Preparation and thermoresponsive properties of helical polypeptides bearing pyridinium salts

Abstract

Water-soluble polypeptides bearing various pyridinium groups (i.e., pyridinium, 2-methylpyridinium, 3-methylpyridinium, 4-methylpyridinium) and Cl⁻ counter-anions have been prepared by nuleophilic substitutions between poly(γ-3-chloropropyl-L-glutamate) (PCPLG) or poly(γ-6-chlorohexyl-L-glutamate) (PCHLG) and pyridine or y-methyl pyridine (y = 2, 3, and 4). Polypeptides bearing pyridinium groups and BF₄⁻ counter-anions were prepared by ion-exchange reactions from the polypeptide–pyridinium conjugates with Cl⁻ counter-anions. CD analysis revealed that water-soluble polypeptides adopted α-helical conformation in solutions with a fractional helicity in the range of 24.7–41.4%. Variable-temperature UV-vis spectroscopy revealed that polypeptides bearing 3-methylpyridinium and BF₄⁻ counter-anions (i.e., P11 and P15) showed upper critical solution temperature (UCST)-type transitions in aqueous solutions. The UCST-type phase transition temperatures increased as increasing the hydrophobicity of the spacer groups, the polymer concentrations, and the concentrations of NaBF₄.

---

Introduction

Water-soluble polymers with upper critical solution temperatures (UCSTs) are a type of thermoresponsive polymers which undergo solution phase separation below their respective UCSTs.^1,2 Comparing to their counterparts, polymers with lower critical solution temperatures (LCSTs),^3–7 UCST-type polymers have been less studied due to the difficult synthesis of monomers and polymers. Zwitterionic polymers, such as poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) and poly(3-(N-(3-methacrylamidopropyl)-N,N-dimethyl)ammo-niopropane sulfonate), represent the most investigated UCST-type polymers, which become insoluble in water at low temperatures due to intra- and intermolecular electrostatic interactions between adjacent zwitterionic groups.^8–13 In addition, polymers with reversible intra- and intermolecular hydrogen bonding interactions also showed UCST-type transitions, such as poly(allylamine-co-allylurea) and poly(acrylamide-co-acrylo-nitrile).^14–16 Currently, the utility of the UCST-type polymers is mainly focused on development of thermoresponsive self-assemble structures (e.g., vesicle and micelle) and hydrogels toward temperature-triggered release systems.^17–19 However, a majority of the UCST-type polymers are polyacrylamide derivatives with non-biodegradable properties, which will hamper their uses in biomedical applications.

Synthetic polypeptides are mimics of natural peptides with excellent biocompatibility and unique secondary structures (e.g., α-helix and β-sheet).^20–22 They have showed immense potential as biomedical materials, such as tissue-engineering scaffolds, drug-delivery carriers and antimicrobial agents.^23–27 While water-soluble LCST-type polypeptides have been developed by incorporation of oligo-ethylene-glycol pendants,^28–34 polypeptides with UCSTs in water have been rarely reported. Recently, we have demonstrated that helical polypeptides bearing 1-butylimidazolium groups and I⁻ or BF₄⁻ counter-anions exhibited UCST-type transitions in water.³⁵ By following that study, we designed a series of polypeptides bearing various pyridinium salts, aiming to expand our understanding of their structure–property relationship.

In this contribution, we report a new family of α-helical polypeptides bearing various pyridinium groups (i.e., pyridinium, 2-methylpyridinium, 3-methylpyridinium, and 4-methylpyridinium) and counter-anions (i.e., Cl⁻ or BF₄⁻) prepared by the combination of post-polymerization (i.e., nuleophilic substitution) and ion-exchange reaction. The molecular structures and conformations have been characterized by a combination of spectroscopic techniques (i.e., NMR, FTIR, and CD). Their UCST-type phase transition behaviors have been investigated by variable-temperature UV-vis spectroscopy.

Experimental section

Materials

Anhydrous tetrahydrofuran (THF) and n-butylamine were dried over calcium hydride and distilled. N,N-Dimethylformamide (DMF) was dried over molecular sieves. Chloroacetyl chloride (98%), pyridine (>99.5%), 2-methylpyridine (>99%), 4-methylpyridine (>99%), NaI (98%), 6-chloro-1-hexanol (>99%), NaBF₄ (98%), and triphosgene (99.5%) were purchased from Energy Chemical. 3-Methylpyridine (98%) was purchased from Tokyo Kasei Kogyo Co. Ltd. Sodium azide (99.5%) was purchased from Sigma-Aldrich. L-Glutamic acid (99%) and other chemicals were purchased from Aladdin and used as received. Deionized water (DI H₂O) was obtained from Aquapro AR1-100L-P11 water-purification-system (Ever Young Enterprises Development Co., LTD, P. R. China). Poly(γ-3-chloropropyl-L-glutamate) (PCPLG) and poly(γ-6-chlorohexyl-L-glutamate) (PCHLG) were synthesized and characterized according to reported procedures.^36,37

Instruments and method

¹H 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 and solvent ¹³C resonances in deuterated solvents. The polymer solutions (∼15 mg mL⁻¹) for ¹H NMR test 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, T = 25 °C). 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 × 300 mm, effective molecular weight range of 0.2–400.0 kg mol⁻¹). 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 molecular weight range of 0.5–7.5 × 10⁷ g mol⁻¹ (PSS, Mainz, Germany) were utilized for calibration. 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 temperatures above respective UCST-type transition temperatures, 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 600 nm. DI H₂O at 25 °C was set to be 100% of transmittance. The solutions were cooled from high temperatures to low temperatures with initial stabilization of 20 min. The solution phase transition temperature (T_pt) was determined at 50% of transmittance in the cooling cycle for UCST-type transition. For ionic strength dependent studies, the salt concentration was adjusted by the addition of NaX (X = Cl or BF₄). Circular dichroism (CD) measurements were carried out on an AVIV 410 CD spectrometer (Biomedical Inc., Lakewood, NJ, USA). The polymer aqueous solutions were prepared at concentrations of 1 mg mL⁻¹ by directly mixing and stirring at room temperature for the samples without phase transitions or at the temperatures above UCST-type T_pts. Then, the above solutions (1 mg mL⁻¹) were diluted to 0.05 mg mL⁻¹ for CD measurements. 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 185–250 nm with a resolution of 0.5 nm. The data were processed by subtracting the solvent (i.e., DI H₂O) background and smoothing with FFT-Filter method with points of window of 8. The CD spectra were reported in mean residue ellipticity (MRE) (unit: deg cm² dmol⁻¹) which was calculated by the equation [θ]_λ = MRW × θ_λ/10 × d × c,³⁸ where MRW is the mean residue weight (MRW = the molecular weight of polypeptide repeating unit), θ_λ 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).

Synthesis of poly(γ-3-propyl-L-glutamate)-N-pyridinium-chloride conjugate (P1), poly(γ-3-propyl-L-glutamate)-N-y-methylpyridinium-chloride conjugate (y = 2, P2; y = 3, P3; y = 4, P4), poly(γ-6-hexyl-L-glutamate)-N-pyridinium-chloride conjugate (P5), and poly(γ-6-hexyl-L-glutamate)-N-y-methylpyridinium-chloride conjugate (y = 2, P6; y = 3, P7; y = 4, P8). A representative post-polymerization for water-soluble products (i.e., P1, P3–P5, P7–P8) is as follows. PCHLG (86.7 mg, 0.35 mmol of chlorine) was dissolved in DMF (2.0 mL) in a 25 mL flask under nitrogen, followed by adding acetonitrile (2.0 mL) solution of NaI (157 mg, 1.05 mmol) and 3-methylpyridine (68.0 μL, 0.7 mmol). The mixture was stirred at 80 °C for 48 h. Then, brine (13.0 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 water 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 (111.0 mg, 92% yield). ¹H NMR of P7 (DMSO-d₆, δ, ppm): 9.28 (s, 1H, –NCHCCH₃–), 9.14 (s, 1H, –CH=CHN–), 8.47–8.48 (d, 1H, –CHCH=CCH₃–), 8.06–8.10 (t, 1H, –CH=CHCH=), 4.65–4.64 (d, 2H, –NCH₂CH₂–), 4.30 (s, 1H, –COCHNH–), 3.95 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.34 (s, 2H, –CH₂CH₂CH₂N–), 1.24–1.94 (m, 10H, –CH₂CH₂CH₂N–, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P1 (D₂O, δ, ppm): 8.89–8.93 (d, 2H, –NCH=CH– and –N=CHCH–), 8.56–8.63 (q, 2H, –CH=CHCH– and –N=CHCH=CH–), 8.11–8.14 (t, 1H, –CCH=CH–), 4.75 (s, 2H, –NCH₂CH₂–), 4.35 (s, 1H, –COCHNH–), 4.23 (s, 2H, –OCH₂CH₂–), 1.87–2.44 (m, 6H, –NCH₂CH₂CH₂–, –OCOCH₂CH₂–). ¹H NMR of P3 (DMSO-d₆, δ, ppm): 9.24 (s, 1H, –NCH=CH–), 9.12 (s, 1H, –CCH=CH–), 8.45 (s, 1H, –CH₃CCH=CH–), 8.06 (s, 1H, –CH=CHCH=), 4.74 (s, 2H, –NCH₂CH₂–), 4.29 (s, 1H, –COCHNH–), 4.03 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.27 (s, 2H, –CH₂CH₂CH₂N–), 1.21–2.05 (t, 4H, –OCOCH₂CH₂–). ¹H NMR of P4 (D₂O, δ, ppm): 8.67–8.72 (t, 2H, –NCH=CH– and –N=CHCH–), 7.89–7.93 (t, 2H, –CH=CHCH– and –N=CHCH=CH–), 4.70 (s, 2H, –NCH₂CH₂–), 4.40 (s, 1H, –COCHNH–), 4.22 (s, 2H, –OCH₂CH₂–), 2.53 (s, 3H, –PyCH₃), 2.41 (s, 2H, –CH₂CH₂CH₂N–), 2.17–2.27 (m, 4H, –OCOCH₂CH₂–). ¹H NMR of P5 (D₂O, δ, ppm): 8.82 (s, 2H, –NCH=CH– and –N=CHCH–), 8.50 (s, 2H, –CH=CHCH– and –N=CHCH=CH–), 8.02 (s, 1H, –CCH=CH–), 4.55 (s, 2H, –NCH₂CH₂–), 3.99 (s, 3H, –COCHNH– and –OCH₂CH₂–), 1.19–1.95 (m, 12H, –NCH₂CH₂CH₂–, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P8 (D₂O, δ, ppm): 8.61 (s, 2H, –NCH=CH–), 7.82 (s, 2H, –CCH=CH–), 4.47 (s, 2H, –NCH₂CH₂–), 3.98 (s, 3H, –COCHNH– and –OCH₂CH₂–), 2.43 (s, 5H, –PyCH₃ and –CH₂CH₂CH₂N–), 1.29–2.17 (m, 10H, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–).

A representative post-polymerization for water-insoluble products (i.e., P2 and P6) is as follows. PCHLG (86.7 mg, 0.35 mmol of chlorine) was dissolved in DMF (2.0 mL) in a 25 mL flask under nitrogen, followed by adding acetonitrile (2.0 mL) solution of NaI (157 mg, 1.05 mmol) and 2-methylpyridine (68.6 μL, 0.7 mmol). The mixture was stirred at 80 °C for 48 h. Then, brine (13.0 mL) was added to the reaction solution, following by stirring at room temperature for 24 h to promote the ion exchange. The product was precipitated immediately and stuck on the glass wall. After that, cold DI water was used to wash the product to remove any solvent residue. Removing the solvent under vacuum afforded a glassy solid (74.0 mg, 63% yield). ¹H NMR of P6 (DMSO-d₆, δ, ppm): 8.98 (s, 1H, –NCH=CH–), 8.45 (s, 1H, –CCH=CH–), 8.03 (s, 1H, –CH₃CCH=CH–), 7.96 (s, 1H, –CH=CHCH=), 4.51 (s, 2H, –NCH₂CH₂–), 4.31–4.36 (d, 1H, –COCHNH–), 3.95 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.31 (s, 2H, –CH₂CH₂CH₂N–), 1.25–1.82 (m, 10H, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P2 (DMSO-d₆, δ, ppm): 9.05 (s, 1H, –NCH=CH–), 8.4 (s, 1H, –CCH=CH–), 8.16 (s, 1H, –CH₃CCH=CH–), 8.05–7.96 (d, 1H, –CH=CHCH=), 4.66 (s, 2H, –CH₂CH₂CH₂N–), 4.09 (s, 3H, –COCHNH– and –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.34 (s, 2H, –CH₂CH₂CH₂N–), 1.65–1.98 (t, 4H, –OCOCH₂CH₂–).

Synthesis of poly(γ-3-propyl-L-glutamate)-N-pyridinium-tetrafluoroborate conjugate (P9), poly(γ-3-propyl-L-glutamate)-N-y-methylpyridinium-tetrafluoroborate conjugate (y = 2, P10; y = 3, P11; y = 4, P12), poly(γ-6-hexyl-L-glutamate)-N-pyridinium-tetrafluoroborate conjugate (P13), and poly(γ-6-hexyl-L-glutamate)-N-y-methylpyridinium-tetra-fluoroborate conjugate (y = 2, P14; y = 3, P15; y = 4, P16). A representative ion–exchange reaction is as follows. P7 (74.0 mg, 0.22 mmol) were dissolved in DI H₂O (5 mL), followed by addition of NaBF₄ (0.37 g, 3.35 mmol). The reaction was stirred at room temperature for 5 min. The product was precipitated immediately and collected by centrifugation. The product was repeated stirring in the aqueous solution (5 mL) of NaBF₄ (0.37 g, 3.35 mmol) to further perform the ion-exchange reaction. Then, cold DI H₂O was used to wash the product to remove any salt residue. Removing the solvent under vacuum afforded a glassy solid (85.1 mg, 99% yield). ¹H NMR of P15 (DMSO-d₆, δ, ppm): 8.95 (s, 1H, –NCHCCH₃–), 8.88 (s, 1H, –CH=CHN–), 8.44–8.45 (d, 1H, –CHCH=CCH₃–), 8.03–8.06 (t, 1H, –CH=CHCH=), 4.50–4.54 (d, 2H, –NCH₂CH₂–), 4.27 (s, 1H, –COCHNH–), 3.97 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.30 (s, 2H, –CH₂CH₂CH₂N–), 1.24–1.91 (m, 10H, –CH₂CH₂CH₂N–, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P9 (DMSO-d₆, δ, ppm): 9.04 (s, 2H, –NCH=CH– and –N=CHCH–), 8.58 (s, 2H, –CH=CHCH– and –N=CHCH=CH–), 8.13 (s, 1H, –CCH=CH–), 4.65 (s, 2H, –NCH₂CH₂–), 4.24 (s, 1H, –COCHNH–), 4.03 (s, 2H, –OCH₂CH₂–), 1.65–2.23 (m, 6H, –NCH₂CH₂CH₂–, –OCOCH₂CH₂–). ¹H NMR of P10 (DMSO-d₆, δ, ppm): 8.94 (s, 1H, –NCH=CH–), 8.45 (s, 1H, –CCH=CH–), 8.02–8.16 (t, 1H, –CH₃CCH=CH–), 7.94 (s, 1H, –CH=CHCH=), 4.60 (s, 2H, –NCH₂CH₂–), 4.21 (s, 1H, –COCHNH–), 4.08 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.30 (s, 2H, –CH₂CH₂CH₂N–), 1.73–1.98 (t, 4H, –OCOCH₂CH₂–). ¹H NMR of P11 (DMSO-d₆, δ, ppm): 8.95 (s, 1H, –NCH=CH–), 8.87 (s, 1H, –CCH=CH–), 8.41–8.43 (d, 1H, –CH₃CCH=CH–), 8.02 (s, 1H, –CH=CHCH=), 4.60 (s, 2H, –NCH₂CH₂–), 4.25 (s, 1H, –COCHNH–), 4.05 (s, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.25 (s, 2H, –CH₂CH₂CH₂N–), 1.66–1.83 (d, 4H, –OCOCH₂CH₂–). ¹H NMR of P12 (DMSO-d₆, δ, ppm): 8.84 (s, 2H, –NCH=CH– and –N=CHCH–), 7.92 (s, 2H, –CH=CHCH– and –N=CHCH=CH–), 4.54 (s, 2H, –NCH₂CH₂–), 4.20 (s, 1H, –COCHNH–), 3.99 (s, 2H, –OCH₂CH₂–), 2.54 (s, 3H, –PyCH₃), 2.20 (s, 2H, –CH₂CH₂CH₂N–), 1.64–1.79 (d, 4H, –OCOCH₂CH₂–). ¹H NMR of P13 (DMSO-d₆, δ, ppm): 9.03 (s, 2H, –NCH=CH– and –N=CHCH–), 8.56–8.58 (d, 2H, –CH=CHCH– and –N=CHCH=CH–), 8.13 (s, 1H, –CCH=CH–), 4.24–4.4.31 (d, 2H, –NCH₂CH₂–), 3.93 (s, 3H, –COCHNH– and –OCH₂CH₂–), 1.27–2.27 (m, 12H, –NCH₂CH₂CH₂–, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P14 (DMSO-d₆, δ, ppm): 8.93 (s, 1H, –NCH=CH–), 8.42–8.44 (d, 1H, –CCH=CH–), 8.16–7.94 (q, 2H, –CH₃CCH=CH– and –CH=CHCH=), 4.49 (s, 2H, –NCH₂CH₂–), 4.23 (s, 1H, –COCHNH–), 3.95–4.04 (d, 2H, –OCH₂CH₂–), 2.49 (s, 3H, –PyCH₃), 2.28 (s, 2H, –CH₂CH₂CH₂N–), 1.28–1.81 (m, 10H, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–). ¹H NMR of P16 (DMSO-d₆, δ, ppm): 8.86 (s, 2H, –NCH=CH–), 7.95 (s, 2H, –CCH=CH–), 4.47 (s, 2H, –NCH₂CH₂–), 4.22 (s, 1H, –COCHNH–), 3.92 (s, 2H, –OCH₂CH₂–), 2.57 (s, 3H, –PyCH₃), 2.27 (s, 2H, –CH₂CH₂CH₂N–), 1.25–1.84 (m, 10H, –CH₂CH₂CH₂CH₂O–, –OCOCH₂CH₂–).

Results and discussion

Poly(γ-3-chloropropyl-L-glutamate) (PCPLG) and poly(γ-6-chlorohexyl-L-glutamate) (PCHLG) were prepared via ring-opening polymerizations (ROPs) of respective N-carboxyanhydrides (NCAs) at room temperature, using DMF as a solvent and hexamethyldisilazane (HMDS) as the initiator^36,37 (Scheme S1†). Prior to the ROPs, γ-3-chloropropyl-L-glutamate- and γ-6-chlorohexyl-L-glutamate-based NCAs (i.e., CP-NCA and CH-NCA) were synthesized by monoesterification of L-glutamic acid with 3-chloropropanol or 6-chlorohexanol in the presence of sulphuric acid and subsequent cyclization of the resulting γ-chloroalkyl-L-glutamates (i.e., CP-Glu and CH-Glu) with triphosgene in room temperature THF (Scheme S1†). The molecular structures of chloro-based polypeptides (i.e., PCPLG and PCHLG) and NCAs were verified by ¹H NMR (Fig. S1†) and FTIR (Fig. S2†). The number average molecular weights (M_n) and molecular distributions of PCPLG and PCHLG were determined by gel permeation chromatography (GPC, Fig. S3†).

Poly(γ-3-propyl-L-glutamate)-N-pyridinium-chloride conjugate (P1), poly(γ-3-propyl-L-glutamate)-N-y-methylpyridinium-chloride conjugates (y = 2, P2; y = 3, P3; y = 4, P4), poly(γ-6-hexyl-L-glutamate)-N-pyridinium-chloride conjugate (P5), and poly(γ-6-hexyl-L-glutamate)-N-y-methylpyridinium-chloride conjugates (y = 2, P6; y = 3, P7; y = 4, P8) were synthesized by post-polymerizations of PCPLG or PCHLG with pyridine or y-methylpyridine (y = 2, 3, and 4) in 80 °C N,N-dimethyl-formamide (DMF) under nitrogen (Scheme 1). Sodium iodide (NaI) was used to promote the reaction. The products were purified by dialyzing against NaCl aqueous solution and DI H₂O to remove the excess reactants and DMF. The molecular structures of P1–P8 (Table 1) were verified by ¹H NMR (Fig. 1) and FTIR (Fig. 2). ¹H NMR spectra of the polymers were consistent with their molecular structures. The grafting density was in the range of 90–94%, which was calculated based on the integration of ¹H NMR resonances of H_d and H_i for P3 (Fig. 1a) or H_k for P7 (Fig. 1c). In the FTIR spectra, P1–P8 showed characteristic peaks at 3286, 2937, and 1720 cm⁻¹, which corresponded to N–H stretching (v_N–H), C–H stretching (v_C–H), and C=O of ester bond stretching (v_C=O), respectively (Fig. 2). The C–H deformation mode (δ_C–H) of pyridinium was in the range of 770–830 cm⁻¹ depending on the types of pyridinium salts. Additionally, the amide I band at ∼1650 cm⁻¹ suggested the α-helical conformation of P1–P8 in the solid-state.³⁹

Scheme 1 The synthetic route of poly(γ-3-propyl-L-glutamate) or poly(γ-6-hexyl-L-glutamate) bearing various pyridinium salts.

Table 1 Molecular structure parameters, fractional helicities and UCST-type transition temperatures of poly(γ-3-propyl-L-glutamate) or poly(γ-6-hexyl-L-glutamate) bearing various pyridinium groups and counter-anions

Name	Mn^a	Mw/Mn^b	DP^c	x	R^d	X^−^e	fH^f	Tpt^g (°C)
a Number-average molecular weight calculated from the degree of polymerization of the precursors (i.e., PCPLG and PCHLG) plus the respective molecular weight of various pyridinium groups added.b Molecular weight distribution of the precursors determined by GPC.c Degree of polymerization of the precursors calculated from the GPC results.d Substituent group of pyridinium.e Counter-anions.f Fractional helicity.g UCST-type transition temperature in DI H2O (20 mg mL^−1).
P1	7700	1.33	27	1	H	Cl^−	24.7	—
P2	8060	1.33	27	1	2-Me	Cl^−	—	—
P3	8060	1.33	27	1	3-Me	Cl^−	26.6	—
P4	8060	1.33	27	1	4-Me	Cl^−	28.1	—
P5	8500	1.57	26	4	H	Cl^−	34.3	—
P6	8860	1.57	26	4	2-Me	Cl^−	—	—
P7	8860	1.57	26	4	3-Me	Cl^−	37.2	—
P8	8860	1.57	26	4	4-Me	Cl^−	40.6	—
P9	9080	1.33	27	1	H	BF4^−	—	—
P10	9450	1.33	27	1	2-Me	BF4^−	—	—
P11	9450	1.33	27	1	3-Me	BF4^−	28.7	25.9
P12	9450	1.33	27	1	4-Me	BF4^−	—	—
P13	9830	1.57	26	4	H	BF4^−	—	—
P14	10 200	1.57	26	4	2-Me	BF4^−	—	—
P15	10 200	1.57	26	4	3-Me	BF4^−	41.4	28.2
P16	10 200	1.57	26	4	4-Me	BF4^−	—	—

---

Fig. 1 ¹H NMR spectra of (a) P3, (b) P11, (c) P7, and (d) P15 in DMSO-d₆.

Fig. 2 FTIR spectra of (a) P1–P4, P9–P12, and (b) P5–P8, P13–P16 in the solid-state.

Polypeptides bearing pyridinium and BF₄⁻ counter-anions, namely, poly(γ-3-propyl-L-glutamate)-N-pyridinium-tetrafluoro-borate conjugate (P9), poly(γ-3-propyl-L-glutamate)-N-y-methylpyridinium-tetrafluoroborate conjugates (y = 2, P10; y = 3, P11; y = 4, P12), poly(γ-6-hexyl-L-glutamate)-N-pyridinium-tetra-fluoroborate conjugate (P13), and poly(γ-6-hexyl-L-glutamate)-N-y-methyl-pyridinium-tetrafluoroborate conjugates (y = 2, P14; y = 3, P15; y = 4, P16) were synthesized by ion-exchange reactions in NaBF₄ aqueous solution at room temperature (Scheme 1). All products were water-insoluble at room temperature and were purified by repeatedly washing with NaBF₄ aqueous solution and DI H₂O. ¹H NMR (Fig. 1) and FTIR analysis (Fig. 2) confirmed the molecular structures of P9–P16 (Table 1) with BF₄⁻ counter-anions. The exchange reactions were nearly quantitative, as confirmed by the ¹H NMR spectra which showed upfield shifts in the signals assignable to the pyridinium ring protons relative to the precursors (P1–P8). For instances, the chemical shift of H_g of pyridinium ring protons shifted from 9.24 ppm for P3 to 8.95 ppm for P11 while the chemical shift of H_j shifted from 9.28 ppm for P7 to 8.95 ppm for P15 (Fig. 1). In the FTIR spectra (Fig. 2), the appearance of B–F vibration mode (v_B–F) at 1037 cm⁻¹ further confirmed the success of ion-exchange reaction. Additionally, P9–P16 adopted α-helical conformation in the solid-states, as verified by the amide I band at ∼1650 cm⁻¹. GPC was used to characterize the molecular weights and molecular weight distributions of P1–P16. However, polypeptides with pyridinium groups were featureless due to the strong absorption between the charged side-chains and the stationary phase of GPC (Fig. S3†). Similar phenomenon has been observed in our previous publication.⁴⁰

The solubility of the obtained polypeptides bearing different pyridinium groups and counter-anions were tested in various solvents ranging from highly polar dimethyl sulphoxide (DMSO) to less polar chloroform (Table 2). P1–P8 with Cl⁻ counter-anions, namely, the Cl⁻-type polypeptides were soluble in polar solvents such as DMSO, water, and methanol except P2 and P6 with 2-methyl-pyridinium groups which showed poor solubility in methanol and water. Additionally, P1–P8 showed poor solubility in less polar solvent (e.g., chloroform and THF). In comparison, P9–P16 with BF₄⁻ counter-anions, namely, the BF₄⁻-type polypeptides were soluble in DMSO except that P11 and P15 exhibited reversible UCST-type phase transitions in water (Fig. 3a). The solubility results suggested that the existence of methyl groups and medium hydrophobicity of the counter-anions (i.e., BF₄⁻) are necessary conditions to show UCST-type solution phase transition behaviors in water. Similar results have been demonstrated in poly(vinyl ether)s with pendant imidazolium salts.⁴¹ Additionally, the specific methyl position in pyridinium groups (i.e., 3-substitution) also plays important role in the UCST-type transitions, which is likely related to the steric hindrance effect.⁴² More research effort is demanded to fully understand the structure–property relationship.

Table 2 Solubility of poly(γ-3-propyl-L-glutamate) or poly(γ-6-hexyl-L-glutamate) bearing various pyridinium groups and counter-anions^a

Polymer	Chloroform	THF	Acetone	Ethanol	Methanol	H2O	DMSO
a S = soluble; I = insoluble; U = UCST-type phase transition; concentration = 10 mg mL^−1.
P1	I	I	I	I	S	S	S
P2	I	I	I	I	I	I	S
P3	I	I	I	I	S	S	S
P4	I	I	I	I	S	S	S
P5	I	I	I	S	S	S	S
P6	I	I	I	I	I	I	S
P7	I	I	I	S	S	S	S
P8	I	I	I	S	S	S	S
P9	I	I	I	I	I	I	S
P10	I	I	I	I	I	I	S
P11	I	I	I	I	I	U	S
P12	I	I	I	I	I	I	S
P13	I	I	I	I	I	I	S
P14	I	I	I	I	I	I	S
P15	I	I	I	I	I	U	S
P16	I	I	I	I	I	I	S

---

Fig. 3 (a) Optical image of P11 solutions at different temperatures. CD spectra of (b) P1, P3, P4, P11, and (c) P5, P7, P8, P15 in DI H₂O (0.05 mg mL⁻¹).

The aqueous solution conformations of the polypeptides bearing different pyridinium salts were characterized by CD spectroscopy (Fig. 3b and c). All polypeptide samples showed characteristic CD bands at 208 and 222 nm, indicating an α-helical conformation in solution.⁴³ The fractional helicity (f_H) of the resulting polypeptides was calculated using eqn (1)⁴⁴ to allow for a quantitative comparison of the relative helical content (Table 1).

f_H = (−[θ]₂₂₂ + 3000)/39 000 (1)

where [θ]₂₂₂ is the mean residue ellipticity at 222 nm.

Polypeptide samples with hexyl spacer groups (i.e., P5, P7, P8, and P15) showed higher helicities (f_H = 34.3–41.4%) than the samples with propyl spacer groups (i.e., P1, P3, P4, and P11, f_H = 24.7–28.7%), suggesting that increasing the hydrophobicity of the spacer groups improved their helical stability. Additionally, the f_H values for the samples with Cl⁻ counter-anions (e.g., P3 and P7) was lower than the samples with BF₄⁻ counter-anions (e.g., P11 and P15), suggesting that counter-anions with smaller ionic radius and higher charge density may further increase the charge repulsions between adjacent pyridinium groups and subsequently destabilize the α-helical conformation.

The thermoresponsive behaviors of P11 and P15 in aqueous solutions were studied by variable-temperature UV-vis spectroscopy (Fig. 4 and 5). P11 and P15 were soluble in DI H₂O at high temperatures, yet the solutions become turbid when cooling the solutions to respective UCST-type transition temperatures (T_pts), which lead to a sharp transition in the transmittance as monitored by UV-vis spectroscopy. The T_pts were determined at 50% of transmittance in the cooling cycle. At 20 mg mL⁻¹, the aqueous solution of P15 with hexyl spacer groups showed a T_pt at 28.2 °C (Table 1) which was 2 °C higher than that of P11 with propyl spacer groups, suggesting that T_pt increased as increasing the hydrophobicity of the spacer groups. The effect of polymer concentrations (Fig. 4) and ionic strength (Fig. 5) on the T_pts was also investigated by UV-vis spectroscopy to further understand the mechanism of their solution phase transition behaviors. The T_pts were significantly affected by polymer concentrations and salts (i.e., NaX, X = Cl and BF₄), suggesting that the mechanism of the thermoresponsive phase transition mainly ascribed to the electrostatic interactions.^35,41,45 For examples, the T_pts increased from 6.4 °C to 25.9 °C for P11 and from 12.6 °C to 28.2 °C for P15 as the polymer concentrations increased from 10 mg mL⁻¹ to 20 mg mL⁻¹. The increment of T_pts for P15 was 4 °C lower than that for P11, indicating that increasing the hydrophobicity of the spacer groups improved their resistance to concentration change. In addition, the T_pts decreased as the NaCl concentration increased and increased as increasing the NaBF₄ concentration, which was likely resulted from the anion exchange reactions and the difference of hydrophobicity between different counter-anions. We also noticed that P15 with more hydrophobic spacer groups (i.e., hexyl groups) showed less dependence on the variation of ionic strength than P11. For example, the concentration of NaBF₄ for P15 (35.7 mM) was ∼2.7 times higher than that of P11 (13.3 mM) as increasing the T_pts to ∼50 °C.

Fig. 4 Variable-temperature UV-vis spectra of (a) P11 and (b) P15 aqueous solutions with different concentrations.

Fig. 5 Variable-temperature UV-vis spectra of (a) P11 and (b) P15 aqueous solutions (10 mg mL⁻¹) with different salt (i.e., NaCl or NaBF₄) concentrations.

Conclusions

We have demonstrated the preparation and thermoresponsive properties of a new family of UCST-type polypeptides based on poly(γ-3-propyl-L-glutamate) and poly(γ-6-hexyl-L-glutamate) bearing various pyridinium groups (i.e., pyridinium, 2-methylpyridinium, 3-methylpyridinium, and 4-methylpyridinium) and counter-anions (i.e., Cl⁻ and BF₄⁻). Polypeptides bearing pyridinium groups and Cl⁻ counter-anions were prepared by the post-polymerizations (i.e., nuleophilic substitutions) between PCPLG or PCHLG and pyridine or y-methyl pyridine (y = 2, 3, and 4). Polypeptides bearing pyridinium groups and BF₄⁻ counter-anions were prepared by ion-exchange reactions from the polypeptide–pyridinium conjugates with Cl⁻ counter-anions. Water-soluble polypeptides showed α-helical conformation in aqueous solutions. Polypeptide samples bearing 3-methylpyridinium and BF₄⁻ counter-anions (i.e., P11 and P15) showed UCST-type phase transitions in aqueous solutions. The UCST-type phase transition temperatures (T_pts) were affected by the polymer concentration and ionic strength, suggesting a mechanism of electrostatic interactions. P15 with more hydrophobic spacer groups (i.e., hexyl groups) showed less dependence on the variation of polymer concentration and ionic strength.

Acknowledgements

This work is supported by the Scientific Research Fund of Hunan Provincial Education Department (13C908), National Natural Science Foundation of China (Grant 21204075), Hunan Provincial Natural Science Foundation of China (13JJ6042), and Xiangtan University start-up fund (12QDZ06).

Notes and references

1. A. B. Lowe and C. L. McCormick, Chem. Rev., 2002, 102, 4177.
2. J. Seuring and S. Agarwal, Macromol. Rapid Commun., 2012, 33, 1898.
3. H. G. Schild, Prog. Polym. Sci., 1992, 17, 163.
4. V. Aseyev, H. Tenhu and F. Winnik, Adv. Polym. Sci., 2011, 242, 29.
5. A. J. L. Costa, M. R. C. Soromenho, K. Shimizu, J. M. S. S. Esperanca, J. N. C. Lopes and L. P. N. Rebelo, RSC Adv., 2013, 3, 10262.
6. J. W. Lapworth, P. V. Hatton and S. Rimmer, RSC Adv., 2013, 3, 18107.
7. E. Zeinali, V. Haddadi-Asl and H. Roghani-Mamaqani, RSC Adv., 2014, 4, 31428.
8. D. N. Schulz, D. G. Peiffer, P. K. Agarwal, J. Larabee, J. J. Kaladas, L. Soni, B. Handwerker and R. T. Garner, Polymer, 1986, 27, 1734.
9. P. Mary, D. D. Bendejacq, M.-P. Labeau and P. Dupuis, J. Phys. Chem. B, 2007, 111, 7767.
10. Y. Terayama, M. Kikuchi, M. Kobayashi and A. Takahara, Macromolecules, 2011, 44, 104–111.
11. F. Polzer, J. Heigl, C. Schneider, M. Ballauff and O. V. Borisov, Macromolecules, 2011, 44, 1654.
12. V. A. Vasantha, S. Jana, A. Parthiban and J. G. Vancso, RSC Adv., 2014, 4, 22596.
13. Q. Zhang, X. Tang, T. Wang, F. Yu, W. Guo and M. Pei, RSC Adv., 2014, 4, 24240.
14. N. Shimada, H. Ino, K. Maie, M. Nakayama, A. Kano and A. Maruyama, Biomacromolecules, 2011, 12, 3418.
15. J. Seuring and S. Agarwal, Macromolecules, 2012, 45, 3910.
16. N. Shimada, S. Kidoaki and A. Maruyama, RSC Adv., 2014, 4, 52346.
17. J. He, B. Yan, L. Tremblay and Y. Zhao, Langmuir, 2010, 27, 436.
18. A. Das and S. Ghosh, Angew. Chem., Int. Ed., 2014, 53, 1092.
19. H. Zhang, X. Tong and Y. Zhao, Langmuir, 2014, 30, 11433.
20. T. J. Deming, Adv. Polym. Sci., 2006, 202, 1.
21. T. J. Deming, Prog. Polym. Sci., 2007, 32, 858.
22. M. Gkikas, H. Iatrou, N. S. Thomaidis, P. Alexandridis and N. Hadjichristidis, Biomacromolecules, 2011, 12, 2396.
23. C. He, X. Zhuang, Z. Tang, H. Tian and X. Chen, Adv. Healthcare Mater., 2012, 1, 48.
24. C. Deng, J. Wu, R. Cheng, F. Meng, H.-A. Klok and Z. Zhong, Prog. Polym. Sci., 2014, 39, 330.
25. C. Lu, B. Li, N. Liu, G. Wu, H. Gao and J. Ma, RSC Adv., 2014, 4, 50301.
26. J. Wang, C. Gong, Y. Wang and G. Wu, RSC Adv., 2014, 4, 15856.
27. T. Xing and L. Yan, RSC Adv., 2014, 4, 28186.
28. M. Yu, A. P. Nowak, T. J. Deming and D. J. Pochan, J. Am. Chem. Soc., 1999, 121, 12210.
29. C. Chen, Z. Wang and Z. Li, Biomacromolecules, 2011, 12, 2859.
30. Y. Cheng, C. He, C. Xiao, J. Ding, X. Zhuang and X. Chen, Polym. Chem., 2011, 2, 2627.
31. C. M. Chopko, E. L. Lowden, A. C. Engler, L. G. Griffith and P. T. Hammond, ACS Macro Lett., 2012, 1, 727.
32. J. Ding, L. Zhao, D. Li, C. Xiao, X. Zhuang and X. Chen, Polym. Chem., 2013, 4, 3345.
33. X. Fu, Y. Shen, W. Fu and Z. Li, Macromolecules, 2013, 46, 3753.
34. Y. Wu, Y. Deng, Q. Yuan, Y. Ling and H. Tang, J. Appl. Polym. Sci., 2014, 131, 41022.
35. Y. Deng, Y. Xu, X. Wang, Q. Yuan, Y. Ling and H. Tang, Macromol. Rapid Commun., 2015, 36, 453.
36. H. Tang and D. Zhang, Biomacromolecules, 2010, 11, 1585.
37. H. Tang, L. Yin, K. H. Kim and J. Cheng, Chem. Sci., 2013, 4, 3839.
38. S. M. Kelly, T. J. Jess and N. C. Price, Biochim. Biophys. Acta, 2005, 1751, 119.
39. H. Susi, S. N. Timasheff and L. Stevens, J. Biol. Chem., 1967, 242, 5460.
40. Q. Hu, Q. Yuan, Y. Deng, Y. Ling and H. Tang, Eur. Polym. J., 2015, 63, 74.
41. H. Yoshimitsu, A. Kanazawa, S. Kanaoka and S. Aoshima, Macromolecules, 2012, 45, 9427.
42. P. Liu, L. Xiang, Q. Tan, H. Tang and H. Zhang, Polym. Chem., 2013, 4, 1068.
43. N. Berova, K. Nakanishi and R. W. Woody, Circular Dichroism: Principles and Applications, Wiley-VCH, New York, 2000.
44. J. A. Morrow, M. L. Segall, S. Lund-Katz, M. C. Phillips, M. Knapp, B. Rupp and K. H. Weisgraber, Biochemistry, 2000, 39, 11657.
45. E. Karjalainen, V. Aseyev and H. Tenhu, Macromolecules, 2014, 47, 7581.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra04541j

---