RESUMEN
The coupling of 5-acetoxy-1,1-dimethoxypent-2-ene with cytosine and thymine trimethylsilyl derivatives, as well as the reaction of 5-acetoxy-1-bromopent-2-ene with adenine sodium salt, yielded acyclic analogues of the corresponding nucleosides containing 5'-acetoxy groups. They were deprotected with a saturated methanolic solution of ammonia to the target analogues of nucleosides, which were characterized with 1H NMR, IR, and UV spectra. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
Asunto(s)
Nucleósidos/química , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Nucleósidos/síntesis química , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
Acyclic analogs of thymidine, adenine, and cytosine where the ribose moiety is replaced with cis-5-hydroxypenthene, were obtained via condensation of cis-5-acetoxy-1-bromo-2-pentene with nucleic acid bases. Triphosphate derivatives of the new nucleosides were also synthesized to evaluate their action as terminators of DNA synthesis catalyzed by DNA polymerases.
Asunto(s)
Nucleósidos/síntesis química , Catálisis , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/química , Ácidos Nucleicos/química , Nucleósidos/química , Regiones Terminadoras GenéticasRESUMEN
Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.
Asunto(s)
ADN/química , Ácidos Nucleicos Heterodúplex/química , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Datos de Secuencia Molecular , Espectrofotometría UltravioletaRESUMEN
Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.
Asunto(s)
ADN/química , Ácidos Nucleicos Heterodúplex/química , Autorradiografía , Secuencia de Bases , Carbodiimidas/química , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia MolecularRESUMEN
A new type of acyclic nucleoside analogs is proposed, containing C5 hydroxyalkyl fragments, where the distance between 5'-hydroxyl group and the heterocyclic moiety corresponds to that in dideoxydidehydronucleosides (as confirmed by computer modelling). Condensation between 5-0-acetyl-1-bromo-2-pentene and persilylated heterocyclic bases (pyrimidines and guanine) or adenine sodium salt gives rise to the acyclic nucleosides (5a-d, 6a-d) with the yields ranging from 40% up to 85%. Deprotection by NH3/MeOH results in the desired nucleosides (1a-d, 2a-d) formation.
Asunto(s)
Nucleósidos/síntesis química , Guanina , Indicadores y Reactivos , Nucleósidos/química , PirimidinasRESUMEN
The mutation system has been developed to study the mutagenic properties of modified oligonucleotide analogs. The mutagenic properties of oligonucleotides containing one ribonucleotide have been examined. The presence of a ribonucleotide is shown not to induce any mutations. But when the oligonucleotide induces two marker deletions detached by 6 nucleotides they may be repaired separately, in this case the deletion bordering with the ribonucleotide is predominantly repaired.
Asunto(s)
Reparación del ADN , Replicación del ADN , Mutagénesis Sitio-Dirigida , Oligonucleótidos/genética , Ribonucleótidos/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Chemical ligation of oligonucleotides in double stranded complexes has been considered in its structural-kinetic aspect. In a study of the reactivity of eleven synthetic water soluble carbodiimides the reaction rate has been found to be strongly affected by the substituents at N1 and N3 atoms. The chemical ligation appears to involve the linear form of carbodiimide. Model systems have been used to assess the rate constant for the phosphate activation. The ratio of kinetic constants for the productive and nonproductive reactions of the activated derivate apparently reflects the reactive site conformation; for a given duplex this parameter is virtually independent of the condensing agent structure.
Asunto(s)
ADN/química , Imidas/química , Ácidos Nucleicos Heterodúplex/química , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , SolubilidadRESUMEN
To characterize structural factors affecting kinetic parameters of chemical ligation, DNA duplexes with various arrangements of reacting groups in the ligation site have been synthesized and studied. The modifications included replacement of a deoxythymidine with a uridine or a nucleoside having reversed configuration at C2' and/or C3' atoms, the modified residue being phosphate donor or acceptor; introduction of an "extra" nucleotide residue; replacement of a G.C pair second to the junction with a noncomplementary G.G pair. Thermal stability of the anomalous nucleotide duplexes has been characterized. Based on analysis of the kinetic parameters of chemical ligation, a suggestion is made about the conformation of the reactive site.
Asunto(s)
ADN/química , Ácidos Nucleicos Heterodúplex/química , Autorradiografía , Secuencia de Bases , Sitios de Unión , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
Structural defects, affecting T4 DNA ligase function, were revealed with the help of synthetic DNA duplexes, containing modifications at single nick. Changes of configuration at C2' and C3' atoms of furanose in the acceptor terminus lead to total blocking of the nick sealing activity of T4 DNA ligase. On the contrary, substitution of 3'-terminal deoxyribonucleotide for ribonucleotide doesn't affect the enzyme's action. The duplex looses all of it's substrate activity if the next from the nick G.C pair is substituted for the noncomplementary G.C pair. In DNA duplexes containing an unpaired base in the nick, elimination of the extrahelical nucleotide proceeds the ligation step. In these cases the duplex substrate activity decreases depending on the extent of extrahelical base stacking into the double stranded DNA.
Asunto(s)
ADN Ligasas/metabolismo , ADN Viral/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , Polinucleótido Ligasas/metabolismo , Fagos T/enzimología , Catálisis , ADN Ligasa (ATP) , Electroforesis en Gel de Poliacrilamida , Especificidad por SustratoRESUMEN
Two types of DNA-duplexes containing the repeating fragments of natural promoters have been obtained starting from synthetic oligodeoxyribonucleotides TGCATTATAA, AACTAGTT, AGTTAACT. Deca- and octanucleotides have been synthesized by solid phase method with stepwise or blockwise chain elongation. UV- and CD-spectroscopy has been used to study the physico-chemical properties of the synthetic oligonucleotides. Polycondensation of oligonucleotides induced by water-soluble carbodiimide (chemical ligation) or T4-polynucleotide ligase allowed to synthesize the promoter models. The degree of polymerization varied from 2 to 8 in case of chemical ligation and from 2 to 30 in case of enzymatic ligation. A new chain length regulation technique has been developed by means of addition of a terminator of polycondensation (unphosphorylated oligonucleotide) in the reaction mixture.