Chemical protein modification strategies are powerful tools for chemical biology. However, there is only a limited number of modular chemical probes available for the delivery of functionally intact native proteins. Here, we introduce a three-component probe that enables bioreversible protein modification for a variety of protein delivery applications. Our probe incorporates a diazo moiety for chemoselective esterification of carboxyl groups, a pyridyl disulfide group for late-stage functionalization with thiolated ligands, and a self-immolative carbonate group for esterase-mediated cleavage. The probe can be synthesized in only three steps from commercially available materials. Using model proteins—Cytochrome c (Cyt c) and superfolder GFP, we showed that our probe can generate a diverse set of protein conjugates modified with cell-penetrating peptides, targeting ligands, or PEG under mild conditions. As a proof of concept, we applied our strategy to deliver proteins into the cytosol of mammalian cells in the presence of serum. Successful delivery of a functional Cyt c, an apoptosis-inducing protein, into cancer cells further highlighted the advantage of our bioreversible strategy over a classic irreversible one. Overall, we demonstrated the modularity and generality of our strategy in traceless protein delivery and highlighted its potential in advancing future basic and biomedical applications.

Chemoselective late-stage modification for traceless protein delivery

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ACS Fall 2022

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