Patent EA-003091-B1 (EA)
Title: Solution phase synthesis of oligonucleotides
Inventor: REESE COLIN BERNARD
IPC: C07D265/33
Filed: 1998-08-10
Abstract: 1. A process for the preparation of a phosphorothioate triester which comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester. 2. A process according to claim 1 wherein the H-phosphonate is a protected nucleoside or oligonucleotide comprising a 3'-H-phosphonate function. 3. A process according to either of claims 1 and 2, wherein the alcohol is a protected nucleoside or oligonucleotide comprising a free 5'-hydroxy function. 4. A process according to any preceding claim, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO)2POCI, in which Ar represents phenyl, 2chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl. 5. A process according to any preceding claim, wherein the sulfur transfer agent has the general chemical formula:   wherein L represents a leaving group, and A represents a substituted or unsubstituted phenyl or naphthyl group, a methyl group, a substituted C1-4 alkyl group or an C1-4 alkenyl group. 6. A process according to claim 5, wherein the leaving group is a morpholine-3,5dione, phthalimide, succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group. 7. A process according to any preceding claim, wherein the H-phosphonate and the alcohol is a ribonucleoside, a 2-O'-(C1-4 alkyl, C1-4 alkoxy a C1-4 alkyl or C1-4 alkenyl)-ribonucleoside, an oligoribonucleotide or a 2-O'-(C1-4 alkyl, C1-4 alkoxy C1-4 alkyl or C1-4 alkenyl)-oligoribonucleotide. 8. An H-phosphonate having the general chemical formula:   wherein each B independently is a base selected from A, G, T, C or U; each Q independently is H or OR' wherein R' is C1-4-alkyl, substituted C1-4-alkyl, C1-4-alkenyl or a protecting group; each R independently is a substituted or unsubstituted phenyl or naphthyl, methyl, substituted C1-4-alkyl or C1-4-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 an integer and is at least 2; 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. 9. An H-phosphonate having the general chemical formula:   wherein each B independently is a base selected from A, G, T, C or U; each Q independently is H or OR' wherein R' is C1-4-alkyl, substituted C1-4-alkyl, C1-4-alkenyl or a protecting group; each R independently is a substituted or unsubstituted phenyl or naphtyl, methyl, substituted C1-4-alkyl or C1-4-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 S; Z is H, a protecting group or an H-phosphonate group of formula   in which M<+> is a monovalent cation; and n is an 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. 10. An H-phosphonate according to claim 9, wherein W represents a protecting group, each X represents O, each Y represents S 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(C1-4-alkylammonium) ion, and n is 1 to 16. 11. A process for the production of an oligonudeotlde H-phosphonate comprising a 3'-terminal or S'-teminal H-phosphonate group, wherein an oligonucleotide comprising a free 3<1>-or 5'- hydroxy functon is reacted with either a) a C1-4-H-phosphonate wherein the alkyl group is substituted by a halo group; or b) with a substituted or unsubstituted phenyl H-phosphonate saft; in the presence of an activator. 12. A process according to claim 11, wherein the oligonucleotide comprising a free 3'- or 5'- hydroxy hydroxy function is a protected oligodeoxyribonucleotide or oligoribonucleotide. 13. A process according to claim 11 or 12, wherein the alkyl or aryl H- phosphonate salt is an ammonium salt of a phenyl, C1-4-alkylphenyl or halophenyl H-phosphonate. 14. A process according to any one of claims 11 to 13, wherein the activator is an aryl phosphorochloridate or an alkyl acid chloride. 15. A process according to claim 14, wherein the activator is pivaloyl chloride. 16. A process according to any one of claims 11 to 15, wherein the free hydroxy function is a 3' hydroxy function. 17. An H-phosphonate according to claim 8, wherein W represents a protecting group, each X represents O, 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(C1-4-alkylammonium) ion, and n is 2 to 16. 18. An H-phosphonate according to either of claims 8 or 17, wherein n is 2 or 3. 19. An H-phosphonate according to either of claims 9 or 10, wherein n is 1, 2 or 3. 20. A process according to any one of claims 1 to 3, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO)2POCl, in which Ar represents phenyl, 2-chlorophenyl, 2,4.6-trichlorophenyl or 2.4,6-tribromophenyl; and the sulfur transfer agent has the general chemical formula:   wherein L represents a leaving group, and A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group. 21. A process according to claim 20, wherein the leaving group is a morpholine-3.5-dione, phthalimide, succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-halophenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nltrophenyl)ethyl group. 22. A process according to claim 20 or 21, wherein the H-phosphonate and the alcohol are selected from the group consisting of deoxyribonucleosides, oligodeoxyribonucleotides, ribonucleosides, 2'-O-(alkyl, alkoxyalkyl or alkenyl)-ribonucleosides, oligoribonucleotides and 2'-O-(alkyl, alkoxyalkyl or alkenyl)-oligoribonucleotides. 23. A process for the preparation of a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected olfgonudeotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages which comprises: a) solution phase coupling of a protected nucleoside or oligonucleotide H-phosphonate comprising a 3'- or 5'-H-phosphonate function with a protected nucleoside or oligonucleotide comprising a free 3'- or 5'-hydroxy function in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester; and b) deprotecting the phosphorothioate triester produced in a) thereby to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate ollgonudeotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothiaote diester internucleotide linkages. 24. A process according to claim 23, wherein the protected nucleoside or oligonucleotide H-phosphonate comprises a 3'-H-phosphonate function and the protected nucleoside or oligonucleotide comprises a free 5'-hydroxy function. 25. A process according to claims 23 or 24, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO2POCl, in which Ar represents phenyl, 2- chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl. 26. A process according to any one of claims 23 to 25. wherein the sulfur transfer agent has the general chemical formula:   wherein L represents a leaving group, and A represents an aryl group, a methyl group, a substituted C1-4-alkyl group or C1-4-an alkenyl group. 27. A process according to claim 26. wherein the leaving group is a morpholine-3,5-dlone, phthalimide. succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group. 28. A process according to any one of claims 23 to 27, wherein an oligonucleotide H-phosphonate and/or an oligonucleotide comprising a free 3' or 5'-hydroxy function is employed and either or both of the oligonucleotide H-phosphonate and the oligonucleotide comprising a free 3' or 5'-hydroxy function comprise one or more phosphorothioate internucleotide linkages. 29. A process according to any one of claims 23 to 28, wherein the deprotected oligonucleotide is subsequently purified. 30. A process according to claim 29, wherein the deprotected oligonucleotide comprises one or more phosphorothioate diester linkages. 31. A process according to any one of claims 11 to 16, wherein the oligonucleotide comprising a free hydroxy group comprises one or more phosphorothioate internucleotide linkages. 32. A process according to any one of claims 2 to 7, wherein the H-phosphonate comprising a 3' H-phosphonate function is a protected oligonucleotide H-phosphonate comprising at least one phosphorothioate internucleotide linkage. 33. A process according to any one of claims 3 to 7, wherein the alcohol comprising a free S'-hydroxy function is an oligonucleotide comprising at least one phosphorothioate intetnucleotide linkage. 34. A process according to claim 33, wherein the H-phosphonate is a protected oligonucleotide H-phosphonate comprising a 3' H-phosphonate fundion and also comprises at least one phosphorothioate internucleotide linkage. 35. A compound of formula:   wherein A represents a substitute