CA2192073C - Novel oligosaccharide derivatives, preparation method therefor and uses thereof - Google Patents

Abstract

The subject of the invention is compounds comprising one or more oligosaccharides, each of said oligosides being covalently attached to one or more molecules, matrices or particles, in particular one, two or three, by means of an intermediate molecule having an atom of nitrogen carried by a carbon in ad C = O group and one or more functional groups, in particular one, two or three, the covalent bond between said intermediate molecule and the oligoside ayan t via the aforesaid atom of nitrogen, and the covalent bond between said intermediate molecule and said molecule, said matrix, said particle, or said molecules, said matrices, said particles occurring through said functional group (s) of said molecule intermediate and appropriate functional groups on the molecule (s), the matrix (s) or the (the) particle (s).

Description

& U 96100229 PCT / FR95 / 00790 NOVEL OLIGOSID DERIVATIVES, THEIR METHOD OF
PREPARATION AND THEIR APPLICATIONS.

The subject of the invention is novel oligoside derivatives, their process preparation and their applications.
Natural oligosides can be prepared in the free state from various physiological fluids such as milk, or extracted from products natural or transfotmated (honey, beer, etc.). Natural oligosides can also be obtained by cutting a glycosidic bond of one of the parts carbohydrates of glycoconjugates (glycolipids, glycoproteins, polysaccharides, proteoglycans, etc.), by hydrolysis using enzymes or by catalysis from said glycoconjugates.
Natural oligosides can be used as substrates, such as inhibitors, as a recognition signal, etc. In most cases, the oligoside should be attached to a molecule, matrix or particle, who can be chosen from:
a matrix as a support for affinity chromatography;
- a gold ball, latex, for histology and cytology;
a protein for visualization, purification, etc., especially 1) receptors specific for osides, receptors called lectins, adhesins, agglutinins etc., or else 2) proteins with enzymatic activity or no, which have an affinity for osides, especially glycosyltransferases, such as sialyltransferase, sulfotransferases, phosphotransferase, exoglycosidases or endoglycosidases;
a lipid for the characterization of the preceding receptors;
oligonucleotides to selectively increase their capture by target cells;
a protein or polymers for targeting drugs, of oligonucleotides or genes, or to produce inhibitors intramolecular.
The synthesis of oligoside derivatives capable of being bound in a covalently to a protein, a template, an oligonucleotide or in a general way at an organic molecule or particle, while preserving the integrity of the structure and functions of each of its constituent oses - this is necessary to preserve the functional capacity of the oligoside - can be obtained essentially in two ways: the total synthesis de novo and the intermediate transformation into glycosylamine. A third way leads to a

two derivative also useful but by destroying the terminal reducing ose, this is the way of amination in a reducing medium.
In terms of total synthesis, this path requires selective protection hydroxyls not involved in a glycosidic bond, steps of condensation, selective deprotection steps, and a step of protection FMALE. Even though in recent decades the returns of some of ~
these steps have been improved, this path is a long way and the yield overall, remains modest, especially since the oligoside to be synthesized is more complex.
The yields are included for each step between 20 and 95% according to the steps considered.
For example, the synthesis of a para-nitrophenyl derivative of a pentasaccharide such as the Lewis determinant x:
Ga1G33 (Fuca4) G1cNAcj33Ga1 (34GIc (3-Op-C6H2 NO2) requires a total of several dozen steps. Each step has a return understood between 50 and 95%, more generally 80%. In total the yield of the product expected is a few%.
The high number of steps comes from that alcohol functions each must be protected in different ways depending on whether the hydroxyl will or will not be involved in a condensation reaction.
An ose such as G1cNAc that will be substituted twice will receive momentarily 3 different substituents to allow selective substitution on the hydroxyl

3 by galactose, on hydroxyl 4 by fncose, hydroxyl 6 remaining protected until final deprotection.
For each condensation step, the yield is affected by the fact that the product formed is generally a mixture of two anomeric forms a or P.
In addition, it should be noted that intermediate products must be purified, either by crystallization or by chromatography, which is a cause important of the duration of the total synthesis. Finally, it should be noted that yields can be very low for some steps per cause steric hindrance, which is particularly the case with branching: 2 oses substituting the same dare.
In the end, this global synthesis is very expensive and very long.
As for the formation of a glycosylamine followed by acylation, this pathway based on a reaction described at the end of the last century: an oside possessing a dose reducer, incubated in the presence of a high concentration of ammonia, a ammonium salt or aromatic amine, reversibly transforms in glycosylamine.
For example, lactose gives a lactosylamine, with ammonia:
Gal (34G1c + NH3 => Ga1p4Glc (3-NHZ + H20 or with aniline:
Ga1R4G1c + NH2-C6II5 = Ga1R4Glc (3-NH-C6H5 + H20 This reaction is however reversible, that is to say that the isolated product, dissolved in a buffer, hydrolyzes to give back the starting materials.
Glycosylamine can, however, be stabilized by acylation, for example by selective N-acetylation:
Ga1R4Glcji-NH2 + (CH3 CO) 2 O => Ga1p4Glc (3-NH-CO-CH3 + CH3-CO2H
On this basis, it has recently been proposed to substitute the amine of oligosylamines with an organic compound having a functional group reagent. Manger et al., 1992, Biochemistry 31, 10724 and 31, 10733.
This path includes the following steps:

1) incubation of the oside having a terminal reducing ose in the presence a very concentrated ammonium salt.
For example, Ga1p4Glc + NH4 + pGa1R4Gicg-NH3 + + H20 C12H22011 + NH4 + q C12H2401oN + H20 2) Purification of glycosylamine by column chromatography for remove the excess ammonium salt.

3) Substitution of the amine of glycosylamine by reaction with a derivative activated monochloroacetic acid, in an alkaline medium.
For example, Gal (34G1c (3NH2 + (Cl-CH2 CO) 2-O => Ga1p4Glc-NH-CO-CH2-Cl

4) Transformation of chloroacetyl residues into glycyl residue.
Chloroacetylglycosylamide is incubated in the presence of a salt highly concentrated ammonium, which makes it possible to introduce an amine function. By example:
Gal (34G1cp-NH-CO-CH2-Cl + NH4 + => Ga1p4Glc (3-NH-CO-CH2-NH3 +, Cl-H +

2. 192073 WO 96/00229 1 '~, PCT / FR95 / 00790

5) Purification of glycyl-glycosylamide by column chromatography to remove ammonium salts.

6) Condensation of glycyl-glycosylamide and a compound capable of selectively react with an amino group of the glycyl residue.
For example:
Ga1 (34G1c (iNH-CO-CH2-NH2 + R-CO-G => Ga1O4Glc (3-NH-CO-CH2-NH-CO-R + HG
wherein G is an activator of the carboxylic function.
This 6-step route involves two intermediate purification steps and the use of a toxic product: the activated derivative of the acid chloroacetic.
The yield of each step is variable and varies between 50 and 959b; the overall yield is less than about 60%.
Another path has also been suggested, but it leads to transformation of the reducing ose into polyol; this way was proposed in 1974 by Gray (Arch Biochem Biophys., 163, 426-428).
The oligoside is condensed with a compound having an amine function (or more) in the presence of sodium cyanoborohydrate in an alkaline medium;
The imine formed between the reducing ose and the amine is reduced by the sodium cyanoborohydride to a secondary amine.
For example:
Gal (#Glc + NH2-R ~
Gal (4-O-CH (CHOH-CH 2 OH) -CHOH-CHOH-CH = NR
+ NaBH3CN = Gal (34-O-CH (CHOH-CH2OH) -CHOH-CHOH-CH2-NH-R
The yield of this reaction varies according to the size of the partners and is usually between 5 and 70%.
There is destruction of the reducing ose, which is undesirable.
French Patent No. 2,277,823 relates to N-osides of the acid Pyrrolidone-2-carboxylic acid-5, derivatives thereof and process for preparation, consisting of condensing L-pyrrolidone-2-carboxylic acid-5 and or L-glutamic acid and / or a salt of these acids with an ose or oside, reducing properties or not.
From the examples, the reducing sugars are all monosaccharides ketoses or aldoses), while non-reducing sugars are, for example, the sucrose; given the reaction conditions, sucrose hydrolyses in glucose and fructose.

q (} `~ ''1'j, O 96100229 ^ PCT / FR95 / 00790 It should also be noted that in the process of the aforesaid French patent n 2 277 823, the condensation is preferably carried out in an aqueous medium, and at a temperature of 50 C to 150 C, preferably around 105 C.
= These operating conditions require working with very concentrates of ose and acid that are inapplicable to the preparation oligosides;
the solubility of oligosides decreases rapidly when the number of oses grows.
Moreover, the condensation reaction between the ose and the acid being a thermal dehydration, it is inapplicable to the preparation of oligosides in because of the fragility of the osidic bonds, which are easily hydrolyzed at hot (see, for example, the case of sucrose).
Thermal condensation has the further disadvantage of leading to obtaining colored products, witnesses of degradation reactions and requiring a subsequent purification step.
This method therefore makes it possible to obtain only monosaccharide derivatives and is not suitable for producing oligoside derivatives.
One aspect of the invention is to provide novel derivatives oligosides in which the oligosides are attached to at least one molecule, a matrix or a particle.
Another aspect of the invention is to propose derivatives of glycosylanines, stable in aqueous medium, capable of being fixed on less a molecule, a matrix or a particle.
One of the aims of the present invention is to provide a method of preparation of oligosides attached to at least one molecule, matrix or particle, simple to implement, having a reduced number of steps and allowing a yield of up to almost 100%.
Another aspect of the invention is to be able to fix oligosides in a covalent on at least one molecule, matrix or particle, while preserving the functional capacity of oligosides.
The subject of the invention is new compounds comprising one or more several oligosaccharides, each of said oligosides being covalently attached sure one or more molecules, matrices or particles, in particular one, two or three, thanks to an intermediate molecule having a nitrogen atom carried by a carbon at a of C = O and one or more functional groups, particular one, two or three, the covalent bond between said molecule intermediate and the oligosaccharide via the aforesaid atom nitrogen, and the covalent bond between said intermediate molecule and the abovementioned molecule, the aforesaid matrix, the aforesaid particle, or the aforesaid molecules, the - --- - - - - - - - -said matrices, the aforesaid particles occurring via the or above functional groups of said intermediate molecule and groups functional appropriate for the molecule (s), the matrix (s) or the particle (s).
The subject of the invention is also compounds comprising one or more oligosides, each of said oligosides being covalently attached to one or more many molecules, matrices or particles, thanks to an intermediary molecule possessing a nitrogen atom carried by a carbon at a of a group C = O, the nitrogen atom being N-acylated, and one or more functional groups, the covalent bond between said molecule intermediate and the oligoside taking place via the aforesaid atom nitrogen, and covalent bond between said intermediate molecule and said molecule, the above matrix, the aforesaid particle, or the aforesaid molecules, the aforesaid matrices, the said particles taking place via the one or more of the aforesaid groups functional of said intermediate molecule and appropriate functional groups on the (the) molecule (s), the matrix (s) or the particle (s).
The term oligosides corresponds to the presence of at least two, and advantageously to the presence of more than two oses, and advantageously from less 4.
The oligosides used in the constitution of the compounds of the invention are such that before the bond with the intermediate molecule, they have a daring terminal reducer. As a result, the covalent bond between the aforementioned molecule intermediate and the oligoside takes place via the nitrogen atom of the molecule intermediate and anomeric carbon of the terminal reducing ose of the oligoside, that is to say of carbon at position 1 of the reducing endose of the series of aldoses, or carbon in position 2 of the terminal reducing ose of the series of ketoses.
The subject of the invention is compounds of general formula (I) oligosidyl - N CO (-D) a (Z) b CH [- (CH 2) p] j (i) COX
in which:
* A = 0or1, * J = 0or1, * b = 0 or 1, 6a * p = 2 to 4, especially 2, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of group (CHz) p, D represents a residue of an organic acid of formula DCO, H, in particular H
or an alkyl chain of 1 to 10 carbon atoms, in particular CH 3, * Z represents

7 ** B, where B is H, alkyl of 1 to 10 carbon atoms, or side chain of an amino acid, natural or synthetic such as CH (CH 3) 2, CH 2 OH, CH 3, and preferably H, or = ** B'-P ', B' being an alkyl chain of 1 to 10 carbon atoms, or a side chain of an amino acid, natural or synthetic, said chains = containing a group derived from a functional group likely to be activated, such as carboxylic, SH, OH or amine, free or protected, preferably protected, P 'having the meanings given below, * X represents:
the group [NH- (A;) - CO] m Q, (P) k or the group [NH- (A;) - CO] m P, (P ") k or the group [NH- (A;) - CO] ,, iRP

(F) k where m is an integer of 0 to 10, preferably 0 to 5 and advantageously 1 or 2, k = 0 or 1 Q represents OH, OCH3, OCHrC6H5, O-C6H5, O-C6F5, O-pC6H4-NO2, ON-CO-CHZ

R representing a group having an alcohol, phenol, thiol or amine, * P being as defined below, * AT; represents an organic radical such as an alkyl chain of 1 to 10 carbon atoms, notanunent (CH2) II W- (CHa) n, n + n 'representing a integer from 0 to 10, W representing CHY, Y being H, an alkyl of 1 to 6 linear or branched carbon atoms, an amino residue (x acid, natural or synthetic, or W representing an aromatic compound, in particular phenyl, * P, P 'and P "are identical or different and represent:
a matrix as a support for affinity chromatography;
a gold ball, latex, for histology and cytology;
a protein for visualization, purification, etc., especially 1) receptors specific for osides, receptors called lectins,

8 adhesins, agglutinins ete, or 2) proteins with enzymatic activity or no, which have an affinity for osides, especially glycosyltransferases, such as sialyltransferase, sulfotransferases, phosphotransferases, exoglycosidases or endoglycosidases;
a lipid for the characterization of the preceding receptors;
oligonucleotides to selectively increase their capture by target cells;
a protein or polymers for targeting drugs, oligonucleotides or genes;
P, P 'and P "having at least one function allowing a reaction of condensation by reaction with an oligopeptide, for example . an amine function (-NH2) allowing the formation of an amide with an ester active ingredient, amidine with imidate, thiourea with isothiocyanate, . a thiol function (-SH) allowing the formation of a disulfide bridge with a oligopeptide containing a thiol, a thioether with an oligopeptide containing a a maleimide group, or a haloalkyl group, or a haloalkanoyl group, . a phenol (-C6H4OH) allowing the formation of an azo with a oligopeptide containing a diazoide, provided that Z represents B'-P ', and / or X includes P and / or P "in its formula.
The invention also relates to compounds of general formula (I) oligosidyl - N - CO (-D) a (Z) b CH [- (CH 2) p] j (I) COX
in which:
* A = 0or1, * J0ou1, * b = 0 or 1, * P2à4, * provided that 8a ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of a group (CH) p, * D represents a residue of an organic acid of formula ECOZH, E being H
or an alkyl chain of 1 to 10 carbon atoms, * Z represents ** B, where B is H, alkyl of 1 to 10 carbon atoms, or a chain side of an amino acid, natural or synthetic, or ** B'-P ', B' being an alkyl chain of 1 to 10 carbon atoms, or a side chain of an amino acid, natural or synthetic, said chains containing a group derived from a functional group capable of being activated, P ' having the meanings given below, * X represents:
. the group [NH- (Ai) -CO] n, - Q, (P ") k . or the group [NH- (A) -CO], n P, (P ") k . or the group [NH- (A;) - CO] ,,, - RP
(P ~~) k where m is an integer of 0 to 10, preferably 0 to 5 and advantageously 1 or 2, k = 0 or 1 Q is OH, OCH3, OCH2-C6H5, O-C6H5, O-CJ5, O-pC6H4-NO2, ON-CO-CH2 R representing a group having an alcohol, phenol, thiol or amine, * P being as defined below, * AT; represents an organic radical, * P, P 'and P "are identical or different and represent:

8b a matrix as a support for affinity chromatography;
- a ball of gold or latex;
a protein;
a lipid;
oligonucleotides;
polymers;
P, P 'and P "having at least one function allowing a reaction of condensation by reaction with an oligopeptide, provided that Z represents B'-P ', and / or X includes P and / or P "in its formula.
In formula I and in those which follow, the oligosidyl group comes from an oligoside whose terminal ose is reducing.
It will be recognized that in the definition of compounds of general formula (I) indicated above, the aforementioned intermediate molecule comprises in its structure the following chemical entity:

- N CO (-D) a (ZI) b-CH [- (CH2) p] j COXI
in which D, a, b, j and p are as defined above, Z 1 and X 1 having one or several functional groups capable of forming at least one bond with one (of) molecule (s), particle (s), or matrix (s), as discussed above, and ZI and Xl being more precisely defined in what follows (cf.
defined below).

0 96100229 21 92 0 ~ J ~ `j PCT / FR95 / 00790

9 ic ~

In the definitions above, the amino acids involved have a L or D configuration, and preferably L.
The compounds of the invention therefore consist of one or more oligosides, (identical or some of which are possibly different) each of these oligosides being attached:
to a molecule, matrix or particle, via either of a functional group of X, or of a functional group of Z, - or to two molecules, matrices or particles, via respective functional groups of X and Z, or via two functional groups of X, or three molecules, matrices or particles, by through functional groups of X and Z.
It should be noted that the general formula (I) represents only the possibilities following:
a single oligoside attached to a molecule, a matrix or a particle, P, a single oligoside attached to a molecule, a matrix or a particle, P ', a single oligoside attached to a molecule, a matrix or a particle, P ", a single oligoside attached to two molecules, matrices or particles, respective P and P ', or P' and P "or P and P", a single oligoside attached to three molecules, matrices or particles, P, P 'and P '.
Such a representation is intended not to complicate the understanding of the general formula (I). But we can consider the possibility that many oligosides (identical or some of which may be different) attached either to a molecule, a matrix or a particle, P, or to a molecule, a matrix or particle, P ', either to a molecule, a matrix or a particle, P ", or that several oligosides (identical or some of which are possibly different) are attached to two molecules, matrices or particles P and P ', or P and P ", or P' and P", or that several oligosides (identical or whose some are possibly different) are attached to three molecules, matrices or particles, P, P 'and P ".
As an example of B ', mention may be made of:
-CHZ S-or CH2-S-or - (CH2) 3-NH-or - (CH2) 4-NH-wo 96ro0229 21920-7 3 PCa / FR95 / 00790 (io As an example of P, or P 'or P ", mention may be made of polylysine, especially gluconoylated polylysine.
According to an advantageous embodiment of the invention, one or more oligosides are linked to the same molecule, matrix or particle, P, constituting compounds which can be represented by the general formula (II) oligosidyl - N-CO (-D) a (B) b-CH [- (CHDp]] - (II) COX
in which:
* A = 0or1, * J0ou1, * B = 0or1, * p 2 to 4, especially 2, * provided that ** a = b = 0, when j 1, which results in the presence of a cyclic molecule, ** or a = b = 1, when j 0 which implies the absence of a group (CHZ) p, D represents a residue of an organic acid of formula DCOZH, especially H or an alkyl chain of 1 to 10 carbon atoms, in particular CH3, B represents H, an alkyl of 1 to 10 carbon atoms, or a chain of an amino acid such as CH (CH3) 2, CHZOH, CH3, and preferably H
* X represents:
either the group [NH- (A;) - CO] m P, either the group [NH- (A;) - CO] m RP
m being an integer of 0 to 10, preferably 0 to 5 and advantageously 1 or 2, R and P being as defined below, Al represents an organic radical such as an alkyl chain of 1 to 10 carbon atoms, especially (CH 2) II W- (CH 2) n, n + n 'representing a integer from 0 to 10, W being CHY, Y being H, alkyl of 1 to 6 linear or branched carbon atoms, an amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenyl, * 096/00229 2192073 `- PCgYFR95 / 00790 R representing a group having an alcohol, phenol, thiol or amine, * P represents a matrix as a support for affinity chromatography;
a gold ball, latex, for histology and cytology;
= a protein for visualization, purification, etc.
receptors specific to osides, so-called receptors lectins, adhesins, agglutinins etc;
a lipid for the characterization of the preceding receptors;
oligonucleotides to selectively increase their capture by target cells;
a protein or polymers for targeting drugs, oligonucleotides or genes.
An advantageous class of compounds according to the invention corresponds to the formula general (III) oligosidyl - N-CO-CH2 (III) -Cox wherein X represents [NH- (Al) -CO] m P or (NH- (A) -CO] m RP, A ;, m, P
and R having the meanings indicated above.
An advantageous class of compounds according to the invention corresponds to the formula oligosidyl - N-CO-CH2 (III) COX

WO 96100229 "3" PCTIFR95100790 in which X represents:
[NH- (A)) - COIm P, P "
or [NH- (A)) - CO] m RP, P "
A, m, P, R and P "having the meanings indicated above.
Another advantageous class of compounds according to the invention is general formula (IV) oligosidyl - N-CO-D (IV) B-CH-COX

in which D and B have the meanings indicated above, and X
represent [NH- (A 1) -CO] m P or [NH- (A 1) -CO] m RP, A 1, m, R and P having the meanings indicated above.
Another advantageous class of compounds according to the invention corresponds to the formula (IV):

oligosidyl - N-CO-D (IV) B-CH-COX
in which D and B have the meanings indicated above, and X
represent:
[NH- (Al) -CO] m P, P "
or [NH- (A;) - CO] ID RP, P "
AI, m, R, P and P 'having the meanings indicated above.

& O 96/00229 PCd '/ FR95 / 00790 Another advantageous class of compounds according to the invention is general formula (V) oligosidyl - N-CO-CH2 (V) COX

wherein X is [NH- (A)) - CO] m P or [NH- (A)) - CO] m RP, P, A; m and R having the meanings indicated above.
The subject of the invention is also new products, capable of in particular to serve as intermediate products for the preparation of compounds of the invention, said products comprising an oligoside bound to a molecule having a nitrogen atom, carried by a carbon in a group C = O, and at least one functional group, especially one, two or three functional groups, the covalent bond between the oligoside and the molecule effected through the aforesaid nitrogen atom.
The products of the invention advantageously correspond to the formula general oligosidyl - N-CO (-D) a (Zi) b CH [- (CH 2) p] j (Ia) COXI
in which:
* A = 0or1, * J = 0or1, * B = 0or1, * p = 2 to 4, especially 2, * provided that ** a = b = 0, when j = 1, which leads to the presence of a cyclic molecule, ** or a = b = 1, when j 0 which implies the absence of a group (CH?) P, D represents a residue of an organic acid of formula DCO2H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in particular CH 3, * Zi represents * B, B being selected from: H, an alkyl chain of 1 to 10 carbon atoms carbon, or a side chain of an amino acid such as CH (CH 3) 2, CH 2 OH, CH3, and preferably H, or B ', B' being chosen from: an alkyl chain of 1 to 10 carbon atoms carbon, or a side chain of an amino acid, said chains containing a functional group such as carboxylic, SH, OH or amine, free or protected, * X ~ represents . the group [NH- (Ai) -CO] ,,, -R, . or the group [NH- (A;) - CO] ,,, - Q

R represents a compound having an alcohol, phenol, thiol or amine, Q represents OH, OCH3, OCH2-C6H5, O-Cf, Hs, O-C6F5, O-pC6H4-NO2, O-1 ~ CO-CH2 where m is an integer of 0 to 10, preferably 0 to 5 and advantageously 1 or 2, * AT; represents an organic radical such as an alkyl chain of 1 to 10 atoms of carbon, especially (CHz), - W- (CH,) ,,,, n + n 'representing a whole number from 0 at 10, where W is CHY, Y is H, alkyl of 1 to 6 carbon atoms linear or branched, an amino acid residue, natural or synthetic, or W
representing a aromatic compound, especially phenyl.
The invention also relates to products of general formula (Ia) oligosidyl - N CO (-D) a (Zi) b-CH [- (CH 2) p] j (la) coxi in which:

14a * A = 0or1, * J = 0or1, * B = 0or1, * P = 2A4, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of a group (CH2) p, * D represents a residue of an organic acid of formula ECO ~ H, E being H
or an alkyl chain of 1 to 10 carbon atoms, * Z, represents * B, B being selected from: H, an alkyl chain of 1 to 10 carbon atoms carbon, or a side chain of an amino acid, or B ', B' being chosen from: an alkylidene chain of 1 to 10 atoms of carbon, or a side chain of an amino acid, said chains containing a functional group, * X, represents . the group [NH- (A) -CO] ,,, -R, . or the group [NH- (Ai) -CO] õ-Q

R represents a compound having an alcohol, phenol, thiol or amine, Q and selected from the group consisting of OH, OCH3, OCH2-C6HS, O-C6H5, O-C6F5, O-pC6H4-NO2, and O-hj-CO-CHZ

where m is an integer from 0 to 10, * Ai represents an organic radical.

These groups are acylated glycosylamine derivatives.
The acylated glycosylamine derivatives are stable in an aqueous medium in a wide pH range on both sides of neutrality. This means that he product 14b less than 1% hydrolysis at pH 5-8, for 24 h, whatever the temperature between 0 and 95 C.
Sudits products of formula (la) can be used as is.
The aforementioned products of formula (Ia) can also be used to prepare the compounds of the invention of formula I.

An advantageous class of products according to the invention, corresponds to the formula General (IIa) oligosidyl - N-CO (-D) a (B) b CH (- (CH7) p] j (IIa) CO [NH-Al-CO] m R
in which:
* A = 0or1, * J = 0or1, * B = 0or1, * p = 2 to 4, especially 2, * provided that ** a = b = 0, when j = 1, which results in the presence of a cyclic molecule, ** or a = b = 1, when j = 0 which implies the absence of a group (CH) p, D represents a residue of an organic acid of formula DCO2H, especially H or an alkyl chain of 1 to 10 carbon atoms, in particular CH3, B represents H, an alkyl of 1 to 10 carbon atoms, or a chain of an amino acid such as CH (CH 3) 2, CH 2 OH, CH 3, and preferably H
where m is an integer of 0 to 10, preferably 0 to 5 and advantageously 1 or 2, * AT; represents an organic radical such as an alkyl chain of 1 to 10 carbon atoms, especially (CHZ) to W- (CH2),, n + n 'representing a integer from 0 to 10, W being CHY, Y being H, alkyl of 1 to 6 linear or branched carbon atoms, an amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenyl, R represents a compound having an alcohol, phenol, thiol or amine.
The above products of formula (IIa) can be used to prepare the compounds of formula (II).

Another advantageous class of products according to the invention corresponds to the general formula (IIla) oligosidyl - N-CO-CH2 (IIIa) f I

CO [NH-A; -CO] m R
in which A, m and R have the meanings indicated above.
The above products of formula (IIIa) can be used to prepare the compounds of formula (III).
Another advantageous class of products according to the invention corresponds to the general formula (IVa) oligosidyl - N-CO-D (IVa) B-CH-CO [NH-A; -CO] m R

wherein D, B, m, A; and R have the meanings indicated above.
The above products of formula (IVa) can be used to prepare compounds of formula (IV).
Another advantageous class of products according to the invention corresponds to the general formula (Va) oligosidyl - N-CO-CH2 (Va) CO [NH-A; -CO] .- R
in which A ;, m and R have the meanings indicated above.
The above products of formula (Va) can be used to prepare the compounds of formula (V).
Products of formula (VI) oligosidyl - NH - CH - CO - [NH- (AJ-CO] m - R

B (VI) 0 96/00229 219 2 0 7 3 PNt95 / 00790 in which B, A ;, m and R have the meanings indicated above, are of the intermediate products obtained during the preparation of the products above and are new.
Products of formula (VII) oligosidyl - NH - CH - CO - [NH- (A) - CO] m - R

(CH2) p (VII) in which p, A, R, and m have the meanings indicated above, are of the intermediate products obtained during the preparation of products defined above and are new.

In all the compounds or products of the invention, R may represent the following radicals:

OH, O-CH 2 -C 6 O 5 O-C 6 H 5 O-C 6 F 5 O-pC 61 14 -NO 2 CO-CHZ
-NH-pC6H4-N = C = S and its precursors:
-NH-pC6H4 NO2 -NH-NH2-PC6Ha -NH-CH2- (CH2) m C (= NH2 +) OCH2 -NH-CH 2 - (CH 2) m CN
-NH-CH, (CH2) m CH2-NH-CO-CH2- (CH2) m C (= NH2) OCH3 -NH-CHa- (CH 2) .- CH 2 -NH-CO-CH 2 (CH 2 CN

these compounds allow an addition to an amino compound.
R can also represent:

-NH-CHZ- (CH2) m CHr-N
derivative of maleimide -NH-CH2- (CH2) m CH2-NH-CO-mC6H4-N
derived from m-maleimidyl benzoyl 0 & 0 96/00229 2192073 PG rIFR95 / 00790 -NH-CH2- (CH2) m CH2-NH-CO-pC6H, o-CH2-N
derived from N-methylmaleimidyl p-cyclohexylcarboxy -NH-CH2- (CH2) m CH2-NH-CO-CH2-T T = Br, I, CI
haloacetyl derivative -NH-CHZ-CHa-NH-CO-CH 2 - (CH 2) m SS-Pyr dithiopyridine derivative NB: -Pyr: -2-pyridine -NH-CH2- (CH2) m CHZ-SS-Pyr dithiopyridine derivative these compounds allow addition to a thiol.
R can also represent:
H
-NH-CH2- (CH2) .- CH2-NH-CO (CH2) 4 NH
biotin derivative this compound comprising a biotin residue allowing the formation of a complex with avidin or streptavidin.
R can also represent:

-NH-CH2- (CH2) m CH2-NH-CO / N3 derived from -4-azidobenzoyl -NH-CH2- (CH2) m CH2-NH-CO. \ / Nz derived from 4-azido 2-nitrobenzoyl xp2 .
WO96 / 00229 2192073 PCT1FR95 / 00790 =

-NH-CHZ- (CH 2 CH 2 -NH-CO-CH - (CH 2) S NH
4-azido derivative 2-nitroanilide NOZ

~ ~ N3 -NH-CH2- (CH2) m CH2 -NH-CO-CH2 c.1i 7-azido 4-methylcoumarin 3-acetyl derivative -NH-CH2- (CH2) m CH2-NH-CO9-N, OH
derived from 4-azidosalicyclic or 4-azido 2-hydroxybenzoyl these compounds permitting covalent attachment to a receptor, a enzyme, an antibody, specific for the carbohydrate moiety by activation photonics.
R can also represent:
-NH- (CH2) m pC6H4OH

-NH-CHa- (CHz) m CHa, -NH-CO- (CH2) m pC6H40H
these compounds for fixing one or two iodine atoms, particularly of a radioactive iodine atom.

R can also represent:

-NH-CH2- (CH2) m CH2-NH-CS-NH, =
. / I

derived from COZ
fluorescein / I \

-NH-CH2- (CH2) n CHZ-NH-SOa derived from dansyl N (CH3) Z
0 0 ~ Nti2 -NH-CH2- (CH2) m CH2-NH-CO-CH2 CIi3 7-amino derivative 4-methylcoumarin 3-acetyl COOH
-NH

derived from amino-fluorescein WO 96/00229 ~ ji ~ Q 7 3 PCT / FR95100790 -NH-CHZ- (CH 2) m CH 2 -NH

NOT/

derived from nitrobenzoxadiazole these fluorescent compounds to visualize the position of the oligosidylpeptide in a cell, tissue, organ, gel or electrophoresis band, etc.
In the compounds of the invention, P may represent an oligopeptide, or a polypeptide, especially gluconoylated polylysine, and P 'or P "may represent an oligonucleotide, or R can represent fluorescein or one of its derivatives or another fluorescent derivative, and P 'or P "may represent a oligonucleotide. P can also represent a therapeutic agent or any molecule of interest.
In the compounds and products of the invention, the oligoside residue has from 2 to 50 oses and in particular is selected from Iacto-N-Tetraose Galp 3 GIcNAcp 3 Gal (3 4 Glc neolacto-N-tetraose Ga1p 4 GIcNAcp 3 Gal (3 4 Glc GroupH Fuca2Ga1p 3Ga1p 4Glc Lewisa Gal5 3 GIcNAcp 3 Gal (3 4 Glc Fuc has 4-T

Lewisx Gal (3 4 GIcNAcp 3 Galp 4 Glc Fuc has 3-I ' OWO 96/00229 PCT / F'R95100790 Lewisb Fuca 2 Ga1p 3 G1cNAc (3 3 Gal (3 4 Glc Fuc a 4-I ' LewisY Fuc has 2 Ga1G3 4 G1cNAc (3 3 Gay (3 4 Glc Fuc has 3-1 Disialolacto-N-Tetraose Neu 5Aca 3 Gal (3 3 G1cNAc j3 3 Gal (3 4 Glc Neu 5Aca 6-I ' Complex type with 3 antennas Gal (34GIcNAcp2 Mana6 Gal (3 4 GIcNAc (3 4 Man (3 4GIcNAc Mana6 Gal 4 G1cNAc 9 2 Sialylactose 3 Neu 5Ac a 3 Ga14 Glc Sialylactose 6 Neu 5Ac a 6 Gal (3 4 Glc Disialylactose 3 Neu 5Ac a 8 Neu 5Ac a 3 Gal (t 4 Glc simple or complex osides recognized by lectins membrane, and selected from:

at. Asialo-oligoside of triantennary lactosamine type: receptor of asialoglycoprotein Gal (3 4GIcNAcg 2 Mana 6 Manp 4GIcNAc (3 2 4G1cNAc (3 -a Gal (3 4G1cNAc (i 4 Mana 3 Gal (i 4GIcNAc (3 2 b. Asialo oligoside tetraanthene-type lactosamine: receptor asialoglycoprotein Ga1R 4G1cNAc (3 6 Mana 6 \
Gal (3 4G1cNAc (3 2 Man (3 4GIcNAc (i 4GIcNAcR -a Gal (3 4G1cNAc (3 4 Mana 3 Gal (3 4GicNAc (3 2 WO 96/00229 ~ õ 1 i ~ 2 0 7 3 PCTIFR95100790 24 vs. Lewis x: LECAM 213 Ga1R 4 G1cNAc (3 3Galp ->
Fuca 3 d. Sialyl Lewis x: LECAM 3/2 Neu5Aca3Ga1R 4 G1cNAcR 3Ga1 (3 ~
Fuca 3 "He ' e. Sulphated Lewis X derivative (âINK1): LECAM 1 (SOg) 3Glc UAj3 3Ga1 (3 4 GIcNAc (3 3Ga1p 4Glc ->
Fuca 3 f. Oligomannoside: mannose receptor Mana. 2Mana 6 \ Mana 6 Mana Manji 4G1cNAcR 4G1cNAc5 ->
Mana 2Mana Mana 3 /

boy Wut. Phosphorylated oligomannoside: mannose 6 phosphate receptor (HP03) 6 - '~ -Mana 6 Mana 2 Mana 6 Mana 3 - "' Ivlan (i 4GIcNAc (3 4G1cNAc (i -a (HP03) 6 Mana 2 Mana 3 Mana 2 - "

h. Sulphated lactosamine oligosaccharide: GaINAc receptor 4 sulfate (S03 ') 4GalNAc (3 4GlcNAc (3 2Mana 6 \
Man (3 4GleNAc (3 4G1cNAcj3 - ~
(S03 ') 4Ga1NAc (3 4G1eNAc ~ 3 2Mana 3 /

To prepare the compounds of the invention, it is first necessary to prepare the products (acylated glycosylamine derivatives), which serve in particular intermediaries the preparation of said compounds of the invention.
The invention also relates to a process for preparing the compounds mentioned above, characterized in that:
- an oligoside having an ose is condensed in a suitable solvent reducer free, on the nitrogen atom of an intermediate molecule, this nitrogen atom belonging to an amine group, attached to a carbon atom placed in a of a C = O group, the intermediate molecule having no side chain containing a group functional, to obtain a derivative of glycosylamine in which the ose terminal of the oligoside retains its cyclic structure, and in which the hydroxyl semiacétalique is replaced by the amine a of one of the starting sudite molecules, the glycosylamine derivative obtained after the previous step is acylated by addition of an organic acid activated by a conventional activator, to obtain a derivative N-acylated glycosylamine.
The invention also relates to a process for preparing the compounds mentioned above, characterized in that:
- an oligoside having an ose is condensed in a suitable solvent reducer free, on the nitrogen atom of an intermediate molecule, this nitrogen atom belonging to an amine group, attached to a carbon atom placed in a of a C = O group, the intermediate molecule having a side chain containing a group functional, to obtain a glycosylamine derivative in which the terminal ose of the oligosaccharide retains its cyclic structure, and in which the semi-acetal hydroxyl is replaced by the amine a of one of the above starting molecules, when the intermediate molecule has a side chain whose group functional is protected or not, the glycosylamine derivative obtained is the outcome of the preceding step by adding an activated organic acid with an activator classic, to obtain an N-acylated glycosylamine derivative, 215a when the intermediate molecule has a side chain containing a carboxylic group, the above-mentioned carboxylic group is activated so that it react intramolecularly with the aforesaid amine a, resulting in cyclization to interior of the above starting molecule to obtain a N-glycosylamine derivative.
acylated when the intermediate molecule has a side chain containing a carboxylic group, the acylating agent can be added in the form of an ester active.
The invention also relates to a method for preparing the products (acylated glycosylamine derivatives) defined above, characterized in that:
an oligoside having a free reducing terminal ose is condensed on atom of an intermediate molecule, this nitrogen atom belonging to a group amine, bound to a carbon atom placed in a of a group C = O, the molecule intermediate possibly having a lateral chavie containing a group such as OH, SH, NH2 or COOH, free or protected, this molecule intermediate being selected from the following intermediate niolecules:
acid a amine, natural or synthetic, amino acid derivative, amino acid in position NOT-terminal of a peptide, or a peptide derivative, possibly in the presence a catalyst such as imidazole, in a suitable solvent to obtain a derived from glycosylamine in which the terminal ose of the oligoside retains its structure cyclic, and wherein the semi-acetal hydrosyl is replaced by the amine a of one of the said starting molecules.
when the intermediate molecule does not have a side chain containing a functional group as defined above, or has a side chain whose functional group is optionally protected, the derivative of glycosylamine obtained at the end of the preceding stage by addition of an organic acid activated by a conventional activator such as carbonyl diimidazole, BOP (benzotriazolyl N-oxy tris (dimethylamino) phosphoniumhexafluorophosphate) or HBTU (O-benzotriazol-1) yl-N, N, N ', N', tetranlehyluronium hexafluorophosphate) to obtain a derivative of N-acylated glycosylamine, possibly followed by deprotection of the group functional of the aforesaid side chain, with a view to a possible substitution, when the intermediate molecule has a side chain containing a carboxylic group, the above-mentioned carboxylic group is activated so that it react intramolecularly with the aforesaid amine a, resulting in cyclization WO 96/00229 PCa / FR95 / 07790 within the aforementioned intermediate molecule, to obtain a derivative of N-acylated glycosylamine, when the intermediate molecule has a side chain containing a carboxylic group, the acylating agent can be added in the form of a active ester.
The aforementioned method for preparing the invention of the derivatives of glycosylamine thus comprises two steps, a condensation step of a oligoside on an intermediate molecule to obtain a derivative of glycosylamine, and an acylation step of the aforesaid glycosylamine derivative.
In the acylation step envisaged in the process of the invention, always use an activator, whether or not the intermediate molecule has a side chain containing a functional group.
Moreover, it should be noted that the step of condensation of the oligoside on the intermediate molecule, to obtain a glycosylamine derivative, as well as the acylation step of the aforesaid glycosylamine derivative are carried out in presence of suitable organic solvents.
One of the advantages of using an organic solvent is especially allow to couple oligosides peptides and derivatives that are few or very not very soluble in water.
These cases can be schematized as follows:
1) the intermediate molecule does not have a side chain containing a functional group, Oligoside + NH2-CH (R1) -CO-R2 = * Oligosyl-N-CH-RI CO-RZ
+ R3-CO2H + activator Oligosyl-N (CO-R3) -CH (RI) -CO-R2 . Ri representing a residue of an organic molecule not comprising protected functional group, or R1 may also represent H;
. R2 representing a residue of an organic molecule such as -CO-R2, either an ester or an amide:
. R3 represents a residue of an organic molecule not containing, preferably, free functional group.
The set R3-CO2H + activator, can be replaced by the product activated or by a hydride.

& 0 96100229 219 the M 073 PCT / FR95100790 In the above, we can also include the case where R3 has a functional group, and in this respect reference is made to paragraph 1 bis below.

1a) the intermediate molecule does not have a side chain containing a functional group, but the acylating agent is bifunctional, oligoside + NH 2 CH (R 1) -CO-RZ => oligosyl-NH-CH (R 1) -CO-R 2 + R3 COZH + activator oligosyl-N-CH (RI) -CO-R2 R3 representing an organic molecule comprising a second functional group such as, in particular SH, free or protected, or -CO2H.
Alternatively, the assembly R3-COZH + activator can be replaced by activation product: R3-CO-activated.
For example, R3 CO-Cl, (R3-CO) 20 or R3 CO-ON-CO-CH2 or else a cyclic anhydride:
CHZ CO-O
1 ~
(CHZ) ,; CO, for example with n integer equal to 1, 2, 3 or 4;
or a thioester:
CHZ CO

(CH2), S, where n is 1, 2, 3 or 4, preferably n = 2 When the acylation of the bound nitrogen on the oligoside is acylated by an anhydride cyclic, the product obtained is of the type:
Oligosyl-N- (CO-CH2 (CH2) n-COZH) -CH (R1) -CO-RZ
The carboxylic group is usable for a coupling reaction on an organic molecule or matrix, or a particle having a functional group (hydroxyl or amine, for example).
When the acylation of the bound nitrogen on the oligoside is acylated by a thio ester cyclic, the product obtained is of the type:

WO 96100229 _ 219 ~ O ~ V PCTIFR95 / 00790 Oligosyl-N- (CO-CH 2 (CH 2) n SH) -CH (R 1) -CO-R 2 The thiol group is usable for a coupling reaction on a soluble or non-soluble molecule, capable of being substituted by a thiol, for example a dithiopyridine or a maleimide derivative.

2) the intermediate molecule has a functional side chain and there is no cyclization, Oligoside + NH2-CH (R4) -CO-R2 =:> Oligosyl-NH-CH (R4) -CO-R2 + R3-CO2H + activator ~
Oligosyl-N (CO-R3) -CH (R4) -CO-R2, R2 and R3 having the meanings indicated above, and R4 representing a residue of an organic molecule having a functional group, in in particular a carboxylic group, R4 especially representing CHZ-CHZ
CO2H.
In this case, a product of activation of R3-CO2H as defined above.
The functional group contained in R4 is available for a condensation or substitution reaction on a soluble molecule or insoluble or on a matrix, or a particle comprising a group capable of giving a covalent bond with the functional group of R4, for example an amine when R4 has a carboxylic group.

3) The intermediate molecule has a side chain containing a carboxylic functional group, and cyclization is carried out, and to stare the molecule on 1 or 2 molecule (s), matrix (s) or particle (s), ib 0 96/00229 2 l ^ Z0 [J PCTIFR95 / 00790 Oligoside + NHZ-CH (RS-COZH) -CO-R2 => Oligosyl-NH-CH (R5-CO2H) -CO-R2 + activator ~
Oligosyl-N-CH-CO-R2 CO-RS
R2 having the meanings indicated above, RS-COZH being a residue of an organic molecule such that - (CH 2) a COZH
. n being an integer of 1 to 10, preferably 2 or 3.
The glycopeptide thus obtained can be used by reacting a functional group existing in the free state or converted into a group active on R2. For example, if R2 represents the para-nitroanilide group, the group NOZ is reduced to N112, then converted to isothiocyanate -N = CS, which is an excellent reagent for amines and alcohols.
Examples of intermediate molecules include:
Oligosyl-N-CH-CO-NH-PC6-H4-NCS
( CO-RS
Oligosyl-N-CH-CO-NH- (CH2) II C (= NHZ +) OCH3 CO-RS

Oligosyl-N-CH-CO-NH- (CH) õ +

Oligosyl-N-CH-CO-NH- (CH?) O +, - SS-Pyr CO-RS

withn = 1A10. _ The invention also relates to the preparation of the compounds of the invention, characterized in that one or more products (derived from acylated glycosylamine) of the invention, having either a group R as defined above, activated or activatable, a group B 'containing an activated functional group or activatable, that is a group A; containing a functional group that can react on a molecule, nlatrice or particle, respectively P, P 'or P ", containing a group functional, to obtain a product of the type:
(glycopeptidyl-R) õ-P, (glycopeptidyl-B ') õ-P' or (glycopeptidyl-A;) nP "
The invention also relates to the preparation of compounds of the invention, characterized in that one or more products (derived from acylated glycosylamine) of the invention, on the one hand carrying a group R such that defined above on, activated or activatable, on the other hand a group B 'containing a group activated or activatable, or a group A; containing a functional group on molecules, matrices or particles P and P ', or molecules, matrices or particles P and P '.
The invention also relates to the preparation of compounds of the invention, characterized in that one or more products (derived from acylated glycosylamine) of the invention, on the one hand carrying a group R such that defined above on, activated or activatable, on the other hand a group B 'containing a group activated or activatable, and a group A; containing a functional group respectively on of the molecules, matrices or particles P, P 'and P ".
The invention also relates to a method for preparing a compound mentioned above, characterized in that the ori makes react one or more products (acylated glycosylamine derivatives) mentioned above above, carrying a Q group, activated or activatable, reacting on a molecule, matrix or particle P, P 'and P ", one or more products (acylated glycosylamine derivatives) mentioned above above, bearing either a group R, activated or activatable, or a group B ' containing a activated or activatable functional group, ie a group A; containing a group functional on a molecule, a matrix or a particle, respectively P, P 'and P ", containing a functional group, to obtain a product of the type:
(glycopeptidyl-R) nP, (glycopeptidyl-B ') õ-P', or (glycopeptidyl-A;) õ-P ", n> 1, 30a one or more products (acylated glycosylamine derivatives) of the invention, carrying on the one hand a group R, activated or activatable, on the other hand a group B ' containing an activated or activatable group, or a group A; containing a group on molecules, matrices or particles P and P ', or molecules, nlatrices or particles P and P ", one or more products (acylated glycosylamine derivatives) of the invention, carrying on the one hand a group R, activated or activatable, on the other hand a group B ' containing an activated or activatable group, and a group A; containing a group functional groups respectively on molecules, matrices or particles P, P 'and P "
R, B ', A, P, P' and P "being defined above.
The molecules, matrices or particles involved in the preparation of compounds of the invention may advantageously be a natural molecule or synthetic, whether or not soluble in an organic solvent, aqueous or hydro-organic, a nanoparticle, a lipid vesicle, a solvent-insoluble matrix organic, aqueous or hydro-organic, a protein, a lipid, a nucleic acid, a oligonucleotide, a polylysine, a polymer that is insoluble in a solvent organic, aqueous or hydroorganic, a ball of latex or a ball of gold etc ...
In a detailed manner, with regard to the process according to the invention, the oligoside, having a free reducing ose is placed in a suitable solvent, the dimethylsulfoxide, N-methylpyrrolidone or dimethylformamide, for example, in the presence of a equivalent quantity or two equivalent quantities of a molecule of departure selected from: a natural or synthetic amino acid, a peptide, a derivative acid amine or a peptide derivative.

& 0 96100229 219 2 0 7 3 PCT / FR95 / 00790 By appropriate solvent, is meant a solvent allowing, on the one hand the solubilization of the compounds to be condensed, and secondly, the solubilization of the compounds resulting from condensation.
The main product of the reaction is the condensation product of the type "glycosylamine derivative": the reducing terminal ose retains its structure cyclic, its semiacetal hydroxyl is replaced by amine a the amino acid, the amino acid derivative, or the amino acid in the N position terminal of a peptide or a peptide derivative.
In a second step, the glycosylamine derivative thus formed is acylated by addition of an activated organic acid or in the case of amino acid wearing a side chain containing a functional group such as a chain lateral carboxylic acid, as in the case of glutamic acid or its homologues, by addition of a carboxylic group activator.
The product thus formed is an N-acylated glycosylamine derivative.
The acylated glycosylamine derivatives are isolated by chromatography of molecular sieving or by any other conventional purification technique known to those skilled in the art.
The acylated glycosylamine derivatives are then used to substitute a compound (protein, lipid, nucleic acid, oligonucleotide, polylysine, insoluble polymer, latex beads, gold ball, etc.).
The amino acid moiety, or peptide or substituent is of the peptide to carry out the condensation reaction, so that the oligoside retains all its properties and accessibility to serve as substrates or recognition signal.
According to the following general scheme:
oligoside + NH2 - CHB - CO - R
_> Oligosidyl - NH - CHB - CO - R
=> oligosidyl - N - CO-D

CHB-CO-R
=> oligosidyl - N - CO-D
CHB-CO-P

WO 96/00229 2 ~ n ~ 0 r7 ~ ~ PCT / FR95 / 00790 =
.7 1 32.
For example, condensation of lactose with glycylparanitroanilide writes GaIP4 Glc + NH2 - CH2 - CO - NH - pC6I-14 - NOZ
=> Gal (34Glcp -NH-CH2-CO-NH-pC61-14 -NOa The addition of acetic acid and an organic acid activator leads to:
=> GaIP4 Glc - N - CO - CH3 CHZ - CO - NH - pCb H4 - NO2 In the example chosen, the nitro group can be reduced quantitatively to amine, then the amine is quantitatively transformed into isothiocyanate (according to Roche and B1, 1983, G. Cell Biochem 22, 131-140, Monsigny et al. 1984, Biol. Cell 21, 187-196).
The compound thus activated can react in a slightly alkaline medium on a amine carried by a protein, a lipid, a polymer, (polylysine by example), a solid support having amine groups, or a amine-substituted oligonucleotide, or on an amine carried by a molecule or a suitable body, such as NH2 - P.
By way of example, the following reaction scheme can be written:
=> GaIP4 Gic - N - CO - CH3 CH2 - CO - NH - pC6 H4 - NOZ
~ H2 GaIP4 Glc - N - CO - CH3 CH - CO - NH - pC6 H4 - NH2 ~ CsCI2 y 73 => Ga1j34Glc-N-COCH3 CHa-CO-NH-pC6ü4-N = C = S
~ PNH2 => Ga1p4 Glc - N- CO CH3 S
CHZ-CO - NH - C - NH - P

In the case where the amino acid or the derivative is an amino acid having a side chain containing a carboxylic group as is the case for glutamate, the reaction is written, for example:
Ga1Gi4 Glc + Glu NH - pC6H4 - NOZ
=> Gal (34Glc (3-NH-CH-CO-NH-pCJ-I4 -NO2 The addition of a carboxylic group activator leads to the product expected:

Gal (34 GIcp - N - CO - CH2 CO-NH-pC, J-14-NO2 In a similar way, this compound can be activated and can react on a amine NHZ - P, to give the final product:

Gal (34 Glcp - N - CO - CHZ

~ S
II
CONH-p-CJ-14-NH-C-NH-P

WO 96/00229 219 2 0 7 3 PCl '/ FR95 / 00790 EXAMPLES
ExamFlel Preparation of an acylated glycosylamine derivative: N-acetyl lactosyl ji glycyl-pNA.
Glycyl amido paranitrophenyl (0.1 mmol) and lactose (0.1 mmol) are dissolved in 1 ml of dimethylsulfoxide.
The solution is maintained at 50 ° C. for 48 hours. 0.1 mmol of gly-pNA in 0.5 ml dimethylsulfoxide at times 12 h, 24 h and 36 h. We cool to 25 C.
0.44 mmol of BOP, hexafluorophosphate, is then added.
benzotriazolyl-1-tris (dimethylamino) phosphonium, and 0.44 mmol of acetate of diisopropylethylamine and stirred for 3 h at 25 ° C.
The expected product is purified by molecular sieving in a column Ultrogel GF05 (90 cm x 2.3) using as solvent 0.1 acetic acid, M containing 3% n-butanol; before the injection, the reaction products are diluted by addition of 7.5 ml of chromatographic solvent.
The expected product comes out ahead, followed by excess reagents and dimethylsulfoxide.
The product: N-acetyl lactosyl (3-gly-pNA is obtained by lyophilization of the solution eluted from the column.

Exemption 2 Preparation of an acylated glycosylamine derivative intramolecularly: the N-lactosyl (3-pyrogiu-pNA.
Glutamyl paranitroanilide (Glu-pNA) (0.1 mmol) and lactose (0.1 mmol) are dissolved in 1 ml of dimethylsulfoxide. The solution is maintained at 50 C for 48 h. 0.1 mmol of glu-pNA dissolved in 0.5 ml of dimethylsulfoxide at times 12 h, 24 h, and 36 h. It is then cooled to 25 C.
0.44 nunol of BOP is then added and the mixture is stirred for 3 h at 25 ° C.
The expected product is purified under the same conditions as in the previous example.

Example 3 Preparation of an organic compound having the side chain of the amine contains a functional group and use of the functional group for form a conjugate with an oligonucleotide.

0 96/00229 ~ PCr / F1295100790 The oligoside is incubated in the presence of two to four equivalents of the derivative S- (2-thio-pyridine) cysteinyl-p-nitroanilide NHa CH-CO-NH p-C6-H NOZ

CH2 SS / \
NOT
in solution in N-methylpyrrolidone (or in dimethylsulfoxide or 1V-dimethylformamide) and in the presence of four equivalents of imidazole 20 h to 50 C. The solution is cooled to 25 C. Ten equivalents are then added acetic acid, imidazole and BOP. The acylation reaction is carried out in a half hour.
The glycopeptide is isolated by molecular sieve chromatography (Ultrogel GF05 column, for example) in 0.1M acetic acid. The fraction containing the purified glycopeptide is frozen and lyophilized.
The glycopeptide dissolved in a 0.1M sodium acetate buffer, pH 6 is reduced by adding an equivalent of TCEP (tris-carboxyethylphosphine) to 25 C for 30 min (see K. Arar et al., 1993, Tetrahedron Letters 34, 8087-8090, JA Burns et al., 1991, J. Org. Chem., 56, 2648-2650). We then add an equivalent of an oligonucleotide substituted on its 5 'end by a substituent terminated with a dithio-2-pyridine group.
The glycopeptide-oligonucleotide conjugate formed has the general structure next:
oligosidyl-NH-CH-CO-X

CH3-CO CH2-SS-5'-Oligonucleotide wherein X is NH-p-C6 H N02, and 5 'is the joining arm the first S of dithio-2-pyridine to the primary hydroxyl of the first nucleotide of the oligonucleotide.

Example 4 Preparation of lactosyl-pyroglutamyl-paranitroanilide (in the dimethylsulfoxide).
= Ga1R4Glc lactose (0.15 mmol) is dissolved in 1.25 ml of dimethylsulfoxide (CH3-SO-CH3). 0.30 mmol of Glu-pNA is added to 1.25 ml dimethylsulfoxide containing 0.6 mmol imidaaole (pNA = para nitro-aniline). The solution is kept at 50 ° C. for 20 hours and then cooled down to WO 96/00229 PCT / FR95 / 00790 =

C. More than 95% of the lactose is converted to glycopeptide: lactosylglucose pNA.
0.33 mmol of BOP and 0.6 mmol of imidazole are added. The solution is stirred for 30 minutes at 25 ° C. More than 95% of the glycopeptide is cyclized in lactosyl-pyroglutamyl-paranitro anilide:
Ga1MG1cj3pGlu pNA
pGlu is: the pyroglutamyl residue -NH-CH-CO-~ CHZ
CO-CHZ
ExeIDFlp5 -Preparation of lactosyl-pyroglutamyl p-nitroanilide (in the N-methylpyrrolidone).
GalG34Glc lactose (0.15 mmol) is dissolved in 1.25 ml of N-methylpyrrolidone of formula: CO-CHZ
CH3-N ~ 1 CHZ CHZ
0.3 mmol of Glu-pNA in 1.25 mmol of N-methyl-pyrrolidone containing 0.6 mmol of imidazole. The solution is kept at 50 C
for 20 hours and then cooled to 25 C. More than 95% of the lactose is converted into glycopeptide: lactosyl-Glu-pNA. 0.33 mmol of BOP and 0.6 mmol are added imidazole. The solution is stirred for 30 min at 25 C. More than 95% of the glycopeptide is cyclized to lactosyl-pyroglutamyl p-nitroanilide.
The analyzes are carried out by high-performance column chromatography pressure on a Dioxex device equipped with an amperometric detector. The yields are calculated relative to an internal control (sorbitol) added to the initial solution of lactose.
The retention times of the compounds under standard conditions, expressed in minutes, are:

Lactose. 9.9 t 0.1 Lactosyl-Glu-pNA. 21.4 0.1 Lactosyl-p-Glu-p-NA. 16.2 0.1 Imidazole. 4.3 t 0.1 Sorbitol. 2.7 0.1 ~

Fxsmgjç 6 Preparation of a compound of formula Oligosidyl glycopeptidyl oligopeptide oligonucleotide The preparation can be done according to the following reaction scheme.
The starting material indicated below can be obtained as indicated previously, about the preparation of the products of the invention.
Glycosyl-N-CH-CO-NH-CH-CO-OCH3 I {
CH2-SS-Pyr ~ NHZ NHa, 4 C
Glycosyl-N-CH-CO-NH CH-CO-NH-NH2 CH2 C {I H2-SS-Pyr CO-eH2 ~ HNO2 4 C, pH 1 Glycosyl-N-CH-CO-NH-CH-CO-N3 ! 2 ~ H2-SS-Pyr pH 8 J ~ NH2 ........ peptide NHa CH-CO NH-CH-CO -OH
II
R; R; not R: side chain of amino acids Glycosyl-N-CH-CO-NH-CHto NH-CH-CO OH

CH2 CHZ SS-Pyr R n + 1 TCEP, pH 5 TCEP = Tris carboxyethylphosphine Glycosyl-N-CH-CO-NH-CH-CO NH-CH-CO OH

CH2 CH2-SH R; n + 1 pH 7 Pyr-SS-oligonucleotide Glycosyl-N-CH-CO-NH-CH-CO NH-CH-CO OH
1 ~
CH2 LR; n +
CO = eH2 CHZ SS-oligonucleotide The example chosen corresponds to the preparation of a derivative oligonucleotide, such as this derivative has a biological activity in relationship with the sequence of the oligonucleotide selected (sense oligonucleotide, meaning, antigen, lure, etc., is specific for a cellular or viral element of nature nucleic acid or protein), the oligoside petmet to the derivative of being selectively recognized by some cells that possess a membrane receptor (Lectin) having an affinity for the chosen oligoside, the peptide allows the derivative - a times inside the endosomes (intracellular vesicles), thanks to the mechanism of endocytosis due to membrane lectin - to enter the compartments cytosolic, and then nuclear.
The oligonucleotide is an oligomer comprising between 10 and 40 nucleotides, preferably 20 to 25. The oligopeptide is an oligomer having between 20 and 40 amino acids, preferably 20 to 25. This kind of derivatives corresponds to a line of compounds that can be used for drugs.

FxempjsZ
Preparation of a compound of formula Glycosyl-N-CH-CO-NH-CH-CO-NH-Flu ~ 2 JH2-SS-Pyr The preparation of this compound can be done according to the reaction scheme next:

Oligoside + NH2-CH-CO-NH CH-CO-NH-Flu COZH-CHZ-CHZ CH2-SS-Pyr Fmoc-NH-CH-CO2H

CH2-CH2-CO-OC (CH3) 3 + NHa-CH-CO-NH-Flu CHZ SS-Pyr I

Fmoc-NH-CH-CO-NH-CH-CO-NH-Flu I
CHZ-SS-Pyr CHZ-CH-CO-OC (CH3) 3 Fmoc-NH-CH-CO-NH-CH-CO-NH-Flu CHZ-SS-Pyr II

~ NH (C2H5) 2 ~~ S ~ = `}. ~ y, r NHZ CH-CO-NH-CH-CO-NH-FIu CH-SS-Pyr 20h, 50C oligoside, N-methylpyrrolidone Oligosidyl-NH CH-CO-NH-CH-CO-NH-FIu COZH ICHa ( CH2-SS-Pyr 30 min, 25 C y BOP, N-methylpyrrolidone Oligosidyl-N 7-CO-NH-CH-CO-NH-FIu çH2 1 CO-CH2 CHZ-SS-Pyr Oligosidyl-N T CO-NH-CH-CO-NH-Flu 1 ~ H2 -CO-CH2 CH2-ss-Pyr TCEP P- (CH2-COZ) 3 Oligosidyl-N T CO-NH-CH-CO-NH-F1u Pyr-SS-5'-oligonucléoflde Oligosidyl-N T CO-NH-CH-CO-NH-FIu = ~ ClH2 i CO-CH2 CH2-SS-5'-oligonucleotide The example chosen corresponds to the preparation of a glycopeptide derivative fluorescent general purpose.
The fluorescein residue allows the use of glycopeptides to location, visualization, generally, to fms analytical, especially in fluorescence microscopy.
The fluorescent glycopeptide derivative linked to an oligonucleotide may also be used as an antiviral or anticancer agent, to allow at both a study of the biological activity of the derivative and its traffic intracellular, as well as pharmacokinetics, in animals.
In this example, the disulfide bridge is present in the parent compound on a residue Ai of the general formula.

Example 8 Preparation of a compound of formula Oligosidyl-glycopeptidyl - Flu oligonucleotide Oligoside + NHZ CH-CO-NH-Flu CH2-SS-Pyr 20h, 50C N-methylpyrrolidone + Imidazole Oligosidyl-NH-CH-CO-NH-Flu CH2-SS-Pyr CH3 COZH, BOP -WO 96/00229 PCT / FR95,00790 Oligosidyl - N - CH-CO-NH-Flu CH3-CO CHZ-SS-Pyr y TCEP
Oligosidyl - N - CH-CO-NH-Flu CH3-CO CHZ-SH

J ~ Pyr-SS-oligonucleotide Oligosidyl - N - CH-CO-NH-Flu CH3 CO CHZ SS-oligonucleotide What has been said about the glycopeptide derivative of Example 7, applies to this example. It should be noted that in example 8 here considered, the disulfide bridge is present in the parent compound on the Z chain of the general formula.

Bxemole 9 Preparation of glycosylated derivatives of gluconoylated polylysine Gluconoylated polylysine is used to transfer genes into animal cells. The substitution of gluconoylated polylysine by one or more glycopeptides helps to make gene transfer selective.
The glycosylated derivatives of the gluconoylated polylysine penetrate preferably (100 to 1000 times) in cells that express on their surface a lectin (oligoside receptor), which specifically recognizes the oligosaccharide glycopeptide bound to the gluconoylated polylysine.
a) Binding of a glycopeptide to the gluconoylated polylysine via a bridge disulfide.

Gluconoylated polylysine (degree of polymerization 190) containing 60 gluconoyl residues), is substituted by a derivative of dithiopyridine; the polymer (20 mg, 0.33 pmol) is dissolved in 0.5 ml of dimethylsulfoxide.
1 mol (312 g) of N-succinimidyl 3- (2-pyridyldithio) propionate and 20 mol (3.6 l) diisopropylethylamine. The The solution is stirred at 20 ° C. for 15 hours. The polymer is precipitated by addition 10 volumes of isopropanol; the precipitate is recovered after centrifugation (1800 g, 15 min).
After washing with isopropanol, the polymer is dissolved in a buffer 0.1 M sodium phosphate pH 7.2 (1 ml).
The glycopeptide: oligosylpyroglutamyl amido ethylidithiopyridine:
Gal (34GICp-pyroglutamyl-NH- (CH2) 2-SS-pyridine (1 mol) is treated with 1 mole of TCEP (triscarboxyethylphosphine: P (CH2-CHZ COZ) 3) in a 0.1 M sodium phosphate buffer (1 ml), for 1 h at 20 C. This solution is added to the gluconoyl-substituted polylysine solution pyridyldithiopropionate. After 1 h at 20 ° C., the polymer is precipitated by addition of 10 volumes of isopropanol. The precipitate is recovered after centrifugation (1800 g, 15 min) and washed in isopropanol and then dissolved in water and freeze-dried.
The yield of the coupling reaction under the conditions used is equal to or greater than 90%.
The reactions used in this preparation are derived from those described in Midoux, P., Mendes, C., Legrand, A., Rammond, J., Mayer, R., Monsigny, M. and Roche, AC, 1993: Specific gene transfer mediated by lactosylated poly-l-lysine into hepatoma cells. Nucleic Acid Research, 21: 871-878, and in Arar, K., Monsigny, M., and Mayer, R., 1993: Synthetis of oligonucleotide peptide conjugates containing a KDEL signal sequence.
Tetrahedron Letters, 34: 8087-8090.
b) Binding of a glycopeptide to the gluconoylated polylysine via a link thiourea.
Gluconoylated polylysine (degree of polymerization 190) containing 60 gluconoyl residues), is substituted by an activated glycopeptide in the form of of phenyl isothiocyanate.
The glycopeptide oligosylpyroglutamyl p-nitroanilide is reduced to a derivative p-aminoanilide which is then activated to a p-cyanatoanilide derivative:
Gal (34GICp-pyroglutamyl-NH-p-C6H4-NCS according to a protocol adapted from that described in Roche, AC, Barzilay, M., Midoux, P., Junqua, S., Sharon, N. and VVO 96100229 ~ 1 ~~ 0 7 3 PCT1FR95,00790 Monsigny, M. (1983): Sugar specific endocytosis of glycoproteins by Lewis lung carcinoma cells., J. Cell. Biochem., 22: 131-140.
The cyanatoanilide derivative (1 mole) is dissolved in dimethylsulfoxide (1 ml) containing 1 mmol of gluconoylated polylysine and 4 moles of diisopropyl ethylamine. The solution is stirred at 20 ° C. for 24 hours. The glycosylated polymer is precipitated by the addition of 10 volumes of isopropanol; the precipitate is recovered after centrifugation, washed with isopropanol, and finally dissolved in water and lyophilized.
Under the conditions described, the glycopeptide coupling efficiency on the gluconoylated polylysine is greater than 95%

Example 10 Preparation of a glycopeptide (oligosylpyroglutamyl p-nitroanilide) in dimethylformamide as solvent.
Glutamyl-p-nitroanilide (0.2 mmol) and lactose (0.1 mmol) are dissolved in 1 ml of dimethylformamide in the presence of 0.2 mmol imidazole. The solution is maintained at 50 ° C. for 8 hours.

It is cooled to 25 ° C., and 0.2 mmol of BOP and 0.2 mmol are added.
imidazole and wait 30 minutes. Under these conditions, more than 95% of the oside of The starting point is converted into a glycopeptide derivative. The purification is identical at that described using the other solvents.

Description of the figures:

FIG. 1 represents the elution profile of G3-lactosyl-pyroGlu-pNA
obtained by high performance anion exchange chromatography.
Figure 2 shows the elution profile of LewisA / LewisX-pyroGlu-pNA obtained by high performance anion exchange chromatography.
Example 11 The purity of the product prepared in Example 2 (N-lactosyl (3-pyroglu-pNA, hereinafter also referred to as (3-lactosyl-pyroGlu-pNA) was verified by high performance anion exchange chromatography and amperometric detection (HPAE-PAD) on a DIONEX branded device.
With regard to this technique, the procedure is as follows:
The osides are ionized in an alkaline medium (0.1M sodium hydroxide) in the form of plurialcoolates. Their separation on a cationic resin (ammonium ions immobilized) is very effective. The detection of the osides is advantageously performed by pulsed current amperometric measurement. The device used (Dionex) was specially designed to perform the chromatography in medium alkaline and amperometric line detection.
The retention times (tr) of the different products have been characterized (see figure 1):
Peak tr (min) Compound 1 4,4 Imidazole 2 10.0 Lactose 3 27.5 R-lactosyl-Glu-pNA
4 22.9 -3-lactosyl-pyroGlu-pNA
In Figure 1:
the first curve (from the top of the board) corresponds to the injection of lactose alone, the second curve corresponds to the injection of the reaction mixture after 12pm the third curve corresponds to the injection of the reaction mixture after 12 hours + 30 minutes of cycling.

Example 12 Preparation of an acylated glycosylamine derivative intramolecularly:
LewisA / L.ewisKpyroGlu-pNA.

LewisA / LewisX (0.06 nunol) is dissolved in 1 ml of diméthylfotmamide. 0.12 mmol of Glu-pNA and then 0.24 mmol are added imidazole. The solution is kept at 50 ° C. for 15 hours. Stabilization of glycopeptide is obtained by adding, to the reaction medium reduced to 20 C, 0.13 mmol of BOP and 0.24 mmol of imidazole. After 30 min, the glycopeptide is cyclized LewisA / LewisX-pyroGlu-pNA. The reaction is followed by HPAE-PAD.

Synthesis of LewisA / LewisKpyroGlu-pNA. Dionex elution profiles.
The retention times (tr) of the different products have been characterized (see figure 2):
Peak tr (min) Compound 1 4,5 Imidazole 2 8.9 / 9.4 LewisA / LewisX
3 22.5 / 22.7 LewisA / LewisX-Glu-pNA
4 17.4 / 17.6 LewisA / LewisX-pyroGlu-pNA
In Figure 2:
- the first curve (from the top of the board) corresponds to the injection of the reaction mixture after 15 minutes, the second curve corresponds to the injection of the reaction mixture after 15 hours, and the third curve corresponds to the injection of the reaction mixture after 15 hours + 30 minutes of cycling.

Purification of the glycopeptide.

After the synthesis described above, the purification is carried out in two steps:

by molecular sieving in a column of Trisacryl GF05 (100 cm x 2.3 cm) with a flow rate of 10 ml / h, eluted with an aqueous solution containing 0.1 M acetic acid and 3% n-butanol; this first step eliminates unreacted oligosaccharides and excess of BOP = benzotriazolyl-oxy-hexafluorophosphate tris (dimethylamino) phosphonium, and Glu pNA.
- by ethanol precipitation (90%), during which the sample is kept at 4 ° C. for 24 hours; this second step eliminates the excess imidazole.
Purification is followed by HPAE-PAD.

In FIG. 2, the fourth curve corresponds to the injection of the product LewisA / LewisX-pyroGlu-pNA purified by molecular sieving followed by ethanolic precipitation (tr: 17.4 / 17.6).

Characterization of the glycopeptide (LewisA / LewisX-pyroGlu-pNA).
1H NMR analysis at 300 MHz was performed.
The LewisA / LewisX-pyroGlu-pNA is dissolved in D20 (6.10-3 mol / l).
LewisA has a 3-linked terminal galactose and a 4-linked terminal fucose sure N-acetylglucosamine. LewisX has a 4-linked terminal galactose and a terminal fucose bound in 3 on N-acetylglucosamine. The spectrum review allowed to identify a number of characteristic protons:
- common to both glycopeptides: 8.33 and 7.79 (4H, 2d, aromatic H);
4.90 (2H, m, H5 aFuc); 4.67 (1H, d, J12, 7.32 Hz, H1 (3G1cNAc);
4.37 (1H, d, J1, 7.42 Hz, H1 (3Galint), 4.16 (1H, s, H4 (3Galmt), 2.83 (2H, m, y CH 2 pyroGlu); 2.33 (2H, m, (3 and (CH 2 pyroGlu), 2.05 and 2.04 (6H, 2s, CH3Ac G1cNAc); 1.22 and 1.21 (6H, 2s, CH3 Fuc);
Lewis A specific: 5.05 (1H, d, H1 aFuc); 4.53 (1H, d, H1 PGal);
specific for LewisX: 5.24 (1H, d, J1.2, 7.2 Hz, H1 aFuc);
4.50 (1H, d, Hi (3Gal).

Example 13 LewisB-pyroGlu-pNA was prepared as in Example 12 and the oligoH-pyroGlu-pNA.

wo 96/00229 ~ T i (1 ~ (1 rt ~ j PCT / FR95 / 00790 fw3e7 and (u 48 The following is a summary of the glycopeptides obtained, including that of β-lactosyl-pyroGlu-pNA (N-lactosyl (3-pyroG (u-pNA) and LEWISA / Lewis x-pyroGlu-pNA.

Analysis of glycopeptides (Dionex apparatus).

Separation by anion exchange chromatography. CarboPac column PAI (4 x 250 mm). Flow: 1 mlfmin. Detection by pulsed amperometry.
Work at room temperature.

It is used to separate well at a sodium acetate gradient:
100 mM NaOH time 100 mM NaOH
(min) CH3COONa 1M

Injection 0.1 100 0 Retention times expressed in minutes:

Imidazole. 4,4 0,1 Lactose. 10.0 0.1 (3-lactosyl-Glu-pNA) 27.5 0.1 (3-lactosyl-pyroGlu-pNA 22.9 0.1 Imidazole. 4.5 0.1 LewisA / LewisX. 8.9-9.4 0.1 LewisA / LewisX-Glu-pNA. 22.5 / 22.7 0.1 LewisA / LewisX-pyroGlu-pNA. 17.4 / 17.6 0.1 Imidazole. 4,4 0,1 LewisB 7.3 t 0.1 LewisB-Glu-pNA. 19.7 0.1 LewisB-pyroGlu-pNA. 16.6 0.1 Imidazole 4.5 t 0.1 OligoH. 8.2 / 8.7 t 0.1 OligoH-Glu-pNA. 23.8 0.1 OligoH-pyroGlu-pNA 18.9 0.1

Claims (27)

Compounds comprising one or more oligosaccharides, each of said oligosaccharides being covalently attached to one or more molecules, matrices or particles thanks to an intermediate molecule having a nitrogen atom carried by a carbon in .alpha. of a C = O group, the nitrogen atom being N-acylated, and one or more groups functional, the covalent bond between said intermediate molecule and the oligoside having by means of the above-mentioned nitrogen atom, and the covalent bond between said intermediate molecule and the aforesaid molecule, the aforesaid matrix, the aforesaid particle, or the aforesaid molecules, the aforesaid matrices, the aforesaid particles having place by through the one or more functional groups of said molecule intermediate and appropriate functional groups on the molecule (s), the matrix (s) or the (the particles). 2. Compounds of general formula (I) in which:
* a = 0 or 1, * j = 0 or 1, * b = 0 or 1, * p = 2 to 4, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of group (CH2) p, * D represents a residue of an organic acid of formula ECO2H, E being H or an alkyl chain of 1 to 10 carbon atoms, * Z represents ** B, where B is H, alkyl of 1 to 10 carbon atoms, or a chain side of a .alpha acid. amine, natural or synthetic, or ** B'-P ', B' being an alkylidene chain of 1 to 10 carbon atoms, or a side chain of an .alpha acid. amine, natural or synthetic, the said chains containing a group derived from a functional group likely to be activated, P ' having the meanings given below, * X represents:
. the group . or the group . or the group where m is an integer from 0 to 10, k = 0 or 1 Q is OH, OCH3, OCH2-C6H5, O-C6H5, O-C6F5, R representing a group having an alcohol, phenol, thiol or amine, * P being as defined below, * A i represents an organic radical, * P, P 'and P "are identical or different and represent:

a matrix as a support for affinity chromatography;
- a ball of gold or latex;

a protein;
a lipid;

oligonucleotides;
polymers, P, P 'and P "having at least one function allowing a reaction of condensation by reaction with an oligopeptide, provided that Z represents B'-P ', and / or X includes P and / or P "in its formula. 3. Compounds according to one of claims 1 or 2, of general formula (II) in which :

* a = 0 or 1, * j = 0 or 1, * b = 0 or 1, * p = 2 to 4, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of group (CH2) p, * D represents a residue of an organic acid of formula ECO2H, E being H or an alkyl chain of 1 to 10 carbon atoms, B represents H, an alkyl of 1 to 10 carbon atoms, or a chain lateral of an acid .alpha. amine, * X represents:
. or the group [NH- (Ai) -CO] mP, , or the group [NH- (Ai) -CO] mRP
m being an integer from 0 to 10, R and P being as defined below, * A i represents an organic radical, R representing a group having an alcohol, phenol, thiol or amine, * P represents a matrix as a support for affinity chromatography;
- a ball of gold or latex;

a protein;

a lipid;
oligonucleotides;
polymers. 4. Compounds according to one of claims 1 or 2, of general formula (III) wherein X is [NH- (Ai) -CO] mP or [NH- (Ai) -CO] mRP, A i, m, P
and R having the meanings indicated in claim 2. 5. Compounds according to one of claims 1 or 2, of general formula (IV) wherein D and B have the meanings given in claim 2, and X
represents [NH- (Ai) -CO] mP or [NH- (Ai) -CO] mRP, A i, m, R and P having the meanings given in claim 2. 6. Compounds according to one of claims 1 or 2, of general formula (V) wherein X is [NH- (Ai) -CO] mP or [NH- (Ai) -CO] mRP, P, A i, m and R having the meanings indicated in claim 2. 7. Products which may be used as intermediates for the preparation of one of the compounds according to any one of claims 1 to 6, said products comprising an oligoside bound to a molecule having a nitrogen atom, carried by a carbon in .alpha. of a group C = O, and at least one functional group, the covalent bond between the oligoside and the molecule being effected by through the aforementioned nitrogen atom. 8. Products of general formula (Ia) in which :

* a = 0 or 1, * j = 0 or 1, * b = 0 or 1, * p = 2 to 4, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, ** or a = b = 1, when j = 0 which implies the absence of group (CH2) p, * D represents a residue of an organic acid of formula ECO2H, E being H or an alkyl chain of 1 to 10 carbon atoms, * Z1 represents * B, B being selected from: H, an alkyl chain of 1 to 10 carbon atoms carbon, or a side chain of an .alpha acid. amine, or B ', B' being chosen from: an alkylidene chain of 1 to 10 carbon atoms carbon, or a side chain of an .alpha acid. amine, said chains containing a group functional, * X1 represents .cndot. the group [NH- (Ai) -CO] mR, or .cndot. the group [NH- (Ai) -CO] mQ

R represents a compound having an alcohol, phenol, thiol or amine, Q is selected from the group consisting of OH, OCH3, OCH2-C6H5, O-C6H5, O-C6F5, where m is an integer from 0 to 10, * A i represents an organic radical. 9. Products according to one of claims 7 or 8, of general formula (IIa) in which :

* a = 0 or 1, * j = 0 or 1, * b = 0 or 1, * p = 2 to 4, * provided that ** a = b = 0, when j = 1, which results in the presence of a molecule cyclic, * * or a = b = 1, when j = 0 which implies the absence of group (CH2) p, * D represents a residue of an organic acid of formula ECO2H, E being H or an alkyl chain of 1 to 10 carbon atoms, B represents H, an alkyl of 1 to 10 carbon atoms, or a chain lateral of an acid .alpha. amine, where m is an integer from 0 to 10, * A i represents an organic radical, R represents a compound having an alcohol, phenol, thiol or amine. 10. Products according to one of claims 7 or 8, of general formula (IIIa) in which A i, m and R have the meanings indicated in the claim 8. 11. Products according to one of claims 7 or 8, of general formula (IVa) in which D, B, m, A i and R have the meanings indicated in claim 8. 12. Products according to one of claims 7 or 8, of general formula (Va) in which A i, m and R have the meanings indicated in the claim 8. 13. Products of formula (VI) in which B, A i, m and R have the meanings indicated in the claim 2. 14. Products of formula (VII) in which p, A i, R, m have the meanings indicated in the claim 2. 15. Compounds or products according to claim 2 or 8, characterized in what Q represents the following radicals:
OH, O-CH2-C6H5, O-C6H5, O-C6F5, Op C6H4-NO2, 16. Compounds or products according to any one of claims 2 to 6 or 8 at 14, characterized in that R represents one of the following radicals:

-NH-p C6H4-N = C = S and its precursors:
-NH-p C6H4-NO2 -NH-p C6H4-NH2 -NH-CH2- (CH2) mC (= NH2 +) OCH3 -NH-CH2- (CH2) m-CN
-NH-CH2- (CH2) m-CH2-NH-CO-CH2- (CH2) mC (= NH2 +) OCH3 -NH-CH2- (CH2) m-CH2-NH-CO-CH2- (CH2) m-CN

-NH-CH 2 - (CH 2) m -CH 2 -NH-CO-CH 2 -T T = Br, I, Cl -NH-CH2-CH2-NH-CO-CH2- (CH2) MSS-Pyr NH-CH2- (CH2) m-CH2-SS-Pyr -NH- (CH2) m-pC6H4OH
-NH-CH2- (CH2) m CH2-NH-CO- (CH2) m -pC6H4OH
m representing an integer from 0 to 10, Pyr representing the 2-pyridine group. 17. Compound according to claim 2, characterized in that P

represents an oligopeptide, or a polypeptide, and P 'or P "represents a oligonucleotide, or R represents fluorescein or a derivative thereof or another derivative fluorescent, and P 'or P "represents an oligonucleotide or P represents an agent therapeutic. 18. Compounds according to any one of claims 1 to 12, characterized in that the oligoside residue is either monosaccharide or an oligosaccharide comprising from 2 to 50 oses and is selected from:
lacto-N-tetraose Gal.beta. 3 GlcNAc.beta. 3 Gal .beta. 4 Glc neolacto-N-tetraose Gal.beta. 4 GlcNAc.beta. 3 Gal .beta. 4 Glc group H Fuc .alpha. 2 Gal.beta. 3 Gal.beta. 4 Glc Lewis has Gal.beta. 3 GlcNAc.beta. 3 Gal 4 Glc Fuc .alpha. 4 ~ .uparw.

Lewis x Gal.beta. 4 GlcNAc.beta. 3 Gal.beta. 4 Glc Fuc .alpha. 3 ~ .uparw.

Lewis b Fuc.alpha. 2 Gal.beta. 3 GlcNAc .beta. 3 Gal.beta. 4 Glc Fuc .alpha. 4 ~ .uparw.
Lewis y Fuc .alpha. 2 Gal.beta. 4 GlcNAc.beta. 3 Gal.beta. 4 Glc Fuc .alpha. 3 ~ .uparw.

Disialolacto-N-Tetraose Neu 5Ac.alpha. 3 Gal .beta. 3 GlcNAc .beta. 3 Gal .beta. 4 Glc Neu 5Ac.alpha. 6 ~ .uparw.

Complex type with 3 antennas Sialylactose 3 Neu 5Ac .alpha. 3 Gal 4 Glc Sialylactose 6 Neu 5Ac .alpha. 6 Gal .beta. 4 Glc Disialylactose 3 Neu 5Ac .alpha. 8 Neu 5Ac .alpha. 3 Gal .beta. 4 Glc simple or complex osides recognized by membrane lectins. 19. Compounds according to claim 18, characterized in that the residue oligoside is selected from the following simple or complex osides, recognized by membrane lectins:

at. Asialo-oligoside of triantennary lactosamine type: receptor asialoglycoprotein b. Asialo oligoside tetraanthene-type lactosamine: receptor of asialoglycoprotein vs. Lewis x: LECAM 2/3 d. Sialyl Lewis X: LECAM 3/2 e. Lewis x derivative sulfated (HNK1): LECAM 1 f. Oligomannoside: mannose receptor boy Wut. Phosphorylated oligomannoside: mannose 6 phosphate receptor h. Sulphated lactosamine oligosaccharide: GalNAc receptor 4 sulfate Compounds according to any one of claims 2 to 6 or 8 to 14, characterized in what A; represents an alkylidene chain of 1 to 10 carbon atoms. 21. Compounds according to claim 20, characterized in that A i meets the formula (CH2) nW- (CH2) n1, n + n1 representing an integer from 0 to 10, W
representing CHY, where Y is H, an alkyl of 1 to 6 linear carbon atoms or branched an amino residue .alpha. acid, natural or synthetic, or W representing a compound aromatic. 22. Process for the preparation of the compounds according to one of Claims 7 to 12, characterized in that - an oligoside having an ose is condensed in a suitable solvent reducer free, on the nitrogen atom of an intermediate molecule, this nitrogen atom belonging to an amine group, attached to a carbon atom placed in .alpha. a group C = O, the molecule intermediate that does not have a side chain containing a group functional, for obtain a glycosylamine derivative in which the terminal ose of the oligoside keep his cyclic structure, and in which the semi-acetal hydroxyl is replaced by the amine .alpha.
one of the above-mentioned starting molecules, the glycosylamine derivative obtained after the previous step is acylated by addition of an organic acid activated by a conventional activator, to obtain a derivative N-acylated glycosylamine. 23. Process for preparing compounds according to one of claims 7 to 12, characterized in that - an oligoside having an ose is condensed in a suitable solvent reducer free, on the nitrogen atom of an intermediate molecule, this nitrogen atom belonging to an amine group, attached to a carbon atom placed in .alpha. a group C = O, the molecule intermediate having a side chain containing a functional group, for obtain a glycosylamine derivative in which the terminal ose of the oligoside keep his cyclic structure, and in which the semi-acetal hydroxyl is replaced by the amine .alpha.
one of the above-mentioned starting molecules, when the intermediate molecule has a side chain whose group functional is protected or not, the glycosylamine derivative obtained is the outcome of the preceding step by adding an activated organic acid with an activator classic, to obtain an N-acylated glycosylamine derivative, when the intermediate molecule has a side chain containing a carboxylic group, the above-mentioned carboxylic group is activated so that it react intramolecularly with the aforesaid amine .alpha., leading to a cyclization inside of the aforementioned starting molecule, to obtain a N-glycosylamine derivative.
acylated when the intermediate molecule has a side chain containing a carboxylic group, the acylating agent can be added in the form of an ester active. 24. Process according to any one of claims 22 or 23, characterized in the intermediate molecule is selected from the following molecules:
acid .alpha.
amino, natural or synthetic, derived from acid .alpha. amine, amino acid in position N-terminal of a peptide or a peptide derivative. 25. Process according to any one of claims 22 to 24, characterized in this that the condensation step is carried out in the presence of a catalyst. 26. A process for preparing a compound according to any one of Claims 1 to 6, characterized in that the reaction is one or more products (acylated glycosylamine derivatives) according to one of the Claims 8 to 12, carrying a Q group, activated or activatable, reacting on a molecule, matrix or particle P, P 'and P ", one or more products (acylated glycosylamine derivatives) according to one of the Claims 8 to 12, bearing either an R group, activated or activatable, or a group B ' containing an activated or activatable functional group, a group A i containing a functional group on a molecule, a matrix or a particle, respectively P, P ' and P ", containing a functional group, to obtain a product of the type:

(glycopeptidyl-R) nP, (glycopeptidyl-B ') n-P', or (glycopeptidyl-A i) nP ", n > = 1, one or more products (acylated glycosylamine derivatives) of the invention, carrying on the one hand a group R, activated or activatable, on the other hand a group B ' containing an activated or activatable group, or a group A i containing a group functional on molecules, matrices or particles P and P ', or molecules, matrices or P particles and P "
one or more products (acylated glycosylamine derivatives) of the invention, carrying on the one hand a group R, activated or activatable, on the other hand a group B ' containing an activated or activatable group, and a group A i containing a group functional respectively on molecules, matrices or particles P, P 'and P ", R, B ', A i, P, P' and P "being defined according to any one of Claims 1 to 6. 27. The method of claim 26 wherein the compound to be substituted is a protein, a lipid, a nucleic acid, an oligonucleotide, a polylysine, a insoluble polymer, a ball of latex or a ball of gold.