Improved synthetic method and conformation studies of polymers and copolymers of l-β-3,4-dihydroxyphenyl-α-alanine with l-glutamic acid

Eight different copolymers of l-β-3,4-dihydroxyphenyl-α-alanine (l-Dopa) and l-glutamic acid with high degrees of polymerization have been synthesized by the treatment of a series of $\text{copoly}\text{(O,O′-}\text{dimethyl-}\text{l}\text{-Dopa}$, γ-benzyl-l-glutamate) with boron tribromide in chloroform. The conformation of poly(l-Dopa) has been established to be a right-handed helix in trimethyl phosphate on the basis of the following observations. The [θ]₂₂₂ and b₀ values of the copolymers were almost linear with composition in trimethyl phosphate. The linear relationship between the rotation properties and composition indicates that poly(l-Dopa) has the same helical sense as that of poly(l-glutamic acid) which is a right-handed α-helix.     

Potentiometric study of poly(l-glutamic acid) in water-dioxane mixtures

The potentiometric behaviour of poly(l-glutamic acid) (PLGA) has been studied in several water-dioxane mixtures and 0.2 M NaCl/dioxane mixtures. The intrinsic ionization constant pK₀ was found to increase with dioxane content and decreasing temperature. This variation of pK₀ with dioxane content can be explained by the variation of the dielectric constant, ? of the solvent. The free energy change ΔF° associated with the helix-coil transition for the uncharged conformers has been determined. At 25°C, the absolute value of ΔF° increases when decreasing the dielectric constant (i.e. when increasing the dioxane content) but the comparison with the experimental results of other authors show that a change in ? is not sufficient to explain the variations of ΔF° in all solvent mixtures. The temperature dependence of ΔF° in different 0.2 M NaCl/dioxane mixtures was determined between 3° and 55°C. The experimental results show that the stabilization of the α helix by dioxane has an entropic origin as it was already found for a 0.1 M KCl/ethanol/PLGA system. In the absence of salt, a sharp change in the variation of ΔF° is observed for the solvent composition at which a solvent induced conformational transition occurs, along with a change in the absolute adsorption of dioxane on the PLGA molecule. The free energy change involved in the adsorption of a molecule of dioxane on a PLGA residue has been evaluated.     

Synthesis and characterization of poly(l-glutamic acid)-block-poly(l-phenylalanine)

Poly(γ-benzyl l-glutamate)-block-poly(l-phenylalanine) was prepared via the ring opening polymerization of γ-benzyl l-glutamate N-carboxyanhydride and l-phenylalanine N-carboxyanhydride using n-butylamine·HCl as an initiator for the living polymerization. Polymerization was confirmed by ¹H-nuclear magnetic resonance spectroscopy and matrix assisted laser desorption ionization time of flight mass spectroscopy. After deprotection, the vesicular nanostructure of poly(l-glutamic acid)-block-poly(l-phenylalanine) particles was examined by transmission electron microscopy and dynamic light scattering. The pH-dependent properties of the nanoparticles were evaluated by means of ζ-potential and transmittance measurements. The results showed that the block copolypeptide could be prepared using simple techniques. Moreover, the easily prepared PGA-PPA block copolypeptide showed pH-dependent properties due to changes in the PGA ionization state as a function of pH; this characteristic could potentially be exploited for drug delivery applications.     

Synthesis and characterization of poly(ethylene glycol)-b-poly (l-lactide)-b-poly(l-glutamic acid) triblock copolymer

A biodegradable amphiphilic triblock copolymer of poly(ethylene glycol)-b-poly(l-lactide)-b-poly(l-glutamic acid) (PEG-b-PLLA-b-PLGA) was obtained by catalytic hydrogenation of poly(ethylene glycol)-b-poly(l-lactide)-b-poly(γ-benzyl-l-glutamic acid) (PEG-b-PLLA-b-PBLGA) synthesized by the ring-opening polymerization (ROP) of N-carboxyanhydride of γ-benzyl-l-glutamate (BLG-NCA) with amino-terminated MPEG-b-PLLA-NH₂ as a macroinitiator. MPEG-b-PLLA-NH₂ converted from MPEG-b-PLLA-OH first reacted with tert-Butoxycarbonyl-l-phenylalanine (Phe-^NBOC) and dicyclohexylcarbodiimide (DCC) and then deprotected the tert-butoxycarbonyl group. MPEG-b-PLLA-OH was prepared by ROP of l-lactide with monomethoxy poly(ethylene glycol) in the presence of stannous octoate. The triblock copolymer and its diblock precursors were characterized by ¹H NMR, FTIR, GPC and DSA (drop shape analysis) measurements. The lengths of each block polymers could be tailored by molecular design and the ratios of feeding monomers. The triblock polymer PEG-b-PLLA-b-PLGA containing carboxyl groups showed obviously improved hydrophilic properties and could be a good potential candidate as a drug delivery carrier.     

Stereocomplex crystallization and spherulite growth behavior of poly(l-lactide)-b-poly(d-lactide) stereodiblock copolymers

The non-isothermally and isothermally crystallized stereodiblock copolymers of poly(l-lactide) (PLLA) and poly(d-lactide) (PDLA) with equimolar l-lactyl and d-lactyl units and different number-average molecular weights (M_n) of 3.9 × 10³, 9.3 × 10³, and 1.1 × 10⁴ g mol⁻¹, which are abbreviated as PLLA-b-PDLA copolymers, contained only stereocomplex crystallites as crystalline species, causing higher melting temperatures of the PLLA-b-PDLA copolymers compared to those of PLLA homopolymers. In the case of non-isothermal crystallization, the cold crystallization temperatures of the PLLA-b-PDLA copolymers during heating and cooling were respectively lower and higher than those of PLLA homopolymers, indicating accelerated crystallization of PLLA-b-PDLA copolymers. In the case of isothermal crystallization, in the crystallizable temperature range, the crystallinity (X_c) values of the PLLA-b-PDLA copolymers were lower than those of the PLLA homopolymers, and were susceptible to the effect of crystallization temperature in contrast to that of homopolymers. The radial growth rate of the spherulites (G) of the PLLA-b-PDLA copolymers was the highest at the middle M_n of 9.3 × 10³ g mol⁻¹. This trend is different from that of the PLLA homopolymers where the G values increased monotonically with a decrease in M_n, but seems to be caused by the upper critical M_n values of PLLA and PDLA chains as in the case of PLLA/PDLA blends (in other papers), above which homo-crystallites are formed in addition to stereocomplex crystallites. The disturbed crystallization of PLLA-b-PDLA copolymers compared to that of the PLLA/PDLA blend is attributable to the segmental connection between the PLLA and PDLA chains, which interrupted the free movement of those chains of the PLLA-b-PDLA copolymers during crystallization. The crystallite growth mechanism of the PLLA-b-PDLA copolymers was different from that of the PLLA/PDLA blend.     

A route to anionic hydrophilic films of copolymers of l-leucine, l-aspartic acid and l-aspartic acid esters

A series of copolymers of l-leucine and β-benzyl-l-aspartate [Leu/Asp(OBz)] covering the range 30–70 mol % of l-leucine, was synthesized by the N-carboxyanhydride (NCA) method. The copolymers were characterized by elemental analysis, infra-red spectroscopy and viscometry. For all compositions high molecular weight copolymers were prepared with excellent film-forming properties. Tercopolymers of l-leucine, β-benzyl-l-aspartate and β-methyl-l-aspartate [Leu/Asp(OBz)/Asp(OMe)] were obtained after an ester interchange reaction (conversion 85–95%) with the original copolymer systems. These tercopolymers were characterized by elemental analysis and i.r. spectroscopy. Films of the tercopolymers, cast from organic solvents, could be converted into hydrophilic films by saponification of the methyl ester groups using alkaline water/organic solvent media. The hydrophilic films, which will be further investigated for their use as haemodialysis membranes were characterized by potentiometric titration and i.r. spectroscopy.     

pH-dependent self-assembly of amphiphilic poly(l-glutamic acid)-block-poly(lactic-co-glycolic acid) copolymers

A novel biodegradable AB-type diblock copolymer poly(L-lactic- co-glycolic acid)-block-poly(l-glutamic acid) (PLGA-b-PGA) was synthesized by a macromolecular coupling reaction between carboxyl-terminated PLGA and amino-terminated poly(γ-benzyl-glutamate) (PBLG) and the subsequent elimination of the protecting benzyl group. The structures of PLGA-PGA and its precursors were confirmed by Fourier transform infrared spectroscopy (FT-IR), ¹H nuclear magnetic resonance (¹H NMR) spectroscopy and gel permeation chromatography (GPC). This synthetic strategy simplified a former synthesis process of polypeptide-poly(l-lactic acid)(PLA); by using this new synthetic route the molecular weight and block ratio of PLGA-PGA could be easily controlled by adjusting the chain length of PLGA/PGA. The pH sensitivity and self-assembly behavior of PLGA-PGA copolymer were investigated by environmental scanning electron microscopy (ESEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed that the copolymer exhibited high pH responses, and the morphologies of the copolymer aggregates underwent four stages orderly with the pH increase (pH = 3-9): a disorganized form, micelles, semi-vesicles with thick walls and vesicles. Such a pH-dependent self-assembly process of the copolymer is promising for drug control release and bio-applications.     

Crystallization behavior of poly(l-lactic acid)

The crystallization behavior of poly(l-lactic acid) was studied in the range of 80-160 °C. The peak crystallization time (τ_p) was defined and obtained from the crystallization isotherm measured with a differential scanning calorimeter (DSC). Isothermal crystallization temperature (T_c) dependence of log(τ_p) discretely changed at 113 °C (= T_b). The linear growth rate of spherulite, G, was measured with a polarizing microscope. The T_c dependence of G and the size of the spherulite also discretely changed at T_b. Crystal structures for samples isothermally crystallized at temperatures which were higher and lower than T_b were orthorhombic (α-form) and trigonal (β-form), respectively. The discrete change of the crystallization behavior was explained by the formation of different crystal.     

13C and 1H n.m.r. dynamic study of poly(γ-benzyl-l-glutamate) poly(β-benzyl-l-aspartate) and poly(l-alanine) in solution

¹H T₁ relaxation times were measured in solutions of poly (γ-benzyl-l-glutamate), PBLG, and poly(β-benzyl-^l-aspartate), PBLA, and ¹³C T₁ relaxation times, NOE factors and line widths in solutions of the two polypeptides and of poly(l-alanine), PLA, over a broad range of solvent composition (CDCI₃—trifluoroacetic acid) including the helix—coil transition. PBLG was also studied through temperature-induced inverted helix—coil transition. Most relaxation parameters change smoothly over the studied range, and the observed changes correspond to increase of mobility with increasing content of trifluoroacetic acid and with increasing temperature except for PBLG which undergoes an inverted helix—coil transition with temperature. Analysis of experimental data by means of the isotropic model yielded, for the backbone carbons of all three polypeptides, τ_eff ～ 2 × 10^?8 sec in the helix and τ_eff ～ 1 × 10^?9 sec in the coil form. For the side-chain carbons, τ_eff decreases in the range 10^?9 ? 10^?10 sec with increasing distance from the backbone, with τ_eff values consistently lower in the coil form. Results of analysis by means of a model with two correlation times are discussed.     

Preferential and absolute adsorption on poly(l-glutamic acid) in water — dioxane mixtures

The preferential adsorption exhibited by poly(l-glutamic acid) (PLGA) in water-dioxane mixtures has been determined by measuring the specific refractive index increments at constant concentration and at constant chemical potential. These measurements have been carried out at two values of pH (12 and 4.5) and in the presence or in absence of a strong electrolyte (NaCl). At pH 12, in the absence of dioxane, the conformation of the PLGA macromolecule is an ionized chain. When adding low concentrations of dioxane (whether NaCl is present or not) a preferential adsorption of dioxane takes place. While increasing the dioxane concentration the preferential adsorption of dioxane increases and then after passing through a maximum, it decreases and an inversion of the preferential adsorption is observed at higher concentrations of dioxane, water being now preferentially adsorbed. The conformation of PLGA is α-helical, showing that a transition takes place from a disordered ionized state to an ordered one by the addition of dioxane. At pH 4.5, in the absence of dioxane, the conformation of PLGA is α-helical. When adding dioxane in the presence of NaCl, a preferential adsorption of dioxane occurs. While increasing the dioxane concentration no change of the preferential adsorption is observed.     

Controlled crystal nucleation in the melt-crystallization of poly(l-lactide) and poly(l-lactide)/poly(d-lactide) stereocomplex

Among the various inorganic nucleators examined, Talc and an aluminum complex of a phosphoric ester combined with hydrotalcite (NA) were found to be effective for the melt-crystallization of poly(l-lactide) (PLLA) and PLLA/poly(d-lactide) (PDLA) stereo mixture, respectively. NA (1.0 phr (per one hundred resin)) can exclusively nucleate the stereocomplex crystals, while Talc cannot suppress the homo crystallization of PLLA and PDLA in the stereo mixture. Double use of Talc and NA (in 1.0 phr each) is highly effective for enhancing the crystallization temperature of the stereo complex without forming the homo crystals. The stereocomplex crystals nucleated by NA show a significantly lower melting temperature (207 °C) than the single crystal of the stereocomplex (230 °C) in spite of recording a large heat of crystallization ΔH_c (54 J/g). Photomicrographic study suggests that the spherulites with a symmetric morphology are formed in the stereo mixture added with NA while the spherulites do not grow in size in the mixture added with Talc. The exclusive growth of the stereocomplex crystals by the melt-crystallization process will open a processing window for the PLLA/PDLA.     

Anion-induced conformational transition of poly(l-arginine) and its two homologues

The interaction of various anions with poly(l-arginine) and its two homologues, poly(l-homoarginine) and poly(l-α-amino-γ-guanidinobutyric acid), has been studied in the neutral pH region. These polypeptides were found to change their conformations from coil to helix due to I^?, ClO^?₄ and SCN^?, and among them poly(l-homoarginine) and poly(l-arginine) were found to change conformation at smaller concentrations of the anions than poly(l-lysine). The helix of poly(l-homoarginine) was induced in the lyotropic series of the counteranions. Using the van't Hoff enthalpies for the transition of the polypeptides and the transition enthalpies obtained from calorimetry, the various thermodynamic parameters of the transitions were calculated by use of a theory based on the non-specific and the specific binding interactions of the anions with the charged sites on the polypeptides. The binding constants of ClO^?₄ and SCN^? with poly(l-homoarginine) and poly(l-arginine) were found to be four times as large as those with poly(l-lysine). The free energy changes of the transitions from coil to helix of poly(l-homoarginine) and poly(l-arginine) were found to be more negative than that of poly(l-lysine). From these results, the guanidinium ion can be concluded to form easily the ion pair with the anions on the polymer surface, thus allowing poly(l-homoarginine) and poly(l-arginine) to change their conformations at smaller concentrations of the anions than poly(l-lysine).     

Synthesis and conformational study of poly(l-β-3,4-dihydroxyphenyl-α-alanine)

High molecular weight poly(l-β-3,4-dihydroxyphenyl-α-alanine) has been synthesized. Both optical rotatory dispersion and circular dichroism spectra of the polypeptide are anomalous and give little information about its conformation. From the total results obtained by a study of the optical rotation, nuclear magnetic resonance and infra-red absorption, poly(l-β-3,4-dihydroxyphenyl-α-alanine) is most probably right-handed helical in trimethyl phosphate, methanol or water/trimethyl phosphate ($\text{1:1}\text{v}\text{v}$) mixed solvents below pH 10.4. It is in the random coil structure in dimethyl sulphoxide or water/trimethyl phosphate mixed solvents above pH 11. The transition midpoint is pH 10.6 in water/trimethyl phosphate mixed solvents. The results were compared with those of poly(l-tyrosine).     

Properties of l-tyrosine based polyphosphates pertinent to potential biomaterial applications

Since their introduction by Kohn and Langer et al. in 1984, l-tyrosine based ‘pseudo’ poly(amino acids) have undergone extensive research in the area of polymeric biomaterials. Starting from l-tyrosine based diphenolic monomers, polyiminocarbonates, polycarbonates and polyarylates have been developed by Kohn and co-workers and are being investigated for potential orthopedic biomaterial applications. Mao et al. have reported development of l-tyrosine based polyphosphates and polyphosphonates in a patent, however, detailed structural and physico-chemical characterization studies on such polymers have not been reported yet. For the purpose of the current paper, using a novel solid phase process for synthesis of l-tyrosine based diphenolic monomers and adapting the polymerization process described by Mao et al., l-tyrosine based polyphosphates were developed and investigated for their pertinent bioengineering properties. The properties investigated consist of chemical solubility, hydrophilicity and hydrolytic degradation. The results of this investigation are crucial to validate further investigation of biomaterial applications of these polymers.     

Syntheses and conformational studies of poly(S-menthyloxycarbonylmethyl l- and d-cysteines)

S-menthyloxycarbonylmethyl l- and d-cysteines were prepared by the reaction of l- or d-cysteine and (?)-menthyl chloroacetate in liquid ammonia and were then polymerized to poly(S-menthyloxycarbonylmethyll- and d-cysteines) by the N-carboxyanhydride (NCA) method. From the results obtained by means of infra-red spectra, X-ray diffractions, optical rotatory dispersions (o.r.d.), and circular dichroisms (c.d.), $\text{poly}\text{(S-}\text{menthyloxycarbonylmethyl-}\text{l}\text{-cysteine}\text{)}$ was found to be a right-handed α-helix in the solid state and in ethyl ether/chloroform and chloroform solutions. Similarly, $\text{poly}\text{(S-}\text{menthyloxycarbonylmethyl-}\text{d}\text{-cysteine}\text{)}$ was a left-handed α-helix. The helix-coil transition of these polymers was observed in the vicinity of 3–4% trifluoroacetic acid (TFA) in chloroform/TFA mixtures.     

High molecular weight methyl ester of microbial poly(β,l-malic acid): Synthesis and crystallization

High molecular weight poly(α-methyl β,l-malate) (M_n ∼ 25,000, PD ∼ 1.7) was prepared from microbial poly(β,l-malic acid) (M_n ∼ 29,000, PD ∼ 1.3) by methylation with diazomethane in dry acetone without substantial cleavage of the polyester main chain. The thermal properties of this poly(malate) were assessed and its crystal structure was preliminary examined. Two crystal forms were identified by X-ray diffraction, their occurrence being dependent on crystallization conditions. The kinetics of nonisothermal and isothermal crystallizations from the melt were studied and modelled using the Avrami approach. Results were compared to those recently reported by us for low molecular weight poly(α-methyl β,l-malate) (M_n ∼ 3000, PD ∼ 1.3).     

Towards the conception of an amperometric sensor of l-tyrosine based on Hemin/PAMAM/MWCNT modified glassy carbon electrode

A novel amperometric sensor was fabricated based on the immobilization of hemin onto the poly (amidoamine)/multi-walled carbon nanotube (PAMAM/MWCNT) nanocomposite film modified glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and ultraviolet visible (UV-vis) adsorption spectroscopy were used to investigate the possible state and electrochemical activity of the immobilized hemin. In the Hemin/PAMAM/MWCNT nanocomposite film, MWCNT layer possessed excellent inherent conductivity to enhance the electron transfer rate, while the layer of PAMAM greatly enlarged the surface average concentration of hemin (Γ) on the modified electrode. Therefore, the nanocomposite film showed enhanced electrocatalytical activity towards the oxidation of l-tyrosine. The kinetic parameters of the modified electrode were investigated. In pH 7.0 phosphate buffer solution (PBS), the sensor exhibits a wide linear range from 0.1 μM to 28.8 μM l-tyrosine with a detection limit of 0.01 μM and a high sensitivity of 0.31 μA μM⁻¹ cm⁻². In addition, the response time of the l-tyrosine sensor is less than 5 s. The excellent performance of the sensor is largely attributed to the electro-generated high reactive oxoiron (IV) porphyrin (O = Fe^IV-P) which effectively catalyzed the oxidation of l-tyrosine. A mechanism was herein proposed for the catalytic oxidation of l-tyrosine by oxoiron (IV) porphyrin complexes.     

Synthesis and characterization of poly(ε-carbobenzoxy-l-lysyl-γ-benzyl-l-glutamate)

An alternating copolypeptide poly(?-carbobenzoxy-l-lysyl-γ-benzyl-l-glutamate) was synthesised by polymerizing the p-nitrophenyl ester of ?-carbobenzoxy-l-lysyl-γ-benzyl-l-glutamate. The highest molecular weight obtained was 570 000 (2370 peptide residues), which had not yet been attained by the active ester method. The polymers were soluble in various organic solvents including those solvents in which the parent polymers were insoluble, i.e. poly(?-carbobenzoxy-l-lysine) and poly(γ-benzyl-l-glutamate), and their conformations were essentially α-helical except in strong acids such as dichloroacetic acid.     

Insertion of novel optically active poly(amide-imide) chains containing pyromellitoyl-bis-l-phenylalanine linkages into the nanolayered silicates modified with l-tyrosine through solution intercalation

In the present investigation, for the first time, functional optically active poly(amide-imide) (PAI)/organonanosilica bionanocomposite films were successfully fabricated through solution intercalation technique. At the start, Cloisite Na⁺ and protonated form of l-tyrosine amino acid were used for the preparation of the novel chiral organoclay via ion-exchange reaction. Then, PAI containing phenylalanine amino acid was synthesized via solution polycondensation of N,N’-(pyromellitoyl)-bis-phenylalanine diacid chloride (5) with 4,4′-diaminodiphenylsulfone (6). This polymer was end-capped with amine end groups near the completion of the reaction to interact chemically with organoclay. Finally, PAI/organ-nanosilica bionanocomposites films containing 5, 10 and 15% of organoclay were prepared via solution intercalation method through blending of organoclay with the PAI solution. The nanostructures and properties of the PAI/organoclay hybrids were investigated using Fourier transform infrared spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and thermogravimetry analysis (TGA) techniques. XRD, FE-SEM and TEM results revealed the formation of exfoliated and intercalated organoclay platelets in the PAI matrix. TGA results indicated that the addition of organoclay into the PAI matrix increases in the thermal decomposition temperatures of the resulted bionanocomposites. The transparency of the nanocomposite films decreased gradually by the addition of organoclay, and the films became semitransparent as well as brittle at high loading of organoclay. Mechanical data indicated improvement in the tensile strength and modulus with organoclay loading. The film containing a 10 wt.%. of organoclay had a tensile strength of the order of 85.24 MPa relative to the 67.52 MPa of the pure PAI.     

Synthesis and characterization of tailorable biodegradable thermoresponsive methacryloylamide polymers based on l-serine and l-threonine alkyl esters

A series of monomers based on the methyl, ethyl, and isopropyl esters of N^α-(methacryloyl)-serine and -threonine were synthesized, and used in an AIBN-initiated radical polymerization reaction to yield polymers with an M_n ranging between 6.6 and 23.8 kDa. The newly synthesized polymers showed LCST behavior in aqueous solution that could be tailored by subtle variations of the hydrophobicity of the monomers to obtain a broad range of cloud points between 1.5 and >100°C. According to HPLC, the hydrolytic t_1/2-values (pH 7.4 at 37°C) of the monomers were found to be 5, 12, and 40 days of the methyl, ethyl, and isopropyl esters, respectively, while the hydrolysis rate of poly[N^α-(methacryloyl)-Ser-OMe] and poly[N^α-(methacryloyl)-Thr-OMe] was found to be significantly lower compared to the corresponding monomers. In order to obtain thermoresponsive nanoparticles, N^α-(methacryloyl)-Thr-OEt was polymerized with (PEG monomethyl ether 5000)₂-ABCPA as macroinitiator to yield an amphiphilic block co-polymer, poly[N^α-(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether 5000), which forms particles of 300 nm at a temperature higher than its cloud point of 24°C. Incubation at physiological conditions induced ester hydrolysis resulting in a destabilization of the particles making these particles suitable for drug delivery purposes.