Postsynthetic modification of nucleic acids has the advantage that the chemical development of only a few building blocks is necessary, each bearing a chosen reactive functional group that is applicable to its reactive counterpart for a variety of different labeling types. The reactive group is either linked to phosphoramidites for chemical synthesis on solid phase or attached to nucleoside triphosphates for application in primer extension experiments and PCR. Chemoselectivity is required for this strategy, together with bioorthogonality to perform these labelings in living cells or even organisms. Currently, the copper-free reactions include strain-promoted 1,3-dipolar cycloadditions, "photoclick" reactions, Diels-Alder reactions with inverse electron demand, and nucleophilic additions. The majority of these modification strategies show good to excellent reaction kinetics, an important prerequisite for labeling inside cells and in vivo in order to keep the concentrations of the reacting partners as low as possible.

In order to establish the Diels-Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4-triazine-modified 2'-deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4-triazine group was attached at the 5-position of 2'-deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2'-deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN-modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4-triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5-tetrazine for postsynthetic labeling by the Diels-Alder reaction with inverse electron demand.

No abstract

Fluoride-catalyzed stereoselective nucleophilic addition of PhSCF(2)SiMe(3) (1) to α-carboethoxycycloalkanones 2 followed by intramolecular radical cyclization of the resulting cis-3 adduct afforded the corresponding gem-difluoromethylenated bicyclic compounds 4, which underwent ring-expansion followed by the Baeyer-Villiger-type oxidation of the resulting macrocyclic ketone intermediates to give gem-difluoromethylenated macrocyclic lactones 5.

Two series of new, water-soluble, membrane-permeable, far-red/NIR emitting benzothiazolium-based fluorescent labels with large Stokes' shifts were synthesized that can be conjugated to alkyne-modified biomolecules through their azide moiety via azide-alkyne cycloaddition. We have used these azide bearing labels to make fluorescent DNA constructs using copper-catalyzed "click" reaction. All dyes showed good or remarkable fluorescence intensity enhancement upon conjugation to DNA. We also investigated the possibility to incorporate the benzocyclooctyne motif through rigid (ethnynyl) or flexible (ethyl) linkers into the DNA, thus enabling copper-free labeling schemes. We observed that there is a marked difference between the two linkers applied in terms of optical properties of the labeled oligonucleotides. We have also tested the in vivo labeling potential of these newly synthesized dyes on HeLa cells previously transfected with cyclooctynylated DNA. Confocal fluorescent images showed that the dyes are all able to cross the membrane and suitable for background-fluorescence free fluorescent tagging of nucleic acids. Moreover, we have observed different accumulation of the two dye series in the endosomal particles, or in the nuclei, respectively.