Claims
- 1. A method for synthesizing oligonucleotides which comprises (a) condensing the 3′-OH or 5′-OH group of a support-bound nucleoside or oligonucleotide with a monomeric nucleoside phosphoramidite having a carbonate-protected hydroxyl group, to provide an intermediate in which the support-bound nucleoside or oligonucleotide is bound to the monomeric nucleoside through a phosphite triester linkage, and (b) treating the intermediate provided in step (a) with a deprotecting reagent effective to convert the carbonate-protected hydroxyl group to a free hydroxyl moiety and simultaneously oxidize the phosphite triester linkage to give a phosphotriester linkage.
- 2. The method of claim 1, wherein steps (a) and (b) are carried out in aqueous solution.
- 3. The method of claim 1, wherein step (b) is an irreversible reaction.
- 4. The method of claim 1, wherein step (b) is conducted at neutral to mildly basic pH.
- 5. The method of claim 4, wherein the deprotecting reagent used in step (b) comprises a nucleophile that exhibits an alpha effect at neutral to mildly basic pH.
- 6. The method of claim 4, wherein step (b) is conducted at a pH of less than about 10.
- 7. The method of claim 1, wherein the nucleophile is a peroxide.
- 8. The method of claim 7, wherein the peroxide is an inorganic peroxide of the formula M+OOH−, wherein M+ is a counterion.
- 9. The method of claim 8, wherein the counterion is selected from the group consisting of H+, Li+, Na+, K+, Rb+ and Cs+.
- 10. The method of claim 7, wherein the peroxide is an organic peroxide of the formula ROOH, wherein R is hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
- 11. The method of claim 10, wherein the peroxide has the structure of formula (V), (VI) or (VII)
- 12. The method of claim 7, wherein the peroxide is one of t-butyl hydroperoxide, m-chloroperoxybenzoic acid, and mixtures thereof.
- 13. The method of claim 5, wherein step (b) is conducted at a pH at which said nucleophile exhibits a maximum alpha effect.
- 14. The method of claim 13, wherein step (b) is conducted at a pH that is greater than the pKa of said nucleophile.
- 15. The method of claim 1, wherein steps (a) and (b) are repeated until an oligonucleotide having a desired sequence and length is provided.
- 16. The method of claim 1, wherein synthesis is conducted in the 3′-to-5′ direction, and step (a) involves condensation at the 5′-OH of the support-bound nucleoside or oligonucleotide.
- 17. The method of claim 16, wherein the monomeric nucleoside phosphoramidite has the structure of formula (II)
- 18. The method of claim 17, wherein X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing β-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.
- 19. The method of claim 18, wherein Y is selected from the group consisting of methyl, β-cyanoethyl and 4-nitrophenylethyl.
- 20. The method of claim 17, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 21. The method of claim 18, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 22. The method of claim 19, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 23. The method of claim 17, wherein R3 comprises a moiety that becomes fluorescent or colored upon cleavage of the carbonate —OCOOR3.
- 24. The method of claim 1, wherein synthesis is conducted in the 5′-to-3′ direction, and step (a) involves condensation at the 3′-OH of the support-bound nucleoside or oligonucleotide.
- 25. The method of claim 24, wherein the monomeric nucleoside phosphoramidite has the structure of formula (X)
- 26. The method of claim 25, wherein X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing β-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.
- 27. The method of claim 26, wherein Y is selected from the group consisting of methyl, β-cyanoethyl and 4-nitrophenylethyl.
- 28. The method of claim 25, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 29. The method of claim 26, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 30. The method of claim 27, wherein R1 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 31. The method of claim 25, wherein R3 comprises a moiety that becomes fluorescent or colored upon cleavage of the carbonate —OCOOR3.
- 32. A method for making an oligonucleotide array made up of array features each presenting a specified oligonucleotide sequence at an address on an array substrate, the method comprising steps of:
providing a hydroxyl-derivatized array substrate and treating the array substrate to protect hydroxyl moieties on the derivatized surface from reaction with phosphoramidites, then iteratively carrying out the steps of (i) applying droplets of an alpha effect nucleophile to effect deprotection of hydroxyl moieties at selected addresses, and (ii) flooding the array substrate with a medium containing a selected monomeric nucleoside phosphoramidite having a carbonate-protected hydroxyl group, to permit covalent attachment of the selected nucleoside to the deprotected hydroxyl moieties at the selected addresses.
- 33. The method of claim 32, wherein steps (i) and (ii) are carried out in aqueous solution.
- 34. The method of claim 32, wherein step (i) is an irreversible reaction.
- 35. The method of claim 31, wherein step (i) is conducted at neutral to mildly basic pH.
- 36. The method of claim 35, wherein step (b) is conducted at a pH of less than about 10.
- 37. The method of claim 1, wherein the nucleophile is a peroxide.
- 38. The method of claim 37, wherein the peroxide is an inorganic peroxide of the formula M+OOH−, wherein M+ is a counterion.
- 39. The method of claim 38, wherein the counterion is selected from the group consisting of H+, Li+, Na+, K+, Rb+ and Cs+.
- 40. The method of claim 37, wherein the peroxide is an organic peroxide of the formula ROOH, wherein R is hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
- 41. The method of claim 40, wherein the peroxide has the structure of formula (V), (VI) or (VII)
- 42. The method of claim 37, wherein the peroxide is one of t-butyl hydroperoxide, m-chloroperoxybenzoic acid, and mixtures thereof.
- 43. A nucleoside monomer having the structure of formula (II) or formula (X)
- 44. The nucleoside monomer of claim 43, wherein X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing p-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.
- 45. The nucleoside monomer of claim 44, wherein Y is selected from the group consisting of methyl, P-cyanoethyl and 4-nitrophenylethyl.
- 46. The nucleoside monomer of claim 43, wherein R3 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 47. The nucleoside monomer of claim 44, wherein R3 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 48. The nucleoside monomer of claim 45, wherein R3 is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′-(α-methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
- 49. The nucleoside monomer of claim 43, wherein R3 comprises a moiety that becomes fluorescent or colored upon cleavage of the carbonate —OCOOR3.
- 50. The nucleoside monomer of claim 43, having the structure of formula (II).
- 51. The nucleoside monomer of claim 43, having the structure of formula (X).
- 52. A kit for synthesizing an oligonucleotide on a solid support, comprising a hydroxyl-derivatized support surface, a protecting group for protecting hydroxyl moieties on said derivatized support surface, at least one protected nucleoside, at least one nucleoside phosphoramidite, a nucleophile that exhibits an alpha effect at neutral to mildly basic pH, and reagents suitable for establishing pH and for carrying out reactions of deprotection, phosphoramidite coupling, and oxidation to form an internucleotide phosphotriester linkage.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent application Ser. No. 09/128,052, filed Aug. 3, 1998, the disclosure of which is incorporated by reference herein.
Divisions (1)
|
Number |
Date |
Country |
Parent |
09338179 |
Jun 1999 |
US |
Child |
09756991 |
Jan 2001 |
US |