Here we report the first use of ligand-directed proximity accelerated bioconjugation chemistry in the tandem delivery and release of a therapeutic payload. To do this we designed a nitrophenol carbonate for ligand-directed in situ bioconjugation of a prodrug payload to a protein. The transient nature of our conjugation chemistry renders the protein a depot for time-dependent release of active drug following hydrolysis and self-immolation. In our model system, using an immunostimulant prodrug, biotin ligand, and avidin protein, we observe time-dependent release of bioavailable immunostimulant both spectroscopically and with an immune cell line over 48 h. Avidin co-crystalized with the biotin directing group verified the binding pose of the ligand and offered insight into the mechanism of in situ bioconjugation. Overall, this scaffold warrants further investigation for the time-dependent delivery of therapeutics and use in protein ligand pairs beyond biotin and avidin used for this work.

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 binding 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