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 VIII.1 An Improved Procedure for Solid-Phase Oligodeoxyribonucleotide Synthesis Using a Controlled Pore Glass Support and Phosphotriester Chemistry

Abstract

An improved method for solid phase oligodeoxyribonucleotide synthesis is described that uses phosphotriester chemistry, controlled pore glass as support, a new and more stable linkage agent and a better protecting group combination.     

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.     

A new method for introducing amidate linkages in oligonucleotides using phosphoramidite chemistry.

Cyanoethyl-protected phosphotriester links in oligonucleotides made with standard pophosporamidite chemistry were converted to pbosphoramidate linkages during oligonucleotide synthesis on solid support. The cyanoethyl group was removed with piperidine, and the resulting phosphodiester was activated with p-tosyl chloride. An amine nucleophile displaced the tosyl to yield a phosphoramidate linkage.     

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.     

Efficient methods for attachment of thiol specific probes to the 3''-ends of synthetic oligodeoxyribonucleotides.

Methodology is described for the synthesis of DNA oligomers containing a free 3'-thiol group which can be selectively crosslinked with a wide variety of probes. This chemistry is compatible with both phosphotriester and phosphoramidite solid phase chemistry. Moreover, the sulphydryl group is introduced into the 3'-nucleoside solid support linkage prior to oligonucleotide synthesis. Consequently, no additional coupling steps are required after oligonucleotide synthesis, and isolation of the 3'-thiol oligonucleotide requires only one additional deprotection step. Cross-linking of the thiol-containing oligonucleotide to a fluorescent probe was carried out with high selectivity, in high yield, and under mild conditions.     

Approach to the synthesis of natural and modified oligonucleotides by the phosphotriester method using O-nucleophilic intramolecular catalysis

An approach to the solid phase synthesis of natural and modified oligonucleotides using phosphotriester technique has been developed. Particularly, this method allows the synthesis of ribo- and deoxyribo-oligonucleotides containing various 2'-modified mononucleotides as well as stereodefined nucleotide phosphorothioate analogues.     

Improved synthesis of oligodeoxyribonucleotide using 3-methoxy-4-phenoxybenzoyl group for amino protection.

3-Methoxy-4-phenoxybenzoyl group has been used for the protection of exocyclic amino group of nucleosides. In case of 2'-deoxycytidine it has been found to be highly selective under controlled conditions. The N-protected derivatives of 2'-deoxyadenosine and 2'-deoxyguanosine have been found to be sufficiently stable towards acids minimising depurination under conditions of synthesis of oligodeoxyribonucleotide on solid support via phosphotriester approach. The high lipophilicity of the group and milder deprotection conditions are additional advantages.     

Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support.

A simple solid phase method for the synthesis of oligodeoxyribonucleotides has been developed using the phosphotriester approach. Mononucleotide coupling units are sequentially added to the polystyrene copolymer with 1% divinylbenzene and two kinds of oligonucleotides, d(CACGACCCCTCCACGT) and d(AACTGGTATTACTGGGCG), are synthesized in a relatively high yield. One cycle of the mononucleotide addition is about 70 minutes, and this method is particularly suitable for the automation of the synthesis upon availability of an automatic synthesizer.     

Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives.

A procedure is described for linking nucleosides covalently to controlled pore glass or cross-linked polystyrene supports by means of an oxalyl anchor. Though stable to triethylamine and diisopropylamine, the nucleoside-oxalyl link can be cleaved within a few minutes at room temperature with ammonium hydroxide in methanol. This new anchor can be used in automated synthesis of conventional oligonucleotides. The primary value, however, is that it enables one to employ solid support methodology to synthesize a variety of base-sensitive oligonucleotide derivatives, as illustrated here by synthesis of oligomers with base protecting groups intact and with methyl phosphotriester groups at the internucleoside links.     

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.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

Utilisation d'Un Nouveau Type de Support Pour la Synthese d'Oligonucleotides en Phase Solide

Abstract

A method of oligonucleotide synthesis was developped on a new type of support by the phosphotriester approach. Using this method the heptanucleotide dT(pT)₆ was synthetized in 68 % yield.     

Gel-phase P-NMR. A new analytical tool to evaluate solid phase oligonucleoside synthesis.

This paper shows Gel-phase ³¹P-NMR spectra of synthetic intermediates obtained during solid-phase oligonucleotide synthesis on polystyrene for the first time. We have demonstrated the application of this technique using the phosphotriester, H-phosphonate and phosphite triester approaches. The use of Gel-phase ³¹P-NMR for monitoring solid phase oligonucleotide synthesis is discussed.     

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.     

Synthese d'Oligodesoxyribonucleotides sur Support de Gel de Silice par la Methode au Phosphotriester

Abstract

A method of oligonucleotide synthesis was developped on a silica gel support by the phosphotriester approach. Using this method, the nonanucleoside octaphosphate dT(pT)₈ was synthetized in 28% yield.     

Automated Synthesis of Cyclic Oligodeoxyribonucleotides via Phosphoramidite Method

Abstract

The functionalized polyethylene glycol/polystyrene copolymer support 4 was shown to be suitable for a completely automated synthesis of small- to medium-sized cyclic oligodeoxy-ribonucleotides. Syntheses of the linear precursors were achieved by the phosphoramidite method, whereas the cyclization reactions were based on the phosphotriester chemistry.     

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.     

The pro-oligonucleotide approach: solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates.

A modified phosphoramidite method has been designed for the solid-phase synthesis of two dodecathymidine phosphotriesters and two dodecathymidine thionophosphotriesters. In these analogs, each internucleoside link bears an S -acyl-2-thioethyl (Me-SATE or tBu-SATE) group removable upon esterase activation. Efficient synthesis of these lipophilic analogs was achieved thanks to the use of a photolabile linker anchored to the solid support in combination with thymidine-3'- O -phosphoramidites having a SATE group in place of the regular 2-cyanoethyl one. Both dodecathymidine phosphotriester and thionophosphotriester having S -acetyl-2-thioethyl groups were found to be stable in the presence of snake venom and calf spleen phosphodiesterases whereas, upon incubation in CEM cell extracts, they were selectively hydrolyzed to the anionic parent dodecathymidylate and dodecathymidine phosphorothioate, respectively. In addition, Me-SATE-protected dodecathymidine thionophosphotriester was stable in mouse and human sera as well as in human gastric juice. These results depict the potential of SATE-protected oligonucleotides as prodrugs of antisense oligonucleotides.     

cDNA文库固相构建法

本文介绍一种cDNA文库的固相构建法。文库构建过程cDNA的合成和修饰全部在固相介质—磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。