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Synthesis of Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Adenosine (aza-ENA-A)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
2007 (English)In: Heterocycles, ISSN 0385-5414, E-ISSN 1881-0942, Vol. 73, no 1, 303-324 p.Article in journal (Refereed) Published
Abstract [en]

The synthesis of conformationally constrained 2'-N,4'-C-ethylene-bridged adenosine(aza-ENA-A), in which the pentose-sugar is cis-fused with the piperidino skeleton at C2' and C4' centres of the sugar ring, is reported. The corresponding phosphoramidite building block will be used for incorporation into oligo-DNA and -RNA by solid phase synthesis to examine their nuclease stability as well as their application in blocking the translation of the target RNA using the antisense and siRNA approach. 2-Aza-6-oxabicyclo[3.2.1]octane skeleton is assembled through multi-step synthetic manipulation of appropriately protected D-arabinose based sugar precursor. The conversion of appropriate arabino precursor to ribo counterpart was achieved by direct nucleophilic displacement of "ara" positioned 2-(trifluoromethanesulfonyloxy) group in the sugar precursor 8. A high regio- and enhanced stereoselectivity with preferential formation of beta anomer in glycosylation reaction was achieved using Vorbruggen conditions in the absence of any 2-participating group. Coupling step was performed using 1-O-acetyl-3,5-di-O-benzyl-(2-deoxy-2-azido)-4-C-(p-toluoyloxyetliyl)-D- ribofur anose (10) as a glycosyl donor and persilylated N-6-benzoyladenine. Finally, the ring-closure giving the North-type conformationally constrained cis-fused bicyclic aza-ENA-A have been confirmed unambiguously by the long range H-1-C-13 NMR correlation (HMBC), TOCSY, COSY and nOe experiments.

Place, publisher, year, edition, pages
2007. Vol. 73, no 1, 303-324 p.
Keyword [en]
antisense, conformationally constrained nucleoside, stereoselactivity, piperidino skeleton, conformational analysis
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97298DOI: 10.1002/chin.200819206ISI: 000253154500026OAI: oai:DiVA.org:uu-97298DiVA: diva2:172170
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
Open this publication in new window or tab >>Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system.

The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence.

Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 440
Keyword
Bioorganic chemistry, antisense oligonucleotides, conformationally constrained nucleosides, azetidine, aza-ENA, target affinity, RNase H, exonuclease stability, pyrene-functionalized nucleotides, fluorescence, mismatch discrimination, Bioorganisk kemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-8887 (URN)978-91-554-7219-1 (ISBN)
Public defence
2008-06-05, C10:301, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2010-03-03Bibliographically approved

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