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Conformationally Co...
Conformationally Constrained Nucleosides : Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
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- Varghese, Oommen P., 1977- (författare)
- Uppsala universitet,Institutionen för biokemi och organisk kemi
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Engman, Lars (preses)
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- Stawinski, Jacek, Professor (opponent)
- Department of Organic Chemistry, Stockholm University
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(creator_code:org_t)
- ISBN 9789155469924
- Uppsala : Acta Universitatis Upsaliensis, 2007
- Engelska 60 s.
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Serie: Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 354
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences (hsv//eng)
Nyckelord
- 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
- Chemistry
- Kemi
Publikations- och innehållstyp
- vet (ämneskategori)
- dok (ämneskategori)
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