A Cleavable C(2)-Symmetric trans-Cyclooctene Enables Fast and Complete Bioorthogonal Disassembly of Molecular Probes

[Image: see text] Bioorthogonal chemistry is bridging the divide between static chemical connectivity and the dynamic physiologic regulation of molecular state, enabling in situ transformations that drive multiple technologies. In spite of maturing mechanistic understanding and new bioorthogonal bond-cleavage reactions, the broader goal of molecular ON/OFF control has been limited by the inability of existing systems to achieve both fast (i.e., seconds to minutes, not hours) and complete (i.e., >99%) cleavage. To attain the stringent performance characteristics needed for high fidelity molecular inactivation, we have designed and synthesized a new C(2)-symmetric trans-cyclooctene linker (C(2)TCO) that exhibits excellent biological stability and can be rapidly and completely cleaved with functionalized alkyl-, aryl-, and H-tetrazines, irrespective of click orientation. By incorporation of C(2)TCO into fluorescent molecular probes, we demonstrate highly efficient extracellular and intracellular bioorthogonal disassembly via omnidirectional tetrazine-triggered cleavage.