Fluorogenic bioorthogonal reactions enable visualization of biomolecules with excellent signal-to-noise ratio. A bicyclononyne-tetrazine ligation that produces fluorescent pyridazine products has been developed. In stark contrast to previous approaches, the formation of the dye is an inherent result of the chemical reaction and no additional fluorophores are needed in the reagents. The crucial structural elements that determine dye formation are electron-donating groups present in the starting tetrazine unit. The newly formed pyridazine fluorophores show interesting photophysical properties the fluorescence intensity increase in the reaction can reach an excellent 900-fold. Model imaging experiments demonstrate the application potential of this new fluorogenic bioorthogonal reaction.
- Klíčová slova
- Diels-Alder reactions, cyclic alkynes, cycloaddition reactions, fluorogenic probes, tetrazines,
- MeSH
- cykloadiční reakce MeSH
- fluorescenční barviva chemie MeSH
- heterocyklické sloučeniny monocyklické chemie MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- můstkové bicyklické sloučeniny chemie MeSH
- nádorové buněčné linie MeSH
- pyridaziny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorescenční barviva MeSH
- heterocyklické sloučeniny monocyklické MeSH
- můstkové bicyklické sloučeniny MeSH
- pyridaziny MeSH
Inverse-electron-demand Diels-Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.
- Klíčová slova
- bioorthogonal chemistry, click chemistry, cycloaddition, heterocycles, tetrazines,
- MeSH
- cykloadiční reakce MeSH
- cyklooktany chemie MeSH
- fluorescenční barviva chemická syntéza chemie MeSH
- fluorescenční mikroskopie metody MeSH
- HeLa buňky MeSH
- heterocyklické sloučeniny bicyklické chemická syntéza chemie MeSH
- heterocyklické sloučeniny monocyklické chemie MeSH
- konfokální mikroskopie metody MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cyklooktany MeSH
- fluorescenční barviva MeSH
- heterocyklické sloučeniny bicyklické MeSH
- heterocyklické sloučeniny monocyklické MeSH
Fluorogenic bioorthogonal reactions enable visualization of biomolecules under native conditions with excellent signal-to-noise ratio. Here, 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.
- Klíčová slova
- Diels-Alder reaction, cell labeling, fluorogenic, tetrazines, trans-cyclooctene,
- Publikační typ
- časopisecké články MeSH
1,2,4-Triazines have recently been identified as versatile dienes participating in the inverse electron-demand Diels-Alder reaction with strained dienophiles. However, their widespread utility in bioconjugation reactions is still limited. Herein, we report a systematic study on the reactivity of various 1,2,4-triazines with trans-cyclooctenes showing that the structure of both the triazine and the dienophile significantly affect the reaction rate. Our kinetic study led to the discovery of novel cationic 1,2,4-triazines with superior properties for bioconjugation reactions. We have developed an efficient method that enables their late-stage functionalization and allows for easy access to various useful heterobifunctional scaffolds. In addition, these charged dienes form unprecedented fluorescent products upon reaction with trans-cyclooctenes and can be used for fluorogenic labeling of subcellular compartments in live cells.
- Publikační typ
- časopisecké články MeSH
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.
- Klíčová slova
- bioorthogonal chemistry, click reactions, cycloaddition, heterocycles, imaging agents,
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
