Polysulfide Reagent in Solid-Phase Synthesis of Phosphorothioate Oligonucleotides: Greater than 99.8% Sulfurization Efficiency

Abstract

A solution of sulfur (0.1 M) and sodium sulfide (0.01 M) in 3-picoline, referred to as polysulfide reagent, rapidly converts trialkyl and triaryl phosphite triesters to the corresponding phosphorothioate derivatives. Greater than 99.8% average stepwise sulfurization efficiency is obtained in the solid-phase synthesis of DNA and RNA phosphorothioate oligonucleotides via the phosphoramidite approach.

Keywords:

• Sulfur
• Polysulfide
• Sulfurization
• Antisense
• Phosphorothioate
• Oligonucleotide

Notes

^aIn August 1998, Isis Pharmaceuticals, Inc., Carlsbad, CA and Ciba Vision, a division of Novartis AG, Switzerland, received FDA approval for VitraveneTM (fomivirsen sodium injectible) for the treatment of cytomegalovirus (CMV) retinitis in patients with AIDS.

^bA class of process-related substances that is frequently observed in PS-oligonucleotides is a group of oligonucleotides that are identical to the main product, except it contains one (or more) phosphate (PO) diester linkage(s) instead of a PS-diester linkage randomly distributed in the sequence [(PO)_n-oligonucleotide, n indicates the number of PO linkages]. Removal of (PO)_n-oligonucleotide on preparative scale using chromatographic separation technology is difficult to achieve without significant yield loss.

^cPolysulfide reagent (0.1 M, 0.5–2.5 mL) was added to a P(OPh)₃ solution (1 M, 0.25 mL). After 15 min, a sample (0.1 mL) was removed, diluted with acetonitrile (0.9 mL), and analyzed by HPLC (injection volume 0.02 mL). HPLC conditions: Phenomenx Luna C18(2), 5 µm, 250 × 4.6 mm, solvent A: ammonium acetate (0.1 M)/1% sodium hydroxide(1 N), solvent B: acetonitrile, linear gradient from 30% to 1% A in 20 min, flow 1.5 mL/min, detector wavelength 260 nm. Retention times: P(OPh)₃: 11.2 min, SP(OPh)₃: 10.0 min. The sulfurization effectiveness is the slope of the linear regression line of a plot of P(III) converted [a × b × 2.34c/(2.34c + d)] versus sulfur added [c(S) × volume] a=volume of P(OPh)₃ solution, b=concentration of P(OPh)₃ solution, c=peak area of SP(OPh)₃, 2.34=extinction factor correction, d=peak area of P(OPh)₃, ³¹P NMR shifts [ppm](CD₃CN): P(OEt)₃: 139.9, P(OPh)₃: 130.0, SP(OEt)₃: 68.5, SP(OPh)₃: 55.2.

^dOligonucleotides were synthesized on lab-scale (0.75 to 1 mmol) using an AKTA 100 solid-phase synthesizer. Primer Support 200 (Amersham, loading=approx. 200 µmol/g) was used as solid support. For detritylation we used dichloroacetic acid in toluene (10%). Standard phosphoramidites (0.2 M in acetonitrile) and 1H-tetrazole (0.45 M in acetonitrile) were used for coupling. The sulfurization reagent was prepared from sulfur and sodium sulfide (see text). For capping we used a mixture of acetic anhydride/pyridine/N-methyl imidazole/acetonitrile. Cleavage of the oligonucleotide (“DMT-on” mode) from the support and base deprotection was performed in conc. ammonium hydroxide at elevated temperature (50 to 60°C) for approximately 12 h. The crude oligonucleotide product was analyzed by LC-MS.