Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

► The inhibitor cystine knotted peptide gurmarin is synthesized using a random oxidation strategy and an orthogonal oxidation strategy. ► A thermolysin cleavage of random oxidized gurmarin shows two possible disulfide combinations. ► The non-native gurmarin disulfide isomer is synthesized using the orthogonal strategy and compared with random oxidized gurmarin using UPLC. ► A synthetic approach is used for the first time to confirm the structure of a random oxidized inhibitor cystine knotted peptide.