OLIGONUCLEOTIDE SYNTHESIS

Abstract

The invention relates to a process for the manufacture of an oligonucleotide comprising at least one non-chiral phosphorothioate intemucleoside linkage of formula (I) wherein R.sup.1 is as defined in the description and in the claims.

##STR00001##

Claims

1. A process for the manufacture of an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) ##STR00022## said process comprising the step of reacting an oligonucleotide comprising an internucleoside linkage of formula (II) ##STR00023## in the presence of iodine, wherein the concentration of iodine is between about 0.001 M and about 0.01 M; and wherein R.sup.1 is a phosphate protecting group.

2. A process according to claim 1, wherein the sulfur atom of the at least one non-chiral phosphorothioate internucleotide linkage of formula (I) or (II) is linked to the 3′ carbon atom or 5′ carbon atom of an adjacent nucleoside of the oligonucleotide.

3. A process according to claim 1, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprises a fragment of formula (III) ##STR00024## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; provided that X.sup.1 and Y.sup.1 are not both sulfur at the same time; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.1 is sulfur, then R.sup.4a is hydrogen; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.3 is a hydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

4. A process according to claim 1, wherein the oligonucleotide comprising an internucleoside linkage of formula (II) comprises a fragment of formula (IV) ##STR00025## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; provided that X.sup.1 and Y.sup.1 are not both sulfur at the same time; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hdydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —OCF, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.1 is sulfur, then R.sup.4a is hydrogen; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —OCF, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.3 is a hydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

5. A process according claim 3, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) is reacted in the presence of acid to arrive at an oligonucleotide comprising a fragment of formula (V) ##STR00026## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; provided that X.sup.1 and Y.sup.1 are not both sulfur at the same time; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.1 is sulfur, then R.sup.4a is hydrogen; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.3 is a hydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

6. A process according to claim 5, wherein the oligonucleotide comprising a fragment of formula (V) is reacted in the presence of a compound of formula (VI) ##STR00027## to arrive at an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprising a fragment of formula (VII) ##STR00028## wherein Y.sup.2 is oxygen or sulfur; R.sup.2c is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4c is hydrogen or hydroxyalkyl; or R.sup.2c and R.sup.4c together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.2 is sulfur, then R.sup.40 is hydrogen; R.sup.5 is dialkylamino; each R.sup.p is independently alkyl; X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; provided that X.sup.1 and Y.sup.1 are not both sulfur at the same time; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —OCF, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.1 is sulfur, then R.sup.4a is hydrogen; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —OCF, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.3 is a hydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase

7. A process according to claim 6, wherein the oligonucleotide comprising a fragment of formula (VII) is reacted in the presence of a thiooxidation agent or iodine, wherein the concentration of iodine is between about 0.001 M and about 0.01 M, to arrive at an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprising a fragment of formula (VIII) ##STR00029## wherein X.sup.2 is oxygen or sulfur; Y.sup.2 is oxygen or sulfur; provided that X.sup.2 and Y.sup.2 are not both sulfur at the same time; X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; provided that X.sup.1 and Y.sup.1 are not both sulfur at the same time; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, —CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; provided that when Y.sup.1 is sulfur, then R.sup.4a is hydrogen; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —OCF, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.3 is a hydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase R.sup.2c is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy, and R.sup.4c is hydrogen or hydroxyalkyl and R.sup.2c and R.sup.4c are as defined in claim 6.

8. A process according to claim 7, wherein the oligonucleotide comprising a fragment of formula (VII) is reacted in the presence of a thiooxidation agent when Y.sup.2 is oxygen.

9. A process according to claim 7, wherein the oligonucleotide comprising a fragment of formula (VII) is reacted in the presence of iodine when Y.sup.2 is sulfur.

10. A process according to claim 1, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprises a fragment of formula (IX) ##STR00030## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4b is hydrogen or hydroxyalkyl; or R.sup.2b and R.sup.4b together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is a hydroxyl protecting group or a thiohydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

11. A process according to claim 1, wherein the oligonucleotide comprising an internucleoside linkage of formula (II) comprises a fragment of formula (X) ##STR00031## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR')—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, —CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN.sub.3, CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4b is hydrogen or hydroxyalkyl; or R.sup.2b and R.sup.4b together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, —CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is a hydroxyl protecting group or a thiohydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

12. A process according to claim 10, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) is reacted in the presence of acid to arrive at an oligonucleotide comprising a fragment of formula (XI) ##STR00032## wherein X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4b is hydrogen or hydroxyalkyl; or R.sup.2b and R.sup.4b together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—13 , —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, CH(CH.sub.2CH)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is a hydroxyl protecting group or a thiohydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

13. A process according to claim 12, wherein the oligonucleotide comprising a fragment of formula (XI) is reacted in the presence of a compound of formula (XII) ##STR00033## to arrive at an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprising a fragment of formula (XIII) ##STR00034## wherein Y.sup.2 is oxygen or sulfur; R.sup.2c is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, —NH.sub.2, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4c is hydrogen or hydroxyalkyl; or R.sup.2c and R.sup.4c together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—13 , —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is a hydroxyl protecting group or a thiohydroxyl protecting group; R.sup.5 is dialkylamino; each R.sup.p is independently alkyl; and X.sup.1 is oxygen or sulfur; Y.sup.1 is oxygen or sulfur; each R.sup.1 is independently is a phosphate protecting group; R.sup.2a is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4a is hydrogen or hydroxyalkyl; or R.sup.2a and R.sup.4a together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.2b is hydrogen, hydroxyl, fluoro, alkyl, alkoxy, alkoxyalkoxy, alkylamino, dialkylamino, alkylcarbonylamino, azido, —SH, —CN, —CF.sub.3, —OCF.sub.3, alkyl sulfanylalkoxy, aminooxyalkoxy, alkylaminooxyalkoxy, dialkylaminooxyalkoxy, aminocarbonylalkoxy, alkylaminocarbonylalkoxy or dialkylaminocarbonylalkoxy; R.sup.4b is hydrogen or hydroxyalkyl; or R.sup.2b and R.sup.4b together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR.sup.p)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH)—, —CHCH.sub.3C(═CH)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is a hydroxyl protecting group or a thiohydroxyl protecting group; each R.sup.p is independently alkyl; and each Nu is independently a nucleobase.

14. A process according to claim 13, wherein the oligonucleotide comprising a fragment of formula (XIII) is reacted in the presence of a thiooxidation agent or iodine, wherein the concentration of iodine is between about 0.001 M and about 0.01 M, to arrive at an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprising a fragment of formula (XIV) ##STR00035## wherein Y.sup.1 is oxygen or sulfur; X.sup.2 is oxygen or sulfur; provided that Y.sup.1 and X.sup.2 are not both sulfur at the same time;

15. A process according to claim 14, wherein the oligonucleotide comprising a fragment of formula (XIII) is reacted in the presence of a thiooxidation agent when Y.sup.1 is oxygen.

16. A process according to claim 14, wherein the oligonucleotide comprising a fragment of formula (XIII) is reacted in the presence of iodine when Y.sup.1 is sulfur.

17. A process according to claim 1, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) comprises 1 to 8 internucleoside linkages of formula (I).

18. A process according claim 1, wherein the concentration of iodine is between about 0.001 M and about 0.005 M, preferably between about 0.002 M and about 0.005 M.

19. A process according to claim 1, wherein R.sup.1 is cyanoethyl.

20. A process according to claim 1, wherein the hydroxyl protecting group or the thiohydroxyl protecting group is bis-(4-methoxy-phenyl)-phenyl-methyl.

21. A process according to claim 6, wherein R.sup.5 is diisopropylamino.

22. A process according to claim 1, wherein each Nu is independently selected from adenine, thymine, uracil, guanine and cytosine.

23. A process according to claim 1, wherein the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) is bound to a solid support for solid phase synthesis.

24. A process according to claim 1, wherein the acid is dichloroacetic acid or trichloroacetic acid.

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. A process according to claim 1, wherein the phosphate protecting group R.sup.1 of the oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) is removed to arrive at an oligonucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (XV) ##STR00036##

30. (canceled)

31. (canceled)

32. An oligonucleotide manufactured according to a process of claim 1.

33. An oligounucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) ##STR00037## wherein le is a phosphate protecting group as defined in claim 1 comprising 7 to 31 nucleotides.

34. (canceled)

35. An oligounucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I) ##STR00038## wherein R.sup.1 is a phosphate protecting group wherein the oligonucleotide comprises at least one nucleotide of formula (XVI) ##STR00039## wherein X is oxygen or sulfur; R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR')—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2-O—CH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; each R.sup.p is independently alkyl; and Nu is a nucleobase.

36. An oligounucleotide comprising at least one non-chiral phosphorothioate internucleoside linkage of formula (I), ##STR00040## wherein R.sup.1 is a phosphate protecting group wherein the oligonucleotide comprises at least one nucleotide of formula (XVII) ##STR00041## wherein X is oxygen or sulfur; R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR')—, —CHCH.sub.3O—, C(CH.sub.3).sub.2O—13 , —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2-O—CH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; each R.sup.p is independently alkyl; and Nu is a nucleobase.

37. (canceled)

38. (canceled)

39. (canceled)

40. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

Description

EXAMPLES

Example 1

Monomer synthesis 5′S-LNA monomer Synthesis

[0181] To a solution of Cl.sub.3CCOOH (2.98 g, 18.23 mmol) in CH.sub.2Cl.sub.2 (150 ml) was added N-[9-(1-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purin-6-yl]benzamide (10 g, 14.58 mmol) at 25° C. Then the reaction mixture was stirred for 3 h at 25° C. Volatiles were removed under reduced pressure and the resulting crude was purified by combiflash (10% MeOH in CH.sub.2Cl.sub.2) to get N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (5 g, 89%) as white solid. (MS: (ESI): m/z=383.8 [M+H].sup.+).

[0182] To an ice cooled solution of triphenyl phosphine (10.26 g, 39.13 mmol) in anhydrous THF (150.0 mL) was added diethyl azodicarboxylate (6.14 mL, 39.13 mmol) and the reaction mixture was stirred at 0° C. for 30 min. PhCOSH (4.62 mL, 39.13 mmol) was added drop wise to the reaction mixture and it was stirred at 0° C. for another 30 min. N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (5.0 g, 13.04 mmol) was added and the reaction mixture was stirred at 0° C. for 2 h followed by stiffing at room temperature for 2 h. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (120 mL×3). The combined organic layers were washed with NaHCO.sub.3 (100 mL), dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure to get N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide (25 g, crude) as a yellow viscous oil. (MS: (ESI): m/z=504.3 [M+H].sup.+).

[0183] An aqueous solution of NaOH (0.5 M, 238 mL) as well as a mixture of THF-MeOH (6:4, 250 mL) were bubbled with argon for 30 min. N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide (20 g, crude) was dissolved in the argon purged solution of THF-MeOH (6:4, 250 mL) under argon and cooled to 0° C. to −5° C. To this solution was added the NaOH solution (0.5 M, 238 mL, 119.16 mmol) and the reaction mixture was stirred at 0° to −5° C. for 30 min. A solution of citric acid (30.04 g, 142.98 mmol) was added at 0° C. The reaction mixture was diluted with a saturated NaHCO.sub.3 solution (300 mL) and extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure to get N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (20 g, crude) as an off white viscous oil. (MS: (ESI): m/z=400.2 [M+H].sup.+).

[0184] To a solution of N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (20 g, crude) in anhydrous pyridine (20 mL, argon purged) was added DMTrCl (5.09 g, 15.02 mmol) at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Volatiles were removed under reduced pressure and the reaction mixture was diluted with CH.sub.2Cl.sub.2 (300 mL). The organic layer was washed with a sat. NaHCO.sub.3 solution (100 mL×2) followed by brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The resulting crude material was purified by combiflash (2% MeOH in CH.sub.2Cl.sub.2 containing 0.5% triethylamine) to get N-{9-[1-({[bis(4-methoxyphenyl) (phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (5 g, 68% over 3 steps) as a pale yellow solid. (MS: (ESI): m/z=702.14 [M+H].sup.+).

[0185] A solution of 5-ethylmercapto-1H-tetrazole (1.3 g, 9.97 mmol, 0.25 M solution in 38.4 mL dry acetonitrile) was added to a stirred solution of N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (3.5 g, 4.99 mmol) in dry CH.sub.2Cl.sub.2 (120 mL) under argon at room temperature followed by the addition of 2-cyanoethyl tetraisopropylphosphorodiamodite (3.17 mL, 9.98 mmol). After stiffing at room temperature for 4 h the reaction mixture was diluted with CH.sub.2Cl.sub.2 (300 mL) and poured into a sat. NaHCO.sub.3 solution (100 mL). The organic layer was separated off and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (70 mL×2). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The resulting crude compound was purified by combiflash (10-20% ACN in DCM) to get (2.7 g) impure. Using the same protocol two further batches were performed at a 1.0 g and a 2.5 g scale resulting in 0.6 g and 2.5 g of impure product respectively. The impure compound thus obtained from different batches was mixed and repurified to get N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (5′-Mercapto-LNA adenosine phosphoramidite, 4.0 g, 44%) as a white solid. (MS: (ESI): m/z=901.6 [M+H].sup.+).

Example 2

5′-S and 3′-S DNA monomer Synthesis

3′S DNA Phosphoramidite

[0186] To an ice-cooled solution of N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide) (25 g, 38.01 mmol) and 4-nitrobenzoic acid (12.70 g, 76.02 mmol) in dry THF (2.0 L) was added triphenyl phosphine (39.89 g, 152.04 mmol) followed by the drop wise addition of diisopropyl azodicarboxylate (30.74 g, 152.04 mmol) and the reaction mixture was stirred at 0° C. for 2 h. Progress of the reaction was monitored by TLC. Volatiles were removed under reduced pressure to get a pale brown thick mass crude compound which was purifiedby combiflash (70-90% EtOAc in hexane) to afford N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (40 g, crude) as an off-white solid. (MS: (ESI): m/z=807.6 [M+H].sup.+).

[0187] To an ice-cooled solution of N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (40 g, crude) in dry methanol (500 mL) was added K.sub.2CO.sub.3 (6.85 g, 49.58 mmol) under an argon atmosphere and the reaction mixture was stirred at 0° C. for 2 h. Volatiles were removed under reduced pressure to get crude compound which was dissolved in ethyl acetate (300 mL) and washed with water (100 mL). The aqueous layer was further extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to get crude compound which was purified by combiflash (5% MeOH in EtOAc) to afford N-{9-[(2R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl) methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (13 g, 52% after two steps) as an off-white solid. (MS: (ESI): m/z=658.4 [M+H].sup.+).

[0188] To an ice-cooled solution of triphenyl phosphine (15.55 g, 59.30 mmol) in dry THF (350 mL) was added diethyl azodicarboxylate (9.30 mL, 59.30 mmol) drop wise and the reaction mixture was stirred at 0° C. under an argon atmosphere for 30 min. Thiobenzoic acid (7.00 mL, 59.30 mmol) was added and the resulting mixture was stirred at 0° C. for 30 min. After the addition of N-{9-[(2R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (13.0 g, 19.77 mmol) in dry THF (150 mL) stirring was continued at 0° C. for 3 h, allowed to warm to room temperature and stirred at 25° C. for 16 h. Volatiles were removed under reduced pressure to get crude compound which was purified by combiflash (50% ethyl acetate in hexane containing 0.5% triethylamine) to afford N-{9-[(2R,4S,5R)-4-(benzoylsulfanyl)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}oxolan-2-yl]-9H-purin-6-yl}benzamide (8.1g, 53%) as yellow solid. (MS: (ESI): m/z=777.6 [M+H].sup.+).

[0189] An aqueous solution of NaOH (0.5 M, 38.5 mL) as well as a mixture of THF-MeOH (6:4, 75 mL) were bubbled with argon for 30 min. N-{9-[(2R,4S,5R)-4-(benzoylsulfanyl)-5-{[bis (4-methoxyphenyl)(phenyl) methoxy]methyl}oxolan-2-yl]-9H-purin-6-yl}benzamide (5.0 g, 6.43 mmol) was added to the solution of MeOH/THF (75.0 mL) under argon and cooled to 0 to −5° C. To this solution was added the above NaOH solution (38.5 mL, 19.28 mmol) and the reaction mixture was stirred at 0 to −5° C. for 30 min. After neutralization with citric acid at 0° C., a saturated NaHCO.sub.3 solution was added to the reaction mixture and the solution was extracted with ethyl acetate (200 mL×3). Combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to get N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-sulfanyloxolan-2-yl]-9H-purin-6-yl}benzamide (5.3 g, crude) as pale yellow solid. (MS: (ESI): m/z=673.6 [M+H].sup.+).

[0190] A solution of 5-ethylmercapto-1H-tetrazole (1.64 g, 12.62 mmol, 0.25 M solution in 50.0 mL dry acetonitrile) was added to a stirred solution of N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-sulfanyloxolan-2-yl]-9H-purin-6-yl}benzamide (8.5 g, crude) in dry CH.sub.2Cl.sub.2 (80.0 mL) under an argon atmosphere at room temperature followed by the addition of 2-cyanoethyl tetra isopropyl phosphorodiamidite (6.004 mL, 18.92 mmol) and the reaction mixture was stirred at 25° C. for 1 h. Then the reaction mixture was diluted with CH.sub.2Cl.sub.2 (300 mL) and poured into a saturated NaHCO.sub.3 solution (200 mL). The organic layer was separated off and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (100 mL×2). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to get crude compound which was purified by combiflash (50% acetonitrile in CH.sub.2Cl.sub.2) to get N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}sulfanyl)oxolan-2-yl]-9H-purin-6-yl}benzamide (3′-Mercapto-DNA adenosine phosphoramidite, 6.3 g, 70% over 2 steps) as an off-white solid. (MS: (ESI): m/z=874.7 [M+H].sup.+).

5′S DNA Phosphoramidite

[0191] To an ice cooled solution of N-{9-[(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (20 g, 30.41 mmol) in CHCl.sub.3 (300 mL) was added a solution of Cl.sub.3CCOOH (2.48 g, 15.20 mmol) in CHCl.sub.3 (50 ml). Then the reaction mixture was stirred for 2 h at 0° C. Triethylamine (8.5 mL, 60.82 mmol) was added and volatiles were removed under reduced pressure. The resulting crude compound was purified by flash column chromatography (10% MeOH in CH.sub.2Cl.sub.2). The solid thus obtained was washed with water and dried to get N-{9-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-9H-purin-6-yl}benzamide (7 g, 65%) as white solid. (MS: (ESI): m/z=356.0 [M+H].sup.+).

[0192] To an ice cooled solution of triphenyl phosphine (10.33 g, 39.40 mmol) in anhydrous THF (200 mL) was added diethyl azodicarboxylate (6.18 mL, 39.40 mmol) and the reaction mixture was stirred at 0° C. for 30 min. PhCOSH (4.65 mL, 39.40 mmol) was added drop wise and stirring was continued at 0° C. for another 30 min. To the resulting reaction mixture was added N-{9-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-9H-purin-6-yl}benzamide (7 g, 19.70 mmol). After stirring at 0° C. for 2 h, the mixture was allowed to warm to room temperature and stirred at 25° C. for 2 h. Volatiles were removed under reduced pressure and the crude compound thus obtained was purified by combiflash (2% MeOH in CH.sub.2Cl.sub.2) to get N-{9-[(2R,4S,5S)-5-[(benzoylsulfanyl)methyl]-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (5 g, 53%) as pale yellow viscous oil. (MS: (ESI): m/z=475.6 [M+H].sup.+).

[0193] An aqueous solution of NaOH (0.5 M, 25.25 mL) as well as a mixture of THF-MeOH (6:4, 75 mL) were bubbled with argon for 30 min. N-{9-[(2R,4S,5S)-5-[(benzoylsulfanyl)methyl]-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (2.0 g, 4.21 mmol) was dissolved in the argon purged solution of THF-MeOH (6:4, 75 mL) under argon and cooled to 0 to −5° C. To this solution was added the NaOH solution (0.5 M, 25.25 mL, 12.62 mmol) and the reaction mixture was stirred at 0 to −5° C. for 30 min. Citric acid was added (3.18 g, 15.14 mmol) at 0° C. A saturated NaHCO.sub.3 solution (70 mL) was added to the reaction mixture and extracted with ethyl acetate (75 mL×3). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure to get N-{9-[(2R,4S,5S)-4-hydroxy-5-(sulfanylmethyl)oxolan-2-yl]-9H-purin-6-yl}benzamide (1.9 g, crude) as colorless viscous oil. (MS: (ESI): m/z=372.1 [M+H].sup.+).

[0194] To a solution of N-{9-[(2R,4S,5S)-4-hydroxy-5-(sulfanylmethyl)oxolan-2-yl]-9H-purin-6-yl}benzamide (1.9 g, crude, azeotropically distilled with pyridine) in dry pyridine (10 mL, argon purged) was added DMTrCl (1.56 g, 4.60 mmol) at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Volatiles were removed under reduced pressure to get crude compound. The resulting crude was purified by combiflash (0.5% MeOH in CH.sub.2Cl.sub.2 containing 0.5% triethylamine) to get N-{9-[(2R,4S,5S)-5-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (1.2 g, 42% after two steps) as off white solid. (MS: (ESI): m/z=674.3 [M+H].sup.+).

[0195] A solution of 5-ethylmercapto-1H-tetrazole (1.93 g, 14.84 mmol, 0.25 M solution in 60 mL dry acetonitrile) was added to a stirred solution of N-{9-[(2R,4S,5S)-5-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-4-hydroxyoxolan-2-yl]-9H-purin-6-yl}benzamide (5 g, 7.42 mmol) in dry CH.sub.2Cl.sub.2 (120 mL) under argon at 25° C. To the resulting reaction mixture was added 2-cyanoethyl tetra isopropylphosphorodiamidite (4.71 mL, 14.84 mmol) and stirring continued at 25° C. for 4 h. Then the reaction mixture was diluted with CH.sub.2Cl.sub.2 (250 mL) and poured onto a sat. NaHCO.sub.3 solution (250 mL), the organic layer was separated off and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (250 mL×2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to get crude compound which was purified by amine silica gel (80% CH.sub.2Cl.sub.2 in hexane) to get impure compound (3.1 g) which was again purified as above to get the desired N-{9-[(2R,4S,5S)-5-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-4-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)oxolan-2-yl]-9H-purin-6-yl}benzamide (5′-Mercapto-DNA adenosine phosphoramidite, 2.32 g, 36%) as white solid. (MS: (ESI): m/z=874.8 [M+H].sup.+).

Example 3

Incorporation of 3′-S DNA Monomers in Oligonucleotide Synthesis

[0196] Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer by Bioautomation. Syntheses were conducted on a 1 μmol scale using a controlled pore glass support (500Å) bearing a universal linker.

[0197] In standard cycle procedures for the coupling of DNA and LNA phosphoramidites DMT deprotection was performed with 3% (w/v) trichloroacetic acid in CH.sub.2Cl.sub.2 in three applications of 200 μL for 30 sec. The respective phosphoramidites were coupled three times with 100 μL of 0.1M solutions in acetonitrile (or acetonitrile/CH.sub.2Cl.sub.2 1:1 for the LNA-.sup.MeC building block) and 110 μL of a 0.1M solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile as an activator and a coupling time of 180 sec. For thiooxidation a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine 1:1 was used (3×190 μL, 55 sec). Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec.

[0198] Synthesis cycles for the incorporation of 2′,3′-dideoxy-3′-mercapto phosphoramidites included DMT deprotection using 3% (w/v) of trichloroacetic acid in CH.sub.2Cl.sub.2 in three applications of 200 μL for 30 sec. Phosphoramidite coupling was performed ten times with 40 μL of 0.1m solutions in acetonitrile and 44 μL of a 0.1m solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile with a coupling time of 900 sec. Oxidation was performed immediately after coupling by applying six times 200 μL of a 2 mm solution of iodine in THF/H.sub.2O/pyridine 77:2:21 for 50 sec. Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec.

[0199] Removal of the nucleobase protecting groups and cleavage from the solid support was achieved under standard conditions using 32% aqueous ammonia at 55° C. for a minimum of 8h. Crude DMT-on oligonucleotides were purified either using a solid phase extraction cartridge or by RP-HPLC purification using a C18 column followed by DMT removal with 80% aqueous acetic acid and ethanol precipitation.

Example 4

Incorporation of 5′-S-DNA Monomers in Oligonucleotide Synthesis

[0200] Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer by Bioautomation. Syntheses were conducted on a 1 μmol scale using a controlled pore glass support (500Å) bearing a universal linker.

[0201] In standard cycle procedures for the coupling of DNA and LNA phosphoramidites DMT deprotection was performed with 3% (w/v) trichloroacetic acid in CH.sub.2Cl.sub.2 in three applications of 200 μL for 30 sec. The respective phosphoramidites were coupled three times with 100 μL of 0.1m solutions in acetonitrile (or acetonitrile/CH.sub.2Cl.sub.2 1:1 for the LNA-.sup.MeC building block) and 110 μL of a 0.1M solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile as an activator and a coupling time of 180 sec. For thiooxidation a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine 1:1 was used (3×190 μL, 55 sec). Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec.

[0202] Synthesis cycles for the incorporation of 2′,5′-dideoxy-5′-mercapto phosphoramidites included coupling of the phosphoramidite building blocks using 100 μL of 0.1m solutions in acetonitrile and 110 μL of a 0.1m solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile with a coupling time of 180 sec. Triple couplings were performed. Depending on the desired sequence the synthesis column was subjected either to thiooxidation using a 0.1m solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine 1:1 (3×190 μL, 55 sec) or oxidation using a 2 mM solution of iodine in THF/H.sub.2O/pyridine 77:2:21 (6×200 μL, 55 sec). Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec. DMT deprotection and liberation of the thiol was conducted with 3% (w/v) trichloroacetic acid in CH.sub.2Cl.sub.2 in 15 applications of 200 μL for 30 sec. After coupling of the subsequent phosphoramidite building block according to the conditions given above, oxidation was performed using a 2 mm solution of iodine in THF/H.sub.2O/pyridine 77:2:21 (6×200 μL, 55 sec).

[0203] DMT deprotection and liberation of the thiol was also advantageously conducted with 3-6 applications of 200 μL for 45 sec of 1-10% (v/v) trifluoroacetic acid or 5-10% (w/v) trichloroacetic acid, in the presence of 5-30% (v/v) triethylsilane in CH.sub.2Cl.sub.2 and/or 2-10% of p-methoxy thiophenol in CH.sub.2Cl.sub.2.

[0204] Removal of the nucleobase protecting groups and cleavage from the solid support was achieved under standard conditions using 32% aqueous ammonia at 55° C. for a minimum of 8 h. Crude DMT-on oligonucleotides were purified either using a solid phase extraction cartridge or by RP-HPLC purification using a C18 column followed by DMT removal with 80% aqueous acetic acid and ethanol precipitation.

Example 5

Incorporation of 5′-S-LNA Monomers in Oligonucleotide Synthesis

[0205] Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer by Bioautomation. Syntheses were conducted on a 1 μmol scale using a controlled pore glass support (500 Å) bearing a universal linker.

[0206] In standard cycle procedures for the coupling of DNA and LNA phosphoramidites DMT deprotection was performed with 3% (w/v) trichloroacetic acid in CH.sub.2Cl.sub.2 in three applications of 200 μL for 30 sec. The respective phosphoramidites were coupled three times with 100 μL of 0.1 m solutions in acetonitrile (or acetonitrile/CH.sub.2Cl2 1:1 for the LNA-.sup.MeC building block) and 110 μL of a 0.1m solution of 5(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile as an activator and a coupling time of 180 sec. For thiooxidation a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine 1:1 was used (3×190 μL, 55 sec). Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec.

[0207] Synthesis cycles for the incorporation of 2′,5′-dideoxy-5′-mercapto LNA-phosphoramidites included coupling of the phosphoramidite building blocks using 100 μL of 0.1M solutions in acetonitrile and 110 μL of a 0.1M solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile with a coupling time of 600 sec. Triple couplings were performed. Depending on the desired sequence the synthesis column was subjected either to thiooxidation using a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine 1:1 (3×190 μL, 55 sec) or oxidation using a 2 mM solution of iodine in THF/H.sub.2O/pyridine 77:2:21 (6×200 μL, 55 sec). Capping was performed using THF/lutidine/Ac.sub.2O 8:1:1 (CapA, 75 μmol) and THF/N-methylimidazole 8:2 (CapB, 75 μmol) for 55 sec. DMT deprotection and liberation of the thiol was conducted with 3% (w/v) trichloroacetic acid in CH.sub.2Cl.sub.2 in 15 applications of 200 μL for 30 sec. After coupling of the subsequent phosphoramidite building block according to the conditions given above, oxidation was performed using a 2 mm solution of iodine in THF/H.sub.2O/pyridine 77:2:21 (6×200 μL, 55 sec).

[0208] DMT deprotection and liberation of the thiol was also advantageously conducted with 3-6 applications of 200 μL for 45 sec of 1-10% (v/v) trifluoroacetic acid or 5-10% (w/v) trichloroacetic acid, in the presence of 5-30% (v/v) triethylsilane in CH.sub.2Cl.sub.2 and/or 2-10% of p-methoxy thiophenol in CH.sub.2Cl.sub.2.

[0209] Removal of the nucleobase protecting groups and cleavage from the solid support was achieved under standard conditions using 32% aqueous ammonia at 55° C. for a minimum of 8 h. Crude DMT-on oligonucleotides were purified either using a solid phase extraction cartridge or by RP-HPLC purification using a C18 column followed by DMT removal with 80% aqueous acetic acid and ethanol precipitation.

Example 6

3′-S Oligonucleotides

[0210] According to the General Procedure outlined above the following molecules were prepared:

TABLE-US-00001 Compound Calculated Found ID No. Sequence mass mass  #1 GAGttacttgccaA.sup.mCT 5275.4903 5275.4745  #2 GAGttacttgccaA.sup.mCT 5275.4903 5275.4760  #3 GAGttacttgccaA.sup.mCT 5275.4903 5275.4826  #4 GAGttacttgccaA.sup.mCT 5275.4903 5275.4858  #5 GAGttacttgccaA.sup.mCT 5275.4903 5275.4706  #6 GAGttacttgccaA.sup.mCT 5275.4903 5275.4716  #7 GAGttacttgccaA.sup.mCT 5275.4903 5275.4743  #8 GAGttacttgccaA.sup.mCT 5275.4903 5275.4892  #9 GAGttacttgccaA.sup.mCT 5275.4903 5275.4947 #10 GAGttacttgccaA.sup.mCT 5275.4903 5275.4845 #11 GAGttacttgccaA.sup.mCT 5275.4903 5275.5082 #12 GAGttacttgccaA.sup.mCT 5275.4903 5275.4948 #13 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4244 #14 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4262 #15 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.2726 #16 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.2687 #17 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4256 #18 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4278 #19 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4266 #20 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4263 #21 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4515 #22 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4214 #23 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4224 #24 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4220 #25 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4560 #26 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4490 #27 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4208 #28 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4210 #29 A.sup.mCTtatggtta.sup.mCG 4322.4292 4322.4224 #30 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4872 #31 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4877 #32 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4872 #33 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4903 #34 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4862 #35 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4898 #36 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4872 #37 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4829 #38 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4904 #39 .sup.mCA.sup.mCattccttgct.sup.mCTG 5230.4927 5230.4879
a, g, c, t represent 2′,3′-Dideoxy-3′-mercapto modifications [0211] A, G, .sup.mC, T represent LNA nucleotides [0212] a, g, c, t represent DNA nucleotides [0213] all oligonucleotides were prepared as full phosphorothioates (with a sulfur atom either at a terminal or in the 3′-position)

Example 7

5′-S Oligonucleotides

[0214] According to the General Procedure outlined above the following molecules were prepared:

TABLE-US-00002 Compound Calculated Found ID No. Sequence mass mass #40 GAGttacttgccaA.sup.mCT 5275.4903 5275.4880 #41 GAGttacttgccaA.sup.mCT 5275.4903 5275.4849 #42 GAGttacttgccaA.sup.mCT 5275.4903 5275.5014 #43 GAGttacttgccaA.sup.mCT 5275.4903 5275.4904 #44 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4227 #45 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4236 #46 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4238 #47 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4228 #48 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4235 #49 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4225 #50 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4226 #51 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4220 #52 G.sup.mCattggtatT.sup.mCA 4322.4292 4322.4256 [0215] a, g, c, t represent 2′,5′-Dideoxy-5′-mercapto modifications [0216] A, G, .sup.mC, T represent LNA nucleotides [0217] a, g, c, t represent DNA nucleotides [0218] all oligonucleotides were prepared as full phosphorothioates (with a sulfur atom either at a terminal or in the 5′-position)