US20130172548A1 - Derivatization of oligosaccharides - Google Patents

Derivatization of oligosaccharides Download PDF

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US20130172548A1
US20130172548A1 US13/809,829 US201113809829A US2013172548A1 US 20130172548 A1 US20130172548 A1 US 20130172548A1 US 201113809829 A US201113809829 A US 201113809829A US 2013172548 A1 US2013172548 A1 US 2013172548A1
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Prior art keywords
lacto
3galβ1
acetyl
fucα1
lactosaminyl
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Inventor
Gyula Dekany
Istvan Bajza
Károly Ágoston
Ignacio Figueroa Pérez
Markus Hedros
Andreas Schroven
Ioannis Vrasidas
Julien Boutet
Lars Kröger
Christoph Röhrig
Imre Kovacs
Péter Trinka
Ágnes Ágoston
Piroska Kovács-Pénzes
Ferenc Horváth
Christian Risinger
Gergely Pipa
Sándor Demkó
László Kalmár
Elise Champion
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Glycom AS
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Glycom AS
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Priority claimed from GBGB1012036.8A external-priority patent/GB201012036D0/en
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Assigned to GLYCOM A/S reassignment GLYCOM A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHAMPION, ELISE, ROHRIG, CHRISTOPH, TRINKA, PETER, HEDEROS, MARKUS, RISINGER, CHRISTIAN, DEKANY, GYULA, KROGER, LARS, VRASIDAS, IOANNIS, BOUTET, JULIEN, KALMAR, LASZLO, BAJZA, ISTVAN, HORVATH, FERENC, KOVACS, IMRE, AGOSTON, AGNES, AGOSTON, KAROLY, DEMKO, SANDOR, KOVACS-PENZES, PIROSKA, PIPA, GERGELY, SCHROVEN, ANDREAS, FIGUEROA-PEREZ, IGNACIO
Publication of US20130172548A1 publication Critical patent/US20130172548A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • HMOs human milk oligosaccharides
  • Mature human milk is the natural milk source that contains the highest concentrations of milk oligosaccharides (12-14 g/l), other milk sources are cow's milk (0.01 g/l), 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.
  • 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 characterized 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.
  • 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.
  • 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.
  • HMO derivatives obtainable by the inventive method to be specified later are known in the prior art: 1-O- ⁇ -benzyl-LNnT (Ponpipom et al. Tetrahedron Lett. 20, 1717 (1978)), 1-O- ⁇ -(4-hydroxymethylbenzyl)-LNnT (Yan et al. Carbohydr. Res. 328, 3 (2000)), 1-O- ⁇ -benzyl-LNT (Malleron et al. Carbohydr. Res. 341, 29 (2006), Liu et al. Bioorg. Med. Chem. 17, 4910 (2009)), 1-O- ⁇ -benzyl-6′-O-sialyl-lactose Na salt (Rencurosi et al.
  • 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.
  • the present invention relates to a method for purifying, separating and/or isolating an oligosaccharide of general formula 1 or a salt thereof
  • protecting group that is removable by hydrogenolysis or “group removable by hydrogenolysis” refers to groups whose C—O 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.
  • protecting groups are well known to the skilled man and are discussed in Protective Groups in Organic Synthesis , P G M Wuts and T W 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.
  • substitution if present, is on the aromatic ring(s).
  • 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, by-product(s), degradation product(s), inorganic salt(s) and/or other contaminations other than organic solvent(s) and/or water.
  • alkyl means a linear or branched chain saturated hydrocarbon group with 1-6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-hexyl, etc.
  • aryl refers to a homoaromatic group such as phenyl or naphthyl.
  • acyl represents an R′—C( ⁇ O)-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.
  • alkyl only for aryl residues
  • halogen nitro, aryl, alkoxy, amino, alkylamino, dialkylamino, carb
  • 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 —NH-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( ⁇ O)-group, such as methoxycarbonyl, t-butoxycarbonyl, etc.
  • Carbamoyl is an H 2 N—C( ⁇ O)-group.
  • N-Alkylcarbamoyl means an alkyl group (see above) attached to the parent molecular moiety through a —HN—C( ⁇ O)-group, such as N-methylcarbamoyl, etc.
  • N,N-Dialkylcarbamoyl means two alkyl groups (see above), either identical or different ones, attached to the parent molecular moiety through a >N—C( ⁇ O)-group, such as N,N-methylcarbamoyl, etc.
  • 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 2+ , Ba 2+ , Fe 2+ and Zn 2+ .
  • 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,
  • succosyl within the context of the present invention means a L-fucopyranosyl group attached to the core oligosaccharide with ⁇ -interglycosidic linkage:
  • N-acetyl-lactosaminyl within the context of the present invention means the glycosyl residue of N-acetyl-lactosamine (LacNAc, Galp ⁇ 1-4GlcNAcp) linked with ⁇ -linkage:
  • Lacto-N-biosyl within the context of the present invention means the glycosyl residue of lacto-N-biose (LNB, Galp ⁇ 1-3GlcNAcp) linked with ⁇ -linkage:
  • sialyl within the context of the present invention means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac) linked with ⁇ -linkage:
  • glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl units
  • glycosyl residue 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.
  • 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.
  • 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.
  • a dipolar aprotic solvent such as DMF, DMSO, N-methylpyrrolidone, hexamethylphosphoramide (HMPA), N,N′-dimethylhexahydropyrimidine-2-one (DMPU), THF, dioxane, acetonitrile,
  • alkylating agents are benzyl or f- 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 chemoselectively 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 t Bu, KO t 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 addition 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.
  • 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.
  • the cesium salt is dissolved in a dipolar aprotic solvent such as DMF, DMSO, N-methylpyrrolidone, hexamethylphosphoramide (HIMPA), N,N′-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.
  • a dipolar aprotic solvent such as DMF, DMSO, N-methylpyrrolidone, hexamethylphosphoramide (HIMPA), N,N′-dimethylhexahydropyrimidine-2-one (DMPU), THF, dioxane, acetonitrile, etc., or mixture thereof.
  • a methylating agent like methyl iodide
  • the resulting methyl ester compound is then subjected to anomeric O-alkylation as specified above.
  • 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.
  • 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.
  • 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.
  • 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 column chromatography, HPLC, reverse phase chromatography, size exclusion chromatography, or ion exchange chromatography. generally used for the separation, isolation and/or purification of carbohydrates.
  • 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 C 1 -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.
  • 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.
  • 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, trifluoroacetic acid, HCl, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • chemical/stereochemical purity of a compound of general formula 1 is directly linked to that of a compound of general formula 2.
  • step a) 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,
  • compounds used and obtained according to the inventive method as defined above are characterized by their linkages and modifications.
  • the compounds used and obtained in step a) of the inventive method and obtained in step c) as defined according to general formulae 1a, 1b, 2a or 2b are characterized in that:
  • the compounds involved in the inventive method are characterized in that general formula 1a 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 1b 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
  • the compounds participating in the inventive method specified above are characterized in that:
  • the following human milk oligosaccharides may be derivatized by the claimed method: 2′-O-fucosyllactose, 3-O-fucosyllactose, 2′,3-di-O-fucosyllactose, 3′-O-sialyllactose, 6′-O-sialyllactose, 3′-O-sialyl-3-O-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, Fuc ⁇ 1-2Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-I), Gal ⁇ 1-3(Fuc ⁇ 1-4)GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-II), Gal ⁇ 1-4(Fuc ⁇ 1-3)GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-III), Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal
  • 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.
  • the present invention provides compounds of general formula 2′ or salts thereof
  • compounds of general formula 2′ are characterized by general formulae 2′ a, 2′ b or 2′ c or salts thereof
  • compounds according general formulae 2′ a or 2′ b as defined above are further characterized by their linkages and modifications.
  • the compounds as defined according to general formulae 2′ a or 2′ b are characterized in that:
  • 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
  • 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 fucosy
  • the compounds participating specified above are further characterized in that:
  • the R-glycosides of the following human milk oligosaccharides are provided: 2′-O-fucosyllactose, 3-O-fucosyllactose, 2′,3-di-O-fucosyllactose, 3′-O-sialyllactose, 6′-O-sialyllactose, 3′-O-sialyl-3-O-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, Fuc ⁇ 1-2Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-I), Gal ⁇ 1-3(Fuc ⁇ 1-4)GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-II), Gal ⁇ 1-4(Fuc ⁇ 1-3)GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc (LNFP-III), Gal ⁇ 1-3GlcNAc ⁇ 1-3G
  • compounds characterized by general formula 2′ can be considered as sole chemical entities such as either a or ⁇ anomers or even an anomeric mixture of ⁇ and ⁇ isomers, preferably as the ⁇ -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.
  • 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 benzylated/substituted benzylated neutral HMO compound in 70-80% yields.
  • 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 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. or crystallized or optionally converted into salt form giving rise to the desired benzylate/substituted benzylated acidic HMO compound in 60-70% yields.
  • 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.

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