Steric requirements at position B12 for high biological activity in insulin.

The alpha-helix formed by the amino acid residues 9-19 of the B-chain of insulin is involved in the stabilization of its three-dimensional structure. We have shown that modification at positions B9, B10, B12, and B16 results in analogues possessing biological activities ranging from ca. 0.2% to ca. 500% relative to that of natural insulin. The lowest potency was displayed by [B12 Asn]insulin, in which the hydrophobic B12 Val residue was replaced by the hydrophilic Asn residue. We now report the synthesis of four insulin analogues in which hydrophobicity is retained, and only the spatial arrangement of atoms in the B12 region is altered. Substitution of B12 Val with alpha-aminoisobutyric acid (Aib), D-Ala, and Phe led to analogues possessing biological activities, in lipogenesis assays, of 8.5%, 2%, and 0.2%, respectively, relative to that of natural insulin. Inversion of the B11-B12 sequence, -Leu-Val-, led to an analogue displaying 3.3% activity. A synthetic B-chain in which the B11 Leu-B12 Val sequence was replaced by B11 Ala-B12 Ile was incapable of combining with the natural A-chain. We conclude that the Val residue in the B12 position in insulin fulfills special side-chain packing requirements involved in the stability of the structure of insulin. Even slight steric alteration at position B12 results in a distortion of the overall conformation of the B-chain which affects its ability to combine with the natural A-chain. This distortion is retained in the corresponding analogue, which is reflected in diminished biological potency.