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

Abstract 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.