Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof

• US 20050277154A1
• Filed: 07/27/2005
• Published: 12/15/2005
• Est. Priority Date: 02/17/2000
• Status: Abandoned Application

• Current Assignee: Glycominds, Ltd. (Storage Drop Storage Technologies Ltd.)
• Original Assignee: Glycominds, Ltd. (Storage Drop Storage Technologies Ltd.)
• Inventors: Dotan, Nir, Dukler, Avinoam
• Application Number:
  US11/189,932
• Publication Number:
  US 20050277154A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 15/02 Acyclic radicals, not substituted by cyclic structures
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C07K 14/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 2333/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 2400/02 involving antibodies to sugar part of glycoproteins lectins from plants G01N2333/42, lectins from mammals G01N2333/4724
• CPC Class Codes: G01N 2400/10 Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

A combinatorial complex carbohydrate library is provided and including a plurality of addressable complex carbohydrate structures.

Claims

1. An addressable carbohydrate library comprising a plurality of carbohydrate structures each being attached at a specific and addressable location of a single solid support, wherein each of said plurality of carbohydrate structures is composed of monosaccharides, such that a stereo-specificity of each bond interconnecting said monosaccharides is defined by said addressable location on said single solid support.

2. The addressable carbohydrate library of claim 1, wherein each of said plurality of carbohydrate structures is attached to said single solid support via a linker.

3. The addressable carbohydrate library of claim 2, wherein said linker is cleavable.

4. The addressable carbohydrate library of claim 2, wherein said linker includes at least two contiguous covalent bonds.

5. The addressable carbohydrate library of claim 2, wherein said linker is selected from the group consisting of an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain and a nucleic acid.

6. The addressable carbohydrate library of claim 2, wherein said linker is at least 20 Angstrom in length.

7. The addressable carbohydrate library of claim 2, wherein said linker comprises at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an aniline group.

8. The addressable carbohydrate library of claim 1, wherein said single solid support is a flat platform.

9. The addressable carbohydrate library of claim 8, wherein said single solid support is a chip and further wherein different carbohydrate structures of said plurality of carbohydrate structures are arranged on said chip in patches spaced not more than 2.25 mm from one another, center to center.

10. The addressable carbohydrate library of claim 1, wherein said single solid support is of a substance selected from the group consisting of polystyrene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, silica, glass, quartz, plastic and cellulose.

11. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least two covalently attached identical saccharide units.

12. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least one branch.

13. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least 4 saccharide units.

14. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least 5 saccharide units.

15. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least 6 saccharide units.

16. The addressable carbohydrate library of claim 1, wherein at least one of said plurality of carbohydrate structures comprises at least 7 saccharide units.

17. The addressable carbohydrate library of claim 1, wherein at least a portion of said plurality of carbohydrate structures are not naturally occurring carbohydrate structures.

18. The addressable carbohydrate library of claim 1, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to naturally occurring carbohydrate structures.

19. The addressable carbohydrate library of claim 18, wherein said naturally occurring carbohydrate structures are present in human cells.

20. The addressable carbohydrate library of claim 18, wherein said naturally occurring carbohydrate structures are derived from tissue, cells and/or body fluids of a human.

21. The addressable carbohydrate library of claim 18, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to domains of at least one naturally occurring carbohydrate structure.

22. The addressable carbohydrate library of claim 21, wherein said at least one naturally occurring carbohydrate is present in human cells.

23. The addressable carbohydrate library of claim 1, wherein said plurality of carbohydrate structures are selected from the group consisting of:

24. The addressable carbohydrate library of claim 1, wherein at least some of said monosaccharides are in alpha configuration.

25. The addressable carbohydrate library of claim 1, wherein at least some of said monosaccharides are in beta configuration.

26. The addressable carbohydrate library of claim 1, wherein at least some of said monosaccharides are sulfated.

27. A method of producing an addressable carbohydrate library, the method comprising:

(a) providing a single solid support having a plurality of addressable locations; and

(b) enzymatically synthesizing a plurality of carbohydrate structures on said single solid support, wherein each of said plurality of carbohydrate structures is composed of monosaccharides, such that a stereo-specificity of each bond interconnecting said monosaccharides is defined by said addressable location on said single solid support, thereby producing the addressable carbohydrate library.

28. The method of claim 27, wherein each of said plurality of carbohydrate structures is attached to said single solid support via a linker.

29. The method of claim 28, wherein said linker is cleavable.

30. The method of claim 28, wherein said linker includes at least two contiguous covalent bonds.

31. The method of claim 28, wherein said linker is selected from the group consisting of an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain and a nucleic acid.

32. The method of claim 28, wherein said linker is at least 20 Angstrom in length.

33. The method of claim 28, wherein said linker comprises at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an aniline group.

34. The method of claim 27, wherein said single solid support is a flat platform.

35. The method of claim 34, wherein said single solid support is a chip and further wherein different carbohydrate structures of said plurality of carbohydrate structures are arranged on said chip in patches spaced not more than 2.25 mm from one another, center to center.

36. The method of claim 27, wherein said single solid support is of a substance selected from the group consisting of polystyrene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, silica, glass, quartz, plastic and cellulose.

37. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least two covalently attached identical saccharide units.

38. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least one branch.

39. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least 4 saccharide units.

40. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least 5 saccharide units.

41. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least 6 saccharide units.

42. The method of claim 27, wherein at least one of said plurality of carbohydrate structures comprises at least 7 saccharide units.

43. The method of claim 27, wherein at least a portion of said plurality of carbohydrate structures are not naturally occurring carbohydrate structures.

44. The method of claim 27, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to naturally occurring carbohydrate structures.

45. The method of claim 44, wherein said naturally occurring carbohydrate structures are present in human cells.

46. The method of claim 44, wherein said naturally occurring carbohydrate structures are derived from tissue, cells and/or body fluids of a human.

47. The method of claim 44, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to domains of at least one naturally occurring carbohydrate structure.

48. The method of claim 47, wherein said at least one naturally occurring carbohydrate is present in human cells.

49. The method of claim 27, wherein said plurality of carbohydrate structures are selected from the group consisting of:

50. The method of claim 27, wherein at least some of said monosaccharides are in alpha configuration.

51. The method of claim 27, wherein at least some of said monosaccharides are in beta configuration.

52. The method of claim 27, wherein at least some of said monosaccharides are sulfated.

53. A method of identifying a carbohydrate capable of binding an entity, the method comprising the steps of:

(a) producing an addressable carbohydrate library by;

(i) providing a single solid support having a plurality of addressable locations; and

(ii) enzymatically synthesizing a plurality of carbohydrate structures on said single solid support, wherein each of said plurality of carbohydrate structures is composed of monosaccharides, such that a stereo-specificity of each bond interconnecting said monosaccharides is defined by said addressable location on said single solid support, thereby producing the addressable carbohydrate library; and

(b) screening said addressable carbohydrate library with the entity for identifying the carbohydrate capable of binding the entity.

54. The method of claim 53, wherein each of said plurality of carbohydrate structures is attached to said single solid support via a linker.

55. The method of claim 54, wherein said linker is cleavable.

56. The method of claim 54, wherein said linker includes at least two contiguous covalent bonds.

57. The method of claim 54, wherein said linker is selected from the group consisting of an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain and a nucleic acid.

58. The method of claim 54, wherein said linker is at least 20 Angstrom in length.

59. The method of claim 54, wherein said linker comprises at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an aniline group.

60. The method of claim 53, wherein said single solid support is a flat platform.

61. The method of claim 60, wherein said single solid support is a chip and further wherein different carbohydrate structures of said plurality of carbohydrate structures are arranged on said chip in patches spaced not more than 2.25 mm from one another, center to center.

62. The method of claim 53, wherein said single solid support is of a substance selected from the group consisting of polystyrene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, silica, glass, quartz, plastic and cellulose.

63. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least two covalently attached identical saccharide units.

64. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least one branch.

65. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least 4 saccharide units.

66. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least 5 saccharide units.

67. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least 6 saccharide units.

68. The method of claim 53, wherein at least one of said plurality of carbohydrate structures comprises at least 7 saccharide units.

69. The method of claim 53, wherein at least a portion of said plurality of carbohydrate structures are not naturally occurring carbohydrate structures.

70. The method of claim 53, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to naturally occurring carbohydrate structures.

71. The method of claim 70, wherein said naturally occurring carbohydrate structures are present in human cells.

72. The method of claim 70, wherein said naturally occurring carbohydrate structures are derived from tissue, cells and/or body fluids of a human.

73. The method of claim 70, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to domains of at least one naturally occurring carbohydrate structure.

74. The method of claim 73, wherein said at least one naturally occurring carbohydrate is present in human cells.

75. The method of claim 53, wherein said plurality of carbohydrate structures are selected from the group consisting of:

76. The method of claim 53, wherein at least some of said monosaccharides are in alpha configuration.

77. The method of claim 53, wherein at least some of said monosaccharides are in beta configuration.

78. The method of claim 53, wherein at least some of said monosaccharides are sulfated.

79. The method of claim 53, wherein said entity is selected from the group consisting of proteins encoded by an EST library and proteins extracted from a natural source.

80. A method of identifying glyco-markers associated with a disease or condition, the method comprising:

(a) enzymatically synthesizing an addressable carbohydrate library representing the glyco-markers, wherein said addressable carbohydrate library comprises a plurality of carbohydrate structures each being attached at a specific and addressable location of a single solid support, wherein each of said plurality of carbohydrate structures is composed of monosaccharides, such that a stereo-specificity of each bond interconnecting said monosaccharides is defined by said addressable location on said single solid support;

(b) reacting said addressable carbohydrate library with a sample derived from a healthy individual to thereby generate an anti-glycan antibody profile of said healthy individual;

(c) reacting said addressable carbohydrate library with a sample derived from a diseased individual to thereby generate an anti-glycan antibody profile of said diseased individual;

(d) identifying antibodies which are present in said anti-glycan antibody profile of said diseased individual and not in said anti-glycan antibody profile of said healthy individual, thereby identifying the glyco-markers associated with a disease or condition.

81. The method of claim 80, wherein each of said plurality of carbohydrate structures is attached to said single solid support via a linker.

82. The method of claim 81, wherein said linker is cleavable.

83. The method of claim 81, wherein said linker includes at least two contiguous covalent bonds.

84. The method of claim 81, wherein said linker is selected from the group consisting of an amino acid, a peptide, a non-glycosylated protein, a lipid, a ceramide, dolicol phosphate, a cyclodextrin, an oligosaccharide, a monosaccharide, an alkyl chain and a nucleic acid.

85. The method of claim 81, wherein said linker is at least 20 Angstrom in length.

86. The method of claim 81, wherein said linker comprises at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an aniline group.

87. The method of claim 80, wherein said single solid support is a flat platform.

88. The method of claim 87, wherein said single solid support is a chip and further wherein different carbohydrate structures of said plurality of carbohydrate structures are arranged on said chip in patches spaced not more than 2.25 mm from one another, center to center.

89. The method of claim 80, wherein said single solid support is of a substance selected from the group consisting of polystyrene cross-linked with divinylbenzene, polyethylene glycol-polystyrene block copolymer, polyamides, polyacrylamide, polymethacrylamide, silica, glass, quartz, plastic and cellulose.

90. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least two covalently attached identical saccharide units.

91. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least one branch.

92. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least 4 saccharide units.

93. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least 5 saccharide units.

94. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least 6 saccharide units.

95. The method of claim 80, wherein at least one of said plurality of carbohydrate structures comprises at least 7 saccharide units.

96. The method of claim 80, wherein at least a portion of said plurality of carbohydrate structures are not naturally occurring carbohydrate structures.

97. The method of claim 80, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to naturally occurring carbohydrate structures.

98. The method of claim 97, wherein said naturally occurring carbohydrate structures are present in human cells.

99. The method of claim 97, wherein said naturally occurring carbohydrate structures are derived from tissue, cells and/or body fluids of a human.

100. The method of claim 97, wherein at least a portion of said plurality of carbohydrate structures are structurally identical to domains of at least one naturally occurring carbohydrate structure.

101. The method of claim 100, wherein said at least one naturally occurring carbohydrate is present in human cells.

102. The method of claim 80, wherein said plurality of carbohydrate structures are selected from the group consisting of:

103. The method of claim 80, wherein at least some of said monosaccharides are in alpha configuration.

104. The method of claim 80, wherein at least some of said monosaccharides are in beta configuration.

105. The method of claim 80, wherein at least some of said monosaccharides are sulfated.

106. The method of claim 80, wherein said sample is a serum sample or a urine sample.

107. The method of claim 80, wherein the disease is selected from the group consisting of cancer, cardiovascular diseases and transplantation.