Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins

Isoaspartate (isoAsp) is a β-linked residue in proteins spontaneously generated through Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish and site-nonselective generation of isoAsp residues in proteins severely impedes in-depth biological investigations as well as the exploitation of its unique β-linkage features. Herein, we introduce a method that allows site-selective and rapid generation of isoAsp residues in proteins. This method leverages the genetic incorporation of a side-chain-esterified Asp derivative (BnD), which undergoes facile intramolecular arrangement to form the key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis of Suc gives rise to isoAsp as the major product. On native sites of proteins, including Cu/Zn superoxide dismutase and calmodulin, we demonstrate that BnD-mediated isoAsp formation is faster than Asn deamidation generally by three orders of magnitude.