Chemoenzymatic Synthesis of Rhamnolipid-Modified Exendin-4: A Dual Approach for Long-Acting Glycemic Control through Endogenous Antibody Recruitment and Albumin Interaction

Effective management of Type 2 diabetes requires therapies that balance potency with sustained action. Glycosylation of glucagon-based drugs improves stability and reduces aggregation but does not significantly enhance pharmacokinetics. This study develops a novel glycosylation strategy─a rhamnolipid conjugation platform─to address this challenge. Using sortase A-mediated enzymatic ligation, we synthesized Ex4–rhamnolipid bioconjugates that prolong action through a dual mechanism: carbohydrate-mediated antibody engagement and lipophilic albumin binding. These conjugates potently activated GLP-1 receptors (EC50 = 0.14–0.21 nM) and elicited rapid blood glucose reduction in diabetic rodent models. In immunized subjects, glucose-lowering effects persisted for 24 h, reflecting a 6.7-fold improvement in circulation half-life. In extended-duration studies, once-daily administration of the lead conjugate achieved glycemic control and pancreatic β-cell protection comparable to semaglutide without adverse effects. This methodology capitalizes on natural immune components and plasma protein interactions to address the challenges of peptide-based treatments, offering a promising platform for sustained-release antidiabetic drugs.