U.S. patent application number 11/189932 was filed with the patent office on 2005-12-15 for combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof.
This patent application is currently assigned to Glycominds Ltd.. Invention is credited to Dotan, Nir, Dukler, Avinoam.
Application Number | 20050277154 11/189932 |
Document ID | / |
Family ID | 25128109 |
Filed Date | 2005-12-15 |
United States Patent
Application |
20050277154 |
Kind Code |
A1 |
Dukler, Avinoam ; et
al. |
December 15, 2005 |
Combinatorial complex carbohydrate libraries and methods for the
manufacture and uses thereof
Abstract
A combinatorial complex carbohydrate library is provided and
including a plurality of addressable complex carbohydrate
structures.
Inventors: |
Dukler, Avinoam; (Modi'in,
IL) ; Dotan, Nir; (Shoham, IL) |
Correspondence
Address: |
Martin MOYNIHAN
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
Glycominds Ltd.
|
Family ID: |
25128109 |
Appl. No.: |
11/189932 |
Filed: |
July 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11189932 |
Jul 27, 2005 |
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09860559 |
May 21, 2001 |
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09860559 |
May 21, 2001 |
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09783083 |
Feb 15, 2001 |
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09783083 |
Feb 15, 2001 |
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PCT/IL00/00099 |
Feb 17, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/6.13; 435/6.14; 435/7.1; 436/518; 536/123 |
Current CPC
Class: |
G01N 33/6842 20130101;
C07B 2200/11 20130101; G01N 33/6845 20130101; C07H 15/02 20130101;
G01N 2333/42 20130101; C07H 15/203 20130101; G01N 33/66 20130101;
G01N 2400/02 20130101; G01N 2400/10 20130101; G01N 33/5308
20130101; G01N 33/543 20130101; C40B 30/04 20130101; C07K 14/59
20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 436/518; 536/123 |
International
Class: |
C12Q 001/68; G01N
033/53; C08B 037/00; G01N 033/543 |
Claims
What is claimed is:
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:
29 Fuc(.alpha.1,2)Gal(.beta.)
NeuAC(a2,3)Gal(.beta.1,3)GlcNAc(.alpha.) NeuAC(a2,3)Gal(.beta.1,3-
)GlcNAc(.beta.) NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.beta.) NeuAC(a2,3)Gal(.beta.1,4)-
GlcNAc(.alpha.) Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.alpha.)
Fuc(.alpha.1,6)[Man(.beta.1,4)GlcNAc(.beta.1,4)]GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.)
GalNAc(.beta.1,4)[NeuAC(a2,3)]Gal(.beta.1,4)GlcNAc(.beta.)
GlcA(.beta.1,3)Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.)
GlcNAc(.beta.1,6)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)GalNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.)
NeuAC(.alpha.2,6)GalNAc(.alpha.) Fuc(.alpha.1,2)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) Fuc(.alpha.1,3)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.) Fuc(.alpha.1,3)GlcNAc(.beta.)
Gal(.beta.1,3)GlcNAc(.alpha.) Gal(.beta.1,3)GlcNAc(.beta.)
Gal(.beta.1,4)Xyl(.beta.) Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)GalNAc(.beta.) GlcNAc(.beta.1,3)GalNAc(.alpha.)
GlcNAc(.beta.1,4)GlcNAc(.alpha.) GlcNAc(.beta.1,4)GlcNAc(- .beta.)
GlcNAc(.beta.1,6)Gal(.alpha.) GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(- .beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4- )Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]Glc(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,4)[Gal(.beta.1,3)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3) [Fuc(.alpha.1,2)Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,4) [Gal(.beta.1,3)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Fuc(.alpha.1,3)[GlcNAc(.beta.1,3)Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,6)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
Fuc(.alpha.1,6)[GlcNAc(.beta.1,4)]Man(.alpha.)
Gal(.beta.1,4)GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
GlcNAc(.beta.1,4)[Gal(.beta.1,4)GlcNAc(.beta.1,2)]Man(.alpha.)
Man(.alpha.1,4)GlcNAc.beta.1,4[Fuc(.alpha.1,6)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.) Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(-
.beta.) Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
NeuAC(.alpha.2,3)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
Glc(.beta.1,3) Glc(.beta.) Glc(.beta.1,2) Glc(.beta.)
Glc(.beta.1,6) Glc(.beta.) Glc(.alpha.1,2) Glc(.alpha.)
Glc(.alpha.1,3) Glc(.alpha.) Glc(.alpha.1,4) Glc(.alpha.)
Glc(.alpha.1,6) Glc(.alpha.) Ara(.alpha.1,2) Ara(.alpha.)
Ara(.alpha.1,5) Ara(.alpha.) Ara(.alpha.1,2) Glc(.beta.)
Ara(.alpha.1,3) Glc(.beta.) Ara(.alpha.1,4) Glc(.beta.)
Ara(.alpha.1,6) Glc(.beta.) Xyl(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.) Man(.alpha.1,3)Man(.alpha.)
Man(.alpha.1,6)Man(.alpha.) Gal(.alpha.1,2)Gal(.alpha.)
Gal(.alpha.1,3)Gal(.alpha.) Gal(.alpha.1,4)Gal(.alpha.)
Gal(.alpha.1,6)Gal(.alpha.) Gal(.beta.1,2)Gal(.beta.)
Gal(.beta.1,3)Gal(.beta.) Gal(.beta.1,6)Gal(.beta.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,6)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)[NeuAc(.alpha.2,6)]GalNAc(.alpha.)
NeuAc(.alpha.2,8)NeuAc(.alpha.2,3)Gal(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.1,4)GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.) Gal (.beta.1,6)Gal
(.beta.1,4)Gal(.beta.1,4)Glc (.beta.) Gal(.beta.1,3)Gal(.beta.1,4-
)Xyl(.beta.) Gal(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
GalNAc(.beta.1,3)Gal(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.) GlcNAc(.beta.1,3)Gal(.bet-
a.1,3)GalNAc(.beta.) GlcNAc(.beta.1,6)Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Xyl(.alpha.1,3)Glc(.beta.) Xyl(.alpha.1,3)Xyl(.alpha.1,3)Glc(.bet-
a.) Gal(.beta.1,3)GalNAc(.beta.) Gal(.beta.1,3)GlcNAc(.bet- a.)
GlcNAc(.beta.1,3)GalNAc(.beta.)
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:
30 Fuc(.alpha.1,2)Gal(.beta.)
NeuAC(a2,3)Gal(.beta.1,3)GlcNAc(.alpha.) NeuAC(a2,3)Gal(.beta.1,3-
)GlcNAc(.beta.) NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.beta.) NeuAC(a2,3)Gal(.beta.1,4)-
GlcNAc(.alpha.) Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.alpha.)
Fuc(.alpha.1,6)[Man(.beta.1,4)GlcNAc(.beta.1,4)]GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.)
GalNAc(.beta.1,4)[NeuAC(a2,3)]Gal(.beta.1,4)GlcNAc(.beta.)
GlcA(.beta.1,3)Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.)
GlcNAc(.beta.1,6)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)GalNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.)
NeuAC(.alpha.2,6)GalNAc(.alpha.) Fuc(.alpha.1,2)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) Fuc(.alpha.1,3)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.) Fuc(.alpha.1,3)GlcNAc(.beta.)
Gal(.beta.1,3)GlcNAc(.alpha.) Gal(.beta.1,3)GlcNAc(.beta.)
Gal(.beta.1,4)Xyl(.beta.) Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)GalNAc(.beta.) GlcNAc(.beta.1,3)GalNAc(.alpha.)
GlcNAc(.beta.1,4)GlcNAc(.alpha.) GlcNAc(.beta.1,4)GlcNAc(- .beta.)
GlcNAc(.beta.1,6)Gal(.alpha.) GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(- .beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4- )Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]Glc(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,4)[Gal(.beta.1,3)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)[Fuc(.alpha.1,2)Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,4)[Gal(.beta.1,3)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Fuc(.alpha.1,3)[GlcNAc(.beta.1,3)Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,6)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
Fuc(.alpha.1,6)[GlcNAc(.beta.1,4)]Man(.alpha.)
Gal(.beta.1,4)GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
GlcNAc(.beta.1,4)[Gal(.beta.1,4)GlcNAc(.beta.1,2)]Man(.alpha.)
Man(.alpha.1,4)GlcNAc.beta.1,4[Fuc(.alpha.1,6)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.) Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(-
.beta.) Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
NeuAC(.alpha.2,3)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
Glc(.beta.1,3) Glc(.beta.) Glc(.beta.1,2) Glc(.beta.)
Glc(.beta.1,6) Glc(.beta.) Glc(.alpha.1,2) Glc(.alpha.)
Glc(.alpha.1,3) Glc(.alpha.) Glc(.alpha.1,4) Glc(.alpha.)
Glc(.alpha.1,6) Glc(.alpha.) Ara(.alpha.1,2) Ara(.alpha.)
Ara(.alpha.1,5) Ara(.alpha.) Ara(.alpha.1,2) Glc(.beta.)
Ara(.alpha.1,3) Glc(.beta.) Ara(.alpha.1,4) Glc(.beta.)
Ara(.alpha.1,6) Glc(.beta.) Xyl(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.) Man(.alpha.1,3)Man(.alpha.)
Man(.alpha.1,6)Man(.alpha.) Gal(.alpha.1,2)Gal(.alpha.)
Gal(.alpha.1,3)Gal(.alpha.) Gal(.alpha.1,4)Gal(.alpha.)
Gal(.alpha.1,6)Gal(.alpha.) Gal(.beta.1,2)Gal(.beta.)
Gal(.beta.1,3)Gal(.beta.) Gal(.beta.1,6)Gal(.beta.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,6)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)[NeuAc(.alpha.2,6)]GalNAc(.alpha.)
NeuAc(.alpha.2,8)NeuAc(.alpha.2,3)Gal(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.1,4)GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.) Gal (.beta.1,6)Gal
(.beta.1,4)Gal(.beta.1,4)Glc (.beta.) Gal(.beta.1,3)Gal(.beta.1,4-
)Xyl(.beta.) Gal(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
GalNAc(.beta.1,3)Gal(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.) GlcNAc(.beta.1,3)Gal(.bet-
a.1,3)GalNAc(.beta.) GlcNAc(.beta.1,6)Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Xyl(.alpha.1,3)Glc(.beta.) Xyl(.alpha.1,3)Xyl(.alpha.1,3)Glc(.bet-
a.) Gal(.beta.1,3)GalNAc(.beta.) Gal(.beta.1,3)GlcNAc(.bet- a.)
GlcNAc(.beta.1,3)GalNAc(.beta.)
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:
31 Fuc(.alpha.1,2)Gal(.beta.)
NeuAC(a2,3)Gal(.beta.1,3)GlcNAc(.alpha.) NeuAC(a2,3)Gal(.beta.1,3-
)GlcNAc(.beta.) NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.beta.) NeuAC(a2,3)Gal(.beta.1,4)-
GlcNAc(.alpha.) Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.alpha.)
Fuc(.alpha.1,6)[Man(.beta.1,4)GlcNAc(.beta.1,4)]GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.)
GalNAc(.beta.1,4)[NeuAC(a2,3)]Gal(.beta.1,4)GlcNAc(.beta.)
GlcA(.beta.1,3)Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.)
GlcNAc(.beta.1,6)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)GalNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.)
NeuAC(.alpha.2,6)GalNAc(.alpha.) Fuc(.alpha.1,2)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) Fuc(.alpha.1,3)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.) Fuc(.alpha.1,3)GlcNAc(.beta.)
Gal(.beta.1,3)GlcNAc(.alpha.) Gal(.beta.1,3)GlcNAc(.beta.)
Gal(.beta.1,4)Xyl(.beta.) Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)GalNAc(.beta.) GlcNAc(.beta.1,3)GalNAc(.alpha.)
GlcNAc(.beta.1,4)GlcNAc(.alpha.) GlcNAc(.beta.1,4)GlcNAc(- .beta.)
GlcNAc(.beta.1,6)Gal(.alpha.) GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(- .beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4- )Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]Glc(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,4)[Gal(.beta.1,3)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3) [Fuc(.alpha.1,2)Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,4) [Gal(.beta.1,3)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Fuc(.alpha.1,3)[GlcNAc(.beta.1,3)Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,6)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
Fuc(.alpha.1,6)[GlcNAc(.beta.1,4)]Man(.alpha.)
Gal(.beta.1,4)GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
GlcNAc(.beta.1,4)[Gal(.beta.1,4)GlcNAc(.beta.1,2)]Man(.alpha.)
Man(.alpha.1,4)GlcNAc.beta.1,4[Fuc(.alpha.1,6)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.) Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(-
.beta.) Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
NeuAC(.alpha.2,3)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
Glc(.beta.1,3) Glc(.beta.) Glc(.beta.1,2) Glc(.beta.)
Glc(.beta.1,6) Glc(.beta.) Glc(.alpha.1,2) Glc(.alpha.)
Glc(.alpha.1,3) Glc(.alpha.) Glc(.alpha.1,4) Glc(.alpha.)
Glc(.alpha.1,6) Glc(.alpha.) Ara(.alpha.1,2) Ara(.alpha.)
Ara(.alpha.1,5) Ara(.alpha.) Ara(.alpha.1,2) Glc(.beta.)
Ara(.alpha.1,3) Glc(.beta.) Ara(.alpha.1,4) Glc(.beta.)
Ara(.alpha.1,6) Glc(.beta.) Xyl(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.) Man(.alpha.1,3)Man(.alpha.)
Man(.alpha.1,6)Man(.alpha.) Gal(.alpha.1,2)Gal(.alpha.)
Gal(.alpha.1,3)Gal(.alpha.) Gal(.alpha.1,4)Gal(.alpha.)
Gal(.alpha.1,6)Gal(.alpha.) Gal(.beta.1,2)Gal(.beta.)
Gal(.beta.1,3)Gal(.beta.) Gal(.beta.1,6)Gal(.beta.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,6)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)[NeuAc(.alpha.2,6)]GalNAc(.alpha.)
NeuAc(.alpha.2,8)NeuAc(.alpha.2,3)Gal(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.1,4)GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.) Gal (.beta.1,6)Gal
(.beta.1,4)Gal(.beta.1,4)Glc (.beta.) Gal(.beta.1,3)Gal(.beta.1,4-
)Xyl(.beta.) Gal(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
GalNAc(.beta.1,3)Gal(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.) GlcNAc(.beta.1,3)Gal(.bet-
a.1,3)GalNAc(.beta.) GlcNAc(.beta.1,6)Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Xyl(.alpha.1,3)Glc(.beta.) Xyl(.alpha.1,3)Xyl(.alpha.1,3)Glc(.bet-
a.) Gal(.beta.1,3)GalNAc(.beta.) Gal(.beta.1,3)GlcNAc(.bet- a.)
GlcNAc(.beta.1,3)GalNAc(.beta.)
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:
32 Fuc(.alpha.1,2)Gal(.beta.)
NeuAC(a2,3)Gal(.beta.1,3)GlcNAc(.alpha.) NeuAC(a2,3)Gal(.beta.1,3-
)GlcNAc(.beta.) NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAC(a2,6)Gal(.beta.1,4)GlcNAc(.beta.) NeuAC(a2,3)Gal(.beta.1,4)-
GlcNAc(.alpha.) Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.alpha.)
Fuc(.alpha.1,6)[Man(.beta.1,4)GlcNAc(.beta.1,4)]GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2) Man(.alpha.)
Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.)
GalNAc(.beta.1,4)[NeuAC(a2,3)]Gal(.beta.1,4)GlcNAc(.beta.)
GlcA(.beta.1,3)Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.)
GlcNAc(.beta.1,6)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)GalNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.) GlcNAc(.beta.1,2)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.)
NeuAC(.alpha.2,6)GalNAc(.alpha.) Fuc(.alpha.1,2)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) Fuc(.alpha.1,3)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.) Fuc(.alpha.1,3)GlcNAc(.beta.)
Gal(.beta.1,3)GlcNAc(.alpha.) Gal(.beta.1,3)GlcNAc(.beta.)
Gal(.beta.1,4)Xyl(.beta.) Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)GalNAc(.beta.) GlcNAc(.beta.1,3)GalNAc(.alpha.)
GlcNAc(.beta.1,4)GlcNAc(.alpha.) GlcNAc(.beta.1,4)GlcNAc(- .beta.)
GlcNAc(.beta.1,6)Gal(.alpha.) GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.alpha.) GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(- .beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4- )Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)Glc(.beta.)
Gal(.beta.1,3)GlcNAc(.beta.1,6)Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,3)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)[Gal(.beta.1,4)]Glc(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,4)[Gal(.beta.1,3)]GlcNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,3) [Fuc(.alpha.1,2)Gal(.beta.1,4)]GlcNAc(.beta.)
Fuc(.alpha.1,4) [Gal(.beta.1,3)]GlcNAc(.beta.1,3)Gal(.beta.)
Fuc(.alpha.1,2)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Fuc(.alpha.1,3)[GlcNAc(.beta.1,3)Gal(.beta.1,4)]GlcNAc(.beta.)
GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.1,4)Glc(.beta.)
Fuc(.alpha.1,3)GlcNAc(.beta.1,6)[GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,3)[Gal(.beta.1,4)]GlcNAc(.beta.1,6)Gal(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)GlcNAc(.beta.1,3)]Gal(.beta.)
Fuc(.alpha.1,6)GlcNAc(.beta.) GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,6)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,4)GlcNAc(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
Fuc(.alpha.1,6)[GlcNAc(.beta.1,4)]Man(.alpha.)
Gal(.beta.1,4)GlcNAc(.beta.1,4)[GlcNAc(.beta.1,2)]Man(.alpha.)
GlcNAc(.beta.1,4)[Gal(.beta.1,4)GlcNAc(.beta.1,2)]Man(.alpha.)
Man(.alpha.1,4)GlcNAc.beta.1,4[Fuc(.alpha.1,6)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.) Gal(.alpha.1,3)Gal(.beta.1,4)GlcNAc(-
.beta.) Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
NeuAC(.alpha.2,3)Gal(.beta.1,4)[Fuc(.alpha.1,3)]GlcNAc(.beta.)
Gal(.beta.1,4)GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
Glc(.beta.1,3) Glc(.beta.) Glc(.beta.1,2) Glc(.beta.)
Glc(.beta.1,6) Glc(.beta.) Glc(.alpha.1,2) Glc(.alpha.)
Glc(.alpha.1,3) Glc(.alpha.) Glc(.alpha.1,4) Glc(.alpha.)
Glc(.alpha.1,6) Glc(.alpha.) Ara(.alpha.1,2) Ara(.alpha.)
Ara(.alpha.1,5) Ara(.alpha.) Ara(.alpha.1,2) Glc(.beta.)
Ara(.alpha.1,3) Glc(.beta.) Ara(.alpha.1,4) Glc(.beta.)
Ara(.alpha.1,6) Glc(.beta.) Xyl(.alpha.1,2) Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.) Man(.alpha.1,3)Man(.alpha.)
Man(.alpha.1,6)Man(.alpha.) Gal(.alpha.1,2)Gal(.alpha.)
Gal(.alpha.1,3)Gal(.alpha.) Gal(.alpha.1,4)Gal(.alpha.)
Gal(.alpha.1,6)Gal(.alpha.) Gal(.beta.1,2)Gal(.beta.)
Gal(.beta.1,3)Gal(.beta.) Gal(.beta.1,6)Gal(.beta.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,6)Gal(.beta.1,3)GalNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,4)GlcNAc(.alpha.)
NeuAc(.alpha.2,3)Gal(.beta.1,3)[NeuAc(.alpha.2,6)]GalNAc(.alpha.)
NeuAc(.alpha.2,8)NeuAc(.alpha.2,3)Gal(.beta.)
Man(.beta.1,4)GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
GlcNAc(.beta.1,4)[Fuc(.alpha.2,6)]GlcNAc(.beta.)
Man(.alpha.1,3)Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,2)Man(.alpha.1,2)Man(.alpha.)
Man(.alpha.1,3)[Man(.alpha.1,6)]Man(.beta.1,4)GlcNAc(.beta.)
Man(.beta.1,4)GlcNAc(.beta.) Gal (.beta.1,6)Gal
(.beta.1,4)Gal(.beta.1,4)Glc (.beta.) Gal(.beta.1,3)Gal(.beta.1,4-
)Xyl(.beta.) Gal(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,4)GlcNAc(.beta.1,6)Gal(.beta.)
GalNAc(.beta.1,3)Gal(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.beta.1,4)Gal(.beta.1,4)Glc(.beta.)
GalNAc(.alpha.1,3)[Fuc(.alpha.1,2)]Gal(.beta.1,4)
Gal(.beta.1,3)Gal(.beta.1,4)Xyl(.beta.) GlcNAc(.beta.1,3)Gal(.bet-
a.1,3)GalNAc(.beta.) GlcNAc(.beta.1,6)Gal(.beta.1,3)GlcNAc(.beta.)
GlcNAc(.beta.1,3)Gal(.beta.1,4)GlcNAc(.beta.)
GlcNAc(.beta.1,6)[Gal(.beta.1,3)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]GalNAc(.beta.)
GlcNAc(.beta.1,3)[GlcNAc(.beta.1,6)]Gal(.beta.)
Xyl(.alpha.1,3)Glc(.beta.) Xyl(.alpha.1,3)Xyl(.alpha.1,3)Glc(.bet-
a.) Gal(.beta.1,3)GalNAc(.beta.) Gal(.beta.1,3)GlcNAc(.bet- a.)
GlcNAc(.beta.1,3)GalNAc(.beta.)
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.
Description
RELATED APPLICATIONS
[0001] This is a Continuation of U.S. patent application Ser. No.
09/860,559, filed on May 21, 2001, which is a Divisional of U.S.
patent application Ser. No. 09/783,083, filed on Feb. 15, 2001, now
abandoned, which is a Continuation-In-Part (CIP) of PCT Patent
Application No. PCT/IL00/00099, filed on Feb. 17, 2000, which
claims the benefit of U.S. patent application Ser. No. 09/251,298,
filed on Feb. 17, 1999, now abandoned.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to combinatorial complex
carbohydrate libraries and methods for the manufacture and use
thereof and, more particularly, to such libraries prepared on a
solid support via stepwise enzymatic synthesis, to thereby provide
a combinatorial array of complex carbohydrate structures. The
combinatorial complex carbohydrate libraries synthesized according
to the present invention can be exploited in a variety of ways,
including, but not limited to, (i) identification of complex
carbohydrate drugs; (ii) identification of complex carbohydrate
associated receptors or proteins as potential new carbohydrate
related targets for drug therapy; (iii) identification of
biologically-active complex carbohydrates; (iv) identification of
specific complex structural carbohydrate elements as potential new
targets for drug therapy; (v) identification of the active sites of
known complex carbohydrate structures; (vi) identification of new
glyco-markers in complex carbohydrate structures; and (vii)
detection of antibodies formed against a cancer-related
glyco-epitope or other disease related glyco-antigens.
[0003] Drug Discovery
[0004] Modern pharmaceutical research and development was instated
with the transition from folklore based medicine to the discovery
and isolation of medicaments using modern chemistry. Since the
1950s drug discovery focused on testing large numbers of candidate
compounds on a variety of animal models in an effort to identify
pharmaceutical active compounds. As such, discovery of new drug
candidates necessitated screening of diverse sources of compounds
for potential therapeutic activities. These sources included, for
example, known chemicals and drugs for which novel therapeutic
activities were searched, fermentation broths and compounds
excreted and/or extracted from plant or marine organisms, etc.
(Granellin, 1992).
[0005] During the 1970s and 1980s, advances in the fields of
biochemistry, molecular biology, cellular biology and structural
(and functional) biology have led to a better understanding of the
biochemical and molecular processes leading to the development and
the progression of various diseases. This, in turn, has led to the
development of protein-based primary screening assays, which
replaced the more cumbersome and time consuming methods of
screening for drug candidates in animal models (Nicholls,
1991).
[0006] In addition, advances in X-ray crystallography and
computational chemistry, shed new light on the physical processes
governing molecular recognition and interaction events of receptors
and their ligands, leading to the development of what is known as
the "rational drug design" approach (Hendrickson, 1991).
[0007] Understanding the three dimensional structure of peptides
and proteins, and having the ability to manipulate such structures
both virtually through molecular modeling and physically through
molecular cloning techniques led researchers to the development of
new drug candidates.
[0008] Unfortunately, due to the need for very highly skilled
personnel and dedicated and complex equipment, "rational drug
design" has failed to provide researchers with the drug design tool
they had hoped for (Jacobs, 1994).
[0009] As such, in the early 1990's, the leading biotechnology and
pharmaceutical firms turned to robotics and automation in an
attempt to supplement the "rational drug design" approach.
Automation enabled a "shotgun" approach to drug discovery, allowing
for rapid screening of hundreds of thousands of compounds for
desired biological activities. The new approach incorporated (i)
combinatorics as a source of novel compounds; (ii) genomics, as a
source of novel targets; and (iii) high throughput screening (HTS),
as a method to cross screen various compounds/targets. As a result,
the "shotgun" approach enabled researchers to disregard the
technical hurdles associated with the previous approach and to
focus on issues such as, "what to make?" or "how much diversity is
required to produce a positive result?" (Hogan Jr., 1997).
[0010] Combinatorial Libraries
[0011] In the search for novel drug candidates, researchers looked
to complement existing natural compounds which have been
extensively screened, with a novel and diversified group of
molecules not found in nature. As such, combinatorial libraries of
newly synthesized novel compounds comprising nucleic or amino acid
sequences were synthesized and screened for potential drug
candidates.
[0012] Combinatorial libraries of such novel compounds or of novel
targets can be categorized into three main categories.
[0013] The first category relates to the matrix or platform on
which the library is displayed and/or constructed (Blondelle,
1996). As such, combinatorial libraries can be provided (i) on a
surface of a chemical solid support, such as microparticles, beads
or a flat platform; (ii) displayed by a biological source (e.g.,
bacteria or phage); and (iii) contained within a solution. In
addition, three dimensional structures of various computer
generated combinatorial molecules can be screened via computational
methods (Gaasterland, 1998).
[0014] Combinatorial libraries can be further categorized according
to the type of molecules represented in the library, which can
include, (i) small chemical molecules; (ii) nucleic acids (DNA,
RNA, etc.); (iii) peptides or proteins; and (iv) carbohydrates.
[0015] The third category of combinatorial libraries relates to the
method by which the compounds or targets are synthesized, such
synthesis is typically effected by: (i) in situ chemical synthesis
(Borman, 1996); (ii) in vivo synthesis via molecular cloning
(Kenan, 1994); (iii) in vitro biosynthesis by purified enzymes or
extracts from microorganisms (Michels, 1998); and (iv) in silico by
dedicated computer algorithms (Sansom, 1997).
[0016] Combinatorial libraries typified by any of the above
synthesis methods can be further characterized by: (i) split or
parallel modes of synthesis; (ii) molecules size and complexity;
(iii) technology of screening; and (iv) rank of automation in
preparation/screening.
[0017] In the split synthesis method, a combinatorial library which
is synthesized, for example, on the surface of microparticles or
beads, is divided into groups in which a unique first synthesis
building block is attached to the beads. Groups of beads are then
combined and separated to form new groups of unique diversity, the
next building block is then added, and the process is repeated
until the desired complexity is achieved. In the parallel synthesis
method each of the compounds of the combinatorial library is
synthesized separately, in a solution or immobilized to a matrix,
requiring a unique and independent synthesis regimen for each of
the compounds of the library.
[0018] The complexity of molecules in a combinatorial library
depends upon the diversity of the primary building blocks and
possible combinations thereof. Furthermore, several additional
parameters can also determine the complexity of a combinatorial
library. These parameters include (i) the molecular size of the
final synthesis product (e.g., oligomer or small chemical
molecule); (ii) the number of bonds that are created in each
synthesis step (e.g., one bond vs. several specific bonds at a
time); (iii) the number of distinct synthesis steps employed; and
(iv) the structural complexity of the final product (e.g., linear
vs. branched molecules).
[0019] Combinatorial libraries can be synthesized of several types
of primary molecules, including, but not limited to, nucleic and
amino acids and carbohydrates. Due to their inherent single bond
type complexity, synthesizing nucleic and amino acid combinatorial
libraries typically necessitates only one type of synthesis
reaction. On the other hand, due to their inherent bond type
complexity, synthesizing complex carbohydrate combinatorial
libraries necessitates a plurality of distinct synthesis
reactions.
[0020] Thus, the simplistic and repetition of both nucleic and
amino acid polymers, allows for a relative simple synthesis method
for combinatorial library of such constituents. On the other hand,
since oligosaccharides are structurally much more complex,
combinatorial libraries of complex carbohydrates are difficult to
synthesize. As a result, the combinatorial carbohydrate libraries
synthesized to date are of a very low complexity and typically
include complex carbohydrate molecules consisting of no more than
three building block constituents.
[0021] The evolution of nucleic and amino acid combinatorial
libraries, has necessitated the utilization of screening techniques
which capitalize on the unique nature of such libraries, such that
rapid screening of diversified libraries can be effected.
[0022] For example, in order to identify specific interactions
between a "probe" and a library constituent, a library can be
constructed such that the identity and location of every single
constituent is known or controlled at the synthesis stage. Such
libraries are known as addressable libraries. By using the process
of photolithography, light-directed--spatially addressable,
parallel synthesizable libraries of peptides or oligonucleotides
can be produced (Ramsay, 1997). In addition, microfabricated array
of closed reaction chambers with micro-fluid systems are used for
"Lab on a chip" oligonucleotides or chemical addressable library
synthesis, (U.S. Pat. Nos. 5,643,738; 5,681,484; and 5,585,069,
which are incorporated herein be reference). Using such an
addressable technology enables the determination, during synthesis,
of the nature and location of the library constituents.
[0023] In comparison, libraries that are synthesized employing the
"one bead-one molecule" approach, in which the diversity is created
by a split-and-pool synthesis, are screened by using probes
conjugated to detectable moiety, e.g., a fluorescent molecule or an
enzyme, such that beads interacting with a labeled probe can be
identified, isolated and analyzed for composition (Schullek,
1997).
[0024] Since such screening methods are time consuming, tagged
libraries approach has evolved mainly for the use with libraries
created by the split-and-pool method. To accelerate the analysis of
the isolated molecule of interest, the tagged libraries approach
combines library members synthesis with parallel orthogonal
synthesis of tagged building block standards (Janda, 1994; Chabala,
1995) or radio frequency tagged memory devices (Borman, 1996).
[0025] In order to screen large arrays, robotics and
miniaturization equipment are utilized in high throughput screening
(HTS) assays. In the past, HTS assays were basically upscaled
laboratory assays. As such, and depending on the diversity of the
screened molecules, the adaptation of a relatively simple assay to
HTS involved miniaturization and automation of liquid handling,
such that a large number of independent molecules can be screened
relatively rapidly. Presently, more integrative approaches to HTS
are developed and implemented. These approaches are referred to as
`Ultra Technologies` (UT, Sittampalam, 1997) or `New Technologies`
(NT, Burbaum, 1997). Such HTS methodologies vary between the
split-and-pool or parallel synthesis methods. In the split-and-pool
synthesis method the microparticles are used both for synthesis and
for screening by pre treatment with assay reagents (Stinson, 1998).
Improving the parallel synthesis methods necessitated additional
miniaturization of the support matrix (Cargill, 1997). For example,
if 10.sup.6 compounds are tested against 200 probes each year, this
translates to 2.times.10.sup.8 assays. If such assays are employed
at a well volume of 100 .mu.l each, containing 10 .mu.l of the test
compound of an approximal molecular weight of 500 g/mol, would
require roughly two million microtiter plates of 96 wells, 20,000
liters of each target solution and 100 grams of each of the
compounds.
[0026] Accordingly, new technologies for the miniaturization of the
support matrix have been proposed. Such technologies are divided
into open and closed vessel formats. The open vessel technology
maintains compatibility with the standard 96-well plates and
follows a geometric series of N=n.sup.2.times.96, where N is the
number of wells, and n is an integer describing the possible
packing density of a rectilinear array. Following this reasoning,
the balance point between a microliter volume limitation and a
suitable packing density is achieved by a plate with 1536 wells
(n=4) having a well volume of 1-2 .mu.l.
[0027] Screening of larger arrays requires further reduction of
well volume to nanoliter amounts which further necessitates the use
of the closed vessel format to prevent evaporation. Using
fabrication techniques pioneered by the semiconductor industry,
synthesis and analysis of nanoliter reaction volumes can be
effected, such that a single four square-inch silicon wafer can
support 10.sup.5 separate synthesis and bioassay reactions (Cheng,
1998).
[0028] Preparation methods of combinatorial libraries of small
chemically synthesized organic molecules, such as nucleic acids
(DNA, RNA and anti-sense RNA), peptides and biomimetics of peptide,
such as peptoids and semi peptoids, etc., are now well established
in the art and as such the technologies in most of the above
categories and divisions (see Table 1 below) have been
demonstrated.
1TABLE 1 Combinatorial libraries categorization Small chemical
Nucleic Amino Category molecules Acids Acids Carbohydrates Platform
Solution + + + + Chemical support + + + + Biological support + + +
- Computer + + + - Synthesis chemically synthesized + + + + in vivo
via molecular - - + - biology in vitro via enzymatic + + + -
synthesis in silico via computer + + + - algorithm Mode of
combinatorial Split-and-pool + + + + Parallel + + + - Complexity
Low Molecular Weight + + + + Oligomer (H.M.W.) - + + + One bond at
a time + + + + Several bonds a time + - - - One reaction type + + +
+ Several reaction types + - - - Linear oligomer - + + + One Branch
in molecule + - - + Highly Branched + - - - molecules Screening
methods Isolation and analysis + + + + Encoded + + + + Spatially
Addressable + + + - Automation a) Microparticles libraries Pre
treatment of the + + + + beads with the screening assay Radio
frequency tags + + + - b) Addressable arrays 96 well-based + + + -
HTS-NT + + + - legend: +: available; -: non available.
[0029] In spite of the abundance of carbohydrates in nature and
their important role in many biological processes, highly
diversified and complex carbohydrate libraries have not been
demonstrated (Borman, 1996). Moreover, although solid phase
chemical synthesis of glycosidic bond products was proposed almost
30 years ago (Frechest, 1971, 1972; Guthrie, 1971), chemical
synthesis of carbohydrate combinatorial libraries has only been
demonstrated in recent years, and due to the limitations of
chemical synthesis as further detailed hereinunder, such libraries
constitute rather simple arrays of soluble untagged trisaccharide
constituents.
[0030] Hindsgaul and co-workers demonstrated a `random
glycosylation` chemical approach to library synthesis which
involves coupling a protected glycosyl donor with a sugar acceptor
containing 3-5 free hydroxyls, to produce a mixture of 6 to 8
distinct carbohydrate products (Kanie, 1995). According to this
method, following a glycosylation step the protecting groups are
removed, and the coupled products are then separated from the
starting monosaccharide building blocks via reverse phase
chromatography.
[0031] Boons and co-workers reported a somewhat more direct
chemical synthesis approach. To ensure the formation of
regiospecific glycosidic linkages, they synthesized ten protected
disaccharide acceptors containing one free hydroxyl group each.
Each protected acceptor was separately reacted with a glycosyl
donor to form 32 defined disaccharides. The disaccharide products
were then mixed, a deprotection procedure was employed and the
mixture was split into four subgroups. Each subgroup was then
reacted with a different donor to give four libraries, each
containing 64 trisaccharides. Finally, the products were separated
by a tedious procedure of size-exclusion chromatography (Boons,
1996).
[0032] Chemical synthesis of a combinatorial carbohydrate library
on a solid support was demonstrated by Kahne and co-workers (Liang,
1996). U.S. Pat. No. 5,700,916 teaches in this respect a
carbohydrate library consisting of 1300 tagged di- and
trisaccharide. This library was synthesized using the
split-and-pool approach by coupling 12 different glycosyl donors to
six different polymer-bound acceptors employing glycosylation
methods incorporating anomeric sulfoxide as a glycosyl donor.
[0033] Chemical methods of preparing combinatorial carbohydrate
libraries were also described by several other research groups
(Rademann, 1996; Rodebaugh, 1997; Liang 1997, reviewed by Kahne,
1997; Arya, 1997, WO 97/34623; WO 97/35202; WO 98/08799; WO
98/40410; and U.S. Pat. No. 5,780,603). The above methods disclose
chemically prepared carbohydrate libraries in which the
carbohydrate constituents are attached to a non-sugar moiety via a
glycosidic bond, or alternatively carbohydrate constituents of low
structural complexity such as long uniform polyvalent chains or
di-tetrasaccharides with 0-1 branching.
[0034] More recently, a synthetic oligosaccharide-mimetic was
demonstrated to be capable of replacing the glycosidic bonds with
amide bonds, thereby forming "carbopeptoids" (Nicolaou, 1995) and
"glycotides" (McDevitt, 1996), or with phosphodiester bonds,
thereby forming "carbonucleotides" (Nicolaou, 1995). Although these
saccharide-mimetic forms are structurally more complex than
previously synthesized carbohydrates, their complexity level is far
from that of naturally occurring complex carbohydrates (for
example, see FIG. 1).
[0035] Wong and co-workers (Wong, 1998) chemically synthesized core
building blocks with four different selectively removable
protecting groups, to yield a pentasaccharide-mimetic with four
orthogonal glycosidic bonds.
[0036] Although carbohydrate libraries of limited complexity have
been synthesized using various chemical methods, a combinatorial
library of complex carbohydrates with a high rank of structural
complexity resembling natural complex carbohydrates (e.g., highly
branched structures) has not yet been produced. Furthermore,
synthesizing such libraries using an addressable parallel synthesis
approach which would enable rapid screening of library constituents
has never been proposed or discussed by the prior art (see Table 1
above).
[0037] This is possibly due to the fact that chemical synthesis
methods are still molded by the classical selective
protection/deprotection strategies, making the length of the
synthesized molecule the major contributor to the complexity
thereof (Grout, 1998). It was calculated that to encompass all the
possible linear and branched isomers of a hexamer oligosaccharide,
more than 1012 distinct structural forms would be needed (Laine,
1994). Synthesis of such an array would be impractical by a
chemical synthesis method utilizing selective
protection-deprotection groups. In such a method the formation of
mixtures of anomers which disable or terminate directed
carbohydrate specific chain formation is unavoidable, and as such
controlling the formation of such anomeric centers generated during
synthesis, is impossible.
[0038] In order to synthesize combinatorial libraries of complex
carbohydrates with high order of complexity and diversity one must
seek alternative, non-chemical, methods of synthesis.
[0039] Enzymes are high fidelity biocatalists which are in
prevalent use in the synthesis of organic compounds. As such,
enzymes can be employed in a carbohydrate synthesis method which
avoids the above mentioned limitations inherent to the prior art
chemical synthesis methods. Enzymatic synthesis of glycosidic bonds
displays high stereo- and regioselectivity, and as such, the
employment of enzymes in the synthesis of complex carbohydrates
abolishes the need for protected monomers and negates the problems
inherent to the incorporation of such protected building blocks
(Grout, 1998).
[0040] Nature employs four types of enzymes for in vivo
biosynthesis of glycosidic bonds (see Table 2 below). The basic
common division segregates these enzymes according to the Leloir
pathway (Leloir, 1971) and the non-Leloir pathway. Leloir pathway
enzymes are responsible for the biosynthesis of most N- and
O-linked glycoproteins and other glycoconjugates in mammalian
systems. The N-linked pathway involves an initial biosynthesis of a
dolichol pyrophosphoryl oligosaccharide intermediate in the
endoplasmic reticulum by mannosyl and N-Acetylglucosyl
transferases. This oligosaccharide structure undergoes further
glucosylation and is then transferred via an
oligosaccharidetransferase to an aspargine residues of a growing
peptide chain (Kornfeld, 1985). Prior to transport into the Golgi
apparatus (GA), the glucose and some mannose residues are removed
by glycosidases to reveal a core pentasaccharide. Additional
monosaccharides are then added sequentially by glycosyltransfrases
in the GA, in a process known as O-linked glycosylation, which is
initiated in the GA by the addition of a monosaccharide to serine
or threonine via a glycosidic bond and continues by the sequential
addition of monosaccharides (Kornfeld, 1985). Glycosyltransferases
of the Leloir pathway utilize only eight nucleoside sugars as
monosaccharide donors for the synthesis of most oligosaccharides
(see Table 3 below).
2TABLE 2 Enzyme types used for in vitro synthesis of glycosidic
bonds Enzyme type Leaving group Reference Phosphorylase
--O--PO.sub.3.sup.2- Hassid, 1950 Glycosidase --OR; F or --OH
Nilsson, 1988 Transglycosidase -O-Sugar or --O--R Schenkman, 1991
Glycosyltransferase -O-UDP; -O-GDP; Hunez, 1980 -O-CMP
[0041]
3TABLE 3 Glycosyltransferases of the Leloir pathway Enzyme Donor
Glucosyltransferase Uridinediphosphate-Glucose (UDP-Glc)
N-Actylglucosaminyltransferas- e Uridinediphosphate-N-
acetylglucoseamine (UDP-GlcNAc) Galactosyltransferase
Uridinediphosphate-Galctose (UDP-Gal)
N-Actylgalctosaminyltransferase Uridinediphosphate-N-
acetylgalctoseamine (UDP-GalNAc) Mannosyltransferase
Guanidinediphosphate-Mannose (GDP-Man) Fucosyltransferase
Guanidinediphosphate-Fucose (GDP-Fuc) Glucoronic acid transferase
Uridinediphosphate-Glucoronic acid (UDP-GlcUA) Sialyltransferase
Cytosinediphosphate-N- acetylneuraminicacid (CMP-NeuAc)
[0042] The non-Leloir pathway employs additional monosaccharides,
such as anionic or sulfated sugars which are also founds in
mammalian cells. A very diverse pool of yet additional
monosaccharides which are not utilized by either pathway (e.g.,
rhamnose and arabinose; see Table 4 below) are also present in
microorganisms, plants and invertebrates (Oths, 1990; Mengling,
1998).
4TABLE 4 Monosaccharide presents only in microorganisms and plants
(Gleeson, 1988) Monosaccharide found in Monosaccharide found in
microorganisms and microorganisms, in plants, but not in animals
plants and in animals Arabinose Glucose Apiose Galactose Fructose
Mannose Galacturonic acid Fucose Rhamnose Xylose Aceric Acid
(3-C-carboxy-5-deoxyl-xylose) N-Acetylglucosamine
N-Acetylgalactosamine Glucuronic acid
[0043] Two main strategies have been proposed for in vitro enzyme
catalyzed synthesis of oligosaccharide. According to the first
strategy, glycosidases or glycosyl hydrolases are employed in a
reverse hydrolysis reaction (WO 87/05936; WO 98/40512; and U.S.
Pat. No. 5,532,147, Nilsson, 1988 and 1996; Watt, 1997), while
according to the second strategy, glycosyltransferases are employed
in a sequential synthesis method (Hunez, 1980; Toone, 1989). Due to
the high yields and stereo- and regioselective specificity
displayed by the second strategy it is considered to be the
preferred approach (David, 1991; Wong, 1992). The second strategy,
which is extensively discussed in the prior art (see, for example,
Grout, 1998; Watt, 1997; Ichikawa, 1997; Wong, 1996; U.S. Pat. No.
5,583,042; and WO 96/32492), was exploited for the synthesis of a
very narrow range of oligosaccharide molecules, ranging from 2 to 5
units in size utilizing seven of the eight Leloir monosaccharide
species formed with only 0-1 branching bonds.
[0044] In nature, the use in combinations of all four types of
enzymes (see Table 2 above) produces a complex array of
oligosaccharides. On the other hand, very few descriptions of
combined methods of enzymatic in vitro glycosidic bond synthesis
were recorded.
[0045] Such methods can incorporate, in combination, a glycosidase
and a glycosyltransferase (.beta.-galactosidase and a
(2,6)sialyltransferase) to produce a narrow range of
oligosaccharide products (Herrmann, 1993; Nilsson 1988).
Alternatively, a transglycosidase can also be used in combination
with the above enzymes (e.g., trans-sialidase from Trypanosoma
cruzi Schenkman, 1991). Utilization of phosphorylase to transfer a
sugar 1-phosphate donor to 2-keto-sugars has also been described as
early as the 1950s (Hassid, 1950) but this strategy was not further
pursued.
[0046] Enzymatic synthesis of complex carbohydrates on solid
support was first proposed in the 1980s by Zehavi et al. (1983 and
1984). Zehavi and co-workers attached a glycosyl unit to
4-hydroxymethyl-3-nitrobenzoate to create a photolabile linker.
This saccharide-linker was coupled to an amino functionalized
water-compatible support, such as polyacrylamide-gel polymer or
polyvinyl alcohol, via an amide linkage (Zehavi, 1984). The
polymer-bound glycoside was then galactosylated using 1,4
galactosyltransferase.
[0047] By combining chemical synthesis steps along with an
enzymatic sugar chain elongation steps, the solid-phase
chemo-enzymatic synthesis of the Lewis X glycopetide antigen
(Halcomb, 1994; Seitz, 1997), as well as synthesis of a sialylated
unodecasaccharide-aspargine conjugate in which the .alpha.-2,6
sialyl residue was enzymatically added to a chemically synthesized
decasaccharide (Unverzagt, 1996) has been effected. In addition,
enzymatic synthesis of a sialylated Lewis X antigen has also been
accomplished on an activated silica support (Schuster, 1994). This
glycopeptide was created in relatively high yields by three
repetitive enzymatic glycosylation steps which resulted in four
attached saccharide units on the peptide. An excellent yield was
also reported for the synthesis of a glycopeptide via enzymatic
glycosylation of a polyethyleneglycol-polyacrylamide copolymer
solid support (Meldal, 1994).
[0048] To date, the discovery of new carbohydrate-derived
pharmaceutical agents still lags far behind that of other classes
of molecules, such as proteins. This lag is mostly attributed to
the unavailability of an efficient and comprehensive synthesis
method applicable for producing diverse and complex carbohydrate
species. Since complex carbohydrates are both difficult to
synthesize and to analyze, it is not feasible to employ the above
described prior art methods for such tasks.
[0049] Despite these limitations, three aspects of carbohydrate
medicinal chemistry and biochemistry were extensively studied: (i)
specific interference with biosynthesis of bacterial cell-wall
(Mengling, 1998); (ii) unique markers to malignant tumors (Orntoft,
1995; Kobata, 1998); and (iii) participation of cell-surface
oligosaccharide markers in cell-to-cell communication, cell
adhesion, cell infection and cellular differentiation (Simon,
1996).
[0050] These aspects of carbohydrate medicinal chemistry and
biochemistry were studied using chemically synthesized complex
carbohydrate species. Synthetic complex carbohydrates have proven
to be an important tool for the developing glyco-therapeutic field,
but the limitations inherent to the chemical synthesis process and
as such to diverse combinatorial libraries produced thereof,
impedes significant progress in this field.
[0051] For example, the CarbBank database (Complex Carbohydrate
Structure Database-CCSD) includes 48,956 records (22,048 unique
structures) which were derived from published articles and were
compiled by the Georgia University project-Complex Carbohydrate
Research Center (CCRC). These carbohydrates are grouped according
to their complexity in FIG. 2. More than 58% of the entries are
branched molecular structures that are practically impossible to
synthesize using present day chemical synthesis methods. FIG. 3 is
a histogram representing the distribution, in percentages, of the
number of sugar residues present in the complex carbohydrates found
in the CarbBank database. Although 44% of all the complex
carbohydrates in the database posses 6 or more residues, chemical
or enzymatic synthesis of such complex carbohydrates has not been
extensively practiced, in particular not in context of a
library.
[0052] Difficulties are further compounded when one wishes to
construct a combinatorial library of such entities, necessitating
parallel synthesis of a multitude of complex carbohydrates. As
such, using present day chemical synthesis methods to synthesize
combinatorial arrays of addressable complex carbohydrate entities
is of a paramount challenge.
[0053] There is thus a widely recognized need for, and it would be
highly advantageous to have, methods for synthesizing and screening
complex carbohydrate combinatorial libraries of substantial
structural complexity and diversity.
SUMMARY OF THE INVENTION
[0054] According to one aspect of the present invention there is
provided a combinatorial complex carbohydrate library comprising a
plurality of addressable complex carbohydrate structures.
[0055] According to another aspect of the present invention there
is provided a method of producing an addressable combinatorial
complex carbohydrate library, the method comprising the steps of
(a) providing a solid support having a plurality of locations; and
(b) enzymatically synthesizing a plurality of complex carbohydrate
structures, each of the plurality of complex carbohydrate
structures being attached to at least one addressed location of the
plurality of locations, thereby producing the addressable
combinatorial complex carbohydrate library.
[0056] According to further features in preferred embodiments of
the invention described below, each of the addressable complex
carbohydrate structures is attached to a solid support.
[0057] According to still further features in the described
preferred embodiments attaching each of the addressable complex
carbohydrate structures to the solid support is effected by a
linker.
[0058] According to still further features in the described
preferred embodiments the linker is cleavable, preferably under
conditions that are harmless to carbohydrates.
[0059] According to still further features in the described
preferred embodiments the linker is selected so as to allow
attaching thereto a p-Nitrophenyl, amine or squaric acid derivative
of a sugar.
[0060] According to still further features in the described
preferred embodiments the linker includes at least two contiguous
covalent bonds.
[0061] According to still further features in the described
preferred embodiments the 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.
[0062] According to still further features in the described
preferred embodiments the linker is of a length of at least 20
Angstrom.
[0063] According to still further features in the described
preferred embodiments the solid support is selected from the group
consisting of addressable microparticles, addressable beads and a
flat platform.
[0064] According to still further features in the described
preferred embodiments the flat platform is selected from the group
consisting of a microtiterplate, a membrane and a chip.
[0065] According to still further features in the described
preferred embodiments the microtiterplate is an addressable
microfabricated array of closed reaction chambers supplemented with
micro-fluid systems. The reaction chambers are preferably of a
density of 4-25 per square cm, each having a volume of 50-1000
nanoliter.
[0066] According to still further features in the described
preferred embodiments the solid support is a chip and further
wherein different complex carbohydrate structures of the plurality
of addressable complex carbohydrate structures are arranged in
patches spaced not more than 2.25 mm center to center
[0067] According to still further features in the described
preferred embodiments the 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.
[0068] According to still further features in the described
preferred embodiments at least one of the plurality of addressable
complex carbohydrate structures includes at least two contiguous
saccharide units of a single species.
[0069] According to still features in the described preferred
embodiments at least one of the plurality of addressable complex
carbohydrate structures includes at least one branch.
[0070] According to still further features in the described
preferred embodiments at least one of the at least one branch is
formed of identical core and branching saccharide units.
[0071] According to still further features in the described
preferred embodiments at least one of the plurality of addressable
complex carbohydrate structures includes at least 5 saccharide
units.
[0072] According to still further features in the described
preferred embodiments the plurality of addressable complex
carbohydrate structures are a representation including non-natural
complex carbohydrates.
[0073] According to still further features in the described
preferred embodiments the plurality of addressable complex
carbohydrate structures are a representation including natural
complex carbohydrates.
[0074] According to still further features in the described
preferred embodiments the natural complex carbohydrates are derived
from a human source.
[0075] According to still further features in the described
preferred embodiments the human source is selected from the group
consisting of a tissue, cells and body fluids.
[0076] According to still further features in the described
preferred embodiments the plurality of addressable complex
carbohydrate structures are a representation of domains of at least
one natural complex carbohydrate.
[0077] According to yet another aspect of the present invention
there is provided a method of identifying a complex carbohydrate
capable of binding an entity, the method comprising the steps of
(a) producing an addressable combinatorial complex carbohydrate
library by (i) providing a solid support having a plurality of
locations; and (ii) enzymatically synthesizing a plurality of
complex carbohydrate structures, each of the plurality of complex
carbohydrate structures being attached to at least one addressed
location of the plurality of locations, thereby producing the
addressable combinatorial complex carbohydrate library; and (b)
screening the addressable combinatorial complex carbohydrate
library with the entity for identifying the complex carbohydrate
capable of binding the entity.
[0078] According to further features in preferred embodiments of
the invention described below, the entity is a candidate for a
biologically active material, the method serves for identifying a
complex carbohydrate which is a target for the candidate for a
biologically active material.
[0079] According to still further features in the described
preferred embodiments the entity is a ligand known to bind a
specific natural complex carbohydrate and further wherein the
addressable combinatorial complex carbohydrate library is a
representation of domains of the specific natural complex
carbohydrate, the method serves for identifying a specific domain
of the domains which binds the ligand.
[0080] According to still further features in the described
preferred embodiments the entity is a potential drug.
[0081] According to still another aspect of the present invention
there is provided a method of diagnosing a disorder characterized
by a self or non-self complex carbohydrate structures and
elicitation of antibodies there against, the method comprising the
steps of (a) producing an addressable combinatorial complex
carbohydrate library representing the self or non-self complex
carbohydrates by (i) providing a solid support having a plurality
of locations; and (ii) enzymatically synthesizing a plurality of
complex carbohydrate structures, each of the plurality of complex
carbohydrate structures being attached to at least one addressed
location of the plurality of locations, thereby producing the
addressable combinatorial complex carbohydrate library; and (b)
reacting the addressable combinatorial complex carbohydrate library
with antibodies derived from a patient suspected of having the
disorder to thereby generate a pattern of the locations to which
the antibodies bind, such that by comparing the pattern with a
known pattern characterizing a healthy individual, a diagnosis of
the disorder is obtainable.
[0082] The present invention successfully addresses the
shortcomings of the presently known configurations by providing a
combinatorial complex carbohydrate libraries and methods for their
manufacture and screening.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0084] In the drawings:
[0085] FIG. 1 is a formula of a complex carbohydrate of 14
monosaccharide units of five different types with three branching
points. This complexity, which can be effected by the method of the
present invention cannot be effected by any chemical or enzymatic
synthesis methods currently known in the art. This Figure shows the
monosaccharide types by name, the anomers (reactive centers) and
bond types, as well as the branching points.
[0086] FIG. 2 represents a statistical study on the occurrence of
branching in the complex carbohydrate records of the CCSD database.
The CCSD database includes 48,956 records which were derived from
published articles and were compiled by the Georgia University
Project-Complex Carbohydrate Research Center-CCRC. Each slice of
the pie shown represents, in percentage, the number of branches
(0-26) occurring in the complex carbohydrates of the database.
Statistical analysis was performed using CarbBank 3.2.009.
[0087] FIG. 3 is a histogram representing the distribution, in
percentages, of the number of sugar residues present in the complex
carbohydrates found in the CarbBank database. Although 44% of all
the complex carbohydrates in the database posses 6 or more
residues, chemical or enzymatic synthesis of such complex
carbohydrates has not been extensively practiced, in particular not
in context of a library.
[0088] FIG. 4 is a table representing the stepwise enzymatic
synthesis of the complex carbohydrate structure of FIG. 1. In each
step, the enzymatic reaction (ER) is listed (see Table 7 for
details) and the added monosaccharide unit is marked by a gray
background. "S" represents the solid support onto which the complex
carbohydrate is immobilized and the synthesis reaction occurs.
[0089] FIG. 5a depicts the synthesis and elongation cycles of a
cleavable linker that enables covalent immobilization of a
p-Nitrophenyl or amine derivative of a sugar (for example,
p-Nitrophenyl-.beta.-D-GlcNAc) to a Covalink NH microtiterplate,
which immobilization constitutes a first step in the synthesis of a
complex carbohydrate library according to the teachings of the
present invention.
[0090] FIG. 5b depicts the synthesis and elongation cycles of a
cleavable linker that enables covalent immobilization of a Squaric
acid derivative of a sugar (for example, Squaric-.beta.-D-GlcNAc)
to a Covalink NH microtiterplate, which immobilization constitutes
a first step in synthesis of a complex carbohydrate library
according to the teachings of the present invention.
[0091] FIG. 6 depicts covalent immobilization of GlcNAc to NHS/EDC
activated Covalink NH.
[0092] FIG. 7a is a graph depicting the binding of WGA to
PNP-GlcNAc coupled to Covalink NH plates in the presence or absence
of cyanuric chloride activation (one elongation cycle); binding was
visualized via a WGA conjugated peroxidase as further described in
the Examples section.
[0093] FIG. 7b is a graph depicting the effect of various blocking
methods on the binding of WGA to GlcNAc-COOH coupled to Covalink NH
plates; binding was visualized via a WGA conjugated peroxidase as
further described in the Examples section.
[0094] FIG. 7c is a graph depicting the binding of WGA to GlcNAc
coupled to cyanuric chloride activated Covalink NH; binding was
visualized via WGA conjugated FITC as further described in the
Examples section.
[0095] FIG. 8a is a graph depicting WGA binding to BSA-GlcNAc which
coats the Maxisorb plates; binding was visualized via a WGA
conjugated peroxidase as further described in the Examples
section.
[0096] FIG. 8b is a graph depicting the binding described in FIG.
8a as a function of the number of wash steps employed.
[0097] FIG. 8c is a graph depicting the transfer of
.beta.-D-Galactose to the plate immobilized phenyl-.beta.-D-GlcNAc
(22 atom linker) as verified using ECorA lectin binding.
[0098] FIG. 9 depicts the enzymatic steps required for the
synthesis of a library consisting of the structures described in
Table 17 immobilized to a plate, outlining the organization of the
microtiter plate, the enzymatic reactions performed at each step
and the lectins/antibody binding assays.
[0099] FIG. 10a is a graph depicting RCA.sub.120 binding following
incubation with a .beta.1,4 galactosyltransferase reaction mixture
(D7) (solid line) or following a control reaction (hatched
line).
[0100] FIG. 10b is a graph depicting BS-1 binding following
incubation with a .alpha.1,3 galactosyltransferase reaction mixture
(D3) (solid line) or following a control reaction (hatched
line).
[0101] FIG. 10c is a graph depicting TGP binding following
incubation with a .alpha.1,3 fucosyltransferase VI reaction mixture
(B2) (solid line) or following a control reaction (hatched
line).
[0102] FIG. 10d is a graph depicting TML binding following
incubation with an .alpha.2,6 sialyltransferase reaction mixture
(A2) (solid line) or following a control reaction (hatched
line).
[0103] FIG. 10e is a graph depicting TML binding following
incubation with a .alpha.2,3 sialyltransferase reaction mixture
(A3) (solid line) or following a control reaction (hatched
line).
[0104] FIG. 10f is a graph depicting Anti-sialyl Lewis X mouse IgM
binding following incubation with fucosyltransferase VI reaction
mixture (B2) (solid line) or following a control reaction (hatched
line).
[0105] FIG. 11 depicts the enzymatic reactions performed in each
step of a library (referred to as library 2) synthesis and the
lectins binding assays that were used to verify the efficiency of
the various enzymatic steps.
[0106] FIG. 12 is a graph depicting the binding of RCA.sub.120 to
BSA-GlcNAc bound to Maxisorb plates at various time points
following incubation with a .beta.1,4 galactosyltransferase
reaction mixture (D7).
[0107] FIG. 13 is a graph depicting the reduction in binding of
WGA/FITC to GlcNAc attached to wells of a microtiter plate
following the cleavage of the linker.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0108] The present invention is of combinatorial complex
carbohydrate libraries and of methods for the synthesis thereof,
which can be used for (i) identification of complex carbohydrate
drugs; (ii) identification of complex carbohydrate associated
receptors or proteins as potential new carbohydrate related targets
for drug therapy; (iii) identification of biologically-active
complex carbohydrates; (iv) identification of specific complex
structural carbohydrate elements as potential new targets for drug
therapy; (v) identification of the active sites of known complex
carbohydrate structures; (vi) identification of new glyco-markers
in complex carbohydrate structures; and (vii) detection of
antibodies formed against a cancer-related glyco-marker or other
disease related glyco-antigens.
[0109] The principles and operation of the combinatorial complex
carbohydrate libraries and the methods for the synthesis thereof
according to the present invention may be better understood with
reference to the drawings and accompanying descriptions.
[0110] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0111] Enzymes for Synthesis of Complex Carbohydrate Libraries:
[0112] The enzymatic synthesis of complex carbohydrate
combinatorial libraries according to the present invention is
effected by glycosyltransferases, glycosidases and
transglycosidases. These enzymes can be obtained from different
sources using different strategies as describe herein below.
[0113] Enzymes derived from natural sources: To date, more than two
hundred different kinds of glycosyltransferases, transglycosidases
and glycosidases active on a large number of substrates and donors
have been extensively characterized. These enzymes are found in
mammalians cells, plant cells, invertebrate cells and
microorganisms, (for references see Palcic, 1994 and Nilsson, 1996
which are incorporated by reference as if fully set forth
herein).
[0114] Recombinant enzymes: The coding sequences of many
glycosyltransferases and transglycosides have been cloned, and the
acceptor substrate specificity of each of the recombinant enzymes
encoded thereby have been characterized. Table 5 below lists some
of the cloned glycosyltransferases and their respective acceptor
substrate specificity. Enzymes for which the coding sequences have
been cloned can be produced in sufficient quantities using standard
recombinant DNA techniques. Since most of these enzymes require
post translational modifications for functionality, expression is
preferably effected in insect cell cultures (Toki, 1997; Tan,
1995). In addition, in the case of soluble enzymes for which the
catalytic domain(s) have been characterized, expression can be
effected for the domain sequence only, providing it retains the
catalytic activity and substrate specificity of the holoenzyme
(Vries, 1997). Other possible expression systems for soluble
glycosyltransferases also include secretion from mammalians
tissue-cultures (see for example U.S. Pat. No. 5,032,519, which is
incorporated by reference as if fully set forth herein).
5TABLE 5 Partial list of cloned glycosyltransferases and their
acceptors. E. C. Enzyme Acceptor number Reference .alpha.2,3 sialyl
D-Gal-.beta.(1,3)-D-GalNAc-R 2.4.99.4 Chang 1995, transferase
Gillespieet 1992, Kurosawa 1995 .beta.2,6 sialyl
D-Gal-.beta.(1,4)-D-GlcNAc-R 2.4.99.1 Grundmann 1990, transferase
Kurosawa 1994, Hamamoto 1993 .alpha.2,3 sialyl
D-Gal-.beta.(1,4)-D-GlcNAc-R 2.4.99.6 Kitagawa and transferase
Paulson 1993, Wen 1992 .alpha.2,8 sialyl
D-NeuAC-.alpha.(2,3)-D-Gal-.beta.-R 2.4.99.8 Nara 1994 transferase
.alpha.1,2 fucosyl D-Gal-.beta.-R 2.4.1.69 Larsen 1990, transferase
Hitoshi 1995 .alpha.1,3 fucosyl [D-Gal-.beta.(1,4)]-D-GlcNAc-R
2.4.1.152 Kudo 1998 transferase .alpha.1,6 fucosyl
[D-Man-.beta.(1,4)-D-GlcNAc- 2.4.1.68 Voynow 1991, transferase
.beta.(1,4)]-D-GlcNAc-R Uozumi 1996, Yanagidani 1997 .beta.1,4
mannosyl D-GlcNAc-.beta.(1,4)-D-GlcNAc-R none Albright and
transferase Robbins 1990 .alpha.1,2 mannosyl
D-Man-.alpha.(1,2)-Man-R 2.4.1.131 Romero 1997 transferase
.alpha.1,3 mannosyl D-Man-.alpha.(1,2)-Man-.alpha.(1,2- )- none Yip
1994 transferase Man-R .alpha.1,3 galactosyl
D-Gal-.beta.(1,4)-D-GlcNAc-R. 2.4.1.151 Joziasse 1989, transferase
Larsen 1989, Strahan 1995 .beta.1,4 galactosyl D-GlcNAc-R 2.4.1.38
Masibay and Qasba transferase 1989 .alpha.1,3 N-
[L-Fuc-.alpha.(1,2)] D-Gal-R 2.4.1.40 Yamamoto 1990,
Acetylgalactose aminyltransferase .beta.1,4 N-
[D-NeuAC-.alpha.(2,3)]D-Gal- 2.4.1.92 Hidari 1994, Nagata
Acetylgalactose .beta.(1,4)-D-Glc-R 1992 aminyltransferase
N-Acetylgalactose Ser/Thr 2.4.1.41 Meurer 1995, aminyltransferase
Hagen 1993 .beta.1,3 glucoronosyl D-Gal-.beta.(1,3)-D-Gal-.beta.(-
1,4)-D- 2.4.1.135 Kitagawa 1998 transferase Xly-.beta.-R .beta.1,6
N- D-Gal-.beta.(1,4)-D-GlcNAc 2.4.1.150 Bierhuizen, 1993
Acetylglucose aminyltransferase .beta.1,6 N-
D-GalNAc-.beta.(3,1)-D-Gal(R) 2.4.1.102 Bierhuizen 1992
Acetylglucos aminyltransferase .beta.1,2 N-
D-Man-.alpha.(1,3)[R1]D-man- 2.4.1.101 Kumar 1990, Acetylglucos
.beta.(1,4)-R2 Pownall 1992, aminyltransferase Sarkar 1991, Fukada
1994 .beta.1,4 N- D-Man-.alpha.(1,3)[R1]-D-Man- 2.4.1.145 Minowa
1998 Acetylglucos .beta.(1,4)-R2 aminyltransferase .beta.1,2 N-
D-Man-.alpha.(1,6)[R1]D-man- 2.4.1.143 Tan 1995 Acetylglucos
.beta.(1,4)-R2 aminyltransferase
[0115] Methods for identifying and cloning new enzymes: In addition
to the presently available natural and recombinant
glycosyltransferase, the identification and isolation of novel
glycosyltransferases can be undertaken. Glycosyltransferases which
are useful for complex carbohydrate library synthesis, can be
identified and isolated from cell types which posses the complex
carbohydrate structures typically synthesized by these desired
glycosyltransferases. Affinity chromatography techniques with an
immobilized acceptor as a ligand are well known in the art and
enable a simple one-step separation of a desired
glycosyltransferase (see in this respect U.S. Pat. No. 5,288,637,
which is incorporated herein by reference). Once a
glycosyltransferase is identified and isolated, it can be partially
sequenced and the gene encoding therefor cloned. Technologies for
cloning glycosyltransferases genes are well-established, and many
examples and strategies for cloning glycosyltransferase genes are
reviewed in the prior art (see, for example, Schachter, 1994 and WO
95/02683, which are incorporated by reference as if fully set forth
herein).
[0116] Another possible source for novel glycosyltransferase
sequences resides within the DNA and Protein databases. With the
rapid accumulation of new DNA and protein sequence data, sequence
alignment techniques can be used for the identification of new
glycosyltransferases. For example, 110 distinct cDNAs and genes
from animal, yeast, plants and bacteria, whose protein products
contain the characteristic "signature sequence" of the UDP
glycosyltransferase gene super family were identified (Mackenzie,
1997). Using these signature sequences or motifs, one skilled in
the art can screen relevant databases for novel
glycosyltransferases. For example, three new arabinosyltransferase
genes were identified in the completely sequenced genome of
Mycobacterium tuberculosis via sequence homology comparison to
arabinosyltransferase genes from Mycobacterium smegmatis (Cole,
1998).
[0117] Utilizing enzymes modified by directed evolution: Enzymes
with modified affinities or altered substrate donor or acceptor
specificities could also be employed in the synthesis of certain
complex carbohydrates. For example, the synthesis of complex
carbohydrate structures composed of identical repeating
monosaccharide units connected in the same regio-specific
orientation, such as, D-man-.alpha.((1,2)-D-Man-.alpha.((1-
,2)-D-man-.alpha.((1,2)-R, requires the use of an .alpha.-1,2
mannosyltransferase with an acceptor specificity to .alpha.-1,2
mannose. Employing the native enzyme in the presence of GDP-Mannose
and the acceptor D-man-.alpha.(1,2)-R immobilized onto a solid
support, would result in an uncontrolled polymerization reaction
which would create long polymer chains of the oligo-mannose,
[D-Man-.alpha.(1,2)].sub.n-R. In order to synthesize an
oligo-mannose with a defined number of mannose units (three in the
above example), a controlled stepwise process is required. The
ability to control undesired polymerization can be achieved by
using a modified glycosyl donor, and a unique glycosyltransferase
with a modified donor specificity. Such a modified enzyme, would be
employed for the addition of a modified GDP-Man to immobilized
acceptor D-man.alpha.(1,2)-R. The modification of the mannoside
moiety of the GDP-Man will then prevent addition of the next
mannose moiety since the acceptor to this manosidetransferase is
D-man-.alpha.((1,2)-R and not D-(modified
man)-.alpha.(1,2)-D-man-.alpha.(1,2)-R. Following this reaction,
any excess of the modified donor and the enzyme is washed out and
the modifying group is removed, thereby enabling the subsequent
repeat of the same enzymatic step. This controlled process is
continued until the desired number of mannose molecules are
assembled into the newly formed carbohydrate. To this effect, the
modifying group can be a chemical residue attached to the donor at
any position, but position 1. This modifying group can then be
selectively removed by either an enzymatic or chemical reaction,
such that the modifying group is released without imposing damage
to the complex carbohydrate molecule.
[0118] Table 6 below lists some of the presently available
saccharide modifying groups, classified by their method of removal
(Kunz, 1997, which is incorporated by reference as if fully set
forth herein, and references cited therein). Additional
monosaccharide can also be used as modifying groups and as such,
removal thereof can be effected by using specific glycosidases that
will not affect the existing complex carbohydrate structure
(Peieto, 1995).
6TABLE 6 Available saccharide modifying groups classified by their
cleavage (Kunz and Schultz, 1997; and references cited therein)
Cleavage principle Cleavage reagents Modifying group Hydrogenolysis
H.sub.2/Palladium benzyloxycarbonyl benzyl ether benzyl ester
methoxybenzyl ether Acidolysis Trifluoroacetic acid
tert-butyloxycarbonyl formic acid tert-butyl ether HCl/ether
tert-butyl ester methoxybenzyl ether Base promoted Morpholine,
9-fluorenylmethoxy cleavage piperidine carbonyl NaOMe/MeOH O-acetyl
NH.sub.2NH.sub.2/MeOH Reductive cleavage Zn/acetic acid 2,2,2-
trichloroethoxycarbonyl Oxidative cleavage Ceric ammonium
methoxybenzyl ether nitrate Metal complex
[(Ph.sub.3p).sub.4Pd.sup.0]/ allyl ester catalyzed cleavage
nucleophile Photolysis h.nu. O-nitrobenzyl Enzymatic cleavage
lipase, esterase, alkyl and protease alkoxyalkylesters
[0119] Thus, enzymes having modified donor and/or acceptor
specificities can be prepared using the directed evolution
approach. With the recent progress in the field of protein
engineering, many examples of enzymes with engineered specificity
obtained via directed evolution were described (for reviews of this
field see: Kuchner, 1997; Harris, 1998, which are incorporated
herein by reference). A directed evolution of an enzyme specificity
is achieved by random sequential generation of region directed or
site directed mutagenesis of the gene or genes encoding the enzyme,
followed by selection or screening for clones exhibiting desired
specificity and activity. For example, Moore and co-workers
performed seven rounds of DNA shuffling to change the substrate
specificity of paranitrobenzyl-esterase to a novel antibiotic
substrate (Moore, 1996). Zhang and co-workers performed directed
evolution of a fucosidase from galactosidase by DNA shuffling
(Zhang, 1997). Shan and co-workers engineered an unnatural
nucleotide specificity for the Rous Sarcoma Virus tyrosine kinase
(Shan, 1997). Paulson and co-workers performed mutation of the
sialyltransferase S-sialyl motif that alters the kinetics of the
donor and acceptor substrates (Datta, 1998).
Parallel Addressable Enzymatic Synthesis of Combinatorial Complex
Carbohydrate Libraries
[0120] The following sections detail a step by step enzymatic
preparation of a combinatorial complex carbohydrate library in
accordance with the teachings of the present invention.
[0121] Enzymatic combinatorial complex carbohydrate library design:
Enzymatic combinatorial complex carbohydrate library design
includes, according to the present invention, determination of the
complex carbohydrate constituents included within a specific
library in accordance with an envisaged application thereof.
[0122] Determination of the complex carbohydrate members included
within a specific library depends upon the desired application for
that specific library.
[0123] For example, in order to utilize an enzymatic combinatorial
complex carbohydrate library of the present invention for
identification of complex carbohydrates functional as drug targets,
the complex carbohydrate members of the library are preferably
derivatives or modificants of complex carbohydrates present in
human cells. Screening such a library against other molecules
derived from other sources, such as specific human cells or
pathogens thereof, enables the identification of novel complex
carbohydrates that function as receptors for these molecules,
functioning in vivo as pathogen receptors, or involved in cell to
cell recognition processes.
[0124] Similarly, in order to utilize an enzymatic combinatorial
complex carbohydrate library of the present invention for
identification of potential drugs, the complex carbohydrate members
of the library are preferably synthesized similar or identical to
natural complex carbohydrates present in human cells. Screening
such a library with drug candidates derived from various natural
and synthetic sources, enables the identification of drug
candidates which bind to one or more of the complex carbohydrate
structures of the library.
[0125] Another specific library according to the present invention
contains complex carbohydrates dedicated for the identification of
novel drug candidates. In this case, a library of maximized complex
carbohydrate diversity which represents, among others, complex
carbohydrate structures not found in nature, is generated. Such a
library is thereafter screened for potential binding of pathogens
or pathogen derived molecules. Alternatively, such a library is
thereafter screened for potential binding of other disease
inflicting molecules.
[0126] To identify active site domains within a known complex
carbohydrate, a library in accordance with the present invention
representing all of the possible domains of the complex
carbohydrate is prepared. Screening this library for binding or
bioactivity enables one to identify the active site domains of the
known complex carbohydrate.
[0127] To detect antibodies generated against, for example,
cancer-related glyco-epitopes (markers), organ transplantation
related glyco-markers, or other glyco-markers in blood serum, a
complex carbohydrate library of specific combinations of
glyco-markers is prepared and screened. For example, one specific
library can represent the glyco-markers of several cancer
conditions. This library is thereafter screened against antibodies
derived from human serum to identify the presence of antibodies
against one or more of these glyco-markers.
[0128] To map glyco-markers related to, for example, cancer or
organ transplantation, a complex carbohydrate library of specific
combinations of carbohydrate members which are structural
variations of the glyco-markers normally associated with such
conditions is prepared and screened.
[0129] Other enzymatic combinatorial complex carbohydrate
libraries, dedicated at other applications are envisaged and are
within the broad scope of the present invention as claimed.
[0130] Enzymatic modules (EMs) construction: EMs construction
includes evaluation of the required enzymatic reactions (ERs),
glycosyl donors and acceptors and enzymes that are required for the
synthesis of each complex carbohydrate of a library. EMs
construction further includes optimization and process development
of the required ERs with considerations given to reaction time,
temperature and reagent concentrations. EMs construction further
includes determination of the specific order in which the ERs
should be utilized for every EM.
[0131] To enable the synthesis reactions employed, a specific
sequence of enzymatic reactions (ERs) is determined for each
complex carbohydrate constituent of a given library. For complex
carbohydrates that have a linear non-branched structure, the ERs
sequence follows that of the monosaccharide sequence of such linear
non-branched structures in a stepwise fashion. For complex
carbohydrates possessing branched structure(s) and/or repetitive
monosaccharide units arranged in a linear assembly, unique
synthesis processes should be designed employing unique EMs.
[0132] The following example provides the rational for selecting
particular ERs to provide an EM tailored for the synthesis of a
distinct complex carbohydrate. The final complex carbohydrate
structure is described by FIG. 1 and the design process is
described by FIG. 4 and Table 7. Such an EM is designed, according
to the present invention, for each complex carbohydrate present in
a given library. As further detailed hereinunder, consideration is
given to efficiency when practically effecting each of the EMs
while constructing a library according to the present
invention.
7TABLE 7 A partial list of ERs including their donors, acceptors
and indexes Index extenslon .alpha./.beta. Pos. acceptor donor E.C.
A1 .alpha. 3 D-Gal-.beta.(1,3)-D-GalN- Ac-R CMP-NeuAC 2.4.99.4 A2
.alpha. 6 D-Gal-.beta.(1,4)-D-GlcNAc CMP-NeuAC 2.4.99.1 A3 .alpha.
3 D-Gal-.beta.(1,4)-D-GlcNAc-R CMP-NeuAC 2.4.99.6 A4 .alpha. 6
D-GalNAc-.alpha.-R CMP-NeuAC 2.4.99.3 A5
[NeuAc-.alpha.(2,3)-D-Gal-.beta.(1,4)] .alpha. 6 D-GalNAc-.alpha.-R
CMP-NeuAC 2.4.99.7 A6 .alpha. 8 D-NeuAC-.alpha.(2,3)-D-Gal-.beta.-R
CMP-NeuAC 2.4.99.8 A7 .alpha. 8 D-NeuAC-.alpha.(2,8)-D-NeuAC-R
CMP-NeuAC(modified) none B1 .alpha. 2 D-Gal-.beta.-R GDP-L-Fuc
2.4.1.69 B2 [D-Gal-.beta.(1,4)] .alpha. 3 D-GlcNAc-R GDP-L-Fuc
2.4.1.152 B3 [D-Man-.beta.(1,4)-D-GlcNAc-.beta.(1,4)] .alpha. 6
D-GlcNAc-R GDP-L-Fuc 2.4.1.68 B4 .alpha. 6 D-GlcNAc-R GDP-L-Fuc
none B5 [D-GlcNac-.beta.-(1,4)] .alpha. 6 D-GlcNAc-R GDP-L-Fuc none
B6 [D-Gal-.beta.(1,3)] .alpha. 4 D-GlcNAc-R GDP-L-Fuc none B7
[D-Gal-.beta.(1,4)] .alpha. 3 D-Glc-R GDP-L-Fuc none B8 .alpha. 3
D-Glc-R GDP-L-Fuc none B9 .alpha. 4 D-GlcNAc-.beta.-R GDP-L-Fuc
none B10 .alpha. 3 D-GlcNAc-.beta.-R GDP-L-Fuc none B11 .alpha. 4
D-GlcNAc-.beta.(1,3)-Gal-R GDP-L-Fuc none B12 .alpha. 3
D-GlcNAC-.beta.(1,6)-Gal-R GDP-L-Fuc none C1 .beta. 4
D-GlcNAc-.beta.(1,4)-D-GlcNAc-R GDP-Man none C2 .alpha. 3
D-Man-.alpha.(1,2)-Man-.alpha.(1,2)-Man-R GDP-Man none C3 .alpha. 2
D-Man-.alpha.(1,2)-Man-R GDP-Man 2.4.1.131 C4 .alpha. 3
D-Man-.beta.(1,4)D-GlcNAc-.beta.(1,4)-D-GlcNAc-R GDP-Man none C5
[D-Man-.alpha.(1,3)] .alpha. 6 D-Man-.beta.(1,4)D-GlcNAc-R GDP-Man
none C6 .beta. dolicol phosphate GDP-Man 2.4.1.83 C7 .alpha. 6
D-Man-.alpha.(1,0)-R GDP-Man none C8 .alpha. 3 D-Man-.alpha.(1,0)-R
GDP-Man none C9 .alpha. 4 D-GlcNAc-.beta.(1,4)-R GDP-Man none C10
.alpha. 3 D-Man-.beta.(1,4)D-GlcNAc-.beta.(1,0)-R GDP-Man none D1
.beta. ceramide UDP-Gal 2.4.1.45 D2 .beta. 6
D-Gal-.beta.(1,4)-.beta.-Ga- l-.beta.(1,4)-D-Glc-ceramide UDP-Gal
2.4.1.154 D3 .alpha. 3 D-Gal-.beta.(1,4)-D-GlcNAc-R. UDP-Gal
2.4.1.151 D4 .beta. 3 D-Gal-.beta.(1,4)-D-Xly-.beta.-P UDP-Gal
2.4.1.134 D5 .beta. 3 D-GalNAc-R UDP-Gal 2.4.1.122 D6
[L-Fuc-.alpha.(1,2)] .alpha. 3 D-Gal-R UDP-Gal 2.4.1.37 D7 .beta. 4
D-GlcNAc-R UDP-Gal 2.4.1.38 D8 .beta. 4 D-Xly-.beta.-P UDP-Gal
2.4.1.133 D9 .beta. 4 D-Glc-R UDP-Gal 2.4.1.38 D10 .beta. 3
D-GlcNAc-R UDP-Gal none D11 .beta. 3 D-GlcNAc-.beta.(1,3)-Gal-R
UDP-Gal none D12 .beta. 4 D-GlcNAc-.beta.(1,6)-Gal-R UDP-Gal none
E1 .beta. 3 D-Gal-(1,4)-D-Gal-(1,4)-D-Glc-ceramide UDP-GalNAc
2.4.1.79 E2 [D-NeuAC-a(2,3)]- .beta. 4
D-Gal-.beta.(1,4)-D-Glc-ceramide UDP-GalNAc 2.4.1.92 E3
[L-Fuc-.alpha.(1,2)] .alpha. 3 D-Gal-R UDP-GalNAc 2.4.1.40 E4
Ser/Thr UDP-GalNAc 2.4.1.41 F1 sphingosine UDP-Glc 2.4.1.80 G1
.beta. 3 D-Gal-.beta.(1,3)-D-Gal-- .beta.(1,4)-D-Xly-.beta.-P
UDP-GlcA 2.4.1.135 H1 .beta. 3 D-Gal-.beta.(1,3)-D-GalNAc-R
UDP-GlcNAc 2.4.1.146 H2 .beta. 6 D-Gal-.beta.(1,4)-D-GlcNAc
UDP-GlcNAc 2.4.1.150 H3 .beta. 3
D-Gal-.beta.(1,4)-D-GlcNAc-.beta.(1,X)R. UDP-GlcNAc 2.4.1.149 H4
[Gal-.beta.(1,3)] .beta. 6 D-GalNAc-R UDP-GlcNAc 2.4.1.102 H5
.beta. 3 D-GalNAc-R UDP-GlcNAc 2.4.1.147 H6 [GlcNAc-.beta.(1,3)]
.beta. 6 D-GalNAc-R UDP-GlcNAc 2.4.1.148 H7 [D-Man-.alpha.(1,3)]
.alpha. 2 D-Man-.alpha.(1,2)-D-Man-.alpha.(1,2)-D-Man UDP-GlcNAc
2.4.1.138 H8 .beta. 2 D-Man-.alpha.(1,3)[R1]D-man-.beta.(1,4)-R2
UDP-GlcNAc 2.4.1.101 H9 .beta. 4 D-Man-.alpha.(1,3)[R1]-D-Man-.be-
ta.(1,4)-R2 UDP-GlcNAc 2.4.1.145 H10 .beta. 2
D-Man-.alpha.(1,6)[R1]D-man-.beta.(1,4)-R2 UDP-GlcNAc 2.4.1.143 H11
.beta. 6 D-Man-.alpha.(1,6)[R1]-D-Man-.beta.(1,4)-R2 UDP-GlcNAc
2.4.1.155 H12 [D-GlcNAc-.beta.(1,2)][D-GlcNAc-.beta.(1,6)] .beta. 4
D-Man-.alpha.(1,6)R UDP-GlcNAc none H13 [D-Man-.alpha.(1,3)][D-M-
an-.beta.(1,6)] .beta. 4 D-Man-.beta.(1,4)R UDP-GlcNAc 2.4.1.144
H14 [D-GlcNAc-.beta.(1,3)] .beta. 6 D-Gal-.beta.(1,4)-D-GlcNAc
UDP-GlcNAc none H15 .beta. 3 D-Gal-.beta.(1,4)-D-Glc UDP-GlcNAc
none H16 [D-GlcNAc-.beta.(1,3)] .beta. 6 D-Gal-.beta.(1,4)-D-Glc
UDP-GlcNAc none H17 .beta. 4 D-GlcNAc-R UDP-GlcNAc none H18 .beta.
4 D-Man-(1,0)R UDP-GlcNAc none H19 [D-GlcNAc-.beta.(1,4)] .beta. 2
D-Man-(1,0)R UDP-GlcNAc none H20 .beta. 3 D-Gal-.beta.(1,4)-D-Glc-
NAc UDP-GlcNAc none H21 .beta. 6 D-Gal-R UDP-GlcNAc none H22 .beta.
3 D-Gal-R UDP-GlcNAc none H23 [D-GlcNAc-.beta.(1,6)] .beta. 3
D-Gal-R UDP-GlcNAc none H24 [D-GlcNAc-.beta.(1,6)] .beta. 3 D-Gal-R
UDP-GlcNAc-(4,1).alpha.-Fuc none I1 .alpha. 3 D-Glc-.beta.-Ser
UDP-Xyl none I2 .alpha. 2
D-Man-.beta.(1,4)D-GlcNAc-.beta.(1,4)-D-GlcNAc-R UDP-Xyl none I3
.alpha. 3 D-Xly-.alpha.(1,3)-D-Glc-.beta.-Ser UDP-Xyl none
[0133] The first step in the synthesis of the complex carbohydrate
shown in FIG. 1 is effected, as shown in FIG. 4, by attachment of a
first building block, GalNAc, onto a solid support (S) via an
appropriate linker which is further described herein below.
[0134] The second step (D5, see Table 7 and FIG. 4 for details) in
the synthesis of the complex carbohydrate shown in FIG. 1 involves
transferring Gal from UDP-Gal to GalNAc-S by a
.beta.(1,3)-galactosyltran- sferases (E.C. 2.4.1.122).
[0135] The third step (H1, see Table 7 and FIG. 4 for details) in
the synthesis of the complex carbohydrate shown in FIG. 1 involves
the utilization of .beta.(1,3)N-acetylglucosaminyltransferase (E.C.
2.4.1.146) to transfer an acetylglucoseamine group from UDP-GlcNAc
to Gal-.beta.(1,3)-GalNAc-S.
[0136] Then, a galactose unit is added to the acceptor (see FIG.
4), rather then a fucose unit because the specificity of the
enzymes .beta.(1,3)-fucosyltransferase (E.C. 2.4.1.152) to the
acceptor Gal-.beta.(1,4)-GlcNAc-R rather than to the naked
GlcNac.
[0137] Thus, in the fourth step (D7, see Table 7 and FIG. 4 for
details) in the synthesis of the complex carbohydrate shown in FIG.
1 galactose is added to GlcNAc-R using the enzyme .beta.(1,4)
galactosyltransferase (E.C. 2.4.1.38).
[0138] Following the fourth synthesis step described above, the
synthesis process complicates since the galactose units branch into
two antennas (branches). Since the structure of these antennas is
identical at the branching point, yet different towards their
non-reducing ends, an identical stepwise synthesis process that
simultaneously forms the identical parts of the two antennas would
not enable the subsequent synthesis of a unique reducing end for
each of the antennas. Therefor, the synthesis of the two unique
portions of each of the antennas proceeds in an independent
stepwise fashion. In any case, in the example given, the
.beta.(1,3) branch has to be synthesized first because this antenna
requires fucosylation. If the synthesis process would initiate with
the other branch, directed fucosylation to the desired branch could
not have been effected.
[0139] Thus, the fifth step (H3, see Table 7 and FIG. 4 for
details) in the synthesis of the complex carbohydrate shown in FIG.
1 is effected using .beta.(1,3)N-acetylglucosaminyltransferase
(E.C. 2.4.1.149) to transfer acetylglucosamine from UDP-GlcNAc to
Gal-.beta.(1,4)-GlcNAc-R.
[0140] In the sixth step (D7, see Table 7 and FIG. 4 for details)
in the synthesis of the complex carbohydrate shown in FIG. 1, a
galactose unit is added to GlcNAc-R using a
.beta.(1,4)galactosyltransferase (E.C. 2.4.1.38).
[0141] In the seventh step (D3, see Table 7 and FIG. 4 for details)
in the synthesis of the complex carbohydrate shown in FIG. 1, an
additional galactose residue is added to Gal-.alpha.(1,4)-GlcNAc-R
using .alpha.(1,3)galactosyltransferase (E.C. 2.4.1.151).
[0142] In the eighth step (H14, see Table 7 and FIG. 4 for details)
in the synthesis of the complex carbohydrate shown in FIG. 1,
branching is effected by using
.beta.(1,6)N-acetylglucosaminyltransferase on the
Gal[GlcNAc-.beta.(1,3)].beta.(1,4)-GlcNAc-R acceptor substrate.
[0143] The ninth step (B2, see Table 7 and FIG. 4 for details) in
the synthesis of the complex carbohydrates shown in FIG. 1 involves
two of the four GlcNAc monomers and is effected by
.alpha.(1,3)fucosyltransferas- e (E.C. 2.4.1.152) which transfers
fucose to Gal-.beta.(1,4)-GlcNAc-R.
[0144] The tenth step (D7, see Table 7 and FIG. 4 for details) in
the synthesis of the complex carbohydrate shown in FIG. 1 effects
further elongation of the second antenna using an .beta.(1,4)
galactosyltransferase (E.C. 2.4.1.38) on the GlcNAc-R acceptor
substrate.
[0145] In the eleventh step (A3, see Table 7 and FIG. 4 for
details) in the synthesis of the complex carbohydrate shown in FIG.
1, a sialic acid monomer is appended to the Gal-.beta.(1,4)GlcNAc-R
of the antenna in an .alpha.(1,6) orientation using an
.alpha.(2,3)Sialyltransferases (E.C. 2.4.99.6) The twelfth step
(A6, see Table 7 and FIG. 4 for details) in the synthesis of the
complex carbohydrate shown in FIG. 1 effects further elongation of
this antenna using an .alpha.(2,8)Sialyltransferases (E.C.
2.4.99.8), thereby adding another NeuAC unit to the
NeuAC-.alpha.(2,3)-Gal-R substrate acceptor.
[0146] Further elongation of this antenna requires the use of a
(2,8) polysialyltransferases with specificity to the
NeuAC-.alpha.(2,8)-NeuAC-R substrate acceptor. Unfortunately, an
enzymatic reaction with such an enzyme will cause uncontrollable
polymerization of multiple sialic acid monomers rather than the
required addition of only a single sialic acid monomer. Achieving a
controllable addition of a single sialic acid monomer in each ER
step necessitates the use of an enzyme with a modified donor
specificity. As such, instead of using CMP-NeuAC as a donor
substrate, this modified enzyme incorporates a modifying group to
the glycan end. The presence of this modifying group prevents the
unwanted polymerization of multiple monomers of sialic acid, since
this enzyme cannot append sialic acid to the acceptor
NeuAC(modified)-.alpha.(2,8)-Ne- uAC-R. In the last step of this ER
the modifying group is removed.
[0147] Thus, in the final step (A7, see Table 7 and FIG. 4 for
details) in the synthesis of the complex carbohydrate shown in FIG.
1, the modified enzyme .alpha.(2,8) polysialyltransferasease is
employed along with the modified donor CMP-NeuAC and the acceptor
NeuAC(modified)-.alpha.(2,8)-Ne- uAC-R, to thereby generate the
complex carbohydrate shown in FIG. 1.
[0148] Table 8 below outlines the stepwise synthesis of the complex
carbohydrate shown in FIG. 1, as outlined in FIG. 4.
8TABLE 8 First immobilized ERs sequence (the ERs details are shown
in EM monosaccharide Table 7 and FIG. 4): EM.sub.1 GalNAc-S D5, H1,
D7, H3, D7, D3, H14, B2, D7, A3, A6,A7
[0149] ERs selection for providing a desired EM is generated using
a computer algorithm taking into account the complex carbohydrate
structure and the available ERs. Such an algorithm can readily be
programmed by one ordinarily skilled in the art, based on the
donor-acceptor specificities of the various glycosyltransferases
glycosidases and transglycosylases available.
[0150] Automated synthesis and screening: The EMs used in the
construction of a combinatorial complex carbohydrate library
according to the present invention are executed to produce a
combinatorial complex carbohydrate library bound to a solid support
using automated technology. Screening to identify bio-active
complex carbohydrates cross reactive to a probe of interest is also
executed, according to preferred embodiments of the invention, via
automated technology.
[0151] The different structural characteristics of every single
complex carbohydrate of a specific library dictates a multitude of
unique synthesis protocols. As such, the libraries described by the
present invention are preferably generated by a parallel synthesis
method, wherein consideration is preferably given to ensure a
minimal number of steps executed. Consider, for example, a robotics
system permitting parallel addressable distribution of reagents
from reagent containers. In this case, a minimal number of steps
implies that each of the reagent containers is detailed a minimal
number of times. Thus, consideration is given to a sequence of
synthesis steps that will ensure completion of the synthesis of all
of the complex carbohydrates of the library with a minimal number
of times each reagent container is detailed. A dedicated algorithm
can be readily developed by one ordinarily skilled in the art to
design automated library synthesis protocol which will comply with
the above requirements. In fact, a similar algorithm has already
been developed for the parallel addressable synthesis of
oligonucleotides (Pease, 1994) and peptides (Fodor, 1991) on
microchips, both are incorporated by reference as if fully set
forth herein).
[0152] Technologies enabling automated high-throughput parallel
addressable synthesis have developed immensely in the past few
years. Automated synthesizers that can control and perform many
different solid phase synthesis protocols at the same time are
commonly available nowadays (Rivero, 1997). These technologies can
be classified according to the type of solid phase substrate that
is utilized for the synthesis, the means of the introduction and
removal of reagents, and the design of the reaction chambers.
[0153] Technologies enabling automated high-throughput solid phase
parallel synthesis can be used in accordance with the present
invention. Such technologies include, for example, (i) opened
reactor systems (e.g., conventional microtiterplates); (ii) closed
reactor systems or semi-closed reactor systems (e.g., lab-on-chip);
(iii) reaction block systems; (iv) synthesis on polymeric pins; (v)
synthesis on polymeric sheets; and (vi) synthesis on a microchip.
For a comprehensive review of these technologies and systems see
Cargil, 1997, which is incorporated by reference as if fully set
forth herein.
[0154] Solid phase support: The libraries according to the present
invention are preferably synthesized on a solid phase support. As
such, the first building block is provided with a suitable
functional group for binding such a support. Suitable binding
groups include hydroxyls, carbonyl, carboxyl, amines, halides,
thiols, esters, boronates, siloxy, aza, oxo, oxiren, or any
unsaturated group.
[0155] Several solid matrix supports are most suitable for
generating carbohydrate libraries according to the present
invention, such as, but not limited to, polysterene cross-linked
with divinylbenzene (Merrifield, 1963), polyethylene
glycol-polystyrene block copolymer (PEG-PS, Bayer, 1991),
polyamides (Dryland, 1986), polyacrylamide (Ashardy, 1979),
polymethacrylamide (Hsiau, 1997) and cellulose (Frank, 1988).
Microfabricated silicon-based arrays produced by standard
semi-conductor processing techniques (Fodor, 1991; Sosnowski, 1997;
Cheng, 1998, U.S. Pat. Nos. 5,643,738; 5,681,484; and 5,585,069)
may also serve as a solid phase support.
[0156] Linking the first saccharide building block to the solid
phase support: The first saccharide building block is preferably
covalently attached to the solid phase matrix via a single atom
(e.g., the solid phase functional group) or a linker. The general
properties of a linker include: having bi-functional groups
enabling attachment to both the solid support and to the initial
building block, and as such to define a structure (Atherton, 1989).
In a preferred embodiment, the linker is designed cleavable, so as
to allow removal of the synthesized oligosaccaride from the matrix
post synthesis. This allows for analysis thereof using, for
example, mass spectroscopy or any other suitable method. Since
enzyme accessibility to the immobilized saccharides is of great
importance, the linker length and flexibility are crucial for high
yield. As such, linkers suitable for synthesis according to the
present invention can include, for example, amino acids, peptides,
non-glycosylated proteins, lipids, lipid A, ceramides, dolicol
phosphates, cyclodextrins, oligosaccharides, monosaccharides, alkyl
chains, nucleic acids, or other spacer molecules. These linkers can
be cleavable or non-cleavable and be composed of simple, complex,
cyclic or branched entities.
[0157] According to presently preferred embodiments of the present
invention the linker is between 3.5 nm and 8 nm in length. It is
preferably selected sufficiently hydrophilic so as to stay in
solution and to avoid none specific interaction with proteins. It
is synthesized by elongation cycles using bi-functional building
blocks molecules that can form bonds between each other. The
starting point can be any functional group which is attached or
attachable to the solid support that can react with a bi-functional
building block. Because the linker can be ended by any one of the
bi-functional building blocks, there are, as shown in FIGS. 5a and
5b, two possible ways to connect the first monosaccharide to the
linker. The linker is preferably selected cleavable under mild
conditions that do not damage carbohydrates.
[0158] Presently preferred cleavable linkers which comply with all
of the above preferred criteria are constructed as described under
Example 11 and shown in FIGS. 5a and 5b.
[0159] Library arrangement: The preferred arrangement of the
library constituents according to the present invention is in an
array synthesized on a solid phase support in various geometric
forms and layouts, such as: two dimensional arrays, multi layer
arrays, three dimensional arrays (e.g., stacked microtiters), and
arrays which are displayed on spherical disks or cone shapes.
Alternatively, the library constituents can be attached to polymer
beads in reaction chambers (opened or closed) and arrayed on a two
dimensional or a three dimensional support. Any arrangement that
enables easy automatic addressable operation of the EMs collection
may be used in accordance with the present invention. Attention is
preferably given to the spatial distribution of complex
carbohydrates of a library to ensure shortest distances among most
similar carbohydrates, so as to ensure efficiency of the automated
synthesis process. Microfluid systems can and are preferably
employed (U.S. Pat. Nos. 5,643,738; 5,681,484; and 5,585,069).
[0160] Automated library screening: There are numerous screening
technologies and procedures currently employed in the art that can
be applied to screen the complex carbohydrate libraries of the
present invention. For reviews see Broach, 1996 and Burbaum, 1997,
which are incorporated by reference as if fully set forth herein.
As such, technologies suitable for high-throughput screening of
binding or bio-activities can be based on the following: (i)
radioactive detection methods; (ii) fluorescence detection methods;
(iii) ELISA based detection methods; and (iv) cell-based assay
systems via reporter genes.
[0161] Radiolabeled probes that bind to complex carbohydrates of a
given library can be detected, for example, by a Scintillation
Proximity Assay (SPA, Cook, 1996). The main advantages to SPA are
that (i) it does not require removal of free radiolabeled
molecules; and (ii) it is readily automated. Additional methods,
such as, fluorescence can also be employed either via direct
fluorescence detection of a fluorescently labeled bound molecule
or, for example, by either the Homogenous Time-Resolved
Fluorescence (HTRF, Mathis, 1995) or the Fluorescence Polarization
Assay (PFA) technologies (Checovich, 1995). The main advantages of
these latter technologies lie in the ability to use "mix and
measure" protocols without the addition of further complicating
steps. In addition, many variations of the Enzyme-Linked
Immunosorbent Assays (ELISA) detection method can also be employed
with accordance to this invention.
[0162] The bioactivity and binding capabilities of each of the
complex carbohydrates of a library according to the present
invention can be evaluated by using cell-based assay systems.
Cell-based assay systems for high-throughput screening have been
extensively studied, and guidelines for selecting appropriate
screening systems have been introduced (Rose, 1996). Assay systems
using mammalians and insect cells, as well as yeast and bacterial
cells, have been thoroughly described (Broach, 1996; Rose, 1996;
Suto, 1997).
[0163] One of the most common methods for detecting interactions
between molecules expressed in cells and ligands capable of binding
such cells is to employ a reporter gene. This involves splicing the
transcriptional control elements of a target gene with a coding
sequence of a reporter gene into a vector and introducing the
vector into a suitable cell line in order to establish a detection
system that responds to modulation of the target, in this case by
an addressable library derived complex carbohydrate. Common
examples of reporter genes are enzymes such as alkaline
phosphatase, chloramphenicol acetyltransferase, firefly and
bacterial luciferases, and .beta.-galactosidase. Low levels of
activity for these enzymes can be detected using calorimetric,
chemiluminescent or bioluminescent detection methods. Non enzymatic
reporter genes such as green, red shifted and blue fluorescent
protein (Phillips, 1997) can be employed as well.
[0164] Thus, according to one aspect of the present invention there
is provided a combinatorial complex carbohydrate library. The
combinatorial complex carbohydrate library according to the present
invention includes a plurality of addressable complex carbohydrate
structures.
[0165] According to another aspect of the present invention there
is provided a method of producing an addressable combinatorial
complex carbohydrate library. The method according to this aspect
of the present invention is effected by enzymatically synthesizing
a plurality of complex carbohydrate structures, each of which is
attached to at least one addressed location of a plurality of
locations of a solid support, resulting in an addressable
combinatorial complex carbohydrate library.
[0166] As used herein in the specification and in the claims
section below the terms "addressable" and "addressed" refer to both
location and identity. Thus, the location and identity
(composition) of a complex carbohydrate structure of a library
according to the present invention are both known in advance and
that carbohydrate structure is therefore addressable. It is
understood that the phrase "a complex carbohydrate structure"
refers to a plurality of complex carbohydrate molecules all having
the same structure and localized at a specific and addressable
location on the solid support.
[0167] The addressable complex carbohydrate structures of a library
according to the present invention are preferably attached to the
solid support via a linker (spacer). The linker according to
preferred embodiments of the invention includes at least two
contiguous covalent bonds and it is of a length of at least 20
Angstroms. Suitable linkers include, but are not limited to, 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 (e.g., an
oligonucleotide).
[0168] The solid support onto which complex carbohydrate structures
of a library according to the present invention are attached can
include addressable microparticles or beads, or a flat platform.
The addressable microparticles or beads are arranged, for example,
within wells of a microtiter plate. Alternatively, a
microtiterplate, a membrane or a chip (e.g., silicone chip) serve
as the flat platform solid support according to the present
invention.
[0169] According to a presently preferred embodiment of the
invention the solid support is a chip and different complex
carbohydrate structures of the plurality of addressable complex
carbohydrate structures are formed in patches spaced not more than
2.25 mm from one another (center to center) over the surface of the
chip, thereby providing a density of at least 20 different
addressable complex carbohydrate structures per square
centimeter.
[0170] The substance of which the solid support is made can be, for
example, polysterene cross-linked with divinylbenzene, polyethylene
glycol-polystyrene block copolymer, polyamides, polyacrylamide,
polymethacrylamide, cellulose, glass, quartz, plastic or
silica.
[0171] According to a preferred embodiment of the present invention
at least one, preferably at least three, more preferably at least
ten, more preferably, at least 100, more preferably at least 1000
of the plurality of addressable complex carbohydrate structures of
a library of the present invention includes at least two, three,
four or at least five or more contiguous saccharide units of a
single species. As further detailed hereinabove and resolved such a
structure is not trivial due to uncontrolled polymerization.
[0172] According to another preferred embodiment of the present
invention at least one, preferably at least three, more preferably
at least ten, more preferably, at least 100, more preferably at
least 1000 of the plurality of addressable complex carbohydrate
structures of a library of the present invention includes at least
one, two, three, four or at least five or more branches.
[0173] According to yet another preferred embodiment of the present
invention at least one, two, three or at least four of the branches
are formed of identical core and branching saccharide units. As
further detailed hereinabove and resolved such a structure is not
trivial especially if the antennas attached to each of the branches
differ in saccharide units composition.
[0174] According to still further features in the described
preferred embodiments at least one, preferably at least three, more
preferably at least ten, more preferably, at leas 100, more
preferably at least 1000, of the plurality of addressable complex
carbohydrate structures includes at least 4 preferably at least 5,
more preferably at least 7, more preferably at least 9, more
preferably at least 10, more preferably at least 12, more
preferably at least 15, 20, 25 or at least 30, more preferably at
least 50 or more saccharide units.
[0175] Depending on its intended use, as further detailed
hereinunder, the plurality of addressable complex carbohydrate
structures of a library according to the present invention can be a
representation including non-natural or natural complex
carbohydrates, e.g., which are derived from a human source, such as
tissue, cells or body fluids of a human-being. Alternatively, the
plurality of addressable complex carbohydrate structures can be a
representation of domains (fragments) of at least one natural
complex carbohydrate. Such a library, as further detailed herein,
can be employed to identify an active site of the natural complex
carbohydrate.
[0176] According to yet another aspect of the present invention
there is provided a method of identifying a complex carbohydrate
capable of binding an entity. The method according to this aspect
of the present invention is effected by providing an addressable
combinatorial complex carbohydrate library according to any of the
embodiments herein described and screening the addressable
combinatorial complex carbohydrate library with the entity for
identifying the complex carbohydrate capable of binding the
entity.
[0177] As further exemplified hereinunder, any entity can be used
to screen an addressable combinatorial complex carbohydrate library
according to the present invention. For example, the entity can be
a candidate for a biologically active material, such as a drug
candidate derived from a natural or synthetic origin. In this case
the method according to this aspect of the invention serves for
identifying a complex carbohydrate which is a target for the
candidate for the biologically active material. Alternatively, the
entity can be a ligand known to bind a specific natural complex
carbohydrate. In this case, the addressable combinatorial complex
carbohydrate library can be a representation of domains of the
specific natural complex carbohydrate, whereas the method serves in
this case for identifying a specific domain of the domains which
binds the ligand to thereby identify the active site of the natural
complex carbohydrate.
[0178] According to still another aspect of the present invention
there is provided a method of diagnosing a disorder characterized
by self or non-self complex carbohydrate structures and elicitation
of antibodies there against. The method according to this aspect of
the present invention is effected by providing an addressable
combinatorial complex carbohydrate library representing the self
and/or the non-self complex carbohydrates by employing any of the
methods described herein for synthesizing such a library. The
addressable combinatorial complex carbohydrate library is
thereafter reacted with antibodies derived from a patient suspected
of having the disorder to thereby generate a pattern of the
locations to which the antibodies bind, such that by comparing that
pattern with a known pattern characterizing a healthy individual, a
diagnosis of the disorder is obtainable.
[0179] Disorders known to be associated with production or
introduction of self and/or non-self complex carbohydrate
structures include, but are not limited to, tumorogenesis,
metastasis, pregnancy, vascular disease, heart disease,
neurodegenerative disease, autoimmune disease and organ
transplantation. Neurodegenerative diseases include, but are not
limited to, Parkinson's disease, Alzheimer's disease, basal ganglia
degenerative diseases, motoneuron diseases, Scrapie, spongyform
encephalopathy and Creutzfeldt-Jakob's disease, infertility,
allergies, embryogenesis, apoptosis and neurodegenerative
disorders.
[0180] Outlined below are some strategies employed while screening
specific libraries generated by the method according to the present
invention.
[0181] Thus, in order to identify complex carbohydrate receptors,
associated proteins, lectins and/or drug targets, an enzymatically
combinatorial complex carbohydrates array composed of known complex
carbohydrate structures from human cells or novel carbohydrate
structures generated from reduction mapping is screened against a
variety of labeled probes from sources such as, labeled human
tissue homogenates, labeled receptors, labeled proteins encoded by
EST collections, labeled recombinant proteins, phage display
libraries, labeled cells from either human tissues, pathogens or
solutions containing mixtures of labeled protein molecules from
human tissue sources or from pathogenic cells. The objective of
such screening is to identify the labeled molecules from the above
mentioned sources which bind specifically to a complex carbohydrate
of the library. Following isolation and characterization, these
molecules can be tested further as potential candidates for drug
therapy or targets for drug therapy. These strategies can also be
employed to identify new receptors, lectins or any proteins or
molecules that binds to specific complex carbohydrate constituents
of the library.
[0182] To identify lead compounds which bind a specific complex
carbohydrate, an enzymatic combinatorial complex carbohydrate
library composed of known complex carbohydrate structures of human
cells or novel carbohydrate structures generated from reduction
mapping of normal and/or pathogenized cells or pathogens are
screened against a diverse group of labeled molecules. The
objective of this screening is to gain a clearer understanding of
the specific interactions between the complex carbohydrate found in
or on these cells and the respective ligand thereof. The isolated
and characterized ligands can then be utilized as modulators of
important biological activities, such as cell-to-cell
communication, cell recognition, cell development and tumor cell
metastasis.
[0183] For the identification of novel bio-active complex
carbohydrates, a complex carbohydrate library, according to the
present invention, is prepared composed of a diverse array of
complex carbohydrate structures, including such structures not
normally found in nature. This complex carbohydrate library is then
screened against cell-based assay systems, or against defined human
or microbial labeled target molecules, such as lectins or
receptors. Such screening leads to the identification of new
complex carbohydrate based drug candidates. Alternatively, such
screening leads to the identification of a disease associated
complex carbohydrate. Such disease associated complex carbohydrate
can be used to elicit antibodies thereto, the antibodies can
thereafter be used to identify and isolate a glycoprotein harboring
the disease associated carbohydrate, to thereby identify new
protein and genes associated with that disease.
[0184] To identify an active site of a known or novel complex
carbohydrate, a library according to the present invention is
prepared composed of all the possible domain fragments of this
particular complex carbohydrate. As such, these domain fragments
can then be screened with a labeled receptor which normally binds
the complex carbohydrate including such domains. The binding
specificity to each of the domain fragments can then be assessed to
enable the isolation of the domain fragment of a particular complex
carbohydrate responsible for the binding activity, i.e., the active
site. This objective can be performed in parallel for a number of
well characterized complex carbohydrate-receptor pairs.
[0185] The present invention also enables mapping of antibodies
against self or non-self glyco-markers found in the blood serum of
a patient. As such, a complex carbohydrate library according to the
present invention is synthesized to include a diverse array of
glyco markers present in the blood serum of a patient. This library
is then screened against labeled pools of serum antibodies from
this patient and a resulting generated antibody profile can be
implemented as a pre-diagnostic tool for cancer and organ
transplantation compatibility.
[0186] Specific arrays of glyco-antigens can also be used for the
identification of new glyco-markers related to cancer,
cardiovascular diseases or organ transplantation. Such
glyco-antigen arrays are screened according to the present
invention against labeled serum antibodies from a diverse
population. The antibody profile of a diseased individual can then
be compared with the profile of the healthy population. This
comparison produces a unique profile of antibodies associated with
the immune response to a disease state and as such, a particular
complex carbohydrate reacting with an antibody of the unique
profile turns into a diagnostic marker for that particular
disease.
[0187] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0188] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0189] The following examples detail the structure of complex
carbohydrates synthesized using a collection of specifically
selected enzymes. Generally, the nomenclature used herein and the
laboratory procedures in biochemistry described below are those
well known and commonly employed in the art. As such, it will be
appreciated that the following synthesis procedures could be
practiced with ease by one ordinarily skilled in the art.
[0190] In view of the findings that sugar residues of glycoproteins
play an important role in the control of cellular function and
cellular recognition, investigation of carbohydrate function in
pathological states has led to the assignment of some complex
carbohydrates as tumor specific markers (Orntoft, 1995). Dramatic
changes in glycosylation of proteins occur in almost every
carcinoma (Hakomori, 1989), which often reflects changes in the
biosynthesis pathways.
[0191] For example, blocking the glycosylation biosynthesis pathway
leads to an overproduction of structures which are typically found
in small amounts in normal cells. Furthermore, alterations in
glycosylation pathways can lead to the utilization of alternate
pathways which, in turn, lead to the formation of new complex
carbohydrate structures not normally present in or on cells. Such
regulation of glycosylation pathways in the cell can often be
attributed to glycosyltransferase activities.
[0192] The altered carbohydrate structures of glycoproteins of
various tumor tissues are considered to be the basis for abnormal
behavior of tumor cells, which behavior includes metastasis and
invasion of the tumor cells into healthy tissues (Kobata,
1998).
[0193] Tumor markers are significant for the diagnosis and
treatment of malignant cells. Potential markers can be any specific
epitopes presented by the tumor cells, such as, peptides,
glycopeptides, glycolipids or any combinations thereof. These
unique epitopes can be specifically identified by monoclonal
antibodies and as such unique glycosylation patterns were and are
intensively investigated as potential tumor markers for cancer
immunotherapy and diagnostics (Ronin, 1998).
[0194] The present invention enables to use tumor markers not only
for immunotherapy or diagnostics but also as potential targets for
drug therapy. To this end, a combinatorial array including both
Tumor Specific Complex Carbohydrates (TSCC) and normal carbohydrate
structures would enable isolation of new drug candidates by
identifying molecules that bind specifically and uniquely to
complex carbohydrate structures associated with a tumorous
conditions.
Example 1
[0195] Lung cancer is a disease of almost epidemic proportion.
Approximately 157,000 new cases causing 142,000 deaths were
recorded in 1990 (Faber, 1991). Squamous Lung Carcinoma (SLC) which
is of the Non-Small Cell Lung Cancer (NSCLC) type, accounts for
approximately 35% of all lung cancers. It is closely correlated
with smoking and diagnosed most frequently in males. Squamous
carcinoma originates in the central or hilar region of the lung and
may cavitate when found in a peripheral location. It is classified
as a severe and malignant form of cancer. Although poorly
differentiated, SLC displays unique complex carbohydrate antigens
associated with both membrane bound glycoproteins and mucine-like
molecules which are released into circulatory system and serve as
serum markers (Martensson, 1988).
[0196] The following tables describe the components and enzyme
modules (EMs) necessary for the synthesis of a complex carbohydrate
library for screening and isolation of chemical compounds, proteins
or other molecules which specifically bind SLC markers. Such
molecules may serve as potential new drug candidates or drugs
useful for the prevention of squamous lung cancer metastasis.
[0197] Tables 9-10 present complex carbohydrates of abnormal
structures of SLC carbohydrate chains released into the circulatory
system (marked A1, A2, A3, A8, A9 and A10), all the possible
fragments derived from such SLC carbohydrate chains (associated
with A1, A2, A3, A8, A9 and A10 by a1, a2, a3, a8, a8 and a10,
respectively), normal blood antigens (marked A4, A5, A6, A7, A11
and A12) and all possible normal blood antigen fragments derived
therefrom (associated with A4, A5, A6, A7, A11 and A12 by a4, a5,
a6, a7, a11 and a12, respectively).
9TABLE 9 Source Carbohydrates EM A1 SLC 1 1 A2 SLC 2 2 A3 SLC 3 3
A4 .alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.f-
wdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-
-Glcp-(1.fwdarw.1)- 4 Normal Ceramide blood HI antigen A5
.alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.fwdarw.4)--
.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-Glcp-(1.-
fwdarw.1)- 5 Normal Ceramide blood HII antigen A6; a7 Normal Lewis
a antigen 4 6 A7 Normal Lewis b antigen 5 7 A8 SLC 6 8 A9 SLC 7 9
A10 SLC 8 10 A11 Normal Lewis x antigen 9 11 A12 Normal Lewis x
antigen 10 12
[0198]
10TABLE 10 Source Carbohydrates sub fragments EM a1; a2; a3;
.beta.-D-Galp-(1.fwdarw.4)-D-Glc 13 a8; a9; a10 a8
.alpha.-L-Fucp-(1.fwdarw.3)-D-Glc 14 a1; a2; a3;
.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw. 15 a4; a6;
a7; a8; a9; a10 a1; a2; a3; .beta.-D-Galp-(1.fwdarw-
.4)-.beta.-D-GlcpNAc-(1.fwdarw. 16 a9; a10 a1; a2; a3;
.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw. 17 a1; a2;
a3; .beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 18 a4;
a5; a6; a7; a8; a9; a10; a11; a12 a4; a5; a7
.alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.fwdarw. 19 a2; a3; a6;
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw. 20 a7; a8;
a10 a1; a2; a3; .alpha.-L-Fucp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(-
1.fwdarw. 21 a9; a10; a11; a12 a1; a2; a3
.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 22
a1; a2; a3;
.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Gl- c 23
a9; a10 a1; a2; a3; .beta.-D-Galp-(1.fwdarw.3)-.beta.-D-
-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 24 a4; a6; a7; a8;
a9; a10 a1; a2; a3 .beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNA-
c-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw. 25 a5; a9; a10;
.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1-
.fwdarw. 26 a11; a12 a1; a2; a3 .alpha.-L-Fucp-(1.fwdarw.3)--
.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw. 27 a2; a3;
a6;
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Ga-
lp-(1.fwdarw. 28 a7; a8 a4; a7 .alpha.-L-Fucp-(1.fwdarw.2)-.-
beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw. 29 a5
.alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(-
1.fwdarw. 30 a9; a10;
.alpha.-L-Fucp-(1.fwdarw.3)-.beta.-D-GlcpNAc--
(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 31 a11; a12 a9; a10
.alpha.-L-Fucp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(-
1.fwdarw.4)-D-Glc 32 a2
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNA-
c-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 33 a1; a2; a3;
.alpha.-L-Fucp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(-
1.fwdarw.4)-D-Glc 34 a9; a10
.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-Gl-
cpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 35 a1; a2; a3;
.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1-
.fwdarw.4)-D-Glc 36 a1; a2; a3;
.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-
-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 37 a8 a4; a7
.alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-Gl-
cpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 38 a5; a12
.alpha.-L-Fucp-(1.fwdarw.2)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(-
1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 39 a9; a10
.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-
-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw. 40 a9, a10
.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-
-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 41 a3
.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-
-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc 42 a3
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(-
1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw.
43 a3
.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Ga-
lp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw.
44 a9; a10
.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.-
beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.f-
wdarw. 45 a3
.beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.-
3)-.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp--
(1.fwdarw.4)-D-Glc 46 a10 *see below 47 a3
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(-
1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.6)-.beta.-D-Galp-(1.fwdarw.4)-D-Glc
48 a9 .beta.-D-Galp-(1.fwdarw.3)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.be-
ta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwd-
arw.4)-D-Glc 49 a8 11 50 a1; a2; a3; a9; a10; a11; a12 12 51 a1;
a2; a3 13 52 a2; a6; a7; a8; a10 14 53 a2 15 54 a9; a10; a11; a12
16 55 a12 17 56 a2; a3; a6; a7; a8; a10 18 57 a7 19 58 a3; a9; a10
20 59 a1; a2; a3 21 60 a1; a2; a3 22 61 a1; a2; a3 23 62 a1; a2; a3
24 63 a1; a2; a3 25 64 a1; a2; a3 26 65 a1; a2; a3 27 66 a1; a2; a3
28 67 a1; a2; a3 29 68 a1; a2; a3 30 69 a1; a2 31 70 a2 32 71 a2;
a8; a9 33 72 a2 34 73 a2 35 74 a2 36 75 a8 37 76 a8 38 77 a12 39 78
a9; a10 40 79 a9; a10 41 80 a7 42 81 a10 43 82 a3; a9; a10 44 83
a1; a2; a3 45 84 a1; a2; a3 46 85 a1; a2; a3 47 86 a1; a2; a3 48 87
a2 49 88 a2 50 89 a2 51 90 a2 52 91 a2 53 92 a2 54 93 a3; a10 55 94
a3 56 95 a3 57 96 a3 58 97 a9; a10 59 98 a9; a10 60 99 a2 61 100 a2
62 101 a2 63 102 a10 64 103 a3 65 104 a3 66 105 a3 67 106 a3 68 107
a3 69 108 a3 70 109 a3 71 110 a3 72 111 a3 73 112 a10 74 113 a3 75
114 a3 76 115 a3 77 116 a3 78 117 a3 79 118 a3 80 119 a3 81 120 a3
82 121 a3 83 122 a3 84 123 a3 85 124 a3 86 125 a3 87 126 a3 88 127
*.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp--
(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.3)-.beta.-D-Galp-(1.fwdarw.4)-D-Gl-
c
[0199] Table 11 includes a list of the EMs required for the
synthesis of the complex carbohydrate collection described in
Tables 9-10
11TABLE 11 First Immobilized ERs sequence EM monosaccharide (the
ERs details are found in Table 7): 1 Glc-S D9, H15, D10, H16, D7,
B2 2 Glc-S D9, H15, D10, B6, H16, D7, B2 3 Glc-S D9, H15, D10, H16,
D7, B2, H20, D10, B6 4 Glc-S D9, H15, D10, B1 5 Glc-S D9, H15, D7,
B1 6 Glc-S D9, H15, D10, B6 7 Glc-S D9, H15, D10, B6, D1 8 Glc-S
D9, H15, D10, B6, B7 9 Glc-S D9, H15, D7, H3, B2, D10 10 Glc-S D9,
H15, D7, H3, B2, D10, B6 11 Glc-S D9, H15, D7, B2 12 Glc-S D9, H15,
D7, B2, B1 13 Glc-S D9 14 Glc-S B8 15 GlcNAc-S D10 16 GlcNAc-S D7
17 Gal-S H21 18 Gal-S H22 19 Gal-S B1 20 GlcNAc-S B9 21 GlcNAc-S
B10 22 Gal-S D9, H21 23 Gal-S D9, H22 24 Gal-S H22, D10 25 Gal-S
H21, D7 26 Gal-S H22, D7 27 Gal-S H21, B10 28 Gal-S H22, B9 29
GlcNAc-S D10, B1 30 GlcNAc-S D7, B1 31 Gal-S H22, B10 32 Glc-S D9,
H22, B10 33 Glc-S D9, H22, B9 34 Glc-S D9, H21, B10 35 Glc-S D9,
H22, D7 36 Glc-S D9, H21, D7 37 Glc-S D9, H22, D10 38 Gal-S H22,
D10, B1 39 Gal-S H22, D7, B1 40 Gal-S H22, D7, H3 41 Glc-S D9, H15,
D7, H3 42 Glc-S D9, H21, D7, H3 43 Gal-S H21, D7, H3, B9 44 Gal-S
H21, D7, H3, D10 45 Gal-S H22, D7, H3, D10 46 Glc-S D9, H21, D7,
H3, D10 47 Glc-S D9, H22, D7, H3, B9 48 Glc-S D9, H21, D7, H3, B9
49 Glc-S D9, H22, D7, H3, D10 50 Glc-S D9, B7 51 GlcNAc-S D7, B2 52
Gal-S H21, H23 53 GlcNAc-S D10, B6 54 Gal-S H21, H23, B11 55 Gal-S
H22, D7, B2 56 GlcNAc-S D7, B2, B1 57 Gal-S H22, D10, B6 58
GlcNAc-S D10, B6, B1 59 GlcNAc-S D7, B2, H22 60 Glc-S D9, H21, H23
61 Gal-S H21, H23, B12 62 Gal-S H21, D7, B2 63 Gal-S H21, H23, D11
64 Gal-S H21, D7, B2 65 Glc-S D9, H21, H23, D11 66 Glc-S D9, H21,
D7, B2 67 Glc-S D9, H21, H23, B12 68 Glc-S D9, H21, H23, D12 69
Gal-S H21, H23, B12, D11 70 Gal-S H21, H23, D11, D12 71 Glc-S D9,
H21, H23, B11 72 Glc-S D9, H22, D11, B6 73 Gal-S H21, H23, D11, B6
74 Gal-S H21, H23, D12, B11 75 Gal-S H21, H23, B11, B12 76 Glc-S
D9, B7, H22, D11 77 Glc-S D9, B7, H22, B11 78 Gal-S H22, D7, B2, B1
79 Gal-S H22, D7, H3, B2 80 Glc-S D9, H22, D7, B2 81 Gal-S H22,
D11, B6, B1 82 GlcNAc-S D10, H3, D11, B6 83 GlcNAc-S D7, B2, H3,
D11 84 Glc-S D9, H21, H23, D11, B12 85 Glc-S D9, H21, H23, D11, D12
86 Glc-S D9, H21, H23, D12, B2 87 Gal-S H21, H23, D12, B2, D11 88
Glc-S D9, H21, H23, D11, B6 89 Glc-S D9, H21, H23, D12, B11 90
Glc-S D9, H21, H23, B11, B12 91 Gal-S H21, H23, B12, D10, B6 92
Gal-S H21, H23, D12, D10, B6 93 Gal-S H21, H23, B11, D7, B2 94
GlcNAc-S D7, B2, H3, D10, B6 95 Gal-S H21, D12, H3, D10, B6 96
Gal-S H21, D12, B2, H3, D10 97 Gal-S H21, D12, B2, H3, B9 98 Glc-S
D9, H15, D7, B2, H22 99 Gal-S H22, D7, B2, H22, D10 100 Glc-S D9,
H21, H23, B12, D11, B6 101 Glc-S D9, H21, H23, D12, D11, B6 102
Glc-S D9, H21, H23, D12, B2, B11 103 Glc-S D9, H22, D7, B2, H22,
B11 104 Glc-S D9, H21, D7, H3, D10, B6 105 Glc-S D9, H21, D7, B2,
H22, D11 106 Glc-S D9, H21, D7, B2, H22, B11 107 Gal-S H21, D7, B2,
H22, D11, B6 108 Gal-S H21, D12, H22, D11, B6, H23 109 Gal-S H21,
H23, D12, B2, D11, H20 110 Gal-S H21, D12, H20, B2, D11, H23 111
Gal-S H21, D12, H20, B2, B11, H23 112 Gal-S H21, D12, H20, B9, H23,
D11 113 Gal-S H22, D7, B2, H22, D11, B6 114 Glc-S D9, H21, D7, B2,
H22, D11, B6 115 Glc-S D9, H21, D12, H22, D11, B6, H23 116 Glc-S
D9, H21, H23, D12, B2, H3, D11 117 Glc-S D9, H21, D12, B2, H3, D11,
H23 118 Glc-S D9, H21, D12, B2, H3, B11, H23 119 Glc-S D9, H21,
D12, H3, B11, H23, D11 120 Gal-S H21, D12, B2, H3, D11, B6, H23 121
Gal-S H21, D12, H3, D11, B6, H23, D11 122 Gal-S H21, D12, B2, H3,
B9, H23, D11 123 Gal-S H21, D12, B2, H3, D11, H23, D11 124 Glc-S
D9, H21, D12, B2, H3, D11, B6, H23 125 Glc-S D9, H21, D12, B2, H3,
D11, B6, H23 126 Glc-S D9, H21, D12, B2, H3, D11, H23, D11 127
Glc-S D9, H21, D12, H3, D11, B6, H23, D11
Example 2
[0200] Human chorionic gonadotropin (hCG) is a glycoprotein hormone
produced by the trophoblast cells of the placenta. High levels of
hCG are detected in blood and urine samples taken from patients of
a variety of trophoblastic diseases. As such, urinary and serum hCG
levels have been employed as useful markers for the diagnosis and
prognosis of trophoblastic diseases, as well as being markers for
pregnancy. A study comparing the complex carbohydrates released
from hCGs purified from the urine of pregnant women with those
purified from urine taken from patients with trophoblastic disease
revealed the existence of several alteration in the sugar chains of
hCGs purified from the latter (Mizuochi, 1983; Endo, 1987).
[0201] Tables 12-14 present the components and EMs necessary to
synthesize a complex carbohydrate library for screening and
isolating molecules that specifically bind to the abnormal hCG
markers or to their subfragments. Tables 12-13 list complex
carbohydrates structures incorporated into an hCG specific arrays.
Such structures include abnormal sugar chains represented in the
hCGs present in malignant trophoblastic diseases (marked B5, B6, B7
and B8), all the possible fragmented sugar chains of such hCGs
present in malignant trophoblastic diseases (associated with B5,
B6, B7 and B8 by b5, b6, b7 and b8, respectively), normal sugar
chains as typically found in the hCGs present in the urine of
pregnant women (marked B1, B2, B3 and B4) and all of their possible
fragments (associated with B1, B2, B3 and B4 by b1, b2, b3 and b4,
respectively). Table 14 presents the collection of EMs required for
the synthesis of the complex carbohydrates of Tables 12-13.
12TABLE 12 Source Carbohydrate EM B1; b5 Normal 89 1 B2; b1; b5; b6
Normal 90 2 B3; b1; b2 Normal 91 3 B4; b1; b5; b7 Normal 92 4 B5
Normal 93 5 B6 Normal 94 6 B7; b5 Abnormal 95 7 B8; b5; b6; b7
Abnormal 96 8
[0202]
13TABLE 13 Source Carbohydrate sub fragments EM b1; b4; b5;
.alpha.-L-Fuc-(1.fwdarw.6)-.beta.-D-GlcpNa- c 9 b7 b1; b2; b3;
.beta.-D-GlcpNAc-(1.fwdarw.4)-.beta.-D-Gl- cpNac 10 b4; b5; b6; b7;
b8 b1; b2; b3;
.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.beta.-D-GlcpNa-
c 11 b4; b5; b6; b7; b8 b1; b2; b3;
.alpha.-D-Manp-(1.fwdarw.6)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc--
(1.fwdarw.4)-.beta.-D-GlcpNac 12 b4; b5; b6; b7; b8 b1; b2; b3;
.alpha.-D-Manp-(1.fwdarw.3)-.alpha.-D-Manp-(1.fwdarw.4)-.beta-
.-D-GlcpNAc-(1.fwdarw.4)-.beta.-D-GlcpNac 13 b4; b5; b6; b7; b8 b1;
b2; b5; .beta.-D-GlcpNAc-(1.fwdarw.2)-.alpha.-D-Manp-(1.fwd-
arw.6)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.beta.-D--
GlcpNac 14 b6 b1; b2; b3; .beta.-D-GlcpNAc-(1.fwdarw.2)-.alp-
ha.-D-Manp-(1.fwdarw.3)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fw-
darw.4)-.beta.-D-GlcpNac 15 b4; b5; b6; b7; b8 b5; b6; b7;
.beta.-D-GlcpNAc-(1.fwdarw.4)-.alpha.-D-Manp-(1.fwdarw.3)-.alpha.-
-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.beta.-D-GlcpNac
16 b8 b1; b2; b5; .beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.f-
wdarw.2)-.alpha.-D-Manp-(1.fwdarw.6)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D--
GlcpNAc-(1.fwdarw.4)-.beta.-D-GlcpNac 17 b6 b1; b2; b3;
.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.2)-.alpha.-D-Manp-(-
1.fwdarw.3)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.bet-
a.-D-GlcpNac 18 b4; b5; b6; b7; b8 b5; b6; b7;
.beta.-D-Galp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.alpha.-D-Manp-(-
1.fwdarw.3)-.alpha.-D-Manp-(1.fwdarw.4)-.beta.-D-GlcpNAc-(1.fwdarw.4)-.bet-
a.-D-GlcpNac 19 b8 b5; b6; b7; b8 97 20 b1; b4; b5; b7 98 21 b5;
b6; b7; b8 99 22 b5; b6; b7; b8 100 23 b1; b4; b5; b7 101 24 b5;
b6; b7; b8 102 25 b5; b6; b7; b8 103 26 b5; b6; b7; b8 104 27 b5;
b6; b7; b8 105 28 b1; b4; b5; b7 106 29 b1; b4; b5; b7 107 30 b5;
b6; b7; b8 108 31 b5; b6 109 32 b5; b6; b7; b8 110 33 b1; b2; b5;
b6 111 34 b1; b4; b5; b7 112 35 b1; b5 113 36 b5; b6; b7; b8 114 37
b1; b5 115 38 b1; b4; b5; b7 116 39 b5 117 40 b5; b6; b7; b8 118 41
b5; b6 119 42 b5; b6 120 43 b5; b6; b7; b8 121 44 b1; b2; b5; b6
122 45 b5; b6 123 46 b5; b6 124 47 b5; b6 125 48 b1; b2; b5; b6 126
49 b5; b6 127 50
[0203] Table 14 includes a list of the EMs required for the
synthesis of the complex carbohydrate collection described in
Tables 12-13.
14TABLE 14 First immobilized ERs sequence EM monosaccharide (ERs
details describe in Table 7) 1 GlcpNac-S H17, C1, C4, H8, C5, H10,
D7, B3 2 GlcpNac-S H17, C1, C4, H8, C5, H10, D7 3 GlcpNac-S H17,
C1, C4, H8, C5, D7, H10 4 GlcpNac-S H17, C1, C4, H8, C5, D7, H10,
D7, B3 5 GlcpNac-S H17, C1, C4, H8, H9, C5, H10, D7, B3 6 GlcpNac-S
H17, C1, C4, H8, H9, C5, H10, D7 7 GlcpNac-S H17, C1, C4, H8, H9,
C5, D7, B3 8 GlcpNac-S H17, C1, C4, H8, H9, C5, D7, B3 9 GlcpNac-S
B4 10 GlcpNac-S H17 11 GlcpNac-S H17, C1 12 GlcpNac-S H17, C1, C7
13 GlcpNac-S H17, C1, C4 14 GlcpNac-S H17, C1, C7, H10 15 GlcpNac-S
H17, C1, C4, H8 16 GlcpNac-S H17, C1, C4, H9 17 GlcpNac-S H17, C1,
C7, H10, D7 18 GlcpNac-S H17, C1, C4, H8, D7 19 GlcpNac-S H17, C1,
C4, H9, D7 20 D-Man-.alpha.-(1,0)-S H18, H19 21 GlcpNac-S H17, B5
22 D-Man-.alpha.-(1,0)-S H18, D7, H19 23 D-Man-.alpha.-(1,0)-S H18,
H19, D7 24 GlcpNac-S H17, C1, B3 25 D-Man-.alpha.-(1,0)-S H18, H19,
D7 26 D-Man-.alpha.-(1,0)-S C8, H8, H9, C7 27 D-Man-.alpha.-(1,0)-S
C8, H8, D7, H9 28 D-Man-.alpha.-(1,0)-S C8, H8, D7, C7 29 GlcpNac-S
H17, C1, C7, B3 30 GlcpNac-S H17, C1, C4, B3 31
D-Man-.alpha.-(1,0)-S C8, H8, H9, D7 32 D-Man-.alpha.-(1,0)-S C8,
H8, H9, C7, H10 33 D-Man-.alpha.-(1,0)-S C8, H8, D7, H9, C7 34
D-Man-.alpha.-(1,0)-S C8, H8, D7, C7, H10 35 GlcpNac-S H17, C1, C4,
H8, B3 36 GlcpNac-S H17, C1, C7, H10, B3 37 GlcpNac-S H17, C1, C8,
H9, B3 38 GlcpNac-S H17, C1, C4, H8, B3, D7 39 GlcpNac-S H17, C1,
C7, H10, B3, D7 40 GlcpNac-S H17, C1, C8, H9, B3, D7 41
D-Man-.alpha.-(1,0)-S C8, H8, H9, C7, D7 42 D-Man-a-(1,0)-S C7,
H10, D7, H8, H9 43 D-Man-.alpha.-(1,0)-S C8, H8, D7, H9, C7, H10 44
D-GlcpNac-.beta.-(1,0)-S C9, C10, H8, H9, D7 45
D-Man-.alpha.-(1,0)-S C7, H8, C8, H10, D7 46 D-Man-.alpha.-(1,0)-S
C7, H8, H9, D7, C8, H10 47 D-Man-.alpha.-(1,0)-S C7, H9, C8, H10,
D7, C8 48 D-Man-.alpha.-(1,0)-S C7, H8, C8, H10, D7, H8 49
D-GlcpNac-.beta.-(1,0)-S C9, C10, H8, C5, H10, D7 50
D-Man-.alpha.-(1,0)-S C7, H8, H9, C8, H10, D7
Example 3
[0204] During the latter half of the century it has been
demonstrated that many bacterial, fungal and plant polysaccharides
posses anti-viral, anti-coagulant, anti-thrombotic,
anti-cardiovascular and anti-tumor activities (Witczak, 1997). It
was also found that a general structural pattern is common to all
of these complex carbohydrates. Most of these complex carbohydrates
include one or two repeating monosaccharide units connected with
one or two types of glycosidic bonds and decorated with branched
points of constant length.
[0205] Table 15 summerizes partial examples of such unique
structures.
15TABLE 15 Common Monosaccharide Source name Activity content
Configuration Reference Nothogenia Xylomannan antiviral
xylose-mannose 1,3-linked mannose (98%) Matulewicz, 1978 fastigiata
sulfated in position 2 and 6 with single stubs of .beta.-1,2-xylose
Agardhiella Galactan antiviral galactose 1,3-linked D- and
L-galactose Rees, 1965 tenera sulfate with 3,6-anhydro-D- and
L-galactose with half ester sulfate Ecklonica Fucoidan anti
coagulant fucose .alpha.-1,2-linked units of L-fucose- Nishino,
1989 kurome 4-sulfate with branching or second sulfate unit in
position 3 Saccharomyces Glucan anti glucose .beta.-1,3-D-glucose
with .beta.-1,6-D- Konis, 1976; cerevisia (Zymosan) tumor glucose
branches Misaki, 1968 Mycobacterium LAM evoking TNF arabinose-
.alpha.-1,6-D-mannose core with- Chatterjee, 1998; bovis and other
mannose 1,2-D-mannose branches Nigou, 1997 cytokines elongate with
linear .alpha.-1,5-D-arabinose chain Alcaligenes Crudlan anti
glucose Linear .beta.-1,3-D-glucose Sasaki, 1978 faecalis var.
tumor Chemical Ara- anti arabinose- .beta.-1,3-D-glucose with
Matuzaki, 1986 synthesis Crudlan tumor glucose
.alpha.-1,5-D-arabinose linked at position 4 or 6
[0206] Viscosity studies as well as X-ray analysis suggested that
possible helical and triple helical structures are responsible for
the abovementioned activities (Misaki, 1997). However, further
studies demonstrated that fragments derived from partial hydrolysis
of these complex carbohydrates also posses some therapeutic
activities (Misaki, 1980).
[0207] As such, the present invention can be utilized to screen
combinatorial oligosaccharide libraries which include short
oligomers derived from the complex carbohydrates listed above. Such
fragments can, for example, include one or two repeating
monosaccharide units attached therebetween through one or two types
of glycosidic bonds. Such fragments can also include moderate
branching, when required. Such combinatorial arrays of
polysaccharides can be utilized for the isolation of new
anti-viral, anti-coagulant, anti-thrombotic, anti-cardiovascular
and anti-tumor agents.
Example 4
[0208] The following example represents synthesized complex
carbohydrates including .beta.(1,3)D-glucose and
.beta.(1,6)D-glucose branches. Each of the oligomers shown includes
7 monomers. It will be appreciated that an oligomer consisting of 2
to 30 units or more can also be synthesized by the method of the
present invention as described herein.
16 1 128 2 129 130 n 131
Example 5
[0209] The following example represents synthesized complex
carbohydrates consisting of a backbone of .beta.(1,3)D-glucose
units and .alpha.(1,5)D-arabinose branches, which are positioned at
any desired location along the backbone. Each of the oligomers
shown below includes 12 monomers. It will be appreciated that an
oligomer consisting of 2 to 40 units or more can also be
synthesized by the method of the present invention.
17 1 132 2 133 134 n 135
Example 6
[0210] The following example represents synthesized complex
carbohydrates consisting of .alpha.(1,2)linked .alpha.-L-fucose or
.alpha.-L-fucose-4-sulfate with branching or secondary sulfate
units positioned at position 3. Each of the oligomers shown below
includes 6 saccharide monomers. It will be appreciated that an
oligomer consisting of 2 to 20 units or more can also be
synthesized by the method of the present invention.
18 1 136 2 137 138 n 139
Example 7
[0211] The following example represents synthesized complex
carbohydrates consisting of (1,3) linked .alpha.-D-mannose or
.alpha.-D-mannose sulfated positioned at position 2 and/or 6, and
including .beta.(1,2)xylose stubs. Each of the oligomers shown
below includes 5 saccharide monomers. It will be appreciated that
an oligomer consisting of 3 to 20 units or more can also be
synthesized by the method of the present invention.
19 1 140 2 141 142 n 143
Example 8
[0212] The following example represents synthesized complex
carbohydrates consisting of .alpha.(1,5)D-arabinose and
.alpha.(1,2)D-mannose units with a core of .alpha.-D-arabinose unit
connected to the .alpha.-D-arabinose unit at position 2 or to the
.alpha.-D-mannose unit at any position. Each of the oligomers shown
below includes 9 saccharide monomers. It will be appreciated that
an oligomer consisting of 2 to 70 units or more can also be
synthesized by the method of the present invention.
20 1 144 2 145 146 n 147
Example 9
[0213] The following example represents synthesized complex
carbohydrates consisting of .alpha.-1,5-D-arabinose and
.alpha.-1,2-D-mannose units attached to a single core
.beta.-D-arabinose unit, which is connected to the
.alpha.-D-arabinose unit at position 2 or to the .alpha.-D-mannose
unit at any position. The complex carbohydrate also include a
branched .beta.-D-arabinose which is positioned at position 3. The
branch antenna includes, as a major oligomer, a single core
.beta.-D-arabinose unit connected to .alpha.-D-arabinose at
position 2 or to .alpha.-D-mannose at any position. Each of the
oligomers shown below includes 14 saccharide monomers. It will be
appreciated that an oligomer consisting of 2 to 70 units or more
can also be synthesized by the method of the present invention.
21 1 148 2 149 150 n 151
Complex Carbohydrate Library Synthesis
[0214] The following examples describe in detail experiments
demonstrating sequential enzymatic synthesis of complex
carbohydrates libraries according to the teachings of the present
invention.
Materials and Methods
[0215] Abbreviations used below: BSA--Bovine Serum Albumin;
GlcNAc--N-acetylglucoseamine;
PNP-GlcNAc--p-nitrophenyl-N-acetyl-.beta.-D- -GlcNAc;
GlcNAc-COOH--2-(2-carboxyethylthio)ethyl 2-.beta.-D-GlcNAc;
NHS--N-hydroxysuccinimide;
EDC--1-Ethyl-3-(3dimethylaminopropyl)-carbodii- mide; O.D.--Optical
Density; WGA--Wheat Germ Agglutinin; RCA120--lectin from Ricinus
communis; BS-I--Lectin from Bandeiraea Simplicifolia; TGP--Lectin
from Tereagonolobus purpureas; TML--Lectin from Tritrichomonas
mobilensis; ECorA--Lectin from Erythrina corallodendron;
FITC--Fluoresceine-iso-thio-cyanate.
[0216] General materials: PBS--0.1M phosphate buffer pH 7.5, 0.15M
NaCl (Sigma S-7653); TBS--50 mM Tris/HCl pH 7.5 (Sigma T-6791),
0.15M NaCl; TBST--TBS, 0.05% Tween 20 (Sigma P-9416); High ionic
strength washing buffer--PBS, 2M NaCl, 60 millimolar MgSO.sub.4,
0.05% Tween 20. A peroxidase substrate solution was prepared by
mixing in water O-Phenylenediamine Dihydrochloride, 0.4 mg/ml, Urea
Hydrogen peroxide, 0.4 mg/ml, Phosphate-Citrate Buffer, 0.05M
(prepared from SIGMA FAST peroxidase substrate kit P-9187).
Covalink NH was obtained from NUNC (Cat. No. 478042). Optical
density and fluorescence were measured using a multi-label counter
VICTOR.sup.2 (Wallac OY, Finland). All microtiter plate incubations
were performed at controlled shaking speed and temperature using a
microtiter plate incubator obtained from Anthos Thermostar
Shaker/Incubator, Rosys Anthos GmbH Salzburg Austria (Cat. No.
8850001).
[0217] General Enzymatic Reaction Mixes and Conditions (ER's):
[0218] D7: 100 .mu.l of 50 mM MOPS (SIGMA M-9027), 10 mg/ml BSA
(SIGMA A-7030), 20 mM MnCl.sub.2 (SIGMA M-9522), 0.5 mg/ml UDP-Gal
(Calbiochem 670111) and 20 milliunits/ml of recombinant .beta.1,4
galactosyltransferase (Calbiochem 345650) at pH 7.4 were added to
each well of a microtiter plate and the plate was shaken at 50 RPM
at 37.degree. C. for 3 hours. Following incubation, the reaction
mixture was removed and the wells were washed three times with 200
.mu.l of TBST, the last wash consisting of a 15 minutes soak. The
TBST was replaced with TBS 0.2% NaN.sub.3 (Sigma S-8032) and the
plate was stored at 4.degree. C. for subsequent enzymatic
reactions.
[0219] A2: 100 .mu.l of 50 mM MOPS (SIGMA M-9027), 10 mg/ml BSA
(SIGMA A-7030), 0.5 mg/ml CMP-NeuAC (Calbiochem 233263), and 5
milliunits/ml of recombinant .alpha.2,3 (N)-sialyltransferase
(Calbiochem 566218) at pH 7.4 were added to each well of a
microtiter plate and the plate was shaken at 50 RPM at 37.degree.
C. for 4 hours. Following incubation, the reaction mixture was
removed and the wells were washed three times with 200 .mu.l of
TBST, the last wash consisting of a 15 minutes soak. The TBST was
replaced with TBS 0.2% NaN.sub.3 (Sigma S-8032) and the plate was
stored at 4.degree. C. for subsequent enzymatic reactions.
[0220] B2: 100 .mu.l of 50 mM MOPS (SIGMA M-9027), 10 mg/ml BSA
(SIGMA A-7030), 20 mM MnCl.sub.2 (SIGMA M-9522), 0.5 mg/ml GDP-Fuc
(Calbiochem 371443) and 5 milliunits/ml of recombinant .alpha.1,3
fucosyltransferase (Calbiochem 344323) at pH 7.2 were added to each
well of a microtiter plate and the plate was shaken at 50 RPM at
37.degree. C. for 4 hours. Following incubation, the reaction
mixture was removed and the wells were washed three times with 200
.mu.l of TBST, the last wash consisting of a 15 minutes soak. The
TBST was replaced with TBS 0.2% NaN.sub.3 (Sigma S-8032) and the
plate was stored at 4.degree. C. for subsequent enzymatic
reactions.
[0221] D3: 100 .mu.l of 100 mM sodium cacodylate buffer (SIGMA
C-4945), 10 mg/ml BSA (SIGMA A-7030), 20 mM MnCl.sub.2 (SIGMA
M-9522), 0.5 mg/ml UDP-Gal (Calbiochem 670111) and 5 milliunits/ml
of recombinant .alpha.1,3 galactosyltransferase (Calbiochem 345648)
at pH 6.5 were added to each well of a microtiter plate and the
plate was shaken at 50 RPM at 37.degree. C. for 4 hours. Following
incubation, the reaction mixture was removed and the wells were
washed three times with 200 .mu.l of TBST, the last wash consisting
of a 15 minutes soak. The TBST was replaced with TBS 0.2% NaN.sub.3
(Sigma S-8032) and the plate was stored at 4.degree. C. for
subsequent enzymatic reactions.
[0222] A3: 100 .mu.l of 50 mM sodium cacodylate buffer (SIGMA
C-4945), 10 mg/ml BSA (SIGMA A-7030), 0.5 mg/ml CMP-NeuAC
(Calbiochem 233263) and 5 milliunits/ml of Recombinant
.alpha.2,6-(N)-sialyltransferase (Calbiochem 566222) at pH 6.0 were
added to each well of a microtiter plate and the plate was shaken
at 50 RPM at 37.degree. C. for 4 hours. Following incubation, the
reaction mixture was removed and the wells were washed three times
with 200 .mu.l of TBST, the last wash consisting of a 15 minutes
soak. The TBST was replaced with TBS 0.2% NaN.sub.3 (Sigma S-8032)
and the plate was stored at 4.degree. C. for subsequent enzymatic
reactions.
[0223] H3: 100 .mu.L of 50 mM sodium cacodylate buffer (SIGMA
C-4945), 20 mM MnCl.sub.2 (SIGMA M-9522) 10 mg/ml BSA (SIGMA
A-7030), 5% dimethylsulfoxide, 0.5 millimolar UDP-GlcNAc
(Calbiochem 670107), 0.75 millimolar of adenosine three phosphate
and 5 milliunits/ml of recombinant .beta.1,3 N
acetylglucoseaminyl-transferase (prepared as described in Zhou et
al., Proc. Natl. Acad. Sci. USA Vol. 96 pp. 406-411) at pH 7.0 are
added to each well of a microtiter plate and the plate is shaken at
50 RPM at 37.degree. C. for 10 hours. Following incubation, the
reaction mixture is removed and the wells are washed three times
with 200 .mu.l of TBST, the last wash consisting of a 15 minutes
soak. The TBST is replaced with TBS 0.2% NaN.sub.3 (Sigma S-8032)
and the plate are stored at 4.degree. C. for subsequent enzymatic
reactions.
[0224] The BSA of the reaction mixture is omitted and the wash
steps are performed using high ionic strength buffer instead of
TBST when the enzymatic reactions are performed in Covalink NH
plates including a covalently coupled monosaccharide.
[0225] Lectin/Antibody Binding Assays:
[0226] WGA: 100 .mu.l of 5 .mu.g/ml WGA lectin conjugated to
peroxidase (SIGMA L-3892, 150 peroxidase units per mg protein) was
added to the plates and incubated for 1 hour at 25.degree. C. in
TBS containing 1% BSA and 10 millimolar of MnCl.sub.2 and of
CaCl.sub.2. The wells were washed 3 times with 200 .mu.l TBST, with
a last wash consisting of a 15 minutes soak. To detect the
peroxidase labeled WGA, 100 .mu.l of fresh peroxidase substrate
solution was added, and an hour later the O.D. of the solution (at
450 nm) was determined.
[0227] RCA120: 100 .mu.l of 10 .mu.g/ml RCA120 lectin conjugated to
peroxidase (SIGMA L-2758, 11 peroxidase units per mg protein) was
added to the plates and incubated for 1 hour at 25.degree. C. in
TBS containing 1% BSA and 10 millimolar each of MnCl.sub.2 and
CaCl.sub.2. The wells were washed three times with 200 .mu.l TBST
with a last wash consisting of a 15 minutes soak. 100 .mu.l of
fresh peroxidase substrate solution was added, and an hour later
the O.D. of the solution (at 450 nm) was determined.
[0228] TGP: 100 .mu.l of 20 .mu.g/ml TGP lectin conjugated to
peroxidase (SIGMA L-1508, 10 peroxidase units per mg protein) was
added to the plates and incubated for one hour at 25.degree. C. in
TBS containing 1% BSA and 10 millimolar of MnCl.sub.2 and of
CaCl.sub.2. The wells were washed three times with 200 .mu.l TBST
with a last wash consisting of a 15 minutes soak. 100 .mu.l of
fresh peroxidase substrate solution was added, and an hour later
the O.D. of the solution (at 450 nm) was determined.
[0229] BS-I: 100 .mu.l of 20 .mu.g/ml BS-I conjugated to biotin
(SIGMA L-3759) was added to the plates and incubated for 1 hour at
25.degree. C. in TBS containing 1% BSA and 10 millimolar of
MnCl.sub.2 and of CaCl.sub.2. The wells were washed three times
with 200 .mu.l TBST with a last wash consisting of a 15 minutes
soak, following which, 100 .mu.l of TBS containing 1% BSA and 5
.mu.g/ml avidin conjugated to peroxidase (SIGMA A-3151, 40
peroxidase units per mg protein) was added to the plates and
incubated for 1 hour at 25.degree. C. The wells were washed three
times with 200 .mu.l TBST with a last wash consisting of a 15
minutes soak. 100 .mu.l of fresh peroxidase substrate solution was
added, and an hour later the O.D. of the solution (at 450 nm) was
determined.
[0230] TML: 100 .mu.l of 20 .mu.g/ml TML conjugated to biotin
(Calbiochem 431803) was added to the plates and incubated for 1
hour at 25.degree. C. in TBS containing 1% BSA. The wells were
washed 3 times with 200 .mu.l TBST with a last wash consisting of a
15 minutes soak, following which, 100 .mu.l of TBS containing 1%
BSA and 5 .mu.g/ml avidin conjugated to peroxidase (SIGMA A-3151,
40 peroxidase units per mg protein) was added to the plates and
incubated for 1 hour at 25.degree. C. The wells were washed 3 times
with 200 .mu.l TBST with a last wash consisting of a 15 minutes
soak. 100 .mu.l of fresh peroxidase substrate solution was added,
and an hour later the O.D. of the solution (at 450 nm) was
determined.
[0231] Anti-Sialyl Lewis X: 100 .mu.l of 10 .mu.g/ml anti-Sialyl
Lewis X IgM from mouse (Calbiochem 565953) was added to the plates
and incubated for 1 hour at 25.degree. C. in TBS containing 1% BSA.
The wells were washed 3 times with 200 .mu.l TB ST with a last wash
consisting of a 15 minutes soak, following which, a solution of TBS
containing 1% BSA and 100 .mu.l of 5 .mu.g/ml goat anti-mouse IgM
conjugated to biotin (SIGMA b-9265) was added to the plates and
incubated for one hour at 25.degree. C. The wells were washed 3
times with 200 ml TBST with a last wash consisting of a 15 minutes
soak, followed by incubation with 100 ml of TBS containing 1% BSA
and 5 .mu.g/ml avidin conjugated to peroxidase (SIGMA A-3151, 40
peroxidase units per mg protein) for 1 hour at 25.degree. C. The
wells were washed three times with 200 .mu.l TBST with a last wash
consisting of a 15 minutes soak. 100 .mu.l of fresh peroxidase
substrate solution was added, and an hour later the O.D. of the
solution (at 450 nm) was determined.
[0232] ECorA: 100 .mu.l of 20 .mu.g/ml ECorA conjugated to biotin
(SIGMA L-0893) were added to the plates and incubated for 1 hour at
25.degree. C. in TBS containing 1% BSA and 10 millimolar of
MnCl.sub.2 and of CaCl.sub.2. The wells were washed three times
with 200 .mu.l TBST with a last wash consisting of a 15 minutes
soak, following which, 100 .mu.l of TBS containing 1% BSA and 5
.mu.g/ml avidin conjugated to peroxidase (SIGMA A-3151, 40
peroxidase units per mg protein) was added to the plates and
incubated for 1 hour at 25.degree. C. The wells were washed three
times with 200 .mu.l TBST with a last wash consisting of a 15
minutes soak. 100 .mu.l of fresh peroxidase substrate solution was
added, and an hour later the O.D. of the solution (at 450 nm) was
determined.
[0233] When the above described binding assays were performed on
covalently coupled monosaccharide acceptors, such as the case with
Covalink NH plates, the TBS incubation solution was replaced with
TBST and the wash steps were performed using high ionic strength
buffer instead of TBST.
Example 10
Acceptor Immobilization--the First Step
[0234] While reducing the present invention to practice several
methods for monosaccharide acceptor immobilization to microtiter
plates were perfected. The Monosaccharide acceptors were bound to a
microtiter plate surface via a linker and lectins or antibodies
directed against the complex carbohydrates synthesized were
utilized to measure binding efficiency as is further described
hereinabove.
[0235] Plate immobilization of the first monosaccharide acceptor
building block was performed by either (i) adsorption of
neoglycoprotein BSA-GlcNAc (.beta.-D-GlcNAc conjugated to BSA) to
the surface of a microtiter plate; or (ii) covalent immobilization
using an appropriate linker. Covalent immobilization was performed
using either cyanuric chloride activation (FIGS. 5a and 5b), or
NHS/EDC activation (FIG. 6) of CovaLink NH plates. The use of
cyanuric chloride activation enabled linker elongation of about 1.5
nanometer in each elongation cycle. The presence of the immobilized
monosaccharide (GlcNAc) was measured according to lectin binding
using WGA lectin conjugated to peroxidase or FITC. Binding was
quantitated using either colorimetric or fluorescent signal
detection as shown in FIGS. 7a-c.
[0236] Materials and methods:
[0237] Adsorption of GlcNAc Conjugated to BSA to Maxisorb
Plates:
[0238] A solution of 0.1 M Na.sub.2CO.sub.3 pH 9.6, including
0-3000 ng of BSA-GlcNAc (prepared as described by Monsigny et al.,
Biol. Cell, 51, 187 1984) was aliquoted in 100 .mu.l aliquots into
wells of a Maxisorb microtiter plate (NUNC Cat. No. 469914) and the
plate was incubated at 4.degree. C. for 16 hours. Following
incubation, the solution was removed and 200 .mu.l of 0.1 M
Na.sub.2CO.sub.3 pH 9.6 including 1% BSA was added to each well and
the plate was incubated for an additional 2 hours in order to block
nonspecific binding of proteins (such as enzymes or lectins) to the
well surface. Following blocking, the BSA-GlcNAc solution was
replaced with TBS buffer/0.2% NaN.sub.3/BSA 1% and the plate was
stored at 4.degree. C.
[0239] The adsorption of BSA-GlcNAc was verified peroxidase
conjugated WGA as described hereinabove.
[0240] Results:
[0241] The surface area of a single 66 kDa BSA molecule
(approximately 50 nm.sup.2) is covered with approximately 25 GlcNAc
groups. On a Maxisorb surface which is fully coated with
BSA-GlcNAc, the average distance between adjacent GlcNAc groups is
approximately 1.0 nm and the diameter of the lectin's carbohydrate
recognition site is about 2 nm. Thus, in order to prevent steric
interference between adjacently bound lectins or between bound
lectin and other monosaccharides, the density of the immobilized
monosaccharides should not exceed 10.sup.14 per cm.sup.2. Since in
this case, the immobilized monosaccharide forms a part of a bound
protein molecule which is approximately 5 nm in diameter, the
monosaccharide groups are positioned approximately 5 nanometers
above the plate surface and thus are available for enzymatic
elongation by the glycosyltransferase. FIG. 8a describes a
saturation curve for BSA-GlcNAc bound to a Maxisorb microtiter
plate.
[0242] Following BSA-GlcNAc binding, plates were washed with TBST,
a buffer that contains a medium ionic strength detergent (0.15 M
NaCl), in order to remove non-specifically bound lectins. This wash
step does not remove the bound BSA and thus allows sequential
enzymatic reactions. As shown in FIG. 8b, the bound BSA was stable
throughout 12 extensive washing cycles. Following each wash step,
the amount of BSA-GlcNAc (starting at 200 ng/well) was measured by
WGA lectin binding as described above.
[0243] Covalent immobilization of GlcNAc to Covalink NH, as well as
the elongation of Covalink NH with 13 additional atoms in each
subsequent elongation cycle was performed as shown in FIGS. 5a-b.
Binding results for cyanuric chloride mediated immobilization of
WGA to .beta.-D-GlcNAc (with a single elongation cycle) or to
NHS/EDC activated CovaLink NH are shown in FIGS. 7a-b. The density
of the amino groups on the Covalink NH surface is 10.sup.14 per
cm.sup.2 and the average distance between the GlcNAc groups is 1 nm
which is sufficient for lectin binding. Following incubation with
reaction mixture D7 (.beta.1,4 galactosyltransferase, described in
FIG. 8c), the transfer of .beta.-D-galactose to the plate
immobilized phenyl-.beta.-D-GlcNAc (22 atom linker) is verified
using ECorA lectin binding assay as described above. The transfer
of .beta.-D-galactose to the plate immobilized .beta.-D-GlcNAc
(NHS/EDC activated plate with a 20 atom linker) was not detected.
This might be due to differences in linker length.
[0244] The above described covalent immobilization methods enable
the use of a very high ionic strength buffer (e.g., 6 M Guanidine
HCl or 100 mM NaOH) in subsequent washing steps thus allowing
accurate "in situ" verification of each enzymatic step utilized by
the process.
[0245] The removal of nonspecifically bound molecules is crucial
for accurate library synthesis. Since glycosyltransferases are
glycoproteins with complex carbohydrates presented on their outer
surface, nonspecific adsorption of the enzyme may interfere with,
or generate errors in, the synthesis. Lectins and antibodies are
also glycoproteins and as such non-specific binding thereof may
lead to inaccurate structural prediction. The standard blocking
agent commonly used in enzymatic reactions, is nonfat milk. Since
nonfat milk contains many glycoconjugated proteins it is not
suitable for enzymatic synthesis of complex carbohydrates. Instead,
synthesis reactions performed according to the present invention
utilized BSA as a blocking agent since it is non-glycosylated. As
shown by FIG. 7b, while chemical blocking agents interfered with
lectin binding, blocking with BSA enabled specific lectin binding
while substantially reducing non-specific binding. During practice,
it was realized that since cyanuric chloride activated amino groups
hydrolyze spontaneously in water there is no need for further
blocking when using this plate coupling procedure.
Example 11
Library Synthesis
[0246] High yield is critical to an iterative solid phase enzymatic
synthesis. Since glycosyltransferases catalyze the transfer of a
sugar moiety from an activated nucleotide phosphate sugar donor to
an appropriate acceptor, degradation of the phosphodiester
energetic bond of the nucleotide sugar is irreversible. As such,
there is no theoretical obstacle hindering the completion of the
synthesis reactions. Preferably, the nucleotide sugar concentration
of the reaction should be approximately 10 to 20 fold higher than a
K.sub.m value of the enzyme to the donor, which ranges from several
to several hundred millimolars. In a single microtiter plate well
which contains approximately 100 .mu.l of solution, 0.2 nanomoles
of well-bound saccharide groups are available for the enzymatic
reaction. As such, a nucleotide sugar concentration equal to or
greater than 1 millimolar is sufficient.
[0247] Materials and Methods:
[0248] NHS/EDC activation and coupling of .beta.-D-GlcNAc: 50 .mu.l
aliquots of a 2-(2-carboxyethylthio)-ethyl 2-.beta.-D-GlcNAc (NNI
SS-01-003) solution were dispensed onto CovaLink NH strips and the
strips were incubated in wells containing 50 .mu.l of a solution
including 3 mg/ml of 1-Ethyl-3-(3dimethylaminopropyl)-carbodiimide
(EDC) (Sigma E-7750) and 3 mg/ml of N-hydroxysuccinimide (NHS)
(Sigma H-7377). The wells were sealed and the plates were shaken at
50 RPM at 37.degree. C. for 24 hours. The wells were washed three
times with distilled water and the unreacted amino groups in the
wells were blocked for 2 hours in 300 ml of a solution containing
methanol/aceticanhydride/water (85/10/5 V/V/V, respectively).
Following four washes with distilled water the plates were air
dried and incubated for 12 hours with a blocking solution which
included 1% BSA in PBS.
[0249] Synthesis of a Cleavable Linker, Coupling the First
Monosaccharide Thereto, and Cleaving the Linker:
[0250] Cyanuric chloride activation: A solution containing 48 mg of
cyanuric chloride (Aldrich, Cat. No. C95501) dissolved in 3 ml of
acetone was added, while stirring, to 45 ml of 0.1 M phosphate
buffer. An aliquot (200 .mu.l) of this solution was quickly added
(within 2 minutes) to each well of a Covalink NH plate. The plate
was incubated at room temperature for 5 minutes following which the
solution was discarded and the plate washed three times with double
distilled water and dried at 50.degree. C. for 30 minutes.
[0251] Amino linker elongation cycle: 100 .mu.l of a 1,8-diamino
3,6 (MERCK 818116) solution (3 ml in 50 ml 0.1 M carbonate buffer
pH 9.6) or 1,8 diaminooctane (ALDRICH D2, 240-1) solution (100 mg
per ml 0.1 M carbonate buffer pH 9.6) was added to each well of a
cyanuric chloride activated plate. The wells were sealed and the
plate was incubated at 25.degree. C. for 12 hours. Following
incubation, the wells were washed four times with water and the
plate was cyanuric chloride activated as described above. Three
elongation cycles of 1,8-diamino 3,6 dioxaoctane were executed
until the desired linker length was achieved.
[0252] Coupling of p-nitrophenyl-.beta.-D-GlcNAc (first
monosaccharide building block): GlcNAc monosaccharide molecules
were linked to the activated plates described above. The following
procedure was utilized to effect linking: a solution containing 60
mg/ml sodium dithionite in 0.1M sodium carbonate was added to each
well of rows B-H of the plate. A 200 .mu.l aliquot of a second
solution containing 20 mg of p-nitrophenyl-N-acetyl-.beta.-D-GlcNAc
(Calbiochem Cat. No. 487052) and 200 mg of sodium dithionite (Fluka
Cat. No. 71700) which were dissolved in 6 ml of double distilled
water and titrated to a pH of 7.5 using 3 ml of 0.1 M sodium
carbonate (pH 9.6) was serially diluted two folds from rows A to H.
The wells were sealed and incubated at room temperature overnight.
Following incubation, the wells were washed four times with double
distilled water and then three with methanol (200 .mu.l/well), the
third wash including a 15 minutes soak at room temperature. The
methanol was discarded, and the plates were air dried and stored at
4.degree. C.
[0253] Coupling of Squaric acid derivative of -D-GlcNAc (first
monosaccharide building block): GlcNAc-Squaric acid derivative was
prepared as follows: A solution of 100 mg D-GlcNAc (Calbiochem
346299), 31.25 mg ammonium bicarbonate (Merck 1.05426.1000) in 1.9
ml of ammonia was incubated at 35 C for 24 hours. The solution was
concentrated in a vacuum centrifuge, water was added and the
solution re-concentrated to 0.45 ml. To this solution 72 .mu.l of
squaric acid (Across Orgencis 30508-0010), 1.6 ml ethanol, 1.6 ml
of 0.1 M Na.sub.2CO.sub.3, pH 9.6, were added. The resulting
solution was incubated for 3 hours at 25 C and concentrated in a
vacuum centrifuge to eliminate the ethanol. The coupling of
GlcNAc-Squaric acid derivative was affected by incubation of 100
.mu.l of GlcNAc-Squaric acid derivative (10 .mu.mol) in 1 ml of
0.1M Na.sub.2CO.sub.3, pH 9.6, with an elongated amine linker (FIG.
5b) for 2 hours followed by washing with methanol. This was
performed in each well of a microtiter plate. Following enzymatic
synthesis of carbohydrates the linker can be cleaved by incubation
with aqueous solution of bromine (0.3 mmol bromine in 4 ml water)
for 30 minutes, and the removed glycan can be transformed to a
reducing sugar by the addition of 15 .mu.l of 0.2 M aquenuse sodium
borate (see below).
[0254] Binding of WGA to covalently coupled .beta.-D-GlcNAc: The
presence of covalently bound .beta.-D-GlcNAc was verified by
binding of WGA conjugated to peroxidase or
fluoresceine-iso-thio-cyanate (FITC). Detection was performed as
follows: 100 .mu.l of 5 .mu.g/ml of peroxidase (SIGMA L-3892, 150
peroxidase units per mg protein) or FITC (SIGMA L-4895) conjugated
WGA prepared in TBST including 10 millimolar of MnCl.sub.2 and of
CaCl.sub.2 was incubated in each well at 25.degree. C. for one
hour. The wells were washed three times with 200 .mu.l high ionic
strength washing solution, with the last wash consisting of a 15
minutes soak. To develop the peroxidase labeled WGA, 100 .mu.l of
fresh peroxidase substrate solution was added and an hour later an
O.D. at 450 nm was measured. The FITC conjugated WGA bound to the
.beta.-D-GlcNAc-white Covalink NH strips (NUNC 453690) was excited
(485 nm) and the fluorescence emission therefrom was measured (520
nm).
[0255] Transferring of .beta.1,4 galactose to covalently bound
.beta.-D-GlcNAc: 100 .mu.l of 50 mM MOPS pH 7.4 (SIGMA M-9027),
0.2% Triton CF 32 (Sigma), 20 mM MnCl.sub.2 (SIGMA M-9522), 0.5
mg/ml UDP-Gal (Calbiochem 670111) and 20 milliunits/ml of a
recombinant .beta.1,4-galactosyltransferase (Calbiochem 345650)
were added to each well of a plate coupled with .beta.-D-GlcNAc.
The plate was shaken at 50 RPM at 37.degree. C. for 12 hours.
Following incubation the reaction mixture was removed and the wells
were washed three times with 200 .mu.l of high ionic strength
washing buffer, the last wash consisting of a 15 minutes soak. The
transfer of .beta.1,4-galactose was detected via biotin conjugated
lectin (Erythourina corallodenron ECorA) as follows: an aliquot
including of 20 .mu.g/ml ECorA conjugated to biotin (SIGMA L-0893,
5 moles of biotin per mole protein) prepared in TBST including 10
millimolar of MnCl.sub.2 and of CaCl.sub.2 was added to each well
and the plate was incubated for one hour at 25.degree. C. The wells
were washed 3 times with 200 .mu.l of high ionic strength washing
buffer, the last wash consisting of a 15 minutes soak. Following
incubation with 100 ml of avidin conjugated to peroxidase (5
.mu.g/ml in TBST) the wells were washed 3 times with 200 .mu.l of
high ionic strength washing buffer, the last wash consisting of a
15 minutes soak. To detect binding, 100 .mu.l of fresh peroxidase
substrate solution was added and an hour later, an O.D. at 450 nm
was measured.
[0256] Measuring the kinetics of .beta.1,4 Galactosyltransferase
(D7) in solid phase: The enzymatic reaction mixture is as described
for D7 with the exception that in this case 3.9 milliunits/ml of
.beta.1,4-Galactosyltransferase were utilized. The solid phase
consisted of Maxisorb plates coated with 3 .mu.g/well of
BSA-GlcNAc. The enzyme mixture was added to each well at 10 minutes
intervals. The wells were then washed with TBST three times and the
binding of RCA.sub.120 was measured as described above.
[0257] Linker Cleavage: The above described linker is cleavable by
bromine. Linker cleavage was therefore effected by the addition of
100 .mu.l aqueous solution of bromine (0.3 mmol bromine in 4 ml
water) into the wells and incubation for a time period of 60
minutes, followed by washing of the wells three times with TBST. To
this end, 100 .mu.l of 0.5 g/ml WGA/FITC in TBST was added to each
well. Following one hour incubation, the wells were washed three
times with a high salt buffer and fluorescence was measured
(Excitation--485 nm; Emission--520 nm). FIG. 13 shows the reduction
in binding of WGA/FITC to GlcNAc following the above procedure.
[0258] Results:
[0259] Results obtained while reducing the present invention to
practice indicate that a solid phase reaction is slower than a
liquid phase reaction. As shown in FIG. 12, enzymatic transfer of
galactose to 0.5 nanomole of bound GlcNAc using 0.5 mU of
.beta.-1,4 galactosyltransferase takes approximately an hour to
complete, as compared to approximately one minute it takes to
complete the same reaction in solution. Therefore, to achieve
maximum yield, 0.5 milliunits of each enzyme were employed for 3-4
hours.
[0260] Nucleotide phosphates (UDP, CMP, GDP) which result from the
break down of nucleotide sugar are by-products of these enzymatic
reactions. It was observed that these by-products inhibit
glycosyltransferase activity. As such, an addition of a phosphatase
to degrade the released nucleotide phosphate(s) can substantially
increase the rate of the solid phase reaction.
[0261] Linker length, flexibility of the complex carbohydrate,
immobilization of carbohydrate groups and steric hindrance are also
important factors effecting synthesis efficiency. As uncovered by
experimentation conducted as part of the present study, a
neoglycoprotein coated Maxisorb surface can be efficiently utilized
to immobilize the first monosaccharide, obtain the
glycosyltransferase enzymatic reaction and avoid steric hindrance
problems. An elongated covalent linker based on cyanuric acid and
p-Nitro phenyl enables coupling of the first monosaccharide to a
2-8 nanometer linker thus avoiding steric hindrance when the first
monosaccharide is covalently bound to the surface and obtain the
glycosyltransferase enzymatic reaction.
[0262] The reduction in fluorescence following incubation with
bromine indicates the cleavage of the linker. The ability to cleave
the linker and remove the glycan after the sequential synthesis is
crucial for structural analysis and verification of the synthesized
oligosaccharide by, for example, mass spectroscopy or high
performance liquid chromatography.
Example 12
Library 1
[0263] FIG. 9 describes the enzymatic steps required for the
synthesis of a library consisting of the structures described in
Table 17 immobilized to a plate, outlining the organization of the
microtiter plate, the enzymatic reactions performed at each step
and the lectins/antibody binding assays. Each enzymatic step is
verified against a control strip which does not contain the added
nucleotide sugar.
[0264] A different set of enzymatic reactions (described in detail
hereinabove) were performed in each strip in accordance with the
procedures developed by the present invention. The Tables below
describe in detail the various reactions and components utilized in
order to generate this library. Table 16 summarizes the enzymatic
reactions used to synthesize the first library, Table 17 describes
the enzymatic modules (EM's, further described in Examples 1-9),
the complex carbohydrate structures formed and the lectin/antibody
binding assays performed for each strip, while Table 18 describes
the lectins/antibodies binding assays that were used to verify the
complex carbohydrate structure formed following each enzymatic
step.
22TABLE 16 Enzymatic reactions utilized in the synthesis of the
first library (donors, acceptors and indexes) index extension /
Pos. acceptor donor Enzyme Cat. No. E.C. A2 3
(1,4)-D-GlcNAc-RD-Gal- CMP-NeuAC Calbiochem 56621 2.4.99.6 A3 6
(1,4)-D-GlcNAc-RD-Gal- CMP-NeuAC Calbiochem 56622 2.4.99.1 B2
(1,4)D-Gal- 3 D-GlcNAc-R GDP-L-Fuc Calbiochem 34432 2.4.1.152 D3 3
(1,4)-D-GlcNAc-R D-Gal- UDP-Gal Calbiochem 34564 2.4.1.151 D7 4
D-GlcNAc-R UDP-Gal Calbiochem 34565 2.4.1.38
[0265]
23TABLE 17 EMs List First Immobilized ERs Lectin/Antibody EM
Monosaccharide sequence Structure Formula Binding Assays 1 GlcNAc-S
D7 1,4 GlcNAc-SGal RCA120+ 2 GlcNAc-S D7, A2 1,4 GlcNAc-S 2,3 Gal
NeuAC TML+, RCA120- 3 GlcNAc-S D7, A3 1,4 GlcNAc-S 2,6 Gal NeuAC
TML+, RCA120- 4 GlcNAc-S D7, D3 1,4 GlcNAc-S 1,3 Gal Gal BS-I+,
RCA120- 5 GlcNAc-S D7, B2 1,3)GlcNAc-S 1,4 (Fuc Gal TGP+ 6 GlcNAc-S
D7, A2, B2 1,3)GlcNAc-S 1,4 (Fuc 2,3 Gal NeuAC IgM Anti-Sialyl
Lewis X+
[0266]
24TABLE 18 Lectin/Antibody binding assays Type of Name Molecule
Specificity Cat. No. Source Labeling WGA Lectin GlcNAc Sigma L-3892
Tritcum vulgaris Peroxidase/FITC RCA120 Lectin -Gal Sigma L-2758
Ricinus communis Peroxidase BS-I Lectin -GalNAc-Gal, Sigma L-3759
Bandeiraea Simplicifolia Biotin TGP Lectin -Fuc Sigma L-1508
Tereagonolobus purpureas Peroxidase TML Lectin Sialic acid Calbio.
431803 Tritrichomonas mobilensis Biotin Anti-Sialyl IgM Sialyl
Lewis X Calbio. 565953 Mouse Goat anti mouse/ LewisX Peroxidase
[0267] Results:
[0268] FIGS. 10a-f describe the lectins/antibodies binding assays
performed on each strip following each enzymatic reaction. The
increase in binding of RCA.sub.120 (FIG. 10a) indicates transfer of
.beta.-D-Galactose to GlcNAc and formation of a stable .beta.-1,4
glycosidic bond. The efficiency of the second enzymatic step was
verified via an increase in BS-I binding (FIG. 10b) which is
indicative of a transfer of .alpha.-D-Galactose to Gal .beta.-1,4
GlcNAc and formation of stable .alpha.-1,3 glycosidic bond
therebetween. The increase in TGP binding (FIG. 10c) is indicative
of a transfer of .alpha.-L-fucose to Gal .beta.-1,4 GlcNAc and
formation of a stable .alpha.-1,3 glycosidic bond therebetween. The
branched oligosaccharide formed by this reaction is a Lewis X
antigen. As shown by FIGS. 10d-e an increase in TML binding
indicates a transfer of .alpha.-D-NeuAC to Gal .beta.-1,4 GlcNAc
forming a stable .alpha.-2,6-2,3 glycosidic bond therebetween. The
increase in anti sialyl Lewis X IgM binding (FIG. 10f) indicates a
transfer of .alpha.-L-fucose to NeuAC .alpha.-2,3 Gal .beta.-1,4
GlcNAc forming a stable .alpha.-1,3 glycosidic and generating
Sialyl Lewis X antigen composed of four different
monosaccharides.
Example 13
Library 2
[0269] The following library exemplifies the ability of the
synthesis method of the present invention to synthesize poly
N-acetyllactose amine type II chain of different lengths. FIG. 11
describes the organization of the microtiter plate, the enzymatic
reactions to be performed in each step and the lectins binding
assays that can be used to verify the efficiency of the various
enzymatic steps. Each enzymatic step is verified against a control
strip which does not contain the added nucleotide sugar. Each strip
is subjected to a different set of enzymatic reactions (Enzymatic
Module--EM) which are performed according to the procedures
developed by the present invention.
[0270] Table 19 describe the enzymatic reaction mixes and
conditions (described in detail hereinabove) that are used for
synthesis of Poly N-acetyllactoseamine library in accordance with
the teachings of the present invention. Table 20 describes the
Enzymatic Modules (EM's) and the complex carbohydrate structures.
RCA.sub.120 binding assay is performed after each enzymatic step as
described above to evaluate the addition of Galactose (RCA.sub.120
binding) or GlcNAc (disappearance of RCA.sub.120 binding) to the
elongating poly N-acetyllactoseaminide chain.
25TABLE 19 A list of Enzymatic Reactions used for synthesis of Poly
N- acetyllactoseaminide library(donors, acceptors and indexes)
index extension / Pos. acceptor donor Enzymes Cat. No. E.C. D7 4
D-GlcNAc-R UDP-Gal Calbiochem 345650 2.4.1.38 H3 3
D-Gal-(1,4)-D-GlcNAc-R UDP-GlcNAc Zhou et al., Proc. Natl. Acad.
Sci. USA Vol. 96 pp. 406-411
[0271]
26TABLE 20 EM List RCA120 Binding EM ERs sequence Structure Formula
Assays 1 D7, H3 GlcNAc 1,3 Gal 1,4 GlcNAc-S RCA120- 2 D7, H3, D7
Gal 1,4 GlcNAc 1,3 Gal 1,4 RCA120+ GlcNAc-S 3 D7, H3, D7, H3 GlcNAc
1,3 Gal 1,4 GlcNAc RCA120- 1,3 Gal 1,4 GlcNAc-S 4 D7, H3, D7, H3,
Gal 1,4 GlcNAc 1,3 Gal 1,4 RCA120+ D7 GlcNAc 1,3 Gal 1,4 GlcNAc-S 5
D7, H3, D7, H3, GlcNAc 1,3 Gal 1,4 GlcNAc RCA120- D7, H3 1,3 Gal
1,4 GlcNAc 1,3 Gal 1,4 GlcNAc-S 6 D7, H3, D7, H3, Gal 1,4 GlcNAc
1,3 Gal 1,4 RCA120+ D7, H3, D7 GlcNAc 1,3 Gal 1,4 GlcNAc 1,3 Gal
1,4 GlcNAc-S
Example 14
Library 3
[0272] The following library exemplifies the ability of the
synthesis method of the present invention to synthesize poly
N-acetyllactose amine type II chain of different lengths and
modifications. This library include oligosaccharide structures with
two branches. The first monosaccharide is bound to the surface via
BSA. Table 21 describes the enzymatic reactions that are utilized
for the synthesis, while Table 22 describes the enzymatic modules
(EM's) utilized and the complex carbohydrate structures formed
thereby. To verify the accuracy of the sequential enzymatic
synthesis, the oligosaccharide bound to the well is released using
a protease and subjected to analysis using HPLC, methylation
analysis or MALD-TOF-MS (Rudd, P. M. Dwek, R. A. (1997) Current
Opinion in biotechnology 8 488-497).
27TABLE 21 Enzymatic reactions utilized in the synthesis of library
3 index extension / Pos. acceptor donor Enzymes Cat. No. E.C. D7 4
D-GlcNAc-R UDP-Gal Calbiochem 345650 2.4.1.38 A2 3
D-Gal-(1,4)-D-GlcNAc-R CMP-NeuAC Calbiochem 566218 2.4.99.6 B2
D-Gal-(1,4) 3 D-GlcNAc-R GDP-L-Fuc Calbiochem 344323 2.4.1.152 H3 3
D-Gal-(1,4)-D-GlcNAc-R UDP-GlcNAc Zhou et al., Proc. Natl. Acad.
Sci. USA Vol. 96 pp. 406-411
[0273]
28TABLE 22 EM List EM ERs sequence Structure Formula 1 D7, H3, D7,
B2 Gal 1,4 (Fuc 1,3) GlcNAc 1,3 Gal 1,4 (Fuc 1,3) GlcNAc-S 2 D7,
H3, D7, A2, B2 NeuAC 2,3 Gal 1,4 (Fuc 1,3) GlcNAc 1,3 Gal 1,4 (Fuc
1,3) GlcNAc-S
[0274] Thus, the present invention provides an efficient and
accurate method for a solid phase synthesis of complex
carbohydrates of branched or unbranched structures.
[0275] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
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