Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups

• US 4,849,513 A
• Filed: 06/24/1986
• Issued: 07/18/1989
• Est. Priority Date: 12/20/1983
• Status: Expired due to Term

• Current Assignee: Perkinelmer Incorporated
• Original Assignee: California Institute of Technology
• Inventors: Smith, Lloyd M., Fung, Steven
• Primary Examiner(s): Brown, Johnnie R.
• Assistant Examiner(s): Tou, Jenny
• Application Number:
  US06/878,045
• Time in Patent Office: 1,120 Days
• Field of Search: 536/27, 536/28, 536/29
• US Class Current: 536/26.6
• 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
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

The invention consists of compounds and methods for the synthesis of oligonucleotides which contain one or more free aliphatic amino groups attached to the sugar moieties of the nucleoside subunits. The synthetic method is versatile and general, permitting amino groups to be selectively placed at any position on oligonucleotides of any composition or length which is attainable by current DNA synthetic methods. Fluorescent dyes or other detectable moieties may be covalently attached to the amino groups to yield the corresponding modified oligonucleotide.

Claims

1. Protected amino nucleosides having the formula:

##STR26## wherein B is selected from the group consisting of adenine, guanine, thymine, cytosine, uracil, and inosine, R₁, R₂ and R₅ are either H, OR, or NHR'"'"', with the proviso that one of R₁, R₂ and R₅ is NHR'"'"', and only R₅ can be OH, wherein R is a monovalent protecting organic group containing from 1 to about 25 carbon atoms, R'"'"' is a nitrogen protecting group;

R₆ is lower alkyl or heterocyclic, and R₇ is lower alkyl, cyano lower alkyl, halo lower alkyl, or nitrophenyl-lower alkyl.

2. Protected 5'"'"'-amino nucleoside phosphoramidites having the formula:

##STR27## wherein B is selected from the group consisting of thymine and uracil;

X₁ is selected from trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, p-anisyl-diphenylmethyl, triphenylmethyl, and protected amino acyl having the formula;

space="preserve" listing-type="equation">Y--NH--(CHR).sub.n --CO

wherein Y is selected from the group consisting of trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, p-anisyldiphenylmethyl, and triphenylmethyl;

R is an amino acid side chain or hydrogen when n=1; and

R is hydrogen when n is an integer from 2 to 100;

R₁ is selected from the group consisting of lower alkyl (in which case m=2) and heterocyclic (in which case m=1), and R₂ is selected from the group consisting of methyl, beta-cyanoethyl, p-nitrophenethyl, o-chlorophenyl, or p-chlorophenyl.

3. The compounds of claim 2 wherein X₁ is trifluoroacetyl.

4. The compounds of claim 2 wherein X₁ is 9-fluorenylmethyloxycarbonyl.

5. The compounds of claim 2 wherein X₁ is p-anisyldiphenylmethyl.

6. The compounds of claim 2 wherein X₁ is triphenylmethyl.

7. The compounds of claim 2 wherein X₁ is N-tri-fluoroacetylglycol.

8. The compounds of claim 2 wherein B is thymine.

9. The compounds of claim 2 wherein B is uracil.

10. The compounds of claim 2 wherein R₂ is methyl.

11. The compounds of claim 2 wherein R₂ is beta-cyanoethyl.

12. The compounds of claim 2 wherein R₁ is methyl.

13. The compounds of claim 2 wherein R₁ is isopropyl.

14. The compounds of claim 2 wherein R₁ is morpholino.

15. The compounds of claim 2 wherein B is thymine and X₁ is trifluoroacetyl.

16. The compounds of claim 2 wherein B is thymine and X₁ is 9-fluorenylmethyloxycarbonyl.

17. The compounds of claim 2 wherein B is thymine and X₁ is p-anisyldiphenylmethyl.

18. The compounds of claim 2 wherein B is thymine and X₁ is triphenylmethyl.

19. The compounds of claim 2 wherein B is thymine and X₁ is N-trifluolroacetylglycyl.

20. The compounds of claim 2 wherein B is uracil and X₁ is trifluoroacetyl.

21. The compounds of claim 2 wherein B is uracil and X₁ is 9-fluorenylmethyloxycarbonyl.

22. The compounds of claim 2 wherein B is uracil and X₁ is p-anisyldiphenylmethyl.

23. The compounds of claim 2 wherein B is uracil and X₁ is triphenylmethyl.

24. The compounds of claim 2 wherein B is uracil and X₁ is N-trifluoroacetylglycyl.

25. 5'"'"'-N-(9-fluorenylmethyloxycarbonyl)-5'"'"'-amino-5'"'"'-deoxythymidine-3'"'"'-O-methyl-N,N-diisopropylamino phosphoramidite having the formula:

##STR28##

26. 5'"'"'-N-p-anisyldiphenylmethyl-5'"'"'-amino-5'"'"'-dioxythymidine-3'"'"'-O-beta-cyanoethyl-N,N-diisopropylamino phosphoramidite having the formula:

##STR29##

27. 5'"'"'-N-(9-fluorenylmethyloxycarbonyl)-5'"'"'-amino-5'"'"'-deoxythymidine-3'"'"'-O-beta-cyanoethyl-N,N-diisopropylamino phosphoramidite.

28. 5'"'"'-N-(9-fluorenylmethyloxycarbonyl)-5'"'"'-amino-5'"'"'-deoxythymidine-3'"'"'-O-methyl-N,N-dimethylamino phosphoramidite.

29. 5'"'"'-N-(9-fluorenylmethyloxycarbonyl)-5'"'"'-amino-5'"'"'-deoxythymidine-3'"'"'-O-beta-cyanoethyl-N,N-dimethylamino phosphoramidite.

30. 5'"'"'-N-p-anisyldiphenylmethyl-5'"'"'-amino-5'"'"'-deoxy-thymidine-3'"'"'-O-methyl-N,N-diisopropylamino phosphoramidite.

31. 5'"'"'-N-p-anisyldiphenylmethyl-5'"'"'-amino-5'"'"'-deoxy-thymidine-3'"'"'-O-beta-cyanoethyl-N,N-dimethylamino phosphoramidite.

32. 5'"'"'-N-p-anisyldiphenylmethyl-5'"'"'-amino-5'"'"'-deoxy-thymidine-3'"'"'-O-methyl-N,N-dimethylamino phosphoramidite.

33. 5'"'"'-N-p-triphenylmethyl-5'"'"'-amino-5'"'"'-deoxythymidine-3'"'"'-O-beta-cyanoethyl-N,N-diisopropylamino phosphoramidite.

34. Protected 5'"'"'-amino nucleoside phosphoramidites having the formula:

##STR30## wherein B is selected from the group consisting of thymine and uracil;

X₁ is selected from trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, p-anisyl-diphenylmethyl, triphenylmethyl, and protected amino acyl having the formula;

space="preserve" listing-type="equation">Y--NH--(CHR).sub.n --CO

wherein Y is selected from the group consisting of trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, p-anisyldiphenylmethyl, and triphenylmethyl;

R is an amino acid side chain or hydrogen when n=1; and

R is hydrogen when n is an integer from 2 to 100;

R₁ is selected from the group consisting of lower alkyl (in which case m=2) and heterocyclic (in which case m=1), and R₂ is selected from the group consisting of methyl, beta-cyanoethyl, p-nitrophenethyl, o-chlorophenyl, or p-chlorophenyl; and

X₂ is a hydroxyl protecting group selected from triphenylmethyl, p-anisyl-diphenylmethyl, di-p-anisylphenylmethyl, and 9-phenylxanthenyl.

35. The compounds of claim 34 wherein X₁ is trifluoroacetyl.

36. The compounds of claim 34 wherein X₁ is 9-fluorenylmethyloxycarbonyl.

37. The compounds of claim 34 wherein X₁ is N-trifluoroacetylglycol.

38. The compounds of claim 34 wherein B is uracil.

39. The compounds of claim 34 wherein R₁ is methyl.

40. The compounds of claim 34 wherein R₁ is isopropyl.

41. The compounds of claim 34 wherein R₁ is morpholino.

42. The compounds of claim 34 wherein R₂ is methyl.

43. The compounds of claim 34 wherein R₂ is beta-cynaoethyl.

44. The compounds of claim 34 wherein B is uracil and X₁ is trifluoroacetyl.

45. The compounds of claim 34 wherein B is uracil and X₁ is fluorenylmethyloxycarbonyl.

46. The compounds of claim 34 wherein B is uracil and X₁ is N-trifluoroacetylglycyl.

47. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N -trifluoacetyl-2 -amino-2'"'"'-deoxyuridine-3'"'"'-O-methyl-N, N-diisopropylamino phosphoramidite having the formula:

##STR31##

48. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-(N-trifluoacetylglycol) -2'"'"'-amino-2'"'"'-deoxyuridine-3'"'"'-O-methyl-N,N-diisopropylamino phosphoramidite having the formula:

##STR32##

49. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-trifluoacetyl -2'"'"'-amino-2'"'"'-deoxyuridine-3 -O-beta-cyanoethyl-N, N-diisopropylamino phosphoramidite.

57. The compounds of claim 49 wherein R₁ is isopropyl.

58. The compounds of claim 49 wherein R₁ is morpholino.

59. The compounds of claim 49 wherein R₂ is methyl.

60. The compounds of claim 49 wherein R₂ is beta-cyanoethyl.

61. The compounds of claim 49 wherein B is uracil and X₁ is trifluoroacetyl.

62. The compounds of claim 49 wherein B is uracil and X₁ is fluorenylmethyloxycarbonyl.

63. The compounds of claim 49 wherein B is uracil and X₁ is N-trifluoroacetylglycyl.

50. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-(N-trifluoacetylglycol) -2'"'"'-amino-2'"'"'-deoxyuridine-3'"'"'-O-beta-cyanoethyl-N, N-diisopropylamino phosphoramidite.

51. Protected 2'"'"'-amino-nucleoside phosphoramidites having the formula:

##STR33## wherein B is selected from the the group consisting of thymine and uracil;

X₁ is selected from trifluoroacetyl, 9-fluorenylmethyloxycarbonyl, p-anisyl-diphenylmethyl, triphenylmethyl, and protected amino acyl having the formula;

space="preserve" listing-type="equation">Y--NH--(CHR).sub.n --CO

wherein Y is selected from the group consisting of trofluoroacetyl, 9- fluorenylmethoxycarbonyl, p-anisylphenylmethyl, and triphenylmethyl;

R is an amino acid side chain or hydrogen when n=1; and

R is hydrogen when n is an integer from 2 to 100;

R₁ is selected from the group consisting of lower alkyl (in which case m=2) and heterocyclic (in which case m=1), and R₂ is selected from the group consisting of methyl, beta-cyanoethyl, p-nitrophenethyl, O-chlorophenyl, or p-chlorophenyl; and

X₂ is a hydroxyl protecting group selected from triphenylmethyl, p-anisyl-diphenylmethyl, di-p-anisylphenylmethyl, and 9-phenylxanthenyl.

52. The compounds of claim 51 wherein X₁ is trifluoroacetyl.

53. The compounds of claim 51 wherein X₁ is 9-fluorenylmethyloxycarbonyl.

54. The compounds of claim 51 wherein X₁ is N-trifluoroacetylglycyl.

55. The compounds of claim 51 wherein B is uracil.

56. The compounds of claim 51 wherein R₁ is methyl.

64. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-trifluoroacetyl -2'"'"'-amino-2'"'"'-deoxy-ara-uridine-3'"'"'-O-methyl-N, N-diisopropylamino phosphoramidite.

65. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-(N-trifluoroacetylglycol)-2'"'"'-amino-2'"'"'-deoxy-ara-uridine-3'"'"'-O-methyl-N,N-diiso-propylamino phosphoramidite.

66. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-trifluoroacetyl-2'"'"'-amino-2'"'"'-deoxy-ara-uridine-3'"'"'-O-beta-cyanoethyl-N,N-diiso-propylamino phosphoramidite.

67. 5'"'"'-O-di-p-anisylphenylmethyl-2'"'"'-N-(N-trifluoroacetylglycol)-2'"'"'-amino-2'"'"'-deoxy-ara-uridine-3'"'"'-O-beta cyanoethyl-N,N-diisopropylamino phosphoramidite.