Enzymatic Synthesis of Polyesters from Lactones, Dicarboxylic Acid

Biomacromolecules 2000, 1, 335-338

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Enzymatic Synthesis of Polyesters from Lactones, Dicarboxylic Acid Divinyl Esters, and Glycols through Combination of Ring-Opening Polymerization and Polycondensation Shuhei Namekawa,† Hiroshi Uyama,‡ and Shiro Kobayashi*,‡ Department of Materials Chemistry, Graduate School of Engineering, Tohoku University, Aoba, Sendai 980-8579, Japan; and Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501, Japan Received April 14, 2000; Revised Manuscript Received June 15, 2000

Enzymatic copolymerization of lactones, divinyl esters, and glycols has been performed using lipase as catalyst to produce ester copolymers. The monomers used in this study were 12-, 13-, and 16-membered lactones, divinyl esters of adipic and sebasic acids, and R,ω-glycols. Candida antarctica and Pseudomonas cepacia lipases showed relatively high catalytic activity for the present copolymerization, yielding the copolymer having relatively high molecular weight in moderate yields. From 13C NMR analysis, the resulting product was not a mixture of homopolymers, but a copolymer derived from the monomers. NMR data and reaction monitoring results indicate that two different modes of polymerization, ring-opening polymerization and polycondensation, simultaneously take place through enzyme catalysis in one-pot to produce ester copolymers. Introduction A combination of different modes of polymerization is a promising way for preparation of polymers with novel structure, which otherwise cannot be achieved by conventional polymerization processes. However, it is generally difficult that more than two modes of polymerization simultaneously take place in one-pot to form copolymers, since the reaction mechanism and conditions are normally different with each other. Recently, polyester syntheses through enzymatic catalysis by various monomer combinations have been extensively investigated.1 By utilizing specific enzymatic catalysis, enantioselective and regioselective polymerizations have been developed. To obtain polymers of higher molecular weight in high yields, activated diesters having 2,2,2-trifluoroethyl or 2,2,2-trichloroethyl leaving groups have been often used as monomer of the enzymatic polycondensation.2 Recently, divinyl dicarboxylates were found to be more effective monomers for the enzymatic polymerization;3 the lipasecatalyzed polycondensation of divinyl adipate with glycols took place under mild conditions to produce polyesters with molecular weight of several thousands; however, polymer formation was not observed from adipic acid or diethyl adipate under the similar reaction conditions.3a Enzymatic syntheses of aliphatic polyesters have been also achieved by ring-opening polymerization and copolymerization of lactones.4 Lipase showed unique catalysis for the lactone polymerization; macrolides (12-, 13-, 16-, and 17membered lactones) having much lower anionic polymerizability than -caprolactone (-CL) were enzymatically † ‡

Tohoku University. Kyoto University.

Scheme 1

polymerized much faster than -CL. This is considered due to the strong recognition of the macrolides by lipase.1h,4h Lipase-catalyzed polycondensation and ring-opening polymerization are believed to proceed via the similar reaction intermediates (“acyl-enzyme intermediates”).1,4b This speculation explored us to combine two modes of polymerization through enzyme catalysis to produce ester copolymers. The present study deals with lipase-catalyzed copolymerization of lactones, divinyl esters, and glycols (Scheme 1). Relevant to this study, single-step synthesis of polyester macromonomers and telechelics has been achieved by lipasecatalyzed ring-opening polymerization of lactones in the presence of a small amount of vinyl esters, in which the condensation of the terminal alcohol group of poly(lactone) and vinyl ester took place.5 Results and Discussion Copolymerization of Lactones, Divinyl Esters, and Glycols. At first, enzyme screening was carried out in the copolymerization of 12-dodecanolide (13-membered lactone 1b), divinyl adipate (2a), and 1,4-butanediol (3b) in diiso-

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Table 1. Lipase Screen for Copolymerizationa lipase

copolymer

entry

origin

code

1 2 3 4 5

Candida antarctica Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas fluorescens e

lipase CA lipase PA lipase PC lipase PF

yieldb

Mn

(%)

(×10-3)

c

47 18 37 17 0

5.9 10 10 10

Mw/Mnc

lactone unitd (%)

1.8 1.4 1.8 1.3

29 1 17 5

a Copolymerization of 1b, 2a, and 3b (2.0 mmol each) using lipase catalyst (180 mg) in diisopropyl ether (10 mL) at 60 °C for 72 h. b Methanolinsoluble part. c Determined by SEC. d Determined by 1H NMR. e Without lipase.

propyl ether at 60 °C for 72 h. In this study, Candida antarctica, Pseudomonas aeruginosa, Pseudomonas cepacia, and Pseudomonas fluorescens lipases (lipases CA, PA, PC, and PF, respectively) were used as catalysts, which are already known to be active for the polycondensation of divinyl esters with glycols3 as well as the ring-opening polymerization of the macrolides.4 In most cases, the monomers were quantitatively consumed. After the copolymerization, the polymeric materials were purified by reprecipitation (chloroform as good solvent, methanol as poor solvent). During the reprecipitation procedure, the oligomers soluble in methanol might be lost. Copolymerization results are summarized in Table 1. Lipase CA produced the polymer in the highest yield (entry 1). Lipases PA and PF showed relatively low catalytic activity toward the present copolymerization (entries 2 and 4). In the copolymerization without lipase (control experiment), the monomers were recovered unchanged (entry 5), indicating that the present copolymerization proceeded through the enzyme catalysis. Molecular weight was determined by size exclusion chromatography (SEC). The polymer obtained by using a Pseudomonas family lipase as catalyst had a higher molecular weight than that obtained by using lipase CA. Copolymer structure was confirmed by 1H and 13C NMR spectroscopies.3e,4d The unit ratio from 1b in the resulting copolymer, determined by 1H NMR, depended to a large extent on the enzyme origin. The calculated ratio is 33.3% in the case of the equimolar feed ratio of three monomers, which was larger than the observed one in all cases. In using lipases PA and PF, the lactone unit ratio was much smaller than that by lipase CA or PC. This may be due to the difference in activity for the two reactions. These data indicate that the lipase origin much affected the copolymerization behaviors. In subsequent experiments, lipase PC was used as catalyst. The solvent effect was examined in the lipase PC-catalyzed copolymerization of 1b, 2a, and 3b under the similar conditions of Table 1. The copolymerization in isooctane produced the copolymer with molecular weight of 4.3 × 103 in 18% yield, the lactone unit of which was only 1.3%. In using acetonitrile or toluene as solvent, no polymeric materials were obtained. From these data, diisopropyl ether was found to be the most suitable for the present copolymerization. Table 2 shows effects of monomer structure on the polymerization behaviors. Lactones used in this study were 11-undecanolide (12-membered 1a), 1b, and 15-pentadecanolide (16-membered 1c). In fixing 2a and 3b as the

Table 2. Effect of Monomer Structurea comonomers

copolymer lactone yieldb (%) Mnc (×10-3) Mw/Mnc unitd (%)

entry

1

2

3

1 2 3 4 5 6 7 8 9

1a 1a 1b 1b 1b 1b 1b 1c 1c

2a 2b 2a 2a 2a 2a 2b 2a 2b

3b 3b 3a 3b 3c 3d 3b 3b 3b

27 40 30 37 51 60 55 40 80

8.2 5.3 7.0 10 10 6.1 5.5 12 6.5

1.7 3.2 1.8 1.8 2.2 3.5 2.8 1.9 2.7

6 10 11 17 16 12 24 15 27

a Copolymerization of 1, 2, and 3 (2.0 mmol each) using lipase PC catalyst (180 mg) in diisopropyl ether (10 mL) at 60 °C for 72 h. b Methanolinsoluble part. c Determined by SEC. d Determined by 1H NMR.

Table 3. Effect of Feed Ratioa copolymer entry

feed ratio 1b:2a:3b

yieldb (%)

Mnc (×10-3)

Mw/Mnc

lactone unitd (%)

1 2 3 4

1:1:1 2:1:1 4:1:1 10:1:1

37 37 54 29

10 13 12 5.8

1.8 2.2 2.2 1.8

17 (33) 22 (50) 37 (67) 44 (83)

a Copolymerization of 1b, 2a, and 3b (total 6.0 mmol) using lipase PC catalyst (180 mg) in diisopropyl ether (10 mL) at 60 °C for 72 h. b Methanolinsoluble part. c Determined by SEC. d Determined by 1H NMR. In parentheses, calculated value based on the feed ratio.

divinyl ester and glycol monomers, respectively, the polymerization results using 1b and 1c were similar to each other (entries 4 and 8), whereas the yield, molecular weight, and lactone unit of the copolymer from 1a were lower than those from other lactones (entry 1). In the combination of divinyl sebacate (2b) and 3b, a similar tendency was observed (entries 2, 7, and 9). As to the divinyl ester monomer, the yield and lactone unit of the copolymer from 2b were higher than those from 2a, on the other hand, the opposite phenomena were found in the molecular weight of the copolymer. The methylene chain length of glycols also affected the polymerization behaviors. The yield increased as a function of the methylene chain length (entries 3-6). In the lipase PC-catalyzed polycondensation of divinyl esters and glycols, similar polymerization results were reported.3e The highest yield (80%) was achieved in the combination of 1c, 2b, and 3b (entry 9). Effects of the feed ratio were examined in the combination of 1b, 2a, and 3b (Table 3). In the feed ratio of 4:1:1, the yield was the highest (entry 3). The molecular weight was relatively high (>1 × 104) except for the feed ratio of 10:

Enzymatic Synthesis of Polyesters

Biomacromolecules, Vol. 1, No. 3, 2000 337

Figure 2. Time vs monomer conversion and polymer molecular weight in the copolymerization of 1b, 2a, and 3b: (0) conversion of 1b; (O) conversion of 2a; (4) conversion of 3b; (b) polymer molecular weight. The copolymerization was performed using lipase PC as catalyst in diisopropyl ether at 60 °C.

Figure 1. Expanded Table 3).

13C

NMR spectrum of copolymer (entry 3 in

1:1. The lactone content increased as a function of the feed ratio of 1b and was lower than the calculated one. Analysis of Microstructure. Microstructure of the copolymers was analyzed by 13C NMR spectroscopy. In the present copolymerization of a combination of three monomers, the equimolar amount of divinyl ester and glycol monomers is assumed to react each other, and hence, the resulting copolymer can be regarded as of binary system (lactone and divinyl ester/glycol). Figure 1 shows an enlarged NMR spectrum of the copolymer from 1b, 2a, and 3b (entry 3 in Table 3) in the region δ 63-65. Four peaks (P-S) due to C(dO)OCH2 were observed, corresponding to four different triads of the statistically binary copolymer, indicating that the product was a copolymer and not a mixture of two homopolymers. As an index of diad sequence distribution, a universal parameter expressed as D ) FiiFjj/FijFji (F, diad relative intensity; i (j), monomer unit) is often used.6 Theoretically, the D value is larger than 1.0 for a block copolymer, whereas an alternative copolymer has the value less than 1.0, and in the case of random copolymer, it approaches 1.0. From the integrated area of the peaks, the D value was calculated as 1.85, indicating that the copolymer was not statistically random. Reaction Monitoring. To examine whether the ringopening polymerization and polycondensation simultaneously take place in the present copolymerization, the reaction was monitored by using gas chromatography (GC) and SEC. Figure 2 shows polymerization time vs the conversion of monomers (1b, 2a, and 3b) and polymer molecular weight. The divinyl ester and glycol monomers were consumed in the early stage of the polymerization, whereas the lactone conversion and molecular weight of the polymer gradually increased. Our previous study on the lipase-catalyzed polycondensation of 2a and 3b showed that the reaction was of typical polycondensation type; the monomers were quickly

Figure 3. WAXD spectra of (A) poly(1b), (B) poly(1,4-butylene adipate), and (C) copolymer (entry 3 in Table 1).

consumed, and thereafter, the molecular weight increased.3e From these data, it is supposed that the polymerization proceeds as follows; at first, the oligomeric products from 2a and 3b are formed. Afterward, the reaction of the resulting oligomers and 1a takes place via the acyl-lipase intermediates to give the ester copolymers. At the initial stage of the copolymerization of 1b, 2b, and 3b, the copolymer unit, e.g., a linkage between 1b and 3b, was observed by 13C NMR, supporting the simultaneous copolymerization of the lactone, divinyl ester, and glycol monomers. Properties of Copolymers. Figure 3 shows wide-angle X-ray diffraction (WAXD) patterns of poly(1b), poly(1,4butylene adipate) from 2a and 3b, and the present copolymer from 1b, 2a, and 3b (entry 3 in Table 1). The copolymer had the (110) and (200) peaks with 2θ ) 21 and 24°, respectively. The spectral pattern of the copolymer was similar to that of poly(1b), rather than that of poly(1,4butylene adipate). The polarization micrograph of the copolymer showed fine crystal aggregates (data not shown), which were also observed in that of poly(1b). The WAXD of the copolymer was slightly broader than that of poly(1b), suggesting that the crystallinity of the copolymer was lower than that of poly(1b). Table 4 summarizes thermal properties of poly(1b), poly(1,4-butylene adipate), and the copolymer, determined by

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Table 4. Thermal Properties of Polyesters samplea

Tgb (°C)

Tmb (°C)

Tcc (°C)

∆Hmb (J/g)

poly(1b) (1.4 × 104) poly(1,4-butylene adipate) (1.6 × 104) copolymerd(1.0 × 104)

-76 -63 -62

84 57 53

72 45 40

200 120 120

a In parentheses is given the molecular weight of each sample. b Data of the second scan of DSC. c Determined by polarization microscopy. d Ester copolymer of entry 3 in Table 1.

differential scanning calorimetry (DSC) under nitrogen and polarization micrography. Melting point (Tm) and crystallization temperature (Tc) of the copolymer were lower than those of both homopolymers. This may be due to the lower crystallinity of the copolymer, which is supported by the lower melting enthalpy (∆Hm) of the copolymer. Thus, the present copolymerization suggests a new method to control these properties of polyesters. Conclusion The lipase-catalyzed copolymerization of lactones, dicarboxylic acid divinyl esters, and glycols produced the corresponding ester copolymers. The polymer yield and molecular weight were dependent on the monomer combination and feed ratio. In the present copolymerization, two different types of polymerization, ring-opening polymerization and polycondensation, simultaneously occurred probably via the same reaction intermediate (“acyl-lipase intermediate”). To our knowledge, this is the first example of such a case, which is accomplished only by the enzymatic catalysis. Further investigations on the enzymatic synthesis of copolymers by two different types of polymerization via the acyl-enzyme intermediates are underway in our laboratory. Experimental Section Materials. Divinyl sebacate was a gift from Shin-etsu Chemical Co. Other liquid monomers and polymerization solvents were commercially available and stored over freshly activated type 4 molecular sieves. Lipases CA and PA were kindly donated by Novo Nordisk Bioindustry Ltd. and Nagase Seikagaku Co., respectively. Lipases PC and PF were gifts from Amano Pharmaceutical Co. Lipases were used without further purification. Enzymatic Copolymerization. A typical rum was as follows. A mixture of three monomers (each 2.0 mmol), lipase (180 mg), and solvent (10 mL) was placed in a dried test tube. The mixture kept at 60 °C under gentle stirring. After 72 h, the solvent was removed under reduced pressure. Chloroform (10 mL) was added to the residue and the part of the organic solution was separated by filtration. The filtrate was concentrated under reduced pressure, and the residue was poured into a large amount of methanol. The resulting precipitates were collected by centrifugation, followed by drying in vacuo to give the copolymer. Measurements. SEC analysis was carried out by using a Tosoh SC8010 apparatus equipped with refractive index (RI) detector at 40 °C under the following conditions: TSKgel G3000HHR column and chloroform 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. GC analysis was carried out using a Shimadzu GC-14B apparatus equipped with an FID detector and a TC-5 column (GL Sciences). DSC measurement was made at a 20 °C/min heating and cooling rates under nitrogen using a Seiko SSC/5200 differential scanning calorimeter calibrated with an indium reference standard. WAXD patterns were recorded on a Rigaku RINT-1400 (40 kV/200 mA) system with the Cu-KR X-ray beams. Polarization microscope measurement was carried out using a Nikon optical microscope equipped with crossed polarizers and a hot stage. Acknowledgment. This work was supported a Grandin-Aid for Specially Promoted Research (No. 08102002) from the Ministry of Education, Science, and Culture, Japan. We acknowledge the gift of divinyl sebacate and lipases from Shin-etsu Chemical Co., Amano Pharmaceutical Co., Nagase Seikiagaku Co., and Novo Nordisk Bioindustry, Ltd. References and Notes (1) (a) Kobayashi, S. J. Polym. Sci., Polym. Chem. Ed. 1999, 37, 3041. (b) Kobayashi, S.; Shoda, S.; Uyama, H. In Catalysis in Precision Polymerization; Kobayashi, S., Ed.; John Wiley & Sons: Chichester, England, 1997; Chapter 8. (c) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1. (d) Kobayashi, S.; Shoda, S.; Uyama, H. In The Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: Boca Raton, FL, 1996; pp 2102-2107. (e) Gross, R. A.; Kaplan, D. L.; Swift, G. ACS Symp. Ser. 1998, 684. (f) Ritter, H. In Desk Reference of Functional Polymers, Syntheses and Applications; Arshady, R., Ed.; American Chemical Society: Washington, DC, 1997; pp 103-113. (g) Kobayashi, S.; Uyama, H.; Namekawa, S. Polym. Degrad. Stab. 1998, 59, 195. (h) Namekawa, S.; Suda, S.; Uyama, H.; Kobayashi, S. Int. J. Biol. Macromol. 1999, 25, 145. (2) (a) Wallace, J. S.; Morrow, C. J. J. Polym. Sci., Polym. Chem. Ed. 1989, 27, 2553. (b) Brazwell, E. M.; Filos, D. Y.; Morrow, C. J. J. Polym. Sci., Polym. Chem. Ed. 1995, 33, 89. (c) Linko, Y.-Y.; Wang, Z.-L.; Seppa¨la¨, J. J. Biotechnol. 1995, 40, 133. (3) (a) Uyama, H.; Kobayashi, S. Chem. Lett. 1994, 1687. (b) Chaundhary, A. K.; Beckman, E. J.; Russell, A. J. Biotechnol. Bioeng. 1997, 55, 227. (c) Chaundhary, A. K.; Beckman, E. J.; Russell, A. J. AIChE J. 1998, 44, 753. (d) Uyama, H.; Yaguchi, S.; Kobayashi, S. Polym. J. 1999, 31, 380. (e) Uyama, H.; Yaguchi, S.; Kobayashi, S. J. Polym. Sci., Polym. Chem. Ed. 1999, 37, 2737. (4) (a) Uyama, H.; Kobayashi, S. Chem. Lett. 1993, 1149. (b) Uyama, H.; Takeya, K.; Kobayashi, S. Bull. Chem. Soc. Jpn. 1995, 68, 56. (c) MacDonald, R. T.; Pulapura, S. K.; Svirkin, Y. Y.; Gross, R. A.; Kaplan, D. L.; Akkara, J. A.; Swift, G.; Wolk, S. Macromolecules 1995, 28, 73. (d) Uyama, H.; Takeya, K.; Hoshi, N.; Kobayashi, S. Macromolecules 1995, 28, 7046. (e) Uyama, H.; Suda, S.; Kikuchi, H.; Kobayashi, S. Chem. Lett. 1997, 1109. (f) Matsumura, S.; Suzuki, Y.; Tsukuda, K.; Toshima, K.; Doi, Y.; Kasuya, K. Macromolecules 1998, 31, 6444. (g) Kobayashi, S.; Takeya, K.; Suda, S.; Uyama, H. Macromol. Chem. Phys. 1998, 199, 1729. (h) Namekawa, S.; Uyama, H.; Kobayashi, S. Proc. Jpn. Acad. 1998, 74B, 65. (5) (a) Uyama, H.; Kikuchi, H.; Kobayashi, S. Chem. Lett. 1995, 1047. (b) Uyama, H.; Kikuchi, H.; Kobayashi, S. Bull. Chem. Soc. Jpn. 1997, 70, 1691. (6) (a) Kamiya, N.; Yamamoto, Y.; Inoue, Y.; Chujo, R.; Doi, Y. Macromolecules 1989, 22, 1676. (b) Cao, A.; Kasuya, K.; Abe, H.; Doi, Y.; Inoue, Y. Polymer 1998, 39, 4801. (c) Namekawa, S.; Uyama, H.; Kobayashi, S.; Kricheldorf, H. R. Macromol. Chem. Phys. 2000, 201, 261.

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