One-Pot Enzymatic ADDing of Click Chemistry Handles for Protein Immobilization and Bioconjugation of Small and...

Site-specifically attaching biorthogonal handles to proteins is an essential tool for chemical biology research and diverse applications, including imaging, protein immobilization, and the development of next-generation therapeutics such as antibody drug-conjugates. Among the available strategies, enzymatic post-translational modification of short protein tags offers precision, stability, and modularity. However, complex substrate syntheses, the requirement of long recognition tags, and limited reaction efficiencies pose remaining limits for application. Here, we present ADDing, a straightforward enzymatic method for functionalizing proteins with click chemistry handles using the flavin transferase ApbE. We discovered that, given a dedicated adenine diphosphate derivative (ADD) substrate, ApbE attaches a phosphoribosyl moiety bearing bioorthogonal handles to DxxxGAT amino acid motifs. ADDing benefits from a streamlined workflow, due to simple enzymatic substrate synthesis from NAD⁺ and inexpensive precursors, allowing one-pot click handle generation and protein conjugation. ADDing enables rapid, high-yield functionalization of proteins featuring the recognition tag at either terminus or internal loops, and is compatible with copper and copper free azide-alkyne cycloaddition. We demonstrate its broad applicability in a wide variety of protein functionalizations, including fluorescent labeling, protein-protein, protein-DNA conjugation, and protein immobilization. This versatile technology thus holds great potential for chemical biology and therapeutic development.