BACKGROUND OF THE INVENTION

1. Field of the Invention.

5 The present invention is directed to methods of employing oligosaccharide glycosides in the treatment of cell-mediated immune responses, as well as to pharmaceutical compositions containing such oligosaccharide glycosides. Specifically, the methods of the present invention are directed to methods of employing oligosaccharide 10 glycosides related to blood group determinants in modulating (eg. suppressing) cell-mediated immune responses, including cell-mediated inflammatory responses.

2. References.

The following references are cited in this application as 15 superscript numbers at the relevant portion of the application:

1. Brandley et al., J. Leukocyte Biol., 40:97-1 11 (1986).

2. Jacobson, Developmental Neurobiology, New York, Plenum Press

20 p. 5-25, (1978)

3. Trinkaus, Cells into Organs, Englewood Cliffs, N.J., Prentice Hall, p.44-68, (1984).

4. Frazier et al., Annu. Rev. Biochem., 25 48:491 (1979).

Glaser, Mediator of Developmental Processes (Subtency, S. and Wessels, N.K., Eds.) New York, Academic Press, p. 79 (1980).

Paulson, In "The Receptors", Vol. II

(Comm., P.M., Ed.), New York, Academic Press, p. 131 (1985).

7. Sharon, Lectin-Like Bacterial Adherence to Animal Cells. In

10 "Attachment of Microorganisms to the

Gut Mucosa" (Boeheker, E.D., Ed.), Boca Raton, Florida, CRC Press, p. 129 (1984).

8. Wassarman, Fertilization. In "Cell 15 Interactions and Development:

Molecular Mechanisms" (Yamada, K.M., Ed.), New York, John Wiley and Sons, p. 1 (1983).

9. Schwartz et al., Immunol. Rev., 20 40_:153 (1978).

10. Coutinho et al., Immunol. Rev., 78_:211 (1984).

1 1. Hoffmann et al., Eds., Membranes in Growth and Development, New York,

25 Alan R. Liss, p. 429-442, (1982).

12. Galeotti et al., Eds., Membranes in Tumor Growth, Amsterdam, Elsevier, p. 77-81 , (1982).

13. Nicolson et al., Invas. Metas., 5_:144 30 (1985).

14. Aplin et al., Biochim. Biophys. Acta 694:375 (1982).

15. Barondes, Developmentally Regulated Lectins. In "Cell Interactions and

35 Development: Molecular Mechanisms"

(Yamada, D.M., Ed.) New York, John Wiley and Sons, p. 185 (1983).

16. Monsigny, M., Ed., Biol. Cell, 5J_ (Special Issue), 1 13, 1984.

17. Springe et al., Nature, 349:196-197 (1991 ).

5 18. Lowe et al., Cell, £2:475-485 (1990).

19. Phillips et al., Science, in press (1990).

20. Walz et al., Science, 25_Q_:1 132 et seq. (1990).

21. Larsen et a!., Cell, £.1:467-474 (1990).

10 22. Smith et al., Immunology, 5j_.:245

(1986).

23. Sleytr et al., Arch. Microbiol., 146: 19 (1986).

24. Ziola et al., J. Neuroimmunol., 7:315- 15 330 (1985).

25. Reuter et al., Glycoconjugate J., 5 133-135 (1988).

26. Campanero et al., J. Cell Biol., 1J0.:2157-2165 (1990).

20 27. Okamoto et al., Tetrahedron, 46, No.

17, pp. 5835-5837 (1990).

28. Ratcliffe et al., U.S. Serial No. 07/127,905 (1987).

29. Abbas et al., Proc. Japanese-German 25 Symp. Berlin, pp. 20-21 (1988).

30. Paulsen, Agnew. Chem. Int. Ed. Eng., 21:155-173 (1982).

31 . Schmidt, Agnew. Chem. Int. Ed. Eng., 25_:212-235 (1986).

30 32. Fύgedi et al., G/ycoconj. J., 4:97-108

(1987).

33. Kameyama et al., Carbohydr. Res., 209^.-0 ₄ 11991 ,.

34. Toone et al., Tetrahedron, No. 17, 45_:5365-5422 (1989).

5 35. Beyer et al.. Advances in Enzymology, pp. 23-175, John Wiley & Sons, New York (1982).

36. Brossmer et al., Biochem. Biophys. Research Commun., 9 >: 1282-1289

10 (1980).

37. Zbiral et al., Monatsh. Chem., 111:127-141 (1988).

38. Hasegawa et al., J. Carbohydr. Chem ^' ., 8_: 135-144 (1989).

15 39. Christian et al., Carbohydr. Res.,

194:49-61 (1989).

40. Higa et al., J. Biol. Chem., 2£Q:8838- 8849 (1985).

41. Kean et al., J. Biol. Chem., 241:5643- 20 5650 (1960).

42. Gross et al., Biochemistry, 28_:7386- 7392 (1989).

43. Lemieux et al., U.S. Patent No. 4,137,401 (1976).

25 44. Lemieux et al., U.S. Patent No.

4,195,174 (1978).

45. Paulsen et al., Carbohydr. Res., 125:21-45 (1984).

46. Sabesan et al.. Can. J. Chem., 30 62:644-652 (1984).

47. Alais et al., Carbohydr. Res., 207:11- 31 (1990).

48. Lemieux et al., U.S. Patent No. 4,767,845 (1987).

49. Mazid et al., U.S. Patent Application Serial No. 07/336,932 (1989).

5 50. Unverzagt et al., J. Amer. Chem. Soc,

112:9308-9309 (1990).

51. Palcic et al., Carbohydr. Res. , 190: 1 - 11 (1989).

52. Weinstein et al., J. Biol. Chem. 10 257:13835-13844 (1982).

53. Smith et al., Infection and Immunity, 3J_: 129 (1980).

54. Ratcliff et al., U.S. Serial No. 07/278,106, filed November 30, 1988.

15 55. Ziola et al., J. of Immunol. Methods, 97:159.

(1987).

56. Ekberg et al., Carbohydr. Res., 110:55-67 (1982).

57. Dahmen et al., Carbohydr. Res., 118:292-301 (1983).

20 58. Rana et al., Carbohydr. Res., 9J_:149-157 (1981 ).

59. Amvam-Zollo et al., Carbohydr. Res., 150:199-212 (1986).

60. Paulsen et al., Carbohydr. Res., 104:195-219 (1982).

25 61. Chernyak et al., Carbohydr. Res., 128:269-282

(1984).

62. Fernandez-Santana et al., J. Carbohydr. Chem., 8_:531-537 (1989).

63. Lee et al., Carbohydr. Res., 3.7:193 et seq. (1974).

30 64. Schmidt, et al., Liebigs Ann. Chem., 121-124

(1991 )

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66. Veeneman, et al., Tetrahedron Lett., 32:6175-6178 (1991 )

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

3. State of the Art.

Important processes involving mammalian cells, such as growth, locomotion, morphological development, and differentiation are partially controlled by extracellular signals acting upon the cells' surfaces ^1"3 .

While some external stimuli reach the cell via extracellular fluids, other signals are received from neighboring or approaching cell surfaces and exert their effects through direct cell-cell contact*- ⁵ .

Evidence suggests that specific cell-surface receptors can "sense" a molecular signal of an apposing cell via specific binding, and biochemical mechanisms exist to translate that binding into a cellular response. For example, complex cell-surface interactions are believed to help direct processes such as binding of pathogens to target tissues ^6,7 , sperm-egg binding ⁸ , interactions among cells in the immune system ⁹ - ¹⁰ , and recognition of cells during embryonic development ¹¹ . In addition, defects in cell-cell recognition are thought to underlie the uncontrolled cell growth and motility which characterize neoplastic transformation and metastasis ^12,13 .

Other evidence suggests that cell-recognition processes are mediated by carbohydrate chains or glycan portions of glycoconjugates ^4,14"16 . For example, the binding of the surface glycoconjugates of one cell to the complementary carbohydrate-binding proteins (lectins) on another cell can result in the initiation of a specific interaction.

One important group of carbohydrate-binding proteins are LEC- CAM proteins (Lectin + EGF + complementary Regulatory Domains- Cell Adhesion Molecules). These or functionally similar proteins or lectins may play a critical role in immune responses (including inflammatory responses) through mediation of cell-cell contact and through extravasation of leucocytes ^17"21 . Specific carbohydrate ligands have recently been identified as part of the putative receptor structures for LEC-CAM proteins ^17"21 . The structures identified include:

Sialyl-Lewis X-Q: σNeu5Ac(2-3)0Gal(1-4)/?GlcNAc1-Q

| (1-3) σFuc Lewis X-Q: 0Gal(1-4)/3GlcNAc1-Q

I (1-3) σFuc

wherein Q represents another suitably bonded sugar or sugars. However, in vivo, such carbohydrates are generally part of naturally- occurring glycoconjugates which are not readily synthesized and cannot be readily isolated in therapeutic amounts. Although certain oligosaccharide glycosides have been heretofore disclosed, their use in suppressing cell-mediated immune responses (including cell-mediated inflammatory responses) has not been taught.

SUMMARY OF THE INVENTION

It has now been found that cell-surface glycoconjugates which contain the above-mentioned Sialyl Lewis X or Lewis X glycan chains

are not the only ligands that can functionally interact with lectins so as to suppress cell-mediated immune responses in a mammal. Specifically, the present invention is directed to the discovery that low molecular weight (MW generally less than about 2000 daltons) oligosaccharide glycosides related to blood group determinants also interact with LEC-CAM proteins and/or other lectins with sufficient strength to suppress, in vivo, mammalian cell-mediated immune responses including cell-mediated inflammatory responses. The present invention is also directed to the discovery that when such oligosaccharide glycosides related to blood group determinants are administered to a mammal in response to an antigen challenge, such administration induces tolerance to additional challenges from the same antigen.

Accordingly, in one of its method aspects, the present invention is directed to a method of suppressing a cell-mediated immune response in a mammal which method comprises administering to said mammal an amount of an oligosaccharide glycoside related to blood group determinants effective in suppressing said immune response.

In a preferred embodiment, the immune response suppressed by this method is an inflammatory response. In a further preferred embodiment, the oligosaccharide glycoside related to blood group determinants employed in this method is further characterized as a binding-inhibitory oligosaccharide glycoside (as defined below). In another of its method aspects, the present invention is directed to a method of treating a cell-mediated immune response to an antigen in a mammal which method comprises administering to said mammal from about 0.5 mg/kg to about 50 mg/kg of an oligosaccharide glycoside related to blood group determinants. Yet in another of its method aspects, the present invention is directed to a method of reducing sensitization of a mammal to an antigen which comprises administration of an effective amount to the mammal of a blood group

determinant oligosaccharide glycoside simultaneously with exposure to the antigen.

In one of its composition aspects, the present invention is directed to a pharmaceutical composition suitable for parenteral administration to a mammal which comprises a pharmaceutically inert carrier and an amount of oligosaccharide glycoside related to a blood group determinant effective in treating a cell-mediated immune response in said mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 10 μg of the L1 1 1 S-Layer protein antigen wherein some of the mice have been treated at 5 hours after the challenge with 100 /yg of different oligosaccharide glycosides related to blood group determinants.

FIG. 2 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 μg of the L1 1 1 S-Layer protein antigen wherein some of the mice have been treated at 5 hours after challenge with various doses of different mono- and oligosaccharide glycosides including oligosaccharide glycosides related to blood group determinants.

FIG. 3 illustrates secondary antibody responses (i.e., as determined by the amount of antibody measured by quantification of o- phenylenediamine O.D. at 490 nm) two weeks after primary immunization and one week after challenge with the L1 1 1 S-Layer protein antigen and the effect different oligosaccharide glycosides related to blood group determinants had on these responses when the mice were treated with these oligosaccharide glycosides 5 hours after challenge.

FIG. 4 illustrates the effect of an oligosaccharide glycoside related to blood group determinants, i.e., the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, on the inflammatory DTH response in immunized mice challenged with the L111 S-Layer protein antigen wherein the mice were treated at various times before or after challenge with 100 μg of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III.

FIG. 5 illustrates the long term (8 weeks) immunosuppression generated in immunized mice after an injection with 5 mg/kg of oligosaccharide glycosides related to blood group determinants 5 hours after challenge with 20 μg of the L111 S-Layer protein antigen on day 7.

FIG. 6 illustrates the long term (6 weeks) immunosuppression generated in immunized mice after an injection with varying amounts of mono- and oligosaccharide glycosides including oligosaccharide glycosides related to blood group determinants 5 hours after challenge with 20 μg of the L11 1 S-Layer protein antigen on day 7.

FIG. 7 illustrates the long term (10 weeks) immunosuppression generated in immunized mice after an injection with 5 mg/kg of the 8- methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, at various times before, at and after challenge with 20 μg of the L111 S- Layer protein antigen on day 7.

FIG. 8 illustrates the cyclophosphamide induced restoration of a DTH inflammatory response in immunized mice previously suppressed by treatment with the 8-methoxycarboπyioctyl glycoside of Sialyl Lewis X, Compound III.

FIG. 9 illustrates that the nature of the antigen used to induce the inflammatory response does not affect the ability of the 8- methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, to regulate the DTH response.

FIG. 10 illustrates that the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, can inhibit binding of U937 or HL60 to TNFσ activated human umbilical vein endothelial cells (HUVECs).

FIG. 1 1 illustrates the "core" structure of preferred oligosaccharide glycosides related to blood group determinants for use in this invention.

FIG. 12 illustrates the structures of specific oligosaccharide glycosides related to blood group determinants for use in this invention wherein R is -(CH ₂ ) ₈ C(0)OCH ₃ . FIG. 13 illustrates the structures of a couple of monosaccharide glycosides and one disaccharide glycoside used in some of the examples wherein R is -(CH ₂ ) ₈ C(0)OCH ₃ .

FIG. 14 illustrates a general synthetic scheme used for the synthesis of derivatives of NeuδAc. FIG. 15 illustrates the structures of mono- and oligosaccharide glycosides 3b_ to 7a..

FIG. 16 illustrates a general reaction scheme for the synthesis of oligosaccharide glycoside 4c. as specified in Example 8 and for the synthesis of monosaccharide glycoside 32 as specified in Example 9. FIG. 17 illustrates the enzymatic transfer of NeuδAc, and of analogues thereof (collectively "sialic acids") by the /SGal(1-»3/4)/3GlcNAcσ(2→3')sialyϊtransferase to a /3Gal(1-*3)/9GlcN Ac- terminal structure. FIG. 17 also illustrates the enzymatic transfer of L- fucose onto the sialylated oligosaccharide glycosides. FIG. 18 illustrates the enzymatic transfer of NeuδAc, analogues thereof (collectively "sialic acids") by the

/SGal(1→3/4)/3GlcNAcσ(2→3')sialyltransferase to a SGal(1→4)/SGIcNAc- terminal structure. FIG. 18 also illustrates the enzymatic transfer of L- fucose onto the sialylated oligosaccharide glycosides. FIG. 19 illustrates the enzymatic transfer of NeuδAc, analogues thereof (collectively "sialic acids") by the

£Gal(1→4)/_?GlcNAcσ(2→6')s.alyltransferase to a 0Gal(1→4)0GlcN Ac- terminal structure.

FIG. 20 illustrates the enzymatic transfer of NeuδAc, analogues thereof (collectively "sialic acids") by the δ /3Gal(1→3/4)/?GlcNAcσ(2→3')sialyltransferase to a /3GaI(1→4)i9Glc- (lactose) terminal structure.

FIG. 21 illustrates the enzymatic transfer of NeuδAc, analogues thereof (collectively "sialic acids") by the 3Gal(1→3)σGalNAcσ(2→3')sialyltransferase to a /SGal(1-»3)σGalNAc- 0 ("T") terminal structure.

FIGS. 22 and 23 illustrate the reaction schemes involved in the synthesis of analogues of Sialyl Lewis A by chemical modification of a sialylated hapten.

FIG. 24 illustrates the reaction schemes involved in the synthesis δ of analogues of Sialyl Lewis X by chemical modification of a sialylated hapten.

FIG. 2δ illustrates the synthetic pathway leading to Sialyl dimeric Lewis* and internally monofucosylated derivatives thereof. In FIG. 2δ, the nomenclature for compoound 61a is 0 3Gal(1-4)/3GlcNAc(1-3)5Gal(1-4)/?GlcNAc-OR sometimes called di-N- acetyliactosaminγl tetrasaccharide. Similarly, the hexasaccharide moiety present in compounds 65a and 65b in FIG. 1 is sometimes called VI -2 epitope or CD-65 and 67a and 67b are called sialyl dimeric Lewis*. δ FIG. 26 illustrates the synthetic pathway leading to the externally monofucosylated derivatives of the sialyl di-N- acetyllactosaminyl hapten.

FIG. 27 illustrates the increase in foot-pad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with HSV antigen, where some of the mice were treated with Sialyl Lewis X, Compound III, at the time of immunization

and some of the mice were treated with Sialyl Lewis X, Compound III, δ hours after the challenge.

FIG. 28 illustrates the secondary antibody responses (i.e., as determined by the amount of antibody measured by quantification of o- δ phenylenediamine O.D. at 490 nm) two weeks after primary immunization and one week after challenge with the HSV antigen and the effect the time of administration of Sialyl Lewis X, Compound III, had on the these responses.

FIG. 29 illustrates the cγclophosphamide (CP) induced 0 restoration of a DTH inflammatory response in immunized mice previously suppressed by treatment with the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III.

FIG. 30 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH δ response in immunized mice challenged with the OVA antigen wherein the mice were treated with Compound III five hours after challenge by a variety of different methods (IV-intravenously; IN- intranasally) FIG. 31 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH response in immunized mice challenged with the OVA antigen wherein the mice were treated with various doses of Compound III five hours after challenge by a variety of different methods (IV-intravenously; IN- intranasally).

FIG. 32 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH response in immunized mice challenged with the OVA antigen wherein the mice were treated with Compound III by a variety of different methods at the time of immunization of the mice (IV-intravenously; IN- intranasally; IM-intramuscularly) FIG. 33 illustrates the effect of various oligosaccaride and monosaccharide glycosides have on the inflammatory response in the

iungs of mice wherein the mice received LPS intranasally and the various compounds five hours later intravenously.

FIG. 34 illustrates the effect of different amounts of Sialyl LewisX and Sialyl LewisA on the lymphoproliferative response.

δ DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed at the discovery that certain low molecular weight oligosaccharide glycosides (MW less than about 2000 daltons) are effective in suppressing cell-mediated immune responses in a mammal, including cell-mediated and immune directed 0 inflammatory responses to an antigen in a mammal (e.g.,- a DTH response). Additionally, treatment with these oligosaccharide glycosides also provides for induction of tolerance to the antigen in the so-treated mammal.

A. Definitions

δ As used herein, the following terms have the definitions given below:

The term "cell-mediated immune response in a mammal" refers to those mammalian immune responses which are mediated by cell-cell interactions. Included within this term are cell-mediated inflammatory 0 responses to an antigen such as delayed-type hypersensitivity (DTH) responses as well as cell-mediated inflammatory responses arising from myocardial infarction, virus-induced pneumonia, shock and sequelae (e.g., multiple organ failure), adult respiratory distress syndrome, and the like. Preferably, the cell-mediated immune response is a leucocyte- δ mediated response.

The term "blood group substances" refer to specific glycoconjugate antigens on red blood cells which serve as the basis for

assigning blood into various classes according to immunological compatibility.

The term "blood group determinant" refers to any naturally occurring oligosaccharide segment of the nonreducing-terminal, 3-9 glycosyl residues that constitute the glycan chains of blood group substances.

The term "oligosaccharide glycosides relating to a blood group determinant" refer to an oligosaccharide glycoside (a) having an oligosaccharide group of from 3 to 9 saccharide units, (b) which is terminated with an aglycon group on the non-reducing sugar, and (c) wherein the oligosaccharide group is a blood group determinant (as defined above) or an analogue thereof.

Analogues of blood group determinants include those wherein one or more of the monosaccharide units of the blood group determinant has or have been chemically modified so as to introduce and/or remove one or more functionalities in one or more of the saccharide unit(s). For example, such modification can result in the removal of an -OH functionality, the removal of saccharide unit(s), the introduction of an amine functionality, the introduction of a halo functionality, the introduction of one or more saccharide unit(s), and the like.

Such oligosaccharide glycosides related to blood group determinants can be represented by the formula:

OLIGOSACCHARIDE- Y-R

wherein oligosaccharide represents a carbohydrate structure of from 3 to about 9 saccharide units which oligosaccharide contains a blood group determinant or analogues thereof; Y is selected from the group consisting of O, S, > NH and a bond; and R represents an aglycon moiety of at least 1 carbon atom.

Oligosaccharide glycosides related to blood group determinants are different from glycoconjugates, including blood group substances, because the aglycon moiety is neither a protein or a lipid capable of forming a micelle or other large aggregate structure. 5 In a preferred embodiment, the aglycone moiety, R, is selected from the group consisting of -(A)-Z' wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the form -(CH ₂ -CR ₄ G) _n - wherein n is an integer equal to 1 to δ; R ₄ is selected from the group consisting of hydrogen, methyl, or ethyl; and G is 0 selected from the group consisting of hydrogen, oxygen, sulphur, nitrogen, phenyl and phenyl substituted with 1 to 3 substituents selected from the group consisting of amine, hydroxyl, halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms; and Z' is selected from the group consisting of hydrogen, methyl, phenyl δ and nitrophenol and, when G is not oxygen, sulphur or nitrogen and A is not a bond, then Z' is also selected from the group consisting of -OH, -SH, -NH ₂ , -NHR ₅ , -N(R ₅ ) ₂ , -C(O)OH, -C(O)OR ₅ , -C(O)NH-NH ₂ , -C(O)NH ₂ , -C(O)NHR ₅ , and -C(O)N(R ₅ ) ₂ , wherein each R ₅ is independently alkyl of from 1 to 4 carbon atoms. 0 Numerous aglycons are known in the art. For example, a linking arm comprising a para-nitrophenyl group (i.e., -YR = -OC _β H ₄ pNO ₂ ) has been disclosed by Ekberg et al. ⁵⁶ At the appropriate time during synthesis, the nitro group is reduced to an amino group which can be protected as N-trifluoroacetamido. Prior to coupling to a support, the δ trifluoroacetamido group is removed thereby unmasking the amino group.

A linking arm containing sulfur is disclosed by Dahmen et al. ⁵⁷ . Specifically, the linking arm is derived from a 2-bromoethyl group which, in a substitution reaction with thio-nucleophiles, has been shown to lead to linking arms possessing a variety of terminal functional groups such as -OCH ₂ CH ₂ SCH ₂ SCθ ₂ Cθ ₃ and -OCH ₂ CH ₂ SC _β H ₄ -pNH ₂ .

Rana et al. ⁵⁸ discloses a 6-trifluoroacetamido)-hexyl linking arm (-O-(CH ₂ ) _β -NHCOCF ₃ ) in which the trifluoroacetamido protecting group can be removed unmasking the primary amino group used for coupling. Other exemplification of known linking arms include the 7- δ methoxycarbonyl-3,6,dioxaheptyl linking arm ⁵⁹

(-OCH ₂ -CH ₂ ) ₂ OCH ₂ C0 ₂ CH ₃ ; the 2-(4-methoxycarbonylbutan- carboxamido)ethyl ^β0 (-OCH ₂ CH ₂ NHC(O)(CH ₂ ) ₄ CO ₂ CO ₃ ; the allyl linking arm ⁶¹ (OCH ₂ CH = CH ₂ ) which, by radical co-polymerization with an appropriate monomer,leads to co-polymers; other allyl linking arms ⁶² 0 [-O(CH ₂ CH ₂ O) ₂ CH ₂ CH = CH ₂ ]. Additionally, allyl linking arms can be derivatized in the presence of 2-aminoethanethiol ^β3 to provide for a linking arm -OCH ₂ CH ₂ CH ₂ SCH ₂ CH ₂ NH ₂ .

Additionally, as shown by Ratcliffe et al. ⁵⁴ , R group can be an additional saccharide or an oligosaccharide containing a linking arm at δ the reducing sugar terminus.

Preferably, the aglycon moiety is a hydrophobic group and most preferably, the aglycon moiety is a hydrophobic group selected from the group consisting of -CH ₂ ) ₈ COOCH ₃ , -(CH ₂ ) ₅ OCH ₂ CH = CH ₂ and -CH ₂ ) ₈ CH ₂ OH. In particular, the use of a hydrophobic group and most 0 especially, a -(CH ₂ ) ₈ COOCH ₃ , or -(CH ₂ ) ₅ OCH ₂ CH = CH ₂ or

-(CH ₂ ) ₈ CH ₂ OH group may provide for some enhancement of the acceptor properties for transfer sialic acid by this siaiyltransferase.

Without being limited to any theory, we believe that oligosaccharide glycosides related to blood group determinants δ effectively interfere with an immune response, particularly an inflammatory immune response to an antigen, so as to provide a suitable means for treating such an immune response.

Preferably, the oligosaccharide glycoside related to blood group determinants is further characterized as a binding-inhibitory 0 oligosaccharide glycoside, i.e., an oligosaccharide glycoside related to blood group determinants which oligosaccharide glycoside binds sufficiently to a cell surface lectin so as to inhibit leucocytes from

binding to another cell. Such binding-inhibitory oligosaccharide glycosides are particularly effective in suppressing leucocyte-mediated immune responses.

Again, without being limited to any theory, we believe that the δ binding of leucocytes to a cell surface (presumably through the LEC- CAM proteins and/or other proteins on the cell surface) is an integral part of a leucocyte-mediated immune response, including a leucocyte- mediated and immune directed inflammatory response. Accordingly, binding-inhibitory oligosaccharide glycosides are preferred in the 0 treatment of leucocyte-mediated immune responses.

The ability of an oligosaccharide glycoside related to blood group determinants to bind sufficiently to a cell surface lectin so as to inhibit leucocytes from binding to that cell, either heterotypically or homotypically, can readily be determined via simple in vitro δ experiments. For example, in vitro lymphoproliferative experiments which measure the ability of lymphocytes to respond to an antigen can be employed to ascertain the ability of an oligosaccharide glycoside to inhibit or enhance this response. An integral part of this response is the ability of lymphocytes to recognize and bind antigen-presenting 0 cells, which recognition event triggers the proliferation of the lymphocytes. Such in vitro experiments are known in the art as disclosed by Ziola et al ⁵⁵ . In addition, other in vitro experiments which measure the ability of leucocytes to bind to the surface of cells can be employed to ascertain the ability of a candidate oligosaccharide δ glycoside to inhibit the ability of such cells to heterotypically or homotypically bind leucocytes to their surfaces. Such in vitro experiments are well known in the art and are disclosed by Campanero et al. ²⁶ which describes procedures for determining the homotypic binding of leucocytes to other leucocytes; and Lowe et al. ¹⁸ which describes procedures for determining the heterotypic binding of leucocytes to the surfaces of other cells.

The in vitro experiments are generally performed by measuring cell binding in the presence or absence (control) of the candidate oligosaccharide glycoside, e.g., at a concentration of about 10 g/mL of a candidate oligosaccharide glycoside. The extent of leucocyte δ binding to the cell surface is measured in both cases and candidate oligosaccharide glycosides which reduce leucocyte binding by at least about 20 percent (and preferably by at least about 30 percent and even more preferably at least about δO percent) compared to control are deemed binding-inhibitory oligosaccharide glycosides. 0 Saccharide units (i.e., sugars) useful in the oligosaccharide glycosides related to blood group determinants employed in this invention include by way of example, all natural and synthetic derivatives of glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine, fucose, sialic acid (as defined below), 3-deoxy-D,L-octulosonic acid and the like. In δ addition to being in their pyranose form, all saccharide units in the oligosaccharide glycosides related to blood group determinants are In their D form except for fucose which is in its L form.

Preferred oligosaccharide glycosides related to blood group determinants are those which contain from 3 to 8 saccharide units 0 especially those containing the ?Gal(1-»4)/-?GlcNAc or

/3Gal(1-*3)/SGIcNAc groups. Particularly preferred oligosaccharide glycosides related to blood group determinants for use in this invention include those set forth in FIGs. 11 and 12. In FIG. 1 1 , R is an aglycon, preferably as defined above, each R, is independently selected from the 5 group consisting of hydrogen, a saccharide and a compatible saccharide; and each R ₂ is independently selected from the group consisting of hydrogen, a saccharide and a compatible saccharide and at least one of R ₁ and R ₂ is a saccharide.

Especially preferred oligosaccharide glycosides related to blood group determinants include those having the

/3Gal(1→4)0GlcNAc-Y-R | (1-3) σFuc

and the

/3GaI(1→3)SGIcNAc-Y-R I (1-4) σFuc

δ groups where Y and R are as defined above. Even more preferred oligosaccharide glycosides related to blood group determinants are those which contain a N-acetylneuraminic acid residue or an analogue thereof particularly as the non-reducing sugar terminus of the oligosaccharide. 0 The term "compatible saccharide" refers to those substituent saccharide groups which when substituted on an existing oligosaccharide glycoside related to a blood group determinant structure still permit the resulting structure to interfere with the immune response so as to reduce or inhibit the degree of immune δ response. Obviously, if substitution of a particular saccharide or a combination of saccharides so alters the characteristics of the oligosaccharide glycoside related to a blood group determinant so as to render the resulting structure incapable of inhibiting an immune response, then such a saccharide substituent or combination of 0 substituents would be deemed an incompatible substituent at least as it relates to substitution at that point on the structure of the oligosaccharide glycoside related to a blood group determinant.

The term "sialic acid" refers to (N-acetylated) δ-amino-3,δ-dideoxy-D-glycero-D-galacto-nonulosonicacid ("NeuδAc") δ and to derivatives thereof. The nomenclature employed herein in describing derivatives of sialic acid is as set forth by Reuter et al. ²⁵

B. Preparation of Oligosaccharide Glycosides

Oligosaccharide glycosides, including oligosaccharide glycosides related to blood group determinants, are readily prepared either by

complete chemical synthesis or by chemical/enzymatic synthesis wherein glycosyltransferases are employed to effect the sequential addition of one or more sugar units onto a saccharide or an oligosaccharide. Chemical synthesis is a convenient method for δ preparing either the complete oligosaccharide glycoside; for chemically modifying a saccharide unit which can then be chemically or enzymatically coupled to an oligosaccharide glycoside; or for chemically preparing an oligosaccharide glycoside to which can be enzymatically coupled one or more saccharide units. 0 Chemical modifications of saccharide units are well known in the art. For example, chemically modified NeuδAc derivatives including 9- azido-NeuδAc, 9-amino-NeuδAc, 9-deoxy-NeuδAc, 9-fluoro-NeuδAc, 9-bromo-NeuδAc, 8-deoxy-NeuδAc, 8-epi-NeuδAc, 7-deoxy-NeuδAc, 7-epi-NeuδAc, 7,8-bis-epi-NeuδAc, 4-O-methyl-NeuδAc, 4-N-acetyl- NeuδAc, 4,7-di-deoxy-NeuδAc, 4-oxo-NeuδAc, 3-hydroxy-NeuδAc, 3- fluoro-NeuδAc acid as well as the 6-thio analogues of NeuδAc are known in the art. Chemical modifications of other saccharide units are also known in the art.

Additionally, chemical methods for the synthesis of 0 oligosaccharide glycosides are also well known in the art which methods are generally adapted and optimized for each individual structure to be synthesized. In general, the chemical synthesis of all or part of the oligosaccharide glycosides first involves formation of a glycosidic linkage on the anomeric carbon atom of the reducing sugar. δ Specifically, an appropriately protected form of a naturally occurring or of a chemically modified saccharide structure (the glycosyl donor) is selectively modified at the anomeric center of the reducing unit so as to introduce a leaving group comprising halides, trichloroacetimidate, thioglycoside, etc. The donor is then reacted under catalytic 0 conditions well known in the art with an aglycon or an appropriate form of a carbohydrate acceptor which possess one free hydroxyl group at the position where the glycosidic linkage is to be established.

A large variety of aglycon moieties are known in the art and can be attached with the proper configuration to the anomeric center of the reducing unit. Appropriate use of compatible blocking groups, well known in the art of carbohydrate synthesis, will allow selective δ modification of the synthesized structures or the further attachment of additional sugar units or sugar blocks to the acceptor structures. A detailed discussion of prior art methods for forming the glycosidic linkage is recited in Venot et al., U.S. Serial No. 07/ , , and U.S.

Serial No. 07/ , , both filed on May 22, 1992, as Attorney Docket 0 Nos. 00047δ-011 and 00047δ-029 and entitled "Modified Sialyl Lewis* Compounds" and "Modified Sialyl Lewis ^x Compounds," respectively. The disclosures of both of these cases are incorporated herein by reference in their entirety.

After formation of the glycosidic linkage, the saccharide δ glycoside can be used to effect coupling of additional saccharide unit(s) or chemically modified at selected positions or, after conventional deprotection, used in an enzymatic synthesis. In general, chemical coupling of a naturally occurring or chemically modified saccharide unit to the saccharide glycoside is accomplished by 0 employing established chemistry well documented in the literature. See, for example, Okamoto et al. ²⁷ , Ratcliffe et al. ²⁸ , Abbas et al. ²⁹ , Paulson ³⁰ , Schmidt ³¹ , FQgedi et al. ³² , Kameyama et al. ³³ and Ratcliff et al ⁵⁴ . The disclosures of each of these references are incorporated herein by reference in their entirety. δ On the other hand, enzymatic coupling is accomplished by the use of glycosγl transferases which transfer sugar units, activated as their appropriate nucleotide donors, to specific saccharide or oligosaccharide acceptors, generally at the non-reducing sugar portion of the saccharide or oligosaccharide. See, for example, Toone et al. ³⁴ . Moreover, it is possible to effect selected chemical modifications of the saccharide or oligosaccharide acceptor, of the sugar donor or the

product of the enzymatic reaction so as to introduce modifications or further modifications into the structure.

Representative of glycosγltransferases are sialγltransferases which constitute a group of enzymes which transfer N- δ acetylneuraminic acid, activated as its cytidine monophosphate (CMP) derivative, to the terminal oligosaccharide structures of glycolipids or glγcoproteins. Specific transferases have been identified which build the following terminal structures on glycoconjugates: σNeuδAc(2-3)/5Gal(1-3)/9GlcNAc- 0 σNeuδAc(2-3,/3Gal(1-4)/3GlcNAc- σNeuδAc(2-6)0Gal(1-4)0GlcNAc- σNeuδAc(2-3)0Gal(1-3)0GalNAc- σNeuδAc(2-6)σGalNAc- σNeuδAc(2-6)0GlcNAc-. δ The enzymatic transfer of NeuδAc and analogues thereof requires the prior synthesis of their nucleotide (CMP) derivatives. Activation of NeuδAc is usually done by using the enzyme CMP-sialic acid sγnthase which is readily available and the literature provides examples of the activation of various analogues of NeuδAc such as 9- 0 substituted NeuδAc, 7-epi-Neu5Ac, 7,8-bis-epi-NeuδAc, 4-O-methyl- NeuδAc,4-deoxy-NeuδAc, 4-acetamido-NeuδAc, 7-deoxy-NeuδAc, 4,7-dideoxy-NeuδAc, and the 6-thio derivatives of NeuδAc. Alternatively, if the analogue of NeuδAc is not amenable to activation by the CMP-sialic acid synthase, then the NeuδAc analogue can be 6 coupled to the oligosaccharide acceptor by chemical means known in the art.

The nucleotide derivative of NeuδAc or of an analogue thereof and the saccharide acceptor are combined with each other in the presence of a suitable sialyltransferase under conditions wherein NeuδAc or an analogue thereof is transferred to the acceptor. As is apparent, the saccharide acceptor employed must be one which functions as a substrate of the particular sialyltransferase employed.

In this regard, the art recognizes that while NeuδAc is usually enzymatically transferred to a natural acceptor [i.e., gtyCo rOteifiS ant. other glycoconjugates having a glycan chain possessing a terminal acceptor disaccharide structure recognized by the enzymes and terminal acceptor disaccharides not possessing an aglycon moiety, R = H in these cases), some sialyltransferases can tolerate certain modifications in the structure of the acceptor whereas other sialyltransferases show strict specificity for one type of acceptor. It has been found that chemically modified acceptors ("artificial acceptors"), such as oligosaccharide glycosides optionally modified in the oligosaccharide portion, are tolerated in some cases by sialyltransferases. On the other hand, not all chemical modifications of the acceptor can be tolerated. For example,

/SGal(1→3/4)/SGIcNAcσ(2→3)sialyltransferase can transfer NeuδAc to a terminal _/ ?Gal(1→4) ?GlcN Ac- disaccharide structure. However, in this situation, it has been found that the hydroxyl groups at the 3, 4 and 6 positions of /S-galactose are critical to recognition by the enzyme and accordingly chemical modification at one or more of these points can result in non-recognition by the enzyme. Likewise, when an analogue of NeuδAc is to be enzymatically transferred, it is necessary that the CMP derivative of the analogue also be recognized by the sialyltransferase. Since sialyltransferases are naturally designed to transfer or donate NeuδAc, any modification to the NeuδAc results in the formation of an "artificial donor". In this regard, the art recognizes that certain sialyltransferases can tolerate some modifications to the NeuδAc and still transfer analogues of NeuδAc to glycoproteins or glycoiipids possessing a suitable terminal acceptor structure.

Surprisingly, it has been found that sialyltransferases possess sufficient recognition flexibility so as to transfer an artificial donor to an artificial acceptor. Such flexibility permits the facile synthesis of numerous sialic acid containing oligosaccharide glycosides.

-2δ-

As noted above, a suitable nucleotide derivative of NeuδAc or an analogue thereof is combined with a suitable acceptor (i.e., a saccharide glycoside or an oligosaccharide glycoside having terminal saccharide unit(s) on the non-reducing end which are recognized by the δ sialyltransferase) in the presence of the sialyltransferase under conditions wherein NeuδAc or an analogue thereof is transferred to the acceptor. Suitable conditions, known in the art, include the addition of the appropriate sialyltransferase to a mixture of the saccharide acceptor and of the CMP-derivative of the sialic acid in a appropriate 0 buffer such as 0.1 M sodium cacodylate in appropriate conditions of pH and temperature such as at a pH of 6.δ to 7.δ and a temperature between 2δ and 4δ°C, preferably 3δ-40°C for 12 hours to 4 days. The resulting oligosaccharide can be isolated and purified using conventional methodology comprising HPLC, gel-, ion exchange-, δ reverse-phase- or adsorption chromatography.

Likewise, other sugars can be transferred onto a saccharide or oligosaccharide structure by use of appropriate glycosyltransferases in a manner similar to that described above for transfer by sialyltransferase. In this regard, sialyltransferases as well as other 0 glycosyltransferases are well known in the art and are described in

Toone et al. ³⁴ and Beyer et al. ³⁵ .

Utility

Without being limited to any theory, it is believed that oligosaccharide glycosides affect the cell mediated immune response in δ a number of ways. Oligosaccharide glycosides can inhibit the ability of the immune response to become educated about a specifc antigen when the oligosaccharide glycoside is administered simultaneously with the first exposure of the immune system to the antigen. Also, oligosaccharide glycosides can inhibit the effector phase of a cell- mediated immune response (eg., the inflammatory component of a

DTH response) when administered after second or later exposures of the immune system to the antigen. Additionally, oligosaccharide glycosides can induce tolerance to antigens when administered at the time of second or later exposures of the immune system to the δ antigen.

The suppression of the inflammatory component of the immune response by oligosaccharide glycosides related to blood group determinants is believed to require the initiation of a secondary immune response (i.e., a response to a second exposure to antigen). The 0 oligosaccharide glycoside related to a blood group determinant is generally administered to the patient at least about O.δ hours after an inflammatory episode, preferably, at least about 1 hour after, and most preferably, at least about δ hours after an inflammatory episode or exacerbation. δ Oligosaccharide glycosides related to blood group determinants are effective in suppressing cell-mediated immune responses to an antigen (eg. the inflammatory component of a DTH response) when administered at a dosage range of from about O.δ mg to about δO mg/kg of body weight, and preferably from about O.δ to about δ mg/kg of body weight. The specific dose employed is regulated by the particular cell-mediated immune response being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like. The oligosaccharide glycosides related to blood group determinants are generally administered parenterally, such as intranasally, intrapulmonarily, transdermally and intravenously, although other forms of administration are contemplated. Preferably, the suppression of a cell-mediated immune response, eg. the inflammatory component of a DTH response, is reduced by at least about 10% as opposed to control measured 24 hours after administration of the challenge to the mammal and 19

hours after administration of the oligosaccharide glycoside as per this invention.

In addition to providing suppression of the inflammatory component of the cell-mediated immune response to an antigen, δ administration of the oligosaccharide glycoside related to a blood group determinant also imparts a tolerance to additional challenges from the same antigen. In this regard, re-challenge by the same antigen weeks after administration of the oligosaccharide glycoside related to a blood group determinant results in a significantly reduced immune response. 0 Administration of the oligosaccharide glycoside related to a blood group determinant simultaneously with first exposure to an antigen imparts suppression of a cell-mediated immune response to the antigen and tolerance to future challenges with that antigen. In this regard the term "reducing sensitization" means that the compound, δ when administered to a mammal in an effective amount along with a sufficient amount of antigen to induce an immune response, reduces the ability of the immune system of the mammal to become educated and thus sensitized to the antigen administered at the same time as the compound. An "effective amount" of the compound is that amount which will reduce sensitization (immunological education) of a mammal to an antigen administered simultaneously as determined by a reduction in a cell-mediated response to the antigen such as DTH responses as tested by the footpad challenge test. Preferably the reduction in sensitization will be at least about 20% and more preferably at least about 30% or more. Generally oligosaccharide glycosides related to blood group determinants are effective in reducing sensitization when administered at a dosage range of from about O.δ mg to about 60 mg/kg of body weight, and preferably from about O.δ mg to about δ mg/kg of body weight. The specific dose employed is regulated by the sensitization being treated as well as the judgement of the attending clinician depending upon the age and general condition of the patient and the like. "Simultaneous" administration of the

compound with the antigen with regard to inhibiting sensitization means that the compound is administered once or continuously throughout a period of time within 3 hours of the administration of an antigen, more preferably the compound is administered within 1 hour δ of the antigen.

The methods of this invention are generally achieved by use of a pharmaceutical composition suitable for use in the parenteral administration of an effective amount of an oligosaccharide glycoside related to a blood group determinant. These compositions comprise a pharmaceutically inert carrier such as water, buffered saline, etc. and an effective amount of an oligosaccharide glycoside related to a blood group determinant so as to provide the above-noted dosage of the oligosaccharide glycoside when administered to a patient. It is ^" contemplated that suitable pharmaceutical compositions can additionally contain optional components such as an adjuvant, a preservative, etc.

It is also contemplated that other suitable pharmaceutical compositions can include oral compositions, transdermal compositions or bandages etc., which are well known in the art. The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention.

In these examples, unless otherwise defined below, the abbreviations employed have their generally accepted meaning:

i.r. = infra red m = multiplet q = quartet s = singlet t triplet t.l.c. = thin layer chromatography U = Units μm = microns

AG 1 x 8 (formate form) = ion exchange resin AG 1 x 8 (formate form) available from Bio-Rad Laboratories,

Richmond, CA Dowex δO x 8 (H ⁺ form) = ion exchange resin Dowex δO x 8 (H ⁺ form) available from Dow Chemical, Midland, Ml IR-CδO resin (H ⁺ form) = ion exchange resin IR-CδO

(H ⁺ form) available from Rohm & Haas, Philadelphia, PA Commercially available components are listed by manufacturer and where appropriate, the order number. Some of the recited manufacturers are as follows: latron = latron Laboratories, Tokyo, Japan Merck = E. Merck AG, Darmstadt, Germany Millipore = Millipore Corp., Bedford, MA Waters = Waters Associates, Inc., Milford, MA

EXAMPLES

In the following examples, Examples 1-19 illustrate the synthesis of numerous oligosaccharide glycosides whereas Examples 20-37 illustrate the suppression of cell-mediated immune responses to an antigen by administration of an oligosaccharide glycoside related to blood group determinants and the induced tolerance to later challenges

with the same antigen. In Examples 1-13, the oligosaccharide glycosides recited are referred to by Arabic numerals which are depicted in figures 14-24 whereas in Examples 20-3δ, the oligosaccharide glycosides are referred to by Roman numerals which are depicted in figures 11-13.

In one or more of Examples 1-13, pre-coated plates of silica gel (Merck, 60-F ₂₅₄ J were used for analytical t.l.c. and spots were detected by charring after spraying with a δ% solution of sulfuric acid in ethanol. Silica gel 60 (Merck, 40-63 μm) was used for column chromatography. latrobeads were from latron (Order No. 6RS-8060).

Millex-GV filters (0.22 μm) were from Millipore. C ₁₈ Sep-Pak cartridges and bulk C ₁₈ silica gel were from Waters Associates.

Commercial reagents were used in chemical reactions and solvents were purified and dried according to usual procedures. Unless otherwise noted, the reaction mixtures were processed by dilution with dichloromethane and washing with a dilute solution of sodium bicarbonate followed by water. After drying over magnesium sulfate, the solvents were removed by evaporation under vacuum with a bath temperature of 35 °C or lower when necessary. ¹ H-n.m.r. were recorded at 300 MHz (Bruker AM-300) with either tetramethylsilane in CDCI ₃ or acetone set at 2.225 in D ₂ O as internal standards, at ambient temperature, unless otherwise noted. The chemical shifts and coupling constants (observed splittings) were reported as if they were first order, and only partial n.m.r. data are reported. ¹³ C-n.m.r. spectra were recorded at 75. δ MHz with tetramethylsilane in CDCI ₃ or dioxane set at 67.4 in D ₂ O as reference.

A. SYNTHESIS OF DERIVATIVES OF NeuδAc

Unless otherwise noted, derivatives of NeuδAc have been prepared following known procedures with suitable substitution of starting materials where necessary. The following derivatives have

been prepared by a convenient modification of procedures reported in the literature: 9-N ₃ -NeuδAc lb., ³⁶ NeuδPr (δ-propionamido) If, 7-d- NeuδAc Id ³⁷ and the C8-NeuδAc i ³⁸ .

FIG. 14 illustrates a general synthetic scheme used for the δ synthesis of derivatives of NeuδAc. Compounds referred to by underlined Arabic numerals in Examples 1-4 below are depicted Table I and in FIG. 14.

Example 1 -- Synthesis of 5-acetamido-9-azido-3,5,9-tri- deoxy-D-glycero-D-galacto-2-nonulopyranosylonic acid (9-N ₃ -Neu5Ac) Ua

Glycosyl chloride 3J£ (2.83 g, 5.57 mmol) in dry ^' dichloromethane (13 mL) was added to the mixture of benzyl alcohol (5.0 mL, 48.2 mmol), molecular sieves 4A (18.5 g, crushed), dry silver carbonate (4.2 g, 15.2 mmol) in dichloromethane (8 mL). The mixture was stirred in the dark for 4 days, diluted with dichloromethane (60 mL) and filtered through Celite. After usual work up, the residue was chromatographed on silica gel using a 3:2 mixture of hexanes and ethyl acetate as eiuant. The product was then eluted with a 4:δ mixture of the same solvents giving (1.96 g, 60%) of pure material and 0.33 g (10%) of material containing a small amount of impurities, ^-n.m.r.: δ.436 (ddd, 1 H, J _{7 B} 8.6, J _s . 2.δ, _{8 9} δ.δHz, H-8), δ.317 (dd, 1 H, J _{β 7} 1.8Hz, H-7), δ.110 (d, 1 H, _5#NH 9.δ Hz, NH), 4.849 (ddd, 1 H, J _3ax 12.0, J _3βqA 4.6, _4#5 9.δHz, H-4), 4.788 and 4.397 (AB, 2H, J _gm 12.0Hz, benzylics), 3.642 (s, CO^ϋ,), 2.629 (dd, 1 H, _{3βq 3ax} 12.δHz, H-3eq), 2.140, 2.1 13, 2.017, 1.997, 1.867,

(δs, 1 δH, 4 OAc, 1 NAc), 1.986 (dd, 1 H, H-3ax).

The above material (1.5 g, 2.58 mmol) was de-O-acetylated in dry methanol (20 mL) containing a catalytic amount of sodium methoxide for 5 hours at 22°C. After de-ionization with Dowex 50 x 8 (H ⁺ form), the solvent was evaporated leaving the product 39 (1.0 g, 94%) which was used in the next step; ¹ H-n.m.r. (CDCI3): 4.81 δ

and 4.619 (AB, 2H, _oern 11.δHz, benzylics), 3.802 (s, CO^ϋ), 3.582 (dd, 1H, ₅ . _β 9.0, _{β 7} 0.5Hz, H-6), 2.762 (dd, 1H, _3βe| . _3« 12.6, _3βq 4.6Hz, H-3eq), 2.039 (s, 3H, N Ac), 1.861 (dd, 1H, J _ZuxA 11.0Hz, H-3ax). δ A solution of para-toluenesulfonyl chloride (0.126 g, 0.66 mmol) in pyridine (0.1 mL) was syringed into a solution 3_9_ (0.248 g, 0.60 mmol), 4-dimethylaminopyridine (0.01 g) in pyridine (1.1 mL) at 0°C. After stirring for 4 hours at 0°C, methanol (0.10 mL) was added and the mixture was co-evaporated with dry toluene. The 0 residue was quickly chromatographed on silica gel using acetonitrile as eluant giving the tosylate (0.21 g, 62%) still containing some impurities. Sodium azide (0.19 g, 2.92 mmol) was added to a solution of this material (0.21 g, 0.37 mmol) in dimethylformamide (O.δ mL). The mixture was stirred at 6δ°C for 18 hours after which it δ was filtered through Celite and the solvent evaporated in vacuo. The residue was chromatographed on silica gel using a 6:1 mixture of ethyl acetate and acetonitrile as eluant giving the product 40. (0.136 g, 8δ%); i.r.tf _m ^Λ 2110 (N ₃ ); ¹ H-n.m.r.: δ.77δ (d, 1 H, _{5 NH} 9.0Hz, NH), 4.816 and 4.470 (AB, 2H, J _am 11.6Hz, benzylics), 3.738 (s, 0 COsCJi,), 2.871 (dd, 1H, J _3βqΛ 4.8, _{3βq 3«} 13.0Hz, H-3eq), 2.086 (s,

3H, NAc), 1.964 (dd, 1H, J _ZκκΛ 11.6Hz, H-3ax).

The above compound 40. (0.106 g, 0.24 mmol) was left for 3 hours at 22°C in 0.26 N sodium hydroxide (2 mL). After bringing the pH to 6 by addition of Dowex 60 x 8 (H ⁺ form) followed by filtration, δ the material, recovered after freeze drying, was chromatographed on latrobeads using a 6δ:3δ:δ mixture of chloroform, methanol and water as eluant. The appropriate fractions gave the product (0.087 g, 86%). This compound (0.100 g, 0.236 mmol) was heated at 80°C for 6 hours in 0.026 N hydrochloric acid (3 mL). The solution was neutralized with sodium hydroxide and then freeze dried. The product was chromatographed on latrobeads (0.60 g) using a 6δ:3δ:δ mixture of chloroform, methanol and water giving lb (0.067 g, 8δ%); ¹ H-

n.m.r.: 4.106 - 3.896 (m, 6H), 3.639 (dd, 1 H, J _BΛ 3.0, J _aΛ . 13.0Hz, H-9), 3.528 (dd, 1 H, J _VιV 6.0Hz, H-9"), 2.249 (dd, 1 H, _3βq>4 4.5, ^ _3« ,. _3« 12.5Hz, H-3eq), 2.090 (s, 3H, NAc), 1.852 (dd, 1 H, _{3ax 4} 1 1.0Hz, H-3ax).

Example 2 - Synthesis of 5-propionamido-3,5-didβoxy-D- glycero-D-galacto-2-nonulopyranosylonic acid (NeuδPr) If

A solution of 33 (0.076 g, 0.18 mmol) in 2 N sodium hydroxide (1 mL) was left for O.δ hours at 22°C followed by 7 hours at 9δ°C. The pH was then adjusted to 7.6 by addition of IR-CδO resin (H ⁺ form). The filtrate obtained after filtration of the resin was evaporated in vacuo and the residue dried over phosphorous pentoxide.

Propionic anhydride (0.12 mL, 0.94 mmol) was then syringed into a suspension of the above product in a mixture of dry methanol (1.5 mL) and triethylamine (0.2 mL) which was stirred at 0°C. After

3 hours, more propionic anhydride (0.025 mL, 0.196 mmol) was added and the mixture stirred for 2 more hours at 0°C. The mixture was co-evaporated with methanol, and a solution of the residue in water (2 mL) was passed through Dowex 50 x 8 (H ⁺ form, 6 g). The recovered fractions were evaporated in vacuo and the residue chromatographed on latrobeads (5 g) using a 3:1 mixture of chloroform and methanol as eluant giving 41 (0.0646 g, 86.5%); ¹ H- n.m.r.: 4.800, 4.578 (AB, 2H, J _aβm 11.0Hz, benzylics), 3.580 (dd, 1 H, J _Λ 9.0, _β . ₇ 1.0Hz, H-6) 2.776 (dd, 1 H, _3βq>4 4.5, _{3βq 3ax} 12.5Hz, H- 3eq), 2.316 (q, 2H, J 7.5 Hz, CH ₂ CO), 1.762 (dd, 1 H, J _3axA 12.0Hz),

1.129 (t, 3H, CH ₃ ).

A solution of the above benzyl glycoside (0.1 15 g, 0.278 mmol) in water (5 mL) was hydrogenated in the presence of 5% palladium on charcoal (10 mg) at atmospheric pressure and 22°C for 5 hours. The eluate obtained after filtration through Celite followed by Millipore filter, was freeze dried leaving compound If (0.074 g, 82.5%); ¹ H-

n.m.r.: 3.72 - 4.10 (m, H-4,-5,-7,-8,-9), 3.614 (dd, 1 H, J _Λ 6.5, ₉ „ _9b 11.75Hz, H-9a), 3.530 (dd, 1H, _5rβ 9.0 _β>7 1.0Hz, H-6), 2.250 - 2.400 [m, 2H incl. CH ₂ C0 (q, 2.315, J 7.5Hz) and H-3eq (dd, J ₃ ^ _3ai 11.5 Hz, _{3βq 4} 4.5Hz)], 1.880 (t, 1H, J _{3tx 3aq} 11.5Hz, H-3ax), 1.130 (t, δ 3H, CH,).

Example 3 - Synthesis of 5-acetamido-3,5-dideoxy-D- galacto-2-octulosonic acid (C8-Neu5Ac) ϋ

The synthesis of ϋ from 33 essentially follows the published procedure of Hasegawa et al. ³⁸ but using a different starting material than the reported one. In particular, a suspension of 33 (0.52 g,

0.126 mmol) in 2,2-dimethoxypropane (3 mL) was stirred for 1.5 hours at 22°C in the presence of paratoluenesulfonic acid (0.5 mg). After neutralization with some triethylamine, the mixture was evaporated and the residue chromatographed on silica gel using a 16:1 mixture of chloroform and methanol giving 42 (0.049 g, 88%).

42 (0.054 g, 0.185 mmol) was acetylated in a 2:1 mixture of acetic anhydride (1 mL) and pyridine kept at 50°C for δ hours. After the usual work up, the residue was chromatographed on silica gel using ethyl acetate as eluant giving the acetylated product (0.091 g, 92%); ¹ H-n.m.r.: δ.420 (dd, 1H, _β>7 1.5, _7>8 3.5Hz, H-7), 5.196 (d,

1H, _{5 NH} 9.0Hz, NH), 5.009 (ddd, 1H, _4>3« 13.0, «/ _4,3βq 5.0, _4#5 10.0Hz, H-4), 4.797 and 4.498 (AB, 2H, J^ 11.5Hz, benzylics), 3.776 (s, 3H, CO^t ), 2.724 (dd, 1 H, v/ _3βt>3βq 13.0Hz, H-3eq), 2.151 , 2.032, 1.895 (3s, 9H, 2 OAc, 1 NAc), 2.032 (t, 1H, H-3ax), 1.363 and 1.350 (2s, 6H, methyls).

The above product (0.091 g, 0.169 mmol) was heated for 4 hours at 40 °C in 70% aqueous acetic acid. The mixture was co- evaporated with toluene in vacuo. The dry residue was dissolved in dry methanol and stirred for 2 hours at 22°C in the presence of sodium metaperiodate (0.069 g, 0.276 mmol). The mixture was

-36- filtered through a pad of Celite which was washed with methanol. The combined filtrate was stirred at 0°C for 26 minutes in the presence of sodium borohydride (0.036 g, 0.95 mmol). The mixture was then stirred at 0°C with some acetic acid (0.2 mL), after which 5 the solvents were evaporated leaving a residue which was dried in vacuo for 15 minutes and then acetylated in a 5:1 mixture of pyridine and acetic anhydride (6 mL) for 20 hours at 22°C. The residue recovered after the usual work up was chromatographed on silica gel using ethyl acetate as eluant to give a product which still contained 0 some non-separable impurities. The dry material (0.074 g, still containing some impurities) was dissolved in dry methanol (5 mL) and stirred at room temperature for 3 hours in the presence of sodium (3 mg). After de-ionization with Dowex 60 x 8 (H ⁺ form) and filtration, the solvent was evaporated in vacuo and the residue chromatographed δ on silica gel using a 15:1 mixture of chloroform and methanol to give a pure product 44 (0.047 g, 78%); ^-n.m.r.: (CD ₃ OD): 4.724 and 4.416 (AB, 2H, J _gβm 1 1.5Hz, benzylics), 3.671 (s, 3H, 3.456 (dd, 1H, _{5 β} 9.5, _β#7 1.0Hz, H-6), 2.642 (dd, 1H, _{3βq 4} 4.5, ^ _3« ,. _3« 12.5Hz, H-3eq), 1.938 (s, 3H, NAc), 1.699 (t, 1 H, J _3txA 12.5Hz, H-3ax).

The above material (0.022 g, 0.057 mmol) was stirred in 0.25 N sodium hydroxide (2 mL) for 5 hours at 22 °C, the solution was neutralized with Dowex 50 X 8 (H ⁺ form) and the filtrate was freeze dried to give a white solid (0.019 g, 90%). This product was dissolved in water (2 mL) and hydrogenated for 3 hours at 22 °C in the presence of 5% palladium on charcoal (4 mg). The mixture was first filtered through Celite and then through a Millipore filter. The filtrate was freeze dried leaving the desired product ϋ (13.3 mg, 94%); ¹ H-n.m.r.: 3.462-4.093 (m,6H), 2.287 (dd, 1 H, J _{3βq 4} 4.6, _{3βq 3} „ 12.6Hz, H-3eq), 2.052 (s, 3H, NAc), 1.853 (t, 1 H, _{3βq 4} 12.5

Hz, H-3ax).

Example 4 - Synthesis of 5-acetamido-3,5,7-trideoxy- ?- D-galacto-2-nonulopyranosylonicacid (7-d-Neu5Ac) Id.

The synthesis of lg_ essentially follows the published procedure of Zbiral et al. ³⁷ but using a different starting material. In particular, imidazole (0.13 g, 1.93 mmol) and tert-butyldimethylsilyl chloride (0.136 g, 0.89 mmol) were added to a solution of 42 (0.11 g, 0.19 mmol) in dimethyl-formamide (2 mL). After 4 hours at room temperature, the solvent was removed in vacuo, the residue dissolved in chloroform and worked up as usual. Chromatography of the product on silica gel using a 1 :1 mixture of ethyl acetate and hexane provided the monosilylated derivative (0.101 g, 92%): [σ] _D = -2.66 (c. 0.6, chloroform); ¹ H-n.m.r.: 5.196 (d, 1 H, J _{5 m} 7Hz, NH), 4.853 and 4.603 (AB, 2H, _gβm 1 1.5Hz, benzylics), 3.736 (s, CO ₂ CH ₃ ), 2.692 (dd, 1H, _3βq 4.5, _3βq#3 „ 13.0Hz, H-3eq), 2.022 (s, 3H, NAc),

1.884 (dd, 1H, _3ax 11.0Hz, H-3ax), 1.405, 1.375 (2s, 6H, methyls), 0.868 (s, 9H, t-butyl), 0.093 and 0.084 (2s, 6H, methyls).

Sec-butyl lithium (1.3 M in cyclohexane, 0.65 mL, 0.85 mmol) followed by carbon disulfide (1.25 mL, 20.8 mmol) were added dropwise to a solution of the above compound (0.437 g, 0.77 mmol) in dry tetrahydrofuran (20 mL) at -30° C. After stirring at -25 °C for 0.5 hours, methyl iodide (1.6 mL, 25.6 mmol) was slowly warmed up to room temperature. After evaporation, the residue was chromatographed on silica gel using a 4:1 mixture of hexanes and ethyl acetate as eluant providing the xanthate (0.327 g, 65%): [σ] _D

93.9 (c. 0.656, chloroform); ¹ H-n.m.r.: 6.388 (dd, 1H J _{e 7} 1.0, _7<8 2.5Hz, H-7), 5.610 (d, 1 H, J _5m 7.0Hz, NH), 4.778, 4.466 (AB, 2H, J _gem 11.6Hz, benzylics), 3.778 (s, CO^h ), 2.662 (dd, 1H, _{3βq 4} 4.6, a _β cs _ax 12.6Hz, H-3eq), 2.684 (s, 3H, OCH ₃ ), 1.883 (s, 3H, NAc), 1.693 (dd, 1 H, J _3axA 1 1.5Hz, H-3ax), 1.31 δ (s, 6H, methyls) 0.825

(9H, t-butyl), 0.025, 0.092 (2s, 6H, methyls).

Azobisisobutyronitrile (0.004 g) and tri-n-butyltin hydride (0.5 mL, 1.86 mmol) were added to a solution of the above xanthate (0.32 g, 0.48 mmol) in dry toluene (3 mL). After heating at 100°C for 7 hours, the solvents were co-evaporated with dry toluene, and the 5 residue chromato-graphed on silica gel using a 3:2 and then 1 :1 mixtures of hexane and ethyl acetate as eluant to give the 7-deoxy product (0.260 g, 70%); ^-n.m.r.: 5.334 (d, 1 H, _5#NH 7.0Hz, NH), 4.740, 4.455 (AB, 2H, J _oβm 11.6Hz, benzylics), 3.690 (s, CO^H , 2.628 (dd, 1 H, _3βq<4 4.2, _{3βq 3} „ 12.9Hz, H-3eq), 1.914 (s, 3H, NAc), 0 1.805 (dd, 1 H, J _3ax 10.9Hz, H-3ax), 1.718 and 1.597 (m, 2H, H-7 and H-7'), 1.325 (s, 6H, methyls), 0.804 (9H, t-butyl), 0.010, 0.009 (2s, 6H, methyls). The above compound (0.260 g, 0.47 mmol) was heated at 75 °C in 70% acetic acid for 7.5 hours. After co- evaporation with toluene, the residue was chromatographed on silica δ gel using a 10:1 mixture of chloroform and methanol giving 43 (0.157 g, 84%); ¹ H-n.m.r.: 4.860 and 4.655 (AB, 2H, J _gm 1 1.5Hz, benzylics), 3.834 (s, CO^ϋ,), 2.806 (dd, 1 H, _{3βq 4} 4.5, _3βq,3ax 12.5Hz, H-3eq), 2.069 (s, 3H, NAc), 1.881 (dd, 1 H, J _3nc 12.5Hz, H- 3ax), 1.698 (m, 2H, H-7 and H-7'). Compound 43 (0.157 g, 0.396 mmol) was kept in 0.25 N sodium hydroxide (6 mL) at room temperature for 5 hours. After neutralization with Dowex 50W x 8 (H ⁺ form) and filtration, the product (0.149 g, 97%) was recovered after lyophilization of the solution. This product (0.146 g, 0.38 mmol) was hydrogenated in water (5 mL) for 5 hours at room temperature in the presence of 5% palladium on charcoal (0.010 g). The mixture was filtered through Celite and through a Millex-GV (0.22 μm) filter. The filtrate was freeze dried to provide Id (0.105 g, 94%); ¹ H-n.m.r.: as reported by Christian ³⁹ . Table 1 below summarizes the derivatives of Neu5Ac prepared.

Table I

I ω o Io

Table 1 cont.

Sialyl Moieties obtained by chemical modification sialylated oligosaccharides:

1IV

B. SYNTHESIS OF CMP DERIVATIVES OF Neu5Ac AND ANALOGUES THEREOF

Example 5 — Synthesis of the CMP-derivatives of NeuδAc

CMP-sialic acid synthase was extracted from calf brain and partially purified at 4°C by a slight modification of the original procedure of Higa et al. ⁴⁰ Routinely, ~ 200 g of brain tissue were homogenized in a Cuisinart blender (three 30 second bursts with 1 minute intervals) with 400 mL of 25 mM Tris/HCI, pH 7.5, 10 mM magnesium chloride, 10 mM sodium chloride, 2.5 mM dithioerythritol, 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was stirred for 1 hour and then centrifuged at 23,000 x g for 15 minutes. The supernatant was decanted and the pellets were extracted once again with 200 mL of the same buffer as above. The supernatants were combined and centrifuged at 28,000 x g for 15 minutes. The supernatant was filtered through glass wool to give the crude extract

(615 mL, 4.7 mg protein/mL, ~90 U of enzyme).

After adjusting salt concentration to 0.4 M with solid potassium chloride, the crude extract was stirred and solid ammonium sulfate was added to 35% saturation (208 g/L) over a period of 15 minutes. The solution was stirred for an additional 15 minutes, kept on ice for 1 hour and centrifuged at 28,000 x g for 30 minutes. The precipitate was discarded and the supernatant was stirred and adjusted to 60% saturation by the addition of solid ammonium sulfate (163 g/L) over 15 minutes. After an additional 15 minutes of stirring, the suspension was left on ice overnight and then centrifuged as above. The resultant pellets were washed with 150 mL of 60% ammonium sulfate solution to remove the co-precipitates. The washed pellets contain 70-80 U of enzyme with a specific activity of 0.08 U/mg protein. The enzyme was assayed as described by Kean et al. ⁴¹ , with one unit of

enzymatic activity defined as one /mol of product formed per minute at 37°C.

The enzyme present in the pellet could be stored for several weeks in the cold room. Before using the enzyme for synthesis, the pellets were suspended in a minimal volume of 50 mM Tris/HCI, pH

9.0, 35 mM magnesium chloride, 3 mM 2-mercaptoethanol (activation buffer) and dialyzed overnight against 100 volumes of the same buffer. The dialyzed enzyme was centrifuged at 9,000 x g for 10 min. The supernatant containing more than 90% of the enzyme activity was used directly for the synthesis.

The CMP-derivatives of sialic acid analogues were synthesized as noted above and purified by a modification of the reported procedures of Higa et al. ⁴⁰ and Gross et al. ⁴² For example, 7-d- NeuδAc Id (Table 1 , 20 mg, 69 //mol) was activated by using 15 U of the above dialyzed enzyme for 5-6 hours at 37°C in 12 mL of the activation buffer in the presence of four fold excess of cytidine triphosphate. When appropriate, the conversion of the sialic acid analogues was estimated by the usual thiobarbituric acid assay for sialic acid after reduction with sodium borohydride as per Kean et al. ⁴³ The product was extracted with cold acetone as per Gross et al. ⁴²

After evaporation of the acetone in vacuo (at ~ 15°C), the concentrated solution was applied to a column of Bio-Gel P-2 (2.5 x 91 cm) equilibrated and eluted with 10 mM ammonium hydroxide at 4°C with a flow rate of 60 mL/h. Fractions (1 mL) were assayed for cytidine by absorbance at 273 nm, and the fractions corresponding to the first peak were pooled, concentrated in vacuo and the residue was freeze-dried leaving the CMP-7-d-Neu5Ac (2d, 30 mg, -94%). This material showed a very small amount of impurities by H-n.m.r. (Table 2) and was used directly for the reaction with sialyltransferases. In some cases (2e, 2g, 2h), ¹ H-n.m.r. spectra showed that the CMP- derivatives contained some of the unreacted sialic acid.

Table 2 below illustrates the CMP-derivatives of analogues of Neu5Ac prepared from the analogues of NeuδAc set forth in Table 1 above, as well as partial ^-n.m.r. data concerning these compounds.

¹ H-n.m.r. Data and Reaction Data for CMP-βialiσ Acid Derivatives ¹

CO c

09 (0

H

H ω PI O X PI PI

H

1. in D,0 with DOH set at 4.80.

2. thiobartiburic assay

3. 2.31 (q, 7.5Hz, CH-) ; 1.33 (t, CH _j )

4. coupling constants not accurately obtained due to poor resolution.

-44- C. SYNTHESIS OF OLIGOSACCHARIDE GLYCOSIDES

Examples 6-7 illustrate the synthesis of oligosaccharide glycosides. The structure of 3Jj to 7j» are illustrated in FIG. 15. Oligosaccharide glycosides 4b_, 5b_, jjjf, 6jι, and 7a. were synthesized according to the procedures of Lemieux et al. ⁴³ , Lemieux et al. ⁴⁴ ,

Paulsen et al. ⁴⁵ , Sabesan et al. ^4β , and Lemieux et al. ⁴⁷ , respectively.

Oligosaccharide glycosides 4d_ and £d. were synthesized following the procedure reported for the synthesis of oligosaccharide glycosides 4b and 5b, but by replacing the 8-methoxycarbonyloctyl by methanol.

Oligosaccharide glycosides 5s and 5fl were synthesized according to the procedures of Paulsen et al. ⁴⁵ and Alais et al. ⁴⁸ but replacing the methanol by 8-methoxycarbonyloctanoI. In all cases, the oligosaccharide glycosides were purified by chromatography on latrobeads with the appropriate solvent mixtures and the recovered materials chromatographed on BioGel P2 or Sephadex LH20 and eluted with water. The recovered materials were lyophilized from water and the products further dried in vacuo over phosphorus pentoxide.

Example 6 - Synthesis of 9-Hydroxynonyl 2-acetamiάo-2- deoxy-[ ?-D-galactopyranosyl-(1-3)-0-]- ?-D- glucopyranoside 4a.

Sodium acetate (0.200 g) and sodium borohydride (0.060 g) were added to a solution of the disaccharide 4b (0.100 g, 0.189 mmol) in a 10:1 mixture of water and methanol (20 mL) cooled at +4°C. After 24 hours, more sodium borohydride (0.020 g) was added to the reaction mixture maintained at +4°C. After 48 hours at the same temperature, the pH was brought to 5-6 by addition of acetic acid. The solution was then co-evaporated with an excess of methanol. The residue was dissolved in water (10 mL) and run

-45- through a column of C ₁₈ silica gel which was further washed with water. After elution with methanol, the solvent was evaporated in vacuo. The residue was dissolved in a 10:1 mixture of water and methanol and the pH brought to 13-14 by addition of 1 N sodium hydroxide. The mixture was left at room temperature until t.l.c.

(65:35:5 - chloroform, methanol and water) indicated the disappearance of the unreacted starting material 4b_. The mixture was then neutralized by addition of Dowex 50 x 8 (H ⁺ form) and the resin filtered off. The resulting solution was run through a column of AG 1 x 8 (formate form). The eluate was freeze dried and the residue was run through Sephadex LH 20 using a 1 :1 mixture of water and ethanol. The appropriate fractions were pooled and concentrated to give 4a (0.060 g, 65%); ¹ H-n.m.r. (D ₂ 0): 4.545 (d, 1 H, J _Λ 8.0Hz, H- 1 ), 4.430 (d, 1 H, J _VΛ ., 7.5Hz, H-1 '), 2.025 (s, 3H, NAc), 1.543 (m, 4H), and 1.304 (m, 10H): methylenes; ¹³ C-n.m.r. (D ₂ 0): 175.3 (Ac),

104.36 (C-T), 101.72 (C-1 ), 67.72, 61.85, 61.60 (three CH ₂ OH).

Example 7 — 9-Hydroxynonyl 2-acetamido-2-deoxy-[ ?-D- galactopyranosyl-(1-4)-O-]- ?-D-gluco- pyranoside 5a

Oligosaccharide glycoside 5ϋ was prepared from 5 _ as indicated above (60%); ¹ H-n.m.r. (D2O): 4.520 (d, 1H, J _Λ 7.5Hz, H-1), 4.473 (d, 1 H, J _Vj . 7.6Hz, H-1 '), 2.033 (s, 3H, NAc), 1.543 (m, 4H) and 1.302 (m,10H):methylenes; ¹³ C-n.m.r. (D2O): 175.23 (Ac), 103.71 and 101.88 (C-1 and C-1 '), 60.93, 61.85 and 62.71 (three CH ₂ OH).

Example 8 - Synthesis of 5-Allyloxypentyl 2-acetamido-2- deoxy-[ ?-D-galactopyranosyl-( 1 -3)-O-]- ?-D- glucopyranoside 4c

The synthetic schemes for this example and Example 9 are set forth in FIG. 16.

-46- A. Synthesis of Allγloxy-5-pentanol 29

Allyl bromide (2.5 mL, 0.029 mol) was added dropwise to the mixture of 1 ,5-pentanediol (3 g, 0.029 mol) and sodium hydride (1.2 g, 80% dispersion in oil) in dry dimethylformamide. Stirring was continued overnight at room temperature. T.l.c. (2:1 - toluene and ethyl acetate) still indicated the presence of some unreacted pentanediol. The unreacted sodium hydride was destroyed by addition of methanol. The mixture was concentrated to 50 mL by evaporation in vacuo. After dilution with methylene chloride (150 mL), the solvents were washed with water (three times), dried over magnesium sulfate and evaporated in vacuo. The residue was chromatographed on silica gel using a 2:1 mixture of toluene and ethyl acetate as eluant. The appropriate fractions gave compound 29 (0.931 g, 30%). ¹ H-n.m.r. (CDCI ₃ ): 5.83 (m, 1 H, -CH = ), 5.20 (m, 2H, = CH ₂ ), 3.95 (dd, 1H, = 5.5 and 1.0Hz, allylics), 3.66 and 3.46 (two t, 2H each,

J= 6.5Hz, O-CH ₂ ), 1.64 (m, 4H) and 1.44 (m, 2H): methylenes); ¹³ C- n.m.r.(CDCI ₃ ): 134.7 and 116.6 (ethylenics), 71.6, 70.1 (£H ₂ -O-C.H ₂ ), 62.1 (£H ₂ OH) 32.2, 29.2 and 22.2 (methylenes).

B. Synthesis of 5-Allyloxypentyl 2-deoxy-2-phthalimido- ?- D-glucopyranoside 32

A solution of 3,4,6-tri-0-acetyl-2-deoxy-2-phthalimido-D- glucopyranosyl bromide 30 (5.0 g, 10.0 mmol) in dichloromethane (5 mL) was added dropwise to a mixture of the alcohol 29 (1.33 mL, 10 mmol), silver trifluoro-methanesulphonate (2.57 g, 10.0 mmol) and collidine (1.23 mL, 9.0 mmol) in dichloromethane (10 mL) at -70°C.

After stirring for 3 hours at -70°, t.l.c. (2:1-toluene and ethyl acetate) indicated that the starting bromide and the reaction product had the same Rf. After addition of some triethylamine, the reaction mixture was diluted with dichloromethane and worked up as usual. The

-47- syrupy residue was chromatographed on silica gel using a 5:1 mixture of toluene and ethyl acetate providing compound 31 (4.0 g, 71 %). ¹ H-n.m.r. (CDCI ₃ ): 5.80 (m, 2H, -CH = and H-3), 5.36 (d, 1H, _1#2 8.5Hz, H-1 ), 5.18 (m, 3H, =CH ₂ and H-4), 2.13, 2.06, 1.87 (3s, 3H each, 3 OAc), 1.40 (2H) and 1.15 (m, 4H): methylenes.

A 0.2 M solution of sodium methoxide in methanol (0.500 mL) was added dropwise to a solution of compound 31 (4.00 g, 7.1 mmol) in dry methanol (30 mL) cooled at 0°C. The mixture was stirred at 0°C for 2 hours until t.l.c. (10:1 - chloroform and methanol) indicated the disappearance of the starting material. The reaction mixture was de- ionized with Dowex 50 (H ⁺ form, dry) at 0°C. Filtration and evaporation of the solvent left a residue which was purified by chromatography on silica gel using a 100:5 mixture of chloroform and methanol as eluant providing compound 32 (2.36 g, 76%). ¹ H-n.m.r. (CDCI ₃ ): 7.70 and 7.80 (m, 4H, aromatics), 5.82 (m, 1H, -CH = ), 5.17

(m, 3H, =CH ₂ and H-1 ), 1.38 and 1.10 (m, 6H, methylenes); ¹³ C- n.m.r. (CDCI ₃ ): 134.9 and 116.6 (ethylenics), 98.3 (C-1 ), 56.6 (C-2).

Synthesis of 5-Allyloxypentyl 4,6-0-benzylidene-2-deoxy-2- phthalimido- ?-D-glucopyranoside33

Paratoluenesulfonic acid monohydrate (0.025 g) was added to a solution of 32 (1.0 g, 2.3 mmol) and σ,σ-di-methoxytoluene (0.690 mL, 4.6 mmol) in dry dimethylformamide. After stirring for 2 h at 40 °C, t.l.c. (10:1 - chloroform and methanol) indicated the completion of the reaction. After addition of a small amount of triethylamine, most of the solvent was evaporated in vacuo and the residue diluted with dichloromethane and worked up as usual. After evaporation of the solvents, the residue was chromatographed on silica gel using a 9:1 mixture of toluene and ethyl acetate giving compound 33 (1.36 g, 90.1 %). [σ] ²⁰ _D +24.1 (c 0.5 chloroform); ¹ H-n.m.r. (CDCI ₃ ): 7.15- 7.90 (m, 9H, aromatics), 5.83 (m, 1H, -CH.=), 5.56 (s, 1H,

-48- benzylidene), 5.10-5.37 [m, 3H, =CH ₂ and H-1 (5.25, d, J _ΛΛ 8.5Hz)], 1 .40 (m, 2H) and 1.17 (m, 4H): methylenes.

D. Synthesis of 5-Allyloxypentyl 4,6-O-bβnzylidene-2-deoxy- [2,3,4,6-tetra-O-acetyl-/5-D-galactopyranosyl- (1-3)-O-]-2-phthalimido-/?-D-glucopyranoside35

A solution of trimethylsilyltrifluoromethanesulfonate (0.1 mL of a solution made from 0.050 mL of the reagent in 1.0 mL of dichloromethane) was syringed into a mixture of compound 33 (1.20 g, 2.29 mmol), 2,3,4,6-tetra-O-acetyl-σ-D-galactopyranosyl acetimidate 34 (1.70 g, 3.50 mmol) and molecular sieves (0.500 g, crushed in a 1 :1 mixture of toluene and dichloromethane (30 mL) cooled to -20°C. The mixture was stirred at -20°C for 0.5 hours and slowly brought to 0°C in 1 hour. T.l.c. (1 :1 hexane and ethyl acetate) indicated the completion of the reaction. Some triethylamine was added and after dilution with methylene chloride and filtration, the solvents were worked up in the usual manner. After evaporation, the residue was applied on a column of silica gel by using toluene and elution was then continued with a 2:1 mixture of hexane and ethyl acetate. The appropriate fractions gave the disaccharide 35 (1 -63 g, 74%). [σ] ²⁰ _D +4.1 (c, 0.5, CHCI ₃ ); H-n.m.r. (CDCI ₃ ): 7.40 -8.00 (m,

9H, aromatics), 5.85 (m, 1 H, -CH = ), 5.58 (s, 1 H, benzylidene), 5.07 - 5.25 (m, 4H, incl. =CH ₂ , H-4' and H-1 ), 5.00 (dd, 1H, J _VΛ . 8.0, J _{r 3} .10.0Hz, H-2'), 2.11 , 1.90, 1.85, 1.58 (4s, 12H, 4 OAc), 1.37 and 1.12 (m, 6H, methylenes); ¹³ C-n.m.r. (CDCI ₃ ): 134.6 and 1 17.0 (ethylenics), 102.1 , 101.2, 99.4 (benzylidene, C-1 and

C-1 ').

-49-

Synthesis of 5-Allyloxypentyl 2-deoxy-[2,3,4,6-tetra-O- acetyl- ?-D-galactopyranosy l-( 1 -3)-O-j-2-phthalimido- ?-D- glucopyranoside 5

A solution of the disaccharide 35 (1.63 g, 1.91 mmol) in 90% aqueous acetic acid (10 mL) was heated at 70°C for 1 h at which time t.l.c. (100:5 - chloroform and methanol) indicated the completion of the reaction. Co-evaporation with an excess of toluene left a residue which was chromatographed on silica gel using a 100:2 mixture of chloroform and methanol as eluant giving compound 36_ (1.12 g, 76%). [σ] ²⁰ _D +9.3 (C, 0.55 CHCI ₃ ); H-n.m.r. (CDCI ₃ ): 7.70-

7.95 (m, 4H, aromatics), 5.82 (m, 1H, -CH = ), 5.33 (dd, 1 H, J ₃ . _A . 3.5, - _,5 - 1.0Hz, H-4'), 5.10 - 5.27 (m, 3H, incl. =CH ₂ and H-2'), 5.07 (d, 1H, J _Λ 8.5Hz, H-1 ), 4.84 (dd, 1H, J _{r 3} . 10.0Hz, H-3'), 2.10, 2.08, 1.90 (3s, 9H, 3 OAc), 1.05 -1.47 (m, 9H, incl. 1 OAc). ¹³ C-n.m.r. (CDCI ₃ ): 100.3 and 97.5 C-1 and C-1 '. Ana/.ca\cό C, 56.88; H,

6.17; N, 1.83. Found: C, 55.59; H, 6.20; N, 1.84.

Synthesis of 5-Allyloxypentyl 2-acetamido-2-deoxy- r/?-D-galactopvranosyl-π-3)-O-l-i3-D-glucopvranoside4c

Sodium borohydride (0.690 g, 18 mmol) was added to the disaccharide 36 (0.700 g, 0.91 mmol) in a 5:1 mixture of isopropanol and water (20 mL). The mixture was stirred for 24 hours at room temperature after which t.l.c. (65:35:5, chloroform, methanol and water) showed the disappearance of the starting material. After addition of acetic acid (8.2 mL) the mixture was heated for 3 hours at 100°C. The mixture was co-evaporated with an excess of toluene and the dried residue acetylated in a 3:2 mixture of pyridine and acetic anhydride (5 mL) in the presence of dimethylamino-pyridine for 24 hours at 22°C. After addition of some methanol, the mixture was diluted with dichloromethane worked up as usual leaving a residue which was co-evaporated with some toluene. The final syrup was

-50- chromatographed on silica gel using a 100:2 mixture of chloroform and methanol giving the peracetylated disaccharide (0.500 g, 71 %). ¹ H-n.m.r. (CDCI3): 5.90 (m, 1 H, -CH = ), 5.77 (d, 1 H, _{2 NH} 7.5Hz, NH), 5.37 (dd, 1 H, J ₃ . _A . 3.5, ₄ . ₅ . 1.0Hz, H-4'), 5.15 - 5.23 (m, 2H, =CH ₂ ), 1.95 - 2.18 (7s, 21 H, 6 OAc, 1 NAc), 1.58 (m, 4H) and 1.41

(m, 2H): methylenes.

A 0.5 N solution of sodium methoxide (0.300 mL) was syringed into a solution of the above compound (0.500 g, 0.623 mmol) in dry methanol (20 mL). After stirring overnight at room temperature, the mixture was de-ionized with Dowex 50 (H ⁺ form, dried) and evaporated in vacuo. The residue was dissolved in methanol and coated on Celite (3 g) by evaporation of the solvent. The Celite was then applied on top of a column of latrobeads (30 g) and the product eluted with a 65:25:1 mixture of chloroform, methanol and water giving the disaccharide 4c (0.266 g, 80%); [a] ^∞ _D -0.164 (c.1 , water);

¹ H-n.m.r. (D20): 5.95 (m, 1 H, -CH = ), 5.30 (m, 2H, =CH ₂ ), 4.548 (d, 1 H, J _{1 2} 7.7Hz, H-1 ), 4.426 (d, 1 H, J _v>2 . 7.7Hz, H-1 '), 4.031 (dd, 1 H, J 1.0, 11.5Hz, allylics), 2.023 (s, 3H, NAc), 1.58 (m, 4H) and 1.38 (m, 2H): methylenes; ³ C-n.m.r. (D ₂ 0): 175.24 (carbonyl), 134.70 and 119.05 (ethylenics), 104.33 (C-T), 101.68 (C-1 ), 55.42

(C-2).

Example 9 - Synthesis of 5-Allyloxypentyl 2-acetamido-2- deoxy-/?-D-glucopyranoside 32

The starting material 32 (0.300 g, 0.689 mmol) was deprotected as indicated previously for compound 35. The crude material recovered after peracetylation was chromatographed on silica gel using a 1 :1 mixture of hexane and ethyl acetate which gave the peracetylated derivative (0.180 g, 55%), [σ] ²⁰ _D + 11.5 (c, 0.7, chloroform); ¹ H-n.m.r. (CDCI3): 5.90 (m, 1H, -CH = ), 5.64 (d, 1 H, J _2,m 8.5 Hz, NH), 4.68 (d, 1 H, J _1>2 7.5Hz, H-1 ), 1.95, 2.03 (two),

-51-

2.05 (3s, 12H, 3 OAc, 1 NAc), 1.58 (m, 4H) and 1.41 (m, 2H): methylenes. Ana f.calcd.: C, 55.8; H, 7.5; N, 2.05. Found: C, 55.82; H, 7.53; N, 2.98.

This material was de-O-acetylated in methanol (5 mL) to which a 0.5 N solution of sodium methoxide in methanol (0.100 mL) was added. After overnight at room temperature, the mixture was de- ionized with IR-C50 resin (H ⁺ form, dry) and the solvents evaporated. The residue was run through latrobeads using a 7:1 mixture of chloroform and methanol giving the pure 32 (0.103 g, 80%), [α] ^∞ _D - 0.17 (c.1 , water); 'H-n.m.r. (D ₂ 0): 5.85 (m, 1H, -CH = ), 5.29 (m, 2H,

-CH = ), 4.50 (d, 1 H, J _1>2 8.5Hz, H-1 ), 4.03 (d, 2H, J 6.0Hz, allylics), 2.033 (s, 3H, NAc), 1.58 (m, 4H) and 1.36 (m, 2H): methylenes; ¹³ C- n.m.r. (D ₂ 0): 175.2 (carbonyl), 134.7 and 119.1 (ethylenes), 101 :9 (C-1 ), 61.6 (C-6), 56.4 (C-2), 29.1 , 23.0 and 22.6 (methylenes).

D. TRANSFER OF SIALIC ACIDS AND OTHER SUGARS TO

OLIGOSACCHARIDE STRUCTURES

Example 10 -- Transfer of Sialic Acids and other Sugars to

Oligosaccharide Structures via Glycosyltransferases

This example demonstrates the enzymatic transfer of NeuδAc, analogues thereof (collectively "sialic acids"), and other sugars onto oligosaccharide glycoside structures via glycosyltransferases. FIGs. 17, 18, 19, 20, and 21 illustrate these transfers and provide structures for the prepared compounds identified by an underlined arabic numeral. In Examples 10a-10e, preparative sialylation and fucosylation were performed as follows:

i. Preparative Sialylation

Sialic acids, activated as their CMP-derivatives (as set forth in Examples 1-5 above), were transferred onto synthetic oligosaccharide

-52- structures containing /9Gal(1-3)_?GlcNAc-, 0Gal(1-4)/3GlcNAc-, /3Gal(1- 3)σGalNAc-, and ?Gal(1-4) ?Glc- terminal sequences by using three mammalian sialyl-transferases (Examples 10a-e). The /SGal(1-3/4)/3GlcNAc-σ(2-3)sialyltransferase (EC 2.4.99.5) and the 0Gal(1-4)i9GlcNAc-σ(2-6)sialyltransf erase (EC 2.4.99.1 ) from rat liver were purified to homogeneity by affinity chromatography according to the procedure of Mazid et al. ⁴⁹ , which is incorporated herein by reference on a matrix obtained by covalently linking the hapten /3Gal(1-3)0GlcNAcO(CH ₂ ) ₈ CO ₂ H ⁴³ (Chembiomed Ltd., Edmonton, Canada) to activated Sepharose by methods known in the art. The _?Gal(1-3)σGalNAc-σ(2-3)sialyltransferase (EC 2.4.99.4) was purchased from Genzyme Corporation, Norwalk, CT.

In all preparative sialylation reactions, the acceptor oligosaccharide (5-20 mg) was incubated with the selected CMP-sialic acids (5-20 mg) in the presence of the appropriate sialyltransferase

(10-50 mU) and calf intestinal alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany) as in the procedure of Unverzagt et al. ⁵⁰ for 37°C for 24-48 hours in 50 mM sodium cacodylate pH 6.5, 0.5% Triton CF-54, 1 mg/mL BSA ("sialyl transfer buffer"). For example, the sialyloligosaccharide 7-d-σNeu5Ac(2-6) ?Gal{1-

4) GlcNAc-0-(CH ₂ ) ₈ -COOCH ₃ (13d. 4.4 mg) was synthesized by incubation of 3Gal(1-4)3GlcNAc-0-(CH ₂ ) ₈ -COOCH ₃ (5b, 4.6 mg) and CMP-7-d-Neu5Ac (2 ] _f 15.6 mg) in the presence of /3Gal(1-4)0GlcNAc- σ(2-6)sialyltransferase (51 mU) and calf intestinal alkaline phosphatase (2.4 U) for 28 hours at 37°C in 2.5 mL of the sialyl transfer buffer

(see Examples 1-5). After completion, the reaction mixture was diluted to 10 mL and passed onto three Sep-Pak C ₁₈ cartridges, conditioned as suggested by the manufacturer. Each cartridge was washed with water (4 x 5 mL) and then with methanol (3 x 5 mL). The methanol eluate was evaporated to dryness in vacuo and the residue was dissolved in a 65:35:3 mixture of chloroform, methanol and water (0.5 mL - solvent I) and applied on to a small column of

-53- latrobeads (500 mg) equilibrated in the same solvent. The column was successively eluted with solvent I followed by a 65:35:5 mixture of chloroform, methanol and water (solvent II) and then by a 65:35:8 mixture of chloroform, methanol and water (solvent III). The appropriate fractions (30 drops) containing the product, as identified by t.l.c. on silica gel plates (with a 65:35:8 mixture of chloroform, methanol and 0.2% calcium chloride solution as eluent), were pooled together and concentrated to dryness in vacuo. The residue was run through a small column of AG 50W-X8 (Na ⁺ form), the eluate freeze- dried and the recovered product characterized by H-n.m.r. which, in all cases, indicated good purity.

ii. Preparative Fucosylation

Sialylated analogues of the type I and II oligosaccharides can be further fucosylated by the human milk /_?GIcNAcσ(1-3/4)fucosyltransferase. The enzyme was purified from human milk according to the methodology using affinity chromatography on GDP-hexanolamine Sepharose described by Palcic et al. ⁵¹ The synthesis and purification of the fucosylated oligosaccharides was carried out by a modification of the procedures of Palcic et al. ^sι For example, the fucosylated structure 9-N ₃ - σNeu5Ac(2-3)0Gal(1 -3)-[σ-L-Fuc(1 -4)_-/3G.cNAc-0-(CH ₂ ) ₈ -CH ₂ 0H VJh was synthesized by incubating GDP-fucose (2.5 mg) and 9-N ₃ - σNeu5AC(2-3)y?Gal(1-3)^GIcNAc-0-(CH ₂ ) ₈ -CH ₂ OH 8b (1.7 mg) with affinity purified /?GIcNAcσ(1-3/4)fucosyltransferase (4.6 mU) in 1.3 mL of 100 mM sodium cacodylate (pH 6.5), 10 mM manganese chloride, 1.6 mM ATP, 1.6 mM sodium azide. After 27 hours at 37°C, 2.5 mg of GDP-fucose and 2.3 mU of the fucosyltransferase were added to the reaction mixture, which was kept at 37° C for an additional 21 hours. The product was isolated as described above for the sialylation reaction. T.l.c. of the crude material (as above)

-54- indicated that the fucosylation was almost complete. After purification and chromatography on AG 50W x 8 (Na ⁺ form), ¹ H- n.m.r. of the product 17b (1.0 mg) indicated a very good purity (Table 5). In some cases where the fucosyltransferase was not highly purified, partial hydrolysis of the methyl ester of the linking arm occurred.

Examples 10a- 10e are as follows:

Example 10a: This example refers to the transfer of modified sialic acids such as 1a-g to the 3-OH of a terminal ?Gal of acceptors possessing a /9Gal(1-3) ?GlcNAc- (Lewis ⁰ or Type I) terminal structure such as 4a and 4b by a sialyltransferase such as the /SGald- 3/4)/5GlcNAcσ(2-3)sialyltransferase from rat liver following the experimental procedure reported above. The ^-n.m.r. data of the reaction products, which were purified as indicated previously, are reported (Tables 3 and 4).

Example 10b: This example refers to the transfer of modified sialic acids such as Hi and l£ to the 3-OH of the terminal ?Gal of acceptors possessing a /SGal(1-4)/3GlcNAc- (LacNAc or Type II) terminal structure such as 5i# b, sLfl by a sialyltransferase such as that used in 10a. In some cases, dimethylsulfoxide (5% volume) may be added to solubilize the acceptor. The ¹ H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Tables 6 and 8). The reaction mixture was worked up in the manner described previously.

Example 10c: This example refers to the transfer of modified sialic acids such as le to the 3-OH structure of the terminal /SGal of acceptors possessing a ?Gal(1-4)3Glc- (lactose) terminal structure such as 6a by a sialyltransferase such as that used in Example 10a

-55- following the same experimental procedure. The ¹ H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Table 6).

Example 10d: This example refers to the transfer of modified sialic acids such as Ua - il to the 6-OH of the terminal ?Gal of acceptors possessing a /-?Gal(1-4) ?GlcNAc- (LacNAc or Type II) terminal unit such as 5J_., z3. by a sialyltransferase such as the ?Gal(1- 4)/9GlcNAcσ(2-6)sialyltransferase reported previously. The ⁿ H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Tables 7 and 8).

Example 10e: This example refers to the transfer of modified sialic acids such as lc_ to the 3-OH of the terminal /SGal of acceptors possessing a ?Gal(1-3)σGalNAc- ("T") terminal unit such as 2 _. .. by a sialyltransferase such as the ?Gal(1-3)σGalNAcσ(2-3)sialyltransferase (Genzyme) following the experimental procedure reported previously.

The ¹ H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Table 9).

Table 3: Η-n.m.r. Data of Sialyloligosaccharides Obtained by Transfer of Sialic Acids on the Acceptor 4a by the 0Gal(1-3/4)0GlcNAc α(2-3)Sialytrans erase, and by Chemical Modification

(1) overlapping signals 1.500 - 1.730

(2) H-9a: 3.110 (dd, 2.8, 13.2); H-9b: 2.792 (dd, 8.3, 13.2)

Table 4: 'll-n.m.r. data of Sialyloligosaccharides Obtained by Transfer of Sialic Acids on the Acceptor 4 by the 0Gal (l-3/4)01cNAc α(2-3) Sialytransferase

^« l

Table 5: Η-n.m.r. Data of Sialyl Lewis' (CA19-9, 12) and of Sialyl Lewis* 1ft Structures.

Table 6: Η-n.m.r. Data of Sialyloligosaccharides Obtained by Transfer of Sialic Acids on the Acceptors ga, fib and 6a. by the 0Gal (1-3/4) GlcNAc α(2-3)Sialytransferase.

(1) interchangeable

(2) overlapping with other signals

Table 7: Η-n.m.r. data of Sialyloligosaccharides Obtained by Transfer of Sialic Acids on the Acceptor 5b by the 0Gal(l-4)0GlcNAc α(2-6)Sialytransferase

( 1) overlapping s ignals 1. 500 - 1 .730

Table 8: 'll-n.m.r. data of Sialylogliogsaccharides Obtained by Transfer of Sialic Acids on the Acceptor _g by the 0Gal(1-3/4)0BlcNAc α(2-3)sialytransferase (I) and the 0Gal (l-4)/ΪGlcHAc a(2-6) sialyltransferase (11)

Acceptor:

5?

C

1) overlapping with other signals

Table 9: Η-n.m.r. Data of Sialyloligosaccharides Obtained by Transfer of Sialic λcids on the Acceptors la by the 0Gal(l-3)0GalNAc α(2-3)Sialytransferase.

(1) overlapping with other signals

-63-

PREPARATION OF ANALOGUES OF OLIGOSACCHARIDE GLYCOSIDES BY CHEMICAL MODIFICATION

OF THE COMPLETED OLIGOSACCHARIDE GLYCOSIDE STRUCTURE

Examples 11-13 below describe the synthesis of analogues of oligosaccharide glycosides by the chemical modification of the completed oligosaccharide glycoside structure (prepared by either enzymatic or chemical means). FIGs. 22-24 illustrate the reaction schemes involved in the preparation of these analogues and provide structures for the prepared analogues which are identified by an underlined arabic numeral.

Example 11 - Synthesis of 9-Hydroxynonyl (5-acetamido-3,5- dideoxy- ?-L-arabino-2-heptulopyranosylonicacid)-(2-3)

-O- ?-D-galactopyranosyl-(1-3)-O-[σ-L-fucopyranosyl-(1 -4)-O-]-2-acetamido-2-deoxy- ?-D-glucopyranoside T _rn

The starting trisaccharide 8_a (1.3 mg) was stirred for 24 hours at +4°C in 1.7 mL of a solution 0.05 M in sodium acetate and 0.010 M in sodium periodate. The excess of sodium periodate was then destroyed by addition of some ethylene glycol. Sodium borohydride (20 mg) was then added and the stirring was continued for 24 hours at 4°C. The pH of the reaction mixture was then brought to 6 by addition of acetic acid and the solvents were co-evaporated with methanol. The residue was dissolved in water (1 mL) and run through a Sep-Pak cartridge which was further washed with water followed by methanol. The methanol eluate was evaporated and the residue chromatographed on latrobeads (200 mg) using a 65:35:5 mixture of chloroform, methanol and water as eluant. The appropriate fractions were pooled and evaporated leaving the product Sm (1 mg); ¹ H-n.m.r.: see Table 3 above. Trisaccharide 8m was enzymatically fucosylated following the procedure reported in Example 10 and the product purified in the same

-64- manner. T.l.c. of the recovered crude material indicated that the transformation of 8jm was almost complete. Purification gave 17m (0.5 mg); ¹ H-n.m.r.: see Table 5 above.

Example 12 - Synthesis of 9-Hydroxynonyl (5,9-diacetamido- 3,5,9-tri-deoxy-σ-D-glycβro-D-galacto-2-nonulo -pyranosy Ionic acid)-(2-3)-O- ?-D-galactopyranosyl-{ 1 -3) -O-lσ-L-fucopyranosyl-(1-4)-O-]-2-acetamido-2-deoxy- ? -D-glucopyranoside 17k

A solution of the trisaccharide 8_b_ (1 mg) in water (0.5 mL) was hydrogenated at 22°C at atmospheric pressure in the presence of

Lindlar catalyst (1.0 mg, Aldrich Chemical Company, Milwaukee, WI) for 15 minutes T.l.c. (65:35:8 - chloroform, methanol and 0.2% calcium chloride), indicated a complete transformation. The mixture was filtered through Celite and the solid extensively washed with water. The filtrate was concentrated, filtered through Millipore filter and the eluate freeze dried leaving the trisaccharide 8j; ¹ H-n.m.r.: see Table 3 above.

Acetic anhydride (about 0.2 mg) in methanol (10 μl) was added to a solution of 8J (about 1 mg) in a 1 :1 solution of 0.002 N sodium hydroxide and methanol (0.300 mL) at 0°C. T.l.c. (solvent as above) indicated a complete reaction and the solvents were then evaporated. The residue was dissolved in water (2 mL) and applied to a Sep-Pak cartridge. The cartridge was washed with water and the product eluted with methanol giving the trisaccharide 8k (about 1 mg); ¹ H- n.m.r.: see Table 3 above.

Trisaccharide jBJc was enzymatically fucosylated following the procedure reported in Example 10 and the product purified in the same manner. T.l.c. of the recovered crude material indicated that the transformation of fik was almost complete. Purification gave 17k (about 0.5 mg); ¹ H-n.m.r.: see Table 5 above.

-65-

Example 13 ~ Synthesis of 8-N-methylamidooctyl (5-acet

-amido-3,5-dideoxy-σ-D-glycero-galacto-2-nonulo -pyranosylonic acid N-methyIamide)-(2-3)-O- ?-D -galactopy ranosy l-( 1 -3)-O-[σ-L-f ucopy ranosy l-( 1 -4)-O-] -2-acetamido-2-deoxy-/^D-glucopyranoside 1_8J

Tetrasaccharide 18a (0.003 g) was applied on Dowex 50 x8 (Na ⁺ form) resin and eluted with water. The appropriate fractions, were freeze-dried, followed by further drying over phosphorous pentoxide. Methyl iodide (0.050 mL) was added to the residue dissolved in dimethyl sulfoxide. After stirring in the dark for

20 hours, the solution was evaporated in vacuo, diluted with water (11 mL) and applied to a Sep-Pak C ₁₈ cartridge. After washing with water (10 mL), the product was eluted with methanol. Evaporation of the appropriate fractions left a residue which was chromatographed on latrobeads (0.5 g) using a 65:35:5 mixture of chloroform: methanol: water providing the methyl ester of compound 18a (0.025 g): ¹ H-n.m.r.: 5.099 (d, 1H, J _Λ 3.75Hz, H-1 σFUC), 4.517 (d, 2H, J _Λ,2 7.5Hz, H-1 0Gal and /SGIcNAc), 3.866 and 3.683 (2s, CO^t ), 2.781 (dd, 1 H, _3aχ,3βcl 12.5Hz, J _3eqA 4.5Hz, H-3eq NeuδAc), 2.032 and 2.018 (2s, 6H, 2 NAc), 1.913 (dd, 1H, J _{3 xA} 12.5Hz, H-3ax NeuδAc),

1.160 (d, 3H, _{5 β} 6.5Hz, H-6 σFuc).

This material was heated at 50°C in a 40% solution of N- methylamine (1 mL) for 3.5 hours. After evaporation in vacuo, the residue was dissolved in water (1 mL) and applied on a Sep-Pak cartridge which was further washed with water. After elution of the product with methanol, the solvent was evaporated and the residue freeze-dried from water providing !8I (0.0025 g); ¹ H-n.m.r.: (Table 5).

-66-

F. SYNTHESIS OF MONOFUCOSYLATED OLIGOSACCHARIDES

TERMINATING IN DI-N-ACETYLLACTOSAMINYL

STRUCTURES

Examples 14-19 below are presented for the purpose of illustrating that analogues of blood group determinants also posess immunogenic and tolerogenic properties. Specifically, the analogue of the blood group determinant employed is CD65, which is a Sialyl Lewis X derivative having a /SGal(1-4) ?GlcNAc-OR disaccharide glycoside attached to the reducing sugar of the Sialyl Lewis X. See further U.S. Serial No. 07/771 ,259, filed October 2, 1991 , entitled

"Methods for the Synthesis of Monofucosylated Oligosaccharides Terminating in Di-N-acetyllactosaminyl Structures," which is incorporated herein by reference. As noted above, such a compound is an analogue of blood group determinants because Sialyl Lewis X is a blood group determinant, as defined herein.

In Examples 14 to 19 below, preparative sialylation was conducted as follows:

The rat liver /3Gal(1-3/4) ?GlcNAcσ(2-3)sialyltransferase was purified by affinity chromatography ⁴⁹ on a matrix obtained by covalently linking the hapten /?Gal(1-3)yffGlcNAcO(CH ₂ ) ₈ C0 ₂ H ⁴³

(Chembiomed Ltd., Edmonton, Canada) to activated Sepharose by methods known in the art. The 3Gal(1-4)-3GlcNAc σ(2- 6)sialyltransferase contained in the flow-through of the above affinity- column, was further chromatographed on CDP-hexanolamine Sepharose as reported. ⁵²

The enzymatic sialylations were carried out at 37° C in a plastic tube using a sodium cacodylate buffer (50 mM, pH 6.5) containing Triton CF-54 (0.5%), BSA (1 mg/mL) and calf intestine alkaline phosphatase. ⁵⁰ The final reaction mixtures were diluted with H ₂ 0 and applied onto C ₁₈ Sep-Pak cartridges as reported. ⁵¹ After washing with

H ₂ 0, the products were eluted with CH ₃ OH and the solvents evaporated. The residue was dissolved in a 65:35:5 mixture of CHCI ₃ ,

-67-

CH ₃ OH and H ₂ 0 and applied on a small column of latrobeads (0.200 to 0.500 g). After washing with the same solvent mixture, the products were eluted with a 65:35:8 and/or 65:40:10 mixtures of the same solvents. The appropriate fractions (t.l.c.) were pooled, the solvents evaporated in vacuo, the residue run through a small column of AG 50W X 8 (Na ⁺ form) in H ₂ 0 and the products recovered after freeze drying in vacuo. In all cases, the 8-methoxycarbonyioctyl glycosides were separated from the corresponding 8-carboxyoctyl glycosides. In Examples 14 to 19 below, preparative fucosylation was conducted as follows:

The /3GlcNAcσ(1-3/4)fucosyltranf erase was purified from human milk, as reported. ⁵¹ The enzymatic reactions were carried out at- 37 °C in a plastic tube using a sodium cacodylate buffer (100 mM, pH 6.5), MnCI ₂ (10 mM), ATP (1.6 mM), NaN ₃ (1.6 mM). The reaction products were isolated and purified as indicated above.

GDP-fucose as employed below is preferably prepared by the method described in U.S. Serial No. 07/848,223, filed March 9, 1992 and entitled "Chemical Synthesis of GDP-fucose", which is incorporated herein by reference in its entirety. Specifically, GDP- fucose was synthesized as follow:

A. Preparation of Bis(tetra-n-butylammonium) hvdroαen phosphate

Tetra-n-butylammonium hydroxide (40% aq. w/w, about 150g) was added dropwise to a solution of phosphoric acid (85% aq, w/w,

18g, 0.155 mmol) in water (150 mL) until the pH reached 7. Water was then evaporated in vacuo to give a syrup which was co- evaporated with dry aceto-nitrile (2 x 400 mL) followed by dry toluene (2 x 400 mL). The resulting white solid (75g) was dried in vacuo and stored over phosphorus pentoxide under vacuum until used.

-68- B. Preparation of fl-L-Fucopvranosvl-1 -phosphate

A solution of bis(tetra-n-butylammonium) hydrogen phosphate (58g, 127.8 mmol) in dry acetonitrile (300 mL) was stirred at room temperature under nitrogen in the presence of molecular sieves (4λ, 20g) for about one hour. A solution of tri-O-acetyl fucosyl-1 -bromide

(freshly prepared from 31 g, 93 mmol of L-fucose tetraacetate in the manner of Nunez et al. ⁶⁵ ) in dry toluene (100 mL) was added dropwise in about 0.5 hour to the above solution, cooled at 0°C. After one more hour at 0°C, the mixture was brought to room temperature and stirred for 3 hour. Tic (1 :1 toluene:ethyl acetate) indicated a main spot on the base line and several faster moving smaller spots.

The mixture was filtered over a pad of Celite (which was further washed with acetonitrile) and the solvents evaporated in vacuo to give a red syrup. This material was dissolved in water (400 mL) and extracted with ethyl acetate (250 mL, twice). The aqueous layer was then evaporated in vacuo leaving a yellowish syrup to which a solution of ammonium hydroxide (25% aq., 200 mL) was added. The mixture was stirred at room temperature for 3 hours after which tic (65:35:8 chloroform:meth-anol:water) indicated a baseline spot. The solvent was evaporated in vacuo to give a yellowish syrup which was diluted with water (400 mL). The pH of this solution was checked and brought to 7, if necessary, by addition of a small amount of hydrochloric acid. The solution was slowly absorbed onto a column of ion exchange resin Dowex 2 X 8 [200-400 mesh, 5 x 45 cm, bicarbonate form which had been prepared by sequential washing of the resin with methanol (800 mL), water (1200 mL), ammonium bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water (1000 mL) was then run through the column followed by a solution of ammonium bicarbonate (0.5 M, 2.3 mL/minute, overnight). The eluate was collected in fractions (15 mL) and the product detected by charring after spotting on a tic plate. Fractions 20 to 57 were pooled and

-69- evaporated in vacuo leaving a white solid which was further co- evaporated with water (3 x 300 mL) and freeze drying of the last 50 mL and then drying of the residue with a vacuum pump to give β-L- fucopyransyl-1 -phosphate (9.5g, 40%) as a 12:1 mixture of β and a anomers containing some ammonium acetate identified by a singlet at δ ^~~~ Λ .940 in the ¹ H-n.m.r. spectrum. This product was slowly run through a column of Dowex 5 X 8 resin (100-200 mesh, triethylammonium form) and eluted with water to provide the bis triethylammonium salt of jff-L-fucopyransyl-1 -phosphate as a sticky gum after freeze drying of the eluate. ¹ H-n.m.r. £.4.840 (dd, J, ₂ =

J _{1 P} = 7.5 Hz, H-1 ), 3.82 (q, 1 H, J _5-β 6.5 Hz, H-5), 3.750 (dd, 1 H, J ₃ 3.5, J ₄₅ 1.0 Hz, H-4), 3.679 (dd, 1H, J ₂ . ₃ 10.0 Hz, H-3) _> 3.520 (dd, 1H, H-2), 1.940 (s, acetate), 1.26 (d, H-6). Integral of the signals at 3.20 (q, J 7.4 Hz, NCH ₂ ) and 1.280 and 1.260 (NCH _j Ch and H-6) indicates that the product is the bis-triethyl-ammonium salt which may loose some triethylamine upon extensive drying. ¹³ C-n.m.r. £:98.3 (d, J _{C 1P} 3.4 Hz, C-1 ), 72.8 (d, J _C . _2P 7.5 Hz, C-2), 16.4(C-6); ³¹ P-nmr δ: + 2.6(s).

/S-L-fucopyransyI-1 -phosphate appears to slowly degrade upon prolonged storage (1 + days) in water at 22°C and, accordingly, the material should not be left, handled or stored as an aqueous solution at 22°C or higher temperatures. In the present case, this material was kept at -18°C and dried in vacuo over phosphorus pentoxide prior to being used in the next step.

C. Preparation of Guanosine 5'-( ?-1 -f ucopy- ranosyD-diphosphate

Guanosine 5 /S-1-fucopyranosyl)-diphosphate was prepared from ff-L-fucopyranosyI-1 -phosphate using two different art recognized procedures as set forth below:

-70- PROCEDURE #1

/?-L-fucopy ranosy 1-1 -phosphate and guanosine 5'-mono- phosphomorpholidate (4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt, available from Sigma, St. Louis, Missouri, "GMP-morpholidate") were reacted as described in a recent modification ^{64 86} of Nunez's original procedure ⁶⁵ . Accordingly, tri-n-octylamine (0.800g, available from Aldrich Chemical Company, Milwaukee, Wisconsin) was added to a mixture of S-L-fucopyranosyl-1 -phosphate (triethyl-ammonium salt, 1.00g, about 2.20 mmol) in dry pyridine (10 mL) under nitrogen the solvent removed in vacuo. The process was repeated three times with care to allow only dry air to enter the flask. GMP morpholidate (2.4g, about 3.30 mmol) was dissolved in a 1 :1 mixture of dry dimethylformamide and pyridine (10 mL). The solvents were evaporated in vacuo and the procedure repeated three times as above. The residue was dissolved in the same mixture of solvents (20 mL) and the solution added to the reaction flask accompanied by crushed molecular sieves (2g, 4A). The mixture was stirred at room temperature under nitrogen. Tic (3:5:2 25% aq. ammonium hydroxide, isopropanol and water) showed spots corresponding to the starting GMP-morpholidate (Rf~0.8, U.V.), guanosine 5'-(/S-1- fucopyranosyD-diphosphate (Rf -0.5, U.V. and charring), followed by the tailing spot of the starting fucose- 1 -phosphate (Rf -0.44, charring). Additional U.V. active minor spots were also present. After stirring for 4 days at room temperature, the yellowish mixture was co- evaporated in vacuo with toluene and the yellowish residue further dried overnight at the vacuum pump leaving a thick residue (2.43g). Water (10 mL) was then added into the flask to give a yellow cloudy solution which was added on top of a column of AG 50W-X12 (from Biorad) resin (100-200 mesh, 25 x 1.5 cm, Na ⁺ form). The product eluted with water after the void volume. The fractions which were active, both by U.V. and charring after spotting on a tic plate, were

-71- recovered and the solution freeze-dried overnight in vacuo providing a crude material (1.96g).

This residue was dissolved in water (10 mL overall) and slowly absorbed onto a column of hydrophobic C ₁₈ silica gel (Waters, 2.5 x 30 cm) which had been conditioned by washing with water, methanol and water (250 mL each). Water was then run through the column (0.4 mL/min) and the eluate collected in fractions (0.8 mL) which were checked by tic (3:5:2 25% aq. ammonium hydroxide, isopropanol and water). 3-L-fucopyranosyl-1 -phosphate, (Rf~0.54, charring) was eluted in fractions 29 to 45. A product showing a strongly U.V. active spot (Rf~0.51 ) eluted mainly in fractions 46 to 65. Other minor U.V. active spots of higher or lower Rf were observed. Fractions 59 to 86, which contained guanosine 5'-(/?-1-fucopyrariosyl)- diphosphate (Rf -0.62), also showed a narrow U.V. active spot (Rf~0.57). Fractions 59 to 86 were pooled and freeze-dried overnight providing 0.353g of material enriched in guanosine 5'-( -1- fucopyranosyU-diphosphate. ¹ H-n.m.r. indicated that this material was contaminated by a small amount of impurities giving signals at δ = 4.12 and δ = 5.05. Fractions 29 to 45 and 47 to 57 were separately pooled and freeze-dried providing recovered ?-L-fuco-pyranosyl-1 -phosphate (0.264g and 0.223g, respectively, in which the second fraction contains some impurities). Occasionally, pooling of appropriate fractions provided some amount of guanosine 5'-( ?-1-fucopyranosyl)- diphosphate in good purity OH-n.m.r.). Generally, all the material enriched in guanosine 5'-(/3-1-fuco-pyranosyl)-diphosphate was dissolved in a minimum amount of water and run on the same column which had been regenerated by washing with large amounts of methanol followed by water. The fractions containing the purified guanosine 5'-(yt?-1-fucopyranosyl)-diphosphate (tic) were pooled and freezed dried in vacuo leaving a white fluffy material (187 mg, 16%). ^-n.m.r. was identical to the previously reported data.

-72- PROCEDURE #2

/ff-L-fucopy ranosy 1-1 -phosphate and guanosine 5'- monophosphomorpholidate (4-morpholine-N,N'-di-cyclohexyl- carboxamidine salt - "GMP-morpholidate") were reacted in dry pyridine as indicated in the original procedure ⁶⁵ . Accordingly, the β- - f ucopy ranosy I- 1 -phosphate (triethyl-ammonium salt, 0.528g, about 1.18 mmol) was dissolved in dry pyridine (20 mL) and the solvent removed in vacuo. The process was repeated three times with care to allow only dry air to enter the flask. GMP-morpholidate (1.2g, 1.65 mmol) and pyridine (20 mL) were added into the reaction flask, the solvent evaporated in vacuo and the process repeated three times as above. Pyridine (20 mL) was added to the final residue and the heterogeneous mixture was stirred for 3 to 4 days at room temperature under nitrogen. An insoluble mass was formed which had to be occasionally broken down by sonication.

The reaction was followed by tic and worked up as indicated in the first procedure to provide the GDP-fucose (120 mg, 16%).

Example 14 - Preparation of 8-Methoxycarbonyloctyl (5-

Acetamido-3,5-dideoxy-σ-D-glycero-D-galacto-2 -nonulopy ranosy Ionic acid )-{2-6)-O- ?-D-galactopy ranosy I

-( 1 -4)-0-2-acetamido-2-deoxy-glucopyranosyl-( 1 -3.-0-/? -D-galactopyranosyl-( 1 -4)-O-2-acetamido-2-deoxy -glucopγranoside (62a)

0Gal(1 -4)3GlcNAc(1-3)/3Gal(1-4)/SGIcNAc-OR (compound 6 La, 6.5 mg), CMP-Neu5Ac (17 mg), 0Gal(1-4)/5GlcNAc σ(2-

6)sialyltransferase (50 mU) and alkaline phosphatase (15 U) were incubated for 48 hours in 2.5 mL of the above buffer. Isolation and purification provided 62a (3.0 mg).

-73-

Example 15 — Preparation of 8-Methoxycarbonyloctyl (5-

Acetamido-3,5-dideoxy-σ-D-glycero-D-galacto-2- nonulopyranosylonic acid)-(2-6)-O- ?-D-galacto-pyranosyI -(1-4)-O-2-acetamido-2-deoxy-glucopyranosyl-(1-3)-O- ? -D-galactopyranosy l-( 1 -4)-O-[σ-L-f ucopyranosy l-{ 1 -3)-O -]2-acetamido-2-deoxy-glucopyranosidei6_3_al and the 8 -carboxyoctyl glycoside (63b)

Compound 62a (3.0 mg), GDP-fucose (5 mg), SGIcNAc σ(1- 3/4)fucosyltransferase (10 mU) were incubated for 68 hours in the buffer (1.3 mL). Isolation and purification provided 63a (1.2 mg) and

63b (0.5 mg).

Example 16 - Preparation of 8-MethoxycarbonyloctyI ?-D-galactopyranosyl-(1-4)-O-2-acetamido-2-deoxy- ?-D -glucopyranosyM 1 -3)-O- ?-D-galactopyranosyl-( 1 -4)-O -[σ-L-fucopyranosyl-(1-3)-O-]2-acetamido-2-deoxy- ?-D

-glucopyranoside (64a) and the 8-carboxyoctyl glycoside (64b)

Compounds 63a and 63b (1.7 mg) were incubated with Clostridium Perfringens neuraminidase immobilized on agarose (Sigma Chemical Company, St. Louis, MO, 1 U) in a buffer of sodium cacodylate (50 mM, pH 5.2, 2 mL) at 37°C. After 24 hours the mixture was diluted with water (10 mL) and filtered through Amicon PM-10 membrane. The flow-through and washings were lyophilized and the residue dissolved in water (3 mL) and applied to two C ₁₈ cartridges. Each cartridge was washed with water (10 mL) prior to elution with methanol (20 mL). After evaporation of the solvent, the residue was chromatographed on latrobeads (210 mg) as indicated above giving (64a. 0.8 mg) and 64b (0.7 mg). 64b was dissolved in dry methanol and treated with diazomethane until t.l.c. indicated the complete conversion into 64a.

-74-

Example 17 - Preparation of 8-Methoxycarbonyloctyl

(5-Acetamido-3,5-dideoxy-σ-D-glycero-D-galacto-2 -nonulopy ranosy Ionic a cid)-( 2-3 )-O-/3-D -galactopyranosyM1-4)-O-2-acetaπ.ido-2-deoxy- ?-D -glucopyranosyl-( 1 -3)-O- ?-D-galactopyranosy l-( 1 -4)-O-[cr -L-fucopyranosyl-(1-3)-O-]2-acetamido-2-deoxy-/?-D -glucopyranoside (65a) and the 8-carboxyoctyl glycoside (65b)

Compound 64a (1.5 mg), CMP-Neu5Ac (8 mg), 5Gal(1- 3/4)/?GlcNAc σ(2-3)sialyltransferase (17 mU), alkaline phosphatase (5

U), were incubated for 40 hours in the sialylation buffer (1.5 mL). Isolation and purification provided 65a (0.7 mg) and 65b (0.55 mg).

Example 18 -- Preparation of 8-Methoxycarbonyloctyl (5-

Acetamido-3,5-dideoxy-σ-D-glycero-D-galacto-2 -nonulopyranosylonic acid)-(2-3)-O- ?-D

-galactopyranosyl-(1-4)-O-2-acetamido-2-deoxy- ?-D

-glucopyranosyl-(1-3)-0- ?-D-galactopyranosyl-(1-4)-0-2

-acetamido-2-deoxy-glucopyranosidej6_6J

Compound 61a (5 mg), CMP-Neu5Ac (15 mg), 0Gal(1- 3/4) _/ 9GlcNAc σ(2-3)sialyltransferase (46 mU), and alkaline phosphatase (15 U) were incubated in the sialylation buffer (2,5 mL) for 48 hours. Isolation and purification of the product gave 66a (2.5 mg).

Example 19 -- Preparation of 8-Methoxycarbonyloctyl (5- Acetamido-3,5-dideoxy-σ-D-glycero-D-galacto-2

-nonulopyranosylonic acid)-(2-3)-O- ?-D -galactopy ranosy l-(1 -4)-O-[σ-L-f ucopyranosy l-(1 -3)-O -]2-acetamido-2-deoxy-glucopy ranosy l-(1-3)-O-/5-D -galactopyranosyl-( 1 -4)-O-[σ-L-f ucopyranosy l-( 1 -3)-O -]2-acetamido-2-deoxy-glucopyranoside (67a)

Compound 6J>a (2.5 mg), GDP-fucose (8 mg) and the /SGIcNAc σ(1→3/4)fucosyltransferase (19 mU) were incubated in the enzymatic

-75- buffer (2.0 mL) for 48 h. Isolation and purification of the product give 67b (1.7 mα).

¹ H-NMR data for the compounds prepared in Examples 1 to 6 above are set forth in the following Table 10:

H ( n r. Stracb-nl P-romeicri

Sugar Unit Hy -oft* <l> 42a a-> 64a <5« (7b

4516 4.516 4J22 4.525 (»Λ) P5) (80) i) H CO ex

4.457 4.455* 4.441 4.436 CD

PJ) PΛ) PJJ) P.71

4.157 4.158 4.050 4.098 (Ϊ O) 04) ( J» P O)

4.698 4.721 4.726 4.698 (12) P.7) (8-2)

4.478 4.462* 4.457 4.480 (10) J) (80) pt)

5.096 5.096 P.7> PJ> 4.818 4.814

(65) (65) 1.153 1.152

NAc (•) 2.033 2032 1024 1032 2.028. 2.016 1021 1027 (dm) (Λr ^β e) 1012

CII.CO, (I) 2.388 2.386 1386 2.387 2.314 P3> P5) P5| OS) P5)

Oθ,H CH, CH, Cllj CH, 1685 3.618 3.616 3.684

Inttxdi-fifeaMe.

-77-

G. IMMUNOSUPPRESSIVE PROPERTIES OF BINDING- INHIBITORY OLIGOSACCHARIDE GLYCOSIDES

Examples 20-34 illustrate the immunosuppressive properties of oligosaccharide glycosides related to blood group determinants. In these examples, the oligosaccharide glycosides employed are illustrated in FIGs. 12 and 13 which employ Roman numerals to identify the structure of these compounds.

Example 20 ~ Inhibition of DTH Inflammatory Response

DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola ²² . Briefly, groups of Balb/c mice were immunized with 10 μg of the L111 S- Layer protein, a bacterial surface protein ²³ from Clostridium thermohydrosυ/furicum L111-69 which has been shown to induce a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 10 μg of L-111 S-Layer protein.

The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.

To assess the effect of oligosaccharide glycosides related to blood group determinants HI- VI I depicted in FIG. 12 on the inflammatory DTH response, groups of mice received 100 μg of

Compounds lll-VII. injected into the tail vein, 5 hours after challenge. Control groups were left untreated or received 100 μ of phosphate- buffered saline (PBS). The results of this experiment are shown in FIG. 1. Mice injected with oligosaccharide glycoside HI had only about 40% of the footpad swelling of control mice. Mice injected with oligosaccharide glycosides IV- VI I (structures related to Sialyl-Lewis X, Compound III of FIG. 12) had between 55 and 70% of the footpad swelling of control mice. As seen in FIG. 2, mice injected with

-78- saccharides VIH and IX and disaccharide X depicted in FIG. 13 had essentially the extent of footpad swelling observed in control mice.

Example 21 - Dose-Dependency of the Suppression of the DTH Inflammatory Response

Six groups of mice were subjected to primary immunization and challenge with L111 -S-Layer protein as described under Example 20, above. Five hours after challenge, groups were injected intravenously with 100 μl solutions containing 10, 25, 50, 75, or 100 μg of oligosaccharide glycoside related to blood group determinant Jϋ depicted in FIG. 12, or with PBS. The DTH responses for each dose group were measured 24 hours after challenge and are shown in FIG. 2. While the groups receiving PBS or 10 μg of oligosaccharide glycoside jϋ showed essentially the same extent of footpad swelling as PBS-treated controls, the groups receiving 25, 50, 75 or 100 μg of oligosaccharide glycoside Hi displayed reduced footpad swelling (78,

69, 75, and 56% of the PBS controls, respectively).

Example 22 - Lack of Suppression of the Antibody Response to the L111 -S-Layer Protein

Secondary antibody responses to the L111 -S-Layer protein were measured two weeks after primary immunization (one week after challenge) in the sera from groups of mice immunized, challenged, and treated intravenously with oligosaccharide glycosides lll-VII as described in Example 20.

Antibody titers were determined using a solid phase enzyme immunoassay (EIA) as described by Ziola et al ²⁴ . Briefly, 2 μg of

L1 1 1 -S-Layer protein was added per well of a Maxisorb EIA plate (Flow Laboratories, Inc., McLean, VA). Following incubation at room temperature overnight, unabsorbed antigen was removed by inverting the wells. Each well then received 200 μ\ of various dilutions of

-79- mouse serum prepared in phosphate-buffered saline containing 2% (w/v) bovine serum albumin and 2% (v/v) Tween 20. After 1 hour at room temperature, the solutions were removed by inverting the wells, and the wells washed four times with distilled, de-ionized water at room temperature. Horse-radish peroxidase-conjugated, goat anti- mouse immuno-globulin antibodies were then added to each well (200 μl of a 1 :2000 dilution prepared in the phosphate-buffered saline/albumin/Tween 20 solution). After 1 hour at room temperature, the wells were again inverted and washed, and each well received 200 μ\ of enzyme substrate solution (3 mg per ml o-phenylene-diamine and 0.02% (v/v) hydrogen peroxide, freshly dissolved in 0.1 M sodium citrate/ phosphate buffer, pH 5.5). After the enzyme reaction had proceeded for 30 minutes in the dark at room temperature, 50 μ\ of 2N hydrochloric acid was added to each well and the OD ₄₉₀ values were measured.

FIG. 3 graphically illustrates the titers determined with six dilution series of sera from the L111 -immunized and challenged mice which were treated with oligosaccharide glycosides lll-VII depicted in FIG. 12 and examined for footpad swelling as described in Example 20 above. The dilution curves shown in FIG. 3 indicate that the development of antibodies against the L111 S-Layer protein has not been inhibited or otherwise affected by the treatments with oligosaccharide glycosides lll-VII.

Example 23 - Time of Administration of Compound III Relative to Challenge with Antigen

Groups of Balb/c mice, immunized and challenged with L111 S- Layer protein as described in Example 20, were injected with a solution of 100 μg of oligosaccharide glycoside related to blood group determinant M, depicted in FIG. 12, in PBS (100 μl) at different time points relative to the time of antigen challenge. One group received

-80- oligosaccharide glycoside ill one hour prior to the antigen challenge; another, immediately after challenge, the third group one hour after challenge, and the fourth group 5 hours after challenge. A control group was included which received PBS (100 μ ) immediately after challenge.

The results of this experiment are shown in FIG. 4. The DTH responses were not suppressed in those mice which had received oligosaccharide glycoside Jϋ one hour before or immediately after the antigen challenge. Those groups which had received oligosaccharide glycoside Mi one or five hours after challenge showed only 68 or 59% of the footpad swelling seen in the PBS treated controls.

Examples 20-23 above establish that treatment with an effective amount of an oligosaccharide glycoside related to blood group determinants after challenge by an antigen suppresses an immune response to the antigen (i.e., a DTH response), in as much as the level of inflammation measured 24 hours after challenge, is reduced by at least 20% in animals treated with an oligosaccharide glycoside related to blood group determinant as opposed to the level of inflammation exhibited by the control animals.

Example 24 - Persistence of Suppression of the DTH Inflammatory

Response at 6, 8, or 10 Weeks After Challenge

i. The identical groups of mice treated with oligosaccharide glycosides related to blood group determinants III- VII in Example 20 were re-challenged with L111 S-Layer protein 8 weeks after primary immunization. Untreated controls responded with the usual degree of footpad swelling whereas all other groups showed reduced footpad swelling, as follows: Oligosaccharide glycoside Jϋ, 59%; oligosaccharide glycoside IV, 69%; oligosaccharide glycoside V, 78%; oligosaccharide glycoside VI, 78%; oligosaccharide glycosdfide Yii, 69%. The anti-inflammatory effect of oligosaccharide glycosides

-81- iϋ/ V, Vi, or Vπ, given 5 hours after the first challenge (one week after primary immunization), had somewhat weakened eight weeks after primary immunization; however, the effect of oligo-saccharide glycoside JV (the only derivative not containing a sialyl group) was equally as strong at the time of re-challenge as at the time of first challenge.

In addition to providing suppression of cell-mediated immune responses, the above data demonstrate that treatment with an oligosaccharide glycoside as per this invention also imparts tolerance to additional challenges from the same antigen.

ii. The identical groups of mice treated in Example 21 with Sialyl-Lewis X, Compound III, (10 g, 25 μg, 50 μg, 75 μg, 100 /7g) or with the a or ?-Sialyl glycosides of 8-methoxycarbonyloctanol (monosaccharides VIII. IX of FIG. 13; 100 μg), or with 10 μg of the 8-methoxycarbonyloctyl glycoside of T-disaccharide (disaccharide X. of

FIG. 13), were rechallenged six weeks after primary immunization. Footpad swelling similar to that of PBS-treated controls was observed with those mice that had been treated with saccharides V|H and IX, and disaccharide X, 5 hours after the first challenge. Mice originally treated with 10-100 μg of Ul showed footpad swelling that ranged from 90 to 65% of that displayed 24 hours after the first challenge.

iii. The identical groups of mice which had been treated in Example 22 with 100 μg of oligosaccharide glycoside Hi at 1 hour before first challenge, or 5 hours after first challenge, were re- challenged with antigen 10 weeks after primary immunization. Within experimental error, footpad swelling of those mice treated before or shortly after challenge was the same as that of PBS-treated mice, whereas those mice originally treated 1 hour or 5 hours after challenge showed only about 66% of the values observed for PBS-treated controls.

-82-

The results of this example are set forth in FIGs. 5-7 which demonstrate that oligosaccharide glycosides related to blood group determinants impart tolerance to challenges with the same antigen for at least 10 weeks after treatment.

Example 25 - Effect Cyclophosphamide Treatment has on the Suppression Induced by 8-methoxy-carbonyloctyl glycoside of Compound III

It has been demonstrated in the literature that suppressor cells can be removed by treatment of mice with cyclophosphamide (CP). An experiment was carried out to determine if CP could modulate the suppression of cell-mediated inflammatory responses induced by the 8-methoxy-carbonyloctyl glycoside of Compound III.

Specifically, this example employs immunized mice which have been previously suppressed and tolerized to DTH inflammatory responses by treatment with the 8-methoxycarbonyloctyl glycoside of

Compound III in a manner similar to that described above. Fourteen days after immunization, the mice were injected with 200 mg/kg of CP and then 17 days after immunization, the mice were challenged with 20 μg of L111 S-Layer protein. 24 hours after the challenge, the extent of the DTH response was ascertained by measuring (mm ^*1 ) the increase in footpad swelling.

The results of this experiment are set forth in FIG. 8 which illustrates that injection with CP prior to challenge with the L1 11 S- Layer protein restores the DTH inflammatory response in mice that have previously under-gone immunosuppressive treatment with the 8- methoxycarbonyloctyl glycoside of Compound III. These results suggest that tolerance induced by the 8-methoxycarbonyloctyl glycoside of Compound III FIG. 3 is mediated by CP sensitive suppressor T-cells.

-83-

Example 26 - Effect the Antigen Driving the DTH Inflammatory

Response has on the Suppressive Effect Induced by the 8-Methoxycarbonyloctyl Glycoside of Compound III

This example assesses the effect that the antigen driving the DTH inflammatory response has on the suppressive effect induced by

Compound III. Mice were immunized as outlined in Example 20 with S-Layer L11 1 , herpes simplex virus 1 (HSV 1 ) and cationized bovine serum albumin (Super Carrier™, Pierce, Rockford, IL). As shown in FIG. 9, the nature of the antigen used to induce the inflammatory response does not appear to affect the ability of Compound III to regulate this response.

Example 27 - Effect Synthetic Compound III has on ELAM-1 Dependent Cell Adhesion to Activated Vascular Endothelium

This example examines whether the synthetic blood group determinant related to oligosaccharide glycoside III could inhibit ELAM- 1 dependent cell adhesion to activated vascular endothelium. Specifically, an in vitro cell binding assay was preformed, as described by Lowe et al ¹⁸ . Briefly, human umbilical vein endothelial cells (HUVECs purchased from Cell Systems, Seattle, WA) were stimulated with TNFσ (10 ng ml) to express ELAM-1 . Human tumor cell lines, U937 or HL60, which have been shown to bind to HUVECs, in an ELAM-1 dependent manner were used to measure the effect that Compound III has on the ELAM-1 dependent binding to the HUVEC. FIG. 10 sets forth the results of this example illustrates that

Compound III inhibits ELAM-1 dependent binding to the HUVECs.

The data in Examples 20-26 above establish the effectiveness of oligosaccharide glycosides related to blood group determinants in treating immune responses to an antigen and in inducing tolerance to the antigen in a mammal (mice). In view of the fact that the immune

-84- system of mice is a good model for the human immune system, such oligosaccharide glycosides will also be effective in treating human immune responses. This is borne out by the fact that Example 27 establishes that an oligosaccharide glycoside related to blood group determinant inhibits ELAM-1 dependent binding to the HUVEC.

Example 28 -- Effect of Timing of Administration of SleX Relative to Immunization or Challenge with Antigen

Four groups of Balb/c female mice were subjected to primary immunization and challenge with HSV antigen as described in Example

20 with the following modifications:

1 ) The first group was immunized with 20 //g/mouse inactivated Herpes Simplex Virus Type I (HSV) and then challenged seven days later with 20 μg HSV.

2) The second group was immunized with 20 //g/mouse HSV and then challenged seven days later with 20 /g/mouse HSV and then 100 /g/mouse of SleX (Sialyl Lewis X, compound III) was injected intraveneousiy five hours after challenge.

3) The third group was immunized with 20 / g/mouse HSV and 100 / g/mouse SleX in 100 μ\ PBS intramusclularly at the same site. Seven days later, the mice were footpad challenged with 20 //g/mouse of HSV alone.

4) The fourth group of mice was immunized with 100 μ\ PBS and then seven days later challenged with 20 //g/mouse HSV. This provides a measure of the background level

-85- of footpad swelling resulting from the physical injury caused to the footpad during the antigen challenge.

The extent of the DTH inflammatory response was measured 24 hours after challenge by measuring footpad swelling with a Mitutoyo

Engineering micrometer.

FIG. 27 shows the degree of footpad swelling observed. Percentage reduction was calculated by the following equation:

100 - 100 X Swelling of "Treated Mice" - background swelling Swelling of "Untreated Mice" - background swelling

"Treated mice" are those mice which receive compound in addition to the antigen. "Untreated Mice" are those mice which do not receive compound. Background swelling is that level of swelling observed in mice immunized with PBS alone without antigen or compound and challenged with antigen.

Mice injected with SleX at the same time as and site of immunization with HSV, showed a 88% reduction in footpad swelling compared to that of mice immunized with HSV and challenged with HSV. Mice injected with SleX 5 hours after the footpad challenge with HSV showed an approximately 86.7% reduction in footpad swelling compared to that of mice immunized with HSV and challenged with HSV.

The results of this example support previous examples which show that oligosaccharide glycoside III can suppress an immune response to an antigen if given to mice 5 hours after challenge by the antigen. This example also shows that oligosaccharide glycoside III given to mice at the time of immunization can inhibit sensitization of the immune system to the antigen. Without being limited to any theory, it is contemplated that SleX (compound III) interferes with the

-86- abiiity of T helper cells to recognize antigen-presenting cells and inhibits the immune system from becoming educated about the antigen.

Example 29 -- Effect of Sialyl LeX on the Antibody Response to HSV

Four groups of mice were treated as described in Example 28.

Secondary antibody responses to the HSV antigen were measured 2 weeks after primary immunization (1 week after challenge) in the sera from groups of mice described in Example 28.

Antibody titres were determined as described in Example 22 except HSV antigen was used in place of L1 11 S-Layer protein.

FIG. 28 graphically illustrates the titres determined with six dilution series of sera from the groups of immunized mice as described in Example 28. The results of the first two groups correlated with the results obtained in Example 22 for the L111 S-Layer Protein. Treatment of mice with SleX five hours after challenge did not affect the antibody response. However, mice treated with SleX at the time of immunization showed significant reduction in the antibody response to the HSV antigen. Without being limited to any theory, it is contemplated that SleX (compound III) interferes with the T helper cells that are involved in the antibody response and inhibits the immune system from becoming educated about the antigen.

Example 30 ~ Effect Cyclophosphamide Treatment has on the Induction of SleX Immunosuppression

As discussed in Example 25, suppressor cells can be removed by treatment of mice with cyclophosphamide (CP). Specifically, this example employs immunized mice which have been previously suppressed and tolerized to DTH inflammatory responses by treatment with L1 1 1 S-Layer antigen. One group of Balb/c mice were

-87- immunized with 20 //g/mouse of the L111 S-Layer protein. Seven days later, this group of mice was footpad-challenged with 20 μg of L111 S-Layer protein. The second group of mice were immunized with 20 μg of the L111 protein and 100 μg of SleX at the same site and seven days later were footpad challenged with 20 μg of L111.

The third group of mice were injected with 200 mg/kg of CP interperitoneally two days before immunization. This group was then immunized and challenged as described for group two. The fourth group of mice were immunized with 20 //g/mouse of L11 1 and 100 g/mouse of the T-disaccharide at the same site and footpad challenged as described for group one. Group five was immunized 100 μ\ of PBS and then footpad challenged as described for group one.

The results are presented in FIG. 29. These results confirm the results discussed in Example 28 that treatment of mice with SleX at the time of immunization can suppress the immune response to an antigen. Furthermore, this Example shows that the suppression of the immune response by treatment with SleX at the time of immunization can be eliminated by cyclophosphamide treatment before immunization suggesting the involvement of cyclophosphamide sensitive suppressor

T-cells.

Example 31 - Effect of Sites of Administration of Compound After Footpad Challenge on Inhibition of DTH Inflammatory Response induced by OVA

Groups of Balb/c female mice, age 8 -12 weeks, weight about

20-25 mg, were immunized with 100 //g of OVA (Albumin, Chicken Egg, Sigma, St. Louis, M0) and 20 μg of DDA (Di ethydioctacylammonium Bromide, Eastman Kodak, Rochester, NY) in 100 μ\ of PBS (Phosphate Buffered Saline) intra-muscularly into the hind leg muscle of the mouse.

-88-

Seven days after immunization, each group of mice was footpad-challenged with 20 μg of OVA in 20 μ\ of PBS. The resulting inflammatory footpad swelling was measured with a Mitutoya Engineering micrometer 24 hours after challenge. To assess the effect of methods of administration of SleX on the suppression of the inflammatory DTH response, compound III (SleX) was administered by different routes. Certain groups of mice received, five hours after footpad-challenge, either SleX 100 //g/mouse in 200 μ\ of PBS intravenously or Sle 100 //g/mouse in 20 μ\ of PBS intranasally at which procedure the mice were under light anathesia.

The method of administering compound intranasally is described in Smith et al. ^e3 which is incorporated by reference. Briefly, mice are anethesitized with Metofane (Pitman-Moore Ltd., Mississauga, Ontario, Canada) and a 50 μ\ drop of compound is placed on the nares of the mouse and is inhaled. Control groups were left untreated or received 200 μ\ PBS intravenously or 50//I of PBS intranasally. The results of this experiment are shown in FIG. 30. This shows that administration of the SleX compound nasally five hours after challenge results in suppression of the immune response.

Example 32 - Time Dependency of Administration After Footpad

Challenge of the Suppression of the OVA Induced DTH . Inflammatory Response

A group of Balb/c female mice, age 8-12 weeks, weight about 20-25 g, were immunized with 100 //g of OVA (Albumin Chicken Egg, Sigma) and 20 μg of DDA in 100 μ\ of PBS, intramuscularly into the hind leg muscle of the mouse. Seven days after immunization, the mice were footpad challenged with 20 μg of OVA in 20 μ\ of PBS. At 5, 7 or 10 hours after footpad challenge, the mice were either given intravenously 100 //g/mouse of SleX in 200 μ\ of PBS or 200 μ\ PBS only or given intranasally 100 //g/mouse of SleX in 50 μ\ of PBS or 50 μ\ PBS only at which procedure the mice were under light anesthesia.

-89-

The footpad swelling was measured 24 hours later with a Mitutoyo Engineering micrometer.

FIG. 31 shows the results of this experiment. SleX given at 7 hours (both intranasally and intravenously) after the OVA challenge showed 70 - 74% reduction in footpad swelling relative to positive control mice as calculated using the formula set forth in Example 28. SleX given intravenously or intranasally at 5 hours after footpad challenge showed 63% and 54% reduction in swelling respectively. SleX given intervenously or intranasally at 10 hours after footpad challenge showed 58% and 32% reduction in swelling respectively.

Example 33 - Effect of Different Sites of Administration of the Oligosaccharide Glycoside at Immunization on Suppression

Groups of Balb/c female mice were immunized with OVA 100 / g/mouse and DDA 20 //g/mouse in 100 μ\ of PBS intramuscularly into the hind leg muscle, while 100 //g/mouse of SleX was simultaneously administered intramuscularly, intranasally or intravenously. Seven day later the mice were footpad challenged with 20 / g/mouse of OVA in 20 μ\ of PBS. The footpad swelling was measured 24 hours later with a Mitutoyo Engineering micrometer.

FIG. 32 shows that administering SleX to the mice at the time of immunization produces the same level of suppression of the DTH inflammatory response regardless of the method by which the SleX is administered. This suggests that the SleX compound can be administered in the treatment of patients by other methods in addition to that of intravenous injection.

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Example 34 - Effect of Oligosaccharide Glycosides on LPS Caused Lung Injury

LPS (lipopolysaccharide) caused lung injury is measured by weighing the lungs of sacrificed mice 24 hours after mice are given LPS intranasally. Briefly, groups of 8-10 week old Balb/c mice were sensitized with 5 μg /mouse of LPS in 50 μ\ of PBS intranasally under light anesthesia. Five hours later, 10 / g/mouse of SLeX, C19-9, T- disaccharide (#27) in 200 μ\ of PBS are given to the mouse intravenously. After 24 hours, the mice are sacrificed and the lungs removed and weighed.

The results in FIG. 33 indicate that SleA (compound VII) and SleX (compound III) provide at least about a 30% reduction in the DTH inflammatory response in lungs. This suggests that the SleA and SleX compounds may be useful in reducing inflamation in lungs exposed to antigen, for example Acute Respiratory Distress Syndrome.

Example 35 — Effect of Different Amounts of SleX and SleA on Lymphoproliferative Response

A group of Balb/c female mice were immunized with 20 / g/mouse of MUMPS (inactivated Mumps virus) and DDA by injection into the hind leg muscle of the mouse. Seven days later, the mice were sacrificed and draining lymph nodes and speen were collected and lymphocytes isolated. The lymphocytes were cultured with varying concentrations of Mumps virus and varying concentrations of compound in RPMI 1640 medium (Gibco, Burlington, Ontario, Canada) supplemented with 5% Hybermix (Sigma, St. Louis, MO) at a density of 2x10 ⁵ cells per well for three days at 37° C in 5% C0 ₂ . After three days the cells were pulsed for 6 hours with 1.0 μ curies of ³ H - thymidine per well (Amersham Canada Ltd., Oakville, Ontario, Canada). The cells were harvested using a PhD cell harvester and cpm determined in a Beckman 3000 scintillation counter. It has been

-91- shown previously that there is a direct correlation between the ability of lymphocytes to respond to an antigen and the amount of ³ H- thymidine incorporation ⁵⁵ .

Lymphoproliferative responses are used in this example to measure T-helper cell responses in vitro. FIG. 34 demonstrates that

SleX and SleA (C19-9) are able to inhibit an antigen specific lymphoproliferative response since the uptake of ³ H-thymidine is reduced relative to the untreated lymphocytes from mice exposed to the antigen. Without being limited to any theory, it is thought that SleX and SleA may interfere with macrophage T-cell interactions required in order to obtain a proliferative response in vitro.

Examples 36 and 37 illustrate the immunosuppressive properties of hexasaccharide glycoside 65a.

Example 36 - Inhibition of DTH Inflammatory Response

DTH inflammatory responses were measured using the mouse footpad swelling assay as described in Example 20. Briefly, groups of Balb/c mice were immunized with 10 μg of the L111 S-Layer protein. Seven days later, each group of mice was footpad-challenged with 10 μg of L-111 S-Layer protein. The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.

To assess the effect of hexasaccharide glycoside 65a synthesized in Example 17, on the inflammatory DTH response, groups of mice received 100 μg of this compound, injected into the tail vein, 5 hours after challenge. Control groups received 100 μL of phosphate-buffered saline (PBS). The results of this experiment are shown in Table 11 below. In this table, smaller increases in footpad swelling, as compared to control, evidence the fact that the tested compound possesses immunosuppressive properties in that it reduces the degree of footpad swelling in response to an antigen.

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COMPOSITION TESTED INCREASE IN FOOTPAD SWELLING (mm-1 .

Control 3.3

Hexasaccharide Glycoside 65a 1.5

The above results indicate that mice injected with hexasaccharide glycoside 65a had less than 50% of the footpad swelling as compared to the control mice.

Example 37 -- Persistence of Suppression of the DTH Inflammatory Response at 11 Weeks After Challenge

i. The identical groups of mice treated with hexasaccharide glycoside 65a in Example 7 were re-challenged with L1 11 S-Layer protein 1 1 weeks after primary immunization. Mice treated with the PBS control responded with the usual degree of footpad swelling whereas mice treated with hexasaccharide glycoside 65a showed a reduction in footpad swelling of about 40%, i.e., the mice treated with hexasaccharide glycoside 65a exhibited only about 60% of the footpad swelling exhibited in mice treated with PBS.

This anti-inflammatory effect pf hexasaccharide glycosides 65a. given 5 hours after the first challenge (one week after primary immunization), had somewhat weakened eleven weeks after primary immunization but nevertheless provided for a significant reduction in inflammation as compared to PBS treated controls.

In addition to providing suppression of cell-mediated immune responses, the above data demonstrate that treatment with a hexasaccharide glycoside as per this invention also imparts tolerance to additional challenges from the same antigen.

The data in Examples 36 and 37 above establish the effectiveness of the hexasaccharide glycosides described herein in treating immune responses to an antigen and in inducing tolerance to

-93- the antigen in a mammal (mice). In view of the fact that the immune system of mice is a good model for the human immune system, such hexasaccharide glycosides will also be effective in treating human immune responses. By following the procedures set forth in the above examples, other oligosaccharide glycosides related to blood group determinants could be used to suppress a cell-mediated immune response to an antigen by mere substitution for the oligosaccharide glycosides described in these examples.