1,2,4-Triazine-Modified 2'-Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA.

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.