Gaining insight into biological processes relies on sensitive analytical techniques. These often require labeling of biomolecules that help visualize them. Selective covalent labeling without preliminary modification of the biomolecules is an advantageous method. For example, this can be achieved by using probes that are capable of in situ quinone methide (QM) formation. The QM can be masked to give a stable precursor, and the highly reactive form is only generated upon activation by a specific trigger. The in situ formed QM then binds covalently to the nucleophilic side chains of either the target protein or a protein in close proximity. Using fluorogenic probes further improves this method by reducing non-specific background signals, thus improving signal-to-noise ratios. In this review we summarize the development of quinone methide-based probes from mechanism-based inactivation to red-emitting, fluorogenic activity probes, focusing on enzyme-triggered activation.

Proof‐of‐concept studies for the bioorthogonally controlled generation of pro‐fluorescent quinone‐methides are presented here. The novel concept relies on a click‐to‐release tetrazine unit that chemically cages a pro‐fluorescent quinone methide precursor. On a synthetically readily accessible model compound we demonstrate that IEDDA reaction of the tetrazine with a TCO leads to the liberation of a pro‐fluorescent quinone methide, which is readily captured by nearby nucleophiles to form a fluorescent scaffold. Unlike in previous approaches to access bioorthogonally activatable fluorogenic probes, where the tetrazine unit acted as a quencher of fluorescence, the role of the tetrazine is redefined here. Such repurposed role of tetrazines is foreseen to address the limitations of tetrazine‐quenched red/NIR excitable fluorogenic probes.

Click‐to‐release (C2R) chemistry is widely used for controlled payload liberation, yet the reactivity of its transient intermediates has remained largely unexplored. Here we show that an intermediate formed during the reaction of trans ‐cyclooctene (TCO)‐appended protein‐targeting ligands with release tetrazines (rTzs), previously regarded as a byproduct rapidly quenched by water, can be exploited as an electrophile for ligand‐directed covalent protein labeling. The bioorthogonally generated dihydropyridazine methide (DHP‐methide) enables affinity‐directed protein labeling, resulting primarily in capture at surface‐exposed histidine residues proximal to the site of electrophile generation and revealing a histidine‐biased covalent modification pathway. Incorporation of functional handles into the rTz platform enables modular reporter installation via an orthogonal conjugation step. A fluorogenic leaving‐group design further allows direct optical reporting of the electrophile‐generating elimination event and real‐time monitoring of DHP‐methide formation. Selective labeling is retained in cell lysate and can be translated to a cellular context, as demonstrated by confocal imaging of surface‐expressed targets. These findings establish a bioorthogonally gated strategy for affinity labeling and reveal a previously unrecognized functional dimension of rTz–TCO click‐to‐release chemistry.

This manuscript provides a comprehensive overview of bioorthogonal click-to-release (C2R) chemistry with a specific focus on its implications for biointerfaces. It surveys the major C2R reaction platforms, outlines their mechanistic principles at a conceptual level, and critically compares their reactivity–release trade-offs and biological performance. The review highlights applications in antibody-, polymer-, nanoparticle-, and hydrogel-based drug delivery, as well as in fluorogenic imaging, biosensing, and diagnostics. Emerging uses in protein activation, tissue engineering, and adaptive biomaterials are also discussed, alongside current limitations and design challenges. Overall, the manuscript positions C2R chemistry as a versatile tool for dynamic and spatially controlled manipulation of biological systems

Herein we report a ligand-directed affinity labeling platform that leverages a previously overlooked electrophilic intermediate of a click-to-release reaction. Upon TCO – release tetrazine (rTz) reaction, an electrophilic dihydropyridazine methide is formed transiently, which covalently captures proximal nucleophilic residues on the protein of interest, enabling robust and selective modification. The meth-od showed strict ligand dependence and covalent target modification, confirmed through displacement assays and MS/MS mapping of the modified protein. Installation of further functional handles onto the rTz platform enabled modular reporter ligation via strain-promoted click chemistry. This bioorthogo-nally triggered strategy provides a valuable alternative to light-based electrophile activation-based approaches, particularly in systems where optical activation is impractical.

Click-to-release (C2R) chemistry is widely used for controlled payload liberation, yet the chemical reactivity of its transient intermediates has remained largely unexplored. Here we show that an intermediate formed during the reaction of trans -cyclooctene (TCO)-appended protein-targeting ligands with release tetrazines (rTzs), previously regarded as a byproduct rapidly quenched by water, can be viewed from a different perspective and exploited as an electrophile for ligand-directed covalent protein labeling. The bioorthogonally generated dihydropyridazine methide (DHP-methide) enables affinity-directed protein labeling that predominantly results in capture at surface-exposed histidine residues proximal to the site of electrophile generation, revealing a histidine-biased covalent modification pathway. Incorporation of functional handles into the rTz platform enables modular reporter installation via orthogonal bioorthogonal reactions. A fluorogenic leaving group design further allows direct optical reporting of the electrophile-generating elimination event, enabling real-time monitoring of DHP-methide formation during the labeling process. Selective labeling is retained in complex protein mixtures and in spiked lysate experiments and can be translated to a cellular context, as demonstrated by confocal imaging of surface-expressed targets following post-labeling fluorophore installation. These findings reframe a mechanistic intermediate of click-to-release chemistry as a functional electrophile for ligand-directed protein modification and establish a previously unrecognized bioorthogonally gated strategy for affinity labeling.