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Protease-Catalyzed Regioselective Polymerization and Copolymerization of Glutamic Acid Diethyl Ester Hiroshi Uyama,† Tokuma Fukuoka,† Izuru Komatsu,‡ Takashi Watanabe,‡ and Shiro Kobayashi*,† Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan, and Department of Materials Chemistry, Graduate School of Engineering, Tohoku University, Aoba, Sendai 980-8579, Japan Received August 27, 2001; Revised Manuscript Received November 19, 2001
Protease-catalyzed polymerization and copolymerization of L-glutamic acid diethyl ester hydrochloride (1) have been performed in a buffer of high concentration. Papain and bromelain showed high catalytic activity toward the polymerization. H-H COSY NMR analysis of the product showed the exclusive formation of poly(R-peptide), which was further confirmed by comparison with NMR spectra of poly(R-methyl γ-Lglutamate). The papain-catalyzed polymerization of γ-methyl L-glutamate did not occur under the similar reaction conditions, supporting the regioselective production of the polymer having an R-peptide linkage from 1. The effects of the reaction parameters have been systematically investigated. The copolymerization of 1 with various amino acid esters took place by the papain catalyst to give peptide copolymers. Introduction As one possible solution to waste-disposal problems associated with traditional petroleum-derived plastics, biodegradable polymeric materials have been increasingly important.1 Poly(amino acid)s are biodegradable and thus used for medical, cosmetic, and fabric materials.2 Poly(Ramino acid)s with high molecular weight are readily synthesized by ring-opening polymerization of R-amino acid N-carboxylic anhydrides (NCAs); however, use of toxic phosgene (or phosgene derivatives) is involved for the monomer synthesis.3 For the past decades, an enzyme-catalyzed polymerization (“enzymatic polymerization”) has been of increasing importance as a new trend in macromolecular science.4 Enzyme catalysis has provided new synthetic strategy for useful polymers, most of which are difficult to produce by conventional chemical catalysts. In vitro enzymatic syntheses of polymers via non-biosynthetic pathways, therefore, are recognized as a new area of precision polymer syntheses. It is generally accepted that an enzymatic reaction is virtually reversible, and hence, the equilibrium can be controlled by appropriately selecting the reaction conditions. On the basis of this view, many of hydrolases, that are enzymes catalyzing a bond-cleavage reaction by hydrolysis, have been employed as catalyst for the reverse reaction of hydrolysis, leading to polymer production by a bond-forming reaction.5 Proteases catalyze not only hydrolysis of peptide bonds but also peptide bond formation under selected conditions.6 It was reported that the reaction of amino acid esters in the presence of some proteases produced water-insoluble prod† ‡
Kyoto University. Tohoku University.
Scheme 1
ucts. Ester hydrochlorides of methionine, phenylalanine, threonine, and tyrosine were polymerized in a buffer by papain catalyst to give poly(R-amino acid)s with a degree of polymerization (DP) of less than 10.7 As for monomers of amino acids having a carboxylic acid group in the side chain, diethyl L-aspartate was polymerized by alkanophilic protease from Streptomyces sp. in bulk to give the polymer in a non-regioselective manner, having a mixed structure of R- and β-peptide linkages.8 It was also claimed that papain and R-chymotrypsin induced the polymerization of diethyl L-glutamate in a buffer9 and the polymerization proceeded even in benzene by using poly(ethylene glycol)-modified papain as catalyst.10 However, the structure of product polymers was not well characterized. Poly(glutamic acid) is expected as a reactive and biodegradable polymer. In this study, a protease-catalyzed polymerization of diethyl L-glutamate hydrochloride (1) in high concentration buffers and structure of the resulting poly(amino acid) have been investigated in detail (Scheme 1). Furthermore, the protease-catalyzed copolymerization of 1 with various amino acid esters has been performed for the
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Figure 1. H-H COSY NMR spectrum of the polymer obtained by the polymerization of 1 (0.50 M) using papain catalyst (40 mg/mL) in phosphate buffer (I ) 2.0 M, pH 7) at 40 °C for 3 h.
first time, which will expand a scope of in vitro enzymatic synthesis of poly(amino acid)s. Results and Discussion Protease-Catalyzed Polymerization of Diethyl L-Glutamate Hydrochloride. At first, the polymerization of 1 was carried out with using papain from papaya latex as catalyst in phosphate buffer (pH 7, ionic strength (I) ) 2.0 M) at 40 °C for 3 h. Papain is a cystein protease. During the polymerization, white solid precipitates were formed, which were separated by centrifugation after the reaction. The polymer yield was 61% and soluble in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) but insoluble in water and common other organic solvents such as methanol, acetone, chloroform, tetrahydrofuran, and toluene. Size exclusion chromatographic (SEC) analysis showed that the number-average molecular weight and its index were 1200 and 1.3, respectively. The specific rotation ([R]589) of the polymer was -5.1° (DMSO, c ) 10). In the polymerization of 1 without the enzyme (control experiment), the monomer was recovered unchanged, indicating that the present polymerization proceeded via the enzyme catalysis. Figure 1 shows H-H correlation spectroscopy (COSY) of the polymer. A strong cross-peak was observed between peaks A and E. As for peak B, there were three cross-peaks with peaks C, D, and F. Peak G was crossed with peak F. From these data, all the peaks were assigned as shown in Figure 1. The DP value of the polymer, estimated from the
ratio of the integrated area of small peak D at δ 3.9 due to methine proton adjacent to terminal ammonium group to that of peak F ascribed to methine proton adjacent to nitrogen atom of the peptide group, was 9.5. The polymer structure was confirmed by 13C and heteronuclear multiple quantum coherence (HMQC) NMR spectroscopies. Furthermore, 1H and 13C NMR spectra of the product polymer were different from those of poly(R-methyl γ-L-glutamate),11 supporting the exclusive regioselective formation of (γ-ethyl R-L-glutamate) from 1. In the IR spectrum of the product, there were two characteristic peaks at 1628 and 1527 cm-1 due to CdO vibration of amide I and N-H vibration of amide II, respectively. Figure 2 shows fast atom bombardment (FAB) mass spectrum of the polymer. Sets of two peaks with regular peak-to-peak distance (157) in the range of the DP value from 7 to 9 were mainly seen. The mass difference of the two set peaks was 28 or 29, suggesting that the terminal group of the polymer was a mixture of an ester and hydrolyzed carboxylic acid groups. For comparison, the papain-catalyzed polymerization of γ-methyl L-glutamate (2) was carried out under the similar reaction conditions, resulting in no occurrence of the polymerization. These data strongly suggest that the R-ester moiety was required for the protease-catalyzed polymerization, leading to the regioselective production of poly(R-amino acid) from 1. In the present polymerization, the high molecular weight polymer was not formed. This may be because papain does not recognize the hydrolyzed carboxylic
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Figure 2. FAB mass spectrum of the polymer obtained by the polymerization of 1 (0.50 M) using papain catalyst (40 mg/mL) in phosphate buffer (I ) 2.0 M, pH 7) at 40 °C for 3 h.
Figure 3. Relationships between the reaction parameters and polymer yield: (A) effect of monomer concentration in the polymerization of 1 using papain catalyst (40 mg/mL) in phosphate buffer (I ) 2.0 M, pH 7) at 40 °C for 3 h; (B) effect of enzyme concentration in the polymerization of 1 (0.50 M) using papain catalyst in phosphate buffer (I ) 2.0 M, pH 7) at 40 °C for 3 h; (C) effect of ionic strength in the polymerization of 1 (0.50 M) using papain catalyst (40 mg/mL) at 40 °C for 3 h.
acid group like 2. The precipitation of the polymer during the polymerization also prevents the increase of the molecular weight. Besides papain, some proteases were screened for the polymerization of 1 under the similar reaction conditions. Bromelain (cystein protease, from pineapple stem) and R-chymotrypsin (serine protease, from bovine pancreas) catalyzed the polymerization to give the polymer in yields of 70 and 38%, respectively. The DP was ca. 9, determined by 1H NMR. On the other hand, no polymerization took place in the presence of Subtilisin Carlsberg (serine protease, from Bacillus licheniformis) and Thermolysin (metal protease, from Bacillus thermoproteolyticus rokko). These data suggest that cystein proteases are suitable for the polymerization of 1. Effects of Polymerization Conditions. Effects of reaction parameters such as monomer, enzyme, and ionic strength on the polymer yield have been systematically examined. As for the monomer concentration, the polymer formation was observed in the range from 0.20 and 0.70 M and the polymer yield slightly depended on the monomer concentration; there was a maximum yield point at 0.50 M (Figure 3A).
The polymer yield increased as a function of the enzyme concentration less than 40 mg/mL, and above this concentration, the yield was almost constant (Figure 3B). In the polymerization in a phosphate buffer of pH 7, the polymer yield increased with increasing the ionic strength of the buffer less than 1.0 M, and the constant yield was observed above this concentration. On the other hand, there was a maximum point at 0.60 M under the alkaline conditions (carbonate buffer of pH 10) (Figure 3C). The DP value scarcely depended on the reaction conditions. In enzymatic polymerizations for production of polysaccharides and polyphenols, polar organic solvents were used as cosolvent.4h The addition of the organic solvent will increase the solubility of the propagating polymer, leading to the increase of the molecular weight. The papain-catalyzed polymerization of 1 was carried out in a mixture of organic solvent and the phosphate buffer. The cosolvents used were DMF and DMSO. The polymerization in the 25% organic solvent produced the polymer in lower yields (less than 50%) than that without the organic solvent, and the addition of the organic solvent hardly affected the DP value. When the ratio of the organic solvent was 50 or 75%, the polymer formation was not observed. Figure 4 shows a time-yield
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Table 1. Papain-Catalyzed Copolymerization of 1 with 3aa copolymerization
Figure 4. Time-yield curve in the papain-catalyzed polymerization of 1 (0.50 M) using papain catalyst (40 mg/mL) in phosphate buffer (I ) 2.0 M, pH 7) at 40 °C.
curve in the polymerization using the phosphate buffer of I ) 2.0 M. The polymerization proceeded relatively fast, and the polymer yield was almost constant after 2 h. Enzymatic Copolymerization of 1 with L-Amino Acid Esters. So far, protease-catalyzed copolymerization of amino acid derivatives has not been examined to our knowledge. At first, the copolymerization of 1 with methionine methyl ester hydrochloride (3a) has been examined in detail (Scheme 2). Copolymerization results are summarized in Table 1. The copolymerization proceeded fast; the polymer yield and DP value of the copolymer obtained for 0.50 h were close to those for 3 h. The yield was larger than that of the homopolymerization of 1 or 3a under the similar reaction conditions, and the DP value was not affected so much by the feed ratio. The composition of the copolymer determined by 1H NMR fairly agreed with the feed ratio. The specific rotation ([R]589) increased as a function of the content of 3a. To confirm the copolymer formation, the modification of the copolymer was examined. Water-insoluble poly(Rmethionine) was known to be converted to water-soluble poly(R-methionine sulfoxide) by the oxidative treatment with hydrogen peroxide.7c The water-insoluble copolymer obtained from an equimolar mixture of 1 and 3a was oxidized with an excess of hydrogen peroxide to give the water-soluble polymeric materials in a few minutes, whereas no such solubility change was observed in the oxidative treatment of the homopolymer of 1 under the similar reaction mixture. The alkaline hydrolysis (1 N NaOH) of the ester group in the copolymer at 60 °C also afforded the water-soluble copolymer without cleavage of the peptide bond; on the other hand, the polymer from 3a did not become water-soluble by the alkaline treatment. These data strongly suggest the formation of the copolymer. Scheme 2
copolymer
feed ratio [1]0/[3a]0
time (h)
yield (%)
DPb
[1]/[3a]b
[R]589c (deg)
100/0 75/25 50/50 50/50 25/75 0/100
3 3 0.5 3 3 3
61 74 78 76 84 70
9.5 11.3 9.8 10.2 12.0 9.9
100/0 74/26 54/46 52/48 28/72 0/100
-5.1 -8.0 -10.1 -9.8 -10.4 -12.1
a Copolymerization of 1 and 3a (total 1.25 or 2.5 mmol) using papain catalyst (200 mg) in phosphate buffer (5 mL, I ) 2.0 M, pH 7) at 40 °C for 3 h. b Determined by 1H NMR. c Determined by polarimeter at c ) 10 using DMSO solvent.
Table 2. Papain-Catalyzed Copolymerization of 1 with 3a copolymer comonomer
yield (%)
DPb
[1]/[3]b
3b 3c 3d 3e 3f
31 70 23 57 38
15.8 8.2 12.2 10.9 8.1
77/23 40/60 33/67 43/57 83/17
a Copolymerization of an equimolar mixture of 1 and 3 (each 1.25 mmol) using papain catalyst (200 mg) in phosphate buffer (5 mL, I ) 2.0 M, pH 7) at 40 °C for 3 h. b Determined by 1H NMR.
Besides 3a, ethyl ester hydrochlorides of alanine (3b), leucine (3c), phenylalanine (3d), and tyrosine (3e) and aspartic acid diethyl ester hydrochloride (3f) as comonomer (Table 2) were used. It was reported that 3c, 3d, and 3e were polymerized by the papain catalyst.7 In all cases examined, the polymeric materials were formed. The copolymer yield strongly depended on the comonomer; the copolymerization with 3c produced the corresponding copolymer in a good yield; however, the yield of the copolymers obtained using 3b, 3d, and 3f was not high. In the homopolymerization under the similar conditions, 3a and 3f were not converted into the polymer; however, the copolymerization of 1 with 3a or 3f took place, although the unit of 3 was much smaller than that of 1. On the other hand, other combinations produced the copolymer containing a smaller content of 1. The copolymer from 1 and 3e and homopolymer of 3e were insoluble in water but soluble in 1 N NaOH solution at 0 °C due to the neutralization of phenolic group, whereas the homopolymer of 1 was insoluble in the alkaline solution at 0 °C. From these data, the copolymer formation was confirmed in the papain-catalyzed copolymerization of amino acid esters.
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Conclusion Proteases, papain, bromelain, and R-chymotrypsin, catalyzed the regioselective polymerization of L-glutamic acid diethyl ester hydrochloride (1) to give poly(γ-ethyl R-Lglutamate) with DP of ca. 9. The exclusive formation of R-linkage poly(amino acid) was confirmed by comparison with NMR spectra of poly(R-methyl γ-L-glutamate). The polymerization efficiently proceeded in high concentration buffers. The protease-catalyzed copolymerization of amino acids has been examined for the first time in this study. The papain-catalyzed copolymerization of 1 with various amino acid esters produced the corresponding peptide copolymers. Although hydrochlorides of L-aspartic acid diethyl ester and alanine ethyl ester were not subjected to the papain-catalyzed polymerization, they were copolymerized with 1 by the same catalyst. Further studies on the enzymatic synthesis and applications of poly(amino acids)s are under way in our laboratory. Experimental Section Materials. L-Aspartic acid diethyl ester hydrochloride was synthesized according to the literature.12 Other monomers were purchased from Tokyo Kasei Co. and were of guaranteed reagent grade. Other reagents were commercially available and used as received. Papain (2.5 units/mg of solid) and bromelain (1000 units/mg of solid) were kindly donated by Nagase Seikiagaku Co. R-Chymotrypsin (50 units/mg of protein), Subtilisin Carlsberg (10 units/mg of solid), and Thermolysin (75 mg/protein) were purchased from Sigma. Enzymatic Polymerization. A typical run was as follows. In a 50 mL flask, a mixture of 1 (0.60 g, 2.5 mmol), papain (200 mg), and 5 mL of phosphate buffer (I ) 2.0 M, pH 7) was placed. The mixture kept at 40 °C under gentle shaking for 3 h at ambient pressure. After the reaction mixture was cooled to room temperature, 20 mL of water was added to the mixture. The precipitates were collected by centrifugation and washed with dilute HCl solution and subsequently with water twice. The resulting white powder was dried in vacuo to give 0.25 g of the polymer (61% yield). 13C NMR (DMSO-d6): δ 14 (OCH2CH3), 27 (CHCH2CH2), 30 (CH2CO), 52 (CHCH2), 59 (OCH2CH3), 171 (NHCO), 172 (CO2CH2). The copolymerization of 1 with other amino acid esters 3 was carried out by similar procedures. 1H NMR of the copolymer from 1 and 3a (DMSO-d6): δ 1.2 (t, OCH2CH3), 1.8 (br, CHCH2CH2), 2.0 (m, SCH3), 2.4 (br, CHCH2CH2), 3.6 (s, OCH3), 3.9 (br, NH3CHCH2), 4.0 (m, OCH2), 4.3 (br, NHCHCH2), 8.4 (br, NH). 1H NMR of the copolymer from 1 and 3b (DMSO-d6): δ 1.2 (t, OCH2CH3), 1.3 (d, CHCH3), 1.8 (br, CHCH2CH2), 2.3 (br, CH2CO), 3.8 (m, NH3CHCH2 and NH3CHCH3), 4.0 (m, OCH2), 4.2 (br, NHCHCH2 and NHCHCH3), 8.3 (br, NH). 1H NMR of the copolymer from 1 and 3d (DMSO-d6): δ 1.1 (m, OCH2CH3), 1.8 (br, CHCH2CH2), 2.3 (br, CH2CO), 2.6 (br, CH2Ar), 3.7 (m, NH3CHCH2), 4.0 (m, OCH2), 4.2 (br, NHCHCH2), 7.2 (d, Ar), 8.3 (br, NH). 1H NMR of the copolymer from 1 and 3f (DMSO-d6): δ 1.2 (t, OCH2CH3), 1.8 (br, CHCH2CH2), 2.3 (t, CH2CH2CO), 2.7 (m, CHCH2CO), 3.8 (m, NH3CHCH2), 4.0 (m,
Uyama et al.
OCH2), 4.2 (br, NHCHCH2CH2), 4.6 (br, NHCHCH2CO), 8.3 (br, NH). Measurements. SEC analysis was carried out by using a Tosoh SC8010 apparatus equipped with refractive index (RI) detector at 60 °C under the following conditions: TSKgel G2500HHR column and DMF containing 0.10 M LiCl eluent at a flow rate of 0.5 mL/min. The calibration curves were obtained using polystyrene standards. NMR spectra were recorded on a Bruker DPX400 spectrometer. IR measurement was carried out with a Shimadzu IR-460 spectrometer. Specific rotation measurement was performed by a JASCO DIP-370 digital polarimeter. FAB mass measurement was carried out using a JEOL high-performance JMS-HX110 mass spectrometer. Acknowledgment. This work was partly supported by Program for Promotion of Basic Research Activities for Innovative Bioscience. We acknowledge the gift of papain and bromelain from Nagase Seikiagaku Co. Supporting Information Available. 1H, 13C, and C-H correlation (HMQC) NMR spectra of poly(1) and 1H, 13C, and H-H COSY NMR spectra of the copolymer obtained from an equimolar feed ratio of 1 and 3a for 3 h. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Mohanty, A. K.; Misra, M.; Hinrichsen, G. Macromol. Mater. Eng. 2000, 276/277, 1. (2) Sanda, F.; Endo, T. Macromol. Chem. Phys. 1999, 200, 2651. (3) Imanishi, Y. In Ring-Opening Polymerization; Ivin, K. J., Saegusa, T., Ed.; Elsevier: London, 1984; Chapter 8. (4) For recent reviews on enzymatic polymerizations, see: (a) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1. (b) Kobayashi, S.; Shoda, S.; Uyama, H. In The Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: Boca Raton, FL, 1996; pp 2102-2107. (c) Kobayashi, S.; Shoda, S.; Uyama, H. In Catalysis in Precision Polymerization; Kobayashi, S., Ed.; John Wiley & Sons: Chichester, 1997; Chapter 8. (d) Ritter, H. In Desk Reference of Functional Polymers, Syntheses and Applications; Arshady, R., Ed.; American Chemical Society: Washington, DC, 1997; pp 103113. (e) Gross, R. A., Kaplan, D. L., Swift, G., Ed. ACS Symp. Ser. 1998, No. 684. (f) Kobayashi, S.; Uyama, H. In Materials Science and TechnologysSynthesis of Polymers; Schlu¨ter, A.-D., Ed.; WileyVCH: Weinheim, 1998; Chapter 16. (g) Joo, H.; Yoo, Y. J.; Dordick, J. S. Korean J. Chem. Eng. 1998, 15, 362. (h) Kobayashi, S.; Uyama, H.; Ohmae, M. Bull. Chem. Soc. Jpn. 2001, 74, 613. (i) Gross, R. A.; Kumar, A.; Kalra, B. Chem. ReV. 2001, 101, 2097. (j) Kobayashi, S.; Uyama, H. In Encyclopedia of Polymer Science and Technology, 3rd ed.; Kroschwitz, J. I., Ed.; John Wiley & Sons: New York, in press. (k) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. ReV. 2001, 101, 3793. (l) Kobayashi, S.; Kimura, S.; Sakamoto, J. Prog. Polym. Sci. 2001, 26, 1525. (5) For recent papers on hydrolase-catalyzed polymerizations, see: (a) Kobayashi, S.; Hobson, L. J.; Sakamoto, J.; Kimura, S.; Sugiyama, J.; Imai, T.; Itoh, T. Biomacromolecules 2000, 1, 168, 509. (b) Sakamoto, J.; Sugiyama, J.; Kimura, S.; Imai, T.; Itoh, T.; Watanabe, T.; Kobayashi, S. Macromolecules 2000, 33, 4155, 4982. (c) Kumar, A.; Gross, R. A. Biomacromolecules 2000, 1, 133. (d) Osanai, Y.; Toshima, K.; Matsumura, S. Chem. Lett. 2000, 576. (e) Nishida, H.; Yamashita, M.; Nagashima, M.; Endo, T.; Tokiwa, Y. J. Polym. Sci., Polym. Chem. Ed. 2000, 38, 1560. (f) Runge, M.; O’Hagan, D.; Haufe, G. J. Polym. Sci., Polym. Chem. Ed. 2000, 38, 2004. (g) Kumar, A.; Kalra, B.; Dekhterman, A.; Gross, R. A. Macromolecules 2000, 33, 6303. (h) Kikuchi, H.; Uyama, H.; Kobayashi, S. Macromolecules 2000, 33, 8971. (i) Namekawa, S.; Uyama, H.; Kobayashi, S. Macromol. Chem. Phys. 2001, 202, 801. (j) Uyama, H.; Inada, K.; Kobayashi, S. Macromol. Biosci. 2001, 1, 40. (k)
Protease-Catalyzed Polymerization Tsujimoto, T.; Uyama, H.; Kobayashi, S. Biomacromolecules 2001, 2, 29. (6) Rozzell, J. D., Wagner, F., Eds. Biocatalytic Production of Amino Acids and DeriVatiVes; Hanser Publishers: Muenchen, 1992. (7) (a) Sluyterman, L. A. E.; Wijdenes, J. Biochim. Biophys. Acta 1972, 289, 194. (b) Anderson, G.; Luisi, P. L. HelV. Chim. Acta 1979, 62, 488. (c) Jost, R.; Brambilla, E.; Monti, J. C.; Luisi, P. L. HelV. Chim. Acta 1980, 63, 375. (8) Matsumura, S.; Tsushima, Y.; Otozawa, N.; Murakami, S.; Toshima, K.; Swift, G. Macromol. Rapid Commun. 1999, 20, 7.
Biomacromolecules, Vol. 3, No. 2, 2002 323 (9) Aso, K.; Uemura, T.; Shiokawa, Y. Agric. Biol. Chem. 1988, 52, 2443. (10) Uemura, T.; Fujimori, M.; Lee, H.-H.; Ikeda, S.; Aso, K. Agric. Biol. Chem. 1990, 57, 2277. (11) Sanda, F.; Fujiyama, T.; Endo, T. J. Polym. Sci., Polym. Chem. Ed. 2001, 39, 732. (12) Gmeiner, P.; Feldman, P. L.; Chu-Moyer, M. Y.; Rapoport, H. J. Org. Chem. 1990, 55, 3068.
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