Bioconjugation is a key technology in fluorescence imaging and in diagnostic applications. Probing the function of biomolecules by modifying their structure is a major driver in biomedical sciences. Two bioconjugation routes, based on activated esters and maleimides to target lysines and cysteines, have become standard methods that are available as commercial conjugation ‘kits’. However, both strategies have some serious drawbacks. Recently, triazolidinedione (TAD)-based protein and peptide conjugation has emerged as a site-selective bioconjugation method. This method stands out because it is very selective towards tyrosine residues and has very fast forward kinetics. It forms physiologically stable adducts, with minimal disruption of protein function. Unfortunately, very few TAD reagents are commercially available, as the shelf life of TAD reagents is very limited. Most reagents have to be generate before use via a hazardous oxidation procedure, which limits the scope. In this project, we will aim at innovative strategies using "protected TAD" moieties that can be deprotected in situ in aqueous medium in the presence of the target protein, without the need for an oxidation step. The overarching goal of this project is to design a new and powerful generation of TAD-based tools for versatile and wide ranging bioconjugation applications such as fluorescent tagging, biotinylation and PEGylation. These will have a great added value for the life science research community.