Nucleic Acid Binding Compounds Containing Pyrazolo[3,4-D]Pyrimidine Analogues of Purin-2,6-Diamine and Their Uses

• US 20110021365A1
• Filed: 04/30/2007
• Published: 01/27/2011
• Est. Priority Date: 08/03/2000
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Seidel, Christoph, Heindl, Dieter, Von Der Eltz, Herbert, Becher, Georg, Seela, Frank, Bergmann, Frank
• Granted Patent: US 8,057,997 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07H 19/16 Purine radicals
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is in the field of nucleic acid binding compounds comprising 7-substituted 7-deaza-8aza-2,6-diamino-purine bases, compounds useful for the preparation of such compounds, various uses thereof and methods for the determination of nucleic acids using said compounds in the field of diagnostics.

Claims

1. A nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a group of the general formula I

2. The nucleic acid binding compound of claim 1

wherein R¹ is alkynyl-amino-C≡

C-E-NR⁵R⁶, alkenyl-amino —

CH═

CH-E-NR⁵R⁶ and -E-NR⁵R⁶ wherein E is —

[(CH₂)_rF]_s(CH₂)_r—

wherein F═

O or S and r and s are independently from one another an integer from 1 to 18,

wherein R⁵ and R⁶ are selected independently from the group consisting of —

H, —

(C₁-C₁₀)-alkyl, —

(C₂-C₁₀)-alkenyl, —

(C₂-C₁₀)-alkynyl, —

(C₆-C₂₂)-aryl and a reporter group,

wherein R¹¹ is selected from the group consisting of —

NHR¹² and OR¹²,

wherein R⁵, R⁶ and R¹² are selected independently from the group consisting of —

H, —

(C₁-C₁₀)-alkyl, —

(C₂-C₁₀)-alkenyl, —

(C₂-C₁₀)-alkynyl, —

(C₆-C₂₂)-aryl and a reporter group,

said alkyl, alkenyl, alkynyl or aryl being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —

SH, —

S—

(C₁-C₆)-alkyl, —

(C₁-C₆)-alkoxy, —

OH, —

NR⁵R⁶, —

COR¹¹, —

NH—

CONR⁵R⁶, —

NH—

CSNR⁵R⁶ and —

(CH₂)_n—

[O—

(CH₂)_r]_s—

NR⁵R⁶, are independently of each other an integer of from 1 to 18 r and s and n is 0 or 1 independently from r and s.

3. The nucleic acid binding compound according to any of the claims 1 to 2, wherein said nucleic acid compound comprises one or more moieties of the general formula II

4. The nucleic acid binding compound according to any of the claims 1 to 3, wherein the backbone comprises a moiety of the general formula III, wherein t is 0 or 1,

5. The nucleic acid binding compound of claim 4, wherein t=0, R¹⁵ is —

H and R¹⁴ is —

H.

6. The nucleic acid binding compound according to any of the claims 1 to 5, wherein said backbone comprises a moiety of the formula V

7. The compound of claim 6, wherein M′

is O, R¹⁶ is H and R¹⁴ is selected from the group consisting of —

H and —

OH.

8. The nucleic acid binding compound according to any of the claims 1 to 7, wherein the backbone comprises sugar and phosphate moieties.

9. The nucleic acid binding compound according to claim 8, wherein the sugar is in a locked conformation.

10. The nucleic acid binding compound according to any of the claims 1 to 9 wherein the nucleic acid binding compound comprises a heterocyclic group which is a pyrrolo-[2,3-d]-pyrimidine, a pyrazolo[3,4-d]pyrimidine or an analogue thereof.

11. The nucleic acid binding compound according to any of the claims 1 to 10 wherein a protecting group substitutes one or two hydrogen atoms of a —

OH, —

SH, —

NH₂, —

NH-alkyl, —

NH-alkenylene, —

NH—

alkynylene, or a NH-aryl group.

12. The nucleic acid binding compound of claims 1 to 11 wherein said nucleic acid binding compound contains a reporter group.

13. The nucleic acid binding compound according to claim 12 wherein the heterocyclic group of formula I as defined in claim 1 is 1 to 5 nucleotides separated from the nucleotide to which the reporter group is attached.

14. The nucleic acid binding compound of any of claims 1 to 11 wherein a heterocyclic group is 7-bromo-7-deaza-8-aza-2,6-diamino-purine or 7-iodo-7-deaza-8-aza-2,6-diamino-purine.

17. A composition according to claim 15 wherein only the nucleic acid binding compounds or the nucleic acid binding compounds and the complementary nucleic acid binding compounds are nucleic acid binding compounds according to any of the claims 1 to 14.

20. The binding product of a first nucleic acid binding compound according to any of the claims 1 to 15 or a composition according to any of the claims 16 to 19 with a second nucleic acid binding compound or a second a nucleic acid binding compound according to any of the claims 1 to 14, wherein the first nucleic acid binding compound or the composition and the second nucleic acid binding compound are bound to each other by base pairing in parallel or antiparallel orientation.

21. A compound of the general formula VIII

22. A compound of claim 21, wherein R¹⁴ is H and B is 7-Bromo-7-deaza-8-aza-2,6-diaminopurine or 7-iodo-7-deaza-8-aza-2,6-diaminopurine.

23. A compound of formula VI

24. A compound of formula VII

25. A compound of general formula IX

26. A compound of general formula X

27. A precursor for the synthesis of a nucleic acid binding compound comprising a backbone, wherein the backbone comprises a moiety of the general formula VI

28. A precursor or intermediate of a nucleic acid binding compound, wherein the backbone comprises a moiety of the general formula III

29. A precursor or intermediate compound comprising a backbone, said backbone having attached heterocyclic groups characterized in that a heterocyclic group is a group of the general formula I

30. A method for the enzymatic synthesis of a nucleic acid binding compound according to any of the claims 1 to 14, comprising reacting a triphosphate subunit with a primer using a nucleic acid as a template for the elongation of the primer, wherein the triphosphate subunit is a compound according to claim 21 or 22.

31. A method for the chemical synthesis of a compound according to any of the claims 1 to 14 using activated subunits, wherein said subunits contain a compound according to any of the claims 23 to 29.

32. A method according to claim 31 comprising the additional step of reacting the synthesized nucleic acid binding compound with a further compound.

33. A method according to claim 32 wherein the further compound is a label.

37. Use according to any of the claims 34 to 36 wherein the nucleic acid binding compound or the first and/or the second nucleic acid binding compound is a nucleic acid binding compound according to any of the claims 1 to 14.

48. Use according to any of the claims 47 wherein the substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof or the 7-substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof has the formula I with the substituents as defined in claim 1.

80. Method according to claim 79 wherein the substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof or the 7-substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof has the general formula I as defined in claim 1.

81. Method according to any of the claims 49 to 80 wherein the nucleic acid binding compound is a nucleic acid binding compound according to any of the claims 1 to 14.

83. A pharmaceutical composition comprising a nucleic acid binding compound according to any of the claims 1 to 14.

84. A nucleic acid binding compound according to any of the claims 1 to 14 for use in medicine.

15. A composition for analyzing interactions between nucleic acid binding compounds comprising an array of a plurality of nucleic acid binding compounds having different sequences, wherein said plurality of nucleic acid binding compounds are coupled to a solid substrate at known locations and are selected to bind to complementary nucleic acid binding compounds

characterized in that

only the nucleic acid binding compounds or the nucleic acid binding compounds and the complementary nucleic acid binding compounds together are nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases, wherein a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.

16. A composition according to claim 15 wherein the substituted pyrazolo[3,4-d]pyrimidine analogue is a pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine, a substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine or a 7-substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine.

18. A composition according to any of the claims 15 to 17, wherein said solid substrate is selected from the group consisting of silica, polymeric materials, glass, porous glass, beads, chips, and slides.

19. A composition according to any of the claims 15 to 18 wherein said composition comprises an array of nucleic acid binding compounds 5 to 20 nucleotides in length.

34. Use of a first nucleic acid binding compound in a hybridization reaction to form a parallel or antiparallel duplex with a second nucleic acid binding compound wherein the first nucleic acid binding compound and/or the second nucleic acid binding compound comprise a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.

35. Use according to claim 34 wherein the hybridization reaction is a multiplex hybridization reaction.

46. Use according to any of the claims 34 to 45 wherein the substituted pyrazolo[3,4-d]pyrimidine analogue is a substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine or a 7-substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine.

47. Use according to any of the claims 34 to 45 wherein the substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof is a substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof or a 7-substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof.

36. Use of a nucleic acid binding compound having a backbone as a capture probe, whereby the backbone has attached thereto heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.

38. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound to increase the melting temperature of a parallel or antiparallel duplex with a second nucleic acid binding compound whereby the increase in melting temperature is compared to the melting temperature of a duplex of the first nucleic acid binding compound with a second nucleic acid binding compound wherein the heterocyclic group in the first nucleic acid binding compound is complementary to a heterocyclic group in the second nucleic acid binding compound.

39. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound used as probe in an amplification reaction, to increase the melting temperature of a duplex with a second nucleic acid binding compound in comparison to the melting temperature of a primer used in the amplification reaction, whereby the increase in melting temperature is compared to the melting temperature of a duplex of the first nucleic acid binding compound with the second nucleic acid binding compound wherein the heterocyclic group in the first nucleic acid binding compound is complementary to a heterocyclic group in the second nucleic acid binding compound.

44. Use according to any of the claims 39 to 43 wherein one or two reporter groups are attached to the nucleic acid binding compound or the first and/or the second nucleic acid binding compound.

45. Use according to claim 44 wherein the substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group is 1 to 5 nucleotides separated from the point of attachment of a reporter group.

40. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound to harmonize the contribution of each base pair to the melting temperature of a parallel or antiparallel duplex with a second nucleic acid binding compound.

41. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound for enhanced detection of sequences in a second nucleic acid binding compound having mismatches in a duplex with the first nucleic acid binding compound.

42. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a nucleic acid binding compound to increase the melting temperature of an intramolecular duplex of the nucleic acid binding compound whereby the increase in melting temperature is compared to the melting temperature of the intramolecular duplex of the nucleic acid binding compound wherein the heterocyclic group in the nucleic acid binding compound is complementary to a heterocyclic group in the hybridizing part of the nucleic acid binding compound.

43. Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a nucleic acid binding compound for enhanced detection of subtypes in a target nucleic acid.

49. A method for the determination of the presence, absence or amount of a nucleic acid comprising the steps

providing a sample suspected to contain the nucleic acid,

providing a nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, which is essentially complementary to a part or all of the nucleic acid,

contacting said sample with the nucleic acid binding compound under conditions for binding the nucleic acid binding compound to the nucleic acid to form a duplex,

determining the binding product or the degree of hybridization between the nucleic acid and the nucleic acid binding compound as a measure of the presence, absence or amount of the nucleic acid.

51. The method according to any of the claims 49 to 50, wherein the nucleic acid binding compound comprises a reporter group.

52. The method according to any of the claims 51 wherein the reporter group is a fluorescent label.

53. The method according to claim 52 wherein the nucleic acid binding compound further comprises a quenching agent which quenches the fluorescence emission of the fluorescent label.

54. The method according to claim 53 wherein the fluorescent label is a fluorescein and wherein the quenching agent is a fluorescent rhodamine or cyanine.

55. The method according to any of the claims 53 to 54, further comprising the step of altering the spatial relationship between the fluorescent label and the quenching agent subsequent to hybridization.

56. The method according to claim 55, wherein alteration of the spatial relationship between the fluorescent label and the quenching agent is accomplished by exonuclease hydrolysis of the nucleic acid binding compound, wherein release of label occurs as a result of exonuclease hydrolysis.

57. The method according to claim 56, wherein the degree of hybridization between the nucleic acid binding compound and the nucleic acid is determined by the quantity of label that is released from the nucleic acid binding compound subsequent to hybridization.

58. The method according to claims 49 to 50, wherein the degree of hybridization between the nucleic acid binding compound and the nucleic acid is determined by the priming ability of the nucleic acid binding compound.

59. The method according to claim 58, wherein priming occurs as part of a polymerase chain reaction.

60. The method according to claim 52, wherein more than one nucleic acid binding compound is used.

61. The method according to claim 60, wherein two nucleic acid binding compounds are used.

62. The method according to claim 61, wherein the first of the two nucleic acid binding compounds comprises a fluorescence donor and the second of the two nucleic acid binding compounds comprises a fluorescence acceptor, and wherein the emission wavelengths of the fluorescence donor overlap the absorption wavelengths of the fluorescence acceptor.

63. The method according to claim 62 wherein the degree of hybridization is measured by the quantity of light transferred between the fluorescence donor and the fluorescence acceptor and emitted by the fluorescence acceptor.

64. The method according to claim 62 wherein the degree of hybridization is determined by the measurement of the melting temperature between the nucleic acid binding compound and the nucleic acid.

78. Method according to any of the claims 49 to 77 wherein the substituted pyrazolo[3,4-d]pyrimidine analogue is a substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine or a 7-substituted pyrazolo[3,4-d]pyrimidine analogue of adenine or guanine.

79. Method according to any of the claims 49 to 77 wherein the substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof is a substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof or a 7-substituted 7-deaza-8-aza-2,6-diamino-purine or a derivative thereof.

82. Method according to any of the claims 49 to 80 wherein a parallel or an antiparallel duplex is formed.

50. A method for distinguishing related nucleotide sequences in a nucleic acid, the method comprising the following steps:

a) providing a nucleic acid binding compound having a defined sequence, wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof;

b) providing a nucleic acid with two related nucleotide sequences, each of which comprises a target sequence, wherein one of the nucleotide sequence is a target sequence that is perfectly complementary to the nucleic acid binding compound and one other of the segments is a related target sequence;

c) incubating the nucleic acid with the nucleic acid binding compound under hybridization conditions to form a duplex; and

d) determining the degree of hybridization between the nucleic acid binding compound and each of the segments.

65. A method for detecting the presence of a target sequence in a nucleic acid, the method comprising the following steps:

a) providing a nucleic acid which is to be tested for the presence of the target sequence;

b) providing a nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, wherein the nucleic acid binding compound has a sequence that is substantially complementary to the target sequence;

c) incubating the nucleic acid and the nucleic acid binding compound under hybridization conditions; and

d) identifying hybridized nucleic acids;

66. The method according to claim 65, wherein multiple nucleic acids are tested for the presence of the target sequence, and wherein the nucleic acids have related target sequences.

67. The method according to claim 65, wherein the nucleic acid binding compound is a primer comprising an extendible 3′

-hydroxyl group.

68. The method according to claim 67, wherein the hybridized nucleic acids are identified by extending the primer with a polymerizing enzyme.

69. The method according to claim 67, wherein the nucleic acid binding compound is a primer in a polymerase chain reaction.

70. A method for primer extension, the method comprising the following steps:

a) providing a nucleic acid containing a target sequence,

b) providing one or more nucleic acid binding compound complementary to the target sequence wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof,

c) providing a polymerizing enzyme and nucleotide substrates, and

d) incubating the nucleic acid, the nucleic acid binding compounds, the enzyme and the substrates under conditions favorable for polymerization.

71. The method according to claim 70, wherein the method is part of a polymerase chain reaction.

72. The method according to claim 70, wherein the method is used in the synthesis of a cDNA molecule.

73. A method for determining the nucleotide sequence of a nucleic acid, the method comprising the following steps:

a) providing an array of nucleic acid binding compounds having different known sequences, wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, with the proviso that the nucleic acid binding compounds do not contain a reporter group;

b) incubating the nucleic acid with the array under hybridization conditions, and

c) determining to which of the nucleic acid binding compounds in the array the nucleic acid hybridizes.

74. A method for determining the nucleotide sequence of a target sequence in a nucleic acid, the method comprising the following steps:

a) providing a nucleic acid comprising the target sequence;

b) providing at least two nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof with a known sequence and wherein one of the at least two nucleic acid binding compounds has a sequence that is perfectly complementary to the target sequence and at least one other of the nucleic acid binding compounds has a related target sequence;

c) incubating the at least two nucleic acid binding compounds with the nucleic acid under hybridization conditions; and

d) determining the degree of hybridization between each of the nucleic acid binding compounds and the nucleic acid.

75. The method according to claim 74, wherein the at least one other nucleic acid binding compounds has a single-nucleotide mismatch with the target sequence.

76. A method for examining gene expression in a cell, the method comprising the following steps:

a) providing a population of nucleic acids representative of the genes expressed in the cell,

b) providing an array of nucleic acid binding compounds having different sequences wherein the nucleic acid binding compounds comprise a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, and with the proviso that the nucleic acid binding compounds do not contain a reporter group;

c) incubating the population of nucleic acids with the array under hybridization conditions, and

d) determining which of the nucleic acid binding compounds in the array become hybridized to nucleic acids.

77. A method for identifying a mutation in a target sequence of a gene of interest, the method comprising the following steps:

a) providing a nucleic acid that comprises the target sequence.

b) providing an array of nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, wherein the nucleic acid binding compounds have different sequences, wherein the different sequences include the wild-type target sequence and different mutant target sequences, with the proviso that the nucleic acid binding compounds do not contain a reporter group;

c) incubating the nucleic acid with the array under hybridization conditions, and

d) determining which of the nucleic acid binding compounds in the array become hybridized to the nucleic acid.

85. A building block for the synthesis of an oligonucleotide comprising the nucleosides adenosine or desoxyadenosine, guanosine or desoxyguanosine, isoguanosine or desoxyisoguanosine, cytidine or deoxycytidine, uridine or desoxyuridine, thymidine or desoxythymidine, or analogues of these nucleosides

wherein a substituent is attached to the base moiety selected from the group of phthalimidoalkyl, phthalimidoalkenyl, or phthalimidoalkynyl groups with the following formulas

86. A building block according to claim 85 wherein the analogue of these nucleosides is a pyrazolo[3,4-d]pyrimidine analogue.

87. A building block according to any of the claims 85 to 86 wherein the building block is a phosphoramidite derivative.

88. Use of a building block according to any of the claims 85 to 87 for the synthesis of an oligonucleotide.

91. Use according to any of the claims 88 to 90 wherein the building block is a phosphoramidite derivative.

92. A method for the synthesis of an oligonucleotide from building blocks according to any of the claims 85 to 87.

89. Use of a phthaloyl group as a protecting group in a method for the synthesis of an oligonucleotide from building blocks comprising the nucleosides adenosine or desoxyadenosine, guanosine or desoxyguanosine, isoguanosine or desoxyisoguanosine, cytidine or deoxycytidine, uridine or desoxyuridine, thymidine or desoxythymidine, or analogues of these nucleosides wherein the amino groups of —

(CH₂)_n—

NH₂, —

CH═

CH—

(CH₂)_n—

NH₂, or —

C≡

C—

(CH₂)_n—

NH₂ attached to the base moiety are derivatized with the phthaloyl group and n is any integer from 1 to 18,

with the proviso that the —

(CH₂)_n—

NH₂ or —

C≡

C—

(CH₂)_n—

NH₂ group is not attached to the C5-atom of deoxyuridine and the proviso that the —

C≡

C—

(CH₂)_n—

NH₂ group is not attached to the C7-atom of 7-deaza-deoxyguanosine.

90. Use according to claim 89 wherein the analogue of these nucleosides is a pyrazolo[3,4-d]pyrimidine analogue.