In the present years manufacture and commercialization of complex carbohydrates including secreted oligosaccharides have increased significantly due to their roles in numerous biological processes occurring in living organisms. Secreted oligosaccharides such as human milk oligosaccharides (HMOs) are carbohydrates which have gained much interest in recent years and are becoming important commercial targets for nutrition and therapeutic industries. In particular the synthesis of these HMOs has increased significantly due to the role of HMOs in numerous biological processes occurring in humans. The great importance of HMOs is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities. Human milk

oligosaccharides are found to act as prebiotics in the human intestinal system helping to develop and maintain the intestinal flora. Furthermore they have also proved to be anti- inflammatory, and therefore these compounds are attractive components in the nutritional industry for the production of, for example, infant formulas, infant cereals, clinical infant nutritional products, toddler formulas, or as dietary supplements or health functional food for children, adults, elderly or lactating women, both as synthetically composed and naturally occurring compounds and salts thereof. Likewise, the compounds are also of interest in the medicinal industry for the production of therapeutics due to their prognostic use as immunomodulators. However, the syntheses and purification of these oligosaccharides and their intermediates remained a challenging task for science.

To date, access to large volumes of human milk oligosaccharides has not been possible by using isolation, biotechnology and synthetic methodologies.

The availability of naturally occurring sialylated human milk oligosaccharides is limited from natural sources. Mature human milk is the natural milk source that contains the highest concentrations of milk oligosaccharides (12-14 g/1), other milk sources are cow's milk (0.01 g/1), goat's milk and milk from other mammals. Approximately 200 HMOs have been detected from human milk by means of combination of techniques including microchip liquid chromatography mass spectrometry (HPLC Chip/MS) and matrix-assisted laser

desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI- FT ICR MS) (Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which to date at least 115 oligosaccharides have been structurally determined (Urashima et al.: Milk

Oligosaccharides, Nova Medical Books, NY, 201 1). These human milk oligosaccharides can be grouped into 13 core units (Table 1). Due to the large number of similar HMOs and their low concentrations in mammalian milk, isolation of HMOs is a difficult task even in milligram quantities. To date only analytical HPLC methodologies have been developed for the isolation of some HMOs from natural sources. It is therefore difficult to provide suitable HMO replacements in foods, particularly in infant formulae which display at least part of the entire spectrum of HMOs.

RECTIFIED SHEET (RULE 91)

ISA/EP Gal 1 -3GlcNAcP 1 -3 [Gaip 1 -3GlcNAcp 1 -3 (Galp 1 - 4GlcNAcp 1 -6)Gai l-4GlcNAc 1 -6]Galp 1 -4Glc

Table 1: 13 different core structures of human milk oligosaccharides

The chemical synthesis of human milk oligosaccharides is one of the most challenging fields of carbohydrate chemistry due to the nature of the glycosyl donors, especially in the case of the manufacture of sialyl HMOs. Careful selection of a glycosyl donor-acceptor match, kinetic and solvent effects is always needed in synthetic methodologies, which in fact comprise numerous reaction steps, protecting group manipulations and chromatographic purification, poor yields, and provide only milligram quantities of HMOs. Thus, these methods do not offer attractive techniques for large scale preparation.

In case of enzymatic production of HMOs, glycosyltransferases and glycosidases are the preferred enzymes used. These complex enzymatic systems represent very expensive methodologies for scale up productions. The use of glycosidases is often characterised by poor yield and moderate regio- and/or stereoselectivity which may cause difficult purification problems. This prevents its use in industrial scale technology developments.

Glycosyltransferases require the presence of nucleotide type glycosyl donors, the availability of which is rather limited,

Some biotechnological methodologies are also described using genetically modified bacteria, yeast or other microorganisms. Such methodologies have serious drawbacks in regulatory processes.

In summary, isolation technologies have never been able to provide large quantities of human milk oligosaccharides due to the large number of oligosaccharides present in human milk. Additionally, the presence of regioisomers characterized by extremely similar structures further made separation technologies unsuccessful. Enzymatic methodologies suffer from such problems as the low availability of enzymes, extremely high sugar nucleotide donor prices and regulatory difficulties due to the use of enzymes produced in genetically modified organisms. The preparation of human milk oligosaccharides via biotechnology has huge regulatory obstacles due to the potential formation of several unnatural glycosylation products. To date, the chemical methods developed for the synthesis of HMOs have several drawbacks which prevented the preparation of even multigram quantities. The most severe drawback of chemical approaches is the lack of design for crystalline intermediates to facilitate low cost purification methodologies and to enhance scale-up opportunities.

RECTIFIED SHEET (RULE 91)

ISA/EP There is still a need for novel methodologies which can simplify preparation and overcome or avoid purification problems encountered in prior art methods. In addition, there is an urgent need in the art for providing complex oligosaccharides and mixtures thereof, which resemble as good as possible or even imitate the variety of complex oligosaccharides in human milk.

The present invention provides a general derivatization method, the result of which are HMO derivatives having beneficial features for overcoming isolation and/or purification problems characterized by the prior art.

Some individual HMO derivatives obtainable by the inventive method to be specified later are known in the prior art: Ι-Ο-β-benzyl-LNnT (Ponpipom et al. Tetrahedron Lett. 20, 1717 (1978)), l-0-p-(4-hydroxymethylbenzyl)-LNnT (Yan et al. Carbohydr. Res. 328, 3 (2000)), Ι-Ο-β-benzyl-LNT (Malleron et al. Carbohydr. Res. 341, 29 (2006), Liu et al.

Bioorg. Med. Chem. 17, 4910 (2009)), l-0-P-benzyl-6'-0-sialyl-lactose Na salt (Rencurosi et al. Carbohydr. Res. 337, 473 (2002)), l-0-p-benzyl-3'-0-sialyl-lactose Na salt (Rencurosi et al. Carbohydr. Res. 337, 473 (2002), WO 96/32492 A2), l-0-p-(4,5-dimethoxy-2-nitro)- benzyl-3 '-0-sialyl-lactose Na salt (Cohen et al. J. Org. Chem. 65, 6145 (2000)).

Whatever route is taken to synthesise or isolate an oligosaccharide, the final target unprotected oligosaccharide is soluble only in water, which presents challenges for the later steps of the synthesis or for isolation/separati on/purification methods. Organic solvents commonly used in synthetic manufacturing processes are not suitable for the reactions of the very final stages of the oligosaccharide synthesis. Additionally, the presence of numbers of similar compounds either in natural source or obtained in enzyme catalyzed syntheses likewise demands powerful chromatographic system having polar aqueous milieu which is difficult to find.

The present invention provides methodology suitable for derivatizing HMOs. The invention is based upon the formation of anomeric O-benzyl/substituted O-benzyl derivatives in a selective anomeric alkylation reaction.

Accordingly, the present invention relates to a method for purifying, separating and/or isolating an oligosaccharide of general formula 1 or a salt thereof

general formula 1 wherein Ri is fucosyl or H,

R ₂ is fucosyl or H,

R ₃ is selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R ₄ is selected from H, or sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

wherein at least one of the Ri, R ₂ , R ₃ or R ₄ groups differs from H,

comprising the steps: a) one or more compounds of general formula 1 is/are subjected to an anomeric O-alkylation reaction in the presence of R-X to yield a mixture comprising one or more compounds of general formula 2 or salts thereof

general formula 2 wherein X is a leaving group such as halogen, alkyl- or arylsulfonyloxy, R is a group removable by hydrogenolysis, and Ri, R ₂ , R ₃ and R ₄ are as defined above, and wherein at least one of the Ri, R ₂ , R ₃ or R ₄ groups differs from H, b) the mixture comprising one or more compounds of general formula 2 obtained in step a) is subjected to chromatography and/or crystallization to give one or more individual compounds of general formula 2 each in substantially pure form,

c) an individual compound of general formula 2 in substantially pure form obtained in step b) is subjected to catalytic hydrogenolysis to yield a compound of general formula 1.

Throughout the present application the term protecting group that is removable by hydrogenolysis" or„group removable by hydrogenolysis" refers to groups whose C-0 bond to the 1 -oxygen can be cleaved by addition of hydrogen in the presence of catalytic amounts of palladium, Raney nickel or another appropriate metal catalyst known for use in

hydrogenolysis, resulting in the regeneration of the OH group. Such protecting groups are well known to the skilled man and are discussed in Protective Groups in Organic Synthesis, PGM Wuts and TW Greene, John Wiley & Sons 2007. Suitable protecting groups include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl or

triphenylmethyl (trityl) groups, each of which may be optionally substituted by one or more groups selected from: alkyl, alkoxy, phenyl, amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, halogenalkyl or halogen. Preferably, such substitution, if present, is on the aromatic ring(s). Particularly preferred protecting groups are benzyl or 1- or 2-naphthylmethyl groups optionally substituted with one or more groups selected from phenyl, alkyl or halogen. More preferably, the protecting group is selected from unsubstituted benzyl, unsubstituted 1- naphthylmethyl, unsubstituted 2-naphthylmethyl, 4-chlorobenzyl, 3-phenylbenzyl, 4- methylbenzyl and 4-nitrobenzyl.

„Compound in substantially pure form", when referring to a compound of general formula 2, means that the compound contains less than 5 w/w% of impurities, preferably less than 3 w/w% of impurities, more preferably less than 1 w/w% of impurities, most preferably less than 0.5 w/w% of impurities, in particular less than 0.1 w/w% of impurities, wherein "impurities" refers to any physical entity different to that compound, such as unreacted intermediate(s) remaining from the synthesis of the compound of general formula 2, byproducts), degradation product(s), inorganic salt(s) and/or other contaminations other than organic solvent(s) and/or water. Throughout the present description, the term "alkyl" means a linear or branched chain saturated hydrocarbon group with 1-6 carbon atoms, such as methyl, ethyl, ^-propyl, / ^' -propyl, «-butyl, / ^' -butyl, s-butyl, t-butyl, «-hexyl, etc.

The term "aryl" refers to a homoaromatic group such as phenyl or naphthyl.

In the present description, the term "acyl" represents an R'-C(=0)-group, wherein R' may be H, alkyl (see above) or aryl (see above), such as formyl, acetyl, propionyl, butyryl, pivaloyl, benzoyl, etc. The alkyl or aryl residue may either be unsubstituted or may be substituted with one or more groups selected from alkyl (only for aryl residues), halogen, nitro, aryl, alkoxy, amino, alkylamino, dialkylamino, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, halogenalkyl or hydroxyalkyl, giving rise to acyl groups such as chloroacetyl, trichloroacetyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4- phenylbenzoyl, 4-benzamidobenzoyl, 4-(phenylcarbamoyl)-benzoyl etc.

The term "alkyloxy" or "alkoxy" means an alkyl group (see above) attached to the parent molecular moiety through an oxygen atom, such as methoxy, ethoxy, t-butoxy, etc.

"Halogen" means fluoro, chloro, bromo or iodo.

"Amino" refers to a -NH ₂ group.

"Alkylamino" means an alkyl group (see above) attached to the parent molecular moiety through an - H-group, such as methylamino, ethylamino, etc.

"Dialkylamino" means two alkyl groups (see above), either identical or different ones, attached to the parent molecular moiety through a nitrogen atom, such as dimethylamino, diethylamino, etc.

"Acylamino" refers to an acyl group (see above) attached to the parent molecular moiety through an -NH-group, such as acetylamino (acetamido), benzoylamino (benzamido), etc.

"Carboxyl" denotes an -COOH group.

"Alkyloxycarbonyl" means an alkyloxy group (see above) attached to the parent molecular moiety through a -C(=0)-group, such as methoxycarbonyl, t-butoxycarbonyl, etc.

"Carbamoyl" is an H ₂ N-C(=0)-group.

'W-Alkylcarbamoyl" means an alkyl group (see above) attached to the parent molecular moiety through a -HN-C(=0)-group, such as N-methylcarbamoyl, etc. 'W,N-Dialkylcarbamoyl" means two alkyl groups (see above), either identical or different ones, attached to the parent molecular moiety through a >N-C(=0)-group, such as N,N-methylcarbamoyl, etc.

In the present description the term "salt" in connection with compounds of general formulae 1 and 2, which contain at least one sialyl residue, means an associated ion pair consisting of the negatively charged acid residue and one or more cations in any

stoichiometric proportion. Cations, as used in the present context are atoms or molecules with positive charge. The cation may be inorganic as well as organic cation. Preferred inorganic cations are ammonium ion, alkali metal, alkali earth metal and transition metal ions, more preferably Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ , Zn ²⁺ , Mn ²⁺ and Cu ²⁺ , most preferably K ⁺ , Ca ²⁺ , Mg , Ba , Fe and Zn . Basic organic compounds in positively charged form may be relevant organic cations. Such preferred positively charged counterparts are diethyl amine, triethyl amine, diisopropyl ethyl amine, ethanolamine, diethanolamine, triethanolamine, imidazol, piperidine, piperazine, morpholin, benzyl amine, ethylene diamine, meglumin, pyrrolidine, choline, tris-(hydroxymethyl)-methyl amine, N-(2-hydroxyethyl)-pyrrolidine, N- (2-hydroxyethyl)-piperidine, N-(2-hydroxyethyl)-piperazine, N-(2-hydroxyethyl)-morpholine, L-arginine, L-lysine, oligopeptides having L-arginine or L-lysine unit or oligopeptides having free amino group on N-terminal, etc., all in protonated form. Such salt formations can be used to modify characteristics of the complex molecule as a whole, such as stability, compatibility to excipients, solubility and ability to form crystals.

Furthermore, the term„fucosyl" within the context of the present invention means a L- fucopyranosyl group attached to the core oligosaccharide with a-interglycosidic linkage:

„N-acetyl-lactosaminyl" group within the context of the present invention means glycosyl residue of N-acetyl-lactosamine (LacNAc, Galppi-4GlcNAcp) linked with β- linkage:

Furthermore, the term„Lacto-N-biosyl" group within the context of the present invention means the glycosyl residue of lacto-N-biose (L B, GalpPl-3GlcNAcp) linked with β-linkage:

The term„sialyl" within the context of the present invention means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac) linked with a-linkage:

Additionally, the term„glycosyl residue comprising one or more N-acetyl- lactosaminyl and/or one or more lacto-N-biosyl units" within the context of the present invention preferably means a linear or branched structure comprising the said units that are linked to each other by interglycosidic linkages.

The term "anomeric O-alkylation" in the present context means the selective alkylation of the anomeric OH group in the presence of non-protected primary and secondary OHs of the starting compound. Particularly, two basic methodologies shall be describedwith respect to the anomeric O-alkylation used in the present invention. When a compound of general formula 1 is devoid of any sialyl residues (neutral oligosaccharides) the alkylation reaction is performed in a dipolar aprotic solvent such as DMF, DMSO, N-methylpyrrolidone, hexamethylphosphoramide (HMPA), N,N'-dimethylhexahydropyrimidine-2-one (DMPU), THF, dioxane, acetonitrile, etc., or mixture thereof, in the presence of a strong base and R-X wherein X is a leaving group selected from halogen, alkylsulfonyloxy like mesyl, triflyl, etc. and arylsulfonyl like benzenesulfonyl, tosyl, etc. Preferred alkylating agents are benzyl or 1- or 2-naphthylmethyl halogenides optionally substituted with one or more groups selected from phenyl, alkyl or halogen. The strong base is able to deprotonate the anomeric OH chemo selectively due to its more acidic character when an equivalent amount or a slight excess (1 to 1.5 equiv.) of base is used. The strong base suitable for activating the anomeric OH is typically taken from the group of alkali metal or alkaline earth metal hydrides or alkoxides such as NaH, KH, CaH ₂ , NaOMe, NaO'Bu, KO'Bu, inorganic hydroxides, potassium carbonate, etc. The alkylation agent is added in an equivalent amount or a slight excess (1 to 1.5 equiv.). The reaction is carried out between -10 and 80 °C, preferably at a low temperature during whole course of the reaction or at a low temperature during the additon of the reagents/reactants and an elevated temperature in the later stages of the course of the reaction. Neutral oligosaccharide benzyl/substituted benzyl glycosides of general formula 2 can be obtained after usual work-up. In case of acidic oligosaccharides, that is, wherein at least one sialyl residue is present, the cesium salt of the starting material, previously formed by treating the acidic compound with cesium carbonate, is used for masking the carboxylate group in methyl ester form before anomeric O-alkylation. To form the methyl ester, the cesium salt is dissolved in a dipolar aprotic solvent such as DMF, DMSO, N- methylpyrrolidone, hexamethylphosphoramide (HMPA), Ν,Ν'- dimethylhexahydropyrimidine-2-one (DMPU), THF, dioxane, acetonitrile, etc., or mixture thereof, and a methylating agent like methyl iodide, methyl triflate, dimethyl sulphate or the like is added in equivalent amount or slight excess (1 to 1.5 equiv.) with respect to the cesium salt. The reaction typically takes place in 4-24 hours. The resulting methyl ester compound is then subjected to anomeric O-alkylation as specified above. At the end of the reaction the mixture is diluted with water giving rise to a basic aqueous conditions under which the methyl ester is cleaved resulting in the formation of an acidic oligosaccharide benzyl/substituted benzyl glycoside of general formula 2.

In step a) a compound of general formula 1 is generally available as a crude product accompanied by unreacted precursors, reagents, by-products and other contaminants from the chemical, enzymatic or chemo-enzymatic synthesis of said compound. Where compounds of general formula 1 are obtained from a natural source, they may be contaminated by organic molecules such as amino acids, oligo- and polypeptides, lipids, lactose, monosaccharides, vitamins, etc., which contamination profile may be characteristic to the natural pool from which the compounds of general formula 1 were obtained.

In step b) the crude mixture comprising one or more compounds of general formula 2 obtained in step a), accompanied by the contaminants mentioned above and derivatives thereof as well as traces of reagents used in the anomeric O-alkylation, is separated by chromatography and/or crystallization to give one or more individual compounds of general formula 2 each in substantially pure form. That is, where more than one compound of general formula 2 is obtained in step (b), each of those compounds is obtained separately from and substantially free of any of the other compounds of general formula 2 also obtained in that step. The chromatographic means can be any suitable separation techniques such as coloumn chromatography, HPLC, reverse phase chromatography, size exclusion chromatography, or ion exchange chromatography, generally used for the separation, isolation and/or purification of carbohydrates.

In step c)„catalytic hydrogenolysis" means the removal of the R-group which typically takes place in a protic solvent or in a mixture of protic solvents. Step (c) is conducted on a single compound of general formula 2 obtained in step (b). A protic solvent may be selected from the group consisting of water, acetic acid or Ci-C ₆ alcohol. A mixture of one or more protic solvents with one or more suitable aprotic organic solvents miscible partially or fully with the protic solvent(s) (such as THF, dioxane, ethyl acetate, acetone, etc.) may also be used. Water, one or more Ci-C ₆ alcohols or a mixture of water and one or more Ci-C ₆ alcohols are preferably used as the solvent system. The solutions containing the carbohydrate derivatives may have any suitable concentration, and suspensions of the carbohydrate derivatives with the selected solvent(s) may also be used. The reaction mixture is stirred at 10-100 °C temperature range, preferably between 20-70 °C, in a hydrogen atmosphere of 1-50 bar in the presence of a catalyst such as palladium, Raney nickel or any other appropriate metal catalyst, preferably palladium on charcoal or palladium black, until reaching the completion of the reaction. Catalyst metal concentrations generally range from 0.1 % to 10 % based on the weight of carbohydrate. Preferably, the catalyst concentrations range from 0.15 % to 5 %, more preferably 0.25 % to 2.25 %. Transfer hydrogenation may also be performed, when the hydrogen is generated in situ from cyclohexene, cyclohexadiene, formic acid or ammonium formate. Addition of organic or inorganic bases or acids and/or basic or acidic ion exchange resins can also be used to improve the kinetics of the

hydrogenolysis. The use of basic substances is especially preferred when halogen substituents are present on the substituted benzyl moieties of the precursors. Preferred organic bases include but are not limited to triethylamine, diisopropyl ethylamine, ammonia, ammonium carbamate, diethylamine, etc. Preferred organic/inorganic acids include but are not limited to formic acid, acetic acid, propionic acid, chloroacetic acid, dichloroacetic acid, triflouroacetic acid, HC1, HBr, etc. The conditions proposed above allow simple, convenient and delicate removal of the solvent(s) giving rise to pure compound of general formula 1. The compound of general formula 1 can be isolated from the reaction mixture using conventional work-up procedures in crystalline, amorphous solid, syrupy form or concentrated aqueous solution.

It should be emphasized that the introduction of the R-group brings numerous advantageous features to compounds of general formula 2 with respect to the isolation, separation and/or purification of a compound of general formula 2. Firstly, the R-group as an apolar moiety changes the polarity of the entire molecule, and thus it enlarges the repertoire of column packings and elution systems that a skilled person has available for selecting the best suitable conditions in order to achieve the best result. For example, due to the more different polarity of the compounds present a reverse phase chromatographic separation could be easily performed when water is used, as compounds of general formula 2 migrate much more slowly than the very polar compounds present in the reaction mixture, thus the polar compounds can be eluted smoothly. Compounds of general formula 2 can be then washed from the column with e.g. alcohol. Secondly, R-groups are aromatic moieties, and thus can serve as

chromophores offering the possibility of UV-detection which eases the identification of the desired objects. Thirdly, with careful selection of the R-groups crystalline materials of general formula 2 may be obtained. Crystallization or recrystallization is one of the simplest and cheapest methods to isolate a product from a reaction mixture, separate it from

contaminations and obtain pure substance. Isolation or purification that uses crystallization makes the whole technological process robust and cost-effective, and thus it is advantageous and attractive compared to other procedures. Fourthly, removal of the R-group from a compound of general formula 2 takes place under delicate conditions nearly quantitatively without the threat of by-product formation. R-groups such as benzyl/substituted benzyl protective groups are converted exclusively into toluene/ substituted toluene under

hydrogenolysis conditions and they can easily be removed even on a multi ton scale from water soluble oligosaccharide products via evaporation and/or extraction processes. Thus the chemical/stereochemical purity of a compound of general formula 1 is directly linked to that of a compound of general formula 2.

In a preferred method a crude mixture comprising one or more compounds of general formulae la, lb or lc, or salts of these compounds, all of which fall within the scope of compounds of general formula 1,

is subjected to derivatization in step a) to give a crude mixture comprising one or more compounds of general formulae 2a, 2b or 2c, or salts of these compounds, all of which fall within the scope of compounds of general formula 2, respectively,

wherein R, Ri and R ₂ are as defined above

R _3a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N- acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R _t o is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N- biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R _3b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue(s),

R _¾ is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N- acetyl-lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residues,

R ₅ is independently H or sialyl,

wherein at least one of Ri, R ₂ or R ₅ differs from H,

the individual compounds of general formulae 2a, 2b or 2c are isolated in substantially pure form by means of chromatography and/or crystallization in step b) and an individual compound of general formulae 2a, 2b or 2c converted into a compound of general formulae la, lb or lc in step c).

Particularly preferably, compounds used and obtained according to the inventive method as defined above are characterized by their linkages and modifications. Preferably, the compounds used and obtained in step a) of the inventive method and obtained in step c) as defined according to general formulae la, lb, 2a or 2b are characterized in that: - the N-acetyl-lactosaminyl group in the glycosyl residue of R _3a in general formulae la or 2a is attached to another N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of R _3a in general formula la or 2a is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of ILta in general formula la or 2a is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the N-acetyl-lactosaminyl group in the glycosyl residue of ILt _b in general formula lb or 2b is attached to another N-acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of ILt _b in general formula lb or 2b is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage.

Preferably, the compounds involved in the inventive method are characterized in that general formula la or 2a represents lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaose derivatives optionally substituted with one or more sialyl and/or fucosyl residue, or salts of these compounds, and general formula lb or 2b represents lacto-N-tetraose, lacto-N-hexaose, lacto- N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose derivatives optionally substituted with one or more sialyl and/or fucosyl residue, or salts of these compounds. More preferably, the compounds participating in the inventive method specified above are characterized in that:

- the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to

^■ the galactose of the lacto-N-biosyl group with a 1-2 interglycosidic linkage and/or ■ the N-acetyl-glucosamine of the lacto-N-biosyl group with a 1-4 interglycosidic

linkage and/or

^■ the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with a 1-3

interglycosidic linkage,

- the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to

^■ the galactose of the lacto-N-biosyl group with a 2-3 interglycosidic linkage and/or ^■ the N-acetyl-glucosamine of the lacto-N-biosyl group with a 2-6 interglycosidic linkage and/or

^■ the galactose of the N-acetyl-lactosaminyl group with a 2-6 interglycosidic linkage.

Most preferably, the following human milk oligosaccharides may be derivatized by the claimed method: 2'-0-fucosyllactose, 3-O-fucosyllactose, 2',3-di-O-fucosyllactose, 3'-0- sialyllactose, 6'-0-sialyllactose, 3'-0-sialyl-3-0-fucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, Fucal-2Galpl-3GlcNAcpl-3Galpl-4Glc (L FP-I), Galp 1-3 (Fucal - 4)GlcNAcp 1-3 Galp l-4Glc (LNFP-II), Galpl-4(Fucal-3)GlcNAcpl-3Galpl-4Glc (LNFP-III), Galp 1 -3 GlcNAcp 1 -3 Galp 1 -4(Fucal -4)Glc (LNFP-V), Neu5 Aca2-3 Galp 1 -3 GlcNAcp 1 - 3Galpl-4Glc (LST-a), Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (LST-b), Neu5Aca2- 6Galp 1 -4GlcNAcp 1 -3 Galp 1 -4Glc (LST-c), Neu5 Aca2-3 Galp 1 -3 (Fucal -4)GlcNAcp 1 - 3Galpl-4Glc (FLST-a), Fucal-2Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (FLST-b), Neu5Aca2-6Galpl-4GlcNAcpl-3Galpl-4(Fucal-3)Glc (FLST-c), Fucal-2Galpl-3(Fucal- 4)GlcNAcp 1 -3 Galp 1 -4(Fucal -3)Glc (LNDFH-I), Galp 1 -3 (Fucal -4)GlcNAcp 1 -3 Galp 1 - 4(Fucal-3)Glc (LNDFH-II), Galpl-4(Fucal-3)GlcNAcpl-3Galpl-4(Fucal-3)Glc (LNDFH- III), Neu5Aca2-3Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (DS-LNT), Neu5Aca2- 3Galpl-3(Neu5Aca2-6)(Fucal-4)GlcNAcpl-3Galpl-4Glc (FDS-LNT I) and Neu5Aca2- 3Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4(Fucal-3)Glc (FDS-LNT II), or salts thereof. The R-glycosides may be alpha- or beta-anomers. Preferably, said R-glycosides are the beta- anomers.

The second aspect of the present invention provides benzyl or substituted benzyl glycosides of human milk oligosaccharides or analogues. These can be prepared from the crude reaction mixture comprising one or more HMOs or analogues thereof characterized by general formula 1 according to step a) of the claimed method as specified above. Thus the present invention provides compounds of general formula 2' or salts thereof

general formula 2' wherein R is a group removable by hydrogenolysis,

Ri is fucosyl or H, R ₂ is fucosyl or H,

R ₃ is selected from H, sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R4 is selected from H, sialyl and N-acetyl-lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

wherein at least one of the Ri, R ₂ , R3 or R4 groups differs from H, and provided that the following compounds are excluded: R-glycosides of LNnT, Ι-Ο-β-benzyl-LNT, R-glycosides of 6'-0-sialyl-lactose and salts thereof, l-0-P-benzyl-3'-0-sialyl-lactose Na salt, 1-0-β-(4,5- dimethoxy-2-nitro)-benzyl-3'-0-sialyl-lactose Na salt.

In a preferred embodiment compounds of general formula 2' are characterized by general formulae 2' a, 2'b or 2'c or salts thereof

wherein R, Ri and R ₂ are as defined above

R _3a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N- acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R _t o is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N- biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R _3b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue, R4b is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N- acetyl-lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,

R ₅ is independently H or sialyl,

wherein at least one of Ri, R ₂ or R ₅ differs from H.

Particularly preferably, compounds according general formulae 2'a or 2'b as defined above are further characterized by their linkages and modifications. Preferably, the compounds as defined according to general formulae 2'a or 2'b are characterized in that: - the N-acetyl-lactosaminyl group in the glycosyl residue of R _3a in general formulae l'a or

2'a is attached to another N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of R _3a in general formula l'a or 2'a is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of R^ _a in general formula l'a or 2'a is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,

- the N-acetyl-lactosaminyl group in the glycosyl residue of R^ _b in general formula l'a or l'b is attached to another N-acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic linkage,

- the lacto-N-biosyl group in the glycosyl residue of R _¾ in general formula l'a or l'b is attached to the N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage.

Preferably, the compounds involved in the inventive method are characterized in that general formula 2'a represents lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaose R-glycosides optionally substituted with one or more sialyl and/or fucosyl residue, or salts thereof, and general formula 2'b represents lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto- N-octaose, lacto-N-decaose and lacto-N-neodecaose R-glycosides optionally substituted with one or more sialyl and/or fucosyl residue, or salts thereof.

More preferably, the compounds participating specified above are further

characterized in that:

- the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to ^■ the galactose of the lacto-N-biosyl group with a 1-2 interglycosidic linkage and/or

^■ the N-acetyl-glucosamine of the lacto-N-biosyl group with a 1-4 interglycosidic

linkage and/or

^■ the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with a 1-3

interglycosidic linkage,

- the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to

^■ the galactose of the lacto-N-biosyl group with a 2-3 interglycosidic linkage and/or

^■ the N-acetyl-glucosamine of the lacto-N-biosyl group with a 2-6 interglycosidic

linkage and/or

^■ the galactose of the N-acetyl-lactosaminyl group with a 2-6 interglycosidic linkage.

Most preferably, the R-glycosides of the following human milk oligosaccharides are provided: 2'-0-fucosyllactose, 3-O-fucosyllactose, 2',3-di-O-fucosyllactose, 3'-0- sialyllactose, 6'-0-sialyllactose, 3'-0-sialyl-3-0-fucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, Fucal-2Galpl-3GlcNAcpl-3Galpl-4Glc (LNFP-I), Galp 1-3 (Fucal -

4)GlcNAcp 1-3 Galp l-4Glc (LNFP-II), Galpl-4(Fucal-3)GlcNAcpl-3Galpl-4Glc (LNFP-III), Galp 1 -3 GlcNAcp 1 -3 Galp 1 -4(Fucal -4)Glc (LNFP-V), Neu5 Aca2-3 Galp 1 -3 GlcNAcp 1 - 3Galpl-4Glc (LST-a), Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (LST-b), Neu5Aca2- 6Galp 1 -4GlcNAcp 1 -3 Galp 1 -4Glc (LST-c), Neu5 Aca2-3 Galp 1 -3 (Fucal -4)GlcNAcp 1 - 3Galpl-4Glc (FLST-a), Fucal-2Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (FLST-b), Neu5 Aca2-6Galp 1 -4GlcNAcp 1 -3 Galp 1 -4(Fucal -3)Glc (FLST-c), Fucal -2Galp 1 -3 (Fucal - 4)GlcNAcp 1 -3 Galp 1 -4(Fucal -3)Glc (LNDFH-I), Galp 1 -3 (Fucal -4)GlcNAcp 1 -3 Galp 1 - 4(Fucal-3)Glc (LNDFH-II), Galpl-4(Fucal-3)GlcNAcpl-3Galpl-4(Fucal-3)Glc (LNDFH- III), Neu5Aca2-3Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4Glc (DS-LNT), Neu5Aca2- 3Galpl-3(Neu5Aca2-6)(Fucal-4)GlcNAcpl-3Galpl-4Glc (FDS-LNT I) and Neu5Aca2- 3Galpl-3(Neu5Aca2-6)GlcNAcpl-3Galpl-4(Fucal-3)Glc (FDS-LNT II), or salts thereof.

It is strongly emphasised that compounds characterized by general formula 2' can be considered as sole chemical entities such as either a or p anomers or even an anomeric mixture of a and P isomers, preferably as the P-anomer. Compounds of general formula 2' can be characterized as crystalline solids, oils, syrups, precipitated amorphous material or spray dried products. If crystalline, compounds of general formula 2' might exist either in anhydrous or in hydrated crystalline forms by incorporating one or several molecules of water into their crystal structures. Similarly, compounds characterized by general formula 2' might exist as crystalline substances incorporating ligands such as organic molecules and/or ions into their crystal structures.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not to be limiting thereof.

EXAMPLES

1. General procedure for the preparation of compounds of general formula 2

a) General procedure for the preparation of a neutral HMO benzyl/substituted benzyl glycoside

A selected neutral HMO (1 equiv.) was dissolved/suspended in 1-10 volumes (g/mL) of DMF, DMSO or a mixture thereof . The reaction mixture was cooled to 0 °C and benzyl bromide/substituted benzyl bromide (1.2-1.4 equiv.) was added. A strong base such as sodium hydride, potassium hydride, calcium hydride, potassium t-butoxide, sodium t-butoxide (1.2- 1.4 equiv) was added at 0-40 °C and the reaction mixture was stirred for 6-24 hours at 0-60 °C. Subsequently, water was added to quench the excess of base and the reaction mixture was stirred at RT for 30 minutes. The resulting reaction mixture was concentrated and purified in reverse phase chromatography, silica gel chromatography, ion-exchange chromatography, size-exclusion chromatography, etc. or crystallized giving rise to the desired

benzyl ated/substituted benzylated neutral HMO compound in 70-80 % yields. b) General procedure for the preparation of an acidic HMO benzyl/substituted benzyl glycoside

A selected acidic HMO is dissolved in water and treated with the H ⁺ form of an acidic ion-exchange resin, such as Amberlite IR-120, Dowex IL50, etc., to liberate the acidic HMO from its potential salt form. The ion exchange resin is filtered off and cesium carbonate was added to reach basic pH, preferably pH 9-10. The resulting solution was lyophilized and dissolved/suspended in 1-10 volumes (g/mL) of DMF, DMSO or a mixture thereof. A methylating agent such as methyl iodide, methyl triflate, etc. in quantities related to the use of cesium carbonate (0.2-0.5 equivalent excess) was added and the reaction mixture was stirred at 0-60 °C for 4-24 hours. The reaction mixture was cooled to 0 °C and benzyl

bromide/substituted benzyl bromide in required quantities (1.2-1.5 equiv.) was added.

Equivalent amount of strong base comparing to the benzylating/substituted benzylating agent used such as sodium hydride, potassium hydride, calcium hydride, potassium t-butoxide, sodium t-butoxide was added at 0-40 °C and the reaction mixture was stirred for 6-24 hours at 0-60 °C. Subsequently, water was added to create an organic solven water ratio of 1 : 10 to 2:5 and the reaction mixture was stirred at 20-80 °C for 4-24 hours. The resulting reaction mixture was concentrated and purified in reverse phase chromatography, silica gel

chromatography, ion-exchange chromatography, size-exclusion chromatography, etc. or crystallized or optionally converted into salt form giving rise to the desired

benzyl ate/substituted benzylated acidic HMO compound in 60-70 % yields.

c) General procedure for the preparation of a mixture of HMO benzyl/substituted benzyl glycosides from a mixture comprising at least one acidic HMO

The chemical composition of a mixture of HMOs was analyzed by LC-MS or any other suitable quantitative analytical method. Subsequently, the HMO mixture is dissolved in water and treated with the H ⁺ form of an acidic ion-exchange resin, such as Amberlite IR-120, Dowex IL50, etc., to liberate the acidic HMOs from their potential salt forms. The ion exchange resin is filtered off and cesium carbonate was added to reach basic pH, preferably pH 9-10. The resulting solution was lyophilized and dissolved/suspended in 1-10 volumes (g/mL) of DMF, DMSO or a mixture thereof. A methylating agent such as methyl iodide, methyl triflate, etc. in quantities related to the use of cesium carbonate (0.2-0.5 equivalent excess) was added and the reaction mixture was stirred at 0-60 °C for 4-24 hours. The reaction mixture was cooled to 0 °C and benzyl bromide/substituted benzyl bromide in required quantities (calculated according to LC-MS composition of the HMO mixture allowing 0.2-0.5 equivalent excess) was added. An equivalent amount of strong base comparing to the benzylating/substituted benzylating agent used such as sodium hydride, potassium hydride, calcium hydride, potassium t-butoxide, sodium t-butoxide was added at 0- 40 °C and the reaction mixture was stirred for 6-24 hours at 0-60 °C. Subsequently, water was added to create an organic solvent: water ratio of 1 : 10 to 2:5 and the reaction mixture was stirred at 20-80 °C for 4-24 hours. The resulting reaction mixture was concentrated and purified in reverse phase chromatography, silica gel chromatography, ion-exchange chromatography, size-exclusion chromatography, etc. giving the individual

benzyl ated/substituted benzylated compounds in 60-70 % yields, respectively.

Ι-Ο-β-benzyl-LNnT

1 ³ C NMR (D ₂ 0) δ: 105.6, 105.5, 105.4, 103.6 (anomeric carbons). Mp. : 284-286 °C.

1 -0-p-(4-methylbenzyl)-LNnT

1H MR (D ₂ 0): 7.3 (dd, 4H), 4.88 (d, 1H), 4.7 (m), 4.54 (d, 1H), 4.48 (d, 1H), 4.42 (d, 1H), 4.34 (d), 4.0-3.5 (m), 3.34 (dd, 1H). 1 ³ C MR (D ₂ 0): 184.2, 177.6, 173.7, 141.5, 136.1, 131.9, 131.4, 105.6, 105.5, 105.4,

103.6, 93.2, 84.7, 81.5, 81.0, 80.8, 78.0, 77.6, 77.4, 77.1, 75.5, 75.2, 74.8, 74.0, 73.6, 72.9, 63.7, 62.8, 58.9, 56.4, 25.9, 22.9.

1 -0-p-(4-chlorobenzyl)-LNnT 1H NMR (D ₂ 0): 7.4 (s, 4H), 4.9 (d, 1H), 4.72 (m), 4.52 (d, 1H), 4.8 (d, 1H), 4.42 (d,

1H), 4.16 (d, 1H), 4.0-3.52 (m).

¹³ C MR (D ₂ 0): 138.9, 177.6, 138.3, 137.9, 136.2, 131.3, 105.6, 105.5, 105.4, 103.7, 93.2, 86.1, 84.7, 81.5, 81.0, 80.8, 78.0, 77.5, 77.4, 77.2, 77.1, 75.5, 75.2, 74.9, 63.7, 58.9, 57.8, 56.4, 24.8.

Ι-Ο-β-benzyl-LNT

1H-NMR (D ₂ 0, 400 MHz) δ 2.03 (s, 3H, CH ₃ CONH), 3.35 (dd, 1H, J= 8.1 8.5 Hz, H- 2), 3.49 (m, 1H, H-5 ), 3.53 (m, H-2 ), 3.65 (m, 1H, H-3 ), 3.57 (dd, 1H, J= 8.1 9.0 Hz, H-4 ), 3.58 (m, 1H, H-5), 3.59 (dd, 1H, J= 7.7 10.0 Hz, H-2 ), 3.62 (m, 1H, H-3), 3.63 (m, 1H, H-4), 3.71 (m, 1H, H-5 ), 3.71 (m, 1H, H-5 ), 3.73 (dd, 1H, J =

3.3 10.0 Hz, H-3 ), 3.76 (m, 2H, H-6ab ), 3.76(m, 2H, H-6ab ), 3.80 (m, 1H, H- 6a ^" ), 3.80 (dd, 1H, J= 5.0 12.2 Hz, H-6a), 3.82 (dd, 1H, J= 8.1 10.5 Hz, H-3 ^" ), 3.90 (m, 1H, H-6b ^" ), 3.90 (dd, 1H, J= 8.4 10.5 Hz, H-2 ^" ), 3.92 (d, 1H, J= 3.3 Hz, H- 4 ^{^} ), 3.98 (dd, 1H, J= 1.6 12.2 Hz, H-6b), 4.15 (d, 1H, J = 3.3 Hz, H-4 ^' ), 4.44 (d, 1H, J= 7.7 Hz, Η-Γ ), 4.45 (d, 1H, J= 7.7 Hz, Η-Γ ^" ), 4.56 (d, 1H, J= 8.1 Hz, H-l), 4.73 (d, lH, J= 8.4 Hz, H-l ), 4.76 (d, 1H, J= 11.7 Hz, CHiPh), 4.94 (d, 1H, J= 11.7 Hz, CHiPh), 7.40-7.50 (m, 5H, Ph).

¹³ C-NMR (D ₂ 0, 100 MHz) δ 24.9 (CH ₃ CONH), 57.4 (C-2 ), 62.8 (C-6), 63.2 (C-6 ), 63 (C-6 ^{^} ), 63.7 (C- ), 71.0 (C-4'), 71.2 (C-4' ), 71.3 (C-4 ), 72.7 (C-2'), 73.4 (C- ^" ), 74.2 (CH ₂ Ph), 75.2 (C-3 ^{^} ), 75.5 (C-2), 77.1 (C-3), 77.5 (C-5'), 77.6 (C-

5' "), 77.9 (C-5), 78.0 (C-5 ), 81.1 (C-4), 84.7 (C-3'), 84.8 (C-3"), 103.7 (C-l), 105.3 (C-l ^" ), 105.6 (C-r), 106.2 (C-r"), 131.1 (Ph), 131.4 (2C, Ph), 131.5 (2C, Ph), 139.2 (Ph), 177.7 (CH ₃ CONH).

M.p. 245 °C (dec). [a] _D ²² = -10.3 (c = 1, H ₂ 0).

1 -0-p-(4-methylbenzyl)-LNT

1H-NMR (D ₂ 0, 300 MHz) δ 1.97 (s, 3H), 2.29 (s, 3H), 3.27 (dd, 1H, J= 8.1 8.5 Hz), 3.39-3.87 (m, 21H), 3.92 (dd, 1H, J= 1.8 12.3 Hz), 4.09 (d, 1H, J= 3.3 Hz), 4.37 (d, 1H, J= 8.1Hz), 4.38 (d, 1H, J= 7.8 Hz), 4.47 (d, 1H, J= 8.1 Hz), 4.65 (d, 1H, J = 11.7 Hz), 4.67 (d, 1H, J= 8.1 Hz), 4.83 (d, 1H, J= 11.7 Hz), 7.22 (d, 2H, J= 8.1 Hz),

7.30 (d, 2H, J= 8.1 Hz).

¹³ C-NMR (D ₂ 0, 75.4 MHz) δ 23.1, 25.0, 57.7, 62.8, 63.2, 63.7, 63.8, 71.0, 71.1, 71.3, 72.7, 73.4, 74.1, 75.2, 75.5, 77.1, 77.5, 77.6, 77.9, 78.0, 81.1, 84.7, 84.8, 103.6, 105.3, 105.7, 106.2, 131.7 (2C), 132.0 (2C), 136.2, 141.5, 177.7.

l-0-P-benzyl-6'-0-sialyl-lactose

1H-NMR (CD ₃ OD) δ (ppm): 1.63 (t, 1H, J=11.9Hz); 2.00 (s, 3H); 2.78 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.7Hz); 7.22-7.37 (m, 3H); 7.38-7.46 (m, 2H).

¹³ C-NMR (CD ₃ OD) δ (ppm): 21.55, 41.26, 52.70, 60.81, 63.21, 63.42, 68.56, 69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55, 73.78, 74.47, 75.28, 75.32, 80.03, 100.29, 101.89, 103.36, 127.36, 127.87, 128.01, 128.12, 137.72, 173.36, 173.88. l-0-P-benzyl-6'-0-sialyl-lactose sodium salt

1H-NMR (CD ₃ OD) δ (ppm): 1.63 (t, 1H, J=11.9Hz); 2.02 (s, 3H); 2.82 (m, 1H); 3.33- 3.45 (m, 4H); 3.46-3.61 (m, 5H); 3.62-3.97 (m, 12H); 4.02 (dd, 1H, J=7.5Hz,

J=10Hz); 4.37 (m, 2H); 4.66 (d, 1H, J=1 1.9Hz); 7.31 (m, 3H); 7.41 (m, 2H).

1 ³ C-NMR (CD ₃ OD) δ (ppm): 21.10, 40.60, 52.04, 60.22, 62.78, 67.90, 68.51, 68.69,

70.11, 70.61, 70.64, 71.44, 72.44, 72.83, 73.17, 73.92, 74.50, 74.70, 79.82, 99.76, 101.23, 103.52, 127.00, 127.44, 127.56, 137.21, 172.68, 173.28.

l-0-P-benzyl-6'-0-sialyl-lactose zinc salt

1 H-NMR (CD ₃ OD) δ (ppm): 1.71 (t, 1H, J=l l . lHz); 2.00 (s, 3H); 2.78 (dd, 1H,

J=4.5Hz, J=12.2Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.7Hz); 7.22-7.37 (m, 3H); 7.38-7.46 (m, 2H).

¹³ C-NMR (CD ₃ OD) δ (ppm): 21.77, 41.09, 52.78, 60.87, 61.32, 63.06, 63.64, 63.21, 68.26, 69.1 1, 70.67, 71.22, 71.36, 72.01, 73.00, 73.37, 73.58, 73.66, 74.39, 75.19,

75.32, 75.87, 79.40, 80.35, 99.92, 101.86, 101.94, 103.86, 104.00, 127.55, 127.99, 128.13, 137.83, 174.03, 174.08.

l-0-P-benzyl-6'-0-sialyl-lactose ethanolammonium salt

1H-NMR (CD ₃ OD) δ (ppm): 1.63 (dd, 1H, J=l 1.7Hz, J=12.2Hz); 2.00 (s, 3H); 2.78

(dd, 1H, J=4.5Hz, J=12.2Hz); 2.92 (m, 4H); 3.33-3.47 (m, 2H); 3.46-3.57 (m, 5H); 3.58-3.78 (m, 9H); 3.78-3.90 (m, 5H); 3.93 (dd, 1H, J=2.5, J=11.7Hz); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.7Hz); 7.22- 7.37 (m, 3H); 7.38-7.46 (m, 2H).

1 ³ C-NMR (CD ₃ OD) δ (ppm): 21.55, 41.26, 44.83, 52.70, 58.85, 60.81, 63.21, 63.42,

68.56, 69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55, 73.78, 74.47, 75.28, 75.32, 80.03, 99.98, 100.89, 102.36, 127.36, 127.87, 128.01, 128.12, 137.72, 173.36, 173.88. l-0-P-benzyl-6'-0-sialyl-lactose tris-(hydroxymethyl)-methyl ammonium salt

1H-NMR (CD ₃ OD) δ (ppm): 1.63 (dd, 1H, J=11.9Hz, J=12.1Hz); 2.00 (s, 3H); 2.78 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.33-3.48 (m, 2H); 3.48-3.79 (m, 19H); 3.93 (dd, 1H, J=2.5, J=11.8Hz); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.7Hz); 7.22-7.37 (m, 3H); 7.38-7.46 (m, 2H).

¹³ C-NMR (CD ₃ OD) δ (ppm): 21.60, 41.22, 52.71, 59.62, 60.89, 61.38, 63.23, 63.39, 68.64, 68.97, 69.25, 70.64, 71.26, 73.68, 75.24, 75.27, 80.16, 100.39, 101.87, 103.94, 127.52, 127.97, 128.00, 128.11, 137.85, 173.40, 173.90.

l-0-P-benzyl-6'-0-sialyl-lactose diethyl ammonium salt

1H-NMR (CD ₃ OD) δ (ppm): 1.28 (t, 6H, J=11.8Hz); 1.65 (dd, 1H, J=11.9Hz,

J=12.1Hz); 2.00 (s, 3H); 2.79 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.00 (q, 4H); 3.33-3.57 (m, 7H); 3.57-3.77 (m, 5H); 3.78-3.89 (m, 4H); 3.93 (dd, 1H, J=11.9Hz, J=2.4Hz); 4.00 (dd, 1H, J=7.1Hz, J=9.7Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.8Hz); 7.22-7.37 (m, 3H); 7.38-7.46 (m, 2H).

¹³ C-NMR (CD ₃ OD) δ (ppm): 10.64, 21.57, 41.29, 42.29, 52.74, 60.89, 63.23, 63.43, 68.63, 68.95, 69.30, 70.62, 71.25, 72.04, 73.06, 73.41, 73.73, 74.47, 75.26, 75.28, 80.15, 100.41, 101.90, 103.95, 127.51, 127.97, 127.99, 128.10, 137.88, 173.35, 173.85.

l-0-P-benzyl-6'-0-sialyl-lactose choline salt

1H-NMR (CD ₃ OD) δ (ppm): 1.63 (dd, 1H, J=11.9Hz, J=12.1Hz); 1.98 (s, 3H); 2.80 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.19 (s, 9H); 3.33-3.56 (m, 8H); 3.56-3.77 (m, 7H); 3.78-3.94 (m, 5H); 3.95-4.04 (m, 4H); 4.34 (m, 1H); 4.39 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.8Hz); 7.22-7.36 (m, 3H); 7.40 (m, 2H).

¹³ C-NMR (CD ₃ OD) δ (ppm): 21.56, 40.95, 41.34, 52.75, 53.48, 53.53, 53.59, 55.92, 60.96, 63.15, 63.50, 63.84, 67.85, 68.65, 68.90, 69.31, 69.47, 70.61, 71.28, 72.05, 73.05, 73.41, 73.72, 74.45, 75.29, 76.89, 80.17, 100.42, 101.92, 103.97, 109.98 127.52, 127.97, 128.11, 137.90, 138.29, 173.26, 173.85. l-0-P-(4-chlorobenzyl)-6'-0-sialyl-lactose

1H- MR (CD ₃ OD) δ (ppm): 1.62 (t, 1H, J=12Hz); 2.00 (s, 3H); 2.77 (dd, 1H, J=4.7Hz, J=12.1Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=l 1.7Hz); 7.24 (m, 2H); 7.32 (m, 2H).

¹³ C- MR (CD ₃ OD) δ (ppm): 21.52, 41.23, 52.72, 60.79, 63.23, 63.42, 68.61, 69.03, 69.31, 70.56, 71.14, 72.13, 73.11, 73.56, 73.79, 74.48, 75.23, 75.31, 80.09, 100.17, 101.91, 103.21, 127.36, 127.89, 133.01, 136.76, 173.29, 173.78.

l-0-P-(4-methylbenzyl)-6'-0-sialyl-lactose

1H- MR (CD ₃ OD) δ (ppm): 1.63 (t, 1H, J=11.9Hz); 2.00 (s, 3H); 2.32 (s, 3H); 2.78 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.66 (d, 1H, J=11.7Hz); 7.21 (m, 4H).

1 ³ C- MR (CD ₃ OD) δ (ppm): 20.02, 21.55, 41.26, 52.70, 60.81, 63.21, 63.42, 68.56,

69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55, 73.78, 74.47, 75.28, 75.32, 80.03, 99.98, 100.89, 102.36, 127.36, 127.87, 128.01, 128.12, 133.26, 137.72, 173.36, 173.88.

1 - 0- β- ( 1 -naphthylmethyl)-6 ' -O-sialyl-lactose

1H (CD3OD) δ (ppm): 1.62 (t, 1H, J=11.8Hz); 2.00 (s, 3H); 2.78 (dd, 1H, J=4.5Hz, J=12.2Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.6Hz, J=10.1Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8Hz); 4.68 (d, 1H, J=11.8Hz); 7.32-7. 54 (m, 4H); 7.7 (m, 2H); 7.95 (m,lH).

1 ³ C (CD ₃ OD) δ (ppm): 21.54, 41.28, 52.72, 60.81, 63.20, 63.44, 68.53, 69.06, 69.38,

70.64, 71.12, 72.14, 73.05, 73.54, 73.65, 74.45, 75.24, 75.33, 80.02, 100.25, 101.87, 103.21, 122.91, 124.82, 125.25, 125.93, 127.06, 128.72, 129.16, 132.80, 133.12, 135.97, 136.88, 173.36, 173.88. l-0-P-benzyl-3'-0-sialyl-lactose

1H- MR (500 MHz, D ₂ 0): δ [ppm] = 7.46-7.42 (m, 4H, H _(aA , _)arom ); 4.91 (d, IH, CH ₂ a- Bn); 4.74 (d, IH, CH ₂ b-Bn); 4.55-4.52 (m, 2H, H-l/H-1'); 4.11 (dd, IH, H-3'); 2.76 (dd, IH, H-3" _eq ); 2.04 (s, 3H, COCH ₃ ); 1.81 (dd, IH, H-3" _ax ). J ₍ a,b)-Bn= 11.8; h',3- = 9.9; h = 2.9; Jr _a r _| 12.4; = 12.1; J ₃ " _eq ,4" = 4.5 Hz.

"C-NMR (126 MHz, D ₂ 0): δ [ppm] = 135.2, 133.5 (quart. C _arom ); 130.2 (CH _a-arom ); 128.6 (CH _b-arom ); 102.6 (C-1'); 101.1 (C-1); 51.7 (C-5"); 39.6 (C-3"); 22.0 (COCH ₃ ).

l-0-P-(4-methylbenzyl)-3'-0-sialyl-lactose

1H- MR (500 MHz, D ₂ 0): δ [ppm] = 7.36 (d, 2H, H-a _arom ); 7.28 (d, 2H, H-b _arom );

4.89 (d, IH, CH ₂ a-Bn); 4.72 (d, IH, CH ₂ b-Bn); 4.54-4.52 (m, 2H, H-l/H-1'); 4.12 (dd, IH, H-3'); 2.76 (dd, IH, H-3" _eq ); 2.35 (s, 3H, CH ₃ -Tol); 2.04 (s, 3H, COCH ₃ ), 1.81 (dd, lH,H-3" _ax ).

J(a,b)arom ⁼ 7.9; J( _3; b)-Bn ⁼ 11.5; J ₂ ' _;3 ' = 9.9; J ₃ ' _;4 ' = 3.0; J ₃ " _ax , ₃ "eq= 12.5; J ₃ " _; 4" = 12.2; J ₃ "eq,4" = 4.6 Hz.

"C-NMR (126 MHz, D ₂ 0): δ [ppm] = 138.8, 133.4 (quart. C _arom ); 129.3 (CH _a-arom ); 129.0 (CH _b-arom ); 102.6 (C-1'); 100.9 (C-1); 99.8 (C-2"), 51.7 (C-5"); 39.6 (C-3"); 22.0 (COCH ₃ ); 20.2 (CH ₃ -T0I).

l-0-p-(2-naphthylmethyl)-3 '-O-sialyl-lactose

1H- MR (500 MHz, D ₂ 0): δ [ppm] = 7.99-7.97 (m, 4H, H-arom); 7.62-7.59 (m, 3H, H-arom); 5.10 (d, IH, CH ₂ a-Bn); 4.95 (d, IH, CH ₂ b-Bn); 4.60 (d, IH, H-1); 4.53 (d, IH, H-1'); 4.12 (dd, IH, H-3'); 2.77 (dd, IH, H-3" _eq ); 2.04 (s, 3H, COCH ₃ ); 1.81 (dd, IH, H-3 "a,). J ₍ a,b)-Bn= 11.9; ,2 = 8.0; J _v ,r = 7.9; J ₂ ^> ₃ ^> = 9.9; J ₃ ^> ₄ ^> = 3.0; Jr _a r _| 12.5; J ₃ ^» _ax , ₄ ^» =

12.1; J: _CC|. , 4.6 Hz.

¹³ C- MR (126 MHz, D ₂ 0): δ [ppm] = 128.3, 127.9, 127.7, 127.6, 126.6, 126.5 (CH _arom ); 102.6 (C-1'); 101.1 (C-1); 51.7 (C-5"); 22.0 (COCH ₃ ). l-0-P-(3-phenylbenzyl)-3'-0-sialyl-lactose

1H- MR (400 MHz, D ₂ 0): δ [ppm] = 7.75-7.44 (m, 9H, H-arom); 4.99 (d, 1H, CH ₂ a- Bn); 4.82 (d, 1H, CH ₂ b-Bn); 4.57 (d, 1H, H-l); 4.53 (d, 1H, H-l '); 4.13 (dd, 1H, H- 3'); 2.78 (dd, 1H, H-3" _eq ); 2.05 (s, 3H, COCH ₃ ); 1.82 (dd, 1H, H-3" _ax ).

J(a,b)-Bn ⁼ 11.8; J _{1 ;2} = 8.0; Ji' _;2 ' = 7.9; J ₂ ' _;3 ^> = 9.9; J ₃ ' _;4 ^> = 3.1; J ₃ " _ax , ₃ " _e q ⁼ 12.5; J ₃ "ax, ₄ " ⁼ 12.0; J ₃ -eq,4" = 4.6 Hz.

"C-NMR (100 MHz, D ₂ 0): δ [ppm] = 140.9, 140.3, 137.5 (quart. C _arom ); 129.4, 129.2, 127.9, 127.8, 127.1, 127.0, 126.9, (CH _arom ); 102.7 (C-l '); 101.2 (C-l); 99.6 (C- 2"); 51.8 (C-5"); 39.7 (C-3"); 22.1 (COCH ₃ ).

l-0-P-benzyl-2'-0-fucosyl-lactose

Characteristic 1H MR peaks (DMSO-d ₆ ) δ: 7.41-7.25 (m, 5 H, aromatic), 5.20 (d, 1 H, Ji ^» 2 ··= 2 Hz, H-l "), 4.82 and 4.59 (ABq, 2H, J _gem = 12.3 Hz, -CH ₂ Ph), 4.32 (d, 1 H, Ji 6.43 Hz, H-6 ^" ).

¹³ C MR (DMSO-de) δ: 137.97, 128.15, 128.15, 127.58, 127.58 and 127.43

(aromatic), 101.86, 100.94 and 100.20 (C-l, C-l 'and C-l "), 77.68, 76.78, 75.36, 75.33, 74.70, 73.79, 73.45, 71.60, 69.72, 69.69, 68.74, 68.20, 66.38, 60.23 and 59.81 (C-2, C-3, C-4, C-5, C-6, C-2', C-3 ', C-4', C-5 ', C-6', C-2", C-3 ", C-4", C-5" and CH ₂ Ph), 16.47 (C-6").

l-0-P-(4-nitrobenzyl)-2'-0-fucosyl-lactose

Characteristic ¹ H- MR peaks (DMSO-d ₆ ) δ: 8.20 and 7.68 (each d, 4 H, aromatic), 5.04 (d, 1 H, Ji ^» 2 ··= 2 Hz, H-l "), 4.97 and 4.76 (ABq, 2H, J _gem = 12.3 Hz, -CH ₂ Ph),

8.04 Hz, H-l), 1.04 (d, 1 H,

¹³ C- MR (DMSO-de) δ: 162.38, 147.68, 127.95, 127.95, 123.33 and 123.33

(aromatic), 102.18, 100.95 and 100.18 (C-l, C-l 'and C-l "), 77.62, 76.74, 75.36, 75.36, 74.61, 73.78, 73.45, 71.59, 69.67, 68.74, 68.67, 68.21, 66.38, 60.22 and 59.77 (C-2, C-3, C-4, C-5, C-6, C-2 ^' , C-3 ^' , C-4 ^' , C-5 , C-6 ^' , C-2 ^" , C-3 ^" , C-4 ^" , C-5 ^" and CH ₂ Ph), 16.45 (C-6").

2. Hydrogenolysis of compounds of general formula 2 a) 40 g (50.1 mmol) of Ι-Ο-β-benzyl-LNnT were dissolved in 200 ml of water, 1.6 g of Pd-C and 400 μΐ of acetic acid was added, and the mixture was stirred at rt. under H ₂ -atmosphere (approx. 40 bars) for 2 days. The catalyst was filtered off, the cake was washed with water, and the filtrate was added dropwise to 1.6 1 of acetone, then chilled, filtered and the collected solid was dried under vacuum to yield 31.5 g of white powder of LNnT (44.5 mmol, 89 %). b) l-O-benzyl-P-LNT (5 g, 6.27 mmol) was suspended in water (20 mL) and the pH was adjusted to 5.8 by addition of 1M aq. HC1. Palladium on charcoal (0.5 g, 10 %) was added and the reaction flask was evacuated and then saturated with H ₂ (4 bar). The reaction temperature was set to 50 °C and after stirring for 1.5 hour the temperature was allowed to reach RT, the catalyst was removed by filtration and water was used for washing (10 mL). The filtrate was concentrated to dryness and 3.46 g (78 %) of white solid LNT was obtained. c) To a solution of 40 g of l-0-P-benzyl-6'-0-sialyl-lactose in a mixture of methanol and water (250 mL + 300 mL) 4 g of Pd/C (10 %) were added. The reaction mixture was stirred 2 d at RT under H ₂ pressure (balloon). The mixture was then filtered through a pad of Celite and the solvent was evaporated in vacuo. The residue was dissolved in 80 mL of H ₂ 0 and dropped to 1200 mL of EtOH. The slurry was filtrated, the solid was washed with EtOH, acetone and a mixture of 1/1 acetone/Et ₂ 0. The solid was dried to give 35 g of 6 ^' -O-sialyllactose.

d) To a solution of l-0-P-benzyl-2'-0-fucosyl-lactose (5.0 g) in methanol (50 ml) 100 mg 10 % palladium on charcoal is added. The suspension is stirred under hydrogen atmosphere at rt for 4 hours. The catalyst is filtered off, washed with water and the filtrate is evaporated to yield 2'-0-fucosyl-lactose as amorphous white solid: 4.2 g.