Novel carbohydrate derivatives as mimetics of blood group A and B antigens.

Abstract

The present invention relates to novel carbohydrate derivatives having activity as mimetics of blood group A and B antigens. The invention also relates to a process for producing said carbohydrate derivatives and to glycoconjugates comprising them bound to a polymeric support. The invention also relates to the use of the glycoconjugates for inhibiting and / or removing anti-A and / or anti-B antibodies from blood and blood derivatives and by-products, and their use, in particular, for avoiding rejection reactions or hemolysis in blood transfusions in subjects receiving organ transplants from incompatible donors.

Description

[Technical field]

[0001] The present invention relates to novel carbohydrate derivatives which have activity as mimetics of blood group A and B antigens. [Background technology]

[0002] The ABO blood grouping is used to indicate the presence of type A, B and O (H) antigens (ABH antigens) on red blood cells.

[0003] Type A and type B antigens are carbohydrate molecules containing a terminal trisaccharide characterized in that the non-reducing terminal sugar of the terminal trisaccharide is an N-acetylgalactosamine residue or a galactose residue, respectively, which is linked to another galactose residue by an α(1→3) glycosidic bond, and the galactose residue has a fucose residue linked to its 2-position by an α(1→2) glycosidic bond.

[0004] ABH antigens can be further divided into subgroups according to the inner core glycan represented for example by type 1 (Galβ1,3GlcNAc), type 2 (Galβ1,4GlcNAc), type 3 (Galβ1,3GalNAcα) and type 4 (Galβ1,3GalNAcβ) chains, the only difference being the linkage between a galactose residue and an N-acetylglucosamine or N-acetylgalactosamine residue. Thus, for example, the sequence of type 2 A antigen is GalNAcα1,3(Fucα1,2)Galβ1,4GlcNAc and type 2 B antigen is Galα1,3(Fucα1,2)Galβ1,4GlcNAc.

[0005] ABO blood group antigens are found on the surface of red blood cells and epithelial cells and are of great medical importance, especially in the field of blood transfusion and transplantation, due to the anti-A and anti-B antibodies that bind to them. Since blood group antigens vary between individuals, antibodies against blood group A and B epitopes are produced in individuals who do not express these antigens and therefore represent the first obstacle in blood, organ or tissue donation between blood type incompatible individuals. These natural antibodies, mainly belonging to the IgM isotype, are carbohydrate-binding proteins (CBPs) that bind to carbohydrate epitopes on cell surfaces, fix complement and cause intravascular hemolysis of red blood cells during transfusion and hyperacute rejection of vascularized organs.

[0006] Due to the chronic shortage of donors, strategies to transfuse blood and transplant organs across the ABO barrier are under development.

[0007] One strategy known in the art to enable ABO-incompatible transplantation is the use of carbohydrate-based therapeutics to remove / inhibit anti-carbohydrate antibodies formed in the recipient during the period before and after transplantation adaptation has occurred. For example, ex vivo anti-carbohydrate removal can be achieved using carbohydrate-based immunoadsorbents, or alternatively (or complementary) anti-carbohydrate antibodies can be inhibited in vivo by injectable antigens, as disclosed, for example, in Holgerson et al., Immunol. Cell Biol., 2005, 83, 694-708.

[0008] For example, the use of blood group A and B trisaccharide epitopes bound to crystalline silica and Sepharose® for removal of anti-A / B antibodies has been disclosed in the art (Holgerson et al., supra).

[0009] For the injection of materials to inhibit antibodies in vivo, the main problems with the use of free oligosaccharides are the low protein-carbohydrate binding affinity and the short serum half-life. Therefore, the use of glycoconjugates in which multiple antigenic oligosaccharides are attached to a biodegradable backbone is advantageous, allowing multivalent binding and thus increased affinity and inhibitory activity, as disclosed, for example, in Duthaler et al., Chimia, 2010, 64, 23-28; Katopodis et al., J. Clin. Invest., 2002, 110, 1869-1877 or Wang et al., J. Am. Chem. Soc., 1999, 121, 8174-8181.

[0010] It has always been disclosed in the art that for in vitro, ex vivo or in vivo inhibition / adsorption of natural antibodies it is important to use inhibitors or adsorbents which, whenever possible, have active epitopes which correspond to the natural carbohydrate antigens.

[0011] Thus, for example, EP 2010920 discloses the preparation of a solid support, in particular microbeads, to which a plurality of A and B antigens are bound, and also its use for determining the presence of blood group reactive antibodies in a serum sample or for removing blood group reactive antibodies in serum. The carbohydrate antigens are in particular selected from antigens A or B of types 1, 2, 3 or 4.

[0012] Similarly, WO 2013 / 062479 discloses a method for the extracorporeal removal of anti-A and / or anti-B antibodies from blood using an adsorbent material comprising a matrix bound to a saccharide which is blood group determinant A or blood group determinant B, i.e. the A-type trisaccharide determinant (GalNAcα1,3(Fucα1,2)Galβ1) or the B-type trisaccharide determinant (Galα1,3(Fucα1,2)Galβ1) further bound to an internal saccharide as subtype 1, 2, 3 or 4. The matrix is ​​for example agarose, dextran, starch or starch derivative, amylose, amylopectin or polyacrylamide.

[0013] WO 2018 / 167230 discloses macromolecules comprising multiple carbohydrate ligands that bind to various types of carbohydrate-binding proteins, including agglutinins, and their use for the diagnosis and treatment of diseases, including those associated with CBP-mediated agglutination.Specific examples 9, 32 and 35 correspond to polylysine glycoconjugates bearing blood group A antigen type 5 tetrasaccharide, blood group A trisaccharide antigen and blood group B trisaccharide antigen, respectively, where the antigens are attached to the polylysine backbone via a linker chain group.

[0014] The type A and type B carbohydrate polymers disclosed so far in the art, which contain natural type A and type B determinants, still suffer from several drawbacks resulting from the low affinity binding between these natural antigens and antibodies.

[0015] Thus, there remains a need to provide new glycoconjugates with improved affinity for anti-A and / or anti-B antibodies for use in various biological and medical applications, e.g., for efficient removal / inhibition of anti-A and / or anti-B antibodies prior to ABO incompatible transplantation.

[0016] Object of the invention The object of the present invention are compounds of formula (I).

[0017] Another aspect of the present invention is a process for the preparation of a compound of formula (I).

[0018] Another aspect of the present invention is a glycoconjugate of the compound of formula (I).

[0019] Another aspect of the invention is the use of said glycoconjugates for inhibiting and / or removing anti-A and / or anti-B antibodies from samples of blood or other blood derivatives and by-products.

[0020] Another aspect of the present invention is the glycoconjugate as described above for use as a medicament.

[0021] Another aspect of the present invention is a pharmaceutical composition comprising said glycoconjugate and at least one pharma- ceutically acceptable excipient. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows Scheme I illustrating a synthetic route to compounds of formula (I). [Diagram 2] FIG. 2 shows Scheme Ia illustrating a route to the synthesis of compounds of formula (I) where R2 is a group represented by formula (II) and R3 is a group represented by formula (III). [Diagram 3] FIG. 3 shows Scheme Ib illustrating a route to the synthesis of compounds of formula (I) where R2 is a group represented by formula (II) and R3 is a group represented by formula (III). [Figure 4] FIG. 4 shows Scheme Ic illustrating a route to the synthesis of compounds of formula (I) where R2 is OH and R3 is a group represented by formula (III). [Diagram 5] FIG. 5 shows Scheme II illustrating the preparation of glycoconjugates of compounds of formula (I) using polyacrylic acid. [Figure 6] FIG. 6 shows Scheme III illustrating the preparation of glycoconjugates of compounds of formula (I) using poly-L-lysine. [Figure 7] FIG. 7 shows Scheme IV illustrating the preparation of glycoconjugates of compounds of formula (I) using poly-L-lysine. [Figure 8] FIG. 8 shows a synthesis scheme of a glycoconjugate using poly-L-lysine (a=25) as disclosed in Example 4. [Figure 9] Figure 9 shows the binding of purified human anti-B (graphs 9-1 and 9-2) and anti-A (graphs 9-3 and 9-4), IgG or IgM, to various glycan antigens, as measured by the ELISA assay disclosed in Example 6.2. The vertical axis shows the optical density (OD) values ​​for the various samples (1-9). [Figure 10]Figure 10 shows the percentage of removal (in %) of purified anti-A antibodies using different poly-L-lysine glycoconjugates disclosed in Example 6.3. Figures 10-1 and 10-2 show the removal of IgG and IgM, respectively. The black bars correspond to the glycoconjugates of Comparative Example 2 (containing the native group A type 2 antigen) and the grey bars correspond to the glycoconjugates of Examples 4-8 (containing the mimetics of the invention) at increasing concentrations (10, 100 and 400 μg / mL). [Figure 11] Figure 11 shows the percentage of removal (in %) of purified anti-B antibodies using different poly-L-lysine glycoconjugates disclosed in Example 6.3. Figures 11-1 and 11-2 show the removal of IgG and IgM, respectively. The black bars correspond to the glycoconjugates of Comparative Example 1 (containing the native B type 2 antigen) and the grey bars correspond to the glycoconjugates of Example 4-1 (containing the mimetics of the invention) at increasing concentrations (10, 100 and 400 μg / mL). [Figure 12] Figure 12 shows the inhibition of purified anti-B antibody binding to red blood cells (type B) by poly-L-lysine glycoconjugates measured by flow cytometry as disclosed in Example 6.4. Inhibitory capacity is expressed as percentage of removal (%; ordinate) relative to baseline. Glycoconjugates analyzed were Comparative Example 1 (black bars, containing native type B type 2 antigen), Example 4.1 (light grey bars, containing the B mimetic of the invention) and a combination of the glycoconjugates of Comparative Example 1 and Example 4.1 (dark grey bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention The object of the present invention is to provide a compound of formula (I):

[0024] [ka]

[0025] [In the formula, R 1 -OH and -NH-CO-R 5 where R 5 is C1 -C 3 is alkyl, R 5 one or more hydrogen atoms may be replaced by a hydroxy group; R 2 is a group consisting of OH and formula (II):

[0026] [ka]

[0027] is selected from the group represented by Where: * indicates the position at which the group of formula (II) is attached to the remainder of the molecule; R 3 is R 4 -Z, formula (III):

[0028] [ka]

[0029] and a group represented by formula (IV):

[0030] [ka]

[0031] is selected from the group represented by Where: * indicates the position at which the group represented by formula (III) or formula (IV) is attached to the remainder of the molecule, R 4 is a spacer group, Z is -NH 2 , -COOH and -SH. It is a carbohydrate derivative represented by the formula:

[0032] The present inventors have surprisingly developed a novel carbohydrate derivative represented by formula (I) which has high affinity for natural anti-A / B antibodies, even though the non-reducing end is a glucose or glucosamine derivative rather than galactose or N-acetylgalactosamine as in natural A and B antigens.

[0033] definition The term "glycan", as commonly used in the art, is a general term referring to carbohydrates, sugars, monosaccharides, oligosaccharides and polysaccharides. In particular, the term "glycan" is used herein to refer to the carbohydrate portion of the carbohydrate derivative of formula (I) of the present invention; in this regard, the terms "glycan", "carbohydrate portion" and "oligosaccharide portion" are used interchangeably and are also used to refer to the carbohydrate portion of a complex carbohydrate.

[0034] The carbohydrate moieties in the compounds of formula (I) are sometimes referred to herein as "epitopes", "glycoepitopes" or "glycotopes" to indicate that they are the active sites recognized and bound by anti-A and / or anti-B antibodies. These carbohydrate moieties, which have a different structure from the natural A / B antigens but are also recognized by the natural A / B antigens, are sometimes referred to as "mimetics", "glycomimetics" or "Glc analogs".

[0035] The carbohydrate derivatives of formula (I) are also referred to herein as "functionalized glycans" or "ligands" and are generally represented by the formula Glyc-sp-Z, where Glyc is the glycan or carbohydrate moiety and sp is R 4 represents a spacer group, and Z is a functional group as previously described); that is, the glycan "Glyc" in Glyc-sp-Z is the following glycan:

[0036] [ka]

[0037] [In the formula, R 1 and R 2are as explained above, and the specific examples of each group are the same. Shows.

[0038] In certain embodiments, Z is -NH 2 In this case, the saccharide derivative represented by formula (I) is Glyc-sp-NH 2 In another particular embodiment, when Z is -SH, the carbohydrate derivative of formula (I) can be represented as Glyc-sp-SH.

[0039] The term "glycoconjugate", as conventionally understood, relates to a carbohydrate covalently linked to another chemical species, in particular a polymeric substance (e.g., a protein, peptide, lipid or polysaccharide, etc.), also referred to as a polymeric backbone or support. In particular, the glycoconjugate of the present invention comprises a functionalized glycan of formula (I) attached to a polymeric backbone. The glycoconjugate of the present invention is also referred to herein as a carbohydrate polymer.

[0040] C n -C m Alkyl means a linear or branched alkyl group having n to m carbon atoms. In particular, C 1 -C 3 Alkyl means an alkyl group of 1 to 3 carbon atoms and thus includes methyl, ethyl, n-propyl and isopropyl groups.

[0041] A linear alkylene group has a variable value of n, i.e., -(CH 2 ) n For example, when n is 2 to 20, the alkylene group is C 2 -C 20 When n is 2 to 10, the alkylene group is C 2 -C 10 It can be represented as alkylene.

[0042] As used herein, the term "alkylamino" refers to a monoalkylamino group (-NH alkyl) or a dialkylamino group (-N(alkyl) 2), that is, an amino group substituted with one or two alkyl groups.

[0043] As used herein, the term "alkoxy" refers to an --O-alkyl group.

[0044] As used herein, the term "alkylthio" refers to an --S-alkyl group.

[0045] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups.

[0046] In the present specification, unspecified "alkyl" is preferably a straight-chain or branched C 1 -C 6 Alkyl, more preferably C 1 -C 3 Refers to alkyl.

[0047] Generally, terms used in this application are to be understood in their ordinary sense as known in the art unless otherwise specified.

[0048] In the description and claims of this invention, generally, the singular forms preceded by the articles "a," "an," or "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0049] As used herein, the terms "about" or "approximately" when describing an amount are meant to include the exact amount as well as a certain deviation from the stated amount, ie, ±5%.

[0050] Numerical ranges disclosed herein are meant to include every number within the range, as well as the lower and upper limits thereof.

[0051] Compounds represented by formula (I) The object of the present invention is to provide a compound of formula (I):

[0052] [ka]

[0053] [In the formula, R 1 , R 2 and R 3 is as explained above.] It is a carbohydrate derivative represented by the formula:

[0054] The compound represented by formula (I) is Glc(R 1 )α1-3(R 2 )Galβ-R 3 which indicates that the non-reducing terminal carbohydrate residue in the compound of formula (I) is an α-glucose (Glc) linked to a β-galactose (Gal) unit by an α(1→3) glycosidic bond.

[0055] In the compound of formula (I), as explained above, R 1 teeth, -OH and the non-reducing end carbohydrate is an α-glucose residue, and the carbohydrate derivative of formula (I) is a mimetic of the blood group B antigen; or - -NH-COR 5 (In the formula, R 5 is C 1 -C 3 is alkyl, R 5 wherein one or more hydrogen atoms may be replaced by hydroxy groups), and the non-reducing end carbohydrate is an α-N-acetylglucosamine residue, so that the carbohydrate derivative represented by formula (I) is a mimetic substance of the blood group A antigen.

[0056] R in β-Gal residue 2 The group may be OH or a fucose residue (formula (II)).

[0057] Glc(R 1 )α1-3(R 2 ) Galβ-group has the formula -R 4 -Z or is itself a linker of the formula -R 4or to another carbohydrate residue attached to the linker -Z, the carbohydrate being selected from β-N-acetylglucosamine attached at the 4-position (Formula III) or β-N-acetylgalactosamine attached at the 3-position (Formula IV).

[0058] In one embodiment, in the compound of formula (I), the group R 1 -OH, -NH-CO-CH 3 (N-acetyl or N-Ac), -NH-CO-CH 2 CH 3 (N-propionyl) and -NH-CO-CH 2 OH(N-glycolyl).

[0059] In one embodiment, in the compound of formula (I), the group R 2 is OH.

[0060] In another embodiment, in the compound of formula (I), the group R 2 is a fucosyl residue (formula (II)).

[0061] The formula -R 4 The linker group of -Z is -NH 2 , -COOH or -SH functional group Z, which covalently attaches the activated hydrocarbon moiety to a suitable support, said functional group being linked to a spacer group R 4 It is bound to carbohydrates via

[0062] In some embodiments, Z is -NH 2 It is.

[0063] In another embodiment, Z is --SH.

[0064] In another embodiment, Z is -COOH.

[0065] Spacer Spacer (R 4) is an arm connecting the oxygen atom at the reducing end of the carbohydrate moiety with the functional group Z. This linking arm provides an appropriate spatial separation between the polymeric support and the active carbohydrate epitope in the glycoconjugate of the compound of formula (I). The selection of a particular spacer is therefore not critical as long as it is suitable for this function.

[0066] Usually, spacer R 4 is a linear chain of, for example, 2 to 20 atoms, which is not immunogenic and is not reactive under the usual conditions under which compounds of formula (I) act, for example to form glycoconjugates with polymeric supports.

[0067] Spacer R 4 is typically a straight chain alkylene, e.g., C, which is unsubstituted or which is, for example, mono-, di- or tri-substituted, e.g., by groups such as alkyl, alkylamino, hydroxy, alkoxy, hydroxyalkyl, halogen, thiol, alkylthio or cyano. 2 -C 20 Linear alkylene, preferably C 2 -C 10 It is a straight-chain alkylene; no consecutive -CH 2 - groups, e.g. up to 5 non-consecutive -CH 2 - groups, preferably up to three non-consecutive -CH 2 The - group may be substituted with other groups, for example by interrupting the alkylene chain with ether, thioether, ketone, amine and / or amide groups.

[0068] In one embodiment, the spacer R in the compound of formula (I) 4 is preferably an unsubstituted linear C 2 -C 10 an alkylene group, wherein up to three non-consecutive -CH 2 The - group is -O-, -S-, -N(H)-, -N(R 5 )-, -N(OR 5 )-, -N(H)-CO-, -CO-N(H)-, -N(R 5 )-CO- and -CO-N(R5 )-(wherein, R 5 is C 1 -C 3 The alkyl group may be substituted with a group selected from the group consisting of alkyl groups.

[0069] In certain embodiments, the spacer R 4 is a compound of the formula -(CH 2 ) 2-8 For example, -R 4 -Z is of the formula -(CH 2 ) 3 -NH 2 is the one used in the manufacturing example, but other spacers discussed above may be suitable as well.

[0070] In one embodiment, the present invention provides a compound of formula (I):

[0071] [ka]

[0072] [In the formula, - R 1 -OH and -NH-COR 5 where R 5 is C 1 -C 3 is alkyl, R 5 may be replaced by a hydroxy group; preferably, R 1 -OH, -NH-CO-CH 3 , -NH-CO-CH 2 CH 3 and -NH-CO-CH 2 -OH; more preferably, R 1 -OH, -NH-CO-CH 3 and -NH-CO-CH 2 CH 3 is selected from the group consisting of - R 2 is a group consisting of OH and formula (II):

[0073] [ka]

[0074] is selected from the group represented by Where: * indicates the position at which the group of formula (II) is attached to the remainder of the molecule; Preferably, R 2 is a group represented by formula (II); - R 3 is R 4 -Z, formula (III):

[0075] [ka]

[0076] and a group represented by formula (IV):

[0077] [ka]

[0078] is selected from the group represented by Where: * indicates the position at which the group represented by formula (III) or formula (IV) is attached to the remainder of the molecule, R 4 is a spacer group; Preferably, R 4 is unsubstituted or substituted, e.g., mono-, di- or tri-substituted, by groups independently selected from alkyl, alkylamino, hydroxy, alkoxy, hydroxyalkyl, halogen, thiol, alkylthio and cyano; 2 -C 20 Linear alkylene, more preferably C 2 -C 10 linear alkylene; wherein the alkylene has up to 5 non-consecutive -CH 2 - groups, preferably up to three non-consecutive -CH 2The - group may be substituted with other groups by interrupting the alkylene chain with ether, thioether, ketone, amine and / or amide groups; More preferably, R 4 is preferably an unsubstituted linear C 2 -C 10 an alkylene group, wherein up to three non-consecutive -CH 2 The - group is -O-, -S-, -N(H)-, -N(R 5 )-, -N(OR 5 )-, -N(H)-CO-, -CO-N(H)-, -N(R 5 )-CO- and -CO-N(R 5 )-(wherein, R 5 is C 1 -C 3 alkyl group; Even more preferably, the spacer R 4 is a compound of the formula -(CH 2 ) 2-8 -, and even more preferably R 4 is the formula -(CH 2 ) 3 - is a group represented by the formula: Z is -NH 2 , -COOH and -SH, preferably -NH 2 is] It is a carbohydrate derivative represented by the formula:

[0079] In certain embodiments of the present invention, R 2 When is OH, R 3 is R 4 -Z.

[0080] Preferred examples of compounds represented by formula (I) are as follows:

[0081] [Table 1]

[0082] R 1Compounds in which R is an N-acyl group are mimetics of the blood group A antigen, and R 1 The OH one is a mimetic of the blood group B antigen.

[0083] Method for preparing the compound of formula (I): The compounds of formula (I) may be prepared by conventional synthetic methods well known to those skilled in the art of carbohydrate synthesis.

[0084] A general synthetic route by which compounds of formula (I) can be prepared is shown in Scheme I (Figure 1).

[0085] In Scheme I, PG is a conventional protecting group, e.g., each is typically acetyl (Ac, CH 3 -CO-), benzyl (Ph-CH 2 -) or benzylidene (Ph-CH=); R 3 ' is R 4 -Z' is selected from a group represented by formula (III) and a group represented by formula (IV), wherein the group represented by formula (III) and the group represented by formula (IV) are as described above, but the OH groups of the hydrocarbon moieties of the groups represented by formula (III) and formula (IV) are protected, for example with the same protecting groups as described above, the Z group is also protected, and Z' is a protected group of formula Z as described above.

[0086] As is well known in the art, a benzylidene protecting group can be used, for example, to simultaneously protect two spatially close OH groups at C-4 and C-6 of the galactose moiety.

[0087] [ka]

[0088] The Z group, as previously described, is -NH 2 , -SH or -COOH. Conventional protecting groups for these functional groups may be used, as is well known in the art. 2And a suitable protecting group for -SH is, for example, an acyl group such as acetyl or trifluoroacetyl, etc. A suitable protecting group for -COOH is an ester, for example, a methyl, t-butyl or benzyl ester, etc.

[0089] In Scheme I, in the starting material of formula A, R a is an azide group (N 3 ), then R 1 (The formula -NH-CO-R 5 Thus, the azide group is usually reduced to an amine group and then modified with a suitable acyl moiety. a When is a protected hydroxy (e.g., benzyl group, i.e., OBn), R 1 It is possible to obtain a compound of formula (I) in which

[0090] In Scheme I, an intermediate compound of formula C (R b =O-PG) is R 2 This results in a compound of formula (I) in which R b =OH), at which a fucose residue is attached via an α(1→2) glycosidic bond, R 2 is a fucose residue (of formula II as previously described).

[0091] Therefore, different R 1 , R 2 and R 3 It will be appreciated that a person skilled in the art will have no difficulty in adapting the above synthetic routes appropriately for the preparation of a particular compound of formula (I), i.e. depending on different options.

[0092] In Scheme I, the compound of formula (I) obtained directly after carbohydrate synthesis may have a spacer and a linking group with a reducing end different from those described above, and then, by conventional organic synthesis methods, the linking group -R described above may be added. 4 It is understood that the compound is converted to -Z.

[0093] Thus, another aspect of the present invention is a process for the preparation of a compound of formula (I), comprising the steps of: a) producing a compound represented by formula C from a compound represented by formula A and a compound represented by formula B:

[0094] [ka]

[0095] [In the formula, PG is a protecting group; R a is azide or O-PG; R 3 ' is R 4 -Z' is selected from a group of formula (III) and a group of formula (IV), wherein the group of formula (III) and the group of formula (IV) are as hereinbefore described, except that the OH groups of the hydrocarbon moieties of the groups of formula (III) and formula (IV) are protected and the Z group is also protected, and Z' is a protected group of formula Z as hereinbefore described; R b is OH or O-PG] b) b1)R b If is OH, a protected fucose residue is attached to this position via an α(1→2) glycosidic bond, and after deprotection, R 2 is a group represented by formula II; b2) R b is O-PG, the compound represented by formula C is deprotected to give R 2 providing a compound of formula (I), wherein the protecting group definitions and conditions are as previously discussed.

[0096] In particular, the preparation of compounds of formula (I) containing a fucose residue can be carried out similarly to the method disclosed in, for example, Kunetskiy et al., Synthesis of blood group A and B (type 2) tetrasaccharides. A strategy with fucosylation at the last stage, Carbohydrate Research, 2020, Vol. 498, 108192, which describes the preparation of analogous derivatives of natural blood group A and B antigens, i.e., derivatives containing α-Gal at the non-reducing end.

[0097] So, for example, R 2 is a group represented by formula (II), and R 3 To prepare compounds of formula (I) in which the group of formula (III) is represented, the route shown in Scheme Ia (Figure 2) can be followed.

[0098] The compound represented by C is then coupled with a fucose residue at the C-2 position of the galactose residue via the route shown in Scheme Ib (Figure 3) to provide, after deprotection, a compound represented by formula (I).

[0099] For example, compounds 1, 5, 6 and 7 in Table 1 can be prepared by this route.

[0100] Similarly, R 2 is a group represented by formula (II), and R 3 To prepare a compound of formula (I) in which the group is represented by formula (IV), a procedure similar to that of Schemes Ia and Ib may be carried out, for example by using as a starting material a compound of formula B:

[0101] [ka]

[0102] The compound represented by the formula:

[0103] In this manner, for example, compounds 4, 14, 15 and 16 in Table 1 can be obtained.

[0104] Similarly, R 2 is a group represented by formula (II), and R 3 is the formula R 4 To prepare a compound of formula (I) having a group represented by -Z, a procedure similar to that of Schemes Ia and Ib may be used. For example, a compound of formula B:

[0105] [ka]

[0106] The compound represented by the formula:

[0107] In this manner, for example, compounds 2, 8, 9 and 10 in Table 1 can be obtained.

[0108] On the other hand, R 2 OH and R 3 Compounds of formula (I) in which the group is of formula (III) can be prepared by the route shown in Scheme Ic (Figure 4).

[0109] In this manner, for example, compounds 17, 18, 19 and 20 in Table 1 can be obtained.

[0110] Similarly, R 2 OH and R 3 In order to prepare a compound represented by formula (I) in which the group represented by formula (IV) is represented, a procedure similar to that of Scheme Ic may be carried out, for example, by using as a starting material a compound represented by the following formula B:

[0111] [ka]

[0112] The compound represented by the formula:

[0113] Similarly, R 2 OH and R 3 is the formula R 4 To prepare a compound of formula (I) having a group represented by -Z, a procedure similar to that of Scheme Ic may be used. For example, a compound of formula B:

[0114] [ka]

[0115] The compound represented by the formula:

[0116] In this manner, for example, compounds 3, 11, 12 and 13 in Table 1 can be prepared.

[0117] In Scheme I, a compound of formula (I) containing fucose (i.e., R 2 is a group of formula II) is prepared in a final step by adding a fucose moiety to a previously prepared intermediate of formula C. It will be appreciated that alternative synthetic routes may be equally suitable, for example first obtaining a fucosylated derivative of intermediate B, followed by coupling with a protected alpha-glucose starting material of formula A. Identifying alternative routes for the synthesis of compounds of formula (I) is within the ability of one skilled in carbohydrate synthesis.

[0118] Glycoconjugates of the compounds of formula (I) Another aspect of the present invention is a glycoconjugate of a compound of formula (I). The glycoconjugate of the present invention comprises a compound of formula (I), specifically a compound of formula (I) and a polymeric backbone or support. In the glycoconjugate, the compound of formula (I) is covalently bound to the polymeric backbone or support, more specifically, the compound of formula (I) is bound to the polymer via the functional group Z.

[0119] The -NH group of the compound represented by formula (I) 2It is understood that the -SH and -COOH functional groups are typically converted to -NH-, -S- and -CO- or -CO-O- groups, respectively, in the glycoconjugate when a covalent bond is formed between the functionalized glycan and the polymer. It is therefore understood that the Z group is modified in this way when a glycoconjugate "comprises" a compound of formula (I).

[0120] As is well known in the art, such covalent bonds can be directly between the ligand and the polymer, or they can be linked via a suitable separate linking agent (or cross-linking agent), or by including reactive groups on the polymer, the ligand, or both, which thus have the purpose of activating either the polymer or the compound of formula (I) to enable or facilitate the reaction between them.

[0121] It is therefore understood that the final glycoconjugate may contain additional linking fragments between the two, as shown below.

[0122] Thus, the glycoconjugates of the present invention can also be defined as comprising the carbohydrate portion of a compound of formula (I) attached to a polymeric support or polymeric backbone via a suitable spacer.

[0123] The glycoconjugates of the present invention can be prepared by attaching the carbohydrate derivatives of formula (I) to a suitable polymeric backbone via covalent bonds. More specifically, the glycoconjugates are formed with multiple compounds of formula (I) covalently attached to multiple functional groups on the polymeric backbone.

[0124] Suitable polymers for use as backbones or supports for the glycoconjugates of the invention are, for example, homogeneous and heterogeneous α-amino acid polymers (homo- and heteropolypeptides), proteins, lipids, nucleic acids, acrylic acid polymers, methacrylic acid polymers, acrylic acid / methacrylic acid copolymers, polysaccharides such as chitosan or agarose, polyethylene, polypropylene, polystyrene, polyvinyl butyrate, poly(vinyl chloride) or dendrimers. Suitable homogeneous α-amino acid polymers are, for example, polylysine, polyarginine, polyornithine, polyglutamine, polyasparagine, polyglutamic acid or polyaspartic acid. The poly-α-amino acids may be linear or branched.

[0125] The polymers may be used as is or may be combined with other materials such as, for example, glasses, ceramics, or metals. In some embodiments, the polymers are contained within magnetic nanoparticles (MNPs), which are well known in the art and are characterized by a core of magnetic material such as iron, nickel, or cobalt, and a polymeric shell of, for example, polyacrylic acid or other polymers with appropriate functional groups to be attached to ligands.

[0126] The selection of the particular polymer or its particular form from which the glycoconjugates of the present invention are made is based on the desired end use of the glycoconjugates.

[0127] In one aspect, the polymer in the glycoconjugate of the invention is selected from polyacrylic acid, polysaccharide resins and alpha amino acid homopolymers, preferably the polymer is selected from polyacrylic acid, agarose and polylysine.

[0128] The polylysine is a linear or branched poly-L-lysine, preferably a linear poly-L-lysine.

[0129] In some embodiments, the macromolecules in the glycoconjugates are contained within magnetic nanoparticles.

[0130] In the glycoconjugates produced with a given polymer, a single compound of formula (I) may be attached to multiple reactive sites on the polymer, or different compounds of formula (I) may be attached to the polymer to give a multifunctional glycopolymer. Preferably, a single compound of formula (I) is included in a given glycopolymer.

[0131] Furthermore, for a given polymer and a particular glycoconjugate comprising a given compound of formula (I), different specific products can be obtained depending on the degree of polymerization of the polymer, i.e., its molecular weight.

[0132] As is widely known in the art, said polymers suitable for preparing glycoconjugates are characterized in that they contain functional groups within their repeating polymer structure, such as hydroxy, carboxy or amino groups, which can form covalent bonds with functional groups on the carbohydrate ligands of the invention, in particular amine, carboxy or thiol functional groups on the ligand. Usually, the polymer needs to be activated with a compound that is reactive towards said functional groups on the ligand. The activated conjugate can then form a covalent bond with said compound, immobilizing the carbohydrate epitope on the polymer and forming the glycoconjugate.

[0133] There are various suitable methods for activating polymeric supports, which vary according to the type of polymer, as is widely known in the art.Various activation methods are described, for example, in the chapter on Immobilization of Ligands on Chromatography Supports in Bioconjugate Techniques, GT Hermanson, Ed., Chapter 15, p: 589-740.

[0134] For example, some well-known activation strategies for coupling a polymer to an amine group on a glycan include, among other options, activation of a carboxylate group on the polymer as an N-hydroxysuccinimide (NHS) ester group resulting in an amide bond to the ligand; an aldehyde-activated polymer resulting in a second amine bond to the ligand; an azlactone-activated polymer resulting in an amide bond to the ligand; activation of a hydroxy group with 1,1'-carbonyldiimidazole (CDI) to form a reactive imidazole carbamate; or activation of a carboxylate group on the polymer as a 4-nitrophenyl ester group resulting in an amide bond to the ligand.

[0135] Another well-known strategy for attaching the ligand of formula (I) to the polymeric support is by using a linker, which is a homobifunctional or heterobifunctional bifunctional moiety capable of linking both moieties. For example, when both the support and the ligand have amine functional groups, a common homobifunctional linker is a succinimidyl diester, such as succinimidyl adipic acid diester or disuccinimidyl suberate, which can form an amide bond at each of the two ends of the molecule to produce a glycoconjugate. In this case, the ligand can be first activated and then reacted with the polymer, or the polymer can be first activated and then reacted with the ligand.

[0136] Another well-known option for coupling thiol-functionalized glycans to amine-functionalized polymeric supports is to first activate the amine groups on the support by chloroacetylation, and then react the chloroacetylated polymer with thiol functional groups on the ligand to produce glycoconjugates. This strategy is disclosed, for example, in Duthaler et al., Chimia, 2010, 64(1-2), 23-28. Similarly, -OH functional groups on polymers can be activated by chloroacetylation and coupled to thiol-functionalized glycans.

[0137] Other suitable activation methods for producing glycoconjugates are also disclosed, for example, in WO 2018 / 167320.

[0138] Various strategies for the preparation of carbohydrate polymers are widely known in the art and it is within the capabilities of the skilled artisan to select the most appropriate method and conditions in each case.

[0139] The content of ligands on the polymer refers to the percentage of functional groups on the polymer that are bound to the active sugar of the present invention. In general, a content of 2% to 90%, preferably a content of 5% to 50%, more preferably a content of 10% to 40% is used. The remaining unbound functional groups on the polymer are usually capped, i.e., reacted with a suitable capping substance with the aim of suppressing the reactivity of the remaining free functional groups on the polymer. The capping agent used depends on the specific functional groups on the polymer. The selection of a specific capping agent is not critical, as long as the specific capping agent can be easily bound to the free reactive moieties on the polymer in high yield and results in a stable non-reactive capped end. The capping molecule is preferably hydrophilic, non-antigenic, and not highly charged, in order to avoid a decrease in solubility in water. It is therefore within the capabilities of the skilled artisan to select the appropriate capping agent in each case. Commonly used capping agents are, for example, glycolic acid, gluconic acid, succinic acid, ethanolamine or other amine derivatives, thio compounds such as thioglycerol, etc.

[0140] In one embodiment, the glycoconjugates of the present invention comprise a compound of formula (I) attached to a polyacrylic acid backbone, thus forming a polyacrylamide.

[0141] In certain embodiments, the glycoconjugate has formula (V) as shown below:

[0142] [ka]

[0143] [wherein "Cap" means a capping agent, "DP" is the degree of polymerization of the polymer expressed as the number of monomers, and "a" is the content of carbohydrate in the glycoconjugate] The DP is preferably 50 to 1100, more preferably 100 to 1000, and the content is preferably 5 to 80, more preferably 10 to 70.

[0144] Any suitable capping agent known in the art may be used. For example, a suitable capping agent is ethanolamine, and the polyacrylamide glycoconjugate has the following formula:

[0145] [ka]

[0146] has.

[0147] In a preferred embodiment, the glycoconjugates of formula (V) and (Vb) are prepared from a compound of formula (I) selected from compounds 1-20 in Table 1.

[0148] To prepare glycoconjugates with a polyacrylic acid backbone, the acidic -COOH groups on the polyacrylic acid can be activated, for example, with N-hydroxysuccinimide (NHS) to form N-succinimide ester groups, or can be activated as 4-nitrophenyl ester groups, for example, as disclosed in Tuzikov et al., 40 years of glyco-polyacrylamide in glycobiology, Glycoconj. J., 2021, 38, 89-100 or Bovin et al. Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides, Glycoconjugate J., 1993, 10, 142-151.

[0149] Glycoconjugates with polyacrylic acid may be prepared, for example, as shown in Scheme II (FIG. 5), in which the -COOH groups of polyacrylic acid are activated as 4-nitrophenyl ester groups and the capping agent is ethanolamine.

[0150] Other methods of activation of polyacrylic acid may also be suitable for the production of glycoconjugates with functionalized glycans of the invention.

[0151] In another embodiment, the glycoconjugate comprises a compound of formula (I) bound to agarose as a polymeric backbone. Agarose, as it is commonly known, is a linear polysaccharide composed of repeating units of agarobiose and is commercially available as resin beads. Cross-linked forms of agarose are commercially available under the name Sepharose®. Activated agarose is usually used for the preparation of glycoconjugates, which is commercially available. These activated forms are available as resin beads of various sizes.

[0152] Suitable activated agarose forms are, for example, N-hydroxysuccinimide (NHS)-activated agarose, aldehyde-activated agarose, azlactone-activated or 1,1'-carbonyldiimidazole (CDI)-activated agarose, etc. For example, the conjugation of a ligand to NHS-activated agarose can be carried out according to the following scheme:

[0153] [ka]

[0154] It may be implemented as shown in:

[0155] A preferred form of agarose is one having a molecular weight of 25-1000 KDa.

[0156] In a preferred embodiment, the glycoconjugate with agarose is prepared from a compound of formula (I) selected from compounds 1-18 in Table 1.

[0157] In another embodiment, the glycoconjugates of the invention comprise a compound of formula (I) attached to a polylysine backbone, in particular poly-L-lysine.

[0158] In one particular embodiment, the glycoconjugate with poly-L-lysine has the following formula:

[0159] [ka]

[0160] [wherein "Cap" means a capping agent, "DP" is the degree of polymerization of the polymer expressed as the number of lysine units, and "a" is the carbohydrate content in the glycoconjugate] has.

[0161] The polylysine may be linear or branched, preferably linear.

[0162] Suitable degrees of polymerization of polylysine are about 50 to 2000, preferably 100 to 1000. Preferred degrees of polymerization of polylysine are about 100, about 600 and about 1000, and are commercially available in the form of hydrobromide. Poly-L-lysine hydrobromide may be synthesized by ring-opening polymerization of N-carboxy-anhydrides using N6-trifluoroacetyl-L-lysine as a precursor, as disclosed, for example, in Hadjichristidis et al., Chem. Rev., 2009, 11, 5528-5578.

[0163] The ligand content on the polylysine is usually 2% to 90%, preferably 5% to 50%, and more preferably 10% to 40%.

[0164] Suitable capping agents known in the art may be used for glycoconjugation with polylysine. For example, a suitable capping agent is glycolic acid, which forms a glycolamide end group, and the glycoconjugate has the following formula:

[0165] [ka]

[0166] has.

[0167] In a preferred embodiment, the glycoconjugates of formula (VI) and (VIb) are prepared from a compound of formula (I) selected from compounds 1-18 in Table 1.

[0168] To prepare the glycoconjugates of formula (VI) and (VIb), 2 The ligand represented by is attached to the amine group on the side chain of polylysine using an N-hydroxysuccinimidyl diester linker, in particular an adipic acid diester. Preferably, the ligand is first activated by reaction with the diester, and then the activated compound is reacted with polylysine (as the hydrobromide salt) as shown in Scheme III (Figure 6). Preparation of the glycoconjugate is depicted in Scheme IV (Figure 7).

[0169] Capping with glycolic acid may be carried out, for example, in two steps by first reacting the free amino groups on the glycoconjugate with activated and protected glycolic acid (glycolic acid acetate N-hydroxysuccinimide ester) and then deprotecting the acetate-protected glycolic acid amide group to give the final capped product.

[0170] In another embodiment, the glycoconjugates of the invention comprise a compound of formula (I) bound to a magnetic nanoparticle. Magnetic nanoparticles (MNPs) are well known in the art and are characterized by comprising a magnetic material in the core, such as iron, nickel or cobalt, and a usually polymeric outer shell.

[0171] In this particular case, the glycoconjugate of the invention comprises a compound of formula (I) as previously described and a polymeric backbone or support, said polymeric support constituting the outer shell of the magnetic nanoparticle.

[0172] The polymeric shell of the magnetic nanoparticles has suitable functional groups that can be coupled to ligands via common coupling methods as described above. For example, N-hydroxysuccinimide-activated magnetic nanoparticles can be prepared by the following reaction:

[0173] [ka]

[0174] The functionalized glycans may be attached according to the method of the present invention.

[0175] NHS-activated magnetic nanoparticles are commercially available, for example from CD Bioparticles, Inc. Other types of activated magnetic nanoparticles and other binding mechanisms with the glycomimetics of the invention may also be suitable, and it is within the ability of one of skill in the art to adapt the method to a particular activated magnetic nanoparticle.

[0176] It is further meant that said glycoconjugates or carbohydrate polymers comprise a functionalized surface with active mimetics of formula (I) which allow the identification of antibodies in the blood.

[0177] Use of complex carbohydrates The glycoconjugates of the invention comprising the glycan of formula (I) surprisingly have high affinity for natural anti-A / B antibodies, despite the fact that they have different active epitopes than the natural A / B antigens, in particular that, in contrast to what has been disclosed so far in the art, they contain glucose or glucosamine derivatives at the non-reducing end, instead of galactose or N-acetylgalactosamine, as in the natural A and B antigens.

[0178] Furthermore, the novel epitopes are not only available for binding of the natural anti-A / B antibodies, but in some cases have a higher activity than the natural antigen.

[0179] In addition, it may be useful to use them in combination with natural antigens.

[0180] This is shown in the assays disclosed in Examples 5-8, in which its effect on inhibition and removal of anti-A / B antibodies and its use for the purification of human polyclonal anti-A / B antibodies were tested on both normal human serum derived from blood groups A or B and pure fractions of human anti-A / B antibodies.

[0181] Examples 6.2 and 6.3 show that glycoconjugates of the invention, particularly those containing novel epitopes, in particular with poly-L-lysine, were effective in removing purified human anti-A and anti-B antibodies.

[0182] This remarkable affinity for anti-A / B antibodies can be used to inhibit and / or remove anti-A and / or anti-B antibodies from blood and blood derivative samples, which is useful, for example, for many applications enabling blood transfusions and organ transplants between incompatible blood donors, including live donor organ transplants, among other applications, or for the inhibition and removal of anti-A and anti-B antibodies during methods of purification and fractionation of blood products.

[0183] The use of glycoconjugates to avoid rejection in organ transplantation is suitable, for example, for kidney, liver, skin, pancreas, cornea, bone marrow or heart transplants. For this purpose, anti-A and / or anti-B antibodies can be inhibited / removed ex vivo or in vivo, or, complementary, ex vivo and in vivo. For ex vivo removal, blood from the organ transplant recipient is drawn from the subject and then contacted with the glycoconjugates of the invention to remove anti-A and / or anti-B antibodies, and the treated blood is then reinfused into the subject. In an alternative in vivo method, the inhibition / removal of anti-A and / or anti-B antibodies is performed in the body by administering appropriate glycoconjugates capable of inhibiting / removing circulating antibodies in the subject.

[0184] For this in vivo use, the polymer in the glycoconjugate is usually a biodegradable and biocompatible polymer. Suitable polymers for this use are polyamino acids, such as polylysine or polyarginine. One particularly preferred glycoconjugate for in vivo use is a poly-L-lysine glycoconjugate, since poly-L-lysine has been proven to be safe and not immunogenic after in vivo administration (Katopodis et al., J. Clin. Invest., 2002, 110, 1869-1877).

[0185] On the other hand, applications relating to the purification and fractionation of blood and blood derivative samples or the identification of antibodies from blood or blood derivative samples are performed in vitro or ex vivo.

[0186] Depending on the antibodies to be removed or detected, suitable glycoconjugates containing suitable glycans that are blood group A antigen mimetics or blood group B antigen mimetics, respectively, may be selected for a particular purpose.

[0187] Therefore, another aspect of the present invention is the use of the glycoconjugates of the present invention for inhibiting and / or removing anti-A and / or anti-B antibodies from samples of blood or other blood derivatives and by-products, such as for example serum, plasma, fractionated plasma or immunoglobulin preparations. According to this aspect, it is understood that the blood and / or blood derivative / by-product samples are treated with the glycoconjugates in vitro or ex vivo and no treatment is performed in the organism.

[0188] Typically, methods for removing anti-A and / or anti-B antibodies from samples of blood or other blood derivatives and by-products include contacting the sample with glycoconjugates for a sufficient time to allow the antibodies to bind to active epitopes on the glycoconjugates, and then separating the glycoconjugates from the sample. The glycoconjugates are typically made from a solid polymeric support, e.g., in the form of solid microbeads to facilitate contact with and subsequent separation from the processed sample.

[0189] The methods for blocking anti-A and / or anti-B antibodies from the sample are similar, but the sample does not have to be separated from any inactivated antibodies remaining in the sample bound to glycoconjugates.

[0190] The high affinity binding of anti-A / B antibodies by the glycoconjugates of the invention can also be used to detect these antibodies in a sample. For some in vitro applications, the glycomimetics of the invention may be bound to a suitable surface or device adapted for antibody detection.

[0191] Another aspect of the invention are the glycoconjugates of the invention for use in medicine, in particular for use in the treatment and / or prevention of rejection or hemolytic reactions in subjects receiving organ transplants or blood transfusions, respectively, from incompatible donors.

[0192] Another aspect of the present invention is a method for treating and / or preventing rejection or hemolysis in a subject in need of receiving an organ transplant or a blood transfusion, respectively, from an incompatible donor, comprising administering a therapeutically effective amount of a glycoconjugate of the present invention.

[0193] Another aspect of the present invention is the use of the glycoconjugates of the present invention for the manufacture of a medicament for the treatment and / or prevention of rejection or hemolytic reactions in a subject receiving an organ transplant or a blood transfusion, respectively, from an incompatible donor.

[0194] By incompatible donor is meant an organ donor or a blood donor.

[0195] According to this aspect, in the context of the treatment or prevention of rejection or hemolytic reactions in humans, the glycoconjugates of the invention comprise a biodegradable and biocompatible polymer. Preferred polymers are, for example, homopoly-α-amino acids selected from polylysine, polyarginine, polyornithine, polyglutamine, polyasparagine, polyglutamic acid and polyaspartic acid. Particularly preferred is polylysine, in particular poly-L-lysine as previously described.

[0196] Therapeutic glycoconjugates are usually administered by intravenous, subcutaneous or intramuscular injection.

[0197] As used herein, "biodegradable" means that the polymer is metabolically biodegradable such that it is fragmented and removed from the body after administration. As used herein, "biocompatible" means that the polymer is safe and non-toxic, and therefore suitable for administration to an organism.

[0198] The terms "inhibition" and "removal" may be used interchangeably in this specification. Inhibition refers to the fact that the antibodies are inactivated after binding to the active epitope on the glycoconjugate of the invention and are unable to further bind to the natural anti-A / B antigens even if they remain present in the sample. Removal refers to the fact that after in vitro or ex vivo processing, where the treated sample is collected, the antibodies are eliminated from the treated sample and the antibodies are retained on the solid carbohydrate polymer or, after in vivo inhibition, the immune complexes are subsequently metabolized and removed from the bloodstream as well.

[0199] As used herein, the subject undergoing an organ transplant or blood transfusion to be treated with the glycoconjugates of the invention refers to a human.

[0200] As used herein, mismatched donor refers to a subject who has ABH mismatched antigens.

[0201] The glycoconjugates of the invention may be administered before a subject undergoes an ABO incompatible transplant or blood transfusion, or, complementary, the treatment may be administered after a subject undergoes an ABO incompatible transplant or blood transfusion.

[0202] Pharmaceutical Compositions Another aspect of the present invention is a pharmaceutical composition comprising the glycoconjugate of the present invention and at least one pharma- ceutically acceptable excipient.

[0203] It is understood that glycoconjugates suitable for preparing pharmaceutical compositions are as described above for medical use, i.e. the polymer of the glycoconjugate is usually biocompatible and biodegradable. Preferably, the polymer is a poly-alpha amino acid, more preferably polylysine, even more preferably poly-L-lysine, even more preferably poly-L-lysine as described above in the section on glycoconjugates.

[0204] The pharmaceutical compositions of the present invention are manufactured according to general procedures well known in the art, using common and widely available additives, for example as described in the reference book RC Rowe, PJ Sheskey and PJ Weller, Handbook of Pharmaceutical Excipients, Sixth Edition, Pharmaceutical Press, 2009. General additives and procedures for manufacturing the compositions are also described in JP Remington and AR Genaro, Remington The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins, Philadelphia, 2000 [ISBN: 0-683-306472] or in the book ME Aulton and KMG Taylor, Aulton's Pharmaceutics, the design and manufacture of medicines, 4th edition, Churchill Livingstone Elsevier, 2013 [ISBN: 978-0-7020-4290-4].

[0205] Pharmaceutical compositions containing the glycoconjugates of the present invention are preferably for parenteral use.

[0206] The composition usually comprises a vehicle such as water or a saline solution. A non-aqueous solvent such as ethanol, glycerol or propylene glycol can be added as a co-solvent to improve solubility. The composition may comprise a preservative, an antioxidant, a pH adjusting agent, an isotonicity adjusting agent, a suspending agent or a combination thereof, as is generally known in the art. EXAMPLES

[0207] Example 1 Preparation of the compound represented by formula (I) Certain compounds of formula (I) listed in Table 2 were synthesized according to Schemes Ia, Ib, and Ic and the synthetic methods described above.

[0208] [Table 2]

[0209] To further illustrate the preparation methods, the synthesis of some of the above compounds (Examples 1.1, 1.5, 1.8, 1.9, 1.11 and 1.12) is disclosed in detail below, the remaining compounds were obtained by similar procedures.

[0210] General Procedure Reactions were carried out using commercially available reagents (Carbosynth Ltd, Acros, Aldrich and Fluka); anhydrous solvents were purified using standard procedures.

[0211] Column chromatography was performed using silica gel 60 0.040-0.063 mm (Merck). The solvent was removed in vacuum at 30-40° C. Thin layer chromatography (TLC) was performed on silica gel 60 F254 aluminum-backed plates (Merck). 3 PO 4 Compound spots were visualized by immersing the TLC plate in an aqueous solution (8%) followed by heating (>150° C.).

[0212] 1H NMR spectra were recorded on a Bruker BioSpin GmbH (700 MHz) spectrophotometer at 30 °C; chemical shifts (δ, ppm) are expressed as internal D 2 O(δ4.79), CD 3 OD(δ3.31), CDCl 3 (δ7.27) and (CD 3 )SO (δ 2.50) as the solvent residual peak; coupling constants (J) were measured in Hz. 13 C NMR spectra were recorded at 150 MHz. Chemical shifts (δ, ppm) were expressed as internal CD 3 OD(δ49.0), CDCl 3 (δ77.16) and (CD 3 ) 2 The peak of SO (δ 39.62) was referenced. 1 H NMR and 13 Signals in the C NMR spectrum were assigned to the corresponding protons using 2D spectroscopy (COSY, HSQC).

[0213] Example 1.1 3-Aminopropyl α-D-glucopyranosyl-(1→3)-[α-L-fucopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (Glcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O-(CH 2 ) 3 -NH 2 )

[0214] [ka]

[0215] The title compounds were prepared according to the routes shown in Schemes Ia and Ib detailed below.

[0216] Ia, Step 1: 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside (Intermediate 1.1.1)

[0217] [ka]

[0218] β-Bn in anhydrous dichloromethane (5 ml) 4 To a solution of GlcSEt (compound represented by formula A, 553 mg, 0.95 mmol) was added bromine (55 μL, 1.0 mmol) at 0° C. The reaction was left at 0° C. for 1 h. The solvent and bromine were evaporated and the residue was coevaporated with toluene (3×5 mL) to remove traces of bromine and to obtain Bn 4 The nearly colorless oil of GlcBr was dried in vacuum at room temperature.

[0219] To a solution of disaccharide B (500 mg, 0.63 mmol) in anhydrous dichloromethane (6 mL) was added freshly dried MS-4Å (2 mL) and tetramethylurea (0.137 mL, 1.15 mmol), the mixture was stirred for 30 min, after which AgOTf (0.27 g, 1.05 mmol) was added and the mixture was stirred for an additional 15 min. The reaction mixture was treated with Bn in anhydrous dichloromethane (3 mL). 4A solution of GalBr was added dropwise with vigorous stirring. The reaction was stirred for 24 hours. The reaction mixture was neutralized with pyridine (0.5 ml), filtered through Celite on a glass filter, and the solid was washed with dichloromethane and a mixture of dichloromethane / methanol 10:1. The resulting solution was concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. Intermediate 1.1.1 was purified by silica gel column chromatography (hexane / ethyl acetate, 1:1 to 1:5), and the fractions mainly containing intermediate 1.1.1 were collected, concentrated using a rotary evaporator, coevaporated with toluene, dried in vacuum, and used in the next step without further purification. Spectroscopic data ( 1 1 H NMR) was consistent with the predicted structure.

[0220] Ia, Step 2: 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (Intermediate 1.1.2)

[0221] [ka]

[0222] Intermediate 1.1.1 was dissolved in anhydrous methanol (30 mL) and treated with 2M NaOMe (3 mL, 6 mmol) and the reaction mixture was left at room temperature for 4 h. The reaction mixture was diluted with Dowex® 50WX4 (6.0 g, PyH + The mixture was neutralized with toluene (formaldehyde), stirred for 15 minutes, and filtered. The resulting solution was concentrated using a rotary evaporator, coevaporated with ethyl acetate, and dried in vacuum. The product intermediate 1.1.2 was purified by silica gel column chromatography (chloroform / methanol, 10:1), and the fractions mainly containing the product intermediate 1.1.2 were collected, concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. The intermediate 1.1.2 was used in the next step without further purification. Spectroscopic data ( 1 1 H NMR) was consistent with the predicted structure.

[0223] Ia, Step 3: 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2-acetamido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (Intermediate 1.1.3)

[0224] [ka]

[0225] The trisaccharide of intermediate 1.1.2 was suspended in dry acetonitrile (5 mL) and then reacted with PhCH(OMe) 2 (0.16 mL, 1.0 mmol) and TsOH H in dry acetonitrile (0.1 mL). 2 A solution of 1.0 (6.0 mg, 0.034 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction was neutralized with pyridine (0.2 mL) and 30 ml of diethyl ether and 30 ml of hexane were added under stirring. The solid was collected on a glass filter, washed with ether / hexane 1:1, pure hexane and dried in vacuum. The crude intermediate 1.1.3 was dissolved in 25 ml of DMF and precipitated with 75 ml of water, and the pure solid intermediate 1.1.3 was collected on a glass filter, washed with water and dried in a fume hood. Overall yield of intermediate 1.1.3 over three steps: 48% (380 mg, 0.3 mmol). Due to the low solubility of intermediate 1.1.3, NMR spectral data is not available.

[0226] Ia, Step 4: 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside (Compound C, Ra=OBn, R4-Z'=(CH 2) 3 NHCOCF 3 )

[0227] [ka]

[0228] To a powder of the trisaccharide of intermediate 1.1.3 (380 mg, 0.3 mmol) was added a solution of chloroacetic anhydride (150 mg, 0.9 mmol) and sym-collidine (0.8 mL, 5.8 mmol) in anhydrous dichloromethane (2 mL) with vigorous stirring. The mixture was stirred at room temperature for 3 h. The reaction was quenched with methanol (1 mL), concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum.

[0229] The resulting oil was dissolved in anhydrous dichloromethane (4 ml) and then sym-collidine (0.9 mL, 6.7 mmol) and acetyl chloride (0.35 mL, 4.9 mmol) were added. The reaction mixture was left at room temperature for 24 hours. The reaction was quenched with methanol (1.4 mL), concentrated using a rotary evaporator and coevaporated with toluene. The residue was diluted with toluene (7 mL), stirred for 15 minutes and the collidinium salt was collected on a glass filter and washed several times with toluene. The filtered solution of intermediate 1.1.4 was concentrated using a rotary evaporator and dried in vacuum.

[0230] [ka]

[0231] The crude intermediate 1.1.4 was dissolved in pyridine (7 mL), water (3.5 mL) was added and the mixture was warmed for 24 h to 60° C. The mixture was concentrated using a rotary evaporator, coevaporated with toluene and dried in vacuum.

[0232] The product was purified by silica gel column chromatography (chloroform / acetone, 5:1 to 3:1, 0.5% pyridine), and the fractions containing mainly the product were collected, concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. White crystals. Yield of C: 70% (270 mg, 0.21 mmol). Spectroscopic data ( 1 1 H NMR) was consistent with the predicted structure.

[0233] Ib, steps 1 and 2: 3-trifluoroacetamidopropyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-(1→2)]-4,6-di-O-acetyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranoside (intermediate 1.1.7)

[0234] [ka]

[0235] β-Bn in anhydrous dichloromethane (2.63 ml) 3 To a solution of FucSEt (0.20 g, 0.42 mmol) was added bromine (24 μL, 0.47 mmol) at 0° C. The reaction was left at 0° C. for 1 h. The solvent and bromine were evaporated and the residue was coevaporated with toluene (3×2 mL) to remove traces of bromine and to give Bn 3 The nearly colorless oil of FucBr was dried in vacuum at room temperature.

[0236] To a solution of compound C (270 mg, 0.21 mmol) in anhydrous dichloromethane (5.25 ml) was added freshly dried MS-4Å (2.6 mL) and tetramethylurea (56 μl, 0.47 mmol), and the mixture was stirred for 30 min, after which AgOTf (0.11 g, 0.42 mmol) was added and the mixture was stirred for an additional 15 min. The reaction mixture was treated with Bn 3The solution of FucBr was added dropwise with vigorous stirring. The reaction mixture was neutralized with pyridine (0.2 ml), filtered through Celite on a glass filter, and the solid was washed with dichloromethane and a mixture of dichloromethane / methanol 10:1.

[0237] The filtered solution was concentrated using a rotary evaporator, co-evaporated with toluene, and dried in vacuum to give crude intermediate 1.1.5.

[0238] [ka]

[0239] The crude intermediate 1.1.5 was dissolved in acetic acid (7 mL), water (1.75 mL) was added under stirring and the mixture was warmed to 80° C. for 3 h. The mixture was concentrated using a rotary evaporator, coevaporated with toluene and dried in vacuum to give intermediate 1.1.6.

[0240] [ka]

[0241] Intermediate 1.1.6 was purified by silica gel column chromatography (toluene / ethyl acetate, 1:1 to 0:1), and the fractions containing mainly the product were collected (TLC control in pure ethyl acetate), concentrated using a rotary evaporator, coevaporated with toluene, dried in vacuum, and used in the next step without further purification.

[0242] After dissolving intermediate 1.1.6 in methanol (10.0 mL), 10% Pd / C (0.1 g) was added and the reaction mixture was degassed in vacuum by filling with hydrogen gas (1 atm) three times. The reaction was stirred at room temperature under hydrogen gas (1 atm) atmosphere for 48 h. The conversion was controlled by TLC (propan-2-ol / ethyl acetate / water 2:3:1). The reaction mixture was filtered through Celite on a glass filter, the solid was washed with methanol, and the resulting solution with 50% aqueous methanol was concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. The residue was dissolved in pyridine (3.5 mL) and AcOEt (2.2 mL). 2 The mixture was dissolved in 20 (1.75 mL) and the reaction mixture was left for 24 h at 40° C. The reaction mixture was concentrated using a rotary evaporator and coevaporated with toluene.

[0243] The obtained intermediate 1.1.7 was purified by silica gel column chromatography (ethyl acetate / propan-2-ol, 50:0 to 50:1), and the fractions mainly containing the product were collected (TLC with ethyl acetate / propan-2-ol 50:1 and chloroform / propan-2-ol 9:1), concentrated using a rotary evaporator, evaporated with toluene, and dried in vacuum.

[0244] Yield of the last four steps of intermediate 1.1.7: 35% (97 mg, 74 μmol).

[0245] Ib, Step 3: 3-Aminopropyl α-D-glucopyranosyl-(1→3)-[α-L-fucopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (Example 1.1)

[0246] [ka]

[0247] The tetrasaccharide of intermediate 1.1.7 (97 mg, 74 μmol) was dissolved in anhydrous methanol (5.0 mL) and treated with 2 M NaOMe (0.26 mL, 0.53 mmol) and the reaction mixture was allowed to stand for 2 h. The mixture was concentrated in vacuo (do not go to dryness), diluted with water (5.0 mL) and the reaction mixture was allowed to stand overnight.

[0248] The target deprotected tetrasaccharide was chromatographed using Dowex® 50X4-400 (H + ) and eluted with water, 1M aqueous pyridine and 1M aqueous ammonia. The carbohydrate-containing fractions were collected, concentrated in vacuo and lyophilized twice to give a white solid material. Yield of Example 1.1: 90% (50 mg, 63 μmol). 1 H NMR(D 2 O+TFA, characteristic signal): δ 1.26 (d, 3H, J 5,6 6.6Hz, H-6 Fuc), 1.93-2.01(m, 2H, OCH 2 CH 2 CH 2 N), 2.08(s, 3H, NHC(O)CH 3 ), 4.51 and 4.63 (2d, 2×1H, J 1,2 8.4Hz, J 1,2 7.7 Hz, H-1 GlcHAcβ and H-1 Galβ), 5.23 and 5.37 (2d, 2 × 1H, J 1,2 3.8Hz, J 1,2 4.2 Hz, H-1 Galα and H-1 Fucα).

[0249] Example 1.5 3-Aminopropyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-[α-L-fucopyranosyl-(1→2)]-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (GlcNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O-(CH 2 ) 3 -NH 2 )

[0250] [ka]

[0251] This product was prepared in a similar manner to Example 1.1, starting from step 2, using intermediate 1.5.1, the preparation of which is described below, instead of intermediate 1.1.1.

[0252] 3-Trifluoroacetamidopropyl 2-azido-3,4,6-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside (Intermediate 1.5.1)

[0253] [ka]

[0254] Disaccharide B (500 mg, 0.63 mmol) and β-Bn in anhydrous ether / dichloromethane 1:1 (20 ml) 3 N 3 GlcO(CN)CCl 3 To a solution of (735 mg, 1.26 mmol) freshly dried MS-4Å (3 mL) was added, and the mixture was stirred for 30 min, followed by the addition of trimethylsilyl trifluoromethanesulfonate (TMSOTf) (12 μL, 63 μmol). The reaction was stirred for 24 h. The reaction mixture was neutralized with pyridine (0.5 ml) and MeOH (1 ml), filtered through Celite on a glass filter, and the solid was washed with dichloromethane and a mixture of dichloromethane / methanol 10:1. The filtrate was concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. The obtained intermediate 1.5.1 was purified by silica gel column chromatography (toluene / acetone, 2:1 to 1:1), and the fractions mainly containing intermediate 1.5.1 were collected, concentrated using a rotary evaporator, coevaporated with toluene, dried in vacuum, and used in the next step without further purification. Yield of intermediate 1.5.1: 50% (395 mg, 0.315 mmol). 1 H NMR (CDCl 3 , characteristic signal): δ 1.76-1.92 (m, 2H, OCH 2 CH 2 CH 2 N), 1.98, 1.98, 2.06, 2.08, 2.20(5s, 15H, C(O)CH 3 ), 4.37, 4.45(2d, 2H, J 1,2 7.9, J 1,2 8.0, H-1 I and H-1 II ), 5.12(d, 1H, J 1,2 3.5, H-1 III ), 5.44(d, 1H, J 1,2 3.1, H-4 II ); 13 C NMR (CDCl 3 , characteristic signals): δ 20.5, 20.7, 20.8, 20.8, 23.2 (C(O)CH 3 ), 28.5(OCH 2 CH 2 CH 2 N), 94.0(C-1 III ), 100.8, 102.0(C-1 I and C-1 II ).

[0255] Example 1.8 3-Aminopropyl 2-O-(α-L-fucopyranosyl)-3-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (GlcNAcα1-3(Fucα1-2)Galβ-O-(CH 2 ) 3 -NH 2 )

[0256] [ka]

[0257] 1,3,4,6 Tetra-O-acetyl-2-azido-2-deoxy-α / β-D-glucose (Intermediate 1.8.1)

[0258] [ka]

[0259] A solution of 2-azido-2-deoxy-α / β-D-glucose (5.0 g, 24.4 mmol) in anhydrous pyridine (5 ml) was added with Ac 2 0 (5 ml) was added and the reaction mixture was kept at room temperature for 24 hours, concentrated in vacuo, coevaporated with toluene (3×20 ml) and then dried in vacuum (1 mmHg) to give 9.0 g of crude product. f 0.3 (toluene-AcOEt, 3:1).

[0260] 3-Trifluoroacetamidopropyl 4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (Intermediate 1.8.2)

[0261] [ka]

[0262] Anhydrous CH 2 Cl 2 To a solution of intermediate 1.8.1 (2 g, 5.3 mmol) in (20 ml) of TiBr 4 (2.65 g, 7.9 mmol) was added. The reaction was stirred at room temperature for 48 h. The mixture was poured onto ice and CHCl 3 (100 ml). The organic extract was washed with water (100 ml), cold saturated NaHCO 3 The resulting 3,4,6 tri-O-acetyl-2-azido-1-bromo-2-deoxy-α-D-glucose, R f 0.4 (toluene-AcOEt, 3:1) in anhydrous CH 2 Cl2 (20 ml) and dissolved in anhydrous CH 2 Cl 2 3-Trifluoroacetamidopropyl 4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-galactopyranoside (2.04 g, 2.4 mmol) in (70 ml), Ag 2 CO 3 (6.61 g, 24.0 mmol), AgOTf (30.8 mg, 0.12 mmol) and molecular sieves 4 Å were added dropwise to the mixture. The reaction mixture was stirred at room temperature in the dark for 24 h. The solid was filtered and dissolved in CHCl 3 (300 ml) and the combined filtrates were concentrated in vacuo. The residue was chromatographed on silica gel with toluene-AcOEt, 2:1 to give 1.61 g (1.5 mmol) of the title compound in 64% yield. f 0.6 (toluene-AcOEt, 1:1).

[0263] 3-Trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2-azido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (Intermediate 1.8.3)

[0264] [ka]

[0265] Intermediate 1.8.2 (1.61 g, 1.5 mmol) was dissolved in 80% aqueous AcOH (30 ml), the solution was kept at 80° C. for 2 h, concentrated in vacuo, coevaporated with toluene (3×50 ml) and then dried in vacuum (1 mmHg). The residue was extracted with n-hexane-CHCl 3Silica gel chromatography with 1,2-dihydro-2,4-tetrahydrofuran and 1,2-dihydro-2-propanol, 4:2:1 gave 1.18 g (1.22 mmol) of 3-trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside in 80% yield. f 0.49 (n-Hexane-CHCl 3 -2-propanol, 4:2:1).

[0266] 3-Trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (1.18 g, 1.22 mmol) was dissolved in anhydrous MeOH (40 ml), 2M NaOMe in MeOH (116 μl, 0.23 mmol) was added and the reaction mixture was kept at room temperature for 2 hours. Then, the cation exchange resin Dowex® 50X4-400 (H + ) (1 ml in MeOH) was added, held for 10 min and filtered. The filtrate was concentrated in vacuo and the residue was extracted with CHCl 3 Silica gel chromatography with 3:1 hexanes / 2-propanol gave 0.971 g (1.16 mmol) of the title compound in 95% yield. f 0.32(CHCl 3 -2-propanol, 4:1).

[0267] 3-Trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (Intermediate 1.8.4)

[0268] [ka]

[0269] To a solution of intermediate 1.8.3 (150 mg, 0.16 mmol) in 2.5 ml of DMF-water (4:1) was added DTT (128 g, 0.8 mmol) and Et 3 N (50 μl) was added. The reaction mixture was kept at room temperature for 2 h, concentrated in vacuo, coevaporated with toluene (3×30 ml) and then dried in vacuum (1 mmHg). The residue was dissolved in anhydrous MeOH (3 ml) and treated with Ac 2 O (500 μl) and Et 3 N (50 μl) was added and the reaction mixture was kept at room temperature overnight. The mixture was then concentrated in vacuo and coevaporated with toluene (3×20 ml) followed by CHCl 3 Silica gel chromatography with 2:1 hexanes / 2-propanol afforded 81 mg (0.085 mmol) of the title compound in 53% yield. f 0.40(CHCl 3 -2-propanol, 2:1).

[0270] 3-Aminopropyl 2-O-(α-L-fucopyranosyl)-3-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside (Example 1.8)

[0271] [ka]

[0272] Intermediate 1.8.4 (81 mg, 0.085 mmol) was dissolved in anhydrous MeOH (5 ml), 10% Pd / C (80 mg) was added, and the reaction mixture was diluted with H 2 The mixture was stirred at room temperature for 12 h in an atmosphere of 0.5%. The catalyst was filtered and washed with MeOH; the combined filtrates were concentrated in vacuo. The residue was purified by C18 HPLC to give 48 mg of 3-trifluoroacetamidopropyl 2-O-(α-L-fucopyranosyl)-3-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-β-D-galactopyranoside, which was diluted with 5% Et 3 The reaction mixture was treated with a 2 ml solution of N in water (12 h, room temperature).+ ) cation exchange resin (2 ml). The column was washed with water (5 ml), 1M aqueous pyridine solution (5 ml) and the title compound was eluted with 1M aqueous ammonia solution. The fractions containing the compound of Example 1.8 were concentrated in vacuo and the residue was dissolved in water (2 ml) and lyophilized. Yield: 40 mg (97%). 1 H NMR(D 2 O+TFA):δ 1.25(d, 2H, J 6.6Hz, H-6 Fuc), 2.02(m, 2H, OCH 2 CH 2 CH 2 N), 2.05(s, 3H, NHC(O)CH 3 ), 3.15(m, 2H, CH 2 N), 3.56(dd≒t, 1H, J 3,4 ≒J 4,5 9.5Hz, H-4 GlcN), 3.68(ddd≒br.dd, 1H, J 5,6a 8.0Hz, J 5,6b 4.3Hz, J 4,5 <1.0Hz, H-5 Gal), 3.76(dd, 1H, J 6a,6b 10.7Hz, J 5,6a 9.0Hz, H-3 GlcN), 3.75-3.91(m, 9H, H-6 a Gal, H-6 b Gal, H-6 a GlcN, H-6 b GlcN, H-2 Gal, H-2 Fuc, H-3 Fuc, H-4 Fuc, OCH), 3.99(ddd, 1H, J 5,6a 2.4Hz, J 5,6b 4.7Hz, J 4,5 10.0Hz, H-5 GlcN), 3.96-4.05(m, 3H, H-2 GlcN, H-3 Gal, CHO), 4.22(dd≒br.d, 1H, J 3,4 3.4Hz, J 4,5 <1Hz, H-4 Gal), 4.38(br.q, 1H, J 5,6 6.6Hz, J 4,5 <1Hz H-5 Fuc), 4.57(d, 1H, J 1,2 7.8Hz, H-1 Galβ), 5.16(d, 1H, J1,2 3.8Hz, H-1 GlcNα), 5.33(d, 1H, J 1,2 3.7Hz, H-1 Fucα).

[0273] Example 1.9 3-Aminopropyl 2-O-(α-L-fucopyranosyl)-3-O-(2-deoxy-2-n-propionylamido-α-D-glucopyranosyl)-β-D-galactopyranoside (GlcNpropionylα1-3(Fucα1-2)Galβ-O-(CH 2 ) 3 -NH 2 )

[0274] [ka]

[0275] 3-Trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-3-O-(2-deoxy-2-n-propionylamido-α-D-glucopyranosyl)-β-D-galactopyranoside (Intermediate 1.9.1)

[0276] [ka]

[0277] To a solution of intermediate 1.8.3 (234 mg, 0.25 mmol) in 5 ml of DMF-water (4:1) was added DTT (192 g, 1.2 mmol) and Et 3 N (100 μl) was added. The reaction mixture was kept at room temperature for 2 h, concentrated in vacuo, coevaporated with toluene (3×50 ml) and then dried in vacuum (1 mmHg). The residue was dissolved in anhydrous MeOH (6 ml) and treated with n-propionyl anhydride (500 μl) and Et 3 N (100 μl) was added and the reaction mixture was kept at room temperature overnight. The reaction mixture was then concentrated in vacuo and coevaporated with toluene (3×20 ml) followed by CHCl 3Silica gel chromatography with 3:1 hexanes / 2-propanol gave 144 mg (0.16 mmol) of the title compound in 67% yield. f 0.30(CHCl 3 -2-propanol, 3:1).

[0278] 3-Aminopropyl 2-O-(α-L-fucopyranosyl)-3-O-(2-deoxy-2-n-propionylamido-α-D-glucopyranosyl)-β-D-galactopyranoside (Example 1.9)

[0279] [ka]

[0280] Intermediate 1.9.1 (103 mg, 0.106 mmol) was dissolved in anhydrous MeOH (10 ml), 10% Pd / C (120 mg) was added, and the reaction mixture was stirred at room temperature under hydrogen atmosphere for 12 h. The catalyst was filtered and washed with MeOH, and the combined filtrate was concentrated in vacuo. The residue was dissolved in anhydrous pyridine (1.5 ml) and added with Ac 2 O (1.5 ml) was added and the reaction mixture was kept at room temperature for 24 h, concentrated in vacuo, coevaporated with toluene (3×5 ml) and then dried in vacuum (1 mmHg). The residue was purified by distillation with n-hexane-CHCl 3 Silica gel chromatography with 1,2-dihydro-2,4-tetrahydrofuran, and 4:2:1 ethanol gave 105 mg (0.102 mmol) of 3-trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-n-propionylamino-α-D-glucopyranosyl)-4,6-di-O-acetyl-β-D-galactopyranoside in 90% yield. f 0.45(n-Hexane-CHCl 3 -2-propanol, 4:2:1).

[0281] 3-Trifluoroacetamidopropyl 2-O-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-n-propionylamino-α-D-glucopyranosyl)-4,6-di-O-acetyl-β-D-galactopyranoside (105 mg, 0.102 mmol) was dissolved in anhydrous MeOH (4 ml), 2M NaOMe in MeOH (20 μl, 0.04 mmol) was added and the reaction mixture was kept at room temperature for 2 hours. The reaction mixture was then concentrated in vacuo and the residue was dissolved in water (2 ml) and the mixture was kept at room temperature for 12 hours before being washed with Dowex® 50X4-400 (H + ) cation exchange resin (2 ml). The column was washed with water (5 ml), 1M aqueous pyridine solution (5 ml) and the title product was eluted with 1M aqueous ammonia solution. The fractions containing the compound of Example 1.9 were concentrated in vacuo and the residue was dissolved in water (2 ml) and lyophilized. Yield: 55 mg (90%). 1 H NMR(D 2 O+TFA):δ 1.13(t, 3H, J 7.7Hz, NHC(O)CH 2 CH 3 ), 1.25(d, 3H, J 6.6Hz, H-6 Fuc), 2.02(m, 2H, OCH 2 CH 2 CH 2 N), 2.32(q, 2H, J 7.6Hz, NHC(O)CH 2 CH 3 ), 3.11-3.18(m, 2H, CH 2 N), 3.56(dd, 1H, J 4,5 10.1Hz, J 3,4 9.1Hz, H-4 GlcN), 3.68(ddd≒br.dd, 1H, J 5,6a 8.0Hz, J 5,6b 4.3Hz, J 4,5 <1.0Hz, H-5 Gal), 3.76(dd, 1H, J 3,4 9.2Hz, J 2,3 7.7Hz, H-3 GlcN), 3.75-3.90(m, 9H, OCH, H-6 a GlcN, H-6b GlcN, H-2 Fuc, H-3 Fuc, H-4 Fuc, H-2 Gal, H-6 a Gal, H-6 b Gal), 3.98(ddd, 1H, J 4,5 10.2Hz, J 5,6a 4.8Hz, J 5,6b 2.9Hz, H-5 GlcN), 4.05-3.95(m, 3H, CHO, H-2 GlcN, H-3 Gal), 4.21(dd≒br.d, 1H, J 3,4 3.4Hz, J 4,5 <1Hz, H-4 Gal), 4.37(br.q, 1H, J 5,6 6.6Hz, J 4,5 <1Hz, H-5 Fuc), 4.56(d, 1H, J 1,2 7.8Hz, H-1 Galβ), 5.16(d, 1H, J 1,2 3.8Hz, H-1 GlcNα), 5.33(d, 1H, J 1,2 3.6Hz, H-1 Fucα).

[0282] Example 1.11 3-Aminopropyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-β-D-galactopyranoside (GlcNAcα1-3Galβ-O-(CH 2 ) 3 -NH 2 )

[0283] [ka]

[0284] This compound may be prepared in a similar manner to Example 1.12 (disclosed below), substituting Intermediate 1.11.1 for Intermediate 1.12.1, but using the method described below for Intermediate 1.12.1 (EtCO) 2 Ac instead of O 2 It is prepared in the same manner except that O is used.

[0285] [ka]

[0286] 1 H NMR(D 2 O+TFA):δ 2.03(m、2H、OCH 2 CH 2 CH 2 N)、2.06(s、3H、NHC(O)CH 3 )、3.19(m、2H、CH 2 N)、3.56(dd、1H、J 3,4 10.1Hz、J 4,5 9.0Hz、H-4 GlcN)、3.64(dd、1H、J 1,2 3.7Hz、J 2,3 10.0Hz、H-2 Gal)、3.68(ddd≒br.dd、1H、J 5,6a 4.3Hz、J 5,6b 7.9Hz、J 4,5 1.0Hz、H-5 Gal)、3.75(dd 1H、J 2,3 10.0Hz、J 3,4 3.3Hz、H-3 Gal)、3.76(dd、1H、J 6a,6b 11.8Hz、J 5,6a 4.3Hz、H-6 a Gal)、3.80(dd、1H、J 6a,6b 11.8Hz、J 5,6b 7.9Hz、H-6 b Gal)、3.81-3.87(m、4H、H-6 a GlcN、H-6 b GlcN、H-3 GlcN、OCH)、3.97(ddd、1H、J 5,6a 2.8Hz、J 5,6b 4.7Hz、J 4,5 8.9Hz、H-5 GlcN)、3.98(dd、1H、J 1,2 3.7Hz、J 2,3 10.7Hz、H-2 GlcN)、4.09(m、1H、CHO)、4.12(dd≒br.d、1H、J 3,4 3.3Hz、J 4,5 <1Hz、H-4 Gal)、4.47(d、1H、J 1,2 7.9Hz、H-1 Galβ)、5.07(d、1H、J 1,2 3.7Hz、H-1 GlcNα)。

[0287] Example 1.12 3-Aminopropyl 2-propionylamido-2-deoxy-α-D-glucopyranosyl-(1→3)-β-D-galactopyranoside (GlcNpropionylα1-3Galβ-O-(CH 2 ) 3 -NH 2 )

[0288] [ka]

[0289] 3-Trifluoroacetamidopropyl 2-azido-3,4,6-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside (Intermediate 1.12.1)

[0290] [ka]

[0291] 3-Trifluoroacetamidopropyl-2,4,6-tri-O-acetyl-β-D-galactopyranoside (422 mg, 1.0 mmol) and β-Bn were obtained from 3-trifluoroacetamidopropyl-2-O-acetyl-3-O-chloroacetyl-4,6-O-benzylidene-β-D-galactopyranoside by successive debenzylidenation, O-acetylation and removal of the chloroacetyl group in anhydrous ether / dichloromethane 1:1 (20 ml). 3 N 3 GlcO(CN)CCl 3To a solution of (735 mg, 1.26 mmol) freshly dried MS-4Å (3 mL) was added, the mixture was stirred for 30 min, and then trimethylsilyl trifluoromethanesulfonate (TMSOTf) (12 μL, 63 μmol) was added. The reaction was stirred for 24 h. The reaction mixture was neutralized with pyridine (0.5 ml) and MeOH (1 ml), filtered through Celite on a glass filter, and the solid was washed with dichloromethane and a mixture of dichloromethane / methanol 10:1. The filtrate was concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. The resulting disaccharide was purified by silica gel column chromatography (toluene / acetone, 5:2 to 2:2), and the fractions mainly containing the product were collected, concentrated using a rotary evaporator, coevaporated with toluene, dried in vacuum, and used in the next step without further purification. Yield 76%.

[0292] 3-Trifluoroacetamidopropyl 3,4,6-tri-O-propionyl-2-propionylamido-2-deoxy-α-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside (Intermediate 1.12.2)

[0293] [ka]

[0294] Intermediate 1.12.1 (192 mg, 0.21 mmol) was dissolved in methanol (10.0 mL) followed by the addition of 10% Pd / C (0.1 g) and the reaction mixture was degassed in vacuo and filled with hydrogen gas (1 atm) three times. The reaction was stirred under an atmosphere of hydrogen gas (1 atm) for 1 h and then cooled to 5° C. (EtCO) 2 O (0.1 mL) was added (through the septum) and stirring was continued for 48 h at room temperature. The conversion was controlled by TLC. The reaction mixture was filtered through Celite on a glass filter, the solid was washed with methanol and the resulting solution with 50% aqueous methanol was concentrated using a rotary evaporator, coevaporated with toluene and dried in vacuum. The residue was purified by distillation with pyridine (3.5 mL) and (EtCO) 2The mixture was dissolved in 20 (1.75 mL) and the reaction mixture was left for 24 h at 40° C. The reaction mixture was concentrated using a rotary evaporator and coevaporated with toluene.

[0295] The product was purified by silica gel column chromatography, and the product was collected, concentrated using a rotary evaporator, coevaporated with toluene, and dried in vacuum. Yield of intermediate 1.12.2: 91%.

[0296] 3-Aminopropyl 2-propionylamido-2-deoxy-α-D-glucopyranosyl-(1→3)-β-D-galactopyranoside (Example 1.12)

[0297] [ka]

[0298] Intermediate 1.12.2 was dissolved in anhydrous methanol (5.0 mL) and treated with 2M NaOMe (0.26 mL, 0.53 mmol) and the reaction mixture was allowed to stand for 2 h. The mixture was concentrated in vacuo (do not go to dryness), diluted with water (5.0 mL) and the reaction mixture was allowed to stand overnight. The target deprotected disaccharide (Example 1.12) was purified by HPLC using Dowex® 50X4-400 (H + The carbohydrate-containing fractions were collected, concentrated in vacuo, and freeze-dried twice. Yield of Example 1.12: 95%. 1 H NMR(D 2 O+TFA):δ 1.13(t, 3H, J 7.7Hz, NHC(O)CH 2 CH 3 ), 2.03(m, 2H, OCH 2 CH 2 CH 2 N), 2.32(q, 2H, J 7.6Hz, NHC(O)CH 2 CH 3 ), 3.16-3.21(m, 2H, CH 2N), 4.56(d, 1H, J 1,2 7.9Hz, H-1 Galβ), 5.06(d, 1H, J 1,2 3.6Hz, H-1 GlcNα).

[0299] Example 2 Preparation of a glycoconjugate containing a compound of formula (I) and agarose The NHS-activated resin (10 mL) was dissolved in 1.25 mL of coupling solution (0.2 M NaHCO 3 The resin was incubated overnight (gentle agitation) with 1.25 mL of 2 mg / mL ligand prepared in Example 1 in 0.1 mM HCl (pH 8.3) and 0.5 M NaCl, pH 8.3) at 4° C. Unreacted groups in the resin were capped with 5 mL of 0.5 M ethanolamine in 0.1 M Tris-Cl, pH 8.5 for 4 hours at 4° C.

[0300] Example 3 Preparation of glycoconjugates containing a compound of formula (I) and magnetic nanoparticles Glycoconjugates containing magnetic nanoparticles were prepared using the same procedure as in Example 2, using NHS-activated magnetic nanoparticles.

[0301] Example 4 Preparation of a glycoconjugate containing a compound of formula (I) and poly-L-lysine Glycoconjugates were prepared with compounds 1-16 as disclosed in Example 1 and linear poly-L-lysine with a degree of polymerization of 1000±100, i.e., an average of 1000 L-lysine units in the chain (DP1000). The glycan content of the glycoconjugates was about 25%. The free amino groups on the lysine were capped with glycolic acid. The glycoconjugates prepared had the following formula:

[0302] [ka]

[0303] [wherein DP is 1000 and a is 25] It can be expressed as:

[0304] The procedure for preparing glycoconjugates with polylysine is described below. The preparation of specific glycoconjugates of glycans in Examples 1.1 and 1.8 is disclosed in detail, but the method is similar for the preparation of glycoconjugates of all glycans in Examples 1.1 to 1.20.

[0305] Ligand activation using succinimidyl adipate diester

[0306] [ka]

[0307] Adipic acid succinimidyl diester, Ad(ONSu) in DMSO (4 mL) 2 To a stirred solution of Glyc-sp-NH-Ad-OSu (452 ​​mg, 1.328 mmol), a solution of the compound of Example 1 (0.166 mmol) in DMSO (1 mL) was added in four portions (approximately 0.25 mL) at 10 min intervals. The resulting solution was kept at room temperature for 75 min, then acidified with 0.1 mL of AcOH and diluted with 50 ml of cold 1% aqueous AcOH. The precipitate of excess succinimidyl adipic acid diester was filtered and washed with 1% aqueous AcOH. The filtrate was evaporated in vacuum (30° C. bath) to a minimum volume and passed through a Sephadex® LH-20 column (180 mL gel, eluted with 1:1 acetonitrile / water+1% AcOH). The fractions containing Glyc-sp-NH-Ad-OSu were combined, evaporated to a volume of approximately 2 mL, and lyophilized.

[0308] Glycolic acid acetate succinimide ester (AcOCH 2 Preparation of (CO)ONSu) (capping agent)

[0309] [ka]

[0310] N-hydroxysuccinimide (992 mg, 8.62 mmol) and Et 3To a stirred, cooled (0 °C) solution of N (1.141 mL, 8.21 mmol) in 1,2-dichloroethane (5 mL) was added acetoxyacetyl chloride, AcOCH 2 A solution of (CO)Cl (1.121 g, 8.21 mmol) was added in several portions. The reaction mixture (Et 3 After stirring at room temperature for 30 min (a precipitate of N-hydrochloride was formed), AcOH (0.47 mL) was added. The mixture was diluted with 20 mL of 1% AcOH, agitated by shaking, and the organic layer was extracted again with 20 mL of 1% AcOH. The organic solution was evaporated and the residue was dried in vacuum. The crystals were dissolved in 20 mL of Et 2 The mixture was scraped with 20 mL of hexane / O / hexane (1:1), decanted, scraped with 20 mL of hexane, decanted, and dried in vacuum. 2 The yield of (CO)ONSu ​​was 1394 mg (79%). TLC:R f = 0.67 (chloroform / methanol, 10:1 by volume). 1 H NMR (CDCl 3 +1%d 4 -AcOH, 700MHz, 30℃)δ 7.258(s, CHCl 3 ), 4.943(s, 2H;OCH 2 CO), 2.847 (s, 4H; CH of succinimide 2 CH 2 ), 2.175(s, 3H;C(O)CH 3 ) ppm.

[0311] Preparation of glycoconjugates with poly-L-lysine (a=25) The synthetic scheme is shown in Figure 8. A stirred solution of polylysine hydrobromide (8.37 mg, 40 μmol of Lys) in DMSO (698 μL, PLys concentration = 12 mg / mL) was added with a solution of Glyc-sp-NH-Ad-ONSu (40 × a / 100 μmol) in DMSO (Glyc-sp-NH-ONSu concentration = 60 mg / mL) and Et 3A solution of N (3.336 μL of 3 × 40 × a / 100 μmol, 5% by volume in DMSO) was added. The solution was kept at room temperature for 1 h, then dried AcOCH 2 (CO)ONSu ​​(2.5 × 40 × (100-a) / 100 μmol, 0.2152 × (100-a) mg) and Et 3 N (3 × 40 × (100-a) / 100 μmol, 0.139 × (120-1.2 × a) μL) was added. After 1 h at room temperature, the solution was diluted with 2 volumes of water and 3 N was added (2% of the total volume). The reaction mixture was kept at room temperature overnight (approximately 16 hours) and then AcOH (total EtOH used) was added. 3 The polylysine conjugates were neutralized with 100 mL of acetonitrile (equivalent to 30:70 N). The polylysine conjugates were separated on a Sephadex® LH-20 column (100 mL of gel, eluted with 30:70 acetonitrile / water). The fractions with pure conjugates were evaporated to a volume of approximately 2 mL and lyophilized. The yield of Glyc(a%)_PLys conjugates was 92-97%.

[0312] The purity and composition (percentage of polylysine modified with Glyc sugars) of the synthesized glycoconjugates were determined by integrating the signals of the trisaccharide and polylysine fragments. 1 Determined by 1 H NMR spectroscopy.

[0313] Preparation of Glycolyl-Poly-L-Lysine (DP1000) (Control)

[0314] [ka]

[0315] To a stirred solution of polylysine (DP100) hydrobromide (11.43 mg, 54.62 μmol Lys) in DMSO (750 μL) was added dry AcOCH 2 (CO)ONSu ​​(29.4 mg, 136.5 μmol) and Et 3 N (22.8 μL, 164 μmol) was added. After standing at room temperature for 2 h, the solution was diluted with 2 volumes of water (1500 μL) and diluted with Et 3N was added (102 μL, approximately 45 μL of excess free Et 3 The total volume of the Glycolyl_PLys_DP100 complex was approximately 2% (corresponding to N). The reaction mixture was stirred at room temperature for 24 h and the small gel-like precipitate was filtered and washed with water. The combined filtrates were evaporated to a volume of approximately 2 mL and the polylysine complex was separated on a Sephadex® LH-20 column (100 mL gel, eluted with 30:70 acetonitrile / water). The fractions with the pure complex were evaporated to a volume of approximately 2 mL and lyophilized. The yield of Glycolyl_PLys_DP100 complex was 8.6 mg (84%). Polylysine (DP100) hydrobromide 1 H NMR(D 2 O, 700MHz, 30℃)δ 4.750(s, HOD), 4.365(dd, 100H, J=8.8Hz, J=5.7Hz;α-CH), 3.051(t, 200H, J=7.6Hz;ε-CH 2 ), 1.770(m, 400H;β-CH 2 and δ-CH 2 ), 1.474(m, 200H; γ-CH 2 ) ppm. Glycolyl_PLys_DP100 complex 1 H NMR(D 2 O+TFA, 14mM / L, 700MHz, 30℃)δ 4.750(s, HOD), 4.107(m, 100H;α-CH), 4.057(s, 200H;C(O)CH) 2 O), 3.253(m, 200H; ε-CH 2 ), 1.969(m, 200H;β-CH 2 ), 1.593 and 1.453 (m, 400H; δ-CH 2 and γ-CH 2 ) ppm.

[0316] Example 4.1 Complex of tetrasaccharide Glcα1-3(Fucα1-2)Galβ1-4GlcNAcβ (Example 1.1) (25%) with polylysine DP1000

[0317] [ka]

[0318] To a stirred solution of polylysine (DP1000) hydrobromide (2.58 mg, 12.34 μmol Lys) in DMSO (430 μL) was added a solution of Glcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-sp-Ad-ONSu (3.01 mg, 3.086 μmol) in DMSO (50 μL) and Et 3 A solution of N (26 μL of 5% by volume in DMSO, 9.3 μmol) was added. The solution was kept at room temperature for 1 h, and then AcOCH in DMSO (80 μL) was added. 2 A solution of (CO)ONSu ​​(4.0 mg, 18.5 μmol) and Et 3 N (3.9 μL, 28 μmol) was added. After 1 h at room temperature, the solution was diluted with approximately twice the volume of water (1172 μL) and diluted with Et 3 N was added (35 μL, 2% of the total volume). The reaction mixture was kept at room temperature overnight (approximately 17 h) and then AcOH (16.5 μL, total Et 3 The polylysine conjugate was neutralized with 100 mL of acetonitrile (equivalent to 30:70 N). The polylysine conjugate was separated on a Sephadex® LH-20 column (100 mL of gel, eluted with 30:70 acetonitrile / water). The fractions with the pure conjugate were evaporated to a volume of approximately 2 mL and lyophilized. The yield of Glcα1-3(Fucα1-2)Galβ1-4GlcNAcβ(25%)-PLys1000-glycoconjugate was 4.5 mg (94%). TLC:R f Approximately 0 (2-propanol / acetonitrile / water, 4:3:2 by volume), ninhydrin negative, free derivatives of Glcα1-3(Fucα1-2)Galβ1-4GlcNAcβ, free of glycolic acid and salts. 1 H NMR Selected Chemical Shifts (D 2O, 700 MHz, 30 °C): δ 5.372 (d, 245H, J = 4.2 Hz; 245 H-1 of Glcα), 5.236 (d, 245H, J = 3.9 Hz; 245 H-1 of Fucα), 4.750 (s, HOD), 4.632 (d, 245H, J = 7.7 Hz; 245 H-1 of Galβ), 4.517 (d, 245H, J = 8.3 Hz; 245 H-1 of GlcNAcβ), 4.345 (q, 245H, J = 6.7 Hz; 245 H-5 of Fucα), 3.291-3.187 (m, 2490H; 245 CH of glycan spacer) 2 N and 1000 CH of lysine 2 N), 1.261 (d, 735H, J = 6.5Hz; 245 6-CH of Fucα 3 ) ppm. Spectroscopic analysis gives 245 glycans per 1000 lysines, approximately 25% molar concentration.

[0319] Example 4.8 Complex of trisaccharide GlcNAcα1-3(Fucα1-2)Galβ (Example 1.8) (25%) with polylysine DP1000

[0320] [ka]

[0321] To a stirred solution of polylysine (DP1000) hydrobromide (3.07 mg, 14.68 μmol Lys) in DMSO (512 μL) was added a solution of GlcNAcα1-3(Fucα1-2)Galβ-sp-Ad-ONSu (2.98 mg, 3.67 μmol) in DMSO (50 μL) and Et 3 A solution of N (31 μL of 5% by volume in DMSO, 11 μmol) was added. The solution was kept at room temperature for 1 h, and then AcOCH in DMSO (95 μL) was added. 2 A solution of (CO)ONSu ​​(4.74 mg, 22 μmol) and Et 3 N (4.6 μL, 33 μmol) was added. After standing at room temperature for 1 h, the solution was diluted with approximately twice the amount of water (1386 μL) and diluted with Et 3N was added (42 μL, 2% of the total volume). The reaction mixture was kept at room temperature overnight (approximately 16 h) and then AcOH (19.8 μL, total Et 3 The polylysine conjugate was neutralized with 100 mL of acetonitrile (equivalent to 30:70 N). The polylysine conjugate was separated on a Sephadex® LH-20 column (100 mL of gel, eluted with 30:70 acetonitrile / water). The fractions with the pure conjugate were evaporated to a volume of approximately 2 mL and lyophilized. The yield of GlcNAcα1-3(Fucα1-2)Galβ(25%)-PLys1000-glycoconjugate was 4.7 mg (93%). TLC:R f Approximately 0 (2-propanol / acetonitrile / water, 4:3:2 by volume), ninhydrin negative, free derivatives of GlcNAcα1-3(Fucα1-2)Galβ, free of glycolic acid and salts. 1 H NMR Selected Chemical Shifts (D 2 O, 700 MHz, 30 °C): δ 5.361 (d, 248H, J = 3.3 Hz; 248 H-1 of GlcNAcα), 5.171 (d, 248H, J = 3.9 Hz; 248 H-1 of Fucα), 4.750 (s, HOD), 4.541 (d, 248H, J = 7.8 Hz; 248 H-1 of Galβ), 4.441 (q, 248H, J = 6.6 Hz; 248 H-5 of Fucα), 3.306-3.187 (m, 2496H; 248 CH of glycan spacer). 2 N and lysine 1000CH 2 N), 1.242 (d, 744H, J = 6.5Hz; 248 6-CH of Fucα 3 ) ppm. Spectroscopic analysis gives 248 glycans per 1000 lysines, approximately 25% molar concentration.

[0322] Comparison of natural anti-A and anti-B antigens with poly-L-lysine glycoconjugates Using the natural antigen, the following glycoconjugates were prepared in a similar manner, where the carbohydrate has an N-acetylgalactosamine or galactose residue at the non-reducing end.

[0323] Comparative Example 1: Complex of Galα1-3(Fucα1-2)Galβ1-4GlcNAcβB(type 2) tetrasaccharide and polylysine DP1000

[0324] [ka]

[0325] Glycan content: 44.7% by weight.

[0326] Comparative Example 2: Complex of GalNAcα1-3(Fucα1-2)GalβA(type 2) trisaccharide and polylysine DP1000

[0327] [ka]

[0328] Glycan content: 38.2% by weight.

[0329] Example 5 Anti-A / B antibody inhibitory capacity of the poly-L-lysine glycoconjugates of the invention using normal human serum of blood group A or B The glycopolymers analyzed were PLys-DP1000 modified with the glycans of the invention (prepared in Example 4) or with the native A / B antigen (glycan content 25%).

[0330] Maxisorp® 96-well plates (Nunc®) were coated overnight at 2-8°C with 10 μg / ml of native antigen A or B complexed with polyacrylamide (PAA) in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Wells coated with 10 μg / ml of PAA alone were considered as background for each serum sample. After coating, plates were washed three times with phosphate-buffered saline (PBS) + 0.1% (v / v) Tween® 20 (Merck) and blocked for 1 h at 2-8°C with 0.5% (v / v) Tween® 20 in PBS + 1% (w / v) BSA (Sigma-Aldrich). Serum samples were pre-incubated with increasing concentrations of carbohydrate polymers (0-1000 μg / ml) for 15-17 h at 2-8°C under controlled gentle orbital shaking (183 rpm). Washing was repeated and serum samples (1:100 for IgG and IgM; 1:50 for IgA) diluted in 0.1% (v / v) Tween® 20 in PBS + 0.3% (w / v) BSA were added to the wells and incubated for 0.5 h at 25° C. with shaking. After washing, the plates were incubated for 0.5 h at 25° C. (shaking) with horseradish peroxidase (HRP)-labeled anti-human IgM, IgA (1:8000) and IgG (1:12000) antibodies diluted in 0.1% (v / v) Tween® 20 in PBS + 0.3% BSA (Invitrogen). After a new wash, the substrate o-phenylenediamine tablets (Sigma-Aldrich) were added to the wells as HRP substrate and incubated for 10 min at 25° C. The reaction was stopped with 3 N HCl, and the absorbance at 492 nm was recorded using a microplate reader (Biotek). For each glycopolymer, the IC was calculated considering the inhibitory capacity (%) obtained at various polymer concentrations relative to the sample treated with vehicle (PBS) only. 50 was calculated.

[0331] Example 6 Anti-A / B antibody inhibitory capacity of the poly-L-lysine glycoconjugates of the invention using a pure fraction of polyclonal human anti-A / B antibodies The glycopolymers analyzed were PLys-DP1000 modified with the glycans of the invention (prepared in Example 4) or with the native A / B antigen (glycan content 25%).

[0332] In this example, pure fractions of human polyclonal anti-A or anti-B antibodies were used instead of normal human serum. These fractions were separated using Sepharose® resin functionalized with native A / B antigens.

[0333] 6.1. Purification of human anti-A / B antibodies Native type A 2 or native type B 2 conjugated with activated polyacrylamide (PAA) was immobilized on aminated Sepharose® 6 FF (Pharmacia Biotech, Austria) (column I) according to the method disclosed by Bovin et al., Glycoconjugate J., 1993, 10, 142-151. Column II was prepared using Sepharose® 6 FF derivatized with activated PAA.

[0334] Columns I and II (polypropylene, 1.5×12 cm, Econo-Pac® Chromatography Columns, BioRad, CA, USA) were packed with 2 mL of affinity adsorbent ligand-Sepharose® 6FF.

[0335] The ligand density in column I was 0.5 μmol per mL of sorbent to ensure elution of antibody fractions exhibiting isotype ratios similar to those present in pooled normal human serum. The purity of the glycans used was 95-97% according to NMR and HPLC data. 1. Initial Wash of Adsorbent Ligand-Sepharose® 6FF 1.1 20mL Milli-Q grade water 1.2 20 mL of 0.1 M NaOH 1.3 20mL Milli-Q grade water 2. PBS + 0.02% NaN 3Adsorption equilibration by passing 20 mL of buffer through the adsorbent. 3. Preparation of pooled serum samples 3.1. Mix sera from healthy donors (n samples) to obtain a donor pool. 3.2. Heat the pooled serum at 56°C for 30 minutes to inactivate complement proteins. 3.3. After heating, leave the serum on the table to reach room temperature. 3.4. Centrifuge (7000g) the serum for 10 minutes at room temperature. 3.5. Transfer the serum from the centrifuge vial to a new vial (leaving no visible precipitate behind). PBS + 0.02% NaN 3 Dilute the serum 1:5 in 4. Load the diluted serum onto the sorbent at 0.4 mL / min (passively pass through the column by gravity) 5. Washing of the sorbent with loaded serum samples 5.1. 20 mL of PBS + 0.02% NaN 3 +0.1% Tween® 20. 5.2. 100 mL of PBS + 0.02% NaN 3 Carefully pipette the last portion of buffer to mix the buffer and adsorbent for a thorough wash. 6. Elution of antibodies bound to the adsorbent ligand 6.1. 0.2M Tris-OH buffer + 0.02%NaN 3 Elute the antibody (Eluent I) at 0.2 mL / min with 0.2 M Gly-HCl + 0.02% NaN (pH 10.4). From the beginning of the elution, add 30 μL of neutralization buffer 0.2 M Gly-HCl + 0.02% NaN for every mL of eluent collected. 3 (pH 2.5) is added. 6.2. Finally, neutralize the eluate to pH 7.4-7.6. 7. PAA crosslinking reaction / removal of non-specifically bound antibodies 7.1. Pack column II with 1.5 mL of sorbent PAA-Sepharose® 6FF. 7.2. Rinse with the following solutions: - 15mL Milli-Q grade water - 15mL 0.1M NaOH - 15mL Milli-Q grade water. 7.3. Equilibrate by passing 15 mL of 0.2 M TrisOH / Gly-HCl (pH 7.4). 7.4. Load eluate I from column I at 0.4 mL / min. Collect eluate II. 8. Concentration of antibodies from eluate II using Microcon® 30 kDa centrifugal filter units with Ultracel® 30 membranes (Merck Millipore, Burlington, MA, USA) at 14000 g for 10 min.

[0336] 6.2. Determination of specificity of purified anti-A / B antibodies by ELISA Polyacrylamide-based glycoconjugates were prepared by the method disclosed in Bovin et al. (supra). A solution of oligosaccharides in DMF and triethylamine (or diisopropylethylamine) was added to a solution of poly(4-nitrophenylacrylate) in DMF. The resulting mixture was diluted with a molar excess of NH 3 The PAA-glycoconjugates were separated on Sephadex® LH-20 using an acetonitrile-water mixture. In this way, polyacrylamide-based glycoconjugates of the following glycans were produced:

[0337] [Table 3]

[0338] PAA-glycoconjugates 2, 6, 7 and 8 were produced using the mimetics of the invention, products 1, 3, 4 and 5 are comparative examples based on the natural antigen, ie all have a Gal residue at the non-reducing end.

[0339] Maxisorp® 96-well flat-bottom plates (Thermo Scientific, Waltham, MA, US) were coated with 50 μL / well of the above polyacrylamide-based glycoconjugates at 10 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) for 18 h at 4° C. Wells coated with 50 μL / well of 10 μg / ml PAA alone were considered as background for each sample.

[0340] After coating, plates were washed 3 times with 200 μL / well of 0.1% (v / v) PBST (phosphate buffered saline with Tween®) using an automated washer and blocked with 100 μL / well of 0.1% (v / v) PBST + 1% (w / v) BSA (bovine serum albumin) for 1 h at 4° C. Washing was repeated and 50 μL / well of purified anti-A or anti-B antibodies diluted in 0.1% (v / v) PBST + 0.3% (w / v) BSA were added to the wells and incubated for 0.5 h at 25° C. in a rocker.

[0341] After washing, the plates were incubated for 0.5 h (with shaking) at 25° C. with the corresponding HRP-labeled antibodies goat anti-human IgG or goat anti-human IgM (Invitrogen, Waltham, MA, USA) diluted in 0.1% (v / v) PBST + 0.3% (w / v) BSA (1:12000 and 1:8000, respectively). After another round of washing, 50 μL / well of OPD (Sigma-Aldrich, St. Louis, MO, USA) as HRP (horseradish peroxidase) substrate was added and the plates were incubated for 14 min at 25° C. in the dark. The reaction was stopped with 50 μL / well of 3N HCl and the absorbance at 490 nm was recorded using a microplate reader.

[0342] The results are shown in Figure 9. The vertical axis represents the optical density (OD) values ​​of the various samples (1-9 on the horizontal axis, sample 9 corresponds to the PAA control).

[0343] Graphs 9-1 and 9-2 show the results of the interaction of purified anti-B antibodies with natural or mimetic B antigens (glycoconjugates 1 to 4 in the table) and, as controls, with natural A antigen (sample 5) and PAA (sample 9). Graphs 9-1 and 9-2 show the reactivity to IgG (0.36 μg / mL) and IgM (0.217 μg / mL) antibodies, respectively.

[0344] Graphs 9-3 and 9-4 show the results of the interaction of purified anti-A antibodies with natural or mimetic A antigens (glycoconjugates 5 to 8 in the table) and, as controls, with natural B antigen (sample 1) and PAA (sample 9). Graphs 9-3 and 9-4 show the reactivity to IgG (0.185 μg / mL) and IgM (0.185 μg / mL) antibodies, respectively.

[0345] These results were unexpected and demonstrated that purified fractions of anti-A / B antibodies bind to other glycan structures, including the mimetics of the present invention, with similar or even greater strength than the native type 2 antigen.

[0346] Without wishing to be bound by any theory, all these findings can be explained by the polyclonal and polyreactive nature of human anti-A / B antibodies and support the inhibition of anti-A / B antibodies using glycan structures different from the natural antigen, particularly using the mimetics of the invention.

[0347] 6.3. Inhibition of anti-A or anti-B antibodies using poly-L-lysine glycoconjugates ELISA: Maxisorp® 96-well flat-bottom plates (Thermo Scientific, Waltham, MA, US) were coated with 50 μL / well of the following PAA glycoconjugates (disclosed in Table 3): 1 (natural B type 2) and 5 (natural A type 2) at 10 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) for 18 h at 4° C. Wells coated with 50 μL / well of 10 μg / ml of PAA alone were considered as background for each sample. After coating, plates were washed three times with 200 μL / well of 0.1% (v / v) PBST using an automatic washer and blocked with 100 μL / well of 0.1% (v / v) PBST + 1% (w / v) BSA for 1 h at 4° C. Washing was repeated.

[0348] Inhibition samples were prepared in separate tubes using a fixed concentration of anti-A or anti-B antibody and increasing concentrations of the following polylysine glycoconjugate inhibitors (0-400 μg / mL) prepared in the examples: - Comparative Example 1 (Contains natural B type 2 antigen) - Example 4.1 (Including analogs of Example 1.1) - Comparative Example 2 (Contains natural type A 2 antigen) - Example 4.8 (Including analogs of Example 1.8)

[0349] As disclosed in Example 4, the content of glycans in the poly-L-lysine conjugates was the same (25%), ensuring a similar number of exposed carbohydrate residues in each compound.

[0350] After incubating the tubes for 18 hours at 4°C, a convenient dilution of inhibition mix was added to the ELISA plate (50 μL / well) and incubated for 0.5 hours at 25°C on a rocker. After washing, the plate was incubated for 0.5 hours (with shaking) at 25°C with the corresponding HRP-conjugated antibody goat anti-human IgG or goat anti-human IgM (Invitrogen, Waltham, MA, USA) diluted in 0.1% (v / v) PBST + 0.3% (w / v) BSA (1:12000 and 1:8000, respectively) to detect the remaining unbound anti-A / B fraction. After another round of washing, 50 μL / well of OPD (Sigma-Aldrich, St. Louis, MO, USA) as HRP substrate was added to the wells and the plate was incubated for 14 minutes at 25°C in the dark. The reaction was stopped with 50 μL / well of 3N HCl and the absorbance was recorded at 490 nm using a microplate reader.

[0351] To quantify antibodies, Maxisorp® 96-well plates were coated overnight at 4° C. with decreasing concentrations of pure human IgG or IgM starting from 1000 ng / mL. In this respect, the ELISA procedure was the same as for the samples, except that only 0.1% (v / v) PBST + 0.3% (w / v) BSA was added at the sample addition step.

[0352] The percentage of removal was calculated with reference to baseline conditions (no inhibitor compounds used). The results are shown in Figures 10 and 11, where the vertical axis represents the percentage of removal, indicating the inhibitory potential of the glycoconjugates tested, and the horizontal axis represents the concentration of glycoconjugates in the sample (μg / mL).

[0353] Figure 10 shows the inhibition of anti-A antibodies. Figures 10-1 and 10-2 show the removal of IgG (0.075 μg / mL) and IgM (0.185 μg / mL) anti-A antibodies, respectively, with increasing concentrations of poly-L-lysine glycoconjugates.

[0354] The black bars correspond to Comparative Example 2 (containing the native group A type 2 antigen) and the grey bars correspond to Examples 4-8 (containing the mimetics of the invention).

[0355] Examples 4-8 of the present invention showed greater ability to inhibit purified human anti-A antibodies than comparators containing 10, 100 and 400 μg / mL of native antigen, a result that was unexpected and indicates the potential use of this Glc analog for the inhibition of anti-A antibodies.

[0356] Figure 11 shows the inhibition of anti-B antibodies. Figures 11-1 and 11-2 show the removal of IgG (0.095 μg / mL) and IgM (0.045 μg / mL) anti-B antibodies, respectively, with increasing concentrations of poly-L-lysine glycoconjugates. The black bars correspond to Comparative Example 1 (containing the native B type 2 antigen) and the grey bars correspond to Example 4-1 (containing the mimetic of the invention).

[0357] In this case, Example 4-1 shows a lower ability to inhibit human purified anti-B antibodies than the natural counterpart. However, when used in combination with the natural antigen (see 6.4), it still showed significant activity. Again, without wishing to be bound by theory, this may be due to the polyclonal nature of the anti-B antibodies.

[0358] 6.4. Inhibition of purified antibodies by poly-L-lysine glycoconjugate mimetics of the invention in combination with poly-L-lysine glycoconjugates of natural antigens Antigen combinations (natural type 2 + mimetic) were evaluated combining the poly-L-lysine of Example 4.1 (B mimetic) with the corresponding poly-L-lysine of the natural B antigen (Comparative Example 1, prepared in Example 4). The ability to inhibit the binding of purified anti-B antibodies to red blood cells (type B) was tested by flow cytometry.

[0359] Flow cytometry assays were performed as follows: 50 μL of red blood cells (RBCs) derived from a healthy donor were diluted to 1.5% (v / v) in PBS, after which the corresponding inhibitors and primary antibodies were added.

[0360] Samples were incubated with 1.0 μg / mL of anti-B antibody and the following inhibitors: - Tube 1: Anti-B at 1.0 μg / mL + Comparator 1 (0.25 μg / mL) - Tube 2: 1.0 μg / mL of Anti-B + Example 4.1 (2.5 μg / mL) - Tube 3: Anti-B at 1.0 μg / mL + Comparative Example 1 (0.25 μg / mL) + Example 4.8 (2.5 μg / mL) were prepared in separate tubes using

[0361] The samples were then incubated for 30 min at 37° C. (gently shaking horizontally at 30-40 rpm in the dark). The samples were washed twice with PBS (centrifugation at 300 g for 3 min) and the supernatant was removed by aspiration.

[0362] Then, 50 μL of the corresponding fluorochrome secondary anti-Ig antibodies (7.5 μg / mL goat anti-human IgG Alexa Fluor 647, Jackson Immunoresearch, West Grove, PA, USA and 20 μg / mL goat anti-human IgM CF488A, Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS were added to the RBCs and incubated for 35 min at 37° C. (in the dark). Samples were washed twice with PBS (centrifuged at 300 g for 3 min) and the supernatant was removed by aspiration. Finally, cells were resuspended in filtered PBS. Cells incubated with PBS only were used as negative control. Cells incubated with secondary antibodies only were considered as background signal. Flow cytometric analysis was performed on a Beckman Coulter Gallios Flow Cytometry analyzer using blue (488 nm-22 mW) and red (635 nm-25 mW) solid-state lasers as excitation sources. Every single FACS run recorded approximately 20,000 total events. Data were analyzed using KALUZA software (Beckman Coulter, CA, USA).

[0363] The results of inhibition of the binding of erythrocytes to anti-B antibody (1 μg / mL) are shown in Figure 12. The inhibitory capacity was expressed as the percentage of removal relative to the baseline (ordinate).

[0364] The first bar (black) corresponds to Comparative Example 1 (0.25 μg / mL): IgM: 26.4%, IgG: 15.12%.

[0365] The second bar (light grey) corresponds to Example 4.1 (2.5 μg / mL): IgM: 6.35%, IgG: 5.64%.

[0366] The third bar (dark grey) corresponds to a mixture of Comparative Example 1 (0.25 μg / mL) and Example 4.1 (2.5 μg / mL): IgM: 63.64%, IgG: 20.24%.

[0367] It can be observed that the combination of Example 4.1 (containing the mimetic of Table 2, Example 1.1) with the compound of Comparative Example 1 (containing the native B type 2) dramatically increased the inhibitory potency for both isotypes (IgM and IgG) compared to the compound of Comparative Example 1 alone. This result was completely unexpected, but it shows that the combination of the mimetic of the present invention with the native type 2 antigen is a strategy that can provide better inhibitory results compared to the native type 2 antigen alone.

[0368] Example 7 Removal effect of anti-A / B antibodies from normal human serum by agarose and MNP glycoconjugates of the present invention The carbohydrate polymers analyzed were Sepharose® functionalized with the glycans of the invention (prepared in Example 2) and magnetic nanoparticles functionalized with the glycans of the invention (prepared in Example 3), and were compared with resins and MNPs functionalized with the native A / B antigens.

[0369] Anti-A / B antibodies in tested normal human sera were quantified by ELISA and printed glycan array technology before (baseline) and after passing the serum over the functionalized resin or functionalized MNPs.

[0370] Example 8 Purification of human polyclonal anti-A / B antibodies by agarose and MNP glycoconjugates of the invention Commercially available NHS-activated Sepharose® and MNPs were functionalized with the glycans of the present invention according to the manufacturer's instructions. NHS-activated resin (5-10 mL) and MNPs (0.5-1 mL) were dissolved in 1.25 mL of coupling solution (0.2 M NaHCO 3 The support was incubated overnight (gentle agitation) at 4° C. with 1.25 mL of 2 mg / mL of the ligand prepared in Example 1 in 0.1 M Tris-Cl, pH 8.3 and 0.5 M NaCl, pH 8.3) and 2.5 mL of 1 mM HCl. Unreacted groups in the support were capped with 5 mL of 0.5 M ethanolamine in 0.1 M Tris-Cl, pH 8.5, for 4 hours at 4° C. After washing (PBS-Tween® 0.1%, 0.5 M NaCl), the support was blocked with PBS-Tween® 0.01% and BSA 1% (w / v). The blocked support was incubated (in batches) with pooled human serum of types A and B (25% v / v, in PBS) (gentle shaking, 4-8° C., 2 hours). For Sepharose®, the resin and serum were then loaded onto a column (20×2 cm) to separate the unbound serum fraction. For MNPs, the unbound serum fraction was separated using a magnetic separator. After a new wash, the bound antibodies (anti-A and anti-B) were eluted from the support with glycine-HCl buffer (0.1 M, pH 3.0). The eluted antibody fraction was immediately neutralized to pH 7 and quantified according to Bradford (1976). Purity was assessed by SDS-PAGE.

Claims

1. Formula (I): 【Chemistry 1】 [In the formula, R １ is OH and —NH—CO—R ５ is selected from Here, R ５ is C １ -C ３ alkyl, and R ５ wherein one or more hydrogen atoms may be replaced by a hydroxy group; R ２ is a group consisting of OH and formula (II): 【Chemistry 2】 and where: ＊ indicates the position at which the group represented by formula (II) is attached to the rest of the molecule; R ３ is R ４ -Z, formula (III): 【Transformation 3】 and a group represented by formula (IV): 【Chemistry 4】 and where: ＊ indicates the position at which the group represented by formula (III) or formula (IV) is attached to the rest of the molecule, and R ４ is a spacer group and Z is -NH ２ , —COOH and —SH] A compound represented by the formula:

2. R １ 2. The compound according to claim 1, wherein is selected from OH, N-acetyl, N-propionyl and N-glycolyl.

3. R ２ When is OH, R ３ is R ４ The compound according to claim 1, characterized in that it is -Z.

4. The compound represented by formula (I) is 1) Glcα1-3 (Fucα1-2)Galβ1-4GlcNAcβ-R 2) Glcα1-3 (Fucα1-2)Galβ-R 3) Glcα1-3Galβ-R 4) Glcα1-3 (Fucα1-2)Galβ1-3GalNAcβ-R 5) GlcNAcα1-3 (Fucα1-2)Galβ1-4GlcNAcβ-R 6) GlcN propionyl α1-3 (Fucα1-2) Galβ1-4GlcNAcβ-R 7) GlcN glycolyl α1-3 (Fucα1-2) Galβ1-4GlcNAcβ-R8) GlcNAcα1-3 (Fucα1-2) Galβ-R 9) GlcN propionyl α1-3 (Fucα1-2)Galβ-R 10) GlcN glycolyl α1-3 (Fucα1-2)Galβ-R 11) GlcNAcα1-3Galβ-R 12) GlcN Propionyl α1-3Gal β-R 13) GlcN glycolyl α1-3Galβ-R 14) GlcNAcα1-3 (Fucα1-2)Galβ1-3GalNAcβ-R 15) GlcN propionyl α1-3 (Fucα1-2)Galβ1-3GalNAcβ-R 16) GlcN glycolyl α1-3 (Fucα1-2)Galβ1-3GalNAcβ-R 17) GlcNAcα1-3Galβ1-4GlcNAcβ-R 18) GlcN propionyl α1-3Galβ1-4GlcNAcβ-R 19) GlcN glycolyl α1-3Galβ1-4GlcNAcβ-R 20) Glcα1-3Galβ1-4GlcNAcβ-R wherein R is —O—R ４ The compound according to claim 1, characterized in that it is -Z.

5. 10. A method for preparing the compound of claim 1, comprising the steps of: a) a step of preparing a compound represented by formula C from a compound represented by formula A and a compound represented by formula B: 【Transformation 5】 [In the formula, PG is a protecting group; R ａ is azide or O-PG; R ３ ' is R ４ -Z', Z according to claim 1, selected from a group represented by formula (III) and a group represented by formula (IV), wherein the group represented by formula (III) and the group represented by formula (IV) are as defined in claim 1, but the OH groups of the hydrocarbon moieties of the groups represented by formulas (III) and (IV) are protected, Z is also protected, and Z' is protected; R ｂ is OH or O-PG] b) b1) R ｂ is OH, a protected fucose residue is attached to this position by an α(1→2) glycosidic bond, and after deprotection, R ２ providing a compound of formula I, wherein b2) R ｂ is O-PG, the compound represented by formula C is deprotected to form R ２ providing a compound of formula I, wherein is OH; A method comprising:

6. A glycoconjugate comprising a compound represented by formula (I) according to any one of claims 1 to 4 and a polymer backbone.

7. The glycoconjugate according to claim 6, wherein the polymer is selected from the group consisting of homo- and hetero-polypeptides, proteins, lipids, nucleic acids, acrylic acid polymers, methacrylic acid polymers, acrylic acid-methacrylic acid copolymers, polysaccharides such as chitosan or agarose, polyethylene, polypropylene, polystyrene, polyvinyl butyrate, polyvinyl chloride, and dendrimers; preferably, the polymer is selected from polyacrylic acid, polysaccharide resins, and alpha-amino acid homopolymers; more preferably, the polymer is selected from polyacrylic acid, agarose, and polylysine.

8. The following formula: 【Transformation 6】 [In the formula, "Cap" means a capping agent; "DP" is the degree of polymerization of the polymer; "a" is the content of carbohydrates in the complex carbohydrates, "sp" represents a spacer group; "Glyc" refers to the group: 【Transformation 7】 where R １ is selected from OH and —NH—CO—R 5 ; wherein R 5 is C 1 -C 3 alkyl, and one or more hydrogen atoms of R 5 may be replaced by a hydroxy group; R 2 is a group selected from OH and formula (II): 【Transformation 8】 and where * indicates the position at which the group of formula (II) is attached to the rest of the molecule. The complex carbohydrate according to claim 7, characterized in that it is a polyacrylamide represented by the formula:

9. 8. The glycoconjugate according to claim 7, characterized in that it comprises a compound of formula (I) bound to agarose as the polymer backbone.

10. The following formula: 【Chemistry 9】 [In the formula, "Cap" means a capping agent; "DP" is the degree of polymerization of the polymer, preferably 50 to 2000, more preferably 100 to 1000; "a" is the content of carbohydrates in the complex carbohydrate, preferably 2 to 90, more preferably 5 to 50; "sp" indicates a spacer group; "Glyc" refers to the group: 【Chemistry 10】 wherein R 1 is selected from OH and —NH—CO—R 5 ; wherein R 5 is C 1 -C 3 alkyl, and one or more hydrogen atoms of R 5 may be replaced by a hydroxy group; R 2 is a group selected from OH and formula (II): 【Chemistry 11】 and where * indicates the position at which the group of formula (II) is attached to the rest of the molecule. The poly-L-lysine glycoconjugate is characterized by being represented by The complex carbohydrate according to claim 7.

11. The glycoconjugate according to claim 6, characterized in that the polymer is present within a magnetic nanoparticle.

12. 10. Use of the glycoconjugates according to claim 6 for inhibiting and / or removing anti-A and / or anti-B antibodies in vitro or ex vivo from samples of blood or other blood derivatives and by-products.

13. 7. The glycoconjugate according to claim 6 for use as a medicine, preferably for the treatment and / or prevention of rejection or hemolysis in subjects receiving organ transplants or blood transfusions from incompatible donors, respectively.

14. The glycoconjugate of claim 13, wherein the glycoconjugate comprises poly-L-lysine as a polymeric support.

15. A pharmaceutical composition comprising the glycoconjugate of claim 10 and at least one pharmaceutically acceptable additive.

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