Copper-Free Click Enabled Triazabutadiene for Bioorthogonal Protein Functionalization

Area of Science:

• Bioconjugation Chemistry
• Chemical Biology
• Organic Synthesis

Background:

• Aryl diazonium ions are reactive bioconjugation agents but have limited stability and require harsh conditions for in situ generation.
• Triazabutadiene (TBD) scaffolds can protect aryl diazonium ions, enhancing their synthetic utility and enabling triggered release under physiological conditions.

Purpose of the Study:

• To synthesize and apply a novel TBD scaffold for installing cyclooctynes onto protein surfaces.
• To enable copper-free click reactions for protein functionalization using the TBD-derived cyclooctyne probe.

Main Methods:

• Synthesis of a novel triazabutadiene (TBD) derivative.
• Protein labeling with the TBD-cyclooctyne probe under various pH conditions.
• Orthogonality assessment using a model protein in the presence of other proteins.
• Cleavage of the azobenzene modification using sodium dithionite.

Main Results:

• The novel TBD probe efficiently labels proteins across a wide pH range.
• Protein modification was achieved conveniently and in a timely manner.
• Orthogonal labeling was demonstrated in a complex protein mixture using an azide-functionalized fluorophore.
• The azobenzene modification was successfully cleaved with sodium dithionite.

Conclusions:

• The developed TBD-based cyclooctyne probe facilitates efficient and orthogonal protein labeling under biologically relevant conditions.
• This method provides a valuable tool for bioconjugation and chemical biology applications, particularly for copper-free click chemistry.
• The TBD scaffold offers a stable and versatile platform for generating reactive intermediates for bioconjugation.