Tetrazine Ligation in Living Systems: Beyond Fast Kinetics to Effective Bioorthogonality

Tetrazine-based bioorthogonal chemistry has become a cornerstone of chemical biology due to the exceptional reaction rate and selectivity of the inverse electron-demand Diels-Alder (IEDDA) reaction. These properties have enabled a wide range of applications, from live-cell labeling to in vivo imaging and chemical manipulation of biomolecules. However, accumulating evidence indicates that rapid reaction kinetics alone are insufficient to ensure reliable performance in complex biological environments. In this Review, we examine tetrazine bioorthogonal chemistry from the perspective of effective orthogonality, defined as the ability to react selectively and productively in living systems. We summarize key failure modes that compromise orthogonality in biological settings and discuss emerging design strategies aimed at mitigating these limitations. Building on this framework, we integrate comparative studies that assess tetrazine performance across different levels of biological complexity, highlighting how the dominant constraints on bioorthogonal ligation evolve from cellular to in vivo and compartment-restricted environments. Together, these insights provide a unified perspective on why strategies optimized in simplified systems often fail upon biological translation and offer guidance for the rational development of bioorthogonal reagents for use in living systems.