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Chapter 2
Polypeptide Diblock Copolymers: Syntheses and Properties of Poly(N-isopropylacrylamide)-b-Polylysine
Abstract
A hydrolysis-resistant amide-linkage hetero-functional initiator was synthesized and used successfully for polymerization of well-defined rod-coil block copolymers poly(N-isopropylacrylamide)-b-poly(Z-L-lysine) (PNIPAm-b-PZLys) by combination of atom transfer radical polymerization (ATRP) and amine hydrochloride mediated ring-opening polymerization (ROP). The ATRP of NIPAm was carried out at 0 °C using CuBr/Me6TREN complex in 2-propanol and resulted in narrow polydispersity and high monomer conversion.
The amine hydrochlorides has been replaced the primary amine in PNIPAm macroinitiator resulting in a well-controlled ROP of Nε-(carbobenzoxy)-L-lysine N-carboxyanhydride in DMF at 20 °C. These amphiphilic block copolymers are able to form universal micelle morphologies of spherical micelles, wormlike micelles, and vesicles by varying the polymer compositions and the helicogenic common solvents. From synchrotron SAXS, WAXS, and TEM results, the PNIPAm-b-PZLys microphase self-assembly morphology in solid state is a hierarchical lamellar-in-hexagonal structure. After removing the protective ε-benzyloxycarbonyl group, the dual stimuli-responsive behaviors of the PNIPAm-b-PLys investigated by nuclear magnetic resonance spectroscopy in aqueous solution resulted in either coil-to-helix or coil-globule transition by changing the environmental condition of elevating the temperature or increasing the pH value.
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2-1 Introduction
Self-assembly of block copolymers, driven by the incompatibility of constituents, into ordered structures in solution and solid state in the sub-micrometer range provide a route to hierarchical nanostructure materials with a variety of potential applications.1 Bio-mimetic stimuli-responsive block copolymers capable of self-assembly in aqueous solution are particularly interesting as their promising potential in variety of applications such as in drug delivery,2 in biotechnology,3 and in the development of sensors.4 These “smart” materials can reversibly change their physicochemical properties in response to variations in temperature,5 pH,6 or ionic strength.7
Block copolymers comprised of polypeptide segments have shown significant advantages in controlling functional and supramolecular structures of bio-inspired self-assemblies in aqueous solution.8 Peptide copolymers provide many advantages over conventional synthetic polymers due to its ability to hierarchically assemble into stable ordered conformations. With different amino acid substitutions, polypeptides can adopt well-defined stable secondary structures such as the α-helix, β-sheet, or coil depending on external environment.9 Therefore the hybrid copolymers (also named molecular ‘’chimeras’’)10 combining various conventional synthetics of specific functionalities with polypeptides have been intensively investigated.11
To be successful in these purposes, it is important that materials can self-assemble into precisely and predictable structures. Since the late 1940s, the ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) initiated by nucleophiles or bases have been the most common technique used for polypeptides preparation.12 However, attempt to prepare polypeptides always results in unmatched compositions with monomer feed ratios and significant homopolymer contaminants because of the nucleophilic/basic duality of the initiator. Elimination of these side reactions has been the major synthetic challenge for this
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polymerization system.13 Several innovative approaches have been proposed for controlling NCA polymerization by different concepts. Deming reported that replacing primary amine initiators with organonickel initiators which are able to give controllable polymerization of a wide range of NCA monomers from pure enantiomers to racemic mixtures.14 Sequential addition different NCA monomers enables preparation of block copolypeptides with defined sequence and composition. Recently, Deming et al. also described the first use of a macroinitiator bearing amido-amidate nickel cycle end groups to synthesize hybrid block copolymers.15 Both Hadjichristidis and Schlaad groups reported the conventional primary amine-initiated polymerization of NCAs with distinct ideals. The former employed their high vacuum technique to create and maintain conditions necessary for the living polymerization of NCAs.16 Their approaches of controlling the reaction conditions resolved the existing problem for more than fifty-year-old challenge presented by the NCA/nucleophile system.
The latter group’s strategy is to allow the reversible dissociation equilibrium between free primary amine and a proton H+(Cl－) to exist only a very short lifetime of reactive amine, the amine chain-end group is immediately protonated and thus to prevent side reactions.17 This approach has been applied by other groups to synthesize well-defined hybrid copolymers.18
In order to incorporate desired functional segments into polypeptides based copolymers, additional polymerization techniques are required. Controlled/living radical polymerization (CRP), developed in the past decade,19 enables the facile synthesis of polymers with controlled molecular weight, low polydispersity, and well-defined architecture. Additionally, desired functionalities can be incorporated into either end group or distributed along polymer backbone. Since ROP and CRP are distinct mechanisms, a dual hetero-functional initiator20 to combine these two routes is important to design and synthesize various hybrid block copolymers.
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Few research activities have been focused on dual stimuli-responsive hybrid block copolymers consisting of pH-sensitive polypeptides and thermo-sensitive segment.21 Poly(N-isopropylacrylamide) (PNIPAm), the most widely studied thermo-responsive polymer, exhibits coil-to-globule transition above the lower critical solution temperature (LCST) at 32
°C in aqueous solution. Below this critical temperature, it is in a hydrophilic state, and above this temperature it is in a hydrophobic state.22 All the major CRP techniques, atom transfer radical polymerization (ATRP),23 nitroxide-mediated polymerization (NMP),24 and reversible addition-fragmentation chain transfer polymerization (RAFT)25 have been successfully used to prepare poly(N-isopropylacrylamide) with low-to-moderate chain length and narrow molecular weight distribution.
In this paper, we report the synthesis of a new amide-linkage phthalimidoethyl 2-bromo-2-methylpropionamide hetero-functional initiator which allows both polymerizations to be conducted consecutively by ATRP then the amine hydrochloride mediated ROP of Nε-(carbobenzoxy)-L-lysine N-carboxyanhydride (Z-Lys-NCA) or γ-benzyl L-glutmate
N-carboxyanhydride (BLG-NCA) to synthesize well-defined poly(N-isopropylacrylamide)-b-polypeptides hybrid block copolymers. An investigation of
these rod-coil hybrid block copolymers’ hierarchical self-assembly nanostructure and dual stimuli-responsive of pH and thermo-sensitive behaviors in aqueous solution and in solid states are detail presented.
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2-2 Experimental Section 2-2.1 Materials
All reagents and solvents were purchased from commercial suppliers and used as received unless otherwise noted, including L-glutamic acid 5-benzyl ester (Fluka, >99%), Nε-(carbobenzoxy)-L-lysine (ACROS, 98%), triphosgene (TCI, >98%), ethanolamine (Tedia, 99%), bromoisobutyryl bromide (ACROS, 98%), diethyl azodicarboxylate solution 40% in toluene (Fluka), triphenylphosphine (Lancaster, 99%), phthalimide (ACROS, 98%), hydrazine monohydrate (Sigma-Aldrich, 98%), 2-propanol (Tedia, 99.5%), uranyl acetate dihydrate (Fluka, 98%), hydrogen bromide 33 wt. % in acetic acid (ACROS), and trifluoroacetic acid (ACROS, 99%). The monomer N-isopropylacrylamide (NIPAm, 99%, TCI) was recrystallized in hexane/toluene, and dried under vacuum before use. Ethyl acetate (Tedia, 99.8%) and N,N-Dimethylformamide (DMF; Tedia, 99.8%) were dried over CaH2 (ACROS, 93%) and distilled under reduced pressure. Tetrahydrofuran (Tedia, 99.8%) was distilled over Na/benzophenone. Deioned (DI) water used in all reactions, solution preparations, and polymer isolations was purified to a resistance of 18 MΩ (Milli-Q Reagent Water System, Millipore Corporation). Nε-(carbobenzoxy)-L-lysine N-carboxyanhydride (Z-L-lysine NCA) and γ-benzyl L-glutmate N-carboxyanhydride (BLG NCA) were synthesized according to the method described by Poché et al. (Fuchs-Farthing method).26 Hexamethylated tris(2-(dimethylamino)ethyl)amine (Me6TREN) was synthesized according to method of Ciampolini.27
2-2.2 Synthesis of 2-bromo-N-(2-hydroxyethyl)-2-methylpropionamide (BrPA) (1) (Scheme 1)
Ethanolamine (5.31 g, 86.9 mmol, 1.0 equiv) and TEA (8.8 g, 173.8 mmol, 2.0equiv) dissolved in THF (400mL) were fed into a 500 mL two-necked round-bottomed flask fitted
41
with a Arinlet and a rubber septum and were cooled in an ice bath. The α-bromoisobutyryl bromide (20 g, 86.9 mmol, 1.0 equiv) was added into the mixture dropwise. White precipitate of triethylammonium bromide was observed and the reaction was allowed to stir at room temperature overnight. After the precipitate was filtered off, THF was removed by rotary evaporation. The residue was purified with column chromatography (silica gel, hexane/ethyl acetate: v/v: 1/1) yield product 1 (11.33 g, 62%) as a viscous oil. 1H NMR (CDCl3, δ, ppm) 7.13 (br, 1H, -NH-), 3.72 (m, 2H, HO-CH2-), 3.42 (m, 2H, HO-CH2-CH2-), 2.32 (br, 1H, HO-CH2-), 1.93 (s, 6H, Br(CH3)2-C-). 13C NMR (CDCl3, δ, ppm): 173.12 (-NH-C(O)-C-), 62.51 (Br(CH3)2-C-C(O)-), 61.77 (HO-CH2-CH2-), 42.97 (HO-CH2-CH2-), 32.48 (Br(CH3)2-C-).
2-2.3 Synthesis of phthalimidoethyl 2-bromo-2-methylpropionamide (PIBrPA), (2) (Scheme 1)
To a THF solution (250 mL) of a mixture of product (1) (9 g, 42.8 mmol, 1.0 equiv), triphenylphosphine (14.6 g, 55.6 mmol, 1.3 equiv), phthalimide (6.3 g, 42.8 mmol, 1.0 equiv) was added dropwise 40 % toluene solution of diethylazodicarboxylic acid (DEAD; 24.3 g, 55.6 mmol, 1.3 equiv), and the resulting mixture was stirred under argon at room temperature for 12h. The reaction mixture was then evaporated to dryness, and the residue was purified with column chromatography (silica gel, hexane/ethyl acetate: v/v: 4/1) and recrystallization with THF/hexane to yield product (2) (6.5 g, 45%) as a white needle-like crystals. 1H NMR (CDCl3, δ, ppm): 7.86 (m, 2H, phthalyl aromatic), 7.70 (m, 2H, phthalyl aromatic), 7.09 (br, 1H, -NH-), 3.87 (m, 2H, -CH2-CH2-NH-), 3.54 (m, 2H, -CH2-CH2-NH-), 1.85 (s, 6H, Br(CH3)2-C-). 13C NMR (CDCl3, δ, ppm): 172.44 (-NH-C(O)-C-), 168.43 (-C-C(O)-N-), 134.12, 131.86, 123.38 (aryl-C), 62.13 (Br(CH3)2-C-C(O)-), 39.82 (-CH2-CH2-NH-), 37.16 (-CH2-CH2-NH-), 32.23 (Br(CH3)2-C-).
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2-2.4 Preparation of phthalimide end-capped poly(N-isopropylacrylamide) (3) by ATRP (Scheme 2)
A typical polymerization procedure of NIPAm at monomer/initiator ratio of 250 was as carried out follows. NIPAm (4.00 g, 35.40 mmol), initiator (0.048 g, 0.14 mmol), and 2-propanol (7.65 mL) were combined and deoxygenated by performing freeze-pump-thaw cycle. Upon equilibration at 20 °C after the third cycle, the flask was immersed into an water or ice bath. To allow the buildup of the complex between the metal and ligand, an oxygen free solution of 2-propanol (2.55 mL) containing CuBr or CuCl (20.5 mg or 14.16 mg, 0.1416 mmol) and Me6TREN (32.7 mg, 38.85 μL) was prepared separately. This solution was then added to the monomer and initiator mixture via an argon-washed syringe to start polymerization. The reaction mixture was exposure to air to stop polymerization, then evaporated to dryness and the residue was dissolved in 150mL of THF, and the copper catalyst was removed by passing through a neutral alumina column. The solution was concentrated and precipitated in n-hexane to yield PNIPAm as white powder. To obtain the actual Mn calculated from the ratio of the integral of the phthalimide and methine protons peak by 1H NMR, the phthalimide end-capped PNIPAm was further purify by dialysis against DI water to exclusively eliminate residual monomer and non-initiated initiator. 1H NMR (500MHz, CDCl3): δ 1.12 (br, CH(CH3)2) 1.33 − 2.24 (br, aliphatic H) 3.98 (br, CH(CH3)2), 6.63 (br, NH), 7.71 (br, phthalyl aromatic), 7.81 (br , phthalyl aromatic).
2-2.5 Hydrazinolysis of phthalimide end-capped PNIPAm to primary amine (4a) and amine hydrochloride-functionalized PNIPAm (4b) (Scheme 2)
Phthalimide end-capped PNIPAm (5 g) and 5-folds excess hydrazine monohydrate were dissolved in ethanol (25 mL) and the mixture was stirred at room temperature under argon for 12 hours. (upon the addition of the hydrazine, the solution became yellowish due to the
43
formation of phthalyl hydrazide.) The mixture was placed in a dialysis bag (MWCO = 3500 Da) and dialyzed against DI water for 48 hours. The DI water was changed every hour for the first five hours. Finally the water solution was freeze-dried. Isolated yield: 2.3 g (46%). To convert primary amine into amine hydrochloride, excess amount of 1 M HCl was added into the mixture solution and stirred for two hours to convert primary amine into amine hydrochloride. The mixture was dialyzed and lyophilized. The amine hydrochloride-functionalized PNIPAm isolated yield: 2.6 g (52%). 1H NMR (500MHz, D2O):
δ 1.05 (br, CH(CH3)2), 1.50 (br, -CHCH2-), 1.92 (br, -CHCH2-), 3.02 (br, -CH2-CH2-NH3+Cl—), 3.39 (br, -CH2-CH2-NH3+Cl—), 3.81 (br, CH(CH3)2).
2-2.6 General procedure for synthesis of poly(N-Isopropylacrylamide-b-peptide) block copolymer (5, 6) by ROP polymerization (Scheme 2)
Typically, the functionalized PNIPAm macroinitiator and N-carboxyanhydride monomer were dried at room temperature at separated dried flask under high vacuum for one hour. Then, two separate DMF solutions were prepared and subsequently combined via transfer needle under argon to give ~9 wt% solution. The mixture was stirred at room temperature for several days under inert argon atmosphere. After polymerization, the solvent was concentrated to a minimum amount under high vacuum. The concentrated DMF solution was precipitated in ether and subsequently dried under vacuum.
2-2.7 Deprotection of the ε-benzyloxycarbonyl (Cbz-group) side chains in PNIPAm-b-PZLys (7) (Scheme 2)
A round-bottom flask was charged with a solution of the appropriate PNIPAm-b-PZLys in trifluoroacetic acid (100 mg/ 3 mL). Then, a 4-folds molar excess of a 33 wt% solution of HBr in acetic acid was added, and the reaction mixture was stirred for one hour at room temperature. Finally, the reaction mixture was precipitated in ether and repeatedly dialyzed
44
against water until the conductivity remains constant. The product was isolated via lyophilization.
2-2.8 Preparation samples of block copolymer micelles assembled in water
Dried solid block copolymer powder was dissolved in DMF or THF (1.0 mL) to give a 0.1%
(w/v) solution. A stir bar was added followed by dropwise addition of DI water (0.1 mL) under constant stirring. The solution was allowed to stir for 48 hours before exhaustive dialysis (Spectra/Por CE (cellulose ester) dialysis membranes, MWCO: 2000) against DI water for another 48 hours to remove DMF or THF.
2-2.9 Preparation of polymer film
Polymer film was prepared by solvent casting 10% polymer solutions in DMF as a non-selective solvent. Liquid samples were placed on Teflon-coated aluminum foil (BYTAC) and were slowly dried within five days at 70 °C. The sample was scratched off the foil and isolated as transparent thin film.
2-2.10 Characterizations
1H NMR and 13C NMR measurements were carried out at room temperature on a Varian Unity inova spectrometer operating at 500 MHz using CDCl3, DMSO-d6 or D2O as solvents.
Monomer conversion was determined from the 1H NMR integration ratio of the monomer double bond at 5.5 ppm to polymer peak at 3.8 ppm in DMSO-d6. The molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC) using a HITACHI PUMP L-7100—equipped with a RI 2000 refractive Index detector, and three Ultrastyragel columns (100, 500, and 103Å) connected in series in order of increasing pore size—using DMF as an eluent at a flow rate of 0.6 mL/min. The molecular weight calibration curve was obtained using polystyrene standards. Fourier Transform Infrared
45
Spectroscopy (FT-IR) spectra of solid samples were recorded at room temperature with a Nicolet AVATAR 320 FT-IR Spectrometer. Transmission electron microscopy (TEM) images were obtained by using a JEOL JEM-2000EXII instrument operated at 120 KV. The sample was ultra-microtomed at room temperature using a diamond knife from Leica Ultracut UCT Microtome to give 70 nm-thick sections then transferred onto carbon-coated copper grids. For a selective staining of poly(Z-L-lysine), specimens were exposed to the vapor of a freshly prepared aqueous RuO4 solution. In micelles solution, one drop of each respective sample was placed on a 200 mesh Formvar coated copper grid and allowed to remain on the grid for 120 seconds. Filter paper was then used to remove the residual sample and liquid.
One drop of 2 % (w/v) uranyl acetate (negative stain) was then placed on the grid, allowed to stain for 60 seconds, and subsequently removed by wicking away excess liquid with filter paper. The resulting samples were imaged by TEM. Wide-angle X-ray scattering (WAXS) spectrum was recorded on film sample using a Rigaku D/max-2500 type X-ray diffraction instrument. The radiation source used was Ni-filtered, Cu Kα radiation (λ = 1.54Å). The sample was mounted on a circular holder, the scanning rate was 0.6°/min from 2θ = 1 to 80.
Data were collected and plotted as intensity versus scattering vector, q, where q = (4π/λ)sin(θ) and θ is the Bragg angle (or 1/2 the scattering angle). Small-angle X-ray scattering (SAXS) measurement was conducted on a dedicated setup at the end-station of the BL17B3 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. We used an X-ray beam of 0.5 mm diameter and a wavelength (λ) of 1.24 Å for the SAXS measurements.
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2-3 Results and Discussion
2-3.1 Synthesis of amide linkage hetero-functional ATRP initiator
Since ester groups are good activating groups, α-haloester-based compounds are commonly used as ATRP initiators. In order to combine mechanistically incompatible initiation group into ATRP initiator compound, usage of the robust amide linkage of α-haloamide-based initiator28 is an alternative method to build up various architecture block copolymers. There have been several examples of using 2-bromo-2-methylpropionamide-based ATRP initiators to prepare various homopolymers and block copolymers.29 In this study, α-bromo amide-based initiator was synthesized by amidation of the amino group of ethanolamine with α-bromoisobutyryl bromide and then the hydroxyl group was substituted by Mitsunobu reaction into phthalimide group which can be converted into amino group (Scheme 2-1).
Accordingly, an new hetero-functional initiator was afforded for consecutive polymerization of N-isopropylacrylamide by ATRP and then ROP of amino acids NCA (Scheme 2-2).
2-3.2 Preparation of phthalimide end-capped poly(N-isopropylacrylamide)
Stöver and co-works recently showed that ATRP of NIPAm in different alcohols, especially in 2-propanol that alleviate catalyst inactivation by hydrogen bonding between amide groups and branched alcohols, led to narrow-disperse PNIPAm with high conversion and good molecular weight control.23 Me6TREN, a branched tetradentate ligand, forms one of the most active catalyst complex among all the ligands has been investigated, particular success in ATRP of acrylamides with good control over polymerization in room temperature.30 In this study, the polymerization of NIPAm was investigated under a range of reaction conditions to investigated the catalysts and reaction temperature effect on ATRP. All reactions were conducted at the ratio of NIPAm/2-propanol = 1/2 (w/w) with Me6TREN as ligand. When the Cu(I)Br was used to form the catalyst complex, the reaction was conducted at 20 °C for 70
47
minutes and yielded PNIPAm in 56.19% conversion with Mn = 17747 g mol-1 and polydispersity (PDI) = 1.31. When the reaction was carried out with Cu(І)Cl at 20 °C, a halide exchange catalyst system,31 polymerization for 250 minutes resulted in higher conversion of 70.25% with Mn=19 846 g mol-1 (PDI = 1.32). At lower polymerization temperature at 0 °C, product with significant narrow molecular weigh distribution was achieved (PDI=1.12) over 720 minutes with conversion of 63.88% and Mn=18 444 g mol-1. These polymerization data are summarized in Table 1. The reaction catalyzed by Cu(І)Br at 20 °C give the fastest polymerization rate and the kinetic plot is non-linear (Figure 2-1, square). This highly active Cu(І)Br/Me6TREN has large equilibrium constant in the ATRP and can rapidly initiate the initiators to generate high radical concentration at early stages of polymerization. Irreversible radical combination occurs continuously and initiators are consumed until a sufficient number of the Cu(II)Br2 deactivators are established to the activation/deactivation equilibrium. As a result, high persistent radical32 concentration of Cu(ІІ)Br2 decreases the polymerization rate and increases the product PDI because active catalysts are insufficient for fast exchange between active and dormant chain ends. In addition, portion of initiators produce low molecular weight polymer chains at the early stage of polymerization during the irreversible radicals combination, leading to lower initiation efficiency and higher PNIPAm molecular weight than theoretical values (Figure 2-2, square). A small number of dead chains in Figure 2-3a suggest low initiation efficiency. In order to alleviate the fast polymerization rate, the halide exchange (R-Br/Cu(І)Cl) catalyst system was performed.Slower polymerization rate
minutes and yielded PNIPAm in 56.19% conversion with Mn = 17747 g mol-1 and polydispersity (PDI) = 1.31. When the reaction was carried out with Cu(І)Cl at 20 °C, a halide exchange catalyst system,31 polymerization for 250 minutes resulted in higher conversion of 70.25% with Mn=19 846 g mol-1 (PDI = 1.32). At lower polymerization temperature at 0 °C, product with significant narrow molecular weigh distribution was achieved (PDI=1.12) over 720 minutes with conversion of 63.88% and Mn=18 444 g mol-1. These polymerization data are summarized in Table 1. The reaction catalyzed by Cu(І)Br at 20 °C give the fastest polymerization rate and the kinetic plot is non-linear (Figure 2-1, square). This highly active Cu(І)Br/Me6TREN has large equilibrium constant in the ATRP and can rapidly initiate the initiators to generate high radical concentration at early stages of polymerization. Irreversible radical combination occurs continuously and initiators are consumed until a sufficient number of the Cu(II)Br2 deactivators are established to the activation/deactivation equilibrium. As a result, high persistent radical32 concentration of Cu(ІІ)Br2 decreases the polymerization rate and increases the product PDI because active catalysts are insufficient for fast exchange between active and dormant chain ends. In addition, portion of initiators produce low molecular weight polymer chains at the early stage of polymerization during the irreversible radicals combination, leading to lower initiation efficiency and higher PNIPAm molecular weight than theoretical values (Figure 2-2, square). A small number of dead chains in Figure 2-3a suggest low initiation efficiency. In order to alleviate the fast polymerization rate, the halide exchange (R-Br/Cu(І)Cl) catalyst system was performed.Slower polymerization rate