Biomacromolecules 2003, 4, 211-215
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Enzymatic Synthesis and Curing of Biodegradable Epoxide-Containing Polyesters from Renewable Resources Hiroshi Uyama, Mai Kuwabara, Takashi Tsujimoto, and Shiro Kobayashi* Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan Received July 3, 2002; Revised Manuscript Received December 6, 2002
Epoxide-containing polyesters were enzymatically synthesized via two routes using unsaturated fatty acids as starting substrate. Lipase catalysis was used for both polycondensation and epoxidation of the unsaturated fatty acid group. One route was synthesis of aliphatic polyesters containing an unsaturated group in the side chain from divinyl sebacate, glycerol, and the unsaturated fatty acids, followed by an epoxidation of the unsaturated fatty acid moiety in the side chain of the resulting polymer. In another route, epoxidized fatty acids were prepared from the unsaturated fatty acids and hydrogen peroxide in the presence of lipase catalyst, and subsequently the epoxidized fatty acids were polymerized with divinyl sebacate and glycerol. The polymer structure was confirmed by NMR and IR, and for both routes, the high epoxidized ratio was achieved. Curing of the resulting polymers proceeded thermally, yielding transparent polymeric films with high gloss surface. Pencil scratch hardness of the present films improved, compared with that of the cured film obtained from the polyester having an unsaturated fatty acid in the side chain. The obtained film showed good biodegradability, evaluated by BOD measurement in an activated sludge. Introduction Recently, polyester syntheses through enzymatic catalysis by various monomer combinations have been extensively investigated.1 By utilizing specific enzymatic catalysis, enantioselective,2 regioselective,3 and chemoselective4 polymerizations have been developed. As for the enzymatic production of polyesters from dicarboxylic acid derivatives, divinyl esters were found to be effective monomers;5 the lipase-catalyzed polycondensation of divinyl adipate with glycols took place under mild conditions to produce polyesters with molecular weights of several thousands; however, the polymer formation was not observed from adipic acid or diethyl adipate under similar reaction conditions.5a Worldwide potential demands for replacing petroleumderived raw materials with renewable plant-based ones in production of polymeric materials are quite significant in the social and environmental viewpoints.6 Using such plantbased raw materials contributes to global sustainability without depletion of scarce resources. Furthermore, these materials are sometimes cheaper than petrochemicals. We have developed production of new functional polymers from plant oils under mild reaction conditions. Cashew nut shell liquid (CNSL) constitutes nearly the one-third of the total nut weight; thus, much of the amount of CNSL is formed as a byproduct from mechanical processes for the edible use of the cashew kernel. Thermally treated CNSL, whose main component is cardanol, a phenol derivative mainly having a meta substituent of a C15 unsaturated hydrocarbon chain with one to three double bonds, has various * To whom correspondence should be addressed. Phone: +81-75-7535608. Fax: +81-75-753-4911. E-mail:
[email protected].
potential industrial utilizations such as resins, friction lining materials, and surface coatings; however, only a small part of CNSL is used in the industrial field.6 We reported that the oxidative polymerization of thermally treated CNSL using Fe-salen as catalyst produced the oily soluble polymer with a molecular weight of several thousands, which was subjected to curing by cobalt naphthenate catalyst or by thermal treatment, yielding the cross-linked film (“artificial urushi”)7 with a high gloss surface. Recently, we have reported the single-step synthesis of cross-linkable polyesters (4) from plant oil-based unsaturated fatty acids, which were synthesized by lipase-catalyzed polymerization of divinyl sebacate (1) and glycerol (2) in the presence of unsaturated fatty acids (3) under mild reaction conditions.8 The thermal treatment of the polymer having linoleic or linolenic acid groups produced the cross-linked film, which was subjected to biodegradation in an activated sludge. Lipase is known to catalyze epoxidation of unsaturated groups in the presence of a catalytic amount of carboxylic acids under mild reaction conditions.9 A conventional epoxidation process utilizes peracetic or performic acid for oxygen transfer to double bonds, resulting in low yields by side reactions such as acid-catalyzed ring opening of oxiranes. In contrast, the enzymatic epoxidation provides a mild and simple alternative, especially for production of sensitive epoxides. This study deals with enzymatic synthesis and curing of polyesters having an epoxide group in the side chain (6). The epoxide-containing polyesters were synthesized using lipase catalyst via two routes (Scheme 1). One is synthesis of aliphatic polyesters containing an unsaturated group in
10.1021/bm0256092 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/07/2003
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Scheme 1
the side chain from divinyl sebacate, glycerol, and the unsaturated fatty acids, followed by an epoxidation of the unsaturated fatty acid moiety in the side chain of the resulting polymer (route A). In another route, epoxidized fatty acids are prepared from the unsaturated fatty acids and hydrogen peroxide in the presence of lipase, and subsequently the epoxidized fatty acids are polymerized with divinyl sebacate and glycerol to give epoxide-containing polyesters 6 (route B). To our knowledge, this is the first example of the enzymatic synthesis of epoxide-containing polymers; the lipase-catalyzed epoxidation of polybutadiene was reported; however, the in situ formed epoxide group was opened during the reaction.12 Experimental Section Materials. Divinyl sebacate was a gift from Shin-etsu Chemical Co. and stored over freshly activated type 4 molecular sieves. Polyesters having an unsaturated fatty acid moiety in the side chain were synthesized according to the literature.8 Other liquid substrates and solvents were commercially available and used as received. Candida antarctica lipase (lipase CA) was a gift from Novozymes Japan Ltd. An activated sludge was kindly donated by a sewage plant in Fushimi-ku, Kyoto. Enzymatic Epoxidation of Polyesters Having an Unsaturated Fatty Acid in the Side Chain. A typical run was as follows. A mixture of polyester having a linoleic acid moiety (0.63 g, 1.2 mmol of polymer unit), linoleic acid (17 mg, 59 µmol), and lipase CA (30 mg) in 3 mL of toluene was placed in 50 mL flask. Hydrogen peroxide (30%, 0.35 mL, 4.4 mmol) was added dropwise to the mixture under gentle stirring and kept for 24 h. After removal of the enzyme by filtration, the residue was washed with saturated NaCl solution, followed by drying the organic layer over Na2SO4. The organic solvent was removed under reduced pressure, and the residue was dried in vacuo to give 0.27 g of the polymer (yield 40%): 1H NMR (CDCl3) δ 0.9 (t, CH3), 1.3 (m, CH2CH2CH2), 1.5 (m, CH2CH2CHO), 1.6 [m, C(dO)CH2CH2], 1.8 (m, OCHCH2CHO), 2.3 [t, C(dO)CH2], 2.9, 3.1 (m, CHO). 4.2 (m, OCH2CHCH2O), 5.3 (m, OCH2CHCH2O).
Uyama et al.
Enzymatic Epoxidation of Unsaturated Fatty Acids. A typical run was as follows. To a mixture of linoleic acid (1.4 g, 5.0 mmol) and lipase CA (100 mg) in 10 mL of toluene was added 30% hydrogen peroxide (1.4 mL, 18 mmol) dropwise under gentle stirring. After 24 h, the enzyme was removed by filtration, and the residue was washed with saturated NaCl solution. The organic layer was dried over Na2SO4, and the organic solvent was removed by evaporation under reduced pressure. The residue was dried in vacuo to give 0.67 g of epoxidized linoleic acid (yield 43%): 1H NMR (CDCl3) δ 0.9 (t, CH3), 1.3 (m CH2CH2CH2), 1.5 (m, CH2CH2CHO), 1.6 [m, C(dO)CH2CH2], 1.8 (m, OCHCH2CHO), 2.3 [t, C(dO)CH2], 2.9, 3.1 (m, CHO). Enzymatic Polymerization of Divinyl Sebacate, Glycerol, and Epoxidized Fatty Acids. A typical run was as follows. Under argon, a mixture of divinyl sebacate (0.51 g, 2.0 mmol), glycerol (0.18 g, 2.0 mmol), epoxidized linoleic acid (0.62 g, 2.0 mmol), and lipase CA (100 mg) was placed in 2 mL of anhydrous toluene. The mixture was gently stirred under reduced pressure (2700 Pa) at 60 °C for 24 h. A small amount of tetrahydrofuran (THF) was added to the mixture, and the part of the organic solution was separated by filtration. The filtrate was poured into a large amount of methanol/water (70:30 vol %). The resulting oily precipitates were collected by centrifugation, followed by drying in vacuo to give 0.95 g of the polymer (yield 86%). Biochemical Oxygen Demand (BOD) Test in an Activated Sludge. A film sample (12.5 mg) and the activated sludge (15 mL) were taken in a bottle, and the oxygen consumption was measured at 20 °C for 50 days by the BOD tester. BOD-based biodegradability was estimated by the percent of the consumed amount of oxygen, corrected for a blank test to the theoretical amount of oxygen required for complete oxidation of the sample. Measurements. SEC analysis was carried out by using a GPC8020 apparatus equipped with a refractive index (RI) detector at 40 °C under the following conditions: TSK gel G3000HHR column and THF eluent at a flow rate of 1.0 mL/ min. The calibration curves were obtained using polystyrene standards. NMR spectra were recorded on a Bruker DPX400 spectrometer. IR spectra were recorded on a Perkin-Elmer Spectrum One spectrometer. Pencil scratch hardness was evaluated by Mitsubishi Uni pencils with different hardness. The gloss value of films was measured at 60 °C by a BYK gardner micro-TRI-gloss meter. BOD measurement was performed by a TITEC BOD tester 200F. Results and Discussion Lipase-Catalyzed Synthesis of Epoxide-Containing Polyesters. C. antarctica lipase (lipase CA) immobilized on macroporous acrylic resin is industrially developed for modification of triglyceride oils. Previously, we first demonstrated highly efficient catalysis of lipase CA for polymerization of lactones;10 a small amount of this enzyme (less than 1%) induced the polymerization of -caprolactone, and the polymerization rate using lipase CA was much larger than that by other commercially available lipases under similar reaction conditions. In this study, lipase CA was used
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Biodegradable Epoxide-Containing Polyesters Table 1. Enzymatic Epoxidation of 4a 4 3
3/1b
Mnc
Mw/Mnc
yield (%)
3a 3b 3c
0.85 0.89 1.03
9100 3600 8700
1.7 1.7 1.8
47 40 34
6 epoxidation ratiob (%)
Mnc
Mw/Mnc
83 96 76
7900 3300 7700
1.6 1.3 1.5
a Epoxidation of 4 (1.2 mmol of polymer unit) using lipase catalyst (30 mg) in toluene (3 mL) at room temperature for 24 h. b Determined by 1H NMR. c Determined by SEC.
as catalyst for the polymerization of divinyl sebacate and glycerol in the presence of unsaturated or epoxidized fatty acids and the epoxidation of the unsaturated groups in 3 and 4. At first, epoxide-containing polyesters 6 were synthesized via route A. In this study, oleic, linoleic, and linolenic acids (3a, 3b, and 3c, respectively) were used as substrate from renewable plant oils. In the enzymatic synthesis of polyesters, the polymerization was often performed under reduced pressure because the removal of the leaving group (water or alcohol) from the reaction mixture leads to the shift of the equilibrium to the polymer.8,11 Thus, polyesters 4 having an unsaturated fatty acid group were synthesized by lipase CAcatalyzed polymerization of an equimolar mixture of 1, 2, and 3 at 60 °C for 72 h under reduced pressure (2700 Pa).8 The polymer structure was confirmed by 1H NMR. The unit ratio between the unsaturated fatty acid and sebacate units (3/1) in 4 determined by 1H NMR was 0.85-1.03 (Table 1),8 which was relatively close to the feed ratio. The molecular weights of 4 estimated by size exclusion chromatography (SEC) were several thousands. In all cases, the yield was not high, since the product was partly lost in the isolation procedures. The enzymatic epoxidation of the unsaturated group in 4 was performed using hydrogen peroxide as oxidizing agent in the presence of a small amount of fatty acid at room temperature for 24 h. The polymer structure was confirmed by NMR and IR spectroscopies. Figure 1 shows 1H NMR spectra before and after the epoxidation of 4b. In the 1H NMR spectrum of 4b, there were characteristic peaks at δ 5.4, 2.8, and 2.0 (peaks a, d, and f, respectively) due to CHdCH proton and its neighboring methylene protons (Figure 1A). On the other hand, these peaks disappeared in epoxidized product 6b, and new peaks (peaks C and D) were seen at δ 3.0 and 3.1 ascribed to methine protons of the oxirane ring (Figure 1B). The epoxidized ratio was calculated by the ratio of integrated area between peaks C (or D) and J. In the FT-IR spectrum of 4b, there was a characteristic peak at 3010 cm-1 ascribed to C-H stretching of the inner olefin moiety. After the epoxidation, this peak disappeared and a new peak was observed at 820 cm-1 due to C-C asymmetric stretching of oxirane group. The microstructural analysis of 6 by 13C NMR showed that the trisubstituted unit was mainly formed (Figure 2A).3b,3d Next, 6 was synthesized via route B. Lipase-catalyzed epoxidation of 3 using hydrogen peroxide as the oxidizing agent was performed in toluene at room temperature for 24
Figure 1. 1H NMR spectra of (A) 4b and (B) 6b synthesized via route A.
Figure 2. Expanded 13C NMR spectra of 6c via (A) route A and (B) route B.
h under air. 1H NMR analysis showed that the epoxidized ratio was ca. 90% for all unsaturated fatty acids 3. The lipase-catalyzed polymerization of an equimolar mixture of 1, 2, and 5 was carried out at 60 °C for 24 h under reduced pressure (2700 Pa) (Table 2). 1H NMR analysis showed that the epoxidized ratio of 6 was close to
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Table 2. Enzymatic Polymerization of 1, 2, and 5a 6 5b,c
yield (%)
5/1c
epoxidation ratioc (%)
Mnd
Mw/Mnd
5a (0.85) 5b (0.93) 5c (0.88)
83 86 66
0.87 0.93 0.97
89 88 94
5100 4200 6500
1.9 1.9 2.1
a Polymerization of 1, 2, and 5 (2.0 mmol each) using lipase CA (100 mg) as catalyst under 2700 Pa in bulk at 60 °C for 24 h. b In parentheses, epoxidized ratio. c Determined by 1H NMR. d Determined by SEC.
Biodegradability of Cured Film. Previously, we reported that the cured film of 4c was subjected to biodegradation. Here, biodegradability of the cured film from 6c was evaluated by BOD measurement in an activated sludge (Figure 3). The degradation gradually took place, and the biodegradability reached higher than 50% after 50 days, indicating the good biodegradability of the present crosslinked film. Conclusion
Table 3. Pencil Scratch Hardness of Cured Filmsa 4a
4b
4c
6a
-b,c
5Bb
2Bb
