Carbohydrate-based amphiphilic diblock copolymers with pyridine for the sensitive detection of protein binding

Article abstract of DOI:10.1166/jnn.2014.9365

Glycopolymers are useful macromolecules for presenting carbohydrates in multivalent form. Here, amphiphilic block copolymers consisting of hydrophilic lactose and hydrophobic pyridine were synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT). RAFT polymerization of 2-O-methacryloyloxyethyl-(-D-lactoseheptaacetate) (2-O-MALac) was performed using cumyl dithiobenzoate (CDB) as the chain transfer agent to give well-defined glycopolymers. The livingness of the process was further demonstrated by successfully chain-extending one of obtained glycopolymers with 4-pyridyl methyl methacrylate affording narrow dispersed diblocks. With the obtained block copolymers, a glycosurface was generated on the gold surface of quartz crystal microbalance (QCM) through self-assembled strategy by the use of gold affinitive pyridine functional group. Furthermore, the resulting glycosurface was used to detect the binding of lactose specific lectin, ricinus communis agglutinin (RCA120) without non-specific protein adsorption.

Full text of DOI:10.1166/jnn.2014.9365

Article
Journal of
Nanoscience and Nanotechnology
Vol. 14, 6764–6773, 2014
Copyright © 2014 American Scientific Publishers
All rights reserved
www.aspbs.com/jnn
Printed in the United States of America
Carbohydrate-Based Amphiphilic Diblock
Copolymers with Pyridine for the Sensitive
Detection of Protein Binding
Hidenori Otsuka1ꢀ2ꢀ∗, Toshiya Hagiwara1, and Sayuri Yamamoto1
1Faculty of Science, Department of Applied Chemistry, Tokyo University of Science,
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
2Department of Chemical Science and Technology, Graduate School of Chemical Science and Technology,
Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Glycopolymers are useful macromolecules for presenting carbohydrates in multivalent form. Here,
amphiphilic block copolymers consisting of hydrophilic lactose and hydrophobic pyridine were syn-
thesized via reversible addition-fragmentation chain transfer polymerization (RAFT). RAFT polymer-
ization of 2-O-methacryloyloxyethyl-(ꢀ-D-lactoseheptaacetate) (2-O-MALac) was performed using
cumyl dithiobenzoate (CDB) as the chain transfer agent to give well-defined glycopolymers. The
livingness of the process was further demonstrated by successfully chain-extending one of obtained
glycopolymers with 4-pyridyl methyl methacrylate affording narrow dispersed diblocks. With the
Delivered by Publishing Technology to: Chinese University of Hong Kong
obtained block copolymers, a glycosurface was generated on the gold surface of quartz crystal
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
microbalance (QCM) through self-assembled strategy by the use of gold affinitive pyridine functional
Copyright: American Scientific Publishers
group. Furthermore, the resulting glycosurface was used to detect the binding of lactose specific
lectin, ricinus communis agglutinin (RCA120) without non-specific protein adsorption.
Keywords: Self-Assembly, Amphiphilic Glycopolymer, Lectin Binding, Specific Recognition,
QCM.
1. INTRODUCTION
multivalent forms of synthetic ligands, either polymers or
dendrimers, often have amplified inhibitory effects over
the monovalent counterparts. In recent years, a great num-
ber of glycopolymers have been designed and synthe-
sized to mimic the multivalent functionalities of natural
glycoconjugates.13–17 Caebohydrate-based surface (glyco-
sylated surface) has been under active development to
study protein–carbohydrate interaction.
Recently functional glycopolymers have been synthe-
sized with surface anchoring groups located along the
polymer backbone to generate glycosurface with poten-
tial utility in bio- and immunochemical assays18–22 as well
as biocapture analysis. Keissling and coworkers prepared
the end-functionalized 3,6-disulfogalactose polymers by
ring-opening metathesis polymerization (ROMP). These
materials were immobilized onto the surfaces for the spe-
cific interaction with soluble P- and L-selectin.23 Chaikof
et al. synthesized biotin chain-terminated glycopolymers
through cyanoxyl-mediated free-radical polymerization for
surface glycoengineering.24 On the other hand, among
Carbohydrates are known to play essential roles in var-
ious biological processes such as cell adhesion, blood
coagulation, viral infection, immune response, and apop-
tosis, because carbohydrates have been found on most cell
surfaces in the forms of glycoproteins, glycolipids, and
polysaccharides.1–9 Indeed, numerous biological phenom-
ena are based on the carbohydrate–protein interactions.
The studies of protein–carbohydrate interaction have been
challenged due to the complexity and heterogeneity of
cell surfaces, the inherent structure complexity of carbo-
hydrates, and the typically weak affinities of the bind-
ing. In the biological context, this limitation has been
overcome by multivalent interactions, i.e., simultaneous
contact between the clustered carbohydrates on cell sur-
face and protein receptors that contain multiple carbohy-
drate recognition domains, which is accepted as “cluster
glycoside effect.”1–4ꢀ10–12 It has been reported that the
∗Author to whom correspondence should be addressed.
6764
J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 9
1533-4880/2014/14/6764/010
doi:10.1166/jnn.2014.9365

Otsuka et al.
Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
the large variety of macromolecular amphiphiles that
exist in nature, many possess carbohydrate residues as
hydrophilic components. Therefore, amphiphilic block
copolymers bearing pendant carbohydrate residues can
serve as mimics of naturally occurring amphiphiles such
as glycolipides and glycoproteins for a better under-
standing of essential biological processes in living sys-
tems. Because amphiphilic block copolymers are known
to exhibit various interesting properties such as sur-
face activity, aggregate formation into micelle, and self-
assembled monolayer on the surface,25–29 glycopolymers
with amphiphilic character is expected to form densely
packed glycosylated surface for suitable multivalent inter-
action. Despite the considerable progress in their devel-
opments, only few researches have been directed to the
synthesis of block copolymers with pendant carbohydrate
residues.
packed glycopolymeric recognition layers which could be
chemo-adsorbed onto gold surface of QCM and generate
multivalent binding cavity. The obtained glycosylated sur-
face was verified by the specific recognition with a lectin
without adsorption of nonspecific proteins.
2. EXPERIMENTAL DETAILS
2.1. Materials
2-Hydroxyethyl methacrylate (HEMA) (Aldrich) was
distilled under reduced pressure before use. Lactose
actaacetate was synthesized by the reaction of lactose
and acetic anhydride according to literature.30ꢀ31 2,2ꢀ-
Azoisobutyronitrile (AIBN) (Aldrich) as initiator was puri-
fied by recrystallization in ethanol. All other chemicals
were of analytical grade and used as received. Water used
in this syudy was purified by passing it through a Milli-Q
System (Nihon MilliporeCo., Tokyo, Japan) until its spe-
We herein describe the amphiphilic block copolymers
consisting of hydrophilic lactose ligands for lectin bind-
ing and hydrophobic pyridines as anchor groups on gold
surface. Our interest is to combine the self-assembly strat-
egy with the specially designed functional glycopoly-
mers (Scheme 1) to engineer self-organized, densely
cific conductivity fell below 0.1 ꢂS cm−1
.
2.2. Preparation of Cumyl Dithiobenzoate (CDB)
The synthesis procedure of CDB has been reported in
the literature.32 Bromobenzene (8.02 mL, 76.4 mmol)
BF3-ethercomplex
O
OAc
OAc
OAc
OAc
HO
OAc
OAc
Delivered by Publishing Technology to: Chinese University of Hong Kong
O
O
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
O
O
O
O
O
O
O
COAocpyright: American Scientific Publishers
AcO
AcO
CH2Cl2
O
S
AcO
AcO
OAc
OAc
OAc
OAc
S
S
m
S
O
O
OAc
OAc
OAc
AcO
AIBN, DMF
O
O
O
O
OAc AcO
OAc
S
S
n
m
O
Poly(2-O-MALac)12
+
O
O
O
O
AIBN, DMF, 70 ºC
O
OAc
OAc
O
OAc
AcO
O
O
O
N
N
OAc
OAc
AcO
Pyridine linker as anchor
group on gold surface
Lactose ligand for
multivalent lectin binding
Scheme 1. Synthesis of glycomonomer and its RAFT polymerization with cumyl dithiobenzoate (CDB). Chain extension of poly(2-O-
methacryloyloxyethyl-(ꢁ-D-lactoseheptaacetate)) with 4-pyridyl mthyl methacrylate and block copolymer obtained by these sequential polymerizations.
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014
6765

Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
Otsuka et al.
was mixed with 120 mL of dried tetrahydrofuran (THF),
and then the solution was added to magnesium turnings
(1.86 g, 76.5 mmol, in diethyl ether) under a nitrogen
atmosphere. The mixture was stirꢁred for 30 min or more
and then heated to reflux at 60 C for 30 min. Carbon
disulfide (5.08 mL, 84 mmol) was added slowly after the
mixture was cꢁhilled with ice water. The reaction mixture
was kept at 0 C for 2 h under stirring and then carefully
poured into ice water (500 mL) followed by the addition
of hydrochloric acid (1N) until the solution showed acidic
pH. The product was extracted with diethyl ether three
times. After removal of the ether, the crude dithiobenzoate
acid was obtained as a dark red-brown oil.
saturated aqueous sodium hydrogen carbonate and dried
over MgSO4. The organic layer was evaporated using
a rotatory evaporator under reduced pressure to get the
residue, which was purified further by column chromatog-
raphy (SiO2) using acetone-dichloromethane (1:30 to 1:25,
v/v) as eluent. The glycomonomer was then obtained by
reprecipitation from methanol-water (5.36 g, 50.2 wt%)
1
as white solid. H-NMR (CDCl3) ꢄ (ppm): 6.13 (1 H, d,
J = 8ꢅ5 Hz), 5.60 (1 H, td, J = 3ꢅ4, 1.5 Hz), 5.35 (1 H,
d, J = 3ꢅ4 Hz), 5.19–4.93 (4 H, m), 4.51–3.77 (13 H, m),
2.15–1.95 (24 H, m).
2.5. Synthesis of Lactose-Based Amphiphilic
Block Copolymer, Poly((2-O-MALac)-b-Py),
Dithiobenzoate acid (10.5 g, 68.1 mmol), ꢃ-methyl-
styrene (13.3 mL, 102 mmol), carbon tetrachloride
(200 mL), and a small amount of acid catalyst (p-toluene-
sulfonic acid, 260 mg, 1.36 mmol) were combined under
nitrogen and heated to reflux at 90 ꢁC for 12 h. After evap-
oration of the solvent and excess monomer using a rota-
tory evaporator, the residue was purified twice by column
chromatography on silica gel with hexane as the eluent
to give cumyl dithiobenzoate as a dark-purple oil (yield
by RAFT Polymerization
Lactose-based block copolymers were synthesized with 2
times of RAFT polymerization step and deprotection step
of hydroxyl group on lactose. In these RAFT polymer-
ization, cumyl dithiobenzoate (CDB) was used as RAFT
agent. On the RAFT polymerization, lactose block was
constructed at first step, and then pyridine block was con-
1
structed stepwise. The conversion was determined by H
1
55 wt%). HNMR (500 MHz, CDCl3) ꢄ (ppm):7.85 (dd,
NMR from relative integration of the peaks correspond-
ing to the protons of the vinyl group and of the polymer
backbone in each polymerization.
2 H, J = 8.2, 1.2 Hz, 1), 7.56–7.54 (2 H, m, 2), 7.47
(t, 1 H, J = 7ꢅ5 Hz, 3), 7.34–7.30 (4 H, m, 4), 7.22 (t,
1 H, J = 7ꢅ3 Hz, 5), 2.01 (s, 6 H, 6).
2.6. Homopolymerization of Lactose Monomer
Lactose monomer (5.0 g, 6.68 mmol) was dissolved
Delivered by Publishing Technology to: Chinese University of Hong Kong
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
2.3. Preparation of 4-Pyridyl Methyl Methacrylate
Copyright: American Scientific Publishers
with a dimethylformamide solution (30 mL) of 2,2ꢀ-
azoisobutyronitrile (AIBN, 11 mg, 0.067 mmol) and cumyl
dithiobenzoate (CDB, 54.6 mg, 0.20 mmol). The reactor
was then sealed, degassed with three freeze-evacuate-thaw
cycles, and transferred to an oil bath preheated to 70 ꢁC. At
the end of the reaction for 6 days, the reaction mixture was
quenched in cold water, then precipitated in isopropylether
twice. The obtained glycopolymer was dried in vacuo for
24 h (Table I) and used as a macro-RAFT agent for the fol-
lowing chain extension with 4-pyridyl methyl methacrylate.
4-pyridyl methyl methacrylate as a pyridine monomer was
synthesized. Methacrylic acid (4.73 g, 55 mmol), 4-pyri-
dine methanol (5.4 g, 50 mmol), and 4-(1-pyrrolidinyl)
pyridine (740 mg, 5 mmol) were dissolved in dry
dichloromethane (100 ml) in a glass vessel. After N,Nꢀ-
dicyclohexylcarbodiimide (11.3 g, 55 mmol) was added to
the solution, the reaction mixture was stirred for 1 h at
room temperature. After the resulting insoluble urea was
removed by filtration, the mixture was washed with satu-
rated aqueous sodium hydrogen carbonate and dried over
MgSO4. The organic layer was evaporated under reduced
pressure to get the residue, which was purified by column
chromatography (SiO2, hexane/ethyl acetate = 1/1) yield-
ing colorless oil (8.1 g, 46 mmol, Y = 91ꢅ5%).
2.7. Chain Extension of Poly(2-O-
Methacryloyloxyethyl-(ꢁ-D-Lactoseheptaacetate))
with 4-Pyridyl Methyl Methacrylate
4-pyridyl methyl methacrylate (114 mg, 0.64 mmol) and
poly(2-O-MALac) (600 mg, 0.064 mmol) was dissolved
with a dimethylformamide solution (4 mL) of 2,2ꢀ-azoiso-
butyronitrile (AIBN, 3.5 mg, 0.021 mmol). The reactor
was then sealed, degassed with three freeze-evacuate-thaw
2.4. Preparation of the Glycomonomer, 2-O-
Methacryloyloxyethyl-(ꢁ-D-Lactoseheptaacetate)
(2-O-MALac)
To a 100 mL three-neck flask cooled with ice-water bath
were added a stirred solution of lactose octaacetate (10.0 g,
14.7 mmol,) and 2-hydroxyethyl methacrylate (2.06 g,
17.7 mmol) in dry dichloromethane and then BF3-etherate
(46–49%, 6.0 mL) was added dropwise. The reaction mix-
ꢁ
cycles, and transferred to an oil bath preheated to 70 C.
At the end of the reaction for 6 days, the reaction mixture
was quenched in cold water, then precipitated in isopropy-
lether twice. Finally, the acetyl group on lactose residue
was deprotected with hydrazine mono-hydrate in dimethyl
sulfoxide, reaction mixture was dialyzed versus water, and
the resulting aqueous solution was freeze dried to give pure
lactose block polymer (poly(Lac-b-Py)).
ꢁ
ture was stirred for 2 h at 0 C. Then the solution was
allowed to warm to room temperature and stirred for an
additional 18 h under under argon atmosphere. The mix-
ture was diluted with chloroform, washed with water and
6766
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014

Otsuka et al.
Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
Table I. Summary of RAFT polymerization experiments.
Reaction
time (h)
Conversion
(wt%)
Polymer
[M]/[CDB]/[I]
Mntha
MnNMRb
MnGPCc
Mw/Mn
Glycopolymer
Block copolymer
100/3/1
30/3/1
12
24
38
84
9815
11302
9409
10782
9364
–
1.18
–
Notes: aDetermined from conversion of glycomonomer (2-O-Methacryloyloxyethyl-(b-D-lactoseheptaacetate)); bDetermined from relative integration of the aromatic chain
end group peaks and polymer bachbone peak; cDetermined from THF GPC (PS calibration).
2.8. Characterization
the deposited mass in 1959. The equation is given as
1H NMR spectra were recorded on a JEOL JNM-AL500
spectrometer operated at 500 MHz with CDCl3 as a sol-
vent and with the tetramethylsilane as an internal standard.
Molecular weights and molecular weight distributions of
the glycopolymers were measured by gel permeation chro-
matography (GPC) analysis on TOSOH HLC-8220 sys-
tem comprising a SD-8022 solvent degasser, DP-8020
pump, CO-8020 column oven, and RI-8020 refractive
index detector. The system was equipped 20 × 4ꢅ6 mm
guard column (SuperHZ-H) and two 150×4ꢅ6 mm linear
columns (SuperHZM-H). THF including 20 mM triethy-
lamine was used as eluent at a flow rate of 1 mL min−1
follows:
2Fo2
ꢆF = −
ꢆm
√
ꢂqꢇq
Where ꢆF is the measured frequency change (Hz), F0 the
intrinsic crystal frequency of the QCM (5 × 106 Hz), Dm
the area mass density of the film (ng·cm−2), ꢂq the shear
modulous of quartz (2ꢅ95×1011 g·cm−1 ·s−2ꢈ, ꢇq the den-
sity of quartz (2.65 g·cm−3).
2.11. Construction of the Glycosylated Surface
After the gold chips were mounted in the QCM cell, milli-
Q water was run through the system until flat baselines
of frequency were achieved. Then, immobiiloization of
poly(Lac-b-Py) on the gold sensor chip surface was per-
formed using a QCM instrument. Milli-Q water solutions
of poly(Lac-b-Py) were injected at a flow rate of 10 ꢂL
ꢁ
while columns temperature was maintained at 40 C.
2.9. Characterization of QCM Gold Chip
Coated with Amphiphilic Block
Copolymers by Static Contact Angle
The wettability of all surfaces was estimated from the
−1
Delivered by Publishing Technology to: Chinese University ofꢁHong Kong
min for 200 s at 20 C under running Milli-Q water.
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
A resonant frequency on the gold sensor chip for this
Copyright: American Scientific Publishers
adsorption-rinsing of poly(Lac-b-Py) was monitored with
static contact angle measurements (CA-W contact angle
meter manufactured by Kyowa Kaimen Kagaku Co.). The
water-in-air system measurement was performed by a ses-
sile droplet technique, where a water droplet (Milli-Q qual-
time, and the amount of immobilized poly(Lac-b-Py) was
assessed by the decrease in resonant frequency. poly(Lac-
b-Py) solutions with different concentrations (0.008, 0.04,
0.2, 0.5, 1.0 mg mL−1) were injected on a sensor chip, and
then the plateau region for poly(Lac-b-Py) immobilization
was determined.
ꢁ
ity) was placed on the film surface at 25 C. The contact
angle of each film sample was measured on at least 15
spots, and the values were averaged.
2.12. Specific Adsorption of Protein on the
2.10. A Quartz-Crystal Microbalance
Glycosylated Surface and Estimation of the
Binding Affinity
(QCM) Analysis
Detection of poly(Lac-b-Py) adsorption and RCA120 bind-
ing to the constructed glycosylated surface was carried out
by a QCM (NAPiCOS, Nihon Dempa Kogyo Co., Ltd.,
Tokyo, Japan). Frequency decreases (mass increases) of
the QCM responding to the addition of glycopolymer and
RCA120 were followed with time. Experiments were car-
PBS buffer solution (pH = 7ꢅ4, 10−2 M PBS, 0.15 M NaCl)
was first slowly injected and flowed through the cell where
the gold chip was mounted with only one side exposed
to the liquid. The resonant frequency of the crystal in
the buffer solution was then recorded. After the frequency
reached a steady state, the resonant frequencies of RCA120
solutions (pH = 7ꢅ4, PBS buffer solution) with concentra-
tions from 0ꢅ084 × 10−7 to 8ꢅ4 × 10−7 M were collected
respectively. To quantitatively measure the binding affinity,
the association saturation conctant was calculated accord-
ing to the resonant frequency curve. Bovine serum albumin
(BSA) in PBS (pH = 7ꢅ4, 10−2 M PBS, 0.15 M NaCl) was
used as a reference protein to characterize the nonspecific
adsorption of the glycosylated surface. Similar detection
process was carried out by QCM in comparison with that
of RCA120 solution.
ꢁ
ried out at 20 C. The QCM employed is commercially
available 9 MHz, AT-cut quartz crystal deposited with gold
electrodes on both sides. The quartz crystal was cleaned
in piranha solution (98% concentrated H2SO4/30% H2O2,
3:1 v/v) for 30 min, followed by rinsing with deionized
water and drying with a stream of nitrogen gas before
measurement.
QCM has been used as a mass-sensing technique since
Sauerbrey first proposed the linear relationship between
the frequency change of the oscillating quartz crystal and
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014
6767

Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
Otsuka et al.
3. RESULTS AND DISCUSSION
3.1. Homopolymerization of Lactose Monomer
RAFT agent. For the vinyl glycomonomer polymerization,
–C(CH3)2Ph as the R group and –Ph as the Z group should
have a stronger activation ability to the glycomonomer.
Thus, cumyl dithiobenzoate (CDB) was used as the CTA
in this study. For the synthesis of CDB, bromobenzene and
dithiobenzoate acid as starting reagents was an effective
way to get the target product, providing the higher yield
of 55 wt%.
The synthesis procedure for glycopolymer is shown
in Scheme 1. In the first step, lactose octaacetate
was used to react with 2-hydroxyethyl methacrylate to
give corresponding lactose containing monomer under
the catalysis of BF3-Et2O. The glycomonomer obtained
was characterized by 1H NMR. The key to controlled
RAFT polymerizations is the suitable combination of
monomer/chain transfer agents (CTA). The effectiveness
of the chain transfer agents depends on their chain trans-
fer constant which is determined by the nature of the
groups X, Z, and R, the monomer, and the polymeriza-
tion conditions. The structures of X, Z, and R on the
RAFT agent play a crucial role in controlling the molec-
ular weight distribution and the rates of polymerization.
The most effective reagents for RAFT polymerization are
dithioester compounds, where X is sulfur, R is a free rad-
ical leaving group that is capable of reinitiating polymer-
ization, and Z is a group that modifies the activity of the
The polymerization of 2-O-MALac in DMF was car-
ried out using CDB as the RAFT agent and AIBN as the
initiator. After polymerization, a few amount of reaction
mixture was concentrated, and conversion was calculated
1
by integral value of monomer residue on HNMR spectra.
The rest of reaction mixture was purified with reprecipi-
tation from isopropylether twice. In this step, the ratio of
monomer and CTA were arbitrarily-changed, and the ratio
of CTA and initiator was fixed on 3:1.
Polymerization results are summarized in Table I. The
glycomonomer and glycopolymer were characterized by
1H NMR spectroscopy. Figure 1 shows the 1H NMR
(a)
f
f
f
OAc
OAc
OAc
e
O
g
O
O
b
c
a
O
f
O
AcO
f
O
AcO
f
d
OAc
f
OAc
f
f
Delivered by Publishing Technology to: Chinese University of Hong Kong
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
Copyright: American Scientific Publishers
a
a
e, g
d
b
c
b
S
(b)
c
S
m
O
O
b
OAc
OAc
O
OAc
O
O
O
AcO
a
AcO
OAc
OAc
b
c
a
Figure 1. 1H NMR spectra of (a) glycomonomer and (b) glycopolymer.
6768
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014

Otsuka et al.
Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
spectra of 2-O-MALac and poly(2-O-MALac) in CDCl3.
All of the saccharide signals can be identified in the region
3.5–5.5 ppm in Figure 1(b) and easily correlated with
those of the starting monomer (Fig. 1(a)). The signals
of the double bond protons (6.1, 5.6 ppm) disappeared
in Figure 1(b). Enlargement of the aromatic area clearly
displays the signals from the end-of-chain dithiobenzoyl
group, whereas polymer backbone alkyl protons can be
seen at 0.6–1.2 ppm. The molecular weight and molecu-
lar weight distribution of the obtained glycopolymers were
showed narrow polydispersity (1.18), which is significantly
lower than the value for a classical free-radical poly-
merization under the same reaction conditions.34 Inter-
mediate molecular weight distributions and conversions
1
were obtained from GPC and H NMR analysis of sam-
ples collected at increasing reaction times, respectively.
Conversion versus time plot for the polymerization of
glycomonomer is given in Figure 2(a). After an initial
inhibition period of 3 h, the homopolymerization of gly-
comonomer was proven to proceed with pseudo-first-order
kinetics consistent with a constant radical concentration.
When the GPC Mn values were plotted against conver-
sion, the Mn values linearly increased up to quantitative
monomer consumption as expected for a well-controlled
living process (Fig. 2(b)). However, the experimental trend
lines are systematically lower than the theoretical lines
with increasing conversion. This is most probably due
to the inadequacy of polystyrene standards to approx-
imate the hydrodynamic volume of poly(2-O-MALac)
in THF. Similar problems with polystyrene-equivalent
molecular weights of glycopolymers have been previously
reported in the literature.35ꢀ36 A further evidence for the
growth of the poly(2-O-MALac) in a controlled fashion
was given by the polydispersity index of the glycopoly-
mers which typically comprised of values between 1.15
and 1.25.
1
measured by GPC and H NMR, respectively. The final
molecular weight of polymer as determined by GPC and
1H NMR are in excellent agreement with the theoretical
values calculated from the formula
ꢉMꢊ0
M ꢉRAFTꢊ0
Mn = M
x +MRAFT
where MM and MRAFT are the molecular weights of
monomer and RAFT agent, respectively, x is the conver-
sion, and ꢉMꢊ0 and ꢉRAFTꢊ0 are the initial concentrations
of monomer and RAFT agent.33 The glycopolymer also
Delivered by Publishing Technology to: Chinese University of Hong Kong
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
3.2. Synthesis of Block Copolymers
Copyright: American Scientific Publishers
Poly((2-O-MALac)-b-Py)
To confirm the livingness of the poly(2-O-MALac)
obtained from these experiments, we investigated the abil-
ity to chain-extend the poly(2-O-MALac) to yield block
copolymers consisting of one water-soluble glycopoly-
mer block and one water-insoluble pyridine block (Py).
The reprecipitated poly(2-O-MALac) was used as macro-
RAFT agent for the polymerization of 4-pyridyl methyl
methacrylate (Scheme 1). The theoretical value was used
as molecular weight of macro-RAFT agent for the sec-
ond block polymerization. The growth of the second
block and purification was conducted with the same man-
ner of poly(2-O-MALac). The experimental molecular
weight of the purified final block copolymer determined
by 1H NMR (Mn(NMR) = 10782 g mol−1, poly((2-O-
MALac)12-b-Py8)) was in good agreement with the the-
oretical value (Mn(th) = 11302 g mol−1) determined by
conversion of pyridine monomer. These results clearly
corroborate the chain extension of poly(2-O-MALac) in
a controlled manner. The deprotection of acetyl groups
of block copolymer was treated with hydrazine mono-
hydrate in dimethyl sulfoxide. Figure 3 shows a typi-
cal 1H NMR spectrum after the hydrolysis. The acetyl
group protons (around 2 ppm) have disappeared after the
hydrolysis, which indicates the deprotection reaction was
complete.
Figure 2. (a) Pseudo-first-order plot obtained by 1H NMR for the
polymerization of 2-O-Methacryloyloxyethyl-(ꢁ-D-lactoseheptaacetate)
in DFM at 70 ꢁC. (b) Evolution of the molecular weight (Mn, GPC) (•)
and polydispersity with conversion for the RAFT polymerization of 2-O-
Methacryloyloxyethyl-(ꢁ-D-lactoseheptaacetate) in DMF at 70 ꢁC. The
reaction condition; [2-O-MALac] = 0ꢅ223 mol·L−1, [CDB] = 6ꢅ66×10−3
mol·L−1, [AIBN]= 6ꢅ66×10−5 mol·L−1. Full line: linear regression of
experimental values; dashed line: theoretical values.
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014
6769

Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
Otsuka et al.
S
c
S
n
m
O
O
O
O
OH
O
OH
O
OH
HO
b
O
O
N
a
HO
OH
OH
a
b
c
Figure 3. 1H NMR spectra of poly((2-O-MALac)-b-Py) after deprotection.
3.3. Construction of the Glycosylated Surface and
Their Biological Characterization
The surface properties of the poly(Lac-b-Py) coating were
of immobilized poly(Lac-b-Py) increased with increases
in the injected copolymer concentrations; saturation was
observed at copolymer concentrations over 0.5 mg mL−1
,
suggesting that the amount of possible immobilization on
studied in detail to estimate specific and nonspecific pro-
tein adsorption on glycosylated surface. First, milli-Q solu-
Delivered by Publishing Technology to: Chinese University of Hong Kong
the gold surface is constant with injection of copolymer
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
Copyright: American ScaibeonvteifitchiPsucbolnischenetrrsation. Therefore, following specific and
tions of various concentrations of poly(Lac-b-Py) were
nonspecific adsorption of protein was studied on the gly-
cosylated surface immobilized with 1 mg mL−1 poly(Lac-
b-Py) solution.
injected onto the gold surface using QCM instrument to
modulate immobilized concentration on a sensor chip. The
decrease in resonant frequency at each concentration of
copolymer are shown in Figure 4. The average value of
frequency shift after coplymer immobilization is also plot-
ted in Figure 5. The results confirmed that the amount
To avoid the influence of the solution properties, the
resonant frequency of PBS buffer solution at the initial
stage was recorded as the baseline for protein adsorp-
tion. When PBS buffer solution was injected into the flow
cell of QCM where a glycosylated surface was exposed,
an obvious resonant frequency shift was not detected
as shown in Figure 6. Then, a frequency decrease was
obtained after the injection of bivalent galactose-binding
Time (s)
0
50
100
150
200
250
20
0
(a)
–20
–40
–60
–80
–100
–120
–140
100
80
60
40
20
0
545
436
327
218
109
0
(b)
(c)
(d)
(e)
0.0
0.2
0.4
0.6
0.8
1.0
Concentration (mg⋅mL–1
)
Figure 4. Typical time course of poly(Lac-b-Py) adsorption on the gold
chip monitored by a QCM. (a) 0.008, (b) 0.04, (c) 0.2, (d) 0.5, and
Figure 5. Saturation adsorption behavior of poly(Lac-b-Py) on gold
chip.
(e) 1.0 mg mL−1
.
6770
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014

Otsuka et al.
Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
(a)
0
–40
0
0
–4
0
(a)
(b)
218
436
654
872
22
44
65
87
–80
–8
(c)
(d)
(b)
–120
–160
–12
(e)
–16
0
0
40
80
Time (s)
120
160
40
80
Time (s)
120
160
Figure 7. Typical time course of RCA120 binding on the poly(Lac-b-
Py) surface monitored by a QCM. (a) 0ꢅ084 × 10−7, (b) 0ꢅ84 × 10−7
,
Figure 6. Time course of (a) nonspecific adsorption of BSA and
(b) specific bindingof RCA120 on the poly(Lac-b-Py) surface monitored
by QCM.
(c) 2ꢅ1×10−7, (d) 4ꢅ2×10−7, and (e) 8ꢅ4×10−7 M.
adsorption of RCA120 on the poly(Lac-b-Py) surface is
due to the specific interaction between RCA120 and lactose
residues.
lectin (R. communis agglutinin, RCA120) solution (0ꢅ084×
10−7 M) and without much change after rinsing with PBS
buffer solution. This result confirms that lactose residues
take part in the specific and highly sensitive interactions
by even small amount of RCA120 addition. It is interest-
ing to note that there exist few reports for the detection
of protein in nmol order. This is because our glycopoly-
mer, poly(Lac-b-Py), was to combine the self-assembly
strategy with the specially designed functional poly(Lac-b-
Py) (Scheme 1) to engineer self-organized, densely packed
glycopolymeric recognition layers which could be chemo-
The binding of RCA120 on the glycosylated surface was
estimated by saturation analysis. After the PBS buffer
solution flowed on the gold chip until no frequency shift,
injections of RCA120 solutions ranging in concentration
from 0ꢅ084×10−7 to 8ꢅ4×10−7 M were performed respec-
tively and the corresponding frequency shift with the time
was collected (Fig. 7). Since we used experiment condition
in which mass transfer is very fast and is not the rate-
limiting step, the apparent binding affinity for RCA120 to
Delivered by Publishing Technology to: Chinese University of Hong Kong
adsorbed onto gold surface via Py segments and generate
the lactose immobilized sensor surface could be estimated
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
multivalent binding cavity. Densely packed glycopolymer
42–44
Copyright: American Scientific Publishers
by using the Langmuir adsorption model.
Indeed, it
could contribute to the particulaer sensitive recognition for
the very small amount of proteins without any nonspecific
proteins.
is found that the binding amount (ꢆm) increases obvi-
ously at a low concentration ([RCA120]). When the con-
centration gets to 4ꢅ2 × 10−7 M, the increase becomes
slower, showing simple saturation curve (Fig. 8(a)). It
seems that this type of adsorption curve is similar to that
of Langmuir adsorption curve, the RCA120 on the lac-
tose surface presents a monolayer adsorption. Reciprocal
plot between [RCA120]/ꢆm and [RCA120] gave straight line
(Fig. 8(b)) according to the following equation
Then, BSA was employed as a reference protein to
evaluate the effect of hydrophilicity/hydrophobicity of gly-
cosylated surface on the nonspecific adsorption. Water
contact angle was used to measure the static wettabil-
ity of the surface in air. In the water-in-air measurement,
coating of the poly(Lac-b-Py) on the QCM sensor surface
increased its wettability, as indicated by a decrease in the
static contact angle. One of the most sensitive methods that
provides information on the outermost polymer surfaces
of a few angstroms is the contact angle measurement.37
It is found that the glycosylated surface presents a rel-
atively hydrophilic surface due to a lower contact angle
value of 26ꢅ3 2ꢅ6ꢁ, compared with that of polyvinylpyri-
dine film (80ꢅ8 4ꢅ1ꢁ). The relatively low contact angles
on the poly(Lac-b-Py) surfaces are most likely attributed to
an complete coverage of the uppermost surface by the lac-
tose chains. Generally, the hydrophilic surface can reduce
the nonospecific adsorption of protein.38–41 In this study,
the slight frequency shift by concentrated BSA injection
(1ꢅ5×10−6 M, Fig. 6) indicates that the hydrophilic glyco-
sylated surface can effectivey resist the nonspecific BSA.
Thus, it is believed that the nonspecific adsorption of
RCA120 can also be negligible on the glycosylated sur-
face. As a consequence, it is reasonable to note that the
ꢉRCA120ꢊ ꢉRCA120ꢊ
1
1
=
+
ꢆmmax Ka
ꢆm
ꢆmmax
where ꢆmmax is a maximum binding amount of RCA120
,
Ka is the binding constant. From the linear correlation
in Figure 8(b), ꢆmmax and Ka were calculated from the
slope and the intercept, respectively. Consequently, Ka for
the binding between RCA120 and lactose was calculated
as 6ꢅ26 × 106
M
−1. This result show that the immobi-
lization of glycopolymeric ligands yields much higher Ka
than that of the monovalent lactose/protein interactions
(103 M−1).45–47 Surface coverage of RCA120 binded on the
gold electrode was also calculated from maximum binding
amount (ꢆmmax) that can be obtained from the reciprocal
plot. The obtained value (196 nm2 molecule−1) suggested
the loosely monolayer packing and assumed Langmuir
type adsorption, considering from the molecular size of
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014
6771

Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
Otsuka et al.
1000
800
600
400
200
0
(a)
specially designed functional glycopolymers to engineer
self-organized, densely packed glycopolymeric recogni-
tion layers which could be chemo-adsorbed onto gold
surface of QCM and generate multivalent binding cav-
ity. Indeed, the glycosylated surface can selectively bind
RCA120, but resist the nonspecific adsorption of BSA.
The binding constant was determined as 6ꢅ26 × 106 M−1
by QCM with the aid of Langmuir adsorption model.
The value is much higher than that of monovalent carbo-
hydrate/protein interaction, which suggests the formation
of multivalent interactions between RCA120 and lactose
residues on the surface. This simple, yet highly effective,
derivatization of gold surface with glycopolymer provide
a convenient method to construct various sensing systems
currently applied in bioassays and biorecognitions.
0
2
4
6
8
10
[RCA120] (10–7 M)
(b)
10
8
References and Notes
1. Y. Zhang, J. Yuan, J. Song, Z. Wang, and L. Huang, Glycobiology
23, 643 (2013).
6
2. L. Laura, L. L. Kiessling, and J. C. Grim, Chem. Soc. Rev. 42, 4476
(2013).
4
3. H. Wanga, W. Huanga, J. Orwenyoa, A. Banerjeeb, G. R. Vastab,
and L. X. Wang, Bioorg. Med. Chem. 21, 2037 (2013).
4. Y. Chen, L. Lin Ding, and H. Ju, Chem. Commun. 49, 862 (2013).
5. N. Kim and I. S. Park, Biosen. Bioelectron. 18, 1101 (2003).
6. C. R. Bertozzi and L. L. Kiessling, Science 291, 2357 (2001).
7. R. D. Vaughan, C. K. Sullivan, and G. G. Guilbault, Enzyme
2
0
0
4
6
8
De2livered by Publishing Technology to: ChMinicerosbe. TUecnhinvoel.r2s9it,y63o5f(H20o01n)g. Kong
[RCA120] (10–7 M)
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
8. R. A. Dwek, Chem. Rev. 96, 683 (1996).
Copyright: American Scientific Publishers
9. Y. C. Lee and R. T. Lee, Acc. Chem. Res. 28, 321 (1995).
10. J. M. Boggs, H. Wang, W. Gao, D. N. Arvanitis, Y. Gong, and
W. Min, Glycoconjugate J. 21, 97 (2004).
Figure 8. (a) Saturation binding behaviors of RCA120 on poly(Lac-
b-Py) surface, and (b) their reciprocal plot for [RCA120]/ꢆm versus
[RCA120].
11. J. J. Lundquist and E. J. Toone, Chem. Rev. 102, 555 (2002).
12. M. M. Mammen, S. K. Choi, and G. M. Whitesides, Angew. Chem.
Int. Ed. 37, 2754 (1998).
13. R. Sunasee and R. Narain, Macromol. Biosci. 13, 9 (2013).
14. V. V. Dorbatt, J. Lee, E. W. Lin, and H. D. Maynard, ChemBioChem
13, 2478 (2012).
15. H. Otsuka, Y. Nagasaki, and K. Kataoka, Adv. Drug Delivery Rev.
64, 246 (2012).
16. M. H. Stenzel, Chem. Commun. 3486 (2008).
17. V. Ladmiral, E. Melia, and D. M. Haddleton, Eur. Polym. J. 40, 431
(2004).
RCA120 lectin (12×6×4 nm). The strong binding affinity
confirms that lactose residues present multivalent interac-
tions (cluster glycoside effect) with RCA120, suggesting
the successful construction of a multivalent glycosylated
surface. Moreover, the value of Ka is in the same order
of magnitude as reported in the literature,48 which indi-
cates this immobilization method is a simple and effec-
tive method for glycosylation with anti-nonspecific protein
binding property.
18. Y. Gou, S. J. Richards, D. M. Haddleton, and M. I. Gibson,
Polym. Chem. 3, 1634 (2012).
19. J. C. J. C. Kim, Y. Rho, G. Kim, M. Kim, H. Kim, I. J. Kim, J. R.
Kimc, and M. Ree, Polym. Chem. 4, 2260 (2013).
20. A. Beaussart, S. E. K. Chatel, P. Herman, D. Alsteens, J. Mahillon,
P. Hols, and Y. F. Dufrêne, Biophys. J. 104, 1886 (2013).
21. V. V. Dorbatt, J. Lee, E. W. Lin, and H. D. Maynard, ChemBioChem
13, 2478 (2012).
22. X. L. Y. Meng, L. S. Fang, X. J. Huang, and Z. K. Xu, Langmuir
28, 13616 (2012).
23. J. E. Gestwicki, C. W. Cairo, D. A. Mann, R. M. Owen, and L. L.
Kiessling, Anal. Biochem. 305, 149 (2002).
24. X. Sun, K. M. Faucher, M. Houston, D. Grande, and E. Chaikof,
J. Am. Chem. Soc. 124, 7258 (2002).
25. Y. Bakkour, V. Darcos, F. Coumes, S. Li, and J. Coudane, Polymer
541, 746 (2013).
26. A. Rösler, G. W. M. Vandermeulen, and H. A. Klok, Adv. Drug
Delivery Rev. 64, 270 (2012).
4. CONCLUSION
In conclusion, we have demonstrated the design and
synthesis of pyridine containing glycopolymer. RAFT
polymerization using CDB as the chain transfer agent
proves to be a viable method for the synthesis of well-
defined glycopolymers with pendant lactose residues. The
glycopolymers as a hydrophilic segments were subse-
quently chain-extended with 4-pyridyl methyl methacry-
late as a hydrophobic segments to obtain well-defined
amphiphilic poly(Lac-b-Py) diblocks confirming the liv-
ing/controlled character of the polymerization. Compared
to other glycopolymer, the novelty of polymer presented
here is to combine the self-assembly property with the
6772
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014

Otsuka et al.
Carbohydrate-Based Amphiphilic Diblock Copolymers with Pyridine for the Sensitive Detection of Protein Binding
27. T. Schnitzler and H. Herrmann, Acc. Chem. Res. 45, 1419
(2012).
39. E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama, and G. M.
Whitesides, Langmuir 17, 5605 (2001).
28. H. Otsuka, M. Fukaishi, T. Ishizuka, and Y. Saito, J. Nanosci. Nan-
otechnol. 537 (2013).
40. H. Otsuka, Y. Nagasaki, and K. Kataoka, Biomacromolecules 1, 39
(2000).
29. H. Otsuka, Y. Nagasaki, and K. Kataoka, Current Opinion in Colloid
& Interface Science 6, 3 (2001).
41. T. Satomi, Y. Nagasaki, H. Kobayashi, H. Otsuka, and K. Kataoka,
Langmuir 23, 6698 (2007).
30. B. Helferich and K. Weis, Chem. Ber. 89, 314 (1956).
31. C. S. Hudson and J. M. Johnson, J. Am. Chem. Soc. 37, 1270 (1915).
32. R. K.Bai, Y. Z. You, and C. Y. Pan, Polym. Int. 65, 985 (2012).
33. G. Moad, E. Rizzardo, and S. H. Thang, Aust. J. Chem. 58, 379
(2005).
42. Y. Ebara, K. Itakura, and Y. Okahata, Langmuir 12, 5165 (1996).
43. Y. Zang, S. Z. Luo, Y. J. Tang, L. Yu, K. Y. Hou, J. P. Cheng, X. Q.
Zeng, and P. G. Wang, Anal. Chem. 78, 2001 (2006).
44. T. Mori, M. Toyoda, T. Ohtsuka, and Y. Okahata, Anal. Biochem.
395, 211 (2009).
34. L. Albertin, M. H. Stenzel, C. B. Kowollik, L. J. R. Foster, and T. P.
Davis, Polymer 46, 2831 (2005).
45. K. Sato, Y. Imoto, J. Sugama, S. Seki, H. Inoue, T. Odagiri, T. Hoshi,
and J. Anzai, Langmuir 21, 797 (2005).
35. K. Ohno, Y. Tsuji, T. Miyamoto, T. Fufuda, M. Goto, K. Kobayashi,
and T. Akaike, Macromolecules 31, 1064 (1998).
36. Y. M. Chen and G. Wulff, Macromol. Chem. Phys. 202, 3426 (2001).
37. E. B. Troughton, C. D. Bain, G. M. Whitesides, R. G. Nuzzo, D. L.
Allara, and M. D. Porter, Langmuir 4, 365 (1988).
38. G. B. Sigal, M. Mirksich, and G. M. Whitesides, J. Am. Chem. Soc.
120, 3464 (1998).
46. K. Mizukami, H. Takakura, T. Matsunaga, and H. Kitano, Colloids
Surf. B: Biointerfaces 66, 110 (2008).
47. S. K. Podder, A. Surolia, and B. K. Bachhawat, Eur. J. Biochem. 44,
151 (1974).
48. L. Yu, M. Huang, P. G. Wang, and X. Zeng, Anal. Chem. 79, 8979
(2007).
49. H. R. Jones and M. K. Wiles, J. Phys. Chem. 78, 8356 (1999).
Received: 17 June 2013. Accepted: 14 July 2013.
Delivered by Publishing Technology to: Chinese University of Hong Kong
IP: 119.1.226.185 On: Thu, 25 Feb 2016 06:34:53
Copyright: American Scientific Publishers
J. Nanosci. Nanotechnol. 14, 6764–6773, 2014
6773