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Chapter 7
Protease-Catalyzed Polymerization of Tripeptide Esters Containing Unnatural Amino Acids: α,α-Disubstituted and N-Alkylated Amino Acids Kousuke Tsuchiya* and Keiji Numata* Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan *E-mail:
[email protected]. *E-mail:
[email protected].
Chemoenzymatic polymerization utilizing the aminolysis reaction catalyzed by proteases in aqueous solution is a useful method to synthesize various types of polypeptides in an environmentally benign manner. Various ester derivatives of amino acids can be polymerized using proteases. However, the polymerization of unnatural amino acid esters generally results in no polypeptide formation because proteases exhibit high substrate specificity for certain amino acid residues. To overcome the poor affinity of unnatural amino acids for proteases, we prepared tripeptide esters containing unnatural amino acids, such as 2-aminoisobutyric acid (Aib) and sarcosine (Sar). The tripeptide esters can be polymerized by papain to obtain polypeptides with a periodic sequence containing these unnatural amino acids. The incorporation of unnatural amino acids in the periodic sequences of polypeptides caused drastic changes in their secondary structure and physical properties.
© 2018 American Chemical Society
Introduction Depending on their amino acid sequences, polypeptides are unique biopolymers with diverse functional and physical properties. Biological or chemical synthetic methods have been developed to synthesize artificial polypeptides and to achieve precise control of their amino acid sequences. A biological synthetic method uses living microbes as host-reaction fields via a genetic transformation technique. Solid-phase polypeptide synthesis is generally used to synthesize polypeptides by a chemical synthetic method. These methods offer sophisticated sequential control in target polypeptides but are costly and produce low-yields. Another synthetic approach, chemoenzymatic polymerization of amino acid ester derivatives using proteases has been developed to synthesize polypeptides in an environmentally benign manner (1, 2). This technique is applicable for the syntheses of various types of polypeptides, including homopolypeptides (3, 4), random/block copolypeptides (5–7), and polypeptides with special structures, such as star and telechelic shapes (8, 9). In addition to DNA-coding amino acids, unnatural amino acids can be incorporated into polypeptide sequences and confer novel functionalities and/or physical properties. A representative example in nature is a variety of antibiotic peptides containing N-alkylamino acid residues, such as sarcosine (N-methylglycine, Sar). The existence of N-alkylamino acids allows the antibiotic peptides to resist proteolytic degradation (10). This feature indicates that unnatural amino acids possess a poor affinity for proteases. Recently, it was demonstrated that the papain-catalyzed copolymerization of amino acid esters with ω-aminoalkanoates (nylon monomers) as an unnatural amino acid unit provided polypeptides containing nylon units in their sequence (Figure 1a) (11, 12). The obtained nylon-containing polypeptides exhibited a melting point. This result indicates that the introduction of nylon units enables polypeptides to be subjected to thermal processing greater than the melting temperature; polypeptides usually decompose before melting. However, the papain-catalyzed copolymerization of nylon monomers is impeded by the extremely poor reactivity of nylon, resulting in low-nylon introduction rates of up to 15%, even with a high-feed ratio of nylon to amino acids. Papain is an extracellular cysteine protease and shows a relatively broad substrate specificity. Therefore, we utilized papain for chemoenzymatic polymerization of various amino acid esters (1, 2). The substrate specificity of papain relies on specific interactions between the substrate amino acids and subsites in a substrate pocket around its catalytic center. Each subsite favors specific amino acid residues, and recognition is regulated by the combination of subsite interactions with the amino acid residues. Early fundamental research on substrate specificity using polyalanines revealed that the subsite next to the catalytic center in papain strictly recognizes l-amino acid residues in polypeptides; whereas the subsites far from the catalytic center are tolerant of d-amino acid residues (13). This feature suggests that an appropriate sequential design of target polypeptides enables polymerization of unfavorable substrates.
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Figure 1. (a) Chemoenzymatic copolymerization of Leu and nylon ethyl esters using papain and (b) general strategy for introduction of unnatural amino acids into polypeptide backbone using tripeptide esters. In contrast to other solution-based polypeptide syntheses, such as the ring-opening polymerization of N-carboxy amino acid anhydrides (NCAs), protease-catalyzed polymerization can adopt oligopeptide esters as a monomer. Various periodic sequences, including alternating polypeptides, are attainable by polymerization of oligopeptide monomers. Dipeptide esters, such as GlyAla and LysLeu ethyl esters, were successfully polymerized using proteases to afford the corresponding polypeptides with alternating sequences (14, 15). Recently, we also performed chemoenzymatic copolymerization of ValProGly tripeptide and ValGly dipeptide ethyl esters in the presence of papain. The resulting polypeptide possessed a sequence similar to the periodic motif in elastin (i.e., ValProGlyValGly) (16). The resulting polypeptide showed an elastin-mimetic, reversible structural transition dependent on the temperature. This method of synthesizing specific periodic sequences can be applied to unnatural amino acids by sandwiching them between natural amino acids (Figure 1b). Modification with natural amino acids at both N- and C-terminals is expected to mitigate the poor affinity of papain according to the previous evaluation of the substrate specificity of subsites in the substrate pocket (13). In this context, two types of unnatural amino acids [namely, 2-aminoisobutyric acid (Aib) and Sar] were incorporated into tripeptide ester monomers (17). The chemoenzymatic polymerization of tripeptide esters using papain successfully afforded polypeptides that consist of periodic sequences containing unnatural amino acids. 97
General Procedure for Chemoenzymatic Polymerization The general procedure for the chemoenzymatic polymerization of amino acid esters is as follows. A solution of amino acid HCl salt in an aqueous buffer is placed in a glass tube equipped with a stir bar and stirred at 40°C until all monomers are dissolved. In most cases, the buffer solution concentration is 1 M and the pH is greater than 7, which activates the monomers. A solution of a protease in the aqueous buffer is then added to this solution in one portion. In the case of papain, the final concentration of papain is usually 50 mg/mL at optimal condition. The mixture is stirred at 800 rpm and 40°C for 2–24 h. As the polymerization proceeds, the precipitation of insoluble polypeptide occurs. After the mixture cools to room temperature, the precipitate is collected by centrifugation at 7000 rpm for 15 min at 4°C. The crude product is washed twice with deionized water and lyophilized to afford the polypeptide as a white solid. The obtained polypeptides are characterized by 1H NMR spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Dipeptide and tripeptide esters are used as well as a monomer for chemoenzymatic polymerization. An optimized monomer concentration is in a range of 0.1–2.0 M depending on the amino acid residues.
Synthesis of Polypeptides Containing Aib Chemoenzymatic polymerization of amino acid esters using papain has been intensively studied for the synthesis of various types of polypeptides. Hydrophobic amino acids, such as alanine and leucine, become water-insoluble polypeptides after papain-catalyzed polymerization, resulting in precipitation of the product during the reaction. The precipitated polypeptides can be easily isolated from papain by centrifugation of the reaction mixture. To investigate the ability to polymerize an unnatural amino acid, Aib, we attempted papain-catalyzed polymerization of an Aib ethyl ester in a phosphate buffer solution under various conditions (Figure 2). No precipitate appeared, even after a prolonged reaction time of 24 h, and only the starting material was obtained. A dipeptide ethyl ester consisting of Aib and Ala units, AibAla-OEt, was also used for polymerization with papain in a phosphate buffer solution. However, alanine modification at the C-terminal of Aib did not improve the reactivity, which resulted in no polymerization. This indicates that Aib has a poor affinity for papain and is less reactive than natural amino acid monomers. The copolymerization of AibAla-OEt with the Ala ethyl ester (1:1 molar ratio in feed) in the presence of papain afforded a polypeptide as a white precipitate in a very low yield (< 5%). The resulting polypeptide contained small amounts of the Aib unit (i.e., up to 5 mol%) revealing the low reactivity of AibAla-OEt. In contrast, the papain-catalyzed polymerization of the tripeptide ethyl ester containing Aib, AlaAibAla-OEt, provided a moderate yield of polypeptides of up to 30%. The incorporation of Aib units in the obtained polypeptides was confirmed by 1H NMR spectroscopy. The chemoenzymatic polymerization of AlaAibAla-OEt at different monomer concentrations was performed. The results are summarized in Figure 98
3. The polymer yield was maximized at a monomer concentration of 0.25 M and reduced with an increasing monomer concentration. The polypeptide was no longer obtained at a concentration of more than 0.5 M. The Aib content in the polypeptide, which was calculated from 1H NMR spectra, was almost comparable to the theoretical value (33 mol%) at the lower monomer feed concentration. However, the Aib content slightly decreased as the feed concentration increased due to the additional Ala insertion in poly(AlaAibAla) by transamidation, which was revealed by MALDI-TOFMS (17). The Ala insertion was suppressed at low-feed concentrations.
Figure 2. Chemoenzymatic polymerization of Aib-containing monomers in the presence of papain.
The MALDI-TOFMS of the products revealed that the tetramer (AlaAibAla)4 and pentamer (AlaAibAla)5 were mainly obtained with a series of small peaks derived from Ala-inserted poly(AlaAibAla). The conversion of the AlaAibAla-OEt monomer to poly(AlaAibAla) during chemoenzymatic polymerization was monitored by a time-course study, as shown in Figure 4. The polypeptide formation rapidly occurred within 30 min, and the conversion of the monomer was saturated at approximately 50%. The chemoenzymatic polymerization reaction was kinetically controlled by using moderately activated ester monomers. Therefore, the tandem aminolysis of amino acid esters catalyzed by an enzyme rapidly proceeds in the early stage of polymerization, whereas the aminolysis reaction competes with hydrolysis in the later stage. The polymerization behavior is similar to the chemoenzymatic polymerization of natural amino acids, revealing that the reaction is regulated by the alanine moiety in the tripeptide, which shows a high affinity for papain. 99
Figure 3. The effect of the monomer concentration on the yield for papain-catalyzed polymerization of AlaAibAla-OEt (square) and the Aib content in poly(AlaAibAla) (circle). (Reproduced with permission from reference (17). Copyright 2017 Royal Society of Chemistry.)
To investigate the effect of Aib units on the secondary structure of the polypeptide, IR and circular dichroism (CD) spectroscopic analyses were performed on the poly(AlaAibAla) prepared by chemoenzymatic polymerization of AlaAibAla-OEt. The IR and CD spectra are shown in Figure 5. In the IR spectra, a peak derived from the stretching vibration mode of the carbonyl group (1700–1600 cm−1) is defined as the amide I region. A peak shift in the amide I region reflects the specific environment of the amide bonds and depends on the secondary structures of the polypeptides (18, 19). Polyalanine (polyAla), which has the natural backbone structure of poly(AlaAibAla), showed a strong, sharp peak at 1630 cm−1, corresponding to a β-sheet structure. Poly(Ala-r-AibAla) containing only 5 mol% of the Aib unit exhibits a similar profile with a slight shoulder at 1660 cm−1, corresponding to an α-helical structure. In contrast, the IR spectrum of poly(AlaAibAla) showed a substantial peak shift to 1660 cm−1, indicating a structural transition from a β-sheet to α-helical conformation. This finding revealed that the periodic introduction of Aib units into the polypeptide backbone effectively induced a helical conformation. The Aib-containing triad, XaaXaaAib, in polypeptide sequences tends to assemble into a helical conformation because of the steric hinderance of the two methyl groups at the α-carbon (20–22). A 5 mol% introduction of Aib in poly(Ala-r-AibAla) was not adequate to induce the α-helix structure. 100
Figure 4. Time-course study of monomer conversion during chemoenzymatic polymerization of AlaAibAla-OEt (0.25 M) in the presence of papain at 40°C. (Reproduced with permission from reference (17). Copyright 2017 Royal Society of Chemistry.)
The secondary structure of poly(AlaAibAla) in a solution state was also investigated by CD spectroscopy in 2,2,2-trifluoroethanol. The polyAla sequence is most likely to adopt a β-strand/sheet structure, even in organic solvents that strongly induce helical conformations (23). The CD profile of polyAla showed a very weak negative peak at 218 nm and a positive peak at 193 nm, indicating the formation of a β-strand (sheet) structure. This β-strand propensity was unchanged with a small amount of Aib, as shown in the CD profile of poly(Ala-r-AibAla). On the other hand, poly(AlaAibAla) exhibited a drastic change in the CD profile compared to polyAla. A strong negative Cotton effect was observed with two negative peaks at 218 and 208 nm and a positive peak at 191 nm. This result revealed that poly(AlaAibAla) adopted an α-helix structure with a right-handed screw direction. 101
Figure 5. (a) IR spectra of Aib-containing polypeptides and (b) CD spectra of Aib-containing polypeptides in a 2,2,2-trifluoroethanol solution (100 mM). (Reproduced with permission from reference (17). Copyright 2017 Royal Society of Chemistry.)
Figure 6. Chemoenzymatic polymerization of Sar-containing tripeptide esters in the presence of papain.
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Synthesis of Polypeptides Containing Sar This tripeptide ester can be applied to other types of unnatural amino acids. Polysarcosine (polySar), also referred to as polypeptoide, is a promising polypeptide-like material for bioapplications due to its biocompatible, hydrophilic nature, which is similar to that of poly(ethylene glycol) (24, 25). We synthesized two types of tripeptide esters containing the Sar unit, namely, GlySarGly and AlaSarAla ethyl esters, as the monomers for chemoenzymatic polymerization. Similar to the Aib-containing monomers, Sar and SarGly dipeptide ethyl esters were inactive in papain-catalyzed polymerization; no polypeptide was obtained by chemoenzymatic polymerization of these monomers. In contrast, GlySarGly-OEt was converted to poly(GlySarGly) using papain in a phosphate buffer solution (Figure 6). Papain-catalyzed polymerization of AlaSarAla-OEt in a phosphate buffer solution also afforded the corresponding polypeptide, poly(AlaSarAla). In the case of the Sar-containing sequences, the resulting polypeptides were water-soluble; therefore, no precipitate was obtained after the chemoenzymatic polymerization. The resulting mixture was subjected to ultrafiltration for the removal of papain followed by dialysis to isolate the polypeptides. Extraction using organic solvents, such as chloroform, was also effective in the case of the isolation of poly(AlaSarAla). Polyalanine and polyglycine are water-insoluble because of hydrophobic aggregates that form via intermolecular hydrogen-bonding. The Sar units in the polypeptide backbone can break the hydrogen bonds by the methyl group on the N atom. Therefore, the introduction of Sar units into polyGly and polyAla caused significant changes in the solubility in water by breaking hydrogen bonds. The resulting polypeptides were totally soluble in water and other organic solvents, such as chloroform and acetonitrile. The formation of poly(GlySarGly) and poly(AlaSarAla) was confirmed by MALDI-TOFMS, as shown in Figure 7. The molecular weights of poly(GlySarGly) and poly(AlaSarAla) were in the range from 400 to 2000, and the maximum degree of polymerization was 10 (30 residues) and 4 (12 residues), respectively. In the case of papain-catalyzed polymerization of GlySarGly-OEt, only poly(GlySarGly) with a free carboxylic acid at the C-terminal was obtained. Because the resulting poly(GlySarGly) was highly water-soluble, the ester group at C-terminal was assumed to be easily hydrolyzed in the presence of papain during the reaction.
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Figure 7. MALDI-TOF mass spectra of (a) poly(GlySarGly) and (b) poly(AlaSarAla) synthesized by papain-catalyzed polymerization.
Conclusions Three types of tripeptide esters containing unnatural amino acids, namely, AlaAibAla-OEt, GlySarGly-OEt, and AlaSarAla-OEt, were rationally designed to mitigate the poor affinity of unnatural amino acids for papain. The papain-catalyzed polymerization of these tripeptide esters successfully provided polypeptides with periodic sequences containing unnatural amino acids. The introduction of Aib units into a polyalanine backbone induced a drastic structural transition from a β-sheet to an α-helix, whereas the introduction of Sar units changed the solubility of the polypeptides. The chemoenzymatic polymerization of tripeptide esters enhances the versatility of the substrates and can be applied to various types of unnatural amino acids. The incorporation of unnatural amino acids into polypeptides will open a way to finetune their material properties and confer novel functionalities.
Acknowledgments This work was financially supported by Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT), JSPS KAKENHI Grant Number JP17K18361, and JST ERATO Grant Number JPMJER1602. 104
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Tsuchiya, K.; Numata, K. Macromol. Biosci. 2017, 1700177. Numata, K. Polym. J. 2015, 47, 537–545. Ma, Y.; Sato, R.; Li, Z.; Numata, K. Macromol. Biosci. 2016, 16, 151–159. Baker, P. J.; Numata, K. Biomacromolecules 2012, 13, 947–951. Tsuchiya, K.; Numata, K. ACS Macro Lett. 2017, 6, 103–106. Numata, K.; Baker, P. J. Biomacromolecules 2014, 15, 3206–3212. Fagerland, J.; Finne-Wistrand, A.; Numata, K. Biomacromolecules 2014, 15, 735–743. Tsuchiya, K.; Numata, K. Macromol. Biosci. 2016, 16, 1001–1008. Ageitos, J. M.; Baker, P. J.; Sugahara, M.; Numata, K. Biomacromolecules 2013, 14, 3635–3642. Agrawal, S.; Adholeya, A.; Deshmukh, S. K. Front. Pharmacol. 2016, 7, 333. Yazawa, K.; Gimenez-Dejoz, J.; Masunaga, H.; Hikima, T.; Numata, K. Polym. Chem. 2017, 8, 4172–4176. Yazawa, K.; Numata, K. Polymers 2016, 8, 194. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157–162. Qin, X.; Xie, W.; Tian, S.; Cai, J.; Yuan, H.; Yu, Z.; Butterfoss, G. L.; Khuong, A. C.; Gross, R. A. Chem. Commun. 2013, 49, 4839–4841. Qin, X.; Khuong, A. C.; Yu, Z.; Du, W.; Decatur, J.; Gross, R. A. Chem. Commun. 2013, 49, 385–387. Gudeangadi, P. G.; Tsuchiya, K.; Sakai, T.; Numata, K. Polym. Chem. 2018, 9, 2336–2344. Tsuchiya, K.; Numata, K. Chem. Commun. 2017, 53, 7318–7321. Huang, W.; Krishnaji, S.; Tokareva, O. R.; Kaplan, D.; Cebe, P. Macromolecules 2014, 47, 8107–8114. Rabotyagova, O. S.; Cebe, P.; Kaplan, D. L. Macromol. Biosci. 2010, 10, 49–59. Demizu, Y.; Doi, M.; Sato, Y.; Tanaka, M.; Okuda, H.; Kurihara, M. Chem. Eur. J. 2011, 17, 11107–11109. Solà, J.; Helliwell, M.; Clayden, J. J. Am. Chem. Soc. 2010, 132, 4548–4549. Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 2299–2300. Shinchuk, L. M.; Sharma, D.; Blondelle, S. E.; Reixach, N.; Inouye, H.; Kirschner, D. A. Proteins 2005, 61, 579–589. Robert, L.; Corinna, F.; Arlett, G. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 2731–2752. Ueda, M.; Makino, A.; Imai, T.; Sugiyama, J.; Kimura, S. Chem. Commun. 2011, 47, 3204–3206.
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