Title of Invention	PROCESS FOR THE PREPARATION OF PHOSPHOROTHIOATE TRIESTERS AND OLIGONUCLEOTIDES
Abstract	A process for the synthesis of a phosphorothioate triester 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 triester.

Title of Invention

PROCESS FOR THE PREPARATION OF PHOSPHOROTHIOATE TRIESTERS AND OLIGONUCLEOTIDES

Abstract

A process for the synthesis of a phosphorothioate triester 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 triester.

Full Text	FORM 2 THE PATENTS ACT 1970 [39 OF 1970] COMPLETE SPECIFICATION [See Section 10] "PROCESS FOR THE PREPARATION of PHOSPHOROTHIOATE TRIESTERS AND OLIGONUCLEOTIDES" AVECIA LIMITED A British company of Hexagon House, Blackley, Manchester M9 8ZS, United Kingdom, The following specification particularly describes the nature of the invention and the manner in which it is to be performed:- The present invention concerns a method for the synthesis of phosphorothioate triesters, and particularly oligonucleotides." In the past 15 years or so, enormous progress has been made iri the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues, see 'Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides 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 monomeric phosphorarnidite 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 oligonucleotides have met a. number of crucial needs in biology and biotechnology. Following Zamecnnik and Stephenson's seminal discovery that a synthetic oligonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamechik, P.; Stephenson, M. Proc. Natl Acad. Sci. USA 1978, 75,. 280-284), the idea that synthetic oligonucleotides 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 oligonucleotides and their phosphbrothioate 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 (Oligonucleotides. Antisense Inhibitors'of Gene Expression, Cohen,,J.S.,. Ed., Macmillan, Basingstoke 1989 Moser, H. E.; Deryan, P.S:-. Science 1987, 238, 645-649), have profoundly affected the requirements for synthetic oligonucleotides. Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide 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), phosphorarnidite (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 oligonucleotides. While the phosphotriester approach has been.used most widely for synthesis in solution, the phosphorarnidite 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 sulphurisation step at the end of the assembly of the desired molecule means that the process cannot readily be employed for the controlled production of chimeric nucleotides.. Furthermore, when an oxidation step isemployed, this is slow, and can cause concomitant degradation, and when a sulphurisation step is employed,'not only are . the reagents toxic, but the reaction is also slow. Additionally, the most common sulphurisation 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 triester 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 triester. 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 present invention relates to a supported H-phosphonate diester subjected to sulfur transfer with a sulfur agent to form a phosphorothioate ester. The prior art documents D1: BRILL W. K-D: discloses a process in which an H-phosphonate diester is reacted with a silylating agent in order to form a silyl phosphite. This silyl phosphite is then subjected to sulfur transfer with a sulfur transfer agent. BRILL W. K-D does not disclose a process in which an H phosphonate diester is subjected to sulfur transfer with a sulfur transfer agent. Accordingly, BRILL W. K-D does not destroy the novelty of the present claims. Regarding the inventive step, the process of the present invention disclosed that a supported H-phosphonate diester is subjected to sulphur transfer with a sulphur transfer agent to form a phosphorothioate trimester. BRILL W. K.-D discloses the solid phase preaparation of a phosphorothioate trimester by a process comprinsing the formation of a solid-supported H-phosphonate diester, silylation to form a silylphosphite, and reaction with a source of a thioalkly group to form the phosphorothioate triester. WO 99 09041 A discloses a solution phase process the preparation of a phosphorothiate triester-by a process comprising the formation of an H-phosphonate diester and reaction with a sulphur transfer reagent to form the phosphorothioate triester. EP-A-0 739 899 discloses that certain solid supports can be employed in the spnthesis of oligonucleotides. The claims of the present invention involve an inventive step over the teaching of BRILL W. K-D, either alone, or in combination with either or both of WO 99 09041 A and EP-A-0 739 899. A skilled person consulting BRILL W. K-D would perceive that the core teaching of BRILL W. K-D is the thioaklylation of H-phosphonates via a silylphosphite intermediate. Thus, the very last change a skilled person would be motivated to make to the process of BRILL W. K-D would be to omit the silylation step. Indeed, it would be perverse of the skilled person to omit the core step of the teaching of BRILL W. K-D. The skilled person reading BRILL W. K-D without benefit of the knowledge of the present invention would not ignore the benefits taught by BRILL W. K-D for the silylation step (page 703 2nd paragraph, last 3 lines). Accordingly, the process of claim 1 would not be obvious to the skilled person in view of BRILL W. K-D. Further, the present invention would not be obvious to one of the ordinary skill in the art in view of the combination of BRILL W. K-D and WO 99 09041 A without the benefit of knowledge of the present invention. The clear teaching of BRILL W. K-D is that the syntheses of phosphorothioate trimesters by the H-phosphonate route on a solid phase requires the intermediate silylation step in order to yield the desired product. It is submitted that it would be extremely perverse of the skilled person to choose to omit such a key teaching from the process of BRILL W. K-D. While, WO 99 09041 A discloses a process in which an H-phosphonate diester is reacted directly with a sulfur transfer agent, the process discloased is a solution phase process and hence does not anticiapate the claimed invention. The skilled person; would recognize that the conducting of reactiosn on solid supports places markedly different constrainst on the process chemistry, especially in terms of reaction kinetics, resulting from the lower freedom of movement of molecules attached to, a solid support, and also from surfaces effects caused by the presence-of the solid support. Accordingly, the skilled person would not expect simply to substitute the process chemistry of WO 99 G9041 A for the process of BRILL W. K-D. In fact, the skilled person aware of the teaching of BRILL W. K-D would, conclude that it was precisely the difficulties associated with conducting reactions on a solid support which resulted in the teaching of BRILL W. K-D concerning the importance of the silylation state. Additionally, EP-A-0 739 899 does not provide a teaching that would motivate the skilled person to ignore the clear teaching of BRILL W. K-D. Therefore, the skilled person, based on a fair teaching of BRILL W. K-D and WO 99 09041 A, and without the benefit of knowledge of the present invention, would simply not consider replacing the chemistry of BRILL W. K-D with the chemistry of WO 99 09041 A. Accordingly, the claims of the present application would not be obvious over BRILL W. K-D, WO 99 09041 A and EP-A-0 739 899, and accordingly involve an inventive step. 'The H-phosphonate employed in the process of the present-invention is ofterran H-phosphonate monoester, and advantageously a protected nucleoside or oligonucleotide. H-phosphonate,, preferably comprising a 5' or a/3' H-phosphonate function, particularly preferably a 3' H-phosphonate function. Preferred nucleosides are 2'-. " deoxyribonucleosides and ribonucleosides; - preferred oligonucleotides are oligodeoxyribonudeotides . and oligoribonucleotides. 2'-deoxyribonucleosides and oligodeoxyribonucleotides may comprise 2'-C-alkyI and 2'-C-alkenyl substituents. When the H-phosphonate building block is a protected deoxyribonudeosrde, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide 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 dimethoxytrityl 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 deoxyribonucleoside, When the substrate comprising a free hydroxy group is a ribonucleoside or an oligoribonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fIuorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and trialkyisilyl groups, often tri(C1-4-alkyl)silyl groups such as a tertiary butyl dimethyl silyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2'-O-alkyl, 2'-O-alkoxyalkyl or 2-'0-alkenyl derivative, commonly a C1-4 alkyl, C1-4 alkoxyC1- 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 saccharides, especially abasic nucleosides such as ribose and deoxyribose, and non-saccharide polyols, especially alkyl polyols, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. 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 ribonucleosides, 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 C1-4 alkyl- or C1-4 alkoxy-, benzoyl; pivaloyl; and amidine, particularly dialkylaminomethylene, preferably di(C1-4 -alkyl) aminomethylene such as dimethyl or dibutyl aminomethylene. Guanine (G) may be protected by a phenyl group, including substituted phenyl, for example 2,5-dichlorophenyl 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 04 by a phenyl group, including substituted phenyl, for exampfe 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl, alkyl or alkoxy substituted benzoyl, such as C1-4 alkyl- or C1-4 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 triester produced by the process of the present invention. When the phosphorothioate triester produced is a protected nucleoside or oligonucleotide 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 oligonucleotide 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 oligonucleotide sequence. The method may then proceed with steps to remove the protecting groups from the internucleotide 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 nucleophiiic acylation catalyst such as 4-(N,N-dimethyl)aminopyridine) 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'-O-(4,4'-dimethoxytrityl)-2'-deoxyribonucleoside 3-H-phosphonate or a protected oligodeoxyribonucleotide 3'-H-phosphonate and a substrate supported on a solid support with a free hydroxy function, most commonly a 2'-deoxyribonucleoside or oligodeoxyribonucleotide, 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 aryi chloroformates, alkyl and aryl chlorosulphites and alkyl and aryl phosphorochloridates, 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted phenyl chlorosulphites. When substituted, from 1 to 3 substituents are often present, particufarly in the case of alkyl and halo substituents. Examples of suitable alkyl phosphorochloridates which may be employed include di(C1 to C6 alkyl) phosphorochloridates. Examples of aryl phosphorochloridates which may be employed include substituted and unsubstituted diphenyl phosphorochloridates, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted diphenyl phosphorochloridates. 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 (benzotriazo-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 carbodiimides that may be employed include alkyl carbodiimides, especially (C1 to C6 alkyl) carbodiimides, such as 1,3 dicyclohexyl carbodiimide, 1,3 diisopropyl carbodiimide, 1 -tert.-butyl-3-ethyl carbodiimide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. One or more of the alkyl groups may be substituted, for example by an alkyl amino moiety. An example of a substituted aikyl carbodiimide is 1-(3-dimethyfaminopropyl)-3-ethyI carbodiimide. Preferred coupling agents are diaryl phosphorochloridates, particularly those having the formula (ArO)2POCI wherein Ar is preferably phenyl, 2-chiorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl. The sulphur transfer agents employed in the process of the present invention introduce a protected thio moiety into the linkage, thereby forming a phosphorothioate triester. The phosphorthioate triesters 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 oligonucleotide, a phosphorothioate analogue or a mixed oligonucleotide/oligonucleotide phosphorothioate is required. Sulphur transfer agents employed in the process of the present invention often have the general, chemical formula: 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 morpholines such as morpho!ine-3,5-dione; imides such as phthalimides, succinimides and maieimides; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles; and triazoles. 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 aryi 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-alkyfphenyl, commonly 4-(C1-4 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-(aryisulphanyl)phthalimide (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 C1-4 alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-position with an electron-withdrawing s'ubstituent such as 2-(4-nitrophenyl)ethyl and 2-cyanoethyl groups. Examples of suitable alkenyl groups are allyl, 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-cyanoethyf)-sulphanyl]morphofine-3,5-drone or a corresponding reagent such as 3-(phthalimidosulphanyl)propanonitrile. A suitable temperature for carrying out the coupling reaction and sulphur transfer is in the range of from approximately -55°C to about 40oC, 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 alky], 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 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 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 dimethoxytrityl group. When Z is a protecting group, the protecting group is preferably a trityl group, particularly a dimethoxytrityl group, or an acyl group, preferably a levulinoyl group. Organic bases which may be represented by B include nucleobases, such as natural and unnatural nucleobases, and especially, purines, such as hypoxanthine, and particularly A and G, and pyrimidines, 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 C1-4 alkenyl group, especially ally!, propargyl or crotyl group. When R' represents alkyl, the alkyl is preferably a C1-4 alkyl group. When R' represents substituted alkyl, the substituted alkyl group includes alkoxyalkyl groups, especially C1-4 alkyoxyC1-4 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-methoxypiperidine-4-yl (Fpmp) or a trialkylsilyl groups, often a tri(C1-4 -alkyl)silylgroup 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 C1-4 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-nitrophenyl)ethyl or a 2-cyanoethyl group; a C1-4 alkenyl group, preferably an allyl and crotyl group; or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially 4-haIophenyl group or a 4-alkylphenyl, commonly a 4-(C1-4 a!kyl)phenyl group, and most preferably a 4-chlorophenyl or a p-tolyi group. M+ preferably represents a trialkyl ammonium ion, such as a tri(C1-4-aikylammonium) ion, and preferably a triethylammonium ion or a cation of a cyclic base such as 1,5-diazabicyio[4.3.0]non-5-ene (DBN) or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). , ' n may be 0 or 1 up to any number convenient for the synthesis of the desired oligonucleotide, 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 oligonucleotide 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 acetorittrlle, amides, such as dimethylformamide and N-methylpyrollidinone, and basic, nucleophilic solvents such as pyridine. Preferred solvents are pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone 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 levulinoyl-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 oligonucleotides 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 oligonucleotides and their phosphorothioate analogues. When deprotecting the desired product once this has been produced, protecting groups on the phosphorus which produce phosphorothioate triester 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-diazabicylo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or thethylamine. Phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the base residues can be removed by oximate treatment, for example with the conjugate base of an aldoxime, preferably that of £-2-nitrobenzaldoxime or pyhdine-2-carboxaldoxime (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 oligonucleotide synthesis by the phosphotriester approach in solution, based on S-phenyl phosphorothioate intermediates; and van Boom and his co-workers in an approach to oligonucleotide synthesis, based on S-(4-methylphenyl) phosphorothioate intermediates (Wreesman, C. T. J. et al, Tetrahedron Left., 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 internucleotide linkages. In the present invention, the unblocking of S-(4-chlorophenyl)-protected phosphorothioates with the conjugate base of E-2-nitrobenzaldoxime proceeds smoothly and with no detectable internucleotide 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 oligonucleotide synthesis, another important consideration of the present invention, is that the removal of trityl, often a 5'-terminal DMTr, protecting group ('detritylation') should proceed without concomitant depurination, especially of any 6-N-acyl-2'-deoxyadenosine. residues. Silyl protecting groups may be removed by fluoride treatment, for example with a solution of an ammonium fluoride, for example a solution of trialkylamine trihydrogen fluoride or a solution of a tetraaikyl 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 oligonucleotides 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 internucleotide 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 oligonucleotides. Examples include -silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly{ethylene glycol) copolymers and polymers such as polyvinylacetate. Additionally, poly(acrylamide) 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(acrylamide) supports are amine-functionalised supports, especially those derived from supports prepared by copoiymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide 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 afkyl 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 weil known in the art for the solid phase synthesis of oligonucleotides, such as urethane, oxalyl, succinyl, and amino-derived linkers. In many embodiments when the substrate is bound to a poiy{acrylamide) 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-derivatised nucleoside; and b) reacting the linker-derivatised nucleoside with an amine-functionalised poiy(acryiamide) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example diisopropylethylamine (DIPEA) or N-methyimorphofine (NMM), or hydroxybenzotriazole; or c) reacting an amine-functionalised poly(acryiamide) support with a linker, preferably succinic anhydride, to form a linker-derivatised support; and d) reacting the linker-derivatised 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 hydroxybenzotriazole; then in either case, removing the 5'-protecting group, which is preferably a trityl or substituted trityl group. However, it will be recognised that it may be desired to retain the 5'-protecting 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 triesters 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-functionalised poly(acrylamide) support include those known in the art of peptide synthesis, see for example those coupling reagents disclosed by Wellings, D.A.; Atherton, E.; in Methods in Enzymoiogy, Pub!., Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N,N'-diisopropylcarbodiimide (DIC), and reagents that form active esters, particularly benzotriazole active esters in situ, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or benzotriazole-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP). An organic solvent such as N.N-dimethylformamide (DMF) or N-methylpyrrolidinone (NMP) is suitably employed for attaching the substrate to an amine-functionalised poly(acrylamide) support. The process for the synthesis of phosphorothioate triesters 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 oligonucleotides typically comprising 3 or more bases. The upper limit will depend on the length of the oligonucleotide it is desired to prepare. Often, oligonucleotides 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 oligonucleotide, 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-N-benzoyl-5'-0-(4,4'-dimethoxytrityl)-2'-deoxycytidine (DMT Cta) supported via a succinimide linker at the 3'-position 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 Cbz was treated as follows: i) with 100ml of 3% v/v dichloroacetic acid in dichloromethane (DCA/DCM) - Wait for 60 seconds, remove DCA/DCM ii) with 100ml of CH2CH2 - Wait for 60 seconds, remove CH2CH2 iii) 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-A/-benzoyl-2'-deoxycytidine (HOCbz-poly) Quantities Equivs Amount 822 DMTAbz (H) 5.0 0.84mmo! - 690mg HOCbz-poly 1.0 0.168mmol 232 CESP 10.0 1.68mmol-389mg 268.5/1/3 Activator* 10.0 1.68mmo! - 0.35ml * As a 1:1 solution in DCM :.use 0.69ml 6-N-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-H-phosphonate (DMTAbz(H), 690mg) was dried by co-evaporation (2x5ml) with anhydrous pyridine, and combined with 2g of HOCbz-poly (in a 100ml florentine). Pyridine was added such that all the DMTAbz(H) was in solution and a heterogeneous slurry was formed. This required 40ml of pyridine. 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/vo! mixture in anhydrous dichloromethane) of diphenylphosphorochloridate 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)su[phanyl phthalimlde (CESP) in 8mf of pyridine 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 pyridine (50ml) and DCM (200ml) and the resin dried under vacuum to produce 5'-(DMT)-Abz-0-P(=O)(SCH2CH2CN)-Cbz-(3'-O-polymer support) ("DMT-AC-pofy"). DMT-AC-po)y (1.4g@71 pmolg-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)-Ab2-O-P(=O)(SCH2CH2CN)-Cbz-(3,-O-polymer support) ("HO-AC-poly"). Equivs Amount 2235 DMTACAC(H) 71 umol/g HO-AC-poly 232 CESP 268.5/13 Activator 5.0 0.46mmol-1.03g 1.0 ' 13g@71 umol/g 10,0 0.923mmol-214mg 10.0 0.923mmol- 0.19ml ■ 1/1 in CH2CH2.-. 0.38ml A 5'-DMT protected nucleotide 3'-H-phosphonate having the base sequence 5-DMT-ACAC, with each internucleotide being protected by a beta-cyanoethylthio moiety (5'-DMT-ACAC-3'-H phosphonate) was prepared from the corresponding tetrameric oligonucleotide comprising a free 3'-hydroxy group. (5'-DMT-ACAC-3'-OH) as follows. Ammonium toluyl-H-phosphonate was dissolved in 50m! methanol and 5ml triethylamine. This mixture is evaporated to form a gum, and the gum redissolved in 100ml pyridine, together with 12.3g 5'-DMT-ACAC-3'-OH, and the pyridine evaporated. The residue was then redissolved in 50ml pyridine. The pyridine 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, 250mf of a solution of 10% v/v methanol in dichloromethane was added, and this was washed with 0.5M triethylammonium phosphate buffer. The organic layer was separated, diluted with 25ml methanol and the wash repeated. The organic (ayer was separated, evaporated from 100ml tofuene, and then evaporated from 200ml 50:50 by vol toiuene/pyridine mixture. The crude gum is re-dissolved 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 pyridine (2x5ml) and re-suspended in 40m/ 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 diphenylphosphorochlondate 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 pyridine) 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 (1ml 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) -Abz-O-P(=O)(SCH2CH2CN)-Cbz-O- P(=O)(SCH2CH2CN) -Abz-O-P(=O)(SCH2CH2CN)-Cbz-O-P(=O)(SCH2CH2CN)-Abz-O- P(=O)(SCH2CH2CN)-Cbz- (3'-O-polymer support) ("DMT-ACACAC-poly"). The cyanoethyl groups can be removed by treatment with anhydrous 1,8-diazabicyclo[5,4,0]undec-7-ene to yield phosphorothioate linkages, and the phosphorothioate oligonucleotide 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(acrylamide) resin produced by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide 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 (900m\; 0-100% ether). The resin was then dried overnight in a stream of nitrogen at 40°C. The resin produced had an amino functionalisation of 973 micromoles per gram ("Amino-PDMA resin"). . A solution of 4-N-benzoyl-5'-0-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-0-succinate ("DMT-Cbz-succinate", 3eqvs, 234 mmol), hydroxybenzotriazole (6 eqvs, 467 mmol) and DIC (diisopropyl carbodiimide;4 eqvs, 311 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 trimethylsilyl 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 amino groups on the resin had reacted with DMT-Cbz-succinate. 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 amino 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-Cbz-PDMA resin loaded to 573 mmol per gramme. A 5'-DMT-deoxycytidine dimer (protected on the internucleoside phsophorus by a p-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-Cbz-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 detritylation 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-Cbz-PDMA resin is then air dried and dried in-vacuo at 40°C overnight. HO-Cbz-PDMA resin (1 mmol; 1.48g), DMT-Cbz-H-phosphonate, (6 eq; 4.79g) were weighed into a 100ml florentine. 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 pyridine (4.36ml) was added. The reaction mixture was then stirred using a small (12x5mm) flea (at maximum stir rate on a Heidolph MR3001K stirrer hotplate). The neat activator, diphenyl phosphorochloridate {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; 1mmol), 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 pyridine containing 4-(N,N-dimethyl)aminopyridine (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-CbzCb2-PDMA, protected on the internucleoside linkage with a (3-cyanoethyl groap. Preparation of 5'-HQ-AbzCbzAbzCbzCbz-PDMA HO-Cbz-PDMA resin prepared as above (1mmol; 1.48g) and 5'-DMT-AbzCbzAbzCbz-H-phosphonate protected on the internucleoside linkages with p-cyanoethyl groups ( 1.2eq; 2.68g) were weighed into a 100ml florentine. Anhydrous DMF (60ml) was added and the resin allowed to swell for 5 mins at room temperature. Pyridine (5mmol; 0.4ml) was added followed by the coupling agent, diphenyl phosphorochloridite (2mmol; 0.41ml) which was added dropwise during 5 mins 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%. We Claim: 1. A process for the synthesis of a phosphorothioate triester 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 triester. 2. A process as claimed in 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 as claimed in claim 1 or claim 2, wherein the H-phosphonate is a 2'-deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide. 4. A process as claimed in claim 3, wherein the 2'-deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide comprises a 3' H-phosphonate function. 5. A process according to any preceding claim, wherein the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide. 6. A process as claimed in claim 5, wherein the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 5'-hydroxy group and is bonded to the solid support via the 3'-position. 7. A process according to any preceding claim, wherein the coupling agent is an alkyl phosphorochloridate or an aryl phosphorochloridates, and preferably diphenyl phosphorochloridate. 8. A process according to any preceding claim, wherein the sulphur transfer agent has the general chemical formula: 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 as claimed in 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, nitriles, amides and basic, nucleophilic solvents, and mixtures thereof. 10. A process as claimed in claim 9, wherein the organic solvent is selected from the group consisting of pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone and mixtures thereof. 11. A process as claimed in 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(acrylamide) supports. 12. A process as claimed in claim 11, wherein the solid support is an amine-functionalised support copolymer of acryloyl-sarcosine methyl ester, N3N-dimethylacrylamide and bis-acryloylethylenediamine. 13. A process according to any preceding claim, wherein the coupling agent is an alkyl phosphorochloridate or an aryl phosphorochloridates, and preferably diphenyl phosphorochloridate- 14. A process according to any preceding claim, wherein the sulphur transfer agent has the general chemical formula-: 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 as claimed in 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, nitriles, amides and basic, nucleophilic solvents, and mixtures thereof. 10. A process as claimed in claim 9, wherein the organic solvent is selected from the group consisting of pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone and mixtures thereof. 11. A process as claimed in 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(acrylamide) supports. 12. A process as claimed in claim 11, wherein the solid support is an amine-functionalised support copolymer of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine. 13. A process as claimed in 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 as claimed in any preceding claim, wherein hydroxy groups which are unreacted after a given coupling are capped by reaction with a capping agent. 15. A process as claimed in any preceding claim, wherein the phosphorothioate triester is an oligonucleotide, and the process comprises the additional steps of deprotecting the phosphorothioate triester, 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. Dated this 8th day of April, 2002. (JAYANTA PAL) OF REMFRY & SAGAR ATTORNEY FOR THE APPLICANT

Full Text

FORM 2
THE PATENTS ACT 1970 [39 OF 1970]
COMPLETE SPECIFICATION
[See Section 10]
"PROCESS FOR THE PREPARATION of PHOSPHOROTHIOATE TRIESTERS AND OLIGONUCLEOTIDES"
AVECIA LIMITED A British company of Hexagon House, Blackley, Manchester M9 8ZS, United Kingdom,
The following specification particularly describes the nature of the invention and the manner in which it is to be performed:-

The present invention concerns a method for the synthesis of phosphorothioate triesters, and particularly oligonucleotides."
In the past 15 years or so, enormous progress has been made iri the development
of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides
(RNA sequences) and their analogues, see 'Methods in Molecular Biology, Vol. 20,
Protocol for Oligonucleotides 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 monomeric phosphorarnidite 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 oligonucleotides have met a. number of crucial
needs in biology and biotechnology.
Following Zamecnnik and Stephenson's seminal discovery that a synthetic oligonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamechik, P.; Stephenson, M. Proc. Natl Acad. Sci. USA 1978, 75,. 280-284), the idea that synthetic oligonucleotides 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 oligonucleotides and their phosphbrothioate 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 (Oligonucleotides. Antisense Inhibitors'of Gene Expression, Cohen,,J.S.,. Ed., Macmillan, Basingstoke 1989 Moser, H. E.; Deryan, P.S:-. Science 1987, 238, 645-649), have profoundly affected the requirements for synthetic oligonucleotides. Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide 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), phosphorarnidite (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 oligonucleotides. While the phosphotriester approach has been.used most widely for

synthesis in solution, the phosphorarnidite 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 sulphurisation step at the end of the assembly of the desired molecule means that the process cannot readily be employed for the controlled production of chimeric nucleotides.. Furthermore, when an oxidation step isemployed, this is slow, and can cause concomitant degradation, and when a sulphurisation step is employed,'not only are . the reagents toxic, but the reaction is also slow. Additionally, the most common sulphurisation 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 triester 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 triester. 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 present invention relates to a supported H-phosphonate diester subjected to sulfur transfer with a sulfur agent to form a phosphorothioate ester. The prior art documents D1: BRILL W. K-D: discloses a process in which an H-phosphonate diester is reacted with a silylating agent in order to form a silyl phosphite. This silyl phosphite is then subjected to sulfur transfer with a sulfur transfer agent. BRILL W. K-D does not disclose a process in which an H phosphonate diester is subjected to sulfur transfer with a sulfur transfer agent. Accordingly, BRILL W. K-D does not destroy the novelty of the present claims.

Regarding the inventive step, the process of the present invention disclosed that a supported H-phosphonate diester is subjected to sulphur transfer with a sulphur transfer agent to form a phosphorothioate trimester. BRILL W. K.-D discloses the solid phase preaparation of a phosphorothioate trimester by a process comprinsing the formation of a solid-supported H-phosphonate diester, silylation to form a silylphosphite, and reaction with a source of a thioalkly group to form the phosphorothioate triester. WO 99 09041 A discloses a solution phase process the preparation of a phosphorothiate triester-by a process comprising the formation of an H-phosphonate diester and reaction with a sulphur transfer reagent to form the phosphorothioate triester. EP-A-0 739 899 discloses that certain solid supports can be employed in the spnthesis of oligonucleotides. The claims of the present invention involve an inventive step over the teaching of BRILL W. K-D, either alone, or in combination with either or both of WO 99 09041 A and EP-A-0 739 899. A skilled person consulting BRILL W. K-D would perceive that the core teaching of BRILL W. K-D is the thioaklylation of H-phosphonates via a silylphosphite intermediate. Thus, the very last change a skilled person would be motivated to make to the process of BRILL W. K-D would be to omit the silylation step. Indeed, it would be perverse of the skilled person to omit the core step of the teaching of BRILL W. K-D. The skilled person reading BRILL W. K-D without benefit of the knowledge of the present invention would not ignore the benefits taught by BRILL W. K-D for the silylation step (page 703 2nd paragraph, last 3 lines). Accordingly, the process of claim 1 would not be obvious to the skilled person in view of BRILL W. K-D. Further, the present invention would not be obvious to one of the ordinary skill in the art in view of the combination of BRILL W. K-D and WO 99 09041 A without the benefit of knowledge of the present invention. The clear teaching of BRILL W. K-D is that the syntheses of phosphorothioate trimesters by the H-phosphonate route on a solid phase requires the intermediate silylation step in order to yield the desired product. It is submitted that it would be extremely perverse of the skilled person to choose to omit such a key teaching from the process of BRILL W. K-D. While, WO 99 09041 A discloses a process in which an H-phosphonate diester is reacted directly with a sulfur transfer agent,

the process discloased is a solution phase process and hence does not anticiapate the claimed invention. The skilled person; would recognize that the conducting of reactiosn on solid supports places markedly different constrainst on the process chemistry, especially in terms of reaction kinetics, resulting from the lower freedom of movement of molecules attached to, a solid support, and also from surfaces effects caused by the presence-of the solid support. Accordingly, the skilled person would not expect simply to substitute the process chemistry of WO 99 G9041 A for the process of BRILL W. K-D. In fact, the skilled person aware of the teaching of BRILL W. K-D would, conclude that it was precisely the difficulties associated with conducting reactions on a solid support which resulted in the teaching of BRILL W. K-D concerning the importance of the silylation state. Additionally, EP-A-0 739 899 does not provide a teaching that would motivate the skilled person to ignore the clear teaching of BRILL W. K-D. Therefore, the skilled person, based on a fair teaching of BRILL W. K-D and WO 99 09041 A, and without the benefit of knowledge of the present invention, would simply not consider replacing the chemistry of BRILL W. K-D with the chemistry of WO 99 09041 A. Accordingly, the claims of the present application would not be obvious over BRILL W. K-D, WO 99 09041 A and EP-A-0 739 899, and accordingly involve an inventive step. 'The H-phosphonate employed in the process of the present-invention is ofterran H-phosphonate monoester, and advantageously a protected nucleoside or oligonucleotide. H-phosphonate,, preferably comprising a 5' or a/3' H-phosphonate function, particularly preferably a 3' H-phosphonate function. Preferred nucleosides are 2'-. " deoxyribonucleosides and ribonucleosides; - preferred oligonucleotides are oligodeoxyribonudeotides . and oligoribonucleotides. 2'-deoxyribonucleosides and oligodeoxyribonucleotides may comprise 2'-C-alkyI and 2'-C-alkenyl substituents.
When the H-phosphonate building block is a protected deoxyribonudeosrde, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide 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 dimethoxytrityl 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 deoxyribonucleoside,

When the substrate comprising a free hydroxy group is a ribonucleoside or an oligoribonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fIuorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and trialkyisilyl groups, often tri(C1-4-alkyl)silyl groups such as a tertiary butyl dimethyl silyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2'-O-alkyl, 2'-O-alkoxyalkyl or 2-'0-alkenyl derivative, commonly a C1-4 alkyl, C1-4 alkoxyC1- 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 saccharides, especially abasic nucleosides such as ribose and deoxyribose, and non-saccharide polyols, especially alkyl polyols, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. 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 ribonucleosides, 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 C1-4 alkyl- or C1-4 alkoxy-, benzoyl; pivaloyl; and amidine, particularly dialkylaminomethylene, preferably di(C1-4 -alkyl) aminomethylene such as dimethyl or dibutyl aminomethylene. Guanine (G) may be protected by a phenyl group, including substituted phenyl, for example 2,5-dichlorophenyl 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 04 by a phenyl group, including substituted phenyl, for exampfe 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl, alkyl or alkoxy substituted benzoyl, such as C1-4 alkyl- or C1-4 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 triester produced by the process of the present invention. When the phosphorothioate triester produced is a protected nucleoside or oligonucleotide 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 oligonucleotide 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 oligonucleotide sequence. The method may then proceed with steps to remove the protecting groups from the internucleotide 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 nucleophiiic acylation catalyst such as 4-(N,N-dimethyl)aminopyridine) 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'-O-(4,4'-dimethoxytrityl)-2'-deoxyribonucleoside 3-H-phosphonate or a protected oligodeoxyribonucleotide 3'-H-phosphonate and a substrate supported on a solid support with a free hydroxy function, most commonly a 2'-deoxyribonucleoside or oligodeoxyribonucleotide, 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 aryi chloroformates, alkyl and aryl chlorosulphites and alkyl and aryl phosphorochloridates, 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 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 C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted phenyl chlorosulphites. When substituted, from 1 to 3 substituents are often present, particufarly in the case of alkyl and halo substituents.
Examples of suitable alkyl phosphorochloridates which may be employed include di(C1 to C6 alkyl) phosphorochloridates. Examples of aryl phosphorochloridates which may be employed include substituted and unsubstituted diphenyl phosphorochloridates, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted diphenyl phosphorochloridates. 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 (benzotriazo-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 carbodiimides that may be employed include alkyl carbodiimides, especially (C1 to C6 alkyl) carbodiimides, such as 1,3 dicyclohexyl carbodiimide, 1,3 diisopropyl carbodiimide, 1 -tert.-butyl-3-ethyl carbodiimide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. One or more of the alkyl groups may be substituted, for example by an alkyl amino moiety. An example of a substituted aikyl carbodiimide is 1-(3-dimethyfaminopropyl)-3-ethyI carbodiimide.
Preferred coupling agents are diaryl phosphorochloridates, particularly those having the formula (ArO)2POCI wherein Ar is preferably phenyl, 2-chiorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.
The sulphur transfer agents employed in the process of the present invention introduce a protected thio moiety into the linkage, thereby forming a phosphorothioate triester. The phosphorthioate triesters 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 oligonucleotide, a phosphorothioate analogue or a mixed oligonucleotide/oligonucleotide phosphorothioate is required. Sulphur transfer agents employed in the process of the present invention often have the general, chemical formula:

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 morpholines such as morpho!ine-3,5-dione; imides such as phthalimides, succinimides and maieimides; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles; and triazoles.
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 aryi 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-alkyfphenyl, commonly 4-(C1-4 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-(aryisulphanyl)phthalimide (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 C1-4 alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-position with an electron-withdrawing s'ubstituent such as 2-(4-nitrophenyl)ethyl and 2-cyanoethyl groups. Examples of suitable alkenyl groups are allyl, 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-cyanoethyf)-sulphanyl]morphofine-3,5-drone or a corresponding reagent such as 3-(phthalimidosulphanyl)propanonitrile.
A suitable temperature for carrying out the coupling reaction and sulphur transfer is in the range of from approximately -55°C to about 40oC, 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 alky],
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

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

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 dimethoxytrityl group. When Z is a protecting group, the protecting group is preferably a trityl group, particularly a dimethoxytrityl group, or an acyl group, preferably a levulinoyl group.
Organic bases which may be represented by B include nucleobases, such as natural and unnatural nucleobases, and especially, purines, such as hypoxanthine, and particularly A and G, and pyrimidines, 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 C1-4 alkenyl group, especially ally!, propargyl or crotyl group. When R' represents alkyl, the alkyl is preferably a C1-4 alkyl group. When R' represents substituted alkyl, the substituted alkyl group includes alkoxyalkyl groups, especially C1-4 alkyoxyC1-4 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-methoxypiperidine-4-yl (Fpmp) or a trialkylsilyl groups, often a tri(C1-4 -alkyl)silylgroup 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 C1-4 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-nitrophenyl)ethyl or a 2-cyanoethyl group; a C1-4 alkenyl group, preferably an allyl and crotyl group; or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially 4-haIophenyl group or a 4-alkylphenyl, commonly a 4-(C1-4 a!kyl)phenyl group, and most preferably a 4-chlorophenyl or a p-tolyi group.
M+ preferably represents a trialkyl ammonium ion, such as a tri(C1-4-aikylammonium) ion, and preferably a triethylammonium ion or a cation of a cyclic base such as 1,5-diazabicyio[4.3.0]non-5-ene (DBN) or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). , '
n may be 0 or 1 up to any number convenient for the synthesis of the desired oligonucleotide, 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 oligonucleotide 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 acetorittrlle, amides, such as dimethylformamide and N-methylpyrollidinone, and basic, nucleophilic solvents such as pyridine. Preferred solvents are pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone 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 levulinoyl-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 oligonucleotides 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 oligonucleotides and their phosphorothioate analogues.
When deprotecting the desired product once this has been produced, protecting groups on the phosphorus which produce phosphorothioate triester 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-diazabicylo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or thethylamine.
Phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the base residues can be removed by oximate treatment, for example with the conjugate base of an aldoxime, preferably that of £-2-nitrobenzaldoxime or pyhdine-2-carboxaldoxime (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 oligonucleotide synthesis by the phosphotriester approach in solution, based on S-phenyl phosphorothioate intermediates; and van Boom and his co-workers in an approach to oligonucleotide synthesis, based on S-(4-methylphenyl) phosphorothioate intermediates (Wreesman, C. T. J. et al, Tetrahedron Left., 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 internucleotide linkages. In the present invention, the unblocking of S-(4-chlorophenyl)-protected phosphorothioates with the conjugate base of E-2-nitrobenzaldoxime proceeds smoothly and with no detectable internucleotide 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 oligonucleotide synthesis, another important consideration of the present invention, is that the removal of trityl, often a 5'-terminal DMTr, protecting group ('detritylation') should proceed without concomitant depurination, especially of any 6-N-acyl-2'-deoxyadenosine. residues.
Silyl protecting groups may be removed by fluoride treatment, for example with a solution of an ammonium fluoride, for example a solution of trialkylamine trihydrogen fluoride or a solution of a tetraaikyl 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 oligonucleotides 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 internucleotide 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 oligonucleotides. Examples include -silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly{ethylene glycol) copolymers and polymers such as polyvinylacetate. Additionally, poly(acrylamide) 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(acrylamide) supports are amine-functionalised supports, especially those derived from supports prepared by copoiymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide 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 afkyl 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 weil known in the art for the solid phase synthesis of oligonucleotides, such as urethane, oxalyl, succinyl, and amino-derived linkers.
In many embodiments when the substrate is bound to a poiy{acrylamide) 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-derivatised nucleoside; and

b) reacting the linker-derivatised nucleoside with an amine-functionalised poiy(acryiamide) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example diisopropylethylamine (DIPEA) or N-methyimorphofine (NMM), or hydroxybenzotriazole; or
c) reacting an amine-functionalised poly(acryiamide) support with a linker, preferably succinic anhydride, to form a linker-derivatised support; and
d) reacting the linker-derivatised 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 hydroxybenzotriazole;
then in either case, removing the 5'-protecting group, which is preferably a trityl or substituted trityl group. However, it will be recognised that it may be desired to retain the 5'-protecting 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 triesters 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-functionalised poly(acrylamide) support include those known in the art of peptide synthesis, see for example those coupling reagents disclosed by Wellings, D.A.; Atherton, E.; in Methods in Enzymoiogy, Pub!., Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N,N'-diisopropylcarbodiimide (DIC), and reagents that form active esters, particularly benzotriazole active esters in situ, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or benzotriazole-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP).
An organic solvent such as N.N-dimethylformamide (DMF) or N-methylpyrrolidinone (NMP) is suitably employed for attaching the substrate to an amine-functionalised poly(acrylamide) support.
The process for the synthesis of phosphorothioate triesters 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 oligonucleotides typically comprising 3 or more bases. The upper limit will depend on the length of the oligonucleotide it is desired to prepare. Often, oligonucleotides 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 oligonucleotide, 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-N-benzoyl-5'-0-(4,4'-dimethoxytrityl)-2'-deoxycytidine (DMT Cta) supported via a succinimide linker at the 3'-position 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 Cbz was treated as follows:
i) with 100ml of 3% v/v dichloroacetic acid in dichloromethane (DCA/DCM) - Wait for
60 seconds, remove DCA/DCM
ii) with 100ml of CH2CH2 - Wait for 60 seconds, remove CH2CH2
iii) 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-A/-benzoyl-2'-deoxycytidine (HOCbz-poly)
Quantities Equivs Amount
822 DMTAbz (H) 5.0 0.84mmo! - 690mg
HOCbz-poly 1.0 0.168mmol
232 CESP 10.0 1.68mmol-389mg
268.5/1/3 Activator* 10.0 1.68mmo! - 0.35ml
* As a 1:1 solution in DCM :.use 0.69ml

6-N-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-H-phosphonate (DMTAbz(H), 690mg) was dried by co-evaporation (2x5ml) with anhydrous pyridine, and combined with 2g of HOCbz-poly (in a 100ml florentine). Pyridine was added such that all the DMTAbz(H) was in solution and a heterogeneous slurry was formed. This required 40ml of pyridine. 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/vo! mixture in anhydrous dichloromethane) of diphenylphosphorochloridate 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)su[phanyl phthalimlde (CESP) in 8mf of pyridine 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 pyridine (50ml) and DCM (200ml) and the resin dried under vacuum to produce 5'-(DMT)-Abz-0-P(=O)(SCH2CH2CN)-Cbz-(3'-O-polymer support) ("DMT-AC-pofy").
DMT-AC-po)y (1.4g@71 pmolg-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)-Ab2-O-P(=O)(SCH2CH2CN)-Cbz-(3,-O-polymer support) ("HO-AC-poly").
Equivs Amount
2235 DMTACAC(H)
71 umol/g HO-AC-poly
232 CESP
268.5/13 Activator
5.0 0.46mmol-1.03g
1.0 ' 13g@71 umol/g
10,0 0.923mmol-214mg
10.0 *0.923mmol- 0.19ml ■
*1/1 in CH2CH2.-. 0.38ml
A 5'-DMT protected nucleotide 3'-H-phosphonate having the base sequence 5-DMT-ACAC, with each internucleotide being protected by a beta-cyanoethylthio moiety (5'-DMT-ACAC-3'-H phosphonate) was prepared from the corresponding tetrameric oligonucleotide comprising a free 3'-hydroxy group. (5'-DMT-ACAC-3'-OH) as follows. Ammonium toluyl-H-phosphonate was dissolved in 50m! methanol and 5ml triethylamine. This mixture is evaporated to form a gum, and the gum redissolved in 100ml pyridine, together with 12.3g 5'-DMT-ACAC-3'-OH, and the pyridine evaporated. The residue was then redissolved in 50ml pyridine. The pyridine 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, 250mf of a solution of 10% v/v methanol in dichloromethane was added, and this was washed with 0.5M triethylammonium phosphate buffer. The organic layer was separated, diluted with 25ml methanol and the

wash repeated. The organic (ayer was separated, evaporated from 100ml tofuene, and then evaporated from 200ml 50:50 by vol toiuene/pyridine mixture. The crude gum is re-dissolved 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 pyridine (2x5ml) and re-suspended in 40m/ 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 diphenylphosphorochlondate 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 pyridine) 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 (1ml 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) -Abz-O-P(=O)(SCH2CH2CN)-Cbz-O-
P(=O)(SCH2CH2CN) -Abz-O-P(=O)(SCH2CH2CN)-Cbz-O-P(=O)(SCH2CH2CN)-Abz-O-
P(=O)(SCH2CH2CN)-Cbz- (3'-O-polymer support) ("DMT-ACACAC-poly").
The cyanoethyl groups can be removed by treatment with anhydrous 1,8-diazabicyclo[5,4,0]undec-7-ene to yield phosphorothioate linkages, and the phosphorothioate oligonucleotide 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(acrylamide) resin produced by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide 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 (900m\; 0-100% ether). The resin was then dried overnight in a stream of nitrogen at 40°C. The resin produced had an amino functionalisation of 973 micromoles per gram ("Amino-PDMA resin"). .
A solution of 4-N-benzoyl-5'-0-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-0-succinate ("DMT-Cbz-succinate", 3eqvs, 234 mmol), hydroxybenzotriazole (6 eqvs, 467 mmol) and DIC (diisopropyl carbodiimide;4 eqvs, 311 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 trimethylsilyl 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 amino groups on the resin had reacted with DMT-Cbz-succinate. 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 amino 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-Cbz-PDMA resin loaded to 573 mmol per gramme.
A 5'-DMT-deoxycytidine dimer (protected on the internucleoside phsophorus by a p-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-Cbz-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 detritylation 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-Cbz-PDMA resin is then air dried and dried in-vacuo at 40°C overnight.
HO-Cbz-PDMA resin (1 mmol; 1.48g), DMT-Cbz-H-phosphonate, (6 eq; 4.79g) were weighed into a 100ml florentine. 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 pyridine (4.36ml) was added. The reaction mixture was then stirred using a small (12x5mm) flea (at maximum stir rate on a Heidolph MR3001K stirrer hotplate). The neat activator, diphenyl phosphorochloridate {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; 1mmol), 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 pyridine containing 4-(N,N-dimethyl)aminopyridine (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-CbzCb2-PDMA, protected on the internucleoside linkage with a (3-cyanoethyl groap.
Preparation of 5'-HQ-AbzCbzAbzCbzCbz-PDMA
HO-Cbz-PDMA resin prepared as above (1mmol; 1.48g) and 5'-DMT-AbzCbzAbzCbz-H-phosphonate protected on the internucleoside linkages with p-cyanoethyl groups ( 1.2eq; 2.68g) were weighed into a 100ml florentine. Anhydrous DMF (60ml) was added and the resin allowed to swell for 5 mins at room temperature. Pyridine (5mmol; 0.4ml) was added followed by the coupling agent, diphenyl phosphorochloridite (2mmol; 0.41ml) which was added dropwise during 5 mins 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%.

We Claim:
1. A process for the synthesis of a phosphorothioate triester 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 triester.
2. A process as claimed in 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 as claimed in claim 1 or claim 2, wherein the H-phosphonate is a 2'-deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide.
4. A process as claimed in claim 3, wherein the 2'-deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide comprises a 3' H-phosphonate function.
5. A process according to any preceding claim, wherein the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide.
6. A process as claimed in claim 5, wherein the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 5'-hydroxy group and is bonded to the solid support via the 3'-position.

7. A process according to any preceding claim, wherein the coupling
agent is an alkyl phosphorochloridate or an aryl phosphorochloridates,
and preferably diphenyl phosphorochloridate.
8. A process according to any preceding claim, wherein the sulphur
transfer agent has the general chemical formula:

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 as claimed in 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, nitriles, amides and basic, nucleophilic solvents, and mixtures thereof.
10. A process as claimed in claim 9, wherein the organic solvent is selected from the group consisting of pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone and mixtures thereof.
11. A process as claimed in 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(acrylamide) supports.
12. A process as claimed in claim 11, wherein the solid support is an amine-functionalised support copolymer of acryloyl-sarcosine methyl ester, N3N-dimethylacrylamide and bis-acryloylethylenediamine.

13. A process according to any preceding claim, wherein the coupling agent is an alkyl phosphorochloridate or an aryl phosphorochloridates, and preferably diphenyl phosphorochloridate-
14. A process according to any preceding claim, wherein the sulphur transfer agent has the general chemical formula-:

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 as claimed in 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, nitriles, amides and basic, nucleophilic solvents, and mixtures thereof.
10. A process as claimed in claim 9, wherein the organic solvent is selected from the group consisting of pyridine, dichloromethane, dimethylformamide, N-methylpyrollidinone and mixtures thereof.
11. A process as claimed in 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(acrylamide) supports.
12. A process as claimed in claim 11, wherein the solid support is an amine-functionalised support copolymer of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine.

13. A process as claimed in 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 as claimed in any preceding claim, wherein hydroxy groups which are unreacted after a given coupling are capped by reaction with a capping agent.
15. A process as claimed in any preceding claim, wherein the phosphorothioate triester is an oligonucleotide, and the process comprises the additional steps of deprotecting the phosphorothioate triester, 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.
Dated this 8th day of April, 2002.
(JAYANTA PAL)
OF REMFRY & SAGAR
ATTORNEY FOR THE APPLICANT

Documents:

in-pct-2002-00431-mum-cancelled pages(30-11-2004).pdf

in-pct-2002-00431-mum-claims(granted)-(30-11-2004).doc

in-pct-2002-00431-mum-claims(granted)-(30-11-2004).pdf

in-pct-2002-00431-mum-correspondence(26-05-2005).pdf

in-pct-2002-00431-mum-correspondence(ipo)-(09-01-2008).pdf

in-pct-2002-00431-mum-form 1a(08-04-2002).pdf

in-pct-2002-00431-mum-form 2(granted)-(30-11-2004).doc

in-pct-2002-00431-mum-form 2(granted)-(30-11-2004).pdf

in-pct-2002-00431-mum-form 3(08-04-2002).pdf

in-pct-2002-00431-mum-form 3(16-12-2004).pdf

in-pct-2002-00431-mum-form 5(08-04-2002).pdf

in-pct-2002-00431-mum-form-pct-isa-210(30-11-2004).pdf

in-pct-2002-00431-mum-power of authority(30-11-2004).pdf

« Previous Patent

Next Patent »

Patent Number

213613

Indian Patent Application Number

IN/PCT/2002/00431/MUM

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

09-Jan-2008

Date of Filing

08-Apr-2002

Name of Patentee

AVECIA LIMITED

Applicant Address

HEXAGON HOUSE, BLACKLEY, MANCHESTER M9 8ZS, UNITED KINGDOM

Inventors:

#	Inventor's Name	Inventor's Address
1	MARK EDWARD DOUGLAS	PO BOX 42, HEXAGON HOUSE, BLACKLEY, MANCHESTER M9 8ZS, UNITED KINGDOM
2	JAMES EN MELLOR	EARLS ROAD, GRANGEMOUTH, STIRLINGSHIRE, FK3 8XG, UNITED KINGDOM
3	JOHN DAVID MOODY	EARLS ROAD, GRANGEMOUTH, STIRLINGSHIRE, FK3 8XG, UNITED KINGDOM
4	KEVIN GERARD SCOTT	4 TENBURY CLOSE, GREAT SANKEY, WARRINGTON, WA5-3RN, UNITED KINGDOM

PCT International Classification Number

C07H 1/00

PCT International Application Number

PCT/GB00/03912

PCT International Filing date

2000-10-12

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	9924285.1	1999-10-14	GB