Understanding High Diastereomeric Discrimination in Formation of Oligoribonucleotide Phosphorothioate Linkages: The First Study of pKa-Dependent Activation in Solid-Supported Coupling of 2′-O-Substituted Ribonucleoside Phosphoramidites

Abstract

Activation of 2′-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.     

Stability of Phosphorothioate Oligonucleotides in Aqueous Ammonia in Presence of Stainless Steel

Abstract

Antisense oligonucleotides as modulators of gene expression represent an exciting new drug technology. Oligodeoxyribonucleotide phosphorothioates are now among the most intensively investigated nuclease-resistant antisense analogs, as evidenced by a number of ongoing clinical trials against several disease targets. Structurally, these differ from natural oligonucleotides by the replacement of one of two nonbridging oxygen atoms by a sulfur atom at each internucleotide linkage. Among factors in the successful development of these complex molecules to support broad clinical trials have been advances made in automation, analysis and purification. The large scale synthesis of oligonucleotide phosphorothioates is presently carried out by initial formation of the internucleotide phosphite linkage using solid-phase phosphoramidite chemistry followed by sulfurization. Efficient synthesis of 20-mer oligophosphorothioates has been achieved on 0.15 mole scale with only 1.75-fold excess of amidite synthons. However, as the scale of synthesis increases to meet future market demands, issues related to fast and efficient synthesis, automation, scalability and product purification are also being investigated. One issue has been the protocol for final product deprotection. Since deprotection involves large quantities of saturated aqueous ammonium hydroxide, one might consider use of stainless steel reactors to withstand resulting vapor pressure at 55°C. A recent report,¹ however, discusses the instability of dimer phosphorothioates in aqueous ammonia in the presence of metal ions. As this is potentially an important issue for phosphorothioate oligonucleotide synthesis, we describe herein our findings regarding deprotection of a 20-mer oligodeoxyribonucleotide phosphorothioate with aqueous ammonia during process development studies.     

Understanding high diastereomeric discrimination in formation of oligoribonucleotide phosphorothioate linkages: the first study of pKa-dependent activation in solid-supported coupling of 2'-O-substituted ribonucleoside phosphoramidites

Activation of 2'-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.     

Influence of a Thiophosphate Linkage on the Duplex Stability - Does Sp Configuration Always Lead to Higher Stability Than Rp?

Abstract

ABSTRACT: In order to design an oligodeoxynucleoside phosphorothioate as an antisense molecule, it is important to establish the structure of the S-oligo with a strong affinity to the target RNA. In these molecules, internucleotide thiophosphate linkages produce diastereomers, the number of which increases in proportion to 2ⁿ (n: number of thiophosphate). To estimate the effect of this linkage on the duplex stability by UV melting curves, oligodeoxynucleotides having a single thiophosphate (referred to Soligo), dGCNsN'CG (s: thiophosphate, N, N′ = A or T), were prepared and their diastereomers isolated by HPLC. As demonstrated previously, the melting temperatures (Tm) for the Sp isomers were higher than those of the Rp when DNA was a target. On the other hand, it was found that for RNA as a target, the Rp isomers of dGCTsTCG and dGCAsTCG had higher stability than the Sp, and that the difference in the Tm values between the diastereomers was smaller than when DNA was a target. With dGCsTsACG, which has two thiophosphates, it was also found that the Tm values decreased with an increase in the number of thiophosphate linkages, and that the difference in Tm between the diastereomers was smaller when RNA was a target. Consequently, in practical clinical applications where RNA is a target, the influence of thiophosphate chirality on the duplex structure is almost negligible and Rp/Sp separation of an S-oligo may be of no major concern.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Diastereomeric Process Control in the Synthesis of 2′-O-(2-Methoxyethyl) Oligoribonucleotide Phosphorothioates as Antisense Drugs

Abstract

Coupling of 2′-O-methoxyethylsubstituted nucleoside phosphoramidites to 5′-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

Synthesis of Oligodeoxyribonucleotide Analogues by Use of Deoxyribonucleoside-3′-yl O-bis(1,1,1,3,3,3-Hexafluoro-2-Propyl) Phosphites as New Key Intermediates

Abstract

The deoxyribonucleoside-3′-yl O-bis(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite units (3) could be converted into the O-nucleosidyl phosphonate, O-2-cyanoethyl O-nucleosidyl phosphonate, and O-1,1,1,3,3,3-hexafluoro-2-propyl O-nucleosidyl phosphonothioate. Compound 3a was activated by methylimidazole to give the dithymidylate derivatives (8). The appropriately protected nucleosidyl phosphonates (3) were applied to the synthesis of oligodeoxyribonucleotides used as antisense oligonucleotides.     

Effects of β1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the β1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [³H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.     

Second Generation Antisense Oligonucleotides—Inhibition of PKC-α and c-raf Kinase Expression by Chimeric Oligonucleotides Incorporating 6″-Substituted Carbocyclic Nucleosides and 2″-O-Ethylene Glycol Substituted Ribonucleosides

Abstract

6′-substituted carbocyclic deoxyribonucleosides and 2′-O-ethylene glycol substituted ribonucleosides have been evaluated as building blocks for antisense oligonucleotides. Within the former class 6′-hydroxy substituted building blocks in combination with internucleoside phosphorothioate linkages have the potential to enhance antisense activity. 2′-O-ethylene glycol substituted ribonucleosides generally allow for the construction of potent antisense oligonucleotides with reduced phosphorothioate content, but differences exist in their effects on biological activity in cell culture in spite of virtually identical effects on RNA-binding affinity. Activity enhancement was most pronounced for a 2′-O-methoxyethyl substituent.     

Synthesis,biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (T_m), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.     

Methylphosphonate Modified DNA Hairpin Loops

Abstract

We synthesized and analyzed DNA hairpin molecules with methylphosphonate linkages of defined stereochemistry in the loop region. Dinucleotide building blocks ApA and TpT (p indicating methylphosphonate linkage with either Rp or Sp configuration) were synthesized, separated into the diastereomers, and incorporated at three positions of the tetraloops 5′-CGCAAAAGCG-3′ and 5′-CGCTTTTGCG-3′. The oligonucleotides were analyzed for their melting behavior. With a Tm of 67.5°C the molecule 5′-CGCAAApAGCG-3′ with a Sp configurated methylphosphonate is distinctly more stable than the Rp configurated one (Tm = 60.5 °C) and the unmodified oligonucleotide (Tm = 64.5 °C). In contrast to double helical DNA where the substitution of a phosphorodiester by a Sp configurated methylphosphonate results in a lower Tm, in DNA hairpin the introduction of Sp and Rp methylphosphonates at specific positions can lead to a stabilization of the structure.     

Synthesis of Dimer Phosphoramidite Synthons for Oligodeoxyribonucleotide Phosphorothioates Using Diethyldithiocarbonate disulfide as an Efficient Sulfurizing Reagent

Abstract

An efficient solution phase synthesis of deoxyribonucleoside phosphorothioate dimers utilizing phosphoramidite approach is described. Diethyldithiocarbonate disulfide (DDD) was found to be an efficient sulfurizing reagent in the conversion of phosphite triesters to phosphorothioate triesters.     

Use of Cationic Lipids to Enhance the Biological Activity of Antisense Oligonucleotides

Abstract

Antisense oligonucleotides bind to specific mRNA or pre-mRNA sequences through Watson-Crick base pairing, resulting in decreased expression of the targeted protein. The use of cationic lipids to enhance cellular uptake of antisense oligonucleotides is reviewed herein. Cationic lipids such as N[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA) were found to enhance the biological activity of phosphorothioate oligonucleotides by at least 1000-fold in cell culture. Cationic lipid preparations enhanced both the rate and amount of oligonucleotide which associated with cells. In addition, DOTMA markedly changed the subcellular distribution of the oligonucleotide. In the absence of lipid, fluorescein labelled phosphorothioate oligonucleotides accumulated in discrete cytoplasmic structures. In the presence of cationic lipids, the oligonucleotides concentrated within the nucleus, were excluded from nucleoli, and localized in punctate cytoplasmic structures. The accumulation of the oligonucleotide in the nucleus was inhibited by incubation of the cells at 4°C and by monensin, but not by chloroquine, ammonium chloride, or nocodazole. Cell lines, both primary and transformed, differ markedly in their sensitivity to inhibition of gene expression with antisense oligonucleotides in the presence of cationic lipids. The differential sensitivity of the cells correlates with the amount of ³⁵S-labelled oligonucleotide associated with the cells and the number of cells in the population which take up the oligonucleotide. Our studies have demonstrated that several types of cationic lipids markedly enhance the activity of phosphorothioate oligonucleotides in cell culture models. We are currently investigating the ability of cationic lipids to enhance activity of antisense oligonucleotides in more complex systems such as organ cultures and in animals.     

Synthesis of Deoxyribonucleoside Phosphorodithioate Dimers by a Triester Method

Abstract

Modified oligodeoxynucleotides have recently received much attention due to their therapeutic applications. Among the more promising are phosphorodithioates where both nonbridging oxygen atoms in the phosphate diesters are replced by sulfur. Deoxynucleoside phosphorodithioate dimers have been prepared in several ways, using H-phosphonate, phosphordiamidite, phosphoramidite, and thiophosphoramidite methods. Reports have also appeared on the synthesis of oligonucleotides with alternating phosphodiester and dithiophosphodiester linkages, as well as one on ribonucleoside dimers. Of the above methods, the thiophosphoramidite method has been applied successfully for the preparation of mixed base oligonucleotides containing contiguous phosphorodithioate linkages. However, this method gives products which contain varying amounts of phosphorothioate linkages (2 ? 10%) due to factors associated with the involvement of trivalent thiophosphorus compounds. In addition, the thiophosphoramidite synthons are difficult to purify on silica gel column, and have a tendency to dismutate in presence of acidic catalysts such as tetrazole. The thiophosphite intermediate which is formed is also unstable to tetrazole. Similarly in the thio- and dithio-H-phosphonate method, the primary coupling products are unstable to catalysts, pivaloyl chloride and iodine. Recently, Dahl et al reported^1–2 synthesis of dimers and oligomers upto octamer which also leads to formation of small amounts of phosphorothioate linkages. In additon, about 1.2% per phosphorodithioate linkage of the oligomer is cleaved during     

In Vitro and In Vivo Anti-influenza A Virus Activity of Antisense Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.     

Mixed-Backbone Oligonucleotides Containing Phosphorothioate and Methylphosphonate Linkages as Second Generation Antisense Oligonucleotide

Abstract

Antisense oligonucleotides are being studied as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have synthesized phosphorothioate oligonucleotides containing methylphosphonate linkages at the 5′-end, the 3′-end, or in the center, and have evaluated the impact of these linkages on the biophysical properties, biological properties, and some of the safety parameters.     

Synthesis of Anti-Sense Phosphorothioate Analogues for Pharmacokinetic and Pre-Clinical Studies

Abstract

Nudease-resistant oligonucleotides (11 to 28-mers) containing stereorandom phosphorothioate linkages have been recently reported to exhibit potent anti-HIV III effects and sequence-specific inhibition of protein synthesis. Relatively large amounts (100 mg 1g) of these analogues. which are needed for further biological testing and initial pharmacokinetic and pre-clinical studies, were readily obtained by automated hydrogen phosphonate chemistry followed by reversed-phase HPLC and further processing. This chemistry features 1 -adamanetanecarbonyl chloride as the activator, capping with isopropyl phosphite, and more complete sulfurization in only one-step following chain assembly. An automated, quantitative. picomole method for analysis of the analogues in blood samples has been developed.