Engineering a Glucose‐Responsive Glucagon Prodrug Through Arginine–Phenylboronic Acid Pendant Modification

Activatable prodrug strategies offer powerful means to control therapeutic presentation in space and time. Here, we report a single-molecule prodrug design that enables glucose-responsive activation of a glucagon analog for hypoglycemia protection. The system conjugates dasiglucagon with a synthetic pendant comprised of alternating arginine and phenylboronic acid (PBA) units, designed to couple peptide solubility to glucose concentration. The pendant modulates net charge through glucose-dependent PBA-diol complexation, driving aggregation under normoglycemia and solubilization under hypoglycemia. The lead pendant contains five arginine-PBA repeats and exhibits optimal glucose-responsive solubility and charge modulation, forming aggregates at high glucose and dissolving as glucose levels decline. Despite a modest reduction in receptor potency relative to native dasiglucagon, this approach provides significant prophylactic protection in a streptozotocin-induced diabetic mouse model of insulin overdose, rescuing mice from hypoglycemia and eliminating mortality events. This work demonstrates a proof-of-concept for molecularly engineered, metabolite-responsive glucagon prodrugs that function as on-demand therapeutic depots. More broadly, it establishes a modular design paradigm for dynamic, self-regulating peptide therapeutics based on charge modulation rather than external carrier systems.