COMPOSITIONS AND METHODS FOR PHOSPHORAMIDITE AND OLIGONUCLEOTIDE SYNTHESIS

• US 20190375774A1
• Filed: 11/22/2017
• Published: 12/12/2019
• Est. Priority Date: 11/23/2016
• Status: Active Grant

• Current Assignee: WAVE Life Sciences Ltd.
• Original Assignee: WAVE Life Sciences Ltd.
• Inventors: Butler, David Charles Donnell, Kandasamy, Pachamuthu, Marappan, Subramanian, Paik, Ik-Hyeon, Kumarasamy, Jayakanthan, Bommineni, Gopal Reddy
• Granted Patent: US 11,873,316 B2
• Time in Patent Office: Days
• CPC Class Codes: C07F 9/65844 the phosphorus atom being part of a five-membered ring which may be condensed with another ring system
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 1/06 Separation; Purification
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

The present disclosure, among other things, provides technologies for oligonucleotide synthesis. In some embodiments, the present disclosure provides phosphoramidites and methods for synthesis thereof. In some embodiments, provided methods provides higher yields and/or purities. In some embodiments, provided methods remove byproducts without contact with an aqueous solution.

Claims

1. A method for preparing a phosphoramidite, comprising steps of:

a) providing a preparation comprising a phosphoramidite product and one or more reaction byproducts of the product'"'"'s synthesis; and

b) performing a filtration step on the preparation to provide a filtrate, wherein the filtrate contains a reduced level of the one or more reaction byproducts relative to the product as compared to that level present in the preparation prior to the filtration step.

2. The method of claim 1, wherein the preparation comprises a solution and one or more solids.

3. The method of claim 2, wherein the solution comprises the phosphoramidite product.

4. The method of claim 3, wherein one or more solids comprise one or more reaction byproducts.

5. The method of claim 4, wherein the phosphoramidite has the structure of formula I:

6. The method of claim 5, wherein SU is

7. The method of claim 6, wherein R^2s is —

H.

8. The method of claim 6, wherein R^2s is —

F.

9. The method of claim 6, wherein R^2s is —

OMe.

10. The method of claim 6, wherein R^2s is —

OCH₂CH₂OMe.

11. The method of claim 6, wherein R^2s is L connecting C2 with C4, wherein L is (C2)-O-(optionally substituted methylene)-(C4).

12. The method of claim 6, wherein R^2s is L connecting C2 with C4, wherein L is (C2)-O-(unsubstituted methylene)-(C4).

13. The method of any one of claims 6-12, wherein R¹, R², and R³ are taken together with their intervening nitrogen, phosphorus and oxygen atoms to form

14. The method of claim 13, wherein Ring A comprises a ring system having the backbone structure of

15. The method of claim 14, wherein

16. The method of claim 14, wherein no solution comprising the phosphoramidite product is contacted with water of more than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 mole equivalents of the phosphoramidite.

17. The method of claim 16, comprising purifying the phosphoramidite product using silica gel column chromatography, wherein the silica gel is treated with an anhydrous acetonitrile.

18. In a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product'"'"'s synthesis, the improvement comprising:

subjecting a preparation comprising the phosphoramidite product and the one or more reaction byproducts to filtration without adding a material amount of water to the preparation after the phosphoramidite product has been formed.

19. In a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product'"'"'s synthesis, the improvement comprising:

subjecting a preparation comprising the phosphoramidite product and the one or more reaction byproducts to filtration without adding a material amount of any aqueous or halogenated solvent to the preparation after the phosphoramidite product has been formed.

20. In a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product'"'"'s synthesis, the improvement comprising:

subjecting a preparation comprising the phosphoramidite product and the one or more reaction byproducts to filtration without adding a material amount of any aqueous solvent, or performing any extraction from an aqueous solvent, after the phosphoramidite product has been formed.

21. A compound having the structure of formula I:

31. A method for preparing an oligonucleotide, comprising reacting a compound of any one of claims 21-30 to incorporate a nucleoside into an oligonucleotide.

32. A method for preparing an oligonucleotide, comprising providing a compound of any one of claims 21-30.

22. A compound having the structure of formula I:

23. A compound having the structure of formula I:

24. The compound of claim 23, wherein BA is

25. A compound or a salt thereof, wherein the compound is an oligonucleotide comprising one or more base moieties BA, wherein BA is protected

27. The compound of claim 25 or 26, wherein the oligonucleotide comprises one or more moieties having the structure of BA-SU—

, wherein;

SU is -L-O—

or

28. The compound of claim 27, wherein BA is

29. The compound of claim 28, wherein the oligonucleotide comprises about or more than 5, 6, 7, 8, 9, 10, 11, 12, 14, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 chiral linkage phosphorus.

26. A compound or a salt thereof, wherein the compound is an oligonucleotide comprising one or more base moieties BA, wherein BA is substituted

30. A compound selected from:

33. A compound or method of any one of embodiments 1-265.