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.     

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.     

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.     

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.     

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.     

An Inverse Approach in Oligodeoxyribonucleotide Synthesis

Abstract

We synthesized 3′-O-dimethoxytrityl-5′O-phosphoramidites and 5′-O-succinates which can be used as monomeric building blocks for the built up of oligodeoxyribonucleotides in the alternative 5′-3′ direction. With this inverse strategy oligonucleotide 3′-conjugates as well as 3′-3′ and 5′-5′ internucleotidic linkages can be easily formed.     

Further Optimization of Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis

Various conditions for optimum detritylation (i.e., the removal of 5′-O-trityl protecting groups) during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic acids of variable concentrations were used to study the removal of the 4,4′-dimethoxytrityl (DMTr) group. It was found that the DMTr group could be completely removed under much milder acidic conditions than what are currently used for automated solid-phase synthesis. The 2,7-dimethylpixyl (DMPx) is proposed as an alternative and more readily removable group for the protection of the 5′-OH functions both in solid- and solution-phase synthesis. The improved detritylation conditions are expected to minimize the waste and offer a protocol for incorporation of acid sensitive building-blocks in oligonucleotides.     

A Simple and Stable Silica Gel Support for the Oligodeoxyribonucleotide Synthesis

Abstract

A simple and efficient synthesis of a solid support with a long chain polyamide spacer has been developed. The spacer was made by successive addition of ethylene-diamine and succinic anhydride. The obtained solid support gives very homogeneous 20 mer oligodeoxyribonucleotides, detected by HPLC and electrophoresis.     

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.     

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.     

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.

     

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.     

Solid Phase Synthesis of Phosphorothioate Oligonucleotides Utilizing Diethyldithiocarbonate Disulfide (DDD) as an Efficient Sulfur Transfer Reagent

Abstract

Diethyldithiodicarbonate (DDD), a cheap and easily prepared compound, is found to be a rapid and efficient sulfurizing reagent in solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach. Product yield and quality based on IP-LC-MS compares well with high quality oligonucleotides synthesized using phenylacetyl disulfide (PADS) which is being used for manufacture of our antisense drugs.      

Sulfurization Efficiency in the Solution Phase Synthesis of Deoxyribonucleoside Phosphorothioates - Comparison of Sulfur Triethylamine with Various Sulfurizing Agents 1

Abstract

Efficient solution-phase synthesis of nucleoside phosphorothioates utilizing phosphoramidite approach is described. Elemental sulfur in combination with triethylamine is the prefered choice for sulfurization of phosphite triester to phosphorothioates.

     

Physicochemical Properties of the Phosphoryl Guanidine Oligodeoxyribonucleotide Analogs

Russian Journal of Bioorganic Chemistry - This work describes the basic physicochemical properties of phosphoryl guanidine oligonucleotides (PGOs)—a new type of DNA analog with a partially or...     

The Use of Hydrogen Peroxide for Closing Disulfide Bridges in Peptides

The use of hydrogen peroxide for the formation of disulfide bridges was studied in 15 peptides of various lengths and structures. The oxidation of peptide thiols by hydrogen peroxide was shown to proceed under mild conditions without noticeable side reactions of Trp, Tyr, and Met residues. Yields of the corresponding cyclic disulfides were high and mostly exceeded those obtained with other oxidative agents, in particular, iodine. It was established that the use of hydrogen peroxide in organic medium also provided sufficiently high yields when large-scale syntheses of oxytocin and octreotide (up to 10 g) were carried out.     

An alternative advantageous protocol for efficient synthesis of phosphorothioate oligonucleotides utilizing phenylacetyl disulfide (PADS)

Phosphorothioate oligonucleotides could be synthesized using a 0.2 M solution of phenylacetyl disulfide (PADS) in a mixture of pyridine and acetonitrile (1:1, v/v) with > 99.9% step-wise efficiency. Unlike most other sulfurizing reagents that need to be stable in solution for performance, PADS needs to degrade and "age" in solution and hence performs efficiently even after storing the solution at room temperature for over a month. High yield and quality of oligonucleotides are produced thereby offering an alternative attractive protocol for use of this efficient sulfurizing reagent.     

Development of siRNA for therapeutics: efficient synthesis of phosphorothioate RNA utilizing phenylacetyl disulfide (PADS)

Efficient synthesis of phosphorothioate RNA (PS-RNA) is demonstrated by using phenylacetyl disulfide (PADS) in a mixture of pyridine and acetonitrile (1:1, v/v) for 3 min. Sulfurization is achieved with >99.8% stepwise efficiency. This reagent also performs efficiently during synthesis of RNA containing PS:PO mixed backbone.     

The 9-Fluorenylmethoxycarbonyl (Fmoc) Group and Its Use in Oligonucleotide Synthesis

Abstract

The introduction of the base-labile 9-fluorenylmethoxycarbonyl (Fmoc) group into the exocyclic amino function of 2′-deoxynucleosides and their dimethoxytritylation and phosphitylation is described. The resulting key intermediates were investigated in the built-up of different oligodeoxyribonucleoside phosphate and thiophosphate chains which were deprotected under mild basic conditions leading to crude oligomers of high purity.     

Synthesis and Biological Properties of 2′-5′ Oligodeoxyribonucleotide,an Isomer of Biologic DNA

Abstract

Oligonucleotide having 2′-5′ phosphodiester linkage has been synthesised on solid support using indigenously prepared 3′-deoxy-2′-phosphoramidites. The 2′-5′ oligonucleotide showed higher half-life when subjected to 3′-exonuclease, SVPD, digestion. This oligonucleotide formed a stable duplex with complementary RNA but not with DNA. Similarly, it did not form triplex as well either with DNA or RNA duplex.