Intramolecular enzymatic peptide synthesis: trypsin-mediated coupling of the peptide bond between B22-arginine and B23-glycine in a split crosslinked insulin.

The resealing of a split peptide bond (B22/23) in a crosslinked insulin derivative by a trypsin-mediated coupling reaction in Tris buffer/glycerol/dimethylsulphoxide 1 : 1 : 1 at pH 6.5 is described. The coupling yield obtained with N alpha A1, N epsilon B29-(carbonyl-bis-methionyl) insulin [OC(Met)2] was 55-60%, and reproducible yields of OC(Met)2-insulin were 40%. After removing the crosslink and purification, crystalline resynthesized insulin of high biological activity was obtained in a yield of 43% and an overall yield, based on split OC(Met)2-insulin, of 18%.