Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display

Area of Science:

• Chemical Biology
• Biotechnology
• Molecular Imaging

Background:

• Bioorthogonal reactions are crucial for tracking biomolecules in living systems.
• Traditional methods for optimizing these reactions are time-consuming and complex.
• Developing faster and more specific bioorthogonal tools is essential for in vivo studies.

Purpose of the Study:

• To streamline the optimization of bioorthogonal reactions using a novel in vivo approach.
• To identify novel bioorthogonal chemical reporters with enhanced kinetics and specificity.
• To demonstrate the utility of these reporters for labeling specific proteins in live cells.

Main Methods:

• Phage-assisted interrogation of reactivity (PAIR) was employed to screen and optimize bioorthogonal reaction conditions.
• Kinetic analysis was performed to determine reaction rates.
• Site-directed mutagenesis was used to understand the role of surrounding residues.
• The optimized reporter was genetically encoded and inserted into the extracellular domain of epidermal growth factor receptor (EGFR).

Main Results:

• A streamlined approach, phage-assisted interrogation of reactivity (PAIR), was established to optimize bioorthogonal reactions.
• A short alkyne-containing peptide sequence was identified with fast kinetics (k2=13,000±2000 M⁻¹ s⁻¹) in a palladium-mediated cross-coupling reaction.
• Residues surrounding the alkyne moiety were found to facilitate the reaction intermediate.
• The peptide sequence enabled specific labeling of EGFR in live mammalian cells when genetically encoded.

Conclusions:

• Phage-assisted interrogation of reactivity (PAIR) offers a rapid and efficient strategy for optimizing bioorthogonal reactions in situ.
• The identified alkyne-containing peptide sequence serves as a highly reactive and specific bioorthogonal reporter.
• This method facilitates targeted protein labeling in live cells, advancing molecular tracking and imaging capabilities.