Boronic acid terminated thermo-responsive and fluorogenic polymer: Controlling polymer architecture for chemical sensing and affinity separation

Article abstract of DOI:10.1021/ma301213f

Thermo-responsive poly(N-isopropylacrylamide) (polyNIPAm) containing terminal boronic acid was synthesized using atom transfer radical polymerization (ATRP) in combination with Cu(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Alkyne-terminated polyNIPAm was first synthesized by ATRP using an alkyne-containing initiator. A fluorogenic boronic acid, 3-(2-azido-acetylamino)phenylboronic acid (APBA) was then linked to the polyNIPAm through CuAAC. The synthesized polymers were characterized by ¹H NMR, FT-IR, UV-vis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and turbidity measurements. The intensity of fluorescence emission of the boronic acid-terminated polyNIPAm (BA-polyNIPAm) was found to increase when increasing amount of a cis-diol compound (i.e., fructose) was added. At physiological pH value, the BA-polyNIPAm effectively bound fructose and could be easily separated from aqueous solution by raising the temperature above its lower critical solution temperature (LCST).

Full text of DOI:10.1021/ma301213f

Article
pubs.acs.org/Macromolecules
Boronic Acid Terminated Thermo-Responsive and Fluorogenic
Polymer: Controlling Polymer Architecture for Chemical Sensing and
Affinity Separation
Zhifeng Xu,†,‡,§ Khan Mohammad Ahsan Uddin,† and Lei Ye*,†
†Division of Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden
‡Department of Chemistry and Material Science, Hengyang Normal University, Hengyang, Hunan 421008, China
§
Key Laboratory of Functional Organometallic Materials, College of Hunan Province, Hengyang, Hunan 421008, China
S
* Supporting Information
ABSTRACT: Thermo-responsive poly(N-isopropylacryla-
mide) (polyNIPAm) containing terminal boronic acid was
synthesized using atom transfer radical polymerization
(ATRP) in combination with Cu(I)-catalyzed alkyne−azide
1,3-dipolar cycloaddition (CuAAC) reaction. Alkyne-termi-
nated polyNIPAm was first synthesized by ATRP using an
alkyne-containing initiator. A fluorogenic boronic acid, 3-(2-
azido-acetylamino)phenylboronic acid (APBA) was then
linked to the polyNIPAm through CuAAC. The synthesized
1
polymers were characterized by H NMR, FT-IR, UV−vis,
matrix-assisted laser desorption/ionization time-of-flight
(MALDI−TOF) mass spectrometry, and turbidity measure-
ments. The intensity of fluorescence emission of the boronic
acid-terminated polyNIPAm (BA-polyNIPAm) was found to increase when increasing amount of a cis-diol compound (i.e.,
fructose) was added. At physiological pH value, the BA-polyNIPAm effectively bound fructose and could be easily separated from
aqueous solution by raising the temperature above its lower critical solution temperature (LCST).
reduce the biofouling of membrane materials.29 Poly(N-
isopropylacrylamide) (polyNIPAm) hydrogel is among the
INTRODUCTION
■
As one type of the primary biological molecules, saccharides
most extensively studied thermo-responsive polymers. The
play fundamental roles in various biological systems. Many
phase transition point of polyNIPAm (at about 32 °C) is near
efforts have been made to construct functional materials that
body temperature,30−34 which makes polyNIPAm very suitable
can selectively bind and respond to saccharides under
physiological conditions.1−5 It is well-known that boronic
acids can bind cis-diol compounds in basic aqueous solution
through reversible cyclic boronic ester bond. Boronic acid
ligands have been immobilized on different materials to offer
to provide effective separation of biologically relevant
molecules.35 To improve the selectivity of polymer-based
separations, it is necessary to introduce affinity ligands into
polyNIPAm structure. Ideally, the affinity ligand should remain
accessible to the solution even at temperature above the LCST,
so that the target molecules can be firmly associated with the
aggregated polymers and be separated through, e.g., a simple
filtration step.
Atom transfer radical polymerization (ATRP) is one of the
effective affinity separation of saccharides and glycosylated
biomolecules.6−10 For practical bioseparation or sensing
purposes, the use of high pH to promote saccharide binding
is undesirable. Although many boronic acid derivatives have
been developed to act as optical sensors for saccharides, most
most powerful and versatile controlled radical polymerization
of the systems do not work in pure aqueous solution at
(CRP) techniques. ATRP enables precise control over
physiological pH. Therefore, the development of new boronic
monomer sequence and molecular weight distribution of
acid-based functional polymers that can bind and respond to
polymers. Additionally, in ATRP, end group functionality can
saccharides under physiological conditions is still a challenging
be introduced either by utilizing functionalized initiators or by a
task.
postpolymerization end group modification.36−38 For these
Thermo-sensitive polymers are hydrophilic at low temper-
ature, and can be turned into hydrophobic to form aggregates if
reasons, ATRP is ideal for synthesis of ligand-tagged thermo-
the temperature is raised above their lower critical solution
temperature (LCST). Thermally modulated on/off-adsorption
materials can be used for bioseparations,11−23 in controlled
release systems,24−26 sensors,27 in a catalytic system28 and to
Received: June 15, 2012
Revised: July 17, 2012
© XXXX American Chemical Society
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controlled cuvette was employed, and the heating rate was 0.2 °C
min−1.
responsive polymers to enable efficient bioseparations. To
ensure the most effective affinity binding for saccharides, we are
particularly interested in low molecular weight polyNIPAm that
contains terminal boronic acids. These boronic acid-tagged
thermo-responsive polymers not only provide convenient
separation, but they also may be used as modular building
blocks to construct other separation systems and biosensing
units, which may be further adjusted through temperature
control.
In a previous work, we developed a fluorogenic boronic acid
that contains a terminal azide.39 The clickable boronic acid can
be easily conjugated to alkyne-functionalized agarose gels using
copper-catalyzed azide−alkyne cycloaddition (CuAAC), a
prototypical example of click reaction that has been recognized
as a facile and versatile chemistry for bioconjugation and
functionalization of polymeric architectures.40,41 In this work,
we take the advantages of ATRP and the CuAAC click
chemistry to synthesize boronic acid-terminated polyNIPAm
(BA-polyNIPAm), and demonstrate that the thermo-responsive
polymer can be used to achieve simple and fast affinity
separation of saccharides under physiological condition.
Because of the interesting fluorescence property of the boronic
acid,39 the BA-polyNIPAm synthesized in this work also
displayed clear fluorescence intensity change upon binding
saccharide at neutral pH. The multifunctional BA-polyNIPAm
developed in this work should be useful in many practical
applications involving recognition of saccharids and glycosy-
lated biological molecules.
Synthesis of Small Molecules and Polymers. The fluorogenic
boronic acid, 3-(2-azidoacetylamino)phenylboronic acid (APBA) was
synthesized according to our previously reported procedure.39
Synthesis of Propargyl 2-Bromo-2-methylpropionamide (BMP).
BMP was synthesized according to a literature method with some
modifications.42 Typically, to a solution of propargylamine (2.00 g,
36.36 mmol) and triethylamine (7.55 mL, 54.6 mmol) in THF (100
mL) cooled to ice−water temperature was slowly added 2-
bromoisobutyryl bromide (8.36 g, 36.36 mmol). The reaction mixture
was warmed to room temperature and then stirred overnight. The
precipitate was filtered off and the solvent was removed using a rotary
evaporator. The crude product was recrystallized twice from n-hexane/
methanol (5:1, v/v) to give BMP as a pale yellow solid. Yield: 72.8%.
FT-IR ν (cm−1): 3327, 3269, 3053, 2980, 2929, 2119, 1642, 1528,
1
1463, 1424, 1369, 1300, 1196, 1108, 1008, 911, 820, 690; H NMR
(DMSO-d6, 400 MHz): δ 8.52 (broad, 1H, NH), 3.33 (q, 2H,
CCH2NH), 3.12 (t, 1H,CHC), 1.87 (s, 6H, C(CH3)2Br).
Synthesis of BA-BMP through Click Reaction. APBA (0.219 g, 1.0
mmol) was dissolved in 6 mL of methanol:water (2:1, v/v). To the
solution, BMP (0.204 g, 1.0 mmol), CuSO4 (16.0 mg, 0.10 mmol) in 1
mL water, and sodium ascorbate (59.4 mg, 0.30 mmol) in 1 mL of
water were added. The solution was fluxed with a stream of nitrogen
gas for 5 min. The reaction mixture was then sealed and stirred at
room temperature for 48 h. The solvent was removed using a rotary
evaporator. The dry product was dissolved in a mixture of ethyl acetate
and water (1:1, v/v). The ethyl acetate phase was collected, washed
with water, and dried over anhydrous sodium sulfate. The crude
product was obtained by removing the solvent using a rotary
evaporator. The crude product was purified through column
chromatography (ethyl acetate/acetone, 2:1) to give BA-BMP as a
powder solid. Yield: 27%. FT-IR ν (cm−1): 3200, 2977, 2927, 1648,
EXPERIMENTAL SECTION
1
■
1530, 1424, 1335, 1238, 1111, 1044, 795, 704; H NMR (DMSO-d6,
400 MHz): δ 10.38 (broad, 2H, NH), 8.05 [s, 2H, B(OH)2], 7.27−
7.90 (m, 4H, C6H4), 5.30 (s, 2H, NHCH2), 4.38 (s, 2H, COCH2),
1.87 (s, 6H, C(CH3)2Br).
Materials. 3-Aminophenylboronic acid hemisulfate, bromoacetyl
bromide, CuSO4, CuBr (98%), sodium ascorbate, sodium azide, tris(2-
dimethylaminoethyl)amine (Me6TREN), Alizarin Red S (ARS),
propargylamine, 2-bromoisobutyryl bromide, D-fructose and (methyl
sulfoxide)-d6 (99.9 atom % D) were purchased from Sigma-Aldrich.
CuBr was stirred overnight in acetic acid, filtered, washed with
acetone, and dried in vacuo before use. N-Isopropylacrylamide
(NIPAm) was purchased from Acros and recrystallized from
toluene/hexane (2:1, v/v). 2-Propanol, tetrahydrofuran (THF) and
N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich
and used without further purification. Ultrapure water (18.2 MΩ cm)
obtained from an ELGA LabWater System (Vivendi Water Systems
Ltd.) was used throughout the experiments. All other solvents
purchased from commercial resources were of analytical grade.
Characterization. Attenuated total reflection (ATR) infrared
spectra were recorded using a Perkin-Elmer FTIR instrument
(Perkin-Elmer Instruments). UV−vis absorption spectra were
recorded with a Beckman Coulter DU 800 UV/vis spectrophotometer.
Fluorescence emission was measured using a QuantaMaster C-60/
2000 spectrofluorometer (Photon Technology International, Lawren-
ceville, NJ). 1H NMR spectra were recorded on a 400 MHz
Superconducting Magnet NMR Spectrometer (Bruker B-ACS60).
MALDI−TOF mass spectra were acquired using a 4700 Proteomics
Analyzer (Applied Biosystems/MDS SCIEX, USA) in the positive
reflector mode. The samples were dissolved in THF and the
concentration was 0.2 mg/mL. The matrix solution consisted of
50% (v/v) acetonitrile in water, 5 mg/mL α-cyano-4-hydroxy
cinnamic acid and 0.1% (v/v) phosphoric acid. The matrix solution
was mixed with sample on a stainless target plate. Typically, 0.5 μL of
sample was mixed with 0.5 μL of matrix solution spiked with two
internal standard peptides (m/z = 904.468 and m/z = 2465.199). The
two internal standards allowed accurate mass calibration with a mass
deviation less than 20 ppm.
Synthesis of polyNIPAm by ATRP Using BMP as Initiator. NIPAm
(1.80 g, 15.9 mmol), CuBr (21.6 mg, 0.15 mmol) and 2-propanol
(10.0 mL) were added to a 100 mL dried flask. The mixture was
deoxygenated by bubbling with nitrogen for 30 min. Me6TREN
(0.0414 g, 0.18 mmol) was added via a syringe, and the solution was
stirred for 20 min to allow formation of the CuBr/Me6TREN complex.
After addition of BMP (61.2 mg, 0.30 mmol), the reaction mixture was
stirred with a magnetic bar for 12 h at room temperature under a slight
positive pressure of nitrogen. After the reaction, the solvent was
removed using a rotary evaporator. The residue was diluted with THF
and then passed through an alumina column to remove the copper
catalyst. The product was precipitated from diethyl ether. Further
dissolution/precipitation procedures by THF/diethyl ether were
repeated three times before the product was dried in vacuo. The
final polymer polyNIPAm was obtained as a white powder. Yield:
48.7%. FT-IR ν (cm−1): 3299, 2968, 2929, 2886, 2119, 1636, 1527,
1
1454, 1363, 1172, 1126, 634; H NMR (DMSO-d6, 400 MHz): δ
6.82−7.60 (broad, NH), 3.70−3.95 [broad, CH(CH3)2], 3.61 (s, 
CCH2), 3.10 (s, CHC), 1.4−2.2 (broad, backbone Hs), 1.05 [broad,
CH(CH3)2].
Synthesis of BA-polyNIPAm through Click Reaction. DMF (10
mL) was placed in a three-neck flask and degassed by bubbling
nitrogen for 1 h before the addition of CuBr (21.6 mg, 0.15 mmol),
APBA (0.219 g, 1.0 mmol) and polyNIPAm (0.15 g). After 15 min,
Me6TREN (41.4 mg, 0.18 mmol) was introduced and the solution was
then heated to 85 °C under nitrogen atmosphere, and magnetically
stirred for 48 h. When the mixture was cooled to room temperature,
DMF was distilled off under reduced pressure. The residue was diluted
with THF and then passed through an alumina column to remove the
copper catalyst. The solid product was then precipitated from diethyl
ether. Further dissolution/precipitation procedures by THF/diethyl
ether were repeated three times before the product (BA-polyNIPAm)
was dried in vacuo. Yield: 64.5%. FT-IR ν (cm−1): 3429, 3200, 2977,
Optical transmittance of aqueous solution of the synthesized
polymers was measured at a wavelength of 700 nm using a Beckman
Coulter DU 800 UV/vis spectrophotometer. A thermostatically
B
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Scheme 1. Synthesis of Boronic Acid Terminated polyNIPAm
1
2927, 1648, 1530, 1424, 1335, 1238, 1111, 1044, 795, 704, 642; H
complex used in the ATRP reaction, possibly through an
interaction with the amine ligand Me6TREN.43 When BMP was
used instead as the initiator (route 2), alkyne-terminated
polyNIPAm was obtained readily by the ATRP. This thermo-
responsive polymer was then successfully conjugated with
APBA to give the desired boronic acid-terminated polyNIPAm
through CuAAC.
NMR (DMSO-d6, 400 MHz): δ 6.82−7.67 (broad, aromatic Hs and
NH), 3.80−3.95 [broad, CH(CH3)2 and COCH2], 3.60 (s, CH2NH),
1.40−2.24 (broad, backbone Hs), 1.05 [broad, CH(CH3)2].
Measurement of Fluorescence Response to Fructose. To a
set of 15 mL calibrated test tubes, 2.0 mg of BA-polyNIPAm, 0.5 mL
of 0.20 M phosphate buffer (PBS) (pH 7.4), and a given concentration
of fructose solution were sequentially added. The mixture was then
diluted to 2.0 mL with ultrapure water and mixed thoroughly. The
fluorescence tests were carried out after the solutions have been
shaken for 2 h.
Separation of Fructose by Affinity Precipitation. To a set of
15 mL calibrated test tubes, 2.0 mg of the synthesized polymers (BA-
polyNIPAm or polyNIPAm), 0.5 mL of 0.20 M PBS buffer (pH 7.4),
and a given concentration of fructose solution were sequentially added.
The mixture was then diluted to 3.0 mL with ultrapure water and
mixed thoroughly. The initial concentrations of fructose were 1.0, 2.0,
3.0, 4.0, 5.0, and 6.0 mM, respectively. After being shaken for 2 h, the
test tubes were left to stand in a 45 °C water bath for 5 min. Then, the
mixture was filtrated through a cellulose filter (with pore size 0.2 μm).
The concentration of fructose in the filtrate was determined by a
competition assay in a three-component system (see Supporting
Information). The amount of fructose bound to the polymer was
calculated by subtracting the concentration of free fructose from the
initial fructose concentration. The results reported are average data
from triplicate independent samples.
FT-IR spectroscopy was used to confirm the successful click
reaction between the alkyne-terminated polyNIPAm and
APBA. After the click reaction, the characteristic IR absorption
band of the terminal alkyne group in polyNIPAm (at 2119
cm−1) almost disappeared, indicating that most of the terminal
alkyne groups were converted into terminal boronic acid
1
(Figure 1). In the H NMR spectra of polyNIPAm and BA-
RESULTS AND DISCUSSION
■
Synthesis of Boronic Acid Terminated polyNIPAm
Using ATRP and CuAAC. As illustrated in Scheme 1, the
alkyne-functionalized ATRP initiator, propargyl-2-bromo-2-
methylpropionamide (BMP) can be synthesized conveniently
through the reaction between propargyl amine and 2-bromo-2-
methylpropanoyl bromide. From BMP, two alternative
synthetic routes were conceived to prepare boronic acid-
terminated polyNIPAm: one is to first convert BMP into
boronic acid-functionalized initiator, BA-BMP, followed by
carrying out ATRP of NIPAm (route 1), and the other is to
first synthesize alkyne-terminated polyNIPAm using the BMP
initiator, then conjugate the clickable boronic acid (APBA) with
the obtained polyNIPAm through the terminal alkyne using
CuAAC (route 2). The first synthetic route turned out to be
unsuccessful, because no polymer product could be obtained
even after prolonged reaction period. One possible reason is
that the terminal boronic acid interfered with the catalytic
Figure 1. FT-IR spectra of polyNIPAm (a) and BA-polyNIPAm (b).
polyNIPAm (see Supporting Information), the integration
ratios between the different protons are close to their
theoretical values, suggesting that the expected molecular
structures were obtained.
The presence of boronic acid moiety in BA-polyNIPAm was
also verified by UV−vis spectroscopy. APBA in H2O exhibited
characteristic absorption bands at 244 and 280 nm, which were
essentially absent in polyNIPAm. After introduction of the
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terminal boronic acid, BA-polyNIPAm displayed absorption
bands similar to APBA (Figure 2).
polyNIPAm is ∼1400 Da, which is very close to the value
calculated from the UV−vis measurements (1358 Da).
Thermal-Responsiveness of polyNIPAm and BA-
polyNIPAm. Thermal phase transition temperature, expressed
as the lower critical solution temperature (LCST) or cloud
point (CP), is one important parameter describing the
properties of thermo-responsive polymers. LCST and CP
values can be defined as the temperature at which 1% and 50%
reduction of transmittance of the polymer solution is observed,
respectively. Figure 4 shows the temperature-dependent
Figure 2. UV−vis spectra of polyNIPAm (0.2 mg/mL), BA-
polyNIPAm (0.2 mg/mL), and APBA (0.50 mM) in H2O.
Because only one single boronic acid was attached to the end
of the polyNIPAm, it was possible to estimate the molecular
weight of BA-polyNIPAm using simple UV−vis measure-
ment.42 In this work, we used BA-BMP as a standard to
establish a calibration curve based on its UV−vis absorption in
THF, which was then used to calculate the number of polymer
chains in a given concentration of BA-polyNIPAm solution in
THF. The average molecular weight of the BA-polyNIPAm was
calculated to be 1358 Da (Supporting Information).
Figure 4. Optical transmittance of polyNIPAm and BA-polyNIPAm vs
temperature for aqueous solution of polymers (1.0 mg/mL). The
transmittance was measured at 700 nm.
transmittance at a wavelength of 700 nm obtained for aqueous
solutions of polyNIPAm and BA-polyNIPAm, measured at a
polymer concentration of 1.0 mg/mL. Here, the optical
absorption at 700 nm is outside the absorption range of the
chromophore BA-BMP, therefore could be used to study the
LCST without interference from the intrinsic UV−vis
absorption of the soluble polymer.
The molecular weight of the synthesized polymers was also
determined by MALDI−TOF mass spectrometry. As shown in
Figure 3, the MALDI−TOF spectrum of polyNIPAm has
As shown in Figure 4, the LCST of polyNIPAm and BA-
polyNIPAm are 28.2 and 27.9 °C, respectively. The CP of
polyNIPAm and BA-polyNIPAm are 32.4 and 32.2 °C,
respectively. Therefore, the introduction of the boronic acid
into the polyNIPAm does not change the thermo-responsive-
ness of polyNIPAm significantly. The minimal effect of the
terminal boronic acid on the thermo responsiveness is different
from what have been observed in previous studies.44,45 The
discrepancy may be explained by the different end groups
involved in these studies. The phase transition of the thermo-
responsive polymers is shown more clearly in Figure 5: while
the aqueous solutions of polyNIPAm and BA-polyNIPAm were
transparent at room temperature, they became turbid and form
Figure 3. MALDI−TOF mass spectrum of polyNIPAm.
systematic peaks centered at ∼1200 m/z, extending from 800
to 3000 m/z. The difference in the m/z values between the
neighboring peaks is 113.09, corresponding to the molar mass
of NIPAm unit. For instance, the peaks at m/z values of 1254.9,
1367.99, and 1481.08 can be assigned to BMP-(NIPAm)9 +
Na+ (1245.44 calcd), BMP-(NIPAm)10 + Na+ (1358.6 calcd),
and BMP-(NIPAm)11 + Na+ (1471.76 calcd), respectively.
Direct determination of the molecular weight of BA-
polyNIPAm was not successful, as this polymer gave a rather
poor ionization results under the experimental condition.
Nevertheless, based on the molecular weight of the polyNIPAm
and APBA precursor, we can conclude that the average
molecular weight of the boronic acid-terminated polymer BA-
Figure 5. Images of 1.0 mg/mL aqueous solution of polyNIPAm (I)
and BA-polyNIPAm (II) at 21 °C (1), 40 °C (2) and 50 °C (3).
D
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strong aggregates when heated to above 40 °C. This visible
phase transition was truly reversible and was observed through
repetitive heating and cooling cycles.
Fluorescence Property of BA-polyNIPAm. In our
previous work, we noticed that the clickable boronic acid
APBA has interesting fluorescence property in aqueous
solution. More interestingly, the fluorescence characteristic
remains even after the terminal azide group is converted into a
triazole after an alkyne−azide click reaction.39 This interesting
feature suggests that APBA may be exploited as a modular
building block for further development of many different
functional fluorogenic materials. In this study, it was found that
when BA-polyNIPAm was dissolved in PBS at pH 7.4, the
solution displayed a maximum fluorescence emission at 515 nm
when excited at 494 nm (Figure 6). This fluorescence emission
Figure 7. Fluorescence spectra of BA-polyNIPAm (1.0 mg/mL) in 50
mM PBS buffer, pH 7.4 at 21 °C, measured in the presence of
increasing amount of fructose (λex = 494 nm).
B−N bond can have strong fluorescence emission. It should be
noted that in this work, the corresponding fluorescence
response was observed under physiological pH conditions,
which makes the thermo-sensitive and fluorescence-responsive
polymer suitable for analysis of biological samples.
Affinity Separation of Fructose Using BA-polyNIPAm.
As can be seen in Figure 5, BA-polyNIPAm is fully soluble in
water and its aqueous solution is transparent when the
temperature is below its LCST. The polymer forms aggregates
and precipitates when the temperature is raised to above its
LCST. This property of BA-polyNIPAm provides a convenient
means to separate saccharides from aqueous solution. We
suggest that by locating the affinity ligand at the end of the
thermo-responsive polymer, the important molecular recog-
nition contributed by the ligand will not be affected by steric
hindrance, and the obtained polymer conjugate should offer the
best molecular binding to the molecular target. In this work, we
investigate the binding performance of BA-polyNIPAm for
saccharides using fructose as a model. The binding isotherm of
fructose to BA-polyNIPAm was determined in the concen-
tration range of 0−6.0 mM of fructose. After being equilibrated
with fructose, BA-polyNIPAm was precipitated from solution
by heating the samples at 45 °C for 5 min. This treatment
allowed the aggregated polymer (together with the bound
fructose) to be easily removed by a simple filtration through a
0.2 μm cellulose filter. The concentration of fructose in the
filtrate was then determined through a three-component
competitive assay, where the fructose caused dose-dependent
reduction of fluorescence intensity of a mixture of ARS and 3-
aminophenylboronic acid) (Supporting Information). The
amount of fructose bound to the polymers was then calculated
by subtracting the concentration of free fructose from the initial
fructose concentration. For comparison, polyNIPAm itself was
used as a control to evaluate the nonspecific binding caused by
the polymer backbone. As shown in Figure 9, the amount of
fructose bound to BA-polyNIPAm increased with the
increasing concentration of fructose. The figure shows that
the binding reached saturation when the initial concentration of
fructose was 3.0 mM, which corresponds to a maximum
binding capacity of 0.502 μmol/mg polymer. Because
polyNIPAm alone did not show obvious binding to fructose,
the saccharide binding achieved by BA-polyNIPAm (Figure 9)
can only be attributed to the specific interaction offered by the
terminal boronic acid.
Figure 6. Fluorescence emission spectra of polyNIPAm and BA-
polyNIPAm (1.0 mg/mL) in PBS buffer at pH 7.4 (λex = 494 nm).
is similar to the materials prepared from the same clickable
boronic acid and alkyne-modified agarose.39 The fluorescence
emission of BA-polyNIPAm further verified the presence of the
boronic acid moiety in the conjugated polymer.
Change of Fluorescence Emission of BA-polyNIPAm
in Response to Fructose. The interesting fluorescence
emission of APBA was previously explained as a result of
possible B−N bond formed between the boron and one of the
nitrogen atoms in the terminal azide.39 Binding of cis-diol
compounds was suggested to stabilize this intra molecular N−B
bond, resulting in an increase of the fluorescence intensity.39
To investigate if the same fluorescence response of APBA to cis-
diol compounds can be achieved in our present thermo-
responsive BA-polyNIPAm system, we collected the fluores-
cence spectra of BA-polyNIPAm in PBS buffer (pH 7.4) in the
presence of different amount of fructose. Figure 7 shows the
change of fluorescence emission when increasing amount of
fructose was added into the polymer solution. Clearly, the
intensity of fluorescence emission of BA-polyNIPAm increased
with the increasing fructose concentration. According to the
literature,2 phenylboronic acid in water can exist as several
interchangeable coordination structures, and the proportion of
the different structures can be influenced by the solution pH
and the concentration of cis-diols added in the solution. On the
basis of our fluorescence measurement results and the literature
information, we propose a possible mechanism to explain the
fluorescence change of BA-polyNIPAm in response to fructose
(Figure 8), where among the different solution structures of the
terminal boronic acid, only the structures containing the dative
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Figure 8. Hypothetical mechanism of fluorescence change in response to binding of cis-diol.
ASSOCIATED CONTENT
■
S
* Supporting Information
Measurement of fructose concentration using a three-
component competitive assay, 1H NMR spectra of polyNIPAm
and BA-polyNIPAm and measurement of molecular weight of
BA-polyNIPAm by UV−vis spectroscopy. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lei.ye@tbiokem.lth.se.
Notes
■
Figure 9. Affinity separation of fructose by BA-polyNIPAm ( ) and
polyNIPAm ( ).
The authors declare no competing financial interest.
▽
ACKNOWLEDGMENTS
■
CONCLUSIONS
■
This work was supported in part by the Swedish Research
Council FORMAS. Z.X. is grateful for a visiting grant from the
China Scholarship Council.
In this work, we have used ATRP in combination with CuAAC
to synthesize a new type of thermo-sensitive, fluorogenic, and
boronic acid-terminated multifunctional poymer, BA-polyNI-
PAm. Using alkyne-modified ATRP initiator, a low molecular
weight polyNIPAm was first synthesized, and then conjugated
to an azide-tagged fluorogenic boronic acid to give the BA-
polyNIPAm. This synthetic route avoided the potential
interference of boronic acid with the ATRP catalyst, and did
not involve complicated protection−deprotection steps for the
involved boronic acid. The multifunctional polymer displayed
does-dependent fluorescence response upon binding fructose at
physiological pH value, and could be easily separated by simple
thermo-precipitation. The controlled location of the affinity
ligand, the interesting fluorescence response and the possibility
to anchor the polymer on different surfaces via the remaining
bromide functional group should open many interesting
applications of the synthesized BA-polyNIPAm. The synthetic
methodology may also be expanded to other polymer systems
to prepare new affinity separation and chemical sensing
materials.
REFERENCES
■
(1) Tuma, P.; Malkova, K.; Samcova, E.; Stulik, K. Anal. Chim. Acta
2011, 698, 1−5.
(2) Nishiyabu, R.; Kubo, Y.; James, T. D.; Fossey, J. S. Chem.
Commun. 2012, 47, 1106−1123.
̈
(3) Okutucu, B.; Onal, S.; Telefoncu, A. Talanta 2009, 78, 1190−
1193.
(4) Gao, S. H.; Wang, W.; Wang, B. H. Bioorg. Chem. 2001, 29, 308−
320.
(5) Manju, S.; Hari, P. R.; Sreenivasan, K. Biosens. Bioelectron. 2010,
26, 894−897.
(6) Schumacher, S.; Gruneberger, F.; Katterle, M.; Hettrich, C.; Hall,
̈
D. G.; Scheller, F. W.; Gajovic-Eichelmann, N. Polymer 2011, 52,
2485−249.
(7) Tan, J.; Wang, H. F.; Yan, X. P. Anal. Chem. 2009, 81, 5273−
5280.
(8) Edwards, N. Y.; Sager, T. W.; McDevitt, J. T.; Anslyn, E. V. J. Am.
Chem. Soc. 2007, 129, 13575−13583.
F
dx.doi.org/10.1021/ma301213f | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
(9) Shabbir, S. H.; Joyce, L. A.; da Cruz, G. M.; Lynch, V. M.; Sorey,
(45) Nakayama, M.; Okano, T. Biomacromolecules 2005, 6, 2320−
2327.
S.; Anslyn, E. V. J. Am. Chem. Soc. 2009, 131, 13125−13131.
(10) Suksrichavalit, T.; Yoshimatsu, K.; Prachayasittikul, V.; Bulow,
̈
L.; Ye, L. J. Chromatogr. A 2010, 1217, 3635−3641.
(11) Song, F.; Wang, X. L.; Wang, Y. Z. Eur. Polym. J. 2011, 47,
1885−1892.
(12) Mizutania, A.; Nagase, K.; Kikuchi, A.; Kanazawa, H.; Akiyama,
Y.; Kobayashi, J.; Annaka, M.; Okano, T. J. Chromatogr. B 2010, 878,
2191−2198.
(13) Ichijo, H.; Kishi, R.; Hirasa, O. Polym. Gels Networks 1994, 2,
315−322.
(14) Nagase, K.; Yuk, S. F.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.;
Kanazawac, H.; Okano, T. J. Mater. Chem. 2011, 21, 2590−2593.
(15) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa,
H.; Okano, T. Biomaterials 2011, 32, 619−627.
(16) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa,
H.; Okano, T. Langmuir 2011, 27, 10830−10839.
(17) Lu, X. H.; Sun, M. Y.; Barron, A. E. J. Colloid Interface Sci. 2011,
357, 345−353.
(18) Saitoh, T.; Sugiura, Y.; Asano, K.; Hiraide, M. React. Funct.
Polym. 2009, 69, 792−796.
(19) Xie, R.; Zhang, S. B.; Wang, H. D.; Yang, M.; Li, P. F.; Zhu, X.
L.; Chu, L. Y. J. Membr. Sci. 2009, 326, 618−626.
(20) Pan, L. C.; Chien, C. C. J. Biochem. Biophys. Methods 2003, 55,
87−94.
(21) Fukuda, N.; Ishii, J.; Tanaka, T.; Fukuda, H.; Ohnishi, N.;
Kondo, A. Biotechnol. Prog. 2008, 24, 352−357.
(22) Zhang, J.; Gassmann, M.; He, W. D.; Wan, F.; Chu, B. Lab Chip
2006, 6, 526−533.
(23) Hermida Merino, D.; Slark, A. T.; Colquhoun, H. M.; Hayes,
W.; Hamley, I. W. Polym. Chem. 2010, 1, 1263−1271.
(24) Chung, J. E.; Yokoyama, M.; Yamato, M.; Aoyagi, T.; Sakurai,
Y.; Okano, T. J. Controlled Release 1999, 62, 115−127.
(25) Lee, J. S.; Zhou, W.; Meng, F. H.; Zhang, D. W.; Otto, C.;
Feijen, J. J. Controlled Release 2010, 146, 400−408.
(26) Duan, L. L.; Chen, M.; Zhou, S. X.; Wu, L. M. Langmuir 2009,
25, 3467−3472.
(27) Mori, T.; Maeda, M. Langmuir 2003, 20, 313−319.
(28) Xie, L.; Chen, M.; Wu, L. M. J. Polym. Sci., Part A: Polym. Chem.
2009, 47, 4919−4926.
(29) Yua, S.; Chen, Z. H.; Liu, J. Q.; Yao, G. H.; Liu, M. H.; Gao, C.
J. J. Membr. Sci. 2012, 392−393, 181−191.
(30) Cammas, S.; Suzuki, K.; Sone, C.; Sakurai, Y.; Kataoka, K.;
Okano, T. J. Controlled Release 1997, 48, 157−164.
(31) Alvarez-Lorenzo, C.; Concheiro, A. J. Chromatogr. B 2004, 804,
231−245.
(32) Saitoh, T.; Sekino, A.; Hiraide, M. Anal. Chim. Acta 2005, 536,
179−182.
(33) Mizutani, A.; Kikuchi, A.; Yamato, M.; Kanazawa, H.; Okano, T.
Biomaterials 2008, 29, 2073−2081.
(34) Idota, N.; Kikuchi, A.; Kobayashi, J.; Akiyama, Y.; Sakai, K.;
Okano, T. Langmuir 2006, 22, 425−430.
(35) Kanazawa, H.; Kashiwase, Y.; Yamamoto, K.; Matsushima, Y.;
Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1997, 69, 823−830.
(36) Coessens, V.; Pintauer, T.; Matyjaszewski, K. Prog. Polym. Sci.
2001, 26, 337−377.
(37) Pyun, J.; Matyjaszewski, K. Chem. Mater. 2001, 13, 3436−3448.
(38) Siegwart, D. J.; Oh, J. K.; Matyjaszewski, K. Prog. Polym. Sci.
2012, 37, 18−37.
(39) Uddin, K. M. A.; Ye, L. J. Appl. Polym. Sci. 2012, in press.
(40) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun.
2007, 28, 15−54.
(41) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun.
2008, 29, 952−981.
(42) Lai, C. T.; Chien, R. H.; Kuo, S. W.; Hong, J. L. Macromolecules
2011, 44, 6546−6556.
(43) Matyjaszewski, K.; Xia, J. H. Chem. Rev. 2001, 101, 2921−2990.
(44) Xia, Y.; Burke, N. A. D.; Stover, H. D. H. Macromolecules 2006,
̈
39, 2275−2283.
G
dx.doi.org/10.1021/ma301213f | Macromolecules XXXX, XXX, XXX−XXX