Xiaohui Fuab,
Yinan Mab,
Jing Sun*b and
Zhibo Li*ab
aSchool of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China. E-mail: jingsun@qust.edu.cn; zbli@qust.edu.cn
bInstitute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
First published on 15th July 2016
A series of poly(L-glutamate) bearing Y-shaped oligo(ethylene glycol)x (OEGx) side-chains (PPLG-g-EGx, x = 2, 3 and 4) were synthesized via a combination of ring opening polymerization (ROP) of γ-propargyl-L-glutamate N-carboxyanhydride (PLG-NCA) with thiol–yne photoaddition. The solubility of the polypeptides in water was firstly investigated. PPLG-g-EG3 and PPLG-g-EG4 were soluble in water and displayed fully reversible thermal-responsive behaviors. Additionally, the polypeptides showed redox-responsive properties along with the conformation associated water solubility due to the presence of thioether groups in the side-chains. In particular, the clouding points (CPs) of the polypeptides are highly tunable depending on the degree of polymerization (DP), the side-chain length, the polymer/salt concentration, and the degree of oxidation/reduction. The key feature of our design is the simultaneous incorporation of thermal-responsive OEG units and redox-responsive thioether linkages by one-step thiol–yne photoaddition, which allows for precisely tuning the phase transition temperature by both stimuli. This synthetic approach offers a facile and efficient way to prepare polypeptide-based biomaterials with highly tunable properties.
Post-polymerization modification has emerged as a versatile and efficient strategy for the functionalization of polymers. The combination of living/controlled ROP of synthetic functional NCA monomers with orthogonal conjugation reactions, i.e., “Click” chemistry, greatly facilitates the preparation of stimuli-responsive polypeptides. Several NCA monomers with clickable substitutes have been prepared for subsequent modifications.12–18 In particular, alkynyl/vinyl groups modified polypeptides allow for the post-modification using thiol–yne/–ene photochemistry, respectively.19 Both reactions proceed rapidly and efficiently under ambient conditions without using metal catalysts, which offer significant advantages in biomedical applications.20,21 More importantly, the resultant thioether groups show redox-responsive behavior under mild conditions that often trigger polarity/conformation transitions of polypeptides. Deming and coworkers reported that the complete oxidation of poly(glyco-L-cysteine) and the partial oxidation of poly(L-methionine) can switch the secondary conformation from α-helix to random coil, and the partially oxidized poly(L-methionine) can be reduced back to the parent polymer.9,22 They also found that methionine-containing peptides can be alkylated by alkyl halides or epoxides into water-soluble disordered coils in high yields.23,24 Some of the dealkylated reactions were fully reversible. Our group previously synthesized a family of thermal-responsive OEGylated poly(L-cysteine)s via thiol–ene Michael addition.25 We further obtained stimuli-responsive self-assemblies that show oxidation-induced polarity/conformation changing.26
Combining the advantages of click chemistry with redox-responsive properties of the resulting thioether groups, we prepared a new class of OEGylated thioether bond-containing polypeptides derived from poly(L-glutamate)s (Scheme 1). The key feature of our design is the simultaneous incorporation of thermal-responsive OEG units and redox-responsive thioether bonds by thiol–yne photoaddition. The thiol–yne photoaddition allows for Y-shaped molecular architecture with two thermal-responsive OEG units on each side-chain. The redox-responsive properties of thioethers further provide an additional stimulus.
Scheme 1 The synthetic route to PPLG-g-EGx by combination of ROP of PLG-NCA and thiol–yne photochemistry. Reagents and conditions: (i) HMDS, THF/DMF 1:1 v/v, rt, 48 h. (ii) DMPA, DMF, hν, 3 h. |
Both thermal-/redox-responsive properties allow access for highly tunable phase transition temperatures. This combination makes the obtained polypeptides promising candidates for multi-stimuli responsive polymeric materials.
Fig. 1 1H NMR spectra of (a) PLG-NCA in CDCl3 (* indicates CDCl3 and ethyl acetate residues); (b) PPLG and (c) PPLG-g-EG3 in CDCl3/CF3CO2D (v/v = 85:15). |
Three samples were synthesized, denoted as PPLG35, PPLG65 and PPLG130, where the subscript represented the DP of PPLG. GPC traces showed relatively narrow molecular weight distributions with dispersity (Ð) < 1.2 (Fig. S1a†), indicative of the well-controlled ROP of NCAs.
To systematically study the relationship of the OEG side-chain length on physical properties of poly(L-glutamate)s, we synthesized three types of thiol-terminated oligo(ethylene glycol) monomethyl ethers (OEGx-SH) with x ranging from 2 to 4. Their chemical structures were confirmed by 1H NMR spectrometry (see ESI†).30 OEGx-SH was then conjugated to PPLG via thiol–yne coupling reaction using DMPA as the photoinitiator in DMF. The reaction solution was purged with N2 to remove oxygen and irradiated with UV light at room temperature for 3 hours to yield the corresponding OEGx grafted homopolypeptides, PPLG-g-EGx.33 The chemical structure of PPLG-g-EGx was confirmed by 1H NMR spectroscopy. Fig. 1c shows a typical 1H NMR spectrum of PPLG-g-EG3. The protons of propargyl group at 4.66–4.76 and 2.46–2.53 ppm completely disappear, indicative of the quantitative conversion of alkynyl groups. The appearance of new peaks at 2.73–3.16, 3.50, and 3.80 ppm correspond to the protons of OEG moiety, further confirming the successful addition. The GPC traces of PPLG-g-EGx (Fig. 2a and S1†) maintain unimodal distribution with an apparent shift to high molecular region, indicative of negligible side reactions. FTIR spectra show that a characteristic –CC– stretching band at 2130 cm−1 completely disappear (Fig. 2b and S2†), confirming the complete conversion of alkynyl groups. The obtained PPLG-g-EGx samples show good solubility in common organic solvents, but behave different in aqueous solution, summarized in Table S1.† PPLG-g-EG2 with the shortest OEG side-chain length is insoluble in water at ambient temperature; however, PPLG-g-EG3 and PPLG-g-EG4 are readily soluble under similar conditions. This is possibly due to the increased hydrophilicity of the polymers with longer OEG side-chains.34,35 We therefore focus on PPLG-g-EG3 and PPLG-g-EG4.
It is known that the secondary structure of polypeptides can be affected by both the main chain length and the side chain structures.36 We thus investigated the conformation of the samples using FTIR and circular dichroism (CD) spectroscopy. All of PPLG samples exhibit strong absorption bands centered at 1653 (amide I) and 1550 cm−1 (amide II), indicative of predominant helical conformation (Fig. S2a†). In addition, a broad band in the range of 1630–1660 cm−1 and a weak band at 1516 cm−1 were observed, indicative of the coexistence of β-sheet or random coil.37 As the PPLG chain length increases from 35 to 130, the absorbance at 1653 and 1550 cm−1 increases while decreasing at 1516 cm−1 in all cases, suggesting the enhanced helical content. This is consistent to previously reported results of OEGylated poly-L-glutamate (poly-L-EGxGlu).38 Interestingly, all PPLG-g-EGx samples show sharp absorption bands at 1653 (amide I) and 1550 cm−1 (amide II) and trivial absorption at 1516 cm−1 (Fig. 2b and S2†). This demonstrates that the introducing of OEGx side-chains is beneficial for stabilization of α-helical structures.29 Note that the conformation shows less dependence on OEG side-chain length. The secondary structures of PPLG-g-EG3 and PPLG-g-EG4 were also characterized by CD spectra, shown in Fig. 2c and S3.† We calculated the α-helical contents based on the CD analysis, and the results are given in Table S2.† CD measurement reveals that both PPLG-g-EG3 and PPLG-g-EG4 adopt α-helix conformation in water with 100% helicity at 20 °C. The helicity is nearly independent on OEG side-chain length and PPLG main-chain length in the case of PPLG-g-EG3 and PPLG-g-EG4, which is consistent with the FTIR results. These results are different from that of poly-L-EGxGlu, which show the increased helical content with increasing OEG side-chain length.29 This is possibly due to the increased OEG moiety in the Y-shape structure of PPLG-g-EGx that can stabilize the helicity of polypeptide backbones.
We then examined the effects of temperature on solution properties of samples PPLG-g-EG3 and PPLG-g-EG4 in water. In both cases, fast transitions from clear to cloudy upon heating and cloudy to clear upon cooling were shown, indicative of reversible LCST behaviors. Turbidimetry was applied to determine the corresponding cloud points (CPs) (Fig. 3a and S4†). Fig. 3a shows the transmittance versus temperature plots of PPLG130-g-EG3 and PPLG130-g-EG4 aqueous solutions (2 mg mL−1). In the case of PPLG130-g-EG3, the transmittance decreases from 100 to 1% when temperature increases from 5 to 45 °C and recovers to 100% upon cooling. If we define the temperature with 50% transmittance as CP, the CP of PPLG130-g-EG3 is about 25 °C for the heating process. Similarly, PPLG130-g-EG4 shows a sharp phase transition with the corresponding CP of 42 °C and complete recovery of transmittance upon cooling. Note that the CP determined from the cooling ramp is about 2 °C lower than that from the heating ramp in both cases. This is probably because rehydration of OEG units requires slight overcooling to overcome the energy barriers.39 We further studied the repeatability of phase transitions, shown in Fig. 3b. A full recovery of transmittance in PPLG130-g-EG3 was achieved after 6 heating–cooling cycles between 5 and 35 °C. Similarly, the phase transition of PPLG130-g-EG4 could be repeated multiple times between 30 and 55 °C (Fig. S4c†).
Fig. 3c shows the CP dependence on DP for PPLGn-g-EG3 and PPLGn-g-EG4. It is revealed that PPLGn-g-EG3 shows a lower CP than that of PPLGn-g-EG4 at the same DP. It is not surprised as the increase of OEG length in the side-chains can increase the hydrophilicity of the polymer, resulting in the increase of CP. With the same OEG side-chain length, the CP decreases with increasing the DP of PPLG backbones. In the case of PPLGn-g-EG3, the CP decreases from 33 to 25 °C as the DP increases from 35 to 130. Similarly, the CP of PPLGn-g-EG4 decreases from 48 to 42 °C with increasing DP. The effects of polymer/salt concentration on the CP were also investigated. As the polymer concentration increases from 0.5 to 2 mg mL−1, the CP of PPLG65-g-EG3 decreases rapidly, accompanied by the reduced transition process (Fig. S5†). This is likely due to the enhanced intermolecular association in concentrated solution.31 When the polymer concentration further increased, the CP reaches a plateau. NaCl was added to investigate the effect of the salt concentration on CPs. As the NaCl concentration increases from 0 to 100 mM, the CP decreases slightly from 32 to 31.4 °C, which is possibly due to the partial dehydration of polymer chains induced by addition of NaCl (Fig. S6†).40 Hence, it is seen that the CP can be finely tuned by the OEGx side-chain lengths, the DP of PPLG backbone and the polymer/salt concentration for different requirements of biomedical applications.
The stability of the secondary structure of polypeptides on temperature was then investigated by temperature-varied CD measurements (Fig. 4 and S7–S9†). When the samples PPLGn-g-EG3 and PPLGn-g-EG4 were heated above the CPs, the CD signals significantly decreased (Fig. 4a and S7a–S9a†). The helicities declined accordingly, shown in Table S2.† This is most likely due to the aggregation when the temperature is above the CP.25 Their conformations are recovered during the cooling ramp (Fig. 4b and S7b–S9b†). The result is consistent with turbidity measurements.
It is known that thioethers can undergo selectively oxidation into either sulfoxide or sulfone groups,9,25 while the partially oxidized sulfoxide polymers can be reversed to thioethers via enzymatic or selective chemical reduction (Scheme 2).7,22 We therefore performed different levels of chemical oxidation to achieve partially oxidized sulfoxide polymers (PPLG-g-OEGx) and fully oxidized sulfone polymers (PPLG-g-O2EGx). The corresponding structures were confirmed by 1H NMR and FTIR spectra. Fig. 5a shows the 1H NMR spectrum of PPLG-g-EG3, in which the broad peaks between 2.73 and 3.16 ppm are attributed to methylene groups (d and e) in β position to sulfur atoms (–CH2SCH(CHCH2)CH2SCH2–). As oxidized to sulfoxides (PPLG-g-OEG3), it is clearly observed that the peaks at 2.73–3.16 ppm disappear and the peaks at 3.11–3.60 ppm appear (Fig. 5b) that can presumably be ascribed to methylene groups in the β position of sulfoxides (–CH2S(O)CH(CHCH2)CH2S(O)CH2–). For PPLG-g-O2EG3, a higher-frequency shift of methylene groups (d and e in Fig. 5c) is observed, which can be assigned to more polar sulfone groups. FTIR spectra show increased νSO band at 1030 cm−1 of PPLG-g-OEG3 (Fig. 6a). In the case of PPLG-g-O2EG3, two types of new FTIR bands at 1291 and 1321 cm−1 appear, likely associated with the symmetric and asymmetric stretching of –SO2– groups. All the data indicate the successful oxidation to different levels.
Fig. 5 1H NMR spectra of (a) PPLG-g-EG3, (b) PPLG-g-OEG3, (c) PPLG-g-O2EG3 and (d) PPLG-g-*EG3 in CDCl3/CF3CO2D (v/v = 85:15). |
Fig. 6 (a) FTIR spectra and (b) GPC chromatographs of PPLG130-g-EG3, PPLG130-g-OEG3, PPLG130-g-O2EG3 and PPLG130-g-*EG3. |
To investigate the reduction of sulfoxides, we initially incubated PPLG-g-OEGx with MSR enzymes (methionine sulfoxide reductase A and methionine sulfoxide reductase B) and DTT according to reported procedures.22 Unfortunately, we did not observe considerable reduction from PPLG-g-OEGx to PPLG-g-EGx. We assume that the synthetic polypeptide is not a good substrate for MSR enzymes. This may be ascribed into two possible reasons. One is that the specificity and selectivity of enzymes excludes the other different structures of substrates from natural methionine sulfoxides. The other possible reason is that the Y-shaped side-chain structure of PPLGn-g-EGx sterically limits the reduction efficiency of the sulfoxide groups. Beyond MSR enzymes, we also used thioglycolic acid to explore the reduction reaction of PPLG-g-OEGx. The structures of the reducing product, denoted as PPLGn-g-*EGx, were confirmed by 1H NMR and FTIR spectra (Fig. 5d, 6a and spectral data in ESI†). Fig. 5d shows a typical 1H NMR spectrum of PPLG130-g-*EG3. Two multiple peaks at 2.73–3.16 ppm and 3.11–3.60 ppm are clearly observed, indicative of the coexistence of sulfoxides and thioethers. We therefore increased the amount of reducing reagent (10 eq. with respect to sulfoxide groups). The degree of oxidation/reduction was quantitatively calculated based on the integrals of methylene peaks at 2.73–3.16 ppm and 3.11–3.60 ppm. FTIR data exhibit a decrease of infrared absorption band at 1030 cm−1 (νSO band, Fig. 6a) after reduction, consistent with the 1H NMR analysis. GPC traces demonstrated that the polypeptide backbones and ester groups on the side-chains remain intact under the oxidation or reduction reaction condition (Fig. 6b and S11†).
It is generally accepted that the thermal-responsive properties of the polymer in water is mainly governed by hydrophilic/hydrophobic balance of polymer subunits.7,9 As the thioether groups are oxidized into sulfoxide or sulfone groups, the molecular polarity and hydrophilicity of the system can be largely increased. We therefore investigated the influence of redox properties on solubility and CPs of PPLG130-g-EGx. PPLG130-g-EG2, insoluble in water at room temperature, shows reversible LCST behavior with the CP of 32 °C after partially oxidation (Fig. S15a†). The degree of oxidation was calculated to be 19% by 1H NMR. Fully conversion to PPLG130-g-OEG2 and PPLG130-g-O2EG2 remains soluble in water at elevated temperature up to 85 °C without showing thermal-responsive properties. This is possibly due to the increased hydrophilicity of polypeptides upon fully oxidation into sulfoxides or sulfones. Fig. 7a shows plots of transmittance versus temperature for PPLG130-g-EG3 at different oxidizing degree. As the OEG side-chain length increased to 3 and 4, the polypeptides are completely soluble in water without showing observable thermal-responsive behaviors up to water boiling point (Fig. S15b†). Similarly, the oxidation of PPLG65-g-EG3 and PPLG65-g-EG4 disrupt the thermal-responsive property of the polymers (Fig. S14†). Reduction of sulfoxide groups can recover thermal-responsive property as the redox reaction between sulfoxide and thioether groups is reversible. As shown in Fig. 7a, PPLG130-g-*EG3 after reduction by thioglycolic acid, denoted as PPLG130-g-*EG3 (1st), regains reversible LCST behavior. The CP of PPLG130-g-*EG3 (1st) is determined to be 38 °C, located between the CP of PPLG130-g-EG3 (25 °C) and the CP of PPLG130-g-OEG3 (above 80 °C), indicative of partial reduction of sulfoxide groups.7 We further calculated the degree of reduction to be 85% by 1H NMR. Similarly, reducing products PPLG130-g-*EG4, PPLG65-g-*EG3 and PPLG65-g-*EG4 also show reversible LCST behavior (Fig. S14 and S15†). To investigate the repeatability of redox process, we performed two oxidation/reduction cycles for PPLG130-g-EG3. It is observed that the reduction product in the second cycle, denoted as PPLG130-g-*EG3 (2nd), still show reversible LCST behavior with a CP at 52 °C, indicative of reversible redox properties. It is estimated that 75% of sulfoxide groups in PPLG130-g-*EG3 (2nd) are reduced to thioethers by 1H NMR.
It is known that the inherent secondary structures of polypeptides can largely influence the thermal-responsiveness.26,29 We therefore investigated the effects of secondary structures on redox properties of PPLGn-g-EGx using FTIR and CD spectroscopy. FTIR was first used to study the conformation in solid state at room temperature (Fig. 7b, S12b and S13b†). Generally, PPLG130-g-OEG3, PPLG130-g-O2EG3 and PPLG130-g-*EG3 show similar infrared absorption bands to that of PPLG130-g-EG3 with strong absorbance at 1653 cm−1(amide I) and 1550 cm−1 (amide II), indicative of predominant α-helical structures. We thus assume that the conformation remains during the redox process. The helicity of the polymers was determined by CD spectra. Fig. 7c shows that PPLG130-g-EG3, PPLG130-g-OEG3 and PPLG130-g-*EG3 exhibit similar CD traces with nearly 100% helicity, summarized in Table S3.† This is not surprised as the sulfoxide functionality is not sufficient to destabilize the ordered helical conformation.9,25 Further oxidation into PPLG130-g-O2EG3 results in the decrease of helicity from 100% to 69%. The possible reason is that conversion from thioethers to sulfones generally destabilizes the α-helices.38,41 The similar results were also observed in PPLG130-g-O2EG2, PPLG130-g-O2EG4, PPLG65-g-O2EG3 and PPLG65-g-O2EG4 (see ESI†). Note that the CD data are not quite in agreement with the FTIR results, possibly because FTIR measurements were performed in dry state. In the case of CD measurements, which were recorded in aqueous solution, the steric effects of solvated OEG side chains may dominate that result in destabilization of the helical conformation.25
Footnote |
† Electronic supplementary information (ESI) available: GPC, CD, UV-vis, FTIR and NMR spectra of the samples. See DOI: 10.1039/c6ra17427b |
This journal is © The Royal Society of Chemistry 2016 |