A super versatile flexizyme system with phenol esters for genetic code reprogramming

Yun-Kyu Choi, Takayuki Katoh (), Adam Beattie and Hiroaki Suga ()
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Yun-Kyu Choi: 7-3-1 Hongo
Takayuki Katoh: 7-3-1 Hongo
Adam Beattie: 7-3-1 Hongo
Hiroaki Suga: 7-3-1 Hongo

Nature Communications, 2025, vol. 16, issue 1, 1-10

Abstract: Abstract Genetic code reprogramming enables ribosomal incorporation of multiple nonproteinogenic amino acids (npAAs), supporting bioactive peptide development. Flexizyme technology with npAA-benzyl(thio)esters (BZEs) as acyl-donors has been crucial for preparing diverse npAA-tRNAs. However, low acylation yields for some npAAs hinder peptide library construction. Here we report a versatile flexizyme system using phenol esters as alternative acyl-donors. Computational pKa predictions guided the synthesis of five phenol esters, which are mild enough to prevent random aminoacylation yet reactive with tRNA 3'-hydroxy groups. Among them, 3-nitrophenol (3NP) was chosen to prepare 18 structurally distinct npAA-3NPs, demonstrating direct tRNAs aminoacylation in 15–41% yields. Moreover, the flexizyme eFx drastically enhances aminoacylation efficiency, yielding near-quantitative conversion for some npAAs, e.g. cyclic β-npAAs. This eFx/npAA-3NP system facilitates access to diverse npAA-tRNAs, expanding ribosomal synthesis of nonstandard peptides, including macrocyclic structures. Thus, our approach provides an efficient tool for constructing peptide libraries with multiple npAA building blocks.

Date: 2025
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DOI: 10.1038/s41467-025-59921-w

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