Note: Descriptions are shown in the official language in which they were submitted.
WO 92/22301 PCS /CA9~/00244
-1-
IMMUNOSUPPRESSIVE AND TOLEROt;ENIC
OLIGOSACCHARIDE DERIVATIVES
BACKGROUND OF THE INVENTION
1. Field Qf the Invention.
The present invent;on is directed to methods of employing
oligosaccharide glycosides in the treatment of cell-mediated immune
responses, as well as to pharmaceutical compositions containing such
oli30saccharide glycosides. Specifically, the methods of the present
invention are directed to methods of employing oligosaccharide
glycosides related to blood ~roup determinants in modulating (eg.
suppressingl ~ell-mediated immune responses, including cell-mediated
inflammatory responses.
2. Refarenc~s.
The following references are cited in this application as
superscript numbers at the relevsnt portion of the application:
1. Brandley et al., J. Leukocyte Biol.,
40:97-1 1 1 (1 986).
2. Jacobson, Developmental
Neuro~iology, New York, Plenum Press
p. 5-25, (1978)
3. Trinkaus, Cells into Organs, Englewood
Cliffs, N.J., Prentic~ Hall, p.44-68,
(~ 984~.
4. Frazier et al., Annu. Rev. Biochem.,
48:491 ~1979).
WO 92/22301 PCI'/CA92/00244
2 ~ 2-
5. Glaser, Mediatorof Developmental
Processes (Subtency, S. and Wessels,
N.K., Eds.) New York, Academic Press,
p. 79 (1980).
6. Paulson, /n ~The Receptors~, Vol. Il
~Comm., P.M., Ed.), New York,
Academic Press, p. 131 119851.
7. Sharon, Lectin-Like Bacterial
Adherence to Animal Cells. In
~Attachment of Microor~anisms to the
Gut Mucosa~ (Boeheker, E.D., Ed.),
Boca Raton, Florida, CRC Press, p. 129
(1984).
8. Wassarman, Fertilkation. In ~Cell
Interactions and Development: -
Molecular Mechanisms~ IYamada,
K.M., Ed.), New York, John Wiley and
Sons, p. 1 (1983).
9. Schwartz et al., Immunol. Rev.,
~Q:153 (1978).
10. Coutinho et al., /mmunol. Rev.,
21 1 (1 984).
11. Hoffmann et al., Eds., Membr~nes jn
: Growth and Development, New York,
Alan R. Liss, p. 429-442, 11982).
12. Galeotti et al., Eds., Membranes in
Tumor Growth, Amsterdam, Elsevier,
p. 77-81, ~1982).
13. Nicolson et al., /nvas. Metas., 5:144
(1980.
14. Aplln et al., ~iochim. Biophys. Acta
694:375 ~1 982).
15. Barondes, Developrnentally Rc~ulated
Lectins. In ~Cell Interactions and
DeveloprMnt: Molecular Mechanisrns~
IYamada, D.M., Ed.) N~wYork, ~lohn
W;ley and Sons, p. 185 11983).
-3-
16. Monsigny, M., Ed., Biol. Cell, 51
(Special Issue), 113, 1984.
17 . Springe et al . , N~ture, 349:196- 197
(1991).
18. Lowe et al., Cel/, 6~:475-485 (1990).
19. Phillips et al., Scjence, in press (1990).
20. Walz et al., Science, 250:1132 et seq.
(1 990).
21. Larsen et al., Cell, 63:467-474 l1990).
22. Smith et al., Immunology, 58:245
( 1 986).
23. Sleytr et al., Arch. Microbiol., 146:19
(1 9867.
24. Ziola et al., J. Neuroimmunol., 7:315-
330 (1985).
25 . Reuter et al ., ~Iycoconjugate J.,
5:133-135 ~1988~.
26. Campanero et al., J. Cel/ Biol.,
1 10:2157-2165 (1990).
27. Okamoto et al., retrahedron, 46, No.
17, pp. 5835-5837 l1990).
28. Ratcliffe et al., U.S. Serial No.
07/1 27,905 ~now U .S . Patent No.
5,079,305, issued January 7, 1992).
29. Abbas et al., Proc. Japanese-German
Symp. Berlin, pp. 20-21 (1988).
30. Paulsen, Agnew. Chem. /nt. Ed. Eng.,
21:155-173 11982).
31. Schmidt, Agnew. Chem. Int. Ed. Eng.,
25:21 2-235 (1 986).
32. F~edi et al., Gtycoconj. J., 4:97-108
ll 987).
WO 92/22301 2 ~ 1 d ~ 9 ~ PCr/CA92/00244
33. ~ Kameyama et al., Carbohydr. Res.,
209:Cl-C~ (1991).
34. Toone et al., Tetrahedron, No. 17,
45:5365-5422 ~1989).
35. Beyer et al., Advances in Enzymology,
pp. 23-175, ~lohn Wiley & Sons, New
York (1982) .
36. Brossmer et al., Biochem. Biophys.
Research Commun., 96:1282-1289
(1980).
37. Zbiral et al., Monatsh. Chem.,
119:127-141 (1988).
38. Hase~awa et al., J. Carbohydr. Chem.,
g~:135-144 (1989).
39. Christian et al., Carbohydr. Res.,
194:49-61 (1989).
40. Higa et al., J. Biol. Chem., 260:8838-
8849 (1985).
41. Kean et al., ~1. Blol. Chem., 241:5643-
5650 (1960).
42. Gross et al., Biochemistry, ~:7386-
7392 (1989).
43. Lemieux et al., U.S. Patent No.
4,137,401 (1976).
44. Lemieuxet al., U.S. Patent No.
4,195,174 (1978).
45. Paulsen et al., Carbohydr. Res.,
21 -45 (1984).
46. Sab~san et al., C~n. J. Chem.,
~:644-652 (1984).
47. Alais et al., C~rbohydr. Res., 207
31 (1990).
48. Lemieux et al., U.S. Patent No. 4,767,845
( 1 g87) .
49. Mazid et al., U.S. Patent Application Serial No.
07/336,932 (now U.S. Patent No. 5,059,535,
issued October 22, 1991) .
50. Unverzagt et al., J. Amer. Chem. Soc., 112:9308-
9309 (1990).
5t. Palcic et al., Carbohydr. Res., 190:1-11 (1989).
52. Wsinstein et al., J. Biol. C~)em. 257:13835-
13844 (1982).
53. Smith et al., Infection and Immvnity, 31: 129
(1980) .
54. Ratcliff et al., U.S. Serial No. 07/278,106, filed
November 30, 1988.
55. Ziola et al., J. of Immunol. Me~hods, 97:159,
(1987) .
56. Ekborg et al., Carbohydr. Res., 1 10:55-67 (1982).
57. Dahmen et al., Carbohydr. Res., 118:292-301
(1983).
58. Rana et al., Carbohydr. Res., 91:1~9-157 (1981).
59. Amvam-~ollo et al., (,`arbohydr. Res., 150:199-
212 (1986).
60. Paulsen et al ., Carbohydr. Res., 104: 195-219
(1982).
61. Chernyaket al., Carbohydr. Res., 12~:269-282
(1984) .
62. Fernandez-Santana et àl., J. Carbohydr. Chem.,
8:531-537 (1989).
63. Lee et al., Carbohydr. Res., 37:193 et seq.
(1974).
64. Schmidt, et aJ., Liebigs Ann. Chem.,121-124
(1991)
~i
WO 92~22301 PCI/CA92/00244
2 i 1 i) ~19 ~
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65. Nunez, et al., Can. ~1. Chem., 59:2086-2095
11981)
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 involvin~ mammalian cells, such as ~rowth,
locomotion, morpholo~ical development, and differentiation are partially
controlled b~ extracellular si~nals actin~ upon the cells' surfaces' 3.
While some external stimuli reach the cell via extracellular fluids, other
signals are received from nei~hboring or approachin~ cell surfaces and
exert their effects throu~h direct cell-cell contact~ 5.
Evidence sug~ests that specific cell-surface receptors can
"sense" a molecular si~nal of an apposin~ cell via specific bindin~, and
biochemical mechanisms exist to translate that bindin~ into a cellular
response. For example, complex cell-surface interactions are believed
to help direct processes such as bindin~ of patho~ens to tar~et
tissues~ ', sperm-e~ bindin~, interactions amon~ cells in the immune
system9 10, and reco~nition of cells durin~ embryonic development'1.
In addition, defects in cell-cell reco~nition are thou~ht to undQrlie the
uncontrolled cQII ~rowth and motility which characterkQ neoplastic
transformation and rMtastasisl2'~.
WO 92/22301 PCI`/CA92/00244
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Other evidence su~gests that cell-reco~nition processes are
mediated by carbohydrate chains or ~Iycan portions of
glycoconju~ates4 ~ . For example, the bindin~ of the surface
glycoconju~ates of one cell to the complementary çarbohydrate-binding
5 proteins ~lectins) on another cell can result in the initiation of a specific
interaction.
One important group of carbohydrate-bindin~ proteins are LEC-
CAM proteins ~Lectin + EGF + complementary Re~ulatory Domains-
Cell Adhesion Molecules). These or functionally similar proteins or
10 lectins may play a critical role in immune responses ~including
inflammatory responses) through mediation of cell-cell contact and
throu~h extravasation of leucocytes'7 2t. Specific carbohydrate ligands
have recently been identified as part of the putative receptor structures
for LEC-CAM proteins17 21. The structures identified include:
Sialyl-Lewis X-Q: aNeu5Ac~2-3)pGal(1-4)~GlcNAc1-Q
I (1-3)
oFuc
Lewis X-Q: ~Gal( 1 -4)~GlcNAc 1 -Q
I ~1-3)
oFuc
wherein Q represents another suitably bonded su~ar or sugars.
However, in vivo, such carbohydrates are ~enerally part of naturally-
occurring ~Iycoconju~ates which are not readily synthesked and
cannot be readily isolated in therapeutic amounts.
Although certain oligosaccharide glycosides have been
heretofore disclosed, their use in suppressin~ ccll-mediated immune
responses (including cell-mediated inflammatory responses) has not
been taught.
SUMMARY OF THE INVENT~ON
It has now been found that cell-surface glycoconju~ates which
contain the above-mentioned Sialyl Lewis X or Lewis X glycan chains
WO 92t22301 PCI/CA92/00244
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are not the only li~ands that can functionally interact with lectins so as
to suppress cell-mediated immune responses in a mammal.
Specifically, the present invention ~s directe~ to the discovery that low
molecular weight (MW ~enerally less than about 2000 daltons)
5 oli~osaccharide ~Iycosides related to blood ~roup determinants also
interact with LEC-CAM proteins and/or other lectins with sufficient
strength to suppress, in vivo, mammalian cell-mediated immune
responses includin~ cell-mediated inflammatory responses. The
present invention is also directed to the discovery that when such
10 oligosaccharide glycosides related to blood ~roup determinants are
administered to a mammal in response to an antigen challenge, such
administration induces tolerance to additional challen~es from the same
antigen.
Accordingly, in one of its method aspects, the present invention
15 is directed to a method of suppressin~ a cell-mediated immune
response in a mammal which method comprises administering to said
mammal an amount of an oli~osaccharide glycoside related to blood
group determinants effective in suppressin~ said immune response.
In a preferred embodiment, the immune response suppressed by
20 this method is an inflammatory response. In a further preferred
embodiment, the oligosaccharide ~Iycoside related to blood group
determinants employed in this method is further characterized as a
binding-inhibitory oli~osaccharide gîycoside (as defined below).
In another of its method aspects, the present im~ention is ~irected to a
25 method of treating a cell-mediated immune response to an antigen in a
mammal which method comprises administerin~ to said mammal from
about 0.5 m~/kg to about 50 mg/kg of an oli~osaccharide ~Iycoside
related to blood group determinants. Yet in another of its method
aspects, the present invention is directed to a method of r~ducing
30 sensitization of 8 mammal to an anti~en which comprises
administration of an effective amount to the mammal of a blood group
WO 92/22301 PCI`/CA92/OOt44
4 '~ ~
g
determinant oligosaccharide glycoside simultaneously with exposure to
the anti~en.
In one of its composition aspects, the present invention is
directed to a pharmaceutical composition suitable for parenteral
5 administration to a mammal which comprises a pharmaceutically inert
carrier and an amount of oli~osaccharide ~Iycosid~ related to a blood
~roup determinant effective in treatin~ a cell-mediated immune
response in said mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the increase in footpad swellin~ of immunized
mice arising from a DTH inflammatory response measured 24 hours
after challenge with 10 ~ug of th~ L111 S-Layer protein anti~en
wherein some of the mice have been treated at 5 hours aner the
challen~e with 100 /ug of different oligosaccharide ~Iycosides related to
15 blood group determinants.
FIG. 2 illustrates the increase in footp~d swelling of immunked
mice arisin~ from a DTH inflammatory response measured 24 hours
after challen~e with 20 ~u~ of the L111 S-Layer protein anti~en wherein
some of the mice have been treated at 5 hours after challen~e with
20 various doses of different mono- and oli~osaccharide ~Iycosides
including oli~osaccharide 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-
25 phenylenediamine O.D. at 490 nm) two weeks after primaryimmunization and one week after challenge with the L111 S-Layer
protein anti~en and the eff0ct different oli~osaccharide ~Iycosides
related to blood group determinants had on these responses when the
mice were treated with these oligosaccharide glycosides 5 hours after
30 challenge.
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FIG. 4 illustrates the effect of an oli~osaccharide ~Iycoside
related to blood ~roup determinants, i.e., the 8-methoxycarbonvloctyl
glycoside of Sialyl Lewis X, Compound lll, on the inflammatorv DTH
response in immunized mice challen~ed with the L111 S Layer protein
5 anti~en wherein the mice were treated at various times before or after
challen~e with 100 /u59 of the 8-methoxycarbonyloctyl ~Iycoside of
Sialyl Lewis X, Compound lll.
FIG. 5 illustrates the lon~ term (8 weeks) immunosuppression
~enerated in immunized mice after an injection with 5 m~/k~ of
10 oligosaccharide ~Iycosides related to blood ~roup determinants 5 hours
after challen5ge with 20 ~ of the L111 S-Layer protein anti5gen on day
7.
FIG. 6 illustrates the lon~ term (6 weeks) immunosuppression
generated in immunized mice after an injection with varyin5g amounts
15 of mono- and oli~osaccharide ~Iycosides includin~ oli~osaccharide
; ~ ~Iycosides related to blood ~roup determinants 5 hours after challen5ge
with 20 ~u~ of the L111 S-Laver protein anti~en on day 7.
FIG. 7 illustrates the lon~ term ~10 weeks) immunosuppression
generated in immunized mice after an injection with 5 m~/k~ of the 8-
20 methoxycarbonyloctyl ~Iycoside of Sialyl Lewis X, Compound lll, atvarious times before. at and after challen~e with 20 ~u59 of the L111 S-
Layer protein anti~en on day 7.
FIG. 8 illustrates the cyclophosphamide induced restoration of a
DTH inflammatory response in immunized mice previously suppressed
25 by treatment with the 8-methoxvcarbonyloctyl 5glycoside of Sial~l
Lewis X, Compound lll.
FIG. 9 illustrates that the nature of the anti~n used to induce
the inflammatory response does not affect the ability of the 8-
methoxycarbonyloctyl ~Iycoside of Sialyl Lewis X, Compoùnd lll, to
30 re5gulate the DTH response.
WO 92~22301 PCI'/CA92/00244
"~ C3
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FIG. 10 illustrates that the 8-methoxycarbonyloctyl ~Iycoside of
Sialvl Lewis X, Compound 111, can inhibit bindin~ of U937 or HL60 to
TNFa activated human umbilical vein endothelial cells (HUVECs).
FIG. 11 illustrates the ~core~ structure of preferred
oli~osaccharide ~Iycosides related to blood ~roup determinants for use
in this invention.
FIG. 12 illustrates the structures of specific oli~osaccharide
glycosides related to blood ~roup determinants for use in this invention
wherein R is -(CH2~"C~OIOCH3.
FIG. 13 illustrates the structures of a couple of monosaccharide
glycosides and one disaccharide ~Iycoside used in some of the
examples wherein R is-(CH2)"C~O)OCH3.
FIG. 14 illustrates a ~èneral synthetic scheme used for the
synthesis of derivatives of Neu5Ac.
FIG. 15 illustrates the structures of mono- and oli~osaccharide
glycosides ;~g to 7a.
FIG. 16 illustrates a ~eneral reaction scheme for the synthesis of
oligosaccharide ~Iycoside 4c as specified in Example 8 and for the
synthesis of monosaccharide ~Iycoside ~ as specified in Example 9.
FIG~ 17 illustrates the enzymatic transfer of Neu5Ac, and of
analo~ues thereof (collectively ~sialic acids") by the
,BGal( 1 ~3/4)pGlcNAc~(2~3')sialyltransferase to a pGal( 1--3)~GlcNAc-
terminal structure. FIG. 17 also illustrates the enzymatic transfer of L-
fucose onto the sialylated oli~osaccharide ~Iycosides.
FIG. 18 illustrates the enzymatic transfer of Neu5Ac, analogues
thereof ~collectively ~sialic acids~) by the
~Gal(1--314)~G1cNAc~(2--3')sialyltransferase to a pGal(1~4)pG1cNAc-
terminal structure. FIG. 18 also Illustrates the enzymatic transfer of L-
fucose onto the sialylated oli~osaccharide ~Iycosides.
FIG. 19 illustrates the enzymatic transfer of Neu5Ac, analo~ues
thereof (collectively ~sialic acids~) by the
WO 92/22301 PCl`/CA92/00244
5 -12-
~Gal(1--4),BGlcNAco~2--6')sialyltransferase to a,BGal(1--4)BGlCNA
terminal structure.
FIG. 20 illustrates the enzymatic transfer of Neu5Ac, analo~ues
thereof (collectively ~sialic ac;ds~) by the
~Gal(1--3/4),BGlcNAco~2--3')sialyltransferase to a pGal~1--4)~G1c-
~lactose) terminal structure.
FIG. 21 ;llustrates the enzymatic transfer of Neu5Ac, analogues
thereof lcollectively ~sialic acids~) by the
,BGal(1--3)aGalNAca(2--3')sialyltransferase to a pGal~1--3)aGalNAc-
(nTn) terminal structure.
FIGS. 22 and 23 illustrate the reaction schemes involved in the
synthesis of analo~ues of Sialyl Lewis A by chemical modification of a
sialylated hapten.
FIG. 24 illustrates the reaction schemes involved in the synthesis
of analogues of Sialyl Lewis X by chemical
modification of a sialylated hapten.
FIG. 25 illustrates the synthetic pathway leading to Sialyl
dimeric LewisX and internally monofucosylated derivatives thereof. In
FIG. 25, the nomenclature for compoound Çl~ is
~Gal~1-4),B~;lcNAc(1-3)pGal~1-4)~GlcNAc-OR sometimes called di-N-
acetyllactosaminyl tetrasaccharide. Similarly, the hexasaccharide
moiety present in compounds ~ and 65b in FIG. 1 is sometimes
called VIM-2 epitope or CD-65 and ~ and 67b are called sialyl
dimeric LewisX.
FIG. 26 illustrates the synthetic pathway leading to the
extbrnally 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 anti~en, where some ot the mice were
treated with Sialyl Lewis X, Compound lll, at the time of immunkation
- 1 3-
and some of the mice were treated with Sialyl Lewis X, Compound 111,
5 hours after the challenge.
flG. 28 illustrates the secondary antibody responses ~i.e., as
determined bv the amount of antibody measured bv quantification of o-
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 lll,
had on the these responses.
FIG. 29 illustrates the cyclophosphamide (CP) induced
restoration of a DTH inflammatory response in immunized mice
previously suppressed by treatment with the 8-methoxycarbonyloct~l
glycoside of Sialyl Lewis X, Compound lll.
FIG. 30 illustrates the effect of the 8-methoxycarbonyloctyl
glycoside of Sialyl Lewis X, Compound lll on the inflammatory DTH
response in immunized mice challenged with the OVA anti~en wherein
the mice were treated with Compound lll fiv~ hours after challen~e by
a variety of different methods (IV-intravenousl-/; IN- intranasally)
FIG. 31 illustrates the effect of the 8-methoxy~:arbonyloctyl
glycoside of Sialyl Lewis X, Compound lll on the inflammatory DTH
response in immunized mice challenged with the OVA anti~en wherein
the mice were treated with various doses of Compound lll five hours,
seven hours or ten hours after challenge by a variety of different
rnethods (IV-intravenously; IN-intranasally).
FIG. 32 illustrates the effect of the 8-methoxycarbonyloctyl
glycoside of Sialyl Lewis X, Compound lll on the inflammatory DTH
response in immunized mice challenged with the OVA anti~en wherein
the mice were treated with Compound lll by a variety of different
methods at the time of Immunization of the mice ~IV-intravenously; IN-
intranasally; IM-lntramuscularly)
FIG. 33 Illustrates the effect of various oli~osaccaride and
monosaccharide ~Iycosides have on the inflammatory response in the
... .
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2110l~9S
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s of mice wherein the mic~ 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 Iymphoproliferative response.
5DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed at the discovery that certain
low molecular weight oli~osaccharide ~Iycosides ~MW less than about
2000 daltons) are effective in suppressin~ cell-mediated immune
responses in a mammal, includin~ cell-mediated and immune directed
10 inflammatory responses to an antigen in a mammal (e.~., a DTH
response). Additionally, treatment with these oligosaccharide
~Iycosides also provides for induction of tolerance to the antigen in the
so-treated mammal.
A. Definitions -
15As used herein, the followin~ terms have the definitions ~iven
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
20 responses to an anti~en such as delayed-type hypersensitivity (DTH)
responses as w811 as cell-mediated inflammatory responses arisin~ from
myocardial infarction, virus-induced pneumonia, shock and sequelae
(e.~., multiple organ failure), adult respiratory distress syndrome, and
the like. Preferably, the cell-mediated immune response is a leucocyte-
25 mediated response.
The term ~blood group substances~ refer to specificglycoconjugate antigens on red blood cells which serve as the basis for
WO 92/22301 2 11 0 ~ j PCJ'/CA92/00244
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assigning blood into various classes accordin~ to immunological
compatibility.
The term ~blood ~roup determinant~ refers to any naturally
occurrin~ oli~osaccharide se~ment of the nonreducin~-terminal, 3-9
5 ~Iycosyl residues that constitute the ~Iycan chains of blood ~roup
substances.
The term ~oli~osaccharide ~I~/cosides relatin~ to a blood ~roup
determinant" refer to an oli~osaccharide ~lvcoside (a) havin~ an
oligosaccharide ~roup of from 3 to 9 saccharide units, (b) which is
10 terminated with an a~lycon ~roup on the non-reducin~ su~ar, and (c)
wherein the oligosaccharide ~roup is a blood group determinant las
defined above) or an analo~ue thereof.
- Analo~ues of blood ~roup determinants include those wherein; one or more of the monosaccharide units of the blood ~roup
15 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 o~ an -OH functionalit~, the removal of saccharide unit(s), the
introduction of an amine functionality, the introduction of a halo
20 functionality, the introduction of one or more saccharide unit(s), and
the like~
Such oli~osaccharide ~Iycosides related to blood ~roup
determinants can be represented by the formula:
OLIGOSACCHARIDE-Y-R
25 wherein oli~osaccharide represents a carbohydrate structure of from 3
to about 9 saccharide units which oll~osaccharide contains a blood
-- group determinant or analo~ues ther~of; Y is selected from the ~roup
consistin~ of O, S, ~NH and a bond: and R represents an a~lycon
;~ moiety of at least 1 carbon atom.
- 1 6-
Oligosaccharide glycosides related to blood group determinants
are different from glycoconju~ates, 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.
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 moietV of the form
-(CH2-CR~G),~- wherein n is an inte~er equal to 1 to 5; R~ is selected
from the ~roup consisting of h~/drogen, methyl, or ethyl; and G is
selected from the group consisting of hydro~en, oxy~en, 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 sebcted from the ~roup consistin~ of hydro~en, methyl, phenyl
and nitrophenol and, when G is not oxy~en, sulphur or nitro~en and A
is not a bond, then Z' is also selected from the ~roup consisting of
OH,-SH,-NH2,-NHRs,-N(R6)2,-C(O)OH,-C(O)OR5,-C(O)NH-NH2,
-C~O)NH2, -C(O)NHR5, and -C~O)N~Us)2, wherein each Rs is
independently alkyl of from 1 to 4 carbon atoms.
Numerous aglycons are known in the art. For example, a linking
arm comprising a para-nitrophenyl group (i.e., -YR = -OC"H~pNO2) has
been disclosed by Ekborg et al.5~ 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
trifluoroacetamido group is remov~d thereby unmasking th~ amino
group.
A linkin~ arm containin~ sulfur is disclosed by Dahmen et al.57.
SpecifTcally, the linkin~ arm is derlved from a 2-bromoethyl ~roup
which, in a substitution reaction with thio-nucleophibs, has been
shown to lead to linkln~ arms posses~in~ a variety of termTnal
functional groups such as -OCH2CH2SCH2SC02CH~ and
-OCH2CH2SC~H~.-PNH2
~ ~. ~
Rana et al.58 discloses a 6-trifluoroacetamido)-hexvl linking arm
PO-(CH2)~,-NHCOCF3) 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-
5 methoxycarbonyl-3,6,dioxaheptvl linking arm59
(-OCH2-CH2)20CH2CO2CH3; the 2-~4methoxyca-bonylbutan-
carboxamido)ethyl~ (-OCH2CH2NHC(O)~CH2)~CO2CH3); the allyl linking
arm~l (OCH2CH=CH2) which, by radical co-pol~merization with an
appropriate monomer,leads to co-polymers; other allyl linking arms~2
l-O(CH2CH20)2CH2CH=CH21. Additionally, allyl linking arms can be
derivatized in the presence of 2-aminoethanethiol~3 to provide for a
linking arm -OCH2CH2CH2SCH2CH2NH2.
Additionally, as shown by Ratcliffe et al.5~, R group can be an
additional saccharide or an oli~osaccharide containin~ a linkin~ arm at
the reducing sugar terminus.
Preferably, the a~lycon moiety is a hydrophobic group and most
preferably, the a~lycon moiety is a hydrophobic ~roup selected from
the group consisting of -(CH2)8COOCH3, -(CH2)50CH2CH=CH2 and
-(CH2)"CH20H. In particular, the use of a hydrophobic ~roup and most
especiallv, a -(CH2),lCOOCH3, or ~CH2)sOCH2CH=CH2 or
-~CH2)8CH20H group may provide for some enhancement of the
acceptor properties for transfer sialic acid by this sialyltransferase.
Without being limited to any theory, we bclieve that
oligosaccharide glycosides related to blood group determinants
effectively interfere with an immune response, particularly an
inflammatory immune response to an antig~n, so as to provide a
suitable means for treatin~ such an immune response.
Preferably, the oli~osaccharide ~Iycoside related to blood ~roup
determinants is further characteri2ed as a bindin~-inhibitory
oli~osaccharide ~Iycoside, i.e., an oU~osaccharide ~Iycoside related to
blood ~roup determinants which oli~osaccharide ~Iycoside binds
sufficiently to a cell surface lectin so as to inhibit leucocytes from
`~ ~
WO 92/2230l PCI'/CA92/00244
9 S -18-
- bindin~ to another cell. Such bindin~-inhibitory oli~osaccharideglycosides are particularly effective in suppressin~ leucocyte-mediated
immune responses.
Again, without bein~ limited to any theory, we believe that the
5 bindin~ of leucocytes to a cell surface (presumably throu~h the LEC-
CAM proteins and/or other proteins on the cell surface~ is an inte~ral
part of a leucocyte-mediated immune response, includin~ a leucocyte-
mediated and immune directed inflammatory response. Accordin~ly,
binding-inhibitory oli~osaccharide glycosides are preferred in the
10 treatment of leucocyte-mediated immune responses.
The ability of an oligosaccharide glycoside related to blood
~roup determinants to bind sufficiently to a cell surface lectin so as to
inhibit leucocytes from bindin~ to that cell, either heterotypically or
homotypically, can readily be determined via simple in vitro
15 experiments. For example, in vftro Iymphoproliferative experiments
which measure the ability of Iymphocytes to respond to an anti~en can
be employed to ascertain the ability of an oli~osaccharide ~Iycoside to
inhibit or enhance this response. An inte~ral part of this response is
the ability of Iymphocytes to reco~nize and bind anti~en-presentin~
20 cells, which reco~nition event tri~ers the proliferation of the
Iymphocytes. Such in vitro experiments are known in the art as
disclosed by Ziola et al55. In addition, other in vi~ro experiments which
measure the ability of leucocytes to bind to the surface of cells can be
employed to ascertain the ability of a candidate oli~osaccharide
25 ~Iycoside 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 disclos~d by Campanero
et al.20 which describes procedures for determinin~ the homotypic
bindin~ of leucocytes to othor leucocytes; and Lowe et al.~ which
30 dcscribes procedures for dcterminin~ the heterotypic bindin~ of
leucocytes to the surfaces of other cells.
. .
~i ,
,
WO 92/22301 PCI`/CA92/00244
~ 1 LO'l'~
- 1 9-
The in vitro experiments are ~enerally performed bv measurin~
cell bindin~ in the presence or absence ~control) of the candidate
oligosaccharide glycoside, e.g., at a concentration of about 10 ~g/mL
of a candidate oli~osaccharide ~Iycoside. The extent of leucocyte
5 bindin~ to the cell surface is measured in both cases and candidate
oli~osaccharide ~Iycosides which reduce leucocyte bindin~ by at least
about 20 percent (and preferably by at least about 30 percent and
even more preferably at least about 50 percent) compared to control
are deemed bindin~-inhibitory oli~osaccharide ~Iycosides.
10 Saccharide units (i.e., su~ars) useful in the oli~osaccharide ~Iycosides
related to blood ~roup determinants employed in this invention include
by way of example, all natural and synthetic derivatives of ~lucose,
galactose, N-acetyl-~lucosamine, N-acetyl-~alactosamine, fucose, sialic
acid ~as defined below), 3-deoxy-D,L-octulosonic acid and the like. In
15 addition to bein~ in their pyranose form, all saccharide units in the
oli~osaccharide ~Iycosides related to blood ~roup determinants are in
their D form except for fucose which is in its L form.
` Preferred oli~osaccharide ~Iycosides related to blood ~roup
determinants are those which contain from 3 to 8 saccharide units
20 especially those containin~ the ~Gai(1~ GlcNAc or
~Gal(1--3)pGlcNAc groups. Particularly preferred oli~osaccharide
glycosides related to blood ~roup determinants for use in this invention
include those set forth in FlGs. 11 and 12~ In FIG. 11, R is an aglycon,
preferably as defined above, each R, is independently selected from the
25 group consisting of hydro~en, a saccharid~ and a compatible
saccharide; and each R~ is independently selected from the ~roup
consistin~ of hydro~en, a saccharide and a compatible saccharide and
at least one of R~ and R2 Is a saccharide.
Especially preterred oli~osaccharide ~Iycosides related to blood
30 ~roup determinants include those havin~ the
pGal( 1--4)pGlcNAc-Y-R
aFuc
WO 92/22301 PCI`~CA92/00244
2 ~ cj
-20-
and the
~Gal( 1~3)~GlcNAc-Y-R
I (1--4)
aFuc
S groups where Y and R are as defined above. Even more preferred
oligosaccharide glycosides related to blood ~roup determinants are
those which contain a N-acetylneuraminic acid residue or an analo~ue
thereof particularly as the non-reducin~ sugar terminus ot the
oli~osaccharide .
The term ~compatible saccharide" refers to those substituent
saccharide groups which when substituted on an existin~
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
15 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 ~roup determinant so as to
render the resultin~ structure incapable of inhibitin~ an immune
response, then such a saccharide substituent or combination of
20 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 IN-acetylated)
5-amino-3,5-dideoxy-D-glycero-D-~alacto-nonulosonicacid (~Neu5Ac~)
25 and to derivatives thereof. The nomenclature employed herein in
describin~ derivatives of sialic acid is as set forth by Reuter et al.25
B. PreearatiQn of OliQosa~h~ide Gly~
Oligosaccharide glycosides, includin~ oli~osaccharide ~Iycosides
related to blood group determinants, are readily prepared either by
WO 92/22301 PCl/CA92/00244
~ i i ~ 3 ~
-21 -
complete chemical synthesis or by chemical/enzymatic synthesis
wherein ~Iycosyltransferases are employed to effect the sequential
addition of one or more sugar units onto a saccharide or an
oli~osaccharide. Chemical synthesis is a convenient method for
5 preparin~ either the complete oli~osaccharide ~Iycoside; for chemically
modifyin~ a saccharide unit which can then be chemically or
enzymatically coupled to an oli~osaccharide ~Iycoside; or for
chemically preparin~ an oli~osaccharide ~Iycoside to which can be
enzymatically coupled one or more saccharide units.
Chemical modifications of saccharide units are well known in the
art. For example, chemically modified Neu5Ac derivatives includin~ 9-
azido-Neu5Ac, 9-amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac,
9-bromo-NeuSAc, 8-deoxy-Neu5Ac, 8-epi-NeuSAc, 7-deoxy-Neu5Ac,
7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-0-methyl-Neu5Ac, 4-N-acetyl-
Neu5Ac, 4,7-di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, 3-hydroxy-Neu5Ac, 3-
fluoro-Neu5Ac acid as well as the ~thio analogues of Neu5Ac are
known in the art. Chemical modifications of other saccharide units are
also known in the art.
Additionally, chemical methods for the synthesis of
20 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 ~Iycosides first involves formation of a
glycosidic linkage on the anomeric carbon atom of the reducin~ sugar.
25 Specifically, an appropriately protected form of a naturally occurrinQ or
of a chemically modified saccharide structure ~the ~Iycosyl donor) is
selectively modified at the anomeric center of the reducin~ unit so as
to introduce a leavin~ ~roup comprisin~ halides, trichloroacetTmidate,
thioQlycos;de, etc. The donor is then reacted under catalytic
30 conditions well known in the art with an a~lycon 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
reducin~ unit. Appropriate use of compatible blocking ~roups, well
known in the art of carbohydrate synthesis, will allow selective
5 modification of the synthesized structures or the further attachment of
additional su~ar units or sugar blocks to the acceptor structures. A
detailed discussion of prior art methods for forming the glycosidic
linka~e is recited in Venot et al., U.S. Serial No. 07/887,747, and U.S.
Serial No. 07/887,746, both filed on May 22, 1992, as Attornev
Docket Nos. 000475-011 and 000475-029 and entitled "Modified
Sialyl Lewis~ Compounds~ and ~Modified Sialyl LewisX Compounds,"
respectively. The disclosures of both of these cases are incorporated
herein by reference in their entirety.
After formation of the ~Iycosidic linkage, the saccharide
~Iycoside can be used to effect couplin~ 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
saccharid~ unit to the saccharide glycoside is accomplished b`~
employin~ established chemistry well documented in the literature.
See, for example, Okamoto et al.27, Ratcliffe et al.28, Abbas et al.29,
Paulson30, Schmidt3~, F~gedi et al.32, Kameyama et al.33 and Ratcliff et
al5~. The disclosures of each of these references are incorporated
herein by reference in their entirety.
On the other hand, enzymatic coupling is accomplished by the
use of ~Iycosyl transferases which transfer sugar units, activated as
their appropriate nucleotide donors, to sp~cific saccharide or
oligosaccharlde acceptors, ~enerally at the non-reducing sugar portion
of the saccharide or oli~osaccharide. See, for example, Toone et al.3~.
Moreover, it is possibb to effect selected chemTcal modifications of the
saccharide or oli~osaccharide acceptor, of the sugar donor or the
WO 92/223~1 PCI'/CA92/00244
21iO49.S
-23-
product of the enzymatic reaction so as to introduce modifications or
further modifications into the structure.
Representative of glycosyltransferases are sialyltransferases
which constitute a ~roup of enzyrnes which transfer N-
acetylneuraminic acid, activated as its cytidine monophosphate ~CMP)
derivative, to the terminal oli~osaccharide structures of ~Iycolipids or
~Iycoproteins. Specific transferases have been identified which build
the following terminal structures on glycoconjugates: ;
oNeu5Ac(2-3)pGal~ 1 -3)~G1cNAc-
oNeu5Ac(2-3)pGal~ 1 ~4)~G1cNAc-
aNeu5Ac(2-6),t~Gal( 1 -4)~GlcNAc-
oNeuSAc(2-3)pGal( 1 -3)~alNAc-
oNeu5Ac(2-6)aGalNAc-
oNeu5Ac(2-6)~G1cNAc- .
l S The enzymatic transfer of Neu5Ac and analogues thereof
requires the prior synthesis of their nucleotide (CMP) derivatives.
Activation of NeuSA-c is usually done by usin~ the enzyme CMP-sialic
acid synthase which is readily available and the literature provides
examples of the activation of various analogues of Neu5Ac such as 9-
substituted Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-0-methyl-
Neu5Ac,4-deoxy-Neu5Ac, ~acetamido-NeuSAc, 7-deoxy-NeuSAc,
4,7-dideoxy-Neu5Ac, and the 6-thio derivatives of Neu5Ac.
Alternatively, if the analo~ue of Neu5Ac is not amenable to activation
by the CMP-sialic acid synthase, then the Neu5Ac analogue can be
coupled to the oligosaccharide acceptor by chemical means known in
the art.
The nucleotide derivative of Neu5Ac or of an analogue thereof
and the saccharide acceptor are combin~d with each other in the
presence of a suitable sialyltransterase under conditions wherein
3~ NeuSAc or an analogue thereof is transferred to the acceptor. As is
appàrent, the saccharide acceptor employed must be one which
functions as a substrate of the particular sialyltransferase employed~
WO 92/22301 PCI`/CA92/00244
2110~9~ -24-
ln this regard, the art recognizes that while Neu5Ac is usually
enzymatically transferred to a natural acceptor ~i.e., ~Iycoproteins and
other ~Iycoconju~ates having a glvcan chain possessin~ a terminal
acceptor disaccharide structure recognized by the enzymes and
terminal acceptor disaccharides not possessin~ an aglycon moiety,
R = H in these cases), some sialyltransferases can tolerate certain
modifications in the structure of the acceptor whereas other
sialvltransferases show strict specificity tor one type of acceptor. It
has been found that chemically modified acceptors (~artificial
acceptors"), such as oligosaccharide ~Iycosides 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,
~Gal(l--3/4~pG1cNAco(2--3)sialyltransferase can transfer Neu5Ac to a
terminal ~ial(1~4)pG1cNAc- disaocharide str~lctwe. However, in this
situation, it has been found that tho hydroxyl ~roups at the 3, 4 and 6
positions of p-~alactose are critical to recognition by the ~nzyme and
accordin~ly chemical modification at one or more of these points can
result in non-reco~nition by the enzyme.
Likewise, when an analogue of Neu5Ac is to be enzymatically
transferred, it is necessary that the CMP derivative of the analogue
aiso be recogni2ed by the sialyltransferase. Since sialyltransferases are
naturally designed to transfer or donate NeuSAc, any modification to
the Neu5Ac results in the formation of an ~artificial donor~ In this
regard, the art recognizes that certain sialyltransferases can tolerate
some modifications to the Neu5Ac and still transfer analo~ues of
Neu5Ac to glycoproteins or glycolipids possessin~ 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~
WO 92/22301 pcr/cAs2/oo244
` 211~49~
-25-
As noted above, a suitable nucleotide derivative of Neu5Ac 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-reducin~ end which are r~co~nized by the
S sialyltransferase) in the presence of the sialyltransferase under
conditions wherein NeuSAc or an analogue thersof 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
10 buffer such as 0.1 M sodium cacodylate in appropriate conditions of
pH and temperature such as at a pH of 6.5 to 7.5 and a temperature
between 25 and 45C, preferably 35-40C for 12 hours to 4 davs.
The resulting oligosaccharide can be isolated and purified using
conventional methodology comprising HPLC, gel-, ion exchange-,
15 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, sialyltransf~rases as well as other
20 ~Iycosyltransferases are well known in the art and ars described in
Toone et al.34 and Beyer et al.35.
C. UtTlity
Without being limited to any theory, it is believed that
oligosaccharide glycosides affect the cell mediated immune response in
25 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-
30 mediated immune response (eg., the inflammatory component of a
WO 92/22301 - pcr/cA92/oo244
2110~95 -26-
DTH response~ when administered after second or later exposures of
the immune svstem to the antigen. Additionally, oli~osaccharide
glycosides can induce tolerance to anti~ens when administered at the
time of second or later exposures of the immune system to the
5 anti~en.
The suppression of the inflammatory component of the immune
response by oligosaccharide ~Iycosides related to blood group
determinants is believed to re~uire the initiation of a secondary immune
response (i.e., a response to a second exposure to anti~en). The
10 oli~osaccharide ~Iycoside related to a blood ~roup determinant is
generally administered to the patient at least about 0.5 hours after an
inflammatory episode, preferably, at least about 1 hour after, and most
preferably, at least about 5 hours after an inflammatory episode or
exacerbation.
l S Oli~osaccharide glycosides related to blood ~roup determinants
are effective in suppressin~ cell-mediated immune responses to an
anti~en ~eg. the inflammatory component of a DTH response) when
administered at a dosa~e ran~e of from about 0.5 m~ to about 50
m~/k~ of body wei~ht, and preferably from about 0.5 to about 5
20 m~/k~ of body wei~ht. The specific dose employed is re~ulated by the
particular cell-mediated immune response being treated as well as by
the judgment of the attendin~ clinician dependin~ upon factors such as
the severity of the adverse immune response, the a~e and ~eneral
condition of the patient, and the like. The oli~osaccharide glycosides
25 related to blood ~roup determinants are ~enerally administered
parenterally, such as intranasally, intrapulmonarily, transdermally and
intravenously, althou~h other forms of administration are
contemplated. Preterably, the suppression of a cell-mediated immune
response, eg. the inflammatory component of a DTH response, is
30 reduced by at least about 10% as opposed to control measured 24
hours after administration of the challenge to the mammal and 19
wo 92/22301 21~ 0 ~ 9 S pcr/cA92/oo244
-27-
hours after administration of the oligosaeeharide glyeoside as per this
invention.
In addition to providing suppression of the inflammatorv
eomponent of the eell-mediated immune response to an antigen,
5 admin;stration of the oligosaeeharide glyeoside related to a blood ~roup
determinant also imparts a toleranee to additional ehallenges from the
same antigen. In this regard, re-ehallenge bV the same antigen weeks
after administration of the oligosaeeharide glyeoside related to a blood
~roup determinant results in a si~nifieantly redueed immune response.
Administration of the oli~osaecharide glycoside related to a
blood group determinant simultaneously with first exposure to an
antigen imparts suppression of a eell-mediated immune response to the
antigen and toleranee to future ehallenges with that antigen. In this
regard the term ~reducing sensitization~ means that the eompound,
15 when administered to a mammal in an effective amount along with a
suffieient amount of antigen to induee an immune response, reduees
the ability of the immune system of the mammal to beeome educated
and thus sensitized to the antigen administered at the same time as the
eompound. An ~effeetive amount~ of the compound is that amount
20 whieh will reduee sensitization ~immunolo~ieal edueation) of a mammal
to an antigen administered simultaneously as determined by a
reduetion in a eell-mediated response to the anti~en such as DTH
responses as tested by the footpad ehallenge test. Preferably the
reduction in sensitization will be at least about 20% and more
25 preferably at least about 30% or more. G~nerally oli~osaccharide
glyeosides related to blood group determinants are effeetive in reducing
sensitization when administered at a dosa~e range of from about 0.5
mg to about 50 mg/k~ of body weight, and preferably from about 0.5
mg to about 5 mg/kg of body wei~ht~ The speeifie dose employed is
30 regulated by the sensitization being treated as well as the judgement of
the attending clinieian dependin~ upon the a~e and ~eneral eondition of
the patient and the like. ~Simultaneous" administration of the
wo 92t22301 pcr/cAs2/oo244
21iQ~ 28- `
compound with the anti~en with re~ard to inhibitin~ sensitization
means that the compound is administered once or continuously
throu~hout a period of time within 3 hours of the administration of an
anti~en, more preferably the compound is administered within 1 hour
of the anti~en.
The methods of this invention a-e ~enerally achieved by use of a
pharmaceutical composition suitable for use in the parenteral
administration of an effective amount of an oli~osaccharide ~Iycoside
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 oli~osaccharide ~Iycoside related to a blood
~roup determinant so as to provide the above-noted dosage of the
oligosaccharide ~Iycoside 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 composltions, transdermal compositions
or banda~es etc., which are well known in the art.
The followin~ 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 ~eneralîy accepted meanin~:
AB = AB pattern
ax = axial
BSA = bovine serum albumin
d = doublet
dd = doubîet of doublets
ddd = doublet of doublets of doublets
DTH = delayed-type hypersensitivity
eq = equatorial
WO 92/22301 pcr/cA92/oo244
2110 ~9 j
-29-
i.r. = infra red
m = multiplet
q = quartet
s = sin~let
t = triplet
t.l.c. = thin layer chromatography
U = Units
m = microns
AG 1 x 8 (formate form) = ion exchan~e resin AG 1 x 8
(formate form) available from Bio-Rad Laboratories,
Richmond, CA
Dowex 50 x 8 (H~ form) = ion exchan~e resin Dowex
50 x 8 ~H~ form) available from Dow Chemical,
Midland, Ml
IR-C50 resin (H~ form) = ion exchan~e resin IR-C50
/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
20 manufacturers are as follows:
latron = latron Laboratories, Tokyo, dapan
Merck = E. Merck AG, Darmstadt, Germany
Millipore = Millipore Corp., Bedford, MA
Waters = Waters Associates, Inc., Milford, MA
2S 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
30 blood group determinants and the induced tolerance to later challenges
WO 92/22301 PCI`/CA92~00244
2110495 30 ;
with the same anti~en. In Examples 1-13, the oligosaccharide
~Iycosides recited are referred to by Arabic numerals which are
depicted in fi~ures 14-24 whereas in Examples 20-35, the
oligosaccharide ~Iycosides are referr~d to by Roman numerals which
5 are depicted in fi~ures 11-1~.
In one or more of Examples 1-13, pre-coated plates of silica ~el
(Merck, 60-F25, ) were used for analytical t.l.c. and spots were
detected by charrin~ after spraying with a 5% solution of sulfuric acid
in ethanol. Silica ~el 60 ~Merck, 40-63 ~m) was used for column
chromato~raphy. Iatrobeads were from latron (Order No. 6RS-8060).
Millex-GV filters (0.22 JJm) were from Millipore. Cl~ Sep-Pak
cart~id~es and bulk C,8 silica ~el were from Waters Associates.
Commercial reagents were used in chemical reactions and
solvents were purified and dried accordin~ 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 dryin~ over magnesium
sulfate, the solvents were removed by evaporation under vacuum with
a bath temperature o~ 35C or lower when necessary.
~H-n.m.r. were recorded at 300 MHz (Bruker AM-300) with
either tetramethylsilane in CDCI3 or acetone set at 2.225 in D20 as
internal standards, at ambient temperature, unless otherwise noted.
The chemical shifts and coupling constants ~observed splittin~s) were
reported as if they were first order, and only partial n.m.r. data are
reported. '3C-n.m.r. spectra were recorded at 75.5 MHz with
tetramethylsilane in CDCI3 or dioxane set at 67.4 in D20 as reference.
A. SYNTI~ESIS OF DERIVATIVES OF Neu5Ac
Unless otherwise noted, derivatives of NeuSAc have been
prepared following known procedures with suitable substitution of
starting materials where necessary. The following derivatives have
WO 92/22301 2 1 1 U ~ PCl`/CA92/00244
, . . . .~
-31 -
been prepared by a convenient modification of procedures reported in
the literature: 9-N,-NeuSAc 1~.3 Neu5Pr ~5-propionamido) 1t, 7 d-
Neu5Ac 1d37 and the C8-Neu5Ac li~.
FIG. 14 illustrates a ~eneral synthetic scherne used for the
5 synthesis of derivatives of Neu5Ac. 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-~alacto-2-nonulopyr~nosylonic
acid ~9-N3-NeuSAc) lb
Glycosyl chloride 38 (2.83 ~, 5.57 mmol) in dry
dichloromethane (13 mL) was added to the mixture of benzyl aicohol
(5~0 mL, 48.2 mmol), molecular sieves 4A (18 5 9, crushed), dry
silver carbonate 14.2 ~, 15.2 mmol) in dichloromethane (8 mL). The
mixture was stirred in the dark for 4 days, diluted with
dichloromethane (50 mL) and filte-ed th-ou~h t:elite. After usual work
up, the residue was chromato~raphed on silica ~el usin~ a 3:2 mixture
of hexanes and ethyl acetate as eluant. The product was then eluted
with a 4:5 mixture of the same solvents ~Ivin~ (1.96 9, 60%) of pure
material and 0.33 ~ (10%) of materTal containin~ a small amount of
impurities~ lH-n.m.r.: 5.436 (ddd, lH, J7.8 8.5, J~,. 2.5, J8.9 5.5Hz,
H-81, 5.317 (dd, lH, Jo,7 1.8Hz, H-7), 5.110 (d, lH, J6N~ 9.5 Hz, NH),
4.849 (ddd, 1H, J3,",4 12.0, J3~,~ 4.5, J~5 9.5Hz, H-4), 4.788 and
4.397 ~AB, 2H, J0.,,~ 12.0Hz, benzylics), 3.642 (s, CO2C~3), 2.629
(dd, lH, J3.~.3.X 12.5Hz, H-3eq), 2.140, 2.113, 2.017, 1.997, 1.857,
(5s, 15H, 4 OAc, 1 NAc), 1.986 (dd, lH, H-3ax).
The above material ~1.5 9, 2.58 mmol) was d~-0-acetylated in
dry methanol (20 mL) containing a catalytic amount of sodium
methoxide for 5 hours at 22C. After de-ionization with Dowex 50 x
8 (H~ form), the solvent was evaporated leavin~ the product 39 (1.0
9, 94%) which was used in the next step; 'H-n.m.r. (CDCI3): 4.815
WO 92/22301 PCI/CA92/00244
2 110 49~ -32-
and 4.619 (AB, 2H, J0.,,,11.5Hz, benzylics), 3.802 ~s, CO2C~3),
3.582 ~dd, 1H, J6." 9.0, J~7 0.5Hz, H-6), 2.752 ~dd, 1H, J,.~ 12.5,
J3."~ 4.5Hz, H-3eq), 2.039 ~s, 3H, N Ac), 1.861 ~dd, 1H, J3.""
11.0Hz, H-3ax).
A solution of para-toluenesulfonyl chloride ~0.125 9, 0.65
mmol) in pyridine ~0.1 mL) was syringed into a solution ;~ ~0.248 ~,
0.60 mmol), 4dimethylaminopyridine ~0.01 9) in pyridine (1.1 mL) at
0C. After stirring for 4 hours at 0C, methanol ~0.10 mL) was
added and the mixture was co-evaporated with dry toluene. The
residue was quickly chromato~raphed on silica ~el usin~ acetonitrile as
eluant giving the tosylate ~0.21 ~, 62%) still containin~ some
impurities. Sodium azide (0.19 9, 2.92 mmol) was added to a
solution of this material (0.21 9, 0.37 mmol) in dimethylformamide
(0.5 mL). The mixture was stirred at 65C for 18 hours after which it
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 ~iving the product 40 (0.136 9,
85%); i.~ .' 2110 (N3); lH-n.m.r.: 5.775 (d, lH, J~.NH 9.0HZ, NH),
4.816 and 4.470 (AB, 2H, J0,,~ 1 1 .5HZ, benzylics), 3.738 (s,
CO2C~), 2.871 (dd, l H, ~13, " 4.8, J3.~,3,~ 13.0Hz, H-3eq), 2.086 (s,
3H, NAc), 1.964 (dd, lH, J3~"~ 11.5Hz, H-3ax~. -
The above compound ~0 (0.105 9, 0.24 mmol) was left for 3
hours at 22C in 0.25 N sodium hydroxide (2 mL). After bringing the
pH to 6 by addition of Dowex 50 x 8 (H~ form) followed by filtration,
the material, recovered after freeze drying, was chromatographed on
latrobeads using a 65:35:5 mixture of chloroform, methanol and water
as eluant. The appropriate fractions ga~e the product (0.087 9,
86%). This compound (0.100 9, 0.235 mmol) was heated at 80C
for 6 hours in 0.025 N hydrochloric acid (3 mL). The solution was
neutraîized with sodium hydroxide and then freeze dried. The product
was chromatographed on latrobeads (0.60 9) using a 65:35:5 mixture
of chloroform, methanol and water giving 1b (0.067 9, 85%); 'H-
W0 92/22301 2 ~ 19 5 PCr/CA92/00244
-33-
n.m.r.: 4.106 - 3.895 (m, 5H), 3.639 ~dd, lH, J"~ 3.0, J,,.13.0Hz,
H-9), 3.528 (dd, lH, J".,. 6.0H2, H-9"), 2.249 ~dd, lH, J3.~ 4.5,
J3.",3.,~ 12.5Hz, H-3eq), 2.090 (s, 3H, NAc), 1.852 (dd, lH, J3.",
11.OHz, H-3ax).
S Example 2 -- Synthesb of 5-propionamldo-3,5-dideoxy-D-
glycero-D-galacto-2-nonulopyranosylontc
acid INeu5Pr) lf
A solution of 39 ~0.075 9, 0.18 mmol) in 2 N sodium hydroxide
~1 mL) was left for 0.5 hours at 22C followed by 7 hours at 95C.
10 The pH was then adjusted to 7.5 by addition of IR-C50 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 0C. After
3 hours, more propionic anhydride ~0.025 mL, 0.195 mmol) was
added and the mixture stirred for 2 more hours at 0C. 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 ~). The
recovered fractions were evaporated in vacuo and the residue
chromatographed on latrobeads (5 9) using a 3:1 mixture of
chloroform and methanol as eluant giving 41 (0.0646 9, 86.5%); 'H-
n.m.r.: 4.800, 4.578 (AB, 2H, J~"" 11.OHz, benzylics), 3.580 (dd, 1 H,
J5~, 9.0, JO~ 1.0Hz, H-6) 2.776 ~dd, lH, J3.~,~ 4~5, J3" 9~",12.5Hz, H-
3eq), 2.316 (q, 2H, J 7.5 Hz, CH2CO), 1.762 (dd, lH, J3,"~ 12.0Hz),
1.129 (t, 3H, CH3).
A solution of the above benzyl glycoside (0.115 9, 0.278 mmol)
in water (5 mL) was hydro~enated in the presence of S96 palladium on
charcoal (10 mg) at atmospheric pressure and 22C for 5 hours. The
eluate obtained after filtration through Celite followed by Millipore
filter, was freeze dried leaving compound 1f (0.074 9, 82.5%); 'H-
wo 92/22301 pcr/cA92/oo244
21104!)5 - - 34
n.m.r.: 3.72 - 4.10 ~m, H-4,-5,-7,-8,-9), 3.614 (dd, 1H, J8.". 6.5, J""~,
11.75Hz, H-9a), 3.530 ~dd, lH, J6.~ 9.0J~ 1.0Hz, H-6), 2.250 -
2.400 Im, 2H incl. CH2CO ~q, 2.315, J 7.5Hz) and H-3eq ~dd, J3.~
11.5 Hz, J3."4 4.5Hz)l, 1.880 ~t, 1H, J3."3." 11.5Hz, H-3ax), 1.130 ~t,
3H, C~
Example 3 -- Synthesis of 5-acetamido-3,5-dideoxy-D-
~atacto-2-octulosonic actd ~C8-Neu5Ac) 1i
The synthesis of li from 39 essentially follows the published
procedure of Hase~awa et al.~8 but usin~ a different startln~ material
than the reported one. In particular, a suspension of 39 (0.52 9,
0.125 mmol) in 2,2-dimethoxypropane ~3 mL) was stirred for 1.5
hours ~t 22C 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 9, 88%).
42 (0.054 9, 0.185 mmol) was acetylated in a 2:1 mixture of
acetic anhydrid~ (1 mL) and pyridine kept at 50C for 5 hours. After
the usual work up, the residue was chromatographed on silica ~el
usin~ ethyl acetate as eluant ~ivin~ the acetylated product (0.091 9,
92%); 1H-n.m.r.: 5.420 (dd, lH, Jo7 1.5, J~.8 3.5Hz, H-7), 5.196 (d,
lH, '16,NH 9.0H~, NHI, 5.009 (ddd, lH, .~,3." 13.0, J~ ,5.0, ~ s
10.t)Hz, H-4), 4.797 and 4.498 ~AB, 2H, ../",~ 11.5Hz, benzylics),
3.776 (s, 3H, CO2C~3), 2.724 (dd, lH, J30"3.~, 13.0Hz, H-3eq), 2.151,
2.032, 1.895 (3s, 9H, 2 OAc, 1 NAc), 2.032 (t, lH, H-3ax), 1~363
and 1.350 (2s, 6H, methyls).
The above product (0.091 9, 0.169 mmol) was heated for 4
hours at 40C 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 22C in the presence of
sodium metaperiodate (0.059 9, 0.275 mmol). The mixture was
.
`:
WO 92/22301 pcr/cA92/oo244
21~ 0~9~
-35-
filtered throu~h a pad of Celite which was washed with methanol.
The combined filtrate was stirred at 0C for 25 minutes in the
presence of sodium borohydride ~0.036 ~, 0.95 mmoi). The mixture
was then stirred at 0C with some acetic acid (0.2 mL), after which
S the solvents were evaporated leavin~ 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 22C. The residue
recovered after the usual work up was chromato~raphed on silica ~el
using ethyl acetate as eluant to give a product which still contained
some non-separable impurities. The dr/ material ~0.074 ~, still
containin~ 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 50 x 8 (H~ form) and filtration,
the solvent was evaporated in vacuo and the residue chromatographed
on silica ~el using a 15:1 mixture of chloroform and methanol to give
a pure product 44 ~0.047 9, 78%); 'H-n.m.r.: ICD~OD): 4.724 and
4.416 (AB, 2H, J0."~ 11.5Hz, benzylics), 3.671 (s, 3H, CO2CH3),
3.456 (dd, lH, J5~, 9.5, J~,7 1.0Hz, H-6), 2.642 (dd, lH, J3",~, 4.5,
J3~,3." 12.5Hz, H-3eq), 1.938 (s, 3H, NAc), 1.699 ~t, lH, J3",4
12.5H2, H-3ax).
The above material (0.022 9, 0.057 mmol) was stirred in 0.25
N sodium hydroxide (2 mL! for 5 hours at 22C, the solution was
neutralized with Dowex 50 X 8 (H~ form) and the filtrate was freeze
dried to give a white solid (0.019 9, 90%). This product was
dissolved in water ~2 mL) and hydrogenated for 3 hours at 22C 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 leavin~ the desired product 1l (13~3 mg,
94%); lH-n~m.r.: 3~462-4.093 (m,6H), 2.287 (dd, lH, ~3~", 4.5,
J3.~,3 x 12.5Hz, H-3eq), 2.052 (s, 3H, NAc), 1.853 (t, lH, J3,~4 12.5
Hz, H-3ax).
WO 92/22301 PCl'/CA92/00244
,~1~,
2110~ 36-
Example 4 -- Synthesis of 5-acetamido-3,5,7-trideoxy-B-
D-~alacto-2-nonulopyrano~ylonic acid
(7-d-Neu5Ac) ld
The synthesis of ld essentially follows the published procedure
of Zbiral et al.3~ but using a different startin~ material. In particular,
imidazole (0.13 9, 1.93 mmol) and tert-butyldimethylsilyl chloride
(0.135 ~, 0.89 mmol) were added to a solution of 42 (0.11 ~, 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 10.101 9, 92%): la]D = -2.66
(c. 0.6, chloroform); lH-n.m.r.: 5.195 ~d, lH, JS.NH 7Hz, NH), 4.853
and 4.603 ~AB, 2H, J~ m 11.5Hz, benzylics), 3.736 ~s, C02CH3),
2.692 (dd, 1H, J3."4 4.5, J3.~,3,~ 13.0Hz, H-3eq), 2.022 (s, 3H, NAc),
1.884 (dd, lH,..13.,~4 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 disulflde (1.25 mL, 20.8 mmol) were added
dropwise to a solution of the above compound (0.437 9, 0.77 mmol)
in dry tetrahydrofuran (20 mL) at -30C. After stirrin~ at -25C for
0.5 hours, methyl iodide (1.6 mL, 25.6 mmol) was slowly warmed up
to room temperature. After evaporation, the residue was
chromato~raphed on silica gel using a 4:1 mixture of hexanes and
ethyl acetate as eluant providin~ the xanthate (0.327 ~, 65%): l01D
93.9 (c. 0.655, chloroform); lH-n.m.r.: 6.388 (dd, lH J~, 1.0, J,,~
2.5Hz, H-7), 5.610 (d, lH, JS.NH 7.0Hz, NH), 4.778, 4.466 (AB, 2H,
J~ m 11.5Hz, benzylics), 3.778 (s, C2C~3~, 2.662 (dd, lH, J~,,4 4.5,
J3.~3", 12.5HZ, H 3eqJ, 2.584 (s, 3H, OCH3~, 1.883 ~s, 3H, NAc),
1.693 (dd, lH, J3.,~ 11.5Hz, H-3ax~,1.315 (s, 6H, methyls~ 0.825
(9H, t-butyl~, 0.025, 0.092 (2s, 6H, methyls~.
WO 92/22301 PCI`/CA92/00244
211~9.S
-37-
Azobisisobutyronitrile (0.004 9) and tri-n-butyltin hydride ~0.5
mL, 1.86 mmol) were added to a solution of the above xanthate ~0.32
~, 0.48 mmol) in dry toluene ~3 mL). After heating at 100C for 7
hours, the solvents were co-evaporated with dry toluone, and the
S residue chromato-~raphed on silica ~el usin~ a 3:2 and then 1 :1
mixtures of hexane and ethyl acetate as eluant to give the 7-deoxy
product ~0.260 ~, 70%); lH-n.m.r.: 5-334 (d, lH, JS.NH 7.0HZ, NH~
4.740, 4.455 ~AB, 2H, J~ 11.6Hz, benzylics), 3.690 (s, CO2C~3),
2.628 (dd, lH, J3."~ 4.2, J3."3.,~ 12.9Hz, H-3eq), 1.914 (s, 3H, NAc),
1.805 (dd, 1H, J3."" 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 75C in 70% acetic acid for 7.5 hours. After co-
evaporation with toluene, the residue was chromatographed on silica
~el usin~ a 10:1 mixture of chloroform and methanol giving 43 (0.157
~, 84%~; 'H-n.m.r.: 4.860 and 4.655 ~AB, 2H, Jo"l,11.5Hz,
benzylics), 3.834 (s, CO2C~), 2.806 (dd, lH, J3."~ 4.5, J3~13~t
12.5Hz, H-3eq), 2.069 ~s, 3H, NAc), 1.881 (dd, lH, J~.~4 12.5Hz, H-
3ax), 1.698 ~m, 2H, H-7 and H-7').
Compound ~3 (0.157 9, 0.396 mmol) was kept in 0.25 N
sodium hydroxide ~6 mL) at room temperature for 5 hours. Af~er
neutralization with Dowex 50W x 8 (H~ form) and filtration, the
product (0.149 9, 97%) was r~covered after Iyophilization of the
solution. This product (0.146 ~, 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 9)~ The mixture was filtered through
Celite and through a Millex-GV (0~22 ~m) filter. The filtrate was
freeze dried to provide ld (0.105 ~, 94%); 'H-n.m.r.: as reported by
Christian39.
Table 1 below summarizes the derivatives of NeuSAc prepared~
Table I
o"l
R5 ~ :
Rs ~ R~ OH
I SidiC Acd D~v~i~
_ r _ . = ~
Compou~ Rl R2 R3 R4 R5 R~ R7 R8 R9
No~ _ ~ _
1~ H H OH NHAc H OH OH H CH20H
.-- .
1b _ _ CH~N~
lc _ . CH~
ld H CH~OH
lc _ _ OH H l
~ _ = ~ NHCOCH~CH~ H OH _ = _ ¦
¦1h _ NHCOCH~OH
li ~ ~ ~ NHAc ~ ~ ~ ~ H
_
~,
-39-
Table I cont.
Sialyl Moieties obtained by chemical modification of sialylated
oligosaccharides: -
OH
~cNH ¦
H \ ~1 CO,H
R, ,
~H 1~ CH2NH2
1k R9.CH2NHAc
OH
U'"'""`~
~ CONHCIl,
~1
OH OH
OH
~t NH ~ --
N \~
CO,~
1m
wo 92/22301 ?PCr/CAg2/00244
2110495
-40-
B. SYNTHESIS OF CMP DERIVATIVES OF N~u5Ac
AND ANALO~;UES THEREOF
Example S -- Synthesis of the CMP~eriv?sti?ve~ of N~?uSAc
CMP-sialic acid synthase was extracted from calf brain and
5 partially pur;fied at 4C by a sli~ht modification of the ori~inal
procedure of Hi~a et al.~ Routinely, - 200 ~ 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,
10 0.5 mM phenylmethylsulfonyl fluoride. The homo~enate was stirred
for 1 hour and then centrifuged at 23,000 x ~ for 15 minutes. The
supernatant was decanted and the pellets were extracted once again
with 200 mL of the same buffe- as above. The supernatants were
combined and centrifuged at 28,000 x 9 for 15 minutes. The
` 15 supernatant was filtered through glass wool to ~ive the crude extract
~515 mL, 4.7 mg protein/mL,--90 U of enzyn~).
After adjusting salt concentration to 0.4 M with soîid 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.
20 The solution was stirred for an additional 15 minutes, kept on ice for 1
hour and centrifu~ed at 28,000 x 9 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
25 was left on ice overnight and then centrifuged as above~ The
resultant pellets were washed with 150 mL of 60% ammonium suîfate
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~", with one unit of
WO 92/22301 2 11 ~ 19 5 PCI`/CA92~00244
enzymatic activit~/ defined as one ~mol of product formed per minute
at 37C.
The enzyme present in the pellet could be stored for several
weeks in the cold room. Before usin~ the enzyme for synthesis, the
5 pellets were suspended in a minimal volume of S0 mM Tris/HCI, pH
9.0, 35 mM ma~nesium chhride, 3 mM 2-mercaptoethanol ~activation
buffer) and dialyzed overni~ht a~ainst 100 volumes of the same
buffer. The dialyzed enzyme was centrifu~ed at 9,000 x ~ for 10
min. The supernatant containin~ more than 90% of the enzyme
10 activit~ was used directly for the synthesis.
The CMP-derivatives of sialic acid analogues were synthesked
as noted above and purified by a modification of the reported
procedures of Hi~a et al.~ and Gross et al.~2 For example, 7-d-
Neu5Ac .~ ~Table 1, 20 m~, 69 ~mol) was activated by using 15 U of
the above dialyzed enzyme for 5-6 hours at 37C 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
analo~ues was estimated by the usual thiobarbituric acid assay for
sialic acid after r0duction with sodium borohydride as per Kean et al.~3
20 The product was extracted with cold acetone as per Gross et al.~2
After evaporation of the acetone in vacuo lat - 15C), 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
4C with a flow rate of 60 mL/h. Fractions (1 mL) were assayed for
25 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 leavin~ the CMP-7 d-Neu5Ac ~, 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
30 some cases (2e, ~, ~h). 'H-n.m.r. spectra showed that the CMP-
derivatives contained some of the unreacted sialic acid.
WO 92/22301 pcr/cA92/oo244
'~11049~ ; ~
42 ~:
Table 2 below illustrates the CMP-derivatives of analo~ues of ~.
Neu5Ac prepared from the analo~ues of NeuSAc set forth in Table 1
above, as well as partial ~ n.m.r. data concernin~ these compounds.
:
-43-
o .~1
~o=~-o ~ ~ _ _ . I
~ 1~
11 ~ ~ _ _ _ _ _ _ s
~ _ : _ : . ~
,~
-43/1-
I
~^ O O 10' ~ O, _ O In. `
_ ~ N N 1'1 ~ ~1
X ~ _ '` _ .~ ~ O _ ~ .~ .~ ~ ~
~11 ~ o ~n ~ o o ~ ~ o o ~1) 0 0 o ~ n ~ ~o o
. ~O ~O . . ~'~ ~ ~O '.0
~ . ~o ~ . ..0 ~ . ~!. ~ ' . ~o ~ . . ~. N . 1
a ~_,, ~__, .,_.~ ~ ~ . .~ .,_., .~
_ _ _ _ _ _ _ _
'O ~ o ~n ~ o ~ ~
_ ~ I ~ /~ ~'1 N ~
O~ 0 0 ~ <~ 1~'1 ~ i C~l O ~ <1~ 1~` a~ o
~~ ~ ~; ~ ~; ~ .~ ~r ~ ~ . .
N-- N-- N--N--N-- N N-- N-- : ~.
_ ~^ ~. ~ _
O O O O ~: ~ O
N ~ ~ N 5~ ;0~_ ~ O
~ b ~ ~ ~ ~ ~! O ~ Oz~ ~i ~J
_ _ 11~
~ ~ ~n ~ ~ ~ ~
_ O~ O O ~ O ~ O~
O C ~ I~ ~ ~ ~` ~` ~` ~ I~ O
_~ ~
_ ~ ~ ~ ~ ~ ~ ~ .~ ~ .
~ ~ ~1 ~1 ~ ~ ~ ~ r~ ~~ X ''~' ~ ~ '.D ~ ~O 'D ~ '
b _ _ . _ ~
O ~1~
O _ 0 0 ~ 0 ,~ ~ 0 1~
n I ~ ~ ~ a~ ~ ~ ~ ~ .a
0~ n _ Y~ ~ u~ u7 ~ ~n ~ ~:~-
~ ~ _ ~
~' ~ o
a ~ 0.~,
Co ~ O O ~ O ~ U) ~ ~ ~ ~0
1 ~-- ~ ~ z r~ ~o ~ ~ O~ a ~, _ c
- ~
_ _ .~.,a ~~' ~ .
:~ t) ~ ~0
~ ~ ~1 ~1 ~1 ~:1 ~1 ~1 ~1
~ ~ _ _ .~
WO 92/22301 PCI'/CA92/00244
`` 2110~95
-44,
C. SYNTHESIS OF OLIGOSACCHARIDE GLY~;OSIDES
Examples 6-7 illustrate the synthesis of oli~osaccharide
~Iycosides. The structure of ;~k to 7a are Illustrated in FIG. 15.
Oli~osaccharide ~Iycosides 4b, ~, ~, ~, and 7a were synthesized
accordin~ to the procedures of Lemieux et ab~3, Lemieux et al.~,
Paulsen et al.~5, Sabesan et al.~, and Lemieux et al.4~, respectiv~ly.
Oligosaccharide ~Iycosides 4d and 5d were synthesized
following the procedure reported for the synthesis of oli~osaccharide
~Iycosides ~ and Sb but by replacin~ the 8-methoxycarbonyloctyl by
1 0 methanol.
Oligosaccharide ~Iycosides Se and g~Q were synthesized
according to the procedures of Paulsen et al.~5 and Alais et al.'~ but
replacing t~ e methanol by 8-methoxycarbonyloctanol. 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 Iyophilized from
water and the products furth~r dried in vacuo over phosphorus
pentoxide.
Example 6 -- Synth~sis of 9-Hydroxynonyl 2-acetamido-2-
deoxy-LB-D-~alactopyranosyl-( 1 -3)-0-l-~-D-
~lucopyranoslde 4a
Sodium acetate (0.200 ~) and sodium borohydride
(0~060 9) were added to a solution of the disaccharid~ 0~100 ~,
0~189 mmol) in a 10:1 mixture of water and methanol ~20 mL) cooled
at +4C~ After 24 hours, more sodium borohvdride (0~020 9) was
added to the reaction mixture maintained at +4C~ Atter 48 hours at
the same temperature, the pH was brou~ht to 5-6 bv 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
wo 92/22301 pcr/cA92/oo244
2 1 1 ~ 5 45
through a column of C", silica ~el 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 brou~ht 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 startin~ material 4b. The mixture was
then neutralized by addition of Dowex 50 x 8 (H~ form) and the resin
filtered off. The resultin~ solution was run throu~h a column ot AG 1
x 8 ~formate form). The eluate was freeze dried and the residue was
run through Sephadex LH 20 usin~ a 1 :1 mixture of water and
ethanol. The appropriate fractions were pooled and concentrated to
~ive 4a (0.060 9, 65%); lH-n.m.r. (D20): 4.545 (d, lH, Jl.2 8.0Hz, H-
1), 4.430 (d, lH, J'2'- 7.5Hz, H-1'), 2.025 (s, 3H, NAc), 1.543 Im,
4H), and 1.304 (m, 10H): methylenes; '3C-n.m.r. (D20l: 175.3 (Ac),
104.36 (C-1'), 101.72 (C-1), 67.72, 61.85, 61.60 (three CH20H).
Example 7 -- 9-Hydroxynonyl 2-acetamido-2-deoxy-U~-D- ..
~alactopyranosyl-~1 4)-0-1-~-D-~luco-
pyranoslde ~
Oli~osaccharide ~Iycoside ~ was prepared from 5b as indicated
above (60%); 1H-n.m.r. (D20): 4.520 (d, 1H, J~.2 7.5Hz, H-1), 4.473
(d, 1H, J1'.2' 7.6H~, H-1'), 2.033 ~s, 3H, NAc), 1.543 (m, 4H) and
1.302 (m,10H):methylenes; '3C-n.m.r. (D20): 175.23 (Ac), 103.71
and 101.88 (C-1 and C-1'), 60.93, 61.85 and 62.71 ~three CH20H~.
Example 8 -- Synthesis of 5-Allyloxypentyl 2-acetamido-2-
deoxy-lp-D-~alactopyranosyl-(1 -3~-0-l-~-D-
glucopyranoside 4c
The synthetic schemes for this example and Example 9 are set
forth in FIG. 16.
WO 92/22301 211~ PCI`/CA92/00244
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A. Synthe~is of Allyloxy-5-pentanol ~
Allyl bromide (2.5 mL, 0.029 mol) was added dropwise to the
mixture of 1,5-pentanediol (3 9, 0.029 mol) and sodium hydride ~1.2
9, 80% dispersion in oil) in dry dimethylformamide. Stirrin~ was
continued overni~ht 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 chromato~raphed
on silica gel using a 2:1 mixture of toluene and ethyl acetate as
eluant. The appropriate fractions ~ave compound 29 ~0.931 9, 30%).
'H-n.m.r. ~CDCI3): 5.83 ~m, tH, -CH=~, 5.20 ~m, 2H, =CH2), 3.95
~dd, lH, J=5.5 and l.OHz, allylics), 3.66 and 3.46 ~two t, 2H each,
J=6.5Hz, O-CH2), 1.64 ~m, 4H) and 1.44 ~m, 2H): rr~thylenes); 13G
n.m.r.~CDCI,): 134.7 and 116.6 (ethylenics), 71.6, 70.1 ~H2-0-~H~),
62.1 ~CH20H) 32.2, 29.2 and 22.2 (methyhnes).
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 9, 10.0 mmol) in dichloromethane 15
mL) was added dropwise to a mixture of the alcohol 2~ (1.33 mL, 10
mmol), silver trifluoro-methanesulphonate (2.57 9, 10.0 mmol) and
collidine (1.23 mL, 9.0 mmol) in dichloromethane (10 mL) at -70C.
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
WO 92/22301 PCI~/CA92/00244
211049S 47
syrupy residue was chromatographed on silica gel using a 5:1 mixture
of toluene and ethyl acetate providing compound ~ 4.0 9, 71%).
'H-n.m.r. (CDC13): 5.80 ~m, 2H, -CH = and H-3), 5.36 Id, 1 H, J1 2
8.5Hz, H-1), 5.18 (m, 3H, =CH2 and H-4), 2.13, 2.06, 1.87 (3s, 3H
each, 3 OAc), 1.40 (2H~ and 1.15 (m, 4H): methvlenes.
A 0.2 M solution of sodium methoxide in methanol (0.500 mL) was
added dropwise to a solution of compound 31 (4.00 ~, 7.1 mmol) in
dry methanol (30 mL) cooled at 0C. The mixture was stirred at 0C
tor 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 0C. 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 co~npound 32 ~2.36 9, 76%). 1H-n.m.r.
(CDCI3): 7.70 and 7.80 (m, 4H, aromatics), 5.82 (m, lH, -CH=), 5.17
(m, 3H, =CH2 and H-1), 1.38 and 1.10 (m, 6H, methylenes); 13C-
n.m.r. (CDCI3): 134.9 and 116.6 (ethvlenics), 98.3 ~C-1), 56.6 ~C-2).
C~ Synthesis of 5-Allyloxypentyl 4,6-0-ben~yUdene-2~eoxy-2-
phthalimido-~-D-glucopyranosids 33
Paratoluenesulfonic acid monohydrate (0.025 9) was added to a
solution of ~ (1.0 9, 2.3 mmol) and o,a-di-methoxytoluene ~0.690
mL, 4.6 mmol) in dry dimethylformamide. After stirring for 2 h at
40C, t.l.c. I10:1 - 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 evsporation of
the solvents, the residue was chromatographed on silica gel using a
9:1 mixture of toluene and ethvl acetate giving compound 33 (1.36 9,
90.1%). Ial200 ~24.1 (c 0.5 chloroform); lH-n.m.r. (CDCI3): 7.15-
7.90 (m, 9H, aromatics), 5.83 ~m, lH, -CH=), 5.56 ~s, lH,
WO92/22301 211 G ~ 9 ~ PCI`/CA92/00244
-48-
benzylidene), 5.10-5.37 Im, 3H, =CH2 and H-1 (5.25, d, J1.2 8.5Hz)l,
1.40 (m, 2H) and 1.17 (m, 4H): methybnes.
D. Synthe~b of 5-Allyloxypentyl 4,6-0-benzylidene-2-deoxy-
l2,3,4,6-tetra-O~acetyl-~-D-galactopyranosyl-
~1 -3)-0-1-2-phthalimldo-~-D-~lucopyranoside 35
A solution of trimethylsilyltrifluoromethanesulfonate (0.1 mL of
a solution made from 0.050 mL of the rea~ent in 1.0 mL of
dichloromethane) was syrin~ed into a mixture of compound 33 (1.20
~, 2.29 mmol), 2,3,4,6-tetra-0-acetyl-a-D-~alactopyranosyl
acetimidate 34 (1.70 ~, 3.50 mmoî) and molecular sieves (0.500 9,
crushed in a 1:1 mixture of toluene and dichloromethane (30 m~!
cooled to -20C. The mixture was stir-ed at -20C for 0.5 hours and
slowly broughtto 0C 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 ~el by usin~ 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 ~,
74%). la]20D +4.1 tc, 0.5, CHCI3); lH-n.m.r. (CDCI3): 7.40 -8.00 (m,
9H, aromatics), 5.85 (m, 1H, -CH=), 5.58 (s, lH, benzylidene), 5.07
- 5.25 ~m, 4H, incl. =CH2, H-4' and H-l), 5.00 ~dd, 1H, Jl..2. 8.0,
J2.3~1 O.OHz, H-2'), 2.11, 1.90, 1.85, 1.58 (4s, 1 2H, 4 OAc), 1.37
and 1.12 (m, 6H, methylenes); '3C-n.m.r. ~CDCI3): 134.6 and 117.0
(ethylenics), 102.1, 101.2, 99.4 ~benzylidene, C-1 and
C-1 ').
wO 92/2230l pcr/cAs2/oo244
211~95 ` 49
E. Synthesb of 5-Allyloxypentyl 2-deoxy-l2,3,4,6-~etra-0-
acetyl-~-D-~alactopyranosyl~ 3)-0-~-2-phthalimido-~-D-
~lucopyranoside 36
A solution of the disaccharide ;~ ~1.63 ~, 1.91 mmol) in 9096
aqueous acetic acid (10 mL) was heated at 70C for 1 h at which
time t.l.c. (100:5 - chloroform and methanol) indicated the compîetion
of the reaction. Co-evaporation with an excess of toluene left a
residue which was chromato~raphed on silica ~el usin~ a 100:2
mixture of chloroform and methanol as eluant ~ivin~ compound 36
(1.12 ~, 76%)~ lal20D +9.3 ~C, 0.55 CHCI~ H-n.m.r. (CDCI3): 7.70-
7.95 (m, 4H, aromatics), 5.82 (m, lH, -CH=), 5~33 (dd, lH, J3~4. 3~5,
J,.5. l.OHz, H-4'~, 5.10 - 5.27 (m, 3H, incl. =CH2 and H-2'~, 5~07 (d,
lH, J~.2 8.5Hz, H-l), 4.84 ~dd, lH, J2`.:~' lO.OHz, H-3'), 2.10, 2.08,
1.90 ~3s, 9H, 3 OAc), 1.05 -1.47 (m, 9H, incl. 1 OAc). l3C-n.m.r.
(CDCI3): 100.3 and 97.5 C-l and C-l'. An~/.calcd: C, 56.88; H,
6.17; N, 1.83. Found: C, 55.59; H, 6.20; N, 1.84.
F. Synthests of S-Allyloxypentyl 2--cetamldo-2-deoxy-
L~ D-~alactopyranosyl-~1-3)-O-I~-D-91ucopy-anoside~h :
Sodium borohydride (0.690 ~, 18 mmol) was added to tt~e
disaccharide ~à (0.700 ~, 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
waterl showed the disappearance of the startin~ material. After
addition of acetic acid ~8.2 mL) the mixture was heated for 3 hours at
100C. 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 22C. 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
WO 92/22301 2 1 10 ~ 9 ~ .
-50-
chromato~raphed on silica ~el usin~ a 100:2 mixture of chloroform
and methanol ~ivin~ the peracetylated disaccharide (0.500 ~, 71 %).
1H-n.m.r. ICDCI3): 5.90 (m, lH, -CH=), 5.77 ~d, 1H, J2.NH 7 5HZ~
NH), 5.37 (dd, lH, J3.4. 3.5, ~.~.1.0Hz, H-4'), 5.15 5.23 (m, 2H,
=CH2),1.95 - 2.18 (7s, 21H, 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 ~, 0.623 mmol) in dry
methanol (20 mL). After stirrin~ overni~ht 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 ~) by evaporation of the solvent. The Celite was
then applied on top of a column of latrobeads (30 ~) and the product
eluted with a 65:25:1 mixture of chloroform, methanol and water
~ivin~ the disaccharide 4c (0.266 9, 80%); tal20D -0.164 (c.1, water);
H-n.m.r. (D20): 5.95 (m, lH, -CH=), 5.30 (m, 2H, -C~2), 4.548 (d,
lH, J~.2 7.7Hz, H-1), 4.426 (d, lH, J1'.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; ~3C-n.m.r. (D20): 175.24 ~carbonyl),
134.70 and 119.05 (ethylenics), 104.33 ~C-1'),101.68 (C-1), 55.42
(C-2) .
Example 9 -- Synthasis of 5-Allyloxypentyl 2-acetamido-2-
deoxy-~-D-~lucopyranoside 37
The starting material 32 (0.300 9, 0.689 mmol~ was
deprotected as indicated previously for compound 36. The crude
material recovered after peracetylation was chromato~raphed on silica
~el using a 1 :1 mixture of hexane and ethyl acetate which gave the
peracetylated derivative (0.180 ~, 55%), Ia]20D ~11.5 (c, 0~7,
chloroform); lH-n.m.r. (CDCI3): 5.90 (m, lH, -C~=), 5.64 (d, lH,
J2N~ 8.5 Hz, NH), 4.68 (d, lH, J~.2 7.5Hz, H-1), 1.95, 2.03 Itwo),
WO 92/22301 ` ; PCr/CA92/00244
2110~9~ -~
-51 -
2.05 ~3s, 12H, 3 OAc, 1 NAcl, 1.58 ~m, 4H) and 1.41 ~m, 2H):
methylenes. Ana/.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
S a 0.5 N solution of sodium methoxide in methanol (0.100 mL) was
added. After overni~ht 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 ~ivin~ the pure 37 (0.103 ~, 80%), lal20D -
100.17 ~c.1, water); IH-n.m.r. (D20): 5.85 (m, lH, -CH=), 5.29 ~m, 2H,
-CH=), 4.50 (d, 1H, Jl.2 8.5Hz, H-1), 4.03 ~d, 2H, J 6.0Hz, allylics),
2.033 (s, 3H, NAc), 1.58 Im, 4H) and 1.36 ~m, 2H): methylenes; 13C-
n.m.r. ~D20): 175.2 ~carbonyl), 134.7 and 119.1 ~ethylenes), 101;9
tC-1), 61.6 (C-6), 56.4 (C-21, 29.1, 23.0 and 22.6 (methylenes).
15D. TRANS~:~OF SIAI~CIDS AND OTHER ~iUGARS TO
O~IGOSACCHARIDE STRUCTURES
Example 10 -- Transfer of Sialic Acids and other Su~ars to
Oligosaccharide Structures via Glycosyltransferases
This example demonstrates the en2ymatic transfer of Neu5Ac,
20 analogues thereof (collectively ~sialic acids~), and other sugars onto
oligosaccharide ~Iyooside structures via ~Iycosyltransferases. FlGs.
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
25 fucosylation were performed as follows:
i. PreDarativQ Sialvlation
Sialic acids, activated as their CMP-derivatives ~as set forth in
Examples 1-5 above), were transferred onto synthetic oligosaccharide ;
WO 92/22301 PCr/C~92/00244
21~a~
-52-
structures containin~ pGal(1 -3)~G1cNAc-, pt;al(1-4)pGlcNAc-, pGal(l -
3)oGalNAc-, and pGal(1-4)~G1c- terminal sequences by usin~ three
mammalian sialyl-transferases (Examples 10a-e). The
pGal(1-3/4)pG1cNAc-o(2-3)sialyltransferase (EC 2.4.99.5) and the
~Gal(1-4)pGlcNAc-a(2-6)sialyltransferase ~EC 2.4.99.1) from rat liver
were purified to homo~eneity by affinity chromato~raphv accordin~ to
the procedure of Mazid et al.~, which is incorporated herein by
reference on a matrix obtained bv covalently linkin~ the hapten
pGal(1-3)~GlcNAcO(CH2)8CO2H~ ~Chembiomed Ltd., Edmonton,
Canada) to activated Sepharose by methods known in the art. The
pGal(1-3~aGalNAc-o(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 m~) was Incubated with the selected CMP-sialic
acids (5-20 m~) in the presence of the appropriate sialyltransferase
(1~50 mU) and calf intestinal alkaline phosphatase ~Boehrin~er
Mannheim, Mannheim, Germany) as in the procedure of Unverza~t et
al.~ for 37C for 2448 hours in 50 mM sodium cacodvlate pH 6.5,
0.5% Triton CF-54, 1 m~/mL BSA (~sialyl transfer buffer~). For
example, the sialyloli~osaccharide 7-d-aNeuSAc~2-6)~Gal(1-
4)pGlcNAc-O-(CH2)~-COOCH3 ~13d, 4.4 mg) was synthesized by
incubation of pGal~1-4)~GlcNAc-~(CH2)~,-COOCH3 (~, 4.6 m~) and
CMP-7-d-Neu5Ac (~, 15.6 m~ in the presence of pGal(1-4)~G1cNAc-
at2 6)sialyltransferase ~51 mU) and calf intestinal alkaline phosphatase
(2.4 U) for 28 hours at 37C 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", cartrid~es,
conditioned as su~gested by the manufacturer. Each cartrid~e 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 chlorotorm, methanol
and water (0.5 mL - solvent 1) and applied on to a small column of
WO 92/22301 PCl`/CA92/00244
i 9 ~ ` 53
latrobeads (500 m~) 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 ll) and then by a 65:35:8
mixture of chloroform, methanol and water ~solvent llll. The
S appropriate fractions (30 drops) containin~ the produet, as identified
by t.l.c. on silica ~el plates (with a 65:35:8 mixture of chloroform,
methanol and 0.2% calcium chloride solution as eluent), were pooled
to~ether and concentrated to dryness in vacuo. The residue was run
throu~h 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.
;i. PreDarative Fucosylation
Sialylated analo~ues of the type I and ll oligosaccharides can be
further fucosylated by the human milk
pGlcNAco(1-3/4)fucosyltransferase. The enzyme was purified from
human milk accordin~ to the methodolo~y usin~ affinity
chromato~raphy on GDP-hexanolamine Sepharose described by Palcic
et al.6l The synthesis and purification of the fucosylated
oli~osaccharides was carried out by a modification of the procedures
of Palcic et al~5l For example, the fucosylated structure 9-N~-
aNeu5Ac~2-3),BGal( 1 -3~-lo-L-Fuc( 1 -4)1-~GlcNAc-O-(CH2)~-CH20H 1 7b
was synthesized by incubatin~ GDP-fucose (2~5 m~) and 9-N~-
oNeu5AC~2-31,BGal(1-3)~GlcNAc-0-(CH2)8-CH20H ~ (1~7 mg) with
affinity purified pGlcNAca(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
37C, 2.5 m~ of GDP-fucose and 2.3 mU of the fucosyltransferase
were added to the reaction mixture, which was kept at 37C 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)
WO 92/22301 PCI`/CA92/00244
21104~3~
-54-
indicated that the fucosylation was almost complete. After
purification and chromato~raphy on AG 50W x 8 (Na~ form), 'H-
n.m.r. of the product 17b ~1.0 m~) indicated a very ~ood purity (Table
5). In some cases where the fucosyltransferase was not hi~hly
5 purified, partial hydrolysis of the methyl ester of the linkin~ arm
occurred.
Examphs 10a-10e ue 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
10 possessing a pGal(1-3UGlcNAc- (LewisC or Type 1) terminal structure
such as 4a and 4b bV a sialyltransferase such as the pGal(l-
3/4)pGlcNAca(2-3)sialyltransferase from rat liver followin~ the
experimental procedure reported above. The 'H-n.m.r. data of the
reaction products, which were purified as indicated previously, are
15 reported (Tables 3 and 4).
Exampb 10b: This example refers to the transfer of modified
sialic acids such as lb and lc to the 3-OH of the terminal pGal of
acceptors possessing a ,aGal(1-4)pGlcNAc- (LacNAc or Type ll)
terminal structure such as ~ by a sialyltransferase such as
20 that used in 10a. In some cases, dimethylsulfoxide (S% volume) may
be added to solubilize the acceptor. The 'H-n.m.r. data of the reaction
products, which were purified as indicated previously, is reported
(Tables 6 and 8). Th~ reaction mixture was worked up in the manner
described previously.
Example 10c: This example refers to the transfer of modified `
sialic acids such as .1~ to the 3-OH structure of the terminal pGal of
acceptors possessing a p~;al(1-4)pG1c- (lactose) terminal structure
such as 6a by a sialyltransferase such as that used in Example 1 Oa
WO 92/22301 PCI'/CA92/00244
21104~ 55
followin~ the same experimental procedure. The 1H-n.m.r. data of the
react;on products, which were purified as indicated pr~viously, is
reported ~Table 6).
Example 10d: This example re~ers to the trans~er of modifled
5 sialic acids such as 1b - h to the 6-OH of the terminal ~Gal of
acceptors possessin~ a pGal~1-4)pG1cNAc- ~LacNAc or Type lll
terminal unit such as 5b, ~ ~ by a sialyltransferase such as the pGal(l-
4)~GlcNAco(2-6)sialyltransferase reported previously. The 'H-n.m~r.
data of the reaction products, which were purified as indicated
10 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 pGal of acceptors
; ~ ~ possessin~ a pGal~1-3)oGalNAc- (~T~) terminal unit such as 7a by a
sialyltransferase such as the PGal~1-3)oGalNAco(2-3)sialyltransferase
15 (Genzyme) followin~ the experimental procedure reported previously.
The lH-n.m.r. data of the reaction products, whkh were purified as
indicated previously, is reported (Table 9).
-56-
~- _ _ ~
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a 1~ ~ ~ ~ ~ ~ ~ ~3
1- .
S ~ _ ~ ~ ^ ~ ~ C ~ C ~ ~ ~ ~ ~ g
r S~ ~ =~ =
3 ~
~ ~ I 1 ~ ~ l ~ g ~- ~ ~
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I ~ ~
~ ~ 11 l e ~ 1 ~ ~ 1 ~
1~ ~ ~ ~ ;;i` ~ ~ ;;i` ~ ;;j ~ o
~; L ~ ~ !~! ~ ~ ~ ~ ~ ~ C ~ C ~ C ~ ~ '^ ~o
~ ~ ~o
~ ~ ~ ~ a ~ ~ ~1 ~ ~
1~ _ _---- a ~1
-57-
~ __ _ _
35 ~ ~; ~ ~;~ , ~ æ
_
:~ ~ ~ 0~ ~ .~.~ ~ :
~ ~ ~ ~ ~ ~ ~ _ ~ ~ . ~ ~ ~,
~ _
: ~ -- O _ ~ _
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~ ~ z ~ ~ 3~1
'~ ~ ~ _ _ I
o ~ X ~ ~,
~ ~ .~.~ ~ ~
7~ ~ ¦ n ~
-58-
1~
?~ ' ~;~ ~; 8 ~ _ li, 8 iii~
~ ___ _ ~^
~i g,l ~g ~' 3
~C~ JC~ F~R ac~ _~
E F~ a E
I '- e~ C ~i C ~. C 8 f 8 =
I ~ ~ ~
E E E E E
S ¦ 3 ~ ¦ E
~ ~ ~ ~, 6 _ , r ~ C
~ - ~ ~.~ ~
~ ~ l ~;- ~ ~ c
~ ~ ~ _ ~ 6 ~, 6 ~ ~ E
I 1 ~ -C
~1~! ~3 ~1 r~ ~3 3
il 31 ,, _----L~ .~
;",
-59-
3 3 ~i ~.e, ~ lc
It~
11 ~ I ~ I Z I Z I Z ~ '
~ ~1 ~ 3 ~ ~ ~ = ~ - E g
s~
~ ~ _ I
~ ~'~
.. ~k ~ -
-60-
_ U_; _ _ :
6 ~
I ~ I ~1 `D 8 1 ~ ~
~ o o .,o' ~ _ ~o o
3 ~ ~ o'o ~o g~ ~^~o ~ ~ -~
~ 6l5~
, 2i'q ~_
~L~ I~ ~
'aO ~ o6 ~6 ^ -6 -6 ~g6 ~-
~ ~ ~ v~ ~ 6 ~ ;;;` ~ ~ I ~
~ ~ ~ ~ r ~ o ~ ~ iO ~ O g ~ a Z
-61 -
6 1..3 1~3 1.,3
~ I ~: .~ ~ ~ ~,
a ~ O~ ~}z ¦ s ¦ ~ ~ o ¦
3 ~ O O
~ ~ T ~ ~; O
~Y~
T ~ i ~ ~ ~ _
Z ~3C~ T _ ~
~ Y I I I ¦ ¦ ~
u~
~ -62-
S
.''
WO 92/22301 PCI`/CA92/00244
211049~
-63-
E. PREPARATION OF ANALOGUES OF OLIGOSACCHARIDE
GLYCOSIDES BY CHEMICAL MQ~ CATION
OF THE COMPLET~
OLIGOSACCHARIDE GLYCOSIDE STRUCTURE
Examples 11-13 below describe the synthesis of analo~ues of
oli~osaccharide ~Iycosides by the chemical rnodification of the
completed oli~osaccharide ~Iycoside structure (prepared by either
enzymatic or chemical means). FlGs. 22-24 illustrate the reaction
schemes involved in the preparation of these analo~ues and provide
10 structures for the prepared analogues which are identified by an
underlined arabic numeral.
Example 11 -- Synthesis of 9-Hydroxynonyl 15-acetamido-3,5-
~ dideoxy-~-L-arabino-2-heptulopyranosylonicacid)-~2-3)
u -O-~-D-~alactopyranosyl-( 1 -3)-O-Io-L-fucoPyranosyl-( 1
4)-0-1-2-acetamido-2-deoxY-~-D-~lucopyranoside17m
The starting trisaccharide 8a (1.3 m~) was stirred for 24 hours
at +4C 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 ~Iycol. Sodium borohydride
(20 m~) was then added and the stirrin~ was continued for 24 hours
at 4C. The pH of the reaction mixture was then brou~ht 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:~5:5 mixture of
chloroform, methanol and water as eluant. The appropriate fractions
were pooled and evaporated leaving the product I~m ~1 m~); 'H-n.m.r.:
see Table 3 above.
Trisaccharide 8m was en~ymatically fucosylated following the
procedure reported in Example 10 and the product purified in the same
WO 92/22301 PCI'/CA92/00244
2 1 1 ~
-64-
manner. T.l.c. of the recovered crude material indicated that the ~-
transformation of ~m was almost complete. Purification gave l~m
(0.5 mg); 'H-n.m.r.: see Table 5 above.
Example 12 -- Synthesis of 9-Hydroxynonyl (5,9-dbcetamido-
3,5,9-tri-deoxy-~-D-~lycero-D-galacto-2-nonulo
-pyranosylonic acid)-12-3)-0-B-D-~alactopyranosyl~ 3)
-0-la-L-fucoPYranosYl-~ 1 4)-0-~-2-acetamido-2-deoxy-~
-D-glucopyranoside 17k
A solution of the trisaccharide 8b (1 m~) in water (0.5 rnL) was
hydrogenated at 22C at atmospheric pressure in the presence of
Lindlar catalyst ~1.0 mg, Aldrich Chemical Company, Milwaukee, Wl)
for 15 minutes T.l.c. (65:35:8 - chloroform, methanol and 0.2%
calcium chloride), indicated a complete transformation. The mixture
was filtered throu~h Celite and the solid extensively washed with
water. The ~iltrate was concentrated, filtered throu~h Millipore filter
and the eluate freeze dried leavin~ the trisaccharide ~; 'H-n.m.r.: see
Table 3 above.
Acetic anhydride (about 0.2 m~) in methanol (10 JuL) was added
to a solution of ~ ~about 1 mg) in a 1:1 solution of 0.002 N sodium
2û hydroxide and methanol ~0.300 mL) at 0C. 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 cartrid~e was washed with water and the product
eluted with methanol giving the trisaccharide ~ ~about 1 mg); 'H-
n.m.r.: see Table 3 above.
Trisaccharide ~k was enzymatically fucosylated followin~ the
procedure reported in Example 10 and the product purified in the same
manner. T.l.c. of the recov~red crude material indicated that the
transformation of 8k was almost complete. Purification gave 17k
~about 0.5 mg); lH-n.m.r.: see Table 5 above.
WO 92/22301 ~ . PCI`/C~92/00244
~110~ 65-
Example 13 -- Synthesis of 8-N-methylamidooctyl (5-acet
-amido~3,5-dideoxy~-D-glycero~galacto-2-nonulo
-pyranosylonic acid N-methybmide)-~2-3)-0-~-D
-~alactopyrano~l-~1-3)-0-la-L-fucop~rano~YI-~14)-0-~
-2-acetamido-2-deoxy-p-D-~lucopyranoside 181
Tetrasaccharide 18a (0.003 9) was applied on Dowex
50 x 8 lNa~ form) resin and eluted with water. The appropriate
fractions, were freeze-dried, followed by further dryin~ 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,8 cartrid~e. 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 9) using a 65:35:5 mixture of chloroform:
methanol:wate- providing the methyl ester of compound .1~ ~0.025
9): 'H-n.m.r.: 5.099 (d, lH, Jl.2 3.75Hz, H-1 oFUC), 4.517 ~d, 2H, J, 2
7.5Hz, H-1 ~Gal and pGlcNAc), 3.866 and 3.683 (2s, CO2C~3), 2.781
~dd, lH, J3,,~ ,." 12.5Hz, J3."4 4.5Hz, H-3eq Neu5Ac), 2.032 and
2.018 ~2s, 6H, 2 NAc), 1.913 (dd, lH, J3,"" 12.5Hz, H-3ax Neu5Ac),
1.160 (d, 3H, J5.~, 6.5Hz, H-6 aFuc).
This material was heated at 50C 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 providin~l (0.0025 9); lH-n.m.r.: (Table 5).
WO 92~22301 2 1 1 ~ '1 9 ~ ~/CA92/00244
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F. SYNTHESIS OF MONOFU~Q~QTED OL~ pEs
IE~MINATING IN D~ ~LLt~CTQSAM~
STRU~TURES
Examples 1419 below are presented for the purpose of
5 illustratin~ that analo~ues of blood ~roup determinants also posess
immuno~enic and tolero~enic properties. Specifically, the analogue of
the blood ~roup determinant employed is CD65, which is a Sialvl
Lewis X derivative havin~ a ~!K;al(1-4)pG1cNAc-OR disaccharide
~Iycoside attached to the reducin~ su~ar 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 Oli~osaccharides
Terminatin~ in Di-N-acetyllactosaminyl Structures,~ which is
incorporated herein by reference. As noted above, such a compound
is an analogue of blood ~roup determinants because Sialyl Lewis X is a
blood ~roup determinant, as defined herein.
In Examples 14 to 19 below, preparative sialylation was
conducted as follows:
The rat liverpGal~1-3/4)pGlcNAco~2-3)sialyltrans~rase was
purified by affinity chromato~raphy~ on a matrix obtained by
covalently linkin~ the h apten pGal~1-3)~GlcNAcO(CH2)"CO2H'3
(Chembiomed Ltd., Edmonton, Canada) to activated Sepharose by
methods known in the art. The pGal(1-4)-~gGlcNAc o(2-
6~sialyltransferase contained in the flow-throu~h of thc above affinity-
column, was further chromato~raphed on CDP-hexanolamine
Sepharose as reported.52
The enzymatic sialylations wer~ carried out at 37C in a plastic
tube using a sodium cacodylate buffer (50 mM, pH 6.5~ containing
Triton CF-54 (0.5%), BSA (1 m~/mL) and calf intestine alkaline
phosphatase.~ The final reaction mixtures were diluted with H20 and
appîied onto Cl" Sep-Pak cartrid~es as reported.5' After washing with
H~O, the products were eluted with CH30H and the solvents
evaporated. The residue was dissolved in a 65:35:5 mixture of CHCI~,
WO 92/22301 PCI`/CAg2/00244
2110495:
-67-
CH30H and H20 and applied on a small column of latrobeads (0.200
to 0.500 9). 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 It.l.c.) were pooled, the
5 solvents evaporated in vacuo, the residue run through a small column
of AG 50W X 8 ~Na~ form) in H20 and the products recovered after
freeze drying in vacuo. In all cases, the 8-methoxycarbonyloctyl
glycos~des were separated from the corresponding 8-carboxyoctyl
glycosides.
In Examples 14 to 19 below, preparative fucosylation was
conducted as follows:
The pGlcNAca~1-3/4)fucosyltranferase was purified from human
milk, as reported.5' The enzymatic reactions were carried out at
37C in a pk,stic tube using a sodium cacodylate buffer (100 mM, pH
6.5), MnCI2 110 mM), ATP (1.6 mM), NaN3 11.6 mM). The reaction
products were isolated and purified as indicated above.
GDP-fucose as emploved 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
20 incorporated herein by reference in its entirety. Specifically, GDP-
fucose was synthesized as follow:
A. Preparation of Bis(tetra-n butylammonium)
hvdrogen QhosDhate
Tetra-n-butylammonium hydroxide ~40% aq. w/w, about 1509)
25 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 l75g) was dried in vacuo and
30 stored ove~ phosphorus pentoxide under vacuum until used.
WO 92/22301 211 0 d~ 9 S PCI/CA92/00244
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B. ~reearation of ~-L-Fucoeyranosyl-l -eh~sehat~
A solution of bisltetra-n-butvlammonium) hydro~en phosphate
(58~, 127.8 mmol) in dry acetonitrile ~300 mL) was stirred at room
temperature under nitrogen in the presence of molecular sieves 14A,
5 209) for about one hour. A solution of tri-O-acetyl fucosyl-1-bromide
(freshly prepared from 319, 93 mmol of L-fucose tetraacetate in the
manner of Nunez et al.~6) in dry toluene (100 mL) was added dropwise
in about 0.5 hour to the above solution, cooled at 0C. After one
more hour at 0C, the mixture was brought to room temperature and
10 stirred for 3 hour. Tlc ~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 acetonitrib) and the solvents evaporated in vacuo to give
a red syrup. This material was dissolved in water (400 mL) and
15 extracted with ethyl acetate ~250 mL, twke). The aqueous laver was
then evaporated in vacuo leavin~ a yellowish syrup to which a solution
of ammonium hydroxide (2596 aq., 200 mL) was added. The mixture
was stirred at room temperature for 3 hours after which tlc (65:35:8
chloroform:meth-anol:water) indicated a baseline spot. The solvent
20 was evaporated in vacuo to ~ive 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 81200-400 mesh, S x 45 cm,
25 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)l. Watcr (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
30 collected in fractions (15 mL) and the product detected by charring
after spotting on a tlc plate. Fractions 20 to 57 were pooled and
WO 92/22301 PCI~/CA92/00244
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-69-
evaporated in vacuo leaving a white solid which was further co-
evaporated with water (3 x 300 mL~ and freeze dryin~ of the last 50
mL and then drying of the residue with a vacuum pump to ~ive p-L-
fucopyransyl-1-phosphate ~9.59, 4C)%) as a 12:1 mixture of ~ and o
5 anomers containin~ some ammonium acetate identified by a singlet at
~=1.940 in the 'H-n.m.r. spectrum. This product was slowly run
through a column of Dowex S X 8 resin ~100 200 mesh,
triethylammonium form) and eluted with water to provide the bis
triethylammonium salt of ~-L-fucopyransyl-1-phosphato as a sticky
gum after freeze drying of the eluate. 'H-n.m~r. ~:4.840 ~dd, J~.2 =
J1P = 7.5 HZ, H-1~, 3.82 (q, lH, J5." 6.5 Hz, H-5), 3.750 ~dd, lH, J3,
3.5, J,,5 1.0 Hz, H-4), 3.679 ~dd, lH, J2.3 10.0 Hz, H-3), 3.520 (dd,
1 H, H-2), 1 .940 ~S, acetate), 1 .26 (d, H-6). Integral of the signa~s at
3.20 (q, J 7.4 Hz, NCH2) and 1.280 and 1.260 (NCH2C!~3 and H-6)
15 indicates that the product is the bis-triethyl-ammonium salt which may
loose some triethylamine upon extensive dryin~. ~3C-n.m.r. ~S:98.3 (d,
Jc 1P 3-4 Hz, C-1), -72-8 (d, JC~2P 7.5 Hz, C-2), 16.4(C-6); 31P-nmr ~:
+ 2.6(s).
p-L-fucopyransyl-1-phosphate appears to slowly degrade upon
20 prolonged storage (1 + days) in water at 22C and, accordingly, the
material should not be left, handled or stored as an aqueous solution
at 22C or higher temperatures. In the present case, this material
was kept at -18C and dried in vacuo over phosphorus pentoxide prior
to being used in the next step.
C. Preparation of Guanosine 5'~ fucopy-
ranosyl)-diphosphate
Guanosine 5'-1B-1-fucopyranosyl)-diphosphate was prepared
from ~6-L-fucopyranosyl-1-phosphate using two different art recognized
procedures as set forth below:
WO 92/22301 PCl'/CA92/00244
2110~S5
-70-
PROCEDURE #1
,~-L-fucopyranosyl-l-phosphate and ~uanosine 5~mono-
phosphomorpholidate (4morpholine-N,N'-di-cyclohexyl-carboxamidlne
salt, available from Sigma, St. Louis, Missouri, ~GMP-morpholidate~)
5 were reacted as described in a recent modification~ of Nunez's
original procedure~S. Accordin~lv, tri-n-octylamine ~o.aoo9, available
from Aldr~ch Chemical Company, Milwaukee, Wisconsin) was added
to a mixture of p-L-fucopyranosyl-l-phosphate (triethyl-ammonium
salt, 1.009, about 2.20 mmol) in dry pyridine (10 mL) under nitro~en
10 the solvent removed in vacuo. The process was repeated three times
with care to allow only dry air to enter the flask~ GMP morpholidate
(2.4~, 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.
15 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 ~2~, 4A). The mixture was stirred at room
temperature under nitro~en. Tlc (3:5:2 25% aq. ammonium
hydroxide, isopropanol and water) showed spots corresponding to the
20 startin~ GMP-morpholidate (Rf-0.8, U.V.), ~uanosine 5'~
fucopyranosyl)-diphosphate (Rf--0.5, U.V. and charring), followed by
the tailin~ spot of the startin~ 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-
25 evaporated in vacuo with toluene and the yellowish residue furtherdried overnight at the vacuum pump leaving a thick residue (2.439).
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~ forml~ The product
30 eluted with water after the void volume. The fractions which were
active, both by U.V. and charring after spotting on a tlc plate, were
WO 92/22301 PCI`/CA92/00244
!
211049~ -71-
-
recovered and the solution treeze-dried overni~ht in vacuo providin~ a
crude material (1.969).
This residue was dissolved in water ~10 mL overall) and slowly
absorbed onto a column of hydrophobic C", silica ~el ~Wate-s, 2.5 x
30 cm) which had been conditioned by washin~ with water, methanol
and water (250 mL each). Water was then run throu~h the column
~0.4 mL/min) and the eluate collected in fractions (0.8 mL) which were
checked by tlc (3:5:2 25% aq. ammonium hydroxide, isopropanol and
water) . ~-L-fucopyranosyl-1 -phosphate, (Rf - 0.54, charring) was
eluted in fractions 29 to 45. A product showin~ 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 ~uanosine 5'~ 1-fucopyrariosyl)-
diphosphate (Rf-0.62), also showed a narrow U.V. active spot
lS (Rf-0.57). Fractions 59 to 86 were pooled and freeze-dried
overni~ht providing 0.3539 of material enriched in ~uanosine S'~
fucopyranosyl)-diphosphate. ~H-n.m.r. indicated that this material was
contaminated by a small amount of impurities ~ivin~ signals at ~S =
4.12andlS = 5.05.
Fractions 29 to 45 and 47 to 57 were separately pooled and
freeze-dried providin~ recovered ~-L-fuco-pyranosyl-l-phosphate
(0.2649 and 0.2239, 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 ('H-n.m.r.l. Generally, all the material
enriched in guanosine 5'-U~-1-fuco-pyranosyl)-diphosphate was
dissolved in a minimum amount of water and run on the same column
which had been regenerated by washin~ with large amounts of
methanol followed by water. The fractions containin~ the purified
guanosine S'-(p-l-fucopyranosyl)-diphosphate (tlc) were pooled and
freezed dried in vacuo leaving a white fluffy material ~187 mg, 16%)~
lH-n~m~r~ was identical to the previously reported data~
WO 92~22301 PCI`/CA92/00244
211C4~
-72-
PROCEDURE #2
~-L-fucopyranosyl-1-phosphate and guanosine 5'-
monophosphomorpholidate (~morpholine-N,N'-di-cyclohexyl-
carboxamidine salt -- ~GMP-morpholidate~) w~re reacted in dry
pyridine as indicated in the original procedure~6. Accordingly, the ~-L-
fucopyranosyl-1-phosphate ~triethyl-ammonium salt, 0.528~, 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 ~1.29, 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. Pyr;dine (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 tlc and worked up as indicated in
the first procedure to provide the GDP-fucose (120 m~, 16%).
Example 14 -- Preparation of 8-Methoxycarbonyloctyl ~5-
Acetamido-3,5-dldeoxy~-D-~lycero-D-galacto-2
-nonulopyranosylonic acid)-(2-6)-0-~D-~alactopyranosyl
-(1 4)-0-2-acetamido-2~eoxy-~lucopyranosyl-l 1-3)-0-~
-D-galactcpyrano~yl-~1 4)-0-2-acetamido-2-deoxy
-9lucopyranostde 1
~Gal( 1 -4),~GlcNAc~ 1 -3)~BGall 1 -4)~GlcNAc-OR ~compound
6.5 mg), CMP-Neu5Ac (17 m~ Gal~1-4)pGlcNAc al2-
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 ~ (3.0 mg).
WO 92/22301 PCI`/CA92/00244
2110495 73
Example 15 -- Preparation of 8-Methoxycarbonyloctyl (5-
Acetamido-3,5-dideoxy-a-D-~lycero-D-~alacto-2-
nonulopyranosylonic acid)-~2-6)-0-~-D-~abcto-pyranosyl
-11 4)-0-2-acetamido-2-deoxy-~lucopyranosyl-(1 -3)-0-~
-D-galactopyranosyl-( 1 4)-0-lo-L-fucopyrano~yl-( 1-3)-0
-12-acetamido-2-deoxY-~lucoPY-anosidel~ and the 8
-carboxyoctyl~ coside (63b)
Compound 62a (3.0 mg), GDP-fucose ~5 mg), pGlcNAc o(1-
3/4)fucosyltransferase (10 mU) were incubated for 68 hours in the
buffer ~1.3 mL). Isolation and purification provided~ ~1.2 m~) and
63b (0.5 mg).
Example 16 -- Preparation of 8-Methoxycarbonyloctyl
~-D-galactopyranosyl-(1 4)-0-2-acctamido-2-deoxy-~-D
-~lucopyranosyl-( 1 -3)-0-~-D-~alactopyrano~yl-( 14)-0
1 5 -la-L-fucoPYranosYl-~1-3)-o-l2-acetamido-2-deoxy-a-D
-glucopyranoside ~ and the 8-carboxyoctyl
~Iycoside IÇ~
Compounds ~ and .~k ~1.7 mg) were incubated with
Clos~ridium Perfringens neuraminidase immobilized on agarose (Sigma
Chemical Company, St. Louis, M0, 1 U) in a buffer of sodium
cacodylate (50 mM, pH 5.2, 2 mL) at 37C~ After 24 hours the
mixture was diluted with water (10 mL) and filtered through Amicon
PM-10 membrane~ The ~low-through and washings were Iyophilized
and the residue dissolved in water ~3 mL) and applied to two C,8
cartridges~ Each cartridge was washed with water (10 mL) prior to
elution with methanol ~20 mL)~ After evaporation of the solvent, the
residue was chromato~raphed on latrobcads (210 mg~ as indicated
above giving ~, 0.8 m~) 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~
WO 92/22301 PCI /CA92/00244
` 21113~5
-74-
Example 17 -- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5-dideoxy-a-D-~lycero-D-~alacto-2
-nonulopyranoaylonic acid)-~2-3)-O*D
-galactopyranosyl-11 4)-0-2-~cetamido-2-deoxy-~-D
-glucopyranosyl-( 1 -3)-0-B-D-galactopyrano~yl-( 1 ~)-0-lo
-L-fucopyranosyl-( 1 -3)-0-12-acetamtdo-2-deoxy-~B-D
-glucopyranoslde U~a) and the 8-carboxyoctyl
glycosid~ (~)
Compound Ç4~ (1.5 mg), CMP-Neu5Ac (8 m~ BGal~1-
3/4)~BGlcNAc a(2-8)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 m~).
Example 18 -- Preparation of 8-Methoxycarbonyloctyl (5-
Acetamido-3,5-dideoxy~r-D-~lycero-D-~alacto-2
-nonulopyranosylonic acid)-(2-3)-0-1~-D
-~alactopyranosyl-( 1 4)-0-2-acetamtdo-2~Qoxy-~B-D
-glucopyrano~yi-( 1 -3)-0-~-D-~alactopyrano~yl-( 14)-0-2
-acetamido-2-deoxy-glucopyranoside~
Compound 61a ~5 mg), CMP-Neu5Ac (15 mg), ~K;alt1-
3/4J~GlcNAc a~2-3)sialyltransferase ~46 mU~, and alkaline
phosphatase tl5 U) were incubateo in the sialylation buffer ~2,5 mL)
for 48 hours~ Isolation and purification of the product ~ave 66a (2~5
mg).
Exampls 19 -- Preparation of 8-Methoxycarbonyloctyl (5-
Acetamido-3,5-dideoxy-o-D-glycero-D-galacto-2
-nonulopyranosylonic acid)-(2-3)-0^~-D
-galactopyranosyl-( 1 4)-0-lo-L-fucopyranosyl-l 1-3)-0
-]2-acetamido-2-deoxy-glucopyranosyl-(1 -3)-0-~-D
-galactopyranosyl-~ 1 4)-0-la-L-fucopyranosyl-( 1-3)-0
-]2-acetamido-2-deoxy-glucopyranoside (67a)
Compound Ç~ (2.5 mg), GDP-fucose (8 mg) and the ~GIcNAc
a(1~3/4)fuoosyltransferase ~19 mU) were incubated in the enzymatic
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buffer (2.0 mL) for 48 h. Isolation and purification of the product ~ive
(1.7 m~).
'H-NMR data for the compounds prepared in Examples 1 to 6
above are set forth in the followin~ Table 10:
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Table 10
o
n ~ ~ ~t ~ o~ 0 O~ o ~ ~
C ~ C~ ~ ~ C~ C X
~ ~ C ~ C~ P ~ C ~ ~
~ ~ C .. 3 .~3`~ C U~ ~,
..
S ~ C ~ C~ . C v~ 3~ _ ~ ~ c ~ ~ ..
~:
:' ~; .~
:c ~ `8'`.-'~ o`~ o~ 2~o
.` c ~ . c ~ P ~ 3~ ` _
~ ;; ~ ~`
S ~, r " ~ , C ~ ~ C t~
~o y ~ y ~ o~
, 3 ~ r ~ ,.,
_~ S i
Z Z Z~U ~ ~ ~ l1Yi
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G. IMMUNOSUPPRESSIVE PROPERTIES OF BINDING-
INHIBITORY OLIGOSACCHARIDE GLYCOSI~ES
Examples 20-34 illustrate the immunosuppressive properties of
oligosaceharide ~Iyeosides related to blood ~roup determinants. In
5 these examples, the oligosaeeharide ~Iyeosides employed are
illustrated in FlGs. 12 and 13 whieh employ Roman numerals to
identify the strueture of these eompounds.
Example 20 -- Inhibition of DTH Inflammatory Response
DTH inflammatory responses were measured using the mouse
10 footpad swelling assay as deseribed by Smith and Ziola22. Briefly~
~roups of Balb/c miee were immunized with 10 ~9 of the L111 S-
Layer protein, a bacterial surfaee protein23 from C/ostridium
: thermohyd~osulfuricum L111-69 whieh has been shown to induee a
strong inflammatory DTH response. Seven days later, each group of
miee was footpad-ehalhn~ed with 10 ~u~ of L-111 S-Layer protein.
The resulting inflammatory footpad swelling was measured with a
Mitutoyo Engineerin~ mierometer 24 hours after ehallen~e.
To assess the effect of oli~osaceharide glycosides related.to
blood group determinants III-VII depicted in FIG. 12 on the
20 inflammatory DTH response, groups of mice received 100 ~ug of
Compounds III-VII, injeeted into the tail vein, 5 hours after ehallenge~
Control groups were left untreated or reeeived 100 ~uL of phosphate-
buffèred saline ~PBS). The results of this experiment are shown in
FIG. 1. Miee injeeted with oli~osaeeharide glyeoside 111 had only about
25 40% of the footpad swelling of eontrol miee. Mice injeeted with
oligosaceharide ~Iyeosides IV-VII (struetures related to Sialyl-Lewis X,
Compound lll of FIG. 12) had between 55 and 70% of the footpad
swelling of eontrol mice. As seen in FIG. 2, miee injected with
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saccharides Vlll 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 Suppr-~lon of the
DTH Inflammator~ Respon~
Six groups of mice were subjected to primary immunization and
challenge with L11 1-S-Layer protein as described under Example 20,
above. Five hours after challenge, ~roups were injected intravenously
with 100 ~uL solutions containing 10, 25, 50, 75, or 100 ~ug of
oligosaccharide glycoside related to blood group determinant Iîl
depicted in FIG. 12, or with PBS. The DTH responses for each dose
group were measured 24 hours after challen~e and are shown in FIG.
2. While the groups receiving PBS or 10 ~ug of oli~osaccharide
glycoside lll showed essentially the same extent of footpad swelling
as PBS-treated controls, the ~roups receivin~ 25, 50, 75 or 100 ~ of
oligosaccharide glycoside îlî displa~ved reduced footpad swelling ~78,
69, 75, and 56% of the PBS controls, respectiv01y).
Example 22 -- Lack of Suppression of the Antibody Response to the
L11 1-S-Layer Protein
Secondary antibody responses to the L11 1-S-Layer protein were
measured two weeks after primary immunization lone week after
challenge~ in the sera from ~roups of mice immunized, challenged, and
treated intravenously with oligosaccharide ~Iycosides Ill-yll as
described in Example 20.
Antibody titers were determined using a solid phase enzyme
immunoassay (EIA) as described by Ziola et al~. Brieflv, 2 ~ug of
L11 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 ~ul of various dilutions of
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mouse serum prepared in phosphate-buffered saline eontaining 2%
(w/v) bovine serum albumin and 2% (v/v) Tween 20. After ~ hour at
room temperature, the solutions were rernoved by inverting the wells,
and the wells washed four tirnes with distilled, de-ionized water at
roomtemperature. Horse-radlsh peroxidase-eonjugated, goat anti-
mouse immuno-globulin antibodies were then added to eaeh well (200
~l of a 1:2000 dilution prepared in the phosphate-buffered
saline/albumin/Tween 20 solution). Aner 1 hour at room temperature,
the wells were again inverted and washed, and eaeh well reeeived
200 /ul of enzyme substrate solution (3 mg per ml o-phenylene-diamine
and 0.02% (v/v) hydrogen peroxlde, freshly dissolved in 0.1 M sodium
eitrate/ phosphate buffer, pH 5.5). After the enzyme reaction had
proceeded for 30 minutes in the dark at room temperature, 50 ~l of
2N hydroehloric acid was added to ea(:h well and the OD,90 values
were measured.
FIG. 3 graphically illustrates the tlters determined with six
dilution series of sera from the L11 1-immunked and ehallen~ed miee
which were treated with oligosaeeharide glycosides Iîî-VII depieted in
FIG. 12 and examined for footpad swellin~ as described in Example 20
above. The dilution eurves shown in FIG. 3 indicate that the
development of antibodies a~ainst the L111 S-Layer protein has not
been inhibited or otherwise affeeted by the treatments with
oli~osaceharide glycosides lll-~.
Example 23 -- Time of Admintstration of Compound lll R~latlve to
Challenge with Antlgon
Groups of Balb/c miee, immuni2ed and ehallenged with L111 S-
Layer protein as described in Example 20, were injected with a
solution of 100 ~ug of oligosaceharide glycoside related to blood group
determinant Lll, depicted in FIG. 12, in PBS (100 /uL) at different time
30 points relative to the time of antigen challenge~ One group received
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oligosaccharide ~Iycoside 111 one hour prior to the anti~en challen~e;
another, immediately after challenge, the third group one hour after
challen~e, and the fourth ~roup 5 hours after ehallenge. A control
~roup was included whieh received PBS ~ L) immedlately after
5 ehallen~e.
The results of this experiment are shown in FIG. 4. The DTH
responses were not suppressed in those mice which had received
oligosaccharide ~Iycoside 111 one hour before or immediately after the
antigen challenge. Those ~roups which had receivcd oligosaccharide
10 ~Iycoside îlî one or five hours after ehallenge showed only 68 or 59%
of the footpad swellin~ 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
15 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
~Iycoside related to blood ~roup determinant as opposed to the level
of inflammation exhibited by the control animals.
20 Example 24 -- Persistence of Suppression of the DTH Inflammatory
Response at 6, 8, or 10 Weeks After Challenge
i. The identical ~roups of mice treated with oli~osacçharide
glycosides related to blood group determinants III-VII in Example 20
were re-challenged with L111 S-Layer protein 8 weeks after primary
25 immunization. Untreated controls responded with the usual de~ree of
footpad swelling whereas all other ~roups showed reduced footpad
swelling, as follows: Oligosaccharide ~Iycoside îll, 59%;
oligosaccharide glycoside IV, 69%; oligosaccharide glycoside V,
78%; oligosaccharide ~Iycoside Vl, 78%; oli~osaccharide glycosdfide
30 Vll, 69,6. The anti-inflammatory effect of oligosaccharide glycosides
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111, y, Yl, or Vll, ~iven 5 hours after the first challen~e ~one week after ~ `
primary immunization), had somewhat weakencd eioht weeks after
primary immunization; however, the effect of oli~o-saccharide
~Iycoside IV ~the only derivative not containin~ a sialyl ~roup) was
equally as stron~ at the timo of re-challen~e as at the tirne of first
challen~e.
In addition to providin~ suppression of cell-mediated immune
responses, the above data demonstrate that treatment with an
oli~osaccharide ~Iycoside as per this invention also imparts tolerance
to additional challen~es from the same anti~en.
ii. The identical ~roups of mice treated in Example 21 with
Sialyl-Lewis X, Compound lll, ~10 JU~, 25 ~u~, 50 ~, 75 ~, 100 ~)
or with the o or ~-Sialyl ~Iycosides of 8-methoxycarbonyloctanol
~monosaccharides ~!!11, !~ of FIG. 13; 100 ~), or with 100 ~ of the
8-methoxycarbonyloctyl ~Iycoside of T-disaccharide (disaccharide X of
FIG. 13), were rechallen~ed six weeks after primary immunization.
Footpad swellin~ similar to that of PBS-treated controls was observed
with those mice that had been treated with saccharides ~111 and IX,
and disaccharide ;!~, S hours after the first challen~e. Mice ori~inally
treated with 10-100 /ug of lll showed footpad swellin~ that ran~ed
from 90 to 65% of that displayed 24 hours after the first challen~e.
iii. The identical ~roups of mice which had been treated in
Example 22 with 100 ~ug of oli~osaccharide ~Iycoside lll at 1 hour
before first challen~e, or 5 hours after first challen~e, were re-
challenged with anti~en 10 weeks after primary immunizatiom Within
experimental error, footpad swellin~ of those mice treated before or
shortly after challen~e was the same as that of P3S-treated mice,
whereas those mice originally treated 1 hour or 5 hours after challenge
showed onîy about 66% of the values observed for PBS-treated
controls.
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The results of this example are set fo-th in FlGs. 5-7 which
demonstrate that oli~osaccharide ~Iycosides related to blood group
determinants impart tolerance to challenges with the same anti~en for
at least 10 weeks after treatment.
5 Example 25 -- Effect Cyclopho-phamlde Treatment has on the
Suppression Induced by 8-methoxy~ bonyloctyl
~Iycostde of Compound lll
It has been demonstrated in the literature that suppressor cells
can be removed by treatment of mice with cyclophosphamide (CP).
10 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 lll.
Specifically, this example employs immunized mice which have
been previously suppressed and tolerized to DTH inflammatory
15 responses by treatment with the 8-methoxycarbonyloctyl ~Iycoside of
Compound lll in a manner similar to that described above. Fourteen
days after immunization, the mice were injected with 200 m~/kg of
CP and then 17 days afte- immunization, the mtce were challenged
with 20 ~g of L111 S-Layer protein. 24 hours after the challen~e, the
20 extent of the DTH response was ascertained by measurin~ (mm-') the
increase in footpad swellin~.
The results of this experiment are set forth in FIG. 8 which
illustrates that injection with CP prior to challenge with the L111 S-
Layer protein restores the DTH inflammatory response in mice that
25 have previously under-~one immunosuppressive treatment with the 8-
methoxycarbonyloctyl ~Iycoside of Compound îll~ These results
sug~est that tolerance induced by the 8-methoxycarbonyloctyl
glycoside of Compound lll FIG~ 3 is mediated by CP sensitive
suppressor T-cells~
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Example 26 -- Effect the Antigen Drivin~ the DTH ~nfbmm~to-y
R~sponse ha~ on the Suppressive Effect Induced by the
8-Methoxycarbonyloctyl Glycoside of Compound lll
This example assesses the effect that the anti~en drivin~ the -~
5 DTH inflammatory response has on the suppressive effect induced by
Compound lll. 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 anti~en used to induce the intlammatory
10 response does not appear to affect the ability of Compound lll to
regulate this response.
Example 27 - Effect Synthetic Compound lll has on ELAM-1
Dependent Cell Adhesion to Activated
Vascuhr Endothelium
This example examines whether the synthetic blood group
determinant related to oli~osaccharide ~Iycoside lll could inhibit ELAM-
1 dependent cell adhesion to activated vascular endothelium.
Specifically, an in ~ ro cell binding assay was preformed, as described
by Lowe et al18. Briefly, human umbilical vein endothelial cells .
20 (HUVECs purchased from Cell Systems, Seattle, WA) were stimulated
with TNFa ~10 n~/ml) to sxpress 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 lll has on the ELAM-1 depend~nt bindin~ to the HUVEC.
25 FIG. 10 sets forth the results of this example illustrates that
Compound lll inhibits ELAM-1 dependent binding to the HUVECs.
The data in Examples 20-26 above establish the effectiveness
of oligosaccharide ~Iycosides related to blood ~roup determinants in
treating immune responses to an anti~en and in inducing tolerance to
30 the antigen in a mammal (mice). In view of the fact that the immune
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system of mice is a ~ood model for the human immune system, such
oligosaccharide glycosides will also be effective in treating human
immune responses. This is borne out by the tact that Example 27
establishes that an oligosaccharide ~Iycoside related to blood ~roup
determinant inhibits ELAM-1 dependent binding to the HUVEC.
Examph 28 -- Effect of T~ming of Admhistration 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
u~/mouse inactivated Herpes Simplex Virus
Type I (HSV) and then challenged seven
days later with 20 ~ug HSV.
2) The second group was immunized with 20
~ug/mouse HSV and then challenged seven
days later with 20 ~ug/mouse HSV and then
100 /lglmouse of SleX ~Sialyl Lewis X,
compound 111) was injected intraveneously
five hours after challenge.
3) The third group was immunized with 20
Jug/mouse HSV and 100 ~ug/mouse SleX in
100 ~ul PBS intramusclularly at the same site.
Seven days later, the mice were footpad
challenged with 20 ~ug/mouse of HSV alone.
4) The fourth group of mice was immunized
with 100 ~ul PBS and then seven days later
challenged with 20 ~ug/mouse HSV. This
provides a measure of the background level
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2110 4 9 5 of footpad swellin~ resultin~ from the
- physical injury caused to the footpad durin~
the anti~en challen~e.
The extent of the DTH infbmmatory response was measured 24
5 hours after challen~e by measurin~ footpad swellin~ with a Mitutoyo
En~ineerin~ micrometer.
FIG. 27 shows the de~ree of footpad swellin~ observed.
Percenta~e reduction was calculated by the followin~ equation:
100 - 100 XrSwellinQ of ~Treated~ ce~ - backoround swellingl -
10L Swellin~ of ~l~ntreated Mice~ - back~round swellin~
~ Treated mice~ are those mice which receive compound in
addition to the anti~en. ~Untreated Mice~ are those mice which do
not receive compound. Back~round swélîin~ is that îevel of swellin~
observed in mice immunized with PBS alone without anti~en or
15 compound and chalhn~ed with anti~en.
Mice injected with SleX at the same time as and site of
immunization with HSV showed a 88% reduction in footpad swellin~
compared to that of mice immunized with HSV and challen~ed with
HSV. Mice injected with SleX 5 hours after the footpad challenge
20 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 oli~osaccharide ~Iycoside 111 can suppress an immune
25 response to an anti~en if ~iven to mice 5 hours after challenge by the
anti~en. This example also shows that oli~osaccharide ~Iycoside 111
given to mice at the time of immunization can inhibit sensitization of
the immune sVstem to the anti~en. Without bein~ limited to any
;~theorv it is contemplated that SleX (compound 111) interferes with the
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ability o~ T helper cells to reco~nize anti~en-presentin~ cells and
inhibits the immune system from becoming educated about the
anti~en.
Example 29 -- Effect of Slalyl LeX on the An~body Response to HSV
Four ~roups of mice were treated as described in Example 28.
Secondary antibody responses to the HSV anti~en were measured 2
weeks after primary immunkation (1 week after challenge) in the sera
from ~roups of mice described in Example 28.
Antibody titres were determined as described in Example 22
except HSV anti~en was used In place of L111 S-Layer protein.
FIG. 28 graphically illustrates the titres determined with six
dilution series of sera from the ~roups of immunized mice as described
in Example 28. The results of the ~irst two groups correlated with the
results obtained in Example 22 for the L111 S-Layer Protein.
Treatment of mice with SleX five hours after challen~e did not affect
the antibody response. However, mice treated with SleX at the time
of immunization showed si~nificant reduction in the antibody
response to the HSV antigen. Without bein~ limited to any theory, it
is contemplated that SleX (compound lll) interferes with the T ~elper
ZO cells that are involved in the antibody response and inhibits the
immune system from becomin~ educated about the anti~en.
Examp!e 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 11 S-Layer antigen. One group of Balb/c mice were
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immunized with 20 ~ug/mouse of the L111 S-Layer protein. Seven
days later, this group of mice was footpad-challen~ed with 20 ~ug of
L111 S-Layer prot~in. The second group ot mice were immunized
with 20 Jug of the L111 protein and 100 ~ug of SleX at the same site
5 and seven days later were footpad challen~ed with 20 ~ of L111.
The third ~roup of mice were injected with 200 m~/kg of CP
interperitoneally two davs before immunization. This group was then
immunized and challen~ed as described for group two. The fourth
group of mice were immunized with 20 ~ug/mouse of L111 and 100
10 /ug/mouse of the T-disaccharide at the same site and footpad
challenged as described for group one. Group five was immunized
100 /ul of PBS and then footpad challlenged as described for group
one.
The results are presented in FIG. 29. These results confirm the
15 results discussed in Example 28 that treatment of mice with SleX at
the time of immunization can suppr~ss the immune response to an
antigen. Furthermore, this Example shows that the suppression of the
immune response by treatment with ShX at the time of immunization
can be eliminated by cyclophosphamide treatment before immunization
20 suggesting the involvement of cyclophosphamide sensitive suppressor
T-cells.
Example 31 -- Effect of Sites of Adminlst-ation of Compound After
Footpad Chalbnge on Inhibition of DTH Intlammatory
Response induced by OVA
Groups of Balb/c f~maie mice, a~e 8 -12 weeks, weight about
20-25 mg, were immunized with 100 JUg of OVA (Albumin, Chicken
Egg, Sigma, St. Louis, MO) and 20 ~ug of DDA
(Dimethydioctacylammonium Bromide, Eastman Kodak, Rochester,
NY) in 100 ~l of PBS (Phosphate Buffered Saline) intra-muscularly into
the hind leg muscle of the mouse.
:
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Seven days after immunization, each group of mice was
footpad-challen~ed with 20 /ug of OVA in 20 ~ul of PBS. The resulting
inflammatory footpad swellin~ was measured with a Mitutoya
Engineering micrometer 24 hours after challen~e.
To assess the effect of methods of administration of SleX on
the suppression of the inflamrnatory DTH rosponse, compound lll
~SIeX) was administered by different routes. Certain groups of mice
received, five hours after footpad-challenge, either SleX 100 /ug/mouse
in 200 /ul of PBS intravenously or Sle~ 100 Jug/mouse in 20 ~ul of PBS
intranasally at which procedure the mice were under light anathesia.
The method of administering compound intranasally is described
in Smith et al.63 which is incorporated by reference. Briefly, mice are
anethesitized with Metofane (Pitman-Moore Ltd., Mississauga,
Ontario, Canada) and a 50 ~I drop of compound is placed on the nares
of the mouse and is inhaled. Control groups were left untreated or
received 200 ~I PBS intravenously or 50l11 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 Suppresston of the OVA Induced DTH
Inflammatory Response
A group of Balb/c female mice, age 8 12 weeks, weight about
20-25 9, were immunized with 100 ~9 of OVA (Albumin Chicken Egg,
Sigma) and 20 ~9 of DDA in 100 ~l of PBS, intramuscularly into the
hind leg muscle of the mouse. Seven days after immunization, the
mice were footpad challenged with 20 /ug of OVA in 20 ~l of PBS~ At
5, 7 or 10 hours after footpad challenge, the mice were either given
intravenously 100 ~ug/mouse of SleX in 200 L~l of PBS or 200 ~ul PBS
only or given intranasally 100 /uglmouse of SleX in 50 ~ul of PBS or 50
~I PBS only at which procedure the mice were under light anesthesia~
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The footpad swellin~ was measured 24 hours later with a Mitutoyo
En~ineerin~ micrometer.
FIG. 31 shows the results of this experiment. SleX ~iven at 7
hours (both intranasally and intravcnously) after the OVA challen~e
5 showed 70 - 74% reduction in footpad swellin~ 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 swellin~ respectively.
SleX given intervenously or intranasally at 10 hours after footpad
10 challenge showed 58% and 32% reduction in swellin~ respectively.
Example 33 -- Effect of Different Sites of Administration of the
Oli~osacch~ride Glycoside at Immunization on
Suppression
Groups of Balb/c female mice were immunized with OVA 100
Lg/mouse and DDA 20 ~u~/mouse in 100 Jll of PBS intramuscularly into
the hind le~ muscle, while 100 ~u~/mouse of SI~X was simultaneously
administered intramuscularly, intranasally or intravenousl-~. Seven day
later the mice were footpad challen~ed with 20 /u~lmouse of OVA in
~0 Jul of PBS. The footpad swellin~ was measured 24 hours later with
20 a Mitutoyo Engineerin~ micrometer.
FIG. 32 shows that administerin~ SleX to the mice at the time
of immunization produces the same level of suppression of the DTH
inflammatory response re~ardless of the method by which the SleX is
` administered. This suggests that the SleX compound can be
25 administered in the treatment of patients by other methods in addition
to that of intravenous injection.
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Example 34 -- Eff0ct of Oligosaccharide Glycosid0s on LPS Caus0d
Lun~ Injury
LPS (lipopolysaccharide) caused lun~ injury is measured by
weighing the lungs of sacrific~d mice 24 hours after mice are given
5 LPS intranasally. Brieflv, groups of 8-10 week old Balb/c mice were
sensitized with 5 ~ /mouse ot LPS in 50 ~ul of PBS intranasally under
light anesthesia. Five hours later, 10 ~g/mouse of SLeX, C19-9, T-
disaccharide ~#27) in 200 ~l of PBS are ~iven to the mouse
intravenously. After 24 hours, the mice are sacrificed and the lungs
10 removed and wei~hed.
The results in FIG. 33 indicate that SleA (compound Vll) and
SleX (compound lll) 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 reducin~ inflamation in lun~s
15 exposed to antigen, for example Acute Respiratory Distress Syndrome.
Example 35 -- Effect of Different Amounts of SbX and SleA on
Lymphoproliferative Response
A group of Balb/c female mice were immunized with 20
~ug/mouse of MUMPS (inactivated Mumps virus) and DDA by injection
20 into the hind leg muscle of the mouse. Seven days later, the mice
were sacrificed and draining Iymph nodes and speen were collected
and Iymphocytes isolated. The Iymphocytes were cultured with
varying concentrations of Mumps virus and varying concentrations of
compound in RPMI 1640 medium ~Gibco, Burlin~ton, Ontario, Canada)
25 supplemented with 5% Hybermix ~Si~ma, St. Louis, MO) at a density
of 2x105 cells per well for three days at 37 C in 5% CO2. After three
days the cells were pulsed for 6 hours with 1.0 /u curies of ~H -
thymidine per well (Amersham Canada Ltd., Oakville, Ontario,
Canadal. The cells were harvested using a PhD cell harvester and
30 cpm determined in a Beckman 3000 scintillation counter. It has been
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shown previously that there is a direct correlatlon between the ability
of Iymphocytes to respond to an antigen and the amount of ~H-
thymidine incorporation~6.
Lymphoproliferative responses are used in this example to
S measure T-helper cell responses in vltro. FIG. 34 demonstrat~s that
SleX and SleA (C19-9) are able to inhibit an antigen specific
Iymphoproliferative response since the uptake of ~H-thymidine is
reduced relative to the untreated Iymphocytes from mice exposed to
the antigen. Without being limited to any theory, it is thought that
10 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 JUg of the L1 11 S-Layer protein.
Seven days later, each group of mice was footpad-challenged with 10
~Jg of L-111 S-Layer protein. The resulting inflammatory footpad
20 swelling was measured with a Mitutoyo Engineering micrometer 24
hours after challenge.
To assess the effect of hexasaccharide ~Iycoside~
synthesized in Example 17, on the inflammatory DTH response,
groups of mice received 100 JUg of this compound, injected into the
25 tail vein, 5 hours after challen~e. Control groups received 100 ~L of
phosphate-buffered saline (P8S). 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
`~ ~ 30 the degree of footpad swelling in response to an antigen.
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TABLE 1 1
l:OMPOSITION TESTED INCREASE IN FOOTPAD SWELLING (mm
Control 3,3
Hexasaccharide Glycoside li~ 1.5
The above results indicate that mice injected with
hexasaccharide glycoside ~ 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 Chalîenge
i. The identical groups of mice treated with hexasaccharide
~Iycoside 65a in Example 7 were re-challenged with L111 S-Layer
protein 11 weeks aner primary immunization. Mice treated with the
PBS control responded with the usuaî degree of footpad swelling
whereas mice treated with hexasaccharide ~îycoside ~ showed a
reduction in footpad swelling of about 40%, i.e., the mke treated
with hexasaccharide glycoside ~ exhibited only about 60% of the
footpad swelling exhibited in mice treated with PBS.
This anti-inflammatory effect of hexasaccharide glycosides 65a,
~iven 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.
în addition to providin~ 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
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the anti~en in a mammal ~mice). In view of the fact that the immune
system of mice is a ~ood model for the human immune system, such
hexasaccharide ~Iycosides will also be effective In tre~tin~ human
immune responses.
By followin~ the procedures set forth in the above oxamples,
other oli~osaccharide ~Iycosides related to blood ~roup determinants
could be used to suppress a cell-mediated immune response to an :
anti~en by mere substitution for the oli~osaccharide ~Iycosides ::
described in these examples.
