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Macromolecules 2007, 40, 2486-2502
Synthesis and Characterization of End-Functionalized Cylindrical Polyelectrolyte Brushes from Poly(styrene sulfonate) Karen Lienkamp, Lucie Noe´ , Marie-He´ le` ne Breniaux, Ingo Lieberwirth, Franziska Groehn, and Gerhard Wegner* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ReceiVed NoVember 7, 2006; ReVised Manuscript ReceiVed January 30, 2007
ABSTRACT: Cylindrical polyelectrolyte brushes from poly(styrenesulfonate) were synthesized by polymer analogous hydrolysis from the corresponding dodecyl and ethyl ester brushes. These brushes were characterized in solution (GPC-MALLS, static and dynamic light scattering, SANS, and 1H NMR), and on solid interfaces (AFM and TEM). It was shown that the cylindrical brushes may form extended aggregates in solution. The aggregation behavior and the size and shape of the aggregates depend on the side chain length and the degree of saponification. For samples with identical backbone and side chain length, but varying degrees of ester hydrolysis, marked differences in the aggregation behavior were observed. Cylindrical end-functionalized polyelectrolyte brushes from poly(styrenesulfonate) were synthesized by a combination of anionic polymerization yielding an end-functionalized ATRP macroinitiator with a positively charged head group, atom transfer radical polymerization, and polymer analogous hydrolysis. The brushes and their precursors were characterized in solution by MALDI-TOF MS, 1H NMR, GPC-MALLS, static and dynamic light scattering, and cryo-TEM. These brushes were found to form complexes with negatively charged latex particles. They may be discussed as models to mimic proteoglycan in the proteoglycan-hyaluronic acid complex.
Introduction Self-organizing systems are ubiquitous in nature, the doublehelix of DNA and the folding of protein structures being common examples. Another important example of selforganization in the human organism is the formation of proteoglycan aggregates with hyaluronic acid (Figure 1).1 These aggregates are found throughout all extracellular compartments. Because of their ability to absorb large amounts of water and form gels, these complexes form a tissue that can undergo reversible compression in articular cartilage, giving elasticity to blood vessels and contributing to the structural integrity of many tissues such as skin or brain.2 The most abundant proteoglycan-hyaluronic acid aggregate found in nature is the aggrecan-hyaluronic acid aggregate. Aggrecan is a linear polypeptide chain carrying a large number of anionic polysaccharide side chains, thus forming an anionic polymer brush. The linker between aggrecan and hyaluronic acid is a positively charged, claw-shaped protein, which is covalently attached to the aggrecan molecule.1,3 Hyaluronic acid (hyaluronan) is a glycosaminoglycan consisting of a disaccharide repeat unit (Dglucuronic acid and N-acetylglucosamine with a molar mass of approximately 106-107 g mol-1. Because of its polyanionic nature, it is a linear rod-like molecule of 2-25 µm length.4 Besides being a structural component of connective tissues, it forms loose hydrated matrices that enable cell division and migration, as well as adhesion of immune cells, and it plays a role in intracellular signaling.2 The importance of hyaluronan in these processes is due to the ability of many proteins to bind to it. One such protein is contained in proteoglycans. These are anionic polymer brushes consisting of a core protein, to which heteropolysaccharide side chains are attached. They are produced in the cell by the Golgi apparatus by glycosilation of the core protein: first, a tetrasaccharide is attached, then glycosyl transferases add one sugar molecule after the other (which is * Corresponding author. Fax: +49 6131 379 100. E-mail: wegner@ mpip-mainz.mpg.de.
much like a “grafting-from” reaction), followed by sulfonation of the sugar side chains.3 The side chains of aggrecan, (the proteoglycan found in human cartilage), consist of chondroitin sulfate and keratane sulfate. A typical aggrecan brush consists of 150 side chains, each having a molar mass of 20 000 g mol-1,1 making a total of 3 × 106 g mol-1 for the whole molecule. In living organisms, aggrecan and hyaluronic acid are synthesized and separately released into the extracellular compartment, where the aggrecan molecules bind to the hyaluronic acid molecule. The aggregates are nondissociating and nondisplaceable under physiological conditions.5 The binding between the link protein and hyaluronan is mainly due to the formation of ionic bonds between about eight basic amino acids in the protein and the carboxylic acid groups of hyaluronan.6 Besides the ionic component of the binding, there is evidence that hydrogen bonding and van der Waals forces also play a role.7 In order to understand the unusual mechanical properties of these aggregates, which act as biological lubricants, and to mimic them in synthetic products, the aim of this work was to produce model compounds for the proteoglycan-hyaluronic acid complex (Figure 1). As a model for the proteoglycan, endfunctionalized poly(styrenesulfonate) (PSS) polyelectrolyte brushes with a positively charged linker were synthesized and characterized. In an attempt to model the proteoglycan-hyaluronic acid aggregates, the complexation behavior of these end-functionalized brushes toward negatively charged species was investigated. The structure of the materials was analyzed by imaging methods (cryo-TEM) and scattering techniques (static and dynamic light scattering). Before attempting to synthesize such a complicated polymeric species, we had to review the existing methods to synthesize cylindrical polymer brushes (“bottle brushes”). These consist of a linear main-chain and polymeric side chains. Because of the highly branched architecture of these materials, their bulk and solution properties are exceedingly different from those of their linear analogues of comparable molecular weight: the
10.1021/ma062569b CCC: $37.00 © 2007 American Chemical Society Published on Web 03/09/2007
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Figure 1. Cartoon representation of the proteoglycan-hyaluronic acid aggregates in human cartilage (left) and a simplified synthetic model system for this structure (right).
hydrodynamic radii are smaller, and the solution conformation depends on the length ratio of the side chains and the backbone.8-11 The backbone being an “entropic spring” that strives for a coiled shape, the excluded volume interaction of the side chains lead to a more or less stretched molecular geometry.12 It was confirmed by SAXS,13 AFM,14 and rheology15 that polymer brushes do not entangle in bulk. Synthetic approaches toward polymer brushes are “grafting onto” ,16-19 “grafting from” ,20-25 and “grafting through” .26-31 Cylindrical PSS brushes thus far reported in the literature were obtained by sulfonation of poly(styrene) (PS) brushes: Schmidt et al.32 obtained PSS brushes by sulfonation of PS poly(macromonomers). Only very recently, Fernyhough et al.33-35 described the sulfonation of PS comb polymers obtained by “grafting onto” . However, polymer analogous sulfonation is never quantitative and often leads to structural imperfection (cross-linking and degradation) due to the harsh reaction conditions, which was also reported by Fernyhough et al. (increased polydispersity after sulfonation). We therefore synthesized cylindrical PSS brushes by hydrolysis of the corresponding esters,36 as described below. The advantage of this method is that the complications of polymer-analogous sulfonation, i.e., incomplete and random sulfonation, cross-linking and degradation are avoided. The first part of this paper is dedicated to the synthesis of these structures, as depicted in Scheme 1. The structure of the materials was investigated by imaging methods (TEM, AFM) and scattering techniques (static and dynamic light scattering, SANS). The second part of the paper covers the end-functionalization (Scheme 2). We would like to point out that the term “polyelectrolyte brush” is used rather indiscriminately in the literature for 1D-, 2D- and 3D-brushes [see ref 36]. The correct term for the type of molecule discussed in this paper is “cylindrical brush” , while “brush” usually refers to 2D- or 3D-brushes made up of chains attached to a 2D surface or a 3D object. In a previous paper, polymerization of styrenesulfonate ethyl and dodecyl ester via ATRP (“grafting from” ) to obtain cylindrical polymer brushes was described.36 The PSS brushes discussed in this paper were derived from these materials. For the reader’s convenience, the molecular parameters determined
for these samples are summarized in Table 1 (Sample Nomenclature: X-Y-DZ or X-Y-EZ, X ) brush backbone (3 ) 570 repeat units, 11 ) 1600 repeat units), Y ) grafting density, D ) dodecyl ester, E ) ethyl ester, Z ) sample identification number)). Depending on the hydrolysis method used (a-d, see Scheme 1), the suffix, a-d, is added to the sample name of the hydrolyzed species to clarify the sample history. Unless otherwise stated, the free acid form is referred to. Experimental Part Synthesis. All chemicals were bought from Aldrich, Fluka, or Acros and were used as received. Styrenesulfonate dodecyl ester was synthesized as described elsewhere.37 Styrenesulfonate dodecyl ester brushes 1 (PSSD) and styrenesulfonate ethyl ester brushes 2 (PSSE) were synthesized as described elsewhere.36 Poly(styrenesulfonate) brushes (3a-3d) were obtained via four hydrolysis procedures: (a) 100 mg (0.29 mmol SSD) of polymer was dissolved in a minimum amount of THF. Then 96 mg (2.9 mmol) of KOH (85%) was dissolved in Milli-Q H2O and added to this solution. The reaction mixture was kept at 60 °C for 3 days. It was then dialyzed vs 10 L Milli-Q water, which was exchanged every 24 h, until the conductivity of the water dropped to 0.1 µS after 12 h (membrane pore size 10 000 g/mol). Freeze-drying yielded the desired polyelectrolyte as the potassium salt. 1H NMR (D2O, 80 °C): δ ) 7.8-8.5 (Hmeta), 7.5-6.7 (Hortho), 4.3-5.5 (HCH2 ester), 1.1-1.4 (HCH3 ester) 1.4-1.9 (Hβ-ω CH2 ester), 0.9-3.0 (Hbackbone). (b) First, 250 mg (0.71 mmol SSD) was dissolved in 20 mL of CHCl3. Then 1 mL (7.10 mmol) of triethylamine was added. The reaction mixture was refluxed for 12 h and cooled to room temperature. The solvent was removed by vacuum evaporation. The reaction product was dissolved in 50 mL of Milli-Q H2O. It was then dialyzed and freeze-dried as described above to obtain the PSS ammonium salt. 1H NMR (D2O, 80 °C): δ ) 7.8-8.5 (Hmeta), 7.56.7 (Hortho), 0.9-3.0 (Hbackbone). (c) First, 250 mg (0.71 mmol SSD) were dissolved in 20 mL of CHCl3. Then, 1 mL (7.10 mmol) of triethylamine was added. The reaction mixture was refluxed for 3 h and cooled to room temperature. The solvent was removed by vacuum evaporation. The reaction product was dissolved in 50 mL Milli-Q H2O. Then, 240 mg (7.10 mmol) KOH (85%) was added. The reaction was refluxed for 48 h. It was then brought to pH ) 1 with HCl and refluxed for 48 h. The solution was cooled to room temperature, dialyzed and freeze-dried as described above to yield the potassium salt. 1H NMR (D2O, 80 °C): δ ) 7.8-8.5 (Hmeta),
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Macromolecules, Vol. 40, No. 7, 2007 Scheme 1. Saponification of Polymer Brushes (1 for Dodecyl Ester and 2 for Ethyl Ester)a
a Hydrolysis with KOH (a) yields polyelectrolyte brush 3a; 3b is obtained by saponification with N(C2H5)3. This is also the first reaction step toward 3c (c-1), which is followed by treatment with NaOH (c-2) to yield the product 3c. 3d is obtained by reaction with TMS-iodide (d-1) followed by hydrolysis with NaOH (d-2). In each case, the product is subject to ion exchange to yield 4.
7.5-6.7 (Hortho), 0.9-3.0 (Hbackbone). (d) First, 150 mg (0.43 mmol SSD) was dissolved in 20 mL of CH2Cl2. Then, 856 mg (4.30 mmol) of trimethylsilyl iodide was added. The reaction mixture was refluxed for 12 h and cooled to room temperature. The solvent was removed by vacuum evaporation. The reaction product was dissolved in 50 mL of Milli-Q H2O. Then 100 mL of 2 M NaOH was added. The reaction was stirred for 4 h. It was then dialyzed and freeze-dried to yield the sodium salt as described above. 1H NMR (D2O, 80 °C): δ ) 7.8-8.5 (Hmeta), 7.5-6.7 (Hortho), 0.93.0 (Hbackbone). PSSE brushes were hydrolyzed analogously: 50 mg
(0.24 mmol SSE) was dissolved in 20 mLof CH2Cl2. Then 960 mg (2.40 mmol) of trimethylsilyl iodide was added. Following procedure d yielded the desired polyelectrolyte sodium salt. 1H NMR (D2O, 80 °C): δ ) 7.8-8.5 (Hmeta), 7.5-6.7 (Hortho), 0.9-3.0 (Hbackbone). Ion exchange to obtain 4: 100 mg of the polyelectrolyte brush salt was dissolved in 100 mL of Milli-Q H2O and passed slowly (1 drop/min) over an ion-exchange resin (Amberlyte IR120(+)). The solution was freeze-dried, redissolved in 50 mL of Milli-Q H2O, and then dialyzed vs 10 L Milli-Q water, which was exchanged every 24 h, until the conductivity of the water dropped
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Scheme 2. Synthesis of End-Functionalized Polyelectrolyte Brushesa
a After initiation of anionic polymerization with a functionalized initiator (5), the macroinitiator precursor 6 is obtained. Quarternization and bromination yields the macroinitiator 8, from which the polymer brush 9 is obtained by ATRP. Saponification yields the end-functionalized polyelectrolyte brush 10.
Table 1. Molecular Parameters of the Parent Esters (Sample Nomenclature: X-Y-DZ or X-Y-EZ, X ) Brush Backbone (3 ) 570 Repeat Units, 11 ) 1600 Repeat Units), Y ) Grafting Density, D ) Dodecyl Ester, E ) Ethyl Ester, Z ) Sample Identification Number)a 1H
GPC-MALLS sample 3-30-D1 11-30-D2 11-20-E1 11-40-E4 11-60-E1 a
Mw × 10-3 [g/mol] PSSD esters do not elute from column 2739 3256 1577
Mn × 10-3 [g/mol] 18 2377 1490 702
Mw/Mn
NMR
Rg,z [nm]
recovery rate
nn, GPC-MALLS
nn, NMR
Mn × 10-3 [g/mol]
51 64 61
1.03 1.03 0.87
32 9 3
19 40 24
1388 4848 4391
1008 1.2 2.1 2.3
Analytical data from GPC-MALLS (DMF, 0.1 M NaNO3) and 1H NMR (CD2Cl2 for D samples, acetone-d6 for E samples)
to 0.1 µS after 12 h (membrane pore size 10 000 g/mol). Freezedrying yielded the desired poly(styrene sulfonic acid) brushes. The functionalized initiator 5 was synthesized as described by Wittig38 and Quirk:39 8.36 g (20.6 mmol) of triphenylphosphonium iodide was dissolved in 100 mL of dry diethyl ether under argon atmosphere. Then 16.1 mL (20.6 mmol) of methyl lithium was slowly added at room temperature. After an intense gas development, a colorless precipitate of lithium iodide appeared. The solution was stirred for 4 h and then filtered with a reverse frit under argon. The yellow solution was cooled to 0 °C. A 5.00 g (18.7 mmol) sample of 4,4′-bis(dimetylamino)benzophenone was suspended in 100 mL of dry THF under argon atmosphere. The blue suspension was added slowly to the cooled ylid solution with a steel transfer needle. The reaction mixture was allowed to warm to room temperature over night. In was then quenched with 30 mL of methanol and 10 mL of water. After cessation of the gas development, the solution was filtered. The solvent was removed by vacuum evaporation. The remaining solid was three times recrystallized from methanol and dried in vacuum. A white solid was obtained. 1H NMR (CDCl3): δ ) 7.25 (d, 4H, H2), 6.65 (d, 4H, H3), 5.15 (s, 2H, H1), 2.90 (s, 12 H, H4); MS (70 eV): 266.6 (M+), 267.6 (M + H+), 268.5 (M + 2H+), 530.9 (dimer); Anal. Calcd for C16H19N2 (239.34): C, 81.16; H, 8.32; N, 10.52. Found: C, 81.05; H, 8.36; N, 10.55. Synthesis of end-functionalized poly(p-methylstyrene) 2: Following standard procedures for anionic
polymerization, p-methylstyrene was and purified by high-vacuum distillation from fluorenyl-lithium. THF (200 mL) was distilled into the reactor. 0.4 mL (c ) 0.13 mol/L, 0.05 mmol) sec-butyl lithium were added to 133 mg (0.5 mmol) 1,1-di(4-dimethylaminophenyl)ethylene) (5). At -78 °C, p-methylstyrene was added to the deep red solution. The reaction completed in 3 h. Precipitation into methanol yielded 6. 1H NMR (CD2Cl2): δ ) 6.6-6.7 (Harom), 6.16.5 (Harom), 1.9-2.2 (HCH3), 0.9-1.9 (Hbackbone). Anal. Calcd for C22H32N2(C9H10)n (324.51 + (118.18)n): C, 91.45; H, 8.53; N, 0.01. Found: C, 91.47; H, 8.64; N,
