Interactive Bioconjugation at N‐Terminal Cysteines by Using O‐Salicylaldehyde Esters towards Dual Site‐Selective Functionalization

N‐terminal Cys modification has been intensively studied to produce homogeneous bioconjugates essentially through two modes of reaction: reversible modification with the equilibrium shifted towards the formation of the desired conjugate or stable and irreversible conjugates. Herein, we report a new method of N‐terminal cysteine modification using O‐salicylaldehyde esters (OSAEs) through fast conjugation and irreversible deconjugation. These reagents can rapidly react with N‐terminal Cys at low‐micromolar concentration to form thiazolidines with subsequent hydrolysis of the ester moiety to the phenolic derivative. These phenolic thiazolidines can be hydrolyzed at acidic pH (≈4.5) to recover the intact N‐terminal Cys. Bioconjugation reactions using OSAEs offer controlled reversibility to as act as a protecting group for N‐terminal cysteines, allowing the modification of in‐chain residues without perturbing the N‐terminal Cys, which can then be deprotected and used as a conjugation site.