Title: High binding oligosaccharide compounds, compositions and uses thereof.

The invention relates to oligosaccharide compositions having affinity for proteins and the medical or biotechnological or diagnostic use of such compositions.

Gangliosides are a class of glycolipids, often found in cell membranes, which consists of three elements. One or more sialic acid residues are attached to an oligosaccharide or carbohydrate core moiety, which in turn is attached to a hydrophobic lipid (ceramide) structure which generally is embedded in the cell membrane. The ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion. Gangliosides, as well as other glycolipids and their structures in general, are discussed in, for example, Lehninger, Biochemistry (Worth Publishers, 1981) pp. 287-295 and Devlin, Textbook of Biochemistry (Wiley-Liss, 1992). Gangliosides are classified according to the number of monosaccharides in the carbohydrate moiety, as well as the number and location of sialic acid groups present in the carbohydrate moiety. Monosialogangliosides are given the designation "GM", disialogangliosides are designated "GD", trisialogangliosides "GT", and tetrasialogangliosides are designated "GQ". Gangliosides can be classified further depending on the position or positions of the sialic aeid residue or residues bound. Further classification is based on the number of saccharides present in the oligosaccharide core, with the subscript "1" designating a ganglioside that has four saccharide residues (Gal-GalNAc-Gal-Glc-Ceramide), "2 designating" disaccharide (Gal-Glc-Ceramide) and "3 designating" monosaccharide (Gaϊ-Ceramide) gangliosides, respectively.

Gangliosides are most abundant in the brain, particularly in nerve endings. They are believed to be present at receptor sites for neurotransmitters, including acetylcholine, and can also act as specific receptors for other biological macromolecules, including interferon, hormones; viruses, bacterial toxins, and the like. Gangliosides have been used for treatment of nervous system disorders,

including cerebral ischemic strokes. See, e.g., Mahadnik et al. (1988) Drug Development Res. 15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz (1988) Adv. Exp. Med. and Biol. 174: 593-600; Karpiatz et al. (1984) Adv. Exp. Med. and Biol. 174: 489-497. Certain gangliosides are found on the surface of human hematopoietic cells (HMebrand et al. (1972) Biochim. Biophys. Acta 260: 272-278; Macher et al. (1981) J. Biol. Chem. 256: 1968-1974; Dacremont et aL Biochim, Biophys. Acta 424: 315-322; Klock et al. (1981) Blood CeIk 7: 247) which may play a role in the terminal granulocytic differentiation of these cells. Nojiri et al. (1988) J. Biol. Chem. 263: 7443-7446. These gangliosides, referred to as the "neolacto" series, have neutral core oligosaccharide structures having the formula [GaLbeta.-

(l,4)GlcNAc.beta.(l,3)].sub.nGal.beta.(l,4)Glc, where n=l-4. Included among these neolacto series gangliosides are 3'-nLM.sub.l

(NeuAc.alpha.(2 ₎ 3)Gal.beta.(l ₎ 4)GlcNAc.beta.(l _> 3)Gal.beta.(l,4)-Glc.beta.- (l,l)- Ceramide) and 6'-nLM.sub.l (NeuAc.alpha.(2,6)Gal.beta.(l,4)GlcNAc.be- ta.(l,3)Gal.beta.(l,4)-Glc.beta.(l,l)-Ceramide).

Ganglioside "mimics" are often associated with some pathogenic organisms. For example, the core oligosaccharides of low-molecular-weight Iipo Poϊy Saccharide (LPS) of Campylobacter jejuni 0:19 strains were shown to exhibit molecular mimicry of gangliosides. Since the late 1970s, Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (SMrrow (1977) Brit. Med. J. 2: 9-11). Epidemiological studies have shown that Campylobacter infections are more common in developed countries than Salmonella infections and they are also an important cause of diarrheal diseases in developing countries (Nachamkin et al. (1992) Campylobacter jejuni: Current Status and Future Trends. American Society for Microbiology, Washington, D.C.). In addition to causing acute gastroenteritis, C. jejuni infection has been implicated as a frequent antecedent to the development of Guillain-Barre syndrome (GBS), a form of neuropathy that is the most common cause of generalyzed paralysis (Ropper (1992) N. Engl. J. Med. 326: 1130-1136). The most common C. jejuni serotype associated with Guillain-Barre syndrome is 0:19 (Kuroki (1993) Ann. Neurol. 33: 243-247) and this prompted detailed study of the lipopolysaccharide (LPS) structure of strains belonging to this serotype (Aspinall et al. (1994a) Infect. Immun. 62: 2122-2125; Aspinall et al. (1994b) Biochemistry 33:

241-249; and Aspinall et al. (1994c) Biochemistry 33: 260-255).

Terminal oligosaccharide moieties identical to those of GDIa, GD3, GMl and GTIa gangliosides have been found in various C. jejuni 0:19 strains. C. jejuni OH4384 belongs to serotype 0:19 and was isolated from a patient who developed the Guillain-Barre syndrome following a bout of diarrhea (Aspinall et al. (1994a), supra.). It was shown to possess an outer core LPS that mimics the tri-sialylated ganglioside GTIa. Molecular mimicry of host structures by the saccharide portion of LPS is considered to be a virulence factor of various mucosal pathogens which would use this strategy to evade the immune response (Moran et al. (1996a) FEMS

Immunol. Med. Microbiol. 16: 105-115; Moran et al. (1996b) J. Endotoxin Res. 3: 521- 531).

Autoimmune neuropathies including Guillain-Barre syndrome are frequently associated with anti-GMl ganglioside antibodies. These are believed to play a pathogenic role and their clearance from the circulation would be predicted to produce therapeutic benefit. Towson et al. (Glycobiology Advance Acces, December 4, 2006) examined the conditions required for effective immunoadsorption of anti-GMl antibodies using glycan-conjugated sepharose as a matrix. In solution inhibition studies using a range of GMl-like saccharides in conjunction with mouse and human anti-GMl antibodies, the whole GMl pentasaccharide, ss-Gal-(l-3)-ss-GalNAc-(l-4)- [alpha-Neu5Ac-(2-3)]-ss-Gal-(l-4)-ss-Glc, was found the favoured ligand for maximal inhibition of antibody-GMl interactions in comparison with monosaccharides, GaI- (l-3)-ss-GalNAc-ssOMe, and synthetic GMl mimics. Immunoadsorption studies comparing binding of mouse monoclonal anti-GMl antibodies to GMl-Sepharose and ss-Gal-(l-3)-ss-GalNAc-Sepharose confirmed the preference seen in solution inhibition studies. GMl-Sepharose columns were then used by Towson et al. to adsorb anti-GMl IgG and IgM antibodies from human neuropathy sera. Anti-GMl antibodies subsequently eluted from the columns often showed a striking monoclonal or oligoclonal pattern, indicating that the immune response to GMl is restricted to a limited number of B cell clones, even in the absence of a detectable serum paraprotein. Based on these data, Towson et al, support the view that immunoadsorption plasmapheresis could potentially be developed for the acute

depletion of serum anti-GMl antibodies in patients with neuropathic disease, and also provide purified human anti-GMl antibodies for analytical studies.

Proteins often hind to their carbohydrate ligands in a multivalent manner (a) R, T. Lee, Y. C. Lee, Glycoconj. J. 2000, 17, 543-551; E. Boy, Trends Glycosά. and

GlyeotechnoL 2003, 15, 291-310;) . The cholera toxin (CT) is a prime example of a multivalent protein and is capable of binding simultaneously to the carbohydrate moieties of five GMl gangliosides. The binding of the five B subunits is of critical importance for the internaHzation and subsequent disease process of this ABδ toxin (E. Pan, et al., Curr, Opin, Struct. Biol. 2000, 10, 680-686). The development of strong binding agents to the toxin is of interest for the development of disease prevention/treatment, but also for the detection of toxin in patient samples or in materials suspect of terrorist origin (S, Ahn-Yoon, et al., Anal. Chem. 2003, 75, 2256- 2261; S. C. Clarke, Br. J. Biomed. Sd. 2005; 62, 40-46) Furthermore, the cholera toxin serves as a benchmark case to test multivalent strategies. As such, several systems have been reported that showed varying degrees of multivalency effects (J. P. Thompson, C-L. Schengrund, Glycoconj. J. 1997, 14, 837-845; I. Vrasidas, et al, Bur. J. Org. Chem. 2001, 4685 - 4692; Z. Zhang, et al., J. Am. Chem. Soc. 2002, 124, 12991-12998; d) D. Arosio, et al, Org. Biomol. Chem. 2004, 2, 2113-2124; Z. Zhang, et al., Org. Lett. 2004, 6, 1377-1380; f) D.

Arosio, et al, J. Am. Chem. Soc. 2005, 127, 3660-3661). For example, a multivalent conjugated modified GMlos was prepared in very small quantities via reductive amination, and found to enhance binding up to 250-fold. Studies of the smaller Shiga-like toxin and the B. coli heat-labile enterotoxin, indicated that long spacers are beneficial for strong binding (P. I. Kitov, et al, Nature 2000, 403, 669-672. ;E. Fan, et al., J. Am. Chem. Soc. 2000, 122, 2663-2664). A pentavalent version of conjugated /n-nitrophenyl α-D-galactopyranoside that contained long spacer arms, surpassed GMlos in affinity (relative potency 2.8), although the compound was unstable in water. While a pentavalent presentation of ligands seemed essential for effective inhibition of ABs toxins, glycodendrimers were shown to be even more effective (P. I. Kitov, D. R. Bundle J. Am. Chem. Soc. 2003, 125, 16271-16284).

The invention provides high binding oligosaccharide compounds and compositions comprising one or more of said compounds. The high binding oligosaccharide compounds provided herein are preferably synthetic ganglioside mimics designed to detect, purify, characterize and/or deplete substances, e.g. bacterial toxins and serum antibodies, that are related to human diseases. They consist of an oligosaccharide structure that is essentially identical to the carbohydrate portion of a ganglioside, attached to aU∑yi spacer molecules, which in turn are covalently linked to multivalent, such as divalent, tetravalent or octavalent dendrimers. Dendrimers are molecules that at least partially consist of repeating units, and which are molecules that have a two- or three- dimensional diverting branching structure starting from the "base" of the dendrimer. The name is derived from the Greek word for tree; dendrimers are (often) tree-shaped molecules.

The compound preferably has a terminal azide, or other type of reactive group, by which it can be covalently bound to a solid carrier or solid surface, such as a bead, ELISA plate, monolithic column, monolitenic particles or other solid carrier that can be used to detect or adsorb ligands for ganglioside-ligands, including bacterial toxins and serum anti-ganglioside antibodies. Accordingly, in one aspect there is provided at least one oligosaccharide compound of the invention, said compound being a ganglioside mimic, immobilized onto a solid carrier or solid surface, e.g. by covalent coupling. The covalent binding of the synthetic ganglioside (mimics) to a solid carrier as provided herein prevents the detachment of these mimics during detection and depletion. Because of this covalent binding the measurements and depletions are highly reproductive, and some of these devices may be reused after proper washing. It is preferred that said carrier is selected from the group of beads, nano-beads or nanoparticles, monoliths, Elisa plates, Si wafers, capillaries and glass slides, and other carriers known in the art. In a preferred embodiment, said carrier comprises a methacrylate-based monolith, such as one comprising polymethacrylate-based polymer. The invention also provides a highly effective and selective immunoadsorption because of the use of the preferred authentic ganglioside oligosaccharide sequences that are the most physiological and relevant targets in diseases. Further the use of an extremely high density of these oligosaccharide

sequences required for multivalent binding and for the conformational epitopes mimicking those in the physiological clusters of gangliosides in lipid rafts at the neural membranes is provided herein, as well as the use of multivalent dendrimeric scaffolds that further increase the epitope density, or the use of elongated spacer arms and in a preferred embodiment the use of the monolith composites resulting in an strong increase of efficient surface area for antibody-ligand interaction. In particular, the invention provides a compound or substance having multivalent binding capacity for a protein, said compound comprising at least two oligosaccharide moieties linked to a dendrimer, preferably having at least three, or more preferably, four oligosaccharide moieties linked to a dendrimer. It is most preferred for authenticity that at least one of said oligosaccharide moieties is a monosialogangliosid (GM), a disialogangliosid (GD), a trisialogangliosid (GT), a tetrasialogangliosid (GQ) or a mimic thereof. It is preferred that at least one of said oligosaccharide moieties has been linked to said dendrimer via 'click' chemistry (Also described in EP 1 733 742).

It is preferred that such a compound according to the invention is linked to a dendrimer carrier. Preferred dendrimers are selected from the group of hydrophilic dendrimers, particularly wherein said dendrimer comprises oxy-ethylene spacer arms. It is preferred that at least one of said oligosaccharide moieties linked via a spacer arm to a dendrimer, preferably selected from the group of spacers consisting of 11 to 80 atoms. In a particularly preferred embodiment, said spacer arm comprises C, H, O, and N atoms and preferably consists of 34 atoms. As is exemplified in the Examples below, a further aspect of the invention relates to a method for providing a multivalent compound of the invention, and to a method for immobilizing said compound to a solid carrier. Also provided are compounds and solid carriers obtainable by such methods.

The invention herewith provides a compound or composition having multivalent binding capacity for a bacterial toxin, preferably an ABs toxin such as Cholera toxin (CT), E. coli heat labile toxin (LT) or Shiga toxin (Stx), and use of such a compound or composition for the detection of a bacterial toxin, preferably an ABs toxin in a sample. The invention also provides use of such a compound for the preparation of a pharmaceutical composition, preferably for treatment of a subject suspected to be suffering of an intoxication with a bacterial toxin such as an ABs toxin. Preventive

treatment of subjects prone to such intoxication is herein also provided. The invention also provides use of such a composition for the preparation of a pharmaceutical composition for treatment of a subject suspected to be suffering from an autoimmune disease, for example for treatment of a subject suspected to be suffering from an autoimmune neuropathy, such as wherein said neuropathy comprises Guillain Barrέ Syndrome. Preventive treatment of subjects prone to such autoimmune disease is herein also provided. The invention thus herewith also provides a pharmaceutical composition comprising one or more compounds according to the invention having multivalent binding capacity for a bacterial toxin, preferably an ABe toxin such as Cholera toxin (CT), E. coli heat labile toxine (LT) or Shiga toxin (Stx). The invention also provides various uses of a compound according to the invention, including for the preparation of a medical device, for diagnostic purposes, for treatment of a subject suspected to be suffering of an intoxication with a bacterial toxin such as an ABs toxin, or for treatment of a subject suspected to be suffering from an autoimmune disease such as an autoimmune neuropathy. It is preferred that said medical device is a plasmapheresis unit, for example provided with a compound having multivalent binding capacity for a bacterial toxin, preferably an AB5 toxin such as Cholera toxin (CT), E. coli heat labile toxine (LT) or Shiga toxin (Stx), for example linked to a carrier, such as a monolith, according to the invention. The invention also provides a diagnostic device comprising for example a compound coupled or linked to a solid carrier, such as an affinity monolith according to the invention equipped for selective detection of antibodies and toxins, like SPR or ESI- MS, and use of a compound or composition according to the invention for the sensitive and fast diagnosis of autoimmune diseases and intoxications with bacterial toxins.

Figure legends

Figure 1. Structures of the functionalized GMlos and GM2os.

Figure 2. Synthesis of heptaol 4. (a) AC2O, NaOAc, reflux, 4 h; (b) SnCU in dry CH2CI2, rt, overnight, 56%; (c) NaOCH ₃ in MeOH, rt, 15 min, 99%.

Figure 3. Enzymatic syntheses of GM3, GM2 and GMl mimics.

Figure 4. Synthesis of the ligands. Eeagents and conditions: a) 1, CuSCU, sodium ascorbate, DMFZH ₂ O, 8O ⁰ C, 20 min. yields: 57% for 4b, 78% for 5b, 76% for 6b. b) 3, CuSO ₄ , sodium ascorbate, DMF/H2O, 8O ⁰ C, 20 min 64% for 5c, 37% for 6c.

Figure 5. Applications of high binding oligosaccharide compositions for diagnostics and treatment.

Figure 6. Luminex beads detecting cholera toxin B-subunit and serum anti-GMl antibodies. Luminex beads covalently coated with GMl-alkyl and detecting serum anti-GMl antibodies (from normal control NC 18 and GBS patient F 102A) and cholera toxin B-subunit.

Figure 7. ELJSA detecting serum anti-GMl antibodies. ELISA plates coated non- covalently with GMl, and covalently with GMl-alkyl and GMl-dimers. Incubation with serum from 2 healthy controls (NC30, BCN23) and GBS patients (F120, F183).

Figure 8. Specific depletion of serum anti-GMl antibodies with dendrimers. Depletion experiment with tetravalent GMl-dendrimers: no depletion of serum anti- GQIb antibodies (F297C), but depletion of serum anti-GMl antibodies (F102A).

Figure 9. Depletion of serum anti-GM2 antibodies with monoliths coated with GM2 mimics. Immunoadsorption of serum with anti-GM2 antibodies: no depletion with blank monolithic column, but depletion with monolithic column coated with GM2 mimics.

Figure 10. Binding of serum anti-GM2 antibodies to GM2-coated monolithic column. Two monolithic columns were incubated with serum from a GBS patient with anti- GM2 antibodies, then washed and then incubated with FITC-conjugated anti-human immunoglubulin. The monolithic columns were blank (A) or coated with a GM2

mimic (B). Observed fluorescence in column B clearly demonstrates homogenous binding of immunoglobulins from the serum to the column where column A demonstrates no binding.

Detailed description

Proteins often bind to their carbohydrate ligands in a multivalent manner. The cholera toxin (CT) is a prime example of a multivalent protein and is capable of binding simultaneously to the carbohydrate moieties of five GMl gangliosides. The binding of the five B subunits is of critical importance for the internalization and subsequent disease process of this ABe toxin. The development of strong binding agents to the toxin is of interest for the development of disease prevention/treatment, but also for the detection of toxin in patient samples or in materials suspect of terrorist origin. Furthermore, the cholera toxin serves as a benchmark case to test multivalent strategies. As such, several systems have been reported that showed varying degrees of multivalency effects. For example, a multivalent conjugated modified GMlos was prepared in very small quantities via reductive amination, and found to enhance binding up to 250-fold. Studies of the smaller Shiga-like toxin and the E. coli heat-labile enterotoxin, indicated that long spacers are beneficial for strong binding. A pentavalent version of conjugated m- nitrophenyl α-D-galactopyranoside that contained long spacer arms, surpassed GMlos in affinity (relative potency 2.8), although the compound was unstable in water. While a pentavalent presentation of ligands seemed essential for effective inhibition of ABs toxins, glycodendrimers were shown to be even more effective. The invention provides strong inhibition of Cholera Toxin by multivalent GMl derivatives. We here report on a highly effective inhibitor of the cholera toxin due to a combination of three factors in the inhibitor design: 1) the use of the authentic GMl oligosaccharide sequence (GMlos) as the optimal monovalent ligand, 2) the use of a tetravalent dendritic scaffold, and 3) the use of elongated spacer arms. The combined effects led to an inhibitor that is unprecedentedly 83,000-fold more potent than a monovalent GMlos conjugate.

GMlos conjugates 1 and 2 and related GM2os conjugate 3 were prepared on a 100 mg scale via a route containing chemical synthesis and enzymatic steps. The CIl tail of 1 and 3 contains an azido group to enable its conjugation via "click" chemistry, a method which extends the range of organic and inorganic substrates to which oligosaccharides can be attached significantly. The dendritic scaffolds 5a, 6a and the

monovalent reference compound 4a were derived from our earlier developed dendrimers ^b that were functionalized with alkynes by coupling to 4-pentynoic acid. In the subsequent step 1 and the alkynes 4a, 5a and 6a were exposed to CUSCM and sodium ascorbate in DMF/H2O (1:1) at 80 ⁰ C with microwave heating for 20 min according to reported conditions.'These conditions facilitated efficient coupling, and provided products 4b, 5b and 6b in good isolated yield after HPLC purification. It is worth noticing that analytical HPLC analysis showed the reaction between 1 and 6a to form 6b to be, in fact, complete after 1 min! GM2os conjugates 5c and 6c were prepared in a similar fashion.

In order to evaluate the inhibitory potency of the inhibitors an ELISA assay was used. In this assay, wells of a 96-well plate were coated with GMl ganglioside, and after blocking with BSA, horseradish peroxidase (HRP)-conjugated CTBe was allowed to bind to the surface with or without inhibitors. In this assay the monovalent GMlos derivative 2 exhibited an IC50 in the micromolar range (19 μM, Table 1). Since the GMlos oligosaccharide is reported to have a Kd of 43 nM, the high inhibitory concentration of the conjugated GMlos 2 indicates that the toxin binds strongly to the ELISA plate due to multivalent binding. The role of the aglycon part beyond the CIl tail was found to be negligible, since monovalent 4b showed a similar ICβoas 2 i.e. in the micromolar range. The divalent 5b was subsequently measured, and found to have an IC∞that was almost 4 orders of magnitude lower, i.e 2.0 nM. Moving to the tetravalent 6b, gave an ICso which was a further order of magnitude lower (0.23 nM). Control experiments with nonfunctional dendrimers (4a, 5a and 6a) showed no measurable inhibition for all concentrations tested.

The prepared compounds exhibited unprecedented affinities, not only because the strongest known CT ligands (GMlos) were used, but also because these ligands were combined with the strongest multivalency effects observed for CT. For tetravalent 6b, each of the GMlos moieties bound more than 20 thousand-fold stronger than monovalent 2. Possibly the multivalency effects could be even larger, since the detection limit of the assay appears to have been reached. Experiments with GM2os support this notion. Monovalent GM2os was not measured because an expected low affinity indicated that it would take too much material to perform the assay.

However, divalent 5c was prepared and its inhibitory potency could just be observed with an IC50 of 2 mM. A large jump in potency was observed when moving to tetravalent 6c (19,000-fold) having an ICso of 0.1 μM. This jump was almost three orders of magnitude larger than in the GMlos series. Considering that the reported affinity of GM2os (Kd 2.0 mM) is ca. 50.000-fold weaker than GMlos, while the difference between the two tetravalent compounds 6b and 6c is less than 500-fold, also suggests that multivalency effects are very large for 6c and that the value obtained for 6b is an underestimation.

In the assay, significant better data fits were observed when a Hill cooperativity coefficient was introduced. Interestingly, for the monovalent compounds the best fit was obtained with negative cooperativity and a Hill coefficient of around 0.5, thus implying that after one molecule of 2 bound to CT, subsequent molecules of 2 bind less strongly. For the divalent 5b the Hill coefficient was close to 1, but the tetravalent 6b and 6c both showed steeper inhibition curves and thus positive cooperativity, with Hill coefficients of around 3. Cooperativity of CT binding has previously been observed. The observations made here indicate cooperative binding of the tetravalent inhibitors to CTB5. In short, multivalent GMlos and GM2os compounds were prepared via efficient 'click' chemistry to dendritic scaffolds with extended arms. The unambiguously characterized compounds clearly reveal strong cooperative binding, with an unparalleled value of at least 83, 000-fold stronger for tetravalent GMlos conjugate 6b than monovalent GMlos derivatives, which are known to have Kd's in the nM range (Kd GMlos = 43 nM). This opens up possibilities in e.g. development of very sensitive sensor applications.

Experimental Section

Synthesis of ω-undecenyl glycosides of GM3, GM2 and GMl The chemoenzymatic synthesis of glycosides with glycosyltransferases from

C. jejuni ¹⁹ was explored in terms of substrate specificity (presence of an ω-undecenyl

chain rather than a hydrogen atom or a short alkyl chain at the aglycone end) and solvent tolerance. The fully unprotected undec-lO-enyl lactoside that was used for the enzymatic modifications was synthesized from D-lactose 1 in three steps. Lactose octaacetate 2, prepared hy acetylation of D-laetose, was glycosidated with 10-undecen-l-ol to give heptaacetyl undecenyl lactoside 3, which after isolation was deprotected according to the standard Zemplen procedure to afford heptaol 4. The latter did not require a purification step and was directly used for further transformations,

Unlike the previously reported synthetic substrates for glycosyltransferases, compound 4 is poorly soluble in water: it forms a gel, and as such is not amenable for enzymatic modification. Compound 4 was first dissolved in 100% MeOH. After addition of the different reaction components we obtained a mixture of methanol/water (20/80 v/v) which was compatible with CST-06, the sialyltransferase used to add NeuAc. The mixture of compound 4, sialyltransferase CST-06 and CMP- NeuAc was kept at 37 °C for 1 h, after which TLC analysis unambiguously showed that lactoside 4 was converted completely. The target GM3-Cπ (5) was subsequently bound on a Sep-Pak column, which was then washed with η2O to elute hydrophilic compounds (such as the buffer and the nucleotide) and finally the GM3-Cn was eluted with MeOH in a 92% yield.

For synthesis of compound GM2-Cπ (6), we added the different components directly to the GM3-Cn (5) reaction mixture which resulted in a final concentration of 10% (v/v) methanol. Compound 5 was in this medium reacted with the in situ- generated UDP-GaINAc in a one-pot mixture containing UDP-GIcNAc, the UDP- GIcNAc 4'-epimerase (CPG- 13) and the β-l,4-iV-acetylgalactosaminyltransferase (CJL-30). The GM2-Cπ (6) was bound to a Sep-Pak column as described above for GM3-Cii (5) and eluted with MeOH. We obtained a 99.3% recovery yield of GM2-Cπ (6). Compound 6 was further elongated in an aqueous solution using UDP-GaI and the β-l,3-galactosyltransferase (CJL-20) to GMl-Cn (T), which, after purification, was recovered in high yield (93.8%).

Click chemistry general procedure: Alkyne dendrimer (1-5 mM), sugar azide (1.5 equiv / alkyne), CuSθ4-5H2θ (1 equiv / alkyne) and sodium ascorbate (1 equiv /

alkyne) were dissolved in an appropriate volume of DMF/H2O (1/1, v/v). The mixture was heated under microwave irradiation at 80°C for 20 min. The reaction mixture was concentrated in vacuo at 60°C and the product was isolated by reversed phase HPLC.

CTBs inhibition assay: A 96-well plate was coated with a solution of GMl (100 μL, 2 μg/mL) in PBS buffer. Unattached ganglioside was removed by washing with PBS and the remaining binding sites of the surface were blocked by BSA (1%) followed by PBS-washing. Samples of toxin-perosddase conjugate (CTB-HRP Sigma) and inhibitor in PBS with BSA (0.1%) and Tween 20 (0.05%) were incubated at r.t. for 2 h and were then transferred to the GMl-coated plate. After 30 min of incubation the solution was removed followed by washing steps with BSA (0.1%)/Tween 20 (0.05%) in PBS. To identify toxin binding to surface-bound GMl the wells were treated with a freshly prepared solution of o-phenylenediamine/H2θ2 in citrate buffer (100 μL) for 15 min. After H2SO4 quenching the absorbance at 490 nm was measured.

Unless stated otherwise, chemicals were obtained from commercial sources and used without further purification. Solvents were purchased from Biosolve (Valkenswaard, The Netherlands). Microwave reactions were carried out in a dedicated microwave oven, i.e. the Biotage Initiator. The microwave power was limited by temperature control once the desired temperature was reached. A sealed vessel of 2 - 5 mL was used. Analytical HPLC runs were performed on a Shimadzu automated HPLC system with a reversed phase column (Alltech, Adsorbosphere C8, 90 A, 5μm, 250x4.6 mm) equipped with an evaporative light scattering detector (PL-ELS 1000, Polymer Laboratories) and a UV7VIS detector operating at 220 and 254 nm. Preparative HPLC runs were performed on a Applied Biosystems workstation. Elution was effected using a gradient of 5% MeCN and 0.1% TFA in H2O to 5% H ₂ O and 0.1% TFA in MeCN. ¹ H NMR (300MHz) and ¹³ C NMR (75MHz) were performed on a Varian G-300 spectrometer.

Meθ2C-[G0](LinkerNHBoc)ι X

To a solution of dendrimer X (591 mg, 2 mmol) in CH2CI2 (5 mL) with a trace of H2O was added TFA (5 ml). The solution was stirred at rt for 1 h followed by concentration to dryness. The product was dissolved in CH ₂ CIs (10 mL) and acid X (960 mg, 2.2 mmol) and HATU (836 mg, 2.2 mmol) were subsequently added followed by DiPEA (0.99 mL, 6.0 mmol). The reaction mixture was stirred for 2 h. The reaction mixture was concentrated in vacuo and silica gel chromatography (CH2Cl2/MeOH, 19/1 → 9/1) was used for purification (1.24 gr, quantitative, small impurity).

Meθ2C-[G0](Linker-Alkyne)i 4a

A solution of X (1.24 gr, 2 mmol) in CH2CI2 (5 mL) with a trace of H2O was added TFA (5 mL). After 1 h the mixture was concentrated to dryness and dissolved in CH2CI2 (10 mL). HATU (836 mg, 2.2 mmol), 4-pentynoic acid (294 mg, 3.0 mmol) and DiPEA (0.99 mL, 6.0 mmol) were subsequently added and the reaction mixture was stirred for 18 h. Silica gel chromatography (CH ₂ Cl ₂ ZMeOH, 19/1 → 9/1) was used for purification followed by size exclusion chromatography (MeOH) to afford pure product (552 mg, 46% over 2 steps). ¹ H NMR (CDCl ₃ , 300 MHz): δ = 7.65 (IH, d,

CHarom-6), 7.55 (IH, S, CHarom-2), 7.35 (IH, t, CHarom-5), 7.10 (IH, dd, CHarom-4), 7.44,

7.20 and 6.60 (each IH, 3 xbt, 3 x C(O)NH), 4.15 - 4.11 (2H, m, OCH ₂ CH ₂ NH), 4.08 and 4.04 (each 2H, s, OCH ₂ C(O)), 3.91 (3H, s, C(O)OCH ₃ ), 3.74 - 3.72 (2H, m,

OCH ₂ CH ₂ NHC(O)), 3.61 - 3.53 (12H, m, OCH ₂ ), 3.41 -3.35 (4H, m, CH ₂ NHC(O)), 2.54 - 2.48 and 2.42 - 2.37 (each 2H, 2 x m, CH ₂ CH ₂ CCH), 2.00 (IH, t, CH ₂ CCH) and 1.83 - 1.72 (8η, m, OCH ₂ CH ₂ CH ₂ NH). ™C NMR (CDCl ₃ , 75.5 MHz): δ = 171.0, 169.0 and 168.4 (C(O)NH), 166.7 (C(O)OCH ₃ ), 158.4 (Carom-3), 131.5 (C _a «,m-1), 129.5 (CHarom-5), 122.4 (CHarom-6), 119.6 (CHarom-4), 114.8 (CHarøn-2), 83.2 (CH ₂ CCH), 71.0

(CH ₂ CCH), 70.3 - 69.1 (OCH ₂ ), 66.6 (OCH ₂ CH ₂ NH), 52.2(C(O)OCH ₃ ), 38.5 (OCH ₂ CH ₂ NH), 37.8 and 37.3 (CH ₂ NHC(O)), 35.3 (CH ₂ CH ₂ CCH), 29.0 and 28.8 (OCH ₂ CH ₂ CH ₂ NH) and 14.8 (CH ₂ CCH). HRMS for C ₂₉ H ₄₃ N ₃ Oi ₀ (M, 593.2948) M + Na found 616.2775, calcd 616.2846.

MeO _z C-[Gl](LinkerNHBoc) ₂

To a solution of dendrimer X (250 mg, 0.55 mmol) in CH2CI2 (10 mL) with a trace of H2O was added TFA (5 ml). The solution was stirred at rt for 1 h followed by concentration to dryness. A solution of acid X (602 mg, 1.38 mmol) in CH2CI2 (10 mL) was added to the dendrimer, subsequently followed by HATU (525 mg, 1.38 mmol) and DiPEA (0.68 mL, 4.13 mmol). The reaction mixture was stirred for 70 h. The reaction mixture was concentrated in vacuo and Silica gel chromatography (CH ₂ Cl ₂ MeOH, 19/1 → 9/1) was used for purification (580 mg, 97 %, small impurity). ¹ H NMR (CDCl ₃ , 300 MHz): δ = 7.40 and 7.24 (4H, 2 x bs, 2 x 2 C(O)NH), 7.16 (2H, d, CHarom-2,6), 6.66 (IH, t, CHarom-4), 5.06 (NHBoc), 4.12 - 4.10 (4H, m, OCH ₂ CH ₂ NH), 4.08 and 4.04 (each 4H, s, OCH ₂ C(O)), 3.89 (3H, s, C(O)OCH ₈ ), 3.73 - 3.68 (4H, m, OCH ₂ CH ₂ NHC(O)), 3.64 - 3.49 (24H, m, OCH ₂ ), 3.43 - 3.37 (4H, m, CH ₂ NHC(O)), 3.23 - 3.17 (4H, m, CH ₂ NHBoc), 1.83 - 1.72 (8H, m, OCH ₂ CH ₂ CH ₂ NH) and 1.43 (18H, s, C(O)OC(CHs) ₃ ). ¹³ C NMR (CDCb, 75.5 MHz): 8 = 169.3 and 168.6 (C(O)NH), 166.4 (C(O)OCH ₃ ), 159.5 (Carom-3,5), 156.1 (C(O)OC(CH ₃ )s), 132.1 (Carom- 1), 108.1 (CHarom-2,6), 106.5 (CHarom-4), 78.9 (C(O)OC(CH ₃ )S), 71.0 - 69.3 (OCH ₂ ), 66.7 (OCH ₂ CH ₂ NH), 52.2 (C(O)OCH ₃ ), 38.4 (CH ₂ NHBoc), 37.5 (OCH ₂ CH ₂ NH), 29.6 and 28.9 (OCH ₂ CH ₂ CH ₂ NH) and 28.4 (C(O)OC(CH ₃ )s).

MeO ₂ C-[Gl](Linker-Alkyne) ₂ 5a A solution of X (300 mg, 0.27 mmol) in CH ₂ Cl ₂ (10 mL) with a trace OfH ₂ O was added TFA (5 mL). After 4 h of stirring the mixture was concentrated to dryness and dissolved in CH2CI2 (5 mL) and DMF (5 mL). HATU (260 mg, 0.68 mmol), 4- pentynoic acid (67 mg, 0.68 mmol) and DiPEA (225 μL, 1.36 mmol) were subsequently added and the reaction mixture was stirred for 18 h. Silica gel chromatography (CH ₂ Cl ₂ ZMeOH, 19/1 → 4/1) was used for purification followed by size exclusion chromatography (MeOH) to afford pure product (170 mg, 60%). ¹ H NMR (CDCl ₃ , 300 MHz): 8 = 7.58 and 7.33 (4H, 2 x t, 2 x 2 C(O)NH), 7.17 (2H, d, CHatom-2,6), 6.67 (3H, bt, CHato∞-4 and 2 x C(O)NH), 4.13 - 4.09 (4H, m, OCH ₂ CH ₂ NH), 4.08 and 4.03 (each 4H, s, OCH ₂ C(O)), 3.90 (3H, s, C(O)OCH ₃ ), 3.71 - 3.63 (4H, m, OCH ₂ CH ₂ NHC(O)), 3.62 - 3.53 (24H, m, OCH ₂ ), 3.42 - 3.32 (8H, m,

CH ₂ NHC(O)), 2.54 - 2.48 and 2.41 - 2.36 (each 4H, 2 x m, CH ₂ CH ₂ CCH), 2.01 (2H, t, CH ₂ CCH) and 1.83 - 1.72 (8η, m, OCH ₂ CH ₂ CH ₂ NH). ∞C NMR (CDCl ₃ , 75.5 MHz): 8

132.1 (Carom- I), 108.1 (CHawm-2,6), 106.6 (CHarom-4), 83.2 (CH ₂ CCH), 70.9 (CH2CCH), 70.3 - 69.2 (OCH ₂ ), 66.7 (OCH2CH2NH), 52.3 (C(O)OCH ₃ ), 38.4 (OCH2CH2NH), 37.7 and 37.2 (CH ₂ NHC(O)), 35.2 (CH ₂ CH ₂ CCH), 29.0 and 28.8 (OCH ₂ CH ₂ CH ₂ NH) and 14.8 (CH ₂ CCH). HRMS for CBoH ₇ SN ₆ Oi ₈ (M, 1050.537) M + Na found 1073.559, cakd 1073.5271.

Me0 ₂ C-[G2](LinkerNHBoc)4 X

To a solution of dendrimer X (540 mg, 1.10 mmol) in CH ₂ Cl ₂ (20 mL) with a trace of H2O was added TFA (10 ml). The solution was stirred at room temperature for 3 h followed by concentration to dryness. A solution of acid X (1.07 gr, 2.46 mmol) in CH2CI2 (10 mL) was added to the dendrimer, subsequently followed by HATU (936 mg, 2.46 mmol) and DiPEA (1.06 mL, 6.4 mmol). The reaction mixture was stirred for 20 h. The reaction mixture was concentrated in vacuo and silica gel chromatography (CH ₂ Cl ₂ ZMeOH, 9/1 — » 4/1) was used for purification (442 mg, 38 %). ⁱ H NME (CDCl ₃ , 300 MHz): δ = 7.83, 7.63 and 7.44 (2,4 and 4H, 3 x bt, 10 C(O)NH),

7.15 (2H, d, CHarom-2,6), 6.98 (2H, d, CBarom-2',6'), 6.68 (IH, t, CHarom-4), 6.51 (IH, t,

CHarom-4'), 5.19 (NHBoc), 4.17 (4H, bs, OCH ₂ CH ₂ NH), 4.04 and 4.00 (each 24H, bs, 8 x OCH ₂ C(O) and 4 x OCH ₂ CH ₂ NH), 3.87 (3H, s, C(O)OCH ₃ ), 3.80 - 3.79 (4H, m, 0CH ₂ CHi?NH), 3.62 - 3.48 (48H, m, OCH ₂ ), 3.39 - 3.33 (8H, m, CaNHC(O)), 3.21 - 3.17 (8H, m, CH*NHBoc), 1.81 - 1.68 (16H, m, OCH ₂ CHsCH ₂ NH) and L43 (36H, s, C(O)OC(CHs) ₃ ). ¹³ C NMR (CDCl ₃ , 75.5 MHz): δ - 169.2, 168.5 and 167.3 (C(O)NH), 166.4 (C(O)OCH ₃ ), 159.5 (Carom-3,5), 159.3 (Carom-3 ^> ,5 ^> ), 156.0 (C(O)OC(CHs) ₃ ), 136.3

(Carom- 1), 131.8 (Carom- V), 107.9 (CHarom-2,6), 106.4 (CHarom-4), 106.1(CHar-m-2',6 ^> ), 104.3 (CHarom-4 ¹ ), 78.7 (C(O)OC(CH ₃ ) ₃ ), 70.7 - 69.1 (OCH ₂ ), 66.5 and 66.3

(OCH ₂ CH ₂ NH), 52.1 (C(O)OCH ₃ ), 39.4 and 38.2 (OCH ₂ CH ₂ NH), 37.0 (CH ₂ NHBoc), 29.5 and 28.8 (OCH2CH2CH2NH) and 28.2 (C(O)OC(CH ₃ ) ₃ ).

MeO ₂ C-[G2](Linker-Alkyne)46a A solution of X (442 mg, 0.186 mmol) in CH ₂ Cl ₂ (10 mL) with a trace of H ₂ O was added TFA (5 mL). After 4 h stirring the mixture was concentrated to dryness and dissolved in CH ₂ Cl ₂ (10 mL) and DMF (5 mL). HATU (354 mg, 0.93 mmol), 4-

pentynoic acid (91 mg, 0.93 mmol) and DiPEA (463 μL, 2.80 mmol) were subsequently added and the reaction mixture was stirred for 20 h. Silica gel chromatography (CH ₂ Cl ₂ MeOH, 9/1 — » 1/1) was used for purification, followed by Sephadex size exclusion chromatography (MeOH) to afford pure product (369 mg, 87%). ¹ H NMR (CDCl ₃ , 300 MHz): 8 = 7.70, 7.61 and 7.39 (2, 4 and 4H, 3 x t, 10 x C(O)NH), 7.17 (2H, d, CHarom-2,6), 6.98 (4H, d, CH _a rom-2 ⁾ ,6 ^> ), 6.75 - 6.71 (5H, m, CHarom-4 and 4 x C(O)NH), 6.54 (2H, t, CHaro _β -4 ^> ), 4.21 - 4.17 and 4.08 - 4.04 (4 and 8H, 2 x m, OCH ₂ CH ₂ NH), 4.04 and 4.00 (each 8H, s, OCH ₂ C(O)), 3.88 (3H, s, C(O)OCH ₃ ), 3.83 - 3.78 and 3.68 - 3.63 (4 and 8H, 2 x m, OCH ₂ CH ₂ NHC(O)), 3.62 - 3.51 (48H, m, OCH ₂ ), 3.39 - 3.29 (16H, m, CHaNHC(O)), 2.51 - 2.46 and 2.40 - 2.35 (each 8H, 2 x m, CH ₂ CH ₂ CCH), 2.02 (4H, t, CH ₂ CCH) and 1.81 - 1.70 (16η, m, OCH ₂ CH ₂ CH ₂ NH). "»C NMR (CDCl ₃ , 75.5 MHz): δ = 171.2, 169.3, 168.5 and 167.4 (C(O)NH), 166.6 (C(O)OCH ₃ ), 159.7 (Carom-3,5), 159.6 (Carom-3',5 ¹ ), 136.6 (0«™»- 1 ¹ ),

132.0 (Carom- 1), 108.2 (CHarom-2',6'), 106.7 (CHarom-2,6), 106.3 (CHarom-4"), 104.5 (CHarom-4), 83.2 (CH ₂ CCH), 70.9 (CH ₂ CCH), 70.3 - 69.2 (OCH ₂ ), 66.7 and 66.5 (OCH ₂ CH ₂ NH), 52.3 (C(O)OCH ₃ ), 39.6 and 38.4 (OCH ₂ CH ₂ NH), 37.7 and 37.2 (CH ₂ NHC(O)), 35.2 (CH ₂ CH ₂ CCH), 29.1 and 28.9 (OCH ₂ CH ₂ CH ₂ NH) and 14.8 (CH ₂ CCH). HRMS for CπoHieeNuOss (M, 2291.149) M + Na found 2314.415, calcd 2314.1385.

Monovalent GMl dendrimer 4b

A solution of 4a (10 mg, 16.8 μmol), pentasaccharide Y (30 mg, 25 μmol), CuSO4.5H ₂ O (4.2 mg, 16.8 μmol) and sodium ascorbate (3.3 mg, 16.8 μmol) in H ₂ O/DMF (1/1, v/v, 4 mL) was heated under microwave irradiation at 80°C for 20 min. Analytical HPLC showed conversion of the Monovalent dendrimer to a new peak. The reaction mixture was concentrated and subjected to preparative HPLC purification. The product was obtained after lyophilization as a glass (17 mg, 57 %). ¹ H NMR (H ₂ O/D ₂ O, 9/1, v/v, 400 MHz): δ = ¹³ C NMR (H ₂ OfD ₂ O, 9/1, v/v, 100 MHz): δ = 175.5, 175.2, 174.9, 174.3, 172.1, 171.6 (COOH and C(O)NH), 168.5 (C(O)OCH ₃ ), 158.5 (Carom-3), 146.3 (Ctna _* >le-4), 131.2 (Carom- 1), 130.5 (CHarom-5), 123.6 (Ctriazolβ-

5),122.9 (CHarom-6), 120.7 (CHarom-4), 115.4 (CHarom-2), 105.1 (Coai-l), 102.9 (Cθal-1),

102.8 (CG.INAC-1), 102.4 (Cαiuc-1), 101.8 (CNeuAc-2), 80.6 (CcaiNAc-3), 79 (Cαiut-4), 77.4

(Cαai-4), 75.2 (Cαai-5), 75.1 (Cαte-5), 74.8 (Coai-3), 74.717 (CGI«C-3), 74.698 (CαaiNAc-5), 74.4 (Cβai-5), 73.4 (C _N enA«r6), 73.1 (CGIUC-2), 72.8 (Coar-3), 72.5, 71 ,70.9 (CH ₂ -0CGI«4),

70.3 (CλnAc-8), 70.2 (OCH ₂ ), 70 (OCH ₂ ), 69.8 (OCH ₂ ), 69 (Coai-2 and 68.9 (OCH ₂ ), 68.7 (OCH ₂ ), 68.4 (CoaiNAc-4), 68.2 (CN _6U AC-4), 67.1 (OCH ₂ CH ₂ NH), 63.1 (CκβuAc-9), 61.4 (CoaiNAc-6), 61.3 (C _Ga r-6), 60.9 (CGI _W -6), 60.5 (CG»I-6), 53.2 (C(O)OCHa), 51.9 (CGaiNAc-2), 51.5 (CNβuAe-5), 50.7 (CH ₂ N(N)CH), 38.9 (CNeuAc-3), 37.3, 36.7 (OCH ₂ CH ₂ NH and CH ₂ NHC(O)) 35.5 (CH ₂ CH ₂ C(N)CH), 29.7, 29.2, 29.12, 29.058, 29, 28.971, 28.6, 28.5, 25.9, 25.5 (all of CH ₂ CH ₂ CHz), 22.9 (CoaiNAc-CHs), 22.4 (CNθUAC- CH ₃ ), 21.3 (CH ₂ C(N)CH).

Divalent GMl dendrimer 5b

A solution of 5a (3.8 mg, 3.6 μmol), pentasaccharide Y (13 mg, 10.8 μmol), CuSO4.5H ₂ O (1.8 mg, 7.2 μmol) and sodium ascorbate (1.4 mg, 7.2 μmol) in H ₂ OZDMF (1/1, vZv, 2 mL) was heated under microwave irradiation at 80"C for 20 min. Analytical HPLC showed conversion of the dendrimer to a new peak. The reaction mixture was concentrated and subjected to preparative HPLC purification. The product was obtained after lyophilization as a white fluffy compound (9.8 mg, 78 %). η NMR (H ₂ OZD ₂ O, 9/1, vZv, 400 MHz): δ = ™C NMR (H ₂ OZD ₂ O, 9Zl, vZv, 100 MHz): δ = 175.5, 175.2, 174.9, 174.3, 172.1, 171.6 (COOH and C(O)NH), 168.5 (C(O)OCH ₃ ), 159.9 (Carom-3,5), 146.4 (Ctna∞ie-4), 132.0

(Caroπrl), 123.6 (Ctnazolβ-5), 108.8 (CHarom-2,6), 107.1 (CHarom-4), 105.1 (Coal-l), 102.9 (CGaI-I), 102.8 (CθalNAc-1), 102.4 (CGIUC-1), 101.8 (CtonM-2), 80.6 (CθalNAc-3), 79 (CGIUC-4),

77.4 (C _G ai-4), 75.2 (C _G ai-5), 75.1 (Cca∞-5), 74.8 (Coai-3), 74.717 (CGI _W -3), 74.698 (CGaiNA«-5), 74.4 (Coai-5), 73.4 (CNeuAa-6), 73.1 (CGIUC-2), 72.8 (CG _P I-3), 72.5, 71 ,70.9 (CH ₂ -OCGIU ₀ I), 70.3 (CNθUAC-8), 70.2 (OCH ₂ ), 70 (OCH ₂ ), 69.8 (OCH ₂ ), 69 (Coai-2 and CNeuA«-7), 68.9 (OCH ₂ ), 68.7 (OCH ₂ ), 68.4 (CoaiNAc-4), 68.2 (CNβuAc-4), 67.1 (OCH2CH2NH), 63.1 61.4 (CoaiNAc-6), 61.3 (Coar-6), 60.9 (Cαi«e-6), 60.5 (Cαai- 6), 53.2 (C(O)OCH ₃ ), 51.9 (CG*INAC-2), 51.5 (C _N euA _C -5), 50.7 (CH ₂ N(N)CH), 38.9 (C _Ne uA _* - 3), 37.3, 36.7 (OCH ₂ CH ₂ NH and CH ₂ NHC(O)) 35.5 (CH ₂ CH ₂ C(N)CH), 29.7, 29.2, 29.12, 29.058, 29, 28.971, 28.6, 28.5, 25.9, 25.5 (all Of CH ₂ CH ₂ CH ₂ ), 22.9 (CcaiNAc- CH ₃ ), 22.4 (CNeuAc-CHs), 21.3 (CH ₂ C(N)CH).

HRMS for Ci46H ₂ 44Niβθ76 (M, 3437.572) M + Na found 2314.415, calcd 2314.1385.

Divalent 6M2 dendrimer 5c

A solution of 5a (9.5 mg, 9.0 μmol), tetrasaccharide Y (28.8 mg, 28 μmol), CuSθ4.5H2θ (2.3 mg, 9 μmol) and sodium aseorbate (1.8 mg, 9.0 μmol) in H2O/DMP (1/1, v/v, 2 mL) was heated under microwave irradiation at 8O ⁰ C for 20 min. Analytical HPLC showed conversion of the dendrimer to a new peak. The reaction mixture was concentrated and subjected to preparative HPLC purification. The product was obtained after lyophilization as a white fluffy compound (19.0 mg, 68 %). ¹ H NMR (H2O/D2O, 9/1, v/v, 400 MHz): δ = ¹³ C NMR (H2O/D2O, 9/1, v/v, 100 MHz): 8 = 175.5, 175.2, 174.9, 174.3, 172.1, 171.6 (COOH and C(O)NH), 168.5

(C(O)OCH ₃ ), 159.9 (Carom-3,5), 146.4 (Ctriasole-4), 132.0 (Carom- 1), 123.5 (Ctriasole-5), 108.9 (CHarom-2,6), 107.2 (CHarom-4), 103.1 (CGaI- 1), 103.0 (CGa]NAo-I), 102.5 (Coiuc-

D.101.9 (CGIUO-4),77.5 (C _Ga i-4),75.1 (CGai-3),75.0 (CGI«C-5),74.8 (CGIUC- 3),74.4 (C _G aiNAc-5),73.5 (CGai-5),73.3 (C _Nβ «Ac-6),72.7 (CGIUO-2),71.8 (CoaiNAc-3), 71.0 (CH ₂ - OCαiucl), 70.5 (C _N euAc-8),70.3 (OCH ₂ ),, 70.0 (OCH ₂ ),, 69.8 (OCH ₂ ),69.1 (2C, C _Nβ uAc- 7+CGai-2), 68.9 (OCH ₂ ), 68.7 (OCH ₂ ), 68.5 (CαaiNAc-4), 68.3 (C _Ne »Ac-4), 67.2 (OCH ₂ CH ₂ NH), 63.4 (CNewk-9), 61.6 (CαaiNAo-6), 61.0 (CGI _UC -6), 60.6 (Coai-6), 53.3 (C(O)OCH ₃ ), 52.8 (CαaiNAc-2), 52.1 (CN_UAC-5), 50.7 (CH ₂ N(N)CH), 39.0 (CNθUAC-3), 37.4, 36.8 (OCH ₂ CH ₂ NH and CH ₂ NHC(O)), 35.6 (CH ₂ CH ₂ C(N)CH), 29.8, 29.3, 29.2, 29.1, 29.0, 28.9, 28.7, 28.5, 26,0, 25.5 (all of CH ₂ CH ₂ CH ₂ ),23.1 (CoaiNAc-CHs), 22.5 (C _N e«Ac- CH ₃ ), 21.4 (CH ₂ C(N)CH). HRMS for Ci ₃₄ H ₂₂₄ Ni ₆ O ₆₆ (M, 3113 ^~ 4664) M + Na found 2314.415, calcd 2314.1385.

Tetravalent GMl dendrimer 6b

A solution of 6a (4.1 mg, 1.8 μmol), pentasaccharide Y (13.0 mg, 10.8 μmol), CuSO ₄ .5H ₂ O (1.8 mg, 7.2 μmol) and sodium ascorbate (1.4 mg, 7.2 μmol) in H ₂ O/DMF (1/1, v/v, 2 mL) was heated under microwave irradiation at 80°C for 20 min. Analytical HPLC showed conversion of the dendrimer to a new peak. The reaction mixture was concentrated and subjected to preparative HPLC purification. The product was obtained after lyophilization as a white fluffy compound (9.7 mg, 76 %). m NMR (H2O/D2O, 9/1, v/v, 400 MHz): δ = ¹³ C NMR (H ₂ OZD ₂ O, 9/1, v/v, 100

MHz): δ = 175.5, 175.2, 174.9, 174.3, 172.1, 171.6 (COOH and C(O)NH),168.5

(C(O)OCH ₃ ), 159.9 (Carom-3,5), 146.4 (Ctriazole-4), 132.0 (Carom- 1), 123.6 (Ctriazolβ-5), 108.8

(CHarom-2,6), 107.1 (CHarom-4), 105.1 (CG ₈ I-I), 102.9 (CG ₈ I-I), 102.8 (CoaiNAc-l), 102.4 (CGIUC-1), 101.8 (CNeuAτ2), 80.6 (CGa]NAc-3), 79 (CGI _U C-4), 77.4 (Coai-4), 75.2 (Coai-5), 75.1 (Coiac-5), 74.8 (Cαai-3), 74.717 (CGIUC-3), 74.698 (CoaiNAc-5), 74.4 (Cαai-5), 73.4 (CNeuAq-6), 73.1 (CGIUC-2), 72.8 (C _G ai-3), 72.5, 71 ,70.9 (CH ₂ -OCGIUCI), 70.3 70.2 (OCH ₂ ), 70 (OCH ₂ ), 69.8 (OCH ₂ ), 69 (C _Ga i-2 and C _N euAc-7), 68.9 (OCH ₂ ), 68.7 (OCH ₂ ), 68.4 (CGSJNAC-4), 68.2 (CNβuAc-4), 67.1 (OCHzCHzNH), 63.1 (CκeuAc-9), 61.4 (CGaiNAe-6), 61.3 (Coai-6), 60.9 (Cαiuc-6), 60.5 (Coai-6), 53.2 (C(O)OCH ₃ ), 51.9 (CoaiNAc-2), 51.5 (CN _B UAC-5), 50.7 (CH ₂ N(N)CH), 38.9 (C _Nβ »Ac-3), 37.3, 36.7 (OCH ₂ CH ₂ NH and CH ₂ NHC(O)) 35.5 (CH ₂ CH ₂ C(N)CH), 29.7, 29.2, 29.12, 29.058, 29, 28.9 (all Of CH ₂ CH ₂ CH ₂ ), 22.9

(CoalNAα-CHs), 22.4 (CNeuAq-CHϋ), 21.3 (CH ₂ C(N)CH)-

HRMS for C30 ₂ H ₄ 9 ₈ N34θi54 (M, 7065.2182) M + Na found 2314.415, calcd 23141385.

Tetravalent GM2 dendrimer 6c

A solution of 6a (7.4 mg, 3.2 μmol), tetrasaccharide Y (20 mg, 19.4 μmol), CuSO4.5H ₂ O (3.2 mg, 12.8 μmol) and sodium ascorbate (2.5 mg, 12.8 μmol) in H ₂ O/DMF (1/1, v/v, 2 mL) was heated under microwave irradiation at 80°C for 20 min. Analytical HPLC showed conversion of the dendrimer to a new peak. The reaction mixture was concentrated and subjected to preparative HPLC purification. The product was obtained after lyophilization as a white fluffy compound (7.6 mg, 37 %). ¹ H NMR (H ₂ O/D ₂ Q, 9/1, v/v, 400 MHz): δ = ¹³ C NMR (H ₂ O/D ₂ O, 9/1, v/v, 100 MHz): 6 = 175,4, 175,2, 174,6, 174.0, 172.0, 171,4 (COOH and C(O)NH),168.9 (C(O)OCH ₃ ),160 (Carom-3,5), 146.4 (Ctriazole-4), 131,8 (Carom-1),123,5 (Ctriazole-5), 115,2 (CHarom-2,6), 106,8 (CHarom-4), 103.1 (Cθal-1), 103 (CoalNAc-l), 102.6 (CGIUC-1), 101.6

(CweuAc-2), 79 (CGIUC-4), 77.3 (Coai-4), 75.1 (Ccai-3), 75 (Cαiuc-5), 74.9 (CGIUC-3), 74.4 (CcaiNAc-5), 73.5 (Coai-5), 73.3 (CNeuAo-6), 72.6 (CGIUC-2), 71.8 (CαaiNAo-3), 70.9 (CH ₂ - OCoiucl), 70.4 (CNβuAc-8), 70.3 (OCH ₂ ), 70 (OCH ₂ ), 69.8 (OCH ₂ ), ? (Cαai-2), 69 (C _NeU Ao- 7), 68.9 (OCH ₂ ), 68.7 (OCH ₂ ), 68.5 (CoaiNAc-4), 68.3 (C _N βuAc-4), 67 (OCH ₂ CH ₂ NH), 63.4

(CNeuAc-9), 61.6 (CθalNAc-6), 61 (Cffluc-6), 60.6 (CGal-6), 53.2 (C(O)OCH ₃ ), 52.9 (CGalNAc-2),

52.1 (CNθUAC-5), 50.8 (CH ₂ N(N)CH), 38.9 (C _N euAc-3), 37.4, 36.9, 36.8 (OCH ₂ CH ₂ NH and

CH ₂ NHC(O)), 35.5 (CH ₂ CH ₂ C(N)CH), 29.9, 29.4, 29.3, 29.2, 29.1, 28.8, 26.1, 25.7 (all Of CH ₂ CH ₂ CH ₂ ), 23.1 (CGaINA ₀ -CH ₃ ), 22.5 (CNeuAc-CHs), 21.4 (CH ₂ C(N)CH).

HRMS for C278H458N34O134 (M, 6417.0069) M + Na found 2314.415, calcd 2314.1385.

GM-I Elisa Experiment

A standard 96 wells plate was coated with a 100 μL solution of GMl (2μg/mL)

(Sigma-Aldrieh) in PBS at 37 °C for 16 h. Unattached ganglioside was removed by washing with PBS (2 x 450 μL) after which remaining binding sites were blocked by incubation with 100 μL 1% (w/v) BSA in PBS for 30 min at 37 "C. After blocking the plate was washed with PBS (3 x 450 μL). Samples of toxin-peroxidase conjugate (Sigma-Aldrich) and inhibitor in 0.1% BSA, 0.05 % Tween 20 in PBS were incubated at rt for 2 h and were then transferred to the coated plate. After 30 min incubation the solution was removed followed by washing steps with 0.1 % BSA, 0.05 % Tween- 20 in PBS (3 x 450 μL). To identify toxin binding to surface bound GMl the wells were treated with a freshly prepared o-phenylenediamine (OPD) solution (100 μL) (25 mg OPD.2HC1, 7.5 mL 0.1M citric acid, 7.5 mL 0.1M sodium citrate and 6 μL of a 30% H ₂ O ₂ solution) and the colour forming reaction was quenched after 15 min with H2SO4 (50 μL) (2.5 M). The absorbance at 490 nm was measured by a μQuant plate reader.

Monolithic carriers. In recent years, monolithic (continuous bed) stationary phases have found wide application, e.g. in analytical columns, capillaries and channels of microfluidic chips. Affinity chromatographic methods using monolithic matrixes have been reported. It has been demonstrated that affinity chromatography using monolithic beds has a high potential for the analysis of biological macromolecules because the macroporous structure of the bed improves mass transport, allows a high mobile phase velocity without losing resolution and they are easily prepared. So far only few publications have appeared [Bedair, M. and El Rassi, Z., J. Chromatogr. A, 1044 (2004) 177-186;

Bedair, M. and El Rassi, Z., Affinity chromatography with monolithic capillary columns - J. Chromatogr. A, 1079 (2005) 236-245, Bedair and El Rassi developed monolithic capillary columns for affinity-based separations of lectins in both nano- LC and CEC modes. The analysis of mannan/mannose-binding proteins in rabbit serum was performed successfully with these affinity monoliths. In general, for preparation of a suitable affinity sorbent for biomolecules, an affinity ligand must be immobilized on the surface of the chromatographic material. Bedair and El Rassi incorporated glycidyl ether in their monolith. Subsequently the mannan was immobilized directly or after attachment of a spacer by a nucleophilic attack on the epoxide ring under basic conditions

Preparation of monolithic carriers

3-(Trimethoxysilyl)propyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), ammonium sulfate, (+)-N _J N'-diallyltartardiamide (DATD), piperazine diacrylamide (PDA), 2,2'-azobis(2-methylpropion amidine) dihydrochloride (AMPA), Concanavalin A (Con A), Arachis hypogaea (PNA), Lens culinaris (LCA) and FITC-labeled Con A were purchased from Sigma (The Netherlands). Polystyrene standards (Mr 707, 1920, 3460, 5610, 12,500, 27,500, 51,500, 125,000, 271,000, 524,000, 864,000, 1,530,000) were from Polymer Standards Service (Mainz, Germany). Fused-silica capillaries with an internal diameter (Ld.) of 75 μm and an outer diameter (o.d.) of 375 μm were from Polymiero Technologies (Phoenix, AZ, USA). THF and benzene were HPLC grade solvents from Sigma (The Netherlands).

Surface modification of fused-silica capillary inner surface (capillary pretreatment) was performed as described in [Ericson, C, Liao, J.L., Nakazato, K, and Hjerten, S., J. Chromatogr. A, 767 (1997) 33-41.] with slight modification: acetone (20 min), distilled water (20 min), 0.1 M HCl (20 min), distilled water (20 min), 0.1 M NaOH (1 hr), distilled water (20 min) and acetone (20 min), followed by drying with nitrogen. Treatment of hydroxyl terminated capillaries by filling them with a 30% (v/v) solution of 3-(trimethoxysilyl)propyl methacrylate in acetone and by closing both ends for 12 h resulted in acrylate groups on the internal surface, which can co- polymerize with the monomers. In situ polymerization was accomplished essentially as described in [Kornysova, O., Jarmalaviciene, R. and Maruska, A., , Electrophoresis, 25 (2004) 2825-2829.] with slight modification. Reaction mixture: 30

μL of HEMA, 20 mg of DATD, 12.5 mg Of(NHi) ₂ SO ₄ and 250 μL of PDA buffer solution (160 mg of PDA was dissolved in 2 mL of 50 mM sodium phosphate buffer, pH 7.0) were taken in an Eppendorf tube and mixed well. The reaction mixture was then degassed by vacuum for 5 min. 10 μL of 10% (w/v) aqueous AMPA solution was added to the above reaction mixture and mixed well. The resulting polymerization mixture was taken in a disposable syringe and injected during 5 min into the capillary by using a syringe pump at a flow rate of 1 μL/min. The capillary ends were closed with a GC septum and placed in an oven CT = 65 "C) for 12 h. The resulting monolithic column was washed with distilled water and acetonitrile successively. in situ preparation of GMl & GM2 monolith beds

Surface modification of fused-silica capillary (150, 180, 250 μm i.d) inner surface was performed as described in [Ericson, C, Liao, J. L., Nakazato, K, Hjerten, S., J. Chromatogr. A, 767, 1997, 33-41] with slight modification: acetone (20 min), distilled water (20 min), 0.1 M HCl (20 min), distilled water (20 min), 1 M NaOH (1 hr), distilled water (20 min) and acetone (20 min). Treatment of hydroxyl terminated capillaries with a 30% (v/v) solution of 3-(trimethoxysilyl)propyl methacrylate in acetone by closing both ends for 12 h resulted in acrylate groups on the surface, which can co-polymerize with the monomers [Kornysova, O., Owens, P. K., Maruska, A., Electrophoresis, 22, 2001, 3335-3338], The monolith solution was prepared with slight modification as described in [Kishore K. R. Tetala, B. Chen, G. M. Visser, A. Maruska, O. Kornysova, T. A. van Beek, E. J. R. Sudholter., J.Biochem. Biophys. Methods, 70, 2007, 63-69.]. Monomer HEMA (30 μL) was taken in an eppendorf tube along with ammonium sulfate (8 mg), DATD (20 mg) and PDA (20 mg in 250 μL phosphate buffer). To this was added a solution "alkene terminated- Cll-spacer- GMl or GM2 and mixed well followed by de-aeration for a period of 5 min. At this point of time, the initiator AMPA (10 μL, 10 % v/v in water) was added to the above solution and sucked into the acrylate terminated capillary. Capillary ends were sealed with GC septum and placed in an oven (T = 65 ⁰ C) for 12 h which resulted in an affinity monolith bed, which was washed with water and acetonitrile:water (50:50) each for 2h (2 μL/min) respectively.

• Preparation of GMl or GM2 solution: GMl (3 mg) & GM2 (4.3 mg) were dissolved in 30 μL of methanol.

Definition of target human diseases

Gangliosides play a crucial role as target molecules for bacterial toxins and serum antibodies in a wide range of human infectious and auto-immune diseases. The specificity and pathogenic effects of these bacterial toxins and serum antibodies has been identified in most of these diseases.

Toxin-related diseases

Some bacteria produce toxins that are highly toxic proteins that may contribute significantly to the severity of certain infectious diseases. Some of these toxins bind to host gangliosides to exert their effects. The aimed toxins in this invention are cholera toxin binding to GMl in patients with cholera, enterotoxm binding to GMl in patient with travellers diarrhoea, diphtheria toxin binding to disialic gangliosides (including GDIb, GD2 and GD3) in patients with diphtheria, tetanus toxin binding to disialic gangliosides (including GDIb, GD2 and GD3) in patients with tetanus, and any other toxins binding to ganglioside or glycoconjugates in other patients with infectious diseases.

Auto-antibody-related diseases

The invention provides treatment and diagnostic methods and means relating to human diseases associated with aύto-antibodies to gangliosides and other glycoeonjugates. Most are post-infectious or autoimmune neural diseases, including Guillain-Barre syndrome (GBS) and all its variants, Miller Fisher syndrome (MFS), acute ophthalmoplegia, acute bulbar palsy, mononeuritis multiplex, (polyneuritis cranialis, acute diplegia facialis, acute brachealis palsy, acute autonomic neuropathy, acute sensory neuropathy, chronic inflammatory demyelinating neuropathy (CIDP), multifocal motor neuropathy (MMN), paraprotein-related polyneuropathy (PP-PNP), other forms of chronic ataxic neuropathy with ophthalmoplegia M-protein ataxia and disialic ganglioside antibodies to (CANOMAD), and all other forms of axonal or demyelinating immune-mediated neuropathies, acute ataxia (being cerebellar and proprioceptic or gnostic),

Bickerstaff encephalitis, acute demyelinating encephalomyelitis (ADBM) and all other forms of immune-mediated encephalitis and myelitis.

Application of high binding oligosaccharide compositions in human diseases The high binding oligosaccharide compounds and compositions can be used in the diagnosis, prevention and treatment of human diseases associated with toxins and antibodies binding to gangliosides and glycoconjugates.

Diagnostic usage Toxin-related diseases

The invention provides the detection of any toxin that specifically binds to oligosaccharide structures, binding oligosaccharides as such, glycolipids and glycoproteins. The high binding oligosaccharide compounds and compositions have a high affinity for toxins, including the cholera toxin. This toxin binds specifically to the ganglioside GMl and binding is crucial to exert its pathogenic effects leading to the clinical manifestations of cholera. Experiments have shown that Luminex beads covalently coated with GMl-ClI alkyl specifically captured the cholera toxin B- subunit. The compounds covalently bound to solid carriers such as beads, monolithic beds and ELISA plates can therefore be used as highly sensitive and highly specific assays to detect bacterial toxins. These techniques can be used in the diagnosis or exclusion of these infectious diseases by demonstrating these toxins in serum, sputum and faeces samples from suspected patients. After diagnosis and treatment of these patients the changes in levels of these toxins can also be τised to monitor and evaluate the effects of therapy.

Auto- antibody-related diseases

The detection of antibodies to gangliosides using a compound or composition as provided herein has several applications in patients with related neurological diseases. Detection of these antibodies by the compositions can be used to (1)

diagnose or exclude one of the aforementioned auto-antibody related diseases, (2) identify patients with a distinct pathogenic and/or clinical subtype of one of these diseases, (3) identify and predict in these patients a distinet type of clinical course (good or poor prognosis after certain types of treatment), (4) identify patients who require distinct forms of specific and/or additional treatments, and (5) monitor the effects of treatment based on the changes in antibody titre and specificity. An example of these applications is the detection of IgG antibodies to the ganglioside GMl in the serum from patients with neuropathies. The presence of these antibodies in serum was found to be highly associated with (ad. 1) the diagnosis GBS, (ad. 2) a distinct pathogenic and clinical subtype of GBS caused by an antecedent

Campylobacter jejuni infection and with severe and specific involvement of motor nerve fibres (and not sensory or cranial nerve involvement), (ad. 3) a poor recovery after treatment with plasmapheresis, (ad. 4) a clinical course that will be improved considerably after treatment with a pure motor variant of the disease GB with poor outcome, patients show a much better outcome after treatment with intravenous immunoglobulins (TVIg), and (ad. 5) a prolonged, chronic or relapsing-remitting form of GBS in those patients in whom the titre does not decrease after treatment. Another example are serum IgG antibodies to the ganglioside GQIb which are highly associated with (ad. 1) the diagnosis of MFS, (ad. 2) a distinct pathogenic and clinical subtype of patients with GBS caused by an antecedent Campylobacter jejuni infection and with severe involvement of oculomotor nerve fibres, (ad, 3) a good recovery after treatment with plasmapheresis, (ad. 4) a possibly better recovery after treatment with FVIg, and (ad. 5) a prolonged, chronic or relapsing-remitting form of neuropathy in those patients in whom the titre does not decrease after treatment. Experiments demonstrated that the compounds according to the invention can be bound covalently to solid carriers including beads, monolithic beds and ELISA plates, in which they can be used to demonstrate anti-ganglioside antibodies in the serum from patients with neuropathy associated with the presence of these antibodies. We found that Luminex beads covalently coated with GMl-CIl alkyl bind with high sensitivity and specificity serum antibodies to GMl in a patient with GBS. Serum from a healthy control without antibodies to GMl in routine ELISA was negative in the bead-based assay. The ratio between this positive and negative

serum was much higher in this assay than in routine GMl ELJSA, indicating that the signal-noise ratio in this assay is significantly better than in routine EUSA. Similar successful results were obtained when ELISA plates were covalently bound with this GMl-CIl alkyl composition, ELISA plates coated with these compositions detected serum anti-GMl antibodies in 2 patients with GBS, but not in serum from 2 healthy blood donors, Again the ratio between the positive and the negative serum samples was better in this assay than in routine ELISA. The ELISA plates were also covalently coated with GMl-dimers, and the usage of these compositions further improved the performance of the assay. In conclusion, these data show that the synthetic compounds are highly effective for the detection of both bacterial toxins and auto-antibodies to gangliosides, a feature that can be used to improve diagnosis in patients with the aforementioned diseases.

Therapeutic usage of high binding oligosaccharide compounds Toxin-related diseases

In order to evaluate the potency of the inhibitors an ELISA type assay was used. In this assay, wells of a 96-well plate were coated with GMl ganglioside, and after blocking with BSA, horseradish peroxidase (HRP)-conjugated CTBs was allowed to bind to the surface in the presence or absence of inhibitors. In this assay the monovalent GMlos derivative 2 exhibited an ICBO in the micromolar range (19 μM, Table 1). The divalent 5b was subsequently measured, and found to have an ICso that was almost 4 orders of magnitude lower, i.e 2.0 nM. The tetravalent 6b gave an ICBO which was an additional order of magnitude lower (0.23 nM). Finally for the octavalent 7b the ICso was lower still at 50 pM. Control experiments with non- functional dendrimers (4a, 5a and 6a) showed no measurable inhibition for all concentrations tested (up to 100 μM). The prepared compounds exhibited unprecedented affinities. For tetravalent 7b, each of the GMlos moieties bound 47, 500-fold stronger than monovalent 2. Experiments with GM2os were performed to confirm the multivalency effects with a similar but weaker ligand. Table 1. Inhibitory potency of the inhibitor

Compound valency ICB ₀ (M)M rel. pot.P> ^] Hill

(per sugar) coefficient

GMlos derivatives

2 1 1.9 (±0.6) x 10" ⁵ KD 0.5

4b 1 7 (±3) x 10- ⁶ 2,7 (2.7) 0.5

5b 2 2 (±1) x 10- ⁹ 9,500 (4,750) 1.0

6b 4 2.3 (±0.7) x 10 ¹⁰ 83,000 (20,750) 3.0

7b 8 5 (±1) x 10- ¹¹ 380,000 (47,500) 1.7

GM2os derivatives

5c 2 2 (±1) x lO- ³ 1 n.d.

6c 4 1.05 (±0.02) x lO ⁷ 19,000 2.8

7c 8 4 (±1) x lO ⁷ 5,000 1.4

[a] Determined in an ELISA experiment with 0.43 nM CTBs-HRP and wells coated with 0.2 μg GMl. [b] relative potency of compounds 2 and 5c were taken as 1.

Auto-antibody-related diseases

Antibodies to various gangliosides are highly neurotoxic and are considered to be the main cause of neurological deficits in the aforementioned diseases. For none of these diseases at present a specific form of treatment exists. Most of these patients are treated with apecific forms of treatment such as plasmapheresis and IVIg, which have only a modest therapeutic effect. Moreover, a major disadvantage of plasmapheresis is that it needs a skilled centre and facilities and may lead to severe cardiovascular complications. Major disadvantages of TVIg are the expenses, the shortage of material, and the possible contaminations in this biological product. The invention intends to deliver a new and rational form of treatment for these diseases. The invention can be applied in several ways to selectively neutralizes the pathogenic effects of the anti-ganglioside antibodies in these diseases.

Depletion of anti-ganglioside antibodies from the blood by plasmapheresis and selective immunoadsorption.

For immunoadsorption, columns are used containing monoliths covalently coated with single or various synthetic ganglioside mimics. The invention provides an highly effective and selective immunoadsorption because of (a) the use of authentic ganglioside oligosaccharide sequences as the most physiological and relevant target, (b) the use of an extremely high density of these oligosaccharide sequences required for multivalent binding and for the conformational epitopes mimicking those in the physiological clusters of gangliosides in lipid rafts at the neural membranes, (c) the use of multivalent dendrimeric scaffold further increasing the epitope density, (d) the use of elongated spacer arms and (e) the use of the monolithic composites resulting in a strong increase of the efficient surface for antϊbody-ligand interaction. The covalent binding of the synthetic ganglioside mimics to the monolith prevents the detachment of these mimics from the column during plasmapheresis and the contamination of plasma returned to the patient. Because of this covalent binding the columns may be reused after regeneration.

In experimental settings it was shown that dendrimers coated with the oligosaccharide structures effectively bind with serum anti-ganglioside antibodies from neuropathy patients. This was illustrated by an experiment in which a solution with tetravalent GMl dendrimers was incubated with the serum from a GBS patient with anti-GMl antibodies. After incubation no residual anti-GMl antibodies could be detected in this serum using routine GMl BLISA. Apparently, all anti-GMl antibodies in this serum were bound to the GMl dendrimers and neutralized. The fact that this binding was not reversed in the proximity of GMl in the routine ELISA indicates that the avidity of the antibodies was higher to the GMl dendrimers than to GMl. This high avidity was predicted based on the structure of the GMl dendrimer. Antibodies to the ganglioside GQIb from another patient with GBS were not neutralized by these GMl dendrimers, illustrating the specificity of the adsorption.

The unprecedented high efficiency in depletion of anti-ganglioside antibodies was achieved by using monolithic immunoadsorption columns covalently coated with the compositions. In a series of experiments serum from various patients with GBS and anti-ganglioside antibodies was added to GMl and GM2 coated monoliths to deplete

the serum antibody activity. Serum from one patient with high titres of IgM antibodies to GM2 was applied to a monolith covalently coated with GM2. Because of the flow of the serum of 1 μl/min in a column with a volume of less than 1.7 μl, the antibodies only had 1.7 minutes to incubate with the bound synthetic ganglioside mimics. Despite this very short incubation time 98% of anti-GMl activity was depleted by this protocol. Staining with FITC conjugated anti-human IgM demonstrated the presence of these bound serum antibodies in the column. Usage of monolithic columns not coated with compositions showed no serum depletion and no binding of serum antibodies in the column. This control experiment demonstrated the selectivity of the binding of serum anti-GM2 antibodies to the synthetic GM2 mimic and not to the monolith itself. Serum from another patient with GBS and high titres of IgM antibodies to GMl applied to a monolith column coated with GMl also showed no depletion of antibodies. However, when this serum was applied to a monolith column covalently coated with GMl more than 95% of antibody activity was depleted from the serum. This experiment further demonstrates the highly selective immunoadsorption of anti-ganglioside antibodies from the serum of patients with autoimmune neuropathy. Alternatively monolithic beads, prepared from grinding a polymerised monolithic mass, and containing synthetic ganglioside mimics, can be administered orally as a pharmaceutical composition to deplete anti- gangloside antibodies in the intestinal tract for the acute treatment of a subject suffering of an intoxication with a bacterial toxin, such as cholera toxin. Alternatively, immunoadsorption columns are used containing beads covalently and densely coated with single or various synthetic ganglioside mimics which have similar advantages as the coated monoliths (although the effective surface for antibody-ligand interaction may be less than in coated monoliths). Monolithic columns, containing synthetic ganglioside mimics according to the invention can be used for sensitive diagnostic purposes, for instance for the detection of GBS antibodies. For such purposes the monolithic affinity column should be connected to a surface plasmon resonance (SPR) device coated with ganglioside mimics or connected to an electrospray ionization mass spectrometer equipped with a nanospray interface capable of specifically detecting antibody proteins or toxins. For this purpose, serum or another biological fluid containing pathogenic antibodies, for

example IgM antibodies against GM2, is infused into the monolithic affinity column. After selective binding of said antibodies, the column is washed and then the antibodies are eluted with a suitable medium. The eluted antibodies are directed in high concentration and pure form to the SPE device. SPE is a powerful technique to measure biomolecular interactions in real-time and in a label-free environment. While the ligand is immobilized on a gold sensor surface, the proteins are free in solution and passed over the surface. Association and dissociation on the gold surface with immobilised ligands can be measured sensitively and rapidly through minute differences in refractive index. As an alternative detection device ESI-MS can be used. Such a combination of affinity chromatography and selective detection, enables fast and sensitive diagnosis of diseases at an early stage and replaces more elaborate ELJSA methods.