Click-enabled heterotrifunctional template for sequential bioconjugations

A heterotrifunctional template was developed that utilizes thiol-maleimide and click chemistries (both copper-free and copper-mediated) to effect sequential biomolecule conjugations in a one-pot process. The breadth of compatible substrates was illustrated through highly efficient conjugations of protein, peptide, sugar, lipid, fluoroalkane, biotin and fluorophore molecules. This template should be useful for the creation of chemically-enhanced/enabled biotherapeutics, especially through the expression of discontinuous (and heterogeneous) epitopes.