IP Library › Granted Patent US 8,148,503

Granted Patent B2

US 8,148,503 · App. 12/483,080 · Granted Apr 3, 2012

Nucleotides and nucleosides and methods for their use in DNA sequencing

Assignee: Lasergen, Inc.

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Quick Facts

Patent No.: US 8,148,503
App. No.: 12/483,080
Granted: Apr 3, 2012
Kind: B2

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Abstract

The present invention relates generally to labeled and unlabled cleavable terminating groups and methods for DNA sequencing and other types of DNA analysis. More particularly, the invention relates in part to nucleotides and nucleosides with chemically cleavable, photocleavable, enzymatically cleavable, or non-photocleavable groups and methods for their use in DNA sequencing and its application in biomedical research.

Claims (147)

1. A compound of formula:

wherein:

Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or —NHC(O)NH—;

R 1 is hydroxy, monophosphate, diphosphate, triphosphate or polyphosphate;

R 2 is hydrogen or hydroxy;

R 3 and R 4 are each independently:

hydrogen, hydroxy, halo or amino; or

alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , aryl (C≦12) , aralkyl (C≦12) , heteroaryl (C≦12) , heteroaralkyl (C≦12) , alkoxy (C≦12) , aryloxy (C≦12) , aralkoxy (C≦12) , heteroaryloxy (C≦12) , heteroaralkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , arylamino (C≦12) , aralkylamino (C≦12) , or a substituted version of any of these groups; or

R 5 , R 6 , R 7 , R 8 and R 9 are each independently:

hydrogen, hydroxy, halo, amino, nitro, cyano or mercapto;

alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , aryl (C≦12) , aralkyl (C≦12) , heteroaryl (C≦12) , heteroaralkyl (C≦12) , acyl (C≦12) , alkoxy (C≦12) , alkenyloxy (C≦12) , alkynyloxy (C≦12) , aryloxy (C≦12) , aralkoxy (C≦12) , heteroaryloxy (C≦12) , heteroaralkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , alkoxyamino (C≦12) , alkenylamino (C≦12) , alkynylamino (C≦12) , arylamino (C≦12) , aralkylamino (C≦12) , heteroarylamino (C≦12) , heteroaralkylamino (C≦12) , alkylsulfonylamino (C≦12) , amido (C≦12) , alkylthio (C≦12) , alkenylthio (C≦12) , alkynylthio (C≦12) , arylthio (C≦12) , aralkylthio (C≦12) , heteroarylthio (C≦12) , heteroaralkylthio (C≦12) , acylthio (C≦12) , thioacyl (C≦12) , alkylsulfonyl (C≦12) , arylsulfonyl (C≦12) , alkylammonium (C≦12) , alkylsulfonium (C≦12) , alkylsilyl (C≦12) , or a substituted version of any of these groups;

a group of formula:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups;

Y is —O—, —NH—, alkanediyl (C≦12) or substituted alkanediyl (C≦12) ;

n is an integer from 0-12; and

m is an integer from 0-12; or

a -linker-reporter;

or a salt, esters, hydrates, solvates, tautomers, prodrugs, or optical isomers thereof.

2. The compound of claim 1 , wherein Z is —O—.

3. The compound of claim 1 , wherein R 2 is hydrogen.

4. The compound of claim 1 , wherein R 3 is hydrogen.

5. The compound of claim 1 , wherein R 3 is alkyl (C≦8) .

6. The compound of claim 5 , wherein R 3 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

7. The compound of claim 1 , wherein R 4 is hydrogen.

8. The compound of claim 1 , wherein R 5 is hydrogen.

9. The compound of claim 1 , wherein R 5 is cyano.

10. The compound of claim 1 , wherein R 6 is hydrogen.

11. The compound of claim 1 , wherein R 6 is a group of formula:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups; and

n is an integer from 0-12.

12. The compound of claim 11 , wherein X is alkynediyl (C2-8) .

13. The compound of claim 12 , wherein X is —C═C—.

14. The compound of claim 11 , wherein n is zero.

15. The compound of claim 1 , wherein R 6 is a group of formula:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups;

Y is —O—, —NH—, alkanediyl (C≦12) or substituted alkanediyl (C≦12) ;

n is an integer from 0-12; and

m is an integer from 0-12.

16. The compound of claim 15 , wherein X is alkynediyl (C2-8) .

17. The compound of claim 16 , wherein X is —C═C—.

18. The compound of claim 15 , wherein Y is —CH 2 —.

19. The compound of claim 15 , wherein n is zero.

20. The compound of claim 15 , wherein m is zero.

21. The compound of claim 1 , wherein R 6 is a -linker-reporter.

22. The compound of claim 21 , wherein the linker is:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups; and

n is an integer from 0-12.

23. The compound of claim 22 , wherein X is alkynediyl (C2-8) .

24. The compound of claim 23 , wherein X is —C≡C—.

25. The compound of claim 22 , wherein n is zero.

26. The compound of claim 21 , wherein the linker is:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups;

Y is —O—, —NH—, alkanediyl (C≦12) or substituted alkanediyl (C≦12) ;

n is an integer from 0-12; and

m is an integer from 0-12.

27. The compound of claim 26 , wherein X is alkynediyl (C2-8) .

28. The compound of claim 27 , wherein X is —C≡C—.

29. The compound of claim 26 , wherein Y is —CH 2 —.

30. The compound of claim 26 , wherein n is zero.

31. The compound of claim 26 , wherein m is zero.

32. The compound of claim 21 , wherein the linker is:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups;

Y is —O—, —NH—, alkanediyl (C≦12) or substituted alkanediyl (C≦12) ;

n is an integer from 0-12; and

m is an integer from 0-12.

33. The compound of claim 32 , wherein X is alkynediyl (C2-8) .

34. The compound of claim 33 , wherein X is —C═C—.

35. The compound of claim 32 , wherein Y is —CH 2 —.

36. The compound of claim 32 , wherein n is zero.

37. The compound of claim 32 , wherein m is zero.

38. The compound of claim 21 , wherein the reporter is based on a dye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, or a squaraine dye.

39. The compound of claim 21 , wherein the reporter is:

40. The compound of claim 1 , wherein R 7 is hydrogen.

41. The compound of claim 1 , wherein R 8 is hydrogen.

42. The compound of claim 1 , wherein R 8 is nitro.

43. The compound of claim 1 , wherein R 8 is alkyl (C≦8) .

44. The compound of claim 43 , wherein R 8 is methyl.

45. The compound of claim 1 , wherein R 9 is hydrogen.

46. The compound of claim 1 , wherein R 9 is nitro.

47. The compound of claim 1 , wherein R 9 is alkyl (C≦8) .

48. The compound of claim 47 , wherein R 9 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

49. The compound of claim 1 , wherein R 9 is aryl (C≦8) .

50. The compound of claim 49 , wherein R 9 is phenyl.

51. The compound of claim 1 , further defined as:

or a salt thereof.

52. The compound of claim 1 , further defined as:

or a salt thereof.

53. The compound of claim 52 , further defined as: