Lipase-Catalyzed Synthesis of Aliphatic Polyesters via

Oct 21, 2008 - Candida antarctica lipase (CALB) has been successfully used as catalyst for copolymerization of dialkyl diester with diol and lactone t...
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Biomacromolecules 2008, 9, 3246–3251

Lipase-Catalyzed Synthesis of Aliphatic Polyesters via Copolymerization of Lactone, Dialkyl Diester, and Diol Zhaozhong Jiang* Biomedical Engineering Department, Yale University, 55 Prospect Street, New Haven, Connecticut 06511 Received July 21, 2008; Revised Manuscript Received September 11, 2008

Candida antarctica lipase (CALB) has been successfully used as catalyst for copolymerization of dialkyl diester with diol and lactone to form aliphatic polyesters. The polymerization reactions were performed using a two stage process: first stage oligomerization under low vacuum followed by second stage polymerization under high vacuum. Use of the two-stage process is required to obtain products with high molecular weights at high yields for the following reasons: (i) the first stage reaction ensures that the monomer loss via evaporation is minimized to maintain 1:1 diester to diol stoichiometric ratio, and the monomers are converted to nonvolatile oligomers; (ii) use of high vacuum during the second stage accelerates equilibrium transesterification reactions to transform the oligomers to high molecular weight polymers. Thus, terpolymers of ω-pentadecalactone (PDL), diethyl succinate (DES), and 1,4-butanediol (BD) with a Mw of whole product (nonfractionated) up to 77000 and Mw/Mn between 1.7 and 4.0 were synthesized in high yields (e.g., 95% isolated yield). A desirable reaction temperature for the copolymerizations was found to be around 95 °C. At 1:1:1 PDL/DES/BD monomer molar ratio, the resultant terpolymers contained equal moles of PDL, succinate, and butylene repeat units in the polymer chains. 1H and 13 C NMR analyses were used to determine the polyester microstructures. The synthesized PDL-DES-BD terpolymers possessed near random structures with all possible combinations of PDL, succinate, and butylene units via ester linkages in the polymer backbone. Furthermore, thermal stability and crystallinity of a pure PDL-DES-BD terpolymer with 1:1:1 PDL to succinate to butylene unit ratio and Mw of 85400 were studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The copolyester was found to be a semicrystalline material with a Tg of -34 °C and a Tm of 64 °C, which degrades in a single weight loss step centered at Tmax ) 408 °C.

Introduction Biodegradable and biocompatible polyesters are important polymeric materials and have been extensively used in biomedical applications, including as biomaterials for constructing absorbable surgical fixation devices, surgical sutures, and controlled release drug carriers.1 Copolymerization of two or more monomers is commonly practiced to synthesize copolyesters with improved properties, such as enhanced mechanical properties and tailored hydrolysis or degradation rates for specific end uses.2 Aliphatic polyesters are produced primarily by condensation copolymerization of dicarboxylic acids with diols, transesterification reaction of diesters with diols, polymerization of hydroxy acids, and ring-opening polymerization of lactones.3 Although various organometallic catalysts were employed for promoting these reactions, enzymes (particularly lipases) have emerged as superior, alternative catalysts due to their higher activity and selectivity, and resultant high purity of products that are also metal-free.4 Numerous lipases are known to be active for condensation polymerization of long chain (gsix carbon chain) aliphatic diacids (or diesters) and diols.5 Recent progress has also rendered it possible to synthesize polyesters from shorter chain substrates. Thus, poly(butylene succinate) (PBS) with a Mw of 38000 and Mw/Mn of 1.39 was prepared from diethyl succinate and 1,4-butanediol using Novozym 435 as catalyst.6 Lactones with different ring sizes have also been employed as substrates in enzymatic ring-opening polymeriza* To whom correspondence should be addressed. Phone: 203-432-7638. Fax 203-432-0030. E-mail: [email protected].

tion.7 Unlike organometallic catalysts,8 lipases show high activity toward large lactones.9 For example, Novozym 435catalyzed polymerization of ω-pentadecalactone (PDL) yielded poly(PDL) with a Mn of 86000.10 Another approach for novel polyester synthesis, which remains largely unexplored, is to use a combined one-step process of ring-opening and polycondensation reactions. For example, copolymerization of β-butyrolactone with 12-hydroxydodecanoic acid was reported to yield a copolyester with a Mn of 2100 and Mw/Mn of 1.2 using porcine pancreatic lipase catalyst.11 On the other hand, Kobayashi, et al. reported copolymerizations of large ring (C11-C15) lactones, divinyl diesters, and various (C2-C12) diols to form copolyesters using various lipases as catalysts.12 After reprecipitation to remove low molecular weight fractions, the copolymers were obtained in 27-80% yields and had a Mn in the range from 5300 to 13000 and Mw/Mn between 1.7 and 3.5. Recently, by employing similar synthesis methods, Thompson et al. synthesized PDL-divinyl adipate-1,3-propanediol and PDL-divinyl adipate-glycerol terpolymers, and successfully processed these polymers into microspheres for potential drug delivery applications.13 However, the divinyl diester monomers are expensive and chemically unstable, and it is a great challenge to synthesize copolyesters using conventional dialkyl diester monomers instead. In this paper, I report CALB-catalyzed synthesis of copolyesters with Mw up to 77000 via copolymerization of three conventional monomers: PDL, diethyl succinate (DES), and 1,4-butandiol (BD). The polymer syntheses were performed using a two-stage process: first stage oligomerization under low vacuum followed by second stage polymerization under high vacuum.14 It needs

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Enzyme-Catalyzed Polyester Synthesis

to be pointed out that there is a fundamental difference in promoting polymer formation using dialkyl diester versus divinyl diester as comonomers for lactone-diester-diol terpolymerization. During copolymerizations of divinyl diester with diol and lactone, the reaction byproduct vinyl alcohol is spontaneously isomerized to acetaldehyde, which serves as a driving force to shift the equilibrium condensation reactions toward polymer formation. Because acetaldehyde does not participate in the equilibrium transesterification reactions, it is not necessary to remove the aldehyde byproduct from reaction mixtures (e.g., by vacuum). In contrast, for copolymerizations of dialkyl diester with diol and lactone, the formed alkanol (e.g., ethanol) byproduct is involved in the equilibrium transesterification reactions and would inhibit the copolymerization reactions if it is not removed upon formation. Thus, the current synthesis method relies on the use of vacuum to facilitate removal of ethanol byproduct and to accelerate polymer chain growth. PDLDES-BD terpolymers are of particular interest because (i) PBS is a well-known biodegradable material1 and (ii) biodegradation rates of the PDL-DES-BD terpolymers are expected to be adjustable based on the polymer compositions. The polymer molecular weights were measured by gel permeation chromatography (GPC) and the polymer structures were characterized by 1H and 13C NMR spectroscopy. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were employed to study the thermal stability and morphology of copolymers.

Experimental Section Materials. Diethyl succinate (DES), 1,4-butanediol (BD), ω-pentadecalactone (PDL), palmitic acid, 1-hexadecanol, and diphenyl ether were purchased from Aldrich Chemical Co. in the highest available purity and were used as received. Immobilized CALB (Candida antarctica lipase supported on acrylic resin) catalyst or Novozym 435, chloroform (HPLC grade), and chloroform-d were also obtained from Aldrich Chemical Co. The lipase catalyst was dried at 50 °C under 2.0 mmHg for 20 h prior to use. Instrumental Methods. 1H and 13C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. The chemical shifts reported were referenced to internal tetramethylsilane (0.00 ppm) or to the solvent resonance at the appropriate frequency. The number and weight average molecular weights (Mn and Mw, respectively) of polymers were measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a model 1515 isocratic pump, a 717 plus autosampler, and a 2414 refractive index (RI) detector with Waters Styragel columns HT6E and HT2 in series. Empower II GPC software was used for running the GPC instrument and for calculations. Both the Styragel columns and the RI detector were heated and maintained at 40 °C temperature during sample analysis. Chloroform was used as the eluent at a flow rate of 1.0 mL/min. Sample concentrations of 2 mg/mL and injection volumes of 100 µL were used. Polymer molecular weights were determined based on a conventional calibration curve generated by narrow polydispersity polystyrene standards from Aldrich Chemical Co. Thermogravimetric analysis (TGA) was carried out using a TA Instruments TGA2950 thermogravimetric analyzer from room temperature to 600 °C, with a heating rate of 10 °C/min, under nitrogen flow. Differential scanning calorimetry (DSC) measurements were performed using a TA Instruments Q100 DSC apparatus equipped with a LNCS (liquid nitrogen cooling system) accessory. Samples (about 5 mg) were heated at 20 °/min from -90 to 180 °C. Glass transition temperature (Tg) and melting temperature (Tm) were taken at half-height of the glass transition heat capacity step and at the peak of the melting endotherm, respectively. General Procedure for CALB-Catalyzed Copolymerization of ω-Pentadecalactone (PDL), Diethyl Succinate (DES), and

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1,4-Butanediol (BD). The ring-opening and condensation copolymerizations were performed in diphenyl ether solution using a parallel synthesizer connected to a vacuum line with the vacuum ((0.2 mmHg) controlled by a digital vacuum regulator. In a typical experiment, reaction mixtures contained (i) PDL, DES, and BD monomers; (ii) 10 wt % Novozym 435 (vs total monomer, dried at 50 °C under vacuum for 20 h prior to use); and (iii) 200 wt % diphenyl ether solvent (vs total monomer). The copolymerization reactions were carried out in two stages: first stage oligomerization, followed by second stage polymerization. During the first stage reaction, the reaction mixtures were stirred at 60-100 °C under 600 mmHg pressure for 18-24 h. Thereafter, the reaction pressure was reduced to 1-3 mmHg and the reactions were continued for an additional 40-60 h. To monitor polymer chain growth, aliquots were withdrawn for analysis during the second stage polymerization. The aliquot samples were dissolved in HPLC-grade chloroform and filtered to remove the enzyme catalyst. Products were not fractionated by precipitation prior to analysis of molecular weight and structure. The filtrates containing whole products were analyzed by GPC using polystyrene standards to measure polymer molecular weights. To determine polymer structures, aliquots were dissolved in chloroform-d. The resultant solutions were filtered to remove catalyst particles and then analyzed by 1H and 13C NMR spectroscopy. PDL-DES-BD terpolymer: 1H NMR (CDCl3; ppm) 1.26 (br, -OCH2CH2-(CH2)10-CH2-CH2-CO-), 1.61 (m, -OCH2-CH2-(CH2)10-CH2CH2-CO-), 1.69 (br, -OCH2-CH2-CH2-CH2O-), 2.29 (t, -OCH2-CH2(CH2)10-CH2-CH2-CO-), 2.61 (s, -OOC-CH2-CH2-COO-), 4.04-4.11 (m, -CH2O-), plus small absorptions at 3.55-3.61 (m) attributable to -CH2OH end groups; 13C NMR (CDCl3; ppm) 24.97, 25.02, 25.22, 25.28, 25.34, 25.88, 25.93, 28.59, 28.66, 29.00, 29.08, 29.17, 29.26, 29.29, 29.48, 29.53, 29.59-29.64 (m), 34.27, 34.37, 63.61, 63.66, 64.09, 64.13, 64.18, 64.35, 64.85, 64.89, 172.20, 172.24, 172.28, 172.31, 173.75, 173.89. Preparation of Pure PDL-DES-BD Terpolymer for Thermal Property Studies. Terpolymerization of PDL, DES, and BD was performed following a procedure analogous to the one as described above. A 1:1:1 PDL/DES/BD monomer molar ratio was employed. The first stage oligomerization was run at 95 °C, 600 mmHg for 18 h, and the subsequent second stage polymerization was carried out at 95 °C, 2.0 mmHg for 48 h. At the end of the reaction, the product mixture was dissolved in chloroform and the resultant chloroform solution was filtered to remove the enzyme catalyst. After being concentrated under vacuum, the filtrate was dropwise added to stirring methanol to cause precipitation of a white solid polymer. The obtained polymer (in 95% yield) was then filtered, washed with methanol three times, and dried under vacuum at 30 °C overnight. The synthesized PDL-DES-BD terpolymer was found to contain 1:1:1 PDL to succinate to butylene unit ratio (by proton NMR analysis) and have a Mw of 85400 and Mw/ Mn of 3.5 (by GPC analysis). Preparation of Butylene Dipalmitate. A reaction mixture containing 1,4- butanediol (0.60 g, 6.66 mmol), palmitic acid (3.41 g, 13.32 mmol), Novozym 435 (0.40 g), and diphenyl ether solvent (6.02 g) was magnetically stirred at 90 °C under 600 mmHg pressure for 18 h. Thereafter, the reaction pressure was reduced to 2.0 mmHg and the reaction was continued at 90 °C for an additional 24 h. At the end of the reaction, the formed product mixture was dissolved in chloroformd, followed by filtration to remove the enzyme catalyst particles. The chloroform-d solution of the product was analyzed by both 1H and 13 C NMR spectroscopy. Except diphenyl ether solvent, butylene dipalmitate was found to be the only compound in the product. Butylene dipalmitate [CH3-(CH2)14-COO-CH2-CH2-CH2-CH2-OOC(CH2)14-CH3]: 1H NMR (CDCl3; ppm) 0.88 (6H, t, -CH3), 1.26 (48H, br, CH3-(CH2)12-CH2-CH2-COO), 1.61 (4H, quintet, CH3-(CH2)12-CH2CH2-COO-), 1.69 (4H, m, -OCH2-CH2-CH2-CH2O-), 2.28 (4H, t, CH3(CH2)12-CH2-CH2-COO-), 4.08 (4H, t, -OCH2-CH2-CH2-CH2O-); 13C NMR (CDCl3; ppm) 14.16 (-CH3), 22.74, 25.00, 25.39, 29.19, 29.33,

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Scheme 1. Two-Stage Process for Copolymerization of PDL, DES, and BD

29.42, 29.53, 29.67, 29.72-29.76 (m), 31.97, 34.24, 63.62 (-OCH2CH2-CH2-CH2O-), 173.54 (-COO-). Preparation of Di(n-hexadecyl) Succinate. Into a round-bottom flask was added 1-hexadecanol (2.92 g, 12.06 mmol), diethyl succinate (1.05 g, 6.03 mmol), Novozym 435 (0.40 g), and diphenyl ether solvent (5.96 g). The resultant mixture was magnetically stirred at 90 °C under 600 mmHg pressure for 18 h. Thereafter, the reaction pressure was reduced to 2.0 mmHg and the reaction was continued at 90 °C for additional 24 h. At the end of the reaction, the formed product mixture was dissolved in chloroform-d solvent. After filtration to remove the lipase catalyst, the chloroform-d solution was analyzed by 1H and 13C NMR spectroscopy. Excluding diphenyl ether solvent, di(n-hexadecyl) succinate was identified to be the only compound present in the product. NMR analysis also showed that, during the reaction, ethanol was formed as a byproduct and was condensed in the dry ice trap between the reaction flask and vacuum pump. Di(n-hexadecyl) succinate [CH3-(CH2)14-CH2-OOC-CH2CH2-COOCH2-(CH2)14-CH3]: 1H NMR (CDCl3; ppm) 0.88 (6H, t, -CH3), 1.26 (52H, br, CH3-(CH2)13-CH2-CH2O-), 1.61 (4H, quintet, CH3-(CH2)13CH2-CH2O-), 2.60 (4H, s, -CH2-COO-), 4.07 (4H, t, CH3-(CH2)13CH2-CH2O-); 13C NMR (CDCl3; ppm): 14.19 (-CH3) 22.77, 25.96, 28.67, 29.19, 29.34, 29.46, 29.62, 29.68, 29.75-29.80 (m), 32.00, 64.84 (-CH2O-), 172.26 (-COO-).

Results and Discussion Two-Stage Process for Copolymerization of ω-Pentadecalactone (PDL), Diethyl Succinate (DES), and 1,4-Butanediol (BD). Because maintaining 1:1 mol/mol stoichiometric ratio of DES to BD is crucial to synthesize high molecular weight copolymers, the polymerization reactions were carried out in two stages: first stage, oligomerization at low vacuum (600 mmHg); and second stage, polymerization under high vacuum. The first stage reaction minimizes monomer loss via evaporation and allows conversion of the monomers to form nonvolatile oligomers. The oligomers are then converted to polymers under high vacuum during the second stage reaction. Scheme 1 illustrates a general reaction for synthesizing PDLDES-BD terpolymers. NMR analysis showed that during the reaction, ethanol was formed as a byproduct, which was condensed in the dry ice trap between the reaction flask and vacuum pump. Temperature Effects on Copolymerization of PDL, DES, and BD. The ring-opening and condensation copolymerizations were studied at different temperatures in diphenyl ether using a 1:1:1 PDL/DES/BD molar ratio and 10 wt % Novozym

Figure 1. Temperature effects on copolymerization of PDL, DES, and BD in diphenyl ether (polymerization conditions: 1:1:1 PDL/DES/BD, 2.0 mmHg pressure).

435 (vs total monomer). After the first stage oligomerization at 600 mmHg for 20 h, polymer chain growth vs reaction time was monitored during the second stage polymerization at 2 mmHg pressure (Figure 1A). The polymer chains grow rapidly during the initial 8 h reaction time. At all reaction temperatures (60, 70, 80, 90, 95 °C), product Mw values reached between 9800 and 25000 by 3 h, increasing up to between 14600 and 44300 by 8 h. From 8 to 52 h reaction time, chain growth was gradual. Among these five reactions at different temperatures, the fastest polymerization occurred at 95 °C. For example, for the copolymerization at 95 °C, Mw values at 3, 8, 25, 35, and 52 h were 25000, 44300, 55200, 66000, and 77100, respectively. In contrast, copolymers formed at 52 h at 60, 70, 80, 90 °C had Mw values of 26000, 35800, 66100, and 66000, respectively. Polydispersity (Mw/Mn) versus Mw for the PDL-DES-BD terpolymers from Figure 1A are shown in Figure 1B. Copolymer polydispersities ranged from 1.7 to 2.5 ((0.1) at Mw e 30000 but increased up to between 2.5 and 4.0 ((0.1) at Mw values from 30000 to 77000. All copolymers showed GPC curves with symmetric and monomodal distributions. The higher polydispersities of the polymers with Mw above 30000 are presumably attributed to high viscosity of the reaction mixtures containing the copolymers and resultant slower diffusivity of oligomer/ polymer chains in the reaction media and between the reaction media and the catalyst particles. At the initial stage of the copolymerizations when the formed polymers had relatively low molecular weights, short polymer chains would diffuse more rapidly within reaction mixtures and from reaction mixtures to

Enzyme-Catalyzed Polyester Synthesis

catalyst particles to form longer polymer chains, yielding low polydispersities.6 However, at the late stage of the polymerizations when the resultant polymers had high molecular weights and reaction mixtures became unusually viscous, the diffusivity difference between long chains and shorter chains could be significantly smaller or even disappear, which would lead to higher product polydispersities. This is consistent with the observation that the copolymers formed at 95 °C versus 80 and 90 °C temperatures had lower polydispersities owing possibly to decreased viscosity of the reaction solution and improved substrate diffusivity at 95 °C (Figure 1B). Furthermore, the two unusually high Mw/Mn values (2.9 and 3.3, Figure 1B) for the copolymerization at 70 °C correspond to the polymer products formed at 35 and 52 h, respectively (Figure 1A). Although exact factors attributing to the high polydispersity values remain unclear, the observed polydispersities of 2.9 and 3.3 could result from polyester hydrolysis side reactions caused by leaking of moisture (from air) into the reactor during sampling. This hypothesis is consistent with the slight decrease in Mw for the polymerization at 70 °C from 25 to 52 h (Figure 1A). To verify that the second stage reaction under high vacuum is essential for synthesis of high molecular weight polymers, the copolymerization of PDL, DES, and BD in 1:1:1 molar ratio was performed at 95 °C under 600 and 760 mmHg pressures, respectively, using 10 wt % Novozym 435 (vs total monomer) in diphenyl ether (200 wt % vs total monomer) for 72 h. GPC analysis showed that the formed products of both reactions had a Mn of less than 1000. Thus, chain growth is driven substantially by high vacuum during the copolymerizations of PDL, DES, and BD. PDL-DES-BD terpolymer composition and unit sequence distribution were analyzed by 1H and 13C NMR spectroscopy. NMR resonance absorptions were assigned by comparing signals of PDL-DES-BD terpolymer to those of two reference polymers, poly(PDL),9a and poly(butylene succinate),6 and by observing changes in signal intensities as a function of variations in the PDL/DES/BD monomer feed ratio used. To further confirm the polymer microstructures, two model compounds, butylene dipalmitate and di(n-hexadecyl) succinate, were synthesized (see Experimental Section) and their 1H and 13C NMR absorptions were also compared with those of PDL-DES-BD terpolymer. The molar ratios of PDL to succinate to butylene units in PDL-DES-BD terpolymers were calculated from proton resonance absorptions: number of PDL units from methylene absorptions at 1.61 or 2.28 ppm, number of succinate units from absorptions at 2.61 ppm, and number of butylene units from absorptions at 1.69 ppm. Figure 2 shows 1H NMR spectrum of the PDL-DES-BD terpolymer formed at 60 °C after 52 h. The resonance absorptions due to terminal -CH2OH groups were observed at 3.55-3.61 ppm. However, the absorptions of expected ethyl ester (-COOEt) termini are not observable due to their overlaps with those of PDL middle methylene groups at 1.26 ppm and -CH2O- groups at 4.06-4.12 ppm. By using this 1H NMR analysis method, all terpolymers formed at 60, 70, 80, 90, and 95 °C after a 52 h reaction time (Figure 1A) were found to have approximately 1:1:1 PDL/succinate/butylene unit ratio. Furthermore, at 60 °C, all polymers formed at 3, 8, 25, 35, and 52 h had the same composition. Thus, neither changes in the reaction temperature after 52 h reaction time nor changes in reaction time at 60 °C altered the terpolymer composition. For the copolymerization of PDL, DES, and BD at 95 °C, the polymer structures were also monitored by 13C NMR spectroscopy. No changes in polymer microstructures were observed between an 8 and 52 h reaction time.

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Figure 2. 1H NMR spectrum of the PDL-DES-BD terpolymer (Mw ) 26000, Mn ) 11300) formed in diphenyl ether at 60 °C after 52 h using 1:1:1 PDL/DES/BD monomer ratio (solvent: CDCl3). Inset: magnification of resonances due to -CH2OH terminal groups.

Figure 3. Selected carbon-13 resonance absorptions of the PDLDES-BD terpolymer formed in diphenyl ether at 60 °C after 52 h using 1:1:1 PDL/DES/BD monomer ratio (solvent: CDCl3): (A) an expansion of the carbon-13 NMR spectrum showing resonances of carbonyl groups in the polymer and (B) an expansion of the carbon-13 NMR spectrum showing absorptions of -CH2O- groups in the polymer.

The distribution of PDL, succinate, and butylene repeat units along polymer chains was analyzed by 13C NMR. Figure 3 displays an expended carbon-13 NMR spectrum of the PDLDES-BD terpolymer formed at 60 °C after 52 h, showing resonances of carbonyl groups (Figure 3A) and -CH2O- groups (Figure 3B) in the polymer. The resonances at 173.89 and 173.75 ppm are attributable to the ester carbonyls [-(CH2)14-COO(CH2)14-COO-, -(CH2)14-COO-(CH2)4-O-] of PDL units adjacent to another PDL and a butylene unit, respectively. The former is identical to the absorption of ester carbonyls in poly(PDL).9a The four resonance absorptions at 172.20, 172.24, 172.28, and 172.31 ppm are due to the ester carbonyls of succinate units. The resonances at 172.20 and 172.31 ppm are ascribable to the carbonyls [-O-(CH2)4-OOC-CH2-CH2COO-(CH2)4-O-, -OOC-(CH2)14-OOC-CH2-CH2-COO-(CH2)14COO-] of succinate units flanked by two butylene units and by two PDL units, respectively. The relative intensity of the former absorption increases with decreasing PDL unit content in the copolymer. The latter resonance matches that of the carbonyls (172.26 ppm) in di(n-hexadecyl) succinate and its relative

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intensity decreases with decreasing PDL unit content in the terpolymer. Synthesis of PDL-DES-BD terpolymers with various ratios of PDL to succinate to butylene repeat units will be reported in a separate publication. On the other hand, the absorptions at 172.24 and 172.28 ppm are presumably due to the presence of succinate units [-OOC-(CH2)14-OOC-CH2-CH2COO-(CH2)4-O-] in between a PDL unit and a butylene unit. The assignments of carbon-13 resonance absorptions of the succinate carbonyls are further supported by the observation that for copolymers with different PDL unit contents, the two absorptions at 172.24 and 172.28 ppm exhibit comparable intensity, while the resonance intensity varies at 172.20 and 172.31 ppm. The resonance absorptions between 63.61 and 64.89 ppm (Figure 3B) are attributable to methylene groups adjacent to an oxygen in both PDL and butylene units. The resonances at 64.13 and 64.35 are likely due to -CH2O- groups in butylene succinatebutylene succinate-butylene succinate [-(CH2)4-OOC-CH2-CH2COO-CH2-CH2-CH2-CH2-OOC-CH2-CH2-COO-(CH2)4-OOCCH2-CH2-COO-] and PDL-PDL [-(CH2)14-COO-CH2-(CH2)13COO-] structures, respectively. The former was the most intense peak for the copolymer with a low (e.g., 19 mol %) PDL content, and the latter was the most intense peak for the copolymer with a high (e.g., 81 mol %) PDL content. The absorptions at 63.66 and 64.85 ppm are ascribable to the presence of butylene units flanked by two PDL units [-O-(CH2)14-COO-CH2-CH2-CH2CH2-OOC-(CH2)14-O-] and succinate units flanked by two PDL units [-OOC-(CH2)13-CH2-OOC-CH2-CH2-COO-CH2-(CH2)13COO-], respectively. The two resonance absorptions appeared substantially weak for the copolymer with a low (e.g., 19 mol %) PDL content. However, their intensities increase with increasing (up to 81 mol %) PDL unit content in the polymer chains. For comparison, the chemical shift of carbon-13 NMR absorption of -CH2O- groups in butylene dipalmitate is 63.62 ppm and that of carbon-13 NMR absorption of -CH2O- groups in di(n-hexadecyl) succinate is 64.84 ppm. Finally, the resonances at 63.61, 64.09, 64.18, and 64.89 ppm are presumably attributed to the presence of butylene succinate-PDL-butylene succinate structural arrangements [CH2-CH2-CH2-fCH2-OOCCH2-CH2-COO-rCH2-(CH2)13-COO-vCH2-CH2-CH2-VCH2-OOCCH2-CH2-COO-], corresponding to the absorptions of four labeled methylene groups vCH2, fCH2, VCH2, rCH2, respectively, in the polymer backbone. Comparisons of chemical shifts for selected carbon-13 resonance absorptions between PDLDES-BD terpolymer and model compounds, butylene dipalmitate, and di(n-hexadecyl) succinate are further delineated in Figure 4. To further elucidate the copolymer unit sequence distribution, statistical analysis was performed and the results were compared to corresponding NMR data. For a completely random PDLDES-BD terpolymer, relative distributions of X-Succinate-Y triads can be calculated by the following equation:

distribution of X-succinate-Y ) fX × fY where X and Y are independently equal to the PDL or butylene unit, fPDL ) molar fraction of PDL units in the polymer chains, and fbutylene ) 2× molar fraction of butylene units in the polymer chains. It needs to be noted that, in the formula to calculate fbutylene, the molar fraction of butylene units is doubled because both ends of butylene units can be adjacent to a succinate unit, while only one end of the PDL units can link to a succinate unit via an ester group. Thus, for the random terpolymer with 1:1:1 PDL to succinate to butylene unit ratio, the relative distributions of

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Figure 4. Comparison of chemical shifts for selected carbon-13 resonance absorptions between PDL-DES-BD terpolymer and model compounds: (A) PDL-butylene-PDL structure in polymer chains, (B) butylene dipalmitate, (C) PDL-succinate-PDL structure in polymer chains, and (D) di(n-hexadecyl) succinate.

PDL-succinate-PDL, PDL-succinate-butylene/butylene-succinate-PDL, and butylene-succinate-butylene triads should be 0.111, 0.222 + 0.222, 0.444, respectively. This number closely matches the ratio 0.11:0.46:0.43 calculated from the corresponding carbonyl carbon-13 absorptions of succinate units at 172.31, 172.28/172.24, 172.20 ppm, respectively (Figure 3A). Therefore, on the basis of these results, the structure of the PDLDES-BD terpolymers can be described as near random arrangements of PDL, succinate, and butylene repeating units in the polymer chains with all possible unit combinations via ester linkages. To determine whether the polymerization reactions were indeed catalyzed by CALB, control experiments were performed without the lipase. The control reaction was performed in diphenyl ether under identical conditions (stage 1: 1/1/1 PDL/ DES/BD, 95 °C, 600 mmHg for 20 h; stage 2: 95 °C, 2.0 mmHg for 52 h). Analysis of the resulting product by GPC showed that its Mn is below 400. This verifies that CALB is the catalyst for terpolymerization of PDL, DES, and BD. It needs to be pointed out that for all reactions at 60, 70, 80, 90, and 95 °C after 3 h reaction time, both GPC and NMR analyses indicated that PDL, DES, and BD monomers were completely converted to polymers. Furthermore, under the twostage reaction conditions employed, loss of the monomers was not observed (e.g., no monomers were found in the dry ice trap between the reactors and vacuum pump). Thus, the polymer yields (unisolated yields) of the reactions are quantitative (∼100%). Thermal Characterization. Thermal stability of PDL-DESBD terpolymer was investigated by thermogravimetric analysis (TGA). Figure 5 shows the TGA curve of the pure PDL-DESBD terpolymer with 1:1:1 PDL to succinate to butylene unit ratio, Mw of 85400, and Mw/Mn of 3.5. The terpolymer is stable up to 300 °C with weight loss of less than 0.1% and degrades

Enzyme-Catalyzed Polyester Synthesis

Figure 5. Thermogravimetric curve of the PDL-DES-BD terpolymer with 1:1:1 PDL/succinate/butylene unit ratio, Mw of 85400, and Mw/ Mn of 3.5.

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tions, use of the two-stage process is crucial to obtain high molecular weight copolymers for the following reasons: (i) the first stage reaction ensures that the monomer loss via evaporation is minimized to maintain 1:1 diester to diol stoichiometric ratio, and the monomers are converted to nonvolatile oligomers; (ii) use of high vacuum during the second stage accelerates equilibrium transesterification reactions to transform the oligomers to high molecular weight polymers. NMR analyses, including statistical analysis on repeat unit sequence distribution, indicate that PDL-DES-BD terpolymers thus synthesized using 1:1:1 PDL/DES/BD molar ratio contain PDL, succinate, and butylene units near randomly distributed in the polymer chains with all possible combinations of the units via ester linkages. TGA and DSC analyses showed that the PDL-DES-BD terpolymer with 1:1:1 PDL to succinate to butylene unit ratio is a semicrystalline material with a Tm of 64 °C and is thermally stable up to 300 °C. PDL-DES-BD terpolymers are biodegradable polymeric materials with potential important biomedical applications. The results on synthesis and solid state properties of PDL-DES-BD terpolymers with compositions other than 1:1:1 PDL/succinate/butylene unit ratio will be published separately. Acknowledgment. This work was supported by Yale University. I also wish to thank Dr. Laura Mazzocchetti at University of Bologna, Italy, for her help in acquiring the DSC and TGA data.

References and Notes

Figure 6. First DSC heating scan of the PDL-DES-BD terpolymer with 1:1:1 PDL/succinate/butylene unit ratio, Mw of 85400, and Mw/ Mn of 3.5. Inset: focus on the glass transition.

in a single weight loss step centered at Tmax ) 408 °C. Differential scanning calorimetry (DSC) was used to study the glass transition and melting behaviours of the copolyester. Figure 6 displays the DSC curve of the PDL-DES-BD terpolymer. The polymer shows a glass transition (Tg) at -34 °C with heat capacity increment (∆Cp) value of 0.42 J/(g °C), which was followed by a sharp and intense melting endotherm (Tm) at 64 °C with melting enthalpy (∆Hm) value of 101 J/g. The observed behaviours indicate that this terpolymer contains a large crystalline fraction. The substantial differences in melting temperature between the PDL-DES-BD terpolymer (Tm ) 64 °C) and poly(PDL) (Tm ) 97 °C)15 or PBS (Tm ) 113 °C)16 reference polymers are consistent with the NMR data, which support random structural arrangements of PDL, succinate, and butylene repeat units in the polymer chains.

Conclusions Dialkyl diester, diol, and lactone monomers have been successfully copolymerized, for the first time, using a lipase catalyst to form aliphatic copolyesters. The polymerization reactions were performed using a two stage process: first stage oligomerization under low vacuum followed by second stage polymerization under high vacuum. During the copolymeriza-

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