Efficiency Of Coupling in the Synthesis of Deoxyribonucleotide Phosphorothioates Via Phosphotriester Approach Utilizing Various Activating Reagents 1

Abstract

The synthesis of deoxyribonucleotide phosphorothioates via phosphotriester approach utilizing various coupling reagents is described.

     

Efforts Toward Synthesis of Oligonucleotides for Commercialization

Abstract

Recent progress on the development of oligonucleotide phosphorothioates to meet market demands is reported.     

Chemoselective Synthesis of Dithymidine Phosphorothioate in Solution Using O-Protected Thiophosphate Monomers

Abstract

Diastereomerically pure O-protected thymine monothioate nucleotide (I) is efficiently coupled to protected thymidine (II) in a chemoselective, but not stereoselective manner, to give dithymidine phosphorothioates (III).     

Enzymatic Synthesis of Stereoregular (All Rp) Oligonucleotide Phosphorothioate and Its Properties

Abstract

A protocol has been established for the synthesis of stereoregular (all Rp) oligonucleotide phosphorothioates. A 25-mer oligodeoxynucleotide phosphorothioate has been synthesized and studied for biophysical and biochemical properties.     

Conjugated Antisense Oligonucleotides for Inhibition of Human Cytomegalovirus In Vitro

Abstract

Several thiono triester containing oligonucleotide phosphorothioates linked with a lipophilic group have been synthesized. Some of these modified antisense oligonucleotides show potent anti-HCMV activity as well as improved cellular association and nuclease resistance.     

Use of Phenylacetyl Disulfide (PADS) in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

Investigations into the use of phenylacetyl disulfide (PADS) as an efficient sulfur transfer agent in the solid phase synthesis of oligodeoxyribonucleotide phosphorothioates showed that under suitable solvent conditions, this relatively inexpensive reagent rapidly and efficiently sulfurizes internucleotide phosphite linkages.     

Improved Deproteciton Procedure for DPSE Protected Phosphorothioates

Abstract

Deprotection of 2-(diphenylmethylsilyl)ethyl (DPSE) protected (O,O,O)-and (O,O,S)-trialkyl phosphorothioates with tetrabutylammonium fluoride affords the corresponding phosphorothioate. Formation of phosphodiester linkages is not observed.     

Use of Phenylacetyl Disulfide (PADS) in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

Oligodeoxyribonucleotide phosphorothioates, where one of the nonbridging oxygen of the internucleotide phosphate is formally replaced by a sulfur atom, are the first class to undergo human clinical trials. Ongoing phosphorothioate clinical trials against several disease targets has necessitated manufacture of very large quantities of oligonucleotide active pharmaceutical ingredient (API). Clinical trial and future market demands have stimulated effort towards developing cost efficient large scale synthesis of these complex bio-molecules. This effort has culminated in the routine synthesis of 20-mer oligodeoxyribonucleotide phosphorothioates at 150 mmole scale using only 1.75-fold molar excess of amidites in less than 8 h total synthesis time.     

The Cytotoxicity of Anti-PAI-I Oligonucleotides and Their Conjugates

Abstract

The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.     

Stability Studies in Biological Media of Dimer Phosphotriesters Bearing Glucuronic Acid Residue

Abstract

Hydrolytic stability of dithymidine phosphorothioates and dithioates bearing a glucuronic acid derivative protecting group on the phosphate linkage were studied in various biological media. We found that the enzymatic hydrolysis was accompanied by another reaction resulting in formation of the dithymidine phosphodiesters. We have proposed several possible mechanisms of hydrolysis.     

Diastereocontrolled Synthesis of Phosphorothioate DNA via an Oxazaphospholidine Approach Using a Novel Class of Activators,Dialkyl(cyanomethyl)ammonium Salts

Abstract

Dialkyl(cyanomethyl)ammonium salts 1 were synthesized and used as a novel class of activators for the stereospecific condensations of diastereopure nucleoside 3′-O-oxazaphospholidines with a nucleoside. This new oxazaphospholidine method could efficiently produce both (Rp)- and (Sp)-dinucleoside phosphorothioates.     

Solution Phase Synthesis of Dithymidine Phosphorothioate by a Phosphotriester Method Using New S-Protecting Groups

Abstract

ABSTRACT

A phosphotriester method for the synthesis of dithymidine phosphorothioates with eight S-protecting groups has been investigated. Three of the S-protecting groups possesed catalytic activity, however side reactions occurred under deprotection. The best S-protecting group was 4-chloro-2-nitrobenzyl which could be removed with a minimum of side reactions (0.3 %). The coupling reagent PyFNOP (11) gave protected dithymidine phosphorothioate in 96% yield after 15 min coupling.     

Enhanced Biological Activity of Antisense Oligonucleotides Containing 5-(1-Hexynyl)-substituted Pyrimidine Nucleotides

Abstract

Antisense oligonucleotides directed against HSV- 1 immediate early gene mRNA were synthesized with replacement of the internal pyrimidine nucleotides by the corresponding 5-(l-hexynyl) analogues and with end-capping by phosphorothioates. These compounds were found to have improved binding affinity, increased stability towards nucleases and enhanced antiviral activity in a cell culture assay.     

Synthesis and Biological Activity of O,O-Dialkyl S-(5-Aryl-l,3,4-oxadiazol-2(3H)-on-3-yl)methyl Phosphorothioates and Phosphorodithioates

Some phosphorus derivatives of oxadiazoles were synthesized to seek insecticidal lead compounds. The l,3,4-oxadiazol-2-ones were converted via the N-methylol derivatives to the corresponding N-chloromethyl derivatives. From these derivatives a variety of O,O-dimethyl phosphorodithioates 4, O,O-dimethyl phosphorothioates 5 and O,O-di-i-propyl phosphorothioates 6 were prepared.

These phosphorus derivatives were examined for insecticidal activity towards houseflies and for anti-acetylcholinesterase (anti-AChE) activity using the housefly heads as an enzyme source. Most of the compounds 4 and 5 showed contact toxicity as high as the analogous methidathion insecticides, which appeared to correlate with the strong anti-AChE activity. On the other hand, all the compounds 6 showed a high activity in AChE inhibition but only a poor insecticidal activity.     

Isolation of Oligodeoxynucleoside Phosphorothioate Diastereomers by the Combination of DEAE Ion-Exchange and Reversed-Phase Chromatography

Abstract

In antisense trials, oligodeoxynucleoside phosphorothioates (S-oligo) stereoisomers arise from the thiophosphate R/S configuration. We have explored effective separation of these isomers by the combination of DEAE ion-exchange (IELC) and reversed-phase (RPLC) liquid chromatography. Twenty-six S-oligos and some of the natural counterparts were examined. In the presence of DMTr at the 5′-end, isomers could be separated well by ELC, while RPLC gave good resolution in the absence of this protecting group. Combination of these two different modes leads to more effective isolation.

     

Synthesis and Incorporation of 5″-Amino- and 5′-Mercapto-5′-Deoxy-2′-O-Methyl Nucleosides Into Hammerhead Ribozymes

Abstract

Novel 5′-amino-5′-deoxy-2′-O-methyl uridine, guanosine and adenosine 3′-O-phosphoramidites 5, 11, and 20, as well as protected 5′-mercapto-5′-deoxy-2′-O-methyl uridine 3′-O-phosphoramidite 23 were synthesized from 2′-O-methyl nucleosides. These analogs were incorporated at the 5′-ends of hammerhead ribozymes to evaluate achiral bridging 5′-N- phosphoramidates and 5′-S-phosphorothioates as alternatives for non- bridging phosphorothioates commonly used for end stabilization against nucleases. Oligonucleotide synthesis and deprotection conditions were optimized for better yields of these modified ribozymes.     

Studies on Nucleoside H-Phosphonothioates as Synthons in the Synthesis of Oligonucleotide Analogues

Abstract

Recently, we have reported that H-phosphonothioate diesters¹ are versatile synthetic intermediates for the preparation of phosphorothioates, phosphorodithioates, or other phosphodiester analogues. The most suitable coupling agents to produce Hphosphonothioate diesters were found to be various chlorophosphates¹ [e.g., 2-chloro-5,5-dimethyl-2-oxo-2λ⁵-1,3,2-dioxaphosphinane (NEP), diphenyl phosphorochloridate]. These reagents secure the required chemoselectivity during the condensation and are unreactive towards the P-H function in H-phosphonothioate diesters^1,2.     

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.     

Oligodeoxyribonucleotide Phosphorothioates: Substantial Reduction of (N-1)-Mer Content Through the Use of Blockmer Phosphoramidite Synthons

Abstract

Sequence-specific modulation of gene expression for the treatment of diseases has come to reality. Multiple examples of oligodeoxyribonucleotide phosphorothioates, in which one nonbridging oxygen atom of the internucleotide phosphate group of DNA is replaced by a sulfur atom are currently in advanced clinical trials. Recent advances in phosphoramidite coupling chemistry and solid phase synthesis methodology, together with current state of the art large-scale synthesizers, allow complete assembly of a 20-mer deoxyribonucleotide phosphorothioate at 150 mmole scale in just 8 h. Very high average coupling efficiencies (>98.5%) have been achieved at these scales with only 1.75-fold molar amidite excess.     

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates,Br, Pt and Hg

Abstract

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH₃)₃]²⁺ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.