Click Chemistry for the Production of Reporter Molecules

• US 20100081137A1
• Filed: 10/31/2007
• Published: 04/01/2010
• Est. Priority Date: 10/31/2006
• Status: Active Grant

• Current Assignee: Baseclick GmbH
• Original Assignee: Baseclick GmbH
• Inventors: Carell, Thomas, Schwogler, Anja
• Granted Patent: US 8,193,335 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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

Abstract

The present invention relates to methods for producing reporter molecules suitable for the detection of analytes, e.g. nucleic acids. Further, the present invention relates to methods and regions for detecting analytes.

Claims

1-29. -29. (canceled)

30. A method for producing a reporter molecule comprising at least two different functional groups, said method comprising:

(a) synthesizing said reporter molecule from a plurality of building blocks, wherein at least one building block comprises a first handle group which is selected from the group consisting of an alkyne group, a protected alkyne group, an azide group, aldehyde group, a protected aldehyde group, a hydrazine group and a hydroxylamino group, wherein at least one building block comprises a second handle group which is selected from the group consisting of an alkyne group, a protected alkyne group, an azide group, aldehyde group, a protected aldehyde group, a hydrazine group and a hydroxylamino group, and wherein said first handle group is different from said second handle group;

(b) coupling a first reaction partner to said first handle group under conditions wherein said first handle group is reactive and said second handle group is not reactive,

wherein said first reaction partner comprises a first functional group; and

subsequently

(c) coupling a second reaction partner to said second handle group wherein said second reaction partner comprises a second functional group and wherein said first functional group is different from said second functional group,

wherein said reporter molecule is selected from the group consisting of nucleic acids and nucleic acid analogues.

31. The method of claim 30, wherein said reporter molecule comprises up to 2000 building blocks.

32. The method of claim 30, wherein said reporter molecule comprises 4-200 building blocks.

33. The method of claim 30, wherein said reporter molecule comprises 10-100 building blocks.

34. The method of claim 30, wherein said functional groups are selected from the group consisting of labelling groups, quencher groups and attachment groups.

35. The method of claim 34, wherein said labelling groups are dyes, and said attachment groups are selected from the group consisting of biotin, biotin derivatives and hapten.

36. The method of claim 30, wherein said first and said second functional groups are a labelling group and a quencher group or wherein said first and said second functional group are a labelling group and an attachment group.

37. The method of claim 30, wherein said handle groups are selected from the group consisting of alkyne groups and protected alkyne groups.

38. The method of claim 37, wherein a reaction partner comprising an azide group is coupled to an alkyne group via Click reaction.

39. The method of claim 30, wherein said first and said second handle group are an alkyne group and a protected alkyne group or wherein said first and said second handle group are a first protected alkyne group and a second protected alkyne group.

40. The method of claim 30, wherein said first reaction partner is coupled to said first handle group under conditions wherein said first handle group is unprotected and said second handle group is protected.

41. The method of claim 30, wherein at least three handle groups are incorporated into said reporter molecule.

42. The method of claim 30, wherein said first handle group is an alkyne group and said second and third handle groups are protected alkyne groups carrying different protection groups.

43. The method of claim 30, wherein the synthesis of said reporter molecule is a chemical synthesis.

44. The method of claim 43, wherein said synthesis is a chemical solid phase synthesis wherein the reporter molecule is synthesized by stepwise assembly of building blocks while being bound to a solid phase.

45. The method of claim 44, wherein at least the first reaction partner is coupled while said reporter molecule is bound to said solid phase.

46. The method of claim 44, wherein said first reaction partner is coupled after said reporter molecule has been cleaved off said solid phase.

47. The method of claim 30, wherein said reporter molecule is synthesized from nucleoside triphosphate building blocks.

48. A method for detecting an analyte in a sample comprising the steps:

(a) providing a sample;

(b) contacting the sample with a reporter molecule comprising at least two different functional groups wherein said functional groups are bound to said reporter molecule via linker groups comprising a 1,2,3-triazole ring and

(c) detecting an interaction of said reporter molecule with said analyte which is indicative for the presence and/or amount of analyte in said sample, wherein said reporter molecule is a nucleic acid or nucleic acid analogue.

49. The method of claim 48, wherein said analyte is a nucleic acid or a nucleic acid cleaving or a binding protein.

50. The method of claim 48, wherein the interaction of said reporter molecule with said analyte is a binding.

51. The method of claim 48, wherein the interaction of said reporter molecule with said analyte is a hybridization.

52. The method of claim 48, wherein said reporter molecule is a Molecular Beacon.

53. A reporter molecule comprising at least two different functional groups wherein said functional groups are bound to said reporter molecule via linker groups comprising a 1,2,3-triazole ring, and wherein said reporter molecule is a nucleic acid or a nucleic acid analogue.

54. The reporter molecule of claim 53, which is a Molecular Beacon.

55. A compound of the Formula (I)

C-S-N

wherein

C is a protected alkyne group,

S is a spacer or a bond, and

N is a nucleic acid or nucleic acid analogue building block such as a nucleosidic or nucleotidic or non-nucleosidic compound.

59. The compound of claim 55, wherein said spacer has a chain length of 3 to 10 atoms.

61. The compound of claim 55, wherein said spacer comprises an alkyne group.

63. The compound of claim 55, wherein said protected alkyne group is a silyl-protected alkyne group.

65. The compound of claim 63, wherein the silyl group is a tris(alkyl/aryl) silyl group such as trimethylsilyl, triethylsilyl, triisopropylsilyl, triphenylsilyl or tert.-butyl-dimethylsilyl, or a cyclic bridged silyl group.

56. A non-nucleosidic compound of the Formula (VI)

57. The compound of claim 56, wherein said non-nucleosidic group is a spacer or a bond.

58. The compound of claim 56, wherein S₁ is a spacer and S₂ is a covalent bond.

60. The compound of claim 56, wherein said spacer has a chain length of 3 to 10 atoms.

62. The compound of claim 56, wherein said spacer comprises an alkyne group.

64. The compound of claim 56, wherein said protected alkyne group is a silyl-protected alkyne group.

66. The compound of claim 64, wherein the silyl group is a tris(alkyl/aryl) silyl group such as trimethylsilyl, triethylsilyl, triisopropylsilyl, triphenylsilyl or tert.-butyl-dimethylsilyl, or a cyclic bridged silyl group.

67. A compound of the Formula (IV)

Z-S-N

wherein

Z is an aldose group, wherein the hydroxy groups are protected with acyl and/or silyl groups, or a protected or an unprotected 1,2 diol group

S is a spacer or a bond, and

N is a nucleic acid or nucleic acid analogue building block.

68. The compound of claim 67, wherein said nucleic acid or nucleic acid analogue building block is a nucleosidic or nucleotidic compound.

69. The compound of claim 67, wherein said acyl groups are selected from the group consisting of acetyl, butyryl and pivaloyl.