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<p>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 p<i>K</i><sub>a</sub> 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, <i>e.g</i>. 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月20日

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  • Yun-Kyu Choi,
  • Takayuki Katoh,
  • Adam Beattie,
  • Hiroaki Suga

摘要

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.