Biomacromolecules 2005, 6, 2275-2280
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Preparation of Aliphatic Poly(thioester) by the Lipase-Catalyzed Direct Polycondensation of 11-Mercaptoundecanoic Acid Makoto Kato, Kazunobu Toshima, and Shuichi Matsumura* Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan Received March 6, 2005; Revised Manuscript Received April 12, 2005
An aliphatic polythioester was enzymatically prepared by the direct polycondensation of mercaptoalkanoic acid using immobilized lipase of Candida antarctica (lipase CA) in bulk. The commercially available 11mercaptoundecanoic acid was polymerized by lipase CA in bulk in the presence of molecular sieves 4A as a water absorbent at 110 °C for 48 h to produce poly(11-mercaptoundecanoate) with an Mw of 34 000 in high yield. The 104.5 °C melting temperature (Tm) of poly(11-mercaptoundecanoate) was about 20 °C higher than that of the corresponding polyoxyester, poly(11-hydroxyundecanoate). The polythioester was readily transformed by lipase into the corresponding cyclic oligomers mainly consisting of the dimer, which were readily repolymerized by the ring-opening polymerization using lipase as a sustainable chemical recycling. Introduction Polythioesters have attracted attention because of the recent discovery of the microbial production of poly(3-mercaptoalkanoate)s. Microbial polyesters containing thioester linkages in their backbone were first reported by Lu¨tke-Eversloh et al. and revealed a novel class of biopolymers.1-4 The first example was the copolymer of 3-hydroxybutyrate and 3-mercaptopropionate. More recently, the microbial homopolymers of poly(3-mercaptoalkanoate)s, such as poly(3-mercaptopropionate) and poly(3-mercaptovalerate), were produced by a recombinant strain of Escherichia coli.5 Though the chemical synthesis of a polythioester was reported 50 years ago, no commercial production of the polythioester has yet been partially established due to the complex preparation methods.1,6 Nowadays, polythioesters are mainly made by the ring-opening polymerization of cyclic monomers of thionolactones,7,8 which are synthesized by complex synthetic routes.9 The ring-opening polymerization of thionolactones, such as -thiocaprolactone or γ-thionobutyrolactone, and the ring-opening polycondensation of 2-stanna-1,3-dithiacycloalkanes with dicarboxylic acid chlorides were recently reported.10,11 However, a more straightforward method, such as the direct polycondensation using commercially available mercaptoalkanoic acid, is now needed for establishing an industrially feasible method that meets the green polymer chemistry requirements. Enzyme-catalyzed polymerization should open a new frontier for more types of polyesters, such as structural and elemental variations. It has been revealed that the hydrolase enzyme promotes the polymerization of a versatile type of monomer forming polyesters, polycarbonates, polyamides, polyphosphates, and polysaccharides.12-16 The enzymecatalyzed polymerization may become one of the environmentally benign methods for industrial applications. How* To whom correspondence should be addressed.
ever, the enzyme-catalyzed preparation of high-molecular weight polythioesters has not yet been reported. Only the thioester formation by the lipase-catalyzed reaction of thiol and carboxylic acid has been reported.17,18 Such examples include the enzymatic synthesis of thioesters by the reaction of oleic acid and butanethiol using lipase.19,20 We previously reported the enzymatic preparation of aliphatic polyesters containing thioester linkages by the copolymerization of a lactone with mercaptoalkanoic acid and by the transesterification of polyesters with mercaptoalkanoic acids.21 However, only the low-molecular weight oligomer of mercaptoalkanoic acid was produced by the direct polycondensation reaction. Polythioesters are expected to show characteristic features, such as a higher melting point, lower solubility, and higher heat stability when compared with polyoxyesters.22 The higher melting point of the polythioester may be one of the most important properties, because the melting points of conventional polyoxyesters, such as poly(-caprolactone) and poly(11-undecanolide), are below 100 °C. It is reported that poly(-thiocaprolactone) had a 45 °C higher melting point than the corresponding polyoxyester, poly(-caprolactone).23 Furthermore, the melting point for poly(3-mercaptopropionate) was 170 °C, which is about 100 °C higher than the equivalent polyoxyester analogue, poly(3-hydroxypropionate).22 In this study, a novel aliphatic polythioester was prepared by the lipase-catalyzed polycondensation of 11-mercaptoundecanoic acid (11MU) in bulk. Also, lipase-catalyzed degradation of the polythioester into a cyclic oligomer and repolymerization was demonstrated as a novel repetitive chemical recycling of polythioesters (Scheme 1). Experimental Section Materials and Measurements. 11-Mercaptoundecanoic acid (11MU) was purchased from Aldrich Chemical Co.
10.1021/bm050168i CCC: $30.25 © 2005 American Chemical Society Published on Web 05/12/2005
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Biomacromolecules, Vol. 6, No. 4, 2005
Scheme 1. Synthesis and Chemical Recycling of Polythioester Using Lipase
(Milwaukee, WI) and purified by silica gel column chromatography using chloroform/methanol (20:1, v/v; Rf ) 0.32) as eluent. Immobilized lipase from Candida antarctica [CA: Novozym 435 (triacylglycerol hydrolase + carboxylesterase) having 10 000 PLU/g (propyl laurate units: lipase activity based on ester synthesis)] and Rhizomucor miehei lipase immobilized on a macroporous anion-exchange resin (RM: Lipozyme RM IM) were kindly supplied by Novozymes Japan Ltd. (Chiba, Japan). Lipase from Candida rugosa (CR) was purchased from Aldrich Chemical Co. Immobilized lipase from Pseudomonas sp. (PS-C “Amano” I) and Pseudomonas fluorescens (AK “Amano” 20) were kindly supplied by Amano Pharmaceutical Co., Ltd. (Nagoya, Japan). The enzymes were used without further purification and were dried in a vacuum over P2O5 at 25 °C for 2 h before use. Thermally deactivated lipase CA was prepared by heating the lipase CA in steam using an autoclave at 120 °C for 15 min and freeze-dried. Molecular sieves 4A was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). The weight-average (Mw) and number-average (Mn) molecular weights as well as molecular weight distribution (Mw/Mn) of polymer were determined by a size exclusion chromatography (SEC) using SEC columns (Shodex K-G + K-804, Showa Denko Co., Ltd., Tokyo, Japan) with a refractive index detector. Chloroform was used as the eluent. The SEC system was calibrated with polystyrene standards of a narrow molecular weight distribution. The 1H and13C NMR spectra were recorded on a Lambda 300 Fourier Transform Spectrometer (JEOL Ltd., Tokyo, Japan) operating at 300 and 75 MHz, respectively. The matrix-assisted laser desorption ionization time-offlight mass spectrometery (MALDI-TOF MS) was measured with Bruker Ultraflex mass spectrometer (Bruker Daltonics, Billerica, MA). The spectrometer was equipped with a nitrogen laser. The detection was in the reflectron mode. 2,5Dihydroxybenzoic acid was used as the matrix and sodium bromide was used as the cation source. Positive ionization was used. The atmospheric pressure chemical ionization mass spectrometry (APCI MS) was measured with Finnigan Mat Inc. LCQ-mass spectrometer system (Finnigan Corp., CA). The crystallization temperature (Tc) and melting temperature (Tm) of the polymer were determined with a differential scanning calorimetry (DSC-60, Shimadzu, Kyoto, Japan). The heating rate was 20 °C/min within a temperature range of -50∼130 °C. The measurements were made with a 3∼5 mg sample on a differential scanning calorimetry (DSC) plate. The polymer samples were heated at a rate of 20 °C/ min from 26 to 130 °C (first scan), rapidly cooled at a rate
Kato et al.
of - 50 to -110 °C and then scanned at the same heating rate and over the same temperature range (second scan). The Tc and Tm values were collected from the second scan. Poly(11-hydroxyundecanoate) [Poly(11HU)] with an Mw of 29 000 was synthesized by the polycondensation of 11HU using lipase CA. That is, 11HU (60 mg) and lipase CA (36 mg) were stirred under a nitrogen atmosphere at 110 °C for 2 days. The polymerization was carried out in a screw-capped vial with molecular sieves 4 Å placed at the top of the vial to absorb the produced water. After the reaction, the reaction mixture was dissolved in hot chloroform (20 mL), and the insoluble enzyme was removed by filtration. The solvent was then evaporated to obtain the crude polymer followed by its purification by the reprecipitation, using chloroform (good solvent) - n-propanol (poor solvent). 1H NMR (300 MHz, CDCl ): δ ) 1.20 ∼ 1.42 (m, 12H, 3 C4-C9), 1.52 ∼1.70 (m, 4H, C-3, C-10), 2.24 ∼ 2.34 (t, 2H, C-2, J ) 7.8 Hz), 4.05 (t, 2H, C-11, J ) 6.3 Hz). 13C NMR (75 MHz, CDCl ): δ ) 25.0 (C-9), 25.9 (C-4), 3 28.6 ∼ 29.4 (C-3, C-5 ∼C-8), 34.3 (C-2), 64.3 (C-11), 173.9 (C-1). General Enzymatic Polymerization Procedure of 11MU. The general procedure for the enzymatic polymerization of 11MU was carried out in a screw-capped vial with molecular sieves 4A placed at the top of the vial to absorb the produced water. The preparation of poly(11-mercaptoundecanoate) [poly(11MU)] with an Mw of 34 000 was described as a typical example. A mixture of 11MU (60 mg) and lipase CA (36 mg) was stirred under a nitrogen atmosphere at 110 °C for 2 d. The reaction mixture was dissolved in hot chloroform (20 mL), and the insoluble enzyme was removed by filtration. The solvent was then evaporated to obtain the crude polymer. The crude polymer was purified by reprecipitation using chloroform (good solvent) - n-propanol (poor solvent). The molecular weight and the molecularweight distribution of the polymer were determined using SEC. The molecular structure was analyzed by MALDI-TOF MS, 1H and 13C NMR spectroscopies. The spectral data of poly(11MU) are shown to be representative. 1H NMR (300 MHz, CDCl ): δ ) 1.15 ∼ 1.40(m, 12H, 3 C-4∼C-9), 1.52 ∼ 1.70(m, 4H, C-3, C-10), 2.53 (t, 2H, C-2, J ) 7.8 Hz), 2.70 (m, 4H, CH2SSCH2), 2.85 (t, 2H, C-11, J ) 7.6 Hz). 13C NMR (75 MHz, CDCl ): δ ) 25.7 (C-9), 28.8∼29.6 3 (C-3∼C-8, C-10), 44.1 (C-2), 199.8 (C-1). General Enzymatic Degradation Procedure. The general procedure for the enzymatic degradation was carried out in a screw capped vial. A mixture of poly(11MU) (100 mg) with an Mw )34 000 (Mw/Mn ) 2.3), lipase CA (300 mg) and n-nonane (20 mL) was stirred using a magnetic stirring bar under a nitrogen atmosphere in a themostated oil bath at 110 °C for 1 day. Hot chloroform was then added to the reaction mixture, and the insoluble enzyme was removed by filtration. The solvent was then evaporated under slightly reduced pressure to obtain the reaction mixture. The crude products were further purified by silica gel column chromatography using ethyl acetate/hexane (1:15, v/v; Rf ) 0.28, 0.19, and 0.09) as the eluent to obtain the cyclic oligo(11MU). The molecular structure of the cyclic oligo(11MU)
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Preparation of Aliphatic Poly(thioester) Table 1. Polymerization of 11MU Using Various Lipases at 70 °C entry
lipase
conv. (%)
Mw
Mw/Mn
1 2 3 4 5 6 7
Candida antarctica (lipase CA) Pseudomonas sp. (PS-C) Rhizomucor miehei (lipase RM) Pseudomonas fluorescens (AK) Candida rugosa (CR) thermally deactivated lipase CA blank
84 47
