WO2010072831A1 - Sulfurizing reagents and their use for oligonucleotides synthesis - Google Patents
Sulfurizing reagents and their use for oligonucleotides synthesis Download PDFInfo
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- WO2010072831A1 WO2010072831A1 PCT/EP2009/067902 EP2009067902W WO2010072831A1 WO 2010072831 A1 WO2010072831 A1 WO 2010072831A1 EP 2009067902 W EP2009067902 W EP 2009067902W WO 2010072831 A1 WO2010072831 A1 WO 2010072831A1
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- OMXHVBULBOFUDS-UHFFFAOYSA-N CCC(C)(N(C=C(C)C(N1)=O)C1=O)O/C(/CO)=[O]\C(CCC(C)=O)=O Chemical compound CCC(C)(N(C=C(C)C(N1)=O)C1=O)O/C(/CO)=[O]\C(CCC(C)=O)=O OMXHVBULBOFUDS-UHFFFAOYSA-N 0.000 description 1
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
- C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
- C07C311/03—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C313/00—Sulfinic acids; Sulfenic acids; Halides, esters or anhydrides thereof; Amides of sulfinic or sulfenic acids, i.e. compounds having singly-bound oxygen atoms of sulfinic or sulfenic groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C313/08—Sulfenic acids; Derivatives thereof
- C07C313/18—Sulfenamides
- C07C313/36—Sulfenamides having nitrogen atoms of sulfenamide groups further bound to other hetero atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
- C07D207/48—Sulfur atoms
Definitions
- the present application claims the benefit of U.S. application No. 61/140391, filed December 23, 2008, the whole content of which being herein incorporated by reference.
- Technical Field The present invention relates to phosphorothioate oligonucleotides, the preparation thereof using novel sulfurizing reagents, said sulfurizing reagents and the preparation thereof.
- Oligonucleotides belong to a class of biopharmaceuticals with a great potential for therapies of various diseases including cancer, viral infections and inflammatory disease to name a few.
- An important approach to advancing oligonucleotides as therapeutics involve modifications of the oligomer backbone to provide, among other things, metabolic resistance, chemical stability and to improve in vivo transport to the site of action. Examples of modified backbone chemistries include: peptide nucleic acids (PNAs) (see Nielsen, Methods MoI.
- Phosphorothioates can be formed by oxidative sulfurization (Oligonucleotide synthesis, methods and applications, P. Herdewijn Methods in Molecular Biology, volume 288, Chapter 4, 51-63). There are basically two approaches to making phosphorothioates depend upon the nature of phosphorous esters used for this reaction and the expected products. One of them involves introduction of the unsubstituted sulfur atom to phosphorus by means of, for example, elemental sulfur, dibenzoyl tetrasulfide, 3-H-l,2- benzodithiol-3-one 1,1 -dioxide (also known as Beaucage reagent, (Iyer et al., J. Org. Chem.
- TETD tetraethylthiuram disulfide
- DTD dimethylthiuram disulfide
- PADS phenylacetyl disulfide
- Stec's reagent bis(O,O-diisopropoxy phosphinothioyl) disulfide
- a second approach to making oligomeric phosphorothioates is used with the H-phosphonate method and involves a reaction between H- phosphonate diester and a sulfur transfer reagent in which the sulfur atom, bearing an aliphatic or aromatic substituent, is transferred to phosphorus.
- the auxiliary substituent at sulfur serves the role of a protecting group during the synthetic operation and usually is cleaved at the final stage of oligonucleotide preparation. This method is particularly suitable for the synthesis of oligonucleotides in solution.
- a critical problem in the solution synthesis of oligonucleotides concerns the necessity to obtain high substrate conversions with excellent specificity at each synthetic step giving high purity products in a form that facilitates simple purification, in particular avoiding chromatography. Given the lack of methods allowing for economical solution phase synthesis, the solution phase technology does not seem to be currently used for commercial scale oligonucleotide synthesis.
- the invention now discloses novel sulfurizing reagents, a process for their manufacture and their use in the economical and convenient synthesis and purification of phosphorothioate oligonucleotides notably in solution. Disclosure of Invention
- the present invention relates in particular to the invention described in the appended claims.
- the invention also relates to processes and reagents substantially described in the present specification, in particular in the examples.
- the invention has a number of advantages over existing methods of P-S linkage formation, in particular in the synthesis of oligonucleotides carried out preferably via the H-phosphonate method.
- the residue R transferred e.g. to an oligonucleotide with the novel reagent can facilitate crystallization or precipitation of oligonucleotides, allowing for simple purification of the products with minimum or no chromatography. It has been found out that the oligonucleotides having from two to at least sixteen nucleotide units made with this method do not necessarily have to be purified by chromatography until after the final deprotection of the required oligonucleotide.
- the intermediate oligomers can be obtained pure enough for optional deprotections at the 5'- and 3' - positions and further coupling of these crude deprotected materials to higher oligonucleotides, if desired.
- the disclosed method provides an access to a variety of sulfurizing reagents which can be used to modify the properties of formed oligonucleotides with respect to maximizing the efficiency of simple, chromatography- free purifications.
- Still another advantage of the method according to the invention is that a simple cleavage of for example the sulfur- protecting acyloxymethylene group RC(O)-OCH 2 can be easily accomplished under mild conditions, for example, with primary or secondary or hindered amines e.g.
- cleavage products can be easily removed from the products by solvent or aqueous wash.
- the stability characteristics for example of the acyloxymethylene group e.g. under basic non-nucleophilic conditions allow for selective deprotection reactions along the synthesis pathways and hence greater flexibility of synthesis schemes, for example by preventing the cleavage of nucleobase protection groups.
- each elongation cycle comprises generally three steps and it is advantageous to remove even small amounts of impurities which would otherwise accumulate along the way. Because of large number of steps, the use of chromatography at each step may not be economically feasible in the practical large scale oligonucleotide synthesis. Therefore, we also disclose a chromatography- free methodology for the purification of oligonucleotides formed during the chain elongation process.
- a first particular object of this invention is to provide oligonucleotides which comprise at least one internucleotide linkage comprising a P-S-R bond and at least two nucleosides, wherein R corresponds to the formula (I)
- A is a geminally substituted alkylene group, preferably CH 2
- X and Y are independently selected from S and O
- Ro is selected from the group consisting of optionally substituted carbon bonded organic residue, such as in particular optionally substituted alkyl or aryl, SRx, ORx and NRxRy wherein Rx and Ry are selected from H and organic residues and at least Rx is a substituent other than H.
- the oligonucleotides according to the invention are valuable synthesis intermediates for synthesis of P-sulfurized oligonucleotides which have advantageous properties as to their solubility characteristics thus allowing for efficient purification which can be effectively accomplished, for example, by a combination of precipitation and extraction techniques.
- the oligonucleotides according to the invention are also believed to be effective as pro-drug, capable to release a phosphorothioate oligonucleotide in vivo, by cleavage of the R-group in the human body or in the body of an animal.
- oligonucleotide in the frame of the present invention, denotes in particular an oligomer of nucleoside monomeric units comprising sugar units connected to nucleobases, said nucleoside monomeric units being connected by internucleotide bonds.
- An "internucleotide bond” refers in particular to a chemical linkage between two nucleoside moieties, such as the phosphodiester linkage typically present in nucleic acids found in nature, or other linkages typically present in synthetic nucleic acids and nucleic acid analogues.
- Such internucleotide bond may for example include a phospho or phosphite group, and may include linkages where one or more oxygen atoms of the phospho or phosphite group are either modified with a substituent or replaced with another atom, e.g., a sulfur atom, or the nitrogen atom of a mono- or di-alkyl amino group.
- Typical internucleotide bonds are diesters of phosphoric acid or its derivatives, for example phosphates, thiophosphates, dithiophosphate, phosphoramidates, thio phosphoramidates.
- nucleoside is understood to denote in particular a compound consisting of a nucleobase connected to a sugar.
- Sugars include, but are not limited to, furanose ring such as ribose, 2'-deoxyribose and non-furanose ring such as cyclohexenyl, anhydrohexitol, morpholino.
- the modifications, substitutions and positions indicated hereinafter of the sugar included in the nucleoside are discussed with reference to a furanose ring, but the same modifications and positions also apply to analogous positions of other sugar rings.
- the sugar may be additionally modified. As non limitative examples of the modifications of the sugar mention can be notably made of modifications at e.g.
- the 2'-or 3'-position, in particular 2'-position of a furanosyl sugar ring including for instance hydrogen; hydroxy; alkoxy such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy; azido; amino; alkylamino; fluoro; chloro and bromo; 2 '-4'- and 3 '-4 '-linked furanosyl sugar ring modifications, modifications in the furanosyl sugar ring including for instance substitutions for ring 4'-0 by S, CH 2 , NR, CHF or CF 2 .
- nucleobase is understood to denote in particular a nitrogen- containing heterocyclic moiety capable of pairing with a, in particular complementary, nucleobase or nucleobase analog.
- Typical nucleobases are the naturally occurring nucleobases including the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U), and modified nucleobases including other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil
- nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4- b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
- Oligonucleotide typically refers to a nucleoside subunit polymer having from about 2 to about 50 contiguous subunits. The nucleoside subunits can be joined by a variety of intersubunit linkages.
- oligonucleotides includes modifications, known to one skilled in the art, to the sugar backbone (e.g., phosphoramidate, phosphorodithioate), the sugar (e.g., 2' substitutions such as 2'-F, 2'-0Me), the base, and the 3' and 5' termini.
- the oligonucleotide comprises from 2 to 30 nucleotides.
- the oligonucleotide contains nucleosides selected from ribonucleosides, 2'-deoxyribonucleosides, 2 '-substituted ribonucleosides, 2'-4'-locked-ribonucleosides, 3 '-amino- ribonucleosides, 3'- amino-2'-deoxyribonucleosides.
- R is selected from a methyleneacyloxy group, a methylene carbonate group and a methylene carbamate group.
- R is a methyleneacyloxy group, it corresponds preferably to formula -CH 2 -O-C(O)-Ro wherein R 0 is a C1-C20, saturated, unsaturated, heterocyclic or aromatic, hydrocarbon residue.
- Ro is a saturated hydrocarbon residue, it is preferably selected from linear, branched or cyclic alkyl residues. Ro can for example be selected from lower alkyl or cycloalkyl (C1-C7) residues. Particular saturated hydrocarbon residues are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert. butyl, cyclopentyl and cyclohexyl.
- a methyl, ethyl or n-propyl group is preferred.
- An ethyl group is more particularly preferred.
- Ro is an aromatic residue, it is suitably selected from aromatic systems having from 6 to 14 carbon atoms.
- Particular aromatic residues are selected from phenyl and naphthyl groups which can be substituted, for example, by aryl or heteroaryl, alkyl, cycloalkyl, heterocycle or heterosubstitutents such as halogens,amines,ethers, carboxylates, nitro, thiols, sulfonic and sulfones.
- a phenyl group is preferred.
- Ro is a heterocyclic residue
- it is often selected from heterocycles containing at least one annular N, O or S atom which are bonded to the carbonyl group through an annular carbon atom.
- heterocyclic residues include pyridine and furan.
- the oligonucleotide comprises at least two internucleotide linkages comprising a P-S-R bond and at least three nucleotides, wherein R is a methyleneacyloxy group as described herein.
- R is a methylene carbamate group
- Rx and Ry are independently selected from alkyl or (hetero)aryl.
- Rx and/or Ry are alkyl groups.
- Rx and/or Ry can for example be selected from lower alkyl or cycloalkyl (C1-C7) residues.
- Particular alkyl groups are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert butyl, cyclopentyl and cyclohexyl.
- Rx and Ry in the methylene carbamate group are both alkyl groups, in particular as described herein before.
- a N,N-dimethyl or N 5 N- diethyl group is more particularly preferred.
- R x and Ry form together a 3 to 8 membered ring optionally containing an additional annular heteroatom selected from O, N and S.
- Particular examples include a N-piperidyl or an N-pyrrolidyl group.
- R is a methylene carbonate group, it corresponds preferably to formula -CH 2 -O-C(O) ORx wherein Rx is selected from optionally substituted alkyl, cycloalkyl and (hetero)aryl groups.
- Rx is an alkyl group.
- Rx can for example be selected from lower alkyl or cycloalkyl (C1-C7) residues.
- Particular alkyl groups are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert butyl, cyclopentyl and cyclohexyl.
- a methyl, ethyl or n-propyl group is preferred.
- An ethyl group is more particularly preferred.
- Rx is an aryl group, it is suitably selected from aromatic systems having from 6 to 14 carbon atoms. Particular aromatic residues are selected from phenyl and naphthyl groups. A phenyl group is preferred.
- Rx is a heterocyclic residue, it is often selected from heterocycles containing at least one annular N, O or S atom which are bonded to the oxycarbonyl group through an annular carbon atom. Particular examples of such heterocyclic residues include pyridine and furan.
- substituents Rx, Ry and Ro given herein before for the case when R is selected from methyleneacyloxy group, a methylene carbonate group and a methylene carbamate group equally apply to the corresponding thioanalogues wherein X and/or Y in formula (I) are sulfur. It is also understood that the mentioned substituents may be optionally substituted, for example by halogen or alkoxy substituents or they may be modified, for example by inclusion of catenary heteroatoms, in particular oxygen into an alkyl chain.
- a second particular object of this invention relates to a sulfurizing agent of formula R"-S-R wherein R is as defined here before in the context of the oligonucleotide according to the invention and R" is a leaving group.
- the sulfurization agent according to the invention allows for particularly efficient sulfur transfer, in particular to form S- protected phosphorthioate internucleotide linkages in oligonucleotides.
- the sulfurizing agent according to the invention introduces a protected sulfur from which the protective group can be cleaved selectively and efficiently.
- the leaving group R" is generally an electrophilic group.
- R" is a group containing an electrophilic nitrogen atom bonded to the sulfur.
- the electrophilic nitrogen atom is suitably substituted with at least one electron- withdrawing group.
- the sulfurizing agent corresponds to formula (II)
- R A R-S'%° wherein R A and R B are equal or different from each other and at least one of R A and R B is selected from substituted sulfonyl or an acyl group, said R A and R B optionally forming together a cyclic substituent.
- R A and R B When at least one, preferably one, of R A and R B is substituted sulfonyl, it is generally selected from alkyl and aryl sulfonyl groups.
- the alkyl substituent therein is preferably selected from lower alkyl or cycloalkyl (C1-C7) residues.
- Particular alkyl groups are selected from methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-Butyl, cyclopentyl and cyclohexyl.
- a methyl, ethyl or n- propyl group is preferred.
- a methyl group is more particularly preferred.
- the aryl substituent therein is, for example, an, optionally substituted, phenyl group.
- the alkyl substituent therein is preferably selected from lower alkyl or cycloalkyl (C1-C7) residues.
- alkyl groups are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert butyl, cyclopentyl and cyclohexyl.
- a methyl, ethyl or n-propyl group is preferred.
- a methyl group is more particularly preferred.
- R A and R B are acyl groups forming together a cyclic substituent, preferably a 4 to 7 membered ring
- the sulfurizing agent corresponds to formula (III)
- Ri, R 3 and R 4 are independently a C1-C20, optionally unsaturated or aromatic, hydrocarbon residue, preferably a linear or branched alkyl group or a cycloalkyl group.
- R" is a dicarboxylamide.
- the sulfurizing agent corresponds to formula (IV)
- Z is an group, chosen among the group of -CH 2 -CH 2 - ,
- a third particular object of the invention relates to a process for the synthesis of the sulfurizing agent according to the invention which comprises (a) reacting a sulfuryl halide, preferably sulfuryl chloride with a thioacetal of formula R-S-C(O)-R 2 wherein R is as described previously and R 2 is an organic residue, preferably selected from a C1-C20 optionally unsaturated or aromatic hydrocarbon residue to produce an intermediate product of formula R-S-W, wherein W is halogen preferably Cl and, (b) reacting said intermediate product with an N-sulfonyl compound or an N-acyl compound.
- the thioacetal is of formula Ri- C(O)-O-CH 2 -S-C(O)-R 2 wherein Ri and R 2 are independently a C1-C20 optionally unsaturated or aromatic hydrocarbon residue and said thioacetal is reacted with sulfuryl chloride to produce an intermediate product of formula Ri-C(O)-O-CH 2 -S-Cl, wherein Ri is independently a C1-C20, optionally unsaturated or aromatic, hydrocarbon residue.
- step (b) the intermediate is reacted with an N-sulfonyl compound of formula R 3 -S (O) 2 -NH-R 4 , wherein R 3 and R 4 are independently organic residues, preferably a C1-C20, optionally unsaturated or aromatic, hydrocarbon residue .
- the reaction of step (a) is generally carried out in an aprotic polar organic solvent such as for example a halogenated hydrocarbon solvent, in particular a chlorinated hydrocarbon solvent such as methylene chloride.
- the reaction of step (a) is generally carried out at a temperature of from -8O 0 C to 30 0 C.
- step (b) the reaction of step (b) is generally carried out in an aprotic polar organic solvent such as for example a halogenated hydrocarbon solvent, in particular a chlorinated hydrocarbon solvent such as methylene chloride.
- an aprotic polar organic solvent such as for example a halogenated hydrocarbon solvent, in particular a chlorinated hydrocarbon solvent such as methylene chloride.
- step (b) the reaction of step (b) is generally carried out at a temperature of from -20 0 C to 50 0 C, preferably from 0 0 C to 30 0 C.
- a fourth particular object of this invention concerns a method for manufacturing an oligonucleotide using the sulfurizing agent according to the invention.
- the method according to the invention comprises at least (a) a coupling step wherein a phosphorus internucleotide linkage is formed between two reactants selected from nucleotides and oligonucleotides and (b) a sulfurization step wherein the sulfurizing agent according to the invention is used to sulfurize said phosphorus internucleotide linkage.
- Steps (a) and (b) can be repeated after 3' or 5' deprotection of the sulfurized oligonucleotide.
- Step (a) of said manufacturing method preferably comprises forming the H-phosphonate diester bond by coupling an H-phosphonate monoester salt with a protected nucleoside or oligonucleotide having a free hydroxy group. The coupling is preferably carried out in solution phase.
- Step (a) is preferably carried out in an aprotic polar organic solvent for example a halogenated solvent or nitrogen containing solvents, more particularly N-heterocyclic solvents or chlorinated hydrocarbon, even more particularly acetonitrile and pyridine and preferably pyridine.
- the reaction to form an H-Phosphonate diester is preferably activated by a carboxylic acid halide, in particular pivaloyl chloride.
- Step (a) is generally carried out at a temperature from -40 0 C to 30 0 C, preferably from 0 0 C to 20 0 C .
- the liquid reaction medium generally contains at least 20% by weight of H-phosphonate oligonucleotide relative to the total weight of the reaction medium. Preferably this content is at least 20% weight.
- the liquid reaction medium generally contains at most 50% by weight of H- phosphonate oligonucleotide relative to the total weight of the reaction medium.
- step (a) may be isolated and subsequently sulfurized in step (b). It may also, preferably, be used without isolation in step (b). Sulfurization of formed diester can be carried by in-situ addition of the sulfurizing reagent, suitably dissolved in an appropriate solvent, or after pre-purifying formed diester from the reaction mixture.
- Step (b) is preferably carried out in an aprotic polar organic solvent such as for example a solvent comprising a halogenated hydrocarbon solvent, in particular a chlorinated hydrocarbon solvent such as methylene chloride.
- step (b) is carried out in a solvent mixture comprising a halogenated hydrocarbon solvent and nitrogen containing solvents, more particularly N-heterocyclic solvents, preferably pyridine.
- a pyridine/methylene chloride mixture is more particularly preferred, in particular when the coupling product of step (a) is sulfurized without isolation.
- Step (b) is generally carried out at a temperature of from -40 0 C to 30 0 C, preferably from 0 0 C to 20 0 C.
- the molar ratio of sulfurizing agent relative to the amount of internucleotide linkages to be sulfurized is generally at least 1, often from 1.5 to 4.0, preferably from 2.0 to 3.0.
- the intermediate H-phosphonate diester is preferably activated by by an activator, in particular a base.
- Suitable bases include alkylamines, in particular tertiary alkylamines, diisopropylethylamine is preferred.
- the invention in a fifth aspect, relates to a method for purifying an oligonucleotide in accordance with the invention having at least one P-S-R linkage as described herein before.
- the method comprises at least precipitating the second oligonucleotide.
- this method further comprise extraction of the second oligonucleotide, in particular from solid material recovered from the precipitation step, with a solvent.
- Suitable solvents for extraction include a polar organic solvent
- the precipitation method generally comprises (a) dissolving the oligonucleotide in a polar organic solvent and (b) adding a non-polar organic solvent until the solution becomes turbid.
- the solvent used to dissolve the oligonucleotide in step (a) is preferably selected from halogenated hydrocarbons such as methylene chloride and chloroform, nitrogen containing solvents such as acetonitrile and pyridine, and carbonyl-containing solvents such as acetone.
- a solvent volume is used ranging from about 0.5 (n+1) mL to about 2.0 (n+1) mL. Preferably, about 1.0 (n+1) niL, where n is the millimoles number of phosphorothioate triester linkages.
- the solution of the second oligonucleotide is treated with a non-polar organic solvent preferably selected from hydrocarbons, for example alkane solvents such as hexane, ether solvent in particular MTBE and their mixtures, such as, preferably hexane/MTBE mixtures until the solution becomes turbid.
- a non-polar organic solvent preferably selected from hydrocarbons, for example alkane solvents such as hexane, ether solvent in particular MTBE and their mixtures, such as, preferably hexane/MTBE mixtures until the solution becomes turbid.
- the turbid solution is subsequently treated with a precipitation aid.
- the precipitation aid is generally selected from inert porous solids preferably selected from Celite, charcoal, wood cellulose and chromatography stationary phases such as silica or alumina.
- the precipitation aid is generally used in an amount ranging from about 0.25 (n+1) g to about 1.5 (n+1) g, preferably, about 0.75 (n+1) g, where n is the millimoles number of phosphorothioate triester linkages.
- the mixture is treated with a second fraction of a non-polar organic solvent as described here before.
- the volume of said fraction generally ranges from about l(n+l) mL to about 4(n+l) mL, preferably, about 2.0 (n+1) mL, wherein n is the millimoles number of phosphorothioate triester linkages.
- the obtained mixture is generally subjected to a solid/liquid separation operation such as, preferably, a filtration.
- a solid/liquid separation operation such as, preferably, a filtration.
- the oligonucleotide is generally recovered from solid recovered from solid/liquid separation operation, in particular from precipitation aid by extraction with a polar organic solvent preferably selected from carbonyl-type solvents such as acetone, from nitrogen-containing solvents such as acetonitrile and from halogenated hydrocarbons such as methylene chloride and chloroform.
- the oligonucleotide obtained from the above precipitation treatment can be further purified by partitioning between an organic solvent and water. This step usually separates polar impurities, which dissolve in aqueous layer, from the product.
- the oligonucleotide is suitably dissolved in a organic solvent, in particular a polar organic solvent such as nitrogen- containing solvents in particular selected from acetonitrile, formamides such as DMF and N-hetero cycles such as pyridine, carbonyl-type solvents such as acetone, or THF or DMSO.
- the volume of organic solvent used is generally ranging from 2.0 (n+1) mL to 8.0 (n+1) mL, preferably, about 4.0 (n+1) mL, where n is the millimoles number of the phosphorothioate triester linkage.
- the solution is treated with an aqueous medium, in particular water.
- the volume of aqueous medium used is generally from about 0.5 volume equivalent of the organic solvent to about 1.5 volume equivalent of the organic solvent, usually about 0.7 volume equivalent of the organic solvent.
- an oligonucleotide-containing layer is generally separated and can be further processed, if appropriate, to obtain purified oligonucleotide.
- a sixth particular object of this invention concerns a method for producing a second oligonucleotide having at least one phosphothioate group, which comprises (a) providing a first oligonucleotide according to the invention and (b) cleaving at least one R group, from said first oligonucleotide to produce said second oligonucleotide having at least one thiophosphate linkage.
- the R group is cleaved by reacting the first oligonucleotide in solution with a base chosen preferably from alkyl, cycloalkyl and aromatic amines, more preferably from primary, for example an alkyl primary amine wherein alkyl group bears identical or different substituents selected preferably from Cl to C8 linear or branched alkyl or secondary alkyl amines, most preferably from n-propyl and tert-butyl amine, preferably the base is a hindered primary amine.
- a base chosen preferably from alkyl, cycloalkyl and aromatic amines, more preferably from primary, for example an alkyl primary amine wherein alkyl group bears identical or different substituents selected preferably from Cl to C8 linear or branched alkyl or secondary alkyl amines, most preferably from n-propyl and tert-butyl amine, preferably the base is a hindered primary amine.
- the cleavage according to the sixth aspect of the invention is carried out in the presence of a sterically hindered base and of an activator which is generally a N-heteroaromatic base.
- activator is 1,2,4-triazole or other triazole and tetrazole derivatives, and more preferably such activator is used with a sterically hindered base, in particular tert-butyl amine.
- the deprotection of S-methylene-ester, -carbonate or -carbamate group can be accomplished for example in a treatment of protected nucleotide with a sterically hindered base such as e.g. t-butylamine.
- a sterically hindered base such as e.g. t-butylamine.
- These bulky amines are particularly selective because they do not react with the nucleobases, particularly those protected at carbonyl oxygen. They allow in fact limiting or substantially avoiding possible side-reactions with the nucleobase moiety.
- an activator may suitably be added.
- activators which are suitable include N- heterocyclic bases such as e.g. diazole, triazole, and their derivatives. This embodiment allows particularly clean, fast and efficient deprotection reactions.
- the deprotection method involves using a substituted aniline as base wherein the aryl group of the aniline contains linear or branched alkyl or aryl substituents at 2 and/or 6 positions such as e.g. 2,6-dimethylaniline and 2,6-diethylaniline.
- the deprotection according to the sixth aspect is preferably carried out in an aprotic polar organic solvent for example a solvent comprising nitrogen containing solvents, more particularly N-heterocyclic solvents, preferably pyridine.
- an aprotic polar organic solvent for example a solvent comprising nitrogen containing solvents, more particularly N-heterocyclic solvents, preferably pyridine.
- the deprotection according to the sixth aspect is generally carried out at a temperature from -10 0 C to 50 0 C, preferably from 0 0 C to 30 0 C.
- the liquid reaction medium generally contains at least 20% by weight of first oligonucleotide relative to the total weight of the reaction medium. Preferably this content is at least 50% weight.
- the amount of base used is generally ranging from 5n mmol to 15n mmol, preferably about 1On mmol, where n is the millimoles number of the phosphorothioate triester linkage.
- the amount of activator used is generally ranging from 0.5n mmol to 3n mmol, preferably, 1.5n mmol, where n is the millimoles number of the phosphorothioate triester linkage.
- Ap, Gp, Tp are the 2-deoxyribose nucleobases as previous described respectively connected to A, G and T nucleobases as previously described wherein A, G and T are protected as follows: Ap is the 2-deoxyribose nucleobase wherein A is N-(purin-6- yl)benzamide, Gp is the 2-deoxyribose nucleobase wherein G is N-(6-(2,5- dichlorophenoxy)-purin-2-yl)isobutyramide and Tp is the nucleobase wherein T is 5-methyl-4-phenoxypyrimidin-2-one.
- Ap(S), Gp(S) and Tp(S) are the corresponding 4'0-P-thiomethyl propionates of respectively Ap, Gp and Tp as previously described.
- Ap(H), Gp(H) and Tp(H) are the corresponding 4'0-P-H phophonates of respectively Ap, Gp and Tp as previously described.
- DMTr is the bis para-methoxy trityl protecting group, known to one skilled in the art, bonded to the 5-0' of the corresponding oligonucleotide as previously described, when linked to it.
- Lev is the pentanl,4-dione protecting group, known to one skilled in the art, bonded to the 3-0' of the corresponding oligonucleotide as previously described, when linked to it.
- Methanesulfonyl chloride 38.7 mL, 500 mmol was added dropwise over 15 min to stirred aqueous methylamine (40% in water, 152 mL,
- N-methyl methanesulfonamide (1.09 g, 10.0 mmol)
- pyridine (1.66 g, 21.0 mmol)
- bis(propionyloxymethyl)disulfide 1.2 g, 5.0 mmol
- anhydrous CH 2 Cl 2 8 mL
- the mixture was stirred under N 2 at room temperature and a solution ofBr 2 (0.882 g, 5.52 mmol) in 4 mL Of CH 2 Cl 2 was added dropwise over 30 min.
- the resulting mixture was stirred at room temperature for 2 hours.
- MTBE (15 mL) was added and the resulting mixture was filtered.
- N-methyl methanesulfonamide (49.5 g, 453.1 mmol)
- molecular sieves (4 A, activated, 5.0 g)
- anhydrous CH 2 Cl 2 200 mL
- anhydrous pyridine 41.9 mL, 517.8 mmol
- the above solution A was added slowly over 15 minutes.
- the resulting mixture was then stirred at room temperature for 1.5 hours.
- Hexane 200 mL was added slowly and the resulting mixture was stirred at room temperature for 10 minutes.
- chloromethyl chloroformate (12.9 g, 100.0 mmol), anhydrous acetonitrile (300 mL).
- the solution is stirred in an ice-water bath, and a mixture of anhydrous ethanol (4.6 g, 100.0 mmol) and anhydrous pyridine (23.7 g, 300 mmol) is added slowly over 20 min.
- the mixture is stirred at room temperature for 1 hour.
- Sodium iodide (1.50 g, 10.0 mmol) is added into the reaction mixture.
- the mixture is stirred in an ice-water bath, and thioacetic acid (7.6 g, 100 mmol) is added over 5 min.
- N-methyl methanesulfonamide (6.0 g, 55.0 mmol), molecular sieves (4 A, activated, 3.0 g) and anhydrous CH 2 Cl 2 (150 mL).
- N-methyl methanesulfonamide 6.0 g, 55.0 mmol
- molecular sieves (4 A, activated, 3.0 g)
- anhydrous CH 2 Cl 2 150 mL
- anhydrous pyridine 5.3 mL, 65.0 mmol
- the above solution A is added slowly over 10 minutes.
- Hexane 200 mL
- the resulting mixture is stirred at room temperature for 10 minutes.
- N-methyl methanesulfonamide (6.0 g, 55.0 mmol), molecular sieves (4 A, activated, 3.0 g) and anhydrous CH 2 Cl 2 (150 mL).
- N-methyl methanesulfonamide 6.0 g, 55.0 mmol
- molecular sieves (4 A, activated, 3.0 g)
- anhydrous CH 2 Cl 2 150 mL
- anhydrous pyridine 5.3 mL, 65.0 mmol
- the above solution A is added slowly over 10 minutes.
- Hexane 100 mL
- the resulting mixture is stirred at room temperature for 10 minutes.
- Example 13 synthesis of fully protected dinucleotide phosphorothioate with isolation of intermediate H-phosphonate
- Example 13-1 A mixture of 1.86g (2.62 mmol, 1.15 eq.) of H- phosphonate 1 and 0.78g (2.28 mmol) of 3 '-protected deoxy-thymidine 2 was co-evaporated with anhydrous pyridine (3x 25mL). The oily residue was dissolved in 1OmL of anhydrous pyridine and cooled to ⁇ 0 0 C under argon atmosphere. A total of 0.56g (4.66 mmol, 2 eq.) of pivaloyl chloride was added dropwise via syringe, and the resulting mixture was allowed to warm up to ambient temperature.
- Example 13-2 By analogy with the method A, an intermediate H- phosphonate 3 was obtained by reaction of H-phosphonate 1 (1.49g, 2.1 mmol, 1.08 eq.) with 3 '-protected deoxythymidine 2 (0.66g, 1.94 mmol) in the presence of 0.49g (4.06 mmol, 2 eq.) of pivaloyl chloride in 2OmL of anhydrous pyridine. After quenching of the reaction mixture with cold water/aq.NaHCO3/brine, the intermediate 3 was isolated by extraction with dichloromethane (3x3 OmL). The organic extract was washed with water (5OmL), aq.
- the reaction mixture was stirred at O 0 C for 5 min and partitioned between methylene chloride (100 mL) and 1.25 N sodium acetate - acetic acid buffer (2 x 100 mL).
- the buffer was made by mixing 190 mL of 1.25 N aqueous sodium acetate solution with 10 mL of 1.25 N aqueous acetic acid solution.
- the organic layer was dried (Na 2 SO 4 ) and concentrated.
- Phosphorous acid 29.8 g, 364.0 mmol was rendered anhydrous by evaporation with pyridine (182 mL).
- DMTr-Gp(s)T-OH 33.0 g, 26.0 mmol was added and the mixture was again rendered anhydrous by evaporation with pyridine.
- the mixture was diluted with anhydrous pyridine (130 mL) and treated with pivaloyl chloride (24.0 mL, 195.0 mmol) which was added over 30 min at 10 0 C. The mixture was stirred for 16 hours at room temperature, concentrated, and the residue was dissolved in 400 mL of methylene chloride.
- the solution was washed sequentially with cold water (400 mL) and triethylammonium hydrogen carbonate (2.0 N, 200 mL x 3).
- the organic layer was dried (anhydrous Na 2 SO 4 ) and concentrated.
- the mixture was filtered and the solid was washed with a solvent mixture made from the solution A and CH 2 Cl 2 in the ratio of 5: 1 (180 mL).
- Phosphorous acid (4.7 g, 57.3 mmol) was evaporated with pyridine (29 mL) and mixed with OMT ⁇ -Gp(s)Tp(s)Gp(s)A-OR (8.7 g, 3.58 mmol). The mixture was rendered anhydrous by was anhydrous by evaporation of added pyridine and diluted with anhydrous pyridine (29.0 mL). To the stirred mixture, pivaloyl chloride (3.75 mL, 30.4 mmol) was added over 5 min at 10 0 C. The mixture was stirred for 6 hours at room temperature and concentrated.
- the mixture was filtered and the solid was washed with a solvent mixturwe made from the solution A and CH2C12 in the ration of 5: 1 (120 mL).
- the solid was extracted with methylene chloride (100 mL x 4) and the extract concentrated.
- the residue was dissolved in 70 mL of acetonitrile and treated with cold water (49 mL) added over 30 min.
- Gp(s) Tp(s) Gp(s)Ap(s) Gp(s) Tp(s) Gp(s)Ap(s) Gp(s) Tp(s) Gp(s)A-Lev (6.1 g, 0.89 mmol) was rendered anhydrous by evaporation with pyridine. The residue was diluted with anhydrous pyridine (10 mL) and treated under N 2 at O 0 C with pivaloyl chloride (0.37 mL, 3.0 mmol) which was added slowly over 3 min. The cold bath was removed and the mixture was stirred at ambient temperature for 1 hour.
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CN2009801552887A CN102307889A (en) | 2008-12-23 | 2009-12-23 | Sulfurizing reagents and their use for oligonucleotides synthesis |
CA2747999A CA2747999A1 (en) | 2008-12-23 | 2009-12-23 | Sulfurizing reagents and their use for oligonucleotides synthesis |
JP2011542834A JP2012513450A (en) | 2008-12-23 | 2009-12-23 | Sulfurizing agents and their use to synthesize oligonucleotides |
EP09799643A EP2382227A1 (en) | 2008-12-23 | 2009-12-23 | Sulfurizing reagents and their use for oligonucleotides synthesis |
US13/165,918 US20120071640A1 (en) | 2008-12-23 | 2011-06-22 | Sulfurizing reagents and their use for oligonucleotides synthesis |
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JP (1) | JP2012513450A (en) |
KR (1) | KR20110099333A (en) |
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WO2012001126A1 (en) * | 2010-06-30 | 2012-01-05 | Girindus America, Inc. | A new method of using n-thio compounds for oligonucleotide synthesis |
GB2471806B (en) * | 2008-04-03 | 2012-12-19 | Spring Bank Pharmaceuticals Inc | Compositions and methods for treating viral infections |
WO2013017469A1 (en) * | 2011-07-29 | 2013-02-07 | Girindus America, Inc. | Sulfurization reagents on solid supports |
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US9394333B2 (en) | 2008-12-02 | 2016-07-19 | Wave Life Sciences Japan | Method for the synthesis of phosphorus atom modified nucleic acids |
US9598458B2 (en) | 2012-07-13 | 2017-03-21 | Wave Life Sciences Japan, Inc. | Asymmetric auxiliary group |
US9605019B2 (en) | 2011-07-19 | 2017-03-28 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
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US10144933B2 (en) | 2014-01-15 | 2018-12-04 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator |
US10149905B2 (en) | 2014-01-15 | 2018-12-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having antitumor effect and antitumor agent |
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WO2016117663A1 (en) * | 2015-01-21 | 2016-07-28 | 味の素株式会社 | Precipitation accelerator and precipitation method in which same is used |
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- 2009-12-23 CA CA2747999A patent/CA2747999A1/en not_active Abandoned
- 2009-12-23 JP JP2011542834A patent/JP2012513450A/en active Pending
- 2009-12-23 KR KR1020117017343A patent/KR20110099333A/en not_active Application Discontinuation
- 2009-12-23 WO PCT/EP2009/067902 patent/WO2010072831A1/en active Application Filing
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- 2009-12-23 EP EP09799643A patent/EP2382227A1/en not_active Withdrawn
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CN102307889A (en) | 2012-01-04 |
JP2012513450A (en) | 2012-06-14 |
CA2747999A1 (en) | 2010-07-01 |
KR20110099333A (en) | 2011-09-07 |
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