To our knowledge we are the first to utilize ligand-directed proximity accelerated bioconjugation chemistry to deliver a therapeutic payload. Here we report a new chemical moiety based on nitrophenol carbonates for ligand-directed in situ labeling of avidin with an immunostimulant prodrug. This renders avidin a depot for time-dependent release of bioavailable immunostimulant following hydrolysis and self-immolation of the transient modification. Structural biology was utilized to verify the

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... ing pose of the modified biotin directing group and offer insight into nucleophilic residues proximal to the nitrophenol carbonate. Time-dependent release of the immunostimulant was monitored with an immune cell line to demonstrate the release of bioavailable immunostimulant over 48 h. We show that this scaffold warrants further investigation for the time-dependent delivery of therapeutics and further investigation of protein ligand pairs beyond biotin and avidin used for this work