Phosphotriester Method Research Articles

ChemInform Abstract: Synthesis of Cytidyl(3′‐5′)adenosine Bearing 2′(3′)‐O‐Leucyl Ester via a Phosphorothioate Triester Intermediate.

Abstract 2'(3')-O-(Leucyl)CpA was synthesized in the phosphotriester method. The phenylthio group was used as a protecting group of the Internucleotidic bond. The P-S bond of the triester intermediate was selectively cleaved by using (Bu3Sn)2O under neutral conditions without cleavage of the leucyl ester bond.

Synthesis of cytidyl(3'-5')adenosine bearing 2'(3')-O-leucyl ester via a phosphorothioate triester intermediate

2'(3')-O-(Leucyl)CpA was synthesized in the phosphotriester method. The phenylthio group was used as a protecting group of the Internucleotidic bond. The P-S bond of the triester intermediate was selectively cleaved by using (Bu 3Sn) 2O under neutral conditions without cleavage of the leucyl ester bond.

Nucleotides, XXXIII: Synthesis and Properties of Inosinate Trimer I2'p5'I2'p5'I and Inosinate Tetramer I2'p5'I2'p5'I2'p5'I

The chemical synthesis of 2'-5' inosinate trimer and tetramer was achieved by the phosphotriester method using the 2-(p-nitrophenyl)ethyl group for phosphate protection. The sugar hydroxyl groups have either been protected by the (tertbutyl)-dimethylsilyl or the benzoyl group. Final deprotection to the unblocked oligomers was achieved in over 85% yield. The inosinate oligomers show some antiviral activity against TMV.

Nucleotides. Part XXXI. Modified Oligomeric 2′–5′ A Analogues: Synthesis of 2′–5′ oligonucleotides with 9‐(3′‐azido‐3′‐deoxy‐β‐D‐xylofuranosyl)adenine and 9‐(3′‐amino‐3′‐deoxy‐β‐D‐xylofuranosyl)adenine as modified nucleosides

AbstractA series of new 2′–5′ oligonucleotides carrying the 9‐(3′‐azido‐3′deoxy‐β‐D‐xylofuranosyl)adenine moiety as a building block has been synthesized via the phosphotriester method. The use of the 2‐(4‐nitrophenyl)ethyl (npe) and 2‐(4‐nitrophenyl)ethoxycarbonyl (npeoc) blocking groups for phosphate, amino, and hydroxy protection guaranteed straightforward syntheses in high yields and easy deblocking lo form the 2′–5′ trimers 21, 22, and 25 and the tetramer 23. Catalytic reduction of the azido groups in [9‐(3′‐azido‐3′‐deoxy‐β‐D‐xylofuranosyl)adenine]2′‐yl‐[2′‐(Op‐ammonio)→ 5′]‐[9‐(3′‐azido‐3′‐deoxy‐β‐D‐xylofuranosyl)adenin]‐2′‐yl‐[2′‐(Op‐ammonio)→ 5′]‐9‐(3′‐azido‐3′‐deoxy‐β‐D‐xylofuranosyl)adenine (21) led to the corresponding 9‐(3′‐amino‐3′‐deoxy‐β‐D‐xylofuranosyl)‐adenine 2′–5′ trimer 26 in which the two internucleotidic linkages are formally neutralized by intramolecular betaine formation.

Chemical synthesis of oligonucleotides. 3: Synthesis and characterization of N,O-protected ribophosphoesters for applications in RNA synthesis

The chemical synthesis of RNA, in contrast to that of DNA, poses problems due to fi) additional requirement of 2′-hydroxyl protection of the ribose moieties, and (ii) high lability of inter-ribophosphate bonds. Herein we report the synthesis and characterisation of N, O-protected ribophosphoesters 1 which are key monomeric derivatives in phosphotriester methodology for RNA synthesis. Both the isomeric 2′ and 3′-O-phosphates have been obtained and characterised. The utility of the t-butyl dimethylsilyl group for 2′-hydroxyl protection in the phosphotriester method is demonstrated by the synthesis of r(AUAU),r (UAUA) andr (CACA).

Synthesis of Nonadeca- and Octadecaribonucleotides Using the Solid-Phase Phosphotriester with Tetrahydropyranyl Groups as the 2′-Hydroxyl-Protecting Group

Abstract Nonadeca- and octadecaribonucleotides corresponding to the D-loop of tRNAPhe from yeast and the leader sequence of phage f1 coat protein mRNA were synthesized by the activated phosphotriester method. Coupling yield in the synthesis of oligoribonucleotides depended on the extent of nucleosides loaded on controlled pore glass beads (CPG). N-Acyl-5′-O-dimethoxytrityl-2′-O-tetrahydropyranyl derivatives were used as fully protected ribonucleotide monomer units. The 18mer and 19mer corresponding to the D-loop did not serve as substrates for tRNA (guanosine-2′-)methyltransferase from T. thermophilus, but inhibited methylation of the 5′-half fragment of tRNAPhe. This indicates that both fragments possess some affinity with the enzyme.

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.

Synthesis and thermal stability of dodecadeoxyribonucleotides containing deoxyinosine pairing with four major bases.

Eight dodecauncleotides, each containing deoxynosine or deoxyguanosine at a pairing position to one of the four major deoxynuclesides, d(CGCIAATTNGCG)or d(CGCGAATTNGCG)(N=dA, dC, dG and dT), were synthesized by the phosphotriester method in solution. Purification methods which were suitable for milligram quantities of oligonucleotides were established.The thermal stabilities of these duplexes and the circular dichroism spectra suggested that the dodecamers formed duplexes similar to B-deoxyribonucleic acid.

Synthesis of wobble pairing oligoribonucleotides.

Hexaribonucleotides containing inosine at a pairing position adjacent to the other four major nucleosides (GGINCC, N=A, C, G, U) were synthesized in solution by the phosphotriester method using 2'-O-tetrahydrofuranyl protecting groups. 5'-O-Dimethoxytrityl-2'-O-tetrahydropyranylinosine 3'-O-(metyl-N, N-diisopropyl)phosphor-amidite was used for solid-phase syntheses of decaribonucleotides (CGNGAUCICG, N=A, C, G, U). Thermal stabilities and circular dichroism spectra for these self-complementary duplexes were measured. The stabilizing effects of wobble base pairs in these duplexes were estimated as IC>IA>IU>IG.

Oligothymidylates Substitues Par Un Derive De L'Acridine en Position 5′, A La Fois En Position 5′ et 3′ Ou Sur Un Phosphate Internucleotidique

Abstract Oligothyraidylates covalently linked to an acridine derivative (2-methoxy-6-chloro-9-aminoacridine) have been synthesized by the phosphotriester method in solution. The substitution has been carried out at the 5′ position, both at the 5′ and 3′ positions or on an internucleotidic phosphate. A polymethylene linker was used to tether the acridine derivative via its 9-amino group to the terminal or internucleotidic phosphate.

Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome.

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.

ChemInform Abstract: Synthesis of Oligoribonucleotides by Using 2′‐O‐(1‐Methyl‐1‐methoxy)ethyl Nucleosides.

ChemInformVolume 19, Issue 14 Natural Products ChemInform Abstract: Synthesis of Oligoribonucleotides by Using 2′-O-(1-Methyl-1-methoxy)ethyl Nucleosides. H. TAKAKU, H. TAKAKU Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this authorK. IMAI, K. IMAI Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this authorK. NAKAYAMA, K. NAKAYAMA Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this author H. TAKAKU, H. TAKAKU Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this authorK. IMAI, K. IMAI Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this authorK. NAKAYAMA, K. NAKAYAMA Lab. Bioorg. Chem., Chiba Inst. Technol., Tsudanuma, Narashino, Chiba 275, JapanSearch for more papers by this author First published: April 5, 1988 https://doi.org/10.1002/chin.198814352AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume19, Issue14April 5, 1988 RelatedInformation

Silica Gel Functionalised with Different Spacers as Solid Support for Oligonucleotide Synthesis

Abstract The phosphotriester method for synthesis of oligonucleotides has been used for evaluating silica with spacer groups. Three different spacers were synthesized of which a long chain alkylamine, a double glycine and a double y-aminobutyric acid unit. Only the double glycine spacer improved the yields of the first condensations and is now routinely used in our laboratory.

Solid phase synthesis of oligoribonucleotides using [formula omitted]-nitrobenzyl protection of 2'-hydroxyl via a phosphotriester approach

Oligoribonucleotides with chain length of 6–9 were synthesized on a polystyrene support using o -nitrobenzyl protection of 2'-hydroxyls via phosphotriester method. The condensation reactions between N -acyl-5'- O -monomethoxytrityl-2'- O -( o -nitrobenzyl) nucleoside 3'-( o -chlorophenyl) phosphate and nucleoside bound to the resin using l-mesitylenesulfonyl-3-nitro-1 H -1,2,4-triazole (MSNT) as a condensing agent were examined. It was found that the condensation reaction using MSNT was promoted in the presence of 1-methylimidazole as a catalyst. A mixture of MSNT and 1-methylimidazole was also found to be effective in the condensation reaction involving a dinucleotide with 3'-phosphodiester. The fully protected oligomer were deblocked and purified by chromatography in total yields of 3–21 % based on initial nucleoside bound to the support.

Synthesis and expression of a gene for human tumor necrosis factor.

A gene for human tumor necrosis factor has been synthesized by joining 60 oligodeoxyribonucleotides of 13 to 20 residues using deoxyribonucleic acid (DNA) ligase. These oligodeoxyribonucleotides were prepared by the solid-phase phosphotriester method. The gene (483 base pairs) was designed to carry a Cla I site plus a start codon at the N-terminal end and stop codons plus a Sal I site at the C-terminal end. The gene was expressed in Escherichia coli under the control of the tryptophan promoter of E. coli, and the expressed product was purified and tested for cytotoxic activity.

Synthesis and proton-NMR studies of oligonucleotides containing an apurinic (AP) site.

In order to elucidate the conformational properties of base-deleted oligodeoxyribonucleotides, the molecules d-CpS(pCpG)n (n = 1,2; S = sugar) were synthesized by the phosphotriester method and characterized by 1H-NMR spectroscopy. Complete assignment of all non-exchangeable proton resonances of both compounds was obtained by 1D- and 2D-NMR techniques. In combination with computer simulation, these spectra yielded proton-proton and proton-phosphorus coupling constants of high accuracy. These data provide valuable information about the sugar and the backbone conformation. It appears that d-Cp1Sp2Cp3G4 does not form a duplex under any of the conditions studied. On the contrary, the base-deleted hexamer d-Cp1Sp2Cp3Gp4Cp5G6 occurs as a right-handed' staggered' DNA duplex at 280 K: the core of this duplex is formed by the residues C(3)-G(6); two 'dangling' residues C(1) and S(2) are located at the two 5'-ends of the duplex. The assignment of the corresponding imino proton resonances for [d-CpS(pCpG)2]2 was based on their thermal behavior: the line broadening of these resonances was studied as a function of temperature. The chemical shift and the number of imino proton resonances accord well with the number and type of Watson-Crick base pairs which can be formed in the staggered duplex described above. Thermodynamic parameters of duplex formation were obtained from an analysis of the chemical shift versus temperature profiles of aromatic base and H-1' protons. It is suggested that the cytosine ring of C(1) stacks, at least part of the time, with the guanine ring on the nucleotide residue, G(6), situated in the complementary strand. The binding of Lys-Trp-Lys to [d-CpS(pCpG)2]2 as well as to [d-CpGpCpG]1 was investigated. It is concluded that the indole ring of the tryptophan residue probably stacks on top of the 3'-terminal guanine base of both duplexes, but not on the nucleic acid bases next to the apurinic (AP) site.

ChemInform Abstract: Recent Developments in Phosphorylation of Nucleosides

The phosphorylation of nucleosides is a fundamental reaction in the synthesis of oligonucleotides. This article reviews recent results on the phosphorylation of nucleosides in the synthesis of oligonucleotides by the phosphotriester method and the phosphite triester method. The new condensing and phosphorylating reagents for modified phosphotriester method, activated phosphotriester method, and modified phosphite triester method were discussed.

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.

Synthesis of Oligoribonucleotides by Using 2′-O-(1-Methyl-1-methoxy)ethyl Nucleosides

Abstract 3′,5′-O-Tetraisopropyldisiloxanylnucleosides smoothly react with 2-methoxypropene to give 2′-O-(1-methyl-1-methoxy)ethyl nucleosides in high yields without formation of diastereoisomers. These nucleosides were used as intermediates for oligonucleotide synthesis by the phosphotriester method. The (1-methyl-1-methoxy)ethyl group was removed rapidly from oligonucleotides by acid treatment.

Preparative synthesis of guanylate‐rich fragments of the terminal sequence of macronuclear DNA from hypotrichous ciliates using the phosphotriester method in solution

AbstractGuanylate‐rich oligodeoxyribonucleotides d(G4T4), d(T4G4), d(T4G4T4), d(G4T4G4), and d(T4G4T4G4) were synthesized in amounts up to 350 mg using the triester method in solution. These oligonucleotides correspond to fragments of the terminal of macronuclear DNA from hypotrichous ciliates. The protected oligonucleotides were obtained by block‐condensation of protected 5′‐hydroxyl and 3′‐phosphate derivatives. Mixtures of 2,4,6‐triisopropylbenzenesulfonyl chloride/N‐methylimidazole or 2,4,6‐trimethylbenzenesulfonyl chloride/N‐methylimidazole were used as condensating agents. Different concepts of protecting the nucleotides were investigated and it was found that yields and purities of the resulting oligonucleotides do not depend essentially on the employed concept. The 36mer d(G4T4G4T4G4T4G4T4G4) was obtained by enzymatic phosphorylation of aliquots of the dodecamers d(G4T4G4) and d(T4G4T4), followed by ligation with T4 DNA ligase.