Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.     

In vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid)s

The in vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid)s was investigated. Benzyl-ether-protected hydroxyl-functionalized dilactones (S)-3-benzyloxymethyl-(S)-6-methyl-1,4-dioxane-2,5-dione (1a) and (S)-3-benzyloxymethyl-1,4-dioxane-2,5-dione (1b) were copolymerized in a melt with various amounts of L-lactide using benzyl alcohol and SnOct2 as the initiator and catalyst, respectively. The benzyl groups were removed by hydrogenation to yield polyesters with hydroxyl functional groups, poly(lactic acid-co-hydroxymethyl glycolic acid) and poly(lactic acid-co-glycolic acid-co-hydroxymethyl glycolic acid) (2a and 2b). Degradation of the hydroxyl-functionalized polyesters and poly(lactic-co-glycolic acid) (50/50) was studied by incubation of pellets of these polymers in phosphate buffer (174 mM, pH 7.4) at 37 degrees C. Polymer degradation was monitored by mass-loss measurements and by gel permeation chromatography, differential scanning calorimetry, and 1H NMR analysis. The degradation times ranging from less than 1 day (for the homopolymer of 2a) to 2 months (copolymer of 25% 2a and 75% lactide) were found. The degradation rates increased with increasing hydroxyl density of the polymers, which was associated with a switch from bulk to surface erosion. NMR and thermal analysis showed that the moieties with the hydroxyl groups were preferentially removed from the degrading polymer. In conclusion, this study shows that the degradation rate of polyesters containing 2a and 2b can be tailored from a few days to 2 months, making them very suitable for biomedical and pharmaceutical applications.     

Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s with functional carbonate building blocks. 1. Chemical synthesis and their structural and physical characterization

This study presents chemical synthesis, structural, and physical characterization of novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s P(BS-co-CC) bearing functional carbonate building blocks. First, five kinds of six-membered cyclic carbonate monomers, namely, trimethylene carbonate (TMC), 1-methyl-1,3-trimethylene carbonate (MTMC), 2,2-dimethyl-1,3-trimethylene carbonate (DMTMC), 5-benzyloxytrimethylene carbonate (BTMC), and 5-ethyl-5-benzyloxymethyl trimethylene carbonate (EBTMC), were well prepared from ethyl chloroformate and corresponding diols at 0 degrees C in THF solution with our modified synthetic strategies. Then, a series of new P(BS-co-CC)s were synthesized at 210 degrees C through a simple combination of poly-condensation and ring-opening-polymerization (ROP) of hydroxyl capped PBS macromers and the prepared carbonate monomers, and titanium tetra-isopropoxide Ti(i-OPr)4 was used as a more suitable catalyst of 5 candidate catalysts which could concurrently catalyze poly-condensation and ROP. By means of NMR, GPC, FTIR, and thermal analytical instruments, macromolecular structures and physical properties have been characterized for these aliphatic poly(ester carbonate)s. The experimental results indicated that novel biodegradable P(BS-co-CC)s were successfully synthesized with number average molecular weight Mn ranging from 24.3 to 99.6 KDa and various CC molar contents without any detectable decarboxylation and that the more bulky side group was attached to a cyclic carbonate monomer, the lower reactivity for its copolymerization would be observed. The occurrences of 13C NMR signal splitting of succinyl carbonyl attributed to the BS building blocks could be proposed due to the randomized sequences of BS and CC building blocks. FTIR characterization indicated two distinct absorption bands at 1716 and 1733 approximately 1735 cm(-1), respectively, stemming from carbonyl stretching modes for corresponding BS and CC units. With regard to their thermal properties, it is seen that the synthesized P(BS-co-CC)s exhibited thermal degradation temperatures 10 approximately 20 degrees C higher than that of PBS. On the basis of the synthesized P(BS-co-BTMC)s, new aliphatic poly(butylene succinate-co-5-hydroxy trimethylene carbonate)s were further synthesized, bearing hydrophilic hydroxyl pendant functional groups through an optimized Pd/C catalyzed hydrogenation. These semi-crystalline new biodegradable aliphatic copolymers with tunable physical properties and functional carbonate building blocks might be expected as potential new biomaterials.     

A new degradable hydroxamate linkage for pH-controlled drug delivery

A new drug delivery system based on a hydrodegradable hydroxamate linkage was evaluated. The carrier support system was poly(N-hydroxyacrylamide), which was synthesized via free radical polymerization of acryloyl chloride in 1,4-dioxane, initiated with 2,2'-azobisisobutyronitrile. The poly(acryloyl chloride) was modified in two steps. First, N-hydroxysuccinimide was added to give the imide ester of poly(acryloyl). In the second step, the imide ester of poly(acryloyl) was reacted with either hydroxylamine or N-methylhydroxylamine to give the corresponding hydroxamic acid. The hydroxamide functionality was then used to link the model drug ketoprofen. All products and intermediates were characterized by elemental analysis and FTIR and 1H NMR spectra. In vitro drug release was performed under specific conditions to elucidate the influence of the pH, polymer microstructure, and temperature on the hydrolysis rate of the amido-ester bond that linked the drug to the macromolecule. The drug release rate from N-methylhydroxamic acid polymers was faster than from hydroxamic acid polymers. All polymers showed higher rates of drug release at higher pH values (9.0 > 7.4 > 2.0) and at higher temperatures (37 degrees C > 20 degrees C).     

Enzymatic preparation of novel thermoplastic di-block copolyesters containing poly[(R)-3-hydroxybutyrate] and poly(epsilon-caprolactone) blocks via ring-opening polymerization

Enzymatic modification of a microbial polyester was achieved by the ring-opening polymerization of epsilon-caprolactone (CL) with low-molecular weight telechelic hydroxylated poly[( R)-3-hydroxybutyrate] (PHB-diol) as initiator and Novozym 435 (immobilized Candida antarctica Lipase B) as catalyst in anhydrous 1,4-dioxane or toluene. The ring-opening polymerization was investigated at different conditions with two different types of PHB-diols: PHB-diol(P), containing a primary OH and a secondary OH end groups, and PHB-diol(M), consisting of 91% PHB-diol(P) and 9% PHB-diol containing two secondary OH end groups. The reactions were followed by GPC analyses of the resulting polymers at different time points, and the optimal conditions were established to be 70 degrees C at a weight ratio of CL/enzyme/solvent of 8:1:24. The ring-opening polymerization of CL with PHB-diol(M) (Mn of 2380, NMR) at the molar ratio of 50:1 under the optimal conditions in 1,4-dioxane gave the corresponding poly[HB(56 wt %)-co-CL(44 wt %)] with Mn (NMR) of 3900 in 66% yield. Polymerization of CL and PHB-diol(P) ( Mn of 2010, NMR) at the same condition in toluene gave the corresponding poly[HB(28 wt %)-co-CL(72 wt %)] with Mn (NMR) of 7100 in 86% yield. Both polymers were characterized by (1)H and (13)C NMR and IR analyses as di-block copolyesters containing a PHB block with a secondary OH end group and a poly(epsilon-caprolactone) (PCL) block with a primary OH end group. NMR analyses and control experiments suggested no formation of random copolymers and no change of the PHB block during the reaction. The enzymatic ring-opening polymerization was selectively initiated by the primary OH group of PHB-diol, whereas the secondary OH group remained as an end group in the final polymers. The thermal properties of the di-block poly(HB-co-CL)s were analyzed by DSC, with excellent T g values for the elastomer domain: poly[HB(56 wt %)- co-CL(44 wt %)] with M n (NMR) of 3900 demonstrated a T g of -57 degrees C, Tm of 145, 123, and 53 degrees C; and poly[HB(28wt%)-co-CL(72wt%)] with Mn (NMR) of 7100 gave a Tg of -60 degrees C, Tm of 147 and 50 degrees C. Thus, the selective enzymatic ring-opening polymerization with PHB-diol as macro-initiator provides a new method for the preparation of PHB-based block copolymers as biomaterials with good thermoplastic properties and novel structures containing functional end groups.     

Functionalized amphiphilic hyperbranched polymers for targeted drug delivery

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(epsilon-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by (1)H NMR, (13)C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug-loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.     

Synthesis, characterization, and lectin recognition of hyperbranched polysaccharide obtained from 1,6-anhydro-D-hexofuranose

1,6-Anhydro-D-hexofuranoses, such as 1,6-anhydro-β-D-glucofuranose (1), 1,6-anhydro-β-D-mannofuranose (2), and 1,6-anhydro-α-D-galactofuranose (3), were polymerized using a thermally induced cationic catalyst in dry propylene carbonate to afford hyperbranched polysaccharides (poly1-3) with degrees of branching from 0.40 to 0.46. The weight-average molecular weights of poly1-3 measured by multiangle laser light scattering varied in the range from (1.02 to 5.84) × 10(4) g·mol(-1), which were significantly higher than those measured by size exclusion chromatography. The intrinsic viscosities ([η]) of poly1-3 were very low in the range from 4.9 to 7.4 mL·g(-1). The exponent (α) in the Mark-Houkwink-Sakurada equation ([η] = KM(α)) of the polymers was 0.20 to 0.33, which is <0.5. The steady shear flow of poly1-3 in an aqueous solution exhibited a Newtonian behavior with steady shear viscosities independent of the shear rate. These viscosity characteristics were attributed to the spherical structures of hyperbranched polysaccharides in an aqueous solution. Poly1-3 contained a high portion of terminal units of 31-43 mol % nonreducing D-hexopyranosyl and D-hexofuranosyl units, in which the D-hexofuranosyl units were 20-44 mol %. Moreover, poly1 and poly2 showed a strong interaction to Concanavalin A due to the cluster effect or multivalent effect of numerous nonreducing saccharide units on their surfaces with binding constants in the range from 1.7 × 10(4) to 2.7 × 10(5) M(-1).     

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).     

Accelerated biodegradation of poly(vinyl alcohol) by glycosidations of the hydroxyl groups or addition of sugars

Biodegradabilities of N-acetyl-d-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 degrees C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP = 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, (1)H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAc-substituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability.     

Effects of residual zinc compounds and chain-end structure on thermal degradation of poly(epsilon-caprolactone)

Effects of chain-end structure and residual metal compounds on thermal degradation of poly(epsilon-caprolactone) (PCL) were investigated by means of thermogravimetric and pyrolysis-gas chromatograph mass spectrometric analyses. Four types of PCL samples with different chain-end structures (alpha-carboxylic acid-omega-hydroxyl-PCL, alpha-dodecyl ester-omega-hydroxyl-PCL, alpha-carboxylic acid-omega-acetyl-PCL, and alpha-dodecyl ester-omega-acetyl-PCL) were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of zinc-based catalyst and by subsequent acetylation reaction of polymers with acetic anhydride. PCL samples with different zinc contents were obtained by washing with acetic acid in chloroform solution of polymer. Thermal degradation behaviors of these PCL samples with different chain-end structures were examined under both isothermal and nonisothermal conditions. From both the isothermal and nonisothermal experiments, the thermal degradation of PCL samples containing high amounts of residual zinc compounds from synthesis process revealed the selective unzipping depolymerization step below 300 degrees C producing epsilon-caprolactone exclusively. In contrast, zinc-free PCL samples were stable at temperatures below 300 degrees C, and the thermal degradation occurred only at temperatures above 300 degrees C regardless of the chain-end structure. From (1)H NMR analysis of the residual molecules after isothermal degradation of zinc-free PCL, it was confirmed that the omega-chain-end of residual molecules was 5-hexenoic acid unit. However, the cyclic monomer and oligomers were detected as the volatile products of zinc-free PCL samples. These results suggest that the dominant reaction of thermal degradation for PCL above 300 degrees C is a competition between the random chain scission via cis elimination reaction and the cyclic rupture via intramolecular transesterification of PCL molecules.     

Freezing denaturation of ovalbumin at acid pH

The effects of rapid freezing and thawing at acid pH on the physiochemical properties of ovalbumin were examined. At low pH (around 2), UV difference spectra showed microenvironmental changes around the aromatic amino acid residues; elution curves by gel permeation chromatography showed decreasing numbers of monomers after neutralization. These changes depended on the incubation temperature (between -196 and -10 degrees C) and the protein concentration (0.5-10 mg/ml), and a low concentration of ovalbumin incubated at around -40 degrees C suffered the most damage to its conformation. With freezing and then incubation at -40 degrees C, three of the four sulfhydryl groups in the ovalbumin molecule reacted with 2,2'-dithiodipyridine. The CD spectra showed these changes in the secondary structure, but they were smaller than those when guanidine hydrochloride was used for denaturation. Supercooling at -15 degrees C or freezing at -196 degrees C had little or no effect on the conformation of the ovalbumin molecule. Thus, irreversible conformational changes of ovalbumin were caused under the critical freezing condition at an acid pH. These changes arose from partial denaturation and resembled those with thermal denaturation of ovalbumin at neutral pH.     

Synthesis and characterization of branched polymers from lipase-catalyzed trimethylolpropane copolymerizations

Lipase-catalyzed terpolymerizations were performed with the monomers trimethylolpropane (B3), 1,8-octanediol (B2), and adipic acid (A2). Polymerizations were performed in bulk, at 70 degrees C, for 42 h, using immobilized lipase B from Candida antartica (Novozyme-435) as a catalyst. To determine the substitution pattern of trimethylolpropane (TMP) in copolymers, model compounds with variable degrees of acetylation were synthesized. Inverse-gated 13C NMR spectra were recorded to first determine the chemical shift positions for mono-, di-, and trisubstituted TMP units and, subsequently, to determine substitution of TMP units along chains. Variation of TMP in the monomer feed gave copolymers with degrees of branching (DB) from 20% to 67%. In one example, a hyperbranched copolyester with 53 mol % TMP adipate units was formed in 80% yield, with Mw 14 100 (relative to polystyrene standards), Mw/Mn 5.3, and DB 36%. Thermal and crystalline properties of the copolyesters were studied by thermogravimetric analysis and differential scanning calorimetry.     

Synthesis and characterization of cyclic and linear helical poly(alpha-peptoids)s by N-heterocyclic carbene-mediated ring-opening polymerizations of N-substituted N-carboxyanhydrides

Cyclic poly(alpha-peptoid)s [a.k.a. poly(N-R-glycine)] with chiral aromatic side-chains [R = (R)- or (S)-CHMePh] have been synthesized by N-heterocyclic carbene-mediated ring-opening polymerization of N-substituted N-carboxyanhydrides (N(R-NCA)). Their linear analogs have been prepared by primary amine-initiated polymerization of the corresponding N(R-NCA). Poly[(R)/(S)-N-CHMePh-glycine] with polymer molecular weights (MWs) in the range of 4-15 kg mol(-1) and low MW distribution (Polydispersity index (PDI) < 1.15) can be readily accessed by these methods. Their high MW analogs were not obtained due to the competitive formation of cyclic oligomeric species that result from intramolecular transamidation. Intrinsic viscosity measurements confirm the architectural difference between the polymers prepared by the two methods and reveals that both cyclic and linear poly[(S)-N-CHMePh-glycine]s behave as random-coil polymers in 0.1M LiBr/Dimethylformamide (DMF) solution. Circular dichroism analysis suggests that the cyclic and linear poly(alpha-peptoid)s retain polyproline I helix conformations, analogously to previously reported linear oligomers. Differential scanning calorimetry analysis reveals that cyclic and linear poly[(S)-N-CHMePh-glycine] are both amorphous with the glass transition temperature of the cyclic polymers (T(g) = 122 degrees C) being notably higher than that of the linear analogs (T(g) = 112 degrees C) with identical MW. These results are consistent with the absence of chain ends, causing the polymers to have reduced segmental mobilities.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).     

Synthesis of two sequential polypeptides by dispersion in benzene and their circular dichroism spectra in aqueous solution: Poly(L-glu-L-lys-L-ala-gly) and poly(L-ala-D-glu-L-lys-D-ala-gly)

The monomers γ-benzylglutamyl-ε-benzyloxycarbonyl-lysylalanylglycine pentachlorophenyl ester and alanyl-γ-benzyl-D -glutamyl-ε-benzyloxycarbonyllysyl-D -alanyl-glycine pentachlorophenyl ester, were polymerized in dilute solutions of dimethylform-amide (DMF) or as dispersions in the same volume of benzene. After deprotection with hydrogen bromide, the products were either chromatographed on Sephadex G-50 or dialyzed. The polymers derived from the polymerization in benzene were considerably larger than those from DMF. The results in benzene indicated that high monomer to solvent ratios are not necessary for the production of high-molecular-weight sequential polypeptides. Circular dichroism spectra of the polymers and monomers at neutral and acid pH indicated that poly(L -Glu-L -Lys-L -Ala-Gly) exists in a random coil configuration and poly(L -Ala-D -Glu-L -Lys-D -Ala-Gly) exists in a β conformation.     

Poly(ether-ester) conjugates with enhanced degradation

When a linear or a four arm star-shaped polyglycidol is used as macroinitiator, densely grafted poly(glycidol-graft-epsilon-caprolactone) and poly(glycidol-graft-L-lactide) and loosely grafted poly[(glycidol-graft-epsilon-caprolactone)-co-glycidol] copolymers have been synthesized by chemical or, in the latter case, by enzymatic catalyzed ring-opening polymerization of epsilon-caprolactone and L-lactide. The well-defined copolymers possess similar molecular weights, but differ in their architecture, microstructure and chemical composition. The hydrolytic degradation behavior was studied in a phosphate buffer solution at pH 7.4 and 37 degrees C for up to 90 days. After different time periods, the mass loss was determined and the degraded copolymers were analyzed by means of NMR, size exclusion chromatography, and scanning electron microscopy. Compared to linear poly(epsilon-caprolactone), poly[(glycidol-graft-epsilon-caprolactone)-co-glycidol] shows a change of the degradation mechanism and a tremendous enhancement of polymer degradation. As this effect is attributed to the high concentration of hydroxy groups at the polyglycidol backbone, this work points out a new possibility to tailor the degradation profiles of polyesters by the introduction of functionality into the polymeric material.     

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers

In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104. The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests. Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information. Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution. By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition. Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics. Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content. The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers. Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV). However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.