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Patent Application

App. No. 13/929,317

Nucleic Acid Labeling Compounds

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Patent No.: US None
App. No.: 13/929,317

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Abstract

Nucleic acid labeling compounds are disclosed. The compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

Claims (104)

1 . A nucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X is O, S, NR 1 or CHR 2 , wherein R 1 and R 2 are, independently, hydrogen, alkyl or aryl;

Y is hydrogen, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is hydrogen, alkyl or aryl;

Z is hydrogen, N 3 , F, OR 9 , SR 9 or NHR 10 , wherein R 10 is hydrogen, alkyl or aryl;

L is a linker moiety; Q is a detectable moiety; and

M is a connecting group, wherein m is an integer ranging from 0 to about 20.

2 . The nucleic acid labeling compound of claim 1 , wherein A is H or H 4 O 9 P 3 —; X is O; Y is H or OR 9 , wherein R 9 is hydrogen, alkyl or aryl; Z is hydrogen, N 3 , F or OR 10 , wherein R 10 is hydrogen, alkyl or aryl; and L is amido alkyl and M is -(M 1 a -(M 2 ) b - wherein a and b are independently integers from 0 to about 5 and the sum of a and b is not greater than 15.

3 . The nucleic acid labeling compound of claim 2 , wherein L is —(CH 2 ) n C(O)NR 3 —, wherein R 3 is hydrogen, alkyl or aryl and n is an integer ranging from about 1 to about 10; M 1 is —(CH 2 ) i O— and M 2 is —(CH 2 ) j NH—, wherein i and j are independently integers from 1 to about 5.

4 . The nucleic acid labeling compound of claim 3 , wherein Y is H or OH; Z is H or OH; -L is —CH 2 C(O)NH—; M 1 is —(CH 2 CH 2 O) 3 — and M 2 is —CH 2 CH 2 NH—; and Q is biotin or a carboxyfluorescein.

5 . The nucleic acid labeling compound of claim 4 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 C(O)NH—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; and Q is biotin.

6 . The nucleic acid labeling compound of claim 4 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 C(O)NH—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; Q is carboxyfluorescein.

7 . The nucleic acid labeling compound of claim 1 , wherein A is H or H 4 O 9 P 3 —; X is O; Y is H or OR 9 , wherein R 9 is hydrogen, alkyl or aryl; Z is hydrogen, N 3 , F or OR 10 , wherein R 10 is hydrogen, alkyl or aryl; and L is amido alkyl and M is -(M 1 ) a -(M 2 ) b -(M 3 ) c -(M 4 ) d - wherein a, b, c, and d are independently integers from 0 to about 5 and the sum of a, b, c, and d is not greater than 15.

8 . The nucleic acid labeling compound of claim 7 , wherein L is —(CH2)nC(O)NR3—, wherein R3 is hydrogen, alkyl or aryl and n is an integer ranging from about 1 to about 10; each M is independently —C(O)(CH2)kO—, —(CH2)iO— or —(CH2)jNH—, wherein i, j and k are independently integers from 1 into about 5.

9 . The nucleic acid labeling compound of claim 8 , wherein L is —CH 2 C(O)NR 3 —, wherein R 3 is hydrogen or, alkyl; M 1 is —(CH 2 ) 2 NH—, M 2 is —C(O)(CH 2 ) 2 O—, M 3 is —(CH 2 CH 2 O) 3 —and M 4 is —(CH 2 ) 2 NH—.

10 . The nucleic acid labeling compound of claim 9 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 C(O)NH—CH 2 CH 2 NH—C(O)CH 2 CH 2 O—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; Q is biotin.

11 . The nucleic acid labeling compound of claim 9 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 C(O)NH—CH 2 CH 2 NH—; m is 1; and Q is carboxyfluorescein.

12 . The nucleic acid labeling compound of claim 8 , wherein L is —(CH 2 ) 2 C(O)NR 3 —, wherein R 3 is hydrogen or, alkyl; M 1 is —(CH 2 ) 2 NH—, M 2 is —C(O)(CH 2 ) 2 O—, M 3 is —(CH 2 CH 2 O) 3 — and M 4 is —(CH 2 ) 2 NH—.

13 . The nucleic acid labeling compound of claim 12 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 C(O)NH—CH 2 CH 2 NH—C(O)CH 2 CH 2 O—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; Q is biotin.

14 . The nucleic acid labeling compound of claim 12 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 CH 2 C(O)NH—CH 2 CH 2 NH—; m is 1; and Q is carboxyfluorescein.

15 . The nucleic acid labeling compound of claim 1 , wherein A is H or H 4 O 9 P 3 —; X is O; Y is H or OR 9 , wherein R 9 is hydrogen, alkyl or aryl; Z is hydrogen, N 3 , F or OR 10 , wherein R 10 is hydrogen, alkyl or aryl; and L is functionalized alkyl and M is -(M 1 ) a -(M 2 ) b -(M 3 ) c -(M 2 ) d - wherein a, b, c, and d are independently integers from 0 to about 5 and the sum of a, b, c, and d is not greater than 15.

16 . The nucleic acid labeling compound of claim 15 , wherein L is —(CH 2 ) n O—, wherein n is an integer ranging from about 1 to about 10; each M is independently —C(O)(CH 2 ) k O—, —(CH 2 ) i O— or —(CH 2 ) j NH—, wherein i, j and k are independently integers from 1 to about 5.

17 . The nucleic acid labeling compound of claim 16 , wherein L is —CH 2 O—, M 1 is —(CH 2 ) 2 NH—, M 2 is —C(O)(CH 2 ) 2 O—, M 3 is —(CH 2 CH 2 O) 3 — and M 4 is —(CH 2 ) 2 NH—.

18 . The nucleic acid labeling compound of claim 17 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 O—CH 2 CH 2 NH—C(O)(CH 2 ) 2 O—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; Q is biotin.

19 . The nucleic acid labeling compound of claim 17 , wherein Y is OH; Z is OH; -L-(M) m - is —CH 2 O—CH 2 CH 2 NH—; m is 1; and Q is carboxyfluorescein.

20 . The nucleic acid labeling compound of claim 15 , wherein L is —(CH 2 ) n NR 3 —, wherein R 3 is hydrogen, alkyl or aryl and n is an integer ranging from about 1 to about 10; each M is independently —C(O)(CH 2 ) k O—, —(CH 2 ) i O— or —(CH 2 ) j NH—, wherein i, j and k are independently integers from 1 to about 5.

21 . The nucleic acid labeling compound of claim 20 , wherein L is —(CH 2 ) 6 NH—; M 1 is —(CH 2 ) 2 NH—, M 2 is —C(O)(CH 2 ) 2 O—, and M 3 is —(CH 2 CH 2 O) 3 —.

22 . The nucleic acid labeling compound of claim 21 , wherein Y is OH; Z is OH; -L-(M) m - is —(CH 2 ) 6 NH—C(O)(CH 2 ) 2 O—(CH 2 CH 2 O) 3 —CH 2 CH 2 NH—; and Q is biotin.

23 . The nucleic acid labeling compound of claim 21 , wherein Y is OH; Z is OH; L is —(CH 2 ) 6 NH—; m is 0; and Q is carboxyfluorescein.

24 . The nucleic acid labeling compound of claim 1 , wherein A is H or H 4 O 9 P 3 —; X is O; Y is H or OR 9 , wherein R 9 is hydrogen, alkyl or aryl; Z is hydrogen, N 3 , F or OR 10 , wherein R 10 is hydrogen, alkyl or aryl; and L is alkenyl alkyl and M is -(M 1 ) a -(M 2 ) b -(M 3 ) c -(M 4 ) d - wherein a, b, c, and d are independently integers from 0 to about 5 and the sum of a, b, c, and d is not greater than 15.

25 . The nucleic acid labeling compound of claim 7 , wherein L is structure —CH═CH—(CH 2 ) n C(O)N(R 6 )—, wherein R 6 is hydrogen, alkyl or aryl and n is an integer ranging from about 0 to about 10; each M is independently —C(O)(CH 2 ) k O—, —(CH 2 ) i O— or —(CH 2 ) j NH—, wherein i, j and k are independently integers from 1 to about 5.

26 . The nucleic acid labeling compound of claim 2 , wherein L is —CH═CH—C(O)N(H); M 1 is —(CH 2 ) 2 NH—, M 2 is —C(O)(CH 2 ) 2 O—, M 3 is —(CH 2 CH 2 O)— and M 4 is —(CH 2 ) 2 NH—.

27 . The nucleic acid labeling compound of claim 12 , wherein -L-(M) m - is —CH═CH—C(O)NH—CH 2 CH 2 NH—C(O)CH 2 CH 2 O—(CH 2 CH 2 O) 3 CH 2 CH 2 NH—; Y is OH; Z is OH; Q is biotin.

28 . The nucleic acid labeling compound of claim 12 , wherein Y is OH; Z is OH; -L-(M) m - is —CH═CH—C(O)NH—CH 2 CH 2 NH—; m is 1; and Q is carboxyfluorescein.

29 . The nucleic acid labeling compound of claim 1 , having the formula:

wherein A, X, Y, Z, L, M, m, and Q are as defined herein.

30 . A nucleic acid labeling compound of claim 1 , having the formula:

wherein A, X, Y, Z, L, M, m, and Q are as defined herein.

31 . A nucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

L Is a linker moiety:

Q is a detectable moiety; and

M is a connecting group, wherein m is an integer ranging from 0 to about 3.

32 . The nucleic acid labeling compound of claim 31 , wherein A is H or H 4 O 9 P 3 —; L is alkanoyl; and Q is biotin or a carboxyfluorescein; M is —NR 3 — where R 3 is hydrogen or alkyl, and wherein m is 1 or 0.

33 . The nucleic acid labeling compound of claim 32 , wherein L is —C(O)(CH 2 ) n — wherein n is an integer ranging from about 1 to about 10; M is —NH—; Q is biotin or a carboxyfluorescein.

34 . The nucleic acid labeling compound of claim 33 , wherein -L-(M) m - is —C(O)(CH 2 ) 6 —NH—; and Q is biotin.

35 . The nucleic acid labeling compound of claim 33 , wherein -L-(M) m - is —C(O)(CH 2 ) 6 —NH—; and Q is carboxyfluorescein.

36 . A nucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X 1 is O, S, NR 1 or CHR 2 , wherein R 1 and R 2 are, independently, hydrogen, alkyl or aryl;

R 15 is hydrogen, alkyl or aryl;

Y 1 is hydrogen, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is hydrogen, alkyl or aryl;

Z 1 is hydrogen, N 3 , F, OR 9 , SR 9 or NHR 10 , wherein R 10 is hydrogen, alkyl or aryl;

wherein one of Y 1 or Z 1 is a group having the formula —X 2 L-(M) m -Q;

X 2 is O, S, or NR 16 , and R 16 is hydrogen, alkyl or aryl;

L is a linker moiety; Q is a detectable moiety; and

M is a connecting group, wherein m is an integer ranging from 0 to about 20.

37 . The nucleic acid labeling compound of claim 36 , wherein A is H or H 4 O 9 P 3 —; X 1 is O; Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen, or alkyl, or —NR 16 -L-(M) m -Q; wherein R 16 is hydrogen, alkyl or aryl; Z 1 is hydrogen OR 10 , wherein R 10 is hydrogen, or alkyl, or —NR 16 -L-(M) m -Q, wherein R 16 is hydrogen, or alkyl; L is N-alkyl amido; R 15 is hydrogen or alkyl; M is —(CH 2 ) n NR 3 — where R 3 is hydrogen or alkyl, and m is from 1 to about 15.

38 . The nucleic acid labeling compound of claim 37 , wherein Y 1 is hydrogen or OH; Z 1 is —NH-L-(M) m -Q; L is —(CH 2 ) n NHC(O)— where n is an integer from 1 to about 10; M is —NH(CH 2 ) p — where p is an integer from 1 to about 10; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

39 . The nucleic acid labeling compound of claim 38 wherein Y 1 is OH: R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is biotin.

40 . The nucleic acid labeling compound of claim 39 , wherein Y 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is a carboxyfluorescein.

41 . The nucleic acid labeling compound of claim 39 wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is biotin.

42 . The nucleic acid labeling compound of claim 39 , wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is a carboxyfluorescein.

43 . The nucleic acid labeling compound of claim 37 , wherein Y 1 is —NH-L-(M) m -Q; Z 1 is hydrogen or OH; L is —(CH 2 ) n NHC(O)— where n is an integer from 1 to about 10; M is —NH(CH 2 ) p — where n is an integer from 1 to about 10; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

44 . The nucleic acid labeling compound of claim 43 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is biotin.

45 . The nucleic acid labeling compound of claim 43 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is a carboxyfluorescein.

46 . The nucleic acid labeling compound of claim 43 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is biotin.

47 . The nucleic acid labeling compound of claim 43 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and Q is a carboxyfluorescein.

48 . The nucleic acid labeling compound of claim 37 , wherein A is H or H 4 O 9 P 3 —; X 1 is O; Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen, or alkyl, or —O-L-(M) m -Q; Z 1 is hydrogen OR 10 , wherein R 10 is hydrogen, or alkyl, or —O-L-(M) m -Q, or alkyl; L is alkylene; and m is from 1 to about 10.

49 . The nucleic acid labeling compound of claim 48 , wherein Y 1 is hydrogen or OH; Z 1 is —O-L-(M) m -Q; L is -(CH 2 ) n — where n is an integer from 1 to about 12; M is —NH—; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

50 . The nucleic acid labeling compound of claim 49 , wherein Y 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is biotin.

51 . The nucleic acid labeling compound of claim 49 , wherein Y 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is a carboxyfluorescein.

52 . The nucleic acid labeling compound of claim 49 , wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is biotin.

53 . The nucleic acid labeling compound of claim 49 , wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is a carboxyfluorescein.

54 . The nucleic acid labeling compound of claim 37 , wherein Y 1 is —O-L-(M) m -Q; Z 1 is hydrogen or OH; L is —(CH 2 ) n — where n is an integer from 1 to about 12; M is —NH—; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

55 . The nucleic acid labeling compound of claim 54 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is biotin.

56 . The nucleic acid labeling compound of claim 54 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is a carboxyfluorescein.

57 . The nucleic acid labeling compound of claim 54 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 6 —NH—; and Q is biotin

58 . The nucleic acid labeling compound of claim 54 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —(CH 2 ) 6 —NHC—; and Q is a carboxyfluorescein.

59 . The nucleic acid labeling compound of claim 37 , wherein A is H or H 4 O 9 P 3 —; X 1 is O; Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen, or alkyl, or —S-L-(M) m -Q; Z 1 is hydrogen OR 10 , wherein R 10 is hydrogen, or alkyl, or —S-L-(M) m -Q; L is alkyl thio; and m is from 1 to about 10.

60 . The nucleic acid labeling compound of claim 59 , wherein Y 1 is hydrogen, or OH; Z 1 is —S-L-(M) m -Q; L is —S—(CH 2 )n- where n is an integer from 1 to about 10; M is —NH—; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

61 . The nucleic acid labeling compound of claim 60 , wherein Y 1 is OH; R 15 is hydrogen; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is biotin.

62 . The nucleic acid labeling compound of claim 60 , wherein Y 1 is OH; R 15 is hydrogen; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is a carboxyfluorescein.

63 . The nucleic acid labeling compound of claim 60 , wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is biotin.

64 . The nucleic acid labeling compound of claim 60 , wherein Y 1 is OH; R 15 is methyl; -L-(M) m - is —S—(CH 2 ) 2 —NR—; and Q is a carboxyfluorescein.

65 . The nucleic acid labeling compound of claim 37 , wherein Y 1 is —S-L-(M) m -Q; Z 1 is hydrogen or OH; L is —S—(CH 2 ) n — where n is an integer from 1 to about 10; M is —NH—; R 15 is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

66 . The nucleic acid labeling compound of claim 65 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is biotin.

67 . The nucleic acid labeling compound of claim 65 , wherein Z 1 is OH; R 15 is hydrogen; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is a carboxyfluorescein.

68 . The nucleic acid labeling compound of claim 65 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is biotin.

69 . The nucleic acid labeling compound of claim 65 , wherein Z 1 is OH; R 15 is methyl; -L-(M) m - is —S—(CH 2 ) 2 —NH—; and Q is a carboxyfluorescein.

70 . A nucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X 1 O, S, NR 1 or CHR 2 , wherein R 1 and R 2 are, independently, hydrogen, alkyl or aryl; X 2 is a bond or alkylene; Q is a detectable moiety; and G is a connecting group.

71 . The nucleic acid labeling compound of claim 70 , wherein A is H or H 4 O 9 P 3 —; X 1 is O; X 2 is a bond; and G is —C(O)NR 3 —(CH 2 ) m —(CH 2 CH 2 O) n —CH 2 CH 2 —C(O)NR 3 —, where R 3 is hydrogen or alkyl, and m and n are independently an integer from 1 to about 15.

72 . The nucleic acid labeling compound of claim 71 , wherein G is —C(O)NH—(CH 2 ) m —(CH 2 CH 2 O) n —CH 2 CH 2 —C(O)NH— where m is from 1 to about 6 and n is from 1 to about 4.

73 . The nucleic acid labeling compound of claim 70 , wherein A is H or H 4 O 9 P 3 —; X 1 is O; X 2 is CH 2 ; and G is —C(O)NR 3 —(CH 2 m —(CH 2 CH 2 O) n —CH 2 CH 2 —C(O)NR 3 —, where R 3 is hydrogen or alkyl, and m and n are independently an integer from 1 to about 15.

74 . The nucleic acid labeling compound of claim 73 , wherein G is —C(O)NH—(CH 2 ) m —(CH 2 CH 2 O) n —CH 2 CH 2 —C(O)NH— wherein m is from 1 to about 6 and n is from 1 to about 4.

75 . A nucleic acid derivative produced by coupling a nucleic acid labeling compound of claim 2 , 7 , 12 , 15 , 20 , 24 , 32 , 37 , or 71 with a nucleic acid.

76 . A hybridization product, wherein the hybridization product comprises the nucleic acid derivative of claim 75 bound to a complementary probe.

77 . The hybridization product of claim 76 , wherein the probe is attached to a glass chip.

78 . A hybridization product, wherein the hybridization product comprises the nucleic acid derivative of claim 75 bound to a complementary probe.

79 . A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound of claim 2 , 7 , 12 , 15 , 20 , 24 , 32 , 37 , or 71 to a nucleic acid.

80 . A method of detecting a nucleic acid comprising incubating a nucleic acid derivative according to claim 75 with a probe.

81 . A method according to claim 80 , wherein the probe is attached to a glass chip.