Site-specific modification of proteins has significantly advanced the use of proteins in biological research and therapeutics development. Among various strategies aimed at this end, genetic code expansion (GCE) allows structurally and functionally distinct non-canonical amino acids (ncAAs) to be incorporated into specific sites of a protein. Herein, we genetically encode an esterified glutamic acid analogue (BnE) into proteins, and demonstrate that BnE can be applied in different types of site-specific protein modifications, including N-terminal pyroglutamation, caging Glu in the active site of a toxic protein, and endowing proteins with metal chelator hydroxamic acid and versatile reactive handle acyl hydrazide. Importantly, novel epigenetic mark Gln methylation is generated on histones via the derived acyl hydrazide handle. This work provides useful and unique tools to modify proteins at specific Glu or Gln residues, and complements the toolbox of GCE.
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