Glen Report 18.13: Sulfurizing Reagent II - Stable in Solution and Optimized for RNA Sulfurization

The use of a sulfurizing reagent during the regular synthesis cycle using phosphoramidite chemistry has revolutionized the production of phosphorothioate oligonucleotide analogues. Undoubtedly, this ease of preparation of phosphorothioates has made this oligonucleotide modification by far the most common in research. Glen Research was one of the first sources of the sulfurizing reagent, 3H-1,2-benzodithiol-3-one 1,1-dioxide, popularly known as Beaucage Reagent (1).1 This sulfurizing reagent has found common use in the face of a plethora of rival reagents over the years because of its high efficiency, fast reaction time, and widespread availability. The one mild flaw we have found with Beaucage Reagent is that, although it is quite stable in acetonitrile solution in a silanized amber bottle, it is has relatively poor stability in solution once installed on the DNA synthesizer. Consequently, we have not been able to supply a sulfurizing solution, preferring to supply the powdered reagent along with an appropriate silanized bottle. The customer then weighs an appropriate amount of reagent into the silanized bottle and adds acetonitrile at a concentration of 1g/100mL. Over the years, we have considered other sulfurizing reagents but we were not able to find another reagent that exhibits the same fast sulfurization kinetics along with improved stability on the synthesizer. RNA Sulfurization

Sulfurizing Reagent (Beaucage) (1)
Sulfurizing Reagent II (DDTT) (2)
Figure 1: Structures of Sulfurizing Reagents

The most common usage for oligonucleotide phosphorothioates has been in the production of antisense oligodeoxynucleotides destined for use in identifying or modifying gene expression. Now, phosphorothioate linkages are popping up in the RNA world and sulfurizing RNA linkages with reagents like Beaucage Reagent has proved to be much more difficult than DNA linkages. The phosphorothioate (PS) linkage is a not-so-expensive way of increasing the stability of nucleic acids and increasing nuclease resistance of RNA. Now, it has been shown2 that fully PS oligos can promote the delivery of siRNA in cell culture. This siRNA uptake is sequence-independent and the length seems to vary between 30 and 70 nucleotides depending on the cell line. Even though this method is not yet as efficient as the cationic lipids, it opens the way to possible new methods. Reasons that may explain this are not understood at this time.

Another paper3 describes a method for the inactivation of micro RNA (miRNA) that may help to elucidate their functions. It uses 2’-OMe-RNA oligonucleotides (23-mers, complementary to a target miRNA) with a cholesteryl group at the 3´terminus and phosphorothioates at positions 1 and 2 at the 5´end and at the last four positions at the 3´end. These oligos are called antagomirs. These molecules promote the cleavage of complementary miRNAs and thus should allow analysis of their function. The role of the PS linkages presumably is the stabilization against degradation in the mouse experiments as it is standard in the antisense field in such in vivo situations. And finally, a recent paper4 shows that PS does not systematically abolish siRNA activity, opening the way for some potentially less expensive stabilization of such molecules. Incorporation of 2’-OMe (in the sense strand) in combination with PS linkages should confer to siRNA increased resistance to degradation by nucleases, as well as prolonged serum retention. And it is also possible that such easy modification of siRNA may increase the specificity by eliminating sense strand recruitment in the RISC complex and thus reducing a source of off-target effect.

Sulfurizing Reagent II

A new sulfurizing reagent must, therefore, exhibit all the good properties of Beaucage Reagent while adding good stability in solution on the synthesizer AND offering strong ability to sulfurize RNA linkages. We are happy to offer Sulfurizing Reagent II, 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT (2).

Use of Sulfurizing Reagent II in RNA Synthesis

Our experiments demonstrate that a 0.05 M solution of Sulfurizing Reagent II is recommended for the synthesis of RNA phosphorothioates. A sulfurizing time of 2-4 minutes generated oligophosphorothioates of high quality. This was true for both TOM-RNA and TBDMS-RNA monomers. As shown in Figure 2, Beaucage Reagent was significantly more sluggish than Sulfurizing Reagent II. Representative HPLC analyses5 of RNA oligos are shown in Figure 3. The chromatogram on the left was obtained from sulfurizing U-TOM-RNA linkages for 60 seconds with Beaucage Reagent. The large n-1 peak is due to incomplete stepwise sulfurization and accumulation of deletions. The chromatogram on the right was an identical synthesis except using Sulfurizing Reagent II. Individual RNA sequences, especially those containing stretches of purine nucleoside residues are more difficult to sulfurize irrespective of the reagent used. To obtain a high degree of sulfurization with those oligonucleotides, a 0.1 M solution of Sulfurizing Reagent II and/or extended contact time may be required.

Figure 2
Figure 2: Cycle efficiency using sulfurizing reagents and U-TOM-RNA monomer
Figure 3
Figure 4

Figure 3: Ion exchange HPLC analysis of RNA oligos sulfurized with sulfurizing reagents

Column: Dionex B100, 4.6mm x 250mm
Buffers: A- 10mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile, pH 7.4 ;
B- 600mM NaClO4, 25mM TRIS-HCl, 20% Acetonitrile,
pH 7.4 Flow rate: 1mL/min

Use of Sulfurizing Reagent II in DNA Synthesis

Sulfurizing Reagent II was compared to Beaucage Reagent in the synthesis of DNA phosphorothioates. The quality of the products was identical with both reagents. In DNA synthesis, the cycle efficiency seemed to be optimal for Sulfurizing Reagent II when using a 60 second sulfurizing time.

Solubility and Stability of DDTT

The solubility of Sulfurizing Reagent II in mixtures of anhydrous pyridine and acetonitrile or anhydrous pyridine and THF is relatively limited and increases with the increasing concentration of pyridine. Some useful compositions are listed in Table 1. Under these conditions, Sulfurizing Reagent II forms stable solutions that do not display any loss of functional activity or precipitation of the reagent for a period of over 6 months. To prepare solutions of the desired concentration, we recommend first dissolving Sulfurizing Reagent II in the calculated amount of pyridine, which may require mild heat, followed by diluting the obtained solution with the required volume of acetonitrile or THF.

Table 1: Solubility of Sulfurizing Reagent II
Concentration of Sulfurizing Reagent II Solvents and their ratio
Pyridine:ACN (v/v) Pyridine:THF(v/v)
0.1M 100:0 40:60
0.06M 50:50 -
0.05M 40:60 20:80
0.03M 30:70 -
0.02M 20:80 0:100
Notes: Dissolve Sulfurizing Reagent II in pyridine first and then add acetonitrile (ACN) or tetrahydrofuran (THF). Molecular Weight is 205.31

We are happy to offer Sulfurizing Reagent II as a powder and as a 0.05M solution in pyridine/acetomitrile.


  1. R.P. Iyer, W. Egan, J.B. Regan, and S.L. Beaucage, J. Amer. Chem. Soc., 1990, 112, 1253-1254.
  2. M. Overhoff and G. Sczakiel, EMBO Rep, 2005, 6, 1176-81.
  3. J. Krutzfeldt, et al., Nature, 2005, 438, 685-9.
  4. B.A. Kraynack and B.F. Baker, RNA, 2006, 12, 163-76.
  5. B.J. Bergot and W. Egan, Journal of Chromatography, 1992, 599, 35-42.

Product Information

Sulfurizing Reagents