CA1150248A - Process for preparing trisaccharide b. - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Novel process for the preparation of linear or branched osides. This process comprises the reaction of an imidoyl group and of an -OH group respec-tively of an ose substituted at the 1-position by an -O-imidoyl group and of another ose having all but one -OH groups protected. Application to the pre-paration of substituted trisaccharides having blood group properties, particu-larly to the preparation of trisaccharide B derivatives.
*

Description

115Q24~

m e invention relates to a new process for the preparation of oside derivatives, the new oside derivatives obtained and their biological applica-tions.
m e study of oside derivatives, that is to say of derivatives contain-ing several ose or monosaccharide units, is æ ousing a considerable interest; in fact, a certain number of them have proved biologically and chemically active or capable of giving rise to such active substances.
It has been shown, inter alia, that scme trisaccharides and tetra-sacch æ ides possess a blo~d group specificity.
Thus, the trisacch æide 2-0-(~-L-fucopyranosyl)-3-0~ D-galacto-pyranosyl)-D-galactopyranose of the formula I:

CH~OH
0~

~40 OH I OH
) ~ CH20H
/ y (I) ~0/
\ ¦ OH
~ 3 O

OH ¦
OH

constitutes the antigenic determinant B of human blood group. Because of this antigenic specificity, this trisaccharide (hereafter referred to as trisac-charide B) finds applications of great importan oe in various biological areas, , ~

~s~

in particular in imn~nohaematology and more especially in the determ m ation of blood groups.
However, these various applications can only be carried out on a large scale if trisaccharide B or oside derivatives possessing its structure can be made available easily and with high yields.
Apart fram the problem of the synthesis of these products, there is the problem of the form in which they are obtained. In this respect, it will be noticed that, deFending on the type of application envisaged, the oside deriva-tive with antigenic specificity can be used as obtained, for example as an anti-genic reagent or for preparing a testing serum, or it must contain a substituentchain, more particularly a coupling arm for fixing to a carrier, in order to form, more especially, an artificial antigen or an imm~noabsorbent. m e problem is therefore to have available an oside derivative with which the various de-sired applications can easily be carried out. Now, the methods proposed hitherto for the synthesis of oside derivatives constituting antigenic determin-ants of blood group, and the yields to which they lead, do not prove entirely satisfactory. Furthermore, the products obtained cannot easily be modified for use in a given application.
m e study of these problems by the inventors has led them to the develcpment of a new method of stereospecific synthesis which makes it possible to obtain, easily and with an excellent purity and high yields, oside derivatives providing access to numerous biological applications. In a particularly advant-ageous manner, this method of synthesis makes it possible to obtain branched osides, that is to say osides having oside side-chains, such as trisaccharide B.
This new method of synthesis is all the more valuable because it leads to the production of new oside derivatives, in particular of substitution deriva-tives of trisaccharide B, same of which can be used, in particular, in the bio-logical applications referred to above.

1~50~

Advantageously, it is shcwn that, by virtue of their inherent chemical characteristics, these new derivatives make it easier to c æ ry out these applica-tions, and, in particNlar, that they can easily be m2dified in accordan oe with the desired application or c~n even be used clirectly.
m e object of the invention is therefore to provide a new process for the synthesis of oside derivatives, whi~l makes it possible easily to make avail-able large am~unts of osides and, in particular, branched osicles such as tri-saccharide B or substitution derivatives of the latter.
According to another aspect, the object of the invention is to provicle new osicles, in particul æ new di and trisacch æ ide clerivatives, and mDre especially oside derivatives possessing the structure of trisaccharicle B.
Acc~rding to a further aspect, it relates to the biological applica-tions of the new oside derivatives.
m e process for the synthesis of oside derivatives according to the invention is characterised in that an oside derivative a) which consists of one or more ose units 1~5~

optionally attached to an organic radical, this ose unit, or at least one of these ose units, being substituted, on the anomeric carbon in the l-position, by a group -O-imidyl or the formula -O-C-(-R2)=N-Rl, in which the substituents R
and R2, which are identical or different from one another, represent an alkyl or aryl radical and preferably an alkyl radical containing 1 to 4 carbon atoms, and the groups -OH of the oside derivative being protected by suitable groups, is reacted with an oside derivative ~b) which consists of one or more ose units optionally attached to an organic radical, only one group -OH of this or these ose units being free and occupying any one of the secondary hydroxyl positions 1 to 4 and 6, the oside derivatives (a) and/or (b) comprising one unit having at least two oses.
A process of this type is advantageously employed for the synthesis of derivatives comprising branched osides.
For this purpose, the derivative (a), defined above, is reacted with an oside derivative which consists of one or more ose units optionally a-ttached to an organic radical, only one group -OH of this or these units being free and occupying a hydroxyl poSitiOn 1 to 4 or 6, the case where the -OH group occupies position 6 permitting the formation of a branched oside chain.
This reaction can advantageously be used for the synthesis of an oside derivative which consists of a branched trisaccharide or comprises such a branched trisaccharide attached, for example, to an organic radical.
According to a preferred method of carrying out the invention, which provides access, in particular, to the production of substances with antigenic properties, an oside .~ .

l~SQ~

derivative a), which consists of a galactopyranose unit, or a fucopyranose unit, substituted in the l-position by a group O-imidyl as defined above, is employed in the various embcdiments of the above process.
According to a oamplementary arrangement, the oside derivative b) oam~
prises an ose unit containing, in the l-position, a substituent -OA consisting of a functional group, which is unreactive under the conditions of the oside synthesis or has been render~d unreactive, or consisting of a group into which functional groups can be introduced, that is to say a group which permits the introduction of functional groups during or at the end of the synthesis.
~his essentially implies the availability, in the l-position, of a group which makes it easy to exploit the properties of the osides obtained, and to do so in the various biological applications disclosed hereinafter.
Particularly suitable groups A consist of radicals containing one or more unsaturated bonds, such as ethylenically unsaturated radicals. Preferably, A then represents an aIkenyl radical containing from 2 to 10 carbon atoms. A
also represents radicals which can be produ oe d, in accordance with the conven-tional techniques of organic synthesis, by the introduction of functional groups into the unsaturated bond or bonds in question. Radicals of this latter type comprise, for example, aIkyl radicals which contain, in particular, 2 to 10 carbon atoms and are substituted by at least one group -CH which, if ne oe ssary, is protected by a blocking group or forms part of a functional ralical.
A is also constituted by a functional gnDup containing at least one ether and/or amine radical.
More especially, the pro oe ss of the invention advantageously employs oside derivatives b) in which the hydroxyl group in the l-position is substituted by a radical O-allyl, sin oe this group provides ac oe ss to the introduction of a large number of chemical groups.

1~l5C~4~

Derivatives b) which correspond to the above characteristics, and com~
prise a galactopyranose and/or fucc~yranose unit are particularly preferred for the preparation of the products of the invention.
In order to prepare, in accordance with the above preferred procedure, a branched trisaccharide derivative which ccmprises a galactopyranose-galacto-pyranose-fucopyranose linkage and corresponds, more especially, to the structure II:

0~0~

I OR

(II) ~0/
O O
~ \J
1~
OR
OR

a l-O-Lmidyl-~-D~galactopyranose of the formula III:

C ~ O ~ C = H

OR/ ~ O
(III) OR

OR
with a disaccharide constituted ky a fucogalactopyranose comprising one free -OH

.~, 1~5~

group, in the 3-position of the galactopyranose moiety, said disaccharide corres-ponding to formula IV.

O ~ O ~OA

(IV) ~0 OR
OR

In these formulae:
- the _ substituents, identical or different each from the others, are protect-ing hydroxyl groups, optionally with a neighbouring substituent and are selected between stable groups, unreactive under the usual osidic synthesis conditions, easily remDvable under mild conditions compatible with the upkeeping of the osidic structure, in particular they represent groups forming benzylic ethers or benzylidenic a oe tals with the oxygen atom of the -OH;
- A represents an organic radical as defined above; and - ~ and R2 have the above meanings.
Preferably, the substituent A of the disaccharide of the formula IV
oonsists of an ethylenically unsaturated radical chosen from amangst alkenyl radicals preferably containing from 2 to 10 carbon atoms. A can also consist of an alkyl radical substituted by at least one group -OH which, if necessary, is protected by a blocking group or forms part of a functional radical.
It is particularly preferred to use groups R which represent a benzyl 1~l50~4~

radical and, in the case of the 4- and 6-positions, a benzylidene radical, and to choose an allyl radical as the substituent A.
The choi oe of these meanings for R and A offers the possibility of re-moving A independently of R or of treating it without affecting R. m us, the allyl group repr~sented by A can be converted, in particular, to a ~-hydroxy-ethyl group, for example under the action of osmium tetroxide and sodium periodate, the value of which group in the biological applications of the pro-ducts formed will be emphasised below. m ere is then free scope to remDve the groups R when desired. Amongst the wide possibilities for treating the allyl group, its conversion by hydroboration to give a y-hydroxypropyl group will also be mentioned by way of example. In addition, such chains can be treated in order to introduce an allyl group which therefore makes it possible to carry out a large number of reactions and to create substitution chains of hydrophilic character, which are particularly valuable for the desired biological applica-tions.
The allyl meaning for A will be noted. It enables to have a group which, while being unreactive with the osidic synthesis conditions, can be further treated depending on the desired application.
According to one variant, in order to prepare the trisaccharide deriva-tive of the formLla II, a digalactopyranose which contains a free group -QH in the 2-position of one of the galactopyranose units and corresponds to the formLla:

1~l50~48 o~o ~o (V) OR I OR
J~CH20R

~0~
OH
is employed as the disaccharide! and it is condensed with the group -O-imidyl of a l-O-imidyl-~-L,fucopyranose of the formLla VI:

-f~ (VI) OR ~ C = H
OR Rl/ \ R2 In these form~lae V and VI, R, ~ , R2 and A possess the meanings givenabove.
The use of this synthesis process leads to the production, with a high yield, of trisaccharide units possessing the desired stereospecificity.
The condensations of the process of the invention are advantageously carried out under anhydrous conditions and at ambient te~perature.
m e reaction will be carried out in the presence of a strong acid of little nucleophilic action and in a solvent which is weakly nucleophilic and inert with respect to reaction products.
Anhydrous p-toluenesulphonic acid is advantageously used as the acid.

_ g _ 1~l5~48 As regards the solvent, it advantageously consists of benzene, ether and, more especially, nitromethane.
The disaccharides employed in the above condensation reactions are new products which, within the scope of the invention, constitute synthesis inter-mediates. These disaccharides are advantageously prepared in accordance with a process which is dependent on the same principle as these condensations.
Thus, in order to obtain the fuco-galactopyranose of the formula (IV), a galactopyranose which contains a free group -OH in the 2-position and corres-ponds to the formula VII:
OR

~ \l ~ (VII) is advantageously reacted with a fucopyranose of the formula VI given above, and the group R in the 3-position of the ose unit originating from the galacto-pyranose VI is then removed in accordance with the conventional techniques for removing the blocking group represented by R.
Similarly, the digalactopyranose of the formula V is advantageously obtained by reacting the galactopyranose of the formula VIII, which contains a free group -OH in the 3-position, CE~2R
O ~ O ~OA

~ OH ~ (VIII) \ ~
OR

with a l-O-imidyl-galactopyranose of the formula III above. The disaccharide l~SOZ4~

thus obtained is then treated, in accordan oe with the conventional methods, so as to remove the group R in the 2-position of the ose unit originating from the galactopyranose VIII.
According to an aspect of the invention which is of great value, the above process permits a total synthesis of trisaccharide B.
For this purpose, after having carried out the condensations of the disaccharide of the formula IV or V with, respectively, the monosaccharide of the formula III or VI, the substituent A is sequentially removed, under mild con-ditions in accordance with the conventional techniques, from the resulting pro-duct of the structure II, and this leads to a pro~uct of the structure IX:

o~ O\

OR o OR

(IX) ~

OR I
OR

the hydrogenolysis of which makes it possible to obtain trisaccharide B.
Each of the steps of the synthesis pro oe ss of the invention is characterised by a high yield. Carrying out these steps thus makes it possible to obtain large amounts of trisaccharide B or of substituted trisaccharides.

1~5C~

According to another aspect of the invention which is of great value, the perfection of the process referred to above leads to the preparation of new oside derivatives.
The study of these new derivatives has shown that, by virtue of their chemical characteristics, they constitute synthesis substances which æe analogous to the blood group antigens B; advantageously, these derivatives also provide access to the prep æ ation of antigenic products.
The new osides of the invention contain at least one galactopyranose unit in which the hydrogen atom of the group -OH in the l-position is sub-stituted by a radical A which represents an alkyl group substituted by at leastone hydroxyl group which, if necessæ y, is protected by a blocking group or forms part of a functional radical, or also represents a group containing one or more unsaturated bonds, in particular an ethylenically unsaturated radical chosen from amongst alkenyl radicals, and more especially aIkenyl radicals hav-ing 2 to 10 carbon atcms, it being possible for A also to contain one or more ether and/or amine groups.
In a preferred group of oside derivatives according to the invention, _ represents an alkyl radical which contains 2 to 10 carbon atoms and is sub-stituted by at least one hydroxyl group, this radical advantageously being chosen from amongst aIkylene glycol or hydroxyalkyl radicals.
Products in which _ represents an ~,~-dihydroxypropyl or ~-hydroxy-ethyl or ~-hydroxypropyl group constitute biological reagents and are also especially valuable in the biological applications which involve fixing these products to a protein or an insoluble carrier, for example for the formation of immunoabsorbents.
In another preferred group of oside derivatives according to the inven-tion, _ represents an alkenyl radical containing 2 to 10 carbon atoms and, in ,~
. .

1~5t~;~,4~i particular, an allyl radical. The reactivity of this radical proves very part-icularly advantageous when carrying out the actual oside synthesis, insofar as A
can be treated independently of the blocking groups used for the hydroxyl groups of the ose units. The value of the meaning allyl for A also lies in the range of possibilities offered for the introduction of a given functional group in accordance with the applications envisaged. Moreover, it will be noted that the allyl derivative can be used, as obtained, as a biological reagent, for example for neutralising haemolysins. Other preferred products contain a group A com-prising at least one ether group and at least one ethylenically unsaturated group which is advantageously an allyl group. In this series of products, A can represent, for example, an alkoxyaIkenyl chain and, in particular, an aIkoxy-allyl chain, the alkoxy group containing a variable number of carbon atcms, which is generally less than 10 and more especially of the order of 3. In a further group of prcducts, _ is a substitution chain of the aIkoxy-alcohol, alkoxy ester, amine or -amide or alkoxy-aIkylamine or amide, or aIkoxy-carboxylic acid type, it being possible for these various chemical groups to additionally contain an inserted amine group.
The galactopyranose units defined above advantageously form part of a linear or branched trisacch æide chain comprising a fucopyranose unit and an-other galactopyranose unit.
In these trisaccharide chains, the hydroxyl groups of the various oseunits are free or protected by blocking groups chosen from amongst stable groups which are unreactive under the usual conditions of oside synthesis and which can readily be removed under mild conditions ccmpat;hle with the retention of the oside structure.
The trisaccharides corresponding to the characteristics defined above, and more especially the branched trisaccharides consisting of a galactopyranose ~5~ 4~

unit which contains a substituent _ as referred to above and is simultaneously substituted by another galactopyranose unit and by a fucopyranose unit, are pre-ferred.
In particular, the invention relates to the branched trisaccharides of the form~la II

0~ o o OR
~ 2 / ~ (II~
OA
l \ O
~,~O~J
~Y
OR
OR

in which: the substituents R, which are identieal or different from one another, represent as necessary with a neighboring substituent of the protective groups of the hydroxyl radical and are chosen from amongst stable groups which are un-reactive under the usual conditions of oside synthesis and can readily be re-moved un~r mild conditions compatible with the retention of the oside structure, and in particular from amongst benzyl or benzylidene groups, or represent a hydrogen atom, and A represents an organie radieal which eontains at least one group into whieh funetional groups ean be introdueed, and whieh eonsists, in partieular, of an ethylenieally unsaturated radieal ehosen from amongst alkenyl 1~l5~4fl radicals, preferably having 2 to 10 carbon atoms, and more particularly consists of the allyl radical, or by a radical in which the group -OH is optionally pro-tected, or also A is an alkoxy-alkyl, alkoxy-ester, -amine or amide, alkoxy-aIkyLamine or amide or alkoxy-carboxylic acid type, it being possible for these various chemical groups to additionally contain an inserted amine group.
As indicated above, these new osides are themselves, constitute inter-mediates of great value for the preparation of products with blood yroup prcper-ties and, in particular, of trisaccharide B, or of products possessiny its struc-ture.
The invention therefore provides the means which make it possible to obtain synthesis homologues of antigen B. It also provides products which con-stitute such homoloyues. In fact, the trisaccharides of the formula II in which R is a hydroyen atam prove particularly valuable in this respect.
m e possibility, afforded by the process of the invention, of having the osides in question available in an extremely pure form imparts even more value to their properties, in particular in their biological applications, especially in immwnohaematology and more especially for the determination of blood groups.
The great value of having such synthesis products available, for example for transfusion purposes, will be appreciated by considering the process used hitherto for the determination of blood groups.
According to the most common techniques, these determinations are carried out using testing sera which are of human origin or produced by the imm~nisation of animals.
Several thousand litres of anti-D antiserum are used each year in France for carrying out the systematic determination of the Rhesus standard group (antigen D). In order for the specificity of this reagent of human origin ~5~4~

to be directed solely against antigen D, it is necessary to absorb the natural anti-A and anti-B antibodies, which are initially present in these sera, onto A
or B red corpuscles of the Rhesus negative group. Several thousand litres of blood of the Rhesus negative group B are used for this pllrpose. This entails not only the handling of very large volumes, but, in particular, involves the use of relatively rare blood.
m us, in France, only 1.2% of the population belong to the Rhesus negative group B. m is has the consequence of causing a "shortage", created artificially, of bottles of blood of this group, which are intended for blood transfusion.
m e new trisacch æ ides, of the formula II of the invention, in which R
represents a hydrogen atcm in fact constitute a solution to this problem of the "shortage" of bottles of blood belonging to groups which are valuable for blood transfusion.
In fact, by fixing these new trisaccharides to a solid carrier, the invention provides an artificial model, which can be regenerated, of Rhesus negative B corpuscles.
m e same type of co~pound can be used for solving other problems which involve an even rarer occurren oe of combinations of blood group systems.
For example, some subjects belong to the exceptional group which is negative to Cellano antigen (Kell system) and are capable of developing an anti-Cellano antibody which can be used as a testing serum for Cellano antigen.
According to the conventional process for the determination of blood groups, thenatural anti-B antibo~ies of these subjects are absorbed by red corpuscles of a subject of Cellano negative group B. The undeniable value of the use of the comrpounds of the invention is appreciated by considering that such a ccmb m ation is only found 1.7 times in 10,000.

1~5(1~

It is clear that the use of the immunoabsorbent, of the invention de-fined above, in plaoe of these red corpuscles, for performing the same f~nction, is of very particular value.
In these applications, it is possible to resort advantageously to the carriers normally used in this type of technique and more especially to insolu-ble polymers such as cellulose. m e osides of the invention and more especially the trisaccharides of formula II defined above, in which A represent an ethyleni-cally unsaturated radical, more particularly, an allyl radical or an hydroxy-alkyl group are useful as such, or eventually modified, as biological reagents.
m e study of these derivatives has shcwn notably their high capacity of neutralizing haemDlysins. These derivatives are hen oe advantageously usable for the detection and neutralizatiRn of anti-B haemolysins by replacing subst-ances of blood groups isolated fram the gastric mucin of the pig or the horse.
Cther characteristics and advantages of the invention become apparent in the remainder of the description given in the examples.
m e melting points given in these examples are measured in capillary tube by means of a Buchi apparatus and are not corrected. m e optical rotations æ e determlned using the model 141 polarimeter marketed by Perkin-Elmer. m e infrared (IR) spectra are recorded using an IR~-l spectrophotometer of Jouan-Jasco and the nuclear magnetic resonance (NMR) spectra are recorded using aPerkin-Elmer R-32 spectrometer (90 MHz). As reg æ ds the results relating to NMR, the chemical shifts (~) æ e ;ndicated relative to internal tetramethylsilane;
the protons of the L,fucopyranose unit bear single prime and those of the non-reducing D-galactopyranose unit bear a double prime. m e colu~n chromatography is carried out using Mercksilica gel (p æ ticle size 0.063-0.200 mm).
EX~MPLE 1.
Preparation of the trisaccharide allyl 2-0-(2,3,4-tri-O-benzyl-~-Lr 'l~lS~7~4~

fucopyranosyl)-3-0-(2,3,4,6-tetra-0-benzyl-~-D-galactopyranosyl)-4,6-0-benzyl-idene-~-D-galactopyranosyl of the formula X:

0~--0 j O ~ 6 5 OBz ¦ O O/

~H2 (X) ~0/

~1-- I
~ C~13 1~
OBz ¦
OBz in which Bz represents the benzyl radical.
In order to prepare the trisaccharide of the formula X, a disaccharide, namely a fucogalactopyranose of the formwla XI (allyl-2-0-(2,3,4-tri-0-benzyl-~-L,fucopyranosyl)-4,6-0-benzylidene-~-D-galactopyranoside):

~ ~ O (XI) 0~

\~ /

~ \l .

OBz Osz ~.

~s~

is reacted with the l-O-imidyl-galactopyranose of the formula XII (l-O-(N-methyl)-aoe timidyl-2,3,4,6-tetra-0-benzyl-~-D-galactopyranose):

~ 3` C N ~ 3 OBa~ ~ O
~ ~ (XII) I

OBz Prior to this condensation reaction, on the one hand the disaccharide XI, and on the other hand the l-O-imidyl-galactopyranose XII, are prepared in accordan oe with the foll^wing pro oe dure.
1. Preparation of the disaccharide XI.
The disaccharide XI is vbtained by condensing the fuccpyranose of the formLla XIII (l-O-(N-methyl)-aoe timidyl-2,3,4-tri-0-benzyl-~-Lrfucopyranose):

o vB~ (XIII) OBz I --C = N
vBz CH3~ ~ CH3 with the allyl galactopyranose of the formula XIV (allyl 3-0-benzoyl-4,6-0-benzylidene-~-D-galactopyranoside):

C6H5~- ^CH2 O ~ O ~
1~ ~ (XIV) \ OBz QH

~l5~?~.4F3 a) Preparation of l-O-(N-methyl)-acetimidyl-2,3,4-tri-O-benzyl-~-L-fucopyranose of the formula XIII.
434 mg of 2,3,4-tri-O-benzyl-a-L-fucopyranose of the formula XV:

~ \J
~ (XV) OBz ¦
OBz are dissolved in 10 ml of methylene chloride, and 2.5 equivalents of dimethyl-chloroformiminium chloride are then added. The reaction mixture is stirred in the absence of moisture and is then filtered, after 40 minutes, through a short column of silica gel and evaporated. This yields 2,3,4-tri-O-benzyl-~-L-fucopyranosyl chloride (yield 96%) which is employed directly in the preparation of l-O-(N-methyl)-acetimidyl-fucopyranose.
434 mg of this fucopyranosyl chloride and 190 mg of ethyldiisopropyl-amine are added to 10 ml of an anhydrous benzene solution, which contains 81 mg of N-methylacetamide and is stirred under dry nitrogen and in the absen oe of light, in the presence of 580 mg of silver oxide and 4 A molecular sieves.
After 20 hours, the reaction medium is filtered on a bed of neutral alumina, which is washed with 250 mg of ether containing 0.1% of triethylamine. The filtrate and the ether phase are evaporated to dryness and the solid residue (462 mg, 94%) is crystallised from hexane, this giving the desired imidate (431 mg, 88%).
Melting poin~ 89-90 &; [~]D0 = -67 (c 1, benzene); NMR (chloroform~d): ~1.18 (3H,d,7Hz,Me-C), 1.84 (3H,s,~e-C), 2.97 (3H,s,Me-N), 5.80 (lH,d,Jl 28Hz,H-l), 7.30-7.35 (15H,m,Ph); analysis: calculated for C30H35NO5: C: 73.59; H: 7-20;
N: 2.86; O: 16.34; found: C: 73.72; H: 7.33; N: 2.92; O: 16.19.

~lS~

b) Preparation of the allyl galactopyranose of the formula XIV.
m e preparation is OE ried out by reacting benzaldehyde, in the pre-sence of zinc chloride, with allyl ~-D-galactopyranoside, and this leads to allyl 4,6-O-benzylidene-~-D-galactopyranoside which is subjected to a benzoyla-tion reaction. m ese steps are carried out as follcws:
~) allyl ~-~-galactopyranoside.
A mixture of 6 g of mercuric oxide, 0.4 g of mercuric bromide and 10 g of drierite is stirred for 30 minutes, at ambient temperature and in the absence of moisture, in the presence of 80 ml of 1,2-dichloroethane and freshly dis-tilled allyl alcohol. 12 g of 1,2,3,4-tetra-O-acetyl-~-D-galactopyranosyl bromide æe then added and stirring is continued for 12 hours. The solids are filtered off and the filtrate is evaporated to dryness. m e residue is dis-solved in 200 ml of chloroform and the resulting solution is washed with a 10%
strength aqueous solution of potassium iodide and with water, dried (CaC12) and evaporated. me residue is dissolved in 100 ml of methanol, and 3 ml of a molar solution of sodium methylate in methanol are added.
After one hour, the react_on medium is neutralised (acid resin) and evaporated. m e residue is crystallised from ethanol, this giving allyl ~-D-galactopyranoside (5.2 g, 81%); melting point 103-104 & ; [~]D0 = -19 (c 1, MeOH).
~) allyl 4,6-O-benzylidene-~ galactopyranoside.
5.96 g of allyl ~-D-galactopyranoside are then stirred vigorously for 12 hours in the presen oe of 80 ml of benzaldehyde and 5.5 g of zinc chloride.
m e reaction medium is then slowly poured, whilst stirring vigorously, into 300 ml of diisopropyl ether. m e precipitate formed is filtered off after a few hours and washed with about 100 ml of ether. The resulting solid is crystal-lised from ethanol, this giving allyl 4,6-O-benzylidene-~ galactopyranoside ~5~2~

(6.92 g, 82~); melting point 178-179 C, [~]D = ~7 (c 1, pyridine);

analysis: calculated for C H O .H O: C: 58.88; H: 6.79; O: 34.31;

fcund: C: 58.94; H: 6.47; O: 34.09.
miS product was characterised by its di-O-a oe tate: melting point 94-95 &; [~]D = +56 (c 1, chloroform);
analysis: calculated for C20H2408: C: 61.22; H: 6.16; O: 32.62; found:
C: 61.31; H: 6.12; O: 32.77.
y) allyl 3-O-benzcy1-4,6-O-benzylidene-3-_-galactopyranoside.
A solution of 457 mg of benzoyl chloride in 2 ml of anhydrous chloro-form is added, at 5 &, to 5 ml of an anhydrous chloroform solution containing443 mg of imidazole. After 10 minutes, the imidazole hydrochloride formed is filtered off and washed with 5 ml of chloroform~ 20 ml of an anhydrous chloro-form solution of allyl 4,6-O-benzylidene-~-D-galactopyranoside (1 g) are then added to the filtrate and the reaction medium is heated under reflux at 75C for about 18 hours. After cooling to ambient temperature, the reaction medium is diluted with 50 ml of chloroform, washed with a dilute aqueous solution of bicarbonate and with water, ~ried (CaC12) and evaporated. m e residue is cry-stallised from an ethyl aoe tate/ether mixture, this giving the compound of the formLla XIV (1.14 g, 85~); melting point 172-173 ; [~]D = +83 (_ 1, chloro-form);

analysis: calculated for C H 4O : C: 66.98; H: 5.87; O: 27.16; found:23 2 7 C: 67.01; H: 5.75; O: 27.37.
c) Condensation of the products obtained under a) and b).
A solution of 619 mg of the derivative of the formula XIV in 20 ml of anhydrous nitromethane is stirred for 3 hours, in the absence of moisture and under dry nitrogen, in the presen oe of powdered 4 A molecular sieves (1 g).
1.222 g of the a oe timidyl fucopyranose XIII and 344 mg of p-toluenesulphonic 1~5~

acid are added to this reaction solution and stirring is continued for 20 hours.
The reaction medium is then neutralised with triethylamine and filtered. m e filtrate is evaporated and the residue is purified by chrcmatography on a column containing 150 g of silica gel, using a hexane/ethyl a oe tate mixture (5:2, volume/volume). Allyl 3-O-benzoyl-2-0-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-4,6-O-benzylidene-~-D-galactopyranoside is obtained in the pure state (1.131 g, 91%); [~]D0 = +13 (c 1, chloroform); NMR (chloroform-d): ~ 1.12 (3H,d,7Hz, Me-C), 7.00-7.55 (20H,m,Ph);
analysis: calculated for C50H52Oll: C: 72.44; H: 6.32; O: 21.23; found:
C: 72 66; H: 6.32; O: 21.27.
This disaccharide is subjected to a selective O~debenzoylation reac-tion in accordan oe with the following procedure:
1.03 g of the galactopyranoside derivative in question are dissolved in 20 ml of methanol, and 1 ml of a molar methanolic solution of sodium methylate is added. After one hour, the reaction medium is concentrated and poured into ice-cooled water. m e phase obtained is extracted with chloroform and the chloroform extracts are then washed with water, dried (CaC12) and evaporated. m e residue is crystallised fram an ethyl acetate/ether~hexane mix-ture, this giving allyl 2-0-(2,3,4-tri-O-~enzyl-~-L-fucopyranosyl)-4,6-O-benzyl-idene-~-D-galactopyranoside (847 mg, 94%); melting point 166-167 &; [~]D0 = -58 (c 1, chloroform); NMR spectrum (chloroform~ 1.12 (3H,d,7Hz,Me-C), 5.26 (lH,d,Jl 2,4Hz,H-1'), 7.15-7.65 (20H,m,Ph);
analysis: calculated for C43H4801o: C: 71.52; H: 6.67; O: 22.07; found:
C: 71.68; H: 6.63; O: 22.15.

2. Preparation of the l-O-(N-methyl)-a oe t;midate of the formula XII.
This imidate is prepared from 2,3,4,6-tetra-O-benzyl-~-D-galacto-pyranose in accordance with the method described under l-b). The 2,3,4,6-tetra-1~5-~4F~

-O-benzyl-~-D-galactopyranosyl chloride formed as an intermediate is obtained with a yield of 93%; [a]20 = +151 (c 2.5, benzene). The imidate i9 obtained with a yield of 92%; [a]D = +25 (c 1, ch]oroform); NMR sFectrum (chloroform-d): ~ 1.84 (3H,s,Me-C), 2.95 (3H,s,Me-N), 5.83 (lH,d,Jl 28Hz,H-1), 7.30 (20H,m,Ph);
analysis: calculated for C37H41NO6: C: 74.60; H: 6.94; N: 2.35; O:
16.12; found: C: 74.68; H: 6.91; N: 2.10; O: 16.31.

3. Condensation of the products under 1 and 2.
A solution of the compound allyl 2-0-(2,3,4-tri-O-benzyl-a-L-fuco-pyranosyl)-4,6-O-kenzylidene-~-D-galactopyranoside (600 mg) and 900 mg of the aoetimidate of the formula XII in 15 m~l of nitrcmethane is stirred for 5 hours in the presence of 4 A molecular sieves (1 g) and under a dry nitrogen atmos-phere. A solution of anhydrous p-toluenesulphonic acid (150 mg) in 5 ml of nitromethane is then added. The reaction mixture is stirred for 48 hours at ambient temperature. After adding 1 ml of triethylamine, the reaction medium is filtered and the filtrate is evaporated to dryness. The residue is dissolved in 50 ml of chloroform and the solution is washed with a saturated aqueous solution of sodium bicarbonate and with water, dried (CaC12) and evaporated. The residue is chromatographed on a column contaming 80 g of silica gel, using a hexane/
ethyl acetate mixture (5:2, volume/volume), this leading to the ccmpound of the formula X (907 mg, 88%) which is crystallised from 95% strength ethanol (865 mg, 84%); melting point 117-118; [~]D0 = +2 (c 1, chloroform), NMR spectrum (chloroform-d): ~ 1.18 (3H,d,7Hz,CH3-C), 7.05-7.55 (40H,m,Ph);
analysis: calculated for C77H82O15: C: 74.13; H: 6.62; O: 19.24; found:
C: 74.22; H: 6.64; O: 19.54.
EX~MPLE 2.
According to a variant, the product of the formula X is prepared by llS~;?~P~

1. condensing the imidyl galactopyranose of the formula XII with the allyl galactopyranose of the form~la XVI (allyl 2-0-benzoyl-4,6-0-~enzylidene-~-D-galactcpyranose):

~ ~ O

V \) (XVT) \ OH

OBz this leading to the disaccharide allyl 2-0-benzoyl-3-0-~2,3,4,6-tetra-0-benzyl-~-D-galactopyranosyl)-4,6-0-benzylidene-~-D-galactopyranoside of the formula:

0~ 0 \ OBZ /1 \~ I I

OBz O ~ (XVII) ~C~I
o OR

and then by 2. condensing the disaccharide of the formula XVII, O~debenzoylated beforehand, with the imidyl fucopyranose of the formLla XIII.
1. Condensation of the imid~l galactopyranose of the formula XII with th~ allyl galactopyranose of the formula XVI
a) Initially, allyl 2-0-~enzoyl-4,6-0-benzylidene-~-D-galactopyranoside is pre-pared in accordan oe with the following procedure:

~15~

50 ml of an ice-cooled solution of sodium hydroxide are added to 15 ml of an acetone solution containing 1.5 g of the compound of the formula XIV.
After two minutes, the reaction medium is extracted with 80 ml of chloroform and the organic extracts are washed with water, dried with CaC12 and evaporated.
The residue is chromatographed on a column containing 80 g of silica gel, using an ethyl acetate/hexane muxture (1:1, volume/volume). m is yields the starting material (782 mg, 52~) and t~e desired derivative (661 mg, 44%); melting point 144-145 &; []D = +10.2 (c 1, chloroform);

analysis: calculated for C 3H 4O : C: 66.98; H: 5.87; O: 27.16; found:

C: 67.04; H: 6.03; N: 27.12.
b) m e galactopyrar.oside thus formed is converted to the galactosyl derivative using the compound of the formula XII. The reaction is carried out in accord-an oe with the technique described above, 285 mg of the galactopyranoside and 596 mg of the imidate being employed.
After purification on a colume containing silica gel (carried out with 40 g of silica and a ~:1 volume/volume ethyl a oe tate/hexane mixture), 594 mg of the desired disaccharide are obtained, this oorresponding to a yield of 95%;
[~]D0 = +80.5 (c 1, chloroform); NMR spectrum (chloroform~ 4.63 (lH,d,Jl 2--.Hz,H-1), 5-13 (lH,d,Jl" 2,4Hz,H-l');

analysis: calculated for C H O : C: 73.21; H: 6.25; O: 20.53; found:

C: 73.06; H: 6.02; O: 20.71.
c) The resulting disacch æ ide is subjected to a selective O-debenzoylation.
580 mg of the disacch æ ide æ e dissolved in 20 ml of methanol and the solution is then treated with 1 ml of a IM methanolic solution of sodium methyl-ate. After 12 hours, the reaction medium is con oe ntrated, diluted with 50 ml of chloroform and then washed with water, dried with CaC12 and evaporated. The residue is crystallised from 95 strength methanol. This yields 479 mg, which 1~5~

is a yield of 93% of the O-debenzoylated disaccharide; melting point 150-151 C;
[~]D = +44 (c 1, chloroform); NMR spectrum (chloroform-_): ~ 2.86 (lH,d,J3Hz,OH), 5.21 (lH,d,Jl, 2,4Hz,H-1'), 7.10-7.60 (25H,m,Ph);
analysis: calculated for C50H54Oll: C: 72.27; H: 6.55; O: 21.18; found:
C: 72.46; H: 6.52; O: 21.29.
d) In accordance with the technique described above, the trisaccharide of the formula X is prepared by condensing the disaccharide of the formula XVII with the crystalline imidate of the formula XIII, these oompounds being employed in respective amounts of 275 and 325 mg. This yields 370 mg of trisaccharide, which corresponds to a yield of 79%; melting point 115-116C; [~]20 = +2 (c 1, chloroform).
EXAMPLE 3. Preparation of 2-0-(2,3,4-tri-O-benzyl-2-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-~-D-galactopyranosyl)-4,6-O-benzylidene-D-galactopyranose of the formula:

0~ o\

\ OBz A

`11 1 ~ C6H5 ~L~&2 (X~:x) o ~o i~ \J
1~
OBz ¦
OBz . ~

115~41q 624 mg of the trisaccharide of the formula X are dissolved in 10 ml of dimethylsulphoxide. The reaction mixture thus formed is stirred for about 4 hours at 100 C in the presen oe of 294 mg of potassium tert.-butoxide.
After cooling to ambient temperature, the reaction medium is poured into a 10% strength i oe -cooled aqueous solution of ammonium chloride; the result-ing solution is extracted with 60 ~1 of chloroform and the chloroform extracts are washed with water, dried with CaC12 and evaporated. The residue is dis-solved in 20 ml of an acetone/water mixture (9:1, volume/volume) and the solu-tion is then stirred for 30 minutes in the presen oe of 428 mg of yellow mercuric oxide and 271 mg of mercuric chloride. After filtration, the reaction medium is washed with a 10% strength aqueous solution of potassium iodide and with water, dried with CaC12 and then evaporated. m e residue is chromatographed on a column containing silica gel (50 g), using a hexane/ethyl a oe tate mixture (3:2, volume/volume). 495 mg of the desired trisaccharide (yield 82~) are thus ob-tained in the form of a colourless foan~ [~]20 = +50 (c 1, methanol); NMR
spectrum (chloroformr~ 2.99 (3H,d,7Hz,CH3-C), 5.56 (lH,s,CEI-Ph), 7.05-7.65 (40H,m,Ph);
analysis: calculated for C74H78O15: C: 73.61; H: 6.51; O: 19.87; found:
C: 73.80; H: 6.58; O: 20.06.
EXAMPLE 4. Preparation of trisaccharide B or 2-O-(~-~-fucopyranosyl)-3-O-(~-D-galactopyranosyl)-D-galactopyranose of the formula:

~15~

OH

~ OH
~0 OH ¦ OH
OH

\ I
~ CH3 OH~

OH ¦
OH

480 mg of the trisaccharide of the formula XIX are subjected to hydro-genolysis for about 20 hours, at a~bient temperature and at atmospheric pressure, in 20 ml of acetic acid, in the presen oe of 500 mg of palladium~on-charcoal.
After filtering off the catalyst, the filtrate is evaporated to dryness at a temperature below 35C, this giving the desired trisaccharide (186 mg, 96%) in the form of a white powder; melting point 135-138C (decomposition); [~]DO = +35 (c 1, water:methanol, 19:1, volume/volume); NMR spectrum (D2O): ~ 1.70 (3Hr d, 7HZ,CH3-C).
This c y d is characterised by its crystalline peracetate; melting point 215-216 &; [~]D0 = +51 (C 1~ chloroform); NMR spectrum (chloroform~d):
1.17 (3H~ d,6. 5Hz,~e-C), l.g0-2.20 (30H,m,Ac), 6.40 (IH,d,Jl 23.5HZrH~lr anomer).

.

-1~5~

EX~MPLE 5. Preparation of ~-hydroxyethyl 2-0-(2,3,4-tri-O-benzyl-~-L-fuco-pyranosyl)-3-0-(2,3,4,6-tetra-O-benzyl-~-D-galactopyranosyl)-4,6-O-benzylidene-~-E~galactopyranoside of the formula XX:

o~ O

\ OBz /

( ~ ~ 6 5 OBz O

/ ~OCH2 (XX) I\
o ~o ~ y 2 2 1~
OBz I
OBz 312 mg (0.25 mol) of trisaccharide X are stirred, at ambient tempera-ture, in 16 ml of a dioxane/water mixture (3-1, volume/volume) to which 1.25 mg of osmium tetraxide (OSO4) are added. After one hour, 112 mg, 2.1 equivalents, of sodium periodate (NaIO4) are added and the mixture is then stirred for about

4 hours.
50 mg of sodium borohydride (NaBH4) are added and then, after 10 minutes, the mixture is evaporated to dryness and the residue is taken up in about 100 ml of chlorofonm. This chloroform solution is washed with a 10%
strength aqueous solution of sodium sulphite (Na2S03) and then with water. It is subsequently dried over calcium chloride (CaC12) and then filtered, and the filtrate is evaporated. This yields a syrup which is treated with 16 ml of a 1~5~

dioxane/water mixture (3:1, volume/volume) and stirred with 52 mg (1 equivalent) of NaIO4.
After 3 hours, 50 mg of NaBH4 are added and then, after 10 minutes, the mixture is evaporated to dryness and the residue is taken up in 100 ml of chloroform. The chloroform solution is washed as indicated above.
The resulting syrup is chromatographed on 20 g of silica (SiO2). A
mixture of ethyl acetate and hexane (3:1, volume/volume) is used as the eluent.
The product collected æ ter evaporating off the eluent is recrystal-lised from an ether~hexane mixture. This yields 247 mg of the desired trisac-10charide; melting point 83-84 & ; yield 79%.
EX~MPLE 6. Preparation of y-hydroxypropyl 2-0-(2,3,4-tri-O-benzyl-~-Lrfuco-pyranosyl)-3-o-(2t3l4~6-tetra-o-benzyl-~-D-galactopyranosyl)-4~6-o-benzylidene ~-D-galactopyranoside of the formula XXI:

B~ O

\OBZ
( ~ ~ C6 5 BzO I O
)~~ ~
y XN

BZO ¦

OBz ` .

312 mg (0.25 mmol) of the trisaccharide X are stirred, at ambient 1~5~2~.4~

temperature, in anhydrous tetrahydrofurane (3 ml), and 0.8 ml of a 0.5M solution of 9-borabicyclo[3.3.1]nonane, or 9-BBN, in tetrahydrofurane is added. After heating under reflux for 4 hours and cooling to ambient temperature, the exoe ss 9-BEN is destroyed by adding ethanol (0.3 ml). A 3M aqueous solution of sodium hydroxide (0.2 ml) is subsequently added and 0.2 ml of a lOM solution of hydrogen peroxide is then added dro~wise, the temEerature being kept at between 40 and 50C. The reaction mLxture is then heated at 50 for 1 hour, whilst stirring. After cooling, solid potassium carbonate (1 g) and methylene chloride (20 ml) are added. The solids are filtered off and the filtrate is evaporated.
m e residue is purified by passing it through a column containing silica gel (15 g) and eluting it using an ethyl a oe tate~hexane mLxture (2:1, volume/volume).
The compc~nd XXI is thus obtained in the pure state (292 mg, 92%); [~]D0 = +2 (c 1, chloroform).
This co~pcund is characterised by its crystalline a oe tate; melting point 112-113; [~]D0 = +1.5 (c 1, chloroform); NMR sFectrum (chloroform~d):
~ 1.20 (3H,d,6.5Hz,Me-C), 2.00 (s,3H,AC), 7.10-7.60 (40H,m,Ph).

,~

~L51~!7,4~

EXPMPLE 7. Preparation of y-allyloxypropyl 2-0-(2-0-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-3-Ç~(2,3,4,6-tetra-O-benzyl-~-D-galactcpyranosyl)-4,6-Q~benzyl-idene-~-D-galactopyranoside of the form~la XXII:

(~H2BZ
B~ O

¦ O ~ 6 5 BzO ~

~ ~ CH2- XXII
~0 \ OBz/
~r~
BzO ¦
OBz 633 mg of the cc~lpound XXI are stirred, at ambient temperature, in N,N-dimethylformamide (10 ml), in the presence of sodium hydride (96 mg of a 50%
strength dispersion in oil). After one hour, allyl brcmide (0.15 ml) is added.
The reaction is oamplete in 1 hour 30 minutes. After adding methanol (2 ml), the reaction medium is evaporated and the residue is taken up in chloroform (50 ml). m e organic phase is washed with a saturated aqueous solution of sodium chloride and with water, dried aver sodium sulphate, filtered and evapor-ated. The oil present in the residue is extracted with boiling hexane (20 ml) and the desired ccmçound is crystallised fram an ethyl acetate/hexane n~xture (588 mg, 90%); melting point 103-104 C; [~]D = +3 (c 1, chloroform); NMR

spectrum (chloroform-~ 1.21 (3H,d,J7Hz,Me-C), 7.10-7.55 (40H,m,Ph).

1~l5~41!~

EXAMPLE 8. Preparation of y~[(y-hydroxy)-pro~yloxy] propyl 2-0-(2,3,4-tri-O-benzyl-~-Irfucopyranosyl)-3-0-(2,3,4,6-tetra-O-benzyl-~-D-galactopyranosyl)-4,6-O-benzylidene-~-D-galactopyranoside of the formula XXIII:

B~ O~
\ ~Bz /
~ I
I O ~ 6 5 BzO ~ ¦

\I XXIII

1 ~0 o O
CH - CH - CH - O - CH - CH - CH - o~
1~
BzO
OBZ
490 mg of the derivative XXII are stirred, at ambient temperature, in anhydrous tetrahydrofurane (4 ml), and 1 ml of a 0.5M solution of 9-BeN in tetra-hydrofurane is added. After heating under reflux for 2 hours 30 minutes and o~oling to ambient temperature, the ex oess 9-BEN is destroyed by adding ethanol (1 ml). A 3M aqueous solution of sodium hydroxide (0.25 ml) is subsequently added and 0.38 ml of a lOM solution of hydrogen peroxide is then added dropwise, the temperature being kept at between 40 and 50C. The reaction mix*ure is then heated at 50 & for 1 hour. After cooling, the aqueous phase is saturated with potassium carbonate (1 g), filtered and evaporated.
m e residue is purified by passing it through a column containing silica gel (30 mg) and eluting it using an ethyl acetate/hexane mixture (2:1, l~SQ~4~

volume/volume). The compound XXIII is thus obtained in the pure state (442 mg, 89~); [~]20 = +1.5 (c 1, chlorofonm); NMR spectrum (chloroform~d): ~ 1.20 (3H,d,6.5Hz,Me-C), 7.60-7.70 (40H,m,Ph).
EXAMPLE 9. Preparatlon of the compound of the formula XXIV:

BzO ~

OBz BzO ¦
I
~ a~IV
~0/

~ ~ CH2 ~ CH ~ ~cH ~ COOMe 1~
BzO ¦
OBz Dry chromium triolide (400 mg) is added, whilst stirring and in the ab-sence of moisture, to a mixture of methylene chloride (15 ml) and pyridine (0.644 ml). After stirring for 20 minutes, a solution of the trisaccharide XXI
(633 mg) in methylene chloride (5 ml) is a~ded. After 20 minutes, the reaction medium is decanted and the residue is extracted with chloroform. m e chloroform phase is washed with a saturated solution of sodium bicarbonate and with water, dried over sodium sulphate, filtered and evaporated. The residue is purified by chrcmatography on silica gel (10 g) using an ethyl a oe tate~hexane mixture (3:1, volume/volume). The resulting aldehyde (575 mg, 91%) is converted into the derivative XXIV by means of a conventional Wittig reaction in benzene (15 ml).

1~5~

m is derivative is crystallized from ether/hexane (524 mg, 88~), melting point 152-153; [~]D0 = _3 (c 1, chloroform); NMR spectrum (chloroform-d): 1.23 (3H,d,6.5Hz,Me-C), 3.69 (3H,s,COOMe), 7.10-7.55 (40H,m,Ph).
EX~qPlE 10.
Application of the products of the invention as imn~noabsorbents:
1) - An activated agarose matrix covered by human albumin serum is used (SAH).
The activation of the agarose is effected with cyanogen bromide CNBr according to the method of Axen et al described in Nature, 214, 1302 (1967) mcdified by March et al according to the technique described in Anal. Biochem, 60 149 (1974).
m e agarose is covered with the human albumin serum according to the method of Parikh et al described in "Methods in enzymology" 35, 77 (1974). The coupling of the trisaccharide XXIV on the matrix is effected by means of a carbodiimide derivative by operating according to the method of Parikh et al con-sidered above.
In this way about 10 to 20 molecules of trisaccharide XXIV per mole-cule of albumin are introduced.
2) - The carrier is constituted by activated agarose derivative by means of ethylene diamine according to the oFerational method described by Cohen in Methods in enzymology 35, 102 (1974). m e coupling of the trisaccharide xxrv is carried out as explained above. The acetylation of the coupling product then follows to remove the excess of amino groups.
3 ) - A derivative of formula II in which R represents a hydrogen atom and A is a -(CH2)3-0-(CH2)4NH2 group is fixed on carboxymethyl cellulose. m e coupling is done by means of the carbodiimide derivative as described above.
EXP~PIE 11.
Use of the trisaccharide of formula XXIV for neutralising anti-B
haemolysins.

'l~l5~?t4?;~

A solution containing 10 mg/ml of the product XXIV is used. sy carry-ing out the reaction in accordance with the techniques of the European Pharma-copoeia, a total inhibiti~n of an agglutinant dose of an anti-s testing serum is observed with a 1/5 dilution of the trisaccharide of the invention.

Claims (32)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Process for the preparation of oside derivatives, characterized in that an oside derivative (a) which consists of one or more ose units optionally attached to an organic radical, this ose unit, or at least one of these ose units, being substituted, on the anomeric carbon in the 1-position, by a group -O-imidyl of the formula -O-C-(-R2)=N-R1, in which the substituents R1 and R2, which are identical or different from one another, represent an aryl radical or an alkyl radical containing 1 to 4 carbon atoms, and the groups -OH of the oside derivative being protected by suitable groups, is reacted with an oside derivative (b) which consists of one or more ose units optionally attached to an organic radical, only one group -OH of this or these ose units being free and occupying any one of the secondary hydroxyl positions 1 to 4 and 6, the osides derivatives (a) and/or (b) comprising one unit having at least two oses.

2. Process for the preparation of oside derivatives, characterized in that, in order to obtain branched osides, the oside derivative (a), according to claim 1, is reacted with an oside derivative which consists of one or more ose units optionally attached to an organic radical, only one group -OH of this or these units being free and occupying a hydroxyl position 1 to 4 or 6, this permitting the formation of a branched oside chain.

3. Process according to claim 1, characterized in that the oside deriva-tive (a) employed consists of a galactopyranose unit or a fucopyranose unit.

4. Process according to Claim 3, characterised in that the oside derivative b) comprises an ose unit containing, in the 1-position, a substituent -OA consisting of a functional group which is unreactive under the conditions of the oside synthesis or has been rendered unreactive, or consisting of a group into which functional groups can be introduced, that is to say a group which permits the introduction of functional groups during or at the end of the synthesis.

5. Process according to Claim 4, characterised in that, in the substituent -OA, A represents a radical containing one or more ethylenically unsaturated radicals, selected amongst the alkenyl radicals containing from 2 to 10 carbon atoms, and also represents any radical which can be produced, in accordance with the conventional techniques of organic synthesis, by the introduction of functional groups into the unsaturated bond or bonds in question.

6. Process according to Claim 5, characterised in that, A
represents an alkyl radical which contains, in particular, 2 to 10 carbon atoms and is substituted by at least one group -OH which, if necessary, is protected by a blocking group or forms part of a functional radical.

7. Process according to Claim 5, characterised in that A
represents the allyl radical.

8. Process according to Claim 5 wherein A is a functional group containing at least one ether and/or amine radical.

9. Process for the preparation of a branched trisaccharide derivative which comprises a galactopyranose-galactopyranose-fucopyranose linkage and corresponds to the structure II:
(II) characterised in that a 1-O-imidyl-.beta.-D-galactopyranose of the formula III:

(III) is reacted with a disaccharide consisting of a fuco-galactopyranose containing a free group -OH in the 3-position of the galacto-pyranose unit, this disaccharide corresponding to the formula IV:

(IV) in which formulae: the substituents R, which are identical or different from one another, represent groups for protecting hydroxyl radicals, these protective groups incorporating a neighbouring substituent if desired, and are chosen from amongst stable groups which are unreactive under the usual conditions of oside synthesis and can readily be removed under mild conditions compatible with the retention of the oside structure, and comprising groups which form, together with the oxygen atom of the hydroxyl, benzyl ethers or benzylidene-acetals, A represents a radical containing one or more ethylenically unsaturated radicals selected from alkenyl radicals containing 2 to 10 carbon atoms or any radical which can be produced, in accordance with conventional techniques of organic synthesis, by the introduction of functional groups into the unsaturated bond or bonds in question and R1 and R2 possess the meanings given in Claim 1.

10. Process for the preparation of the trisaccharide derivative of the formula II, according to Claim 9, characterised in that a digalactopyranose which contains a free group -OH in the 2-position of one of galactopyranose units and corresponds to the formula V:

(V) is employed, and in that it is condensed with the group -O-imidyl of a 1-O-imidyl-.beta.-L-fucopyranose of the formula VI:

(VI) R1, R2, R and A possessing, in these formulae, the meanings given in Claim 9.

11. Process according to Claim 9 or 10, characterised in that the substituent A of the disaccharide of the formula IV is the allyl radical.

12. Process according to Claim 1, characterised in that the reaction is carried out in the presence of anhydrous p-toluenesulfonic acid and nitro-methane.

13. A process according to Claim 9 further comprising, in view of obtaining trisaccharide B, the sequential removal of the substituent A, from the resulting product of the structure II, under mild conditions, in accordance with the conventional techniques, which leads to a product of the structure IX:

(IX) followed by hydrogenolysis.

14. A process for preparing an oside which comprises reacting an oside derivative a) which consists of one or more ose units optionally attached to an organic radical, this ose unit, or at least one of these ose units, being substituted, on the anomeric carbon in the 1-position, by a group -O-imidyl of the formula -O-C(-R2)=N-R1, in which the substituents R1 and R2, which are identical or different from one another, represent an aryl radical or an alkyl radical containing 1 to 4 carbon atoms, and the groups -OH of the oside derivative being protected by suitable groups, with an oside derivative b) which consists of one or more ose units optionally attached to an organic radical, only one group -OH of this or these ose units being free and occupying any one of the secondary hydroxyl positions 1 to 4, the oside derivatives a) and/
or b) comprising one unit having at least two oses, which contain at least one galactopyranose unit in which the hydrogen atom of the group -OH in the 1-position is substituted by a radical A
which represents an alkyl group substituted by at least one hyd-roxyl group which, if necessary, is protected by a blocking group or forms part of a functional radical, which represents a group containing one or more unsaturated bonds and able to include also an intercalary amine function characterized in that the said gala-ctopyranose unit forms part of a linear or branched trisaccaride chain comprising a fucopyranose unit and another galactopyranose unit.

15. A process according to claim 14 wherein A is an alkenyl radical containing 2 to 10 carbon atoms.

16. A process according to claim 14 wherein A is an alkoxy-alkenyl, alkoxy-alcohol, alkoxy-ester, -amide or -amine, alkoxy-alkylamine or alkyl-amide or alkoxy-carboxylic acid radical.

17. A process according to claim 9 in which the various substituents R represent a hydrogen atom.

18. A process according to claim 9 in which A is an alkyl group.

19. An oside derivative which contains at least three ose units, at least one of which is a galactopyranose unit in which the hydrogen atom of the group -OH in the 1-position is substituted by a radical A which represents an alkyl group substituted by at least one hydroxyl group which, if necessary, is pro-tected by a blocking group or forms part of a function radical or which repre-sents a group containing one or more unsaturated bonds and able to include also an intercalary amine function, when prepared by a process according to claim 14 or an obvious chemical equivalent thereof.

20. Novel oside derivatives of formula II as defined in claim 9 when pre-pared by a process according to claim 9 or an obvious chemical equivalent thereof.

21. A process for preparing allyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fuco-pyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside which comprises reacting allyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside with 1-O-(N-methyl)-acetimidyl-2,3,4,6-tetra-O-benzyl-.beta.-D-galactopyranose.

22. The compound allyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galacto-pyransoside when prepared by a process according to claim 21 or an obvious chemical equivalent thereof.

23. A process according to claim 21 which comprises the further steps of reacting the allyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside with osmium tetroxide, followed by treatment with sodium periodate, then reducing the product with sodium borohydride to obtain .beta.-hydroxyethyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside.

24. The compound .beta.-hydroxyethyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside when prepared by a process according to claim 23 or an obvious chemical equivalent thereof.

25. A process according to claim 21 which comprises the further step of reacting the allyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside with 9-borabicyclo[3.3.1]nonane in tetrahydrofuran, followed by reaction with hydrogen peroxide and aqueous sodium hydroxide to form .gamma.-hydroxypropyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranose.

26. The compound .gamma.-hydroxypropyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fuco-pyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranose when prepared by a process according to claim 25 or an obvious chemical equivalent thereof.

27. A process as claimed in claim 23 which comprises the further step of reacting the .beta.-hydroxyethyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galacto-pyranoside with sodium hydride, followed by addition of allyl bromide, to form .gamma.-allyloxypropyl 2-O-(2-O-2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside.

28. The compound .gamma.-allyloxypropyl 2-O-(2-O-2,3,4-tri-O-benzyl-.alpha.-L-fuco-pyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside when prepared by a process according to claim 27 or an obvious chemical equivalent thereof.

29. A process as claimed in claim 27 which comprises the further step of reacting the .gamma.-allyloxypropyl 2-O-(2-O-2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside with 9-borabicyclo[3.3.1]in tetrahydrofuran, followed by reaction with hydrogen peroxide and aqueous sodium hydroxide, to obtain .gamma.-[.gamma.-hydroxy-propyloxy]-propyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galacto-pyranoside.

30. The compound .gamma.-[.gamma.-hydroxy-propyloxy]-propyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galactopyranoside when prepared by a process according to claim 29 or an obvious chemical equivalent thereof.

31. A process according to claim 25 which comprises the further step of reacting the .gamma.-hydroxypropyl 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galacto-pyranoside with chromium trioxide in the presence of methylene chloride and pyridine and subjecting the aldehyde product to a Wittig reaction to obtain 4-methoxycarbonyl-3-butene 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-.beta.-D-galacto-pyranoside.

32. The compound 4-methoxycarbonyl-3-butene 2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-4,6-O-benzyli-dene-.beta.-D-galactopyranoside when prepared by a process according to claim 31 or an obvious chemical equivalent thereof.