Transforming Aryl-Tetrazines into Bioorthogonal Scissors for Systematic Cleavage of trans-Cyclooctenes

02 July 2024, Version 3

Abstract

Bioorthogonal bond-cleavage reactions have emerged as a powerful tool for precise spatiotemporal control of (bio)molecular function in the biological context. Among these chemistries, the tetrazine-triggered elimination of cleavable trans-cyclooctenes (click-to-release) stands out due to high reaction rates, versatility, and selectivity. Despite an increasing understanding of the underlying mechanisms, application of this reaction remains limited by the cumulative performance trade-offs (i.e., click kinetics, release kinetics, release yield) of existing tools. Efficient release has been restricted to tetrazine scaffolds with comparatively low click reactivity, while highly reactive aryl-tetrazines give only minimal release. By introducing hydroxyl groups onto phenyl- and pyridyl-tetrazine scaffolds, we have developed a new class of ‘bioorthogonal scissors’ with unique chemical performance. We demonstrate that hydroxyaryl-tetrazines achieve near-quantitative release upon accelerated click reaction with cleavable trans-cyclooctenes, as exemplified by click-triggered activation of a caged prodrug, intramitochondrial cleavage of a fluorogenic probe (turn-on) in live cells, and rapid intracellular bioorthogonal disassembly (turn-off) of a ligand-dye conjugate.

Keywords

bioorthogonal chemistry
click chemistry
click-to-release
cleavage reactions
tetrazines

Supplementary materials

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Supporting Information
Description
general methods, synthesis, UV-Vis spectroscopy, reaction kinetics, release and cleavage experiments, tetrazine stability, prodrug activation, cell imaging, NMR spectra
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