Nitric Oxide (NO) Endows Arylamine-Containing Block

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Nitric Oxide (NO) Endows Arylamine-Containing Block Copolymers with Unique Photoresponsive and Switchable LCST Properties Jinming Hu,†,‡ Michael R. Whittaker,‡ John F. Quinn,*,‡ and Thomas P. Davis*,‡,§ †

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China ‡ ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia § Department of Chemistry, University of Warwick, Coventry ULCV4 7AL, U.K. S Supporting Information *

ABSTRACT: The fabrication of materials that are responsive to endogenous gasotransmitter molecules (i.e., nitric oxide, hydrogen sulfide, and carbon monoxide) has emerged as an area of increasing research interest. In the case of nitric oxide (NO), o-phenylenediamine derivatives have traditionally been employed due to their ability to react with NO in the presence of oxygen (O2) with the formation of benzotriazole residues. Herein, we report the synthesis of a novel NO-responsive polymer containing aromatic primary amine groups derived from p-phenylenediamine groups (i.e., isomers of o-phenylenediamine). A new NO-responsive monomer, N-(4-aminophenyl)methacrylamide (p-NAPMA), was first synthesized via the amidation of one of the primary amine groups in the pphenylenediamine with methacrylic anhydride. Notably, the p-NAPMA monomer can efficiently react with NO in aqueous solution in the presence of O2 with the generation of phenyldiazonium groups rather than benzotriazole moieties. While the resultant phenyldiazonium residues were relatively stable in aqueous solution, they were highly sensitive to UV irradiation (i.e., λmax = 365 nm) which gave the formation of phenol derivatives. After incorporation into a thermoresponsive block copolymer using reversible addition−fragmentation chain transfer (RAFT) polymerization, the resulting diblock copolymer, poly(ethylene glycol)-b-(N-isopropylacrylamide-co-p-NAPMA) (PEG-b-P(NIPAM-co-p-NAPMA)), was rendered with unique NO- and UVresponsive characteristics. Specifically, the NO-triggered transformation of p-NAPMA moieties into phenyldiazonium residues dramatically elevated the lower critical solution temperature (LCST) of the block copolymer due to increased water solubility of phenyldiazonium residues at neutral pH (i.e., pH 7.4). Further, subsequent UV irradiation significantly decreased the LCST due to the formation of relatively hydrophobic phenol derivatives from the hydrophilic phenyldiazonium intermediate. These results demonstrate, for the first time, that NO-responsive polymers can be synthesized without the necessity of incorporating ophenylenediamine groups and that a further solubility switch can be stimulated by irradiation with ultraviolet light.

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lower than 1 μM,26 has been identified as one of the most important endogenous transmitters in biological systems, serving as a broad-spectrum signaling molecule throughout the nervous and immune systems.27−29 The physiological importance of NO has spurred the invention of robust strategies to probe NO in situ precisely and efficiently, leading to the successful development of a variety of fluorescent probes that are highly sensitive to NO over other biologically derived reactive oxygen species (ROS).30−37 Of these probes, the most popular design strategy has been to incorporate o-phenylenediamine moieties, which can react with NO in the presence of O2 to yield benzotriazole derivatives and therefore turn on a fluorescence emission due to a suppressed photoinduced

INTRODUCTION The preparation of stimuli-responsive materials has been an area of increasing research focus in recent years due to their potential application in a number of fields.1−5 In addition to materials that exploit conventional stimuli such as changes in pH value,6 temperature,7 redox conditions,8,9 or incident light,10,11 or the presence of certain enzymes12 or electromagnetic fields,13,14 recent investigations have explored gaseous stimuli such as carbon dioxide (CO2)15−18 and oxygen (O2).19,20 By taking advantage of chemical and/or physical interactions between these gaseous stimuli and specific functional groups, a variety of CO2- and O2-responsive materials have been developed. Such materials have potential applications in controlled release, oil/water separation, and water-solubility regulation.21−25 Nitric oxide (NO), a diatomic, lipophilic free radical synthesized in the body with a typical concentration range © XXXX American Chemical Society

Received: January 9, 2016 Revised: March 3, 2016

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Scheme 1. Schematic Representation of the Chemical Structure Transitions of Thermoresponsive PEG-b-P(NIPAM-co-pNAPMA) Diblock Copolymer in the Presence of NO Followed by UV Irradiation (λmax = 365 nm)a

a

The aniline residues are transformed into phenyldiazonium when subjected to NO purging, these moieties being further converted to phenol derivatives when subjected to UV irradiation.

Scheme 2. (a) Consecutive Transition of p-NAPMA Monomer in the Presence of NO and Subsequent UV Irradiation and (b) Synthetic Route Employed for the Preparation of PEG-b-P(NIPAM-co-p-NAPMA) Diblock Copolymer via RAFT Polymerization

electron transfer (PET) mechanism. Inspired by this intricate chemistry, we have developed a category of NO-responsive polymers and conducted preliminary investigations into their potential application in NO-triggered self-assembly and anion sensing.38−41 However, in comparison with the substantial advances made in the development of NO probes based on diverse fluorophores, the exploration of moieties that are highly sensitive to NO stimulus is far less common. Indeed, the identification of novel NO-reactive substances would not only advance the field of NO sensing but also could enable better understanding of the biological functions of NO.32,34 It is important to note that the reaction between NO and an o-phenylenediamine derivative yields the benzotriazole via an intramolecular nucleophilic displacement on the diazohydroxide by the neighboring amino group.42 As such, the reaction between o-phenylenediamine and NO initially involves only one primary amine group with the formation of a phenyldiazonium intermediate. We thus hypothesized that a category of novel NO-responsive materials might conceivably be derived from aromatic compounds with only a single primary group.

Interestingly, there were a few early studies regarding the reaction of aniline toward NO, and these reactions were reported to proceed via a deamination mechanism in organic solvents.43−45 Recently, the NO-induced deamination reaction has also been performed in an aqueous milieu, and this chemistry has been successfully employed to construct novel NO probes using a fluorescein-based fluorophore.46 Herein, we report the fabrication of a new NO-responsive material based on an aniline moiety which exhibits a unique NO-responsive behavior in purely aqueous medium and which does not proceed via the widely suggested deamination route. Starting from p-phenylenediamine (an isomer of o-phenylenediamine), one of the primary amine groups was functionalized with a polymerizable double bond by reaction with methacrylic anhydride, yielding the target monomer N-(4-aminophenyl)methacrylamide (p-NAPMA) which contains an aromatic primary amine. The p-NAPMA monomer was then incorporated into a polymeric system via reversible addition− fragmentation chain transfer (RAFT) copolymerization with N-isopropylacrylamide (NIPAM) using a poly(ethylene glycol) B

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Figure 1. 1H NMR spectra recorded in D2O for (a) p-NAPMA monomer at pH 1, (b) p-NAPMA after reaction with NO (p-NDPMA), and (c) pNAPMA after reaction with NO followed by irradiation with UV 365 nm (p-NHPMA). thioylthio)pentanoic acid using a similar protocol as reported previously.47,48 NO was prepared according to a literature procedure, and the concentration of NO solution was quantified by Griess reagent.46 Synthesis. The preparation of N-(4-aminophenyl)methacrylamide (p-NAPMA) monomer and subsequent chemical transitions in the presence of NO and under UV irradiation are shown in Scheme 2a. The synthesis of PEG-b-P(NIPAM-co-p-NAPMA) via RAFT polymerization using a PEG45-based macromolecular RAFT agent is shown in Scheme 2b. Synthesis of N-(4-Aminophenyl)methacrylamide (p-NAPMA, Scheme 2a). The preparation of p-NAPMA monomer was conducted via the amidation reaction between p-phenylenediamine and methacrylic anhydride. In a typical experiment, p-phenylenediamine (5.4 g, 50 mmol) was dissolved in pyridine (150 mL), and methacrylic anhydride (7.7 g, 50 mmol) was added into the solution dropwise via a dropping funnel. After the addition, the reaction was stirred at room temperature overnight. The insoluble salt was then removed via vacuum filtration, and pyridine was removed under reduced pressure. The solid residue was washed three times with diethyl ether and further purified by column chromatography using ethyl acetate/ petroleum ether (v/v = 1:2) as the eluent. The target p-NAPMA monomer was obtained as a white solid (6.78 g, yield: 77%). 1H NMR (Figure 1a, D2O, δ, ppm): 7.56−7.40 (2H), 7.36−7.19 (2H), 5.69 (1H), 5.46 (1H), 1.86 (3H). 13C NMR (Figure S1, CDCl3, δ, ppm): 166.44, 143.43, 140.92, 129.12, 122.11, 119.43, 115.41, 18.82. Treatment of p-NAPMA Monomer with NO To Generate pNDPMA Monomer (Scheme 2a). Typically, p-NAPMA monomer (176 mg, 1 mmol) was dissolved in 20 mL of deionized water, and the solution pH was adjusted to approximately 1 with hydrochloric acid (HCl, 37%) to fully dissolve the p-NAPMA monomer. The solution was then purged with NO gas for 1 h and subsequently stirred at room temperature for 12 h. The resulting aqueous solution was lyophilized, and the resultant solid was collected without further purification. 1H

(PEG)-based macromolecular RAFT agent, thus affording a thermoresponsive diblock copolymer, PEG-b-P(NIPAM-co-pNAPMA). The presence of p-NAPMA moieties in the polymer chain imparted the resulting block copolymer with distinct NOresponsive behavior. Upon exposure to NO in aqueous solution, the primary amine residues were efficiently transformed into phenyldiazonium derivative (abbreviated to pNDPMA) which had long-term stability at room temperature. However, under UV irradiation (λmax = 365 nm), the phenyldiazonium moieties were readily converted to phenol derivatives (abbreviated to as p-NHPMA) with the liberation of nitrogen. Importantly, the NO-induced formation of phenyldiazonium residues followed by the generation of phenol moieties under UV light irradiation dramatically switched the lower critical solution temperature (LCST) of the block copolymers, leading to increased and decreased LCST, respectively (compared to the intact block copolymer without NO treatment) (see Scheme 1).

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EXPERIMENTAL SECTION

Materials. p-Aminophenol, p-phenylenediamine, methacrylic anhydride, methacryloyl chloride, sodium nitrite (NaNO2), and poly(ethylene glycol) methyl ether (PEG45-OH, Mn = 2 kDa) were purchased from Sigma-Aldrich and used as received. N-Isopropylacrylamide (NIPAM) was purchased from Sigma-Aldrich and purified by crystallization from a mixture of toluene and petroleum ether (v/v = 1:3) twice prior to use. Sulfuric acid (H2SO4, 98%) was obtained from RCI Labscan Ltd. All solvents were purchased from EMSURE and used as received unless otherwise noted. PEG45-based macromolecular RAFT agent was synthesized through the esterification reaction between PEG 45 -OH and 4-cyano-4-(phenylcarbonoC

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Macromolecules NMR (Figure 1b, D2O, δ, ppm): 8.44−8.37 (2H), 8.06−7.96 (2H), 5.84 (1H), 5.67 (1H), 1.93 (3H). Treatment of p-NDPMA Monomer with N,N-Dimethylaniline (Figure S2). p-NDPMA monomer (205 mg, 1 mmol) was dissolved in 5 mL of N,N-dimethylaniline, and 15 mL of deionized water was then added. The mixture was stirred at room temperature overnight, and the successful transformation of p-NDPMA monomer into azobenzene-containing derivatives was evidenced by the formation of an undissolved yellowish solid. After removing the water and excess N,Ndimethylaniline by filtration, the solid was washed with an excess of diethyl ether and was dried in a vacuum oven at room temperature, yielding a yellowish solid. 1H NMR (Figure S2a, CDCl3, δ, ppm): 7.94−7.83 (4H), 7.75−7.67 (2H), 7.67−7.55 (1H), 6.79 (2H), 5.85 (1H), 5.52 (1H), 3.11 (6H), 2.11 (3H). 13C NMR (Figure S2b, CDCl3, δ, ppm): 166.49, 152.32, 149.67, 143.64, 140.87, 138.88, 124.88, 123.16, 120.09, 120.05, 111.59, 40.34, 18.78. Exposure of p-NDPMA Monomer to UV Irradiation (λmax = 365 nm) To Generate p-NHPMA Monomer (Scheme 2a). The p-NDPMA monomer (41 mg, 0.2 mmol) was dissolved in 5 mL of deionized water, and the resulting solution was irradiated with UV light (λmax = 365 nm) in a 10 mL glass vial for a predetermined time. After irradiation, the resulting solid was recovered by lyophilization and was analyzed by NMR. 1H NMR (Figure 1c, D2O, δ, ppm): 7.24 (2H), 6.81 (2H), 5.69 (1H), 5.43 (1H), 1.89 (3H). Synthesis of N-(4-Hydroxyphenyl)methacrylamide (p-NHPMA, Figure S7). p-Aminophenol (5.46 g, 50 mmol) and K2CO3 (13.8 g, 100 mmol) were dissolved in 150 mL of dry THF (dried over molecular sieves), and the solution was cooled to 0 °C in an ice−water bath for 10 min. To the cooled 4-aminophenol/K2CO3 solution was added dropwise a THF solution (20 mL) of methacryloyl chloride (4.84 mL, 50 mmol). After the addition, the reaction was gradually warmed up to room temperature and stirred overnight at room temperature. After removing insoluble salt by filtration, THF was removed under vacuum, and the crude product was purified by column chromatography using ethyl acetate/petroleum ether (v/v = 1:2) as the eluent. The target p-NHPMA monomer was obtained as a white crystal (3.01 g, yield: 34%). 1H NMR (Figure S7b, D2O, δ, ppm): 7.31−7.08 (2H), 6.91−6.73 (2H), 5.73 (1H), 5.47 (1H), 1.93 (3H). Synthesis of Thermoresponsive PEG-b-P(NIPAM-co-p-NAPMA) Diblock Copolymer (Scheme 1b). PEG-b-P(NIPAM-co-p-NAPMA) diblock copolymer was synthesized via RAFT copolymerization of NIPAM and p-NAPMA monomers using a PEG-based macromolecular RAFT agent. Typically, PEG45-based macromolecular RAFT agent (22.6 mg, 0.1 mmol), NIPAM (905 mg, 8 mmol), pNAPMA (352 mg, 2 mmol), AIBN (2 mg, 12 μmol), and DMF (3 mL) were charged into a 20 mL polymerization vial with a rubber septum. The mixture was deoxygenated via purging with nitrogen for 30 min. The polymerization vial was then transferred to a hot plate set at 70 °C to commence the polymerization. After 16 h polymerization, the reaction was quenched into an ice−water bath, and the obtained diblock copolymer was purified by precipitating three times into an excess amount of diethyl ether. After drying in a vacuum oven overnight, the diblock copolymer was obtained as a pale red solid (0.54 g). The molecular composition of the diblock copolymer was determined to be PEG45-b-P(NIPAM0.74-co-p-NAPMA0.26)30 as determined by analysis of the 1H NMR spectrum (Figure S10a). The number-average molecular weight (Mn) and polydispersity (Mw/ Mn) of the diblock copolymer were determined to be 12.2 kDa and 1.26, respectively, based on GPC measurement using N,N-dimethylacetamide (DMAc) as the eluent (Figure 4C) and PS calibration standards. Characterization. Nuclear Magnetic Resonance (NMR) Spectra. 1 H and 13C NMR spectra were recorded on a Bruker AC400F (400 MHz) spectrometer. Deuterated chloroform (CDCl3) and deuterium oxide (D2O) were used as the solvents, depending on the particular substances being analyzed. Gel Permeation Chromatography (GPC). GPC analyses of polymer samples were performed in N,N-dimethylacetamide (DMAc with 0.03% w/v LiBr using a Shimadzu modular system comprising a DGU-12A degasser, an SIL-10AD automatic injector, and a 5.0 μm

bead-size guard column (50 × 7.8 mm) followed by three KF-805L columns (300 × 8 mm, bead size: 10 μm, pore size maximum: 5000 Å), a SPD-20A ultraviolet detector, and an RID-10A differential refractive index detector. The temperature of columns was maintained at 40 °C using a CTO-20A oven, and the flow rate was kept at 1 mL/ min using a LC-10AT pump. A molecular weight calibration curve was produced using commercial narrow molecular weight distribution polystyrene standards with molecular weights ranging from 500 to 106 g/mol. Polymer solutions at 2−3 mg/mL were prepared in the eluent and filtered through 0.45 μm filters prior to injection. UV−Vis Spectrophotometry. All UV−vis spectra were acquired on a Shimadzu UV-3600 UV−vis−NIR spectrophotometer. Quartz cuvettes of 10 mm path length were employed for all the measurements. For the turbidity measurements, the optical transmittance of the aqueous PEG-b-P(NIPAM-co-p-NAPMA) solutions (2.0 g/L in all cases) before and after reaction with NO and further treatment with UV irradiation were acquired on the same spectrophotometer equipped with a Shimadzu temperature controller and Tm Analysis software. The recorded wavelength was set at 500 nm with a heating rate of 0.5 °C/min. The LCST was defined as the temperature leading to a 50% decrease in transmittance.39 Fluorescence Spectroscopy. All fluorescence spectra were acquired on a Shimadzu RF-5301 fluorescence spectrometer. The excitation wavelength was set at 495 nm and the slit widths for both excitation and emission were 3 nm. Attenuated Total Reflectance−Fourier Transform Infrared Spectroscopy (ATR-FTIR). ATR-FTIR measurements were performed using a Shimadzu IRTracer-100 Fourier transform infrared spectrometer by averaging 128 scans with a resolution of 4 cm−1.

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RESULTS AND DISCUSSION NO-responsive monomer containing a single primary amine, pNAPMA, was first synthesized via the monoamidation reaction between p-phenylenediamine and methacrylic anhydride in a molar ratio of 1:1. The chemical structure of p-NAPMA monomer was confirmed by both 1H and 13C NMR spectroscopy analysis (Figure 1a and Figure S1). Subsequently, the reactivity of p-NAPMA monomer toward NO was evaluated by monitoring the UV−vis absorption changes of p-NAPMA in the presence of varying amounts of NO. Initially, the p-NAPMA monomer in aqueous solution in its nonprotonated form possessed a maximum absorption peak at 273 nm. Upon progressive addition of NO to the solution, the absorption peak initially blue-shifted to 261 nm in the presence of 0.125 equiv of NO (relative to p-NAPMA monomer). It should be noted that this initial hypochromic shift was ascribed to the lowering of the solution pH and the resulting protonation of p-NAPMA moieties, since NO is easily oxidized to nitric acid and nitrous acid in air.40 Subsequently, the absorption intensity at 261 nm gradually dropped, while a new absorption peak centered at 340 nm appeared and steadily intensified upon increasing the NO concentration in the range of 0.25−1.5 equiv (relative to p-NAPMA moieties) (Figure 2a,b). The absorption intensities stabilized when the NO concentration was greater than 1.5 equiv of p-NAPMA monomer, suggesting a high reactivity of p-NAPMA toward NO in aqueous solution. This high reactivity was comparable to that observed for the N-(2-aminophenyl)methacrylamide counterpart, which possess the typical o-phenylenediamine motif frequently used in NO reactive materials.38 However, for the N-(2-aminophenyl)methacrylamide monomer, only a slight shift in the UV−vis absorbance spectra (from 288 to 302 nm) was observed upon NO addition as a result of the formation of amide-functionalized benzotriazole moieties.38 Therefore, this unique variation in the UV−vis absorption not only revealed a high reactivity of p-NAPMA monomer toward NO but also D

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vinylic protons. This result suggested that the reaction between p-NAPMA monomer and NO was highly selective and was compatible with other functional groups (e.g., polymerizable double bonds). Although we could safely conclude that the pNAPMA monomer, after reaction with NO, afforded a new compound with a para-substituted phenyl ring structure, unravelling the exact chemical structure was not straightforward using only the 1H NMR result. It should be noted that in previous studies the reaction between aniline derivatives and NO has been performed in various organic solvents such as tetrahydrofuran and chloroform. In these studies the result of NO treatment was the formation of deaminated products, likely through an aryl radical mechanism. In these cases an unstable intermediate product (a phenyldiazonium salt) was suggested, which can be further transformed into an aryl radical in the presence of NO, after which proton abstraction from the solvents was suggested to give the deaminated product.44,45 However, this deamination mechanism cannot be reconciled with the current experimental results because the 1H NMR spectrum clearly demonstrated the generation of para-substituted product (Figure 1b). This discrepancy could be a result of the differing solvents used.45 We postulated that the aqueous milieu may impede the deamination reaction of p-NAPMA monomer in the presence of NO. However, after treating the NO-reacted product recovered from water with N,N-dimethylaniline, a yellowish solid was obtained and its chemical structure was identified by 1 H and 13C NMR spectroscopy (Figure S2).49 The formation of azo-containing derivatives revealed that the product of reacting p-NAPMA with NO was a phenyldiazonium derivative, which was in accord with the previously assumed intermediate product achieved in organic solvents.45 On the basis of the above results, we concluded that the p-NAPMA monomer comprising an aromatic primary amine could efficiently react with NO to generate a phenyldiazonium product in aqueous solution (Scheme 2a). Although the phenyldiazonium product

Figure 2. (a, c) Normalized UV−vis absorbance spectra and (b, d) relative absorption intensity changes of (a, b) p-NAPMA monomer (40 μM) in the presence of varying amounts of NO and (c, d) pNAPMA monomer after NO treatment (p-NDPMA) under UV 365 nm irradiation. Note: UV−vis spectra were recorded immediately after the NO introduction without additional standing time.

implied a distinct reaction mechanism between p-NAPMA and NO compared to that of the N-(2-aminophenyl)methacrylamide analogue with an o-phenylenediamine motif.38 To identify the resulting product obtained by treating pNAPMA monomer with NO and elucidate the underlying reaction mechanism, the product was recovered from aqueous solution through lyophilization and was analyzed by 1H NMR spectroscopy (Figure 1b). In comparison with the initial pNAPMA monomer, the signals originating from the phenyl ring shifted to downfield after NO treatment, but the reaction did not significantly affect the chemical shifts corresponding to the

Figure 3. 1H NMR spectra recorded in D2O for p-NDPMA monomer (10 mg/mL) under UV 365 nm irradiation for varying times (0−70 min). E

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Macromolecules was reported to be labile in organic solvent,44,45 we found that it was relatively stable in aqueous solution. Specifically, there were no noticeable changes in the absorbance spectra after 15 h standing in PBS buffer solution at pH 7.4 at room temperature (Figure S3), which was much longer than that needed to obtain the deaminated product in organic solvents.45 It is worth noting that this characteristic is quite unique as the phenyldiazonium salts are typically unstable at room temperature with the liberation of nitrogen. In spite of the unexpected stability at room temperature, we found the as-synthesized phenyldiazonium monomer was highly sensitive to UV irradiation. Upon UV irradiation with a hand-held UV light (λmax = 365 nm), the UV−vis absorbance spectra underwent a dramatic change within only 3 min. Specifically, the absorbance assigned to the phenyldiazonium salt (at 339 nm) gradually attenuated, while a new absorption peak at 263 nm steadily increased (Figure 2c,d). Moreover, there were three isosbestic points observed at 220, 240, and 288 nm for the spectra measured during the UV irradiation (Figure 2c). The UV light-responsive feature was further confirmed by examining the time-dependent evolution of the 1H NMR spectrum (Figure 3). Upon constant UV irradiation, the signals due to the protons on the phenyl ring of the phenyldiazonium gradually shifted to higher field over 70 min. The corresponding conversion of the phenyldiazonium could be quantified from the NMR analysis, and this data is shown in Figure S4. This UV-induced conversion of phenyldiazonium derivatives followed first-order kinetics, and the half-life (τ1/2) was determined to be approximately 17.3 min (Figure S4b). Interestingly, there were no changes in the signals associated with the vinylic protons, which were similar to those observed after NO addition. That said, it is acknowledged that the double bonds are potentially susceptible to UV irradiation and UVmediated coupling reactions could occur.50 The current very clean result can be attributed to the very low UV irradiation dosage applied (hand-held UV lamp; ∼1.5 mW/cm2) and indicates that the phenyldiazonium is highly sensitive toward UV irradiation. It is worth noting that the UV irradiation time applied for the 1H NMR analysis was significantly longer than for the UV−vis absorption measurement, and this variation was unsurprising due to the different monomer concentrations used in each experiment (40 μM for UV−vis vs 48.7 mM for 1H NMR). Importantly, the initial p-NAPMA monomer with a free primary amine group (i.e., without NO treatment) was essentially inert to the UV irradiation. With the same time period of UV irradiation (i.e., 70 min), no shifts in the 1H NMR spectrum were evident (Figure S5). Therefore, the emergence of UV light-responsive behavior for the p-NAPMA monomer after NO treatment is considered to be a consequence of forming a phenyldiazonium derivative. From the 1H NMR spectra (Figures 1c and 3f), it is apparent that a new product with another para-substituted phenyl ring structure was achieved after UV irradiation. Interestingly, bubbles were observed during the UV irradiation, suggesting that a gas was produced and released from the aqueous solution due to the UV exposure. To confirm that the gas was not NO, the fluorescence spectra of a mixture of the phenyldiazonium and 4,5-diaminofluorescein (DAF-2, a fluorescent probe of NO)33 was irradiated with the UV lamp. This experiment revealed no significant fluorescence turn-on under UV light irradiation (Figure S6), demonstrating that the generated gas was not NO. Given the chemical structure of the phenyldiazonium derivative, we surmised that the generated gas could

be nitrogen (N2) rather than NO under UV irradiation. As a result, the final product subjected to UV irradiation was postulated to be N-(4-hydroxyphenyl)methacrylamide (pNHPMA) (Scheme 2a). To verify this hypothesis, p-NHPMA was prepared through the amidation reaction between p-aminophenol and methacryloyl chloride, and the 1H NMR spectrum of the product formed is shown in Figure S7b. The spectrum obtained concurred quite well with the 1H NMR spectrum of the phenyldiazonium derivative subjected to UV irradiation. Further, the absorbance spectrum of as-synthesized pNHPMA was identical to that of the product obtained from the phenyldiazonium salt after UV irradiation (Figure S8). These results confirmed that the final photodegraded product yielded from the phenyldiazonium derivative after UV light irradiation was p-NHPMA (Scheme 2a). Further, the consecutive transition of p-NAPMA monomer in the presence of NO and UV light irradiation to form phenyldiazonium (pNDPMA) and phenol (p-NHPMA) derivatives was further confirmed by ATR-FTIR spectroscopy (Figure S9). It should be noted that although we did not visually discern the changes in water solubility after the formation of the phenyldiazonium derivative, the UV light irradiation of the phenyldiazonium derivative led to significantly decreased water miscibility, which was evident by the appearance of insoluble solid that was clearly visible to the naked eye. We thus inferred that the cascade reaction of p-NAPMA monomer caused by NO and UV light irradiation could lead to a drastic transition in the water solubility if the monomer was incorporated into a thermoresponsive polymeric system. In other words, the temperatureinduced volume phase transition (VPT) of p-NAPMA containing thermoresponsive polymers could potentially be manipulated by both NO gas and UV irradiation. As a proof-of-principle, p-NAPMA monomer was incorporated into a thermoresponsive diblock copolymer, PEG-bP(NIPAM-co-p-NAPMA), through RAFT polymerization using a PEG-based macromolecular RAFT agent (Scheme 2b). Importantly, the incorporation into a polymeric system did not compromise the NO-responsive behavior of p-NAPMA moieties as evidenced by 1H NMR spectroscopy. As shown in Figure S10, the signals corresponding to the phenyl ring protons shifted to downfield after NO treatment, presumably due to the formation of phenyldiazonium moieties (Figure S10b), as was observed for the p-NAPMA monomer (Figure 1). Interestingly, even with an extra 15 h standing in aqueous solution, there were no evident changes in 1H NMR spectra of PEG-b-P(NIPAM-co-p-NAPMA) block copolymer after reaction with NO, suggesting the stability of newly generated phenyldiazonium moieties within the polymeric chains (Figure S11). This result concurred quite well with the stability of pNAPMA monomer after NO treatment (Figure S3). Importantly, this result demonstrated that the aniline derivative-containing monomer can be successfully polymerized via RAFT polymerization without loss of the NO reactivity. Further, the resulting diblock copolymer after NO treatment underwent a comparable UV−vis spectral evolution to the monomer when exposed to UV irradiation at λmax = 365 nm (Figure 4A). The absorption corresponding to phenyldiazonium moieties at 336 nm gradually decreased, while the absorption of corresponding to phenol residues (i.e., at 247 nm) steadily increased within 360 s of UV irradiation (Figure 4A,B). The extended irradiation time required for the diblock copolymer (compared to that of the p-NAPMA small F

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hydrophilic PEG block.39,53 However, after reaction with NO, the LCST was switched to approximately 57 °C due to the formation of water-soluble phenyldiazonium moieties. This result was in accordance with the 1H NMR result (Figure S10b) and the significant bathochromic shift of the maximum absorbance from 258 to 336 nm in the UV−vis absorption spectra (Figure S12). Upon UV irradiation, a pronounced drop in the LCST from 57 to 33 °C was observed as a result of the formation of relatively hydrophobic phenol residues and hydrogen bonding interaction between phenol and amide groups.51,52 This pronounced transition in the LCST was accompanied by a hypochromatic shift in the maximum absorption peaks from 336 to 246 nm (Figure 4A and Figure S12).

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CONCLUSIONS In summary, we have successfully prepared a new NOresponsive monomer starting from p-phenylenediamine rather than conventional o-phenylenediamine. After converting to a polymerizable form with methacrylic anhydride, the resulting pNAPMA monomer can selectively react with NO with the formation of a phenyldiazonium derivative instead of a benzotriazole (as observed with o-phenylenediamine derivatives). Moreover, although the phenyldiazonium residues were unexpectedly stable in aqueous solution at room temperature, they were inherently responsive to UV light irradiation, with the generation of p-NHPMA monomer. This cascade transition under NO treatment and UV light irradiation was characterized by a significant shift in the water solubility, thus providing the opportunity to regulate the VPTs of thermoresponsive polymers. After incorporating p-NAPMA monomer into a diblock copolymer (PEG-b-P(NIPAM-co-p-NAPMA)), the LCST of the copolymer could be regulated by exposure to NO and subsequent UV light irradiation. This work opens a new window toward fabricating novel gasotransmitter-responsive materials by exploiting the unique reaction of primary amines with NO. The exploration of the potential application of this novel dual-responsive material in a biological milieu is currently underway.

Figure 4. (A) UV−vis spectra and (B) relative absorbance intensity changes of 0.1 g/L of PEG-b-P(NIPAM-co-p-NAPMA) in PBS buffer solution (0.1 mM, pH 7.4) after treatment with NO and UV 365 nm irradiation. (C) GPC elution traces for (a) PEG45-based macroRAFT agent and PEG-b-P(NIPAM-co-p-NAPMA) diblock copolymer (b) before and (c) after reaction with NO and (d) after reaction with NO followed by UV 365 nm irradiation. (D) Temperature-dependent transmittance at 500 nm for an aqueous solution of PEG-b-P(NIPAMco-p-NAPMA) diblock copolymer (2.0 g/L in PBS buffer, pH 7.4) before and after reaction with NO and after reaction with NO followed by UV 365 nm irradiation.

molecule) could be attributed to macromolecular effects deriving from a relatively high local concentration of pNAPMA moieties. Moreover, the blue-shift of the maximum absorbance associated with the phenol derivatives in the polymer chain was tentatively attributed to the hydrogen bonding interactions among pendant phenol and acrylamide moieties.51,52 After NO treatment followed by UV irradiation, the 1H NMR spectrum of PEG-b-P(NIPAM-co-p-NAPMA) diblock copolymer revealed a shift to high-field for the phenyl ring protons, which is in accord with the result for the p-NAPMA monomer after UV irradiation (Figure S10c). Despite using the same concentration (i.e., 20 mg/mL), the signal intensities corresponding to the protons of the phenyl rings significantly dropped when subjected to UV irradiation compared to those observed before UV light irradiation. This is likely a result of the decreased solubility of the phenol derivatives in aqueous solution and is consistent with the formation of insoluble solid after UV irradiation for the p-NAPMA small molecule. Moreover, the changes in chemical composition in the presence of NO and after UV irradiation could also be evidenced by the GPC elution traces (Figure 4C), in which a much boarder elution trace was achieved for the diblock copolymer after both NO and UV irradiation. Finally, the temperature-induced VPT of the thermoresponsive block copolymer was further examined by following turbidity changes with variation in temperature. Specifically, prior to NO treatment and UV irradiation, the lower critical solution temperature (LCST) was determined to be 43 °C (if the LCST is defined as the temperature corresponding to a 50% decrease in the transmittance at the wavelength of 500 nm). The LCST of the current diblock copolymer was higher than for PNIPAM homopolymers due to the presence of the

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b00054. Additional 1H NMR spectra, UV−vis spectra, and fluorescence spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (T.P.D). *E-mail [email protected] (J.F.Q.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was conducted within the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (Project CE140100036). T.P.D. gratefully acknowledges the award of an Australian Laureate Fellowship. G

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DOI: 10.1021/acs.macromol.6b00054 Macromolecules XXXX, XXX, XXX−XXX