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Title:
NOVEL BINDING EPITOPES FOR HELICOBACTER PYLORI AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2004/065400
Kind Code:
A1
Abstract:
The present application describes binding epitopes to Helicobacter pylori, namely substances comprising oligosaccharide sequences Hex2(NAc)β4Glc(A)(NAc) and Hex1(A)(NAc)&agr /β3Hex2(NAc) (where Hex1 = Gal, Glc or Man, Hex2 = Gal or Glc, the contents of the brackets are optional, and GlcA is a glucuronic acid derivative), and the use thereof in pharmaceutical and nutritional compositions for the treatment of conditions due to the presence of Helicobacter pylori in a subject, e.g. gastritis, gastric ulcers, gastric adenocarcinoma, liver disease, pancreatic disease, skin disease, heart disease, autoimmune diseases and sudden infant death syndrome. The invention is also directed to the use of the receptor for diagnostics of Helicobacter pylori, to a method of producing chondroitin oligosaccharides from chondroitin sulphates, and a method for production of amidated glucuronic acid comprising oligosaccharides and monosaccharides from glucuronic acid comprising polysaccharides.

Inventors:
Miller-podraza, Halina (Lantmilsgatan 20, Västra Frölunda, S-42137, SE)
Teneberg, Susann (Postbox 1639, Hindas, S-43063, SE)
Ångström, Jonas (de Geersgatan 12, Göteborg, S-41657, SE)
Karlsson, Karl-anders (Nilssonsberg 37, Göteborg, S-41143, SE)
Natunen, Jari (Oolannintie 10 E 18, Vantaa, FIN-01520, FI)
Helin, Jari (Askistontie 2B, Vantaa, FIN-01680, FI)
Application Number:
PCT/FI2004/000027
Publication Date:
August 05, 2004
Filing Date:
January 20, 2004
Export Citation:
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Assignee:
GLYKOS FINLAND OY (Viikinkaari 4, Helsinki, FI-00790, FI)
Miller-podraza, Halina (Lantmilsgatan 20, Västra Frölunda, S-42137, SE)
Teneberg, Susann (Postbox 1639, Hindas, S-43063, SE)
Ångström, Jonas (de Geersgatan 12, Göteborg, S-41657, SE)
Karlsson, Karl-anders (Nilssonsberg 37, Göteborg, S-41143, SE)
Natunen, Jari (Oolannintie 10 E 18, Vantaa, FIN-01520, FI)
Helin, Jari (Askistontie 2B, Vantaa, FIN-01680, FI)
International Classes:
A61K31/702; A61K31/7028; A61K31/715; A61K31/726; A61P1/04; A61P1/16; A61P1/18; A61P9/00; A61P17/00; A61P31/04; A61P35/00; A61P37/00; C07H1/00; C07H3/06; C07H15/04; (IPC1-7): C07H15/04; A61K31/702; A61K31/7028; A61K31/715; A61K31/726; A61P1/04; A61P1/16; A61P1/18; A61P9/00; A61P17/00; A61P31/04; A61P35/00; A61P37/00; C07H1/00; C07H3/06
Attorney, Agent or Firm:
OY JALO ANT-WUORINEN AB (Iso Roobertinkatu 4-6 A, Helsinki, FI-00120, FI)
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Claims:
What is claimed:
1. A Helicobacterpylori binding substance comprising an oligosaccharide sequence [Hex 1 (A) ql (NAc) ria/p3], jHex2 (NAc) r2 [p4Glc (A) q2 (NAc), 3] s2 wherein ql, q2, rl, r2, r3, sl and s2 are each independently 0 or 1, so that at least r2 or q2 is 1, at least sl or s2 is 1, or Hex2 is Glc, with the proviso that when s2 is 0, then Hexl is Glc and preferably rl is 0; Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc ; and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for the prophylaxis or treatment of any condition due to the presence of Helicobacterpylori in a subject.
2. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Hex 1 (A) ql (NAc) rl alß3] slHex2 (NAc) r2ß4Glc (A) q2 (NAc) r3 wherein ql, q2, rl, r2, r3, and sl are each independently 0 or 1, so that at least r2 or q2 is 1; Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc.
3. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Hexl (A) ql (NAc) rlα/ß3]slGal(NAc)r2ß4Glc (A) q2 (NAc) r3 wherein ql, q2, rl, r2, r3, and sl are each independently 0 or 1, so that at least r2 or q2 is 1 ; Hexl is galactose (Gal), glucose (Glc) or mannose (Man).
4. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Glc (A) qip3] siHex2 (NAc) r2p4Glc (A) q2 wherein ql, q2, r2, and sl are each independently 0 or 1, so that at least r2 or q2 is 1; Hex2 is Gal or Glc.
5. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Glc (A) qlß3]slHex2NAcß4Glc (A) q2 wherein ql, q2, and sl are each independently 0 or 1 ; Hex2 is Gal or Glc.
6. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Glc (A) qip3LiGalNAcp4Glc (A) q2 wherein ql, q2, and sl are each independently 0 or 1.
7. A Helicobacter pylori binding substance according to claim 1 comprising oligosaccharide sequence [Glc (A) qlß3]slGlcNAcß4Glc (A) q2 wherein ql, q2, and sl are each independently 0 or 1.
8. The Helicobacter pylori binding substance according to claim 1 further comprising ß6Hex3 (A) r4 (NAc) rs or (33Hex3 (A) r4 (NAc) r5 structure in the reducing end of the oligosaccharide sequence forming the following structure [Hex 1 (A) ql (NAc) ria/p3] siHex2 (NAc) r2 [p4Glc (A) q2 (NAc) r3] s2p6/p3Hex3 (A) r4 (NAc) r 5 wherein ql, q2, rl, r2, r3, sl, s2, Hexl and Hex2 are as defined in claim 1; r4 and r5 are independently 0 or 1 ; Hex3 is mannose (Man), galactose (Gal) or glucose (Glc).
9. The Helicobacter pylori binding substance according to claim 1 further comprising p3Gal (NAc)r5[ß4Glc (A) q3 (NAc) r6] u structure in the reducing end of the oligosaccharide sequence forming the following structure [Hexl (A) ql (NAc) rlα/ß3]slHex2(NAc)r2[ß4Glc (A) q2 (NAc) r3] 2p3Gal (NAc) b [ß4Glc (A) q3 (NAc ) r6] u wherein ql, q2, rl, r2, r3, sl, s2, Hexl and Hex2 are as defined in claim 1; r5 is as defined in claim 8, q3, r6, and u are independently 0 or 1.
10. The Helicobacter pylori binding substance according to claim 1 further comprising p6 {B} Hex4 (NAc) rs structure in the reducing end of the oligosaccharide sequence forming the following structure [Hexl (A) ql (NAc) ria/p3] siHex2 (NAc) r2 [ 34Glc (A) q2 (NAc) r3] 2p6 {B} Hex3 (NAC), 5 wherein ql, q2, rl, r2, r3, sl, s2, Hexl and Hex2 are as defined in claim 1; B is branch structure Hex3 (NAc) r4p3, Hex3 and Hex 4 are independently mannose (Man), galactose (Gal) or glucose (Glc), r5 is independently 0 or 1.
11. The Helicobacter pylori binding substance according to claim 1 further comprising ß6{B]Hex4 (NAc) rs [a4Glc (A) q3 (NAc) r6] u structure in the reducing end of the oligosaccharide sequence forming the following structure [Hexl (A) ql (NAc) rlα/ß3]slHex2 (NAc) r2 [ß4Glc (A) q2 (NAc) ß] s2ß6 {B} Hex4 (NAc)r5[ß4Glc (A) q3 (NAc) r6 ] u wherein ql, q2, rl, r2, r3, sl, s2, Hexl and Hex2 are as defined in claim 1; B and Hex4 is as defined in claim 10; q3, r5, r6, and u are independently 0 or 1.
12. The Helicobacter pylori binding substance according to claim 1, wherein said oligosaccharide sequence is a natural type chondroitin sequence according to the following structure [GlcAß3]slGalNAcß4GlcA[ß3GalNAc]u wherein s 1 and u are independently 0 or 1 with the proviso that either s or u is 1.
13. The Helicobacterpylori binding substance according to claim 1, wherein said oligosaccharide sequence is a natural type chondroitin sequence according to the following structure [GlcAp3] siGlcNAcp4GlcA [p3GlcNAc] u wherein s 1 and u are independently 0 or 1 with the proviso that either s or u is 1.
14. The Helicobacter pylori binding substance according to claim 1 comprising one or several of the following oligosaccharide sequences: Glcß3GalNAcß4Glc, Glcp3GalNAcp4Glcp3GalNAcp4Glc, Glcß3GalNAcß4Glcß3NAcß4Glcß3GalNAcß4Glc, Glcß3GalNAcß4Glcß3NAc, Glcß3GalNAcß4Glcß3NAcß4Glcß3GalNAc, Glcp3GalNAcp4Glcp3GalNAcp4Glcp3GalNAcp4Glcp3GalNAc ; Glcß3GlcNAcß4Glc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAcß4Glc, Glc3GlcNAcp4Glcp3GlcNAc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAc, and Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAc.
15. The Helicobacter pylori binding substance according to claim 1 comprising one or several of the following oligosaccharide sequences GlcAp3GalNAcp4GlcA, GlcAß3GalNAcß4GlcAß3GalNAcß4GlcA, GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAcß4GlcA, GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAcß4GlcA ; GlcAß3GlcNAcß4GlcA, GlcAß3 GlcNAcß4GlcAß3 GlcNAcß4GlcA, GlcAß3GlcNAcß4GlcAß3GlcNAcß4GlcAß3GlcNAcß4GlcA, and GIcAp3GlcNAcp4GlcAp3GlcNAcp4GlcAp3GlcNAcp4GlcAp3GlcNAcp4GlcA.
16. The Helicobacterpylori binding substance according to claim 1 comprising one or several of the following oligosaccharide sequences GalNAcß4GlcAß3GalNAcß4GlcA, GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAcß4GlcA, GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcA ; GalNAcß4GlcAß3GalNAc, GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAc, GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAc ; GlcNAcß4GlcAß3GlcNAcß4GlcA, GIcNAc 4GlcAp3GlcNAcp4GlcAp3GlcNAcp4GlcA, GlcNAcp4GlcAp3GlcNAcp4GlcAp3GlcNAcp4GlcA (33GlcNAcp4GlcA ; GlcNAcß4GlcAß3GlcNAc, GlcNAcß4GlcAß3GlcNAcß4GlcAß3GlcNAc, and GlcNAcß4GlcAß3GlcNAcß4GlcAß3GlcNAcß4GlcAß3GlcNAc.
17. The Helicobacter pylori binding substance according to claim 1 comprising a disaccharide structure and preferably comprising at least one of the following subepitope oligosaccharide sequence GalNAcß4Glc, Galß4GlcA, GalNAcß4GlcA ; GlcNAcp4Glc, GlcNAcß4GlcA, Glcp4GlcA, Glc (34G1c, Glcp4GlcNAc, GlcNAcß4GlcA, <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> Glcp3GalNAc,<BR> <BR> <BR> <BR> <BR> <BR> <BR> GlcAß3GalNAc,<BR> <BR> <BR> <BR> <BR> <BR> <BR> Glc (33G1c, Glcp3GlcNAc, and GlcAß3GlcNAc.
18. The Helicobacter pylori binding substance according to any one of claims 1 17, wherein the substance is conjugated to a polysaccharide, preferably to a polylactosamine chain or a conjugate thereof.
19. The Helicobacterpylori binding substance according to any one of claims 1 17, wherein the substance is a glycolipid.
20. The Helicobacter pylori binding substance according to any one of claims 1 17, wherein the substance is an oligomeric molecule containing at least two or three oligosaccharide chains.
21. The Helicobacter pylori binding substance consisting of a micelle comprising one or more of the substances of any one of claims 120.
22. The Helicobacter pylori binding substance according to any one of claims 1 21, wherein the substance (s) is/are conjugated to a carrier.
23. The Helicobacter pylori binding substance according to any one of claims 122, wherein the substance is covalently conjugated with an antibiotic effective against Helicobacter pylori, preferably a penicillin type antibiotic.
24. The Helicobacter pylori binding substance or a mixture of substances according to any one of claims 122, wherein position C 1 of reducing end terminal monosoccharide (such as Glc, GIcA, GlcNAc, GlcANAc, Man, Gal, or GaINAc) of the oligosaccharide sequence (OS) is oxygen linked (O) to an oligovalent or a polyvalent carrier (Z), via a spacer group (Y) and optionally via a monosaccharide or oligosaccharide residue (X), forming the following structure [OS0 (X) nY] mZ where integers m, and n have values m = 1, and n is independently 0 or 1; X is preferably lactosyl, galactosyl, polyNacetyllactosaminyl, or part of an Oglycan or an Nglycan oligosaccharide sequence, Y is a spacer group or a terminal conjugate such as a ceramide lipid moiety or a linkage to Z; or an analog or a derivative of the substance of said structure having binding activity to Helicobacter pylori.
25. The substance according to any one of claims 124 for use as a Helicobacter pylori binding or inhibiting substance.
26. The substance according to any one of claims 124 for use as a medicament.
27. Use of the substance according to any one of claims 124 for the production of a composition having Helicobacter pylori binding or inhibiting activity.
28. Use of the substance according to any one of claims 124 for the production of a pharmaceutical composition for the treatment of any condition due to the presence of Helicobacter pylori.
29. A pharmaceutical composition comprising the substance according to any one of claims 124 for the treatment of any condition due to the presence of Helicobacter pylori.
30. The pharmaceutical composition according to claim 29, for the treatment of chronic superficial gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, nonHodgkin lymphoma in human stomach, liver disease, pancreatic disease, skin disease, heart disease, or autoimmune diseases including autoimmune gastritis and pernicious anaemia and nonsteroid antiinflammatory drug (NSAID) related gastric disease, or for prevention of sudden infant death syndrome.
31. A method for the treatment of a condition due to presence of Helicobacter pylori, wherein a pharmaceutically effective amount of the substance according to any one of claims 124 or the composition according to claims 29 or 30 is administered to a subject in need of such treatment.
32. The method according to claim 31, when said condition is caused by the presence of Helicobacter pylori in the gastrointestinal tract of a patient.
33. The method according to claim 31, for the treatment of chronic superficial gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, nonHodgkin lymphoma in human stomach, liver disease, pancreatic disease, skin disease, heart disease, or autoimmune diseases including autoimmune gastritis and pernicious anaemia and nonsteroid antiinflammatory drug (NSAID) related gastric disease, or for prevention of sudden infant death syndrome.
34. Use of the substance according to any one of claims 124, for the diagnosis of a condition due to infection by Helicobacterpylori.
35. A nutritional additive or composition containing the substance according to any one of claims 124.
36. The nutritional additive or composition according to claim 35 for use in infant food.
37. Use of the substance according to any one of claims 124, for the identification of bacterial adhesin.
38. Use of the substance according to any one of claims 124 or a substance identified according to claim 32, for the production of a vaccine against Helicobacter pylori.
39. Use of the substance according to any one of claims 124, for typing Helicobacter pylori.
40. The substance according to any one of claims 124, for use in Helicobacter pylori binding assays.
41. A Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula 9 Asaccharide Bsaccharide wherein integers 1, m, and n have values m = 1,1 and n are independently 0 or 1 ; Rl is H and R2 is OH, or RI is OH and R2 is H, or Ri is H and R2 is a monosaccharidylor oligosaccharidylgroup, preferably a beta glycosidically linked galactosyl group ; Ri 1 is H and RI 2 is OH, or Ri 1 is OH and R12 is H ; R3 is independentlyOH or acetamido (NHCOCH3) or an acetamido analogous group, R7 is acetamido (NHCOCH3) or an acetamido analogous group; when 1 = 1, R4 is H and R5 is oxygen linked to bond R6 and forms a beta anomeric glycosidic linkage to saccharide B, or Rs isH and R4 is oxygen linked to bond R6 and forms an alpha anomeric glycosidic linkage to saccharide B; when 1 = 0, R6 isOH linked to B; X is monosaccharide or oligosaccharide residue, X is lactosyl, galactosyl, polyN acetyllactosaminyl, or part of an Oglycan or an Nglycan oligosaccharide sequence, or Glc, GlcNAc OR GaINAc or part of chondroitin or hyaluronic acid oligosaccharide sequence; Y is a spacer group or a terminal conjugate such as a ceramide lipid moiety or a linkage to Z; Z is an oligovalent or a polyvalent carrier; the oxygen linkage (O) between position Cl of the B saccharide and saccharide residue X or spacer group Y can be replaced by carbon (C), nitrogen (N) or sulphur (S) linkage; Rg and Rg are independently carboxylic acid amide, such as methylamide or ethyalamide, hydroxymethyl (CH2OH) or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester; R3, R7, and Rio are independently hydroxyl, acetamido or acetamido group mimicking group, such as Cul6 alkyl amides, arylamido, secondary amine, preferentially Nethyl or Nmethyl, 0acetyl, or 0alkyl for example Oethyl or 0methyl, with the provisio that Rio is acetamido or acetamido group mimicking group, or Rg is independently carboxylic acid amide, such as methylamide or ethyalamide, or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester, or R12 is OH; preferably so that Rio is acetamido or acetamido group mimicking group or R12 is OH or so that Rio is acetamido or acetamido group mimicking group and R12 is OH or preferably so that two of the groups Rg, Rio and Ru docs not comprise OH as described above.
42. A Helicobacterpylori binding substance comprising to claim 1, wherein said oligosaccharide sequence is a natural type chondroitin or hyaluronic acid sequence or carboxylix acid derivatized or carboxylix acid reduced form thereof according to the following structure [Glc (A) qlß3] sl [Hex2NAcß4Glc (A) q2] s2 [P3Hex2NAc] u wherein ql, q2, u and sl are each independently 0 or 1, and s2 is independly an integer from 1 to 10, preferably from 1 to 4, and most preferably from 1 to 3 and Hex2 is Gal or Glc and any mixture of such oligosaccharides preferably with different values of S2.
43. A Helicobacter pylori binding nonacidic polyvalent substance comprising the oligosaccharide sequence as defined in any one of claims 123, wherein said oligosaccharide sequence (OS) is a part of structure [OS0 (X) nY] mZ as defined in claim 24, Y being a hydrophilic spacer, more preferably a flexible hydrophilic spacer, and Helicobacter pylori binding analogs and derivatives thereof.
44. The Helicobacter pylori binding nonacidic polyvalent substance according to claim 43, wherein linker structure Y is [OSO (X) nLzCH (H/{CH 2OH} p) {CHlOH} p2 {CH (NHR) p3 {CHiOH} p4L Z wherein Ll and L2 are linking groups comprising independently oxygen, nitrogen, sulphur or carbon linkage atom or two linking atoms of the group forming linkages such as O, S, CH2, N, N (COCH3) , amide groupsCONHorNHCOor NN (hydrazine derivative) or an amino oxylinkages ON and NO; l1 is linkage from carbon 1 of the reducing end monosaccharide of X or when n =0, LI replacesOand links directly from the reducing end C1 of OS ; pl, p2, p3, and p4 are independently integers from 07, with the proviso that at least one of pi, p2, p3, and p4 is at least 1 ; CH12OH in the branching term {CHI 2OH} pl means that the chain terminating group is CH2OH and when the pl is more than 1 there is secondary alcohol groupsCHOHlinking the terminating group to the rest of the spacer; R is preferably acetyl group (COCH3) or R is an alternative linkage to Z and then L2 is one or two atom chain terminating group, in another embodiment R is an analog forming group comprising Cl 4 acyl group comprising amido structure or H or Cl 4 alkyl forming an amine; and m > 1 and Z is polyvalent carrier; OS and X are as defined in claim 24.
45. A Helicobacter pylori binding substance according to claim 42, wherein ql and q2 are 0 as a nonreducing end terminal sequence, and Helicobacter pylori binding analogs and derivatives thereof.
46. The substance according to any one of claims 4245 for use in binding bacteria, toxins or viruses.
47. The substance according to any one of claims 4245 for use as a medicament.
48. A method for the treatment of a condition due to presence of Helicobacter pylori, wherein a pharmaceutically effective amount of the substance as defined in any one of claims 124 or 4245 is administered to a subject in need of such treatment.
49. The method according to claim 48, when said condition is caused by the presence of Helicobacter pylori in the gastrointestinal tract of a patient.
50. The method according to claim 48, for the treatment of chronic superficial gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, nonHodgkin lymphoma in human stomach, liver disease, pancreatic disease, skin disease, heart disease, or autoimmune diseases including autoimmune gastritis and pernicious anaemia and nonsteroid antiinflammatory drug (NSAID) related gastric disease, or for prevention of sudden infant death syndrome.
51. The method of treatment according to any one of claims 4850, wherein said substance is a nutritional additive or a part of a nutritional composition.
52. The substance according to any of the claims 124, wherein A is carboxylic acid derivative, preferably an ester or amide.
53. The substance according to any one of claims 4547, wherein said oligosaccharide sequence is ß 16 linked from the reducing end to GaINAc, GIcNAc, Gal or Glc.
54. The substance according to any one of claims 4547, wherein said oligosaccharide sequence is according to claim 5.
55. A method of screening Helicobacter pylori binding substances comprising modifying at least one hydroxyl or acetamido group of an oligosaccharide sequence as defined in any one of claims 124 into another chemical group determining Helicobacter pylori binding or inhibiting substances from the modified oligosaccharide sequences.
56. A functional food comprising substances according to any of the claims 124 or 45.
57. A functional food comprising substances according to any of the claims 1317.
58. A functional food according to claim 56 or 57, wherein said food is a beverage.
59. A functional food according to claim 56 or 57, wherein said food is an infant formula.
60. A functional food according to claim 56 or 57, wherein said food is animal feed.
61. A method of producing chondroitin oligosaccharides from chondroitin sulphates comprising removing sulphates from chondroitin sulphate by chemical hydrolysis specifically hydrolyzing glycosidic bonds between GaINAc and GIcA.
62. The method of claim 61, wherein the hydrolysis is performed by acid hydrolysis, preferably by a strong carboxylix acid.
63. The method of claim 61, wherein said strong carboxylix acid is trifluoroacetic acid.
64. The method of claim 61 further comprising a step of purification involving anion exchange chromatography and/or gel filtration chromatography (size exclusion chromatography).
65. A method for production of amidated glucuronic acid comprising oligosaccharides and monosaccharides from glucuronic acid comprising polysaccharides, the method comprising the steps of optionally oxidating of 6hydroxyls of a polysaccharide to carboxylic acid groups, when the substate does not comprise uronic acid groups, or does contain oxidatable 6 hydroxyl groups. amidating of glucuronic acid residues of the glucuronic acid comprising polysaccharide hydrolysing the polysaccharide to fragments optionally isolating oligosaccharide by chromatographic means.
66. The method according to claim 65, wherein said polysaccharides are selected from the group consisting of pectin, desulphated chondroitin sulphate, hyaluronic acid and bacterial exopolysaccharide comprising glucuronic acid.
67. The method according to claim 65, wherein the amidation is performed from the polysaccharide activated by uronium type amide bond synthesis activator.
68. The method according to claim 65, wherein the carboxylic acid is activated by methyl ester.
69. The method according to claim 65, wherein said fragments are either oligosaccharides or monosaccharides.
70. Helicobacter pylori binding substance [Hex 1 (A) ql (NAc) rly3] slHex2 (NAc) r2p4Glc (A) q2 (NAc) r3 wherein ql, q2, rl, r2, r3, and sl, are each independently 0 or 1, and Hexl, and Hex2 is a hexose structures, preferably galactose (Gal) or glucose (Glc), which may be further modified by the A and/or NAC groups; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that at least r2 is 1 or q2 isl or Hex2 is Glc and that A indicates a glucuronamide when at least ql or q2 is 1 or when sl is 0, then q2 is 1 and r2 is 0 or q2 and r2 and r3 are 1 or q2 and r2 are 1, r3 is 0 and A indicates a glucuronamide; or when s is 1 then when r2 is 1 then at least ql is 1 or q2 is 1 with the provision that the molecule does not comprise two nonderivatized plinked glucuronic acid units.
71. A method of screening Helicobacter pylori binding substance analogs comprising docking by molecular modeling a carbohydrate binding molecule of Helicobacter pylori in silico designing binding active analogues by allowing determination of binding interactions and positions for possible additional binding interactions determining Helicobacter pylori binding or inhibiting substances from the modified carbohydrate binding molecules.
72. The method according to claim 71, wherein said carbohydrate binding molecule of Helicobacterpylori is a Helicobacterpylori binding oligosaccharide sequence according to claim 1.
Description:
Novel binding epitopes for Helicobacter pylori and use thereof FIELD OF THE INVENTION The present invention describes a substance or receptor binding to Helicobacter pylori, and use thereof in, e. g. , pharmaceutical and nutritional compositions for the treatment of conditions due to the presence of Helicobacter pylori. The invention is also directed to the use of the receptor for diagnostics of Helicobacter pylori.

BACKGROUND OF THE INVENTION Helicobacter pylori has been implicated in several diseases of the gastrointestinal tract including chronic gastritis, non-steroidal anti-inflammatory drug (NSAID) associated gastric disease, duodenal and gastric ulcers, gastric MALT lymphoma, and gastric adenocarcinoma (Axon, 1993; Blaser, 1992; DeCross and Marshall, 1993; Dooley, 1993; Dunn et al., 1997; Lin et al., 1993; Nomura and Stemmermann, 1993; Parsonnet et al. 1994; Sung et al., 2000 Wotherspoon et al., 1993).

Helicobacter pylori is also involved in totally or partially non-gastrointestinal diseases including sudden infant death syndrome (Kerr et al., 2000 and US 6,083, 756), autommune diseases such as autoimmune gastritis and pernicious anaemia (Appelmelk et al., 1998; Chmiela et al, 1998; Clayes et al., 1998; Jassel et al., 1999; Steininger et al., 1998) and some skin diseases (Rebora et al., 1995), pancreatic disease (Correa et al., 1990), liver diseases including adenocarcinoma (Nilsson et al., 2000; Avenaud et al., 2000) and heart diseases such as atherosclerosis (Farsak et al., 2000). Multiple diseases caused or associated with Helicobacter pylori has been reviewed (Pakodi et al., 2000). Of prime interest with respect to bacterial colonization and infection is the mechanism by which this bacterium adheres to the epithelial cell surfaces of the gastric mucosa.

Glycoconjugates, both lipid-and protein-based, have been reported to serve as receptors for the binding of this microorganism, e. g., sialylated glycoconjugates (Evans et al., 1988), sulfatide and GM3, NeuNAca3LacßCer, (Saitoh et al., 1991), Leb determinants, Fuca2Galp3 (Fuca4) GlcNAc (Boren et al., 1993), polyglycosylceramides, heterogenous branched and linear fucosylated and sialylated structures with sialic acid as part of receptor (Miller-Podraza et al., 1996; 1997a), lactosylceramide, LacpCer (hydroxyl fattyacids) (Angstrom et al., 1998) and gangliotetraosylceramide (Lingwood et al., 1992; Angström et al., 1998). Other potential receptors for Helicobacter pylori include polysaccharide heparan sulphate,

(GIcNAca4GlcAp/IdoAa4)n comprising various sulphates, (Ascensio et al., 1993) as well as the phospholipid phosphatidylethanolamine (Lingwood et al., 1992).

These sequences are different from the epitopes described by the present invention.

The polysaccharides chondroitin sulphate, chondroitin, and hyaluronic acid, and even non-specified fragmnets derived from these as polyvalent conjugates, have been also described as receptors for Helicobacter pylori (FI20011403). However, FI20011403 does not describe minimum size of the oligosaccharide effective for inhibition or binding of H. pylori (if any possible in monovalent form) and does not give effective binding structure. Moreover, the exact sulphation status of the chondroitin oligosaccharides was not described nor possible natural variations of the oligosaccharide sequences or the role of these in specific binding to H. pylori.

Numerous patent documents describe therapheutic compositions comprising chondroitin sulphate, chondroitin sulphate or hyaluronic acid polysaccharides or fragments thereof (e. g. W009827988, EP0704216, US5894070, US4524066, W09407505) in various therapheutic compositions. W09106303 claims chondroitin sulphate as molecules comprising chondroitin sulphate disaccharide, but specific oligosaccharides or non-sulphated oligosaccharides were not claimed. Chondroitin sulphate oligosaccharides has not been described as medicines and specific molecular structures has not been indicated. Some applications describe hyaluronic acid oligosaccharides in medical applications (WO0204471, W09957301), the latter application describes ester derivatives of hyaluronic acid. W003002128 describes the use of hyaluronic acid and chondroitin (sulfate) against H. pylori. However, W003002128 does not reveal the binding mode of H. pylori towards oligosaccharide sequences, actual specific oligosaccharide sequence based H. pylori binding epitopes, especially effective terminal epitopes, or the size of the binding epitopes.

Gangliotetraosylceramide has sequence Gal 3GalNAc 4Galp4GlcpCer. An international application WO 02056893 claims similar terminal sequences Galp3GalNAc of gangliotetraosylceramide and disaccharide terminal Galp3GlcNAc of lactotetraosylceramide Galß3GlcNAcß3Galß4GlcßCer as H. pylori binding sequences. However, the minimal disaccharide of the present invention GalNAcp4Glc (A) ou (NAc) on has different monosaccharides and different linkage configuration compared to Galp3GalNAc.

US patents of Zopf et al. : 5,883, 079 (March 1999), 5,753, 630 (May 1998) and 5,514, 660 (May, 1996) describe Neu5Aca3Gal-containing compounds as inhibitors of the H. pylori adhesion. The sialyl-lactose molecule inhibits Helicobacter pylori

binding to human gastrointestinal cell lines (Simon et al., 1999) and is also effective in a rhesus monkey animal model of the infection (Mysore et al., 2000). The compound is in clinical trials.

US patent Krivan et al. 5,446, 681 (November 1995) describes bacterium receptor antibiotic conjugates comprising an asialo ganglioside coupled to a penicillin antibiotic. Especially is claimed the treatment of Helicobacterpylori with an amoxicillin-asialo-GMl conjugate. The oligosaccharide sequences/glycolipids described by the invention do not belong to the ganglioseries of glycolipids.

US patents of Krivan et al. : 5,386, 027 (January 1995) and 5,217, 715 (June 1993) describe use of oligosaccharide sequences or glycolipids to inhibit several pathogenic bacteria, however the current binding specificity is not included and Helicobacter pylori is not among the bacteria studied or claimed.

The saccharide sequence GlcNAcp3Gal has been described as a receptor for Streptococcus (Andersson et al., 1986). Some bacteria may have overlapping binding specificities, but it is not possible to predict the bindings of even closely related bacterial adhesins. In case of Helicobacter pylori the saccharide binding molecules, except the Lewis b binding protein are not known.

SUMMARY OF THE INVENTION The present invention relates to a substance or receptor binding to Helicobacter pylori comprising the oligosaccharide sequence [Hexl (A) ql (NAc) ria/p3], iHex2 (NAc) r2 [p4Glc (A) q2 (NAc) r3] s2 wherein ql, q2, rl, r2, r3, sl and s2 are each independently 0 or 1, so that at least r2 or q2 is 1, at least sl or s2 is 1, or Hex2 is Glc, with the proviso that when s2 is 0, then Hexl is Glc and preferably rl is 0; Hex1 is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc and analogs or derivatives of said oligosaccharide sequence for binding or inhibiting Helicobacter pylori.

Among the objects of the invention are the use of the Helicobacter pylori binding oligosaccharide sequences described in the invention as a medicament, and the use of the same for the manufacture of a pharmaceutical composition, particularly for the treatment of any condition due to the presence of Helicobacter pylori in a subject.

The present invention also relates to the methods for the treatment of conditions due to the presence of Helicobacter pylori. The invention is also directed to the use of the receptor (s) described in the invention as Helicobacter pylori binding or inhibiting substance for diagnostics of Helicobacter pylori.

Another object of the invention is to provide substances, pharmaceutical compositions and nutritional additives or compositions containing Helicobacter pylori binding oligosaccharide sequence (s).

Other objects of the invention are the use of the above-mentioned Helicobacter pylori binding substances for the identification of bacterial adhesin, the typing of Helicobacter pylori, and the Helicobacter pylori binding assays.

Yet another object of the invention is the use of the above-mentioned Helicobacter pylori binding substances for the production of a vaccine against Helicobacter pylori.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A and 1B. Thin-layer chromatogram with separated glycosphingolipids detected with 4-methoxybenzaldehyde (1A) and autoradiogram after binding of radiolabeled Helicobacter pylori strain 032 (1B). The glycosphingolipids were separated on aluminum-backed silica gel 60 HPTLC plates (Merck) using chloroform/methanol/water 60: 35: 8 (by volume) as solvent system. The binding assay was done as described in the"Materials and methods"section.

Autoradiography was for 72 h. The lanes contained: lane 1) Galp4GlcNAcp3Galp4GlcplCer (neolactotetraosylceramide), 4 tg ; lane 2) Gala3Galp4GlcNAcp3Gal 4Glc (3lCer (B5 glycosphingolipid), 4 pg ; lane 3) Gala3Galp4GlcNH3Galp4GlcplCer, 4 pg ; lane 4) Gala3 (Fuca2) Galß4GlcNAcß3Galß4Glcß1Cer (B6 type 2 glycosphingolipid), 4 gag ; lane 5) GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer, 4 µg ; lane 6) Galp4GlcNAcp3Galp4GlcNAcp3Galp4GlcplCer, 4 j g ; lane 7) GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (x2 glycosphingolipid), 4 zig ; lane 8) NeuAca3GalNAcp3Gal (34GlcNAc (33Gal (34GlcplCer (NeuAc- ), 4 ag ; lane 9) Fuca2Galp4GlcNAcp3Galp4GlcplCer (H5 type 2 glycosphingolipid), 4 ? ;

lane 10) NeuAca3Galp4GlcNAcp3Galp4GlcplCer (sialylneolactotetraosylceramide), 4 p-g. The sources of the glycosphingolipids are the same as given in Table 1.

Fig. 2. MALDI-TOF mass spectrum of the purified amidation product of GlcAß3Galß4GlcNAcß6GlcNAc. A sample of the product was deposited on a DHB matrix and analyzed in the reflector positive ion mode.

Fig. 3. MALDI-TOF mass spectrum of the purified amidation product of chondroitin tetrasaccharide (Glc [ANH2] ß3GalNAcß4Glc[A-NH2]ß3GalNAc) : m/z 797.2 ([M+Na] +) and 813.2 ([M+K] +). A-NH2 indicates the glucuronamides-CO-NH2 Fig. 4. MALDI-TOF mass spectrum of the purified amidation product of chondroitin hexa- saccharide (Glc [A-NH2] ß3GalNAcß4Glc [A-NH2] p3GalNAcp4Glc [A-NH2] (33Ga1NAc) : m/z 1175.2 ([M+Na]+) and 1191.2 ( [M+K]).

Fig. 5. MALDI-TOF mass spectrum of the purified monosulphated GlcAß1,3GalNAcß1,4GlcAß1,3GalNAc : m/z 855.0 [M-H]-, m/z 877.0 [M-2H+Na]', m/z 892.9 [M-2H+K]-and m/z 914.9 [M-3H+Na+K]'.

Fig. 6. MALDI-TOF mass spectrum of the purified DAP-16 dendrimer conjugate of Glc [ANH2] ßl, 3GalNAcßl, 4Glc [ANH2] ßl, 3GalNAc. The broad signal around 12000-17000 Da represents a nonspecific dimer, i. e. [2M+H] +, of the dendrimeric conjugate.

Fig. 7. MALDI-TOF mass spectrum of the reduced hyaluronic acid oligosaccharide mixture.

See text for peak details.

DETAILED DESCRIPTION OF THE INVENTION WO 02056893 showed optimal trisaccharide binding sequences for Helicobacter pylori having the oligosaccharide sequence Gal (A) ql (NAc) rl/Glc (A) q2 (NAc) r2a3/p3Galp4GIc (NAc) u wherein ql, q2, rl, r2, and u are each independently 0 or 1, and wherein lactose based sequences are present in polyvalent form or in high concentration. Also non- reducing-end elongated variants were shown to be effective. Moreover, the studies showed that the oligosaccharide sequence can vary in many positions without losing its H. pylori binding activity.

The present invention allows adding specific modifications to the sequence adding possible modifications of the reducing end side monosaccharide residue and additional variations to the middle and reducing end monosaccharide residues.

Furthermore, the present invention represents novel derivatives of uronic acids and method for their preparation. Moreover the present invention is directed to the use of the oligosaccharide backbone in screening assays, in design of combinatorial chemistry libraries and in molecular modeling for design of better analogues or derivatives of the oligosaccharide sequences.

The present invention shows that Gal (34 residue can be replaced in the oligosaccharide sequences by GalNAcß4. The examples show that GaINAc supports the H. pylori binding activity of lacdiNAc type oligosaccharide sequences such as GaINAcp4GlcNAc, and chondroitin oligosaccharide sequeces Glc (A-NH2) (33GalNAc (34Glc (A-NH2) ß3GalNAc, wherein A-NH2 indicates the glucuronamides, and GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAc. The chondroitin structures show the usefulness of the uronic acid also in the middle or at the reducing end of the oligosaccharide chain. The examples further include a library of uronic acid amide derivatives, from methyl amine to octadecylamine, from which smaller structures were found to be more active than larger structures. Furthermore, the novel binding structures share a common conformation. The putative conformation indicates that there are possible areas of the molecules which can be modified such as position number two of Gal/GalNAcß4 and reducing end Glc/GlcA/GlcNAc, which both obviously tolerate amine derivative group and hydroxylstructures.

The present invention further discloses that hyaluronic acid oligosaccharides with similar sequences as chondroitin oligosaccharides, but containing GlcNAc residue in the place of GaINAc do also bind to Helicobacter pylori. Therefore a general formula of H. pylori binding saccharide epitopes can be drawn as Formula A.

The present invention provides a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula A [Hex 1 (A) ql (NAc) ia/p3], iHcx2 (NAc), 2 [p4Glc (A) q2 (NAc) 3lys2 wherein ql, q2, rl, r2, r3, sl and s2 are each independently 0 or 1, so that at least r2 or q2 is 1, at least sl or s2 is 1, or Hex2 is Glc, with the proviso that when s2 is 0, then Hexl is Glc and preferably rl is 0; and Hexl is galactose (Gal), glucose (Glc)

or mannose (Man), Hex2 is Gal or Glc ; and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for the prophylaxis or treatment of any condition due to the presence of Helicobacterpylori in a subject.

The present invention further provides a Helicobacter pylori binding substance comprising oligosaccharide sequence according to Formula B [Hex 1 (A) ql (NAc) rla/ß3] slHex2 (NAc) r2ß4Glc (A) q2 (NAc) r3 wherein ql, q2, rl, r2, r3, and sl are each independently 0 or 1, so that at least r2 or q2 is 1 ; and Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc.

More preferably disaccharide or trisaccharide oligosaccharide sequences according to the formula B are used. The trisaccharide epitopes are especially preferred, most preferably the trisaccharide epitope is a terminal oligosaccharide sequences.

The terminal monosaccharide unit is preferably (33-linked as in chondroitin, chondroitin sulphate, hyaluronic acid or previously described N-acetyllactosamine oligosaccharide sequences. Large scale cost effective availability of these materials makes these very useful and preferred raw materials for production of the oligosaccharide sequences according to the invention. The present invention is thus further preferably directed to the Helicobacter pylori binding substance comprising oligosaccharide sequence according to Formula C [Glc (A) ql ß3] slHex2 (NAc) r2p4Glc (A) q2 wherein ql, q2, r2, and sl are each independently 0 or 1, so that at least r2 or q2 is 1; and Hex2 is Gal or Glc.

Hyaluronic acid and chondroitin are preferred raw material for production of the oligosaccharide sequences according to the invention. The present invention is further directed to N-acetylhexosamine based sequences derived from chondroitin or hyaluronic acid. More preferred embodiment includes thus Helicobacter pylori binding substance comprising oligosaccharide sequence according to the Formula D [Glc (A) qip3] siHcx2NAcp4Glc (A) q2

wherein ql, q2, and sl are each independently 0 or 1 ; and Hex2 is Gal or Glc.

The two most preferred embodiments of the invention, chondroitin and hyaluronic acid oligosaccharide sequences are shown in Formulas E and F. The formulas include carboxylic acid derivatives of the oligosaccharide sequences, especially amides and esters as described by the invention and partially or totally carboxylic acid reduced analogs thereof. In a preferred embodiment the reducing end of the oligosaccharide sequence is reduced, especially preferred, when the oligosaccharide sequence is a carboxylic acid reduced oligosaccharide sequence or oligosaccharide.

The oligosaccharide sequences are in a preferred embodiment sulphated, or in another preferred embodiment partially suphated chondroitin oligosaccharides. In a preferred embodiment the sulphated oligosaccharide sequence is sulphated so that one sulphate group is linked by natural linkage type and position to the GaINAc residue or residues of the oligosaccharide sequence.

Thus, the present invention is preferably directed to a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula E [Glc (A) qi (33], iGalNAc (34Glc (A) q2 wherein ql, q2, and sl are each independently 0 or 1.

The present invention is also preferably directed to a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula F [Glc (A) qip3] siGlcNAcp4Glc (A) q2 wherein ql, q2, and sl are each independently 0 or 1.

The present invention is further directed to novel disaccharide subepitope structures which contributes to the H. pylori binding. The subepitopes may be used according to the invention, but in some case high concentration or polyvalent presentation is needed. The subepitopes are preferably disaccharide sequences according to the Formula A. The present invention is preferably directed to the use of the subepitope structures for design and/or synthesis of molecular libraries and analogs and/or derivatives of the oligosaccharides according to the invention. In a preferred embodiment, preferably a natural or enzymatically synthesized, disaccharide sequence subepitope is used for synthesis of an oligosaccharide sequence according

to the invention, and in another embodiment for the synthesis of an analogue or derivative of an oligosaccharide sequence according to the invention.

More preferred disaccharide subepitopes include the following subepitope oligosaccharide sequences <BR> <BR> <BR> <BR> <BR> <BR> <BR> GalNAcp4Glc,<BR> <BR> <BR> <BR> <BR> Galß4GlcA, GalNAcp4GlcA ; GlcNAcp4Glc, GlcNAc (34GlcA, Glcp4GlcA, Glc (34G1c, Glcp4GlcNAc, GIcNAcp4GlcA, Glcp3GalNAc, GlcA (33GalNAc, Glcp3Glc, Glcp3GlcNAc, and GIcAp3GlcNAc.

Additionally in another embodiment preferred disaccharide subepitopes include saccharides comprising GlcANAc in place of GIcA in any preferred sequences above.

The present invention also shows that for the optimal binding interaction the reducing end monosaccharide of the trisaccharide epitope should be in beta configuration. The optimal presentation models can be used with similar reducing end structures for saccharide sequences according to Formulas A-F. An example shows strong binding to reductively aminated GIcNAcp3Galp4GlcNAcp6GlcNAc, while corresponding alpha anomer GlcNAca3Galp4GlcNAca6GlcNAc was inactive or very weakly active under the experimental conditions. High binding activity was observed when the reducing end monosaccharide is Gal or GlcNAc. These monosaccharides have similar conformation except for N-acetyl substituent in the place of hydroxyl at carbon 2 and epimeric hydroxyls at carbon 4. GalNAc and Glc are preferred homologs having the same modifications. The preferred specificity for

(36-type reducing end terminal was thus determined to be (36Hex (Nac) o/,, wherein Hex is preferably Gal or Glc.

Surprisingly, the binding activity was strongly reduced, when the reducing end (36- structure is further elongated by another monosaccharide by (34-linkage (GlcNAcp3Galp4GlcNAcß6Galß4Glc), therefore it is preferred that there is only one monosaccharide in the reducing end ( (36Hex (NAc) o/i-structure).

Preferably the reducing end monosaccharide residue is in open chain derivative form. Preferred open chain forms of the reducing end of the substance include 1. reducing form of the reducing end monosaccharide, 2. reduced reducing end monosaccharide, 3. reduced conjugated reducing end monosaccharide, for example reductively aminated reducing end, and 4. open chain conjugated reducing end such as an oxime of an aminooxy structure, NH2-O-R where R is the rest of the conjugation agent.

Reduced reducing ends are in aqueous environment balanced in alpha, beta and open chain forms. For example, the oxime of aminooxy acetic acid is usually in alpha, beta, and open chain forms in aqueous solutions.

Furthermore, the present invention is especially directed to favourable (36-linked structures at the reducing end which are cost effective to produce and increase the binding to H. pylori. The disaccharide GlcNAcß6Gal can be produced effextively by organic synthesis or by enzymatic means and the substrate monosaccharides are relatively cheap compared to GaINAc. GlcNAc (36GlcNAc can be synthesized even more effectively chemically by traditional methods or by acid reversion chemistry. A suitable bacterial transferase for the synthesis of the substance is known.

Structural formulas of the preferred embodiments The present invention is directed to the following disaccharide to pentasaccharide oligosaccharide sequences for the treatment of Helicobacter pylori.

The present invention is further directed to the oligosaccharides as Helicobacter pylori binding substances for treatment of infectious disease, preferably H. pylori dependent diseases. In a specific embodiment the present invention is directed to the chemically modified or analogous oligosaccharide sequences to obtain lower biological degradation and/higher or more specific activity towards H. pylori and/or other pathogens. Especially analogs comprising glucuronylamide are preferred. The present invention is separately directed to the sequences according to the invention

for use as medicine, preferably at least trisaccharide sequences are used. The present invention is further directed to pharmaceutical composition comprising terminal oligosaccharide sequences according to the invention.

In the formulas 1-9 the structural element A indicates the presence or absence of uronic acid form of a monosaccharide residue, furthermore A indicates the presence of derivatized uronic acid, preferably as amides of ammonia, -CO-NH2 or organic amines of the carboxylic acid group at the 6 position. Organic amides include preferably alkylamides, cycloalkylamides, and arylamides, including preferably amides from C1-C22 amines, more preferably C 1-C6 amines, even more preferably from C1-C3 amines and most preferably amides from methylamine or ethylamine. Preferably alkylamines of various sizes are aliphatic 1-alkylamines. Preferred arylamide has benzoic acid like benzyl-ring structure, preferred alkylamides include linear 1-amino alkanes. The amides of ammonia or methylamine or ethylamine, and amide from ammonia are more preferred when the molecule comprises uronic acid derivative other than non-reducing end uronic acid derivative or when the same oligosaccharide sequence comprises several uronic acid derivatives. Other uronic acid derivatives include esters.

The preferred structures of the novel Helicobacter pylori binding oligosaccharide sequences comprise the terminal trisaccharide or disaccharide structures according to the Formula la [Hex 1 (A) ql (NAc) rly3] Gal (NAc) r2p4Glc (A) q2 (NAc) r3 [p3/6Hex3 (NAc) r5] w or when w is 0, the minimal structure according to the Formula lb [Hex 1 (A) ql (NAc) riy3] sGal (NAc) r2p4Glc (A) q2 (NAc) r3 wherein ql, q2, rl, r2, r3, r5 and s, and w are each independently 0 or 1, and Hexl, and Hex2 is a hexose structures, preferably galactose (Gal) or glucose (Glc) or mannose (Man), most preferably Gal or Glc, which may be further modified by the A and/or NAC groups; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that when w is 0, then at least one of the integers r2, ql (when s is also 1), or q2 is 1, preferably at least r2 or q2 is 1 and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for binding or inhibiting Helicobacter pylori.

Brackets [] or {} indicate herein that the structure is either present or absent, () indicates presence or absence of a monosaccharide residue derivatizing structure.

Preferably, the reducing end monosaccharide is p-linked, when w is 0. In a preferred embodiment the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit. Preferably the structure is not linked to a ceramide, more preferably the structure is not linked to a ceramide comprising a hydroxyl fatty acid.

In a preferred embodiment w is 1 and the reducing end monosaccharide is not glycosidically conjugated to another monosaccharide unit, more preferably the reducing end monosaccharide unit is in an open chain reducing end derivative.

In a preferred embodiment A in the Formula is amide, methylamide or ethylamide of the carboxylic acid group of the glucuronic acid residue.

One embodiment of the present invention is a substance or a receptor binding to Helicobacter pylori comprising the oligosaccharide sequence according to separately preferred formulas: Formula 2 [Hex 1 (A) ql (NAc) riy3] Gal (NAc) r2ß4Glc (A) q2 (NAc) r3, 86Hex3 (NAc) r5 or Formula 3 [Hex 1 (A) ql (NAc) rly3] Gal (NAc) r2p4Glc (A) q2 (NAc) r3p3Hex3 (NAc) rs or Formula 4 [Hex 1 (A) ql (NAc) rly3] Gal (NAc) r2p4Glc (A) q2 (NAc) r3p3Gal (NAc) rs [p4Glc (A) q3 (NAc) r6] u wherein ql, q2, q3, rl, r2, r3, r5, r6, s, t, and u are each independently 0 or 1, and Hexl, and Hex3 are hexose structures, preferably mannose (Man), galactose (Gal) or glucose (Glc), and most preferably Gal or Glc, which may be further modified by A and/or NAc groups; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that at least one of the integers r2, q2, or q3 is 1 or r5 is 1 when u is 1, preferably r2 is 1 or q2 is 1,

and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for binding or inhibiting Helicobacter pylori.

The reducing end monosaccharide structure can be a conjugated derivative of the monosaccharide residue indicated by the formula, preferably an open chain reducing end derivative of the monosaccharide unit. In a preferred embodiment the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit. In another preferred embodiment s is 1.

One embodiment of the present invention is a substance or a receptor binding to Helicobacter pylori comprising the oligosaccharide sequence according to separately preferred formulas: Formula 5 [Hexl (A) ql (NAc) r, Gal (NAc) r2p4Glc (A) q2 (NAc) r3J33/6 {B} yHex3 (NAc) r5 wherein ql, q2, rl, r2, r3, r4, r5, s, and v are each independently 0 or 1, and Hexl, Hex2 and Hex3 are hexose structures, preferably mannose (Man), galactose (Gal) or glucose (Glc), and most preferably Gal or Glc, which may be further modified by A and/or NAC groups; B is a branch structure Hex2 (NAc) r4p3, which can be present or absent, v is 0 or v is 1, only when the structure indicated by t is present (t is 1) in 86-linked form; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that at least one of the integers r2 is 1 or q2 is 1 and that when t = 0 then u = 1, then either q3 =1 and r6 is 1 ; and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for binding or inhibiting Helicobacter pylori.

The reducing end monosaccharide structure can be a conjugated derivative of the monosaccharide residue indicated by the formula, preferably an open chain reducing end derivative of the monosaccharide unit. In a preferred embodiment the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit. In another preferred embodiment s is 1.

A in the above oligosaccharide sequences indicates uronic acid of the monosaccharide residue or carbon 6 derivative of the monosaccharide residue, most preferably the derivative of carbon 6 is an amide of the uronic acid.

GalNAcp4GlcNAc (LacdiNAc) and GaINAcp4Glc (reduced chondroitin) type structures Preferred oligosaccharide sequences according to Formula 1 a, when r is 1 and q2 is 0 Formula 6, [Hexl (A) ql (NAc) rly3] Gal (NAc)r2ß4Glc(NAc)r3ß[ß3/6Hex3(NAc)r5]w The following oligosaccharide sequences are among the preferable Helicobacter pylori binding substances for the uses of the invention. <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>GalNAca3GalNAcp4GlcNAc, GalNAcp3GalNAcp4GlcNAc, <BR> <BR> <BR> <BR> <BR> <BR> GlcNAca3GalNAc GlcNAcp3GalNAcp4GlcNAc, Gala3GalNAcp4GlcNAc, Galp3GalNAcp4GlcNAc Glca3GalNAcp4GlcNAc, <BR> <BR> <BR> Glcp3GalNAcp4GlcNAc, GlcAp3GalNAcp4GlcNAc, GlcAa3GalNAcp4GlcNAc, and GalNAcp4GlcNAc Preferred carboxylic acid reduced chondroitin type oligosaccharides include [Glcp3] s [GalNAcp4Glc] t [p3GalNAc] u wherein s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10. Preferably t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3.

The present invention is directed to the substances as medicines, preferred medicines include substance according to the above structure when sl. Glucuronic acids of natural type chondroitin oligosaccharide sequences can be reduced from carbodiimide conjugate or from methylesters by methods known in the art (see for example WO 0123398). Preferred substances includes carboxylic acid reduced forms of preferred chondroitin oligosaccharides described below, for example Glcß3GalNAcß4Glc, Glcß3GalNAcß4Glcß3GalNAcß4Glc, and Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAcß4Glc Glcß3GalNacß4Glcß3GalNAc, Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAc,

and Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAc The present invention is directed to optimal receptor size glucuronic acid reduced chondroitin oligosaccharides as substances. The preferred reduced oligosaccharide substance contains at least four, more preferably at least five monosaccharide residues. In another embodiment the preferred oligosaccharide has less than 11 monosaccharide residues, more preferably less than nine monosaccharide residues.

The present invention is further directed to composition comprising mixtures of the preferred oligosaccharides, which may further comprise smaller similar oligosaccharides. Preferably the mixtures comprise at least 20 %, more preferably at least 50 % and most preferably at least 80 % and even more preferably at least 90 % tetra-to octasacharides.

Preferred natural chondroitin type oligosaccharide sequences The present invention is especially directed to specific natural type chondroitin oligosaccharide sequences according to Formula 8 [GlcAp3] s [GalNAcp4GlcA] t [p3GalNAc] u wherein s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10. Preferably t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3.

In a more preferred embodiment GIcA in the formula indicates glucuronic acid not derivatized at carboxylic acid. The non-derivatized oligosaccharide sequences or oligosaccharides are especially preferred for various uses. These are derivatives of natural structures and are therefore especially preferred for functional foods, food additives, nutritional additives, and animal feeds. The present invention is specifically directed to chondroitin oligosaccharides. Preferably, the chondroitin type oligosaccharide sequence is for use as a medicine for infectious diseases.

Chondroitin oligosaccharides is considered also useful for therapheutical uses in foods, feeds, nutrients, self medication products, nutritional additives, food additives, and/or feed additives.

In another preferred embodiment GIcA in the formula and specific structures below represent glucuronic acid which is derivatized to an amide, preferred amides includes amides formed from carboxylic acid group and ammonium/ammonia

forming structure-CONH2, organic amides including alkylamides, cycloalkylamides, arylamides, including preferably amides from C1-C22 amines, more preferably C1-C6 amines, even more preferably from C1-C3 amines and most preferably amides from methylamine or ethylamine. Preferred arylamide has benzoic acid like benzyl-ring structure. Preferred alkylamides include linear 1-amino alkanes.

When s is 1, specifically preferred chondroitin oligosaccharide sequences include oligosaccharides with non-reducing end terminal GIcA due to their high affinity towards H. pylori : <BR> <BR> <BR> <BR> <BR> <BR> GlcAp3GalNAcp4GlcA,<BR> <BR> <BR> <BR> GIcAp3GalNAcp4GlcAp3GalNAcp4GlcA, GIcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcA, and GIcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcA A specifically preferred group of oligosaccharide sequences includes structures comprising non-reducing-end terminal GIcA and reducing end terminal GalNAc.

Such oligosaccharides can be produced effectively from natural chondroitin sulphates by desulfation and hyaluronidase digestion (or by hyalurondase digestion and desulphation : GlcAß3GalNAcß4GlcAß3GalNAc, GIcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAc, and GIcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAc Yet another preferred embodiment includes oligosaccharide structures, when s is 0, contains terminal GalNAc : <BR> <BR> <BR> <BR> <BR> <BR> GaINAcp4GlcA,<BR> <BR> <BR> <BR> <BR> GalNAcp4GlcAp3GalNAcp4GlcA, GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcA, and GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcA GaINAcp4GlcAp GaINAc, GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAc, and GaINAcp4GlcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAc The present invention is in its preferred embodiments directed to the natural type oligosaccharide sequences such as chondroitin oligosaccharides as practically pure

single components or as mixtures comprising 2-5 major component comprising more than 90 % of the oligosaccaride mixture. The chondroitin sulphate oligosaccharides are preferably devoid of sulphate ester modifications. The invention is further directed to analogous H. pylori binding oligosaccharide sequences which have non- reducing terminal delta uronic acid residue (double bond between C4 and C5), which can be created by lyase type enzymes cleaving the corresponding polysaccharide.

Analogous hyaluronic acid oligosaccharide sequences Previously it was demonstrated that H. pylori binds to hyaluronic acid polysaccahride, Present invention allows to determine the binding active oligosaccharide epitopes. The present invention is directed to the hyaluronic acid sequences for preparation medicines for the treatment of infectious diseases, especially due to the presence of H. pylori.

The preferred oligosaccharide sequences may also have the structure according to Formula 9b [GlCAß3] s [GlcNAß4GlcA] t [p3GlcNAc] u and carboxylic acid reduced variants produced as described above for chondroitin oligosaccharides according to Formula 9c [Glcp3] s [GlcNAcp4Glc] t [p3GlcNAc] u wherein s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10. Preferably t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3. The invention is further directed to analogous H. pylori binding oligosaccharide sequences which have non-reducing terminal delta uronic acid residue (double bond between C4 and C5), which can be created by lyase type enzymes cleaving the corresponding polysaccharide. The invention is further directed to H. pylori binding analog substances and screening higher affinity variants from the analogs based on formulas 1-8 wherein Gal is replaced by Glc.

The present invention is also directed to the hyaluronic acid derivative substances wherein A is amide as described for other oligosaccharide sequences according to the invention, preferably the glucuronic acid is derivatized to amides of ammonia, methylamine, or ethylamine. The invention is further directed to the substances as medicines.

The present invention is directed to optimal receptor size glucuronic acid reduced hyaluronic acid oligosaccharides as substances. The preferred reduced oligosaccharide substance contains at least four, more preferably at least five monosaccharide residues. In another embodiment the preferred oligosaccharide has less than 11 monosaccharide residues, more preferably less than nine monosaccharide residues. The present invention is further directed to composition comprising mixtures of the preferred oligosaccharides, which may further comprise smaller similar oligosaccharides. Preferably the mixtures comprise at least 20 %, more preferably at least 50 % and most preferably at least 80 % and even more preferably at least 90 % tetra-to octasacharides.

Methods to produce chondroitin oligosaccharides with non-reducing terminal GlcA The present invention is further directed to a method producing chondroitin oligosaccharides from chondroitin sulphates. The method steps involve 1. Removal of sulphates from chondroitin sulphate by chemical hydrolysis. There are several useful methods in the art for example incubating at 80-100 degrees of Celsius in dimethylsulfoxide in the presence of methanol or water.

2. Specifically hydrolyzing (34-glycosidic bonds between GaINAc (GIcNAc) and GlcA. The specific hydrolysis is preferably performed by acid hydrolysis, more preferably by a strong carboxylix acid, more preferably the strong carboxylic acid is trifluoroacetic acid. In another preferred embodiment the present invention is directed to specific hydrolysis of hyaluronic acid to oligosaccharides according to step 2. The method steps can be performed in any order. Preferably, step 1 is performed first. The purification of the product oligosaccharides involves anion exchange chromatography for the separation of the oligosaccharides. More preferably the purification involves anion exchange and gel filtration chromatography (size exclusion chromatography). Preferably oligosaccharides are from 3-mers to 14-mers, more preferably 4-mers to 10-mers.

The present invention is further directed to methods for the production of amidated glucuronic acid comprising oligosaccharides and monosaccharides from glucuronic acid comprising polysaccharides. Examples of the preferred polysaccharides includes pectin, desulphated chondroitin sulphate, and hyaluronic acid as well as bacterial exopolysaccharide comprising glucuronic acid. Alternatively, 6-hydroxyls of a polysaccharide can be specifically oxidized to carboxylic acid groups.

The method involves following steps: 1. Optional oxidation of 6 hydroxyls of a polysaccharide to carboxylic acid groups

when the substrate does not comprise uronic acid groups, or does contain oxidatable 6-hydroxyl groups.

2. Amidation of glucuronic acid residues of the glucuronic acid comprising polysaccharide. Preferably the amidation is performed from the polysaccharide activated by uronium type amide bond synthesis activator. In another embodiment the carboxylic acid is activated by methyl ester.

3. Hydrolysing the polysaccharide to fragments In separately preferred embodiments the fragments are either oligosaccharides or monosaccharides.

4. optionally isolating oligosaccharide by chromatographic means.

In a preferred embodiment chondroitin or hyaluronic acid polysaccharide is amidated and then hydrolyzed to oligosaccharides according to the invention.

Preferably hydrolysis is performed by a carboxylic acid which is more acidic than acetic acid, preferably by trifluoroacetic acid. Preferably oligosaccharides are from 3-mers to 14-mers, more preferably 4-mers to 10-mers.

Preferred substances Present invention is further directed to novel substances which are useful for multiple therapheutical and other uses: (Hexl (A) ql (NAc) rly3] slGal (NAc) r2p4Glc (A) q2 (NAc) 3 wherein ql, q2, rl, r2, r3, r5 and s, and w are each independently 0 or 1, and Hexl, and Hex2 is a hexose structures, preferably galactose (Gal) or glucose (Glc), which may be further modified by the A and/or NAC groups; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that when s is 0, q2 is 1 and A indicates an amide, when s is 1 both, ql, q2 are 1 with the provision that the molecule does not comprise two p- linked glucuronic acid units, or ql is 1 and q2 is 0 then y is alpha or r2 is 1, preferably A indicates glucuronic acid amide, methylamide or ethylamide.

Characterization of the overlapping oligosaccharide binding specificties The present invention describes a family of specific oligosaccharide sequences binding to Helicobacter pylori. The structures of the glycosphingolipids used were characterized by proton NMR and mass spectrometric experiments. Numerous naturally occuring glycosphingolipids have been previously screened (in FI 20010118) by thin-layer overlay assay (Table 1).

In this invention the terms"analog"and"derivative"are defined as follows.

According to the present invention it is possible to design structural analogs or

derivatives of the Helicobacter pylori binding oligosaccharide sequences. Thus, the invention is also directed to the structural analogs and derivatives of the substances according to the invention. The structural analogs according to the invention are molecules different from original oligosaccharide structures studied and comprise the structural elements important for the binding of Helicobacter pylori to the oligosaccharide sequences. The analogue is such molecule which can not be produced by chemical derivatization reaction in a few steps from the original oligosaccharide backbone while derivatives can be produced by chemical manipulation of the oligosaccharide structures of the invention, derivative can be naturally produced also using derivatized monosaccharide building blocks or by other means.. For example derivatives can be easily produced from amino groups after de-N-acetylation of one or several N-acetylgroups of the molecule. Derivatives can also be effectively produced from carboxylic acids groups of uronic acids and even hydroxyl groups can be derivatized for example by ether or ester groups.

Design of preferred structural analogs and derivatives The present invention is also specifically directed to the design of structural analogs and derivatives for the Helicobacter pylori binding oligosaccharide sequences. For design of effective structural analogs and derivatives it is important to know the structural element essential for the binding between Helicobacter pylori and the saccharides. The important structural elements are preferably not modified or these are modified by very close mimetic of the important structural element. These elements preferably include the 4-, and 6-hydroxyl groups of the Gal (34 residue in the trisaccharide and disaccharide epitopes. Also the positioning of the linkages between the ring structures is an important structural element. For a high affinity binding the acetamido group or acetamido mimicking group is preferred in the position corresponding to the acetamido group of the reducing end-GIcNAc of the di-or trisaccharide epitopes. Acetamido group mimicking group may be another amide, such as alkylamido, arylamido, secondary amine, preferentially N-ethyl or N- methyl, 0-acetyl, or 0-alkyl for example O-ethyl or 0-methyl. For high affinity binding amide derivatives from carboxylic acid group of the terminal uronic acid and analogues thereof are preferred. The activity of non-modified uronic acid is considered to rise in lower pH.

The structural derivatives according to the invention are oligosaccharide sequences according to the invention modified chemically so that the binding to the Helicobacter pylori is retained or increased. According to the invention it is preferred to derivatize one or several of the hydroxyl or acetamido groups of the oligosaccharide sequences. The invention describes several positions of the

molecules which could be changed when preparing the analogs or the derivatives.

The hydroxyl or acetamido groups which tolerate at least certain modifications are indicated by R-groups in Formula 9. The present invention is in a preferred embodiment directed for the production of analogues or derivatives according to the Formula 9.

More preferably the analogues are tested for binding or inhibition of Helicobacter pylori and best binding sequences are selected for development of a product. In another embodiment the molecules according to the invention or analogues or derivatives are tested for binding of other microbes or viruses, preferably for binding to toxin A of Clostridium difficile.

Bulky or bulky acidic substituents having size of a monosaccharide residue and other structures, such as monosaccharide residues, are not preferred at least when linked in the position of the C2, C3 or C6-hydroxyls of the Gal-residue Galp4GlcNAc and on C3-hydroxyl of the non-reducing terminal monosaccharide of the minimal trisaccharide epitopes. Preferably, the analogues designed do not comprise substituents or bulky substituents at these positions.

Methods to produce oligosaccharide analogs for the binding of a lectin are well known. For example, numerous analogs of sialyl-Lewis x oligosaccharide has been produced, representing the active functional groups different scaffold, see page 12090, Sears and Wong 1996. Similarily analogs of heparin oligosaccharides has been produced by Sanofi corporation and sialic acid mimicking inhibitors for the sialidase enzyme, such as accepted medical sialidase inhibitors against influenza by Hofmann-La Roche or Glaxo-Wellcome, by numerous groups.

The present invention is specifically directed to the design of analogues of the oligosaccharide structures according to the invention comprising ring structures analogous to the monosaccharide residues of the oligosaccharide residues. More preferably the analogues are tested for binding or inhibition of Helicobacter pylori and best binding sequences are selected for development of a product. In another embodiment the molecules according to the invention or analogues or derivatives are tested for binding of other microbes or viruses, preferably for binding to toxin A of Clostridium difficile. Preferably the oligosaccharide analog is build on a molecule comprising at least one six-or five-membered ring structure, more preferably the analog contains at least two ring structures comprising 6 or 5 atoms. A preferred analogue type of the oligosaccharide comprise a terminal uronic acid amide or analogue or derivative thereof linked to Gal/GaINAcp4GlcNAc-saccharide

mimicking structure. According to the invention 2 and 4 hydroxyl groups of the terminal monosaccharide residue are not important for binding and 6-hydroxyl can be modified to structures actually increasing the affinity of the molecule, high affinity analogs can be produced when these positions are modified. The data shows that it is possible to design analogs which do not comprise all hydroxyl groups of the terminal monosaccharide residue. The present invention is specifically directed to attaching various organic derivatization molecule such as aromatic or aliphatic cyclic organic residue for the 3-position of terminal Gal/GalNAc production functional analog design. The derivatization may be produced by special linker chemistry allowing linking the cyclic organic residues to 3-position of Gal/GaINAcp4. The geometry and even length of linking structures may be different from glycosidic bond structures provided that the cyclic organic residue can have at least some of the positive binding interactions of the corresponding terminal monosaccharide residue, especially close to the position of 6- hydroxyl/carboxyl/amide of the terminal monosaccharide residue.

In a specific embodiment the analogs are produced from amine group by replacement of hydroxyl group at position 3 of Gal/GaINAcp4.

The present invention is specifically directed to the screening of analogs comprising terminal cyclic molecule on terminal 3-position of Gal/GaINAc in the trisaccharide epitope for binding to Helicobacterpylori. The terminal cyclic molecule is preferably a six membered organic residue, and more preferably it also comprises a carboxylic acid, an amide or alkyl amide structure similar to the structures terminal and/or in the middle hexuronic acids in the formulas according the invention.

Alternatively terminal uronic acid amide or analogue or derivative is 1-3-linked to Gal, which is linked to the GlcNAc mimicking structure. In mimicking structures monosaccharide rings may be replaced by rings such as cyclohexane or cyclopentane, aromatic rings including benzene ring, heterocyclic ring structures may comprise beside oxygen for example nitrogen and sulphur atoms. To lock the active ring conformations the ring structures may be interconnected by tolerated linker groups. Typical mimetic structure may also comprise peptide analog- structures for the oligosaccharide sequence or part of it. The present invention is also directed to the design and/or screening of peptide analogs for the oligosaccharide sequences. Furthermore the present invention is directed for screening of DNA or RNA-based analogues, for example so called aptamers, of the oligosaccharide sequences according to the invention. The effects of the active groups to binding activity are cumulative and lack of one group could be compensated by adding an active residue on the other side of the molecule.

Molecular modelling, preferably by a computer, can be used to produce analog structures for the Helicobacter pylori binding oligosaccharide sequences according to the invention. The results from the molecular modelling of several oligosacharide sequences are given in examples and the same or similar methods, besides NMR and X-ray crystallography methods, can be used to obtain structures for other binding oligosaccharide sequences or analogues or derivatives according to the invention. In a preferred embodiment the analogues or derivatives of the oligosaccharide structures having same or similar conformations with the oligosaccharide structures according to the invention are selected from computer assisted molecular modeling results or screening database or databases containing three dimensional structures of molecules, these methods are referred as computerized screening methods.

The present invention is further directed to the testing oligosaccharide structures and analogues and derivatives thereof which are selected by the computerized screening methods for binding to other pathogenic microbes or viruses or toxins having a binding specificity similar to the binding specificity of Helicobacter pylori with regard to one or several oligosaccharide sequences according to the invention. In a preferred embodiment the oligosaccharide structures and analogues and derivatives selected by the computerized screening methods are tested for binding to toxin A of Clostridium difficile.

The analogue molecules can be synthetically produced or obtained from natural sources. Molecules can also be produced virtually in computers and part of the screening of the active molecules can also be performed in silico. The present invention is also directed to the searching of Helicobacterpylori-binding and/or inhibiting analogues and/or derivatives for the oligosaccharide structures according to the invention by computerized fitting of a carbohydrate structure, analogue or derivative to a carbohydrate binding site on H. pylori.

The Helicobacterpylori-binding oligosacchride sequence, analogues or derivatives thereof are"docked"by methods of molecular modeling to the carbohydrate binding molecule (s) of Helicobacter pylori, most probably to lectins of the bacterium and additional binding interactions are searched. The computerized docking of a three dimensional structure of the oligosaccharide sequence on a three dimensional model of a carbohydrate binding site further helps the design of binding active analogues by allowing determination of binding interactions and positions for possible additional binding interactions. The method is also directed to the comparison of the

binding of the oligosaccharide structures and analogues and derivatives thereof by the computerized docking methods.

The present invention is further directed to the testing of oligosaccharide structures and analogues and derivatives thereof by the computerized docking methods for binding to other pathogenic microbes or viruses or toxins having a binding specificity similar to the binding specificity of Helicobacterpylori with regard to one or several oligosaccharide sequences according to the invention. In a preferred embodiment the oligosaccharide structures and analogues and derivatives thereof are tested for binding to toxin A of Clostridium difficile by the computerized docking methods.

It is also noted that the monovalent, oligovalent or polyvalent oligosaccharides can be activated to have higher activity towards lectins by making a derivative of the oligosaccharide by combinatorial chemistry. When a library is created by substituting one or few residues in the oligosaccharide sequence, it can be considered as a derivative library. Alternatively, when the library is created from the analogs of the oligosaccharide sequences described by the invention, it can be considered as an analog library. A combinatorial chemistry library can be built on the oligosaccharide or its precursor or on glycoconjugates according to the invention. For example, oligosaccharides with variable reducing end can be produced by so called carbohydrid technology. The present invention is directed to the design and production of a combinatorial chemistry library, a multide of chemical analogues and/or derivatives of the oligosaccharide structures according to the invention, and testing these for binding or inhibition of Helicobacter pylori, The present invention is further directed to the testing of the combinatorial chemistry library for binding to other pathogenic microbes or viruses or toxins having a binding specificity similar to the binding specificity of Helicobacter pylori with regard to one or several oligosaccharide sequences according to the invention. In a preferred embodiment the combinatorial chemistry library is tested for binding to toxin A of Clostridium difficile.

In a preferred embodiment a combinatorial chemistry library is conjugated to the Helicobacter pylori binding substances described by the invention. In a more preferred embodiment the library comprises at least 6 different molecules. Preferably the combinatorial chemistry modifications are produced by different amides from carboxylic acid group on R8 or Rg according to Formula 9. Group to be modified in R8 may also be an aldehyde or amine or another type of reactive group. Such library is preferred for use of assaying microbial binding to the oligosaccharide sequences

according to the invention. Amino acids or collections of organic amides are commercially available, which substances can be used for the synthesis of combinatorial library of uronic acid amides. A high affinity binder could be identified from the combinatorial library for example by using an inhibition assay, in which the library compounds are used to inhibit the bacterial binding to the glycolipids or glycoconjugates described by the invention. Structural analogs and derivatives preferred according to the invention can inhibit the binding of the Helicobacterpylori binding oligosaccharide sequences according to the invention to Helicobacter pylori.

In the following the Helicobacter pylori binding sequence is described as an oligosaccharide sequence. The oligosaccharide sequence defined here can be a part of a natural or synthetic glycoconjugate or a free oligosaccharide or a part of a free oligosaccharide. Such oligosaccharide sequences can be bonded to various monosaccharides or oligosaccharides or polysaccharides on polysaccharide chains, for example, the saccharide sequence is expressed as part of a bacterial polysaccharide. Moreover, numerous natural modifications of monosaccharides are known as exemplified by 0-acetyl or sulphated derivatives of the oligosaccharide sequences. In a limited embodiment the oligosaccharide sequence means terminal non-reducing end oligosaccharide sequence which is not modified by any other monosaccharide residue, except optionally at the reducing end. Preferably in broadest sense the term oligosaccharide sequence includes structural analogues and derivatives of the oligosaccharide structures according to the invention, preferably as described by the invention, having same or similar binding activity with regard to the H. pylori. The Helicobacter pylori binding substance defined here can comprise the oligosaccharide sequence described as a natural or synthetic glycoconjugate or part thereof or a corresponding free oligosaccharide or a part of a free oligosaccharide. The Helicobacter pylori binding substance can also comprise a mixture of the Helicobacter pylori binding oligosaccharide sequences.

The Helicobacter pylori binding oligosaccharide sequences can be synthesized enzymatically by glycosyltransferases, or by transglycosylation catalyzed by glycosidase or transglycosidase enzymes (Ernst et al., 2000). Specifities of these enzymes and the use of co-factors can be engineered. Specific modified enzymes can be used to obtain more effective synthesis, for example, glycosynthase is modified to perform transglycosylation only. Organic synthesis of the saccharides and the conjugates described herein or compounds similar to these are known (Ernst et al., 2000). Saccharide materials can be isolated from natural sources and modified chemically or enzymatically into the Helicobacter pylori binding compounds.

Natural oligosaccharides can be isolated from milks of various ruminants.

Transgenic organisms, such as cows or microbes, expressing glycosylating enzymes can be used for the production of the saccharides.

The uronic acid monosaccharide residues described in the invention can be obtained by methods known in the art. For example, the hydroxyl of the 6-carbon of N- acetylglucosamine or N-acetylgalactosamines can be chemically oxidized to carboxylic acid. The oxidation can be performed to a properly protected oligosaccharide or monosaccharide or even to a non-protected carbohydrate.

In a preferred embodiment a non-protected polymer or oligomer comprising hexoses, N-acetylhexosamines or hexosamines, wherein the linkage between the monosaccharides is not between carbon 6 atoms, is 1. oxidized to corresponding polymer of uronic acid residues, or to polymer comprising monomers of 6-aldehydomonosaccharides 2. optionally derivatized from the carboxylic acid group or 6-aldehydo group, preferentially to an amide or an amine and 3) hydrolysed to the uronic acid monosaccharides or uronic acid derivative monosaccharides.

Methods to oxidize monosaccharide residues to uronic acids and to hydrolyse amine or uronic acid polymers chemically or enzymatically are well-known in the art. It is especially preferred to use the method for oligomers or polymers of cellulose, starch or other glucans with 1-2 or 1-3 or 1-4 linkages, chitin (GIcNAc polymer) or chitosan (GlcN polymer), which are commercially available in large scale or N- acetylgalactosamine/galactosamine polysaccharides (for example, ones known from a bacterial source) is oxidized to a corresponding 1-4-linked saccharide. This method can also be applied to galactan polymers. Derivatives of uronic acid can be produced also from natural polymers comprising uronic acids such as pectins or glucuronic acid containing bacterial polysaccharides including N-acetylheparin, hyaluronic and chondroitin type animal or bacterial polysaccharides. This method involves 1. derivatization of the carboxylic acid groups of the polysaccharide, preferably by an amide bond and 2. hydrolysis of the polysaccharide to the uronic acid monosaccharides or uronic acid derivative monosaccharides.

Chemical and enzymatic methods are also known to oxidize primary alcohol on carbon 6 of the polysaccharide to aldehyde or to carboxylic acid. An aldehyde can be further derivatized, for example, to amine by reductive amination. Preferably

terminal Gal or GalNAc is oxidized by a primary alcohol oxidizing enzyme-like galactose oxidase and can then be further derivatized, for example, by amines.

The uronic acid residues can be conjugated to disaccharides or oligosaccharides by standard methods of organic chemistry. Alternatively GIcA can be linked by a glucuronyl transferase transferring a GIcA from UDP-GIcA to terminal Lac (NAc).

Monosaccharide derivatives mimicking N-acetylhexosamines could be produced from a polymer or an oligomer comprising hexosamines or other monosaccharides with free primary amine groups by method involving: 1. derivatization of the amine groups to a secondary or tertiary amine or amide 2. hydrolysing the polymer to corresponding monosaccharides.

Chitosan and oligosaccharides thereof are an example of an amine comprising polymer or oligomer.

In general the method to produce carboxylic acid containing, 6-aldehydo comprising, amine and/or amide comprising monosaccharide/monosaccharides involves following steps 1. optionally introducing an carboxylic acid or 6-aldehydo group to a carbohydrate polymer wherein primary hydroxyl is available for modification 2. derivatization of carboxylic acid groups or 6-aldehydo groups or primary amine groups of the polymer to secondary or tertiary amines or to amides, when step 1 is applied, step 2 is optional.

3. hydrolysis of the polymer to corresponding monosaccharides.

The hydrolysis to monosaccharides may also be partial and produce useful disaccharide or oligosaccharide to produce analog substances. Preferably the hydrolysis produces at least 30 % of monosaccharides. Methods to produce the chemical steps are known in the art. For example oxidation of the polysaccharides to corresponding monoaccharides can be performed as described by Muzzarelli et al 1999 and 2002. These methods are preferred to the use of non-protected monosaccharides, because the protection or reactive reducing ends of the monosaccharides is avoided.

In a preferred embodiment the oligosaccharide sequences comprising GIcAp3Lac or GlcAp3LacNAc are effectively synthesised by transglycosylation using a specific glucuronidase such as glucuronidase from bovine liver. It was realized that the enzyme can site-specifically transfer from pi-3 linkage to Galp4GlcNAc and

Galp4Glc with unexpectedly high yields for a transglycosylation reaction. In general such selectivity and yields close 30 % or more are not obtained in transglycosylation reactions.

Another embodiment of the invention is described in Formula 9. The formula describes preferrable derivatives and analogues of the structures according to the formula 1. The formula shows also preferable modifications for producing structural analogues of the sequences.

Formula 9: A-saccharide : B-saccharide Among the preferable Helicobacterpylorl binding substances or mixtures of the substances of the invention and for the uses of the invention are the oligosaccharide structures according to Formula 9, wherein integers 1, m, and n have values m = 1, 1 and n are independently 0 or 1; Rl is H and R2 is OH, or RI is OH and R2 is H, or RI is H and R2 is a monosaccharidyl-or oligosaccharidyl-group, preferably a beta glycosidically linked galactosyl group; RI 1 is H and R12 is OH, or Ri 1 is OH and R12 is H; R3 is independently-OH or acetamido (-NHCOCH3) or an acetamido analogous group, R7 is acetamido (-NHCOCH3) or an acetamido analogous group; when 1 = 1, R4 is-H and Rs is oxygen linked to bond R6 and forms a beta anomeric glycosidic linkage to saccharide B, or R5 is-H and R4 is oxygen linked to bond R6 and forms an alpha anomeric glycosidic linkage to saccharide B; when l = 0, R6 is- OH linked to B; X is monosaccharide or oligosaccharide residue, X is lactosyl-, galactosyl-, poly-N-acetyl-lactosaminyl, or part of an O-glycan or an N-glycan oligosaccharide sequence, or Glc, GlcNAc OR GalNAc or part of chondroitin or hyaluronic acid oligosaccharide sequence; Y is a spacer group or a terminal conjugate such as a ceramide lipid moiety or a linkage to Z; Z is an oligovalent or a polyvalent carrier ; the oxygen linkage (-O-) between position C 1 ~of the B saccharide and saccharide residue X or spacer group Y can be replaced by carbon (-C-), nitrogen (-N-) or sulphur (-S-) linkage; Rs and Rs are independently carboxylic acid amide, such as methylamide or ethyalamide, hydroxymethyl (-CH2-OH) or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester; R3, R7, and

Rio are independently hydroxyl, acetamido or acetamido group mimicking group, such as C 1-6 alkyl-amides, arylamido, secondary amine, preferentially N-ethyl or N- methyl, 0-acetyl, or 0-alkyl for example O-ethyl or 0-methyl, with the provisio that Rio is acetamido or acetamido group mimicking group, or Rg is independently carboxylic acid amide, such as methylamide or ethyalamide, or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester, or R12 is OH; preferably so that Rio is acetamido or acetamido group mimicking group or R12 is OH or so that Rio is acetamido or acetamido group mimicking group and R12 is OH or preferably so that two of the groups Rg, Rio and Rl does not comprise OH as described above.

In Formula 9 R8 is preferably carboxylic acid amide, such as-CO-NH2, methylamide or ethyalamide, hydroxymethyl (-CH2-OH) or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester. The carboxylic acid amide may comprise an alternative linkage to the polyvalent carrier Z comprising an amine such as chitosan or galactosamine polysaccharide or Z comprising a primary amine containing spacer, preferably a hydrophilic spacer. The structure in R8 can be also a mimicking structure known in the art to ones described above. For example secondary or tertiary amines or amidated secondary amine can be used.

In Formula 9 Rg is preferably hydroxymethyl or carboxylic acid amide, but it can be used for derivatisations as described for R8.

R3 is hydroxyl, acetamido or acetamido group mimicking group, such as Ci-6 alkyl- amides, arylamido, secondary amine, preferentially N-ethyl or N-methyl, 0-acetyl, or 0-alkyl for example O-ethyl or 0-methyl. R7 and Rio are same as R3 but more preferentially acetamido or acetamido mimicking group. Ra may also comprise preferentially a six-membered ring structure mimicking Gal (34- terminal.

The bacterium binding substances are preferably represented in clustered form such as by glycolipids on cell membranes, micelles, liposomes, or on solid phases such as TCL-plates used in the assays. The clustered representation with correct spacing creates high affinity binding.

According to the invention it is also possible to use Helicobacter pylori binding epitopes or naturally occurring, or a synthetically produced analogue or derivative thereof having a similar or better binding activity with regard to Helicobacter pylori.

It is also possible to use a substance containing the bacterium binding substance such as a receptor active ganglioside described in the invention or an analogue or derivative thereof having a similar or better binding activity with regard to

Helicobacter pylori. The bacterium binding substance may be a glycosidically linked terminal epitope of an oligosaccharide chain. Alternatively the bacterium binding epitope may be a branch of an oligosaccharide chain, preferably a polylactosamine chain.

The Helicobacter pylori binding substance may be conjugated to an antibiotic substance, preferably a penicillin type antibiotic. The Helicobacter pylori binding substance targets the antibiotic to Helicobacter pylori. Such conjugate is beneficial in treatment because a lower amount of antibiotic is needed for treatment or therapy against Helicobacter pylori, which leads to lower side effect of the antibiotic. The antibiotic part of the conjugate is aimed at killing or weaken the bacteria, but the conjugate may also have an antiadhesive effect as described below.

The bacterium binding substances, preferably in oligovalent or clustered form, can be used to treat a disease or condition caused by the presence of the Helicobacter pylori. This is done by using the Helicobacter pylori binding substances for anti- adhesion, i. e. to inhibit the binding of Helicobacter pylori to the receptor epitopes of the target cells or tissues. When the Helicobacter pylori binding substance or pharmaceutical composition is administered it will compete with receptor glycoconjugates on the target cells for the binding of the bacteria. Some or all of the bacteria will then be bound to the Helicobacter pylori binding substance instead of the receptor on the target cells or tissues. The bacteria bound to the Helicobacter pylori binding substances are then removed from the patient (for example by the fluid flow in the gastrointestinal tract), resulting in reduced effects of the bacteria on the health of the patient. Preferably the substance used is a soluble composition comprising the Helicobacter pylori binding substances. The substance can be attached to a carrier substance which is preferably not a protein. When using a carrier molecule several molecules of the Helicobacter pylori binding substance can be attached to one carrier and inhibitory efficiency is improved.

The target cells are primarily epithelial cells of the target tissue, especially the gastrointestinal tract, other potential target tissues are for example liver and pancreas. Glycosylation of the target tissue may change because of infection by a pathogen (Karlsson et al., 2000). Target cells may also be malignant, transformed or cancer/tumour cells in the target tissue. Transformed cells and tissues express altered types of glycosylation and may provide receptors to bacteria. Binding of lectins or saccharides (carbohydrate-carbohydrate interaction) to saccharides on glycoprotein or glycolipid receptors can activate cells, in case of cancer/malignant cells this may be lead to growth or metastasis of the cancer. Several of the oligosaccharide epitopes

described herein, such as GlcNAcp3Galp4GlcNAc (Hu, J. et al., 1994), Gala3Galp4GlcNAc (Castronovo et al., 1989), and neutral and sialylated polylactosamines from malignant cells (Stroud et al., 1996), have been reported to be cancer-associated or cancer antigens. Oligosaccharide chains containing substances have also been described from lymphocytes (Vivier et al., 1993).

Helicobacter pylori is associated with gastric lymphoma. The substances of the invention can be used to prevent binding of Helicobacter pylori to premalignant or malignant cells and activation of cancer development or metastasis. Inhibition of the binding may cure gastric cancer, especially lymphoma. The Helicobacter pylori binding oligosaccharide sequence has been reported in the structure GIcNAcp3Galp4GlcNAc (36GalNAc from human gastric mucins. This mucin epitope and similar O-glycan glycoforms are most probably natural high affinity receptors for Helicobacter pylori in human stomach. This was also indicated by high affinity binding of an analogous sequence GIcNAc 3Gal 4GlcNAc (36GlcNAc as neoglycolipid to Helicobacter pylori and that the sequence GlcNAcß3Galß4GlcNAcß6Gal has also some binding activity towards Helicobacter pylori in the same assay. Therefore the preferred oligosaccharide sequences includes O-glycans and analogues of O-glycan sequences such as GlcNAcß3Galß4GlcNAcß6GlcNAc/GalNAc/Gal, <BR> <BR> <BR> GlcNAcß3Galß4GlcNAcß6GlcNAc/GalNAc/GalaSer/Thr,<BR> <BR> <BR> <BR> <BR> GlcNAcß3Galß4GlcNAcß6 (GaVGlcNAcß3) GlcNAc/GalNAc/GalaSer/Thr and glycopeptides and glycopeptide analogs comprising the O-glycan sequences. Even sequences lacking the non-reducing end GlcNAc may have some activity. Based on this all the other Helicobacter pylori binding oligosaccharide sequences (OS) and especially the trisaccharide epitopes are also especially preferred when linked from the reducing end to form structures OS (36Gal (NAc) o or OSß6Glc (NAc) o-l or OSp6Gal (NAc) o laSer/Thr or OSp6Glc (NAc) o-laSer/Thr. The Ser or Thr- compounds or analogue thereof or the reducing oligosaccharides are also preferred when linked to polyvalent carrier. The reducing oligosaccharides can be reductively linked to the polyvalent carrier.

Target cells also includes blood cells, especially leukocytes. It is known that Helicobacter pylori strains associated with peptic ulcer, as the strain mainly used here, stimulates an inflammatory response from granulocytes, even when the bacteria are nonopsonized (Rautelin et al., 1994a, b). The initial event in the phagocytosis of the bacterium most likely involves specific lectin-like interactions resulting in the agglutination of the granulocytes (Ofek and Sharon, 1988).

Subsequent to the phagocytotic event oxidative burst reactions occur which may be

of consequence for the pathogenesis of Helicobacterpylori-associated diseases (Babior, 1978). Several sialylated and non-acid glycosphingolipids having repeating N-acetyllactosamine units have been isolated and characterized from granulocytes (Fukuda et al., 1985 ; Stroud et al., 1996) and may thus act as potential receptors for Helicobacter pylori on the white blood cell surface. Furthermore, also the X2 glycosphingolipid has been isolated from the same source (Teneberg, S. , unpublished). The present invention confirms the presence of receptor saccharides on human erythrocytes and granulocytes which can be recognized by an N- acetyllactosamine specific lectin and by a monoclonal antibody (x2, GalNAcp3Gal (34GlcNAc-). The Helicobacter pylori binding substances can be useful to inhibit the binding of leukocytes to Helicobacter pylori and in prevention of the oxidative burst and/or inflammation following the activation of leukocytes.

It is known that Helicobacter pylori can bind several kinds of oligosaccharide sequences. Some of the binding by specific strains may represent more symbiotic interactions which do not lead to cancer or severe conditions. The present data about binding to cancer-type saccharide epitopes indicates that the Helicobacter pylori binding substance can prevent more pathologic interactions, in doing this it may leave some of the less pathogenic Helicobacter pylori bacteria/strains binding to other receptor structures. Therefore total removal of the bacteria may not be necessary for the prevention of the diseases related to Helicobacter pylori. The less pathogenic bacteria may even have a probiotic effect in the prevention of more pathogenic strains of Helicobacter pylori.

It is also realized that Helicobacter pylori contains large polylactosamine oligosaccharides on its surface which at least in some strains contains non- fucosylated epitopes which can be bound by the bacterium as described by the invention. The substance described herein can also prevent the binding between Helicobacter pylori bacteria and that way inhibit bacteria for example in process of colonization.

According to the invention it is possible to incorporate the Helicobacterpylori binding substance, optionally with a carrier, in a pharmaceutical composition, which is suitable for the treatment of a condition due to the presence of Helicobacter pylori in a patient or to use the Helicobacter pylori binding substance in a method for treatment of such conditions. Examples of conditions treatable according to the invention are chronic superficial gastritis, gastric ulcer, duodenal ulcer, non-Hodgkin lymphoma in human stomach, gastric adenocarcinoma, and certain pancreatic, skin, liver, or heart diseases, sudden infant death syndrome, autoimmune diseases

including autoimmune gastritis and pernicious anaemia and non-steroid anti- inflammatory drug (NSAID) related gastric disease, all, at least partially, caused by the Helicobacter pylori infection.

The pharmaceutical composition containing the Helicobacterpylori binding substance may also comprise other substances, such as an inert vehicle, or pharmaceutically acceptable carriers, preservatives etc, which are well known to persons skilled in the art. The Helicobacter pylori binding substance can be administered together with other drugs such as antibiotics used against Helicobacter pylori.

The Helicobacter pylori binding substance or pharmaceutical composition containing such substance may be administered in any suitable way, although an oral administration is preferred.

The term"treatment"used herein relates both to treatment in order to cure or alleviate a disease or a condition, and to treatment in order to prevent the development of a disease or a condition. The treatment may be either performed in a acute or in a chronic way.

The term"patient"or"subject", as used herein, relates to any human or non-human mammal in need of treatment according to the invention.

It is also possible to use the Helicobacterpylori binding substance to identify one or more adhesins by screening for proteins or carbohydrates (by carbohydrate- carbohydrate interactions) that bind to the Helicobacter pylori binding substance.

The carbohydrate binding protein may be a lectin or a carbohydrate binding enzyme.

The screening can be done for example by affinity chromatography or affinity cross linking methods (Ilver et al., 1998).

Furthermore, it is possible to use substances specifically binding or inactivating the Helicobacter pylori binding substances present on human tissues and thus prevent the binding of Helicobacter pylori. Examples of such substances include plant lectins such as Erythrina cristagalli and Erythrina corallodendron (Teneberg et al., 1994). When used in humans, the binding substance should be suitable for such use such as a humanized antibody or a recombinant glycosidase of human origin which is non-immunogenic and capable of cleaving the terminal monosaccharide residue/residues from the Helicobacter pylori binding substances. However, in the

gastrointestinal tract, many naturally occuring lectins and glycosidases originating for example from food are tolerated.

Furthermore, it is possible to use the Helicobacter pylori binding substance as part of a nutritional composition including food-and feedstuff. It is preferred to use the Helicobacter pylori binding substance as a part of so called functional or functionalized food. The said functional food has a positive effect on the person's or animal's health by inhibiting or preventing the binding of Helicobacter pylori to target cells or tissues. The Helicobacter pylori binding substance can be a part of a defined food or functional food composition. The functional food can contain other acceptable food ingredients accepted by authorities such as Food and Drug Administration in the USA. The Helicobacterpylori binding substance can also be used as a nutritional additive, preferably as a food or a beverage additive to produce a functional food or a functional beverage. The food or food additive can also be produced by having, e. g. , a domestic animal such as a cow or other animal produce the Helicobacter pylori binding substance in larger amounts naturally in its milk.

This can be accomplished by having the animal overexpress suitable glycosyltransferases in its milk. A specific strain or species of a domestic animal can be chosen and bred for larger production of the Helicobacter pylori binding substance. The Helicobacter pylori binding substance for a nutritional composition or nutritional additive can also be produced by a micro-organisms such as a bacteria or a yeast.

It is especially useful to have the Helicobacter pylori binding substance as part of a food for an infant, preferably as a part of an infant formula. Many infants are fed by special formulas in replacement of natural human milk. The formulas may lack the special lactose based oligosaccharides of human milk, especially the elongated ones such as lacto-N-neotetraose, Galp4GlcNAcp3Galp4Glc, and its derivatives. The natural type oligosaccharide derived from desulphated chondroitin, and hyaluronic acid are preferred for infant formulas, other functional foods and food additive. Also the carboxylic acid reduced hyaluronic acid and chondroitin oligosaccharides according to the invention are considered useful for use in infant formulas as close analogs of the natural type polylactosamines and lactose based milk oligosaccharides, the chondroitin saccharides has also homology with core 1 structures of O-linked glycans, moreover disaccharide GaINAcp4Glc is a component of bovine milk. For example following oligosaccharides are preferred for infant foods, other functional foods, feeds and nutritional additives:

GaINAc 4Glc, Glcp3GalNAcp4Glc, Glcß3GalNAcß4Glcß3GalNAcß4Glc, <BR> <BR> <BR> Glcp3GaINAcp4Glcp3GalNAcp4Glcp3GalNAc4Glc,<BR> <BR> <BR> <BR> <BR> <BR> <BR> Glc (33Ga1NAc (34G1c (33GalNAc, Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAc, Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAcß4Glcß3GalNAc, Glcß3GlcNAcß4Glcß3GlcNAc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAc, Glc ß3GlcNAcß4Glc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glc, Glcß3GlcNAcß4Glcß3GlcNAcß4Glcß3GlcNAcß4Glc.

These can be considered as safe additives or ingredients in an infant food.

Helicobacter pylori is especially infective with regard to infants or young children, and considering the diseases it may later cause it is reasonable to prevent the infection. Helicobacter pylori is also known to cause sudden infant death syndrome, but the strong antiobiotic treatments used to eradicate the bacterium may be especially unsuitable for young children or infants.

Furthermore, it is possible to use the Helicobacter pylori binding substance in the diagnosis of a condition caused by an Helicobacter pylori infection. Diagnostic uses also include the use of the Helicobacterpylori binding substance for typing of Helicobacter pylori. When the substance is used for diagnosis or typing, it may be included in, e. g., a probe or a test stick, optionally constituting a part of a test kit.

When this probe or test stick is brought into contact with a sample containing Helicobacter pylori, the bacteria will bind the probe or test stick and can be thus removed from the sample and further analyzed.

The results also show that the non-reducing end terminal monosaccharide residue in the preferred trisaccharide sequences of the invention can contain a carboxylic acid group on the carbon 6 (terminal monosaccahride residue is a uronic acid, HexA or HexANAc, wherein Hex is Gal or Glc) or a derivative of the carbon 6 of the HexA (NAc) residue or a derivative of the carbon 6 of the corresponding Hex (NAc) residue. Such terminal residues includes preferably ß3-linked glucuronic acid and more preferably 6-amides such as methylamide thereof. Therefore analogs and derivatives of the sequence can be produced by changing or derivatising the terminal 6-position of the trisaccharide epitopes.

Preferred Helicobacter zaylori binding substances The oligosaccharide sequences according to the invention were found to be unexpectedly effective binders when presented on thin layer surface. This method allows polyvalent presentation of the glycolipid sequences. The surprisingly high activity of the polyvalent presentation of the oligosaccharide sequences makes polyvalency a preferred way to represent the oligosaccharide sequences of the invention.

The glycolipid structures are naturally presented in a polyvalent form on cellular membranes. This type of representation can be mimicked by the solid phase assay described below or by making liposomes of glycolipids or neoglycolipids.

The present novel neoglycolipids produced by reductive amination of hydrophobic hexadecylaniline were able to provide effective presentation of the oligosaccharides.

Most previously known neoglycolipid conjugates used for binding of bacteria have contained a negatively charged groups, such as phosphor ester of phosphadityl ethanolamine neoglycolipids. Problems of such compounds are negative charge of the substance and natural biological binding involving the phospholipid structure.

Negatively charged molecules are known to be involved in numerous non-specific bindings with proteins and other biological substances. Moreover, many of these structures are labile and can be enzymatically or chemically degraded. The present invention is directed to the non-acidic conjugates of oligosaccharide sequences meaning that the oligosaccharide sequences are linked to non-acidic chemical structures. Preferably, the non-acidic conjugates are neutral meaning that the oligosaccharide sequences are linked to neutral, non-charged, chemical structures.

The preferred conjugates according to the invention are polyvalent substances.

In the previous art bioactive oligosaccharide sequences are often linked to carrier structures by reducing a part of the receptor active oligosaccharide structure.

Hydrophobic spacers containing alkyl chains (-CH2-) n and/or benzyl rings have been used. However, hydrophobic structures are in general known to be involved in non- specific interactions with proteins and other bioactive molecules.

The neoglycolipid data of the examples below show that under the experimental conditions used in the assay the hexadecylaniline parts of the neoglycolipid compounds do not cause non-specific binding for the studied bacterium. In the neoglycolipids the hexadecylaniline part of the conjugate forms probably a lipid layer like structure and is not available for the binding. The invention shows that reducing a monosaccharide residue belonging to the binding epitope may destroy the

binding. It was further realized that a reduced monosaccharide can be used as a hydrophilic spacer to link a receptor epitope and a polyvalent presentation structure.

According to the invention it is preferred to link the bioactive oligosaccharide via a hydrophilic spacer to a polyvalent or multivalent carrier molecule to form a polyvalent or oligovalent/multivalent structure. All polyvalent (comprising more than 10 oligosaccharide residues) and oligovalent/multivalent structures (comprising 2-10 oligosaccharide residues) are referred here as polyvalent structures, though depending on the application oligovalent/multivalent constructs can be more preferred than larger polyvalent structures. The hydrophilic spacer group comprises preferably at least one hydroxyl group. More preferably the spacer comprises at least two hydroxyl groups and most preferably the spacer comprises at least three hydroxyl groups.

According to the invention the hydrophilic spacer group is preferably a flexible chain comprising one or several-CHOH-groups and/or an amide side chain such as an acetamido-NHCOCH3 or an alkylamido. The hydroxyl groups and/or the acetamido group also protects the spacer from enzymatic hydrolysis in vivo. The term flexible means that the spacer comprises flexible bonds and do not form a ring structure without flexibility. A reduced monosaccharide residues such as ones formed by reductive amination in the present invention are examples of flexible hydrophilic spacers. The flexible hydrophilic spacer is optimal for avoiding non- specific binding of neoglycolipid or polyvalent conjugates. This is essential optimal activity in bioassays and for bioactivity of pharmaceuticals or functional foods, for example.

A general formula for a conjugate with a flexible hydrophilic linker has the following Formula 10: <BR> <BR> <BR> <BR> <BR> <BR> <BR> [OS-0- (X).-LI-CH (H/ICHI-20HIpi)-ICHIOH} p2-ICH (NH-R) l P3-f CHIOHI p4-L2].-Z wherein L, and L2 are linking groups comprising independently oxygen, nitrogen, sulphur or carbon linkage atom or two linking atoms of the group forming linkages such as -O-, -S-, -CH2-, -N-, -N (COCH3) -, amide groups-CO-NH-or-NH-CO-or -N-N- (hydrazine derivative) or amino oxy-linkages-O-N-and-N-O-. L1 is linkage from carbon 1 of the reducing end monosaccharide of X or when n =0, LI replaces- O-and links directly from the reducing end Cl of OS. pl, p2, p3, and p4 are independently integers from 0-7, with the proviso that at least one of pl, p2, p3, and p4 is at least 1. CHl-20H in the branching term {CHs 20H} pl

means that the chain terminating group is CH20H and when the pi is more than 1 there is secondary alcohol groups-CHOH-linking the terminating group to the rest of the spacer. R is preferably acetyl group (-COCH3) or R is an alternative linkage to Z and then L2 is one or two atom chain terminating group, in another embodiment R is an analog forming group comprising Cl-4 acyl group (preferably hydrophilic such as hydroxy alkyl) comprising amido structure or H or C1 4 alkyl forming an amine. And m > 1 and Z is polyvalent carrier. OS and X are defined in Formula 9.

In a preferred embodiment of the invention pl is 0. The invention shows that open chain forms of a monosaccharide are effective H. pylori binding structures at the reducing end. These structures can mimic ceramides with hydroxylfattyacid. In a preferred embodiment of the invention the hydrophilic linker according to Formula 10 comprise a hydroxyl group at the same distance from the reducing end oligosaccharide sequence as the corresponding hydroxyl group is on ceramide from the reducing end oligosaccharide in natural glycosphingolipids with hydroxylfattyacid.

Preferred polyvalent structures comprising a flexible hydrophilic spacer according to formula 2 include Helicobacterpylori binding oligosaccharide sequence (OS) (31-3 linked to Gal (34Glc (red) -Z, and OSß6GlcNAc (red) -Z and OSß6GalNAc (red) -Z., where" (red)" means the amine linkage structure formed by reductive amination from the reducing end monosaccharides and an amine group of the polyvalent carrier Z.

In the present invention the oligosaccharide group is preferably linked in a polyvalent or an oligovalent form to a carrier which is not a protein or peptide to avoid antigenicity and possible allergic reactions, preferably the backbone is a natural non-antigenic polysaccharide.

The novelty of the glucuronic acid derivative containing oligosaccharide sequences makes them especially preferred. There are no known glycosidases cleaving such sequences. Therefore, the sequences are especially stable and preferred under biolological conditions. The natural type of the sequences described by the invention can be cleaved by glycosidase enzymes which reduces usefulness of these especially when used in human and animal body. Glycosidase enzymes cleaving the sequences are known to be active in human gastrointestinal tract. Several glycosidases such as N-acetylhexosaminidases or galactosidases has been described as digestive enzyme and are also present in food stuffs.

Ex vivo uses of the present invention It is realized that the present invention can be used for inhibition of pathogens especially Helicobacter pylori ex vivo and such method have use in disinfection and preservevation type applications.

Glycolipid and carbohydrate nomenclature is according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (Carbohydrate Res. 1998, 312,167 ; Carbohydrate Res. 1997,297, 1; Eur. J. Biochem. 1998, 257,29).

It is assumed that Gal, Glc, GlcNAc, and Neu5Ac are of the D-configuration, Fuc of the L-configuration, and all the monosaccharide units in the pyranose form.

Glucosamine is referred as GlcN or GlcNH2 and galactosamine as GaIN or GalNH2.

Glycosidic linkages are shown partly in shorter and partly in longer nomenclature, the linkages of the Neu5Ac-residues a3 and a6 mean the same as a2-3 and a2-6, respectively, and with other monosaccharide residues al-3, (31-3, (31-4, and (31-6 can be shortened as a3, (33, (34, and (36, respectively. Lactosamine refers to N- acetyllactosamine, Galß4GlcNAc, and sialic acid is N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) or any other natural sialic acid.

Term glycan means here broadly oligosaccharide or polysaccharide chains present in human or animal glycoconjugates, especially on glycolipids or glycoproteins. In the shorthand nomenclature for fatty acids and bases, the number before the colon refers to the carbon chain lenght and the number after the colon gives the total number of double bonds in the hydrocarbon chain. Abbreviation GSL refers to glycosphingolipid. Abbreviations or short names or symbols of glycosphingolipids are given in the text and in Tables 1 and 2. Helicobacter pylori refers also to the bacteria similar to Helicobacter pylori.

In the present invention hex (NAc) -uronic acid and their derivatives and residues are indicated as follows: GlcA is glucuronic acid and derivatives of carbon 6 of glucose or glucuronic acid, GalA is galacturonic acid and derivatives of carbon 6 of galactose or galacturonic acid, GlcANAc is N-acetylglucuronic acid and derivatives of carbon 6 of N-acetylglucosamine or is N-acetylglucosamine uronic acid and GalANAc is N-acetylgalactosamine uronic acid and derivatives of carbon 6 of N- acetylgalactosamine or N-acetylgalactosamine uronic acid.

The expression"terminal oligosaccharide sequence"indicates that the oligosaccharide is not substituted to the non-reducing end terminal residue by another monosaccharide residue.

The term"a3/ß3"indicates that the adjacent residues in an oligosaccharide sequence can be either a3-or (33-linked to each other.

The present invention is further illustrated by the following examples, which in no way are intended to limit the scope of the invention: EXAMPLES Example 1. General materials and methods Materials-TLC silica gel 60 (aluminum) plates were from Merck (Darmstadt, Germany). Ham's F12 medium from Gibco (U. K.), 35S-methionine from Amersham (U. K. ) and FCS (fetal calf serum) was from Sera-Lab (England). The clinical isolates of Helicobacterpylori (strains 002 and 032) obtained from patients with gastritis and duodenal ulcer, respectively, were a generous gift from Dr. D.

Danielsson, Orebro Medical Center, Sweden. Type strain 17875 was from Culture Collection, University of Göteborg (CCUG).

Glycosphingolipids. The pure glycosphingolipids of the experiment shown in Figs. 7A and 7B were prepared from total acid or non-acid fractions from the sources listed in Table 1 as described in (Karlsson, 1987). In general, individual glycosphingolipids were obtained by acetylation (Handa, 1963) of the total glycosphingolipid fractions and separated by repeated silicic acid column chromatography, and subsequently characterized structurally by mass spectrometry (Samuelsson et al., 1990), NMR (Falk et al., 1979a, b, c; Koerner Jr et al., 1983) and degradative procedures (Yang and Hakomori, 1971; Stellner et al., 1973).

Glycolipids derived from rabbit thymys are described below.

De-N-acylation. Conversion of the acetamido moiety of GlcNAc/GalNAc residues into an amine was accomplished by treating various glycosphingolipids with anhydrous hydrazine as described previously (Angstrom et al., 1998).

Bacterial growth. The Helicobacter pylori strains were stored at-80 °C in tryptic soy broth containing 15% glycerol (by volume). The bacteria were initially cultured on GAB-CAMP agar (Soltesz et al., 1988) under humid (98%) microaerophilic conditions (02 : 5-7%, COa : 8-10% and N2 : 83-87%) at 37 °C for 48-72 h. For labeling colonies were inoculated on GAB-CAMP agar, except for the results presented in Figs. lA and 1B where Brucella agar (Difco, Detroit, MI) was used instead, and 50 pCi 35S-methionine (Amersham, U. K. ), diluted in 0.5 ml phosphate- buffered saline (PBS), pH 7.3, was sprinkled over the plates. After incubation for 12- 24 h at 37 °C under microaerophilic conditions, the cells were scraped off, washed three times with PBS, and resuspended to lx108 CFU/ml in PBS. Alternatively,

colonies were inoculated (1x105 CFU/ml) in Ham's F12 (Gibco BRL, U. K.), supplemented with 10% heat-inactivated fetal calf serum (Sera-Lab). For labeling, 50 uCi 35S-methionine per 10 ml medium was added, and incubated with shaking under microaerophilic conditions for 24 h. Bacterial cells were harvested by centrifugation, and purity of the cultures and a low content of coccoid forms was ensured by phase-contrast microscopy. After two washes with PBS, the cells were resuspended to 1x108 CFU/ml in PBS. Both labeling procedures resulted in suspensions with specific activities of approximately 1 cpm per 100 Helicobacter pylori organisms.

TLC bacterial overlay assay. Thin-layer chromatography was performed on glass-or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany) using chloroform/methanol/water 60: 35: 8 (by volume) as solvent system.

Chemical detection was accomplished by anisaldehyde staining (Waldi, 1962). The bacterial overlay assay was performed as described previously (Hansson et al., 1985). Glycosphingolipids (1-4 pg/lane, or as indicated in the figure legend) were chromatographed on aluminum-backed silica gel plates and thereafter treated with 0.3-0. 5% polyisobutylmethacrylate in diethylether/n-hexane 1: 3 (by volume) for 1 min, dried and subsequently soaked in PBS containing 2% bovine serum albumin and 0.1% Tween 20 for 2 h. A suspension of radio-labeled bacteria (diluted in PBS to 1 x 108 CFU/ml and 1-5x106 cpm/ml) was sprinkled over the chromatograms and incubated for 2 h followed by repeated rinsings with PBS. After drying the chromatograms were exposed to XAR-5 X-ray films (Eastman Kodak Co., Rochester, NY, USA) for 12-72 h.

Molecular modeling was performed as described in the priority application and in Teneberg et al-96.

Synthesis of oligosaccharides. The oligosaccharide GlcNAcß3Galß4GlcNAc was synthesised from Galp4GlcNAc (Sigma, St. Louis, USA), GalNAcp4GlcNAcp3Galp4GlcNAc was synthesized from the trisaccharide by transferring GalNAc from vast excess of UDP-GaINAc (Sigma) by large amounts of ß4Galactosyltransferase (bovine milk, Calbiochem., CA, USA) and GIcNAcp3Gal 4GlcNAcp6GlcNAc, GlcNAcp3Galp4GlcNAcp6Galp4Glc, GlcNAcp3Galp4GlcNAca6GlcNAc, GlcNAcp3Galp4GlcNAcp3Man were synthesised from Galß4GlcNAcß6GlcNAc, Galß4GlcNAcß6Galß4Glc, Galp4GlcNAca6GlcNAc, and Galj34GlcNAcp3Man, respectively, by incubating the acceptor saccharide with human serum ß3-N-acetylglucosaminyltransferase and UDP-GlcNAc in presence of 8 mM MnCl2 and 0.2 mg/ml ATP at 37 degree of Celsius for 5 days in 50 mM TRIS-HCl pH 7.5. Galß4GlcNAcß6GlcNAc, Galp4GlcNAcp6Galp4Glc, Galp4GlcNAca6GlcNAc, and Gal (34G1cNAc i3Man

were obtained from GIcNAcp6GlcNAc (Sigma, St Louis, USA), GlcNAcß6Galß4Glc (Sigma), GlcNAca6GlcNAc (HC1 vapor catalyzed acid reversion rection for solid GlcNAc (Sigma) in dessicator and chromatographic purification, NMR, and mass spectrometric analysis), and GlcNAcß3Man (DextraLabs Reding, UK) by incubating the acceptor saccharide with ß4Galactosyltransferase (bovine milk, Calbiochem. , CA, USA) and UDP-Gal in presence of 20 mM MnCl2 for several hours in 50 mM MOPS-NaOH pH 7.4.

Hexasaccharide Galß3GlcNAcß3Galß4GlcNAcß3Galß4Glc (1 mg, from Dextra labs, UK) ) was treated with 400 mU ß3/6-galactosidase (Calbiochem. , CA, USA) overnight as suggested by the producer to obtain GlcNAcß3GlcNAcß3Galß4GlcNAcß3Galß4Glc, GlcAß3Galß4GlcNAcß6GlcNAc, GlcAp3Galp4GlcNAcp3Galp4Glc were produced from Galp4GlcNAcp6GlcNAc, Galp4GlcNAcp3Galp4Glc by transglycosylation catalysed by glucuronidase enzyme (bovine testes, Sigma) using GIcA-paranitrophenyl as donor substrate. Glc (A- methylamide) ß3Galß4GlcNAcß6GlcNAc was obtained by methylamidation of the corresponding glucuronyloligosaccharide as described. The oligosaccharides were purified chromatographically and their purity was assessed by MALDI-TOF mass spectrometry and NMR. Gala3Galp4GlcNAcp3Galp4Glc was from Dextra laboratories, Reading, UK. The glycolipid GlcAp3Galp4GlcNAcp3Galp4GlcpCer (Wako Pure Chemicals, Osaka, Japan) was reduced to Glcp3Galp4GlcNAcp3Galp4GlcpCer as described in Lanne et al 1995. The glycolipid derivative Glc (A-methylamide) p3Galp4GlcNAcp3Galp4GlcpCer was produced by amidatation of the carboxylic acid group of the glucuronic acid of GlcAß3Galß4GlcNAcß3Galß4GlcßCer as described in Lanne et al 1995.

Beta3-glucuronidation and amidation of Galß4GlcNAcß6GlcNAc Gal (34G1cNAc (36G1cNAc was ß3-glucuronidated by transglycosylation using paranitrophenylglucuronide as the donor and bovine ß l, 3-glucuronidase as the enzyme. The product was purified by solid-phase extraction using a C-18 material, anion-exchange chromatography and gel filtration.

The glucuronic acid unit in GlcAß3Galß4GlcNAcß6GlcNAc was converted to glucuronmethylamide as follows: The tetrasaccharide was dissolved in pyridine containing 10% water, and 5-fold molar excess of HBTU (Benzotriazole-1-yl- 1,1, 3,3,-tetramethyluroniumhexafluorophosphate) and DIPEA (N, N,- diisopropylethylamine) were added. 50-fold molar excess of methylamine was then added and the reaction was allowed to proceed for 48 h at room temperature. The methylamidated tetrasaccharide was puridied by gel filtration, anion-exchange chromatography as well as hydrophilic interaction chromatoraphy. MALDI-TOF

mass spectrum of the purified product (Fig. 2) shows the expected signals: m/z 798.2 ([M+Na] +) and 814.2 ( [M+K] +). The structure of the molecule was confirmed NMR- spectormetry.

The glucuronic acid unit in GlcAß3Galß4GlcNAcß6GlcNAc was also converted to glucuronamide. Essentially similar method was used as above, except that ammonia was used as the base. The structure of the molecule was confirmed by MALDI-TOF mass spectrometry and NMR-spectormetry- Example 2 Production of amidated chondroitin type oligosaccharides Chondroitin sulphate A (Sigma) was converted to pyridinium salt by running the sample through cation exchange resin (hydrogen form), after which the solution was titrated to slightly basic pH with pyridine, and then dried in a vacuum sentrifuge.

Desulphation was carried out by dissolving the dry sample to DMSO containing 10% methanol, and incubating this solution for 4h at 80° C. DMSO was removed by extensive dialysis against water.

Oligosaccharides were produced from the desulphated material by acid hydrolysis in 0.5 M TFA at 60° C for 18 h. In these conditions tetra-and hexasaccharides with GaINAc at their reducing end are effectively produced. Pure tetra-and hexasaccharides were isolated by use of gel filtration and anion-exchange chromatography. NMR-spectorometry reavealed oligosaccharide with non-reducing terminal GIcA and reducing termial GaINAc. The hydrolysis method revealed to be surprisingly selective in cleaving GaINAcp4GlcA-bond. The use of volatile acid allows use of oligosaccharide mixrures even without desalting, which is another benefit of using trifluoroacetic acid or similar strong carboxylic acids.

Amide groups were introduced to glucuronic acid residues of chondroitin tetrasaccharides and hexasaccharides by using HBTU activator and ammonia as described above. Pure amidated tetra-and hexasaccharides were isolated by use of gel filtration and anion-exchange chromatography. The MALDI-TOF mass spectra of the purified products (Figs. 3 and 4) show the expected signals.

Alternatively, amide groups can be introduced to the desulphated chondroitin sulphate polymer, using the methodology essentially as described above. When using polymer in a preferred method amidation reagents are effectively removed by extensive dialysis, and modified oligosaccharides are produced by acid hydrolysis, as described above.

The methods to degradate polysaccharide and amidate glucuronic acid residues described above are also used be (without desulphation step) for commercial hyaluronic acid (for example Sigma) or oligosaccharides such as GlcAp3GlcNAcp4GlcAp3GlcNAc and GlcAß3GlcNAcß4GlcAß3GlcNAcß4GlcAß3GlcNAc derived from hyaluronic acid (as above). Alternatively hyaluronidase (bovine, Sigma) can be used for production of chondroitin or chondroitin sulphate or hyaluronic acid oligosaccharides.

Example 3 The binding of Helicobacterpylori (strain 032, several other strains were used in other experiments) to purified glycosphingolipids separated on thin-layer plates using the overlay assay is shown in Figs. 1 panels A and B. These results together with those from an additional number of purified glycosphingolipids are summarized in Table 1. The binding of Helicobacter pylori to neolactotetraosylceramide (lane 1) and the five-and six-sugar glycosphingolipids (lanes 5 and 6) derived from NeuGca3Galp4GlcNAcp3Galp4GlcNAcp3Galp4GlepCer is identical to results above. Unexpectedly, however, binding was also found for GalNAcß3Galß4GlcNAcß3Galß4GlcßCer (x2 glycosphingolipid, lane 7) and the de- fucosylated A6-2 glycosphingolipid GalNAca3Galp4GlcNAcp3Galp4GlcpCer (no.

12, Table 1). Together with the finding that Gala3Galp4GlcNAcp3Gal) 34GlcpCer (B5 glycosphingolipid, lane 2) also is binding-active, these results suggest the possibility of cross-binding rather than the presence of multiple adhesins specific for each of these glycosphingolipids (see below). Furthermore, the only extension of the different five-sugar-containing glycosphingolipids just mentioned that was tolerated by the bacterial adhesin was Gal (34 to the thymus-derived GIcNAcp3-terminated compound (lane 6). Other elongated structures, as the NeuAc-x2 (lane 8) and GaINAcp3-B5 (no. 25, Table 1), were thus all found to be non-binding. It may be further noticed that the acetamido group of the internal GIcNAcp3 in B5 is essential for binding since de-N-acylation of this moiety by treatment with anhydrous hydrazine leads to complete loss of binding (lane 3) as is the case also when neolactotetraosylceramide is similarly treated (no. 6, Table 1).

Cross-binding offive-sugar glycosphingolipids. In order to understand the binding characteristics of the different neolacto-based glycosphingolipid molecules used in this study the conformational preferences of active as well as inactive structures were investigated by molecular modeling. The four active structures all have neolacto cores which thus are terminated by GaINAcp3, GalNAca3, Gala3 and GIcNAcp3, respectively. The minimum energy conformers of these structures were generated as descibed previously (Teneberg et al., 1996). Other minimum energy structures according to the invention are based on earlier results found in the literature (Bock et al., 1985; Meyer, 1990 ; Nyholm et al., 1989).

As mentioned above, the fact that there are four binding-active five-sugar glycosphingolipids (nos. 10-13, Table 1), all having a neolacto core, suggests that cross-binding to the same adhesin site may be the reason behind these observations.

At first glance, however, it might seem surprising that the B5 glycosphingolipid, which differs at the terminal position in comparison with the five-sugar compound obtained from rabbit thymus, the former having a Gala3 and the latter a GIcNAcp3, is equally active and should be included within the binding specificity of the neolacto series. Despite the fact that these two terminal saccharides differ also in their anomeric linkage it is seen that the minimum energy structures topographically are very similar, the differences being that Gala3 lacks an acetamido group, has the 4-OH in the axial position and its ring plane raised slightly above the corresponding plane in the five-sugar compound. However, neither the 4-OH position nor the absence/presence of an acetamido group appear to be crucial for binding to occur, since also the x2 and defucosylated A6-2 glycosphingolipids, which are terminated by GalNAcp3 and GalNAca3, respectively, have similar affinities for the Helicobacter pylori adhesin. In the light of these findings also Galß3Galß4GlcNAcß3Galß4GlcßCer, known from human erythrocytes, would be expected to bind the bacterial adhesin and also three other terminal monosaccharides in Helicobacter pylori binding epitopes are trisaccharide binding epitopes, namely GlcNAca3Galp4GlcNAc, Glcp3Galp4GlcNAc and Glca3Galp4GlcNAc. Such compounds are not known from human tissues so far, but could rather represent analogues of the natural receptor. Finally similar sequences with changes in the terminal monosaccharide position 6, (for example carboxylic acid, desulfo HNK-1, or amide derivative) and N-asetyl group on position 2 of Gal (34 were included to the family of Neolacto type receptors for H. pylori.

Example 4.

Analysis of neoglycolipids The oligosaccharides GlcNAcß3Galß4GlcNAc, GlcNAcp3Galp4GlcNAcp6GlcNAc, Gala3Galp4GlcNAcp3Galp4Glc, Glc (A- methylamide) ß3Galß4GlcNAcß6GlcNAc, Galß4GlcNAcß6GlcNAc, <BR> <BR> <BR> GIcNAcp3Galp4GlcNAcp6Gal, GIcNAcp3Galp4GlcNAcp6Galp4Glc <BR> <BR> <BR> <BR> <BR> <BR> GlcNAcß3Galß4GlcNAca6GlcNAc, GlcNAcß3Galß4GlcNAcß3Galß4Glc, <BR> <BR> <BR> <BR> <BR> <BR> <BR> GIcA (33Galp4GlcNAcp6GlcNAc, GIcAp3Galp4GlcNAcp3Galp4Glc, GlcNAcp3Galp4GlcNAcp3Man, GalNAcp4GlcNAc) 33Galp4GlcNAc, Glc (A- NH2) p3GalNAcp4Glc (A-NH2) p3GalNAc, GIcAp3GalNAcp4GlcAp3GalNAcp4GlcAp3GalNAc and maltoheptaose (Sigma, Saint Louis, USA), wherein A-NH2 indicates the glucuronamides-CO-NH2 and (A- methylamide) methylamide of the 6-position of glucuronic acid,-CO-NH-CH3, were reductively aminated with 4-hexadecylaniline (abbreviation HDA, from Aldrich,

Stockholm, Sweden) and/or with another lipid anchor NH2-CH2-CH2-CH2-amide- lysineamidated to two palmitates (custom product, Rapp polymere, Germany) by cyanoborohydride (Halina Miller-Podraza, to be published later). The products were characterized by mass spectrometry and were confirmed to be conjugates reductively aminated at the reducing end of the molecules to the lipid anchor/anchors. The neoglycolipids from oligosaccharides Gala3Galp4GlcNAcp3Galp4Glc, and GlcNAcß3Galß4GlcNAcß3Galß4Glc, Galß4GlcNAcß6GlcNAc , GlcAp3Galp4GlcNAcp6GlcNAc, GlcAp3Galp4GlcNAcp3Galp4Glc, GIcNAcp3Galp4GlcNAcp3Man, GaINAcp4GlcNAcp3Galp4GlcNAc, GlcAß3GalNAcß4GlcAß3GalNAcß4GlcAß3GalNAc had clear binding activity and GIcNAcp3Galp4GlcNAcp6GlcNAc, Glc (A- methylamide) p3Galp4GlcNAcp6GlcNAc, GlcNAcp3Galp4GlcNAcp6Gal and Glc (A-NH2) p3GalNAcp4Glc (A-NH2) p3GalNAc derivatives had strong binding activity with regard to Helicobacter pylori in TLC overlay assay described above, while the GlcNAcp3Galp4GlcNAc, GlcNAcp3Galp4GlcNAcp6Galp4Glc GlcNAcp3Galp4GlcNAca6GlcNAc and maltoheptaose neoglycolipids were weakly binding or inactive. The example shows that the terminal tri-or disaccharide epitopes are preferably represented (36-HexA (Gal, GIcNAc) linked as structures binding to Helicobacter pylori. The reducing end Glc-residue is probably not needed for the binding because the reduction destroys the pyranose ring structure of the Glc- residue. In contrast, the intact ring structure of reducing end GlcNAc is needed for good binding of the trisacharide GlcNAcp3Galp4GlcNAc.

Example 5. Production of partially sulphated chondroitin oligosaccharides, hyaluronic acid oligosaccharides, polyvalent oligosaccharide and carboxylic acid reduction of an oligosaccharide.

Isolation of monosulphated GlcAß3GalNAcß4GlcAß3GalNAc The acid hydrolysis product of partially desulphated chondroitin sulphate was subjected to gel filtration chromatography. Fractions containing a high proportion of monosulphated GlcAp3GalNAcp4GlcAp3GalNAc were pooled, and subjected to anion-exchange chromatography. Pure monosulphated GlcAp3GalNAcp4GlcAp3GalNAc was obtained as indicated by MALDI-TOF mass spectrometry (Fig. 5).

Isolation of sulphated chondroitin 8-12-mers Chondroitin sulphate A (500 mg) was dissolved in 50 ml of sodium phosphate buffer, pH 6.0, containing 150 mM NaCI. 10 kU hyaluronidase (bovine testes) was added, and the reaction was allowed to proceed at 37 °C. The reaction was monitored by running aliquots in gel filtration chromatography. A fraction containing primarily monosulphated chondroitin octa-, deca-and dodecamers was isolated.

Synthesis of a dendrimeric Glc[ANH2]ß3GalNAcß4Glc[ANH2]ß3GalNAc conjugate A sample of chondroitin tetrasaccharide amide Glc [ANH2] ß3GalNAcß4Glc [ANH2] ß3GalNAc (44 umol) was dissolved in 1.5 ml of 0.1 M sodium borate buffer, pH 8.5, containing 1 M sodium cyanoborohydride. 500 mnol of DAP-AM-16 dendrimer (Aldrich 46,907-6) was added to the solution, and the mixture was reacted for 4 days at room temperature.

The oligosaccharide-dendrimer conjugate was purified by gel filtration chromatography, and the purified product was subjected to MALDI-TOF mass spectrometry (Fig. 6). As the dendrimer has MW of 1687 Da and the increment in size per tetrasaccharide attached to the dendrimer is 776 Da, the product was found to contain on average 7.7 tetrasaccharide units per dendrimer.

Reduction of hyaluronic acid oligosaccharide mixture An oligosaccharide mixture was isolated from a hyaluronic acid hydrolysis reaction by gel filtration chromatography. This fraction contained hexa-, octa-, deca-and dodekasaccharides. A sample of this mixture containing 4 Ilmol of carboxyl groups was methylesterified in a solution containing 1.6 mmol of CH3I (102 p. l), 4 pmol of DIPEA, 31 1 H20 and 173 u. l DMSO. The reaction was allowed to proceed for 48 h at RT in the dark. The methylesterified oligosaccharide mixture was purified by extensive dialysis.

To reduce the methylesterified GIcA units to glucose units, the methylesterified sample was dissolved in 100 1 of 50 mM ammonium bicarbonate and 3.2 mg of sodium borohydride was added. The reaction was allowed to proceed for 2 h at RT, and the product was isolated by gel filtration chromatography.

The MALDI-TOF mass spectrum of the reduced sample is shown in Fig. 7. The major signals in the spectrun are as follows:

M/z Ion type Suggested composition 1138. 5 [M+Na] + (Glc) 3 (GlcNAc) 3-ol 1152.4 [M+Na] + (Glc) 2 (GlcA) z (GlcNAc) 3-ol 1174.4 [M-H+2Na] + (Glc) 2 (GlcA) l (GlcNAc) 3-ol 1300. 6 [M+Na] + (Glc) 4 (GlcNAc) 3-ol 1314.4 [M+Na]+ (Glc) 3 (GlcA) l (GlcNAc) 3-ol 1336.5 [M-H+2Na] + (Glc) 3 (GlcA)1(GlcNAc)3-ol 1503.6 [M+Na] + (Glc) 4 (GlcNAc) 4-ol 1517.5 [M+Na] + (Glc) 3 (GlcA)1(GlcNAc)4-ol 1531.5 [M+Na] + (Glc) 2 (GlcA) 2 (GlcNAc) 4-ol 1539.6 [M-H+2Na]+ (Glc) 3 (GlcA)1(GlcNAc)4-ol 1553.5 [M-H+2Na] + (Glc) 2 (GlcA) 2 (GlcNAc) 4-ol 1575.5 [M-2H+3Na] + (Glc) 2 (GlcA) 2 (GlcNAc)4-ol 1868.7 [M+Na] + (Glc) s (GlcNAc)5-ol 1882.6 [M+Na] + (Glc) 4 (GlcA)1(GlcNAc)5-ol 1896.6 [M+Na] + (Glc) 3 (GlcA) 2 (GlcNAc) 5-ol 1904.7 [M-H+2Na] + (Glc) 4 (GlcA) I (GlcNAc) 5-ol 1918.6 [M-H+2Na] + (Glc) 3 (GlcA) 2 (GlcNAc) 5-ol 1940.6 [M-2H+3Na] + (Glc) 3 (GlcA) 2 (GlcNAc) 5-ol Example 6. Partially sulphated chondroitin oligosaccharides GIcA 3GalNAcp4GlcAp3GalNAc, GlcAß3GalNAcß4[GlcAß3GalNAcß4]2-4GlcAß3GalNAc (mixture of octa-, deca-, and dodeca-saccharides), Glc (A) 0-1ß3GlcNAcß4 [Glc (A) 0-1ß3GlcNAcß4]1-3Glc (A) 0-1ß3GlcNAcOl (mixture of carboxylic acid reduced (and reducing end reduced) hyaluronic acid oligosaccharides containing a few uronic acid groups), GlcAß3GlcNAcß4[GlcAß3GlcNAcß4]2-5GlcAß3GlcNAc hyaluronic acid 8-, 10-, 12-and 14-meric oligosaccharides, and Glc (A-NH2) ß3GalNAcß4Glc (A-NH2) ß3GalNAc, amidated chondroitin oligosaccharide dendrimer, were tested in TLC overlay assay, the saccharides and dendrimer were spotted on TLC plate, with acidic oligosaccharides Starburst (PAMAM)-dendrimer was used as carrier as described in W003002128 and were observed to be binding active with regard to Helicobacter pylori. The dendrimer bound to the plate without a carrier.

No binding was observed to the carrier or negative glycolipid control globoside, strong binding was observed with positive control glycolipid

Galp4GlcNAcp3Galp4GlcNAc 3Galp4GlcpCer. Especially strong binding was observed with hyaluronic acid oligosaccharides.

Table 1<BR> Binding of Helicobacter pylori to glycosphingolipids separated on thin-layer chromatograms.<BR> <P>No. Trivial name Glycosphingolipid structurea H. pylori Sourcec References<BR> bindingb<BR> 1 Lactotri GlcNAcß3Galß4Glcß1Cer - RT (Miller-Prodraza et al., 2001)<BR> 2 GgO3 GalNAcß4Galß4Glcß1Cer (+) GPE (Yamakawa, 1966)<BR> 3 GgO3 (de-N-acylated) GalNH2ß4Galß4Glcß1Cer - GPEe (Ångström et al., 1998)<BR> 4 Ley-6 Fucα2Galß4(Fucα3)GlcNAcß3Galß4Glcß1Cer - DSI (McKibbin et al., 1982)<BR> 5 Neolactotetra Galß4GlcNAcß3Galß4Glcß1Cer (+) HEf<BR> 6 Neolactotetra (de-N-acylated) Galß4GlcNH2ß3Galß4Glcß1Cer - HEe<BR> 7 GgO4 Galß3GalNAcß4Galß4Glcß1Cer + HBg<BR> 8 GgO4 (de-N-acylated) Galß3GalNH2ß4Galß4Glcß1Cer - HBe (Ångström et al., 1998)<BR> 9 Lex-5 Galß4(Fucα3)GlcNAcß3Galß4Glcß1Cer - DSI (Teneberg et al., 1996)<BR> 10 GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer + RTd (Miller-Podraza et al., 2001)<BR> 11 x2 GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer + HE (Teneberg et al., 1996; Thorn et al., 1992)<BR> 12 GalNAcα3Galß4GlcNAcß3Galß4Glcß1Cer + HEh<BR> 13 B5 Galα3Galß4GlcNAcß3Galß4Glcß1Cer + RE (Eto et al., 1968)<BR> 14 B5 (de-N-acylated) Galα3Galß4GlcNH2ß3Galß4Glcß1Cer - REe<BR> 15 P1 Galα4Galß4GlcNAcß3Galß4Glcß1Cer - HE (Naiki et al., 1975)<BR> 16 H5-1 Fucα2Galß3GlcNAcß3Galß4Glcß1Cer - HM (Karlsson and Larson, 1981a)<BR> 17 Leb-6 Fucα2Galß3(Fucα4)GlcNAcß3Galß4Glcß1Cer - HM (Karlsson and Larson, 1981b)<BR> 18 H5-2 Fucα2Galß4GlcNAcß3Galß4Glcß1Cer - HE (Koscielak et al., 1973)<BR> 19 NeuAcα3-SPG NeuAcα3Galß4GlcNAcß3Galß4Glcß1Cer - HE (Ledeen and Yu, 1978) Table 1 (continued)<BR> 20 NeuAcα6-SPG NeuAcα6Galß4GlcNAcß3Galß4Glcß1Cer - HM (Nilsson et al., 1981)<BR> 21 Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (+) RTd (Miller-Podraza et al., 2001)<BR> 22 A6-2 GalNAcα3(Fucα2)Galß4GlcNAcß3Galß4Glcß1Cer - HE (Laine et al., 1974)<BR> 23 B6-2 Galα3(Fucα2)Galß4GlcNAcß3Galß4Glcß1Cer - HE (Koscielak et al., 1973)<BR> 24 NeuAc-x2 NeuAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer - HE (Watanabe and Hakomori, 1979)<BR> 25 GalNAcß3Galα3Galß4GlcNAcß3Galß4Glcß1Cer - RCC (Thurin et al., 1989)<BR> 26 Galß4GlcNAcß6 (Galß4GlcNAcß3)Galß4Glcß1Cer - BB (Teneberg et al., 1994)<BR> 27 NeuGcα3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (+) RT (Lanne et al., 2001)<BR> 28 Galα3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (+) RT (Lanne et al., 2001)<BR> 29 A7-2 GalNAcα3(Fucα2)Galß4(Fucα3)GlcNAcß3Galß4Glcß1Cer - DSI (Falk et al., 1979c)<BR> 30 B7-2 Galα3(Fucα3)Galß4(Fucα3)GlcNAcß3Galß4Glcß1Cer - HE<BR> Footnotes to Table 1<BR> a The glycosphingolipid shorthand nomenclature follows recent recommendations (Nomenclature of glycoproteins, 1988).<BR> b The following abbreviations are used for the glycosphingolipid sources: RT, rabbit thymus; HE, human erythrocytes; RE, rabbit<BR> erythrocytes; HM, human meconium; RCC, rat colon carcinoma; BB, bovine buttermilk; DSI, dog small intestine.<BR> c Definition of binding strength is as follows: + denotes a significant darkening of the autoradiogram with 4 µg applied on the TLC plate, (+)<BR> indicates a weak to intermediate darkening while a minus sign signifies no binding.

Footnotes to Table 1 (continued)<BR> d Prepared from No. 27 by mild acid hydrolysis and No. 10 by subsequent treatment with ß-galactosidase.<BR> e Glycosphingolipid Nos. 3, 6, 8 and 14 were prepared from Nos. 2, 5, 7 and 13, respectively, by treatment with anhydrous hydrazine.<BR> f Prepared from no. 19 by neuraminidase treatment.<BR> g Prepared by mild acid hydrolysis of GM1 ganglioside from human brain.<BR> h Prepared from No. 22 by incubation in 0.05 M HCl at 80°C for 2 h.

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