ABSTRACT Expanding the chemical diversity of DNA through functionalization of purine‐purine base pairs by bioorthogonal reactions offers new opportunities for site‐specific labeling. Herein, the bioconjugation of 7‐functionalized 7‐deaza‐2′‐deoxyxanthosine (c 7 X d ) by inverse electron‐demand Diels‐Alder (iEDDA) reaction in oligonucleotides is reported. For this, 7‐iodo, 7‐vinyl, and 7‐cyclopropenyl nucleoside derivatives of c 7 X d were synthesized by Stille ‐ and Sonogashira cross‐coupling reactions performed on bis ‐NPE protected nucleosides. Oligonucleotide synthesis was accomplished on solid‐phase using iodinated and vinylated phosphoramidites of c 7 X d , whereas cyclopropenyl side chains were unstable even under mild conditions. Hybridization of modified oligonucleotides with complementary strands containing 2‐amino‐2′‐deoxyadenosine or 2‐amino‐7‐deaza‐2′‐deoxyadenosine furnished Watson‐Crick duplexes containing single or consecutive incorporations of 7‐functionalized (iodo, vinyl) purine‐purine base pairs. 7‐Vinylated and 7‐alkynylated oligonucleotides served as effective substrates for iEDDA cycloaddition with 3,6‐dipyridyl‐1,2,4,5‐tetrazine, producing oligonucleotide pyridazine adducts under mild, copper‐free conditions. Thermal denaturation studies demonstrated that 7‐iodinated and 7‐vinylated duplexes retained base‐pair stability, whereas duplexes with pyridazine conjugates showed reduced stability. Studies on mismatch discrimination disclosed almost identical duplex stability among 7‐functionalized and non‐functionalized 7‐deazaxanthine 2’‐deoxyribonucleosides in DNA opposite canonical nucleosides. The results of this study demonstrate the proof of concept that 7‐vinylated and 7‐alkynylated 7‐deazaxanthine 2’‐deoxyribonucleosides incorporated in oligonucleotides are applicable to functionalization by bioorthogonal iEDDA reaction.
Chandankar et al. (2026) studied this question.