uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Conformationally Constrained Nucleosides: Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 354
Keywords [en]
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
Keywords [sv]
Organisk kemi
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-8266ISBN: 978-91-554-6992-4 (print)OAI: oai:DiVA.org:uu-8266DiVA, id: diva2:170846
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
List of papers
1. Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides
Open this publication in new window or tab >>Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides
Show others...
2006 (English)In: J. Am. Chem. Soc., Vol. 128, no 47, p. 15173-15187Article in journal (Refereed) Published
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-96310 (URN)10.1021/ja0634977 (DOI)
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2015-05-19Bibliographically approved
2. Synthesis and Structure of Novel Conformationally Constrained 1‘,2‘-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and Thermal Stability of the Heteroduplexes
Open this publication in new window or tab >>Synthesis and Structure of Novel Conformationally Constrained 1‘,2‘-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and Thermal Stability of the Heteroduplexes
Show others...
2006 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 71, no 1, p. 299-314Article in journal (Refereed) Published
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-96311 (URN)10.1021/jo052115x (DOI)
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14Bibliographically approved
3. Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides
Open this publication in new window or tab >>Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, p. 5635-5646Article in journal (Refereed) Published
Abstract [en]

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing NorthEast conformationally constrained 1‘,2‘-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2‘,4‘-ethylene-bridged (aza-ENA-T) nucleoside, and 2‘-alkoxy modified nucleosides (2‘-O-Me-T and 2‘-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (ΔTm = 2.5−4 °C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4−5.5 °C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of 5−6 °C per modification. The 2‘-O-Me-T and 2‘-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 °C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (1.4−2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (1.7−8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (1.1−3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2‘-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2‘-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3‘-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3‘-exonuclease preferentially hydrolyzed the 3‘-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3‘-exonuclease cleaved at the 3‘-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5‘-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5‘-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2‘-O-Me-T, and 2‘-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-96312 (URN)10.1021/bi0620205 (DOI)000246283600002 ()17411072 (PubMedID)
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14
4. The chemical nature of the 2' substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA
Open this publication in new window or tab >>The chemical nature of the 2' substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA
Show others...
2006 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 4, no 9, p. 1675-1686Article 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.    

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-96313 (URN)10.1039/B601460G (DOI)000236970100010 ()
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14Bibliographically approved

Open Access in DiVA

fulltext(674 kB)972 downloads
File information
File name FULLTEXT01.pdfFile size 674 kBChecksum MD5
277c0c28a8bab80d64313b414d65a49a7840d4419d9092b1df681de0f4e137ca02881086
Type fulltextMimetype application/pdf
cover(575 kB)29 downloads
File information
File name COVER01.pdfFile size 575 kBChecksum MD5
cfe0399414afb41d8fa2a6214d056f25f68423821927717507510f83283b1418af3a400d
Type coverMimetype application/pdf
Buy this publication >>

By organisation
Department of Biochemistry and Organic Chemistry
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 972 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 964 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf