Two-Factor Fluorogenic Cyanine-Styryl Dyes with Yellow and Red Fluorescence for Bioorthogonal Labelling of DNA.

An orange- and a red-emitting tetrazine-modified cyanine-styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. Both dyes use the concept of the "two-factor" fluorogenicity for nucleic acids: (i) The dyes are nucleic-acid sensitive by their non-covalent binding to DNA, and (ii) their covalently attached tetrazine moiety quench the fluorescence. As a result, the reaction with bicyclononyne- and spirohexene-modified DNA is significantly accelerated up to k =280,000 M  s , and the fluorescence turn-on is enhanced up to 305.

Metabolic labelling of DNA in cells by means of the "photoclick" reaction triggered by visible light.

Two pyrene-tetrazole conjugates were synthesized as photoreactive chromophores that allow for the first time the combination of metabolic labelling of DNA in cells and subsequent bioorthogonal "photoclick" modification triggered by visible light. Two strained alkenes and three alkene-modified nucleosides were used as reactive counterparts and revealed no major differences in their "photoclick" reactivity. This is a significant advantage because it allows 5-vinyl-2'-deoxyuridine to be applied as the smallest possible alkene-modified nucleoside for metabolic labelling of DNA in cells.

Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.