Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS-Activated Acrylamides

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Organic Synthesis

Background:

• Michael acceptors and N-hydroxysuccinimide (NHS) activated esters are key peptide functionalization reagents.
• Chemoselective modification of peptides and proteins is crucial for bioconjugate development.

Purpose of the Study:

• To develop a novel reagent for efficient and chemoselective amino-sulfhydryl stapling on native peptides and proteins.
• To demonstrate the versatility and applicability of this new bioconjugation strategy.

Main Methods:

• Synthesis and application of NHS-activated acrylamides for peptide and protein modification.
• Kinetic analysis of N-terminal cysteine functionalization.
• Demonstration of cross-linking with lysine residues and orthogonal dual-modifications.

Main Results:

• NHS-activated acrylamides enable fast functionalization of N-terminal cysteines with high selectivity.
• Successful cross-linking of internal or C-terminal cysteines with lysine residues.
• Demonstrated late-stage functionalization of peptides and proteins with PEG, fluorescent probes, and cytotoxic agents.

Conclusions:

• NHS-activated acrylamides represent a powerful new tool for amino-sulfhydryl stapling.
• This strategy offers a high level of molecular control for designing functional bioconjugates.
• The versatility of this method is expected to advance bioconjugation technologies.