Advancements and applications of click chemistry in protein labeling and bioconjugation

Click chemistry has emerged as a versatile and efficient chemical strategy for constructing complex molecules under mild conditions. Its core reaction is copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides high yields, stereoselectivity, and biocompatibility. Click reactions, particularly the copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC), were highlighted for their high selectivity, efficiency, and bioorthogonality. In addition to these, other click reactions reported in the study included thiol-ene reactions, Diels-Alder cycloadditions (particularly inverse electron-demand Diels-Alder, IEDDA), and electro-click chemistry, all of which expanded the click chemistry toolbox for diverse biological applications. Recent advancements in both CuAAC and copper-free click strategies are explored, emphasizing their applications in protein tagging, imaging, proteomics, and drug development. Various innovative methodologies, such as bioorthogonal non-canonical amino acid tagging (BONCAT), click chemistry-assisted RNA interactome capture (CARIC), electro-click chemistry, and cross-linking mass spectrometry, demonstrate the versatility of click reactions in studying cellular processes and biomolecular interactions. Furthermore, the review highlights the use of click chemistry in live-cell labeling, biomaterials development, enzyme profiling, and disease-related studies through protein tagging. Copper-free strategies were emphasized for overcoming toxicity limitations. Overall, click chemistry was presented as a versatile and rapidly evolving platform for precise biomolecular modification.