WO2014167537A1 - Synthèse d'oligosaccharides sialylés/fucosylés - Google Patents

Synthèse d'oligosaccharides sialylés/fucosylés Download PDF

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WO2014167537A1
WO2014167537A1 PCT/IB2014/060647 IB2014060647W WO2014167537A1 WO 2014167537 A1 WO2014167537 A1 WO 2014167537A1 IB 2014060647 W IB2014060647 W IB 2014060647W WO 2014167537 A1 WO2014167537 A1 WO 2014167537A1
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compound
formula
moiety
hydrogenolysis
group
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Nikolay Khanzhin
Pierre CHASSAGNE
Markus Hederos
Elise Champion
Martin MATWIEJUK
Gyula Dekany
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Glycom A/S
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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

Definitions

  • This invention relates to a chemo-enzymatic synthesis of oligosaccharides having important biological activities and significant commercial value for the pharmaceutical and food industry.
  • HMOs oligosaccharides
  • mucin oligosaccharides oligosaccharides
  • Lewis type oligosaccharides have gained much interest and have become important commercial targets for nutrition and therapeutic applications.
  • the synthesis of these oligosaccharides has increased significantly due to their role in numerous biological processes occurring in humans.
  • oligosaccharides contain sialosides, mainly N-acetyl-neuraminic acid, that are most frequently found at the terminal end of oligosaccharides.
  • the linkages of sialic acids, in which they are bound to galactose, N-acetyl-galactosamine and N-acetyl-glucosamine, are most commonly a- 2,3- and a-2,6-ketosidic bonds.
  • Their terminally exposed positions allow sialoconjugates to be recognized by receptors of cells, viruses and bacteria, thus to be involved in a wide variety of biological processes, such as tumour metastasis, cell differentiation, cell-cell interactions, etc.
  • N- Acetyl-glucosamine or -galactosamine to which a galactose is bound with ⁇ 1-3 interglycosidic linkage (that is lacto-N-biose and Gaip(l-3)-GalNAc, respectively), can be substituted by a sialic acid at its 6-position.
  • These trisaccharide motifs often substituted with further sialic acid and/or fucosyl residue(s), can be found naturally in various glycoconjugates having diverse biological roles.
  • Aminopropyl glycoside of Neu5Aca(2-3)-Gaip(l-3)-[Neu5Aca(2-6)-]GalNAc has been shown to bind to Sclerotium rolfsii lectin (SRL) (Chachadi et al. Glycoconj. J. 28, 49 (2011)).
  • the pentasaccharide 6" '-0-sialyl-LNT (LST b, Galp(l-3)-[Neu5Aca(2-6)-]GlcNAcp(l-3)-Galp(l- 4)-Glc) is a human milk oligosaccharide and represents a common structural element in some other sialylated human milk oligosaccharides (Urashima et al. : Milk Oligosaccharides, Nova Medical Books, NY, 2011); they are listed in Table 1.
  • DS-LNT has recently been found to be preventive against necrotising enterocolitis (NEC) in rat model (Jantscher-Krenn et al. Gut 61, 1417 (2012), WO 2012/106665).
  • the first aspect of the invention relates to a method for making a sialylated or fucosylated 3-0- galactosyl-GlcNAc or -GalNAc derivative of formula 1 and salts thereof
  • R is selected from -OH, -N 3 and -OR 5 wherein 5 is selected from: allyl optionally substituted by one or more methyl, propargyl optionally
  • n is an integer of 1 to 10, preferably 2 or 3
  • Ri is selected from sialyl moiety, -SO 3 H and -CH(R 5 )-COOH wherein R 5 is selected from H, alkyl and benzyl,
  • R 2 is selected from H and fucosyl
  • R 3 is selected from H and sialyl
  • R 4 is selected from H and fucosyl, provided that one but only one of R 3 and R 4 is H, and
  • A is selected from a bond and a divalent carbohydrate linker, comprising the steps of: a) sialylation, sulfation or carboxymethylation of a compound of formula 2
  • R' is selected from -N 3 and -OR' 6 wherein R' 6 is selected from allyl optionally substituted by one or more methyl, propargyl optionally
  • n is an integer of 1 to 10
  • R 7 is independently acyl
  • Y is selected from -NHAc, haloalkanoylamido, -NAc 2 , haloalkoxycarbonylamino, 2,3- diphenylmaleimido, 2,3-dimethylmaleimido and -N 3 , and
  • B is selected from a bond and a divalent carbohydrate linker in protected form, b) optional fucosylation of the sialylated, sulfated or carboxymethylated compound of formula 2 obtained in step a), c) de-O-acylation and/or basic hydrolysis, optional mild acidic hydrolysis and optional transformation of Y to -NHAc of the compound obtained in step a) or step b), d) sialylation or fucosylation of the compound obtained in step c), and e) optional catalytic hydrogenolysis and/or anomeric deprotection of the compound
  • the second aspect of the invention provides a compound of formula 11 and salts thereof
  • R' is selected from -N 3 and -OR' 6 which R' 6 is selected from allyl optionally substituted by one ore more methyl, propargyl optionally substituted by one ore more methyl, a group removable by hydrogenolysis, 2-trimethylsilyl-ethyl and -(CH 2 ) n -N3 wherein integer n is 1 to 10, preferably 2 or 3,
  • A' is a divalent lactosyl moiety having the R' group on its C-l (anomeric) carbon atom and attached via the 3'-OH group to the lacto-N-biosyl residue of the compound of formula 11, A' being optionally substituted by a fucosyl moiety on its 3-OH or by a N-acetyllactosaminyl moiety on its 6'-OH, which N-acetyllactosaminyl moiety can optionally be further substituted by an N-acetylneuraminyl moiety on its 6-OH, or by a fucosyl on its 3 -OH or 2' -OH,
  • Ri is selected from sialyl moiety, -SO 3 H and -CH(R 5 )-COOH wherein R 5 is selected from H, alkyl and benzyl,
  • R 3 is selected from H and sialyl moiety
  • R 4 is selected from H and fucosyl moiety, provided that at least one of R 3 and R 4 is H, and Ri 6 is selected from H and moiety C, preferably H,
  • Rn and Ris independently, are selected from H and a group removable by hydrogenolysis, provided that the disodium salt of the following compound is excluded:
  • R' is 2-trimethylsilyl- ethyloxy
  • A' is unsubstituted lactosyl
  • Ri and R 3 are sialyl moieties
  • R 4 and Ri 6 are H.
  • the alkyl and aryl residues can be unsubstituted or substituted one or several times, preferably 1-5 times, more preferably 1-3 times.
  • the substituents can preferably be alkyl (for aromatic acyl), hydroxy, alkoxy, carboxy, oxo (for alkyl, forming a keto or aldehyde function), alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono- and dialkylamino, carbamoyl, mono- and dialkyl-aminocarbonyl, alkylcarbonylamino, cyano, alkanoyloxy, nitro, alkylthio and/or halogen (F, CI, Br, I).
  • aryl and alkyl moieties of acyl groups can modify the general chemical characteristics of the acyl group, and thereby the characteristics, such as stability, solubility and the ability to form crystals, of a molecule as a whole.
  • group removable by hydrogenolysis preferably means a protecting group whose C-0 bond can be cleaved by hydrogen in the presence of a catalytic amount of palladium, Raney nickel or any other conventional hydrogenolysis catalyst to regenerate the protected -OH group.
  • Such protecting groups are described in Wuts and Greene: Protective Groups in Organic Synthesis, John Wiley & Sons, 2007, and include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2- naphthylmethyl and triphenylmethyl (trityl) groups, each of which can be optionally substituted by one or more of the following groups: alkyl, alkoxy, phenyl, amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N- dialkylcarbamoyl, azido, halogenalkyl or halogen.
  • a preferred protecting group is benzyl optionally substituted with one or more of the following groups: phenyl, alkyl and halogen, particularly unsubstituted benzyl, 4-chlorobenzyl, 3-phenylbenzyl and 4-methylbenzyl groups.
  • phenyl, alkyl and halogen particularly unsubstituted benzyl, 4-chlorobenzyl, 3-phenylbenzyl and 4-methylbenzyl groups.
  • These preferred and particularly preferred protecting groups have the advantage that the by-products of their hydrogenolysis are exclusively toluene or substituted toluene. Such by-products can easily be removed, even in multi-ton quantities, from water-soluble oligosaccharide products via evaporation and/or extraction processes.
  • a-sialyl or “sialyl” preferably means - in accordance with Chen and Varki ACS Chem. Biol. 5, 163 (2010) - a glycosyl moiety of any naturally occurring or modified neuraminic acid or sialic acid derivative or an analogue thereof having an a-glycosidic linkage.
  • Preferred neuraminic acid derivatives are N-acetyl- (Neu5Ac), N-glycolyl- (Neu5Gc) and deamino- neuraminic acid (3-deoxy-D-glycero-D-galacto-nonulosonic acid, KDN), as well as Neu5Ac, Neu5Gc and KDN derivatives that are derivatized with linkers, reactive functional groups, detectable labels or targeting moieties, and/or substituted at C-4, C-7-, C-8 and/or C-9, especially at C-9, with acyloxy, alkoxy, halogen or azido.
  • the preferred sialyl moiety is the glycosyl residue of Neu5Ac (see Scheme 1). It should be noted that the term "sialyl" in the generally accepted trivial names of human milk oligosaccharides and Lewis type oligosaccharides always refers to Neu5Ac.
  • succosyl preferably means a L-fucopyranosyl group (see Scheme 2) attached to a core oligosaccharide with a-interglycosidic linkage.
  • salt in connection with sialylated, sulfated and carboxymethylated compounds described in the application preferably means an associated ion pair consisting of the negatively charged acid residue and one or more cations in any stoichiometric proportion.
  • Cations which are atoms or molecules with a positive charge, can be inorganic or organic.
  • 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+ , more preferably K + , Ca 2+ , Mg 2+ , Ba 2+ , Fe 2+ and Zn 2+ .
  • Preferred basic organic compounds in positively charged form are diethyl amine, triethyl amine, diisopropyl ethyl amine, ethanolamine, diethanolamine, triethanolamine, imidazole, piperidine, piperazine, morpholine, 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 salts can be used in a conventional manner to modify the characteristics of the compounds of this invention, such as their stability
  • alkyl preferably means a linear or branched hydrocarbon group with 1-6 carbon atoms, such as methyl, ethyl, ⁇ -propyl, / ' -propyl, «-butyl, / ' -butyl, s-butyl, t-butyl, etc.;
  • haloalkanoylamido preferably means a halogen substituted Ci-C 6 -alkanoylamido such as chloroacetamido, trichloroacetamido, trifluoroacetamido, etc.;
  • haloalkoxycarbonylamino preferably means a Ci-C 6 -alkyloxycarbonyl- H-group substituted by one or more halogen atoms such as 2,2,2-trichloroethoxycarbonylamino, etc.;
  • optionally substituted phenyl and "optionally substituted benzyl
  • the present invention provides an efficient approach to a diverse group of sialylated and/or fucosylated 3'-0-galactosyl-6-0-sialyl-GlcNAc or -GalNAc derivatives and analogs thereof based on the unique combination of chemical and enzymatic glycosylation steps.
  • the claimed method is attractive for scale-up developments and therefore may imply a potential industrial process.
  • the first aspect of the invention relates to a method for making a sialylated or fucosylated 3-0- galactosyl-GlcNAc or -GalNAc derivative of formula 1 and salts thereof
  • R is selected from -OH, -N 3 and -OR 6 wherein R 6 is selected from: allyl optionally substituted by one or more methyl, propargyl optionally
  • n is an integer of 1 to 10, preferably 2 or 3
  • Ri is selected from sialyl moiety, -SO 3 H and -CH(R 5 )-COOH wherein R 5 is selected from H, alkyl and benzyl,
  • R 2 is selected from H and fucosyl
  • R 3 is selected from H and sialyl
  • R 4 is selected from H and fucosyl, provided that one but only one of R 3 and R 4 is H, and
  • A is selected from a bond and a divalent carbohydrate linker, comprising the steps of: a) sialylation, sulfation or carbox methylation of a compound of formula 2
  • R' is selected from -N 3 and -OR' 6 wherein R' 6 is selected from allyl optionally substituted by one or more methyl, propargyl optionally
  • n is an integer of 1 to 10
  • R 7 is independently acyl
  • Y is selected from -NHAc, haloalkanoylamido, -NAc 2 , haloalkoxycarbonylamino, 2,3- diphenylmaleimido, 2,3-dimethylmaleimido and -N 3 , and
  • B is selected from a bond and a divalent carbohydrate linker in protected form, b) optional fucosylation of the sialylated, sulfated or carboxymethylated compound of formula 2 obtained in step a), c) de-O-acylation and/or basic hydrolysis, optional mild acidic hydrolysis and optional transformation of Y to -NHAc of the compound obtained in step a) or step b), d) sialylation or fucosylation of the compound obtained in step c), and e) optional catalytic hydrogenolysis and/or anomeric deprotection of the compound
  • a compound of formula 2 is sialylated, sulfated, or carboxymethylated.
  • B is a bond
  • a compound of formula 2 is a partially protected disaccharide of formula 2A
  • the secondary OH-group of the deoxy-aminohexose moiety of the disaccharide of formula 2A can be in equatorial orientation giving rise to a partially protected lacto-N-biosyl derivative of formula
  • the disaccharide of formula 2 A is a GalpPl- 4GalNAc derivative of formula 2C
  • the compound of formula 2 is a compound of formula 2B wherein Y is - HAc and R 7 groups are identical and are acetyl or benzoyl.
  • Compounds of formula 2A can be made in a conventional manner by standard carbohydrate chemistry, for example by reacting a protected glucos- or galactosamine acceptor having a free 3- OH with a tetraacyl galactosyl donor followed by selective deprotection of the 4- and 6-OH groups (see e.g. Hendel at al. Carbohydr. Res. 343, 2914 (2008), Jain et al. Carbohydr. Res. 275, 231 (1995), WO 95/29927, Bao et al. Bioorg. Med. Chem. 18, 3760 (2010), Rana et al. Carbohydr. Res.
  • B is a divalent carbohydrate linker in protected form.
  • the term "carbohydrate linker in protected form” preferably means any mono-, di- or oligosaccharide glycosyl residue which is attached to the R' group by the C-l (aldoses) or C-2 (ketoses) anomeric carbon, and is also attached, via one of its non-glycosidic OH-groups, to the galactosylated aminodeoxy-hexose residue of the compound of formula 2.
  • this divalent glycosyl residue B may represent a linear or branched structure consisting of monosaccharide units that are linked to each other by interglycosidic linkages.
  • the monosaccharide unit(s) of the divalent glycosyl residue B can be selected from any 5- 9 carbon atom containing sugars consisting of aldoses (e.g. D-glucose, D-galactose, D-mannose, D- ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g.
  • D-fructose, D-sorbose, D-tagatose, etc. deoxysugars (e.g. L-rhamnose, L-fucose, etc.), deoxy-amino sugars (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, ketoaldonic acids (e.g. sialic acid) and like.
  • the divalent glycosyl moiety B is a lactosyl moiety or an oligosaccharide moiety that consists of a lactosyl moiety and at least one monosaccharide unit selected from the group consisting of galactose, N-acetylglucosamine, fucose and N-acetyl neuraminic acid.
  • the divalent lactosyl moiety is attached to the R' group by its C-1 anomeric carbon atom, and it is also attached, via one of its non-glycosidic OH-groups, preferably via its 3'-OH group to the
  • the divalent lactosyl moiety defined above can be optionally substituted by other glycosyl residues, preferably on its 3- OH group by a fucosyl moiety, or on its 6' -OH group by a N-acetyllactosaminyl moiety, which N- acetyllactosaminyl moiety can optionally be further substituted by an N-acetyl neuraminyl moiety on its 6-OH, or by a fucosyl on its 3-OH or 2'-OH.
  • These oligosaccharide substructures can be found in human milk oligosaccharides.
  • the functional groups of the divalent glycosyl residue B are protected, preferably the free OH groups are acylated (e.g. acetylated, benzoylated) and the carboxy group of the optional N-acetyl neuraminyl moiety is blocked in ester form (e.g. methyl, ethyl or benzyl ester).
  • ester form e.g. methyl, ethyl or benzyl ester
  • the R' group has ⁇ -orientation.
  • the moiety B is attached to the parent carbohydrate backbone by ⁇ -linkage.
  • step a) the primary free OH group of a compound of formula 2 is regioselectively sialylated, sulfated or carboxymethylated, without affecting the neighbouring secondary OH-group, to give a compound of formula 4
  • R', R 7 , B and Y are as defined above, and
  • P is selected from protected sialyl moiety, -SO 3 H and -CH(R 5 )-COORio wherein R 5 is selected from H, alkyl and benzyl, R 10 is selected from alkyl and benzyl.
  • sialyl donors For the chemical sialylation suitably protected and activated sialyl donors are used.
  • functional group protection of the sialyl donor an array of protecting groups, mainly esters, ethers and acetals, are available to the skilled person.
  • optionally substituted acyls such as acetyl, benzoyl, chloroacetyl or chlorobenzoyl, and ether-type groups such as benzyl are of synthetic usefulness;
  • the carboxyl group can be protected by an ester, typically by a methyl, ethyl or benzyl ester;
  • the amino function can be masked by diacetyl, trifluoroacetyl, trichloroacetyl, Troc or Fmoc group, or as a cyclic carbamate with the adjacent 4-OH.
  • the anomeric centre activation can be varied among halo, alkyl- or arylthio, dialkyl, dibenzyl or diaryl phosphite, or trihaloacetimidate, each of which is commonly used in sialoglycosidation methods.
  • the protective group introduction and anomeric centre activations mentioned above can be carried out by known processes (see e.g. Ress et al. Curr. Org. Synth. 1, 31 (2004), Chen et al. ACS Chem. Biol. 5, 163 (2010) and references cited therein).
  • the preferred sialyl donors are those disclosed in WO 2011/100979 and WO 2012/113404, among which the N-acetyl neuraminyl phosphite donors of formula 3
  • R 8 is acyl, preferably acetyl
  • Q is selected from alkyl and benzyl, preferably methyl, ethyl and benzyl, and
  • R9 is optionally substituted phenyl or benzyl, are especially preferred.
  • the coupling reaction runs in aprotic solvent, preferably in
  • the sialylated product can be characterized by the following formula 4 A
  • R', R 7 , R 8 , B, Q and Y are as defined above.
  • step a) is a sulfation step
  • a compound of formula 2 defined above is treated with SCVpyridine complex in a non-protic solvent to form a compound of formula 4B or a salt thereof
  • a compound of formula 2 can be carboxymethylated to give rise to a compound of formula 4C
  • R 5 is selected from H, alkyl and benzyl
  • R 10 is selected from alkyl and benzyl
  • R', R 7 , B and Y are as defined above.
  • step b) the sialylated, sulfated or carboxymethylated compound obtained in step a) is optionally fucosylated with a suitably protected and activated fucosyl donor.
  • the fucosyl moiety is introduced at the remaining free secondary OH-group (4-OH) of the compound of formula 4.
  • X is selected from a halogen, -O-pentenyl, -OAc, -OBz and -SRi 3 , in which Ri 3 is alkyl or optionally substituted phenyl,
  • Rii is selected from acyl and a group removable by hydrogenolysis
  • Ri 2 is selected from a group removable by hydrogenolysis, acyl or two R 12 groups together form a moiety R-14 C R 15j wherein R14 and R15 independently are alkyl or phenyl, or wherein R14 and R15 together with the carbon atom, to which they are attached, form cycloalkylidene, can be used in this reaction to give a compound of formula 6
  • R', R 7 , Rn, Ri 2 , B, P and Y are as defined above.
  • the fucosylation can be carried out in a conventional manner in an aprotic solvent or in a mixture of aprotic solvents in the presence of an activator, see Demchenko (Ed.): Handbook of Chemical Glycosylation Wiley (2008).
  • the fucosylation reaction is generally promoted by heavy metal ions, mainly mercury or silver, and Lewis acids such as trimethylsilyl triflate or BF 3 -etherate.
  • a fucosyl halide i.e., X is F, CI, Br or I
  • X is F, CI, Br or I
  • anomeric halides follow the reactivity order F ⁇ Cl ⁇ Br ⁇ I for nucleophilic displacement.
  • Glycosyl fluorides can be prepared by treating the appropriate precursors such as hemiacetals, glycosyl halides, glycosyl esters and ⁇ -glycosides with fluorinating reagents such as HF, AgF, AgBF 4 , tetrabutyl ammonium fluoride, diethyl amino sulfur trifluoride, 2- fluoro-l-methylpyridinium tosylate, Selectfluor, Deoxo-Fluor or 4-methyl(difluoroiodo)-benzene.
  • fluorinating reagents such as HF, AgF, AgBF 4 , tetrabutyl ammonium fluoride, diethyl amino sulfur trifluoride, 2- fluoro-l-methylpyridinium tosylate, Selectfluor, Deoxo-Fluor or 4-methyl(difluoroiodo)-benzene.
  • the resulting glycosyl donor can be activated by a catalytic amount of a Lewis acid, such as trimethylsilyl triflate or BF 3 -etherate, for the glycosylation reaction.
  • Fucosyl acetates or benzoates are preferably first subjected to electrophilic activation to provide a reactive intermediate and then treated with a nucleophilic OH-acceptor.
  • Typical activators of choice are Bronsted acids (e.g., /?-TsOH, HC10 4 or sulfamic acid), Lewis acids (e.g., ZnCl 2 , SnCl 4 , triflate salts, BF 3 -etherate, trityl perchlorate, A1C1 3 or triflic anhydride) or a mixture thereof.
  • Pentenyl fucosides can be transglycosylated with appropriate glycosyl acceptors in the presence of a promoter such as NBS and NIS.
  • a promoter such as NBS and NIS.
  • Protic or Lewis acids triflic acid, Ag-triflate, etc. can enhance the reaction.
  • the pentenyl glycosides can be prepared with the aid of «-pentenol by standard Fischer glycosylation of hemiacetals under acidic condition, by silver(I) salt promoted coupling of glycosyl bromides (Koenigs-Knorr method), or by glycosylation of 1 -acetyl glycosides in the presence of tin(IV) chloride.
  • Thiofucosides i.e., X is alkylthio- or optionally substituted phenylthio-group
  • thiophilic promoters such as mercury(II) salts, Br 2 , 1 2 , NBS, NIS, triflic acid, triflate salts, BF 3 - etherate, trimethylsilyl triflate, dimethyl-methlythio sulphonium triflate, phenylselenyl triflate, iodonium dicollidine perchlorate, tetrabutylammonium iodide or mixtures thereof, preferably by Br 2 , NBS, NIS or triflic acid.
  • thiophilic promoters such as mercury(II) salts, Br 2 , 1 2 , NBS, NIS, triflic acid, triflate salts, BF 3 - etherate, trimethylsilyl triflate, dimethyl-methlythio sulphonium tri
  • Aprotic solvents such as toluene, THF, methyl-THF, DCM, chloroform, dioxane, acetonitrile, chlorobenzene, ethylene di chloride, DMSO, DMF or N-methylpyrrolidone or mixtures thereof, preferably DMF, toluene, DCM or mixtures thereof, more preferably toluene or DMF-DCM mixture can be used in this glycosylation reaction at -20 to 20 °C, preferably at -10 to 5 °C, with reaction time of 5 min to 2 hours.
  • Br 2 , NBS or NIS can be used, optionally in the presence of triflic acid or a triflate derivative.
  • a slight excess of donor 1.1-1.2 eq.
  • water or a Ci-C 6 alcohol is generally used, preferably an aqueous or alcoholic solution of a base like sodium carbonate, sodium bicarbonate, ammonia or triethyl amine, more preferably an aqueous Na 2 S 2 0 3 /NaHC0 3 solution.
  • the fucosyl donor is a compound of formula 5 wherein Rn is as defined above, R 12 is acyl or a group removable by hydrogenolysis, and X is phenylthio optionally substituted with one or more alkyl. More preferably Rn is benzyl, 4-methylbenzyl, naphthylmethyl, 4-phenylbenzyl, 4- chlorobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4,6-trimethylbenzyl or 2,3,4,5,6- pentamethylbenzyl, R 12 is benzyl, 4-methylbenzyl, naphthylmethyl, 4-phenylbenzyl, 4- chlorobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4,6-trimethylbenzyl, 2,3,4,5,6- pentamethylbenzyl or benzoyl optionally substituted by one or more halogens and X is
  • Rn is benzyl or 4-methylbenzyl and R 12 is benzoyl or 4-chlorobenzoyl.
  • step b) a compound of formula 4A, 4B or 4C defined above is reacted with a fucosyl donor of formula 5 giving rise to a compound of formula 6A, 6B or 6C, respectively, and salts thereof
  • R', R 5 , R 7 , R 8 , Rio, Rn, R 12 , B, Q and Y are as defined above.
  • step c) the protecting groups of a compound obtained in step a) or step b) are removed except for the anomeric protection R', and optionally Y is converted into a - HAc group.
  • protecting groups are O-acyls (R 7 , R 8 , the OH-protections in moiety B, and optionally Rn and R 12 ).
  • Acyl protective groups can be removed by a conventional base catalysed
  • transesterification deprotection reaction wherein the acyl groups are removed in an alcohol solvent such as methanol, ethanol, propanol or t-butanol in the presence of an alcoholate such as NaOMe, NaOEt or KO*Bu at 20-100 °C.
  • an alcohol solvent such as methanol, ethanol, propanol or t-butanol
  • an alcoholate such as NaOMe, NaOEt or KO*Bu at 20-100 °C.
  • a co- solvent such as toluene or xylene can be beneficial in order to control particle size of the product and to avoid gel formation.
  • a catalytic amount of NaOMe is used in methanol (Zemplen de-O-acylation). Under this condition only O-acyls can be deprotected.
  • Y is -NAc 2
  • one of the acetyl groups can also be removed to make the Y group -NHAc.
  • Y is: haloalkanoylamido, haloalkoxycarbonylamino, 2,3-diphenylmaleimido, 2,3-dimethylmaleimido or -N 3 , and ester groups (see group -COOQ or -COORio) remain intact under these conditions.
  • acyloxy groups to OH, esters to carboxyl see group -COOQ or -COORio
  • Y-groups to -NH 2 haloalkanoylamido, halocarboxylamino, 2,3-diphenylmaleimido and 2,3-dimethylmaleimido
  • basic hydrolysis which is a base catalysed hydrolysis in water, alcohol or water-organic solvent mixtures, in homogeneous or heterogeneous reaction conditions at 0-100 °C.
  • a strong base such as Li OH, NaOH, KOH, Ba(OH) 2 , K 2 C0 3 , a basic ion exchange resin or tetraalkylammonium hydroxide, is used, but the base can also be in an aqueous solution as well.
  • the base is NaOH and the solvent is methanol. If Y is -NAc 2 , one of the acetyl groups can also be removed to make the Y group -NHAc. Azido Y group is not affected in this reaction.
  • R' is not azido or R' 6 is not -(CH 2 ) n -N 3 ).
  • the free amino group obtained by one of the deprotective methods can then be acetylated without acetylating the free OH-groups.
  • Selective N-acetylation in the presence of one or more hydroxyls can be carried out in a conventional manner with a slight excess of acetic anhydride or acetyl chloride (-1.5-3 equiv.) at about 0-35 °C with or without added base. Any resulting overacetylated by-product(s) can be readily transformed into the desired compounds with e.g. NaOH/MeOH or NaOMe/MeOH treatment.
  • the deprotected compound having a free amino group can be peracetylated (that is the amino group and all available OH groups are acetylated) followed by base catalysed transesterification (see above).
  • trichloroacetyl amide Y-group can be transformed in one step to -NHAc with tributyltin hydride.
  • step c) an acetal/ketal group optionally present on the compounds of formulas 6A, 6B and
  • 6C (i.e. when two Ri 2 groups together form a moiety ⁇ 14 C R 15 ) ca n be selectively deprotected by acid catalysed mild hydrolysis, i.e. by reacting the compounds with water or an alcohol in the presence of acid at pH>l-2 to produce OH-groups on the compounds.
  • Acyl protective groups will not be affected because they can be deprotected only by extremely strong acidic hydrolysis (pH ⁇ l).
  • the interglycosidic linkage and anomeric protecting groups of the compounds of formulas 6A, 6B and 6C can also be sensitive to acids, they can be split in the compounds of formulas 6A, 6B and 6C only by acidic hydrolysis at pH ⁇ l-2.
  • Water which is a reagent, can also serve as solvent or co-solvent.
  • Organic protic or aprotic solvents which are stable under acidic conditions and fully or partially miscible with water, such as Ci-C 6 alcohols, acetone, THF, dioxane, ethyl acetate or MeCN, can be also used in a mixture with water.
  • the acid used is generally a protic acid, such as acetic acid, trifluoroacetic acid, HC1, formic acid, sulphuric acid, perchloric acid, oxalic acid, ⁇ -toluenesulfonic acid,
  • benzenesulfonic acid or a cationic exchange resin can be present in from a catalytic amount to a large excess.
  • the hydrolysis can be carried out at between 20 °C and reflux until completion of the reaction which can take from about 2 hours to 3 days depending on temperature, concentration and pH.
  • an organic acid such as acetic acid, formic acid, chloroacetic acid or oxalic acid
  • a Ci-C 6 alcohol-acetonitrile or Ci-C 6 alcohol-water mixture is used in the presence of HC1 or a sulfonic acid such as ⁇ -toluenesulfonic acid or camphorsulfonic acid.
  • an anhydrous Ci-C 6 alcohol such as methanol, ethanol, propanol and butanol
  • an anhydrous Ci-C 6 alcohol such as methanol, ethanol, propanol and butanol
  • Catalytic amount of hydrogen chloride, sulphuric acid, perchloric acid, ⁇ -toluenesulfonic acid, acetic acid, oxalic acid, camphorsulfonic acid or a strong acidic ion-exchange resin can be used at temperatures of 20 °C to reflux.
  • any compound of formula 6A, 6B and 6C, wherein at least one of the Rn and R 12 substituents is a group removable by hydrogenolysis can be subjected to catalytic hydrogenolysis in order to remove
  • R' is as defined above, moiety A is a bond or is a divalent carbohydrate linker in deprotected form,
  • Ri is selected from sialyl, -SO 3 H and -CH(R 5 )-COOH, wherein R 5 is as defined above,
  • Ri6 is selected from H and a moiety of formula C
  • Rn and Ris independently, are selected from H and a group removable by hydrogenolysis.
  • divalent carbohydrate linker in deprotected form preferably means any mono-, di- or oligosaccharide glycosyl residue which is attached to the R' group by the C-l (aldoses) or C-2 (ketoses) anomeric carbon, and in the same time it is also attached, via one of its non-glycosidic OH-groups, to the galactosylated aminodeoxy-hexose residue of the compound of formula 7, as defined for moiety B above, in deprotected from, which means that the OH-protective groups and the ester protective group of the N-acetyl neuraminyl moiety (if present) in the divalent
  • the divalent glycosyl moiety A is a lactosyl moiety or an oligosaccharide moiety that consists of a lactosyl moiety and at least one monosaccharide unit selected from the group consisting of galactose, N-acetylglucosamine, fucose and N-acetyl neuraminic acid.
  • the divalent lactosyl moiety is attached to the R' group by its C-l anomeric carbon atom, and at the same time it is also attached, via one of its non-glycosidic OH-groups, preferably via its 3'-OH group to the galactosylated aminodeoxy-hexose residue of the compound of formula 7.
  • the divalent lactosyl moiety defined above can be optionally substituted by other glycosyl residues, preferably on its 3 -OH group by a fucosyl moiety, or on its 6' -OH group by a N-acetyllactosaminyl moiety, which N-acetyllactosaminyl moiety can optionally be further substituted by an N-acetyl neuraminyl moiety on its 6-OH, or by a fucosyl on its 3 -OH or 2' -OH.
  • step d) a compound obtained in step c), that is a compound of formula 7 defined above, is brought into a glycosidase mediated transsialylation or transfucosylation reaction and a compound of formula 10 and salts thereof can be obtained wherein R', Ri, Ri 6 and A are as defined above, R 3 is selected from H and a sialyl, and
  • R 4 is selected from H and a fucosyl moiety, provided that one but only one of R 3 and R 4 is H.
  • a compound of formula 7 is reacted with a sialyl donor, preferably a N-acetyl neuraminyl donor, under the catalysis of an enzyme having a2-3- transsialidase activity.
  • Typical natural sialyl donors can be selected from, but are not limited to, 3'- O-sialyl-lactose, fetuin, gangliosides, O- or N-linked glycopeptides, all of which contain a sialic acid a-2,3-linked to a terminal ⁇ -galactoside residue, or polysialic acid with a-2,8-linkage.
  • the sialyl donor used in step d) can be characterized by formula 8 and salts thereof,
  • Z is selected from the group consisting of azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group D, group E, group F and group G
  • alkylhydrazino dialkylhydrazino or trialkylhydrazino.
  • sialyl donors 4- methylumbelliferyl and optionally substituted phenyl N-acetyl-a-neuraminosides and 3'-0-sialyl- lactose, more commonly /?-nitrophenyl N-acetyl-a-neuraminoside and 3'-0-sialyl-lactose, are of high preference.
  • Enzymes having a2-3-transsialidase activity are preferably selected from a sialidase or
  • transsialidase as described in the following, e.g. from sialidases (EC 3.2.1.18) and transsialidases (EC 2.4.1.-) as classified according to the the GH33 family. They are retaining enzymes. Sialidases and transsialidases are widely distributed in nature. They are found particularly in diverse virus families and bacteria, and also in protozoa, some invertebrates and mammals. These enzymes differ in their biochemical properties, e.g., kinetics, binding affinity or substrate preference. Nevertheless, they possess conserved domains and structural similarities.
  • Transsialidases differ from sialidases since they can transfer sialic acids, preferably a-2, 3 -bonded sialic acids, from a donor molecule to an acceptor derivative, which is preferably a terminal galactose moiety with a ⁇ -interglycosidic linkage. As a result of this transfer, an a-glycosidic bond is formed between the sialic acid and the acceptor. However, if there is no suitable acceptor, the transsialidase hydrolyses the sialic acid.
  • TcTS transsialidase
  • transsialidases have been detected in several other trypanosome types such as Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma brucei brucei and Trypanosoma congolense. Moreover, the existence of transsialidases has been shown in Endotrypanum types, in Corynebacterium diphtheriae and even in human plasma.
  • Sialidases can be classified into two different subgroups, endo- and exo-sialidases.
  • the endo- sialidases hydrolyse sialic acid linkages internal to macromolecules, while the exo-sialidases attack terminal sialic acid linkages, and desialylate glycoproteins, glycopeptides, gangliosides,
  • sialidases from Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis have been identified, cloned and characterized. These sialidases can cleave and so recognize both a-2, 3- and a-2,6-linked sialosides.
  • Sialidases from Bifidobacterium longum subsp. infantis have a consistent preference for a-2,6-linkage whereas sialidases from Bifidobacterium bifidum have a consistent preference for a-2, 3 -linkage.
  • Sialidases which may be employed in the context of the present invention, may also comprise engineered sialidases. Based on sequence and structure comparisons, sialidase from Trypanosoma rangeli may be mutated at six positions, wherein the resulting mutant is able to display a significant level of transsialidase activity (see Paris et al. J. Mol. Biol. 345, 923 (2005)).
  • the enzyme having a sialidase and/or transsialidase activity may be selected from sialidases or transsialidases derived from Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium bifidum JCM1254, Bifidobacterium bifidum S17, Bifidobacterium bifidum PRL2010, Bifidobacterium bifidum NCIMB 41171, Trypanosoma cruzi, etc.
  • enzyme having a sialidase and/or transsialidase activity may be selected from sialidases or transsialidases as defined according to the following deposit numbers: gi
  • siab2 (Bifidobacterium bifidum JCM1254), further sialidases or transsialidases from Bifidobacterium bifidum JCM1254, gi
  • PRL2010 gi
  • sialidases with sialidase/transsialidase activity are listed in the following Table 2:
  • R 3 is a sialyl, preferably N-acetyl-neuraminyl moiety.
  • a compound of formula 7 is reacted with a fucosyl donor under the catalysis of an enzyme having al-2-transfucosidase activity.
  • Typical fucosyl donors can be selected from, but are not limited to, 2'-0-fucosyl-lactose, difucosyl-lactose, and fucose donors of formula 9 wherein Z is selected from the group consisting of azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group D, group E, group F and group G
  • the enzyme exhibiting al-2-transfucosidase activity is preferably selected from fucosidases, transfucosidases and fucosynthases as classified according to EC 3.2.1.38 and 3.2.1.51.
  • a-L- Fucosidases are widely spread in living organisms such as mammals, plants, fungi and bacteria. These enzymes belong to the families 29 and 95 of the glycoside hydrolases (GH29 and GH95) as defined by the CAZY nomenclature (http://www.cazy.org).
  • Fucosidases from GH29 are retaining enzymes (3D structure: ( ⁇ / ⁇ ) 8 ) whereas fucosidases from GH95 are inverting enzymes (3D structure: ( ⁇ / ⁇ ) 6 ).
  • the substrate specificity of the GH29 family is broad whereas that of the GH95 family is strict to al,2-linked fucosyl residues.
  • a-L-Fucosidases generally hydrolyse the terminal fucosyl residue from glycans.
  • Fucosidases which may be employed in the context of the present invention, may also comprise engineered fucosidases.
  • Such engineered fucosidases preferably comprise engineered a-L- fucosidases, preferably engineered fucosidases derived from fucosidases as described above, e.g. an engineered a-l,2-L-fucosynthase from Bifidobacterium bifidum, a-L-fucosynthases from Sulfolobus solfataricus and Thermotoga maritime, etc.
  • Such engineered fucosidases show an acceptor dependent regioselectivity and are devoid of product hydrolysis activity.
  • engineered fucosidases preferably comprise a-L-fucosidase from Thermotoga maritima, which has also been recently converted into an efficient a-L-transfucosidase by directed evolution (see Osanjo et al. Biochemistry 46, 1022 (2007)).
  • the enzyme having a fucosidase and/or trans-fucosidase and/or fucosynthase activity may be selected from a-L-fucosidases derived from Thermotoga maritima MSB 8,
  • Bifidobacterium dentium Bdl or Lactobacillus casei BL23, etc.
  • the enzyme having a fucosidase and/or trans-fucosidase and/or fucosynthase activity may be selected from following a-L-fucosidases as defined according to the following deposit numbers gi
  • 4980806 Thermotoga maritima MSB8
  • 13816464 Sulfolobus solfataricus P2
  • 34451973 Bifidobacterium bifidum JCM 1254
  • 310867039 (Bifidobacterium bifidum PRL2010), gi
  • step d) provides a compound of formula 10B and salts thereof
  • R', Ri, Ri 6 and A are as defined above.
  • the enzymes comprising transsialidase or transfucosidase activity as defined above may also comprise engineered enzymes comprising transsialidase or transfucosidase activity. It is particularly envisaged that wild type or mutated glycosidases displaying a-transsialidase or a- transfucosidase activity can be used in the present invention to produce the target oligosaccharides. Preparation of such enzymes is preferably carried out via site directed mutagenesis approaches or directed evolution.
  • mutants are created via site directed mutagenesis approaches, preferably by introduction of point mutations. This technique generally requires reliance on the static 3D protein structure.
  • the mutations generally affect the active site of the enzymes such that they lose their ability to degrade their transglycosylation products but remain capable of synthesis.
  • a preferred strategy consists of the replacement of the catalytic nucleophile by a non-nucleophilic residue. This modification results in the formation of an inactive mutant or an altered enzyme with reduced transglycosylation activity due the lack of appropriate environment for the formation of the reactive host-guest complex for transglycosylation.
  • a more active glycosyl donor e.g.
  • glycosynthase e.g. fucosynthase
  • their development represents one of the major advances in the use of glycosidases for synthetic purposes.
  • the glycosynthase concept can be applied to all GH specificities and offer a large panel of enzymes potentially able to synthesize various oligosaccharides with very high yields, up to 95%.
  • the second preferred technique is called directed evolution.
  • This strategy comprises random mutagenesis applied to the gene of the selected glycosidase and generates thus a library of genetically diverse genes expressing glycosidase. Generation of sequence diversity can be performed using well-known methodologies, the most preferable being the error prone polymerase chain reaction (epCR) method.
  • epCR error prone polymerase chain reaction
  • This gene library may be inserted into suitable microorganisms such as E. coli or S. cerevisiae for producing recombinant variants with slightly altered properties.
  • Clones expressing improved enzymes are then identified with a fast and reliable screening method, selected and brought into a next round of mutation process.
  • the recursive cycles of mutation, recombination and selection are continued as far as mutant(s) with the desired activity and/or specificity is/are evolved.
  • different high-throughput screening methodologies for glycosidases including glycosynthases have been developed. Applying these approaches, effective engineered transglycosidases, including new and more efficient glycosynthases can and have been created and isolated.
  • An a-L-fucosidase from Thermotoga maritima has been recently converted into an efficient a-L-transfucosidase by directed evolution.
  • the transferase/hydrolysis ratio of the evolved enzyme was 30 times higher than the native enzyme (see Osanjo et al. Biochemistry 46, 1022 (2007)).
  • Proteins comprising a transglycosidase and/or a glycosynthase activity as defined above may also comprise fragments or variants of those protein sequences.
  • Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned proteins sequences of at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, 98% or 99% as compared to the entire wild type sequence on amino acid level.
  • “Fragments” of proteins or peptides in the context of the present invention may also comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide.
  • fragments of nucleic acids in the context of the present invention may comprise a sequence of a nucleic acid as defined herein, which is, with regard to its nucleic acid molecule 5'-, 3'- and/or intrasequentially truncated compared to the nucleic acid molecule of the original (native) nucleic acid molecule.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire nucleic acid as defined herein.
  • “Variants” of proteins or peptides as defined in the context of the present invention may be encoded by the nucleic acid molecule of a polymeric carrier cargo complex.
  • a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property.
  • “Variants” of proteins or peptides as defined in the context of the present invention may also comprise conservative amino acid substitution(s) compared to their native, i.e. non- mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g.
  • CD spectra circular dichroism spectra
  • variants of proteins or peptides as defined herein may also comprise those sequences, wherein nucleotides of the nucleic acid are exchanged according to the degeneration of the genetic code, without leading to an alteration of the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • nucleic acid sequences or amino acid sequences as defined herein preferably the amino acid sequences encoded by a nucleic acid sequence of the polymeric carrier as defined herein or the amino acid sequences themselves
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position.
  • the percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. Proc. Natl. Acad. Sci. USA 90, 5873 (1993) or Altschul et al. Nucleic Acids Res. 25, 3389 (1997). Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • the enzymes used in step d) may be provided in a free form or alternatively be bound to or are immobilized onto a surface. Binding to or immobilization onto a surface may be carried out e.g. via electrostatic bonds, van der Waals-bonds, covalent bonds, etc. Binding to or immobilization onto a surface may be furthermore carried out, using a covalent linker or a crosslinker, or a tag, as known to a skilled person for purification of proteins. Such tags comprise, inter alia, e.g. affinity tags or chromatography tags. Affinity tags may include e.g.
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • Strep-Tag Strep-Tag
  • the poly(His) tag is a widely- used protein tag, that binds to metal matrices. Chromatography tags are used to alter
  • the surface may be the surface of a bioreactor, or any suitable reaction chamber.
  • the enzymatic reaction can be carried out as described in WO 2012/007588, WO 2012/127410 or WO 2012/156897, preferably occurs with a concentration of respective enzyme in a concentration of 1 mU/1 to 1000 U/l, preferably 10 mU/1 to 100 U/l, when the activity capable of forming 1 ⁇ of specific product for a defined protein starting from a defined educt is defined as 1 unit (U), e.g. for a glycotransferase the production of a glycose-containing complex carbohydrate at 37 °C in 1 minute.
  • the activity of each enzyme as defined herein may be assessed with respect to its naturally occurring or engineered substrate.
  • the incubation may be carried out in a reaction medium, preferably an aqueous medium, comprising the compound obtained according to step c) and optionally water; a buffer such as a phosphate buffer, a carbonate buffer, an acetate buffer, a borate buffer, a citrate buffer and a tris buffer, or combinations thereof; alcohol, such as methanol and ethanol; ester such as ethyl acetate; ketone such as acetone; amide such as acetamide; and the like. Furthermore, the incubation may be carried out in a reaction medium as defined above, wherein optionally a surfactant or an organic solvent may be added, if necessary.
  • a surfactant or an organic solvent may be added, if necessary.
  • any surfactant capable of accelerating the formation of a complex carbohydrate as defined according to the present invention as a possible product of the invention can be used as the surfactant.
  • examples include non-ionic surfactants such as poly oxy ethylene octadecylamine (e.g., Nymeen S-215, manufactured by Nippon Oil & Fats); cationic surfactants, such as cetyltrimethylammonium bromide and alkyldimethyl benzylammoniumchloride (e.g., Cation F2-40E, manufactured by Nippon Oil & Fats); anionic surfactants such as lauroyl sarcosinate; tertiary amines such as alkyldimethylamine (e.g., Tertiary Amine FB, manufactured by Nippon Oil & Fats); and the like, which are used alone or as a mixture of two or more.
  • non-ionic surfactants such as poly oxy ethylene octade
  • the surfactant may be used generally in a concentration of 0.1 to 50 g/1.
  • the organic solvent may include xylene, toluene, fatty acid alcohol, acetone, ethyl acetate, and the like, which may be used in a concentration of generally 0.1 to 50 ml/1.
  • the incubation may be furthermore carried out in a reaction medium as defined above, preferably having a pH 3 to 10, pH 5 to 10, preferably pH 6 to 8.
  • the incubation may be furthermore carried out at a temperature of about 0 °C to about 100 °C, preferably at a temperature of about 10 to about 50 °C, e.g. at a temperature of about 20 °C to about 50 °C.
  • inorganic salts such as MnCl 2 and MgCl 2 , may be added, if necessary.
  • the incubation according to step d) of the method of the present invention may be carried out in a bioreactor.
  • the bioreactor is preferably suitable for either a continuous mode or a discontinuous mode. If carried out in a continuous mode, the method preferably provides for a continuous flow of compounds and/or enzymes as necessary, preferably by continuously providing educts of the reaction to the reaction mixture and continuously removing products from the reaction mixture, while maintaining the concentration of all components, including enzymes at a predetermined level.
  • the enzymes used in a continuous mode may be added either in free form or as bound or immobilized to a surface.
  • step e) of the present invention a compound obtained in step d) is subjected to catalytic hydrogenolysis and/or anomeric deprotection.
  • the catalytic hydrogenolysis is performed when, in a compound of formula 10 obtained in step d), R' is -N 3 or -OR' 6 wherein R' 6 is a group removable by hydrogenolysis or - (CH 2 ) n -N3, or when Ri 6 is a moiety C wherein at least one of the Rn and Ris groups is a group removable by hydrogenolysis and R' is 2-trimethylsilyl-ethyloxy.
  • Such catalytic hydrogenolysis typically takes place in a protic solvent or in a mixture of protic solvents.
  • a protic solvent may be selected from the group consisting of water, acetic acid or Ci-C 6 alcohols.
  • a mixture of one or more protic solvents with one or more suitable aprotic organic solvents partially or fully miscible with the protic solvent(s) may also be used.
  • suitable aprotic organic solvents partially or fully miscible with the protic solvent(s) (such as THF, dioxane, ethyl acetate or acetone) 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. Solutions containing the carbohydrate derivatives in any concentration or suspensions of the carbohydrate derivatives in the solvent(s) used are also applicable.
  • the reaction mixture is stirred at a temperature in the range of 10-100 °C, preferably between 20-50 °C, in a hydrogen atmosphere of 1-50 bar absolute (100 to 5000 kPa) 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.
  • a catalyst such as palladium, Raney nickel or any other appropriate metal catalyst, preferably palladium on charcoal or palladium black
  • Transfer hydrogenolysis 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 and/or acidic ion exchange resins can also be used to improve the kinetics of the hydrogenolysis.
  • anomeric azide group (R' is -N 3 ) in a the above-mentioned compound of formula 10 is reduced to amino under the conditions disclosed above, and the thus formed glycosyl amine easily undergoes hydrolysis to give the anomerically unprotected compound of formula 1 (R is OH).
  • R' -0-(CH 2 ) n -N3 in a compound of formula 10 is converted to -0-(CH 2 ) n - H 2 under these conditions.
  • R' group being -N 3 or -OR' 6 wherein R' 6 is selected from allyl optionally substituted by one or more methyl or 2-trimethylsilyl-ethyl in a compound of formula 10 obtained in step d) - provided that in moiety C, if it is present in a compound of formula 10, both Rn and Ri 8 groups are H - can be converted to OH in an anomeric deprotection reaction.
  • the R' group being -N 3 in a compound of formula 10 can be reduced by complex metal hydrides like NaBH 4 , or by PPh 3 or Cu/Zn.
  • 2- Trimethylsilyl-ethyl glycosides (R' is -OR' 6 wherein R' 6 is 2-trimethylsilyl-ethyl) can be deprotected with fluoride ion (from BF " ), an anion of a strong acid (e.g. trifluoroacetate) or a Lewis-acid (BF 3 -etherate, ZnCl 2 , SnCl 4 , FeCl 3 ) followed by careful hydrolysis of the resulting glycosyloxy derivative substituted with the remains of the reagent that is used (see Jansson et al. J. Org. Chem. 53, 5629 (1988)).
  • fluoride ion from BF "
  • an anion of a strong acid e.g. trifluoroacetate
  • a Lewis-acid BF 3 -etherate, ZnCl 2 , SnCl 4 , FeCl 3
  • R' is -N 3 or -OR' 6 wherein R' 6 is selected from allyl optionally substituted by one or more methyl or 2-trimethylsilyl-ethyl, and in moiety C at least one of the Rn and Ri 8 groups is a group removable by hydrogenolysis
  • the anomeric deprotection reaction is followed by catalytic hydrogenolysis to give compounds of formula 1 wherein R is -OH and R 2 is fucosyl moiety.
  • R is selected from -OH, -N 3 and -OR 5 wherein 5 is selected from allyl optionally substituted by one or more methyl, propargyl optionally substituted by one or more methyl, 2-trimethylsilyl-ethyl, -(CH 2 ) n -NH 2 and -(CH 2 ) n -N3 wherein integer n is i to 10, preferably 2 or 3,
  • Ri is selected from sialyl moiety, -SO 3 H and -CH(R 5 )-COOH wherein R 5 is selected from H, alkyl and benzyl,
  • R 2 is selected from H and fucosyl moiety
  • R 3 is selected from H and sialyl moiety
  • R 4 is selected from H and fucosyl moiety, provided that one but only one of R 3 and R 4 is H, and
  • A is selected from a bond and a divalent carbohydrate linker, can be effectively synthesized.
  • Both the anomerically deprotected derivatives (R is -OH), and the glycosides listed above have synthetic usefulness.
  • the unsaturated bond of glycosides having -N 3 , -0-(CH 2 ) n -N 3 , -O-allyl or -O-propargyl aglycon or the -0-(CH 2 ) n -NH 2 group can be functionalized by a wide variety of selective and mild water-compatible chemical reactions. Thus e.g.
  • the azido group of a compound of formula 1 can be brought into "click chemistry" with an alkyne to form bioconjugates (and so can the propargyl derivatives of formula 1 with an azido reagent).
  • the allyl functionality can be converted to other functional groups by addition reactions to the double bond or by ozonolysis.
  • the amino function can be used to make an amide or urea linkage to bind a compound of formula 1 to (bio)macromolecules/polymers.
  • the present method readily provides the synthesis of complex sialylated and/or fucosylated 3'-0- galactosyl-6-O-sialyl-GlcNAc or -GalNAc derivatives, glycosides and analogs thereof, by a unique combination of chemical and enzymatic glycosylation steps in the reaction sequence.
  • the process opens the possibility, via the permutation of the mandatory and optional glycosylation steps, to obtain a number of compounds being substituted by sialyl and/or fucosyl moiety/moieties in various positions.
  • sialyl residue at position 6 can be replaced by simpler charged residues like carboxymethyl or sulfate while maintaining the biological effect of the parent sialylated compounds (see e.g. Chachadi et al. Glycoconj. J. 28, 49 (2011), WO 2011/130332, Liao et al. J. Am. Chem. Soc. 132, 14849 (2010), Wang et al. Proc. Nat. Acad. Sci. USA 106, 18137 (2009), Schwardt et al. J. Med. Chem. 52, 989 (2009)).
  • the method disclosed above gives the chance for the synthesis of these kinds of analogs as well when the chemical sialylation in step a) is replaced by a chemical carboxymethylation or sulfation.
  • the present method is advantageous in that these various groups of compounds can be obtained in a simpler manner and/or in fewer steps.
  • Ando et al. ⁇ Carbohydr. Res. 338, 503 (2003)) taught that 6-O-sialylation of "bulky" acceptors where the OH-group to be sialylated belonged to an internal GlcNAc residue was not favourable because only low yields could be achieved. Therefore it was proposed to sialylate acceptors having a terminal GlcNAc moiety.
  • the present inventors found that compounds of formula 2 having internal GlcNAc can be sialylated effectively.
  • all the prior art methods (Ando et al. J. Carbohydr. Chem. 20, 425 (2001) and
  • the present method avoids the fabrication and use of this type of difficult to obtain disaccharide donor, and instead introduces a direct enzymatic sialylation of intermediates of formula 7 obtained in step c) of the present method that takes place in good regio- and stereoselectivity.
  • the intermediate of formula 7 can be a substrate for enzymatic fucosylation as well, and therefore the method is able to provide a diverse group of variously sialylated and/or fucosylated 3'-0-galactosyl-6-0-sialyl-GlcNAc or -GalNAc derivatives and analogs thereof.
  • compounds of formula 1 wherein A is a divalent carbohydrate linker are preferred. More preferably, compounds of formula 1 are human milk oligosaccharides listed in Table 1 above and derivatives, even more preferably F-LST b, DS-LNT or FDS-LNT I and derivatives. Accordingly, a compound of formula 2D falling into the scope of compounds of formula 2 wherein Y and R 7 are as defined above,
  • R' is as defined above, preferably R' is OR" wherein R" is a group removable by
  • B' is a divalent lactosyl moiety having the R' group on its C-l anomeric carbon atom and attached via its 3'-OH group to the lacto-N-biose residue of the compound of formula 2D, optionally substituted by a fucosyl moiety on its 3-OH or by a N-acetyllactosaminyl moiety on its 6'-OH, which N-acetyllactosaminyl moiety can optionally be further substituted by an N-acetylneuraminyl moiety on its 6-OH, or by a fucosyl on its 3-OH or 2' -OH, and the functional groups of the divalent lactosyl residue B' are protected, preferably the free OH groups are acylated (e.g.
  • R', B', Y and R 7 are as defined above, and Ri9 is selected from -S0 3 H, -CH(R 5 )-COORi 0 and moiety H
  • R20 is independently acyl
  • R21 is selected from H and acyl, more preferably a compound of formula 2E wherein Y is selected from - HAc and
  • R 7 groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl
  • R20 groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl
  • R21 is selected from H, acetyl, benzoyl or 4-chlorobenzoyl, is reacted with a sialyl donor of formula 3 defined above to give a compound of formula 4E
  • Y is selected from -NHAc, haloalkanoylamido, -NAc 2 , haloalkoxycarbonylamino, 2,3- diphenylmaleimido, 2,3-dimethylmaleimido and azido, preferably -NHAc and
  • R 7 is independently acyl, preferably R 7 groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl,
  • R 8 is independently acyl, preferably Rs groups are acetyl,
  • R 2 o is independently acyl, preferably R 2 o groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl,
  • R 21 is selected from H and acyl, preferably R 2 i is selected from H, acetyl, benzoyl or 4- chlorobenzoyl, and
  • Q is selected from alkyl and benzyl, preferably methyl, ethyl and benzyl.
  • a compound of formula 4D preferably of formula 4E, is optionally fucosylated with a fucosyl donor of formula 5A
  • XA is selected from alkylthio and optionally substituted phenylthio, preferably -SPh,
  • R 22 and R 23 are, independently, selected from a group removable by hydrogenolysis and acyl, preferably acetyl, pivaloyl, benzoyl and 4-chlorobenzoyl, to give rise to a compound of formula 6D wherein R', B', Y, R 7 and R19 are as defined above, and R24 is a moiety I
  • R" is a group removable by hydrogenolysis
  • Y is selected from -NHAc, haloalkanoylamido, -NAc 2 , haloalkoxycarbonylamino, 2,3- diphenylmaleimido, 2,3-dimethylmaleimido and azido, preferably -NHAc and
  • R 7 is independently acyl, preferably R 7 groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl,
  • R 8 is independently acyl, preferably Rs groups are acetyl,
  • R 2 o is independently acyl, preferably R 20 groups are identical and selected from acetyl, benzoyl and 4-chlorobenzoyl,
  • R 2 i is selected from H and acyl, preferably R 2 i is selected from H, acetyl, benzoyl or 4- chlorobenzoyl, and Q is selected from alkyl and benzyl, preferably methyl, ethyl and benzyl, and
  • R-24 is moiety I as defined above.
  • step c) a compound of formula 4D obtained in step a) or a compound of formula 6D obtained in step b) is subjected to deprotective treatment and, where necessary, transformation of Y to
  • moiety A' is a divalent lactosyl moiety having the R' group on its C-1 anomeric carbon atom and attached via its 3'-OH group to the lacto-N-biose residue of the compound of formula 7A, optionally substituted by a fucosyl moiety on its 3-OH or by a N-acetyllactosaminyl moiety on its 6'-OH, which N-acetyllactosaminyl moiety can optionally be further substituted by an N-acetylneuraminyl moiety on its 6-OH, or by a fucosyl on its 3-OH or 2' -OH,
  • Ri is selected from sialyl, -SO 3 H and -CH(R 5 )-COOH, wherein R 5 is as defined above, and
  • Ri6 is selected from H and a moiety of formula C
  • Rn and Ris independently, are selected from H and a group removable by hydrogenolysis, comprising the steps of i) base catalysed transesterification to remove O-acyl groups,
  • step c) of the method a compound of formula 4E or a compound of formula 6E defined above is converted into a compound of formula 7B
  • R" is a group removable by hydrogenolysis
  • Ri6 is selected from H and moiety C defined above, preferably H comprising: i) base catalysed transesterification deprotection, preferably NaOMe/MeOH treatment, ii) basic hydrolysis, preferably NaOH/MeOH treatment, and
  • haloalkoxycarbonylamino, 2,3-diphenylmaleimido, 2,3-dimethylmaleimido and azido, preferably trichloroacetamido, is deprotected to amino under the conditions used in step ii) or by other azido-amino transformations disclosed above.
  • a compound of formula 7A is sialylated or fucosylated under the action of a sialidase or fucosidase having transsialidase or transfucosidase/fucosynthase activity, respectively, to give a compound of formula IOC
  • R', A', Ri, R 3 , R 4 and Ri 6 are as defined above, provided that one but only one of the R 3 and R 4 is H.
  • a compound according to formula 7B is reacted with a sialyl donor, preferably a N- acetyl neuraminyl donor selected from 3'-0-sialyllactose or /?-nitrophenyl N-acetyl-a- neuraminoside in the presence of transsialidase, preferably a transsialidase listed in Table 2 above, to make a compound of formula 10D
  • a compound of formula 7A is subjected to enzymatic fucosylation under the action of a fucosidase having transfucosidase/fucosynthase activity, preferably those listed in Table 3, in the presence of a fucosyl donor selected from 2-O-fucosyllactose or fucosyl fluoride to give a compound of formula 10E
  • R" and Ri 6 are as defined above.
  • step e if it is desired, a compound of formula IOC is subjected to catalytic
  • the hydrogenolysis of a compound of formula 10D provides a compound of formula IB
  • R 2 is selected from H and fucosyl, that is DS-LNT and FDS-LNT I, respectively.
  • R 2 is selected from H and fucosyl, that is DS-LNT and FDS-LNT I, respectively.
  • R 2 is as defined above, is easily accessible.
  • R 2 is H in a compound of formula 1C that corresponds to F-LST b.
  • the method according to the first aspect of the invention involves useful novel synthetic intermediates for the synthesis of sialylated/fucosylated oligosaccharides.
  • the second aspect of the invention provides a compound of formula 11 and salts thereof
  • R' is selected from -N 3 and -OR' 6 which R' 6 is selected from allyl optionally substituted by one ore more methyl, propargyl optionally substituted by one ore more methyl, a group removable by hydrogenolysis, 2-trimethylsilyl-ethyl and -(CH 2 ) n -N3 wherein integer n is 1 to 10, preferably 2 or 3,
  • A' is a divalent lactosyl moiety having the R' group on its C-l anomeric carbon atom and attached via its 3'-OH group to the lacto-N-biose residue of the compound of formula 11, A' being optionally substituted by a fucosyl moiety on its 3-OH or by a N-acetyllactosaminyl moiety on its 6'-OH, which N-acetyllactosaminyl moiety can optionally be further substituted by an N-acetylneuraminyl moiety on its 6-OH, or by a fucosyl on its 3-OH or 2' -OH,
  • Ri is selected from sialyl moiety, -SO 3 H and -CH(R 5 )-COOH wherein R 5 is selected from H, alkyl and benzyl,
  • R 3 is selected from H and sialyl moiety
  • R 4 is selected from H and fucosyl moiety, provided that at least one of R 3 and R 4 is H, and Ri 6 is selected from H and moiety C, preferably H,
  • Rn and Ris independently, are selected from H and a group removable by hydrogenolysis provided that the di sodium salt of the following compound is excluded: wherein R' is 2- trimethylsilyl-ethyl, A' is unsubstituted lactosyl, Ri and R 3 are sialyl moieties, and R 4 and Ri 6 are H.
  • R' is -OR" wherein R" is a group removable by hydrogenolysis.
  • a compound of formula 11 can be either an a- or ⁇ -anomer or an anomeric mixture of a- and ⁇ - anomers. It can be crystalline solid, oil, syrup, precipitated amorphous material or spray dried product. If crystalline, a compound of formula 11 can exist either in an anhydrous or hydrated crystalline form by incorporating one or several molecules of water into its crystal structure.
  • a compound of formula 11 can exist as a crystalline substance, incorporating ligands such as organic molecules and/or ions into its crystal structure.
  • a preferred compound of formula 11 is a compound of formula IOC being a precursor for DS-LNT, FDS-LNT I and F-LST b and analogs thereof
  • R', A', Ri, R 3 , R 4 and Ri 6 are as defined above, provided that one but only one of R 3 and R 4 is H, particularly a compound of formula 10D
  • Ri6 is selected from H and moiety C defined above, preferably H, and also particularly a compound of formula 10E wherein R" is a group removable by hydrogenolysis, and Ri 6 is selected from H and moiety C defined above, preferably H.
  • a compound of formula 11 is characterized by formula 7A
  • a preferred compound of formula 7A is a compound of formula 7B
  • R" is a group removable by hydrogenolysis
  • Ri 6 is selected from H and moiety C defined above, preferably H.
  • Step 1 A solution of the compound from example 1 (6.57 g) in dry MeOH (50 ml) was treated with 25% (w/w) methanolic MeONa (0.5 ml) and stirred at r.t. for 21 hours, quenched with AcOH and evaporated in vacuo. The obtained residue (6.14 g) was sonicated with Et 2 0 and filtered (5x40 ml). Alternatively, methyl benzoates could be extracted by partitioning between water and organic solvent such as ether or hexane. The compound was dried in vacuum oven to give 3.90 g of a crude material.
  • Step 2 The obtained solid from step 1 was taken up in water (30 ml) containing NaOH (0.514 g) and stirred at r.t. for 3 days and then at 60 °C for 2 hrs. The mixture was neutralized with AcOH and extracted with ether. The aqueous phase gave 5.52 g of solid after evaporation.
  • LC-HRMS calc. for [C 4 2H 66 N 2 028-H] " 1045.3729, found 1045.3732.
  • Step 3 The product from step 2 was taken up in pyridine and Ac 2 0 mixture (50 ml each) and stirred at 50 °C for 30 min then at r.t. for 18 hours. Extra amount of acetic anhydride (20 ml) was added and the mixture was stirred at 40 °C for 2 hours. The volatiles were removed in vacuo and the residue was coevaporated with toluene then partitioned between CH 2 C1 2 (200 ml) and 0.5 M HC1 (50 ml). The aqueous phase was re-extracted with CH 2 C1 2 (3x20 ml). Combined organic solution was dried and evaporated to give 5.0 g of crude product as white solid. It was chromatographed on silica to give 3.89 g of the title compound (80.5 %).
  • reaction mixtures were stirred at 20 °C for 24 hours and the conversion of the acceptor to the desired product was determined by HPLC. Conversion yield was estimated to be from 70 to 80 % when using 3'-0-sialyllactose or from 40 to 50 % when using /?-nitrophenyl N-acetyl-a-neuraminoside.
  • Reaction mixtures were loaded on a Dowex 1 (HC0 3 " -form) column. After washing with distilled water, the mono-acidic compounds (3'-0-sialyllactose or sialic acid and the remaining acceptor) were eluted with a 60 mM NaHC0 3 solution. The product was then eluted with a 125 mM NaHC0 3 solution. The eluted fractions containing acidic oligosaccharides were analysed by HPLC and pooled. The NaHC0 3 was removed by treating with Amberlite IR120 (H + form) until pH 3.0 was reached. The pH was then adjusted to 6.0 with NaOH. The mono-acidic fractions were freeze-dried and reused with another enzymatic run. The fractions containing the products were freeze-dried.

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Abstract

Cette invention concerne une synthèse chimio-enzymatique d'oligosaccharides de formule 1, dans laquelle R est choisi parmi -OH, ‑N3 et -OR6, où R6 est choisi parmi un allyle facultativement substitué par un ou plusieurs méthyle, propargyle facultativement substitué par un ou plusieurs méthyle, 2-triméthylsilyl-éthyle, -(CH2)n -NH2 et -(CH2)n -N3, où l'entier n va jusqu'à 10, étant de préférence 2 ou 3, R1 est choisi parmi une fraction sialyle, -SO3H et -CH(R5)-COOH où R5 est choisi parmi H, alkyle et benzyle, R2 est choisi parmi H et fucosyle, R3 est choisi parmi H et sialyle, R4 est choisi parmi H et fucosyle, à la condition qu'un mais seulement un de R3 et R4 représente H, et A est choisi parmi une liaison et une séquence de liaison glucide divalent, ayant d'importantes activités biologiques et une valeur commerciale significative pour l'industrie pharmaceutique et alimentaire.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021061991A1 (fr) 2019-09-24 2021-04-01 Prolacta Bioscience, Inc. Compositions et procédés de traitement de maladies inflammatoires et immunitaires
EP3645146A4 (fr) * 2017-06-30 2021-05-05 Glycom A/S Synthèse d'oligosaccharides
WO2022036225A1 (fr) 2020-08-14 2022-02-17 Prolacta Bioscience, Inc. Compositions d'oligosaccharide de lait humain destinées à être utilisées avec des bactériothérapies
WO2022155201A1 (fr) 2021-01-12 2022-07-21 Prolacta Bioscience, Inc. Régimes de traitement symbiotique

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EP0319253A2 (fr) * 1987-12-02 1989-06-07 Alberta Research Council Glycosides de l'acide sialique, antigènes, immunoadsorbants et procédé de leur préparation
WO2003089574A2 (fr) * 2002-04-15 2003-10-30 Biomira, Inc. Glycolipopeptides de synthese utilises comme vaccins
WO2005116088A2 (fr) * 2004-05-25 2005-12-08 The Johns Hopkins University Methodes et compositions pour traiter des maladies et des troubles associes a des cellules exprimant siglec-8
EP1650226A1 (fr) * 2003-07-28 2006-04-26 Yasuhiro Kajihara Derives amines a chaine de sucre de type complexe et procede de realisation associe
WO2011100979A1 (fr) * 2010-02-19 2011-08-25 Glycom A/S Production de 6'-o-sialyllactose et intermédiaires
CN103555796A (zh) * 2013-10-24 2014-02-05 山东大学 双唾液酸化四糖的合成方法

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EP0319253A2 (fr) * 1987-12-02 1989-06-07 Alberta Research Council Glycosides de l'acide sialique, antigènes, immunoadsorbants et procédé de leur préparation
WO2003089574A2 (fr) * 2002-04-15 2003-10-30 Biomira, Inc. Glycolipopeptides de synthese utilises comme vaccins
EP1650226A1 (fr) * 2003-07-28 2006-04-26 Yasuhiro Kajihara Derives amines a chaine de sucre de type complexe et procede de realisation associe
WO2005116088A2 (fr) * 2004-05-25 2005-12-08 The Johns Hopkins University Methodes et compositions pour traiter des maladies et des troubles associes a des cellules exprimant siglec-8
WO2011100979A1 (fr) * 2010-02-19 2011-08-25 Glycom A/S Production de 6'-o-sialyllactose et intermédiaires
CN103555796A (zh) * 2013-10-24 2014-02-05 山东大学 双唾液酸化四糖的合成方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3645146A4 (fr) * 2017-06-30 2021-05-05 Glycom A/S Synthèse d'oligosaccharides
US11214588B2 (en) 2017-06-30 2022-01-04 Glycom A/S Synthesis of oligosaccharides
US11919919B2 (en) 2017-06-30 2024-03-05 Glycom A/S Synthesis of oligosaccharides
WO2021061991A1 (fr) 2019-09-24 2021-04-01 Prolacta Bioscience, Inc. Compositions et procédés de traitement de maladies inflammatoires et immunitaires
WO2022036225A1 (fr) 2020-08-14 2022-02-17 Prolacta Bioscience, Inc. Compositions d'oligosaccharide de lait humain destinées à être utilisées avec des bactériothérapies
WO2022155201A1 (fr) 2021-01-12 2022-07-21 Prolacta Bioscience, Inc. Régimes de traitement symbiotique

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