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

Grace H PhamWeijia OuBadry BursulayaMichael DiDonatoAnanda HerathYunho JinXueshi HaoJon LorenGlen SpraggonAnsgar BrockTetsuo UnoBernhard H GeierstangerSusan E Cellitti
Published in: Chembiochem : a European journal of chemical biology (2018)
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.
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