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[17] Synthesis of ganglioside analogs containing sulfur in place of oxygen at the linkage positions
[17]
GANGLIOSIDE
ANALOGS
CONTAINING
SULFUR
183
Chemoenzymatic Synthesis of Ganglioside G~3 The combined chemical enzymatic approach has been applied to the synthesis of natural ganglioside Gm .49,5o The key synthetic step is the regio- and a-stereoselective glycosylation of sialic acid to Y-OH of the lactose moiety by use of CMP-Neu5Ac and a-2,3-sialyltransferase. However, this method is usually restricted to the synthesis of natural gangliosides, and in addition both the sugar nucleotide and glycosyltransferase are still not readily available. 49 y . lto and J. C. Paulson, J. Am. Chem. Soc. 115, 1603 (1993). t0 K. K.-C. Liu and S. J. Danishefsky, J. Am. Chem. Soc. 115, 4933 (1993).
[ 17] S y n t h e s i s of G a n g l i o s i d e A n a l o g s C o n t a i n i n g S u l f u r in P l a c e of O x y g e n at t h e L i n k a g e Positions
By HIDEHARU ISHIDh, MhgOTO KISO, and AKIRA HASEGAWA Introduction
Chemical synthesis of naturally occurring gangliosides as well as a variety of ganglioside analogs is becoming stimulating and rewarding as more and more biological functions 1-6 of sialoglycoconjugates are reported. Among these analogs, thioglycosides are of interest as inhibitors of the corresponding glycosides (mainly via competitive inhibition). 7.8 For instance, ganglioside analogs containing the a-thioglycoside of sialic acid are expected to affect the activity of sialidases, and analogs containing thioglycosidically linked ceramide could be an inhibitor of ceramide glycanase 9 In this chapter we describe the established methods for the synthesis I G. Walz, A. Aruffo, W. Kolanus, M. Bevilacqua, and B. Seed, Science 250, 1132 (1990). 2 M. L. Phillips, E. Nudelman, F. C. A. Graeta, M. Perez, A. K. Singhal, S. Hakomori, and J. C. Paulson, Science 250, 1130 (1990). 3 j. B. Lowe, L. M. Stoolman, R. P. Nair, R. D. Larsen, T. L. Berhend, and R. M. Marks, Cell (Cambridge, Mass.) 63, 475 (1990). 4 M. J. Polley, M. L. Phillips, E. Wayner, E. Nudelman, A. K. Singhal, S. Hakomori, and J. C. Paulson, Proc. Natl. Acad. Sci. U.S.A. 88, 6224 (1991). 5 H. Nojiri, M. Stroud, and S. Hakomori, J. Biol. Chem. 266, 4531 (1991). 6 I. Eggens, B. Fenderson, T. Toyokuni, B. Dean, M. Stroud, and S. Hakomori, J. Biol. Chem. 264, 9476, (1989). 7 D. Horton and J. D. Wander, in "The Carbohydrates, Chemistry/Biochemistry" (W. Pigrnan and D. Horton, eds.), 2nd Ed., Vol. IB, p. 803. Academic Press, New York, 1980. 8 p. j. Deschavanne, O. M. Viratelle, and J. M. Yon, J. Biol. Chem. 253, 833 (1978). 9 M. Ito and T. Yamagata, this series, Vol. 179, p. 488.
METHODS IN ENZYMOLOGY, VOL. 242
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
NEOGLYCOLIPIDS
[17]
of ganglioside analogs containing thioglycosidic linkages in the following positions: (1) between the sialic acid and hexopyranoside (compounds A-D); (2) between the oligosaccharide chain and ceramide (compounds E-H); (3) between both the sialic acid-hexopyranoside and oligosaccharide chain-ceramide (compounds I-K); and (4) between sialic acid and sialic acid (compound L).
Synthesis of S-(a-Sialyl)-(2--~6)-fl-D-hexopyranosyl and S-(a-Sialyl)(2---)6')-fl-D-lactosyl Ceramides (A-D) In this section, the synthesis of S-(o~-sialyl)-(2--~6)-/3-D-lactosyl ceramide (D), shown in Fig. 1, is described as an example of the synthesis of the title compounds. Other compounds (A-C) are synthesized essentially by the same method described for D. ~°,~1 For the synthesis of D, we employ the sodium salt of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2nonulopyranosonate (3) 52 as glycosyl donor and per-O-acetylated 2-(trimethylsilyl)ethyl 6'-bromo-6'-deoxy-fl-D-lactoside (9) l° as glycosyl acceptor, respectively. The intermediates are coupled and converted, by introduction of ceramide moiety, to the end product. Treatment of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro2,3,5-trideoxy-o-glycero-fl-D-galacto-2-nonulopyranosonate (1) 13 with potassium thioacetate in dry dichloromethane gives a-2-S-acetyl derivative 2 in 90% yield. Selective S-deacetylation of 2 with the amount of sodium methoxide equivalent to 2 in dry methanol at -40 ° gives the sodium salt 3, which is used for the next reaction without further purification. Treatment of 2-(trimethylsilyl)ethyl/3-D-lactoside 414 with 2-methoxypropene in N,N-dimethylformamide (DMF) in the presence of p-toluenesulfonic acid monohydrate and subsequent acetylation give 6 in 93% yield. The de-O-isopropylidenation of 6 by heating with 80% aqueous acetic acid for 10 hr at 45° gives crystalline 7 in 91% yield. Selective C-6' bromination of 7 with N-bromosuccinimide in the presence of triphenylphosphine and subsequent acetylation afford compound 9 as crystals in good yield. Condensation of 3 with 9 in DMF under a nitrogen atmosphere leads to the i0 A. Hasegawa, M. Morita, Y. Ito, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 369 (1990). II A. Hasegawa, T. Terada, H. Ogawa, and M. Kiso, J. Carbohydr. Chem. 11, 319 (1992). 12A. Hasegawa, J. Nakamura, and M. Kiso, J. Carbohydr. Chem. 5, 11 (1986). t3 R. Kuhn, P. Lutz, and D. L. MacDonald, Chem. Ber. 99, 611 (1966). J4 K. P. R. Kartha, A. Kameyama, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 8, 145 (1989).
[17]
GANGLIOS1DE ANALOGS CONTAINING SULFUR HOL..pOH
COOH
~(CH~)I6CH3
H&~"-O-~',. R~
AcHN~ ' ~ HO
185
O~
n2" S ~ ' ~ 0 Nil R ~ O ~ R 10VAN~C
13H2.~
OH
A RI = OH, R2 = H, R3 = OH (Gal) OH, R2 = OH, R3 = H (GIc) C R1= NHAc,R2 = OH, R3 ffiH (GIcNAc) B R1=
A : ~ ~ 3 0 ! ~ ~ ) O
O~~HO D
R2 ~oa
o - ( (ca2h+cas
RI.~...O
-
.OH
OH
/(CH2)I6CH3
OH
OH
HO
HO .OH
S
C H
13 27
OH
COOH
~(CH2)tsCH 3
o~
OH (Gal) J R1= OH, R2 = H (Glc) 1 R l = H, R2 =
OH
COOH
.o~o-~.~..d OH
COOH . . ~
~
~
'+"'
.o,+~--o-~,7, a~
H~.~H
O-(,
O
O
HO
HO"~-~'~ 0 ~ A c H N ~
NH
G
O
HO .OH
°=((ca2)l~cns Q
HO
t:Jou
HO"'
oH
HO. ~ E ~ O " ~ . ~
....
#
AcH
O--~(CH2)i+CH 3
oH
NH
OH (~H ER1 = H, R2= OH (Gal) F Rt = OH, R2 = H (GIc)
HO
ON H ~ C I 3 H ~
"'OH -
K . #un
COOH
.
"f
TM
NH
O ~x(CH2)t6CH3 . r~H
(CHz)I6CH3 au
O~
L
Fro. 1. Ganglioside analogs containing thioglycosides.
186
NEOGLYCOLIPIDS
[ 17]
corresponding a-thioglycoside 10 in 87% yield. Selective removal 15,16 of the 2-(trimethylsilyl)ethyl group in 10 is performed by treatment of 10 with boron trifluoride etherate in dichloromethane for 2 hr at - 2 0 °, to give 11 in 84% yield. When treated with trichloroacetonitrile 15,~7,18in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) for 2 hr at 0°, compound 11 gives the trichloroacetimidate 12 as the a-anomer in 92% yield, after column chromatography. The glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene1,3-diol (13) 19'2° with 12 in the presence of boron trifluoride etherate for 6 hr at - 2 0 ° yields only the expected/3-glycoside 14 in 82% yield. Selective reduction ~5'2~of the azide group in 14 with hydrogen sulfide in 5 : 1 (v/v) pyridine-water gives the amine 15, which on condensation with octadecanoic acid using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DAC) in dichloromethane gives the S-(c~-N-acetylneuraminyl)(2-~6')-O-(6'-thio-fl-D-lactosyl)-(1--~l)-ceramide derivative 16 in 94% yield. Finally, de-O-acetylation of 16 with sodium methoxide in methanol, followed by saponification of the methyl ester group, yields almost quantitatively the end product D. Compounds A, B, and D show potent inhibition against sialidases from several kinds of influenza virus, acting as competitive inhibitors. The order of decreasing inhibition is D > A > B . 22
Detailed Procedures Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2thio-D-glycero-c~-o-galacto-2-nonulopyranosonate (2). To a solution of 112 (3.3 g) in dry dichloromethane (30 ml) is added potassium thioacetate (3.5 g) (Scheme 1). The mixture is stirred overnight at room temperature and concentrated, the residue is extracted with chloroform, and the extract is washed with water, dried (Na2SO4) , and evaporated to a syrup. The product is chromatographed on a column of silica gel (200 g) with 100 : 1 (v/v) chloroform-methanol, to give 3.2 g (90%) of compound 2 as an amorphous mass; mp 74o-76 °, [a] D -15.6 ° (c 0.75, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-4,6-O-isopropylidene-fl-Dgalactopyranosyl)-(1--~4)-2,3,6-tri-O-acetyl-[3-D-glucopyranoside (6). To a 15T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 188, 71 (1989). 16 K. Jansson, T. Frejd, J. Kihlberg, and G. Magnussun, Tetrahedron Lett. 27, 753 (1986). 17 M. Numata, M. Sugimoto, K. Koike, and T. Ogawa, Carbohydr. Res. 163, 209 (1987). 18R. R. Schmidt and J. Michel, Angew. Chem., Int. Ed. Engl. 19, 731 (1980). 19 M. Kiso, A. Nakamura, Y. Tomita, and A. Hasegawa, Carbohydr. Res. 158, 101 (1986). 20 R. R. Schmidt and P. Ziminermann, Angew. Chem., Int. Ed. Engl. 25, 726 (1986). Zl T. Adachi, Y. Yamada, I. Inoue, and M. Saneyoshi, Synthesis, 45 (1977). 22 y. Suzuki, K. Sato, M. Kiso, and A. Hasegawa, Glycoconjugate J. 7, 349 (1990).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR AcO .OAc
AcO
CI
OAc
187
COOMe
A c O ~ O ~ o ~ A c H N ~ at~
AcO" 0 A c H ~ C O O M e AcO 1
2R=Ac 3R=Na OR l
/ ORL
s,o o - os JR2~ R 3
OR
4RI=H,R2=R3=OH 5 R 1 = H, R2,R3 = OCMe20 6 R 1 = Ac, R2,R 3 = OCMe20 7 R I = Ac, R2 = R3 = OH 8 R I = Ac, R2 = OH, R3 = Br 9 R 1 = Ac, R 2 = O A c , R 3 = Br
10 R 1 = OSE, R 2 = H 11 R1,R2 = H, OH 12 R 1 = H, R2 = OC(=NH)CCI 3
N3 HON/A~CI3Hz7 OBz
13 R 3 0 .OR 3 R30"t~ R~O
COOR 4 O ""~
OR~3
~3
R3O - ~ . . ~ . ~
~ t~ ~ ~!P'm~ t'~q
" ~-~ - - - - < ' O R ~"
OR2 _ ~
l
v
t 131rl127
RI
14 R I = N 3, R 2 = Bz, R 3 = Ac, R4 = Me 15 R 1 = NH 2, R 2 = Bz, R 3 = Ac, R 4 = Me
16 R l = NHCO(CH2)I6Me, R2 = Bz, R 3 = Ac, R 4 = Me D R 1 = NHCO(CH2)16Me, R 2 = R3 = R4 = H SE = 2-(trimethylsilyl)ethyl, Bz = benzoyl
SCHEME 1. Synthesis of ganglioside analogs containing the c~-thioglycoside of sialic acid.
solution of 41° (1.5 g, 3.39 mmol) in DMF (15 ml), cooled to 0 °, are added, with stirring, 2-methoxypropene (0.6 ml) and p-toluenesulfonic acid monohydrate (30 mg), and the mixture is stirred for 2 hr at 0 °, the progress of the reaction being monitored by thin-layer chromatography (TLC). Acetic anhydride (7 ml) and pyridine (10 ml) are added to the mixture, and this is stirred for 5 hr at room temperature and concentrated to a syrup, which is chromatographed on a column of silica gel (150 g)
188
NEOGLYCOLIPIDS
[ 17]
with 1 : 4 (v/v) ethyl acetate-hexane to give 6 (2.14 g, 93%) as an amorphous mass, [a]D + 14.0 ° (c 1.0, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-fl-o-galactopyranosyl)(1-~4)-2,3,6-tri-O-acetyl-fl-D-glucopyranoside (7). A solution of 6 (2.34 g, 3.38 mmol) in 80% aqueous acetic acid (30 ml) is kept for 10 hr at 45 °, then concentrated to a syrup, which is chromatographed on a column of silica gel (200 g) with 1 : 1 (v/v) ethyl acetate-hexane to give 7 (2.0 g, 91%) as crystals. Recrystallization from ether-hexane gives needles: mp 197 °, [a] D -8.1 ° (c 0.6, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-6-bromo-6-deoxy-fl-o-galactopyranosyl)-(1---~4)-2,3,6-tri-O-acetyl-fl-o-glucopyranoside (8). To a solution of 7 (500 mg, 0.77 mmol) in DMF (10 ml), cooled to 0°, are added, with stirring, Nobromosuccinimide (267 mg, 1.5 mmol) and triphenylphosphine (262 mg, 1 mmol), and the mixture is stirred for 2 days at 0°. Methanol (1 ml) is added to the mixture, and this is stirred for 10 min, then concentrated. The residue is chromatographed on a column of silica gel (60 g) with 2 : 1 (v/v) ethyl acetate-hexane to give 8 (380 mg, 69%) as an amorphous mass, [a]D --2.8 ° (C 0.86, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3,4- Tri-O-acetyl-6-bromo-6-deoxy-fl-D-galactopyranosyl)-(l--~4)-2,3,6-tri-O-acetyl-fl-D-glucopyranoside (9). Acetylation of 8 (329 mg, 0.46 mmol) with acetic anhydride (l ml) in pyridine (2 ml) overnight at room temperature gives 9 (345 mg, quantitative) as crystals. Recrystallization from ether-hexane gives needles: mp 208°-209 °, [o~]D - 1 1 . 9 ° (c 1.0, chloroform).
2-(Trimethylsilyl)ethyl S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl3,5-dideoxy-o-glycero-a-o-galacto-2-nonulopyranosylonate)-(2-->6)-O(2,3,4-tri-O-acetyl-6-thio-fl-D-galactopyranosyl)-(1--~4)-2,3,6-tri-O-acetylfl-o-glucopyranoside (lO). A solution of 9 (280 mg, 0.37 mmol) and 3 (400 mg, 0.76 mmol) in dry DMF (3 ml) is stirred overnight at room temperature under a nitrogen atmosphere, the course of the reaction being monitored by TLC. Acetic anhydride (2 ml) and pyridine (4 ml) are added to the solution, and the mixture is stirred overnight at room temperature. Dichloromethane (200 ml) is added, and the solution is successively waslled with 1 M sodium carbonate, 2 M hydrochloric acid, and water, dried (Na2SO4), and concentrated. The residue is chromatographed on a column of silica gel (100 g) with 1 : 1 (v/v) ethyl acetate-hexane to give 10 (380 mg, 86.5.%) as an amorphous mass, [a] D + 1.5 ° (c 0.67, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1--~4)-2,3,6-tri-O-acetyl-D-glucopyranose (11). To a solution of 10 (904 mg, 0.76 mmol) in dry dichloromethane (10 ml), cooled to - 2 0 °, is added boron trifluoride etherate (1.2 ml), and the mixture
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
189
is stirred for 2 hr at - 2 0 °, the progress of the reaction is monitored by TLC. Dichloromethane (100 ml) is added to the mixture, and the solution is successively washed with 1 M sodium carbonate and water, dried (NazSO4), and concentrated to a syrup, which is chromatographed on a column of silica gel (250 g) with 100 : 1 (v/v) dichloromethane-methanol to give 11 (695 mg, 84%) as an amorphous mass, [odd +2.1 ° (c 1.9, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroO~-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(l--->4)-2,3,6-tri-O-acetyl-D-glucopyranosyl Trichloroacetimidate (12). To a stirred solution of 11 (403 mg, 0.37 mmol) in dry dichloromethane (5 ml), cooled to 0°, are added trichloroacetonitrile (0.075 ml) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.03 ml). The mixture is stirred for 2 hr at 0 ° and then concentrated. The residue is chromatographed on a column of silica gel (20 g) with 120 : 1 (v/v) dichloromethane-methanol, to give 12 (420 mg, 92%) as an amorphous mass, [a]D +52.4 ° (c 0.9, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2--*6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1--~4)-O-(2,3,6-tri-O-acetyl-fl-D-glucopyranosyl)(1--H)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-l,3-diol (14). To a solution of 12 (453 mg, 0.37 mmol) and 13 (320 mg, 0.74 mmol) in dry dichloromethane (15 ml) is added Molecular Sieves 4A powder (MS-4A; 600 mg), and the mixture is stirred for 1 hr at room temperature, then cooled to - 2 0 °. Boron trifluoride etherate (0.06 ml) is added to the cooled mixture, and this is stirred for 6 hr at - 2 0 °, the progress of the reaction being monitored by TLC. The precipitate is filtered off and washed with chloroform. The filtrate and washings are combined, and the solution is successively washed with 1 M sodium carbonate and water, dried (Na2SO4), and concentrated. The residue is chromatographed on a column of silica gel (60 g) with 120 : 1 (v/v) dichloromethane-methanol to give 14 (452 mg, 82%) as an amorphous mass, [a]D +0.9 ° (c 0.56, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1---~4)-O-(2,3,6-tri-O-acetyl-fl.D-glucopyranosyl)(l--* l )-(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-octadecene-1,3-diol (16). Hydrogen sulfide is bubbled through a solution of 14 (I00 mg, 0.069 mmol) in pyridine (2.5 ml) and water (0.5 ml) for 2 days while the solution is stirred at room temperature. The mixture is concentrated to give the syrupy amine 15, which is used for the next reaction without further purification. To a solution of 15 in dry dichloromethane (2.5 ml) are added octadecanoic acid (40 mg, 0.14 mmol) and DAC (35 rag, 0.19 mmol), and
190
NEOGLYCOLIPIDS
[17]
the mixture is stirred overnight at room temperature. After completion of the reaction, dichloromethane (15 ml) is added to the mixture, and the solution is washed with water, dried (Na2SO4) , and concentrated to a syrup that is chromatographed on a column of silica gel (5 g) with 90 : 1 (v/v) dichloromethane-methanol, to give 16 (106 rag, 94%) as an amorphous mass, [a]D + 8 . 0 ° (C 0.4, chloroform).
S-(5-Acetamido-3 ,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2-->6)-O-(6-thio-fl-D-galactopyranosyl)-(1-->4)-O-(fl-D-glucopyranosyl)-(1--> l )-(2S,3R,4E)-2- octadecanamido-4- octadecene-l ,3-diol (19). To a solution of 16 (121 rag, 0.072 mmol) in dry methanol (5 ml) is added sodium methoxide (25 mg), and the mixture is stirred overnight at room temperature. After completion of the reaction, water (0.2 ml) is added to the mixture at 0°, and this is stirred for 1 hr, then treated with Amberlite IR-120 (H-') resin to remove the base. The solution is concentrated, and the residue is chromatographed on a column of Sephadex LH20 (100 g) with 1 : l (v/v) chloroform-methanol to give compound D (79.6 mg, 97%) as an amorphous mass, [a] D +16.5 ° (c 1.5, chloroform-methanol). Synthesis of Cerebroside, Lactosyl Ceramide, and Ganglioside GM3 Analogs Containing fl-Thioglycosidically Linked Ceramide In this section, we describe the synthesis of a ganglioside GM3 analog carrying fl-thioglycosidically linked ceramide (H). Other thioglycolipids (E-G) were synthesized by the same procedure. 23 Compound H is prepared by the coupling of the sodium salt of O-(a-sialyl)-(2--->3')-fl-D-1thiolactose (19) and the tosylate derivative of azidosphingosine (20), and the coupled product is readily converted to the target compound H. Treatment of per-O-acetylated O-(a-sialyl)-(2--->3')-a-D-lactosyl trichloroacetimidate (17) 15 with thioacetic acid in the presence of boron trifluoride etherate gives the required fl-thioacetate derivative 18 in 91% yield. (2S,3R,4E)-2-Azido-3-O-benzoyl-l-O-(p-toylysulfonyl)-4-octadecene-1,3-diol (20) is derived byp-tolylsulfonylation of(2S, 3R, 4E)-2-azido3-O-benzoyl-4-octadecene-l,3-diol (13), 19,2° which is used as the glycosyl acceptor. Treatment of sodium salt 19, freshly prepared from 18 by selective S-deacetylation with sodium methoxide, with 20 in DMF under nitrogen affords the desired fl-thioglycoside (21) in 41% yield. The 1H nuclear magnetic resonance (NMR) data for 21 demonstrate it to be a fully blocked glycoside. The intermediate 21 is converted, by selective reduction of the 23A. Hasegawa, M. Morita, Y. Kojima, H. Ishida, and M. Kiso, Carbohydr, Res. 214, 43 (1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
191
azide group, condensation with octadecanoic acid, de-O-acylation, and saponification of the methyl ester group, to the end product H as described for the conversion of 14 to D. B~ir and Schmidt z4 synthesized fl-lactosyl 1-thioceramide (G) in an alternative manner, namely, by the coupling of lactosyl bromide with the sodium salt of the 1-thio derivative of azidosphingosine. Detailed Procedure O - ( M e t h y l 5 - A c e t a m i d o - 4 , 7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa- D- g alacto- 2- n o n u l o p y r a n o s y l o n a t e )-( 2-->3 ) - O - ( 2 , 4 - d i - O - a c etyl-6- Obe nzoyl- fl- D- g a l a c t o p y r a n o s y l ) - ( l--->4)- 3 - O - a c etyl- l - S - a c etyl- 2 , 6 - d i - O b e n z o y l - l - t h i o - f l - o - g l u c o p y r a n o s e (18). To a solution of 17 (100 mg, 71.5
/xmol) in dry dichloromethane (5 ml) are added thioacetic acid (10/xl) and boron trifluoride etherate (10/.d), and the mixture is stirred overnight at room temperature (Scheme 2). The mixture is successively washed with 1 M sodium carbonate and water, dried (Na2SO4) , and concentrated to a syrup that is chromatographed on a column of silica gel (50 g) with 90 : 1 (v/v) dichloromethane-methanol to give 18 (85 mg, 91%) as an amorphous mass, [a]D +0.5° (C 0.2, chloroform). O - ( M e t h y l 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~3)-O-(2,4-di-O-acetyl-6-O-benzoyl-fl- D-galactopyranosyl)-(1--~4 ) - S - ( 3 - O - a c e t y l - 2 , 6 - d i - O - b e n z o y l - f l - Dglucopyranosyl) - (1---~1) - (2R,3R,4E) - 2 - azido - 3 - b e n z o y l o x y - 4 - o c t a d e c ene-l-thiol (21). To a stirred solution of 18 (30 rag, 0.023 mmol) in dry
methanol (0.5 ml) and chloroform (0.1 ml), cooled to - 2 0 °, is added a solution of sodium metal (5.0 mg) in dry methanol (0.2 ml). Stirring is continued for 5 min at - 2 0 °, and the mixture is concentrated to give 19 as an amorphous mass, which is used for the next reaction without purification. A solution of 19 and 20 (26 mg, 0.045 mmol) in dry DMF (2 ml) is stirred for 24 hr at 40 ° under a nitrogen atmosphere, the course of the reaction being monitored by TLC. Acetic anhydride (0.5 ml) and pyridine (1 ml) are added to the mixture, which is stirred overnight at room temperature, then concentrated. The residue is dissolved in dichloromethane (20 ml), and the solution is successively washed with 2 M hydrochloric acid, 1 M sodium carbonate, and water, and then dried (Na2SO4) and evaporated to a syrup that is chromatographed on a column of silica gel (10 g) with 6 : 1 (v/v) hexane-ethyl acetate to afford 21 (15.5 mg, 41%) as an amorphous mass, [a]D -64.5 ° (c 0.73, chloroform).
24 T. B~ir and R. R. Schmidt, Liebigs Ann. Chem., 185 (1991).
192
NEOGLYCOLIPIDS AcO~OAc
COOMe
AcO
OAc
[17]
.OB~)
AcO "OBz-
OBz I
OxyCCl3 NH
17
AcO..~Ac
COOMe
OAc
A c ° " - ~ - Z " o " AZ.,...F-~ 'o~ n AcHN .j~,.~)~,,~ AcO
AC(~ '~OBz~
¢OB~)
~'X'~'~-.~ ~', s~ ACU-~w..~ -
OBz
.OR 2
Rt
ISR=Ac 19R=Na
N3 ROvAy~CI3H27
OBz 13R=H 20R =Ts
R30~.OR 3
.COOR4
OR 3
21Rl=N3.R2=Bz, R3=Ac,R4=Me 22Rl=NH2, R2=Bz,R3=Ac,R4=M¢ 23 R 1 = NHCO(CH2)I6Me, R 2 = Bz, R 3 = Ac, R 4 = Me H R 1 = NHCO(CH2)I6Me, R 2 = R 3 = R 4 = H
Ts = p-tolylsulfonyl
SCHEME2. Synthesis of ganglioside analogs containingthioglycosicallylinked ceramide. Synthesis of S-(a-Sialyl)-(2--->6)-/3-hexopyranosyl and S-(a-Sialyl)-(2-->6' )fl-lactosyl Ceramides Containing/3-Thioglycosidically Linked Ceramides (I-K) In this section, the synthesis of S-(a-sialyl)-(2--->6')-/3-1actosyl 1-thioceramide (K) is described as an example of the synthesis of the title compounds (I-K), all of which are synthesized in the same manner. 25 For the synthesis of the target thio analog ofganglioside, we employ the sodium salt of the per-O-acetylated S-a-sialyl-(2-->6' )-l,6'-dithio-fl-D-lactose (26) as the glycosyl donor, for coupling with (2S,3R,4E)-2-azido-3-O-benzoyl25A. Hasegaw, H. Ogawa, and M. Kiso, J. Carbohydr. Chem. 10, 1009 (1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
193
1-O-(p-tolylsulfonyl)-4-O-octadecene-l,3-diol (20). Treatment of S(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-
g alac to- 2-nonulop yranos ylonate )-(2~6)-O-( 2,3 ,4-tri- O-acetyl-6-thio-[3-Dgalactopyranosyl)-(1---~4)-2,3,6-tri-O-acetyl-D-glucopyranose (11) with methane sulfonyl chloride in dichloromethane in the presence of 2,4,6collidine for 1 hr at -15 ° gives the 1-chloro derivative 24, which is converted to the desired [3-thioacetate 25 in 53% yield. Coupling of the sodium salt 26, freshly derived from 25 by selective S-deacetylation with sodium methoxide at - 3 0 °, with the tosylate of azidosphingosine 211in DMF under a nitrogen atmosphere overnight at 45 ° yields the expected [3-glycoside 27 (38%) after column chromatography. Compound 27 is converted to the final compound K by the set reaction sequence, that is, selective reduction of the azide group, condensation with octadecanoic acid, and final deprotection, as described for the preparation of D.
Detailed Procedures S-(Methyl 5-Acetamido-4, 7,8, 9-tetra-O-acetyl-3,5-dideoxy-D-glyceroot-D-galacto-2-nonulopyranosylonate )-(2--~6). 0-(2,3,4-tri-O-acetyl-6-thio[3-D-galactopyranosyl)-(1---~4 )-2,3 ,6.tri-O-acetyl- l-S-acetyl- l-thio-[3- Dglucopyranose (25). To a solution of 11 (500 mg, 0.46 mmol) in dichloromethane (6 ml) is added 2,4,6-collidine (0.6 ml), and the mixture is cooled to -15 ° (Scheme 3). Methane sulfonyl chloride (0.3 ml, 3.88 mmol) is added to the mixture, and the mixture is stirred for 20 min at -15 °, then for 1 hr at room temperature; progress of the reaction is monitored by TLC. Dichloromethane (60 ml) is added, and the solution is successively washed with 2 M hydrochloric acid and water, dried (Na2SD4), and concentrated to the crude 24, which is used for the next reaction without further purification. To a solution of 24 in acetone (10 ml) is added Drierite (2 g), and the mixture is stirred for 2 hr at room temperature, after which potassium thioacetate (315 mg, 2.76 mmol) is added. The mixture is stirred overnight at 45 °, then filtered, and the precipitate is washed with dichloromethane. The filtrate and washings are combined and concentrated to a syrup. The residue is chromatographed on a column of silica gel with 120:1 (v/v) dichloromethane-methanol, to give 25 (279 mg, 53%) as an amorphous mass, [a]D +0.26 ° (C 0.75, chloroform).
S-(Methyl 5-Acetamido-4, 7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroOt-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thio[3-D-galactopyranosyl)-(1--~4)-S-(2,3,6-tri-O.acetyl-[3-o-glucopyranosyl)(1--~l )-(2R,3R,4E)-2-azido-3-benzoyloxy-4-octadecene-l-thiol (27). To a stirred solution of 25 (250 rag, 0.22 mmol) in dry methanol (2 ml), cooled to - 3 0 °, is added a solution of sodium metal (15 mg) in dry methanol (0.2
194
NEOGLYCOLIPIDS
A A c A ~ ~
O ~ ' ~ O
[ 17]
AcO UAC
R2 OAc
11 RI,R2 = H, OH 2 4 R 1 = CI, R2 = H 2 5 R 1 = H, R2 = SAc 26 Rl = H, R2 = SNa
N3 TsO.,./~CI3H27 OBz 20
R30 ~OR 3 R3
COOR'S OR s
2
,o
27 RI = N3, R2 ffi Bz, R3 = AC, R4 = Me 28 R 1 = NH2, R 2 = Bz, R 3 = Ac, R 4 = Me 29 R 1 = NHCO(CH2)I6Me, R 2 = Bz, R 3 = Ac, R 4 = Me K R 1 = NHCO(CH2)I6Me, R 2 = R 3 = R 4 = H
SCHEME 3. Synthesis of ganglioside analogs containing both the a-thioglycoside of sialic acid and thioglycosidicallylinked ceramide. ml). Stirring is continued for 5 min at - 3 0 °, and the mixture is concentrated to give 26 as an amorphous mass, which is used for the next step without purification. A solution of 26 and 20 (410 mg, 0.7 mmol) in dry DMF (4.5 ml) is stirred overnight at 45 ° under nitrogen. Acetic anhydride (2 ml) and pyridine (4 ml) are added to the mixture, which is stirred overnight at room temperature, then concentrated. The residue is chromatographed on a column of silica gel (50 g) with 100 : 1 (v/v) dichloromethane-methanol, to give 27 (124 mg, 38%) as an amorphous mass, [a] D - 6 . 4 ° (c 0.95, chloroform). Synthesis of S-(a-Sialosyl)-(2--->9)-O-(a-sialosyl)-(2--~3' )-fl-lactosyl Ceramide (g) For the synthesis of the target ganglioside analog, 26 we set out to synthesize the per-O-acylated 2-(trimethylsilyl)ethyl S-(methyl a-N-ace26A. Hasegawa, H. Ogawa, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 10, 1009(1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
195
tylneuraminyl)-(2~9)-O-(methyl o~-N-acetyl-9-thioneuraminyl)-(2~3' )-/3lactoside (39) as the intermediate. Compound 39 could then, by introduction of the ceramide moiety, be transformed to the end product. Treatment of methyl (methyl 5-acetamido-3,5-dideoxy-2-thio-o-glycero-~-o-galacto-2-nonulopyranosid)onate (30) with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid monohydrate gives the 8,9O-isopropylidene derivative 31 in 70% yield, which is acetylated with acetic anhydride in pyridine to give 32 (Scheme 4). Removal of the isopropylidene group from 32 with 80% aqueous acetic acid for 3.5 hr at 45° gives compound 33 in 89% yield, without acetyl migration. In the NMR spectrum, H-7 appears as a doublet of doublets at 8 5.08 (J6,7 = 2.0, J7.8 = 9.3 Hz). Selective bromination 27of 33 with carbon tetrabromide-triphenylphosphine in pyridine gives methyl (methyl 5-acetamido-4,7-di-O-acetyl-9-bromo-3,5,9-trideoxy-2-thio-D-glycero-o~-o-galacto-2-nonulopyranosid)onate (34) in 72% yield, which is converted to the 4,7,8-tri-O-acetyl derivative 35 quantitatively. The glycosylation15'28of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-fl-o-galactopyranosyl)-(l---~4)-2,6-di-O-benzoyl-fl-o-glucopyranoside15 (36) with 35 in acetonitrile for 20 hr at - 15° in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) as the glycosyl promoter gives the expected o~-glycoside 37 of the sialic acid at 0-3' in compound 36, in 37% yield. Acetylation of 37 with acetic anhydride in pyridine gives the acetate 38 in 93% yield. Condensation of the sodium salt 3 with compound 38 in DMF under nitrogen affords the desired tetrasaccharide 39 in 80% yield. The intermediate 39 is transformed into the final product I, by the procedures described for the conversion of 10 to D.
Detailed Procedures Methyl (Methyl 5-Acetamido-3,5-dideoxy-8,9-O-isopropylidene-2-thioo-glycero-o~-o-galacto-2-nonulopyranosid)onate (31). To a solution of 30 (500 mg, 1.4 mmol) in dry DMF (5 ml) are added 2,2-dimethoxypropane (0.87 ml), p-toluenesulfonic acid monohydrate (15 mg), and Drierite (1.0 g), and the mixture is stirred for 1 hr at 0°, then neutralized with solid sodium bicarbonate (Scheme 4). The precipitate is filtered off and washed with methanol. The filtrate and washings are combined, then concentrated. The residue is chromatographed on a column of silica gel (50 g) with 40 : 1 (v/v) dichloromethane-methanol, giving compound 31 (390 mg, 70%) as an amorphous mass; [a] D + 12.6° (c 0.58, chloroform). 27 A. Kashem, M. Anisuzzaman, and R. L. Whistler, Carbohydr. Res. 61, 511 (1978). 28 T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 188, 71 (1989).
196
NEOGLYCOLIPIDS
H?_.pOH COOMe HO~,.L-~ O . ~ A c a l q ~ SMe
~f~.~ COOMe RO~"L7"~O " ~ A c H N ~ -SMe
30
31R=H 32 R = Ac
0 RI COOMe AcOT..~..o.Tt.. AcH~I~ SMe
__ O14
Br .OAc
] "~OBz HO
COOMe
OR
"~..~b 0 E
RO
AC~L_~C
COOMe
A c H N ~
~rca
OBz
37R=H 38 R = Ac
AcO .OAc
OSE
OBz 36
0
AcO
OBz /--O
HO o'o'o'o'o'o'o'o'o'o
33R1 =H, R2=OH 34R1 = H, R2 = Br 35 R 1 = AC, R2 = Br
AcO"
[ 17]
3
COOMe
AcO"" P"'L~ O" ~ A c H N ~
S [ pOAc
COOMe
ACU
OAc
_
¢OBz~
AcO
R2
39 R 1 = OSE, R2 = H 40 RI, R2 ffi H, OH 41 R 1 = H, R2 = OC(=NH)CCI3 R30 A
R
.OR 3 ¢
~
COOR 4 ~
S ~'OR3
R-~O
COOR4
R30
OR'
-OK"
OR2
-1
v
OR"
42RI=N3,R2=Bz,R3=Ac,R4=M¢ 43 R 1 = Nit2, R2 = Bz, R3 = AC, R4 ffiMe 44 R 1 = NHCO(CH2)I6Me, R2 = Bz, R3 = Ac, R4 = Me L R 1 = NHCO(CH2)I6Me, R2 = R3 ffiR4 = H
SCHEME4. Synthesis of ganglioside GD3analogs containingthe a-thioglycoside of sialic acid.
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-3,5-dideoxy-8,9-O-isopropylidene-2-thio-D-glycero-c~-D-galacto-2-nonulopyranosid)onate (32). Compound 31 (390 mg, 0.99 mmol) is acetylated with acetic anhydride (2 ml) in pyridine (4 ml) overnight at room temperature. The product is
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
197
purified by chromatography on silica gel (60 g) with 60 : 1 (v/v) dichloromethane-methanol, to give 32 (470 mg, quantitative) as an amorphous mass, [ot]D + 14.6° (c 0.8, chloroform).
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-3,5-dideoxy-2-thio-Dglycero-a-o-galacto-2-nonulopyranosid)onate (33). A solution of 32 (2.12 g, 4.44 mmol) in 80% aqueous acetic acid (20 ml) is heated for 3.5 hr at 45°, then concentrated. The residue is chromatographed on a column of silica gel (100 g) with 80:1 (v/v) dichloromethane-methanol to give 33 (1.73 g, 89%) as an amorphous mass, [a]D +33.8 ° (c 0.7, chloroform).
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-9-bromo-3,5,9-trideoxy2-thio-D-glycero-a-D-galacto-2-nonulopyranosid)onate (34). To a solution of 33 (2.0 g, 4.57 mmol) in pyridine (40 ml) is added carbon tetrabromide (3.03 g, 9.14 mmol), and the solution is cooled to 0°. Triphenylphosphine (2.4 g, 9.15 mmol) is added, and the mixture is stirred for 5 hr at room temperature. Methanol (1 ml) is added to the mixture, which is concentrated to a syrup and then chromatographed on a column of silica gel (100 g) with 2 : 1 (v/v) ethyl acetate-hexane, giving 34 (1.65, 72%) as an amorphous mass, [a]D +38.5 ° (C 0.94, chloroform).
Methyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-bromo-3,5,9-trideoxy-D-glycero-a-o-galacto-2-nonulopyranosid)onate (35). Compound 34 (1.35 g, 2.7 mmol) is acetylated with acetic anhydride (5 ml) in pyridine (10 ml) overnight at room temperature. The product is purified by column chromatography on silica gel (60 g) with 2 : 1 (v/v) ethyl acetate-hexane, to give 35 (1.45 g, quantitative) as an amorphous mass, [a]D +28.0 ° (c 0.88, chloroform).
2-(Trimethylsilyl)ethyl O-(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9bromo-3,5 ,9-trideoxy-D-glycero-o~- D-galacto-2-nonulopyranosylonate )(2--*3)-O-(6-O-benzoyl-fl-D-galactopyranosyl)-( l---~4)-2,6-di-O-benzoyl-flD-glucopyranoside (37). To a solution of 3615'29 (800 rag, 1.06 mmol) and 35 (1.15 g, 2.12 mmol) in dry acetonitrile (10 ml) is added MS-3,~ (2.5 g). The mixture is stirred for 10 hr at room temperature and then cooled to -25 °. To the cooled mixture is added, with stirring, a mixture (2.94 g; 74.6% DMTST by weight) of D M T S T 7'19 and MS-3,~, and the stirring is continued for 20 hr at -15 °. The precipitate is filtered off, then washed thoroughly with dichloromethane. The filtrate and washings are combined, and the solution is successively washed with 1 M sodium carbonate and water, dried (Na2SO4), and evaporated to a syrup that is chromatographed on a column of silica gel (80 g) with 3 : 1 (v/v) ethyl acetate-hexane, to give 37 (490 mg, 37%) as an amorphous mass, [aid +9.1 °, (C 0.78, chloroform).
2-(Trirnethylsilyl)ethyl O-(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-929 p. Fiigedi and P. J. Garegg, Carbohydr. Res. 149, c9 (1986).
198
NEOGLYCOLIPIDS
[ 18]
bromo-3,5,9-trideoxy-o-glycero-a-o-galacto-2-nonulopyranosylonate)(2---~3)-O-(2,4-di-O-acetyl-6-O-benzoyl)-fl-o-galactopyranosyl)-(1---~4)-3O-acetyl-2,6-di-O-benzoyl-fl-o-glucopyranoside (38). Compound 37 (540 rag, 0.43 mmol) is acetylated with acetic anhydride (3 ml) in pyridine (6 ml) overnight at room temperature. The product is purified by chromatography on silica gel (50 g) with 2 : 1 (v/v) ethyl acetate-hexane, giving 38 (550 rag, 93%) as an amorphous mass, [a]D +5.0 ° (C 0.6, chloroform).
2-(Trimethylsilyl)ethyl S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl3,5-dideoxy- D-glycero-a- D-galacto-2-nonulopyranosylonate )-(2--.9)-O(methyl 5-acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-9-thio-D-glycero-a-Dgalacto-2-nonulopyranosylonate)-(2--*3)-O-(2,4-di-O-acetyl-6-O-benzoylfl- D-galactopyranosyl)- (1--.4)-3-O-acetyl-2,4-di- O-benzoyl-fl- o-glucopyranoside (39). A solution of 38( (400 rag, 0.29 mmol) and the sodium salt 3 (308 mg, 0.58 mmol) in DMF (5 ml) is stirred overnight at room temperature under a nitrogen atmosphere, then concentrated. The residue is chromatographed on a column of silica gel (60 g) with ethyl acetate to give 39 (421 mg, 80%) as an amorphous mass, [O~]D +9.0 ° (c 0.58, chloroform).
[18] R e p l a c e m e n t o f G l y c o s p h i n g o l i p i d C e r a m i d e R e s i d u e s b y G l y c e r o l i p i d for M i c r o t i t e r P l a t e A s s a y s
By RENI~ ROY, ANNA ROMANOWSKA, and FREDRIK O. ANDERSSON Introduction Glycosphingolipids and gangliosides in particular constitute well-defined antigenic carbohydrate structures which have been implicated in a wide range of biological and immunological activities. When monoclonai antibodies are used for determining the fine chemical specificities involved in protein-carbohydrate interactions, solid-phase enzyme-linked immunosorbent assays (ELISA) are frequently used as a standard technique.l,2 The prime event in the ELISA is a nonspecific lipophilic adsorption of coating antigens on the surface on the plastic wells, which are generally made of either polystyrene or polyvinyl chloride (PVC). Although the exact structures of the ceramide portions have been suggested as being involved in monoclonal antibody-ganglioside (GM3 , 2 in Scheme 1) interactions, 3 the structure of the lipid component is not critically important for I p. F r e d m a n , S. J e a n s s o n , E. L y c k e , and L. Svennerholm, FEBS Lett. 189, 23 (1985). 2 j. Miyoshi, Y. Fujii, and M. Naiki, J. Biochem. (Tokyo) 92, 89 (1982). 3 S. Itonori, K. Hidari, Y. Sanai, M. Tanigushi, and Y. Nagai, Glycoconjugate J. 6, 551 (1989).
METHODS IN ENZYMOLOGY. VOL. 242
Copyright © 1994by AcademicPress, Inc. All fights of reproduction in any form reserved.
GANGLIOSIDE
ANALOGS
CONTAINING
SULFUR
183
Chemoenzymatic Synthesis of Ganglioside G~3 The combined chemical enzymatic approach has been applied to the synthesis of natural ganglioside Gm .49,5o The key synthetic step is the regio- and a-stereoselective glycosylation of sialic acid to Y-OH of the lactose moiety by use of CMP-Neu5Ac and a-2,3-sialyltransferase. However, this method is usually restricted to the synthesis of natural gangliosides, and in addition both the sugar nucleotide and glycosyltransferase are still not readily available. 49 y . lto and J. C. Paulson, J. Am. Chem. Soc. 115, 1603 (1993). t0 K. K.-C. Liu and S. J. Danishefsky, J. Am. Chem. Soc. 115, 4933 (1993).
[ 17] S y n t h e s i s of G a n g l i o s i d e A n a l o g s C o n t a i n i n g S u l f u r in P l a c e of O x y g e n at t h e L i n k a g e Positions
By HIDEHARU ISHIDh, MhgOTO KISO, and AKIRA HASEGAWA Introduction
Chemical synthesis of naturally occurring gangliosides as well as a variety of ganglioside analogs is becoming stimulating and rewarding as more and more biological functions 1-6 of sialoglycoconjugates are reported. Among these analogs, thioglycosides are of interest as inhibitors of the corresponding glycosides (mainly via competitive inhibition). 7.8 For instance, ganglioside analogs containing the a-thioglycoside of sialic acid are expected to affect the activity of sialidases, and analogs containing thioglycosidically linked ceramide could be an inhibitor of ceramide glycanase 9 In this chapter we describe the established methods for the synthesis I G. Walz, A. Aruffo, W. Kolanus, M. Bevilacqua, and B. Seed, Science 250, 1132 (1990). 2 M. L. Phillips, E. Nudelman, F. C. A. Graeta, M. Perez, A. K. Singhal, S. Hakomori, and J. C. Paulson, Science 250, 1130 (1990). 3 j. B. Lowe, L. M. Stoolman, R. P. Nair, R. D. Larsen, T. L. Berhend, and R. M. Marks, Cell (Cambridge, Mass.) 63, 475 (1990). 4 M. J. Polley, M. L. Phillips, E. Wayner, E. Nudelman, A. K. Singhal, S. Hakomori, and J. C. Paulson, Proc. Natl. Acad. Sci. U.S.A. 88, 6224 (1991). 5 H. Nojiri, M. Stroud, and S. Hakomori, J. Biol. Chem. 266, 4531 (1991). 6 I. Eggens, B. Fenderson, T. Toyokuni, B. Dean, M. Stroud, and S. Hakomori, J. Biol. Chem. 264, 9476, (1989). 7 D. Horton and J. D. Wander, in "The Carbohydrates, Chemistry/Biochemistry" (W. Pigrnan and D. Horton, eds.), 2nd Ed., Vol. IB, p. 803. Academic Press, New York, 1980. 8 p. j. Deschavanne, O. M. Viratelle, and J. M. Yon, J. Biol. Chem. 253, 833 (1978). 9 M. Ito and T. Yamagata, this series, Vol. 179, p. 488.
METHODS IN ENZYMOLOGY, VOL. 242
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
NEOGLYCOLIPIDS
[17]
of ganglioside analogs containing thioglycosidic linkages in the following positions: (1) between the sialic acid and hexopyranoside (compounds A-D); (2) between the oligosaccharide chain and ceramide (compounds E-H); (3) between both the sialic acid-hexopyranoside and oligosaccharide chain-ceramide (compounds I-K); and (4) between sialic acid and sialic acid (compound L).
Synthesis of S-(a-Sialyl)-(2--~6)-fl-D-hexopyranosyl and S-(a-Sialyl)(2---)6')-fl-D-lactosyl Ceramides (A-D) In this section, the synthesis of S-(o~-sialyl)-(2--~6)-/3-D-lactosyl ceramide (D), shown in Fig. 1, is described as an example of the synthesis of the title compounds. Other compounds (A-C) are synthesized essentially by the same method described for D. ~°,~1 For the synthesis of D, we employ the sodium salt of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2nonulopyranosonate (3) 52 as glycosyl donor and per-O-acetylated 2-(trimethylsilyl)ethyl 6'-bromo-6'-deoxy-fl-D-lactoside (9) l° as glycosyl acceptor, respectively. The intermediates are coupled and converted, by introduction of ceramide moiety, to the end product. Treatment of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro2,3,5-trideoxy-o-glycero-fl-D-galacto-2-nonulopyranosonate (1) 13 with potassium thioacetate in dry dichloromethane gives a-2-S-acetyl derivative 2 in 90% yield. Selective S-deacetylation of 2 with the amount of sodium methoxide equivalent to 2 in dry methanol at -40 ° gives the sodium salt 3, which is used for the next reaction without further purification. Treatment of 2-(trimethylsilyl)ethyl/3-D-lactoside 414 with 2-methoxypropene in N,N-dimethylformamide (DMF) in the presence of p-toluenesulfonic acid monohydrate and subsequent acetylation give 6 in 93% yield. The de-O-isopropylidenation of 6 by heating with 80% aqueous acetic acid for 10 hr at 45° gives crystalline 7 in 91% yield. Selective C-6' bromination of 7 with N-bromosuccinimide in the presence of triphenylphosphine and subsequent acetylation afford compound 9 as crystals in good yield. Condensation of 3 with 9 in DMF under a nitrogen atmosphere leads to the i0 A. Hasegawa, M. Morita, Y. Ito, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 369 (1990). II A. Hasegawa, T. Terada, H. Ogawa, and M. Kiso, J. Carbohydr. Chem. 11, 319 (1992). 12A. Hasegawa, J. Nakamura, and M. Kiso, J. Carbohydr. Chem. 5, 11 (1986). t3 R. Kuhn, P. Lutz, and D. L. MacDonald, Chem. Ber. 99, 611 (1966). J4 K. P. R. Kartha, A. Kameyama, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 8, 145 (1989).
[17]
GANGLIOS1DE ANALOGS CONTAINING SULFUR HOL..pOH
COOH
~(CH~)I6CH3
H&~"-O-~',. R~
AcHN~ ' ~ HO
185
O~
n2" S ~ ' ~ 0 Nil R ~ O ~ R 10VAN~C
13H2.~
OH
A RI = OH, R2 = H, R3 = OH (Gal) OH, R2 = OH, R3 = H (GIc) C R1= NHAc,R2 = OH, R3 ffiH (GIcNAc) B R1=
A : ~ ~ 3 0 ! ~ ~ ) O
O~~HO D
R2 ~oa
o - ( (ca2h+cas
RI.~...O
-
.OH
OH
/(CH2)I6CH3
OH
OH
HO
HO .OH
S
C H
13 27
OH
COOH
~(CH2)tsCH 3
o~
OH (Gal) J R1= OH, R2 = H (Glc) 1 R l = H, R2 =
OH
COOH
.o~o-~.~..d OH
COOH . . ~
~
~
'+"'
.o,+~--o-~,7, a~
H~.~H
O-(,
O
O
HO
HO"~-~'~ 0 ~ A c H N ~
NH
G
O
HO .OH
°=((ca2)l~cns Q
HO
t:Jou
HO"'
oH
HO. ~ E ~ O " ~ . ~
....
#
AcH
O--~(CH2)i+CH 3
oH
NH
OH (~H ER1 = H, R2= OH (Gal) F Rt = OH, R2 = H (GIc)
HO
ON H ~ C I 3 H ~
"'OH -
K . #un
COOH
.
"f
TM
NH
O ~x(CH2)t6CH3 . r~H
(CHz)I6CH3 au
O~
L
Fro. 1. Ganglioside analogs containing thioglycosides.
186
NEOGLYCOLIPIDS
[ 17]
corresponding a-thioglycoside 10 in 87% yield. Selective removal 15,16 of the 2-(trimethylsilyl)ethyl group in 10 is performed by treatment of 10 with boron trifluoride etherate in dichloromethane for 2 hr at - 2 0 °, to give 11 in 84% yield. When treated with trichloroacetonitrile 15,~7,18in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) for 2 hr at 0°, compound 11 gives the trichloroacetimidate 12 as the a-anomer in 92% yield, after column chromatography. The glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene1,3-diol (13) 19'2° with 12 in the presence of boron trifluoride etherate for 6 hr at - 2 0 ° yields only the expected/3-glycoside 14 in 82% yield. Selective reduction ~5'2~of the azide group in 14 with hydrogen sulfide in 5 : 1 (v/v) pyridine-water gives the amine 15, which on condensation with octadecanoic acid using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DAC) in dichloromethane gives the S-(c~-N-acetylneuraminyl)(2-~6')-O-(6'-thio-fl-D-lactosyl)-(1--~l)-ceramide derivative 16 in 94% yield. Finally, de-O-acetylation of 16 with sodium methoxide in methanol, followed by saponification of the methyl ester group, yields almost quantitatively the end product D. Compounds A, B, and D show potent inhibition against sialidases from several kinds of influenza virus, acting as competitive inhibitors. The order of decreasing inhibition is D > A > B . 22
Detailed Procedures Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2thio-D-glycero-c~-o-galacto-2-nonulopyranosonate (2). To a solution of 112 (3.3 g) in dry dichloromethane (30 ml) is added potassium thioacetate (3.5 g) (Scheme 1). The mixture is stirred overnight at room temperature and concentrated, the residue is extracted with chloroform, and the extract is washed with water, dried (Na2SO4) , and evaporated to a syrup. The product is chromatographed on a column of silica gel (200 g) with 100 : 1 (v/v) chloroform-methanol, to give 3.2 g (90%) of compound 2 as an amorphous mass; mp 74o-76 °, [a] D -15.6 ° (c 0.75, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-4,6-O-isopropylidene-fl-Dgalactopyranosyl)-(1--~4)-2,3,6-tri-O-acetyl-[3-D-glucopyranoside (6). To a 15T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 188, 71 (1989). 16 K. Jansson, T. Frejd, J. Kihlberg, and G. Magnussun, Tetrahedron Lett. 27, 753 (1986). 17 M. Numata, M. Sugimoto, K. Koike, and T. Ogawa, Carbohydr. Res. 163, 209 (1987). 18R. R. Schmidt and J. Michel, Angew. Chem., Int. Ed. Engl. 19, 731 (1980). 19 M. Kiso, A. Nakamura, Y. Tomita, and A. Hasegawa, Carbohydr. Res. 158, 101 (1986). 20 R. R. Schmidt and P. Ziminermann, Angew. Chem., Int. Ed. Engl. 25, 726 (1986). Zl T. Adachi, Y. Yamada, I. Inoue, and M. Saneyoshi, Synthesis, 45 (1977). 22 y. Suzuki, K. Sato, M. Kiso, and A. Hasegawa, Glycoconjugate J. 7, 349 (1990).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR AcO .OAc
AcO
CI
OAc
187
COOMe
A c O ~ O ~ o ~ A c H N ~ at~
AcO" 0 A c H ~ C O O M e AcO 1
2R=Ac 3R=Na OR l
/ ORL
s,o o - os JR2~ R 3
OR
4RI=H,R2=R3=OH 5 R 1 = H, R2,R3 = OCMe20 6 R 1 = Ac, R2,R 3 = OCMe20 7 R I = Ac, R2 = R3 = OH 8 R I = Ac, R2 = OH, R3 = Br 9 R 1 = Ac, R 2 = O A c , R 3 = Br
10 R 1 = OSE, R 2 = H 11 R1,R2 = H, OH 12 R 1 = H, R2 = OC(=NH)CCI 3
N3 HON/A~CI3Hz7 OBz
13 R 3 0 .OR 3 R30"t~ R~O
COOR 4 O ""~
OR~3
~3
R3O - ~ . . ~ . ~
~ t~ ~ ~!P'm~ t'~q
" ~-~ - - - - < ' O R ~"
OR2 _ ~
l
v
t 131rl127
RI
14 R I = N 3, R 2 = Bz, R 3 = Ac, R4 = Me 15 R 1 = NH 2, R 2 = Bz, R 3 = Ac, R 4 = Me
16 R l = NHCO(CH2)I6Me, R2 = Bz, R 3 = Ac, R 4 = Me D R 1 = NHCO(CH2)16Me, R 2 = R3 = R4 = H SE = 2-(trimethylsilyl)ethyl, Bz = benzoyl
SCHEME 1. Synthesis of ganglioside analogs containing the c~-thioglycoside of sialic acid.
solution of 41° (1.5 g, 3.39 mmol) in DMF (15 ml), cooled to 0 °, are added, with stirring, 2-methoxypropene (0.6 ml) and p-toluenesulfonic acid monohydrate (30 mg), and the mixture is stirred for 2 hr at 0 °, the progress of the reaction being monitored by thin-layer chromatography (TLC). Acetic anhydride (7 ml) and pyridine (10 ml) are added to the mixture, and this is stirred for 5 hr at room temperature and concentrated to a syrup, which is chromatographed on a column of silica gel (150 g)
188
NEOGLYCOLIPIDS
[ 17]
with 1 : 4 (v/v) ethyl acetate-hexane to give 6 (2.14 g, 93%) as an amorphous mass, [a]D + 14.0 ° (c 1.0, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-fl-o-galactopyranosyl)(1-~4)-2,3,6-tri-O-acetyl-fl-D-glucopyranoside (7). A solution of 6 (2.34 g, 3.38 mmol) in 80% aqueous acetic acid (30 ml) is kept for 10 hr at 45 °, then concentrated to a syrup, which is chromatographed on a column of silica gel (200 g) with 1 : 1 (v/v) ethyl acetate-hexane to give 7 (2.0 g, 91%) as crystals. Recrystallization from ether-hexane gives needles: mp 197 °, [a] D -8.1 ° (c 0.6, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3-Di-O-acetyl-6-bromo-6-deoxy-fl-o-galactopyranosyl)-(1---~4)-2,3,6-tri-O-acetyl-fl-o-glucopyranoside (8). To a solution of 7 (500 mg, 0.77 mmol) in DMF (10 ml), cooled to 0°, are added, with stirring, Nobromosuccinimide (267 mg, 1.5 mmol) and triphenylphosphine (262 mg, 1 mmol), and the mixture is stirred for 2 days at 0°. Methanol (1 ml) is added to the mixture, and this is stirred for 10 min, then concentrated. The residue is chromatographed on a column of silica gel (60 g) with 2 : 1 (v/v) ethyl acetate-hexane to give 8 (380 mg, 69%) as an amorphous mass, [a]D --2.8 ° (C 0.86, chloroform).
2-(Trimethylsilyl)ethyl O-(2,3,4- Tri-O-acetyl-6-bromo-6-deoxy-fl-D-galactopyranosyl)-(l--~4)-2,3,6-tri-O-acetyl-fl-D-glucopyranoside (9). Acetylation of 8 (329 mg, 0.46 mmol) with acetic anhydride (l ml) in pyridine (2 ml) overnight at room temperature gives 9 (345 mg, quantitative) as crystals. Recrystallization from ether-hexane gives needles: mp 208°-209 °, [o~]D - 1 1 . 9 ° (c 1.0, chloroform).
2-(Trimethylsilyl)ethyl S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl3,5-dideoxy-o-glycero-a-o-galacto-2-nonulopyranosylonate)-(2-->6)-O(2,3,4-tri-O-acetyl-6-thio-fl-D-galactopyranosyl)-(1--~4)-2,3,6-tri-O-acetylfl-o-glucopyranoside (lO). A solution of 9 (280 mg, 0.37 mmol) and 3 (400 mg, 0.76 mmol) in dry DMF (3 ml) is stirred overnight at room temperature under a nitrogen atmosphere, the course of the reaction being monitored by TLC. Acetic anhydride (2 ml) and pyridine (4 ml) are added to the solution, and the mixture is stirred overnight at room temperature. Dichloromethane (200 ml) is added, and the solution is successively waslled with 1 M sodium carbonate, 2 M hydrochloric acid, and water, dried (Na2SO4), and concentrated. The residue is chromatographed on a column of silica gel (100 g) with 1 : 1 (v/v) ethyl acetate-hexane to give 10 (380 mg, 86.5.%) as an amorphous mass, [a] D + 1.5 ° (c 0.67, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1--~4)-2,3,6-tri-O-acetyl-D-glucopyranose (11). To a solution of 10 (904 mg, 0.76 mmol) in dry dichloromethane (10 ml), cooled to - 2 0 °, is added boron trifluoride etherate (1.2 ml), and the mixture
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
189
is stirred for 2 hr at - 2 0 °, the progress of the reaction is monitored by TLC. Dichloromethane (100 ml) is added to the mixture, and the solution is successively washed with 1 M sodium carbonate and water, dried (NazSO4), and concentrated to a syrup, which is chromatographed on a column of silica gel (250 g) with 100 : 1 (v/v) dichloromethane-methanol to give 11 (695 mg, 84%) as an amorphous mass, [odd +2.1 ° (c 1.9, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroO~-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(l--->4)-2,3,6-tri-O-acetyl-D-glucopyranosyl Trichloroacetimidate (12). To a stirred solution of 11 (403 mg, 0.37 mmol) in dry dichloromethane (5 ml), cooled to 0°, are added trichloroacetonitrile (0.075 ml) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.03 ml). The mixture is stirred for 2 hr at 0 ° and then concentrated. The residue is chromatographed on a column of silica gel (20 g) with 120 : 1 (v/v) dichloromethane-methanol, to give 12 (420 mg, 92%) as an amorphous mass, [a]D +52.4 ° (c 0.9, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2--*6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1--~4)-O-(2,3,6-tri-O-acetyl-fl-D-glucopyranosyl)(1--H)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-l,3-diol (14). To a solution of 12 (453 mg, 0.37 mmol) and 13 (320 mg, 0.74 mmol) in dry dichloromethane (15 ml) is added Molecular Sieves 4A powder (MS-4A; 600 mg), and the mixture is stirred for 1 hr at room temperature, then cooled to - 2 0 °. Boron trifluoride etherate (0.06 ml) is added to the cooled mixture, and this is stirred for 6 hr at - 2 0 °, the progress of the reaction being monitored by TLC. The precipitate is filtered off and washed with chloroform. The filtrate and washings are combined, and the solution is successively washed with 1 M sodium carbonate and water, dried (Na2SO4), and concentrated. The residue is chromatographed on a column of silica gel (60 g) with 120 : 1 (v/v) dichloromethane-methanol to give 14 (452 mg, 82%) as an amorphous mass, [a]D +0.9 ° (c 0.56, chloroform).
S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thiofl-D-galactopyranosyl)-(1---~4)-O-(2,3,6-tri-O-acetyl-fl.D-glucopyranosyl)(l--* l )-(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-octadecene-1,3-diol (16). Hydrogen sulfide is bubbled through a solution of 14 (I00 mg, 0.069 mmol) in pyridine (2.5 ml) and water (0.5 ml) for 2 days while the solution is stirred at room temperature. The mixture is concentrated to give the syrupy amine 15, which is used for the next reaction without further purification. To a solution of 15 in dry dichloromethane (2.5 ml) are added octadecanoic acid (40 mg, 0.14 mmol) and DAC (35 rag, 0.19 mmol), and
190
NEOGLYCOLIPIDS
[17]
the mixture is stirred overnight at room temperature. After completion of the reaction, dichloromethane (15 ml) is added to the mixture, and the solution is washed with water, dried (Na2SO4) , and concentrated to a syrup that is chromatographed on a column of silica gel (5 g) with 90 : 1 (v/v) dichloromethane-methanol, to give 16 (106 rag, 94%) as an amorphous mass, [a]D + 8 . 0 ° (C 0.4, chloroform).
S-(5-Acetamido-3 ,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2-->6)-O-(6-thio-fl-D-galactopyranosyl)-(1-->4)-O-(fl-D-glucopyranosyl)-(1--> l )-(2S,3R,4E)-2- octadecanamido-4- octadecene-l ,3-diol (19). To a solution of 16 (121 rag, 0.072 mmol) in dry methanol (5 ml) is added sodium methoxide (25 mg), and the mixture is stirred overnight at room temperature. After completion of the reaction, water (0.2 ml) is added to the mixture at 0°, and this is stirred for 1 hr, then treated with Amberlite IR-120 (H-') resin to remove the base. The solution is concentrated, and the residue is chromatographed on a column of Sephadex LH20 (100 g) with 1 : l (v/v) chloroform-methanol to give compound D (79.6 mg, 97%) as an amorphous mass, [a] D +16.5 ° (c 1.5, chloroform-methanol). Synthesis of Cerebroside, Lactosyl Ceramide, and Ganglioside GM3 Analogs Containing fl-Thioglycosidically Linked Ceramide In this section, we describe the synthesis of a ganglioside GM3 analog carrying fl-thioglycosidically linked ceramide (H). Other thioglycolipids (E-G) were synthesized by the same procedure. 23 Compound H is prepared by the coupling of the sodium salt of O-(a-sialyl)-(2--->3')-fl-D-1thiolactose (19) and the tosylate derivative of azidosphingosine (20), and the coupled product is readily converted to the target compound H. Treatment of per-O-acetylated O-(a-sialyl)-(2--->3')-a-D-lactosyl trichloroacetimidate (17) 15 with thioacetic acid in the presence of boron trifluoride etherate gives the required fl-thioacetate derivative 18 in 91% yield. (2S,3R,4E)-2-Azido-3-O-benzoyl-l-O-(p-toylysulfonyl)-4-octadecene-1,3-diol (20) is derived byp-tolylsulfonylation of(2S, 3R, 4E)-2-azido3-O-benzoyl-4-octadecene-l,3-diol (13), 19,2° which is used as the glycosyl acceptor. Treatment of sodium salt 19, freshly prepared from 18 by selective S-deacetylation with sodium methoxide, with 20 in DMF under nitrogen affords the desired fl-thioglycoside (21) in 41% yield. The 1H nuclear magnetic resonance (NMR) data for 21 demonstrate it to be a fully blocked glycoside. The intermediate 21 is converted, by selective reduction of the 23A. Hasegawa, M. Morita, Y. Kojima, H. Ishida, and M. Kiso, Carbohydr, Res. 214, 43 (1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
191
azide group, condensation with octadecanoic acid, de-O-acylation, and saponification of the methyl ester group, to the end product H as described for the conversion of 14 to D. B~ir and Schmidt z4 synthesized fl-lactosyl 1-thioceramide (G) in an alternative manner, namely, by the coupling of lactosyl bromide with the sodium salt of the 1-thio derivative of azidosphingosine. Detailed Procedure O - ( M e t h y l 5 - A c e t a m i d o - 4 , 7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa- D- g alacto- 2- n o n u l o p y r a n o s y l o n a t e )-( 2-->3 ) - O - ( 2 , 4 - d i - O - a c etyl-6- Obe nzoyl- fl- D- g a l a c t o p y r a n o s y l ) - ( l--->4)- 3 - O - a c etyl- l - S - a c etyl- 2 , 6 - d i - O b e n z o y l - l - t h i o - f l - o - g l u c o p y r a n o s e (18). To a solution of 17 (100 mg, 71.5
/xmol) in dry dichloromethane (5 ml) are added thioacetic acid (10/xl) and boron trifluoride etherate (10/.d), and the mixture is stirred overnight at room temperature (Scheme 2). The mixture is successively washed with 1 M sodium carbonate and water, dried (Na2SO4) , and concentrated to a syrup that is chromatographed on a column of silica gel (50 g) with 90 : 1 (v/v) dichloromethane-methanol to give 18 (85 mg, 91%) as an amorphous mass, [a]D +0.5° (C 0.2, chloroform). O - ( M e t h y l 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroa-D-galacto-2-nonulopyranosylonate)-(2---~3)-O-(2,4-di-O-acetyl-6-O-benzoyl-fl- D-galactopyranosyl)-(1--~4 ) - S - ( 3 - O - a c e t y l - 2 , 6 - d i - O - b e n z o y l - f l - Dglucopyranosyl) - (1---~1) - (2R,3R,4E) - 2 - azido - 3 - b e n z o y l o x y - 4 - o c t a d e c ene-l-thiol (21). To a stirred solution of 18 (30 rag, 0.023 mmol) in dry
methanol (0.5 ml) and chloroform (0.1 ml), cooled to - 2 0 °, is added a solution of sodium metal (5.0 mg) in dry methanol (0.2 ml). Stirring is continued for 5 min at - 2 0 °, and the mixture is concentrated to give 19 as an amorphous mass, which is used for the next reaction without purification. A solution of 19 and 20 (26 mg, 0.045 mmol) in dry DMF (2 ml) is stirred for 24 hr at 40 ° under a nitrogen atmosphere, the course of the reaction being monitored by TLC. Acetic anhydride (0.5 ml) and pyridine (1 ml) are added to the mixture, which is stirred overnight at room temperature, then concentrated. The residue is dissolved in dichloromethane (20 ml), and the solution is successively washed with 2 M hydrochloric acid, 1 M sodium carbonate, and water, and then dried (Na2SO4) and evaporated to a syrup that is chromatographed on a column of silica gel (10 g) with 6 : 1 (v/v) hexane-ethyl acetate to afford 21 (15.5 mg, 41%) as an amorphous mass, [a]D -64.5 ° (c 0.73, chloroform).
24 T. B~ir and R. R. Schmidt, Liebigs Ann. Chem., 185 (1991).
192
NEOGLYCOLIPIDS AcO~OAc
COOMe
AcO
OAc
[17]
.OB~)
AcO "OBz-
OBz I
OxyCCl3 NH
17
AcO..~Ac
COOMe
OAc
A c ° " - ~ - Z " o " AZ.,...F-~ 'o~ n AcHN .j~,.~)~,,~ AcO
AC(~ '~OBz~
¢OB~)
~'X'~'~-.~ ~', s~ ACU-~w..~ -
OBz
.OR 2
Rt
ISR=Ac 19R=Na
N3 ROvAy~CI3H27
OBz 13R=H 20R =Ts
R30~.OR 3
.COOR4
OR 3
21Rl=N3.R2=Bz, R3=Ac,R4=Me 22Rl=NH2, R2=Bz,R3=Ac,R4=M¢ 23 R 1 = NHCO(CH2)I6Me, R 2 = Bz, R 3 = Ac, R 4 = Me H R 1 = NHCO(CH2)I6Me, R 2 = R 3 = R 4 = H
Ts = p-tolylsulfonyl
SCHEME2. Synthesis of ganglioside analogs containingthioglycosicallylinked ceramide. Synthesis of S-(a-Sialyl)-(2--->6)-/3-hexopyranosyl and S-(a-Sialyl)-(2-->6' )fl-lactosyl Ceramides Containing/3-Thioglycosidically Linked Ceramides (I-K) In this section, the synthesis of S-(a-sialyl)-(2--->6')-/3-1actosyl 1-thioceramide (K) is described as an example of the synthesis of the title compounds (I-K), all of which are synthesized in the same manner. 25 For the synthesis of the target thio analog ofganglioside, we employ the sodium salt of the per-O-acetylated S-a-sialyl-(2-->6' )-l,6'-dithio-fl-D-lactose (26) as the glycosyl donor, for coupling with (2S,3R,4E)-2-azido-3-O-benzoyl25A. Hasegaw, H. Ogawa, and M. Kiso, J. Carbohydr. Chem. 10, 1009 (1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
193
1-O-(p-tolylsulfonyl)-4-O-octadecene-l,3-diol (20). Treatment of S(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-
g alac to- 2-nonulop yranos ylonate )-(2~6)-O-( 2,3 ,4-tri- O-acetyl-6-thio-[3-Dgalactopyranosyl)-(1---~4)-2,3,6-tri-O-acetyl-D-glucopyranose (11) with methane sulfonyl chloride in dichloromethane in the presence of 2,4,6collidine for 1 hr at -15 ° gives the 1-chloro derivative 24, which is converted to the desired [3-thioacetate 25 in 53% yield. Coupling of the sodium salt 26, freshly derived from 25 by selective S-deacetylation with sodium methoxide at - 3 0 °, with the tosylate of azidosphingosine 211in DMF under a nitrogen atmosphere overnight at 45 ° yields the expected [3-glycoside 27 (38%) after column chromatography. Compound 27 is converted to the final compound K by the set reaction sequence, that is, selective reduction of the azide group, condensation with octadecanoic acid, and final deprotection, as described for the preparation of D.
Detailed Procedures S-(Methyl 5-Acetamido-4, 7,8, 9-tetra-O-acetyl-3,5-dideoxy-D-glyceroot-D-galacto-2-nonulopyranosylonate )-(2--~6). 0-(2,3,4-tri-O-acetyl-6-thio[3-D-galactopyranosyl)-(1---~4 )-2,3 ,6.tri-O-acetyl- l-S-acetyl- l-thio-[3- Dglucopyranose (25). To a solution of 11 (500 mg, 0.46 mmol) in dichloromethane (6 ml) is added 2,4,6-collidine (0.6 ml), and the mixture is cooled to -15 ° (Scheme 3). Methane sulfonyl chloride (0.3 ml, 3.88 mmol) is added to the mixture, and the mixture is stirred for 20 min at -15 °, then for 1 hr at room temperature; progress of the reaction is monitored by TLC. Dichloromethane (60 ml) is added, and the solution is successively washed with 2 M hydrochloric acid and water, dried (Na2SD4), and concentrated to the crude 24, which is used for the next reaction without further purification. To a solution of 24 in acetone (10 ml) is added Drierite (2 g), and the mixture is stirred for 2 hr at room temperature, after which potassium thioacetate (315 mg, 2.76 mmol) is added. The mixture is stirred overnight at 45 °, then filtered, and the precipitate is washed with dichloromethane. The filtrate and washings are combined and concentrated to a syrup. The residue is chromatographed on a column of silica gel with 120:1 (v/v) dichloromethane-methanol, to give 25 (279 mg, 53%) as an amorphous mass, [a]D +0.26 ° (C 0.75, chloroform).
S-(Methyl 5-Acetamido-4, 7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glyceroOt-D-galacto-2-nonulopyranosylonate)-(2---~6)-O-(2,3,4-tri-O-acetyl-6-thio[3-D-galactopyranosyl)-(1--~4)-S-(2,3,6-tri-O.acetyl-[3-o-glucopyranosyl)(1--~l )-(2R,3R,4E)-2-azido-3-benzoyloxy-4-octadecene-l-thiol (27). To a stirred solution of 25 (250 rag, 0.22 mmol) in dry methanol (2 ml), cooled to - 3 0 °, is added a solution of sodium metal (15 mg) in dry methanol (0.2
194
NEOGLYCOLIPIDS
A A c A ~ ~
O ~ ' ~ O
[ 17]
AcO UAC
R2 OAc
11 RI,R2 = H, OH 2 4 R 1 = CI, R2 = H 2 5 R 1 = H, R2 = SAc 26 Rl = H, R2 = SNa
N3 TsO.,./~CI3H27 OBz 20
R30 ~OR 3 R3
COOR'S OR s
2
,o
27 RI = N3, R2 ffi Bz, R3 = AC, R4 = Me 28 R 1 = NH2, R 2 = Bz, R 3 = Ac, R 4 = Me 29 R 1 = NHCO(CH2)I6Me, R 2 = Bz, R 3 = Ac, R 4 = Me K R 1 = NHCO(CH2)I6Me, R 2 = R 3 = R 4 = H
SCHEME 3. Synthesis of ganglioside analogs containing both the a-thioglycoside of sialic acid and thioglycosidicallylinked ceramide. ml). Stirring is continued for 5 min at - 3 0 °, and the mixture is concentrated to give 26 as an amorphous mass, which is used for the next step without purification. A solution of 26 and 20 (410 mg, 0.7 mmol) in dry DMF (4.5 ml) is stirred overnight at 45 ° under nitrogen. Acetic anhydride (2 ml) and pyridine (4 ml) are added to the mixture, which is stirred overnight at room temperature, then concentrated. The residue is chromatographed on a column of silica gel (50 g) with 100 : 1 (v/v) dichloromethane-methanol, to give 27 (124 mg, 38%) as an amorphous mass, [a] D - 6 . 4 ° (c 0.95, chloroform). Synthesis of S-(a-Sialosyl)-(2--->9)-O-(a-sialosyl)-(2--~3' )-fl-lactosyl Ceramide (g) For the synthesis of the target ganglioside analog, 26 we set out to synthesize the per-O-acylated 2-(trimethylsilyl)ethyl S-(methyl a-N-ace26A. Hasegawa, H. Ogawa, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 10, 1009(1991).
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
195
tylneuraminyl)-(2~9)-O-(methyl o~-N-acetyl-9-thioneuraminyl)-(2~3' )-/3lactoside (39) as the intermediate. Compound 39 could then, by introduction of the ceramide moiety, be transformed to the end product. Treatment of methyl (methyl 5-acetamido-3,5-dideoxy-2-thio-o-glycero-~-o-galacto-2-nonulopyranosid)onate (30) with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid monohydrate gives the 8,9O-isopropylidene derivative 31 in 70% yield, which is acetylated with acetic anhydride in pyridine to give 32 (Scheme 4). Removal of the isopropylidene group from 32 with 80% aqueous acetic acid for 3.5 hr at 45° gives compound 33 in 89% yield, without acetyl migration. In the NMR spectrum, H-7 appears as a doublet of doublets at 8 5.08 (J6,7 = 2.0, J7.8 = 9.3 Hz). Selective bromination 27of 33 with carbon tetrabromide-triphenylphosphine in pyridine gives methyl (methyl 5-acetamido-4,7-di-O-acetyl-9-bromo-3,5,9-trideoxy-2-thio-D-glycero-o~-o-galacto-2-nonulopyranosid)onate (34) in 72% yield, which is converted to the 4,7,8-tri-O-acetyl derivative 35 quantitatively. The glycosylation15'28of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-fl-o-galactopyranosyl)-(l---~4)-2,6-di-O-benzoyl-fl-o-glucopyranoside15 (36) with 35 in acetonitrile for 20 hr at - 15° in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) as the glycosyl promoter gives the expected o~-glycoside 37 of the sialic acid at 0-3' in compound 36, in 37% yield. Acetylation of 37 with acetic anhydride in pyridine gives the acetate 38 in 93% yield. Condensation of the sodium salt 3 with compound 38 in DMF under nitrogen affords the desired tetrasaccharide 39 in 80% yield. The intermediate 39 is transformed into the final product I, by the procedures described for the conversion of 10 to D.
Detailed Procedures Methyl (Methyl 5-Acetamido-3,5-dideoxy-8,9-O-isopropylidene-2-thioo-glycero-o~-o-galacto-2-nonulopyranosid)onate (31). To a solution of 30 (500 mg, 1.4 mmol) in dry DMF (5 ml) are added 2,2-dimethoxypropane (0.87 ml), p-toluenesulfonic acid monohydrate (15 mg), and Drierite (1.0 g), and the mixture is stirred for 1 hr at 0°, then neutralized with solid sodium bicarbonate (Scheme 4). The precipitate is filtered off and washed with methanol. The filtrate and washings are combined, then concentrated. The residue is chromatographed on a column of silica gel (50 g) with 40 : 1 (v/v) dichloromethane-methanol, giving compound 31 (390 mg, 70%) as an amorphous mass; [a] D + 12.6° (c 0.58, chloroform). 27 A. Kashem, M. Anisuzzaman, and R. L. Whistler, Carbohydr. Res. 61, 511 (1978). 28 T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 188, 71 (1989).
196
NEOGLYCOLIPIDS
H?_.pOH COOMe HO~,.L-~ O . ~ A c a l q ~ SMe
~f~.~ COOMe RO~"L7"~O " ~ A c H N ~ -SMe
30
31R=H 32 R = Ac
0 RI COOMe AcOT..~..o.Tt.. AcH~I~ SMe
__ O14
Br .OAc
] "~OBz HO
COOMe
OR
"~..~b 0 E
RO
AC~L_~C
COOMe
A c H N ~
~rca
OBz
37R=H 38 R = Ac
AcO .OAc
OSE
OBz 36
0
AcO
OBz /--O
HO o'o'o'o'o'o'o'o'o'o
33R1 =H, R2=OH 34R1 = H, R2 = Br 35 R 1 = AC, R2 = Br
AcO"
[ 17]
3
COOMe
AcO"" P"'L~ O" ~ A c H N ~
S [ pOAc
COOMe
ACU
OAc
_
¢OBz~
AcO
R2
39 R 1 = OSE, R2 = H 40 RI, R2 ffi H, OH 41 R 1 = H, R2 = OC(=NH)CCI3 R30 A
R
.OR 3 ¢
~
COOR 4 ~
S ~'OR3
R-~O
COOR4
R30
OR'
-OK"
OR2
-1
v
OR"
42RI=N3,R2=Bz,R3=Ac,R4=M¢ 43 R 1 = Nit2, R2 = Bz, R3 = AC, R4 ffiMe 44 R 1 = NHCO(CH2)I6Me, R2 = Bz, R3 = Ac, R4 = Me L R 1 = NHCO(CH2)I6Me, R2 = R3 ffiR4 = H
SCHEME4. Synthesis of ganglioside GD3analogs containingthe a-thioglycoside of sialic acid.
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-3,5-dideoxy-8,9-O-isopropylidene-2-thio-D-glycero-c~-D-galacto-2-nonulopyranosid)onate (32). Compound 31 (390 mg, 0.99 mmol) is acetylated with acetic anhydride (2 ml) in pyridine (4 ml) overnight at room temperature. The product is
[17]
GANGLIOSIDE ANALOGS CONTAINING SULFUR
197
purified by chromatography on silica gel (60 g) with 60 : 1 (v/v) dichloromethane-methanol, to give 32 (470 mg, quantitative) as an amorphous mass, [ot]D + 14.6° (c 0.8, chloroform).
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-3,5-dideoxy-2-thio-Dglycero-a-o-galacto-2-nonulopyranosid)onate (33). A solution of 32 (2.12 g, 4.44 mmol) in 80% aqueous acetic acid (20 ml) is heated for 3.5 hr at 45°, then concentrated. The residue is chromatographed on a column of silica gel (100 g) with 80:1 (v/v) dichloromethane-methanol to give 33 (1.73 g, 89%) as an amorphous mass, [a]D +33.8 ° (c 0.7, chloroform).
Methyl (Methyl 5-Acetamido-4,7-di-O-acetyl-9-bromo-3,5,9-trideoxy2-thio-D-glycero-a-D-galacto-2-nonulopyranosid)onate (34). To a solution of 33 (2.0 g, 4.57 mmol) in pyridine (40 ml) is added carbon tetrabromide (3.03 g, 9.14 mmol), and the solution is cooled to 0°. Triphenylphosphine (2.4 g, 9.15 mmol) is added, and the mixture is stirred for 5 hr at room temperature. Methanol (1 ml) is added to the mixture, which is concentrated to a syrup and then chromatographed on a column of silica gel (100 g) with 2 : 1 (v/v) ethyl acetate-hexane, giving 34 (1.65, 72%) as an amorphous mass, [a]D +38.5 ° (C 0.94, chloroform).
Methyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-bromo-3,5,9-trideoxy-D-glycero-a-o-galacto-2-nonulopyranosid)onate (35). Compound 34 (1.35 g, 2.7 mmol) is acetylated with acetic anhydride (5 ml) in pyridine (10 ml) overnight at room temperature. The product is purified by column chromatography on silica gel (60 g) with 2 : 1 (v/v) ethyl acetate-hexane, to give 35 (1.45 g, quantitative) as an amorphous mass, [a]D +28.0 ° (c 0.88, chloroform).
2-(Trimethylsilyl)ethyl O-(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9bromo-3,5 ,9-trideoxy-D-glycero-o~- D-galacto-2-nonulopyranosylonate )(2--*3)-O-(6-O-benzoyl-fl-D-galactopyranosyl)-( l---~4)-2,6-di-O-benzoyl-flD-glucopyranoside (37). To a solution of 3615'29 (800 rag, 1.06 mmol) and 35 (1.15 g, 2.12 mmol) in dry acetonitrile (10 ml) is added MS-3,~ (2.5 g). The mixture is stirred for 10 hr at room temperature and then cooled to -25 °. To the cooled mixture is added, with stirring, a mixture (2.94 g; 74.6% DMTST by weight) of D M T S T 7'19 and MS-3,~, and the stirring is continued for 20 hr at -15 °. The precipitate is filtered off, then washed thoroughly with dichloromethane. The filtrate and washings are combined, and the solution is successively washed with 1 M sodium carbonate and water, dried (Na2SO4), and evaporated to a syrup that is chromatographed on a column of silica gel (80 g) with 3 : 1 (v/v) ethyl acetate-hexane, to give 37 (490 mg, 37%) as an amorphous mass, [aid +9.1 °, (C 0.78, chloroform).
2-(Trirnethylsilyl)ethyl O-(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-929 p. Fiigedi and P. J. Garegg, Carbohydr. Res. 149, c9 (1986).
198
NEOGLYCOLIPIDS
[ 18]
bromo-3,5,9-trideoxy-o-glycero-a-o-galacto-2-nonulopyranosylonate)(2---~3)-O-(2,4-di-O-acetyl-6-O-benzoyl)-fl-o-galactopyranosyl)-(1---~4)-3O-acetyl-2,6-di-O-benzoyl-fl-o-glucopyranoside (38). Compound 37 (540 rag, 0.43 mmol) is acetylated with acetic anhydride (3 ml) in pyridine (6 ml) overnight at room temperature. The product is purified by chromatography on silica gel (50 g) with 2 : 1 (v/v) ethyl acetate-hexane, giving 38 (550 rag, 93%) as an amorphous mass, [a]D +5.0 ° (C 0.6, chloroform).
2-(Trimethylsilyl)ethyl S-(Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl3,5-dideoxy- D-glycero-a- D-galacto-2-nonulopyranosylonate )-(2--.9)-O(methyl 5-acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-9-thio-D-glycero-a-Dgalacto-2-nonulopyranosylonate)-(2--*3)-O-(2,4-di-O-acetyl-6-O-benzoylfl- D-galactopyranosyl)- (1--.4)-3-O-acetyl-2,4-di- O-benzoyl-fl- o-glucopyranoside (39). A solution of 38( (400 rag, 0.29 mmol) and the sodium salt 3 (308 mg, 0.58 mmol) in DMF (5 ml) is stirred overnight at room temperature under a nitrogen atmosphere, then concentrated. The residue is chromatographed on a column of silica gel (60 g) with ethyl acetate to give 39 (421 mg, 80%) as an amorphous mass, [O~]D +9.0 ° (c 0.58, chloroform).
[18] R e p l a c e m e n t o f G l y c o s p h i n g o l i p i d C e r a m i d e R e s i d u e s b y G l y c e r o l i p i d for M i c r o t i t e r P l a t e A s s a y s
By RENI~ ROY, ANNA ROMANOWSKA, and FREDRIK O. ANDERSSON Introduction Glycosphingolipids and gangliosides in particular constitute well-defined antigenic carbohydrate structures which have been implicated in a wide range of biological and immunological activities. When monoclonai antibodies are used for determining the fine chemical specificities involved in protein-carbohydrate interactions, solid-phase enzyme-linked immunosorbent assays (ELISA) are frequently used as a standard technique.l,2 The prime event in the ELISA is a nonspecific lipophilic adsorption of coating antigens on the surface on the plastic wells, which are generally made of either polystyrene or polyvinyl chloride (PVC). Although the exact structures of the ceramide portions have been suggested as being involved in monoclonal antibody-ganglioside (GM3 , 2 in Scheme 1) interactions, 3 the structure of the lipid component is not critically important for I p. F r e d m a n , S. J e a n s s o n , E. L y c k e , and L. Svennerholm, FEBS Lett. 189, 23 (1985). 2 j. Miyoshi, Y. Fujii, and M. Naiki, J. Biochem. (Tokyo) 92, 89 (1982). 3 S. Itonori, K. Hidari, Y. Sanai, M. Tanigushi, and Y. Nagai, Glycoconjugate J. 6, 551 (1989).
METHODS IN ENZYMOLOGY. VOL. 242
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