Stabilisation of DNA by Incorporation of Phosphothioate Groups

Abstract

The synthesis of Rp- and Sp-d (GGsAATTCC), an octanucleotide containing the recognition sequence for the restriction endonuclease Eco R1 as well as a phosphorothioate group at the cleavage site is described. Only the Rp-diastereomer is a substrate for Eco R1. Viral DNA, containing exclusively phosphate groups in the (+)strand, and phosphorothioate groups 5′-to deoxyadenosine in the (-)strand yields nicked DNA on cleavage by Eco R1 as an isolatable intermediate. Other restriction enzymes show a similar pattern of hydrolysis. - The influence of phosphorothioate groups on the B Z conformational change is demonstrated on phosphorothioate analogues of d(C-G)₄ and d(G-C)₄.     

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (2¹⁹) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.     

Synthesis of P-Thioadenylyl (2′-5′) Adenosine and P-Thioadenylyl (2′-5′)-P-Thioadenylyl-(2′-5′)-Adenosine

Abstract

Dimer and trimer adenylates with 2′-5′ phosphorothioate linkages were synthesized via the phosphoramidite method using p-nitrophenyl-ethyl group for phosphate protection and followed by sulfur oxidation. The various diastereoisomers were separated and characterized.     

Disulfide-linked oligonucleotide phosphorothioates: Novel analogues of nucleic acids

Summary The synthesis of phosphorothioate analogues of oligonucleotides by the oxidation of deoxyadenosine 3,5-bisphosphorothioate (3) was attempted. Cyclization of3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5-terminal phosphorothioate group undergoes efficient chain-extension when oxidized in the presence of3.     

Synthesis of the 3′-Derivatives of Phosphorothioate Oligonucleotide Analogues

Abstract

3′-Derivatives of phosphorothioate (PS) oligonucleotide analogues have been synthesized by a selective activation of a 3′-terminal phosphate group of the deprotected PS oligonucleotides using a mixture of triphenylphosphine and 2,2′-dipyridyldisulfide.     

Pharmacokinetic Properties of Phosphorothioate Oligonucleotides

Abstract

The pharmacokinetic parameters determined for different phosphorothioate oligonucleotides were compared. The data suggest that phosphorothioate pharmacokinetics are primarily determined by chemical class. The pharmacokinetics are consistent across species, show dose-dependency, and are independent of sequence.     

Solution Phase Synthesis of an Oligodeoxyribonucleotide Phosphorothioate for Therapeutic Applications

Abstract

Solution phase synthesis of an oligodeoxyribonucleotide phosphorothioate Octamer (5′-TTGGGGTT) using phosphorothioate triester method is reported.     

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.     

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

Abstract

Use of fully protected trimeric phosphoramidite synthons in the synthesis of oligonucleotide phosphorothioate shows a substantial reduction (>85%) in (n-1)-mer content as compared to oligomers synthesized through coupling of standard phosphoramidite monomers. A 20-mer oligodeoxyribonucleotide phosphorothioate which is in phase I clinical trials was chosen as an example for the studies.     

The Derivatives of Phosphorothioate Oligonucleotides

Abstract

Derivatives of phosphorothioate oligonucleotide analogues bearing alkylating N-methyl-4-(N-2-chloroethyl)-N-methylamino)benzylamine or stabilizing complementary complexes N-(2-hydroxyethyl)phenazinium residues at the 3′-terminal phosphate group were synthesized and investigated.     

Stereocontrolled Synthesis of Dithymidine Phosphorothioates by Use of a Functionalized 5′- Protecting Group Bearing an Imidazole Residue†

Abstract

Diastereoselective formation of internucleotidic phosphorothioate triester bonds was achieved by use of 3-(imidazol-1-ylmethyl)-4′,4″-dimethoxytrityl (IDTr) as a 5′-hydroxyl protecting group in the phosphotriester approach. After removal of all the protecting groups, stereochemistry of the major product was determined as the Spconfiguration by enzymatic digestion.

     

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.     

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.     

Stability of Stereoregular Oligo(Nucleoside Phosphorothioate)s in Human Cells. Diastereoselectivity of Cellular 3′-Exonuclease

Abstract

Stability of oligo(nucleoside phosphorothioate)s (PS-oligos) in HUVEC (human umbilical vein endothelial cells) has been studied. Cytosolic fraction of HUVEC possesses 3′-exonucleolytic activity which is responsible for degradation of natural oligomers and PS-oligos. The enzyme is R_p-specific, i.e. it cleaves internucleotide phosphorothioate function of R_p- and not S_p-configuration at phosphorus atom.     

Inhibition of 5-α-Reductase (TYPE-II) Expression by Antisense 3′-Deoxy-(2′-5′) Oligonucleotide Chimeras

Abstract

ABSTRACT: 3′-Deoxy-(2′-5′) oligonucleotides bind selectively to complementary RNA but not to DNA. 3′-Deoxy-(2′-5′) phosphorothioate ODN chimeras embedded with a short stretch of 3′-5′ phosphorothioate cassette are potent inhibitors of steroid 5-α-reductasc expression with significantly less non-specific interactions in cell culture.     

Evaluation of Sulfur-Transfer Reagents for Large Scale Synthesis of Oligonucleotide Phosphorothioate Analogues

Abstract

Several sulfur-transfer reagents have been evaluated for large scale synthesis of oligonucleotide phosphorothioate analogues, in which 3-ethoxy-1, 2-dithiazoline-5-one (EDITH, 5) shows potential as an alternative to Beaucage reagent.     

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.     

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 and Properties of Novel 5′-Linked Oligos

Abstract

We have synthesized normal and phosphorothioate oligos with 5′-linked groups, using either a phosphoramidite with the linked group attached or a mercaptopropanol linker. These linked oligos have been studied for cellular uptake as fluorescent labels, and for inhibition of gene expression in a cell free expression system, and in several other biological systems.