Philippe Guerin

Optically active poly (\-malic-acid)

SummaryA simple and reproducible method of synthesizing enantiomers of benzyl malolactonate is described starting from optically active aspartic acid. Chiral benzyl malolactonate is a β-substituted β-lactone monomer which can be readily polymerized anionically using triethylamine as the initiator to yield poly(benzyl β-malate) which is an optically active, semicrystalline polymer. Cleavage of protecting benzyl ester groups yields optically active poly(β-malic acid). The properties of the racemic and optically active monomers and polymers are compared. Optically active (−)poly(β-malic acid) shows one accessible positive CD band in the far UV.

Bacterial poly-3-hydroxyalkenoates with epoxy groups in the side chains

Pseudomonas oleovorans was grown on 10-undecenoic acid and on a mixture of sodium octanoate and 10-undecenoic acid. With either or both of these substrates, cells produced polyesters containing unsaturated side chains in the repeating units. In the case of 10-undecenoic acid as the sole nutrient, the microorganism produced a polymer containing only repeating units with unsaturated side chains. A second bacterium, Rhodospirillum rubrum was grown on 4-pentanoic acid alone and it produced polyesters with shorter pendant groups in the repeating units, including both saturated and unsaturated side chains. Epoxidation of these different bacterial polyesters with m-chloroperbenzoic acid, as 4 chemical reagent, yielded to quantitative conversions of the unsaturated groups into epoxy groups as determined by 1H- and 13C-NMR, no side reaction was observed on the macromolecular chain by molecular weights measurements. More important, it has been possible to produce new functional bacterial polyesters containing terminal epoxy groups in the side chains, in variable proportions up to 37% by growing P. oleovorans on a 10-epoxyundecanoic acid and sodium octanoate culture feed mixture.

Polymers of malic acid and 3-alkylmalic acid as synthetic PHAs in the design of biocompatible hydrolyzable devices.

Poly(beta-malic acid) and poly(beta-3-alkylmalic acid) derivatives, as synthetic polyhydroxyalkanoates (PHAs), present several advantages as macromolecular materials for temporary biomedical applications. Indeed, such polymers, which can be synthesized through different chemical and biological routes, have cleavable ester bonds in their backbone for hydrolytic degradation, stereogenic centres in the monomers units for controlling the macromolecular structure. bioassimilable or non-toxic repeating units and lateral chemical functions which can be adapted to specific requirements. The strategy for building such complex architectures, with one or several specific pendant groups, is based on the anionic ring-opening polymerization or copolymerization of the large family of malolactonic and 3-alkylmalolactonic acid esters. Because we are able to control the monomer synthesis and the polymerization step, we have been able to prepare different degradable materials for the biomedical field, such as: degradable associating networks made up by the association of random copolyesters containing a small percentage of hydrophobic moieties and beta-cyclodextrin copolymers; degradable macromolecular micelles constituted by degradable amphiphilic block copolymers of poly(beta-malic acid) as hydrophilic segments and poly(beta-alkylmalic acid alkyl esters) as hydrophobic blocks; and degradable nanoparticles made up by hydrophobic poly(beta-malic acid alkyl esters) derivatives. We have also prepared a terpolymer which exhibits growth factor-like properties in vivo. Finally, poly(beta-malic acid) has been used as an additive in the preparation of peritoneal dialysis bags.

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g.mol−1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol−1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a β-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 μM CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.

Polymers of malic acid conjugated with the 1-adamantyl moiety as lipophilic pendant group

Abstract Ethyladamantyl malolactonate and butyladamantanamide malolactonate were prepared, starting from malic acid, following the usual synthesis route described for different malolactonic acid esters. Despite the steric hindrance of both adamantyl groups, the three-step synthesis led to the corresponding lactones with a quite good yield and high purity. Otherwise, ethyladamantyl malolactonate has been obtained by chemical modification of the lateral car☐ylic acid function of malolactonic acid. Both ethyladamantyl malolactonate and butyladamantanamide malolactonate were homopolymerized by anionic ring opening polymerization using tetramethylammonium benzoate as initiator. Expected high molecular weight homopolymers were obtained and characterized by 1 H nuclear magnetic resonance (n.m.r.) and size exclusion chromatography (s.e.c.). Furthermore, ethyladamantyl malolactonate was copolymerized with benzyl malolactonate in the molar ratio 5/95. The resulting copolymer was studied by 1 H and 13 C n.m.r., s.e.c. and differential scanning calorimetry. After deprotection of the benzyl protecting groups by catalytic hydrogenolysis, the corresponding poly(β-malic acid- co -ethyladamantyl β-malate) displayed a water solubility.

A study of the biocide release from antifouling paints

Abstract To evaluate the impact of antifouling paints on medium, it is critical to set up test methods which quantify the release of biocides from coatings. Many tests and models have been developed for this estimation, but few of them have been checked experimentally. We compared an European standard with two other protocols and showed that the models of the standard seemed to be inadequate. A static test was chosen because of its easiness of use and its accuracy in the determination of the cumulative release. The developed protocol was a powerful tool to study the effects of formulation on the leaching. The release was relatively independent from the extenders used but strongly dependent on the nature of the binder. Three phenomena generally involved in controlled release from delivery drug systems were considered to explain this effect. But no corroboration was made between hydration, degradation, erosion and the leaching of biocides.

New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester

To study the behaviour of polymeric materials under in-vivo conditions, degradable macromolecular micelles based on amphiphilic block copolymers of poly(β-malic acid) as hydrophilic units and poly(β-malic acid alkyl esters) as hydrophobic blocks are studied. First three β-substituted β-lactones, benzyl malolactonate, butyl malolactonate, and butyl 3-methylmalolactonate were prepared, starting from aspartic acid. A prepolymer based on benzyl malate units was synthesized by anionic ring-opening polymerization of benzyl malolactonate. Then the carboxylic end groups of this prepolymer were used as initiator for the polymerization of the second lactone, e. g. butyl malolactonate or butyl 3-methylmalolactonate. The prepolymer and block copolymers have been characterized by 1H NMR and size exclusion chromatography (SEC). Degradable macromolecular micelles were prepared from the block copolymers by two different methods and characterized by dynamic light scattering and fluorescence measurements using pyrene as a fluorescence probe. It was shown that these amphiphilic degradable copolymers form stable micelles under physiological conditions (10–2M phosphate buffered solution, PBS, pH 7.4 with 0.15 M NaCl). Moreover, it was displayed that the characteristics of these macromolecular micelles, especially the critical micellar concentration (cmc), are depending on the chain length of both blocks and on the chemical structure of the hydrophobic block. A very important conclusion of this study is, that micelle formation is dependent on the pH of the medium. Therefore, besides the fact that such micelles are potentially degradable into non-toxic low molecular weight molecules, their properties and stability were proven to be pH-dependent. This property can lead development of an “intelligent” drug carrier able to release the entrapped biologically active molecule depending on the pH values.

Preparation of a novel artificial bacterial polyester modified with pendant hydroxyl groups.

The Poly(hydroxyalkanoate) (PHA) chemical modifications represent an alternative route to introduce functional groups, which cannot be introduced by bioconversion. PHAs containing unsaturated chains were readily converted into polyesters containing a terminal hydroxyl group on the side chains. With the use of the borane-tetrahydrofuran complex, the pendant side chain alkenes were quantitatively transformed into hydroxyl functions. The conversion proceeded to completion without a significant decrease in molecular weight. The introduction of hydroxyl groups in the products was confirmed from Fourier transform infrared and 1H NMR analysis. The presence of repeating units containing pendant hydroxyl groups in the proportion 25 mol % caused an increase in hydrophilicity of these new PHAs because they were soluble in polar solvents such as ethanol. Besides, these reactive PHAs can be used to bind bio-active molecules or to prepare novel graft copolymers with desired properties.

Bioactive functionalized polymer of malic acid for bone repair and muscle regeneration.

A bioactive poly(β-hydroxyalkanoate) derived from malic acid was prepared and tested on bone repair and muscle regeneration. This functionalized and hydrolyzable polymer was obtained after several steps, the first one being the anionic copolymerization of three malolactonic acid esters. Chemical modifications were carried out on the terpolymer to turn benzyl-protecting groups into carboxyl groups and allyl groups into sulfonate groups. The resulting polymer bore carboxylate, sulfonate, and sec-butyl pendent groups in 65/25/10 molar proportions and were aimed at interacting with heparan binding growth factors. This polymer did not present any toxic effect in cell viability of HepG2 cells, over a large range of concentrations (0.01-0.25 mg l-1). Its ability to improve wound healing was tested in vivo and positive results are reported. Furthermore, the bioactivity of this polymer was evaluated using the regeneration model of Extensor digitorum longus (EDL) rat muscle. The study displayed a significant increase in the muscle regeneration and maturation.

Poly(β -malic acid) derivatives with unsaturated lateral groups: epoxidation as model reaction of the double bonds reactivity

Poly(β-malic acid) derivatives bearing a lateral allyl or 3-methyl-3-butenyl ester group have been prepared by anionic ring opening polymerization or copolymerization of 4-allyloxycarbonyl-2-oxetanone or 4-[3-methyl-3-butenyloxycarbonyl]-2-oxetanone and 4-benzyloxycarbonyl-2-oxetanone as comonomer. These new chiral β-substituted-β-lactones with unsaturated lateral groups have been synthesized from aspartic acid as precursor and by using allylic alcohol or 3-methyl-3-buten-1-ol for opening bromosuccinic acid anhydride, an intermediate compound in the monomer synthesis route. The proportion of unsaturated lateral groups in the polymer was strictly controlled by the proportion of the corresponding lactone in the initial monomers mixture and can vary up to 100%. Functionalized polyesters have been prepared and characterized, and to test the reactivity of the present double bonds, epoxidation has been carried out by using m-chloroperbenzoic acid and dimethyldioxirane as chemical reagents. The activation of the double bond was depending on its chemical environment and on the polymer composition, but quantitative epoxidation has been achieved.