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WO2013091660A1 - Procédé pour obtenir des précurseurs de lacto-n-tétraose et de lacto-n-neotétraose cristallin et des mélanges de ceux-ci - Google Patents

Procédé pour obtenir des précurseurs de lacto-n-tétraose et de lacto-n-neotétraose cristallin et des mélanges de ceux-ci Download PDF

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Publication number
WO2013091660A1
WO2013091660A1 PCT/DK2012/050502 DK2012050502W WO2013091660A1 WO 2013091660 A1 WO2013091660 A1 WO 2013091660A1 DK 2012050502 W DK2012050502 W DK 2012050502W WO 2013091660 A1 WO2013091660 A1 WO 2013091660A1
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Prior art keywords
formula
mixture
compound
lnnt
lnt
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Inventor
Markus Hederos
Gyula Dekany
Martin MATWIEJUK
Rémy DUREAU
Ignacio FIGUEROA PÉREZ
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Glycom AS
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Glycom AS
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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
    • 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
    • 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
    • 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    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen

Definitions

  • the present invention relates to a process for producing a mixture of LNT (lacto-N- tetraose, Gal( 31 -3)GlcNAc( 31 -3)Gal( 31 -4)Glc) and LNnT (lacto-N-neotetraose, Gal( 31 -4)GlcNAc( 31 -3)Gal( 31 -4)Glc) and for separating the mixture and its
  • HMOs Human milk oligosaccharides
  • LNT (Scheme 1 ) and LNnT (Scheme 2) are considered to be among the more important HMOs.
  • This invention relates to a mixture consisting essentially of LNT and LNnT.
  • Another aspect of this invention relates to a mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2:
  • R is a group removable by hydrogenolysis and R 3 ' is a group removable by hydrogenolysis or H.
  • a further aspect of this invention relates to a method of obtaining a crystalline compound of formula 2 and optionally a separate crystalline compound of formula 1 by: a) dissolving a a mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2 in water, b) adding 2.5-3.5 fold volume of a CrC 4 alcohol, preferably methanol, or a solvent mixture of C C 4 alcohols, to the solution obtained in step a), c) crystallizing the compound of formula 2 from the solution obtained in step b), and forming a mother liquor containing the compound of formula 1 , and then optionally d) removing the C C 4 alcohol from the mother liquor to give an aqueous solution, e) adding 3-5 fold volume of a ketone type solvent to the solution obtained in step e), and f) then crystallizing the compound of formula 1 from the mixture obtained in step e).
  • a further aspect of this invention relates to a method of obtaining a crystalline compound of formula 1 and optionally a separate crystalline compound of formula 2 by: a) dissolving a mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2 in water, b) optionally adding 1 /5 to 1 /10 parts of a CrC 4 alcohol, or a solvent mixture of Ci-C 4 alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a C C 4 alcohol, in proportion to the volume of the aqueous solution obtained in step a), c) crystallizing the compound of formula 1 from the aqueous solution
  • step a) or optionally from the solution obtained in step b), and forming a mother liquor containing the compound of formula 2, and then optionally d) treating the mother liquor obtained in step c) with at least a double
  • a still further aspect of this invention relates to a method for obtaining a crystalline mixture, preferably a crystalline mixture consisting essentially of, compounds of formulas 1 and 2 by: a) dissolving a mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2 in water, b) adding at least fivefold volumes of a C C 4 alcohol, or a solvent mixture of Ci-C 4 alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a CrC 4 alcohol, to the aqueous solution obtained in step a), c) crystallizing a mixture of, preferably a mixture consisting essentially of, compounds of formulas 1 and 2 from the mixture obtained in step b).
  • Still another aspect of this invention relates to method for the preparation of a mixture of, preferably a mixture consisting essentially of, compounds of formulas 1 and 2 by deprotecting a mixture of, preferably a mixture consisting essentially of, compounds of formulas 3 and 4:
  • R is a group removable by hydrogenolysis
  • Ri is acyl
  • R 2 is selected from acyl and H
  • R 3 is selected from a group removable by hydrogenolysis
  • R 4 is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAC 2 , benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino,
  • Yet another aspect of this invention relates to a compound of formula 7
  • R 6 is, independently, H or a residue of formula A
  • R, R-i, R 2 , R 3 ,R 4 and Y are as above, particularly a compound of formula 3
  • R, R-i, R 2 , R3, R 4 and Y are as defined above.
  • a further aspect of this invention relates to a method for the preparation of a mixture of, preferably a mixture consisting essentially of, compounds of formulas 3 and 4, by: reacting a donor of formula 6
  • R 4 is acyl
  • Still another aspect of this invention relates to process for the preparation of a mixture consisting essentially of LNT and LNnT, comprising a step of subjecting a mixtureof, preferably a mixture consisting essentially of, compounds of formulas 1 and 2 to catalytic hydrogenolysis.
  • composition composition and nutritional formulation.
  • group removable by hydrogenolysis preferably means a protective group whose C-O bond to the 1 -oxygen can be cleaved by hydrogen in the presence of a catalytic amount of palladium, Raney nickel or any other conventional hydrogenolysis catalyst to regenerate a 1 -OH group.
  • protective groups are described in Wuts and Greene: Protective Groups in Organic Synthesis, John Wiley & Sons, 2007, and include benzyl, diphenylmethyl
  • benzyloxymethyl or benzyloxycarbonyl groups each of which can optionally be substituted by one or more of the following substituent groups: alkyl, alkoxy, phenyl, amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl, alkoxycarbonyl, carbamoyl, A/-alkylcarbamoyl, A/,A/-dialkylcarbamoyl, azido, halogenalkyl or halogen.
  • any such substituent group if present, is on an aromatic ring.
  • a preferred protective group is benzyl optionally substituted with one or more of the following substituent groups: phenyl, alkyl and halogen, particularly unsubstituted benzyl, 4- chlorobenzyl, 3-phenylbenzyl and 4-methylbenzyl groups.
  • substituent groups phenyl, alkyl and halogen, particularly unsubstituted benzyl, 4- chlorobenzyl, 3-phenylbenzyl and 4-methylbenzyl groups.
  • oligosaccharide products via evaporation and/or extraction processes.
  • alkyl preferably means a linear or branched chain saturated hydrocarbon group with 1 -6 carbon atoms, such as methyl, ethyl, n-propyl, /-propyl, n-butyl, /-butyl, s-butyl, f-butyl or n-hexyl.
  • aryl preferably means a homoaromatic group such as phenyl or naphthyl.
  • Benzyl preferably means phenylmethyl.
  • alkyloxy or “alkoxy” preferably means an alkyl group attached to a parent molecular moiety through an oxygen atom, such as methoxy, ethoxy or f-butoxy.
  • Halogen preferably means fluoro, chloro, bromo or iodo.
  • Haloalkyl preferably means alkyl substituted by at least one halogen, such as chloromethyl, trichloromethyl, trifluoromethyl or 2,2,2-trichloroethyl.
  • Amino refers to a -NH 2 group.
  • Alkylamino preferably means an alkyl group (see above) attached to a parent molecular moiety through an -NH-group, such as methylamino, ethylamino, etc.
  • “Dialkylamino” preferably means two alkyl groups, either identical or different, attached to its parent molecular moiety through a nitrogen atom, such as dimethylamino or diethylamino.
  • Acylamino or “acylamido” or “amido” preferably mean an acyl group attached to its parent molecular moiety through an -NH-group, such as acetylamino (acetamido) or benzoylamino (benzamido).
  • Alkanoylamido” or “alkanoylamino” preferably means an acylamino group having alkyl chain, such as formamido, acetylamino (acetamido), propionyalmino (propionamido), etc.
  • “Haloalkanoylamido” or “haloalkanoylamino” preferably means an acylamino having an haloalkyl chain, such as
  • Carboxyl preferably means an -COOH group.
  • alkyl or aryl (e.g., phenyl) moieties in any of the above-mentioned groups can either be unsubstituted or substituted with one or more of the following groups, alkyl (only for aryl moieties), halogen, nitro, aryl, alkoxy, amino, alkylamino, dialkylamino, acylamino, carboxyl, alkoxycarbonyl, carbamoyl, A/-alkylcarbamoyl, N,N- dialkylcarbamoyl, azido, halogenalkyl or hydroxyalkyl, to provide groups such as chloroacetyl, trichloroacetyl, 4-chlorobenzoyl, 4-methylbenzyl and 4-chlorobenzyl.
  • sil group preferably means a protective group containing a silicon atom covalently bonded to the oxygen atom of a hydroxy group to be protected (silyl ethers).
  • This kind of group is well-known, and many examples are referred to by P.G.M. Wuts and T.W. Greene: Protective groups in organic synthesis John Wiley & Sons (2007), such as trimethylsilyl, triisopropylsilyl, f-butyldimethylsilyl and f-butyldiphenylsilyl.
  • the silyl ethers are labile under mild acidic conditions.
  • acetal type group means a protective group that, with the oxygen atom of a hydroxyl group to be protected, forms the following general structure with two singly bonded oxygens attached to the same carbon atom: OH-group to be protected
  • R a , R b and R c are carbon-bonded groups.
  • acetal type protective groups are labile under mild acidic conditions.
  • the term "consisting essentially of” preferably means at least 90%, especially at least 95%, particularly at least 98% of the relevant content of a material.
  • One aspect of this invention relates to a mixture of, preferably a mixture consisting essentially of, an LNT precursor of formula 1 and an LNnT precursor of formula 2:
  • R is a group removable by hydrogenolysis
  • R3' is selected from a group removable by hydrogenolysis and H.
  • R is benzyl, 4- methylbenzyl or 4-chlorobenzyl, more preferably benzyl
  • R 3 ' is preferably H
  • the -OR is in ⁇ -orientation.
  • the mixture consists essentially of LNT- OBn (a compound of formula 1 wherein R is benzyl and R 3 ' is H) and LNnT-OBn (a compound of formula 2 wherein R is benzyl and R 3 ' is H), both in the form of ⁇ - glycoside.
  • the mixture preferably consisting essentially of compounds of formulas 1 and 2, can be used as a starting material for making a mixture consisting essentially of LNnT and LNT or providing LNnT or LNT or both as discreet individual HMOs.
  • Another aspect of the invention relates to a method of providing a mixture consisting essentially of LNnT and LNT comprising the step of subjecting the mixture of, preferably a mixture consisting essentially of, compounds of formulas 1 and 2 to catalytic hydrogenolysis.
  • the catalytic hydrogenolysis that is removal of the R-group and R3'-group if present, can be carried out in a protic solvent or in a mixture of protic solvents.
  • the protic solvent can be water, acetic acid or a CrC 6 alcohol.
  • a mixture of one or more protic solvents with one or more aprotic organic solvents that are partially or fully miscible with the protic solvent(s), such as THF, dioxane, ethyl acetate or acetone, can also be used.
  • Water, one or more C 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 compounds of formulas 1 and 2 in any concentration or suspensions of the compounds of formulas 1 and 2 with the solvent(s) can also be used.
  • the reaction mixture can be stirred at 10-100 °C temperature range, preferably between 20-70 °C in 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 can 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 can also be carried out by generating hydrogen in situ from cyclohexene, cyclohexadiene, formic acid or ammonium formate.
  • Addition of an organic or inorganic base or acid and/or a basic and/or acidic ion exchange resin 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 compounds of formula 1 and 2.
  • Preferred organic bases include triethylamine, diisopropyl ethylamine, ammonia, ammonium carbamate and diethylamine.
  • Preferred organic/inorganic acids include formic acid, acetic acid, propionic acid, chloroacetic acid, dichloroacetic acid, triflouroacetic acid, HCI and HBr.
  • the conditions described above allow for simple, convenient and delicate removal of the solvent(s), to leave a pure LNnT and LNT mixture.
  • the mixture can then be isolated from the reaction milieu in crystalline, amorphous solid or syrupy form or in a concentrated aqueous solution, using conventional work-up procedures.
  • a mixture of 1 -O-benzyl LNnT (LNnT-OBn) and 1 -0- benzyl LNT (LNT-OBn) is subjected to catalytic hydrogenolysis to give a mixture of, preferably a mixture consisting essentially of, LNnT and LNT.
  • the hydrogenation can be performed in water or in aqueous alcohol, preferably in water/methanol or water/ethanol mixture (alcohol content: 10-50 v/v %) at 15-65 °C, preferably between 50-65 °C.
  • the concentration of the starting materia 1 can be between 140 and 230 g/l, and the catalyst concentration can be between 0.4 % and 1 .2 % (weight of the metal content based on the weight of the carbohydrates).
  • Both solid forms of the resulting LNnT/LNT mixture such as amorphous, freeze dried, or spray dried forms and liquid forms of the LNnT/LNT mixture such as aqueous solutions or syrups have very high purity, suitable for pharmaceutical, therapeutic or nutritional use, especially for use in food supplements and for infant nutritional use in, for example, infant formulas, infant cereals and clinical infant nutritional products.
  • a further aspect of the invention relates to a method of providing the mixture of, preferably the mixture consisting essentially of, compounds of formulas 1 and 2, comprising the step of deprotecting the OR-i, OR2, OR3, OR 4 groups, but not the OR group, and not the OR 3 group when R 3 means a group removable hydrogenolysis, and of deprotecting or transforming Y group, of the compounds of formulas 3 and 4 in a mixture thereof, preferably in a mixture consisting essentially thereof
  • R is a group removable by hydrogenolysis
  • Ri is acyl
  • R2 is acyl or H
  • R3 is selected from a group removable by hydrogenolysis
  • R 4 is acyl and Y is selected from alkanoylamido
  • haloalkoxycarbonylamino benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3-diphenylmaleimido and 2,3-dimethylmaleimido.
  • ORi , OR 2 , OR 3 and OR 4 groups can be deprotected to form hydroxy groups, and the Y group can be converted into an acetamido group as follows.
  • Acyl protective groups of ORi , OR2, OR3 and OR 4 can be removed by a conventional base catalyzed transesterification deprotection reaction wherein the acyl groups are removed in an alcohol solvent such as methanol, ethanol, propanol or f-butanol in the presence of an alcoholate such as NaOMe, NaOEt or KO ⁇ u at 20-100 °C.
  • an alcohol solvent such as methanol, ethanol, propanol or f-butanol
  • the alcohol solvent and the alcoholate should be matched.
  • 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 these conditions of base catalyzed transesterification deprotection, only O-acyls can be deprotected. If Y is -NAc 2 , under these conditions, one of the acetyl groups can also be removed to make the Y group -NHAc. The -NH- haloacyl and cyclic imide Y protective groups remain intact under these conditions.
  • Acyloxy groups to OH and the following Y-groups: acylamino, alkoxycarbonylamino, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide to -NH 2 can be deprotected by basic hydrolysis, which is a base catalyzed hydrolysis in water, alcohol or water- organic solvent mixtures, in homogeneous or heterogeneous reaction conditions at 0-100 °C.
  • a strong base such as LiOH, NaOH, KOH, Ba(OH) 2 , K 2 CO 3 , a basic ion exchange resin or tetraalkylammonium hydroxides, is used, but the base can also be in an aqueous solution as well.
  • Y is -NAC2
  • one of the acetyl groups can also be removed to make the Y group -NHAc.
  • the base is NaOH and the solvent is methanol.
  • a 2,2,2-trichloroethoxycarbonylamino Y-group can also be converted into free amino groups by means of Zn/HCI.
  • Benzyloxycarbonylamino and azido Y-groups can also be easily transformed into amino groups by catalytic hydrogenolysis. It should be noted, in this regard, that the benzyloxycarbonylamino and azido Y-groups are much more reactive under hydrogenolysis conditions than the -OR group and optionally - OR3 group, wherein R 3 means a group removable hydrogenolysis, on the compounds of formulas 3 and 4.
  • the different kinetic behaviours of these groups allows one to drive the deprotection to reduce the benzyloxycarbonylamino and azido groups to amino groups without affecting -OR group and optionally -OR3 group, wherein R 3 means a group removable hydrogenolysis, for example, by running the reaction for shorter time and stopping the reduction before -OR group and optionally -OR3 group tend to be split.
  • R 3 means a group removable hydrogenolysis, for example, by running the reaction for shorter time and stopping the reduction before -OR group and optionally -OR3 group tend to be split.
  • the azido Y-group can be easily reduced to amino by a complex metal hydride, such as NaBH 4 , or by PPh 3 .
  • transformations can then be acetylated without acetylating the OH-groups on the compounds of formulas 3 and 4.
  • Selective AAacetylation 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.
  • Acetal and silyl R3-groups on the compounds of formulas 3 and 4 can 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>1 -2 to produce OH-groups on the compunds.
  • Acyl protective groups on the compounds of formulas 3 and 4 will not be affected because they can be deprotected only by extremely strong acidic hydrolysis ( ⁇ 1 ).
  • the interglycosidic linkage and anomeric protecting groups of the compounds of formulas 3 and 4 can also be sensitive to acids, they can be split in the compounds of formulas 3 and 4 only by acidic hydrolysis at pH ⁇ 1 -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 C 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, HCI, formic acid, sulphuric acid, perchloric acid, oxalic acid, p-toluenesulfonic acid, benzenesulfonic acid or a cationic exchange resin, and 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 competion 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, is used.
  • a C C6 alcohol-acetonitrile or Ci-C6 alcohol-water mixture is used in the presence of HCI or a sulfonic acid such as p-toluenesulfonic acid or champhorsulfonic acid.
  • a sulfonic acid such as p-toluenesulfonic acid or champhorsulfonic acid.
  • an anhydrous CrC 6 alcohol such as methanol, ethanol, propanol and butanol, can be used for the cleavage of cyclic acetal/ketal R3-groups via acid catalysed trans-acetalization/trans-ketalization processes.
  • Catalytic amount of hydrogen chloride, sulphuric acid, perchloric acid, p- toluenesulfonic acid, acetic acid, oxalic acid, champhorsulfonic acid or a strong acidic ion-exchange resin can be used at temperatures of 20 °C to reflux. Under such conditions, a f-butoxycarbonylamino Y-group can als be deprotected to amino.
  • a f-butoxycarbonylamino Y-group can als be deprotected to amino.
  • R is benzyl, 4-methylbenzyl or 4-chlorobenzyl
  • R is acetyl or optionally substituted benzoyl
  • R 2 is acetyl, optionally substituted benzoyl or H
  • R 3 is acyl or silyl
  • R 4 is acetyl or optionally substituted benzoyl
  • Y is alkanoylamido or haloalkanoylamido
  • -OR is in ⁇ -orientation.
  • R is benzyl
  • Ri is benzoyl or 4- chlorobenzoyl
  • R 2 is H or benzoyl
  • R 3 is pivaloyl
  • R 4 is acetyl
  • Y is acetamido or trichloroacetamido.
  • the mixture of these more preferred embodiments of compounds of formula 3 and 4 is converted in one step to a mixture of LNT-OBn and LNnT-OBn by NaOMe/MeOH treatment (when Y is acetamido), or in three consecutive steps of NaOMe/MeOH treatment, basic hydrolysis (in aqueous NaOH-solution) and selective N-acetylation.
  • a still further aspect of the invention relates to a method of providing the mixture of, preferably the mixture consisting essentially of, compounds of formulas 3 and 4, comprising the step of reacting an acceptor of formula 5
  • R is a group removable by hydrogenolysis
  • Ri is acyl
  • R 2 is acyl or H
  • R 3 is selected from a group removable by hydrogenolysis
  • Y is selected from alkanoylamido, haloalkanoylamido, -NAc 2 , benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino,
  • This glycosidation reaction is preferably carried out with a glycosyl halides (Xi is F, CI, Br, I) because of its easy accessibility and satisfactory reactivity.
  • the anomeric halides typically obey the following reactivity order, F ⁇ CI ⁇ Br ⁇ l, for nucleophilic displacement.
  • the glycosylation reaction can be promoted by a heavy metal ion, such as mercury or silver, or a Lewis acid, such as trimethylsilyl triflate or BF 3 -etherate.ln this glycosylation reaction with a glycosyl acetate or benzoate (Xi is -OAc or -OBz), the glycosyl acetate or benzoate is first subjected to electrophilic activation providing a reactive intermediate, then treated with the nucleophilic OH-acceptor of formula 5.
  • a heavy metal ion such as mercury or silver
  • a Lewis acid such as trimethylsilyl triflate or BF 3 -etherate.
  • Typical activators include Bronsted acids, such as TsOH, HCIO 4 or sulfamic acid, and Lewis acids, such as ZnC , SnCI 4 , triflate salts, BF 3 -etherate, trityl perchlorate, AICI 3 or triflic anhydride, and their mixtures.
  • Thioglycosides (Xi is alkylthio- or phenylthio- group) can be activated by thiofilic promoters such as mercury(ll) salts, Br 2 , I 2 , NBS, NIS, triflic acid, triflate salts, BF 3 -etherate, trimethylsilyl triflate, dimethyl-methylthio sulphonium triflate, phenylselenyl triflate, iodonium dicollidine perchlorate,
  • thiofilic promoters such as mercury(ll) salts, Br 2 , I 2 , NBS, NIS, triflic acid, triflate salts, BF 3 -etherate, trimethylsilyl triflate, dimethyl-methylthio sulphonium triflate, phenylselenyl triflate, iodonium dicollidine perchlorate,
  • tetrabutylammonium iodide or mixtures thereof preferably by Br 2 , NBS, NIS or a triflate salt.
  • This glycosylation reaction is carried out to be non-regioselective, so that either the
  • 3- OH or the 4-OH group of the trisaccharide of formula 5 is galactosylated and a mixture of compounds of formulas 3 and 4 is obtained.
  • the proportion of the two compounds of formulas 3 and 4 can subsequently be determined in each case by conventional detection and spectroscopic methods, such as TLC, H-NMR, C-NMR or HPLC.
  • a galactosyl donor of formula 6 wherein R 4 is acetyl or benzoyl, preferably acetyl, and Xi is trichloroacetimidate is reacted with an acceptor of formula 5 wherein R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, preferably benzyl, Ri is acetyl or optionally substituted benzoyl, preferably benzoyl or
  • R 2 is acetyl, optionally substituted benzoyl or H, preferably benzoyl or H
  • R 3 is acyl or silyl, preferably pivaloyl, benzoyl or 4-chlorobenzoyl
  • Y is alkanoylamido or haloalkanoylamido, preferably acetamido or trichloroacetamido
  • -OR is in ⁇ -orientation.
  • Compounds involved in this glycosidation reaction are considered to be crucial elements of this invention.
  • this invention also relates to compounds of formula 7
  • R 6 is, independently, H or a residue of formula A
  • R is a group removable by hydrogenolysis
  • Ri is acyl
  • R 2 is acyl or H
  • R 3 is selected from a group removable by hydrogenolysis
  • R 4 is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAc 2 , benzamido, alkoxycarbonylamino,
  • haloalkoxycarbonylamino benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3-diphenylmaleimido and 2,3-dimethylmaleimido.
  • Novel compounds of formula 7 can be crystalline solids, oils, syrups, precipitated amorphous material or spray dried products. If crystalline, they can be either in anhydrous or in hydrated crystalline forms incorporating one or several molecules of water into their crystal structures. Similarly, compounds of formula 7 can exist as crystalline substances incorporating ligands such as organic molecules and/or ions into their crystal structures. The crystalline form of a compound of formula 7 can be considered as an anomeric mixture of a- and ⁇ -anomers or even pure form of one of the anomers.
  • Preferred compound of formula 7 are those of formula 3
  • R is benzyl, 4-methylbenzyl or 4-chlorobenzyl
  • Ri is acetyl or optionally substituted benzoyl
  • R 2 is acetyl, optionally substituted benzoyl or H
  • R 3 is acyl or silyl
  • R 4 is acetyl or optionally substituted benzoyl
  • Y is alkanoylamido or haloalkanoylamido
  • -OR is in ⁇ -orientation.
  • R is benzyl
  • Ri is benzoyl or 4-chlorobenzoyl
  • R2 is H or benzoyl
  • R3 is pivaloyi
  • R 4 is acetyl
  • Y is acetamido or trichloroacetamido.
  • R is benzyl, 4-methylbenzyl or 4-chlorobenzyl
  • Ri is acetyl or optionally substituted benzoyl
  • R2 is acetyl, optionally substituted benzoyl or H
  • R 3 is acyl or silyl
  • R 4 is acetyl or optionally substituted benzoyl
  • Y is alkanoylamido or haloalkanoylamido
  • -OR is in ⁇ -orientation.
  • R is benzyl, R is benzoyl or 4-chlorobenzoyl, R 2 is H or benzoyl, R 3 is pivaloyi, benzoyl or 4-chlorobenzoyl, R 4 is acetyl and Y is acetamido or trichloroacetamido.
  • R is benzyl, 4-methylbenzyl or 4-chlorobenzyl
  • Ri is acetyl or optionally substituted benzoyl
  • R 2 is acetyl, optionally substituted benzoyl or H
  • R 3 is acyl or silyl
  • Y is alkanoylamido or haloalkanoylamido
  • -OR is in ⁇ -orientation. More preferably, R is benzyl, Ri is benzoyl or 4-chlorobenzoyl, R 2 is H or benzoyl, R 3 is pivaloyi, benzoyl or 4-chlorobenzoyl and Y is acetamido or trichloroacetamido.
  • R is a group removable by hydrogenolysis
  • Ri is acyl
  • R 2 is H
  • Y is selected from alkanoylamido, haloalkanoylamido, -NAc 2 , benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino, benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3- diphenylmaleimido and 2,3-dimethylmaleimido, by selective protection of the primary hydroxyl group.
  • Such selective 6-O-substitution can be achieved, for example, with a base catalysed reaction using an R 3 -halide to give a compound of formula 5 wherein R 3 is a group removable by hydrogenolysis.
  • Both inorganic and organic bases such as sodium hydride, potassium tert-butoxide, potassium hydroxide, sodium hydroxide, potassium carbonate, diisopropyl ethylamine or 1 ,8-diazabicyclo[5.4.0]undeco-7-ene, are suitable to catalyse such a selective 6-O-substitution reaction of a triol of formula 8.
  • This reaction can be carried out either in a homogeneous solution using a solvent such as DMF, THFor dioxane, or in an aqueous phase.
  • a solvent such as DMF, THFor dioxane
  • NaH or potassium tert-butoxide is used in dioxane or DMF at 20-80 °C.
  • the selective 6-O-acylation can be carried out with conventional acylating agents such as acyl halides, anhydrides and active esters, in the presence of, for example, pyridine, triethylamine or diisopropyl ethylamine using organic solvents such as DCM, chloroform, THF, dioxane or acetonitrile, or a mixture thereof at -20-80 °C to yield compounds of formulas wherein R 3 is acyl.
  • acylating agents such as acyl halides, anhydrides and active esters
  • Selective acyclic acetal formation on the 6-position can be performed with, for example, methoxymethyl, f-butoxymethyl, 2-methoxyethyl, benzyloxymethyl or 2- tetrahydrofuranyl halogenides, in the presence of, for example, triethylamine, morpholine, diisopropyl ethylamine or pyridine, or with, for example, dihydropyran, 1 ,4-dihydrodioxin, dihydrofuran, 2-methoxypropene or 2-phenoxypropene in the presence of acids in organic solvents, such as DMF, THF, dioxane or acetonitrile, at 0-60 °C to yield compounds of formulas wherein R 3 is an acetal type group.
  • a selective primary OH-silylation reaction of a compound of formula 8 with a silyl chloride in the presence of an amine base, such as imidazole or triethyl amine, at room temperature or with a silyl triflate with a hindered amine base, such as 2,6- lutidine, at a low temperature can yield compounds of formula 5 wherein R 3 is silyl.
  • an amine base such as imidazole or triethyl amine
  • the reaction of the lactose acceptor of formula 10 (R, Ri and R 2 are as defined above) with the glucosaminyl donor of formula 11 (Y is as defined above or Y with the vicinal X 2 forms a 2-alkyl-, 2-haloalkyl- or 2-phenyl-oxazoline, X 2 is selected from halogen, -OC( NH)CCI 3 , -OAc, -OBz and -SR 5 , wherein R 5 is selected from alkyl, substituted phenyl and unsubstituted phenyl) can be carried out in aprotic solvent or in a mixture of aprotic solvents in the presence of an activator, so as to yield a glycosylated product of formula 9.
  • the new interglycosidic linkage in the compund of formula 9 is formed by the nucleophilic displacement of the leaving group X 2 of the donor of formula 11 with the 3'-OH group of the acceptor of formula 10.
  • regioselective glycosylation can be achieved on an acceptor of formula 10, wherein R 2 is H, with the donor of formula 11 .
  • the reactivity of the equatorial 3'-OH and the axial 4'-OH is different: the equatorial OH- group can act as stronger nucleophile under glycosylation conditions.
  • the reaction can be driven to the formation of the desired 1 -3 interglycosidic linkage instead of a 1 -4 coupling.
  • stereoselectivity because the stereochemical outcome oft eh reaction can be affected by factors, such as the presence or absence of a participating group at C-2 of the donor of formula 11 , the nature of the leaving group X 2 on the donor, the solvent, the nature of the protective groups on both the donor and acceptor, the nature of the promoters or catalysts the temperature and pressure of the reaction and steric interactions between the donor and acceptor. See, for example, Demchenko (Ed.): Handbook of Chemical Glycosylation, Wiley (2008).
  • Oxazoline derivatives (Y with the vicinal X 2 forms 2-alkyl-, 2-haloalkyl- or 2-phenyl-oxazoline) can be promoted in such a glycosylation reaction with TsOH, camphorsulfonic acid, TMSOTf, FeCl 3 , CuC or pyridinium p-toluenesulfonate.
  • Compounds of formula 8 can be obtained from compounds of formula 9 by selectively removing the acetyl groups on its glucosaminyl potion when its lactose portion is protected by acyls different from acetyl. This is due to the higher reactivity of acetyls towards acidic transesterification than other acyls.
  • An acetyl group from the galactose 4-position R 2 is acetyl
  • This deprotection step can be carried out in a CrC 6 alcohol or a mixture of CrC 6 alcohols, preferably methanol or ethanol, in the presence of a protic acid, such as acetic acid,
  • trifluoroacetic acid HCI
  • formic acid sulphuric acid, perchloric acid, oxalic acid, p- toluenesulfonic acid, benzenesulfonic acid or a cationic exchange resin, preferably a strong inorganic acid which can be present in from catalytic amount to excess.
  • Aprotic co-solvents such as dichloromethane, chloroform, dioxane or THF
  • the reaction can be carried out between 0 and 25 °C, preferably at 5-20 °C, until TLC shows the reactionto complete or nearly complete which takes from about 2 hours to 3 days depending on temperature, concentration and pH.
  • Acceptors of formula 10 in Scheme 1 can also be synthesized in a conventional manner by, for example, as described in WO 201 1 /100980.
  • Donors of formula 11 in Scheme 1 can also be synthesized in a conventional manner.
  • the amino Y-group of the compunds of formula 11 can be protected with, for example, a phthalyl, tetrachlorophthalyl, trichloroacetyl, dimethylmaleolyl or diphenylmaleolyl group.
  • These groups can provided by reacting the amine with an activated acyl derivative such as an anhydride, halogenide or active ester in the presence of a base.
  • the AAprotected glucosamine derivative obtained can then have its OH-groups protected by, for example, with an acylating agent such as a halogenide, anhydride or active derivative of a carboxylic acids (e.g. an imidazolide, thioester, silyl ester, vinyl ester, tetrazolide, ortoester or hydroxy- benztriazolyl ester) in the presence of a base, such as pyridine, triethylamine, diisopropyl ethylamine or dimethylaminopyridine ina organic solvent, such as DCM, chloroform, THF, dioxane or acetonitrile or a mixture thereof at -20-80 °C.
  • an acylating agent such as a halogenide, anhydride or active derivative of a carboxylic acids (e.g. an imidazolide, thioester, silyl ester, vinyl ester, te
  • peracylated derivatives can also be prepared from glucosamine via peracylation followed by amine protection.
  • Selective removal of the 1 -O-acyl group e.g. with water in the presence of a Lewis or Bronsted acid
  • fluorinating reagent such as HF, AgF, AgBF 4 , tetrabutyl ammonium fluoride, diethylaminosulfur trifluoride, 2-fluoro-1 -methylpyridinium tosylate
  • Oxazoline-type donors can be synthesized from the appropriate acylamido derivative having any of the X 2 leaving group mentioned above when treated with a conventional activator.
  • a compound of formula 12 (wherein R 6 is acetyl or two R6 groups form a cyclic acetal/ketal type protective group, such as isopropylidene, and R 7 is a group removable by hydrogenolysis or R 7 is acyl except for acetyl, X 2 and Y are as defined above) can be reacted with a compound of formula 10.
  • the resulting compound of formula 13 (wherein R, R-i , R 2 , R6, R7 and Y are as defined above) can then be subjected to mild acidic deprotection to selectively remove R6 and thus to form the compound of formula 5.
  • Another aspect of this invention relates to the use of a mixture consisting essentially of LNnT and LNT.
  • both solid and liquid forms of such a mixture of LNT and LNnT produced by the methods of this invention are suitable for general nutritional use for infants, toddlers, children, adults and the elderly.
  • Both solid and liquid forms of mixture of LNnT and LNT of this invention can also be used as food additives, dietary supplements, ingriedient of alcoholic and non alcoholic beverages such as soft drinks, fruit juices, bottled water, wine, and beer.
  • Both solid and liquid forms of the mixture of LNnT and LNT of this invention can also be used as a therapeutic agents, for example to prevent bacterial and viral infections, avoid diarrhoea, enhance human immune systems and enhance human brain
  • Both solid and liquid forms of the mixture of LNnT and LNT of this invention can also be used in veterinary applications, swuch as to fight against infectious diseases of domesticated animals.
  • the mixture of LNnT and LNT of this invention can also be used for pharmaceutical and nutritional purposes, for example:
  • sialic acid containing human milk oligosaccharides to aid in the development and/or maturation of the immune system of neonatal
  • infections such as influenza.
  • probiotics such as Lactobacillus and Bifidobacterium species, in
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a mixture consisting essentially of LNnT and LNT as the pharmaceutically active ingredient and preferably one or more pharmaceutically acceptable carriers, additives, adjuvants, excipients and/or diluents, such as water, gelatine, talc, sugars, starch, gum arabic, vegetable gums, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, lubricants, colorants, fillers, and/or wetting agents as described in J. P. Remington: Remington's Pharmaceutical Sciences, Mack Pub. Co.; 17th edition.
  • the dosage form for administration of this pharmaceutical composition includes, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions, capsules, syrups, injections, liquids, elixirs, extracts and tinctures as described in J. P. Remington: Remington's Pharmaceutical Sciences, Mack Pub. Co.; 17th edition.
  • a still further aspect of the invention relates to the preparation of a pharmaceutical composition
  • a pharmaceutical composition comprising a mixture consisting essentially of LNnT and LNT as the pharmaceutically active ingredient.
  • Such a pharmaceutical composition can be made in a convenentional manner, for example as described in in J. P. Remington:
  • a yet further aspect of invention relates to the preparation of a nutritional formulation for humans comprising a mixture consisting essentially of LNnT and LNT as the nutritionally active ingredient.
  • the nutritional formulation can also contain edible micronutrients, vitamins, minerals and/or trace elements, but the amounts of these ingredients can vary depending on whether the formulation is intended for use with normal, healthy infants, children, adults or subjects having specialized needs (e.g. suffering from metabolic disorders).
  • Micronutrients can include, for example, edible oils, fats, fatty acids (such as coconut oil, soy-bean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid or linolenic acid.), carbohydrates (such as glucose, fructose, sucrose, maltodextrin, starch or hydrolysed corn starch) and proteins (such as from casein, soy-bean, whey, skim milk, or their hydrolysates). Vitamins can include, for example, vitamin A, B1 , B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid.
  • fatty acids such as coconut oil, soy-bean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid or linolenic acid.
  • carbohydrates such as glucose, fructose
  • a preferred nutritional formulation of this invention is an infant formula, i.e., a foodstuff intended for use by infants during the first 4-6 months of life and satisfying by itself the nutritional requirements of the infants. It can contain one or more probiotic Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk, carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof, lipids (e.g.
  • the infant formula also contains a mixture consisting essentially of LNT and LNnT in a total amount of 0.1 to 3.0 g/100 g of infant formula.
  • Another preferred nutritional formulation of this invention is a food supplement wherein the mixture consisting essentially of LNnT and LNT as the nutritionally active ingredient is crystalline.
  • the food supplement can also contain one or more probiotics, vitamins, minerals, trace elements and other micronutrients.
  • the food supplement can be in the form of, for example, tablets, capsules, pastilles or liquids, and can contain conventional additives such as binders, coatings, emulsifiers, solubilising agents, encapsulating agents, film forming agents, adsorbents, carriers, fillers, dispersing agents, wetting agents, jellifying agents and gel forming agents.
  • the daily dose of the LNnT-LNT mixture ranges from 0.1 to 3.0 g.
  • a preferred food supplement of this invention is a digestive health functional food which can serve to enhance and preserve digestive health.
  • Another aspect of this invention relates to a method of obtaining a mixture of, preferably a mixture consisting essentially of, compounds of formulas 1 and 2, preferably a mixture consisting essentially of LNT-OBn and LNnT-OBn, in solid form from an aquous solution containing a mixture of, preferably a mixture consisting essentially of, these compounds.
  • a volume of the aqueous solution containing the mixture of, preferably the mixture consisting essentially of, compounds of formulas 1 and 2 is added at least fivefold volumes of a C C 4 alcohol, preferably methanol, or a solvent mixture of CrC 4 alcohols, or a ketone type solvent, preferably acetone, or a solvent mixture of a ketone type solvent with a CrC 4 alcohol.
  • a C C 4 alcohol preferably methanol, or a solvent mixture of CrC 4 alcohols, or a ketone type solvent, preferably acetone, or a solvent mixture of a ketone type solvent with a CrC 4 alcohol.
  • the volume of the organic solvent(s) mentioned above is at least sixfold that of the aqueous solution mentioned above, more preferably at least sevenfold than of the aqueous solution mentioned above.
  • the aqueous solution is heated before the organic solvent(s) is/are added, and it is further preferable to use a preheated hot organic solvent(s) in this step.
  • pH of the aquous solution can be adjusted to be basic, preferably to pH 8-1 1 , more preferably 8.5-9.5, by adding an aqueous solution of a base such as NaOH, KOH, LiOH, Na2CO3 or K2CO3 to it.
  • a further aspect of this invention relates to a method of obtaining a crystalline compound of formula 1 and optionally a separate crystalline compound of formula 2 from an aqueous solution containing a mixture of, preferably a mixture consisting essentially of, these compounds.
  • a compound of formula 1 preferably LNT-OBn
  • the concentration of the starting aqueous solution can vary around 15 m/V% to around 60 m/V%.
  • a Q-C 4 alcohol preferably methanol, or a solvent mixture of d- C 4 alcohols, or a ketone type solvent, preferably acetone, or a mixture of a ketone type solvent with a C C 4 alcohol, preferably preheated.
  • the ratio of organic solvent(s) to the aqueous solution should be about 1 /5 to 1 /10 (in volume).
  • the resulting aqueous-organic solution is cooled (if it has been heated) and preferably stirred, so that the compound of formula 1 , preferably LNT-OBn, crystallizes out from the solution.
  • This crystallization process can be facilitated by adding seed crystals of the compound of formula 1 to the aqueous/organic solution.
  • This crystallization process can also be facilitated by adjusting the pH of the aqueous solution:
  • an organic or inorganic acid such as acetic acid, formic acid, hydrochloric acid, sulfuric acid or perchloric acid, preferably HCI
  • - to be basic preferably 8-9, by adding an aqueous solution of a base such as NaOH, KOH, LiOH, Na2CO3 or K2CO3, when the ratio of the compound of formula 1/ compound of formula 2 is about 2:1 or higher, preferably about 3:1 or higher, before adding the above organic solvent(s). Also preferably, when the ratio of the compound of formula 1/compound of formula 2 is about 7:1 or higher, no addition of organic solvent(s) is needed to crystallize the compound of formula 1 from the aqueous solution. Crystals of the compound of formula 1 , preferably LNT-OBn, in substantially pure or at least in enriched form can be separated form the aqueous solution by filtration.
  • a base such as NaOH, KOH, LiOH, Na2CO3 or K2CO3
  • a compound of formula 2 can be crystallized out from the mother liquor, especially if it contains a considerable amount of a compound of formula 2 (i.e. the initial ratio of the compound of formula 1/compound of formula 2 before crystallization is about 1 :4 to 2:1 ). Doing so, the pH of the mother liquor, enriched in compound of formula 2, preferably LNnT-OBn, is then adjusted, if needed, to 8-9.
  • a volume of the mother liquor containing the compound of formula 2 can then be added at least a double volume, preferably at least a triple volume, of Ci-C 4 alcohol solvent, preferably methanol, or a solvent mixture of C C 4 alcohols, or a ketone type solvent, preferably acetone, or a mixture of a ketone type solvent with a Ci-C 4 alcohol, preferably while heating to about 45-50 °C.
  • the compound of formula 2 crystallizes out in substantially pure or at least in enriched form. This crystallization process can be facilitated by adding seed crystals of the compound of formula 2.
  • a compound of formula 2 in another method relating to obtaining a crystalline compound of formula 1 and/or a separate crystalline compound of formula 2 from an aqueous solution containing a mixture of, preferably a mixture consisting essentially of, these compounds, a compound of formula 2, preferably LNnT-OBn, can be crystallized out when the mixture of the compounds of formulas 1 and 2 contains no more than about 20 % of the compound of formula 1 , preferably LNT-OBn, and at least about 80 % of the compound of formula 2.
  • the starting aqueous solution is about 40-60 m/V%.
  • the pH of the aqueous solution is adjusted to be acidic, preferably 4-6, more preferably 5.0-6.0, by adding an organic or inorganic acid such as acetic acid, formic acid, hydrochloric acid, sulfuric acid or perchloric acid, preferably HCI.
  • an organic or inorganic acid such as acetic acid, formic acid, hydrochloric acid, sulfuric acid or perchloric acid, preferably HCI.
  • a CrC 4 alcohol solvent preferably methanol, or a solvent mixture of C C 4 alcohols, preferably while heating to about 45-50 °C.
  • This crystallization process can be facilitated by adding seed crystals of the compound of formula 2 to the acidic aqueous-alcoholic solution. Crystals of the compound of formula 2 in substantially pure or at least in enriched form are then separated from the acidic aqueous-alcoholic solution by filtration.
  • a compound of formula 1 can be crystallized out from the mother liquor, especially if it contains a considerable amount of a compound of formula 1 (i.e. the initial ratio of the compound of formula
  • 2/compound of formula 1 before crystallization is about 4:1 to 8:1 ).
  • the organic solvent(s) are then evaporated from the resulting mother liquor.
  • the pH of the resulting aqueous solution is then adjusted to be basic, preferably 8-10, by adding an aqueous solution of a base such as NaOH, KOH, LiOH, Na 2 CO 3 or K 2 CO 3 .
  • a base such as NaOH, KOH, LiOH, Na 2 CO 3 or K 2 CO 3 .
  • a ketone type solvent preferably acetone
  • This crystallization process can be facilitated by adding seed crystals of the compound of formula 1.
  • the so-separated compounds of formulas 1 and 2 can then be converted to LNnT and LNT, respectively, by means of catalytic hydrogenolysis as described above and in, e.g., WO 201 1 /100980.
  • Phenyl 2-azido-2-deoxy-3,4,6-tri-0-acetyl-1-thio-a/ -D-glucopyranoside To a solution of tetraacetyl 2-azido-2-deoxyglucopyranose in DCM (100 imL), tiophenol was added and the mixture was cooled with ice bath, then BF 3 OEt 2 was added drop wise and the reaction mixture stirred for 3 days at RT. The reaction mixture was neutralized with saturated bicarbonate solution and was washed with water, dried with sodium sulfate and the solvent was removed in vacuo. The product was purified by flash chromatography (petroleum ether/EtOAc 2:1 ) to give the title compound (12.3 g, 84%). The data were in coincidence with those reported in the literature.
  • Phenyl 2-azido-2-deoxy-3,4,6-tri-O-acetyl-1 -thio-a/3-D-glucopyranoside (12.00 g, 28.34 mmol) was dissolved in MeOH (250 imL) and 4 imL of a 0.2 M NaOMe solution was added. The reaction mixture was stirred for 1 h, and after this time the reaction mixture was neutralized with ionic exchange resin (IR120 H + form) and the solvent was evaporated in vacuo.
  • the intermediate product was dried 1 h in high vacuum, and was redissolved in DMF (100 imL), and dimethoxybenzaldehyde (6.4 imL, 42.52 mmol) waswas added to the solution followed by addition of pTsOH (0.488g, 2.83 mmol).
  • the reaction mixture stirred overnight at 40 °C; after this time the mixture was poured into water and extracted with EtOAc twice. The organic extracts were unified, washed with water and brine, dried with sodium sulfate, and the solvent was evaporated in vacuo.
  • the product was purified by flash chromatography (petroleum ether/EtOAc 3/1 -- 1/1 ) to give the title compound (10.12 g, 93%).
  • Phenyl 2-azido-2-deoxy-4,6-O-benzylidene-1 -thio-a/3-D-glucopyranoside was dissolved in pyridine/Ac 2 O (2/1 mixture 150 imL). The reaction mixture stirred for 2h and the solvent was removed in vacuo. The residue was dissolved in EtOAc and was with 1 N HCI solution and water, dried with MgSO 4 and the solvent was removed in vacuo. Flash chromatography (petroleum ether/EtOAc 3:1 ) yielded the title compound (5.90 g, 97%).
  • Phenyl 2-azido-2-deoxy-3,4-di-0-acetyl-6-0-benzyl-1-thio-a/B-D- glucopyranoside NaBH 3 CN 19.03 mmol, 8 equiv.uiv. was added to a cooled (0 °C) suspension of phenyl 2-azido-2-deoxy-4,6-O-benzylidene-1 -thio-a/3-D-glucopyranoside (1 1 .3 mmol), molecular sieves (4 A, 12 g) and a spatula tip of methyl orange in THF (40 m L). A satd.
  • Phenyl 2-azido-2-deoxy-3,4-di-0-acetyl-6-0-triisopropylsilyl-1-thio-a/B-D- glucopyranoside Phenyl 2-azido-2-deoxy-3,4,6-tri-O-acetyl-1 -thio-a/3-D-glucopyranoside (7.4 mmol) was dissolved in MeOH (60 imL) and 1 imL of a 0.2 M NaOMe solution was added. The reaction mixture was stirred for 1 h, and after this time the reaction mixture was neutralized with ionic exchange resin (IR120 H + form) and the solvent was
  • MeOH/ water mixture (apprx. 1 :1 ) gave white crystals (54 g, 75%). M.p. 195 °C; changed structure; 205-207 °C; melted.
  • Phenyl 2-aminobenzyloxycarbonyl-2-deoxy-3,4,6-tri-O-acetyl-1 -thio-a/3-D- glucopyranoside (28.34 mmol) was dissolved in MeOH (250 mL) and 4 mL of a 0.2 M NaOMe solution was added. The reaction mixture waswas stirred for 1 h, and after this time the reaction mixture was neutralized with ionic exchange resin (IR120 H + form) and the solvent was evaporated in vacuo. The intermediate product was dried 1 h in high vacuum, and was redissolved in DMF (100 mL), and
  • Phenyl 2-deoxy-2-A/-trichloroacetamido 3-1 -D-thioglucosamine 75.6 g, 0.181 mol
  • 2,2-dimethoxypropane 70 mL
  • p-TsOH x H 2 O 2.6 g, 0.1 eq
  • Phenyl 2-deoxy-4,6-0-isopropylidene-2-A/-trichloroacetamido 3-1 -D-thioglucosamine 50 g, 0.1 138 mol was dissolved in pyridine (150 mL) and cooled to 0-5 °C
  • R 7 Piv, CIBz, Bz
  • the starting material (1 .0 g, 0.78 mmol) was poured in one portion into a mixture of DCM (10 mL), MeOH (10 mL) and cc. H 2 SO 4 (0.3 mL, 5.63 mmol) at room temperature
  • tetrasaccharide mixture obtained according to Example 3 having R 3 : acetal type group is dissolved in DCM and cooled to 0-5 °C ushg.
  • Diluted aq. HCIO 4 (1 part 70% aq. HCIO 4 diluted with 1 part water) is added drop wise and the solution is stirred vigorously at 0-5 °C for 2 hours when aq. sat. NaHCO 3 is added and stirred for half an hour. Water is added and the phases are separated. The lower organic phase is washed with brine / water mixture and concentrated.
  • the protected LNT/LNnT product mixture was suspended in MeOH and heated to 50 °C. Catalytic amount of methanolic NaOMe solution was added (25% w/w). After 6 hrs the reaction was quenched by the addition of glacial AcOH to adjust the pH to 6. Nearly all MeOH was evaporated off and then equal volumes of water and hexane was added. The biphasic mixture was stirred at the rotational evaporator at 45 °C for 10 minutes and then the phases were separated in a funnel. The aqueous phase was taken to the rotational evaporator and vacuum was applied to remove residual MeOH and hexanes. The obtained aq. extract containing the mixture of LNT/LNnT NHTCA- OBn tetra-saccharides was used directly in the next step without further purification.
  • the mixture of tetrasaccharide amines was suspended in water and to the mixture was added acetic anhydride (1 .3 eq.) dropwise controlling the inner temperature not to exceed 30 °C. In less than 10 minutes a clear sdution was obtained.
  • the solution was stirred for 30 minutes at RT (-22-25 °C) whereupon aq. 50% NaOH solution was added to neutralize the pH.
  • Another portion of acetic anhydride (0.2 eq) was added dropwise and the mixture was stirred additional 30 minutes.
  • Aq. NaOH (50%) was added to adjust the pH above 1 1 and the solution was heated to 45 °C for 30 minutes.
  • MeOH (4V) was added in one portion and the light yellow solution was heated to 50- 55°C.
  • Example 4 The crude mixture obtained in Example 4 is dissolved in water (1 g sugar content in 2 imL water). The pH is optionally adjusted to around 9 by adding aq. NaOH and then the mixture solution is heated to 45 °C and 5 volumes of pre-heated MeOH (25 imL to 1 g sugar content) is added under vigorous stirring. The clear solution is allowed to cool to RT and then placed with stirring at 4°C overnight. The formed crystals are filtered and washed once with cold MeOH. The crystalline solids, consisting
  • Example 4 The crude mixture obtained in Example 4 (LNnT-OBn:LNT-OBn is about 4:1 ) is dissolved in water (1 g sugar content in 2 imL water). The pH is adjusted to around 5- 5.5 by adding aq. HCI and then the mixture is heated to 45°C and 5 volumes preheated MeOH (5 imL to 1 g sugar content) is added under vigorously stirring. The clear solution is allowed to cool to RT and then seeded with LNnT-OBn and then stirred at RT overnight. The formed crystals of LNnT-OBn are filtered and washed once with cold MeOH.
  • the mother liquid is placed on a rotary evaporator, and the MeOH is evaporated off under reduced pressure.
  • the pH is checked and adjusted to around 9-10 by aq. NaOH and the solution is heated to 50 °C.
  • hotacetone (3 volumes, in proportion to the volume of the solution to wich it is added) is added to the solution, and it is allowed to cool to RT slowly.
  • the solution is stirred at RT overnight, and then the white crystalline solid, that is formed, is filtered off and washed with cold acetone/water (3:1 ) to provide a solid LNT-OBn product.
  • Example 4 The crude mixture obtained in Example 4 (LNnT-OBn:LNT-OBn is about 4:1 ) is dissolved in water (1 g sugar content in 2 mL water). The pH is adjusted to around 5- 5.5 by adding aq. HCI and then the mixture is heated to 45 °C and 3 volumes pre- heated MeOH (3 mL to 1 g sugar content) is added under vigorously stirring. The clear solution is allowed to cool to RT and then seeded with LNnT-OBn and then stirred at RT for 2 hours. The formed crystals of LNnT-OBn are filtered, mother liquid separated, and the solid washed once with cold MeOH.
  • the mother liquid is placed on a rotary evaporator, and the MeOH is evaporated off under reduced pressure.
  • the pH is checked and adjusted to around 9-10 by aq. NaOH and the solution is stirred at RT for 3 hours when seeded with LNT-OBn.
  • the aq. mixture is stirred another 3 hours when hot acetone (3 volumes, in proportion to the volume of the solution to wich it is added) is added to the solution, and it is allowed to cool to RT slowly.
  • the solution is stirred at RT overnight, and then the white crystalline solid, that is formed, is filtered off and washed with cold
  • LNT-OBn/LNnT-OBn 360 mg, 5:1
  • pH was adjusted to 8-9 by adding aq. NaOH and the mixture was heated to 50 °C for 1 hour when hot acetone (0.4 imL) was added.
  • the thick mixture was stirred for 2 hours at ambient temperature before filtrated.
  • the obtained solid was washed with cold acetone/water (3:1 ) and dried to give LNT-OBn (HPLC analysis: from 80% to > 98%; relative purity by UV detection). Yield 200 mg.
  • LNT-OBn/LNnT-OBn a mixture of LNT-OBn (250 mg) and LNnT-OBn (250 mg) was added 1 imL of water. The suspension was stirred at RT for 2 hours when filtrated. HPLC analysis: LNT-OBn/LNnT-OBn ratio in the solid is 71 :29 (relative purity by UV detection); LNT- OBn/LNnT-OBn ratio in the mother liquor is 25:75 (relative purity by UV detection). 7. Hvdroqenolvsis of LNnT-OBn/LNT-OBn mixture
  • a mixture of 1 -O-benzyl-LNT (0.6 g) and 1 -O-benzyl-LNnT (0.6 g) is dissolved in 4.8 ml of water.
  • 0.12 g of 10% Pd/C is added, followed by the addition of concentrated HCI until the pH of the solution is pH 4.
  • the suspension is treated with hydrogen gas (5-6 bar) at 54 °C while being stirred.
  • the resulting suspension is filtered to provide a solid product consisting essentially of a mixture of LNT and LNnT.

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  • Saccharide Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne un mélange de, de préférence un mélange essentiellement constitué de, un précurseur de lacto-N-tétraose (LNT) (1) et un précurseur de lacto-N-neotétraose (LNnT) (2), (formule 1, 2), R étant un groupe éliminable par hydrogénolyse et R3 étant soit un groupe éliminable par hydrogénolyse ou H, un procédé de cristallisation de 1 et/ou 2 à partir dudit mélange, et l'utilisation dudit mélange pour fabriquer un mélange essentiellement constitué de LNnT et LNT pour utilisation en tant que substance pharmaceutiquement ou nutritionnellement active. Les précurseurs peuvent être préparés par réaction d'un accepteur de formule 5, (formule 5), R étant un groupe éliminable par hydrogénolyse, R1 étant acyle, Ri étant acyle ou H, R3 étant choisi parmi un groupe éliminable par hydrogénolyse, acyle, silyle et un groupe de type acétal et Y étant choisi parmi alcanoylamido, halogéno-alcanoylamido, -NAc2, benzamido, alcoxycarbonylamino, halogéno-alcoxycarbonylamino, benzyloxycarbonylamino, azido, phtalimido, tétrachlorophtalimido, 2,3-diphénylmaléimido et 2,3-diméthylmaléimido, avec un donneur de formule 6, (formule 6), R4 étant acyle et Xi étant choisi parmi halogène, -OC(=NH)CCl3, -OAc, -OBz ou -SR5, R5 étant choisi parmi alkyle, phényle substitué et phényle non substitué, suivie par une ou plusieurs étapes de déprotection.
PCT/DK2012/050502 2011-12-23 2012-12-21 Procédé pour obtenir des précurseurs de lacto-n-tétraose et de lacto-n-neotétraose cristallin et des mélanges de ceux-ci Ceased WO2013091660A1 (fr)

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WO2016063262A1 (fr) 2014-10-24 2016-04-28 Glycom A/S Mélanges d'oligosaccharides de lait humain (hmo)
WO2016066174A1 (fr) 2014-10-29 2016-05-06 Glycom A/S Composition synthétique et procédé pour favoriser la cicatrisation des muqueuses
WO2016091265A1 (fr) 2014-12-08 2016-06-16 Glycom A/S Composition synthétique pour le traitement de troubles métaboliques
WO2016138911A1 (fr) 2015-03-05 2016-09-09 Glycom A/S Composition et méthode pour le traitement d'infections aiguës des voies respiratoires
WO2016157108A1 (fr) 2015-03-31 2016-10-06 Glycom A/S Mélange d'oligosaccharides de lait humain comprenant du 3'-o-sialyllactose
WO2017071715A1 (fr) 2015-10-28 2017-05-04 Glycom A/S Composition synthétique et procédé de modulation de la fonction cérébrale et du comportement
WO2017071716A1 (fr) 2015-10-28 2017-05-04 Glycom A/S Composition synthétique et procédé de modulation des troubles émotionnels et de l'humeur
WO2017084673A1 (fr) 2015-11-17 2017-05-26 Glycom A/S Composition synthétique permettant le traitement de complications associées aux antibiotiques
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WO2017190754A1 (fr) 2016-05-05 2017-11-09 Glycom A/S Composition comprenant du hmos pour le traitement de la diarrhée non infectieuse
WO2017198276A1 (fr) 2016-05-19 2017-11-23 Glycom A/S Composition synthétique
WO2019071021A2 (fr) 2017-10-04 2019-04-11 The Regents Of The University Of California Oligosaccharides immunomodulateurs
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WO2019111115A2 (fr) 2017-12-05 2019-06-13 Glycom A/S Oligosaccharides du lait humain pour le traitement de la migraine
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WO2020128947A1 (fr) 2018-12-19 2020-06-25 Glycom A/S Composition et procédé de traitement d'humains à régime faible en fodmap
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US10857168B2 (en) 2016-02-24 2020-12-08 Glycom A/S Synthetic composition for microbiota modulation
US10881674B2 (en) 2014-12-08 2021-01-05 Glycom A/S Synthetic composition for treating metabolic disorders
US10987368B2 (en) 2014-12-08 2021-04-27 Glycom A/S Synthetic composition for preventing or treating CVD
US11026959B2 (en) 2014-10-29 2021-06-08 Glycom A/S Synthetic composition and method for treating irritable bowel syndrome
US11040049B2 (en) 2014-10-29 2021-06-22 Glycom A/S Composition comprising HMSs/HMOs and use thereof
US11040050B2 (en) 2014-10-29 2021-06-22 Glycom A/S Composition comprising HMSs/HMOs and use thereof
US11278558B2 (en) 2017-03-01 2022-03-22 Glycom A/S Synthetic composition for microbiota modulation
US11291677B2 (en) 2017-05-09 2022-04-05 Glycom A/S Synthetic composition for microbiota modulation
US11304966B2 (en) 2017-12-22 2022-04-19 Glycom A/S Composition comprising HMOs for preventing or reducing nociception
WO2022078859A1 (fr) 2020-10-16 2022-04-21 Société des Produits Nestlé S.A. Composition nutritionnelle comprenant des oligosaccharides de lait humain
WO2022101222A1 (fr) 2020-11-10 2022-05-19 Société des Produits Nestlé S.A. Composition nutritionnelle
US11419884B2 (en) 2016-06-24 2022-08-23 Glycom A/S Compositions comprising HMOS, their production and use for the prevention and/or treatment of viral and/or bacterial infections
US11432578B2 (en) 2015-12-15 2022-09-06 Glycom A/S Mixture of HMOs
WO2022223430A1 (fr) 2021-04-19 2022-10-27 Dsm Ip Assets B.V. Composition d'enzymes et d'oligosaccharides de lait humain
US11524019B2 (en) 2017-08-21 2022-12-13 Glycom A/S Synthetic composition for reducing allergy symptoms
US11541069B2 (en) 2017-11-02 2023-01-03 Glycom A/S One or more HMOs for reducing or preventing fatigue and/or improving focus or concentration
US11541067B2 (en) 2017-05-24 2023-01-03 Glycom A/S HMO compositions and methods for reducing detrimental proteolytic metabolites
US11541068B2 (en) 2017-05-24 2023-01-03 Glycom A/S HMO compositions and methods for reducing autism spectrum disorder symptoms
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WO2014187464A1 (fr) * 2013-05-22 2014-11-27 Glycom As Mélange synthétique d'oligosaccharides pour le traitement d'un microbiote de mammifère
US10364449B2 (en) 2013-09-06 2019-07-30 Glycom A/S Fermentative production of oligosaccharides
WO2016063262A1 (fr) 2014-10-24 2016-04-28 Glycom A/S Mélanges d'oligosaccharides de lait humain (hmo)
US10946032B2 (en) 2014-10-24 2021-03-16 Glycom A/S Mixtures of HMOs
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