EZ-Link TFP Ester-PEG12-DBCO is a hetero-bifunctional crosslinker that contains an amine-reactive tetrafluorophenyl (TFP) group and a dibenzylcyclooctyne (DBCO) group with a water-solubilizing long PEG12 spacer in between. This crosslinker can be used to label any primary-amine-containing molecule (such as a protein) with a distantly attached DBCO group. Once a protein or (bio)molecule is DBCO-labeled, it can undergo a copper-free azide-alkyne cyclo-addition reaction to form a stable triazole linkage with an azide-labeled coupling partner.

EZ-Link TFP Ester-PEG12-DBCO features include:
• Bioorthogonal—DBCO and azides react selectively to form triazole linkages
• Heterobifunctional—reactive towards amine and azide
• Soluble—reagent readily dissolves in water-miscible solvents for dilution into aqueous reaction mixtures
• Stable—TFP esters are more hydrolytically stable than NHS esters

EZ-Link TFP Ester-PEG12-DBCO can be used to create diverse conjugates by means of the bioorthogonal coupling between an alkyne and azide (supplied separately). The coupling or crosslinking procedure is typically done in two steps. First, a protein- or amine-containing molecule is labeled with a DBCO group and excess crosslinker is removed. Second, an azide-containing moiety (prepared independently) is introduced into the same system as the DBCO moiety and labeling occurs under biological conditions without the introduction of any additional reagents such as a copper catalyst.

Classic click reactions involve a copper-catalyzed azide-alkyne cyclo­addition to label or conjugate molecules. A drawback of this approach is that copper ions can impair protein function, harm cells, and reduce the fluorescence of fluorophores. DBCO is distinct in that it doesn’t require copper to catalyze the reaction with an azide for triazole formation. The strain in the eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts, preventing copper-induced damage to cells or proteins.