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Biomacromolecules 2004, 5, 169-174

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Improved Synthesis with High Yield and Increased Molecular Weight of Poly(r,β-malic acid) by Direct Polycondensation Tetsuto Kajiyama,*,† Hisatoshi Kobayashi,*,† Tetsushi Taguchi,† Kazunori Kataoka,†,‡ and Junzo Tanaka† Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan, and Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received August 12, 2003; Revised Manuscript Received October 3, 2003

The development of synthetic biodegradable polymers, such as poly(R-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW-PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of L-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized R,β-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain R,β-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 °C for 45 h with a molecular weight of 5300. The method for R,β-PMA synthesis established here will facilitate production of R,β-PMA of various molecular weights, which may have a potential utility as biomaterials. 1. Introduction The fabrication and evaluation of functional polymers are of interest in polymer science and biomedical fields. Biodegradable materials can be obtained from natural polysaccharides, polypeptides, and bacterial polyesters, or from synthetic polyanhydrides, poly(amino acids), polyorthoesters, polyorganophosphazenes, and polyesters.1,2 Among these biodegradable polymers, poly(R-hydroxy acid) has attracted a great deal of attention for use in medical applications. Poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers are commercially available for medical use,3-5 for example, as surgical sutures and scaffolds for tissue engineering. In addition, changing the molecular weight and chemical composition of these polymers can provide delivery systems for controlled drug release.6,7 Poly(malic acid) (PMA), the L-malic acid polymer contained in grapes and apples,8 is well-known as a watersoluble, biodegradable, and bioabsorbable polymer,9,10 and has two remarkable advantages for use in medical applications: it is metabolized in the mammalian tri-carboxylic acid (TCA) cycle11 and can be modified by the pendant carboxy group present in the molecule that can be readily react with other functional groups, allowing the introduction of drugs into its polymer chain.29,31,33 Therefore, it is important to develop a simple synthetic method of this functional polyester in a high yield. Most PMA compounds are synthetic, although PMA is also available from natural or bacterial resources.12-19 PMA * To whom correspondence should be addressed. Phone: +81-29-8513354. Fax: +81-29-854-7037 e-mail: [email protected] (T.K.); [email protected] (H.K.). † National Institute for Materials Science. ‡ The University of Tokyo.

is synthesized by two methods: ring-opening polymerization and direct polycondensation. Ouchi et al. synthesized poly(R-malic acid) ester by ring-opening polymerization of malide dibenzyl ester.20 Many researchers have synthesized poly(β-malic acid) ester by polymerizing benzyl or the other substituents, such as malo-lactonate, through ring-opening polymerization.10,21-35 Cammas et al. (1996) reported the synthesis of β-PMABz with the unusually HMW of 174 000 with an improved synthetic route, using repeated purification of the intermediate products.22 However, synthesis of these polymers by this route is not easy because of the number of steps involved in the reaction cycle, including repeated purification steps. In contrast, direct polycondensation is possible in a 1-step reaction. It also has the advantage of being “green” chemistry, as it uses fewer organic solvents than other polymerization methods, such as ring-opening polymerization. This feature makes this method attractive in terms of environmental conservation. Ohtani et al. synthesized R,β-PMA with a molecular weight of 1900 by direct polycondensation of L-malic acid and detailed its physicochemical properties.36 This synthesis is not yet practical, because the molecular weight of the resultant synthesized polymer is too low for medical applications, and its yield is insufficient for industrial use. Therefore, further basic studies are needed to establish a method for the synthesis of HMW R,β-PMA. Previously, we investigated the reaction conditions of R,β-PMA (solvents, catalysts, temperatures, and reaction times) by direct polycondensation, and reported a higher production of R,βPMA with HMW than that obtained by conventional methods.37,38 These results opened a new possibility of

10.1021/bm0342990 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/11/2003

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directly condensed PMA for the use in biomedical application, such as drug delivery systems (DDS). Here, we present a substantial improvement on R,β-PMA synthesis by direct polycondensation and conclude that the best approach allows the synthesis of higher molecular weight R,β-PMA (g5000). 2. Experimental Section 2.1. Materials. L-Malic acid (Aldrich, Milwaukee, USA, optical purity of 97%), citric acid (Wako Chemicals, Osaka, Japan, optical purity of 98+%), diethyl ether (Wako Chemicals, optical purity of 99.5+%), petroleum ether (Kanto Chemicals, Tokyo, Japan, the highest quality), tetrahydrofuran (THF) (Wako Chemicals, optical purity of 97+%), tin(II) chloride (Wako Chemicals, optical purity of 97+%), tin(II) oxide (Wako Chemicals, optical purity of 95+%), tin(II) acetate (Aldrich), and acetone-d6 (Cambridge Isotope Laboratories, Andover, USA, optical purity of 99.9%) were used without further purification. 2.2. Methods. To determine the chemical structure of the products, the 1H NMR and 13C NMR spectra were measured by a nuclear magnetic resonance spectrometer (Bruker ARX300) using tetramethylsilan (TMS) as the internal reference. The infrared (IR) spectra were also measured by an IR spectrometer (Perkin-Elmer SPECTRUM 2000). The molecular weight of the synthesized PMA was estimated in 0.1M citric acid/THF solution by gel permeation chromatography (GPC) (apparatus: Tohso HLC-8220GPC, column: TSK Gel SuperHz2000 and SuperHz4000, standard: polystyrenes). 2.3. Synthesis of r,β-PMA. 0.2 mol of L-malic acid (28 g) was placed in the reaction vessels and purged by pure N2 gas. Three different kinds of Tin(II) catalyst (tin(II) chloride, tin(II) oxide, and tin(II) acetate) were tested in the range of 0.036 to 3.6 wt % (0.01-1.0 g). The mixture was stirred at 110-130 °C for 5-55 h in a vacuum under a stream of N2 (1 mmHg) in a direct polycondensation reaction. The resultant polyester was then dissolved in 150 mL of THF solution to further purify it. The polyester solution was precipitated in a mixture of 1000 mL of diethyl ether and 1000 mL of petroleum ether. The precipitates were characterized by 1H NMR (acetone-d6, δppm): 5.5-5.4 (d, 1 H, methylidine unit), 3.1-2.9 (d, 2 H, methylene unit); 13C NMR (acetone-d6, δppm): 36.3 (methylene unit), 70.0 (methylidine unit), 168.5 (R-carbonyl ester carbon), 169.3 (β-carbonyl ester carbon), 170.2 (R-side chain carboxylic carbon), 171.1 (β-side chain carboxylic carbon); and IR (ATR): 3300 (OH), 3000 (CH2, CH), 1730-1710 cm-1 (ester and COOH CdO). 3. Results First, the optimum reaction temperature and time to obtain the higher molecular weight of R,β-PMA were investigated. The weight average molecular weight (Mw), number average molecular weight (Mn), molecular weight distribution (Mw/Mn), and yield (amount of recovery) of R,β-PMAs synthesized at 110-130 °C for 5-55 h using tin(II) chloride

Kajiyama et al. Table 1. Direct Polycondensation of L-Malic Acid for Synthesizing R,β-PMAa temp (°C)

time (h)

Mw

Mn

Mw/Mn

yield (%)

110 110 110 110 110 110 110 110 110 110 110 120 120 120 120b 120 120 120 120 120 120 120 130c 130c 130c 130b 130c

5 10 15 20 25 30 35 40 45 50 55 5 10 15 20 25 30 35 40 45 50 55 5 10 15 20 25

500 1000 1300 1800 2600 2800 3800 5000 5300 4800 4000 1300 2200 2800 3100 3900 4400 4500 5100 4700 4500 2800 1700 3200 3300 3600 2800

400 800 900 1100 1500 1600 2000 2500 2800 2600 2100 1000 1300 1500 1600 2100 2200 2300 2700 2400 2300 1700 1100 1800 1800 2000 1600

1.3 1.3 1.4 1.6 1.7 1.8 1.9 2.0 1.9 1.8 1.9 1.3 1.7 1.8 1.9 1.9 2.0 2.0 1.9 2.0 2.0 1.7 1.5 1.8 1.8 1.8 1.8

70 88 95 98 95 98 98 98 95 88 81 88 98 95 92 98 98 98 98 92 92 63 99 89 75 75 71

a The reaction was carried out using tin(II) chloride as catalyst. Molecular weights were determined by GPC (column: TSK Gel SuperHz4000 + SuperHz2000 + SuperHz2000) in citric acid 0.1 M THF solution, polystyrene standards. The yield is equal to the amount of recovery. b Taken from ref 37. c Taken from ref 38.

(0.36 wt %) are shown in Table 1. It was found that the Mw of the resultant R,β-PMA was in the range from 500 to 5300 and the Mn was from 400 to 2,800. In these conditions, Mw/ Mn was in the range from 1.3 to 2.0 and the yield was from 63 to 99%. R,β-PMA of considerably high molecular weight (Mw ) 5300, Mn ) 2800) was obtained at 110 °C. Second, the optimum catalyst system for the direct polycondensation to obtain the higher molecular weight of R,βPMA was investigated. The relationship between the molecular weight and catalyst type for the synthesis of R,βPMA is shown in Table 2. The synthesis of the R,β-PMAs were carried out at 110 °C for 45-55 h using tin(II) chloride, tin(II) oxide, or tin(II) acetate (each 0.36 wt %) or with no catalyst. It was found that the Mw of the resultant R,β-PMA was in the range from 4100 to 5300 and the Mn was from 2200 to 2800. In these conditions, Mw/Mn was in the range from 1.7 to 2.1, and the yield was from 82 to 96%. The optimum reaction catalyst for the synthesis of R,β-PMA in the present study was found to be tin(II) chloride. The relationship between the molecular weight and the amount of tin(II) chloride for the synthesis of R,β-PMA is summarized in Table 3, where the synthesis of R,β-PMAs was carried out at 110 °C for 45 h. It was found that the Mw of the resultant R,β-PMA was in the range from 4800 to 5300 and the Mn was from 2400 to 2800. In these conditions, the Mw/Mn was in the range from 1.9 to 2.0 and the yield was as higer as 96%. An 18 mol amount of tin(II) chloride was enough to obtain R,β-PMA with higher than 5000.

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Improved Synthesis of Poly(R,β-malic acid) Table 2. Direct Polycondensation of L-Malic Acid for Synthesizing R,β-PMA by Different Catalyst Typesa catalyst

time (h)

Mw

Mn

Mw/Mn

Yield (%)

40 45 50 40 45 50 40 45 50 40 45 50

4900 5100 4700 4800 4900 5200 5000 5300 4800 4200 4800 4600

2400 2800 2600 2400 2600 2700 2500 2800 2600 2400 2400 2500

2.1 1.8 1.8 2.0 1.8 1.9 2.0 1.9 1.8 1.7 2.0 1.9

96 93 82 93 93 93 98 95 88 93 96 82

SnO(II)

Sn(CH3COO)2(II)

SnCl2(II)

non

a Reaction temperatures were 110 °C. Molecular weights were determined by GPC (column: TSK Gel SuperHz4000 + SuperHz2000 + SuperHz2000) in citric acid 0.1 M THF solution, polystyrene standards. The yield was equal to the amount of recovery.

Table 3. Direct Polycondensation of L-Malic Acid for Synthesizing R,β-PMA by Different Catalyst Volume tin (II) chloride (mol)

Mw

Mn

Mw/Mn

Yield (%)

0.72 0.36 0.18 0.036 0

5300 5300 5300 4900 4800

2800 2800 2800 2400 2400

1.9 1.9 1.9 2.0 2.0

96 96 96 96 96

a The reaction was carried out at 110 °C for 45 h using Tin (II) chloride. Molecular weights were determined by GPC (column: TSK Gel SuperHz4000 + SuperHz2000 + SuperHz2000) in citric acid 0.1 M THF solution, polystyrene standards. The yield was equal to the amount of recovery.

Figure 1. Time-course of the molecular weight alteration in resultant R,β-PMA at each reaction temperature, 110, 120, and 130 °C. Reactions were carried out for 5-55 h.

4. Discussion 4.1. Effects of Reaction Time and Temperature. Figure 1 shows the molecular weights of the synthesized R,β-PMA compounds plotted against the reaction time at each reaction temperature (110, 120, and 130 °C) on the same basis as the results shown in Table 1. There was observed an optimum reaction time to obtain the sample with highest molecular

Figure 2. Yield changes versus the reaction time at each reaction temperature, 110, 120, and 130 °C. Reactions were carried out for 5-55 h. The yield is equal to the amount of recovery.

weight at each reaction temperature. The polymerization reaction proceeded faster at 130 °C, but the molecular weight increase was limited to 3600. In contrast, polymerization at 110 °C gave a higher molecular weight than that at 130 °C, even though the longer reaction time was required. The polycondensation reaction of R,β-PMA persisted through the intermolecular dehydration of L-malic acid and is a competitive reaction against the intramolecular dehydration of L-malic acid and the depolymerization reaction of synthesized R,β-PMA. In general, intramolecular dehydration becomes the dominant reaction at temperatures over 150 °C, which produces fumaric acid as a main product. This is an undesirable side reaction for the polycondensation reaction. The fumaric acid production was observed in the reaction system, even at 110-130 °C, and the amount of resultant fumaric acid increased with the temperature increase. Furthermore, the equilibrium of polymerization shifted toward the depolymerization at a higher reaction temperature, resulting in a decrease in the maximum molecular weight of R,β-PMA with an increase in the reaction temperature. Figure 2 shows the yields of the R,β-PMA versus the reaction time at each reaction temperature on the same basis as the results shown in Table 1. Focusing on the plots at 110 °C, the yield was 70% in the early stages of the reaction (5 h). The molecular weight of the resultant polyester was still very low at this stage, and thus, the lower molecular weight fragment of polyester was removed in the reprecipitation process. The yield of R,β-PMA was kept in the range of 90% by 45 h and then gradually decreased to 81% by 55 h. Although the reaction times differed, the plots at 120 and 130 °C showed the same behavior as those at 110 °C, except in the early stages of the reaction. These observations were concluded to be due to the depolymerization of synthesized R,β-PMA, as mentioned above. Moreover, we assume that depolymerization occurred from the terminal group because the decreases in molecular weight and yield followed the same time course. This assumption may be

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Figure 4. 1H NMR spectra of R,β-PMA. The R,β-PMA was synthesized at 110 °C for 45 h using tin(II) chloride as a catalyst. Mw ) 5300 and Mn ) 2800.

Figure 3. Chromatograms of R,β-PMA synthesized at 110 °C for different reaction times (column: TSK Gel SuperHz4000 + SuperHz2000 + SuperHz2000). The eluent contained citric acid.

supported by the characteristics of the bulk R,β-PMA to have a strong intermolecular-hydrogen-bond, and thus, the main chain scission of R,β-PMA might have occurred from a relatively free terminal group. In our previous study, we demonstrated that the byproducts of the R,β-PMA synthesis at 130 °C for 20 h using tin(II) chloride as a catalyst are fumaric acid and malic acid.37 The synthesis of R,β-PMA at 110 and 120 °C was considered to be accompanied by generation of the same byproducts as that at 130 °C for 20 h using tin(II) chloride as a catalyst, although their levels were reduced as the yields of R,β-PMA were increased. The chromatograms of R,β-PMA synthesized for different reaction times (10, 20, 30, 45, and 55 h) at 110 °C are shown in Figure 3. The peaks at 20 h or later showed shorter retention times than that at 10 h. The rapid increase in the molecular weight of the main R,β-PMA fraction occurred by increasing a reaction time from 10 to 20 h. As for the higher molecular weight fraction, a small broad peak developed on the shoulder of the main peak (30 h) and became a larger sharp peak (45 h). The sharp peak was assigned as R,β-PMA of the molecular weight around 10 000. Previously, we reported a higher molecular weight R,β-PMA fraction synthesized by direct polycondensation, though the peaks were small and broad.37 In the present study, the higher molecular weight fraction of R,β-PMA was remarkably increased compared with the result of the previous study. 4.2. Structural Determination of r,β-PMA by NMR Spectroscopy. Figure 4 shows the 1H NMR spectra of R,βPMA, and Figure 5 shows the 13C NMR spectra of R,βPMA, synthesized at 110 °C for 45 h, using tin(II) chloride.

The spectra at 5.5 ppm show two peaks of near equivalent intensity (Figure 4). The peak at 5.6 ppm is an R-type, whereas that at 5.5 ppm is a β-type. The 13C NMR spectra in Figure 5 shows a carbonyl carbon of an R-type ester (168.6 ppm), a carbonyl carbon of a β-type ester (169.3 ppm), a carbonyl carbon with an R-type side chain group (170.4 ppm), and a carbonyl carbon with a β-type side chain group (171.0 ppm). From these results, we concluded that these polymers have random sequences of both R-and β-type units in almost equivalent ratios. This result was supported by previous reports.36,37 For the terminal group of the resultant R,β-PMA, a peak of olefinic hydrogen was observed at ∼6.8 ppm (Figure 4). The peak was nearly assigned to some terminal fumaric acid group in the resultant polyester, part of which was considered to be the terminal fumaric acid group of R,β-PMA. The peak assigned to the terminal hydroxylic hydrogen was slightly observed at ∼4.7 ppm. However, no terminal carboxylic hydrogen peak was observed, probably because it overlapped with the main chain methylidine peak (∼5.5 ppm). These results suggested some amount of fumaric acid type terminal groups exist in the resultant polyester. 4.3. Effects of Catalyst: Investigation of Different Catalysts and Addition Volume. The optimum catalyst system for the direct polycondensation to obtain the higher molecular weight of R,β-PMA was then investigated. First, we discussed the optimum reaction catalyst. Figure 6 shows the time course of the change in the molecular weights of the obtained R,β-PMA compound, using different ones at 110 °C. A bell-type relationship was observed, with maxima at 45 h except for the case of tin(II) acetate. Synthesis with tin(II) chloride, tin(II) oxide, or no catalyst showed similar profiles, although the molecular weights of the products were different. In the case of tin(II) acetate, the molecular weight of R,β-PMA was monotonically increased for 50 h. Therefore, it is unlikely that a marked increase of polycondensation will be obtained, as this catalyst is similar to other tin(II) compounds, although it remains possible that further polycondensation may occur. Tin(II) chloride seems to be the best catalyst in terms of the preparation of R,β-PMA with high molecular weight.

Improved Synthesis of Poly(R,β-malic acid)

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Figure 5. 13C NMR spectra of R,β-PMA. The R,β-PMA was synthesized at 110 °C for 45 h using tin(II) chloride as a catalyst. Mw ) 5300 and Mn ) 2800.

Figure 6. Effects of the catalysts on the molecular weight of resultant R,β-PMA. SnCl2(II) ([), SnO(II) (9), Sn(CH3COO)2(II) (2), and non (-) catalysts were used.

The yields of the obtained R,β-PMA compound using different catalysts at 110 °C were summarized in Table 2. High yields in the order of 80-90% were achieved, which is important from a practical viewpoint. The yield was slightly decreased at 50 h, suggesting the depolymerization of R,β-PMA similar to the results shown in Figure 1. The molecular weights of the obtained R,β-PMA compounds synthesized using different amounts of catalysts at 110 °C for 45 h were summarized in Table 3. The purpose of this experiment was to optimize all amounts of catalyst. In general, catalysts, such as tin(II) chloride, tin(II) oxide, tin(II) acetate, and other catalysts of this type are highly toxic. Therefore, the development of a synthetic route with no or little catalyst is very important for biomedical use. R,β-PMA with the highest molecular weight of 5300 was obtained with the addition of tin(II) chloride ranging from 0.18 to 0.72 mol, but in the reactions with only 0.036 mol, the amount of catalyst yielded a product of almost the same molecular weight as that with no catalyst. These observations indicated that the amount of catalyst added can be reduced by 0.18 mol. The synthesis of R,β-PMA by direct polycondensation is still at the preliminary stage, and to date, there have been

no reports of methods for obtaining this product with HMW. As HMW-R,β-PMA is expected to have many applications, even with the use of catalysts, the synthesis of R,β-PMA by direct polycondensation will be very important. In this nonsolvent reaction system, the encounter probability of the reacting point in the polycondensation decreases markedly though the reaction proceeds. Therefore, we expected that increasing the encounter probability would result in an increase in the molecular weight of the product. However, the molecular weight of R,β-PMA did not increase in comparison to that in the control reaction (using 0.36 mol of catalyst). Although the data are not shown here, we also examined the effect of using 3.6 mol of catalyst. Although the data are not accurate because churning of this system became impossible in mid-reaction, the molecular weight of R,β-PMA increased compared to that obtained in this study (Table 3). As the reaction proceeded, the product became very hard, and churning was difficult. Therefore, future studies should examine more efficient stirring, for example, that using an extruder and a blender or other methods. Conclusions We investigated the reaction conditions for synthesis of R,β-PMA with HMW and high yield. The molecular weight of synthesized R,β-PMA is dependent on both the reaction temperature and time. The optimum reaction conditions for the synthesis of R,β-PMA were 110 °C for 45 h using tin(II) chloride as a catalyst. Therefore, we expect the synthesized R,β-PMA compound to be applied for the use in biomedical application, such as DDS. Acknowledgment. This study was supported in part by grant-in-Aid for Scientific Researches 1301B/15300176 and the Leading Project “Human Bodybuilding” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors are grateful to their colleagues at the Biomaterials Center, National Institute for Materials Science, for their useful advice.

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