Fast and Stable N‐Terminal Cysteine Modification through Thiazolidino Boronate Mediated Acyl Transfer

Abstract We report a novel conjugation of N‐terminal cysteines (NCys) that proceeds with fast kinetics and exquisite selectivity, thereby enabling facile modification of NCys‐bearing proteins in complex biological milieu. This new NCys conjugation proceeds via a thiazolidine boronate (TzB) intermediate that results from fast ( k 2 : ≈5000 m −1 s −1 ) and reversible conjugation of NCys with 2‐formylphenylboronic acid (FPBA). We designed a FPBA derivative that upon TzB formation elicits intramolecular acyl transfer to give N‐acyl thiazolidines. In contrast to the quick hydrolysis of TzB, the N‐acylated thiazolidines exhibit robust stability under physiologic conditions. The utility of the TzB‐mediated NCys conjugation is demonstrated by rapid and non‐disruptive labeling of two enzymes. Furthermore, applying this chemistry to bacteriophage allows facile chemical modification of phage libraries, which greatly expands the chemical space amenable to phage display.