Design and Synthesis of Aza‐Bicyclononene Dienophiles for Rapid Fluorogenic Ligations

Fluorogenic bioorthogonal reactions enable visualization of biomolecules under native conditions with excellent signal-to-noise ratio. Herein, we present the design and synthesis of conformationally-strained aziridine-fused trans-cyclooctene (aza-TCO) dienophiles, which lead to the formation of fluorescent products in tetrazine ligations without the need for attachment of an extra fluorophore moiety. The presented aza-TCOs adopt the highly strained ‘half-chair’ conformation, which was predicted computationally and confirmed by NMR measurements and X-Ray crystallography. Kinetic studies revealed that the aza-TCOs belong to the most reactive dienophiles known to date. The potential of the newly developed aza-TCO probes for bioimaging applications is demonstrated by protein labeling experiments, imaging of cellular glycoconjugates and peptidoglycan imaging of live bacteria.