RESUMO
The aminoacyl-tRNA synthetase (aaRS) class of enzymes is a validated target for antimicrobial development. Aminoacyl analogues of 5'-O-(N-L-aminoacyl)-sulfamoyladenosines are known to be potent inhibitors of aaRS, but whole cell antibacterial activity of these compounds is very limited, and poor penetration into bacteria has been proposed as the main reason for this. Aiming to find derivatives that better penetrate bacteria, we developed a simple and short method to prepare dipeptidyl-derivatives of 5'-O-(N-L-aminoacyl)-sulfamoyladenosines, and used this method to prepare 18 5'-O-(N-dipeptidyl)-sulfamoyladenosines. The antibacterial activity of these derivatives and a number of reference compounds against S. aureus, E. faecalis and E. coli was determined. Several of the new derivatives showed improved antibacterial activity and an altered spectrum of antibacterial activity.
Assuntos
Adenosina/análogos & derivados , Antibacterianos/síntese química , Sulfonamidas/farmacologia , Adenosina/farmacologia , Antibacterianos/farmacologia , Dipeptídeos , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Sulfonamidas/químicaRESUMO
Modified synthetic oligodeoxyribonucleotides containing 2'-O-beta-D-ribofuranosyladenosine were used as primers in the RNA-dependent DNA synthesis catalyzed by HIV-1 reverse transcriptase. The degree of elongation of the primers depends on the position of the additional ribose unit, its presence in the specific position of the primer (-4) (and only in it) completely preventing elongation. Computer-modeled binding of the modified primers to the active site of reverse transcriptase demonstrated that steric hindrances arising from the interaction of the additional ribose residue with the reverse transcriptase region 262-270 interacting with the minor groove of the DNA substrate prevents elongation in the above mentioned case.
Assuntos
Adenosina/análogos & derivados , Adenosina/química , Primers do DNA/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/química , Adenosina/metabolismo , Sítios de Ligação , Primers do DNA/metabolismo , Replicação do DNA , Transcriptase Reversa do HIV/metabolismo , Humanos , Modelos Químicos , Ligação Proteica , Inibidores da Transcriptase Reversa/metabolismo , Replicação ViralRESUMO
Several ribavirin congeners containing a hexitol moiety were prepared via ring opening of two different epoxides with the methylcarboxylate ester of triazole and further elaboration. Unfortunately, none of the newly synthesized compounds displayed appreciable antiviral activity.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Ribavirina/análogos & derivados , Álcoois , Indicadores e Reagentes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Ribavirina/síntese química , Ribavirina/farmacologia , TriazóisRESUMO
The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.
Assuntos
Adenosina/análogos & derivados , Oligorribonucleotídeos/síntese química , RNA de Transferência/química , Indicadores e ReagentesRESUMO
The efficient synthesis of oligonucleotides containing 2'-O-beta-D-ribofuranosyl (and beta-D-ribopyranosyl)nucleosides, 2'-O-alpha-D-arabinofuranosyl (and alpha-L-arabinofuranosyl)nucleosides. 2'-O-beta-D-erythrofuranosylnucleosides, and 2'-O-(5'-amino-5-deoxy-beta-D-ribofuranosyl)nucleosides have been developed.
Assuntos
Dissacarídeos/química , Nucleosídeos/química , Oligodesoxirribonucleotídeos/síntese química , Oligorribonucleotídeos/síntese química , Indicadores e ReagentesRESUMO
In an effort to further improve the hybridisation potential of anhydro-hexitol nucleoside analogues, the 1'-methoxyl and 3'-methoxyl substituents were introduced and evaluated for their antisense potential. In view of the selectivity of pairing with RNA, especially the introduction of a 3'-O-alkyl moiety deserves further study.
Assuntos
Álcoois , Ácidos Nucleicos/síntese química , Alquilação , Metilação , Nucleosídeos/síntese químicaRESUMO
A convenient method of regioselective introduction of 1-beta-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymic reaction of formation and hydrolysis of internucleotide bonds. The English version of the paper.
Assuntos
Bioquímica/métodos , Galactose/química , Galactose/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Timina/química , Timina/metabolismo , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Galactose/análogos & derivados , Hidrólise , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Relação Estrutura-Atividade , Timina/análogos & derivadosRESUMO
Oligodeoxynucleotides containing 2'-O-beta-D-ribofuranosyladenosine were prepared and used as modified primers in RNA-templated DNA synthesis catalyzed by HIV reverse transcriptase. It was shown that the additional 2'-ribofuranose residue in specific position of primer prevents its elongation.
Assuntos
Primers do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Ribose , Domínio Catalítico/efeitos dos fármacos , Primers do DNA/química , Primers do DNA/farmacologia , Transcriptase Reversa do HIV/metabolismo , Humanos , Cinética , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.
Assuntos
Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Álcoois Açúcares/metabolismo , Metilação , Oligonucleotídeos Antissenso/síntese química , Polirribonucleotídeos/metabolismo , RNA/metabolismo , Estabilidade de RNARESUMO
1,5-Anhydrohexitol nucleoside congeners with alkoxy substituents, were prepared, resulting in a further improvement of their RNA affinity and antisense potential.
Assuntos
Nucleosídeos/síntese química , RNA/metabolismo , Álcoois Açúcares/química , Cinética , Nucleosídeos/metabolismo , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , RNA/química , Relação Estrutura-Atividade , Álcoois Açúcares/síntese química , Álcoois Açúcares/metabolismoRESUMO
Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against degradation in serum. A CeNA/RNA hybrid is able to activate E. Coli RNase H. resulting in cleavage of the RNA strand.
Assuntos
Cicloexanos/química , Ácidos Nucleicos/química , RNA/química , Ribonuclease H/metabolismo , Dicroísmo Circular , Cicloexanos/metabolismo , Cicloexenos , DNA/química , Ativação Enzimática , Escherichia coli/enzimologia , Ácidos Nucleicos/metabolismo , RNA/metabolismo , Álcoois Açúcares/químicaRESUMO
The title compound, 9(R)-[6(R)-hydroxymethyl-1-oxa-4-thiacyclohexan-2-yl]-1,9-dihydro-6H-purin-6-one-water (4/3), C10H12N4O3S-0.75H2O, crystallizes in the triclinic space group P1 with four molecules in the asymmetric unit and 0.75 waters of hydration per molecule. The structure was refined to an R value of 0.072 for 3382 observed reflections. The four crystallographically independent molecules are designated A, B, C and D. All four oxathiane rings adopt the chair conformation and the purine bases are in an anti orientation with respect to the sugar moieties. Molecules A and D and molecules C and B are base paired by a single hydrogen bond of the type N-H...N. These base pairs are again hydrogen bonded to their translated pairs in the direction of a cell diagonal.
Assuntos
Hipoxantina/química , Nucleosídeos/química , Nucleosídeos de Purina/química , Antivirais/química , Cristalografia por Raios X , Hipoxantinas , Estrutura MolecularRESUMO
Cross-talking between nucleic acids is a prerequisite for information transfer. The absence of observed base pairing interactions between pyranose and furanose nucleic acids has excluded considering the former type as a (potential) direct precursor of contemporary RNA and DNA. We observed that alpha-pyranose oligonucleotides (alpha-homo-DNA) are able to hybridize with RNA and that both nucleic acid strands are parallel oriented. Hybrids between alpha-homo-DNA and DNA are less stable. During the synthesis of alpha-homo-DNA we observed extensive conversion of N6-benzoyl-5-methylcytosine into thymine under the usual deprotection conditions of oligonucleotide synthesis. Alpha-homo-DNA:RNA represents the first hybridization system between pyranose and furanose nucleic acids. The duplex formed between alpha-homo-DNA and RNA was investigated using CD, NMR spectroscopy, and molecular modeling. The general rule that orthogonal orientation of base pairs prevents hybridization is infringed. NMR experiments demonstrate that the base moieties of alpha-homo-DNA in its complex with RNA, are equatorially oriented and that the base moieties of the parallel RNA strand are pseudoaxially oriented. Modeling experiments demonstrate that the duplex formed is different from the classical A- or B-type double stranded DNA. We observed 15 base pairs in a full helical turn. The average interphosphate distance in the RNA strand is 6.2 A and in the alpha-homo-DNA strand is 6.9 A. The interstrand P-P distance is much larger than found in the typical A- and B-DNA. Most helical parameters are different from those of natural duplexes.
Assuntos
Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Dicroísmo Circular , DNA/química , Hexoses , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Pentoses , RNA/química , TemperaturaRESUMO
Within the polyA:polyT recognition system, cross-pairing between several nucleic acids with a phosphorylated six-membered carbohydrate (mimic) as repeating unit in the backbone structure has been observed. All investigated nucleic acids (except for beta-homo-DNA) hybridize with RNA, leaving RNA as a versatile biopolymer for informational transfer. [reaction: see text]
Assuntos
RNA/química , Conformação de Ácido NucleicoRESUMO
The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/química , Nucleosídeos/química , Álcoois Açúcares/química , Cromatografia Líquida de Alta Pressão , Primers do DNA/química , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/isolamento & purificação , Hexosefosfatos/química , Cinética , Oligodesoxirribonucleotídeos/química , Moldes GenéticosRESUMO
Two major hurdles to the widespread use of synthetic nucleic acids as drugs are the biological stability of the compounds and efficiency of cellular penetration. Recent advances in the chemistry of nucleic acids has given rise to highly stable derivatives with an anhydrohexitol backbone. This report addresses the cellular uptake of these molecules. We show that the uptake of HNA in the absence of a carrier is very low but HNA is efficiently internalized with a range of transfection reagents.
Assuntos
Hexosefosfatos/metabolismo , Ácidos Nucleicos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Corantes Fluorescentes , Células HeLa , Humanos , Ribonuclease H/biossínteseRESUMO
Liquid chromatography was used to follow the degradation of hexopyranosylated cytosine nucleosides in buffers of acid, neutral and alkaline pH and of constant ionic strength. The compounds were found to degrade by hydrolysis to cytosine and/or by deamination to the corresponding uracil nucleosides. Degradation in acid is influenced by the number of sugar hydroxyl groups, presence of sugar double bonds and the type of anomer. Stability of some of the compounds was compared with that of related thymine nucleosides. Temperature studies support a unimolecular mechanism of hydrolysis at pH 1.22.
Assuntos
Citosina/química , Nucleosídeos/química , Soluções Tampão , Cromatografia Líquida , Citosina/análogos & derivados , Desaminação , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Concentração OsmolarRESUMO
The synthesis and in vitro anti human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activities of some unnatural beta-L-nucleoside enantiomers related to the anti-HIV compound 2', 3'-dideoxy-3'-fluoro-5-chlorouridine (beta-D-3'Fdd5ClU) are reported. In contrast to beta-D-3'Fdd5ClU, beta-L-3'Fdd5ClU and the other L-congeners were devoid of significant anti-HIV effects, but beta-L-2',3'-dideoxy-5-chlorocytidine (beta-L-dd5ClC) and beta-L-2', 3'-dideoxy-3'-fluoro-cytidine (beta-L-3'FddC) showed a distinct anti-HBV activity. Three mononucleoside phosphotriester derivatives with S-pivaloyl-2-thioethyl (t-BuSATE) groups as biolabile phosphate protective groups were also synthesized. The bis(t-BuSATE) derivative of beta-D-3'Fdd5ClU retained anti-HIV activity in thymidine kinase deficient (TK(-)) CEM cells.
Assuntos
Antivirais/farmacologia , Didesoxinucleosídeos/farmacologia , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Antivirais/síntese química , Linhagem Celular , Didesoxinucleosídeos/síntese química , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Estrutura Molecular , EstereoisomerismoRESUMO
Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.
Assuntos
DNA/química , Oligonucleotídeos/química , RNA/química , Álcoois Açúcares/química , Química/métodos , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/química , Ácidos Nucleicos/química , Moldes GenéticosRESUMO
Hexitol nucleic acid (HNA) is a new steric blocking oligonucleotide, hybridizing sequence selectively with RNA. The biological activity of HNA was evaluated in an in vitro translation arrest system targeting Ha-ras mRNA and in a cellular system targeting intracellular adhesion molecule-1 (ICAM-1) expression. HNA very efficiently and selectively inhibited Ha-ras mRNA translation (IC(50) of 50 nM) when targeted at the translation initiation region. When targeting at the 12th codon region, a gap-mer approach was needed to inhibit mRNA translation. Similarly, HNA inhibited ICAM-1 expression in keratinocytes when targeting at codon sequences. In this test system, HNA is less active but more selective than phosphorothioates, but needs lipofection to become active in keratinocytes. This new steric blocker may be an efficient antisense agent providing that enough material can be brought into cells.