Click-to-Release Reactivity of Quaternary Anilinium Tetrazines Enables Cascade Assembly of Multifunctional Biomolecular Scaffolds.

Click chemistry has evolved from its origins in forming covalent connections to also include breaking bonds, cleaving linkers, and liberating cargo, creating broad applications in chemical biology and biomedicine. The reaction sequences that drive these tools are complex, and thus prone to an array of dead-end intermediates that reduce their release/cleavage efficiency. Here, we redirect that complexity to explore yet another aspect of tetrazine (Tz)/trans-cyclooctene (TCO) click-to-release. By exchanging the carbamate linkage of established release Tzs for an anilinium leaving group, post-click tautomerization is accelerated and temporally synchronized, generating an intermediate capable of trapping thiols rapidly and in high yields. Further integration of an orthogonal azide into the Tz hub enables a one-pot multi-component reaction that allows straightforward combinatorial synthesis of densely functionalized architectures. Leveraging both click and release, the resulting process converts a Tz/TCO pair into a ternary scaffold on demand, readily incorporating fluorophores, chelators, cleavable drugs, proteins, oligonucleotides, and more. This modular cascade assembly offers a rapid and versatile platform for functionalization of biomolecules and small molecule targeting vectors, with immediate potential in imaging, theranostics, and multivalent bioconjugate design.