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WO2001027126A1 - Preparation de phosphorothioate triesters et d'oligonucleotides - Google Patents

Preparation de phosphorothioate triesters et d'oligonucleotides

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Publication number
WO2001027126A1
WO2001027126A1 PCT/GB2000/003912 GB0003912W WO0127126A1 WO 2001027126 A1 WO2001027126 A1 WO 2001027126A1 GB 0003912 W GB0003912 W GB 0003912W WO 0127126 A1 WO0127126 A1 WO 0127126A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
process according
phosphonate
phosphorothioate
alkyl
Prior art date
Application number
PCT/GB2000/003912
Other languages
English (en)
Inventor
Mark Edward Douglas
Kevin Gerard Scott
David John Moody
Ben James Mellor
Original Assignee
Avecia Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Avecia Limited filed Critical Avecia Limited
Priority to EP00968075A priority Critical patent/EP1224193A1/fr
Priority to CA002386867A priority patent/CA2386867A1/fr
Priority to JP2001530344A priority patent/JP2003517467A/ja
Priority to AU78035/00A priority patent/AU7803500A/en
Publication of WO2001027126A1 publication Critical patent/WO2001027126A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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 concerns a method for the synthesis of phosphorothioate tnesters, and particularly ohgonucleotides
  • a process for the synthesis of a phosphorothioate t ⁇ ester comprising the reaction, in the presence of a coupling agent, of an H-phosphonate with a substrate comprising a free hydroxy group and bonded to a solid support, thereby forming a supported H-phosphonate diester, and subjecting the H- phosphonate diester to sulphur transfer with a sulphur transfer agent thereby forming a phosphorothioate tnester
  • a plurality of coupling and sulphur transfer steps are carried out, with a sulphur transfer step being carried out after each coupling step
  • the H-phosphonate employed in the process of the present invention is often an
  • nucleosides are 2'- deoxy bonucleosides and nbonucleosides
  • preferred ohgonucleotides are o godeoxyribonucleotides and ohgoribonucleotides 2'-deoxyr ⁇ bonucleos ⁇ des and ohgodeoxyribonucleotides may comprise 2'-C-alkyl and 2'-C-alkenyl substituents
  • the H-phosphonate building block is a protected deoxynbonucleoside, nbonucleoside, ohgodeoxyribonucleotide or ohgoribonucleotide derivative comprising a 3' H-phosphonate function
  • the 5' hydroxy function is advantageously protected by a suitable protecting group
  • suitable protecting groups include acid labile protecting groups, particularly trityl and substituted trityl groups such as dimethoxyt ⁇ tyl and 9-phenylxanthen-9-yl groups, and base labile-protecting groups such as FMOC
  • Further protecting groups that may be employed include silyl ether groups
  • the 3' hydroxy function is advantageously protected by a suitable protecting group Suitable protecting groups include those disclosed above for the protection of the 5' hydroxy functions of 3' H-phosphonate building blocks and acyl, such as levulinoyi and substituted levuhnoyl, groups
  • the 2'-hydroxy function is advantageously protected by a suitable protecting group, for example an acid-labile acetal protecting group, particularly 1-(2- fluorophenyl)-4-methoxyp ⁇ per ⁇ d ⁇ ne-4-yl (Fpmp), and alkyl and aryl silyl protecting groups such as t-butyldiphenyl silyl groups, commonly tnalkylsilyl groups, often t ⁇ (C 1 4 -alkyl)s ⁇ lyl groups such as a tertiary butyl dimethylsilyl group
  • the ribonucleoside or oligonbonucleotide may be a 2'-O-alkyl, 2'-O-alkoxyalkyl or 2'-O-alkenyl derivative, commonly a C 1 4 alkyl, C 4 alkoxy ⁇ 4 alkyl or alkeny
  • the substrate comprising a free hydroxy group employed in the process of the present invention is commonly a protected nucleoside or ohgonucleotide comprising a free hydroxy group, preferably a free 3' or 5' hydroxy group, and particularly preferably a 5' hydroxy group
  • preferred nucleosides are deoxy ⁇ bonucleosides and ⁇ bonucleosides and preferred ohgonucleotides are ohgodeoxyribonucleotides and ohgo ⁇ bonucleotides
  • the substrate comprising a free hydroxy group is a deoxynbonucleoside, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide derivative comprising a free 5'-hydroxy group
  • the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 3'-hydroxy function
  • the substrate comprising a free hydroxy group is a deoxynbonucleoside, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide derivative comprising a free 3'-hydroxy group
  • the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 5'-hydroxy function
  • the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fluorophenyl)-4-methoxyp ⁇ per ⁇ d ⁇ ne-4- yl (Fpmp), and t ⁇ alkylsilyl groups, often t ⁇ (C 1 4 -alkyl)s ⁇ lyl groups such as a tertiary butyl dimethyl silyl group
  • the ribonucleoside or oligonbonucleotide may be
  • substrates comprising a free hydroxy group that may be employed in the process according to the present invention are saccha ⁇ des, especially abasic nucleosides such as ⁇ bose and deoxy ⁇ bose, and non-saccha ⁇ de polyols, especially alkyl polyols, and preferably diols or t ⁇ ols
  • alkyl diols include ethane-1 ,2-d ⁇ ol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400
  • alkyl t ⁇ ols include glycerol and butane t ⁇ ols
  • suitable protecting groups such as those disclosed hereinabove for the protection at the 5' or 2' positions of nbonucleosides, or being employed to bond the substrate to the solid support
  • more than one free hydroxy group may be present if it is desired to perform
  • bases present in nucleosides/nucleotides employed in present invention are also preferably protected where necessary by suitable protecting groups
  • protecting groups employed are those known in the art for protecting such bases
  • adenine (A) and/or cytosine (C) can be protected by benzoyl, including substituted benzoyl, for example alkyl- or alkoxy-, often C 1 4 alkyl- or C 1 4 alkoxy-, benzoyl, pivaloyl, and amidine, particularly dialkylaminomethylene, preferably d ⁇ (C 1 4 -alkyl) aminomethylene such as dimethyl or dibutyl aminomethylene
  • Guanine (G) may be protected by a phenyl group, including substituted phenyl, for example 2,5-d ⁇ chlorophenyl and also by an isobutyryl group G may also be protected by diphenylcarbamoyl and glyoxal type protecting groups thymine (T)
  • the phosphorothioate tnester produced is a protected nucleoside or ohgonucleotide having protected hydroxy groups
  • one of these protecting groups may be removed after carrying out the process of the first invention Commonly, the protecting group removed is that on the 5'-hydroxy function
  • the ohgonucleotide thus formed may then proceed through further stepwise or block coupling and sulphur transfers according to the process of the present invention in the synthesis of a desired ohgonucleotide sequence
  • the method may then proceed with steps to remove the protecting groups from the intemucleotide linkages, the 3' and the 5'- hydroxy groups and from the bases, and to cleave the product from the solid support
  • the process according to the present invention may comprise a capping step, where hydroxy groups which are unreacted after a given coupling are capped to prevent further reaction in later couplings
  • Capping agents which may be employed are those known in the art for such a step, and include for example acylating agents such as acetic anhydride, (preferably in the presence of a nucleophihc acylation catalyst such as 4-(N,N- d ⁇ methyl)am ⁇ nopy ⁇ d ⁇ ne) and lower, eg up to C 4 , alkyl H-phosphonates, such as ethyl H- phosphonate, and 2-cyanoethyl H-phosphonate
  • the invention provides a method comprising the coupling of a 5'-0-(4,4'-d ⁇ methoxytr ⁇ tyl)-2'-deoxy ⁇ bonucleos ⁇ de Z'-H- phosphonate or a protected ohgodeoxyribonucleotide 3'-H-phosphonate and a substrate supported on a solid support with a free hydroxy function, most commonly a 2'- deoxy ⁇ bonucleoside or ohgodeoxyribonucleotide, in the presence of a suitable coupling agent and subsequent sulphur transfer in the presence of a suitable sulphur-transfer agent
  • any suitable coupling agents and sulphur- transfer agents available in the prior art may be used
  • suitable coupling agents include alkyl and aryl acid chlorides, alkane and arene sulphonyl chlorides, alkyl and aryl chloroformates, alkyl and aryl chlorosulphites and alkyl and aryl phosphorochlondates, and carbodiimides
  • suitable alkyl acid chlorides which may be employed include up to C 12 alkyl acid chlorides, including adamantyl carbonyl chloride, and especially C 2 to C 7 alkanoyl chlorides, particularly pivaloyl chloride
  • aryl acid chlorides which may be employed include substituted and unsubstituted benzoyl chlorides, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C 1 4 alkyl, substituted benzoyl chlorides When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents
  • alkanesulphonyl chlorides which may be employed include C 2 to C 7 alkanesulphonyl chlorides
  • arenesulphonyl chlorides which may be employed include substituted and unsubstituted benzenesulphonyl chlorides, such as C-, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C-, 4 alkyl, substituted benzenesulphonyl chlorides When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents
  • alkyl chloroformates examples include C 2 to C 7 alkyl chloroformates
  • aryl chloroformates which may be employed include substituted and unsubstituted phenyl chloroformates, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C 1 4 alkyl, substituted phenyl chloroformates When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents
  • suitable alkyl chlorosulphites which may be employed include C 2 to
  • C 7 alkyl chlorosulphites examples include substituted and unsubstituted phenyl chlorosulphites, such as C 1 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C 1 4 alkyl, substituted phenyl chlorosulphites When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents
  • alkyl phosphorochlo ⁇ dates which may be employed include d ⁇ (C, to C 6 alkyl) phosphorochlo ⁇ dates
  • aryl phosphorochlo ⁇ dates which may be employed include substituted and unsubstituted diphenyl phosphorochlo ⁇ dates, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C, 4 alkyl, substituted diphenyl phosphorochlo ⁇ dates When substituted, from 1 to 5 substituents on each phenyl group may be present, particularly in the case of alkyl and halo substituents
  • alkyl carbodiimides especially (C, to C 6 alkyl) carbodiimides, such as 1 ,3 dicyclohexyl carbodiimide, 1 ,3 dnsopropyl carbodiimide, 1-tert -butyl-3-ethyl carbodnmide and 1-(3- d ⁇ methylam ⁇ nopropyl)-3-ethyl carbodiimide
  • alkyl carbodiimides especially (C, to C 6 alkyl) carbodiimides, such as 1 ,3 dicyclohexyl carbodiimide, 1 ,3 dnsopropyl carbodiimide, 1-tert -butyl-3-ethyl carbodnmide and 1-(3- d ⁇ methylam ⁇ nopropyl)-3-ethyl carbodiimide
  • One or more of the alkyl groups may be substituted, for example by an alkyl ammo moiety
  • Preferred coupling agents are diaryl phosphorochloridat.es, particularly those having the formula (ArO) 2 POCI wherein Ar is preferably phenyl, 2-chlorophenyl, 2,4,6- t ⁇ chlorophenyl or 2,4,6-tr ⁇ bromophenyl
  • the sulphur transfer agents employed in the process of the present invention introduce a protected thio moiety into the linkage, thereby forming a phosphorothioate tnester
  • the phosphorthioate t ⁇ esters are commonly subsequently converted to phosphodiester or phosphorothioate diesters, and the sulphur transfer agent can be selected accordingly
  • the nature of the sulphur-transfer agent will depend on whether an ohgonucleotide, a phosphorothioate analogue or a mixed ohgonucleotide/ohgonucleotide phosphorothioate is required
  • Sulphur transfer agents employed in the process of the present invention often have the general chemical formula
  • L represents a leaving group
  • D represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group
  • the leaving group is selected so as to comprise a nitrogen-sulphur bond
  • suitable leaving groups include morphohnes such as morphohne-3,5-d ⁇ one, imides such as phthahmides, succinimides and maleimides, indazoles, particularly indazoles with electron-withdrawing substituents such as 4-n ⁇ tro ⁇ ndazoles, and t ⁇ azoles
  • the sulphur transfer agent is commonly selected such that the moiety D represents an aryl group, such as a phenyl or naphthyl group
  • suitable aryl groups include substituted and unsubstituted phenyl groups, particularly halophenyl and alkylphenyl groups, especially 4-halophenyl and 4-alkylphenyl, commonly 4-(C ⁇ 4 alkyl)phenyl groups, most preferably 4-chlorophenyl and p-tolyl groups
  • An example of a suitable class of standard phosphodiester-directing sulphur-transfer agent is an N- (arylsulphanyl)phthahm ⁇ de (succinimide or other imide may also be used)
  • the moiety D commonly represents a methyl, substituted alkyl or alkenyl group
  • suitable substituted alkyl groups include substituted methyl groups, particularly benzyl and substituted benzyl groups, such as alkyl-, commonly C, 4 alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-pos ⁇ t ⁇ on with an electron-withdrawing substituent such as 2-(4- n ⁇ trophenyl)ethyl and 2-cyanoethyl groups
  • suitable alkenyl groups are ally], propargyl and crotyl
  • Examples of a suitable class of phosphorothioate-directing sulphur- transfer agents are, for example, (2-cyanoethyl)sulphanyl derivatives such as 4-[(2- cyanoethyl)-sulphanyl]
  • a suitable temperature for carrying out the coupling reaction and sulphur transfer is in the range of from approximately -55°C to about 40°C, such as from 0 to 30°C, and preferably about room temperature (commonly in the range of from 10 to 25°C, for example approximately 20°C)
  • Preferred nucleoside or nucleotide H-phosphonates employed in the process of the present invention have the general chemical formula wherein each B independently is an organic base, each Q independently is H, CH 2 R' or OR' wherein R' is alkyl, substituted alkyl, alkenyl or a protecting group, each R independently is an aryl, methyl, substituted alkyl or alkenyl group,
  • W is H, a protecting group or an H-phosphonate group of formula
  • M + is a monovalent cation
  • each X independently represent O or S
  • each Y independently represents O or S
  • Z is H, a protecting group or an H-phosphonate group of formula
  • M + is a monovalent cation
  • n is zero or a positive integer
  • W or Z is an H-phosphonate group, commonly only Z being an H-phosphonate group
  • the protecting group may be one of those disclosed above for protecting the 3' or 5' positions respectively
  • the protecting group is preferably a trityl group, particularly a dimethoxyt ⁇ tyl group
  • the protecting group is preferably a trityl group, particularly a dimethoxytntyl group, or an acyl group, preferably a levuhnoyl group
  • Organic bases which may be represented by B include nucleobases, such as natural and unnatural nucleobases, and especially pu ⁇ nes, such as hypoxanthine, and particularly A and G, and pynmidines, particularly T, C and U
  • the bases may be protected, with A, G and C preferably being protected Suitable protecting groups include those described hereinabove for the protection of bases
  • the alkenyl group is often a C, 4 alkenyl group, especially allyl, propargyl or crotyl group
  • the alkyl is preferably a C 1 4 alkyl group
  • the substituted alkyl group includes alkoxyalkyl groups, especially C-, 4 alkyoxy ⁇ 4 alkyl groups such as methoxyethyl groups
  • the protecting group is commonly an acid-labile acetal protecting group, particularly 1-(2- fluorophenyl)-4-methoxyp ⁇ per ⁇ d ⁇ ne-4-yl (Fpmp) or a t ⁇ alkylsilyl groups, often a t ⁇ 4 - alkyl)s ⁇ lyl group such as a tertiary butyl dimethylsilyl group
  • X represents O
  • Y represents S and each
  • H-phosphonates wherein n represents 1 , 2 or 3 can be employed when it is desired to add small blocks of nucleotide, with correspondingly larger values of n, for example 4, 5, or 6, being employed if larger blocks of ohgonucleotide are desired to be coupled
  • H-phosphonates, coupling agents and sulphur transfer agents can employed as a solution, although the coupling agent or sulphur transfer agent may employed as a neat liquid or solid as appropriate
  • Organic solvents which can be employed include haloalkanes, particularly dichloromethane, esters, particularly alkyl esters such as ethyl acetate, and methyl or ethyl propionate, nitriles, such as acetonit ⁇ le, amides, such as dimethylformamide and N-methylpyrolhdinone, and basic, nucleophihc solvents such as py ⁇ dine Preferred solvents are py ⁇ dine, dichloromethane, dimethylformamide, N- methylpyrolhdinone and mixtures thereof
  • Protecting groups can be removed using methods known in the art for the particular protecting group and function
  • transient protecting groups particularly gamma keto acids such as levuhnoyl-type protecting groups
  • hydrazine for example, buffered hydrazine, such as the treatment with hydrazine under very mild conditions disclosed by van Boom J H , Burgers, P M J Tetrahedron Lett , 1976, 4875-4878
  • the resulting partially-protected ohgonucleotides with free 3'-hydroxy functions may then be converted into the corresponding H- phosphonates which are intermediates which can be employed for the block synthesis of ohgonucleotides and their phosphorothioate analogues
  • a cyanoethyl group can be removed by treatment with anhydrous, strongly basic amine such as DABCO, 1 ,5-d ⁇ azab ⁇ cylo[4 3 0]non-5-ene (DBN), 1 ,8-d ⁇ azab ⁇ cyclo[5 4 0]undec-7-ene (DBU) or tnethylamine
  • Phenyl and substituted phenyl groups on the phosphorothioate intemucleotide linkages and on the base residues can be removed by oximate treatment, for example with the conjugate base of an aldoxime, preferably that of E-2-n ⁇ trobenzaldox ⁇ me or py ⁇ d ⁇ ne-2-carboxaldox ⁇ me (Reese et al, Nucleic Acids Res 1981 ) Kamimura, T et al in
  • Trityl groups present can be removed by treatment with acid
  • acid With regard to the overall unblocking strategy in ohgonucleotide synthesis, another important consideration of the present invention, is that the removal of trityl, often a 5'-term ⁇ nal DMTr, protecting group ('det ⁇ tylation') should proceed without concomitant depu ⁇ nation, especially of any 6- ⁇ /-acyl-2'-deoxyadenos ⁇ ne residues
  • Silyl protecting groups may be removed by fluoride treatment, for example with a solution of an ammonium fluoride, for example a solution of t ⁇ alkylamine t ⁇ hydrogen fluoride or a solution of a tetraalkyl ammonium fluoride salt such as tetrabutyl ammonium fluoride
  • Fpmp protecting groups may be removed by acidic hydrolysis under mild conditions
  • the substrate is commonly bound to the solid support via a cleavable linker
  • linkers that may be employed include those well known in the art for the solid phase synthesis of ohgonucleotides, such as urethane, oxalyl, succinyl, and amino- de ⁇ ved linkers
  • the substrate is attached to the support by a process comprising either a) reacting a 5'-protected nuceloside having a free 3'-hydroxy group with a linker, preferably succinic anhydride, to form a linker-de ⁇ vatised nucleoside, and b) reacting the linker-de ⁇ vatised nucleoside with an amine-functionahsed poly(acrylam ⁇ de) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example
  • Coupling agents used for amide bond formation that can be employed in the process for attaching the substrate to an amine-functionahsed poly(acrylam ⁇ de) support include those known in the art of peptide synthesis, see for example those coupling reagents disclosed by Wel ngs, D A , Atherton, E , in Methods in Enzymology, Publ , Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N,N'- diisopropylcarbodnmide (DIC) and reagents that form active esters particularly benzot ⁇ azole active esters in situ, such as 2-(1 H-benzot ⁇ azole-1-yl)-1 , 1 ,3,3- tetramethyluronium tetrafluoroborate (TBTU) or benzot ⁇ azole-1-yloxy-t ⁇ s-
  • DIC diisopropylcarbodnmide
  • An organic solvent such as N,N-d ⁇ methylformam ⁇ de (DMF) or N- methylpyrrohdinone (NMP) is suitably employed for attaching the substrate to an amine- functiona sed poly(acrylam ⁇ de) support
  • DMF N,N-d ⁇ methylformam ⁇ de
  • NMP N- methylpyrrohdinone
  • the process for the synthesis of phosphorothioate t ⁇ esters according to the present invention can be carried out by stirring a slurry of the substrate bonded to the solid in a solution of the H-phosphonate and coupling agent or sulphur-transfer agent
  • the solid support can be packed into a column, and solutions of H- phosphonate and coupling agent, followed by sulphur transfer agent can be passed sequentially through the column
  • the process according to the present invention is preferably employed to produce ohgonucleotides typically comprising 3 or more bases
  • the upper limit will depend on the length of the ohgonucleotide it is desired to prepare Often, ohgonucleotides produced by the process of the present invention comprise up to 40 bases, commonly up to 30 bases and preferably from 5 to 25, such as from 8 to 20, bases
  • the coupling and sulphur transfer steps of the process of the present invention are repeated sufficient times to produce the desired length and sequence
  • the product may be cleaved from the solid support, preferably following deprotection of the product
  • the desired product is an ohgonucleotide
  • the product will be a phosphate diester, phosphorothioate diester or chimera comprising both phosphate diester and phosphorothioate diester moieties
  • Cleavage methods employed are those known in the art for the given solid support
  • cleavage methods appropriate for the linker are employed following cleavage, the product can be purified using techniques known in the art, such as one or more of ion-exchange chromatography, reverse phase chromatography, and precipitation from an appropriate solvent Further processing of the product by for example ultrafiltration may also be employed
  • the invention will now be illustrated without limitation by the following examples
  • DMT C z 4- ⁇ /-benzoyl-5'-0-(4,4'-d ⁇ methoxytr ⁇ tyl)-2'-deoxycyt ⁇ d ⁇ ne (DMT C z ) supported via a succinimide linker at the 3'-pos ⁇ t ⁇ on to a polystyrene support (commercially available under the trade name Pharmacia Primer Support 30HL, loading 84 umol/g, 2g) was poured into a sintered vessel, wetted with 100ml of CH 2 CH 2 , and aerated with house nitrogen The solvent was removed Following this wash procedure, the supported DMT C bz was treated as follows i) with 100ml of 3% v/v dichloroacetic acid in dichloromethane (DCA/DCM) - Wait for 60 seconds, remove DCA/DCM n) with 100ml of CH 2 CH 2 - Wait for 60 seconds, remove CH 2 CH 2 in) with 100ml of 3% DCA/DCM - Wait for
  • a 5'-DMT protected nucleotide 3'-H-phosphonate having the base sequence 5'- DMT-ACAC, with each intemucleotide being protected by a beta-cyanoethylthio moiety (5'-DMT-ACAC-3'-H phosphonate) was prepared from the corresponding tetrame ⁇ c ohgonucleotide comprising a free 3'-hydroxy group (5'-DMT-ACAC-3'-OH) as follows Ammonium toluyl-H-phosphonate was dissolved in 50ml methanol and 5ml t ⁇ ethylamine This mixture is evaporated to form a gum, and the gum redissolved in 100ml pyndine, together with 12 3g 5'-DMT-ACAC-3'-OH, and the pyndine evaporated The residue was then redissolved in 50ml pyndine The pyndine solution is cooled to -30°C and pivaloyl chloride (2 2
  • the cyanoethyl groups can be removed by treatment with anhydrous 1 ,8- d ⁇ azab ⁇ cyclo[5,4,0]undec-7-ene to yield phosphorothioate linkages, and the phosphorothioate ohgonucleotide could be cleaved from the support by treatment with concentrated aqueous ammonia containing 10% vol mercaptoethanol
  • a poly(acrylam ⁇ de) resin produced by copolyme ⁇ sation of acryloyl-sarcosine methyl ester, N,N-d ⁇ methylacrylam ⁇ de and bis-acryloylethylenediamine (PDMA resin, 69g) was treated with ethylene diamine (700ml) in a 2L round bottomed flask which was sealed and allowed to stand at room temperature overnight The slurry was then transferred to a sinter funnel and washed with DMF (12x 700ml) This produced DMF washings containing no trace of amine The resin was then washed with DMF containing an increasing gradient of DCM (2 5L, 0-100% DCM) then an increasing gradient of ether in DCM (900ml, 0-100% ether) The resin was then dried overnight in a stream of nitrogen at 40°C The resin produced had an ammo functionahsation of 973 micromoles per gram ("Ammo-PDMA resin”)
  • a 5'-DMT-deoxycyt ⁇ d ⁇ ne dimer (protected on the mternucleoside phsophorus by a ⁇ -cyanoethyl group) was also attached to the resin at a similar loading using identical conditions to above
  • the loaded resin was prepared for coupling by having the DMT group removed as follows DMT-C bz -PDMA resin is poured into a sintered funnel (7cm, porosity 3) and a positive pressure of nitrogen is applied A 3% solution of DCA in DCM (15ml/g resin) is then added to the resin and left to bubble gently for 5 minutes This was repeated (twice, quantities same as above) All of the orange colour was removed from the resin at this point indicating that det ⁇ tylation is complete Residual acid was removed by washing with

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Abstract

Cette invention concerne un procédé de synthèse d'un phosphorothioate triester. Ce procédé consiste à faire réagir, en présence d'un agent de couplage, un H-phosphonate avec un substrat qui comprend un groupe hydroxy libre et qui est lié à un support solide. Le H-phosphonate diester supporté ainsi obtenu est soumis à un transfert du soufre au moyen d'un agent de transfert de soufre.
PCT/GB2000/003912 1999-10-14 2000-10-12 Preparation de phosphorothioate triesters et d'oligonucleotides WO2001027126A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00968075A EP1224193A1 (fr) 1999-10-14 2000-10-12 Preparation de phosphorothioate triesters et d'oligonucleotides
CA002386867A CA2386867A1 (fr) 1999-10-14 2000-10-12 Preparation de phosphorothioate triesters et d'oligonucleotides
JP2001530344A JP2003517467A (ja) 1999-10-14 2000-10-12 ホスホチオエーテトリエステル及びオリゴヌクレオチドの調製方法
AU78035/00A AU7803500A (en) 1999-10-14 2000-10-12 Process for the preparation of phosphorothioate triesters and oligonucleotides

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GBGB9924285.1A GB9924285D0 (en) 1999-10-14 1999-10-14 Process
GB9924285.1 1999-10-14

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WO2001027126A1 true WO2001027126A1 (fr) 2001-04-19

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CN (1) CN1409719A (fr)
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CA (1) CA2386867A1 (fr)
GB (1) GB9924285D0 (fr)
WO (1) WO2001027126A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064702A1 (fr) * 2000-03-01 2001-09-07 Avecia Limited Procede de preparation de triesters de phosphorothioate
JP2006512336A (ja) * 2002-12-18 2006-04-13 アベシア・リミテッド オリゴヌクレオチド・シントン類を精製する方法
US7227017B2 (en) * 2000-12-05 2007-06-05 Avecia Limited Process for the preparation of phosphorothioate oligonucleotides
US8569476B2 (en) 2008-09-23 2013-10-29 Suzhou Ribo Life Science Co., Ltd. Method for preparing oligonucleotide
WO2014012081A2 (fr) 2012-07-13 2014-01-16 Ontorii, Inc. Contrôle chiral
WO2015107425A2 (fr) 2014-01-16 2015-07-23 Wave Life Sciences Pte. Ltd. Conception chirale
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
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
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US10149905B2 (en) 2014-01-15 2018-12-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10322173B2 (en) 2014-01-15 2019-06-18 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries

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WO2001064702A1 (fr) * 2000-03-01 2001-09-07 Avecia Limited Procede de preparation de triesters de phosphorothioate
US7022833B2 (en) 2000-03-01 2006-04-04 Avecia Limited Process for the preparation of phosphorothioate triesters
US7227017B2 (en) * 2000-12-05 2007-06-05 Avecia Limited Process for the preparation of phosphorothioate oligonucleotides
JP4824931B2 (ja) * 2002-12-18 2011-11-30 アベシア・バイオテクノロジー・インコーポレーテッド オリゴヌクレオチド・シントン類を精製する方法
JP2006512336A (ja) * 2002-12-18 2006-04-13 アベシア・リミテッド オリゴヌクレオチド・シントン類を精製する方法
US8569476B2 (en) 2008-09-23 2013-10-29 Suzhou Ribo Life Science Co., Ltd. Method for preparing oligonucleotide
US9394333B2 (en) 2008-12-02 2016-07-19 Wave Life Sciences Japan Method for the synthesis of phosphorus atom modified nucleic acids
US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US9695211B2 (en) 2008-12-02 2017-07-04 Wave Life Sciences Japan, Inc. Method for the synthesis of phosphorus atom modified nucleic acids
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US9744183B2 (en) 2009-07-06 2017-08-29 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
US10280192B2 (en) 2011-07-19 2019-05-07 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9605019B2 (en) 2011-07-19 2017-03-28 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
WO2014012081A2 (fr) 2012-07-13 2014-01-16 Ontorii, Inc. Contrôle chiral
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US9982257B2 (en) 2012-07-13 2018-05-29 Wave Life Sciences Ltd. Chiral control
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
US9598458B2 (en) 2012-07-13 2017-03-21 Wave Life Sciences Japan, Inc. Asymmetric auxiliary group
US10590413B2 (en) 2012-07-13 2020-03-17 Wave Life Sciences Ltd. Chiral control
EP4219516A2 (fr) 2012-07-13 2023-08-02 Wave Life Sciences Ltd. Controle chiral
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
US10322173B2 (en) 2014-01-15 2019-06-18 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design
WO2015107425A2 (fr) 2014-01-16 2015-07-23 Wave Life Sciences Pte. Ltd. Conception chirale
EP4137572A1 (fr) 2014-01-16 2023-02-22 Wave Life Sciences Ltd. Conception chirale

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AU7803500A (en) 2001-04-23
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CN1409719A (zh) 2003-04-09
GB9924285D0 (en) 1999-12-15
CA2386867A1 (fr) 2001-04-19

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