AsiaChem | Chemistry in Japan | December 2021 Volume 2 Issue 1

44 | December 2021 www.facs.website to mimic their functionality using artificial polypeptide materials. The chemoenzymatic polymerization of amino acids or short oligopeptide motifs can be used to realize the facile synthesis of biomimetic artificial polypeptides for these structural proteins. Silk proteins are produced by some animals, such as silkworms and spiders, to construct silk fibers for multiple tasks. Spider silk exhibits excellent mechanical properties that occasionally compete with those of artificial high strength fiber or even steel.6 A dragline silk, which is the most well-studied type of spider silk, primarily consists of major ampullate spidroins (MaSp) and shows high strength and toughness.7 Polyalanine motifs repeatedly appear in the amino acid sequences of MaSp proteins and form β-sheet crystallites in silk fibers. The β-sheet nanocrystals align along the fiber axis during the spinning process, resulting in the high tensile strength of the silk fiber. On the other hand, glycine-rich complex motifs alternate with polyalanine motifs in the highly repeating domain, which are responsible for the extensibility of silk fibers. We have focused on these motifs to assemble multiblock polypeptides that possess a sequence similar to that of spider dragline silk proteins (Figure 2).8 Chemoenzymatic polymerization of alanine ethyl ester was performed in the presence of papain, a cysteine protease with broad substrate specificity, in phosphate buffer at mild temperature, and polyalanine was obtained as a precipitate within an hour. Structural analysis revealed that the obtained polyalanine spontaneously forms a β-sheet structure similar to that of natural spider silks. Similarly, the glycine-rich motif in spider silk proteins was imitated by a random sequence of glycine and leucine obtained by papain-catalyzed copolymerization of these amino acid monomers. Ligation of the resulting polypeptide motifs with condensing agents afforded a specific amino acid sequence in which crystalline polyalanine and amorphous poly(glycine-random-leucine) motifs are tandemly flanked. For a crystalline/ random composition ratio that is similar to that of natural spider silk, the multiblock polypeptide was found to exhibit a secondary structure containing β-sheet crystallites and the ability to simultaneously form nanofibers. A great advantage of the chemoenzymatic synthesis approach used to construct spider silk-mimetic sequences is easy tuning of the polypeptide motifs. Random polypeptides of glycine with other amino acids, such as serine and tyrosine, are also available for an amorphous motif, and reactive hydroxy side groups of these motifs can be exploited for further modification or cross linking. Another example of the chemoenzymatic synthesis of biomimetic polypeptides is elastin. Elastin repeatedly contains a valine-proline-glycine-valine-glycine (ValProGlyValGly) motif in the hydrophobic region of its amino acid sequence, and the high elastic property of elastin arises from the tandem sequence of these moti fs with cross l inking.9 The ValProGlyValGly motifs in the elastin sequence undergo a temperature-induced reversible phase transition above a transition temperature, which has tremendous potential for use in thermoresponsive biomaterials.10 To construct the repetitive sequence of elastin, solid phase peptide synthesis is generally used for the synthesis of elastin-mimetic polymers. We utilized a chemoenzymatic polymerization technique to synthesize a repetitive sequence containing the thermoresponsive motif of elastin (Figure 3).11 Papain-catalyzed copolymerization of ValProGly tripeptide and ValGly dipeptide ester monomers in aqueous buffer affords a polypeptide with an elastin-mimetic sequence. The polypeptide with a tandem ValProGly-random-ValGly sequence showed a temperature-dependent structural transition. Interestingly, when the valine residue was substituted with a glycine residue, the resulting analogous sequence of GlyProGly-randomValGly did not show any temperature-dependent structural transition. Such a trivial difference in sequences drastically changes the physical properties of the polypeptides, indicating the significant importance of specific motifs that structurally and physically determine the properties of proteins. Unnatural polypeptides In addition to 20 proteinogenic amino acids, some nonproteinogenic amino acids also exist in the amino acid sequences of proteins and broaden the scope of protein functionality. Such nonproteinogenic residues are generally assembled in proteins not by enzymatic ligation in the central dogma but by epigenetic postmodification of proteinogenic amino acid residues in protein sequences. Although the assembly of unnatural amino acids into polypeptides via chemoenzymatic polymerization is fascinating, things are not Figure 3. Papain-catalyzed polymerization of VPG and VG monomers for the synthesis of elastin-mimetic polypeptide. The polypeptide possessing VPGVG repetitive motifs exhibits a reversible structural transition in a temperature-dependent manner, as shown by the circular dichroism (CD) spectra. Reproduced from Ref. 11 with permission from the Royal Society of Chemistry. Figure 2. Synthesis of a multiblock polypeptide mimicking a repetitive sequence of spider silk proteins via chemoenzymatic polymerization. Reproduced from Ref. 8 with permission from the American Chemical Society.

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