Note: Descriptions are shown in the official language in which they were submitted.
~O 93/24505 2 1 1 ~ ~ 2 ~ PC~/US93/04909
-- 1 --
REDUCING INFLAMMATION BY IIME DEPENDENT
ADMINISTRATION OF OLIGOSACCHARIDES GLYCOSIDES
RELATED TO BLOOD
GROUP DI~R~ANT~
BACXGROUND OF THE INVENTION
1. Field of_the Inventio~.
This invention is directed to methods for
reducing the de~ree of inflammation arising from a
secondary immune response in a mammal due to antigen
5 ~xposure ~challenge) by the time dependent
administration of an oligosaccharide glycoside
related to blood group determinants having a type I
~Gal(1-3)GlcNAc] or a type II ~Gal(1~4)GlcNAc]
core structure.
The methods of this invention are directed
to the discovery that the reduction in anti~en
induced inflammation in sensitized mammals by
administration of oligosaccharide glycosides related
to blood group determinants having a type I or a
type II core structur~ is critically dependent on
the point in time when that oligosaccharide
glycoside is administered.
20. 2. Re~erence~.
The following publications and patent
applications are cited in this application as
superscript num~ers at the relevant portion of the
application:
1. Gaeta, et al., U.S. Patent Application
Serial No. 07/538,853, filed 15 June 1990.
2. Paulson, et al., U.S. Patent Application
Serial No. 07/619,319, filed 28 November
1990.
2113.~2 ~
WO 93J2450~ PCr/US93/0490
----2 ----
3. Paulson, et al., U.S. Patent Application
Serial No. 07/632,390, filed 21 Decem~er
1990. ...
4. Brandley, et al., PCT International Patent
Application No. PCT~US91/05416; published
20 February 1992.
5. Lowe, PCT International Patent Application
No. PCT/US91/07678; published 14 May 1992.
6. McEver, PCT International Patent
hpplication No. PCT/US91/05059, published
6 February 1992.
7. Furie, et al., PCT International Patent
Application No. PCT/US92/C1915, published
l October 1992.
8. Seed, et al., PCT International Patent
Application No. PCT/USgl/08685, published
11 June 1992.
9. Venot, et al., U.S. Patent Application
Serial No. 07/771,007, filed 2 October
1991.
10. Kashem, et al., U.S. Patent Application
Serial No. 07/914,172, filed 14 July 1992.
11. Venot, et al., U.S. Patent Application
- Serial No. 07/887,747~ filed 22 May 1992.
12. Ratcliffe, et al., U.S. Patent No.
5,079,353, issued 7 January 1992.
13. Ippolito, et al., U.S. Patent Application
Serial No. 07/895,930, filed 9 June 1992.
14. Verlot, et al., U.S. Patent Application
Serial No. 07/887,746, filed 22 May 1992.
15. Ekborg, et al., Carbohydr~ Res., 110:55-67
(1982~.
16. Dahmen, et al., Car~ohydr. Res., 118:292-
301 (1983).
17. Rana, et al., Carbohydr. Res., 91:149-157
5û (1981).
18. Amvam-Zollo, et al., Carbohydr. Res.,
150:199-212 (1986).
WO 93/24505 2 ~ 2 2 PCI/US93/U4909
13. Paulsen, et al., CarboAydr. Res., 104:195-
219 (1982).
20. Chernyak, et al., Carbohydr. Res.,
128:269_282 (1984).
21. Fernandez-Santana, et al., J. Carbohydr.
Chem., 8:531-537 (1989).
22. Lee, et al., Carbohydr. Res., 37:193 et
seq. ~1974).
23. Ratcliffe, et al., U.S. Serial No.
07/278,106, filed November 30j 1988.
24. Reuter, et al., Glycoconjugate J., 5:133-
13S (1988). ~-
2S. Palcic, et al., Carbohydr. Res., 190:1-11 -
(198~).
26. Prieels, et al., J. Biol. Chem.,
256:1045~-10~633 (1981).
27. Eppenberger-Castori, et al., Glycoconj.
J., 6:101-114 (lg89~.
28. Lemieux, et al., Can. J. Chem., 60: 63-67
(1982).
-
29. Nicolaou, et al., J. Amer. Chem. Soc.,
112:3693-3695 (1990). --
30. Hindsgaul, et al., Carbohydr . Res .,
109:109-142 (1982).
31. Okamoto, et al., Tetrahedron, 46, No. 17,
pp. 5835-5&37 (lg90).
32. Abbas, et al. & Proc. Japanese-German Symp.
Berlin, pp. 20 21 (1988).
33. Paulsen, Agnew. Chem. Int. Ed . Eng.,
21:155-173 (1~82).
34. Schmidt, Agnew. Chem . Int. Ed. Eng .,
25:212-235 (1986).
35. Fugedi, et al., Glycoconj. J., 4: 97-108 -
(1987).
36. Kameyama, et al ., Carbohydr. Res ., 209:C~-
C4 (1991)-
2 ~ 2 ~
W093/24505 PCT~U~93/0490
37. Ratcliffe, et al., U.S. Patent ApplicationSerial No. 07/278,106 filed November 30,
1988.
38. Matta, et al., Carbohydro. Res., 208:51-58
(19~0~.
3g. Norberg, et al., Carbohydr. Res., 183:71
et seq. (1988)
40. Richardson, et al., Carbohydr. Res.,
216:271-287 (1991)
41. Kukowskaa-Latallo, et al., Genes and
Development, 4:1288-1303 (1990).
42. Jiang, et al., U.S. Patent Application
Serial No. 07/848,223, filed March 9,
1992.
43. Inazu, et al., Bull. Soc. Chim., Jap.,
611:4467 (1988).
44. Bernotas, et al., Biochem. J., 270:539-540
(1990).
45. Wollenberg, et al., U.S. Patent No.
4,612,132, issued September 21, 1986.
46. Greig, et al., J. Chem. Soc., p. 879
(1961).
47. Piekarska-Bartowzewicz, et al., Carbohydr.
Res., 203:302-307 (1990).
48. Petitou, et al., Caxbohydr. Res., 147:221-
236 (1986).
49. Trumtez, et al., Carbohydr. Res., 19l:2
52 (1989).
50. Lemieux, et al., J. Amer. Chem. Soc.,
97:4076-4083 (1975).
51. Bodanszky, et al., The Practice of Peptide
Synthesis, Springer-Verlag (1984).
52. Higa, et al., J. Biol. Chem., 260:~838-
8849 (1985).
53. 8rossmer, et al., Biochem. Biophys.
Research Commun., 96:1282-1289 (1980).
54. Gross, et al., Eur. J. Biochem., 168:595-
602 (1987~.
W~93/24505 2 1 ~ PCT/US93/04909
55. Gross, et al., Eur. J. Bio~hem., 177:583-
589 (1988).
56. Christian, et al., Car~ohydr. Res.,
194:49-61 (1989~.
57. Conradt, et al., FEBS Lett., 170:295-300
(1984).
58. Christian, et al., Carbohydr. Res., 162:1-
11 (1987).
59. Haverkamp, et al~, Hoppe-Seyler's Z.
Physiol. Chem., 360:159-166 (1979).
60. Gross, et al., Glycoconj. J., 4:145-156
(1987).
61. Hagedorn, et al., XIIIth Carbohydr. Symp.
Ithaca (1986) A4.
62. Zbiral, et al., Carbohydr. Res., 194:C15- -
C18 (19~9).
63. Belkhouya, et al., Tetrahedron Letters,
3971-3980 (1991).
64. Sialic Acids in "Cell Biology Monographs",
Schauer, Editor, Vol. 10 (1982).
65. Kukowska-Latallo, et al., Genes and
Development, 4:1288-1303 (1990).
66. Dumas, et al., Bloorg. Med. Letters,
1:425-428 (1991).
67. ~okhale, et al., Can. J. Chem., 68:1063-
1071 (19gO).
68. Smith, et al., Immunology, 58:245 (lg86).
69. Sleytr, et al., Arch. Microbi~l., 146:19
~ (1986~.
70. Ziola, et al., J. Neuroimmunol., 7:315-330
(1985).
71. Smith, et al., Infection and Immunity, 31:
12g (lg~O).
72. Petrakova, et al., Can. J. Chemistry,
70:233-24~ (19g2).
73. Ichikana, et al., Anal. Biochem., 202:215-
138 (1992)
2~1~'J2._, ~
W093~24~05 PCT/US93/0490Q -
74. Weinstein, et al., J. Biol. Chem.
257:13835-1384~ (19~2).
75. Sticher, et al., Biochem. J., 253:577-S80
(198~).
76. Dean, et al., Chromatogr., 165:301-319
(1979).
77. Mazid, et al., U.S. Patent No. 5,059,535,
"Process for the Separation and Purification
of Sialyl Transferases", issued Oct. 22,
1992.
78. Matsumoto, et al., Anal. Biochem., 116:103-
110 (1981).
79. Schmidt, et al., Liebigs Ann. Chem., 121-124
(1991)
80. Nunez, et al., Can. J. Chem., 59:2086-2095
(1981)
81. Veeneman, et al., Tetrahedron Lett., 32:6175-
6178 (1991)
82. Hanessian, Carbohydr. Res., 2:86-88 (1966).
83. Zbiral, et al., Monatsh. Chem., 119:127-141
(lg88).
84. Hasegawa, et al., J~ Carbohydr. Chem., 8:135-
144 (1989).
85. Kean, et al., J. Biol. Chem., 241:5643-5650
(1960).
86. Gross, et al., Biochemistry, 28:7386-7392
(1989).
87. Lemieux, et al., U.S. Patent No. 4,137,401
(1976).
88. Lemieux, et al., U.S. Patent No. 4,195,174
(1978).
89. Paulsen, et al., Car~ohydr. Res., 125:21-45
- ~1984).
90. Sabesan, et al., Can. J. Chem., 62:644-652
(1984).
91. Lemieux, et al., U.S. Patent No. 4,767,845
(1987).
; W093/24505 2 1 1 8 ~ i 2 ~ PCT/US93/04909
92. Alais, et al., Carbonydr. Res., 207~ 31
(1990).
93. Unverzagt, et al., J. Amer. Chem. Soc.,
112:9308-9309 (1990).
94. Toone, et al., Tetrahedron, No. 17, 45:5365-
5422 (1989).
All of the above publications and patentapplications 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 AFt.
The administration to mammals of different
oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure,
such as ~Neu5Ac(2~3)~Gal(1-4)-~a-L-Fuc(1-3)]-~GlcNAc-OR
("Sialyl LewisX-OR"), ~Neu5Ac(2-3)~Gal(1-3)-[~-L-Fuc(1-
4)~-~GlcNAc-OR (Sialyl LewisA-OR), ~NeuSAc(2-6)~Gal-
(1-4)-[~-L-Fuc(1-3)]-~GlcNAc-OR, and the like, has been
disclosed by Gaeta, et al.~, Paulson, et al. 2~3,
Brandley, et al. 4, Lowe5, McEver6, Furie, et al. 7, among
others, to reduce infla~mation in the mammal arising
from a variety of conditions such as injury, infection,
exposure to an antigen, etc. These disclosures are
based on the fact that an integral step in the
inflammatory process in a ma~mal is the adherence of
leukocytes to one or more selectins and the discovery
that such oligosaccharide glycosides adhere/bind to one
or more selectins involved in the inflammatory response
thereby interfering with the binding of the leukocyte
to those selectins.
2~1$ ~ ~
W093/24505 PCT/US93/04909``
-- 8 --
Specifically, the presence of selectins such
as ELAM-l (Endothelium keUcocyte Adheslon Molecule-l)
on the vascular endothelium and/or the presence of
PADGEM (also referred to as GMP-140) on either the
vascular endothelium or on activated platelets and/or
the presence of ~-selectin on high endothelial venuels
(HEV) in the peripheral and mesenteric lymph nodes is
believed to be stimulated by an inflammatory event such
as exposure to an antigen, myocardial infarction, lung
injury, etc. In turn, adhesion of circulating
leukocytes (e.g., neutrophils, monocytes, etc.) to
ELAM-1 and/or PADGEM located on the stimulated vascular
endothelium and/or to PADGEM located on activated
platelets are believed to be primary events of the
inflammatory response. Add.itionally, the L-selectin is
believed to be present on neutrophils and, accordingly,
may play some role in the inflammatory process.
Based on in-vitro data demonstrating that
certain oligosaccharide glycosides related to blood
group determinants having a type I or type II core
structure (e.g., Sialyl LewisX and Sialyl LewisA) bind
to the ELAM-ll~2.3,4,5,6 selectin and possibly other
selectins and that certain oligosaccharide glycosides
2S related to blood group determinants having a type I or
type II core structure (e.g., ~Neu5Ac(2~6)~Gal(1-4)-~-
L-Fuc(1-3)]-~GlcNAc) bind to the ELAM-14 and PADGEM7
selectins, it was postulated that these oligosaccharide
glycosides would reduce inflammation when administered
hylacticallYl~2~3~4 or therapeutically1234s
to a mammal by interferring with adhesion between
ELAM-1, PADGEM, and/or other selectins and the
circulating leukocytes.
In disease/inflammatory conditions, the
Gaeta, et al.1, Paulson, et al. 2~3 and Brandley, et al.4
references recite that the therapeutic administration
,: W O 93/2450~ 211 ~ ` 2~ P ~ /US93/04909
__ g __
of the therein disclosed oligosaccharide glycosides can ~`
be conducted on a patient already suf~ering from the
disease/inflammatory condition. According to the
Gaeta, et al.1 and Paulson, et al. 2~3 references, the
therein disclosed oligosaccharide glycosides can be
used to treat a variety of disease/inflammatory
conditions including disease conditions arising from
antigen exposure such as rheumatoid arthritis, asthma,
dermatitis, psoriasis, and inflammatory bowel disease
as well as inflammatory conditions arising from an
injury including frost-bite injury, reperfusion injury,
acute leukocyte-mediated lung injury, etc. The Gaeta,
et al.1 and Paulson, et al.Z~3 references specifically
recite that for inflammation arising from reperfusion
or other injury, the therein disclosed oligosaccharide
glycoside should ideally be administered as soon as
possible after the injury. However, no guidance is
provided by these or by the Brandley, et al. 4, Lowes,
McEver6 or Furie, et al. 7 references as to when such
oligosaccharide glycosides specifically should be
therapeutically administered to a mammal sensitized to
an antigen after subsequent antigen exposure.
This invention is directed to the discovery
~5 that, in order to reduce inflammation in the case of
antigen challenge (exposure) in a sensitized mammal,
the oligosaccharide glycoside related to blood group
de~erminants must be administered after initiation of
the mammal's secondary immune response to the antigen
challenge but at ~r prior to one-half that period of
time where the mammal experiences maximal inflammatory
response~
Specifically, the data set forth in the
examples below evidence that administration of the
oligosaccharide glycoside related to a blood group
determinant having a type I or type II core structure
2~ ~85 2~
W093~24~05 PCT/US93/0490g
-- 10 ----
prior to initiation of the mammal's immune response,
provides no reduction in inflammation. Additionally,
administration of the oligosaccharide glycoside at a
point in time after one-half that period of time where
the mammal experiences maximal inflammatory re~ponse to
the antigen exposure results in minimal reduction in
inflammation. In fact, the data evidence that it is
only when the oligosaccharide glycoside related to
blood group determinants having a type I or type II
core structure is administered after the sensitized
mammal's secondary immune response has been initiated
to antigen challenge but at or prior to about one-half
that period of time where the mammal experiences
maximal inflammatory response to the antigen challenge
does significant reduction in inflammation occur.
These findings are particularly surprising in
view of the fact that the Gaeta, et al.1, Paulson, et
al. 2~3, Brandley, et al. 4 references state that the
therein disclosed oligosaccharide glycoside can be
administered either prophylactically or therapeutically
and further in Vi2W of the fact that none of the Gaeta,
et al~, Paulson, et al. 2~3, Brandley, et al. 4, Lowe5,
McEver6, Furie, et al.7 references disclose any
criticality with regard to the point in time when such
oligosaccharide glycosides should be therapeutically
administered to a mammal in order to reduce the degree
of inflammation arising from the mammal's secondary
immune response to an antigen challenge.
8~NM~RY OF T~E INVENTION
In view of the above, in one of its method
aspects, this invention is directed to a method for
reducing the degree of inflammation in a mammal arising
from the initiation of a mammal/s secondary immune
response due to antigen exposure which method comprises
W093/24505 2 t 1 8 'j 7 ' PCT/US93/04909
---- 11 ----.
administering to said mammal an inflammation reducing
effective amount of an oligosaccharide glycoside
related to blood group determinants having a type I or
type II core structure wherein said administration is ~.
5 after initiation of the mammal's secondary immune
response to the antigen exposure but at or prior to .
one-half that period of time required for maximal
inflammatory response to the antigen exposure.
In another of its method aspects, this
invention is directed a method for reducing the degree
of inflammation in a mammal arising from initiation of
a mammal's secondary immune response due to antigen
exposure which method comprises administering to said
15 mammal from about 0.5 to about 50 mg/kg of an
oligosaccharide glycoside selected from the group
consisting of an oligosaccharide glycoside of Formula I
H0 X R2
~ Xo ~ OyR
X~ ~ 0 ~ 0
R
and Formul~ II
H0
X20~_0~Y~R II
wherein said administration is after initiation of the
mammal's secondary immune response to the antigen
exposuré but at or prior to one-half that period of
time required for maximal inflammatory response to th~
antigen exposure,
~1-L3J~
WO 93/24505 PCr~US93/04909
-- 12 --
where R is selected from the group consisting of
hydrogen, a saccharide-OR1~, an oligosaccharide-ORl9 of
from 2 to 7 saccharide units, and an aglycon having at
least one carbon atom where R19 is hydrogen or an
aglycon of at least one carbon atom;
Y is selected from the group consisting of oxygen,
sulfur, and -NH-;
R1 is selected from the group consisting of
hydrogen, -NH2, -N3, -NHSO3H, -N~C(O)R4, -N=C(F~) 2 '
-NHCH(Rs)2, -NHR6, -N(R6)2, -OH, -OR6, -S(O)R6, -S(0)2R6
and sulfate,
wherein R4 is selected from the group consisting
of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
-OR7 wherein R7 is alkyl of from 1 to 4 carbon
atoms, or alkyl of from 2 to 4 carbon atoms substituted
with a hydroxyl group, and
-NR8R~ wherein R8 and ~ are independentiy
selected from the group consisting of hydrogen and
alkyl of from 1 to 4 carbon atoms,
each ~ is selected from the group consisting of
hydrogen and alkyl of from 1 to 4 carbon atoms,
each R6 is alkyl of from 1 to 4 carbon atoms,
R2 is selected from the group consisting of
hydrogen, -N3, -NH2, -NHS03H, -NR11C(O)Rlo, -N=C(R11) 2 '
NHCH(Rll)2~ -NHR12j -N(R12)Z~ -OH and -ORl2,
wherein Rlo is selected from the group consisting
of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
- -OR13 wherein Rl3 is alkyl of from 1 to 4
carbon atoms, or alkyl of from 2 to 4 carbon atoms
substituted with a hydroxyl group, and
-NR14Rl5 wherein Rl4 and R1s are independently
selected from the group consisting of hydrogen and
alkyl of from 1 to 4 carbon atoms,
W O 93/24~05 2 1 1 8 5 2 ~ PC~r/US93/04909
13 --
each R~1 is selected from the group consisting of
hydrogen and alkyl of from l to 4 carbon atoms;
each R12 is alkyl of from 1 to 4 carbon atoms,
R3 is selected from the group consisting of
hydrogen, fluoro, sulfate and hydroxy;
X is selected from the group consisting of
hydrogen, L-fucosyl, 4-sulfo-L~fucosyl, and 4-phospho-
L-fucosyl; - :
X1 is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR18COOH
where R~8 is selected from the group consisting of
hydrogen, alkyl of from 1 to 7 carbon atoms and -COOH;
X2 is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR~8COOH
where R18 is selected from the group consisting of
hydrogen, alkyl of from 1 to 7 carbon atoms and -COOH;
and
pharmaceutically acceptable salts thereof; and
with the proviso if X is hydrogen, then either at
least one of X~ or X2 is not hydrogen or R3 is sulfate,
and
with the further proviso that only one of X1 and X2
is sialyl.
In yet another of its method aspects, this
invention is directed to a method for reducing the
degree of inflammation in a mammal arising from
initiation of a mammal's secondary immune response due
to antigen exposure as well as inducing tolerance in
the mammal to later exposure to the antigen which
method comprises administering to the mammal from about
0.5 to about 5 mg/kg of an oligosaccharide glycoside
selected from the group consisting of an
oligosaccharide glycoside of Formula I
211~3~32 ~
W093~24505 PCT/US93/04909
HO R
X2O ~ O ~ OY-R
R3 ~,
and Formula II
HO~ oX~Y-R II
R~ R
wherein said administration is after initiation of the
mammal's secondary immune re~ponse to the antigen
exposure but at or prior to one-half tha~ period of
time required for maximal inflammatory response to the
antigen exposure,
where R is selected from the group consisting of
hydrogen, a~saccharide-OR19, an oligosaccharide-OR~g of
from 2 to 7 saccharide units, and an aglycon having at
least one carbon atom where R19 is hydrogen or an
aglycon of at least one carbon atom;
Y is selected from the group consisting of oxygen,
sulfur, and -NH-;
R1 is selected from the group consisting of
hydrogen, -NH2, ~N3, -NHSO3H, -N~C(O)R4, ~N=C(Rs)2,
NHCH~R5j2, NHR6, -N(R6)2, -OH, -OR6, -S(O)R6, -S(O)2R
and sulfate,
wherein R4 is selected from the group consisting
of
hydrogen,
3S alkyl of from l to 4 carbon atoms,
211.~S '~
: W093/2450~ PCT/US93/049~9
15 --
-OR7 wherein R7 is alkyl of from l to 4 carbon
atoms, or alkyl of from 2 to 4 carbon atoms substituted
with a hydroxyl group, and
-NR8R9 wherein R8 and ~ are independently
selected from the group consisting of hydrogen and
alkyl of from l to 4 carbon atoms,
each ~ is selected from the group consisting of
hydrogen and alkyl of from 1 to 4 carbon atoms,
each R6 is alkyl of from 1 to 4 carbon atoms,
R2 is selected from the group consisting of
hydrogen, -N3, -NH2, -NHS03H, -NRl 1 C ( 0 3 R~ o, N ( 11 ) 2 r
-NHCH (Rl1 ) 2 ~ -NHR12, -N (R,Z) 2, -OH and -OR~2,
wherein R10 is selected from the group consisting
of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
-OR13 wherein R13 is alkyl of from l to 4
carbon atoms, or alkyl of from 2 to 4 carbon atoms
substitutPd with a hydroxyl group, and
-NR14R1s wherein R14 and R15 are independently
selected from the group consisting of hydrogen and
alkyl of from 1 to 4 carbon atoms,
- each Rl1 is selected from the group consisting of
hydrogen and alkyl of from 1 to 4 carbo~ atoms;
each R12 is alkyl of from 1 to 4 carbon atoms,
R3 is selected from the group consisting of
hydrogen, fluoro, sulfate and hydroxy;
X is selected from the group consisting of
hydrogen, L-fucosyl, 4-sulfo-L-fucosyl, and 4-phospho-
L-fucosyl;
X~ is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR18COOH
where R18 is selected from the group consisting of
hydrogen, alkyl of from 1 to 7 carbon atoms and -CQOH:
X2 is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR18COGH
where R~8 is selected from the group consisting of
2113~2~
W093/24~05 PCT/US93/0490S`~`
16 --
hydrogen, alkyl of from 1 to 7 carbon atoms and -COOH;
and
pharmaceutically acceptable salts thereof; and
with the proviso if X is hydrogen, then either at
least one of X1 or X2 is not hydrogen or R3 is sulfate,
and
with the further proviso that only one of X~ and X2
is sialyl.
Preferably, R2 is -NH2, -N3, -NHC(0)R1o and R3
is preferakly -OH or sulfate.
Preferably, X2 is sialyl or a sulfate group.
When X2 is a sialyl grcup, X is preferably a
fucosyl group.
In Formula I, when X2 is a sulfate group, X
is preferably hydrogen.
In one preferred embodiment, the
oligosaccharide glycoside related to blood group
determinants having a type I or type II core structure
is selected from the group of oligosaccharide
glycosides A-N set forth in Example A hereinbelow.
Preferably, the oligosaccharide glycoside
related to blood group determinants having a type I or
type II core structure is administered to the mammal at
least 1 hour after the mammal has been exposed to the
antigen, more preferably from about 1 to 10 hours after
the mammal has been exposed to the antigen. In another
preferred embodiment, the oligosaccharide glycoside
related to blood group determinants having a type I or
type II core structure is administered to the mammal
from about 1 to 5 hours after the mammal has been
exposad to the antigen.
. WO93/245~5 2 11~3'.~ 2 7 PCl/US93/049~9
BRIEF DESCRI~?TION OF THE DR~WING~;
Figure 1 illustrates the increase in footpad
swelling of immunized mice arising from a DTH
inflammatory response measured 24 hours after challenge
with 10 ~g of the L111 S-Layer protein antigen wherein
some of the mice have been treated at 5 hours after the
challenge with 100 ~g of different oligosaccharide
glycosides related to blood group determinants having
type I or type II core structures.
Figure 2 illustrates the increase in footpad
swelling of immunized mice arising from a DTH
inflammatory response measured 24 hours after challenge
with 20 ~g of the L111 S-Layer protein antigen wherein
some of the mice have been~treated at 5 hours after
challenge with various doses of different mono- and
oligosaccharide glycosides including ollgosaccharide
glycosides related to blood group determinants having
type I or type II core structures.
- 20
~-~- ; Figure 3 illustrates secondary antibody
responses (i.e., as determined by the amount of
antibody measured by quantification of o-phenylene-
- diamine O.D. at 490 nm) two weeks after primary
immu izatlon and one week after challenge with the Llll
S-Layér protein antigen and the effect different
oligosaccharide glycosides related to blood group
determinants had on these responses when the mice were
treated with these oligosaccharide glycosides 5 hours
after challenge.
;~ Figure 4 illustrates the effect of an
oligosaccharide glycoside related to blood group
determinants having type I or type II core structures,
i.e, Sialyl LewisX-OR where R = 8-methoxycarbonyloctyl,
on the inflammatory DTH response in immunized mice
challenged with the Llll S-Layer protein antigen
2 1 1 :3 S 2 ~
W093/2450~ PCT/US93/0490S
18 --
wherein the mice were treated at various times before
or after challenge with 100 ~g of Sialyl Lewis~-OR.
Figure 5 illustrates the long term (8 weeks)
immunosuppressio~ generated in immunized mice after an
injection with 5 mg/kg of oligosaccharide glycosides
related to blood group determinants having type I or
type II core structures, 5 hours after challenge with
20 ~g of the L111 S-Layer protein antigen on day 7.
Figure 6.illustrates the long term (6 weeks)
immunosuppression generated in immunized mice after an
injection-with varying amounts of mono- and
oligosaccharide glycosides including an oligosaccharide
glycoside related to blood group determinants having
type I or type II core structure 5 hours after
challenge with 20 ~g of the Llll S-Layer protein
antigen on day 7.
.20 Figure 7 illustrates the long term (10 weeks)
immunosuppression generated in immunized mice after an
injection:with 5 mg/kg of the 8-methoxycarbonyloctyl
glycoside of Sialyl LewisX at various. times before, at
and after challenge with 20 ~g of the Llll S-Layer
protein antigen on day 7.
Figure 8 illustrates the cyclophosphamide
induced restoration of a DTH inflammatory response in
immunized mice previously suppressed by treatment with
the 8-methoxycarbonyloctyl glycoside of Sialyl LewisX.
Figure 9 illustrates that the nature of the
antigen used to induce the inflammatory response does
not affect the ability of the 8-methoxycarbonyloctyl
glycoside of Sialyl LewisX to regulate the DTH
response.
` W093/2450~ PCT/USg3/04909
19 ----
Figure 10 illustrates the increase in foot-
pad swelling of immunized mice arising from a DTH
inflammatory response measured 24 hours after challenge
with HSV antigen, where some of the mice were treated
with Sialyl LewisX at the time of i~munization and some
of the mice were treated with Sialyl LewisX 5 hours
after the challenge.
Figure 11 illustrates the secondary antibody
responses (i.e., as determined by the amount of
antibody measured by quantification of o-phenylene-
diamine 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
1$ Sialyl LewisX had on the these responses.
.
Figure 12 illustrates the cyclophosphamide
(CP) induced restoration of a DTH inflammatory response
in immunized mice previously suppressed by treatment
with the 8-methoxycarbonyloctyl glycoside of Sialyl
LewisX .
:.
Figure 13 illustrates the effect of the
8-methoxycarbonyloctyl glycoside of Sialyl LewisX on
the inflammatory DTH response in immunized mice
challenged with the OVA antigen wherein the mice were
treated with 8-methoxycarbonyloctyl glycoside of Sialyl
LewisX five hours after challenge by either intravenous
(IV) or intranasal (IN) administration.
Figure 14 illustrates the effect of the
8-methoxycarbonyloctyl glycoside of Sialyl LewisX on
the inflammatory DTH response in immunized mice
challenged with the OVA antigen wherein the mice were
treated with various doses of the 8-methoxycarbonyl-
octyl glycoside of Sialyl LewisX five hours after
2~ 135 2 ~
W093/24S05 PCT/US93/0490
-- 20 -
challenge by either intravenous (IV) or intranasal (IN)
administration.
Figure 15A illustrates the increase in
footpad swelling of immunized mice arising from a DTH
inflammatory response measured 24 hours after challenge
with 20 ~g of the OVA antigen and compared to a control
group of mice wherein different groups of the mice were
treated at 5 hours, 10 hours, 12 hours, 15 hours, 18
hours, and 24 hours after the challenge with 200 ~g of
an oligosaccharide glycoside related to blood group
determinants having a type II core structure [sialyl
LewisX-OR, R - -(CH~)8CO2CH3].
Figure l5B illustrates percent reduction in
maximal inflammation in the sensitized mice of Figure
15A by administration of sialyl LewisX-OR where maximal
inflammation is taken as the inflammation occuring at
24 hours after OVA challenge with administration of
only PBS immediately after challenge.
Figure 16 illustrates the residual
inflammation (as measured by an increase in footpad
swelling) in the sensitized mice of Figure l5A at
48 hours after antigen challenge.
Figure 17 illustrates reaction schemes for
the synthesis of partially blocked N-acetylglucosamine
derivatives which are then used to prepare
oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure.
Figure 18 illustrates reaction schemes for
the synthesis of blocked fucose derivatives which are
then used to prepare oligosaccharide glycosides related
to blood group determinants having a type I or type II
core structure.
W093/24~05 2 1 1 8 5 2 } PCT/US93/04909
-- 21
Figure 19 illustrates reaction schemes for
the synthesis of partially blocked galactose
derivatives which are then used to prepare
oligosaccharide glycosides related to blood group
S determinants having a type I or type II core structure.
Figure 20 illustrates the synthesis of
modified LewisX compounds having a sulfate substituent
in the 3 position of the galactose unit. In this
scheme, the 2,3 positions of galactose are
differentially blocked so that the 3-position oan be
selectively deblocked and then selectively converted to
the sulfate substituent.
Figure 21 illustrates the synthesis of
modified LewisX compounds having a sulfate substituent
in the 3 position of the galactose unit. In this
scheme, the 2,3 positions of galactose are not
differentially blocked. Accordingly, deprotection of
the 3-position of the galactose unit also results in
deprotection of the 2-position and subsequent reaction
to form the sulfate at the 3-position does not proceed
with 100% yield but rather some of the product has a
sulfate substituent a~ the 2-position of the galactose
which is then separated by chromatography.
Figure 22 illustrates the synthesis of
modified LewisX deriva~ives bearing a sulfate
substituent at the 3-position of the galactose and
which utilize a 6-benzyl and 2-N-phthaloyl blocked
glucosamine which can be later deblocked to provide for
a glucosamine derivative.
In this figure, because the 3,4-dihydroxyl
groups of the 6-benzyl and 2-N-phthaloyl blocked
glucosamine 15 are not blocked, reaction with l-bromo-
2,3,4,6-tetraacetyl galactose will result in formation
wo 93,24~o2 1 1 8 S 2 ~ PCT/US93/0490~
-- 22
of both the blocked ~Gal~l~4)~GlcNH2-OR derivative 48
and the blocked ~Gal(1-3)~GlcNH2-OR derivative (not
shown). In turn, these materials can be further
derivatized at an appropriate point in the synthesis so
as to provide for N-functionalized derivatives of
oligosaccharide glycosides related to blood group
determinants having a type I or type II structure.
Figure 23 illustrates a second synthesis of
modified LewisX compounds bearing a sulfate substituent
at the 3-position of the galactos~ and which utilize a
different N-phthaloyl blocked glucosamine intermediate
that allows for the sele~tive preparation of 2-amino or
N-functionalized LewisX derivatives. In this figure,
only the 4-position of the glucosamine is not blocked
so that only the blocked ~Gal~1~4)~GlcNH2-OR derivative
70 is formed.
Figure 24 illustrates the preparation of
modified LewisA analogues having a sulfate substituent
in the 3 position of the galactose unit. In this
scheme, the 2,3 positions of galactose are
differentially blocked so that the 3-position can be
selectively deblocked and then selecti~ely converted to
the sulfate substituent.
Figure 25 illustrates the synthesis of the
6-azido derivative of GlcNAc-OR.
Figure 26 illustrates the synthesis of the
6-alkoxy derivatives, 6-bromo derivatives, and the
6-deoxy derivatives of GlcNAc.
Figures 27A and 27B illustrate general
reaction schemes for the chemo-enzymatic synthesis of
the analogues of sialyl LewisX ~compound 112b-d) and
- W093/24505 2 1 1 ~` S 2 ~ PCT/US93/04909
23 --
Sialyl ~ewisA (compounds 108a-d~ wherein Ac represent
acetyl, Bn represents benzyl, and R represents
--( CH2 ) 8C02CH3 .
Figure 28 illustrates an alternative chemo-
enzymatic synthesis of analogues of sialyl LewisX
modified at the C-2 and/or C-6 positions of the
N-acetylglucosamine unit.
Figures 29 and 30 illustrate general schemes
for the synthesis of type I and type II structures.
Figure 31 illustrates a general reaction
scheme for the chemo-enzymatic synthesis of analogues
of sialyl LewisX and sialyl LewisA modified at the C-6
position of the N-acetylglucosamine unit.
Figure 32 illustrates a general reaction
scheme for the total chemical synthesis of analogues of
sialyl LewisX and sialyl LewisA modified at the C-2
position of the N-acetylglucosamine unit.
Figure 33 illustrates a general synthetic
scheme used for the synthesis of derivatives of Neu5Ac.
Figure 34 illustrates the structures of mono-
and oligosaccharide glycosides 203b to 207a.
Figure 35 illustrates a general reaction
scheme for the synthesis of oligosaccharide glycoside
204c as specified in Example 38 and for the synthesis
of monosaccharide glycoside 237 as specified in Example
39.
Figure 36 illustrates the enzymatic transfer
of Neu5Ac, and of analogues thereof (collectively
"sialic acids") by the ~Gal(1-3/4)~GlcNAc~(2-3')-
21 1~2~
W093/24~0s PCT/US93/0490S~
24
sialyltransferase to a ~Gal(1-3)~GlcNAc- terminal
structure. Figure 36 also illustrates the enzymatic
transfer of L-fucose onto the sialylated
oligosaccharide glycosides.
Figure 37 illustrates the enz~matic transfer
of Neu5Ac, analogues thereof (collectively "sialic
acids") by the ~Gal(1~3/4)~GlcNAc~(2~3')-
sialyltransferase to a ~Gal(1-4)~GlcNAc- terminal
structure. Figure 37 also illustrates the enzymatic
transfer of L-fucose onto the sialylated
oligosaccharide glycosides.
Figure 38 illustrates the enzymatic
transfer of Neu5Ac, analogues thereof by the
~Gal(1-4)~GlcNAc~(2-6')sialyltransferase to a
~al(1-4)~GlcNAc- terminal structure.
Figure 39 illustrates the enzymatic
transfer of Neu5Ac, analogues thereof by the
~Gal(1-3/4)~GlcNAc~(2~3')-sialyltransferase to a
~&al(1-4)~Glc- (lactose) terminal structure.
Figure 40 illustrates the enzymatic
transfer of Neu5Ac, analogues thereof by the
~Gal(1~3)~GalNAc~(2~3')sialyltransferase to a
~Gal(1~3)~GalNAc- t"T") terminal structure.
Figure 41 and 42 illustrate the reaction
schemes involved in the synthesis of analogues of
sialyl LewisA by chemical modification of a sialylated
hapten.
Figure 43 illustrates the reaction schemes
involved in the synthesis of analogues of sialyl LewisX
by chemical modification of a sialylated hapten.
- W093/24~05 2~l~ i2 ~ PCT/US93/0490g
-- 25 -
Figure 44 illustrates the synthetic pathway
leading to Sialyl dimeric LewisX and internally
monofucosylated derivatives thereof. In Figure 44, the
nomenclature for compound 301a is ~Gal(1-4)~GlcNAc(l-
3)~Gal(1-4)~GlcNAc-OR, sometimes called di-N-
acetyllactosaminyl tetrasaccharide. Similarly, the
hexasaccharide moiety present in compounds 305a and
305b in Figure 44 is sometimes called VIM-2 epitope or
CD-655 and 307a and 307b are called sialyl dimeric
10 LewisX.
Figure 45 illustrates the synthetic pathway
leading to the externally monofucosylated derivatives
of the sialyl di-N-acetyllactosaminyl hapten.
Figure 46 illustrates an enzymatic pathway
leading to monofucosylated and monosialylated
compounds.
Figure 47 illustrates an alternative chemical
synthesis of trisaccharide 319 which can then be used
as per Figure 46 to prepare monofucosylated and
monosialylated compounds.
Figure 48 illustrates that the enzymatic
pathway set forth in Figure 46 can be used to extend
the structure of the hexasaccharides glycosides.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As noted above, this invention is directed to
the discovery that, in order to reduce antigen induced
inflammation in sensitized mammals, the oligosaccharide
glycoside related to blood group determinants having a
type I or a type II core structure must be administered
after initiation of the mammal's secondary immune
response to the antigen challenge but prior to one-half
211~52
W~93/24~05 PCT/US93/0490g'
26 --
that period of time where the mammal experiences
maximal inflammatory response.
However, prior to discussing this invention
in further detail, the following terms will first be
defined.
A. Definition~
As used herein, the following terms have the
definitions given below:
The term "sensitized mammal" refers to those
mammals which have been previously exposed to an
antigen and, accordingly, their immune systems have
- 15 become educated to that antigen. Typically, initial
exposure of an antigen to a mammal primes or educates
the mammal's immune response to later exposure to that
antigen with minimal inflammation during-such initial
exposure.
The term "secondary immune response" refers
- to the effector phase of a mammal's immune response to
an antigen to which it has been previously been
sensitized. A mammal's secondary immune response is
typically accompanied by inflammation at the point of
antigen exposure.
The term "antigen" refers to any protein,
peptide, carbohydrate, nucleic acid or other non-
endogenous substance which when exposed to a mammalinduces an immune response in that mammal.
-Disease conditions believed to be caused by
antigen exposure include, by way of example, psoriasis,
asthma, dermatitis, rheumatoid arthritis, delayed type
hypersensitivity, inflammatory ~owel disease, multiple
- . ~
i- 2118S2~
W093/24~05 PCT/US93/04gO9
27 --
scelorsis, viral pneumonia~ bacterial pneumonia, and
the like.
The te~m "period for maximal inflammation"
refers to the period of time typically required to
achieve m~ximal inflammation in a sensitized mammal due
t~ exposure to a specific antigen. This period of time
depends on several factors such as the specific antigen
to which the mammal has been exposed, the particular
mammalian species exposed to the antigen, etc.
Accordingly, the period of time required to effect
maximal antigen induced inflammation in a sensitized
mammal will vary for, by way of example, asthma as
opposed to rheumatoid arthritis.
Moreover, while the specific time required to
effect maximal inflammation will vary somewhat in a
given mammalian species, the timP typically required to
effect maximal inflammation for different antigen
exposures in human and other mammals resulting in
asthma, rheumatoid arthritls, psorasis, DTH, etc. is
known in the art or are readily ascertainable by the
skilled artisan. For example, in the case of a DTH
response in mice, maximal inflammation is typically 24
hours after antigen exposure.
.
The term "blood group determinants having a
type I or a type II core structure" re~ers to an oligo-
saccharide glycoside (a) having a core type I
disaccharide struct~re of ~Gal(1-3)~GlcNAc or a core
type II disaccharide structure of ~Gal(1~4)~GlcNAc or
analogues thereof; (~) having from 2 to 9 saccharide
units provided that if the oligosaccharide glycoside
has only 2 saccharide units then the oligosaccharide
glycoside has at least one substituent which carries a
charge at physiological pH such as a sulfate group, a
phosphate group or a carboxyl group (e.g., -CHR18COOH)
2 1 1 ~ ~ 2 ~ v
W~93~24~05 PCT/US93/04909;
. .
2~ --
at either the 2, 3 or 6 position of the galactose unit;
~c) which is terminated with a -YR group on the
reducing sugar.
Oligosaccharides of the formula
~Gal(1~3)~GlcNAc and ~Gal(1-4)~GlcNAc are core
structures of human type I and type II blood group
determinants respectively because all type I and type
II blood group determinants contain such core
disaccharide structures.
Analogues of blood group determinants having
the core type I or type II structures include those
wherein one or both of the monosaccharide units of
these disaccharide structures has been chemically
modified so as to introduce and!or remove one or morP
functionalities. For example, such modification can
result in the removal of an -OH functionality (i.e.,
: the formation of a deoxy substituent), the introduction
of: an amine functionality, a halo functionality, an
azide functionality, an amide functionality, a
carbamate functionality, a sulfate functionality, a
phosphate functionality, a carboxyl functionality
~-~ (e.g.,~-~R~8COOH), and the like.
Preferred oligosaccharide glycosides relatèd
: to:blood group determinants having a core type I or
type II structure are represented by Formula I and II:
;HO
X~
~ R,
~ XO1 ~ II
X20~ 0
R~ R2/
~.~ W093/24505 21~g 2 ~. PCT/US93/04909
29
where R is selected from the group consisting of
hydrogen, a saccharide-OR19, an oligosaccharide-OR~9 of
from 2 to 7 saccharide units, and an aglycon having at
least one carbon atom where R~ is hydrogen or an
aglycon of at least one carbon atom;
Y is s~lected from the group consisting of oxygen,
sulfur, and -NH-;
R1 is selected from the group consisting of
hydrogen, -NH2, -N3, -NHSO3H, -N~C(O)R4, -N=C(~) 2 '
~~HCH(Rs)z, -NH~6, -N(R6)z, -OH, -OR6, -S(O)R6, -S~O)2R6
and sulfate,
wherein R4 is selected fxom the ~roup consisting
of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
-OR7 wherein R7 is alkyl of from l to 4 carbon
atoms, or alkyl of from 2 to 4 carbon atoms substituted
with a hydroxyl group, and
-NR~R9 wherein R8 and R9 are independently
selected from the group consisting of hydrogen and
alkyl of from l to 4 carbon atoms,
each F~ is selected from the group consisting of
hydrogen and alkyl of from 1 to 4 carbon atoms,
each R6 is alkyl of from 1 to 4 carbon atoms,
R2 is selected from the group consisting of
hydrogen, -N3, -NH2, -NHSO3H, -NR11C(O)R1o,
-N=C(R11)2~ ~NHCH(R11)2~ -NHR12~ N(R12)2'
-ORl2 ~
wherein R10 is selected from the group consisting
of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
-OR13 wherein R13 is alkyl of from l to 4
carbon atoms, or alkyl of from 2 to 4 carbon atoms
substituted with a hydroxyl group, and
2l l ~?~f ~ s.
W093~24505 PCT/US93/~4909'
-- 30 --
-NR14R1s wherein R14 and R15 are independently
selected from the group consisting of hydrogen and
alkyl of from l to ~ carbon atoms,
each R~1 is selected from the group consisting of
hydrogen and alkyl of from l to 4 carbon atom~;
each R12 is alkyl of from l to 4 carbon atoms,
R3 is selected from the group consisting of
hydrogen, fluoro, sulfate and hydroxy;
X is selected from the group consisting of
hydrogen, L-fucosyl, 4-sulfo-L-fucosyl, and 4-phospho-
L-fucosyl;
X1 is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR18COOH
where R18 is selected from the group consisting of
hydrogen, alkyl of from l to 7 carbon atoms and -COOH;
X2 is selected from the group consisting of
hydrogen, sialyl, sulfate, phosphate, and -CHR18COOH
where R18 is selected from the group consisting of
hydrogen, alkyl of from l to 7 carbon atoms and -COOH;
and
pharmaceutically acceptable salts thereof;
with the proviso if X is hydrogen, then either at
least one of X1 or X2 is not hydrogen or R3 is sulfate,
and
with the further pro~iso that only one of Xl and X2
is sialyl.
The term "aglycon of at least one carbon
atom" refers to non-saccharide containing residues
having at least one carbon atoms. In a preferred
embodiment, the aglycon moiety, R, is selected from the
group consisting of -(A)-Z' wherein A represents a
bond, an alkylene group of from 2 to l0 carbon atoms,
and a moiety of the form -(CH2-CR20G) n~ wherein n is an
integer equal to l to 5; R20 is selected from the group
consisting of hydrogen, methyl, or ethyl; and G is
selected from the group consisting of hydrogen,
¦ ``W093/2450~ 2 11 '3 -~ 2 2 PCT/US93/04909
-- 31 --
¦ halogen, oxygen, sulphur, nitrogen, phenyl and phenyl! substituted with 1 to 3 substituents selected from the
group consisting of amine, hydroxyl, halo, alkyl of
from 1 to 4 carbon atoms and alkoxy of from 1 to 4
carbon atoms; and Z' is selected from the group
consisting of hydrogen, methyl, phenyl, aminophenol,
- nitrophenol and, when G is not oxygen, sulphur or
nitrogen and A is not a bond, then Z' is also selected
from the group consisting of -OH, -SH, -NH2, -NHR21,
-N(R21)z, -C(O)OH, -C(O)OR21, -C(O)NH-NH2, -C(O)NH2,
-C(O)NHR21, -C(O)N(R21)2, and OR22 wherein each R21 is
independently alkyl of from 1 to 4 carbon atoms and R22
is an alkenyl group of from 3 to 10 carbon atoms.
Numerous aglycons are known in the art. For
example, an aglycon comprising a para-nitrophenyl group
(i.e., -YR = -OC6H4pN02) has been disclosed by Ekborg,
et al . 15 At the appropriate time during synthesis, the
nitro group can be reduced to an amino group which can
be protected as N-trifluoroacetamido. The trifluoro-
acetamido group can later be removed thereby unmasking
the amino group which can be used to further
functionalize the aglycon group.
-.~
An aglycon group containing sulfur is
disclosed by Dahmen, et al.16. Specifically, this
aglycon group is derived from a 2-bromoethyl group
which, in a substitution reac~ion with thio-
nucleophiles, has been shown to lead to aglycons
possessing a variety of terminal functional groups such
as -ocH2cH2scH2sco2cH3 and -OcH2cH2sc6H4 pNH2.
Rana, et al.17 discloses a 6-trifluoro-
acetamido)hexyl aglycon (-O-(CH2)6-NHCOCF3) in which the
3S trifluoroacetamido protecting group can be removed
unmasking the primary amino group which can then be
used to further functionalize the aglycon.
W093/2450~ 2 ~ 1 3 ;, 2 ~ PCT/US93/~4909`~-
-- 32 --
Other exemplifications of known aglycons
include the 7-methoxycarbonyl-3,6,dioxaheptyl aglycon18
(-OCH2-CH2)2OCH2CO2CH3; the 2-(4-methoxycarbonylbutane-
carboxamido)ethyl19 aglycon (-OCH2CH2NHC~O)(CH2)4CO2CH3);
an allyl aglycon20 (-OCH2CH=CH2) which, by radical
co-polymPrization with an appropriate monomer, leads
to co-polymers; other allyl aglconsZ1 are known [e~g.,
-O(CH2CH20)2cH2cH=cH2]- Additionally, allyl aglycons
can be dexivatized in the presence of 2-aminoethane-
thiol22 to provide for an aglycon -OCH2CH2CH2SCH2CH2NH2.
Still other aglycons are illustrated hereinbelow.
Additionally, as shown by Ratcliffe et al.37,
R group can be an additional saccharide-OR19 or an
lS oligosaccharide-OR19 containing an aglycon at the
reducing sugar terminus.
Preferably, the aglycon moiety is a
hydrophobic group and most preferably, the aglycon
- 20 moiety is a hydrophobic group selected from the group
consisting of -(CH2)8COOCH3, -(CH2)sOCH2CH=C~2 and
- ( CH2 ) 8CH20H .
Saccharide units (i.e., sugars) useful in the
oligosaccharide glycosides related to blood group
determinants ha~ing a type I or type II core structure
include by way of example, all natural and synthetic
derivatives of glucose, galactose, N-acetylglucosamine,
, N-acetyl-galactosamine, fucose, sialic acid (as defined
below), 3-deoxy-D,L-octulosonic acid and the like. In
- addition to being in their pyranose form, all
saccharide units in the oligosaccharide glycosides
related to blood group determinants are in their D form
except for fucose which is in its L form.
The term "sialic acid" or "sialyl" means all
naturally occurring structures of sialic acid and
`W O 93/24505 2 L 1 ~ 2 ~ ` P ~ /US93/04909
-- 33 --
analogues of sialic acid which, as their CMP-
derivatives, are compatible with the ~Gal(1~3/4)~GlcNAc
(2~3)sialyltransferase and/or the ~Gal(1~4)~GlcNAc
~(2-6)sialyltransferase. In this regard, any sialic
acid which, as its CMP-derivative, is recognized by
either of these sialyltransferases so as to bind to the
enzyme and is then available for transfer to an
oligosaccharide glycoside having a type I or type II
structure is said to be compatible with these
sialyltransferases.
Naturally-occurring structures of sialic acid
include, by way of example, 5-acetamido-3,5-dideoxy-D-
glycero-D-galacto-nonulopyranosylonic acid ("Neu5Ac"),
N-glycoyl neuraminic acid (Neu5Gc) and 9-O-acetyl
neuraminic acid (NeuS,9Ac2). ~ complete list of
naturally occurring sialic acids known to date are
provided by Schauer~.
- 20 Analogues of sialic acid refers to analogues
~ of naturally occurring structures of sialic acid
-~ ~ including those wherein the sialic acid unit has been
~- chemically modified so as to introduce and/or remove
one or more functionalities from such structures. For
example, such modification can result in the removal of
- an -OH functionality, the introduction of an amine
functionality, the introduction of a halo
functionality, and the like.
,
Certain analogues of sialic acid are known in
the art and include, by way of example, 9-azido-NeuSAc,
9-amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac, 9-
bromo-Neu5Ac, 7-deoxy-NeuSAcj 7-epi-Neu5Ac, 7,8-bis-
epi-Neu5Ac, 4-O-methyl-Neu5Ac, 4 N-acetyl-Neu5Ac, 4,7-
di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, as well as the 6-thio
analogues of Neu5Ac. The nomenclature employed herein
2118~ ~ '
W09~24s05 PCT/US~3/0490g-
34 __
in describing analogues of sialic acid is as set forth
by Reuter et al. Z4
CMP-nucleotide derivative of sialic acid
refers to the cytidine-5-monophosphate derivative of a
naturally occurring sialic acid or an analogue thereof.
In the case where the sialic acid is Neu5Ac, the CMP
derivative has the formula:
o NH2
OH
ACN~?/CO2H (~
lS OH O P - O- ~ I O
CMP Neu5Ac ~
HO OH
The term "fucose" or "fucosyl" refers to L-
fucose and analogues thereof which, as their GDP-
derivatives, are compatible with ~Gal(1-3/4)~GlcNAc
~ 3/4)fucosyltransferase. As noted below, this
fucosyltransferase is readily isolated from human milk.
Additionally, it is contemplated that these fucose or
fucosyl compounds will be compatible with other
fucosyltransferases of appropriate specificity such as
cloned fucosyltransferases65~.
In regard to the above, any fucose compound
which, as its GDP-derivative, is recognized by the
~Gal~1~3/4)~GlcNAc ~ 3/4)fucosyltransferase so as to
bind to the enzyme and is then available for transfer
to a compound of Formula I and Formula II above (X = H)
is said to be compatible with this fucosyltransferase.
093/24505 ~ 2 ~? PCT/US93/04909
-- 35 --
Analogues of fucose refer to naturally
occurring and synthetic analogues of fucose including
those where the fucose unit has ~een chemically
modified so as to introduce and/or remove one or more
functionalities from this structure. For example, such
modification can result in the removal of an -OH func-
tionality, the introduction of an amine functionality,
the introduction of a halo functionality, and the like.
Certain compatible analogues of fucose are
known in the art and include, by way of example, 3-
deoxyfucose, arabinose, and the like. 67
. The GDP-derivative of fucose refers to
guanosine 5'-(~-L-fucopyranosyl)diphosphate and any and
all compatible salts thereof which has the formula:
,~ ' '
O O <
HO P-O''P\ ~ N N~
HO OH
Methods for preparing GDP-fucose are known in the art.
However, GDP-fucose is preferably prepared by the
method described by Jiang et al. 42 in U~S. Patent
Application,Serial No. 07/~48,223 which is incorporated
herein by reference in its entirety.
The term "amino acid or polypeptidyl residue"
refers to product obtained by reacting an appropriate
form of an amino acid or a polypeptide with an
oligosaccharide glycoside related to blood group
determinants having a type I or type II core structure
and which has an amine functionality (-NH2) at the 2 or
211~'J2 i
W093/2450~ PCT/US93/0490
-- 36
6 positions of the GlcNAc unit under conditions where
the ami~e reacts with a carboxyl group or activated
car~oxyl gr~up on the amino acid or polypeptide to form
an amide bond. The particular amino acid or
polypeptide employed is not critical. However, in a
preferred embodiment, the polypeptide contains from
about 2 to about 5 amino acids and preferably from
about 2 to 3 amino acids.
The term 'Ipharmaceutically acceptable salts"
includes the pharmaceutically acceptable addition salts
of oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure
capable of forming salts and are derived from a variety
of organic and inorganic counter salts well known in
the art and 1nclude, by way of example only~ sodium,
potassium, calcium, magnesium, ammonium, tPtralkyl-
ammonium, and the like.
The term "removable blocking group" or
"blocking group" refers to any group which when bound
to one or more hydroxyl groups of the galactose,
N-acetylglucosamine, the sialic acid (including the
hydroxyl group of the carboxylic acid moiety), the
fucose, etc., units of oligosaccharide glycosides
related to blood group determinants having a type I or
type II core structure prevents reactions from
occurring at these hydroxyl groups and which protecting
group can be removed by conventional chemical or
enzymatic steps to reestablish the hydroxyl group. The
particular removable blocking group employed is not
critical and preferred removable hydroxyl blocking
groups include conventional substituents such as
benzyl, acetyl, chloroacetyl, benzylidine, t-butyl-
diphenylsilyl and any other group that can be
introduc~d either enzymatically or chemically onto a
hydroxyl functionality and later selectively removed
- W093/24505 2 ~ 1 S ) ~ ~ PCT/US93/04909
37 __
either by enzymatic or chemical methods in mild
conditions compatible with the nature of the product.
One such additional contemplated blocking group ls a
~-galactose which can be removed en~ymatically with an
~-galactosidase.
The term "sulfate" such as used to define the
substituents -X, -X1, and -X2 refers to substituents
which, with the oxygen of a hydroxyl group of the
galactose unit and/or fucose group, form a sulfate
group (i.e., -O-S(O)2-OH). Thus, when X, X1 or X2 is a
su1fate~ the resulting -OX, -OX1 and/or -OX2 group is
-O-S(0)2-OH, which readily forms pharmaceutically
acceptable salts thereof (e.g., -O-S(O)2-O Na+).
Contrarily, the term "sulfate" as it is used for R3
refers to the -O-S(O)2-OH group, which also readily
forr,s pharmaceutically acceptable salts thereof (e.g.,
-O-S () 2- Na j.
The term "phosphate" such as used to define
the substituents -X, -X1, and -X2 refers to substituents
which, with the oxygen of a hydroxyl group of the
galactose unit and/or fucose group) form a phosphate
group (i.e., -O-P(O)-(OH)2. Thus, when X, X1 or X2 is a
phosphate, the resulting -OX, -OX1 and/or
-OX2 group is -0-P(O)-~OH)2, which readily forms
pharmaceutically acceptable salts thereof (e.g.,
_O_p(O)-(O~Na~)z
B. Methodoloqy
As shown below in the examples,
oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure
are effective in reducing the degree of antigen induced
inflammation in a sensitized mammal provided that such
~113 J~? ~
W093/24505 PCT/US93/0490
-- 38
oligosaccharide glycosides are administered after
initiation of the mammal's secondary immune response
and at or prior to one-half the period required for
maximal inflammation induced by the antigen exposure.
The data in Examples A-L substantiate the criticality
of when these oligosaccharide glycosides are adminis-
tered and demonstrate that if the oligosaccharide
glycoside related to blood group determinants having a
type I or type II core structure are administered
before initiation of the mammal's secondary immune
response, no reduction in inflammation is achieved.
Likewise, these examples also demonstrate that if the
oligosaccharide glycoside related to blood group
determinants having a type I or type II core structure
a.e administered after one-half the period of time
required for the mammal to effect maximal inflammation,
then minimal reduction in inflammation is achieved.
Additionally, Examples A-L demonstrate that
- 20 oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure
can induce tolerance to still later exposure to the
antigen when administered during the critical period
after exposure of the immune system to the antigen.
In view of the above, the oligosaccharide
glycosides related to blood group determinants having a
type I or type II core structure are preferably
administered to the patient at least about 0.5 hours
after exposure to an antigen; more preferably, from at
least about 1 to 10 hours after exposure,to the antigen
and still more preferably from at least about 1 to 5
hours after antigen exposure.
Oligosaccharide glycosides related to blood
group determinants are effective in reducing antigen
induced inflammation in a sensitized mammal when
r ~
W093/24505 2 1 1 $ '-~ 2 ~ Pcr/usg3/o4909
-- 39 --
administered at a dosage range of from about 0.5 mg to
about 50 mg/kg of body weight, and preferably from
about 0.~ to about 5 mg/kg of body weight. The
specific dose employed is regulated by the particular
antigen induced inflammation being treated as well as
by the judgment of the attending clinician depending
upon factors such as the severity of the inflammation,
the age and general condition of the patient, and the
like. The pharmaceutical compositions described herein
can be administered in a single dose or in multiple
doses or in a continuous infusion over the critical
time frame up to one-half the period required for
maximal inflammation.
Oligosaccharide glycosides related to blood
group determinants having a type I or type II core
structure are preferably administered parenterally,
intranasally, intrapulmonarily, transdermally and
intravenously, although other forms of administration
are contemplated.
In addition to providing suppression of
antigen induced inflammation in a sensitized mammal,
administration of oligosaccharide glycosides related to
blood group determinants having a type I or a type II
core structure also imparts tolerance to still later
challenges from the same antigen. In this regard, re-
challenge by the same antigen weeks after
administration of such oligosaccharide glycosides
results in a significantly reduced immune response.
The methods of this invention are preferably
achieved by use of a pharmaceutical composition
suitable for use in the parenteral administration of an
effective amount of an oligosaccharide glycoside
related to blood group determinants having a type I or
type II core structure. These compositions comprise a
W O 93/24505 2 1 1 8 5 2 2 PC~r/US93/0490~
-- 40
pharmaceutically inert carrier such as water, buffered
saline, etc. and an e~ective amount of an
oligosaccharide glycoside related to blood group
determinants having a type I or a type II core
structure (or mixtures thereof) so as to provide the
above-noted dosage of the oligosaccharide glycoside
when administered to a patient. It is contemplated
that suitable pharmaceutical compositions can
additionallyicontain optional components such as a
preservative, etc.
It is further contemplated that other
suitable pharmaceutical compositions can include oral
compositions, transdermal compositions or bandages
etc., which are well known in the art.
It is still further contemplated that tAe
oligosaccharide glycoside related to a blood group
determinant having a type I or a type II core structure
can be incorporated as a part of a liposome or a
micelle which can then be formulated into a
pharmaceutical composition.
C. PreParation of Oliqosacchar de Glvcosides
The oligosaccharide glycosides related to
blood group determinants having a core type I or type
II structure are readily prepared by complete chemical
' syntheses, by chemical/enzymatic syntheses wherein
glycosyltransferases are employed to effect the
sequential addition of one or more of sugar units onto
a GlcNAc-OR saccharide structure, a LewisC-OR
disaccharide structure, a LacNAc-OR disaccharide
structure, or onto derivatives of such structures and
chemical syntheses are employed to effect modifications
on one or more of the saccharide structures, or by
W093/24505 2118 ~ 2 2 PCT/US93/04909
-- 41 --
complete enzymatic synthesis starting with the
GlcNAc-OR saccharide glycoside.
Specifically, enzymatic means to prepare
oligosaccharide glycosides related to blood group
determinant having a type I or type II core structure
can be used at different steps. For example, L-fucose
can be enzymatically transferred onto LewisC, lactose,
N-acetyllactosamine (LacNAc), sialylated LewisC,
sialylated lactose, sialylated N-acetyllactosamine,
suitable derivatives thereof, and the like, by an
appropriate fucosyltransferase such as the
~Gal(1~3/4)~GlcNAc ~ 3/4)fucosyltransferase which is
¦ readily obtained from human milk25~26~27.
1~
The LacNAc-OR disaccharide can be made
enz~latically from an N-acetyl glucosamine glycoside
(~GlcNAc-OR) and the known bovine milk
~-galactose(1~4)transferase. The LewisC glycoside
(i.e., ~Gal(1~3)~GlcNAc-OR~ can be made chemically.
Additionally, it is contemplated that
sulfotransferases may be used to effect sulfation at
the 3-position of galactose on either the type I or
type II structures. As is apparent and if desired,
suIfotransferation can be followed by transfer of
fucose using an appropriate fucosyltransferase as
described above.
Alternatively, chemical and enzymatic means
can be coupled wherein, for example, the sulfated,
phosphorylated, or -CHR~8COOH substituted LacNAc-OR
structure or sulfated, phosphorylated, or -CHR18COOH
substituted ~Gal(1~3)~GlcNAc-OR structure is made
chemically and the fucosyl group, if desired, can be
transferred enzymatically.
W093/24soS 2 1 1 8 5 2 2 PCT/US93/0490~ -~
-- 42 --
Chemicai ~ynthesis is a convenient method for
preparing either the complete oligosaccharide
glycoside; for chemically modifying a saccharide unit
which can then be chemically or enzymatically coupled
to an oligosaccharide glycoside; or for chemically
preparing an oligosaccharide glycoside to which can be
enzymatically coupled one or more sacsharide units.
Several chemical syntheses of blocked
intermediates exist282930. These intermediates are
suitable for the preparation of oligosaccharide
glycosides related to blood group determinants having a
type I or a type II core structure using methods known
in the art.
Chemical modifications include introduction
of the sulphate or phosphate group or a -OCHR~8COOH at
the 3 and/or 6 position of the terminal galactose,
introduction of modification at the 2- and 6- positions
of N-acetylglucosamine, introduction of functionality
at the 2-position of the galactose and the like as well
as modifications of sialic acid and/or fucose. Methods
for the preparation of such oligosaccharide glycosides
related to blood group determinants is set forth in
Venot, et al. ,9; Kashem, et al. ,10; Venot, et al.,~1;
Ratcliffe, et al.,12 Ippolito, et al.13, and Venot, et
al.14, each of which is incorporated herein by reference
in their entirety.
Examples 1 to 53 hereinbelow and Figures 17
to 48 attached hereto elaborate on a variety of
synthetic schemes which result in the preparation of
oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure.
Well known modifications of these procedures will lead
to other such oligosaccharide glycosides.
V0~3/2450~ 2 1 1 8 5 2 ~ PCT/US93/04909
-- 43 --
In the description below as well as in the
examples and figures, reference is made to the -OR
group at the reducing sugar. However, it is understood
that this group could also be -NHR or -SR the
preparation of which is well known in the art.
Cl. CHEMICAL AND CHEMICAL~ENZYMATIC SYNTHESIS OF
SACCHARIDE MONOMERS
Chemical methods for the synthesis of
oligosaccharide glycosides related to blood group
determinants containing a type I or type II core
structure are known in the art. These materials are
generally assembled using suitably protected individual
monosaccharides including glucosamine, fucose and
.galactose, and suitably protected individual disaccha-
rides such as lactose-OR, N-acetyllactosamine-OR or
~Gal(1-3)~GlcNAc-OR intermediates.
The specific methods employed are generally
adapted and optimized for each individual structure to
be synthesized. In general, the chemical synthesis of
all or part of the oligosaccharide glycosides related
to blood group determinants having a type I or a type
II core structure first invoIves formation of a
glycosidic linkage on the anomeric carbon atom of the
reducing sugar. Specifically, an appropriately
protected form of a naturally occurring or of a
chemically modified saccharide structure (the glycosyl
donor) is selectively modified at the anomeric center
of the reducing unit so as to introduce a leaving group
comprising halides, trichloroacetimidate, acetyl,
thioglycoside, etc. The donor is then reacted under
catalytic conditions well known in the art with an
aglycon or an appropriate form of a carbohydrate
acceptor which possess one free hydroxyl group at the
position where the glycosidic linkage is to be
2118~2~
W093/24~5 PCT/US93/049Q~
;
i~ 44
established. A large variety of aglycon moieties are
known in the art and can be attached with the proper
configuration to the anomeric center of the reducing
unit. Appropriate use of compatible blocking groups,
w~ll known in the art of carbohydrate synthesis, will
allow selective modification of the synthesized
- structures or the further attachment of additional
sugar units or sugar blocks to the acceptor structures.
After formation of the glycosidic linkage,
the saccharide glycoside can be used to effect coupling
of additional saccharide unit(s) or chemically modified
at selected positions or, after conventional
deprotection, used in an enzymatic synthesis. In
general, chemical coupling of a naturally occurring or
chemically modified saccharide unit to the saccharide
glycoside is accomplished by employing established
chemistry well documented in the literature. See, for
example, Okamoto et al . 31, Abbas et al.32, Paulsen33,
Schmidt34, Fugedi et al.35~, Kameyama et al. 36 ànd
Ratcliffe, et al. 37
Similarly, the use of enzymatic methods for
;~ the preparation of oligosaccharide glycosides is also
documented25,94
~ .
Cl(i) -- Preparation of Oligosacccharide Glycosides
related to Blood Group Determinants having a
type I or a type II core structure with
sulfate, phosphate or carboxyl substitution
on the aalactose unit
Figure 17 illustrates the synthesis of
numerous blo~ked derivatives of glucosamine and
N-acetylglucosamine which are useful in the preparation
of blocked LacNH2-OR, LacNAc-OR, ~Gal(1~3)~GlcNAc-OR,
~Gal(1~3)~GlcNH2-OR, etc. structures which, in turn,
can be used to prepare oligosaccharide glycosides
~3 ~0 93/24505 2 1 1 8 5 2 2 PCT/US93/04909
-- 45 --
~ related to blood group determinants having a type I or
¦ type II core structure particularly those containing
¦ sulfate, phosphate or carboxyl substitution on the
galactose unit.
Specifically, in Figure 17, glucosamine
¦ hydrochloride is slurried in dichloroethane containing
an equivalent of anhydrous sodium acetate to which
acetic anhydride is added dropwise and, after addition
is completed, the solution is refluxed for a period of
from about 12-16 hours to provide for the peracylated
compound lO (about 3:1 ratio of ~
Alternatively, the glucosamine hydrochloride
is first taken up in methanol and then treated with 1
equivalent of metallic sodium to neutralize the HCl.
Phthalic anhydride is then added quickly to the
reaction mixture followed shortly thereafter by
triethylamine to provide for the phthalimido
derivative. This compound is then isolated and
acetylated with acetic anhydride/pyridine using
conventional techniques to provide for peracylated
compound 1 having a phthalimide blocking group
protecting the amine.
Afterwards, the aglycon i5 formed by
conventional techniques. For example, compound 10 is
converted to l-~-chloro compound 2 by well known
chemistry which involves bubbling saturating amounts of
hydrogen chloride directly into a dichloroethane
solution of compound 10. In this regard, the solution
used to prepare compound 10 can be used in this
reaction after that solution has been quenched into
water to remove ace~ic anhydride and sodium acetate,
dried and recovered. The reaction generally proceeds
over a period of about 4-6 days and hydrogen chloride
is bubbled into the solution periodically (e.g., about
21185~2
W093/24s0~ PCT/US93/04~0
46 --
once every 1-2 days). A~ter reaction completion, the
solution is quenched in aqueous sodium bicarbonate at
about 0-5C and the product is recovered after drying
the organic layer and stripping the solution to provide
for compound 2 (one spot on t.l.c.)
Compound 2 is then converted to the
1-~-(CH2)8COOCH3 aglycon by well known chemistry which
involves reaction of compound 2 with HO(CH2)8COOCH3 in
anhydrous dichloromethane containing molecular sieves
in the presence of an equivalent amount of mercuric
cyanide. The reaction is generally conducted at room
temperature for a period of about 12 to 24 hours. Upon
reaction completion (as evidenced by t.l.c.), the
reaction soIution is filter through silica and the
resulting solution is quenched by adding the reaction
solution to cold water. The organic layer is recovered
and the washed twice with an aqueous potassium iodide
~5 weight/vol percent) solution and then with a
saturated aqueous sodium bicarbonate solution. The
resulting organic solution is then dried and the
solvent removed by stripping to provide for
compound 3.
The 3, 4, and 6 hydroxyl groups of compound 3
are then deprotected by reaction with sodium methoxide
in methanol to provide for N-acetylglucosamine-OR,
compound 4. This compound can reacted with C6H5CH(OCH3) 2
' in, for example, an acidic medium in an appropriate
sol~ent at around 40-50C for about 4-6 hours to
provide for the 4,6-O-diprotected benzylidine compound
5. In turn, compound 5 can be reacted with p-methoxy-
benzyl trichloroacetimidate in an appropriate solvent
(e.g., DMF, dichloromethane) in the presence of a
catalytic amount of an acid (e.g., p-toluenesulfonic
acid--pTSA) to provide for the p-methoxybenzyl
protected 3-hydroxy compound 6. Treatment of compound
:J :
;~:
!, ~0 93/24505 2 1 1 8 5 2 2 PCT/US93/04909
,.
-- 47 --
6 with sodium cyanoborohydride in tetrahydrofuran
followed by the dropwise addition of HCl saturated
~ther at about 0C leads to compound 7.
Alternatively, compound 5 can be blocked at
the 3-hydroxyl group by reaction with, for example,
allyl bromide and base (e.g., barium hydroxide/barium
oxide) to provide for compound 8. Treatment of
compound 8 with sodium cyanoborohydride in
tetrahydrofuran followed by the dropwise addition of
HCl saturated ether at about 0C leads to compound 9.
Because compounds 7 and 9 contain only a
¦ free hydroxyl group at the 4-position of the blocked
GlcNAc-OR saccharide, subse~uent reaction with an
appropriately blocked galactose will result in
formation of a blocked type II LacNAc-OR structure
~Gal(1~4)~GlcNA~-OR].
Because compound 5 contains a free hydroxyl
group only at the 3-position of the bloc~ed GlcNAc OR
saccharide, subsequent reaction with an appropriately
blocked galactose will result in formation of a blocked
type I structure [~Gal(1~3)~GlcNAc-OR].
Alternatively, compound 1 can be converted to
compound 11 by reaction of compound 1 with an
equivalent of p-chlorothiophenol in dichloromethane at
room temperature in the presence of 2 equivalents of
boron trifluoride etherate (BF3-etherate) to provide for
compound 11.
In yet another embodiment, compound 1 is
converted to compound 12 (or the bromo analogue) by
following similar procedures set forth above for
compound 2.
211~2~
W093t24505 PCT/US93tO490
48 --
Compound 12 is converted to compound 13 by
reaction with an alcohol (e.g., ethanol -- R = CH2CH3)
in a manner similar to that of compound 3 with the
exception that the alcohol replaces Ho(CH2)8COOCH3.
Compound 13 is then converted to compound 14 with
sodium methoxide/methanol and is then converted to
compound 15 by reaction with bisEtributyltin] oxide in
refluxing toluene containing tetraethylammonium bromide
followed by reaction with ben2yl bromide.
Because compound 15 contains free hydroxyl
groups at the 3- and 4-positions of the blocked GlcNAc-
OR saccharide, subsequent reaction with an appropriate-
ly blocked galactose will result in formation of both a
type I structure [~Gal(1-3)~GlcNAc-OR] and a type II
structure ~Gal(1-4)~GlcNAc-OR] which are readily
separated by conventional tec~niques including
chromatography.
Compound 16 is prepared by treating p-chloro-
thiophenol with 0.95 equivalents of potassium hydroxide
in ethanol followed by heating the solution to absut
40-50C and then adding about 0.5 equivalents of
compound 2 to the reaction solution. The reaction is
maintained at 40-50C for about 1-2 hours and the
product 16 precipitates upon cooling the solution and
is recovered by filtration.
In Figure 18, the synthesis of compounds
17 - 20 are set forth in the examples hereinbelow. The
process to produce the highly crystalline fucose
intermediate 20 from L-fucose as shown in Figure 18 is
noval. This procedure optimizes the production of ~-
fucopyranose tetraacetate 17 by adding acetic anhydride
(AcOAc) dropwise to a slurry of fucose and about
equimolar amounts (e.g., about 1.1 equivalents) of
sodium acetate (NaOAc) maintained at about 50-55C in
~ W093/2450~ 2 1 1 8 ~ 2 2 PCT/US93/04909
. -- 49
i dichloroethane (DCE3 and stirred at this temperature
for a sufficient period of time to result in formation
of compound 17 (e.g., for about 2-3 days). The
reaction mixture is treated with water, quenched into
ice water, extracted with additional dichloromethane
and dried and partially concentrated to provide the
¦ peracylated compound 17 (about 4~ ratio of 1-
acetate).
Compound 17 is then reactêd with an
approximately equivalent amount of p-chlorothiophenol
(p-Cl-Ph-SH) and approximately 1 to 3 (preferably 2)
equivalents of boron trifluoride etherate (BF30Etz) in
a suitable solvent (e.g., dichloromethane) to provide
for the p-chlorophenyl 2,3,4-tri-O-acetyl-~-thiofuco-
pyranoside, compound 18. The reaction conditions
employed are not critical and temperatures of from
about oo to about 25C tprefe~ably at room temperature)
and reaction times of about 3 to about 16 hours can be
used.
Compound 18 is quickly deacetylated under
Zemplen conditions (NaOMe, MeOH~ to yield p-chloro-
phenyl B-thiofucopyranoside 19 as a crystalline product
in 55-65% overall yield from fucose after
recrstallization f rom an appropriate sol~ent (e.g.,
isobutanolj. Again, the reaction conditions employed
are not critical and temperatures of from about 15 to
about 30~C and reaction times of about 1 to about 10
hours can be used.
Compound l9 is, in turn, readily benzylated
with benzyl chloride or benzyl bromide to yield p-
chlorophenyl 2,3,4-tri-O-benzyl-B-thiofucopyranoside,
compound 20, in 45-50% overall yield from fucose. As
before, the reaction conditions employed are not
critical and temperatures of from about 15 to about
211~`522
W093/24505 PCT/US93/0490
,"., ~, ...
-- 50
30OC and reaction times of about 24 to about 48 hours
can be used. In general, at least 3 equivalents of
benzyl chloride or bromide are employed and the
reaction is generally conducted in the presence of at
S least about 4-5 equivalents of a suitable base (e.g.,
potassium hydroxide -- KOH) in a suita~le inert solvent
(e.g., dimethoxysulfoxide -- DMSO).
In a preferred embodiment, about 3 equivalent
of base are added to the reaction system prior to
addition of about 3 equivalents benzyl chloride or
benzyl bromide. After about 18 hours, an additional
1.5 equivalents of base and an additional equivalent of
ben~yl chloride are added.
The simple reagents, easy processing and
highly crystalline products eliminate the
chromatography that frequently has been required using
heretofore described methodology.
The synthesis of compounds 21-24 are
conducted by following known techniques, for example
those described by Matta et al.38 In the procedure of
Matta et al., compound 23 can be converted to either a
3-acetyl (compound 24) or the 4-acetyl blocking group
(not shown). In turn, ~oth of these compounds are then
blocked at the remaining hydroxyl group with a
chloroacetyl blocking group by acetylation with
chloroacetylchloride in pyridine/dichloromethane at
about 0C. This results in compounds which have
differentially protected 3,4-hydroxy groups. The
chloroacetyl blocking group in either compound can be
selectively removed at the appropriate point in the
synthesis by treatment with thiourea in
pyridine/ethanol (6:1) and then reacted to form the
sulfate or phosphate in the manner described below.
~ . .
- W093/24~5 2 1 1 8 ~ 2 2 PCT/US93/04909
The synthesis of compounds 26 31 are
depicted in Figure 19 and are set forth in the examples
hereinbelow. In this figure, the synthesis of
compounds 26, 27, 28, and 36 parallels that of
compounds 17, 18, 19, and 20 as set forth above and
illustrated in Figure 18. In this regard, benzyl 4,6-
O-~enzylidene-2-O-benzoyl-3-O-chloroacetyl-~-D-
thiogalactopyranosidP (compound 31) has been produced
without the necessity of chromatography. D-Galactose
pentaacetate 26 is produced by slurring D-galactose and
about an equimolar amount (e.g., about 1.1 equivalents)
of sodium acetate (NaOAc) in dichloroethane (DCE),
heating to reflux and adding at least 5 equivalents of
acetic anhydride (AcOAc) dropwise to the refluxing
solution (about 80-85C) and then maintaining the
reaction system at this temperature for a sufficient
period of time (about 16-32 hours) to result in
formation of compound 26. This procedure optimizes the
yield of B-D-galactose pentaacetate 26 and controls the
exotherm of heretofore known procedures.
After workup of the solution in a similar
manner to that described above for compound 17, the
product is treated with approximately eouimolar amounts
of benzyl mercaptan (Ph-CH2-SH) and from about 1-3
(preferably two) equivalent of boron trifluoride
etherate (BF3OEt2) in dichloromethane. The reaction
conditions are not critical and the reaction is
preferably conducted at from about 0C to about 30C
for a period abouut 6 to 16 hours to yield after
crys allization from hot methanol or hot isopropanol
55-65% of benzyl 2,3,4,6-tetra-O-acetyl B-D-
thiogalactopryanoside, compound 27.
Deacetylation under Zemplen conditions
(sodium methoxide/methanol) leads to compound 28.
Deacetylation reaction conditions are not critical and
211~ 5 2~
W093/2450~ PCT/US93/0490
-- 52 --
the reaction is generally conducted at room temperature
for a period of from about 2 to about 15 hours. After
the deacetylation reaction is complete (as judged by
t.l.c.), the solution is neutralized with an acid ion
S exchange resin, filtered and evaporated to dryness to
provide for compound 28. The residue is crystallized
from hot acetone and the product is taken up in
dimethylformamide or acetonitrile and treated with from
1 to 2 equivalents (preferably 1.4 equivalents) of
benzaldehyde dimethyl acetal and about 0.25 to 3 weight
percent of p-toluenesulphonic acid (based on compound
28). The reaction condition~ are not critical and
preferably the reaction is condu~ted at room
temperature and is generally complete in about 12 to 24
hours. After neutralization, the benzyl 4,6-O-
benzylidene B-D-thiogalactopyranoside, 29, is isolated
and crystal1ized from hot isopropanol.
Benzyl 4,6-O-benzylidene-3-O-chloroacetyl-
~-D-thiogalactopyranoside 30 is prepared by chloro-
,.
acetylation using from about 1 to 3 (preferably 2)equivalents of chloroacetylchloride;which is added to a
dimethylformamide (DMF) solution containing benzyl 4,6-
O-benzylidene B-D-thiogalactopyranoside 29. The
chloroacetylchloride is added dropwise while
maintaining the DMF solution at from about -40 to
about -15C (preferably at -25C). Under these
conditions, it is unexpectedly been found that the use
of DMF permits selective chloroacetylation of compound
29 without the need for additional base. The reaction
is generally complete in about 10-24 hours.
-
Benzyl 4,6-O-benzylidene-3-O-chloroacetyl-~-D-
thiogalactopyranoside (compound 30) is benzoylated with
at least 1 equivalent (and preferably about 2
equivalents) of benzoyl chloride in a suitable solvent
containing a base (e.g., pyridine/methylene chloride)
~ ` :
NO93/24505 211~ 5 2 2 PCT/US93/04909
- 53 -~
with from about 0.1 to about 1 weight percent of
dimethylaminopyridine [DMAP] as a catalyst. The
reaction conditions are not critical and preferably the
reaction is conducted at from about 0C to about 30C
and for about 1 to about 4 hours (preferably room
temperature for 2 hours) to give crystalline benzyl
4,6-0-benzylidene 2-0-benzoyl-3-0-chloroacetyl-~-D-
thiogalactopyranoside, compound 31, in approximately
10-20% overall yield from galastose.
The advantage of this approach is that after
subsequent assembly, the blocked intermediates will be
simply deblocked and modified by sulfation or
phosphorylation. The material is crystalline and the
process obviates the need for chromatography.
The sulfates and phosphates of the galactose
moiety of oligosaccharide glycosides related to blood
group determinants having a type I or a type II core
structure can also be made using compound 32 in the
synthesis of these compounds. This compound is made by
direct benzoylation of both the 2,3-hydroxyl groups of
compound 29. However, after deblocking, both the 2 and
3 hydroxyl groups of galactose are then available for
sulfation and phosphorylation and the selectivity is
not as efficient. Selectivity can be improved by, for
example, conducting the sulfation reaction at a low
temperature (e.g., -50C).
Compound 29 can be converted to the 2,3-
dibenzoyl protected compound 32 in a manner similar to
that described above for the preparation of compound
31. In this case, 3-5 equivalents of benzoyl chloride
are generally employed.
Compounds 31 and 32 are converted to
compounds 33 and 32a (shown in Figure 21) via known
W093/2450s 2 1 1 ~ 5 ~ ~ P~T/US93/04~0~ `
.. .. . . ..
-- 54 --
methodology (Norberg, et al.39) using br~mine
tetraethylammonium bromide.
Alternatively, compound 31 can be converted
to compound 34 by contacting compound 31 with 80%
acetic acid/water at approximately 50C for about 1-2
hours. Compound 34 is then converted to compound 35 by
treatment with acetic anhydride/pyridine in
dichloromethane.
In another embodiment, compound 32 is treated
with sodium cyanoborohydride and ceric chloride to
provide for the benzyl-2,3-O-dibenzoyl-4-O-benzyl-~-D-
thiogalactopyranoside (not shown). In turn, this
lS compound is chloroacetylated at the 6-hydroxyl group.
After formation of the type I or type II backbone, the
chloroacetyl group can be selectively remo~ed (as
described above) and then either phosphorylated or
sulfated so as to provide for the 6-phosphate or
6-sulfate derivative.
SYNTHESIS OF TYPE II STRUCTURES
Z5 ~igure 20 illustrates one method for
synthesizing blocked type II backbones which can be
used to prepare oligosaccharide glycosides related to
blood group determinants having type II core structures
which can optionally be converted to blocked LewisX
type structures.
Specifically, in Figure 20, the 2,3 hydroxyl
groups of the galactose are differentially blocked so
that at the appropriate point in the synthetic scheme,
the chloroacetyl protecting group at the 3-position of
galactose is selectively removed and then converted to
the sulfate, phosphate or -OCHR18COOH group. Also, as
~ V093/24505 2 1 1 8 ~ ~ 2 PCT/US93/04909
noted above, the chloroacetyl protecting group can be
selectively placed at the 6-position of the galactose
and then selectively removed so as to allow for the
formation of the sulf~te, phosphate or -OCHR18COOH group
at the 6-position of galactose.
Specifically, in Figure 20, compound 7 and
compound 33 are combined to form compound 37. This is
accomplished by dissolving compound 7 and approximately
1.5 equivalents of compound 33 in dichloromethane
containing molecular sieves to which is added about 1
equivalent (based on compound 7) of 2,6-di-t-butyl-4-
methylpyridine. The reaction is stirred for 30 minutes
at room temperature and then cooled to -50C. An
anhydrous toluene solution containing approximately a
slight exc~ss (e.g., about 1.2 equivalents) of silver
trifluoromethane sulfonate is then added to the
solution and the reaction is allowed to warm to -15C
over 2 hours and maintained at that temperature for an
additional 5 hours.
At this time, the molecular sieves are
removed by filtration by passing through celite and the
recovered solution is quenched by addition to a
saturated sodium bicarbonate solution. The organic
extract is then washed with water, with aqueous 0.5N
HCl, and then with water. The organic solution is then
dried and concentrated in vacuo to provide a crude
product of compound 37. This is then purified by
conventional techniques such as column chromatography
using silica gel and hexane-ethyl acetate (1:1) as the
eluant.
When desired, a LewisX structure can be
prepared from compound 37. Specifically, to a
dichloromethane solution containing compound 37 can be
added an excess of dichlorodicyanoquinone (DDQ) which
2118522
W093/24505 PCTJUS93/04~0
-- 56 --
selectively removes the p-methoxybenzyl protecting
group to provide compound 38~ This compound is
fucosylated wlth an excess of compound 20 (about 1.3-
1.5 equivalents) in dichloromethane containing mercuric
bromide or cupric bromide and about l-1.5 volume
percent DMF to give blocked LewisX compound 39. After
work-up and chromatography compound ~9 is treated with
thiourea to remove the chloroacetyl group and the
compound is sulfated with sulphur trioxide/pyridine
complex in DMF at 0C for 2 hours to provide compound
41. The blocking groups on compound 41 are then
removed by conventional techniques to provide for the
LewisX analogue having a sulfate group at the 3-
position of the galactose unit. However, in this
embodiment, it has been found that removal of the
benzoyl group (Bz) in compound 41 is accompanied by
some deacylation of the -NHAc group on the GlcNAc unit
and possibly some other side reactions. Accordingly,
chromatography is necessary to obtain pure compound 41.
In any event, it is that fucosylation in the above
reactions, fucosylation is not necessary.
Alternatively, compound 25 (or the 3-
chloroacetyl analogue of compound 25 described above--
nst shown) can be used in place of compound 20 in theabove synthesis. Removal of the chloroacetyl blocking
groups on the 3-hydroxyl of the galactose and the 4-
hydroxyl of the fucose provides an facile route to the
preparation of a disulfated or diphosphorylated LewisX
derivatives.
In another embodiment, compound 40 can then
be alkylated by first adding an appropriated base
(e.g., silver oxide, barium hydroxide, sodium hydride)
and then adding benzyl bromoacetate (BrCH2COOBn) or
other similar acetates (e.g., BrCHRl8,COOBn -- where
R18, is alkyl of from 1 to 7 carbon atoms or -COOBn) to
W O 93/24505 2 1 1 8 5 2 2 P ~ /US93/04909
57 __
the reaction medium in an appropriate solvent such as
DMF. After reaction completion, the benzyl ester(s) is
(are) readily removed by conventional hydrogenatlon
techniques which additionally removes the other benzyl
protecting groups and the benzylidine protecting group.
Treatment with sodium methoxide/methanol provides for a
-OCH2COOH (or -OCHRl8C0OH where R18 is alkyl of from 1
to 7 carbon atoms or -COOH) substituted to the 3-
position of galactose. Similar type chemistry can be
performed at the 6-hydroxyl group of the galactose or
at the 4-hydroxyl group of the fucose by use of
appropriate blocking groups.
In another embodiment, compound 40 can be
treated by known methods3~ to provide for the 3-
phosphate compound. Specifically, compound 40 can be
treated with diphenylphosphorochloridate and 4-
dimethylaminopyridine (1:1) in pyridine at 0C. The
solution is allowed to warm to room temperature over
0.5 hours and stirred for 15 hours. The resulting
compound is then hydrogenated under conventional
conditions (first with H2 in EtOH with Pd on carbon for
15 hours and then with H2 in EtOH with Pt02 for 3 hours)
to provide for the phosphate derivative at the
3-position of galactose. Further deprotection leads
to the modified LewisX compound having a phosphate
substituent at the 3-position of galactose) which is
purified and converted to its disodium salt by
contacting a solution of this compound with a sodium
form of Dowex 50 x 8.
- As is apparent, the procedures set forth
above can also be used to introduce a phosphate or a
-OCHR18C0OH group at the 6-position of ~alactose or a
phosphate group on the fucoise.
2 1 1 13 ~ 2 2
W093/24505 PCT/US93/0490 A
-- 58 -
Figure ~l illustrates another method for
synthesizing blocked ty~e II backbones and the optional
conversion of these blocked backbones to blocked LewisX
structures. In this figure, the 2,3 hydroxyl groups of
s the galactose are not differentially blocked and,
accordingly, while the resulting compound 4s (and the
type I analogue) is useful for preparing the 3-sulfate
(as part of a mixture with the 2-sulfate and 2,3
disulfate which can be purified by chromatography) it
is not as versatile as the synthetic scheme set forth
in Figure 20. -
In any event, in Figure 21, compound 7 andapproximately 1.6-1.7 equivalents of compound 32a are
dissol~ed in dichloromethane containing molecular
sieves to which is added about 1 equivalent (based on
compound 7) of 2,6-di-t-butyl-4-methylpyridine. The
reaction is stirred for 30 minutes at room temperature
and then cooled to -50~C. An anhydrous toluene
solution containing approximately a slight excess
(e.g., about 1.2 equivalents) of silver trifluoro-
methane sulfonate is then added to the solution and the
reaction was allowed to warm to -15C over 2 hours and
maintained at that temperature for an additional S
~5 hours.
After reaction completion, the reaction
system was worked up to provide a crude product of
compound 42. This is then purified by conventional
techniques such as column chromatography using silica
gel and toluene-ethyl a~etate (1:1) as the eluant.
When desired, a LewisX structure can be
prepared from compound 42. Specifically, to a
dichloromethane solution containing compound 42 is
added an excess of dichlorodicyanoquinone (DDQ) which
selectively removes the p-methoxybenzyl protecting
~0 93/24505 2 1 1 8 ~ 2 2 PC~r/US93/04909
group to provide compound 43. This compound is
fucosylated with an excess of compound 20 (about~1-3
equivalents and preferably about 1.3-1.5 equivalents)
in dichloromethane containing mercuric bromide or
cupric bromide and about 12 volume percent DMF to give
blocked LewisX compound 44. After work-up and
chromatography compound 44 is treated with sodium
methoxide/methanol to remove the benzoyl blocking
groups at the 2,3-positions of the galactose so as to
provide for compound 45. This compound is then
sulfated with sulphur trioxide/pyridine complex in DMF
at from -50 to 0C for 2 hours to provide compound 46.
Compound 46 is produced as a mixture of the 3-sulfate,
the 2-sulfate, and the 2,3-disulfate which is separated
by chromatography (e.g., column chromatography on
silica). Conventional deprotection of the removable
protecting groups provides for the sulfate derivative
at the 3-position of galactose for LewisX, compound 47,
which can be passed onto an anion exchange resin
(sodium form) to generate the sodium salt.
In order to improve the selectivity for the
generation of the 3-sulfate in this step, lower
temperatures, e.g., -50 to -30C, can be employed.
Alternatively, compound 4S can be chloroacetylated
under typical conditions to provide for a mixture of
the 2- and 3-chloroacetyl protecting groups. This
mixture can be separated by chromatography and the
resulting purified components can be used to prepare
3U 2- or 3-sulfated products selectively.
Additionally, lactose can be used in the
methods of this invention in place of LacNAc by merely
placing a suitable blocking group at the 2-hydroxy of
the glucose moiety of the lactose structure40.
Differential blocking of the lactose provides for a
composition having a selectively removable blocking
2 1 1 ~ ~ 2
W093/24505 PCT/US~3/~490~'~
-- 60
group at the 3 and/or',,6,~osition of the galactose.
This compound is then selectively deblocked at the 3
and/or 6 position and then derivatized to the 3 and/or
6 sulfate, phosphate or -OCHR18COOH. Afterwards, the
remaining blocking groups are removed and the fucosyl
unit added enzymatically (see below).
SYNTHESIS OF TYPE I ST~UCTURES
While Figures 20 and 21 illustrate the
synthesis of oligosaccharide glycosides related to
blood group determinants having a type II core
structure, oligosaccharide glycosides related to blood
group determinants having a type I core structure are
readily prepared in a similar manner, as illustrated in
Figure 24, using appropriately blocked GlcNAc-OR
structures. The ~Gal(1~3)~GlcNAc-OR type I structures,
and derivatives thereof, can be prepared, for example,
from compounds 5 and 35. Specifically, compound 35 is
first converted to the 1-~-bromo derivative via known
, methodology (Norberg et al.3~) using bromine (Br2) and
tetraethylammonium bromide fEt4N~Br') at about 0C.
' About 1.5 equivalents of this compound and compound 5
are dissolved in dichloromethane (C12CHz) containing
molecular sieves to which is added about 1 equivalent
(based on compound 5) of 2,6-di-t-butyl-4-methyl-
pyridine. The reaction is stirred for 3Q minutes at
room temperature and then cooled to -50C. An
anhydrous toluene solution containing approximately a
slight excess (e.g., about 1.2 equivalents) of silver
trifluoromethane sulfonate (silver triflate) is then
added to the solution and the reaction is allowed to
warm to -15C over 2 hours and maintained at that
temperature for an additional 5 hours~ Afterwards, the
solution is allowed to come to room temperature and
stirred overnight.
~093/24~05 2 1 1 8 5 2 2 PCT/US93/04gO9
-- 61 --
At this time, pyridine and dichloromethane
are added and the molecular sieves are removed by~
filtration by passing through celite and the recovered
solution is quenched by addition to a saturated sodium
bicarbonate solution. The organic extract is then
washed with water, with aqueous 0.5N HCl, and then with
water. The organic solution is then dried and
concentrated in vacuo to provide a crude product which
is then purified by conventional techniques such as
column chromatography using silica gel and hexane-ethyl
acetate (l:1) as the eluant to provide for compound 80.
The benzylidine protecting group of compound 80 is then
selectively removed by treatment with 80% acetic acid
(AcOH) in water (H20) to provide for compound 81.
Compound 81 is selectively acetylated at the 6-hydroxy
group of the GIcNAc unit by treatment with acetic
anhydride (AcOAc) in pyridine at about -20C to provide
for compound 82 (i.e., 8-methoxycarbonyloctyl-2-
~ acetamido-3(2-0-benzoyl-3-chloroacetyl-4,6-di-0-acetyl-
~-D-galactopyranosyl)-6-0-acetyl-2-deoxy-~-D-
glucopyranoside. This compound is then fucosylated
with,~for example, compound 20 in the manner similar to
compound 38 as described above to provide for compound
83 and then deblocked and sulfated in the manner
described above for compounds ~0, ~1, and 47 to provide
for compounds 84, 85, and 86.
,
Alternatively, compound 32 is converted to
the 1-~-bromo derivative via known methodology (Norberg
et al.39) as described above and the resulting compound
is then treated with sodium cyanoborohydride and ceric
chloride to provide for the benzyl-2,3-0-dibenzoyl-4-0-
benzyl-~-D-thiogalactopyranoside (not shown). In turn,
this compound is chloroacetylated at the 6-hydroxyl
group and then reacted with compound 5 in the manner
described above to provide for the 8-methoxy-
carbonyloctyl-2-acetamido-3(4-0-benzoyl-6-chloroacetyl-
21185~
W093/24505 PCT/US93/0490
-- 62 --
2,3-di-O-benzoyl-~-D-galacto~yranosyl)-6-O-acetyl-2-
deoxy-~-D-glucopyranosidë.;-This compound is then
treated in the manner described above for compound 82
so as to provide for a type I derivative having a
sulfate, phosphate or a -O(CHR18COOH) substituent at the
6-position of the galactose~
.
In yet another embodiment, both type I and
type II structures can be made simultaneously by
combining compound 15 and compound 33 under appropriate
conditions well known in the art. For example,
compound 15 and approximately 1.5 equivalents of
compound 33 are added to dichloromethane containing
molecular sieves to which is added about 1 equivalent
(based on compound 15) of 2,6-di-t-butyl-4-
methylpyridine. The reaction is stirred for 30 minutes
at room temperature and then cooled to -50C~ An
anhydrous toluene solution containing approximately a
slight excess (e.g., about 1.2 equivalents) of silver
trifluoromethane sulfonate is then added to the
solution and the reaction is allowed to warm to -15C
over 2 hours and maintained at that temperature for an
additional S hours. Afterwards, the solution ls
allowed to come to room temperature and stirred
overnight.
At this time, pyridine and dichloromethane
are added and the molecular sieves are removed by
filtration by passing through celite and the recovered
solution is quenched by addition to a saturated sodium
bicarbonate solution. The organic extract is then
washed with water, with aqueous O.SN HCl, and then with
water. The organic solution is then dried and
concentrated in vacllo to provide a crude product which
contains both the type I and type II structures which
are separated and purified by conventional techniques
~ ~093/24505 2 1 1 8 5 2 2 PCT/US93/04909
~ -- 63 --
~'
~ such as column chromatography using silica gel and
¦ hexane-ethyl acetate (l:l) as the eluant.
The ratio of type I structure to type II
structure resulting from this reaction can be improved
by using the 2-NAc derivative of GlcNH2 compound 15.
This compound can be readily pr~pared by reacting
compound 15 with hydrazine, acetylating the resulting
product with acetic anhydride/pyridine and then
deacetylating the 3,4-hydroxyl groups by treatment with
sodium methoxide/methanol.
i 3D. ENZYMATIC REACTIONS
1 15 In addition to the chemical syntheses of
¦ oligosaccharide glycosides related to blood group
determinants having a type I or a type II core
structure as described above, the appropriately blocked
type I [~Gal(1~3)~GlcNAc-OR] and type II
[~Gal(l~4)~GlcNAc-OR~ structures and derivatives
ther~of can be selectively deblocked to provide for a
hydroxyl group at the 3-position of galaçtose and then
sulfated, phosphorylated, or converted to -OCHR18COOH
(each of which are des~ribed above). The resulting
compound is then totally deblocked and can be
fucosylated by using, for example, ~Gal(1-3/4)~GlcNAc
~ 3/4) fucosyltransferase~1. It being understood that
the when such a fucosyltransferase is employed, the
galactose unit of this disaccharide must contain 2
6-hydroxyl substituent.
The enzymatic transfer of fucose onto the 4-
position of GlcNAc to form LewisA structures and to the
3-position of GlcNAc to form LewisX structures re~uires
the prior synthesis of its nucleotide (GDP)
derivatives. Synthesis of GDP-fucose is preferably
Wog3~24so~ 2 `~
PCT/VS~3/0490~`
-- 64
accomplished in the manner recited by Jiang et al. 42 and
which is exemplified in the examples hereunder. ~
,
GDP-fucose (GDP-Fuc~ is then combined with
~Gal(1-4)~GlcNAc-OR or ~Gal(1-3)~GlcNAc-OR (including
derivatives thereof) in the presence of a suitable
fucosyltransferase (e.g;, ~Gal(1-3/4)~1cNAc
~(1-3/4)fucosyltransferase3 under conditions wherein
fucose is transferred to the 3 position of GlcNAc of
the derivatized ~Gal(1 4)~GlcNAc-OR or the 4-position
of the derivatized ~Gal(1-3)~GlcNAc-OR compound to form
a LewisX or LewisA structures respectively.
Suitable fucosylations conditions, known in
the art, include the addition of the fucosyltransferase
to a mixture of the derivatized ~Gal(1-4)~GlcNAc-OR (or
alternatively the derivatiæed ~Gal(1-3)~GlcNAc-OR
compound~ and the GDP-fucose in a appropriate buffer
such as 50 mM sodium cacodylate in appropriate
conditions of pH and temperature such as at a pH of 6.5
and a temperature between 30 and 45C, preferably 35
to 40C while incubating for about 12 hours to 4 days~
The resulting fucosylated product can be isolated and
purified using conventional methodology comprising
HPLC, ion exchange-, gel-, reverse-phase- or
adsorption chromatography.
It is also contemplated that the deblocked
type I and II structures can be sulfated by use of an
appropriate sulfotransferase.
Alternatively, type II structures containing
a sulfate, phosphate or carboxyl substituent at the 2
and/or 3-positions of the galactose unit can be
sialylated to form the 6-sialyl derivative on the
galactose unit by use of known ~Gal(1~4)~GlcNAc
~(2-6)sialyltransferase. However, as noted above, such
~ -W093/24505 2 1 1 8 ~ 2 2 PCT/USg3/04909
- 65 --
sialylated structures cannot then be used to form
fucosyl derivatives at the 3-position of the GlcNAc
unit by use of the ~Gal(1-3/4)~GlcNAc ~(1~3/4)fucosyl-
transferase.
2E. MODIFICATION ON THE 2 AND/OR 6 POSITIONS OF
GlcNAc
Figures 22 and 23 illustrate two different
synthesPs for the retention of the 2-amino substituent
on the glucosamine unit of oligosaccharide glycosides
related to blood group determinants having a type I or
a type II core structure (e.g., derivatives of
15 ,BGal ( 1 ~ 3 ) ~GlcNAc-OR or ,BGal ( l ~ 4 ) ,BGlcNAc-OR where the
NAc group of GlcNAc has been converted to an amine).
As shown below, the retention of the amino group on the
glucosamine unit allows for the facile preparation of
different 2-substituted derivatives. Also, as is
apparent, the methodology found in Figures 22 and 23
also permit the selective formation of the 3-sulfate,
3-phosphate, 3-CHR18COOH, 6-sulfate, 6-phosphate, and
6-CHR,8COOH while retaining an NHAc group on the GlcNAc
unit.
In Figure 22, compounds 1, 13, 14, and 15 are
prepared in the manner described above and illustrated
in Figure 17. Likewise, l-a-bromo-2,3,4,6-tetracetyl-
galactose is prepared by first forming the
3 0! peracetylated derivative of galactose, compound 26.
Compound 26 is then converted to the 1-~-bromo
derivative via known methodology (HBr/Acetic acid -- at
- about 0C to 20C) so as to provide for l-~-bromo-
2,3,4,6-tetracetylated galactase.
The 1-~-bromo-2,3,4,6-tetracetylated
galactose (about 1.2 to about 1.5 equivalents) is added
dropwise to a solution of compound 15 in dichloro-
W093/24~05 PCT/US93/0490
66 --
methane at about -50C i~ the presence of excess
calcium sulfate, about 4 equivalents of silver
carbonate and about o.5 equivalents of silver t~iflate.
The reaction is then allowed to warm to -30~C and
maintained there for a~out 1-3 days. The reaction is
then quenched by the addition of methanol, warmed to
room temperature, and filtered through celite. The
filtrate is washed with aqueous sodium ~icarbonate and
aqueous ethylene diamine tetraacetic acid (EDTA). The
recovered solution is dried and then stripped in vacuo
to provide a crude product containing both the type I
structure (not shown) and the type II structure
(compound 48). The residue is chromatographed on a
silica gel column eluted with toluene:acetone:methanol
(20:3:1) to give compound 48 as well as the type I
analogue (not shown).
For convenience sake, further reactions are
shown on compound 48, it being understood however, that
the same reactions could be conducted on the type I
analogue.
If~these type II (or type I) structures are
to be fucosylated so as to provide for LewisX
structures (or LewisA structures), then this can be
ac¢omplished as follows. Compound 20 is reacted with
one equivalent of~bromine in dichloromethane at -20C
for about 1 hour to provide for the l-~-bromo
~, derivative of compound 20. The solution is then
quenched with a cold aqueous sodium bicarbonate
solùtion. The organic solution is dried and
concentrated to approximately half the original volume
in vacuo at room temperature. About 2 equivalents of
this compound are then add to a dichloromethane
solution of compound 48 that further contains about 2
equivalents of mercuric bromide (HgBr2), molecular
sieves and tetraethylammonium bromide. The reaction is
~ 093/24505 2118522 PCT/US93/04909
-- 67 --
stirred at room temperature for approximately 48 hours
and the solution is filtered through celite and the
filtrate washed with water, a 5% EDTA solution, ~
saturated aqueous sodium bicarbonate, and then water.
~ 5 The organic layer is then dried and the solvent removed
I in vacuo to provide for compound 49 which is purified
¦ by chromatography on silica gel.
Compound 49 is converted to compound 50 by
conventional Zemplen conditions and compound 50 is then
converted to compound 51 by conventional methodology
(e.g., benzaldehyde dimethylacetal, DMF, pTSA). In
turn, compound 51 is treated with hydrazine acetate in
methanol at room temperature for about 1 - 5 hours to
provide for compound 52 which is converted to compound
53 by contacting with trifluoroacetic anhydride in
methanol. Alternatively, compound 52 serves as a
convenient point in the synthesis to convert this amine
to an amide, a carbamate, a urea, a -NHSO3H group, etc.
in the manner described below.
Compound 53 can then be sulfated in the same
manner as described above for compound 45.
Alternatively, compound 53 can be differentially
blocked at the 2,3 hydroxyl groups of the galactose by
converting the 3-hydroxyl group of compound 53 to a
chloroacetyl group which is achieved in the manner
described above for compound 29 so as to provide for
compound 54. Compound 54 is then treated under
conditions described above for blocking the remaining
free hydroxyl yroup with a benzyl group so as to
provide for compound 55. In turn, compound 55 is
selectively deblocked with thiourea to provide for
compound S6 in the same manner described above for
compound 39 (to provide compound 40). Compound 56 is
then selectively sulfated in the manner described above
to provide for compound 57. Alternatively, compound 56
21~ 2 7
W093/24~0s PCT/US93/0490~````
-- 68 --
can be converted to ~h~,;3-phosphate group on the
galactose by reaction~with diphenylphosphorochloridate
and 4-dimethylaminopyridine (1:1) in pyridine at 0C.
The solution is allowed to warm to room temperature
over 0.5 hours and stirred for 15 hours. The resultin~
compound is then hydrogenated under conventional
conditions (first with H2 in EtOH with Pd on carbon for
15 hours and then with H2 in EtOH with PtO2 for 3 hours)
to provide for the phosphate derivative at the 3-
position of galactose. Further deprotection leads tothe modified LewisX compound having a phosphate
substituent at the 3-position of galactose) which is
purified and converted to its disodium salt by
contacting a solution of this compound with a sodium
form of Dowex 50 x 8. Compound 56 can also be
converted to the -CHR18COOH in the manner described
above.
Lastly, compound 57 is deblocked by
conventional techniques to provide for compound 60
which is a LewisX analogue having a 2-amino glucose
saccharide unit instead of a GlcNAc saccharide and
further having a sulfate or other substituent at the
3-position of the galactose saccharide unit.
In the above case, use of the benzyl blocking
group on the 2-position provides for a more effective
synthesis since this group as well as the other benzyl
protecting groups are readily removed under
hydrogenation conditions.
Figure 23 parallels somewhat the chemistry
depicted in Figure 22 but, because the 3-hydroxyl group
of the GlcNH2 derivative is blocked (compound 69), this
synthesis results only in type II structures. In
particular, in Figure 23, compound 13 is prepared by
the methods described above. This compound is then
~ 093/24~05 2 1 1 8 5 2 2 PCT/US93/04909
-- 69
deacetylated by conventional techniques (sodium
methoxide/methanol) to provide for compound 1~ which is
then benzylidenated under conventional techniques to
provide compound 66. Compound 66 is then treated with
benzyl chloride and sodium hydride in dimethylformamide
at about -20C to 20C to provide for compound 67. The
benzylidine group of compound 67 is then removed with
80% aqueous acetic acid at about 80C for about 1-4
hours to provide for compound 68. Thi compound is
then selectively acetylated at the 6-position by use of
approximately equimolar amounts of acetyl
chloride/pyridine-in dichloromethane at about -10C to
provide for compound 69. Approximately 1.2 - 1.5
equivalents of the 1-~-bromo-2,3,4,6-tetraacetylated
galactose (described above) are added dropwise to a
solution of compound 69 in dichloromethane maintained
at about -30C in the presence of about 1.3 equivalents
of 2,6,-di-t-butyl-4-methylpyridine and about 1.3
equivalents of silver triflate. The reaction is then
maintained at -30C for l hour and then allowed to warm
to 5C and maintained there for about 2 hours. The
reaction is then quenched by the addition of methanol,
warmed to room temperature, and filtered through
celite. The filtrate is washed with aqueous sodium
bicarbonate. The recovered solution is dried and the~
stripped in vacuo to provide a crude product containing
compsund 70 which is purified by chromatography on a
silica gel column eluted with ethyl acetate:hexanes
~1:2) to give compound 7~.
The benzyl protecting group is then removed
by hydrogenolysis (H2/Pd on C) to provide for compound
71. Compound 71, in turn, is fucosylated in the same
manner as described above for compound 48 (to provide
for compound 49 as illustrated in Figure 6) so as to
provide for compound 72. Compound 72 is deacetylated
by conventional techniques described above to provide
211~2~ ~
W O 93/24505 P ~ /US93/04909' `
-- 70 --
for compound 73. Compound 73 is then converted to
compound 74 by conventlonal methodology (e.g.,
benzaldehyde dimethylacetal, DMF, pTSA), followed by
selective acetylation at the 6-position of the
partially protected GlcNH2 derivative by an
approximately equivalent amount of acetyl
chloride/pyridine in dichloromethane maintained at
about -50 to about -20C). Compound 74 is then
converted to compound 79 in the same manner (described
above) as compound 53 was treated to provide for
compound 60.
Alternatively, the free hydroxyl groups of
compound 74 can be acetylated with acetyl
chloride/pyridine in the manner described above and the
benzylidine group selectively opened by sodium
cyanoborohydride and ceric or aluminum chloride to give
the 2,3-diacety~-4-benzyl-6-hydroxy derivative on the
galactose moiety (not shown). This compound is then
functionalized at the 6-position of the galactose so as
to contain a sulfate, phosphate or -CHR18COOH group at
this position.
In addition to the above, the 2,6 positions
of the GlcNAc unit can be modified prior to coupling so
as to provide for type I and type II structures
modified at these positions which can optionally be
further modified in the manner described above to
; prepare the sulfated, phosphorylated or -CHR18COOH
substituted structures. As shown by Venot et al., U.S.
Patent Application Serial No. 07/887,747, filed May 22,
1992 as Attorney Docket No. 000475-011 and entitled
"MODIFIED SIALYL LEWISA COMPOUNDS" and by Venot et al.,
U.S. Patent Application Serial No. 07/887,746, filed
May 22, 1992 as Attorney Doc~et No. 000475-029 and
entitled "MODIFIED SIALYL LEWISX COMPOUNDS",
modification at the 2 and/or 6-positions of the GlcNAc
` -~093/24505 2 1 1 8 S ~ ~ PCT/US93Jo4909
-- 71
moiety of type I structures [~Gal(1-3)~GlcNAc-OR] and
on type II structures [~Gal(1-3)~GlcNAc-OR -- LacNAc-
OR] still permit the use of the ~Gal(l~3/4)~GlcNAc
a(l~3/4)fucosyltransferase on the deblocked compound.
The disclosures of both of these applications are
incorporated herein by reference in their entirety.
¦ i. Modification at the 2-position of GlcNAc
Modification at the 2-position of GlcNAc can
I be accomplished by a variety of ways. For example, the
¦ known37 2-azido-2-deoxy-glucose-OR compound (prepared,
¦ for example, by azidonitration of 4,5,6-
¦ triacetylglucal) can be protected at the 6 position
with a removable protecting group (i.e., Si(C6Hs)2tBu)
by conventional techniques37 and then combined with an
appropriate blocked galactose compound in the manner
described above to provide a mixture of blocked
~Gal(l~3)GlcN3-OR and ~Gal(l~4)GlcN3-OR derivati~es
20 which are readily separated by conventional techniques.
.
At the appropriate time during synthesis of
oligosaccharide glycosides related to-blood group
determinants having type I or type II core structures,
the azido group is reduced to an amino group which can
be protected as N-trifluoroacetamido. In turn, the
trifluoroacetamido group is removed at the appropriate
point in the synthesis thereby unmasking the amino
group.
The amino group can also be derivatized by
con~entional methods to provide for -NR11C(O)R1o,
-NHSO3H, -N=C(R1~)2~ -NHCH(R11) 2 ~ -NH~,2, and -N(R12) 2
groups. For example, the -NH2 group can be reacted,
using conventional techniques:
with a carboxylic acid, anhydride or chloride to
provide for amides. Alternatively, the desired acid
2118~)22
W093J24505 PCT~US93/0490
-- 72
can be activated, as reported by Inazu et al43 and then
reacted with the amino group. The carboxylic acid,
anhydride, chloride, or activated acid is selected so
as to provide for an R10 group (i.e., as part of the
-NR11C(O)R,o substituent) which is hydrogen or alkyl of
from 1 to 4 carbon atoms,
with an aldehyde or ketone (of from 1 to 4 carbon
atoms) at controlled pH to form an imine ~-N=C(R11) 2]
which upon reduction (e.g., with sodium cyanoboro-
hydride) provides for an alkylamine substituent [i.e.,-N~CH(R11)2~ as reported by Bernotas et al. 44,
with a cyclic carbonate such as ethylene carbonate
or propylene carbonate which ring opens upon reaction
with the amine to form a carbamate group having an
HO-alkylene-OC~O)NH- substituent where alkylene is from
2 to 4 carbon atoms as reported by Wollenberg et al. 45,
- U.S. Patent No. 4,612,132,
with a chloroformate [i.e., ClC(O)OR13] in the
manner disclosed by Greig et al. 46 . In this case, the
chloroformate has an R13 group which is alkyl of from 1
to 4 carbon atoms,
with O=C(O-C6H4-pNO2)2 which leads to an activated
intermediate which is then reacted with an amine
(HNR14R15) to provide for ureas [-NHC(O)NR14R1s] as
described by Piekarska-Bartoszewicz et al. 47,
with trimethylamine, sulfur trioxide (S03) SO as
to form the -NHSO3H group as described by Petitou48, and
with derivatized formic acid or other materials to
form a formamide (-NH-CHO) 49 which can be further
functionalized to the isocyano (-N=C=O) and reduced to
the deoxy derivative by tributyltin hydride (Bu3SnH) 49 .
- Alternatively, the 2-deoxy (R2 = H) and 2-
alkoxy glucose derivatives [i.e., derivatives of GlcNAc
where the NAc has been replaced by -H (deoxy) or by an
-OR-2 (alkoxy)] are prepared using a synthetic scheme
similar to that recited by Trumtez et al. 49
~ W O 93/24505 2 1 1 8 5 2 2 P ~ /US93/04909
-- 73
Specifically, the known 3,4, 6-triacylated 1,2-ortho
ester of glucose is deacylated under conventional
conditions to give the 1,2-ortho ester of glucose.
This compound is then converted to the 3,4,6-tribenzyl
S 1,2-ortho ester of glucose using conventional
techniques. The 1,2-ortho ester of the resulting
compound is then opened by conventional techniques to
provide a protected glycosyl donor such as the
1-~-bromo-2-acetyl-3,4,6-tribenzyl derivative of
glucose. This l-~-bromo derivative is then converted
to the glycoside (-OR) by conventional techniques and
the 2-acetyl group is then removed. The 2-position is
now ready for formation of the 2-deoxy by conventional
methods such as first treating with carbon disulfide
and methyl iodide in the presence of one equivalent of
a base to ~orm the -C(S)SCH3 derivative, followed by
reaction with tributyltin hydride or for the
preparation of the 2-al~oxy. The remaining protecting
groups are removed so as to provide for 2-deoxyglucose
glycoside or a 2-alkoxyglucose 71ycoside which can then
be derivatized in the manner described above and
illustrated in Figure 1 without the need to form ~he
aglycon.
~, .
ii. Modification at the 6-Position of GlcNAc
As shown in Figure 26, the 6-deoxy derivative
of GlcNAc-OR is synthesized from a known benzylidene
ring blocked saccharide (8-methoxycarbonyloctyl-2-
! acetamido-4,6-0-benzylidene-2-deoxy-~-D-gluco-
pyranoside) 50 which is protected at the 3-hydroxy
position with a removable benzoyl blocking group (Bz)
by reaction with benzoic anhydride or benzoyl chloride
in pyridine. Further canversion of this compound by
reaction with N-bromosuccinimide and barium carbonate
in carbon tetrachloride (CCl4) at 65C leads to the
3,4-dibenzoyl-6-bromo-GlcNAc-OR compound. This
compound is, in turn, converted to the 3,4-dibenzyl-6-
f
Wog3/~4505 211 ~ ~ 2 ~ PCT/US93/04909~
-- 74 --
deoxy-GlcNAc-OR by,,~eaction with (C4H9)3SnH in the
presence of AIBN (azo bis-isobutyronitrile) at 110C
followed by treatment with methanol/sodium methoxide.
This compound can then be deprotected by conventional
techniques to provide for the 6-deoxyGlcNAc-OR
glycoside which can then be derivatized in the manner
described above and illustrated in Figure 17 without
the need to form the aglycon as shown in Figure 17.
The 6-azido derivatives of GlcNAc-OR can be
prepared in the manner described in Figure 25.
Specifically, GlcNAc-OR, compound 87, is converted to
the p-methoxybenzylidine blocked compound 88 by
reaction with (CH3O~2CH-C6H4-p-OCH3. This compound is
then protected at the 3-hydroxyl position by reaction
with 4-CH3O-C6H4-CH2Br to provide for compound 89 where
X is 4-CH3O-C6H4-CH2-. Compound 89 is partially
deprotected at the 4 and 6 positions by reaction with
acetic acid (AcOH) in water at about 45~C to provide
for compound 90. The 6-mesylate, compound 91, is
prepared by reacting compound 90 with mesyl chloride in
: . pyridine (MsCl/py). The 6-azido derivative, compound
92, is then formed by reaction with sodium azide in
dimethylformamide (DMF) and removal of the 3-blocking
group with dichlorodicyanoquinone ~DDQ) yields compound
93.
The 6-mesyl compound 91 can also be
, derivatized to any of a number of 6-substituents
including alkoxy substituents, and the like by well
known chemistry.
The 6-azido compound 92 can be derivatized to
the 6-amino at an appropriate point in the synthesis of
the oligosaccharide glycosides related to blood group
determinants having a type I or type II core structure
in the manner described above. The 6-amino derivative
:'W093/24505 2 1 1 ~ S 2 2 ~CT/U~93/04909
can then be further functionalized by conventional
methods to provide for -NR5C (O) R4, -NHSO3H, -N=C (~) 2
-NHCH(~)2, -NHR6 and -N ( R6 ) 2 . For example, the~-NH2
group can be reacted, using conventional techniques:
a carboxylic acid, anhydride or chloride to
provide for amides. Alternatively, the desired acid
can be activated, as reported by Inazu et al43 and then
reacted with the amino group. The carboxylic acid,
anhydride, chloride, or activated acid is selected so
as to provide for an R4 group ~i.e., as part of the
-N~C(O)R4 substituent) which is hydrogen or alkyl of
from 1 to 4 ~arbon atoms,
with an aldehyde or ketone (of from 1 to 4 carbon
atoms) at controlled pH to form an imine [-N=C(R5) 2]
which upon reduction (e.g., with sodium cyanoboro-
hydride) provides for an alkylamine substituent [i.e.,
-NHCH(F~)2~ as reported by Bernotas et al. 44, `
with a cyclic carbonate such as ethylene carbonate
or propylene carbonate which ring opens upon reaction
with the amine to form a carbamate group having an
HO-alkylene-OC(O)NH- substituent where alkylene is from
2 to 4 carbon atoms as reported by Wollenberg et al. 45, -
U.S. Patent No. 4,612,132,
with a chloroformate [i.e., ClC(O)OR7] in the
manner disclosed by Greig et al.46. In this case, the
chloroformate has an R7 group which is alkyl of from 1
to 4 carbon atoms,
with O=C(O-C6H4-pNO2)2 which leads to an activated
intermediate which is then reacted with an amine
(HN~R9) to provide for ureas [-NHC(O)NR8R~] as
described by Piekarska-Bartoszewicz et al. 47,
with trimethylamine, sulfur trioxide (SO3) at pH
9.5 so as to form the -NHSO3H group as described by
Petitou48~ and
with derivatized formic acid or other materials to
form a formamide (-NH-CHo)49 which can be further
o5 2 1 1 ~ Pcr/usg3/o49~9t ~/-
A 76
. ,.
functionalized to the isocyano (-N=C=O) and reduced to
the deo'xy derivative by tributyltin hydride (Bu3SnH) 49 .
The 6-alkoxy derivatives of GlcNAc can be
prepared in the manner described in Figure 26.
Specifically, GlcNAc~OR, compound 87, is reacted with
C6HsCH(OCH3)2 in an acidic medium in acetonitrile to
provide for the 4,6-di~rotected benzylidine compound
94. In turn, compound 9~ can be reacted with benzyl
(Bn) bromide and sodium hydride in the presence of
dimethylformamide at around OC to provide for a benzyl
protecting group at the 3-~osition, i.e., compound 95.
Deprotection at the 4,6 positions by contacting
compound 95 with acetic acid and water at about ~0~-
90C provides for compound 96. Reaction of compound 9
with dibutyltin oxide [(Bu)2SnO] and R6Br provides for
the 6-alkoxy compound 97. Conventional deprotection of
the benzyl group with hydrogen in palladium/carbon
yields compound 9~.
In another embodiment, compound 94 can be
reacted with tC6H5C(0)]20 in pyridine to provide for a
benzoyl protecting group (Bz) at the 3-position, i.e.,
compound 99. Reaction of compound 99 with N-bromo-
succinimide in carbon tetrachloride yields the 6-bromo
compound 100. Compound 100 can be reacted with
tributyltin hydride ~(Bu)3SnH] in toluene to provide
for the 6-deoxy compound lOOb which after conventional
deprotection of the benzoyl groups with sodium
methoxide in methanol gives the 6-deoxy compound lOOc.
The 6-SR6 compounds are prepared from the 6-
mesyl derivative, compound 91, by reaction with
potassium thioacetate, CH3C(O)S-~, to give the
thioacetate derivative at the 6-position. This
derivative is then treated with mild base to produce
the 6-SH derivative. The 6-SH can be reacted with an
:- W093/24505 2 1 1 8 5 2 2 - - PCT/US93/04909
alkyl halide (e.g., CH3Br) to provide the -SR6
derivatives which, in turn, can be partially or fully
oxidized to the 6-sulfone or the 6-sulfoxide
derivatives, -S(O)R~ and -S(O)2R6 where R6 is alkyl of
from 1 to 4 car~on atoms.
Cl(ii) Alternative Methods For Preparing Oligo-
saccharide Glycosides Related to Blood Group
Determinants Having a Type I or Type II
_ Core Structure
Alternative methods for preparing
oligosaccharide glycosides related to blood group
determinants having type I or type II core structures
lS can be employed using known chemistry. Additionally,
certain of the type I or type II core structures can be
enzymatically converted to LewisA and LewisX structures,
to sialylated type I or type II structures, and to
- sialyl LewisA and sialyl LewisX structures.
The following discussion is directed to the
- preparation of sialyl LewisA and sialyl LewisX
structures modified at the 2 and/or 6 positions of the
N-acetylglucosamine (GlcNAc)~ unit and/or at the 2
position of the galactose unit of these ~structures. It
being understood, however, that one skilled in the art
c~uld readily prepare modified LewisA and LewisX
structures or sialylated type I or type II structures
merely by omitting the sialylation step in the case of
LewisA and LewisX and merely by omitting the
fucosylation step in the case of sialylated type I or
type II structures.
Derivatives of sialyl LewisX, modified at the
2 and/or 6 positions of the N-acetylglucosamine unit
and/or at the 2-position of the galactose unit are
prepared by first synthesizing the ~Gal(1~4)~GlcNAc-OR
W093/24505 2 1 1 ~ 5 2 ~ PCT/US93J04909'~`-
-- 78 --
-
backbone or the ~Gal(~3)~GlcNAc-OR backbone
derivatized at the Z and/or 6 positions of the
N-acetylglucosamine unit and/or at the 2 positio~ of
the galactose unit. These backbones are then
sequentially sialylated and fucosylated using the
~Gal(1-3/4)~GlcNAc ~(2~3)sialyltransferase and the
~Gal(1~3/4)~GlcNAc ~ 3/4)fucosyltransferase or other
suitable sialyl- or fucosyltransferases. In this
regard, it has been previously disclosed that this
sialyltransferase requires the presence of a hydroxyl
group at the 3, 4, and 6 positions of galactose, and a
hydroxyl group at the 4-position of the GlcNAc unit in
type I structures or at the 3-position of the GlcNAc
unit in type II structures~ 4. Likewise, it has been
previously disclosed that this fucosyltransferase
requires the presence of hydroxyl groups at the 6-
position of the galactose unit and at the 4-position of
the GlcNAc unit for type I structures and at the 3-
position of the GlcNAc unit for type II structures11~14.
However, both the ~Galt1~3/4)~GlcNAc ~(2-3)sialyltrans-
ferase and the ~Gal(1-3/4)~GlcNAc ~ 3/4~fucosyltrans-
: ferase tolerate substitution at the 2,6 positions of
the GlcNAc unit and some substitution at the 2 position
of the galactose unit in type I and type II
structuresl1 '14 .
The use of such sialyltransferases and
fucosyltransferase provides for the facile synthesis of
analogues of sialyl LewisX and sialyl LewisA including
those having modification on either the sialyl and/or
fucosyl groups. For example, use of such
sialyltransferases permits the transfer of Neu5Ac or
compatible analogues of Neu5Ac to the backbone
structure9; whereas the use of such fucosyltrnsferases
permits the transfer of fucose and compatible analogues
thereof to these backbone structures.
~1185~2
W093/24505 ! PC~US93t04909
-- 79
General schemes for these alternative methods
for preparing sialyl LewisX derivatives and, in some
cases, sialyl LewisA derivatives are set farth in
Figures 27A-33. It being understood that where only
sialyl LewisX is disclosed, similar methods can be used
to prepare sialyl LewisA derivatives as evidenced by
the Examples.
Specifically, trisaccharide 104 set forth in
Figure 27 is a known compound and is disclosed by
Ratcliffe, et al12~37. This compound is then derivatized
by conventional steps well known in the art to provide
for a trisaccharides lllb, lllc, and llld described in
the Examples.
Specifically, hydrogenation (H2) of the
benzyl ester (-COOBn) of trisaccharide 104 at
atmospheric pressure in ethyl acetate (CH3C02C2Hs) in
the presence of 5~ palladium on carbon (Pd/C), followed
by de-~-acetylation with sodium methoxide in methanol
(CH30Na, CH30H) provided trisaccharide lllb. The use of
~-~ ethyl acetate as solvent is recommended~in the first
step in order to leave the 2-azido group untouched.
Only a very small amount of impurity is formed in this
step which can be separated by conventional separation
techniques (e.g., chromatography).
~ .
Alternatively, reduction of the 2-azido group
; of tetrasaccharide 104 by hydrogen sulfide (H2S) in a
mixture of pyridine, water and triethylamine provided
the 2-amino trisaccharide 109. Reduction of the benzyl
ester (-COOBn) followed by de-O-acetylation (as
described above) lead to trisaccharide lllc.
Trisaccharide llld is prepared by first
conducting N-propionylation of trisaccharide 109 using
propionic anhydride [(CH3CH2CO)20] in methanol (CH30H)
o93/245052 l 1 8 a 2 ~ PCT~US93/04909,;
-- 80 --
to provide for trisacchar de ll0. Trisaccharide ll0
was accompanied by a small amount of the corresponding
4-O-propionylated material which can be separat~d by
conventional separation techniques (e.g.,
chromatography). Removal of the acetyl and benzyl
protecting groups, as indicated above, provided the
trisaccharide l~ld.
Trisaccharide lllc can also be derivatized by
conventional methods to provide for -NR11C(O)R1o,
NHSO H -N=C(R11)2, -NHCH(R11)2, -NHR12~ N(R12)2~
an amino acid or polypeptidyI residue derivatives by
conventional methods. For example, the -NH2 group can
be reacted, using conventional techniques:
with a carboxylic acid, anhydride or chloride to
provide for amides. Alternatively, the desired acid
can be activated, as reported by Inazu et al43 and then
reacted with the amino group. The carboxylic acid,
anhydride, chloride, or activated acid is select~d so
as to provide for an R10 group (i.e., as part of the
-NR11C(O)R10 substituent) which is hydrogen or alkyl of
from l to 4 carbon atoms,
with an aldehyde or ketone ~of from l to 4 carbon
atoms) at controlled pH to form an imine [-N=C(R11) 2]
which upon reduction ~è.g., with sodium cyanoboro-
hydride) provides for an alkylamine substituent [i.e.,
-NHCH(R11)2] as reported by Bernotas et al.44,
with a cyclic carbonate such as ethylene carbonate
! or propylene carbonate which ring opens upon reaction
with the amine to form a carbamate group having an
HO-alkylene OC(O)NH- substituent where alkylene is from
2 to 4 carbon atoms as reported by Wollenberg et al. 4S,
U.S. Patent ~o. 4,612,132,
wi~h a chloroformate [i.e., ClC(O)OR13] in the
3 manner disclosed by Greig et al. 46 . In this case, the
chloroformate has an R13 group which is alkyl of from l
to 4 carbon atoms,
~ ` WO 93/24505 2 1 1 3 5 2 ~ PCr/US93/04gO9
---- 8 1 ----
with O=C tO-C6H4-pNO2) 2 which leads to an activated
intermediate which is then reacted with an amine
(HNR,4Rl5) to provide for ureas [-NHC(O)NRl4Rl5] as
described by Piekarska-Bartoszewicz et al. 47,
with trimethylamine, sulfur trioxide (SO3) so as
to form the -NHSO3H group as described ~y Petitou48, and
with derivatized formic acid or other materials to
form a formamide (-NH-CHO) 49 which can be further
functionalized to the isocyano (-N=C=O) and reduced to
the deoxy derivative by tributyltin hydride (Bu3SnH) 49 .
with an appropriate form of an amino acid or
polypeptide moiety activated at the acid group as
reported by Bodanszky et al.Sl;
Trisaccharides lllb, lllc, and llld and
derivatives derived therefrom can then be fucosylated
by contacting the appropriate trisaccharide with
~Gal(1~3/4)~GlcNAc ~ 3/4)fucosyltransferase in the
presence of GDP-fucose (GDP-Fuc) so as to provide
tetrasaccharides 112b, 112c, and 112d which are
analogues of sialyl LewisX.
Figure 28 illustr~tes a general scheme for
preparing the sialyl LewisX analogues from an
appropriately derivatized ~Gal(1-4)~GlcNAc-OR structure
by the sequential enzymatic sialylation and
~ucosylation of this structure. Figure 28 only
illustrates modification at the 2 or 6 position of the
N-acetyl-glucosamine (GlcNAc) structure. However, it
is understood that the modifications can be combined to
provide for modification at both the 2 and 6 position
of the N-acetylglucosamine. It is further understood
that while Figure 28 illustrates only a 2-hydroxyl
group at the 2 position of the galactose, this position
may also be substituted with hydrogen or fluoro. Such
substituted galactose compounds are known in the art.
Substitution of these galactose compounds in the
2118~2 ~
W093/24505 PCT/US93/04909~;-
-- ~2
. ,
reactions depicted in the figures lead to these
modified galactose units in the sialyl LewisX
analogues.
Enzymatic Si~l~lation
In Figure 28, sialylation is accomplished by
use of the ~Gal(1-3/4)~GlcNAc ~(2~3)sialyltransferase
~i.e., ~Gal(1-3/4)~GlcNAc ~(2~3)ST]. The enzymatic
transfer of sialic acid onto the 3-position of
galactose to form ~-sialyl(2-3)~Gal- requires the prior
synthesis (i.e., activation) of its nucleotide (CMP)
derivatives. Activation of sialic acid is usually done
by using the enzyme CMP-sialic acid synthase which is
readily available and the literature pro~ides examples
of the activation of various analogues of sialic acid
such as 9-substituted Neu5Ac52~53~54~55~57, 7-epi-
Neu5Ac58, 7,8-bis-epi-Neu5Ac58, 4-O-methyl-Neu5Ac59, 4-
deoxy-Neu5Ac6, 4-acetamido-NeuSAc62, 7-deoxy-Neu5Ac56,
4,7-dideoxy-Neu5Ac56, the 6-thio derivatives of Neu5Ac
ZO and Neu50H (KDN).
The resulting CMP-sialic acid analogue,
illustrated in Figure 28 as the CMP deriYative of
Neu5Ac (i.e., CMP-Neu5Ac), is then combined with the
derivatized ~Gal(1~4)~GlcNAc-OR compound in the
presence of the ~Gal(1~3/4)~GlcNAc ~(2-3)sialyltrans-
ferase under conditions wherein sialic acid is
transferred to the 3 position of the galactose to form
a ~Neu5Ac(2~3)~al- linkage. Suitable conditions,
known in the art r include the addition of the
sialyltransferase to a mixture of the derivatized
~Gal(1~4)~GlcNAc-OR compound and of the CMP-sialic acid
in a appropriate buffer such as 0.1 ~ 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, while incubating for 12 hours
to 4 days. The resulting sialylated product can be
--W093/2450~ 2 1 1 ~ 5 2 2 PCT/US93/04909
-- 83
isolated and purified using conventional methodology
comprising HPLC, ion exchange-, gel-, reverse-phase-
or adsorption chromatography.
Enzvmatic fucosYlation
In Figure 28, fucosylation is accomplished by
use of ~Gal(1-3/4)~GlcNAc ~ 3/4)fucosyltransferase
[i.e., ~Gal(1-3/4)~GlcNAc ~(1~3/4)FT]. The enzymatic
transfer of fucose onto the 3-position of GlcNAc to
form ~Fuc(1~3)~GlcNAc rPquires the prior synthesis of
its nucleotide (GDP) derivatives. Synthesis of GDP-
fucose is preferably accomplished in the manner recited
by Jiang et al.42 and which is exemplified in the
examples hereunder.
GDP-fucose (GDP-Fuc) is then combined with
the sialylated ~Gal(1-~4)~GlcNAc-OR compound in the
presence of the ~Gal(1~3J4)~GlcNAc (1~3/4)fucosyl-
trans~erase under conditions wherein fucose istransferred to the 4 position of the GlcNAc unit of the
sialylated ~al(1~4)~GlcNAc-OR compound so as to form a
~Neu5Ac(2-3)~Gal(1l4)[aFuc(1-3)]~GlcNAc-OR compsund
~when the sialic acid is aNeuSAc) derivatized in the
~Gal(1~4)~GlcNAc backbone. Suitable conditions, known
in the art, include the addition of the fucosyl-
transferase to a mixture of the derivatized
~Neu5Ac(2~3)~al(1~4)~GlcNAc-OR compound (when the
sialic acid is ~Neu5Ac) and of the GDP-fucose in a
appropriate buffer such as 50 mM sodium cacodylate in
appropriate conditions of pH and temperature such as at
a pH of 6.5 and a temperature between 30 and 45C,
preferably 35-40C, while incubating for 12 hours to 4
days. The resulting sialylated and fucosylated product
ca~ be-isolated and purified using conventional
methodology comprising HPLC, ion exchange-, gel-,
reverse-phase- or adsorption chromatography.
W093/24505 2 1 1 8 5 2 2 PCT/US93/04909~
-- 84
In the case of trisaccharides lllb-d,
preparative fucosylation of these trisaccharides ~as
performed according to Palcic et al. ~5 The products
were purified as indicated therein. The structures of
trisaccharides l1lb-d were con~irmed by 1H-n.m.r. at
300 MHz and those of the resulting sialyl LewisX
compounds ll~b-d by lH-n.m`.r. at 500 MHz
Figure 29 illustrates the chemical synthesis
of specific disaccharide derivatives of
~Gal(1~3)~GlcNAC-OR and ~Gal(1~4)~GlcNAc-OR structures
starting with saccharide monomers. In this regard, the
chemical coupling of the galactose and GlcNAc-OR units
results in the formation of both ~Gal(1-3)~GlcNAc-OR
ttype I backbone) and ~Gal(1~4)~GicNAc-OR (type II
backbone) which can be separated by conventional
purification techniques (i.e., chromatography).
- Specifically, in Figure 29, the known12~37
2-azido compound 116 is protected at the 6 position
with a removable protecting group (i.e., Si(C6Hs)2tBu)
by conventional techniques12~37. This derivative 117 is
then combined with a fully acylated derivative of
galactose 118 in the presence trimethylsilyltrifluoro-
methanesulfonate (TMSOTf) and afterwards ammonium
chloride (NH4Cl), potassium fluoride (KF) in
tetrahydrofuran are added. The reaction yields a
mixture of type I and type II derivatives (i.e.,
~Gal(1-3)~GlcNAc-OR and ~Gal(1~4)~GlcNAc-OR
derivatives), compounds 119 and 121, which are
separated by conventional methods such as
chromatography.
~ither derivative 119 or 121 is then
deprotected with a mixture of sodium methoxide in
methanol (CH3ONa/CH3OH) to provide for derivative 120b
or 122~ respectively which can be converted to either
W093/24505 2 1 1 ~ 5 2 ~ PCT/US93/04909
~- 85 --
the amine derivative 120c or 122c respectively or the
propionate (Pr) derivative 120d or 122d respectively
following similar procedùres set forth above for
trisaccharides lllc and llld.
Alternatively, derivative ll9 or 121 can be
tosylated by conventional techniques to provide for a
tosyl group at the 6-position of the GlcNAc derivativec
The tosyl derivative can then be used to form a 6-halo
substituent by a substitution reaction using the
appropriate nucleophilic reagent or a 6-alkoxy
substituent by alkylation with an alkyl halide in the
presence of bis-tributyltin hydride, and the like.
Additionally, while not shown in Figure 29,
the 2-deoxy (R2 = H) and 2-alkoxy glucose derivatives
are prepared using a synthetic scheme similar to that
recited ~y Trumtez, et al. 49 Specifically, the known
3,4,6-triacylated 1,2-ortho ester of glucose is
deacylated under conventional conditions to give the
1,2-ortho ester of glucose. This compound is then
converted to the 3,4,6-tribenzyl 1,2-ortho ester of
glucose using conventional techniques. The 1,2-ortho
ester of the resul~ing compound is then opened by
conventional techniques to pr~vide a protected glycosyl
donor such as the 1-~-bromo-2-acetyl-3,4,6-tribenzyl
derivati~e of glucose. This 1 ~-bromo derivative is
then converted to the glycoside (-OR) by conventional
techniques and the 2-acetyl group is then removed. The
2-position is now ready for formation of the 2-deoxy by
conventional methods (e.g., first treating with carbon
disulfide and methyl iodide in the presence of one
equivalent of a base to form the -C(S)SCH3 derivative,
followed by reaction with tributyltin hydride) or for
the preparation of the 2-alkoxy.
WOg3/24~ 2 ~ PCT/US93/0490?-~ i
-- 86 -
Figure 30 illustrates the synthesis of the
6-deoxy derivatives on the GIcNAc unit of
~Gal(1~3)~GlcNAc-OR and-~Galt1-4)GlcNAc-OR, compounds
114~ and 115b, and the 6-bromo derivative on the GlcNAc
unit of ~Gal~1-4)GlcNAc-OR, compound 115a. The 6-deoxy
compounds 114b and 115b are synthesized from a known
benzylidene ring blocked saccharide (8-methoxycarbonyl-
octyl 2-acetamido-4,6-O-benzylidene-2-deoxy-~-D-gluco-
pyranoside) which is protected at the 3-hydroxy
position with a removable benzoyl blocking group (Bz)
by reaction with benzoic anhydride in pyridine.
Further conversion of this compound by reaction with
N-bromosuccinimide and barium carbonate in carbon
tetrachloride (CCl4) at 6SC leads to the 3,4-
dibenz.oyl-6-bromo-GlcNAc compound. This compound is,
in turn, converted to the 3,4-dibenzoyl-6-deoxy-GlcNA
by reaction with (C4H9)3SnH in the presence of AIBN (azo
bis-isobutyronitrile) at 110C followed by treatment
with methanol/sodium methoxide. The resulting 6-deoxy-
GlcNAc glycoside is reacted with a known 2,3,4,6-
tetraacylated derivative of galactose having an
appropriate leaving group at the 1 position to permit
for~.ation of a ~ linkage. Suitable leaving groups
incll1de ~-bromo and ~-trichloroacetamidate
[~-C(=NH)CCl3]. The reaction is conducted in the
presence of a catalyst which facilitates ~ linkage
formation. Suitable catalysts include silver
trifluoromethane sulfonate in the presence of tetra-N-
methyl urea when the precursor is a galactosyl bromide;
and boron trifluoride athereate when the donor is
galactosyl trichloroacetamidate. The reaction leads to
a mixture ~Gal(1-3)~GlcNAc-OR and ~Gal(1-4)GlcNAc-OR
protected compounds which c~n be isolated and separated
by conventional ~echniques (e.g., chromatography).
Removal of the removal protecting groups then leads to
compound 114b or 115b.
~ V093/24505 2 1 1 ~ 5 2 ~ PCT/US93/04909
-- 87 --
As also shown in Figure 30, the 6-bromo-
GlcNAc glycoside precursors can be reacted with a known
2,3,4,6-tetraacylated derivative of galactose having an
appropriate leaving group at the 1 position to permit
S formation of a ~ linkage so as to provide for a route
to the 6-bromo compounds. Suitable leaving groups
include ~C(=NH)CCl3 and the reaction is conducted in
the same manner as that employed to prepare compound
115a (i.e., ~Gal(1~4)~GlcNAc~OR having a bromo group at
the 6-position of the GlcNAc unit).
Figures 31 and 32 illustrate the chemical
synthesis of ~-sialyl(2~3)~Gal(1~4)~GlcNAc-OR and
~-sialyl(2~3)~Gal(1-3)~GlcNAc-OR derivatives modified
at the 6-position (Figure 31) or the 2-position (Figure
32) of the GlcNAc deriv~.tive by using one of the
procedures described in Ratcliffe et al.12~37
Specifically, as illustrated in Figure 31,
the appropriate 6-substituted derivatives of GlcNAc-OR
are prepared as above from either known50 glycoside 127
or from the known benzylidene ring blocked saccharide
protected form depicted in Figure 30 (which is derived
from glycoside 127) as.describ~d in detail above. The
6-derivatized blocked material (as depicted in Figure
5~ is then deblocked using conventional methods to
provide for compound 128 which is a 6-derivative of
GlcNAc.
Compound 128 is then combined with
disaccharide 129b in a manner known in the art12~37 to
provide for trisaccharides 130 and 131 having
conventional removable blocking groups on the Neu5Ac
and the galactose units. Specifically, compound 129b
is synthesized from the disaccharide 129a by known
methods and is then reacted with compound 128 in the
presence of an appropriate catalyst such as
W093/24so~ 2 ~ PcT/us93~04909 -
-- 88 --
[BF30(C2H5)2] to give a mixture of the corresponding
trisaccharides 130 and 131, respectively. The ratio of
compounds 130.131 will depend upon~the nature of the
substituent R1 and on the reaction conditions. In any
event, trisaccharides 130 and i31 are typically
separated and purified by conventional techniques
including chromatography. Removal of the blocking
groups on trisaccharides 130 and 131 is also conven-
tional (i.e., addition of hydrogen in the presence of
palladium on carbon followed by treatment with sodium
methoxide in the presence of methanol) and leads to the
trisaccharide ~Neu5Ac(2~3)~Gal(1~3)~Gl~cNAc-OR 123 and
~Neu5Ac(2~3)~Gal(1-4)~GlcNAc-OR 125. Fucosylation of
trisaccharides 123 and 125 is prefera~ly conducted with
GDP-fucose (GDP-Fuc) in the presence of
~Gal(1-3/4)~GlcNAc ~ 3/4)fucosyltransferase
[~Gal(1~3/4)~GlcNAc ~ 3/4)FT] to lead to sialyl
LewisA analogues 124 or to sialyl LewisX analogues 126
modified at the 6-position of the GlcNAc unit.
When the R1 substituent is azido (-N3 -- the
synthesis of which is described below), this substi-
tuent can be further functionalized to other
appropriate R1 substituents as described above either
at the monosaccharide level ~as shown in Figure 31) or `
at the trisaccharide 130 or 131 level. For example, if
the R1 group of trisaccharide 130 or 131 is an azido
group, then this group can be functionalized in
trisaccahride 130 or 131 to provide for the amino,
amido, imino, etc. substituents de~cribed above.
In any event, functionalization i generally
at a point in the synthesis where the to-be formed
functional group does not interfere with any of the
further intended reactions. For example, if an R
functional group in monosaccharide 128 would interfere
with the coupling reaction between disaccharide 129b
W093/24505 ~ 3 2 2 PCT/USg3/04909
-- 89 --
and monosaccharide 128 then this functional group can
~e introduced into trisaccharide 130 or 131.
In Figure 32, the appropriate 2-substituted
6-protected derivatives of GlcNAc-OR, compound 132, are
prepared, for example, from the known bloc~ed
saccharide 117 depicted in Figure 29.
Compound 132 is then combined with
disaccharide '29a or 129b using methods known in the
art such as those described by Ratcliffe et al.12~37 to
provide for trisaccharides having conventional
removable blocking groups on the Neu5Ac, on the Gal,
and on the 6-position of the GlcNAc units.
Specifically, compound 12Sb is synthesized from the
disaccharide 129a and is then reacted with compound 132
in the presence of an appropriate catalyst such as
[BF30(C2H5)z] to give a mixture of the corresponding
type I or type II linked trisaccharides,~respectively.
The ratio of the type I to type II compounds will
depend upon the nature of the substituent R2 and on the
reaction conditions. In any event, these
trisaccharides are typically separated and purified by
conventional techniques including chromatography.
Fucosylation of either of these protected type I or
type II trisaccharides is then accomplished by reaction
of the trisaccharide with an appropriate fucosyl donor
such as tetra-0-benzyl-fucopyranosyl bromide as recited
by Ratcliffe et al.12~37 Removal of the ~locking groups
on the re-~ulting tetrasaccharide is also conventional
and leads to sialyl LewisX and sialyl LewisA analogues
modified at the 2-position of the GlcNAc unit.
Alternati~ely and in a pref~rred embodiment,
fucosylation is accomplished by contacting the
deprotected trisaccharide with GDP-fucose (GDP-Fuc) in
the presence of ~Gal(1-3/4)~GlcNAc a(1~3/4)-
W093/24~ a 2 2 PCT~US93/04g09.
__ 90
fucosyltransferase [~Gal(1~3/4)~GlcNAc ~ 3/4)FT] tolead to sialyl LewisA or sialyl LewisX analogues
modified at the 2-position of the GlcNAc unit. ~
As noted above, when the R2 substituent is
azido (-N3), this substituent can be further
functionalized to other appropriate R2 substituents as
described above either at the monosaccharide level or
at the protected trisaccharide level. For example, if
the R2 group of the protected trisaccharide is an azido ;
group, then this group can be functionalized in this .~
trisaccahride to provide for the amino, amido, imino, :-.
etc. substituents described above. Functionalization ~:
is generally at point in the synthesis where the to-be
lS formed functional group does not interfere with any of
the further intended reactions. For example, if an R2 :
functional group in monosaccharide 132 would interfere
with the couyling reaction between disaccharide 129b
and monosaccharide 132 then this functional group can
be introduced into the protected trisaccharide.
Other derivatives at the 6-position of the
GlcNAc can be prepare by art recognized methods and
then ~hese compounds can be coupled to the galactose to
form ~Gal(1~3t~GlcNAc-OR derivatives and
~Gal(1~4)~GlcNAc-OR derivatives which can be separated
by conventional techniques (e.g., chromatography). The
~Gal(1~3)~GlcNAc-OR and ~Gal(1~4)~GlcNAc-OR
derivatives, in turn, can be sialylated and fucosylated
as described above, to provide the sialyl LewisA and
sialyl LewisX derivatives modified at the 6-position.
In regard to the above, compound 128 having a
chloro, bromo or iodo substituent at the 6 position can
be prepared by direct halogenation of the unmodified
~lcNAc-OR using the methods reported by Belkhouya et
al 63
W O 93/24505 ~ 1 1 S 3 2 2 P ~ /US93/049~9
---- 91
ThP 6-azido derivatives of GlcNAc-OR can be
prepared in the manner described earlier in Figure 25.
As also described earlier, the 6-azido compound ~an be
derivatized to the 6-amino at an appropriake point in
the synthesis of the oligosaccharide glycoside related
to blood group determinants having a type I or type II
core structure in the manner described above for ;~
trisaccharide 103. Additionally, as still further
described earlier, the 6-amino derivative can then be
further functionalized by conventional methods to
provide for -NHSO3H, -NR5C(O)R4, -N=C(R5)2, -NHCH(R5)2,
-NHR6, and -N(R6)z or an amino acid or polypeptidyl
residue derivatives by conventional methods.
The 6-alkoxy, 6-bromo, and 6-deoxy
derivatives of GlcNAc can be prepared in the manner
described in Figure 26.
The 6-fluoro compound is prepared from known
chemistry72 by reacting compound 49 with mesyl chloride
in pyridine to form the 6-mesylate which upon reaction
with tetraethylammonium fluoride provides for the
6-fluoro derivative. Deprotection of the 3 benzyl
group by hydrogen and palladium on carbon gives the
6-deoxy 6-fluoro derivative of compound 40.
- The above reaction schemes depict a number of
2- or 6- substituted deri~atives of GlcNAc. However,
it is apparent that these modifications can be combined
to provide for substituents at both the 2- and 6-
positions. When disubstitution is desired, the
modifications are conducted at an appropriate point in
the synthesis so as to be compatible with each other.
That is to say that modification at the 2-position must
be made with respect to the modification at the 6-
position. This is within the ordinary skill of the
art.
W093/24s0s 211~ ~ 2 ~ PCT/US93/04909
-- 92 --
Additionally, as noted above, the desired
modifications to the 2 and/or 6 derivatized materials
(especially of the 2-azido) are done at appropri~ate
point in the synthetic route so as not to introduce a
functionality that is incompatible with subsequent
reactions. However, in the case of the 6-substituted
derivatives of GlcNAc, the ~Gal~1-4) linkage can be
formed by using UOP-galactose and the commercial GlcNAc
~(1-4)galactosyl transferase, which is known to accept
modification at the 6 position~.
The following examples are offered to -
illustrate this invention and are not to be construed
in any way as limiting the scope of this invention.
Unless otherwise stated, all temperatures are in ~-
degrees Celsius. Also, in these examples, unless
otherwise defined below, the abbreviations employed
have their generally accepted meaning:
A = : Angstroms ~
~B = AB pattern -`
ATP = adenosine tri-phosphate
ax = axial
bs = broad singlet
BSA = bovine serum albumin
bt = broad triplet
CDP = cytidine di-phosphate
13C-n.m.r = C3 nuclear magnetic resonance
d = doublet
dd = doublet of doublets
ddd = doublet of doublets of doublets
- DDQ = dichlorodicyanoquinone
DTH = delayed-type hypersensitivity
eq = equatorial
= gram
35 H-n.m.r. = proton nuclear magnetic resonance
i.r. = infra red
kg - kilogram
L = liter
m = multiplet
mL = milliliter
q = quartet
s = singlet
t = triplet
t.l.c. = thin layer chromatography
U = Units
~m = microns
W093/2450s 2 1 1 ~ S 2 2 PCT/US93/04909
93 --
AG 1 X 8 (formate form) = ion exchange resin AG 1 x 8
(format~ form) available from Bio-Rad
Laboratories, Richmond, CA
Dowex 50W X 8 (H' form) = ion exchange resin Dowex 50 X
8 (H' form) available from Dow Chemical,
Midland, MI
IR-120 resin ~H~ form) = amberlite resin available from
Rohm & Haas, Philadelphia, PA
IR-C50 resin (H' form) = ion exchange resin IR-C50 (H+
form) available from Rohm & Haas,
Philadelphia, PA
- 20 Commercially available components are listed
by manufacturer and where appropriate, the order
number. Some of the recited manufacturers are as
follows:
Amersham = Amersham Canada Limited, Ontario, Canada
BioRad = Bio-Rad Laboratories, Richmond, California
Iatron - Iatron Laboratories, Tokyo, Japan
Merck = E. Merck AG, Darmstadt, Germany
Millipore = Millipore Corp., Bedford, MA.
Pel-Freeze Biologicals = Pel-Freez, Rogers, Ar~ansas
Pharmacia = Pharmacia Biosystems, Inc., Piscataway, NJ
Serva = Serva Felnbiochemica, Heidelberg, Germany
Sigma = Sigma Chemical Company, St. Louis, MS.
Waters = Waters Associates, Inc., Milford, MA.
EXAMP~ES
WO93t24~0s 2 1 1 ~ ~ 2 ~ PCT/US93/04909
-- 94 -- :-
In the following:examples, Examples A-L ::
illustrate the suppression of antigen-induced ;
inflammation in a mammal by administration of a~
oligosaccharide glycoside related to blood group :.
determinants having a type I or type II core structure
and the induced tolerance to later challenges with the
same antigen and Examples 1- illustrate the synthesis
of oligosaccharide glycosides related to blood group -
determinants having a type I or type II core structure
as well as components thereof.
Example A -- In~ibition of DTX Inflammatory Re~ponse
DTX inflammatory responses were measured :~
using the mouse footpad swelling assay as described by
Smith and Ziola~ Briefly, groups of Balb/c mice
(about 19-20 grams each) were immunized with 10 ~g of
the Ll~1 S-Layer protein, a bacterial surface protein69
from Clostridium thermohvdrosulfuricum L111-69 which
has been shown to induce a strong inflammatory DTH .~-
response or with 100 ~g of the OVA antigen containing
20~g of the adjuvant (DDA -- dimethyldioctadecyl-
ammonium bromide) which also induces a strong
infla~matory DTH response. Seven days later, each
group of mice was footpad-challenged with either 10 ~g ;
- of L-111 S-Layer protein or with 20 ~g of the OVA
antigen (without adjuvant). The resulting inflammatory
footpad swelling was measured with a Mitutoyo
Engineering micrometer 24 hours after challenge.
To assess the effect of oligosaccharide
glycosides related to blood group determinants having
type I and type II core structures on the inflammatory
DTH response, groups of mice received 100 ~g of the
following oligosaccharide glycosides related to blood
group determinants having a type I or type II core
structure:
W093/24505 2 1 1 8 S 2 ~ : PCT/US93/04909
---- g s
1. ~Neu5Ac(2~3)~Gal(1-3)-[~-L-Fuc(1-4)]-~GlcNAc-OR
(Sialyl LewisA or C19.9)
2. aNeu5Ac(2~3)~Gal(1-3)~GlcNAc-OR (Sialyl LewisC or
Sialyl Le C)
3. ~Neu5Ac(2~3)~Gal(1-4)~GlcNAc-OR (Sialyl LacNAc)
4. ~Gal(1-4)-[~-L-Fuc(1-3)]-~GlcNAc-OR (LewisX-OR or
Cl9.g)
5. ~Neu5Ac(2~3)~Gal(1-4)-[~-L-Fuc(1-3)]-~GlcNAc-OR
(Sialyl LewisX or Sialyl Le X)
R = -(CHz)gCO2CH3
These compounds were injected as a solution into the
tail vein, 5 hours after challenge. Control groups
were left untreated or received 100 ~L of phosphate-
buffered saline (PBS). The results of this experiment
are shown in Figure 1. Mice injected with sialyl
LewisX-OR had the most reduction in the footpad
swelling compared to control mice. Mice injected with
sialyl LewisA-OR, sialyl LewisC-OR, LewisX-OR, and
sialyl LacNAc-OR (structures related to Sialyl-LewisX)
also exhibited reductions in swelling compared to the
footpad swelling of control mice. As shown in Figure
2, mice injected with ~-sialyl-OR, ~-sialyl-OR or l'T"
disaccharide ~Gal(1~3)~GalNAc-OR], which is neither a
type I or type II structure, had essentially the extent
of footpad swelling observed in control mice.
An additional oligosaccharide glycoside was
tested for its ability to reduce antigen induced
inflammation (DTH response) in sensitized mammals in
the manner set forth above using 20~g of the OVA
antigen as the antigen challenge. The results of these
experiments are set forth below:
211852~ ~
WOg3/24505 PCT/US93/0440~
`::
-- 96 -- ~
. . ,
Timea of % Reductionb
Antiqen Com~ound _ Admlnistration in Inflam. -.
OVA ¦ A '.~ 5 hrs ¦ -76
., ,~
a hours after challenge with antigen
b % reduction determined as per Example H
below.
..
Compd A = ~Neu5Ac(2-3)~Gal(1-4)~GlcNAc(1-3)~Gal(1-4)[~- -
L-Fuc(1-3)]~GlcNAc-OR (CD 65)
In a side-by-side analysis, 100 ~g of several
oligosaccharide glycosides related to blood group
determinants having a type I or a type II core
structure were tested for their ability to reduce
antigen induced inflammation (DTH response) in
~: ~sensitized mammals in the manner set forth above using
20~g of the OVA antigen as the antigen. Administration
of these oligosaccharides glycosides was conducted 5
hours after antigen challenge. The results of these
experiments are set forth below:
.W093t24505 ~118522 PCT/~IS93/04~09
97 --
% Reductionb
Compound in Inflammation
C -55
D -60
E -24
F ~31
G ~17
H -45
I ~53
J ~14
Compound C = Sialyl LewisX-OR (~Neu5Ac(2~3)~Gal(1-4)-
[~-L-Fuc(1-3)]-~GlcNAc-OR)
Compound D - SO3-LewisX-OR (sulfate substituent on the
3-position of the galactose of LewisX-OR)
Compound E ~ SO3-LacNAc-OR (sulfate substituent on the
3-position of the galactose of LacNAc-OR)
Compound F = Sialyl LewisA-OR (~Neu5Ac(2-3)~Gal(1-3)-
[~-L-Fuc(1-4)]-~GlcNAc-OR)
Compound G = SO3-LewisA-OR (sulfate substituent on the
3-position of the galactose of LewisA-OR)
Compound H = Sialyl LewisC-OR (~Neu5Ac(2-3)~Gal(1-3)-
~GlcNAc-OR~
Compound I = SO3-~ewisC-OR (sulfate substituent on the
3-position of the galactose of LewisC-O~)
Compound J = Sialyl LacNAc-OR (~Neu5Ac(2~3)~Gal(1-4)-
~GlcNAc-O~) (~70% pure -- contains about
30% of 3 sulfate and 2,3-disulfate)
R = -(CH2~8C02C~3
b % reduction as per Example H below.
In another side-by-side analysis, 100 ~g of
several oligosaccharide glycosides related to blood
group determinants haYing a type I or a type II core
structure were tested for their ability to reduce
antigen induced inflammation (DTH response) in
sensitized mammals in the manner set forth above using
lO~g of the OVA antigen as the antigen. Administration
211~S2;)
W093/24505 PCT/US~3/04909 -
-- 98 -- :
. . .~,
of these oligosaccharides glycosides was conducted 5 ;
hours after antigen challenge. The results of these :
experiments are set forth below:
% Reduction
Compound in Inflammation
A ~47
C -49
D -44
F -45
K -44
L ~25
M -27
N -36
Compd A = CD-65
Compd C = Sialyl LewisX-OR (~Neu5Ac(2-3)~Gal(1-4~-
[~-L-Fuc(1-3)]-~GlcNAc-OR)
Compd D = SO3-LewisX-OR (sulfate substituent on the
3-position of the galactose of LewisX-QR)
Compd F = Sialyl LewisA-OR (~Neu5Act2~3~Gal(1-3)-
~-L-Fuc(1~4)]-~GlcNAc-OR)
Compd K = 2-N3-Sialyl LewisX-OR (~Neu5Ac(2~3)~Gal(1-4)-
[~-L-Fuc(1-3)]-~GlcN3-OR)
Compd L = 2-N3-Sialyl LewisA-OR (~Neu5Ac(2~3)~Gal(1-3)-
. [-~-Fuc(1~4)]-~Glc~3-OR)
Compd M - 2-NH2-Sialyl LewisX-OR (aNeu5Ac(2-3)~Gal(1~4)-
[~-L-Fuc(1-3)]-~GlcNH2-OR)
Compd N = 2-NH2-Sialyl LewisA-OR ~Neu5Ac(2~3)~Gal(1~3)-
[~-L-Fuc(1~4)]-~GlcNH2-OR)
R = ~(CH2)scO2cH3
b % reduction as per Example H below
The above results demonstrate that
oligosaccharide glycosides related to blood group
determinants having a type I or a type II core
structure are effective in reducing antigen induced
inflammation in a sensitized mammal.
~ W093/24~0~ 2 1 1 ~ S 2 2 PCT/US93~049~9
__ 99 __
Example B -- Dosa-Depe~dsncy of the Suppression of the
DTH Inflammatory Re~pon~e
- Six groups of mice were subjected to primary
immunization and challenge with Llll-S-Layer protein as
described under Example A, above. Five hours after
challenge, groups were injected intravenously with 100
~L solutions containing 10, 25, 50, 75, or 100 ~g of
sialyl LewisX~OR [R = -(CH2)8CO2CH3] or with PBS. The
DTH responses for each dose group were measured 24
hours after challenge and are shown in Figure 2. While
the groups receiving PBS or 10 ~g of sialyl LewisX
showed essentially the same extent of footpad swelling
as PBS-treated controls, the groups receiving 25, 50,
75 or 100 ~g of sialyl,LewisX displayed reduced footpad
swelling (78, 69, 75, and 56% of the PBS controls,
respectively).
Exnmple C -- Lack of ~uppression of the ~tibody
Recponse to the L111-S-~ayer Protein
Secondary antibody responses to the Llll-S-
Layer protein were measured two weeks after primary
immunization (one week after challenge) in the sera
from groups of mice immunized, challenged, and treated
intravenously with sialyl LewisX-QR, sialyl LewisA-OR,
sialyl LewisC-OR, LewisX-OR, and sialyl LacNAc-OR
(oligosaccharide glycosides related to blood group
determinants having a type I or type II backbone
structure).
Antibody titers were determined using a solid
phase enzyme immunoassay (EIA) as described by Ziola et
al70. Briefly, 2 ~g of Llll-S-Layer protein was added
per well of a Maxisorb EIA plate (Flow Laboratories,
Inc., McLean, VA). Following incubation at room
~emperature overnight, unabsorbed antigen was removed
by inverting the wells. Each well then received 200 ~l
of various dilutions of mouse serum prepared in
W093/24~05 2 1 1~J ~ PCT/US93/04909 -
-- 100 --- .~
phosphate-buffered saline containing 2% (w/v) bovine :
serum albumin and 2% (v~v) Tween 20. After 1 hour at
room temperature, the solutions were removed by~
in~erting the wells, and the wells washed four times
with distilled, de-ionized water at room temperature.
Horse-radish.peroxidase-conjugated, goat anti-mouse
immuno-globulin antibodies were then added to each well
(200 ~1 of a 1:2000 dilution prepared in the phosphate-
buffered saline/albumin/Tween 20 solution). After 1 :~
hour at room temperature, the wells were again invertedand washed, and each well received 2Q0 ~1 of enzyme
substrate solution (3 mg per ml o-phenylene-diamine and
0.02% (v/v) hydrogen peroxide, freshly dissolved in 0.1
M sodium citrate/ phosphate buffer, pH 5.5). After the
enzyme reaction had proceeded for 30 minutes in the
dark at room temperature, 50 ~1 of 2N hydrochloric acid
was added to each well and the OD49~ values were
measured.
Figure 3 graphically illustrates the titers
determined with six dilutlon series of sera from the
L111-immunized and challenged mice which were treated
with sialyl LewisX-OR, sialyl LewisA-OR, LewisX-OR,
sialyl LewisC-OR, and sialyl LacNAc-OR and examined Lor
footpad swelling as described in Example A above. The .
- dilution curves shown in Figure 3 indicate that the
development of antibodies against the L111 S-Layer
protein has not been inhibited or otherwise affected by
, i the .treatments with.such compounds.
:: Ex~mple D -- Time of Administration of Compound III
Relative to Challenge with Antigen
A. The purpose of this part of Example D was
to determine whether oligosaccharide glycosides related
to blood group determinants having a type I or type II
core structure could be administered to a sensitized
~ W093/2450~ 2 1 1 ~ 5 2 2 ~CT/US93/04909
---- 101 ----
mammal prophylactically or therapeutically in order to
reduce antigen induced inflammation.
Specifically, groups of Balb/c mice,
immunized and challenged with L111 S-Layer protein as
described in Example A, were injected with a solution
of 100 ~g of sialyl LewisX-OR [R = -(CH2)8CO2CH3] in PBS
(100 ~L) at different time points relative to the time
of antigen challengeO One group received sialyl
LewisX-OR one hour prior to the antigen challenge;
another, immediately after challenge, the third group
one hour after challenge, and the fourth group 5 hours
after challenge. A control group was included which
received PBS (100 ~L) immediately after challenge.
The results of this experiment are shown in
Figure 4. The DTH responses were~not suppressed in
those mice which had received sialyl LewisX-OR one hour
before or immediately after the antigen challenge.
Those groups which had received sialyl LewisX-OR one or
five hours after challenge showed only 68 or S9% of the
footpad swelling seen in the PBS treated controls.
Accordingly, this data demonstrates that in order to
- reduce antigen induced inflammation in a sensitized
mammal, it is necessary to administer the
oligosaccharide glycoside related to blood group
determinants having type I or type II core structures
after initiation of the immune response.
B. The purpose of this part of Example D was to
determine at what point in time oligosaccharide glyco-
sides related to blood group determinants having a type
I or type II core structure could be therapeutically
administered to a sensitized mammal in order to reduce
antigen induced inflammation. In this regard, the
antigen induced inflammation used in this experiment
was a DTH response in mice which is art recognized to
211~a2~
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-- 102 --
provide for maximal inflammation at 24 hours after
antigen exposure.
Specifically, groups of Balb/c mice,
immunized and challenged with OVA antigen in a manner
similar to that described in Example A, were injected
with a solution of 200 ~g per mouse of sialyl LewisX-OR
[R = -(CH2)8COzCH3] in PBS (100 ~L) at different time
points relative to the time of antigen challenge. One
group of mice re~eived sialyl LewisX-OR five hours
after antigen challenge; another, at 10 hours after
challenge; a third group at 12 hours after challenge; a
fourth group at 15 hours after challenge; a fifth group
at 18 hours after antigen challenge; and a sixth group
at 24 hours after antigen challenge, and a control
group was included which received P3S (100 ~L)
immediately after challenge.
The results of this experiment are shown in
FIGs. 15A, 15B, and 16. Specifically, Figure 15A
illustrates the increase in footpad swelling arising
from the DTH response to the OVA antigen challenge.
The results illustrated in this figure are graphically
represented in Figure 15B to show the reduction in
2S inflammation arising at the point in time sialyl
LewisX-OR is administered to the mice as compared to
the increase in inflammation for mice challenged with
the OVA antigen and treated with PBS. Specifically,
Figure 16B illustrates that significant reduction (>20%
reduction in inflammation~ occurs only when the
oligosaccharide glycoside is administered to the mice
at or prior to 12 hours after antigen challenge. The
reduction at lS hours of about 12% is not considered
meaningful because of the high dose (200 ~g) of sialyl
LewisX-OR used and the proportionally small reduction
in inflammation. The reduction at 18 and 24 hours was
less than 10%.
- W093t24505 2 1 1 8 5 ~ 2 PCT/US93/04909
-- 103 --
Since the maximal DTH inflammation occurs in
mice about 24 hours after antigen challenge, the
results of this part of Example D demonstrate that the
oligosaccharid glycoside related to blood group
determinants having a type I or type II core structure
must be administered to the mammal at or prior to one-
half that period of time required for maximal
inflammatory response to antlgen challenge.
Figure 16 illustrates that the degree of
residual inflammation in the challenged mice at 48
hours. In this regard, it is noted that the
inflammation arising from a DTH responses is generally
completed in 72 hours after antigen challenge.
Taken together, Examples A-D above establish
that in order to effectively reduce antigen induced
inflammation in a sensitized mammal, treatment with an
effective amount of an oligosaccharide glycoside
related to blood group determinants having a type I or
type II core structure must be after initiation of the
mammal's secondary immune response to the antigen and
at or prior to one-half that period required to effect
maximal inflammation to the antigen challenge.
Example E -- Per~istence of suppre~ion of the DTX
I~flammatory Re~ponse at 6, 8, or 10 Weeks
~fter Challe~ge
i. The identical groups of mice treated with
- sialyl LewisX-OR, sialyl LewisA-OR, LewisX-OR, sialyl
LewisC-OR, and sialyl LacNAc-OR in Example A were
re-challenged with Llll S-Layer protein 8 weeks after
primary immunization~ Untreated controls responded
with the usual degree of footpad swelling whereas all
other groups showed reduced footpad swelling.
Specifically, the degree of swelling in the treated
211~S~
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-- 104 --
mice relative to the degree of swelling in the control
mices were as follows~ sialyl LewisX-OR, 59%; LewisX-
OR, 69%; sialyl LacNAc-OR, 78%; sialyl LewisC-OR, 78%;
and sialyl LewisA-OR, 69~. (See Figure 5).
The anti-inflammatory effect of sialyl
LewisX-OR, sialyl LewisA-OR, LewisX-OR, sialyl
LewisC-OR, and sialyl LacNAc-OR, given 5 hours after
the first challenge (one week after primary
immunization), had somewhat weakened eight weeks after
primary immunization; however, the effect of LewisX-OR
(the only derivati~e not containing a sialyl group) was
equally as strong at the time of re-challenge as at the
time of first challenge.
In addition to providing suppression of
antigen induced inflammation in a sensitized mouse, the
above data demonstrate that treatm.ent with an
oligosaccharide glycoside related to blood group
determinants having a type I or type II core structure
as per this invention also imparts tolerance to still
later challenges from the same antigen.
ii. The identical groups of mice treated in
Example B with sialyl LewisX-OR, (10 ~g, 25 ~g, 50 ~g,
75 ~g, 100 ~g) or with the ~ or ~-sialyl-OR (R =
8-methoxycarbonyloctanol (100 ~g), or with 100 ~g of
the T-disaccharide-OR (R = 8-methoxycarbonyloctyl) were
rechallenged six weeks after primary immunization.
Footpad swelling similar to that of PBS-treated
controls was observed with those mice that had been
treated with ~-sialyl-OR, ~-sialyl-OR or the T-disac-
charide-OR 5 hours after the first challenge. Mice
originally treated with 10-100 ~g of sialyl LewisX-OR
showed footpad swelling that ranged from 90 to 65% of
that displayed by the control mice (Figure 6).
W093/24~05 2 1 1 ~ S 2 ~ PCT/US93/~4~09
-- 105 --
iii. The identical group of mice which had
been treated in ~xample C with 100 ~g of sialyl
LewisX-OR at l hour before first challenge, or 5~hours
aft~r first challenge, were re-challenged with antigen
10 weeks after primary immunization. Within
experimental error, footpad swelling of those mice
treated before or shortly after challenge was the same
as that of PBS-treated mice, whereas those mice
originally treated 1 hour or 5 hours after challenge
showed only about 66% of the values observed for PBS-
treated controls (figure 7).
The results of this example are set forth in
FIGs. 5-7 which demonstrate that oligosaccharide
glycosides related to blood group determinants having a
type I or type II core structure impart tolerance to
challenges with the same antigen for at least 10 weeks
after treatment.
Additional oligosaccharide glycosides were
tested for their ability to induce tolerance to antigen
induced inflammation (DTH response) in sensitized
mammals in the manner set forth above. The results of
- these experiments are set forth below:
Timea of Rechall. % Reductionb
Antiaen Compound Adm. at ~ weeks in infl~m.
Llll l A ¦ 5 hrs ¦ 1 ¦ 40~
SC B 5 hrs 6 58%
a supra-
b SUpra-
SC = 20~g of SuperCarrier
L111 = 10~g of L-111 S-Layer protein
Compd A = aNeu5Ac(2-3)~Gal(1-4)~GlcNAc(1~3)~Gal(1~4)[a-
L-Fuc(1-3)]~GlcNAc-OR (CD-65~
Compd B = SO3-LewisX-OR (sulfate substituent on the
3-position of the galactose of LewisX-OR)
W093/24s05 2 1 1 ~ ~ 2 PCT/US93/0490~ - ~
-- 106 --
Example F -- Effect Cyclophosphamide Treatment ha~ on
the ~uppression Induced by 8-methoxy-
carbonyloctyl glyco~ide of Compound III
It has been demonstrated in the literature
that suppressor cells can be removed by treatment of
mice with cyclophosphàmide (CP). An experiment was
carried out to determine if CP could modulate the ~-
suppression of cell-mediated inflammatory responses
induced by the ~-methoxycarbonyloctyl glycoside of
sialyl LewisX.
Specifically, this example employs immunized
mice which have been previously suppressed and
tolerized to DTH inflammatory responses by treatment
with the sialyl LewisX-OR (R = 8-methoxycarbonyloctyl)
in a manner similar to that described above. Fourteen
days after immunization, the mice were injected with
200 mg/kg of CP and then 17 days after immunization,
the mice were challenged with 20 ~g of L111 S-Layer
protein antigen. 24 hours after the challenge, the
extent of the DTH response was ascertained by measuring
(mm-l) the increase in footpad swelling.
:: :
The results of this experiment are set forth
in Figure 8 which il-lustrates that injection with CP
prior to challenge with the L111 S-Layer protein
antigen restores the DTH inflammatory response in mice
that have previously undergone immunosuppressive treat-
ment with sialyl LewisX-OR. These results suggest that
tolerance induced by this oligosaccharide glycoside is
mediated by CP sensitive suppressor T-cells.
Example G -- Effect the Antigen Driving the DTH
Inflammatory ~espon~e ha~ on the
suppre~ivQ Eff~ct Induced by the
8-Methoxy¢arbonyloctyl Glycoside of
Sialyl LewisX
This example assesses the effect that the
antigen driving the DTH inflammatory response has on
-: W093/2450~ 2 1 1 8 ~ 2 2 PCT/US93/04909
-- 107 --
the suppressive effect induced by sialyl LewisX OR
[R = -(CH2)8C02CH3] Mice were immunized as outlined in
Example A with S-Layer Llll, herpes simplex vir~s 1
(HSV 1) and cationized bovine serum albùmin (Super
CarrierTM, Pierce, Rockford, IL). As shown in Figure 9,
the nature of the antigen used to induce the
inflammatory response does not appear to affect the
ability of sialyl LewisX-OR to regulate this response.
~ample H -- Effect of Timi~g of Admini~tration of
Sialyl Lewis~ Relative to Immunization or
Challenge with Antige~
Four groups of Balb/c female mice were
subjected to primary immunization and challenge with
HSV antigen as described in Example A with the
following modifications:
1) The first group was immunized with
20 ~g/mouse inactivated Herpes
Simplex Virus Type I (HSV) and
then challenged seven days later
with 20 ~g HSV.
2) The second group was immunized with
20 ~g/mouse HSV and then challenged
seven days later with 20 ~g/mouse
HSV and then 100 ~g/mouse of sialyl
LewisX-OR ~ (CHz)8CO2CH3] which
was injected intraveneously five
hours after challenge.
3) The third group was immunized with
20 ~g/mouse HSV and 100 ~g/mouse of
sialyl LewisX-OR in 100 ~l PBS
injected intramusclularly at the
same site. Seven days later, the
mice were footpad challenged with
20 ~g/mouse of HSV alone.
4~ The fourth group of mice was
immunized with 100 ~l PBS and then
seven days later challenged with 20
~g/mouse HSV. This provides a
measure of the background level of
~ootpad swelling resulting from the
physical in~ury caused to the
footpad during the antigen
challenge.
211~522
W093~24505 PCT/~S93/~4909
-- 108 --
The extent of the DTH inflammatory response
was measured 24 hours after challenge by measuring
footpad swelling with a Mltutoyo Engineering -
~micrometer.
Figure 10 shows the degree of footpad
swelling observed.
Percentage reduction for this and Example L
was calculated by the following equation:
100 - 100 X Swellinq of Treated Mice -bkq swellinq
Swelling of Untreated Mice - bkg swelling
'ITreated mice" are those mice which receive
compound in addition to the antigen. "Untreated Mice"
are those mice which do not receive compound.
Background tbkg) swelling is that level of swelling
observed in mice immunized with P~S alone without
antigen or compound and challenged with antigen.
Mice injected with sialyl LewisX-OR at the
same time as and site of immunization with HSV showed a
50% reduction in footpad swelling compared to that of
mice immunized with HSV and challenged with HSV. Mice
injected with sialyl LewisX-OR 5 hours after the
footpad challenge with HSV showed an approximately
86.7% reduction in footpad swelling compared to that of
mice immunized with HSV and challenged with HSV.
The results of this example support previous
examples which show that oligosaccharide glycosides
related to blood group determinants having a type I or
type II core structure can suppress an immune response
to an antigen if given to mice 5 hours after challenge
by the antigen. This example also shows that
oligosaccharide glycosides related to blood group
W093/24505 2 1 1 8 5 2 2 PCT/US93/04909
---- 109 ----
determinants having a type I or type II core structure
given to mice at the time of immunization can inhibit
sensitization of the immune system to the antigen.
Without being limited to any theory, it is contemplated
that such compounds interfer2 with the ability of T
helper cells to recognize antigen-presenting cells and
inhibits the immune system from becoming educated about
the antigen.
Example I -- Effect of Sialyl LeX on the Antibody
Response to HS~
Four groups of mice were treated as described
in Example H. Secondary antibody responses to the HSV
antigen were measured 2 weeks after primary
immunization (1 week after challenge) in the sera from
groups of mice described in Example H.
Antibody titres were determined as described
in Example C except HSV antigen was used in place of
L111 S-Layer protein.
Figure 11 graphically illustratés the titres
determined with six dilution series of sera from the
groups of immunized mice as described in Example H.
The results of the first two groups correlated with the
results obtained in Example C for the Llll S-Layer
protein antigen. Treatment of mice with sialyl
LewisX-OR five hours after challenge did not affect the
antibody response. However, mice treated with sialyl
LewisX-OR at the time of immunization showed
significant reduction in the antibody response to the
HSV antigen. Without being limited to any theory, it
is contemplated that the above results are explained by
the fact that oligosaccharide glycosides related to
blood group determin~nts containing a type I or type II
core structure (e.g., sialyl LewisX-OR) interfere with
Wog3/~4505 2 1 1 ~ ~ 2 2 PCT/US93/04909
---- 110 ----
the T helper cells that are involved in the anti~ody
response and inhibits the immune system from becoming
educated about the antigen.
Example J -- Effect Cyclophosphzmide Treatme~t ha~ on
the Inductio~ of SleX Immuno uppre~sio~
As discussed in Example F above, 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 Llll S-Layer protein antigen. One
group of Balb/c mice were immunized with 20 ~g/mouse of
the Llll S-Layer protein. Seven days later, this group
of mice was footpad-challenged with 20 ~g of L111
S-Layer protein. The second group of mice were
immunized with 20 ~g of the L111 protein and 100 ~g of
sialyl LewisX-OR [2 = -(C~2)8CO2CH3] at the same site
and seven days later were footpad challenged with 20 ~g
of Llll. The third group of mice were injected with
200 mg/kg of CP interperitoneally two days before
immunization. This group was then immunized and
challenged as described for group two. The fourth
group of mice were immunized with 20 ~g/mouse of L111
and 100 ~g/mouse of the T-disaccharide-OR at the same
site and footpad challenged as described for group one.
Group five was immunized 100 ~l of PBS and then footpad
challlenged as described for group oneO
The results are presented in Figure 12.
These results confirm the results discussed in Example
H that treatment of mice with an oligosaccharide
glycoside related to blood group determinants having a
type I or type II core structure (e.g. sialyl LewisX-
OR) at the time of immunization can suppress the immune
response to an antigen. Furthermore, this example
W093/24505 21 I 8 5 2 2 PCT~US93/04909
----111 ----
shows that the suppression of the immune response by
treatment with such compounds at the time of
immunization can be eliminated by cyclophosphamide
treatment before immunization suggesting the
involvement of cyclophosphamide sensitive suppressor T-
cells.
E~ample K -- Eff~ct of ~ites of Administration of
Compound After Footpad ChAlle~ge on
Inhibition of DT~ Inflammatory
Response induced by OVA
Groups of Balb/c female mice, age 8 -12
weeks, weight about 20-25 g, were immunized with 100 ~g
of OVA ~Albumin, Chicken Egg, Sigma, St. Louis, MO) and
20 ~g of DDA (Dimethydioctacylammonium Bromide, Eastman
Kodak, Rochester, NY) in 100 ~l of PBS (Phosphate
Buffered Saline) intramuscularly into the hind leg
muscle of the mouse.
Seven days after immunization, each group of
mice was footpad-challenged with 20 ~g of OVA in 20 ~l
of PBS. The resulting inflammatory footpad swelling
was measured with a Mitutoya Engineering micrometer 24
: 25 hours after challenge.
To assess the effect of methods of
administration of an oligosaccharide glycoside related
to blood group determinants having a type I or a type
II core structure on the suppression of the
inflammatory DTH response, sialyl LewisX-OR tR =
-(CH2)8CO2CH3] was administered by different routes.
Certain groups of mice received, five hours after
footpad-challenge, either 100 ~g/mouse of sialyl
LewisX-OR in 200 ~1 of PBS intravenously or 100
~g/mouse of sialyl LewisX-OR in 20 ~l of PBS
intranasally at which procedure the mice were under
light anathesia.
2 1 1 ~ ~ 2 2
W093t2450S PCT/US93/~4909--
-- 112 --
The method of administering compound
intranasally is described in -Smith et al.71 which is
incorporated by reference. Briefly, mice are
anethesitized with Metof~e (Pitman-Moore Ltd.,
Mississauga, Ontario, Canada) and a 50 ~l drop of
compound is placed on the nares of the mouse and is
inhaled. Control groups were left untreated or
received 200 ~l PBS intravenously or 50~1 of PBS
intranasally. The results of this experiment are shown
in Figure 13. These results show that administr~tion
of an oligosaccharide glycoside related to blood group
determinants having a core type I or type II structure
nasally five hours after challenge results in
suppression of the ~mmune response.
E~ample L -- Time Dependency of Administration After
Footpad Challenge of the ~uppreR ion of
the OVA Induced DT~ In~lammatory Respon~e
A group of Balb/c female mice, age 8-12
weeks, weight about 20-25 g, were immunized with 100 ~g
of OVA (Albumin Chicken Egg, Sigma) and 20 ~g of DDA in
100 ~l of PBS, intramuscularly into the hind leg muscle
of the mouse. Seven days after immunization, the mice
2S were footpad challenged with 20 ~g of OVA in 20 ~l of
PBS. At 5, 7 or 10 hours after footpad challenge, the
mice were either given intravenously 100 ~g/mouse of
sialyl LewisX-OR tR = -(CH2)8~02CH3~ in 200 ~l of PBS or
200 ~1 PBS only or given intranasally 100 ~g/mouse of
sialyl LewisX-OR in 50 ~1 of PBS or 50 ~l PBS only at
which procedure the mice were under light anesthesia.
The footpad swelling was measured 24 hours later with a
Mitutoyo Engineering micrometer.
Figure 14 shows the results of this
experiment. Specifically, administration of sialyl
LewisX-OR given at 7 hours (both intranasally and
intravenously) after the OVA challenge showed 70 - 74%
W093/2450~ 2 1 1 8 ~ 2 ~ PCT/US93/04909
-- 113 -
reduction in footpad swelling relative to positive
control mice as calculated using the formula set forth
in Example H~ Sialyl LewisX-OR given intravenously or
intranasally at S hours after footpad challenge showed
63% and 54% reduction in swelling respectively. Sialyl
LewisX-OR given intervenously or intranasally at 10
hours after footpad challenge showed 58% and 32%
reduction in swelling respectively.
The data in Examples A-L above establish the
effectiveness of oligosaccharide glycosides related to
blood group determinants having a type I or a type II
core structure in treating immune responses to an
antigen and in inducing tolerance to still later
challenges by that antigen in a sensitized mammal
(mice). In view of the fact that the immune system of
mice is a good model for the human immune system, such
oligosaccharide glycosides will also be effective in
treating human immune responses.
By following the procedures set forth in the
above examples, o~her oligosaccharide glycosides
related to blood group determinants having a type I or
type II core structure could be used to suppress a
cell-mediated immune response to an antigen by mere
substitution for the oligosaccharide glycosides
described in these examples.
SYNT~ESIS
Examples 1 - 53 below illustrate the
synthesis of numerous oligosaccharide glycosides
related to blood group determinants having a type I or
a type II core structure.
211~3~2~
W093/24~05 PCT/US93/04909
- .
-- 114 --
Examples 1-24 illustrate the synthesis of
monosaccharides, disaccharides, and trisa charides used
in preparing oligosaccharide glycosides related~to
blood group determinants having a type I or a type II
core structure as depiGted'ln Figures 17 to 26.
EXAMPLE 1 -- Synthesis of Benzyl-2~0-benzoyl-4,5-O-
benzylidene-3-O-chloroacetyl-~-D-
thiogalactopyranoside (compound 31)
Dry a 20 L stirred reactor equipped with
reflux condenser, heating mantle and 1 L addition
funnel. Charge to this reactcr 10 L of dichloroethane.
Begin to stir the reactor then charge 1 kg D-galactose
and 500 g anhydrous sodium acetate to the
dichloroethane. Heat this slurry to reflux. Add
dropwise 4 L of acetic anhydride to the reaction
mixture using the 1 L addition funnel on the reactor.
Reflux is to be maintained during the 2-4 hour addition
period. Continue to stir and heat the mixture at
reflux overnight.
When the reaction is complete as determined
by t.l.c., turn off the heat to the reactor and add 250
mL of water in slow dropwise fashion using the addition
funnel. This reaction is quite vigorous but is
controlled by slowing the addition of the water. Stir
the reaction for 1-2 hours. Charge 30 L of cold water
to a 50 L stirred reactor and begin stirring. Drain
the contents of the 20 L reactor into a 20 L
polyethylene pail and pour into the stirring ice water
in the 50 L reactor. Stir this mixture for twenty
minutes. Drain the lower organic layer into a 20 L
polyethylene pail. Extract the aqueous layer in the 50
L reactor with an additional 5 L of dichloromethane.
Combine the dichloromethane extract with the first
organic layer. Drain the aqueous layer to polyethylene
pails and discard as aqueous waste.
W093/2450~ 2 1 1 ~ 5 2 2 PCT/U~93/04909
-- llS --
Return the combined organic layers to the 50
L reactor and extract twice with 5 L portions of ice
water for lO minutes. Drain the organic layer to a
clean 20 L polyethylene pail. Drain the aqueous to
waste, return the organic layer to the 50 L reactor,
stir and add 1 kg of anhydrous sodium sulfate. Stir
for 1-2 hours and then drain the solution into a clean
20 L polyethylene pail and filter the solution using a
4 L vacuum filtration set [or large Buchner attached to
a collector].
Concentrate the filtratP to 8 L then transfer
into a clean 20 L reactor equipped with stirrer, 1 L
addition funnel and cooling bath. Additional solvent
can be added if the level of the solution is below the
thermowell. Cool the organic solution to 0C using a
cooiing ~ath. Charge to this cool solution 724 g of
benzyl mercaptan. Add a total of 1.1 L of colorless
boron trifluoride etherate in slow dropwise fashion
over 2 hours using the 1 L addition funnel. Stir the ~
reaction 3-4 hours after the addition is complete ;
maintaining the temperature at 0C. The reaction is
checked for completion by t.l.c. on silica gel. ~The
reaction can be left to sit overnight].
The reaction mixture is drained into a clean
20 L polyethylene pail. The 50 L reactor is charged
with 15 L of saturated sodium car~onate solution. The
20 L polyethylene pail is 510wly transferred into the
slowly stirring carbonate solution at such a rate that
the gas evolution is not overly vigorous. Stir the
solution for 20 minutes then increase the rate of
stirring. When gas evolution ceases bubble air through
the entire solution for 24-36 hours. Drain the organic
layer into a clean 20 L polyethylene pail and store in
a hood. Extract the sodium carbonate solution with 3-5
W093/Z4s0s ~ 22 PCT/US93/04909~-~
-- 116 --
L of dichloromethane and drain this solution into the
same 20 L polyethylene pail.
,, ~.. .
Once the smell has been reduced the organic
solution can be filtered using a 4 L vacuum filtration
set and the fi;trate evaporated under reduced pressure
on the 20 L rotovap. 7 L of methanol is introduced
into the rotavap flask and the residue heated with the
rotavap bath till the residue dissolves in the warm
methanol. The flask is rotated and allowed to cool.
Cool ice water is added to the rotavap bath and the
flask slowly rotated for several hours. The flask is
removed from the rotovap and the white crystalline
product filtered using a 4 L vacuum filtration set.
The benzyl 2,3,4,6-tetra-O-acetyl-~-D-
thiogalactopyranoside (-1.3 kg) is charged into a clean
dry 20 L reactor with stirring motor and 7 L of dry
methanol is added to dissolve the material. The
solution is treated with 3 g of freshly surfaced sodium
and stirred for two hours. The reaction is checked by
t.l.c~ on silica gel using a retained sample of the
benzyl 2,3,4,6-tetra-O-acetyl-~-D-thiogalactopyranoside
with 80:20 ethyl acetate: methanol (v/v) the eluant.
The absence of starting material indicates the reaction
is complete.
50 g of fresh methanol washed H~ ion exchange
resin is added, the reaction stirred for 15 minutes.
The pH is checked using pH paper to ensure a neutral
solution. The resin is filtered off under reduced
pressure and the methanol is removed under reduced
pressure using the 20L rotovap. To the residue, 5 L of
acetone is added to the 20 L flask and the solution
warmed to reflux. The residue dissolves and is allowed
to cool to room temperature at which time ice is added
to the bath, the solution rotated with cooling
W093/24505 21~ 8 ~ 2 2 PCT/US93/04909
-- 117 --
overnight. 800-900g of benzyl B-D-thiogalacto-
pyranoside crystallizes and is filtered and dried under
vacuum.
To 8 L of dry acetonitrile is added 800 g of
benzyl B-D-galactopyranose, 600 g of benæaldehyde
dimethyl acetal and 2-5 g of p-toluenesulphonic acid.
The solution is stirred at room temperature overnight.
The reaction progress is checked by t.l.c. When
complete, the reaction is brought to pH 7 by the
addition of triethylamine. The volume of acetonitrile
is reduced to a minimum, 7 L of isopropanol is added
and the mixture is heated to near reflux. Most of the
product goes into the hot isopropanol after warming for
several hours. The mixture is cooled and ice added to `
the bath and cooling continued overnight to give a
precipitate. After filtering and drying the
precipitate, 760 g of benzyl-4,6-0-benzylidene-~-D-
thiogalactopyranoside is obtained.
180 g of benzyl-4,6-O-benzylidene-~-D-
thiogalactopyranoside was dissolved in dry DMF and -
placed in a jacketed reactor. The reactor was cooled
using a recirculating cooling bath maintained at a
temperature of -25C and treated dropwise with 108 g of `
chloroacetyl chloride over 3 hours while stirring the
reaction mixture. Stirring was continued 24 hours at
this temperature then the reaction was quenched into
several volumes of cold bicarbonate solution. The
product was extracted into methylene chloride, water ;;
washed several times, dried over sodium sulphate and
evaporated to dryness. The product was crystallized
from isopropanol. Yield: 125 g of benzyl 4,6-O-
benzylidene-3-O-chloroacetyl-B-D-thiogalacto-
pyranoside.
211g~22
W O 93~24~05 PC~r/US93/04909
-- 118 --
5 g Benzyl 4,6-0-benzylidene-3-0-
chloroacetyl-B-D-thiogalactopyranoside was benzoylated
at room temperature in methylene chloride/pyridine
using 3 equivalents of benzoyl chloride and a catalytic
amount of dimethylaminopyridine. The solution is
quenched into cold sodium bicarbonate solution, the
organic layer is washed with saturated copper sulphate
solution to remove the pyridine the organic layer dried
and evaporated. The residue is taken up in hot
isopropanol and benzyl 4,6-0-benzylidene-2-O~benzoyl-3-
O-chloroacetyl-B-D-thiogalactopyranoside crystallizes
from solution. 1H-n.m.r. (CDCl3): ~ = 7.96, 7.4 t2m~
15H, aromatic, 5.79 (t, lH, H-2), 5.5 (s, lH, CH), 5.2
(q, lH, H-4, J2,3 9-9 Hz, J34 3.3 ~z), 4.5 (m, 2H), 4.4
(d, lH), 3.9~ (m, 5H), 3.55 (s, lH).
Example 2 -- Synthesis of 4,~-0-benzylidene-2,3-di-0-
benzoyl-B-D-galactopyranosyl bromide
(compound 32A~
Benzyl-4,6-0-benzylidene-~-D-thiogalacto-
pyranoside (10 g) was dissolved in 100 mL
dichloromethane and 6.35 g of pyridine was added. To
the solution was added 9 g of benzoyl chloride in
dropwise fashion and after 1 hour, 50 mg of
dimethylaminopyridine was added to the solution and the
mixture was stirred for an addition 2 to 4 hours. The
progress of the reaction was chec~ed by t.l.c. on
silica gel~ Benzyl-4,6-0-benzylidene-2,3-di-0-benzoyl-
~-D-thiogalactopyranoside (compound 32) was isolated by
quenching the reaction mixture into saturated sodium
bicarbonate solution and washing the organic extract
with water, 5% copper sulfate solution, water, drying
and evaporating the solvent. The residue was
crystallized from isopropanol to give 10.7 g of
compound 32.
W093/24505 2 1 1 8 5 2 ~ - PCT/US93/~4909
---- 119 ---
Compound 32, benzyl-4,6-O-benzylidene-2,3-di-
O-benzoyl-~-D-thiogalactopyranoside (9.89 g), was
dissolved in 100 mL of dichloromethane, cooled to 0C,
and treated with a solution of bromine ~2.85 g) in 10
mL of dichloromethane. After 15 minutes, 1.8 grams of
tetraethylammonium bromide was added to the mixture and
the mixture stirred for 2-3 hours at room temperature
(followed by t.l.c. on silica gel). A small quantity
of cyclohexane was added to quench excess bromine and
the reaction mixture was quenched into cold saturate
sodium bicarbonate solution, washed with water, dried
and volume of the solution reduced to 30 mL. This
dichloromethane solution of compound 32a was used
directly in the synthesis of compound 42 without
further isolation and/or purification.
Example 3 -- Synthesis of p-Chlorophenyl 2,3,4-tri-O-
benzyl-B-L-thiofucopyranoside (compound
20)
Dry a 2 L three neck round bottomed flask,
reflux condenser and 500 mL addition funnel. Then cool
under a flow of nitrogen. Charge to the flask 1000 g
of L-fucose, 500 g of anhydrou~ sodium acetate and 800
mL of dry dichloroethane. Heat the mixture with
stirring to 50C. Charge to the addition funnel 400 mL
of acetic anhydride. Add the acetic anhydride to the
stirring, warm (50-55C) slurry in dropwise fashion at
30, a rate that does not ool the reaction appreciably.
Upon completion of the addition stir the mixture for 72
hours at this temperature, removing aliquots from the
reaction mixture every 24 hours to chec~ the progress
of the reaction by t.l.c.
When the reaction appears to be complete add
200 mL of water to the warm stirring mixture dropwise
over 30 min. and stir for 1 hour at this temperature.
. . . . , ... , . . , . . . . .. ... , . ,,,, ~ .. . ~ , .... . , ~,. ... . . ..
211~2~
W093/24~05 PCT/US93/04909--
-- 120 --
This converts the remaining acetic anhydride to acetic
acid. The reaction mixture is quenched into 3-4
volumes of water.' The organic layer is remove~ and the
aqueous layer is`extracted with 4 L dichloromethane. 5 The combined organic layers are backwashed three times
with 2 L portions of water. The organic layers are
dried over sodium sulphate and concentrated under
reduced pressure to approximately 5 L.
To the organic layer is added 925 g of
- p-chlorothiophenol. The organic layer is cooled with
cold water. To the mixture of p-chloro-thiophenol and
fucose acetates is added 1.72 kg of boron trifluoride
etherate in dropwise fashion. The mixture is then
stirred for 6 hours (overnight is acceptable) allowing -
the reaction mixture to come to ambient temperature. A
small aliquot is removed from the reaction mixture and ~;
quenched into sodium bicarbonate so~ution. Once CO2
evolution has ceased, the reaction is checked for
completion by t.l.c. If complete, the whole reaction
mixture is quenched into l L of saturatsd sodium
bicarbonate and the organic layer separated after CO2
evolution has finished. The organic layer is separated
and air bubbled through this layer for 1 hour.
The separated organic layer is then dried
over sodium sulphate and evaporated to dryness. The
residue is taken up in 1 L of dry methanol in a 2 L
round bottom flask and treated with 1 g of freshly
surfac~d sodium. The reaction is kept under nitrogen
~or several hours then checked by t.l.c. for removal of
the acetate groups. The reaction is neutralized with
H~ ion exchange resin and filtered and evaporated under
reduced pressure. The residue is taken up in a minimum
3S of hot isobutanol and the p-chlorophenyl-~-L-
thiofucopyranoside crystallizes from solution after
cooling overnight at 0C. Yield: 1060 g.
W093J24505 211 8 a 2 ~ PCT/US93/0490~
-- 121--
p-Chlorophenyl-~-L-thiofucopyranoside is
dissolved in 7 L of dry dimethylsulphoxide. To the
solution is added 600 g of powdered KOH and the
reaction mixture stirred for 30 minutes. Benzyl
S chloride (1.275 L) is added dropwise to the stirring
solution and the mixture stirred overnight at room
temperature. T.l.c. indicates incomplete reaction so
an additional 300 g of powdered KOH is added to the
reaction mixture followed 30 minutes later by 425 mL of
benzyl chloride. The solution is stirred at room
temperature until t.l.c. indicates the reaction is
complete. If the reaction is not complete after 24
hours, powdered KOH is added followed by 200 mL of
benzyl chloride. The reaction is quenched into several
volumes of water, extracted with methylene chloride,
backwashed twice with water, dried and evaporated. The
residue is taken up in hot hexanes. p-Chlorophenyl-
2,3,4-tri-0-benzyl-~-L-thiofuco-pyranoside crystallizes
- and is filtered and dried under vacuum. Yield: 1.3 kg.
1H-n.m.r. (CDCl3): ~ = 7.57 (m, 19H, aromatic), 4.99
(d, lH), 4.65 (m, 5H), 4.55 (d, lH, J-2 9-5 Hz), 3.98
- (t, lH), 3.55 (m, 3H), 1.26 (d, 3H, J5 6 6.2 Hz, H-6). -
Example 4 -- Synthesis of 8-methoxycarbonylo~tyl 2-
acetamido-4,6-di-0-benzylidene-2-deoxy-
B-D-glucopyranoside (Compound 5)
A 2OL glass reactor was charged with 8 L of
dichloroethane, 1 L of acetic anhydride and 1 kg of
anhydrous sodium acetate. To the stirring mixture was
added 1 kg of glucosamine hydrochloride and the mixture
was brought to reflux. A further 3.5 L of acetic
anhydride was added dropwise to the refluxing solution
over 3-4 hours and the solution maintained at reflux
for 3~ hours. During the last hour of reflux 200 mL of
water was added dropwise to the solution. The reaction
was then coo~ed and added to 35 L of ice water in a 50
W093/24~05 2 1 1 8 ~a 2 2 PCT/US93/04909~
-- 122 --
L stirred reactor. The organic layer was removed and
then water washed a second time with an additional 20 L
of water. The organic layer ;was dried over sodlum
sulphate, filtered, and s:a,turated with anhydrous
gaseous HCl for 2 hours.''' The reaction was allowed to
sit for 6 days being saturated with HCl for 1 hour
every second day. 2-acetamido-2-deoxy-3,4,6-tri-O-
acetyl-B-D-glucopyranosyl chloride was isolated by
quenching into ice cold sodium bicarbonate solution.
The organic layer was dried over sodium suIphate and
evaporated to a brown solid.
Four hundred grams of 2-acetamido-2-deoxy-
3,4,6-tri-O-acetyl-~-D-glucopyranosYl chloride was
dissolved in 2 L of anhydrous dichloromethane
containing 200 g of activated molecular sieves. 266 g
of 8-methoxycarbonyloctanol was charqed to the reaction
mixture along with 317 g of mercuric cyanide. The
solution was stirred rapidly at room temperature for 24
hours. After checking for reaction completion by t.l.c.
the reaction mixture was filtered through a buchner
funnel of silica and the organic layer washed twice
with water, twice with a 5~ solution of potassium
iodide and twice-with a saturated solution of sodium
bicarbonate. The solution was dried over sodium
sulphate and evaporated to dryness. The residue was
- taken up in anhydrous methanol and treated with 1 g of
freshly cut sodium then stirred at room temperature
overnight. The solution of 8-methoxycarbonyloctyl 2-
acetamido-2-deoxy-B-D-glucopyranoside was neutralized
with acid ion exchange resin and filtered and
evaporated to yield 218 g of product after
crysta}lization from isopropanol /diisopropyl ether.
Two hundred grams of 8-methoxycarbonyloctyl
2-acetamido-2-deoxy-B-D-glucopyranoside was dissolved
in 1.2 L of anhydrous dimethylformamide and treated
W093/24505 2 1 1 8 5 2 2 PCT/US93/04909
-- 123 -~
with 169 mL of dimethoxytoluene ~benzylaldehyde
dimethyl acetal~ and 1-2 g of p-toluenesulphonic acid.
The reaction was stirred and heated to 40C for 5
hours, then checked for completion ~y t.l.c. When the
reaction appears complete the mixture was neutralized
with triethylamine and quenched into several volumes of -
ice water, extracted into dichloromethane and -
backwashed several times with water. The organic layer
was dried over sodium sulphate, evaporated to dryness
and taken up in hot isopropanol. After cooling 8-
methoxycarbonyloctyl-2-acetamido-4,6-0-benzylidene-2-
deoxy-~-D-glucopyranoside precipitates. It is filtered
and dried to yield 106 g of product. 1H-n.m.r.
(CDCl3): ~ = 7.41 (m,5H, aromatic), 6.11 (d, lH, NH),
lS 5.5 (s, lH, C~-), 4.63 (d, lH, H-1, J1 2 7-4 Hz)~ 2-29
(t, 2H), 1.99 (s, 3H, Ac), 1.58 (m, 4H), 1.29 (bs, 8H). --
..-.
Example 5 -- Synthesis of 8-Methoxycarbonyloctyl 2-
acetamido-3-0-p-methoxybenzyl-4,6-o-
benzylidene-B-D-glucopyranoside
(compound 6).
To a stirred solution of compound 5 (17.5 g,
-3 mmol) in dry dichloromethane (100 mL) and catalytic
amount of p-toluenesulfonic acid (0.25 to 3 weight
percent based on compound S) was added dropwise a
solution of p-methoxybenzyl trichloroacetimide (10 g in
25 mL CH2Cl2). The reaction mixture was~stirred at
room temperature overnight. Triethylamine was added to
quench the reaction, the organic layer was washed with
sodium bicarbonate solution and the organic layer dried
and evaparated to dryness, Crystallization in hot
- ethanol gave 20 g of the desired product. 1H-n.m.r.
(CDCl3): ~ 7.56-6.90 (m, 9H, aromatic), 5.60 (d, lH, I
NH), 5.30 (s, lH, PhCH), 4.94 (d, lH, J12 8.0Hz, H-l), '
3.80 (s, 3H, CH3), 3.60 (s, 3H, CH3Ph), 2.30 (t, 2H,
CH2C0), 1.90 (s, 3H, AcNH), 1.80-1.10 (m, 12H, (CH2)6).
W093/24505 2 1 1 ~ 5 ~ ~ PCT/US93/0490g
-- 124 --
Example 6 -- Synthesis of 8-Methoxycarbonyloctyl-2-
acetamido-2-deoxy-3-O-p-methoxybenzyl-6-
O-benzyl-2-~-D-glucopyranoside (compound
To a stirred so~ution of compound 6 (15.0 g,
~3 mmol) in 200 mL of dry THF were added, 11.0 g of
sodium cyanoborohydride, 10 g of molecular sieves 4A
and 5 mg of methyl orange. The solution was cooled to
-10 a C and then ethereal hydrochloric acid was added
dropwise until the solution remained acidic. On
completion of the reaction, it was diluted with
dichloromethane (200 mL), filtered through celite and
washed su~cessively with aqueous sodium bicarbonate (2
x 100 mL) and water ~2 x 100 mL) and then the solvent
dried and evaporat~d to give a syrup. Purification of
the mixture on column chromatograp~y using silica gel
as adsorbent and eluting with hexane:ethyl
acetate:ethanol (20:10:1) gave 7 in 70% yield.
- 20 1H-n.m.r. (CDCl3): ~ 7.40-6.90(m, 9H, aromatic~,
5.70(d, lH, NH), 4.64(d, lH, J-2 8.0Hz, H-1), 3.86(s,
3H, CH30), 3.68(s, 3H, CH30Ph), 2.30(t, 2H, CH2CO),
1.90(s, 3H, NHAc), 1.80-1.10(m, 12H, (CH2)6).
~xample 7 -- Synthesis of 8-Methoxycarbonyloctyl 2-
acetamido-2-deoxy-3-O-p-methoxybenzyl-4-
- 0-(4,6-O-benzylidene-~,3-O-dibenzoyl-~-
D-galactopyranosyl)-6-O-benzyl-2-deoxy-
*-D-glucopyranoside (compound 42)
A solution of compound 7 (10.61 g, 19.7 mmol)
and compound 32A (1.6-1.7 equivalents based on compound
7) and 2,6-di-t-butyl-4-methyl pyridine (3.11 g, lS.2
mmol) in 250 L of dichloromèthane and 40 g of molecular
sieves ~4A) was stirred at room temperature for 30
minutes, and then cooled to _50 D C under nitrogen. A
dry solution of silver triflate (4.47 g, 17.3 mmol) in
toluene (40 mL) was added to the stirred mixture. The
mixture was warmed to -15C during two hours and kept
W093/24~05 2 1 1 8 5 2 2 PCT/USg3/04909
-- 125 --
at -15~C for an additional 5 hours. At the end of
which the mixture was warmed to room temperature and
stirred overnight. 3 mL of pyridine and 250 mL of ;
dichloromethane were added to the mixture and was
filtered over celite, filtrate was washed with
saturated aqueous sodium hydrogen carbonate (200 mL)
and then with water (200 mL), aqueous hydrogen chloride
(0.SN, 200 mL) and water (200 mL), concentrated in
vacuo. 6.0 g of compound 8 was crystallized as white
crystals from ethyl acetate-diethyl ether-hexane. The
mother liquor was concentrated, purified over chromato-
graphy (300 g silica gel, toluene:ethyl acetate, (1:1)
to give 4.5 g pure compound 42. Total yield was 10.5 g
(68%) Rf 0.48 (methanol:dichloromethane, 4:96).
lH-n.m.r. (CDCl3): ~ 5-80(t, lH, J2 3 11.0Hz, H-2 ),
5.52(s, lH, CHPh), 5.25(dd, lH, J34 4.OHz, H-3 ),
4.88(d, lH, J~ 2 11.0Hz, H-1 ), 4.70(d, lH, J12
9.0Hz, H-l), 3.78(s, 2H, CH30), 3.64(s, 3H, CH30Ph~.
Example 8 -- Synthesis of 8-Methoxycarbonyloctyl 2-
acetamido-4-O-(4,6-O-benzylidene-2 3 -
di-O-benzoyl-~-D-ga1acto-pyranosyl)-6-O-
benzyl-2-deoxy-B-D-glucopyranoside
(compound 43)
DDQ tl26 mg, 0.5 mmol) was added to a stirred
solution of compound 42 (350 mg, 18S ~mol) in
dichloromethane (10 mL) saturated with water. After 2
hours at room temperature, the reaction was complete,
and organic layer was successively washed with aqueous
sodium bicarbonate and water, dried and concentrated.
Column chromatography gave the desired compound 43 in
85~ yield. 1H-n.m.r. (CDCl3): ~ 5.65(dd, lH, J2 3
10.8Hz, H-2 ), 5.61(d, lH, J3 4, 4.0Hz, H-3 ), 4.68(d,
lH, J1 z 1l.0Hz, H-l ), 4.62(d, lH, J12 10Hz, H-1),
3.60(s, 3H, COOCH3).
211~32~ ~
W093/24505 PCT~US93/04909
-- 126 --
~xample 9 -- Synthesis of 8-methoxycarbonyloctyl 2-
acetamido-3-O-(2,3,4-tri-O-benzyl-~-L-
fucopyranosyl)-4-0-(4,6-O-benzylidene-
2,3-di-O-benzoyl-~-D-galactopyranosyl)~
6-O-benzyl~ deoxy-B-D-
glucopyra,,noside(compound 44)
To a mixture of copper (cupric) bromide (40
g, 17.7 mmol) and 5 g of molecular sieves 4A in 10 mL
of dry dichloromethane were added 1.2 mL of dry DMF and
tetraethylammonium bromide (1.85 g, 8.B mmol). The
mixture was stirred at room temperature for 1 hour and
then a solution of compound 43 (5.0 g, 5.75 mmol3 and
the thiofucoside 20 (7.5 g, 11.8 mmol) in 30 mL dry
dichloromethane was added dropwise at 0C for 30
minutes. The mixture was stirred at room temperature
for 4& hours, at the end of which time 5 mL of methanol
was added and stirred for 30 minutes. Further, 3 mL of
pyridine, 100 mL of ethyl acetate and 100 mL of toluene
were added to the reaction mixture. The mixture was
filtered over celite pad and the solvent evaporated to
give a brown syrup. Purification over column
chromatography with silica gel and eluted with
toluene:ethyl acetate (2:1~ ga~e the compound 44 in 86%
yield. 1H-n.m.r. tCDCl3): ~ 5.80~dd, lH, J2 3
ll.OHz, ~-2'), 5.60(s, lH, CHPh), S.50(d, lH, NH),
5.10(dd, lH, J3, 4~ 4.0Hz, H 3'), 3.60(s, 3H, OCH3~,
1.20(d, 3H, CH3, fucose).
Example 10 -- Synthesis of 8-methoxycarbonyloctyl 2-
acetamido-3-O-(2,3,4,-tri-D-benzyl-~-L-
fucopyranosyl)-4-0-(4,6-O-benzylidene-B-
D-galactopyranosyl)-6-O-benzyl-2-deoxy-
~-D-glucopyranoside (compound 45)
Compound 44 (200mg) was treated with 20 mL of
sodium methoxide in methanol. After 3 hours, t.l.c.
(toluene-ethyl acetate, 1:1) indicated the
disappearance of the starting material and the
appearance of a slower moving spot. The solution was
2~1~522
W093~24~0~ PCT/US93/04909
-- 127 --
neutralized with amberlite resin IR-120 H' and the
solvent evaporated under reduced pressure to yive a
quantitative yield of crude compound ~5. The ~roduct -
was purified on silica gel using toluene-ethyl acetate
S (2:1) as eluant. 1H-n.m.r. lCDCl3): ~ 7.15-7,S5
(aromatic, 2SH), 5.62 (d, lH,NH~, 5~58 (s, lH, CH-
benzylidene), 5.06(d, lH, J1"2" 7.0Hz, H-l"), 4.95 (d,
lH, J1~ 2' 3.8Hz, H-l') 4.85 (d, lH, J12=9.OHz, H-l)
3.62 (s, 3H, COOCE3) 1.0(d, 2H, Fuc-C~3).
'
Example 11 -- Synthesis of 8-methoxycarbonyloctyl 2-
acetamido-3-O~ L-fucopyranosyl)-4-O-
(3-O-sulphate-~-D-galactopyranosyl)-2-
deoxy-~-D-glucopyranoside (compound 47)
Diol ~100 mg -- compound 45) was dissolved in
Sm~ dry dimethylformamide. Pyridine:sulfur trioxide
complex (120 mg) was added to the solution and the
reaction mixture stirred ar room temperature for 1
hour. The reaction was followed by t.l.c. to monitor
the disappearance of the diol (Rf =0~ 28 in EtOAc: MeOH
80:20). Solvent was evaporated to dryness and taken up
in 50mL of methanol then treated with Na' resin to
convert it to the sodium salt. Purification by column
chromatography on silica gel gave 65 mg of compound 46
which was immediately hydrogenated with 10% Pd(OH)2 on
carbon to give 35 mg of compound 47. 13C-n.m.r. (D2O):
~ 103.94~C-l, Gal), 103.44(Cl, GlcNAc), 101.07(C-l,
Fuc), 82.7(C-3, Gal), 63.83(C-6, Gal), 62.2(C6,
GlcNAc), 54.55(C-N, GlcNAc), 17.75(C6-Fuc).
Example 12 -- Synthesis of 2-O-benzoyl-4,6-O-
benzylidene-3-O-chloroacetyl-~-D-
galactopyranosyl bromide (compound 33)
Compound 32, benzyl 4,6-O-benzylidene-2-o-
benzoyl-3-chloroacetyl-~-D-thiogalactopyranoside (8~87
g) was dissolved in 100 mL of dichloromethane, cooled
211~ .~ 2 ~
W093/24505 PCT/US93/04909 -
-- 128 --
to ooc and treated with a solution of bromine (2.7 g)
in lO mL of dichloromethane. After 15 minutes, 1.7 g
of tetraethylammonium bromide was added to the mixture
and the mixture stir~ëd for 2 to 3 hours at room
temperature ~followed by t.l.c. on silica gel). A
small quantity of cyclohexene was added to quench
excess bromine and the reaction mixture was quenched
into cold saturate sodium bicarbonate solution, washed
with water, dried, and the volume of the solution
reduced to 30 mL so as to provide a dichloromethane
solution of compound 33. This solution was used
directly in the synthesis of compound 38.
Example 13 -- Synthesis of 8-methoxycarbonyloctyl 2-
acetamido-4-O-(2'-O-benzoyl-4',6'-o-
benzylidene-3 -O-chloroacetyl-B-D-
galactopyranosyl)-6-O-benzyl-2-deoxy-3-
o-p-methoxybenzyl-B-D-glucopyranoside
(compound 37)
A solution of the compound 7 (5.0 g, 0.9
mmol) and compound 33 (1.4 to 1.5 equivalents -- from
example 12) and 2,6-di-t-butyl-4-methyl pyridine (1.78
g, 1.0 mmol) in 50 mL of dichloromethane and 20 g of
molecular sieves (4A) was stirred at room temperature
for 30 minutes, and then cooled to -50~C under
nitrogen. A dry solution of silver triflate (3.3 g,
1.5 mL) in toluene (10 mL) was added to the stirred
mixture. The mixture was warmed to -15~C over two
hours and kept at -15 D C for an additional 5 hours,
then allowed to warm to room temperature and stirred
overnight. 1 mL of pyridine and lOO mL of
dichloromethane were added to the mixture and it was
fil~ered over celite, the filtrate was washed with
aqueous sodium bicarbonate (100 mL) and then with water
(100 mL), aqueous hydrogen chloride ~0.5 N, 100 mL) and
water (lO0 mL), then concentrated in vacuo.
W0~3/24505 2 1 1 8 S 2 ~ PCT/US93/04909
-- 129 --
Purification of the crude mixture on column
chromatography with silica gel as adsorbent eluted with
hexane:ethyl acetate (l:l) gave 5.2 g of pure c~mpound
37. tH-n.m.r. (CDCl3): ~ 5.85(d, lh, NH), 5.62(t, lH,
J2 3- 10.8Hz, H-2 ), 5.52(s, lH-CH-benzylidene), 5.08
(dd, lH, J3 4 4Hz, H-3 ), 4.85 (d, lH, J1 2. 11.0Hz,
H-1 ), 4.68 (d, lH, J-2 9.0Hz, H-l), 3.72 and 3.64
(2s, 6H, OCH3 and COOCH3); 13C-n.m.r.: 159.0(aromatic
c-p-methoxyl) 165.15(c=0, chloroacetyl), 167.12(c-0,
acetyl), 174.2(c=0, COOCH3), 99.64(c-1), 100.26(c-1 ),
101.0(PhCH).
Compound 37 was then treated with DDQ in the same
manner as Example 8 to give compound 38 in near
quantitative yields.
Example 14 -- Synthesis of 8-methoxycarbonyloctyl 2-
acetamido-3-O-(2,3,4-tri-O-benzyl-~-L-
fucopyranosyl-4-0-(2-O-benzoyl-4,6-O-
benzylidene-3-0-chloroacetyl-B-D-
galactopyranosyl)-6-O-benzyl-2-deoxy-~-
D-glucopyranoside (compound 39~
Thiofucoside 20 (4 g) was stirred in dry
dichloromethane (50mL) and bromine (0.60g) was added.
The mixture was cooled to -20C. The conversion to the
bromide was complete in 1 hour and the reaction mixture
was washed with cold aqueous sodium bicarbonate, dried
and concentrated to 10mL and syringed into a flask
containing the alcohol 38 (2.g7 g, 3.56 mmol~, CuBr2
~2.39 g), tetraethyl-ammonium bromide (2.24 g),
molecular sieves 4 A (4g) in dimethylformamide (1 mL)
in dry dichloromethane (75 mL). The mixture was
stirred at room temperature for 48 hours after which
the t.l.c. showed the disappearance of the alcohol 38
and a faster moving spot (Rf 0.56 -- toluene:ethyl
acetate 2:1). After the usual work up, the crude
mixture was purified by column chromatography to give
W093/2450~ 2 ~ 1 ~ S 2 .~ PCT/US93/0490~ ~
-- 130 --
compound 39 (4.2 g, about 80% yield). 1H-n.m.r.
(CDCl3): ~ 7.1-8.0 (m,aromatic-30H), 5.58, 5.61 (m, 2H,
NH and CH-benzylidene, overlapping), 5.56 (d, l~, ~ 2~
7.0 Hz, H-l"), 4.98 (d, lH, J12 8.0 Hz, H-l), 4.95 (d,
lH, ~1~ 2~ 3.8 HZ, H-l'), 3.65 (s, 3H, COOCH3) and l.l
(d, 3H, CH3-FuC).
Compound 39 is then dechloroacetylated by
treatment with thiourea and the compund is sulphated
with sulfur trioxide/pyridine complex in dimethyl-
formamide at 0C for 2 hours to provide for compound
~l. The blocking groups on compound 41 are then
removed by conventional techniques to provide for
compound 47.
Example 15 -- Synthesis of 2-deoxy-2-phthalimido-
l,3,4,6-tetra-0-acetyl-~-D-
glucopyranoside (compound l)
(D+) Glucosamine hydrochloride (lO0 g, 0.46
mol) was added to a solution of sodium methoxide in
methanol which was prepared from equimolar amount of
sodium metal in methanol (o.s L). The resultant
mixture was treated with equimolar equivalent of
phthalic anhydride and triethylamine (80 mL). The
mixture was then stirred for 2 hours, filtered and the
solid was dried in vacuum for 12 hours. The dry solid
was dissolved in pyridine t300 mL) and treated with
acetic anhydride (200 mL, 2.l mol). The mixture was
then stirred at room temperature for 48 hours and
quenched in an ice-water mixture, and the resultant
precipitate was filtered, concentrated and crystallized
from diethylether to 98.3 g (45%) of the title
compound. 1H-n.m.r. (CDCl3): ~ 7.75 (m, 4h, aromatic),
6.45 (d, lH, H-l, J1 2 9.0Hz), 5.85 (t, lH), 5.15 (t,
lH), 4.4 (t, lH), 4.3 (q, lH), 4.1 (q, lH). 4.00 (m,
lH), 2.05, 2.00, l.9S, 1.80 (4s, 12H, 4Ac). 13C-n.m.r.
W093/24505 2 1 1 ~ 5 2 ~ PCT/US93/04909
-- 131 ~-
(CDCl3) ~ 89.7 (C-1), 72.6, 70.5, 68.3 ~3C, C-3, C-4,
C-5), 61.45 (C-6), 53.42 ~C-2).
Example 16 -- Synthesis of 2-deoxy-2-phthalamido-
3,4,6-tri-0-acetyl-~-D-glucopyranosyl
bromide (compound 12)
2-deoxy-2-phthalamido-1,3,4,6-tetra-0-
acetyl-~-D-glucopyranoside 1 (20g, 41.9mmol) was
treated with hydrogen bromide solution in acetic acid
(30%, 200mL) and stirred at room tPmperature for 2 hrs.
The mixture was then poured into an ice water mixture
and extracted with dicloromethane. The extract was
washed with NaHC03 solution and water followed by MgS0
drying. The mixture is filtered, dried and
concentrated in vacuo to give compound 12 as a dry
syrup (compound 12
Example 17 -- 5ynthesis of Ethyl 2-deoxy-2-
phthalimido-3,4,6-tri-0-acetyl-B-D~
glucopyranoside (compound 13)
2-Deoxy-2-phthalamido-3,4,6-tri-0-acetyl-~-D-
glucopyranosyl bromide (compound 12) from example 16
was ta~en up in dry ethanol and treated directly with
dry ethanol (200 mL), mercuric cyanide (13.7 g. 55
mmol~ and stirred at room temperature for 48 hr. The
- 3 0 mixture was then filtered and concentrated. The
residue was taken up in 200 mL of dichloromethane and
washed with a solution of 10% potassium iodide, 5%
sodium bicar~onate, water, dried over MgS04 and
concentrated to a syrup.
Example 18 -- Synthesis of Ethyl 2-deoxy-2-
phthalimido-B-D-glucopyranoside
(Compound 14)
Ethyl 2-deoxy-2-phthalamido-3,4,6-tri-o-
acetyl-~-D-glucopyranosyide (compound 13) from example
W093/245~5 2 1 1 ~ S 2 ~ PCT/US93/~490g -
-- 132 --
17 was taken up in 100 mL of dry methanol and treated
with loo mg of sodium metal. The solution was stirred
at room temperature ~f~r 24 hours and then neutralized
with Amberlite [R-120(H+)~ resin, filtered, and
evaporated to dryness in vacuo. This compound was used
in the preparation of compound 15 and campound 66.
Example 19 -- Synthesis of Ethyl 2 deoxy-2-
phthalimido-6-0-henzyl-~-D-
glucopyranoside (Compound 15)
Compound 14 (2.1 g, 6.23 mmol) was taken up
in 100 mL of toluene. To it was added bis(tributyl
tin) oxide (2.22 mL, 4.35 mmol) and tetrabu~ylammonium
bromide (0.983 g, 3.05 mmol). The mixture was heated
at 150C for 4 hours and then toluene ~50 mL) was
distilled off from the mixture. The reaction mixture
was cooled to room tempera~ure and benzyl bromide (2.17
mL, 18.27 mmol) was added and the reaction heated to
110C for 36 hours. Toluene was evaporated and the
residue taken up in ethyl acetate (22 mL), washed
successively with aqueous sodium bicarbonate, saturated
sodium chloride solution and water. The organic layer
was dried and evaporated to dryness to give a crude
solid. Purification by column chromatography on silica
gel gave a crystalline solid 15 (1.4 g, 70%).
H-n.m.r.(CDCl3) ~ 7.3-8.1 (9H, aromatic), 4.5 (dd, 2H,
CH2Ph), 5.18 (d, lH, J1 2 lO-HZ~ H-l), 4.36 (dd, lH,
H-3), 4-25 (dd~ H~ J2- lO.OHz, ~2 3 8.0Hz, H-2) and
l.O(t, 3H, CH3).
Example 20 -- Synthesis of Ethyl 6-0-benzyl-2-deoxy-2-
phthalimido-3-0-(2,3,4,-tri-0-benzyl-~-
L-fucopyranosyl)-4-0-(2,3,4,6-tetra-0-
acetyl-~-D-galactopyranosyl)-~-D-
glucopyranoside (Compound 49)
To a stirred solution of compound 15 (2.49,
5.71 mmol) in dry dichloromethane (50 mL) was added dry
- W093/24~05 2 1 1 8 S 2 2 PCT/I~S93tO4909
-- 133 --
CaS04 (7.S g), silver triflate (0.73 g, 2.8 mmol~ and
silver carbonate (7.0 g, 25.7 mmol) and the reaction
mixture cooled to -50C. 2,3,4,6-tetraacetyl~
~romogalactose (3.5 g, 8.5 mmol) in dry dichloromethane
(15 mL) was added dropwise through a dropping funnel.
The reaction mixture was warmed to -30C and stirred
for 48 hours and then methanol (5 mL) was added to
cease the reaction and the mixture allowed to warm to
room temperature. After filtration through a celite
pad and the filtrate was washed with aqueous
bicarbonate and 5% EDTA solution. Evaporation of the
solvent in vacuo gave a reddish brown syrup which was
chromatographed on silica with toluene: acetone:MeOH
(20:3:1) as eluant to give compound 48 (Rf 0.528) as
the major compound.
Thiofucoside 20 (1.5 g, 2.8 mmol) was stirred
in dry dichloromethane ~50 mL) cooled to -20C and
bromine (0.40 g) was added. The conversion to bromide
was complete in 1 hour and the reaction mixture was
washed with cold aqueous bicarbonate, dried and
concentrated to 50 ml and syringed into a flask
containing compound 48 (1 g, 1.4 mmol), HgBrz (1.08 g,
- 3 mmol), molecular sieves 4A (2g) and tetraethyl-
ammonium bromide (1 g) in dry dichloromethane (SO mL).The mixture was stirred at room temperature for 48
hours. T.l.c. showed a faster moving spot. The
reaction mixture was filtered through celite, and the
filtrate washed with water, 5% EDTA, saturated aqueous
sodium bicarbonate, water, then dried over sodium
sulphate, filtered and evaporated to dryness in vacuo.
Purification of the crude product by silica gel
chromatography gave the title compound 49 (1.2 g, 70%,
Rf 0.669 in toluene; acetone; MeOH 20:3:1).
1H-n.m.r.(CDC13): ~ 7.00 - 7.8 (aromatic 24
H) 5-35(d~1H~ Jl,2 9.0Hz, H-l), 5.15 (d, lH, J1 2 3.8Hz,
211~22
W O 93/24505 P ~ /US93/04909
-- 134 --
H--1-Fuc), 4.35(dd, lH, J2 ~3 lO.OHz, H-3 ) 2.1(s, 3H,
acetyl CH3) 1.95(s, 6H, acetyl CH3), l.90(S, 3H, acetyl
CH3), l.l(t, 3H, CH3), and~.O. 6 (d, 3H, CH3~Fuc).
13C-n.m.r.: ~ 168, 170 (~C-O, phthalimido and acetyl),
101.0 (C-l, Gal), 100.0(C-l, GlcNPhth) 97~7(C-1-Fuc),
20.6~ CHz-CH3) and 15.98(C-6-Fuc).
Example 21 -- Synthesis of Ethyl 2-acetamido-6-o-
acetyl-3-O-benzyl-2 deoxy-B-D-
glucopyranoside
A solution of compo~nd 90 (described below-2
g, 4.68 mmol) in aqueous acetic acid (80%, 150 mL) was
heated at 80C for 2 hours. The mixture then was
evaporated and the resultant solid was dried over P2Os
in high vacuum. The dry solid was selectively
acetylated with acetyl chloride (O.33 m~, 4.7 mmol) and
pyridine (10 mL) in dichloromethane tl00 mL) at -10C
to 5C. The mixture was then diluted with
dichloromethane (50 mL), washed with aqueous NaHC03,
dried over MgSO4 and evaporated. The residue was
chromatographed on a silica gel column usinq EToAc:
hexanes, 3:1 (v:v~ as eluant to give 0.82 g (4~) of
the title compound: 1H-n.m.r. (300 MHz, CDCl3):
~7.3(m, 5H, aromatic), 5.67(bs, lH, NH), 4.86(d, lH,
H-l), 4.75(m, 2H), 4.48(q, lH), 4.27(d, lH), 4.1(t,
lH), 3O85~m/ lH3, 3.5(m, 3H), 3.16(m, lH~, 2.70(bs, lH,
OH), 2.1(s, 3H, Ac), l.9(s, 3H, Ac), 1.18(t, 3H, CH3),
3C-n.m.r. (CDCl3): ~ 99.45(C-1), 79.85, 74.5(CH2ph),
73.7, 71.09, 65.25 (C-6), 63.36(CH2-), 57.7(C-2),
23.6(Ac), 20.86~Ac), 15.06(CH3).
Example 22 -- Synthesis of Ethyl 6-O-acetyl-3-O-
benzyl-2-deoxy-2-phthalimido-B-D-
glucopyranoside (compound 69)
A solution of ethyl 2-deoxy-2-phthalimido-~-
D-glucopyranoside (compound 14) from Example 18 was
: W O 93/24505 2 1 1 8 S 2 ~ PC~r/VS93/04909
-- 135 --
taken up in dry acetonitrile tl00 mL) and treated with
benzaldehyde dimethylacetal (9.6 g) and a catalytic
amount of p-toluenesulphonic acid ~100 mg). T~e
mixture was stirred for 17 hours at room temperature
and then neutralized to pH 7 with triethylamin The
mixture was evaporated and crystallized from hot
hexanes to give 12.7 grams of ethyl 4,6-O-benzylidene-
2-deoxy-2-phthalimido-~3-D-glucopyranoside compound 66.
Compound 66 (10 g) was dissolved in dry
dimethylformamide (DMF) at -5~C and treated with 1.1 g
(46.6 mol) sodium hydride and benzyl bromide (5.46 mL,
22 mmol). The mixture was stirred at 0C for 2 hours
and then treated slowly with 20 mL methanol then slowly
brought to room temperature and treated with HCl (lN)
to pH 7 and then extracted three times with
dichloromethane. The organic layer was dried over
anhydrous magnesium suIfate then filtered, concentrated
to dryness and taken up in hot ethanol to give 7.2 g of
Compouhd 67. Compound 67 (S.43 g, 10.50 mmol) in
aqueous acetic acid (80%, 200 mL) was heated at 80C
for 2 hours. The mixture was evaporated and the
resultant solid was dried over P2O5 in high vacuum.
The dry solid was selectively acPtylated with acetyl
chloride (0.8 mL, 11.0 mmol) and pyridine (10 mL) in
dichloromethane (200 mL~ at -10C to 0-C. The mixture
was then diluted with dichloromethane (l 0 mL), washed
with aqueous NaHC03, dried over MgSO4 and e~raporated.
, ~ The residue was chromatographed on a silica gel column
using EtOAc:hexane, 1:2 (v:v) as eluant to give 3.5 g
(71%) of the compound 69: 1H-n.m.r. (300 MHz, CDCl3):
~ 7.7(m, 4H, aromatic), 7.0(m, 5H, aromatic), 5.16(d,
lH, H-l), 4.7(d, lH), 4.5(m, 2H), 4.2(m, 3H), 3.8(m,
lH), 3.6(m, 2H), 3.45(m, lH), 2.9(bs, lH, OH), 2.1~s,
3H, Ac), 1.95(t, 3H, CH3). 13C-n.m.r. (CDCl3):
~ 98.09(C-1), 78.45, 74.5, 73.9, 71.7, 65.1, 63.1,
55.5, 20.87 (Ac), 14.92 (CH3).
211~ 2~
W093/2450s PCT/US93~04~09j--
-- 136 --
Example 23 -- Synthesis of Ethyl 6-O-acetyl-3-benzyl-
2-deoxy-2-phthalimido-4-O (2,3,4,6-
tetra-O-acetyl-B-~-galactosyl)-~-D-
glucopyranoside (compound 70)
S , .
To a stirred solution of compound 9 (80 mg,
0.17 mmol) in dichloromethane (10 mL) containing
molecular sieves (3A, 1 g), 2,6-di-tert-butyl-4-
methylpyridine (45 mg, 0.22 mmol) and silver triflate
10 (57 mg, 0.22 mmol) was added, at -30C under nitrogen,
2,3,4,6-tetra-O-acetyl~a-D-galactosyl bromide in
dichloromethane (S mL). The mixture was stirred at
this temperature for 1 hour and then warmed up to 5C
over ~ hours. The mixture was then diluted with
lS dichloromethane (10 mL), filtered and the insoluble
material was washed with dichloromethane (5 mL). The
combined filtrates were washed with saturated aqueous
sodium hydrogen carbonate and water, dried over MgSO4,
and concentrated. The residue was chromatographed on a
20 silica gel column using ethyl acetate: hexanes, 1:2
(v:v) as eluant to give 120 mg (80%) of the title
compound: 1H-n.m.r. (300 MHz, CDCl3): ~ 7.68,
6.96(2m, 9H, aromatic), 5.3(m, 2H), 5.13(d, lH, H-1 ,
J1.z 8.0Hz), 4.99(q, lH), 4.82~d, lH), 4.62(d, lH,
H-1, J1 2 7.7Hz), 4.S4(d, lH), 4.42(d, lH), 4.3(q, lH),
4.15(m ,2H), 3099(m, 2H), 3.87(m, 2H), 3.72(m, 2H),
3.46(m, lh), 2.15, 2.12, 2.09, 2.00, 1.98(5s, 15H,
- 5XAc), l.OO(t, 3H, CH3). 13C-n.m.r. (CDC13): ~ 101.2,
97.8(C-1, C-l ), ~4.85(C~3).
The 2-amine of Compound 67 above can be
regenerated ~y contacting this compound with hydrazine
acetate and then acetylated with acatic
anhydride/pyridine or other acetylating agents to
provide for a saccharide (~ompound 90)
- W093/~4505 2 ~ 1 8 5 2 ~ PC~/US93/049~9
-- 137 --
Example 24 -- Synthesis of Ethyl 2-acetamido-6-0-
acetyl-2-deoxy-4-0-(2,3,4,6-tetra-O-
acetyl-B-D-galactosyl)-3-0-(2,3,4-tri-0-
benzyl-~-L-fucosyl)-B-D-glucopy~anoside
To a stirred solution of the disaccharide 90
(80 mg, 0.129 mmol) in dichloromethane ~2 mL)
containing molecular sieves (3A, 1 g), tetraethyl-
ammonium bromide (41 mg, 0.195 mmol), dimethylformamide
10(0.1 mL) and diisopropylethylamine (0.087 mL, 0.5 mmol)
was added, at room temperature under nitrogen, a
solution of 2,3,4-tri-0-benzylfucosyl bromide (130 mg,
0.26 mmol -- as per Example 9) in dichloromethane (2
mL). The mixture was stirred at room temperature under
nitrogen for 72 hours and then filtered, and the
insolu~le material was washed with dichloromethane (10
mL~. The combined filtrates were washed with saturated
aqueous sodium hydrogen carbonate and water, dried over
MgS04, and concentrated. The residue was
chromatographed on a silica gel column using ethyl
acetate:hexanes, 3:1 (v:v) as eluant to give 115 mg
(90%) of the title trisaccharide: 1H-n.m.r. (300 MHz,
CDCl3): ~ 7.30(m, 15H, aromatic), 6.00(d, lH, NH, J
8.0Hz), 5.38(d, lH, H-l Fuc, J12 3.3Hz), 5.14(d, lH,
25H-l Glc, J12 7.8Hz), 5.1(m, lH), 4.98(m, 2H), 4.80(m,
6H3, 4.40(m, 2H), 4.33(q, lH), 4.1(m, 5H), 3.77(m, 7H),
3.48(m, lH). 2.09, 2.07, 2.01, 2.00, 1.97(5XAc, 15H),
1.80(s, 3H, NAc), 1.18(d, 3H, H-6 Fuc, J56 6.6Hz),
1.087(t, 3H, CH3OfEt). 13C-n.m.r. (CDCl3): ~ 99.87
30(C-1 Gal), 99.19~C-1 Glc), g7.18(C-1 Fuc), 16.67(C-6
Fuc), 14.79(C~3OfEt3.
Examples 25-30 illustrate the synthesis of
modified sialyl LewisX and sialyl LewisA structures as
depicted in Figures 27 to 32. Sialyl LewisA contains a
core type I structure whereas sialyl LewisX contains a
core type II structure.
211~
W O 93/24505 PC~r/US93/04909 -
-~ 138 --
General methods used in Examples 25-30 as
well as methods for preparation the sialyltransferase
and the fucosyltransferase used in these examples are
set forth below: -
General Methods
Pre-coated plates of silica gel (Merck, 60-
F254) were used for analytical t~l.c. and spots were
detected by charring after spraying with a 5% solution
of sulfuric acid in ethanol. Silica gel 60 (Merck,
230-400 mesh) was used for column chromatography.
Iatrobeads were ~rom Iatron (Order No. 6RS-8060)o
Millex-GV filters (0.22 ~m) were from Millipore. C18
Sep-Pak cartridges and bulk Cl8 silica gel were from
Waters Associates.
Commercial reagents were used in chemical
reactions and solvents were purified and dried
according to usual procedures. Unless otherwise notPd,
the reaction mixtures were processed by dilution with
dichloromethane and washing with a dilute solution of
sodium bicarbonate followed by water. After drying over
magnesium sulfate, the solvents were removed by
evaporation under va~uum with a bath temperature of
35C or lower when necessary.
H-n.m.r. were recorded at 300 MHz or 500 MXz
with either tetramethylsilane in CDCl3 or acetone set
at 2.225 in D20 as internal standards, at ambient
temperature, unless otherwise noted. The chemical
shifts and coupling constants (observed splitting) were
reported as if they were first order, and only partial
n.m.r. data are reported. 13C-n.m.r. spectra were
recorded at 75.5 MHz with tetramethylsilane in CDCl3 or
dioxane set at 67.4 in D20 as reference.
W~93/2450~ 2 1 1 ~ S ~ 2 PCT/US93/04909
~- 139 --
Frozen rat livers were from Pel-Freeze
Biologicals. Sepharose 6B, Dowex l-X8 were from
Pharmacia, CDP and CMP-Neu5Ac were from Sigma.
GDP-fucose was obtained by chemical synthesis as
descri~ed below. All other chemicals were of
analytical grade and of commercial origin.
Preparative Example A -- Preparation of
~Gal(1~3/4)~GlcNAc ~(2-3)sialyltransferase
The ~Gal~1~3/4)~GlcNAc ~(2~3)sialyl-
transferase [(EC 2.4.99.5) -- sometimes referred to as
"~(2-3)ST"] and the ~Gal(1-4)~GlcNAc ~(2-6)sialyl-
transferase [(EC 2.4.99.1) - sometimes referred to as
"~(2~6)ST" were extracted from rat liver (600 g) using
Triton CF-54 (Sigma) according to Weinstein et al. 74
The enzymes from the Triton extract were partially
purified and concentrated on Cibacron Blue F3GA-
Sepharose by a reported modification~ of Sticher et
al.'~s process.~ The detergent extract (3L, 3.5 mg
protein/ML) was loaded onto a column (8 x 20 cm) of
Cibacron Blue F3GA (Serva) linked to Sepharose 6B
(prepared according to Dean and Watson76) equilibrated
in 10 mM sodium cacodylate (pH 6.5), 0.15 M NaCl, 25%
glycerol, 0.1~ Triton CF-54 (buffer A) in two portions,
with a wash step in between with buffer A. The column
was washed with the same buffer until no further
protein was eluted, and was then eluted with buffer A
containing 2.0 M NaCl. Active fractions containing
sialytrans~erases were pooled, concentrated by
ultrafiltration on an Amicon PM 30 membrane and
dialyzed against 200 volumes of buffer A. The ~(2~3)ST
was separated from the ~(2~6)ST and purified by
affinity chromatography on a matrix ~LeC-Sepharose)
3S obtained by covalently linking the hapten
~Gal(1-3)~GlcNAcO(CH2)8COOH disclosed by Mazid et al.
to activated Sepharose described by Matsumoto78 using
art recognized techniques involving the N-succinimidyl
2 1 1 ~ ~ 2 ~
W093/24~05 PCT/US93/04909
-- 140 --
ester of the hapten. The sialytransferases, partially
purified by the above dye chromatography, containing
-160 mU of ~(2~3)ST and 2.4 U of ~t2~6)ST (about 860 mg
protein~ were diluted with an equal volume of buffer A
containing 2.5 mM CDP at a flow rate of 5 mL/hour. The
column was washed with the eq~ilibrating buffer to
remove any loosely bound protein. Enzyme activity
determination indicted that the ~(2~3)ST adsorbed
strongly to the column during application and
subsequent wash steps, while the bulk of the inert
protein and the ~(2-6)ST eluted unretarded. The
~(2~3)ST was then eluted from the column with buffer A
containing 0.2 M lactose. Fractions (2 mL each)
containing the ~(2~3)ST were pooled and concentrated to
- 15 a small column (~1 mL) on an Amicon PM 30 membrane.
The concentrate was dialyzed against 200 volumes of 50
mM sodium cacodylate (pH 6.5), 0.25 M NaCl, 50%
glycerol, 0.1~ Triton CF-54 and stored at -20~C. This
preparation, 82,000-fold purified to a specific
activity of 2.7 U/mg protein, was devoid of ~(2~6)ST
activity when preparative sialylation using
~Gal(1-4)~GlcNAc-O-(CH2)8C~OCH3 (compound 22a) as the
acceptor9 was carried out and the product analyzed by
1H-n.m.r. spectroscopy and by t.l.c.
Preparative Example B -- Preparation of the ~Gal(1~3/4)
~GlcNAc ~ 3~4)fucosyltransferase from Human Milk
(EC 2.4.1.65)
The enzyme was purified from human milk
obtained ~rom Lewisa~b~ donors, according to the
methodology using affinity chromatography on GDP-
hexanolamine Sepharose described by Palcic et al. 25 .
Example 25 -- Synthesis of the starting materials:
Synthesis of Acceptors: Compounds 20b, 20c,
22b, 22c, 22d (Figure 29)
::- YVO 93/24505 P ~ /VS93/04909
211~S 2 ~
- 141 --
A. Preparation of 8-Methoxycarbonyl 2,3,4,6- :
tetra 0-acetyl-~-D-galactopyranosyl-(1~3)-O-
2-azido-2-deoxy-~-D-gluGopyranoside (compound
119) and 8-methoxycarbonyl 2,3,4,6-t~tra-0-
acetyl-~-D-galactopyranosyl-(1-4)-0-2-azido-
2-deoxy-~-D-glucopyranoside (compound 121).
A solution of trimethylsilyltrifluoromethane-
sulfonate (0.460 g, 1.94 mmol) in dry methylene
chloride (5 mL) was syringed dropwise to a mixture of
compound 171Z (1.20 g, 1.94 mmol), compound 18 (0.757
g, 1.94 mmol) and molecular sieves 4A (2 g) in
dichloromethane stirred at 22C. After 2 h, the
reaction was stopped by addition of triethylamine, the
mixture filtered and worked up as usual. The recovered
material was chromatographed on silica gel (150 g)
using a 3:1 mixture of hexanes and ethyl acetate as
eluant providing a mixture of the ~ 3) and the ~ 4)
disaccharide (1.10 g, 60%) which could not be separated
at this stage.
Tetraethylammonium chloride (0.196 g, 1.18
mmol) and anhydrous potassium fluoride (0.299 g, 5.15
mmol) were added to a solution of the mixture of the
above disaccharides (0.460 g, 0.487 mmol3 in dry
acetonitrile (10 mL). After 24 hours at 22~C, acetic
acid (1-5 mL) was added and the solvents were
evaporated in vacuo. The residue was dissolved in
chloroform (20 mL), washed with a dilute solution of
sodium bicar~onate followed by water. The recovered
crude material was chromatographed on silica gel (36 g)
using a 1:1 mixture of ethyl acetate and hexane as
eluant providing the (1-4) disaccharide 121 (157 mg,
46%) and the (1-3) disaccharide 119 (96 mg, 28~).
Disaccharide 121: [~]D20 +6.9~(c 1.0, CHCl3)
~-n.m.r. (CDCl3) S.381 (d, lH, J3,4, 3.5Hz, H-4'),
5.222(dd, lH, J~2~ 8.0Hz, ~2, 3, lO.OHz,H-2'), 5.014
(dd, lH, H-3'), 4.628(d, lH, H-1'), 4.257[m, incl. H-1
.
2118~2'~
W093/24505 PCT/US9~/04909;-
-- 142 --
(d, J12 8.0Hz)), 3.420-3.640[m, incl. CO2CH3(s,
3.650~], 2.227~t,2H, J 7.5Hz, CH2CO2), 2.150, 2.100
(two), 1.950(3s, 12H, 4 OAc), 1.600(m, 4H, met~ylenes),
1.300(m, 8H, methylenes);
. ~
Disaccharide 119; [~]D20 +7,8 (C 1, CHCl3)
H-n.m.r. (CDCl3) 5.359 (d,lH, J3, 4, 3.2Hz), 5.225(dd,
lH, J~ 2 8.0, J2 31 10 Hz; H-2'), 5.013 (dd, lH,
H-3~), 4.524 ~d, lH, H-l'), 4-300 (d~ J12 8-0HZ, H~
3.628(s, 3H, CO2CH3), 2.230 (t, 2H, J 7.5 Hz, CH2CO2),
2.150, 2.080, 2.000, 1.920 (4s, 12H, 4 OAc), 1.600(m,
4H, methylenes), 1.300(m, 8H, methylenes).
For identification purposes both
disaccharides were peracetylated in a mixture of
pyridine and acetic anhydride.
P~racetylated derivative of 121: 5.314(dd,
lH, J3,4, 3.5, J4,5, <lHz,H-4'), 5.047(dd, lH, Jl,z,
8.0, J2 3~ lO.OHz, H-2'), 4.870-4.970~m,2H, incl. H-3,
4-923(dd~ J2~3~ ~ J3"4, lO.OHz) and H-3'(4.903, dd)]
4.420tm, 2H, incl. H-l'(d)~, 4.300(d, J12 8.0Hz, H-l),
3.627[m, incl. CO2CH3(s, 3.627)], 3.335(dd, lH, H-2),
2.230(t,J 7.5Hz, CH2CO2) 2.080, 2.070, 2.050, 2.010,
1.980, 1.936(5s, 18H, 6 OAc), 1.570(m, 4H, methylenes),
1.210(m, 8H t methylenes).
Peracetylated derivative of compound 119: ;
5.120(dd, lH, J3, 4, 3.5, J4,5, l.OHz, H-4'), 5.080
(dd, 1~ J1"2, 7-8, J2~,3~ lO-OHz, H-2'), 4.980 (dd,
lH, H-3'), 4.875(dd, lH, J3 4 - J4,5 - lO-OHz, H-4),
4.715(d, lH, H-l'), 4.257(d, lH, J12 8.0Hz, H-l),
3.627(s, 3H, CO2CH3), 3.320(dd, lH, J23 lo.oHz, H-2),
2.230(t, J 7.5Hz, CH2CO2), 2.080, 2.050, 2.020, 2.010,
1.970, (6s, 18H, 6 OAc), 1.600(m, 4H, methylenes),
1.250(m, 8H, methylenes).
~ W093~24505 2 1 1 8 5 2 ~ PCT/US93/04909
-- 143 --
B. Preparation of 8-Methoxycarbonyloctyl ~-D-
galactopyranosyl-(1-3)-0-2-azido-2-deoxy-~-D-
glucopyranvside (compound 120b)
A catalytic amount of a dilute solutlon of
sodium methoxide in methanol was added to a solution of
compound 119 (0.045 g, 0.064 mmol) in methanol (2 mL).
After 5 hours at 22C, neutralization with Dowex 50W X
8 (H~ form) and filtration, the solvent was evaporated
in vacuo providing the pure 120b (30 mg, 88%);
[~]D20 - 11.7 (C 0.65, H20) lH-n.m~r. (CD30D,DOH: 4.80):
~ 4.45(d, lH, J 7.0Hz) and 4.34(d, lH, J 7.5Hz): H-l
and H-l', 3.61(s, CO~CH3), 2.27(t, 2H, J 7.5Hz,
CH2CO2), 1.58(m, 4H) and 1.30 (m, 8H): methylenes.
CO Preparation of 8-Methoxycarbonyloctyl ~-D-
galactopyranosyl-(1~3)-0-2-amino-2-deoxy-~-D-
glucopyranoside (compound 120c)
Compound 120b (0.018 g, 0.034 mmol) was
hydrogenated in the presence of 5% palladium on carbon
(5 mg) in methanol (2 mL) at atmospheric pressure for 6
hours. After filtration through`Celite, the solvent
was evaporated and the residue chromatographed on
Iatrobeads (2 g~ using a mixture of chloroform and
methanol as the eluant providing the pure compound
120C; ~a]20D -4.2o (c 0.48 H20); 1H-n.m.r. (DzO, DOH at
4.80): ~ 4.56 and 4.48(2d, lH each, J 7.5Hz): H-l and
H-l', 3.70(s, CO2CH3), 2.90(-t, lH, J 9.5Hz, H-2)
2.39(t, 2H, J 7.5Hz, CHzCOz), 1062(m, 4H) and 1.35 (m,
8H): methylenes.
D. Preparation of 8-Methoxycarbonyloctyl ~-D-
galactopyranosyl~ 4)-0-2-azido-2-deoxy-~-D-
glucopyranoside (compound 122b)
A catalytic amount of a dilute solution of
sodium methoxide in methanol was added to a solution of
compound 121 (0.027 g, O. 38 mmol) in methanol (2 mL).
After 5 hours at 22C, neutralization with Dowex 50W X
8 (H' form) and filtration, the solvent was evaporated
211~52~
W093/24505 PCT/US93/04909
-- 144 --
in vacuo. The residue was chromatographed on
Iatrobeads using a 65:35 mixture of chloroform and
methanol as eluant to give compound 122b (O.Ol~ g,
92%); [~]20D -12.4 (c 0.73 CH30H); 1H-n.m.r. (CD30D,
DOH at ~.80): ~ 4.32(d, lH, J 7.5Hz) and 4.30(d,1H, J
8.0Hz): H-l and H-l', 3;60(s, C02CH3), 3.13(dd, J1 2 ~-
J23 lO.O~z, H-2) 2.27(t, 2H, J 7.5Hz, CH2CO2), 1.56(m,
4H~ and l.29(m, 8H): methylenes.
E. Preparation of 8-Methoxycarbonyloctyl ~-D-
galactopyranosyl-(1-4)-0-2-amino-2-deoxy-~-D-
glucopyranoside (compound l22c)
lS Compound 122~ ~0.016 g, 0.29 mmol) was
hydrogenated in the presence of 5% palladium on carbon
(lO mg) in methanol (5 mL) for 5 hours at 22C. After
filtration through Celite, the solvent was evaporated
and the residue chromatographed on Iatrobeads (0.25 g)
using a 8:2 mixture of chloroform and methanol as
eluant providing the pure ~22c (0.013 g, 86~).
[~20 2.8 (c 0.42, H20)-
F. Praparation of 8-Methoxycarbonyloctyl ~-D-
galactopyranosyl~ 4)-0-2-deoxy-2-propion-
amido-~-D-glucopyranoside (compound 122d)
Compound 12i (0.017 g, 0.032 mmol) was
hydrogenated in the presence of 5% palladium on carbon
~5 mg) in methanol (8 mL) at atmospheric pressure for 8
hours. After filtration through Celite and evaporation
of the sol~ent, the residue was dissolved in dry
methanol (3 mL) containing some triethylamine (0.150
mL). Propionic anhydride (0.150 mL) was added and the
mixture was stixred for 4 hours at 22C after which the
solvents were evaporated to dryness. The residue was
acetylated in a 2:l mixture of pyridine and acetic
anhydride (4 mL) at 22C for 18 hours. After addition
of methanol, the mixture was worked up as usual and
W093/2450~ 2 1 l ~ S 2 2 PCT/US93/04909
-- 145 --
after evaporation of the solvents, the residue was
chromatographed on silica gel using a 1:1 mixture of
ethyl acetate and hexane as eluant providing the pure
hexa-O-acetate of compound 122d; 1H-n.m.r. (CDCl3):
5.50(d, lH, J 9.5Hz, NH), 5.32( d, J3, 4, 3~5Hz, H-4'),
5.07(m, 2H, H-2' and H-3), 4.93~dd, lH, J2~3~ 10.0Hz,
~-3'), 4.4S(m, 3H, incl. H~1 and H-1'), 3.63(s,
CO2CH3), 2.257(t, 2H, J 7.5, CH2COz), 2.137(dq, J 1.0
and 7.5Hz, NHCH2), 2.11, 2.07, 2.02 (three), 1.93t4s,
12H, 6 OAc), 1.540(m, 4H) and 1.25(m, 8H): methylenes,
1.09 (t, 2H, NHCH2CH3).
.
The above disaccharide was de-O-acetylated in
dry m~thanol (1 mL) containing a catalytic amount of a
solution of sodium methoxide. After neutralization
with Dowex 50W X 8 (H~ form) resin and filtration,
evaporation of the solvents left the pure 122d; [a] 20D
-18.0 (c 0.43, CH30H); lH-n.m.r. (CD30D, DOH at 4.80~:
~ 4.36(d, lH, J 8.0Hz) and 4.33(d, lH, J 7.5Hz~: H-l
and ~-1', 3.60(s, CO2CH3), 2.26(t, 2H, J 7.5Hz,
CH2CO2), 2.18(q, 2H, J 7.5Hz, NHCOCH2), 1.51(m, 4H) and
1.26(m, 8H): methyl nes, 1. 09 (t, 3H, NHCOCH2CH3).
Example 26 -- Synthesis of Sialylated Trisaccharides
(CQmpounds lllb, lllc, and llld, Figure 27A)
A. Preparation of 8-methoxycarbonyloctyl (5-
acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
2-nonulopyranosylonic acid)-(2~3)-O~ D-
galactopyranosyl)-(1-4)-0-(2-azido-2-deoxy-~-
D-glucopyranoside (Trisaccharide l1lb)
Known12 trisaccharide 10~ (60.8 mg, 0.05
mmol) is hydrogenated in ethyl acetate (1.5 mL) at 22C
in the presence of 5% palladium on carbon for 1 hour to
obtain the intermediate free acid (sialic acid~.
~a]D2-18.6 (c,0.3, chloroform). This product is
211~S2~
W093/2450~ PCT/US93/04909
-- 146 -~
de-O-acetylated using a catalytic amount of sodium
methoxide in methanol for 16 hours at 22 J C and the
recovered material is chromatographed on BioGe~ P2
providing trisaccharide 111b ;(10.4 mg, 55%~,
[a]D2-6~5 (c,0.17, water).` 1H-n.m.r. data are
reported below.
B. Preparation of 8-Methoxycarbonyloctyl
(benzyl-5-acetamido-4,7,8,9-tetra-0-acetyl-
3,5-dideoxy-D-glycero-~-D-galacto-2-
nonulopyranosylonate)-(2-~3)-0-(2,4,6-tri-0-
acetyl-~-D-galacto-pyranosyl)~ 4)-0-(6-0-
acetyl-2-amino-2-deoxy-~-D-glucopyranoside)
(trisaccharide 109)
Hydrogen sulfide is bubbled through a
solution of trisaccharide 104 (400 mg, 0.32 mmol) in a
mixture of pyridine (32 mL), water (4.8 mL) and
triethylamine (1.3 mL). After 16 hours at 22C, the
mixture is evaporated to dryness and co-evaporated with
toluene to give a crude trisaccharide (430 g). Some of
this material (85.9 mg, 0.07 mmol) is chrom~tographed
(10:1, toluene:ethanol) providing 109 (55 mg, 70%).
ta]D+25.9 tc,0.22, chloroform); 1H-n.m.r. (CDCl3~:
~ 7.25-7.45 (m,~ 5H, aromatics), 5.480(m, H-8",
overlapping with 5.42 (d, J 12.5Hz, benzylic), 5.340 (dd,
lH~ J6~ 2 5, J7~ 8~ 8.5Hz t H-7"), 5.052(m, incl.
benzylic (d) and H-2' dd(Jl, 2l 8.0, J2~ 3,10.0Hz)
5-000(dd, lH, J3, 4, 3.5Hz, H-4'3, 4.904(d, lH, J
10.0HZ, NH), 4.860(ddd~ lH, J3"~,4" 4-5, J3,.ax,41, 12-5
J4" 5" ll.OHz, H-4"), 4.640tm, 2H, incl., H-l' and H-
~, 3~), 3.660(s, 3H, OCH3), 2-780(dd, Jl,2~J2,3 8-5Hz H 2)~
2.604(dd, lH, J3--~,3~ax 13 .OHz, H-3~), 2. 300 (t,J 7.5Hz,
: CH2CO2), 2.260, 2.170, 2.115, 2.080(three), 2.050,
1.985, 1.830(7s,27H, 8 OAc, 1 NAc), 1.670 (t, lH, J
H-3eq), 1.600(m, 6H, methylenes), 1.240(m, 8H,
methylenes).
W093/24505 2 1 1 8 5 2 .~ PC~/US93/04909
-- 147 --
C. Preparation of 8-Methoxycarbonyloctyl (5-
acetamido-3,5-dideoxy-D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-(2~3)-o-(~-D-
galacto-pyranosyl)-(1~4)-0-(2-amino~2-deoxy-
~-D~glucopyranoside (trisaccharide lllc)
A solution of the pure 109 (53 mg, 0.04 mmol~
is hydrogenated in methanol for 1 hour at 22~C in the
presence of 5% palladium on carbonO Filtration of the
catalyst and evaporation of the methanol provides the
sialic acid intermediate t44 mg), [~D~11.3
(c/0.22,water). This compound is de-O-acetylated using
a catalytic amount of sodium methoxide in met:hanol for
24 hours at 22C. Evaporation of the solution obtained
after neutralization with acetic acid left a material
which is purified by chromatography on BioGel P2 to
provide for trisaccharide llc (29.5 mg, 99%) ~ [~]D~ 5~5
(c, 0.22, water). 1H-n.m.r. data are reported below.
D. Preparation of 8-Methoxycarbonyloctyl
(benzyl-5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-~-D-galacto-2-
nonulopyranosylonate~-(2-3)-0-(2,4,6-tri-O-
acetyl-~-D-galacto-pyranosyl)-(1-4)-0-(6-O-
acetyl-2-deoxy-2-N-propionamido-~-D-
glucopyranoside) ~trisaccharide 110)
The crude amino compound 109 (98 mg, 0.08
mmol) is N-propionylated by adding propionic anhydride
dropwise over 10 min to a solution of the crude amino
trisaccharide 109 in a mixture of pyridine and water
(about a 10:1 ratio of pyridine to water). The mixture
is stirred overnight at 22C, evaporated in vacuo and
co-evaporated with toluene leaving a residue which is
chromatographed (100:10, toluene:ethanol) providing
trisaccharide 110 (74.4 mg, 71%). [~]D+10.3 (c,0.17,
- chloroform); 1H-n.m.r. (CDCl3): ~ 7.400 (m, 5H,
aromatics), S.S43(d, lH, J 7.5Hz, NH), 5.480(m, lH,
H-8'~), overlapping with 5.440(d,lH, J 12.5Hz,
benzylic), 5.341 (dd, lH, J6~,7~ 2-5~ J7~,8~ 8-5Hz~
h ~ 2 ~
W O 93/2450~ PC~r/US93/04909
~ 8 --
H-7"), 4.490-5.100~m, 3H, incl. benzylics (5.051, d, J
12.5 Hz), H-2'(5.038r dd, J~. 2~ 8-09 J2' 31lo-OHz)~
4.859 (ddd, lH, J3l'eq,4 4-6~ J3l~ax4~l 12-5, J4" 5" 10.
H-4"), 4.610-4.69[m, 2H, incl., H-l'~d) and H-3'(dd)],
3.580-3.700[m, 2H, incl.,-OCH3(s, 3.668)], 2.602(dd,
lH, J3 eq 3"aX12.5HZ, H-3"eq), 2.150-2.330 [m, lOH,
incl., CH2CO2(t, J 7.5Hz), NHCOCH2(q, J 7 .5Hz) and
acetyls (2.260, 2.180, 2s)], 2.088(four), 2.068, 1.987,
1.838(4s, 21H, acetyls), 1.662(t, lH, H-3"ax), 1.570(m,
6H, methylenes), 1.240(m, 8H, methylenes), 1.130(t, 3H, -
CH2C~I3 ) -
E. Preparation of 8-methoxycarbonyloctyl (5-
acetamido-3,5-dideoxy-D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-~2-3)-O-(B-D-
galactopyranosyl)-(1~4)-0-(2-deoxy-2-N-
propionamido-B-D-glucopyranoside. (ll~d)
Trisaccharide 110 (71 mg, 0.055 mmol) is
hydrogenated in the same manner as indicated in the
synthesis of trisaccharide lllc to obtain the
intermediate product (64 mg, 97%), [~]~-22.6~ (c, 0.23,
chloroform). This material was de-O-acetylated as
usual and the recovered material chromatographed on
BioGel P2 giving llld (39 mg, 83%), [~D -8.5 (C, 0.2,
water). lH-n.m.r. data are reported below.
Example 27 -- Synthesis of Sialylated Trisaccharides
(Compounds 107b, 107~, and 107d, Figure 27B)
A. Preparation of 8-methoxycarbonyloctyl ( 5-
acetamido-3,5-dideoxy-D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-(2~3)-O~
galactopyranosyl)-(1-3)-0-(2-azido-2-deoxy-~-
D-glucopyranoside (Trisaccharide 107b)
Known12 trisaccharide 103 (60.8 mg, 0.05
mmol) was hydrogenated in ethyl acetate (1.5 mL) at
22C in the presence of 5% palladium on carbon (60 mg)
for 1 hour to obtain the intermediate free acid (sialic
~ W093~24505 2 1 1 ~ S 2 ~ PCT/US93/04909
-- 149 --
acid) (57.6 mg, 98.8%), [~]D-14.7 (c, 0.18,
chloroform); i.r. (chloroform~: 2120 cm1 (N3). This
intermediate was de-O-acetylated by using a ca~alytic
amount of sodium methoxide in methanol for 16 hours at
22C. After neutralization with BioRex-70 tBioRad)
weak acid resin (H+ form) and evaporation of the
filtrate, the residue was chromatographed on BioGel P2
providing trisaccharide 107b (36.6 mg. 88%~ D-10.1
(C,0.32, water). 1H-n.m.r. data are reported below.
B. Preparation of 8-methoxycarbonyloctyl
(benzyl-5-acetamido-4,7,8,9-tetra-0-acetyl-
3,5-dideoxy-D-glycero-~-D-galacto-2-
nonulopyranosylonate)-(2~3)-0-(2~4,6-tri-0-
acetyl-~-D-galactopyrano-syl)-(1-3)-0-~6-0-
acetyl-2-amino-2-deoxy-~-D-glucopyranoside)
(trisaccharide ~05~
Hydrogen sulfide was bu~bled through a solution of
trisaccharide 103 (500 mg, 0.4 mmol) in a mixture of
pyridine (40 mL), water (6 mL) and triethylamine (1.5
mL). After 16 hours at 22C, the mixture was
evaporated to dryness and co-evaporated with toluene to
give a crude product (4SO mg). Some of this material
(105 mg) was chromatographed (10:1, toluene-ethanol)
providing tetrasaccharide 105 (86 mg, 75%), ~]D20
-21.1 (c,0.02, chloroform). 1H-n.m.r. (CDCl3):
~ 7.32-7.45(m, 5H, aromatics), 5.484(ddd, lH, J7"8"8.5,
J8~9~8 2.5, J8.. 9.. b 5.5Hz,H-8 "), 5.418(d, lH, J 12.2Hz,
benzylic), 5.291(dd, lH, J6~7~ 2.7Hz, H-7''), 5.080(dd,
lH~ J1~,2~ 8-0~ J2',3' lO-OHz, H-2'), 5.022(d, lH, J
12.2Hz, benzylic), 4.980(bd, lH, J3, 4, 3.5Hz, H-4'),
4-890 (d, lH, J5~,NH 10.5Hz, NH~, 4.829(ddd,1H,
J3"~4"4 5~ J3.,aX4"12.5, J4.5. 10.5Hz,H-4"), 4.739(d,
lH, J~2 8.0Hz, H-l'), 4.670(dd, lH, J2 3~ 10.3,
J3,4, 3.5Hz, H-3'), 3.635(s, 3H, OCH3), 2.851(dd, lH,
J12 8.5, J23 9.5Hz, H-2), 2.586(dd, lH, J3~'ax 3~
13.OHz, H-3"eq), 2.265(t, lH, 7.5Hz, CH2C02), 2.222,
211~5~
W093/24~05 PCT/US93/0490g -
-- 150 --
2.150, 2.060, 2.050, 2.04S, 2.040, 2.018, l.9S8,
1.805(9s, 3H each, 8 OAc, 1 NHAc), 1.650(t, lH,
H-3"ax), 1.564(m, 6H, methylenes), 1.300(m, 8H
methylenes).
C. Preparation of 8-methoxycarbonyloctyl (5-
acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
2-nonulopyranosylonic acid~-(2~3)-O-(~-D-
galacto-pyranosyl)-(1~3)-0-(2-amino-2-deoxy- ~-~
~-D-glucopyranoside (tetrasaccharide 107c)
A solution of pure trisaccharide 105 (82 mg,
0.07 mmol) in methanol (1 mL) was hydrogenated for 1
hour at 22C in the presence of 5% palladium on carbon
(82 mg). Filtration of the catalyst and evaporation
left the intermediate (76 mg), t~]o20 +6.0 (c,0.4,
chloroform). This compound (72 mg, 0.06 mmol) was
de-O-acetylated by using a catalytic amount of sodium
methoxide in methanol (3 mL) for 24 hours at 22C. -
Evaporation of the solution obta ned after
neutralization with acetic acid left a material which -~
was purified ~y chromatography on BioGel P2 providing -
trisaccharide 107c (45.5 mg) 88%, [~] D-6.3 (CI.3 5
water). 1H-n.m.r. data are reported below.
;
D. Preparation of 8-Methoxycar~onyloctyl
(benzyl-5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-(2~3)-0-(2,4,6-tri-O- -~
a~etyl-~-D-galactopyranosyl)-(1-3)-0-(6-O-
acetyl-2-deoxy-2-N-propionamido-~-D-
glucopyranoside) (trisaccharide 106)
Propionic anhydride (0.5 mL) was added
dropwise in 10 min to a solution of the crude amino
trisaccharide 105 (140 mg, 0.11 mmol) in a mixture of
pyridine (9.5 mL) and water 0.9 mL). The mixture was
~tirred overnight at 22C, evaporated in vacuo and
co-evaporated with toluene leaving a residue which ~as
chromatographed (100:10, toluene:ethanol) providing
.
~ . W O 93/24505 2 1 1 ~ 5 2 2 PC~r/US93/04909
-- 151 --
trisaccharide 106 (110 mg, 7~%) which contained a small
amount of the 4-0-propionylated trisaccharide.
H-n.m.r. (CDC13): ~ 7.310-7.450(m, 5H, aromatics),
5.812(d, lH, J 8.0Hz, NH), 5.45[m, incl., H-8" and
benzylic (d, J 12.5 Hz)], 5.280(dd), J~"7" 2.7, J7~8
8.5Hz, H-7"), 5.300~m, incl. benzylic (d)], 3.630(s,
3H, OCH3), 2.578(dd, lH~ J3~,4 4-5, J3~l~,3llax 13 . 0Hz ,
H-3~eq), 2 . 262(t, 2H, J 7.5Hz, CH2CO2),
2.200(q,NHCOCH2), 2.147, 2.060, 2.045 (five), 1.955,
1.800(5s, 27H, 8 OAc, 1 NAc), 1.630(t, lH, J3~ax 4~l 13.0
Hz, H-3~ l.S70(m, 6H, methylenes), 1.240(m, 8H,
methylenes), 1.130(t, 7.5Hz, COCH2CH3).
E. Preparation of 8-methoxycarbonyloctyl (5-
acetamido-3,5 dideoxy~D-glycero-~-D-galacto-
2-nonuIopyranosylonic acid)-(2-3)-0-B-D-
galacto-pyranosyl-(1-3)-0-(2-deoxy-2-N-
propionamido-(~-D-glucopyranoside
~trisaccharide 107d)
Trisaccharide 106 (105 mg, O.082 mmol) was
deprotected using the same procedure as indicated in
the synthesis of 107c. Chromatography on BioGel P2
provided the pure 107d (60.8 mg, 95%), [~]D2-16.6
(c,0.32, water). 1H-n.m.r. ~ata are reported below.
Example 28 -- Pre~arative Fucosylation
i. Synthesis of GDP-Fucose.
A. Preparation of Bis(tetra-n-butylammonium)
hydrogen phosphate
Tetra-n-~utylammonium hydroxide (40% aq. w/w,
about lSOg) 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 acetonitrile (2 x 400 mL) followed
.
211~2~
W093/2450~ PCT/~S93/0~909
-- 152 -_
by dry toluene (2 x 400 mL). The resulting white solid ~-
(75g) was dried in vacuo and stored over phosphorus
pentoxide under vacuum until used.
.. ~.
B. Preparation of ~-L-Fucopyranosyl-1-phosphate
A solution of bis(tetra-n-butylammonium)
hydrogen phosphate (S8g, 127.8 mmol) in dry
acetonitrile (300 mL~ was stirred at room temperature
under nitrogen in the presence of molecular sieves (4A, -~
20g) for about one hour. A solution of tri-0-acetyl
fucosyl-1-bromide (freshly prepared from 31g, 93 mmol -
of L-fucose tetraacetate in the manner of Nunez et
al.80) 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 OoC, the mixture was brought to
room temperature and stirred for 3 hour. T.l.c. (1:1
toluene:ethyl acetate) indicated a main spot on the
base line and several faster moving smaller spots~
The mixture was filtered over a pad of Celite
(which was further washed with acetonitrile) and the
solvents evaporated in vacuo to gi~e a red syrup. This
material was dissolved in water (400 mL) and extracted
with ethyl acetate (250 mL, twice). The aqueous layer
was then evaporated in vacuo leaving a yellowish syrup
to which a solution of ammonium hydroxide (25~ a~., 200
mL~ was added. The mixture was stirred at room
temperature for 3 hours after which t.l.c. (65:35:8
chloroform:methanol:water) indicated a baseline spot.
The solvent was evaporated in vacuo to give a yellowish
syrup which was diluted with water (400 mL). The pH of
this solution was checked and brought to 7, if
necessary, by addition of a small amount of
hydrochloric acid. The solution was slowly absorbed
onto a column of ion exchange resin Dowex 2 X 8
~-~ W~93/24505 2 1 ~ ~ ~ 2 2 PCT/US93/04909
-- 153 --
[200-400 mesh, 5 x 45 cm, bicarbonate form which had
been prepared by sequential washing of the resin with
methanol (800 mL), water (1200 mL), ammonium
bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water
(1000 mL) was then run through the column followed by a
solution of ammonium bicarbonate (0.5 M, 2.3 mL/minute,
overnight). The eluate was collected in fractions (15
mL) and the product detected by charring after spotting
on a t.l.c. plate. Fractions 20 to 57 were pooled and
evaporated in vacuo leaving a white solid which was
further co-evaporated with water (3 x 300 mL) and
free~e drying of the last 50 mL and then drying of the
residue with a vacuum pump to give ~-L-fucopyransyl-l-
phosphate (9.5g, 40%) as a 12:1 mixture of ~ and ~
anomers containing some ammonium acetate identified by
a singlet at ~=1.940 in the 1H-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-phosphate as a sticky gum after
freeze drying of the eluate. lH-n.m.r. ~:4.840 (dd,
J-2 = J~p = 7.5 Hz, ~-1), 3.82 ~q, lH, Js6 6.5 Hz,
H-5), 3.7S0 (dd, lH, J3 4 3.5, J4 5 1.0 Hz, H-4), 3.679
(dd, lH, J2 3 10-0 Hz, H-3), 3.520 (dd, lH, H-2), 1.940
(s, acetate), 1.26 (d, H-6). Integral of the signals at
3.20 (q, J 7.4 Hz, NCHz) and 1.280 and 1.260 (NC~2C~3
and H-6) indicates that the product is the bis-
triethylammonium salt which may loose some
triethylamine upon extensive drying. 13C-n.m.r. ~:98.3
(d, Jc 1P 3.4 Hz, C-l), 72-8 (d~ Jc 2P 7-5 Hz~ C-2)~
16.4(C-6); 31P-nmr ~: +2.6(s).
~-L-fucopyransyl-l-phosphate appears to
slowly degrade upon prolonged storage (1+ days) in
water at 22C and, accordingly, the material should not
be left, handled or stared as an aqueous solution at
22C or higher temperatures. In the present case, this
211~52;~
W093/24~,0~ PCT/US93/~4909
-- 154 --
material was kept at -18C and dried in vacuo over
phosphorus pentoxide prior to being used in the next
step.
C. Preparation of Guanos~ne 5'-(~-1-fucopy- -
ranosyl)-diphosphate
Guanosine 5'-(~ fucopyranosyl)-diphosphate
was prepared from ~-L-fucopyranosyl-1-phosphate using
two different art recognized procedures as set forth
below:
'~
PROCED~RE #1 ~
- -
~-L-fucopyranosyl-1-phosphate and guanosine
5'-mono-phosphomorpholidate (4-morpholine-N,N'-di- -
cyclohexyl-carboxamidine salt, available from Sigma,
St. Louis, Missouri, "GMP-morpholidate") were reacted
as described in a recent modification~81 of Nunez's
original procedure80. Accordingly, tri-n-octylamine
(0.800g, available from Aldrlch Chemical Company,
Milwaukee, Wisconsin) was added to a mixture of ~-L-
fucopyranosyl-~-phosphate (triethylammonium salt,
1.00g, about 2.20 mmol) in dry pyridine (10 mL) under
nitrogen the solvent removed in vacuo. The process was
repeated three times with care to allow only dry air to
enter the flask. GMP morpholidate (2.4g, about 3.30
mmol) was dissolved in a 1:1 mixture of dry
dimethylformamide and pyridine (10 mL). The solvents
were evaporated in vacuo and the procedure repeated
three times as above. The residue was dissolved in the
same mixture of solvents (20 mL) and tha solution added
to the reaction flask accompanied by crushed molecular
sieves (4A, 2g). The mixture was stirred at room
temperature under nitrogen. T.l.c. (3:5:2 25% aq.
ammonium hydroxide, isopropanol and water) showed spots
corresponding to the starting GMP-morpholidate (Rf~0.8,
U.V.), guanosine 5'~ 1-fucopyranosyl)-diphosphate
W093/24505 2 1 1 8 ~ ~ 2 PCT/US93/04909
-- 155 --
(Rf~0.5, U.V. and charring), followed by the tailing
spot of the starting fucose-1-phosphate ~Rf-0.44,
charring). Additional U.V. active minor spots ~ere
also present. After stirring for 4 days at room
temperature, the yellowish mixture was co-evaporated in
vacuo with toluene and the yellowish residue further
dried overnight at the vacuum pump leaving a thick
residue (2.43g). Water (19 mL) was then added into the
flask to give a yellow cIoudy solution which was added
on top of a column of AG 50W-X12 (from Biorad) resi~
(100-209 mesh, 25 x 1.5 cm, Na+ form). The product
eluted with water after the void volume. The fractions
which were active, both by U.V. and charring after
spotting on a t.l.c. plate, were recovered and the
solution freeze-dried overnight in vacuo providing a
crude material (1.96g).
This residue was dissolved in water (10 m~
overall) and slowly absor~ed onto a column of
hydrophobic C18 silica gel (Waters, 2.5 x 30 cm) which
had been conditioned by washing with water, methanol
and water (250 mL each). Water was then run through
the column (0.4 mL/min) and the eluate collected in
fractions (0.8 mL) which were checked by t.l.c. (3:5:2
25% aq. ammonium hydroxide, isopropanol and water).
~-L-fucopyranosyl-l-phosphate, (Rf~0.54, charring) was
eluted in fractions 29 to 45. A product showing a
strongly U.V. active spot (Rf-0.51) eluted mainly in
fractions 46 to 65. Other minor U.V. active spots of
higher or lower Rf were observed. Fractions 59 to 86,
which contained guanosine 5'~ 1-fucopyranosyl)-
diphosphate (Rf-0.62), also showed a narrow U.V. active
spot (Rf~0.57). Fractions 59 to 86 were pooled and
freeze-dried overnight providing 0.353g of material
enriched in guanosine 5'-(~-l-fucopyranosyl)-
diphosphate. 1H-n.m.r. indicated that this material
211852~ - ~
W093/24505 PCT/US93/0490~-
-- 156 --
was contaminated by a small amount of impurities giving
signals at ~ = 4.12 and ~ = 5.05.
::~
Fractions 29 to 4S and 47 to 57 were
separately pooled and freeze-dried providing recovered --~
~-L-fuco-pyranosyl-l-phosphate (0.264g and 0.223g, --
respectively, in which the second fraction contains
some impurities). Occasionally, pooling of appropriate
fractions provided some amount of guanosine 5'~
fucopyranosyl)-diphosphate in good purity (1H-n.m~r.).
Generally, all the material enriched in guanosine 5'-
(~-1-fucopyranosyl)-diphosphate was dissolved in a
minimum amount of water and run on the same column
which had been regenerated by washing with large
amounts of methanol followed by water. The fractions
containing the purified guanosine 5'-(~ fuco-
pyranosyl)-diphosphate (t.l.c.) were pooled and freezed
dried in vacuo leaving a white fluffy material (187 mg,
- 16%). 1H-n.m.r. was identical to the previously
reported data67.
PROC~URE #2
~-L-fucopyranosyl-l-phosphate and guanosine
5'-monophosphomorpholidate (4-morpholine-N,N'-di- -
cyclohexyl-carboxamidine salt -- "GMP-morpholidate")
were reacted in dry pyridine as indicated in the
original procedure of Nunez, et al80. Accordingly, the
~-L-fucopyranosyl-1-phosphate ~triethylammonium salt,
0.528g, about 1.18 mmol) was dissolved in dry pyridine
(20 mL) and the solvent removed in vacuo. The process
was repea~ed three times with care to allow only dry
air to enter the f~as~. GMP-morpholidate (1.2g, 1.65
mmol) and pyridine (20 mL) were added into the reaction
flask, the solvent evaporated in vacuo and the process
repeated three times as above. Pyridine (20 mL) was
W0~3/24505 2 ~ 1~ 3 2 2 PCT/US93/04909
-- 157 -
added to the final residue and the heterogeneous
mixture was stirred for 3 to 4 days at room temperature
under nitrogenO An insoluble mass was formed ~hich had
to be occasionally broken down by sonication.
The reaction was followed by t.l.c. and
worked up as indicated in the first procedure to
provide the GDP-fucose (120 mg, 16%).
ii. Enz~matic Conditions
~Gal(1~3/4)~GlcNAc(1~3/4) fucosyltransferase was
purified from human milk according to the methodology
using affinity chromatography on GDP-hexanolamine
Sepharose described by Palcic et al. 25 The enzymatic
reactions were carried out at 37C in a plastic tube
using a sodium cacodylate buffer (lO0 mM, pH 5.5),
MnC12 (10 mM~, ATP (1.6 mM) NaN3 (1.6 mM). The final
reaction mixture was diluted with H20 (S mL) and
applied onto C18 Sep-Pak cartridges as reported25.
After washing with H20 (30 mL) the products were eluted
with CH30H and ~he solvents evaporated. The residue
was dissolved in a 65:35:5 mixture of CHCl3, CH30H, and
H20 and applied on a small column of Iatrobeads (0.200
to 0.500 g). After washing with the same solvent
mixture, the products were eluted with a 65:35:8 and/or
60:40:10 mixtures of the same solvents. The
appropriate fractions (t.l.c.) were pooled, the
solvents evaporated in vacuo, the residue run through a
small column of AG 50W X 8 (Na~ form) (BioRad) in H20
- and the products recovered after freeze drying in
vacuo. 1H-n.m.r. data of the tetrasaccharides are
reported below:
21~2~ .~
W093/245~5 PCT/U~93/04909
-- 158 --
-'.~' .''
iii. FucosYlation Reactions ~.
A. Preparation of 8-Methoxycarbonyloctyl(S- .~
acetamido-3,5-di-deoxy-D glycero-~-D-galacto- - -~:
2-nonulopyranosylonic acid)-(2-3)-O-~-D- ~:
galactopyranosyl-(1-4)-O-[~-L-fucopyranosyl-
(1-3)-0~-(2-azido-2-deoxy-B-D-
glucopyranoside) (tetrasaccharide 112b) ~
1 0 ' ' '
Trisaccharide lllb 17.7 mg), GDP-fucose (18
mg), the.fucosyltransferase (20 mU) and calf intestine
alkaline phosphatase (10 U) were incubated for 72 hours ~.
in the buffer (2 mL). Isolation and purificafion
provided 12b (2~84 mg).
B. Preparation of 8-Methoxycarbonyloctyl(5-
acetamido-3,S-di-deoxy-D-glycero-~-D-galacto- ~:
2-nonulopyranosylonic acid)-(2-3)-O-~-D- .:
galacto-pyranosyl-(1-4)-O-[~-L-fucopyranosyl-
(1-3)-03-(Z-amino-2-deoxy-B-D-
glucopyranoside) (tetrasaccharide 112~)
Trisaccharide lllc (8.4 mg), GDP-fucose (18
mg), the fucosyltransferase (20 mU) and calf intestine
alkaline phosphatase (10 U) were incubated for 67 hours
in the buffer (2 mL). Isolation and purification
provided 112c (2.46 mg).
~ , .
C. Preparation of 8-Methoxycarbonyloctyl(S-
acetamido-3,5-di-deoxy-D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-(2-3)-O-B-D-
galacto-pyranosyl-(1-4)-O-[~ fucopyranosyl-
(1-3)-0]-(2-N-propionamido-2-deoxy-~-D-
glucopyranoside) (tetrasaccharide 112d)
Trisaccharide ll1d (8.3 mg), GDP-fucose (18
mg), the fucosyltransferase (18 mU) and.calf intestine
alkaline phosphatase (10 U) were incl~bated for 72 hours
in the buffer (2 mL). Isolation and purification .
provided 112d (6.17 mg). -~
wo g3/24s0s 2 1 1 8 ~ 2 ~ PCT/US93/04909
159 --
D. Preparation of 8-methoxycar~onyloctyl (5-acet
amido-3,5-di-deoxy-D-glycero-~-D-galacto-2-
nonulopyranosylonic acid)-(2~3)-O-~-D~-
galactopyranosyl~ 3)-O-[~-L-fucopyranosyl-
(1-4)-0]-(2-azido-2-deoxy-B-~
glucopyranoside) (Tetrasaccharide 108b)
The trisaccharide 107b (8.7 mg), GDP-fucose
(18 mg), the fucosyltransferase (18 mU) and calf
intestine alkaline phosphatase (10 U) were incubated
for 68 hours in the buffer (2 mL). Isolation and
purification provided tetrasaccharide ~08b (4.42 mg).
E. Preparation of 8-Methoxycarbonyloctyl (5-
acetamido-3,5-di-deoxy-D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-(2-3)-O-B-D-
galacto-pyranosyl-(1-3)-O-[~-L-fucopyranosyl-
(1~4)-0]-(2-amino-2-deoxy-~-D-
glucopyranoside) (tetrasaccharide 108c)
The trisaccharide 107c (9.5 mg), GDP-fucose
(18 mg), the fucosyltransferase (18 mU) and calf
intestine alkaline phosphatase (10 U) were incubated
for 60 hours in the buffer (2.8 mL). Isolation and
purifi- cation provided for tetrasaccharide 108c (4.94
mg).
F. Preparation of 8-methoxycar~onyloctyl(5-
acetamido-3,5-di-deoxy D-glycero-~-D-galacto-
2-nonulopyranosylonic acid)-(2-3)-O-~-D-
galacto-pyranosyl~ )-O-[~-L-fucopyranosyl-
(1-4)-0]-(2-N-propionamido-2-deoxy-B-D-
glucopyranoside) (tetrasaccharide 108d)
Trisaccharide 107d (8.4 mg), GDP-fucose (18
m~), the fucosyltransferase (19.4 mU) and calf
intestine alkaline phosphatase (10 U) were incubated
for 72 hours in the buffer (2 mL). Isolation and
purification provided for tetrasaccharide 108d (5.gl
mg).
2 1 1 ~ ~ 2 ~ PCr/us93/0490c ~ ~ ~
---- 160 ----
Table A provides 1H-n.m. r . data for compounds
lllb, lllc, llld, 112b, ~112c, and 112d; whereas Table B
provides 1H-n.m.r. ~da~a for compounds 107b, 107c, 107d,
108b, 108c, and 108d. .
WO 93/24505 2 1 1 ~ PC~/USg3/0490g
~ C ~ ~ C C, ~ ;
_ ,~ 8 ~ o ~ ,o .~
~, ., - ~o _'' C
~ I ~
~ o~ ~ô "~ ~
~ ~ o o Q ~ O ~ `
~a O ,~ O O v~
IU Io ¦-- e~ ~r~ i O r"0
~olllôl 6 6
X _ v~ o~o
_ ol~ ~ ~Y C
o _ ~
~ _ ~ ~ Z ~~ t
s ~ J
o ~ ¦o ~L ~ 2 a
21~852~ : ~
WO 93/24505 PCI/USg3/04909. -
~~ 162 ~~
¦ ~: ~ c r~ c n 3 ~ n o æ ~ ~ ~O O ~
l ~ ~ ~ c ~, ~O 1` n ~ n
o ~oO 0~ x~
z n _ c ~ c7~ O O c `~ ~:~
¦ ~ ¦~ n G ô ô r C
~ ~ ~ 0~ æ " ~
l I n 0 0 ~ ~ C
. Z 0' ~ o~o ~
Y I l~o~ F~
r . o r~ " _
~ I I F. - 1~ - o o ~ 9~ o
o 1~ ¦-~ o o _ ~ az ~ o ¦ -~
ol ~ u
_~ ~ O U ~ Z U
- W093/24505 2 1 1 ~ S 2 2 PCT/~S93/04909
-- 163 --
Example 29 -- ~ynthe~is of Acceptor 114a
A. Preparation of 8-Methoxycarhonyloctyl ~-D-
galactopyranosyl~ 3)-0-2-acetamido-6-bromo-
2,6-di-deoxy-~-D-glucopyranoside (compound
ll~a)
A mixture of the compound 1135 (0.061 g,
0.075 mmol), barium carbonate (0.020 g, 0.1 mmol) and
N-bromosuccinimide ~O.016 g, 0.09 mmol) in carbon
tetrachloride (2 mL) was refluxed for 2 hours as
indicated82. After the appropriate work up, the
recovered crude material was chromatographed on silica
gel (6 g) using a 1:1 mixture of hexanes and ethyl
acetate as eluant providing the pure bromo derivative
(36 mg, ~4%).
Some of this material (0.019 g, 0.021 mmol)
in dry methanol was de-O-acetylated using a catalytic
amount of sodium methoxide for 3 hours at 22C. After
neutralization with Dowex SOW X 8 (H~ farm) and
filtration, the solvents were evaporated in vacuo and
the residue chromatographed on Iatrobeads (2 g) using a
10:1 mixture of chloroform and methanol as eluant
providing a pl~re disaccharide (0.012 g, 99%), which was
poorly soluble..in water. This material was saponified
using a 0.25 N solution of sodium hydroxyde for 1 hour
at 22C. After neutralization with Dowex 50W X 8 (H~
form) and filtration, the solution was freeze dried in
vacuo providing the disaccharide 114a; ~]20D -11.4
(c 0-12, H2O).
As noted above, the oligosaccharide
glycosides related to blood group determinants can be
administered to a mammalian patient as part of a
liposome. The following example sets forth one method
for preparing such liposomes.
21 i ~S2'~
W093/24505 PCT/US93/04909~-
-- 164 --
Example 30 -- SYNTHESIS OF AGGREGATES CONTAINING SIALYL
LEWISX ANALOGUES
Aggregates such as liposomes and micelles can
be prepared so as to incorporate oligosaccharide
glycosides related to blood group determinants having a
type I or a type II core structure. Specifically,
incorporation of such an oligosaccharide glycoside into
such aggregates requires that the aglycon moiety be
sufficiently hydrophobic to be incorporated into such
aggregates. It is contempla~ed that such hydrophobic
aglycons can include the -(CH2)XCOOCH3 (x ~ 2) which
has been extended by various moieties such as naphthyl,
substituted naphthyl, octyl, and the like which would
improve the ability to incorporate the saccharide into
the aggregate.
In such aggregat~s, the hydrophobic aglycon
group of the oligosaccharide glycoside becomes
partitioned in the lipid portion of the aggregate
whereas the oligosaccharide group is generally
partitioned in the aqueous phase.
Methods of preparing such aggregates are well
known in the art. See, for instance, U.S. Patent No.
4,522,803 which is incorporated herein by reference.
Examples 31-43 illustrate the synthesis of
modified sialyl LewisX and sialyl LewisA structures as
depicted in Figures 33 to 43.
General methods used in Examples 31-43 as
well as methods are as follows:
W093/24~05 2 1 1 8 ~ 2 2 PCT/US93/04909
-- 165 --
General Methods
In one or more of Examples 31-43, pre~coated
plates of silica gel (Merck, 60-F2s4) were used for
analytical t.l.c. and spots were detected by charring
after spraying with a 5% solution of sulfuric acid in
ethanol. Silica gel 60 (Merck, 40-63 ~m) was used for
column chromatography. Iatrobeads were from Iatron
(Order No. 6RS-8060). Millex-GV filters (0.22 ~m~ were
from Millipore. C18 Sep-Pak cartridges and bulk C18
silica gel were from Waters Associates.
Commercial reagents were used in chemical
reactions and solvents were purified and dried
a~cording to usual procedures. Unless otherwise noted,
the reaction mixtures were processed by dilution with
dichloromethane and washing with a dilute solution of
sodium bicarbonate followed by water. After drying
over magnesium sulfate, the solvents were removed by
evaporation under vacuum with a bath temperature of
35~C or lower when necessary.
lH-n.m.r. were recorded at 300 MHz (Bruker
AM-300) with either tetramethylsilane in CDCl3 or
acetone set at 2.225 in D2O as internal standards, at
ambient temperature, unless otherwise noted. The
chemical shifts and coupling constants (observed
splittings) were reported as if they were first order,
and only partial n.m.r. data are reported. 13C-n.m.r.
spectra were recorded at 75.5 MHz with
tetramethylsilane in CDC13 or dioxane set at 67.4 in
DzO as reference.
A. SYNT~ESIS OF DEXIV~TIVES OF Neu5Ac
Unless otherwise noted, derivatives of Neu5Ac
have been prepared following known procedures with
suitable substitution of starting materials where
211~ ~ 2 ~
W093/24505 PCT/US93/04909 --
-- 166 --
necessary. The following derivatives have been
- prepared ~y a convenient modification of procedures
reported in the literature: 9-N3-Neu5Ac 201b,53 Neu5Pr
(5-propionamido) 201f, 7-d-Neu5Ac 201d83 and the
C8-Neu5Ac 201i~.
Figure 33 illustrates a general synthetic
scheme used for the synthesis of derivatives of Neu5Ac.
Compounds referred to by underlined Arabic numerals in
Examples 31-34 below are depicted Table I and in Figure
33.
Example 31 -- ~ynthesi3 of 5-acetamido-9-azido-3,5,9-
trideoxy-D-gly~ero-D-galacto-2-nonulo-
pyrano~ylo~ic acid ~9-N3-Neu5Ac) 201b
Glycosyl chloride 238 (2.83 g, 5.57 mmol) in ~
dry dichloromethane (13 mL) was added to the mixture of `
benzyl alcohol (5.0 mL, 48.2 mmol), molecular sieves 4A
(18.5 g, crushed), dry silver ca~bonate (4.2 g, 15.2
mmol) in dichloromethane (8 mL). The mixture was
stirred in the dark for 4 days, diluted with
dichloromethane (50 mLj and filtred through Celite.
After usual work up, the re5idue was chromato~raphed or.
silica gel using 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 giving (1.96 g, 60%)
of pure material and 0.33 g (10~) of material
containing a small amount of impurities. lH-n.m.r.:
5.436 (ddd, lH, J7 8 8.5, J8 9~ 2.5, J8 9 5.5Hz, H-8),
5.317 (dd, lH, J6 7 1.8Hz, H-7), 5.110 (d, lH, J5 NH 9-5
HZ, NH) ~ 4.849 (ddd, lH~ J3ax,4 12-0~ J3~,4 ~ 4~5
9.5Hz, H-4), 4.788 and 4.397 (AB, 2H, Jg~ 12.0Hz,
benzylics), 3.642 (s, CO2CH3), 2.629 (dd, lH, J3,~ 3ax
i2.5Hz, H-3eq), 2.140, 2.113, 2.017, 1.997, 1. 8S7, (5s,
15H, 4 OAc, 1 NAc), 1.986 (dd, lH, H-3ax).
- W093/24~5 2 1 1 ~ 5 2 ~ PCT/US93/04909
~ 167 --
The above material (1.5 g, 2.58 mmol) was
de-O-acetylated in dry methanol (20 mL) cuntaining a
ca~alytic amount of sodium methoxide for 5 hours at
22C. After de-ionization with Dowex 50 x 8 (H~ form),
the solvent was evaporated leaving the product ~39 (l.O
g, 94%) which was used in the next step; 1H-n.m.r.
(CDCl3): 4.815 and 4.619 (AB, 2H, Jg~ 11.5Hz,
benzylics), 3.802 (s, CO2C_3)~ 3.582 (dd, lH, J5 6 9-0
J6 7 0.5Hz, H-6), 2.752 (dd, lH, J3~,3ax 12 . 5 ~ J3~,4
4.5Hz, H-3eq), 2.039 (s, 3H, N Ac), 1.861 (dd, lH,
J3ax,4 11- OHz, H-3ax).
A solution of para-toluenesulfonyl chloride
(0.125 g, 0.65 mmoi~ in pyridine (0.1 mL) was syringed
into a solution 239 (0.248 g, 0.60 mmol), 4-dimethyl-
aminopyridine (0.01 g) in pyridine (l.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 quic~ly chromatographed on
silica gel using acetonitrile as eluant giving the
tosylate (0.21 g, 62%) still containing some
impurities. Sodium azide (0.19 g, 2.92 mmol) was added
to a solution of this material (0.21 g, 0.37 mmol) in
dimethylformamide (O.S 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
giving the product 240 (0.136 g, 85%); i.r.~ cm'l 2110
(N3); 1H-n.m.r.: 5.775 (d, lH, J5 NH 9.0Hz, NH), 4.&16
and 4.470 (A8, 2H, Jg~ 11.5Hz, benzylics), 3.738 (s,
CO2Ca3), 2.871 (dd, lH, J3~,4 4-8~ J3~,3ax 13 - 0HZ ~ H
3eq), 2.086 ~s, 3H, NAc), 1.964 (dd, lH, J3ax 4 ll.SHz,
H-3ax).
The above compound 240 (0.105 g, 0.24 mmol)
was left for 3 hours at 22C in 0.25 N sodium hydroxide
2118a2~
W093~24505 PCT/US93/04909~-
-- 168 --
(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 Iatrobeads using a 65:35:5 mixture
of chloroform, methanol and water as eluant. The
appropriate fractions gave the product (0.087 g, 86%).
This compound (0.100 g, 0.235 mmol) was heated at 80C
for 6 hours in 00025 N hydrochloric acid (3 mL). The
solution was neutralized with sodium hydroxide and then
freeze dried. The product was chromatographed on -~
Iatrobeads (0.60 g) using a 65:35:5 mixture of
chloroform, methanol and water giving 201b (0.067 g,
85%); 1H-n.m.r.: 4.106 - 3.89S (m, 5H), 3.639 (dd, lH,
J89 3-0, J9 9, 13.0Hz, H-9), 3.528 (dd, lH, J8~ 9
6.0Hz, H-9''), 2.249 (dd, lH, J3~,4 4-5~ J3~,3ax
12.5Hz, H-3eq), 2.090 (s, 3H, NAc~, 1.852 (dd, lH,
J3ax,4 ll-OHz, H-3ax).
~xample 32 -- ~ynthesis of 5-propiona~ido-3,5-didaoxy-
D-glycero-D-galacto-2-nonulopyranosylonic
acid (Neu5Pr) 201f
A solution of 239 (0.075 g, 0.18 mmol) in 2 N
sodium hydroxide (1 mL) was left for 0.5 hours at 22C
followed by 7 hours at 95C. The pH was then adjusted
to 7.5 by addition of IR-C50 resin (H~ form). The
filtrate obt~ined after filtration of thP 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 (O.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
- W093/245U5 2 1 1 8 5 2 ~ PCT/US93/04909
-- 169 --
50 x 8 (H+ form, 6 g). The recovered fractions were
evaporated in vacuo and the residue chromatographed on
Iatrobeads (5 g) using a 3:1 mixture of chloro~orm and
methanol as eluant giving 241 (0.0646 g, 86.5%); 1H-
n.m.r.: 4.800, 4.578 (AB, 2H, Jg~ ll.~Hz, benzylics),
3-580 (dd~ lH~ J5 6 9-0r 367 l.OHz, H-6) 2.776 (dd, lH,
J3~4 4 5r J3~ 3ax 12.5Hz, H-3eq), 2.316 (q, 2H, J 7.5
Hz, Ca?C0), 1.762 (dd, lH, J3ax 4 12.0Hz), 1.129 (t, 3H,
CH3 ) -
A solution of the above benzyl glycoside
(0.115 g, 0.278 mmol) in water (5 mL) was hydrogenated
in the presence of 5% 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 compcund
201f (0.074 g, 82.5%); 1H-n.m.r.: 3.72 - 4.10 (m, H-4,
-5,-7,-8,-9), 3.614 (dd, lH, J8 9a 6.5~ J9~ 9b 11.75Hz,
H-9a), 3.530 (dd, lH, J5,6 9-0 J6,7 l-OHZ~ H-6) ~
2.250 - 2.400 [m, 2H incl. CH2Co (q, 2.315, J 7.5Hz)
and H-3eq (dd, J3eq 3ax 11-5 Hz, J3~4 4-5Hz)], 1-880
(t~ lH, J3ax,3eq 11.5Hz, H-3ax), 1.130 (t, 3H, CH3).
Example 33 -- Synthe~is of 5-ac~tamido-3,5-dideoxy-D-
galacto-2-octuloso~ic acid (C8-Neu5Ac) li
The synthesis of 201i from 239 essentially
follows the published procedure of Hasegawa et al.
but using a different starting material than the
reported one. In particular, a suspension of 239 (0.52
g, 0.125 mmol) in 2,2-dimethoxypropane (3 mL) was
stirred for 1.5 hours at 22 C in the presence of
paratoluenesulfonic acid (0.5 mg). After
neutralization with some triethylamine, the mixture was
evaporated and the residue chromatographed on silica -~
gel using a 16:1 mixture of chloroform and methanol
giving 242 (0.049 g, 88%'.
. . .
.
2~
W093/24505 P~T/US93/04909;~^
-- 170 --
242 (0.054 g, 0.185 mmol) was acetylated in a ~-
2:1 mixture of acetic anhydride (l mL) and pyridine
kept at 50C for 5 hours. After the usual work up, the
residue was chromatographed on silica gel using ethyl
acetate as eluant giving the acetylated product (0.091
g, 92%); 1H-n.m.r.: 5.420;(dd, lH, J6 7 1.5, J7 8 3.5Hz,
H-7), 5.196 (d, lH, J5 bH 9.0Hz, NH), 5.009 (ddd, lH,
J4 3aX 13-0~ J43~ 5-0, J4 5 10.0Hz, H-4), 4.797 and
4.498 (AB, 2H, Jg~ 11.5Hz, benzylics), 3.776 (s, 3H,
C02CH3), 2-724 (dd, lH, J3ax,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 g, 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 g, 0.275 mmol). The mixture was
filtered through 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
g, 0.95 mmol). The mixture was then stirred at 0C
with some acetic acid (0.2 mL), after which the
solvents were evaporated leaving a residue which was
dried in vacuo for 15 minutes and then acetylated in a
5:1 mixture of pyridine and acetic anhydride (6 mL) for
20 hours at 22C. The residue recovered after the
usual work up was chromatographed on silica gel using
ethyl acetate as eluant to give a product which still
contained some non-separa~le impurities. The dry
material (0.074 g, still containing some impurities)
was dissolved in dry methanol (5 mL) and stirred at
room temperature for 3 hours in the presence of sodium
(3 mg). After de-ionization with Dowex 50 x 8 (H~
~orm) and filtration, the solvent was evaporated in
vacuo and the residue chromatographed on silica gel
~ W 093f24505 2 1 1 8 ~ ~ ~ P ~ /US93/04909
-- 171
using a 15:1 mixture of chloroform and methanol to give
a pure product 244 (0.047 g, 78%); ~H-n'~ m.r.: (CD30D):
4.724 and 4.416 (AB, 2H, Jg~ 11.5Hz, benzylics), 3.671
(s, 3H, CO2CH3), 3.456 (dd, lH, J5,6 9 5~ J6,7 l-OHz~
H-6), 2.642 (dd, lH, J3eq,4 4 5~ J3eq,3ax 12.5Hz, H 3eq),
1 938 (s, 3H, NAc), 1.699 (t, lH, J3ax,4 12.5Hz, H 3ax).
The above material (0.022 g, 0.057 mmol) was
stirred in 0.25 N sodium hydroxide (2 m~) 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 g, 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 leaving the desired product 201i (13.3 mg, 94%);
H-n.m.r.: 3.462-4.093 (m,6H), 2.287 (dd, lH, J3~4
4 - 5 ~ J3~ 3ax 12.5Hz, H-3eq), 2.052 (s, 3H, NAc), 1.853
(t, lH, J3~ 4 12.5 HZ, H-3ax).
Example 34 -- 8ynthesis of 5-~c~ta~ido-3j5,7-trideoxy-
~-D-galacto-2-nonulopyrano~ylonic acid
~7-d-NeuSAc) 201d
The synthesis of 20~d essentially follows the
published procedure of Zbiral et al.37 but using a
different starting material. In particular, imidazole
(0.13 g, 1.93 mmol) and tert butyldimethylsilyl
chloride (O.135 g, 0.89 mmol) were added to a solution
of 242 (0.11 g, 0.19 mmol) in dimethylformamida (2 mL).
After 4 hours at room temperature, the solvent was
removed in vacuo, the residue dissolved in chloroform
and worked up as usual. Chromatography of the product
on silica gel using a 1:1 mixture of ethyl acetate and
hexane provided the monosilylated derivative (0.101 g,
92%): ~a]D = -2.66 (c. 0.6, chloroform); tH-n.m.r.:
5.195 (d, lH, J5 NH 7Hz, NH), 4.853 and 4.603 (AB, 2H,
211~5Z~
W093/2450~ PCT/US93/04909-~-
-- 172 ~
Jg~ 11.5Hz, benzylics), 3.736 (s, C~2CH3), 2.692 (dd,
lH, J3~ 4 4 5~ J3~3ax 13.0Hz, H-3eq), 2.022 (s, 3H,
NAc), 1.884 (dd, lH, J3ax4 ll.OHz, H-3ax), 1.405, 1.375
(2s, 6H, methyls), 0.868 ~s, 9H, t-butyl), 0.093 and
0.084 (2s, 6H, methyls).~
Sec-butyl lithium (1.3 M in cyclohexane, 0.65
mL, 0.85 mmol) followed by carbon disulfide (1.25 m1,
20.8 mmol) were added dropwise to a solution of the
above compound (0.437 g, 0.77 mmol) in dry tetra-
ydrofuran (20 mL) at -30C. After stirring 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 chromatographed on silica
gel u~ing a 4:1 mixture of hexanes and ethyl acetate as
eluant providing the xanthate (0.327 g, 65%): [~]D 939
(c. 0.655, chloroform); 1H-n.m.r.: 6.388 (dd, lH J67
1.0, J78 2.5Hz, H-7), 5.610 (d, lH, J5 ~H 7.0Hz, NH),
4~778, 4.466 (AB, 2H, Jg~ 11.5Hz, benzylics), 3.778
(5~ CO2CH3), 2.662 (dd, lH, J3~,4 4-5, J3~,3ax 12-5Hz,
H-3eq), 2.584 (5~ 3H, OCH3) ~ 1-883 (S~ 3H, NAc), 1.693
(dd, lH, J3ax 4 11.5Hz, H-3ax), 1.315 (s, 6H, methyls)
0.825 (9H, t-butyl), 0.025, 0.092 (2s, 6H, methyls!.
Azobisisobutyronitrile (0.004 g) and tri-n-
butyltin hydride (0.5 mL, 1.86 mmol) were added to a
solution of the above xanthate (0.32 g, 0.48 mmol) in
dry toluene (3 mL). After heating at 100C for 7
hours, the solvents were co-evapoxated with dry
toluene, and the residue chromato-graphed on silica gel
using a 3:2 and then 1:1 mixtures of hexane and ethyl
acetate as eluant to give the 7-deoxy product (0.260 g,
70%); 1H-n.m.r.: 5.334 (d, lH, J5 NH 7.0Hz, NH), 4.740,
4.45S (AB, 2H, Jg~ 11.6Hz, benzylics), 3.690 (s,
CO2CH3), 2.628 (dd, lH, J3~,4 4-2~ J3~,3ax 12.9Hz,
H-3eq), 1.9;4 (s, 3H, NAc), 1.805 (dd, lH, J3ax4
lO.9Hz, H-3ax), 1.718 and 1.597 (m, 2H, H-7 and H-7'),
W093/24~05 2 ~ PCT/US93/04909
-- 173 --
1.325 (s, 6H, methyls), 0.804 (9H, t-butyl), 0.010,
0.009 (2s, 6H, methyls). The above compound (0.260 ~,
0.47 mmol) was heated at 7SC in 70% acetic aci~d for
7.5 hours. After co-evaporation with toluene, the
residue was chromatographed on silica gel using a 10:1
mixture of chloroform and methanol giving 243 (0.157 g,
84%); lH-n.m.r.: 4.860 and 4.655 (AB, 2H, Jg~ 11.5Hz,
benzylics), 3.834 (s, C02CH3), 2.806 (dd, lH, J3~ 4
4.5~ J3~ 3ax 12.5Hz, H-3eq), 2.Q69 (s, 3H, NAc), 1.881
(dd, lH, J3ax 4 12.5Hz, H-3ax), 1.698 (m, 2H, H-7 and
H-7').
Compound 243 ~0.157 g, 0.396 mmol) was kept
in 0.25 N sodium hydroxide t6 mL) at room temperature
for 5 hours. After neutralization with Dowex 50W x 8
(H~ form) and filtration, the product (0.149 y, 97%)
was reco~ered after lyophilization of the solution.
This product (0.146 g, 0.38 mmol) was hydrogenated in
water (5 mL) for 5 hours at room temperature in the
presence of 5% palladium on charcoal (0.010 g). The `
mixture was filtered through Celite and through a
Millex-GV (0.22 ~m) filter. The filtrate was freeze
dried to provide 201d (0.105 g, 94%); ~H-n.m.r.: as
reported by ChristianS6.
Table 1 below summarizes the derivatives of Neu5Ac
prepared.
~V~ 93~2450~ PCI/US93/04909 -
---- 174 ----
Tab1e I~
.
R3 R~ :
RS __~R~ 1H
R~
R7
Sblic Acid Danv~t~v~s
~ _ . _ , ~
ComDound ¦ R1 ¦ R2 ¦ R3 ¦ R4 ¦ R5 ¦ R6 ¦ R7 R8 R9
~ _ I
l2~ ¦ H ! H ¦ O~ NHAo ¦ H ¦ OH ¦ OH ¦_H ~ CH,OH
1~ ~ !
. . ¦ H _ CH,OH
2~ ~ . ¦~ H ¦ _
~d ~. ¦~ l NHCOCH.CH~ H OH ¦ ' _ _
~.~ I . .OH _ l l _ _ .
~o1 h I . NHCOCH~OH ~ ' l ¦
1~ ~ _ ~ I
W093/24505 ~l 1 8 j 2 ~ PCT/US93/~4~09
-- 175 --
Table 1 (con't)
Sialyl Moieties obtained by chemical modification
of sialylated oligosaccharides:
o~ . I
~~
ACE~ 3_~
~ \ ~0;~
Rlo~
~O lj R~D = C~NE~
= C~c
O~I I
~c= ~ '
~
~- ~O~C~
0~ 1
~~~~\"" ~ .
AcBN ~ o ~ O
-' Co~ ,
PCl'/US93/0490~--
---- 176 ----
B. S~rNTHESI~; OF CMP DE~IVATIVES OF NeuSP.c
AND ANALOGUES THEREOF
Example 35 - 8ynthesis of the CMP-deri.vative~ o~ NeuSAc
CMP-sialic acid synthase was extracted from
calf brain and partially pu~ ed at 4C by a slight
modification of the originàl- procedure of Higa et al. 52
Routinely, ~200 g of brain tissue were homogenized in a
Cuisinart blender (three 30 second bursts with 1 minute
intervals) with 400 mL of 25 mM Tris/HCl, pH 7.5, 10 mM
magnesium chloride, 10 mM sodium chloride, 2.5 mM
dithioerythritol, 0.5 mM phenylmethylsulfonyl fluoride.
The homogenate was stirred for 1 hour and then
centrifuged at 23,000 x g for 15 minutes. The
supernatant was decanted and the pellets were extracted
once again with 200 mL of the same buffer as above.
The supernatants were combined and centrifuged at
28,000 x g for 15 minutes. The supernatant was
filtered through glass wool to give the crude extract
(515 mL, 4.7 mg protein/mL, ~90 U of enzyme).
After adjusting salt concentration to 0.4 M
with solid potassium chloride, the crude extract was
stirred~and solid ammonium sulfate was added to 35%
- saturation (208 g/L) over a period of 15 minutes. The
solution was stirred for~an additional 15 minutes, kept
on ice for 1 hour and centrifuged at 28,000 x g for 30
minutes. ~The precipitate was discarded and the
supernatant was stirred and adjusted to 60% saturation
by the addition of solid ammonium sulfate (163 g/L)
over 15 minutes. After an additional 15 minutes of
stirring, the suspension was left on ice overnight and
then centrifuged as above. The resultant pellets were
washed with 1~0 mL of 60% ammonium sulfate solutian to
remové 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
- W093/24505 2 L I ~ ~ 2 2 PCT/US93/04909
-- 177 --
et al. 85, with one unit of enzymatic activity defined
as one ~mol of product formed per minute at 37OC.
The enzyme present in the pellet could be
stored for several weeks in the cold room. Before
using the enzyme for synthesis, the pellets were
suspended in a minimal volume of 50 mM Tris/HCl, pH
9.O, 35 mM magnesium ~hloride, 3 mM 2-mercaptoethanol ~-
(acti~ation buffer) and dialyzed overnight against 100
volumes of the same buffer. The dialyzed enzyme was
centrifuged at 9,000 x g for 10 min. The supernatant -
containing more than 90% of the enzyme activity was -
used directly for the synthesis.
The CMP-derivatives of sialic acid analogues -;
were synthesized as noted above and purified by a
modification of the reported procedures of Higa et --
alO 52 and Gross et al.~ For example, 7-d-Neu5Ac 201d
(Table 1, 20 mg, 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
analogues was estimated by the usual thiobarbituric
acid assay for sialic acid after reduction with sodium
borohydride as per Kean et al. 85 The product was
extracted with cold acetone as per Gross et al.~
After evaporation of the acetone in vacuo (at -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 4~C with a flow rate of 60 -`
mL/hour. Fractions (1 mL) were assayed for cytidine by
absorbance at 273 nm, and the fractions corresponding
to the first peak were pooled, concentrated in vacuo
and the residue was freeze-dried leaving the CMP-7-d-
Neu5Ac (202d, 30 mg, -94%). This material showed a
very small amount of impurities by ~H-n.m.r. (Table 2)
21l~2~ ~
W093/24505 PCT/US93/04909
-- 178 --
and was used directly for the reaction with
sialyltrans-ferases. In some cases (202e, 202~, 202h),
H-n.m.r. spectra showed that the CMP-derivatives
contained some of the unreacted sialic acid.
Table 2A below illustrates the CMP-
derivatives of analogues of Neu5Ac prepared from the
analogues of Neu5Ac set forth in Table 1 above, and
partial 1H-n.m.r. data concerning these compounds are
set forth in Table 2B.
.,~
. WO 9:~/24505 2 1 1 ~ 2 PCr/US93/04909
1 7 9 ----
__.--~ ~=__
_ ~ r
O . OT1~ IO~ . _ _' _
~n o, O. O, O. o, I
_ o, ~ ~n ~ r~ _
~ x ~ ~ r~ _ ~ r~ c~ I
~ ~ ~9 I~o ~ui o~ ~o o ~In ~
û~ O U~ ~ O U~ ~ I
~q ~i ~ ~ ~ ~ ~ I
o ~7 e ~ ~ u~ Yi x * al - ~
- 1~ ~ = : , r~7 rl ~7 ~7
o O O O _ I S O
Ci: T I T I r O _ Z
1~ ~ m ~ I ~ ~ I ~D r~ c
O ~o O O. O ~ 0ol a) a~ . -~
cc ~ r~ c~ ~ r~ I~I~ ~ I~ o
.~ .. _ _ _ . ~ :
ln Ir 1~~1- 1- I
I 0 ~ ~ o ¦ 'D ¦ o
E ~ ~ I ~ .
T ~, ~ ¦ 0 ¦ ~ ¦ ~n ¦ O¦ 0 ~ t~ ¦ 01 I ~ .
~c :r u~ I Iti I 10 1 U7 1 U'lI U~ I It~ I I .
¦ I 1 - I I I 1- I --
o,~ I I I I I I I lo"-,
~, I o I z I ,~ ~o ~~ -
~ . ~ ~ l ¦ ¦¦O 11 ~ _
L~ I ~ yl I ~ `~
~ O ~ C _ N o
l~o~ L~
211~2;~ ~
WO 93/24~05 PCI/US93/0490~ :
---- 1 8 0 -- -
TABT .E 2 B
R~ i
R~ . /~;;' . R~ - NH2
~/ ~O~H \ ~
s~R~I --Pl ~N
R7
HO OH
,,
CMP-Sialic Acid Doriv~ivo~
_ - 1'' 1 1 .
~ _ _ I
¦ H ¦ H ¦ O~ ¦ NH~c l ~ H 1 OH ¦ OH j~L,~
CH~
_ I_ ¦ _ I . ¦ H l¦~ I C:H?OH ¦ :102d
. I . ¦ . ¦ OH ¦ ~ l v ~o20
r i,~ ~ i ~
z
11~ l I NHCOCH;OH ¦
~-- - WO 93/24505 2 1 1 ~ 5 2 2 PCI`/US93/04~09
---- 181 ---- -
C. SYNTHESIS OF OLIGOSACC}IARIDE ~;LYCOSIDES
Examples 36-37 illustrate the synthesis of
oligosaccharide glycosides. The structure of Zo3b to
207a are illustrated in Figure 3~. Oligosaccharide
glycosides 204b, 205b, 205f, 206a, and 2~7a were
synthesized according to the procedures of ~emieux et
al.87, Lemieux et al.~, Paulsen et al.89, Sabesan et -
al. 90, and Lemieux et al. 91, respectively.
'.
Oligosaccharide glycosides 204d and 205d were
synthesized following the procedure reported for the
synthesis of oligosaccharide glycosides 204b and 205b
but by replacing the 8-methoxycarbonyloctyl by
methanol.
Oligosaccharide glycosides 205e and 205q were
synthesized according to the procedures of Paulsen et
al.89 and Alais et al.92 but replacing the methanol by
8-methoxycarbonyloctanol. In all cases, the
oligosaccharide glycosides were purified by
chromatography on Iatrobeads with the appropriate
solvent mixtures and the recovered materials
chromatographed on BioGel P2 or Sephadex LH20 and
eluted with water. The recovered materials were
lyophilized from water and the products further dried
in vacuo over phosphorus pentoxide.
Example 36 -- Synthe is of 9-Xydroxynony7 2-ac~tamido-
2-deoxy-t~-D-galactopyranosyl-(1-3)-0-]-~-D-
glucopyranoside 204a
Sodium acetate (0.200 g) and sodium
borohydride (0.060 g) were added to a solution of the
disaccharide 204b (0.100 g, 0.189 mmol) in a 10:1
mixture of water and methanol (20 mL) cooled at +4C.
After 24 hours, more sodium borohydride (0.020 g) was
added to the reaction mixture maintained at +4C.
After 48 hours at the same temperature, the pH was
211~ a 2 ~
WO 93/2450~ PCl/US93/04909 `-
----182 --
brought to 5-6 by addition of acetic acid. The
solution was then co-evaporated with an excess of
methanol. The residue was dissolved in water (10 mL)
and run through a column of Cl8 silica gel which was
S further washed with water. After elution with
methanol, the solvent was evaporated in vacuo. The
residue was dissolved in a 10:1 mixture of water and
methanol and the pH brought to 13-14 by addition of 1 N
sodium hydroxide. The mixture was left at room
temperature until t.l.c. (65:35:5 - chloroform,
methanol and water) indicated the disappearance of the
unreacted starting material 204b. The mixture was then
neutralized by addition of Dowex 50 x 8 (H~ form) and
the resin filtered off. The resulting solution was run
through a column of AG 1 x 8 (formate form~. The
eluate was freeze dried and the residue was run through
Sephadex LH 20 using a l:l mixture of water and
etnanol. Tlle appropriate fractions were pooled and
concentrated to give 204a (0.060 g, 65%); 1H-n.m.r.
(D20): 4.545 (d, lH, J1 2 8.0Hz, H-1), 4.430 (d, lH,
J1~ 2'' 7.5Hz, H-l'), 2.02S (s, 3H, NAc), 1.543 (m, 4H),
and 1.304 (m, lOH~: methy]enes; 13C-n.m.r. (DzO): 175.3
(Ac), 104.36 (C-l'), 101.72 (C-1), 67.72, 61.85, 61.60
(three CH20H~.
Example 37 -- 9-}Iydroxynonyl 2-acetamido-2-deoxy-~ D-
galactopyranosyl-~1-4)-0-]-,~-D-gluco-
pyr~nosid~ 20Sa
Oligosaccharide glycoside 205a was prepared
from 205b as indicated above (60%); 1H-n.m.r. (D20):
4.520 (d, lH, J1 2 7.5Hz, H-l), 4.473 (d, lH, J1, 2~
7.6Hz, H-l'), 2.033 (s, 3H, NAc), 1.543 (m, 4H) and
1.302 (m,lOH):methylenes; 13C-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).
-W093/~4505 2 l 1 ~ S 2 2 P~T/US93/04909
- 183
Example 38 - æynthe~is of 5-~llyloxypentyl 2-acetamido-
2-deoxy-t~-D-galactopyranosyl-(1-3)-Q-3-~-D-
glucopyranoside 204c -
The synthetic schemes for this example and
Example 39 are set forth in Figure 35.
. Synthe~is of Al~yloxy-5-penta~ol 229
Allyl bromide (2.5 mL, 0.029 mol) was added -
dropwise to the mixture of 1,5-pentanediol (3 g, 0.029
mol) and sodium hydride (1.2 g, 80% dispersion in oil)
in dry dimethylformamide. Stirring was continued
overnight at room temperature. T.l.c. (2:1 - toluene
and ethyl acetate) still indicated the presence of some
unreacted pentanediol. The unre~cted sodium hydride
lS was destroyed ~y addition of methanol. The mixture was
concentrated to 50 mL by evaporation in vacuo. After
dilution with methylene chloride (150 mL), the solvents
were washed with water (three times), dried over
magnesium sulfate and evaporated in vacuo. The residue
was chromatographed on silica gel using a 2:1 mixture
of toluene and ethyl acetate as eluant. The
appropriate fractions gave compound 229 (0.931 g, 30%).
1H-n.m.r. (CDCl3): 5.83 (m, lH, -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, 0-CH~), 1.64 (m, 4H)
and 1.44 (m, 2H): methylenes); 13C-n.m.r.(CDCl3): 134.7
and 116.6 (ethylenics), 71.6, 70.1 (CH2-0-SH2), 62.1
(CH20H) 32.2, 29.2 and 22.2 (methylenes).
B. Sy~thesis of 5-Allyloxypentyl 2-deoxy-2-
phthalimido-~-D-glucopyra~oside 232
A solution of 3,4,6-tri-0-acetyl-2-deoxy-2-
phthalimido-D-glucopyranosyl bromide 230 (5.0 g, 10.0
mmol) in dichloromethane (5 mL) was added dropwise to a
mix~ure of the alcohol 229 (1.33 mL, 10 mmol), silver
trifluoromethanesulphonate (2.57 g, 10.0 mmol) and
collidine (1.23 mL, 9.0 mmol) in dichloromethane (10
mL) at -70C. After stirring for 3 hours at -700,
211'3S2`~
W O 93/2450~ PC~r/~S93/04909`~`
-- 184 --
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 syrupy residue was
chromatographed on silrca gel using a 5:1 mixture of
toluene and ethyl acetate providing compound 231 (4.0
g, 71%). 1H-n.m.r. (CDCl3): 5.80 (m, 2H, -CH= and
H-3), 5.36 (d, lH, J12 8.~Hz, H-l), 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): methylenes. A 0.2 M solution of
sodium methoxide in methanol (0.500 mL) was added
dropwise to a solution of compound 231 (4.00 g, 7.1
mmol) in dry methanol (30 mL) cooled at 0C. The
mixture was stirred at 0~ for 2 hours until t.l.c.
(10:1 - chloroform and methanol) indicated the
disappearance of the starting material. The reaction
mixture was de-ionized with Dowex 50 (Ht 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 compound 232 (2.36 g, 76%). 1H-n.m.r.
- (CDC13): 7.70 and 7.80 (m, 4H, aromatics), 5.82 (m, lH,
-C~=), 5.17 (m, 3H, =CH2 and H-l), 1.38 and 1.10 (m,
6H, methylenes); 13C-n.m.r. (CDCl3): 134.9 and 116.6
(ethylenics), 98.3 (C-l), 56.6 (C-2).
C. ~y~the is of S-Allyloxypentyl 4,6-0-
benzylidene-2-deoxy-2-phthalimido-~-D-
glucopyranoside 233
Paratoluenesulfonic acid monohydrate (0.025
g) was added to a solution of 232 (1.0 g, 2.3 mmol) and
a,~-di-methoxytoluene (0.690 mL, 4.6 mmol) in dry
dimethylformamide. After stirring for 2 hours at 40C,
t.l.c. (10:1 - chloroform and methanol) indicated the
completion of the reaction. After addition of a small
amount of triethylamine, most of the solvent was
W093/2450~ 2 ~ 1 ~ 5 2 2 PCT/US93/0~09 ~
-- 185 -- ~
.
evaporated in vacuo and the residue diluted with
dichloromethane and worked up as us~al. After
evaporation of the solvents, the residue was
chromatographed on silica gel using a 9:1 mixture of
toluene and ethyl acetate giving compound 233 (1.36 g, ~-
90.1%). [~]20D +24.1 (C 0.5 chloroform); 1H-n.m.r.
(CDCl3): 7.15-7.90 (m, 9H, aromatics), 5.83 (m, lH,
-CH=), 5.56 (s, lH, benzylidene), 5.10-5~37 ~m, 3H,
=CH2 and H-1 (5.25, d, J~z 8.5Hz)], 1.40 (m, 2H) and
1.17 (m, 4H): methylenes.
D. Synthe~i~ of 5-Allyloxypentyl 4,6-O-
be~zylide~e-2-deoxy-t2,3,4,6-tetra-O~acetyl-
~-D-galactopyra~o~yl-~1-3)-0-]-2-phthalimido-
~-D-glucopyra~o ide 235
A solution of trimethylsilyltrifluoromethane- ~-
sulfonate ~0.1 mL of a solution made from 0.050 mL of
the reagent in 1.0 mL of dichloromethane) was syringed
into a mixture of compound 233 (1.20 g, 2.29 mmol),
2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl acetimidate
234 (1.70 g, 3.50 mmol) and molecular sieves (0.500 g,
crushed in a 1:1 mixture of toluene and dichloromethane
(30 mL) cooled to -20C. The mixture was stirred at
-20C for 0.5 hours and slowly brought ~o 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 gel by using toluene and elution was
then continued with a 2:1 mixture of hexane and ethyl
acetate. The appropriate fractions gave the
disaccharide 235 (1.63 g, 74%). [~]20D +4.1 (c, 0.5,
CHCl3); 1H-n.m.r. (CDCl3): 7.40 -8.00 (m, 9H,
aromatics), 5.85 (m, lH, -CH=), 5.58 (s, lH,
benzylide~e), 5.07 - 5.25 (m, 4H, incl. =CH2, H-4' and
H-1), 5.00 (dd, lH, J1~2, 8.0, J2,3,10.0Hz, H-2'),
2.11, 1.90, 1.85, 1.58 (4s, 12H, 4 OAc), 1.37 and 1.12
'Zll~S22
W093/24505 PCT/US93/04909,-
-- 186 --
~m, 6H, methylenes); 13C-n.m.r. (CDCl3): 134.6 and
117.0 (ethylenics), 102.1, 101.2, 99.4 (benzylidene,
C-l and C-1').
E. 8y~thesi~ of 5-Allyloxypentyl 2-deoxy-
~,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl-
(l-3)-o-]-2-phthalimido-~-D-gl~copyranoside
236
A solution of the disaccharide 235 (1.63 g,
1.91 mmol) in 90~ aqueous acetic acid (10 mL) was
heated at 70C for 1 hour at which time t.l.c.
(100:5 - chloroform and methanol) indicated the
completion of the reaction. Co-evaporation with an
excess of toluene left a residue which was
chromatographed on silica gel using a 100:2 mixture of
chloroform and methanol as eluant giving compound 236
(1.12 g, 76%). [~20D +9.3 (C, 0.55 CHCl3), lH-n~m.r.
(CDCl3): 7.70-7.95 (m, 4H, aromatics), 5.82 (m, lH,
-CH=), 5.33 ~dd, lH, J3, 4, 3.5, Jt"5, 1.0Hz, H-4'),
5.10 - 5.27 (m, 3H, incl. -CHz and H-2'), 5.07 (d, lH,
J12 8.5Hz, H-1), 4.84 (dd, lH, J2~3~ 10.0Hz, H-3'),
2.10j 2.08, 1.90 (3s, 9H, 3 OAc~, 1.05 -1.47 (m, 9H,
incl. 1 OAc). 13C-n.m.r. (CDCl3): 100.3 and 97.5 C-l
and C-1'. Anal.calcd: C, 56.88; H, 6.17; N, 1.83.
Foun~: C, 55.59; H, 6.20; N, 1.84.
F. Sy~th~is o~ 5-Allyloxype~tyl 2-acetamido-2-
deoxy~ D-gal~ctopyra~osyl~ 3)-O~ D-
glucopyrano~ide 204c
Sodium borohydride (0.690 g, 18 mmol) was
added to the disaccharide 236 (0.700 g, 0.91 mmol) in a
5:1 mixture of isopropanol and water (20 mL). The
mixture was stirred for 24 hours at room temperature
after which t.l.c. (65:35:5, chloroform, methanol and
water) showed the disappearance of the starting
material. After addition of acetic acid (8.2 mL) the
mixture was heated for 3 hours at 100C. The mixture
was co-evaporated with an excess of toluene and the
W093/24~0~ 2 1 1 ~ ~ 2 ~ PCT/US93/04909 ~
~- 187 --
~: .
dried residue acetylated in a 3:2 ~ixture 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 chromatographed on silica gel using a 100:2 -~
mixture of chloroform and methanol giving the
peracetylated disaccharide (0.5Q0 g, 71%). 1H-n.m.r.
(CDC13): 5.90 (m, lH, -CH=), 5.77 (d, lH, J2 NH 7.5Hz,
NH), 5.37 (dd, lH, J3,4, 3.5, J4,5, 1.0Hz, H-4'), 5.15
- 5.23 (m, 2H, =CH2), 1.95 - 2.18 (7s~ ZlH, 6 OAc, 1
NAc), 1.58 (m, 4H) and 1.41 (m, 2H): methylenes.
A 0.5 N solution of sodium methoxide (0.300
mL) was syringed into a solution of the above compound ``
(0.500 g, 0.623 mmol) in dry methanol (20 mL). After
stirring overnight at room~temperature, the mixture was
de-ionized with Dowex 50 (H' form, dried) and
evaporated in va~uo. The residue was dissolved in
methanol and coated on Celite (3 g) by evaporation of ~`
the solvent. The Celite was then applied on top of a
column of Iatrobeads (30 g) and the product eluted with
a 65:25:1 mixture of chloroform, methanol and water
2S gi~ing the disaccharide 204c (0.266 g, 80%);
t~20D -0.164 (c.l, waterj; 1H-n.m.r. (D20): 5.95 ~m,
lH, -CH=), 5.30 (m, 2H, =CH2), 4.548 (d, lH, J1 2
7.7Hz, H-1), 4.426 (d, lH, J1, 2~ 7.7Hz, H-l'), 4.031
(dd, lH, J 1.0, 11.5Hz, allylics), 2.023 (s, 3H, NAc),
1.58 (m, 4H) and 1.38 (m, 2H): methylenes; 13C-n.m.r.
~2): 175.24 (carbonyl), 134.70 and 119.05
(ethylenics), 104.33 (C-1'), 101.68 (C-1~, 55~42 (C-2).
Example 39 -- Synthe is of 5-Allyloxypentyl 2-a~et-
amido-2- deoxy-~-D-glucopyrano~ide 237
The starting material 232 (0.300 g, 0.689
mmol) was deprotected as indicated previously for
211S~22
W093/2450~ PCT/US93/0490~-
-- 188 --
compound 236. The crude material recovered after
peracetylation was chromatographed on silica gel using
a 1:1 mixture of hexane and ethyl acetate whic~ gave
the peracetylated derivative (0.180 g, 5S%)j [~]20D
+11.5 ~c, 0.7, chloroform); ~H-n.m.r. (CDC13): 5.90 (m,
lH, -CH=), 5.64 (d, lH, J2 NH 8.5 Hz, NH), 4.68 (d, lH,
J12 7.5Hz, H-1), 1.95, 2.03 (two), 2.05 (3s, 12H, 3
OAc, 1 NAc), 1.58 (m, 4H) and 1.41 (m, 2H): methylenes.
Anal.calcd.: C, 55.8: H, 7.5; N, 2.05. Found: C,
55.82; H, 7.53; N, 2.98.
This material was de-O-acetylated in methanol
(5 mL) to which a 0.5 N solution of sodium methoxide in
methanol (0.100 mL) was added. After overnight at room
temperature, the mixture was de-ionized with IR-C50
resin (H+ form, dry) and the solvents evaporated. The
residue was run through Iatrobeads using a 7:1 mixture
of chloroform and methanol giving the pure 237 (0.103
g, 80%), I~]20D -0.17 (c.1, water~; lH-n.m.r. (D2O):
5.85 (m, lH, -CH=), 5.29 (m, 2H, -CH=), 4.50 (d, lH,
J12 8.5Hz, H-1), 4.03 (d, 2H, J 6.0Hz, allylics),
2.033 (S, 3H, NAc), $.58 (m, 4H) and 1.36 (m, 2H):
methylenes; 13C-n.m.r. (D20): 175.2 (carbonyl), 134.7
and 119.1 (ethylenes), 101.9 (C-l), 61.6 (C-6), 56.4
(C-2), 29.1, 23.0 and 22.6 (methylenes).
D. ~RAN8FER OF SIALIC ACIDS ~ND OT~ER 8UGARS TO
OLIGOSACCEARIDE S~R~CTURES
Ex~mple 40 -- Transfer of 8ialic Acids and other
Sugars to Oligosaccharide 8tructure3 via
Glycosyltr~nsferases
This example demonstrates the enzymatic
transfer of Neu5Ac, analogues thereof ~collectively
"sialic acids"), and other sugars onto oligosaccharide
glycoside structures via glycosyltransferases. FIGs.
36, 37, 38, 39, and 40 illustrate these transfers and
provide structures for the prepared compounds
identified by an underlined arabic numeral. In
--.W093/~4505 2 ~ 1 ~ S ~ 2 PCT/US93~04909
-- 189 -
Examples 40a-40e, preparative sialylation and
fucosylation were performed as follows:
i. Preparative ~ialylation
Sialic acids, activated as their CMP-
derivatives (as set forth in Examples 31-35 above),
were transferred onto synthetic oligosaccharide
structures containing ~Gal(1-3)~GlcNAc-,
~Gal(1-4~GlcNAc-, ~Gal(1-3)~GalNAc-, and
~Gal(1-4)~Glc- terminal sequences by using three
mammalian sialyltransferases (Examples 40a-e). The
~Gal(1-3/4)~GlcNAc-~(2-3)sialyltransferase (EC
2.4.99.5) and the ~Gal(1-4)~GlcNAc-~(2-6)sialyl-
transferase (EC 2.4.99.1) from rat liver were purified
to homogeneity by affinity chromatography according to
the procedure of Mazid et al.~, which is incorporated
herein by reference on a matrix obtained by covalently
linking the hapten ~Gal(1-3)~GlcNAcO(CH2)8CO2H87 to
acti~ated Sepharose by methods known in the art. The
~Gal(1-3)~GalNAc-~(2-3)sialyltransferase (EC 2.4.99.4)
was purchased from Genzyme Corporation, Norwalk, CT.
In all preparative sialylation reactions, the
acreptor oligosaccharide (5-20 mg) was incubated with
the selected CMP-sialic acids (5-20 mg) in the presence
of the appropriate sialyltransferase (10-50 mU) and
calf intestinal alkaline phosphatase (Boehringer
Mannheim, Mannheim, Ger~any) as in the procedure of
Unverzagt et al.93 for 37C for 24-48 hours in 50 mM
sodium cacodylate pH 6.5, 0.5% Triton CF-54, 1 mg/mL
BSA ("sialyl transfer buffer"). For example, the
sialyloligosaccharide 7-d-~NeuSAc(2-6)~&al(1-4)~GlcNAc-
0-(CH2)8-COOCH3 t213d, 4.4 mg) was synthesized by
incubation of ~Gal(1-4)~GIcNAc-0-(CH2)8-C~OCH3 t205~,
4.6 mg) and CMP-7-d-Neu5Ac (202d, 15.6 mg) in the
presence of ~Gal(1-4)~GlcNAc-~(2-6)sialyltransferase
(51 mU) and calf intestinal alkaline phosphatase (2.4
211~a2`~
WQ93/2450~ PCT/US93/04909 --
---- 190 ----
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 C18 cartridges, conditioned as
suggested by the manufacturer. Each cartridge was
washed with water (4 x 5 m~) and then with methanol (3
x 5 mL). The methanol eluate was evaporated to dryness
in vacuo and the residue was dissolved in a 65:35:3
mixture of chloroform, methanol and water (0.5 mL -
solvent I) and applied on to a small column ofIatrobeads (500 mg) equilibrated in the same solvent.
The column was successively eluted with solvent I
followed by a 65:35:5 mixture of chloroform, methanol
and water (solvent II) and then by a 65:35:8 mixture of
chloroform, methanol and water ~solvent III). The
appropriate fractions (30 drops) containing the
product, as identified by t.l.c. on silica gel plates
(with a 65:35:8 mixture of chloroform, methanol and
0.2~ calcium chloride solution as eluent), were pooled
together and concentrated to dryness in vacuo. The
residue was run through a small column of AG 50W-X8
(Na' form), the eluate freeze-dried and the recovered
product characterized by 1H-n.m.r. which, in all cases,
indicated good purity.
ii. Pre~arative_FucosYlation
- Sialylated analogues of the type I and II
oligosaccharides can be further fucosylated by the
human milk ~GlcNAc~ 3/4)fucosyltransferase. The
enzyme was purified from human milk according to the
methodology using affinity chromatography on GDP-
hexanolamine Sepharose described by Palcic et al. 25
The synthesis and purification of the fucosylated
oligosaccharides was carried out by a modification of
the procedures of Palcic et al. 25 For example, the
fucosylated structure 9-N~-~Neu5Ac(2-3)~Gal(1-3)-[~-L-
Fuc(1-4)]-~GlcNAc-0-(CHz)8-CH2OH 217b was synthesized
W093/24505 2 1 ~ ~ S 2 2 PCT/US93/04909
---- 191 ----
by incubating GDP-fucose (2.5 mg) and 9-N3-aNeu5AC(2-
3~Gal(l-3)~GlcNAc-0-(CH2)8-CH2OH 208b (1.7 mg) with
affinity purified ~GlcNAc~(1-3/4)fucosyltransferase
(4.6 mU) in 1.3 mL of 100 mM sodium cacodylate (pH
5 6.5), lO mM manganese chloride, 1.6 mM ATP, 1.6 mM
sodium azide. After 27 hours at 37C7 2.5 mg of GDP-
fucose and 2.3 mU of the fucosyltransferase were added
to the reaction mixture, which was kept at 37OC 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) indicated that the
fucosylation was almost complete. After purification
and chromatography on AG 50W x 8 (Na+ form), 1H n.m.r.
of the product 217b (1.0 mg3 indicated a very good
purity (Table 5). In some cases where the
fucosyltransferase was not highly purified, partial
hydrolysis of the methyl ester of the linking arm
occurred.
E~ample~ 40a-40e are a~ follows:
Example 40a: This example refers to the
transfer of modified sialic acids such as 201a-~ to the
3-OH of a terminal ~Gal of acc~ptors possessing a
~Gal(1-3)~GlcNAc- (LewisC or Type I) terminal structure
such as 204a and 204b by a sialyltransferase such as
the ~Gal(l-3/4)~GlcNAc~(2-3)sialyltransferase from rat
liver following the experimental procedure reported
above. The 1H-n.m.r. data of the reaction products,
which were purified as indicated previously, are
reported (Tables 3 and 4).
Example 40b: This example refers to the
transfer of modified sialic acids such as 201b and 201c -
to the 3-OH of the terminal ~Gal of acceptors
possessing a ~Gal(1-4)~GlcNAc- ~LacNAc or Type II)
terminal structure such as 205a, k, d-a by a
2118-J2 ''
W O 93/24505 Pc~r/~s93/04909!` :
-- l9Z --
sialyltransferase such as that used in 4Oa. In some
cases, dimethylsulfoxide (5% volume) may be added to
solubilize the acceptor. The 1H-n.m~r. data of the
reaction products, which were purified as indicated
previously, is reported (Tables 6 and 8). The reaction
mixture was wor~ed up in the manner described
previously.
Example 40c: This example refers to the
transfer of modified sialic acids such as 201c to the
3-OH structure of the terminal ~Gal of acceptors
possessing a ~Gal(1-4)~Glc- (lactose) terminal
structure such as 206a by a sialyltransferase such as
that used in Exampie 40a following the same
experimental procedure. The ~H-n.m.r. data of the
reaction products, which were purified as indicated
previously, is reported (Table 6).
Example 40d: This example refers to the
transfér of modified sialic acids such as 201b - h to
the 6-OH of the terminal ~Gal of acceptors possessing a
~Gal(1-4)~GlcNAc- (LacNAc or Type II) terminal unit
such as 20Sb, d-a by a sialyltransferase such as the
~Gal(1-43~GlcNAc~(2-6jsialyltransferase reported
previously. The 1H-n.m.r. data of the reaction
products, which were purified as indicated previously,
is reported (Tables 7 and 8).
Example 40e: This example refers to the
transfer of modified sialic acids such as 201c to the
3-OH of the terminal ~Gal of acceptors possessing a
~Gal(1-3)~GalNAc- ("T") terminal unit such as 207a by a
sialyltransferase such as the ~Gal(;-3)~GalNAc~(2-
3)sialyltransferase (Genzyme) following the experi-
mental procedure reported previously. The 1H-n.m.r.
data of the reaction prd~ucts, which were purified as
indicated previously, is reported (Table 9).
W O 93/24505 ~ 2 ~ P ~ /US93/04909
-- 193 --
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W O 93~24505 PC~rJUS93/04909 :
-- 194 --
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WO 93/24505 2 1 1 ~ ~ ~ 2 PCI /US93/04909
---- 195 ---- ~
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WO 93/24~0~ 211 ~ ~ 2 ~ PCI/US93/04909
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WO 93~24505 2 1 1 ~J a ~ ~ PCr/USg3/04909
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W O 93/24505 P ~ /U~93/04909
-- 200 --
.............. ..........PREPARATION OF ANALOGUE8 OF OLIGOS~CCXARIDE
LYCOSIDES BY CHEMICAL MODIFICATION
OF THE COMPLETED
OLIGOS~CCHARIDE GLYCOSIDE STRUCT~RE
Examples 11-13 below describe the synthesis
of analogues of oligosaccharide glycosides by the
chemical modification of the completed oligosaccharide
glycoside structure (prepared by either enzymatic or
chemical means). FIGs. 22-24 illustrate the reaction
schemes involved in the preparation of these analogues
and provide structures for the prepared analogues which
are identified by an underlined arabic numeral.
Example 41 -- Synthe~is of 9-Hydroxy~onyl ~5-
acetamido--3,5-dideoxy~ -arabino-2-
heptulopyranosylonic acid)-(2-3)
-O-~-D-galactopyranosyl~ 3)-O-t~
fucopyrano~yl-~1-4)-0-]-2-acet.~mido-2-
deoxy-~-D-glucopyra~oside 217m
The starting trisaccharide 208a (1.3 mg) 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 o some ethylene glycol. Sodium
borohydride (20 mg) was then added and the stirring was
continued for 24 hours at 4C. The pH of the reaction
mixture was then brought to 6 by addition of acetic
acid and the solvents were co-evaporated with methanol.
The residue was dissolved in water (1 mL) and run
through a Sep-Pak cartridge which was further washed
with water followed by methanol. The methanol eluate
was evaporated and the residue chromatographed on
Iatrobeads (200 mg) using a 65:35:5 mixture of
chloroform, methanol and water as eluant. The
appropriate fractions were pooled and evaporated
leaving the product 208m (1 mg); lH-n~m.r.: see Table
3 above.
W093/~4505 2 1 1 ~ ~ 2 2 PCT/US93/04909
-- 201 --
Trisaccharide 208m was enzymatically
fucosylated following the procedure reported in Example
lO and the product purified in the same manner.~ T.l.c.
of the recovered crude material indicated that the
transformation of 20~m was almost complete.
Purification gave 217m (0.5 mg); 1H-n.m.r.: see Table 5
above .
E~ample 42 -- Synthe~is of 9-Hydroxynonyl (5~-
diacetamido-3~5,9-tri-deoxy-~-D-glyc2ro-
D-galacto-2-nonulo-pyrano~ylo~ic acid~-
~2-3)-o-~-D-galactopyra~o~y~ -3)-o-t~-
L-fucopyrano~yl~ 4~-0~-2-~cetamido-2-
deoxy-~-D-glucopyranoside 217k
A solution of the trisaccharide 208b (l mg)
in water (0.5 mL) was hydrogenated at 22C at
atmospheric pressure in the presence of Lindlar
catalyst (l.0 mg, Aldrich Chemical Company, Milwaukee,
WI~ for 15 minutes T.l.c. (65:35:8 - chloroform,
methanol and 0.2~ calcium chloride), indicated a
complete transformation. The mixture was filtered
through Celite and the solid extensively washed with
water. The filtrate was concentrated, filt~red through
Millipore filter and the eluate freeze dried leaving
the trisaccharide 208i; 1H-n.m.r.: see Table 3 above.
Acetic anhydride (about 0.2 mg) in methanol
(lO ~L3 was added to a solution of 208t (about l mg) in
a l:l solution of 0.002 N sodium hydroxide and methanol
(0.300 mL) at 0C. T.l.c. (~olvent as above) indicated
a complete reaction and the solvents were then
evaporated. The residue was dissolved in water (2 mL)
and applied to a Sep Pak cartridge. The cartridge was
washed with water and the product eluted with methanol
giving the trisaccharide 208k (about l mg); 1H-n.m.r.:
see Table 3 above.
a 2~
W093/24505 PCT/US93/04909-
-- 202 --
Trisaccharide 208k was enzymatically
fucosylated following the procedure reported in Example
10 and the product purified in the same manner. T.l.c.
of the recovered crude material indicated that the
transfo~mation of 208k was almost complete.
Purification gave _17k (about 0.5 mg); 1H-n.m.r.: see
Table 5 above.
Example 43 -- Synthesis of ~-N-methylamidooctyl (5-
acetamido-3,5-dideoxy-~-D-glycero-
galacto-2-nonulo-pyranosylonic acid N-
methylamide)-~2-3)-0-~-D-galacto-
pyrano~yl-~1-3)-0-[~-L-fucopyrano~yl-(1-
4)-0-]-2-acetamido-2-deoxy-~-D-
lS glucopyra~o-~ide 2181
Tetrasaccharide 218a (0.003 g) was applied on
Dowex SOx8 (Na~ form) resin and eluted with water. The
appropriate fractions, were freeze-dried, followed by
further drying over phosphorous pentoxide. Methyl
iodide (0.050 mL) was added to the residue dissolved in
dimethyl sulfoxide. After stirring in the dark for 20
hours, the solution was evaporated in vacuo, diluted
with water ~11 mL) and applied to a Sep-Pak C18
cartridge. After washing with water (10 mL), the
product was eluted with methanol. Evaporation of the
appropriate fractivns ieft a residue which was
chromatographed on Iatrobeads (O.S g) using a 65:35:5
mixture of chloroform: mekhanol:water providing the
methyl ester of compound ~333 (0.025 g) lH n.m.r.:
5.099 ~d, lH, J12 3.75Hz, H-l ~FUC), 4.517 (d, 2H, J1 2
7.5Hz, H-1 ~Gal and ~GlcNAc), 3.866 and 3.683 (2s,
CO2C_3~ ~ 2-781 (dd, lH~ J3ax 3eq 12.5Hz, J3~ 4 4.5Hæ,
H-3eq Neu5Ac), 2.032 and 2.018 (2s, 6H, 2 NAc), 1.913
(dd, lH, J3ax 4 12.5Hz, H-3ax Neu5Ac), 1.160 (d, 3H,
Js6 6.5Hz, H-6 ~Fuc).
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
WOg3/24~5 ~ a ~ 2 PCT/US93/04909
-- 203 --
water (1 mL) and applied on a Sep-Pak cartridge which
was further washed with water. After elution of the
product with methanol, the solvent was evaporated and
the residue freeze-dried from water providing 2181
(0.0025 g~; 1H-n.m.r.: (Table 5).
SYNTHESIS OF MONOFUCOSYLATED OLIGOSACCHARIDES
TERMINATING IN DI-N-ACETYLLACTOSAMINYL
STRUCTURES
Examples 44-51 below are presented for the
purpose of illustrating different methods for
synthesizing an oligosaccharide glycoside related to
~lood group determinants having a type II core
structure, i.e., CD65, which is a sialyl LewisX
derivative having a ~Gal(1~4)~GlcNAc-OR disaccharide
glycoside attached to the reducing sugar of the sialyl
LewisX. See further Kashem, et al.~, and U.S. Serial
No. 07/771,2S9, filed October 2, 1991, entitled
"Methods for the Synthesis of Monofucosylated
Oligosaccharides Terminating in Di-N-acetyllactosaminyl
Structures," both of which are incorporated herein by
reference.
The following examples illustrate the
preparation of Compounds 305a and 305b which
preparation is illustrated in Figure 44. The
synthethic pathway in Examples 44-Sl utiliæed the
following general methods:
General Methods: All organic solvents used were re-
distilled reagent grade. Pre-coated silica gel plates
(60-F254, E. Mexck, Darmstadt) were run in 65:35:5,
65:35:8 and/or 60:40:10 mixtures of CHC13, CH30H, and
0.2% CaC12 solution, and detection was by charring
after spraying with a 5% solution of sulphuric acid
(H2SO4) in ethanol. Sep-Pak C18 cartridges (Waters
211~ )2~
W093/24505 PCT/US93/Q4909
-- 204 --
Associates, Milford, MA) were conditioned as indicated
by the supplier. Iatrobeads (6RS-8060) were from
Iatron Laboratories, Tokyo, Japan and the AG 50W X 8
ion exchange resin was purchased from BioRad, Richmond,
California. CMP-Neu5Ac was purchased from Sigma
Chemical Company (St. Louis, Missouri) and GDP-fucose
was obtained by chemical synthesis.4Z ~Gal(1-
4)~GlcNAc(1-3)~Gal(1-4)~GlcNAc-OR was obtained by
following the procedures of Alais et al92 with the
appropriate substitution of the aglycon. Evaporation
of organic solvents was done at 20-25C using a rotory
evaporator connected to a water aspirator. 1H-n.m.r.
spectra have been run on at 300 and 500 MHz using
internal acetone t~=2.225) as reference and samples
were freeze dried twice from D20 and dissolved in
99.99% D20. The spectra of compounds obtained as
8-methoxycarbonyloctyl glycosides all show a singlet at
~-3.686 ~Co2cH3j and a triplet at ~=2.387 (7.5Hz,
CH2CO2). The spectra of compounds obtained as the
8-carboxyoctyl glycosides differ from the respective
8-methoxycarbonyloctyl glycosides by the absence of the
singlet due to CO2CH3 and the presence of a triplet at
~=2.314 ~t, 7.5Hz) for CH2CO2H.
In Examples 44 to 51 below, preparative
sialylation was conducted as follows:
The rat liver BGal(1-3/4)~GlcNAc
~(2-3)sialyltransferase (EC 2.4.99.5) was purified by
affinity chroma~ography according to the procedure of
Mazid, et al.77 but using a matrix obtained by
covalently linking the hapten
BGal(1~3)~GlcNAcO(CH2)8CO2H87 activated as in its
N-succinimidyl ester to epichlorohydrin activated
Sepharose. 78
The ~Gal(1~4)~GlcNAc ~(2~6~sialyltransferase
contained in the flow-through of the above affinity-
W093/245~ 352 ) PCT/US93/04909
-- 205 --
column, was further chromatographed on CDP~hexanolamine
Sepharose as reported. 74
,~
The enzymatic sialylatlons were carried out
at 37~C in a plastic tube using a sodium cacodylate
buffer (50 mM, pH 6.5) containing Triton CF-54 tO.5%),
BSA (1 mg/m~) and calf intestine alkaline
phosphatase.~93 The final reaction mixtures were
diluted with H20 and applied onto C~8 Sep-Pak
cartridges as reported. 25 After washing with H20, the
products were eluted with CH30H and the solvents
~-vaporated. Thç residue was dissolved ln a 65:35:5
mixture of CHCl3, CH30H and H20 and applied on a small
column of Iatrobeads (0.200 to 0.500 g). After washing
with the same solvent mixture, the products were eluted
with a 6S:35:8 and/or 65:40:10 mixtures of the same
solvents. The appropriate fractions (t~l.c.) were
pooled, the solvents evaporated in vacuo, the residue
run through a small column of AG SOW X 8 (Na~ form) in
H20 and the products recovered after freeze drying in
~acuo. In all cases, the 8-methoxycarbonyloctyl
glycosides were separated from the corresponding 8-
carboxyo~tyl glycosides.
In examples 44 to 51 below, preparative
fucosylation was conducted as follows:
The BGlcNAc ~ 3/4)fucosyltranferase ~EC
2.4~1.65) was purified from human milk, as reported.25
The enzymatic reactions were carried out at 37C in a
plastic tube using a sodium cacodylate buffer (100 mM,
pH 6.5), MnCl2 (10 mM~, ATP (1.6 mM), NaN3 (1.6 mM).
The reaction products were isolated and purified as
indicated above.
Z 1 1 ~ 2 ~
W093/24505 PCT/US93/04~09 -
-- 206 --
Example 44~- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5-dideoxy-~-D-glycero-D-
galacto-2-nonulopyranosylonic acid)-(2-6)-0-
B-D-galactopyranosyl-(1-4)-0-2-acetamido-2-
deoxy glucopyranosyl-(1-3)-0-~-D- -
galactopyranosyl~(l-4)-0-2-acetamido-2-deoxy-
glucopyranoside (30~a)
Compound 30~a (6.5 mg), CMP-Neu5Ac ~17 mg),
~Gal(1-4)~GlcNAc ~(2-6)sialyltransferase (50 mU) and
alkaline phosphatase (15 U) were incubated for 4~ hours
in 2.5 mL of the above buffer. Isolation and -~
purifiration provided 30z (3.0 mg).
:
Example 45-- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5-dideoxy-~-D-glycero-D- ~
galacto-2-nonulopyranosylonic acid)-(2-6)-0- ::
~-D-galacto-pyranosyl-(1-4)-0-2-acetamido-2-
deoxy-glucopyranosyl-(1-3)-0-B-D-
galactopyranosyl-tl-4)-0-[~-L-fucopyranosyl-
~1-3)-0-]2-acetamido-2-deoxy-glucopyranoside :~
~3Q3a) and the 8-carboxyoctyl glycoside ~:
(303b)
Compound 302a (3~0 mg), GDP~fucose (5 mg~, :
BGlcNAc ~(1-3/4)fucosyltransferase (10 mU) were
incubated for 68 hours in the buffer (1.3 mL).
Isolation and purification provided 303a (1.2 mg) and
303b (0.5 mg).
Example 46-- Preparation of 8-Methoxycarbonyloctyl
~-D-galactopyranosyl-(1-4)-0-2-acetamido-2-
deoxy-~-D-glucopyranosyl-(1-3)-0-B-D-
galactopyranosyl-(1-4)-0-[~-L-fuco-pyranosyl-
~1-3)-0-]2-acetamido-2-deoxy-B-D-
glucopyranoside (304a) and the 8-carboxyoctyl
glycoside ~30~b)
Compounds 303a and 303b (l G 7 mg) were
incu~ated with Clostridium Perfringens neuraminidase
immobilized on agarose (Sigma Chemical Company, 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) an~ filtered through Amicon PM-10
W O 93/24505 ~ 5 2 2 PC~r/US93/04909
-- 207 --
membrane. The flow-through and washings were
lyophilized and the residue dissolved in water (3 mL)
and applied to two Cl8 cartridge. Each cartridge was
washed with water (10 mL) prior to elution with
methanol (20 mL). After evaporation of the solvent,
the residue was chromatographed on Iatrobeads (210 mg)
as indicated above giving (304a, 0.8 mg) and 304b (0.7
mg). 304b was dissolved in dry methanol and treated
with diazomethane until t.l.c. indicated the complete
conversion into 304a.
Example 47-- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5-dideoxy-~-D-glycero-D-
galacto-~-nonulopyranosylonic acid)-(2-3)-o-
B-D-galactopyranosyl-(1-4)-0-2-acetamido-2-
deoxy-~-D-glucopyranosyl-(1-3)-O-~-D-
galactopyranosyl-(1-4)-0-[~-L-fucopyranosyl-
(1-3)-0-]2-acetamido-2-deoxy-B-D-
glucopyranoside (305a) and the 8-carboxyoctyl
glycoside ~305b)
-~ Compound 304a (1.5 mg), CMP-Neu5Ac (8 mg), BGal(l-
3/4)BGlcNAc a(2-3)sialyltransferase (17 mU), alkaline
phosphatase (5 U), were incubated for 40 hours in the
sialylation buffer (1.5 mL). Isolation and
purification provided 305a (0.7 mg) and 305b (0.55 mg~.
Example 48-- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5-dideoxy-~-D-glycero-D-
galacto-2-nonulopyranosylonic acid)-(2-3)-0-
B-D-galactopyranosyl-(1-4)-0-2-acetamido-2-
deoxy-~-D-glucopyranosyl-(1-3)-0-~-D-
galactopyranosyl-(1-4)-0-2-acetamido-2-deoxy-
glucopyranoside(306)
Compound 301a (5 mg), C~P-Neu5Ac (15 mg), BGal(1-
3/4)BGlcNAc ~(2-3)sialyltransferase (46 mU), and
alkaline phosphatase (15 U) were incubated in the
sialylation buffer (2,5 mL) for 48 hours. Isolation
and purification of the product gave 306a (2.5 mg).
2 11~2;~
W093/24505 PC~/US93/04909 -
-- 208 --
Example 49-- Preparation of 8-Methoxycarbonyloctyl
(5-Acetamido-3,5~dideoxy-~-D-glycero-D-
galacto-2-nonulopyranosylonic acid)-(2-3)-o-
B-D-galactopyranosyl-(1-4)-O-[~-L- ~.
S fucopyranosyl-(1-3)-0-]2-acetamido-2-deoxy- -
glucopyranosyl-(1-3)-O-B-D-galactopyranosyl-
(1-4)-O-~-L-fuc~pyranosyl-(1-3~-0-]2-
acetamido-2-deoxy-glucopyranoside (307a)
Compound 306a (2.5 mg), GDP-fucose (8 mg) and
the BGlcNAc ~ 3/4~fucosyltransferase (19 mU) were
incubated in the en~ymatic buffer (2.0 mL) for 48 h.
Isolation and purification of the product give 307b
(1.7 mg). :~
1H-NMR data for the compounds prepared in
Examples 44 to 49 above are set forth below:
WO 93/24505 ~ 2 2 PCI`/US93/04909
---- 209 --
~ I ~ a 1~ ;~
I $~ ~
,~, .~ ~
e ~
~ ~1
~ ~ -o l _ 5' ~ 1
ll ~ t .;~
'211~52~
W~93/24505 PCT/US93/04909
-- 210 --
The following Examples 50 and 51 illustrate
alternative methods for preparing for compounds of
Formula I.
:
- 5 In these examples, during the chemical
synthesis, unless otherwise specially indicated, the
work up generally included extraction with
dichloromethane followed by the normal sequential
washings of the organic phase with water, a dilute
solution of sodium carbonate and water. The organic
solvent were then dried over magnesium sulfate, the
solid filtered and the solvent evaporated in vacuo as
indicated.
''~
Evaporation of organic solvents was done at
20-25C using a rotory evaporator connected to a water
aspirator. lH-n.m.r. spectra were run at 300 MHz using
internal acetone (~=2.225) as reference and samples
were freeze dried twice from D2O and dissolved in
99.99% D2O. The spectra of compounds obtained as
8-methoxycarbonyloctyl all show a singlet at ~=3.686
(CO2CH3) and a triplet at -~=2.387 (7.5Hz, CH2CO2). The
- spectra of compounds obtained as the 8-carboxyoctyl
glycosides differ from the respective 8-methoxy-
carbonyloctyl glycosides by the absence of the singlet
due to CO2CH3 and the presence of a triplet at ~=2.314
~t, 7.5Hz) for CH2CO2H.
PreParative Enz~matic Galactos~lation
The bovine milk ~GlcNAc ~ 4~
galactosyltransferase (EC 2.4.1.22, specific activity
6.5 units/mg of protein) and UDP-Gal were obtained from
Sigma. The enzymatic reactions were carried out at
37C in a plastic tube using a sodium cacodylate buffer
(100 mM, pH 7.5) containing 20 mM manganese dichloride.
The reaction products were purified as indicated above
in the case of the preparative sialylation.
W 0 93/24505 211~a2 ~ PC~r/US93/04909
- 211 --
In some cases, depending upon the enzymatic
preparation, it may happen that the terminal methyl
ester of the aglycone is hydrolyzed. As a result, the
final products may possibly be isolat~d as saccharides
possessing the aglycone terminated by a methyl ester or
a free acid group. These two saccharides are separated
during the step of the chromatography on Iatrobeads as
indicated above. The two forms of the aglycone of the
saccharide are identified by 1H-n.m.r.
Example 51 -- Synthesis of Compound 319
A. Synthesis of 8-Methoxycarbonyloctyl (2,3,4,6-
15 - tetra-O-acetyl-~-D-galactopyranosyl)~ 4)-O-
3,6-di-O-acetyl-2-deoxy-2-phthalimido-B-D-
glucopyranoside (Compound 312)
A solution of trimethylsilyltrifluoro-
methanesulfonate (0.504 mL, 2.6 mmol) in
dichloromethane (4 mL) was added to the mixture of the
disaccharide donor 31192 (2.0 g, 2.6 mmol), drierite
(4.0 g, crushed) and B-methoxycarbonyloctanol (1.9 g,
10.0 mmol) in dichloromethane (30 mL) at 4C. After
stirring for 0.5 h at 4~, the mixture was slowly
warmed up to room temperature for 1 h. After cooling
to 4C, a second portion of the catalyst (0.250 mL, 1.3
mmol) in dichloromethane (2 mL) was added. After
slowly warming up and stirring at room temperature for
1 h, the reaction was stopped ~y addition of
' triethylamine. After filtration, the crude product
recovered after the usual work up was dried in vacuo,
and acetylated in a 2:1 mixture of pyridine and acetic
anhydride. After addition of methanol, the mixture was
worked up as usual, and the solvents co-evaporated with
an excess of toluene. The residue was chromatographed
on silica gel using a 2:1 mixture of toluene and ethyl
acetate providing compound 312 (1.40 g, 60%).
.
2 ~hQ~3~242~os PCT/US93/04909
-- 212 --
H-n.m.r.(CDCl3): ~ 7.90-7.70(m, 4H, aromatics~
( , lH~ J23 10.5 J3 4 3.5Hz, H-3), 5.34(m 2H
incl. H-l and H-4 ), 5.15(dd, lH, J~ 2 8.0, J2 3
10.5Hz, H-2 ), 4.97(dd, lH, H-3 ), 3.67~s, 3H, CO2CH3),
2.25(t, 2H, J 7.5Hz, CH2CO2), 2.19-1.94~6s, 18H, 6
OAc), 1.45(m, 4H) and 1.08(m, 8H): methylenes. ~
B. Synthesis of Compound 315 -- 8-Methoxy-
carbonyloctyl (2,6-di-O-acetyl-3,4-O-
isopropylid~ne-A-D-galactopyranosyl)-
(1-4)-0-(3,6-di-O-acetyl-2-deoxy-2-
phthalimido-B-D-glucopyranoside (315)
A lM solution of sodium methoxide in methanol
(0~200 mL) was added to a solution of compound 312
(1.40 g, 1.65 mmol) in methanol (40 mL) cooled at 4C.
After 1.5 h at 4OC, the solution was deionized using
IRC-50 resin (H' form). The resin was filtered, the
solvent evaporated and the product dried in vac~o (1.0
g, 94%).
A solution o~ the above material (0.776 g,
1.2 mmol) and paratoluene sulfonic acid monohydrate (60
- mg) in dry acetone (60 mL) was refluxed for 3 h. After
neutralization with triethylamine, the solvent was
evaporated and the residue chromatographed on silica
gel using a 100:1 mixture of ethyl acetate and methanol
providing compound 314 (0~575 g, 70%); 1H-n.m.r.
(C~OD, DOH at 4.80): 7.80-7.6Q(m, 4H, aromatics),
5.10(d, lH, J12 8.0Hz, H-1), 4.38(m, 2H, H-1 and H-3),
3.70~s, 3H, CO2CH3), 2.31(t, ~ 7.5Hz, CH2CO2), 1.65-
1.00[m, incl. 1.57 and 1.45 (2s, C(CH3)2~. Further
elution provided the 4,6-isopropylidene derivative
(0.200 g, 24~).
Compound ~14 (0.515 g, 0.84 mmol) was
acetylated in a 2:1 mixture of pyridine and acetic
anhydride for 24 h at 22. After addition of methanol
and the usual work up, the solvents were co-evaporated
W093/24~05 2 1 1 8 5 2 ~ PCT/US93104909
-- Z13 --
with an excess of toluene and the residue
chromatographed on sillca gel using a 100:3 mixture of
chloroform and methanol providing compound 315 `~0.646
g, 90%); [~]D ~ 13.8 (c, 1 chloroform); lH-
n.m.r.(CDCl3); ~ 7.90-7.70(m, 4H, aromatics), 5.74(J1 2
8.5, J23 10.5Hz, H-3), 5.34(d, lH J12 8.5Hz, H-1),
4-88(dd, lH, J1-2 ~J2 3 6.5Hz, H-2 ), 3.67(s, 3H,
CO2CH3), 2.23(t, J 7.5Hz, CH2CO2), 2.14, 2.13, 2.10,
1.91(4s, 12H, 4 OAc), 1.30-1.54[m, incl. 1.53 and
1.32(2s, C~CH3)2]-
C. Synthesis of Compound 316 -- 8-Methoxycarbonyl-
octyl (2,6-di-O-acetyl-~-D-galactopyranosyl)~
4)-O-3,6-di-O-acetyl-2-deoxy-2-phthalimido-~-D-
glucopyranoside
Compound 315 (0.575 g, 0.68 mmol) in 90%
acetic acid (12 mL) was heated at 80C for 2 hours.
After dilution with dichloromethane, the solvent was
washed with water, a solution of sodium bicarbonate and
water. After drying over magnesium sulfate, the
solvents were evaporated in vacuo, and the residue
chromatographed on silica gel providing compound 316
( 2 g, 82%); ~]D +12.1 (c, 1.`03 chloroform)
D. Synthesis of Compound 318 -- 8-Methoxy-
carbonyloctyl (3,4,6-tri-O-acetyl-~-deoxy-2-
phthalimido-~-D-glucopyranosyl) (1-3;~-0-(2,6-di-O-
acetyl-B-D-galactopyranosyl)-(1-4)-0~3,6-di-O-
acetyl-2-deoxy-2-phthalimido-B-D-glu_opyranoside
(318)
Trimethylsilyltrifluoromethanesulfonate (0.036 mL,
0.060 mmol) in methylene chloride (0.5 mL) was added to
a solution of compound 316 (0.100 g, 0.123 mmol) in
methylene chloride (5 mL). A solution o~ the imidate
17 (0.102 g, 0.18S mmol) in methylene chloride (4 mL)
was slowly added to the above solution cooled at -70.
211~5 2~
W093/24~05 PCTtUS93/04909
-- 214 --
`''
The mixture was further stirred at that temperature for
0.5 h. An additional portion of the catalyst (0.018
mL, 0.030 mmol) in methylene chloride (0.5 mL) was
further added. After 0.5 h at -70~, the reaction was
stopped by addition of triethylamine, and the mixture
worked up as usual. The recovered residue was
chromatographed on silica gel using a 100:2 mixture of
chloroform and methanol pro~iding compound 318 (0.120
g, 80%); 1H-n.m.r.(CDCl3): ~ 7.95-7.60 (m, 8H,
aromatics~, 5.74(dd, lH, J2 3 10.5 J3 4 9~OHz, H-
3 ), 5.61(dd, lH, J23 10.5, J34 8.5Hz, H-3), 5.48(d,
lH, J~-- 2 ' 8.5Hz, H-l ), 5.27(d, lH, J1 2 8.5Hz, H- ;
1), 5.14(dd, lH, J4 5 lO.OHz, H-4 ), 4.90(dd, lH,
J2 3 8.0 J3 4 lO.OHz, H-2 ), 3~68(s, CO2CH3), 2.22(t,
J 7.5Hz, CHzCO2), 2.12(two), 2.10, 2.04, 1.86, 1.85,
1,56(6s, 21H, 7 OAc), 1.40(m, 4H), and 1.20(m, 8H):
methylenes.
E. Synthesis of Compound 319 - 8-Methoxycarbonyl-octyl
(2-acetamido-2-deoxy-~-D-glucopyranosyl)-(1-3)-0-
(B-D-~alactopyranosyl)-(1-4)-0-2-acetamido-2-
-~ deoxy-B-D-glucopyranoside
- Hydrazine acetate (1.27 g, 13.8 mmol) was added to compound 318 (0.120 g, 0.098 mmol) in anhydrous ethanol
(15 mL). The mixture was refluxed for 18 h. The
solvents were then co-evaporated with an excess of
toluene. After drying in vacuo, the residùe was
acetylated in a 2:1 mixture of pyridine and acetic
anhydride for 48 h. After quenching the excess of
acetic anhydride with some methanol, the reaction
mixture was worked up as usual. The recovered solvents
were evaporated in vacuo and the residue co-evaporated
with an excess of toluene. The residue was
chromatographed on silica geI using a 100:9 mixture of
chloroform and methanol as eluant provided the
peracetylated trisaccharide intermediate. This
material was de-O-acetylated in anhydrous methanol (S
W093/24505 2 1 ~ 8 ~ 2 2 PCT/USg3/04909
-- 215 -~
mL) in the presence of 0.2 M sodium methoxide in
methanol (0.200 mL). After overnight at 22OC, de-
ionization with Dowex 50 X 8 and filtration, the
solvent was evaporated in vacuo. The recovered product
was chromatographed on BioGel P-2 and eluted with a 1:1
mixture of water and ethanol which provided the pure
trisaccharide 319 (0.044 g, 60%); [~D -4.8O (c, 0.48,
water); 1H-n.m.r. (D20): data provided below.
Example 51 - Synthesis of 8-Methoxycarbonyloctyl (5-
acetamido-3,5-dideoxy-~-D-glycero-D-galacto-
2-nonulopyranosylonic acid)-(2-3)-O-(B-D-
galactopyranosyl)-(1-4)-0-(2-acetamido-2-
deoxy-~-D-glucopyranosyl)-(1-3)-O-(~-D-
galactopyranosyl)-(1-4)-0-[~-L-fucopyranosyl-
(1-3)-O]-2-acetamido-2-deoxy-~-D-
glucopyranoside (322) (Compound 322 -- the
CD-65/VIM-2 Saccharide)
A. Synthesis of Compound 320 -- 8-Methoxy-
carbonyloctyl (2-acetamido-2-deoxy-~-D-
glucopyranosyl)-(1-3)-O-(B-D-
galactopyranosyl)-(1-4)-0-~-L-fucopyranosyl-
2S (1-3)-03-2-acetamido-2-deoxy-~-D-
glucopyranoside
Compound 319 (15 mg), GDP-fucose (33 mg) and the
~GlcNAc ~1-3/4)fucosyltransferase (56 mU) were
incub~ted for 72 hours in the buffer (4 mL) as
indicated above. Isolation and purification provided
the compound 320 (14.0 mg). 1H-n.m.r. data is set
forth below.
B. Synthesis of Compound 321 -- 8-Methoxycarbonyloctyl
(~-D-galactopyranosyl)-(1-4)-0-(2-acetamido-2-
deoxy-B-D-glucopyranosyl)-(1-3)-O~ D-
galactopyranosyl)-(1-4)-O-[~-L-fucopyranosyl-(l-
3)-0]-2-acetamido-2-deoxy-B-D-glucopyranoside
Compound 320 (14.0 mg), UDP-Gal (25 mg), BGlcNAc
B(1-4) galactosyltransf,erase (14.5 U, Sigma) were
incubated for 48 hours in the buffer described above
(3.2 mL). Isolation and purification provided compound
321 (13.2 mg). 1H-n.m.r. data is set forth below.
211~52~ :
W093/24505 PCr/VSg3/04909--
-- 216 --
C. Synthesis of Compound 3~2 - 8-Methoxycarbonyloctyl
(5-acetamido-3,5-dideoxy-~-D-glycero-D-galacto-2-
nonulopyranosylonic acid)-(2-3)-O-(~-D~ ~
galactopyranosyl)-(1~4)-0-(2-acetamido-2-deoxy-B-
D-glucopyranosyl)-(1-3)-O-(B-D-galacto-pyranosyl)- ~ :
(1-4)-O-[~-L-fucopyranosyl-(1-3)-0]-2-acetamido-2-
deoxy-f~-D-glucopyranoside
Compound 322 was synthesized from compound 321 as
indicated above10.
1H-n.m.r. data for compounds 319, 320, 321,
and 322 are set forth below.
In addition to the above methodology, Figures
45, 46 and 48 illustrate alternative methods to prepare
some of the above compounds, to prepare alternative
structures and to prepare extended chains.
WO ~3/24505 2 1 1 ~ 5 2 2 P~/US93/04909
--~ 217 ----
H-n . m. r . Structural Parameters
_ . .~ ~ _ .. ,. ,. ~, ~ I
SUUn~9tar ¦ Hydrogen . 31'~ ~ 20~,b 3a~ b 3
_ ~ . . ___ - - --.. ~
BGtcNAc 1 ~d) 4.50e (7 5) 4.52 (7.5~ 4.52 (8.0~ 4.53 (8 0)
. . . . . ~-
~Gal (B) 1 (d) 4.4~' (8.0) 4.43 (7.0) 4.43 (7-5) 4.43 (8-0)
4 (d) 4.15 (3.0~ 4.09 (3.5) 4.10 ~3.2) 4.10 (3.0)
_ _. . .
BGlcNAc 1 ~d) 4.66 (8.53 4.67 (8.5) 4.70 (7-8) 4.69 (8-~)
. . , . , ~
3Gal (D) 1 (d~ 4.48 (7.8~ 4.46 (7 7)
3 (d) . . - . _ . . . .
~F~c ~ (d) 5.09 (4.0) 5.10 (3.8) 5.09 (3.8~
5 (q) 4.81 ~6.5~ 4.81 (6.5) 4.81 (6.5)
6 (d~ 1.14 1.15 1 15 _
NeuShc 3" (dd) 2.76 (~.5. 13.0)
. 3~ ) . _ . 1.7g (1~.0)
NHAc s. 2.02, 2.01 2.02, 2.0i 2.03. 2.02 2.02 (~ree)
C~?CO2 1 2.38 (7.5~ 2.38 (7.5) 2.38 (7.5) 2.38 (7.5)
. . . . . .
CO2C~ s 3.6~ 3.~9 3.69 3.69
9, lO, 11 and 12 show multiplets around l.49-l.63 (4H) and
1 . 3 0 ( ~H~: methylenes
b ;r in }{z
c interchangeable
211~ ~ 2;~
W093/24s0~ PC~`/US93/04gO9-
-- 218 --
Examples 52-53 below are offered to
illustrate the synthesis of 2-sulfated and 3-sulfated
LewisC-OR derivatives and the synthesis of 3-sulfated
and LacNAc-OR. Such compounds and further derivatives
thereof are described in detail in U.S. Patent
Application No. 07/g88,254 filed concurrently herewith
as Attorney Docket No. 000475-050 and entitled
"IMMUNOSUPPRESSIVE AND TOLEROGENIC MODIFIED LEWISC AND
LacNAc COMPOUNDS" which application is incorporated
herein by reference in its entirety.
In the following examples, unless indicated
otherwise, R = -(CH2)8C02CH3
~xample 52 -- Synthesis of 8-Methoxycarbonyloctyl-3-0-
(4,6-0-benzylidene-3-0-sulfo-fl-D-
galactopyranosyl)-2-acetamido-4,6-o-
benzylidene-2-deoxy-fl-D-glucopyranoside
The title compound was prepared by first
generating 8-methoxycarbonyloctyl 3-0-(2,3-di-o-
benzoyl-4,6-0-benzylidene-fl-D-galactopyrnosyl)-2-
acetamido-4,6-0-benzylidene-2-deoxy-~-D-glucopyranoside
which, in turn, was generated by coupling the 8-
methoxycarbonyloctyl 2-acetamido-4,6-0-benzylidene-2-
deoxy-fl-D-glucopyranoside and compound 32, the
synthesis of which is exemplified in example 2 above.
The 8-methoxycarbonyloctyl 2-acetamido-4,6-0-
benzylidene-2-deoxy ~-D-glucopyranoside can be prepared
; by reacting N-acetylglucosamine-OR (e.g., compound 4 in
Figure 1) with about 1.5 equivalents of C6H5CH(OCH3) 2
in an acidic (p-toluene sulfonic acid~ acetonitrile or
dimethylformamide (DMF) medium at from about 0 to
about 50C over 6-48 hours to provide for the 4,6-0-
protected benzylidine compound.
W093/24505 ~ 11 8 S 2 ~ PCT/USg3/04909
-- 219 -
Coupling of compound 32 with the
8-methoxycarbonyloctyl 2-acetamido-4,6-O-benzylidine-2-
deoxy-~-D-glucopyranoside ("compound A") can be
achieved by first combining about 1 equivalent of N-
S iodosuscinimide with about 1 equivalent oftrifluoromethanesulfonic acid in methylene chloride
containing molecular sieves which remove any water
present. The reaction mixture is cooled to -50C and
then compound A is added followed by the addition of
approximately, 1 to 1.1 equivalents of compound 32
(based on compound A). When large amounts of
trifluoromethanesulfonic acid are employed, the
reaction is preferably cooled to -50C prior to the
addition of the trifluoromethanesulfonic acid.
The reaction is allowed to equilibrate to
about -20 to O~C over about 1-3 hours. The reaction
solution is then quenched by cooling to -50 followed
by the addition of triethylamine until neutral pH is
reached. The solution is filtered through Celite and
then washed with a saturated sodium bicarbonate
solution and water. The organic layer was dried and
stripped in vacuo to provide for 8-methoxycarbonyloctyl
3-O-(2,3-di-O-benzoyl-4,6-O-benzylidene-~-D-
galactopyranosyl~-2-acetamido-4,6-0-benzylidine-2-
deoxy-~-D-glucopyranoside ("compound 9").
The ~enzoyl groups on compound B can be
removed under Zemplen conditions (NaOMe/MeOH) to
provide for 8-methoxycarbonyloctyl 3-0-(4,6-O-
benzylidene-~-D-galactopyranosyl)-2-acetamido-4,6-O-
benzylidene-2-deoxy-~-D-glucopyranoside ("compound C").
Compound C (1 gram, 1.37 mmol) was dissolved
in dry dimethylformamide (5.0 mL) and sulfur trioxide-
pyridine complex (267.1 mg, 1.64 mmol) was added at
211~2;~
W093~24505 PCT/US93/04909
-- 220 --
-30C. The resulting solution was stirred at -30C for
5 hours and then 15 hours at ooc. Excess reagent was
destroyed by the addition of me~hanol (2 mL). The
The reaction mixture was converted into sodium salt by
passage through Dowex 50-X8~(Na+) resin in methanol.
Evaporation and co-evapoTation with toluene left a
while solid which was purified by chromat~graphy on
silica gel using dichloromethane-methanol-pyridine
(9:1:0.1) as eluant to provide the title compound as a
white solid. This material was converted into sodium
salt by passage through Dowex 50-X8 (Na~) resin in
methanol to give the title compound (850 mg, 74.6%).
Example 53 -- Synthesis of 8-Methoxycarbonyloctyl 3-o-
(3-O-sulfo-~-D-galactopyranosyl)-2 acetamido-
2-deoxy-~-D-glucopyranoside
The product of Example 52 (800 mg, 0.96 mmol)
was dissolved in methanol (10 mL) containing 5%
palladium on carbon ~800 mg) and was stirred under
hydrogen ~1 atmosphere) for S hours at room
temperature. Catalyst was removed by filtration,
washed with methanol (500 mL) and the solvent was
evaporated to dryness. The residue was then purified
by chromatography on silica gel using dichloromethane-
methanol-water-pyridine (80:20:2:0.2) as eluant. The
title compound (488 mg, 77.5~) was obtained as a white
solid after BioGel P-2 (200-400 mesh) filtration and
conversion into its sodium salt. lH.n.m.r. ~D20)
~: 4.520-4.570 [m, 2h, incl. H-l(d, 4-550, J12 8.0Hz
and H1'(d, 4.542, J~2~ 7.7 Hz)], 4.277-4.343 [m, 2H,
incl. H-3'(dd, 4.310, J23 lO.OHZ, J3 4 3.5Hz) and
H-4'(4.296)~, 3.687(s, 3H~ OH3), 2.388(t, 2H, J7.5Hz,
CH2C00),2.025(s, 3H, NHAc), 1.570(m,4H) and 1.30(m,
8H~.
W093/24505 2 1 1 ~ S 2 2 PCT/US93/04909
-- 221 --
The 2,3-disulfate of Example 53 was prepared
in a similar manner except that sulfation was conducted
with 6 equivalents of sulfur trioxide/pyridine complex
and the reaction was conducted at room temperature for
24 hours. The resulting product was a mixture of 2-,
3-sulfate and predominantly 2,3-disulfate. The mixture
was purified by chromatography in the manner described
and ion exchange of the resulting product provided for
the 2,3-disulfate of LewisC-OR as the disodium salt.
The 2-sulfate of LewisC-OR was prepared from
compound C by benzoylation under conditions described
above. The resulting product contained predominantly
the 3-benzoyl group on the galactose unit and a small
amount of the 2-benzoyl and 2,3-dibenzoyl derivatives.
The products were separated by chromatography on silica
gel to provide for both the 2-ben~oyl and the 3-benzoyl
derivatives as pure products.
The 3-benzoyl derivative was sulfated in the
mann~r described above and then deprotected to provide
for the 2'-sulfo-LewisC-OR which upon ion exchange as
described above provided for the sodium salt of this
product.
The 3'-sulfo-LacNAc derivative was prepared
from compound 42 (prepared in Example 7) wherein the
dibenzoyl groups are removed via Zemplen conditions
(sodium methoxide/methanol) and then sulfated under the
conditions described above to provide for
3'-sulfo-LewisC-OR.
In the following examples, enzymatic
fucosylation was conducted as follow:
h ~ 2 ~
W093/~4505 P~T/US93/04909j-
-- 222 --
Enz~matic Fucosvlation
~Gal(1-3~4)~GalcNAc(1-3/4) fucosyltrans-
ferase was purified from human milk according to the
methodology using affinity chromatography on GDP-
hexanolamine Sepharose described by Palcic et al. 22
The cloned fucosyltransferases, Fuc T III and Fuc T IV,
immobilized on rabbit IgG-sepharose (Sigma) were
provided by Glycomed, Alameda, CA. The enzyamatic
reactions were carried out at room temperature or 37C
in a plastic tube using a sodium cacodylate buffer (100
mM, pH 6.5), MnC12 (10 mM), ATP (1.6 mM) NaN3 (1.6 mM).
The final reaction mixture was diluted with H2O (5 mL)
and applied onto C18 Sep-Pak cartridges as reported22.
After washing with H2O (30 mL) the products were eluted
with CH30H and the solvents evaporated. The residue
was dissolved in a 65:35:5 mixutre of CHC13, CH30H and
H2O and applied on a small column of Iatrobeads (0.200
to 0.S00 g). After washing with the same solvent
mixture, the products were eluted with a 65:35:8 and/or
60:40:10 mixtures of the same solvents. The
appropriate fractions (t.l.c.) were pooled, the
solvents evaporated in vacuo, the residue run through a
small column of AG 50W x 8 (Na form) (BioRad) in H2O
and the products recovered after freeze drying in
2S vacuo.
iii. Fucosvlation ~eactions
A. Preparation of fucosylated derivative of the sodium
salt of the 3-sulfo-LewisC-OR (3-sulfo-~Gal(1-3)-
[~-L-Fuc(1-4)]-~GlcNAc-OR)
The product prepared in Example 53 above
(19.2 mg) GDP-fucose (17.2 mg), milk ~Gal(1- -
3/4)~GalcNAc(1-3/4) fucosyltranferase (25 mL) and calf
intestine alkaline phosphatase (20 U) were incubatPd
for 68 hours in the buffer (3.5 mL) at 37~C. Isolation
and purification provided the title compound (10.8 mg).
W093/24505 ~ ) 2 2 PC~/US93/04909
-- 223 --
B. Preparation of fucosylated derivative of the sodium
salt of the 2-sulfo-LewisC-OR (2-sulfo-~Gal(1-3)-
[~-L-Fuc(1-4)]-~GlcNAc-OR)
The 2-sulfo-LewisC-OR compound, described
above, (9.5 mg), GDP-fucose (9.5 mg), cloned
fucosyltranferase FucT-III (75 ~L of beads) and calf
intestine alkaline phosphatase (20 U) were incubated
for 72 hours in the buffer (2.0 mL) at room
temperature. Isolation and purification provided the
title compound (5.8 mg).
C. Preparation of fucosylated-derivative of the sodium
salt of the 3-sulfo-LacNAc-OR (3-sulfo-~Gal(1-4)-
lS [a-L-Fuc(1-3)]-~GlcNAc-OR)
The 3-sulfo-LacNAc-OR compound, described above
~6.8 mg), GDP-fucose (6.8 mg), cloned fucosyltranferase
Fuc T-IV (50 mL beads) and calf intestine alkaline
phosphatase (20 U) were incu~ated for 72 hours in the
buffer (2.0 mL) at room temperature. Isolation and
purification provided the title compound.
