Site-Specific In Vivo Bioorthogonal Ligation via Chemical Modulation.

A critical limitation of bioorthogonal click chemistry for in vivo applications has been its low reaction efficiency due to the pharmacokinetic barriers, such as blood distribution, circulation, and elimination in living organisms. To identify key factors that dominate the efficiency of click chemistry, here a rational design of near-infrared fluorophores containing tetrazine as a click moiety is proposed. Using trans-cyclooctene-modified cells in live mice, it is found that the in vivo click chemistry can be improved by subtle changes in lipophilicity and surface charges of intravenously administered moieties. By controlling pharmacokinetics, biodistribution, and clearance of click moieties, it is proved that the chemical structure dominates the fate of in vivo click ligation.