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

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Genetic code reprogramming allows incorporating nonproteinogenic amino acids (npAAs) into peptides.
• Flexizyme technology using npAA-benzyl(thio)esters (BZEs) is vital for npAA-tRNA synthesis.
• Low acylation yields with BZEs limit the construction of diverse peptide libraries.

Purpose of the Study:

• To develop a versatile flexizyme system using alternative acyl-donors for improved npAA-tRNA synthesis.
• To overcome limitations in peptide library construction caused by low acylation yields.

Main Methods:

• Designed and synthesized five phenol esters as alternative acyl-donors based on computational pK_a predictions.
• Utilized the flexizyme enhanced factor (eFx) to boost aminoacylation efficiency.
• Prepared 18 distinct npAA-phenol esters, including 3-nitrophenol (3NP) derivatives.

Main Results:

• Phenol esters demonstrated mild reactivity, enabling specific tRNA 3'-hydroxy group acylation.
• The 3NP-based system achieved direct tRNA aminoacylation yields of 15-41% for various npAAs.
• The eFx/npAA-3NP system significantly enhanced aminoacylation, reaching near-quantitative conversion for some npAAs, including cyclic β-npAAs.

Conclusions:

• The novel eFx/npAA-3NP system provides efficient access to diverse npAA-tRNAs.
• This approach expands the ribosomal synthesis of nonstandard peptides, including complex macrocyclic structures.
• The developed system is a valuable tool for constructing peptide libraries with multiple npAA building blocks.