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Temporal and reversible control over protein and cell conjugations holds great potential for traceless release of antibody-drug conjugates (ADCs) on tumor sites as well as on-demand altering or removal of targeting elements on cell surface. We herein developed a bioorthogonal and traceless releasable reaction on proteins and intact cells to fulfill such purposes. A systematic survey of transition metals in catalyzing the bioorthogonal cleavage reactions revealed that copper complexes such as Cu(I)-BTTAA and dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc) moieties offered a bioorthogonal releasable pair for reversible blockage and rescue of primary amines and phenol alcohols on small molecule drugs, protein side chains, as well as intact cell surface. For proof-of-concept, we employed such Cu(I)-BTTAA/dsProc and Cu(I)-BTTAA/dsPra pairs as a "traceless linker" strategy to construct cleavable ADCs to unleash cytotoxic compounds on cancer cells in situ and as a "reversible modification" strategy for cell surface engineering. Furthermore, by coupling with the genetic code expansion strategy, we site-specifically modulated ligand-receptor interactions on live cell membranes. Together, our work expanded the transition-metal-mediated bioorthogonal cleavage tool kit from terminal decaging to internal-linker breakage, which offered a temporal and reversible conjugation strategy on therapeutic proteins and cells.

Chemical de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-molecule reagents. The previously reported chemical de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, we developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chemical rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymatic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the post-translational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. Our strategy provides a general platform for chemical rescue of Tyr-dependent protein activity inside cells.