Improved Impurity Profile of Phosphorothioate Oligonucleotides Through the Use of Dimeric Phosphoramidite Synthons

Abstract

Phosphorothioate oligonucleotides synthesized through assembly of dimeric phosphoramidite synthons show a significantly improved impurity profile compared to oligomers synthesized through coupling of standard monomer phosphoramidites. A greater than 70% reduction of the (n-1)-mer population and a ca. 50% reduction of phosphodiester linkages has been achieved.

Notes

Nomenclature: T₁₉ denotes a nonadecathymidylate phosphorothioate, (T₂)₉T and (TdC)₉T indicate monomer assembly, (T ₂)₉T, (TdC)₉T indicate dimer assembly, TdC indicates S-CE protection Cruachem, Inc., Glasgow, Scotland

Columns: dT-CPG (1μmol) from Glen Research. Standard detritylation, capping and 1H-tetrazole solutions (Applied Biosystems). Amidites (0.1 M in CH₃CN), coupling time 200 s. Sulfurization: 3H-1, 2-benzodithiol-3-one-1, 1-dioxide (0.2 M in CH₃CN, R. I. Chemical, Orange, CA), 900 s (not optimized)

Precise quantitation below 1% is difficult due to lack of baseline resolution. The actual value may be significantly lower.