Boronic Acid Terminated Thermo-Responsive and Fluorogenic

Aug 7, 2012 - Boronic Acid Terminated Thermo-Responsive and Fluorogenic Polymer: Controlling Polymer Architecture for Chemical Sensing and Affinity ...
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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-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-(2azido-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).

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reduce the biofouling of membrane materials.29 Poly(Nisopropylacrylamide) (polyNIPAm) hydrogel is among the most extensively studied thermo-responsive polymers. The phase transition point of polyNIPAm (at about 32 °C) is near body temperature,30−34 which makes polyNIPAm very suitable 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 most powerful and versatile controlled radical polymerization (CRP) techniques. ATRP enables precise control over monomer sequence and molecular weight distribution of polymers. Additionally, in ATRP, end group functionality can be introduced either by utilizing functionalized initiators or by a postpolymerization end group modification.36−38 For these reasons, ATRP is ideal for synthesis of ligand-tagged thermo-

INTRODUCTION As one type of the primary biological molecules, saccharides play fundamental roles in various biological systems. Many efforts have been made to construct functional materials that 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 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 of the systems do not work in pure aqueous solution at physiological pH. Therefore, the development of new boronic acid-based functional polymers that can bind and respond to saccharides under physiological conditions is still a challenging task. Thermo-sensitive polymers are hydrophilic at low temperature, and can be turned into hydrophobic to form aggregates if 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 © XXXX American Chemical Society

Received: June 15, 2012 Revised: July 17, 2012

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controlled cuvette was employed, and the heating rate was 0.2 °C min−1. 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 2bromoisobutyryl 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, 1463, 1424, 1369, 1300, 1196, 1108, 1008, 911, 820, 690; 1H 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, 1530, 1424, 1335, 1238, 1111, 1044, 795, 704; 1H 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). 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, 1454, 1363, 1172, 1126, 634; 1H 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,

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 glycosylated biological molecules.

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EXPERIMENTAL SECTION

Materials. 3-Aminophenylboronic acid hemisulfate, bromoacetyl bromide, CuSO4, CuBr (98%), sodium ascorbate, sodium azide, tris(2dimethylaminoethyl)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, Lawrenceville, 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. 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

2927, 1648, 1530, 1424, 1335, 1238, 1111, 1044, 795, 704, 642; 1H 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 (BApolyNIPAm 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.

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 thermoresponsive polymer was then successfully conjugated with APBA to give the desired boronic acid-terminated polyNIPAm through CuAAC. 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 (Figure 1). In the 1H NMR spectra of polyNIPAm and BA-

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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-2methylpropionamide (BMP) can be synthesized conveniently through the reaction between propargyl amine and 2-bromo-2methylpropanoyl bromide. From BMP, two alternative synthetic routes were conceived to prepare boronic acidterminated 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 C

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polyNIPAm is ∼1400 Da, which is very close to the value calculated from the UV−vis measurements (1358 Da). Thermal-Responsiveness of polyNIPAm and BApolyNIPAm. 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

terminal boronic acid, BA-polyNIPAm displayed absorption bands similar to APBA (Figure 2).

Figure 2. UV−vis spectra of polyNIPAm (0.2 mg/mL), BApolyNIPAm (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 measurement.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). 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

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. As shown in Figure 4, the LCST of polyNIPAm and BApolyNIPAm 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-responsiveness 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 thermoresponsive 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 BApolyNIPAm 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 recognition 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 concentration 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 3aminophenylboronic 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 BApolyNIPAm (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 cisdiol compounds can be achieved in our present thermoresponsive BA-polyNIPAm system, we collected the fluorescence 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 E

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Figure 8. Hypothetical mechanism of fluorescence change in response to binding of cis-diol.

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ASSOCIATED CONTENT

S Supporting Information *

Measurement of fructose concentration using a threecomponent 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.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

Figure 9. Affinity separation of fructose by BA-polyNIPAm (■) and polyNIPAm (▽).

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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.

CONCLUSIONS 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-polyNIPAm. 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 BApolyNIPAm. 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.

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REFERENCES

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dx.doi.org/10.1021/ma301213f | Macromolecules XXXX, XXX, XXX−XXX