Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

Abstract

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.

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

“…[8] As eries of experimentsa nd calculations confirmed the identityo f1,4-dihydropyridazines as the actual fluorescent speciesf ormed during the reaction( see the Supporting Information).W ed etermined the photophysical properties of the correspondingc lick products,w hich are summarized in Ta ble 1. [8] As eries of experimentsa nd calculations confirmed the identityo f1,4-dihydropyridazines as the actual fluorescent speciesf ormed during the reaction( see the Supporting Information).W ed etermined the photophysical properties of the correspondingc lick products,w hich are summarized in Ta ble 1.…”

“…Am uch less common situation represents fluorogenic ligations in which the formation of the fluorophore is an inherentr esult of the chemicalr eaction. [8] Althoughw ecould demonstrate the powero ft his chemistry for bioimaging, we also found that by modifying the hydroxyl groupo ft he ax-TCO-ol, the fluorogenic properties of the reaction alter or even completely disappear.F or many reasons,i t wouldb ea dvantageous to maintain the fluorogenic nature of the reactionw hilee nabling attachmento fv ariousu seful functionalg roups to, for example, biomolecules. [6] Shang et al recently reported ac onceptually similars tyrene-tetrazinec ycloaddition leadingt of luorescent dihydropyridazines.…”

“…[8] These calculations showt hat the conformation of the intermediate 4,5-dihydropyridazine promotes hydrogen migration to the hydroxyl oxygen,w hich results in the formation of the final 1,4-dihydropyridazine fluorophore. [8] These calculations showt hat the conformation of the intermediate 4,5-dihydropyridazine promotes hydrogen migration to the hydroxyl oxygen,w hich results in the formation of the final 1,4-dihydropyridazine fluorophore.…”

Bioorthogonal Cycloadditions with Sub‐Millisecond Intermediates

“…[4] Ag ood match between the LUMO tetrazine and HOMO dienophile endows tetrazine-based iEDDAr eactions with fast bimolecular kinetics compared to other click reactions.T he rate constants are as high as 10 6 m À1 s À1 under physiological conditions. [6,7] Recently,a nother class of bioorthogonal click reactions,namely between sydnones and strained alkynes,h as been developed. Forc yclooctene dienophiles,slow tautomerisation ensues in protic solvents to yield the 1,4-dihydro isomer.…”

“…60 fs). [6,7] b) Cycloaddition between asydnone and astrained alkyne. [16] An internal residue within the transmembrane b barrel of the aHL pore is modified with ar eactant of interest.…”