www.asiachem.news December 2021 | 45 that simple. Thanks to the substrate specificity, we can chemoselectively polymerize natural amino acid monomers by chemoenzymatic polymerization. However, unnatural amino acid monomers are tightly excluded during the enzymatic reaction. Our initial attempt to polymerize unnatural amino acid esters in the presence of various proteases was not satisfactory: the amount of unnatural amino acids that can be incorporated into polypeptide sequences is quite limited,12 which motivated us to design unnatural amino acid-containing oligopeptide ester monomers. Sandwiching the unnatural amino acids amid natural amino acids allows proteases to recognize such species in the substrate pocket, leading to successful polymerization of these oligopeptide monomers (Figure 4).13-15 A typical unnatural amino acid, 2-aminoisobutyric acid (Aib), is an α,α-disubstituted amino acid with bulky side groups. Introduction of Aib residues in polypeptides is known to strongly induce a helical conformation due to the bulky structure. We prepared a tripeptide ethyl ester with an alanine-Aib-alanine (AlaAibAla) sequence for the monomer for chemoenzymatic polymerization.15 Neither Aib nor a Aib-containing dipeptide monomer was found to be able to polymerize in the presence of papain because the Aib unit has a poor affinity for papain. In contrast, the papain-catalyzed polymerization of the Aib-containing tripeptide monomer afforded a polypeptide that periodically contains Aib every three residues. The resulting polypeptide adopts an α-helix conformation, whereas polyalanine with no Aib units shows a β-strand structure in circular dichroism spectroscopic analysis. We expanded this “tripeptide” strategy to various types of unnatural amino acids from N-alkyl amino acids to monomer units of synthetic polyamides such as nylon and aramide polymers. The periodic introduction of nylon units (ω-aminoalkanoic acid) provides polypeptides with melting behavior below their decomposition temperature, promising improvement in the processability of polypeptide materials with thermal plasticity.13 On the other hand, aromatic monomers can also be inserted in polypeptides via chemoenzymatic polymerization of tripeptide monomers containing 4-aminobenzoic acid residues.14 Fusing the polypeptide backbone with an aromatic structure can increase the thermal stability of polypeptides, which is reminiscent of the thermal properties of synthetic aromatic polyamides such as Kevlar. Notably, periodic introduction of such an aromatic structure, by which an unnatural secondary structure distinct from natural polypeptide forms, is important for improving the physical properties. The random introduction of 4-aminobenzoic acid in the polypeptide backbone was found to conversely deteriorate the thermal stability. Therefore, the introduction of unnatural amino acids shows synergy with secondary structures derived from periodic sequences to improve the physical properties of polypeptide materials. Functional polypeptides for plant modification Aligning functional side groups of polypeptides by rationally designed sequences can lead to assembly into specific higher-order structures with unique functionality. Such polypeptides have been applied to pharmaceutical and biomedical fields, including drug delivery systems, due to their physiological functions. In particular, our interest lies in plantbased sustainable bioproduction of bulk polymeric materials. Material production using plants has been studied in a broad range of fields, such as drug discovery, energy production, food production, and materials synthesis. Figure 5. Material delivery into plant cells mediated by peptide carriers. Cationic polypeptides are used as carriers to complex with cargo materials (DNA, proteins), and the complex is further functionalized by various peptides to overcome several barriers for internalization into desired organelles. Figure 4. 2-Aminoisobutyric acid (Aib), an unnatural amino acid showing a poor affinity for proteases, can be recognized in the catalytic center of papain by “sandwiching” with natural amino acids. Various unnatural amino acids can be introduced in periodic sequences via chemoenzymatic polymerization using tripeptide esters flanking natural amino acids. Reproduced from Ref. 15 with permission from the Royal Society of Chemistry.
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