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1.
Selective 2''-benzoylation at the cis 2'',3''-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures. 总被引:2,自引:2,他引:0
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T Kempe F Chow W I Sundquist T J Nardi B Paulson S M Peterson 《Nucleic acids research》1982,10(21):6695-6714
5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA. 相似文献
2.
A novel cationic building nucleoside building block designed for antisense and siRNA oligonucleotides is presented. Protected L-lysine was coupled to 2'-O-aminohexyluridine and the resulting nucleoside was phosphitylated for automated oligonucleotide synthesis. An increasing number of these 2'-O-lysylaminohexyl nucleosides lowered the melting temperature of desoxy-thymidine homododecamers, but the decrease was lower than that for DNA/RNA hybrids. Incubation with an exonuclease showed the exceptionally high resistance against enzymatic degradation. CD spectrometry revealed a gradual transition towards an A-type oligonucleotide structure. Based on these data, the cationic building block is particularly suited for gapmer antisense as well as siRNA oligonucleotides. 相似文献
3.
Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.
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Oligoribonucleotides containing 3'-S-phosphorothiolate linkages are valuable probes in nucleic acid biochemistry, but their accessibility has been limited because 3'-thioribonucleoside phosphoramidites have not been available. We synthesized 3'-thioribonucleoside derivatives (C, G, and U) via glycosylations of nucleoside bases with 3-S-thiobenzoyl-5-O-toluoyl-1,2-O-diacetylfuranose 5, which was obtained from 1 ,2-O-isopropylidene-5-O-toluoyl-3-trifluoromethane-sulfonyl-alpha-D-x ylofuranose 2 by SN2 displacement with sodium thiobenzoate. Additionally, a 3'-thioinosine derivative was prepared from inosine via direct modification of the ribose, analogous to the previously reported synthesis of 3'-thioadenosine, except that the intermediate 2',3'-epoxide 9 was first protected as the 5'-O-tert-butyldiphenylsilyl ether prior to subsequent synthetic steps. This hydrophobic silyl group facilitated extraction and isolation of synthetic intermediates. After removal of the protecting groups, the 3'-thionucleosides (C, G, U, and I) were treated with 2,2'-dipyridyl disulfide to protect the free thiol group as a disulfide. The 3'-thionucleosides were converted to the corresponding phosphorothioamidites using procedures analogous to those for standard phosphoramidites. The amino groups of 3'-thiocytidine and 3'-thioguanosine were protected as benzoyl and isobutyryl amides, respectively, and the 5'- and 2'-hydroxyl groups of each nucleoside were protected as dimethoxytrityl and tert-butyldimethylsilyl ethers, respectively. The 3'-thiol group was deprotected by reduction with DTT and phosphitylated to afford analytically pure 3'-S-phosphorothioamidites 15, which were incorporated into oligoribonucleotides by solid-phase synthesis. Chemical assays and mass spectrometry of the synthetic RNA showed that ribose-3'-S-phosphorothiolate linkages were installed correctly and efficiently into RNA oligonucleotides using phosphoramidite chemistry. 相似文献
4.
Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides. 总被引:3,自引:2,他引:1
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K Augustyns G Godard C Hendrix A Van Aerschot J Rozenski T Saison-Behmoaras P Herdewijn 《Nucleic acids research》1993,21(20):4670-4676
Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not. 相似文献
5.
《Bioorganic & medicinal chemistry》2020,28(16):115611
An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-β-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides. 相似文献
6.
Angeles Canol Myron F. Goodman Ramon Eritja 《Nucleosides, nucleotides & nucleic acids》2013,32(1-3):501-509
Abstract The preparation of a new protected derivative of 2,6-diaminopurine 2′-deoxyriboside carrying two phenoxyacetyl groups is described. The new derivative is useful to prepare oligonucleotides containing 2,6-diaminopurine and it is deprotected at the same time as the standard protecting groups of the natural bases. 相似文献
7.
Reverse DNA oligonucleotide synthesis (i.e. from 5′→3′) is a strategy that has yet to be exploited fully. While utilized previously for the construction of alternating 3′-3′- and 5′-5′-linked antisense oligonucleotides, the use of nucleoside 5′-phosphoramidites has not generally been used for the elaboration of (modified) oligonucleotides. Presently, the potential of reverse oligonucleotide synthesis for the facile synthesis of 3′-modified DNAs is illustrated using a phosphoramidite derived from tyrosine. The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligonucleotide resulting from the proteinase K digestion of the vaccinia topoisomerase I–DNA covalent complex. The results confirm the nature of the structure previously assigned to this product, and establish the facility with which proteinase K is able to complete the digestion of the polypeptide backbone of the DNA oligonucleotide-linked topoisomerase I. 相似文献
8.
Muthiah Manoharan Andrew M. Kawasaki Thazha P. Prakash Allister S. Fraser Marija Prhavc Gopal B. Inamati 《Nucleosides, nucleotides & nucleic acids》2013,32(6-7):1737-1746
Abstract Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety1 has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming30. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics. 相似文献
9.
10.
K Kamaike H Takahashi K Morohoshi N Kataoka T Kakinuma Y Ishido 《Acta biochimica Polonica》1998,45(4):949-976
A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met). 相似文献
11.
Qiu X Fernando L Melito PL Audet J Feldmann H Kobinger G Alimonti JB Jones SM 《PLoS neglected tropical diseases》2012,6(3):e1575
Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3-4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 μg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3-4 MAbs completely protected the majority of animals, while administration at 2-3 dpi achieved 50-100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection. 相似文献
12.
Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl. 总被引:11,自引:9,他引:2
The synthesis of protected nucleoside phosphoramidites bearing various markers such as biotinyl, dinitrophenyl, dansyl and pyrenyl groups are reported. These labelled deoxynucleosides phosphoramidites were used for solid phase oligonucleotide synthesis in the same way than the usual protected phosphoramidities without any change in the synthetic cycle and the deprotection step. The new labelled building blocks described herein have been used in conjunction with the labile base protected phosphoramidites ('PAC phosphoramidites') which allowed mild ammonia deprotection, especially recommended for the dinitrophenyl-labelled oligonucleotides. Multiple labelling (i.e. 10 to 20 biotins) can be efficiently and easily performed, on the same oligonucleotide which results in an increase of sensitivity. The polylabelled oligonucleotides are chemically well defined and gave increased signal and low background coloration for in situ hybridisation. The modified oligonucleotides can still be kinased in the normal way as the reporter groups are on the heterocycles. 相似文献
13.
Králíková S Budĕsínský M Masojidková M Rosenberg I 《Nucleosides, nucleotides & nucleic acids》2000,19(7):1159-1183
In this report we present a novel, simple way for the synthesis of 3'-C-phosphonate derivatives of all four basic 2'-deoxynucleosides in both fully protected and deprotected forms. The reactivity of the geminal hydroxy phosphonate moiety located at the 3'-carbon atom of the nucleoside was studied with respect to the use of this type of nucleoside phosphonic acid for the preparation of short oligonucleotides, namely, dinucleoside monophosphate analogues. 相似文献
14.
An orthogonal oligonucleotide protecting group strategy that enables assembly of repetitive or highly structured DNAs 总被引:1,自引:1,他引:0
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A general problem that exists in the assembly of large and organized DNA structures from smaller fragments is secondary structure that blocks or prevents it. For example, it is common to assemble longer synthetic DNA and RNA fragments by ligation of smaller synthesized units, but blocking secondary structure can prevent the formation of the intended complex before enzymatic ligation can occur. In addition, there is a general need for protecting groups that would block reactivity of some DNA bases in a sequence, leaving others free to react or hybridize. Here we describe such a strategy. The approach involves the protecting group dimethylacetamidine (Dma), which we show to remain intact on exocyclic amines of adenine bases while other bases carrying commercially available ‘ultra mild deprotection’ protecting groups are removed by potassium carbonate in methanol. The intact Dma groups prevent unwanted hybridization at undesired sites, thus encouraging it to occur where intended, and allowing for successful ligations. The Dma group is then deprotected by treatment with ammonia in methanol. Other common amine protecting groups such as benzoyl and allyloxycarbonyl were not successful in such a strategy, at least in part because they did not prevent hybridization. We demonstrate the method in the synthesis of a circular 54mer oligonucleotide composed of nine human telomere repeats, which was not possible to assemble by conventional methods. 相似文献
15.
Novel oligonucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) group have been described. Solid-phase synthesis of these compounds using an automated DNA synthesizer has been performed for the first time, including the Staudinger reaction between methanesulfonyl azide (mesyl azide) and 3′,5′-dinucleoside 2-cyanoethyl phosphite within an oligonucleotide immobilized on the polymer support, which is a product of phosphoramidite coupling. The mesyl phosphoramidate group is stable to the conditions of oligonucleotide synthesis, in particular, during acidic detritylation and subsequent removal of protecting groups and cleavage of an oligonucleotide from the polymer support by concentrated aqueous ammonia or methylamine at 55°C. It has been shown that the stability of complementary duplexes of oligodeoxynucleotides containing the mesyl phosphoramidate group with a single-stranded DNA is not inferior to the stability of native DNA:DNA duplex. Furthermore, mesyl phosphoramidate oligonucleotides are able to form a complementary duplex with RNA, which is only slightly less stable than the equivalent DNA:RNA duplex. This raises the possibility of their application as potential antisense therapeutic agents. 相似文献
16.
We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. 相似文献
17.
Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker is described. The branched oligonucleotides were synthesized on a DNA/RNA synthesizer using a controlled pore glass (CPG) with a pentaerythritol linker carrying 4,4'-dimethoxytrityl (DMTr) and levulinyl (Lev) groups. The stability of the triplexes between the branched oligonucleotides and the target single-stranded DNA or RNA was studied by thermal denaturation. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with the single-stranded DNA and RNA. Furthermore, the branched oligonucleotides containing 2'-O-methylribonucleosides, especially the oligonucleotide composed of 2'-deoxyribonucleosides and 2'-O-methylribonucleosides, stabilized the triplexes with the single-stranded DNA or RNA. Thus, the branched oligonucleotide containing 2'-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation. 相似文献
18.
Śárka Králíková Miloś Buděšínský Milena Masojídková Ivan Rosenberg 《Nucleosides, nucleotides & nucleic acids》2013,32(7):1159-1183
Abstract In this report we present a novel, simple way for the synthesis of 3′-C-phosphonate derivatives of all four basic 2′-deoxynucleosides in both fully protected and deprotected forms. The reactivity of the geminal hydroxy phosphonate moiety located at the 3′-carbon atom of the nucleoside was studied with respect to the use of this type of nucleoside phosphonic acid for the preparation of short oligonucleotides, namely, dinucleoside monophosphate analogues. 相似文献
19.
Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells 总被引:2,自引:0,他引:2
Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin. 相似文献
20.
Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA. 相似文献