Used in bioconjugation to link biomolecules via the strain-promoted inverse electron-demand Diels-Alder reaction (SPIEDAC) between trans-cyclooctene (TCO) and tetrazine, a copper-free 'click chemistry' variant. The TCO group rapidly reacts with tetrazine-modified molecules, enabling fast and selective ligation in biological systems. The PEG3 spacer improves water solubility and reduces steric hindrance, enhancing reaction efficiency. The NHS ester reacts with primary amines on proteins, peptides, or amine-modified surfaces, allowing stable amide bond formation. Commonly applied in labeling, crosslinking, and functionalizing biomolecules for assays, imaging, drug delivery, and surface modification, especially where copper-free conditions are essential, such as live-cell labeling or in vivo applications.