The pro-oligonucleotide approach: solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates

Area of Science:

• Medicinal Chemistry
• Oligonucleotide Synthesis
• Prodrug Strategies

Background:

• Antisense oligonucleotides (ASOs) are a promising therapeutic modality.
• Developing stable and efficiently delivered ASOs remains a challenge.
• Prodrug approaches can enhance ASO stability and cellular uptake.

Purpose of the Study:

• To develop a modified phosphoramidite method for synthesizing SATE-protected thymidine phosphotriesters and thionophosphotriesters.
• To evaluate the stability and hydrolysis of these lipophilic oligonucleotide analogs in various biological environments.
• To assess the potential of SATE-protected oligonucleotides as prodrugs for antisense therapeutics.

Main Methods:

• Solid-phase synthesis using a modified phosphoramidite approach.
• Incorporation of S-acyl-2-thioethyl (SATE) protecting groups on internucleoside linkages.
• Stability studies in the presence of phosphodiesterases, cell extracts, sera, and gastric juice.
• Esterase-mediated hydrolysis was investigated.

Main Results:

• Efficient synthesis of lipophilic dodecathymidine phosphotriesters and thionophosphotriesters with SATE groups was achieved.
• The SATE-protected analogs demonstrated stability against snake venom and calf spleen phosphodiesterases.
• Selective hydrolysis to parent compounds occurred in CEM cell extracts.
• Me-SATE-protected dodecathymidine thionophosphotriester exhibited stability in human and mouse sera and human gastric juice.

Conclusions:

• SATE-protected oligonucleotides can be efficiently synthesized using a modified phosphoramidite method.
• These analogs exhibit favorable stability profiles in various biological matrices.
• Selective esterase-mediated hydrolysis suggests potential for targeted prodrug activation.
• SATE-protected oligonucleotides represent a promising strategy for developing effective antisense oligonucleotide prodrugs.