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The chemical nature of the 2' substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA
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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2006 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 4, no 9, 1675-1686 p.Article in journal (Refereed) Published
Abstract [en]

We here show that the pK(a) (error limit: 0.01 to 0.03 pK(a) unit) of a nucleobase in a nucleotide can be modulated by the chemical nature of the 2'-substituent at the sugar moiety. This has been evidenced by the measurement of nucleobase pK(a) in 47 different model nucleoside 3',5'-bis- and 3'-mono-ethylphosphates. The fact that the electronic character of each of the 2'-substituents ( Fig. 1) alters the chemical shift of the H2' sugar proton, and also alters the pKa of the nucleobase in the nucleotides has been evidenced by a correlation plot of pK(a) of N3 of pyrimidine (T/C/U) or pK(a) of N7 of 9-guaninyl with the corresponding delta H2' chemical shifts at the neutral pH, which shows linear correlation with high Pearson's correlation coefficients ( R = 0.85 - 0.97). That this modulation of the pK(a) of the nucleobase by a 2'-substituent is a through-bond as well as through-space effect has been proven by ab initio determined pK(a) estimation. Interestingly, experimental pK(a)s of nucleobases from NMR titration and the calculated pK(a)s (by ab initio calculations utilizing closed shell HF6-31G** basis set) are linearly correlated with R = 0.98. It has also been observed that the difference of ground and protonated/de-protonated HOMO orbital energies (Delta HOMO, a. u.) for the nucleobases (A/ G/ C/ T/ U) are well correlated with their pK(a)s in different 2'-substituted 3', 5'-bis-ethylphosphate analogs suggesting that only the orbital energy of HOMO can be successfully used to predict the modulation of the chemical reactivity of the nucleobase by the 2'-substituent. It has also been demonstrated that pKa values of nucleobases in 3',5'-bis-ethylphosphates ( Table 1) are well correlated with the change in dipole moment for the respective nucleobases after protonation or de-protonation. This work thus unambiguously shows that alteration of the thermodynamic stability (T-m) of the donor - acceptor complexes [ref. 20], as found with various 2'-modified duplexes in the antisense, siRNA or in triplexes by many workers in the field, is a result of alteration of the pseudoaromatic character of the nucleobases engineered by alteration of the chemical nature of the 2'-substitution.    

Place, publisher, year, edition, pages
2006. Vol. 4, no 9, 1675-1686 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-96313DOI: 10.1039/B601460GISI: 000236970100010OAI: oai:DiVA.org:uu-96313DiVA: diva2:170845
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2015-05-19Bibliographically approved
In thesis
1. Conformationally Constrained Nucleosides: Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
Open this publication in new window or tab >>Conformationally Constrained Nucleosides: Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring.

The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.

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 ; 354
Keyword
Organic chemistry, chemically modified oligonucleotides, azetidine, aza-ENA-T, cyanomethylene locked, free energy of activation, diaxial interactions, chair conformation, stable duplex, human serum, snake venom phosphodiesterase, antisense agents, RNase H, Organisk kemi
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-8266 (URN)978-91-554-6992-4 (ISBN)
Public defence
2007-11-09, B7:113a, BMC, Husargatan 3, Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2012-08-03Bibliographically approved

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Plashkevych, Oleksandr

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