Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon

N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-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. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 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 N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 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.