Ligand-Directed Approaches to Site-Selective Protein Modification

Precision bioconjugation techniques are essential for constructing well-defined protein-based therapeutics such as antibody-drug conjugates (ADCs). Among native amino acids, cysteine is particularly attractive for site-selective modification due to its high nucleophilicity and low natural abundance. However, most proteins contain multiple free cysteines, making it challenging to target specific residues without affecting protein structure or function.

Current site-selective protein modification strategies often rely on genetic engineering to introduce tags (protein tags, peptide tags, or unnatural amino acids). An alternative approach uses affinity-driven bioconjugation, where a ligand; either a small molecule or peptide binds reversibly to the target protein through noncovalent interactions, positioning a reactive warhead near a specific amino acid side chain for covalent bond formation. Despite progress in this field, achieving site selectivity among multiple cysteines in complex proteins remains difficult, particularly when genetic modification is undesirable.

Recently, Waser and coworkers addressed this challenge by developing ligand-directed cysteine bioconjugation using ethynylbenziodoxolone (EBX) reagents conjugated to peptide ligands. Targeting the KELCH domain of KEAP1, a protein containing nine free cysteines, they achieved site-selective modification of Cys434 even in the presence of cell lysate. This approach exploits the dual reactivity of EBX reagents: silyl-EBX derivatives enable traceless alkyne transfer, yielding minimally modified alkynylated cysteine, while alkyl- and aryl-EBX reagents form stable vinyl-benziodoxolone (S-VBX) adducts that retain the peptide ligand. Both pathways provide orthogonal handles for subsequent bioorthogonal chemistry, demonstrating how combining peptide affinity with hypervalent iodine chemistry enables precision protein modification on native, untagged proteins.

References:

1. Marty, C.; Ji, X.; Nicolai, S.; Heinis, C.; Waser, J. Ligand-Directed Site-Selective Cysteine Bioconjugation of the KELCH Domain of KEAP1 with Hypervalent Iodine Reagents. J. Am. Chem. Soc.2025. https://doi.org/10.1021/jacs.5c13391.
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