CA1265132A - Process for the organic synthesis of oligosaccharides and derivatives thereof - Google Patents

Abstract

A B S T R A C T

Process for the organic synthesis of oligosaccharides and derivatives thereof.

The invention relates to a process for the organic synthesis of oligosaccharides constituting or comprising fragments of acid mucopolysaccharides comprising the reac-tion of two compounds constituted or terminated by units of glucosamine structure and of uronic acid structure respectively, said units being specifically substituted.
This process particularly enables valuable anticoagulant drugs to be obtained.

(No figure)

Description

Sl~3;2 , 1.
P~OCES~ FOR THE ORGANIC SYNTHESIS OF O~IGOSACCHARIDES
AND DERIVATIVES 'rHEREO~

~ield of the Invention The invention relates to a process ~or -the organic synthesis of oligosaccharides constituting or com-prising fragments of acid mucopolysaccharides. It also relates to the synthesis of derivatives of these oligo-saccharides .
The invention relates, in addition, to novel oligosaccharides of the above-indicated type and to -their 10 derivatives, po~sessing, particularly, biological proper-ties conferring on them, in particular, interest as medi-caments and/or useful, for example, as laboratory reagents.
It is directed also to their uses particularly their biological and biochemical uses.
By the term "acid mucopolysaccharide", is meant derivatives also currently called glycosamino-glycuronoglycane~. It concerns oligosaccharides and polysaccharides encountered more especially in chains of biologically active derivatives such as derivatives of the heparin and heparane-sulphate~ype~

In natural products, the mucopolysaccharides concerned are essentially formed of alternate amino-sugar -uronic acid units, or conversely. In these units, -the amino-sugar, denoted below by~A, has more especiaIly a ' ~ . .

i5~3~

D-glucosamine s t r u c t u r e . The uronic acid, which will be called U, has, more especially, a D-glucuronic acid or ~-iduronic acid structure.
The basic structure for A correspon~ respect-S ively to -the formula. a and for U to the formulae b and c below :
6 r OH -~

HO ~
-~lino derivati~e (a) D,glucosamine COOH

~ ~ OH

0~1 OH
~b) ~-glucuronic acid . (c) L-]duronic acid ~ ~n the natural products concerned, these various unit,s are linked to one another stereo-speciæically generally by 1 ~ , 4, and .1 ----, 4 .
Thus, for example in heparin, linkages of the type 1 ~ 4 (between the c and a, a and b, and a and c units) and of the type 1 ~ ,~4 (between the b and a units), are to be found.

~,6~i~1L3~

It will be noted, also, still with reference to na-tural products, tha-t the above units comprise specific substitutions, that is to say certain substitu-tions a-t given posi-tions. The chains of natural products contain, thus, for example, -0-substitu~ed units 2-0-sulphate-~-iduronic acid, 3-0-sulphate-D-glucosamine, 3,6-di-0-sulpha-te-D-glucosamine, 6-0-sulphate-D-glucosamine, andn~-0-substituted units, like, for example, units D-glucuronic acid, L-iduronicacidandD-glucosamine~
In addition, the unit a is N-substituted at the 2 position of the -N-acetyl and/or -N-sulphate group 9 .
Description of the Prior A~t The importance of the therapeutic uses of the above acid mucopolysaccharides is known, in particular, for the prevention and treatment of disorders of clot-ing and of the vascular wall, and in particular throm-bos~s and atheroscleroses and arterioscleroses.

Moreover the numerous researches of Applicant are known for the obtaining of fragments of high affinity , ~165~3~

for AT III and biologically active fragments from hepa-rin chains. The inventions developed on the basis of these researches are the subject of various patent applications among which are patent application EP No.
80 40 1425.6 of 6 October 1980 and patent applica-tion FR No. 81 08604 of 29 April 1981.
It is recalled -that in the EP application, there is described in particular an octasaccharide called ABCDEFGH possessing anti-thrombotic properties of great interest, corresponding to the structure:
COO OR OR COO OR r OR

H ~ ~ ~ o ~ ~ OII
OSO3- NHSO3 OH NHAc OH 3 3 NHSO3 A B C D E F G H
In this formula, R represents a SO3 group or a hydrogen atom.
In the above FR patent application of Appli-cant, a homogeneous hexasaccharide composition of the structure C'DEFGH is described, also possessing high anti-thrombotic properties. This structure corresponds to the formula:
OR COO OR OR

HO ~ O ~ O ~ OH
H NHAc OH NHSO3 OSO3 NHSO3 C' D E F G H

5 ~265~32 in which R represents an S03 group or a hydrogen atom The methods propo~ed until now to obtain this -type of product bring into play extraction techniques from heparin or from products obtained in the course of the preparation of heparin, or again depolymerisation techniques of heparin chains under the action of a chemical or enzymatic agent, followed by specific frac-tionation par-ticularly by affinity chromatography.
The progress of the researches of~ pplicants in -this field has led them to inve~tigate novel means enabling this type of product to be obtained and more especially study of the possibilities of obtaining them synthetically.
In this respect, it is appropriate to measure the number of problems raised by such synthesis. In fact, on the ore hand, these products contain in their chains several types of A and U units. On the other hand, the linkages between these units correspond to a given stereo-chemistry and are of the 1,4 type, of which the particular difficulties of production are well-known. In addition, each unit comprises one or several specific substitutions according to the type of product concerned. It is to be considered also that the glucosamine units in natural prQducts comprise two nitrogenous groups different from one another, namely an N-acetyl group and an -N-sulphate group.
It follows that such syntheses have practically never been contemp7ated until now i~ the scientific lit~erature, more particularly, as regards ~-iduronic acid.

6 ~ 51~2 All these elements highlight the restrictive requirements of which it is easy to appreciate the dif-ficulties that they involve for the development of a general process and of the process of synthesis.
By researching conditions of oside synthesis suitable for the development of this type of compound, Applican-ts have developed a strategy by selecting cer-tain particular types of protection for the substances utilised.

The work carried out has then shown tha-t with such so-protected substances, it was possible to produce a stereo-specific chain formation and then to introduce, if desired, into the sequences formed, given substitut-ions at predetermined positions.

According to one aspect presenting an interest of which the impor-tance will be measured, the process developed has great flexibility. It is thus possible to arrive at, with the advantages in particular of specificity and purity associated with a synthetic pro-cess t numerous oligosaccharide derivatives including the specific substitutions encountered with natural products, 23 or even different substitutions and/or again units of similar structure with different configurations.
Due to this process, Applicants have obtained oligosaccharides endow~-sd in particular with medicinal properties of great value and more especially high anti-thrombotic activity. ~he process of the invention also permits accesa to a large number of particularly valuable 7 ~6~132 oligosaccharides, in particular for biological reagentsand/or for reference compounds for structure studies~
It is therefore an object of the invention to provide a process for producing, synthetically, oligo-saccharides and their derivatives or the like, includ-ing or corresponding -to fragments of acid mucopolysaccha-rides.
It is also an object to provide means enabling the es-tablishment between A and U type units of glycoside linkages in the desired stero-specificity.
It is also an object to provide means enabling the introduction into the units of the glycoside chain of given functional groups, in particular of specific substituents such as encountered in the chains of biolog-ically active molecules, particularly those of the heparin and heparane-sulphate t~pe.

It is also an object to provide means enabling the production of oligosaccharides such as mentioned above, but of which the substituentsand/or the chemical nature of the sugars and/or the position and configuration of the inter-glycoside linkages and/or the configuration of the monosaccharides and/or the order of the enchain-ments are different from those of natural products.
According to another aspect, it is also an object of the invention to provide novel oligosaccharides constituting intermediate products of the process of synthesis concerned in which all the -OH groups of the various units are blocked by protective groups and the 8 1265~32 precurs~r groups of the functional radicals possibly present ; if necessary, these radicals themselves are also protected.
According to yet another aspect, the invention is aimed at providing novel oligosaccharides having the structure of the above natural produc-ts as well as oligosaccharides corresponding to fragments of these products.
It is also directed at providing novel oligo-saccharides possessing specific substitutions of natural products.
It is also an object of the in~ention to pro-vide novel oligosaccharides bearing subs-titutions dif-ferent ~rom the specific substitutions ` concerned and/or including different units with respect to the natural products considered above.
~he invention ~so relates to the biological uses of these oligosaccharides, particularly as active medicinal substances, laboratory agents or reference sub-qtances for the study, in particular, of compounds includ-ing this type of structure.
General Description of the Invention The process of synthesis of the invention is characterised in that it brings about the reaction of two compounds :
- constituted or terminated respectively by A units of glucosamine structure, in particular D-glucosamine, g ~ ;53L3Z

and U units of glucuronic acid structure~ in particular D-glucuronic, or iduronic acid, in particular L-iduronic acid ;
- one of the units A or U being an alcohol in which the -OH group of the alcohol function occupies any one of the positions 3, 4 or 6 in the case of unit A and

2, 3 or 4 in the case of unit U, the other unlt possess- .
ing an activated anomeric carbon, that is to say compris-ing a reactive group capable of establishing with the -OH
group o~ the alcohol the desired glycosylation -0~
linkage, in the desired s-tereo-chemistry, to form a -A-U or -U-A sequence , - the reactive group of A and U being compat-ible with the protective groups and/or functional groups pre~ent on the units ;
- all the position of A and U excep-ted those of which the anomeric carbon is activated bearing ~OH, amino or carboxyl groups, or precursors of such groups, the groups themselves, when they are present being blocked by one or advantageously several types of pro-2~ tective groups, these various groups being compatible with one another and with the above precurgors, these protective groups and precurso.rs being inert with respect to the glycosylation reaction and with the reactive groups, permitting the positioning, in the course of subsequent operations, of given substituents at the various positions, and this, as the case may be, sequentially, the conditions of application to cause . ~

1 o ~;~65~

the starting substances to react being selected so as not to alter the structure of the units o~ these substances and the nature of the various substituents present, pro-vided that the establishment of the interglycoside link-age does not lead -to the production of a disaccharide with a ~ 2 N-sulphate or (2-N-acetyl)-6-0-sulphate-D-glucosamine ~ ~methyl-D-glucuronic acid~ structure.
Due to the above arrangements, it is thus possible to form a covalent bond between the units of structure A and U and this, in the stereo-chemistry which this type of enchainment presents in the biolog-ically active molecules already considered.
I-t is even possible by means of the invention to carry out the desired ohain formations in a given order and/or possessing a given stereo-specificity.
The means proposed according to the invention ~hus enable the establishment particularly of a 1 ~ 4 type linkage between a D-glucosamine unit and either D-glucuronic acid, or ~-iduronic acid, a 1 -~ 4 type linkage between a D-glucuronic acid unit and a D-glucosamine unit and a 1 ~ 4 type linkage between an ~-iduronic acid unit and a D-glucosamine unit.
The mono- or oligo-saccharidic intermediatesOf this synthesis are semi-open or open products. A com-pound will be called semi-open on the right when it is a compound activated or potentially activatable on its anomeric carbon, thus permitting its transfer to the non-reducing end of a monosaccharide or of an 1 1 1265~ 3,'2 oligosaccharide. The expression "compound semi-open on the left" will denote a monosaccharide or an oligosacch-aride possessing a single free or potentially free -OH
function, enabling its specific glycosylation. By way of illustration, there is indicated below the formula 1 of an example of a compound semi-open on the left and that 2 of an example of a compound semi-open on the right :

o ~c C

1 0~ ~ 0~

l 2 ~ B~

It follows that derivatives will be called.
open when they relate to a derivative semi-open both on the right and on the left according to the above defini-tion, such derivatives permi-t-ting elongation of -the chain in both directions. A derivative of this -type corresponds for example to formula 3 :

~/lCA~( ~
r ~e ~e ~265i~2 As for closed derivatives, they are substances whose units cannot give rise to chain elongation by rea-son of the nature of their substituents.
According to an additional feature to be able -to add units to the A-U or U-A sequence formed in the preceding step, the A and U units of the sequence formed must include temporary protective groups, that is to say groups capable of select~ely blocking a position of the A or U unit intended to take part in a novel glycosy-lation reaction. These groups are removable in the pre-sence of other groups present on the units of the start-ing products by recreating an alcohol, which permits in repeating the preceding step of glycosylation elongation of the gluoid skeleton.
The inven-tion hence provides access to the synthesis of oligosaccharides with varied enchainments, whether it relates to ~ or ~ stereo-specificity and/or the order of enchainmen-t between the a and c ~nd/or b, units, lengthening being producible as desired.
According to ye-t another feature of the process of the inven-tion, the developed glucid chain is subject to one or several chemical reactions in order to intro-duce a given type of functional group or, successively, several types of groups, then to form, if desired, deriv-atives of these functional groups.
This functionalisation step may be effec-ted by elinlinating only certain protective groups and/or certain . .

13 ~ 2 ~ S~3 2 precurs~r groups of the amino derivatives or again the whole of the protective groups and/or of the precursor groups and by introducing in their place a given type of substituent or successively different subs-tituents7 -then by releasing a portion or all of the -OH groups still blocked, if desired.
It is understood then that the various groups present on the units of the chain are compatible with the substi-tuent introduced at each step.
The one or more chemical reactions applied in the course of the functionalisation steps are carried out so as not -to alter the struc-ture of the chain and the groups that it is desired if necessary to maintain and/or those which have already been introduced.
According -to a preferred embodimen-t of the invention, -to obtain oligosaccharides with speci~ic substitutions as defined above, starting materials are advantageously used containing several types of pro-tective groups, namely (1) one or several semi-permanent groups and (2) one or several permanent groups.
By semi-permanent groups, is meant groups removable in the firs-t place after the reac-tions of glycosylation when the glucid skeleton includes the number of desired units, without removal or alteration of the other groups present, then enabling the intro-duction of the desired functional gro~ps at the positions that they occupy.

~Z6~13~

The permanent groups are groups capable of main-taining -the protection of the -OH radicals during the introduction of the functional groups in place of the semi-permanent groups.
These groups are selected from among those cQmpatible with the functional groups introduced after removal of -the semi-permanent groups. It concerns, in addi-tion,groups inert with respect to the reactions carried out for the posi-tioning of these functional groups and which are removable without the functional groups being altered.
Advan-t~geously, the practising of these arrangements enables the development of a glucid chain in which the A and U units are selectively substituted.
To prepare more particularly oligosaccharldes containing ~ and/or U units of the biologically active molecules men-tionsd above, recourse is advantageously had to protective groups such as acyl, alkyl possibly substituted or aryl radicals.
~he units of -the products employed of type A
comprise, at the 2 position, a nitrogen group permitting the maintenance of the presence of a nitrogen function during the operations applied in the process. This nitrogen group is advantageously constituted by groups such as N3 or -NHcoo-cH2-c6H5~ or any other group con-stituting a precursOr of the amine functiQn or of an amine deriva-tive, in par-ticular -NHS03 or -NH-acyl, more especially -NH-COCH3.

.

12~i5132 As for the carboxyl functions of the U units, they are blocked by groups inert with respect to reactions used for the replacement of the protective groups and removal at the end of the synthesis to liberate the carboxyl groups, possibly for the purposes of salt for~a-tion. These protective groups of carboxyl function areselected advan-tageously from among alkyl radicals or aryl radicals.
The structure of the product employed in the glycosylation reac-tion is selectcd as a func-tion of the units of the glucide skeleton desired as well as of the desired substitutions.
To form, for example, a disaccharide of -U-A-type, two compounds respec-tively with uronic acid and amino sugar structure, corresponding, in addition to the above-men-tioned definitions, are used.
For chain lengthening, these compounds as employed to form -the disaccharide concerned, contain, in addition, a temporary group on the position intended~to be involved in the new glycosylation reaction. For U-A
disaccharide lengthening towards -the left, this temporary group is present on the U unit and for lengthening to the right on the A unit.
It is thus possible to obtain, in particul~r, enchainments Uw Ax Uy Az in which the sum of the indices is comprised between 2 and 12, these values being included in the range , where w and y cannot be nil simultaneously.

Regular enchainments are of the type U (AU)n, (AU~n A, (UA)n or again (AU)n with n 1 to 6.

"

16 ~26513~:

According to a modification of the process of the invention, the alternation of A-U or U-A type encountered in the structuresof natural products can be modified by using, in place of the one or several A or U
units~ a sugar constitu-ting a structural analog of an A or U unit, such as a ncutral sugar or a desoxy-sugar, or again other uronic acid uni-ts or amino sugars U or A o~
different configurations.
In a preferred embodiment of the process of the invention, the above alcohol is reacted with a reactive derivative such as a halide, an imidate or an orthoester. These condensations are carried GUt under anhydrous conditions.
The condensation reaction between the halide and the alcohol is advantageously of -the Koenigs-Knorr type. The halide is advantageously constituted by a bromide or a chloride by reason ~ the ease of production.
Operations are in a solvent medium, more especially in an organic solvent, particularly of the dichloromethane or dichloroethane type.
Advantageously a catalyst is used, generally a silver or mercury salt, for example, silver trifluoro-methane sulphonate, commonly called silver triflate, silver carbonate, silver oxide, merouric bromide or mercuric ~yanide. Also a proton accept~r is used such as sym-collidine in the same way as an extractor for the water possibly present and/or for the halohydric acid formed, for example 4 A molecular si~ves.

~26~1~2 Study of the reaction conditions show that it is appropriate to operate a-t room temperature or again at a lower temperature which can reach 0C or less, in an atmosphere of an inert gas such as nitrogen or argon.
~ hese conditions enable the units of structure a and b or c (or -the reverse), t o b e c o n d e.n s e d, i n t h e d e s 1 r e d s t e r e o - c h e m i s t r y .
They also permit the establishment of covalent bonds with neutral sugars or desoxy-sugars.
A modification comprising the use, as catalyst, of mercuric derivatives, in particular of cyanide and/or or mercuric bromide, is established to be suitable for forming covalen-t bond~ between alcohols of various structures and an ~-idose precursor of the unit of c structure (~-iduronic acid). According to this modifica-tion, 4 A molecular sieves are also used. The organicsolvent is selected according to the reactivity of the alcohol. ~hus advantageously there is used a solvent of the type of nitrobenzene when the condensation requires a temperature higher than 100C. For lower temperatures, 2~ solvents such as benzene or dichloromethane are used.
~ixtures of solvents are also suitable to carry out the condensation reaction.
With units of type U, in particular c units, it is advantageous to use, as reagent group an orthoester.
The reaction is then preferably carried out at a temper-ature above 100C.

~, 1 8 ~6513~

~he solvent medium is of the chloro~enzene type or any other solvent ~Jhose boiling point exceeds 100C
and it is advantageously between 100 and 150C. To activate the reaction, a catalyst such as 2,6-dimethyl pyridinium perchlorate is used.
This embodiment of the condensation step is found to be of great interest to form an interglycoside linkage between a uni-t o~ structure (~-iduronic acid) and a unit of structure _ (D-glucosamine) The use of the orthoester group has in parti-cular a double advantage.
On the one hand, it permits conferring on the anomeric carbon of c the necessary reactivity for the glycosylation reaction. On the other hand, the opening of this group ensures the positioning at the 2 position of c of a protective group, selectively removable, there-by permitting the introduction in its place, of a speci-fic substituent group.
~ hus, by -the reaction of a 1,2-0-methoxy-ethylidene group of a c unit with the -OH radical of an a unit, it is possible at the same time to establibh an interglycoside linkage between the two products used and to have at the 2 position of c an -OAc group (Ac representing an acetyl group) which could be removed selectively for the purposes of introduction of a given functional group, for example -S03 . This feature also permits full liberty to be left for treating the 4 posi-tion of the c unit.

,................................... .

. . .

1;~6513~

These features, particularly advantageous, enable the provision of a 2-0-sulphate ~-iduronic unit to be made, such a~ exis~ for example, in heparin chains.
When an imidoyl group is used as the reagent group, it is found to be appropriate to operate at low temperature, more especially at a temperature below or equal to about 0C, in a solvent medium, such as dichloro-methane, in the presence of a 4 A molecular sieve and a catalyst such as boron trifluoride etherate.

In -the starting alcohol, the free -OH group occupies the posi-tion that it is desired to engage in the glycosylation linkage.
By selec-ting the alcohol suitably, it is thus possible to form linkages of the 1-2, 1-3, 1-4 or 1-6 type.
From the sequence formed at the end of the con-densation reaction, a chain is developed including the desired number of units by repeating the glycosylation step.
The alcohol function of one of the units A or U involved in the glucide sequence already constituted is then advantageously liberated from its temporary pro-tective group. The choice of this group will be easily determined by the technician s~illed in the art accord-ing to the nature of the other groups present on the glucide chain.
Among the various groups which can be used, is mentioned the allyl group which, by treatment, for example ~265132 first with an isomerising agent such as Pd, Rh and Ir derivatives, in particular rhodium tris-triphenylphos-phine chloride (I), or again potassium tertio-butoxide, then under acid conditions, in particular with a mixture of mercuric oxide and mercuric chloride, enable the recreation easily of an alcohol at the position -that it occupies.
In -the same way, it is possible to obtain an -OH group by saponification from an -O-acyl group, in particular -O-ace-tyl or O-chloroacetyl.
These radicals can be removed to liberate an -OH func-tion, for example, by means of thiourea in a solven-t medium, advantageously at a temperature higher than 80C, preferably of the order of 100C.
The foregoing arrangements enable -the product-ion of a glucide chain wi-th alternate A-U or U-A units.
This regular alternation can be modified by applying suitable substances in the glycosylation reaction. It is thus possible to develop an irregular structure with the incorpora-tion ofunits other than U or A, in par-ticular neutral sugars or again desoxy-sugars. Another type of irregular structure can be obtained by adding several consecutive A units or U units between two A-U or U-A structural units.
It is understood that the various arrangements of the invention relating to the A and U units are applied equally to other units which can include the glucide chain,such as neutral sugars or desoxy-sugars.

, 2 1 ~Z6~132 As has already been indicated, the various groupspresent on the A and U units are selected so as to confer on the la-tter sufficient reactivity to pro-duce the glycoside linkage concerned.
The -OH radical protective groups, apart from the temporary groups already considered, are generally selected from -the group comprising acyl radicals (par-ticularly acetyl, alkyl , substituted alkyl such as benzyl), and for two neighbouring posi-tions, among the acetal groups or Ketals, for example benzylidene. Another form of protection consists of carrying out blocking of two -OH groups in epoxide form or of 1,6-anhydro bridge.
Advan-tageously, the produc~ used in the glycosylation reaction contain several types of pro-tective groups, which permits in the course of the stepof -functionalisation the successive introduction of one or several functional groups and the liberation of one or several -OH radicals if desired.
In general, the protective groups may already 2~ occupy certain positions on the products applied in the glycosylation reaction.
They may also be introduced from other groups once the glucide skeleton is constituted. This modifi-cation comprises, for example, the use for glycosylation of a substance A in which the -OH groups at the 2 and 3 positions and at the 1 and 6 positions are blocked in anhydrous form, respectively 2,3-epoxide and 1,6-anhydro.

22 l 2 ~ ~l 32 Due to thi~ blocking, during the development of the glucide skeleton there is available an element con-stituting potentially an A unit but not interfering with -the reactions applied in the synthesis. This arrangement has the advantage of allowing wide liberty to carry ou-t desired reactions on the groups of the other uni-ts.
I-t will be noted, in addition, in the case concerned, that the opening of the epoxide function by the sodium a~ideenables the introduction, at the 2 position, of an N3 group which hence constitutes a precursor of an amine function.
Preferably, to have available.a glucide chain permitting the introduction successively of one or several types of substituents in the course o~ the functionalisation step, in particular the specific substitutionsabove, products are applied com~rising several types of protective groups~ namely the semi-permanent groups and the permanent groups defined above.
As already indicated, the substitutions of the natural products concerned, apart from those of the 2 positions of the A units, are essentially consti-tuted by sulphate groups.
Applicants researches to perfect the suitable sulphation conditions have shown that it is possible a.nd even advantageous to carry out a sulphation reaction in t~e presence of benzyl groups. Contrary to . .

.

23 ~:6~

opinions accepted in this field, the removal of benzyl permanent groups, in the presence of -O-sulphate groups, can be effected.
Preferably, the -OH radicals of the starting materials intended to be sulphated are then pro-tected by acyl groups, in particular acetyl, whilst the -OH
radicals intended to be liberated at the end of the synthesis are protected by a permanent group such as the benzyl group.
By the high flexibility of the process of the invention, it is possible to subject all of the glucide chain *ormed to a given chemical reaction in order to introduce a particular type of substituen-t.
This treatment can consist, for example, of esterification, particularly sulphation by means of a suitable agent, carried out under conditions not chang-ing the oside s-tructure. This sulphation can be carried speci*ically or not, as necessary on the fully protected glycoside.
In a preferred embodiment of the invention, 2~ the functionalisation step is however effected select-ively so as to introduce on the chain, successively, several types of substituent and then certain -OH
radicals to be libera-ted.
By particularly advantageous conditions, enabling the introduc-tion of the sulphate groups on the predetermined positions of the units, to free the 24 1~6S~32 -OH radicals at other positions, to form at the 2 position of -the A units an amino derivative and in the 6 position U units of the acid derivatives, units cor-responding to the following characteristics are applied.
The semi-permanent groups of these units occupy posi-tions intended to be sulphated and are con-stituted by O-acetyl groups.
As for -the positions corresponding to an ~OH
group intendedto be liberated, they are occupied by semi-permanent groups constituted by benzyl groups.
The 2 positions of the A units are substitu-ted by groups such as N3 or NH-COO-CH2-C6H5 and the 6 posi-tions of the U units are occupied by carboxyl groups protected by an alkyl radical, in particular methyl.
This set of conditions enables the realisation of the func-tionalisation step, for example as follows :
First there is introduced selectively the sulphate groups after having eliminated the -O-acetyl blocking groups. This reaction is carried out so as not to affect the benzyl groups and the nitrogen and carboxyl ~0 groups present.
In this respect, advantageously a saponification reaction is carried out by means of a strong base such as soda.
This reaction is carried out preferably at a ~5 temperature below ambient temperature and more especially close to 0C.

~26~
2~

The produc-t resulting from the hydrolysis is subjected -to the action of an alkylation agent in order to introduce, on the carboxyl group, the protected alkyl groups which are found to be removed on hydrolysis.
By reaction with a sulphation agent, the intro-duction of sulphate groups at the posi-tions released by hydrolysis and left free after the action of the alkyla-tion agent, is then obtained.
Satisfactory reaction conditions for the sul-phation comprise the utilisation of a sulphation agent, such as a trimethylamine/S03 complex. This reaction is advantageously carried out in a solvent medium, more especially in a solvent such as dimethylformamide.
Preferably operation is at a temperature higher than room temperature, generally in -the vicinity of 50C, which corresponds to a reactlon time of about 12 hours.
After the introduction of the sulphate groups on the alcohol functions, the liberation of the -OH
groups blocked by the benzyl radicals follows.
The removal of benzyl groups is advantageously done by catalytic hydrogenation under conditions compat-ible with the maintenance of the sulphate groups and the conversion of the nitrogenous groups into amino function-al groups.
Preferably the operation is carried out under hydrogen pressure in the presence of a catalyst of the Pd/C type.

This reaction is advantageously carried out in an organic solvent medium, in particular alcoholic, :

.

26 ~ 2 6 5l3 2 supplemented with water.
~o ob-tain hydrogenation of -the precursor nitrogenous groups and the removal of the protective radicals from the -OH groups, the reaction is advan-tageously carried out over a period of about 3 -to 4 days.
As already indicated, the amino functional groups are in the form of derivatives of the N-acetyl or N-sulphate type in the biologically active molecules concerned, To form N-acetyl groups, the product resulting from the hydrogenation reaction is subjected to an acetylation agent. In this respect, acetic anhydride constitutes a particularly suitable agent.
To carry out this selective acetylation reaction without a~fecting the other substituents present on the units, it is appropriate, in particular, to operate at a basic pH, in particular close to ~ in an aqueous medium.
It may also be desired to form N-sulphate groups which may be done by means of a sulphation agent cO of the above-indicated type. pHs higher -than 9, advantageously of the order to 9-10, are used for the sulphation.
After the sulphation reaction, the addition of a strong base enables the liberation of the carboxyl groups.
The products formed may easily be salted by exchange resins with an appropriate cation. In natural products, -the ca-tion in particular is constituted by ~2~S~2 sodiwn. Hence exchange resins with sodium cations are advantageously used.
I-t is also possible to form sal-ts of potassium, li-thium,magnesium, calcium. A proton exchange resin is then used, and then the acid formed is neutralised with the base of the cation.
The inven-tion is also directed to oligosaccharides constituting intermedia-tes in the various steps of the pro-cess of synthesis defined above.
In one family, these oligosaccharides include at least one binary A-U and U-A unit completely protected and possessing either a reactive group on the anomeric carbon of the unit at the reducing end, or a single free -OH group on the unit at the non-reducing end, this -OH
group occupying the 3, 4 or 6 position in the case of an A unit and the 2,3 or 4 positions in the case of U units.
In ano-ther family, the oligosaccharides are constituted by completely protected units such as obtained at the end of the glycosylation step. Another family again comprises products in-which one or several -OH
groups are liberated.
zo These various oligosaccharides comprise a chain based on binary units of structure (A-U)n or (U-A)n in which _ is a number from 1 to 6.
These oligosaccharides correspond to an enchain-ment of the type _-b o r a-c In one group of intermediate oligosaccharides of the invention, the glycoside chain is cons-tltuted by 51~;~

a single -type of these binary enchainments.
In another group, several of these types are present.
Corresponding oligosaccharides include in a their chalns a-b and a-c.

It is understood that the order of the enchain-ments concerned above in one or several of the binary units,can be reversed according to the invention.
According to one modification, the intermediate oligosaccharides defined above contain one or several consecutive a or b or again c unlts .
According to ano-ther modification, the inter-mediate oligosaccharides contain one or several units of neutral sugars and/or several desoxy-sugars in their structure. The various protec-tive groups of these sugars corespond to the definitions given above or the A and U
units.
In these oligosaccharides, the constituent units are connected to one another by linkages of 1-2, 1-3, 1-4, or 1-6 type according to the nature of the alcohol utilised in the glycosylation step.
The oligosaccharides possessing the structure of heparin or heparane-sulphate fragments include c 1 ~ ~4a, a 1 ~ >4b, _ 1 ~ 4c and b 1 ~ 4_ 2 a linkages.

~126~3~

One group of preferred oligosaccharides con-tains at least one binary unit possessing a structure of the type ~ 4a, that is -to say ~ D-glucuronic acid ~ 1 ~ 4 ~ D-glucosamine~ corresponding to formula I :

COOM 0~

~ ~ 0 ~ ~ 0~ (I) TO ~
~R1 h in which :
- the R1 radicals, identical or dif~erent from one another, if necessary conjointly with R, represent a protective group, in particular a ~ semi-permanent group or a ~ permanent group, - ~, a temporary group t, or a permanent group ~? or a hydrogen atom, - N, is a nitrogenous group amine or ~nine deri~ative precursor.
- R, an aliphatic or aromatic radical, partic-ularly an alkyl radical comprising from 1 to 4 carbon atoms, where OR represents a reactive group such as a halide or again R an alkyl radical and 30 ~2~;5~13;~

- M, a group blocking the acid function, these various symbols having the above-given meanings.
In a sub-group, all the radicals ~, R1 and T
are identical and represent a p or sp group.
In another sub-group, the radicals R1 are dif-ferent from one another, one at least representing asp type group, possibly conjointly with R, -the one or more other radicals R1 representing a ~ group.
It will be noted that the general meanings of the symbols of formula I are applisd also to the form-ulae of the various groups considered below. In thesame way, there is to be found again in each of these groups particularly, the -two sub-groups mentioned above.
Preferred oligosaccharides correspond to the formulae (II), (III), or (I~) :

~0 ~ _o~ ~ (III) P

31 ~ 2 ~

C~ rP~OsP

R t IV ) in which the various symbols have the above-indicated meanings.
Preferably, in the formulae (II) to (IV), the symbols given have independantly, or in combination, the following meanings :
- M represents a hydrogen atom or an alkyl radical, particularly methyl, - sp an acyl group, in particular acetyl, - ~, a substituted alkyl group, in particular benzyl, - R, an acyl group at a or ~ , in particular an acetyl group, an alkyl radical, in particular methyl or substituted alkyl, particularly benzyl, or -OR a halogen, in particular a bromide, or again an imidoyl radical, ~0 - N, an azide group 9 - T, the group t representing an acyl radical, in particular acetylJ a halogenated acyl radical, in particular, a monochloro or trichloroacetyl radical, or the group ~ representing a substituted alkyl radical in particular the benzyl.radical, as the case may be i~.telf paramethoxy or again a hydrogen atom.

Another preferred group of oligosaccharides includes at least oIIe unit of the type A 1 ~ 4b, that is to say ~ D-glucosamine~ 1 ----~ 4 ~ D-glucuronic acid~ corresponding to formula (V) :

r ~ G~ o~
O ~ I

.

1U Preferred oligosaccharides correspond to the following formulae (VI) or (VII) :

~sp ~o~
~5 ~ ~

DS p f ~ o o D~

. . .
In these formulae (VI and (VII), symbols M, N, sp, ~ have? preferably, the particular r~eanings given above with respect to the -formulae (Il) to (IV), and R represents, in addition, preferably, a propenyl, .

~L2~51~;2 allyl, imidoyl, or -H group~ with N representing then more especially a -HN-acetyl group.
It will be recalled that the order of chain formation of the units may be reversed.
In another preferred group, the oligosaccharides contain at least one binary unit of type c 1 ~ ~ 4a, that is to say ~ ~-iduronic acid~ 1- ~ ~ 4 ~ D-glucosamine~
corresponding to the formula (VIII) :

0~ 1 ~ ~ (VIII) oR, ~

Preferred oligosaccharides correspond to -the following formulae (IX and (X) -~ s p c~OC~
R (IX) Osp I _op~
T O ~ L~) (x) P ~ .

~26;S~3~

In preferred manner, the symbols figuring in theses formulae ~IX) and (X) have the following meanings :
- the various sp and ~ groups may be identical and represent an acyl radical, in particular acetyl , or different, as selec-ted from among acyl radicals, in particular acetyl or benzoyl and aryl or substituted alkyl radicals, - N represents a precursor nitrGgen group, possibly different from that present in compounds of formulae (I) to (V), in particular a NHC00-(substituted alkyl group), particularly a -NH-C00-CH2-C6H5 group, which permits subjecting the nitrogenous groups to dif-feren-t treatments and to form different amino derivatives at 2 position of -the A units, - T represents the acetyl, halogenated acyl radical, in particular, monochloro or trichloroacetyl, p-methoxybenzoyl, the symbols p, M and R having advan-tageously the preferred meanings given above in respect to the formulae (II) to (IV).
Another type of binary unit of preferred oligosaccharides has a 1 ~ 4d structure, that is to say ~ D-glucosamine~ , 1 ~ 4 ~ ~-iduronic acid~
- corresponding to the following formula (XI) :
oR, ~ ~ ~ 0 ~

1\/' O t~ I

Par-ticular oligosaccharides correspo~ to the formulae (XII) and (XIII) :
sp ~ ~ ~ (XII) ~ ~sp ~P
, 3 . O R (XIII) ~ P
in which the preferred meanings correspond to those give above for formulae (II) to (IV).

~ ~3~

Ano-ther preferred family of intermediate oli-gosaccharides entering into the scope 'of the invention corresponds -to the product,s from which the protective groups have been partially removed in the course of synthesis.
In par-ticular, such products include an -OH group in place of the sp groups.
Preferred intermedia-te products correspond to oligosaccharides having the structure of the complete octasaccharide (ABCDEFGH) or hexasaccharide (C'DEFGH) sequence mentioned above.
Preferably, they are disaccharides AB, BC, CD, etc.... trisaccharides ABC, BCD..., tetrasaccharides i ~2~i~13~:

ABCD, BCDE.~.., pentasaccharides, ABCDE...., hexasaccharides, ABCDEF.... heptasaccharides ABCDEFG or BCDEFGH or the octasaccharide i-tself.
Among these oligosaccharides, may be mentioned the s-tructures,BC, DE, DEF, EF, GH, FGH, EFG, EFGH, DEFGH
and CDEFGH.
Preferred intermediates disaccharides correspond to the binary units of formulae (I) to (XIII).
A preferred group of intermediate -trisaccharides has a s-tructure DEF and correspond to one of the formulae XVIII to XXI.

r~ W (XVIII) ~/ P

LC, ~or,\ ~1 >

~ O Sp ~M

~ -~ ~y t~;) ) p /~/~

.

38~L26~;132 Sr Preferably, N1 and N2, identical or different from one another, represent an azide or -NH-acyl group in particular -NH-acetyl.
O-ther preferred trisaccharides possess a struct-1 ure of the type FHG of formula L~S~ OSp .~ _ ~ ,r :, ~ ~P ~æ

in which the various symbols have the above-given meanings, the two substituents N1 and N2 of the two glucosamine units of structure F and H being identical or again advan-20 tageously different, as in the ca.se of natural produc-ts, and selected from among the azide or -NH-COO-acyl group, in particular -NH-COO-acetyl-.~ or -NH-COO-CH2-C6H5.
Other preferred intermediate oligosaccharides are constituted by tetrasaccharides. More especially 25 advantageous tetrasaccharides possess the structure EFGH
and correspond -to the following formula ~i51~Z

CO O ~ Osp _ Os~

0 ~ ~ ~ ~O ~ 0~ (XXIII) ~ ~
op ~ Osp ~2 in which the preferred meanings of the different symbols correspond to those indicated for formula XXII.
Another family of intermediate oligosaccharides is cons-ti-tuted by pentasaccharides, in pa.rticular~ by those of structure DEFGH of formula O;p CO 0~ O._p 05p ~Op' ~o~O~ J/sp )~o)~)J/op ( ~ (XXIV) N ~ O p N ~ Osp N 3 in which the various symbols have the above-preferred meanings, and N1, N2, N3 can be identical or different from one another selected from among the meanings already given.
As mentioned above for binary units, the invention relates also to the above oligosa.ccharides in which one, several or, as -the case may be, a.ll of the -OH groups are liberated in the course of synthesis.
The invention is aimed, in addition, as novel products, ~t the oligosaccharides corresponding respect-ively to the various definitions given above, but including one or several functional groups, with the 4~ ;1;32 exclusion of the disaccharide ~ 2-N-sulpha-te (or 2-N-acetyl)-6-0-sulpha.te-D-glucosamine~-methyl-D-glucuronic acid.
These functional groups are constituted pre-ferably, by esters, and occur more especially in the form of inorganic anions.
Pa.rticularly preferred esters, by rea.son of their presence in biologically active molecules of the type of hepa.rin ar heparane-sulphate are constituted Dy sulphate esters.

Other advan-tageous esters correspond to phosphate esters.
These functional groups are borne by one or several primary alcoholc- and/or secondary alcohol and/or 15~ primary amine functions.
A preferred family of oligosaccharides of the invention thus includes a unit comprising such an anion as defined above at the 6 and/or 3 position .

A partlcularly preferred family contains a.n a unit comprising an ester, in particular a sulphate group, at the 6 position and at the 3 position.
Oligosaccharides of this family contain, at the 2 position of a~ a primary amine functional group advan-tageously substituted by a sulphate or by another substituent group.

~;~6~

In the oligosaccharides of the invention con-taining at least two units a, the amine functional groups a-t the 2 position may be substituted by the same group or by different groups.
A preferred group of oligosaccharides of the family concerned includes units _ comprising sulphate groups on the secondary alcohol and especially the primary alcohol function.
Preferred oligosaccharides of this group com-prise at the 2 position of these units an -NHS03- group.
Other oligosaccharides include an -NH-acyl group, in particular -NH-acetyl.
Preferably, the esters below occur in the form of salt wlth an inorganic or organic cation, in particular a metal cation, particularly an ~ali cation, or again a cation derived from a nitrogenous organic base, for example -triethylammonium.
The cations used are constituted by sodium.
Other cations are suitable such as the potassium, magnesium or calcium cations.
In another preferred family of oligosaccharides of the invention, the carboxyl groups of units ~ or c are free or are preferably in the form of salt with an organic or inorganic cation such as defined above~
They may also be protected as reported above.
- Preferred products contain units c comprising a sulpha-te group at the 2 position.

, 4~ 3~

Other preferred products have sulphates on the ~ unit.
In these various families of oligosaccharides, -the hydroxyl fu~tions ofthep~a nrings are either free, or pro-tected by permanent groups of the alkyl type, in particular by methyl groups.
Preferred products of these various families contain, in combination, the units A and U correspond-ing to -the above characteristics.
~ aking into account their presence in the biologically active molecules above and particularly in the octasaccharide ABCDEFGH or the hexasaccharide CDEFGH, the preferred oligosaccharides correspond to the products of fonmulae (I) to (XII~ and (X~III) to (XXIV) above, but in which the -sp groups are replaced by anions. Preferred products corres-pond to salts o~ the above-defined products.
Other preferred oligosaccharides include in addition to the place of the N groups of the a units, an NH-acyl group, in particular -NHCOCH3, -NHS03 .
Preferred disaccharides of this type have a structure of the type BD, DE, EF or GH and correspond respectively to the following formulae (XXV to (XXVIII) :

OSo3 25 ~; J\r~ (XX'`I) ~ ~- S~3 p 1~65132 C o~

~ ~- 53 P
o F ~ c y l , coo (XXVII) o3 r-~05 3 o ~ (XXVIII) 0,o~ S~ :~
O-ther preferred oligosaccharides of -the invention con-tain or are constituted by an enchainment of the structure DEF or FGH respectively of -the follow-ing formulae (XXIX) and (XXX) :

(xax) ~Y I ~ o3 cr. ~ S ~ ~

UU~ 5~, ~JHS~

, ~26 ~4 O-ther preferred oligosaccharides contain or are constituted by tetra.saccharides of structure EFGH
corresponding to the following formula (XXXI) :

COO OS~, ~ ' 5 ~ oJ(~
p ~H~o~~ O~o~~ ~H SO~

Other oligosaccharides also specially preferred contain or are consti-tuted by pentasaccharides of the following formula (XXXII) :

OSO ~' CO 0' , 050~, , O SO~,~
\ / ~0 ~ \ / CO~
J~J~ ~/~~ J\5)~

~ISo~- op NHS(~ OSO, NHSO, o~ acyl Oligosaccharides of the invention which are particularly preferred comprise or are constituted by hexasaccarides of -the structure CDEFGH corresponding to the following formula (XXXIII) :

rOS03- ~ roso3- roso3-25 o~~O ~ ~ ~O ~ o~

N~3 0~ NHS03- OSO3- NHSo ol.N~-acyl (X~

.

~26~i~3 O-ther oligosaccharides correspond to one of the formulae (XX~ to (XXXIII) above, but contain free -OH groups i.n pla.ce of the -Op groups. These products are then completely depro-tected .
In o-ther oligosa.ccharides again a portion of the -OS03 groups may be replaced by -OH groups.
Preferably, the oligosaccharides of the invention include salts, possibly double salts, of the above anions with the already defined ca-tions. Due to their s-tructure, -the products of the invention con-stitu-te synthesis in-termediates of great interest enabling the production of given fragments, or deriva-tives of fra.gments, of biologically active molecules.
They constitute, particularly, reference com-pounds for structure studies.
Pharmacological study of the oligosaccharides of the invention has shown in certain of -these compounds biological a.ctivities enabling them to control specific-ally certain steps in blood coagulation. Interesting products are constituted, for example, by trisaccha.rides of formula (XXIX), su.lphated and deprotected and more particul.arly the deriva.tives of Example 13bis In a remarkable way, the pentasaccharides of formula (XXX~I~) sulphated and deprotected and very especially the deriva-tive 50 show -themselves to be : 25 endowered particularly with high affinity for A~ III
and very high selective inhibi-tion activity of -the activated X factor or Xa factor of the blood.

.
::
, ~ . . , ~2~3~

The invention -therefore relates also to their use in the constitution of biological reagents, useful in laboratory, particularly as comparison elements for the study of other substances of which it is desired to test the anti-coagulant activity, particularly at the level of the inhibition of the Xa factor and of the determination of antithrombin III.
The trisaccharide of formula (XXIX) with the structure DEF in which the D unit includes an N-sulphate group has, for example, an anti-Xa activity measured by the Yin-Wessler test, of the order of 7 u/mg.
The pentasaccharide 50 of Example 9 is charact-erised by distinctly higher Yin-Wessler titres than those of heparin.

More especially, this pentasaccharide is endowe with an anti-Xa activity (Yin-Wessler) equal to or greater than 2000 u/mg and a high affinity for AT III.
In a test using a chromogen substrate, this activity has even been 4000 anti-Xa units/mg (m~thod of TEIEN A.M. and ~IE modified ; Thrombosis Research No 10,1977, 388-410).
This test consists of using the Xa factor marketed by the SIG~ company in solution at 8 u/ml in physiological serum7 the concentration of the subs-trate being 1. 33 mMr To carry out -this test it is possible to proceed as follows.
10 ~l of solu-tion to be determined and 300 ~1 '``' 1~J4~S1~2 of human plasma dilu-ted with Tris maleate buffer 0.02 M, pE~ 5 are mixed. I
It is left to incubate one minu-te at 37C .
100 ~l of -the above-said Xa factor (8 u/ml) are added and one minute later, the solution obtained is injected into the substrate.
The overall anticoagulant activity of this product is very low, 4 u/mg in the APTT test.
These properties enable them -to check specifically~
certain steps in blood coagulation.
The study of these products shows that they are capable of exer-ting a powerful antithrombotic activityO In addition, derivatives according -to the invention have great interest for combatting disorders of the vascular wall, (athero -scleroses and arterioscleroses).
In addition,they have -the advantage of not having the effect of activation on platelet aggregation and not resulting in thrombocy-topenia. They have also the advantage of being practically devoid of effect on bleeding time, which eliminates the risks of hemor~age. These two properties are extremely 2~ important for medical uses.
In addition, there is observed particularly by the subcutaneous route a prolonged pharmacokinetic reaction which prOcures also a considerable interest in the product.
The oligosaccharides of the invention are, in addltion, 25 advantageously devoid of toxicity.
These products are hence particularly valuable for 48 1265~32 developing useful medicaments, particularly for the prevention and trea-tmen-t of thomboses.
The lnvention hence relates also to pharmaceutical preparations which contain said oligosaccharides wi-th high anti-Xa ac-tivi-ty, more especially the pentasaccharides con~
sidered above.

It relates also par-ticularly to pharmaceutical pre parations devoid of pyrogenic substances containing an effective amount o~ active principles in association with pharmaceutical excipients.
It also relates to the compositions in which the pharmaceutical vehicle is suited for administration orally.
Suitable adminis~trative forms of -the inven-tion for oral adminis-tration may advantageously be gastroresistant capsules, pellets ~5 or tablet~ pills, or again presented in liposome form.
Other pharmaceutical compositions comprise these oligosaccharidss in association with suitable excipients for rectal administration. Corresponding administrative forms are constituted by suppositories.
Other administrative forms of the invention are constituted by aerosols or pommades.
The invention relates also to sterile or sterili-zable injectable pharrnaceutical compositions for administration both intravenously and intramuscularly or subcutaneously.
These solutions contain advantageously 1000 to 100 000 u (Yin-Xessler) /ml o~ oligosaccharides, preferably ~rom 5000 49 ~s~

to 50 000, for example from 25 000 u/ml, when these solutions are intended for subcutaneous injection. ~hey may containm for example from 500 to 10 000 particularly 5000 u/ml of oligosaccharides when they are intended for injection intra-venously or by perfusion.
Advantageously, such pharmaceutical preparations are presented in the form of ready-for-use discardable syringes.
~he invention relates also to the pharmaceutical compositions containing said oligosaccharides in association with another active principle, useful in particular for prophylaxis 10 and treatment of thrombosis, such as a veinotonic agent like dihydroergotamine, nicotinic acid salt or a thrombolytic agent like urokinase.
The pharmaceutical compositions of the invention are par-ticularly adapted for the control (preventive or curative) 15 of certain stages of blood coagulation in man or in the anima1 , particularly in the case where the patent is subject to risks of hypercoagulability resul-ting particularly from surgical opera--tions, from atheroma-tous processes, fr~ the development of tumors and disorders of blood clot~ing by bacterial or enzymatic activators etc.

. . .

In order to illustrate the invention, there is indicated, below, an example of the posology usable in man : this posology comprises, for example, the administration to the patient ~265~ 2 of 1000 -to 25 000 u (~in and Xessler) subcutaneously, once to thrice daily, according to the level of -the risks of hyper-coagulability or the thrombotic condition of the patient1 or from 10~ to 25 000 u/ 24 hours, intravenously, in dis-continuous adminis-tration at regular in-tervals, or continuous by perfusion, or again from 1000 to 25 000 u (three -times weekly) intramuscularly or subcutaneously (-these titers being expressed in Yin_Xessler units). These doses can naturally be adjusted for each patient according to results and blood analyses carried ou-t previously, the nature of the disorders from which he suffers and, generally, his state of health.
Besides the pharmaceu-tical compositions containing the oligosaccharides as such, the invention is aimed also at pharmaceu-tical compo~itions containing at least one oligo-saccharide as defined above, conjugated, by a covalent bond, -to a soluble support or an insoluble support, advantageously by means of the reducing terminal sugar.
~ onjugates fixed to preferred soluble supports are consti-tu-ted by oligosaccharides conjugated wi-th AT III
A conjugate of this type including the pentasac-charide 49 is very especially preferred. Such products con-stitute particularly interesting medicaments in the prevention of thromboses, in the case of deficiencies of AT IIIo Other preferred conjugates with soluble supports are formed from an oligosaccharide fixed to a vehicle such as a pro-tein, particularly polylysine, or bovin albumin serum.
These products are useful as immunogens themselves 5 1 1~651~

sources of circula-ting antibodies produced in vivo or of monoclonal antibodies cloned in vi-tro by suitable techniques.
In other preferred conjugates the oligosaccharides of the invetion are conjugated to insoluble supports. Ad-vantageously conventional supports are utilized.
These conjuga-tes are useful as immunoabsorbents, for example for purification of high speeificity of AT III
and for i-ts estimation or for the developmen-t by fixing to bioeompa-tible polymers, of novel athrombotie hemocompatible polymers.
The invention is direeted also to the use of the oligosaccharides eoneerned in nuelear medieine, as radiophar-maceutical products. ~'hese produets are then labelled by traeers selected from among those currently used in this field~ and partieularly by means of technetium 99 m.
To this end, -the technetium 99m obtained from commercial generators is converted, in the form of sodium pertech n e-tate of unreactive valency 7, into technetium reduced to valeney 4 whieh would be the mos-t reactive fo~ of techne-tium. This conversion is carried out by means of a reducing system produced from certain tin salts (stannous chloride), iron salts (ferrous sulfate), and titanium salts (titanium trichloride) or other salts.
Most of the time, this simple reduction of the technetium suffices, under given pH conditions, to effect the fixing of the technetium to the molecule concerned.
It is possible to use the products of the inventlon .
.

52 l ~ 6 ~l 3~

which constitute in a way a support, at doses of the order of 100 to 200 u Yin-~essler.
For -the development of these radiopharmaceutical reagents, it is possible to operate in accordance with the method of -the P,V, KULKARNI e-t al. in The Journal of Nuclea~
~edbcine 21, N 2, p. 117-121.
The so-marked products are advantageously used in in ~ivo tes-ts for the detection and extended diagnosis of thromboses and of thrombo-tic states.
The oligosaccharides of the invention may also be ~0 used for the determination of the specificity of ~umerous enzymes involved in the metabolisme of the glycosaminoglu-curonoglycans.
0-ther alvantageous characteristics of the invention will appear from the examples which follow and with reference to Figures 1 -to 32 illustrating the products employed in the syntheses described.
In these Figures, the numerical references of the formulae are used also in the ~xamples to denote the same pro-ducts.
2~ The abreviations used in these formulae have the following meanings :
Ac : an acetyl group ; Me : methyl ; Bn : benzyl ;
Bz : benzoyl ; MCA0 : monochloroacetyl ; Tr : trityl ; but~ :
butyl and S an S03 group.

EXAMPLE I Synthesis of the derivative L~ namely methyl (prop~ enyl 2.3-di-O-benzyl-~-D-glucopyranoside)uronate of the formula COOM e ~ 0 ~,OBn ~
H O ~ OP
OBn The synthesis is carried out from glucose by the following steps a) to m) :
a) preparation of the allvl derivative ;
b) blocking of the 4 and 6 nositions of the allyl deri-vative by a benzylidene group;
c) introduction of benzyl groups at 2 and 3 positions d) unblocking of the 4 and 6 positions by removal of the benzylidenegroup;
e) introduction of a trityl group at the 6 position, followed by an acetylation reaction of the 4 position;
f) removal of the trityl group at the 6 position;
g) oxidation of the primary alcohol group at the 6 posi-tion;
h) methylation of the carboxvl group at the 6 position;
i) introduction of the propenyl group at the 1 position;
j) removal of the acetyl group at the 4 position.
These steps are carried out as follows (see Eigures 1 and 2) :
a~ re~aration of a11y10~-D-glucopyranoside (compound 1) ~i5~;~2 A solution of gaseous hydrochloric acid (18 g) in allyl alcohol (600 ml) is heated to 70 C. Then anhydrous glucose(300 a) is added and it is keptat this temperature for 3 hours.
The reaction may be followed by thin layer chroma-tography (t.l.c) in the solvent methanol/chloroform (1/4 v/v). The brown solution obtained after 3 hours is concentrated to dryness, under vacuum, neutralized with a concentrated ammoniac solution (50 ml) then concentrated again to dryness.To the residue obtained, acetone (500 ml) is added; it is brought to boiling and kept there until complete solution. After cooling, the liauid is decanted.
The residue is again subjected to the same treatment until the t.l.c. analysis of the extract shows exhausting of the residue in derivative 1 or indeed a very high conta-mination of the extract with impurities.
A portion of the first fraction extracted (12 g) is chromatographed on silicate. The derivative 1 which can be crystallized in an aceto ~ ~her mixture (6.5 g; m.p. 95-99C) ls recovered. The rest of the product may be purified by the same process.
b) blocking of the 4 and -6 positions of the allyl deri-vative leading to ~.6-0-benzylidene-~ -D-glucopyranoside (compound 2) Compound 1 (37 g) is dissolved in dimethylforma-mide (200 ml). Dimethoxytoluere (41 g) is then added followed by hydrated paratoluene sulfonic acid (130 mg).
After 2 hours heating (water-bath) under vacuum .. .

;13~:

and reflux, the reaction is terminated (t.l.c methanol/
chloroform 2/25 v/vj The solvent is evaporated. The svrup is dissolved in methanol (the minimum), this solution is poured drop by drop into an aqueous sodium bicarbonate solution (6.3 g in 320 ml water). The precipitate obtained is recrystallized in ethanol (21 g; m.p. 120 - 121C). The mother-li~uors also yield product 2. Total yield (37 g;
71. 4%).
c) I troduction of the benz 1 grou~ leadinq to allyl n y _ _ 2~3-di-0-benzyl-4,6-0-benzylidene~ -D-glucopyranoside (compound ~) Compound 2 (45g) is dissolved in anhydrous D~F
(500 ml).Sodium hydride (28 g of a 50~ dispersion in oil) is added.
After 30 minutes, the mixture is cooled to 0C and then, drop by drop, benzyl bromide (52 ml) is added. The reaction is followed by t.l.c. (ether/hexane, 1/1, v/v).
Then slowly methanol (150 ml) is added, evaporated to dryness and taken up again with chloroform. The chloroform phase is washed with water, dried over sodium sulfate. After evaporation of the solvent, the residue is crystallized in an ether/hexane mixture (36.5 g MP 83-84C).
This product is slightly contaminated with an impurity migrating higher in t.l.c. (ether/hexane :1/1;
v/v).
d) Removal o the benzylldenegroup leading to allyl 2.3-di-O-benzyl-~ -D-glucopyranoside-(compound 4) To a solution of compound 3 (56 g) in methanol 56 ~26~i~!L3~

(1 L) is added water (450 ml) and then hydrated parato-luene sulfonic acid (17 g).
After 2 hours at 80C, the mixture is allowed to cool, the solvent is evaporated and residue is taken up 5 again with chloroform (1 L). The chloroform solution is washed with water until pH neutral, then dried over sodium sulfate. In this way a pale yellow syrup is obtained (48 g) which is engaged in the following step (synthesis of compound 5).
10 e) Introduction of a trityl grouP at the 6 position followed by an acetylation reaction of the 4 position leading succes-sively to allyl 2.3-di-O-benzyl-6-O-trityl-.~ -D glucopyrano-side (com ound 5) and its 4-O-acetylated analoq (comr~ound P
6a)-The derivative 4 obtained (48 g) is dissolved in pyridine (250 ml) and trityl chloride (38.5 g) is added.
After one hour at 100C, the reaction is terminated (t.l.c.
ether/hexane, 1/1, v/v). To the preceding solution, is added acetic anhydride (200ml). After one night, the reac-20 tion is complete (t.l.c.ether/hexane), 1/2, v/v). It is e~aporated to dryness, the residue taken up again with chloroform (500 ml), the chloroform phase is washed with a 10 g6 acid potassium sulfate solution with water and dried over sodium sulfate.
The Chloroform is evaporated. In this way compound 6a is obtained which is engaged as such in the reaction for the preparation of compound 7a.
.
f) removal of the trityl group leading to aIlYl 4-0-acetyl-~;~6~

2,3-di-O-benzyl-~ -D-glucopYranoside (com~ound 7a) The derivative 6a obtained is dissolved in chloro-form (500ml). To this solution, cooled to 0C, is added, drop by drop,under stirring, a solution of boron trifluo-ride in methanol (20~, 120 ml). The reaction is followed byt.l.c. (toluene/acetone, 10/2, v/v~.
The reaction mixture is decanted in a separating funnel.The chloroform phase is washed with water (twice 100 ml) with a saturated solution of sodium bicarbonate, and then with water until pH neutral. After drying, and evaporation, the residue obtained is introduced onto a silical gel column (500 g) equilibrated in toluene. After dilution of the majority of the impurities with pure toluene, the product is eluted with a toluene/acetone mixture (10/2, v/v). In this way 48 g. of compound 7a is obtained which will be engaged directly in the synthesis of the compound .
A portion of compound 7a was obtained pure :
D0 = + 11 (chloroform).Its IR and NMR spectra , just as elementary analysis, confirm the structure.
g) oxidation of the primary alcohol group at the 6 position leading to (allyl-4 O-acetyl-2j3-di-O-benzyl-~ -D-gluco-pyranoside) uronic acid (compound 8a) A solution of compound 7a (48g) in acetone (800 ml) is cooled to -5C. Then,drop by drop, a solution of chromium trioxide (30 g) in sulfuric acid (3.5 M; 125 ml) is added.
The mixture is allowed to come back to room temperature.
Reaction is checked by t.l.c (methanol/chloroform~/lO, v/v).

58 1;26;~ 3;~

At the end of reaction,the reaction nixture is poured into water (500 ml). The product is extracted with chloroform`
(3 -times 250 ml). The chloroform phase is washed with water until pH neutraL, dried over sodium sulphate and concen-trated to dryness.
The syrup obtained (83g) is used as such for thepreparation of the compound 9a.
h) Methylation of the carboxyl group at 6 position leadin~
to methyl (allyl-4-0-acetyl-2J3-di-0-benzyl- ^~ -D-gluco-p~ranoside)uronate (com~ound 9a) The syrup obtained in the step of preparing compound __ 8a is dissolved in ether (300 ml). An ether solution of diazomethane is then added until disappearance of the com-pound 8a ~t.l.c ether/hexane , 1/1, v/v) , After acidifi-cation with acetic acid, the solvents are evaporated.
The residue obtai~ (53g) is dissolved in hot ethanol.
Thederiva~ive ~'iscrystallized on cooling. After recrys-tallization, this pure compound 9a is obtained (18,4g)-mp 85-86C ~0~ 20 = ~ 12 (1,2 chloroform).
This product is characterized by its IR,N~ spectra and by its elementary analysis.
From the crystallization filtrate, a further 7.6 g of compound 9a is obtained.
The overall yield of 9a from the compound 2 is 38%~
i) - Introduction of the prop-enyl group a't the''l' ~osition leading to methyl (prop-l'-e~yl 4-0-acetyl-2.3-di-0-benzyl-o~ -D-glucopyranoside) uronate (compound lOa) 59 ~2~s~32 ~ ne derivative 9a (4 g) is dissolved in a mixture of ethanol (119 ml) benzene (51 ml) and water (17 ml).
Then diazabicyclo octane (170 mg) is added and it is brought to reflux. To the boiling solution is added tris (triphenylphosphine)-rhodium (I) (550 mg) chloride.
The boiling is maintained for 4 hours (t.l.c, ether/hexane, 1/1, v/v~.
At the end of the reaction, the solution is fil-tered and the solvents are removed. Residue is chromato-graphed on silica gel (150 g) in an ethyl acetate /chloro-form mixture (1.50, v/v). Compound lOa is obtained (3.25g;
81%) which crystallizes in ethanol ~O~ 20 = ~ 12.(1, chloroform). MP 90C. The structure is confirmed by elemen-tary anaIysis and the NMR and IR spectra.
j) The removal of the acetyl group at 4 position leading to methyl (prop-1'-enyl- 2,3-di-O-benzyl- ~--D-glucopvrano-' side) uronate (compound-~ ) The derivative lOa (350 mg) is dissolved'in methanol (5 ml). Sodium methanolate (0.2 ml, 2M) is added. After 1 hour at room temperature, the reaction is stopped by addition of dowex resin 50-H+. After filtration, product 13 is obtained, contaminated by little product resulting from the ~ -~ elimination.
According to a modification of step e) instead of carrying out an acetylation reaction, a benzoylation reaction is carried out, which leads to :
allyl-4-0-benzoyl, 2.3-di-0-benzyl-4-0-benzoyl, 6-0-trityl-~ -D-glucopyranoside (compound 6b) from which the trityl group is then removed, which enables allyl-~iS~32 4-0-benzoyl-2~3-di-0-benzyl- ~ -D- glucopyranoside (compound 7b) to be obtained.
These reactions are carried out as follows :
- Preparation of compounds 6b and 7b.
To the pyridine solution of the compound 5 is then added benzoyl chloride (1.5 equivalents) and the reaction is followed by t.l.c. (ethyl acetate/benzene,l/20, v/v). The excess of benzoyl chloride is destroyed by the addition of an excess of methanol. After evaporation to dryness, the residue, taken up again with chloroform, is washed with a 10 % KHS04 solution, with water, dried and concentrated to dryness. The syrup obtained is engaged as such in the synthesis of the compound 7b This syrup (105 g, obtained from 30 g of compound 3) is dissolved in chloroform (300 ml). Paratoluene sulfonic acid (76g of monohydrate in 100 ml of methanol) is added.
After one night, the reaction is terminated (t.l.c., ethyl acetate/chloroform, 1/20, v/v). The chloroform phase is washed with water until pH neutral, dried and concentrated to dryness. The syrup obtained (98 g) is chromatographed on a silicate gel column (1.2 kg), eluted with chloroform ( 0.6 1 ) then with an ethyl acetate/chloroform m~xture (1/20, v/v). Thus a pure derivative 7b (30 g) is obtained which is engaged as such in the step of preparation of compound 8b. From compound _~ it is possible to oxidize the -CH2OH group at the 6 position and then to introduce a methyl group on the carboxyl group obtained, by forming successively (allyl-O-benzoyl-2~3-di-0-benzyl- ~-D-~:6~32 glycopyranoside) uronic acid (compound 8b) and the cor-responding methyk~ter (compound 9b) .
These derivatives are prepared by proceeding as fol-lo~s :
- Preparation of-the comx~d ~h and of the ester ~b C~und 7b (27 g) is treated as described for 7a in the preparation of ~ me syrup obtainedat ~he end of the treat~nen~ cont-~i~Scompound 8b which is methylated with diazomethane as described for the compound ~.
The residue obtained at the end of methylation is purified on silica gel (200 g; ether l/hexane 1). In this way the compound 9b is obtained (21 g; 77.5%). Its IR
and NMR spectra confirm its structure.
From compound 9b, the corresponding propenyl deriva-tive lOb is prepared, by operating as for lOa.
The derivative 13 is then obtained from 10 b as by the Teaction given for lOa.
According to another modification, (allyl 2.3-di-0-benzyl-~ -D-glucopyranoside) uronic acid an~-~ethyl(2.3-di-20 0-benzyl-~ -D-glucopyranoside)uronate (compounds 11 and 12).
are prepared.
Compound 8b (1.9g) is dissolved in methanol (40 ml). Then, 5N soda is added in a sufficient amount to have a concentration of 1 M of soda. The reaction is followed by t.l.c.(methanol/chloroform, 1/4, v/v).
When it is ended, water (100 ml) is added. It is washed with ether, acidified and the product extracted with ether. The acid ether phase is washed with water until :~65~L32 pH neutral. The derivative 11 is not isolated. It is methylated with an ether solution of diazomethane , thus giving the compound 12 (900 mg; 56%) which is then puri-fied on a silicate gel column (ether/hexane 1/l, v/v) ~ ~ ~ 20 = ~ 35.2 (1~3 chloroform). Its IR and NMR
spectra and its elementary analysis confirm its structure.
In the same manner, the derivative 11 and hence 12 may be obtained from 2a or 9b, The compound l3 may be obtained from compound 12 by operating as follows :
The derivative 12 is treated with the rhcdium complex as described for 9a. The com ound 13 is obtai-ned with a yield of 90 %. It is characterized by its IR and NMR spectra. In addition, treated with acetic 15 anhydride (1 ml for 180 mg of 9a) it gives compound ,lOa.
According to another modification, the derivative 13 may be obtained from lOa or lOb by operating as described for the production of l7 from 9a or 9b.

EXAMPLE 2' : Synthesis of the disaccharide ~Q or methyl 20 (1-bromo-3~6-di-0-acetyl-2-azido-4-0C~,3-~ O-benzyl-4-O-chloroacetyl-~-D-glucopyranosyl)uronate~-D-glucopyranose of the formula :

CO O ~ O A c ~ /)~
01~ N

, , 63 ~5~

--This synthesis includes the ollowing steps (see f.igures 3 and 4):
A) - the preparation from the derivative 13 of Example 1 of the monosaccharide 16 or .methyl (l-bromo-2~3-di-0-benzyl-4-0-chloracetyl-~-D-glucopyranosyde)uronate;
B) - the condensation of compound 16 with the mono-saccharide 17 leading to the disaccharide 18;
C) - the acetolysis of the compound l leading to the disaccharide 13 and, D) - bromi~ation giving the disaccharide 20.
A) - Pre.par.ation o.f..the. monosac.char.ide 1.7 .
This synthesis is carried out from the monosaccha-ride 13 or ~ethyl (prop-l'-enyl 2~3-di-0-benzyl-oC-D-glucopyranoside)uronate, by the three following steps :

1 : chloroacetylation of compound 13;

2 : unblocking of the anomeric carbon;

3 : bromination of the anomeric carbon 1 : Chloroacetylation of the compound 13 leading to com-pound 14, namely methyl (prop-l'-enyl 2~3-di-0-benzyl-

4-0-chloroacetyl-~C-D-glucopyranoside) uronate.

2.8 g of compound 13 is dissolved in 30 ml of pyridine (6.56 mmoles). After cooling to 0C, drop by drop,10 ml of a solution of 2ml of chloroacetyl chloride in 20 ml of dichloromethane is added. After 30 minutes, it is evaporated to dryness, the residue is taken up again with 200 ml of chloroform, washed with a 10 %
~H SO4 solution, then with water, it is dried and 6~ ~265~L3~:

concentrated. The syrup obtained is chromatographed on silicate gel (200 g; eluent AcO-Et/hexane; 1/3; v/v).
In this way 2.7 g of pure compound 14are obtained in the form of syrup (yield 80 %). ~ 7 D = +2 (c = 1 5;

5 chloroform).
Elementary analysis and the NMR spectrum confirm the expected structure.
2 : unblocking of the anomeric carbon leading compound 15 or methyl (2~3-di-0-benzyl-4-0-chloroacetyl-D-gluco-pyranoside) uronate.
2.7g (5.3 mmoles) of derivative 14 in 80 ml of an acetone/water mixture (5/1; v/v) is dissolved. Mercuric oxide (3.lg) is added followed by a solution of mercuric chloride (3.9 g) in acetone (27 ml). After 5 minutes, salts are removed by filtration. After concentration to dryness, the residue is taken up again with chloroform.
The chloroform phase is washed with a 10 % KI solution then with water After evaporation, the product is crystallized in a methyl acetate/hexane mixture. 2 g of a solid of mp 105-107C are obtained; ~ 7 DO = 4 7o (eq. 1 chloroform).
Elementary analysis and the NMR spectra confirm the structure (yield 80%~
3 : brom-n~tion of the anomeric carbon leading to the compound 16 or methyl (1-bromo-2~3-di-0-benzyl-4-0-chloroacetyl-~-D-glucopyranoside) uronate.
2 g(4.30 ~r~oles) of the compound 15 are dissolved in 50 ml of dichloromethane, 4.8 ml (34.4 mmoles) of sym-collidine at 0C is added, followed by bromomethylene ~ .

.... .

65 12Ç~5~-32 dimethyl ammonium bromure (17 mmoles) prepared according to HF.PBURN D.R. and HUDSON H . R . J . Chem. Soc. Perkin I
(1976) 754-757.
After 4 hours o reaction, the mixture is diluted 5 with 100 ml of dichloromethane, then poured into ice water.
After washing withice water, the solvent is evaporated.
After chromatography on silicate gel (20 g; eluting hexane/ethyl acetate, 2/1; v/v) 2.06g of compound 16 is obtained in the form of a syrup (yield 90 %).
~ ~ 7 D = ~ 82.5 (c = 1.5; chloroform~ .
analysis and the NMR study confirm the structure.
B) -.Preparation .of the disacchar.i.de 18 or 3-0-ac.e.tyl-1~6-.anhy.dr.o-.2.-.az.ido-4-.0 . 2~3-di- 0.--benæyl.-4-0-chl.oroac.e.tyl- ~-D-glucopyranosyl methyl uronate~ -D-glucopyranose This synthesis is based on the condensation of the monosaccharides 16 and 17 of 870 mg (3.8 mmoles).
To a solution of 870 mg (3.8 mmoles) of compound 17, in dichloromethane, is added 1 g of drierite 0.5 g of molecular sieve 4 A, in powder, and 0.525 g of freshly prepared silver carbonate. After 2 hours stir-ring, compound 16 is added drop by drop at 0C (670 mg) (1.3 mmoles). After 6 days, the solids are removed by filtration. The syrup obtained after concentration is chromatographed on silica gel (50 g; eluent :

chloroform/ethyl acetate; 4/1, v/v). The disaccharide 18 is obtained in the form of a foam (421 mg; 50 ~).

,,, ' 66 ~ ~ 6 ~ 3 2 r~C~ D0 = -17 (c = 1, chloroform),Elementary analysis confirms the structure. NMR study confirms the configura-tion of the interglycosidic linkage.
C) - Preparation o.f the disaccharides of stru.cture 19 by ace.tolysis of.. the.disaccharide.l8 The disaccharides 19 are prepared by subjecting the disaccharide 18 to an acetolysis reaction as follows.

300 mg of c ~ ound 18 are dissolved in a mixture of 4 ml of acetic anhydride and 0.5 ml of freshly distilled trifluoracetic acid. The reaction mixture is sub~ected to stirring for 10 hours ~ 18C, then evaporated to dryness and co-evaporated with toluene. The residue is chromatogra-phed on a column of silica gel (lS g). By elution with a dichloromethan ethyl acetate mixture (19 : 1, v/v),282 mg of a mixture of anomeric acetates of structure 19 are obtained in the form of a colorless syrup (yield 86%). The ration of the forms to the forms, determined by NMR
analysis, is 4/1.

NMR spectrum confirms the expected structure.
D) -.Prepar.at.i.on'.o.f.,di.sac.char.i.de''20 by .br.omi.dati,on..of..the.'di.s.ac.charide.s~

. The mixture of acetates of structure 19 is subjected to the action of TiBr4: A solution of 140 mg of the acetates mixture 19 in a mixture of 3 ml of dichloromethane and of 0.3 ml of ethyl acetate is sub-jected to stirring at 17-18C, under a dry argon atmos-phere, in the presence of 140 mg of TiBr ( ~ 2 equivalents) 67 126~132 for 20 hours. After cooling to 0C and dilution with 30 ml of dichloromethane, the mixture is washed with ice water, then with an aqueous 5 % solution of potassium bromide and then with water and dried over sodium sulphate, iltered and evaporated. The residue is chromatographed on a silica gel column (10 g). By elution with et~vl- dichloromethane acetate mixture (19 : 1, v/v) , there is recovered, in order of elution :
- bromide 20 (74 mg; yield 50 %) in the form of a colorless syrup, nstable (immediately engaged in the following reaction);
NMR spectrum confirms the expected structure.
- a fraction (28 mg; yield 20 %) corresponding to the unreacted starting material, - a fraction hardly migrating corresponding to products with partial O-debenzylation.
EX~PLE ~ : Synthesis of the monosaccharide 22 or benzyl

6-0-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-desoxy-~ -D-glucopyranoside of the formula :
~ ~c N~Coo B ;~

This derivative is prepared from benzyl-3-0-benzyl-2-benzyloxy-carbonylamino-2-desoxy-c~ -D-glucopy-ranoside (derivative 21) by proceeding as follows (see figure 5) :

A suspension of compound 21 (987 mg, 2mM), this compound is prepared according to P.C. ~SS and J. KISS, Helv. Chim. Acta, 58 (1975) 1833-1847) in anhydrous 1~2-dichloroethane (15 ml) is stirred under reflux for 30 h in the presence of N-acetyl-imidazole (freshly prepared 2.5 mM). After a cooling and dilution with chloroform (50 ml), the organic phase is washed with an M chlorid acid solution, with water, with a saturated aqueous solution of hydrogen carbonate, with water, dried (sodium sulphate)~filtered and evaporated. The residue is chromatographed on a silica gel column (50 g). Elution with the mixture dichloromethane-acetone (15 : 1, v/v) gives the derivative 22 in the form of a syrup crystalli-zing in a mixture of ethyl acetate-hexane (759 mg, 71 %), M.P; 114-115C; ~ ~D = + 88 (c = 1, chloroform).

. . ~

69 ~65~

EXA~LE 4 : Synthesis of the monosaccharide 33 of the formula ~O~OH
HO~/
OH
(33) The synthesis is carried out from compound by the following steps (see Figure 6) :
1) introduction of a benzoyl group at the 5 posi-tion, 2) methy lation of the carboxyl functi~n at position 6, 3) isomerisation of the OH group at posi-tion 5, 4) - forma-tion of the pyran ring.
1) benzoylation reaction -63g of 3-0-benzyl-1,2-0-isopropylidene ~-D-glucofuranoside (compound 23) are dissolved in 500 ml of anhydrous pyridin~
85 g of trityl chloride is added and it is heated to 80C
for one hour. In th~s way the compound 24 is obtainedr Rotatory Power : ~ ~ DO = _ 34.7, chloroform.
The structure of this compound has been con-firmed by ltS IR and NMR spectra, its elementary analysis i9 correct.
The mixture is then cooled to 0C and 45 ml of benzoyl chloride added. After one night, the excess of re~gents are destroyed by the addition of 300 ml of methanol. q'he mixture obtained, evaporated -to dryness is taken up again with chloroform. The chloroform phase ~2 EiS~32 is washed with water, dried over sodium sulphate and concentrated. In this way the compound 25 is obtained.
The syrup obtained is dissolved in 400 ml of chloroform. After addition of 100 ml of a 5 M
paratoluene-sulphonic acid solution in methanol, the solution is left a-t 4C overnight. After washing the organic phase with water, 215 g of a mixture is obtain-ed. The compound is obtained by chromatography of this mixture on silica gel in the solvent ether-hexane 2/1 (v/v). In this way 36 g of compound 26 is obtained.
Rotatory power : [~]20 = 65.3, chloroform.
The structure of the compound 26 has been confirmed by its IR and NMR spectra.

2) - methylation of the carboxyl function at 6 position -The compound 26 (1.88 g) is dissolved in acetone (20 ml). Drop by drop at 50C, 3.5 ml of a solution of CrO3 (13 g) in H2SO4, 3.5 M (29 ml) is added. The temperature is allowed to rise again and it is left for one hour under these conditions. The reac-tion mixture is then poured into ice and the product is extracted with chloroform. After washing with water and drying, it was evaporated to dryness. The compound 27 was obtained.
The mixture obtained is dissolved in methanol (20 ml), then 10 ml of 1 N soda added and it is left overnight at room temperature. The reaction mixture is then passed through a column (25 ml) of Dowex 5 esin

7 1 ~i513;~

in -the H+ form previously rinsed with methan~. The product is obtained by concentration of the eluate.

In -this way -the compound _ is obtained.

This compound is dissolved in ether and methylated con-ventionally with diazome-thane. After evaporation, the compound 29 (1.08 g ; 70.4~) is obtained.

Rotatory power : ~ DO = _ 27, chloroform.

Elementary analysis found of the compound 29 is correct.

It s-tructure is moreover confirmed by its IR and NMR

spectra.

3) - isomerisation of the -OH group at the 5 ~osition -..
~ o a solution of triflic anhydride (0.8 ml) in dichloromethane (16 rnl), cooled to -20C, is added drop by drop a solution of pyridine (0.8 ml) in dichloromethane (8 ml). Then a-t -10C, drop by drop, is added 800 mg of compound 29 dissolved in dichloromethane (8 ml). Af-ter one hour at -50C, the reaction mixture is poured into a mixture of water and ice (8 ml) containing 160 mg of sodium bicarbonate. It is stirred un-til separation of the two organic and aqueous phases. ~he organic phase is washed with 3% EICl, H20, saturated NaCl, dried and concentrated. In this way the compound 30 is obtained.
The syrup is taken up again with DME` (10 ml).
Sodium trifluoroa¢etate (1.6 g) is added and i5 heated to 80C for three hours. In this way the compound 31 is obtained. ~fter evaporation, taking up again with dichloromethane, washing with water and drying, the 72 1~6513;~

residue is taken up again with methanol and then the solvent is evapora-ted af-ter one hour~ After chromo-tography on a colurnn in -the solvent ether-hexane 2/1 the comp~und 32 is obtained (450 mg : 56~2%)o Rota-tory power : ~cX~ DO = _33~ chloroform The structure of compound 32 is confirmed by i-ts IR
and NMR spectra. Elementary analysis found is correct 4) ~ formation of the pyran ring This synthesis is carried out from the compound 32 ~ The compound 32 (200 mg) is dissolved in a mixture of trifluoroacetic acid/water 9/1). After 15 minutes, the s~vents are evaporated. The residue is crystallised in ethyl acetate/hexane. In this way 110 mg of compound 33 are obtained.
The charac-teristics of this derivative are as follows :
- IR spectrum : in CHCl3, in cm 1 : 3450(0H) ~3080~3060 3030 (CH2 benzyl) and 1740 (COOCH3) - NMR spectrum : ~ in ppm with respect to TMS : 3~75 (s~3H~ COOMe) 4~98 (1H+), 7~30 (s~ 5H+~ C6Hs) - ro-tatory power : ~ 20= + 13 ~ methanol, - elementary analysis for C14 H18 7 calculated found C................................. 56~37 56~17 H~ 6~08 5~85 - M.P............................... 125- 126C ~

, .

73 ~ 3~

Example 5 : S~nthesis of the derivative 38 or 3-0-ben~ 4-0 chloro~cet~l-1,2-0- tertO butoxyethylidene -r~-~-methyl ido-p~ranurona-te of the formula ~ 0 I\OBn j\
MC~O~ O
O~O~-bu~
~ CH
(38) 3 This synthesis (see Figure 7) is carried out from the derivative 33 with an iduronic acid structure by subjecting ~ ) the derivative 33 -to an acetylation reaction, ~ ) the mix-ture of anomeric acetates 34 and 35 obtained, to the action of a brominating agent in order to introduce a bromine atom onto the anomeric carbon, ~ ) by forming an orthoester at the 1, 2 positions and S ) by carrying out a monochloroacetylation a-t the 4 posi-tion of the or-thoester.
~ ~ acet la-tion reaction leading to 1,?,4-tri-0-ac ~
0-benzyl- ~,~ -~-me-thyl ido~yranuronates (derivati~es 34 and ~5).
A solution of compound 33 (3g) in a mixture of anhydrous pyridine (20 ml) and acetic anhydride (10 ml) is stirred at 0C, protected from moisture, for 5 h. The re-action mixture is evaporated to dryness, evaporated with tolu-ene (4x20 ml), and dried under vacuum. The residue is chroma-tographed on a silica gel column (150 g). Elution by -the mix-ture toluene : ethyl aceta-te (4:1 v/v) gives, in order of elution :
- a head fraction composed of furane derivatives, - the compound 34, ( ~ anomer), syrup, (170 mg, 4%), .

2 ~ 5 1~ ~

7D = ~ 43 ; (c : 1, chloroform), N.M.R~ (CDC13):
: 6.23 (Sf 1E~, H-1).
- the compound 35 (~ anomer), crystallizing in a mixture e-ther-hexane, (2.688 g, 63~), MoP~ 112-113C, ~ 7D - ~ 9 (c : 1, chloroform) N.M.R. (CDCl3) : ~: 6.08 (d, 1H, H-1, J1 2 : 1.5 Hz).
The ~ and ~ anomers 34 and 35 are not separated when pro-ceding with the sequence of synthesis described. Their mixture is used direotly in the form of a syrup for -the subsequent reactions.
) bromuration reac-tion leading to the compound 36 or 2,4-di-0-acetyl-3-0-benzyl- ~- ~-methyl idopyranuronyl bromide A mixture os acetates 34 and 35 (212 mg; 0.5 mM) is dissolved in anhydrous dichlorome-thane (5 ml) and anhydrous e-thyl acetate (0.5 ml). Titanium -tetrabromide (250 mg, 0.7 mM) is added in one lot, and the reaction mixture is stirred for 24 h at room temperature protected from moisture. After cooling to 0C and dilution with dichloromethane, the organic phase is washed with ice wa-ter (3 times), dried (sodium sulphate), filtered and evaporated -to give the derivative36 in the form of a light coloured syrup (217 mg, 96%)~ N.M.~. (CDa13) : ~ : 6.41 (s, 1H, H~ his compound, very unstable is immediately engaged in the following reaction.
~ ) preparation of the orthoester of 4-0-acetyl-3-0-benzyl-1,2-0-tert-bu-toxyethylidene- ~ -L-methyl idopyranuronate.
A solution of bromide 36 (freshly prepared from 2.122 g, 5 mM, 1~:65~3;~

of a mixture of acetates 34 and 35 in anhydrous dichlorome-thane (20 ml) is stirred at room -temperature under a dry argon atmosphere. Sym-collidine (2.65 ml, 20 mM) and anhydrous tert-butanol (3 ml; 30 mM) are successively added and the reaction mix-ture is stirred for 15 h under these conditions.
After dilution with dichloromethane (50ml) the organic phase is washed wi-th a satura-ted aqueous solution of sodium hydrogen-carbonate, with wa-ter, dried over sodium sulphate, filtered and evapo~ated. The residue is chromatographed on a silica gel column (120 g). Elution by the mixture hexane:ethyl aceta-te (2:1, v/v, containing 0.5~ of trie-thy~amine) gives compound 37 in the form of a pure syrup (1.542 g, 70~0 from 34 and 35) ~ = -23 (c : 1, chloroform), N.M.R. (CDC13) : ~ :

5 48 (d, 1~, H-1, J1 2 : 2.5 Hz).
S ) monochloroacetylation of the orthoester 37 A solution of the or-thoester 37 (220 mg, 0.5 mM) in anhydrous me-thanol (10 ml) is cooled to -20C with stirring and under a dry argon atmosphere. Anhydrous potassium carbonate (40 mg) is added and -the reaction mixture is stirred for 5 h under these condi-tions. The solids are drained, the filtrat evaporated and the residue is taken up again in chloroform (50 ml). The organic phase is washed rapidly with ice water (3 times), dried (sodium sulphate), filtered and evaporated.
The residue is immediately dissolved in anhydrous pyridine (4 ml) and anhydrous dichloromethane (2 ml). After cooling to -20C under a dry argon atmosphere, a solution of chloro-acetyl chloride (0.1 ml, 1.24 mM, freshly distilled) in anhydrous dichloromethane (1 ml) is added drop by drop. The reaction mixture is stirred under these conditlons for 30 min, then poured into water-ice mixture (100 ml). After 76 ~L26~13;~

stirring for 15 min, the mixture is extracted with chloroform 3x20 ml). rrhe organic phases are washed with ice water, wi-th an aqueous solution of 2% sodium hydrogencarbonate, with wa-ter, dried (sodium sulphate), filtered and evaporated. The 5 residue is rapidly chromatographed on a si~ca gel column (12 g). Elu-tion with the mixture hexane:ethyl acetate (5:2, v/v, containing 0.2~ o~ triethylamine) gives in order of elution :
an unsatura-ted compound 39 (15 mg, 8~o) ~
the or-thoester 38 syrup (145 mg, 61~o from 12), D- ~19 (c:1, chloro~orm), N.M.R. (CDC13) :~ : 5.45 (d~ 1H~ H-1~ J1 2 2.5Hz), 5.24 (d.de d., 1H, H-4, J3 4 : 2.5Hz, J4 5 : 1.5Hz), 4.00 (s, 2H; Cl-CH2-C00-).
EXAMP~E 6 : ~yn-thesis of the disaccharide 41 or benzyl 15 6-0 acetyl-3-0-benzyl-2-benxyloxycarbonylamino -2-desoxy-4-0-(2-0-ace-tyl-3-0-benzyl- ~-~-methyl idopyranuronly)- ~ -D-glucoPyranoside OAc OBn (41) O~n Firs-tly the disaccharide 40 is prepared according -to step ~ ) by condensation of the monosaccharides 38 and 22, then the monochloroacetyl group is removed at the 4 position in step~ , which leads to -the desired disaccharide 41 (see Figure 8).

.

s-tep ~ : preparation of the disaccharide 40 or benz~l 6-0-ace tyl-3-0-benzyl-2-ben~yloxycarbonylamino-2-desoxy-4-0 (?-0-acetyl-3-0-benzyl-~-0-chloroacetyl- 0~ -L-inethylidopy:ranuron-yl )- G~ -D-glucopyranosi.de .
~ 901u tion of the or thoester 38 ( 284 mg, 0 . 6 m~
and alcDhol 22 ( 214 Mg, 0.4 mra) in anhydrous chlorobenzene ( 12 ml~ is heat to 140C with stirring and a slight current oE dry argon. Af ter slow dis-tillation of 10 ml of solvent a solution of 2,6-dirnethylpyridinium perchlora.te (0~006 ml~, freshly prepared) in chlorobenzene (4ml) is added drop by drop in 30 min with simul-taneous dis-tillation of solvent (4 ml) .
Thereactionmixture is stirred 1h, with the addition of fresh solvent (10 ml) and simultaneous distillation so -that the reaction volume rernains constant and equal to 4ml. Af-ter cooling and dilution with chloroform, the organic phase is washed with a sa.turated solution o:f sodium hydrogen carbonate, with wa.ter, dried over sodium sulfate, filtered and eva.pora.te rl'he residue is chroma togra.phed on a column of silica gel (40 g). Elution by the mixture hexa.ne:ethyl acetate (4:3, v/v) gives, in order of elution ~ -the product 22, ( 120 mg, 56 q'0) - the disaccharide 40, crystallised in a rnixture ether-hexane ( 112 Mg, 30 jO, ~P: 144-145C, r<7D20= ~35 ( c: 1, chloroform), NlI~ (CDCl3) : in accordance wi th -the ex-pected structure.
step/~: removal of -the monochloroacetyl group.
A mix ture of the disaccha.ride 40 ( 56 mg, 0 . 06 mM) a.nd of thiourea. (7 rng, 0.1rnM) in pyridine t2.5 ml) and absolute ethanol (0.5 ml) is stirrel at 100C for 30 min. After cooling 78 ~65132 and evaporatlon to dryness, the residue is taken up again with a mixture of water-chloroform (1 : 1, v/v 40 ml). The organic phase is washed with water, dried (sodium sulfa.te), filtered and eva.porated. The residue is chromatographed on a silica.
gel column (2g). Elution by the mixture ethyl acetate : hexane (2 : 1, v/v) gives the disaccharide 41, crysta.llised in ether (46 mg, 30 ~), M.P. : 146-147C ~ 7D = 44 (c : 1, chloro-form), NMR (CDCl3) : in accordance wi-th expected s-tructure.
Example 7 : Syn-thesis of -the tetrasa.ccharide 43 of -the formula CO O ~ Ot\. O~c ~ ~ J~ OAn O~n N 3 Ac NHCOOBn (4.3) The -tetrasaccharide 43 is prepared by carrying out :
- in step a) the condensa-tion of the disa.ccharides 20 and 41 whose synthesis is described in Examples 2 and 6 and by subjecting in -the course of step b) the -tera.saccharide 42 formed to a selective - 0 - dimonochloroace-tylæ.-tion reaction at the 4 position (see Figure 9) :
a) Condensa-tion reaction A mix-ture of 64 mg (80 M) of -the bromide 20 freshly prepared, 51 mg (60 ~M) of the compound 41 and 80 mg of molecular si-~ve 4 ~ in powder in 1.5 ml of .anhydrous dichloroethane is subjected -to stirring for a haIf-hour at 79 ~.2~i5~

:~oorn telnperature, under a dry argon atmosphere, then cooled to -20C. Succcs ivcly 20 ml (150 Iil) o:E sym-collidine at 31 mg (120 M) of silver -trifla.te are added. The rreaction mixture is subjec-tcd for 1 h to s-tirrin~ a-t -20C, -t;hen the tempera-5 ture is allowed to rise a.gain to ambien-t tempera-ture of 15 h.
~fter dilu-tion wi-th 50 ml of dichlorometha.ne, the solids a.re drained and -the fil~rate i5 washed with an iced aqueous solution of 1 M hydrochloric a.cid with water (twice). It is -then dried over sodiurn sul:fate, :~iltered and then evaporated.
The residue is chromatographed on a silica gel clumn (8 g, gel 230-400 mesh). Elution by the mixture hexane-ethyl a.cetate (4:3, v/v) enables recovery of 37 mg of tetra-saccharide 42 (yield 39 ¦0) in the form of a colorless glass ~7D2r= ~ 56 (c = 0.6 ; C~ICl3) ; the NMR spectrum confirms the expec-ted struc-ture.
By elu-tion of the coluinn with the mixture ethyl a.cetate-hexa.ne (2:1, v/v), 23 mg of the starting product 41 is recovered (yield 44 ~).
(b) -0-dechloroace-tyla.-tion reaction A solution of 36 mg (23 M) of -the tetrasaccha.ride 42 in 1.25 ml of a. mixture of pyridine and 0.25 ml of absolute ethanol is hea-ted -to 100C in the presence of 7 mg (100~M) of thiourea for 20 min. After cooling and evapora-tion to dryness, -the solid residue is taken up again wi-th 20 ml of water and extra.cted wi-th chloroforrn (5 times 5 ml). The orga.nic phases are washed with a.n aqueous 10 ~ sodium hydrogen sul-fate solution, with water, dried over sodium sulfate, fil-~65~

tered and eva.porated. The residue is chromatogr~p.hed on a Gilica gel column (3 g). By elution with an ethyl a.cetate-hexa~e mixture (3:2, v/v), 27 mg of the derivative 43 is obtained (yield 80 ~0) in the form of a colorless gla.ss ~ 7D20 = + 61 (c = 0.8 ; chloroform) ; the N~ spectrum confirms the expected structure. (~ ~e31).
EXAMPLE 8 (see Figure 10) :
Syn-thesis of -the pentasaccharide 45 of the formula 0 ~ C0 0 ~e 0~

BnOf~ OI'~n N ~ O ~ n ~ 3 OPc N ~COOBn (45) A condensation reaction is carried out between the tetrasaccharide 43 and the monosaccharide 44, which lead to the pentasaccharide 45.
A mixture of 27 mg (54 ~ M) of bromide 48 prepared according -to H. PAU~SEN und W.S~ENZE~, Chem. Ber., 111 (1978) 20 2334-2347, of 26 mg (18 ~M) of tetra.saccharide 43 a.nd 50 mg of molecular sieve 4 ~ in powder in o.8 ml of dichloroethane is subjected to stirring for 1/2 h at room tempera.ture under a dry argon atmosphere, then cooled to -20C. 16 ml ( 120 r5) of sym-collidine and 26 mg (100 ~M) of silver trifate are 25 added successively and the reaction mixture is subjected to stirring for 18 h allowing the tempera~re to rise slowly to room temperature.

81 ~ ;~65~32 After dilution with 50 ml of dichloromethane, the solids a.re dralned a.nd the filtrate is washed with a 1M aque-ous iced hydrochloric acid solution -then with water (-twice).
It is then dried over sodium sulfate, filtered, and -then evaporated.
The resi.due is chroma-tographed on silica gel column (5 g, 230-400 mesh gel). By elution with a hexane-ethyl aceta-te (4:3, v/v), mixture, 30mg of pentasaccha.ride 45 is recovered in the for of a. colorless glass (yield 90 ~0) r~ 7D20 = + 67 (c 1 : chloroform). The NMR spectrum con-firms the expec-ted s-truc~ture. In par-ticular, for the anomeric protons of the glucosamine units displacements ( ~ , ~MS) of 5.36 and 5.52 ppm are fround for -the pro-tons belonging -to H, F and D respectively.
EXAI~E 9 :
Prepara.tion of -the penta.sa.ccharide 50 (see Figures 10 and 11) Recourse is had to the following s-teps :
(a) removal of the acetyl groups (pentasaccharide _~ .
(b) sulfation of the thus-liberated -OH groups (pentasa.ccharide 47) (c) hydrogenation -to liberate the -OH groups pro-tected by the benzyl groups and -to convert the -N3 group into a -NH2 group (pentasaccharide 4~) . d) sulfation of the NH2 groups (pentasa.ccharide 49 then saponification of the -COOMe groups at the 6 position (pentasaccharide 50) 1~65~3~

These s-teps are carried out as follows :
a) removal of -khe acetyl groups of the derivative A solu-tion of 28 mg of the pentasaccharide 45 in a.
mixture of 2.5 ml of 1,2-dimethoxyethane and 0.8 ml of methanol is cooled to 0C with stirring. Then 1 ml of a. 1M soda solu-tion is added drop by drop, in 10 minutes. The reac-tion mix is subjec-ted to stirring 1 hour a.-t 0C, then 12 hours at room tempera-ture. After cooling to 0C, 3 ml of 1M hydro-chloric acid is a.dded and the milky mixture is immediatelyextracted wi-th chloroform ( 5 times 5 ml). The organic pha.ses are washed with water, dried over sodium sulfate, fil-tered and evaporated. The residue is taken up again in 2 ml of meth-anol and treated with an ether solu-tion of diazome-tha~e (excess until-the persistance of -the yellow color) for a half-hour.
Af-ter evapora,tion to dryness, -the residue is chro-matographed on a. silica. gel column (2 g, 230-400 mesh gel).
~lution by the mixture dichloromethane-methanol (15:1, v/v) enables the recovery of 18 mg of the pen-tasaccharide 46 (yield 72~) in -the form of a colorless glass. ~ 7D 20 = ~57o (c = 1; chloroform) ;
The expected structure is confirmed by the NMR
spectrum.
b) sulfation of the -OH groups To a solutiTo a solu-tion of compound 46 (22 mg) in di ethyl-formamide (0.5 ml), is added the complex trimethylamine/S03 (22 mg, 2.5 e ~OH). The reaction mixture is heated to 50C

~:65~3~

for about 14 h. Then the complex trimethylamine/S03 (10 mg) is again added and the reaction is allowed to develop for 24 hours. To the reaction mixture are then added methanol (0.5 ml) and chloroform (0.5 ml). rhe solution is intro-duced at the top of a Sephadex ~H20 ~column equilibra~ed in aCHCl3/CH30H (1/1 ; v/v) mixture. The Iractions conta.ining the sulfa.ted produc-t are grouped together and the solvent is evaporated. In this way a. glass is obtained ~30 mg). This glass is then chroma.togxa.phed on silica gel (10 g) in a solvent constituted by 3 par-ts of -the mixture ethyl a.cetate/pyridine/
acetic acid/water, (6/2/0,6/1, v/v/v/v) and ~ parts of the mix-ture ethyl aceta.te/pyridine/acetic acid/wa.ter, (5/5/1/3, v/v/v/v ) .
The fractions containing the desired product a.re collected and concentrated. ~fter evaporation of the solvents, the residue obtained is dissolved in methanol suplemen-ted with water, then passed through a. Dowex 50 W x 4,Na+ column, equili-brated in a mixture methanoljwater(50/50, v/v). In this way the sodium salt (compound 47 is obtained).
c) hydrogenation The product obtained above is dissolved in methanol (3.7 ml) supplemented with water (0.3 ml).
To this solution, is added the catalyst (Pd/C,5%,40mg) and it is stirred under a. hydrogen atmosphere for 5 days.
After removal of the catalyst by filtration, analysis of the U.V. spectrum of the solution obtained shows the complete disappearance of the absorption due to the benzyl groups. The solvent is then evaporated, leaving a residue, namely the compound 48.
d) sulfation of the -NH~ groups, then sapQ~ifi-ca-tion of -the carboxyl groups Compound 48 is dissolved in water (4 ml). The pH
is -then a.djusted to 9.5 then -the complex trimethylarnine/S03 ( 54 mg) is added to the solution. The pH is kep-t at 9.5 through-out the reaction by the addition of 0.1N soda.
~fter one nigh-t, a fur-ther addition of sulfating agent is made (27 mg). A last addition is carried out after 24 h.
After 48 h, soda is a.dded ~ 3M, 34 ml) to the com-pound 49 forrned, then -the solution is subjected -to stirring for 3 hours at room temperautre so a.s -to hydrolyse the me-thyl-.
esters of the uronic acid type uni-ts. The reaction mixture is then neutralized and then concentrated to a volume of about 2 ml. The solu-tion so-obtained is placed a-t the top of a. Sephadex G 25 column (100 ml) eluted with water. The fractions collected are ana.lysed by W absorp-tion ( 206 nm) and by polarometry ( 265 nm). The fractions havi~g optical activity are grouped, the solven-t is removed and the residue taken up again by about 2 ml of water and freeze-dried.
In this way the derivative 50 is obtained in -the form of a white powder ( 5 .6 mg, 25 % with respect to the pro-duct 45 ) .
The N~R study confirms -the expected struc~ture.
I-t is found in par-ticula.r for the anomeric protons of -the : glucosamine uni-ts, tha.t the disp~acements are (S , 'l'MS) ~, ~ 3~

5.36, 5.45 and 5.52 ppm for the protons belonging to H, F
arld D respectively.

EXA~LE_10 - Synthesis of -the disaccharide 51, namely methyl 1 prop-1~ yl 2,3-di-0-benzyl-4-0~-azido~
3,4~di-0-benzyl-6-0-acetyl- -~ -D-glucopyrano-side7 uronate of the formula O~c COOMe /CO )~0 I'\OBn ~OJ~OBn ~
Bn O `L~ \ / O-C~I= CH-Me N3 (51 ) bBn Reference will be made to ~igure 12.
To a solution of monosaccharide 13 (0.215 g ; 0.5 mmole) in dichloromethane (3 ml), are added the monosaccharide 44 (0.49 g ; 1 mmole) in dichlorome-thane (3 ml) and then 4 R sieve in powder form. The mix-ture is cooled to 0C, then there is added sym-collidine (0.16 ml) and silver triflate (0.3 g). After 1 hour, the rnixture is diluted wi-th dichloro-2~ methane (50 ml). The solids are drained, and then the solutionis washed wi-th a 5 o/O solution of sodium bicarbonate, with water, then with 10 ~0 acid po-tassium sulfate and again with water.
In -this way, -there is obtained after evaporation 591 mg of residue. After purification on silica in a -toluene/acetone 30/1 ~v/v) mix-ture, 211 mg of pure disaccharide 51 are recovered.
This product is characterised by its elemen-tary analysis.

~.~651~3;~
~6 E~Al~LE 11 - Synthesis of the disaccha.ride 54, namely methyl (1-trichloroa.ce-timidyl-2,3-di-0-benzyl-4-0-r2-acetylamido-2-desoxy-3,4-di-0-benzyl-6-0-ace-tyl- ~-D-glucopyranoside7uronate of the formula.

O~c COOMe ~0 ~0 ~OBn ~ o ~OBn ~ N,H
Bn O\L~/ \L ( O-C-CCl3 NHAc OBn Re:Eerence will be made to Figure 12 for the syn-thesis diagram.
'~o a solution of the disaccha.ride 51 (180 mg) in 6 ml of a mixture acetone/wa-ter (5/1 ; v/v), are a.dded suc-cessively mercuric oxide (232 mg) and then drop by drop a solu-tion of mercuric chloride in an acetone/water mixture (292 mg/2 ml).
After filtra-tion, evapDration, taking up again with chloroform a.nd washing with a 10 % potassium iodide solu-tion and wi-th wa.-ter, the disaccharide 52 is obtained (140 mg).
100~mg of the disaccharide 52 is dissolved in 0~6 ml of methanol. To this solution , are added ammoniurn formia-te (160 mg~ and 10 /~ Pd/C cata.lyst (100 mg). After 5 minutes, the ca.ta.lyst i5 removed a.nd acetic anhydride added (10 drops).
After eva.poration, the product ob-tained is purified on silica.
in a. toluene/acetone mixture (4/1 ; v/v). In -this wa.y 61 mg of disaccharide 53 are obtained.

.

12:6~L3;;~

The disaccharide 53 is characterised by its Rf on a silica plate Merck (~) reference 5719) in two different solvents: chloroform/ethyl acetate, 3/2,v/v Rf = 0.40 an toluene/acetone, 4,/1, v/v ; P.I = O.20.
The derivative 53 (60 mg) is dissolved in dichloro-methane (1.5 ml). Then t:richloroacetonitrile (75 l) and sodium hydride (1~5 mg) are a,dded. After 15 minutes, the derivative 53 had disappeared -to the advantage of the deriva-tive 54. Af-ter filtration and eva.poration, 5 is obtained -(67 mg). The derivative 54 is characterised by its ~f on a silica plate Merck(~) reference 5719) chloroform/ethyl acetate 2/1, v/v; Rf = 0.59 (0.37 for the compound 53).
\
\

\
:

88 126~i132 EXAMPLE ?2 Synthesis o~ the derivative 57, namely methyl 1,6-anhydro-2, 3-epoxy-4-0 t2,3-di-0-benzyl-uronate~ -~-D-glucopyranoside of the Pormula :
COOMe ~c o"~l``~ ~Y
OBn (57) This synthesis is carried out from derivatives 55 and 56 (see figure 13).
a) Preparation of methyl (bromo 2,3-di-0-benzyl-4-0-acetyl-D-glucopyranoside) uronate -(compound 55)-SYN~HESIS OF COMPOUND ld To a solution of la (32 g ; 85,5 mmoles) in pyridine (250 ml), is added trityl chloride (28,6 g ; 1,2 eq) and it is then heated to 80C. A further addition of trityl chloride (4,6 g ; 0,2 eq) i5 made after 3 hours of reaction. When the ormation of 1b is complete (t.l.c. silica ; methanol~chloroform, 1/20~ v/v) the solution is cooled again to 0C, then benzoyl ch~ride is added (15 ml ; 1.5 eq). After one night, 1c is formed quantatively. Methanol (1 5 ml) is then added drop by drop to reaction mixture and then concentrated to dryness.
The residue obtained is taken up again in methanol (500 ml) containing paratolucne-sulPonic acid (95 g). After 2 hours reaction, the reaction mixture is transPerred to a separating funnel containing ice water (21).

89 i2Çi51~32 The product 1d is extracted with chloroform then used as such in the Pollowing step.
A portion of this product was purified. Analysis o~ the IR spectrum confirms the structure. It is a colorless gum. ~ ~D ~ 61 (chloroorm).

SYNTHESIS OF THE COMPOUND 1j The syrup obtained in the preceeding step (95 g) is disolved in acetone (1 l), then to the solution cooled to 0C, is added drop by drop a solution of chromic oxide (52 g) in sulfuric acid 3,5 M (220 ml). A~ter 2 hours reaction, the reaction mixture i5 pourred into iced water (1 l).
The pr~duct le is extracted with chloroform (5 x 200 ml).
The chloroform phase is washed until pH neutral, dried and concentrated to dryness.
To the residue obtained above, disolved in methanol (650 ml), is added drop by drop soda in aqueous phase is then (20 g in 50 ml), then the mixture is heated to 50 C, After one night, the solution obtained is partly concentratedt then poured into water (1 5 l). The aque~us! phase is then washed with ether, then, after acidification with hydrochloric acid, the prdduct 1f is extracted with ether. The ether phase is dried with sodium sulfate, then concentrated to dryness, giving a yellow mass (50 g) which contains 1e.
This residue (50 g) isdissolved in a mixture of acetic acid and trifluoroacetic acid (15/1, v/v, 615 ml). To this solution, stirred at 100 C, is added water (160 ml).

90 ~ ~ ~ S~ 32 After one night it is evaporated to dryness and the traces of acetic acid removed, and toluene evaporated, The residue formed in part rom unhydrolised 1~ and ~rom 1g is disolved in ether (400 ml).
To this solution, is added at 0C, an ether solution of diazomethane until complete methylation (t.l.c. silica, etherhexane, ~1, v/v). The excess diazomethane is then destroyed by acetic acid then the reaction mixture is concentrated to dryness.
The residue is purified on a silica gel column (200 g) eluted first with purechloroform, then by a chloroform/ehter mixture, 3/1, v/v. In this way 1k is obtained (8.6 g ; 22.2 mmoles, 26 o/. with respect to 1a).
The derivative 1k is crystallisedm.p. 122-123C.
Elementary analysis and the NMR spectrum confirms its structure.

S~NTHESIS OF COMPOUND 1l To a solution of 1k (3.9 g ; 10 mmoles) in pyridine (50 ml), is added acetic anhydride(4 ml, 42 mmoles). After 2 hours, the reaction mixture is evaporated to dryness. In this way 1 is obtained (4~62 g ; 98 o/.).

To a solution of 11 (1.4 g) in dichloromethane 30 ml and ethyl acetate (3 ml), is added tltanium tetrabromide (1.5 g). The solution is stirred over-night 9 1 ~2~ 2 at room temperature. After dilution with dichloromethane~
reaction mixture is poured into iced water. The organic phase is washed with 5 ot. bicarbonate in water~ dried and concentrated. The residue is chromatographed on silica (50g, ether/hexane, 1/1, v/v).
In this way the compound 55 is obtained (920 mg, 62 o~) ; it is a colourless syrup ~ ~20 = + 97,5 (c = 1, chloroform). Elementary analysis and the NMR spectrum confirm the structure.
b) A solution of the derivative 56 (432 mg, 3 mmoles) in dichloromethane (10 ml) is stirred at 0C in the presence of a 4 A molecular sieve (0,5 g), drierite (1 g) and freshly prepared sil~er carbonate (0.42 g). After cooling to 0C, is added~ drop by drop, a solution of the compound 55 (490 mg, 1 mmole) in dichloromethane (6 ml). The reac~ion lasts two hours, the reaction mixture is then filtered.
After evaporation to dryness and chromatography on silica gel of the residue~ (solvant : ethyl acetate/chloro~orm, 1/6, v/v), the derivative 57 is obtained (285 mg ; 51 ~.3.
The structure of deri~ative 57 is confirmed by its elementary analysis and its NMR spectrum. Rotatory y~er :
[~] 2~ = 39o ; chloroform MP = 156-159C.
EXAM~LE 13 Synthesis of the deri~ati~e 59, of the formula :

OAc COOMe O

BnO~O~\~ Y J\,~
N3 ( 59 ) OBn 92 ~265~3~:

The trisaccharide 59 i5 prepared by the reaction o~ the disaccharide 58 (obtained by removal of the acetyl group at the 4 position of the compound 57 o~ Example 12), with the monosaccharide 4~ by operating as follows (see Figure 13) :
- deacetylation reactionof the compound 57 :
To a solution of the disaccharide 57 (260 mg) in methanol (25 ml), is added, at 0C, a solution o~ 1 N soda (25 ml)0 A~-ter one hour, the mixture is acidiied by t~e addition o~ 1 N hydrochloric acid (30 ml). The product is extracted with chloro~orm. A~er evapo~ion, the residue is crystallisedin a ethyl acetate/hexane mixture. 167 mg are obtained (yield 7010) of the derivative 58.
Rotatory power : t~] 20 = -31 ; chloroform, MP =
169-170C. The analysis ound is correct. The structure o~ the derivative 58 is more~er conirmed by its NMR spectrum.
- condensation o the disaccharide 58 with the monosaccharide 44 To a solution o~ compounds 44 (300 mg) and 58 (155 mg) in dichloromethane (5 ml), are added successively 4A sieve in powder ~onn (500 mg), then collidine (100 ~l) and silver 2~ triflate. After 15 minutes, the so~ion is diluted with dichloromethane (50 ml), ~iltered, washed successively ~ith water, a solution with 10/. o~ acid potassium sul~ate and with water. Ater drying and concentration, the residue is chrom~ographedon silica gel in an et~l acetate/chloro~orm mixture (1/10, v/v). In this way the derivative 59 is obtained in the orm o white ~oarn.
This derivative 59 is caracterised by its elementary analysis, its NMR spectrum and its rotatory power (~] 20D =
25 ; chloro~rm).

.

93 ~ 65~32 EXAMPLE 13 A : Synthesis oP the trisaccharide oP formula CO O-N ~

~ 0~O~ ~4~ \~t0H
N ~I~sO,,~Ja~ 0 H N H 50,,7~

The monosaccharide 20 is reacted ~ith methanol under the conditions described f~r the synthesis of tetrasaccharide EFGH above. In this way a~ -methylglycoside is obtained.
The MCA group is removed conventionally and then the disaccharid~
is subjected to the action oP the monosaccharide 44 under the conditions described above for the development of the pentasaccharide.
The trisacchar~de obtained is then subjected ~o conVentional reactions for the purposes of protection and functionalisation~ The structure is confirmed by the NMR
spectrum.

Synthesis of the trisaccharide 62 o~ the Pormula :

O~c COOMe O~c n ~--O~\O~OAc NHAc OBn N3 (62) This trisaccharide 62 is prepared by the Pollowing steps (see Piyure 14) :
.

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

:

94 ~ 65~3;~:

a)con~1ersion of the N3 group at 2 position of the glusosamine unit into an -NHAc group.
b) opening of the 2,3 epoxy bridge o~ the unit at the reducing end.
c) opening of the anhydro-1,6 bridge oP the same unit.
a) Passage from -~3 to -NHAc :
To a solution of the derivati~e 59 ( 10 mg) in a mixture of DMF/ethanol (1/1 ; 1 ml), are added the cataly~
Pd/CaC03 , 5 /. (5 mg) . The suspension is stirred under hydrogen pressure of 1 atmosphere for 96 hours.
After filtration of the catalyst and evaporation, the residue is dissolvedin methanol and acetylated by addition of a drop of acetic anhydride. The derivative 60 is obtained quantitati~ely.
The deri~ative 60 ischaracterised by its NMR
specrum, its elementary analysis, its rotatory power :
[~] 20 = ~ 35.5 ; ch~roform . MP : 147-149C.
b) Opening of the epoxy bridge :
The deri~ati~e is first saponified as indicated for the synthesis of the deri~ative 58, and this in order to remo~e the acyl group at the 6 position of the non-reducing terminal unit and the methyl-ester group at the position of the intermediate unit.
After extraction, the residue is disol~ed in DMF
and is heated to 120C, in the presence of sodium azide, for 48 hours~ After e~aporation, extraction with chloroform, washing with 0,1 N HCl, with water, drying and evaporation of the sol~ent, a residue is obtained which is treated with 12Çi5~3;~

diazomethane, then acetylated (pyridine acetic anhydride), gi~ing thus the compound 61.
c) Opening of the anhydro bridge :
The compound 61 is acetolysed under the usual conditions (acetic anhydride~ sulfuric acid) at ~20~C.
After trea~nen-t o the reaction mixture, the derivati~e is obtained.
EX~MPLE 15 Synthesis of the derivati~e 63 of ~ormula :
OAc COOMe rO~c ~O -~ ~B
NH~c OBn N3 (63) Treatment of the derivati~e 62 obtained in Example 14 with titanium tetrabromide in a solution of dichloromethane and ethyl acetate leads to the halogen 63 of which the structure is confi~ned by its NMR spectrum. Its elementary analysis is correct (see Figure 14).

Synthesis o~ the monosaccharide 68 or methyl 2-acetamido-3-6-di O-benzyl-2-desoxy-~-D-glucopyranoside of the f~rmula :

OBn ,~0 H O~OMe (6a? NH~c . .

This synthesis was carried out in the following 4 steps from the monosaccharide 64 prepared by the techni~e o~ A. Neuberger, Journal of Chemical Society 1941, pages 50-51 :
1 - benzylation of the -OH group at the 3 position, 2 - elimination o~ the benzylidene radical to liberate the -OH groups at the 4 and 6 positions, 3 - tosylation ~ the -OH GROUP at the 6 position, 4 - displacement o~ the -OTs group at the 6 position by a benzylate (see figure 15).
Step 1 : benzylation reaction.
To a solution of the compound 64 (6.5 g, 20~10 m~l) in dimethylormamide (120 ml), barium hydroxide octa-hydrate (3~6 g~ and barium oxide (166g), were added. After 10 minutes stirring at room temperature, benzyl bromide(4.5 ml) was added drop by drop. The reaction continued over_night. Ater dilution with chloroform (100 ml) the reaction mixture was filtered over Celite ~.The filtra~ was concentrated to dryness, and in this way a white residue is obtained whose analysis by thin layer chromatography indicates that ~0 it contains a single substance, namely the derl~ative 65, - vhich is used as such in the ollowing step.
Step 2 ~ removal of the benzylidene group.
The residue obtained above is disolved in a mixture of methanol (370 ml) and water (130 ml). To this solution, is added paratoluene sulfonic monohydrate (3 g), then the mixture is taken to re~lux for ~ne hour. After cooling, the major portion of the methanol was evaporated, then water (250 ml) was added~ Ater washing with a small amount o chloroform (100ml) 97 ~i51~

The aqueous phase was subjected to the following treatment :
1) precipitation of barium salts with sulfuric acid ;
2) Filtration of the barium sulfate formed ;

3) removal of the excess of acid by means o~
an IRA resin 45 (OH ).
After removal of the resin and concentration, a slightly yellow residue (5.7 g) is obtained, namely the derivative 66. This derivative is employed as such in the preparation of the compound 67.
Step 3 : Tosylation reaction.
This derivative 66 is disolved in a mixture of dichloromethane (150 ml) and DMF (10 ml). To this solution, i5 added tosyl chloride (5.6 g, 30 mM), then dimethylamino-pyridine (121 mg) and finally triethylamine (5 ml). The reactiondevelops protected from moisture and under a dry nitro~en flow.
After 18 hours reaction, iced water is added and then the mixture is left with stirring for 14 hours approximately-The reaction mixture was then diluted with dichloro-2~ methane a~d then the dichloromethane phase was washed successively with 2M hydrochloricacid, saturated sodium bicarbonate, then with water until pH neutral. After drying ~ver sodium sulate and filtration~ the solvant was evaporated, The residue obtained was puri~ied on a silica gel column (200 g) d luted with an ethyl acetate-hexane mixture (4/1~
The fractions containing the derivative 67 were grouped together.

98 ~5~32 After removal of the solvants, a solid residue is obtained (4.6 g) which is used directly in the synthesis of compound 68.
~ benæylation reaction~
The derivative 67 obtained above is disolved in anhydrous dimethylformamide (50 ml). To this solution, is added a molar solution o~ sodium benzylate in benzyl alcohol(30 ml). The mixture is then heated to 90C for one hour. After cooling to room temperature, the mixture is then concentrated to dryness.
It is then ta~en up again with chloroform (400 ml), the chloroform phase is washed with water, saturated sodium chloride, dried, th~ concentrated to dryness.
The residue ~s chromatographed on a silica gel column (200 g, chloroform/ethyl acetate, 1/1, v/v).
This way the derivative 68 is obtained (2.3 g).
The yield with respect to compound 64 is 27.6 ~..
The compound 68 is crystalline, MP 149-150C, C~ 20 = 87 (C~1, chloroform). Analysis of the infrared spectrum and elementary analysis confirm the expectedstructure 2~ ~or the product 68.
Example 17 Syn~hesis of the disaccharide 73 (see Figure 15).
This SYnthesis includes :
(1) Condensation of derivatives 68 and 69 leading to the disaccharide 70.
(2) Removal of the benzyl groups leading to the derivative 71.

99 ~;~65~

(3) Sul~ation of the -OH group of the derivative 71, leading to the drivative 72, Pollowed by salif~ation of the anionic groups and removal of the acetyl groups.
1) Synthesis of the disaccharide 70.
This synhhesis is carried out from the monosaccharides 68 and 69.
The halide is prepared by the techni~ ~f G.N~ ~ollenback Journal of American Chemical Society, 77 (1955), p. ~312.
To a solution of monosaccharide 68 t450 mg, 1,1 mM), in dichloroethane (30 ml) is added mercuric bromide (400 mg~
1 1 mM). After distillation of about 10 ml of dichloroethane,to the reaction mixture is added molecular sieves in powder (4 A).
The halide 69 (1,1 g, 2~75 mM) in dichloroethane (10 ml) is then added. ~ter distillation o 10 ml of dichloroethane, the reaction mixture is left to reflux for about 14 hours at a temperature o~ 90-100C. After cooling, the reaction mixture is diluted with dichloromethane (100 ml), then the solids are removed by filtration over pleated filters. The organic phase is washed with a solution of 10/. potassium iodide (2 X 25 ml), then with a 5 o/. solution of sodium bicarbonate (2 x 25 ml) and finally with water until pH neutral. After drying over sodium sulfate, filtration and concentration, the residue is purified on a silica gel c lumn (150 g) eluted, successively, with three acetone-ether mixtures (1/5 then 1/4, then 1/2, v/v).
In this way the disaccharide _ is obtained pure (390 mg) in the form of c~ystals. MP = 189-190C ; ~ D20= ~ 60 (c= 0~4 chloroform). The infrared spectrum, the same as the NMR spectrum and the ~ementary analysis confirm the expected 100 ~L265~

structure.
2) Synthesis of the disaccharide 71.
To a solution of the derivative 70 (100 mg) in methanol (20 ml), is added catalyst (Pd/C, 5~.~ 100 mg) and the suspencion so obtained is stirred under hydrogen flow for three days.
The catalyst is then re oved by filtration.
After evaporation, a residue is obtained ~73 mg, 97 ~.
constituted by the disaccharide 71. The NMR spectrum confirms the expected structure of this compound.
It will be noted that the disaccharide 71 is the precursor of the basic unit of heparane - sulfate. It suffices to deprotect it to submit it to a saponification reaction, as reported below for producing the derivative 73 from the derivative 72.
3) Synthesis of the disaccharide 73.
To a solution of compound 71 (70 mg) in dimethyl-formamide (2 ml), is added -the sulfation agent (trimethyl-- amide-sulfur-trioxide complex) (75 mg). After one night, a further addition of complex (35 mg) is made. After 6 hours, the reaction is terminated, the mixture is evaporated to dryness, taken up again with chloroform, neutralised with triethylamine and evaporated.
Chromatography on a silica gel column (20g, methanol/
chloroform, 1/2, v/v) enables isolation of the pure sulfated derivative 72 which is in the form of a white powder. This derivative is used directly in the synthesis of the deprotected disaccharide 73.

3~

To a solution of the derivative 72 (71 mg) in methanol (9 ml), is added water (4 ml) then, drop by drop, a 1 M soda solution (1 ml). After 4 hours stir-ring at room temperature, -the reaction mixture is pass-ed over an Amberlite IR 120 H column. The solution soobtained is neutralised and then salts are removed by passage over a Sephadex G25 column diluted with water.
The fractions containing the sulfated disaccharide are grouped together.
After freeze drying, the derivative 73 is obtained in the form of a white powder (46 mg) [~]2D0 =
34.5 (c = 1, water).
Conductimetric analysis indicates for this derivative a ratio sulfate/carboxyl equal to 2. Ele-mentary analysis, the same as NMR analysis of carbon 13, confirm the expected structure for this product.
EXAMPLE 18 : Synthesis of the compounds 75, 76 and 77 with a D-glucosamine structure (see Figure 16).
Compound 75 : Methyl 3-O-benzyl-4, 6-O-benzylidene-2-benzyloxycarbonylamino-2-desoxy-~-D-glucopyranoside A solution of compound 74 (prepared according to ZU YONG KYI, Sci. Sinica (Peking), 5 (1956) 461-467, CA 52 (1958) 3694) (415 mg, 1 mM) in anhydrous N,N-di-methylformamide (10 ml) is stirred at room temperature protected from moisture for 5 h in the presence of an-hydrous barytes (613 mg), barium hydroxide octahydrate ~s~
lOla (158 mg) and benzyl bromide (0,15 ml). The reaction mixture is then diluted with chloroform (50 ml), the organic phase is washed with 50~ iced acetic acid, with water, dried (sodium sulEate), Eiltered and evaporated.
The solid residue is recrystallised in ethanol (461 mg, 91%); MP : 202-203C; [a]D = ~46 (c : 1, chloroEorm).

102 ~65~3;~

Compound 76 : Methyl-3-0-benzyl-2-benzyloxycarbonylamino-2-desoxy-~ -D-glucopyranoside~
A suspension o the compound 76 (300 mg~ in 60% acetic acid (10 ml) is stired at 100C for 30min. The solution is then cooled, evaporated to dryness, evaporated with water (4x10 ml), The solid residue is dried under vacuum and recrystallised in 2-propanol to give the compound 76 (220 m~, 890/.), MP : 151-152C, [~D-+94 (c : 1,methanol) Compound 77 : Methyl 6-0-benzoyl-3-0-benzyl-2-~enzyloxycarbo-carbonylamino-2-desoxy-~-D-glucopyranoside.
A solution of compound 76 (835 mg, 2mM) in a mixture of an-hydrous pyridine (5 ml) and dichl~romethane (12 ml) is stirred at room temperature protected from moisture inthe presence of benzoyl cyanide (400mg, 3~M) for 5 h. The excess of reagent is then destroyed by the addition of methanol (5ml)andstirring fo 30rnin. The reaction mixture is evaporated to dryness, evaporated with toluene and dried under ~acuum.Thesolid residueis recrystall-ised in a e~yl acetate hexane mixture to give the compound 77 (935 Mg) goo~.) M.P. : 154-155 ~]D = ~ 74 (c : 1, chloroform).
EXAMPLE 19 :Synthesis of the compound ~8 and 79 with L-iduronic acid structure (see Figure 16).
Co~pound 78 : 4-0-acetyl-3-0-benzyl-1,2-0-methoxyethylidene-~ -L-methyl idopyranuronate.
A solution of bromide 36 obtained by Example 5, in stepp (freshly prepared from 0.425g, 1mM, of a mixture of acetates(34 and35) in anhydrous dichloromethane (10ml) is stirred at room temperature under a dry argon atmosphere. Sym-collidine (0.66ml, 5 n~)and anhydrousmethanol (0.40m1~ 10n~) are successi~
ly added, the reactian mixture is stirred 20 h under these conditions. After dilution with dichloromethane (50ml), the organic phase is washed with a saturated aqueous solution 103 ~;~6~132 of sodium Lydrogenenocarbonate, with water, dried, (sodium sul~ate~, ~iltered and e~aporated. The residue is chromatographed on a silica gel column (20g). Elution by the mixture hexane ethyl acetate (3:2 ~l/v, containing 0.50,~ o~ triethylamine) 5 gives the compound _ in the ~rm of a pure syrup. (30 2mg, 76~. from acetates 34 and 35), ~1C]D = -21 (c: 1, chloroform) ~R (CDC13): ~: 5,52 (d,1H, H--1, J1 2: 3Hz).
Compound_79 : 0-benzyl-1,2-0-tert-butoxyethylidene-~-L-methyl idopyranuronate.
10 A solution of the orthoester 37 obtained in Example 5 in step ~ (484mg, 1,1mM)inanhydrous methanol (15ml) is cooled to -20C with stirring and in a dry argon atmosphere. An-hydrous potassium carbonate (60 mg) is added, and the reaction mixture is stirred 5 h under these conditions. The solids are 15 drained,the filtrate e~tap~ated and the residue taken up again in chloro~orm (50ml). The organic phase is washed with iced water (three times) dried (sodium sulfate), filtered and e~ta~ated. The residue is chromatographed rapidly on a silica gel column (25g). Elution with a mixture hexane: ethyl acetate 20 (2~ , containing O.S/. of triethylamine), gives, in order of elution:
- the unsaturated compound 39 (31mg, 70/.) syrup, [~]D=~103 (C: l? chloroform), ~R (CDC13): S: 6,27 (d.ded.9 1H, H-4, J3 4 :5Hz, J2 4 :1Hz), 5,67 (d, 1H,H--1, J1 2: 4 Hz).
- A principal fraction (271mg, 620,~) which is crystallized in a hexane-ether mixture to gi~e the compound 79 (123mg, 280/.), MP : 68-69 ; ~~]D = -19 (c : 1, chloro~orm), NMR (CDC13) : ~:

. , . ~ . .

104 iL2~5~3;~:

In the course of chromatography on silica~ and during the crystallisation tests ~ 79, a novel compound of Rf slightly higher than that o~ 79 appears. Chromatography on silica gel of the mother liquors from c~ystall~sation of 79 enabled the isola~on o~ some pure fractions of this novel compound 80 (~1 mg, 11/.), syrup, [~]D=~21 (c : 1, chloro~onn), MMR (CDC13) :~ : 5,83 (d, IH, H-1, J1 2 : 4-5 Hz).
In the scope of the succession o~ syntheses envisaged according to the invention, in order to avoid the formation of 80, the crude syrup o 37 is not chromatographed, but used iMmediately for the following reaction:
EXAMPLE 20 : Preparation ~ the disaccharides 81, 82 and 83 (see Figure 17) Compound 81 : Methyl 6-0-benzoyl-3-0-benzyl-2-benzyloxycar-bonyl~nino-2-desoxy-4-0-(2,4-di-0-acetyl-3-0 benzyl- ~-k-methyl idopyranuronyl)- ~-D-glucopyranoside.
A solution of the orthoester 78 (80mg, 0,2mM) obtained in Exarnple 19 and the alcohol77 (52mg, On1mM) obtained in 2~ Example 18 inanhydrous chlorobenzene (8ml) is heated to 140C
with stirring and a slight ~low of dry aryon. A~ter slow distillation o~ 6ml of solvant, a solution of 2,6 dimethyl pyridinium perchlorate (0.002 mM freshly prepared according to N.K. KOCHETKOV,A.F. BOCHKOV, T.A. SOKOkOVSKAIA and V.~.
SNTATKOVA, Carbonhdr. Res., 16 (1971) 17-27, in chlorobenzene (2ml) is added, drop by drop, in 15min. with simultaneous distillation of solvent (2 ml). The reaction mixture is then sti~
red for 1 h, under these conditions, with the addition of fresh solvent (10ml) and siu~ltaneous distillation so that the reaction volwne remains constant and equal to 2 ml.

105 lZ6513;;~:

After cooling and dilu~ion with chloroforrll, the organic phase is washed with a saturated sodium hydrogenocarbonate solution, with water~ dried (sodium sulfate), filtered and evaporated. The residue is chromatographed on a silica gel 5 column (S5g)0 Elution by the mixture hexane: ethyl acetate (4:3,v/v) gives7 in order of elution - the starting substance 77 (20mg, 380/.), - a homogeneous fraction in thin layer chromatography (54mg). The NMR spec~rum of this fraction shows the presence of several 0-methyl ( ~: 3,35-3,50) signals due to the methyl glycosides derived from the rearrangement of the orthoester 78. This fraction is crystalli~ed in an ethanol-water mixture and recrystallised in a ethyl acetate hexane mixture to give 81 (44 mg, 500/.), MP: 120-121C, ~D = +17 15 (c: 1, chloroo~m), NMR (CDC13): in accordance with the expected structure.
Compound 81 : Methyl 6-0-benzoyl-3-0-benzyl-2-benzyloxycar-bonylamino-2-desoxy-4-0-(2-0-acetyl~3-0-benzyl-4-0-chloroacetyl-(X -k~methyl idopyranuronyl)- ~( -~glucopyranoside.
A solution of the orthoester 38 (120mg, 0,25rnM) obtained in Example S and the alcohol 77 (66mg, 0,125mM) in anhydrous chlorobenzene (8ml) is heated to 140C with stirring and a slight flow of dry argon. Ater slow distlllation of 6 ml 25 of solvent~ a solution of 2,6-dimethylpyridinium perchlorate (0.00 25mM) in chlorobenzene is added drop by drop in 15 min.
with simultaneous distillation of solvent ( anl). The reaction mix~ure is s~irred for 1 h and then treated under the conditions described for the preparation aE 81~ The residue is . , :
..

106 ~L2~;5132 \

\

.. ,, ., .. . , .......... , _ _ . _ . . _ _ chromatographed on a column of silica gel (15 g). Elu-tion with the mixture hexane : e:thyl aceta-te (7 : 4, v/v) gives;, in order of elution :
- the product 77 (40 mg, 60~), - the disaccharide 82, crystallized in a ether-hexane mix ture, (26 mg, 30 ~), MP : 143-144C, ~ 7 :D = ~ (c : 1, chloroform), N~MtR. (CDC13) : in accordance wi-th the expected structure.

Compound 83 : 0-dechlGroace-tylation and acetylation of the disaccharide 82.
A mixture of the disaccharide 82 (12 mg) and of thi-ourea (5 mg) in pyridine (1.2 ml) and absolute ethanol (0.3 ml) is stirred a-t 100C for 30 min. After cooling, the reaction mix-ture is evaporated to dryness and the residue is taken up again with a wa-ter-chloroform mixture (1:1, v/v, 20 ml).

,, .

107 12~;5132 The organic phase is washed wi-th water, dried (soaium sulfate), filtered an~ evapora-ted. The residue is washed on a silica gel column (1 g). Elution with the mixture ethyl acetate :
hexane (1:1, v/v) gives the disaccharide 83 (3 mg) in the form of a pure syrup which has not been analysed, but acetylated immediately (pyridine : ace-tic anhydride 2:1, v/v, 1.5 ml).
After 15 h at ambient temperature, the reaction mixture i5 evaported to dryness and the residue is applied to a silica gel column (0.5 g). Elu-tion by the mixture ethyl aceta-te :
hexane (1~ /v) gives the disaccharide ~ (7 mg), crystal-lized in an ether-hexane mixture ~P: 120-120.5C, MP of the mix-ture with 81; 120-121C.
Ex~r~ 21 - Syn-thesis of the trisaccharide 85 (see Figure ~8).

A solution of the bromide 84 (prepared according to H. PAULSEN and W. STENZEL, Chem. Ber 111 (1978) 2334-2347, 110 mg, 0.25 mM) and of the alcohol 41 (prepared according to Example 6, 113 mg, 0.13 mM) in anhydrous dichloromethane (2.5 ml) is s-tirred protected from light under a dry argon atmosphere in the presence of a 4 R molecular sieve (powder 100 mg) for 30 min. After ~ooling to 20C, symcollidine (70 ~l, 055 ml~) and silver triflate (78 mg, 0. 30 mM) were added successively and s-tirring was maintained under these conditions for 2 hours. The reaction mixture was then diluted wi-th dichloromethane (50 ml3, the solids ~ere drained, and the filtrate was washed with 0.1 M solution of iced hydrochloric acid with water, with a saturated auqueous sodium hydrogen-carbonate solu-tion, with water, dried (sodium sulfate), filtered 108 ~ 5~3~

and evapora-ted.
The residue was chromatographed on a silica gel column (18 g). Elution by -the mixture hexane : ethyl acetate (4:3, v/v) gives the trisaccharide 85 in the form of a color-less glass which i-t has not been possible to crystallize (139 mg, 88 %) ; ~ 7D = + 83 (cl, chloroform) ; NMR spectrum (90 Mhz, CDC13) :~ : 7~25 (~ 25H, 5Ph.) ;
5.44 (d. de d., 1H, H'3, J2 3" 10-5 Hz~ J3"~4" 9 Hz) ;
( ~ ~ H1~ J1 ' 2 3 : 3.5 Hz) ; 3.59 (s, 3H,COOMe) ;
3006 (d. de d-, 1H~ H2~ J1 , 2 3 ' 2 3 2.12, 2.08, 2.01 and 12.97 (4s, 12H, 4 OAc).
EXAMP~E 22 - Syn-thesis of the trisaccharide _ (see Figure 18).
By the four following steps :
a) removal of the acetyl groups, b) sulfation, c) h~drogenation, d) sulfation of -the amino functions a) Removal of the methyl groups leading to the trisaccharide 89 :
2~ A solution of trisaccharide 85 (122 mg) in a mixture of 1,2-dimethoxyethane (6 ml) and me-thanol (2 ml) is stirred a-t 0C. An aqueous 1 M solution of sod~ ( 2ml) is added drop by drop in 10 min and the reaction mixture is stirred 6 hours at 0C. 1M hydrochloric acid is then added drop by drop until pH - O (appearance of a white precipitate). The mixture is poured into ice water (100 ml) and extracted with chloro-form (5 tlmes 10 ml). The organic phases are washed with log ~L2ÇiS~L3X

ice water, dried (sodium sulfa-te), filtered and evaporated.
The syrupy residue is dissolved in methanol (2 ml) and trea-ted v~i-th an ~ther solution o~ diazomethane until persistance of the yellow color. Af'ter 30 min., -the reaction mixture is evaporated to dryness. The residue is chromatographed on a silica gel column (10 g). Elution by the mixture e'thyl acetate/hexane (2:1, v/v) gives -the trisaccharide 86 in the form of a color-less foam which i-t has not been possible to crys-tallize (85 mg 81 ~o) ; r~7D = -~ 77 (cl, chlDroform), ~ spectrum (90 rl~I
10 CDCl3) : absence of O~c signals (towards ~ = 2 ppm).
Elementary analysis : in accordance wi-th the struc-ture sought.
b) Sulfation leading to the trisaccharide 87 To a solution of the derivative 86 (41 mg) in D~ ( 2ml) is added the complex trimethylamine/sul~ur tri-oxide (TrL~/S03 ; 60 mg ; 2.5 equivalents per OH).
~fter one night at 50C, the reaction is comple-te. Methanol ~0.5 ml) is added and -then the solution is placsd on a Sepha-dex LH-20 column (1.5 x 25 cm) equilibrated in a chloroform/
me-thanol mix-ture (1:1 ; v/v). ~lution by the same mixture enables the product of the reaction to be separated from the excess of reagent and from the reaction solvent. The residue obtained is '~hromatographed on a silica gel column (10 g), eluted by an ethyl ace-ta-te/pyridine/ acetic acid/water mixture (98 : 56 : 13 : 32 ; v/v/v/v). The pure product obtained is dissolved in methanol, then passed through a Dowex 50~r~ x 4, Na (5 ml) resin ¢olumn. ~fteb evaporation and drying, the derivative 87 (58 mg, 100 %) is obtained. It is homogeneous . .

1 10 ~513i~

in tlc (ethyl ace-tate/pyridine/acetic acid/water ; 5 : 5 : 1 :

3 ; v/v/v/v a~d ethyl aceta-te/methanol/ace-tic acid ; 7 : 3 :
0 . 1: v/v/v ) .
r~7D20 = + 55 (methanol). The N~ spectrum is compa-tible with the structure sought c) Hydrogena-tlon leading -to the trisaccharide 88.
A solu-tion of -the compound 87 (20 mg) in a mixture of me-thanol (2 ml) and wa-ter (0.5 ml) is stirred for 96 hours at a hydrogen pressure of 0.2 bar, in the presence of 5 c,~
Pd/C (20 mg). The catalyst is -then removed by filtration.
Ultra-viole-t analysis confi~ms the absence of aromatic nuclei.
After evaporation, the product is employed in the synthesis of the trisaccharide 89.
d) Sulfa-tion leading to -the -trisaccharide 89.
The derivative 88 ob-tained previously is dissolved in water (2 ml). The pH of the solution is adjusted to 9.5 then it is kept at this value by means of a pH-stat~ The complex TMA/S03 (14 mg ; 5 eq./NH2). Af-ter one night, the same amount of complex is added. After 48 hours, the pH is brought to 12 20 by means of 2r~ soda, then it is kept a-t this value for 2 hours.
After neu-tralization with hydrochloric acid, the reaction mix-ture is chroma-tographed on a Sephadex G-25 c~lumn, eluted with water. The compound 89 is detected by a color reaction with carbazole, characteristic of uronic acids (Bitter et 25 Muir, Anal. ~iochem. 4 (1962) 330-334). These frac-tions con-taining 89 are grouped -toge-ther and passed through a Dowex resin column 50W x 47 Na+ elu-ted with water. After freeze-11 1 ~265~3;~

drying, 89 is obtained (4.5 mg).
Colorime-tric analysis of the glucide constituents gives 2.55 mole..of uronic acid per 5.15 moles of glucosamine (ratio 1/2).
The Nr~ spec-trum of this product confirms the struc-ture (sequence, anornerism of the linkages, substitu-tions by sulfates).
~XA~E 23 : Sythesis of the disaccharlde 92 of the formula :
CoO~e _ BnO ¦ ¦
O~n N3 (92) , . . ., .,, , , , . _ . _ . .
Reference will be made to Figure 19.
The produc-t 91 (1 g), in solution in dichlorome-thane (50 ml), is stirred in the presence of drierite (6 ~) and freshly prepared silver carbonate (4.5 g), for 1 hour in an argon atmosphere. Then the halide 90 (2.8 g) dissolved in dichloromethane (10 ml) is a.dded, A-Ster 1 1/2 hours 2.8 g o~
the halide 90 is again added. After one night ~the solids are removed by fil-tration.and the residue ob-tained after evaporation of the solven-ts is purified on a silica column in the solvent ethyl acetate/chloroform (1/30 ; v/v).
In this way the product 92 is obtained (866 mg ;
yield 42 /~). It is crys-tallised in a h~xane/e-thyl aceta-te mixture.

6~L32 MP : 10~-106C ; ~ jD20 _ 0 ( C=1 ; chloroform).
Elementary analysis and the N~R spectrum are in agree~en-t with -the desired structure.
EXAMP~E 24 : Synthesis of the derivative 94 of the formula :
S fOOM~ G
~! ~CY
BnO
(94) N3 Reference will be made to Figures19 and 20.
The derivative 92 (1.5 g) is dissolved in a mixture of chloroform and methanol (1/1 ; v/v). Then 2 ml of sodium methanolate is added (2M in methanol). After 20 minutes, the solution is neutralized by addition of Dowex 50 resin leading to the deriva-tive 93 which is not isolated. After filtration and evaporation, conventional methylation by diazomethane in ether enables the frac-tion of carboxylic acid possibly lib-erated to be reesterified. After evaporation the residue is treated with a mix-ture of pyridine (20 ml) and acetic anhy-dride (2 ml) over night. After evaporation, the residue is crystallized in ethyl acetate/hexane giving the product 94 (1.125 g ; yield 81.6 %).
M.P. : 103- 105 C ; ~ 7D20 = + 5.2 (c = 1 ;
chloroform).
Elementary analysis and the NMR spec-trum are in agreement wi-th the desired structure.
As a modification, the derivative 94 is prepared . ., 113 ~L265~32 by operating as described above but by employing the deriva-tive 95 ins-tead of -the deriva-tive 91.
EXAMP~E ?~: Synthesis of the derivative 97 (see Figure 20).
In a firs-tstep, opening of the anhydride bridge is carried out;then in a following s-tep, a bromination reaction is carried out.
1 : Opening of the 1,6-anhydro bridge.
The compound 94 (1 g) is dissolved in acetic an-hydride (10 ml) then cooled -to -20C under argon. To the cold solution is added concen-trated sulfuric acid (100 ~l). After 30 minutes, the reaction mix-ture is diluted with chloroform (150 ml) then poured into an aqueous sodium bicarbonate solution (26.5 g in 400 ml). Af-ter the release of gas -the chloroform phase is washed twice with a saturated solution of NaCl then dried and concen-trated. Af-ter chromatography on silica (50 g) in a mixture of ethyl aceta-te and chloroform 1/20 v/v, the compou~ 96 is ob-tained (995 mg ; yield 86.7 %).
rrhis compound is in the form of a white foam.
The spectrum and elementary analysis confirm the production of the desired structure.
2 : Bromination .
To titanium tetrabromide (233 mg) is added a solu-tion of the derivative 96 (0.2 g) in dichloromethane/ethyl acetate (9/1 ;v/v 4 ml). After one night with stirring ~ollowed by dilution with dichloromethane, it is poured on-to a mixture of water and ice (50 ml), and then washed with two times 50 ml of ice water. After drying and evaporation the syrup obtained is chromatographed on silica in the solvent ethyl ace-tate/chloro-2~i5~32 form 1/20 ; v/v. In this way the derivative 97 is obtained wi-th a yield of 25 to 50 ~o.
NMR spectrum : (ppm, CDCl3) : 2.04 ; ?~
2 single-ts of 3 protons 2-OAc ; 3.7 : 1 single-t of / protons COOMe ; 6.33 : 1 doublet bf 1 prot~n H1 ; J1~2 3 5 EX~MPLE 26 : Syn-thesis of the -te-trasaccharide 98 (see Figure 20 20).
A solution of bromide 97 (50 mg, 60 M) and alcohol 41 prepared by 13xamp]e 6 (43 mg, 50 ~m) in anhydrous di-chlorome-thane (1 ml) is stirred protected from light in a dry argnn atrnosph~re in the presence of a molecular sieve 4 R(powde~, 100 mg) for 15 minu-tes. After cooling to -10C, sym-collidine (11 ~ , 80 ~M) and silver -trifluoromethanesul-fonate is added ( Ag triflate, 18 mg, 70 ~M) successively, and stirring is maintained under -these conditions for 3 hours.
The reaction mix-ture is then dilu-ted with dichlorome-thane (30 ml), the solids are drained, and -the filtrate iB washed with an 0.1 M solution of iced hydrochloric acid, wi-th wa-ter, with a saturated aqueous solu-tion of sodium h~rogencarbonate, with water, dried (sodium sulfate), filtered and evaporated.
The residue is chromatographed on a silica gel column (7 g). Elution by the mixture hexane-ethyl acetate (4 : 3, v/v) gives 56 mg of te-trasaccharide 98 (yield 70 %) in the form of a colorless glass which it has no-t been pos-sible to crystallize.
Characteristics of the NMR spectrum :

11 5 ~ 53L32 (~70 l~lHz~ CDCl3) : S: 7.25 (~ 35 H, 7 Ph) 5.35 (d.ded., 1 ~I, H3 J 2 ~ 3 ~Iz~ J3 ' 4 5 27 (d., 1H,~I1 J1"' 2" : 3.5H) ; 5.31 (d., 1H, H1 J1 2 7.5 Ilz) ;
5 3.68 (s, 3H, COO~e I~O); 3.59 (s, 3H, COOMe gluco);3.37 (d.de d., 1H, H2 J'1~ 2" 7-5 Hz, J2"' 3"~ 9DSHz) 3.18 (d.de d., 1H, Il'2, J1~ 2~ 3 5 Hz, J2~ 3": 11Hz);
~.06 ~ND 1.97 (2s, 9 and 3 H, 4 OAc).

This spectrum is reported in ~igure 33.
10 EX~IPI,E 27 - Synthesis of the tetrasaccharide 99 (see Figure 21).
A solu tion of -tetrasacch~ride 9~3 (28 mg) in anhydrous methanol (3 ml) is cooled to -15C under a dry argon atmosphere.
Anhydrous po-tassium carbonate (12 mg) is added and the mix-ture 15 is stirred 6 hours under these condi-tions. The solids are then drained, the Eil-tra-te was evapora-ted and the residue is taken up again with chloroform (15 ml). The organic phase is washed with a ~aturated aqueous solution of sodium chloride, washed with water, dried (sodium sulfate), filtered and evaporated.
~0 The residue is chromatographed on a colun~l gel (2 g). Elution with ethyl acetate/hexane (3~2, v/v) gives the tetrasaccharide 99 in the form oE a colorless glass (22 mg, 85 ~/0).
NMR spectrum (270 MHfz~ CDCl3): S 7.30 (m, 35H, 7Ph);
5.37 (d, 1H~ J1 ' 2 3 5 5.29 ( d. de d., 1H~ H3~ J2~ 3 3 4 5.09 (d, 1H, ~I1' J1 2: 3.5 Hz) 3.57 (s,3H, COO~Iie ido) 3.43 (s, 3H, COOMe gluco) ; 2.06 (s, 3H, OAc), 116 ~ i5132 This compound 99, which is a derivative mono-O-acetylated ~t the 3 position on the 2nd unit~ of the tetrasaccharide 103 is a potential intermediatefor the synthesis o~ an analogue o~ this tetrasaccharide which will not be sulfated at the 3 position o~ the second unit.
EXAJ~kE 28 - Synthesis of the tetrasaccharide 103 (see Figure 21) The following steps a) to d) are resorted to :
a) removal ~ the acetyl groups, b) sul~ation, c) hydrogenation, d) sul~ation o~ the amino groups, a) removal of the acetyl groups resulting in the derivative 103 :
A solution of the tetrasaccharide 98 (40mg) in a mixture of 1,2-dimethoxyethane (3 ml) and methanol (1 ml) is cooled to -15C. An M aqueous solution o~ soda (1 ml) is added drop by drop in 1~nin.;the reaction mixture is stirred 5 hours at 0C. M hydrochloric acid is then added drop by drop to pH = O and the mixture is poured into iced water (50 ml)O
After extraction with chkroform (5 times 5 ml), the organic phases are washed with water, dried (sodium sulfate) filtered and evaporated.
The residue is dissolved in methanol (1 ml) and treated with an ether solution ~ diazomethane until persistance of the yellow colour. A~ter 30min. the reaction mixture is evaporated to dryness. The residue is chromatographed on a silica gel column (3 g). Elution by the mixture ethyl acetate/
hexane (2 :1, v/v) gives the tetrasaccharide 100 (27 mg, 75~) ;
MP 125-127C (ethanol) ; ~] D= + 55 (cl, chloro~orm).
NM~ spectr~l (90 MHz, CDC13) : total absence o~ signals.OAc (towards ~ =2).

l 17 126~1;3Z

Elerllentary analysis : in accordance with the desired structure.
b) Sulfation leading to the derivative 101.
To a solution o~ the derivative 100 (2.4 mg) in DMF (1 ml) is added the complex TMA/S03 (24 mg.). After one night at 500C., the sulfation reaction is complete.
Methanol (0.5 ml) is added to the reaction mixture and then the latter is deposited on a Sephadex LH-20 column equilibrated in chloroform methanol (1:1, v/v). The fractions containing 101 are grouped together. After evaporation to dryness, the residue is chromatographed on silica gel (10 g) in the mixture ethyl acetate/pyridine/acetic acid/water (160:77:19:42 ; v/v~v/v). The~pure ~ractions are grouped together. After concentrating to dryness, the residue is passed through a Dowex 50W x 4, Na+ column eluted with water.
The product obtained (30 mg) i5 homogeneous in thin layer chromatography in the above solvent. Its NMR spectrum confirms the structure. ~]D0 = + 39 (1, methanol)~
c) Hydrogenation leading to the derivative 102.
cO A solution of the derivative 101 ( 10 mg) in a mixture of rrle-thanol (1.8 ml) and water (0.2 ml) is stirred under hydrogen pressure of 0.2 bar in the presence of 50/, Pd/C
(10 mg~. After 96 hours, the catalyst is removed by filtration.
Ultra-Violet analysis confirms the absence of aromatic rings.
After evaporation, the derivative 102 is used as such for the preparation of the derivative 103.
d) Sulfation leading to the derivative 103.
The derivative 102 obtained in the preceeding step, ~;~65~32 is dissolved in water (2 ml). The pH of this solution is ajusted to 9.5; it is kept to this value through-out the sulfation. rhe complex TMA/S03 (14 mg) is addedO A second addition is made after 24 hours (14 mg). After 48 hours7 5 the pH is brought to 12, then to 7, two hours later. The reaction rnix-ture is then chromatographed on a Sephadex G-25 column (50 ml). The fractions containing the derivative 103 (detection by colour reaction of the uronic acids) are grouped together, passed through a Dowex 50 W x 4 Na~ resin 10 column then lyophilised. In this way the tetrasaccharide 103 (2 mg) is obtained.
Colorimetric analysis of the constituents of the deri~1ati~e 103 gives 1.84 moles of glucosamine for 2 06 uronic acid moles.
The structure of derivative 103 (sequence, anomerism, position of the sulfate groups) is confirmed by the NMR
spectrum (270 MHz, TMS) : ~ for the anomeric protons respectively of the 1st, 2nd, 3rd and 4th units, 4.72; 5.30; 5.55; and 5.67.
20 EXAMPLE 29.
Synthesis of the monosaccharide 115 (see Figure 22).
This synthesis is carried out by the following steps 1 to 7.
Step 1: synthesis of the monosaccharide 105.
This monosaccharide is prepared from the compound 104 obtained by the technique of N.L Holder and B. Fraser-Reid, Canadian Journal of Chemistry, 51 (1973) page 3357. To a solution of the compound 104 (1 g, 12.67 mM) in dichloromethane (20 ml), is added tosyl chloride (0.55 g), then dimethyllamino-pyridine (16 mg) and finally triethylamine (0.7 ml). After .

~;~65~X 1 19 stirring un~er a flow of nitrogen protected from moisture, for about 14 ~ours, the reaction is stoped by the addition o ice and wa-ter~ ~fter dilution of the reaction mixture with dichloromethane (50 ml), the dichloromethane phase is washed with 2M hydrochloric acid, then a saturated solution of sodium bicarbona-te, and finally with water until pH neutral. After drying and evaporationt a residue is obtained, namely the derivative 105 (1.4 g, g7 o/.) which is used as such in the synthesis of the derivative 106.
Ste~ 2 : SYnthesis of the derivative 106.
.
The monosaccharide 105 (31.8 g) and sodium iodide (39 g) are dissolved in acetQnitrile (250 ml), th~ the solution is brought to reflux for 3 hours. Ater cooling the reaction mixture, the white precipitate Pormed is filtered.
The filtrateisconcentrated, the residue is taken up again with chloroorm, then the chloroform phase is washed with water until pH neutral, dried over sodium sulate and concentrated to dryness. A syrup is obtained which is chromatographed on a column of silica gel (200 g, ether-hexane, D/1, v/v). In this way the iodised derivative is obtained (24.7 g, 71.5 %)- [~ 20 = 24 (1, chlorofo~n).
The inra-red spectrurn, the NM~ spectrwn a~d elementary analysis conirm the structure of 106.
Step 3 synthesis o the derivative 107.
.

To a solution o the derivati~e 106 in anhydrous pyridine (200 ml), is added acetic anhydride (43 ml).
Ater about 14 hours stirring, the reaction is te~ninated The reaction mixture is concentrated to dryness,then the residue ~, 120 ~2 ~5~ 32 is purified on a silica gel column, under pressure, in an ethyl acetate/hexane solvent (1/6, v/~). The pure fractions are grouped together. In this way the product 107 is obtained (16.4 g, 700/,). This product is in the Porm of a sy~lp. [~3 D20 ~ + 4-5 (1.3, chloroform) Elementary analysis as well as analysis of the infrared spectrum confirm the structure.
Step 4 : sy nthesis of the derivative 108.
To a solution of the deri~ative 107 (4 g) in pyridine (100 ml), cooled to 0C, is added silver fluoride (AgF, ~.9 g). After two and one hal hours, the reaction ùixture is poured into a mixture containing chloroform and ether (1/4, v/v, 1 l). The suspension obtained is passed through a folded filter. The iltrateis concentrated to dryness, then the residue is taken up again with chloroform (500 ml). The chloroform phase is washed with acid potassIum sulfate in 10/. solution in water, then with water until pH neutral. After drying over sodium sulfate and concentration to dryness, a residue is obtained (2.7 g), which is chromato-2~ graphed on a silica column (200 g) (eluent : ethyl acetate-hexane, 1/4, v/v. The fractions containing the product 108 are grouped together and ater evaporation of the sol~ents, a crystalline product is obtained (1.62 g, 54 /.).
MP : 81-82C, ~]25 = ~ 2~ (1, chloroform).
Analysis o the infrar~d spectrum, elementary analysis and analysis of the nuclear magnetic resonnance spectrum confirm the structure of the compound 108.

12~13;~

Step 5 : synthesis of the derivative 109.
Product 108 (2 g) is dissolved in methanol (20 ml) and chloroform (20 ml). To this solution, is added sodium methanolate (2 M, 2 ml). Af-ter 1.5 hour, the de-acetylation reaction is terminated. The reaction mixture is diluted with chloroform. The chloroform phase is washed with water until pH neutral, dried, then evaporated to drynessO In this way a residue i5 obtained, the compound 108 (1.8 g, 100 ~)0 It is immedi~ely dissolved in tetrahydrofurane (50 ml), then borum hydride (BH3, 1M) in tetrahydrofurane ; (10 ml) is then added. After one hour of reaction, the excess bor~n hydride is destroyed by the addition of ethanol. At the end of gaseous release, the reaction mixture is diluted by the addition ~ tetrahydrofurane (100 ml). 3 M soda (12 ml) is then added, ~ollowed by hydrogen peroxide (120 ~olumes, 8 ml).
After two hours heating to 50C, the reaction is stopped.
The solution is poured into chloroform (500 ml)~ then the organic phase so obtained is washed with water, with 2 M
hydrochloric acid, finally with water until pH neutral.
In this way a very milky chloroform phase is obtained which becomes limpid in the course of drying o~er sodium sulfate. After filtration, the chloroform is e~aporated and then the residue obtained is chromatographed on silica (200 g chloroform-methanol, 30/1, ~/v).
In this way the derivative o~ the idose 109 (1.05 g, 55 o/~) is obtained. This product is in the form of a syrup.
~] ZQ = ~ 85.5 (1, chloroform).

~265~3~

Elementary analysis as well as NMR ~n~lysis con~irm the expected structure.
Step_6 : Synthesis of the derivative 112.
This synthesis is carried out frorn derivative 109 in a single step (the intermediates 110 and 111 are not isolated). To a solution o~ derivative 109 (2.25 g, ~ mM) in dichloromethane (50 ml), are add~d succesively dimethylaminopyridine (60 mg ; 0.24 mM) triethylamine (1.7 ml ; 12 mM) and trityl chloride (2.5 g ; 9 mM).
After about 14 hours, the reaction is terminated~ In this way the deri~ative 110 is obtained in solu~ion. Then to the reaction mixture dimethylarninopyridine (150 mg), is added and triethylamine (1.7 ml) and then benzoyl chloride (1.05 ml).
After 6 days, dichloromethane is removed by passage of a current of nitrogen and replaced by dime-thylformamide (40 ml).
The reaction mixture is heated to 70C during one night.
Then benzoyl chloride (1 ml) and triethylamine (1.7 ml) are again added, and heating is then maintained at 70C
for two days. Dimethylformamide is then evaporated, then the residue is taken up again with chloroform,the chloroform phase is washed with water, with a saturated sodiwn bicarbonate solution, then with a~2M hydrochloric acid solutlon, and finally with water until pH neutral. After drying, the chloroform is evaporated, which pe~nits the compound 111 to be obtained.
The latter is immediately subjected to a reaction to eliminate the trityl group in order to obtai~ the derivative 112. The residue containing the derivative 111 is dissolved in 25 ml chloroform and to this solution is 65~

added 10 mlo~aparatoluenesul~onic acid monohydrate solution in methanol (1 M). ~fter 4 hours of reaction at room temperature, the reaction is terminatedO The reaction mixture is then diluted with chloroform, washed with water, dried and then evaporated to dryness. The residue obtained is chromatographed on silica gel (200 g, ether-hexane, 3/1, v/v). The derivative ~1-2 is thus obtained in a pure state (1.5 g ; 52 /.). This derivative is in the form o~ a syrup ~ Do = _ 8 (1, chloroform`
Analysis o~ the in~rared spectrum and o~ the MMR spectrum con~rm the structure oP the expected product.
Step 7 : synthesis o~ the compound 115.
This synthesis is carried out directly Prom the derivative 112 without isolating the intermediates 113 and 114.
To a solution oP the compound 112 (1.2 ~) in acetone (20 ml), 15 is added, drop by drop, after cooling to-0C, a solution (2.9 ml) of chromiwn oxide (CrO3; 1.17 g) in sulfuric acid 3.5 M (5 ml). After 30 minutes stirring at 0C, the temperature is brought back to room temperature. Reaction develops over three hours. The reaction mixure is then poured into a separating ~ ~unnel containing iced water (100 ml). The product formed is extracted with chloro~or~ (3 x 50 ml). The chloroform phase is washed with water until pH neutral, then dried over sodium sulfate, filtered and concentrated to dryness. The residue obtained (compound 113) is dissolved in methanol (130 ml).
To this solut1on soda 3 M (17 ml) is added then the mixture is left under stirring for about 14 hours. After acidi~ication with sulfuric acid, the compound 114 is extracted with ether, then immediately methylated with diazomethane by the conven~iona 124 ~65~Z

method to give the compound 115. After evaporation of the ether, the compound 115 is ~tained ~ure by mean of silica gel chromatography (50 g ; ether-hexane ; 4/1 ; ~/~). The pure fractions containing the derivative 115 are grouped together and the solvents are rem~ved. In this way the derivati~e 115 of iduronic acid is obtained (587 mg, 59 o/. with respect to derivative 112). This product is in the form of a syrup.
~]25 ~ ~98 (2.65, chloroform).
NMR analysis, infrared analysis and elernentary analysis confirm the expected structure.
EXAMPLE 30.
SynthesiS of the disaccharide 117 (see Figure 22 and 23).
This synthesis is carried out from the monosaccharde 115 prepared as above and from the monosaccharide 44 prepared by the technique o H. Paulsen and W. Stenzel, chemische Berichte 111 (1978) 2234-2247.
To a solution of the compound 115 (200 mg, 0.5 mM) in dichloromethane (10 ml), are added successively the compound 44 (0.450 g) sym-collidine (150 ml) and silver triflate (260 mg.) The reaction mixture is kept at 0C under a nitrogen flow and with stirring protected from moisture and from light for 3 hours.
It is then diluted with dichloro ethane (100 ml) then the solids are eliminated by filtration on pleated ilters. The solution obtained is washed with a saturated solution of sodium bicarbonate with water and with 2 M sulfuric acid, then again with wa~er until pH neutral.

125 ~i51;~2 APter drying over sodium sulfate and evaporation of the dichloromethane, the residue obtained is chromatographed on silica gel (50 g ; chloroform/ethyl acetate ; 15/1 ; ~/v).
In this way the pure derivative 117 is obtained (327 mg, 82 /.). The product is in the form of a syrup.
~ ] 20 = ~ 57 (1, chloroform).
NMR analysis the same way as elementary analysis confirm the structure and the anomerism o~ the disaccharide 117.
EXAMPLE 31.
Synthesis of the disaccharide 122 (see Figure 23)o The o'lowing ~eps are applied :
- rernoval of the acetyl groups, - sulfation~
- hydrogenation, _ sulfation oP the primary amine group.
- removal o the Me group from the -COOH radical resulting in the compound 118.
The disaccharide 117 (260 mg) is dissolved in methanol (5 ml)and 1 M soda (1 ml) is added drop by drop. At the end ~ of the reaction, the reaction mixture is introduced to the top of a Dowex 50 resin colwnn in the HT form ( 5 ml). The effluent is concentrated to dryness, taken up again with methanol, and the free acid product, obtained at the end of the saponific-ation of the deri~ati~e 117 is methylated by the addition of diazomethane. In this way the deri~ati~e 118 is obtained which is puri~ied by mean of a silica gel column (20 g ; ether/ hexane8/1 ; v/~). The yield of compound 118 is 92 mg. This product is engaged directly in the synthesis ~ deri~ative 119.

:

126 12~ 3~

- Sulfation leading to ~he disaccharide 119.
The product 118 obtained above (92 mg) is dissolved in dimethylformamide (5 ml) then trimethylamine/sul~ur trioxide complex (25 mg) is added. The solution is brought to 50C
for about 14 hours. After evaporation to dryness, the residue is taken up again with chloroform, then the chloroform phase is washed with water, dried a~d concentrated to dryness. The solid obtained is purified on a silica gel column (15 g ; eluent :
methanol/ chloroorm ; 1/4 v/v). After evaporation o~ the pure fractions, the sulfated disaccharide 119 is obtained (58 mg ;
55.6 /.).
- Hydrogenation leading to the disaccharide 120.
The disaccharide 119 (58 mg) is dissolved in a methanol-water mixture (15 ml ~ 2 ml). Then catalyst (Pd/C 5 ~. 60 mg) is added and this suspension is then subjected to stirring under a hydrogen atmosphere f`or 48 hours. At this stage, the complete disappearance of the benzyl groups borne by the derivative 119 is noted,inthesam~way as reduction ~ the azide group of the derivative 119 to an amino group. The catalyst is removed by filtration, then the reaction mixture is concentrated to dryness.
In this way the disaccharide 120 iscbtained which will be treated directly to obtain the product 121 and 122.
- Sulfation of the group -NH2 leading to the disaccharide 122.
The disaccharide 120 is dissolved in water (6 ml).
To this solution the complex trimethylamine/sulfur trioxide (25 mg), is added, whilst keeping the pH at 9.5 by addition of soda (0.1 N). After ~5 hours reaction 1 N soda is added to bring the pH to 12. Then it is kept to this value for one h~ur.

127 ~L2~ Z

The solution of 121 is then neutralized with 1 N hydrochloric acid, then passed to a Dowex 50 colw;ln (5 ml) in the Na+ fo~n.
The eluate from this column is introduced into a column G1 x 2 (16 ml, 1.6 x 8 cm). The column is eluted with a gradient of 5 sodiwll chloride of O to 3 M. The fractions containing the disaccharide 122 in the fo~l of sodiwn salts are collected together, concentrated, then the product is desalted by passage o~er a Sephadex G25 colwnn (50 ml) eluted with water. In this way the disaccharide 122 is obtained (27 mg, 680/.). Apres 10 lyophilisation, the product is in the form of a white powder.
[~]DO = + 95.5 (1.3; water).
The NMR analysis ~ carbon 13 confirm the exprected st~ucture for the product 122.

EXAMPLE 3 2 : Preparation of the 2-0=(d-k-idoPyranosyl)-D-galactose (compound 128) (see Figure 24)~
This synthesis is carried out by the following 4 steps a) to d)-a) Preparation of 2,3,4,6-tetra-0-acetyl~O(-L- idopyranosyl bromide (compound 124) 20 A solution of 5 g of penta-O-acetyl- D( -~k idopyranose (compound 19 prepared according to P. PERCH~lLIDES, T.OSA~7A,E.A DAVIDSON
and R.W. JEANkOZ, Carbohydr.Res.,3 (1967) 463), in anhydrous dichloromethane (100 ml) was saturated at 0C with hydrobromic gas. After 2 hours at room temperature, the reaction rî;edium was 25 poured on to ice and extracted with chloroform. The organic phase was washed with water, dried (calcium chloride) and evaporated. The residue was crystallized in the dichloroethane-ether-pentane, mixture giving the bromide 124(5 g, 95 o~., MP

126-127 C ~(] D = ~ 120 (c; 0.75, chloroform).

~;5~32 /~g b) Preparation o~ benzyl 3,4,6-tri-0-benzyl-2-0_(2,3?4,6-tetra-0-acetyl- 0C-L-idopyronosyl)-/3 -D-galactopyranoside (compound 126) A solution of 200 mg of benzyl-3,4,6-tri-0-benzyl-f~ -D-5 galactopyranoside (compound 125 prepared by the method ofJ.C. JACQUINET and P. SINAY described in Tetrahedron, 32 (1976) 1963) in anhydrous dichloromethane (10 ml) was stirred under dry nitrogen at 91~C in the presence of a molecular sieVe 4 A (300 mg) and mercuric bromide (80 mg), until the Volume was reduced to hal. A solution of bromide 124 (300 mg) in dichloroethane (10 ml) was added over 3 hours, the volume o~ the reaction mixture being kept constant by a continuous distillation of the dichloroethane. 3 hours a~ter the end of the addition, the reaction medium was cooled to ambient temperature, 15 diluted with chloro~orrn (100 ml), ~iltered, washed successively with an aqueous solution 10C/. of potassiurn lodide, with a dilute aqueous solution of sodium hydrogenocarbonate, with water, dried (sodiurn sulfate) and evaporated. The residue is puri~ied by chromatography on a silica gel colwlln (30 g) 20 by means o~ a mixture ethyl-hexane acetate (1 : 1, v/~t), giving the disaccharide 126 in the state of a syrup (290 mg, 90/)~ ~(] D = 57 (c: 1,3 chloroform).

Analysis calculated ~o~ C48H54015 ..
0,27,55. Found: C: 65,98; H, 6,13; 0~27, 35/..

c) Preparation of benzyl-3~4~6-tri-0-benzyl-2-0-(o~-L-=

idopyranosyl)-~-D- galactopyranoside (cornpound 127) The disaccharide 126 (200 mg) was dissolved in anhydrous methanol (100 ml) and a ~I solution of sodium methylate in anhydrous methanol (0.2ml) was added. After one hour, the ~Z65 /~ ~
reaction mediurn was neutralised by means of Dowex 50 (H+) resin, filtere;}!ed and evaporated. The residue was purified by chromatography on a silica gel column (10 g) by means oP the mixture dichloroethane-acetone (7:3, v/~) giving the disaccharide _ (154 mg, 950/.) in a pure state, [~ D=-43 (c : 1,4 chloroPorm).
Analysis -~alculated for C40H460~
O, 25.04; ~ound: C, 68.38; H, 6.64; O, 25~27/.).
d) Preparation oP 2-0-( -k-idopyranosyl)-D-giLactose =

(compound 128) The disaccharide 127 (200 mg) was hydrogenated ~or 48 hours in ethanol (10 ml) containing acetic acid (0.1 ml), in the presence oP 10/. palladiwn on charcoal (50 mg) The product was puriPied by chromatography on a silica gel colurnn (15 g), by means oP the mixture methanol-chloroform (4:1, ~ t), giving the Pree disaccharide 6 in the Porm of a hygroscopic white powder (97.5 mg, 1000/o)~ ~]D=~280 (c: 1,2, methanol) ~ 15 (10min) ~ ~ 93 (2 h) (c 1.2 water) Analysis calculated for C12H22011:
found: C, 41.71; H, 6.480/
EX~:IPLE 33 : Synthesis oP 1,2 . 3,4-di-0-isopropylidene -6-O (~-L-idopyranosyl)-~-D~galacto pyranose (aompound 131) Firstly the 1,2: 3,4-di-0-isopropylidene-6-0 (2,3~4,6-25 tetra-C acetyl-O(-k~idOpyronosyl~-~(-D-galactopyranose (~compound 130) was prepared.
A solution of 300 mg oP 1,2 : 3,4-di-0-isopropylidene~ D-galactopyranose (compound 129) prepared according to R.C. HOCKET~

130 1;~:651;3Z

H.G. FLETCHER and J.B. AMES in J. Am. Chem. Soc., 63 (1941) 2516? in anhydrous dichLoethane (20 ml) was stirred under dry nitrogen at 90~C in the presence o~ a niolecular sieve 4 A (500 rng) and mercuric bromide (300 mg), until the volunie 5 was reduced to half. A solution of bromide 124 (550 mg) in dichloroe-thane (10 ml) was added; and after 24 hours, a ~urther addition of bromide 124 (125 mg) in dichloroethane (2ml).
24 h aÇter the latter addition, the reaction medium is treated as described above for the preparation of the disaccharide 126.
10 Purification by chromatography on a silica gel column (50 g) by means o~ the mixture dichloroethane-acetone (9:1,v/v) leads to the disaccharide 130, which is crystallized in the mixture dichloroethane/pentane (650 mg, 950~.), MP 160-161~C, [~C]D= -86 (c : 1, chloro~o~n).
Analysis calculated Çor C26H38015: C, 52.88; H, 6.48;
0, 40.6~.
Found : C, 52.89 ; H, 6.41 ; 0, 40.630/..
The deri~/ative 130 is then applied for the preparation of 1,2: 3,4-di-0-isopropylidene-6-0 (oC -k-idopyranosyl-o~ -20 D-galactopyranose (compound 131), by proceeding as Çollows:
The disaccharide 130 (300 mg)is desacetylated by the technique previously described Çor the preparation of~ the disaccharide.
PuriÇication by chromatography on a silica gel colunmn (15 n g) by means oÇ the mixture ~ethanol-chloro~orm(4:1, v/v) leads 25 to the disaccharide 131 obtained in the Çorm o~ an amorphous hygroscopic pov~der (204 mg, 95,!), [D~]D = - 63 (c: 0,7, methanol ) .
Analysis calculated Çor C18H30011: C, 51.18; H, 7.16;

O, 41.66.
~ound: C, 50.86 (see Figures 24 and 25).
EXA~lPkE 34 : Preparation of benzyl-2~3,4-tri-0-benzyl-6-0-( ~C-k-idopyranosyl)-~-D-galactopyranoside (compound 134) First benzyl-2,3,4-tri-0-benzyl-6-0 (2,3,4,6-tetra-0-acetyl-~(- L-idopyranosyl)-p-D-galactopyranoside (compound 133) was prepared.
A solution of 200 mg oP benzyl -2,3,4-tri-0-benzyl-10 ~-D-galactopyranoside (compound 132, prepared according to K. MIYAT and R.W. JE~NLOZ, Carbohydr. Res., 21 (1972) 45), in anhydrous dichloroethane (15 ml) was stirred under dry nitrogen 90C in the presence of a 4 A molecular sie~re (300 mg) and rrlercuric bromide (80 mg), until the volume was reduced to 15 5 ml. A solution of bromide 124 (160 mg) in dichloroethane (10 ml) was added and the reaction medium was stirred at 90C
for 24 h. Treatment similar to that described previously for the preparation of the disaccharide 126 resulted in a residue which was purified by chromatography on a silica gel column (30 g) by means o~ the mixture dichloethane-acetone (12:1,v/v), giving the disaccharide 133 (227 rrg; 70~ ~] D= ~ 30 (c:1, chloro ~rm ) .
Analysis calculated for C48H54015:
O, 27.55. Found: C, 65.96; H, 6.23; Or27 660/
25 The compound 133 was then applied for the preparation of benzyl-2i3~4-tri-o-benzyl-6-o-(ol~-L-idopyranosyl)-~l3-D-galactopyranoside (compound 134) by proceeding as follows:
The disaccharide 133 (200 mg) was desacetylated by the technique described previously for the preparation of the disaccharide 127.
:.

5~

Purlfication by chroma-tography on a column of silica gel (10 g) by means of the mixture chloroform-methanol (9:1, v/v) leads to the disaccharide 134 obtained in amorphous form (147 rng, 90%)~ [a]D = -88 (c : 0~8, chloroform).
Analysis calculated for C40H46Oll : C, 68.36 ; H, 6.60;
0, 25.04.
Found : C, 68.74 ; H, 6.68 ; O, 25.37% (see Figure 25).
EXAMPLE 35 : Preparation of 2-acetamido-1,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-a-L-idopyranosyl-2-desoxy-~-D-glucopyranose (compound 138) (Figure 26) =
The preparation of this compound was carried out by -the following steps a) to c).
a) Preparation of 2-acetamido-3-O-acetyl-1,6-anhydro-2-desoxy-4,0-(2,3,4,6-tetra-O-acetyl-a-L-ido-pyranosyl-~-D-glucopyranose (compound 136) A solution of 1 g of 2-acetamido-3-O-acetyl-1,6-anhydro-2-desoxy-~-=D-glucopyranose (compound 135 prepared according to F. SCHMITT and P. SINAY, Carbohydr. Res., 29 (1973) 99.) in anhydrous nitro-2Q benzene (40 ml) was stirred for 2 h at 130C in the presence of a 4 A molecular sieve in powder form (1 g), previously activated for 48 h at 250C. A solution of bromide 12 (1.43 g) in dichloroethane (10 ml) is added and the reaction medium is kept at 130C for 10 h. A
further addition of bromide 124 (0.7 g) in dichloro-ethane (5 ml) is then made and the reaction continued - iL2~i5~
132a for 24 h. Treatment similar to that described for the preparation of disaccharlde 126 leads to a compound which is purified by chromatography on a silica gel column (200 g) by means of a mixture ethyl-acetate-ether (S:1, v/v), giving the disaccharide 136 (1.8 g, 85~), [~¦D = 70.6 (c : 1, chloroform).
/

~,, ~L26S~3~

Analysis calculated ~or C24H33014N: , N, 2.50; 0, 40.03~ Found C, 51.35; H, 5.89; N, 2.51;
0, 40.05/.-b) Preparation o~ 2-acetamido 1,6-anhydro-2-desoxy-4-0-(~-k-ido ranos l)-P~D-~lucopyranose (compound 137) PY Y /, =

The disaccharide 136 (500 mg) is de-acetylated by the technique described previously for the preparation o~
disaccharide 127. A puri~ication by chromatography on a silica gel column (40 g) by means o the mixture ethyl aceta-te-10 Methanol(2:1, v/v) leads to the disaccharide 137 (300 mg, gOo/.), [ ]D- -65 (c: 1,6~ methanol).
Analysis calculated ~ C14H2310N~ 0~5 2 H, 6.46; N, 3.74. Found: C, 44.95; H, 6.61; N, 4.27~
c) Preparation o~ 2-acetam_o-1,3,6-tri-0-acetyl-4-0-(2, 3,4y6-tetra-0-acetyl-~ -L-idopyranosyl) 2-desoxy-/3 -D-glucopyranose (corr,pound 138) The disaccharide 136 (150 Mg) iS acetolysed at ambient temperature ~or 12 hours in the presence o~ a n~xture (5 ml) o~ acetic anhydride-acetic acid and concentrated sulfuric 20 acid (7:3:0,1,v/v). The reaction medium is then poured into iced water and stirred for 4 hours, then extracted with chloro~orm (100 ml). The chloro~ornl phase is washed with dilute aqueous solution o~ sodium hydrogenocarbonate, with water, dri ed (sodium sul~ate) and evaporated. The residue is 25 puri~ied by chromatography on a silica gel column (10 g) by means o~ the nlixture ethyl acetate-ether (5:1,v/v) giving the disaccharide 136 which is crystallized in the mixture ethyl acetate-pentane (120 mg, 640/,), Ml? 120C, [O(]D=40 (c: 1, chloro~orm).

: , ~L265 5L32 Elementary analysis calculated for C28H39018N C,49.63 ;

H, 5.80 ; 0,42.50 ; N, 2.07 ;

Found : C, 49.68 ; H, 5.91 ; 0, 42.16 ; N,2.12/.

EX~ ~LE 3~ : Synthesis of 2-acetamido-2-desoxy-4-0-(K -L-idopyranosyl)-D-glucopyranose (conipound 139) (Figure 26).

The disaccharide 138 (100 mg) is desacetylated by the previously descrlbed technique for the preparation of the disaccharide 127. Purification by chromatography ~n a silica gel column (5 g) by n,eans of the rr,ixture methanol-chloroform (3:2, v/~) results in the disaccharide 139 which is crystallized in aqueous ethanol (48 ng, 85 MP 143-145C, [~ ~D ~ -20 - 31 (c 0.8, water-methanol, 19:1, v/v, after 14h.

Analysis : calculated for : C14H25N011, 0.5 H20 : C,42,86 ;

H, 6.68 ; N, 3~57 Found : C, 42.83 : H, 6.68 ; N, 3.59 /. .

EX~kE 3~ : modification in the preparation of the compound 138 by the step 1 to 6 (see Figure 27) 1 : Preparation of benzyl 2-acetarnido-3,6-di-0-benzyl-2-desoxy-4-0-(6-0-tosyl-~ -D-glucopyranosyl)-~ -D-glucopyranoside (corlpound 1~0) .

A solution of the cornpound 139 (0.2 g) in pyridine (5 ml) is cooled to 0C. Tosyl chloride (0.07 g) dissolved in pyridine (2Inl) is then added. The reaction is left at room tempera~ure ~r 24 hours. After addition of several dropsof water, ~he mixture is stirred for half-hour before being poured onto ice. A~ter taking up again with chloroform (0.2 l), the chloroform phase is washed successively with a 100/. aqueous solution ofKHS04, with water, a saturated solution of NaHC03 135 ~2651;~2 and water. After drying over sodium sulfate and concentrating to dryness, the residue was chroniatographed on silica gel (20 g) in a ethyl acetate/ methanol mixture (15/1l v/v). In this way pure cormpound 140 was obtained (150 mg ; 600,~ ]2~ _ + 74 (1,1 chloroform).
Elemen-tary analysis calcul ~or C42H49013N S (807,912) C, 62.40 ; H, 6.11 ; N, 1.73 ; 0, 25.74 ; S, 3.97~
Found : C, 62.77 ; }I, 6.13 ; N, 1.73 ; 0, 24.98 ; S, 3.48. The N~IR spectrurn confirms the desired structure.
2 : Preparation of benzyl 2-acetamido-3,6-di-0-benzyl-2-desoxy-4-0-(2,3,4-tri-O-acetyl-6-O-tosyl-~ -D-glucopyranosy~ -D-~lucopyranoside (cornpound 141) To a solution of compound 139 (200 g) in pyridine (5 ml), acetic anydride (5 ml) is added. AEter one night at room tenlperature, the reac-tion nlixture was concentrated to dryness. The resi~ue was chromatographed on a silica gel colwnn (25 g) in an ethyl acetate/hexane mixture (3/1, v/~).
This way the conlpound 141 was obtained (208 mg, 90k) in the form of a syrup.~X]20D= + 70 (1, chloro~o~m).
Elernentary analysis calcul for C48H55NS 16 (934~ 023) C, 61~78 ; H, 5.94 ; N, 1.5 ; 0, 27.4l ; S, 3.43.
Found : C, 61.58 ; H, 5.91 ; N 1.27 ; S, 3.23.
The N~IR spect~wn confi~;s the desired structure~
3 : Prepara-tion o~ benzyl 2-acetalnido-3,6-c1i-0-benzyl-2-desoxy-4-0(2,3,4,-tri-0-ac_ty_-6-desoxy-6--iodo-~ -D-glucopyr-a-n-o-s~l)- ~-D-31ucopyranoside (compound 142) 1) Fro~ co~pound 141 -To a solution of conlpound 141 150 rng) in acetone (5 ml),sodium iodide (150 mg) is added. The mixture is heated to 1;~65~32 70C in a seal~d tube for 7 hours. After evaporation to dryness, the residue is taken up again with water and chlorofor~..
The chlorofo~l phase was washed with water and dried over sodiw,l sulfate. ~fter evaporation to dryness, the residue was crystallized in a chloroform/pentane mixture (102 ~g, 700/.).
m.p 173-174C C~] ~- ~ 78.5 (1.2 chloroform).
Elementary analysis calcul.for : C41H48013 Nl (889,733) C, 55.34 ; H, 5.44 ; N, 1.57 ; 0, 23.38 ; I, 14.28.
Found : C, 54.98 ; H, 5.52 ; N, 1.45 ; 0, 23.57 ; I 14~10.
The NMR spectrwn corresponds to the desired structure.
2) From compound 139 via the cor,pound 1~3 -A solution of compound 139(1 g) and N-iodo-succinimide (1 g) in D~IF (50 ml) was stirred at 0C for 30 minutes.
Triphenylphosphine (1,2 g) was then added slowly in one hour.
After heating at 50C for one hour, methanol (1 ml) was added and then the reaction mixture was concentrated to dryness.
The product was extracted with chloroform. The chlorofo~n phase washed with water, with a solution of sodium triosulfate then again with water. After drying and evaporation ~ the chloroform, the residue was deposited on a silica gel column (50 g). The ~ compound 143 contaminated with triphenylphosphine was eluted by an ethyl acetate methanol mixture (15/1, v/V).
After evaporation of the chromatography solvent and drying the derivative 143 is dissolved in pyridine (10 ml) then acetylated with acetic anhydride (10 ml). After conventional treatment, the derivative 142 is crystallized in a mixture chloro~onn/pentane. The yield with respect to compound 139 ~ L265~32 is 85 ~.. T~i5 conlpound is in every respect similar to that obtained rom compound 141.
4 : Preparation oP benzyl 2-acetamido-3,6-d_0-benzyl-2-desoxy-4-0-(2,3~4-tri-0-acetyl-6 desoxy-/3 -D-xylo-hex-5-(enopyranosyl)~
ylucopyranoside-2 (conl~ound 14~) -To a solution of compound 142 (400 mg) in anhydrous pyridine (5 ml), silver Pluoride (400 mg) is added. The suspensionis stirred in the dark ~r 48 hours. The mixture is then poured with stirring with ether (200 ml). APter filtration, the ether phase is washed with a 10~ solution o~ NaHSO~, then with a 10/. solution o NaHC03 Pinally with water. APter drying and concentrating to dryness, The residue is crystallized in chloroPorm/ether mixture (206 mg; 600/.) m.p. 184-185C.
~~`] 20D = ~ 700 (1.4 chloroPorm).
Elementary analysis calcul for: C41H47N 13 (761,821):
C, 64.69; H, 6.22; N, 1.84.
Found: C~ 64.5; H, 5.96; N, 1.79.
The MIR spectrum is in accordance with the desired structure.
2~ 5 : Preparation of benzyl 2-acetamido-3,6-di-0-benzyl-2-desoxy-4-0~ dopyranosyl- D~-D-glucopyranoside (compound 145) The compound 144 (380 g) is dissolved in reshly distille~
tetrahydrourane (8 ml). APter cooling to 0C in a nitrogen 25 atmosphere, boron hydride (BH3, 1M in THF, 1 ml) is added and then the temperature is allowed to rise again to room~
temperature. After one hour oP reaction, a ~urtOer addition ~:
. ,', :

, ~ , , ~ 5~32 o~ hydride is made (1 ml). ~fter 30 minutes, ethanol is added c3rop by dropD When the release o~ gas has ceased, the mixture is diluted with THI; (10 l)o Soda (3 M, 1.2 ml) is added ~ollowed by oxygen peroxide (120 Vol ; O .8 ml). After two hours 5 at 50C, the solution is poured into chloro~o~n. The chloroform phase is washed with aqueous hydrochloric acid solution (0.1N) then with water. A~ter drying (Na2S04) and concentration to dryness, the residue is chromatographed on a silica gel colunln (45 g) in a ethyl acetate/methanol mixture (15/4; v/~).
The derivative 1~5 is ~irst eluted (63 n g; 15/.) followed by the derivative 139 (225 my ; 540/.). The derivative 145 is crystallized in a mixture o~ ethyl acetate/methanol rn.p. 191C
[~] 2~_ ~ 64.4 (1, methanol).
Elenlentary analysis cal~ul ~or C35H43N011~ ~l20: C, 62.57 H, 6.75; N, 2.08.
Found C~ 62.42; H, 6.55 ; N, 1.88.
6: Preparation o~ 2-acetar,lido-1,3J6-tri-O-acetyl-2-desoxy-4-0 (2,3,4,6-tetra-0-acetyl~
idopyranosyl)-~lucopyranose -(compound 138) A solution of the derivative 145 (35 mg) in methanol (10 ml) is stirred in the presence oE a catalyst (Pd/C, 5/.
25 mg) in hydrogen atmosphere ~or 48 hours. I~ter ~iltration and evaporation, the residue (17 mg~ is acetylated with a pyridine/
acctic anhydride mixture (2 ml/1ml). ~ter conventional 25 treatment,the residue is chromatographed on a silica gel colun~n (10 g) eluted with ethyl acetate. t~fter crystallisation, compound 138 is obtained (14 nl~g; 32/.). m.p. 191C ~] ~)- ~ 8 (0.6, chloro~o~sn).

~ "'``' .
..

~26S13;~:

E~ll}'LE 3~ - Synthesis of the trisaccharide 149 o~ -the COOM formula ~OAc ~'\~--o J~
OBn N3 OAc (149) This synthesis is carried out in 3 steps (see Figure 28).
First, glycosylation of the orthoester of a derivative of L-idu~
1~ ronic acid is carried out. Then selectively the rllonochloro-acetyl group, then one of the alcohols formed is reac-ted zith a disaccharide.
1) - Glycosylation o~ the orthoester_38 ~qith ben~yl alcohol A solution of the orthoester 38 (118 rlg 0.25 n~) ob-tained according to Example 5 and benzyl alcohol (0.15 ml, 15 ~, freshly distilled) in anhydrous chlorobenzene (10 ~nl) is heated to 140C protected from moisture. After slo~ distilla-tion of 8 ml of solvent, a solution of 2,6-dimethylpyridiniul~l perchlorate (2.5~iM) in chlorobenzene (2 ml) is added drop by drop in 30min with sinultaneous distillation ~f solvent (2 ml).
The reaction m,i~;ture is then stirred for 30min under these condi-tions, ~ith the addition drop by dr p of fresh solvent and simultaneous distillation, so that the reaction volunie remains constant and equal to about 2 nll. After cooling the dilution with chloroform (50 ml), the organic phase is washed ~ith 5 /.
aqueous solution of sodiwn hydrogen carbonate, with water, dried (sodiun;~ sul~ate~ filtered and evaporated.

The residue is chrol atographed on a silica gel colur;ln (8 g). ~lution by the Irixture hexane-ethyl acetate (2: 1, v/v) enables a Praction to be obtained ~ontaing the mixture 146 o~ gly~osides ancl which has not been separated at this stage (102 r;g, 81 /.),N.~i.R. (90 MHz, CDCl3): ~: 7.30 (m, 10~l, 2 Ph, 3.98 (s, 2H, Cl-CH2-CO), 3.74 (s, 3H, COOMe), 3.08 an~1 2.03 (2s, 3H in total, OAc fornl~ and o~; ~/3: ^' 2 1).
2) - Seleotive O-dernonochloroacetylation A solution o~` the preceding mixture 146 (102 r,!g) in pyridine (5ll) and absolute ethanol (1 Ir~l) is heated to 100C
~or 20 min in the presence of thiourea (25 rng). After cooling, the reaction n;ixture is evaporated to dryness and the residue is taken up again with a water-c:hloroform mixture (1:1, v/v, 50 ml). The organic phase is washed ~ith water, dried (sodium sulfate), Çiltered and evaporated.
The residue is chromatographed on a silica gel ~olulrn (10 g). Elution with the nlixture ethyl acetate-hexane (4:3, v/v ) enables the isolation (in ~order of elution) oP
- the glycoside 148 (26 mg, 25 /. ), colorless syrup, ~~D ~ 70 (c 1, chloroform) N.~I~R. (9OMHz, CdC13) : S
7.30 (nl, 10H, 2 E~h); 5.05 'm, 1H, H2); 4~90 (d, 1H, H1, 1.2 J = 2Hz); 3.78 (s, 3H,COO~Ie); 3.12 (1H,OH, exchanged with D20) ; 2.05 (s, 3H, OAc).
- the ~ glycoside 147 (54 mg, 50 o/. from 38) colorless syrup, ~~ 7D ~ 65 (c1, chloroforrn) N.ll.R. (90 MHz, CdCl3) : ~: 7.30 (m, 10H, 2 Ph); 5/05 (~H, H1 and H~, very weak coupling constants ~or J1 2 ~ 1Hz); 3.78 (s, 3H, COOMe);
2.80 (1H,OH, exchanged-vith D 0) ; 2.06 (s, 3H, OAc).

~65~3~

3) - Glycosylation o~ the alcohol 147 by Means o~ the disaccha-_ ride 97 A solution o~ -the alcohol 147 (22 mg, 50 M), and of the bromide 97 obtained according to example 6 (57 mg, 70 ~I) in anhydrous dichlomethane (1.5 ml) is stirred protected from light and moisture in the presence of 4 ~ molecular sieve (powder, 50 mg). The reaction mi~ture is cooled to -20C and sym-collidine (110 l) and silver triflate (26 mg, 100 M) are added successively. The reaction mixture is stirred 2h under these conditions, diluted with dichlororlethane (50 ml) the solids are drained and the filtrate is washed with an iced aqueous solution o~ 0.1 M HCl, with water, with a 5 /. aqueous solution o~ sodium hydrogencarbonate, with water, dried (sodium sul~ate), ~iltered and evaporated.
The residue i5 chromatographed on a silica gel column (8 g, gel 230-400 mesh~). Elution by the mixutre -toluene-ace ethyl acetate (5:1, v/~) enables the isolation o~ the trisaccha-ride 149 in the ~orm o~ a colorless syrup (50 mg, 86 /~).
The N.M.R. spectrum (270 MHz, CDCl3) is in accordance D ~qth the expected structure. This spectrum is shown in Figure 3'.
EX~LE 3 9 : Fixation of the tetrasaccharide on BSA :
-COONa 0503Na 0503Na UO~ ~ ~ ` ~OH
O NHS03Na 53Na NH503Na ~2ÇiS~

To a solution ofbovine serum al~nin (BSA : 7 mg ;0.1 ~ple) and of tetrasaccharide (15 mg ; 10 ~moles) in a sodium phosphate buffer (0.15 M ; pH 7.0 ; 2.5 ml ), is added sodiurn cyanoborohydride (13 ~g ; ~0 ~moles) and the solu-tion taken to 37C for ive days. The reaction mixtureis then chromatographed on a Sephadex G-50 (1 x 100 cm) column, eluted with water, so as to separate the salts and the pentasaccharide not fixed to the protein-oligosaccharide conjugate. Under these conditions, a fixation of 12 moles of tetrasaccharide per mole o~ BSA is obtained.
The same reaction can be carried out on an insoluble support usuch as 2-aminoethyl-polyacryla-nide or 2-aminoethyl celluloSe, or any other support containing a primary amine function~
In the same way, by operating in the presence of antithrombin III (instead of BSA), under the conditions defined above, a fixation of the oligosaccharide on antithrombin III, and through this a permanently acti~ated antithrombin III is obtained (BJORK et al., FEBS Letters, 20 143 (1982), 96-100).

To a solution of compound 2 (5.6 g) and of mercuric cyanide (3.5 g) in dichloroethane (40 ml), are added~ after distillation of about nll of sol~ent, 4 A molecular sieve 25 (1 g~ then the compound 1 (3.44 g ; 8.82 mmoles). After one night with stirring the solids are remo~ed by ~iltration, ~ .

~L2/65~32 1~3 then washed with dichloromethane. The latter is then joined with the solution and then the organic phase obtained is washed with a saturated potassi~m iodide solution, then with water.
After drying and concentrating to dryness, the syrup obtained (10 g) is deace-tylated in the presence o~ sodiwn methanolate (2 M, 1 ml) in methanol (20 ml). The compound 163 obtained (2.7 g) a~ter chromatography on silica gel (50 g chloro~orm/
methanol ; 20/1 ; v/v), is a syrup (~] 20 _ 12 (1,1 chloro~orm) which is used as such in the synthesis of 5.
10 SYNTHESI S OF THE COl!lPOUND 164 The disaccharide 163 (2.7 g) is dissolved in anhydrous DMF (27 ml) then successively to this solution are added trityl chloride (4.42 g) dimethylaminopyridine (135 mg) then tri~
ethylamine (2.7 ml). After two days at room temperature, the reaction mi~ture is concentrated under vacuum.Then the residue is chromatographed on silica gel (50 g ; hexane then hexane/
ethyl acetate ; 2/1 then 1/1 ; v/v). In this way 164 is obtained (2.6 g). It is a syrup ; [~] D0- 16.3 (1,3 ; chloroform).
SYN7rHESIS OF THE DISACC~IARIDE 166 The syrup obtained at the end of the preparation 164 (2.4 g) is dissolVed in DMF (40 ml). Then bariwll hydroxide octahydrate (1.64 g), bariurn oxide (7.08 g) and flnally benzyl brornide (2 ml), are added. After 4 hours of reaction, - methanol is added followed by chlorofo~n (100 ml). The solids are drained and then the chloroforrn phase is concentrated to dryness. The disaccharide 165 obtained at this stage is directly converted into 166. For this~ the residue is taken up l~4 ~6513~
again in dichlorollletllane (20 ~,~l) then a solution o BF3 in lilethanol ~2~ ) is added, at 0C, protected from nloisture.
~ter 4 hours o~ reaction, the reaction mixture is diluted with d-ichlororrlethane then washed with an aqueous sodiur~
bicarbonate solu-tion. After drying and concentration, the rcsidue is chro~i;atographed on a silica gel colulin (100 g ;
hexane/ethyl acetate ; 4/1 then 1/1 ; v/v). T n this way 166 is obtained [~ ~ 2 (0,7 chloroforl-l).
~}IESIS OF TIIE CO~POUI~ID 168 The derivative 166 is dissolved in acetone (~0 nil).
Then there is added, at 0C, a chromic oxide (VI) (670 mg) solution in 3.5 M sulfuric acid (3 ml). After 1.5 hours, ice and water are added to the reaction Mixture, then the oxidized product is extracted with chloroforr~. The chloroform phase is washed with water, dried, and concentrated to dryness.
The residue, dissolved in ether is methylated by the addition of' diazoll~ethane thus yielding 168 which is puriÇied on silica gel (hexane/ethyl acetate ; 4/1 then 1/1 ; v/v). ~t is a syrup [~]20 _ 8.5 (1, chloroform). The elementary analysis and the IR spectrurl, con~irrl the expected structure ~r 168.

PE~RK : 168 nay be acetolysed and converted into a halide in the r~anner described in Ex~!iple ~5 (passage from 94 to 97).

Claims (32)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the synthesis of oligosaccha-rides of the heparinic type, having [D-glucosamine] or uronic acid (D-glucuronic acid or L-iduronic acid) alternate moieties, or the reverse, comprising 2 to 12 of said moieties, which comprises :
- condensing a D-glucosamine moiety (I) with an uronic acid moiety (II) corresponding to a D-glucuronic acid or a L-iduronic acid structure, of formulae :

I II

or, an uronic acid moiety (III) with a D-glucosamine moiety (IV) having formulae :

III IV
wherein :
- the anomeric carbon is substituted by a reactive group X capable of reacting with the -OH group at the position 4 of (II) to give . a linkage with an .alpha. stereospecificity, between a D-glucosamine and a D-glucuronic or an L-iduronic acid, and between an L-iduronic acid and a D-glucosamine, or . a linkage with a .beta. stereospecificity, between a D-glucuronic acid and a glucosamine, said reactive group-X being an halogenide or an -O-imidoyl group or, together with the adjacent -OR1 group, forming an orthoester (except in the case of glucosamine) - the R1 groups represent -OH protecting groups, at least one of the R1 groups being different from the others, said groups being selected from an acyl, an alkyl, a substituted alkyl or an aryl radical, or for two R1 groups next to each other, a cetal or an acetal group, or R1 and R together form a 1,6-anhydro bridge and/or two -OR1 groups in the glucosamines of formula IV
form an epoxy function, - N is N3 or NHCOO-alkyl or substituted alkyl group, - M is an alkyl or an aryl group, - R and R', identical or different, are selected from . said R1 meanings, . a mono or an oligosaccharidic moiety with D-glucosa-mine or uronic acid units as given above, whose anomeric carbon in the case of R, or position 4 in the case of R' are blocked by a group -OT removable in the presence of the other groups present on the units of the starting products to recreate an alcohol, T being selected from an allyl, a propenyl, an acyl, an halogenated acyl or a p-methoxybenzoyl group, with the proviso that R+R' ? 10 saccharidic moieties, . said T meanings, or . -OR is a symbol representing a reactive group-X, - sequentially removing the R1 groups, first to intro-duce functional groups on specific positions as encountered in heparin, second to make free -OH groups on other specific positions, which simultaneously result in converting -N3 or -NH-COO-alkyl or substituted alkyl into -NH2, introducing a functional group on the amino radical, and removing M, with the proviso that the condensation reaction does not lead to the production of the disaccharide N-acetyl-glucosamine-6-sulfate 1?4 D-glucuronic acid 1 ?CH3 or the disaccharide N-sulfate glucosamine 6-sulfate 1?4 D-glucuronic acid 1?CH3.

2. A process according to Claim 1, wherein R' or R represents T or a mono or oligosaccharide respectively with an -OT group at the 4 or 1 position, comprising removing the protective T groups to make free the -OH groups, substituting the 1 position with a reactive group and condensing the mono- or oligosaccha-ride unit having activated 1 position with the mono- or oligosaccharide unit having a free OH group in the 4 position, and repeating said steps and the condensation reaction until the desired saccharide moiety is ob-tained.

3. A process according to Claim 1, wherein R1 is acetyl when the corresponding position is intend-ed to be functionalized and is benzyl, when the corre-sponding position is intended to be occupied by an -OH
group.

4. A process according to Claim 1, wherein T
is selected from an allyl, a chloroacetyl or a p-methoxybenzoyl group.

5. A process according to Claim 3, wherein the introduction of the functional group is carried out by saponifying with a strong base alkylating the carb-oxyl group and treating with a sulphation or an acyl-ation agent.

6. A process according to Claim 3, wherein the removing of the benzyl group is carried out by catalytic hydrogenation which results simultaneously in the conversion of -N3 to -NH2.

7. A process according to Claim 6, wherein -NH2 is converted to -NH-SO3- or -NH-acyl, by treatment respectively with a sulphation or an acylation agent.

8. A process according to claim 7, which further comprises M removal by saponifying with a strong base to give -COO-.

9. A process according to claim 8, wherein the -COO- group is salified with an alkaline metal.

10. A process according to claim 1, comprising the use in place of one or several D-glucosamine or uronic acid (I) to (IV), of a structural analog selected from a neutral sugar, a desoxy-sugar, or uronic acid units or D-glucosamine units of different configurations.

11. A process according to claim 1, wherein the condensation reaction between the halide and the alcohol is carried out in a solvent medium.

12. A process according to claim 11, wherein the solvent is an organic solvent selected from the dichloro-methane or dichloroethane and the catalyst is selected from a silver or a mercury salt.

13. A process according to claim 12, wherein the silver or mercury salt is silver trifluoromethane sul-phonate, commonly called silver triflate, silver carbon-ate, silver oxide, mercuric bromide or mercuric cyanide.

14. A process according to claim 1, wherein L-idose is used instead of a L-iduronic acid of formula (III), the condensation reaction between the L-idose and glucosamine (IV) being carried out by using as catalyst mercuric derivatives selected from cyanide and/or mer-curic bromide.

15. A process according to claim 1, wherein the condensation with - a 1,2-O-methoxy-ethylidene group is carried out in a solvent whose boil-ing point exceeds 100°C in the presence of a catalyst.

16. A process according to claim 1, wherein the condensation with an imidate is carried out at a temper-ature below or equal to about 0°C, in the presence of a catalyst.

17. The process according to claim 2, wherein R1 represents an acyl group.

18. The process according to claim 17, wherein R1 is -O-acetyl or -O-chloracetyl.

19. A process according to claim 1 or 2, wherein at least one protective group R1 is introduced from other groups once the saccharide skelaton is formed, this modification comprising the use for the conden-sation reaction of a saccharidic unit of formula:

the opening of the epoxide function by the sodium azide enabling the introduction, at the 2 position, of an N3 group.

20. A process according to claim 1 or 2, wherein at least one protective group R1 is introduced from other groups once the saccharide skeleton is formed, this modification comprising the use for the condensation reaction of a D-glucosamine unit of formula I but having a 1,6-anhydro bridge.

21. A process for the organic synthesis of oligosaccharides of the heparinic type, comprising - condensing a D-glucosamine unit of formula:

or an oligosaccharide terminated by said unit, with an uronic acid unit of formula:

or an oligosaccharide terminated by said unit or con-densinq an uronic acid unit (D-glucuronic or L-iduronic acid) of formula:

or an oligosaccharide terminated by said unit, with a D-glucosamine unit of formula:

or an oligosaccharide terminated by said unit, the substituents having the following meanings Ac : acetyl Bn : benzyl X : halogan M : alkyl or aryl T : allyl, propenyl, acyl, halogenated acyl or p-methoxybenzoyl group - saponifying with a strong base, alkylating the carboxy group and treating with a sulphation agent to introduce SO3- groups instead of the Ac groups, - catalytically hydrogenating to remove the benzyl group and simultaneously convert -N3 to -NH2, - treating with either a sulphating or an acylating agent to convert -NH2 into NH-SO3- or -NHAc, - saponifying with a strong base to remove M and give COO-.

22. A process according to claim 21, comprising preparing a disaccharidic unit of formula :

wherein the condensation step is carried out by using an uronic acid unit of formula :

and a D-glucosamine unit of formula :

23. A process according to claim 19, comprising preparing a disaccharidic unit of formula :
wherein the condensation step is carried out by using a O-glucosamine unit of formula :
and an uronic acid unit of formula:

24. A process according to claim 19, comprising preparing a trisaccharidic unit of formula:
wherein first a disaccharidic unit is prepared having a group -OT, either on the glucosamine unit or on the uronic acid, and, after removal of T to make free an OH group, according to the same scheme, the disaccharidic unit is condensed with another sugar unit, and the other steps are performed on the trisaccaridic chaim.

25. A process according to claim 21, compris-ing preparing a tretrasaccharidic chain of the formula :

comprising - condensing a O-glucuronic acid unit with a O-glucos-amine unit, having respective formulae :
said condensation step resulting in the disaccharidic unit of formula :
- condensing a L-iduronic acid unit with a O-glucosamine unit of respective formulae :
said condensation step resulting in the disaccharidic unit of foruula :
- condensing said two disaccharidic units to obtain the following tetrasaccharidic structure :
and the other steps are performed on the tetrasaccharidic chain.

26. A process according to Claim 21, wherein said tetrasaccharide obtained after the condensation step is condensed with a D-glucosamine unit of formula:
the condensation step resulting in the following penta-saccharide:

and the other steps are performed on the pentasaccha-ridic chain.

27. Oligosaccharides obtained by the process of Claim 1.

28. Oligosaccharides having the following formula:

29. Oligosaccharides having a disaccharidic unit with the following structure:

30. Oligosaccharides having the formula:

31. Oligosaccharides having the formula:

32. Oligosaccharides having the formula: