Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages (original) (raw)
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Nucleic Acids Research, 2020
Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically ...
Nature biotechnology, 2017
Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA ...
Nucleic Acids Research, 2019
The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3′ thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3′ and 5′ thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this appr...
Bioorganic & Medicinal Chemistry Letters, 1998
Antisense oligonucleotides are being evaluated in clinical trials as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have designed mixed-backbone oligonucleotides (MBOs) that contain phosphorothioate segments at the 3 and 5 ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligonucleotide. Some of these MBOs indicate improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H activation, and anti-HIV activity. In addition, more acceptable pharmacological, in vivo degradation and pharmacokinetic profiles were obtained with these MBOs.
Efficient synthesis of antisense phosphorothioate oligonucleotides using a universal solid support
Tetrahedron, 2006
It is demonstrated that solid support containing a novel universal linker could be efficiently used to synthesize both phosphorothioate oligodeoxyribonucleotides and second-generation 2 0 -O-methoxyethyloligoribonucleotides with high yield and quality as judged by ion-pair-liquid chromatography-electrospray mass spectroscopy, 31 P NMR and reversed phase HPLC. Analysis of oligonucleotides shows quality being superior to that produced with standard succinyl-linker solid supports, without contamination of materials resulting from linker or support backbone decomposition. q
Molecular Therapy - Nucleic Acids
The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS 2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS 2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS 2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.
Journal of Medicinal Chemistry, 2010
To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2 0 ,4 0 -BNA NC [NMe], and 2 0 ,4 0 -BNA NC bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2 0 ,4 0 -BNA NC [NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2 0 ,4 0 -BNA NC [NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2 0 ,4 0 -BNA NC [NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.
Molecular Therapy — Nucleic Acids, 2012
We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-l-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-l-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-l-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-l-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-l-LNA nucleotides in the 3′-and 5′-flanks with R-5′-Me-α-l-LNA but not R-6′-Me-or 3′-Me-α-l-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.
We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2′-5′-ribo-and 3′-5′-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2′-5′ linkages at each end) with the complementary 3′-5′-DNA and -RNA target strands suggest that 2′-5′-ribonucleoside incorporation into 3′-5′-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3′-5′-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2′-5′ linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996) Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A-and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2′-5′ modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3′-5′-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3′-5′ linkages. The current results suggest that a limited number of 2′-5′ linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents.