WO1997029116A1 - Sulphur containing dinucleotide phosphoramidites - Google Patents
Sulphur containing dinucleotide phosphoramidites Download PDFInfo
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- WO1997029116A1 WO1997029116A1 PCT/GB1997/000327 GB9700327W WO9729116A1 WO 1997029116 A1 WO1997029116 A1 WO 1997029116A1 GB 9700327 W GB9700327 W GB 9700327W WO 9729116 A1 WO9729116 A1 WO 9729116A1
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- phosphorothioate
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- the present invention relates to dinucleotide phosphoramidites having a non-bridging sulphur group attached to the phosphorus moiety, the synthesis of these compounds and their use in the synthesis of phosphorothioate oligonucleotides.
- oligonucleotide synthesis relies upon solid phase chemistry.
- phosphoramidites are added in a stepwise manner to an initial immobilised nucleoside, with protecting and deprotecting steps as necessary in each cycle.
- the process is now automated and is normally able to produce IO "6 mol quantities of the desired end product.
- a suitable methodology is described by Beaucage in Methods in Molecular Biology, Vol 20, Protocols for Oligonucleotides and Analogues, ed Agrawal, Humana Press, Totawa, 1993, pages 33-61.
- Phosphorothioate oligonucleotides are regarded as the first generation of antisense oligonucleotide analogues which have been successfully tested in vi tro and in vivo as inhibitors of gene expression (see, "Oligonucleotides: Antisense Inhibitors of Gene Expression”, Ed. Cohen, Macmillan, London, 1989 and “Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS", Ed. ickstrom, Wiley-Liss, New York, 1992) . At present, a few uniformly modified phosphorothioate oligonucleotides are in human clinical trials and have the potential to be used as approved drugs.
- Phosphorothioate oligonucleotides are isoelectronic analogues of natural oligonucleotides in which one of the non-bridging intemucleotide oxygen atoms is replaced by a sulphur atom.
- the solid phase synthesis of phosphorothiate oligonucleotides has been achieved using H-phosphonate chemistry (see, Froehler et al , Tetrahedron Lett. 5575-5578 [1986]) where only one sulphur transfer step is required after assembling the desired sequence to convert all the intemucleotide linkages to phosphorothioates, or the phosphoramidite approach (see, Stec a t al , J.
- the solid phase monomeric phosphoramidite chemistry is routinely used to synthesize phosphorothioate oligonucleotides (on micromole to milli ole scale) as considerable efforts have been expended in enhancing the efficiency of the synthesis such as (i) the use of improved synthetic cycle protocols and solid supports (see, Ravikumar et al , Bioorganic & Medical Chemistry Lett., 2017 [1994]) (ii) sulphur transfer reagents (see Rao et al , Tetrahedron Lett., 6741 (1994) and references cited therein), (iii) capping and deblocking reagents (see, Agrawal et al , Tetrahedron Lett., 8565 [1994]).
- n- 1 and n+1 impurities are very similar to the full length product "n" and vary from batch to batch, especially when reduced excesses of monomeric nucleoside phosphoramidite synthons are used in each synthetic cycle.
- n-1 and n+l impurities are very similar to the full length product "n" and vary from batch to batch, especially when reduced excesses of monomeric nucleoside phosphoramidite synthons are used in each synthetic cycle.
- the solid phase phosphoramidite approach (useful for micromole to millimole scale synthesis) can be improved by the addition of a dimeric phosphoramidite synthon instead of a monomeric phosphoramidite synthon during the synthetic cycle and this forms the basis of the present invention.
- the dimeric phosphoramidite approach would achieve an increased yield (as the number of steps required to produce a particular oligonucleotide will be reduced) and enhanced separation of the desired oligonucleotide from the impurities (as their use results in n-2 and n+2 impurities instead of n-1 and n+1 impurities) due to the greater difference in size.
- the present invention provides an improved process for the solid phase synthesis of phosphorothioa-ce oligonucleotides using dinucleotide phosphoramidite synthons containing the S-protected phosphorothioate ester inte ucleotide linkage and a 3 -phosphoramidite functional group.
- the present invention provides novel compounds of formula I
- B represents a heterocyclic amine base or a derivative thereof
- R represents an acid labile protecting group
- R represents a protecting group, preferably selected from the group consisting of 2-cyanoethyl, 2- chlorophenyl, 2,4-dichlorophenyl and 4-nitrophenyl;
- R 2 represents a blocking or protecting group;
- Rj represents a blocking or protecting group
- A represents a hydrogen atom, or an alkoxy, allyloxy or suitably protected hydroxy group.
- the dinucleotide phosphoramidite of formula I can be used in conventional automated solid phase synthesis to produce phosphorothioate oligonucleotides .
- the present invention also provides a process for producing an oligonucleotide having at least one phosphorothioate linkage, said process comprising providing a compound of formula I above for reaction with the terminal nucleoside of the nudeotide chain located at the solid phase to assemble the nudeotide chain.
- nudeotide chain includes a single nucleoside located at the solid phase which will itself be the terminal group available for reaction.
- Group R is desirably 4 ,4 '-dimeth ⁇ xytrityl, but any other suitable protecting group may also be used.
- Groups R 2 and R 3 may each independently be an alkyl or aryl group.
- the heterocyclic base of group B may be, for example a purine, such as adenine, guanine or derivatives thereof, or a pyrimidine, such as cytosine, uracil, thymine or derivatives thereof.
- a purine such as adenine, guanine or derivatives thereof
- a pyrimidine such as cytosine, uracil, thymine or derivatives thereof.
- derivatives may be mentioned alkylated derivatives (especially methylated derivatives) and halogenated derivatives, but are not specially limited thereto. Uracil and derivatives thereof may be especially convenient for use.
- Triethylammonium salt of 5 -0-(4,4 - dimethoxytrityl)thymidine S-(2-cyanoe hyl) 3 -phosphorothioate see Reese et al, J. Chem. Soc. Perkin Trans. 1: 1605 [1995]
- N-(2- Cyanoethylthio)phthalimide (3.09g, 13.3 mmol) was added, followed by N-methylmorpholine (6.67ml, 60 mmol) and chlorotri ethylsilane (5.07ml, 40 mmol). The mixture was allowed to stir at ambient temperature. After 3 hours, the reaction mixture was poured into 0.5M triethylammonium bicarbonate (200ml). The organic layer was sepaiated and the aqueous layer was extracted with dichloromethane (200ml). The combined organic layers (dried over MgS0 4 ) were evaporated.
- the foam was then dissolved in dichloromethane (20ml) and purified on a silica chromatography column with a silica/product ratio of 10:1.
- the column was first packed with 1% pyridine in dichloromethane, then once the product had been loaded onto the column it was eluted with dichloromethane (100ml), MeCN (1000ml), and 10% MeOH in dichloromethane (250ml) to strip the column. The appropriate fractions were combined and evaporated under reduced pressure to a foam.
- the product was then dissolved in dichloromethane (50ml) and added dropwise to pentane (500ml) to give a precipitate.
- the material was treated with a solution of DBU (1,8- Diazabicyclo[5, 4 , 0 ]-undec-7-ene) in anhydrous pyridine (5:95, v/v 1.0ml) for 2 hours at 30°C.
- the solvents were then removed and the residue was then treated with concentrated aqueous ammonia (1.0ml) at 55°C for 12 hours.
- the ammoniacal solution was evaporated to a pellet under reduced pressure and the unpurified (crude) oligonucleotides were analysed.
- Fig 2 shows a comparison of anion-exchange (NucleoPac PA-100) chromatograms of unpurified 5'-0-DMT-on phosphorothioate oligomers (TC) 10 T 21-mer (Seq ID Nos 1 and 4) .
- Fig 2A gives the results for the 21-mer synthesised with monomeric phosphoramidites (Seq ID No 1) which has a product purity of 68.5%.
- Fig 2B gives the results for the 21-mer synthesised with dimeric phosphoramidites (Seq ID No 4) which has an increased product purity of 78.0%.
- Fig 3 shows a comparison of anion-exchange (NucleoPac PA-100) chromatograms of unpurified 5 ' -0-DMT-on product purity of 78.0%.
- Fig 3 shows a comparison of anion-exchange (NucleoPac PA-100) chromatograms of unpurified 5'-0-DMT-on phosphorothioate oligomers (CT) 10 A 21-mer (Seq ID Nos 2 and 5) .
- Fig 3A gives the results for the 21-mer synthesised with monomeric phosphoramidites Seq ID No 2) which have a product purity of 74.0%.
- Fig 3B gives the results for the 21-mer synthesised with dimeric phosphoramidites (Seq ID No 5) which has an increased product purity of 83.0%.
- Fig 4 shows a comparison of anion-exchange (NucleoPac PA-100) chromatograms of unpurified 5'-0-DMT-on phosphorothioate oligomers (TCC TTC TCT CCT CTC TTC CTA) 21-mer (Seq ID Nos 3 and 6).
- Fig 4A gives the results for the 21-mer synthesised with monomeric phosphoramidites (Seq ID No 3) which have a product purity of 73.8%.
- Fig 4B gives the results for the 21- mer synthesised with dimeric phosphoramidites (Seq ID No 6 ) which has an increased product purity of 85.5%.
- Fig 5 is a comparison of 31 P NMR spectra of unpurified 5'-0-DMT-on phosphorothioate oligomers for Seq ID Nos 3 and 6.
Abstract
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GB9602326.2 | 1996-02-06 | ||
GBGB9602326.2A GB9602326D0 (en) | 1996-02-06 | 1996-02-06 | Compounds |
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005010042A1 (en) | 2003-07-18 | 2005-02-03 | Charité-Universitäts- Medezin Berlin | 7a5/prognostin and use thereof for the diagnostic and therapy of tumors |
EP2068152A1 (en) | 2007-12-06 | 2009-06-10 | Max-Delbrück-Centrum für Molekulare Medizin (MDC) | c-Kit as a novel target for the treatment of pain |
EP2151248A1 (en) | 2008-07-30 | 2010-02-10 | Johann Bauer | Improved pre-mRNA trans-splicing molecule (RTM) molecules and their uses |
EP2275116A1 (en) | 2003-07-18 | 2011-01-19 | Sanofi-Aventis Deutschland GmbH | Use of a Pak inhibitor for the treatment of a joint disease |
WO2012080460A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012080461A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012080459A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012084709A1 (en) | 2010-12-17 | 2012-06-28 | Sanofi | Mirnas in joint disease |
EP2532755A1 (en) | 2011-06-10 | 2012-12-12 | Sanofi-Aventis | Methods and uses based on Slfn2 expression and relating to the identification and profiling of compounds for use in the treatment or prevention of pain |
WO2012168453A1 (en) | 2011-06-10 | 2012-12-13 | Sanofi | Methods and uses relating to the diagnosis or prognosis of pain-related tissue states or pain-related diseases such as pain |
EP2857501A1 (en) | 2013-10-03 | 2015-04-08 | ETH Zurich | Reprogramming of pluripotent stem cells for improved control of their differentiation pathways |
WO2015049277A1 (en) | 2013-10-01 | 2015-04-09 | Ruprecht-Karls-Universität Heidelberg | S100 based treatment of cardiac power failure |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4668777A (en) * | 1981-03-27 | 1987-05-26 | University Patents, Inc. | Phosphoramidite nucleoside compounds |
US5151510A (en) * | 1990-04-20 | 1992-09-29 | Applied Biosystems, Inc. | Method of synethesizing sulfurized oligonucleotide analogs |
WO1995014029A1 (en) * | 1993-11-18 | 1995-05-26 | Beckman Instruments, Inc. | Protected oligonucleosides and their use in high yielding synthesis of dna and antisense dna |
WO1995032980A1 (en) * | 1994-05-26 | 1995-12-07 | Isis Pharmaceuticals, Inc. | Synthesis of oligonucleotides |
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1996
- 1996-02-06 GB GBGB9602326.2A patent/GB9602326D0/en active Pending
-
1997
- 1997-02-06 WO PCT/GB1997/000327 patent/WO1997029116A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4668777A (en) * | 1981-03-27 | 1987-05-26 | University Patents, Inc. | Phosphoramidite nucleoside compounds |
US5151510A (en) * | 1990-04-20 | 1992-09-29 | Applied Biosystems, Inc. | Method of synethesizing sulfurized oligonucleotide analogs |
WO1995014029A1 (en) * | 1993-11-18 | 1995-05-26 | Beckman Instruments, Inc. | Protected oligonucleosides and their use in high yielding synthesis of dna and antisense dna |
WO1995032980A1 (en) * | 1994-05-26 | 1995-12-07 | Isis Pharmaceuticals, Inc. | Synthesis of oligonucleotides |
Non-Patent Citations (1)
Title |
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ZBIGNIEW J LESNIKOWSKI: "THE FIRST STEREOCONTROLLED SYNTHESIS OF THIOOLIGORIBONUCLEOTIDE: (RPRP)- AND (SPSP)-UPSUPSU", NUCLEOSIDES & NUCLEOTIDES, vol. 11, no. 9, 1 January 1992 (1992-01-01), pages 1621 - 1638, XP000564715 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005010042A1 (en) | 2003-07-18 | 2005-02-03 | Charité-Universitäts- Medezin Berlin | 7a5/prognostin and use thereof for the diagnostic and therapy of tumors |
EP2275116A1 (en) | 2003-07-18 | 2011-01-19 | Sanofi-Aventis Deutschland GmbH | Use of a Pak inhibitor for the treatment of a joint disease |
EP2068152A1 (en) | 2007-12-06 | 2009-06-10 | Max-Delbrück-Centrum für Molekulare Medizin (MDC) | c-Kit as a novel target for the treatment of pain |
EP2151248A1 (en) | 2008-07-30 | 2010-02-10 | Johann Bauer | Improved pre-mRNA trans-splicing molecule (RTM) molecules and their uses |
WO2012080459A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012080461A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012080460A1 (en) | 2010-12-17 | 2012-06-21 | Sanofi | Mirnas in joint disease |
WO2012084709A1 (en) | 2010-12-17 | 2012-06-28 | Sanofi | Mirnas in joint disease |
EP2532755A1 (en) | 2011-06-10 | 2012-12-12 | Sanofi-Aventis | Methods and uses based on Slfn2 expression and relating to the identification and profiling of compounds for use in the treatment or prevention of pain |
WO2012168452A1 (en) | 2011-06-10 | 2012-12-13 | Sanofi | Methods and uses based on slfn2 expression and relating to the identification and profiling of compounds for use in the treatment or prevention of pain |
WO2012168453A1 (en) | 2011-06-10 | 2012-12-13 | Sanofi | Methods and uses relating to the diagnosis or prognosis of pain-related tissue states or pain-related diseases such as pain |
WO2015049277A1 (en) | 2013-10-01 | 2015-04-09 | Ruprecht-Karls-Universität Heidelberg | S100 based treatment of cardiac power failure |
EP2857501A1 (en) | 2013-10-03 | 2015-04-08 | ETH Zurich | Reprogramming of pluripotent stem cells for improved control of their differentiation pathways |
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