Intramolecular Catalysis of Hydrazone Formation of Aryl-Aldehydes via ortho-Phosphate Proton Exchange.

Bioorthogonal site-specific chemical reaction to label biomolecules in vitro and in living cells is one of the most powerful and convenient tools in chemical biology. A reactive pairs frequently used for chemical conjugation are aldehydes/ketones with hydrazines/hydrazides/hydroxylamines. Although the reaction is generally specific for the two components, even in a cellular environment, the reaction is very slow under physiological conditions. Addition of a phosphate group at the ortho-position of an aromatic aldehyde increases the reaction rate by an order of magnitude and enhances the aqueous solubility of the reagent and the product. We have synthesized phosphate-substituted aldehyde synthetic models to study kinetics of their reactions with hydrazines and hydrazides that contain a fluorophore. This rapid bioorthogonal reaction should therefore be potentially a very useful reaction for routine site-specific chemical ligations to study and image complex cellular processes in biological systems.