Total Chemical Synthesis of Glucagon Glycoforms Reveals the Remarkable Influence of Natural Glycosylation

Glycosylation plays a critical role in modulating protein and peptide properties, yet the impact of recently discovered natural mucin-type O-glycosylation on therapeutic peptides like glucagon remains underexplored due to challenges in obtaining homogeneous research samples. Here, we address this challenge by developing a streamlined multistep synthesis-one purification protocol, enabling the production of 21 glucagon glycoforms with systematically varied glycosylation patterns. Investigation of these glycoforms revealed a remarkable impact of natural glycosylation on two properties critical for glucagon: solubility increased by over 870-fold, and fibrillation was completely inhibited, even under stringent conditions, while biological activity in elevating blood glucose levels was preserved. Computational analyses indicated that sialylated glycans disrupt intermolecular associations in the solid state, enhancing solubility, and shift secondary structures from β-sheets to α-helices, mitigating fibrillation. These findings suggest that glycosylation may have evolved as a natural mechanism to optimize undesirable properties, offering valuable insights for the development of therapeutics based on glucagon and other proteins and peptides with similar challenges.