Solid-phase RNA synthesis

This group was developed for the protection of the 5-hydroxyl for solid-phase RNA synthesis. It is introduced with the silyl chloride, and pyridine and can he cleaved with TBAF in THE The trityl group introduces a chromophore for analytical purposes. ... 

Lyttle MH, Wright PB, Sinha ND, Bain JD, Chamberlin AR. New nucleoside phosphoramidites and coupling protocols for solid-phase RNA synthesis. J Org Chem 56 4608-4615, 1991. 

Figure 2.8 Solid Phase RNA Synthesis. 5 -dimethoxytrityl (DMT)-deprotection of resin bound 3 -terminal nucleoside residue is effected with trichloroacetic acid (TCA) (see Fig. 2.5). Thereafter the first coupling reaction is enabled by phosphoamidite activation with tetrazole (see Fig. 2.5) followed by oxidation of the newly formed diester linkage to a phosphodiester link. The process of 5 -DMT deprotection, phosphoramidite coupling and then diester oxidation, continues for as many times as required (n-times), prior to global deprotection and resin removal under basic conditions. RNA synthesis requires that 2 -hydroxyl groups are protected during the synthesis by tc/t-butyl dimethyl silyl (TBDMS) protecting groups labile only to fluoride treatment from tetra butyl ammonium fluoride (TBAF) (mechanism shown).

Reetz et al. described the solid-phase enzymatic synthesis of oligonucleotides on Kieselguhr-PDMA-resins via T4 RNA ligase. Goncomitantly, they found that RNase A selectively cleaves the last bound nucleotide at the ribose sugar leaving a 3, 5 - diphosphorylated ohgomer on the resin, but application in synthesis has not yet been undertaken [22]. 

This section examines the synthesis of nucleosides that contain seven-membered sugar analogues in place of the deoxyribose component. Nucleosides from the last group have been further incorporated into ONs via solid-phase DNA synthesis. A physical and biochemical investigation of the oligomers thus prepared continues in the next section. The study under review culminated in the assessment of the ability of the oligomers to complex with single-stranded RNA and for the heteroduplexes so formed to serve as substrates of RNAseH. 

A solid phase enzymatic synthesis of ODNs has been reported. After attaching a trinucleotide to a solid support, chain extension is carried out using T4 RNA ligase and nucleoside 3, 5 -diphosphates. The terminal phosphate is removed with alkaline phosphatase ready for the next extension. 

Whereas enzymes synthesize DNA and RNA in a 5 to 3 direction, chemical oligonucleotide synthesis is carried out in the opposite, 3 to 5 direction using a solid phase approach. Solid phase polynucleotide synthesis requires stepwise creation of a phosphate ester intemucleotide linkage. Currently, phosphoramidite... 

S. An efficient synthesis of enantiomeric ribonucleic acids from D-glucose. Helv. Chim. Acta 1997, SO 2286—2314). The protected enantiomeric cytidine was produced in 94% yield by the above reaction. After adjusting protecting groups, solid-phase oligonucleotide synthesis methods (Section 25.7) were used with this compound and the other three nucleotide monomers (also derived from L-ribose) for preparation of the unnatural RNA enantiomer. See also Vorbriiggen, H. Ruh-Pohlenz, C., Handbook of Nucleoside SynlhestsTy Wiley Hoboken, NJ, 2001. 

However, conversion of the dinucleoside //-phosphonate diester 10 to the phosphorothioate analogue 11 effected by a 0.02 M solution of 2 in 2% aqueous pyridine is relatively slow it reportedly takes 3 h for a complete reaction. While a solution of 2 in 2% aqueous acetonitrile containing triethylamine can completely transform 10 into 11 within 30 s, the use of this sulfurization mixture is incompatible with automated solid-phase oligonucleotide synthesis given the rapid formation of a yellow precipitate caused by triethylamine. In the absence of triethylamine, 10 is not sulfurized under these conditions. Nonetheless, 10 is completely converted to 11 within 20 min when a 0.02 M solution of 1 in 2% aqueous pyridine is used for the sulfurization reaction. Thus, 3i/-l,2-benzodithiol-3-one in aqueous pyridine is compatible with automated solid-phase synthesis of both DNA and RNA oligonucleoside phosphorodithioates or phosphorothioates from appropriate //-phosphonate derivatives. ... 

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