Induced Solution Phase Transition of Water ... - ACS Publications

Apr 26, 2016 - Key Laboratory of Polymeric Materials and Application Technology of Hunan Province, Key Laboratory of Advanced Functional. Polymer Mate...
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SO2‑Induced Solution Phase Transition of Water-Soluble and α‑Helical Polypeptides Mengxiang Zhu, Yan Wu, Chenglong Ge, Ying Ling, and Haoyu Tang* Key Laboratory of Polymeric Materials and Application Technology of Hunan Province, Key Laboratory of Advanced Functional Polymer Materials of Colleges and Universities of Hunan Province, College of Chemistry, Xiangtan University, Xiangtan, Hunan 411105, China S Supporting Information *

ABSTRACT: Water-soluble random copolypeptides bearing pyridinium tetrafluoroborate (PyBF4) and oligo-ethylene glycol (OEG) pendants (PPLG-PyBF4-r-OEG) showed SO2induced solution phase transition. They were prepared by sequential postpolymerizations including nucleophilic substitution of poly(γ-3-chloropropyl-L-glutamate) with sodium azide to partially convert chloro groups into azido groups, copper-mediated [2 + 3] alkyne−azide 1,3-dipolar cycloaddition to conjugate OEG pendants, another nucleophilic substitution to conjugate pyridinium chloride, and ion-exchange reaction. FTIR and CD analysis revealed that PPLG-PyBF4-r-OEG samples with suitable molar content of PyBF4 (x ≤ 0.4) were water-soluble and adopted α-helical conformation in both the solid-state and aqueous solution. UV−vis spectroscopy and dynamic light scattering (DLS) results revealed that PPLG-PyBF4-rOEG aqueous solution underwent a reversible solution phase transition by sequentially bubbling SO2 and N2. 1H NMR analysis suggested that SO2 interacted with triazole groups and induced the solution phase transition.

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INTRODUCTION Stimuli-responsive polymers are interesting polymeric materials that can undergo conformational or chemical changes on receiving external triggers,1 such as temperature,2−4 pH,5 light,6,7 and so forth. The properties of the polymers (e.g., solubility, permeability, and mechanical and optical properties) vary according to the changes of polymer conformations or chemical structures, which makes stimuli-responsive polymers useful in extensive applications, such as smart membranes,8 sensors,9 tunable catalysis,10 drug delivery,11 and tissue engineering.12,13 Among all external triggers, gas has showed the advantage of easy input and removal with accumulation of unwanted products (e.g., salts which produced after pH variation). Currently, gas-responsive polymers are mainly based on carbon dioxide (CO2).14−25 The mechanism of CO2 responsiveness usually involves the protonation of tertiary amines, amidines, or guanidines upon CO2 bubbling in polymer aqueous solution.26−28 However, gasresponsive polymers based on other gases have been less investigated and reported.29−32 The development of new types of gas-responsive polymers will not only broaden their applications but also provide guidance for future molecular design of stimuli-responsive polymers. Polypeptides are promising biomimetic materials with many attractive properties, such as biocompatibility, ordered conformations (e.g., α-helix and β-sheet), and unique self-assembly structures.33−36 Stimuli-responsive polypeptides with responsiveness to pH,37,38 temperature,39−42 light,43,44 and oxidation45−47 © 2016 American Chemical Society

have been prepared by (1) ring-opening polymerization of side-chain modified N-carboxyanhydride (NCA) monomers or (2) postpolymerization of reactive polypeptides.48 They have exhibited great potential in various biotechnological and biomedical applications.49−51 In this contribution, we report the first example of SO2induced solution phase transition of water-soluble polymers. The polymers are random copolypeptides bearing pyridinium tetrafluoroborate (PyBF4) and oligo-ethylene glycol (OEG) pendants which were prepared by a multistep postpolymerization. Their molecular structures were confirmed by a combination of spectroscopic and chromatographic techniques, such as 1H NMR, FTIR, and GPC. The SO2-induced solution phase transition behavior was investigated by UV−vis spectroscopy, dynamic light scattering (DLS), and 1H NMR.

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EXPERIMENTAL SECTION

Materials. Poly(γ-3-chloropropyl-L-glutamate) (PCPLG, Mn = 9900, PDI = 1.11, DP = 48) and propargyl-functionalized oligo-ethylene glycol (Pr-OEG) were synthesized according to reported procedures.52−54 Anhydrous N,N-dimethylformamide (DMF, 99.9%) was dried over molecular sieves before use. Chloroform-d (CDCl3, D.99.8%) + silver foil was purchased from Cambridge Isotope Laboratories, Inc. Copper bromide (CuBr, 99%), N,N,N′,N″,N″-pentamethyldiethylenetriamine Received: January 19, 2016 Revised: March 24, 2016 Published: April 26, 2016 3542

DOI: 10.1021/acs.macromol.6b00116 Macromolecules 2016, 49, 3542−3549

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Macromolecules Scheme 1. Synthetic Route of PPLG-PyBF4-r-OEG

of polypeptide repeating unit), θλ is the observed ellipticity (mdeg) at the wavelength λ (i.e., 222 nm), d is the path length (mm), and c is the concentration (mg mL−1).55 The fractional helicity (f H) of the polypeptides was calculated using the equation f H = (−[θ]222 + 3000)/ 39000 to allow for a quantitative comparison of the relative helical content, where [θ]222 is the mean residue ellipticity at 222 nm.56 Ultraviolet−visible (UV−vis) spectra were measured using an Agilent Cary 100 spectrometer. The polymer aqueous solutions were prepared by directly mixing and stirring at room temperature and then placed in a quartz cell with a path length of 1.0 cm. The transmittances of solutions were collected at the wavelength of 500 nm. DI-H2O at 25 °C was set to be 100% of transmittance. Dynamic light scattering (DLS) measurements were conducted on a BI-200 SM scattering system (Brookhaven Instruments Corporation, USA) equipped with a digital time correlator (BI-9000), a laser (532 nm) at a scattering angle of 90°, and a controller (BI-TCD) for precisely adjusting the solution temperature. Field emission scanning electron microscopy (FESEM) images were acquired with a XL 30 ESEM FEG SEM (FEI Company). The polymer solutions were dropped on a silicon wafer and free-dried. The samples were goldsputtered prior to FESEM imaging. Synthesis of Poly(γ-3-chloropropyl-L-glutamate)-randomPoly(γ-3-azidopropyl-L-glutamate) (PCPLG-r-PAPLG). PCPLG (211.0 mg, 1.02 mmol of chlorine, Scheme 1) was dissolved in DMF (4.0 mL) in a glass vial (10 mL) under nitrogen, followed by adding NaN3 (55.0 mg, 0.84 mmol). The mixture was stirred at 60 °C for 16 h (x = 0.2), 12 h (x = 0.4), or 8 h (x = 0.6). Then, the DMF solution was slowly added to a 10-fold DI-H2O to precipitate the product. Filtration and drying afforded the production as a pale yellow solid (200.0 mg, 95% yield). 1H NMR (400 MHz, CDCl3, δ, ppm) of PCPLG-r-PAPLG (x = 0.2): 8.32 (s, 1H, −NH−), 4.16 (s, 4H, −CH2CH2CH2Cl and −CH2CH2CH2N3), 3.96 (s, 1H, −COCHNH−), 3.61 (s, 2H, −CH2Cl), 3.38 (s, 2H, −CH2N3), 2.65−2.09 (d, 2H, −CH2CH2CO−), 2.37 (s, 2H, −CH2CH2CO−), 2.09 (s, 2H, −CH2CH2Cl), 1.91 (s, 2H, −CH2CH2N3). FTIR (neat, cm−1) of PCPLG-r-PAPLG (x = 0.2): 3277, 2931, 2096, 1727, 1650, and 1545. Synthesis of Poly(γ-3-chloropropyl-L-glutamate)-randomPoly(γ-propyl- L -glutamate)-graf t-Oligo-Ethylene Glycol (PCPLG-r-PPLG-OEG). A representative copper-mediated [2 + 3] alkyne−azide 1,3-dipolar cycloaddition is as follows. PCPLG-r-PAPLG (50.0 mg, 0.19 mmol of azido groups, x = 0.2, Scheme 1), Pr-OEG (93.0 mg, 0.46 mmol), CuBr (66 mg, 0.46 mmol), and PMDETA (96 μL, 0.46 mmol) were mixed in DMF (4.5 mL) under nitrogen. The mixture was stirred at room temperature for 24 h. The product was purified by dialysis against DI-H2O (HClaq was added to remove the copper ions, pH = 5−6) in a dialysis bag with a molecular weight

(PMDETA, 99%), deuterium oxide (D2O, D.99.9%), pyridine (>99.5%), 2-methylpyridine (>99%), 4-methylpyridine (>99%), NaI (98%), and NaBF4 (98%) were purchased from Energy Chemical. 3-Methylpyridine (98%) was purchased from Tokyo Kasei Kogyo Co. Ltd. Sodium azide (NaN3, 99.5%) was purchased from Sigma-Aldrich. All other chemicals were purchased from Aladdin and used as received. Deionized water (DI-H2O) was obtained from an Aquapro AR1-100L-P11 water-purification system (Ever Young Enterprises Development Co., Ltd., P. R. China). Instrumentation. 1H nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker ARX400 MHz spectrometer at room temperature. Chemical shifts (δ) were reported in the units of ppm and referenced to the protio impurities. The polymer solution with concentrations of ∼15 mg mL−1 for 1H NMR test prepared by directly mixing and shaking at room temperature. Gel permeation chromatography (GPC) measurements were performed on a PL-GPC120 setup equipped with a column set consisting of two PL gel 5 μm MIXED-D columns (7.5 mm × 300 mm, effective molar mass range of 0.2−400.0 kg mol−1) and PL-RI differential refractive index (DRI) detector. DMF containing 0.01 M LiBr was used as the eluent at 80 °C at a flow rate of 1.0 mL min−1. Narrowly distributed polystyrene standards in the molar mass range of (0.5−7.5) × 104 kg mol−1 (PSS, Mainz, Germany) were utilized for calibration. Polymer solutions for the GPC test with a concentration of 5 mg mL−1 in 0.01 M LiBr/ DMF were prepared by directly mixing and shaking at room temperature. FTIR spectra were recorded on a Thermo Scientific Nicolet 6700 FTIR spectrometer equipped with an attenuated total reflection (ATR) sample holder. Solid samples were placed on the diamond crystal window and pressed with a metal probe. Spectral measurements were carried out in the transmittance mode (scan range = 4000−600 cm−1, resolution = 2 cm−1, number of scans = 2, 25 °C). Circular dichroism (CD) measurements were carried out on an AVIV 410 CD spectrometer (Biomedical Inc., Lakewood, NJ). The polymer aqueous solutions were prepared at concentrations of 1 mg mL−1 by directly mixing and stirring at room temperature. Then, the above solutions (1 mg mL−1) were diluted to 0.05 mg mL−1 for CD measurement. The solution was placed in a quartz cell with a path length of 0.2 cm. CD data were collected with the high tension voltage (i.e., the voltage applied to the photomultiplier) less than 600 V. Two scans were conducted and averaged between 185 and 250 nm with a resolution of 0.5 nm. The data were processed by subtracting the solvent (i.e., DI-H2O) background and smoothing with the FFT-Filter method with points of window of 8. The CD spectra were reported in mean residue ellipticity (MRE) (units: deg cm2 dmol−1) which was calculated by the equation [θ]λ = MRW × θλ/10 × d × c, where MRW is the mean residue weight (MRW = the molecular weight 3543

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−CH2CH2Py+BF4−). FTIR (neat, cm−1) of P1: 3286, 2930, 1728, 1651, 1548, 1083, and 1024. 1H NMR of P2 (D2O, δ, ppm): 8.70−8.27 (m, 4H, −PyH), 8.07 (s, 1H, triazole), 4.66 (s, 2H, −O−CH2−triazole−), 4.54 (d, 4H, −CH2CH2-triazole− and −CH2N+BF4−), 4.10 (m, 5H, −COCHNH−, −CH2CH2CH2−triazole−, and −CH2CH2CH2N+BF4−), 3.64−3.37 (m, 15H, −CH2CH2OCH2CH2OCH2CH2O− and −OCH3), 2.65−2.23 (m, 11H, −CH2CH2COO−, −CH2COOCH2−, −CH2CH2− triazole−, −CH2CH2Py+BF4− and −Py−CH3). FTIR (neat, cm−1) of P2: 3287, 2931, 1730, 1651, 1548, 1083, and 1025. 1H NMR of P4 (D2O, δ, ppm): 8.68−8.42 (m, 4H, −PyH), 8.06 (s, 1H, triazole), 4.67 (s, 2H, −O−CH2−triazole−), 4.54 (d, 4H, −CH2CH2-triazole− and −CH2N+FB4−−), 4.11 (m, 5H, −COCHNH−, −CH2CH2CH2− triazole−, and −CH 2 CH 2 CH 2 N + BF 4 − ), 3.65−3.37 (m, 15H, −CH2CH2OCH2CH2OCH2CH2O− and −OCH3), 2.65−2.25 (m, 11H, −CH2CH2COO−, −CH2COOCH2−, −CH2CH2−triazole−, −CH2CH2Py+BF4− and −Py−CH3). FTIR (neat, cm−1) of P4: 3286, 2932, 1729, 1651, 1547, 1083, and 1022. Test of SO2 Responsiveness. A representative procedure is as follows. Polymer aqueous solution (e.g., P3, 20 mg mL−1) was prepared by directly mixing and stirring at room temperature. Then, it was transferred to a glass tube, followed by bubbling SO2 for 5 min. The SO2 gas was produced from the reaction of Na2SO3 and sulfuric acid.

cutoff of 3000 g mol−1 for 3 days. Removing the solvent under vacuum afforded a glassy solid (70.0 mg, 73% yield). 1H NMR (400 MHz, CDCl3, δ, ppm) of PCPLG-r-PPLG-OEG (x = 0.2): 8.32 (s, 1H, −NH−), 7.75 (s, 1H, triazole), 4.65 (s, 2H, −OCH2−triazole), 4.49 (s, 4H, −CH2Cl and −CH2CH2CH2−triazole), 4.18−3.97 (m, 5H, −CH2CH2CH2Cl, −CH2CH2CH2−triazole and −COCHNH−), 3.63−3.34 (m, 15H, −CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O− and −OCH3), 2.66−2.07 (m, 8H, −CH2CH2COO−, −CH2COO−, −CH2CH2−triazole and −CH2CH2Cl). FTIR (neat, cm−1) of PCPLG-r-PPLG-OEG (x = 0.2): 3285, 2920, 1728, 1651, 1547, and 1086. Synthesis of Poly(γ-propyl-L-glutamate)pyridinium Chloride-random-Oligo-Ethylene Glycol Conjugate (PPLG-PyCl-rOEG). A representative nucleophilic substitution is as follows. PCPLG-r-PPLG-OEG (30.0 mg, 0.016 mmol of chlorine, x = 0.2, Scheme 1) was dissolved in DMF (2.0 mL) in a round-bottomed flask (25 mL) under nitrogen, followed by adding acetonitrile (2.0 mL), NaI (13 mg, 0.16 mmol), and 3-methylpyridine (8.0 μL, 0.16 mmol). The mixture was stirred at 80 °C for 48 h. Then, brine (5.0 mL) was added to the reaction solution, followed by stirring at room temperature for 24 h to promote the ion exchange. The product was purified by dialysis against DI-H2O for 48 h in a dialysis bag with a cutoff molecular weight of 3000 g mol−1. Removing the solvent under vacuum afforded a glassy solid (25.0 mg, 80% yield). Synthesis of Poly(γ-propyl-L-glutamate)pyridinium Tetrafluoroborate-random-Oligo-Ethylene Glycol Conjugate (PPLG-PyBF4-r-OEG). A representative ion-exchange reaction is as follows. PPLG-PyCl-r-OEG (20.0 mg, 0.010 mmol of chlorine, R = 3-Me, x = 0.2, Scheme 1) and NaBF4 (49.4 mg, 0.45 mmol) were dissolved in DI-H2O (5 mL). The aqueous solution was stirred at room temperature for 5 min. Then, it was dialyzed against the aqueous solution (50 mL, 0.1 mol L−1) of NaBF4 in a dialysis bag with a cutoff molecular weight of 3000 g mol−1 for 48 h (the outside solution was changed twice per day). After that, it was dialyzed against DI-H2O for another 48 h. Removing the solvent under vacuum afforded a glassy solid (17.0 mg, 85% yield). 1H NMR (D2O, δ, ppm) of P3 (the molecular parameters of P1−P4 are summarized in Table 1): 8.71

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Table 1. Molecular Parameters and Water Solubility of PPLG-PyBF4-r-OEG Samples samples P1 P2 P3 P4 P5 P6

Mna 19 100 19 300 19 300 19 300 18 700 18 000

Mw/Mnb 1.13 1.13 1.13 1.13 1.13 1.13

Rc H 2-Me 3-Me 4-Me 3-Me 3-Me

xd

X− e

water solubility

0.2 0.2 0.2 0.2 0.4 0.6

BF4− BF4− BF4− BF4− BF4− BF4−

yes yes yes yes yes no

RESULTS AND DISCUSSION

Random copolypeptides bearing oligo-ethylene glycol and pyridinium tetrafluoroborate (i.e., PPLG-PyBF4-r-OEGs, Scheme 1) were prepared via a four-step postpolymerization from poly(γ-3-chloropropyl-L-glutamate) (PCPLG) which was prepared according to reported procedures. Poly(γ-3-chloropropyl-L-glutamate)-random-poly(γ-3-azidopropyl-L-glutamate)s (PCPLG-r-PAPLGs) with constant main-chain length and various molar content of chloro groups (x) were obtained by reacting PCPLG with sodium azide (NaN3) in DMF at 60 °C. Different x values were achieved by regulating the reaction time. Poly(γ-3-chloropropyl-L-glutamate)-random-poly(γ-propyl-Lglutamate)-graf t-(oligo-ethylene glycol)s (PCPLG-r-PPLGOEGs) were synthesized by copper-mediated [2 + 3] alkyne− azide 1,3-dipolar cycloaddition between propargyl functionalized oligo-ethylene glycol (Pr-OEG) and PCPLG-r-PAPLG. The molecular structures of PCPLG-r-PAPLG and PCPLGr-PPLG-OEG were characterized by 1H NMR (Figure 1) and FTIR (Figure 2). The chemical shifts in the 1H NMR spectra were consistent with the polymer structures. The appearance of chemical shifts at 3.39 and 1.91 ppm ascribed to Hf′ and He′ (Figures 1a and 1b), which confirmed the formation of azido groups. The integration of Hf/Hf′ corresponded to the molar ratio of chloro groups to azido groups which was used to calculate the x values. The appearance of chemical shift at 7.83 ppm suggested the formation of triazole groups after copper-mediated [2 + 3] alkyne−azide 1,3-dipolar cycloaddition (Figure 1c). The chemical shift in the range of 3.2−3.8 ppm corresponded to the OEG pendants. In the FTIR spectra (Figure 2, curves 1 and 2), PCPLG and PCPLG-r-PAPLG showed similar characteristic bands at 3277, 2931, 1727, 1650, and 1545 cm−1, corresponding to vN−H, vC−H, vCO, amide I, and amide II, respectively. Nevertheless, PCPLG-r-PAPLG exhibited characteristic peak at 2096 cm−1 due to vNNN, suggesting the formation of azido groups. In the FTIR spectrum of PCPLG-r-PPLG-OEG (Figure 2, curve 3), the appearance of the vC−O−C band at 1086 cm−1 and the disappearance of the vNNN band suggested the successful preparation of PCPLG-r-PPLG-OEG with high grafting efficiency. Moreover, the molar masses and molar mass distributions of PCPLG, PCPLG-r-PAPLG, and PCPLG-r-PPLG-OEG

a

Number-average molar mass calculated from the degree of polymerization of PCPLG-r-PPLG-OEG (DP = 48) times the molar mass of repeating unit. bMolar mass distribution of PCPLG-r-PPLG-OEG determined by GPC. cThe substituent of pyridinium groups. dThe molar content of pyridinium groups. eCounteranions. (d, 2H, −NCHCH− and −NCHCH2−), 8.38 (s, 1H, −NCHCH−), 8.05 (m, 1H, triazole), 7.95 (s, 1H, −NCHCHCH−), 4.64 (s, 2H, −O−CH 2−triazole−), 4.53 (d, 4H, −CH2CH2−triazole− and −CH2N+BF4−), 4.10 (m, 5H, −COCHNH−, −CH2CH2CH2− triazole−, and −CH 2 CH 2 CH 2 N + BF 4 − ), 3.66−3.34 (m, 15H, −CH2CH2OCH2CH2OCH2CH2O− and −OCH3), 2.55−2.16 (m,, 11H, −CH2CH2COO−, −CH2COOCH2−, −CH2CH2−triazole−, −CH2CH2−Py−, and −Py−CH3). FTIR (neat, cm−1) of P3: 3289, 2932, 1729, 1651, 1547, 1083, and 1022. 1H NMR of P1 (D2O, δ, ppm): 8.90−8.45 (m, 5H, −PyH), 8.07 (s, 1H, triazole), 4.67 (s, 2H, −O−CH2−triazole−), 4.55 (d, 4H, −CH2CH2−triazole− and −CH2N+BF4−), 4.12 (m, 5H, −COCHNH−, −CH2CH2CH2− triazole−, and −CH 2 CH 2 CH 2 N + BF 4 − ), 3.69−3.38 (m, 15H, −CH2CH2OCH2CH2OCH2CH2O− and −OCH3), 2.68−2.27 (m, 8H, −CH2CH2COO−, −CH2COOCH2−, −CH2CH2−triazole−, and 3544

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which further verified the successful preparation of PCPLG-rPPLG-OEG. The nucleophilic substitution of PCPLG-r-PPLG-OEG in the presence of pyridine or methylpyridine in acetonitrile/DMF solution afforded poly(γ-propyl-L-glutamate)pyridinium chroriderandom-oligo-ethylene glycol conjugate (PPLG-PyCl-r-OEG) which reacted with NaBF4 in DI-H2O to yield poly(γ-propyl-Lglutamate)pyridinium tetrafluoroborate-random-oligo-ethylene glycol conjugate (PPLG-PyBF4-r-OEG). The resulting polypeptide was purified by first dialyzing against NaBF4 aqueous solution to ensure high efficiency of ion exchange and then dialyzing against DI-H2O to remove any salt residue. PPLG-PyBF4-r-OEG samples with constant main-chain length, various molar content of pyridinium groups (x), and different substituent position of pyridinium were successfully prepared as verified by 1H NMR (Figure 4, Figures S1−S3 of Supporting Information) and FTIR

Figure 1. 1H NMR spectra of (a) PCPLG, (b) PCPLG-r-PAPLG, and (c) PCPLG-r-PPLG-OEG in CDCl3.

Figure 4. 1H NMR spectra of P3 in D2O (a) before and (b) after SO2 bubbling and (c) after N2 bubbling.

(Figure 2, curves 4−7). For example, the appearance of characteristic chemical shifts in the range of 7.8−8.7 ppm corresponded to the pyridinium rings. 1H NMR spectra also showed high grafting efficiency of pyridinium groups (∼90%), which was calculated according the integration of chemical shifts of protons on pyridinium (e.g., Hm in Figure 4a) and triazole groups (e.g., Hg in Figure 4a). In the FTIR spectra (Figure 2, curves 4 and 5), the appearance of B−F vibration mode (vB−F) at about 1025 cm−1 confirmed the success of ion-exchange reaction. Moreover, the amide I band at 1650 cm−1 and amide II band at 1545 cm−1 suggested an α-helical conformation of polypeptides in the solid state.57−59 Because of the absorption onto the columns, the polypeptides with pyridinium pendants cannot be detected by GPC. Nevertheless, the number-average molar mass can be calculated from the degree of polymerization of PCPLG-r-PPLGOEG times the molar mass of repeating unit. The molecular structure parameters of PPLG-PyBF4-r-OEG are summarized in Table 1. The water solubility of PPLG-PyBF4-r-OEG was tested by mixing 10 mg of polymer with 1 mL of DI-H2O at room temperature under violent stirring. The polypeptides showed good water solubility when x value was not greater than 0.4 (Table 1, P1−P5). The presence of methyl groups or the substituent position of pyridinium showed no effect on their water solubility. The aqueous solution conformation of watersoluble PPLG-PyBF4-r-OEG was characterized by CD spectroscopy at 25 °C. All polypeptides exhibited α-helical conformation in DI-H2O, as verified by the characteristic CD bands at 208 and 222 nm (Figure 5). The fractional helicity (f H) was in the range of 71−81%. The calculated method was described

Figure 2. FTIR spectra of PCPLG (curve 1), PCPLG-r-PAPLG (curve 2), PCPLG-r-PPLG-OEG (curve 3), and P1−P4 (curves 4−7) in the solid state (x = 0.2).

Figure 3. GPC chromatographs of PCPLG, PCPLG-r-PAPLG, and PCPLG-r-PPLG-OEG (x = 0.2).

were characterized by GPC (Figure 3). A noticeable peak shift to short elution time (i.e., high molar mass) was observed after copper-mediated [2 + 3] alkyne−azide 1,3-dipolar cycloaddition, 3545

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DP ≥ 30.60 The decrease of f H for PPLG-PyBF4-r-OEG likely resulted from the side-chain charge repulsion which destabilized the α-helix. Additionally, the presence of methyl groups of pyridinium groups slightly decreased the f H value due to the steric hindrance effect. The substituent position of pyridinium showed less effect on the f H value. The SO2-induced solution phase transition of PPLG-PyBF4r-OEG aqueous solutions was tested by directly bubbling SO2 gas into polymer aqueous solutions. At room temperature, the polypeptide (e.g., P3) aqueous solution was clear and transparent with 100% transmittance at λ = 500 nm as characterized by UV−vis spectroscopy (Figure 6). After SO2 bubbling, the polymer aqueous solution became turbid with only ∼45% transmittance, indicating a solution phases transition. After N2 bubbling, the solution became clear and transparent again and showed 100% transmittance, suggesting a reversible solution phase transition behavior. The solution phase transitions can be repeated multiple times (Figure 6). The aggregation sizes of PPLG-PyBF4-r-OEG aqueous solutions with or without SO2 treatment were investigated by dynamic light scattering (DLS). The diameters of P1−P4 were 333.5, 239.9, 488.3, and 440.9 nm, respectively, before SO2 bubbling, and they were 2918.1, 1564.7, 782.0, and 3036.4 nm, respectively, after SO2 bubbling (Figure 7, Figures S4−S6). The increased aggregation sizes after SO2 treatment resulted in a solution phase transition, which was consistent with the UV−vis analysis. The initial aggregation size of all samples was relatively high (200−500 nm) due to the amphiphilic side chains, namely, hydrophobic pyridinium tetrafluoroborate pendants and hydrophilic OEG pendants. After N2 treatment, the aggregation size decreased and closed to the diameters before SO2 treatment, which further suggested a reversible solution phase transition behavior. Additionally, the polymer aggregates first suspended in DI-H2O after SO2 treatment and eventually precipitated after ∼11 h (Figure S7). CD results revealed a noticeable decrease of f H value after

Figure 5. CD spectra of P1−P4 in DI-H2O (0.05 mg mL−1).

Figure 6. Plot of transmittance at λ = 500 nm versus gases (i.e., SO2 and N2) for the aqueous solution (20 mg mL−1) of P3 at 25 °C and the optical images of P3 solution at different conditions.

in the Experimental Section. Previously, we have demonstrated that poly(γ-propyl-L-glutamate)-graf t-oligo-ethylene glycol (PPLG-g-OEG) showed high f H values (>90%) when

Figure 7. DLS size distribution plots of P3 in DI-H2O at 1 mg mL−1 (a) before and (b) after SO2 bubbling and (c) after N2 bubbling (25 °C). 3546

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Figure 8. (a) Optical images of P3 aqueous solution (20 mg mL−1) before and after addition of Na2SO3 salt. Molecular structures of (b) P7 and (c) PPLG-g-OEG. (d) Proposed mechanism of SO2-induced reversible solution phase transition.

ion-exchange reaction. Besides the essential role of triazole groups which can interact with SO2, the molar contents of hydrophilic pendants and amphiphilic pendants are also important to construct SO2-responsive polymers with solution phase transition. Our study sheds some light on the molecular design of SO2-responsive polymers and will broaden the application of gas-responsive polymers.

SO2 treatment due to the solution phase transition (Figure S8). FESEM results revealed spherical structures with a diameter of about 200 nm before SO2 treatment (Figure S9). The polymer aggregates showed featureless morphology consisting of irregular nanoparticles after SO2 treatment. Nevertheless, more research effort and better characterization facilities are demanded to further understand the evolution of polymer morphologies after SO2 treatment. On the basis of the DLS results, we suspected that mechanism of SO2-induced reversible solution phase transition was originated from the “cross-linking effect” of SO2. In order to exclude the effect of SO32−, we added Na2SO3 solid to the P3 aqueous solution (Figure 8a), which showed no solution phase transition. We also used 1H NMR analysis to discover which group interacted with SO2. A noticeable peak shift of Hg was observed after SO2 bubbling, and the peak shifted back after N2 bubbling (Figure 4, Figures S1−S3), indicating that SO2 interacted with triazole groups and induced polymer aggregation and consequently solution phase transition. To further demonstrate the importance of triazole groups, an analogue of P3, namely P7 (Figure 8b), was prepared via ring-opening polymerization, nucleophilic substitution, and ion-exchange reaction (Scheme S1). The molecular structure of P7 was confirmed by 1 H NMR (Figure S10). The aqueous solution of P7 showed no SO2-induced solution phase transition, which further confirmed the importance of triazole groups. Additionally, PPLG-g-OEG (Figure 8c) with triazole groups and OEG pendants also showed no SO2-induced solution phase transition, indicating the essential of pyridinium tetrafluoroborate pendants. A proposed mechanism is shown in Figure 8d.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b00116. 1 H NMR spectra (Figures S1−S3) and DLS size distribution plots (Figures S4−S6) of P1, P2, and P4, optical images (Figure S7), CD spectra (Figure S8), and FESEM images (Figure S9) of P2 aqueous solutions before and after SO2 treatment, synthetic procedures, synthetic route (Scheme S1), and 1H NMR spectrum of P7 (Figure S10) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (H.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (Grant 21204075), Scientific Research Fund of Hunan Provincial Education Department (14B175), and Xiangtan University start-up fund (12QDZ06). The authors thank Prof. Shifang Luan at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, for assisting with the FESEM experiments.

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CONCLUSIONS We developed a new family of water-soluble, α-helical, and SO2-responsive random copolypeptides bearing pyridinium tetrafluoroborate (PyBF4) and oligo-ethylene glycol (OEG) pendants (PPLG-PyBF4-r-OEG). The resulting polymers with high grafting efficiency can be readily prepared by a multistep postpolymerization, including nucleophilic substitutions, coppermediated [2 + 3] alkyne−azide 1,3-dipolar cycloaddition, and

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