It is empirically discovered what residue ends up reacting with the probe therefore it is hard to assess which target would be amenable to the chemistry. Second, the nucleophile itself is not rationally selected. First, the size of the required activating groups and/or linkers is substantial and precludes the labeling of residues very close to the active site. (34) Although these methods enabled prominent applications and could retain target protein activity, (35,36) some challenges remain. In these bioconjugation methods, the ligand leaves the active site after forming a covalent bond with a nucleophilic residue on the POI. have pioneered ligand-directed chemistries, which include ligand-directed, tosyl (LDT), (29) acyl imidazole (LDAI), (30) bromo benzoate (LDBB), (31) sulfonylpyridine, (32) and N-acyl- N-alkyl sulfonamide (LDNASA) (33) chemistries. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC 50 < 100 nM), which installed a CRBN binder onto BTK. Using an environment-sensitive “turn-on” fluorescent probe, we were able to monitor ligand binding to the active site of BTK. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket.

For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Using this approach, we were able to label various proteins such as BTK, K-Ras G12C, and SARS-CoV-2 PL pro with different tags. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells.

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more.

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