Solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s

Area of Science:

• Oligonucleotide Chemistry
• Medicinal Chemistry
• Biotechnology

Background:

• Oligo(ribonucleoside phosphorothioate)s are important therapeutic agents.
• Stereochemistry of phosphorothioate linkages significantly impacts biological activity.
• Existing synthesis methods often yield mixtures of stereoisomers, complicating purification and application.

Purpose of the Study:

• To develop the first solid-phase synthesis method for all-Rp-oligo(ribonucleoside phosphorothioate)s.
• To enhance the stereoselectivity of the key condensation and sulfurization steps.
• To produce stereochemically pure phosphorothioate oligonucleotides for potential therapeutic use.

Main Methods:

• Solid-phase synthesis utilizing H-phosphonate intermediates.
• Stereoselective sulfurization of H-phosphonate diesters.
• Enzymatic cleavage of Sp-phosphorothioate linkages using Nuclease P1.
• High-performance liquid chromatography (HPLC) for purification.

Main Results:

• Successful synthesis of mixed-sequence all-Rp-oligo(ribonucleoside phosphorothioate)s up to heptamer length.
• Achieved high stereoselectivity in the H-phosphonate diester formation (72-89% Sp under standard conditions, improved with modifications).
• Stereospecific sulfurization and enzymatic cleavage yielded products enriched with Rp-phosphorothioate linkages, with near-complete selectivity (>99%) achieved in optimized conditions.

Conclusions:

• A robust solid-phase method for synthesizing all-Rp-oligo(ribonucleoside phosphorothioate)s has been established.
• Optimized reaction conditions and building block modifications significantly improved stereoselectivity.
• This methodology provides access to stereochemically pure phosphorothioate oligonucleotides, advancing their therapeutic potential.