Sulfoxide‐Mediated Cys‐Trp‐Selective Bioconjugation that Enables Protein Labeling and Peptide Heterodimerization

<jats:title>Abstract</jats:title><jats:p>A method was developed that enables the magnesium chloride (MgCl<jats:sub>2</jats:sub>)‐activated <jats:italic>S</jats:italic>‐acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp<jats:sup>2</jats:sup>(C−H) sulfenylation of the indole of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid to give the Trp‐sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to various peptides, including lysozyme. Furthermore, we achieved the Trp‐modification of a monoclonal antibody by a MgCl<jats:sub>2</jats:sub>‐mediated reaction in an acidic ionic liquid. The resulting antibody exhibited antibody performance comparable to the parent protein. The amide moiety in the Acm group contributes to the difference in chemical behaviors between <jats:italic>S</jats:italic>‐Acm and <jats:italic>S‐p</jats:italic>‐methoxybenzyl (MBzl)‐protected cysteine sulfoxides. This is because the <jats:italic>S</jats:italic>‐Acm sulfoxide is converted to <jats:italic>S</jats:italic>‐chlorocysteine responsible for Trp‐sulfenylation under less acidic conditions than those required for the reaction of <jats:italic>S</jats:italic>‐MBzl sulfoxide. Based on this rationale, we prepared a linker possessing <jats:italic>S</jats:italic>‐Acm and <jats:italic>S</jats:italic>‐MBzl oxide moieties and subjected the linker to heterodimerization of DNA‐binding MYC and MAX peptides containing a Trp handle. The one‐pot/stepwise Cys‐Trp conjugation between the linker and DNA‐binding peptides allowed the generation of a heterodimeric MYC/MAX DNA binder.</jats:p>