MXPA00001408A - Solution phase synthesis of oligonucleotides - Google Patents

Abstract

A process for the synthesis in solution phase of a phosphorothioate triester is provided. The process comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent to form an H-phosphonate diester. The H-phosphonate diester is oxidised in situ with a sulfur transfer agent to produce the phosphorothioate triester. Preferably, the H-phosphonate and alcohol are protected nucleosides or oligonucleotides. Oligonucleotide H-phosphonates which can be used in the formation of phosphorothioate triesters are also provided.

Description

SYNTHESIS IN SOLUTION OF SOLUTION OF OLIGONÜCLEOTIDOS Field of the Invention The present invention provides a method of synthesis of oligonucleotides and phosphorothioates of oligonucleotides in solution based on the binding of the H-phosphonate and the transfer of sulfur in situ, carried out at low temperature. The invention further provides a process for the gradual synthesis of oligonucleotides and phosphorothioates of oligonucleotides in which a nucleoside residue is aggregated at a time, and block synthesis of the oligonucleotides and phosphorothioates of oligonucleotides in which two or more Nucleotide residues are aggregated at the same time.

Background of the Invention About 15 years ago, enormous progress had been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues "Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogs ", Agrawal, S. Ed., Humana ßf.032749 Press, Totowa, 1993. The majority of the work has been carried out on a micromolar or even smaller scale, and the automated solid phase synthesis involving blocks of construction of idyllic monophonic phosphor Beacage, SL; Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862 has proved that it will be the most convenient approach. Actually, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared with commercially available synthesizers. These synthetic oligonucleotides have to satisfy several crucial needs in biology and biotechnology. Following the pivotal discovery of Zamecnik and Stephenson that a synthetic oligonucleotide could selectively inhibit the expression of genes in the sarcoma virus of 'Rous, (Zamecnik, P .; Stephenson, M. Proc. Nati. Acad. Sci. USA. 1978, 75, 280-284), the idea that synthetic oligonucleotides or their analogs could find good application in chemotherapy has attracted considerable attention in both academic and industrial laboratories. For example, the possible use of oligonucleotides and their phosphorothioate analogs in chemotherapy has been enhanced in the report by Gura, T. Science, 1995, 270, 575-577. The so-called antisense and antigen approaches to chemotherapy (Oligonucleotides, Antisense Inhibitors of Gene Expression, Cohen, JS, Ed., Macmillan, Basingstoke 1989 Moser, HE, Dervan, PB Science 1987, 238, 645-649), have profoundly affected the requirements of the synthetic oligonucleotides. Although milligram quantities have generally been sufficient for molecular biology purposes, larger quantities of one gram to greater than 100 grams are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are new 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, it will probably require a number of kilograms or more of multikilograms of a specific sequence or sequences. A few years ago, a great deal of work was done on the upward scaling of oligonucleotide synthesis. Virtually all of this work has been involved in building larger and larger synthesizers and the same chemistry of the phosphoramidite on a solid support. The applicant does not know of any recent improvement in the methodology of the phosphotriester approach to the synthesis of oligonucleotides in solution, which makes synthetic work on a large scale and even more moderate than solid phase synthesis more suitable. The main advantages that the synthesis in solid phase has on the synthesis in solution are: (i) that it is much faster, (ii) that the yields of the union are generally higher, (iii) it is easily automated and (iv) it is completely flexible with respect to the sequence. Therefore, solid phase synthesis is particularly useful if relatively small amounts of a large number of oligonucleotide sequences are required, say, for combination purposes. However, if a particular sequence of moderate size has been identified and approved as a drug and quantities of kilograms are required, speed and flexibility become relatively unimportant, and solution synthesis is likely to be highly advantageous. Solution synthesis also has an advantage over solid phase synthesis where block binding (ie the addition of two or more nucleotide residues at the same time) is more feasible and scaling up at any level is unlikely to present a problem. It is much easier and certainly much cheaper to increase the size of a reaction vessel than it is to produce larger and larger automatic synthesizers. In the past, the synthesis of oligonucleotides in solution has been carried out mainly by the conventional phosphotriester approach that was developed in the 1970s (Reese, C. B., Tetrahedron 1978, 34, 3143-3179; Kaplan, B. E .; Itakura, L. in "Synthesis and Applications of DNA and RNA", Narang, S.A., Ed., Academic Press, Orlando, 1987, pp. 9-45). This approach can also be used in solid phase synthesis but the binding reactions are somewhat faster and the binding yields are somewhat larger when phosphoramidite monomers are used. This is because the automated solid phase synthesis has been based largely on the use of the phosphoramidite building blocks; perhaps also because workers who require relatively large amounts of the synthetic oligonucleotides have decided to attempt the upward scaling of phosphoramidite-based solid phase synthesis. Three main methods, especially the phosphotriester approaches (Reese, Tetrahedron, 1978), phosphoramidite (Beaucage, SL in Methods in Molecular Biology, Vol. 20, Agra al, S. Ed., Humana Press, Totowa, 1993, pp. 33 -61) and H-phosphonate (Froehler, BC in Methods in Molecular Biology, Vol 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp. 63-80, see also W094 / 15946 and Dreef, CE in Rec. Trav. Chim Pays-Bas, 1987, 106, p512) have proved that they will be effective for the chemical synthesis of oligonucleotides. Although the phosphotriester approach has been used more widely for solution synthesis, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid-phase synthesis. Two different synthetic strategies have been applied to the phosphotriester approach in solution. Perhaps the most widely used strategy for the synthesis of oligodeoxyribonucleotides in solution involves a binding reaction between a protected 3'- (2-chlorophenyl) phosphate or protected oligonucleotide (Chattopadhyaya, JB; Reese, CB Nucleic Acids Res., 1980 , 8, 2039-2054) and a nucleoside or oligonucleotide protected with a free 5'-hydroxy function to give a phosphotriester. A binding agent such as 1- (mesitylene-2-sulfonyl) -3-nitro-1,2,4-H-triazole (MSTN) (Reese, CB; Titmas, R.C; Yau, L. Tetrahedron Lett ., 1978, 2727-2730) is required. The binding is then carried out in the same manner between a 3'-S- (2-cyanoethyl or, for example, 4-nitrobenzyl) phosphorothioate nucleoside or protected oligonucleotide (Liu, X., CBJ Chem. Soc., Perkin Trans. 1, 1995, 1685-1695) and a protected nucleoside or oligonucleotide with a free 5'-hydroxy function. The main disadvantages of this conventional phosphotriester approach are that some 5'-concomitant sulfonation of the second component occurs (Reese, CB, Zhang, P.-ZJ Chem. Soc., Perkin Trans. 1, 1995, 2291-2301) and that the binding reactions generally proceeded relatively slowly. The lateral sulfonation reaction both leads to lower yields and prevents the purification of the desired products. The second strategy for the synthesis of oligodeoxyribonucleotides in solution involves the use of a bifunctional reagent derived from an aryl phosphorodichloridate (usually 2-chlorophenyl) and two molar equivalents of an additive such as 1-hydroxybenzotriazole (van der Marel, et al, Tetrahedron Lett., 1981, 22, 3887-3890). A bifunctional reagent, derived from 2,5-dichlorophenyl phosphorodichloridothioate (Scheme Ib), has been used in a similar manner (Kemal, 0 et al, J. Chem. Soc., Chem. Commun., 1983, 591-593) in the preparation of oligonucleotide phosphorothioates. The main disadvantages of the second strategy result directly from the involvement of a bifunctional reagent. Accordingly, there is the possibility that symmetrical bonding products are formed, and the presence of small amounts of moisture can lead to a significant decrease in bond yields. It is an object of certain aspects of the present invention to provide a new binding method for the synthesis of oligonucleotides in solution which in many embodiments (a) is extremely efficient and does not lead to side reactions, (b) proceeds relatively quickly, and (c) is equally suitable for the preparation of oligonucleotides, their phosphorothioate analogs and chimeric oligonucleotides containing internucleotide linkages of both phosphodiester and phosphorothioate. According to a first aspect of the present invention, there is provided a process for the preparation of a phosphorothioate triester which comprises the in-solution binding of an H-phosphonate with an alcohol in the presence of a binding agent so wherein an H-phosphonate diester is formed and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester.

The H-phosphonate used in the process of the present invention is advantageously a protected nucleoside or oligonucleotide H-phosphonate, preferably comprising a 5 'or 3' H-phosphonate function, particularly preferably a 3 'H function. -phosphonate. The preferred nucleosides are 2'-deoxyribonucleosides and ribonucleosides; the preferred oligonucleotides are oligodeoxyribonucleotides and oligoribonucleotides. When the H-phosphonate building block is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 3'H-phosphonate function, the 5'-hydroxy function is soldly protected by a suitable protection group. Examples of such suitable protecting groups include the acid labile protecting groups, particularly the substituted triphenyl and triflyl groups such as the dimethoxytrityl and 9-phenylxanten-9-yl groups; and basic labile protective groups such as FMOC. When the H-phosphonate building block is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 5'H-phosphonate function, the 3'-hydroxy function is advantageously protected by a suitable protecting group. Suitable protecting groups include those described above for the protection of the 5'-hydroxy functions of the 3'-H-phosphonate and acyl building blocks, such as the substituted levulinoyl and levulinoyl groups. When the H-phosphonate is a protected ribonucleoside or a protected oligoribonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, for example an acid labile acetal protecting group, particularly 1- (2-fluorophenyl) -4 -methoxypiperidin-4-yl (Fpmp); and trialkylsilyl groups, frequently tri (C? 4-alkyl) silyl groups such as the tertiary dimethylsilyl butyl group. Alternatively, the ribonucleoside or oligoribonucleotide can be a 2'-0-alkyl, 2'-O-alkoxyalkyl or 2'-O-alkenyl derivative, commonly an alkyl derivative with C? -4,, C? _44 alcoalkylC? ?44alkyl or alkenyl, in this case, the 2 'position does not need additional protection. Other phosphonates which can be used in the process according to the present invention are derivatives of other polyfunctional alcohols, especially alkyl alcohols and preferably diols or triols. Examples of alkyl diols include ethan-1,2-diol, and low molecular weight poly (ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl thiols include glycerol and butan triols. Commonly, only a single H-phosphonate function will be present, the remaining hydroxy groups are protected by suitable protecting groups, such as those described herein above for protection at the 5 'or 2' positions of the ribonucleosides. The alcohol employed in the process of the present invention is commonly a protected nucleoside or oligonucleotide comprising a free hydroxy group, preferably a free 3 'or 5' hydroxy group, and particularly preferably a 5 'hydroxy group. When the alcohol is a protected nucleoside or a protected oligonucleotide, the preferred nucleosides are the deoxyribonucleotides and the ribonucleosides and the preferred oligonucleotides are the oligodeoxyribonucleotides and the oligoribonucleotides. When the alcohol is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 5'-hydroxy group, the 3'-hydroxy function is advantageously protected by a suitable protecting group. Examples of such protecting groups include acyl groups, commonly comprising up to 16 carbon atoms, such as those derived from the keto-keto acids, such as the levuniloyl groups and the substituted levulinoyl groups. Substituted levulinoyl groups include particularly 5-halo-levulinoyl, such as the 5, 5, 5-trifluorolevulinoyl and benzoylpropionyl groups. Other such protecting groups include the fatty alkanoyl groups, including particularly linear or branched C6-i6 alkanoyl groups, such as the lauroyl groups; substituted benzoyl and benzoyl groups, such as alkyl substituted benzoyl groups, commonly C? -alkyl, and halo, commonly chloro or fluoro; and silyl ethers, such as alkyl, commonly C 4 alkyl, and aryl ethers, commonly phenyl, silyl, particularly tertiary dimethyl silyl butyl and diphenyl silyl butyl tertiary groups. When the alcohol is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide comprising a 3'-hydroxy group, the 5'-hydroxy function is advantageously protected by a suitable protecting group. Suitable protecting groups are those described above for the protection of the 5'-hydroxy group of the 3'-H phosphonates of deoxyribonucleosides, ribonucleosides, oligodeoxyribonucleotides and oligoribonucleotides. When the alcohol 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-fluorophenyl) -4-methoxypiperidin-4-yl (Fpmp); and trialkylsilyl groups, frequently tri (C? 4-alkyl) silyl groups such as the tertiary dimethyl silyl butyl group. Alternatively, the ribonucleoside or oligoribonucleotide can be a 2'-0-alkyl, 2'-O-alkoxyalkyl or 2'-O-alkenyl derivative, commonly an alkyl derivative with C? -4, C? _4 alkoxyC? _4 alkyl or alkenyl, in such a case, the 2 'position does not need additional protection. Other alcohols which can be used in the process according to the present invention are polyols other than saccharides, especially alkyl polyols, and preferably diols or triols. Examples of the alkyl diols include ethane-1,2-diol, and poly. { ethylene glycols) of low molecular weight, such as those having a molecular weight of up to 400. Examples of the alkyl triols include glycerol and butan triols. Commonly, only one free hydroxy group will be present, the remaining hydroxy groups are protected by suitable protecting groups, such as those described herein above for protection at the 5 'or 2' positions of the ribonucleosides. However, more than one free hydroxy group may be present if desired to form identical linkages on more than one hydroxy group. When the H-phosphonate and the alcohol are both protected nucleosides or oligonucleotides, the invention provides an improved method for block synthesis and gradual solution in oligodeoxyribonucleotides, oligoribonucleotides and analogs thereof, based on H-linked reactions. -phosphonate. According to a preferred aspect of the present invention, the nucleosides or oligonucleotides protected with a 3'-terminal H-phosphonate function and the nucleosides or oligonucleotides protected with a 5'-terminal hydroxy function are linked in the presence of an suitable linkage to form an intermediate of dinucleoside H-phosphonate or protected oligonucleotide, wherein the intermediates undergo trer of the sulfur in situ in the presence of a suitable sulfur trer agent. In addition to the presence of hydroxy protecting groups, the bases present in the nucleosides / nucleotides used in the present invention are also preferably protected where necessary by suitable protecting groups. The protective groups employed are those known in the art to protect such bases. For example, A and / or C can be protected by benzoyl, including substituted benzoyl, for example alkyl- or alkoxy-, frequently C? _4 alkyl- or C? _4alkoxy-, benzoyl; pivaloyl; and amidine, particularly dialkylaminomethylene, preferably di (C? 4-alkyl) aminomethylene such as dimethyl or dibutyl aminomethylene. G can be protected by a phenyl group, including substituted phenyl, for example 2,5-dichlorophenyl and also by an isobutyryl group. T and U generally do not require protection, but in certain embodiments they can be advantageously protected, for example in 04 by a phenyl group, including substituted phenyl, for example 2,4-dimethylphenyl or in N 3 by a pivaloyloxymethyl, benzoyl, benzoyl substituted with alkyl or alkoxy, such as C? _4 alkyl- or C? _4 alkoxybenzoyl. When the alcohol and / or the H-phosphonate is a protected nucleoside or oligonucleotide having protected hydroxy groups, one of these protecting groups can be removed after carrying out the process of the first invention. Commonly, the protective group removed is that on the 3'-hydroxy function. After the protecting group has been removed, the oligonucleotide thus formed can be converted to an H-phosphonate and then proceed through further stepwise steps or the block binding and sulfur transfers according to the process of the present invention in the synthesis of a desired oligonucleotide sequence. The method can then proceed with steps to remove the protecting groups of the internucleotide bonds, the 3 'and 5' -hydroxy groups and the bases. A similar methodology can be applied to the binding of the 5 'H-phosphonates, where the protective group removed is that on the 5' hydroxy function. In a particularly preferred embodiment, the invention provides a method comprising the binding of the 3'-H-phosphonate of the 5'-0- (4, '-dimethoxytrityl) -2"-deoxyribonucleoside or a 3'-H-phosphonate of protected oligodeoxyribonucleotide and a component with a free 5'-hydroxy function in the presence of a suitable binding partner and subsequently the transfer of the sulfur in situ in the presence of a suitable sulfur transfer agent In the process of the present invention, any of the binding agents and sulfur transfer agents available in the prior art can be used Examples of suitable bonding agents include acid alkyl and aryl chlorides, alloy and aren sulfonyl chlorides, alkyl and aryl chloroformates, chlorosulfites alkyl and aryl and alkyl and aryl phosphorochloridates Examples of suitable alkyl acid chlorides which may be employed include alkanoyl chlorides n C2 to C, particularly pivaloyl chloride. Examples of the acid aryl chlorides which may be employed include substituted and unsubstituted benzoyl chlorides, such as alkoxy with C ?4, halo, particularly fluoro, chloro and bromo, and alkyl with C? -4, benzoyl chlorides. replaced. When substituted, from 1 to 3 substituents are frequently present, particularly in the case of the alkyl and halo substituents. Examples of suitable alkanesulfonyl chlorides which may be employed include alkanesulfonyl chloride with C2 to C7. Examples of the arenesulfonyl chlorides which may be employed include substituted and unsubstituted benzenesulfonyl chlorides, such as C 1 - alkoxy, halo, particularly fluoro, chloro and bromo, and C 1 -4 alkyl, substituted benzenesulfonyl chlorides. When substituted, from 1 to 3 substituents are frequently present, particularly in the case of the alkyl and halo substituents. Examples of suitable alkyl chloroformates which may be employed include alkyl chloroformates with C2 to C7. Examples of the aryl chloroformates which may be employed include the substituted and unsubstituted phenyl chloroformates, such as alkoxy with C? _, Halo, particularly fluoro, chloro and bromo, and alkyl with C? _4, substituted phenyl chloroformates . When substituted, from 1 to 3 substituents are frequently present, particularly in the case of the alkyl and halo substituents. Examples of suitable alkyl chlorosulfites which may be employed include alkyl chlorosulfites with C2 to C. Examples of the aryl chlorosulfites which may be employed include substituted and unsubstituted phenyl chlorosulfites, such as alkoxy with C? -4, halo, particularly fluorine, chlorine and bromine, and alkyl with C? _4, substituted phenyl chlorosulfites. When substituted, from 1 to 3 substituents are frequently present, particularly in the case of the alkyl and halo substituents. Examples of suitable alkyl phosphorochloridates which may be employed include di (C1 to C6 alkyl) phosphorochloridates. Examples of the aryl phosphorochloridates which may be employed include substituted and unsubstituted diphenyl phosphorochloridates, such as C 1 -4 alkoxy, halo, particularly fluoro, chloro and bromo, and diphenyl phosphorochloridates substituted with C 1 -4 alkyl. When substituted, from 1 to 3 substituents are frequently present, particularly in the case of the alkyl and halo substituents. Additional binding agents which can be used are the chloro-, bromo- and (benzotriazol-1-yloxy) -phosphonium and carbonium compounds described by Wada et al, in J.A.C.S. 1997, 199, pp. 12710-12721 (incorporated herein for reference). Preferred binding agents are the diaryl phosphorochloridates, particularly those having the formula (Ar0) 2P0Cl wherein Ar is preferably phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.

The nature of the sulfur transfer agent will depend on whether an oligonucleotide, a phosphorothioate analog or an oligonucleotide phosphorothioate / mixed oligonucleotide is required. The sulfur transfer agents employed in the process of the present invention frequently have the general chemical formula: L s A wherein L represents a separation group, and A represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group. Commonly, the separation group is selected to comprise a nitrogen-sulfur bond. Examples of suitable separation groups include morpholines such as morpholin-3,5-dione; imides such as phthalimides, succinimides and maleimides; indazoles, particularly indazoles with electron withdrawing substituents such as the 4-nitroindazoles; and triazoles. Where a standard phosphodiester linkage is required in the final product, the sulfur transfer agent, the A portion represents an aryl group, such as a phenyl or naphthyl group. Suitable aryl groups include substituted and unsubstituted phenyl groups, particularly the halophenyl and alkylphenyl groups, especially 4-halophenyl and 4-alkylphenyl, commonly groups of 4- (C con -4 alkyl) phenyl, more preferably groups of 4 -chlorophenyl and p-tolyl. An example of a suitable class of the transfer agent which directs the standard phosphodiester is an N- (arylsulfanyl) phthalimide (succinimide or another may also be used). Where a phosphorothioate diester linkage is required in the final product, portion A 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 benzyl alkyl-, commonly C- _4alkyl- and halo-, commonly chloro-, substituted, and ethyl groups. substituted, especially ethyl groups substituted in the 2-position with an electron withdrawing substituent such as the 2- (4-nitrophenyl) ethyl and 2-cyanoethyl groups. Examples of suitable alkenyl groups are allyl and crotyl. Examples of a suitable class of sulfur transfer agents that direct phosphorothioate are, for example, (2-cyanoethyl) sulfanyl derivatives such as 4- [(2-cyanoethyl) sulfanyl] morpholin-3,5-dione. or a corresponding reagent such as 3- (phthalimidosulfa il) propanenitrile. A suitable temperature for carrying out the binding reaction and the transfer of the sulfur is in the range of about -55 ° C to room temperature (commonly in the range of 10 to 30 ° C, for example about 20 ° C) , and preferably from -40 ° C to 0 ° C. Organic solvents which may be employed in the process of the present invention include haloalkanes, particularly dichloromethane, esters, particularly alkyl esters such as ethyl acetate, and methyl and ethyl propionate, and basic, nucleophilic solvents, such as pyridine. Preferred solvents for the sulfur binding and transfer steps are pyridine, dichloromethane and mixtures thereof. The molar ratio of the H-phosphonate to the alcohol in the process of the present invention is preferably selected to be in the range of from about 0.9: 1 to 3: 1, commonly from about 1: 1 to about 2: 1, and preferably from about 1.1: 1 to about 1.5: 1, such as about 1.2: 1. However, where the bonds on more than one free hydroxyl are being carried out at the same time, the molar ratios will be proportionally increased. The molar ratio of the coupling agent to the alcohol is often selected to be in the range of from about 1: 1 to about 10: 1., commonly from about 1.5: 1 to about 5: 1 and preferably from about 2: 1 to about 3: 1. The molar ratio of the sulfur transfer agent to the alcohol is often selected to be in the range of from about 1: 1 to about 10: 1, commonly from about 1.5: 1 to about 5: 1 and preferably from about 2: 1 to about 3: 1. In the process of the present invention, the H-phosphonate and the alcohol can be premixed in solution, and the binding agent added to this mixture. Alternatively, the H-phosphonate and the binding agent can be premixed, often in solution and then added to a solution of the alcohol, or the alcohol and the binding agent can be mixed, commonly in solution, and then added to a solution of H-phosphonate. In certain embodiments, the H-phosphonate, optionally in the form of a solution, may be added to a solution comprising a mixture of the alcohol and the binding agent. After the binding reaction is substantially complete, the sulfur transfer agent is then added to the solution of the H-phosphonate diester produced in the binding reaction. Additions of the reagent are commonly carried out continuously or increasingly during an addition period. In the process of the present invention, it is possible to prepare oligonucleotides containing phosphorothioate diester and phosphodiester internucleotide linkages in the same molecule by the selection of the appropriate sulfur transfer agents, particularly when the process is carried out in a manner gradual or step by step As previously established, the method of the invention can be used in the synthesis of RNA sequences, 2'-O-alkyl-RNA, 2'-O-alkoxyalkyl-RNA and 2'-0-alkenyl-RNA. The 3'-H-phosphonates of 2'-0- (Fmpm) -5 '-0- (4,4-dimethoxytrityl) -ribonucleoside 1 and 3'-H-phosphonates of 2'-0- (alkyl, alkoxyalkyl or alkenyl) -5 '-0- (4,4-dimethoxytrityl) -ribonucleoside 2a-c can be prepared, for example, from the corresponding nucleoside building blocks, H-ammonium p-phosphonate and p-cresyl chloride and pivaloyl. b: R = CH2 = CHCH2 c: R = MeOCH2CH2 The same protocols are used as in the synthesis of the DNA and phosphorothioate DNA sequences (Schemes 2-4). Following the standard unblocking procedure (Scheme 2, steps v and vi), the Fpmp protecting groups are removed under mild conditions of acid hydrolysis which do not lead to a detectable segmentation or migration of the internucleotide bonds.

(Capaldi, D.C. Reese, C.B. Nucleic Acids Res. 1994, 22, 2209-2216). For chemotherapeutically useful ribozyme sequences, the synthesis of RNA on a relatively large scale in solution is a matter of considerable practical importance. The incorporation of 2'-0-alkyl, 2'-O-substituted alkyl and 2'-O-alkenyl [especially 2'-O-methyl, 2'-0-allyl and 2'-0- (2-methoxyethyl) ] -ribonucleosides (Sproat, BS in "Methods in Molecular Biology, Vol. 20. Protocols for Oligonucleotides and Analogs", Agrawal, S., Ed. Humana Press, Totowa, 1993) in oligonucleotides, is a commonly very important issue because these modifications confer both resistance to nuclease digestion and good hybridization properties on the resulting oligomers. The step of the sulfur transfer is carried out on the product of the binding of the H-phosphonate in situ, ie the separation and purification of the intermediate compound produced by the binding reaction. Preferably, the sulfur transfer agent is added to the stirred mixture resulting from the binding reaction. In addition to the fact that it is carried out in homogeneous solution, the present joining procedure differs from that followed in the H- approach. phosphonate for solid phase synthesis (Froehler et al., Methods in Molecular Biology, 1993) in at least two other important aspects. First, it can be carried out at a very low temperature. The side reactions that can accompany the binding of the H-phosphonate (Kuyl-Yeheskiely et al, Rec .. Trav. Chim., 1986, 105, 505-506) can therefore be avoided even when di (2-chlorophenyl) phosphorodichloridate ) instead of pivaloyl chloride (Froehler, B. C; Matteucci, MD Tetrahedron Lett., 1986, 27, 469-472) is used as the binding reagent. Secondly, the sulfur transfer is carried out after each binding step instead of just once following the binding of the complete oligomer sequence. The protecting groups can be removed using methods known in the art for the particular protective group and function. For example, temporary protecting groups, particularly the gamma keto acids such as the protecting groups of the levulinoyl group, can be removed by the hydrazine treatment, for example, the buffered hydrazine, such as the hydrazine treatment under very mild conditions described. by van Boom. ' J.H .; Burgers, P.M.J. Tetrahedron Lett., 1976, 4875-4878. The resulting partially protected oligonucleotides with the free 3'-hydroxy functions can then be converted into the corresponding H-phosphonates which are intermediates which can be used for the block synthesis of the oligonucleotides and their phosphorothioate analogues. When the desired product is deprotected once it has been produced, the protective groups on phosphorus which produce the phosphorothioate triester bonds are first commonly removed. For example, a cyanoethyl group can be removed by treatment with a strongly basic amine such as DABCO, 1,5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,8-diazabicyclo [5.4.0] ] undec-7-ene (DBU) or triethylamine. The phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the basic residues can be removed by treatment with oximate, for example with the conjugate base of an aldoxime, preferably that of E-2-nitrobenzaldoxime or pyridine. 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 the intermediate S-phenyl phosphorothioate compounds; and van Boom and their collaborators in an approach to the synthesis of oligonucleotides, based on the intermediate phosphorothiate compounds of S- (4-methylphenyl) (Wreesman, CTJ et al., Tetrahedron Lett, 1985, 26, 933-936) all have shown that the deblocking of S-phenylphosphorothioates with oxymate ions (using the method of Reese et al., 1978; Reese, CB; Zard, L. Nucleic Acids Res., 1981, 9, 4611-4626) leads to natural phosphodiester internucleotide bonds. In the present invention, the release of S- (4-chlorophenyl) -protected phosphorothioates with the conjugate base of E-2-nitrobenzaldoxime proceeds smoothly and without detectable internucleotide cleavage. Other basic protection groups, for example the benzoyl, pivaloyl and amidine groups can be removed by the treatment with concentrated aqueous ammonia. The trifyl groups present can be removed by treatment with an acid. With respect to the strategy of complete unblocking in the synthesis of oligodeoxyribonucleotides, another important consideration of the present invention, is that the removal of the trifile, frequently a 'protecting group of DMTr 5' -terminal ("detritylation") must be processed without depurination concomitant, especially of any of the residues of 6-N-acyl-2'-deoxyadenosine. According to one embodiment of the invention, the present inventors have found that such depurination, which is perhaps difficult to completely prevent in solid-phase synthesis, can be completely suppressed by effecting "detritylation" with a dilute solution of hydrogen chloride at low temperature, particularly hydrogen chloride ca. 0.45 M in dioxane dichloromethane solution (1: 8 v / v) at -50 ° C. Under these reaction conditions, "detritylation" can be complemented rapidly, and in certain cases after 5 minutes or less. For example, when 6-N-benzoyl-5 '-0- (4,4-dimethoxytrityl) -2'-deoxyadenosine is treated with hydrogen chloride in dioxane-dichloromethane under such conditions, "detritylation" was supplemented after 2 minutes, but no debridement was detected even after 4 hours. The silyl protecting groups can be removed by the fluoride treatment, for example with a solution of a tetralkyl ammonium fluoride salt such as tetrabutyl ammonium fluoride. The protective groups of Fpmp can be removed by acid hydrolysis under mild conditions. This new approach to the synthesis of the oligonucleotides in solution is suitable for the preparation of the sequences with (a) only one phosphodiester, (b) only the diester of phosphorothioate and (c) a combination of internucleotide bonds of both phosphodiester and diester of phosphorothioate. The invention also relates to the development of the block connection (as illustrated for example in Scheme 4b). In this regard, the examples provide an illustration of the synthesis of the Tp (s) Tp (s) Gp (s) Gp (s) Gp (s) Gp (s) Gp (s) Tp (s) T] (ISIS 5320 Ravikuma, VT; Cherovallath, Z: S: Nucleosides &Nucleosides 1996, 15, 1149-1155), a heptaphosphorothioate of octadeoxyribonucleosides, from tetramer blocks. This oligonucleotide analogue has properties as an anti-HIV agent. Other targets of block synthesis include sequences with therapeutic effects, for example, human thrombin inhibitors and anti-HV agents. The method of the invention can be used in the synthesis of larger sequences. It will be apparent that when the process of the present invention is applied to block synthesis, a number of alternative strategies are available in terms of the route for the desired product. These will depend on the nature of the desired product. For example, an octamer can be prepared by preparing the dimers, linked to produce tetramers, which are then linked to produce the desired octamer. Alternatively, a dimer and a trimer can be linked to produce a pentamer, which can be linked with an additional trimer to produce the desired octamer. The choice of strategy is at the discretion of the user. However, the common characteristic of such block binding is that an oligomer H-phosphonate comprising two or more units is linked with an oligomer alcohol also comprising two or more units. Most commonly, 3'-H-phosphonates of oligonucleotides are coupled with oligonucleotides having free 5'-hydroxy functions. The process of the present invention can also be used to prepare the cyclic oligonucleotides, especially the cyclic oligodeoxyribonucleotides and the cyclic ribonucleotides. In the preparation of the cyclic oligonucleotides, an oligonucleotide comprising an H-phosphonate function, frequently a 3 'or 5' H-phosphonate is prepared, and a free hydroxy function is introduced by the appropriate deprotection. The position of the free hydroxy function is usually selected to correspond to the H-phosphonate, for example a 5'-hydroxy function could be linked with a 3'H-phosphonate, and a 3'-hydroxy function could be linked with a 5'H -phosphonate. The hydroxy and H-phosphonate functions can then be coupled intramolecularly in solution in the presence of a binding agent and this reaction is followed by sulfur transfer in situ.

According to a further aspect of the present invention, novel oligomeric H-phosphonates having the general chemical formula are provided: wherein each B independently is a selected base of A, G, T, C or U; each Q independently is H or OR 'where R' is alkyl, substituted alkyl, alkenyl or a protecting group; each R is independently an aryl, methyl, substituted alkyl or alkenyl group; W is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; each X independently represents O or S; each Y independently represents O or S; Z is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; and n is an integer and is at least 2; provided that when W is H or a protecting group, this Z is a group of H-phosphonate, and that when Z is H or a protecting group, this is a group of H-phosphonate. Oligomeric H-phosphonates having the general chemical formula are also provided: wherein each B independently is a selected base of A, G, T, C or U; each Q independently is H or OR 'where R' is alkyl, substituted alkyl, alkenyl or a protecting group; each R is independently an aryl, methyl, substituted alkyl or alkenyl group; w is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; each X independently represents 0 or S; each Y represents S; Z is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; and n is a positive integer; provided that when W is H or a protective group, this Z is a group of H-phosphonate, and that when Z is H or a protective group, this W is a group of H-phosphonate. Preferably, only one of W or Z is a group of H-phosphonate, commonly only Z is a group of H-phosphonate. When W or Z represents a protecting group, the protecting group may be one of those described above to protect the 3 'or 5' positions respectively. When W is a protecting group, the protecting group is a trifyl group, particularly a dimethoxytrityl group. When Z is a protecting group, the protecting group is a trifyl group, particularly a dimethoxytrityl group, or an acyl, preferably a levulinoyl group. The bases A, G and C represented by B are preferably protected, and the bases T and U can be protected. Suitable protecting groups include those described hereinbefore for the protection of the bases in the process according to the first aspect of the present invention. When Q represents a group of OR ', and R' is alkenyl, the alkenyl group is frequently an alkenyl group of C? ~, Especially an allyl or crotyl group. When R 'represents alkyl, the alkyl is preferably an alkyl group with C? -4. When R 'represents the substituted alkyl, the substituted alkyl group includes the alkoxyalkyl groups, especially C? _4 C? Alkyl groups such as the methoxyethyl groups. When R 'represents a protective group, the protecting group is commonly a labile-acid acetal protecting group, particularly l- (2-fluorophenyl) -4-methoxypiperidin-4-yl (Fpmp) or trialkylsilyl groups, frequently a group of tri (C? _4-alkyl) silyl such as a tertiary butyl dimethyl silyl group. Preferably, X represents 0.

In many embodiments, Y represents S and each R represents the methyl, substituted alkyl, alkenyl or aryl group remaining of the sulfur transfer agent (s) employed in the process of the present invention. Preferably, each R independently represents a methyl group; a substituted methyl group, particularly a substituted benzyl or benzyl group, such as a benzyl alkyl-, commonly C 1-4 alkyl- or halo-, commonly chloro-, substituted group; a substituted ethyl group, especially an ethyl group substituted in the 2-position with a substituent for removal of the electrons such as a group of 2- (4-nitrophenyl) ethyl or a 2-cyanoethyl group an alkenyl group with C? _4 , preferably an allyl or crotyl group; or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially a 4-halophenyl group or a 4-alkylphenyl, commonly a 4- (C 4 -4 alkyl) phenyl group, and more preferably a 4-chlorophenyl group or a p-tolyl group. M + preferably represents a trialkyl ammonium ion, such as a tri (C 4 -alkylammonium) ion, and preferably a triethylammonium ion. n can be 1 up to any number depending on the oligonucleotide which is proposed to be synthesized, particularly up to about 20. Preferably n is 1 to 16, and especially 1 to 9. The H-phosphonate wherein n represents 1, 2 or 3 can to be used when it is desired to add small blocks of nucleotides, with values of correspondingly larger n, for example 5, 6 or 7 or more that are used if it is desired that larger blocks of oligonucleotides are joined. The H-phosphonates according to the present invention are commonly in the form of solutions, preferably those employed in the process of the first aspect of the present invention. These H-phosphonates are also useful intermediates in the synthesis of blocks of the oligonucleotides and phosphorothioates of oligonucleotides. As indicated above, block binding is much more feasible in the synthesis in phase in solution than in the synthesis in solid phase. The oligonucleotide H-phosphonates can be prepared using the general methods known in the art for the synthesis of the nucleoside H-phosphonates. Accordingly, in a further aspect of the present invention, there is provided a process for the production of an oligonucleotide H-phosphonate wherein an oligonucleotide comprising a free hydroxy function, preferably a 3 'or 5' hydroxy function, is it reacts with an alkyl or aryl H-phosphonate salt in the presence of an activator. Preferably, the oligonucleotide is a protected oligonucleotide, and more preferably a protected oligodeoxynucleotide or a protected oligoribonucleotide. The H-phosphonate salt is frequently an ammonium salt, including the mixed alkyl, aryl and alkyl ammonium salts. Preferably, the ammonium salt is a salt of (NH4) + or tri (C4-4alkyl) ammonium. Examples of the alkyl groups which may be present in the H-phosphonate are alkyl with C? _4, especially C2_4 alkyl, groups substituted with strongly electron withdrawing groups, particularly halo, and preferably fluoro groups, such as the groups of "2,2,2-trifluoroethyl and 1,1,3,3,3-hexafluoropropan-2-yl Examples of the aryl groups that may be present include phenyl and substituted phenyl, particularly alkylphenyl, commonly alkylphenyl with C.sup.4 and halophenyl, commonly chlorophenyl groups.Preferably, a substituted phenyl group is a 4-substituted phenyl group.The particularly preferred H-phosphonates are the p-cresyl ammonium and triethylammonium H-phosphonates. can be employed include those compounds described herein for use as binding agents, and particularly diaryl phosphorochloridates and acid alkyl and cycloalkyl chlorides, such as 1-adamantan chloride carbonyl, and preferably pivaloyl chloride. The production of H-phosphonates is preferably carried out in the presence of a solvent, frequently those solvents described for use in the process of the first aspect of the present invention, preferably pyridine, dichloromethane and mixtures thereof. An advantage of the present invention for the synthesis only of phosphorothioate diesters is that, whenever care is taken to avoid desulfurization during the deblocking steps [particularly during heating with aqueous ammonia (for example Scheme '3, step viii (a))], synthesis of oligonucleotide phosphorothioates should not lead to products that are contaminated with the standard phosphodiester internucleotide linkages. In the case of solid phase oligonucleotide phosphorothioate synthesis, the incomplete transfer of sulfur in each synthetic cycle usually leads to a contamination of the residual phosphodiester (Zon, G.; Stec, W. J. in "Oligonucleotides and Analogs, A Practical Approach", Eckstein, F., Ed., IRL Press, Oxford, 1991, pp. 87-108). The synthesis in solution as proposed by the present invention has another enormous disadvantage on the synthesis in solid phase in which there exists the possibility of controlling the selectivity of the reactions working at low or even very low temperatures. This advantage extends to the detritylation step (Scheme 3, step i) which can proceed rapidly and quantitatively below 0 ° C without detectable depurination. After the detritylation step, a relatively quick and efficient purification can be effected by what has been previously described as the "filtration" approach (Chaudhuri, B,; Reese, C.B .; Wecla ek, K. Tetrahedron Lett., 1984, 25, 4037-4040). This depends on the fact that the intermediates of the phosphotriester (and the phosphorothioate triester), but not any of the remaining detritylated charged monomers, are eluted very rapidly from the short silica gel columns by the THF-pyridine mixtures. The method according to the invention will now be illustrated with reference to the following examples which are not proposed to be limiting. In the Examples, it should be noted, that where the nucleoside residues and the internucleotide bonds are in italic letters, this indicates that they are protected in some way. In the present context, A, C, G, and T represent protected 2'-deoxyadenosine on N-6 with a benzoyl group, 2'-deoxycytidine protected on N-4 with a benzoyl group, 2'-deoxyguanosine protected on N-2 and about 0-6 with isobutyryl and 2, 5-dichlorophenyl groups and unprotected thymine. For example, as indicated in scheme 3, p (s) and p (s') represent phosphorothioates of S- (2-cyanoethyl) and S- (4-chlorophenyl), respectively, and p (H), which is not protected and therefore not italicized, represents a monoester of H-phosphonate if it is placed at the end of a sequence or attached to a monomer but otherwise represents an H-phosphonate diester.

Examples Reaction Scheme for the Preparation of Dinucleoside Phosphates.

With particular reference to the preparation of dinucleoside phosphates, Scheme 2 describes in more detail the method of the invention for the preparation of oligodeoxyribonucleotides and phosphorothioate analogs thereof. 16 Scheme 2 .Reactives and conditions: (i) 18, C5H5N, CH2Cl2, -40 ° C, 5-10 minutes; (ii) 19, C5H5N, CH2Cl2, -40 ° C, 15 minutes, b, C5H5N -H20 (1: 1 v / v), -40 ° C at room temperature; (iii) HCl / 4M dioxane, CH2C12, -50 ° C, 5 minutes; (iv) Ac20, C5H5N, temp. environment, 15 h; (v) 20, TMG, MeCN, temp. environment, 12 h; (vi) a, NH3 aq. conc. (d 0.88), 50 ° C, 15 h, b, Amberlite IR-120 (more), Na + form, H20; (vii) a, 21, C5H5N, CH2C12, -40 ° C, 15 minutes, b, C5H5N -H20 (1: 1 v / v), -40 ° C at temp. ambient; (viii) DBU, Me3SiCl, CH2C12, temp. environment, 30 min; (ix) 20, DBU, MeCN, room temperature, 12 h.

From Scheme 2, the synthesis of the oligonucleotides proceeds through intermediate compounds 8, 9, 10 and 11 and the preparation of the phosphorothioate analogues proceeds through intermediate compounds 8, 9, 12 and 13. The bases 14, 15 and 16 correspond to protected adenine, protected cytosine and protected guanine. Base 17 corresponds to thymine, which does not require protection. Any protective group conventionally used can be used. In the synthesis of RNA, thymine will be replaced by uracil. Compound 18 is a suitable coupling agent, and compounds 19 and 21 are suitable sulfur transfer agents. These compounds are referred to more fully herein below. The monomeric building blocks required in the coupling process according to the invention, illustrated in Scheme 2 are the triethylammonium 3'-H-phosphonates 5 '-0- (4-4'-dimethoxytrityl) -2'-deoxyribonucleosides (Bases B and B '= 14-17) which can be easily prepared in quasi-quantitative yields from the corresponding protected nucleoside derivatives by a recently reported procedure (Ozola, V., Reese, CB, Song Q. Tetrahedron Lett ., 1996, 37, 8621-8624). By way of illustration, the 3'-H-phosphonates of triethylammonium 5 '-0- (dimethoxytrityl) -2'-deoxyribocleoside 8 were prepared as follows: 4-methylphenyl ammonium phosphonate 30 (2.84 g, 15.0 mmol) , the derivative of 5 '-0- (dimethoxytrityl) -2'-deoxyribonucleoside (5.0 mmol), triethylamine (4.2 ml, 30 mmol) and dry pyridine (20 ml) are evaporated together under reduced pressure. The residue is coevaporated again with dry pyridine (20 ml). The residue is dissolved in dry pyridine (40 ml) and the solution is cooled to -35 ° C (industrial methylated alcoholic substances / dry ice bath). Pivaloyl chloride (1.85 ml), 15.0 mmol) is added dropwise to the stirred solution over a period of 1 minute, and the reagents are kept at -35 ° C. After 30 minutes, water (5 ml) is added, and the stirred mixture is allowed to warm to room temperature. A buffer solution of potassium phosphate (1.0 mol din-3, pH 7.0, 250 ml) is added to the products, and the resulting mixture is concentrated under reduced pressure until all of the pyridine has been removed. The residual mixture is partitioned between dichloromethane (250 ml) and water (200 ml). The organic layer is washed with a triethylammonium phosphate buffer solution (0.5 mole drrf3, pH 7.0, 3 x 50 ml), dried (MgSO4) and then evaporated under reduced pressure. The residue is fractionated by short column chromatography on silica gel (25 g). The appropriate fractions, eluted with dichloromethane-methanol (95: 5 to 90:10 v / v), were evaporated to give the 3 '-H-phosphonate of (5' -O- (dimethoxytrityl) -2'-deoxyribonucleoside) When the 3'-H-phosphonate of triethylammonium 6-N-benzoyl-5 '-O- (4,4'-dimethoxytrityl) -2'-deoxyadenosine (DMTr-Ap (H)) (Ozola et al., Tetrahedron, 1996), 8 (B-14), 4-N-benzoyl-3'-O-levulinoyl-2'-deoxycytide (HO-C-Lev ) 9 (B '= 15) and di- (2-chlorophenyl) phosphorocloridate 18 were allowed to react together in the pyridine-dichloromethane solution at -40 ° C, the corresponding fully protected dinucleoside H-phosphonate (DMTr- Ap (H) C-Lev) was apparently obtained with a quantitative yield within the range of 5-10 minutes. The protocol used in this particular example was the dropwise addition of a solution of the di- (2-chlorophenyl) phosphorochloridate (2.03 g, 6.0 mmol) in dichloromethane (4 mL) for 5 minutes to a dry, stirred solution of the triethylammonium salt of DMTr-Ap (H) 8 (B = 14) (3.95 g, ca., 4.8 mmol) and 4-N-benzoyl-3'-0-levulinoyl-2'-deoxycytide 9 (B '= 15 ) (1.72 g, 4.0 mmol) in pyridine (36 ml), maintained at -40 ° C (bath of industrial methylated alcoholic substances + dry ice). After an additional period of 5 minutes, only one nucleotide product, assuming it to be DMTr-AP (H) C-Lev, and some of the remaining H-phosphonate monomer 8 (B = 14) could be detected by HPLC from reverse phase). However, it should be noted that these reaction conditions can be varied appropriately. It is particularly remarkable that such high bonding efficiency was achieved with only ca. 20% excess of the H-phosphonate monomer. No attempt was made to isolate the intermediate dinucleoside H-phosphonate (DMTr-Ap (H) C-Lev). N- [(4-chlorophenyl) sulfanyl] phthalimide 19 (2.32 g, 8.0 mmol) (Behforouz, M.; Ker ood, JEJ Org Che, 1969, 34, 51-55) was added to the stirred reagents which They were maintained at -40 ° C. After 15 minutes, the products were worked up and subjected to chromatography on silica gel and the corresponding S- (4-chlorophenyl) dinucleoside DMTr-Ap (s ') C-Lev 10 phosphorothioate (B = 14, B' = 15) was obtained with an isolated yield of 99%. Accordingly, the steps of both sulfur binding and transfer proceeded relatively rapidly and virtually quantitatively at -40 ° C. The four-step procedure (Scheme 2, steps iii-vi) for deblocking the DMTr-Ap (s ') C-Lev 10 (B = 14, B' = 15) preferably involves the "detritylation", the acetylation of the 5'-hydroxy-terminal function, the treatment with the oximate, and finally the treatment with concentrated aqueous ammonia to remove the acyl protecting groups from the basic residues and the 3'- and 5'-terminal hydroxy functions. In this way, the extremely pure 'd [ApC] 11 (B = adenin-9-yl, B' = cytosin-1-yl) was obtained without further purification and isolated as its sodium salt. The monomeric building blocks 8 (B = 17) and 9 (B '= 16) were joined together in the same way and to the same scale. After transfer of the sulfur with N- [(4-chlorophenyl) sulfanyl] phthalimide 19, the dinucleoside phosphorothioate DMTr-Tp (s ') G-Lev fully protected 10 (B = 17, B' = 16) was isolated with a yield of almost 98%. Again, very pure d [TpG] 11 (B = timin-1-yl, B '= guanin-9-yl) was obtained when this material was unblocked by the above procedure (Scheme 2, steps iii-vi). The protocol for the preparation of the phosphorothioates of fully protected oligonucleotides differs from that used for the synthesis of oligonucleotides only in that the transfer of the sulfur is effected with 4 - [(2-cyanoethyl) sulfanyl] morpholin-3,5-dione. or 3- (phthalimidosulfanyl) propanenitrile. However, 4 - [(2-cyanoethyl) sulfanyl] morpholin-3, 5-dione has the advantage that the morpholin-3, 5-dione produced in the course of sulfur transfer is more soluble in water than phthalimide. The 3'-H-triethylammonium phosphonate 6-0- (2,5-dichlorophenyl) -5 '-0- (4,4'-dimethoxytrityl) -2-N-isobutyryl-2'-deoxyguanosine (DMTr-Gp ( H)) '8 (B = 16) [ca. 4.8 mmol], 6-N-benzoyl-3'-O-levulinoyl-2'-deoxyadenosine (HO-A-Lev) 9 (B = 14) [4.0 moles] and di- (2-chlorophenyl) phosphorochloridate. [6.0 mmole] were allowed to react together in pyridine-dichloromethane solution at -40 ° C for 5-10 minutes. 4- [(2-Cyanoethyl) sulfanyl] morpholin-3, 5-dione 21 [8.0 mmole] (Scheme 2, step vii) was then added while the reagents were maintained at -40 ° C. After 15 minutes, the products were worked up and fractionated by chromatography on silica gel to give the fully protected dinucleoside phosphorothioate (DMTr-Gp (s) A-Lev) 12 (B = 14, B '= 16) with a yield 99% isolated This material was unlocked by a 5 step procedure (Scheme 2, steps iii, iv, viii, ix and vi). Following the steps of "detritylation" and acetylation, the product was treated with 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) under strictly anhydrous conditions to remove the protective group of S- (2- cyanoethyl). The 6-0- (2,5-dichlorophenyl) protecting group was then removed from the guanine residue by the oximate treatment, and finally all of the acyl protecting groups were removed by ammonolysis. The treatment step with oximate can be omitted if the phosphorothioate of the oligonucleotide does not contain any 2'-deoxyguanosine residue. The extremely pure [Gp (s) A] 13 (B = guanin-9-yl, B '= adenin-9-yl) was obtained without further purification, and was isolated as its sodium salt.

Preparation of 4- [(2-cyanoethyl) sulfanyl] morpholin-3,5-dione S- (2-Cyanoethyl) isothiouronium chloride was prepared as follows. The thiourea (304 g) was dissolved with heating in concentrated hydrochloric acid (500 ml). The resulting solution is evaporated under reduced pressure and the residual colorless solid is dissolved in boiling absolute ethanol (1300 ml). The solution is cooled to room temperature and acrylonitrile (400 cm3) is added in portions with stirring. The reagents were heated, under reflux, for 2 hours. The cooled products were filtered and the residue was washed with cold ethanol and then dried in vacuo over calcium chloride. The di- (2-cyanoethyl) disulfide was then prepared as follows. Dichloromethane (400 ml) was added to a stirred solution of S- (2-cyanoethyl) isothiouronium chloride (83.0 g) in water (500 ml) at 0 ° C (ice-water bath). Sodium perborate tetrahydrate (44.1 g) was added, and then a solution of sodium hydroxide (30.0 g) in water (250 ml) was added dropwise. The reagents were kept at 0 ° C (ice-water bath). After 5 hours, the products were separated and the aqueous layer was extracted with dichloromethane (3 x 50 ml). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give a solid which was recrystallized from methanol (30 mL) to give colorless crystals. Di- (2-cyanoethyl) disulfide (4.51 g) and morpholin-2,6-dione (5.75 g) were suspended in acetonitrile (10 ml), dichloromethane (20 ml) and 2,6-lutidine (17.4 ml). ) and cooled to 0 ° C (ice-water bath). A solution of bromine (4.28 g) in dichloromethane (20 ml) is added for 30 minutes. The reaction mixture is allowed to stir at 0 ° C for 1.5 hours. The product was then precipitated by the addition of cold-ice methanol (50 ml) for 30 minutes and the title compound was filtered (8.23 g, 82%). Recrystallization from ethyl acetate gave 4- [(2-cyanoethyl) sulfanyl] morpholin-3,5-dione as colorless needles, m.p. 121-122 ° C.

Reaction Scheme for the Preparation of Chimeric Oligonucleotides The stepwise synthesis of d [TpGp (s) ApC] 25 which has a phosphorothioate diester and two phosphodiester internucleotide linkages is illustrated in an exemplary description in Scheme 3. (a) DMT pfs' C-Lev - 1- HO-? p (.s' C-Lev + D Tr-Gp (H)? rgto, HO-Gp (s) Ap (s') C ~ Le \ r 10 (B = 14, B '= 15) 22 8 (B = 16) ca aD 0 23 vjji (b) 23 + DMTr-Tp (H) J ^ f Ac-TpMGptsμpMC-Lev? dL d [TpGp (s) ApC] 8 (B = 17) ca- y¿ / 0 24 25 O- Scheme 3 Reagents and conditions: (i) HCl / 4M dioxane, CH2C12, -50 ° C, 5 minutes; (ii) 18, C5H5N, CH2C12, -40 ° C, 5-10 minutes; (iii) a, 21, C5H5N, CH2C12, -40 ° C, 15 minutes, b, C5H5N -H20 (1: 1 v / v), -40 ° C at temp. ambient; (iv) 19, C5H5N, CH2C12, -40 ° C, 15 minutes, b, C5H5N -H20 (1: 1 v / v), -40 ° C at room temperature; (v) Ac20, C5H5N, temp. environment, 15 h; (vi) DBU, Me SiCl, CH2C12, temp. environment, 30 min; (vii) 20, DBU, MeCN, room temperature, 12 h. (viii) a, NH3 aq. conc. (d 0.88), 50 ° C, 15 h, b, Amberlite IR-120 (more), Na +, H20 form.

No limitation on the scale is anticipated. The reactions shown in scheme 3 are not proposed to be limiting and the method of the invention is equally suitable for the synthesis of the RNA, 2'-O-alkyl-RNA and other oligonucleotide sequences. All of the reactions involved were previously used either in the preparation of d [ApC] 11 (B = adenin-9-yl, B '= cytosin-1-yl or d [Gp (s) A] 13 ( B = guanin-9-yl, B '= adenin-9-yl) (Scheme 2). First, the phosphorothioate of the fully protected dinucleoside DMTr-Ap (s ') C-Lev 10 (B = 14, B' = 15) [ca. 0.75 mmole] becomes four steps and a total isolated yield of ca. 96% (Scheme 3a) in partially protected trimer 23. In each joining step, an excess of ca. 20% of the monomer of H-phosphonate 8 was used, but the excess of the binding agent 18 depends on the scale of the reaction. In addition, a two-fold excess of the sulfur transfer agent 19 or 21 was used in this example. The products were subjected to chromatography on silica gel after each step of "detritylation". This material was then bound to the DMTr-Tp (H) 8 (B = 17) and the product was converted into three steps and a total yield of ca. 93% (Scheme 3b) in tetramer 24 fully protected. This latter material was unblocked to give d [TpGp (s) ApC] 25 which was isolated without further purification as its relatively pure sodium salt (ca. 96.5% by HPLC). The tetranucleoside triphosphate d [TpGpApC] and the tetranucleoside triphosphorothio d [Cp (s) Tp (s) Gp (s) A] were also prepared by step synthesis in a very similar manner. The protocols followed differed from those described in Scheme 3 only in the step synthesis in an almost identical manner. The protocols followed differed from those described in Scheme 3 only in that the sulfur transfer agent 19 was used exclusively in the preparation of d [TpGpApC] and the sulfur transfer agent 21 was used exclusively in the preparation of the d [Cp (s) Tp (s) Gp (s)].

Reaction Scheme for the Union by Blocks By way of illustration, Scheme 4 given hereinafter illustrates an example of a block joint which is part of the invention. (a) Ac-Tp (s) Tp (s) Gp (s) G-Lev I, II »Ac-Tp (s) Tp (s) Gp (s) Gp () 26 27 (b) Ac-Tp (s) s) Tp (s) Gp (s) Gp () + HO-Gp (s) Gp (s) Tp (s) T-Bz II IV 27 28 Ac-Tp ($) Tp (s) Gp (s) 30 Scheme 4 Reagents and conditions: (i) N 2 H 4 H 20, C 5 H 5 N - AcOH (3: 1 v / v), 0 ° C, 20 min; (ii) a, 30, Me3C-C0Cl, C5H5N, -35 ° C, 30 min, b, Et3N, H20; (iii) 18, C5H5N, CH2C12, -35 ° C, (iv) a, 21, C5H5N, CH2C12, -35 ° C, 10 minutes, b, C5H5N -H20 (1: 1 v / v), -35 ° C to temp. ambient; Tqtally protected octadeoxinucleoside heptaphosphorothioate 29 which was obtained with an isolated yield of 91% is a precursor of d [Tp (s) Tp (s) Gp (s) Gp (s) Gp (s) Gp (s) Tp ( s) T]. As indicated above, block bonding is much more feasible in solution than in solid phase synthesis. This approach of course is not limited in any way to the binding of the tetramer. Actually, it is anticipated that this approach of the H-phosphonate will be suitable for the binding of very large (eg, 10 + 10) oligonucleotide blocks together.

Reaction scheme for the preparation of H-phosphonates of Blocks For example, partially protected oligonucleotides 33a and corresponding phosphorothioates 33b which can be prepared by the conventional phosphotriester approved in solution (Chattopadhyaya, JB; Reese, CB Nucleic Acids Res., 1980, 8, 2039-2054; Kemal, 0. , Reese, CB; Serafino ska, HTJ Chem. Soc., Chem. Commun., 1983, 591-593) can be similarly converted to their 3'-H-phosphonates (34a and 34b, respectively) as indicated in the Scheme. 5. a, .X = O, Ar = 2-CIC6H4; b, X = S, Ar = 2,5-C ^ CßßHpβ.

Scheme 5 Reagents and conditions. (i) a, 30. Me3C. COCl, C5H5N, -35 ° C, b, Et3N, H20. Example 1 üc- p < s) Tp < s) Gp < s) G-OSL The H0-Tp (s) Tp (s) Gp (s) G-Lev (5.82 g, 3 immoles) was coevaporated with anhydrous pyridine (2x20 ml) and redissolved in anhydrous pyridine (30 ml). Acetic anhydride (1.42 ml, 15 mmol) is added and the reaction solution allowed to stir at room temperature for 12 hours. Water (1.5 ml) is added to quench the reaction. After 10 minutes, the mixture is cooled to 0 ° C (water-ice bath) and hydrazine hydrate (1.50 g, 30 mmol) in pyridine (15 mL) and glacial acetic acid (15 mL) is added. The mixture is stirred at 0 ° C for 20 minutes and then partitioned between water (100 mL) and CHCl2 (1QQ mL). The two layers were separated and the organic layer washed with water (3x50 ml). The organic layer is dried (MgSO4) and evaporated. The residue is purified by silica gel chromatography. The impurities were eluted with metansl-dichloromethane or (4: 96 v / v) the main product was eluted with acetone. Evaporation of the appropriate fractions gave partially protected tetradeoxynucleoside triphosphorothioate as a colorless solid (5.30 g, 93%).

Example 2 Ac-Tp. { s) Tp (s) Gp. { S) Gp (H) The ammonium salt of the 4-methylphenyl H-phosphonate (1.42 g, 7.5 mmol) is dissolved in the mixture of methanol (15 ml) and tritylamine (2.1 ml, 15 mmol). The mixture is evaporated and coevaporated with pyridine (2x10 ml) under reduced pressure. Add the Ac-Tp (s) Tp (s) Gp (s) G-OH (4.71 g, 2.5 mmol) and coevaporate with dry pyridine. (20 mi). The residue is dissolved in dry pyridine (20 ml) and pivaloyl chloride (1.23 ml, 10 mmol) is added at -35 ° C in 1 minute. After 30 minutes at the same temperature, water (5 ml) is added and the mixture is allowed to warm to room temperature and is stirred for 1 hour. The solution is partitioned between water (100 ml) and dichloromethane (100 ml). The organic layer is separated and washed with a triethylammonium phosphate buffer (pH 7.0, 0.5M, 3x50 ml), dried (MgSO), and then filtered and applied to a column of silica gel (ca. g). The appropriate fractions, which were eluted with methanol-dichloromethane (20:80, v / v), were evaporated to give the Tp (s) Tp (s) Gp (s) G? (H), as a colorless solid (4.85 g, 94%).

Example 3 Ac-Tp. { s} Tp. { s) Gp. { s} Gp. { a) Gp. { s} Gp (s) Tp. { s} T-Bz The Ac-Tp (s) Tp (s) Gp (s) Gp (H) (1229 g, 0.6 mmoles) and HO-Gp (s) Gp (s) Tp (s) T-Bz (0.973 g, 0.5 mmoles) ) were coevaporated with anhydrous pyridine (2 x 10 ml) and the residue was dissolved in anhydrous pyridine (10 ml). The solution is cooled to -35 ° C (dry ice bath-industrial methylated alcoholic substances) and di- (2-chlorophenyl) phosphorocloridate (0.84 g, 2.5 mmol) is added in dry diclsrometaps (1 ml) for 10 minutes. 4- [(2-Cyanoethyl) sulfanyl] morpholin-3., 5-dione (0.20 g, 1.0 mmol) is added and the mixture is allowed to stir for 10 minutes at the same temperature. Then water-pyridine (0.2 ml, 1: 1 v / v) is added and the mixture is stirred for an additional 5 minutes. The reaction mixture is then evaporated under reduced pressure. The residue is dissolved in dichloromethane (100 ml) and the solution is washed with a saturated aqueous sodium bicarbonate solution (3 x 50 ml). The organic layer is dried (MgSO 4) and concentrated under reduced pressure. The residue is purified by chromatography on silica gel. First, the lipophilic impurities were removed with methanol-dichloromethane (4:96 v / v), and then the main product was eluted with acetone. Evaporation of the appropriate fractions gave the heptaphosphorothioate of the fully protected octadeoxynucleoside as a colorless oil (1.81 g, 91%). The fully protected octadeoxinucleoside heptaphosphorothioate which was obtained with an isolated yield of 91% is a precursor of the Tp (s) Tp (s) Gp (s) Gp (s) Gp (s) Gp (s) Tp (s) ) T].

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (14)

1. A process for -the pxepax-ation -of a phosphorothioate triester, characterized in that it comprises the binding in the solution phase of an H-phosphonate with an alcohol in the presence of a binding agent whereby a diester is formed. H-phosphonate and, in situ, reacting the diester of H-phosphonate with a sulfur transfer agent to produce a phosphorothioate triester.

2. A process according to claim 1, characterized in that the H- "fos" fonate is a protected oligonucleotide or nucleoside comprising a function of 3'-H-phosphonate.

3. A process according to any of claims 1 and 2, characterized in that the alcohol is a protected oligonucleotide or nucleoside comprising a 5'-free hydroxy function.

4. A process according to any preceding claim, characterized in that the binding agent is a diaryl phosphorochloridate of the formula (Ar0) 2P0Cl, in which Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2, 4 , 6-tribromophenyl.

5. A process according to any preceding claim, characterized in that the sulfur transfer agent has the general chemical formula: L s A wherein L represents a separation group, and A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

6. A process according to claim 5, characterized in that the separation group is a morpholin-3, 5-dione, -phthalimide, succinimide, maleimide or indazole, and A represents a group of 4-halophenyl, a group of 4 alkylphenyl, a methyl group, a benzyl group, an alkylbenzyl group, a halobenzyl group, an allyl group, a crotyl group, a 2-cyanoethyl group or a 2- (4-nitrophenyl) ethyl group.

7. A process according to any preceding claim, characterized in that the H-phosphonate and the alcohol is a ribonucleoside, a 2-0 '- (alkyl, alkoxyalkyl or alkenyl) -ribonucleoside, an oligoribonucleotide or a 2-0' - (alkyl, alkoxyalkyl or alkenyl) -oligorribonucleotide.

An H-phosphonate, which has the general chemical formula characterized in that: each B independently is a selected base of A, G, T, C or U; each Q independently is H or OR 'where R' is alkyl, substituted alkyl, alkenyl or a protecting group; 61 each R is independently an aryl, methyl, substituted alkyl or alkenyl group; W is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; each X independently represents O or S; each Y independently represents 0 or S; Z is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; and n is an integer and is at least 2; provided that when it is H or a protecting group, this Z is a group of H-phosphonate, and that when Z is H or a protecting group, this W is a group of H-phosphonate.

9. An H-phosphonate that has the general chemical formula: characterized in that: each B independently is a selected base of A, G, T, C or U; each Q independently is H or OR 'where R' is alkyl, substituted alkyl, alkenyl or a protecting group; each R is independently an aryl, methyl, substituted alkyl or alkenyl group; is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; each X independently represents 0 or S; each Y represents S; Z is H, a protecting group or a group of H-phosphonate of the formula wherein M + is a monovalent cation; and n is a positive integer and at least 2; provided that when W is H or a protective group, this Z is a group of H-phosphonate, and that when Z is H or a protective group, this W is a group of H-phosphonate.

10. An H-phosphonate according to claim 9, characterized in that W xepxespresents a protecting group, each X represents 0, and each R represents a methyl group, a benzyl group, a 2-cyanoethyl group, an unsubstituted phenyl group or a 4-halophenyl group, M + represents a tri (C? -alkylammonium) ion, and n is 1 to 16.

11. A process for the production of an oligonucleotide H-phosphonate, characterized in that an oligonucleotide comprising a free hydroxy function, preferably a 3 'or 5' hydroxy function, is reacted with an alkyl or aryl H-phosphonate salt in the presence of an activator.

12. A process according to claim 11, characterized in that the oligonucleotide is a protected oligodeoxyribonucleotide or oligoribonucleotide.

13. A process according to claim 11 or 12, characterized in that the H-phosphonate salt is an ammonium salt of a phenyl, alkylphenyl or halophenyl H-phosphonate.

14. A process according to any of claims 11 to 13, characterized in that the activator is an aryl phosphorochloridate or an alkyl acid chloride, preferably pivaloyl chloride.