Uncovering the Key Role of Distortion in Bioorthogonal Tetrazine Tools That Defy the Reactivity/Stability Trade-Off

Abstract

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 concentrations. 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 the 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 repulsive N−N interaction plays a key role in accelerating the cycloaddition step. We show that the limited stability of tetrazines in biological media strongly correlates with the electron-withdrawing effect of the substituent, while intramolecular repulsion increases the reactivity without reducing the stability. These fundamental insights reveal thus far overlooked mechanistic aspects that govern the reactivity/stability trade-off for tetrazines in physiologically relevant environments, thereby providing a new strategy that may facilitate the rational design of these bioorthogonal tools.

Recent Advances in the Development of Tetrazine Ligation Tools for Pretargeted Nuclear Imaging

“…Steric effects are the main reasons why monosubstituted 3-phenyl-1,2,4,5-tetrazines (H-Tzs) typically have 70-fold-increased activity compared to methyl-or phenyl-substituted Tzs [43]. Recently, Svatunek and al. showed that distortion effects between the aromatic and the tetrazine rings can increase the reactivity of the molecule [44]. Increased distortion led to faster rate constants (Figure 3C) [44].…”

“…Recently, Svatunek and al. showed that distortion effects between the aromatic and the tetrazine rings can increase the reactivity of the molecule [44]. Increased distortion led to faster rate constants (Figure 3C) [44].…”

“…Recently, Svatunek and al. showed that distortion effects between the aromatic and the tetrazine rings can increase the reactivity of the molecule [44]. Increased distortion led to faster rate constants (Figure 3C) [44]. The addition of substituted phenyl rings to the Tz is frequently carried out, for example, to be able to label the Tz.…”

“…Shortly after the pioneering work of Rossin et al, other radiometal PET isotopes such as 64 Cu, 89 Zr, 68 Ga, or 44 Sc, linked to different chelating agents, were used to radiolabel Tzs [73,79,83,84,87,88]. One of the first PET radionuclides used for Tz-radiolabeling was 64 Cu by Lewis et al in 2011.…”

“…In 2019, Edem et al developed a 44 Sc-Tz analogue of [ 111 In]1 [83]. The half-life of scandium-44 (3.97 h) is long enough for its production and transport while minimizing radiation dose.…”