Design, Synthesis, and Cellular Uptake of Oligonucleotides Bearing Glutathione-Labile Protecting Groups.

Glutathione-labile protecting groups for phosphodiester moieties in oligonucleotides were designed, synthesized, and incorporated into oligonucleotides. The protecting groups on the phosphodiester moieties were cleaved in a buffer containing 10 mM glutathione, which was used as a model of intracellular fluid. Cellular uptake of oligonucleotides bearing glutathione-labile protecting groups was strongly affected by the location and number of the protecting groups.

Conjugatable and Bioreduction Cleavable Linker for the 5'-Functionalization of Oligonucleotides.

An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5'-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5'-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.

Bioreductive deprotection of 4-nitrobenzyl group on thymine base in oligonucleotides for the activation of duplex formation.

Oligonucleotides containing 4-O-(4-NO2-benzyl)thymine residues were synthesized to assess potential prodrug-type action against hypoxic cells. These modified oligonucleotides were incapable of stable duplex formation under non-hypoxic conditions. However, following deprotection of the thymine residues under bioreductive conditions, the deprotected oligonucleotides were able to form stable duplexes with target oligonucleotides.