[16] Synthesis of ganglioside GM3 and analogs containing modified sialic acids and ceramides

[16]

S Y N T H E S I S OF G A N G L I O S I D E GM3 A N D A N A L O G S

173

glycosyl donors (75, 79, and 83) in good yields. Glycosylation of the trisaccharide acceptor 29 with these donors (1.45 equivalents with respect to the acceptor), in dichloromethane for 48 hr at 5° in the presence of DMTST and MS-4.A, gives the expected/3-glycosides, 84 (53%), 88 (53%), and 92 (49%), respectively. Catalytic hydrogenolysis (10% Pd-C) in ethanol-acetic acid for 3 days at 45° of the benzyl groups in 84, 88, or 92, and subsequent O-acetylation gave the per-O-acyl derivatives, 85 (85%), 89 (87%), and 93 (83%), after column chromatography. Compounds 85, 89, and 93 are converted to the corresponding ~-trichloroacetimidates, 87, 91, and 95, according to the method described above, and these, on coupling with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, give the required/3-glycosides, 96 (68%), 99 (43%), and 102 (46%). Finally, these are transformed via selective reduction of the azido group, condensation with octadecanoic acid, O-deacylation, and deesterification of the methyl ester group into the target sLe x ganglioside analogs, 98, 101, and 104, in good yields, as described for the synthesis of sLe x ganglioside. Acknowledgments This work was supported in part by Grants-in-Aid (No. 04250102 and No. 03660132) for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan.

[16] S y n t h e s i s of G a n g l i o s i d e GM3 a n d Analogs C o n t a i n i n g M o d i f i e d Sialic Acids a n d C e r a m i d e s By MAKOTO KISO a n d AKIRA HASEGAWA Introduction G a n g l i o s i d e GM3 , first isolated from horse erythrocytes by Yamakawa and Suzuki ~ in 1952, is the major ganglioside component in erythrocytes of many animal species. In addition to the known biological functions of

T. Yamakawa and S. Suzuki, J. Biochem. (Tokyo) 39, 383 (1952).

METHODS IN ENZYMOLOGY, VOL. 242

Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.

174

NEOGLYCOLIPIDS

[ 16]

GM3,2-6 much attention has been focused 7,8 on ganglioside GM3 and its degradation products because of their involvement in cell proliferation, differentiation, oncogenesis, modulation of transmembrane signaling, and so on. Ganglioside Gin3 has also been found to be a potential immunosuppressor 9 and a substrate of the trans-sialidase of T r y p a n o s o m a cruzi. ~° In view of these facts, it is of interest to investigate the relationship between the structures o f sialic acid and ceramide moieties and the biological functions of GM3 at the molecular level. This chapter describes a facile, preparative synthesis not only of natural ganglioside GM3 but also of various types of analogs containing a variety of modified sialic acids and ceramides. Synthesis of Ganglioside G~a Using 2-Thioglycoside of N - A c e t y l n e u r a m i n i c Acid The first synthesis of ganglioside Gin3 was achieved 11 by the use o f methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-Dg l y c e r o - f l - D - g a l a c t o - 2 - n o n u l o p y r a n o s o n a t e but proceeded in very low yield. The problematic step in ganglioside synthesis is the a-stereoselective glycosylation of sialic acid, and a variety of approaches 12 have been attempted. Particularly annoying is the formation of the 2,3-dehydro derivative of sialic acid (compound 7 in Scheme 1) and the thermodynamically favored/3-glycosides. More efficient methods for the synthesis of ganglioside Gin3 by overcoming these difficulties have been reported.13'~4 A highly efficient, regio- and a-stereoselective glycosylation of sialic acid to the 3-OH o f the galactose moiety has been achieved 15-~8by using the 2 G. J. M. Hooghwinkel, P. F. Barri, and G. W. Bruyn, Neurology 16, 934 (1966). 3 N. Handa and S. Handa, J. Exp. Med. 35, 331 (1965). 4 K. Uemura, M. Yuzawa, and T. Takemori, J. Bioehem. (Tokyo) 83, 463 (1978). 5 A. Gorio, G. Carmignoto, F. Facci, and M. Finesso, Brain Res. 197, 236 (1980). 6 y. Suzuki, M. Matsunaga, and M. Matsumoto, J. Biol. Chem. 260, 1362 (1985). 7 S. Hakomori, J. Biol. Chem. 265, 18713 (1990). 8 W. Song, M. F. Vacca, R. Welti, and D. A. Rintoul, J. Biol. Chem. 266, 10174 (1991). 9 S. Ladisch, H. Becker, and L. Ulsh, Biochim. Biophys. Acta 1125, 180 (1992). l0 S. Schenkman, J. Man-Shiow, G. W. Hart, and V. Nussenzweig, Cell (Cambridge, Mass.) 65, 1117 (1991). 11M. Sugimoto and T. Ogawa, Glycoconjugate J. 2, 5 (1985). 12K. Okamoto and T. Goto, Tetrahedron 46, 5835 (1990). t3 T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 188, 71 (1989). 14M. Numata, M. Sugimoto, Y. Ito, and T. Ogawa, Carbohydr. Res, 203, 205 (1990). i5 T. Murase, H. Ishida, M. Kiso, and A. Hasegawa, Carbohydr. Res. 184, cl (1988). 16A. Hasegawa, H. Ohki, T. Nagahama, H. Ishida, and M. Kiso, Carbohydr. Res. 212, 277 (1991). 17A. Hasegawa, T. Nagahama, H. Ohki, K. Hotta, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 10, 493 (1991). t8 A. Hasegawa, H.-K. Ishida, and M. Kiso, J. Carbohydr. Chem. 12, 371 (1993).

[16]

175

SYNTHESIS OF GANGLIOSIDE GM3 AND ANALOGS

A c o " " 7 ~ o - 7 CCOOMe ~

AcO'°'T~_ 0 " 7 Lsa

I

2~,[~ (R = Me, Ph, etc)

o.

/

HO[ ~OBz

OAc

A ~ o N ~ 0 ['~'':" "'~M A c O "OAc ~'o,.J e

\

.o

AcO j.OAc AcO~" 0 AcH AcO

OSE OH

5

COOMe

OH

.OBz.

0 HO uBz

0 SE

_

6 AcO OAc AcO~,. ~ 0~ AcHN~ AcO 7

/ COOMe

Bz = benzoyl SE = 2-(trimethylsilyl)cthyl

SCHEME 1. Regio- and c~-stereoselective glycoside synthesis of sialic acids.

2-thioglycosides (2a,/]) of N-acetylneuraminic acid (Neu5Ac) as glycosyl donors and the 2-(trimethylsilyl)ethyl (SE)19 glycosides (3 and 4) of"lightly protected" galactose or lactose as glycosyl acceptors in the presence of thiophilic promoters such as dimethyl(methylthio)sulfonium triflate 2° (DMTST) or N-iodosuccinimide-trifluoromethanesulfonic acid21,22 (NIS-TfOH) in acetonitrile (Scheme 1). In the initial studies, ~3'~5'23 the methyl a-2-thioglycoside of Neu5Ac (2a, R = Me) was employed as a glycosyl donor. Glycosylation of lightly protected 2-(trimethylsilyl)ethyl 6-O-benzoyl-13-D-galactopyranoside 15'z3 19 G. Magnusson, Trends Glycosci. Glycotechnol. 4, 358 (1992). 2o p. Ftigedi, P. J. Garegg, H. L6nn, and T. Norberg, Glycoconjugate J. 4, 97 (1987). 2~ G. H. Veeneman, S. H. van Leeuwen, and J. H. van Boom, Tetrahedron Lett. 31, 1331 (1990). 22 p. Konradsson, U. E. Udodong, and B. Fraser-Reid, Tetrahedron Lett. 31, 4313 (1990). 23 T. Murase, A. Kameyama, K. P. R. Kartha, H. Ishida, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 8, 265 (1989).

176

NEOGLYCOLIPIDS

[ 16]

or 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-fl-D-galactopyranosyl)(1--*4)-2,6-di-O-benzoyl-fl-o-glucopyranoside 13,15(4) with 2.0 molar equiv(3)

alents of 2 a in acetonitrile at -15 ° in the presence of DMTST and 3,~ Molecular Sieves (MS) affords the desired a-glycoside 5 or 6 in about 50% yield, respectively, together with 2,3-dehydro derivative (7) of Neu5Ac (a major by-product) and unreacted acceptors. It is noteworthy that neither the/3-glycoside nor a positional isomer is isolated in the reactions. This method has been successfully extended '6 to the large-scale preparation of a-sialyl-(2---~3)- and a-sialyl-(2---~6)-Gal derivatives by using 2 a or even the anomeric mixture (2a,fl) obtained almost quantitatively in one step from methyl 5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosonate (1). Iodonium ion-promoted glycosylation of 3 or 4 with 2c~,18 was then examined 17 using NIS-TfOH as a promoter, and the desired a-glycoside 5 or 6 is obtained in 60-70% yield under milder reaction conditions than those of the DMTST-promoted glycosylation described before. Therefore, taking into account the ready availability, lower toxicity, and easy handling of glycosyl promoter, the NIS-TfOH method seems to be superior to the DMTST method. A proposed reaction mechanism '7 of the DMTST- or NIS-TfOH-promoted glycosylation of Neu5Ac using its 2-thioglycoside in acetonitrile is shown in Fig. 1. COOMe S+-SMe

L0 ~ - ~

/

DMTST 1~

2COO"e Ot0-~-A~OAco cF's°3-

~

2a,~ OMe

~0~ CH3 C +~

CH3CN

J~Nu"

ct

- glycoside|

Nu"~COOMe m

"CH~

~

- glycoside

FIG. 1. Proposed reaction mechanism of the DMTST- or NIS-TfOH-promoted glycosylation of Neu5Ac using the 2-thioglycoside in acetonitrile.

[16l

SYNTHESIS OF GANGLIOSIDE GM3 AND ANALOGS

177

Acetylation of 6 with acetic anhydride in pyridine (94%) and selective removal24 of the SE group in 8 with BF3.OEt 2 give the corresponding 1-hydroxy derivative 9 (-90%), which is then treated with trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford the trichloroacetimidate 10 (94%). Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl4-octadecene-1,3-diol25-27 (11) by 10 yields the expected/3-glycoside 12 in high yield (92%). Selective reduction of the azide group with H2S in 5 : 1 pyridine-water gives the amine 13, which is converted stepwise, by condensation with fatty acids in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (DAC) in CH2C12 followed by O-deacylation and saponification of the methyl ester, to the desired ganglioside GM313(Scheme 2). Detailed Procedures

Preparation of 2-Thioglycosides of Neu5Ac (2ot,[3) Methyl 2-Thioglycoside of Neu5Ac. The methyl a- and methyl/3-2thioglycosides of Neu5Ac are each prepared 28by methylation of the corresponding sodium salt of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5dideoxy-2-thio-D-glycero-a- or -/3-D-galacto-2-nonulopyranosonate. A large amount of the anomeric mixture (37.7 g, a :/3 - 1 : 1) is obtained 16 in one step from the acetate z9 1 (40 g) by treatment with (methylthio)trimethyisilane and trimethylsilyltrifluoromethanesulfonatein 1,2-dichloroethane. Phenyl 2-Thioglycoside o f Neu5Ac. The phenyl 2-thioglycoside of Neu5Ac is readily prepared 3° by treatment of 1 with thiophenol in the presence of boron trifluoride etherate in dichloromethane. Other 2-thioglycosides are also obtainable by this method. Preparation of"Lightly Protected" Lactose Acceptor (4) Partial benzoylation la of 2-(trimethylsilyl)ethyl O-(3-O-benzyl-/3-D-galactopyranosyl)-(1-->4)-/3-D-glucopyranoside13 (14) or 2-(trimethylsilyl) ethyl 0-(3,4-O-isopropylidene-/3-D-galactopyanosyl)-(1--*4)-B-D-gluco24K. Jansson, S. Ahlfors,T. Frejd, J. Kihlberg,G. Magnusson,J. Dahmen,G. Noori, and K. Stenvall,J. Org. Chem. 53, 5629 (1988). 25R. R. Schmidtand P. Zimmerman,Angew. Chem., Int. Ed. Engl. 25, 725 (1986). 26M. Kiso, A. Nakamura, Y. Tomita, and A. Hasegawa, Carbohydr. Res. 158, 101 (1986). 27y. Itoh, M. Kiso, and A. Hasegawa,J. Carbohydr. Chem. 8, 285 (1989). 2aO. Kanie, M. Kiso, and A. Hasegawa,J. Carbohydr. Chem. 7, 501 (1988). z9N. Baggettand B. J. Marsden, Carbohydr. Res. 110, 11 (1982). 30A. Marra and P. Sinag, Carbohydr. Res. 187, 35 (1989).

178

NEOGLYCOLIPIDS Ac20 pyr.

Ac? .OAc

COOMe

AcO

OAc

AcO

[ 16] .OB~D

OBz

OBz

94%

BFa OEt2 CH2CI2

CCIBCN/DBU 2 steps94 %

AcO .OAc

COOMe

OAc

Ac~ ~OBz-

AcO

~OB~D

Oaz I O'~(CC13

10

NH

N3 HO~Cj3Hz7

OBz 11

AcOL.~OAc

BF 30Et 2 CH2CI2 92 %

AcO

OAc

COOMe

R

COBb

Ac~ ~OBz"

u~z

~Bz

12R=N 3 H2S/Pyr'H20

I • 13 R = NH2

i) R-COOH/WSC

HO ~OH

COOH

OH

OH

O'~(CHgnCH3

86 - 92 %

NaOMe,/M~OH - H20 quant.

(n = 12,16,22) Gangliosid¢ GM 3

SCHEME 2. Synthesis of ganglioside Gin.

pyranoside 31 (15) gives the corresponding 2,6,6'-tri-O-benzoyl derivative as a major product, which is then converted, either by hydrogenolytic removal of the benzyl group or by hydrolytic cleavage of the isopropylidene group, to the title compound 4 as needles, mp 106°-108 °, [t~]D + 11° (c 0.6, chloroform). 31 A. Hasegawa, K. Fushimi, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 12, 1203 (1993).

[16]

SYNTHESIS OF GANGLIOSIDE

B

OH a O ~ ~ HO vii

O

OHO S E OH

GM3 AND o

ANALOGS

179

° n ~ o s ~

14

15

Glycosylation Promoters The DMTST 3z (CF3SO3.MezS+SMe) reagent is prepared by treatment of methyl trifluoromethanesulfonate (CF3SO3Me) and a small excess of dimethyl disulfide (MeSSMe) in dichloromethane for 48 hr at 20°. Anhydrous ether is added, and the resulting crystals of DMTST are filtered with powdered MS-3A. The DMTST content is adjusted to 60% by weight with MS-3A, and the mixture is used for glycosylation. Both N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) are commercially available.

Synthesis of ot-Sialyl-(2---~3)-Lactose Derivative 6 DMTST Method. ~5-17 The DMTST-promoted glycosylation of 4 (2.6 mmol) with the methyl 2-thioglycoside of Neu5Ac (2a,/L R = Me; 5.2 mmol) is achieved in acetonitrile (20 ml) at - 15° in the presence of DMTST (15 mmol; see glycosylation promoters above) and additional MS-3A. (3 g) as described, ~3,16'17the reaction being monitored by thin-layer chromatography (TLC; silica gel 60 F254, 15:1 to 25:1 CHzClz-methanol). The desired o~-glycoside 6 (50% average yield) is isolated, on a column of silica gel with 4 : 1 (v/v) ethyl acetate-hexane, as an amorphous mass, [a]D + 11° (c 1.74, chloroform). NIS-TfOH Method. To a stirred mixture of 4 (1.9 mmol), the methyl or phenyl 2-thioglycoside of Neu5Ac (2~,/L R = Me or Ph; 3.2 mmol), and MS-3.~ (3 g) in 10:1 acetonitrile-dichloromethane (15 ml) is added, at -40 °, powdered NIS (6.4 mmol) and TfOH (0.64 mmol). 17,18The mixture is stirred overnight at - 4 0 ° and then neutralized by triethylamine. The precipitate is filtered off and washed with dichloromethane. The filtrate and washings are combined, successively washed with 5% Na2S203, saturated NaHCO3, and water, dried (NazSO4), and concentrated. The residue is chromatographed on a column of silica gel to give 6 in 60-70% yield. In both methods, the 2,3-dehydro derivative of Neu5Ac (7) is formed as a major by-product, but neither the/3-glycoside nor any positional isomers 32 M. Ravenscroft, R. M. G. Roberts, and J. G. Tillett, J. Chem. Soc. Perkin Trans. 2, 1569 (1982).

180

NEOGLYCOLIPIDS 9-deoxy 9-OMe

8-deoxy 8-epi

/

HO ~OH

1-CH2OH

/ COOH

o<(CH~I,CH3 OH - v

/

[16]

.,.OH O

NH -

\

S-OH

4-deoxy

(KDN)

4-OMe

FIG. 2. Ganglioside Gm analogs containing modified sialic acids.

are isolated. An alternative approach using phosphites and phosphates of Neu5Ac as glycosyl donors has also been reported. 33

Synthesis of Ganglioside GM3from 6 The conversion of 6 into ganglioside GM3is carried out 13by acetylation and removal of the SE group (6 --~ 9, 94%), imidate formation (94%) and coupling with azidosphingosine derivative 11 (92%), reduction of the azide group and N-acylation (86-92%), and final deprotection reactions (quantitative), yielding a series of natural ganglioside GM3molecules containing three kinds of N-fatty acyl groups (n = 12, 16, and 22) (Scheme 2; for experimental details, see Ref. 13). Synthesis of Ganglioside GM3 Analogs Containing Modified Sialic Acids Various G m analogs containing modified sialic acids (Fig. 2) have been synthesized 34-37 by employing the same procedure just described for the natural GM3. Chemical modifications of Neu5Ac are performed starting from methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-D-glyceroa-o-galacto-2-nonulopyranosid]onate 38 (16) or methyl (methyl 5-acetamido-3,5-dideoxy-2-thio-D-glycero-t~-D-galacto-2-nonulopyranosid) onate 39 (17), and the corresponding methyl 2-thioglycoside derivatives of 33 T. J. Martin, R. Bresceilo, A. Toepfer, and R. R. Schmidt, Glycoconjugate J. 10, 16 (1993). 34 A. Hasegawa, T. Murase, K. Adachi, M. Morita, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 181 (1990). 35 A. Hasegawa, K. Adachi, M. Yoshida, and M. Kiso, J. Carbohydr. Chem. 11, 95 (1992). 36 A. Hasegawa, K. Adachi, M. Yoshida, and M. Kiso, Carbohydr. Res. 230, 273 (1992). 37 A. Hasegawa, K. Adachi, M. Yoshida, and M. Kiso, Biosci. Biotech. Biochem. 56, 445 (1992). 38 A. Hasegawa, Y. Ito, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 8, 125 (1989). 39 A. Hasegawa, T. Murase, M. Ogawa, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 415 (1990).

[16]

181

S Y N T H E S I S O F G A N G L I O S I D E GM3 A N D A N A L O G S

modified Neu5Ac (C7- and C8-Neu5Ac, 34 4-0- or 9-O-methyl-Neu5Ac,35 8-epi-Neu5Ac, 35 and a series of deoxy-Neu5Ac4° derivatives) are used as the glycosyl donors for 4. The neutral GM3 analog containing 1-hydroxymethyl Neu5Ac is synthesized37 by a mild reduction of the methoxycarbonyl group in 6 with sodium borohydride. De-N-acetyl-G~43 and some analogs are synthesized41 by a similar approach using methyl [methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-2-nonulopyranosid] onate (18). HO ~OH

COOMe

Ho"%"Oos

A c H N ~ HO 16

HO ~OH

,,H o

A

COOMe

AcO ~OAc

SMe

COOMe

BocH,.~., AcO

17

SMe

18

The KDN-ganglioside GM3 derivative, 4z which contains 3-deoxy-Dglycero-a-D-galacto-2-nonulopyranosylonic acid (KDN) in place of Neu5Ac, was first isolated43from rainbow trout sperm. The KDN glycosyl donor 19 is prepared by treatment of methyl 3-deoxy-2,4,5,7,8,9-hexaO-acetyl-D-glycero-D-galacto-2-nonulopyranosonate 44 with thiophenol in 92% yield as described for Neu5Ac. Coupling of 19 and 4 is achieved42 in acetonitrile in the presence of NIS and a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMS.OTf), and the resulting trisaccharide 20 (46% isolated yield) is converted42 to KDN-ganglioside GM3 as described for Neu5Ac ganglioside GM3. AcP'~OAc AcOAc"-~'r""~r

__~SOOM' --

A,O .O~c AcO

COOme HO

OH 0Bz

.O% OBz

(KDN) 19

20

Synthesis of Ganglioside GM3 Analogs Containing Modified Ceramides A series of G~43 analogs containing modified ceramides (Fig. 3) are synthesized45 either by N-acylation of the spingosine derivativel3 13 (analogs A) or by glycosylation of 3-benzyloxycarbonylamino-l-propanol, 4o A. Hasegawa, K. Adachi, M. Yoshida, and M. Kiso, Carbohydr. Res. 2,311, 257 (1992). 4x S. Fujita, M. Numata, M. Sugimoto, K. Tomita, and T. Ogawa, Carbohydr. Res. 22,8, 347 (1992). 42 T. Terada, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 12, 425 (1993). 43 S. Yu, K. Kitajima, S. Inoue, and Y. Inoue, J. Biol. Chem. 266, 21929 (1991). 44 R. Shirai and H. Ogura, Tetrahedron Lett. 30, 2263 (1989). 45 A. Hasegawa, T. Murase, M. Morita, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 201 (1990).

182

NEOGLYCOLIPIDS H%OH HO"

[]

COOH 0

OH

[ 16]

OH0

0

0

R1

(3) O

OH

RI= H, OH

RI = N3, NI-12,NI-IAc O

0

N

OH OH

N

~

FIG. 3. Ganglioside GM3 analogs containing modified ceramides.

and (2RS)-3-benzyloxycarbonylamino-2-O-benzoyl-l,2-propanediol with trisaccharide imidate 13 10 (analog B). Synthesis of a-Neu5Ac-(2---->6)-Isomer of Ganglioside

GM3

(22)

The trisaccharide derivative 46 21, obtained by glycosylation of 2-(trimethylsilyl)ethyl O-(2-O-acetyl-3-O-benzyl-fl-D-galactopyranosyl)-(1-->4)2,3,6-tri-O-acetyl-fl-D-glucopyranoside 47 with the methyl a-2-thioglycoside of Neu5Ac (2a, R = Me), is converted 48 stepwise, by hydrogenolysis and O-acetylation (93%), removal of the SE group (96%), imidate formation and coupling with 11 (71%), reduction of the azide group and N-acylation (95%), and de-O-acylation and saponification of the methyl ester (quantitative), to the desired GM3 positional isomer 22. AcOL.~OAc

COOMe

Acd,"'-r'-- n --'/',. OAc A ~ ~ "o-L'k o Aco ~oo~oA:t~-os

OA_c

E

21 H O~-T.OH

COOH yv

.o,..~--o-~/,,,° o~

oH

.o

OH

o

OH

OH

NH 0 ~(CHgI6CH3

22

46 A. Hasegawa, M. Ogawa, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 9, 393 (1990). 47 K. P, R. Kartha, A. Kameyama, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 8, 145 (1989). 48 A. Hasegawa, M. Ogawa, and M. Kiso, Biosci. Biotech. Biochem. 56, 536 (1992).

[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

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