Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation.

Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics. Herein we report a novel method that allows for fast and selective labeling of proteins with an N-terminal cysteine. Although N-terminal cysteines are well known to conjugate with aldehydes to give thiazolidines, the reaction requires acidic conditions and suffers from slow kinetics. We show that benzaldehyde with an -boronic acid substituent readily reacts with N-terminal cysteines at neutral pH, giving rate constants on the order of 10 M s. The product features a thiazolidono boronate (TzB) structure and exhibits improved stability due to formation of the B-N dative bond. While stable at neutral pH, the TzB complex dissociates upon mild acidification. These characteristics make the TzB conjugation chemistry potentially useful for the development of drug-protein conjugates that release the small molecule drug in acidic endosomes.