RESUMEN
Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.
Asunto(s)
Adenosina/química , Compuestos Aza/química , Compuestos Organofosforados/química , ARN Interferente Pequeño/química , Ribonucleósidos/química , Adenosina/farmacología , Compuestos Aza/farmacología , Estabilidad de Medicamentos , Células HeLa , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos Organofosforados/farmacología , Desnaturalización Proteica , ARN Interferente Pequeño/efectos de los fármacos , Ribonucleósidos/farmacología , TemperaturaRESUMEN
Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2'-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory 'hot spots' within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation.
Asunto(s)
Materiales Biomiméticos/efectos adversos , Materiales Biomiméticos/química , Sistema Inmunológico/efectos de los fármacos , MicroARNs/genética , ARN Bicatenario/efectos adversos , ARN Bicatenario/química , Ribosa/química , Animales , Secuencia de Bases , Materiales Biomiméticos/síntesis química , Línea Celular Tumoral , Citocinas/metabolismo , Técnicas de Silenciamiento del Gen , Sistema Inmunológico/metabolismo , Ratones , ARN Bicatenario/síntesis química , ARN Bicatenario/genéticaRESUMEN
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in a double-stranded structure found in various RNA substrates, including mRNAs. Here we present recent efforts to define structure/activity relationships for the ADAR reaction. We describe the synthesis of new phosphoramidites for the incorporation of 7-substituted-8-aza-7-deazaadenosine derivatives into RNA. These reagents were used to introduce the analogues into mimics of the R/G-editing site found in the pre-mRNA for the human glutamate receptor B subunit (GluR B). Analysis of the kinetics of the ADAR2 reaction with analogue-containing RNAs indicated 8-aza-7-deazaadenosine is an excellent substrate for this enzyme with a deamination rate eight times greater than that for adenosine. However, replacing the C7 hydrogen in this analogue with bromine, iodine, or propargyl alcohol failed to increase the deamination rate further but rather decreased the rate. Modeling of nucleotide binding in the enzyme active site suggested amino acid residues that may be involved in nucleotide recognition. We carried out a functional screen of a library of ADAR2 mutants expressed in S. cerevisiae that varied the identity of these residues to identify active deaminases with altered active sites. One of these mutants (ADAR2 R455A) was able to substantially overcome the inhibitory effect of the bulky C7 substituents (-Br, -I, propargyl alcohol). These results advance our understanding of the importance of functional groups found in the edited nucleotide and the role of specific active site residues of ADAR2.
Asunto(s)
Adenosina Desaminasa/química , Adenosina Desaminasa/metabolismo , Dominio Catalítico , Edición de ARN , ARN/química , ARN/metabolismo , Secuencia de Bases , Desoxiadenosinas/química , Humanos , Ligandos , Modelos Moleculares , Oligonucleótidos/metabolismo , ARN/genética , Proteínas de Unión al ARNRESUMEN
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2'-deoxy-2'-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2'-OH with a 2'-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2'-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2'-OH substrate.
Asunto(s)
Adenosina Desaminasa/metabolismo , Adenosina/análogos & derivados , Oligorribonucleótidos/metabolismo , Edición de ARN , Adenosina/síntesis química , Adenosina/química , Adenosina/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Sitios de Unión/genética , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Proteínas de Unión al ARN , Especificidad por SustratoRESUMEN
Dimethylthexylsilyl 2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside was prepared by reduction of the corresponding 4,6-O-(4-methoxybenzylidene) acetal with sodium cyanoborohydride and trifluoroacetic acid. This alcohol was coupled to 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-glucopyranosyl trichloroacetimidate to give a beta-glucoside that was converted to dimethylthexylsilyl 3,4,6-tri-O-benzyl-beta-D-mannopyranosyl-(1-->4)-2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside by saponification, Dess-Martin oxidation, and sodium borohydride reduction. Sulfonylation then gave dimethylthexylsilyl 2-O-(benzylsulfonyl)-3,4,6-tri-O-benzyl-beta-D-mannopyranosyl-(1-->4)-2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside.
Asunto(s)
Glicósidos/química , Glicósidos/síntesis química , Manósidos/química , Manósidos/síntesis química , Secuencia de Carbohidratos , Glucósidos/síntesis química , Glucósidos/química , Glicosilación , Estructura MolecularRESUMEN
An as yet unknown beta-(1-->3)-mannohexaose has been synthesized by a block route involving the coupling of two trisaccharides. Comparison of three closely related attempted mannohexaose syntheses reinforces the influence of subtle matching and/or mismatching interactions on the outcome of convergent oligosaccharide synthesis.
Asunto(s)
Manosa/química , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Mananos/síntesis química , Mananos/química , Manosa/síntesis química , Oligosacáridos/químicaRESUMEN
[reaction: see text] 2-O-Propargyl ethers are shown to be advantageous in the 4,6-O-benzylidene acetal directed beta-mannosylation reaction. The effect is most pronounced when the O3 protecting group is a bulky silyl ether or a glycosidic bond; however, even with a 3-O-benzyl ether, the use of a 2-O-propargyl ether results in a significant increase in diastereoselectivity. The beneficial effect of the propargyl ether is thought to be a combination of its minimal steric bulk, as determined by a measurement of the steric A-value and of its moderately disarming nature, as reflected in the pKa of propargyl alcohol. Conversely, the application of a 3-O-propargyl ether in the benzylidene acetal directed mannosylation has a detrimental effect on stereoselectivity, for which no explanation is at present available. Deprotection is achieved by base-catalyzed isomerization of the propargyl ether group to the corresponding allenyl ether, followed by oxidative cleavage with N-methylmorpholine N-oxide and catalytic osmium tetroxide.
Asunto(s)
Éteres/química , Manósidos/química , Pargilina/análogos & derivados , Pargilina/química , Estructura Molecular , EstereoisomerismoRESUMEN
[reaction: see text] Phenyl 4,6-di-O-benzyl-2,3-O-carbonyl-beta-D-glucothiopyranoside and the regiosiomeric phenyl 2,6-di-O-benzyl-3,4-O-carbonyl-beta-D-glucothiopyranoside were prepared and studied as glucosyl donors at -60 degrees C in dichloromethane with preactivation by 1-benzenesulfinyl piperidine before addition of the acceptor alcohol. The 2,3-O-carbonate protected donor showed moderate to excellent beta-selectivity under these conditions depending on the acceptor employed, thereby providing a means for 1,2-trans-equatorial glycosidic bonds without recourse to neighboring group participation and its associated problem of ortho ester formation. In contrast, the 3,4-O-carbonate protected donor showed moderate to no beta-selectivity under the conditions employed. The results obtained in this study with carbonate protected glucopyranosyl donors are contrasted with those obtained previously in the manno- and rhamnopyranosyl series when the 2,3-O-carbonate protected is alpha-selective and the 3,4-O-carbonate is beta-selective.
Asunto(s)
Glucósidos/síntesis química , Carbonatos/química , Conformación MolecularRESUMEN
[reaction: see text]. The use of 2-O-propargyl ethers as protecting groups in 4,6-O-benzylidene-protected mannopyranosyl donors bearing either bulky silyl groups or glycosidic linkages on O3 overcomes the poor stereoselectivity achieved with the corresponding 2-O-benzyl ethers, due to a reduction of steric buttressing. Deprotection is conducted by treatment with potassium tert-butoxide followed by catalytic osmium tetroxide and N-methylmorpholine N-oxide.