Double-Click Strategy Combining CuAAC and (Thia-) Diels-Alder Reactions; Application Toward Peptide Labeling.

Area of Science:

• Chemical Biology
• Organic Synthesis
• Polymer Chemistry

Background:

• Click chemistry strategies are essential for efficient molecular assembly.
• Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and Diels-Alder (DA) reactions are widely used bioorthogonal reactions.
• Developing versatile platforms for sequential or concurrent application of these reactions is crucial for complex bioconjugation.

Purpose of the Study:

• To develop and evaluate a novel double-click strategy integrating CuAAC and (thia-)Diels-Alder reactions.
• To design and synthesize heterobifunctional platforms with orthogonal clickable groups for this strategy.
• To demonstrate the efficiency and versatility of this methodology in bioconjugation applications.

Main Methods:

• Design and synthesis of heterobifunctional platforms (alkyne-dithioester, alkyne-maleimide, azide-diene).
• Evaluation of one-pot sequential protocols (CuAAC/DA, CuAAC/thia-DA) and three-component reactions (3CR).
• Utilizing a highly reactive s-cis-constrained exocyclic diene for rapid cycloadditions.

Main Results:

• Successful implementation of a double-click strategy combining CuAAC with DA or thia-DA reactions.
• Good isolated yields achieved for desired conjugates under mild, biocompatible conditions (37°C, aqueous solvent mixture, 1-2 hours).
• Demonstrated efficiency in bioconjugating small peptides with a fluorophore or biotin.

Conclusions:

• The developed heterobifunctional platforms and double-click strategy offer an efficient and versatile method for bioconjugation.
• The methodology exhibits excellent functional group tolerance and compatibility with biologically relevant molecules.
• This approach provides a powerful tool for constructing complex biomolecular conjugates.