RESUMO
Pseudo-complementary oligonucleotide analogues and mimics provide novel opportunities for targeting duplex structures in RNA and DNA. Previously, a pseudo-complementary A-T base pair has been introduced. Towards sequence unrestricted targeting, a pseudo-complementary G-C base pair consisting of the unnatural nucleobases n6-methoxy-2,6-diaminopurine (previously described in a DNA context) and N4-benzoylcytosine is now presented for design of pseudo-complementary PNA oligomers (pcPNAs).
RESUMO
An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (DeltaT(m) per modification -1 to -5 degrees C).
Assuntos
Nucleosídeos/química , Oligonucleotídeos/síntese química , Organofosfatos/química , Organofosfonatos/química , Amidas , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/isolamento & purificação , Fosforamidas , Ácidos Fosfóricos , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
The tendency of a series of acyclic nucleoside analogues 1a-f to undergo intramolecular cyclization reactions was investigated. All compounds, when treated with NaOD, were in equilibrium with the bicyclic compounds 2a-f, arising from Michael addition of a hydroxy group to the C(5)=C(6) bonds. Derivatives of 2,4-pyrimidinediones (1a,b) had the highest tendency to undergo intramolecular Michael addition when treated with triethylamine, whereas the cyclization of 4-amino-2-pyridones (1c-f) proceeded best with acid. The exocyclic double bond of was essential for the cyclization to occur. Commonly used N-protecting groups as the benzoyl- and the dibutylaminomethylene group enhanced cyclization. Under acidic anhydrous conditions 1b and 1e cyclized to the 2,4'-anhydro compounds 1b and 1e.
Assuntos
Nucleosídeos de Pirimidina/química , Ciclização , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Água/químicaRESUMO
Acyclic, achiral nucleoside derivatives 1b-e of adenine, cytosine, 5-methylcytosine, and guanine, containing a 3-hydroxy-2-(hydroxymethyl)prop-1-enyl group on N-1 or N-9, have been prepared analogously to the previously described thymine derivative 1a. In contrast to the adenine and guanine derivatives, the cytosine derivative 9 was unstable, and was obtained in a low yield due to side reactions. These include cleavage of the propenyl group from the base, and the formation of a bicyclic compound. The thymine derivative, although stable under neutral conditions, likewise underwent a reversible cyclization reaction (Michael addition) in the presence of acids or bases. The 5-methylcytosine derivative was stable under neutral and basic conditions. Four other nucleoside derivatives 26a-d containing a 2,3-dihydroxy-2-(hydroxymethyl)propyl group on N-1 or N-9, three of which are new, have likewise been prepared. All compounds were evaluated as antiviral agents against HIV-1 and HSV-1 but were devoid of antiviral activity.
Assuntos
Antivirais/síntese química , Nucleosídeos/síntese química , Adenina/análogos & derivados , Adenina/síntese química , Adenina/farmacologia , Animais , Antivirais/farmacologia , Células Cultivadas , Citosina/análogos & derivados , Citosina/síntese química , Citosina/farmacologia , Guanina/análogos & derivados , Guanina/síntese química , Guanina/farmacologia , HIV-1/efeitos dos fármacos , Estrutura Molecular , Nucleosídeos/farmacologiaRESUMO
A novel conformationally constrained pyridazinone E(ag)-base PNA-monomer 2 capable of binding thymine in a triplex motif was designed and synthesised. A bis-PNA with the E(ag)-base incorporated in the Hoogsteen strand was hybridised with a complementary DNA. Thermal stability studies revealed an increase in T(m) (4.3 degrees C per mod.) compared to a no-base unit, but showed no improvement over a previously described unconstrained analogue (E, 1). Surprisingly, no significant difference was found in the thermodynamic parameters (DeltaH degrees, DeltaS degrees and DeltaG degrees ) for PNA-DNA triplex formation involving 2 or the unconstrained analogue 1.
Assuntos
Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Piridazinas/química , Timina/química , Avaliação Pré-Clínica de Medicamentos/métodos , Estrutura Molecular , Ácidos Nucleicos Peptídicos/genética , TermodinâmicaRESUMO
The conformations of an acyclic, achiral enamide thymidine analogue 1 have been studied by model building and geometry calculations, as well as by NMR NOE and UV experiments. The results indicate that there are no significant barriers to rotation around any of the sigma bonds, in particular the N1-C1' enamide bond, and that the analogue should be able to accommodate conformations that mimic the conformations of natural nucleosides in A- and B-type helices quite well. For comparison the saturated analogue 2 has been prepared and built into oligonucleotides. It is shown that incorporation of 2 in oligonucleotides results in a much larger depression of the melting temperature (deltaTm -10 to -12.5 degrees C) than does incorporation of 1 (deltaTm -5 to -6.5 degrees C).
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/síntese química , Carbono/química , Nucleotídeos/química , Nucleotídeos/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Análise Espectral , Estereoisomerismo , Timidina/químicaRESUMO
Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by Tm measurements showing a deltaTm of -2 to -6.5 degrees C per modification. An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage by a 3'-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.
Assuntos
Nucleosídeos/química , Nucleosídeos/síntese química , Oligodesoxirribonucleotídeos/síntese química , Sítios de Ligação , DNA/química , Indicadores e Reagentes , Conformação Molecular , Oligodesoxirribonucleotídeos/química , RNA/química , EstereoisomerismoRESUMO
The new monomer 1 seems to be an excellent mimic of nucleosides with different sugar conformations (north, south, and envelope), because of the relatively free rotation around gamma, delta, and chi. The rotation around chi is primarily controlled by the repulsion between H6 and the two hydrogen atoms on C4' and not pi conjugation between the double bond and the nucleobase. A viable synthesis of the guanine monomer 8 is described.
Assuntos
Nucleosídeos/química , Nucleosídeos/farmacologia , Guanina/análogos & derivados , Guanina/síntese química , Isomerismo , Modelos Moleculares , Conformação Molecular , Nucleosídeos/síntese química , RotaçãoRESUMO
Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.
Assuntos
Compostos Aza/síntese química , Éteres de Coroa/síntese química , DNA/química , Oligodesoxirribonucleotídeos/síntese química , Amidas/química , Aminoácidos/química , Compostos Aza/química , Sítios de Ligação , Éteres de Coroa/química , Metais/química , Oligodesoxirribonucleotídeos/química , Ácidos Fosfóricos/químicaRESUMO
To expand the triplex recognition repertoire of Nucleic Acids, novel nucleobases that recognize thymine in a T-A base pair are still required. A novel conformationally constrained PNA-monomer (II) capable of binding T in a triplex motif was designed and synthesized in 7 steps starting from commercially available dimethyl 2-oxoglutarate.
Assuntos
Ácidos Nucleicos Peptídicos/síntese química , Timina , Pareamento de Bases , Sítios de Ligação , Conformação de Ácido NucleicoRESUMO
A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.
Assuntos
Fucose/farmacocinética , Galactose/farmacocinética , Manose/farmacocinética , Ácidos Nucleicos Peptídicos/farmacocinética , Animais , Feminino , Fucose/síntese química , Galactose/síntese química , Masculino , Manose/síntese química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/farmacocinética , Ácidos Nucleicos Peptídicos/síntese química , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/fisiologiaRESUMO
Synthesis of an asymmetrically substituted triaza crown ether, its incorporation into the 3'-end and 5'-end of ninemer oligonucleotides, and the influence of various alkanediamine ligands on duplex thermostabilities are reported.
Assuntos
Compostos Aza/química , DNA/química , Éteres Cíclicos/química , Oligonucleotídeos/química , Sequência de Bases , Sítios de Ligação , Diaminas/química , Temperatura Alta , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/químicaRESUMO
A Boc-protected amino acid containing an ester function, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, has been synthesized and incorporated into peptide nucleic acid (PNA) oligomers. In model experiments it is found that the ester is fairly stable in aqueous solution at pH 7.4 and 37 degrees C (t(1/2) = 6 h), whereas it is rapidly cleaved in mouse serum and in kidney and liver homogenates (t(1/2) = 0.1-0.5 min). Furthermore, ester-linked fatty acid PNA conjugates targeted to an aberrant splice site in luciferase mRNA were prepared and shown to be twice as potent for inducing active luciferase as the corresponding conjugate not containing the linker. Thus, a PNA prodrug approach may be useful for both ex vivo as well as in vivo applications.
Assuntos
Ácidos Nucleicos Peptídicos/síntese química , Pró-Fármacos/síntese química , Animais , Ésteres , Feminino , Células HeLa , Humanos , Camundongos , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/metabolismo , Pró-Fármacos/metabolismo , Transfecção/métodosRESUMO
An achiral, acyclic nucleoside analogue has been incorporated once or twice in oligodeoxyribonucleotides by the phosphoramidite method, and conditions found which allow deprotection of the oligonucleotides containing a sensitive modified allylic unit. The binding affinity of the modified oligonucleotides towards complementary DNA and RNA was reduced compared to unmodified DNA (DeltaT(m) -2 to -6.5 degrees C). An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage with a 3'-exonuclease.
Assuntos
Oligonucleotídeos/química , Timina/análogos & derivados , Animais , Sequência de Bases , DNA Complementar/química , DNA Complementar/metabolismo , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico , Diester Fosfórico Hidrolases/metabolismo , RNA/química , RNA/metabolismo , Venenos de Serpentes , Temperatura , Timina/metabolismoRESUMO
Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.