Insulin analogues with a glucose-regulated conformational switch

A method of preparation of an insulin analogue containing a modified A-chain polypeptide and a modified B-chain polypeptide, wherein the A-chain is modified with a monomeric glucose-binding moiety at or within 5 residues of the A-chain N-terminus, and wherein the B-chain is modified with a diol-containing modification or an alpha-hydroxycarboxylate modification at or within 6 residues of the B-chain C-terminus, the method comprising

• (a) providing a des-octapeptide[B23-B30] fragment of insulin or insulin analogue;
• (b) providing a saccharide ester of phenylboronic acid;
• (c) incubating the des-octapeptide[B23-B30] fragment of insulin or insulin analogue and saccharide ester of phenylboronic acid under conditions conducive to coupling of phenylboronic acid to the des-octapeptide[B23-B30] fragment of insulin or insulin analogue to provide a phenylboronic acid coupled to the N-terminus of the insulin A-chain of the des-octapeptide[B23-B30] fragment of insulin or insulin analogue;
• (d) providing a synthetic peptide of length 6-10 amino-acid residues whose N-terminal residue is Glycine and which upon modification contains no tryptic cleavage site, and wherein the peptide incorporates an N-linked or O-linked saccharide;
• and
• (e) incubating the synthetic peptide with the des-octapeptide[B23-B30] fragment of insulin or insulin analogue in the presence of trypsin under conditions conducive to the enzymatic activity of trypsin such that trypsin catalyzes the formation of a bond between synthetic peptide and the des-octapeptide[B23-B30] fragment of insulin or insulin analogue.