Norman Metanis

Insulin has long provided a model for studies of protein folding and stability, enabling enhanced treatment of diabetes mellitus via analogue design. We describe the chemical synthesis of a basal insulin analogue stabilized by substitution of an internal cystine (A6–A11) by a diselenide bridge. The studies focused on insulin glargine (formulated as Lantus® and Toujeo®; Sanofi). Prepared at pH 4 in the presence of zinc ions, glargine exhibits a shifted isoelectric point due to a basic B chain extension (Arg B31 −Arg B32 ). Subcutaneous injection leads to pH‐dependent precipitation of a long‐lived depot. Pairwise substitution of Cys A6 and Cys A11 by selenocysteine was effected by solid‐phase peptide synthesis; the modified A chain also contained substitution of Asn A21 by Gly, circumventing acid‐catalyzed deamidation. Although chain combination of native glargine yielded negligible product, in accordance with previous synthetic studies, the pairwise selenocysteine substitution partially rescued this reaction: substantial product was obtained through repeated combination, yielding a stabilized insulin analogue. This strategy thus exploited both (a) the unique redox properties of selenocysteine in protein folding and (b) favorable packing of an internal diselenide bridge in the native state, once achieved. Such rational optimization of protein folding and stability may be generalizable to diverse disulfide‐stabilized proteins of therapeutic interest.