Synthesis of Prodrug‐Type Oligonucleotides Modified With a Galactosylated Self‐Immolative Linker Cleavable by β‐Galactosidase

Abstract This protocol describes procedures for the preparation of a modified thymidine conjugated with galactose via a self‐immolative linker at the O 4‐position, and the synthesis of β‐galactosidase‐responsive prodrug‐type oligodeoxynucleotides (ONs) containing these modified thymidines. These prodrug‐ONs are designed to be activated in response to β‐galactosidase, enabling targeted activation in specific cells or tissues and potentially contributing to the reduction of adverse effects. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of O 4‐modified thymidine phosphoramidite 7 Basic Protocol 2 : Preparation of O 4‐modified thymidine phosphoramidite 15 Support Protocol 1 : Preparation of triazolyl thymidine derivative 1 Support Protocol 2 : Preparation of benzyl alcohol derivative 2 Basic Protocol 3 : Preparation of β‐galactosidase‐responsive ODNs 1 to 5