Biomacromolecules 2005, 6, 891-896
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Preparation of a Novel Artificial Bacterial Polyester Modified with Pendant Hydroxyl Groups Estelle Renard,* Adrien Poux, Laurianne Timbart, Vale´ rie Langlois, and Philippe Gue´ rin Laboratoire de Recherche sur les Polyme` res, CNRS UMR 7581, 2 rue Henri Dunant, 94320 Thiais, France Received October 21, 2004; Revised Manuscript Received December 8, 2004
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. Introduction Poly(hydroxyalkanoates) (PHAs) comprise a large class of polyesters that are synthesized by many bacteria as intracellular carbon and energy compounds.1,2 Particular interest in PHAs is focused on their biodegradability and biocompatibility3,4 and their physical properties that range from thermoplastic to elastomeric. Mechanical and thermal properties depend on the monomer composition that can be adjusted through appropriate fermentation conditions. A variety of applications have been suggested, such as for controlled release, for biomedical devices, for tissue engineering, and in the environment area. Medium-chain-length (mcl) PHAs consisting of 3-hydroxyalkanoate containing 6-12 carbon atoms are generally produced by several Pseudomonas strains. More than 100 different 3-hydroxyalkanoate monomers have been tailored with either specific substituents or reactive groups.5,6 Pseudomonas sp Gpo1 has been most extensively investigated. This bacterium is very versatile for mcl-PHA synthesis because it can produce polymers containing various side chains.7-11 Biosynthetic efforts have been produced to prepare a variety of copolymers with the greatest majority containing long alkyl side chains. As a result, these polymers are very hydrophobic, which is not favorable for biological applications because polymers with enhanced hydrophilicity are usually more biocompatible in therapeutic and biomedical applications. Functional PHAs with hydrophilic groups such as carboxylic, amine, or hydroxyl groups on the side chains were prepared by biotechnological syntheses, but the fermentation conditions are difficult and generally polymers are obtained with very low yields.9 Recently, the biosynthesis of PHAs with alkoxy groups has been described.12 However, functional PHAs have * Corresponding author. E-mail:
[email protected].
been commonly prepared through post-polymerization modification. Double bonds may be considered as stable precursors of other functional groups. Consequently, polyesters bearing unsaturated side chains have been used as precursors of polymers containing maltosyl,13 hydroxy,14 dihydroxy,15 carboxylic,16-19 and acrylamide20 groups in the side chains. Besides, the presence of polar groups such as acid or hydroxyl can improve the physical properties of polymers and modulate their solubility, hydrophilic/hydrophobic balance, and bio-availability. Thus, Lee et al.15 have recently reported the conversion of double bonds to dihydroxy by oxidation with basic KMnO4. We report here an alternative method for the conversion of olefin side chains to hydroxyl groups. This method is based on the hydroborationoxidation of alkenes. The reaction proceeded to complete conversion without important degradation of the macromolecular chain. The reaction provided the ability to vary the number of hydroxyl groups in the range 10-25% in a controlled manner. Experimental Section Materials. Synthesis of poly(3-hydroxyoctanoate-co3-hydroxyundecenoate) (PHOU) was carried out using Pseudomonas sp Gpo1 as described in a previous report.21 PHOU was produced from a mixture of sodium octanoate and 10-undecenoic acid (80:20). The percentage of unsaturated units corresponds to the proportion of alkenoic acid in the mixture of substrates. PHOU prepared contains 25% reactive olefinic groups. Bacterial polyesters were extracted from lyophilized cells with chloroform in a Soxhlet extractor and then purified by precipitation in methanol. The borane-tetrahydrofuran (BH3-THF) complex in solution in THF (1 M) was purchased from Aldrich and used
10.1021/bm049337+ CCC: $30.25 © 2005 American Chemical Society Published on Web 01/12/2005
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Biomacromolecules, Vol. 6, No. 2, 2005
Figure 1.
1H
Renard et al.
NMR spectrum of PHOU in CDCl3.
without any further purification. THF was dried by refluxing over a Na/benzophenone complex and distilled just before use. Hydrogen peroxide (30%) was purchased from VWR. Hydroboration-Oxidation of PHOU. A typical hydroboration procedure of PHOU for a molar ratio unsaturated units/BH3-THF (1:3) was carried out as follows: 0.1 g of PHOU (1.64 × 10-4 mol of unsaturated units) was dissolved in 10 mL of dried THF. Once PHOU was totally dissolved, 493 µL of BH3-THF (3 equiv per unsaturated units in PHOU) was added dropwise. The mixture was stirred for 15 min at 0 °C. The hydroboration of polyfunctional PHOU yielded trialkylboranes that became insoluble, indicating that cross-linking occurred. The alkylboranes were converted into alcohols by oxidation with alkaline peroxide. A total of 493 µL of a NaOH solution (1 M; 3 equiv based on unsaturated units of PHOU) were added followed by 50 µL of H2O2 (30%; 3 equiv based on unsaturated units of PHOU). The gel removal suggests quantitative oxidation of the trialkylboranes. The solution was acidified with a HCl solution (1 M) until pH 5-6 was reached. THF was evaporated under a vacuum. CH2Cl2 was added to extract the product. Then, the organic layer was washed twice with water and then dried over anhydrous MgSO4. The product was isolated by drying under controlled a vacuum and low T (
