Four triphosphates of 2'-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic-chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels-Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the "photoclick" cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine-oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45-55 °C. The cyclopropene-oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The "photoclick" cycloaddition between tetrazole-oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2'-deoxyuridine triphosphate.

This review ventures to summarize the latest developments in bioorthogonal fluorescent imaging labels with a special focus on bioimaging applications. We briefly summarize the most preferred means of bioorthogonal tagging schemes for the labeling of specific biomolecular structures. The review is structured by the type of the fluorescent labels that can address the problems that most commonly compromise fluorescent imaging techniques, i.e. the autofluorescence of biomolecules, the background fluorescence of unreacted reagents, and photobleaching. Thus, we present (i) far-red/near-infra-red emitting dyes, (ii) fluorogenic scaffolds, and (iii) nanoparticle-based signaling platforms.

Two different and small functions for inverse electron demand Diels–Alder reactions were applied for dual labeling of DNA: the 1,2,4-triazine was attached to the 5-position of 2′-deoxyuridine, and the 1-methylcyclopropene to the 7-position of 7-deaza-2′-deoxyadenosine.