Well-Defined Poly(γ-benzyl-l-glutamate)-g-Polytetrahydrofuran

Aug 14, 2014 - The synthesis of well-defined graft copolymers of poly(γ-benzyl-l-glutamate)-g- polytetrahydrofuran, PBLG-g-PTHF, has been achieved vi...
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Well-Defined Poly(γ-benzyl‑L‑glutamate)‑g‑Polytetrahydrofuran: Synthesis, Characterization, and Properties An-ru Guo, Wei-xi Yang, Fan Yang, Rui Yu, and Yi-xian Wu* State Key Laboratory of Chemical Resource Engineering, Key Laboratory of carbon fiber and functional polymers (Ministry of Education), Beijing University of Chemical Technology, Beijing 100029, China

ABSTRACT: The synthesis of well-defined graft copolymers of poly(γ-benzyl-L-glutamate)-g- polytetrahydrofuran, PBLG-gPTHF, has been achieved via controlled termination of living PTHF branch chains with −NH− functional groups along PBLG macromolecular backbone. The PBLG backbone with different molecular weights (Mn = 2000−45000 g·mol−1) were prepared by anionic ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydrade (BLG-NCA). Living PTHF chains with predictable chain length (Mn = 720−7000 g·mol−1) were prepared by living cationic ring-opening polymerization of THF with methyl triflate (MeOTf) as an initiator. The grafting efficiency (GE) of living PTHF chains onto PBLG backbone via controlled termination reached to near 100%. The grafting density (GD) along PBLG backbone and average number of PTHF branches (Nb,PTHF) in PBLG-g-PTHF graft copolymers could be mediated by changing the molar ratio of living PTHF chains to −NH− functional groups. Circular dichroism (CD) and FTIR spectra show that some of the graft copolymers maintain α-helical structure from PBLG, and the strength of CD signals for α-helical structure of the graft copolymers also decreased with increasing GD. The crystallization degree and spherulitic growth rate of the PBLG-g-PTHF graft copolymers decreased with increasing GD. The obvious phase separation and reticular state of aggregation morphology in PBLG-g-PTHF graft copolymers could be observed. PBLG-g-PTHF graft copolymers have no cytotoxicity and even conducive for cell survival. These graft copolymers had extremely low bibulous rate, and all the water absorption ratios were kept around 1.02 to maintain the shape and dimensional stability.

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INTRODUCTION

PALG, PBLG-b-PEG-b-PBLG, and PBLG-b-PTHF-b-PBLG block copolymers could be synthesized via anionic ring-opening polymerization of γ-alkyl-L-glutamate N-carboyanhydride (ALG-NCA) or γ-benzyl-L-glutamate N-carboyanhydride (BLG-NCA) by using monofunctional or bifunctional aminoterminated PEG and PTHF as macroinitiators.22−25 The dendron-like PBLG/line PEO block copolymer was also synthesized by using the propargyl focal point dendron Dm having 2m terminal primary amines groups as a multifunctional initiator for ring-opening polymerization of BLG-NCA to produce “clickable” dendron-like PBLG, followed by coupling with azide-terminated PEO.26 On the other hand, macromolecules with different architectures containing hard backbone and soft branches have been built for the advanced polymer materials with an excellent integrated performance. However, there are few reports on the graft copolymers of

Polypeptides behave various conformations, such as α-helix and random coil, to form morphological structures and possess excellent properties, such as low toxicity, biodegradability and biocompatibility.1−10 As well-known, polypeptide with excellent tissue and blood compatibilities can be used as biomaterials for clinical applications, such as drug delivery systems, surgical sutures. Recently, the copolymers consisting of polypeptide segments and other polymer segments, such as polyether, polyester, chitosan, polysiloxane etc. have received a great interest for their rich morphological structures4,11−18 and potential biomedical applications.11,19−22 The most important copolymers are those of polypeptide with polyether, such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO) and polytetrahydrofuran (PTHF), for their good stability in aqueous and organic solvents, excellent biocompatibility, immunogenicity and ease of excretion from living organisms.7 The block copolymers of polypeptide with polyether were synthesized via anionic ring-opening polymerization by the initiation from functional polyether macroinitiators. PEG-b© 2014 American Chemical Society

Received: May 22, 2014 Revised: August 3, 2014 Published: August 14, 2014 5450

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93−94 °C.32−34 The anionic ring-opening polymerization of BLGNCA was conducted by using triethylamine as an initiator in chloroform at 25 °C for 72 h. The reaction mixture was kept under nitrogen atmosphere until the polymer was isolated by precipitation in ethanol. PBLG product was filtrated and dried under vacuum at 30 °C. Synthesis of Living Polytetrahydrofuran (PTHF) Chains via Living Cationic Ring-Opening Polymerization. Living PTHF chains were prepared by living cationic ring-opening polymerization of THF. THF was added into chloroform with stirring under nitrogen atmosphere, and then MeOTf was added as an initiator at 25 °C. After a predetermined time, PTHF living chains with designed molecular weights and narrow MWD were obtained in the reaction systems, and the concentration of PTHF living chains ([PTHF+]) was in the range of 0.0154−0.314 mol·L−1 according to the initiator concentration. Synthesis of PBLG-g-PTHF via Controlled Termination. The above PBLG solution in CHCl3 was added into the living PTHF solution in CHCl3 at 25 °C by setting the molar ratio of living PTHF chains to amino bonds along PBLG macromolecular chains from 0.01−0.9. The above polymer solutions were dialyzed in CHCl3 with concentration of 0.02 mol·L−1 through dialysis bag for 2 days to remove the possible unreacted homopolymer (PTHF or PBLG) chains. The dialyzate containing the possible unreacted homopolymer (PTHF or PBLG) was further concentrated and then detected by 1H NMR characterization. Finally, the resulting PBLG-g-PTHF graft copolymers were purified by precipitation into frozen diethyl ether (−20 °C). Characterization. Melting points (mp) were determined with X-4 electrothermal digital melting point apparatus (Beijing Taike Company). The number-average molecular weights (Mn), weight-average molecular weight (Mw) and molecular weight distribution (MWD, Mw/Mn) of polymers were measured by gel permeation chromatography (GPC) using a Waters 1515 isocratic HPLC pump connected to four Waters Styragel HT3, HT4, HT5, and HT6 columns and a Waters 2414 refractive index detector at 30 °C. THF was used as a solvent for PTHF and PBLG (PBLG dissolved in THF to form homogeneous solution after 6 h). THF was eluted at a flow rate of 1.0 mL·min−1. The columns were calibrated against standard polystyrene samples. Nuclear magnetic resonance (NMR) spectra of copolymers dissolved in CDCl3 in a 5 mm (o.d.) NMR tube were recorded by Bruker AV400 MHz NMR spectrometers operating at 25 °C. Chemical shifts (δ) were referenced to tetramethylsilane (TMS) as an internal standard in CDCl3 and calculated by using the residual isotopic impurities of the deuterated solvents. All NMR chemical shifts are reported in ppm. The following parameters were used to acquire the 1H NMR spectra: relaxation time = 2 s; acquisition time = 2.73 s; flip angle = π/4; number of transients = 16. The following parameters were used to acquire the 13C NMR spectra: relaxation time = 2 s; acquisition time = 0.54 s; flip angle = π/4; number of transients = 22K. The average grafting efficiency (GE) of PTHF branches grafting onto PBLG backbones was defined as the percentage of living PTHF chains reacted with an −NH− group along PBLG chains and then grafted onto PBLG main chains. If all the living PTHF chains reacted with −NH− groups along PBLG chains, GE reached 100%. GE could be determined according to eq 1

polypeptide with polyether. The graft copolymer could be normally synthesized via three different routes: (1) grafting from, (2) grafting onto, and (3) macromonomer or grafting through.27,28 P(L-glutamates)-g-PEG with very short PEG branches (Mn = 200 g·mol−1) was synthesized via “graft through” by ring-opening polymerization of ethylene glycol-Lglutamate-N-carboxyanhydride.29 PEG absorbs moisture easily, which greatly decreases its mechanical properties and limits its application despite of its excellent biocompatibility, lower antigenicity and nontoxicity.6 PTHF has been widely used in artificial vessel and heart, packing material, and duct due to its biocompatibility, hydrophobicity and safety.30,31 Therefore, the novel class of branched polymeric structures in which the hydrophobic PTHF side chains are covalently linked to a helic PBLG polymer main chain might behave unique property for biomaterials application and to maintain the shape and dimensional stability during their use. However, there is no report on the synthesis, characterization and property of graft copolymer of PBLG-g-PTHF until now. In this paper, the PBLG-g-PTHF graft copolymers were synthesized via “grafting onto” by combination of living cationic ring-opening polymerization of THF with controlled termination of living PTHF chains with −NH− functional groups in structural units along PBLG macromolecular chains. The length of PBLG backbone was designed by BLG-NCA anionic ringopening polymerization. The living PTHF chains with different molecular weights and narrow molecular weight distribution could be synthesized and then grafted onto PBLG main chains to build the graft architecture. The grafting efficiency (GE) was investigated. The grafting density (GD) of PTHF branches was mediated by changing the molar ratio of living PTHF chains to −NH− functional groups in the reaction system. The crystallization behavior, water absorbance and cytotoxicity of PBLG-g-PTHF graft copolymers were also investigated.

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EXPERIMENTAL PART

Materials. Tetrahydrofuran (THF, A.R. Beijing Chemical Company) was dried on sodium wire under reflux in the presence of a small amount of benzophenone until a blue color persisted and then was directly used after distillation. Triethylamine (NEt3, A.R. Tianjin Yongda Company), chloroform (CHCl3, A.R. Beijing Yili Fine Chemicals), n-hexane (n-Hex, A.R. Beijing Yili Fine Chemicals) and ethyl acetate (A.R. Beijing Yili Fine Chemicals) were dried prior to use by distilling from calcium hydride. Methyl triflate (MeOTf, Acros, 98%) was purified by distillation under dry nitrogen. Triphosgene (C.P. Weihai Jinwei Company), γ-benzyl-L-glutamate (BLG, Sigma− Aldrich) and ethanol (A.R. Beijing Yili Fine Chemicals) were used without further purification. Procedures. All the manipulation, reactions, anionic ring-opening polymerizations, and cationic ring-opening polymerizations were carried out under a dry nitrogen atmosphere. Synthesis of Poly(γ-benzyl-L-glutamate) (PBLG) via Anionic Ring-Opening Polymerization. γ-Benzyl-L-glutamate N-carboyanhydride (BLG-NCA) monomer was synthesized by the reaction of γbenzyl-L-glutamate (BLG) with triphosgene under a dry nitrogen atmosphere according to references.32,33 Typically, 8 g of freshly recrystallized BLG was suspended in 160 mL of anhydrous THF at 65 °C, and 4.2 g of triphosgene was added with stirring. A clear pale yellow solution was obtained after 2 h, and nitrogen was bubbled into the solution to remove excess phosgene. The solvent was removed under vacuum at 45 °C, and the resulting solid was dissolved in chloroform. BLG-NCA was precipitated from the solution in CHCl3 by adding n-Hex at 0 °C. BLG-NCA was filtrated and further purified by recrystallization from solution in ethyl acetate/n-hexane (1:2 v/v) three times at 45 °C. The yield of BLG-NCA was 78%. Melting point of BLG-NCA was 91−92 °C, which is similar to the reported data of

GE =

i u mPTHF − mPTHF × 100% i mPTHF

(1)

where miPTHF refers to the initial mass of living PTHF chains before grafting reaction and muPTHF refers to the “un-reacted” PTHF chains after grafting reaction. Here, miPTHF − muPTHF stands for the actual mass of PTHF chains grafted onto PBLG backbone in the copolymer. The grafting density (GD) of PTHF branches was defined as the percentage of PTHF branches on the PBLG backbone based on the total −NH− groups in every structural units along PBLG macromolecular chain in PBLG-g-PTHF copolymers. If all the −NH− groups along PBLG chains reacted with living PTHF chains, GD reached 100%. GD could be determined by 1H NMR characterization 5451

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The cytotoxicity of the graft copolymers was evaluated using the MTT assay in HEK 293 cell lines. They were cultured in Dulbecco’s Modified eagle medium (DMEM), supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 units·mL−1 of penicillin, and 100 μg·mL−1 of streptomycin at 37 °C, under 5% of CO2 and 95% of relative humidity atmosphere. The cells were seeded in a 96-well microtiter plate at a density of 104 cells/well and incubated in 100 μL of DMEM/well for 24 h. The culture media were replaced with fresh culture media containing serial dilutions of polymers, and the cells were incubated for 24 h. Then, 10 μL of sterile-filtered MTT stock solution in PBS (5 mg·mL−1) was added to each well, reaching a final MTT concentration of 0.5 mg·mL−1. After 5 h, the unreacted dye was removed by aspiration. The produced formazan crystals were dissolved in DMSO (100 μL/well). The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at a wavelength of 570 nm. For each sample, the final absorbance was the average of those measured from six wells in parallel. Surface static contact angles were performed detected on a Dataphysics OCA-20 with 5 μL of distilled water droplet being placed on the treated film by a microsyringe, and observed through a traveling microscope fitted with a goniometer eyepiece, All measurements were at 25 °C and were the average of at least six readings at different positions across the surface.

according to eq 2 or eq 3 based on the structural units along PBLG macromolecular chain in PBLG-g-PTHF copolymers.

⎛ A9 ⎞ ⎟⎟ × 100% G D1 = ⎜⎜1 − A 6,6 ′/5 ⎠ ⎝ DD2 =

A 2′ × 100% A 2′ + A 2

(2)

(3)

The average number of PTHF branches (Nb,PTHF) in PBLG-gPTHF graft copolymers was defined as the average number of PTHF chains grafted onto PBLG backbone. Nb,PTHF equaled to the average number of structural units along PBLG chains when GD reached 100%. Nb,PTHF could be determined on the basis of 1H NMR characterization according to eq 4.

Nb,PTHF =

A 2′ × DPPBLG A 2′ + A 2

(4)

where A9 is the integral value of characteristic resonance protons for the proton in the −N(H)−C(H)< group at δ = 8.41. A6,6′ is the integral value of characteristic protons resonances for the phenyl group at δ = 7.28−7.48. A2 and A2′ are the integral values of characteristic resonances at 2.62 and δ = 3.92 for protons of C−H in −N(H)−C(H) < and >N−C(H)< groups in PBLG before and after grafting reaction. DPPBLG is the polymerization degree of PBLG or the average number of structural units along PBLG chain. Fourier transform infrared spectroscopy (FTIR) spectroscopic measurements on polymers were performed on a Nicolet 6700 infrared spectrometer equipped with a DTGS detector at room temperature (25 °C) from 4000 to 400 cm−1 region with a resolution of 1 cm−1. All samples were scrawled on potassium bromide (KBr) flakes for FTIR measurements. The living cationic ring-opening polymerization process of THF was monitored by immersing the diamond tipped attenuated total reflection (ATR) probe (Axiom DMD-270X-LT) into the polymerization solution and the real-time FTIR spectra were recorded by a Nicolet 6700 spectrophotometer. Experimental setup for the in situ monitoring of THF concentration during polymerization has been built as shown in our previous publications.35 FTIR data collection and processing were performed with Nicolet’s OMNIC Series software. The FTIR spectrum of solvent (CHCl3) was chosen as the background for spectrum record. Each spectrum was collected every 32 s by accumulating 32 scans with an instrument resolution of 4 cm−1 over the spectral range of 600−1800 cm−1. Circular dichroism (CD) spectra on polymers in CHCl3 (1 mg· mL−1) were recorded on a Jasco-810 spectropolarimeter at room temperature (25 °C). In each case, a quartz cuvette of 1 mm path length was used. The films of PTHF and PBLG-g-PTHF for polarized optical microscope (POM) or differential scanning calorimeter (DSC) measurement were prepared on slide under a constant pressure of 10 kg·cm−2 for about 30 s from melting temperature to room temperature. The micrograph of POM for films was recorded using Leitz SM-LUX-POL POM. DSC curves on polymers were recorded on polymers by Q200 MDSC (TA Instruments Company) at a scan rate of 5 K·min−1 by modulated-heat procedure under nitrogen. For the method of measuring spherulitic growth rate, the coverslip containing the sample film was then placed under a Leitz SM-LUX-POL polarized optical microscope equipped with a camera system. Pictures of spherulites were taken at different time intervals and recorded, and their radii were measured with ProgRes CapturePro 2.7 software tool. The surface morphology of PTHF, PBLG, and PBLG-g-PTHF films was studied by using an Agilent Technologies 5500 atomic force microscope (AFM, Agilent Technologies Co. Ltd., U.S.) at room temperature in air. The images were obtained by means of tapping mode (height and phase) with a silicon cantilever having a spring constant of 20−30 N/m and a resonating frequency of 320−350 kHz, and the scanning rates varied from 2 to 5 μm·s−1.

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RESULTS AND DISCUSSION Synthesis of PBLG via Anionic Ring-Opening Polymerization of BLG-NCA. The synthetic route for PBLG preparation via anionic ring-opening polymerization of γbenzyl-L-glutamate-N-carboxy-anhydrides (BLG-NCA) using triethylamine (NEt3) as an initiator is shown in Scheme 1. Scheme 1. Synthetic Route for PBLG Preparation via Anionic Ring-Opening Polymerization

The anionic ring-opening polymerizations of BLG-NCA initiated with NEt3 were carried out at 25 °C, and monomer conversion reached ca. 90% within 72 h at various ([M]0[M]t)/[I]0, as shown in Figure 1(a). The number-average molecular weight (Mn) of PBLG increased linearly from 2000 to 45000 g·mol−1 and polydispersity index (Mw/Mn) kept around 2.1 with increasing the feeding molar ratio of BLGNCA to NEt3, as shown in Figure 1b. Synthesis of Living PTHF Chains via Cationic RingOpening Polymerization of THF. The strategy for synthesis of PTHF living chains via living cationic ring-opening polymerization of THF is illustrated in Scheme 2. CHCl3 was selected as a cosolvent for both PBLG and PTHF, providing a condition for the subsequent preparation of PBLG-g-PTHF graft polymers in one pot after completing the living cationic ring-opening polymerization of THF. In situ monitoring of THF polymerization process initiated with MeOTf via attenuated total reflectance (ATR)-FTIR spectroscopy was performed in CHCl3 at 25 °C and FTIR spectrum of CHCl3 was taken as the background. The waterfall plots of real-time FTIR spectra of THF during polymerization in the reaction system with reaction time collected by immersing the diamond tipped ATR probe into the polymerization solution are given in Figure 2. It can be seen from Figure 2 that the intensity of the 5452

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Figure 1. (a) PBLG yield obtained at different ([M]0 − [M]t)/[I]0 ratios; (b) Relationship between Mn or Mw/Mn and ([M]0 − [M]t)/[I]0 for anionic ring-opening polymerization of BLG-NCA at 25 °C using NEt3 as an initiator. [BLG-NCA] = 0.2 mol·L−1, Tp = 25 °C, tp = 72 h, solvent = CHCl3.

Mn of the resulting PTHF theoretically increased with ([M]0 − [M]t)/[I]0 ratio. The living PTHF chains carrying oxonium ion end with desired molecular weights (Mn) from 700 g·mol−1 to 25000 g·mol−1 could be obtained via living cationic ringopening polymerization of THF at different [THF]0 / [MeOTf]0 ratios from 30 to 600 in CHCl3 at 25 °C. Synthesis and Structural Characterization of PBLG-gPTHF via Controlled Termination of Living PTHF Chains with −NH− Groups along PBLG Chains. The strategy for synthesis of PBLG-g-PTHF graft copolymers via controlled termination of living PTHF chains with the secondary amines groups (−NH−) in every structural unit along PBLG macromolecular chains is shown in Scheme 3. The termination of living PTHF chains was carried out by adding the living PTHF chains in CHCl3 into PBLG solution in CHCl3 at 25 °C by setting the various molar ratios of living PTHF to −NH− groups along PBLG from 0.01 to 0.9. The representative FTIR spectra of PBLG, PTHF and PBLG-g-PTHF copolymers with different numbers of PTHF branches (Nb,PTHF) are shown in Figure 4. It can be observed from Figure 4a that the characteristic absorbance at 3350 and 1730 cm−1 are assigned to the stretching vibration for −NH−

Scheme 2. Synthetic Strategy for Living PTHF Chain via Cationic Ring-Opening Polymerization of THF in CHCl3

corresponding signal at 908 cm−1 for THF gradually decreased, indicating that THF concentration gradually decreased during the living cationic ring-opening polymerization. The monomer conversion versus polymerization time for the living cationic ring-opening polymerization of THF in CHCl3 at 25 °C is shown in Figure 2b. The representative GPC profiles of the resulting PTHFs at various [M]0/[I]0 from 600 to 30 and the relationship between Mn or Mw/Mn and ([M]0 − [M]t)/[I]0 before equilibrium are given in Figure 3. It can be seen from Figure 3(a) that all the GPC traces of polymers demonstrate unimodal narrow molecular weight distributions (Mw/Mn ∼ 1.2) and Mn increased with increasing [M]0/[I]0. As shown in Figure 3b,

Figure 2. (a) Waterfall plots (FTIR spectra at 908 cm−1) of THF during its living cationic ring-opening polymerization in CHCl3. The FTIR spectrum of CHCl3 was taken as the background. (b) Conversion−time curve for living cationic ring-opening polymerization of THF in CHCl3. [THF]0 = 9.24 mol·L−1, [MeOTf]0 = 61.6 mmol·L−1, Tp = 25 °C. 5453

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Figure 3. (a) Representative GPC traces of PTHFs obtained at different [M]0/[I]0. (b) Relationship between Mn or Mw/Mn of PTHF and ([M]0 − [M]t)/[I]0. The theoretical line was plotted by setting Mn = 72.1([M]0 − [M]t)/[I]0. [THF]0 = 9.24 mol·L−1, Tp = 25 °C, tp = 60 min, and solvent = CHCl3. The monomer conversions were 38%, 45%, 50%, 54% and 60% for [M]0/[I]0 of 30, 150, 225, 300, and 600 respectively.

Scheme 3. Synthetic Strategy for Preparing PBLG-g-PTHF via Termination of Living PTHF Chains by −NH− Functional Groups along PBLG Macromolecular Chains

and stretching vibration for −CO groups in PBLG respectively. The characteristic absorbance at 1120 cm−1 is attributed to the antisymmetric stretching vibration for C−O− C band in PTHF. It can be clearly seen from Figure 4b that a new characteristic absorbance at 1030 cm−1 assigned to the stretching vibration for the −N< groups36 generated after termination via reaction of living PTHF chains with −NH− groups along PBLG macromolecular chains. The characteristic absorbance at 3350 cm−1 for −NH− gradually decreased while the characteristic absorbance at 1030 cm−1 for −N< groups simultaneously enhanced with increasing the molar ratio of living PTHF chains to −NH− functional groups from 0.06 to 0.53. The plots of absorbance at 1030 cm−1 for the −N< group and at 3350 cm−1 for the −NH− group versus the molar ratio of PTHF to −NH− is quantitatively given in Figure 4c. It can be clearly observed from parts b and c of Figure 4 that the absorbance at 1030 cm−1 for the −N< group in copolymers increased while the absorbance at 3350 cm−1 for −NH− left in PBLG macromolecular backbone decreased with increasing the ratio of [PTHF+]/[−NH−] from 0.06 to 0.53. The results of FTIR analysis show that the controlled termination of living PTHF chains by the secondary amine groups (−NH−) was successfully implemented, which is similar to the result reported by Richards and Goethals that living PTHF chains could be terminated by primary amine, secondary amine and tertiary amine.37 Besides, the absorbance peaks at 1655 and 1548 cm−1 assigning to α-helix structure12 were still maintained in some of the PBLG-g-PTHF graft copolymers. The representative circular dichroism (CD) spectra for PBLG207, PBLG207-g52-PTHF45, PBLG207-g103-PTHF45 and

PTHF45 are demonstrated in Figure 5. It can be seen from Figure 5 that there was no CD signal for PTHF and PBLG207 adopted α-helical structure at 238 nm, which is similar to the reports in references.3,38−40 The CD spectra of PBLG207-g52PTHF45 and PBLG207-g103-PTHF45 in CHCl3 solutions show a minimum at 238 nm, suggesting that PBLG backbone still kept α-helical secondary structure in the PBLG-g-PTHF graft copolymers, which is consistent with the above-mentioned FTIR results. The strength of the peak at 238 nm gradually decreased with an increase in grafting density (GD) or average number of PTHF branches (Nb,PTHF) in the PBLG-g-PTHF graft copolymers. NMR characterization on PBLG-g-PTHF graft copolymer was further performed to evaluate the detailed microstructure. The representative 1H NMR spectrum of PBLG-g-PTHF graft copolymer in CDCl3 is given in Figure 6. The characteristic resonances at δ = 1.62 (4H, 2 × −CH2−, PTHF) and δ = 3.42 (4H, −CH2−O−CH2−, PTHF) are for PTHF segments.41 The characteristic resonances at δ = 3.92 (1H, −CH−CO−, PBLG) and δ = 5.05 (2H, −CO−CH2−, PBLG) are assigned to the corresponding protons along PBLG chain.33 The weak proton signal 7″ at δ = 2.13 attributed to the connecting point (2H, −CH2−NN−C(H)