WO2001027126A1

WO2001027126A1 - Process for the preparation of phosphorothioate triesters and oligonucleotides

Info

Publication number: WO2001027126A1
Application number: PCT/GB2000/003912
Authority: WO
Inventors: Mark Edward Douglas; Kevin Gerard Scott; David John Moody; Ben James Mellor
Original assignee: Avecia Limited
Priority date: 1999-10-14
Filing date: 2000-10-12
Publication date: 2001-04-19
Also published as: EP1224193A1; GB9924285D0; CN1409719A; AU7803500A; CA2386867A1; JP2003517467A

Abstract

A process for the synthesis of a phosphorothioate triester is provided. The process comprises 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.

Description

PROCESS FOR THE PREPARATION OF PHOSPHOROTHIOATE TRIESTERS AND

OLIGONUCLEOTIDES

The present invention concerns a method for the synthesis of phosphorothioate tnesters, and particularly ohgonucleotides

In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyπbonucleotides (DNA sequences), oligoπbonucleotides (RNA sequences) and their analogues, see 'Methods in Molecular Biology, Vol 20, Protocol for Ohgonucleotides and Analogs', Agrawal, S Ed , Humana Press, Totowa, 1993 Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeπc phosphoramidite building blocks Beaucage, S L , Caruthers, M H Tetrahedron Lett , 1981 , 22, 1859-1862 has proved to be the most convenient approach Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers These synthetic ohgonucleotides have met a number of crucial needs in biology and biotechnology

Following Zamecnik and Stephenson's seminal discovery that a synthetic ohgonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamecnik, P , Stephenson, M Proc Natl Acad Sci USA 1978, 75, 280-284), the idea that synthetic ohgonucleotides or their analogues might well find application in chemotherapy has attracted a great deal of attention both in academic and industrial laboratories For example, the possible use of ohgonucleotides and their phosphorothioate analogues in chemotherapy has been highlighted in the report of Gura,

T Science, 1995, 270, 575-577 The so-called antisense and antigene approaches to chemotherapy (Ohgonucleotides Antisense Inhibitors of Gene Expression, Cohen J S , Ed , Macmillan, Basingstoke 1989 Moser, H E , Dervan, P B Science 1987, 238, 645- 649), have profoundly affected the requirements for synthetic ohgonucleotides Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials Several ohgonucleotide analogues that are potential antisense drugs are now in advanced clinical trials If as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram or more probably multikilogram quantities of a specific sequence or sequences will be required

Three main methods, namely the phosphotriester (Reese, Tetrahedron, 1978) phosphoramidite (Beaucage, S L in Methods in Molecular Biology, Vol 20, Agrawal, S Ed , Humana Press, Totowa, 1993, pp 33-61) and H-phosphonate (Froehler, B C in Methods in Molecular Biology, Vol 20, Agrawal, S , Ed , Humana Press, Totowa, 1993, pp 63-80) approaches have proved to be effective for the chemical synthesis of ohgonucleotides While the phosphotriester approach has been used most widely for synthesis in solution, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid phase synthesis The conventional H-phosphonate synthesis approach has been found to have a number of disadvantages First, the process involves the use of an intermediate chain comprising a plurality of reactive H- phosphonate internucleotide linkages The reactivity of these linkages can cause degradation, and hence lower yields and purities Additionally, the use of a single oxidation or sulphunsation step at the end of the assembly of the desired molecule means that the process cannot readily be employed for the controlled production of chimenc nucleotides Furthermore, when an oxidation step is employed, this is slow, and can cause concomitant degradation, and when a sulphunsation step is employed, not only are the reagents toxic, but the reaction is also slow Additionally, the most common sulphunsation reagent, the so-called "Beaucage Reagent", can introduce a significant, and unpredictable, proportion of oxygen, in place of the expected sulphur Attempts to improve the conventional H-phosphonate approach, for example that taught in EP-A-0 219 342 using acylating agents themselves have problems, for example the low coupling yields achieved with acylating agents

According to the present invention, there is provided a process for the synthesis of a phosphorothioate tπester 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 In many highly preferred embodiments, 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

H-phosphonate monoester, and advantageously a protected nucleoside or ohgonucleotide H-phosphonate, preferably comprising a 5' or a 3' H-phosphonate function, particularly preferably a 3' H-phosphonate function Preferred nucleosides are 2'- deoxy bonucleosides and nbonucleosides, preferred ohgonucleotides are o godeoxyribonucleotides and ohgoribonucleotides 2'-deoxyrιbonucleosιdes and ohgodeoxyribonucleotides may comprise 2'-C-alkyl and 2'-C-alkenyl substituents

When 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 Examples of such suitable protecting groups include acid labile protecting groups, particularly trityl and substituted trityl groups such as dimethoxytπtyl 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

When the H-phosphonate building block is a protected deoxynbonucleoside ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide derivative comprising a 5' H-phosphonate function, 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

When the H-phosphonate is a protected ribonucleoside or a protected oligonbonucleotide, 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-methoxypιperιdιne-4-yl (Fpmp), and alkyl and aryl silyl protecting groups such as t-butyldiphenyl silyl groups, commonly tnalkylsilyl groups, often tπ(C_{1 4}-alkyl)sιlyl groups such as a tertiary butyl dimethylsilyl group Alternatively, 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^ ₄alkyl or alkenyl derivative, in which case, the 2' position does not need further protection Other H-phosphonates that may be employed in the process according to the present invention are derived from polyfunctional alcohols, especially alkyl alcohols, and preferably diols or tπols Examples of alkyl diols include ethane-1 ,2-dιol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400 Examples of alkyl tπols include glycerol and butane tnols Further polyfunctonal alcohols include sacchaπdes, especially abasic nucleosides, for example πbose and deoxyπbose Commonly, only a single H-phosphonate function will be present, the remaining hydroxy groups being protected by suitable protecting groups, such as those disclosed hereinabove for the protection at the 5' or 2' positions of πbonucleosides

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

When the substrate comprising a free hydroxy group is a protected nucleoside or a protected ohgonucleotide, preferred nucleosides are deoxyπbonucleosides and πbonucleosides and preferred ohgonucleotides are ohgodeoxyribonucleotides and ohgoπbonucleotides

When 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

When 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 When the substrate comprising a free hydroxy group is a ribonucleoside or an oligonbonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fluorophenyl)-4-methoxypιperιdιne-4- yl (Fpmp), and tπalkylsilyl groups, often tπ(C_{1 4}-alkyl)sιlyl groups such as a tertiary butyl dimethyl silyl group Alternatively, the ribonucleoside or oligonbonucleotide may be a 2'- O-alkyl, 2'-O-alkoxyalkyl or 2-'O-alkenyl derivative, commonly a C, ₄ alkyl, C, ₄ alkoxy^ ₄alkyl or alkenyl derivative, in which case, the 2' position does not need further protection

Other substrates comprising a free hydroxy group that may be employed in the process according to the present invention are sacchaπdes, especially abasic nucleosides such as πbose and deoxyπbose, and non-sacchaπde polyols, especially alkyl polyols, and preferably diols or tπols Examples of alkyl diols include ethane-1 ,2-dιol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400 Examples of alkyl tπols include glycerol and butane tπols Commonly, only a single free hydroxy group will be present, the remaining hydroxy groups being protected by 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 However, more than one free hydroxy group may be present if it is desired to perform identical couplings on more than one hydroxy group

In addition to the presence of hydroxy protecting groups, 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 For example, 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 dι(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-dιchlorophenyl and also by an isobutyryl group G may also be protected by diphenylcarbamoyl and glyoxal type protecting groups thymine (T) and uracil (U) generally do not require protection, but in certain embodiments may advantageously be protected, for example at O4 by a phenyl group, including substituted phenyl, for example 2,4-dιmethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl, alkyl or alkoxy substituted benzoyl, such as ^ ₄ alkyl- or C-, ₄ alkoxybenzoyl

After the coupling and sulphur transfer steps, and when it is desired to carry out further coupling and sulphur transfer steps, it is often necessary to introduce a free hydroxyl group into the phosphorothioate tnester produced by the process of the present invention When 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 After the protecting group has been removed, 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- dιmethyl)amιnopyπdιne) and lower, eg up to C₄, alkyl H-phosphonates, such as ethyl H- phosphonate, and 2-cyanoethyl H-phosphonate

In a particularly preferred embodiment, the invention provides a method comprising the coupling of a 5'-0-(4,4'-dιmethoxytrιtyl)-2'-deoxyπbonucleosιde 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'- deoxyπbonucleoside or ohgodeoxyribonucleotide, in the presence of a suitable coupling agent and subsequent sulphur transfer in the presence of a suitable sulphur-transfer agent In the process of the present invention, any suitable coupling agents and sulphur- transfer agents available in the prior art may be used

Examples of 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 Examples of suitable alkyl acid chlorides which may be employed include up to C₁₂ alkyl acid chlorides, including adamantyl carbonyl chloride, and especially C₂ to C₇ alkanoyl chlorides, particularly pivaloyl chloride Examples of 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

Examples of suitable alkanesulphonyl chlorides which may be employed include C₂ to C₇ alkanesulphonyl chlorides Examples of 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

Examples of suitable alkyl chloroformates which may be employed include C₂ to C₇ alkyl chloroformates Examples of 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 Examples of suitable alkyl chlorosulphites which may be employed include C₂ to

C₇ alkyl chlorosulphites Examples of aryl chlorosulphites which may be employed 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

Examples of suitable alkyl phosphorochloπdates which may be employed include dι(C, to C₆ alkyl) phosphorochloπdates Examples of aryl phosphorochloπdates which may be employed include substituted and unsubstituted diphenyl phosphorochloπdates, such as C, ₄ alkoxy, halo, particularly fluoro, chloro and bromo, and C, ₄ alkyl, substituted diphenyl phosphorochloπdates When substituted, from 1 to 5 substituents on each phenyl group may be present, particularly in the case of alkyl and halo substituents

Further coupling agents that may be employed are the chloro-, bromo- and (benzotπazo-1-yloxy)- phosphonium and carbonium compounds disclosed by Wada et al, in J A C S 1997, 119, pp 12710-12721 (incorporated herein by reference) Examples of suitable carbodnmides that may be employed include alkyl carbodiimides, especially (C, to C₆ alkyl) carbodiimides, such as 1 ,3 dicyclohexyl carbodiimide, 1 ,3 dnsopropyl carbodiimide, 1-tert -butyl-3-ethyl carbodnmide and 1-(3- dιmethylamιnopropyl)-3-ethyl carbodiimide One or more of the alkyl groups may be substituted, for example by an alkyl ammo moiety An example of a substituted alkyl carbodiimide is 1-(3-dιmethylamιnopropyl)-3-ethyl carbodiimide

Preferred coupling agents are diaryl phosphorochloridat.es, particularly those having the formula (ArO)₂POCI wherein Ar is preferably phenyl, 2-chlorophenyl, 2,4,6- tπchlorophenyl or 2,4,6-trιbromophenyl

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 tπesters are commonly subsequently converted to phosphodiester or phosphorothioate diesters, and the sulphur transfer agent can be selected accordingly For example 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 s D wherein L represents a leaving group, and D represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group Commonly the leaving group is selected so as to comprise a nitrogen-sulphur bond Examples of suitable leaving groups include morphohnes such as morphohne-3,5-dιone, imides such as phthahmides, succinimides and maleimides, indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nιtroιndazoles, and tπazoles

Where a standard phosphodiester linkage is required in the final product, the sulphur transfer agent is commonly selected such that the moiety D represents an aryl group, such as a phenyl or naphthyl group Examples of suitable aryl groups include substituted and unsubstituted phenyl groups, particularly halophenyl and alkylphenyl groups, especially 4-halophenyl and 4-alkylphenyl, commonly 4-(Cτ 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)phthahmιde (succinimide or other imide may also be used)

Where a phosphorothioate diester linkage is required in the final product, the moiety D commonly represents a methyl, substituted alkyl or alkenyl group Examples of suitable substituted alkyl groups include substituted methyl groups, particularly benzyl and substituted benzyl groups, such as alkyl-, commonly C, ₄alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-posιtιon with an electron-withdrawing substituent such as 2-(4- nιtrophenyl)ethyl and 2-cyanoethyl groups Examples of 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]morphohne-3,5-dιone or a corresponding reagent such as 3-

(phthahmιdosulphanyl)propanonιtrιle

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)

The coupling and sulphur transfer steps of the process according to the present invention are carried out as often as is necessary to synthesise the desired number of phosphorothioate linkages

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, CH₂R' 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

O

-H

°θM⁺ in which 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

O

— H

°©M⁺ in which M⁺ is a monovalent cation, and n is zero or a positive integer, provided that when W is H or a protecting group, that Z is an H-phosphonate group, and that when Z is H or a protecting group, that W is an H-phosphonate group

Preferably, only one of W or Z is an H-phosphonate group, commonly only Z being an H-phosphonate group

When W or Z represents a protecting group, the protecting group may be one of those disclosed above for protecting the 3' or 5' positions respectively When W is a protecting group, the protecting group is preferably a trityl group, particularly a dimethoxytπtyl group When Z is a protecting 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 puπnes, 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

When Q represents a group of OR', and R' is alkenyl, the alkenyl group is often a C, ₄ alkenyl group, especially allyl, propargyl or crotyl group When R' represents alkyl, the alkyl is preferably a C_{1 4} alkyl group When R' represents substituted alkyl, the substituted alkyl group includes alkoxyalkyl groups, especially C-, ₄ alkyoxy^ ₄ alkyl groups such as methoxyethyl groups When R' represents a protecting group, the protecting group is commonly an acid-labile acetal protecting group, particularly 1-(2- fluorophenyl)-4-methoxypιperιdιne-4-yl (Fpmp) or a tπalkylsilyl groups, often a tπ^ ₄- alkyl)sιlyl group such as a tertiary butyl dimethylsilyl group Preferably, X represents O In many embodiments, Y represents S and each R independently represents methyl, substituted alkyl, alkenyl or aryl Preferably, each R independently represents a methyl group, a substituted methyl group, particularly a benzyl or substituted benzyl group, such as an alkyl-, commonly C_{1 4}alkyl- or halo-, commonly chloro-, substituted benzyl group, a substituted ethyl group, especially an ethyl group substituted at the 2- position with an electron-withdrawing substituent such as a 2-(4-nιtrophenyl)ethyl or a 2- cyanoethyl group, a C_{1 4} alkenyl group, preferably an allyl and crotyl group, or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially 4-halophenyl group or a 4-alkylphenyl, commonly a 4-(C, ₄ alkyl)phenyl group, and most preferably a 4-chlorophenyl or a p-tolyl group M+ preferably represents a tπalkyl ammonium ion, such as a trι(C_{1 4}- alkylammonium) ion, and preferably a tπethylammonium ion or a cation of a cyclic base such as 1 ,5-dιazabιcylo[4 3 0]non-5-ene (DBN) or 1 ,8-dιazabιcyclo[5 4 0]undec-7-ene (DBU) n may be 0 or 1 up to any number convenient for the synthesis of the desired ohgonucleotide, particularly up to about 20 Preferably n is 0 to 9, and especially 0 to 7

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

The 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 acetonitπle, amides, such as dimethylformamide and N-methylpyrolhdinone, and basic, nucleophihc solvents such as pyπdine Preferred solvents are pyπdine, 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 For example, transient protecting groups, particularly gamma keto acids such as levuhnoyl-type protecting groups, can be removed by treatment with 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

When deprotecting the desired product once this has been produced, protecting groups on the phosphorus which produce phosphorothioate tnester linkages are commonly removed first For example, a cyanoethyl group can be removed by treatment with anhydrous, strongly basic amine such as DABCO, 1 ,5-dιazabιcylo[4 3 0]non-5-ene (DBN), 1 ,8-dιazabιcyclo[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-nιtrobenzaldoxιme or pyπdιne-2-carboxaldoxιme (Reese et al, Nucleic Acids Res 1981 ) Kamimura, T et al in

J Am Chem Soc , 1984, 106 4552-4557 and Sekine, M Et al, Tetrahedron, 1985, 41 , 5279-5288 in an approach to ohgonucleotide synthesis by the phosphotriester approach in solution, based on S-phenyl phosphorothioate intermediates, and van Boom and his co-workers in an approach to ohgonucleotide synthesis, based on S-(4-methylphenyl) phosphorothioate intermediates (Wreesman, C T J et al, Tetrahedron Lett , 1985, 26,

933-936) have all demonstrated that unblocking S-phenylphosphorothioates with oximate ions (using the method of Reese et al , 1978, Reese, C B,, Zard, L Nucleic Acids Res , 1981 , 9, 4611-4626) led to natural phosphodiester intemucleotide linkages In the present invention, the unblocking of S-(4-chlorophenyl)-protected phosphorothioates with the conjugate base of E-2-nιtrobenzaldoxιme proceeds smoothly and with no detectable intemucleotide cleavage

Other protecting groups, for example benzoyl, pivaloyl and amidine groups can be removed by treatment with concentrated aqueous ammonia

Trityl groups present can be removed by treatment with 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'-termιnal DMTr, protecting group ('detπtylation') should proceed without concomitant depuπnation, especially of any 6-Λ/-acyl-2'-deoxyadenosιne residues Silyl protecting groups may be removed by fluoride treatment, for example with a solution of an ammonium fluoride, for example a solution of tπalkylamine tπhydrogen 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

This new approach to the synthesis of ohgonucleotides is suitable for the preparation of sequences with (a) solely phosphodiester, (b) solely phosphorothioate diester and (c) a combination of both phosphodiester and phosphorothioate diester intemucleotide linkages Solid supports that are employed in the process according to the present invention are substantially insoluble in the solvent employed, and include those supports well known in the art for the solid phase synthesis of ohgonucleotides Examples include silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly(ethylene glycol) copolymers and polymers such as polyvinylacetate Additionally, poly(acrylamιde) supports, especially microporous or soft gel supports, such as those more commonly employed for the solid phase synthesis of peptides may be employed if desired Preferred poly(acrylamιde) supports are amine-functionahsed supports, especially those derived from supports prepared by copolymeπsation of acryloyl-sarcosine methyl ester, N,N-dιmethylacrylamιde and bis-acryloylethylenediamine, such as the commercially available (Polymer Laboratories) support sold under the catalogue name PL-DMA The procedure for preparation of the supports has been described by Atherton, E , Sheppard, R C , in Solid Phase Synthesis A Practical Approach, Publ , IRL Press at Oxford University Press (1984) The functional group on such supports is a methyl ester and this is initially converted to a primary amine functionality by reaction with an alkyl diamine, such as ethylene diamine

The substrate is commonly bound to the solid support via a cleavable linker Examples of 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- deπved linkers In many embodiments when the substrate is bound to a poly(acrylamιde) support via a cleavable linker and comprises a nucleoside, 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-deπvatised nucleoside, and b) reacting the linker-deπvatised nucleoside with an amine-functionahsed poly(acrylamιde) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example dnsopropylethylamine (DIPEA) or N-methylmorphohne (NMM), or hydroxybenzotπazole, or c) reacting an amine-functionahsed poly(acrylamιde) support with a linker, preferably succinic anhydride, to form a hnker-denvatised support, and d) reacting the hnker-denvatised support with a 5'-protected nuceloside having a free 3'- hydroxy group in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example DIPEA or NMM, or hydroxybenzotπazole, then in either case, removing the 5'-protectιng group, which is preferably a trityl or substituted trityl group However, it will be recognised that it may be desired to retain the 5'-protectιng group in which case its removal may be omitted In this case, the 5'- protecting group can be removed when desired prior to use of the supported substrate in the process for the synthesis of phosphorothioate tπesters according to the present invention

Coupling agents used for amide bond formation that can be employed in the process for attaching the substrate to an amine-functionahsed poly(acrylamιde) 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 benzotπazole active esters in situ, such as 2-(1 H-benzotπazole-1-yl)-1 , 1 ,3,3- tetramethyluronium tetrafluoroborate (TBTU) or benzotπazole-1-yloxy-tπs-

(dιmethylamιno)phosphonιum hexafluorophosphate (BOP)

An organic solvent such as N,N-dιmethylformamιde (DMF) or N- methylpyrrohdinone (NMP) is suitably employed for attaching the substrate to an amine- functiona sed poly(acrylamιde) support The process for the synthesis of phosphorothioate tπesters 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 Alternatively 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

On completion of the assembly of the desired product, the product may be cleaved from the solid support, preferably following deprotection of the product When the desired product is an ohgonucleotide, it will be recognised that 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 When the product is bound to the solid support via a cleavable linker, 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

Example 1

4-Λ/-benzoyl-5'-0-(4,4'-dιmethoxytrιtyl)-2'-deoxycytιdιne (DMT C ^z) supported via a succinimide linker at the 3'-posιtιon 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 CH₂CH₂, 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 CH₂CH₂ - Wait for 60 seconds, remove CH₂CH₂ in) with 100ml of 3% DCA/DCM - Wait for 60 seconds, remove acid iv) Wash with 100ml CH₂CH₂ - Wait for 60 seconds, and finally v) Wash with more CH₂CH₂ (100ml) and dry resin with N₂ to produce supported 4-Λ/-benzoyl-2'-deoxycytιdιne (HOC^bz-poly)

Quantities Equivs Amount

822 DMTA^bz (H) 5 0 0 84mmol - 690mg

HOC^bz-poly 1 0 0 168mmol

232 CESP 10 0 1 68mmol - 389mg 268 5/1/3 Activator* 10 0 1 68mmol - 0 35ml

^* As a 1 1 solution in DCM use 0 69ml 6-Λ/-benzoyl-5'-O-(4,4'-dιmethoxytrιtyl)-2'-deoxyadenosιne-3'-H-phosphonate (DMTA^bz(H), 690mg) was dried by co-evaporation (2x5ml) with anhydrous pyndine, and combined with 2g of HOC^bz-poly (in a 100ml florentine) Pyndine was added such that all the DMTA^bz(H) was in solution and a heterogeneous slurry was formed This required 40ml of pyndine The mixture was cooled to -40°C and this was maintained during both coupling and sulphur transfer stages The reaction mixture was purged with argon, and the reaction was carried out under a blanket of this gas 0 35ml (0 69ml of 1 1 vol/vol mixture in anhydrous dichloromethane) of diphenylphosphorochloπdate was added drop- wise over 5 minutes to the cooled, stirred slurry and left stirring for a further 15 minutes At this point 389mg 2-(2-cyanoethyl)sulphanyl phthahmide (CESP) in 8ml of pyndine was added in one aliquot The mixture was initially stirred at -40°C and then allowed to warm to room temperature The slurry was then transferred to a sinter, washed with pyndine (50ml) and DCM (200ml) and the resin dried under vacuum to produce 5'-(DMT)-A^bz-O- P(=O)(SCH₂CH₂CN)-C^bz-(3'-O-polymer support) ("DMT-AC-poly")

DMT-AC-poly (1 4g@71 μmolg ¹ loading) was treated for 60 seconds at room temperature with 200ml of a 3% solution of dichloroacetic acid in CH₂CH₂ The resin was washed with clean CH₂CH₂ and dried in a stream of N₂ to produce 5'-(HO)-A ^z-O- P(=O)(SCH₂CH₂CN)-C^bz-(3'-O-polymer support) ("HO-AC-poly")

Equivs Amount

2235 DMTACAC(H) 5 0 0 46mmol - 1 03g

71 μmol/g HO-AC-poly 1 0 13g@ 71 μmol/g

232 CESP 10 0 0 923mmol - 214mg

268 5/13 Activator 10 0 *0 923mmol - 0 19ml

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 tetrameπc 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 tπethylamine 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 2ml) added over 1 minute The mixture was stirred for 30 minutes, and 15ml water is added After 10 minutes stirring, 250ml of a solution of 10% v/v methanol in dichloromethane was added, and this was washed with 0 5M tπethylammonium phosphate buffer The organic layer was separated, diluted with 25ml methanol and the wash repeated The organic layer was separated, evaporated from 100ml toluene, and then evaporated from 200ml 50 50 by vol toluene/pyπdine mixture The crude gum is redissolved in dichloromethane, and purified by column chromatography to yield 5'-DMT- ACAC-3'-H phosphonate The 5'-DMT-ACAC-3'-H phosphonate was dried by co-evaporation from anhydrous pyndine (2x5ml) and re-suspended in 40ml of the same solvent The mixture was combined with 1 3g of HO-AC-poly in a 100ml florentine The heterogeneous mixture was stirred, blanketed in Argon and cooled to -40°C 0 38ml of 1 1 vol/vol diphenylphosphorochloπdate in CH₂CH₂ was added drop-wise over 5 minutes, and the mixture stirred for a further 15 minutes At this point CESP (214mg in 5ml pyndine) was added in one aliquot The mixture was initially stirred at -40°C and then allowed to warm to room temperature over 20 minutes prior to quench (1 ml of water)

The slurry was transferred to a sinter, and was filtered and washed (5x100ml of CH₂CH₂) and dried over nitrogen to produce 5'-(DMT) -A^bz-O-P(=O)(SCH₂CH₂CN)-C^bz-O- P(=O)(SCH₂CH₂CN) -A^bz-O-P(=O)(SCH₂CH₂CN)-C^bz-O-P(=O)(SCH₂CH₂CN)-A^bz-O-

P(=O)(SCH₂CH₂CN)-C^bz- (3'-O-polymer support) ("DMT-ACACAC-poly")

The cyanoethyl groups can be removed by treatment with anhydrous 1 ,8- dιazabιcyclo[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

Example 2. General Methodology for H-Phosphonate Coupling and Sulfur Transfer on Poly(dimethylacrylamide) (PDMA)

A poly(acrylamιde) resin produced by copolymeπsation of acryloyl-sarcosine methyl ester, N,N-dιmethylacrylamιde 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 solution of 4-Λ/-benzoyl-5'-0-(4,4'-dιmethoxytrιtyl)-2'-deoxycytιdιne-3'-O- succmate ("DMT-C^bz-succιnate", 3eqvs, 234 mmol), hydroxybenzotπazole (6 eqvs, 467 mmol) and DIC (diisopropyl carbodiimide, 4 eqvs, 31 mmol) was prepared in 1300ml of DMF The solution was prepared in a 2L flask which had previously been silanised (this was achieved by simply swirling tπmethylsilyl chloride around the flask) in order to prevent the resin from adhering to glass The Amino-PDMA resin (1 eqv, 78 mmol) was then added to the solution and left to stand overnight at room temperature This resulted in a stiff non-mobile gel After 22 hrs a few beads were removed and washed with fresh DMF The resin was found to be Kaiser negative, indicating that all the ammo groups on the resin had reacted with DMT-C^bz-succιnate The reaction mixture was then transferred to a sintered funnel (18cm diameter, porosity 3) and washed (5 x 800ml) After the final DMF wash a similar diethyl ether treatment as described for the ammo PDMA was carried out in order to shrink the resin After blowing dry with N₂ for 24 hours and then drying in the vacuum oven overnight the resin was weighed This gave a weight increase of 55 7g from the original 80g of Amino-PDMA This resulted in DMT-C^bz-PDMA resin loaded to 573 mmol per gramme

A 5'-DMT-deoxycytιdιne dimer (protected on the mternucleoside phsophorus by a β-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 detπtylation is complete Residual acid was removed by washing with

DCM and further washing carried out with ether in DCM, starting at 20%v/v ether and continuing to 100% ether The HO-C^bz-PDMA resin is then air dried and dried m-vacuo at 40°C overnight

HO-C^bz-PDMA resin (1 mmol, 1 48g), DMT-C^bz-H-phosphonate (6 eq, 4 79g) were weighed into a 100ml florentme This was then suspended in 60ml of anhydrous grade

DMF The mixture was left for 5 minutes to allow the resin to swell to its maximum capacity at room temperature and pyndine (4 36ml) was added The reaction mixture was then stirred using a small (12x5mm) flea (at maximum stir rate on a Heidolph MR3001 K stirrer hotplate) The neat activator, diphenyl phosphorochloπdate (6 eq 1 24ml) was added dropwise, during a 5 minute period, using the syringe pump (Razel

A99FZ) Immediately after the addition was complete, the sulfur transfer reagent (CESP 232mg, 1 mmol), was added to the mixture as a solid single aliquot After 5 minutes more the resin mixture was poured into a sintered funnel (porosity 3) The resin was then washed for 5 minutes, with stirring, using 50ml of DMF This process was repeated a further two times At this point any un-reacted hydroxyl groups were capped by treatment with 35ml of a 1 4M solution of acetic anhydride in pyndine containing 4-(N,N- dιmethyl)amιnopyrιdιne (DMAP, 0 5mmol) for 20 mins, followed by further washing with DMF ( 3x 30ml) At the end of the final wash as much DMF as possible was removed prior to washing the resin with 100ml of diethyl ether The resin was stirred during the ether addition and the solvent allowed to drop through the sinter under gravity The ether wash was repeated twice more After the final wash the remaining ether was removed by passing N₂ through the resin for 30 minutes The resin was then dried overnight in vacuo at 40°C The weight increase of the resin and trityl assay indicated quantitative coupling to form DMT-C^bzC^bz-PDMA, protected on the internucleoside linkage with a β-cyanoethyl group

Preparation of 5'-HO-A ^zC^bzA^bzC^bzC^bz-PDMA

HO-C^bz-PDMA resin prepared as above (1 mmol, 1 48g) and 5'-DMT-A^bzC^bzA^bzC ^z- H-phosphonate protected on the internucleoside linkages with β-cyanoethyl groups ( 1 2eq, 2 68g) were weighed into a 100ml florentme Anhydrous DMF (60ml) was added and the resin allowed to swell for 5 mms at room temperature Pyndine (5mmol, 0 4ml) was added followed by the coupling agent, diphenyl phosphorochloπdite (2mmol, 0 41 ml) which was added dropwise during 5 mms using a syringe pump as above On completion of this addition, sulfur transfer agent (CESP, 2 5mmol, 0 58g) was added solid as a single aliquot and the suspension stirred for one hour The solid was then washed with DMF followed by DCM and the DMT group removed by treatment with dichloroacetic acid as above The resin was then washed with DCM, DMF and finally ether before being dried in a stream of nitrogen and then in vacuo at 40°C overnight The weight increase and a trityl assay indicated a yield of 75%

Claims

1 A process for the synthesis of a phosphorothioate tnester 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

2 A process according to claim 1 , wherein a plurality of coupling and sulphur transfer steps are carried out, with a sulphur transfer step being carried out after each coupling step

3 A process according to claim 1 or claim 2, wherein the H-phosphonate is a 2'- deoxyπbonucleoside, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide

4 A process according to claim 3, wherein the 2'-deoxyπbonucleosιde, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide comprises a 3' H- phosphonate function

5 A process according to any preceding claim, wherein the substrate comprising a free hydroxy group is a deoxynbonucleoside, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide

6 A process according to claim 5, wherein the substrate comprising a free hydroxy group is a deoxynbonucleoside, ribonucleoside, ohgodeoxyribonucleotide or oligonbonucleotide derivative comprising a free 5'-hydroxy group and is bonded to the solid support via the 3'-posιtιon

7 A process according to any preceding claim, wherein the coupling agent is an alkyl phosphorochloπdate or an aryl phosphorochloπdates, and preferably diphenyl phosphorochloπdate

8 A process according to any preceding claim, wherein the sulphur transfer agent has the general chemical formula

L s D

wherein L represents a leaving group, and D represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group 9 A process according to any preceding claim wherein the reaction between the H- phosphonate and the substrate comprising a free hydroxy group and the reaction between the H-phosphonate diester and the sulphur transfer agent takes place in the presence of an organic solvent selected from the group consisting of haloalkanes, esters, nitnles, amides and basic, nucleophihc solvents, and mixtures thereof

10 A process according to claim 9, wherein the organic solvent is selected from the group consisting of pyndine, dichloromethane, dimethylformamide, N-methylpyrolhdinone and mixtures thereof

11 A process according to any preceding claim, wherein the solid support is selected from the group consisting of silica, controlled pore glass, polystyrene, copolymers comprising polystyrene, polyvinylacetate and poly(acrylamιde) supports

12 A process according to claim 11 , wherein the solid support is an amine- functiona sed support copolymer of acryloyl-sarcosine methyl ester, N,N- dimethylacrylamide and bis-acryloylethylenediamine

13 A process according to any preceding claim, wherein the process is carried out at a temperature in the range of from approximately -55°C to about 40°C, and preferably from 0 to 30°C

14 A process according to any preceding claim, wherein hydroxy groups which are unreacted after a given coupling are capped by reaction with a capping agent

15 A process according to any preceding claim wherein the phosphorothioate tnester is an ohgonucleotide, and the process comprises the additional steps of deprotectmg the phosphorothioate tnester, and cleaving the product thereof from the solid support thereby to form a phosphate diester, phosphorothioate diester or chimera comprising both phosphate diester and phosphorothioate diester moieties, optionally followed by one or more purification processes