This research proposal targets innovative tools for cysteine conjugation based on double Michael acceptor cross-linkers. These types of reagents have already found applications in the controlled design of macromolecular thiol-yne based networks, in which the reactivity of the double Michael additions and retro-additions can be fine tuned. In this project, we will expand the scope of such double Michael acceptor reagents into the field of conjugation of bio(macro)molecules. The double Michael acceptors allow control over the relative kinetics of the two forward addition reactions, so that mixed adducts of two thiols can be formed. The hydrolytic and physiological stability of these adducts will also be fine tuned by changing the substitution pattern. Moreover, triggered thiol release through the addition of suitable bis-nucleophiles will be investigated. We have already gathered encouraging proof-of-principle studies with a range of double Michael acceptors, and will also explore some more innovative options, in which the thiol-yne chemistry is combined with azide-alkyne or hydrazone linking chemistries, both of which are used in bioconjugation. The developed bioconjugation tools will be applied towards peptide functionalization, peptide cyclization and peptide disulfide rebridging experiments. The double Michael reactions allow attaching a thiol-functional payload onto a cysteine. Later stage applications will target protein conjugation, including monoclonal antibodies.