Random Copolypeptide Research Articles

Impact of polypeptide sequence on thermal properties for diblock, random, and alternating copolymers containing a stoichiometric mixture of glycine and valine

This paper describes the experimental elucidation for the impact of polypeptide sequence on the solution structure and the thermo-responsivity, having considered the corresponding theoretical prediction using a binary pattern of hydrophobic and hydrophilic units. We prepare diblock, random, and alternating copolymers consisting of a stoichiometric amount of valine as a hydrophobic unit and glycine as a hydrophilic unit. Through the thermo-responsivity measurements of aqueous polymer solutions, it is found that the UV spectra of diblock copolypeptide exhibit a cloud point upon heating, which indicate that the polymer adopts a random coil structure at low temperature. On the other hand, the alternating peptide shows an upper critical solution temperature (UCST) behavior indicating a globule-to-coil transition upon heating. The opposite behavior of alternating peptide to that of diblock copolypeptide is found to be dependent on not the chirality of repeating units but the alternating binary pattern of hydrophobic and hydrophilic units. The random copolypeptide exhibits not only coil-to-globule transition at low temperature but also globule-to-coil transition at high temperature owing to the presence of both segregated and alternating polymer sequences in the polymer. The thermo-responsive structures of the polymers in an aqueous media are also discussed by determining the thermodynamic parameters for the transitions.

Using Diphenylphosphoryl Azide (DPPA) for the Facile Synthesis of Biodegradable Antiseptic Random Copolypeptides.

A facile method has been developed for the large-scale synthesis of random copolypeptides composed of multiple (i.e., cationic, hydrophobic, and hydrophilic) amino acids and their relative ratios have been optimized for broad-spectrum antibacterial effect. The copolypeptides obtained have measured compositions close to the design ratios in spite of the differing reactivities of the different amino acids. An optimized random copolypeptide of lysine, leucine, and serine (denoted as KLS-3) mimicking the composition of LL-37 host defense peptide gives broad spectrum antibacterial activity against clinically relevant Gram-negative and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) with minimum inhibitory concentrations (MICs) of 32-64 μg mL-1 , as well as good MICs against multidrug resistant Gram-negative bacteria of Escherichia coli EC 958 (64 μg mL-1 ) and Klebseilla pneumoniae PTR3 (128 μg mL-1 ). This method can be applied to the facile large-scale copolymerization of multiple amino acids, including unnatural amino acids, to make effective antibacterial copolypeptides.

Macroporous Hydrogels Composed Entirely of Synthetic Polypeptides: Biocompatible and Enzyme Biodegradable 3D Cellular Scaffolds.

Synthetic polypeptides are a class of bioinspired polymers with well demonstrated biocompatibility, enzyme biodegradability, and cell adhesive properties, making them promising materials for the preparation of macroporous hydrogels as 3D cellular scaffolds. Three-dimensional macroporous hydrogels composed entirely of biocompatible and enzyme biodegradable synthetic polypeptides have thus been prepared. Under cryoconditions, macroporous hydrogels in the form of macroporous cryogels were prepared using a single copolymer component through direct EDC/sulfo-NHS zero-length cross-linking between poly(l-glutamic acid) (PLG) and poly(l-lysine) (PLL) residues on a PLG-r-PLL random copolypeptide chain. The resulting macroporous cryogels were found to contain large interconnected pores (≥100 μm) highly suitable for tissue engineering applications. Tuning the relative ratios of the amino acid components could result in cryogels with very different pore structures, swelling, and mechanical properties, suitable for developing gels for a range of possible soft tissue engineering applications. These cryogels were shown to be enzymatically biodegradable and demonstrated excellent biocompatibility, cell attachment and cell proliferation profiles with mammalian fibroblast (NIH-3T3) cells, demonstrating the appeal of these novel cryogels as highly suitable cellular scaffolds.

Poly(ornithine-co-arginine-co-glycine-co-aspartic Acid): Preparation via NCA Polymerization and its Potential as a Polymeric Tumor-Penetrating Agent.

A novel random copolypeptide of ornithine, arginine, glycine, and aspartic acid [Poly(ornithine-co-arginine-co-glycine-co-aspartic acid), Poly(O,R,G,D)] has been prepared through ring-opening polymerization of N-δ-carbobenzoxy-l-ornithine N-carboxyanhydride [Orn(Cbz)-NCA)], l-glycine N-carboxyanhydride (Gly-NCA) and β-benzyl l-aspartate N-carboxyanhydride [Asp(Bn)-NCA], following by subsequent deprotection and guanidization. The structure of Poly(O,R,G,D) was confirmed by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). Low cytotoxicity of Poly(O,R,G,D) was confirmed from MTT assay. The Poly(O,R,G,D) contain some internal sequences of RXXR (X = O, R, G, or D) that could be proteolytically cleaved to expose the cryptic CendR element and bind to Neuropilin-1. This would lead to vascular and tissue permeabilization. Therefore trypsin-cleaved Poly(O,R,G,D) increase the vascular leakage of Evans blue from dermal microvessels at the injection site in vivo skin permeability assay. The intratumoral injection of the Poly(O,R,G,D) significantly enhanced the concentration of cisplatin-loaded nanoparticles in MCF-7 solid tumors. These results show that Poly(O,R,G,D) could increase the vascular leakage and tissue penetration of nanoparticles in a solid tumor and can be used as a potential polymeric tumor-penetrating agent.

Stimuli-Responsive Zwitterionic Block Copolypeptides: Poly(N-isopropylacrylamide)-block-poly(lysine-co-glutamic acid)

Synthesis of novel zwitterionic block copolypeptides, poly(N-isopropylacrylamide)-block-poly(L-glutamic acid-co-L-lysine) [PNiPAM(n)(PLG(x)-co-PLLys(y))m , where n is the number-average degree of polymerization (DP(n)) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DP(n) of the peptide block], and their stimuli-responsiveness to temperature and pH variation in aqueous solutions are described. Initiated with the amino-terminated poly(N-isopropylacrylamide) (PNiPAM(n)-NH2), ring-opening polymerization (ROP) of a mixture of gamma-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), and Boc-L-lysine N-carboxyanhydride (BLLys-NCA) afforded the block copolypeptides PNiPAM(n)(PBLG(x)-co-PBLLys(y))m, with a poly(N-isopropylacrylamide) block together with a random copolypeptide block, which was then deprotected with HBr/trifluoroacetic acid into the double hydrophilic block copolypeptides, PNiPAM(n)(PLG(x)-co-PLLys(y))m. Their block ratios and lengths, as well as the amino acid residue ratios in the random copolypeptide block are varied (n = 360, x = 0.4-0.5, y = 0.4-0.6, and m = 220-252). The secondary structures of the copolypeptides in aqueous solution at different pH conditions were examined. Phase transitions in aqueous solutions induced by both pH and temperature variation were investigated by (1)H NMR spectroscopy. The transitions induced by temperature were also explored by turbidity measurements using UV/vis spectroscopy for their lower critical aggregation temperature (LCAT) determination. Furthermore, these aggregation processes were followed by dynamic light scattering measurements.

Solution Property and Irradiation Effect of Random Copolypeptides Composed of Ala and Pro Residues

Poly(proline) and random copolypeptide composed of Pro and Ala residues were synthesized, and their solution properties and molecular conformation were investigated. Aqueous solutions of the polypeptide were irradiated with γ-rays above the transition temperature. It was shown that the transition temperature of the aqueous solution of the copolypeptide is influenced by Ala-residue content and γ-ray irradiation.

Biodegradation of random co-polypeptide hydrogels consisting of N-hydroxypropyl l-glutamine as one component

Two component random co-polypeptide hydrogels consisting of N-hydroxypropyl l-glutamine and l-alanine (Ala) or l-phenylalanine (Phe) were prepared by performing aminolysis reactions with 3-amino-1-propanol together with crosslinking reaction with 1,8-octamethylenediamine on hydrogels of the starting co-polymers consisted of γ-benzyl l-glutamate and Ala or Phe. The relationship between their bulk structure and properties was evaluated with regard to the swelling ratio in water ( q), the rate of water vapor permeability ( V f), tensile properties, and enzymatic degradation behaviors of hydrogels in a pseudo-extracellular fluid (PECF). The tensile property of the hydrogels was highly dependent on q in PECF, and on the hydrophobicity of the side chains. A relationship was obtained between the V f and q of hydrogels in PECF regardless of the differences in the nature of the side chains. Biodegradation of the hydrogels in vitro by bromelain indicated that degradation took place in bulk rather than on the surface, and that the rate of degradation was also highly dependent on q in the samples as well as on the hydrophobicity of the side chains of the samples.

Structural Analyses on Aggregates of Graft Copolypeptides Containing Tryptophan in Aqueous Media and Efficiency of Drug Release

The amphiphilic graft copolypeptide, consisting of poly (N-hydroxyethyl L-glutamine) (PHEG) and poly (L-tryptophan (L-Trp)) PTrp (denoted as PHEG-g-PTrp), was synthesized. At the L-Trp composition of 10-25mol%, L-Trp residues aggregated to form the hydrophobic cores in the aqueous solution. The hydrophobic substance could be incorporated in this L-Trp core. This function had not been seen for the random copolypeptide consisting of the same constituent molecules. The aggregation of PHEG-g-PTrp was observed by dynamic light scattering, transmission electron microscopy and small-angle X-ray scattering. The film specimen was prepared from PHEG-g-PTrp by modifying L-Trp residues in trifluoroacetic acid. The hydrophobic substances were incorporated in the high pH region, and the control of the release was achieved by changing pH through the dissociation of the indole rings in the L-Trp residues.

Enzymatic Hydrolysis of Copoly (N-hydroxypropyl-L-glutamine/L-alanine) Fibers in vitro.

Two-component random copolypeptide P(HPG-ran-LA) fibers consisting of N-hydroxypropyl-L-glutamine and L-alanine with different copolymer composition were prepared by an aminolysis reaction of poly(γ-methyl-L-glutamate/L-alanine) fibers with 3-amino-1-propanol, followed by crosslinking reaction with 1, 8-octamethylene diamine. The in vitro hydrolysis was carried out using ficin, one of proteases. The tensile properties and the in vitro hydrolysis of these hydrophilic fibers were highly dependent on the degree of swelling in phosphate buffer solution. The study of the weight loss of the fibers upon the enzymatic hydrolysis suggested that the degradation of the fibers occurred gradually from their surface into their core. Scanning electron microscopy also supported the surface hydrolysis. The weight loss of the fibers in the course of hydrolysis took place almost in parallel with the strength loss of the fibers.

Preparation and properties of biodegradable copoly( N-hydroxyalkyl- d, l-glutamine) membranes

Random copolypeptide (PHADLG) membranes consisting of N-hydroxyalkyl- d-glutamine (HADG) and N-hydroxyalkyl- l-glutamine (HALO) covering the whole range of d, l-copolymer composition were prepared by carrying out aminolysis reactions with 2-amino-1-ethanol (E) or 5-amino-1-pentanol (Pe), together with a crosslinking reaction with 1,8-octamethylenediamine (OMDA) on membranes of the starting random copolymers (PMDLG) consisting of γ-methyl- d-glutamate and γ-methyl- l-glutamate. The relationships between their bulk structure and membrane properties were investigated, such as the swelling ratio in water, tensile properties, water vapor permeability, and enzymatic degradation behavior of the membranes in a pseudo-extracellular fluid (PECF), as a function of d, l-copolymer composition. The tensile properties of d, l-copolymer membranes were highly dependent on the swelling ratio of PECF. Biodegradation of these membranes in vitro by bromelain indicated that degradation was a bulk rather than a surface phenomenon, and that the rate of degradation of membranes was also highly dependent on the hydrophobicity of side chains, d, l-copolymer composition as well as on the swelling ratios of the d, l-copolymer samples.

Molecular designing of biodegradable copolypeptide membranes

AbstractTwo‐component random copolypeptide membranes consisting of N‐(hydroxyalkyl)‐ L‐glutamine and L‐leucine or L‐alanine were prepared by carrying out aminolysis reactions with 3‐amino‐l‐propanol (P) together with crosslinking reaction with octamethylenediamine (OMDA) on membranes of the starting copolymers consisting of γ‐methyl L‐glutamate (M) and L‐leucine (L) or L‐alanine (A). The relation between their bulk structure and properties was investigated with regard to the swelling ratio in water (q), the rate of water vapor permeability (Vf), tensile properties, and enzymatic degradation behavior in a pseudo‐extracellular fluid (PECF). The tensile property of the membranes was highly dependent on q in PECF, and on the hydrophobicity of the side chains. It was shown that a common relation was obtained between Vf and q of membranes in PECF regardless of the difference of the nature of side chains. Biodegradation of the membranes in vitro by bromelain indicated that degradation took place in bulk rather than on a surface, and that the rate of degradation was also highly dependent on q of samples as well as on the hydrophobicity of side chains of samples.

Enzymatic hydrolysis of copoly(N-hydroxypropyl-L-glutamine/L-leucine) hydrogels in vitro.

Two component random copolypeptide hydrogels consisting of N-hydroxyalkyl L-glutamine and L-leucine were prepared by carrying out aminolysis reactions with 3-amino-1-propanol(P) together with cross-linking reactors with 1,8-octamethylenediamine (OMDA) on hydrogels of the starting copolymers consisting of gamma-methyl-L-glutamate(M) and L-leucine(L). The relation between their bulk structure and properties was investigated with regard to the swelling ration in water, aqueous vapor permeability, tensile properties, and enzymatic degradation behavior in a pseudoextracellular fluid (PECF). The tensile property of the hydrogels was highly dependent on the swelling ratio in PECF, and on the hydrophobicity of the side chains, whose behavior was typical of an elastomer. It was shown that a common relation was obtained between the rate of water vapor permeabilities and the swelling ratio of hydrogels in PECF regardless of the difference of the nature of side chains. Biodegradation of the hydrogels in vitro by bromelain indicated that the degradation took place in bulk rather than on surface, and that the rate of degradation was also highly dependent on the swelling ratio of samples as well as on the hydrophobicity of the side chains of samples.

Preparation and properties of copoly(n-hydroxyalkyl- d,l-glutamine) membranes

Random copolypeptide (PHADLG) membranes consisting of N-hydroxyalkyl- d-glutamine (HADG) and N-hydroxylalkyl- l-glutamine (HALG) covering the whole range of d,l-copolymer composition were prepared by carrying out aminolysis reactions with 3-amino-1-propanol (P) or 5-amino-1-pentanol (Pe), together with a crosslinking reaction with 1,8-octamethylenediamine (OMDA) on membranes of the starting random copolymers (PBDLG) consisting of γ-benzyl- d-glutamate and γ-benzyl- l-glutamate. The relationships between their bulk structure and membrane properties were investigated, such as the swelling ratio in water, tensile properties, water vapour permeability, and enzymatic degradation behaviour of the membranes in a pseudo-extracellular fluid (PECF), as a function of d,l-copolymer composition. The tensile properties of d,l-copolymer membranes were highly dependent on the swelling ratio of PECF. Biodegradation of these membranes in vitro by papain indicated that degradation was a bulk rather than a surface phenomenon, and that the rate of degradation of membranes was also highly dependent on the hydrophobicity of side chains, d,l-copolymer composition as well as on the swelling ratios of the d,l-copolymer samples.

Biodegradation of random copolypeptide membranes consisting of [formula omitted] as one component

Two component random copolypeptide membranes, consisting of N-hydroxyalkyl l-glutamine and l-alanine or l-leucine were prepared by carrying out aminolysis reactions with 2-amino-1- ethanol (E) or 5-amino-1-pentanol (Pe), together with a cross-linking reaction with 1,8- octamethylenediamine (OMDA) on membranes of the starting copolymers consisting of γ-benzyl l-glutamate (B) and l-alanine (A) or l-leucine (L). The relationships between their bulk structure and membrane properties were investigated, such as the swelling ratio in water, aqueous vapour permeability, tensile properties and enzymatic degradation behaviour of the membranes in a pseudo-extracellular fluid (PECF). The tensile properties of the hydrophilic membranes were highly dependent on the swelling ratio of PECF, and the hydrophobicity of the side chains, whose behaviour was typical of an elastomer. We showed that a common relation was obtained between the rate of water vapour permeability and the swelling ratio of membranes in PECF despite the difference of the nature of the side chains. Biodegradation of these membranes in vitro by bromelain indicated that the degradation was a bulk rather than a surface phenomenon, and that the rate of degradation was also highly dependent on the swelling ratio of samples and on the hydrophobicity of the side chains of samples.

Conformational Structure of an L-Asparagine/L-Aspartate Random Copolypeptide as Revealed by Vibrational and CD Spectroscopy

The infrared and Raman spectra of an L-asparagine/L-aspartate random copolypeptide with an L-asparagine residue content of 69% mol have been measured in aqueous solution and in the solid state and analyzed for the first time. The infrared amide I, I', and II bands have been related to the conformational structure of the polypeptide backbone with the aid of CO spectroscopy, whereby it has been concluded that this biopolymer adopts a random coiled secondary structure. The backbone conformation remains unordered even when side-chain carboxyl groups are in the undissociated form (-COOH). The fact that this polypeptide takes this secondary structure in preference to α-helices and β-sheets can be explained by weak hydrophobic bonding between side chains and/or by hydrogen bonding between peptide CONH groups and side-chain polar groups. Another interesting site is the amide III Raman region, which in spite of the above results shows a band in the range usually attributed to α-helical structure. This finding is due to contributions from methine bonds and CH2 groups and indicates that the side chains can influence the position of the bands in the amide III region. Consequently, this result reveals that associating polypeptide backbone conformation with characteristic frequency ranges in the amide III region may be incorrect.

Channel Forming Activity of an Anionic Amphiphilic Sequential Polypeptide in a Cationic Bilayer Membrane

Abstract A channel forming activity of hydrophobic polypeptide, hydrophobic and anionic random copolypeptide, and anionic amphiphilic sequential copolypeptide, respectively, was investigated in a cationic bilayer membrane composed of dimethyldioctadecylammonium chloride (DOACl), and correlated with their location in the membrane estimated by fluorescence spectroscopic and microscopic measurements. A pure hydrophobic polypeptide, poly(γ-methyl l-glutamate) (PMG), was incorporated into DOACl bilayer membrane resulting from their hydrophobic interaction. The incorporation, however, allowed PMG to exhibit very little channel forming activity for sodium ion. A hydrophobic and anionic random copolypeptide, composed of γ-methyl l-glutamate and l-glutamic acid containing 30 mol% of l-glutamic acid (70/30 MG/GA) could hardly penetrate the bilayer membrane and was almost localized at the cationic membrane surface because of Coulombic interaction. 70/30 MG/GA did not effectively control the sodium ion permeability through the membrane. On the other hand, an anionic amphiphilic sequential polypeptide composed of γ-methyl l-glutamate and l-glutamic acid (am. -MG/GA) was incorporated into the cationic DOACl membrane to form the transmembrane bundle ca. 40 Å in diameter. This transmembrane bundle consisting of am. -MG/GA acted as a channel for sodium ion. However, lithium ion having larger hydration size than sodium ion could not penetrate through the channel.

Synthesis and properties of hydrophilic copolypeptide membranes containing L‐aspartic acid as one component

AbstractThree‐component random copolypeptide consisting of N‐hydroxypropyl‐L‐glutamine, L‐aspartic acid, and L‐lysine, related two component random copolypeptides and homopolypeptides, were prepared by carrying out aminolysis reaction with 3‐amino‐1‐propanol, followed by crosslinking reaction with 1,8‐octamethylenediamine on starting polymer membranes consisting of γ‐methyl‐L‐glutamate, L‐aspartic acid, and L‐lysine. The effective crosslink density was shown to be proportional to the content of the crosslinker in the reaction mixture. The tensile properties of these hydrophilic membranes were highly dependent on the degree of swelling in the pseudoextracellular fluid, hydrophobicity of the side chains, and the effective charge density of membranes, and their behavior was typical of an elastomer. A higher rate of water permeability was obtained with charged membranes than noncharged and/or compensated charged membranes with the same order of the degree of swelling in the pseudo‐extracellular fluid. Biodegradation of the samples in vitro by papain indicated that the degradation could be regarded as a bulk rather than a surface phenomenon. The rate of degradation was also highly dependent on the degree of swelling of membranes, as well as on the hydrophobicity and the effective charge density of side chains of sample membranes.

Nuclear magnetic resonance study on the relationship between the helicity and L‐alanine content of L‐alanine/β‐alanine random copolypeptides in connection with silk fibroin conformation

AbstractThe helicities of six kinds of L‐alanine/β‐alanine random copolypeptides having different L‐alanine contents were determined using 1H NMR. Next, the theoretical helicities of these copolypeptides were calculated on the basis of a statistical thermodynamic analysis of the helixcoil transition of the random copolypeptide — using Monte Carlo methods — as a function of the L‐alanine content. The statistical weight parameters used in the model were determined from a comparison of the observed and calculated results. The experimental and theoretical results are in agreement with the fact that Bombyx mori silk fibroin with 30,0 mol‐% L‐alanine content assumes exclusively a random‐coil conformation and that Philosamia cynthia ricini silk fibroin with 48,8 mol‐% L‐alanine content locally assumes an α‐helical conformation in the sequence of the L‐alanine residues, both in aqueous solution.

The interaction of cultured cells with membranes composed of random and block copolypeptides.

Random copolypeptides and block copolypeptides were synthesized, and an interaction between these polypeptide membranes and the cells was studied by a cell culture method (cell line, Ca. 9.22). In random copolypeptides composed of gamma-methyl L-glutamate and gamma-benzyl L-glutamate, cell attachment and cell growth depended on the monomer composition, and showed a maximum at around 70 mole % of benzyl glutamate. Block copolypeptide composed of L-methionine and oxyethylene exhibited low cell attachment and cell growth even at 10 mole % of oxyethylene content, compared to L-methionine homopolymer. ESCA study of the membrane suggested this result to be due to concentration of the poly(oxyethylene) block chain of the polymer on the surface of the membrane. Block copolypeptide composed of N5-(3-hydroxypropyl) L-glutamine and L-leucine exhibited low cell attachment and cell growth, while the corresponding random copolypeptide exhibited high cell attachment and cell growth. This difference is attributable to the microheterophase structure with the hydrophilic domains embedded in the hydrophobic matrix in the block copolypeptide membrane.

Enzymatic degradation of synthetic poly(.ALPHA.-amino acid) fibers. [2]. Copoly(N-hydroxyethyl-L-glutamine/.GAMMA.-methyl-L-glutamate) fiber.

Two component random copolypeptide (PHEG-co-PMLG) fibers consisting of N-hydroxyethyl-L-glutamine and γ-methyl-L-glutamate with different copolymer composition were prepared by a partial aminolysis reaction of poly (γ-methyl-L-glutamate) fibers with 2-amino-1-ethanol, followed by crosslinking reaction with 1, 8-octamethylenediamine. The tensile properties of these hydrophilic fibers were highly dependent on the degree of swelling Qw in PBS solution (pH=7.4), and showed a typical elastomeric behavior in wet-state. Biodegradation behaviors of PHEG-co-PMLG fibers were performed in vitro, including enzymatic degradation and mechanical properties to simulate in vivo fiber degradation. The rate of degradation of these hydrophilic fibers in vitro by pronase E was also highly dependent on Qw of fibers. It was pointed out that the enzymatic degradation of these fibers was processed on the surface area of the fibers, and that the rate of degradation of these fibers was analyzed by the first order reaction rate concerning the effective surface area of the fiber. This fact was also demonstrated from the direct observation by scanning electron microscopy. The behavior of the decrease in the tensile strength as a function of the decrease in the fiber weight was characteristic for the enzymatic degradable fibers and quite different from that for the non-enzymatic degradable fibers such as polyglycolic acid fiber. The tissue reaction was observed to be extremely slight for these hydrophilic fibers, which have been replaced by the connective tissue quite safely during the absorption process.