Effects of Selenocysteine Substitution on the Cyclotide Kalata B2

Cyclotides are peptides with a head‐to‐tail cyclic backbone and six cysteine residues that are linked in a knotted arrangement. Their unique structure confers exceptional stability and various biological activities, resulting in numerous studies on their synthesis and development for a range of applications. One challenge in cyclotide synthesis is the formation of the correct disulfide bonds that fold the peptide into the native conformation. Replacement of a native disulfide bond with the faster‐forming diselenide bond by substituting cysteine residues with selenocysteine is a strategy that can be implemented to overcome this issue. In this study, we synthesized selenocysteine analogs of the cyclotide kalata B2 and determined the effect of the substitution on folding, structure, and biological activity. The analogs folded faster than their native counterpart, implying that the diselenide bond assists in the formation of the correct structure. HPLC and nuclear magnetic resonance (NMR) analysis revealed a native‐like structure for all analogs. Cytotoxicity and membrane binding assays showed that biological activity was maintained upon selenocysteine substitution. Overall, this study demonstrates the conservative nature of diselenide replacement while improving the folding of kalata B2.