Thiolation Reagents

Sulfurizing reagents for synthesis of phosphorothioates:

Modified oligonucleotides as modulators of gene expression are currently in great demand. Development of novel oligonucleotides is under intense investigation as therapeutic agents of high specificity through antisense and antigene mechanism of action. Among the various oligonucleotide modifications reported to date, phosphorothioate (PS) oligonucleotides are the first class of antisense therapeutics.

• Phosphorothioate (PS) oligonucleotides are the first class of antisense therapeutics among the various oligonucleotide modifications reported to date.
• Phosphorothioates are isoelectronic analogues of natural phosphodiesters in which one of the oxygen atoms that does not participate in the internucleotidic linkage is replaced by a sulfur atom.
• A large number of PS-oligonucleotide drugs are currently being evaluated in pre-clinical and clinical studies as treatment for a wide range of diseases including cancer, cardiovascular disease, autoimmune diseases, diabetes, and infectious diseases.
• Thus, there is a great need to develop high yield, economical and robust methods for commercial scale production of high quality PS oligonucleotides.

To address the increased commercial need, for a highly efficient sulfurization method, has led to the development of a variety of sulfurizing reagents.

ChemGenes offer two different efficient sulfurizing reagents a) Beaucage Reagent and b) DDTT (Figure 1) as prepared formulated solutions as well as in bulk quantities as solid powdered material. Here we summarized practical guidelines on formulation conditions of various sulfurization reagents that ChemGenes offer for sulfurization step.

Note: Before attaching the any sulfurizing reagent bottle to the synthesizer, it is important to thoroughly wash the oxidation line with anhydrous acetonitrile then with the sulfurizing solution to remove traces of the previous solution.

Figure 1: Chemical structures of a) Beaucage Reagent (RN-1535) and b) DDTT (RN-1588) sulfurizing reagents.

Beaucage Reagent (3H-1,2-Benzodithiol-3-one-1,1-dioxide, RN-1535):¹

• Beaucage sulfurizing reagent served as a reagent of choice for several years due to its solubility in common organic solvents, its high efficiency as a sulfur-transfer agent in addition to its rapid sulfurization kinetics and facile adaptation to automation.
• Beaucage reagent is readily soluble in acetonitrile. However, it has displayed limited stability in solution (at least 1 month). This reagent can be stored indefinitely as a crystalline material in amber glass bottles at ambient temperature.
• The reagent decomposes rapidly when in contact with inorganic and organic bases. Beaucage reagent displays a fast reaction time of 30 seconds and >96% reaction efficiency (Table 1).
• Recommended concentration of Beaucage reagent for the sulfurization is 0.05M in acetonitrile (1 gr/100 ml) for 30 sec.

DDTT [((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione,RN-1588]:²

• An efficient sulfurizing reagent with quantitative (greater than 99%) PS conversion in oligonucleotide synthesis as compared to Beaucage reagent.
• A number of 1,2,4-dithiazolines are described in the literature^3,4 and demonstrate high sulfurizing efficiency during oligonucleotide synthesis.
• Our data has also shown superior sulfurization by DDTT reagent in solution phase. Unlike Beaucage reagent, DDTT did not yield any detectable level of phosphodiester by-product.
• DDTT has further advantage of greater stability in the formulated solutions.
• In solution phase we observed that DDTT (1-8 eq.) in Pyridine:acetonitrile (60:40, v/v) converts amidite fully into phosphorothioate within 2 minutes of reaction time. The conversion was found to be almost quantitative >99% to P-S and negligible amount of P-O formation occurred, as analyzed by 31P NMR.
• Recommended formulations for the synthesis of phosphorothioate oligonucleotides (DNA/RNA) are
  shown in Table 1.

Table 1: Our recommended formulation for the synthesis of phosphorothioates using DDTT.

• Analytical data of the 24-mer 2′-Fluoro phosphorothioate oligo (5′-GAGUUUCUUCCAAAGCAGCCUCUC-3′) synthesized using DDTT as sulfurizing reagent is shown in Figures 2 and 3.

Figure 2: CE analysis of 24-mer 2′-Fluoro phosphorothioate oligo (5′-GAGUUUCUUCCAAAGCAGCCUCUC-3′) using DDTT as sulfurizing reagent (purity greater than 98%).

Figure 3: ESI MS analysis of 24-mer 2′-Fluoro phosphorothioate oligo (5′-GAGUUUCUUCCAAAGCAGCCUCUC-3′) using DDTT as sulfurizing reagent (Target MS 7966).

References:
1. a) Beaucage, S. L. J. Amer. Chem. Soc. 1990, 112, 1253-1254; b) Iyer et al. J. Org. Chem. 1990, 55, 4693.
2. Guzaev, A. P. U.S. Pat. Number 7,723,528; Guzaev, A. P. WO2009148719 A1.
3. Xu, Q.; Musier-Forsyth, K.; Hammer R. P.; Barany G. Nucleic Acids Res. 1996, 24, 1602-1607.
4. Xu, Q.; Barany G.; Hammer R. P.; Musier-Forsyth, K. Nucleic Acids Res. 1996, 24, 3643-3645.