Biomacromolecules 2005, 6, 2352-2361
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Nanodomain-Induced Chain Folding in Poly(γ-benzyl-L-glutamate)-b-polyglycine Diblock Copolymers P. Papadopoulos and G. Floudas* Department of Physics, University of Ioannina P.O. Box 1186, 451 10 Ioannina, Greece and Foundation for Research and Technology-Hellas, Biomedical Research Institute (FORTH-BRI)
I. Schnell Max-Planck Institut fu¨r Polymerforschung, P.O. Box 3148, 55021 Mainz, Germany
T. Aliferis, H. Iatrou, and N. Hadjichristidis* University of Athens, Department of Chemistry, 15771 Athens, Greece Received March 10, 2005; Revised Manuscript Received May 10, 2005
We report on the self-assembly mechanism and dynamics in a series of poly(γ-benzyl-L-glutamate)-b-poly(glycine) (PBLG-b-PGly) diblock copolymers within the composition range 0.67 e fPBLG e 0.97 and the temperature (T) range 303 < T < 433 K. Small- and wide-angle X-ray scattering, 13C NMR, and differential scanning calorimetry are used for the structure investigation coupled with dielectric spectroscopy for both the peptide secondary structure and the associated dynamics. These techniques provide not only the nanophase morphology but also the type and persistence of peptide secondary structures. The thermodynamic confinement of the blocks within the nanodomains and the disparity in their packing efficiency results in multiple chain folding of the PGly secondary structure that effectively stabilize a lamellar morphology for high fPBLG. Nanoscale confinement proves to be important in controlling the persistence length of secondary peptide motifs. Introduction The phase behavior of coil-coil or coil-crystallizable diblock copolymers, driven by the thermodynamic incompatibility of the unlike blocks, is now well established.1,2 In recent years, there is a strive toward the design of macromolecules that combine the nanometer scale self-assembly of block copolymers with biocompatibility and biofunctionality.3 Proteins are known to form hierarchically ordered structures, with R-helices and β-sheets being their fundamental secondary motifs.4 In this respect, the synthesis of block copolymers based on polypeptides can produce new biomaterials with potential applications in drug delivery, tissue engineering, and biomimetic composite formation. Recently, the use of peptidic block copolymers as structuredirecting agents for nanoparticle assembly has started to be explored. Examples include the assembly of magnetic nanoparticles,5 quantum-confined semiconductors,6 and porous oxides with controlled size and shape,7,8 all with the use of specifically tailored peptidic block copolymers. Despite the successful synthesis of such copolymers, their solid state phase behavior and the interplay between nanophase separation and peptide secondary structure is largely unexplored. In an early study,9 diblock copolymers composed from a polyvinyl (polystyrene (PS) and polybutadiene (PB)) and a polypeptidic block (poly(-carbobenzoxy-L-lysine) and poly(γ-benzyl-L-glutamate)) were synthesized and investigated by scattering methods. The number of folds of the * To whom correspondence should be addressed.
polypeptide was found to increase with the molecular weight of the polyvinyl and polypeptide blocks. More recently, rodcoil diblock copolymers based on a synthetic (coil) block and a polypeptide (rod) block with an R-helical conformation were investigated, namely polystyrene-b-poly(γ-benzyl-Lglutamate) (PS-b-PBLG),10 polyisoprene-b-(-benzyloxycarbonyl-L-lysine) (PI-b-PZLys), and polyisoperene-b-poly(Llysine) (PI-b-PLys).11 For a specific diblock (PS10-b-PBLG20), a cylinder-in-hexagonal (peptidic cylinders within a nanophase with hexagonal symmetry) structure has been proposed.10 Linear and bottlebrush-shaped PS-b-PLys copolymers were also investigated.12,13 Cylinder-in-lamellar and undulated lamellae were found, respectively. In the former case, and for polydisperse samples, a fluctuating thickness of the individual peptide domains has been proposed to account for the final zigzag lamellar morphology. In a recent study,14 we reported on the self-assembly mechanism in a series of poly(γ-benzyl-L-glutamate)-b-poly(ethylene glycol)-b-poly(γ-benzyl-L-glutamate) (PBLG-bPEG-b-PBLG) triblock copolymers composed solely from biodegradable and biocompatible blocks. For low peptide volume fraction, fPBLG, nanophase separation produced phases rich in all secondary structures (R-helices, β-sheets, and chain-folded PEG) whereas for fPBLG > 0.4 interfacial mixing destabilized the less coherent secondary structures (β-sheets), with possible biomedical and biophysical applications. In a subsequent study,15 we investigated the self-assembly and the associated dynamics of a series of PBLG peptide melts with varying molecular weight, using wide-angle X-ray
10.1021/bm0501860 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/03/2005
Nanodomain-Induced Chain Folding
scattering (WAXS) and dielectric spectroscopy (DS), respectively. The large dipole moment per peptide residue (3.46 D) makes DS a very sensitive probe of the persistence length of R-helical peptides. In this respect, R-helical polypeptides can be considered as perfect type-A polymers (following Stockmayer’s classification, type A-polymers possess dipoles along the chain contour). The DS study revealed three processes above the calorimetric glass temperature (Tg): (i) an R-process attributed to the relaxation of amorphous segments located between and at the end of helically ordered segments, (ii) a weak intermediate process with an M2 molecular weight (M) dependence, associated with the dynamics of amorphous-like chains, and (iii) a strong slower process reflecting the migration of helical sequences along the chains. The intensity of the slower process provided the means of calculating an effective persistence length of the R-helical segments, and therefore aid in identifying characteristics of the peptide secondary structure that are not accessible to other “static” probes (i.e., X-rays). A step forward in the design of hierarchically ordered structures with bio-functionality has been the recent reports on the synthesis of block copolymers based on polypeptides. In the first such report, organonickel initiators16 rather than amines were used to avoid the unwanted R-amino acid N-carboxyanhydrides (NCA) side reactions, which had, for more than 50 years, hampered the formation of well-defined copolypeptides. This approach gave rise to various peptidicbased block copolymers including diblocks,16,17 triblocks,16 and pentablocks.18 A second approach19 addressed the side reaction problem directly by using amines in combination with high vacuum techniques, to ensure the necessary conditions for the living polymerization of NCA’s. PBLGb-PGly were prepared for the first time with this methodology and are the subject of this work. Despite these important synthetic efforts, the solid state morphology of all peptidic block copolymers is largely unexplored. The present investigation deals with the self-assembly of a series of near monodisperse poly(γ-benzyl-L-glutamate)b-poly(glycine) (PBLG-b-PGly) diblock copolymer melts synthesized with the latter method. PBLG is the well-known R-helical polypeptide, whereas PGly, composed of the simplest amino acid, can exist in two forms depending on the casting conditions: β-sheets (known as PGly-I form that is the most common) and the very rare helical form (known as PGly-II with 3 residues per turn, translation per residue of 0.3 nm and a pitch of 0.9 nm). The two blocks differ not only in their secondary structures but also in their packing efficiency (Gly bears no side group as opposed to the long side group of BLG) and segmental mobility. The system is ideal for studying the effect of thermodynamic block confinement on the type and persistence of the peptidic secondary structures. For the self-assembly on the level of the blocks, we have employed small-angle X-ray scattering (SAXS) and DS, which are probing the global organization and local environment, respectively. For the selfassembly on the level of the secondary peptide structure, we have employed 13C NMR, wide-angle X-ray scattering (WAXS), and differential scanning calorimetry (DSC). Furthermore, we continue employing DS to probe the
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persistence of R-helical structures in PBLG. We found a strong influence of the thermodynamic confinement and of the packing efficiency on the persistence length of peptide secondary structures, with β-sheets to be more affected. Nanopatterned structures could be used for controlling the persistence length of secondary peptide motifs. Experimental Section Materials. DMF (99.9+ %), special grade for peptide synthesis with less than 50 ppm of active impurities, the polymerization solvent, was further purified by short-path fractional distillation under vacuum in a custom-made apparatus. The middle fraction was always used. Ethyl acetate was dried over CaH2. n-Hexane was dried overnight over CaH2 and then distilled to a flask containing n-BuLi. n-hexylamine (99.9%) the initiator which is a highly hygroscopic compound, was left to dry over a sodium mirror for 24 h. Then was diluted with purified DMF, subdivided into ampules, and stored under high vacuum at room temperature. Synthesis of NCA’s. The NCAs of γ-benzyl-L-glutamate (Glu-NCA) and glycine (Gly-NCA) were synthesized from the corresponding R-amino acid and triphosgene in ethyl acetate at 343 K under inert atmosphere according to the literature.20 The triphosgene can decompose to phosgene which is highly toxic, and therefore, the preparation of the anhydrides should be performed in the hood, using gas masks with a filter for organic chemicals. The unreacted species along with the HCl and the amino acid salts are removed by extraction with an aqueous alkali solution and water. The organic phase was introduced into a specially designed homemade apparatus for extreme purification by three times crystallization with ethyl acetate and n-hexane under high vacuum conditions. Finally the pure NCA was diluted with purified DMF, subdivided into ampules, and stored under high vacuum at 253 K. Details are given elsewhere.19 In the case of Gly-NCA, a slight different procedure was followed. Instead of washing with aqueous alkali solution and water, the NCA was dissolved and dried several times with ethyl acetate under high vacuum, to remove the excess triphosgene, along with the remained HCl. Synthesis of PBLG-b-PGly. A homemade glass apparatus was used without ground joins in order to create and maintain the conditions necessary for the living polymerization of NCAs. The polymerization reactors were designed to have a volume at least three times larger than the volume of the CO2 generated by each polymerization. The combination of high vacuum and large volume forces the polymerization to completion. Polymerizations carried out with n-hexylamine as the initiator. After complete consumption of Glu-NCA the second monomer Gly-NCA was added. The PBLG precursor along with the soluble diblock copolypeptides were characterized by SEC, combined with a two-angle laser light scattering (TALLS) and UV detectors. The instrument was operating at 333 K with a 0.1N LiBr solution in DMF for the determination of the polydispersity index (I) and the number average molecular weight (Mn) of the samples. The
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Papadopoulos et al.
Figure 1. Schematic of a PBLG18-b-PG12 diblock copolymer in the “idealized” fully extended conformation (red, O; blue, N; gray, C). Table 1. Molecular Characteristics of the Diblock Copolypeptides PBLG-b-PGly sample PBLG-b-PGly 15-1 PBLG-b-PGly 15-2 PBLG-b-PGly 15-3 PBLG-b-PGly 15-5 PBLG-b-PGly 15-8 PBLG-b-PGly 15-15
MnPBLG IPBLG ) Mw/Mn
MnPGly
I ) Mw/Mn
25300a
1.08a
29800a
1.10a
27500a 22400a 22800a 18500a
1.11a 1.14a 1.11a 1.13a
1050a 2500a 3450b 4800b 7600b 11500b
1.12a 1.12a c c c 1.13d
a Obtained by SEC-LALLS detector in DMF/LiBr 0.1N at 333 K. Obtained by stoichiometry. c Could not be obtained by SEC due to insolubility reasons. d NMR.
b
Table 2. Glass Transition Characteristics of the Copolypeptides and the PBLG Homopolymer
Figure 2. SEC chromatograph of PBLG precursor (black) and a PBLG-b-PGly diblock copolypeptide (red).
dn/dc value, for both soluble polymers, was 0.108 ( 0.002 mL/g as determined by differential refractometry (Chromatix, KMX-16) under the same conditions. The polyglycine block is insoluble in DMF. Consequently, the low content PGly (
