ARTICLE pubs.acs.org/Langmuir
Influence of the Charge Relay Effect on the Silanol Condensation Reaction as a Model for Silica Biomineralization Tatsuya Kuno,†,‡ Takayuki Nonoyama,†,‡ Kiyoshi Hirao,†,‡ and Katsuya Kato*,‡ †
Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi, 466-8555 Japan ‡ Advanced Manufacturing Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimo-Shidami, Moriyama-ku, Nagoya, Aichi 463-8560 Japan
bS Supporting Information ABSTRACT: The catalytic effect of various sequential peptides for silica biomineralization has been studied. In peptide sequence design, lysine (K) and histidine (H) were selected as the standard amino acids and aspartic acid (D) was selected to promote the charge relay effects, such as in the enzyme active site. Therefore, homopolypeptides (K10 and H10), block polypeptides (K5D5 and H5D5), and alternate polypeptides [(KD)5 and (HD)5] were designed, and the dehydration reaction ability of trimethylethoxysilane was investigated as a quantitative model of silica mineralization. The catalytic activity per basic residue of alternate polypeptide was the highest because of the charge relay effects at the surface of the peptide. In silica mineralization using tetraethoxysilane, spherical silica particles were obtained, and their size is related to the catalytic activities of the peptides in the model systems. From these results, the effect of the functional group combination by the peptide sequence design enables the control of the efficiency of mineralization and preparation of specific inorganic materials.
1. INTRODUCTION In nature, biominerals, such as bones, shells, and teeth, have good mechanical properties and are composed of organic inorganic composites controlled at the nanometer scale. Moreover, these specific structural materials are formed under ambient conditions.1 4 Thus, biomineralization to form biominerals in living organisms is a low-energy process and has been recently studied as an environmentally friendly process. In biomineralization, the inorganic crystalline phase and morphology are controlled by an organic substance; therefore, it is expected to be able to create novel functional inorganic materials.5 12 It is well-known that functional groups of biomolecules (hydroxyl, carboxyl, amino groups, etc.) have catalytic activity for the biomineralization process.12 15 For example, carboxyl groups can mineralize calcium phosphate and calcium carbonate, and amino groups induce silica mineralization.5 7,10,16 Moreover, biomolecules, such as enzymes, antibodies, and proteins, show synergetic effects by a specific sequence of functional groups in nature. The enzyme active site is the best example in the synergy of functional group combinations.17 19 In biomineralization, the glycine proline hydroxyproline sequence of collagen is also considered an advantageous sequential unit of osteogenesis.20,21 This so-called “charge relay effect” by a combination of functional groups is favorable for forming biominerals in mineralization. From these concepts, the effect of the functional group combination of peptide on silica biomineralization has been r 2011 American Chemical Society
studied. Silica biomineralization is observed in marine organisms, such as diatoms, sponges, and radiolarian,22 24 and silica has been researched and evaluated as a tissue-engineering material.25 Therefore, the dehydration reaction of trimethylethoxysilane is demonstrated using simple sequential peptides as the model of silica biomineralization. Because trimethylethoxysilane has only one ethoxyl group, it is easy to quantify the dehydration reaction progress. In general, trimethylethoxysilane is hydrolyzed to trimethylsilanol in an aqueous environment, and then, two trimethylsilanols are dehydrated to hexamethyldisiloxane (Scheme 1a). In peptide sequence design, lysine (K) and histidine (H) were selected as the standard amino acids because the amino and imidazole groups have highly catalytic ability for silica mineralization.26,27 In addition, to promote the charge relay effect, aspartic acid (D) having a carboxyl group side chain was incorporated into the peptide sequence (Scheme 1b). From these concepts, homopolypeptides (K10 and H10), block polypeptides (K5D5 and H5D5), and alternate polypeptides [(KD)5 and (HD)5] were designed and synthesized by combinatorial solid-phase peptide synthesis. The process of trimethylethoxysilane dehydration reactions by these peptides were measured by gas chromatography (GC). The results of these model systems not only support the fundamental theory of charge relay effects in biomolecules, such Received: July 7, 2011 Revised: September 19, 2011 Published: September 22, 2011 13154
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Scheme 1a
a
(a) The reaction formula of trimethylethoxysilane. Trimethylethoxysilane is hydrolyzed to trimethylsilanol in the aqueous environment, and then, two trimethylsilanols are dehydrated to hexamethyldisiloxane. (b) The schematic image of the charge relay effect by functional group combination. The number of the charge relay sites on the surface of (c) block polypeptide and (d) alternate polypeptide.
Table 1. Peptide Sequences Synthesized by Combinatorial Solid-Phase Peptide Synthesisa
a
Lysine (K) and histidine (H) are selected as the standard amino acids because the amino and imidazole groups have highly catalytic ability of silica mineralization. In addition, to promote the charge relay effect, aspartic acid (D) having a carboxyl group at the side chain is incorporated in the peptide sequence.
as enzyme and collagen, but also are expected to be applicable to biomineralization for industrial inorganic synthesis.
2. EXPERIMENTAL SECTION 2.1. Materials. CLEAR amide resin and 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids were from the Peptide Institute, Osaka, Japan. 1,3-Diisopropylcarbodiimide was from the Sigma-Aldrich Co., St. Louis, MO. 1-Hydroxy-7-azabenzotriazole was from Watanabe Chemical Industries, Hiroshima, Japan. Dichloromethane (DCM), N,N-dimethylformamide (DMF), piperidine, pyridine, trifluoroacetic acid, thioanisole, ethylenedithiol, and diethylether were from Wako Pure Chemical Industries, Osaka, Japan. Acetic anhydride was from Kanto Chemical Co., Tokyo, Japan. Tetraethoxysilane (TEOS) and trimethylethoxysilane were from Shin-Etsu Chemical Company, Tokyo, Japan. 2.2. Peptide Synthesis. Six different kinds of 10-mer peptides, K10, H10, K5D5, H5D5, (KD)5, and (HD)5, were synthesized by combinatorial solid-phase peptide synthesis28 (Table 1). These peptides were prepared as follows: 500 mg of CLEAR amide resin was swollen in
5 mL of DCM for 30 min, and the resin were then rinsed 5 times with 5 mL of DMF. Prior to the condensation with amino acid, Fmoc groups of resin were removed under basic conditions, using 20 vol % piperidine DMF solutions. After the reactions, the piperidine was removed by rinsing with DMF. To create the bound formation of amino acids on resin (condensation), 0.69 mmol of Fmoc amino acid in 3 mL of DMF, 0.69 mmol of 1-hydroxy-7-azabenzotriazole in 1 mL of DMF, and 0.69 mmol of 1,3-diisopropylcarbodiimide in 1 mL of DMF were added to the resins. The mixture was stirred for 2 h to allow for the condensation of amino acids and resins, and then resins were rinsed with DMF. Prior to the next condensations, Fmoc groups were removed again, as described above. Deprotected amino groups at the terminal of the resin were condensed with Fmoc amino acid, as described above. These deprotection and condensation processes were repeated to reach objective sequences. After condensations of the 10-mer sequence, Fmoc groups of the amino acid terminal were removed, as described above. To compensate for the terminal polar group, N-terminal amino groups were acetylated by treatment with 33 vol % acetic anhydride in 6 mL of pyridine for 2 h. The resins were then rinsed with DMF and DCM. To remove the peptides from the resins and to remove the protection of the side chain, aqueous solutions containing 9.5 mL of trifluoroacetic acid (TFA), 0.85 mL of ethaneditiol, 0.5 mL of thioanisole, and 0.5 mL of water were added to the resin. These mixtures were slowly stirred for 1 h. A total of 100 mL of diethylether was added to TFA solutions, which were filtered to obtain the solid samples. The peptide synthesis was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) with α-cyano-4-hydroxycinnamic acid as a matrix (see Figure S1 of the Supporting Information).
2.3. Measurement of the Secondary Structure of Peptide. The circular dichroism (CD) spectrum of the peptide was measured on a Jasco J-820K spectropolarimeter to investigate the secondary structures. The CD spectra were recorded at room temperature in the region of 190 260 nm, with an integration number of 16. The CD measurements were carried out where the concentrations of peptides were 0.2 and 1 mM and pH of solution was 7, which is similar to the pH of the trimethylethoxysilane dehydration reaction system. In addition, peptides in 13.8 wt % trimethylethoxysilane containing aqueous solution were also measured.
2.4. Quantification of Trimethylethoxysilane Dehydration Reaction by GC. In peptide-containing aqueous environments, trimethylethoxysilane (substrate) is hydrolyzed to trimethylsilanol (intermediate product), and then, two trimethylsilanols are dehydrated to 13155
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Figure 1. Ratio of the substrate and products. The peptide concentration and reaction time are 1 mM and 30 min, respectively. hexamethyldisiloxane (final product) (Scheme 1a). Therefore, the progress of the hydration reaction was measured to quantify the amounts of trimethylethoxysilane, trimethylsilanol, and hexamethyldisiloxane by GC.29 A substrate of 80 μL of trimethylethoxysilane was added to 0.5 mL of peptide solutions with concentrations of 0.2, 1, and 5 mM, and these mixtures were stirred for 60, 30, and 10 min, respectively. After stirring, 1 mL of diethylether was added to each solution to extract the substrate and products and the diethylether and aqueous phases were separated. GC analysis was carried out on 2 μL of diethylether using a Shimazu GC-17A chromatographic system equipped with a flame ionization detector (FID) and HP-5MS (30 m 0.25 mm, 0.25 μm film), J&W Scientific, Folsom, CA. A high-purity helium carrier gas was used at a constant flow rate (1 mL/min) at 50 °C. The temperature of the injector and detector was 180 °C. The retention times of the substrate, trimethylethoxysilane, the intermediate product, trimethylsilanol, and the final product, hexamethyldisiloxane, were 2.8, 2.5, and 3.4 min, respectively. 2.5. Silica Mineralization by Peptides. To substantiate the above model system of silica mineralization, the silica precipitation was demonstrated using TEOS. A total of 1.5 mL of TEOS was mixed with 1 μmol of peptide in 1.5 mL of ethanol containing 10 vol % distilled water. The mixture was stirred for 2 weeks at room temperature. The resulting precipitate was rinsed with ethanol, and the precipitate lyophilized. The morphology of the silica particles was observed by field emission scanning electron microscopy (FESEM, S4300 Hitachi Co., Japan). A non-peptide system was also analyzed as a control. In this experiment, only three kinds of lysine-containing peptides [K10, K5D5, and (KD)5] were analyzed because the histidine-containing peptides were not dissolved in 10 vol % distilled water in ethanol.
3. RESULTS AND DISCUSSION 3.1. Catalytic Activity of Peptide for the Trimethylethoxysilane Dehydration Reaction. The CD spectra of the synthe-
sized peptides at pH 7 (see Figure S2 of the Supporting Information) show that all of the spectra indicated a negative maximum peak at 197 nm, and the secondary structures of the peptides, except for (HD)5, were assigned a random coil configuration. Secondary structural contents of (HD)5 were α/β/ random = 30:0:70% from wave separation of the CD spectrum (1 mM). Because the main secondary structure of (HD)5 was also random coil, we did not consider the influence of the secondary structure in this study. CD spectra of peptide solutions of high concentration (5 mM) cannot be measured because of the saturation of HT voltage. Therefore, the influence of the
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Figure 2. Catalytic activity of the peptide per basic amino acid, in which each amount of the final product in Figure 1 is divided by the number of basic amino acids of each peptide.
other secondary structures, such as α helix and β sheet, was nearly ignorable in this study. Figure 1 shows the abundance ratios of the substrate and products measured by GC and a non-peptide system measured as the control. The peptide concentration and reaction time were 1 mM and 30 min, respectively. Although the trimethylethoxysilane was hydrolyzed to trimethylsilanol efficiently in the control system, trimethylsilanol was not transformed into hexamethyldisiloxane. On the other hand, trimethylsilanol was dehydrated in all peptide-containing systems, although to different extents. Therefore, the catalytic activity of the synthesized peptides on the trimethylsilanol condensation was indicated. When lysine systems are compared to histidine systems, lysine-containing peptides had higher dehydration activity than histidine-containing peptides.26,27 To estimate the effect of the sequence of the peptide on the trimethylethoxysilane dehydration reaction, the final product data in Figure 1 are converted to the percentage per basic residue (Figure 2). In both lysine and histidine systems, alternate polypeptides [(KD)5 and (HD)5] showed the highest catalytic activity. Therefore, a charge relay effect between the basic functional group and the carboxyl group of the side chain of aspartic acid on the peptide surface is indicated (Scheme 1b). In particular, all basic amino acids are located adjacent to acidic amino acids in alternate polypeptide. On the other hand, in block polypeptide, only one basic amino acid is located adjacent to an acidic amino acid (panels c and d of Scheme 1). Therefore, it is easy for the charge relay effect to occur on the surface of the alternate polypeptide. To support the intramolecular charge relay effect by the amino acid combination, the dehydration reaction with the various peptide concentrations was determined. When the peptide concentration is high, the distance between peptide molecules in the solution is very small and the charge relay effect also occurs intermolecularly. Therefore, the catalytic activities between block polypeptide and alternate polypeptide did not show a big difference at high peptide concentrations. When the peptide concentration is low, the peptide molecules have almost no contact with each other and the charge relay effect is limited intramolecularly. Therefore, we speculate that the condensation activity remains, despite the low peptide concentration in the case of the alternate polypeptide system. The trimethylethoxysilane dehydration reaction by alternate polypeptide and block polypeptide was carried out at a low peptide concentration of 0.2 mM for 60 min and a high peptide concentration of 5 mM for 10 min. Figure 3 shows the relative amount of the final product 13156
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Figure 3. Dependence of the peptide concentration for the final product formation: (a) 5 mM for 10 min (high concentration) and (b) 0.2 mM for 60 min (low concentration).
Figure 4. Relative catalytic activity of the alternate polypeptides when block polypeptide catalytic activities were calculated as 100%.
Figure 5. FESEM images and average diameters of the spherical silica particles: (a) K10, (b) K5D5, (c) (KD)5, and (d) size distributions of silica particles.
per basic amino acid. In both the lysine and histidine systems, no significant difference was observed between the block polypeptide catalytic activity and the alternate polypeptide catalytic activity at the 5 mM peptide condition. On the other hand, at 0.2 mM peptide concentration, the activity of alternate polypeptide is clearly higher than the activity of block polypeptide. Figure 4 shows the relative catalytic activity of the alternate polypeptides when block polypeptide catalytic activities were recalculated
as 100%. Because the peptide concentration was low, the relative amount of final product per basic amino acid of the alternate polypeptide compared to block polypeptide was increased more than the case of the high peptide concentration. At low peptide concentrations, the contact frequency between peptide molecules was decreased and the influence of the intermolecular charge relay effect was decreased. Therefore, the intramolecular charge relay effect was particularly prominent for the alternate 13157
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Langmuir polypeptide system, and the amount of final product per basic functional group depended upon the number of functional group combinations. The relative activity of the histidine system was higher than the relative activity of the lysine system. Originally, the catalytic activity of H10 is low because trimethylsilanol dehydration is assisted by a collaboration of basic and acid functional groups (Scheme 1b). Therefore, the relative activity of (HD)5 was increased. 3.2. Silica Mineralized by Peptides. To substantiate the above model system of silica mineralization, silica precipitation was carried out using TEOS. Secondary structures of lysine-containing peptide in TEOS containing ethanol were assigned α helix, and these results were different from the model system (data not shown). However, because all peptide conformations were α helix, the influence of the secondary structure could also be ignored. The silicates were prepared from the lysine-containing peptides as the catalyst under ambient conditions, and silica morphologies were observed by FESEM (panels a c of Figure 5). The FESEM images showed that spherical silica precipitates were obtained. In the nonpeptide system as the control, no silica precipitation occurred. Therefore, the importance of the peptide for the model system was also indicated. In addition, obtained silica had different sizes. We calculated the size distribution of silica particles (Figure 5d), and the average diameters of silica particles from K10, K5D5, and (KD)5 were 128, 430, and 649 nm, respectively. These diameters correlated well with the catalytic activity per basic functional group (Figure 2). From these results, the effect of the functional group combination by the peptide sequence design enables the control of the efficiency of mineralization and is expected to create specific inorganic materials under ambient conditions.
4. CONCLUSION The influence of functional group combination by the peptide sequence design for the dehydration reaction of trimethylethoxysilane is demonstrated as the model of silica mineralization. In both lysine and histidine systems, the relative activity of alternate polypeptide was higher than homo- and block polypeptides. In alternate polypeptides, many basic amino acids are located adjacent to acidic amino acids and the intramolecular charge relay effect occurs frequently. These results are similar to the charge relay effect of the enzyme active site or the repeated sequence of collagen in nature. To the best of our knowledge, this paper is the first report about the charge relay effect on biomimetic mineralization. In silica precipitation, the importance of the catalytic activity on the silica mineralization was indicated. We believe that the strategy of the peptide sequence design not only supports the fundamental theory of the charge relay effect in a biomolecule, such as enzyme and collagen, but also is expected to be applicable to the biomineralization of industrial inorganic synthesis. ’ ASSOCIATED CONTENT
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’ ACKNOWLEDGMENT This work was partly supported by a Grant-in-Aid for Scientific Research (C) 23560925 from the Japan Society for the Promotion of Science (JSPS). ’ REFERENCES (1) Mann, S.; Archibald, D. D.; Didymus, J. M.; Douglas, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286–1292. (2) Mann, S. Nature 1993, 365, 499–505. (3) Shen, X.; Belcher, A. M.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. J. Biol. Chem. 1997, 272, 32472–32481. (4) Stupp, S. I.; Braun, P. V. Science 1997, 277, 1242–1148. (5) Nonoyama, T.; Kinoshita, T.; Higuchi, M.; Nagata, K.; Tanaka, M.; Sato, K.; Kato, K. Langmuir 2011, 27 (11), 7077–7083. (6) Nonoyama, T.; Tanaka, M.; Kinoshita, T.; Nagata, F.; Sato, K.; Kato, K. Chem. Commun. 2010, 46, 6983–6985. (7) Nagata, F.; Miyajima, T.; Yokogawa, Y. Chem. Lett. 2003, 32, 784. (8) Kim, H. J.; Kim, U. J.; Kim, H. S.; Li, C.; Wada, M.; Leisk, G. G.; Kaplan, D. L. Bone 2008, 42, 1226. (9) Forbes, M. L.; Goodwin, P. A.; Cha, N. J. Chem. Mater. 2010, 22, 6524–6528. (10) Cao, B.; Mao, C. Langmuir 2007, 23, 10701–10705. (11) Chen, C.; Qi, J.; Zuckermann, R. N.; DeYoreo, J. J. J. Am. Chem. Soc. 2011, 133, 5214–5217. (12) Chen, C.; Rosi, N. L. Angew. Chem., Int. Ed. 2010, 49, 1924. (13) Huang, Z.; Yan, D.; Yang, M.; Liao, X.; Kang, Y.; Yin, G.; Yao, Y.; Hao, B. J. Colloid Interface Sci. 2008, 325, 356. (14) Masuda, Y.; Kinoshita, N.; Koumoto, K. Electrochim. Acta 2007, 53, 171–174. (15) Lee, M.; Ku, S. H.; Ryu, J.; Park, C. B. J. Mater. Chem. 2010, 20, 8848. (16) Sugarawa, A.; Ishii, T.; Kato, T. Angew. Chem., Int. Ed. 2003, 42, 5299–5303. (17) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428–1441. (18) Ishida, T.; Kato, S. J. Am. Chem. Soc. 2003, 125, 12035–12048. (19) Zhang, Z.; Huang, L.; Shulmeister, V. M.; Chi, Y. I.; Kim, K. K.; Hung, L. W.; Crofts, A. R.; Berry, E. A.; Kim, S. H. Nature 1998, 392, 677–684. (20) Bhattacharjee, A.; Bansal, M. IUBMB Life 2005, 57, 161–172. (21) Almora-Barrios, N.; Austen, K. F.; De Leeuw, N. H. Langmuir 2009, 25, 5018. (22) Hildebrand, M. Chem. Rev. 2008, 108, 4855–4874. (23) Martin-Jezequel, V.; Hildebrand, M; Brzezinski, M. J. Phys. (Paris) 2000, 63, 821. (24) Uriz, M. J. Can. J. Zool. 2006, 84, 322–356. (25) Orita, T.; Tomita, M.; Kato, K. Colloids Surf., B 2011, 84, 187–197. (26) Kroger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276, 26066. (27) Delak, M. K.; Sahai, N. Chem. Mater. 2005, 17, 3221–3227. (28) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161–214. (29) Kato, K.; Nakagaki, S.; Nishida, M.; Hirao, K. J. Ceram. Soc. Jpn. 2011, 119, 140–143.
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Supporting Information. MALDI TOF MS spectra of the synthesized peptides (Figure S1) and CD spectra of the synthesized peptide (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. 13158
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