Article pubs.acs.org/Macromolecules
A Perfect Match: Fast and Truly Random Copolymerization of Glycidyl Ether Monomers to Thermoresponsive Copolymers Silke Heinen, Simon Rackow, Andreas Schaf̈ er, and Marie Weinhart* Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Takustr. 3, 14195 Berlin, Germany S Supporting Information *
ABSTRACT: Thermoresponsive and highly biocompatible poly(glycidyl ether) copolymers of glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) with adjustable molecular weight and defined end groups are synthesized by a monomer-activated anionic ring-opening polymerization with NOct4Br as initiator and i-Bu3Al as activator. In contrast to a conventional oxyanionic (nonactivated) copolymerization, higher molecular weights and a truly random incorporation of the monomers are accomplished. The monomer reactivity ratios were determined by the Kelen−Tüdõs approach to be rGME = 0.98 and rEGE = 0.95. The thermoresponsive properties of these copolymers with varying molecular weight were characterized by UV−vis transmittance and dynamic light scattering. Conformational changes of the copolymer during the phase transition on the molecular level were studied by 1H and 13C NMR spectroscopy in D2O and revealed only a partial dehydration during the collapse of the copolymer affecting both side chains and polymer backbone.
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INTRODUCTION Water-soluble linear poly(glycidyl ether)s such as polyglycerols or derivatives thereof are of particular interest due to their inherent biocompatible properties.1−4 Some of these watersoluble poly(glycidyl ether)s derived from glycidyl methyl ether (GME), ethyl glycidyl ether (EGE), and ethoxyethyl glycidyl ether (EEGE) monomers exhibit thermoresponsive properties in water. The phase transition temperature of a 1 wt % aqueous solution of these homopolymers (3 kDa) is located at 14.6 °C for p(EGE), 40.0 °C for p(EEGE), and 57.7 °C for p(GME) and thus outside the physiologically relevant temperature regime.5 Random copolymerization is a general approach to adjust the phase transition temperature to a desired target temperature for designated applications. This, however, can be a challenging task when comonomers are incompatible due to vastly different reactivity ratios, which leads to a compositional gradient formation along the growing polymer chain. In extreme cases the resulting properties of such copolymers then resemble more the properties of block than random copolymers. In the case of alkyl glycidyl ethers, GME-containing linear copolymers (random or gradient) with ethylene oxide (EO),6 glycidyl methacrylate (GMA),7 and EGE8 as the comonomer have been © XXXX American Chemical Society
prepared successfully. For the comonomer pair GME and EO the adjustable cloud point temperature (CPT) regime of the resulting copolymer at 5 mg mL−1 in water ranged from 55 to 98 °C.6 In addition, a decrease in CPT was observed with increasing molecular weight of the copolymer. The CPT range of copolymers from GME and GMA was tuned from approximately 60 to 40 °C by incorporation of the more hydrophobic GMA comonomer.7 For copolymers of GME and EGE the cloud point temperature is finely tunable by the comonomer composition between 15 and 60 °C and thus spans the physiologically relevant temperature regime.8,9 Low molecular weight (5 kDa) copolymers based on GME and EGE exhibit a strictly linear relation between CPT and comonomer ratio, a sharp phase transition, and no hysteresis of the CPT at concentrations of 2.5 mg mL−1, as determined by UV−vis transmittance measurements.8 Because of their excellent biocompatibility and CPT in the physiological range, thermoresponsive poly(glycidyl ether)s like p(GME-ran-EGE)s are well suited for biomedical applications Received: September 1, 2016 Revised: December 5, 2016
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DOI: 10.1021/acs.macromol.6b01904 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules such as surface coatings for temperature-induced cell sheet fabrication9,10 or smart hydrogels.8 The synthesis of low molecular weight (up to 5 kDa) p(GME-co-EGE)s is generally accomplished by a nonactivated11 anionic ROP (Scheme 1).8−10 Schmalz and co-workers determined the reactivity Scheme 1. Synthesis of Linear Thermoresponsive P(GMExco-EGEy) via (a) Monomer-Activated and (b) Nonactivated Anionic ROP; Chain Transfer (c), a Typical Side Reaction in the Nonactivated Approach, Results in Allylic Head Groups and a Limitation of the Accessible Molecular Weight
Figure 1. Representative GPC traces of samples taken at different time points during the course of the monomer-activated polymerization of 1 (H, 1:3) performed in toluene at 0 °C with [Act]/[Ini] = 4.
ROP of glycidyl ethers are thus overcome, which offers access to high molecular weight poly(glycidyl ether)s of up to 100 kDa in short reaction times of only a few hours, suppressing both side reactions and nonuniform functional end groups.16−18 The monomer-activated ROP mechanism requires an excess of the aluminum compound with respect to the ammonium salt ([i-Bu3Al]/[NOct4Br] > 1) in order to yield high monomer conversions at high speed.17,19,20 Moreover, the reactivity ratios of comonomers can be balanced by increasing the activator-to-initiator ratio.7 Detailed mechanistic investigations on the monomer-activated anionic ROP of glycidyl ethers in general are reported in the literature.6,7,21 Herein, we demonstrate the fast and truly random copolymerization of GME and EGE by this monomer-activated anionic ROP approach. It yielded copolymers with defined functional end groups, adjustable molecular weightswithout molecular weight restrictions as observed in the nonactivated approachand narrow polydispersity indices (PDIs) within a convenient time frame.
ratios of GME and EGE for this nonactivated copolymerization at 50 °C with t-BuOK as initiator, finding a weak preferential incorporation of GME into the copolymer at the beginning of the polymerization and thus a slight compositional gradient along the polymer chain.8 A more severe drawback of this kind of nonactivated anionic ROP of glycidyl ethers, besides the gradient comonomer composition, is chain transfer reactions as indicated in Scheme 1. The latter lead to nonuniform end-groups with up to 50% allylic end groups at high reaction temperatures of 120 °C.12 This drastically limits the accessible molecular weight and unity of the end groups of the obtained poly(glycidyl ether)s.13 Yet chain transfer reactions can be reduced by a decrease in reaction temperature to 60 °C, which is accompanied by a very slow polymerization kinetic and incomplete conversion even after 48 h.12 Alternatively, a monomer-activated anionic ROP of glycidyl ether monomers (Figure 1a), first reported by Carlotti, Deffieux, and co-workers in 2004, can be performed at lower temperatures (−30 °C to rt).14 The activation energy for the ring-opening of the epoxide monomers during such polymerizations is reduced by the presence of a monomer activating Lewis acid. Triisobutylaluminum (i-Bu3Al) is typically used as an activator, and a bulky tetraalkylammonium salt (e.g., NOct4Br) serves as the initiator. The formation of an aluminate complex between the generated alcoholate through initiation and the activator reduces the reactivity of the active chain end. This complex formation yields control over the polymerization and the chain transfer reaction induced by proton abstraction, which otherwise leads to allylic head groups.15 Replacing alkali metals as counterions of the growing oxyanionic chain end by bulky ammonium counterions (NR4+) increases the polymerization rate constant about 5−50 times, depending on the respective counterion. At the same time hydride initiation is significantly reduced, which results in almost quantitative initiation of the ammonium salt counterion, e.g., halides or azides.15 Hence, major drawbacks of the nonactivated anionic
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EXPERIMENTAL SECTION
Materials. All chemicals and solvents were purchased from SigmaAldrich (Steinheim, Germany) and used without further purification unless stated otherwise. GME and EGE were purchased from TCI GmbH (Eschborn, Germany), dried and distilled over CaH2, and stored over 3 Å molecular sieve purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Triisobutylaluminum (i-Bu3Al, 1.1 M in toluene), dry dimethylformamide (DMF, 99.8%), and benzophenone (99%) were purchased from Acros Organics (Geel, Belgium). Tetrahydrofuran (THF) and diethyl ether (Et2O) were supplied by VWR Chemicals (Fontenay-sous-Bois, France or Leuven, Belgium), and the latter was distilled prior to use in order to remove the stabilizer. Magnesium sulfate (99%) and sodium hydrogen carbonate were supplied by Grüssing GmbH (Filsum, Germany); sodium azide was purchased from Merck KGaA (Darmstadt, Germany). Toluene predried via the solvent system MB SPS-800 from MBraun GmbH (Garching, Germany) and 1,2-dimethoxyethane (DME) were both refluxed with elemental sodium and distilled on 3 Å molecular sieve prior to use. Prewetted regenerated cellulose dialysis tubes (molecular weight cutoff (MWCO) 1000 and 3500 g mol−1, Spectra/Por 6 Dialysis membrane) from Spectrumlabs were purchased from Carl Roth GmbH + Co. KG. Methods. 1H and 13C NMR spectra were recorded on a Bruker Avance 3 operating at 700 MHz or on a Jeol ECX at 400 and 500 MHz, respectively. Concentrations of 100 mg mL−1 in CDCl3 were applied for routine spectra acquisition, and 10 mg mL−1 in D2O was B
DOI: 10.1021/acs.macromol.6b01904 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
Table 1. Specification of Poly(glycidyl ether) Copolymers Synthesized by Both Nonactivated and Monomer-Activated Anionic ROP formulaa p(GME72-ran-EGE193) Br/OH p(GME126-ran-EGE122) Br/OH p(GME175-ran-EGE58) Br/OH p(GME6-ran-EGE17) N3/OH p(GME5-co-EGE14) MeO/OH p(GME23-ran-EGE73) Br/OH p(GME62-ran-EGE181) Br/OH
polymerb 1 2 3 4 5 6 7
(H) (H) (H) (L) (L) (M) (H)
Mn,expc (g mol−1)
Mn,thc (g mol−1)
PDIc
GME:EGEd
[Act]/[Ini]
26000 24000 21000 2200 1800 9000 24000
25000 25000 25000 2000 2000 10000 25000
1.08 1.07 1.08 1.22 1.23 1.23 1.05
1.0:2.7 1.0:1.0 3.1:1.0 1.0:3.1 1.0:3.1 1.0:3.2 1.0:2.9
4 4 4 1.5 −e 4 6
a
Both terminal groups of the copolymers are indicated after the formula, e.g., Br/OH. bAnnotations: L, M, and H indicate low, medium, and high molecular weight polymers, respectively. cMn values and PDIs were determined by GPC measurements of the respective polymers in THF with polystyrene standards. dComonomer ratios were obtained from 1H NMR spectra. eNonactivated polymerization at 110 °C. used for temperature-dependent resolution experiments. Temperaturedependent NMRs were acquired 3 min after the air stream, which surrounded the NMR sample tube, and reached the target temperature. This ensured the equilibrium state within the sample as determined from time-dependent measurements (data not shown). Chemical shifts are reported in δ (ppm) and referenced to the respective solvent. Gel permeation chromatography (GPC) was conducted on an Agilent 1100 Series instrument in THF as the eluent solvent with concentrations of 3.5 mg mL−1 and a flow rate of 1 mL min−1 at 25 °C. Three columns PLgel mixed C with the dimensions 7.5 × 300 mm and particle size 5 μm from Agilent (Waldbronn, Germany) were used in-line with a refractive index detector. Calibration was performed with polystyrene standards from PSS (Mainz, Germany). IR measurements were conducted on a Nicolet Avatar 32 FT-IR with Smart iTR accessory. DLS measurements were performed on a Malvern Zetasizer Nano-ZS analyzer (Malvern Instruments) equipped with a 4 mW He−Ne laser (λ = 633 nm), at concentrations of 10 mg mL−1 in MQ water at 10, 20, and 37 °C and in ethanol at 20 °C. Measurements were performed in PS-latex cuvettes with a refractive index (RI) of 1.590 and low absorption of 0.01, and each probe was equilibrated for 180 s (10 and 20 °C) or 20 min (37 °C) prior to the measurement. UV/vis transmittance measurements at λ = 500 nm were recorded on a PerkinElmer Lambda 950 UV/vis spectrometer with a PTP 6 Peltier temperature programmer (PerkinElmer). Measurements were performed in MQ water at varying concentrations between 0.05 and 20 mg mL−1 at heating rates of 0.5 °C min−1 with recording of data points every 0.2 °C. The temperature-dependent transmittance of the aqueous polymer solution was measured for at least three heating and cooling cycles per sample. The CPT was defined as the temperature at the inflection point of the normalized transmittance versus temperature curve. Polymer Synthesis. Low (L) molecular weight (2 kDa), linear copolymers of GME and EGE with a comonomer ratio of 1:3 (GME/ EGE) and potassium methanolate as the initiator were synthesized by a nonactivated anionic ring-opening copolymerization as published previously.2,10 Medium (M) and high (H) molecular weight (9 kDa, ∼24 kDa) copolymers were synthesized by anionic, monomeractivated ROP according to the literature with slight modifications using tetraoctylammonium bromide (NOct4Br) as initiator and i-Bu3Al as activator.7,17,21 General Procedure 1 (GP 1). Monomer-Activated Anionic ROP. NOct4Br was dried at 110 °C under high vacuum (HV) and dissolved in freshly distilled, dry toluene. The monomers GME and EGE were added to the solution and cooled in an ice bath. By the addition of iBu3Al (1.1 M in toluene) at 0 °C the polymerization was started. After 15 min at 0 °C the reaction temperature was allowed to raise to room temperature, while the reaction mixture was stirred for at least an additional 45 min. After quenching with 2 mL of water, two spoons of magnesium sulfate were added in order to capture the hydrolyzed aluminum compound together with traces of water. After filtration of the solid and concentration of the filtrate under reduced pressure, the raw product was dissolved in Et2O in order to precipitate residual aluminum hydroxide and the majority of the tetraoctylammonium
salts. In the cold, the mixture was centrifuged and the supernatant was decanted. The latter two steps were repeated, after storing of the supernatant in the fridge overnight to provoke further precipitation, followed by concentration of the purified supernatant. For further purification the product was dialyzed in methanol in order to remove traces of impurities. Afterward, the pure polymer solution was concentrated under reduced pressure and dried in HV to yield a clear and viscous oil. Bromide, azide, or methoxide initiated polymers with one terminal bromide/azide/methoxide and one secondary hydroxyl group are indicated by the annotation Br, N3, or MeO/OH. Purified polymers were characterized by 1H and 13C NMR, GPC, and IR measurements. For kinetic and reactivity ratio studies samples (0.5−1.0 mL) were taken every few seconds within the first 10 min after the start of the polymerization, induced by addition of i-Bu3Al, and were quenched immediately after sampling with ethanol (1 mL). All volatile components (toluene, ethanol, unconsumed monomers) have been removed by rotary evaporation and HV drying of the crude residue. 1H NMR and GPC measurements of these samples were performed without further purification. Detailed synthetic methods as well as full characterization of the polymers are given in the Supporting Information.
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RESULTS AND DISCUSSION On the basis of preceding studies of Deffieux, Carlotti, and coworkers,7,17,21 we performed copolymerizations of GME and EGE with [i-Bu3Al]/[NOct4Br] ≥ 4 in toluene at low temperature (0 °C) in order to prepare thermoresponsive polymers with adjustable molecular weight in a broader range than currently accessible via an alternative nonactivated polymerization approach. Additionally, kinetic and reactivity ratio studies were performed in order to determine the rate of conversion and the comonomer reactivity ratios which allowed us to deduce the molecular structure of the copolymer. The monomer-activated anionic ROP was the method of choice because it yields copolymers with narrow molecular weight distribution and defined end groups, both desirable properties for sophisticated applications. In Table 1 specifications of the prepared copolymers are summarized. Kinetics and reactivity ratios were studied on copolymers with high molecular weight (approximately 20−25 kDa), as their polymerization proceeded within a reasonable time frame to allow sampling even in a low conversion regime. To investigate the relation between CPT and molecular weight as well as concentration, we synthesized copolymers with molecular weights of 2, 9, and 24 kDa, since it is known from other thermoresponsive polymers that molecular weight dependence is more pronounced for molecular weights below 30 kDa.22,23 Previously, the CPT of p(GME-co-EGE) with a comonomer ratio of 1:3 and a molecular weight of 2 kDa was found to be within the physiologically relevant range at 28.1 ± 0.7 and 33.6 ± 0.2 °C for the respective concentrations C
DOI: 10.1021/acs.macromol.6b01904 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules of 5 and 1 mg mL−1.9 Starting from there, we decided to keep the comonomer ratio fixed at 1:3 (GME:EGE) for the investigation of the impact of varying molecular weights and concentrations. For comparison of the polymerization methods the lowest 2 kDa copolymer was accessed by both the monomer-activated and nonactivated anionic ROP. The obtained comonomer ratio in the resulting polymers matched the monomer feed ratios set to 1:3, 1:1, and 3:1. Molecular weights were close to the theoretically expected values of 2, 10, and 25 kDa with narrow PDIs ranging from 1.05 to 1.23. The molecular weight distribution was found to be narrower for polymers with molecular weights around 25 kDa compared to medium and low molecular weight polymers. This is a general observation we made with this type of monomeractivated polymerization. An explanation might be an initial higher concentration of activated monomer when targeting low molecular weight polymers compared to higher molecular weight polymers. The higher starting concentration of activated monomers is associated with a highly exothermic reaction at the beginning of the polymerization which might yield more transfer reactions and thus results in a broader dispersity. Low molecular weight polymers could be achieved equally well by both monomer-activated and nonactivated anionic ROP. The nonactivated one, however, is beneficial in the way that it avoids tetraalkylammonium hydroxide removal after quenching, which can be quite challenging due to its amphiphilic nature. Suggested procedures in the literature to remove the bulky, amphiphilic salt from poly(glycidyl ether)s rely on either column chromatography,6 dialysis,6 filtration,6 or precipitation of the polymer in sodium hydroxide solution.24 The removal of the aluminum activator has not been addressed explicitly so far, which is a severe problem in the case of water-soluble polymers determined for biomedical applications. Our purification sequence includes complete hydrolysis of triisobutylaluminum by quenching with a small amount of water and vigorous stirring for at least 30 min and addition of magnesium sulfate to capture the evolving aluminum hydroxide together with water, followed by filtration of the solids. Residual traces of aluminum hydroxide and/or the initiator salt are precipitated in diethyl ether and removed by centrifugation. Polymers of high molecular weight can also be quenched by addition of methanol and subsequent dialysis with 3.5 kDa MWCO membranes in order to remove both activator and initiator. Reactivity Ratios of the Comonomers GME and EGE Polymerized by the Monomer-Activated Approach. Reactivity ratios of GME and EGE in the monomer-activated copolymerization were calculated from data sets obtained during the synthesis of 1, 2, and 3 by the Kelen−Tüdõs, Fineman−Ross, and Mayo−Lewis approach. All three mathematical approaches rely on the assumption of a constant monomer feed ratio and thus are only valid for very low conversions below 10%. Therefore, samples were taken every few seconds during the first 10 min of the course of the polymerizations in order to investigate the progress of conversion. The latter is defined as the ratio of the molecular weight of the polymer at an early time point to the molecular weight at the 1 h time point, which is set as 100% conversion. In Figure 1, representative GPC traces of the crude samples which contain polymer, initiator, and activator are plotted indicating a fast progress of the polymerization of 1 (H, 1:3) within the first 10 min.
The GPC traces of polymerizations 2 (H, 1:1) and 3 (H, 3:1) are compiled in Figure S1. Within the first 10 min of the copolymerization 70%−78% conversion was reached for polymers 1, 2, and 3, while reactions were completed within 1 h, as indicated by the matching theoretical and experimental molecular weights. Very low conversions (
