Techniques for obtaining oligonucleotides

Abstract

FIELD: medicine; chemistry.SUBSTANCE: method for producing an oligonucleotide. The method comprises two or more cycles, each of which independently comprises a coupling stage, a capping stage before modification, a modification stage, a capping stage after modification, and a deblocking stage, wherein in each cycle the coupling stage independently comprises reacting a free hydroxyl group of an oligonucleotide or a nucleoside with a compound that is a coupling partner and has a structure specified in the claims, wherein the modification stage comprises sulfurization, wherein the sulfurization comprises converting a phosphorus atom in the -P(-)- bond into a phosphorus atom in the -P(=S)(-)- bond, wherein the modification stage comprises converting a phosphorus atom in the -P(-)- bond into a phosphorus atom in the -P(=N)(-)- bond; BA is 5-Me-C(N-4-Ac or Bz), U, T, A(N-6-Bz), or G(N-2-iBu); R2s is −H, −F, −OCH3, or −OCH2CH2OCH3; R4s is −H; R2 is −CH2SO2R', where R' represents a C1-6 aliphatic group or a C6 aryl group optionally substituted with −CN; and R'O− is DMTrO−.EFFECT: improvement or facilitation of obtaining compositions based on oligonucleotides with controlled chirality in order to provide significant advantages, for example, in relation to therapeutic oligonucleotides.24 cl, 5 dwg, 48 tbl, 15 ex

Claims

1. A method for producing an oligonucleotide comprising two or more cycles, each of which independently comprises the following steps: (1) the combination stage; (2) the capping stage before modification; (3) modification stage; (4) the capping stage after modification and (5) the unblocking stage, and wherein in each cycle the coupling step independently comprises reacting the free hydroxyl group of the oligonucleotide or nucleoside with a compound which is a coupling partner and has the structure or its salts; wherein the modification step comprises sulfurization, wherein the sulfurization comprises the conversion of a phosphorus atom in the −P(−)− bond into a phosphorus atom in the −P(=S)(–)– bond; where the modification step involves converting a phosphorus atom in the −P(−)− bond into a phosphorus atom in the −P(=N)(–)– bond; BA is 5-Me-C(N-4-Ac or Bz), U, T, A(N-6-Bz) or G(N-2-iBu); R 2s represents −H, −F, −OCH3, or −OCH2CH2OCH3; R 4s represents −H; R 2 is −CH2SO2R', where R' is C 1-6 an aliphatic group or C6aryl, optionally substituted with -CN; and R'O− is DMTrO−.

2. The method according to claim 1, wherein the cycle includes a modification step in which an internucleotide bond is formed* P S or * P R, Where * P S independently has the formula or is its salt form, * P R independently has the formula or is its salt form, where −X−L s -R 5 represents where R 6 represents −C(O)R', where R' represents C 1-6 aliphatic group.

3. The method according to claim 1 or 2, wherein the cycle includes a modification step in which an internucleotide linkage is formed* N S or * N R, where * N S independently has the formula or is its salt form, * N R independently has the formula or is its salt form, where −X−L s -R 5 represents where P=W N represents P N ; P N represents where each of R 1 and R s independently represents –H or C 1-6 aliphatic group; Q - is an anion; R 6 represents −C(O)R', where R' represents C 1-6 aliphatic group.

4. The method according to paragraph 3, where in the partner compound P N represents Q - .

5. The method according to any of the preceding claims, wherein in the partner compound R 2s represents –H.

6. The method according to any one of paragraphs 1-4, where in the partner compound R 2s represents −F.

7. The method according to any one of paragraphs 1-4, where in the partner compound R 2s is –OCH3.

8. The method according to any one of paragraphs 1-4, where in the partner compound R 2s is −OCH2CH2OCH3.

9. The method according to any of the preceding paragraphs, where R 2 is −CH2SO2R', where R' is C 1-6 aliphatic group.

10. The method according to any one of paragraphs 1-8, where in the partner compound R 2 is –CH2SO2R', where R' is optionally substituted phenyl.

11. The method according to any one of paragraphs 1-8 or 10, where in the partner compound R 2 is –CH2SO2R', where R' is phenyl.

12. The method according to any one of paragraphs 1-9, where in the partner compound R 2 is –CH2SO2R', where R' is tert-butyl.

13. The method according to any one of the preceding claims, comprising removing the chiral auxiliary group by contacting oligonucleotides containing the chiral auxiliary group with a base under anhydrous conditions.

14. The method of claim 13, wherein the base is DEA.

15. The method according to any of the preceding claims, wherein the oligonucleotide product comprises a phosphorothioate internucleotide linkage.

16. The method according to any one of the preceding claims, wherein the oligonucleotide product comprises a sugar containing 2'-OH.

17. The method according to any of the preceding claims, wherein the oligonucleotide product further comprises an internucleotide linkage that is not negatively charged.

18. The method according to any of the preceding claims, wherein the oligonucleotide product comprises an internucleotide linkage that is not negatively charged and that contains a phosphoryl guanidine moiety.

19. The method according to any one of the preceding claims, wherein the internucleotide linkage that is not negatively charged is 20. The method according to any of the preceding claims, wherein the oligonucleotide product further comprises a natural phosphate bond.

21. The method according to any one of the preceding claims, wherein in at least one ring the partner compound does not contain a chiral auxiliary group.

22. The method according to any one of the preceding claims, wherein in at least one ring the partner compound is 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.

23. The method according to any one of the preceding claims, wherein the partner connection is or its salt.

24. The method according to any one of paragraphs 1-22, wherein the partner compound is or its salt.

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