4'-phosphate analogs and oligonucleotides comprising the same
Disclosed herein are oligonucleotides, such as nucleic acid inhibitor molecules, having a 4′-phosphate analog and methods of using the same, for example, to modulate the expression of a target gene in a cell. The oligonucleotide of the disclosure comprises a 5′-terminal nucleotide represented by Formula III: wherein R a , R b , B, X 2 and Y are as defined in the specification. The phosphate analogs are bound to the 4′-carbon of the sugar moiety (e.g., a ribose or deoxyribose or analog thereof) of the 5′-terminal nucleotide of an oligonucleotide. Typically, the phosphate analog is an oxymethylphosphonate, where the oxygen atom of the oxymethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. An illustrative 5′-terminal nucleotide of an oligonucleotide of the disclosure may have the following chemical structure:
1. An oligonucleotide comprising a 5′-terminal nucleotide, wherein the 5′-terminal nucleotide is represented by Formula III:
wherein R a and R b is each independently selected from hydrogen, CH 3 , CH 2 CH 3 , CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si(CH 3 ) 3 , or a protecting group;
wherein B is a natural nucleobase, a modified nucleobase, a universal base or absent;
wherein Y is an internucleotide linking group attaching the 5′-terminal nucleotide to an oligonucleotide; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent or wherein X 2 is O and R 8 is a glutathione-sensitive moiety.
2. The oligonucleotide of claim 1 , wherein the glutathione sensitive moiety comprises a sulfonyl group or a disulfide bond.
3. The oligonucleotide of claim 1 , wherein X 2 is OH, F, or OCH 3 and R 8 is absent.
4. The oligonucleotide of claim 1 , wherein R a and R b are hydrogen; R a is CH 3 or CH 2 CH 3 and R b is hydrogen; or R a and R b are each CH 3 or CH 2 CH 3 .
5. The oligonucleotide of claim 1 ,
wherein R a and R b are hydrogen; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent.
6. The oligonucleotide of claim 1 ,
wherein R a is CH 3 and R b is hydrogen; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent.
7. An oligonucleotide comprising a 5′-terminal nucleotide, wherein the 5′-terminal nucleotide is represented by Formula VI:
wherein R a and R b is each independently selected from hydrogen, CH 3 , CH 2 CH 3 , CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si(CH 3 ) 3 , or a protecting group;
wherein V is O;
wherein Z is a nucleoside comprising a sugar moiety;
wherein Y is an internucleotide linking group attaching the 5′-terminal nucleotide to an oligonucleotide; and
wherein V is bound to the 4′-carbon of the sugar moiety.
8. The oligonucleotide according to claim 7 , wherein the sugar moiety is a furanose.
9. The oligonucleotide of claim 3 , wherein the 5′-terminal nucleotide comprises a 4′-oxymethylphosphonate and wherein X 2 is OCH 3 or F.
10. The oligonucleotide according to claim 1 , wherein the oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a first strand and a second strand, wherein the first strand is a sense strand and the second strand is an antisense strand.
11. The oligonucleotide according to claim 10 , wherein the double stranded RNAi inhibitor molecule comprises a region of complementarity between the sense strand and the antisense strand of 15 to 45 nucleotides.
12. The oligonucleotide according to claim 11 , wherein the region of complementarity between the sense strand and the antisense strand is 20 to 30 nucleotides.
13. The oligonucleotide according to claim 12 , wherein the region of complementarity between the sense strand and the antisense strand is 21 to 26 nucleotides.
14. The oligonucleotide according to claim 11 , wherein the region of complementarity between the sense strand and the antisense strand is 19 to 24 nucleotides.
15. The oligonucleotide according to claim 14 , wherein the region of complementarity between the sense strand and the antisense strand is 19 to 21 nucleotides.
16. The oligonucleotide according to claim 10 , wherein the 5′-terminal nucleotide is located on the antisense strand.
17. The oligonucleotide according to claim 10 , wherein the 5′-terminal nucleotide is located on the sense strand.
18. The oligonucleotide according to claim 10 , wherein the double-stranded RNAi inhibitor molecule contains a tetraloop.
19. The oligonucleotide according to claim 1 , wherein the oligonucleotide is a single stranded oligonucleotide.
20. The oligonucleotide according to claim 19 , wherein the single stranded oligonucleotide is a single stranded RNAi inhibitor molecule.
21. The oligonucleotide according to claim 19 , wherein the single-stranded oligonucleotide is a conventional antisense oligonucleotide, a ribozyme or an aptamer.
22. The oligonucleotide according to claim 20 , wherein the single stranded RNAi inhibitor molecule is 14-50 nucleotides in length.
23. The oligonucleotide according to claim 22 , wherein the single stranded RNAi inhibitor molecule is about 16-30, 18-22, or 20-22 nucleotides in length.
24. The oligonucleotide according to claim 1 , wherein the oligonucleotide is a naked oligonucleotide.
25. The oligonucleotide according to claim 1 , further comprising at least one delivery agent, wherein the at least one delivery agent is conjugated to the oligonucleotide to facilitate transport of the oligonucleotide across an outer membrane of a cell.
26. The oligonucleotide according to claim 25 , wherein the at least one delivery agent is selected from the group consisting of carbohydrates, peptides, lipids, vitamins and antibodies.
27. The oligonucleotide according to claim 25 , wherein the at least one delivery agent is selected from N-Acetylgalactosamine (GalNAc), mannose-6-phosphate, galactose, oligosaccharide, polysaccharide, cholesterol, polyethylene glycol, folate, vitamin A, vitamin E, lithocholic acid and a cationic lipid.
28. A pharmaceutical composition comprising the oligonucleotide according to claim 10 and a pharmaceutically acceptable excipient.
29. A method for reducing expression of a target gene in a subject comprising administering the pharmaceutical composition of claim 28 to a subject in need thereof in an amount sufficient to reduce expression of the target gene.
30. The method of claim 29 , wherein the administering comprises systemic administration.
31. A nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by Formula XII:
wherein R c and R d is each independently selected from CH 3 , CH 2 CH 3 , CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si(CH 3 ) 3 , or a protecting group;
wherein B is a natural nucleobase attached to a protecting group, a modified nucleobase attached to a protecting group, a universal nucleobase attached to a protecting group, or absent;
wherein R 10 is a phosphoramidite; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent or wherein X 2 is O and R 8 is a glutathione-sensitive moiety.
32. The nucleoside phosphoramidite of claim 31 , wherein X 2 is O and R 8 is a glutathione-sensitive moiety.
33. The nucleoside phosphoramidite of claim 31 , wherein X 2 is F, OCH 2 CH 2 OCH 3 or OCH 3 and R 8 is absent.
34. The nucleoside phosphoramidite of claim 31 , wherein R c and R d are each CH 3 or wherein R c and R d are each CH 2 CH 3 .
35. The nucleoside phosphoramidite of claim 31 , wherein the
glutathione sensitive moiety comprises a sulfonyl group or a disulfide bond.
36. The nucleoside phosphoramidite of claim 31 , wherein R c and R d is each independently selected from CH 3 , CH 2 CH 3 , or a protecting group.
37. The nucleoside phosphoramidite of claim 31 , wherein X 2 is F or OCH 3 and R 8 is absent.
38. The nucleoside phosphoramidite of claim 31 ,
wherein R c and R d are CH 2 CH 3 ; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent.
39. The nucleoside phosphoramidite of claim 31 ,
wherein R c and R d are CH 3 ; and
wherein X 2 is OH, F, OCH 3 , or OCH 2 CH 2 OCH 3 and R 8 is absent.
40. A nucleoside phosphoramidite, wherein the nucleoside phosphoramidite is represented by Formula XV:
wherein R c and R d is each independently selected from CH 3 , CH 2 CH 3 , CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si(CH 3 ) 3 , or a protecting group;
wherein V is O;
wherein Z 1 is a nucleoside comprising a phosphoramidite and a sugar moiety; and
wherein V is bound to the 4′-carbon of the sugar moiety.
41. The nucleoside phosphoramidite according to claim 40 , wherein the sugar moiety is a furanose.
42. The nucleoside phosphoramidite according to claim 40 , wherein R c and R d are each CH 3 or wherein R c and R d are each CH 2 CH 3 .
43. The oligonucleotide according to claim 1 , wherein the oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.
44. The oligonucleotide according to claim 43 , wherein the first strand and the second strand of the double-stranded oligonucleotide is each 15-100 or 15-50 nucleotides in length.