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Chemical and Structural Implications of 1‘,2‘- versus 2‘,4‘- Conformational Constraints in the Sugar Moiety of Modified Thymine Nucleosides
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
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2007 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 72, no 13, 4716-4726 p.Article in journal (Refereed) Published
Abstract [en]

In order to understand how the chemical nature of the conformational constraint of the sugar moiety in ON/RNA(DNA) dictates the duplex structure and reactivity, we have determined molecular structures and dynamics of the conformationally constrained 1‘,2‘-azetidine- and 1‘,2‘-oxetane-fused thymidines, as well as their 2‘,4‘-fused thymine (T) counterparts such as LNA-T, 2‘-amino LNA-T, ENA-T, and aza-ENA-T by NMR, ab initio (HF/6-31G** and B3LYP/6-31++G**), and molecular dynamics simulations (2 ns in the explicit aqueous medium). It has been found that, depending upon whether the modification leads to a bicyclic 1‘,2‘-fused or a tricyclic 2‘,4‘-fused system, they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and the backbone torsions as well as by characteristic North-East type sugar conformation (P = 37° ± 27°, φm = 25° ± 18°) of the 1‘,2‘-fused systems, and (ii) pure North type (P = 19° ± 8°, φm = 48° ± 4°) for the 2‘,4‘-fused nucleosides. Each group has different conformational hyperspace accessible, despite the overall similarity of the North-type conformational constraints imposed by the 1‘,2‘- or 2‘,4‘-linked modification. The comparison of pKas of the 1-thyminyl aglycon as well as that of endocyclic sugar-nitrogen obtained by theoretical and experimental measurements showed that the nature of the sugar conformational constraints steer the physicochemical property (pKa) of the constituent 1-thyminyl moiety, which in turn can play a part in tuning the strength of hydrogen bonding in the basepairing.

Place, publisher, year, edition, pages
2007. Vol. 72, no 13, 4716-4726 p.
National Category
Chemical Sciences Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96274DOI: 10.1021/jo070356uISI: 000247246100014OAI: oai:DiVA.org:uu-96274DiVA: diva2:170793
Available from: 2007-10-16 Created: 2007-10-16 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Structural and Biophysical Studies of Nucleic Acids
Open this publication in new window or tab >>Structural and Biophysical Studies of Nucleic Acids
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (pKa) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic NE-type (P = 37° ± 27°, Φm = 25° ± 18°) for 1′,2′-constrained nucleosides, and N-type (P = 19° ± 8°, Φm = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 349
Keyword
Organic chemistry, nucleic acids, Nuclear Magnetic Resonance, molecular dynamics, sugar modified nucleosides, pKa, Organisk kemi
Identifiers
urn:nbn:se:uu:diva-8245 (URN)978-91-554-6982-5 (ISBN)
Public defence
2007-11-06, 113a, B7, Husargatan 3, Uppsala, 09:30
Opponent
Supervisors
Available from: 2007-10-16 Created: 2007-10-16Bibliographically approved
2. Physicochemical and Structural Aspects of Nucleic Acids
Open this publication in new window or tab >>Physicochemical and Structural Aspects of Nucleic Acids
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis consists of seven research publications concerning (i) pKa studies of nucleobases in model nucleotides to understand why RNA duplexes are more stable than DNA duplexes (Paper I), (ii) the role of Me(T)-π interactions in the relative stability of DNA-RNA heteroduplexes (Paper II), (iii) pKa measurements in nucleotides with different 2′-substituents (paper III), (iv) a conformation study of constrained sugars and a pKa study of 1-thyminyl to reveal effect of sugar constraints on the pKa of the nucleobase (paper IV), (v) NMR and MD studies of 1′, 2′-oxetane constrained thymidine incorporated Dickerson Drew dodecamer (paper V), (vi) the sequence dependent pKa perturbation of 9-guaninyl moeity in single stranded (ss) DNA and RNA (paper VI), (vii) the non identical chemical nature of internucleotidic phosphates in (ss) RNA using 31P NMR (paper VI), and an alkaline hydrolysis study of phosphodiesters in ssRNAs (paper VII). The architecture of DNA and RNA molecules is determined by (a) hydrogen bonding (b) base stacking (c) a variety of additional non-covalent interactions. In paper (I) we showed that A-U and G-C base pairings in RNA are more stable than A-T and G-C base pairings in DNA by 4.3 and 1 kJ mol-1 respectively. Me(T)-π interaction plays a dominant role in the relative stability of DNA-RNA duplexes (paper II). In paper III and IV, we have shown that 1′ , 2′- oxetane and azetidine rings have strong inductive effect on pyrimidine bases, and that the H2′-sugar proton can be the marker to understand the pseudoaromaticity of pyrimidine bases, as well as increasing constraints in sugar reducing the basicity of nucleobases. A 1′, 2′-oxetane locked thymidine (T) moiety deforms the local structure of Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2- investigated by High resolution NMR and MD study, as is discussed in the paper V. In papers VI and VII, we showed sequence context dependent pKa (N1) of 9-guaninyl perturbation in (ss) DNAs and RNAs and the non identical chemical nature of inter-nucleotidic phosphate groups in single stranded RNAs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 379
Keyword
Organic chemistry, pKa, Hydrogen bonding, Stacking, NMR, MD, DNA, RNA, Pyrimidine, Organisk kemi
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-8360 (URN)978-91-554-7056-2 (ISBN)
Public defence
2008-01-11, 101a, B7, Husargatan 3, Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2007-12-20 Created: 2007-12-20 Last updated: 2013-09-26Bibliographically approved
3. 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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Plashkevych, Oleksandr

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