Synthesis of cyclic oligonucleotides by a modified phosphotriester approach.

Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].     

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).     

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.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides

20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R') TGATCCGTA, where R' = R' = H (20), F' = Et, R' = H (20-Et), or R' = R' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.     

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.     

Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.     

Synthesis of chimeric oligonucleotides containing internucleosidic phosphodiester and S-pivaloylthioethyl phosphotriester residues

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.     

Synthesis of oligoribonucleotides containing 2'-O-methoxymethyl group by the phosphotriester method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 '-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 '-О-methoxymethyl-containing oligoribonucleotides were examined.     

Synthesis of phosphorothioate-containing DNA fragments by a modified hydroxybenzotriazole phosphotriester approach.

The phosphorothioylating agent which was obtained by treating 2,5-dichlorophenyl phosphorodichloridothioate with 1-hydroxy-6-nitrobenzotriazole proved to be very effective for the synthesis in solution and on a solid support of phosphorothioate-containing DNA fragments.     

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.     

Synthesis of ppTppp via phosphotriester intermediates.

The bifunctional and crystalline phosphorylating agent morpholino-0,0-bis[1-benzotriazolyl]phosphate has been used for the preparation of a 3',5'-bis-phosphotriester intermediate of thymidine. The latter has been converted into ppTppp by the following consecutive steps; removal of the benzotriazolyl group followed by the addition of phosphoric acid and removal of the 2-(4-nitrophenyl)-ethyl group followed by the addition of pyrophosphoric acid.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo

A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.     

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.     

N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligonucleotides

An effective modification of phosphotriester method for automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and 2-(azidometil)benzoyl group to protect amino groups of heterocyclic bases of nucleotides is described.     

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.     

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 oligodeoxyribonucleotide with aliphatic amino or phosphate group at the 5'' end by the phosphotriester method on a polystyrene support.

Lipophilic protecting groups mTrNH(CH2)n X (mTr:monomethoxytrityl, X = NH,O,S, n = 2,3,4,6) were attached to the 5'-phosphoryl group of 3'-O-protected thymidine. When the diamine derivatives (X = NH2) were used, the time course of the stability of mTr groups on the amino group and the phosphoramidate linkage with 80% aq. AcOH was measured. It was found that the mTr group was removed from the amino group rapidly and that the phosphoramidate linkage was more stable. It's stability depended upon the length of the CH2 linker. Oligonucleotides with an aliphatic amino group at their 5'-ends were synthesized by the phosphotriester method on a polystyrene support using protected nucleotides with P-O or P-S linkages. In the case of product with a P-S linkage, 5'-O-phosphorylated nonadecanucleotide was also prepared by I2-H2O treatment.     

Synthesis of phosphotriester by DCC and its biological activities

The synthesis of a phosphotriester, an inhibitor of acetylcholinesterase, was performed by the coupling reaction of diethylphosphate with various phenolic compounds using dicyclohexylcarbodiimide (DCC). All of the compounds synthesized inhibited housefly acetylcholinesterase activity. Derivatives including an electronegative part as a nitro group in the phenol ring showed strong inhibition towards housefly acetylcholinesterase, but those with hydrophobic derivatives of the phenol group, such as cresol, naphthol and biphenol, showed relatively low inhibition. In experiments with housefly, the value of LD50 for each chemical correlated with the I50 value for acetylcholinesterase except alpha-naphthyl diethylphosphate, beta-naphthyl diethylphosphate and p,p'-biphenyl diethylphosphate.