Critical role of the A2 amino acid residue in the biological activity of insulin: [2-glycine-A]- and [2-alanine-A]insulins.

We report the synthesis of [2-glycine-A]insulin ([ Gly2 -A]insulin) and [2-alanine-A]insulin ([ Ala2-A]insulin) in which the indicated amino acid has been substituted for isoleucine found in this position in the natural hormone. The circular dichroic (CD) spectra of the analogues were obtained, and their properties were examined in several biological assays. CD studies suggested that the analogues remain monomeric at concentrations at which insulin is partly or mostly dimeric. Both analogues are extremely weak full agonists. [ Gly2 -A]-insulin displays 0.05% of the potency of bovine insulin, whereas [Ala2-A]insulin assays at 0.4% of the activity of the natural hormone. We conclude that the presence of the side chain of isoleucine at position A2 is a critical requirement for high biological activity in insulin. The data, together with previous observations, are discussed in connection with an interaction between the side chain of isoleucine-A2 and the phenolic ring system of tyrosine-A19, which are in van der Waals contact in crystalline insulin. This interaction may be required to permit the molecule to assume a conformation consistent with dimerization and with binding to the insulin receptor.