Rapid synthesis of oligonucleotides by the phosphotriester method

An efficient phosphotriester methodology based on the use of condensing agents in the presence of several O-nucleophilic catalysts has been developed.     

Further improvements on the phosphotriester synthesis of deoxyribooligonucleotides and the oligonucleotide directed site-specific mutagenesis of E. coli lipoprotein gene.

Two improvements that greatly enhance the rate of phosphotriester oligonucleotide synthesis are described: 1) use of hindered primary amines, e.g. t-butyl amine for decyanoethylation of oligonucleotide triester intermediates, and 2) a simplified isolation procedure that eliminates the tedious bicarbonate extraction after each condensing reaction. Using the improved procedures, oligonucleotide fragments can be synthesized as rapidly as using solid phase chemistry. The final products are purer than those obtained by solid phase chemistry since each intermediate block is purified by chromatography. The technique has been used to synthesize five oligonucleotide fragments (size 15 to 20) for the purpose of performing guided site-specific mutagenesis on a cloned E. coli lipoprotein gene.     

Rapid synthesis of oligodeoxyribonucleotides VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support

A new kieselguhr-polydimethylacrylamide support has been used in a continuous flow, column system for solid phase synthesis of oligodeoxyribonucleotides by a phosphotriester procedure. Using only protected mononucleotides a 14-mer, 20-mer and 27-mer were assembled in high repetitive yield using a simple manually operated, bench top apparatus.     

Polymer supported synthesis of oligonucleotides by a phosphotriester method

A new polymer supported synthesis of deoxyribooligonucleotides which can be adapted to automation is described. The method is based on the elongation of an oligomer chain in the 5′- to 3′ -end direction using the modified phosphotriester chemistry. The approach is exemplified by the synthesis of a nonanucleotide d(TTCGTCTTG).     

Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA.

A method for phosphorylating a protected deoxyribooligonucleotide containing phosphotriester linkages is described. The modified phosphotriester method of chemical synthesis is further refined in terms of (i) better final deblocking conditions and (ii) new chromatography solvent systems containing acetone-water-ethyl acetate to yield pure oligomers. The effectiveness of these improvements has been demonstrated in the rapid and efficient synthesis of seventeen fragments constituting the sequence of human insulin C-chain DNA.     

New effective method for the synthesis of oligonucleotides via phosphotriester intermediates.

A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.     

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.     

Solid-phase synthesis of polynucleotides. II. Synthesis of polythymidylic acids by the block coupling phosphotriester method.

Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.     

Pre-activation strategy for oligodeoxyribonucleotide synthesis using triaryloxydichlorophosphoranes in the phosphotriester method.

Triaryloxydichlorophosphoranes were tested as condensing agents for oligodeoxyribonucleotide synthesis in the phosphotriester method. Tris(2,4,6-tribromophenoxy)dichlorophosphorane (BDCP) was found to be a relatively stable crystalline material which could be used as a chemical reagent. A notable feature of the BDCP-promoted condensation reaction was studied by 31P-NMR. A small amount of BDCP compared to the conventional condensing agent was effective for the generation of active nucleotide intermediates and BDCP itself was quantitatively converted into an inert material, tris(2,4,6-tribromophenyl)phosphate (2). Thus, BDCP enabled us to separate the activation step from the condensation process in the phosphotriester method. This preactivation method was applied to the solid-phase synthesis.     

An azidomethyl protective group in the synthesis of oligoribonucleotides by the phosphotriester method

A rapid and effective method of an automatic oligoribonucleotide synthesis alternative to the phosphoramidite one was developed. This method is based on the phosphotriester approach to internucleotide bond formation under intramolecular O-nucleophilic catalysis and the use of an azidomethyl group for protection of a nucleotide 2′-hydroxyl function.     

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method.

A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.     

Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method.

Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGpTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The synthesis were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3'-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.     

Synthesis of oligonucleotides on cellulose by a phosphotriester method.

The synthesis of oligothymidilic acids, (dT)m (where m = 4, 7, 10, 13, 16, 19, 22, and 25), was carried out using a solid phase approach in combination with the modified phosphotriester methodology developed in solution. Cellulose was used as the solid support after its functionalization with a specially featured dinucleoside diphosphate, 5'-0-p-chlorophenylphospho-2'(3')-0-acetyluridilyl-[2'(3')-3']-5'-0-dimethoxytritylthymidine p-chlorophenylester. The fully protected trideoxynucleoside triphosphate containing only thymidine was repeatedly used to elongate the oligonucleotide chain in the 3'-5' direction. Individual coupling yields ranged from 45% to 75%. The total time needed to prepare (dT)25 was four days. Similarly, the tridecanucleotide d(AGAAGGTACTTTT) was synthesized in good yield. The results show that this approach can be used for a fast and economic way to synthesize oligodeoxynucleotides.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in the synthesis of oligoribonucleotides by the phosphotriester method

An efficient synthetic method for monomer ribonucleotide synthons containing 2'-O-methoxymethyl and 2'O-(p-nitrobenzyloxy)methyl groups used for oligonucleotide phosphotriester method with O-nucleophilic intramolecular catalysis at the stage of formation of internucleotide bond is developed. It is shown that synthons containing protecting 2'-O-(p-nitrobenzyloxy)methyl group may be used for automatic synthesis of phosphotriester oligoribonucleotides with high yields and synthons containing methoxymethyl group--to get 2'-O-modified oligonucleotides.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.     

Arylsulfonyltetrazoles, new coupling reagents and further improvements in the triester method for the synthesis of deoxyribooligonucleotides.

The modified triester approach has been further improved and refined to the synthesis of defined sequences of deoxyribo-oligonucleotides. Improvements include arylsulfonyltetrazoles as faster and milder condensing agents, benzenesulfonic acid to avoid depurination during deblocking of trityl protecting groups and improved chromatographic procedures for purification of triester intermediates and purification of the final product containing 3'-5' phosphodiester linkages.     

Monomers containing 2'-o-alkoxymethyl groups as synthons for the synthesis of oligoribonucleotides by the phosphotriester method

A general scheme for the synthesis of ribonucleotide monomers containing alkoxymethyl group in 2'-O-position for the solid-phase phosphotriester oligonucleotide synthesis using O-nucleophilic intramolecular catalysis has been developed. In particular, the monomers containing 2'-O-modifying 2-azidoethoxymethyl, propargyloxymethyl, or 3,4-cyclocarbonatebutoxymethyl groups has been prepared.     

A general method for the synthesis of 5'-monophosphates of DNA fragments via phosphotriester intermediates.

A new and attractive phosphorylation procedure which allows the introduction, via phosphotriester intermediates, of 5'-phosphate functions of DNA fragments is described. The method is based on the activation of bifunctional phosphorylating agents with 1-hydroxybenzotriazole. The approach will be exemplified by the synthesis of pACGC using four different 5'-phosphotriester intermediates.