A fast bioorthogonal reaction, tetrazine ligation, is described based on inverse-electron-demand Diels-Alder reactivity between s-tetrazine and trans-cyclooctene derivatives. This reaction proceeds with very fast rates (k₂ ≈ 2000 M⁻¹s⁻¹) and tolerates a broad range of functionalities, working efficiently in organic solvents, water, cell media, or cell lysate. The reaction is highly selective and enables protein modification at low concentrations. Unlike normal-electron-demand Diels-Alder reactions, inverse-electron-demand reactions have not been widely used in bioconjugation. Tetrazines are highly reactive dienes in such reactions, and the only byproduct is N₂. Sauer previously demonstrated the rapid reactivity of electron-deficient tetrazines with strained alkenes, with trans-cyclooctene being the most reactive dienophile. However, Sauer’s conditions were not suitable for bioconjugation due to tetrazine reactivity with water. Substituted s-tetrazines, such as 3,6-diaryl-s-tetrazines, were identified as suitable for bioorthogonal reactivity. A model study showed that 1b and trans-cyclooctene form product 3 in quantitative yield after epimerization. The reaction was tested in cell media and aqueous solutions containing rabbit reticulocyte lysate, achieving yields over 80%. The second-order rate constant for 1b + trans-cyclooctene at 25°C is 2000 M⁻¹s⁻¹ in 9:1 methanol:water. The reaction is fast and efficient, even at low concentrations. The reaction between 1b and trans-cyclooctene is much faster than background reactivity toward water or exogenous nucleophiles. The practicality of the tetrazine ligation is enhanced by the ease of access to starting materials. Trans-cyclooctene derivatives can be synthesized from cis-cyclooctene via a photochemical protocol. Unsymmetrical tetrazines can be prepared in gram quantities from commercially available reagents. The amino group of tetrazines provides a handle for functionalization via acyl transfer. To illustrate compatibility with proteins, thioredoxin was functionalized with a trans-cyclooctene derivative. The reaction between the functionalized thioredoxin and 1b formed the conjugate 12 within 5 minutes. Control experiments showed that the cis-cyclooctene analog did not react with 1b even after 24 hours. HPLC confirmed the fast and high-yield formation of 12. In summary, a new method for bioconjugation based on inverse-electron demand Diels-Alder chemistry is described. The reaction proceeds with very fast rates and tolerates aA fast bioorthogonal reaction, tetrazine ligation, is described based on inverse-electron-demand Diels-Alder reactivity between s-tetrazine and trans-cyclooctene derivatives. This reaction proceeds with very fast rates (k₂ ≈ 2000 M⁻¹s⁻¹) and tolerates a broad range of functionalities, working efficiently in organic solvents, water, cell media, or cell lysate. The reaction is highly selective and enables protein modification at low concentrations. Unlike normal-electron-demand Diels-Alder reactions, inverse-electron-demand reactions have not been widely used in bioconjugation. Tetrazines are highly reactive dienes in such reactions, and the only byproduct is N₂. Sauer previously demonstrated the rapid reactivity of electron-deficient tetrazines with strained alkenes, with trans-cyclooctene being the most reactive dienophile. However, Sauer’s conditions were not suitable for bioconjugation due to tetrazine reactivity with water. Substituted s-tetrazines, such as 3,6-diaryl-s-tetrazines, were identified as suitable for bioorthogonal reactivity. A model study showed that 1b and trans-cyclooctene form product 3 in quantitative yield after epimerization. The reaction was tested in cell media and aqueous solutions containing rabbit reticulocyte lysate, achieving yields over 80%. The second-order rate constant for 1b + trans-cyclooctene at 25°C is 2000 M⁻¹s⁻¹ in 9:1 methanol:water. The reaction is fast and efficient, even at low concentrations. The reaction between 1b and trans-cyclooctene is much faster than background reactivity toward water or exogenous nucleophiles. The practicality of the tetrazine ligation is enhanced by the ease of access to starting materials. Trans-cyclooctene derivatives can be synthesized from cis-cyclooctene via a photochemical protocol. Unsymmetrical tetrazines can be prepared in gram quantities from commercially available reagents. The amino group of tetrazines provides a handle for functionalization via acyl transfer. To illustrate compatibility with proteins, thioredoxin was functionalized with a trans-cyclooctene derivative. The reaction between the functionalized thioredoxin and 1b formed the conjugate 12 within 5 minutes. Control experiments showed that the cis-cyclooctene analog did not react with 1b even after 24 hours. HPLC confirmed the fast and high-yield formation of 12. In summary, a new method for bioconjugation based on inverse-electron demand Diels-Alder chemistry is described. The reaction proceeds with very fast rates and tolerates a