The linkage-type and the exchange molecule affect the protein-labeling efficiency of iminoboronate probes

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N-hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.