12 research outputs found

    A 6'-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF-HC Pseudohydrogen Bonds

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    The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 °C compared to thymidine and +1-3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF-HC hydrogen bond. In contrast, structural investigations in solution by (1)H and (13)C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside

    Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

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    We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides

    A 6′-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF–HC Pseudohydrogen Bonds

    No full text
    The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6′ (6′F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an <i>N</i>-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. <i>T</i><sub>m</sub> measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in <i>T</i><sub>m</sub> were in the range of +1–2 °C compared to thymidine and +1–3 °C compared to a standard bc-T residue. Structural investigations of the 6′F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF–HC hydrogen bond. In contrast, structural investigations in solution by <sup>1</sup>H and <sup>13</sup>C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF–HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6′F-bcT nucleoside

    A 6′-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF–HC Pseudohydrogen Bonds

    No full text
    The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6′ (6′F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an <i>N</i>-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. <i>T</i><sub>m</sub> measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in <i>T</i><sub>m</sub> were in the range of +1–2 °C compared to thymidine and +1–3 °C compared to a standard bc-T residue. Structural investigations of the 6′F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF–HC hydrogen bond. In contrast, structural investigations in solution by <sup>1</sup>H and <sup>13</sup>C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF–HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6′F-bcT nucleoside

    The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

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    Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes

    Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers

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    International audienceAntisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment
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