• Soluble and stable in anhydrous acetonitrile

  • Mixed bases 25-mer of DNA phosphorothioates gave less than 1% P=O using 60 second sulfurizing time

  • Smell less crystalline powder with formula weight 195.27

U.S. Patent #: 6,500,944

3-phenyl 1,2,4-dithiazoline-5-one* (POS) is a novel sulfurizing reagent, highly soluble and stable in anhydrous acetonitrile.

POS is a yellow crystalline product with no foul smell. 0.15M solution of POS in anhydrous acetonitrile is recommended for the synthesis of DNA phosphorothioates using contact time 60 second.
 

Solid phase synthesis of oligonucleotides involves the same generalized protocol. The approach comprises anchoring the 3’-position  of most  nucleoside to a solid support functionalized with amino/or hydroxyl moities and subsequently adding the additional nucleoside in stepwise manner. Internucleoside linkages are formed between the 3’-functional group of the incoming nucleoside and the 5’-hydroxyl group of the 5’-position of most nucleoside of the nascent, support-bound oligonucleotides. The internucleoside in phosphoramidite is a phosphite linkage. To create the sulfur-containing phosphorothioate internucleoside linkage, the phosphite linkage must be treated by an appropriate sulfur transfer reagent. The sulfurization takes place after each coupling, thereby providing the capability to control the state of each linkage in a site-specific manner

It is imperative that an efficient sulfur transfer reagent is used for the synthesis of oligonucleotides phosphorothioate via the phosphoramidite approach. Elemental sulfur is not efficient due to poor solubility and slow sulfurization reaction. A number of more efficient sulfurizing reagents have been reported in recent years. These includes phenylacetyl disulfide (PADS), H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), tetraethylthiuram disulfide (TETD), 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH), 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) and some others. PADS, Beaucage reagent and DDTT are commercially available.

Beaucage reagent and PADS have been widely used; however, its synthesis and stability are not optimal. In addition, the by product formed by Beaucage reagent during sulfurization, 3H-2,1-benzoxanthiolan-3-one-1-oxide, is a potential oxidizing agent that can lead to undesired phosphodiester linkage under certain condition. Therefore, its large scale synthesis of oligonucleotides phosphorothioate may not be particularly suitable. Both PADS and DDTT need pyridine as a solvent to dissolve. Pyridine has unpleasant odor and exposure limit 8H TWA 5 PPM (15 MG/M3). PADS also has very unpleasant odor to work. Therefore, its use at large scale synthesis of oligonucleotides phosphorothioate may not be particularly suitable.

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