Synthesis of glycosphingolipids and corresponding neoglycolipids

Advanced Drug Delivery Reviews, 13 (1994) 267 284

267

© 1994 Elsevier Science B.V. All rights reserved. / 0169-409X/94/$27.00 ADR 00157

Synthesis of glycosphingolipids and corresponding neoglycolipids G6ran Magnusson Organic Chemistry 2, Chemical Center, The Lund Institute of Technology, University of Lund, Lund, Sweden (Received March 25, 1993) (Accepted March 30, 1993)

Key words: Anomeric blocking; Sphingosine synthons; Glycosylation with sialic acid derivatives; Synthesis of gangliosides; Single- and double-chain neoglycolipids

Contents Summary .................................................................................................................

267

i. Introduction ....................................................................................................

268

II. General functional consequences of glycolipid structure ............................................

269

III. Synthesis of glycosphingolipids ............................................................................ I. The anomeric blocking group ........................................................................ 2. Glycosphingolipid synthesis ...........................................................................

270 270 272

IV. Synthesis of neoglycolipids ................................................................................. 1. Single-chain neoglycolipids ............................................................................ 2. Double-chain neoglycolipids .......................................................................... 3. Steroid-based neoglycolipids .......................................................................... 4. Other neoglycolipids ....................................................................................

276 278 279 280 281

References ................................................................................................................

281

Summary Glycolipids of animal origin have many important functions based on molecular recognition in vivo and the over-all shape of glycolipids has a profound influence on their aggregation behavior. This review discusses current methods of glycosphingolipid and neoglycolipid synthesis with special reference to the choice of Correspondence: Dr. G. Magnusson, Organic Chemistry 2, Chemical Center, The Lund Institute of Technology, University of Lund, P.O. Box 124, 221 00 Lund, Sweden.

268

G. M A G N U S S O N

anomeric blocking groups and sphingosine synthons, as well as ways to create glycosidic bonds with sialic acid. Different unnatural glycolipid aglycons for the synthesis of neoglycolipids are also discussed. I. Introduction

Glycolipids of animal origin have many important functions based on molecular recognition in vivo. Thus, many glycolipids are tumor-associated antigens that may be recognized by the immune system [1]; bacteria and viruses use glycolipids at epithelial surfaces as anchoring points and perhaps as ports-of-entry [2]; circulating leukocytes are recruited via their cell-surface glycolipids to vascular endothelial cells present at local sites of inflammation [3,4]. Consequently, the design and synthesis of glycolipid analogs, as well as their naturally occurring counterparts, are of importance in the study of molecular recognition and in the creation of novel systems for cell and surface coating, liposome formation, and targeted drug delivery based on carbohydrate-protein recognition. The aglycon part of the glycosylceramides contains glycosidically bound sphingosine with long-chain, saturated or non-saturated, fatty acids attached via an amide linkage (Fig. 1). These glycolipids may consist of neutral saccharides, as in the globo-, lacto- and neo-lacto-series, or acidic saccharides as in the gangliosides and sulfatides. The latter two glycolipid types contain various numbers of the acidic monosaccharides sialic acid and sulfated sugar, respectively, and numerous compounds have been found for each class. In addition to the glycosphingolipids, trace amounts of glycosyl glycerides (typical plant lipids) have been found in the central nervous system of some animals. Furthermore, glycosyl phosphoryl polyprenols (glycosyl dolicholphosphates) are important glycolipids that transfer saccharide units to proteins or to other saccharides during the biosynthesis of glycoproteins and polysaccharides, respectively. The synthesis of glycolipids has been thoroughly reviewed by Gigg, covering much of the work done up to the end of 1987 [5,6]. The main topic of the present review is the synthesis of glycosphingolipids of importance in mammalian systems and analogs (neoglycolipids) that carry functional elements similar to the ceramide portion. Such glycolipids are useful inter alia as constituents of micelles and liposomes and for coating of cells and surfaces in connection with, for example, the development of new types of diagnostics, immunogens, and drug delivery systems.

O

OHO .H ~C)

HO

H

H0~0"~,"'~.Z~~AA

N .

.

.

.

~ .

.

A .

A

OH Fig. 1. Structure of lactosyl ceramide, one of the most c o m m o n glycosphingo|ipids.

SYNTHESIS OF GLYCOSPHINGOLIPIDS A N D NEOGLYCOLIPIDS

269

II. General functional consequences of glycolipid structure The over-all shape of glycolipids has a profound influence on their aggregation behavior [7,8]. Thus, the relative size of the polar head group (sugar) and the nonpolar hydrocarbon chain(s) determines the mode of packing: a conical shape (large head-group) leads to spherical micelles, whereas cylindrical molecules (similar crosssection for both polar and non-polar parts) give planar bilayers (cf. Fig. 2). Such simple geometrical considerations may be sufficient as a platform for designing lipids that are to perform a certain function when presented as a multimolecular aggregate. It should be stressed that many naturally occurring glycolipids are recognized by proteins (lectins) and, therefore, an aggregate which exposes a saccharide on its surface has the very real capacity to be targeted to lectin sites in the body. Thus, (neo)glycolipids are potentially valuable for both the entrainment and the targeting of drugs.

~ -,...:..-..~}~.-~.,._

:..==.~'L'

"~...,,~;.~

~.'a'~.,~.;"

~.'-~- "~

~'~:;"

,~4~,~;.'~'.'-:r. ''. ' ~ . . ' ~ - - p , s - ~ , . ' . - ' - a . A = = t • . ~ - ' t ,

% ~ ~. ",'.:'2(.'o:..~-.'.'.~,-

``~-~:~!~;~;~9~``:.`,`~<``~``:~:~,,.~,.~;~,~:,~`~,``~``~`~`~.~:~`,~,~g~:~,~::~;.L~;~.~.~

-"',°"~" ";~';i~'~,~'~"-1-'#'~l ",~;'.~.,','& ~'" ~e",;'~',";.'-~';',;'=','~',X',']':';'";",~':.;'-',:';~','?'~=~ ;';',':'I." ~.~...~_,.=',a;--"!,_.'-,;,:-;-~.-. , ~ ~ . . . $ ~ , ~ : ; , . .~,~le.<;-',-i-~'¥"-'-~ ~,~,~,~.~.~..,;. ¢'.'-,-,hf=...-: ~.1;.', ~- %;,..-,T:'...-v..-;:., -~

.,: . . . . . . .

.-=

-~.~_

~...... ~..-

,~

., .......

, ~ ......

~. ........

,~ ......... : ........:-.,.,.,.,-=

Fig. 2. Structure of 1-fl-D-galactopyranosylphytosphingosine and 1-fl-D-galactopyranosyl-N-octadecanoylsphingosin highlighting their over-all conical and cylindrical shapes.

270

G. M A G N U S S O N

III. Synthesis of glycosphingolipids The recent rapid development of efficient methods for the synthesis of complex oligosaccharides [9,10], separation of difficult diastereomeric mixtures, and structure determination with powerful modern N M R (and MS) instruments, has permitted the synthesis of complex glycolipids of relevance to medicinal research. Following the early days of glycosphingolipid synthesis, when synthetic chemists had to depend partly on sphingosine-like materials isolated from natural sources as starting materials, de novo synthesis of enantiomerically enriched compounds is now a practical preparative approach. The development of reliable methods for the synthesis of sphingosine and derivatives in conjunction with efficient methods for the creation of intersaccharidic glycosidic bonds has made possible the total synthesis of very complex glycolipids containing in some cases as many as 15 monosaccharide units. The use of azidosphingosine [11,12] (Fig. 6) as a building block in late steps of long synthetic sequences is an attractive feature, especially in conjunction with trichloroacetimidate activation [13] of the anomeric position. However, when using large oligosaccharides for glycosylation of sphingosine derivatives, separation of the resulting ~//~-mixture of glycosides may be difficult due to the structural similarity of the two diastereomers. During the last few years, methods for the synthesis of gangliosides (sialic-acidcontaining glycosphingolipids) have been greatly improved. As recent as the last decade, a yield of 20% in a sialylation reaction was considered to be quite acceptable, whereas today these reactions have been reported to proceed in the range of 60-80%. As a complement to the organochemical methods, enzymic synthesis based on sialyltransferases is useful for stereoselective glycosylation with sialic acid derivatives [14]. Although the general construction of glycosidic bonds is at the heart of oligosaccharide synthesis, this vast area of synthetic methodology is outside the scope of the present review. III.1. The anomeric blocking group

Most of recent glycosphingolipid syntheses were based on coupling of a sphingosine derivative to a synthetic oligosaccharide obtained from the corresponding anomerically blocked sugar. The choice of anomeric blocking group is important. It should be stable under the reaction conditions used in the synthetic sequences that lead to complex oligosaccharides, and yet be amenable to selective removal or transformation by conditions that are mild enough not to break glycosidic bonds. This is especially important when deblocking has to be performed late in a long synthetic sequence, since the saccharide usually represents a very high value at this final stage; mild, selective, and high-yielding methods are thus desired. Anomerically blocked saccharides of general use are alkyl, aryl, and alkenyl Oand S-glycosides as well as 1-O-acyl sugars, 1,2-orthoesters, 1,2-O-cyanoethylidene derivatives, and 1,2-oxazolines. Some of these blocking groups are however not stable under all the conditions used in oligosaccharide synthesis and their deblocking is sometimes ineffective. A bad choice of anomeric blocking strategy may mean that the anomeric position cannot be modified efficiently and therefore the final oligosaccharide cannot be transformed into the desired functional derivative. The

SYNTHESIS OF GLYCOSPHINGOLIPIDS AND NEOGLYCOLIPIDS

271

final product would thus (often unnecessarily!) be of limited value in subsequent investigations. Efficient routes for removal of the anomeric blocking group and activation of the anomeric center are therefore necessary. Selective removal of the anomeric blocking group leads to the corresponding hemiacetal saccharide. This reaction can be performed with several of the glycoside types normally used in anomeric blocking. Hemiacetal saccharides can then be activated for glycosylation by transformation into, for example, 1-O-acyl-, 1halogeno-, and 1-trichloroacetimidato-saccharides [13a,b]. The most useful anomeric protecting groups are benzyl, allyl, and 2-(trimethylsilyl)ethyl, since they can be removed in high yield when other reactions have been completed. Alkyl and aryl glycosides are often too stable to permit selective cleavage in high yield. The benzyl group can be removed by hydrogenolysis, but in many cases selectivity problems arise due to the presence of benzyl groups in non-anomeric positions. The allyl group has been preferred for anomeric blocking for a long time, mainly due to its ease of removal with, for example, palladium chloride in methanol [15]. The 2-(trimethylsilyl)ethyl (TMSEt) group can be removed in very high yield by treatment of the glycoside with a Lewis acid [16]. Based on 39 examples of mono --, hexasaccharides, the average yield of isolated hemiacetal product was 92%. [17]. It should be noted that in contrast to the cleavage of other glycosides, cleavage of TMSEt glycosides progresses by fission of the bond between the anomeric oxygen and C- 1 of the aglycon. The reaction is compatible with most protecting groups and can also be performed with unprotected TMSEt glycosides. As described in a recent review [17], the TMSEt group was used for anomeric blocking in a large number of syntheses of natural glycosphingolipids (see below). Activation of the anomeric position, directly from the blocked derivative, is highly desirable. This can be performed with thioglycosides, using various thiophilic reagents. A summary of the reagents used to date has recently been published [18]. TMSEt glycosides can be transformed directly into the corresponding l-O-acyl- and 1-chloro-saccharides, which are both useful glycosyl donors [16,19]. The anomeric 1,2-trans-configuration is normally conserved (as is the anomeric oxygen) in the formation of the 1-O-acyl derivatives, whereas the l-chloro-saccharides are obtained in the a-configuration [19,20]. Based on 34 and 16 examples of mono tetrasaccharides, the average yield of isolated 1-O-acyl- and 1-chloro-sugars was 97%, and 96%, respectively [17]. A summary of the most important reactions so far of TMSEt glycosides is shown in Fig. 3.

CF COOH/CHeCIJO-22"C/lO-30min BF3Et20/CHaCN/-20-22°C/I-lO h

Average yield from 39 exps: 92% [16,17]

R OH

(RCO o

:!O/BFEt20/toluene/22-55°C/1-4 h or

R~Ov~s~Me.

Ac~O/F~CI:,/,~1S°C/H2.

O

R/ ~ - -

o.,.. R'

Average yield from 34 exps:

97% [ 16,17]

o

CI2CHOMelZnCI2/2WC/I-25 h

R/

Average yield from 16 exps: 96% [ 19, 20J

~ cI

Fig. 3. Transformationsof 2-(trimethylsilyl)ethyl(TMSEt)glycosides.

272

G. M A G N U S S O N

111.2. Glycosphingolipid synthesis Construction of complex glycolipids relies on thorough planning of the synthesis regarding choice of building blocks, protecting groups, and activating agents. A number of potential problems may be identified and solutions are usually found during the execution of the synthetic route. However, in most cases the overall yield of desired product is low due to the multitude of steps normally needed to complete the synthesis. As an illustration of the complexity of total synthesis, a collection of potential problems in connection with construction of the ganglioside GM3 is shown in Fig. 4. The total synthesis of this and some other glycolipids is discussed below with special emphasis on the modern glycosylation methods that have been developed over the last 10 years. As indicated above, a few different types of sphingosine derivatives have been employed in the total synthesis of glycosphingolipids (Fig. 5). Ogawa and his coworkers coupled ceramide to trifluoroacetimidato- or fluoro-activated globotriose [21] in 13 and 29% yield, respectively. The low yield was attributed to the formation of a 1,2-orthoester when the 2-position carried a participating acetyl group. With a sterically more hindered 2-pivaloyl group, the yield increased to 37% [22]. In addition, orthoester formation and also complexation between ceramide and glycosylation promoter was offered as an explanation for the low yield obtained on glycosylation of the ceramide molecule [23,24]. The introduction, by Schmidt [11] and Hasegawa [12] and also by Nicolaou [24], of protected azidosphingosines (Fig. 5) seems to have solved the problem of low yields in the glycosylation reaction. However, this route requires additional steps (reduction of azide and N-acylation) in order to reach the desired ceramide, thereby lowering the over-all yield somewhat. An advantage though is the possibility to use fatty acids with different chain length in order to obtain glycosyl ceramides with varying properties. A general scheme for the synthesis of glycosphingolipids via ceramide and azidosphingosine derivatives is shown in Fig. 5. Reduction of the azide group in the sphingosine aglycon has been performed successfully with hydrogen sulfide [23,25], triphenylphosphine [24], and [Et3N][Sn(SPh)3] [26,27], thus providing the corresponding 'lyso'-ceramide derivatives ready for N-acylation. The route via azidosphingosine derivatives has been

~ anomeric ]

group

]

during I saccharide I~,..

] Glycosylation ] yield? ] OrthoesterI formation? [1 "~ ~/

/OH~oH

COoH?H#H

0

HO .,..t...... 0~.~,.~ H 0

Synthesis via ceramide or

I derivative? /,/ 0 HN"

v

~

.

v

.

v

.

.

v

.

v

.

v

.

.

v

.

Fig. 4. Potential problems encountered in synthesis of a ganglioside,

v

v

v

SYNTHESIS OF GLYCOSPHINGOLIPIDS

AND NEOGLYCOLIPIDS

273

used repeatedly by Hasegawa [28], Schmidt [29], and Nicolaou [30] as exemplified by their syntheses of glycolipids of the Lewis blood group family. These compounds have generated considerable interest among chemists and biologists lately due to their ability to bind endothelial-cell selectins [3,4], thereby recruiting leukocytes to sites of inflammation. Several impressive syntheses of natural glycosyl ceramides have been reported recently. Thus, large synthetic oligosaccharides were coupled to ceramide [31]. The sialyl-Lewis x ceramide was prepared via azidosphingosine [32], which was also used for the synthesis of trimeric Lewis x ceramides [27]. In the latter example the azidosphingosine aglycon was introduced already at the lactose level and carried through a number of block-synthesis steps. In a synthesis of the tetrameric Lewis x ceramide, the azidosphingosine residue was coupled to the synthetic tetradecasaccharide [29]. Fig. 6 shows these recent glycolipid syntheses, illustrating how very far synthetic carbohydrate chemistry has developed until now. Gangliosides (sialic-acid-containing glycosphingolipids) comprise a very large group of glycolipids. Their synthesis has been hampered by the low yields obtained in the sialylation step, in addition to the problems connected with coupling of the ceramide part to the saccharide portion as discussed above. However, many solutions have been offered and the methods employed until the end of the last decade have been reviewed [33,34]. During the last few years a large number of synthetic variants on two themes (with or without a stereocontrolling auxiliary group in the 3-position) have emerged for the construction of sialic acid glycosides. Two Japanese groups [35,36] initiated the use of sialic acid glycosyl donors carrying an auxiliary 3-phenylthio group. These O

0 R

HN~ O OBz

H

X

~

~

X = OC(=NH)CCI 3, HaI, SR 1. Glycosyla til~ 2. Deprotection O

H

~---~...~,,~O

N

~

OH 11. Reduction ofN 3 N-Acylation i Deprotection

OR' t Glycosylation

R

+

X

X = OC(=NH)CCI 3, Hal, SR

H

O

T

' v OR'

~

"

,

~

~

R' = Bz, SitBuMe2

Fig. 5. General scheme for the synthesis of glycosyl ceramides.

274

G. M A G N U S S O N

donors seem to give a consistently high yield and ~//~ ratio in the glycosylations. The phenylthio group must subsequently be removed by reduction, which is normally done by tin hydrides in high yield. The method was used in a synthesis of GD3 ganglioside, where a disialic acid synthon was employed [37]. The same synthon was OAc

AcO

0

OAc

OAc OAc AcO OBz ~o[--=.,~o.~_co: l'~'o ', , ~ -PivO , - - ~ -N:]o . ~ ~ . ~ . ~

,nl4~ OPrv

0

NPhth I

i

Me

O

O "Ik""~"4"~ O f ~ - % ~

P"I ~ 0

OAc

Me

A

iv

O " ~ ' ~ "'O" z

0

OAc A¢O

OAe

•

|

-

-

-

Sn(SPh)J I Z" PhSH/Et3N 1 89%

2. BOPCI/ Et3N, 85%

o

]

HO~ o

PivO AcO

~...~OAc COOMe

A=O

AcO

OAc 0

OBz

i,,.

0

0

0

BZ//~OBz

~

Ac

O"~C AcO

0

0

OBz

1. BFaEt20 I HO N~ 56% ~ OABz 2. HzS

AcO 0

AcO

O O

"~'~ A¢O

OAc ,,0o

°-7-.°

_

O #" OAc

,o~o

O

PhthN /

0 c O

AcO~

NPhth O

O

NPhth

C°'o\

AcO

OAc

OAc

O "OAc

OPw O

0

Ac(~/~OPiv

NH

OPiv O

CCI:}

o

OAe 0 -~)~'7~0 A ~ - - - " ~ O /~._. NPhth AcO OA¢

T T H MSO [ [ ? CHOI3 I H

ACO L~J~"O,

ACO

[3~]

OAc O

Ac~LOAc OA¢

~

0 HN ~

OAc

O

NPhth

O

i271

~'OAc

c

ACO

OA

Oez

Cl3

3. carbo- / diimide I H 81%

AcO

HN ~ 7

AcO

OAe

NHAc

Me'~OA A/oXc

0

~ 20%

~

N O

~ ~

OBz [31]

O

OPiv

~ OBz

OA¢

AcO

.oo o.,<-.o Me

AcO

OAc

0

0

t O

OAc

NH'°l.~r'C'o. ° OAc

L~

OAc

NH.°''~:~ ,

~:~-~_o,0 AclOAc

Me

AcO

O

OA

c

O

AcO

CCI

--770"0.°

J~

1. TMSOTIJ HO~ 2. H2S

l

N~ O OBz

Me2HN+M

N =C =NEtCI

O AcO

i _ ~ o

HN

o .--

OAc

Fig. 6. Recent examples of glycosphingolipid syntheses.

_

Ogz

[29i

SYNTHESIS OF GLYCOSPHINGOLIPIDS AND NEOGLYCOLIPIDS

275

used for the synthesis of tetrasialic acid-containing ganglioside GQlb [38]. The 3phenylthio-sialic acid was also used in a synthesis of the sialyl Lewis x tetrasaccharide, where the sialylation step proceeded with very high stereoselectivity in 63 % yield [39]. Examples of syntheses with 3-phenylthio-substituted sialic acids is depicted in Fig. 7. Sialic acid donors without an auxiliary group in the 3-position have been thoroughly investigated (Fig. 8). The methyl ester of fl-l-chlorosialic acid was coupled in a tetrabutylammonium-bromide-mediated reaction to the 6-position of unprotected TMSEt galactoside, giving the ~- and fl-glycosides in 36 and 23% yield, respectively [40]. In an attempt to avoid expensive and toxic catalysts, thereby permitting large-scale synthesis, zinc bromide was shown to produce a NeuAc-(2 --, 6)-Gal saccharide in 66% yield and an ~/fl-ratio of 68:32, using the methyl ester offl1-chlorosialic acid [41]. In another attempt at large-scale synthesis, an easily prepared 1:1 anomeric mixture of methyl 2-thioglycoside of NeuAc was shown to glycosylate a 6-protected galactoside selectively in the 3 - p o s i t i o n in 52% yield, using dimethyl(methylthio)sulfonium triflate (DMTST) as promoter. Neither fi-glycoside nor positional isomers were isolated. Similarly, a NeuAc-(2 --, 6)-Gal saccharide was

OMe

N•C CI

AcOu~ AcH

O

0 /oA~c OAc

OH

COOMe B n O n o ~ O h + B

AcO

BnO" ~ Bno

COOMe

8nO

0 4o% [351

~nu OMe OMe

.~OBn 13r BnO, ~ " ~ 7 ~ O --~COOMe + AcHN. ~ . . , ~ r ~ SPh BnO ......................................

noBn COOMe B n O ' ~ _L B.o~o" EIg(CN)2/HgBr2B n O = " ' - 7 ~ O ~ 1 --0~._....~./ A~HN-J...q'..iLSp,

_._

,ff•OH --"--

1 Hg(CN)2/HgBr l (64%)

+ BA:ON" ~ ' ~ " 0 *'~?~--CO0Me ~ihBS~ K

~OBn

AcEHN"t~' ~

OBn C

"Z o.n o

BnO

...................................... OH/OBn

O

~

OBn

7] % {361

/OB~nOBn

OH OBn COOMeL.=~. _ 0

/OBn 0

BnOe"~T~O'/~'COOMe ~,,-z.q"..-~sp, B.o

+

OH ~" -- Ac~INJ Me -7"'0"--/ 0 OBn oB(%~j-n OB° BnO

AcHN~-,.~)'~g---Sph s.o

0

OH i ~ Ac~INJ ~."7""o ~ oBo OBt"~'~n °B" ~ m ] BnO

Fig. 7. Synthesis of gangliosides via sialic acid derivatives carrying an auxiliary 3-phenylthio group.

276

G. MAGNUSSON

obtained in 70% yield without formation of isomers [42]. Arylthio-glycosides of NeuAc reacted with primary alcohols depending on the presence of electron-donating or -withdrawing groups in the para-position of the aglycon. Thus, a p-methoxy or p-acetamido group permitted coupling to the 6position of diisopropyl-protected galactose in 89% yield (c¢///2.6:1), whereas a pnitro group rendered the NeuAc derivative inactive [43]. A crystalline S-glycosyl xanthate of acetyl-protected NeuAc was employed for glycosylation of the 6- and 3-positions of galactose, using DMTST as promoter in acetonitrile, which gave the corresponding glycosides in 64% (~///3:1) and 30% (c~/// 6:1) yield, respectively [44]. A systematic study of the influence of temperature and solvent on sialylation with S-glycosyl xanthate showed that low temperature ( - 7 0 ° C ) and a solvent mixture consisting of acetonitrile and dichloromethane gave the highest ~/// ratio of products. Thus, using a xanthate of benzoyl-protected NeuAc as donor produced 3and 6-sialylated galactosides in 48% (~///4:1) and 59% (e///9:1) yield respectively, while suppressing the formation of sialyl glycal. Under the same conditions, a lactoside having HO-2', -3', and -4' unprotected was sialylated by S-glycosyl xanthate of acetyl-protected NeuAc at the 3'-position in an exceptional 82% yield; no //-glycosidic product was reported [45a,b,c]. In a synthesis of GM3-ganglioside lactam, sialylation under similar conditions of a 3',4'-unprotected 2'-azido-lactoside with the S-glycosyl xanthate of acetyl-protected NeuAc gave the corresponding NeuAc trisaccharide in 75% yield (~///20:1) [46]. In all the sialylation methods discussed above, a full equivalent or more of (often expensive) glycosylation promoter was used. Recently, //-sialyl phosphites were shown to function as glycosyl donors under activation by a catalytic amount of trimethylsilyl triflate (TMSOTf). Thus, partially protected N-acetyllactosamine was sialylated in the 3'-position with a //-sialyl dibenzyl phosphite in acetonitrile at - 4 2 ° C to give the trisaccharide backbone of the sialyl Lewis x tetrasaccharide in 78% yield (~///6:1) [47]. In a similar approach, a partially benzylated lactoside was sialylated in the 3'-position with a//-sialyl diethyl phosphite in acetonitril at - 4 0 ° C to give the GM3-ganglioside trisaccharide in 38% yield. A galactoside unprotected in the 6-position furnished the NeuAc-(2 --* 6)-Gal saccharide in 70% yield (~///4:1) [48].

IV. Synthesis of neoglycolipids Studies on the recognition of glycoprotein-related saccharides by lectins or microorganisms is usually performed by hydrophobic coating of a microtiter plate by the glycoprotein, followed by binding and detection of the lectin or micro-organism. However, many glycoproteins show a heterogeneous glycosylation pattern, which means that the binding specificity obtained from these experiments normally needs to be confirmed via binding to pure saccharides, corresponding to those present on the glycoprotein. In other words, the saccharide portion needs to be synthesized in such a form that its immobilisation on, for example, a plastic surface will emulate the situation on the glycoprotein. Neoglycolipids can be used for hydrophobic coating of surfaces, by evaporating the solvent from a solution of the lipid (often mixed with an auxiliary lipid such as phosphatidyl choline). Neoglycolipids can also

277

SYNTHESIS OF GLYCOSPHINGOLIPIDS A N D NEOGLYCOLIPIDS

OACOAc

Cl

1BuzSnO

HO OH

/OA~COAc

COOMe

°-L-x Ac

HO

AcO

O~/~S~M%

OH

=

Ac

O~/~SiM %

COOke

/OA~coAc

HO OH --.--

~L_ 0 HO

A~ov'--7"~o"7~ ~'X -o 66% AcHNA.~,,,,,~' ~ BzC)~ C ~ V I e

+

BzO

OMe

Z.nB,~

OH COOMe

HO OBz

+

~.~<.o

HO OBz

D~T

..'O~oAc COOM,L<-LO

OH

AcHN-.~/.,~7""~,dr~

...................................................

O OH ~ O ~ o

~O~OAc

COOMe S

AcHN.~,J~m,,,,,~r

AcHNA ~,~o '~

OEI

B9% [4,3l

o '[~- - ~O~ o ~

/ • O A c COOMe

HO OH +

OH

OACOAc COOMe 0 O

NIS/TfOH

.

~== NO-.,:inactive

...................................................

52%1421

li:== ..... {

,:o

OACOAc COOMe

1411

OH

................................................... ~cH~SMeC

s~ 1401

-o

HO

O BzO

CH3CN

OM*

AcO

OH

OH

. o.o coo=_C:o

HO OBn

,~o..7-~o.-.~.o ~ o , , o

D~T OBn CH3CN

HO

,,o~ o - _ . ~ c . - . ~

OBn

o,,o

...................................................

_._

OH OBz ~.~0

+

Br

. 0 ~ - ~ 0 / ~

OA

,-'-'~o~

.o_

AcO

OH ............................................. "0, _B.Z.

+

OH OBn

~ON

O

O

,o.-.r.~.13~o r''

= C 3CN/CH2CI 2 Ac

At"

Br

O

CH

B n O ~ OBn O

82% [4Sl

SiM%

o . OBn

/ ~OAc oAo

coo~E<, Lo 0

A c O ~"

Na

oH OBz

oooM,,L'<. L o

0

OBn /*%~SiM% Boo~r~../ii~o 0

75% 146]

CH3CN/CH2CI2AeH

~OBn

................................................... OBn J /O~OAc oJP'oBn AcO - ~ 7 ~ / ~ o . ~ C O O M e

AcO

TMSOTf ] (02equiv • ,,_

~ OH

OACoAc

OEI O "*'P'oEI

"

HO

OH.OTBDPS

COOMeL <,. -0

. ,# HO'"~ /NH-~H '~ ' ACo/~I'

[47]

0

xOTBDPS

Ph ~"'O

coo,,°. A~O

NHAco/~'~f CH3CN ~ 0

HO

...................................................

OAc /-'~OAc

.L.o

OBn

O OB~ ~

TMSOTf (0 l equ,v C" N" "J ~ OAc

~'~0

o.c coo,,o L_o .oOT>'-'OBnOHo O

AcOr

, OBn

Ph

0

A~HN-J'r"~

AcO

Fig. 8. Sialylations using various sialyl donors.

0

OBn

~XO ~ o I~l

278

G. M A G N U S S O N

be used for coating of cells and for the formation of liposomes, suitable for targeted drug delivery. The synthetic methods used for the preparation of natural glycolipids are highly relevant also to neoglycolipid synthesis, since in the latter, either the saccharide part or the lipid part is often the same as in the natural material (see section above on glycosphingolipid synthesis). Very few syntheses have been reported of neoglycolipids where the lipid part was deliberately designed to mimic the structure of that of natural glycolipids. Synthetic neoglycolipids consisting of a natural sphingosine residue but an unnatural saccharide part [49] are also scarce. On attempting such mimicking, it is important to realize that the overall shape of the lipid determines the type of aggregate formed in solution as was discussed in Section II above. The different neoglycolipid types (single-chain, double-chain, steroid-based, and others) are discussed below.

IV.1. Single-chain neoglycolipids The detergent-like properties of single-chain glycolipids is demonstrated by the wide-spread use of commercially available octyl /~-D-glucopyranoside in biological and other investigations. Similar effects should be obtained with alkylthioethyl glycosides, obtained by reaction of alkylthiols with (we-spacer) 2-bromoethyl glycosides [50,51], and with fatty 1-O-acyl sugars [16]. The sulfur-containing lipids are easily transformed into the corresponding sulfones, thereby providing compounds with altered properties. Single-chain lipids can be used for coating of surfaces and are therefore of value in saccharide receptor mapping. 2-Bromoethyl glycosides have also been coupled to carriers having more than one thiol group. A derivative having two galabiose units linked via a bis-thiol spacer was synthesized for use as an inhibitor of bacterial adhesion [52,53]. Some single-chain neoglycolipids are shown in Fig. 9.

_~-2....I o

R",x-'~,-,&_. o , ~ v v ~...~o m~-~-'~,.~--,,.~O.~.S ~ R

[so, 51]

o ~,,,,,'"-~.~ 0 .~- S~ 0

~.....~o o

R~O~,~

[52,53]

Fig. 9. Examples of single-chain neoglycolipids.

279

SYNTHESIS OF GLYCOSPHINGOLIPIDS A N D NEOGLYCOLIPIDS

IV.2. Double-chain neoglycolipids Double-chain neoglycolipids have been shown to emulate the receptor function displayed by natural glycosphingolipids. Thus, a bis-sulfone lactoside, prepared by treatment of 3-bromo-(2-bromomethyl)propyl (dibromoisobutyl or DIB) fl-Dlactoside with alkyl thiols and oxidation of the sulfide groups, was recognized by Sendai virus with the same efficiency as natural lactosyl ceramide [54]. Furthermore, the corresponding bis-sulfone (and bis-sulfide) globotriosides bind verotoxin with high affinity when adhered to microtiter plates [55]. In addition to variation of the chain length, unsymmetrical aglycons were prepared, where one of the alkyl chains contained a terminal ester function, useful for coupling to carriers. Hydrolysis of the ester group gave a carboxylic acid derivative that was soluble in water at pH > 7, in sharp contrast to the glycolipid counterpart, which is normally insoluble [54]. Such neoglycolipids and neoglycolipid analogs may still be recognized by a lectin in virtually the same manner as the original lipid. This approach might be of value for the preparation of water-soluble analogs of glycosphingolipids, including gangliosides. A useful property of the (pre-spacer) DIB glycosides described here is their relative stability towards Lewis acid-induced anomerisation, which makes the synthesis of pure /%glycosides easier [54]. Synthetic bis-sulfide neoglycolipids dissolve in DMSO (1% solution) by heating to ~ 100°C. On cooling, a clear gel was formed that slowly disintegrated into solid particles. The same phenomenon was observed with natural galactosyl ceramide [56]. Reductive amination of maltotriose with a long-chain primary amine, followed by 0 R ~,,~,",,,,.&~O

~-....._..~0 a~.,~i~,~

~...,.....~OH

•

Glycosylceramide

"~. OH

O, ~

S O 2 ~ SO2~ ' ~ J ~ J ~ , J ~ J ~ J ~ J

~•

~

[57]

O ~,..~.,,.~OH R"""~,~,~",~ NH

O O~ .=

O ..

-/'o-P-O~O~ o

o

R~ - ~ ' ~

(541

[58]

o

o

NH

~

[60]

O R~ - ~ ~ O ~

N

H

.

I

(

~

O

Fig. 10. Examples of double-chain neoglycolipids.

[61]

280

G. M A G N U S S O N

[s2-~l Fig. 11. Examples of cholesterol-based neoglycolipids.

acylation of the resulting secondary amine with a fatty acid derivative, gave a double-tail neoglycolipid that was recognized by Concanavalin A when presented in a glycolipid monolayer [57]. A method has been developed that permits the synthesis of a neoglycolipid from a small amount of saccharide isolated from a natural source [58]. Thus, reductive amination of a saccharide with dipalmitoylphosphatidylethanolamine and sodium cyanoborohydride gave lipids of use in biological investigations. A recent paper deals with the binding of E-selectin to neoglycolipids obtained by the reductive amination route, and to synthetic glycosyl ceramides [59]. It should be noted, however, that the saccharide unit at the reducing end of an oligosaccharide will be reductively opened in this type of conjugation reaction. Therefore, there is still a need for reliable, stereoselective methods for the formation of neoglycolipid glycosides from small amounts of oligosaccharides isolated from natural sources, while retaining the structure of the reducing-end monosaccharide unit. Glycopeptides have been modified to give neoglycolipids that formed monomolecular layers as demonstrated by surface balance studies and liposome formation [60]. Sialic-acid-containing neoglycolipids have been prepared via a pre-spacer strategy [61]. Thus, 2-azidoethyl glycosides were sialylated and the aglycon of the resulting disaccharide was reduced to amine. Coupling with 2-tetradecylhexadecanoic acid gave the corresponding double-tail neoglycolipid. A selection of double-chain neoglycolipid aglycons are depicted in Fig. 10 together with a natural ceramide, which clearly shows the structural similarity between them.

IV.3. Steroid-based neoglycolipids Cholesterol-containing neoglycolipids have been synthesized by coupling of different saccharides via a spacer to the cholesterol residue as depicted in Fig. 11 [62,63]. Sonication of these lipids gave vesicles having a sugar structure-dependent

~ _M--:-.,,--o FI.~-~,,,,,~,,.~0 ~

o

O~

N H ~

o

NMe2

HN."s°2x'%-J

N H ~

NH [651 0 ~NH~(oH2)nCH3 S~'(CH2)mCH3

Fig. 12. Examples of neoglycolipids that bind influenza hemagglutinin.

SYNTHESIS OF GLYCOSPHINGOLIP1DS AND NEOGLYCOLIPIDS

281

half-life when administered subcutaneously in mice [64]. IV.4. Other neoglycolipids The agglutination of erythrocytes by influenza virus was inhibited by liposomes containing a sialyl ganglioside neoglycolipid [65]. A fluorescent e-sialoside had a very high affinity constant towards influenza hemagglutinin, which makes it useful in competition binding assays [66]. Structures of these neoglycolipid types are shown in Fig. 12. References 1 Hakomori, S. (1991) Possible functions of tumor-associated carbohydrate antigens. Curr. Opin. lmmunol. 3, 646-653. 2 Karlsson, K.-A. (1989) Animal glycosphingolipids as membrane attachment sites for bacteria. Annu. Rev. Biochem. 58, 309 350. 3 Aruffo, A. (1992) Selectins: adhesion molecules with multiple carbohydrate ligands. Trends Glycosci. Glycotechnol. 4, 146 151. 4 Karlsson, K.-A. (1991) Glycobiology: a growing field for drug design. Trends Pharm. Sci. 12, 265272. 5 Gigg, R. (1980) Synthesis of glycolipids. Chem. Phys. Lipids 26, 287-404. 6 Gigg, J. and Gigg, R. (1990) Synthesis of glycolipids. Topics Curr. Chem. 154, 77 139. 7 Israelachvili, J.N., Marcelja, S. and Horn, R.G. (1980) Physical principles of membrane organization. Q. Rev. Biophys. 13, 121 200. 8 Israelachvili, J.N. (1992) Intermolecular & Surface Forces. Academic Press, London. 9 Paulsen, H. (1982) Advances in selective chemical syntheses of complex oligosaccharides. Angew. Chem. Int. Ed. Engl. 21, 155-173. l0 Paulsen, H. (1990) Syntheses, conformations and X-ray structure analyses of the saccharide chains from the core regions of glycoproteins. Angew. Chem. Int. Ed. Engl. 29, 823-938. 11 Zimmerman, P. and Schmidt, R.R. (1988) Synthese von Erythrosphingosinen fiber die Azidoderivate. Liebigs Ann. Chem. 663 667. 12 Kiso, M., Nakamura, A., Nakamura, J., Tomita, Y. and Hasegawa, A. (1986) A convenient synthesis of sphingosine and ceramide from D-xylose or D-galactose. J. Carbohydr. Chem. 5, 335 340. 13 a Schmidt, R.R. (1986) New methods for the synthesis of glycosides and oligosaccharides - are there alternatives to the Koenigs-Knorr method? Angew. Chem. Int. Ed. Engl. 25, 212 235. b Schmidt, R.R. (1989) Recent developments in the synthesis of glycoconjugates. Pure Appl. Chem. 61, 1257- 1270. 14 Ichikawa, Y., Lin, Y.-C., Dumas, D.P., Shen, G.-J., Garcia-Junceda, E., Williams, M.A., Bayer, R., Ketcham, C., Walker, L.E., Paulsen, J.C. and Wong, C.-H. (1992) Chemical-enzymatic synthesis and conformational analysis of sialyl Lewis x and derivatives. J. Am. Chem. Soc. 114, 9283 9298. 15 Ogawa, T. and Yamamoto, H., (1985) Synthesis of a model linear mannohexaose for the backbone structure of fruit body polysaccharide of Tremella fuc(formis and Dichtyophora indusiata FISCH. Agric. Biol. Chem. 49, 475-482. 16 Jansson, K., Ahlfors, S., Frejd, T., Kihlberg, J., Magnusson, G., Dahm6n, J., Noori, G. and Stenvall, K. (1988) 2-(Trimethylsilyl)ethyl glycosides. Synthesis, anomeric deblocking, and transformation into 1,2-trans l-O-acyl sugars. J. Org. Chem. 53, 5629-5647. 17 Magnusson, G. (1992) 2-(Trimethylsilyl)ethyl (TMSEt) glycosides; anomeric blocking, deblocking and activation in the synthesis of oligosaccharides. Trends Glycosci. Glycotechn. 4, 358-367. 18 Ito, Y., Nunomura, S., Shibayama, S. and Ogawa, T. (1992) Studies directed toward the synthesis of polysialogangliosides: the regio- and stereocontrolled synthesis of rationally designed fragments of the tetrasialoganglioside GQlb. J. Org. Chem. 57, 1821-1831. 19 Jansson, K., Noori, G. and Magnusson, G. (1990) 2-(Trimethylsilyl)ethyl glycosides. Transformation into glycopyranosyl chlorides. J. Org. Chem. 55, 3181-3185, 20 Kartha, K.P.R. and Jennings, H.J. (1990) A facile, one-step procedure for the conversion of 2-

282

G. MAGNUSSON

(trimethylsilyl)ethyl glycosides to their glycosyl chlorides. Tetrahedron Lett. 31, 2537-2540. 21 Koike, K., Sugimoto, M., Sato, S., Ito, Y., Nakahara, Y. and Ogawa, T. (1987) Total synthesis of globotriaosyl-E and Z-ceramides and isoglobotriaosyl-E-ceramide. Carbohydr. Res. 163, 189-208. 22 Sato, S., Nunomura, S., Nakano, T., Ito, Y. and Ogawa, T. (1988) An efficient approach to stereoselective glycosylation of ceramide derivatives: use of pivaloyl group as a stereocontrolling auxiliary. Tetrahedron Lett. 29, 4097-4100. 23 Zimmermann, P., Bommer, R., B~ir, T. and Schmidt, R.R. (1988) Azidosphingosine glycosylation in glycosphingolipid synthesis. J. Carbohydr. Chem. 7, 435-452. 24 Nicolaou, K.C., Caulfield, T.J. and Kataoka, H. (1990) Total synthesis of globotriaosylceramide (Gb3) and lysoglobotriaosylceramide (lysoGb3) Carbohydr. Res. 202, 177 191. 25 Murase, T., Ishida, H., Kiso, M. and Hasegawa, A. (1989) A facile, regio- and stereo-selective synthesis of ganglioside GM3. Carbohydr. Res. 188, 71 80. 26 Bartra, M., Urpi, F. and Vilarrasa, J. (1987) New synthetic 'tricks'. [Et3N][Sn(SPh)3] and Bu2SnH> two useful reagents for the reduction of azides to amines. Tetrahedron Lett. 28, 5941-5944. 27 Nicolaou, K.C., Caulfield, T.J., Kataoka, H. and Stylianides, N.A. (1990) Total synthesis of the tumor-associated Lex family of glycosphingolipids. J. Am. Chem. Soc. 112, 3693 3695. 28 Hasegawa, A., Ando, T., Kameyama, A. and Kiso, M. (1992) Synthetic studies on sialoglycoconjugates 40: stereocontrolled synthesis of sialyl Lewis X epitope and its ceramide derivative. J. Carbohydr. Chem. l l, 645-658. 29 Toepfer, A. and Schmidt, R.R. (1992) An efficient synthesis of the Lewis x (Lex) antigen family. Tetrahedron Lett. 33, 5161 5164. 30 Nicolaou, K.C., Hummel, C.W. and lwabuchi, Y. (1992) Total synthesis of sialyl dimeric Le x. J. Am. Chem. Soc. 114, 3126 3128. 31 Matsuzaki, Y., lto, Y. and Ogawa, T. (1992) Synthesis of triantennary blood group I antigens: neolacto-glycopentadecaosyl ceramide. Tetrahedron Lett. 33, 6343 6346. 32 Kameyama, A., Ishida, H., Kiso, M. and Hasegawa, A. (1991) Synthetic studies on sialoglycoconjugates 22: total synthesis of tumor-associated ganglioside sialyl Lewis x. J. Carbohydr. Chem. 10, 549 560. 33 Okamoto, K. and Goto, T. (1990) Glycosidation of sialic acid. Tetrahedron 46, 5835 5857. 34 Prabhanjan, H., lshida, H., Kiso, M. and Hasegawa, A. (1991) Chemical synthesis of gangliosides and their analogs. Trends Glycosci. Glycotechnol. 3, 231 xxx. 35 Kondo, T., Abe, H. and Goto, T. (1988) Efficient synthesis of 2c~-glycoside of N-acetylneuraminic acid via phenylsulfenyl chloride adduct of 2-deoxy-2,3-dehydro-N-acetylneuraminicacid methyl ester tetra-O-acetate. Chem. Lett. (Japan) 1657 1660. 36 Ito, Y. and Ogawa, T. (1988) Highly stereoselective glycosylation of N-acetylneuraminic acid aided by a phenylthio substituent as a stereocontrolling auxiliary. Tetrahedron Lett. 29, 3987 3990. 37 lto, Y., Numata, M., Sugimoto, M. and Ogawa, T. (1989) Highly stereoselective synthesis of ganglioside GD3. J Am. Chem. Soc. 111, 8508-8510. 38 lto, Y., Nunomura, S., Shibayama, S. and Ogawa, T. (1992) Studies directed toward the synthesis of polysialogangliosides: the regio- and stereocontrolled synthesis of rationally designed fragments of the tetrasialoganglioside GQ~b. J. Org. Chem. 57, 1821 1831. 39 Nicolaou, K.C., Hummel, C.W., Bockovich, N.J. and Wong, C.-H. (1991) Stereocontrolled synthesis of sialyl LeX, the oligosaccharide binding ligand to ELAM-1 (sialyl = N-acetylneuramin). J. Chem. Soc. Chem. Commun. 870-872. 40 Murase, T., Kartha, K.P.R., Kiso, M. and Hasegawa, A. (1989) Synthesis of the c~-NeuAc-(2 --. 6)Gal structure. Facile 6-O-sialylation following stannylene activation of an unprotected Dgalactopyranoside. Carbohydr. Res. 195, 134-137. 41 Higashi, K., Miyoshi, S., Nakabayashi, S., Yamada, H. and Ito, Y. (1992) New methods suitable for large-scale preparation of sialoglycosides. Chem. Pharm. Bull. 40, 2300 2303. 42 Hasegawa, A., Ohki, H., Nagahama, T., Ishida, H. and Kiso, M. (1991) A facile, large-scale preparation of the methyl 2-thioglycoside of N-acetylneuraminic acid, and its usefulness for the ctstereoselective synthesis of sialoglycosides. Carbohydr. Res. 212, 277 281. 43 Roy, R., Andersson, F.O. and Letellier, M. (1992) 'Active' and 'latent' thioglycosyl donors in oligosaccharide synthesis. Application to the synthesis of c~-sialosides. Tetrahedron Lett. 33, 6053 6056. 44 Marra, A. and Sinai, P. (1990) A novel stereoselective synthesis of N-acetyl-~-neuraminosyl-galactose

SYNTHESIS OF GLYCOSPHINGOLIPIDS AND NEOGLYCOL1PIDS

283

disaccharide derivatives, using anomeric S-glycosyl xanthates. Carbohydr. Res. 195, 303 308. 45a Birberg, W. and L6nn, H. (1991) ~-Selectivity and glycal formation are temperature dependent in glycosylation with sialic acid. Synthesis of a Neu5Ace(2 ~ 6)Gal thioglycoside building block. Tetrahedron Lett. 32, 7453 7456. 45b Birberg, W. and L6nn, H. (1991) Glycosylation with sialic acid at HO-3 of three different O-protected D-galactosides in acetonitrile/dichloromethane at low temperature. Tetrahedron Lett. 32, 7457 7458. 45c L6nn, H. and Stenvall, K. (1992) Exceptionally high yield in glycosylation with sialic acid. Synthesis of a GM3 glycoside. Tetrahedron Lett. 33, 115 116. 46 Ray, A.K., Nilsson, U. and Magnusson, G. (1992) Synthesis and conformational analysis of GM3 lactam, a hydrolytically stable analogue of GM3 ganglioside lactone. J. Am. Chem. Soc. 114, 2256 2257. 47 Kondo, H., Ichikawa, Y. and Wong, C.-H. (1992) fl-Sialylphosphite and phosphoramidite: synthesis and application to the chemoenzymatic synthesis of CMP-sialic acid and sialyloligosaccharides. J. Am. Chem. Soc. 1!4, 8748--8750. 48 Martin, T.J. and Schmidt, R.R. (1992) Efficient sialylation with phosphite as leaving group. Tetrahedron Lett. 33, 6123 6126. 49 Plewe, M., Sandhoff, K. and Schmidt, R.R. (1992) Synthesis of 4-C-methyl analogues of glucosylceramide. Carbohydr. Res. 235, 151-161. 50 Dahmbn, J., Frejd, T., Magnusson, G., Noori, G. and Carlstr6m, A.-S. (1984) Synthesis of spacerarm, lipid, and ethyl glycosides of the terminal trisaccharide [~-D-GaI-(1 ~ 4)-fl-D-Gal-(l ~ 4)-fl-DGIcNAc] portion of the blood-group P1 antigen: preparation of neoglycoproteins. Carbohydr. Res. 129, 63 71. 51 Magnusson, G. (1986) Synthesis of neo-glycoconjugates. In: D. Lark, (Ed.), Protein-Carbohydrate Interactions in Biological Systems. Academic Press, London, pp. 215 228. 52 Dahmdn, J., Frejd, T., Gr6nberg, G., Lave, T., Magnusson, G. and Noori, G. (1983) 2-Bromoethyl glycosides: application in the synthesis of spacer-arm glycosides. Carbohydr. Res. 118, 292-301. 53 Kihlberg, J., Hultgren, S.J., Normark, S. and Magnusson, G. (1989) Probing of the combining site of the PapG adhesin of uropathogenic Escherichia coli bacteria by synthetic analogues of galabiose. J. Am. Chem. Soc. 111, 6364 6368. 54 Magnusson, G., Ahlfors, S., Dahm+n, J., Jansson, K., Nilsson, U., Noori, G., Stenvall, K. and Tj6rnebo, A. (1990) Prespacer glycosides in glycoconjugate chemistry. Dibromoisobutyl glycosides for the synthesis of neoglycolipids, neoglycoproteins, neoglycoparticles, and soluble glycosides. J. Org. Chem. 55, 3932 3946. 55 Lingwood, C.A. and Magnusson, G. Unpublished results. 56 Magnusson, G. Unpublished observation. 57 Read, B.D., Demel, R.A., Wiegandt, H. and van Deenen, L.L.M. (1977) Specific interactions of Concanavalin A with glycolipid monolayers. Biochim. Biophys. Acta 470, 325 330. 58 Stoll, M.S., Mizuochi, T., Childs, R.A. and Feizi, T. (1988) Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides. Biochem. J. 256, 661 664. 59 Larkin, M., Ahern, T., Stoll, M.S., Shaffer, M., Sako, D., O'Brien, J., Yuen, C.-T., Lawson, A.M., Childs, R.A., Barone, K.M., Langer-Safer, P.R., Hasegawa, A., Kiso, M., Larsen, G.R. and Feizi, T. (1992) Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelialleukocyte adhesion molecule E-selectin. J. Biol. Chem. 267, 13661 13668. 60 Waldmann, H., Mfirtz, J. and Kunz, H. (1990) Synthesis of 2-acetamido-2-deoxyglucosylasparagine glyco-tripeptides and -pentapeptides by selective C- and N-terminal elongation of the peptide chain. Carbohydr. Res. 196, 75 93. 61 Hasegawa, A., Terada, T., Ogawa, H. and Kiso, M. (1992) Studies on the thioglycosides of Nacetylneuraminic acid 10: Synthesis of S-(~-sialosyl)-(2 -~ 6)-O-2-acetamido-2-deoxy-fi-D-hexopyranosyl ceramide and its related compounds. J. Carbohydr. Chem. 11,319 331. 62 Ponpipom, M.M., Bugianesi, R.L. and Shen, T.Y. (1980) Cell-surface carbohydrates for targeting studies. Can. J. Chem. 58, 214-220. 63 Slama, J. and Rando, R.R. (1981) The synthesis of glycolipids containing a hydrophilic spacer-group. Carbohydr. Res. 88, 213 221. 64 Mauk, M.R., Gamble, R.C. and Baldeschwieler, J.D. (1980) Vesicle targeting: timed release and specificity for leukocytes in mice by subcutaneous injection. Science 207, 309 311.

284

G. MAGNUSSON

65 Kingery-Wood, J.E., Williams, K.W., Sigal, G.B. and Whitesides, G.M. (1992) The agglutination of erythrocytes by influenza virus is strongly inhibited by liposomes incorporating an analog of sialyl gangliosides. J. Am. Chem. Soc. 114, 7303 7305. 66 Weinhold, E.G. and Knowles, J.R. (1992) Design and evaluation of a tightly binding fluorescent ligand for influenza A hemagglutinin. J. Am. Chem. Soc. 114, 9270 9275.