Core Degradable Star RAFT Polymers: Synthesis, Polymerization, and

Nov 21, 2013 - Kinetic study of RAFT homopolymerization and copolymerization in emulsion. Jianying Ma , Huixuan Zhang. Iranian Polymer Journal 2015 24...
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Core Degradable Star RAFT Polymers: Synthesis, Polymerization, and Degradation Studies Julien Rosselgong,†,* Elizabeth G. L. Williams,† Tam P. Le,† Felix Grusche,‡ Tracey M. Hinton,‡ Mark Tizard,‡ Pathiraja Gunatillake,† and San H. Thang†,* †

CSIRO Materials Science and Engineering, Bag 10, Clayton South Victoria 3169, Australia Australian Animal Health Laboratory, CSIRO Animal, Food and Health Sciences, 5 Portarlington Road, Geelong, Victoria 3220, Australia

‡

ABSTRACT: This study reports on the synthesis and characterization of a novel four-arm reagent and its use in the synthesis of core degradable star polymers and block copolymers using the reversible addition−fragmentation chain transfer (RAFT) polymerization process. The star RAFT polymers prepared from methyl methacrylate (MMA), styrene (ST) and N,N-dimethylacrylamide (DMA) were characterized by size exclusion chromatography (SEC). The PMMA star polymer was further polymerized with poly(ethylene glycol) methyl ether methacrylate (POEGMA8−9) to produce a star block copolymer. The core degradability of the star polymers (PMMA40)4, (PMMA80)4, (PS40)4, (PS80)4 and star block copolymer P[MMA46-b-(POEGMA8−9)46]4 under reductive conditions to cleave the disulfide linkages was demonstrated. The results demonstrated the complete degradation of the star polymer to produce linear polymer and confirmed the near equal degree of polymerization in each of the arms. The star polymer (PDMA80)4 degraded slowly under acidic and enzymatic conditions demonstrating that the ester linkage can also be degraded.

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INTRODUCTION Free radical polymerization is one of the most widely used processes for the production of high molecular weight polymers.1 The emergence of techniques for implementing reversible deactivation radical polymerization (RDRP),2 which serve to impart living characteristics to the process, has provided a new set of tools for polymer chemists that allows the control over the polymerization process while retaining much of the versatility of conventional radical polymerization. The polymerization techniques that are receiving most attention are nitroxidemediated polymerization (NMP),3 atom transfer radical polymerization (ATRP),4 and reversible addition−fragmentation chain transfer (RAFT).5 RAFT polymerization,5 is arguably the most versatile method for providing living characteristics to radical polymerization. This method is now well-established for providing exceptional control over molecular weight, molecular weight distribution, composition and architecture.6−9 The process can be applied to most monomers that are polymerizable by free radical polymerization and offers a convenient route to well-defined homo, gradient, diblock, triblock, and star polymers as well as more complex architectures that include microgels and polymer brushes. Moreover, RAFT polymerization, like conventional radical polymerization, is tolerant to a range of functional groups and solvent (it can be carried out in aqueous media); it is compatible with bulk, solution, suspension, and emulsion processes.5−12 Figure 1 illustrates the versatility of the RAFT process in the design and synthesis of different polymer architectures. For Published 2013 by the American Chemical Society

these, the RAFT agent is the crucial ingredient that allows a living character to this polymerization process. Recently, RAFT polymerization has been expanded to the use of universal (switchable) RAFT agents.13 This allows the synthesis of block copolymer containing both more activated monomers (MAMs) [e.g., (meth)acrylates, styrene, and (meth)acrylamides] and less activated monomers (LAMs) (e.g., vinyl acetate, N-vinylpyrrolidone), previously unachievable.14−18 In the course of our ongoing research into the RAFT-derived polymers as efficient nonviral carriers for siRNA delivery, we have developed a series of linear ABA triblock copolymers19 to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability, and gene silencing. Results of this study indicated that the length of the central cationic block is the key structural parameter determining cytotoxicity and gene silencing efficiency.19 To further explore siRNA delivery with different polymer architectures, we have extended our previous study to include cationic star−copolymer architectures with degradable cores. We envisage that such star copolymers should degrade upon entry into the cell to produce shorter cationic arms, which allows the more efficient release of bound siRNA. This would in turn decrease the toxicity from cell membrane interactions and damage to the kidneys during renal filtration which are the common problems with cationic polymers. In this Received: October 14, 2013 Revised: November 13, 2013 Published: November 21, 2013 9181

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Figure 1. Schematic representation of a selection of fundamental characteristics offered by applying a RAFT chain transfer agent during a free radical polymerization: (I) control of the molecular weight distribution and reduction of the dead chain by increasing the amount of dormant chain versus active chains;8 (II) control of the polymer composition in achieving block, alternating, statistical, or grafted polymers and also control of the polymer functionality in introducing α,ω telechelic, site specific, end group, acceptor/donor or polymerizable (macromonomer) functionalities;23 (III) access to three-dimensional architectures by using a multivinyl monomer/branching agent/cross-linker;24 (IV) access to three-dimensional architectures in controlling the hydrophilic/hydrophobic balance of block copolymers.25 was recrystallized from chloroform and stored in the freezer (−10 °C) until used. (S)-4-Cyano-4-(dodecylthiocarbonothioylthio)pentanoic acid (1) was prepared according to a literature procedure described previously.26 Compound 1 can also be purchased from Sigma-Aldrich Co. or Strem Chemicals Inc.27 2-(Pyridin-2-yldisulfanyl)ethanol (2) was prepared according to procedure previously described by S. Thayumanavan et al.28 Esterase from porcine liver enzyme was purchased from Aldrich and used as received. Analytical thin layer chromatography (TLC) was performed on Merck Silica Gel F254 TLC plates. Preparative column chromatography was performed using Merck Silica Gel 60 (70−230 mesh). Characterization. Nuclear magnetic resonance (NMR) spectra were obtained with a Bruker Avance 400 MHz spectrometer (1H 400 MHz and 13C 100.6 MHz). Size exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a CMB-20A controller system, a SIL-20A HT autosampler, a LC-20AT tandem pump system, a DGU-20A degasser unit, a CTO-20AC column oven, a RDI-10A refractive index detector, and four Waters Styragel columns (HT2, HT3, HT4, HT5 each 300 mm × 7.8 mm, providing an effective molar mass range of 100 to 4 × 106). This SEC system uses N,N-dimethylacetamide (DMAc) (with 2.1 g·L−1 of lithium chloride (LiCl)) as eluent with a flow rate of 1 mL.min−1 at 80 °C. The molar mass of the samples was obtained from a calibration curve constructed with PMMA or PS standards (Polymer Laboratories) of low dispersity (Đ) value. A third order polynomial was used to fit the log Mp vs time calibration curve, which was linear across the molar mass ranges. Synthesis of Four-Arm Disulfide and Ester Core RAFT Agent. Step 1: Synthesis of (S)-2-(Pyridine-2-yldisulfanyl)ethyl 4-Cyano-4(((dodecylthio)carbonothioyl)thio) Pentanoate (3). (S)-4-Cyano-4(dodecylthiocarbonothioylthio)pentanoic acid (1) (4.60 g, 11.39 mmol), 2-(pyridine-2-yldisulfanyl)ethanol (2) (2.13 g, 11.39 mmol),

regard, a suitable star-shaped RAFT agent whose core contains disulfide and/or ester linkages is highly desirable for degradation in the physiological and/or acidic environment within the cell. Examples of three-arm and six-arm star-shaped RAFT agents containing disulfide linkages are already known in the literature.20−22 In this paper, we report the synthesis of a new four-arm star RAFT agent 5 and its use in the polymerizations of well-defined star polymers based on methacrylic, styryl or acrylamide monomers, and also extended to block copolymer showing the retention of the living character. In addition, the dual biodegradability properties of the disulfide and the ester linkages will also be illustrated.

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EXPERIMENTAL SECTION

General Data. All solvents used were of analytical (AR) grade unless otherwise stated. Monomers [styrene (S), methyl methacrylate (MMA), N,N-dimethylacrylamide (DMA) and poly(ethylene glycol) methyl ether methacrylate (OEGMA8−9)] were obtained from Sigma-Aldrich, respectively and were filtered through alumina (to remove inhibitors) and flash distilled (for S and MMA) immediately prior to use. 2,2′Azobis(isobutyronitrile) (AIBN) was obtained from TCI chemicals and 9182

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Table 1. Summary of Final Monomer Conversions, Theoretical Molecular Weight (Based on Monomer Conversion), Experimental Degree of Polymerization (DP), DMAc SEC Molecular Weights, and Dispersities of Four-Arm Stars Polymer Targeted (PMMA40)4, (PMMA80)4, (PS40)4, (PS80)4, (PMMA46-b-POEGMA46)4, and (PDMA80)4 entry

targeted composition

convn (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(PMMA40)4 PMMA46c (PMMA80)4 PMMA91c (PS40)4 PS40c (PS80)4 PS80c (PMMA46-b-POEGMA46)4 PMMA46-b-POEGMA42c PMMA46-b-POEGMA42d PMMA46-b-POEGMA42e (PDMA80)4 PDMA64f PDMA64g PDMA64h PDMA64i

99 99 89 84 95

81

DP (1H NMR)a (PMMA46)4 S−S cleaved (PMMA91)4 S−S cleaved (PS32)4 S−S cleaved (PS59)4 S−S cleaved (PMMA46-b-POEGMA42)4 S−S cleaved S−S cleaved S−S cleaved (PDMA64)4 ester core cleaved ester core cleaved ester core cleaved ester core cleaved

Mn(theoretical)b

Mn(SEC)

Đ

20 750 4600 38 800 9100 15 700 3300 26 900 6100 100 550 24 750 24 750 24 750 27 700 6350 6350 6350 6350

17 400 5600 35 600 10 050 14 850 4750 26 350 7000 70 000 21 700 21 300 22 400 22 400 9600 12 000 13 400 19 500

1.16 1.08 1.09 1.06 1.18 1.08 1.37 1.07 1.27 1.21 1.18 1.17 1.13 1.28 1.34 1.36 1.26

a DP calculated by 1H NMR end group analysis. bTheoretical Mn based on the calculated DP. cS−S bonds cleaved using Bu3P. dS−S bonds cleaved using TCEP. eS−S bonds cleaved using DTT. fEster core cleavage at pH = 0.96 after 3 h at 60 °C. gEster core cleavage at pH = 0.96 after 48 h at 25 °C. hEster core cleavage after warming for 3 h at 60 °C in the presence of esterase enzyme. iEster core cleavage after stirring for 48 h at 25 °C in presence of esterase enzyme.

DIC (diisopropylcarbodiimide, 1.58 g, 12.53 mmol) in dichloromethane (50 mL), and DMAP (N,N-dimethylaminopyridine, catalytic amount) were allowed to stir at room temperature for 3 h. After removal of solvent, the crude reaction mixture was purified by column chromatography on a silica column using ethyl acetate: n-hexane 1:3 (v/v) as the eluent to give the title product 3 (5.5 g, 84% yield) as a yellow oil. 1H NMR (CDCl3): δ (ppm) 0.85 (t, 3H, CH3); 1.27 (br s, 18H); 1.68 (m, 2H); 1.85 (s, 3H, CH3); 2.35−2.60 (m, 4H, CH2CH2); 3.05 (t, 2H, CH2SS); 3.35 (t, 2H, CH2S); 4.35 (t, 2H, CH2O); 7.10 (m, 1H, ArH); 7.65 (m, 2H, 2 × ArH); 8.45 (m, 1H, ArH). 13C NMR (CDCl3): δ (ppm) 14.1; 22.7; 24.9; 27.6; 28.9; 29.0; 29.3; 29.4; 29.5; 29.6 (2C); 31.9; 33.7; 37.1(2C); 46.3; 62.7; 118.9; 119.9; 120.9; 137.0; 149.8; 159.5; 171.1; 216.0. Step 2: Synthesis of 5. (S)-2-(Pyridin-2-yldisulfanyl)ethyl 4-cyano-4(((dodecylthio)carbonothioyl)thio) pentanoate (3) (0.47 g, 8.22 × 10−4 mol) from step 1 above was allowed to react with pentaerythritol tetrakis(3-mercaptopropionate) (4) (0.10 g, 2.04 × 10−4 mol) in dichloromethane solvent (25 mL) with two drops of glacial acetic acid. The reaction was stirred at room temperature overnight. After removal of solvent, the crude reaction mixture was purified by column chromatography on a silica column (Merck 60, 70−230 mesh) first using ethyl acetate: n-hexane 1:3 (v/v) as the eluent to remove some unreacted 3, then using ethyl acetate: n-hexane 2:3 (v/v) solvent to isolate the desired title product, four-arm star RAFT agent 5: (0.36 g, 75.5% yield) as a yellow oil. It is worth noting that for successful purification by column chromatography, a gradient solvent as eluent is required. First, starting from a less polar solvent [ethyl acetate: n-hexane 1:3 (v/v)] to remove some unreacted 3, after that switching to a more polar solvent [ethyl acetate: n-hexane 2:3 (v/v)] the product 5 can be isolated cleanly. 1H NMR (CDCl3): δ (ppm) 0.86 (t, 12H, 4 × CH3); 1.27−1.40 (br s, 72H, 4 × (CH2)9); 1.70 (m, 8H, 4 × CH2); 1.86 (s, 12H, 4 × CH3); 2.35 (dd, 4H, 4 × CHCCN); 2.55 (dd, 4H, 4 × CHCCN); 2.65 (m, 8H, 4 × CH2); 2.76 (m, 8H, 4 × CH2); 2.90 (m, 16H, 4 × CH2SSCH2); 3.30 (t, 8H, 4 × CH2SC(S)); 4.15 (s, 8H, 4 × OCH2C); 4.35 (t, 8H, 4 × CH2O). 13C NMR (CDCl3): δ (ppm): 14.32; 22.86; 25.05; 27.85; 29.12; 29.25; 29.51; 29.60; 29.72; 29.79; 29.87; 32.08; 33.22; 33.96; 33.98; 36.93; 37.26; 42.27; 46.51; 62.49; 63.01; 119.15(CN); 171.19(CO); 171.42(CO), and 217.08(CS). Synthesis of Four-Arm Star (Co)Polymers. Preparation of FourArm Star PMMA. The following procedure is typical for a targeted

(PMMA80)4. A stock solution comprising four-arm star RAFT agent 5 (72.8 mg), methyl methacrylate (1.0 g), and VAZO-88 (3.05 mg) in toluene (1.24 mL) was prepared in a vial then transferred into an ampule. The ampule was degassed with three freeze−evacuate−thaw cycles, sealed and heated at 90 °C for 20 h. The samples were cooled to room temperature using cold water, opened then analyzed by 1H NMR and DMAc SEC (using PMMA standards calibration) for monomer conversion and molecular weight determination. The polymer was finally precipitated twice into heptanes then dried using high vacuum before end group analysis by 1H NMR. Preparation of Four-Arm Star PS. The following procedure is typical for a targeted (PS40)4. A stock solution comprising four-arm star RAFT agent 5 (70.0 mg), styrene (0.5 g), and VAZO-88 (2.93 mg) in toluene (0.66 mL) was prepared in a vial then transferred into an ampule. The ampule was degassed with three freeze−evacuate−thaw cycles, sealed and heated at 90 °C for 20 h. The samples were cooled to room temperature using cold water, opened then analyzed by 1H NMR and DMAc SEC (using PS standards calibration) for monomer conversion and molecular weight determination. The polymer was finally precipitated twice into methanol then dried using high vacuum before end group analysis by 1H NMR. Preparation of Four-Arm Star Block Copolymer P(MMA-bPOEGMA8−9). The following procedure is typical for a targeted P[MMA46-b-(POEGMA8−9)46]4. A stock solution comprising fourarm star macro RAFT agent (cf. entry 1 in Table 1, 1H NMR calculated Mn = 20,750 g·mol−1, 109.2 mg), poly(ethylene glycol) methyl ether methacrylate (OEGMA8−9, Mn = 475 g·mol−1, 0.50 g) and VAZO-88 (0.13 mg, weighted from a 0.1 wt % solution in DMF) in DMF (0.66 mL) was prepared in a vial then transferred into an ampule. The ampule was degassed with three freeze−evacuate−thaw cycles, sealed and heated at 90 °C for 24 h. The sample was cooled to room temperature using cold water, opened then analyzed by 1H NMR and DMAc SEC (using PMMA standards calibration) for monomer conversion and molecular weight determination. The polymer was finally precipitated twice into diisopropyl ether and then dried using high vacuum before end group analysis by 1H NMR. Preparation of Four-Arm Star PDMA. The following procedure is typical for a targeted (PDMA80)4. A stock solution comprising DMA (1.50 g), 4,4′-azobis(4-cyanovaleric acid) (0.88 mg), four-arm star RAFT agent 5 (111.0 mg), and dioxane (1.45 mL) was prepared in a vial 9183

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Figure 2. DMAc SEC traces of (I) two different four-arm star polymers, (PMMA46)4 on the top and (PMMA91)4 at the bottom, plotted with their counterparts cleaved using tributylphosphine represented by a dashed line and (II) the block copolymer [PMMA46-b-(POEGMA8−9)42]4 and its cleaved counterpart using either TCEP, Bu3P, or DTT represented by colored lines.

Figure 3. SEC traces resulting from the cleavage of the RAFT CTA core in a (PDMA64)4 polymer: (top) series of various pH from (left from 5.65 to 2.48 and right from 2.27 to 0.96), after heating the solution for 3 h at 60 °C; (bottom) series of various pH from (left from 5.65 to 2.48 and right from 2.27 to 0.96), left under stirring for 48 h at 25 °C. determination. The crude viscous polymer was finally dissolved in a water acetone mixture (3:1) then dialyzed (dialysis membrane MWCO 1 kDa from Spectrum Laboratories) against water/acetone for 1 day and against water for 2 days before freeze-drying and end group analysis by 1 H NMR.

then transferred into an ampule. The ampule was degassed with three freeze−evacuate−thaw cycles and sealed. The ampule was heated at 80 °C for 60 min, then cooled to room temperature using cold water, opened and analyzed by 1H NMR and DMAc SEC (using PMMA standards calibration) for monomer conversion and molecular weight 9184

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Scheme 1. Synthesis of Dual (Bio-)Degradable Four-Arm Star RAFT Agent 5

Scheme 2. RAFT (Co)Polymerization Using (Bio-)Degradable Four-Arm Star RAFT Agent 5 of Methyl Methacrylate (MMA), Styrene (S), N,N-Dimethylacrylamide (DMA), and Poly(ethylene glycol) Methyl Ether Methacrylate (OEGMA8‑9)

of tributylphosphine (60 μL per mL of GPC sample solution) was added. The sample was filtered using a 200 μm PTFE filter prior SEC analysis but without any other purification (cf. Table 1 for SEC characterizations before and after cleavage).

Degradation Studies. Degradation of the Disulfide Bonds Using Tributylphosphine in Organic Solution. Four-arm star polymer (PMMA46)4, (PMMA91)4 and (PMMA46-b-POEGMA42)4 (cf. Table 1) were prepared in DMAc as SEC samples (5 mg·mL−1) before an excess 9185

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Degradation of the Disulfide Bonds in Aqueous Media. Four-arm star polymer (PMMA46-b-POEGMA42)4 was dispersed in deionized water at a concentration of 4 mg·mL−1. The sample obtained was not fully soluble and showed some cloudiness. Then a 20 mM solution of tris(2-carboxyethyl)phosphine (TCEP) or DL-dithiothretiol (DTT) was added in an equivalent volume to the polymer dispersion. After an overnight stirring, the solutions were freeze-dried and then prepared in DMAc as SEC samples (2 mg·mL−1) (cf. Figure 2). Degradation of the Ester Core Using Acid. A 60 mg sample of fourarm star (PDMA64)4 polymer was dissolved in a mixture of acetone and demonized water (1:3 w/w) at a concentration of 5 mg.mL−1. The solution was acidified using a 0.1 M HCl to gradually bring the pH from 5.65 to 0.96. Eight samples were prepared in this way and separated into two sets. The first set was heated at 60 °C for 3 h when the second set was left stirring at room temperature for 48 h (cf. Figure 3). The samples were then freeze-dried and analyzed by DMAc SEC. Degradation of the Ester Core Using Enzyme Esterase. A 15 mg sample of the four-arm star CTA was mixed with 15 mg of the esterase in a mixture of acetone-d6 and deuterium oxide (1 mL, 1:3 w/w). 1H NMR analysis was carried out on this sample. Second, 30 mg of the four-arm star (PDMA64)4 polymer was mixed with 30 mg of the esterase in a mixture of acetone and deionized water (2 mL, 1:3 w/w): this sample was split in two, half of it was warmed to 60 °C for 3 h, and the other half was left under stirring at room temperature for 48 h. After removal of the solvent by freeze-drying these samples were analyzed using the DMAc SEC.

1.06, respectively. The experimental molecular weight results obtained with linear PMMA calibration were close to the predicted (theoretical). The experimental degree of polymerization (DP) calculated by 1H NMR end group analyses shows a high CTA efficiency of around 88% (the CTA efficiency was calculated from being the ratio of the theoretical Mn = 18,350 g· mol−1 (corresponding to a (PMMA40)4 with a 100% CTA efficiency) and the calculated Mn = 20 750 g·mol−1obtained by 1 H NMR end group analysis). The SEC traces of these polymers and their cleaved products are represented in Figure 2 and summarized in Table 1 (cf. entries 1 to 4). A noticeable difference in retention time was observed for the stars and for their cleaved products, confirming the cleavage of the arms from the core by reduction of the S−S bonds using Bu3P. The molecular weight of the degradation product was close to the predicted molecular weight of one arm of the star, confirming near complete degradation of the core yielding linear polymers as degradation products. Similarly, the two star polymers (PS40)4 and the (PS80)4 were prepared from styrene and treated with Bu3P. The SEC results shown in Table 1 are based, for these star polymers and their cleaved counterpart, on calibration against linear PS standards. Once again the experimental data for the cleaved polymers correlated well with the theoretical (cf. entry 5−8 in Table 1). It can be noted that a slight increase of the Đ for the (PS80)4 (Đ = 1.37, cf. entry 7 in Table 1), presumably due to some termination by recombination. The molecular weight of the cleaved products was close to that of the arm of the star, confirming the cleavage of the S−S core under reductive conditions. The (PMMA46)4 star was used for the block extension polymerization with the hydrophilic monomer POEGMA8−9 to produce [PMMA46-b-(POEGMA8−9)42]4 block copolymer. The Đ of the polymer was high due to the oligomeric nature of POEGMA8−9 (cf. entry 5 in Table 1). The star block copolymer [PMMA46-b-(POEGMA8−9)42]4 of Mn = 70 000 g·mol−1 was degraded under two different test conditions: first in DMAc solution and second in aqueous medium using TCEP and DTT. As illustrated by the results in Table 1 and Figure 2, under both test conditions the polymer molecular weight of the degraded products was approximately one-fourth of the molecular weight of the non degraded polymer, confirming core degradation of each of the four arms to yield linear block copolymers. Core Degradation under Acidic Conditions. The aim of this study was to degrade the ester core of the star polymers under acidic and enzymatic (esterase) conditions. An acrylamide star polymer (PDMA64)4 was synthesized for this purpose (cf. entry 13 in Table 1). Methacrylic polymers would be expected to rapidly degrade under acidic conditions, undergoing ester cleavage to give methacrylic acid, while acrylamide polymers, with amide groups would be more resistant.31 The hydrolytic degradation of the block copolymer (PDMA64)4 was investigated under a range of pH conditions, stepping from an initial value of pH = 5.65 to that of pH = 0.96. Two sets of data were generated: for the first series, different pH polymer solutions were heated at 60 °C for 3 h while the second series was left under stirring at 25 °C for 48 h. Figure 3 shows the SEC traces at time zero after decreasing progressively the pH for these two series. The appearance of a peak at longer retention time for each of the pH values tested clearly indicates that the star copolymer is degrading. The rate of degradation increased with the increase of the polymer solution acidity. This rate was also faster at 60 °C rather than at room temperature. This is confirmed by analyzing the corresponding SEC data at the lowest

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RESULTS AND DISCUSSION Synthesis of Four-Arm RAFT Agent. In this study, the dual (bio)-degradable four-arm star RAFT agent 5 was synthesized by a facile two-step process employing pyridyl disulfide 2 as shown in Scheme 1. In the first step, the pyridyl disulfide containing RAFT agent 3, was synthesized by the reaction of RAFT agent, (S)-4-cyano-4-(dodecylthiocarbonothioylthio)pentanoic acid (1) with 2-(pyridine-2-yldisulfanyl)ethanol (2) in the presence of diisopropyl carbodiimide (DIC) as a coupling agent and a catalytic amount of N,N-dimethylaminopyridine (DMAP) in dichloromethane at room temperature. Similar chemistry using pyridyl disulfide functionality was previously described by Boyer et al.29,30 After removal of the N,N-diisopropylurea byproduct and purification by column chromatography, 3, was obtained in 84% yield. The synthesis of 5 was furnished by a reaction of pentaerythritol tetrakis(3-mercaptopropionate) (4) with a slight excess of 3 in the presence of glacial acetic acid. An acidic condition is necessary to effect the disulfide bond formation between the pyridyl disulfide functional group and a sulfhydryl. The desired product 5 was obtained in 75% yield after silica gel column chromatography. The structure of 5 was unambigously assigned by its NMR spectra (1H, 13C NMR). Polymerizations and Cleavage of the S−S Core. Scheme 2 presents the general homopolymerization process using our four-arm RAFT agent. To test its efficiency, PMMA and PS star polymers were synthesized and analyzed by SEC using PMMA and PS linear calibration standards (theoretically, the linear polymers resulting from the cleavage of the core can be more accurately analyzed by SEC than the actual stars polymers that would have a different radius of gyration due to their architecture). For the polymerization of MMA, two four-arm PMMA polymers with target degrees of polymerization (DP) of 40 and 80 per arm were prepared. The polymers were then degraded as described in the Experimental Section using Bu3P to cleave the S−S linkages. The results are summarized in Figure 2 and Table 1 (cf. entries 1 to 4). The SEC results in Table 1 show that the targeted molecular weights for (PMMA40)4 and (PMMA80)4 were closely approximated with low Đ of 1.16 and 9186

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Figure 4. 1H NMR spectrum representing: the four-arm star RAFT agent without (black) and with (red) the esterase enzyme after 3 h at 60 °C, in a mixture of D2O and acetone-d6 (3/1); (PDMA64)4 without (green) and with (blue) the esterase enzyme after 3 h at 60 °C, in a mixture of D2O and acetone-d6 (3/1).

pH = 0.95. The Mn of the sample warmed at 60 °C (Mn = 9600, cf. entry 14 in Table 1) is closer to the theoretical value of 6300 (corresponding to a fully decomposed core), where the Mn of the sample left at room temperature for 2 days is higher (Mn = 12 000, cf. entry 15 in Table 1). Degradation Studies Using an Esterase Enzyme. To demonstrate that the core of the star can also be cleaved under enzymatic conditions, the polymer was exposed to an esterase enzyme. First the four-arm star RAFT agent was treated with esterase and monitored by 1H NMR at 60 °C. The splitting of the peak C−CH2−O can be observed at 4.2 ppm (cf. Figure 4 bottom), confirming the degradation of the ester core. Second, a similar experiment was conducted with the (PDMA64)4 and the degradation of the ester peak corresponding to the core of the four-arm star RAFT agent was evident based on the observed chemical shift change (cf. Figure 4 top). The analogous SEC data are presented in Table 1 (entry 16 and 17). The corresponding Mn (Mn = 13 400 and Mn = 19 500, cf. entries 16 and 17 in Table 1) are lower than the original Mn for the (PDMA64)4 four-arm star (Mn = 22 400, cf. entry 13 in Table 1) but still far from the theoretical value of 6,350 (corresponding to a fully decomposed core), signifying that the decomposition of the core in the influence of the esterase enzyme is only partial.

core can also be cleaved, under either acidic or enzymatic conditions but at a slower rate.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge the postdoctoral fellowships awarded to E.G.W., F.G., and J.R. by the CSIRO’s Office-of-theChief Executive.

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CONCLUSION This study demonstrated the synthesis of a novel four-arm RAFT agent 5 with two types of cleavable groups (disulfide and ester) and its successful use in the synthesis of four-am star polymers and block copolymers. The synthesis of polymers with this architecture from several different monomers indicated the wide applicability of this RAFT agent in designing polymers for different applications. The disulfide bonds of the central core can be cleaved under reductive conditions to yield linear polymer with narrow dispersity. These results also confirmed the near equal degree of polymerization of each of the arms in star polymers and block copolymers. The ester linkage of the central 9187

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