Protecting-group-free synthesis of glycopolymers bearing thioglycosides via one-pot monomer synthesis from free saccharides

Article abstract of DOI:10.1002/pola.27417

Polyacrylamides having pendant thioglycosides were successfully synthesized from thioglycosidic monomers that were readily prepared by one-pot method without any protection of the hydroxy groups on the starting free saccharides. The glycomonomers were synthesized by the direct synthesis of thioglycosides using 2-chloro-1,3-dimethylimidazolinium chloride and 4-aminobenzentiol, and the following acrylamidation. They were co-polymerized with acrylamide into glycopolymers by reversible addition-fragmentation chain transfer polymerization using a trithiocarbonate derivative as a chain transfer agent. The gold nanoparticles and gold-coated quartz crystal microbalance sensor immobilized with the thiol-terminated glycopolymers exhibited high affinity for the corresponding lectins due to multivalent interaction between saccharides and protein in aqueous solution.

Full text of DOI:10.1002/pola.27417

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Protecting-Group-Free Synthesis of Glycopolymers Bearing
Thioglycosides Via One-Pot Monomer Synthesis from Free Saccharides
Tomonari Tanaka,1 Genri Inoue,1 Shin-Ichiro Shoda,2 Yoshiharu Kimura1
1Department of Biobased Materials Science, Graduate School of Science and Technology, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku, Kyoto 606–8585, Japan
2Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba, Sendai, Miyagi 980–8579,
Japan
Correspondence to: T. Tanaka (E-mail: t-tanaka@kit.ac.jp)
Received 5 June 2014; accepted 7 September 2014; published online 3 October 2014
DOI: 10.1002/pola.27417
ABSTRACT: Polyacrylamides having pendant thioglycosides
were successfully synthesized from thioglycosidic monomers
that were readily prepared by one-pot method without any pro-
tection of the hydroxy groups on the starting free saccharides.
The glycomonomers were synthesized by the direct synthesis
of thioglycosides using 2-chloro-1,3-dimethylimidazolinium
chloride and 4-aminobenzentiol, and the following acrylamida-
tion. They were co-polymerized with acrylamide into glycopol-
ymers by reversible addition-fragmentation chain transfer
polymerization using a trithiocarbonate derivative as a chain
transfer agent. The gold nanoparticles and gold-coated quartz
crystal microbalance sensor immobilized with the thiol-
terminated glycopolymers exhibited high affinity for the corre-
sponding lectins due to multivalent interaction between sac-
C
V
charides and protein in aqueous solution.
2014 Wiley
Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52,
3513–3520
KEYWORDS: cluster effect; glycomonomer; glycopolymer;
gold nanoparticle; lectin; one-pot synthesis; quartz crystal
microbalance; RAFT polymerization
ducing a polymerizable group such as vinyl and norbornene
at the anomeric position,15,16 and finally deprotection. The
resulting glycomonomers are used in the polymerization
reaction, giving rise to glycopolymers. It is possible to per-
form the deprotection step after the polymerization reac-
tion.17–19 In another route for synthesizing glycopolymers,
glycosyl derivatives are attached to the polymer chain by
“click chemistry” such as copper(I) catalyzed azide–alkyne
cycloaddition20–24 and thiol-ene reaction.25,26 These methods
also require multiple steps to synthesize the saccharide
derivatives having functional groups such as azide, acetylene,
and thiol. Several protecting-group-free syntheses of glyco-
monomers have been reported until now. For example, Kita-
zawa et al. synthesized vinyl glycomonomers from methyl
glycoside at high temperature using phosphomolybdic acid
as a catalyst.27 This method, however, gave a mixture of a-
and b-glycosyl monomers because of low regioselectivity at
the anomeric position. In other methods, gluconolactone and
glycosylamine were synthesized and used for obtaining sty-
rene derivatives having saccharides although their synthetic
procedure was rather complicated.28,29 Therefore, a more
efficient synthetic method has been required for the
INTRODUCTION Saccharides on the cell surface play signifi-
cant roles in many important cellular recognition processes
including cell growth regulation, differentiation, adhesion,
cancer cell metastasis, cellular trafficking, inflammation by
bacteria and viruses, and immune response. Although sac-
charide–protein interactions are generally weak, multivalent
forms of saccharides are usually involved in the biological
processes to amplify these interactions, which is called the
“glycocluster effect.”1,2 Many synthetic glycoclusters, for
example, glycopolymers,3–5 glycodendrimers,6,7 and glycona-
noparticles,8–12 have been reported to amplify the saccharide
signals as the natural saccharide ligands such as glycopepti-
des13 and glycoproteins14 on the cell surface. In particular,
glycopolymers containing pendant saccharides have received
considerable attention in the fields of polymer chemistry,
material science, and biomedicine. However, the synthesis of
these glycopolymers requires multistep processes and labori-
ous tasks, including the protection and deprotection of
hydroxy groups on saccharides. Typically, the glycomono-
mers for the preparation of glycopolymers were synthesized
by first protecting all the hydroxy groups of the starting free
saccharides, then activating their anomeric position, intro-
Additional Supporting Information may be found in the online version of this article.
C
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preparation of glycomonomers and glycopolymers starting
from free saccharides.
flow rate of 0.5 mL/min at 30 ꢀC. Pullulan was used as a
standard.
Recently, we developed a one-step synthesis protocol for var-
ious glycosyl derivatives using the dehydrative condensing
agent, 2-chloro-1,3-dimethylimidazolinium chloride (DMC),
without any protection of the hydroxy groups in water.30–34
This approach is called “Shoda activation.”35 Using this
method, various kinds of aryl 1-thioglycosides have been
synthesized using DMC and arylthiol agents in aqueous solu-
tion. In this article, we report on a one-pot protecting-group-
free synthesis of glycomonomers 2a and b starting from free
saccharides via the direct synthesis of 4-aminophenyl 1-thio-
glycosides 1a and b and the following acrylamidation.
One-Pot Synthesis of Glycomonomers
4-(N-Acrylamide)phenyl 1-thio-a-D-mannopyranoside 2a
DMC (169 mg, 1.0 mmol) was added to a mixture of Man
(36 mg, 0.2 mmol), TEA (415 mL, 3.0 mmol), and
4-aminobenzentiol (125 mg, 1.0 mmol) in water/acetonitrile
(1.0 mL, 1/1 [v/v]). The resulting mixture was stirred for
1.5 h at 0 ꢀC, then extracted with chloroform to remove
excess 4-aminothiophenol. The aqueous layer containing 1a
was diluted with water (0.5 mL) and tetrahydrofuran (THF)
(1.0 mL), and added with TEA (250 mL, 1.8 mmol) and acryl-
oyl chloride (73 mL, 0.9 mmol) at 0 ꢀC. The mixture was
stirred overnight at room temperature. The product was
purified by preparative HPLC (column; COSMOSIL 5C18-MS-
II [20 3 250 mm, Nacalai Tesque], eluent; acetonitrile/
water 5 10/90, flow rate; 12 mL/min, column oven; 40 ꢀC,
detection; UV [254 nm]), then concentrated in vacuo and
freeze-dried to give 4-(N-acrylamide)phenyl 1-thio-a-D-
mannopyranoside (ManAAm) (41 mg, 0.61 mmol, 61%).
In addition, we demonstrate
a
reversible addition-
fragmentation chain transfer (RAFT) living radical polymer-
ization36,37 of 2a and b to obtain glycopolymers 3 having
pendant thioglycosides. The glycopolymers having a thiol ter-
minal are also prepared by RAFT polymerization of 2 in the
presence of a trithiocarbonate derivative as a chain transfer
agent (CTA) and the following reduction of 3, and can be
immobilized on gold surfaces. Using the resulting
glycopolymer-modified gold nanoparticles (GNPs) and gold-
coated quartz crystal microbalance (QCM) sensors, we dem-
onstrate the detection of protein–saccharide interactions for
which UV–visible spectroscopy and QCM analysis38 are effi-
ciently used.
1H NMR (600 MHz, DMSO-d6, d): 7.61 (d, 2H, Ph, J 5 7.8 Hz),
7.41 (d, 2H, Ph, J 5 7.8 Hz), 6.40 (m, 1H, CH2@CH), 6.24 (d,
1H, CH2@CH, J 5 17.4 Hz), 5.75 (d, 1H, CH2@CH, J 5 10.2
Hz), 5.22 (s, 1H, H-1 of Man), 3.85 (s, 1H, H-2), 3.78 (s, 1H,
H-5), 3.64 (d, 1H, H-6, J5,6 5 12.0 Hz) 3.50–3.44 (3H, H-3, H-
4, and H-6); 13C NMR (150 MHz, DMSO-d6, d): 163.7 (C@O),
138.7, 133.1, and 132.0 (Ph), 128.9 and 127.7 (vinyl), 120.3
(Ph), 89.7 (C-1 of Man), 75.6 (C-5), 72.1 (C-2), 71.8 (C-3),
67.4(C-4), 61.2 (C-6); ESI-MS: [M 1 Na]1 calcd for
EXPERIMENTAL
Materials
C15H19NO6S, 364.083; found, 364.046.
D-Mannose (Man), lactose (Lac) monohydrate, triethylamine
(TEA), acrylamide (AAm), a,a0-azobisisobutyronitrile (AIBN),
and sodium borohydride (NaBH4) were purchased from
Nacalai Tesque (Kyoto, Japan). AAm was used after recrystalli-
zation from chloroform/methanol 5 10/3. 2-Chloro-1,3-
dimethylimidazolinium chloride (DMC), 4-aminobenzentiol,
and acryloyl chloride were purchased from Tokyo Chemical
Industry (Tokyo, Japan). A CTA, 2-(benzylsulfanylthiocarbonyl-
sulfanyl)ethanol, was synthesized using 2-mercaptoethanol,
carbon disulfide, and benzyl bromide according to the litera-
ture.39 Organic solvents were commercially supplied. Conca-
navalin A from Canavalia ensiformis (ConA) and peanut lectin
from Arachis hypogaea (PNA) were purchased from J-Oil Mills
(Tokyo, Japan). Gold nanoparticle (GNP) solution (particle
size: 40 nm) was purchased from Tanaka Kikinzoku Kogyo
K.K. (Tokyo, Japan).
4-(N-Acrylamide)phenyl 1-thio-b-lactoside 2b
DMC (237 mg, 1.4 mmol) was added to a mixture of Lac
monohydrate (72 mg, 0.2 mmol), TEA (610 mL, 4.4 mmol),
and 4-aminobenzentiol (250 mg, 2.0 mmol) in water/aceto-
nitrile (1.0 mL, 4/1 [v/v]). The resulting mixture was stirred
for 1.5 h at 0 ꢀC, then extracted with chloroform to remove
excess 4-aminothiophenol. The aqueous layer containing 1b
was diluted with water (0.4 mL) and THF (0.8 mL), and
added with TEA (250 mL, 1.8 mmol) and acryloyl chloride
ꢀ
(73 mL, 0.9 mmol) at 0 C. The mixture was stirred overnight
at room temperature. The product was purified by prepara-
tive HPLC (column; COSMOSIL 5C18-MS-II [20 3 250 mm,
Nacalai Tesque], eluent; acetonitrile/water 5 8/92, flow rate;
12 mL/min, column oven; 40 ꢀC, detection; UV [254 nm]),
then concentrated in vacuo and freeze-dried to give 4-
(N-acrylamide)phenyl 1-thio-b-lactoside (LacAAm) (57 mg,
0.56 mmol, 56%).
Characterization
NMR spectra were recorded using a Bruker BioSpin AV-600
spectrometer. ESI mass spectra were recorded using a
Bruker Daltonics micrOTOF Q-III spectrometer. Gel permea-
tion chromatography (GPC) measurements were conducted
using a system consisting of a JASCO PU-2089 pump, a CO-
2065 column oven, an RI-2031 refractive index detector, and
a Shodex OHpak SB-804 HQ (8.0 3 300 mm) column. Phos-
phate buffer (20 mM, pH 7.0) was used as the eluent at a
1H NMR (600 MHz, DMSO-d6, d): 7.60 (d, 2H, Ph, J 5 7.8 Hz),
7.37 (d, 2H, Ph, J 5 7.8 Hz), 6.40 (q, 1H, CH2@CH), 6.21 (d,
1H, CH2@CH, J 5 16.8 Hz), 5.75 (d, 1H, CH2@CH, J 5 10.2
Hz), 5.43, 5.10, 4.77, 4.70, and 4.63 (OH of Lac), 4.54 (d, 1H,
H-1 of Lac, J1,2 5 9.6 Hz), 4.19 (d, 1H, H-1’ of Lac, J1’,2’ 5 6.6
Hz), 3.73 (d, 1H, H-6, J5,6 5 11.4 Hz), 3.59–3.20 (m, 10H,
Lac), 3.04 (t, 1H, H-2); 13C NMR (150 MHz, DMSO-d6, d):
163.7 (C@O), 138.4, 132.5, and 132.0 (Ph), 128.1 and 127.7
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SCHEME 1 One-pot synthesis of glycomonomers 2a and b.
(vinyl), 120.2 and 120.1 (Ph), 104.1 (C-1’ of Lac), 87.4 (C-1
of Lac), 80.5 (C-4), 79.2 (C-3), 76.5 (C-5), 75.9 (C-3’), 73.5
(C-5’), 72.3 (C-2), 70.9 (C-2’), 68.5 (C-4’), 60.9 (C-6’), 60.8
(C-6); ESI-MS: [M 1 Na]1 calcd for C21H29NO11S, 526.136;
found, 526.116.
(100,000g, 30 min). The resulting solid was washed with
water, centrifuged in the ultracentrifuge, and freeze-dried to
give glycopolymer-modified GNP.
Protein Recognition of Glycopolymer-Modified GNPs
The glycopolymer-modified GNP was dispersed in PBS and
assayed using lectins, ConA, and PNA. UV–vis absorption
spectra were recorded using a JASCO V-550 spectrometer at
room temperature.
Synthesis of Glycopolymers 3
All polymerization reactions were performed at 1.0 M in all
monomers concentration. AAm, glycomonomer 2a and b,
AIBN, and CTA were dissolved in 0.25 mL dimethyl sulfoxide
(DMSO) in a glass tube. The resulting solution was degassed
by three freeze–thaw cycles, then the glass tube was sealed
under vacuum and heated at 70 ꢀC for 24 h. The products
were purified by dialysis (Spectra/Por 7 MWCO 3500)
against deionized water and freeze-dried to give glycopoly-
mers 3.
Detection of Glycopolymer–Protein Interaction by QCM
A PBS solution of thiol-terminated glycopolymer 4 (0.1 mg/mL)
was dropped on the gold-coated QCM sensor. After 90 min,
the QCM sensor was washed with water. The QCM analysis
was conducted in PBS using an Initium AFFINIX Q4
instrument.
Polyacrylamide Bearing 4-Phenyl 1-thio-a-D-
mannopyranoside
RESULTS AND DISCUSSION
1H NMR (600 MHz, D2O, d): 7.5–7.1 (Ph), 5.4 (H-1 of Man),
4.2–4.0 (H-2 and H-5), 3.8–3.6 (H-3, H-4, and H-6), 2.5–1.4
(polyAAm).
Synthesis of Glycomonomers
Thioglycosidic monomers 2a and b having an acryl amide
group at the anomeric position of saccharide were synthe-
sized by a one-pot procedure involving the direct synthesis
of thioglycosides 1a and b and the following acrylamidation
in which no protection of the hydroxy groups was necessary
(Scheme 1). Table 1 summarizes the first step of the synthe-
sis, the reaction of Man or Lac with a mixture of DMC,
4-aminobenzentiol, and TEA in water containing acetonitrile
to obtain 1a and b. Man was preferentially converted to a-
thioglycoside 1a. It was found out that the yield of thioglyco-
side 1a was quantitative when five equivalent amounts of
both DMC and 4-aminobenzentiol relative to free saccharide
(entry 2). When the amount of DMC was decreased, the yield
significantly decreased to 63% (entry 1). At least three
equivalent amounts of TEA were required relative to DMC
for the reaction because two equivalents of TEA are used as
Polyacrylamide Bearing 4-Phenyl 1-thio-b-lactoside
1H NMR (600 MHz, D2O, d): 7.5–7.1 (Ph), 4.4 (H-1 of Lac),
3.9–3.3 (Lac), 2.5–1.4 (polyAAm).
Preparation of Glycopolymer-Modified GNPs
The glycopolymer 3 (9 mg) and NaBH4 (2 mg) were dis-
solved in water (100 mL) and stirred for 1 h at room temper-
ature. The reaction mixture was purified by dialysis
(Spectra/Por 7 MWCO 3500) against deionized water, and
the thiol-terminated glycopolymer 4 was obtained by freeze-
drying. Then 4 (1 mg) was dissolved in citric acid solution
containing GNP (1 mL) and shaken overnight in the dark.
The solution was centrifuged in an ultracentrifuge
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TABLE 1 Synthesis of Thioglycosides 1a and b Using DMC
Free
DMC/Thiol/TEA
(equiv.)
Yield
(%)a
a/b
Ratioa
Entry
Saccharide
1
2
3
Manb
Manb
Lacc
2/5/6
5/5/15
7/10/22
63
93/7
96/4
4/96
Quant.
Quant.
a
Determined by 1H NMR.
b
c
200 mM in H2O/MeCN 5 1/1.
200 mM in H2O/MeCN 5 4/1.
the acid scavenger for neutralizing the hydrogen chloride
produced from DMC. The remaining one equivalent of TEA is
required as a base for promoting the deprotonation of the
anomeric hydroxy group. Acetonitrile was used as a co-
solvent for dissolving 4-aminobenzentiol. Lac was also con-
verted to b-thioglycoside 1b quantitatively although a large
amount of DMC was necessary (entry 3). Because Lac was
reacted in a medium containing a higher ratio of water than
that in the case of Man, the intermediate may have easily
been hydrolyzed by water. A hydroxy group at the 2-position
of the saccharide strongly affects the anomeric structure of
the product because one of the intermediates of this reaction
is 1,2-anhydro sugar.32 Man has an axial hydroxy group
(X1 5 OH) whereas Lac has an equatorial hydroxy group
(X2 5 OH) at the 2-position, resulting in production of the a-
thioglycoside and b-thioglycoside, respectively, after the
nucleophilic attack of the arylthiol.
The second step of the synthesis, the coupling between 1a
and b and acryloyl chloride, was also conducted in aqueous
solution without isolating 1a and b. The amino group selec-
tively reacted with acryloyl chloride in aqueous solution
because the nucleophilicity of amino group was higher than
those of hydroxy group and water.40,41 After the first step
reaction, the reaction mixture was washed with chloroform
to thoroughly extract excess 4-aminobenzentiol and was
directly added with acryloyl chloride to yield 2a and b
(Table 2). A small amount of stereoisomeric by-product was
removed by preparative HPLC to provide glycomonomers 2a
and b in good yields for both Man and Lac. The 1H NMR
spectra of the isolated products showed signals at 7.6–7.4
and 6.4–5.7 ppm, which were assigned to the aromatic pro-
tons and vinyl protons, respectively. The 13C NMR spectra
SCHEME 2 Synthesis of glycopolymers 3 by RAFT polymeriza-
tion and their immobilization on gold surfaces.
showed signals at 89.7 and 87.4 ppm, which were assigned
to the anomeric carbons of thioglycosides from Man and Lac,
respectively. This one-pot approach from free saccharides is
facile and effective for the synthesis of glycomonomers, and
can be applied not only to mono- and di-saccharides but also
to oligosaccharides.
Synthesis of Glycopolymers
The glycomonomers 2a and 2b were subjected to RAFT
polymerization to obtain glycopolymers 3. The polymeriza-
tion reaction was performed in DMSO at 70 ꢀC using AIBN
and 2-(benzylsulfanylthiocarbonylsulfanyl)ethanol as the ini-
tiator and CTA, respectively (Scheme 2, Table 3). The conver-
sions of polymerization reactions reached over 80%.
Glycopolymers 3 bearing Man or Lac residues were obtained
as monomodal distribution polymers in good yields with
their dispersities maintained relatively low (<1.3) analyzed
by GPC measurement. The saccharide unit ratios in the prod-
uct polymers exactly agreed with the feed ratios of the glyco-
monomers. When the RAFT copolymerization was performed
on 10% Lac-AAm feed, the saccharide unit ratios in polymer
TABLE 2 One-Pot Synthesis of Glycomonomers 2a and b from
Free Saccharidea
Free
Acryloyl
Yield
(%)b
Entry
Saccharide
Chloride/TEA (equiv.)
1
2
Man
Lac
4.5/9
4.5/9
61 (a)
56 (b)
a
First step reactions were performed using the conditions in Table 1,
entries 2 and 3.
b
Isolated yield from free saccharide using preparative HPLC.
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were kept around 10% on wide range conversions (data
were shown in Supporting Information). These results sug-
gested that the reactivities of glycomonomer and AAm were
almost equal. These reactions certainly produced random
co-polymerization products. When the polymerization reac-
tion was performed under the mole ratio, monomer/
CTA 5 450/1 (Table 3, entry 1), the analytical Mn value of
obtained glycopolymer was higher compared with the case
of mole ratio, monomer/CTA 5 150/1 (Table 3, entries 2–
4). This RAFT co-polymerization reaction using the glyco-
monomer controlled the molecular weight by changing the
mole ratio of agents. The 1H NMR spectra of the polymer
products showed the aromatic proton signals at 7.5–7.1
and the polymer main-chain signals at 2.5–1.4 ppm in addi-
tion to the saccharide residue signals at 5.4–3.2 ppm. The
trithiocarbonate terminal group introduced to the glycopol-
ymers was easily and quantitatively reduced to a thiol
group using NaBH4 to obtain the thiol-terminated glycopol-
ymers 4. We checked the removal of the end group using
homopolymer of AAm (Table 3, entry 5), which had no sac-
charide, by 1H NMR spectroscopy because the signals of
removed ethanol group shown 3.6 and 3.8 ppm were over-
lapped with saccharide signals of glycopolymers. After the
treatment of trithiocarbonate-terminated polymers by
NaBH4, the signals of terminal ethanol group were perfectly
disappeared (data were shown in Supporting Information).
This reduction was also occurred in the case of glycopoly-
mers, giving rise to thiol-terminated glycopolymers quanti-
tatively. They were easily immobilized on the surface of
GNPs and gold-coated QCM sensor via AuAS bond forma-
tion in aqueous solution.
Protein Recognition of Glycopolymer-Modified GNPs
The synthesized glycopolymers were immobilized on
GNPs via AuAS bond formation to investigate the glyco-
cluster effect. The immobilization was performed using
the thiol-terminated glycopolymers 4 obtained by the
reduction of 3 with NaBH4. The recognition of the sac-
charide residues immobilized on GNPs was investigated
with lectins, ConA, and PNA. When ConA recognizing a-
Man residues was added to poly(ManAAm(5.3)-co-AAm)-
modified GNP dispersed in phosphate buffered saline
(PBS), the absorbance of GNP gradually decreased with
increasing the amount of ConA added [Fig. 1(a)], suggest-
ing that the lectin induced aggregation of glycopolymer-
modified GNP due to the lectin–saccharide interactions.
Conversely, the absorbance little decreased upon the
addition of PNA that recognizes b-galactoside residues
[Fig. 1(b)]. These results indicated that ConA strongly
interacted with Man residues of the glycopolymer. Con-
versely, the Lac-containing polymer, poly(LacAAm(9.8)-co-
AAm), was selectively recognized by PNA [Fig. 1(c)].
When ConA and PNA were added to polyAAm-modified
GNP as a control, the absorbance did not decreased (data
were shown in supporting information). ConA and PNA
recognized corresponding saccharides without nonspecific
adsorption on the GNPs. Here, the aggregates of poly(La-
cAAm(9.8)-co-AAm)-modified GNP formed by the addition
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FIGURE 1 UV–vis spectra of glycopolymer-modified GNPs interacted with lectins, (a) poly(ManAAm(5.3)-co-AAm)-modified GNP
and ConA, (b) poly(ManAAm(5.3)-co-AAm)-modified GNP and PNA, (c) poly(LacAAm(9.8)-co-AAm)-modified GNP and PNA, (d)
addition of a saturated solution of free Lac to a mixture of poly(LacAAm(9.8)-co-AAm)-modified GNP and 100 nM PNA.
of 100 nM PNA were re-dispersed by the addition of a
saturated solution of free Lac in about 60% relative to
the initial concentration [Fig. 1(d)]. This result indicated
that the specific recognition of lectin by glycopolymer-
modified GNPs is much stronger than that by free saccha-
ride because of the multivalent effect of saccharide resi-
dues on the synthetic polymer chain.
Detection of Glycopolymer–Protein Interaction by QCM
The interaction between the glycopolymers and the lectins
was also investigated by QCM. Thiol-terminated glycopoly-
mers 4 were easily immobilized on a gold-coated QCM
sensor in aqueous solution in a concentration of around
50 ng/cm2. When 200 nM PNA was added to the poly(La-
cAAm(9.8)-co-AAm)-immobilized QCM sensor in PBS, a
significant decrease in frequency (DF) was detected indi-
cating mass increase due to the saccharide-bound lectin
[Fig. 2(a)]. Conversely, when 200 nM ConA was added to
the poly(LacAAm(9.8)-co-AAm)-immobilized QCM sensor,
no decrease in frequency was observed [Fig. 2(b)]. These
results clearly indicated that the glycopolymer bearing Lac
residue strongly attracts PNA on the gold surface without
nonspecific adsorption. We estimated the association
constant (Ka) for the lectin–saccharide interaction using
a
Langmuir-type isotherm determined by the QCM
FIGURE 2 Interaction between poly(LacAAm(9.8)-co-AAm) and
200 nM lectin by QCM, (a) PNA, (b) ConA.
analysis. This provided only the Ka, due to the complex
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FIGURE 3 QCM analysis of the interaction of the immobilized glycopolymers and lectins, (a) time-course of the frequency shift for
poly(ManAAm(9.4)-co-AAm) and ConA, (b) equilibrium frequency shift of poly(ManAAm(9.4)-co-AAm) using ConA, (c) equilibrium
frequency shift of poly(LacAAm(9.8)-co-AAm) using PNA.
CONCLUSIONS
micro-equilibrium involved in multivalent interac-
tions.42–46 Figure 3(a) shows the time-course of DF for
We succeeded in synthesizing glycopolymers without any
protection of hydroxy groups on saccharide. The thioglycosi-
dic monomers were readily prepared by one-pot reaction of
free saccharide with 4-aminobenzentiol in the presence of
DMC and 4-aminobenzentiol, and the following acrylamida-
tion. They were polymerized by RAFT polymerization into
glycopolymers, which were easily immobilized on gold surfa-
the interaction of poly(ManAAm(9.4)-co-AAm) and ConA
increasing concentration of the lectin. A large change in
DF was observed upon the first addition of lectin, even at
a low concentration of lectin. As more ConA was added,
the change in DF became significantly smaller. It was
therefore clear that the glycopolymer on the gold-coated
QCM sensor interacted with ConA. The equilibrium fre-
ces via AuAS bond formation. Strong interactions between
quency shift obtained from this result allowed us to esti-
the clustered saccharides and lectins were confirmed by the
mate Ka as 2.16 3 107 M21 [Fig. 3(b)]. In the cases of
aggregation of glycopolymer-modified GNPs and by mass
poly(LacAAm(9.8)-co-AAm)/PNA and poly(LacAAm(20.4)-
co-AAm)/PNA, the Ka values were also estimated to be
1.97 3 107 and 0.73 3 107 M21, respectively [Fig. 3(c)].
The increase of saccharide unit in polymer did not effect
for Ka value, suggesting that 10% saccharide unit in poly-
mer was satisfied for having glycocluster effect. It has
been reported that the Ka value for the interaction
between lectin and free saccharide molecule is around 103
M21. This value is not large compared with other biomo-
lecular interactions, for example, antigen–antibody and
DNA–DNA. In contrast, glycopolymers such as polyacryl-
amides having pendant thiophenyl saccharides exhibit a
large Ka value, supporting strong interaction with the cor-
responding lectin because of the multivalent effect of the
saccharides on the synthetic polymer chain.
increase of the glycopolymer-immobilized gold-coated sensor
by QCM. The glycopolymers were strongly and specifically
recognized by the corresponding lectins, with estimated Ka
values of around 107 M21. These results indicated that the
synthesized glycopolymers amplified saccharide–protein
interactions due to the multivalency of the saccharides. The
present facile and efficient synthetic method for glycopoly-
mers in aqueous solution from free saccharides can be
applied not only to mono- and di-saccharides but also to var-
ious oligosaccharides with higher molecular weight and/or
neuraminic acid moieties such as N-linked and O-linked oli-
gosaccharides, contributing to the development of various
biomaterials such as pathogen inhibitors, cell cultivation
agents for tissue engineering, and biosensors for the detec-
tion of viruses and toxins.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2014, 52, 3513–3520
3519

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POLYMER SCIENCE
ARTICLE
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ACKNOWLEDGMENT
22 S. Slavin, J. Burns, D. M. Haddleton, C. R. Becer, Eur.
Polym. J. 2011, 47, 435–446.
This work was supported by JSPS KAKENHI No. 25810075.
23 S. Beghdadi, I. A. Miladi, D. Addis, H. Ben Romdhane, J.
Bernard, E. Drockenmuller, Polym. Chem. 2012, 3, 1680–1692.
24 N. Vinson, Y. Z. Gou, C. R. Becer, D. M. Haddleton, M. I.
Gibson, Polym. Chem. 2011, 2, 107–113.
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