Block Copolypeptides Research Articles

Rheology of Block Polyelectrolyte Solutions and Gels: A Review

We review recent experimental results on the rheology of block polyelectrolyte solutions, focusing mainly on diblock and triblock architectures and the effects of block polyelectrolyte concentration, block composition, solution pH, and ionic strength. Micellar solutions of diblock polyelectrolytes are typically viscoelastic liquids at a low concentration; however, gel formation can occur at higher concentrations when micelle corona chains begin to interpenetrate or interact. The rheology can be tuned by varying pH or ionic strength or through the addition of hydrophobic groups along the corona chain. Triblock architectures form elastic gels, analogous to neutral telechelic associative polymers. However, the charged nature of the backbone promotes the formation of a highly networked structure, enabling gel formation at lower concentrations than those observed with neutral telechelics. Finally, we briefly describe recent work on creating block copolypeptide gels with unique rheological and self-assembly characteristics and new architectures for creating thermoreversible block polyelectrolyte gels.

Synthesis of nanoporous silica with interior composite cells with synthetic block copolypeptide as template

Nanoporous silica with unusual interior composite cells was synthesized with synthetic block copolypeptide Phe20-b-PBLG50 as template for the first time. Anilino-methyl triethoxy silane (AMTS) was used as an intermedium to interact with block copolypeptide Phe20-b-PBLG50 through π-π interaction between the phenyl groups of block copolypeptide and those of AMTS. Meanwhile, AMTS co-condenses together with tetraethoxylsilane (TEOS) after hydrolysis. The structure of composite vesicles due to the self-assembly of block copolypeptide in the organic solvent was immobilized and transcribed by the formation of silica. The formation of nanopores could be ascribed to the secondary structure of block copolypeptide and small molecular amine. Our results provide a new avenue to synthesize porous oxide materials with novel interior structures templated by the copolypeptide self-assembly under ambient conditions.

Construction of Self-Organized Interface via Monodisperse Block Copolypeptide Amphiphile

In order to construct an intelligent functional nano-interface, the self-organized structure of Langmuir-Blodgett (LB) film was investigated by atomic force microscopy (AFM). As a building block, a monodisperse loop-helix-helix triblock copolypeptide containing the helix of which hydrophobic L-leucine residues align on a single face, “leucine zipper”, was synthesized by recombinant DNA techniques with a designed amino acid sequence. AFM observation on the surface structure of LB films constructed at surface pressures of 2, 5 and 18 mN/m revealed that structural transition from ring- or disk-like aggregations to a lane-like fabrication occurred with compression of the copolypeptide membrane on the water of pH 4. The leucine zipper in the copolypeptide was suggested to perform as an important basis for nanostructure formation through a hydrophobic interaction. Exploitation of the recognition and specific binding sites in the copolypeptide will allow the self-assembling mechanism of the copolypeptide to be developed to a novel nano-template.

Synthesis and self-assembly of dendritic-helical block copolypeptides.

Dendritic-helical diblock copolypeptides, dendritic poly(-lysine)--poly(γ-benzyl--glutamate) (PBLG-Lys) were synthesized up to 4th generation of dendritic poly(-lysine). PBLG was synthesized by conventional ring-opening polymerization of γ-benzyl--glutamate--carboxyanhydride with heptyl amine as an initiator. The -terminus of this PBLG was used for further coupling reactions with ,-bis(-butoxycarbonyl)--lysine pentafluorophenylester. These block copolypeptides possess well-defined 3-D structures in solution, and they self-assemble into a fibrous structure a nanoribbon mechanism in toluene. Amphiphilic block copolypeptides were obtained after deprotecting BOC groups at the periphery of the lysine dendrimer block. Due to the well-defined size and structure of the dendritic lysine block and well-defined α-helical conformation of the PBLG block, the block copolypeptides showed a generation-dependent self-assembly behavior in aqueous solution.

Control of calcium carbonate crystallization utilizing amphiphilic block copolypeptides

Control over the morphology of calcium carbonate (CaCO 3) crystals has been achieved through the use of anionic, amphiphilic block copolypeptides during crystallization. Microspheres of CaCO 3 can be synthesized by the introduction of preformed organic, amphiphilic block copolypeptide templates, poly( l-aspartate sodium salt) 100- block-[poly( l-phenylalanine) 25- random-( l-leucine) 25] ( 1) and poly( l-glutamate sodium salt) 100- block-[poly( l-phenylalanine) 25- random-( l-leucine) 25] ( 2). When cationic amphiphiles are used in lieu of the anionic amphiphiles, only CaCO 3 rhombohedra are produced. The self-assembling amphiphile controls the precipitation of the microspheres by acting as a template to form giant CaCO 3 microspheres. These microspheres are composed of nanocrystals ranging in size from 10 to 60 nm using 1 and 20 to 100 nm using 2.

Aggregate formation and release behaviour of model substances with block co-polypeptide containing tryptophan

In this study, amphiphilic block co-polypeptide consisting of hydrophilic poly( N-hydroxyethyl l-glutamine) (PHEG) and hydrophobic poly( l-tryptophan) (poly(Trp)), PHEG- block-poly(Trp)-T, was prepared by aminolysis of poly(γ-benzyl l-glutamate)- block-poly(Trp) with 2-amino-1-ethanol and subsequent treatment with trifluoroacetic acid (TFA). By using the block co-polypeptide, aggregate formation and sustained-release behaviour of model substances were investigated. The block co-polypeptide formed aggregates in aqueous medium and showed the ability to uptake hydrophobic substances into their hydrophobic moiety. The block co-polypeptide exhibited pH-response, the critical aggregate concentration of PHEG- block-poly(Trp)-T and the sizes of the aggregates depended on pH and decreased at pH 2.0. Moreover, fluorescence studies indicated a loose aggregate structure at pH 2.0 and the release rate of the model substances from the polypeptide aggregates was higher at pH 2.0 than at pH 5.0. These results could be explained by dissociation of Trp residues in the hydrophobic cores of the aggregates.

Charged Polypeptide Vesicles with Controllable Diameter

We report the preparation and characterization of charged, amphiphilic block copolypeptides that form stable vesicles and micelles in aqueous solution. Specifically, we prepared and studied the aqueous self-assembly of a series of poly(L-lysine)-b-poly(L-leucine) block copolypeptides, KxLy, where x ranged from 20 to 80 and y ranged from 10 to 30 residues, as well as the poly(L-glutamatic acid)-b-poly(L-leucine) block copolypeptide, E60L20. Furthermore, the vesicular assemblies show dynamic properties, indicating a high degree of membrane fluidity. This characteristic provides stimuli-responsive properties to the vesicles and allows fine adjustment of vesicle size using liposome-based extrusion techniques. Vesicle extrusion also provides a straightforward means to trap solutes, making the vesicles promising biomimetic encapsulants.

Biomimetic Synthesis of Inorganic Nanospheres

The synthesis of silica, silver bromide, and composite nanospheres made in the presence of block copolypeptide vesicles is reported. Hollow silica nanospheres of controllable size can be made with Lys:Phe 1:1 block copolypeptides, whereas at higher block ratios, uniform silica nanospheres are formed that are not hollow. Silver bromide nanospheres of controllable size in the range of 25−250 nm can also be formed, as well as silver bromide/silica core−shell particles. The silver bromide nanospheres can also be assembled into hollow rods in the presence of rhodamine 6G, and this process is reversible. The unique feature of this work is the ability to translate the information of an individual biomimetic supramolecular structure (e.g., vesicle) as a template into an inorganic material. The results show that block copolypeptides offer unique opportunities for assembling nanostructured materials.

Synthesis and Characterization of Random and Block Copolypeptides Derived from γ-Methylglutamate and Leucine N-Carboxyanhydrides

The synthesis of random and block copolypolyeptides derived from gamma-methylglutamate and leucine N-carboxyanhydrides using Al-Schiff's base complexes and allylamine as initiators is here reported. The copolymer structures were confirmed by (1)H and (13)C NMR. The calculation of the statistical average block lengths reveals the presence of longer methylglutamate units in the copolymer. The determination of the reactivity ratios indicated a slightly higher reactivity of gamma-methylglutamateNCA as compared to leucineNCA. Block copolypeptides containing glutamate and leucine units were obtained by sequential polymerization of the two NCAs using Al-Schiff's base complexes or allylamine in dioxane as solvent. Based on (13)C NMR spectra of copolymers exhibiting two signals corresponding to peptide linkages, we confirmed the block structure and concluded that the copolymerization proceeds by attack of an amino group present on a glutamate chain end onto a LeuNCA. The copolymerization with allylamine was also shown, from calculation of the average block lengths of sequences, to exhibit living behavior. Viscometry analysis further showed that molar masses of the copolypeptides obtained with Al-Schiff's base were quite close to those derived from allylamine, supporting the proposed mechanism of copolymerization.

Quantitative analysis of helix–coil transition of block copolypeptide, Glu12–Ala12, by combined use of CD and NMR spectroscopy

To investigate helix-coil transition mechanisms, conformations of Glu12-Ala12, EA, in aqueous solution have been studied in detail over the pH range from 2 to 8 and the temperature range from 20 to 60 degrees C using CD and NMR spectroscopy. The 750-MHz NMR spectra displayed excellent dispersion of the backbone amide proton signals, and permitted essentially complete sequence-specific resonance assignments. These assignments, together with short- and medium-range nuclear Overhauser effect (NOE) constraints and coupling constants, enable us to analyze conformational characteristics of all the residues in the EA peptide individually. A combined use of CD and NMR techniques reveals that the EA peptide assumes a stable alpha-helix from Glu12 to Ala19 in 0.1 M NaCl solution at 20 degrees C above pH 7. The alpha-helix is getting longer as decreasing pH. Below pH 4, the peptide assumes the longest alpha-helix from Glu3 to Ala23. The important observation of the present study is that the helix-coil transition occurs stepwise, residue by residue, from both the N- and C-termini of the alpha-helix. No conformational equilibrium between the helical and random-coil states is detected for the residues in the central region of the alpha-helix. Quantitative analysis of temperature-induced helix-to-coil transitions at various pHs provides a pH-independent residual enthalpy change delta H(r) = 0.95 kcal res(-1). Similar values have been reported for a 50-residue alanine-rich peptide (1.2 kcal res(-1)), poly-L-glutamate (1.1 kcal res(-1)), poly-L-lysine (1.1 kcal res(-1)), and poly-L-alanine (0.86 kcal res(-1)). Those investigations, along with our present result, suggest that delta H(r) is mainly determined by the transformation of the backbone associated with the disruption of the intramolecular hydrogen bond. These results should increase our understanding of the helix-coil transition.

Living polypeptides.

Block copolypeptides, which combine the self-assembly of block copolymers and the highly ordered 3D structures of proteins, are potential candidates for novel supramolecular structures and biotech applications, such as biosensors, tissue engineering, and selective drug delivery. The synthesis of model block copolypeptides through living nucleophilic/basic polymerization of alpha-amino acid N-carboxyanhydrides (NCAs) has been a challenge for more than fifty years, most probably due to traces of impurities in the system. This problem has been overcome, using high vacuum techniques in order to create and maintain the conditions necessary for the living polymerization of NCAs with primary amines. This method is a general one and opens avenues leading to novel, well-defined polypeptides with various architectures.

Rheology of Block Copolypeptide Solutions: Hydrogels with Tunable Properties

Amphiphilic block copolypeptides were prepared through transition-metal-mediated polymerization of amino acid N-carboxyanhydrides. In aqueous solution these materials form strong hydrogels at low concentrations. The self-assembly process that is responsible for gelation was investigated by measuring the rheological properties of the gels for a variety of molecular architectures: poly-l-lysine-b-poly-l-leucine diblock and poly-l-lysine-b-poly-l-leucine-b-poly-l-lysine triblock copolypeptides. Experiments showed that the rodlike helical secondary structure of enantiomerically pure poly-l-leucine blocks was instrumental for gelation at polypeptide concentrations as low as 0.25 wt %. The hydrophilic polyelectrolyte segments have stretched coil configurations and stabilize the twisted fibril assemblies by forming a corona around the hydrophobic core. The self-assembly of hydrophobic blocks is highly specific and sensitive to the chirality of the helices. It was found that mechanical properties of the gels can...

Design of a doubly-hydrophilic block copolypeptide that directs the formation of calcium carbonate microspheresElectronic supplementary information (ESI) available: circular dichroism spectrum of 1 and a table summarizing the effects of different block copolypeptides on the morphology of calcium carbonate crystallization. See http://www.rsc.org/suppdata/cc/b4/b403211j/

The crystallization of calcium carbonate into microspheres has been accomplished using the rationally-designed, doubly-hydrophilic block copolypeptide poly(Nepsilon-2[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine)(100)-b-poly(L-aspartate sodium salt)30 as a structure-directing agent.

Cooperative Assembly of Magnetic Nanoparticles and Block Copolypeptides in Aqueous Media

We demonstrate that highly crystalline, monodisperse maghemite (γ-Fe2O3) nanoparticles, synthesized in organic solvents, can be effectively transferred into an aqueous medium using an ammonium salt and stabilized at neutral pH. The nanoparticles remain monodisperse, as characterized by TEM and XRD, as well as superparamagnetic, as determined by SQUID magnetometry. When the aqueous maghemite is combined with the block copolypeptide poly(EG2-lys)100-b-poly(asp)30, the nanoparticles assemble into uniform clusters comprised of approximately 20 nanoparticles, resulting in a water soluble block copolypeptide−nanoparticle composite structure.

Microrheology as a tool for high-throughput screening

Microrheology can be used for high-throughput screening of the rheological properties of sample libraries of complex fluids. Two passive techniques are particularly suitable: video microscopy and diffusing-wave spectroscopy. The techniques complement each other very well and can be applied to samples that offer different experimental challenges. We offer a thorough analysis of the strengths and limitations of microrheology with the emphasis on high-throughput applications. To illustrate the potential of microrheology, results are presented for two representative cases: the rheological screening of aqueous solutions of a block copolypeptide library and the rheological phase diagram of a water/surfactant/salt system.

Grafting of Homo- and Block Co-polypeptides on Solid Substrates by an Improved Surface-Initiated Vapor Deposition Polymerization

Grafting of poly(γ-benzyl l-glutamate) (PBLG) on silicon native oxide surfaces by the surface-initiated vapor deposition polymerization (SI-VDP) method was first demonstrated by Chang and Frank (Langmuir 1998, 14, 326). In the current study, we have further improved this method by redesigning the reaction chamber and optimizing the reaction parameters, including monomer concentration, substrate temperature, and reaction time. Through the process optimization, we can fabricate end-grafted α-helical PBLG films with tunable thicknesses from a few nanometers to hundreds of nanometers in less than 1 h of reaction time. For example, a 187 nm PBLG grafted film was synthesized at 95 °C monomer evaporating temperature, 75 °C substrate temperature, and 0.1 Pa in 30 min. In this study, the SI-VDP process was also applied to synthesize other homo-polypeptide and block co-polypeptide thin film systems. The successful syntheses of poly(γ-methyl l-glutamate) (PMLG), poly-l-phenylalanine (PLPA), poly(β-benzyl l-aspartate) (PBLA), poly(Nε-carbobenzyloxy l-lysine) (PCBL), PLPA-b-PBLG, and PBLG-b-PCBL grafted films were demonstrated. Their chemical compositions, secondary structures, thicknesses, refractive indices, and water contact angles were characterized by Fourier transform infrared spectroscopy (FTIR), ellipsometry, and contact angle goniometry, respectively.

Methodologies for preparation of synthetic block copolypeptides: materials with future promise in drug delivery

This article summarizes recent developments in the synthesis of copolypeptides, focusing on the realization of living polymerizations that allow preparation of materials with excellent attributes for drug delivery. Traditional methods used to polymerize α-amino acid- N-carboxyanhydrides (NCAs) are described, and limitations in the utility of these systems for the preparation of polypeptides are discussed. A system utilizing transition metal catalysis for polypeptide synthesis is described that allows preparation of materials that begin to rival biologically produced counterparts in terms of complexity and purity. Overall, new developments in NCA and β-lactam polymerizations hold tremendous promise now that block copolypeptides of controlled dimensions (molecular weight, sequence, composition, and molecular weight distribution) can be prepared. Such well-defined polymers should greatly assist in the development of new drug carriers with increased function.

Assembly of Nanoparticles into Hollow Spheres Using Block Copolypeptides

Various modes are being explored for the construction of functional materials out of nanoparticles. Despite these efforts, the assembly of nanoparticles remains challenging with respect to the requirement of multiple component organization on varying dimensions and length scales. We report here a room-temperature, wet chemical-based synthesis route in which silica and gold nanoparticles (∼10 nm) are cooperatively assembled with lysine−cysteine diblock copolypeptides into robust hollow spheres (diameter ∼ microns). The walls of the hollow spheres are composed of two distinct layers of silica and gold nanoparticles, and the hollow center is created without the use of a sacrificial core, emulsion phase, or hollow preformed substrate. Block copolypeptides designed with specific recognition sites for nanoparticles of various compositions provide a versatile approach for the hierarchical organization of nanoparticles into multidimensional composite arrays.

Surface Grafting of Poly(l-glutamates). 3. Block Copolymerization

This paper describes for the first time the synthesis of surface-grafted AB-block copolypeptides, consisting of poly(γ-benzyl l-glutamate) (PBLG) as the A-block and poly(γ-methyl l-glutamate) (PMLG) as the B-block. Immobilized primary amine groups of (γ-aminopropyl)triethoxysilane (APS) on silicon wafers initiated the ring-opening polymerization of N-carboxyanhydrides of glutamic acid esters (NCAs). After removal of the BLG-NCA monomer solution after a certain reaction time, the amine end groups of the formed PBLG blocks acted as initiators for the second monomers. This method provides the possibility of making layered structures of surface-grafted block copolymers with tuned properties. Ellipsometry and small-angle X-ray reflection (SAXR) measurements revealed the thickness of the polypeptide layers ranging from 45−100 Å of the first block to 140−270 Å for the total block copolypeptides. The chemical composition of the blocks was determined by X-ray photoelectron spectroscopy (XPS). In addition, Fourier transform infrared transmission spectroscopy (FT-IR) revealed that the polypeptide main chains of both blocks consisted of pure α-helices. The average orientation of the helices ranging from 22−42° with respect to the substrate within the first block to 31−35° in the second block could be derived with FT-IR as well.

Living polymerization of α-amino acid-N-carboxyanhydrides

This article reviews recent developments in the polymerization of α-amino acid- N-carboxyanhydrides (NCAs) to form polypeptides. Traditional methods used to polymerize these monomers are described, and limitations in the utility of these systems for the preparation of polypeptides with controlled molecular weights and narrow molecular weight distributions are discussed. The development of transition-metal-based initiators, which activate the monomers to form covalent active species, permits the formation of polypeptides via the living polymerization of NCAs. In these systems, polymer molecular weights are controlled by monomer-to-initiator stoichiometry, polydispersities are low, and block copolypeptides can be prepared. The scope and limitations of these initiators and their key features and mode of operation are described in detail in this highlight. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3011–3018, 2000