Oligonucleotides Post‐Synthetic Modifications: An Overview of Clickable Nucleoside Phosphoramidites Suitable for Solid‐Phase Synthesis

Chemical modification of oligonucleotides has become an essential strategy to improve their properties and expand their functional utility in chemical biology, molecular imaging, and therapeutics. Among the various modification strategies, post-synthetic bioorthogonal click chemistry enables efficient, rapid, and biocompatible site-specific conjugation of functional moieties. Central to these advances is the development of clickable nucleoside phosphoramidites, namely nucleoside building blocks containing reactive bioorthogonal click handles that are compatible with the harsh conditions of solid-phase oligonucleotide synthesis (SPOS). This review provides a comprehensive overview of bioorthogonal cycloaddition-based nucleoside phosphoramidites reported to date that are compatible with SPOS. We highlight their synthetic accessibility and deviations from conventional SPOS protocols when required, as well as the stability and hybridization behavior of the resulting clickable oligonucleotides. Furthermore, we examine the performance of subsequent bioorthogonal reactions used for post-synthetic functionalization, with particular attention to their kinetics and efficiencies. Representative applications are also discussed, including the development of labeled probes, multifunctional assemblies, and targeted delivery systems.