Phosphorothioate oligonucleotide synthesis

Vu, H. and Hirschbein, B. L. (1991) Internucleotide phosphite sulfunzation with tetraethylthiuram disulfide phosphorothioate oligonucleotide synthesis via phosphoramidite chemistry. Tetrahedron Lett. 32, 3005-3008. 

A supported sulfide source has been demonstrated by Zhang et al.16 to synthesize oligonucleotide phosphorothioates (entry 12). Aminodithiazole-thione attached to a methacrylate-ethyleneglycol copolymer is used to efficiently convert a nucleotide phosphite to a phosphorothioate. The product nucleotide possesses protecting groups suitable for solid-phase oligonucleotide synthesis and thus is a valuable building block for nucleic acid therapeutics. 

An additional reason for the inferior performance of the H-phosphonate method compared to the phosphoramidite lies in its mechanism, which makes the synthesis very sensitive to pre-activation of the H-phosphonate monoesters with the conventional coupling agents, which must be avoided [138], However, the main appeal of the H-phosphonate approach lies in the ease it offers for the synthesis of backbone analogues modified at the phosphorus atom phosphorothioates [139], phos-photriesters [126] and especially phosphoramidates [126, 140, 141]. Now it is rarely used for routine oligonucleotide synthesis but very often for the preparation of variously modified analogues. Further developments in the H-phosphonate method have been made in the last decade by Polish [142] and Japanese researchers [143]. 

Scremin CL, Bonora GM. Liquid phase synthesis of phosphorothioate oligonucleotides on polyethylene glycol support. Tetrahedron Lett 34 4663-4666, 1993. 

The influence of diastereomeric ratios of deoxyribonucleoside phos-phoramidites on the synthesis of phosphorothioate oligonucleotides has been investigated. Almost diastereomerically-pure phosphoramidites were found to give an almost equal mixture of diastereomers of a monophosphorothioate decamer. A facile synthetic route to dimeric phosphorothioate building blocks 222 has been developed. Dinucleoside phosphite triesters were obtained in a one-pot procedure by sequential coupling to PCI3 of protected nucleosides with... 

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. ... 

In the area of modified intemucleosidic links, phosphorothioate oligonucleotides have been prepared by automated synthesis using the chemistry of Scheme 21,211 and Caruthers group have described two routes to di(deoxynucleoside)phosphorodithioates.2l2 Separable diastereomers of... 

Prior to 1990, this step was commonly performed with an elemental sulfur solution in a pyridine-carbondisulfide mixture. The utilization of [3H]-1,2-benzodithiol-3-one-1,1 -dioxide by Iyer et al. (15) for converting phosphorus (III) triester into pentavalent phosphorothioate has simplified the process of large-scale polyphosphorothioated oligonucleotide synthesis following phosphoramidite chemistry. This reagent, however, fails to convert H-phosphonate diester linkages into phosphorothioate diester, which is still achieved by treating with 5% elemental sulfur solution in pyridine-carbon disulfide off the instrument. 

Guga P, Stec WJ (2003) Synthesis of phosphorothioate oligonucleotides with stereodefined phosphorothioate linkages. Curr Protoc Nucleic Acid Chem 4.17 1-18 doi 10.1002/ 0471142700.nc0417sl4... 

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

---