Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs

Formylglycine‐generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C α ‐formylglycine (FGly). This noncanonical electrophilic amino acid can subsequently be addressed selectively by bioorthogonal hydrazino‐ iso ‐Pictet‐Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to proteins to obtain a defined payload‐to‐protein ratio. However, the disadvantages of these conjugation...