Semisynthetic Derivatives of Glucagon: (Des-His¹)Nε-acetimidoglucagon and Nα-Biotinyl-Nε-acetimidoglucagon

Nε-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole e-amino group. Nε-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. Nγ-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic Nα-biotinyl-Nε-acetimidoglucagon, prepared by reacting (A-hydroxysuccinimido)biotin with Nε-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (p7 = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of Nα-biotinyl-Nε-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein- labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)Nε-acetimidoglucagon, prepared via a manual Edman degradation of Nε-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (p7 = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.