galactopyranoside (387 mg,
4.1.4.2. Synthesis of 10-azidodecyl 3,4-O-isopropyl-
idene-b-D-galactopyranoside. Treatment of the above
compound (708 mg, 1.34 mmol) with 0.1 M NaOMe for
3 h followed by neutralization with Dowex 50 WXHC resin
afforded deacetylated product, 10-azidodecyl b-D-galacto-
pyranoside. To a solution of crude 10-azidodecyl b-D-
galactopyranoside in dry acetone (20 mL), 2,2-dimethoxy-
propane (0.86 mL, 7 mmol) and camphorsulfonic acid
(255 mg, 1.1 mmol) were added. The reaction mixture
was stirred at 25 8C for 2.5 h and then neutralized with Et3N.
The reaction mixture was then diluted with EtOAc. The
organic layer was washed with water, dried (MgSO4),
filtered and concentrated. Column chromatography on silica
gel with EtOAc–hexane (8:2) gave 10-azidodecyl 3,4-O-
isopropylidene-b-D-galactopyranoside (392 mg, 73%); [a]D
C11.58 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 4.18
(d, H-1, JZ8.4 Hz), 3.25 (t, –CH2–N3, JZ6.8 Hz), 1.52 and
1.35 (2!Me of isopropylidene); 13C NMR (100 MHz,
CDCl3): d 110.9 (Me2C), 102.9 (C-1), 79.5, 74.5, 74.2, 74.1,
70.7, 62.9, 52.0 (–CH2–N3), 30.2, 29.97, 29.95, 29.93, 29.7,
29.4, 28.7 (Me2C), 27.3, 26.9 (Me2C), 26.5; IR (liquid film):
3313, 2986, 2933, 2857, 2095, 1456, 1381, 1372, 1241,
1218, 1161, 1144, 1096, 1077, 1036, 964, 904, 891, 873,
808, 738 cmK1; HRMS calcd for C19H35N3O6Na [MC
Na]C 424.2424, found 424.2424.
1
84%); [a]D C5.28 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): d 7.38–7.25 (m, aromatics, 10H), 4.97 (d, CH2–
C6H5, JZ11.6 Hz), 4.67 (d, CH2–C6H5, JZ11.6 Hz), 4.59
(s, CH2–C6H5, 2H), 4.36 (d, H-1, JZ7.6 Hz), 4.00–3.92 (m,
–(CH2)– and H-4, 2H), 3.82–3.71 (m, H-6 and H-60, 2H),
3.63–3.46 (m, H-3, H-5, H-2 and –(CH2)–, 4H), 3.24 (t,
–CH2–N3, JZ7.2 Hz, 2H), 1.71–1.22 (m, –(CH2)–, 16H);
13C NMR (100 MHz, CDCl3): d 139.1, 138.6, 129.1, 129.0,
128.7, 128.4, 128.33, 128.30, 104.3 (C-1), 79.7, 75.1, 74.2,
73.9, 73.8, 70.5, 70.0, 69.6, 52.0 (–CH2–N3), 30.3, 30.03,
29.98, 29.96, 29.7, 29.4, 27.3, 26.7; IR (liquid film): 3401,
3064, 3034, 2929, 2856, 2096, 1454, 1368, 1288, 1260,
1077, 736, 697 cmK1. Anal. Calcd for C30H43O6N3: C,
66.52; H, 8.00; N, 7.76. Found C, 66.47; H, 8.13; N, 7.57.
4.1.4.5. Synthesis of 3. To a solution of the above de-O-
isopropylidenated compound (157 mg, 0.29 mmol) in
toluene (10 mL), Bu2SnO (79.7 mg, 0.32 mmol) was
added and the mixture was refluxed with azeotropic removal
of water for 6 h. The reaction mixture was allowed to 25 8C.
Benzyl bromide (0.1 mL, 0.87 mmol) and Bu4NBr (48 mg,
0.15 mmol) were added and stirred at 25 8C for 3 h. The
reaction mixture was then diluted with EtOAc. The organic
layer was washed with water, dried (MgSO4), filtered and
concentrated. Column chromatography of the product on
silica gel with 4:6 EtOAc–hexane gave pure 3 (171 mg,
4.1.4.3. Synthesis of 10-azidodecyl 2,6-di-O-benzyl-
3,4-O-isopropylidene-b-D-galactopyranoside. A mixture
of isopropylidene compound synthesized in Section 4.1.4.2
(338 mg, 0.84 mmol) and NaH (52 mg, 2.2 mmol) in dry
DMF (20 mL) was stirred at 25 8C for 30 min and benzyl
bromide (0.26 mL, 2.2 mmol) was added. The reaction
mixture was further stirred at 25 8C for 2 h. The reaction
mixture was then diluted with EtOAc. The organic layer
was washed with water, dried (MgSO4), filtered and
concentrated. Column chromatography of the product on
silica gel with 4:6 EtOAc–hexane gave 10-azidodecyl 2,6-
di-O-benzyl-3,4-O-isopropylidene-b-D-galactopyranoside
1
93%); [a]D K7.58 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): d 7.39–7.25 (m, aromatics, 15H), 4.92 (d, CH2–
C6H5, JZ10.8 Hz), 4.72 (d, CH2–C6H5, JZ10.8 Hz), 4.72
(s, CH2–C6H5, 2H), 4.59 (s, CH2–C6H5, 2H), 4.35 (d, H-1,
JZ7.6 Hz), 4.02 (br s, H-4), 3.98–3.91 (m, –CH2O–, 1H),
3.80 (dd, H-6, JZ6.0, 10.0 Hz), 3.72 (dd, H-60, JZ6.0,
10.0 Hz), 3.63 (dd, H-2, JZ8.0, 9.2 Hz), 3.55 (br t, H-5, JZ
6.0 Hz), 3.53–3.47 (m, –(CH2)–, 1H), 3.49 (dd, H-3, JZ3.2,
9.2 Hz), 3.24 (t, –CH2–N3, JZ7.2 Hz, 2H), 1.71–1.22 (m,
–(CH2)–, 16H); 13C NMR (125 MHz, CDCl3): d 139.3,
138.6, 138.5, 129.03, 129.01, 128.9, 128.7, 128.41, 128.36,
128.32, 128.2, 104.2 (C-1), 81.0, 79.4, 75.6, 74.1, 73.6,
72.8, 70.4, 69.7, 67.4, 52.0 (–CH2–N3), 30.3, 30.0, 30.0,
29.7, 29.4, 27.3, 26.7; IR (liquid film): 3536, 3065, 3031,
2929, 2857, 2095, 1454, 1366, 1300, 1264, 1210, 1159,
1096, 1077, 1029, 917, 736, 697 cmK1. Anal. Calcd for
1
(530 mg, quant.); [a]D C18.78 (c 1.0, CHCl3); H NMR
(400 MHz,CDCl3): d 7.42–7.22 (m, 10H, aromatic), 4.85 (d,
CH2–C6H5, 11.6 Hz), 4.78 (d, CH2–C6H5, JZ11.6 Hz),
4.64 (d, CH2–C6H5, JZ11.6 Hz), 4.55 (d, CH2–C6H5, JZ
11.6 Hz), 4.3 (d, H-1, JZ8.0 Hz), 4.16–3.88 (m, H-3, H-4,
H-5, –(CH2)–), 3.82–3.74 (m, H-6 and H-60), 3.54–3.46 (m,
–(CH2)–, 1H), 3.41–3.35 (m, H-2, 1H), 3.23 (t, –CH2–N3,

5902
H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
C37H49O6N3: C, 70.34; H, 7.82; N, 6.65. Found C, 70.60; H,
7.82; N, 6.47.
temperature under atmospheric pressure for 4 h. The
reaction mixture was filtered through a Celite pad and
concentrated to give fully deprotected 5 (18 mg, 85%). [a]D
1
C64.68 (c 0.5, CH3OH); H NMR (600 MHz, CD3OD): d
4.1.5. 10-Azidodecyl O-(2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-benzyl-b-D-galac-
topyranoside 4. A solution of compound 3 (116 mg,
0.18 mmol) and tetra-O-benzyl-a-D-galactopyranosyl chlor-
ide13 (203 mg, 0.36 mmol) in dry diethyl ether (20 mL)
4.97 (d, H-10, JZ2.9 Hz), 4.29 (t, H-50, JZ6.2 Hz), 4.26 (0d,
H-1, JZ7.3 Hz), 3.99 (d, H-4, JZ2.9 Hz), 3.92 (br s, H-4 ),
3.87 (dt, –CH2O–, JZ7.0, 9.5 Hz, 1H), 3.83 (dd, H-6a, JZ
7.7, 11.0 Hz), 3.79–3.75 (m0, H-20, H-30), 3.75–3.70 (m,
H-6b, H-60a), 3.68 (dd, H-6 b, JZ5.5, 11.0 Hz), 3.60 (t,
H-5, JZ8.4 Hz), 3.56 (dt, –CH2O–, JZ6.6, 9.5 Hz, 1H),
3.53 (dd, H-3, JZ2.9, 9.9 Hz), 3.46 (dd, H-2, JZ7.3,
9.9 Hz), 2.90 (t, –CH2–NH2, JZ7.3 Hz), 1.65–1.28 (m,
–(CH2)8–, 16H); 13C NMR (100 MHz, CD3OD): d 106.0
(C-1), 103.3 (C-10), 79.7, 76.9, 75.5, 73.7, 73.4, 72.2, 71.9,
71.6, 63.5, 61.7, 41.7 (–CH2–NH2), 31.7, 31.4, 31.3, 31.2,
31.0, 29.4, 28.3, 27.9; IR (liquid film): 3349, 2930, 2856,
1615, 1376, 1150, 1075, 807 cmK1; HRMS calcd for
C22H44NO11 [MCH]C 498.2914, found 498.292.
˚
containing freshly activated 4 A MS (1 g) was stirred under
N2 for 1 h and cooled at 0 8C, AgClO4 (112 mg, 0.54 mmol)
was then added. The reaction temperature was gradually
elevated to 25 8C, and stirring was continued in the dark for
22 h at the temperature. The reaction mixture was diluted
with EtOAc and then filtered through a Celite pad, washed
with satd aq NaHCO3, water, respectively. The organic
layer was dried (MgSO4), filtered and concentrated. Column
chromatography of the product on silica gel with 2:8
EtOAc–hexane afforded disaccharide 4 (134 mg, 67%) and
b1-4 isomer (14 mg, 7%). Compound 4; [a]D C47.58 (c
0.71, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.37–7.16 (m,
aromatics, 35H), 5.03 (br s, H-10), 4.92–4.87 (m, CH2–
C6H5, 3H), 4.80–4.76 (m, CH2–C6H5, 4H), 4.68 (d, CH2–
C6H5, JZ11.7 Hz, 1H), 4.55 (d, CH2–C6H5, JZ11.4 Hz,
1H), 4.54 (d, CH2–C6H5, JZ12.8 Hz, 1H), 4.46–4.42 (m,
H-50, 1H), 4.31 (d, H-1, JZ7.7 Hz), 4.26 (d, CH2-C6H5, JZ
12.0 Hz, 1H), 4.22 (d, CH2–C6H5, JZ12.0 Hz, 1H), 4.15 (d,
CH2–C6H5, JZ12.0 Hz), 4.13 (d, CH2–C6H5, JZ12.0 Hz),
4.10 (br, H-20, H-30, H-40, 3H), 4.01 (bd, H-4, JZ2.9 Hz,
1H), 3.98–3.90 (m, H-6a and –OCH2–, 2H), 3.66 (dd,
H-2, JZ7.7, 9.9 Hz), 3.56–3.46 (m, H-60a, H-6b, H-5 and
–OCH2–, 4H), 3.38 (dd, H-3, JZ2.9, 9.9 Hz), 3.26–3.22 (m,
H-60b and –CH2–N3, 3H), 1.71–1.26 (m, –(CH2)–, 16H);
13C NMR (100 MHz, CDCl3): d 138.9, 138.8, 138.7, 138.6,
138.1, 138.0, 128.3, 128.21, 128.16. 128.06, 128.0, 127.8,
127.6, 127.5, 127.4, 127.3, 103.9 (C-1), 100.4 (C-10), 80.8,
78.9, 76.5, 75.0, 74.8, 74.7, 74.6, 73.7, 73.5, 73.1, 73.0,
72.3, 72.2, 70.1, 69.2, 68.0, 67.9, 51.4 (–CH2–N3), 29.7,
29.4, 29.3, 29.1, 28.8, 26.6, 26.1; IR (liquid film): 3062,
3030, 2929, 2858, 2095, 1497, 1454, 1365, 1269, 1208,
1097, 1056, 1028, 735, 697 cmK1. Anal. Calcd for
C71H83O11N3: C, 73.87; H, 7.25; N, 3.64. Found C, 74.05;
H, 7.18; N, 3.74; FAB-MS(pos): [MCNa]C 1176.8. b1–4
4.1.7. Polyanionic glycopolymer 6. The reaction con-
ditions are summarized in Tables 1 and 2. A typical
procedure is as follows. Sugar monomer, 5 (10 mg,
0.02 mmol) and poly(ethylene-alt-maleic anhydride)
(1.3 mg, 0.01 mmol as maleic anhydride unit) were
dissolved in dry DMF (1 mL), and stirred at 70 8C for
32 h. The reaction mixture was concentrated, the precipitate
was dissolved in water (20 mL), dialyzed for 3 days in water
(Mw 2000 cut-off) and further purified with NAPe-10
Column (1.3!2.6 cm) to afford the polyanionic glyco-
polymer 6 (4.1 mg). The sugar content was determined by
H2SO4-phenol method to be ca. 43%. [a]D K74.08 (c 0.2,
H2O); 1H NMR(400 MHz,D2O): d5.02(br, H-10), 4.40 (br, H-
1 and H-50, 2H), 1.7–1.1 (br, –(CH2)–); IR (KBr): 3400, 2930,
2857, 1713, 1643, 1566, 1465, 1406, 1226, 1151, 1075 cmK1
.
4.1.8. O-(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)-
(1/4)-1,2,3,6-tetra-O-acetyl-a/b-D-galactopyranoside
8. O-(a-D-galactopyranosyl)-(1/4)-a/b-D-galactopyrano-
side 7 (50 mg, 0.15 mmol) was dissolved in dry pyridine
(4 mL), and acetic anhydride (340 mL, 3.6 mmol) and N,N-
dimethylaminopyridine (8.9 mg, 0.073 mmol) was added.
The reaction mixture was stirred at 40 8C for 20 h. The
mixture was then diluted with EtOAc, and washed with satd
aq NaHCO3, water, respectively. The organic layer was
dried (MgSO4), filtered and concentrated. Column chro-
matography of the product on silica gel with 6:4 EtOAc–
hexane afforded peracetylated disaccharide 8 (97.3 mg,
98%) of a/bZca. 3:2; 1H NMR (400 MHz, CDCl3): d 6.38
(d, Ha-1, JZ3.6 Hz), 5.71 (d, Hb-1, JZ8.0 Hz), 5.59 (dd,
Hb-40, JZ1.2, 3.2 Hz), 5.57 (dd, Ha-40, JZ1.6, 3.2 Hz),
5.32 (dd, Hb-2, JZ8.0, 10.4 Hz), 5.01 (d, Ha-10, JZ3.6 Hz),
5.00 (d, Hb-10, JZ3.6 Hz), 4.87 (dd, Hb-3, JZ2.8, 10.8 Hz);
13C NMR (100 MHz, CDCl3): d 99.5, 99.1, 92.0, 89.8; IR
(KBr): 2934, 1752, 1650, 1436, 1373, 1231, 1134, 1069,
1015, 938, 905 cmK1. Anal. Calcd for C28H38O19: C, 49.56;
H, 5.64. Found C, 49.68; H, 5.52.
1
isomer; [a]D C128 (c 0.5, CHCl3); H NMR (400 MHz,
CDCl3): d 7.47–7.18 (m, aromatics, 35H), 5.12 (d, CH2–
C6H5, JZ10.8 Hz, 1H), 4.99 (d, CH2–C6H5, JZ12.0 Hz,
1H), 4.91 (d, H-10, JZ7.6 Hz), 4.81–4.69 (m, CH2–C6H5,
6H), 4.59–4.49 (m, CH2–C6H5, 3H), 4.42 (d, CH2–C6H5,
JZ10.8 Hz, 1H), 4.35 (d, H-1, JZ8.0 Hz), 4.34 (s, CH2–
C6H5, 2H), 4.24 (br d, H-40, JZ2.4 Hz), 3.98–3.93 (m, –O–
CH2–(CH2)–, 1H), 3.86 (br d, H-4, JZ2.8 Hz), 3.84–3.67
(m, 0H-2, H-20, H-60a and H-60b, 4H), 3.56–3.42 (m, H-3,
H-3 , H-5, H-50, H-6a, H-6b and –O–CH2–(CH2)–, 7H),
3.23 (t, –CH2–N3, 2H), 1.67–1.25 (m, –(CH2)–, 16H); 13C
NMR (100 MHz, CDCl3): d 138.8, 137.9, 137.7, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9. 127.8, 127.7, 127.6, 127.5, 127.3,
103.9 (C-1), 102.8 (C-10), 82.0, 81.9, 80.0, 79.9, 75.2, 74.9,
74.5, 74.2, 73.8, 73.5, 73.4, 73.2, 72.7, 70.7, 70.0, 69.9, 68.7,
51.5 (–CH2–N3), 29.8, 29.5, 29.4, 29.3, 28.8, 26.7, 26.2.
4.1.9. 10-Aminodecyl O-(2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-b-D-galacto-
pyranoside 9.
4.1.9.1. Synthesis of O-(2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-a/b-D-
galactopyranose. The disaccharide 8 (103 mg, 0.15 mmol)
4.1.6. 10-Aminodecyl O-(a-D-galactopyranosyl)-(1/4)-
b-D-galactopyranoside 5. A solution of 4 (49 mg,
0.042 mmol) and a catalytic amount of Pd(OH)2 in
EtOAc–MeOH (2:5, 7 mL) was hydrogenated at room

H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
5903
was treated with hydrazine acetate (27 mg, 0.30 mmol) in
DMF (4 mL) at 0 8C for 4 h. The mixture was then diluted
with CHCl3, and washed with brine. The organic layer was
dried (MgSO4), filtered and concentrated. Column chroma-
tography of the product on silica gel with 8:2 EtOAc–
hexane gave O-(2,3,4,6-tetra-O-acetyl-a-D-galactopyrano-
syl)-(1/4)-2,3,6-tri-O-acetyl-a/b-D-galactopyranoside
1H), 3.77 (br t, H-5, JZ6.8 Hz), 3.49–3.43 (m, –O–CH2–
(CH2)–, 1H), 3.41 (t, –CH2–Br, JZ6.8 Hz, 2H), 2.13 (Ac),
2.10 (Ac), 2.08 (Ac), 2.07 (Ac), 2.04 (2!Ac), 1.98 (Ac),
1.89–1.81 (m, –CH2–CH2Br, 2H), 1.61–1.54 (m, –OCH2–
CH2–, 2H), 1.44–1.26 (m, –(CH2)–, 12H); 13C NMR
(100 MHz, CDCl3): d 170.5, 170.4, 170.3, 170.2, 169.9,
169.5, 168.9, 101.1 (C-1), 99.2 (C-10), 72.6, 71.7, 69.9, 68.7,
68.4, 67.7, 67.2, 66.9, 61.8, 60.4, 33.9, 32.6, 29.3, 29.3,
29.2, 29.1, 28.6, 28.0, 25.7, 20.8, 20.6, 20.55, 20.53, 20.48;
IR (liquid film): 2931, 2856, 1750, 1434, 1371, 1225, 1180,
1132, 1070, 903, 600 cmK1. Anal. Calcd for C36H55O18Br:
C, 50.53; H, 6.48. Found C, 50.33; H, 6.35.
1
(88.5 mg, 93%). Selective H NMR (400 MHz, CDCl3): d
5.51 (d, HaK1, JZ3.2 Hz), 5.12 (dd, Hb-2, JZ8.0,
10.8 Hz), 5.03 (d, Hb-10, JZ3.6 Hz), 5.00 (d, Ha-10, JZ
4.0 Hz), 4.91 (dd, Hb-3, JZ2.4, 10.8 Hz), 4.70 (br d, Hb-1,
JZ7.6 Hz); 13C NMR (100 MHz, CDCl3): 98.9 (C-10), 95.5
(Cb-1), 90.4 (Ca-1); IR (KBr): 3469, 2973, 1751, 1660,
1437, 1374, 1235, 1157, 1131, 1070, 979, 908, 758,
600 cmK1; HRMS calcd for C26H36O18Na [MCNa]C
659.1800, found 659.1776.
4.1.9.4. Synthesis of 10-azidodecyl O-(2,3,4,6-tetra-O-
acetyl-a-D-galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-
b-D-galactopyranoside. A solution of the 10-bromodecyl
glycoside (59 mg, 0.069 mmol) and NaN3 (8 mg,
0.12 mmol) in dry DMF (4 mL) was stirred at 80 8C for
5.5 h. The reaction mixture was then processed in the same
way described for the Section 4.1.4.1 to give 10-azidodecyl
O-(2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-(1/4)-
2,3,6-tri-O-acetyl-b-D-galactopyranoside (55 mg, 99%).
4.1.9.2. Synthesis of O-(2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-a-D-galacto-
pyranosyl trichloroacetimidate. To a mixture of the above
selectively deacetylated compound (87 mg, 0.14 mmol) and
trichloroacetonitrile (400 mL, 4.0 mmol), 1,8-diazabicyclo
[5.4.0]undec-7-ene (30 mL, 0.20 mmol) was added at 0 8C
and stirred for 2 h. The reaction mixture was then subjected
to a silica gel column chromatography with 4:6 EtOAc–
hexane to afford O-(2,3,4,6-tetra-O-acetyl-a-D-galacto-
pyranosyl)-(1/4)-2,3,6-tri-O-acetyl-a-D-galactopyranosyl
trichloroacetimidate (95 mg, 90%). [a]D C148.68 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 8.68 (s, NH), 6.61
(d, H-1, 3.6 Hz), 5.57 (dd, H-40, JZ1.2, 3.2 Hz), 5.42 (dd,
H-2, JZ3.6, 11.2 Hz), 5.38 (dd, H-30, JZ3.2, 11.2 Hz), 5.31
(dd, H-3, JZ2.8, 11.2 Hz), 5.24 (dd, H-20, JZ3.6, 11.2 Hz),
5.03 (d, H-10,03.2 Hz), 4.55–4.51 (m, H-50), 4.37–4.31 (m,
H-5 and H-6 ), 4.28 (br d, H-4, JZ2.4 Hz), 4.16–4.08
(m, H-6a, H-6b and H-60), 2.15 (Ac), 2.12 (2!Ac), 2.04
(Ac), 2.03 (2!Ac), 1.99 (Ac); 13C NMR (100 MHz,
CDCl3): d 170.5, 170.3, 170.1, 169.8, 169.7, 160.8
(C]NH), 109.7 (C-1), 98.9 (C-10), 93.6 (CCl3), 70.6, 69.4,
68.2, 67.7, 67.3, 67.1, 66.7, 61.8, 60.7, 20.9, 20.7, 20.6,
20.5, 20.4; IR (liquid film): 3320, 2977, 1748, 1677, 1434,
1
[a]D C70.48 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
d 5.56 (dd, H-40, JZ1.2, 3.2 Hz), 5.39 (dd, H-30, JZ3.2,
10.8 Hz), 5.21–5.15 (m, H-20 and H-2), 5.00 (d, H-10, JZ
3.6 Hz), 4.81 (dd, H-3, JZ2.4, 11.2 Hz), 4.55–4.52 (m,
H-500), 4.48–4.44 (m, H-6 and H-1), 4.20–4.07 (m, H-60a,
H-6 b and H-6), 4.05 (br d, H-4, JZ2.0 Hz), 3.91–3.85
(m, –O–CH2–(CH2)–, 1H), 3.77 (br t, H-5, JZ6.8 Hz),
3.49–3.43 (m, –O–CH2–(CH2)–, 1H), 3.26 (t, –CH2–N3, JZ
6.8 Hz), 2.13 (Ac), 2.10 (Ac), 2.08 (Ac), 2.07 (Ac), 2.04
(2!Ac), 1.98 (Ac), 1.63–1.54 (m, –(CH2)–, 4H), 1.38–1.28
(m, –(CH2)–, 12H); 13C NMR (100 MHz, CDCl3): d 170.5,
170.4, 170.3, 170.2, 169.9, 169.5, 168.9, 101.0 (C-1), 99.2
(C-10), 72.6, 71.7, 69.8, 68.6, 68.4, 67.7, 67.2, 66.9, 61.8,
60.3, 51.3 (–CH2N3), 29.3, 29.2, 29.1, 28.9, 28.6, 26.5, 25.6,
20.8, 20.6, 20.51, 20.48, 20.43; IR (liquid film): 2933, 2857,
2098, 1751, 1371, 1225, 1133, 1067, 903, 668 cmK1. Anal.
Calcd for C36H55O18N3: C, 52.87; H, 6.78; N, 5.14. Found:
C, 52.59; H, 6.42; N, 4.77.
1372, 1225, 1134, 1068, 971, 904, 837, 797, 752, 644 cmK1
.
4.1.9.3. Synthesis of 10-bromodecyl O-(2,3,4,6-tetra-O-
acetyl-a-D-galactopyranosyl)-(1/4)-2,3,6-tri-O-acetyl-b-
D-galactopyranoside. A solution of the above trichloro-
acetimidate compound (61 mg, 0.077 mmol) and 10-bromo-
1-decanol (182 mg, 0.76 mmol) in dry CH2Cl2 (2 mL)
4.1.9.5. Synthesis of 9. A mixture of the above azide
compound (55 mg, 0.068 mmol) and a catalytic amount of
Pd(OH)2 (ca. 5 mg) was stirred in MeOH (5 mL) at room
temperature under hydrogen atmosphere for 3 h. The reac-
tion mixture was then processed in the same way described
for compound 5 to give 9 (49 mg, 91%). [a]D C70.48 (c 1.0,
˚
containing freshly activated 4 A-MS (400 mg) was stirred
1
for 1 h at 25 8C under N2. The reaction mixture was then
cooled to 0 8C and Me3SiOTf (9 mL, 0.05 mmol) was added.
The mixture was stirred at 0 8C for 2.5 h and then
triethylamine was added. The mixture was filtered through
a Celite pad, then diluted with CHCl3 and washed with satd
aq NaHCO3, water, respectively. The organic layer was
dried (MgSO4), filtered and concentrated. Column chromato-
graphy of the product on silica gel with 4:6 EtOAc–hexane
afforded 10-bromodecyl glycoside (59 mg, 90%); [a]D
C60.38 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 5.56
(dd, H-40, JZ1.2, 3.2 Hz), 5.39 (dd, H-30, JZ3.6, 11.2 Hz),
5.21–5.15 (m, H-20 and H-2), 5.00 (d, H-10, JZ3.6 Hz), 4.81
(dd, H-3, JZ2.8, 10.8 Hz), 4.55–4.52 (0m, H-500), 4.48–4.44
(m, H-6 and H-1), 4.20–4.07 (m, H-6 a, H-6 b and H-6),
4.05 (br d, H-4, JZ2.4 Hz), 3.91–3.85 (m, –O–CH2–(CH2)–,
CH3OH); H NMR (400 MHz, CD3OD): d 5.54 (dd, H-40,
JZ1.2, 3.2 Hz), 5.40 (dd, H-30,0JZ3.2, 11.2 Hz), 5.22–5.14
(m, H-20 and H-2), 5.06 (d, H-1 , 4.0 Hz), 5.01 (dd, H-3, JZ
2.4, 10.8 Hz), 4.63 (d, H-1, JZ8.0 Hz), 4.58–4.54 (m, H-50),
4.43 (dd, H-6, JZ7.2, 11.2 Hz), 4.21–4.11 (m, H-4, H-60a,
H-60b and H-6), 4.00–3.96 (m, H-5), 3.90–3.85 (m, –O–
CH2–(CH2)–, 1H), 3.57–3.51 (m, –O–CH2–(CH2)–, 1H),
2.92 (t, –CH2–N3, JZ7.6 Hz, 2H), 2.13 (Ac), 2.11 (Ac),
2.08 (Ac), 2.05 (Ac), 2.04 (Ac), 2.02 (Ac), 1.96 (Ac), 1.69–
1.33 (m, –(CH2)–, 16H); 13C NMR (100 MHz, CD3OD): d
172.2, 172.0, 171.9, 171.6, 171.3, 102.3, 100.4, 78.5, 73.8,
73.5, 71.0, 70.6, 69.6, 69.5, 69.0, 68.4, 64.1, 62.0, 40.8,
30.6, 30.5, 30.4, 30.3, 30.2, 28.5, 27.4, 27.0, 21.0, 20.81,
20.76, 20.7, 20.6, 20.5; IR (KBr): 3458, 2935, 2859, 1753,
1631, 1435, 1373, 1230, 1134, 1072, 904, 601 cmK1
;

5904
H. Uzawa et al. / Tetrahedron 61 (2005) 5895–5905
HRMS calcd for C36H58NO18 [MCH]C 792.3654, found
792.3629.
Acknowledgements
This work was partly supported by subsidies from the
Ministry of Economy, Trade and Industry of Japan.
4.1.10. Synthesis of acetylated glycopolymer 10. A
solution of 9 (33.1 mg, 0.043 mmol) and poly(ethylene-
alt-maleic anhydride) (2.5 mg, 0.02 mmol as maleic anhy-
dride unit) were dissolved in dry DMF (1 mL), and was
stirred at room temperature for 45 h. A few drops of water
were added, and the mixture was concentrated and then
purified with Ultrafree-MC filter (DMF as an eluent) to afford
the acetylated glycopolymer 10(9.2 mg).[a]D K38.98(c0.61,
DMF); 1H NMR (400 MHz, DMF-d7): d 7.4 (br, NHCO), 5.58
(br, H-40), 5.35 (br, H-30), 5.25 (br dd, JZ3.4, 11.0 Hz, H-20),
5.2–5.1 (0 br, H-2, H-10, H-3), 4.77 (br d, JZ6.8 Hz, H-1), 4.62
(br, H-5 ), 3.85 (br, –OCH2CH2–), 2.17, 2.16, 2.12, 2.09, 2.07,
2.04, 1.99 (7!br s, Ac).
References and notes
1. (a) Esko, J. D. Microbial carbohydrate-binding proteins. In
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Acad. Sci. U.S.A. 2002, 99, 225–232.
4.1.11. Conversion of acetylated glycopolymer 10 to fully
deprotected 6. The above polymer 10 (3 mg) was
deacetylated with 1 M NaOH aq. (3 mL) in DMF (1 mL)
at room temperature for 3 h. The reaction mixture was then
processed in the same way described for the Section 4.1.7 to
afford polyanionic glycopolymer 6 (1.3 mg).
´
2. (a) Rojo, J.; Morales, J. C.; Penades, S. Top. Curr. Chem. 2002,
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4.1.12. Synthesis of glycopolymer 13. A solution of
commercially available pNP lactoside 11 (683.2 mg,
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1
H2O); H NMR (400 MHz, D2O): d 7.2 (br, aromatics),
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.
4.1.13. Synthesis of glycopolymer 16. A solution of
commercially available pNP a-D-mannopyranoside 14
(1 g, 3.3 mmol) and a catalytic amount of Pd(OH)2 (ca.
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reaction mixture was filtered through a Celite pad and
concentrated to give 15 (890 mg, 99%). Then, monomer 15
(100 mg, 0.36 mmol) and poly(ethylene-alt-maleic anhy-
dride) (12.8 mg, 0.1 mmol as maleic anhydride unit) were
dissolved in dry DMF (3 mL), and the mixture was stirred at
room temperature for 22 h. The reaction mixture was then
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H2O); H NMR (400 MHz, D2O): d 7.12 (br, aromatics),
5.52 (br, H-1), 4.0–3.5 (br, H-2, H-3, H-4, H-5, H-6 and
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1228, 1123, 1065, 1010, 975, 831 cmK1
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