Thermoresponsive Dendronized Polypeptides Showing Switchable

Jan 13, 2016 - Because of the dendritic structures and stable oxime linkage, these .... masses in the range of Mp = 2580–981 000 (Polymer Standards ...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Thermoresponsive Dendronized Polypeptides Showing Switchable Recognition to Catechols Jiatao Yan,* Kun Liu, Wen Li, Huang Shi, and Afang Zhang* Laboratory of Polymer Chemistry, Department of Polymer Materials, College of Materials Science and Engineering, Shanghai University, Materials Building Room 447, Nanchen Street 333, Shanghai 200444, China S Supporting Information *

ABSTRACT: A new class of thermoresponsive dendronized polypeptides was prepared through highly efficient oxime ligation between oxyamino-substituted polylysines and aldehyde-cored oligoethylene glycol (OEG) dendrons. Their secondary structures and thermoresponsive behavior were investigated. Because of the dendritic structures and stable oxime linkage, these OEG-based dendronized polypeptides exhibited fast and fully reversible phase transitions in neutrally aqueous solutions, and their phase transition temperatures can be controlled around physiological temperatures. The effect of OEG dendronization on secondary structures of polypeptides were examined to check their prominent dendritic shielding effect, steric hindrance, and thermally driven phase transitions. To further extend the functions and potential applications of these stimuli-responsive dendronized polypeptides, phenylboronic acid moieties were introduced to achieve the corresponding dendronized copolymers, which were utilized to specifically recognize catechol-containing compounds such as alizarin red S or dopamine. These copolypeptides showed a significant enhancement to bind to catechols when comparing to monomeric phenylboronic acid. Furthermore, this enhanced binding can be switched surprisingly by thermally driven phase transitions or through addition of competitive catechols, which makes this class of dendronized polypeptides as unique scaffolds for selective and reversible recognition of catechols.

1. INTRODUCTION Thermoresponsive polypeptides are one of most important intelligent polymers which possess ordered secondary structures such as α-helix and β-sheet as well as enzymatic degradability and thermoresponsiveness. These fantastic biomimetic structures and functions make them highly interesting for versatile biomedical applications including controlled drug delivery, smart biointerface, and tissue engineering.1 One intriguing class of thermoresponsive polypeptides are those based on elastin-like polypeptides (ELPs).2 They exhibit excellent biocompatibility and characteristic thermoresponsive properties but also suffer from tedious genetic synthesis and broad phase transitions. Recently, synthetic polypeptides from N-carboxyanhydride (NCA) polymerization have drawn considerable attention on the basis of their facile preparation and further functionalization.3 Modification of these polypeptides with linear oligoethylene glycol (OEG) pendants has presented a robust methodology to realize their thermoresponsiveness.4 Various polypeptides such as poly(L-lysine)s, poly(L-glutamate)s, and poly(L-cysteine)s carrying linear OEG pendants exhibit thermoresponsive behavior in aqueous solutions, and their phase transition temperatures can be tuned by varying OEG length, ratios of comonomers, and even chirality of polypeptides.5 Furthermore, incorporation of other responsive units into these OEGmodified thermoresponsive polypeptides could even lead to multiple responsiveness for more sophisticated applications. © 2016 American Chemical Society

For example, modification of poly(L-glutamate)s simultaneously by OEG and diisopropylamine units afforded dual response to both temperature and pH inputs.6 Through incorporation of thioether or disulfide linkage,7 OEG-modified poly(L-cysteine)s exhibit additional oxidation responsiveness or abnormal irreversible phase transition behavior, respectively. Despite the significant progress, most synthetic thermoresponsive polypeptides reported to date mainly relied on the utilization of short linear OEGs, where structures are quite simple and property tunabilities thus limited. On the other hand, dendritic OEGs have been recently developed by our group to mediate versatile polymers with superior thermoresponsive properties such as sharp and reversible phase transitions and negligible hystereses.8 This unique thermoresponsive behavior arose from their dendritic topologies and prominent shielding effects.9 We therefore have recently exploited incorporation of these dendritic OEGs to afford thermoresponsiveness to proline- and lysine-based polypeptides through triazole and imine linkage, respectively.10 However, their synthesis suffered from either toxic copper catalyst or very basic reaction condition (pH 11), and furthermore, imine bonds are very sensitive to concentration or pH variation. It remains an important challenge to develop highly efficient and Received: October 13, 2015 Revised: December 30, 2015 Published: January 13, 2016 510

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

measurements were carried out on a Waters GPC e2695 instrument with a three-columns set (Styragel HR3 + HR4 + HR5) equipped with a refractive index detector (Waters 2414) and DMF (containing 1 g L−1 LiBr) as eluant at 45 °C. The calibration was performed with poly(methyl methacrylate) standards with molar masses in the range of Mp = 2580−981 000 (Polymer Standards Service USA). AFM measurements were performed on a Bruker Nanoscope VIII MultiMode microscope with an “E” scanner (scanning range 10 μm × 10 μm) and operated in peak force mode at room temperature in air. Bruker silicon Tip on Nitride Lever cantilevers (T: 0.65 mm; L: 115 mm; W: 25 mm; f 0: 70 mm; k: 0.4 N/m) were used. UV/vis turbidity measurements were carried out on a PE UV/vis spectrophotometer (Lambda 35) equipped with a thermo-controlled bath. Sample solutions were placed in the spectrophotometer (path length 1 cm) and heated or cooled at a rate of 0.2 °C min−1. The absorptions of the solution at λ = 500 nm were recorded every 5 s. The cloud point temperature (Tcp) was determined as the one at which the transmittance had reached 50% of its initial value. Circular dichroism (CD) measurements were performed on a JASCO J-815 spectropolarimeter with a thermo-controlled 1 mm quartz cell (five accumulations; “continues scanning” mode; scanning speed: 100 nm min−1; data pitch: 0.5 nm; response: 1 s; bandwidth: 2.0 nm). The fluorescence spectra were recorded using a Horiba Jobin Yvon Fluorolog-3 spectrophotometer equipped with a Peltier temperature controller. All samples were kept at equilibrium at a predetermined temperature for 2 min before data collection. 2.3. Synthesis. Compound 1. Boc-Lys-OH (0.50 g, 2.03 mmol) and diisopropylethylamine (DIPEA) (0.90 g, 6.96 mmol) were dissolved in a mixture of DMF (35 mL) and water (10 mL), and then Boc-AOA-pfp (0.90 g, 2.52 mmol) was added at −15 °C. The mixture was stirred overnight at 25 °C and carefully acidified with KHSO4. After extraction with dichloromethane, the organic phase was dried over MgSO4 and concentrated under vacuum. Purification by column chromatography with DCM/MeOH (30:1, v/v) afforded the product as a colorless solid (0.69 g, 81%). 1H NMR (d6-DMSO): δ = 1.20−1.48 (m, 22H, CH3 + CH2), 1.48−1.70 (m, 2H, CH2), 3.08− 3.12 (q, 2H, CH2), 3.79−3.84 (m, 1H, CH), 4.14 (s, 2H, CH2), 7.03 (d, 1H, α-NH), 8.00 (s, 1H, ε-NH), 10.31 (s, 1H, ONH), 12.42 (s, 1H, COOH). 13C NMR (d6-DMSO): δ = 23.5, 28.4, 28.7, 29.1, 30.9, 38.4, 53.8, 75.2, 78.4, 81.1, 156.1, 157.4, 168.2, 174.7. HR-MS (ESI): m/z calcd for C18H33N3NaO8 [M + H]+ 420.2340; found 420.2344. NCA Monomer 2. Et3N (0.24 g, 2.37 mmol) was added into a mixture of compound 1 (0.52 g, 1.24 mmol) and triphosgene (0.16 g, 0.54 mmol) in ethyl acetate at 0 °C. The solution temperature was then elevated to 25 °C. After stirring for 10 h, the reaction mixture was cooled to 0 °C and filtered. The organic phase was washed successively with cold saturated NaHCO3 solution and brine and then dried and condensed in a vacuum. The residue was purified by precipitation from hexane three times to afford the title compound as a white solid (0.16 g, 37%). 1H NMR (d6-DMSO): δ = 1.21−1.49 (m, 13H, CH3 + CH2), 1.60−1.79 (m, 2H, CH2), 3.10−3.13 (q, 2H, CH2), 4.14 (s, 2H, CH2), 4.41−4.44 (m, 1H, CH), 8.01 (d, 1H, ε-NH), 9.10 (s, 1H, α-NH), 10.30 (s, 1H, ONH). 13C NMR (d6-DMSO): δ = 21.2, 28.4, 28.9, 31.1, 38.3, 57.4, 75.2, 81.1, 152.4, 157.4, 168.3, 172.1. General Procedure for Polymerization of NCA To Form Polypeptide PnBoc. The freshly prepared NCA monomer 2 (0.40 g, 1.15 mmol) was immediately transferred to a Schlenk tube and dissolved in DMF. After deaeration with nitrogen for several times, amine (6.44 μmol; benzylamine for P44Boc, hexylamine for P56Boc, triethylamine for P205Boc) was added to initiate ring-opening polymerization of NCA. The mixture was stirred for 72 h at 25 °C and then precipitated twice with ethyl ether. The precipitates were freeze-dried from dioxane to yield PnBoc as a white powder (64%). 1H NMR (d6-DMSO) of P44Boc: δ = 1.17−1.89 (m, 15H, CH3 + CH2), 3.09 (br, 2H, CH2), 3.90−4.41 (m, 3H, CH2 + CH), 7.71−8.21 (m, 2H, NH), 10.27 (br, 1H, ONH). General Procedure for Deprotection of PnBoc To Form Polypeptide PnONH2. PnBoc (103.00 mg) was dissolved in TFA (4 mL), and the solution was stirred for 6 h at 25 °C. Methanol was added to terminate the reaction, and the solvent was evaporated in

environment-friendly synthetic methods and simultaneously afford polypeptides with new functions such as biomolecular recognition. Two general synthetic strategies have been established in the past years to prepare thermoresponsive polypeptides: (a) polymerization of side-chain modified NCA monomers and (b) postpolymerization of polypeptide precursors by grafting functional pendent groups.11 The former method gives polypeptides with well-defined and controllable structures but often involves tedious synthesis and challengeable purification of NCA monomers. Instead, postmodification presents a moldable pathway to make functional polypeptides especially with complex structures.12 The key to success for this methodology is to find efficient and selective strategies for functional pendants to couple with side-chain reactive groups in polypeptide precursors. Azide−alkyne cycloaddition and thiol− ene reactions have been extensively employed toward this goal and may be the most popular tools based on their high efficiency and mild reaction conditions. However, these click reactions always need the presence of copper ion or ultraviolet light/heat that may be harmful to environment and human health. Oxime linkages have received significant attention recently as click chemistry for polymer or biomacromolecule functionalization.13 They can be quantitatively formed from the specific condensation between oxyamines and aldehydes or ketones under mild reaction conditions (ambient temperature, aqueous environment, and with no other auxiliaries such as metallic catalyst and UV light). In addition, oximes possess superior hydrolytic stability under physiological conditions as compared to imines or hydrazones,14 which are particularly attractive for bioconjugation applications. Herein, we utilized oxime chemistry to prepare for the first time thermoresponsive dendronized polypeptides with tunable properties. Aldehydecored first (G1) and second generation (G2) OEG dendrons were quantitatively grafted onto oxyamino-substituted polylysines, which were fully characterized with 1H NMR, GPC, and AFM measurements. The thermoresponsive behavior and secondary structures were investigated by UV−vis and circular dichroism spectroscopies. Based on the unique dendritic architecture and thermoresponsive properties, these dendronized polypeptides were also exploited for biomolecular recognition such as dopamine by introduction of phenylboronic acids, and their scaffold effect was examined in particular.

2. EXPERIMENTAL SECTION 2.1. Materials. Aldehyde-cored OEG dendrons (G1 and G2) were synthesized according to the previous reports.10b (S)-6-Amino-2-((tertbutoxycarbonyl)amino)hexanoic acid (Boc-Lys-OH) was purchased from GL Biochem (Shanghai) Ltd. Perfluorophenyl 2-[{(tert-butoxycarbonyl)amino}oxy]acetate (Boc-AOA-pfp) was prepared from EDCpromoted condensation of 2-[{(tert-butoxycarbonyl)amino}oxy]acetic acid and pentafluorophenol. Ethyl acetate and hexane were refluxed over CaH2, while triethylamine (Et3N) was dried over NaOH pellets. 50 mM phosphate buffer (pH 7) was self-made and calibrated with a Metler Toledo seven compact S220-B pH meter. Other reagents and solvents of reagent grade purchased and used without further purification. All reactions were run under a nitrogen atmosphere. Macherey-Nagel precoated TLC plates (silica gel 60 G/UV254, 0.25 mm) were used for thin-layer chromatography (TLC) analysis. Silica gel 60 M (Macherey-Nagel, 0.04−0.063 mm, 200−300 mesh) was used as the stationary phase for column chromatography. 2.2. Instrumentation and Measurements. 1H and 13C NMR spectra were recorded on a Bruker AV 500 (1H: 500 MHz; 13C: 125 MHz) spectrometer. Gel permeation chromatography (GPC) 511

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules Scheme 1. Synthetic Procedure of OEG-Based Dendronized Polypeptidesa

Reagents and conditions: (a) Boc-AOA-pfp, DIPEA, DMF/H2O, −15 to 25 °C, overnight (81%); (b) triphosgene, TEA, ethyl acetate, 0 to 25 °C, 10 h (37%); (c) benzylamine, hexylamine or TEA, DMF, 25 °C, 72 h (64%); (d) TFA, 25 °C, 6 h (76%); (e) aldehyde-cored dendron (G1 or G2) with or without 4-formylphenylboronic acid, H2O, pH = 4.5−5, 24 h (73−76%). Abbreviations: Boc-AOA-pfp = perfluorophenyl 2-[{(tertbutoxycarbonyl)amino}oxy]acetate, DIPEA = diisopropylethylamine, TFA = trifluoroacetic acid, TEA = triethylamine. Molecular structure of monomeric phenylboronic acid (MB) was included. a

vacuo. The residue was freeze-dried from water to afford PnONH2 as a white solid (76%). 1H NMR (D2O) of P44ONH2: δ = 1.16−1.37 (m, 2H, CH2), 1.37−1.54 (m, 2H, CH2), 1.55−1.88 (m, 2H, CH2), 3.13 (br, 2H, CH2), 4.18 (br, 1H, CH), 4.42 (br, 2H, CH2). Homopolypeptide PnG1. Aldehyde-cored G1 dendron (26.34 mg, 41.50 μmol) was mixed with an acidic solution of P44ONH2 (10.60 mg, 33.62 μmol) in 20 mM HCl (3 mL). Then, the solution pH was adjusted to be among 4.5−5 with 3 M NaOH solution. After shaking for 24 h at 25 °C, the mixture was dialyzed against deionized water for 3 days. Freeze-drying from water afforded P44G1 as a viscous liquid (20 mg, 73%). P56G1 and P205G1 were also synthesized in a similar way. 1H NMR (D2O) of P44G1: δ = 0.96−1.11 (m, 9H, CH3), 1.12− 1.96 (m, 6H, CH2), 3.09 (br, 2H, CH2), 3.32−4.22 (m, 43H, CH2 + CH), 4.50 (br, 2H, CH2), 6.76 (br, 2H, CH), 8.06 (br, 1H, CH). Homopolypeptide PnG2. Aldehyde-cored G2 dendron (37.24 mg, 15.47 μmol) was mixed with an acidic solution of P44ONH2 (3.98 mg, 12.62 μmol) in 20 mM HCl (2 mL). Then, the solution pH was adjusted to be among 4.5−5 with 3 M NaOH solution. After shaking for 24 h at 25 °C, the mixture was dialyzed against deionized water for 1 week. Freeze-drying from water afforded P44G2 as a viscous liquid (25 mg, 76%). P205G2 was also prepared in the same way. 1H NMR (D2O) of P44G2: δ = 0.90−1.10 (m, CH3), 1.10−2.05 (m, CH2), 3.05 (br, CH2), 3.26−4.37 (m, CH2 + CH), 4.48 (br, CH2), 6.40−6.88 (m, CH), 8.04 (br, CH). Copolypeptide P56(G10.89-co-B0.11). 4-Formylphenylboronic acid (0.92 mg, 6.14 μmol) and aldehyde-cored G1 dendron (43.70 mg, 68.85 μmol) were dissolved in water (2 mL). This solution was then mixed with the acidic solution of P56ONH2 (20.04 mg, 63.57 μmol) in 20 mM HCl (2 mL). The solution pH was adjusted to be among 4.5− 5 with 3 M NaOH solution. The mixture was kept at 0 °C for 24 h and then dialyzed against deionized water for 3 days. Freeze-drying from water afforded the title polypeptide as a viscous liquid (32 mg). 1H NMR (D2O): δ = 0.96−1.11 (m, CH3), 1.11−2.11 (m, CH2), 3.07 (br, CH2), 3.30−4.26 (m, CH2 + CH), 4.48 (br, CH2), 6.72 (br, CH), 7.25 (br, CH), 7.46 (br, CH), 8.03 (br, CH). Copolypeptide P56(G20.88-co-B0.12). 4-Formylphenylboronic acid (0.24 mg, 1.60 μmol) and aldehyde-cored G2 dendron (42.00 mg, 17.45 μmol) were dissolved in water (2 mL). This solution was then mixed with an acidic solution of P56ONH2 (5.07 mg, 16.07 μmol) in

20 mM HCl (1 mL). The solution pH was adjusted to be among 4.5− 5 with 3 M NaOH solution. The mixture was kept at 0 °C for 24 h and then dialyzed against deionized water for 1 week. Freeze-drying from water afforded the title polypeptide as a viscous liquid (26 mg). 1H NMR (D2O): δ = 0.90−1.10 (m, CH3), 1.10−2.05 (m, CH2), 3.06 (br, CH2), 3.26−4.40 (m, CH2 + CH), 4.49 (br, CH2), 6.35−6.93 (m, CH), 7.26 (br, CH), 7.48 (br, CH), 8.06 (br, CH). Model Phenylboronic Acid MB. 4-Formylphenylboronic acid (79.17 mg, 0.53 mmol) was mixed with equivalent 2-(oxyamino)acetic acid in methanol, and the solution was stirred overnight at 25 °C. After evaporation of methanol, the residue was dissolved in alkaline aqueous solution (pH = 8−9), which was washed twice with DCM. Then, the water phase was carefully acidified with HCl solution and extracted with DCM to yield the pure product as a white solid (80 mg, 68%). 1H NMR (D2O, pH = 7): δ = 4.45 (s, 2H, CH2), 7.55 (d, 2H, CH), 7.69 (d, 2H, CH), 8.23 (s, 2H, CH).

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of OEG-Based Dendronized Polypeptides. Dendronized polypeptides were prepared via “graft-to” strategy, and their synthesis is outlined in Scheme 1. Amide coupling of Boc-Lys-OH with Boc-AOApfp afforded the modified lysine 1 in a good yield. tertButoxycarbonyloxyamino-substituted N-carboxyanhydride 2 was obtained by treatment of 1 with triphosgene in the presence of Et3N. Amines were then used to initiate the ringopening polymerization of the NCA monomer, and the polypeptides PnBoc with three different chain lengths (n = 44, 56, 205; n was calculated according to the molecular weights of PnBoc from GPC measurements; Table 1) were prepared. Subsequent deprotection of PnBoc with TFA afforded polypeptide precursor PnONH2, which was coupled with excessive aldehyde-cored OEG dendrons (G1 and G2) to yield corresponding oxime-linked dendronized polypeptides PnG1 or PnG2. All new compounds were characterized by TLC, 1H NMR, and 13C NMR spectroscopy to confirm their high purities, and the 1H NMR spectra for P44ONH2, P44G1, 512

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

the splits more complicated. The dendron coverages for P44G1 and P44G2 were calculated to be around 98% (similar as the case in D2O) and 90%, respectively, demonstrating the high efficiency of oxime ligation. GPC results also indicate the successful grafting of OEG dendrons onto polypeptides (Table 1). As expected, molecular masses of polypeptides with the same chain length increased with side-group modification from Boc to G1 dendron and then to G2 dendron. AFM measurements were further conducted for imaging the single chains of these dendronized polypeptides.15 It was found that P205G1 and P205G2 exhibit worm-like conformations on mica substrates, with the latter being much thicker than the former (for images, see Figure S2). These OEG-based dendronized polypeptides are watersoluble at room temperature but exhibit characteristic thermoresponsive behavior at elevated temperature; thus, their macroscopic phase transitions were followed by turbidity measurements (Figure 2). All polypeptides display quite fast

Table 1. Characterization Results of Polypeptides PnBoc, PnG1, and PnG2 GPC results polypeptides

Mn × 10−3

PDI

OEGa (wt %)

α-helixb (%)

Tcp (°C)

P44Boc P44G1 P44G2 P56Boc P56G1 P205Boc P205G1 P205G2

13.5 19.5 33.5 16.9 24.7 61.9 71.5 85.8

1.10 1.16 1.16 1.30 1.40 3.82 3.19 2.22

0 73.8 91.0 0 73.8 0 73.8 91.0

N/A 43.2 25.4 N/A 59.3 N/A 100 62.8

N/A 35.2 36.4 N/A 33.8 N/A 31.0 34.4

a

OEG wt % represents the weight fraction of OEG-based dendrons in polypeptides. bHelical contents were calculated by using the following equation: α-helix % = (−[θ]222 + 3000)/39000. [θ]222 was measured in conditions at pH 7 and 20 °C. PnBocs are not soluble in water; therefore their α-helix contents cannot be determined in aqueous solutions.

and P44G2 are shown in Figure 1. For P44G1, all characteristic proton signals from both peptide (peaks 2−6) and dendron

Figure 2. Plots of transmittance versus temperature for OEG-based dendronized polypeptides in buffer of pH 7. Polypeptide concentration = 0.1 wt %.

phase transitions and small hysteresis especially in the case of G2 dendron (50 °C), while the shorter OEG chains caused broader phase transition and worse reversibility due to strong intermolecular interaction of polypeptide backbones.5c,e Thus, dendritic architectures afford polypeptides with prominent thermoresponsive properties, which are dominated by OEG-based dendron units due to their large sizes and shielding effect (the weight fractions of G1 and G2 dendrons in polypeptides reach as high as 73.8 and 91.0% as shown in Table 1). Another point should be mentioned that the oxime linkage should not be dynamic during thermally driven phase transition processes based on their high stability in neutral condition,14,16 which is completely different from the previously reported case where imine linkages were used for preparation of OEGylated polypeptides.10b

Figure 1. 1H NMR spectra of homopolypeptides P44ONH2, P44G1, and P44G2 in D2O and copolypeptides P56(G10.89-co-B0.11) and P56(G20.88 -co-B0.12) in alkaline D2O. Composition ratios for copolypeptides were calculated as follows: the molar content of phenylboronic acid (B) units was calculated by comparing the integration of proton signals 11 and 12 with those of proton signals 8 or 9 and 10, assuming the total coverage of B and dendron on polypeptides was 100%. Dotted lines are guides for the eye.

units (peaks 7−8, HOEG) were clearly present, but their splits are relatively broad in deuterium oxide medium. The dendron coverage was estimated to be around 92% by comparing the integration of proton signal 5 (δ = 3.09 ppm) from lysine moieties with that of proton signal (δ = 0.96−1.11 ppm) from the terminal methyl group of G1 dendron. A similar NMR spectrum was also obtained for P44G2 with much broader signals of peptide moieties (peaks 2−5), indicative of high rigidity of polypeptides when larger G2 dendrons were attached. The signal broadness makes it difficult to calculate G2 dendron grafting efficiency. To avoid this, the 1H NMR measurements was further performed in good solvent DMSO (Figure S1), where the proton signals are better resolved but 513

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

Figure 3. (a) CD (top) and UV (bottom) spectra of P44ONH2, P44G1, P44G2, P205G1, and P205G2 in pH 7 buffer at 20 °C. (b) Plots of [θ]222 versus temperature for P44G1 and P205G1.

Figure 4. (a) UV−vis spectra of ARS titrated with P56(G10.89-co-B0.11) at 25 °C. Photographs of ARS solution and its complex with P56(G10.89-coB0.11) were inserted. (b) Dependence of ΔA515 on [B]. [B] represents the concentration of phenylboronic acid unit in copolypeptides or MB. [ARS] = 50 μM, pH = 7.

with a high helicity. Once the solution temperature was cooled down to room temperature, OEG units start to rehydrate, and the polypeptide chains completely refolded into α-helical conformation (Figure S3). The above results demonstrate (1) OEG dendronization can effectively enhance α-helical conformations of polypeptides as a result of the balance of their shielding effect and steric hindrance and (2) thermally driven phase transitions of the dendronized polypeptides switched reversibly their secondary structures from ordered to disordered. This is quite different from linear OEG-modified polypeptides, where the secondary structures keep nearly unchanged during phase transitions.5c,e The probable reason may be that the collapse and aggregation of dendritic OEG lead to large crowdedness and enhanced solubility to weaken intramolecular hydrogen bonding of polypeptide backbone.10b 3.2. Switchable Recognition of Phenylboronic AcidAppended Dendronized Copolypeptides toward Catechol-Containing Compounds. Based on their dendritic structures and characteristic thermoresponsive properties, these dendronized polypeptides were further exploited as unique scaffolds for biomolecular recognition. It is well-known that phenylboronic acids can specifically recognize various diolcontaining biomolecules such as glucose, dopamine, and sialic acid, which has shown promising applications in biosensing, insulin delivery, and cancer therapies.19 The introduction of phenylboronic acid into thermoresponsive polypeptides will not only expand their functions and potential applications but also provide a way to tune their recognition ability through

Secondary structures of these polypeptides were investigated by circular dichroism (CD) spectroscopy (Figure 3a). Oxyamino-substituted polypeptide P44ONH2 adopts mainly a random-coil conformation at pH 7 (above pKa ∼ 4.5 for the oxyamino group17). Upon grafting of G1 dendron to form P44G1, its secondary structure changed to α-helix as indicated by characteristic Cotton effects with a maximum at λ = 190 nm and two minima at λ = 206 and 222 nm. The mean residual molar ellipticity at λ = 222 nm ([θ]222) was determined to be around −13850° cm2 dmol−1, corresponding to a helicity of 43%. P44G2 also adopts partially α-helical conformation with a helicity of about 25%. For the polypeptides with long chain length such as P205G1 and P205G2, dendronization increases significantly their helicities into 100% and 63%, respectively, demonstrating the high dependence of α-helix formation on peptide chain length. The lower helicity of G2 polypeptides than that of the G1 counterpart is supposed to be due to larger steric hindrance18 and enhanced dissolution of peptidic hydrogen bonding from larger OEG dendrons. The temperature influence on secondary structures of these polypeptides was further examined. P44G1 and P205G1 were taken as the examples, and their [θ]222s versus temperature are plotted in Figure 3b. Below its phase transition temperature, the [θ]222 of P44G1 kept nearly constant when temperature increased from 20 to 35 °C. As the temperature reached 37 °C, which is right above its Tcp (35.2 °C), [θ]222 decreased significantly and underwent great drop with further increase of solution temperature. Similar helix disruption was observed for P205G1 514

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

Figure 5. (a) UV−vis spectra of the complex ARS/P56(G10.89-co-B0.11) at 25 and 45 °C. (b) Dependence of F0/F on temperature for the complexes of ARS with copolypeptides or MB. F0 and F represent fluorescence intensity at 20 °C and given temperature, respectively. [ARS] = 50 μM, [B] within copolypeptides is 50 μM, [MB] = 0.182 mM. Tcp of the complex ARS/P56(G10.89-co-B0.11) is around 36 °C.

co-B0.12), demonstrating phenylboronic acid in copolypeptide scaffolds possesses stronger binding affinity than that in monomeric MB. This result might be originated from the fact that phenylboronic acid located within the dendritic OEG surroundings could be efficiently shielded from bulk water phase, which facilitates boronate formation. Certainly, the cooperative binding of OEG units toward guest dyes should be another reason to contribute partially to the enhancement.22 The influence of thermally driven phase transitions on the binding behavior was further examined. The complex from ARS and P56(G10.89-co-B0.11) displays a clear yellow color and red fluorescence in aqueous solutions at room temperature, whereas it changes to be turbid red and nearly nonfluorescent once heated above its Tcp. UV−vis measurements (Figure 5a) showed that its λmax red-shifts significantly from 478 nm at 25 °C to around 508 nm at 45 °C, suggesting the dissociation of boronate during heating. This decomplexation was further verified by temperature-varied 1H NMR spectroscopy (for spectra, see Figure S8), and the result indicates that ARS molecules gradually changed from the complexed to free state with the increase of solution temperature. Thermally driven fluorescence quenching was also followed with fluorescence spectroscopy (for spectra, see Figure S9) and evaluated by the ratio F0/F, where F0 and F represent fluorescence intensity at 20 °C and given temperature, respectively. The plot of F0/F against solution temperature is shown in Figure 5b. Both complexes from ARS with P56(G10.89-co-B0.11) and P56(G20.88co-B0.12) show similar fluorescence quenching behavior: F0/F increases slowly during initial heating process, then starts to rise at around 40 °C (above Tcps of the complexes), and increases sharply with further increase of solution temperature. For comparison, the monomeric complex from ARS and MB without thermoresponsiveness was also checked, and the results show that F0/F increases slowly as expected during the whole heating process. The above contrast clearly demonstrates that the dehydration and collapse of surrounding OEG dendrons led to significant steric hindrance and accelerated heating driven boronate dissociation. Once cooled, copolypeptides start to rehydrate and complex with ARS again as indicated by the recovery of yellow color and strong red fluorescence. Therefore, the dissociation and association processes are fully reversible by just changing the solution temperature. In a word, dendronized polypeptide scaffolds not only facilitate the boronate formation below Tcp but also provide a thermally

external stimuli. However, phenylboronic acid-incorporated polypeptides have scarcely been reported to date,20 especially in the case of thermoresponsive polypeptides. We here facilely incorporated 4-formylphenylboronic acid into dendronized polypeptides during oxime formation between PnONH2 and aldehyde-cored OEG dendrons. By controlling the feed ratios of dendrons to 4-formylphenylboronic acid, two copolypeptides P56(G10.89-co-B0.11) and P56(G20.88-co-B0.12) with around 11− 12 mol % phenylboronic acid units were obtained (Scheme 1), and their structures were proven by 1H NMR spectra (Figure 1). Both copolypeptides exhibit characteristic thermoresponsiveness similar as their homopolypeptide counterparts, and their Tcps are determined to be 29.1 and 35.8 °C for P56(G10.89co-B0.11) and P56(G20.88-co-B0.12), respectively (Figure S4). The secondary structure of copolypeptide P56(G10.89-co-B0.11) was further examined, which also adopts enhanced α-helical conformation (Figure S5). Alizarin red S (ARS)21 was first selected as a model catecholcontaining compound to evaluate the binding affinity of these copolypeptides. Its complexation with copolypeptides was monitored by UV−vis titration, and the spectra are shown in Figure 4a. The sole ARS solution is red colored with a maximum wavelength λmax at 515 nm. Upon successive addition of P56(G10.89-co-B0.11), the color gradually turned yellow with a significant blue-shift of λmax. This result demonstrates that the phenylboronic acid units within copolypeptide bound efficiently with catechol to form cyclic boronates. The isosbestic point at around 492 nm suggests 1:1 stoichiometry for the complexation between boronic acid and catechol moieties. Similar binding behavior was also observed for P56(G20.88-coB0.12), irrespective of its larger OEG dendron (for UV−vis titration curves, see Figure S6). The boronate formation from G1- or G2-based copolypeptides with ARS was also confirmed by the great fluorescence enhancement of ARS (Figure S7). To examine the possible scaffold effects from dendronized polypeptides on recognition of phenylboronic acid, a monomeric compound MB containing a phenylboronic acid unit was synthesized (Scheme 1), and its complexation with ARS was examined for comparison (for its UV−vis titration curves, see Figure S6). The absorbance change of ARS at λ = 515 nm (ΔA515) versus concentration of MB was plotted, which is compared with those of copolypeptides as shown in Figure 4b. ΔA515 displayed a notably smaller variation with addition of MB than with P56(G10.89-co-B0.11) and P56(G20.88515

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

tides are capable to reversibly “catch” or “release” dopamine through thermally driven phase transitions,25 which can be utilized to regulate the level of dopamine in vivo, thus showing promising applications in dopamine-related diagnostics or disease treatments.26

switchable barrier through phase transitions to reversibly regulate the recognition of phenylboronic acid to catechol. However, no big recognition and dissociation differences were observed between G1 and G2 polypeptides, which suggests the thickness effect in these dendronized copolypeptides have minor contribution to their binding behavior either below or above Tcps. The switchable recognition of these copolypeptides toward ARS promoted us to further evaluate other catechol-containing compounds such as dopamine. Dopamine is one of famous neurotransmitters and plays an important role in a variety of central nervous system functions. Various receptors based on phenylboronic acids have been developed so far, but none was capable to switch the recognition through external stimuli.23 The utilization of thermoresponsive polypeptide scaffolds may provide this possibility. The copolypeptide P56(G10.89-co-B0.11) was selected as an example, and its complexation with dopamine was first investigated by 1H NMR spectroscopy. Their binding in neutrally aqueous solutions was evidenced by the presence of several new broad proton signals which correspond to the formed boronate (Figure S10). Then, ARS was introduced as a fluorescent reporter to further monitor the complexation of P56(G10.89-co-B0.11) with dopamine.24 The stock solution of the complex from ARS and P56(G10.89-coB0.11) is highly fluorescent. With successive addition of dopamine, its fluorescence intensity gradually decreased (Figure 6), indicating the reporter ARS was replaced by

4. CONCLUSION A new class of thermoresponsive dendronized polypeptides constituted through oxime ligation have been reported, and their switchable recognition of catechol-containing compounds have been demonstrated in the present work. These polypeptides were efficiently synthesized through quantitative oxime ligation between oxyamino-substituted polylysines and aldehyde-cored OEG dendrons. The dendronization of polypeptides afforded them characteristic thermoresponsive behavior including fast and reversible phase transitions around physiological temperatures, as well as small hysteresis, which are quite unique as compared to linear OEG-modified polypeptides. At the same time, dendronization of the polypeptides enhanced α-helix formation as a result of balance between shielding effect and steric hindrance from pendanted dendrons, and the enhanced secondary structures can be switched from order to disorder through thermally driven phase transitions. Furthermore, introduction of phenylboronic acids afforded dendronized copolypeptides to specifically recognize catechol-containing compounds such as alizarin red S and dopamine. Based on the positive scaffold effect, phenylboronic acid units in copolypeptides exhibit enhanced binding ability as compared to monomeric counterpart, and the recognition can be reversibly switched on or off through thermally driven phase transitions. To our knowledge, this stimuli-controlled recognition of bioactive dopamine has not been reported previously. Present work provides an efficient and versatile methodology for preparation of stimuli-responsive polypeptides, and the reported findings enrich the understanding of structural effects on secondary structures and switchable recognition ability of the polypeptides, which may lead to the formation of promising intelligent peptide-based materials for various biomedical applications.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b02259. Details of supplemental 1H NMR and UV−vis spectra, turbidity curves, etc. (PDF)

Figure 6. Fluorescence quenching of the complex ARS/P56(G10.89-coB0.11) with the addition of dopamine, glactose, and glucose. F0′ and F′ represent fluorescence intensity without and with addition of analysts, respectively. [ARS] = [B] = 50 μM, pH = 7.

■

dopamine to become nonfluorescent free state. Importantly, the fluorescence quenching occurred only in the presence of dopamine, neither in the cases of glucose nor glactose. This recognition selectivity is ascribed to the combination of solution pH and pKa’s of both phenylboronic acid (pKa ∼ 9) and diol in analytes.21 At physiological pH 7 condition, phenylboronic acid units are in neutral and trigonal form and prefer to bind the catechol group in dopamine due to its lower pKa as compared to aliphatic diol groups in monosaccharides. When solution temperature was increased above Tcp, the copolypeptide collapsed and dissociated with dopamine just as the case for ARS. This dissociation was verified by temperaturevaried 1H NMR spectroscopy (Figure S11), where the proton signals from boronate gradually disappeared with increase of solution temperature. Therefore, these dendronized copolypep-

AUTHOR INFORMATION

Corresponding Authors

*Ph +86-21-66138053; Fax +86-21-66138039; e-mail [email protected] (J.Y.). *Ph +86-21-66138053; Fax +86-21-66138039; e-mail azhang@ shu.edu.cn (A.Z.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We sincerely thank Prof. Toshio Masuda for his kind help with the manuscript. Dr. Hongmei Deng from the Instrumental Analysis and Research Center of Shanghai University is thanked for her assistance with NMR measurements. Financial support 516

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517

Article

Macromolecules

(15) Guo, Y.; van Beek, J. D.; Zhang, B.; Colussi, M.; Walde, P.; Zhang, A.; Kröger, M.; Halperin, A.; Schlüter, A. D. J. Am. Chem. Soc. 2009, 131, 11841−11854. (16) The dynamic nature of oxime linkage was only activated through exchange reaction in given conditions: (a) Jackson, A. W.; Fulton, D. A. Macromolecules 2010, 43, 1069−1075. (b) Mukherjee, S.; Bapat, A. P.; Hill, M. R.; Sumerlin, B. S. Polym. Chem. 2014, 5, 6923− 6931. (c) Mukherjee, S.; Hill, M. R.; Sumerlin, B. S. Soft Matter 2015, 11, 6152−6161. (17) Bissot, T. C.; Parry, R. W.; Campbell, D. H. J. Am. Chem. Soc. 1957, 79, 796−800. (18) Lee, C. C.; Fréchet, J. M. J. Macromolecules 2006, 39, 476−481. (19) (a) Nishiyabu, R.; Kubo, Y.; James, T. D.; Fossey, J. S. Chem. Commun. 2011, 47, 1106−1123. (b) Guo, Z.; Shin, I.; Yoon, J. Chem. Commun. 2012, 48, 5956−5967. (c) Ma, R.; Shi, L. Polym. Chem. 2014, 5, 1503−1518. (d) Peters, J. A. Coord. Chem. Rev. 2014, 268, 1− 22. (20) (a) Kobayashi, H.; Nakashima, K.; Ohshima, E.; Hisaeda, Y.; Hamachi, I.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 2000, 997−1002. (b) Friggeri, A.; Kobayashi, H.; Shinkai, S.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2001, 40, 4729−4731. (c) Zhao, L.; Ding, J.; Xiao, C.; He, P.; Tang, Z.; Pang, X.; Zhuang, X.; Chen, X. J. Mater. Chem. 2012, 22, 12319−12328. (d) Liu, G.; Ma, R.; Ren, J.; Li, Z.; Zhang, H.; Zhang, Z.; An, Y.; Shi, L. Soft Matter 2013, 9, 1636−1644. (21) Tomsho, J. W.; Benkovic, S. J. J. Org. Chem. 2012, 77, 2098− 2106. (22) (a) Liu, L.; Li, W.; Yan, J.; Zhang, A. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1706−1713. (b) Mao, Q.; Liu, K.; Li, W.; Yan, J.; Zhang, A. Polym. Chem. 2015, 6, 1300−1308. (23) Pradhan, T.; Jung, H. S.; Jang, J. H.; Kim, T. W.; Kang, C.; Kim, J. S. Chem. Soc. Rev. 2014, 43, 4684−4713. (24) (a) Springsteen, G.; Wang, B. Chem. Commun. 2001, 1608− 1609. (b) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291− 5300. (25) A similar concept for reversible binding of glucose has been reported in poly(N-isopropylacrylamide)-based systems: (a) Kataoka, K.; Miyazaki, H.; Bunya, M.; Okano, T.; Sakurai, Y. J. Am. Chem. Soc. 1998, 120, 12694−12695. (b) Matsumoto, A.; Kurata, T.; Shiino, D.; Kataoka, K. Macromolecules 2004, 37, 1502−1510. (c) Pasparakis, G.; Vamvakaki, M.; Krasnogor, N.; Alexander, C. Soft Matter 2009, 5, 3839−3841. (26) Whyte, G. F.; Vilar, R.; Woscholski, R. J. Chem. Biol. 2013, 6, 161−174.

from the National Natural Science Foundation of China (Nos. 21304056, 21374058, 21474060, and 21574078) and the Ph.D. Programs Foundation of Ministry of Education of China (No 201331081100166) is acknowledged.

■

REFERENCES

(1) (a) Löwik, D. W. P. M.; Leunissen, E. H. P.; van den Heuvel, M.; Hansen, M. B.; van Hest, J. C. M. Chem. Soc. Rev. 2010, 39, 3394− 3412. (b) Huang, J.; Heise, A. Chem. Soc. Rev. 2013, 42, 7373−7390. (c) Deng, C.; Wu, J.; Cheng, R.; Meng, F.; Klok, H.-A.; Zhong, Z. Prog. Polym. Sci. 2014, 39, 330−364. (d) Shen, Y.; Desseaux, S.; Aden, B.; Lokitz, B. S.; Kilbey, S. M.; Li, Z.; Klok, H.-A. Macromolecules 2015, 48, 2399−2406. (2) (a) Daamen, W. F.; Veerkamp, J. H.; van Hest, J. C. M.; van Kuppevelt, T. H. Biomaterials 2007, 28, 4378−4398. (b) Simnick, A. J.; Lim, D. W.; Chow, D.; Chilkoti, A. Polym. Rev. 2007, 47, 121−154. (c) Nuhn, H.; Klok, H.-A. Biomacromolecules 2008, 9, 2755−2763. (3) (a) Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakellariou, G. Chem. Rev. 2009, 109, 5528−5578. (b) Lu, H.; Wang, J.; Song, Z.; Yin, L.; Zhang, Y.; Tang, H.; Tu, C.; Lin, Y.; Cheng, J. Chem. Commun. 2014, 50, 139−155. (4) Shen, Y.; Fu, X.; Fu, W.; Li, Z. Chem. Soc. Rev. 2015, 44, 612− 622. (5) (a) Yu, M.; Nowak, A. P.; Deming, T. J. J. Am. Chem. Soc. 1999, 121, 12210−12211. (b) Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Zhuang, X.; Chen, X. Polym. Chem. 2011, 2, 2627−2634. (c) Chen, C.; Wang, Z.; Li, Z. Biomacromolecules 2011, 12, 2859−2863. (d) Liao, Y.; Dong, C.-M. J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 1834−1843. (e) Fu, X.; Shen, Y.; Fu, W.; Li, Z. Macromolecules 2013, 46, 3753− 3760. (6) Chopko, C. M.; Lowden, E. L.; Engler, A. C.; Griffith, L. G.; Hammond, P. T. ACS Macro Lett. 2012, 1, 727−731. (7) (a) Kramer, J. R.; Deming, T. J. J. Am. Chem. Soc. 2014, 136, 5547−5550. (b) Ma, Y.; Fu, X.; Shen, Y.; Fu, W.; Li, Z. Macromolecules 2014, 47, 4684−4689. (8) (a) Li, W.; Zhang, A.; Feldman, K.; Walde, P.; Schlüter, A. D. Macromolecules 2008, 41, 3659−3667. (b) Li, W.; Zhang, A.; Schlüter, A. D. Chem. Commun. 2008, 5523−5525. (c) Yan, J.; Li, W.; Liu, K.; Wu, D.; Chen, F.; Wu, P.; Zhang, A. Chem. - Asian J. 2011, 6, 3260− 3269. (9) (a) Junk, M. J. N.; Li, W.; Schlüter, A. D.; Wegner, G.; Spiess, H. W.; Zhang, A.; Hinderberger, D. Angew. Chem., Int. Ed. 2010, 49, 5683−5687. (b) Liu, L.; Li, W.; Liu, K.; Yan, J.; Hu, G.; Zhang, A. Macromolecules 2011, 44, 8614−8621. (c) Junk, M. J. N.; Li, W.; Schlüter, A. D.; Wegner, G.; Spiess, H. W.; Zhang, A.; Hinderberger, D. J. Am. Chem. Soc. 2011, 133, 10832−10838. (d) Yan, J.; Li, W.; Zhang, A. Chem. Commun. 2014, 50, 12221−12233. (10) (a) Zhang, X.; Li, W.; Zhao, X.; Zhang, A. Macromol. Rapid Commun. 2013, 34, 1701−1707. (b) Yan, J.; Liu, K.; Zhang, X.; Li, W.; Zhang, A. J. Polym. Sci., Part A: Polym. Chem. 2015, 53, 33−41. (11) Deming, T. J. Chem. Rev. 2015, DOI: 10.1021/acs.chemrev.5b00292. (12) (a) Lu, H.; Bai, Y.; Wang, J.; Gabrielson, N. P.; Wang, F.; Lin, Y.; Cheng, J. Macromolecules 2011, 44, 6237−6240. (b) Quadir, M. A.; Martin, M.; Hammond, P. T. Chem. Mater. 2014, 26, 461−476. (c) Brosnan, S. M.; Schlaad, H. Polymer 2014, 55, 5511−5516. (d) Xu, Q.; He, C.; Xiao, C.; Yu, S.; Chen, X. Polym. Chem. 2015, 6, 1758− 1767. (e) Liu, H.; Xiao, Y.; Xu, H.; Guan, Y.; Zhang, J.; Lang, M. Chem. Commun. 2015, 51, 10174−10177. (13) (a) Vázquez-Dorbatt, V.; Tolstyka, Z. P.; Maynard, H. D. Macromolecules 2009, 42, 7650−7656. (b) Hill, M. R.; Mukherjee, S.; Costanzo, P. J.; Sumerlin, B. S. Polym. Chem. 2012, 3, 1758−1762. (c) Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Chem. Eur. J. 2014, 20, 34−41. (14) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523− 7526. 517

DOI: 10.1021/acs.macromol.5b02259 Macromolecules 2016, 49, 510−517