Combining Ring-Opening Multibranching and RAFT Polymerization

Article pubs.acs.org/Macromolecules

Combining Ring-Opening Multibranching and RAFT Polymerization: Multifunctional Linear−Hyperbranched Block Copolymers via Hyperbranched Macro-Chain-Transfer Agents Lutz Nuhn,† Christoph Schüll,†,‡ Holger Frey,† and Rudolf Zentel*,† †

Institute of Organic Chemistry, Johannes Gutenberg-University Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany Graduate School Materials Science in Mainz (MAINZ), Staudingerweg 9, D-55128 Mainz, Germany

‡

S Supporting Information *

ABSTRACT: The synthesis of a hyperbranched macro-chaintransfer agent for RAFT polymerization of functional methacrylate or methacrylamide monomers was achieved by selectively attaching one single CTA onto hyperbranched polyglycerol dendron analogues. The combination of ringopening multibranching polymerization of glycidol and subsequent RAFT polymerization of the hyperbranched macro-chain-transfer agents created a new route to a variety of multifunctional linear−hyperbranched block topologies. All linear−hyperbranched block copolymers could be synthesized with controlled molecular weight (Mn = 3.2−43.7 kg/mol) and low polydispersity (PDI = 1.15−1.34). As first examples for this universal approach, we present block copolymer syntheses with thermoresponsive methacrylate (tri(ethylene glycol) methyl ether methacrylate) and biocompatible methacrylamide (2hydroxypropylmethacrylamide). Because of the presence of dithiobenzoate esters at the end of each linear polymer chain end, selective end-group modification with functional methanethiosulfonates for bioconjugation to proteins (via the biotin−avidin interaction) or drugs (and dyes as model compounds, respectively) could be achieved. This expands the scope of this class of polymer architectures and renders the obtained multifunctional linear−hyperbranched block copolymers applicable as topologically advanced polymeric drug delivery systems.

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INTRODUCTION Next generations of synthetic polymers as “smart” materials for future application require versatile combinations of topology, composition, and functionality.1 Thus, establishing universal strategies to gain rapid access to complex functional topologies is one of the major challenges of today’s polymer chemistry. Especially in the field of “nanomedicines”,2 advanced functional polymer architectures may serve as promising tools for demanding applications in the field of polymer-based diagnostics or therapeutics.3 Within the past decades controlled polymerization techniques have opened pathways to novel well-defined structures by e.g. living anionic polymerization4 or controlled radical polymerization techniques like atom transfer radical polymerization (ATRP),5,6 nitroxide-mediated polymerization (NMP),7 and reversible addition−fragmentation chain transfer (RAFT) polymerization.8 These techniques have improved the synthesis of complex polymer compositions and topologies from linear to branched and dendritic architectures. Among these geometries, hyperbranched polymers serve as interesting topological building blocks with promising multifunctionalities.9,10 In contrast to precisely defined dendrimers, they can be synthesized easily by convenient one-step syntheses, in large scales, proper yields, and high purities.11 Hyperbranched polyglycerol (hbPG)12,13 is one of the most popular hyper© 2013 American Chemical Society

branched polymers synthesized by ring-opening multibranching polymerization (ROMBP) of glycidol in a broad range of molecular weights with moderate to narrow polydispersities.14,15 Moreover, its excellent biocompatibility16 enables novel biomedical applications. Expanding hbPG’s functionality, a combination with alternative polymerization techniques may lead to further advanced topologies. For example, RAFT polymerization17 is one of the most versatile polymerization methods that enables polymerization of a variety of functional monomers under mild conditions.18−21 The combination of RAFT and ROMBP may expand the application of hbPG tremendously. This has so far only been reported for some less-defined multiarm starpolymers, where hbPG was modified with several RAFT chain transfer agents (CTA) to create star polymers.22−25 Regarding hyperbranched polymers with precisely one single linear polymer chain attached,26,27 a combination with RAFT polymerization has not been reported yet. Only well-defined dendrimers bearing several carbohydrate structures on their surface were attached to linear polymers synthesized by the Received: February 7, 2013 Revised: February 11, 2013 Published: April 5, 2013 2892

dx.doi.org/10.1021/ma4002897 | Macromolecules 2013, 46, 2892−2904

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RAFT technique to date, affording linear-glycodendritic architectures as suitable carriers for drug delivery.28,29 Alternatively, single CTA functionalities could be attached onto dendrimers possessing a single focal functionality. Such dendritic structures could serve as macro-CTAs to graft one linear polymer via RAFT polymerization from each dendrimer.30−33 However, these structures are mostly based on hydrophobic dendritic poly(benzyl ethers) or poly(2,2-bis((methylol)propionic acid) esters that are not water-soluble and cannot be applied e.g. as drug carriers in physiologically relevant media. As for hyperbranched polymers, only hyperbranched polyethylene has been used as a building block for grafting one linear chain from it to date,34−36 yet polyethylene’s limited solubility and functionality do not allow any challenging application either. Consequently, a combination of multifunctional water-soluble hyperbranched topologies (e.g., hbPG) together with robust polymerization techniques for functional monomers (e.g., RAFT) in graf ting-f rom approaches has not been reported yet. Linear−hyperbranched structures containing a hbPG block have to date only been obtained via the so-called “hypergraf ting” strategy: From a linear polymer with multiple initiation groups glycidol could be polymerized by ROMBP from the end of a linear chain.37,38 Wurm et al., for example, used these isolated structures for carbon nanotube solubilization,39 stealth-liposome preparation,40 and protein conjugation.41 However, all initiators and monomers had to be chemically inert toward the basic anionic polymerization conditions. A graf ting-f rom strategy of the linear block from a hbPG core with a single initiating site would eliminate this requirement and thus expand the scope for novel monomers in the linear block facilitating advanced functionalities in general. Recently, we described the synthesis of hyperbranched polyglycerol dendron analogues with a single focal amino functionality, which were used for the synthesis of linear− hyperbranched graft copolymers.42 We were able to show that each single amino group could be selectively addressed by pentafluorophenyl esters. Pentafluorophenyl esters of 4-cyano4-(phenylcarbonothioylthio)pentanoic acid have been reported as useful chain transfer agents for heterotelechelic α,ω-endgroup-functionalized RAFT polymers.43 The resulting linear structures were used for polymer conjugation with nanoparticles44 and dyes45 or bioconjugation to various proteins.46,47 Roth et al. demonstrated that the pentafluorophenyl ester reacts preferentially with primary amines. Under stoichiometric conditions the dithiobenzoate group of the CTA is not affected and can therefore be used for ω-end-group modification or block copolymerization. In this work we introduce the attachment of one single CTA onto hbPG using hyperbranched polyglycerol dendrons with a single focal amino functionality that can be addressed by a pentafluorophenyl ester of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid selectively in the presence of multiple hydroxyl groups of hbPG. The resulting hbPGs containing a single dithiobenzoate group serve as novel hyperbranched macro-CTAs to generate various functional linear−hyperbranched topologies via graf ting-f rom by RAFT polymerization (Figure 1). Consequently, this strategy can be seen as a universal approach for multifunctional linear− hyperbranched block copolymers, where the linear block can be adjusted to a large variety of functional monomers amenable to the RAFT process, e.g., thermoresponsive methacrylates48−50 or biocompatible methacrylamides.51−53 Moreover,

Figure 1. Synthetic concept for novel multifunctional linear− hyperbranched block copolymers via combination of ring-opening multibranching polymerization (ROMBP) and reversible addition− fragmentation chain transfer (RAFT) polymerization.

due to the presence of dithiobenzoate esters at the end of each linear ω-chain end, aminolysis of fully biocompatible linear− hyperbranched block copolymers in presence of methanethiosulfonates also allows selective and reversible orthogonal ω-end group modification44−46 for bioconjugation to proteins or drugs (and dyes as model compounds, respectively) that are applicable for advanced drug delivery.

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EXPERIMENTAL SECTION

Materials. All reagents were purchased from Acros Organics or Sigma-Aldrich and used as received, unless otherwise stated. BiotinMTS (N-biotinyl aminoethyl methanethiosulfonate) and Texas Red methanethiosulfonate (Texas Red-2-sulfonamidoethyl methanethiosulfonate) were purchased from Toronto Research Chemicals, pentafluorophenol was obtained from Fluorochem, and 2,2′-azobis(4methoxy-2,4-dimehthylvaleronitrile) (V-70) was obtained from Wako Chemicals. Glycidol, diethylene glycol dimethyl ether (diglyme), and dichloromethane were freshly distilled from CaH2 prior to use. Anhydrous THF was freshly distilled from a sodium/potassium mixture. Anhydrous dimethyl sulfoxide (DMSO) was stored over activated molecular sieves (4 Å). Phosphate buffered saline (PBS) was obtained from Fisher BioReagents containing 137 mM NaCl, 11.9 mM phosphates, and 2.7 mM KCl. Thin layer chromatography (TLC) was 2893


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performed on TLC aluminum sheets (20 × 20 cm, silica gel 60 F254) purchased from Merck. Column chromatography was carried out using silica obtained from Macharey-Nagel (0.063−0.2 mm/20−230 mesh). Preparative size exclusion chromatography (SEC) was done using Sephadex G25 and Millipore water as eluent. Instrumentation. 1H NMR and 13C NMR spectra were recorded at 300 and 75 MHz, respectively, on a Bruker AC300 or at 400 and 100 MHz, respectively, on a Bruker Avance-II 400 and were referenced internally to residual proton signals of the deuterated solvent. 19F NMR spectra were recorded on a Bruker 400 MHz FT NMR spectrometer. For analytical SEC measurements in dimethylformamide (DMF) (containing 0.25 g/L of lithium bromide), an Agilent 1100 series was used as an integrated instrument including a PSS HEMA column (porosity 1 000 000 Å/10 000 Å/100 Å) and an RI as well as a UV detector. Calibration was achieved with poly(ethylene glycol) standards provided by Polymer Standard Services (PSS). For analytical SEC measurements in hexafluoroisopropanol (HFIP) (containing 3.0 g/L of potassium trifluoroacetate), a PU 2080+ pump, an autosampler AS1555, and an RI detector RI2080+ from JASCO were used. Columns packed with modified silica were obtained from MZAnalysentechnik: PFG columns, particle size 7 μm, porosity 100 and 1000 Å. Calibration was carried out with poly(methyl methacrylate) standards purchased from PSS. For analytical SEC measurements in buffered aqueous solution (containing 0.05 M sodium phosphate, 0.15 M sodium chloride, pH 7.0) a PU 2086+ pump, a UV detector UV2077+, and an RI detector RI2031+ from JASCO were used. The flow rate was set to 0.4 mL/min using a SuperoseTM 6 10/300 GL column, and calibration was done using commercially available protein standards. In this case, sample preparation was performed by incubation of a 1.2 μg/μL avidin solution in PBS with an excess of hbPG21-b-P(HPMA)9-Biotin (15.2 μg/μL−50 equiv) for a few hours before injecton into the aqueous SEC system. Electrospray ionization mass spectroscopy (ESI-MS) was performed using a Navigator Instrument from Thermoelectronics with sample concentrations of 0.1 mg/mL in methanol, 0.75 mL/min flow rate, cone voltage 70, 45, or 35 V, and nitrogen flow rate 300 L/min. Cloud points measurements for lower critical solution temperature analysis were determined by optical transmittance of a light beam (λ = 632 nm) through a 1 cm sample quartz cell at temperatures from 20 to 80 °C and a heating rate of 1 °C/min using a Jasco V-630 spectrophotometer equipped with a Jasco ETC-717 Peletier element. The sample concentration in water was 10 mg/mL, and the observed cloud point was defined as 50% transmittance during the measurement. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) was performed following the general protocol of Lämmli.54 Samples were dissolved in PBS buffer and incubated for 12 h before treating with 2 × Lämmli buffer (6% SDS, 0.02% bromophenol blue, 0.125 M Tris, 40% glycerol). 10 μg of Avidin, 64 μg of hbPG-bP(HPMA)-Biotin (about 125 equiv), or a mixture of both were incubated for 12 h, loaded on a 10% separation gel (with 5% stacking gel), and run at 150 V for 2 h before visualization by Coomassie Blue treatment. For comparison, a protein ladder (10 μL of SeeBlue Plus2 Pre-Stained Standard) purchased from Invitrogen was used. Syntheses of Pentafluorophenyl 4-Cyano-4((phenylcarbonothioyl)thio)pentanoate, PFP-CTA, Tri(ethylene glycol) Methyl Ether Methacrylate, MEO3MA, and 2-Hydroxypropylmethacrylamide, HPMA. Procedures are described in detail in the Supporting Information. Synthesis of Benzyl-Protected Hyperbranched Polyglycerol Dendron Analogues (hbPG-NBn2). The initiator N,N-dibenzyltris(hydroxymethyl)aminomethane was synthesized according to a previously described procedure.42 For the preparation of 1 g of polymer, 0.3 mol % (compared to the number of hydroxyl groups) of cesium hydroxide monohydrate was added to a suspension of the initiator in dry benzene. The mixture was stirred for 1 h at room temperature, and the solvents were removed under high vacuum at 80 °C for several hours. The initiator salt was dissolved in 1 mL of diethylene glycol dimethyl ether (diglyme), and a solution of glycidol in diglyme (15 vol %) was added using a syringe pump at 100 °C. The reaction was terminated by the addition of 0.5 mL of degassed

methanol. The slightly yellow and highly viscous product was dissolved in methanol, stirred with a cation exchange resin for 30 min, filtrated, concentrated, and precipitated into an excess of cold diethyl ether. Yields: 90%. 1H NMR (300 MHz, CD3OD): δ (ppm) = 7.32−7.00 (m, 10H, aromatic); 4.10−3.96 (br, 4H, benzyl −CH2−); 3.83−3.40 (m, polyether backbone) (compare Supporting Information Figure S8). 13C NMR: The detailed peak assignment of the hyperbranched polymers and their degree of branching can be found in the literature42 as well as in Figure S9 and Table S1 (Supporting Information). Synthesis of Hyperbranched Polyglycerol Dendron Analogues with Exactly One Amino Group (hbPG-NH2). A 0.5 g sample of hbPG-NBn2 was dissolved in 50 mL of a mixture of methanol, water, and THF (3:1:1) and 50 mg (10 wt %) of palladium hydroxide on charcoal, and a catalytic amount of hydrochloric acid was added. The reaction vessel was flushed with hydrogen (70−80 bar), and the mixture was allowed to stir at room temperature. The reaction was monitored by analyzing aliquots via 1H NMR spectroscopy. After the reaction was complete (disappearance of the aromatic signals at 7.32−7.00 ppm), the solution was filtered, concentrated, and precipitated into cold diethyl ether. Yields: 90%. 1H NMR (300 MHz, CD3OD): δ (ppm) = 3.83−3.40 (m, polyether backbone) (compare Supporting Information Figure S10). Synthesis of Hyperbranched Polyglycerol Dendron Analogues with Exactly One CTA Functionality (hbPG-CTA). A 280 mg sample of hbPG21-NH2 (0.18 mmol) was dissolved in 2.5 mL of anhydrous DMSO with 232 mg of PFP-CTA (0.52 mmol) and triethylamine (72.4 μL; 0.52 mmol) under an argon atmosphere. The reaction mixture was stirred for 12 h at room temperature while its color turned dark red. It was finally precipitated into cold diethyl ether three times and obtained as a red oil. To remove further salts of pentafluorophenol triethylamine, the precipitate was dissolved in a few milliliters of Millipore water and purified by size exclusion chromatography (SEC) with Sephadex G25 and Millipore water as an eluent. All red-colored fractions were combined and lyophilized affording hbPG21-CTA as red viscous oil. Yields: 281 mg (0.14 mmol), 80%. 1H NMR (400 MHz, CD3OD): δ (ppm) = 7.96 (d, J = 8 Hz, 2H, o-ArH); 7.63 (t, J = 6 Hz, 1H, p-ArH); 7.47 (t, J = 6 Hz, 2H, m-ArH), 3.90−3.40 (m, polyether backbone); 2.80−2.38 (m, 4H, −CH2− CH2−CO−); 1.97 (s, 3H, −CH3) (compare Figure S11). Synthesis of Hyperbranched Polyglycerol-block-poly(tri(ethylene glycol) methyl ether methacrylate) Copolymers (hbPG-b-P(MEO3MA)). For typical RAFT polymerizations as reported earlier,55 a Schlenk tube equipped with a stir bar was loaded with hbPG21-CTA (30 mg; 15.5 μmol), tri(ethylene glycol) methyl ether methacrylate (MEO3MA) (373 mg; 1.60 mmol), and 2,2′azobis(4-methoxy-2,4-dimethylvaleronitrile) (0.5 mg; 1.55 μmol). All compounds were dissolved in Millipore water (0.8 mL). Following three freeze−pump−thaw cycles, the tube was immersed in an oil bath at 30 °C for about 67 h. The monomer conversion was determined by analyzing an aliquot by 1H NMR spectroscopy. The resulting polymer was isolated by size exclusion chromatography (SEC) with Sephadex G25 and Millipore water as eluent. All red colored fractions were combined and lyophilized affording hbPG21-b-P(MEO3MA)81 as a red viscous oil. Yields: 273 mg (13.2 μmol), 85%. 1H NMR (400 MHz, CD3OD): δ (ppm) = 4.13 (br, 2H, −COO−CH2− P(MEO3MA)); 3.83−3.43 (m, polyether backbone hbPG and P(MEO3MA)); 3.40 (br, 3H, −CH3 P(MEO3MA)); 2.35−1.47 (br, 2H, −CH2− polymer main chain P(MEO3MA)); 1.30−0.60 (br, 3H, −CH3 polymer main chain P(MEO3MA)) (compare Figure S12). Synthesis of Hyperbranched Polyglycerol-block-poly(2-hydroxypropylmethacrylamide) Copolymers (hbPG-b-P(HPMA)). For typical RAFT polymerizations as reported earlier,55 a Schlenk tube equipped with a stir bar was loaded with hbPG91-CTA (21 mg; 3.0 μmol), 2-hydroxypropylmethacrylamide (145 mg; 1.01 mmol), and 4,4′-azobis(4-cyanovaleric acid) (0.1 mg; 0.3 μmol). All compounds were dissolved in Millipore water (2.4 mL). Following three freeze− pump−thaw cycles, the tube was immersed in an oil bath at 70 °C for about 82 h. The monomer conversion was determined by analyzing an aliquot by 1H NMR spectroscopy. The resulting polymer was isolated 2894


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Scheme 1. Synthesis of Hyperbranched Polymer Macro-Chain-Transfer Agents Based on End-Group-Modified hbPG

P(HPMA)); 2.55−1.50 (br, 2H, −CH2− polymer main chain P(HPMA)); 1.50−0.60 (br, 6H, −CH(OH)−CH3 P(HPMA) and −CH3 polymer main chain P(MEO3MA))due to comparably high molecular weight of the polymer, the signals of the bound dye cannot be detected by 1H NMR spectroscopy (compare Figure S16).

by size exclusion chromatography (SEC) with Sephadex G25 and Millipore water as eluent. All red-colored fractions were combined and lyophilized affording hbPG91-b-P(HPMA)194 as a red powder. Yields: 80 mg (2.3 μmol), 76%. 1H NMR (400 MHz, CD3OD): δ (ppm) = 7.52 (br, 1H, −CO−NH− P(HPMA)); 4.23−3.40 (m, polyether backbone hbPG and −CH(OH)− P(HPMA)); 3.25−2.73 (br, 2H, −CO−NH−CH2− P(HPMA)); 2.35−1.30 (br, 2H, −CH2− polymer main chain P(HPMA)); 1.30−0.60 (br, 6H, −CH(OH)−CH3 P(HPMA) and −CH3 polymer main chain P(MEO3MA)) (compare Figure S13). End-Group Modification of Hyperbranched Polyglycerolblock-poly(2-hydroxypropylmethacrylamide) Copolymers with Biotin (hbPG-b-P(HPMA)-Biotin). Modified from a literature procedure,46 15 mg of hbPG21-b-P(HPMA)9 (4.6 μmol) was dissolved in 1 mL of anhydrous DMSO with 6.8 mg of N-biotinylaminoethyl methanthiosulfonate (Biotin-MTS) (17.8 μmol) under an argon atmosphere. By adding 15 μL of n-propylamine (180 μmol), the red solution turned colorless. After stirring for 12 h at room temperature the reaction mixture was precipitated into cold acetone three times, redissolved in a few milliliters of Millipore water, and lyophilized affording hbPG21-b-P(HPMA)9-Biotin as a white powder. Yield: 12.4 mg (3.6 μmol), 79%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) = 7.20 (br, 1H, −CO−NH− P(HPMA)); 6.42 (s, −CO−NH− Biotin) 6.36 (s, −CO−NH− Biotin); 4.8−4.4 (br, −OH, hbPG and P(HPMA)); 4.30 (s, −NH−CH− Biotin); 4.13 (s, −NH−CH− Biotin); 4.10−3.13 (m, polyether backbone hbPG and −CH(OH)− P(HPMA)); 3.10 (s, −S−CH−CH2− Biotin); 3.05−2.70 (br, 2H, −CO−NH−CH2− P(HPMA)); 2.65−2.51 (d, −S−CH−CH− Biotin); 2.08 (br, −NH−CH2− Biotin); 2.00−0.6 (br, 8H, −CH(OH)− CH3 P(HPMA), −CH2− and −CH3 polymer main chain P(MEO3MA)) (compare Figure S14). End-Group Modification of Hyperbranched Polyglycerolblock-poly(2-hydroxypropylmethacrylamide) Block Copolymers with Texas Red (hbPG-b-P(HPMA)-TxRed). Modified from a literature procedure,45 40 mg of hbPG91-b-P(HPMA)194 (1.1 μmol) was dissolved in 2 mL of anhydrous DMSO with 1.5 mg of Texas Red2-sulfonamidoethyl methanethiosulfonate (2.0 μmol) under an argon atmosphere. After addition of n-propylamine (1.7 μL, 20.7 μmol) the reaction mixture was stirred at room temperature for 24 h in the dark. It was then first dialyzed against Millipore water for several days (the solvent was changed twice a day) and afterward lyophilized. To remove further nonattached dye, the lyophilizate was dissolved in a few milliliters of Millipore water and precipitated into cold acetone three times. The dried precipitate was afterward again dissolved in a few milliliters of Millipore water and further purified by size exclusion chromatography (SEC) with Sephadex G25 and Millipore water as an eluent. All purple-colored high molecular weight fractions were combined and lyophilized, affording hbPG91-b-P(HPMA)194-TxRed as a purple powder. Yield: 10.0 mg (0.2 μmol), 20%. 1H NMR (400 MHz, CD3OD): δ (ppm) = 4.23−3.40 (m, polyether backbone hbPG and −CH(OH)− P(HPMA)); 3.25−2.73 (br, 2H, −CO−NH−CH2−

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RESULTS AND DISCUSSION We have developed a synthetic access to a novel hyperbranched macro-chain-transfer agent (CTA) based on polyfunctional, hyperbranched polyglycerol (hbPG), which can be applied to RAFT polymerization of methacrylates and methacrylamides affording multifunctional linear−hyperbranched block copolymers. Their ω-end groups can additionally be modified for protein or dye conjugation as drug model compounds, which are redox-cleavable under physiological conditions (cytoplasm) using the redox-buffer glutathione (GSH). A. Hyperbranched Polymer Macro-Chain-Transfer Agents. We established a selective procedure to modify hyperbranched polyglycerol dendrons with exactly one chain transfer agent group within three steps affording a hyperbranched macro-chain-transfer agent (CTA) that can be applied for RAFT polymerization of a variety of monomers affording multifunctional linear−hyperbranched block copolymers (Scheme 1). As previously reported,42 we have developed a route for the synthesis of hbPG containing exactly one focal amino functional group using a benzyl-protected initiator system (N,Ndibenzyltris(hydroxymethyl)aminomethane) for the anionic ring-opening multibranching polymerization (ROMBP) of glycidol. The three hydroxyl groups of the initiator are important to guarantee controlled polymerization of glycidol with moderate polydispersities.56 The initiator system was partially deprotonated by cesium hydroxide (30 mol % of the hydroxyl groups) to ensure sufficient solubility in diglyme for subsequent polymerization. ROMBP was performed by the established slow monomer-addition (SMA) procedure using highly diluted glycidol. After purification we obtained hbPGNBn2 with molecular weights in the range of 1800−7000 g/mol and polydispersities below 1.57 (Table 1, nos. 1−3). The resulting hyperbranched dendron analogues contain two benzyl protective groups at the focal amino position (confirmed by 1H NMR as well as ESI-MS (Figure 2, Figure 3, and Figure S8), ensuring the quantitative incorporation of the initiator and exactly one amino group into hbPG. All polymers exhibit degrees of branching (DB) in the range 0.53−0.58, determined by quantitative inverse gated 13C NMR spectroscopy using an 2895


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Table 1. Characterization Data of All Hyperbranched Poly(glycerol) Dendrons (hbPG-NBn2, hbPG-NH2, and hbPG-CTA) no.

compositiona

Mna (g/mol)

Mnb (g/mol)

PDIb

1 2 3 4 5 6 7 8 9

hbPG21-NBn2 hbPG32-NBn2 hbPG91-NBn2 hbPG20-NH2 hbPG32-NH2 hbPG91-NH2 hbPG21-CTA hbPG32-CTA hbPG91-CTA

1860 2670 7040

1040 2130 3590 910 1450 3660 1320 1720 4580

1.55 1.42 1.57 1.76 1.61 1.63 1.48 1.30 1.37

1940 2750 7120

a

Determined by 1H NMR. bDetermined by SEC (eluent: DMF, PEG standard).

established equation57 (compare Figure S9 and Table S1). These values are typical for the synthesis of hbPG by the slow monomer-addition (SMA) technique and confirm control over the branching mechanism during ROMBP of glycidol using the protected initiator system. Compared to our previous work,42 the removal of the benzyl protective groups could be accelerated by a using a mixture of methanol, water, and THF as solvent and a catalytic amount of hydrochloric acid. Quantitative removal of the benzyl protective groups for each hbPG-NH2 was observed by 1H and as well as SEC showing a shift in elution volume (Figure 2 and Figure S10). The characterization data of all dendrons bearing exactly one primary amine attached to each focal point are summarized in Table 1, nos. 4−6. HbPG-NH2 could be modified with exactly one RAFT chain transfer agent onto each dendron analogue’s focal point. As reported previously, despite the multitude of hydroxyl group of hbPG-NH2, its primary amine can selectively be addressed by pentafluorophenyl esters.42 The pentafluorophenyl ester of 4cyano-4-(phenylcarbonothioylthio)pentanoic acid has been reported as a useful chain transfer agent for heterotelechelic α,ω-end-group-functionalized polymers.43 The synthesis of pentafluorophenyl 4-cyano-4-((phenylcarbonothioyl)thio)pentanoate PFP-CTA as well as its spectroscopic data are described in the Supporting Information. We used ∼3 equiv of PFP-CTA in the presence of triethylamine to ensure complete conversion of each primary amine with a pentafluorophenyl

Figure 3. ESI-MS spectrum of hbPG21-NBn2 and derived hbPG21-CTA as hyperbranched polymer chain transfer agent.

ester. After complete conversion, the resulting CTA-modified dendrons could be isolated by precipitation in cold diethyl ether. The salts of pentafluorophenol triethylamine were isolated as a byproduct but could be removed by preparative size exclusion chromatography in water affording hbPG-CTA with molecular weights from 1900 to 7100 g/mol and polydispersities below 1.48 (Table 1, nos. 7−9). The color of the viscous hbPG polymers changed from colorless to purple

Figure 2. SEC elugram (solvent: DMF, RI-detector signal) and corresponding 1H NMR spectra (400 MHz, CD3OD) of all dendron analogues containing hbPG21: the benzyl-protected species hbPG-NBn2, its deprotected species hbPG21-NH2, and the corresponding macro-chain-transfer agent hbPG21-CTA. 2896


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Scheme 2. RAFT Polymerization of Tri(ethylene glycol) Methyl Ether Methacrylate (MEO3MA) and 2Hydroxypropylmethacrylamide (HPMA) with hbPG-CTA as Hyperbranched Macro-Chain-Transfer Agent

the chemically labile dithiobenzoate group. Nonetheless, again two major distribution modes can be detected representing sodium and protonated triethylamine as a counterion (hbPGCTA may contain traces of triethylamine according to the synthesis procedure). Again, the mass differences between the signals (74.04 g/mol) indicate the glycerol repeating units of the hbPG dendron. The selected signals in Figure 3b with m/z = 1293.57 and m/z = 1372.70 represent the 12-mers of hbPGCTA with sodium ([(C 3 H 6 O 2 ) 12 C 17 H 22 O 4 N 2 S 2 + Na] + calculated isotopic mass = 1293.53) and protonated triethylamine ([(C3H6O2)12C17H22O4N2S2 + C6H16N]+ calculated isotopic mass = 1372.67). The combined data of SEC, 1H NMR, and ESI-MS confirm that the primary amine of hbPGNH2 can selectively be addressed using pentafluorophenyl esters. Moreover, successful conversion to multifunctional hyperbranched macro-chain-transfer agents is evidenced; thus, each dendron is modified with exactly one single 4-cyano-4(phenylcarbonothioylthio)pentanoic amide group. The obtained hyperbranched macro-chain-transfer agents of hbPG can be used for RAFT polymerization of methacrylates or methacrylamides. Because of their excellent solubility in water, they can even be applied to RAFT polymerization in aqueous solutions.58−60 Therefore, we chose two different biocompatible monomers (tri(ethylene glycol) methyl ether methacrylate MEO3MA) and (2-hydroxypropyl methacrylamide HPMA) that are water-soluble and afford different multifunctional polymers (Scheme 2). Monomer syntheses as well as their spectroscopic data are described in the Supporting Information. B. Hyperbranched Polyglycerol-block-poly(tri(ethylene glycol) Methyl Ether Methacrylate) Copolymers with Adjustable Thermoresponsive Behavior. HbPG21-CTA was selected as initial chain-transfer agent for

after attachment of the CTA, which is typical for RAFT CTAs containing dithiobenzoate groups. To confirm the attachment of exactly one 4-cyano-4(phenylcarbonothioylthio)pentanoic acid onto each dendron, we performed three independent characterization methods: (i) SEC traces of hbPG-CTA show a change in elution volume compared to its precursor hbPG-NH2 supporting a gain of mass for the modified dendron (Figure 2). (ii) The presence of five protons of the dithiobenzoate group of hbPG-CTA at 7.96, 7.63, and 7.47 ppm can be identified by 1H NMR (Figure 2). Integration of the proton signals confirms stoichiometric attachment of exactly one single 4-cyano-4-(phenylcarbonothioylthio)pentanoic amide onto each hbPG-NH2 (Figure S11). Additionally, (iii) mass spectrometry is a crucial key method for the end-group analysis of functional polymers. In our case, ESI-MS has turned out to be the most useful and successful method for end-group analysis under mild conditions because it seemed to retain the sensitive dithiobenzoate group. Figure 3 shows the ESI mass spectra of hbPG21-NBn2 and its CTA modified analogue hbPG21-CTA. For the benzyl protected precursor dendron we can detect two major distribution modes that correspond to sodium and a proton as counterion. The mass difference between the signals (74.04 g/mol) corresponds to the glycerol units incorporated into the hyperbranched polymer. As an example, the highlighted signals for hbPG-NBn2 in Figure 3a at m/z = 1212.63 and m/z = 1190.65 correspond to the 12-mers of hbPG-NBn2 with sodium ([(C3H6O2)12C18H23O3N + Na]+ calculated isotopic mass = 1212.60) or proton ([C 18 H 20 O 3 N(C 3 H 6 O 2 ) 12 H 3 + H] + calculated isotopic mass = 1190.62). However, the occurrence of background signals in the spectra of hbPG21-CTA (Figure 3b) can be explained due to non-neglectable decomposition of 2897


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Table 2. Characterization Data of Linear−Hyperbranched Block Copolymers of hbPG-b-P(MEO3MA)

a

polymer

compositiona

mono:hbPG-CTA:initiator

pa (%)

Mtheora (g/mol)

Mna (g/mol)

Mnb (g/mol)

PDIb

A B C

hbPG21-b-P(MEO3MA)36 hbPG21-b-P(MEO3MA)46 hbPG21-b-P(MEO3MA)81

20:1:0.1 50:1:0.1 100:1:0.1

75 73 76

5 420 10 420 19 590

10 300 12 620 20 750

8 650 10 460 18 040

1.24 1.23 1.34


RAFT polymerization of MEO3MA, affording unusual polyether structures of both hbPG and linear poly(ethylene oxide) (PEG) brush-type ethers. For both block structures excellent biocompatibility has been reported in the literature.16,49 Moreover, polyMEO3MA possesses an additional thermal stimuli-responsive property.48−50 Because of its lower critical solution temperature (LCST) behavior, polymerization had to be performed at temperatures below its cloud point (52 °C) to avoid precipitation. Therefore, we chose 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) as a suitable radical initiator for polymerization at 30 °C because under these conditions, 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) has a similar half-life as AIBN at 80 °C and can still enable RAFT polymerization with efficient monomer conversion and controlled polymer chain growth.61 We observed similar results for the RAFT polymerization of MEO3MA using hbPG21-CTA as initial chain transfer agent in water. The reaction mixture was homogeneous during the polymerization. The molecular weights of the resulting linear−hyperbranched block copolymers could be adjusted by varying the monomer: hbPG-CTA ratio affording good correspondence between the targeted molecular weight calculated by monomer conversion and the observed molecular weights determined by 1H NMR and SEC (Table 2). Polymers B and C follow this trend very well. Both molecular weights determined by 1H NMR and SEC are in the regime of the theoretical molecular weight (only polymer A shows a slight deviation due to a small high molecular weight fraction according to its shouldered SEC trace; Figure 4). Nonetheless, all SEC traces of hbPG-bP(MEO3MA) block copolymers show complete conversion of the hyperbranched macro-chain-transfer agent into linear− hyperbranched block copolymers with no remaining noninitiated hbPG-CTA byproduct (Figure 4). Their molecular weights range between 10 000 and 20 000 g/mol, and their polydispersities are below 1.34, which is moderate for RAFT polymerization according to the rather disperse hbPG chain transfer agent (Table 2). Moreover, 1H NMR studies confirmed the compositions of these structures, too (Figure 4 and Figure S12). The intensities of proton signals of hbPG between 3.83 and 3.43 ppm are increased by the additional signals of the ethylene oxide side chains, and the signals of the ester protons at 4.13 ppm, the methyl ether at 3.40 ppm, and the signals of the methacrylate backbone of the P(MEO3MA) block between 2.35 and 0.60 can be detected as well (Figure 4 and Figure S12). They all support successful incorporation of both monomers into the obtained structure. Besides its demanding topologic composition, we were also interested in the stimuli-responsive properties of the hbPGP(MEO3MA) block copolymers. While the hyperbranched polyglycerol block exhibits ideal solubility in water, we observed its influence on the solubility behavior of the P(MEO3MA) block while increasing the temperature of a 10 mg/mL aqueous solution (Figure 5). Interestingly, all block copolymers A, B, and C had a cloud point with a sharp transition temperature.

Figure 4. 1H NMR spectra (400 MHz, CD3OD) and SEC elugrams (solvent: DMF, RI-detector signal) of the hbPG21-CTA and resulting hbPG21-b-P(MEO3MA) block copolymers.

Figure 5. Cloud point measurements of hbPG21-b-P(MEO3MA) (heating trace) and their corresponding cloud points (defined as 50% transmittance).

We defined 50% transmittance of the temperature-dependent turbidity measurement in this study as cloud point, which is indicative for an LCST, and found that the values for the polymers A−C are all in the regime of the LCST for the P(MEO3MA) homopolymers as reported by the literature (52 °C).48 It is well-known that the cloud point for a given type of polymer depends to some degree on molecular weight, main2898


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Table 3. Characterization Data of Linear−Hyperbranched Block Copolymers of hbPG-b-P(HPMA) polymer D E F G a

compositiona hbPG21-b-P(HPMA)9 hbPG32-b-P(HPMA)93 hbPG91-b-P(HPMA)194 hbPG91-b-P(HPMA)325

mono:hbPG-CTA:initiator 20:1:0.1 120:1:0.1 350:1:0.1 450:1:0.1

p (%) a

38 67a 43b 62b

Mtheora (g/mol)

Mna (g/mol)

Mnc (g/mol)

PDIc

3090 14 260 28 670 47 100

3230 16 070 34 860 43 650

4200 22 890 44 810 54 560

1.34 1.15 1.24 1.28

Determined by 1H NMR. bDetermined gravimetrically. cDetermined by SEC (eluent: DMF, PEG standard).

monomer:hbPG-CTA ratio, affording good correspondence between the theoretical molecular weight calculated by monomer conversion and the observed molecular weights determined by 1H NMR and SEC (Table 3). The hbPG-CTAs could be used for growing both short and long linear P(HPMA) polymer chains from hbPG-CTA affording molecular weights between 3000 and 44 000 g/mol and polydispersities below 1.34. From the SEC traces we observed complete conversion of the used hbPG-CTAs to the resulting block copolymers (Figure 6). Moreover, the 1H NMR spectra

chain end groups, tacticity, concentration, and ionic strength. However, the phase transitions for poly(oligo ethylene glycol) methyl ether methacrylates are reported to be rather insensitive to external physical conditions.49 Only their molecular composition as block copolymers may influence their solubility behavior effectively. That is what we observed for the resulting linear-hyperbranched hbPG-b-P(MEO3MA) block copolymers in Figure 5. While keeping the proportion of the nonthermoresponsive hbPG block at the same level, but increasing the ratio of P(MEO3MA), we detect a decrease of the determined cloud point. For lower ratios of the soluble hbPG block to the thermoresponsive P(MEO3MA) block there are fewer hydroxyl groups of the molecule available for hydrogen bonding with water molecules in the aqueous phase. Therefore, the molecule is less stabilized in water at elevated temperature, which leads to minor solubility in water and finally to macroscopic turbidity. Consequently, the cloud point for polymer A with a composition of hbPG21-b-P(MEO3MA)36 is higher (52.6 °C) than for polymer C with a composition of hbPG21-b-P(MEO3MA)81 (47.6 °C). We can correlate the dependency between the proportion of the two blocks and the resulting clouding behavior approximately linearly, as polymer B with a composition of hbPG21-b-P(MEO3MA)46 has a cloud point at 50.6 °C that is right in between the two other species. Consequently, these data not only ensure the synthesis of multifunctional linear-hyperbranched hbPG-b-P(MEO3MA) block copolymers by the RAFT polymerization technique but also underline the possibility to tune the resulting block copolymer’s cloud point precisely by adjusting the composition of the block structure. Thus, one may tailor the physicochemical properties of thisaccording to its monomer compositionhighly biocompatible material for the desired application. C. Hyperbranched Polyglycerol-block-poly(2hydroxypropyl methacrylamide) Copolymers and Their End-Group Modification for Protein or Dye Conjugation. In a second study hbPG-CTAs with molecular weights in the range of 1800−7000 g/mol were selected and applied to the RAFT polymerization of methacrylamide monomers. We chose HPMA as another biocompatible monomer, which also enables polymerization in water62 and affords interesting linear−hyperbranched structures that can be seen as novel multifunctional alternatives to poly(ethylene glycol).52 For both homopolymers there is no LCST behavior known in water. Thus, water could be used as solvent for the reaction, and as radical initiating system 4,4′-azobis(4-cyanovaleric acid) was chosen for polymerization at 70 °C. Complete initiation with efficient monomer conversion and controlled polymer chain growth was observed according to the monomer conversions, and the resulting linear-hyperbranched block copolymers D−G were isolated after purification. All block copolymers had a rather voluminous powdery morphology with a red color that differed from the hbPG-CTA precursor polymers obtained as viscous red oils. Again, we were able to adjust the molecular weights of the resulting block copolymers by varying the

Figure 6. 1H NMR spectra (400 MHz, CD3OD) and SEC elugrams (solvent: DMF, RI-detector signal) of the hbPG21-CTA and hbPG91CTA together with the resulting hbPG-b-P(HPMA) linear−hyperbranched block copolymers.

of hbPG-b-P(HPMA) also show additional signals at 3.25−2.73 ppm that derive from the α-amide protons of the P(HPMA) block. Additionally, proton signals of the methacrylamide backbone together with the methyl group of the P(HPMA) block between 2.35 and 0.60 ppm are detected, and the intensities of proton signals of hbPG between 4.23 and 3.40 ppm are increased by the signals of the hydroxymethylene group of the P(HPMA) block (Figure 6 and Figure S13). Summing up, this data confirms that the hbPG−CTA system we synthesized is also useful for RAFT block copolymerization using methacrylamide monomers, 2-hydroxypropylmethacrylamide in our case, affording linear−hyperbranched block copolymer architectures. Both blocks are well-known as highly 2899


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Scheme 3. End-Group Modification of Linear−Hyperbranched hbPG-b-P(HPMA) Block Copolymers with Biotin and Texas Red

Figure 7. 1H NMR spectra (400 MHz, DMSO-d6) of the hbPG21-b-P(HPMA)9 with its dithiobenzoate end group and after modification with BiotinMTS as hbPG21-b-P(HPMA)9-Biotin.

applications.44−46 We applied this method to the linear− hyperbranched structures, enabling bioconjugation to proteins and reversible attachment of dyes as drug model compounds, which may open further application possibilities in the field of advanced polymer drug delivery systems (Scheme 3). In a first study, hbPG21-b-P(HPMA)9 was modified at its end group with Biotin-MTS for subsequent conjugation to avidin. The biotin−avidin system is a universal approach, as it represents a general tool for conjugation to further functional proteins, which may act as recognition domains or therapeutically active compounds and can be produced recombinantly as fusion proteins with the biotin-binding domain of avidin.64−66 We chose hbPG21-b-P(HPMA)9 having the shortest linear block attached to the hyperbranched topology as a synthetically challenging topology for this purpose. We aimed at the question of whether the length of the polymer’s linear block is already sufficient for the biotin end group to access the binding domain of avidin, despite the sterically bulky hbPG block. Following a literature procedure,46 the aminolysis of the

biocompatible structures for various biomedical applications. This unusual combination opens new pathways for e.g. polymer therapeutics with unconventional topologies, which were not accessible to date. Taking this approach into account, a possibility to introduce bioactive compounds onto the hbPG-b-P(HPMA) structure is required to serve as novel drug carrier. For a precise design, the selective modification of e.g. one functionality should be preferred instead of conjugation to the multiple hydroxyl groups, which cannot be discriminated between the two blocks. In this case, end-group modification of RAFT block copolymers opens another pathway for selective introduction of additional bioactive moieties on the linear−hyperbranched architecture. Because of the presence of dithiobenzoate esters at the end of each linear ω-chain end, aminolysis of the linear−hyperbranched block copolymers allows selective orthogonal ω-endgroup modification.63 This method has been used widely for heterobifunctional α,ω-end-group modification of linear block copolymers derived by the RAFT technique for various 2900


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dithiobenzoate group of hbPG21-b-P(HPMA)9 with 40 equiv of n-propylamine was performed in presence of about 4 equiv of Biotin-MTS. The conversion of this reaction was followed by the loss of the red color of the dithiobenzoate group. After purification end-group-modified hbPG21-b-P(HPMA)9-Biotin was characterized by SEC and 1H NMR (Figure S15 and Figure 7). The data obtained by SEC in DMF revealed a slight increase of molecular weight after attachment of the biotin group onto hbPG21-b-P(HPMA)9 (Table 4). In the SEC trace,

corresponding integrals can be found in Figure S14, supporting the targeted end-group modification. Having confirmed the molecular structure of hbPG21-bP(HPMA)9-Biotin, its applicability for the interaction with avidin as its counterpart was investigated. Here, native avidin (derived from chicken egg white was preferred over recombinant streptavidine with a 103 higher affinity constant (10−15 M).67 Avidin has four binding sites for biotin and biotin conjugates, respectively. The resulting bioconjugates were analyzed using two independent characterization methods (Figure 8b). Aqueous size exclusion chromatography (SEC) in phosphate buffered sodium chloride solution (PBS) is a versatile method in biochemistry to determine a protein’s molecular weight in its native state. Because of the high affinity constant of the biotin−avidin system, the hbPG21-b-P(HPMA)9-Biotin−avidin conjugate can elute together at decreased elution volumes compared to the native avidin protein itself (Figure 8a). An additional signal at higher elution volume was detected because an excess of biotinylated hbPG21b-P(HPMA)9 was used to ensure quantitative polymer attachment to avidin. To further confirm the obtained results, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as a routine method for protein molecular weight characterization in biochemistry54 was applied. The presence of SDS breaks down the conformational structure of proteins, affording linearized proteins with negative charge-tomass ratios. Avidin can be applied to SDS PAGE without losing its binding affinity to biotin68 (for its recombinant derivative streptavidin, it has even been proposed that the biotin molecule stabilizes its binding pocket, thus making it inert toward the denaturing conditions of the detergents).69 To this end, we performed SDS PAGE with the biotinylated hbPG21-bP(HPMA)9-avidin conjugate using a 10% separation gel with 5% stacking gel (Figure 8c). Native avidin moves during gel electrophoresis and gives rise to a rather diffuse band probably due to different glycosylation pattern in the natural product derived from chicken egg white.70 When conjugated to hbPG21b-P(HPMA)9-Biotin, a sharp band is detected in the stacking gel, which does not move into the separating gel during gel

Table 4. Characterization Data of End-Group-Modified Linear−Hyperbranched Block Copolymers of hbPG-bP(HPMA) polymer

compositiona

Mna (g/mol)

Mnb (g/mol)

PDIb

H I

hbPG21-b-P(HPMA)9-Biotin hbPG91-b-P(HPMA)194TxRed

3 410 35 400

3 730 46 130

1.28 1.21

a


a small shoulder can be detected at higher elution volumes (Figure S15). We propose that this effect is due to nonideal interaction of the biotinylated linear−hyperbranched block copolymer with the HEMA column material of the SEC in DMF. Additional SEC experiments using HFIP as eluent and modified silica as column material revealed a narrow, monomodal molecular weight distribution without any shoulder (Figure S15). 1H NMR analysis of the end-groupmodified polymer also demonstrated successful removal of the dithiobenzoate end group and incorporation of one single Nbiotinyl aminoethyl disulfide moiety at the linear end group of the hbPG21-b-P(HPMA)9 polymer. A detailed peak assignment of all characteristic signals from the 1H NMR spectrum can be found in Figure 7. The two cyclic α-amide protons of the biotin group at 4.30 and 4.13 ppm show quantitative attachment of one single biotin group at the linear end of the hbPG21-bP(HPMA)9 polymer. Additionally, a detailed analysis of the

Figure 8. Bioconjugation of hbPG21-b-P(HPMA)9-Biotin bearing one single biotin group at the end of the each P(HPMA)-block to avidin. (A) Aqueous size exclusion chromatography in phosphate-buffered sodium chloride solution confirms conjugation of hbPG21-b-P(HPMA)9-Biotin to the protein (3) by shift of the protein trace (1) to higher elution volume in presence of excess hbPG21-b-P(HPMA)9-Biotin (2). (B) Scheme for the conjugation of hbPG21-b-P(HPMA)9-Biotin with avidin. (C) SDS-PAGE of avidin in presence of hbPG21-b-P(HPMA)9-Biotin reveals a partly charged polymer−protein conjugate that cannot penetrate into the separating gel (lane 3) compared to the free avidin (lane 1) (diffuse band due to glycosylation of avidin from chicken egg white). The polymer itself (lane 2) cannot be detected by Coomassie Blue staining. The junction between stacking gel (top) and separating gel (bottom) is indicated by a dashed line. 2901


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electrophoresis. This is additional evidence for successful conjugation of avidin to the biotinylated polymer (size increase). As an excess of hbPG21-b-P(HPMA)9-Biotin was used, no free avidin can be detected. Summing up, characterization by SEC and SDS PAGE of the polymer−protein conjugate in its native and denatured state prove successful polymer end-group modification of hbPG-b-P(HPMA)-Biotin and its conjugation to avidin. This approach opens new pathways for protein conjugation with advanced linear− hyperbranched topologies as novel protein delivery systems. Moreover, drug delivery is another promising biomedical application for linear−hyperbranched block copolymers. Therefore, end-group modification using hbPG91-b-P(HPMA)194 for attaching Texas Red as drug model compound onto the polymer was performed in a second study. In contrast to other therapeutically relevant compounds, Texas Red enables simple detection. HbPG91-b-P(HPMA)194 with a rather high molecular weight was selected for this model reaction because therapeutic active polymers with high molecular weight can show an enhanced circulation time.71 To obtain hbPG-b-P(HPMA)TxRed, the synthesis of the end-group-modified linear− hyperbranched block copolymer was performed with Texas Red-MTS in analogy to the modification with biotin. To prevent contamination of the final product with free dye, several purification methods were applied with tolerable loss of yield. These procedures are mandatory with respect to biomedical applications, since unbound drugs may cause severe adverse effects.72 HbPG91-b-P(HPMA)194-TxRed was obtained as a purple powder with similar molecular weight and PDI compared to the precursor polymer (Table 4). The SEC traces of both polymers in DMF overlap perfectly (Figure S17), assuring no influence on the molecular architecture of the polymer during the end-group modification reaction. Because of its high molecular weight, detailed 1H NMR analysis of hbPG91-b-P(HPMA)194-TxRed in analogy to hbPG21-b-P(HPMA)9-Biotin is not possible (Figure S16). Well-defined signals of the Texas Red molecule in the spectrum cannot be detected. The obtained 1H NMR data reveal retention of the polymer composition after end-group modification. Still, optical appearance (purple color) and 1H NMR results from the biotin modification (Figure S14) ensure the effectiveness of the modification reaction with MTS derivatives. Moreover, additional thin layer chromatography (TLC) experiments in acetone as an eluent have been applied to show successful attachment of the dye (Figure 9). HbPG91-b-P(HPMA)194TxRed remains at the starting point because the polymer is not soluble in this solvent. The fluorescence of the acetone soluble Texas Red is detected at the starting point, too, showing successful conjugation. MTS derivatization results in the formation of disulfides as presented in Scheme 3. Disulfides are biologically relevant degradable moieties. They show high stability in the oxidative milieu of the extracellular environment but can be degraded intracellularly by glutathione, the most abundant reducing agent in almost all cell types.73,74 Thus, polymer−drug conjugates linked by disulfide can be a useful tool for selective release in the cytosol or glutathione rich buffers.75,76 This opens options for advanced drug release systems after cell uptake.77 Applying cytosolic redox conditions to hbPG-b-P(HPMA)-TxRed systems, a sample of hbPG91-b-P(HPMA)194-TxRed was incubated with 2 mM glutathione. In the subsequent TLC experiment the red fluorescent dye does not remain at the starting point but elutes with the solvent (Figure 9). To this

Figure 9. Redox-cleavable modification of hbPG91-b-P(HPMA)194TxRed bearing Texas Red as drug model compound via a disulfide bonde at the end of the P(HPMA)-block (A). In the presence of 2 mM glutathione (in the regime of typical intracellular concentrations of glutathione) the disulfide bound can be cleaved, and the fluorescent dye is released as visualized by thin layer chromatography (TLC, elunet: acetone, UV visualization at 365 nm). While the dye covalently attached to the polymer remains at the baseline of the spotted sample (A), the sample treated with 2 mM glutathione releases the dye (C). No fluorescence can be detected at the baseline (B).

respect, hbPG-b-P(HPMA)-TxRed is able to release its covalently attached dye quantitatively, as no remaining fluorescence can be visualized at the starting point. These studies support reversible end-group modification of the linear−hyperbranched block copolymers synthesized by hbPG-CTA. After modification with MTS reagents, the obtained disulfide-connected moieties can selectively be released by the intracellular glutathione redox system. Moreover, they open new pathways for possible application as advanced drug delivery systems for controlled intracellular drug release.

■

CONCLUSION We have presented the synthesis of a novel monofunctional hyperbranched macro-chain-transfer agent (CTA) based on hyperbranched polyglycerol (hbPG). Using the pentafluorophenyl ester of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, we can selectively attach the CTA functionality to hbPG with a single amino functionality, affording the first hyperbranched CTA reported to date. With its bulky topology this macro-CTA can be applied to RAFT polymerization of functional methacrylates and methacrylamides affording multifunctional linear−hyperbranched block copolymers, e.g., hbPGb-P(MEO3MA), whose cloud point can be adjusted according to the block copolymer composition, or hbPG-b-P(HPMA), whose blocks are well-known for their high biocompatibility. Moreover, the focal end group at the linear block can be modified selectively using functional methanethiosulfonates (MTS) providing disulfide linkages. Clearly, the excellent biocompatibility of hbPG and P(HPMA) opens options for biomedical applications using such complex polymer architectures. MTS end-group modification enabled precise attachment of one biotin group onto the linear−hyperbranched polymer for protein conjugation to avidin onto hbPG-b-P(HPMA). Under similar conditions, the attachment of Texas Red as a 2902


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drug model compound was achieved. Because of the disulfide linkage, Texas Red was redox-cleavable under physiological conditions (cytoplasm) by glutathione (GSH). Summing up, the universal applicability of the hyperbranched macro-chaintransfer agent represents an important progress for the multifunctional linear−hyperbranched block copolymers. Based on the first examples presented in this work, this concept may serve as promising platform for demanding applications in the field of polymer-based diagnostics or therapeutics.

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ASSOCIATED CONTENT

S Supporting Information *

Syntheses of pentafluorophenyl 4-cyano-4((phenylcarbonothioyl)thio)pentanoate, PFP-CTA, tri(ethylene glycol) methyl ether methacrylate, MEO3MA, and 2-hydroxypropylmethacrylamide, HPMA; additional characterization data in Table S1 and Figures S1−S17. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS L.N. gratefully acknowledges financial support by the Max Planck Graduate Center (“MPGC”) and the Fond der Chemischen Industrie (“FCI”). C.S. thanks the Graduate School of Excellence Materials Science in Mainz (“MAINZ”) for funding. Both authors also acknowledge technical assistance by Iris Overhoff and Julia Brübach.

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Combining Ring-Opening Multibranching and RAFT Polymerization

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