RESUMEN
We analyzed the effect of modified nucleotides within gapmer antisense oligonucleotides on RNase H mediated gene silencing. Additionally, short hairpins were introduced into antisense oligonucleotides as structural motifs, and their influence on biological and physicochemical properties of pre-structured gapmers was investigated for the first time. The results indicate that two LNA residues in specified positions of the gap flanking regions are sufficient and favorable for efficient knock-down of the ß-actin gene. Furthermore, the introduction of other modified nucleotides, i. e. glycyl-amino-LNA-T, 2'-O-propagyluridine, polyamine functionalized uridine, and UNA, in specified positions, also increases the inhibition of ß-actin expression. Importantly, the presence of hairpins within the gapmers improves their silencing properties.
Asunto(s)
Actinas , Oligonucleótidos Antisentido , Expresión Génica , Nucleótidos , Oligonucleótidos Antisentido/química , Ribonucleasa H/genética , Ribonucleasa H/metabolismoRESUMEN
The low binding affinity of unmodified triplex-forming oligonucleotides (TFO) is the main drawback to their promising utilization in gene therapy. In the present study, we have synthesized DNA intercalator 5-(pyren-1-ylethynyl)indole Y, known as twisted intercalating nucleic acid (TINA), by a Cu-mediated Sonogashira palladium-catalyzed coupling reaction of 1-ethynylpyrene with 5-iodoindole at a high temperature under anaerobic conditions. Coupling with indole C-5 was far more preferable in obtaining stable TINA-indole than enamine site C-3, as neither hydration of the triple bond to ketones nor competitive Glaser-type homocoupling of acetylenes was observed. The insertion of the new TINA monomer Y as a bulge in the middle or at the 5'-end of the oligodeoxynucleotide sequence via a flexible butane-1,2-diol linker showed extraordinary binding potential, resulting in excellent thermal stabilization of Hoogsteen-type triplex- and duplex-deoxyribonucleic acid (DNA) structures which was detected by thermal denaturation studies and supported by circular dichroism (CD). Molecular dynamics AMBER* revealed the lowest energy conformation in which a pyrenyl residue of the TINA monomer Y stacks in the dsDNA part, while an indolyl unit intercalates between the nucleobases of the TFO pattern. Overall the torsionally rigid conjugated TINA system with a decent twisting of 15.1° around acetylene is confirmed here as a requirement for the best fit inside the intercalation site of the triplex, resulting in high TFO-dsDNA affinity.
Asunto(s)
Sustancias Intercalantes , Ácidos Nucleicos , Temperatura , Sustancias Intercalantes/química , Oligonucleótidos/química , Pirenos/química , ADN/química , Ácidos Nucleicos/química , Indoles , Conformación de Ácido Nucleico , Desnaturalización de Ácido NucleicoRESUMEN
Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.
Asunto(s)
Oligonucleótidos , Poliaminas , ADN/química , ADN de Cadena Simple/química , Oligonucleótidos/químicaRESUMEN
Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNaseâ H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNaseâ H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNaseâ H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.
Asunto(s)
Silenciador del Gen , Nucleótidos/química , Oligonucleótidos Antisentido/química , Ribonucleasa H/metabolismo , Secuencia de Bases , Estabilidad de Enzimas , Células HeLa , Humanos , Concentración 50 Inhibidora , Desnaturalización de Ácido Nucleico , Nucleótidos/metabolismo , Oligonucleótidos Antisentido/metabolismo , ARN/química , ARN/metabolismo , Temperatura , TransfecciónRESUMEN
Synthesis of the novel thiophenyl carbazole phosphoramidite DNA building block 5 was accomplished in four steps using a Suzuki-Miyaura cross-coupling reaction from the core carbazole and it was seamlessly accommodated into a 9-mer DNA-based oligonucleotide by incorporation at the flanking 5'-end in combination with a central insertion of an LNA-T nucleotide. The carbazole-containing oligonucleotide was combined in different duplex hybrids, which were characterized by thermal denaturation, circular dichroism and fluorescence studies. The carbazole monomer modulates the duplex stability in various ways. Thus, monomer Z increased the thermal stability of the 9-mer towards the complementary 9-mer/15-mer DNA duplex by 4.2 °C. Furthermore, indications of its intercalation into the duplex were obtained by modeling studies and robust decreases in fluorescence emission intensities upon duplex formation. In contrast, no clear intercalating tendency was corroborated for monomer Z within the DNA/RNA hybrid duplex as indicated by moderate quenching of the fluorescence and similar duplex thermal stabilities relative to the corresponding control duplex. The recognition efficiencies of the carbazole modified oligonucleotide toward single nucleotide mismatches were studied with two 15-mer model targets (DNA and RNA). For both systems, mismatches positioned at the juxtaposition of the carbazole monomer showed pronounced deceases in thermal denaturation temperature. Steady-state fluorescence emission studies of all mismatched duplexes with incorporation of Z monomer typically displayed efficient fluorescence quenching.
Asunto(s)
OligonucleótidosRESUMEN
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
Asunto(s)
ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Glicina/análogos & derivados , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/antagonistas & inhibidores , ADN Bacteriano/química , ADN Superhelicoidal/química , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Plásmidos/química , Plásmidos/metabolismo , Técnicas de Síntesis en Fase Sólida , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.
Asunto(s)
ADN/química , Galactosa/química , Oligonucleótidos/química , Fenómenos Biofísicos , Espectroscopía de Resonancia Magnética con Carbono-13 , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.
Asunto(s)
ADN Superhelicoidal/química , Oligonucleótidos/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Tampones (Química) , ADN/química , División del ADN , Enzimas de Restricción del ADN/química , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Oligonucleótidos/síntesis química , Plásmidos/química , Temperatura de TransiciónRESUMEN
Huntington's disease is a neurodegenerative, trinucleotide repeat (TNR) disorder affecting both males and females. It is caused by an abnormal increase in the length of CAGâ¢CTG TNR in exon 1 of the Huntingtin gene (HTT). The resultant, mutant HTT mRNA and protein cause neuronal toxicity, suggesting that reduction of their levels would constitute a promising therapeutic approach. We previously reported a novel strategy in which chemically modified oligonucleotides (ONs) directly target chromosomal DNA. These anti-gene ONs were able to downregulate both HTT mRNA and protein. In this study, various locked nucleic acid (LNA)/DNA mixmer anti-gene ONs were tested to investigate the effects of varying ON length, LNA content, and fatty acid modification on HTT expression. Altering the length did not significantly influence the ON potency, while LNA content was critical for activity. Utilization of palmitoyl-modified LNA monomers enhanced the ON activity relatively to the corresponding nonmodified LNA under serum starvation conditions. Furthermore, the number of palmitoylated LNA monomers and their positioning greatly affected ON potency. In addition, we performed RNA sequencing analysis, which showed that the anti-gene ONs affect the "immune system process, mRNA processing, and neurogenesis." Furthermore, we observed that for repeat containing genes, there is a higher tendency for antisense off-targeting. Taken together, our findings provide an optimized design of anti-gene ONs that could potentially be developed as DNA-targeting therapeutics for this class of TNR-related diseases.
Asunto(s)
Enfermedad de Huntington , Oligonucleótidos , Masculino , Humanos , Oligonucleótidos/genética , Oligonucleótidos/farmacología , Oligonucleótidos/química , Oligonucleótidos Antisentido/farmacología , ADN/uso terapéutico , Expresión Génica , ARN Mensajero/metabolismo , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/terapiaRESUMEN
A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.
Asunto(s)
Imidazoles/química , Indoles/química , Sustancias Intercalantes/química , Oligonucleótidos/química , ADN/química , Sustancias Intercalantes/síntesis química , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Rayos UltravioletaRESUMEN
A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation.
Asunto(s)
Oligonucleótidos/síntesis química , Piranos/química , Secuencia de Bases , Modelos Moleculares , Oligonucleótidos/química , Ozono , Transición de Fase , Temperatura de TransiciónRESUMEN
Oligonucleotides with the sequences 5'-GTG AUPA TGC, 5'-GCA TAUP CAC and 5'-GUPG ATA UPGC, where UP is 2'-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide-alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection-deprotection strategy that allows efficient separation of the resulting polyamine-oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine-oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. Tm measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature.
RESUMEN
This is the first report exploring the capability of twisted intercalating nucleic acid (TINA) and naphthalene-functionalized non-nucleosidic linkers to stabilize and engage in double-helical structures. Four designs were studied with respect to the formation of duplexes and/or other types of self-assemblies. One of the constructs involving TINA provides a thermostable duplex. The biophysical properties of the individual constructs were investigated by UV thermal melting experiments, circular dichroism, and fluorescence emission spectroscopy. Molecular modeling studies were performed in attempts of explaining the biophysical measurements for the duplex based on the TINA-containing oligonucleotide strands.
Asunto(s)
Sustancias Intercalantes/química , Conformación de Ácido Nucleico , Ácidos Nucleicos/química , Oligonucleótidos/química , Dicroismo Circular , Modelos Moleculares , Estructura Molecular , Desnaturalización de Ácido Nucleico , Espectrometría de FluorescenciaRESUMEN
When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stability of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high discrimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms intercalation as the important factor for triplex stabilization and it rules out an alternative complexation of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y were obtained via a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate. The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotection of the hydroxy groups followed by 4,4'-dimethoxytritylation and phosphitylation.
Asunto(s)
ADN/química , Imidazoles/química , Sustancias Intercalantes/química , Oligonucleótidos/química , Secuencia de Bases , Imidazoles/síntesis química , Imidazoles/metabolismo , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/metabolismo , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/metabolismo , Termodinámica , Temperatura de TransiciónRESUMEN
Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.
RESUMEN
The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3'-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3'-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.
Asunto(s)
ADN/química , ADN/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes myc , Proteínas de Unión a Hierro/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , FrataxinaRESUMEN
This paper reports the synthesis and the antiviral activities of new double-prodrugs against HIV based on the known mixed SATE (S-acyl-2-thioethyl) prodrug approach. The monophosphate of the nucleoside reverse transcriptase inhibitor (NRTI) d4T was masked with one SATE group and one aromatic group through which a nonnucleoside reverse transcriptase inhibitor (NNRTI) was linked. Double-prodrug 1 was a hybrid between d4T monophosphate and the known NNRTI MKC-442, which were linked through a labile p-hydroxybenzoyl protection group in the N-3 position of MKC-442. Double-prodrugs 2 and 3 were conjugates between d4T monophosphate and the new NNRTIs 15 and 19 linked through a stable phenolic linker that was a part of the N-1 substituents of the NNRTIs. The double-prodrugs 1, 2, and 3 all had good activities against wild-type HIV-1, Y181C mutant, and also against a HIV-2 strain that was resistant to NNRTIs.
Asunto(s)
Profármacos/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Estavudina/química , Sulfuros/síntesis química , Timidina Monofosfato/análogos & derivados , Timidina Monofosfato/síntesis química , Uracilo/análogos & derivados , Uracilo/química , Línea Celular , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-2/efectos de los fármacos , VIH-2/genética , Humanos , Mutación , Profármacos/química , Profármacos/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfuros/química , Sulfuros/farmacología , Timidina Monofosfato/química , Timidina Monofosfato/farmacologíaRESUMEN
This paper reports the synthesis and the antiviral activities of a series of 6-arylmethyl-1-(allyloxymethyl)-5-alkyluracil derivatives, which can be viewed as analogues of the anti-HIV-1 drug emivirine (formerly MKC-442) from which they differ in the replacement of the ethoxymethyl group with variously allyloxymethyl moieties. The most active compounds N-1 allyloxymethyl- and N-1 3-methylbut-2-enyl substituted 5-ethyl-6-(3,5-dimethylbenzyl)uracils (12 and 13) showed activity against HIV-1 wild-type in the picomolar range with selective index of greater than 5 x 10(6) and activity in the submicromolar range against the clinically important Y181C and K103N mutant strains known to be resistant to emivirine. Structure-activity relationship studies established a correlation between the anti-HIV-1 activity and the substitution pattern of the N-1 allyloxymethyl group.
Asunto(s)
Fármacos Anti-VIH/síntesis química , VIH-1/efectos de los fármacos , Uracilo/análogos & derivados , Uracilo/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , VIH-1/genética , Humanos , Espectroscopía de Resonancia Magnética , Mutación , Estereoisomerismo , Relación Estructura-Actividad , Uracilo/química , Uracilo/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
N-(Pyren-1-yl)-(3R,4S)-4-[(1S,2R)-1,2,3-trihydroxypropyl]pyrrolidin-3-ol (4) was obtained in 36% yield from 3-deoxy-3-C-formyl-1,2:5,6-di-O-isopropylidene-alpha-D-allofuranose (3) by combined hydrolysis and aminoalkylation reactions with 1-aminopyrene in a one-pot reaction. Cleavage reactions of the exocyclic triol chain in 4 with NaIO4 and NaBH4 resulted in iminosugars 7 and 8, which are analogues of the furanose forms of 2-deoxy-D-allose and of 2-deoxy-d-ribose, the latter analogue N-(pyren-1-yl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol (8) being formed in 83% yield.
Asunto(s)
Nucleósidos/síntesis química , Pirenos/síntesis química , Pirrolidinas/síntesis química , Alquilación , Aminación , Hidrólisis , Espectroscopía de Resonancia Magnética , Estructura Molecular , Nucleósidos/química , Pirenos/química , Pirrolidinas/químicaRESUMEN
Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a similar wild type triplex of the same total length.