Improved Method for the Solid-Phase Synthesis of Oligoribonucleotide 5′-Triphosphates

Abstract

We have developed an improved solid-phase method for the synthesis of 5′-triphosphates (5′-TPs) of oligoribonucleotides. The method is based on the use of salicyl phosphorochloridite as the phosphitylating reagent and the improvement is characterized by the use of the highly reactive pyrophosphorylating reagent tris(tetra-n-butylammonium) hydrogen pyrophosphate instead of the conventional tri-n-butylammonium salt for the nucleophilic substitution reaction to form the cyclic ester intermediate. The improved method can be used to generate oligoribonucleotide 5′-TPs efficiently and reproducibly.Key words 5′-triphosphate; oligoribonucleotide; solid phase; RNA; triphosphorylation 5′-Triphosphates (5′-TPs) of nucleosides and nucleic acids play an important role in biochemical reactions and have many research and medical applications. Thus, nucleoside 5′-TPs have long been known to be substrates for enzymatic DNA and RNA synthesis. A more recent discovery is the fact that the immune response triggered by the binding of 5′-TPs of RNA oligomers to retinoic acid-induced protein I (RIG-I) works together in synergy with gene silencing mediated by small interfering RNAs to promote apoptosis in tumor cells. 1)As for applications, 5′-TPs of DNA and RNA oligomers are used as substrates for ligation in vitro, while modified nucleoside triphosphates are used as antiviral medicines and diagnostic reagents. The demand for 5′-TPs of DNA and RNA is increasing and will most likely be met by chemical synthetic rather than enzymatic methods because of advantages resulting from the ability to introduce chemically modified nucleosides at any point in the sequence as well as the practicality of scaling up the synthesis. Chemical methods for the synthesis of 5′-TPs of RNA in particular have the additional advantage that they allow the production of highly pure RNA.A number of chemical syntheses of 5′-TPs of DNA and RNA have been reported. 2-9) Among these, a liquid-phase synthesis of 5′-TPs by reaction of the oligonucleotide with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl phosphorochloridite) developed by Ludwig and Eckstein 10) is well known.11) However, there are few reports of the solid-phase synthesis of DNA and RNA 5′-TPs. 4,9,11) A solid-phase adaptation of the salicyl phosphorochloridite method of Ludwig and Eckstein 10) has been developed by Schmidt et al.,12) who used it to synthesize the 5′-TP of a 25-mer RNA.For triphosphorylation experiments in the present study, we synthesized a 25-mer RNA oligomer by the 2-cyanoethoxymethyl (CEM) method of RNA synthesis developed in our laboratory. [13][14][15] In this method, the CEM group is used as the 2′-hydroxyl protecting group. Because the CEM group is small, it offers minimal steric hindrance and so allows an increased yield at each coupling step. As a result, and also because of the ease of removal of the CEM group under mild conditions after synthesis, RNA can be synthesized more simply and easily by the CEM method than by methods involving conventional 2′-hydroxyl protect...

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

“…Following the methodd eveloped by Ludwiga nd Eckstein, [12] Nagata et al showed that as olid-phase-bonded 25 mer RNAoligonucleotide could be phosphitylated using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl phosphorochloridite method) which was subsequently reactedw ith pyrophosphate to yield a5 '-cyclotriphosphitylated oligonucleotide followed by oxidation to give the 5'-triphosphate. [15] When tris(tetra-n-butylammonium) hydrogen pyrophosphate was used, the major product was the desired 5'-triphosphate with still as ignificant amount of 5'-OH and5 '-phosphonate oligonucleotide. However,w hen bis(tri-n-butylammonium)d ihydrogen pyrophosphate was used only al ow triphosphorylation efficiency was observed.I nt his case high amounts of the 5'-phosphonate were obtained.…”