Structure-activity Studies on the N-terminal Region of Glucagon

Using solid-phase methodology and preparative medium- and high-performance reverse-phase liquid chromatography, we have synthesized glucagon and its Arg12 analogue in approximately 5% yields. The synthetic glucagon was fully active relative to natural material, and the Arg12 peptide exhibited 50% activity. Since perhaps the most critical part of the glucagon-family peptides is the N-terminal hexapeptide region, both batches of resin were split during synthesis in order to prepare two series of analogues based on glucagon and [Arg12]glucagon with changes in the His-Ser-Gln-Gly-Thr-Phe sequence. The following new analogues were tested for their effects on blood glucose levels in normal male rats relative to glucagon and gave the following activities: [Ac-His1,Arg12]glucagon, 46%; [3-Me-His1,Arg12]glucagon, 30%; [Phe1,Arg12 )glucagon, 31%; [Des-His1,Arg12]glucagon, 4%; [D-Ala2,Arg12]glucagon, 44%; [D-p-Cl-Phe1,D-Ala4,Arg12]glucagon, 9%; [D-Phe4]glucagon, 655%; [Ala2]glucagon, 9%. These data indicate that the amino or imidazole nitrogens of the histidine residue are not essential for biological activity. However, an aromatic group in position 1 may be important, since the Phe1 analogue is almost as active as glucagon in our bioassay. The superagonist activity with [D-Phe4]glucagon, which was synthesized to test the hypothesis that a beta-bend conformation occurs at this position in glucagon by analogy with luteinizing hormone-releasing hormone and other Gly-containing peptides, indicates that this is indeed the case and has important implications for the receptor-recognition requirements of the glucagon-secretin-vasoactive intestinal peptide family of peptides.