uu.seUppsala University Publications
Change search
Refine search result
1 - 30 of 30
CiteExportLink to result list
Permanent 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
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Acharya, P
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Electrostatic Cross-modulation of the Pseudoaromatic Character in Single Stranded RNA by Nearest-neighbor interactions2005In: Pure and Applied Chemistry, Vol. 77, no 1, p. 291-311Article in journal (Refereed)
  • 2.
    Bogucka, M.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Nauš, P.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Pathmasiri, W.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, J.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Facile preparation of the Oxetane-Nucleosides2005In: Org. Biomol.Chem, Vol. 3, p. 4362-4392Article in journal (Refereed)
  • 3. Bramsen, Jesper B.
    et al.
    Pakula, Malgorzata M.
    Hansen, Thomas B.
    Bus, Claus
    Langkjaer, Niels
    Odadzic, Dalibor
    Smicius, Romualdas
    Wengel, Suzy L.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Engels, Joachim W.
    Herdewijn, Piet
    Wengel, Jesper
    Kjems, Jorgen
    A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 17, p. 5761-5773Article in journal (Refereed)
    Abstract [en]

    Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

  • 4. Changalov, M M
    et al.
    Ivanova, G D
    Rangelov, M A
    Acharya, P
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Acharya, S
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Minakawa, N
    Földesi, A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Stoineva, I B
    Yomtova, V M
    Roussev, C D
    Matsuda, A
    Chattopadhyaya, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Petkov, D.D.
    2'/3'-O-peptidyl Adenosine as a General Base Catalyst of its Own External Peptidyl Transfer: Implications for the Ribosome Catalytic Mechanism.2005In: Chembiochem, ISSN 1439-4227, Vol. 6, no 6, p. 992-996Article in journal (Other scientific)
  • 5.
    Chatterjee, S.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Pathmasiri, W.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, J.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    The 5-Me of thyminyl (T) interaction with the Neighboring Nucleobases dictate the relative stability of isosequential DNA/RNA hybrid duplexes2005In: Org. Biomol. Chem, Vol. 3, no 3, p. 3911-3915Article in journal (Refereed)
  • 6.
    Chatterjee, Subhrangsu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Pathmasiri, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Sequence-specific Solution Structures of the Four Isosequential Pairs of Single-stranded DNAs and RNAs2008In: Nature PrecedingsArticle in journal (Other academic)
    Abstract [en]

    The role of the sequence-context in the self-organization of four single-stranded (ss) isosequential pairs of DNAs (1 – 4) and RNAs (5 – 8), [d/r-(5’C1A2X3G4Y5A6C7): X3 = A or C, Y5 = A or C; sequence variations: 22 = 4], has been elucidated by NMR-constrained Molecular Dynamics (MD) simulations (2 ns). Following sequence-specific observations have been made from the solution NMR and the NMR constrained MD simulation study: (i) Analysis of the NOESY footprints, mainly (H8/H6)n to (H1’ and H3’)n-1 contacts, of ssDNAs (1 – 4) and ssRNAs (5 – 8) in the aqueous medium have shown that all ssDNAs (1 – 4) and ssRNAs (5 – 8) adopt right handed stacked helical structures in the NMR time scale. (ii) Intra-residual cross-peak intensities for the H(8/6)n- H(1’/2’/2’’/H3’)n contacts in ssDNAs and ssRNAs are stronger at the 3’-ends in comparison with those at the 5’-ends, suggesting that the dynamics of the nucleobases at the 3’-end are more restricted, whereas those at the 5’-end are more flexible. (iii) This relative NMR found mobility is consistent with the final RMSd calculations of the final NMR-MD structures of ssDNAs and ssRNAs. They show that the 5’-end nucleobases have higher RMSd values compared to those at the 3’-end, except for the sequence d/r(5’C1A2A3G4A5A6C7). (iv) Relative nOe intensities of inter-residual H(8/6)n – H(1’)n-1 and H(8/6)n – H(3’)n-1 contacts, as well as NMR observed fluctuations in the sugar conformations, for ssDNAs (1 – 4) and ssRNAs (5 – 8) show that no ssDNA or ssRNA adopts either a typical B-type DNA or A-type RNA form. (v) In the final NMR-MD structures all the [H8/6N(n)—H1’N(n-1)/ H3’N(n-1), N = A, G, C] distances in different isosequential pairs of ssDNA (1 – 4) and ssRNA (5 – 8) change depending upon the sequence context of the single-stranded nucleic acids. Both in the deoxy and ribo series, it is the purine-rich sequences [d/r-(5’C1A2A3G4A5A6C7) which form the most stable self-organized right-handed helical structures because of the favorable purine-purine stacking interactions. (vi) Stacking pattern at each of the dinucleotide steps show that the base-base nearest neighbor stacking interactions depend solely upon the sequence contexts of the respective ssDNAs (1 – 4) and ssRNAs (5 – 8). See pages 47 – 145 for Supplementary Information for detailed spectroscopic data.

  • 7. Chatterjee, Subhrangsu
    et al.
    Pathmasiri, Wimal
    Plashkevych, Oleksandr
    Honcharenko, Dmytro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Varghese, Oommen P
    Maiti, Mohitosh
    Chattopadhyaya, Jyoti
    The Chemical Nature of the 2'-Substituent in the Pentose-Sugar Dictates the Pseudoaromatic Character of the Nucleobase (pKa) in DNA/RNA2006In: Organic & Biomolecular Chemistry, ISSN 1477-0520, Vol. 4, p. 1675-1686Article in journal (Refereed)
  • 8.
    Chattopadhyaya, Jyoti
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Charge Transfer in DNA: From Mechanism to Application by Hans-Achim Wagenknecht: Charge Transfer in DNA: From Mechanism to Application2006In: ChemBioChem, Vol. 7, no 2, p. 389-Article, book review (Other (popular scientific, debate etc.))
  • 9.
    Chuanzheng, Zhou
    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.
    Conformationally Constrained Nucleic Acids as Potential RNA Targeting Therapeutics2010Doctoral thesis, comprehensive summary (Other academic)
    List of papers
    1. Fine Tuning of Electrostatics Around the Internucleotidic Phosphate through Incorporations of Functionalized 2', 4'-Carbocyclic-LNAs and –ENAs Lead to Significant Modulation of Antisense Properties
    Open this publication in new window or tab >>Fine Tuning of Electrostatics Around the Internucleotidic Phosphate through Incorporations of Functionalized 2', 4'-Carbocyclic-LNAs and –ENAs Lead to Significant Modulation of Antisense Properties
    Show others...
    2009 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 74, no 1, p. 118-134Article in journal (Refereed) Published
    Abstract [en]

    In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression   through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the intemucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic pan and their   integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH3) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resisitance without of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreassed cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or CS') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.

    National Category
    Biological Sciences Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-106719 (URN)10.1021/jo8016742 (DOI)000262004000015 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13
    2. Unusual Radical 6-endo Cyclization to the Carbocyclic-ENA and Elucidation of its Solution Conformation by 600 MHz NMR and ab initio Calculations
    Open this publication in new window or tab >>Unusual Radical 6-endo Cyclization to the Carbocyclic-ENA and Elucidation of its Solution Conformation by 600 MHz NMR and ab initio Calculations
    2008 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 6, no 24, p. 4627-4633Article in journal (Refereed) Published
    Abstract [en]

    In our previous paper (J. Am. Chem. Soc., 2007, 129, 8362), we reported the synthesis of 7'-Me-Carba-LNA and 8'-Me-Carba-ENA thymidine through 5-hexenyl or 6-heptenyl radical cyclization. Both 5-hexenyl and 6-heptenyl radical cyclized exclusively in the exo form, giving   unwanted exocyclic C7'-methyl group. In the present study, we showed that the regioselectivity of the 5-hexenyl radical cyclization could be favorably tuned by introduction of a hydroxyl group beta to the olefinic double bond, yielding about 9% of the 6-endo cyclization product. Possible pathways to give 6-endo cyclization product 9 compared to the intermediates responsible to give the 5-exo cyclization product 5 has been discussed. Based on this unique 6-endo cyclization strategy, a carbocyclic ENA modified thymidine (carba-ENA) has been successfully synthesized, which also enabled us to perform its full solution conformation analysis by using NMR (H-1 at 600 MHz) observables for the first time

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-106716 (URN)10.1039/b813870b (DOI)000261744900021 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13
    3. Synthesis and Structure of New Methylene-bridged hexopyranosyl nucleoside (BHNA)
    Open this publication in new window or tab >>Synthesis and Structure of New Methylene-bridged hexopyranosyl nucleoside (BHNA)
    Show others...
    2009 (English)In: Heterocycles, ISSN 0385-5414, E-ISSN 1881-0942, Vol. 78, no 7, p. 1715-1728Article in journal (Refereed) Published
    Abstract [en]

    A new member of hexopyranosyl nucleoside family, methylene-bridged   hexopyranosyl nucleoside (BHNA), has been synthesized through   generation of carbon radical at C6' in [6'S-Me, 7'S-Me]-carba-LNA T  nucleoside, followed by rearrangement to C4' radical which was quenched   by hydrogen atom to give BHNA. The stereoelectronic requirement for   this unusual radical rearrangement has been elucidated by chemical   model building and ab intio calculations to show that the coplanarity of the single electron occupied p-orbital at C6' with sigma*(O4'-C4')   plays an important role for the rearrangement reaction to take place.   The solution structure of BHNA has also been studied using NMR as well   as by ab initio calculations. The new six-membered pyranosyl ring in  BHNA, unlike other known hexopyranosyl nucleosides, adopts a twist   conformation, with base moiety occupying the axial position while  3'-hydroxymethyl and 4'-hydroxyl occupying the equatorial position.

    Keywords
    Conformationally Constrained Nucleoside, Hexopyranosyl Nucleoside, Radical Rearrangement, Conformational Analysis, ab initio Calculation
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-106720 (URN)10.3987/COM-09-11660 (DOI)000268441500003 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13
    4. New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies
    Open this publication in new window or tab >>New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies
    2009 (English)In: ARKIVOC, ISSN 1424-6376, no Part 3, p. 171-U46Article in journal (Refereed) Published
    Abstract [en]

    The hyper-constrained nucleoside, methylene-bridged hexopyranosyl nucleoside (BHNA) was incorporated into the antisnese oligonucleotides (AON), which show more preference for binding toward the complementary RNA (T-m loss by ca 5 degrees C) than that with the complementary DNA (T-m loss by 10 C), vis-a-vis corresponding native duplex. The origin of reduction of T-m of the duplexes formed by the BHNA incorporated AON and the complementary RNA or DNA was further investigated by thermal denaturation study with the single-mismatched DNA or RNA, CD spectroscopy, RNase H digestion study, as well as by molecular model   building. These studies showed that the introduction of BHNA causes only a limited local conformational perturbation in the AON/RNA heteroduplex, whereas it affects the global conformation in the AON-DNA duplex. BHNA incorporated AONs also show improved stability in the human blood serum, which may prove to have some potential therapeutic application.

    Keywords
    Conformationally-constrained nucleotide, hexopyranosyl nucleotide, antisense oligonucleotides (AON), nuclease stability, molecular modeling
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-106718 (URN)000265191400014 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13Bibliographically approved
    5. Double Sugar and Phosphate Backbone-constrained Nucleotides: Synthesis, Structure, Stability and Their Incorporation into Oligodeoxynucleotides
    Open this publication in new window or tab >>Double Sugar and Phosphate Backbone-constrained Nucleotides: Synthesis, Structure, Stability and Their Incorporation into Oligodeoxynucleotides
    2009 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 74, no 9, p. 3248-3265Article in journal (Refereed) Published
    Abstract [en]

    Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S-p)- or (R-p)-D-2-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio   calculations showed that in (S-p)- and (R-p)-D-2-CNA the Mowing occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)],   epsilon (cis), zeta[anticlinal(+)] for (S-p)-D-2-CNA, and delta [gaitche(+)], epsilon(cis), zeta[anticlittal(-)] for (R-p)-D-2-CNA. It   has been found that F- ion can catalyze the isomerization of pure (S-p)-D-2-CNA or (R-p)-D-2-CNA to give an equilibrium mixture (K =   1.94). It turned out that at equilibrium concentration the (S-p)-D-2-CNA isomer is preferred over the (R-p)-D-2-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered   dioxaphosphorinane ring in D-2-CNA was found to be dependent on the   internucleotidic phosphate stereochemistry. Thus, both (Sp)- and  (Rp)-D2-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximate   to 70-90%, carba-LNA-3',5'-phosphodiester (22) approximate to 10%, and   carba-LNA-6',3'-phosphodiester (23) < 10%. In contrasts the (S-p)-D-2-CNA was about 2 times more stable than (Rp)-D2-CNA under hydrazine hydrate/pyficfine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S-p)-D-2-CNA incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S-p)-D-2-CNA-modified AON by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally   characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S-p)-D-2-CNA   niodification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes   the AON/DNA and AON/RNA duplex by about -6 to -9 degrees C/modification. Treatment of (Sp)-D-2-CNA-modified AON with concentrated aqueous ammonia gave cwba-LNA-6',5'-phosphodiester modified AON (similar to 80%) plus a small amount of carba-LNA-3',5'-Phosphodiester-modified AON (similar to 20%). It is noteworthy that Carba-LNA-3',5'-phosphodiester modification stabilized  the AON/RNA duplex by +4 degrees C/modificafion (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6', 5'-phosphodiester modification   destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 degrees C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6'.5'-phosphodiester modified AONs are much more weakly   stacked than carba-LNA-3',5'-phosphodiester-modified AONs.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-106721 (URN)10.1021/jo900391n (DOI)000265554900002 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13
    6. 2-(4-Tolylsulfonyl)ethoxymethyl (TEM)-a new 2'-OH protecting group for solid-supported RNA synthesis
    Open this publication in new window or tab >>2-(4-Tolylsulfonyl)ethoxymethyl (TEM)-a new 2'-OH protecting group for solid-supported RNA synthesis
    2007 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 5, no 2, p. 333-343Article in journal (Refereed) Published
    Abstract [en]

    The 2-(4-tolylsulfonyl) ethoxymethyl (TEM) as a new 2'-OH protecting group is reported for solid-supported RNA synthesis using phosphoramidite chemistry. The usefulness of the 2'-O-TEM group is exemplified by the synthesis of 12 different oligo-RNAs of various sizes ( 14 - 38 nucleotides long). The stepwise coupling yield varied from 97 - 99% with an optimized coupling time of 120 s. The synthesis of all four pure phosphoramidite building blocks is also described. Two new reliable parameters, delta(C2') - delta(C3') and delta(H2') - delta(H3'), have been suggested for the characterization of isomeric 2'-O-TEM and 3'-O-TEM as well as other isomeric mono 2'/3'-protected ribonucleoside derivatives. The most striking feature of this strategy is that the crude RNA prepared using our 2'-O-TEM strategy is sufficiently pure (> 90%) for molecular biology research without any additional purification step, thereby making oligo-RNAs easily available at a relatively low cost, saving both time and lab resources.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-22696 (URN)10.1039/b614210a (DOI)000243269300019 ()17205178 (PubMedID)
    Available from: 2007-05-08 Created: 2007-05-08 Last updated: 2017-12-07Bibliographically approved
    7. High-quality oligo-RNA synthesis using the new 2′-O-TEM protecting group by selectively quenching the addition of p-tolyl vinyl sulphone to exocyclic amino functions
    Open this publication in new window or tab >>High-quality oligo-RNA synthesis using the new 2′-O-TEM protecting group by selectively quenching the addition of p-tolyl vinyl sulphone to exocyclic amino functions
    Show others...
    2007 (English)In: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 85, no 4, p. 293-301Article in journal (Refereed) Published
    Abstract [en]

    During the F--promoted deprotection of the oligo-RNA, synthesized using our 2′-O-(4-tolylsulfonyl)ethoxymethyl (2′-O-TEM) group [Org. Biomol. Chem. 5, 333 (2007)], p-tolyl vinyl sulphone (TVS) is formed as a by-product. The TVS formed has been shown to react with the exocyclic amino functions of adenosine (A), guanosine (G), and cytidine (C) of the fully deprotected oligo-RNA to give undesirable adducts, which are then purified by HPLC and unambiguously characterized by 1H, 13C Heteronuclear Multiple Bond Correlation (HMBC) NMR and mass spectroscopic analysis. The relative nucleophilic reactivities of the nucleobases toward TVS have been found to be the following: N6-A > N4-C > N2-G > > N3-U. This reactivity of TVS toward RNA nucleobases to give various Michael adducts could, however, be suppressed by using various amines as scavengers. Among all these amines, morpholine and piperidine are the most efficient scavenger for TVS, which gave highly pure oligo-RNA even in the crude form and can be used directly in RNA chemical biology studies.

    Keywords
    RNA synthesis, RNA alkylation, p-tolyl vinyl sulphone, Michael addition
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-13163 (URN)10.1139/V07-025 (DOI)000246576200008 ()
    Available from: 2008-04-15 Created: 2008-04-15 Last updated: 2017-12-11Bibliographically approved
    8. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
    Open this publication in new window or tab >>A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
    Show others...
    2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 9, p. 2867-2881Article in journal (Refereed) Published
    Abstract [en]

    The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

    Place, publisher, year, edition, pages
    Oxford University Press, 2009
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-106722 (URN)10.1093/nar/gkp106 (DOI)000266354600010 ()
    Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13Bibliographically approved
  • 10. Gagnon, Keith T.
    et al.
    Pendergraff, Hannah M.
    Deleavey, Glen F.
    Swayze, Eric E.
    Potier, Pierre
    Randolph, John
    Roesch, Eric B.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Damha, Masad J.
    Bennett, C. Frank
    Montaillier, Christophe
    Lemaitre, Marc
    Corey, David R.
    Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 47, p. 10166-10178Article in journal (Refereed)
    Abstract [en]

    Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

  • 11.
    Honcharenko, D.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Varghese, O.P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Barman, J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Synthesis and Structure of Novel Conformationally-constrained 1'2'-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and the Thermal Stability of the Heteroduplexes2006In: Journal of Organic Chemistry, Vol. 71, p. 299-314Article in journal (Refereed)
  • 12.
    Honcharenko, Dmytro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties2008Doctoral 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.

    List of papers
    1. 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, Vol. 71, p. 299-314Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97297 (URN)
    Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2015-05-19Bibliographically approved
    2. Synthesis of Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Adenosine (aza-ENA-A)
    Open this publication in new window or tab >>Synthesis of Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Adenosine (aza-ENA-A)
    2007 (English)In: Heterocycles, ISSN 0385-5414, E-ISSN 1881-0942, Vol. 73, no 1, p. 303-324Article 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.

    Keywords
    antisense, conformationally constrained nucleoside, stereoselactivity, piperidino skeleton, conformational analysis
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-97298 (URN)10.1002/chin.200819206 (DOI)000253154500026 ()
    Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2017-12-14Bibliographically approved
    3. Chemical and Structural Implications of 1‘,2‘- versus 2‘,4‘- Conformational Constraints in the Sugar Moiety of Modified Thymine Nucleosides
    Open this publication in new window or tab >>Chemical and Structural Implications of 1‘,2‘- versus 2‘,4‘- Conformational Constraints in the Sugar Moiety of Modified Thymine Nucleosides
    Show others...
    2007 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 72, no 13, p. 4716-4726Article 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.

    National Category
    Chemical Sciences Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-96274 (URN)10.1021/jo070356u (DOI)000247246100014 ()
    Available from: 2007-10-16 Created: 2007-10-16 Last updated: 2017-12-14Bibliographically approved
    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 & Biomolecular Chemistry, ISSN 1477-0520, Vol. 4, p. 1675-1686Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97300 (URN)
    Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2015-05-19Bibliographically approved
    5. 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
    6. Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide
    Open this publication in new window or tab >>Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide
    2008 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 73, no 7, p. 2829-2842Article in journal (Refereed) Published
    Abstract [en]

    The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-97302 (URN)10.1021/jo702747w (DOI)000254544800043 ()18331060 (PubMedID)
    Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2017-12-14Bibliographically approved
  • 13.
    Honcharenko, Dmytro
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Varghese, Oommen P
    Plashkevych, Oleksandr
    Barman, Jharna
    Chattopadhyaya, Jyoti
    Synthesis and Structure of Novel Conformationally Constrained 1',2'-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and Thermal Stability of the Heteroduplexes2006In: Journal of Organic Chemistry, ISSN 0022-3263, Vol. 71, p. 299-314Article in journal (Refereed)
  • 14.
    Honcharenko, Dmytro
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Zhou, Chuanzheng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide2008In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 73, no 7, p. 2829-2842Article in journal (Refereed)
    Abstract [en]

    The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.

  • 15.
    Isaksson, J
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    A uniform mechanism correlating dangling-end stabilization and stacking geometry.2005In: Biochemistry, ISSN 0006-2960, Vol. 44, no 14, p. 5390-401Article in journal (Other scientific)
  • 16.
    Kuljus, Kristi
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Mathematics and Computer Science, Department of Mathematics. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Mathematics and Computer Science, Department of Mathematics, Mathematical Statistics. Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    von Rosen, Dietrich
    Sand, Salomon
    Victorin, Katarina
    Comparing Experimental Designs for Benchmark Dose Calculations for Continuous Endpoints2006In: Risk Analysis, Vol. 26, no 4, p. 1031-1043Article in journal (Refereed)
  • 17.
    Li, Qing
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Upadhayaya, RamShankar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Deshpande, Sachin Gangadhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Földesi, Andras
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    The Physicochemical Properties of DNA-RNA Duplexes Containing Pure 7′R-Me- or 7′S-Me-Carba-LNA Derivatives of A, G, 5-MeC or T in the DNA Strand: Diastereomer Specific Comparison of The 3′-Exonuclease Stability and RNase H ElicitationIn: Article in journal (Refereed)
    Abstract [en]

       Recently, the intramolecular 5-exo-5-hexenyl free-radical cyclization gave access to 2′, 4′-locked carba-LNAs with different nucleobase moieties, i.e. 7′S- and 7′R-Me-cLNA-A, -G, -MeC and -T nucleosides (J. Am. Chem. Soc. 2007, 129, 8362-8379; J. Org. Chem. 2011, 76, 4408-4431). In these studies, diastereomeric mixtures of 7′S/R-Me-cLNA-MeC and -T and diastereomerically pure 7′R-Me-cLNA-A and -G have been incorporated into antisense oligonucleotides (AONs) for biological evaluations. These cLNA modified oligos have shown to have comparable RNA affinity and highly improved nuclease and blood serum stabilities relative to that of their LNA modified counterparts. In order to fully understand the spatial effect of diastereomeric orientation of 7′-methyl group in cLNA-A/G/MeC/T on the RNA affinity, nuclease stability and RNase H elicitation efficiency, we have synthesized and preparatively HPLC separated and tested each of the 7′S- (minor) and 7′R- (major) pure diastereomer of 7′S/R-Me-cLNA-A/G/MeC/T nucleosides. Incorporation into oligos of each pure diastereomer of cLNA led to higher RNA affinity (1-4°C/mod).  Tm increase was found to be dependent both on the modification site in the AON as well as whether it is 7′S or 7′R modified cLNA is incorporated. RNA selectivity (DDTm) was found to be in the range of 3.1-6.7°C compared to DDTm of 2.7°C for the native counterpart. The Tm variations modulated by 7′S- and 7′R-Me-cLNAs in the AON have been found to be sequence and position-dependent. Molecular dynamics (MD) simulations of DNA-RNA duplexes with AON2 and AON5, with pure diastereomer incorporated at the 7th position of the AON strand from 3′-end, revealed that both 7′S- and 7′R-Me-cLNA-A modifications have only small local effect on stacking and hydrogen bonding within the duplexes, with Watson-Crick base-pairing remained intact during 98-100% duration of the MD simulations. It has been however found that the Tm of each of the modified heteroduplex is dictated by the individual solvation energy (CPCM) of the 7′S- or 7′R-Me-cLNA diastereomer of A, G, MeC or T nucleobase. This demonstrates that the major factor behind variation in the thermal stabilities of the 7′R- or 7′S-Me-cLNA modified AON-RNA duplexes lies in the intrinsic hydrophobicity, hence its relative solvation energy, inherent in the 7′R- vis-a-vis 7′S-Me-cLNA modified monomer blocks, compared to those of the native and LNA counterparts. We have also found that AONs containing 7′S- and 7′R-Me-cLNA-MeC modifications exhibited unprecedented nuclease stabilities: the most stable AON is the one that contains 7′S-Me-cLNA-MeC, which is ~40 times more stable towards 3′-exonuclease (SVPDE) than 7′S- and 7′R-Me-cLNA-T modified AONs, which was in turn much more stable than 7′S- and 7′R-Me-cLNA-A and G modified counterparts. It is noteworthy that 7′S-methyl group of cLNAs endows the AON strand with more nuclease stability than that of 7′R configured counterpart when compared within the same nucleobase. Thus the carba-LNA modified AONs show nucleobase-dependent activity in the following order: MeC > T > A > G, regardless of 7′S- or 7′R-configurations in the carba-LNA. All of the cLNA and LNA modified AON/RNA hybrids can elicit RNase H activity with similar or even more enhanced rates of digestion by E. coli RNase H1 compared to that of the native AON/RNA. The cleavage rates and patterns of modified AON/RNA hybrids by E. coli RNase H1 are only correlated with the modification site in AON sequence of AON/RNA hybrids, but irrelevant to the structural features of incorporated modifications.

  • 18.
    Li, Qing
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Yuan, Fengfeng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Zhou, Chuanzheng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Free-Radical Ring Closure to Conformationally Locked α-l-Carba-LNAs and Synthesis of Their Oligos:: Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H2010In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 18, p. 6122-6140Article in journal (Refereed)
    Abstract [en]

    A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the β-D-LNA-T or β-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.

  • 19.
    Liu, Yi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Xu, Jianfeng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Karimiahmadabadi, Mansoureh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Zhou, Chuanzheng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Synthesis of 2',4' -Propylene-Bridged (Carba-ENA) Thymidine and Its Analogues: The Engineering of Electrostatic and Steric Effects at the Bottom of the Minor Groove for Nuclease and Thermodynamic Stabilities and Elicitation of RNase H2010In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 21, p. 7112-7128Article in journal (Refereed)
    Abstract [en]

    2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH2/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C=N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON: RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance hut 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH2 substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase HI with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

  • 20. Upadhayaya, Ram Shankar
    et al.
    Lahore, Santosh V.
    Sayyed, Aftab Y.
    Dixit, Shailesh S.
    Shinde, Popat D.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Conformationally-constrained indeno[2,1-c]quinolines: a new class of anti-mycobacterial agents2010In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 8, no 9, p. 2180-2197Article in journal (Refereed)
    Abstract [en]

    The design, synthesis and anti-mycobacterial activities of 23 conformationally-constrained indeno[2,1-c]quinolines against Mycobacterium tuberculosis H37Rv is reported. Based on a structural comparison with the anti-TB TMC207 we have devised a synthetic methodology for making new conformationally-constrained indeno[2,1-c] quinoline analogs (Fig. 1), by retaining the biologically significant quinoline and the phenyl rings in the SW and NW hemispheres, respectively. This new class of conformationally-constrained compounds has been designed such that their conformational flexibility across C4-C2' is diminished to nil by covalently locking the C4 center of the quinoline moiety in the SW hemisphere with the C2' center of the phenyl ring in the NW hemisphere, thereby decreasing the entropic penalty for their complex formation within the target protein, which will in turn give improved free-energy of stabilization of the complex. The efficacies of these anti-TB compounds were evaluated in vitro for 8/9 consecutive days using the BACTEC radiometric assay upon administration of a single-dose on day one. Compounds 11, 13, 16, 24, 30, 32 and 34 showed 85-99% growth inhibition of Mycobacterium tuberculosis. Compounds 13 and 34 however have inhibited the mycobacterial growth more effectively than others in the series, with minimum inhibitory concentrations (MIC) of 0.39 mu g mL(-1) (1 mu M) and 0.78 mu g mL(-1) (2 mu M) respectively.

  • 21. Upadhayaya, Ram Shankar
    et al.
    Vandavasi, Jaya Kishore
    Kardile, Ramakant A.
    Lahore, Santosh V.
    Dixit, Shailesh S.
    Deokar, Hemantkumar S.
    Shinde, Popat D.
    Sarmah, Manash P.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Novel quinoline and naphthalene derivatives as potent antimycobacterial agents2010In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 45, no 5, p. 1854-1867Article in journal (Refereed)
    Abstract [en]

    We have designed and synthesized both the quinoline and naphthalene based molecules influenced by the unique structural make-up of mefloquine and TMC207, respectively. These compounds were evaluated for their anti-mycobacterial activity against drug sensitive Mycobacterium tuberculosis H37Rv in vitro at single-dose concentration (6.25 mu g/mL). The compounds 22,23, 26 and 27 inhibited the growth of M. tuberculosis H37Rv 99%, 90%, 98% and 91% respectively. Minimum inhibitory concentration of compounds 22, 23, 26 and 27 was found to be 6.25 mu g/mL.. Our molecular modeling and docking studies of designed compounds showed hydrogen bonding with Glu-61, Tyr-64 and Asn-190 amino acid residues at the putative binding site of ATP synthase, these interactions were coherent as shown by Mefloquine and TMC207, where hydrogen bonding was found with Tyr-64 and Glu-61 respectively. SAR analysis indicates importance of hydroxyl group and nature of substituents on piperazinyl-phenyl ring was critical in dictating the biological activity of newly synthesized compounds. (C) 2010 Elsevier Masson SAS. All rights reserved.

  • 22. Upadhayaya, R.S.
    et al.
    Kishore, V.J.
    Nageswar Rao, V.
    Sharma, V.
    Dixit, S.S.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Design, Synthesis, Biological Evaluation and Molecular modeling studies of Novel Quinoline Derivatives Against Mycobacterium tuberculosis2009In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 17, no 7, p. 2830-2841Article in journal (Refereed)
    Abstract [en]

    We herein describe the synthesis and antimycobacterial activity of a series of 27 different derivatives of 3-benzyl-6-bromo-2-methoxy-quinolines and amides of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]- malonic acid monomethyl ester. The antimycobacterial activity of these compounds was evaluated in vitro against Mycobacterium tuberculosis H37Rv for nine consecutive days upon a fixed concentration (6.25 mu g/mL) at day one in Bactec assay and compared to untreated TB cell culture as well as one with isoniazide treated counterpart, under identical experimental conditions. The compounds 3, 8, 17 and 18 have shown 92-100% growth inhibition of mycobacterial activity, with minimum inhibitory concentration ( MIC) of 6.25 mu g/mL. Based on our molecular modelling and docking studies on well-known diarylquinoline antitubercular drug R207910, the presence of phenyl, naphthyl and halogen moieties seem critical. Comparison of docking studies on different stereoisomers of R207910 as well as compounds from our data set, suggests importance of electrostatic interactions. Further structural analysis of docking studies on our compounds suggests attractive starting point to find new lead compounds with potential improvements.

  • 23.
    Wenska, Małgorzata
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Honcharenko, Dmytro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Pathmasiri, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Synthesis of Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Adenosine (aza-ENA-A)2007In: Heterocycles, ISSN 0385-5414, E-ISSN 1881-0942, Vol. 73, no 1, p. 303-324Article in journal (Refereed)
    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.

  • 24.
    Xu, Jianfeng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Liu, Yi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Dupouy, Christelle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Synthesis of Conformationally Locked Carba-LNAs through Intramolecular Free-Radical Addition to C=N. Electrostatic and Steric Implication of the Carba-LNA Substituents in the Modified Oligos for Nuclease and Thermodynamic Stabilities2009In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 74, no 17, p. 6534-6554Article in journal (Refereed)
    Abstract [en]

    The syntheses of the hitherto unavailable parentfully unsubstituted carba-LNA and its C7'-amino and/or C6'-hydroxyl substituted derivatives, have been accomplished by the intramolecular 5-exo free-radical addition to a C4'-tethered C=N to give carba-LNAs with variable hydrophilic substituents at C6'/C7' (amino and/or hydroxyl). They have been introduced into isosequential antisense oligonucleotides (AONs) to examine how their relative electrostatic and steric effects in the minor groove of a putative AON-RNA duplex affect the target affinity, nuclease resistance, and RNase H elicitation. We show that 2'-oxygen in LNA is important in stabilizing the DNA/DNA and DNA/RNA duplexes vis-a-vis the unsubstituted carba-LNA and its other derivatives and that hydrophobic groups at C6'/C7' in both carba-LNA and carba-ENA relatively destabilize the AON/DNA duplex more profoundly than those in the AON/RNA duplexes. Two main factors affect the relative stabilities of AON/DNA versus AON/RNA duplexes: (i) hydration in the minor groove depending upon hydrophilicity vis-a-vis hydrophobicity of the substituents, and (ii) the relative size of the minor groove in the AON/DNA versus AON/RNA duplexes dictates the steric clash with the substituents depending upon their relative chiralities. We also show how the chirality and chemical nature of the C6'/C7' substituents affect the nuclease stability as well as the thermal stability and the RNase recruitment by AON/RNA duplexes.

  • 25.
    Zhou, Chuanzheng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies2009In: ARKIVOC, ISSN 1424-6376, no Part 3, p. 171-U46Article in journal (Refereed)
    Abstract [en]

    The hyper-constrained nucleoside, methylene-bridged hexopyranosyl nucleoside (BHNA) was incorporated into the antisnese oligonucleotides (AON), which show more preference for binding toward the complementary RNA (T-m loss by ca 5 degrees C) than that with the complementary DNA (T-m loss by 10 C), vis-a-vis corresponding native duplex. The origin of reduction of T-m of the duplexes formed by the BHNA incorporated AON and the complementary RNA or DNA was further investigated by thermal denaturation study with the single-mismatched DNA or RNA, CD spectroscopy, RNase H digestion study, as well as by molecular model   building. These studies showed that the introduction of BHNA causes only a limited local conformational perturbation in the AON/RNA heteroduplex, whereas it affects the global conformation in the AON-DNA duplex. BHNA incorporated AONs also show improved stability in the human blood serum, which may prove to have some potential therapeutic application.

  • 26.
    Zhou, Chuanzheng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    The synthesis of therapeutic locked nucleos(t)ides2009In: Current opinion in drug discovery & development, ISSN 1367-6733, E-ISSN 2040-3437, Vol. 12, no 6, p. 876-898Article, review/survey (Refereed)
    Abstract [en]

    This review highlights progress made during the past 2 to 3 years in the field of therapeutic locked nucleos(t)ides. Synthetic strategies to construct the conformationally locked nucleos(t)ides, their properties and potential therapeutic applications as antiviral compounds, and modified oligonucleotides to modulate antisense and RNAi properties are described.

  • 27.
    Zhou, Chuanzheng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Liu, Yi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Andaloussi, Mounir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Badgujar, Naresh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Fine Tuning of Electrostatics Around the Internucleotidic Phosphate through Incorporations of Functionalized 2', 4'-Carbocyclic-LNAs and –ENAs Lead to Significant Modulation of Antisense Properties2009In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 74, no 1, p. 118-134Article in journal (Refereed)
    Abstract [en]

    In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression   through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the intemucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic pan and their   integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH3) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resisitance without of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreassed cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or CS') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.

  • 28.
    Zhou, Chuanzheng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Double Sugar and Phosphate Backbone-constrained Nucleotides: Synthesis, Structure, Stability and Their Incorporation into Oligodeoxynucleotides2009In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 74, no 9, p. 3248-3265Article in journal (Refereed)
    Abstract [en]

    Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S-p)- or (R-p)-D-2-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio   calculations showed that in (S-p)- and (R-p)-D-2-CNA the Mowing occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)],   epsilon (cis), zeta[anticlinal(+)] for (S-p)-D-2-CNA, and delta [gaitche(+)], epsilon(cis), zeta[anticlittal(-)] for (R-p)-D-2-CNA. It   has been found that F- ion can catalyze the isomerization of pure (S-p)-D-2-CNA or (R-p)-D-2-CNA to give an equilibrium mixture (K =   1.94). It turned out that at equilibrium concentration the (S-p)-D-2-CNA isomer is preferred over the (R-p)-D-2-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered   dioxaphosphorinane ring in D-2-CNA was found to be dependent on the   internucleotidic phosphate stereochemistry. Thus, both (Sp)- and  (Rp)-D2-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximate   to 70-90%, carba-LNA-3',5'-phosphodiester (22) approximate to 10%, and   carba-LNA-6',3'-phosphodiester (23) < 10%. In contrasts the (S-p)-D-2-CNA was about 2 times more stable than (Rp)-D2-CNA under hydrazine hydrate/pyficfine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S-p)-D-2-CNA incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S-p)-D-2-CNA-modified AON by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally   characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S-p)-D-2-CNA   niodification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the therm