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Sulfurization Strategies for Phosphorothioate (PS) Oligonucleotide Synthesis

Phosphorothioate (PS) oligonucleotides are widely used due to their enhanced nuclease resistance and improved biological stability.

During synthesis, PS linkages are formed by replacing the standard oxidation step with a sulfurization step, making the choice of sulfurization reagent and reaction conditions critical to final product quality.

This article reviews key considerations for sulfurization chemistry, including reagent performance, reaction time, and analytical verification of complete sulfur incorporation.

Role of Sulfurization in Oligonucleotide Synthesis

In standard phosphoramidite chemistry:

  • Each coupling cycle forms a phosphite triester

  • This intermediate must be converted into a stable backbone linkage

For PS oligos:

  • Sulfurization replaces oxidation

  • Incomplete sulfurization results in mixed PO/PS backbones, which can negatively impact stability and performance

Sulfurization Reagent Performance

Different sulfurization reagents vary in:

  • Reaction kinetics

  • Stability during use on an instrument

  • Effectiveness across DNA and RNA chemistries

DDTT Performance

DDTT has been shown to perform very well as a sulfurization reagent, particularly in RNA workflows, delivering strong conversion at practical conditions and timeframes. When compared to more traditional sulfurization chemistries (such as Beaucage-type reagents), DDTT demonstrated more efficient sulfur transfer under shorter reaction times, especially for RNA synthesis.

Key observations

  • Modern sulfurization reagents can achieve efficient PS formation at relatively low concentrations

  • Short reaction times (on the order of minutes) are sufficient when kinetics are favorable

  • RNA chemistries (both TOM- and TBDMS-protected) are more sensitive to sulfurization efficiency than standard DNA

  • DDTT performed strongly compared to other common sulfurization approaches, making it a solid option when consistency and completeness matter.

For RNA synthesis in particular, reagents with faster sulfur transfer kinetics tend to produce more consistent results at shorter reaction times.

Reaction Time Considerations

Sulfurization time must be long enough to ensure full conversion of the phosphite intermediate, but not so long that it:

  • Reduces throughput

  • Increases exposure to harsh chemistry

General guidance:

  • 3 minutes is typically sufficient for effective sulfurization with modern reagents for DNA synthesis

  • 6 minutes for RNA oligos is generally recommended

  • As oligos increase in length, additional dwell time may be beneficial to overcome steric hindrance

What Happens When Sulfurization Is Insufficient

Incomplete sulfurization can produce a partially oxidized (PO) backbone mixed into an otherwise PS product. This can create multiple issues, including:

1) Reduced nuclease resistance

PO linkages are more susceptible to degradation, which can reduce stability and shorten functional lifetime in downstream applications.

2) Mixed product populations

Even a single missed sulfurization event can generate closely related species that may behave differently. This can lead to inconsistent results, broader or distorted chromatographic profiles or unexpected purity challenges


3) Lower yield of the intended full-PS product

If sulfurization is incomplete across multiple cycles, the proportion of “perfect” full-PS product decreases, lowering usable yield after purification.

4) More difficult troubleshooting

Sulfurization failures can look like other synthesis problems (sequence-dependent issues, reagent aging, or fluidic inconsistencies), which may extend troubleshooting time unless sulfurization is evaluated directly.

Practical Recommendations

  • Select sulfurization reagents with fast kinetics and good stability for on-instrument use

  • Optimize sulfurization time based on:

    • DNA vs RNA chemistry

    • Oligo length

    • Instrument dwell capabilities

  • Use ion exchange HPLC or equivalent methods to detect incomplete sulfurization

  • Re-evaluate sulfurization conditions when scaling length or switching chemistries

Summary

Efficient sulfurization is a critical step in producing high-quality phosphorothioate oligonucleotides. Proper reagent selection, optimized reaction time, and robust analytical verification help ensure complete sulfur incorporation, consistent performance, and reliable downstream results—especially for RNA oligos and longer sequences.