Synthesis of Adhesive Peptides Similar to Those Found in Blue

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Synthesis of Adhesive Peptides Similar to Those Found in Blue Mussel (Mytilus edulis) Using Papain and Tyrosinase Keiji Numata* and Peter James Baker Enzyme Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: The blue mussel (Mytilus edulis) foot protein 5 (Mefp-5) is an adhesive protein that is mainly composed of glycine, L-lysine, and 3,4-dihydroxy-L-phenylalanine (DOPA). Thousands of adhesive pads have been analyzed in previous studies, whereby it has been found that adhesion is largely achieved by the redox-chemistry of DOPA, and that L-lysine (approximately 20 mol %) affects the formation of molecular networks. While DOPA and lysine are essential for biomimetic adhesive design, the synthesis of copolymers containing DOPA is limited, in terms of yield, by the multiple reaction steps required. Here, we synthesized adhesive peptides containing DOPA and L-lysine via two enzymatic reactions, namely, chemoenzymatic synthesis of copolypeptides of L-tyrosine and L-lysine by Papaya peptidase I (papain), as well as the enzymatic conversion from L-tyrosine to DOPA by tyrosinase. The synthesis was characterized in terms of yield, degree of polymerization, and composition of the polypeptide. In addition, the conversion of tyrosine to DOPA by tyrosinase was evaluated quantitatively by nuclear magnetic resonance and amino acid analysis. The adhesive properties of the resulting peptides, consisting of DOPA, L-lysine, and L-tyrosine, were evaluated at various pH levels with different protonation/deprotonation states. Our results show that deprotonated DOPA is required for adhesive function, and the deprotonated primary amine group of lysine induces molecular networks by varying the elastic moduli of the adhesives. In this study, we demonstrate the benefit of combining multiple enzymatic reactions, including chemoenzymatic polymerization, in obtaining new types of peptide-based materials.

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groups.3,5 From these reports it is suggested that DOPA and lysine are essential for biomimetic adhesive design; however, the application of these adhesive copolymers is limited, in terms of yield, because their synthesis requires multiple reaction steps. Other studies have shown that chemoenzymatic synthesis is one possible solution for the efficient synthesis of polypeptides, with respect to yield and reaction time.11−16 The synthesis is either a thermodynamically- or a kinetically controlled process. The latter reaction is more selective in that the enzyme needs to react with an ester compound to form an acyl-enzyme intermediate. In competition with water molecules to proceed hydrolysis, it will react with an amino acid-derived nucleophile and form a new peptide bond.11,17 There are several advantages of kinetically controlled chemoenzymatic synthesis over other synthetic methods, such as solid-phase polypeptide synthesis, ring-opening polymerization of the α-amino acid N-carboxyanhydride, and recombinant DNA methods. The enzymatic synthesis produces a relatively high yield without the need for multiple purifications, has a short reaction time, and requires mild reaction conditions. However, the disadvantage of this method is that there is less control over the molecular weight and amino acid sequence of the polypeptide produced. The

INTRODUCTION Adhesive substances are essential materials in various commercial sectors such as the automobile and construction industries. In particular, biologically based and biodegradable adhesives, which can be used in different materials, are required for the recycling of various component parts of cars and houses. One of the candidate adhesive materials is a protein-mimic adhesive, which has recently been highlighted as an attractive biologically based and highly functional adhesive. The blue mussel (Mytilus edulis) foot protein 5 (Mefp-5) is an adhesive protein that can be found on the surface of the mussel adhesive plaque, and is mainly composed of glycine, L-lysine, and 3,4dihydroxy-L-phenylalanine (DOPA).1,2 According to previous studies, which have analyzed thousands of adhesive pads, adhesion is mainly achieved by the redox-chemistry of DOPA.3,4 Artificially designed materials containing DOPA have also been developed and have demonstrated that adhesive function was directly correlated to DOPA content.5 Without lysine, linear and branched DOPA and polyethylene glycol copolymers were shown to have adhesive functions.6−8 The effects of the sequence of mussel proteins have also been investigated using random copolypeptides containing DOPA,9,10 suggesting that DOPA is the main factor underlying adhesive function. In addition to DOPA, Mefp-5 is composed of L-lysine (approximately 20 mol %), which is necessary to form a network of structures between catechol and amine © 2014 American Chemical Society

Received: June 19, 2014 Revised: July 15, 2014 Published: July 16, 2014 3206

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Figure 1. Reaction schemes for the enzymatic synthesis of poly(L-tyrosine-r-3,4-dihydroxy-L-phenylalanine-r-L-lysine) [P(Tyr-DOPA-Lys)], via poly(L-tyrosine-r-L-lysine) [P(Tyr-Lys)], from L-tyrosine ethyl ester (Tyr-Et) and L-lysine ethyl ester (Lys-Et). The images show the reaction intermediate, (a) P(75%Tyr-25%Lys), and products, (b) P(50%Tyr-25%Lys-25%DOPA) and (c) P(45%DOPA-30%Tyr-25%Lys). (pH 9.5) and was prepared in an EYELA ChemiStation (Tokyo Rikakikai Co., Tokyo, Japan) at 40 °C. The activated papain (7.0 mg/ mL) was added to the reaction. For time-course experiments, 100 μL of the reaction mixture was taken after 0.5, 1, 3, 6, 12, and 24 h. Subsequently, the samples were characterized by nuclear magnetic resonance (1H NMR) spectroscopy and amino acid analysis. Following the reaction, the reaction mixture was centrifuged at 15000g and the resultant pellet was washed twice with cold Milli-Q (4 °C). For the characterization of P(Tyr) and P(Tyr-Lys), the washed pellet was lyophilized and directly used for the next reaction, that is, the tyrosinase catalyzed reaction. Enzymatic Conversion to Poly(L-tyrosine-r-3,4-dihydroxy-Lphenylalanine-r-L-lysine). To convert tyrosine to 3,4-dihydroxy-Lphenylalanine (DOPA), 10 mg of the synthesized polypeptides, P(Tyr) and P(Tyr-Lys), were dissolved in a modified phosphate buffer (20 mM boric acid, 150 mM NaCl, 0.1 M ascorbic acid, and 0.1 M phosphate buffer, pH 7). Tyrosinase from mushroom (final concentration: 1300 U/mL) was used as the enzymatic catalyst. The reaction was performed at 25 °C for 0.5−12 h, as described previously.18,19 The reaction mixture was washed twice with cold Milli-Q (4 °C), to remove the enzyme, and was then lyophilized to remove any traces of water. The converted peptide, poly(L-tyrosine-r3,4-dihydroxy-L-phenylalanine-r-L-lysine) [P(Tyr-DOPA-Lys)], was then analyzed by 1H NMR and amino acid analysis and was subsequently tested for adhesive properties. Characterization of the Polypeptide Products. NMR spectra of the polypeptide products were obtained, using a Varian system 500 NMR spectrometer (500 MHz), with VnmrJ software (Agilent Technologies, Santa Clara, CA). Dimethyl sulfoxide-d6 (DMSO-d6) was used to prepare the samples, in glass tubes (5 mm), at concentrations of 5 mg/mL. The samples were analyzed at 25 °C. Tetramethylsilane (ppm: 0.0) was used as the internal standard for the DMSO-d6 samples. Amino acid composition analysis was performed using the ninhydrin method. The hydrolyzed amino acids were characterized using High-Speed Amino Acid Analyzers, L-8900 and L8500A (Hitachi-HighTech, Tokyo, Japan). In addition to the natural amino acids, DOPA and 2,4,5-trihydroxyphenylalanine (6-hydroxyDOPA, TOPA; Sigma-Aldrich) were used to calibrate the analyzer, and accordingly, the peaks were assigned as DOPA and TOPA (see Figure S3). Conjugation with Fe3+ Ion. Catechol functional groups are reported to conjugate with Fe3+ ions, resulting in a change of the solidstate and color of copolypeptides.7 Accordingly, to confirm the presence of a functional catechol group in P(Tyr-DOPA-Lys), the peptide was treated with a solution containing Fe3+. The peptides were mixed with a FeCl3 solution at pH of 6, 8, 10, and 12 in a DOPA/Fe molar ratio mixture of 3:1. The final concentration of the peptide was

yields resulting from the synthesis of polypeptides containing DOPA by solid-phase polypeptide synthesis, or by ring-opening polymerization of the α-amino acid N-carboxyanhydride, were not adequate to be used for the production of bulk material. Furthermore, recombinant DNA methods require post-translational modification in order to synthesize peptides containing DOPA. Previously, our studies on the chemoenzymatic syntheses of peptides have demonstrated an enhanced molecular weight and yield of L-alanine homopolypeptides.12 Additionally, we have reported the synthesis of amphiphilic diblock copolypeptides of 13 L-alanine and L-lysine. Here, we synthesized adhesive peptides containing DOPA and L-lysine via two enzymatic reactions, namely, chemoenzymatic polymerization of L-tyrosine and Llysine by Papaya peptidase I (papain), as well as the enzymatic conversion of tyrosine to DOPA by tyrosinase (Figure 1). The synthesis was characterized by the yield, degree of polymerization, and the composition of the polypeptide. Furthermore, the conversion of tyrosine to DOPA by tyrosinase was evaluated quantitatively by nuclear magnetic resonance (1H NMR) spectroscopy and amino acid analysis. The peptides produced, consisting DOPA, L-lysine, and L-tyrosine, were evaluated for adhesive function in terms of adhesion strength and the Young’s modulus. In our present study, we examine the potential benefit of combining multiple enzymatic reactions, including chemoenzymatic polymerization, on the efficient production of new types of peptide-based materials.

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EXPERIMENTAL SECTION

Materials. L-Tyrosine ethyl ester (Tyr-Et) hydrochloride, L-lysine ethyl ester (Lys-Et) hydrochloride, L-cysteine, and tyrosinase from mushroom were purchased from Sigma-Aldrich (St. Louis, MO). Papaya peptidase I (papain) was purchased from Wako Pure Chemicals (Osaka, Japan). Papain was activated and purified as follows: the enzyme was suspended in Milli-Q water, followed by centrifugation at 12000g for 30 min. The precipitate was then separated from the enzyme, frozen, and lyophilized for 24 h. Chemoenzymatic Synthesis of Copolypeptides. Chemoenzymatic syntheses of poly(L-tyrosine) [P(Tyr)] and poly(Ltyrosine-r-L-lysine) [P(Tyr-Lys)] were performed using the activated papain as a catalyst to monitor degree of polymerization (DP), number-average molecular weight (Mn), and composition for 24 h, based on our previous reaction conditions.13,16 Briefly, a 0.6 M solution of an amino acid ethyl ester mixture (Tyr-Et/Lys-Et ratio of 1:0, 5:1, 1:1, and 1:5) was made up in a 1.0 M borate buffer solution 3207

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Figure 2. Results for chemoenzymatic synthesis of poly(L-tyrosine-r-L-lysine) P(Tyr-Lys) and poly(L-tyrosine) P(Tyr) by Papaya peptidase I (papain) as a catalyst. The products were characterized as a function of the reaction time, h, as follows: (a) the degree of polymerization; (b) yield (%); (c) tyrosine content (mol %), as determined by 1H NMR (solvent: DMSO-d6); and (d) tyrosine content (mol %), as determined by amino acid analysis. The different monomer ratios used are given as follows: 0.6 M tyrosine ethyl ester (Tyr-Et), black square; 0.5 M Tyr + 0.1 M lysine ethyl ester (Lys-Et), striped square; 0.3 M Tyr-Et + 0.3 M Lys-Et, gray square; and 0.1 M Tyr-Et and 0.5 M Lys-Et, white square. In (c), 0.6 M Tyr-Et was overlapped with 0.5 M Tyr + 0.1 M Lys-Et. *Significant differences between groups at p < 0.05. 10 wt %. The color and solid-state of the peptides, with and without a catechol functional group at different pH levels were characterized. Adhesion Test. The different P(Tyr-DOPA-Lys) peptide solutions (10 wt %), consisting of variable DOPA content and made up with different pH levels of 6, 10, and 12, were applied in equal volumes between two freshly cleaved sheets of mica, which were subsequently pasted together. After 24 h, adhesive shear-strength of the pasted sample was characterized by a mechanical testing apparatus (EZ-Test, Shimadzu, Kyoto, Japan), as shown in Figure S6. Based on the resultant stress−strain curves, the adhesive shear-strength and the Young’s moduli of the adhesive peptides were obtained. Statistical Analysis. The significance of differences in studies of synthesis and mechanical properties of the peptides were determined by unpaired t-tests with a two-tailed distribution. Differences were considered statistically significant at p < 0.05.

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carboxyanhydrides, but was higher than that by solid-phase synthesis.20 The tyrosine content, determined by 1H NMR and amino acid analysis, was variable, especially when a monomer content of 0.5 M Tyr + 0.1 M Lys was used (Figure 2c,d). This is because the high molecular weight P(Tyr) and P(Tyr-Lys) polypeptides were insoluble in the DMSO-d6 used for 1H NMR measurement, implying that the tyrosine content from 1H NMR was based on the lower molecular weight fractions, whereas that from the amino acid analyses represented the overall amino acid compositions. In Figure 2c, 0.5 M Tyr + 0.1 M Lys showed quick conversion, while 0.1 M Tyr + 0.5 M Lys demonstrated almost no conversion of Tyr-Et. This is because hydrophilic components were removed during the purification process and 0.1 M Tyr-Et were not enough to be copolymerized as hydrophobic peptides. Also, due to the papain’s substrate specificity, 0.5 M Tyr was preferentially polymerized rather than 0.1 M Lys, resulting with the Tyr content in Figure 2c almost 100 mol % by using 0.5 M Tyr + 0.1 M Lys. To mimic mussel-derived adhesive peptides, a synthesized adhesive peptide should contain approximately 20 mol % lysine and 20 mol % DOPA.5 Therefore, we carried out subsequent synthetic reactions using the following experimental conditions: 0.3 M Tyr + 0.3 M Lys for 12 h, providing an average DP of 8, a yield of 42 wt %; and a composition of 75 mol % tyrosine and 25 mol % lysine, P(75%Tyr-25%Lys). P(Tyr-Lys) and P(Tyr) were treated with tyrosinase from mushroom (final concentration: 1300 U/mL) in a modified phosphate buffer (20 mM boric acid, 150 mM NaCl, 0.1 M ascorbic acid, 0.1 M phosphate buffer, pH 7.0) at 25 °C to convert tyrosine to DOPA. Tyrosinase has been reported to hydroxylate tyrosine to DOPA and then to 2,4,5-trihydroxyphenylalanine (TOPA) and dopaquinone (see the chemical structures of TOPA and dopaquinone in Figure S3).21 To

RESULTS AND DISCUSSION

Poly(L-tyrosine-r-L-lysine) [P(Tyr-Lys)] was chemoenzymatically synthesized from L-tyrosine ethyl ester (Tyr-Et) and Llysine ethyl ester (Lys-Et) using activated papain as a catalyst at 40 °C in 1 M borate buffer (pH 9.5). To obtain P(Tyr-Lys) at different monomeric compositions and poly(L-tyrosine) [P(Tyr)], four types of monomer ratios were used in the reaction media: 0.6 Tyr-Et; 0.5 M tyrosine (Tyr) + 0.1 M lysine (Lys); 0.3 M Tyr + 0.3 M Lys; and 0.1 M Tyr + 0.5 M Lys. The average degree of polymerization (DP, molecular weight), yield (%), and tyrosine content were characterized as shown in Figures 2 and S1. The DP and the tyrosine content (amino acid compositions) were determined by 1H NMR and amino acid analysis (Figure S2). The monomer feeding ratios significantly affected the DP, yield, and monomer content, with 0.5 M Tyr + 0.1 M Lys showing the highest DP (13.9) and 0.6 M Tyr demonstrating the highest yield (74.2%). The yield was similar to the yield by ring-opening polymerization of amino acid N3208

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Figure 3. 1H NMR spectra of poly(L-tyrosine-r-3,4-dihydroxy-L-phenylalanine-r-L-lysine) [P(Tyr -DOPA-Lys)] in DMSO-d6.

maximize the adhesive function of mussel-mimic peptide, the hydroxylation of DOPA to TOPA needs to be prevented due to catechol interactions of DOPA with various surfaces.7,22,23 The effect of the reaction time on the hydroxylation of L-tyrosine by tyrosinase was characterized by 1H NMR (Figure 3) and amino acid analysis (Figures 4 and S4). The reaction was performed

using P(75%Tyr-25%Lys) for 1 to 12 h. The reaction mixture was washed with cold Milli-Q twice to remove the enzyme and then was lyophilized to remove water. The DOPA compositions of the converted peptide, poly(L-tyrosine-r-3,4dihydroxy-L-phenylalanine-r-L-lysine) [P(Tyr-DOPA-Lys)], was approximately 20 mol % after the reaction for 1 h with tyrosinase and then increased to over 40 mol % at 6 h (Figure 4a). On the other hand, the content of tyrosine decreased with the reaction time from approximately 75 to 30 mol % (Figure 4a). The tyrosinase reaction for 6 h was sufficient to convert the tyrosine in the peptide to DOPA and there was no simultaneous conversion from DOPA to TOPA, that is, excess oxidation/hydroxylation, detected by amino acid analysis (Figure S4). A previous study has shown that the hydroxylation of monomeric tyrosine to DOPA and TOPA occurs within 1 h.21 However, according to our amino acid analysis, the hydroxylation of tyrosine-composing peptide, for over 1 h, yielded only DOPA without TOPA. To clarify the effect of various amino acid compositions on the conversion of tyrosine, P(Tyr), P(93%Tyr-7%Lys), and P(75%Tyr-25%Lys) were treated with tyrosinase for 3 h and the conversions from tyrosine to DOPA were evaluated. The resultant copolypeptides were P(74%Tyr-26%DOPA), P(63%Tyr-30%DOPA-7% Lys), and P(50%Tyr-25%DOPA-25%Lys) (Figure 4b), indicating that the conversion efficiency to DOPA was not influenced by the content of lysine. Our data confirmed the presence of functional catechol groups in synthesized copolypeptides, when the copolypeptide, P(50%Tyr-25%DOPA-25%Lys) was treated with iron(III) oxide (Fe/DOPA = 1:3 molar ratio, 10 mg/mL of the peptide) at pH of 6, 8, 10, and 12 (Figure 5), as described previously.7 At pH 6, the peptide solution containing Fe3+ was in a liquid state with a yellow color (Figure 5a). The peptide solution became viscous at pH 8 (Figure 5b), and a solid state at pH 10 (Figure 5c) due to more interactions between Fe3+ and the functional deprotonated catechol groups. At pH 12 it was possible that

Figure 4. (a) Conversion of the tyrosine (Tyr), ■, of poly(L-tyrosiner-L-lysine) [P(75%Tyr-25%Lys)] to 3,4-dihydroxy-L-phenylalanine (DOPA), □, by tyrosinase as a function of reaction time (h). (b) The amino acid compositions of poly(L-tyrosine) [P(Tyr)], P(93% Tyr-7%Lys), and P(75%Tyr-25%Lys) 3 h after the catalytic reaction with tyrosinase. 3209

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Figure 5. Complex formation between 3,4-dihydroxy-L-phenylalanine (DOPA) and Fe3+ at different pH values. The physical state, color, and estimated chemical interactions of poly(L-tyrosine-r-3,4-dihydroxy-Lphenylalanine-r-L-lysine) [P(50%Tyr-25%DOPA-25%Lys)] with Fe3+ at pH values of (a) 6, (b) 8, (c) 10, and (d) 12 were determined.

Figure 6. (a) Chemical interactions, (b) physical state and color, and (c) adhesive strengths, measured at different pH levels and using super glue as a control, of poly(L-tyrosine-r-3,4-dihydroxy-L-phenylalanine-rL-lysine) [P(Tyr-DOPA-Lys)] and poly(L-tyrosine-r-L-lysine) [P(TyrLys)]. Data represent means ± standard deviation from three independent experiments. *Significant differences between groups at p < 0.05.

there could have been too much hydroxyl ion to form ionic interactions (Figure 5d). The changes of the peptide solution, in terms of color and solid state, indicated that the functional catechol groups were synthesized via the enzymatic conversion from tyrosine. To evaluate the adhesive functions of P(Tyr-DOPA-Lys) peptides, made up with different DOPA content, we placed equal volumes of the peptide solutions, prepared at different pH levels of 6, 10, and 12, between sheets of mica (Figures S5 and S6). The results show that the adhesive function of P(50% Tyr-25%DOPA-25%Lys) increased significantly with increasing pH, with no adhesiveness detected under acidic conditions, but notably higher adhesive function at pH 12 (Figure 6). The polypeptide P(50%Tyr-25%DOPA-25%Lys) was insoluble in water at pH 6, but was dissolved at pH 10 as a white solution. At pH 12, the solution of P(50%Tyr-25%DOPA-25%Lys) was brown in color and had an adhesive strength of approximately 0.95 MPa, which is higher than that of Super Glue (Figure 6). In addition, to clarify the effects of the content of DOPA, the adhesion strength of P(Tyr) and P(45%DOPA-30%Tyr-25% Lys) were characterized at pH 12 (Figure 6). The resultant adhesive strengths show that DOPA was required for adhesive function, however, 45 mol % DOPA appeared to be too high a content to yield the best adhesive strength against mica substrates. We suggest that this may be because the molar balance between DOPA and lysine is one of the most important factors in the adhesive properties of mussel-like adhesive peptides. Also, the adhesion strength of the peptide containing lysine and DOPA is dependent on pH, because deprotonated

states of functional groups under alkaline conditions are needed to realize adhesion function. In nature, the seawater exhibits basic pH, suggesting the formation of network between DOPA and lysine is preferred in seawater and the marine mussel also may use this chemistry to adhere. In nature, mussel-derived adhesion protein, Mefp-5, is composed mainly of 20.7 mol % glycine, 19.5 mol % L-lysine, and 25.5 mol % DOPA, that is, the molar ratio between DOPA and lysine is approximately 5:4.5 Furthermore, the effect of the molar ratio of DOPA to lysine on adhesion strength has been studied; the adhesion strength against steel has been reported to be highest when the molar ratio of DOPA/lysine was 1:4.20 Based on these studies, excess DOPA does not appear to contribute significantly to adhesive strength, while the presence of deprotonated lysine (deprotonated at the primary amine group) positively affected the adhesion resulting from DOPA interactions. Conversely, it has also been reported that peptides containing DOPA, without lysine also have adhesive properties, whereas lysine functions to form a network of structures between the amine group of lysine and the catechol group of DOPA.3,5 To clarify the influence of lysine on adhesive function, we evaluated the Young’s modulus (the elastic modulus) of the adhesive peptides. The network structure, in particular, the molecular weight between cross-linking points, affects the 3210

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elastic modulus.24,25 Cross-linked polymeric materials with a higher molecular weight between cross-linking points show lower elastic modulus. The adhesive peptides containing 25% and 45% DOPA at pH 12 demonstrated similar elastic moduli, which were around 6.2 MPa (Figure 7a). Without DOPA, the

L-tyrosine,

at various pH, with different protonation/deprotonation states, we have proposed a mechanism whereby deprotonated DOPA can interact with the surface materials, thus functioning as an adhesive molecule, while on the other hand, the primary amine group of lysine induces molecular networks under deprotonated conditions. In this study, we conclude that the combination of multiple enzymatic reactions, including chemoenzymatic polymerization, increases the efficiency of synthesizing new types of peptide-based materials such as Mefp-5-mimicking adhesive peptides.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Reaction solutions of chemoenzymatic synthesis; Figure S2: 1H NMR spectra; Figure S3: Chemical structures of TOPA and DOPA-quinone; Figure S4: Typical amino acid analysis data by HPLC; Figure S5: Physical states and color of the peptides in water at pH 6, 10, and 12; Figure S6: Schematic diagram of the mechanical test. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +81-48-467-9525. Fax: +81-48-462-4664. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank RIKEN Biomass Engineering Program (K.N.) and Sweden-Japan Foundation’s Gadeliusstipendium (J.F.) for the financial support of this work.

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Figure 7. Determination of Young’s moduli, MPa, of poly(L-tyrosine-r3,4-dihydroxy-L-phenylalanine-r-L-lysine) [P(Tyr-DOPA-Lys)] and poly(L-tyrosine-r-L-lysine) [P(Tyr-Lys)] at pH 10 and 12 (a). The suggested chemical interactions between side-chain groups with and without lysine are illustrated (b). *Significant differences between groups at p < 0.05.

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peptides did not have adhesive function or elastic moduli. On the other hand, the peptide with 25% DOPA and 25% lysine at pH 10 showed lower Young’s modulus, suggesting that the alkaline (deprotonated) condition induced more molecular network formation via the deprotonation of the primary amine group of lysine (Figure 7b). The elastic modulus increased with an increase in the lysine content, indicating that increased lysine formation of more molecular networks, thus, resulting in a higher modulus. On the other hand, free radical was reported to induce the formation of cross-link between catechol groups without lysine (Figure 7b).3 Based on these results, DOPA is an essential component adhesive function, that is, molecular interactions between peptides and surfaces, whereas lysine is the component to form the molecular networks of the peptide.

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CONCLUSION We successfully synthesized adhesive peptides containing DOPA and L-lysine via two enzymatic reactions, namely, chemoenzymatic synthesis of copolypeptides of L-tyrosine and L-lysine by papain as well as the enzymatic conversion of Ltyrosine to DOPA by tyrosinase. Based on the adhesion tests using the synthesized peptides, consisting DOPA, L-lysine, and 3211

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(18) Garciacarmona, F.; Cabanes, J.; Garciacanovas, F. Biochim. Biophys. Acta 1987, 914, 198−204. (19) Duckwort, Hw; Coleman, J. E. J. Biol. Chem. 1970, 245, 1613− &. (20) Wang, J.; Liu, C.; Lu, X.; Yin, M. Biomaterials 2007, 28, 3456− 3468. (21) Burzio, L. A.; Waite, J. H. Anal. Biochem. 2002, 306, 108−114. (22) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426−430. (23) White, J. D.; Wilker, J. J. Macromolecules 2011, 44, 5085−5088. (24) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (25) Numata, K.; Katashima, T.; Sakai, T. Biomacromolecules 2011, 12, 2137−2144.

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