O⁴-Modified thymidine analogues with photolabile o-nitrobenzyl group derivatives for modulating polymerase primer...

Photocaging has emerged as a robust and versatile strategy for achieving precise spatiotemporal control of complex biological processes through UV-induced photoactivation. Here, we sought to develop molecules that allow highly controlled modulation of polymerase function by employing sterically bulky photocaged compounds that are also synthetically accessible. Using the o-nitrobenzyl group, readily amenable to chemical modification, as a fundamental scaffold, we synthesized a series of derivatives with varying sizes and steric profiles, each conjugated at the C4 position of thymidine. These derivatives were incorporated into ODNs, and their duplex-forming abilities were evaluated. In all cases, a decrease in melting temperature was observed. Polymerase primer-extension assays further revealed that, although chain elongation was halted, derivatives bearing aromatic substituents, including the currently used NPOM group, underwent primer degradation due to exonuclease activity. Collectively, these results indicate that the newly synthesized derivatives featuring bulky substituents hold considerable promise for future applications.