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Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A‐T and G‐C base‐pairs are therefore uniquely suitable to facilitate position‐specific labeling of nucleic acids. Here, we employ the orthogonal kappa‐xanthosine‐base‐pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy‐version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA molecules with fluorophores or other labels., Orthogonal nucleic acid building blocks to the canonical A‐T and G‐C base‐pairs are uniquely suitable to facilitate position‐specific labeling. Here, the orthogonal kappa‐xanthosine‐base‐pair for in vitro transcription is employed. Through NMR spectroscopy, it is shown that xanthosine introduces only minor structural changes. A clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA, is also synthesized.

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1+/-0.2% and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons.