Organic Chemistry

Uncovering the key role of distortion in tetrazine ligations guides the design of bioorthogonal tools that defy the reactivity/stability tradeoff


  • Dennis Svatunek Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria ,
  • Martin Wilkovitsch Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria ,
  • Lea Hartmann Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria ,
  • Kendall Houk UCLA, Los Angeles, USA ,
  • Hannes Mikula Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria


The tetrazine/trans-cyclooctene ligation stands out from the bioorthogonal toolbox due to its exceptional reaction kinetics, enabling multiple molecular technologies in vitro and in living systems. Highly reactive 2-pyridyl-substituted tetrazines have become state-of-the-art for time-critical processes and selective reactions at very low concentration. It is widely accepted that the enhanced reactivity of these chemical tools is attributed to the electron-withdrawing effect of the heteroaryl substituent. In contrast, we show that observed reaction rates are way too high to be explained on this basis. Computational investigation of this phenomenon revealed that distortion of the tetrazine caused by intramolecular N-N repulsion plays a key role in accelerating the cycloaddition step. While we show that the limited stability of tetrazines under physiological conditions strongly correlates with the electron-withdrawing effect of the substituent, intramolecular destabilization increases the reactivity without reducing stability. Guided by these fundamental insights we demonstrate application in the design of highly reactive tetrazines with superior stability, finally evading the reactivity/stability trade-off for bioorthogonal tetrazine tools.

Version notes

(1) All quantum-chemical calculations were performed at a higher level of theory. (2) Distortion/Interaction analysis was done at consistent geometry, in addition to calculations at the transition states. (3) Text and figures were updated, corrected and revised (e.g., the labels for Me2Pyr and Me3Pyr in Fig. 5b were wrong in the initial version).


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Supplementary material

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Supporting Information
Experimental details on computational methods, organic synthesis, reaction kinetics, compound stability and characterization, copies of NMR spectra
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xyz-coordinates of all calculated geometries