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

Activated esters are widely used to label proteins at lysine sidechains 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, fluoro-phenyl esters are shown to preferentially label human kappa antibodies at a single lysine (K188) within the light chain constant domain. Neighboring residues H189 and D151 contribute to the accelerated rate of labeling at K188 relative to ~40 other lysine sites. Enriched K188 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 demonstrate that activated esters with fluoro-substituted aromatic leaving groups, including a fluoro-napthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.