Prodrug‐Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment

• Published in: Chembiochem : a European journal of chemical biology Vol. 24; no. 24; pp. e202300526 - n/a
• Main Authors: Sugimoto, Norihito, Hayashi, Junsuke, Funaki, Ryohei, Wada, Shun‐ichi, Wada, Fumito, Harada‐Shiba, Mariko, Urata, Hidehito
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 14.12.2023
• Subjects: Antisense oligonucleotides; Disulfides; Endocytosis; linear disulfide promoieties; Nuclease; Oligonucleotides; Oligonucleotides - chemistry; Oligonucleotides, Antisense - chemistry; Phosphorothioate; phosphotriester modification; prodrug; Prodrugs; Prodrugs - chemistry; Prodrugs - pharmacology; Reagents; reductive activation; Synthesis; Transfection; prodrug; reductive activation; linear disulfide promoieties; phosphotriester modification; antisense oligonucleotides
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300526

Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug‐type phosphotriester‐modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular‐mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding to the fully phosphorothioate‐modified AON. In this study, we synthesized several types of PTE ONs containing linear disulfides (linear SS PTE ONs) and evaluated their conversion rates under reducing conditions. From the results, the structural requirements for the conversion of the synthesized linear SS PTE ONs were elucidated. Linear SS PTE ON with promising promoieties showed a nuclease resistance up to 4.8‐fold compared to unmodified ON and a cellular uptake by endocytosis without any transfection reagent. In addition, although the knockdown activity of the linear SS PTE gapmer AON is weaker than that of the fully phosphorothioate‐modified gapmer AON, the knockdown activity is slightly stronger than that of the cyclic SS PTE gapmer AON. These results suggest that the conversion rates may be related to the expression of the antisense activity. The prodrug‐type phosphotriester‐modified oligonucleotide bearing linear disulfide promoiety can be converted quickly into an unmodified oligonucleotide in a glutathione‐based intracellular mimetic reducing environment. The modification enhances the stability in serum and promotes the cellular uptake of oligonucleotide. In addition, the gapmer bearing linear disulfide promoieties can exhibit gene silencing activity.