Polymerization of Polyfunctional Macromolecules: Synthesis of a New

Apr 6, 2004 - Oxidative coupling of phenol-containing precursor poly(amino acid)s, poly(α-glutamine), poly and poly(γ-glutamine) derivatives, has be...
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Biomacromolecules 2004, 5, 977-983

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Polymerization of Polyfunctional Macromolecules: Synthesis of a New Class of High Molecular Weight Poly(amino acid)s by Oxidative Coupling of Phenol-Containing Precursor Polymers Tokuma Fukuoka, Hiroshi Uyama, and Shiro Kobayashi* Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Received November 25, 2003; Revised Manuscript Received February 25, 2004

Oxidative coupling of phenol-containing precursor poly(amino acid)s, poly(R-glutamine), poly(R/βasparagine), and poly(γ-glutamine) derivatives, has been examined to produce a new class of soluble poly(amino acid)s. Under appropriate reaction conditions, the Fe-salen and HRP catalysts efficiently induced the oxidative coupling without formation of insoluble gels, yielding the soluble polymers of high molecular weight. The oxidative coupling behaviors were greatly influenced by the structure and phenol content of the precursor polymer. The selection of the substrate concentration and catalyst amount was crucial for the production of soluble polymers of high molecular weight. Introduction Recently, there has been much interest in a variety of naturally occurring polymers derived from renewable resources for material applications.1 Among them, polypeptides and related artificial poly(amino acid)s have significantly been developed owing to their specific properties such as biocompatibility, biodegradability, etc.2 Poly(γ-glutamic acid) (γ-PGA) is a biosynthesized polymer consisting of a nylon-3 structure with pendant R-carboxyl groups. An industrial synthetic process of γ-PGA was established by fermentation utilizing Bacillus subtilis F-2-01.3 Poly(R/β-aspartate), a poly(amino acid) with carboxylate side chains, was synthesized by thermal polymerization of aspartic acid, followed by hydrolysis, which is a biodegradable water-soluble polymer.4 It received much attention as an alternative to poly(acrylic acid) for use of chelators and dispersants.5 Recently, γ-PGA and poly(R/β-aspartate) derivatives have been developed for biomedical applications.6 For the past decades, in vitro enzyme-catalyzed polymerization via nonbiosynthetic pathways (“enzymatic polymerization”) has been extensively investigated.7 Specific enzyme catalysis provides a novel synthetic route for functional and useful polymeric materials, many of which are difficult to synthesize by conventional methodologies. It was reported that some proteases induced the polymerization of amino acid esters to give poly(amino acid)s in good yields.8 We found that diethyl L-glutamate hydrochloride was regioselectively polymerized by papain catalyst to produce the polymer consisting of an R-peptide unit exclusively.9 Peroxidase has been frequently used as a catalyst for an oxidative polymerization of phenol derivatives under mild reaction conditions.7 The resulting polyphenols have normally structures directly linked at the aromatic ring by C-C and * To whom correspondence should be addressed. Phone: +81-75-3832459. Fax: +81-75-383-2461. E-mail: [email protected].

C-O coupling of phenols. The peroxidase catalysis was highly useful for chemoselective polymerization of phenol monomers having unsaturated groups to produce reactive polyphenols.10 In a nonaqueous medium, catalytic activity of peroxidase greatly diminishes. Thus, we have developed a new catalyst for the oxidative coupling in organic solvents; iron-N,N′ethylenebis(salicylideneamine) (Fe-salen), a model complex of peroxidase, showed high catalytic activity for oxidative polymerization of phenolic monomers.11,12 The first synthesis of a crystalline fluorinated poly(1,4-phenylene oxide) was achieved by the Fe-salen-catalyzed polymerization of 2,6difluorophenol.11c Cardanol was polymerized by Fe-salen to give a cross-linkable polyphenol in high yields, which was cured by thermal treatment, yielding a cross-linked film with good hardness and viscoelasticity (“Artificial Urushi”).12 These polyphenols obtained by using the enzyme or enzymemodel complex as catalyst are believed to be formed via the coupling of free radical intermediates of phenols.13 Very recently, we have expanded the scope of the oxidative polymerization of phenols by the enzyme or enzyme model catalyst to production of high molecular weight polymers from phenol-containing precursor polymers as a starting substrate; Fe-salen catalyzed the oxidative coupling of poly(R-glutamine) or poly(R/β-asparagine) having a phenol moiety in the side chain to give a new class of soluble poly(amino acid)s without the formation of an insoluble gel under appropriate conditions.14 Furthermore, ultrahigh molecular weight polymers (Mw >106) were prepared by the oxidative coupling of enzymatically synthesized polyphenols with a molecular weight of several thousands.15 From these results, we proposed a new concept “polymerization of polyfunctional macromolecules” for synthesis of high molecular weight polymers. In this study, we have examined the oxidative coupling behaviors of phenol-containing poly(amino acid)s with

10.1021/bm034490+ CCC: $27.50 © 2004 American Chemical Society Published on Web 04/06/2004

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Scheme 1

Fukuoka et al. Table 1. Oxidative Coupling of 2 Catalyzed by the Fe-salen Complex in DMFa product Fe-salen, solvent, conversion,b Mn × entry substrate mol% mL 10-4b Mw/Mnb %

different polymer structures and phenol content by using the enzyme and enzyme model catalyst. For the water-soluble precursor polymers, peroxidase efficiently catalyzed the oxidative coupling in an aqueous medium, whereas the waterinsoluble precursor polymers were converted to the high molecular weight poly(amino acid)s by using Fe-salen as catalyst in an organic solvent. Results and Discussion Oxidative Coupling of Poly(r-L-N-(2-(4-hydroxyphenyl)ethyl)glutamine). Previously we reported that poly(Ltyrosine), a phenol-containing R-type polypeptide, was not to be subjected to an oxidative coupling by the Fe-salen catalyst.14 This may be due to the short spacer length between the polymer backbone and the phenol group. Thus, we designed a new R-polypeptide having a phenol group in the side chain; poly(γ-ethyl R-L-glutamate) (1) with a degree of polymerization (DP) ) 8, which was obtained by the papaincatalyzed polymerization of L-glutamic acid diethyl ester,9 was modified by the reaction with tyramine to give poly(R-L-N-(2-(4-hydroxyphenyl)ethyl)glutamine) (2; Scheme 1).14 The structure of 2 was confirmed by 1H NMR spectroscopy. The introduced ratio of the phenol group was determined from the integrated area of peaks due to CH2CH2C(dO) and aromatic protons. The phenol content of 2 could be controlled by altering the feed ratio of tyramine for 1. Three samples with different phenol content (2a: phenol content of 20 mol %, 2b: 50 mol %, 2c: 80 mol %) were prepared, and the oxidative coupling was performed by Fe-salen catalyst in the presence of a small amount of pyridine in N,N-dimethylformamide (DMF). Table 1 summarizes effects of the substrate concentration and the catalyst amount. The molecular weight of the product was estimated by size exclusion chromatography (SEC). There were two peaks in the SEC chart after the oxidative coupling: a peak at lower elution volume due to the higher molecular products and a peak at higher elution volume due to the lower molecular products including the starting substrate. The conversion of 2 was estimated from the area ratio of these peaks, and Mn and Mw/Mn values of the product were determined from the peak at the lower elution volume. The high molecular weight product was obtained in good yields when 2b and 2c were used as a substrate, but on the other hand, the efficient oxidative polymerization of 2a did not occur. The molecular weight of the product increased

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

2a 2a 2a 2a 2a 2a 2b 2b 2b 2b 2b 2b 2c 2c 2c 2c 2c

1.0 2.0 2.0 2.0 2.0 4.0 1.0 2.0 2.0 2.0 2.0 4.0 1.0 2.0 2.0 2.0 4.0

5.0 1.0 2.5 5.0 10 5.0 5.0 1.0 2.5 5.0 10 5.0 2.5 1.0 2.5 5.0 2.5

12 c 29 24 ∼0 35 42 c 80 59 52 79 64 c 64 57 79

1.0 c 1.7 1.4

1.3 c 1.7 1.6

2.8 1.5 c 8.4 3.2 1.4 42 4.8 c 14 2.3 29

2.4 1.6 c 3.8 3.5 1.6 1.5 2.3 c 1.7 1.5 1.6

a Oxidative coupling of 2 (0.2 mmol phenol units, 2a: 175 mg, M ) n 1500, 2b: 81 mg, Mn ) 1600, 2c: 57 mg, Mn ) 1300) using the Fe-salen complex and hydrogen peroxide (0.2 mmol) as catalyst and oxidizing agent, respectively, in 0.4 vol % pyridine/DMF at room temperature under air. b Determined by SEC using 0.1 M LiCl/DMF as eluent with polystyrene standards. c Gelation took place.

with decreasing the solvent amount (increasing the substrate concentration) and increasing the catalyst amount. Gelation took place when a small amount of the solvent was used (entries 2, 8, and 14). These data clearly indicate that the selection of the substrate concentration and catalyst amount was very important for the efficient production of the soluble high-molecular weight poly(amino acid)s via the intermolecular oxidative coupling. A plausible way of linking between phenol moieties in the side chain is given in Figure 1. We measured the absolute molecular weight of the product (entry 12) by a combination of SEC with an on-line viscometer (VISC) and a right angle laser light scattering (RALLS) detector (SEC-VISC-RALLS analysis). The weight-average molecular weight was 2.7 × 106. The parameters obtained by Mark-Houwink-Sakurada equation ([η] ) KMa) are often used for the molecular conformation of polymers in solution. The a value of the product was 0.50, which is lower than that of a linear polymer (0.6∼0.8),16 suggesting the branched structure of the resulting poly(amino acid) with ultrahigh molecular weight. Under the conditions of entry 12 in Table 1, the coupling reaction of 2b was monitored by SEC (Figure 2). The SEC traces recorded with an RI detector (Figure 2A) revealed that the peak of 2b was converted into the peak due to the coupling product in the range of high molecular weight (>105), and a small amount of low-molecular weight (∼1 × 103) products remained. The SEC traces measured with a UV detector at 340 nm (Figure 2B) showed that the intensity of the peak at the lower elution volume increased as a function of the amount of hydrogen peroxide, suggesting that the oxidative coupling efficiently takes place with the addition of hydrogen peroxide. The 1H NMR spectrum of the product showed that only the peaks of the aromatic

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Figure 1. Outline of an oxidative coupling of the polymer having phenol moieties in the side chain.

Figure 3. SEC traces of oxidative coupling products of 4 (0.25 mmol of phenol unit) with RI detector under the conditions of entry 3 in Table 2.

Figure 2. SEC traces of oxidative coupling products of 2b (0.2 mmol of phenol unit) with (A) RI detector and (B) UV detector (340 nm) under the conditions of entry 12 in Table 1.

moiety became smaller and broader (data not shown), strongly supporting that the phenol moiety of 2 was consumed by the oxidative coupling. Oxidative Coupling of Poly(r/β-N-(2-(4-hydroxyphenyl)ethyl)asparagine). When 2 was used as the substrate for the oxidative coupling, the quantitative conversion was not achieved. This may be because of the low molecular weight of 2. Thus, we designed a new phenol-containing poly(amino acid) with higher molecular weight (Scheme 2). In the course of preparation of biodegradable thermal poly(aspartate), poly(succinimide) (3) is obtained as an intermediate product. It can easily be reacted with various nucleophiles. Alkaline hydrolysis of 3 was reported to produce poly(aspartate) with a mixed structure of R and β units.4 In this study, a phenol-containing asparagine polymer, poly(R/β-N-(2-(4-hydroxyphenyl)ethyl)asparagine) (4), was synthesized by the reaction of 3 with tyramine.14 By using an

excess of tyramine, the quantitative introduction of the phenol moiety was achieved. The oxidative coupling of 4 was conducted by using Fesalen catalyst in DMF. As shown in Figure 3, the soluble polymer of high molecular weight (Mn >105) was produced without the formation of insoluble gel under the appropriate conditions, and no precursor polymer remained after the coupling. The reaction behaviors of 4 were similar to those of 2 (Table 2). SEC-VISC-RALLS analysis of the product (entry 3) showed the formation of the ultrahigh molecular weight poly(amino acid) (Mw ) 1.9 × 106) with highly branched structure. For the enzymatic oxidative coupling of the precursor polymers, a water-soluble phenol-containing poly(amino acid) was designed and synthesized; 3 was reacted with tyramine, and subsequently, the residual succinimide group was hydrolyzed by NaOH to give 5 possessing phenol and carboxylate groups in the side chain (Scheme 2). This polymer was soluble only in water. The oxidative coupling of 5 with different phenol content (5a: phenol content of 10 mol %, 5b: 50 mol %) was carried out in the presence of horseradish peroxidase (HRP) catalyst in a phosphate buffer (Table 3). The HRP-catalyzed oxidative coupling of 5 proceeded quantitatively, and no starting polymer remained after the reaction. The oxidative coupling of 5b gave a water-soluble polymer with high molecular weight (Mn > 105) without the formation of insoluble gel under the selected conditions. The molecular weight of the polymer produced by the coupling of 5a was lower than that from 5b. A similar behavior was observed in the Fe-salen-

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Fukuoka et al.

Scheme 2

Table 2. Oxidative Coupling of 4 Catalyzed by the Fe-salen Complex in DMFa

Table 3. Oxidative Coupling of 5 Catalyzed by HRP in Phosphate Buffera

product entry

Fe-salen, mol%

solvent, mL

Mn ×10-4 b

Mw/Mnb

entry

1 2 3 4 5

0.50 1.0 1.0 1.0 2.0

5.0 2.5 5.0 10 5.0

7.5 c 34 12 26

2.9 c 1.4 2.2 1.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14

a Oxidative coupling of 4 (0.25 mmol phenol units, 58.5 mg, M ) n 37000) using the Fe-salen complex and hydrogen peroxide (0.25 mmol) as catalyst and oxidizing agent, respectively, in 0.4 vol % pyridine/DMF at room temperature under air. b Determined by SEC using 0.1 M LiCl/ DMF as eluent with polystyrene standards. c Gelation took place.

catalyzed coupling of 2; the precursor polymer of higher phenol content was subjected to the oxidative coupling more efficiently, yielding the soluble poly(amino acid) of higher molecular weight. In the case of the large amount of HRP, gelation took place. Another type of hydrophilic phenol-containing poly(R/βasparagine) derivative (6) was synthesized by the reaction of 3 with a mixture of tyramine and 2-aminoethanol (Scheme 2). This precursor polymer was soluble in both water and hydrophilic organic solvents. The oxidative coupling of 6 with a phenol content of 10 mol % was performed by using Fe-salen catalyst in DMF (Table 4). The soluble poly(amino acid) of high molecular weight was obtained, although the

substrate, mg (µmol)b 5a 5a 5a 5a 5a 5a 5a 5a 5a 5b 5b 5b 5b 5b

14.7 (10) 14.7 (10) 14.7 (10) 14.7 (10) 29.3 (20) 29.3 (20) 29.3 (20) 34.0 (30) 34.0 (30) 9.3 (25) 9.3 (25) 9.3 (25) 18.5 (50) 18.5 (50)

HRP, mg 0.01 0.10 0.50 1.0 0.01 0.10 0.20 0.01 0.05 0.01 0.10 0.50 0.10 0.20

product Mn ×10-4 c Mw/Mnc 5.5 9.9 15 d 2.4 3.8 d 3.2 d 14 31 20 20 d

3.2 7.1 6.2 d 2.3 5.6 d 4.6 d 3.0 2.8 2.8 6.3 d

a Oxidative coupling of 5 (5a: M ) 24000, 5b: M ) 16000) using n n HRP and hydrogen peroxide (equimolar to phenol unit) as catalyst and oxidizing agent, respectively, in 0.1 mol/L phosphate buffer (pH 7, 2.5 mL) at room temperature under air. b In parentheses, phenol unit c Determined by SEC using 0.1 M NaCl as eluent with poly(ethylene glycol) standards. d Gelation took place.

conversion of 6 was not high. The reaction behaviors were similar to those of 2 in DMF. The oxidative coupling of 6 also proceeded in water by using HRP as catalyst (Table 5). The quantitative consumption of 6 to the water-soluble poly(amino acid) of high

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Polymerization of Polyfunctional Macromolecules Table 4. Oxidative Coupling of 6 Catalyzed by Fe-salen Complex in DMFa entry

solvent, mL

Fe-salen, mol%

conversion,c %

1 2 3 4 5

5.0 2.5 2.5 2.5 1.25

10 5.0 10 20 5.0

40 50 62 66

product Mn × 10-4 c Mw/Mnc 11 18 28 26 d

1.5 2.1 2.2 2.2 d

a Oxidative coupling of 6 (50 µmol phenol unit, 82.8 mg, M ) 9000) n using the Fe-salen complex and hydrogen peroxide (50 µmol) as catalyst and oxidizing agent, respectively, in 0.4 vol % pyridine/DMF at room temperature under air. b In parentheses, phenol unit c Determined by SEC using 0.1 M LiCl/DMF as eluent with polystyrene standards. d Gelation took place.

Table 5. Oxidative Coupling of 6 Catalyzed by HRP in Phosphate Buffera entry

substrate, mg (µmolb)

HRP, mg

1 2 3 4 5 6

41.4 (25) 41.4 (25) 41.4 (25) 41.4 (25) 82.8 (50) 82.8 (50)

0.01 0.05 0.10 1.0 0.01 0.05

product Mn ×10-4 c Mw/Mnc 5.6 9.4 18 d 6.5 d

2.2 4.1 4.4 d 2.5 d

a Oxidative coupling of 6 (M ) 25000) using HRP and hydrogen n peroxide (equimolar to phenol unit) as catalyst and oxidizing agent, respectively, in 0.1 mol/L phosphate buffer (pH 7, 2.5 mL) at room temperature under air. b In parentheses, phenol unit. c Determined by SEC using 0.1 M NaCl as eluent with poly(ethylene glycol) standards. d Gelation took place.

Scheme 3

molecular weight was observed under the selected conditions. These data strongly suggest that the appropriate design of precursor polymers and the detailed screening of reaction conditions provide a new class of high molecular weight poly(amino acid)s with different solubility. Oxidative Coupling of Poly(γ-N-(2-(4-hydroxyphenyl)ethyl)glutamine). For development of a new class of poly(amino acid)s by the oxidative coupling, a naturally occurring polymer (PGA) was modified and subjected to the oxidative coupling; a phenol-containing γ-glutamine-based polymer, poly(γ-N-(2-(4-hydroxyphenyl)ethyl)glutamine) (8), was synthesized by the reaction of γ-PGA (7) and tyramine in the presence of N,N′-carbonyldiimidazole (CDI) as a dehydration agent (Scheme 3). The structure of 8 was confirmed by 1H NMR. The oxidative coupling of 8 with different phenol content (8a: phenol content of 25 mol %, 8b: 50 mol %) was performed by using Fe-salen catalyst in DMF (Table 6). Though the oxidative coupling reactivity of 8 was lower than that of 4 probably due to the low solubility of 8 against DMF

Table 6. Oxidative Coupling of 8 Catalyzed by Fe-salen Complex in DMFa substrate, mg (µmolb)

entry 1 2 3 4 5 6 7 8 9 10

8a 8a 8a 8a 8a 8b 8b 8b 8b 8b

15.9 (25) 15.9 (25) 15.9 (25) 31.8 (50) 31.8 (50) 18.9 (50) 18.9 (50) 18.9 (50) 37.7 (100) 37.7 (100)

Fe-salen, mol %

product Mn × 10-4 c Mw/Mnc

10 20 40 5.0 10 5.0 10 20 2.5 5.0

5.1 9.6 15 5.9 d 10 16 17 9.2 d

2.1 2.1 7.9 2.3 d 2.1 2.3 6.8 4.1 d

a Oxidative coupling of 8 (8a: M ) 54000, 8b: M ) 86000) using n n the Fe-salen complex and hydrogen peroxide (equimolar to phenol unit) as catalyst and oxidizing agent, respectively, in 0.4 vol % pyridine/DMF (2.5 mL) at room temperature under air. b In parentheses, phenol unit. c Determined by SEC using 0.1 M LiCl/DMF as eluent with polystyrene standards. d Gelation took place.

and/or the steric factor of the phenol moiety in 8, a soluble polymer of high molecular weight (Mn > 105) was quantitatively obtained under selected conditions (entries 3, 7, and 8). Conclusion Generally, cross-linking takes place in polymerizations of polyfunctional macromolecules. For example, it was reported that the oxidative coupling of phenol-containing polymers by laccase or Fe-salen catalyst produced the insoluble crosslinked gels.17 On the other hand, we have found that soluble high molecular weight poly(amino acid)s were exclusively formed by the precise design of the phenol-containing precursor polymer and by the detailed selection of reaction conditions in the polymerizations of polyfunctional macromolecules. An oxidative coupling of phenol derivatives by using the enzyme or enzyme-model catalyst is an environmentally benign synthetic process of polymers, owing to excellent economy on atoms (normally, only water as byproduct) and mild reaction conditions with low energy input. The present study clearly demonstrates that the oxidative coupling of phenol-containing precursor polymers is highly useful for development of new functional polymeric materials from renewable resources. Experimental Section Materials. Fe-salen and HRP (100 units per mg) were purchased from Tokyo Kasei Inc. and Wako Pure Chemical Industries, Inc., respectively, and used without further purification. Poly(succinimide) (3) and poly(γ-glutamic acid) (7) were gifts from Prof. Toyoji Kakuchi (Hokkaido University, Japan) and Meiji Seika Inc., respectively. Poly(γethyl R-L-glutamate) (1)9 and poly(R/β-N-(2-(4-hydroxyphenyl)ethyl)asparagine) (4)14 were synthesized according to the literatures. Other reagents and solvents were commercially available and were used as received. Synthesis of 2c. A mixture of 1 (1.6 g, 10 mmol of monomer unit) and tyramine (1.5 g, 11 mmol) was dissolved

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in anhydrous dimethyl sulfoxide (DMSO) (40 mL) under argon atmosphere. The solution was kept at 90 °C for 72 h under gentle stirring. The mixture was poured into a large amount of water. The formed precipitates were collected by centrifugation and washed with water, followed by drying in vacuo to give 2c as a light-brown powder (1.6 g, 51% yield). Mn ) 1300; Mw/Mn ) 1.3. 1H NMR (DMSO-d ): δ 0.8-1.4 (br, CH ), 1.7-2.0 (br, 6 3 C(dO)CH2CH2), 2.2-2.4 (br, C(dO)CH2), 2.6-3.0 (br, ArCH2CH2), 4.0-4.1 (br, OCH2), 4.1-4.3 (br, CH), 6.57.3 (br, Ar), 7.5-8.5 (br, NH), 9.0-9.5 (br, ArOH). Similarly, 2a and 2b were synthesized, and their structures were confirmed by 1H NMR. Synthesis of 5a. A mixture of 3 (9.7 g, 100 mmol of monomer unit) and tyramine (1.4 g, 10 mmol) was dissolved in anhydrous DMF (60 mL) under argon atmosphere. The solution was kept at 60 °C for 24 h under gentle stirring. The polymer was isolated by reprecipitation using DMF as solvent and water as nonsolvent. The obtained polymer (11 g) was dissolved in 2.0 mol/L NaOH solution (45 mL) at 0 °C and the solution was kept at room temperature for 24 h under gentle stirring. The mixture was poured into a large amount of methanol. The formed precipitates were collected by centrifugation and washed with methanol, followed by drying in vacuo to give 5a as a white powder (9.2 g, 83% yield). Mn ) 2.4 × 104; Mw/Mn ) 2.2. 1H NMR (D O): δ 2.2-2.8 (br, ArCH ), 3.0-3.3 (br, 2 2 ArCH2CH2), 4.2-5.0 (br, C(dO)CH2CHC(dO)), 6.5-7.3 (br, Ar). Similarly, 5b was synthesized, and its structure was confirmed by 1H NMR. Synthesis of 6. A mixture of 3 (4.9 g, 50 mmol of monomer unit), tyramine (0.75 g, 5.5 mmol), and 2-aminoethanol (3.0 g, 49.5 mmol) was dissolved in anhydrous DMF (30 mL) under argon atmosphere. The solution was kept at 60 °C for 24 h under gentle stirring. The mixture was poured into a large amount of acetone. The formed precipitates were collected by centrifugation and washed with acetone, followed by drying in vacuo to give 6 as a white powdery product (8.2 g, 100% yield). Mn ) 9.0 × 103; Mw/Mn ) 1.9. 1H NMR (DMSO-d ): δ 2.6-2.8 (br, ArCH ), 3.0-3.3 6 2 (br, ArCH2CH2 and CH2CH2OH), 3.3-3.6 (br, C(dO)CH2CHC(dO) and CH2OH), 4.2-5.0 (br, C(dO)CH2CHC(d O)), 6.5-7.3 (br, Ar), 7.5-8.5 (br, NH), 9.0-9.5 (br, ArOH). Synthesis of 8b. Poly(γ-glutamic acid) (5.2 g, 40 mmol) was dispersed in anhydrous DMF (250 mL) under argon atmosphere at 0 °C, and subsequently, N,N′-carbonyldiimidazole (CDI) (3.6 g, 22 mmol) was added to the solution at 0 °C. After 2 h, tyramine (2.7 g, 20 mmol) was added to the mixture under gentle stirring at 0 °C, and thereafter, the mixture was gently stirred for 72 h at room temperature. The insoluble part of the mixture was filtrated and the filtrate was subjected to dialysis (cutoff molecular weight: 1000) against distilled water with three changes of the dialysis solution. The remaining solution was lyophilized to give 3.8 g of 8b (yield 50%). Mn ) 8.6 × 104; Mw/Mn ) 2.0. 1H NMR (DMSO-d ): δ 1.5-2.1 (br, CHCH CH CdO), 6 2 2 2.1-2.4 (br, CH2CH2CdO) 2.4-2.7 (br, ArCH2), 3.0-3.3

Fukuoka et al.

(br, ArCH2CH2), 4.1-4.3 (br, C(dO)CHNH), 6.5-7.3 (br, Ar), 7.5-8.5 (br, NH), 9.0-9.5 (br, ArOH). Similarly, 8a was synthesized, and its structure was confirmed by 1H NMR. Fe-salen-Catalyzed Oxidative Coupling of PhenolContaining Poly(amino acid)s in DMF. The following is a typical procedure for the Fe-salen-catalyzed oxidative coupling of the phenol-containing poly(amino acid)s in DMF (entry 9 in Table 1). A mixture of 2b (81 mg, 0.20 mmol of phenol unit) and Fe-salen (1.29 mg, 4.0 µmol) was dissolved in DMF (2.5 mL) containing 0.4 vol % pyridine. The coupling was started by the addition of a quarter equivalents of hydrogen peroxide (30%) with respect to 2b under air. The same amount of hydrogen peroxide was added three more times every 15 min. After 24 h, the reaction mixture was analyzed by SEC without purification. HRP-Catalyzed Oxidative Coupling of Phenol-Containing Poly(amino acid)s in Buffer. The following is a typical procedure for the HRP-catalyzed oxidative coupling of the phenol-containing poly(amino acid)s in a buffer (entry 2 in Table 3). A mixture of 5a (14.7 mg, 10 µmol of phenol unit) and HRP (0.10 mg) was dissolved in 0.1 mol/L phosphate buffer (pH 7, 2.5 mL). The coupling was started by the addition of a quarter equivalents of hydrogen peroxide (30%) with respect to 5a under air. The same amount of hydrogen peroxide was added three more times every 15 min. After 24 h, the reaction mixture was analyzed by SEC without purification. Measurements. SEC analysis was carried out by using a Tosoh GPC-8020 apparatus equipped with refractive index (RI) and UV detectors under the following conditions: TSKgel R-3000 and R-M columns and DMF containing 0.10 M LiCl eluent at a flow rate of 1.0 mL/min at 60 °C, or TSKgel R-3000 and R-M columns and distilled water containing 0.10 M NaCl eluent at a flow rate of 1.0 mL/min at 40 °C. The calibration curves were obtained by using polystyrene (DMF eluent) or poly(ethylene oxide) (water eluent) as the standard. SEC-VISC-RALLS analysis was performed by using a Tosoh GPC-8020 apparatus equipped with refractive index (RI) and UV detectors on-line combined with a TriSEC Dual Detector model 270 apparatus (Viscotek Co.) under the same conditions of SEC analysis. NMR spectra were recorded on a Bruker DPX400 spectrometer. Acknowledgment. This work was partly supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience, by the 21st century COE program, COE for a United Approach to New Materials Science, and by the Japan-US Cooperative Research Program of JSPS. We acknowledge the gift of poly(succinimide) and poly(γglutamate) from Prof. Toyoji Kakuchi (Hokkaido University, Japan) and Meiji Seika Kaisha, Ltd., respectively. References and Notes (1) Mohanty, A. K.; Misra, M.; Hinrichsen, G. Macromol. Mater. Eng. 2000, 276/277, 1. (2) (a) Erhan, S. In Desk Reference of Functional Polymers, Syntheses and Applications; Arshady, R., Ed.; American Chemical Society: Washington, DC, 1997; pp 261-270. (b) Kaplan, D. L., Ed., Biopolymers from Renewable Resources, Springer: Berlin, 1998. (c) Sanda, F.; Endo, T. Macromol. Chem. Phys. 1999, 200, 2651.

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