Methods for Synthesis of Phosphorodithioate RNA

Requirement

Methods for Synthesis of Phosphorodithioate RNA Assignment

Solution

Introduction

The isoelectronic analogs of the natural oligonucleotides where one of the non-bridging internucleotide oxygen atoms were replaced by a single sulfur atom can be regarded as phosphorodithioate RNA. These analogs are found to be useful towards the antisense studies owing to their resistance towards the endonuclease degradation in vivo as compared to the other corresponding phosphodiester. These phosphorothioate bonds are placed throughout the whole oligonucleotide or within specific positions. The structure of phosphorodithioate (PS2) linkages have been depicted in figure 1.

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Many types of research have shown that the phosphorodithioate linkages bind proteins firmly with higher affinity as compared to phosphodiester analogues that have suggested that the phosphorodithioate RNA has additional utility towards the therapeutic as well as diagnostic applications in the form of their sulfur modified phosphate ester aptamers also known as thioaptamers. 
Many studies also indicated that these oligomers and RNAs can bind firmly to the active site of primer-template and thereby interfere with the enzyme function resulting in the very low and even subnanomolar inhibitory constants. Phosphorodithioate deoxyoligonucleotide inhibitors are found to have most potential in doing the function and can bind to the primer-template active site of HIV-1 RT thereby helps in the synthesis of potential therapeutic drug or agent against HIV. This paper discusses the various methods for synthesis of phosphorodithioate RNA. 

Discussion

Phosphorothioate linkages are indicated as (-OP2O-) and the phosphorodithioate RNA can be prepared by various methods including synthesis of phosphorodithioates via thioamidites (Greef, 1996), synthesis of phosphorodithioates RNA using H-phosphonothioate method (Brill et al., 1989). 
For the synthesis of phosphorodithioate RNA using the deoxynucleoside 3'-pyrrolidinyl- or deoxynucleoside 3’-(dimethyl-amino) phosphorothioamidites were used. The protected deoxynucleoside is condensed with S-(4-chlorobenzyl)-N, N, N’, N’-tetraisopropylphosphorothiodiamidites. The various famous thiophosphoramidites can be used for the preparation of phosphorodithioate RNA.
For the preparation of phosphorodithioate RNA, the Thiophosphoramidites (thioPA) should be dissolved thoroughly. The dried form of the thioPA is extremely stable at the low temperature of around -20°C and can remain unaffected for at least one hour. 
For this process, the most recommended synthesis conditions involve:

  • A diluent preferably DCM in acetonitrile;

  • Activator : 0.25M ETT or 0.25M DCI

  • Coupling: 5 to 10 minutes at the one µmole synthesis scale  

  • Sulfurization: Sulfurizing Reagent II (DDTT) 

  • Cleavage and Deprotection: Ammonium hydroxide: Ethanol (3:1, V/V).

Most commonly used Thiopa are given below:

 In this preparation method, (dimethylamino) chlorophosphine or dipyrrolidinylchlorophospine (0.6 mmol) was added to protected deoxynucleoside (0.5 mmol) that was placed in exactly 4ml of acetonitriletriethylamine (2:1, v/v). As soon as they were added, precipitation occurs. The precipitate was kept untouched for 5 to 10 minutes. Later, to the precipitate one mmol of 2, 4-dichloro-benzylmercaptan or 4-cholorobenzylmercaptan was added in vacuo. Some reaction took place, and the reaction products were then dissolved in 4 mL of acetonitriletriethylamine (2:1, v/v). The TLC and PNMR were taken for the product indicating that the product was formed without formation of any assumed side products like bis (deoxynucleoside 3’)- phosphorothioite or deoxy-nucleoside 3’-phosphorodithioite. Hence, it can be seen that with the help of a suitable as well as appropriate an amine salt as the acid catalyst, the selective activation of a solitary phosphorous-nitrogen bond present in phosphorodiamidites is possible. The possible steps towards preparation of the phosphorodithioate RNA can be seen below:

 In H-phosphonothioate method, the ribonucleoside 3′-H-phosphonothioates were utilized for the synthesis of phosphorodithioate RNA. The steps for the preparation of the phosphorodithioate RNA with the same method has been included below in figure 2:

 The use of the in H-phosphonothioate method for preparation of the phospohorodithioate RNA can be done by taking the synthons as 2'-deoxy and ribonucleoside H- phosphonothioate monoester. In the figure 2, a general scheme has been shown. For removing the 5'-dimethoxytrityl protecting group in the acidic medium, 2'-deoxy or ribonucleoside H- phosphonothioate monoester was condensed and along with it an activating agent was added. The activating agent can be adamantoyl chloride or pivaloyl chloride. The corresponding mixed phosphonocarboxylic anhydride was produced by reacting the activating agent- acid chloride with the H- phosphonothioate monoester. This so produced mixed anhydride then undergoes the nucleophilic attack at the position with the phosphorous via the nucleosides’ 5’-hydroxyl that is joined to the polymer support.
This reaction produces H- phosphonothioate diester. This linkage is stable compared to the phosphite triester, especially towards the acidic medium (3% of the trichloroacetic acid in dichloromethane) that is required for removing the 5'-dimethoxytrityl group. Therefore, it becomes unnecessary for carrying out the process of oxidation during every cycle. When the chain has to be elongated for phospohorodithioate RNA, two steps are followed in order to achieve that. The phosphodiesters oxidized using aqueous iodine for the synthesis of phospohorodithioate RNA. Alternatively, various reagents can be used during the process of the reaction so as to oxidize the phosphodiesters and is beneficial for synthesizing a large number of modified RNAs and DNAs. Once the oxidation step is completed, the aqueous ammonia is added for removing the nucleobase amide that was used as a protecting group and for cleaving the oligonucleotide from the basic support. 
Further, the phospohorodithioate RNA can also be prepared by reacting the RNA synthesizer with the thiophosphoramidites to form phosphorodithioate. 

Conclusion

The paper discusses various methods for the preparation of the phosphorodithioate RNA. The paper gave insight into the importance of phosphorothioate RNA and its use in the sciences and research field. The paper has discussed the methods for the synthesis of the phosphorodithioate RNA. And gives a thorough detail on the preparation of phosphorodithioate RNA using the thiophosphoramidites (thioPA) and H- phosphonothioate diester methods. 
The synthesis of phosphorodithioate RNA can be a beneficial step towards identification of various antisense drugs and against the HIV-1.

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References

  • Brill, W., Tang, J., Ma, Y. and Caruthers, M. (1989). Synthesis of oligodeoxynucleoside phosphorodithioates via thioamidites. J. Am. Chem. Soc., 111(6), pp.2321-2322.

  • Cummins, L., Graff, D., Beaton, G., Marshall, W. and Caruthers, M. (1996). Biochemical and Physicochemical Properties of Phosphorodithioate DNA †. Biochemistry, 35(26), pp.8734-8741.

  • WT, W. (1993). Synthesis and purification of phosphorodithioate DNA. Methods Mol Biol., 20(191).

  • Roy, S. and Caruthers, M. (2013). Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries. Molecules, 18(11), pp.14268-14284.

  • JL, T. (1994). Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: a comparative study. Antisense Res Dev, 4(78).

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