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

• US 6,994,966 B2
• Filed: 05/21/2001
• Issued: 02/07/2006
• Est. Priority Date: 02/17/2000
• Status: Expired due to Term

• Current Assignee: Glycominds Diagnostics Ltd.
• Original Assignee: Glycominds, Ltd. (Storage Drop Storage Technologies Ltd.)
• Inventors: Dotan, Nir, Dukler, Avinoam
• Primary Examiner(s): Celsa, Bennett
• Assistant Examiner(s): Epperson, Jon D.
• Application Number:
  US09/860,559
• Publication Number:
  US 20020094541A1
• Time in Patent Office: 1,723 Days
• Field of Search: 435/6, 435/41, 435/72, 435/DIG.38, 435/45, 435/46, 435/49, 435/DIG.46, 435/DIG.45, 435/DIG.49, 435/DIG.7, 536/1.11, 536/123.1
• US Class Current: 435/6.14
• 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
• 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

The present invention provides methods of utilizing a complex carbohydrate library which includes a plurality of complex carbohydrate structures each being attached to a specific site of a solid support such as a platform for screening and isolating complex carbohydrate structures capable of specifically and uniquely binding with an entity such as a polypeptide.

Claims

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

(a) enzymatically synthesizing an addressable carbohydrate library including a plurality of carbohydrate structures each being attached at a specific and addressable location of an array, wherein each of said plurality of carbohydrate structures is composed of naturally occurring monosaccharides, such that a stereo-specificity of each bond interconnecting said monosaccharide units of said plurality of carbohydrate structures is defined by said addressable location thereof on said array and wherein each of said plurality of carbohydrate structures is composed of at least 2 and no more than 20 monosaccharide units each being naturally occurring;

wherein each of said plurality of carbohydrate structures is attached to said array via a linker, said linker including at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an anilino group and

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

2. The method of claim 1, wherein said addressable location of said carbohydrate also defines a stereo-specificity thereof.

3. The method of claim 1, wherein said entity is selected from the group consisting of a polypeptide encoded by a polynucleotide and a protein extracted from a natural source.

12. The method of claim 1, wherein said monosaccharide units are each independently selected from the group consisting of D-Glucose, D-Galactose, D-Mannose, L-Fucose, D-Xylose, D-N-Acetylglucoseamine, D-N-Acetylgalactoseamine, D-Glucuronic acid, D-Galacturonic acid, L-Rhamnose, L-Arabinose, D-Fructose, D-Apiose, (3-C-(Hydroxymethyl)-D-glycero-tetrose), L-Aceric acid (3-C-Carboxy-5dexoyl-xylose) Sialic Acid, and 2-sulfo-D-Mannose, 4-sulfo-L-Fucose, 2,6di-sulfo-D-Mannose and 6-sulfa-D-Mannose.

15. The method of claim 1, wherein said monosaccharide units are in alpha or beta configurations.

18. The method of claim 1, wherein said monosaccharide units are sulfated.

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

(a) enzymatically synthesizing an addressable carbohydrate library including a plurality of carbohydrate structures each being attached at a specific and addressable location of an array, wherein each of said plurality of carbohydrate structures is composed of at least 2 and no more than 20 monosaccharide units each being naturally occurring and whereas each of said plurality of carbohydrate structures is attached to said array via a linker, said linker including at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an anilino group; and

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

5. The method of claim 4, wherein said linker includes three ethylenglycol derivatives, four cyanuric chloride derivatives and an anilino group.

6. The method of claim 4, wherein at least one carbohydrate structure of said plurality of carbohydrate structures is a branched carbohydrate having a single structure defined by said addressable location thereof on said array.

7. The method of claim 4, wherein said addressable location of each of said carbohydrate structures defines a stereo-specificity thereof.

8. The method of claim 4, wherein said entity is selected from the group consisting of a polypeptide encoded by a polynucleotide and a protein extracted from a natural source.

13. The method of claim 4, wherein said monosaccharide units are each independently selected from the group consisting of D-Glucose, D-Galactose, D-Mannose, L-Fucose, D-Xylose, D-N-Acetylglucoseamine, D-N-Acetylgalactoseamine, D-Glucuronic acid, D-Galacturonic acid, L-Rhamnose, L-Arabinose, D-Fructose, D-Apiose, (3-C-(Hydroxymethyl)-D-glycero-tetrose), L-Aceric acid (3-C-Carboxy-5dexoyl-xylose) Sialic Acid, and 2-sulfo-D-Mannose, 4-sulfo-L-Fucose, 2,6di-sulfo-D-Mannose and 6-sulfo-D-Mannose.

16. The method of claim 4, wherein said monosaccharide units are in alpha or beta configurations.

19. The method of claim 4, wherein said monosaccharide units are sulfated.

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

(a) enzymatically synthesizing an addressable carbohydrate library including a plurality of carbohydrate structures each being attached at a specific and addressable location of an array, wherein each of said plurality of carbohydrate structures is composed of at least 2 and no more than 20 monosaccharide units each being naturally occurring and whereas a stereo-specificity of all carbohydrate structures in given addressable locations on said array is identical; and

(b) screening said addressable carbohydrate library with the entity thereby identifying the carbohydrate capable of binding the entity; and

wherein each of said plurality of carbohydrate structures is attached to said array via a linker, said linker including at least one ethylenglycol derivative, at least two cyanuric chloride derivatives and an anilino group.

10. The method of claim 9, wherein at least one carbohydrate structure of said plurality of carbohydrate structures is a branched carbohydrate having a single structure defined by said addressable location thereof on said array.

11. The method of claim 9, wherein said entity is selected from the group consisting of a polypeptide encoded by a polynucleotide and a protein extracted from a natural source.

14. The method of claim 9, wherein said monosaccharide units are each independently selected from the group consisting of D-Glucose, D-Galactose, D-Mannose, L-Fucose, D-Xylose, D-N-Acetylglucoseamine, D-N-Acetylgalactoseamine, D-Glucuronic acid, D-Galacturonic acid, L-Rhamnose, L-Arabinose, D-Fructose, D-Apiose, (3-C-(Hydroxymethyl)-D-glycero-tetrose), L-Aceric acid (3-C-Carboxy-5dexoyl-xylose) Sialic Acid, and 2-sulfo-D-Mannose, 4-sulfo-L-Fucose, 2,6di-sulfo-D-Mannose and 6-sulfo-D-Mannose.

17. The method of claim 9, wherein said monosaccharide units are in alpha or beta configurations.

20. The method of claim 9, wherein said monosaccharide units are sulfated.