Tuning a Protein‐Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site‐selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light‐chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50–70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro‐substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site‐selective lysine labeling of antibodies and other immunoglobulin‐type proteins. Expanding the bioconjugation toolbox: Fluorophenyl esters can be used to preferentially label human κ antibodies at a single lysine (Lys188) among ≈40 unique lysine sites. Lys188 is positioned in the light‐chain constant domain, and site‐selective labeling can be enhanced by tuning reaction conditions or the surrounding protein environment.