CA2099306A1 - Polymeric lewis x saccharides and a process for their preparation - Google Patents

Abstract

Abstract The invention relates to a process for the preparation of polymeric Lewis X saccharides of the formula wherein X is OC(=NH)CCl3 (TCAI), Z or S, Z is OCH2CH(NHCOCuH2u+1)CH(OH)CH=CHCvH2v+1, S is (CH2)qCOOR'', R is OH or R?g, R?g is QAc, OBn or OBz, R' is OH, OAc or OPiv R'' is C1 to C4-alkyl, Ac is acetyl, Bn is benzyl, Bz is benzoyl, Piv is pivaloyl, n is an integer from 0 to 7, q is an integer from 4 to 12, U is an inteqer from 13 to 23 and v is an integer from 11 to 17, a process for the preparation of corresponding inter-mediate products, and new polymeric Lewis X saccharides, in particular tetrameric to octameric Lewis X antigen.
The polymeric saccharides can be synthesised stereoselectively in high yields.

Description

- 2~9~3~8 Merck Patent Ge~ellschaft mit beschrankter Haftung 6100 Darmstadt Polymeric Lewi~ 2 Saccharides and a process ~n~:~h~ 5--b~r~tion The invention relates to polymeric Lewis X
saccharides and a new effective process for their prepar-5 ation. The invention particularly relates to di- ~o octameric Lewis X derivatives.
The biological Lmportance of glycocon~ugates, in particular glycolipids and glycosphingolipidR in their role as tumour-a~sociated antigç~ns in the field of tumour therapy and tumour diagnostics (for example Hakomori (1985), Cancer Res. 45, 2405), has aroused great interest in chemical synthese~, abo~e all in the oligosaccharide content. The oligosaccharide epitopes can function as haptens and initiate antibodies which can be used as potential candidate~ or the therapeutic or diaqnostic treatment of carcinomas and their meta~tase~ or for the stimulation of endogenou~ defence again~t tumour cells (for example Castronovo et al. (1989), J. Natl. Cancer Inst. 81 (3), 212).
A known tumour-associated antigen is the Lewis antigen X (Le~), the oligosaccharide component of which is composed of Gal~ 4)[Fuc~3)]GlcNAc. Lewi~ antigen X is known in the monomeric, dimeric and trimeric form (for example Holmes et al. (1987), J. Biol. Chem. 262, 11331 and 15649).
Previous syntheses of the Le~ antigen have con-centrated on various synthesis stra~egie6, the use of different s~nthesi~ units and the use of different protective groups. Thus, for example, 4-O-unprotected glucosamine ac~ceptors have been reacted with galactosyl donors and~ after remo~al of tha 3-O protection, the product has been linked to a fucosyl radical (for example; Nicolaou et al. (1990), J. Am. Chem. Soc.
112, 3693; Nic:olaos et al. (1992), J. ~m. Chem. Soc.
114, 3126; Sato et al. ~1988), Tetrahedron Lett. ~9, , , 2 0 ~ 6 - 2 ~
5267; Sato et al. (1987), Carbohydr. Re~. 167, 197;
Hindsgaul et al. (1982), Carbohydr. Res . 109, 109; L~nn (19B5), Carbohydr. Res. 139, 115; and Classon et al.
(1989), J. Carbohydr. Chem. 8, 543). Alternatively, chain lengthening of corresponding lactosamine derivatives with subsequent fucosylation has also been carried out (for example: Nillson et al. (1987), Glycokon~ugate J. 4, 219, and Nillson et al. (1988), Carbohydr. Res. 183, 183), or a strategy based on azidolactose has been developed (for example Bommer et al. (1990), Liebigs Ann. Chem. 425). It was possible to achieve further improvements by alpha-fuco~ylation with fuco~yl donors using the ~Inverse Procedure" (for example Schmidt et al. (1991), Tetra-hedron Lett. 32, 3353) or by ~-glycosidic bonding by mean~ of 2-azidoglycosyl donors using ~he ~Nitrile Effect" ~for example Schmidt et al. (1990), Synlett 694;
and Vankar et al. (1391) Tetrahedron Lett. 47, 9985).
This method achieved, inter alia, a high Rtereo-selectivity, especislly in the glyco~ylation step. This is necessary so that the predicted biological activity i8 retained.
In spite of the optimising mea~ures de~cribed, it is found that the yields during synthesis of known tetra-, hexa- and octasaccharides a~ a rule decrease significantly as the chain length increases. ~ccordingly, it wa~ to be expected that application of the kn~wn method to ~ynthesi~ of higher polymeric oligo~accharides, which are likewise of great interest for the applications mentioned, gives even lower yields and therefore an inadequate profitability.
~ bove all, it was not to be expected that tri-, tetra-, hexa- or even octEmeric Lewis ~ derivatives, that is to say oligosaccharides which are composed of eleven, fourteen, twenty or twenty-six monosaccharide units, are also accessible in satisfactory yields by the known method.
The ob~ect was thus to modify and optimise the method proven for lower LeX ~lnits such that derivatives .
,: ~
' - ' ' ' 2~93~6 b~yond octasaccharides are al~o acce~sibl~ by ~ynthesis in a practicable manner.
It has now been found that polymeric Lewis X
saccharides can be prepared by any desired chain lengthening starting from LeX hexasaccharides using corresponding donor and acceptor units, using as the donor units trichloroacetLmidates (TCAI) and as th~
acceptor units sacch~ride3 !having sterically bulky protective groups, in particuLar O-tert-butyldimethyl-silyl compounds (OTBS) of multi.ples of LeX trisaccharideel~ment~, if desired additionally with a di-saccharide which functions as an acceptor.
According to the invention, the acceptor com-pounds men~ioned are distinguished by arylidene, in particular 4,6-O-benzylidene, protective groups, above all on the terminal galactopyranose rings, so that easy selective access to the particular 3-O-position, which is re~ponsible for the glycosyl acceptor properties, i8 possible.
It has moreover been found that the Le~
hexasacchaxides can be prepared effectively via corresponding trisaccharides from the readily accessible tert-butyldimethylsilyl 2-azido-4,6 O-benzylidene-glucopyranoside or a corresponding 2-azido-glucopyrano~ids silylated and~or arylidenated elsewhere, which likewise has ~he abovementioned pat~ern of protective groups with the coxresponding proper~ies for providing donor and acceptor functions.
Polymeric LeX derivatives are thus obtained with stereoselectivity rates of 90 to 100~, preferably 95 to 99%, in yields of be~ween 65 and 85~, preferably be~ween 70 and 80%, ~ased in each case on one chain lengthening cycle. Surprisingly, the yields do not decrease as the chain length increases, for example from dimeric via tetrameric to octameric Lewis X derivatives o~ the com-pounds accord;ing to the invention. ~n even greater chain lengthening (n > 7, Formula I, IV) gives analogous results and ]Leads to ths conclusion that even highly :
- . ~ ` ` ' ' ~':
.

-1 . ~ - ,` ., . . , ~ , .

20~9306 polymeric Lewi~ X saccharides can be provided with the aid of the process according to the invention.
The invention thus relates to a proces~ for the preparation of polymeric Lewis X saccharide~ of the formula I
~ ~ ~
R R. R~ ~ HN~c ~ R
~~ ,o~,o~o~,~
HllAc ~ ~ J ~ R R' wherein X is OC~=NH)CC13 (TCAI), ~ or S, Z is OCH2CH(NHCOCUH2u+l~cH(OH~cH=
S is (CH2)gCOOR~ ~ t 10 R i~ OH or R~6, R98 is OAc, OBn or OBz, R' is OH, OAc or OPiv R'' is Cl to C~-alkyl, Ac i~ acetyl, Bn is benzyl, Bz i5 benzoyl, 15 Piv is pivaloyl, n is an integer from 0 to 7, q is an integer from 4 to 12, u is an integer from 13 to 23 and v i~ an integer from 11 to 17, starting from a hexa~accharide unit, characterised in that the following ~tep~ are carried out:

(a) glycosidation of a donor ~accharide unit activated on the l-O-po~ition (II [TCAI/(x)]), chosen from one of the compound~ of the formula II

:--.
.

. . .- ~ .

~0993a~

~e ~ R ~ S ~ ~oM ~ o~ R
R

wherein Y is OC(NH)CCl3 (TCAI~ or W, W is O-tert-butyldimethylsilyl (OTBS), O-thexyl-dimethylsilyl (OTDSl or O-tert-butyldiphenyl-silyl (OTDPS), R is as defined, Rl is R, Ar is aryl, Ne is methyl and k and 1 are integer~ from 0 to S, and wherein, if Y is TCAI, Rl is OAc, OBn or OBz, and if Y is W, R1 is OH, with an acceptor saccharide unit o~ the formula V
(V[(y)]), A~ Me ~ ~ _ _ ~0 0~ 0~0~

~ ~ W
wherein R, Ar, ~e and W have the meanings given and m is an i:nteger from 0 to 5, : ~ . - , . :~ . . : .

20~9306 to give a saccharide of the formula II of higher chain length (II [w ~x + y)], wherein x i8 6, 9, 12, 15, 18 or 21 and y i8 3, 6, 9, 12, 15 or 18 and represent the number of monosaccharide element~ in the polysaccharide in que~tion, and x + y may not be greater than 24;

(b) conversion into the corresponding donor saccharide of the formula II of higher chain length (II [TCAI/(x + y)]J by suhstitution of the free OH
group(s) by a corresponding protective group and of the W radical by the TCAI radical;

(c) glycosidation of ~he donor saccharidQ from (b~, which ha~ been activated on the l-O-position, with a disaccharide of the formula III which acts as an acceptor, R R~
~j,o~R

wherein R and R' have the meanings given, to give a saccharide of the formula IV [~x ~ y + 2)], ~R~R

wherein R, R', Ar, Ma and n have the meaning~ given;
and 20993~6 (d) reduction of the a~ido groups to acetamido group~ of the compound IV, splitting off of th~ arylidene a~d benzyl radicals and peracetylation of the radicals R and R , substitution of the glycosidic acetyl group by OH and reaction to give the corresponding trichloroacetimidate (X = TCAI) of the formula I, and if desired modification of the radical ~ to Z or S and removal of the protectiv2 groups (R, R = OH~.

The invention furthermorQ relates to a procQs~
for the preparation of Lewis X hexa~3accharidas of the formula II (II ~TCAI/~/(x)], k = 0, 1 = 1, x = 6), characterised in that an arylidene-2-azido-glucopyran-oside equipped with a sterically bulky silyl protectiv~
group, and in particular tert-butyldimethylsilyl 2-azido-4,6-O-benzylidene-glucopyrano4ide, i8 reacted with trichloroacetLmidates o corre~ponding monosaccharides to give a ~risaccharide of the formula II (II lW~(xl], k = 0, 1 - 0, x = 3), and a donor trisaccharide of $he formula II (II [TC~I~(x), x = 3~ and an acceptor tri-saccharide of the formula V (v t(y)], m = 0, y = 3~ areprspared from thi~ and are reacted with one ano~h2r to give the corresponding hexasaccharide.
~ ew polymeric hewi3 X saccharides, ln par~icular the tetrameric , hexameric and octameric Lewis X antigen, can also be ~3yn~hesised chemically with the aid of the process according to the invention.
The inven~ion thu~3 also relate~ to polymeric Lewis X saccharides of the formula I with the a~ove mentioned meanings, but wherein n i~3 an integer from 2 to 7, with the proviso that if n is ~, X may not be Z where u = 15 and v = 13 and R and R may not be OH. These are, in particular: `

tetrameric Lewis X antigen of the formula I (n = 3), hexameric Lewis ~ antigen of the formula I (n = 5) and octameric hewis x an~igen of th~ formula I (n = 7).

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

- 8 - 2~39306 The invention furthermore relate~ to polymeric Lewis X saccharides of the formula II with the above-mentioned meanings, but wherein k is 1 and 1 iB an integer from 1 to 5, and to polymeric Lewis X saccharides of the formula IV with the meaning8 given, but wherein n is an integer from 2 ko 7, and Pinally to polymPric Lewi4 saccharides of the formula V with the meanings given, but wherein m is an integer from 1 to 5.
The compounds of the formulae I, II, IV and V
according to Claims 7 to 11 are suitable for use in tumour dia~nostics and for the preparation of antibodies for tumour therapy. Suitable tumours here are those which carry the corresponding polymeric Lewis X antigens on their surface. ~xamples of such tumour~ can be: gastro-inte~tinal tumours, mammary carcinoma, ovarian carcinoma,hepatic carcinoma and gastric or pancreatic carcinoma.
Such antibodies are prepared here by the customary standard method~ known from the literature (for example Harlow, Lane: Antibodie3, A Laboratory Manual; Cold Spring Harbor 1988).
Finally, the invention thus relates to the U8Q of the compound~ of the formulae I, II, IV and V according to Claims 7 to 11 for the preparation of antibodie~ for tumour therapy and tumour diagnostics.
The following abbreviations are used above and below:

TCAI trichloroacetimidate OTBS O-tert-butyldimethylsilyl TBAF tetrabutyla~moniumfluoride TMSOTf trLmethyl~ilyl ~rifluoromethanesulfonate DBU 1,8-diazabicylo[5.4.0]undec-7-ene OTDS O-theY~ldimethylsilyl OTDPS O-tert:-butyldiphenylsilyl S i9 a spacer radical (CH2~qCOO~'', wherein R'' is Cl to C4-alkyl and q can a~suma the values mentioned.
Alkyl here c~n be straight-chain or branched and i8, :

-: : . : ~ , : .:
- , :
' ~ - ' 2~9~3~S
g specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec- or tert-butyl. R'' i~
preferably methyl or ethyl. q is preferably 6 to 10, but in particular 8. The particularly preferred spacer radical is -(CH2)~COOEt.
Z has the meaning given, but is preferably OCH2CHtNHCOCl5H3l)CH(OH)CH=CHCl3H2, (ceramide)O u can assume the values 13 to 23, preferably 15 to 20, but in par-ticular 15, and v can assume thle value3 11 to 17, prefer-ably 13 to 15, but in particular 13. W i~ a sterically bulky (large) 8ilyl protective group, such as, for example, OTBS, OTDS or OTDPS. OTBS i8 particularly preferred. Ar i~ substituted or unsubstituted aryl or heteroaryl, preferably phenyl. Th~ preferred protective group on the particular galactopyranoside ring is thu3benzylidene. In addition to the arylidene protective groups according to the invention, isopropylidene can also he used, although wi~h limitations.
R8~ is a protective group and i8 O-acetyl, O-benzyl or O-benzoyl.
Figure 1 (1.1 to 1.5) reprasents the most important compound~ of the proces~ according to the invention. The numbers relate to the correspondinq identification in the Examples.
The proce~s accordiny to the invention is des-cribed in a general fonm below. Process step~ which correspond to ~tandard method~ of carbohydrate chemi~try, such as, for example, the introduction and spli~ting-off of suitable protective groups, are not explained in more detail. Reference is made to the standard literature in this respect (for example T.W. Greene, Protective Groups in Organic Synthesis, 1981, John Wiley ~ Sons; and Method~ in Carbohydrate Chemistry, Vol. I-VIII, Academic Press).
The process according to the invention is prefer-ably carried out with the protective groups mentioned above and belo~w ~OAc, OBn, OBz, N3, OPiv); however, other known protect:ive ~roups which are adequate in their - lO 209~30~

introduction and splitting-off actLon can also be used.
The trichloroacetimidate method in general and for synthesis of defined oligosaccharides is described adequately in the abovementioned literature references.
Details of the process according to the invention are to be found in the examples.
Starting compounds for the syntheses according to the invention are the saccharides of the compounds of the formula II, from which donor and also acceptor units (formula V) are prepared according to the invention and from which the polymeric chains are built up in recurring synthesis cycles. The azido-tri~accharides of the formula II (k = O, 1 = O) are the smallest unit. These central base element~, which compri~e a galactopyranose, fucopyranose and a glucopyranose ring~ are prep~red according to the invention from corresponding monosaccharide units by methods which are known per se, readily accessible tert-butyldimethylsilyl 2-azido-4,6-O-benzylidene-glucopyranoside preferably being u~ed as the starting substance. However, a 2-azido-glucopyranoside correspondingly protected el~ewhere is also suitable. Details are given in Examples 1 to 8.
Table I (A, ~ provides a classification of the compounds of the formulae II and V, wherein N i~ OTBS by way of example.

' - ~ ` .

. .

2~9~30~

Table I (~3:

Compounds of the type II [Y/(x)]. Y = TCAI or OTBS;
x = number of monosaccharide elements, z = number of trisaccharide elements; A = number of the compound synthesised (Example No.); R~8, k and l are a~ defined.

Type x/z Rl k 1 ~
. . ~
II [OTBS] 3/1 - 0 0 S
II [TCAI] 3/l - O 0 10 II [OTBS] 6/2 OH 0 1 13 II [TCAI~ 6J2 R~8 0 1 16 II [OTBS]1) 9/3 OH 1 1 38 II [TCAI~l~ 9/3 R~6 1 1 41 II [OTBS]l) 12/4 OH l 2 52 II [TCAI]l) 12/4 R98 1 2 55 II [OTBS]1) 15/5 OH l 3 II [OTBS]l) 18/6 OH 1 4 II ~OTBS]1) 21/7 OH 1 5 II [~TBS]l) 24/8 OH 1 6 II tOTBS32) 24/8 OH 6 II [oTBS]3) 24/8 OH 3 3 The compound types designated 1) relate to those compounds which can be prepared from a hexasaccharide donor and a corresponding acceptor unit.
As shown in Table I, combination3 of donors of higher chain length with acceptor saccharides of cor-respondingly lower chain length are also possible (for example ~): in this case, a 21-saccharide donor i8 linked with a trisaccharide accep~or; 3): linking o~ two dodeca-saccharides)~

:
. ~ ~

Table I (B):

Compounds of the type V [(y)]. y - numb~r of monosaccharide elements; z = number of trisaccharide elements; A = number of the compound synthesised (Example No.), m is as defined.

Type y/z m A
. . .
V [OTBS] 3/1 0 12 V [OTBS] 6/2 1 51 10 V [OTBS~ 9/3 2 V [OTBS]12/4 3 V [OTBS]15/5 4 V [OTBS]18/6 5 Table 2 show~ the reactions of various compound~
of the formula II (II tTCAI/OTBS (x3]~ and of the formula V (V rOTBS (y)]), in which W in turn i~ OTBS merely by way of example.

,:

.
.

; ' . : , .

, 2~993~6 Table II:

The ~alues in () correspond to the number of mono-saccharide units in the particular compound; ~: number of the compound synthesised (Exlample No.); B: reaction conditions (Table III); C: y.ield to be expected (%) (+ 5~; X is as defined (formu].a I).

St~rting Prcduct~sl A B C
co~pound( 8 ) _ _ II~OTBS(3)~ II[TCAI(3)] 10 a 95 IItOTBS(3)~ V[OTBS(3)] 12 b 90 II[TCAI(3)]~VlOTBS(3)] II~OTBS(6)] 13 c 80 IIrOTBS(6)] IIlTCAI(6)] 16 d 95 II[TCAI(6)] + III(2) IV(8) 26 c 75 IV(8) It~(8)] 27-37 e-k 75 II[TCAI(6)]+V~OTBS(3)] IItOTBS(9)] 38 c 75 II[OTBS(9)] II[TCAI(9~] 41 d 90 II[TCAI(9)] + III(2) IV(ll~ 42 c 70 IV(11) I[x(ll)] 42-49 e-k 75 . . _ . . . _ II[OTBS(6)] V[OTBS(~)] 51 b 85 [TCAI(6) ]~V[OTBS(6)] II[OTBS(12)] 5~ c 80 II[OTBS(12)] II[TCAI(12)] 55 d 95 II[TCAI(12)~ + III(2) IV(14) 56 c 75 IY(14) I[x(l4)] 57-63 e-k 75 II[OTBS(9)] V~OTBS(9)] ~ b 85 II[TCAI(9) ]+V[OTBS(9)] II[OTBS(18)] - c 80 II[OTBS(18)] II[TCAI(18)] ~ d 90 II[TCAI(18)] + III(2) IV(20) _ c 80 IV(20) I[~20)] ~ e-k 75 . . . _ . .

.

.. . .
-.

2~9~3~

Table II (continued):
Starti~g Product(s) A B G
Compound(s) . . _ .
II[OTBS(12)] V[OTBS(12)] - b 85 II~TCAI(12)]+V[OTBS(12)] II[OTBS(24)] - c 75 II[OTBS(24)] II[T~I(24)] ~ d 90 II[TCAI(24)] + III(2) IV(26) _ c 70 IV(25) I[X(2b)] _ e k 75 II[OTBS(9)] V[OTE~S(9)] ~ b 85 II[OTBS(15)] V[oTE~s(l5)] ~ b 80 II[OTBS(18)] V[OTBS(l8)] - b 80 IIrOTBS(15)] II[OTBS(15)] - d 95 15 IItOTBS(21)] II[OTBS(21)] ~ d II[TCAI(9)]+V[OTBS(3)] II~OTBS(12)J - c 85 II[TcAI(6)]+v[oTBs(9)] II~OTBS(15)] - c 85 II[TCAI(9)]+v[OTBs(6)] II[OTBS(15)~ - c 80 II[TCAI(12)]+Y[OTBS(3)] II~OTBS(15)~ - c 85 II[TCAI(6)]+V[OTBS(12)] II[OTBS(1~)] - c 80 II[TCAI(12)]+V[OTBS(6)~ II[OTBS(18)] - c 80 II[TCAI(15)]+Vt(OTBS(3)] II[OT~S(18)] - ~ 85 [TCAI(6)]+V[OTBS(15)] II[OTBS(21)] - c IIrTCAI(9)]~V[OTBS(12)~ II[OTBS(21)] c 2S II[TCAI(12)]+VlOTBS(9)] II[OTBS(21)] - c ~ c~ 5)]+v[oTBs(6)] II[OTBS(21)] - c II[TC~I(18)~+VtOTBS(3)~ II[OTBS(?l)] - c II[TCAI(6)]+V~OTBS(18)] II[OTBS(24)] - c 80 II[TC~I(9)]+V[OTBS(15)] II[OTBS(24)~ - c 80 II[TCAI(15)]+V[OTBS(9)~ II[OTBS(24)] - c 80 II[TcAI(l8~]+v[oTBs(6)] IItoTBs(24)] ~ c 80 II[TCAI(15)~ + III(2) IV(17) _ c 75 II[TcAI(2l)] + III(2) IY(23) - c IV(17) I[x(17)] ~ e-k 75 IV(23) I[X(~3)~ - e-k 70 Table III specifiQs the reaction conditions shown in Tab.le II. The reagents li~ted, unless stated - .~

;: - : - ~ - :

2~993~
- 15 _ otherwise, in particular the solvent~, catalysts and reducing agents, can be replaced by other corresponding equivalents which are custo:mary for this purpos0 in carbohydrate chemistry. The conditions also apply when compounds of the formulae II and V, in which W i8 not OTBS and Z is not ceramide (u = 15, v = 13) are used.
The individual reaction 8tep8 are preferably carried out as specified in the table and as described in detail in the examples.

.
.
' ' ~,, - ` ' ' ' . :, : , 2~1~9306 T~ble III:
Reaction Step Reagents~Condition a Introdu~tion of TCAI (l) l) T~AF;
2) CCl3CN, DBU
b Arylidenation l~ NaOCH3/CH30H;
2~ ArCH(OCH3)2, p-T80H
c Glycosidation CH3CN, T~SOTf (-40C) d Introduction of TCAI (2) l~ Ac20/pyridine;
2) T~AF, THF;
3~ CCl3CN, DBg e N3 HNAc conversion l) H2S, pyridine, ~2 2) Ac20/pyridine f Peracetylation l)Pd/C;H2,AcOH/CH30Hdioxane 2) Ac20/pyridine g Glycosidic OH NzH4 x AcOH
h Introduction of TCAI
(formula I) as a i Introduction of Z, S S: S-O~, TNSOTf, CH2Cl2 Z: (ceramide)s 1) 2-azido-3-ben~oyloxy-4-octadecen-l-ol, CH2Cl2, TMSOTf;
2) H2S, pyridine, H20;
(3-dLmethylaminopropyl)-N'-ethylcarbodiLmide, Cl5H3lCOOH
k Remo~al of protective groups NaOCH3, CH30H.

.

, . ; -. , ~ ~ , ' . :
.
.

.

20~93~6 In a preferred embodiment, the glyco~idations treaction c) are carried ou~ in nitrile~ a~ solvents, for example acetonitrile, which means that the donor units no longer havP to be prepared in an anomerically pure form.
This as a rule results in a quilntitative synthesis of th~
trichloroacetimidates. A catalyst, for example trimethyl-silyl trifluorome~hanesulfonate (TMSOTf), i9 expediently added to the batch at a react:ion temperature of -30 to -80C, preferably 40C.
Only very ~mall amounts of ba~e (for example DBU, 0.1-1 %) are necessary for preparation of particular trichloroacetimidate~ (reaction a, d), so that no notice-able isomerisation of the trichloroacetimidates occur~.
~he disaccharide compound~ of the formula III, in particular compound 25, can be easily ~ynthesised in accordance with the instructions of Sato et al. (1988, Tetrahedron Lett. 29, 4097).
Hexasaccharide 13 (Example 13) i8 obtained in crystalline form by the process described, and i~ thus particularly suitable for u~e as a quantitatively pure intermediate stage, which can easily be isolated, for building up the polymeric saccharide chain.
If de~ired, the trichloroacetLmidate radical of the compounds of the formula I can be replaced by a spacer S or Lewis X antigen radical Z defined above by methods which are known per se ~reaction i, k). The spacer radical S is preferably introduced as S-OH in yields of 75 to 90 % at room temperature under the catalytic action of, for example, TM~OTf. The radical Z
is preferably introduced in two steps, and in particular, if it is ceramide, via the compounds (2S,3R,4~)-2-azido-3-benzoyloxy-4-octadecen-1-ol, palmitic acid and N-(3-diaminopropyl)-N'-sthyl-carbodiimide (hydrochloride).
After complete removal of the protective groups in the particular polysaccharide part, the corresponding di- to octameric Lewis X antigens of the formula I (n = 1-7) are obtained.

.

2~993~

The following Examples are intended to illustrate the invention in de~ail.

Example 1 tert-sutyldimethylsilyl 2-azido-2-deoxy-~-D-gluco-pyranoside (1):
Compound 1 is prepared in accordance with the instructions of Kinzy and Scllmidt (Liebigs Ann. Chem.
1985, 1537-1545).

~ample 2 tert-Butyldimethylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-~-D-glucopyranoside (2):
Ben~aldehyde dLmethyl acetal (10~3 g, 67.5 mmol) and p-toluenesulfonic acid (80 mg) are added to (14.37 g, 45 mmol) in absolute acetonitrile (359 ml).
After 1 hour at room temperature, anhydxou~ potassium carbonate (2 g) i~ added and thP mixture i8 shaken for 30 minutes. It i~ filtered and concentrated in vacuo. Flash chromatography of the residue [petroleum ether/methyl acetate (5:1)] gives 2 (17.06 g, 93 %) as a colourless syrup. Thin layer chromatography [petroleum ether/methyl acetate (5:1)]: Rf ~ 0.34.

~xample 3 2,3,4-Tri-O-ben~yl~ L-fucopyranose (3)s Compound 3 i~ prepared in ac~ordance with the instructions of De~ter-Juszynski and Flowers (Carbohydr.
Res. 18 (1971) 219-226).

~ample 4 0-(2,3,4-Tri-O-benzyl-~/~-fucopyranosyl)-trichloro-acetimidate (4):
Trichloroacetonitrile (10 g) and DBU (7 drops) are added to a solution of 3 (10.0 g, 23.0 mmol) in absolute methylene chloride (50 ml). After 30 minutes, the slightly yellowish solution is concentra~ed Ln vacuo and the residue is purified using a short silica gel .

:

- 19 2~993~
column [petroleum ether/methyl acetate t3~ 1 g of triethylamine]. 4 (11.85 g, 89 ~) is obtained in a ratio of ~:~ = 104 as a colourless oil, which gradually solidi-fies; thin layer chromato~raphy [petroleum ether/methyl acetate (3:1) + 1 % of triethylamine]: Rf = O.76 for the ~-trichloroacetLmidate, Rf = O.50 for the ~-trich.loro-acetimidate. The following values result for the ~-trichloroacetimidate: [~20 = -16.0 (c = 1, CHCl3).

E~ample S
tert-Butyldimethylsilyl 0-(2,3,4-tri-0-benæyl-~-L-fuco-pyranosyl)~ 3) 2-azido-4,6-0-benzylidene-2-deoxy-~-D-glucopyranoside (5):
Compound 2 (2.93 g, 7.19 mmol) is dissolved in the smallest possible amount of absolute diethyl e~her under an argon atmosphere, and 0.1 M trimethylsilyl trifluoromethanesulfonate solution (1 ml) is added. A
concentrated solution of compound 4 (6.5 g, 11.25 mmol~
in absolute diethyl ether i8 slowly added dropwise, while stirring. 5 minutes after the end of the addition of ~he trichloroacetimidate, the mixture i8 neutralised with sodium bicarbonate (about 0.5 g), filtered and concen-trated in vacuo. Purification by chromatography using petroleum ether/methyl ~ce~ate (7:1) as the mobile phasa gives compound 5 (5.35 g, 85 ~) as a colourless foam;
thin layer chromatography tpetroleum ether/me$hyl acetate ~521)]: p~ = 0.54 [~]20 = -76.0 (c = 1, ~HCl3).

Bsample 6 ter~-Butyldimethylsilyl 0-(2,3,4-tri-0-benzyl-~-L-fucopy-ranosyl)-(l. 3)-2-azido-6-0-benzyl-2-deoxy ~-D-gluco-pyranoside (6):
A solution of compound 5 (10 g, 12.13 mmol) andsodium cyanoborohydride (7.6 g, 121 mmol) in ab~olute tetrahydrofuran (250 ml) is stirred with a molecular sieve (4 A). A saturated solution of hydrogen chloride in diethyl ether is now added dropwise until the mixture becomes acid.ic. For working up, solid sodium bicarbonate, - ~

-` 2099~06 diethyl ether (400 ml) and saturated sodium bicarbonate solution (lOO ml) are introduced into the reaction ve~sel and the mixture is filtered over glass wool. The organic phase is separated off, concentrated in vacuo and puri-fied by flash chromatography tpetroleum ether/methylacetate (6:1~5.5:1)]. (6.72 g, 67 %) are obtained as a colourless foam; thin layer chromatography [petroleum ether/methyl acetate (4:1)]: R~ = 0.48, [~20 = _30.0 (c = 2, CHCl3).
Example 7 0-(2,3,4,6-Tetra-0-acetyl-~-D-galactopyrano~yl)-tri-chloroacetimidate (7):
Compound 7 is prepared by a proce3s of Schmidt, Michel and Roos (Liebigs Ann. Chem. lg84, 1343 1357).

~ample 3 tert-Butyldimethylsilyl 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)-(1~ 4)-t(2,3,4-tri-0-benzyl-~-L-fucopy-ranosyl)-(1~ 3~]-2-azido-6-0-benzyl-2-deoxy-~-D-gluco-pyranoside (8):
Acceptor 6 (9.0 g, 10.89 mmol) and trichloro-acetimida~e 7 (10.72 g, 21.78 mmol) are di~olved in the smallest possible amount of diethyl ether. A solution of trimethylsilyl trifluoromethanesulfonate (0.15 ml) in diethyl ether (4 ml) is added dropwise, while stirring.
20 minutes after the addition of the cataly3t has ended, the mixture is neutralised with sodium bicarbonate (~bout 2 g~, filtered and concentrated in vacuo. Fla~h chromato-graphy t~oluene/acetone (16sl~ 15:1~ 14:1)] give~ com-pound 8 (8.94 g, 71 %); thin layer chromatography ttoluene/acetone (10:1)]: Rf= 0.47, [~]Z = 19.0 (C = 1, CHCl3).

.

-, - 21 - 209930e Example 9 2,3,4,6-Tetra-O-acetyl-~-D-galactopyranosyl~ 4~-[(2,3,4-tri-O-benzyl-~-L-fucopylanosyl)-(1~ 3)]-2-azido-6-O-benzyl-2-deoxy-~/~-D-glucopyranose (9):
Glacial acetic acid (0.25 ml, 4.32 mmol) is added to a solution of compound 8 (5.0 g, 4.32 mmol) in ab-solute tetrahydrofuran (50 ml) at -45C, while stirring, and a 1 M solution of ~etrabutyl ammonium fluoride in tetrahydrofuran (S ml) i8 then added dropwi~e. The mixture is warmed to 0C and dliluted with diethyl ether (S00 ml), and the organic phas~ is washed with saturated sodium chloride solution ( 3 x 80 ml). Concentration in vacuo and subsequent flash chromatography tpetroleum ether/methyl ace~ate (5 r 4)] gives compound 9 (4.32 g, 95 %) as a colourless foam; thin layer chromatography [petroleum ether/methyl acetate (5:4)]: R~ = 0.35, r~]2o = -7.5 (c = 1, CHC13)-Ex~mple l~
0-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyrano~yl)~ 4)-20 t(2,3,4-tri-O-benzyl--L-fucopyranosyl)-(1~ 3)]-2-azido-6-O-benzyl-2-deoxy-~ D-glucopyranosyl-trichloro-acetimidate tlO):
Trichloroacetonitrile (10 g) and DBU (3 drops) are added to a solution of compound 9 (2.19 g, 2.08 mmol) 25 in absolute methylene chloride (100 ml). After 15 minutes, the mixture is concentrated in vacuo and ~he residue is eluted over a silica gel colu~n using petroleum ether/methyl acetate (3:2) + 1 ~ of triethyl-amine a8 the mobile phase. Compound 10 (2.47 g, 100 %) is obtained in a ratio of ~:~ = 4:~ a~ a colourless foam;
F~ = O.36 for the ~-anomer, R~ = 0~26 for the ~-anomer, [~20 = +27.0 (c = 1, CHC13) for ~he ~-anomer, [~]20 = -24.0 (c = 1, CHC13) for the ~-anomer.

~: :
:: ~` '' :
- ~ . . ~ :

- 22 _ 2~ 3 0 Example 11 tert-Butyldim~thylsilyl O-~-D-galactopyranosyl-(l~ 4)-[2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(1-~ 3)]-2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranoside (11):
S Compound 8 (4.2 g, 3.63 mmol) is dissolved in absolute methanol (about 200 m].), and 2 M sodium methano-late solution (0.5 ml) is added. The solution i~ left to stand overnight and is neutralised the following day with Amberlite~ IR 120 (Rohm and Haas). The ion exchange is filtered off, the filtrate is concentrated i~ vacuo and the residue is coevaporated twice with toluene. Compound 11 (3.22 g) is obtained quantitatively and is analyti-cally pure without further purification; thin layer chromatsgraphy [toluene~acetone (1:1)3 ~ R~ = o . 6, [ ~ ] 20 = -47.0 (c = 1, CHCl3).

~xamp~e 12 tert-Butyldimethylsilyl 0-(4,6-O-benzylidene-~-D-galacto-pyranosyl)-(1- 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl~-(1. 3)]-2-azido-6-o-benzyl-2-deoxy-~-D-glucopyranoside (12):
Benzaldehyde dimethyl acetal (1.1 g, 7 mmol) and p-toluenesulfonic acid (200 mg) are added to a ~olution of compound 11 (3~5 g, 3.5 mmol) in absolute acetonitrile (150 ml). After 1 hour at room temperature, anhydrous potassium carbonate (abo~7t 2 g) is added and the mixture is shaken for 30 minute~. It is filtered and concentrated in vacuo, and the residue is eluted over a ~ilica gel column u~ing petroleum etherJmethyl acetate (7:4-7:5~7:6~1:1) as the mobile phase. Compound 12 (3.22 g, 88 %) is obtained as a colourless foam; thin layer chromatography [petroleum ether/methyl acetate ~ R~ = 0.46, t~]20 = -96.0 (c = 1, CHCl3).

,. , ~ . .
. , : .

2~99306 E~ample 13 tert-ButyldLmethylsilyl 0-t2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopy-xanosyl)-tl~ 3)~ azido-6-o-benzyl-2-deoxy-~-D ~lucopy-ranosyl)~ 3)-(4~6-o-benzylidene-~-~-galactop~ranosyl~-(1- 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyxanosyl)-(1~ 3)]-2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranoside (13)s ~ O.05 M trimethylsilyl trifluoromethanesulfonate solution (0.8 ml) is added dropwise to a ~olution of compound 10 (4.75 g, 4.0 mmol) and 12 (4.84 g, 4.5 mmol) in absolute acetonitrile (60 ml~ at -40C. After 10 minutes, sodium bicarbonate (2~0ut 0.5 g) is added to the solution and the mixture i~ warmed to room temperature.
After filtration and concentration in vacuo, the residue is chromatographed over a short silica gel column usin~
petroleum ether/methyl ace~a~e (3:2). The foam formed on concentration i~ dissolved in diethyl ether (60 ml) and crystallised out with petroleum bsnzine (20 ml). Colour-less crystals (6.72 g, 80 ~) are obtained; hin layer chromatography ~toluene/methyl acetate (4:1)]: ~ = 0.38 t~]20 = -56.5 lc = 1, CHCl3), m.p. = 195C.

~xample 1~
tert-Butyldimethylsilyi 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)~ (2,3,~-tri-0-benzyl-~-L-fuco-pyranosyl)-(l~ 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-gluco-pyrano~yl)-(ll 3)-(2, 0-acetyl-4,6-0-benzylidene-~-D-galactopyrano~yl)-(1~ 4)-t(2,3,4-tri-0-henzyl-~-L-fuco-pyranosyl)-(13)]-2-azido-6-0-benzyl-2-deoxy-~-D-gluco-pyranoside (14)o Compound 13 (6.20 g, 2.95 mmol) is dissolved in pyridine/acetic anhydride (1:1, 30 ml) and the solution is left to stand overnight. The following morning it is concentrated in vacuo and the residue is coevaporated twice with toluene. Flash chromatography [petroleum ether/methyl acetate (5:3~] gives compound 14 (6.13 g, 97 %); thin layer chromatography [petroleum ether/methyl acetate (2:1)]: R~ = 0.22, 1~12 = -70.5 (c = 1, CHCl3).

.
~ ~ . ' . ... ' .
.

2~93~6 Example 15 0-(2,3,4,6-Tetra-0-acetyl-~-D-galactopyrano~yl)~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosy )-(1_ 3)]-(2-a~ido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(1~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(1~3)]-2~azido-6-0-benzyl-2-deoxy-~t~-D-gluCopyrclnOSe (15).
A solution of compouncl 14 (5.83 g, 2.72 mmol) in absolute tetrahydrofuran (50 Dll) i8 reacted with glacial acetic acid (0.16 ml, 2.72 mmol) and a 1 M solution of tetrabutyl ammonium fluoride in tetrahydrofuran (3.4 ml) as described for compound 9. Purifica ion is carried out by means of a short silica gel column using petroleum ether/methyl acetate (1:1. 4:5) as the mobile phase.
Compound 15 (~.41 g, ~8 %) is obtained as a colourle~s foam; thin layer chromatography [petroleum ether/methyl acetate (lrl) ] Rf = 0.30~ [~]20 = _55,5 ~C = 1~ CHCl3).

Example ~
O-(2,3,4,6-Tetra-0-acetyl-~-D-galactopyranosyl)-(1~ 4)-[(2,3,4-0-benzyl-~-L-fucopyranosyl)~ 3)]-~2-azido-6-0-ben~yl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2-0-acetyl-4,6-0-ben2ylidene-~-D-galactopyrano~yl)-(1 D 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(1. 3)]-2-azido-6-0-benzyl-2-deoxy~ D-glucop~ranosyl-trichloroacetimidate (16~s Trichloroac~tonitrile (20 g) and DBU ( 5 drop8 ) are added to a solution of compound 15 (5.41 g, 2.67 ~mol) in absolute methylene chloride (150 ml). Af~er 30 minutes, the mixture i~ concentrated in vacuo and the residue i~ purified by means of ~la~h chromatography tpetroleum ether/ methyl acetate (l:lJ + 1 % of tri-ethylamine]. Compound 1~ (5.8 g, 100 %) is obtained in a ratio of ~ 2 as a colourless foam. Thin layer chromatography [petroleum ether~methyl acetate (1~ R~
= 0.56 for the ~-trichloroacetimidato, R~ = 0.62 for the ~-trichloroacetLmidate, [~] 20 = -46.0 ~c = 1, CHC13) for th~ ~-trichloroacetimidate, [~320 = -62.0 (c = 1, CHCl3) ., . ~ .
,' - , ... . . . : : .: -- : ~
.. . . .- -, . : ~ .

., , .. . . , . : - . , . .. -:

~,~9~306 - 25 _ for the ~-trichloroacetimidate.

~xample 17 Benzyl 0-(2,4,6-tri-O-benzyl-~-D-galactopyranosyl)-(1~ 4)-3,6-di-O-benzyl-2-O-pi~aloyl-~-D-glucopyranoside (~5):
Compound 25 is synthesised by the method of S~to et al. ~Tetrahedron Lett. 29 (:L988) 4097-4100).

E~ampl~ 18 Benzyl 0-(2/3~4r6-tetra-o-acety~ D-galactopyranosyl)-(1- 4)-[(2,3,4-tri-O-benzyl-~ -fucopyrano~yl)~ 3)]-(2-a~ido-6-O-benzyl-2 deoxy-~-D-glucopyranosyl)~ 3)-(2-O-acetyl-4,6-O-benzylidene-~-D-galactopyranosyl)-tl~ 4)-~(2,3,4-tri-O-benzyl-~-L-fucopyranosyl]-(1~ 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-0-benzyl-~-D-galactopyranosyl)~ 4)-3,5-di-0-benzyl 2-0-pivaloyl-~-D-glucopyranoside (2fi):
~ O.05 M trLmethylsilyl trifluoromethanesulfonate solution (0.3 ml) i8 added dropwise to a solution of co~pound 16 as an u/~ mixture (1.35 g, 623 ~mol) and acceptor 25 (1.21 g, 1.25 mmol) in acetonitrile (5 ml) at a temperature of -40C. After 15 minutes, the mixture is neutralised with sodium bicarbonate (about 0.3 g). When the solution has come to room temperature, the sodium bicarbonate i8 filtered off. Purification i8 carried out by means of flash chromatography using petroleum ether,~m thyl acetate (2;1~3:2) as the mobile phase.
Compound 26 (1.37 g, 74 %) is obtained as a colourless foam; thin layer chromatography [petroleum ether~methyl acetate (3:2)]: R~ = 0.42, [~20 = -71.0 (c = 1, CHCl3).

~ample 19 Benzyl 0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-(1~ 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(1~ 3)~-(2-acetamido-~6-O-benzyl-~-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2-O-acetyl-4,6-0-benzylidene-~-D-galacto-pyranosyl)-(1~ 4)-~2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-. .
, ' ,. ~

:

. .
.-'~

(1~ 3)]-(2-acetamido-6-O-benzyl-2-deoxy-~-D-gluco-pyranosyl)-~l, 3~-(2,4,6-tri-~-benzyl-~-D-galacto-pyranosyl)-(l~ 4)-3,6-di-O-benzyl-2-O-pivaloyl-~-D-gluco-pyranoside (27):
Azido compound 26 (1.37 g, 416 ~mol) is reacted with hydrogen sulphide in a pyridine~water mixture (about 100 ml) for 30 minutes and the mixture is left to stand at room temperature for several days. Subsequent N-acetylation in pyridine/acetic anhydride (1:1) gives, after flash chromatography [to]uene/acetone (4:1~3:1)~, a colourless foam (1.23 g, 88 ~); thin layer chromato-g~phy~toluen~a~tone(5:2)] : ~ = 0.43, 1~]2 = -44.0 (c = 1, CHCl3).

Exam~le 20 Acetyl O-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-(1 4)-t(2,3,4-tri-O-acetyl-~-L-fucopyranosyl)-(1~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1 3~-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1~ 4)-1(2,3,4-tri-O-acetyl-~-L-fucopyranosyl~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1~ 4~-3,6-di-O-acetyl-2-O-pivaloyl-~/~-D-glucopyranoside (28):
Compound 27 (1.34 g, 406 ~mol) is hydrogenated with palladium-on-charcoal (10 % of Pd, 800 mg) in a l lol mixture of acetic acid, methanol and dioxane (90 ml) under a hydrogen atmosphere of 4 bar for 48 hours. The solution is decanted off from the palladium-on-charcoal and filtered over kieselguhr. The palladium-on-charcoal is suspended several times in methanol and ~he solution is decanted off again and filtered. Sub-sequent reaction in pyridine/acetic anhydride (1:1~
gives, after flash chromatography with toluene/acetone/
methanol (11:6:0.3~10:6:0.3), a white powder (769 mg, 80 ~); thin layer chromatography [toluene/acetone/
methanol (7:6:0.3)]: ~ = 0.45, [~20 = _35.0 (c = 1, CHCl3).

.
~, , : -,. . . . .. .
. ,. ~, .
. .
' :~ : ' . ; ' ' "' ' ,:~ .

930~

Example 2 1 2,3,4,6-Tetra-0-acetyl-~~D-galactopyrano~yl~ 4)-[(2,3,4-tri-0-acetyl-~-L-fucopyranosyl)~ 3)3-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)-(1- 3)-(2,4,6-tri-0-acetyl-~-D-galacto]pyranosyl)-(1) 4)-[(2,3,4-tri-0-acetyl-~-L-fucopyrano~yl)~ 3~]-(2-acetamid~-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)-(1, 4)-3,6-di-0-acetyl-2-0-pivaloyl-/~-Dglucopyranose (29)s Peracetylated compound 28 (758 mg, 320 ~mol~ i8 dissolved in absolute dLmethylformamide (20 ml)~ and hydrazine acetate (59 mg, 640 ~mol) i~ added at 50C.
After 30 minutes, the mixture is diluted with ethyl acetate (150 ml) and washed with sa~urated ~odium chloride solution (30 ml). The aqueouR phas~ i~ re~
extracted with ethyl acetate (2 x 60 ml). The combined organic extracts are concen~rated in vacuo and purified by means of flash chromatogra~hy ~toluene/methanol (10:1~9:1)]. Compound 29 (670 mg, 90 %) i8 obtained as a white powder; thin layer chromatography ttoluene/acetone/
methanol (5:6:0.3~: R~ = 0.47, 1U]20 = _37.0 (c = 1, CHC13).

Ex~mple 22 0-(2,3,4,6-Tetra-0-acetyl-~-D-galactopyranosyl)~ 4)-[(2~3,4-tri-0-acetyl-~-L-fucopyranosyl~ 3)~-(2-acetamido-6-0-acetyl-2-deQxy-~-D-glucopyranosyl)-(1, 3)-(2,4,6-tri-0-acetyl-~-D-galactopyrano~yl)-(1, 4)-[(2,3,4-tri-0-acetyl-~-L-fucopyranosyl)-(1, 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl~ 3)-(2,4,6-tri 0-acetyl-~-D-galactopyranosyl)-(1- 4)-3,6-di-0-acetyl-2-0-pivaloyl-~-D-glucopyranosyl-trichloroacetimidate (30~s Compound 29 (658 mg, 283 ~mol) is dis~olved in ab~olute methylene chloride (20 ml), and trichloroaceto-nitrile (1 ml) and DBU (1 drop) are added. After 30 minutes, t~he mixturQ i~ concentrated in vacuo and the residue is pu:rified over a short silica gel column using tolueneJmethanol (10:1) as the mobile phase. A white -.

2~306 powder (bO8 mg, 87 ~) is obtained; thin layer chromato~
graphy [toluene/acetone/methanol (7:6:0.3)]; R~ = 0.48, ~ ~20 = -25.~ (c = 1, CHC13)-Example 2 3 8-Ethoxycarbonyl-octan-l-ol ~3~
Compound 31 is synthesiised from methyl oleate in accordance with the instructions of Gerlach et al. (1978 Heov. Chim. Acta, 61 (4), (1978~ 1226).

Example 24 8-Ethoxycarbonyloctyl O-t2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl-(1~ 4)-[(2,3,4-tri-0-acetyl-~-L-fuco pyrano~yl)-(1~ 3)~-(2-acetamldo-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-0-acetyl-~-D-galacto-pyrano~yl~ 4~-t(2,3,4-tri-0 acetyl-~-L-fuco pyranosyl)-(1~ 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)-(l, 3)-(2,4,6 tri-O-acetyl-~-D-galacto-pyranosyl)-~l~ 43-3,S-di-O-acetyl-2-0-pivaloyl-~-D-glucopyranoside (32):
Trichloxoacetimidate 30 (233 mg, 94.5 ~mol) and the compounds according to Example 23 used as spacer~
(5 drops, about 0.23 mmol) are dis~olved in ab~olut~
methylene chloxide (2 ml) and the ~olution is stirred with a molecular ~ieve (4 A) for 3 hour~. A 0.01 M
trimethylsilyl trifluoromethanesulfonate ~olution (0.5 ml) is added dropwise. A~ter 30 minutes, the mixture is neutralised with sodium bicarbonate, filtered and concentrated in vacuo. Fla~h chromatography ltolueneJ
methanol (12:1~10:1.9:1)~ gives compound 32 tl75 mg, 75 ~); thin layer chromatogxaphy ttoluene/acetone/
~0 methanol (7s6:0.3)~: R~ = 0.47, t~]20 = -48.0 ~c = 1, CHCl3).

,, .. ~ .
.

20~93~

E~ample 25 8-EthoxycarbonyloctylO~ D-galactopyranosyl~ 4)-[(~
L-fucopyranosyl)-(1~ 3)]-(2-aceta~ido-2-deoxy-~-D-gluco-pyranosyl)-(l~ 3)-(~-D-galact~pyranosy~ 4)-[t~-L-fucopyranosyl)-(l- 3)]-(2-acetamido-2-deoxy-~-D-gluco-pyranosyl)~ 3)-(~-D-galactopyranosyl)-(1 4)-~-D-glucopyranoside (33):
Compound 32 (168 mg, 68.2 ~mol) i~ dissolved in absolute methanol ~40 ml), 1 ~1 sodium methanolate solu-tion (O.S ml) i8 added and the mixture is stirred at 45C
fer 24 hours. After neutralisation with Amharlite~ IR
120, the mixture i8 filtered and concentrated in vacuo.
The residue is taken up in absolute ethanol (100 ml), 0.5 N sodium methanolate solution (1 ml) i8 added and the mixture is ~irred at 45C for 24 hours. Meutralisation with Amberlite~ IR 120, filtration and concantration in vacuo gi~e an almost colourless compound. The small amount of Lmpurities can be separatQd off by a short flash column packed with RP 18 ~ilica gel using methanol/water (1:1) as the mobile phase. ~thoxycarbonyl-oct-l-yl-dimeric Lewis X (105 mg, 90 %) i~ obtained; thin layer chromatography lmethyl acetate/isopropanol/water (3:3:2)]: Rt = O.43, RP 8 thin layer chromatography tmethanol/water (1~ F~ = 0.37, [~] 20 = _57.0 (c = 1, MeOH).

~xa~ple 26 (2S,3R,4E)-2-~zido-3-benzoyloxy-4-octadecen-1-ol (34) Compound 34 is syn~hesised in accordance with the in~truction~ of ZLmmermann (Tetrahedron Le~t. 27 (1986) 481; Angew. Chem. 98 (1986) 722).

~xam~le 27 0-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyrano~yl)-(1~ 4)-~(2,3,4-tri-0-acetyl-~-L-fucopyranosyl)-(1 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-qlucopyranosyl)-~1~ 3)-l2,4,6-tri-0-alce~yl-~-D-galactopyrano~yl)-(1~ 4)-[~2,3,~-tri-O-acetyl-~-L-fucopyranosyl)-(l~ 3)]-(2-acetamido-6-.. ' ~ .

2ass306 O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1- 3) (2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1~ 4)-(3,6-di~O-ac~tyl-2-0-pivaloyl-~-D-glucopyranosyl)-(1~ 1)-(2S,3R,4E)-2-azido-3-0-benzoyl-4-octadecene-1,3-diol (35):
A 0.01 M trimethyl~ilyl trifluoromethanesulfonate solution (0.3 ml) is added to a solution of trichloro~
acetLmidate 30 (137 mg, 55.5 ~mol) and acceptor 34 (48 mg, 111 ~mol) in absolute methyl~ne chloride (1 ml) as described in E~ample 24. Flash chro~atography [toluene/methanol (13~ 1)] gives compound 35 (114 mg, 75 ~) as a white powder; thin layer chromatography [toluene/acetone/methanol ~7:6:0.3)]: R~ = 0.55, ~]Z =
-44.0 (c = 1, CHC13).

Example 28 0-(~,3,4,6-Tetra-0-acetyl-~-D-galactopyranosyl)-~1~ 4~-[(2,3,4-tri-O~acetyl-~ L-fucopyranosyl)~ 3)J-(2-acetamido-6-0-ac~tyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3~-(2,4,6-tri-0-acetyl-~-D-galactopyrano3yl)~ 4)-~(2,3,4-tri-0-acetyl~ -fucopyranosyl)-(1~ 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2,4,6-tri-0-acetyl-~-D-galactopyxanosyl)-(1~ 43-(3,6-di-0-acetyl-2-0-pivaloyl-~-D-glucopyrano6yl~-(l. 13-(2S,3R,4E)-3 0-benzoyl-2-hexadecanamido-4-octadecene-1,3-diol (3S):
Hydrogen sulphide is pas~ed through a solution of 34 (114 mg, 41.6 ~mol) in pyridine (10 ml) and water (2 ml). After 3 day~, the mixture i8 concentrated at 20C
under a high vacuum. The ~olid residue i~ Lmmediately dissolved in absolute methylene chloride (S ml) and reacted with palmitic acid (25 mg, 100 ~mol) and ~-13-di-methylaminopropyl)-~-ethylcarbodiLmide hydrochloride ~E. Merck, Darmstadt, FRG) (29 mg, 150 ~mol). After 18 hours, the mixtur~ i~ dilu~ed with methylene chloride (50 ml) and washed with wa~er (20 ml). The organic phase i8 concentrated in vacuo. Flash chromatography ~toluene/
methanol (12:1.11:131 give~ 36 (105 mg, 85 %) as a white powder; thin layer chromatography [toluene/aceto~e/
m~thanol (7:6::0.3)1: ~ = 0.55, [~]20 = _35,0 (c = 1, CHC13)-:, `
'' . `

- 31 - ~993~6 E~ample 2~
O-~-D-Galactopyranosyl)~ 4)-[(~-L-fucopyrano~yl)-(1, 3)]-(2-acetamido-2-deoxy ~-D-glucopyranosyl)-(1-~3)-(~~D~galaCtPYransy~ 4)-[(~-L-fucopyranosyl)-3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)~ 3)~
D-galactopyranosyl)-(l~ 4)-(~-D-glucopyranosyl)-(l~ 1)-(2S,3R,4E)-2-hexadecanamido-4-octadecene-1,3-diol (37):
1 M sodium methanolate solution (0.5 ml) is added to a solution of 36 (100 mg, 33.7 ~mol) in absolu~e methanol (30 ml) and the mixture is stirred at 45C for 24 hour3. It i8 neutralised with Amberlite~ IR 120, filtered and concentrated in vacuo. The residue i~
introduced onto an RP 18 silica gel column and eluted with methanol/wa~er (12:1~14sl~16:1). Glycosphingolipid lS 37, from which the protective groups have been removed, (62 mg, 90 ~) is dis~olved in water and lycphilised; thin layer chromatography tmethyl acetate/isopropanol/ water (7:6:4)]: R~ = O.5, RP 8 thin layer chromatography tmethanoliwater ~9:1)]: ~ = 0.33, [~32 a -46.0 (c = 1, MeOH).

~xample 30 tert-Butyldimethyl~ilyl 0-(2~3~4~6-tetra-o-acetyl-~-D-galactopyranosyl)~ 4~-[(2,3,4-tri-0-benzyl-~-L-fuco-pyranosyl)-(1~ 3)]-(2-azido-6-O~benzyl-2-deoxy-~-D
glucopyranosyl~ 3)-(2-o-acetyl-4~6-o-benzylidene-~-D-galactop~ranosyl~-(l. 4)-[(2,3,4-tri-0-benzyl-~-L-fuco-pyranosyl)-(l- 3)]-(2-azido 6-0-benzyl-2-deoxy-~-D-glucopyranosyl~ 3~-(4,6-0-benzylidene-~-D-qalacto-pyranosyl)-(l~ 4j-[(2,3,~-tri-0-benzyl-~-L-fuco~
pyranosyl)~ 3)]-2-azido-6-0-benzyl-2-deo~y-~-D-gluco-pyrano~ide (38):
A O.05 M trLmethylsilyl trifluoromethanesulfonate solution (O.~ ml) is added dropwise to trichloro-acetimidate 16 (1.72 g, 790 ~mol) (Example 16) and acceptor 12 (1.13 g, 1.05 mmol) ~Example 12) in absolute acetonitrile (20....) at -40C. The mixture i~ worked up in the customary manner. Flash chromatography [toluene/

: `
.

20~930~

acetone (5sl-4:1)] gives compound 38 (1.73 g, 71 ~); thin layer chroma~ography [petroleum ether/methyl acetate (4:3)~: R~ = 0.35, t~]20 = -83.0 (c = 1, CHCl3).

~xam~le 31 tert-Butyldimethylsilyl 0 (2,3,4,6-tetra-0-acetyl~-D-glactopyranosyl)-(l~ 4)-~(2,3,4-tri-0-benzyl-~-L-fuco-pyranosyl)-(l- 3)]-(2-azido-6-0-benzyl-~-deoxy-~-D-glucopyranosyl)-(2-0-acetyl-4,6-0-benzylidene-~D-galac-topyranosyl)~ 4)-[(2,3 r 4-txi~0-ben~yl-~-L-fuco-pyranosyl)-(1- 3)]-(2-aziclo-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(l- 3)-(1~ 3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(l~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(l~ 3)~-2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosida (3g~ 2 Compound 33 l1.70 g, 551 ~mol) i8 treated with pyridine/acetic anhydride (1:1, 10 ml) for 24 hours. The solution is concentrated in vacuo and the residue is coevaporated with toluena. Flash chromatography [petroleum ether/methyl acetate (4:3)] gives compound 39 (1.62 g, 94 %); thin layer chromatography rpetroleu~
e~her/methyl acetate (4:3)]: R~ = 0.35, ~]20 = -84.0 (c = 1, CHCl3).

~xample 32 0-(2,3,4,6-Tetra-0-acetyl-~-D-ga~actopyranosyl)-(l. 4)-[(2,3,~-tri-0-benzyl-~L-fucopyranosyl)-(l- 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2-0 acetyl-4,6~0-benzylidene-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl~ 3~-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(l~ 3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyrano~yl)-(1~ 4)-[(2,3,4-tri-0-benzyl~ -fucopyrano~yl)-(1.3)]-2-a~ido-6-0-benzyl-2-~deoxy-~ D-glucopyranose (40):
A solution of compound 39 (1.40 g, 448 ~mol~ in absolute tetrahydrofuran (20 ml) is rea~ed with glacial acetic acid (26 ~l, 448 ~mol) and a 1 M solution of tetrabutylammonium fluoridQ (1 ml) a~ described in ' '.~

:: , - ' . ~ ' - ~ " ' . ' - . ~ .

209~306 Example 9. Flash chromatography [petroleum ether/methyl acetate (4:5)] ~ive~ compound 40 (1.23 g, 91 ~); thin layer chromatography [petroleum ethar/methyl acetate (1:1)]: R~ = 0.23, [~]20 = _73 o (c = 1, CHCl3).

Example 33 0-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyranosyl)~ 4L)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~1~ 3)]-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2-O-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(l, 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(lt 3)]-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranosyl)~ 3~-(2-O-acetyl-4,6-0-benzylidene-~-D-galactopyrano~yl)-~1- 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl) (1~3)]-2-azido-6-O-benzyl-2-deoxy~ D-glucopyranose (41):
Trichloroacetonitrile (3.0 ml) and DBU (1 drop) are added to a solution of compound 40 (980 mg, 325 ~mol) in absoluts methylene chloride (30 ml). After 30 minutes the mixture is concentrated in vacuo. Flash chromato-graphy ttoluene/acetone (5:1) + 1 % of triet~ylamine]
gives trichloroacetimidate 41 (9sL3 mg, 92 %) in a ratio of ~ 1:2; thin layer chromatography ttoluene/acetone (5:1)]: R~ = 0.22 for the ~-trichloroacetLmidate, R~ =
0.29 for the ~-trichloroacetimidate, ~20 = -62.0 (c = 1, CHCl3) for the ~-trichloroacetimidate, [~]20 = _73.5 (c =
1, CHC13) for the ~-trichloroacet~midate.

~xa~ple 34 Benzyl 0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyrano~yl)-(l 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(l~ 3)~-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(l~ 3)-(2-O-acetyl-4,6-O-benzylidPne-~-D-galactopyrano6yl)-(1- 43-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(l~ 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2-O-acetyl-4L,6-O-benzylidene-~-D-galac~opyranosyl)-(l~
4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranoRyl)-(1 33]-(2-azido-6-O-ben~yl-2-deoxy-~-D-glucopyranosyl)-(1~ 3~-(2,4,6-tri-O-benzyl-~-D-galactopyranosyl)-(1-4)-3,6-di-:

. : - . . . .

2~99~

O-benzyl-2-o-pivaloyl-~-D-glucopyranoside (42):
TrichloroacetLmidate 41 (662 mg, 210 ~mol) and acc~ptor 25 (305 mg, 315 ~mol) are reacted in aceto-nitrile (3 ml) at -40C as described in Exampl~ 18. Flash chromatography [toluene/acetone (8:1~7:1)] give~ compound 42 (598 mg, 72 ~); thin layer chromatography [toluene/
acetone (6:1)]: Rf = 0.34, [~12 = -84.0 (C = 1, CHC13).

Example 35 Benzyl 0-(2,3,4,6-tetra-0-acetyl-fl-D yalactopyranosylJ-(1~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-tl~ 3)]-(2-acetamido-6-0-benzyl-2-deoxy-~-d-glucopyranosyl)-(1~
3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(1~ 4)-~(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(1~ 3)]-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~
3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galaetopyTanosyl)-(1. 4)-[(2,3~4-tri-0-benzyl-~-L-fucopyranosyl)~ 3)]-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(l~
3~-~2,4,S-tri-0-benzyl-~-D-galactopyrano~yl)~ 4)-3,6-di-0-benzyl-2-0-pivaloyl-~-D-glucopyranoside ~43~:
~ydrogen sulphide i~ passed through a solution of compound 42 (591 mg, 149 ~mol) in pyridine (40 ml) and water ~14.5 ml) as described in Example 19. Subseguent N-acetylation in pyridine/ace~ic anhydride (1:1) gives, after flash chromatography ~pPtroleum ether/m~thyl acetate (6:7~5:6~4:5)], compound 43 (520 mg, 86 %); thin layer chromatography tpetroleum ether~methyl acetate (4:5)] Rk = 0-52, [~]' = -84.0 (c = 1, CHCl3).
Example 36 Acetyl 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl~- :
(1~ 4)-[(2,3,4-tri-0-acetyl-~-L-fucopyranosyl~ 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)~
3)-(2,4,6-tri-0-acetyl-~-D-galactopyrano~yl)-(1~ 4)-r ( 2,3,4-tri-0-acetyl-~-L-fucopyranosyl~-(1. 3)]-(2-acetamido-6-lD-acetyl-2-deoxy-~-D-glucopyranosyl)-~1.3)-(2,4,6-tri-0--acetyl-~-D-galactopyrano~y~ 4)-t(2~3~4-tri-0-acetyl-~-L-fucopyrano~yl)~ 3)]-(2-acetamido-6-: ''' :- ' -: . .
.

. '' ~ . ~

_ 35 _ 209~306 O-acetyl-2-deoxy-~B-D-glucopyranO~yl3~ 3)-(2~4~6-tri-O-acetyl-~-D-galactopyranosyl)~ 4)-3,6-di-0-acatyl-2-O-pival~yl-~/~-D-glucopyranoside (44):
Compound 43 (346 ms~, 85.5 ~,mol) i hydrogenated 5 with palladium-on-charcoal (10 % Pd, 200 mg) in acetic acid/methanol/dioxane (1:1:1, 30 ml) und~3r a hydrogen atmosphere of 4 bar for 48 hours as described in Example 20. Subsequent treatment with pyridine/acetic anhydride (1:1) gives, after fla~h chromatography ttoluene/acet-10 one/methanol (9:6,0.3~8:6:û.3~], compound 44 (21~ mg, 78 96~ as a white powder; thin layer chromatography [toluene/acetone/methanol (7:6:0.3)]: Rr = 0.31, ttl] 20 -45.0 (c = 1, CHCl3).

E~ample 37 15 2,3,4,6-Tetra-0-ace~yl-B-D-galactopyrarlosyl)~ 4)-~(2,3,4-tri~0-acetyl--L-fucopyranosyl)~ 3)]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)-(1- 3)-(2,4,6-tri-0-acetyl~ D-galactopyranosyl~ 43-[(2,3,4-tri-O-acetyl-~-L-fucopyranosyl)-(1. 3~]-(2-acet~nido-6-~0 0-acetyl-2-deoxy-,~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-0-acetyl-B-D-galactopyranosyl)~ 4)~1(2,3,4-tri-0-acetyl--L-fucopyranosyl~ 3)]-(2-acetamido-6-0-acetyl-2-deoxy-,~-D-glucopyranosyl)-(1. 33-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(l. 4)-3~6-di-0-acetyl-2-0-25 pivaloyl-/8-D-glucopyranose (45):
Compound 44 (145 mg, 45.1 ~6mol) i~ reacted wi~h hydrazine acetate (9.2 mg, 100 umol) in absolute dimethylforma~ide (5 ml) as described in Example 21.
~orking up i8 carried out in the usual mamler. Flash 30 chromatography [ toluene/acetone/methanol (8.5:6:0.3.7.5:6:0.3)] gives compound 45 (125 mg, 87 ~6);
thin layer chromatography tCH2Cl2/MeOH (20~ = 0.10, ~~20 = -48.0 (c = 1, CHC13)-. .
.

~ ' 9930~
- 3b -Examp1e 38 0-(2,3,4,6-Tetra-O-acetyl-~-D-galaCtopyrano~yl)~ 4)-[(2,3,4-tri-O-acetyl-~-L-fucopyranosyl)-(1~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyrano~yl)~ 3~-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1~ 4)-[(2/3,4-tri-O-acetyl-~-L-fucopyranosyl)~ 3)~-(2-acetamido-6-O-acetyl-2-daoxy-~-D-glucopyranosyl)~ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)~-(l. 4~ r ( 2,3,4-tri-O-acetyl-~-L-fucopyranosyl)~ 3~]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyrano~yl)-(1~ 3)-(2,4,6-trî-O-acetyl-~-D-galactopyranosyl)-(:l~ 4)-3,6~di-O-acetyl-2-O-pivaloyl-~/~-D-glucopyrano~yl-trichloroacetimidate (46):
Trichloroacetonitrile ~1 ml) and DBU (1 drop~ are added to a solution of compound 45 (114 mg, 35.8 ~mol) in absolute methylene chloride (5 ml). After 30 minutes, tha mixture i8 concentrated in vacuo and the residue i~
purified by means of flash chromatography lCHzCl2/~eOH
(20:1~17.5:1~15sl)]. Compound 46 (109 mg, 89 ~) is obtained; thin layer chromatography ttoluene/acetone/
methanol (7s6:0.3)] ~ = 0.31t [~]20 = -29.5 (c = 1, CHCl3~.

~xample 39 0-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyrano~yl~ 4)-~(2,3,4-tri-O-acetyl-~-L-fucopyranosyl)-(1- 3)] (2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1- 3)-(2,4,6-tri-O-acetyl-g-D-galactopyranosyl)-(1. 4)-1(2,3,4 tri-O-acetyl-~-L-fucopyranogyl)-(l. 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-~lucopyranosyl)-(1~ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(l~ 4)-[(~,3,4-tri-O~
acetyl-~-L-fucopyranosyl)-(l-~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl) (1~ 4)-3,6-di-O-acetyl-2-O-pi~aloyl ~-D-glucopyranosyl)-(l~ 1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (47):
A solution of trichloroacetimidate 46 ~60.4 mg, 17.7 ~mol~ ancl acceptor 34 (15 mg, 35.4 ~mol) in absolute methylene chloride (1 ml) is ~tirred with a molecular 2~9~3~6 sieve (4~). After 4 hour~, a 5 mM trLm~thyl~ilyl tri-fluoromethanesulfonate solution (0.35 ml) i~ added dropwiseO Working up is carried out a~ described in Example 24. Fla~h chromatography [toluene~acetone/
methanol (10.5:6:0.4)] gives compound 47 (46.5 mg, 73 ~);
thin layer chromatography [toluene/acetone/methanol (7:6:0.3~]: R~ = 0.40, [~]20 = -53.0 (c = 1, CHC13).

Exampl~ 40 0-(2,3,4,6-Tetra-0-acetyl-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-0-acetyl ~-L-fucopyrano3yl)~ 3~]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyrano~yl)~ 3)-(2,4,6-tri-0-acetyl-~-D-galactopyrano~yl)-(1~ 4)-[(2,3,4-tri-0-acetyl-~-L-fucopyranosyl)-(1~ 3)J-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyrano~yl)~ 33-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)~ 41- L ( ~, 3,4-tri-0-acetyl-~-L-fucopyranosyl)~ 3)3-~2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyrano~yl)-(1. 3)-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)-(l. 4)-3,6-di-0-acetyl 2-0-pivaloyl-~-D-glucopyranosyl~ 13-(2S,3R,4E)-3-0-b~nzoyl-2-hexadecan,3mido-4~octadecene-1,3-diol (48):
Hy~rogen ~iulphide is passed through a solution of 47 (27.6 mg, 7.63 ~mol) in pyridine ~10 ml) and water (2 ml). After 3 days, the mixture is concentrated at 20C
under a high vacuum. ~he residue i8 dissolved in a~solute methylene chloride ~2 ml) and reacted with palmitic acid (5 mg, 20~mol) and N-(3-dLmethylaminopropyl)~ ethylcar-bodiimide hydrochloride (5 mg, 30 ~mol). Working up is carried out as de~cribed in Example 28. Fla~h chroma~o-graphy rtoluene~acetone/me~hanol (10. 5 s 6 ~ O . 3 ) ] gives compound 48 (24.2 mg, 84 %); ~hin lay~r chromatography [toluene/acetone~methanol (7:6s0.3)]: R~ = ~.40, t~20 =
_45.0 (c = 1, CHCl3)-.

- : : , ' :

EXamD1e 4 1 O-(~-D-Galactopyranosyl)-~l~ 4)-[(n-L-fucopyranosyl)~
3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)~ 3)-(~-D-galactopyranosyl)-(l 4)-[~-L-fucopyranosyl)-(1-~ 3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(~-D-galact~pyranosyl)~ 4)[(~-L-fucopyranosyl)~ 3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)-(1. 3)-(fl-D-gal-actopyranosyl)-(l~ 4)-(~-D-glucopyranosyl)-~1~ 1)-(2S,3R,4EJ-~-hsxadecane-amido-4-octadecene-1~3-diol (49):
0.2 N sodium methanolate solution (0.25 ml) i8 added to a ~olution of 48 (20.6 mg, 5.45 ~mol) in ab-solute methanol (6 ml) and the mixture is ~tirred at 45C
for 24 hours. It i8 neutrali~ed with Amberlite~ IR 120, filtered and concentrated in vacuo. Purification of the residue by chromatography with RP 18 ~ilica gel and methanol/water (9~ 12.5:1) give~ the trimeric ~e~
antigen (12.3 mg~ 88 %); thin layer chromatography ~methyl acetata/isopropanol/water (7:6:4)]8 R~ = 0.23, RP
8 thin layer chromatography ~methanol/water ~10:1)]: R~ =
0.42, ~20 = _55.0 ~c = 1~ MeOH)-~ample 42 tert-Butyldimethyl~ilyl O-(~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-~-~enzyl-~-L-fucopyranosyl)-(1> 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(4,6-0-benzylidene-~ D-galac~opyranosyl~ 4)-t(2~3~4-tri-0-benzyl-~-L-fucopyranosyl)-(l. 3)]-2-azido-6-0-ben~yl-2-deo~y-~-D-glucopyranoside (50):
Compnund 13 (819 mg, 390 ~mol) (Example 13) i8 suspended in ab~o~ute methanol (100 ml). A 1.5 ~ sodium methanolate ~olution (10 drops) is added dropwi~e at a temperature o~E 45C. After 2 hours, the clear solution is treated with Amberlite~ IR 120 and filtered. Concentra-tion in vacuo gives compound 50 (764 mg, 100 3); thin layer chromatography ttoluene/acetone (Ss3)]: R~ = 0.37, [n]20 = -82.0 (c = 1, CHCl3).

~9~3~6 Example 4 3 tert-sutyldimethylsilyl 0-(4,6-0-benzylidene-p-D galacto-pyranosyl)-(l~ 4)-[(2,3,4-tri-0-benzyl-~-L-fuCo-pyranosyl)-(l~ 3)]-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(l-~ 3)-4,6-0-benzylidene-~-D-gal~cto-pyranosyl)-(1- 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyr~n-osyl)~ t 3~]-(2-azido-6-0-benzyl-2-deoxy-~-D-gluco-pyranoside (51):
A solution of compound 50 (758 mg, 387 ~mol) in ab olute acetonitrile 150 ml) is reacted with benzal-dehyde dimethyl acetal (147 mg, 968 ~mol) and p-toluene sulfonic acid (30 mg) as described in Exa~ple 1~. The mixture is worked up in the customary manner. Flash chromatography [petroleum ether/methyl acetate ~3:2-5:~-1sl)~ qives compound 51 (627 mg, 86 ~); thin layer chromatography [petroleum ether/mathyl acetate (lsl)]: R~ = 0.3~, t~]20 = -112.0 (c = 1, CHCl3).

~x ~l~ 44 tert-ButyldL~ethylsilyl 0-(2,3,4,fi-tetra-0-acetyl-~-D-galactopyranosyl)-(l. 4)-[(2,~,4-tri-O~benzyl-~-L-fuco-pyranosyl)-~l- 3)~-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyrano~yl)-ll~ 3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(l. 4)-[~2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(l, 3)~-2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl~ 3)-(4,6-0-benzylidene-~-D-galacto-pyranosyl)-(1. 4) ~(2,3,4-tri-0-benzyl-~-L-fucopyran-osyl)-(l t 3)~-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyra~-osyl)-(1. 3)-(4,6-0-benzylidene-~-D-galactopyranosyl~
4)-t(2,3,4-tri-0-be~zyl-~-L-fucopyranosyl)-(1~ 3)]-2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranoside (523:
A 0.05 M trimethylsilyl trifluoxomethanesulfonate solution (O.15 ml) is added dropwise ~o a solution of trichloroacet:Lmidate 16 (626 mg, 288 ~mol) and acceptor 51 (727 mg, 360 ~mol) (Exa~ple 43) in absolute aceto-nitrile (12 ml) at -40C. Working up i8 carried out as described a~ve. Flash chromatography ~petroleum ether/methyl acetate (3:2~5:4)] give~ compound 52 , . ., ~, .. .. : .

-2~9~6 (859 mg, 77 %); thin layer chromatography [petroleum ether/methyl acetate (1:1~]: R~ = 0.63, [~]Z = -101.0 (c = 1, CHCl3).
~xample 4~
tert-Butyldimethylsilyl-0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-(l~ 4)-[(2,3,4-tri-O-benzyl-~-L-fuco-pyranosyl)-(l~ 3)~-(2-azido-6-O-benzyl-2-deoxy-~~D-glucopyranosyl)~ 3)-(2-O-acetyl-4,6-O-ben~ylidene-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(1~ 3)]-(2-azido-5-O-benzyl-2-deoxy-~-D-glucopyranosyl)-(l~ 3)-(2-o-acetyl-4~6-o-benzylidene D-galactopyranosyl)-(l. 4)-t(2~3~4-tri-o-benzyl-~-L-fucopyranosyl)-(1~ 3)]-2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranosyl)-(l~ 3)-(2-O-acetyl-4,6-O-benzylidene-~-D-galactopyranosyl)-(1~ 4)-t(2,3,4-tri-O~ben yl-~-L
fucopyranosyl)-(l> 3)~-2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranoside ~53):
Compound 52 (579 my, 144 ~mol) is treated with pyri~ine~acetic anhydride (1:1, 6 ml) for 2~ hour~. The mixture is concentrated in vacuo and the residue is coevaporated with toluene. Flash chromatography [petroleum ether/methyl acetate (3:2~5 4)] gives compound 53 (594 mg, 100 ~); thin layer chromatography rpetroleum ether/methyl acetate (5:4)]: ~ = 0.4C, [~] 20 = _ 9 7 . 0 (c = 1, CHC13).

~xampl~ 46 0-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyranosyl~ 4)-~(2,3,4-tri-O-benzyl-~-L-fucopyTanosyl)-(1~ 3)~-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2-O-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(1- 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(1> 3)]-(2-azido-6--O-benzyl-2-d~oxy-~-D-glucopyranosyl)-(1~ 3)-(2-O-acetyl-4,6-O--benzylidene-~-D-galactopyrano~yl)-(1-~ 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyrano~yl)-(1~ 3)]-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyranosyl)-(1-~ 3)-(2-O-acetyl-4,6-O~benzylidene-~-D-galactopyranosyl)-(1~ 4)-- 2~3~6 [(2,3,4-tri-O-benzyl-~-L-fucopyranOsyl)~ 3)J 2-azido-6-O-benzyl-2-deo~y-~/~-D-glucopyranose (543:
~ solution of compouncl 53 (582 mg, 141 ~mol) in absolute tetrahydrofuran (10 ml) is reacted with glacial S acetic acid (8 ~l, 141 ~mol) and a 1 ~ 801ution of tetrabutyl ammonium fluoride (O.3 ml) as described in Example 9. Flash chromatography [petroleum ether~methyl acetate (1:1~5:6~4:5) gives compound 54 (531 mg, ~4 ~);
thin laysr chromatography Lpetroleum ether/methyl acetate (4:5)]: R~ = 0.58, [~32 = -91.0 (C = l, CHC13).

~xample 47 0-(2,3,4,6-T~tra-O-acetyl-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-O-benzyl--L-fucop~rano~yl)-(1~ 3)]-~2-azido-6-O-benzyl-2-deoxy~ -glucopyranosyl)~ 3~-(2-O-acetyl-4,6-0-benzylidæne-~-D-galac~opyranosyl)-(1~ 4~
[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)~ 3)~-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyrano~yl)~(l. 3)~ O-acetyl-4,6-O-benzylidene-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-(1 t 3)]-(2-azido-6-O-benzyl-2-deoxy-~-D-glucopyxanosyl)-(1~ 3)-~-O-acetyl-4,6-O-benzylidene-~-D-galactopyranosyl)-(1- 4)-t(2,3,4-tri-0-benzyl-~-L-fucopyrano~yl)-(1~ 3)~-2-azido-6-O-benzyl-2-deoxy-~/~-D-glucopyranosyl-trichloxo-acetimidate (55):
Trichloroacetonitrile tl.0 ml) and DBU ~l drop) are ad~ed to a solution of compound 54 (525 mg, 131 ~mol~
in absolute methylene chloride (20 ml~. After 30 minut~s, the mixture is concentrated in Yacuo. Flash chromato-graphy of the residue ~toluene/acetone (5:114:1) + l %
of trie~hylaminel gives compound 55 (495 mg, 91 %) as an ~/~ mixture in a ratio of ~o~ ~ 1:2; Rf = 0.47 (both anomers).

Example 48 Benzyl 0-(2,3,4,6-te~ra-O-acetyl-~-D-galactopyranosyl)-(1. 4)-[(2,3,,4-tri-0-benzyl-~-L-fucopyranosyl) (1~ 3)~-(2-azido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-.~ ,', , " 20~30~

(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(l~ 4)-[(2,3,4-tri-0-benzyl-~-h-fucopyranosyl)~ 3)]-(2-a~ido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(1~
4)-[(2,33,4-tri-0-benzyl-~ -L-fucopyranosyl ) - t 1~ 3 ) ] - ( 2-azido-6-0-benzyl-2-deoxy-~-D-qlucopyranosyl)-(1~ 3)-(2-0-acetyl-4,5-0-benzylidene-~-1)-galactopyranosyl)-(1~ 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranQsyl3-(1, 3)3-(2-azido-6-o-benzyl-2-deoxy-~-D-glucop~-anosy~ 3)-(2,4,6-tri-0-ben~yl-~-D-galactopyranosyl~ 4)~3,6-di-0-bQn2yl-2-0-pivaloyl-~D-glucopyranoside (56)s Trichloroacetimidate 55 (389 mg, 93.9 ~mol) and acceptor 25 (136 mg, 141 ~mol) are reacted in aceto-nitrile (2 ml) at -40~C as described in Example lB. Fla~h chromatogr~phy [toluene/acetone (9sl~7sl~6:1)3 gives compound 56 (358 mg, 77 ~); thin layer chromatography [toluene/acetone (5~ ~ = 0.37, 1~]2 = -92.5 (c = 1, CHCl3).

Example 49 Ben~yl 0-(2,3,4,6-~etra-0-acetyl-~-D-galactopyranosyl)-(1) 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)~ 3)]-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~
3)-(2-0-acetyl-4,6-0-benzylidene-~-D~galactopyranosyl)-(1. 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyrano~yl)-(1- 3)~-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-(1~
3)-(~-0-acetyl-4,6-0-benzylidene-~-D-galactopyranosyl)-(1. 4)-[(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-(1~ 3)]-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyranosyl)-~1-3)-(2-0-acetyl-4,6-0-benzylidene-~-D-galactopyrano~yl)-(1. 4)-[(~,3,4-tri-0-benzyl-~-L-fucopyrano~yl)-(1~ 3)~-(2-acetamido-6-0-benzyl-2-deoxy-~-D-glucopyrano~yl)-(1~
3)-(2,4,6-tri-0-bsn2yl-~-D-galactopyranosyl)-(1~4)-3,6-di-0-benzyl-2-0-pivaloyl-~-D-glucopyranoside (57):
Hydrogen sulphide is pa~ed through a solution of compound 56 li349 mg, 70.6 ~mol) in pyridine (40 ml) and water (15 ml.~ as described in ~xample 19. Subsequent N-acetylation in pyridine/acetic anhydrida (1~1) gives, -, . . . .
- ~

.. . .

-" 2~19306 after flash chromatography [petroleum ethex/methyl acetate ~4:5~3:4~2:3)]t compound 57 (305 mg, 85 %); thin layar chromatography [petroleum eth~r/methyl acetate (3:4)]: R~ = 0.26, [~20 = 55.0 (c = l, CHCl3).

Example 50 Acetyl 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyr~nosyl)-(l~ 4~-[(2~3~4-tri-o-acety~ J-fucopyranosyl)~ 3)J-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyrano~yl)-(1-3) (2,4,6-tri-0-acetyl-~-D-galactopyranosyl) (l~ 4~-~(2,3,4-tri-0-acetyl-~-L-fucopyranosyl) (l~ 33]-(2 acetamido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)-(1~ 4~-[(2,3,4 tri-0-acetyl-~-L-fucopyranosyl)~ 3)]-(2-acetamido-6-0-acetyl-2-dsoxy-~-D-glucopyranosyl)-~l~ 3)-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-0-acetyl-c~-L-fucopyranosyl)-(l~ 3~]-(2-acetamido-6-0-acetyl-2-deoxy-~-D-glucopyrano~yl~-(1 3)-(2,4,6 tri-0-acetyl-~-D-galactopyranosyl)-(l. 4~-3,6-di-0-acetyl-2-0-pivaloyl-c~/~-glucopyranoside (58):
Compound 57 (300 mg, 59.0 ~mol) i5 hydrogena~ed with palladium-on-charcoal (10 ~ of Pd, 150 mg) in acetic acid/methan`ol/dioxane (1:1:1, 30 Dl ) under a hydrogen atmosphere of 4 bar for 48 hours as described in Example ~O. Sub~equent treatment with pyridine/acetic anhydride (1:1) gives, after flash chromatography ~methylene chloxide/methanol (97:3t96:4~95:5~94:6)~, compoun~ 58 (193 mg, 81 %), thin layer chromatograph~ Ltoluene/
acetone~methanol (6:6:0.3)]: R~ = 0.41, [C~20 = -48.0 (c = l, CHCl3).

Example 51 2,3,4,6-Tetra--0-acetyl-~-D-galactopyranosyl)-(l~ 4)-[(2,3,4-tri-0-acetyl-c~-~-fucopyranosyl)~ 3)]-(2-ace~amido-6-0--acetyl-2-deoxy-~-D-glllcopyranosyl)-~1- 3)-(2,4,6-tri-0-acetyl-~-D-galactopyranosyl)-(1~ 4)-[(2,3,4-tri-0-acetyl-c~-L-fucopyranosyl)-(l, 3)]-(2-aceta~ido-6-0-acetyl-2-deoxy-~-D-glucopyranosyl)~ 3)-(2,4,6-tri-- .

- .", . : , .
., . . : . .- . :

- 2~9930~

O-acetyl-~-D-galactopyranSoyl)-(1~4)-[(2,3~4-tri-O-acetyl-~-L-fucopyranosyl)-(1- 3)]-(2-acetamido-6-O-ac~tyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1- 4)-1(2,3,4-tri-O-ace~yl-~-L-fucopyranosyl)-(1- 3)]-(2-acetamido-6-O-ace~yl-2-deoxy-~-D-glucopyranosyl)-(1. 3)-t2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-tl-4)-3,6-di-0-acetyl-2~0-pivaloyl-~/~-D-glucopyrano~e t59) Compound 58 t186 mg, 46.0 ~mol) i~ reacted with hydrazine acetate t9.2 mg, 100 ~mol) in ab~olute dimethylformamide (6 ml) as de~cribed in ~xample 21. The mixtura is worked up in the usual manner. Flash chroma-tography [methylene chloride/methanol t25:1-20:1-15:1)]
gives compound 59 t163 mg, 88 %); thin layer chroma-tography ~CH2Cl2/MeOH ~19:1)]: R~ = 0-12, t~]20 = ~53 5 (c = 1, CHCl3).

~xample 52 O-(2,3,4,6-Tetra-O-acetyl-~-D-galactopyranosyl~ 4)-[(2,3,4-tri-O-acetyl-~-L-fucopyranosyl~ 3~_t2_ acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1. 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl) (1-~ 4)-t(2,3,4-tri-O-acetyl--L-fucopyranosyl)-(1~ 3)3-(2-acetamido-6-O-acetyl-2-deoxy-~-D-ylucopyranosyl)-(1\ 3)-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(l~ 4)-~(2,3,~-tri-O-acetyl--L-fucopyranosyl)-(l. 3)]-(2-acetam~do-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)~(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(l-~ 4)-t(2,3,4-tri-O-acetyl-~-L-fucopyrano~yl)-(l~ 3)J-(2-acetamido-6-O-acetyl-2-deo~y-~-D-glucopyranosyl)-(l. 3)-(2,4,6-tri-O-acetyl-~-D-~alactopyranosyl~-(l. 4)-3~6-di-o-acetyl-2-o-plva /~-D-glucopyranosyl-trichloroacetimidate (60):
Trichloroacetonitrile (1 ml) and DBU (1 drop) are added to a solution of compound 59 (156 mg, 38.8 ~mol) in absolute methylene chloride (6 ml). After 30 minute~, the mixture is concentrated in vacuo and the residue is purified by meanR of flash chromatography [methylene chlorida/methanol (20:1~17.5~15sl)]. Compound 60 (141 mg, _ 45 _ 2~306 89 %) is obtained as an ~/~ mixture in a ratio of a~
3:1; thin layer chromatography [methylene~ chloridet methanol (16:1)]: R~ = 0.31 (both anomers).

Example 53 0-(2,3,4,6-tetra-O-acetyl-~-D-galacto~yranosyl)-(1, 4)-[~2,3,4-tri-0-acetyl-~-L-fucopyranosyl)~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~--D-glucopyranosyl)~ 3~-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl3~ 4)-~2,3,4-tri-O-acetyl-~-L-fuCopyranOsyl~ 3)]-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyrarlosyl)-(1~ 3)-(2,4,6-tri-O-acetyl-~-D-qalactopyrano6yl)~ 4)-~(2,3,4-tri-0-acetyl-a~-L-fucopyranosyl)~ 3)J-(2-acetamido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl~ 3~-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)-(1. 4)-~t2,3,4-tri-O-acetyl -L-fucopyranosyl)-(1. 33]-(2-acetamido-6-O-acetyl 2-deoxy-~-D-glucopyranosyl~ 3)-(2,416-tri-O-acetyl-~-D-galactopyranosyl)-(l~ 4)-(3,6-di-O-acetyl-2-O-pivaloyl-~-D-glucopyranosyl)-(l~ 1)-l2S,3R,4~)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (61):
A solution of ~richloroacetimidate 60 (134 mg, 32.8 ~mol) and acceptor 34 (28.2 mg, 65.6 ~mol) in absolute methylene chloride (2 ml) is stirred with a molecular si~ve (4~. After 6 hours, a 0.01 ~ trLmethyl-silyl trifluoromethanesulfonate solution (0.33 ml) i8 added dropwise. Working up is carried out as de~cribed in Example 24. Flash chromatography tmethylene chloride/
methanol [25:1~22.5:1~20:1)] gives compound 61 (110 mg, 76 %); thin layer chromatography ttoluene/acetone/
methanol (7:6:0.3)~: R~ = 0.45, ta~l20 = -54.0 (c = 1, CHCl3).

~æample 54 0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)~ 4)-[(2~3~4-tri-0-acetyl-a~-L-fucopyranosyl)-(1~ 3)]-~2-acetamido-6-0 acetyl-2-deoxy-~-D-glucopyranosyl)-(1~3)-(2,4,6-tri-O-acetyl-~-D-~alactopyranosyl)-(1- 4)~(2,3,4-tri-o-acQtyl-al-L-fucopyranosy~ 3)]-(2-acetamido-6-` ' ' , - , ':, .

. . .
., : : -.: : ' 2~306 O-acetyl-2-deoxy-~-D-glucopyranosyl)-~ )-(2,4,6-tri-O-acetyl-~-D-galactopyranosyl)~ 43-[(2,3,4-tri-O-acetyl-~-L-fucopyranoYyl)~ 3)]-(2-aceti~mido~-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(2,4,6-t~i-O-acetyl-~-D-galactopyranosyl)~ 4)-[(2,3,4-tri-O-acetyl-~-L-fucopyranosyl)-(1, 3)]-~2-acetiImido-6-O-acetyl-2-deoxy-~-D-glucopyranosyl)-(l- 3)-(2,4,6-tri-O-acetyl-~-D-galactopyrinosyl)~ 4)-(3,6-di-O-acetyl-2-O-pivaloyl ~-D-glucopyrianosyl)-(1~ 1)-(2S,3R,4E)-3-O-benzoyl-2-hexa-decaniamido-4-or~adecene-1,3-diol (62):
Hydrogen sulphide iQ passed through a solution of 61 (104 mg, 23.7 ~mol) in py:ridine (10 ml) and water (2 ml). Af~er 3 days, the mixture is concentrated at 20C
under a high vacuum. The residue is immediately dissolved in absolute methylene chlorid~ (5 ml) and reacted with palmitic acid (20 mg, 80 ~mol) and N-(3-dimethyliamino-propyl)-N~-ethylcarbodiimide hydrochloride (23 mg, 120 ~mol). Working up is carried out as described in Example ~3. Fla3h chromatography tmethylene chl~ride/
methanol (25:1. 23.1. 20sl)] gives compound 62 t92.3 mg, 85 %); thin layer chromatography [toluene/acetone~
methanol (6:7:0.3)]: R~ = 0.64, t~]20 = -47.0 (c = 1, C~Cl3).

~xample 55 O~ D-galactopyranosyl)-(l- 4)-l(u-L-fucopyranosyl 3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(~
D-galac~opyranosyl~-(l- 4)-[(~-L-fucopyranosyl)~ 3)]-(2-acetiamido-2-deoxy-~-D-glucopyranosyl)-(1- 3)-t~-D-galactopyranosyl)-(1-~4)-[(~-L-fucopyranosyl)-(1~3)~-(2-ace~amido-2-deoxy-~-D-glucopyranosyl)-(1- 3)-(~-D-galactopyranosyl)-(l~ 4)-[~-L-fucopyranosyl)~ 3)]-(2-acetamido-2-deoxy-~-D-glucopyranosyl)-(1~ 3)-(~-D-galactopyranosyl)-(1~ 4)-(~-D-glucopyranosyl)-(l- 1)-(2S,3R,4E)-2-hexadecaniamido-4-octadecene-1,3-diol (63):
1 ~ sodium methanolate solution (0.25 ml) is added to a ,301ution of 62 (87.5 mg, 19.1 ~mol~ in absolute methi~nol (30 ml) and the mixture is stirred at .

' ~ ~ - ;, ', ~
. .

~93~

45C for 24 hours. It is neutralised with Amberlite~ IR
120, filtered and concentrated in vacuo. Purification of the re~idue by chromatography using RP 18 silica gel and methanol/water (8:1~12:1) , methanol gives the tetrameric LeX antigen (54.2 mg, 91 %~; thin layer chromatography [methylene chloride/methanol/water (7:7:2)]: R~= O.35, RP
8 thin layer chromatography [methanol/water (9:1)3: ~ =
0 32 [~]20 = _57.5 (c a 1~ MeOH~-- , --:, '~ . :

.' ' . :': ~: ' .

Claims (17)

1. Process for the preparation of polymeric Lewis X
saccharide of the formula I
wherein X is OC(=NH)CCl3 (TCAI), Z or S, Z is OCH2CH(NHCOCuH2u+1)CH(OH)CH=CHCvH2v+1, S is (CH2)qCOOR'', R is OH or R?g, R?g is OAc, OBn or OBz, R' is OH, OAc or OPiv R'' is C1 to C4-alkyl, Ac is acetyl, Bn is benzyl, Bz is benzoyl, Piv is pivaloyl, n is an integer from 0 to 7, q is an integer from 4 to 12, U is an integer from 13 to 23 and v is an integer from 11 to 17, starting from a hexasaccharide unit, charac-terised in that the following steps are carried out:

(a) glycosidation of a donox saccharide unit activated on the 1-O-position (II [TCAI/
(x)]), chosen from one of the compounds of the formula II

wherein Y is OC(=NH)CCl3 (TCAI) or W, W is O-tert-butyldimethylsilyl (OTBS), O-thexyldlmethylsilyl (OTDS) or O-tert butyldiphenylsilyl (OTDPS), R is as defined, R1 is R, Ar is aryl, Me is methyl and k and l are integers from 0 to 6, and wherein, if Y is TCAI, R1 is OAc, OBn or OBz, and if Y is W, R1 is OH, with an acceptor saccharide unit of the formula V (V[(y)]), wherein R, Ar, Me and w have the meanings given and m is an integer from 0 to 5, to give a saccharide of the formula II of higher chain length (II [W (x + Y)]), wherein x is 6, 9, 12, 15, 18 or 21 and y is 3, 6, 9, 12, 15 or 18 and represent the number of monosaccharide elements in the polysaccharide in question, and x + y may not be greater than 24;

b) conversion into tha correspondinq donor saccharide of the formula II of hiqher chain length (II [TCAI/(x + y)]) by substitution of the free OH group(s) by a corresponding pro-tective group and of the W radical by the TCAI
radical;

(c) glycosidation of the donor saccharide from (b), which has been activated on the 1-0-position, with a disaccharide of the formula III which acts as an acceptor, wherein R and R' have the meanings given, to give a saccharide of the formula IV [(x + y +

2)]
wherein R, R', Ar, Me and n have the meanings given; and (d) reduction of the azido groups to acetamido groups of the compound IV, splitting off of the arylidene and benzyl radicals and peracetyla-tion of the radicals R and R', substitution of the glycosidic acetyl group by OH and reaction to give the corresponding trichloroacetimidate (X = TCAI) of the formula I, and if desired modification of the radical X to Z or S and removal of the protective yroups (R, R' = OH).
2. Process according to Claim 1, characterised in that compounds of the formula II in which the terminal galactopyranose ring is acetylated are employed.

3. Process according to Claim 1 or 2, characterised in that a pivaloylated compound of the formula II
( R' = OPiv) is employed.

4. Process according to one of Claims 1 to 3, characterised in that the glycosidations are carried out in nitriles as the solvent.

5. Process according to one of Claims 1 to 4, characterised in that compound 13 (Figure 1 (1.1)) is employed as the hexasaccharide unit.

6. Process according to one of Claims 1 to 5, characterised in that corresponding compounds in which W
is O-tert-butyldimethylsilyl (OTBS), Ar is phenyl, u = 15 and v = 13 are employed.

7. Polymeric Lewis X saccharides of the formula I
having the meanings given in Claim 1, but wherein n is an integer from 2 to 7, with the proviso that if n is 2, x may not be Z and R and R' may not be OH.

8. Tetrameric Lewis X antigen of the formula I
(n = 3);
hexameric Lewis X antigen of the formula (n = 5);
octameric Lewis X antigen of the formula (n = 7).

9. Polymeric Lewis X saccharides of the formula II
having the meanings given in Claim 1, but wherein k is l and l is an integer from 1 to 5.

10. Polymeric Lewis X saccharides of the formula IV
having the meanings given in Claim 1, but wherein n is an integer from 2 to 7.

11. Polymeric Lewis X saccharides of the formula V
having the meanings given in Claim 1, but wherein m is an integer from 1 to 5.

12. Process for the preparation of hexameric Lewis X
saccharides of the formula II (II [TCAI/W/(x)], k = O, l = 1, x = 6), characterisad in that a 2-azido-arylidene-glucopyranoside equipped with a sterically bulky silyl protective group is reacted with trichloroacetimidates of corresponding monosaccharides to give a trisaccharide of the formula II (II [W/(x)], k = 0, l = 0, x = 3), and a donor trisaccharide of the formula II (II [TCAI/(x)], x = 3) and an acceptor trisaccharide of the formula V
(v [(y)], m = 0, y = 3), are prepared from this and are reacted with one another to give the corresponding hexasaccharide.

13. Process according to Claim 12, characterised in that tert-butyldimethylsilyl 2-azido-4,6-O-benzylidene-glucopyranoaide is employed.

14. Process for the preparation of Lewis X saccha-rides of the formula IV, characterised in that a donor saccharide of the formula II (II [TCAI/(x)]) is reacted in accordance with Claim 1 (c).

15. Process for the preparation of Lewis X saccha-rides of the formula V, characterised in that the protec-tive groups of the terminal galactopyranose ring of the compound of the formula II (II [W/(x)], k = 0, l = 0,) is split off and arylidenation, preferably benzylidena-tion, is carried out on positions 4 and 6.

16. Process for the praparation of Lewis X saccha-rides of the formulae I, IV and V according to one of Claims 1 to 6 or 14 and 15, characterised in that hexa-saccharides of the formula II which have been prepared by a process according to Claim 12 are employed.

17. Use of the compounds of the formulae I, II, IV
and V according to Claims 7 to 11 for the preparation of antibodies for tumour therapy and tumour diagnostics.