Enzyme-Catalyzed Polycondensation of Polyester Macrodiols with

Nov 17, 2009 - (PHO) blocks were enzymatically prepared by one- or two-step lipase-catalyzed polycondensation. Novozym. 435-catalyzed reaction of ...
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Communications Enzyme-Catalyzed Polycondensation of Polyester Macrodiols with Divinyl Adipate: A Green Method for the Preparation of Thermoplastic Block Copolyesters Shiyao Dai,† Liang Xue,† Manfred Zinn,‡ and Zhi Li*,† Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, and Laboratory of Biomaterials, EMPA, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland Received October 11, 2009; Revised Manuscript Received November 7, 2009

Enzyme-catalyzed polycondensation for the synthesis of block copolymers was reported for the first time. Thermoplastic block copolyesters containing poly[(R)-3-hydroxybutyrate] (PHB) and poly[(R)-3-hydroxyoctanoate] (PHO) blocks were enzymatically prepared by one- or two-step lipase-catalyzed polycondensation. Novozym 435-catalyzed reaction of PHB-diol (Mn of 3100 g/mol, GPC), PHO-diol (Mn of 3200 g/mol, GPC), and divinyl adipate gave block poly(HB-co-HO) (Mn of 9800 g/mol, GPC) with randomly arranged blocks in 55% yield. In two-step polycondensations, Novozym 435-catalyzed reaction of PHB-diol and divinyl adipate afforded 73% of PHB containing two vinyl ester end groups (Mn of 2700 g/mol, GPC), which was further reacted with PHO-diol in the presence of Novozym 435 to give block poly(HB-co-HO)s (Mn of 8800-14 200 g/mol, GPC) with A-Btype arranged blocks in 55-62% yield. The enzymatically prepared block copolyesters demonstrated Tm of 136-142 °C and 142-153 °C and Tg of -37 to -39 °C and were potentially useful thermoplastic biodegradable and biocompatible materials.

1. Introduction The preparation of block copolymers containing hard and soft segments represents an efficient way to engineer thermoplastic materials. The use of enzyme for such a preparation is of big importance because of the nontoxicity and high selectivity of the enzymatic polymerizations. Great successes have been achieved in several types of enzymatic polymerizations such as lipase-catalyzed ring-opening polymerization and polycondensation.1-3 Whereas these reactions are now well established for the preparation of homopolymers and random copolymers,4-8 approaches for the enzymatic preparation of block copolymers are rather rare. Lipase-catalyzed ring-opening polymerization of a lactone with a polymer contacting one hydroxyl ending group, such as monoprotected poly(ethylene glycol) (PEG)9 or two hydroxyl ending groups such as PEG10 or a polyester macrodiol,11 afforded the corresponding block copolymers. This method is useful, but its wide application may be limited because of the fact that only a few lactones can be enzymatically polymerized. Lipase-catalyzed polycondensation has a much broader substrate range, and many dicarboxylic acids or their derivatives, glycols, and oxyacids or their esters were successfully used for such polymerization.1-3,12-22 Therefore, the method based on lipase-catalyzed polycondensation for the synthesis of block copolymers could be more general, but it has not been reported so far. Here we want to develop such a method for the engineering of

thermoplastic block copolymer by using two macrodiols as starting materials: one as hard block and another as soft block. The preparation of block copolyester derived from microbial poly[(R)-3-hydroxyalkanoates] (PHAs) was selected as a target. Poly[(R)-3-hydroxybutrate] (PHB) and poly[(R)-3hydroxyoctanoate] (PHO) are the most prominent PHAs and could be produced in large quantities. They are excellent candidates for some biomedical applications because of good biodegradability and biocompatibility;23-27 however, they cannot be directly used as thermoplastic biomaterials because PHB is hard brittle23,24 and PHO is soft sticky.28 Nevertheless, both PHB and PHO can be easily transformed to telechelic PHB-diol and PHO-diol as the hard and soft segments, respectively, for further polymerization to improve the physical and mechanical properties.29-33 Block copolymers containing PHB block and one of the other blocks such as poly(ε-caprolactone) (PCL),30,31 poly(lactide acid),31 PEG,32 or PHO33 were prepared by metal-catalyzed polycondensation using the corresponding macrodiols. However, the use of toxic metal catalysts is not desirable for the preparation of biomedical polymers. Here we report a novel and green approach for the preparation of block copolymers based on enzyme-catalyzed polycondensation using two polyester macrodiols as substrates and the first enzymatic synthesis of thermoplastic block copolyesters containing PHB and PHO blocks.

2. Experimental Section * To whom correspondence should be addressed. E-mail: [email protected]. Tel: +65-65168416. Fax: +65-67791936. † National University of Singapore. ‡ EMPA.

2.1. Materials. Novozym 435 (immobilized Candida antarctica lipase B, 10 000 PLU/g) was purchased from Novozymes and stored at 4 °C. Divinyl adipate (99.5%) was purchased from TCI, Japan.

10.1021/bm9011634  2009 American Chemical Society Published on Web 11/17/2009

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Scheme 1. Preparation of Block Copolyesters by Enzyme-Catalyzed Polycondensation of PHB-diol, PHO-diol, and Divinyl Adipate via OneOr Two-Step Reactions

Toluene (99.8%) was purchased from Aldrich, and chloroform (HPLC, 99.9%) and methanol (HPLC, 99.9%) were obtained from TEDIA. Telechelic hydroxylated poly-[(R)-3-hydroxybutrate] (PHB-diol, Mn of 2170 g/mol, 1H NMR; 3100 g/mol, GPC) and poly-[(R)-3-hydroxyoctanoate] (PHO-diol, Mn of 1400 g/mol, 1H NMR; 3200 g/mol, GPC) were prepared according to the published procedures.29,33 Novozym 435, PHB-diol, and PHO-diol were dried in a vacuum oven at 40 °C for 12 h before use, and toluene was dried by refluxing over sodium/ benzophenone under argon. 2.2. Synthesis of Block Copolyester Poly[(R)-3-hydroxybutyrateco-(R)-3-hydroxyoctanoate] by One-Step Enzymatic Polycondensation. Novozym 435 (40 mg), PHB-diol (98 mg, 0.0452 mmol), and PHO-diol (62.3 mg, 0.0445 mmol) were added to a dry Schlenk containing a magnetic stirring bar and then dried under vacuum for 1 h. Under an argon atmosphere, divinyl adipate (17.7 µL, 0.0894 mmol) and freshly distilled toluene (2 mL) were added to the Schlenk using a dry syringe, and the solution was stirred at 70 °C for 8 h. The reaction was terminated by the addition of 10 mL of chloroform, followed by the removal of Novozym 435 via filtration through filter paper (retention characteristics of 6 µm). Toluene and chloroform were removed under reduced pressure with a rotary evaporator. The raw product was dissolved in 2 mL of chloroform, treated with 18 mL of methanol, and then precipitated at 4 °C for 8 h. After filtration, the precipitates were dried under vacuum at first with a rotary evaporator and then in a vacuum oven at 40 °C for 24 h. Block poly(HB-co-HO) (87.8 mg) was isolated in 55% yield with a Mn of 9800 g/mol (GPC). 2.3. Synthesis of Block Copolyester Poly[(R)-3-hydroxybutyrateco-(R)-3-hydroxyoctanoate] by Two-Step Enzymatic Polycondensation. 2.3.1. Enzymatic Condensation Of PHB-diol with DiVinyl Adipate. Novozym 435 (100 mg) and PHB-diol (998 mg, 0.446 mmol) were added to a dry Schlenk containing a magnetic stirring bar and then dried under vacuum for 1 h. Under an argon atmosphere, the freshly distilled toluene (5 mL) and divinyl adipate (884 mg, 4.46 mmol) were added to the Schlenk by the use of a dry syringe. The mixtures were stirred under an argon atmosphere at 70 °C for 8 h. The reaction was terminated by the addition of 25 mL of chloroform. Novozym 435 was separated by filtration, and the solvent was removed by the use of a rotary evaporator. The crude product was dissolved in chloroform (2 mL) and precipitated by the addition of methanol (18 mL) at 4 °C for 8 h to remove unreacted divinyl adipate. PHB-vinyl ester (862 mg) was isolated in 73% yield with a Mn of 2700 g/mol (GPC). 2.3.2. Enzymatic Polycondensation of PHO-diols with PHB-Vinyl Ester. PHB-vinyl ester (Mn of 2650 g/mol, 1HNMR; 99 mg, 0.037 mmol), PHO-diol (74 mg, 0.053 mmol), and Novozym 435 (43 mg) were stirred in toluene (2 mL) under anhydrous conditions at 70 °C for 8 h. The reaction was terminated by the addition of 10 mL of

chloroform. Novozym 435 was separated by filtration, and the solvent was removed by the use of a rotary evaporator. The crude product was dissolved in chloroform (2 mL) and precipitated by the addition of methanol (18 mL) at 4 °C for 8 h. Block poly(HBco-HO) (102 mg) was isolated with a Mn of 14 200 g/mol (GPC) in 59% yield. 2.4. Measurements. 2.4.1. Gel Permeation Chromatography (GPC). We performed molecular weight analysis (Mn and polydispersity index Mw/Mn) by using a Waters instrument with Waters 510 pump, Waters 410 refractive index detector, and Waters HR4E, HR5E, and HR6 columns placed in series. THF was used as the eluent for PHB-diol, PHO-diol, and poly(HB-co-HO)s measurement at a flow rate of 1.0 mL/min and at 30 °C. Sample concentration was ∼0.1% (w/v), and the injection volume was 100 µL. Polystyrene standards with molecular weights of 1310, 2970, 13 900, 30 200, 197 000, and 696 000 g/mol were used to generate a calibration curve. 2.4.2. Nuclear Magnetic Resonance (NMR). 1H NMR (500 MHz) spectra were recorded with a Bruker AMX500 NMR instrument in DMSO-d6 at 333 K or CDCl3 at room temperature. Chemical shifts were referred to TMS at 0 ppm. 2.4.3. Differential Scanning Calorimetry (DSC). The thermal properties of polymers were measured on a Mettler Toledo DSC 822 system. Nitrogen was used as purge gas with a flow rate of 20 mL/min. Samples of 10 mg were prepared in aluminum foils, where the aluminum weights of the sample and reference were closely matched. The samples were heated from room temperature to 180 °C with a rate of 20 °C/min, cooled down to -100 °C with a rate of -20 °C/min, and heated again from -100 to 180 °C at a rate of 20 °C/min. Tm and Tg of the samples were obtained from the second heating curves.

3. Results and Discussion 3.1. One-Step Enzymatic Polycondensation for the Preparation of Block Copolyesters Containing Randomly Arranged PHB and PHO Blocks. PHB-diol with Mn of 3100 g/mol (GPC) and PHO-diol with Mn of 3200 g/mol (GPC) were prepared according to the previously reported procedure.11,29,33 To avoid the possible transesterification between the polyester-diols, enzymatic polycondensation was carried out by the use of the two polyester macrodiols with an active divinyl ester. One-step polycondensation of PHB-diol, PHOdiol, and divinyl adipate with Novozym 435 as the catalyst was first investigated (Scheme 1). PHB-diol and PHO-diol have similar structures and thus similar chances to react with vinyl adipate, giving rise to the formation of poly(HB-coHO)s with randomly arranged PHB and PHO blocks. The reactions were carried out in dry toluene at 70 °C under an argon

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Table 1. Preparation of Block Copolyester Poly(HB-co-HO) by Novozym 435-Catalyzed Polycondensation of PHB-diol, PHO-diol, and Divinyl Adipate via One-Step or Two-Step Reactions

entry

oligomer 1

1 2 3 4

PHB-diolg PHB-vinyl esterj PHB-vinyl esterj PHB-vinyl esterj

feed ratio Mn Mn Mn Mw a a b (g/mol) oligomer 2 (g/mol) DA 3 1:2:3c (1 + 2):Ed (g/mol)e (g/mol)e Mw/Mn yield (%) HB/HOf 2170h 2650 2650 2650

PHO-diol PHO-diol PHO-diol PHO-diol

1400i 1400 1400 1400

DA

1:1:2 1:1:1:1.5:1:2:-

4:1 4:1 4:1 4:1

9800 9200 14 200 8800

16 200 16 300 25 100 15 800

1.66 1.76 1.77 1.80

55 62 59 55

Tm (°C)

Tg (°C) code

1/0.68 142/153 -37 1/0.77 139/149 -37 1/1.00 136/142 -39 1/1.28

A B C

a Calculated from 1H NMR. b DA for divinyl adipate. c Molar ratio. d E for enzyme, weight ratio. e From GPC. f Molar ratio calculated from 1H NMR spectra. g One-step polycondensation in toluene at 70 °C for 8 h. h 3100 from GPC. i 3200 from GPC. j Two-step polycondensation: PHB-diol and divinyl adipate were reacted in the presence of Novozyme 435 in toluene at 70 °C for 8 h to give PHB-vinyl ester, which was further polycondensed with PHO-diol in the presence of Novozyme 435 in toluene at 70 °C for 8 h.

Figure 1. GPC spectra of (i) PHB-diol, (ii) PHO-diol, (iii) block poly(HBco-HO) prepared from one-step polycondensation (sample A in Table 1), (iv) PHB-vinyl ester prepared from the first step reaction in the two-step polycondensation, and (v) block poly(HB-co-HO) prepared from two-step polycondensation (sample C in Table 1).

atmosphere at a molar ratio of PHB-diol, PHO-diol, and vinyl adipate of 1:1:2 (Table 1, entry 1). The generated acetaldehyde was easily released at this temperature to drive the reaction to the formation of polymer, and the released acetaldehyde could be recovered by passing the off gas through a cooling system. After 8 h of reaction, the solvent in the reaction mixture was removed by evaporation under vacuum, the residue was dissolved in chloroform (2 mL), treated with methanol (18 mL), and precipitated at 4 °C for 8 h. The product was dried in a vacuum oven at 40 °C for 24 h to give a yield of 55%. The molecular weight (Mn) of the polymer (Sample A, Table 1) was determined by GPC to be 9800 g/mol, which is much higher than those of the starting materials PHB-diol and PHOdiol (Figure 1). This also confirmed the occurrence of enzymatic polycondensations. 1 H NMR spectra of PHO-diol, PHB-diol, and polymer sample A are given in Figure 2i-iii. The number of HO repeating unit x of PHO-diol was established to be 9.3 on the basis of the signal ratio of protons b and f in Figure 2i, whereas the number of HB repeating unit (n - 1) in PHBdiol was deduced to be 23.6 from the signal ratio of protons m and u in Figure 2ii. In Figure 2iii, signals of protons t′ and tt from the backbone of the junction unit were clearly observed, whereas signals of the protons of the vinyl ester end group were hardly visible. This indicates that divinyl adipate was reacted and the backbone was incorporated into the polymers. Signals of OH groups (protons c and d) of

PHB-diol and PHO-diol disappeared, a small amount of the signals of protons f, g, and h remained, and a new peak of proton e was clearly observed in Figure 2iii. These data suggest that PHB-diol and PHO-diol were reacted and incorporated into the polymers, and part of the OH end groups of PHO-diol or PHB-diol severed as the end groups of the final polymer. The other signals from the backbones of PHB (proton m, a, and j) and PHO (protons b′′, b′, b, a′, and j) were easily assigned, as shown in Figure 2iii. The signal intensity for b′′ (3 H) was 7.2, and thus the intensity of a′ (1 H) should be 2.4; the total signal intensity for a and a′ is 11.8; therefore, the intensity of a should be 9.4. Considering n of 24.6 for the PHB block and x of 9.3 for the PHO block, the ratio of the PHB/PHO block can be calculated to be (9.4/ 24.6)/(2.4/9.3) ) 1/0.68. Combining with the results on molecular weights determined by GPC, it could be concluded that the final polymer contains on average three randomly arranged PHA blocks with a ratio of PHB and PHO blocks of 1:0.68. 3.2. Two-Step Lipase-Catalyzed Polycondensation for the Preparation of Block Copolyesters Containing A-B-Type Arranged PHB and PHO Blocks. To control the structure of the block copolymers, we conducted two-step lipase-catalyzed polycondensation (Scheme 1). Theoretically, block copolyester with A-B type arranged blocks should be produced. Reaction of PHB-diol with 10-fold divinyl adipate was performed in the presence of Novozym 435 in dry toluene under an argon atmosphere at 70 °C for 8 h. The unreacted divinyl adipate was effectively washed out with chloroform, and the product was precipitated in chloroform/methanol (2 mL/18 mL) at 4 °C. This gave the corresponding product PHB-vinyl ester (Mn of 2700 g/mol, GPC) in 73% yield. Reaction of PHB-vinyl ester with PHO-diol in a molar ratio of 1:1 to 1:2 was carried out with Novozym 435 in toluene at 70 °C for 8 h. The reaction conditions and results were summarized in entries 2-4 of Table 1. After the same workup procedure as that described above for one-step polycondensation, poly(HB-co-HO)s were obtained in 55-62% yield. GPC analysis showed a significant increased molecular weight of the products (Mn of 8800-14 200 g/mol) compared with the starting materials (Figure 1). The use of 1:1.5 ratio led to higher Mn of the final polymer than the use of either 1:1 ratio or 1:2 ratio. The 1H NMR spectrum of PHB-vinyl ester is shown in Figure 3i. The signals of OH group (protons c and d) and the protons f, g, and h of PHB-diol disappeared, whereas a new peak of proton e was observed. This suggested that the OH groups were reacted with divinyl adipate. The signals of protons t′, t′′, and tt of the vinyl ester part split into multiple peaks, thus being significantly different from those of divinyl adipate. This suggested the happening of the condensation of divinyl adipate with PHB-diol. Other protons (p, q, s) of the vinyl ester part

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Figure 2. 1H NMR spectrum in DMSO-d6 at 333K of (i) PHO-diol, (ii) PHB-diol, and (iii) block poly(HB-co-HO) prepared from one-step polycondensation (sample A in Table 1).

were clearly observed, showed the signal ratio of 1:1:1, and were proportional to the signals of t′, t′′, tt. Signals of protons a, j, and m from the backbone of PHB remained in the spectrum. On the basis of the signal intensity of protons a and p, the ratio of PHB and the vinyl ester part can be established to be (9.6/ 24.6)/0.87 ) 1:2.2. Combined with the results from GPC, it could be concluded that the obtained product is PHB containing two vinyl ester end groups. The 1H NMR spectrum of poly(HB-co-HO) (sample C, Table 1) is shown in Figure 3ii. The signals of protons p, q, s from the PHB-vinyl ester totally disappeared, and the signals of protons c, d, f, h, and g from the ending group of PHO diol either disappeared or were largely reduced. This indicated that the OH group from the PHO block mostly reacted with the vinyl ester groups forming block copolyester and partially remained as the end group of the final polymer. The protons (m, a, j, b′′, b′, b, and a′) from the backbone of PHB and PHO absorbed in the expected areas. Similarly, the ratio of PHB/PHO block can be calculated on the basisi of proton a and a′: the signal intensity for b′′ (3 H) was 10, and

thus the intensity of a′ (1 H) should be 3.3; the total signal intensity for a and a′ was 12.0, and thus the intensity of proton a should be 8.7; and the ratio of the PHB/PHO block can be estimated to be (8.7/24.6)/(3.3/9.3) ) 1:1. Because the Mn was determined by GPC to be 14 200 g/mol, the polymer sample C contains on average about 5 PHA blocks at a ratio of PHB and PHO blocks of 1:1 with A-B-A-B-A type structure. In the 13C NMR spectrum, signals at 168.5 and 168.3 ppm were observed, which belong to the carboxyl group of PHB and PHO, respectively. No other signals were found between 168.3 and 168.5 ppm, indicating no random copolymer generated between PHB and PHO. 3.3. Physical Properties of Block co-poly(HB-co-HO)s. The melting temperature (Tm) and glass-transition temperature (Tg) of block poly(HB-co-HO)s were analyzed by DSC (Figure 4) and summarized in Table 1. All block copolymers have Tm of 142-153 and 136-142 °C and Tg from -37 to -39 °C. The enzymatically prepared block co-poly(HB-co-HO) demonstrated good thermal properties, and thus is a potentially useful thermoplastic material for biomedical applications.

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Figure 3. 1H NMR spectrum in DMSO-d6 at 333K of (i) PHB-vinyl ester prepared from the first step reaction in two-step polycondensation and (ii) block poly(HB-co-HO) prepared from two-step polycondensation (sample C in Table 1).

Figure 4. DSC spectra of (1) PHO-diol, (2) PHB-diol, (3) block poly(HB-co-HO) prepared from one-step polycondensation (sample A in Table 1), (4) block poly(HB-co-HO) prepared from two-step polycondensation (sample B in Table 1), and (5) block poly(HB-co-HO) prepared from two-step polycondensation (sample C in Table 1).

4. Conclusions A new, green, and efficient method for the preparation of block copolyesters via Novozym 435-catalyzed polycondensation of polyester macrodiols and divinyl adipate was successfully demonstrated. Thermoplastic copolyesters containing microbial PHB and PHO blocks were enzymatically prepared for the first time by either one- or two-step enzymatic polycondensation of PHB-diol (Mn of 3100 g/mol,

GPC) or PHO-diol (Mn of 3200 g/mol, GPC) with divinyl adipate. Whereas one-step synthesis is simpler and gave block copolyester poly(HB-co-HO) (Mn of 9800 g/mol, GPC) with randomly arranged blocks, two-step polycondensation is more controllable and afforded block copolyester poly(HB-co-HO) (Mn of 8800-14 200 g/mol, GPC) with A-B-type arranged blocks. The enzymatically prepared block copolymer poly(HBco-HO)s demonstrated good thermal properties with Tm of

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136-142 and 142-153 °C and Tg of -37 to -39 °C, thus being potentially useful thermoplastic biodegradable and biocompatible materials. Acknowledgment. Financial support by the Ministry of Education of Singapore through an AcRF Tier 1 grant (project no. R-279-000-225-112) is gratefully acknowledged.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)

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