Synthesis Of Phosphorothioate Oligonucleotides Research Articles

Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support.

5'-O-(2-Methoxyisopropyl) (MIP)-protected 2'-deoxynucleosides as chiral P(V)-building blocks, based on the limonene-derived oxathiaphospholane sulfide, were synthesized and used for the assembly of di-, tri-, and tetranucleotide phosphorothioates on a tetrapodal pentaerythritol-derived soluble support. The synthesis cycle consisted of two reactions and two precipitations: (1) the coupling under basic conditions, followed by neutralization and precipitation and (2) an acid catalyzed 5'-O-deacetalization, followed by neutralization and precipitation. The simple P(V) chemistry together with the facile 5'-O-MIP deprotection proved efficient in the liquid phase oligonucleotide synthesis (LPOS). Ammonolysis released nearly homogeneous Rp or Sp phosphorothioate diastereomers in ca. 80% yield/synthesis cycle.

Formation of the N2-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping

The acetyl ‘capping’ reaction routinely employed during phosphorothioate oligonucleotide synthesis has been implicated in the formation of an impurity species with a mass 41amu greater than the expected oligonucleotide molecule. The impurity has been found to arise by conversion of a protected guanine nucleobase to N2-acetyl-2,6-diaminopurine. A two-part mechanism is proposed consisting of transamidation of the protecting group on guanine and substitution of guanine’s O6 atom.

ChemInform Abstract: Internucleotide Phosphite Sulfurization with Tetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.

Abstract A new sulfurizing reagent has been developed for the automated synthesis of phosphorothioate oligonucleotides 1 via the phosphoramidite method. Tetraethylthiuram disulfide (TETD) converts internucleotide cyanoethyl phosphite 2 to the phosphorothioate triester 3 within 15 minutes at room temperature after each coupling step. With an optimized synthesis cycle, the efficiency of phosphorothioate oligonucleotide synthesis is comparable to that of phosphodiester oligonucleotides. Large scale synthesis (200μmole), oligonucleotides up to 100 bases long, and mixed phosphorothioate/phosphodiester DNA are demonstrated.

A two-step sulfurization for efficient solution-phase synthesis of phosphorothioate oligonucleotides

A solution-phase approach for the synthesis of phosphorothioate oligonucleotides that circumvents the use of chromatographic purifications of protected phosphorothioate intermediates was developed. Implementation of a two-step sulfurization protocol in the phosphoramidite method allows efficient isolation of the intermediate phosphorothioates by extractions. The viability of this approach is demonstrated by the synthesis of a hexameric phosphorothioate oligonucleotide fragment.

A Novel Universal Linker for Efficient Synthesis of Phosphorothioate Oligonucleotides

A versatile and conformationally preorganized universal linker molecule is reported here for efficient synthesis of phosphorothioate oligonucleotides. With respect to nucleoside loaded support, comparable yield and quality based on ion-pair LC-MS are obtained for both deoxy and 2′-O-methoxyethyl modified phosphorothioate oligonucleotides. No 3′-phosphate or phosphorothioate monoester or any modification of universal molecule still attached to oligonucleotide was observed.

Solution‐Phase Synthesis of Phosphorothioate Oligonucleotides Using a Solid‐Supported Acyl Chloride with H‐Phosphonate Chemistry

AbstractThe synthesis of oligonucleotides from dimers to a hexamer by a H‐phosphonate approach in solution using solid‐supported acyl chloride is reported herein. This strategy avoids the use of chromatography. The work‐ups have been simplified and involve filtration, aqueous extraction and precipitation. The synthesis was scaled up to 1 mmol for the dimers and 340 μmol for the hexamer. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Polysulfide Reagent in Solid-Phase Synthesis of Phosphorothioate Oligonucleotides: Greater than 99.8% Sulfurization Efficiency

A solution of sulfur (0.1 M) and sodium sulfide (0.01 M) in 3-picoline, referred to as polysulfide reagent, rapidly converts trialkyl and triaryl phosphite triesters to the corresponding phosphorothioate derivatives. Greater than 99.8% average stepwise sulfurization efficiency is obtained in the solid-phase synthesis of DNA and RNA phosphorothioate oligonucleotides via the phosphoramidite approach.

Formation of 4,4′-dimethoxytrityl- C-phosphonate oligonucleotides

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr- C-phosphonate (DMTr = 4,4′-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr- C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr- C-phosphonate moiety located internally or at the 5′terminal hydroxy group. DMTr- C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.

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

AbstractFor Abstract see ChemInform Abstract in Full Text.

Chemistry of Tetrathiomolybdate and Tetraselenotungstate: Applications in Carbohydrate and Peptide Chemistry

The reagents, benzyltriethylammonium tetrathiomolybdate, [BnNEt3]2MoS4 1 [1] and tetraethylammonium tetra­selenotungstate [Et4N]2WSe4 2 have been shown to be useful for sulfur and selenium transfer reactions respectively in organic synthesis. Preparation of disulfides from alkyl halides, [2] ring opening of epoxides, [3] tandem sulfur transfer-reduction-Michael addition [4] in one step, reduction of aryl azides to amines [5] and alkyl azides to imines [5] have been reported from our laboratory using the reagent tetrathiomolybdate 1. Tetrathiomolybdate 1 is also used for the selective deprotection of propargyloxycarbonyl (Poc) protective group for amines [6] in peptides and for alcohols [7] in carbohydrate chemistry. Recently, regio­selective reduction of anomeric azides [8] to amines using the reagent 1 has been reported. Reagent 1 has also been used, as a sulfur transfer reagent, for the synthesis of phosphorothioate oligonucleotides. [9] Large quantities of 1 can be prepared from ammonium tetrathiomolybdate and benzyltriethylammonium chloride. [2a]

A Short, Novel, and Cheaper Procedure for Oligonucleotide Synthesis Using Automated Solid Phase Synthesizer

AbstractFor Abstract see ChemInform Abstract in Full Text.

A Short, Novel, and Cheaper Procedure for Oligonucleotide Synthesis Using Automated Solid Phase Synthesizer

Dimethylthiuram disulfide (DTD) has been developed as an efficient thiolation reagent during automated synthesis of oligonucleotides using phosphoramidite chemistry. Simultaneous thiolation and capping was accomplished by mixing DTD with capping solution B, which saved 20% of solvent consumption and compressed the four-step synthesis cycle to three. Large-scale (1 mmol) synthesis of phosphorothioate oligonucleotides has been demonstrated with excellent yield and purity.

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.

Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

A method for solid-phase synthesis of stereodefined PS-oligos via an oxathiaphospholane approach using pure P-diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5'-O-DMTr-N-protected deoxyribonucleosides with 2-chloro-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O-labeled mercaptoalcohol, the corresponding 18O-labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS-oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s into their P-diastereomers, and for manual solid-phase synthesis of PS-oligos. Oxidation of 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s with selenium dioxide yields their 2-oxo-analogs, which are suitable either for elongation of stereodefined PS-oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O-labeled PO-oligos of predetermined P-chirality.

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

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.

Development of 2‘-O-Methoxyethyl Phosphorothioate Oligonucleotides as Antisense Drugs under Stereochemical Control

A systematic investigation of the stereoreproducibility of 2‘-O-methoxyethylribonucleoside phosphoramidite-based phosphorothioate oligonucleotide synthesis reveals that 1H-tetrazole-catalyzed phosphoramidite coupling followed by sulfurization with phenylacetyl disulfide (PADS) is under inherent stereochemical control. Analyses of single phosphorothioate linkages in oligonucleotides as well as in dimers by 31P NMR spectroscopy reveal reproducible stereochemical control in deoxy/2‘-O-methoxyethylribo- and 2‘-O-methoxyethylribo/deoxydinucleotide phosphorothioates and in 2‘-O-methoxyethylribo/2‘-O-methoxyethylribonucleotide phosphorothioate linkages. Particularly interesting is the fact that the R,S ratio of the phosphorothioate linkage is reproducible (within limits of experimental error) between syntheses and is independent of diastereomeric composition of phosphoramidites, synthesis scale, solid support, reactor, concentration during coupling or synthesis conditions. Activators, however, play a major role ...

Reusable solid-phase supports for oligonucleotides and antisense therapeuticsElectronic supplementary information (ESI) available: CGE data for octathymidine phosphodiester sequences on supports P1–5, P7, P9, and P11–13 PAGE data for phosphorothioate products, dimethoxytrityl analysis and UV measurements on supports P1–7, P9 and P15, step listing for ABI394 DNA synthesizer and CGE and HPLC data for phosporothioate syntheses on support P3. See http://www.rsc.org/suppdata/p1/b1/b105089n/

A general method for oligonucleotide synthesis on reusable solid-phase supports has been developed which will significantly lower the cost of large-scale synthesis. It consists of five steps: 1, nucleoside attachment to an hydroxy derivatized support through a Q-linker (hydroquinone-O,O′-diacetic acid) linker arm; 2, chloro-acetylation of unreacted surface groups; 3, conventional phosphodiester or phosphorothioate oligonucleotide synthesis; 4, cleavage from the support with aqueous or anhydrous ammonia; and 5, support regeneration with methanolic potassium carbonate. The recycling process is fast, fully automatable, and does not require removal of the support from the synthesis column. Fifteen solid-phase supports were evaluated with glycerol-CPG providing the best results. Consecutive syntheses of ISIS 2302, a dGCCCAAGCTGGCATCCGTCA phosphorothioate sequence, on the same synthesis column were performed. A glycerol-CPG synthesis column was satisfactorily used six consecutive times when NH4OH was the cleavage reagent. However, anhydrous NH3 allowed twelve consecutive syntheses without any deterioration in support loading, product quality, or amount of product produced. An improved method for preparing the essential nucleoside-3′-hemiesters of the Q-Linker and an unexpectedly slower rate of cleavage for phosphorothioate DNA vs. phosphodiester DNA are also described.

Influence of Diastereomeric Ratios of Deoxyribonucleoside Phosphoramidites on the Synthesis of Phosphorothioate Oligonucleotides

Extensive investigations on the influence of diastereomeric ratios of deoxyribonucleoside phosphoramidites on stereo-reproducibility of solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach indicate that the process is stereoreproducible and under inherent process control.

Is It Essential to Use Anhydrous Acetonitrile in the Manufacture of Phosphorothioate Oligonucleotides?

It is demonstrated that acetonitrile containing up to at least 200 ppm water can be used as the wash solvent and to prepare the capping and sulfur transfer reagent solutions used in large-scale solid-phase phosphorothioate oligonucleotide synthesis, thus replacing most of the expensive anhydrous acetonitrile previously used. No compromise in yield or product quality is observed, compared to a control synthesis performed using anhydrous acetonitrile throughout.

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

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.