WO2013091660A1 - A method for obtaining crystalline lacto-n-tetraose and lacto-n-neotetraose precursors and mixtures thereof - Google Patents

Abstract

A mixture of, preferably a mixture consisting essentially of, an lacto-N-tetraose (LNT) precursor (1) and an lacto-N-neotetraose (LNnT) precursor (2), (formula 1, 2), where R is a group removable by hydrogenolysis and R₃ is either a group removable by hydrogenolysis or H, a method of crystallizing 1 and/or 2 from said mixture, and the use of said mixture for making a mixture consisting essentially of LNnT and LNT for use as a pharmaceutically or nutritionally active ingredient. The precursors can be made by reacting an acceptor of formula 5, (formula 5), wherein R is a group removable by hydrogenolysis, R₁ is acyl, Ri is acyl or H, R3 is selected from a group removable by hydrogenolysis, acyl, silyl and an acetal type group and Y is selected from alkanoylamido, haloalkanoylamido, -NAc2, benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino, benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3- diphenylmaleimido and 2,3-dimethylmaleimido, with a donor of formula 6, (formula 6), wherein R₄ is acyl and Xi is selected from halogen, -OC(=NH)CCl₃, -OAc, -OBz or -SR₅, wherein R₅ is selected from alkyl, substituted phenyl and unsubstituted phenyl, followed by one or more deprotection steps.

Description

A METHOD FOR OBTAINING CRYSTALLINE LACTO-N-TETRAOSE AND LACTO-N-NEOTETRAOSE PRECURSORS

AND MIXTURES THEREOF

FIELD OF THE INVENTION

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

components by crystallization.

BACKGROUND OF THE INVENTION

Human milk oligosaccharides (HMOs) have become of great interest in the past few years due to their important functions in human development. To date, the structures of at least 1 15 HMOs have been determined, and considerably more are probably present in human milk.

LNT (Scheme 1 ) and LNnT (Scheme 2) are considered to be among the more important HMOs.

Scheme 2. Lacto-N-neotetraose, LNnT

This is because of their nutritional value, as well as their potential to serve as building blocks for synthesizing other HMOs.

Low cost ways of producing them, for example for use as food additives and supplements, have therefore been sought. Hovever, it been difficult to synthesize large amounts of LNT and/or LNnT by conventional chemical or biotechnical processes. The isolation of LNT and/or LNnT from human milk has also been rather difficult, even in milligram quantities due to the presence, in human milk, of a large number of similar oligosaccharides.

Methods for synthesizing LNT have been known [for example from Takamura et al. Chem. Pharm. Bull. 27, 1497 (1979) and 28, 1804 (1980), Aly et al. Carbohydr. Res. 316, 121 (1999) and Malleron et al. ibid. 343, 970 (2008)]. Likewise, methods for synthesizing LNnT have been known [for example from Zurabyan et al. Soviet J. Bioorg. Chem. 4, 679 (1978); Paulsen et al. Carbohydr. Res. 169, 105 (1987) and Aly et al. Carbohydr. Res. 316, 121 (1999)]. Such syntheses have not been considered attractive ways of synthesizing LNT or LNnT. This is because they have involved many reaction steps, protective group manipulations and chromatographic purifications and have provided only poor yields and small amounts of LNT or LNnT. Although, a method for the large scale chemical synthesis of LNnT has recently been published in WO 201 1 /100980, no such method for making LNT has been known.

As a result, methods for industrially producing LNT have been sought, and alternative methods for industrially producing LNnT which could potentially provide lower costs and/or production advantages have been sought. In addition, ways have been sought for industrially producing mixtures of LNT and LNnT.

SUMMARY OF THE INVENTION 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:

wherein R is a group removable by hydrogenolysis and R₃' 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 CrC₄ alcohol, preferably methanol, or a solvent mixture of C C₄ 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 C₄ 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 CrC₄ alcohol, or a solvent mixture of Ci-C₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a C C₄ 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

obtained in 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

volume, preferably at least a triple volume, of CrC₄ alcohol solvent, or a solvent mixture of CrC₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a C C₄ alcohol, and, e) then crystallizing the compound of formula 2 from the mixture obtained in step d).

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 C₄ alcohol, or a solvent mixture of Ci-C₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a CrC₄ 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:

wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is selected from acyl and H, R₃ is selected from a group removable by hydrogenolysis, acyl, silyl and an acetal type group, R₄ is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAC₂, benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino,

benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3- diphenylmaleimido and 2,3-dimethylmaleimido.

Yet another aspect of this invention relates to a compound of formula 7

wherein R₆ is, independently, H or a residue of formula A

wherein R, R-i, R₂, R₃,R₄ and Y are as above, particularly a compound of formula 3

or formula 4

or formula 5

5 wherein R, R-i, R₂, R3, R₄ 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

6 wherein R₄ is acyl, and Xi is selected from halogen, -OC(=NH)CCl₃, -OBz and -SR₅, wherein R₅ is selected from alkyl, substituted and unsubstituted phenyl, with an acceptor of formula 5.

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.

Further aspects of this invention relate to the use of a mixture consisting essentially of LNT and LNnT as a pharmaceutically active ingredient for the preparation of a pharmaceutical composition and as a nutritionally active ingredient for the

preparation of a nutritional formulation and to the resulting pharmaceutical

composition and nutritional formulation.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this invention, the term "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. Such protective 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 triphenylmethyl (trityl),

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. Preferably, 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. These preferred and particularly preferred protective 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.

Also in this invention, the term "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. The term "aryl" preferably means a homoaromatic group such as phenyl or naphthyl. The term "acyl" preferably means a Q-C(=O)-group, wherein Q can be H, alkyl or aryl, such as formyl, acetyl, propionyl, butyryl, pivaloyl or benzoyl. "Benzyl" preferably means phenylmethyl. The term "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 -NH₂ 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

trichloroacetamido or triflouroacetamido. "Carboxyl" preferably means an -COOH group. "Alkyloxycarbonyl" preferably means an alkyloxy group attached to its parent molecular moiety through a -C(=O)-group, such as methoxycarbonyl or t- butoxycarbonyl. "Haloalkyloxycarbonyl" preferably means an haloalkyloxy group attached to the parent molecular moiety through a -C(=O)-group, such as 2,2,2- trichloroethoxycarbonyl. "Carbamoyl" preferably means an H₂N-C(=O)-group. "N- Alkylcarbamoyl" preferably means an alkyl group attached to its parent molecular moiety through a -HN-C(=O)-group, such as A/-methylcarbamoyl. "N,N-

Dialkylcarbamoyl" preferably means two alkyl groups, either identical or different, attached to the parent molecular moiety through a >N-C(=O)-group, such as N,N- methylcarbamoyl.

The 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. Also in this invention, the term "silyl 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.

Further in this invention, the term "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

acetal type protective group

wherein R_a, R_b and R_c are carbon-bonded groups.

This kind of group is also 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 methoxymethyl, f-butoxymethyl, 2-methoxy-ethoxymethyl,

benzyloxymethyl, tetrahydropyranyl, tetrahydrofuranyl, 1 ,4-dioxan-2-yl, 1 -methyl-1 - methoxyethyl, 1 -methyl-1 -phenoxyethyl and 2-trimethylsilylethoxy-methyl. The acetal type protective groups are labile under mild acidic conditions.

Still further in this invention, 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:

wherein R is a group removable by hydrogenolysis, and R3' is selected from a group removable by hydrogenolysis and H. Preferably, R is benzyl, 4- methylbenzyl or 4-chlorobenzyl, more preferably benzyl, R₃' is preferably H, and the -OR is in β-orientation. According to the most preferred embodiment the mixture consists essentially of LNT- OBn (a compound of formula 1 wherein R is benzyl and R₃' is H) and LNnT-OBn (a compound of formula 2 wherein R is benzyl and R₃' is H), both in the form of β- 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 CrC₆ 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 C₆ alcohols or a mixture of water and one or more Ci-C₆ 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. Preferably, 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.

In a preferred embodiment, 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 materia¹ 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.

Because catalytic hydrogenolysis is a clean reaction, the purity of the mixture and the LNnT-LNT ratio in the mixture are proportional to those of the mixture consisting essentially of compounds of formulas 1 and 2.

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, OR₄ groups, but not the OR group, and not the OR₃ group when R₃ 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

4 wherein R is a group removable by hydrogenolysis, Ri is acyl, R2 is acyl or H, R3 is selected from a group removable by hydrogenolysis, acyl, silyl and acetal type group, R₄ is acyl and Y is selected from alkanoylamido,

haloalkanoylamido, -NAc₂, benzamido, alkoxycarbonylamino,

haloalkoxycarbonylamino, benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3-diphenylmaleimido and 2,3-dimethylmaleimido.

The ORi , OR₂, OR₃ and OR₄ 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 OR₄ 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. 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. Preferably, 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 -NAc₂, 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 -NH₂ 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. Preferably, a strong base, such as LiOH, NaOH, KOH, Ba(OH)₂, K₂CO₃, a basic ion exchange resin or tetraalkylammonium hydroxides, is used, but the base can also be in an aqueous solution as well. If Y is -NAC2, one of the acetyl groups can also be removed to make the Y group -NHAc. Preferably, the base is NaOH and the solvent is methanol.

Under the conditions of aminolysis, i.e., AAacyl transfer based deprotection, treatment with ammonia, hydrazine, substituted hydrazine, ethylene diamine or primary amines in water, alcohol or water-organic solvent mixtures at 20-120 °C, all of the O- and N- protective acyl groups, including cyclic imides, can be readily removed.

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 R₃ means a group removable hydrogenolysis, on the compounds of formulas 3 and 4. Hence, 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 R₃ 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. Alternatively, the azido Y-group can be easily reduced to amino by a complex metal hydride, such as NaBH₄, or by PPh₃.

The free amino group obtained by one of the deprotective methods or

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 (ρΗ<1 ). Although 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. The skilled person is able to distinguish which deprotective condition affects the acetal and/or silyl groups while the acyl groups and the interglycosidic bonds remain intact. 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 C₆ 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. Preferably, an organic acid, such as acetic acid, formic acid, chloroacetic acid or oxalic acid, is used. Preferably, 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. Alternatively, an anhydrous CrC₆ 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. In a preferred embodiment, in a mixture of compounds of formulas 3 and 4,

preferably in a mixture consisting essentially of compounds of formulas 3 and 4, R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, R is acetyl or optionally substituted benzoyl, R₂ is acetyl, optionally substituted benzoyl or H, R₃ is acyl or silyl, R₄ is acetyl or optionally substituted benzoyl, Y is alkanoylamido or haloalkanoylamido, and -OR is in β-orientation. More preferably, R is benzyl, Ri is benzoyl or 4- chlorobenzoyl, R₂ is H or benzoyl, R₃ is pivaloyl, benzoyl or 4-chlorobenzoyl, R₄ is acetyl and 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

5 wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is acyl or H, R₃ is selected from a group removable by hydrogenolysis, acyl, silyl and acetal type group, and Y is selected from alkanoylamido, haloalkanoylamido, -NAc₂, benzamido, alkoxycarbonylamino, haloalkoxycarbonylamino,

benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3- diphenylmaleimido and 2,3-dimethylmaleimido, with a galactosyl donor of formula 6

6 wherein R₄ is acyl and Xi is selected from halogen, -OC(=NH)CCI₃, -OAc, -OBz or -SRs, wherein R₅ is selected from alkyl, substituted phenyl or unsubstituted phenyl.

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.) A glycosyl trichloroacetimidate (Xi is -OC(=NH)CCl3) can be prepared by the addition of the free anomeric OH compound, corresponding to the compound of formula 6, to trichloroacetonitrile with inorganic or organic base catalysis. 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 BF₃-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. Typical activators include Bronsted acids, such as TsOH, HCIO₄ or sulfamic acid, and Lewis acids, such as ZnC , SnCI₄, triflate salts, BF₃-etherate, trityl perchlorate, AICI₃ or triflic anhydride, and their mixtures. Thioglycosides (Xi is alkylthio- or phenylthio- group) can be activated by thiofilic promoters such as mercury(ll) salts, Br₂, I₂, NBS, NIS, triflic acid, triflate salts, BF₃-etherate, trimethylsilyl triflate, dimethyl-methylthio sulphonium triflate, phenylselenyl triflate, iodonium dicollidine perchlorate,

tetrabutylammonium iodide or mixtures thereof, preferably by Br₂, 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.

In a preferred glycosylation step, a galactosyl donor of formula 6 wherein R₄ 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

4- chlorobenzoyl, R₂ is acetyl, optionally substituted benzoyl or H, preferably benzoyl or H, R₃ is acyl or silyl, preferably pivaloyl, benzoyl or 4-chlorobenzoyl, Y is alkanoylamido or haloalkanoylamido, preferably acetamido or trichloroacetamido, and -OR is in β-orientation. Compounds involved in this glycosidation reaction are considered to be crucial elements of this invention. Thus, this invention also relates to compounds of formula 7

7 wherein R₆ is, independently, H or a residue of formula A

A wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is acyl or H, R₃ is selected from a group removable by hydrogenolysis, acyl, silyl and acetal type group, R₄ is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAc₂, 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 β-anomers or even pure form of one of the anomers.

Preferred compound of formula 7 are those of formula 3

3 formula 4

4 and formula 5

5 wherein R, Ri , R₂, R3, R4 and Y are as defined above.

In a preferred compound of formula 3 R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, Ri is acetyl or optionally substituted benzoyl, R₂ is acetyl, optionally substituted benzoyl or H, R₃ is acyl or silyl, R₄ is acetyl or optionally substituted benzoyl, Y is alkanoylamido or haloalkanoylamido, and -OR is in β-orientation. More preferably, R is benzyl, Ri is benzoyl or 4-chlorobenzoyl, R2 is H or benzoyl, R3 is pivaloyi, benzoyl or 4-chlorobenzoyl, R₄ is acetyl and Y is acetamido or trichloroacetamido.

In a preferred compound of formula 4 R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, Ri is acetyl or optionally substituted benzoyl, R2 is acetyl, optionally substituted benzoyl or H, R₃ is acyl or silyl, R₄ is acetyl or optionally substituted benzoyl, Y is alkanoylamido or haloalkanoylamido, and -OR is in β-orientation. More preferably, R is benzyl, R is benzoyl or 4-chlorobenzoyl, R₂ is H or benzoyl, R₃ is pivaloyi, benzoyl or 4-chlorobenzoyl, R₄ is acetyl and Y is acetamido or trichloroacetamido. In a preferred compound of formula 5 R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, Ri is acetyl or optionally substituted benzoyl, R₂ is acetyl, optionally substituted benzoyl or H, R₃ is acyl or silyl, Y is alkanoylamido or haloalkanoylamido, and -OR is in β-orientation. More preferably, R is benzyl, Ri is benzoyl or 4-chlorobenzoyl, R₂ is H or benzoyl, R₃ is pivaloyi, benzoyl or 4-chlorobenzoyl and Y is acetamido or trichloroacetamido.

Acceptors of formula 5 can be synthesized from compounds of formula 8

8 wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is H and Y is selected from alkanoylamido, haloalkanoylamido, -NAc₂, 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 R₃-halide to give a compound of formula 5 wherein R₃ 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. Preferably, 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 R₃ is acyl. 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 R₃ 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 R₃ is silyl.

According to a preferred embodiment, a compound of formula 8 wherein R is benzyl, 4-methylbenzyl or 4-chlorobenzyl, preferably benzyl, Ri is acetyl or optionally substituted benzoyl, preferably benzoyl or 4-chlorobenzoyl, R2 is acetyl, optionally substituted benzoyl or H, preferably H, Y is alkanoylamido or haloalkanoylamido, preferably acetamido and -OR is in β-orientation, is silylated, preferably t- butyldimethylsilylated to afford a compound of formula 5 wherein R is benzyl, 4- methylbenzyl or 4-chlorobenzyl, preferably benzyl, Ri is acetyl or optionally substituted benzoyl, preferably benzoyl or 4-chlorobenzoyl, R₂ is acetyl, optionally substituted benzoyl or H, preferably H, R₃ is silyl, preferably t-butyldimethylsilyl, Y is alkanoylamido or haloalkanoylamido, preferably acetamido and -OR is in β- orientation. Compounds of formula 8 can be synthesized in a conventional manner by, for example, a pathway as shown in Scheme 1 .

Scheme 1 .

The reaction of the lactose acceptor of formula 10 (R, Ri and R₂ are as defined above) with the glucosaminyl donor of formula 11 (Y is as defined above or Y with the vicinal X₂ forms a 2-alkyl-, 2-haloalkyl- or 2-phenyl-oxazoline, X₂ is selected from halogen, -OC(=NH)CCI₃, -OAc, -OBz and -SR₅, wherein R₅ 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 X₂ of the donor of formula 11 with the 3'-OH group of the acceptor of formula 10.

Surprisingly, regioselective glycosylation can be achieved on an acceptor of formula 10, wherein R₂ is H, with the donor of formula 11 . In such a dihydroxy acceptor, 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. Thus with careful selection of the conditions such as donor reactivity, solvent, temperature, nature of promoter, means of addition of reactants/promoters and the like, the reaction can be driven to the formation of the desired 1 -3 interglycosidic linkage instead of a 1 -4 coupling. However, particular care has to be taken with regard to the 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 X₂ 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 X₂ forms 2-alkyl-, 2-haloalkyl- or 2-phenyl-oxazoline) can be promoted in such a glycosylation reaction with TsOH, camphorsulfonic acid, TMSOTf, FeCl₃, 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 (R₂ is acetyl) can also be removed in this way. This deprotection step can be carried out in a CrC₆ alcohol or a mixture of CrC₆ 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, can also be used in this reaction. 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. For example, 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. These 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) can produce a protected glycosyl hemiacetal which can be converted to a trichloroacetimidate donor with

trichloroacetonitrile under inorganic or organic base catalysis. Glycosyl iodides, bromides and chlorides (X₂= I, Br, CI) can be synthesized by treatment of the 1 -O- acyl derivative with an appropriate halogenating agent (e.g. hexamethyl-disilazane/l₂, trimethyl iodosilane, Et₃SiH/l₂, HBr, PBr₃, thionyl chloride, PCI₅/BF₃-etherate or TiCI₄) The glycosyl fluorides (X₂= F) can be prepared by treatment of the appropriate precursors such as a hemiacetal, glycosyl halide (I, Br, CI), glycosyl ester or S- glycoside with fluorinating reagent, such as HF, AgF, AgBF₄, tetrabutyl ammonium fluoride, diethylaminosulfur trifluoride, 2-fluoro-1 -methylpyridinium tosylate,

Selectfluor, Deoxo-Fluor or 4-methyl(difluoroiodo)-benzene, etc. Thioglycosides (X₂= -SR₅, in which R₅ is alkyl or substituted or unsubstittued phenyl) can be made by thiolysis of the 1 -O-acyl derivatives or glycosyl halides with R₅SH in the presence of a Lewis acid. Oxazoline-type donors can be synthesized from the appropriate acylamido derivative having any of the X₂ leaving group mentioned above when treated with a conventional activator.

Alternatively, certain acceptors of formula 5 (wherein R₃ is a group removable by hydrogenolysis or R₃ is acyl except for acetyl and R, Ri , R₂ and Y are as defined above) can be synthesized in a conventional manner by, for example, a pathway as shown in Scheme 2.

5

Scheme 2.

In this regard, a compound of formula 12 (wherein R₆ is acetyl or two R6 groups form a cyclic acetal/ketal type protective group, such as isopropylidene, and R₇ is a group removable by hydrogenolysis or R₇ is acyl except for acetyl, X₂ 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 , R₂, 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. In general, 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

development. 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:

- with other A/-acetyllactosamine and/or lacto-N-biose and/or fucose

and/or sialic acid containing human milk oligosaccharides to aid in the development and/or maturation of the immune system of neonatal

infants, particularly against secondary infections following viral

infections such as influenza.

- as a prebiotic to enhance the beneficial effects and efficiency of

probiotics, such as Lactobacillus and Bifidobacterium species, in

promoting the development of an early bifidogenic intestinal microbiota in infants,

- in reducing the risks of the development of allergies and/or asthma in infants, and

- in preventing and treating pathogenic infections such as diarrhoea in infants.

A further aspect of the invention relates to 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 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:

Remington's Pharmaceutical Sciences, Mack Pub. Co.; 17th edition. 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. Minerals and trace elements can include, for example,: Ca, P, K, Na, CI, Mg, Mn, Fe, Cu, Zn, Se, Cr and I. 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. palm olein, sunflower oil, safflower oil) and vitamins and minerals essential in a daily diet. 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. In this method, to 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 C₄ alcohol, preferably methanol, or a solvent mixture of CrC₄ alcohols, or a ketone type solvent, preferably acetone, or a solvent mixture of a ketone type solvent with a CrC₄ alcohol.

Preferably, 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. Also preferably, 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. Before addition of the organic solvent(s) he 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. After cooling, a mixture consisting essentially of compounds of formulas 1 and 2 precipitates or preferably crystallizes out. 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. In this method, a compound of formula 1 , preferably LNT-OBn, can be crystallized out when the mixture of the compounds of formulas 1 and 2 contains at least about 20 % of the compound of formula 1 , and up to a maximum about 80 % of the compound of formula 2, preferably LNnT-OBn. In carrying out the method, the concentration of the starting aqueous solution can vary around 15 m/V% to around 60 m/V%. To the aqueous solution, preferably heated to 45-50 °C, is added a Q-C₄ alcohol, preferably methanol, or a solvent mixture of d- C₄ alcohols, or a ketone type solvent, preferably acetone, or a mixture of a ketone type solvent with a C C₄ alcohol, preferably preheated. The ratio of organic solvent(s) to the aqueous solution should be about 1 /5 to 1 /10 (in volume). Then 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:

- 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, when the ratio of the compound of formula 1/ compound of formula 2 is about 1 :3 to 2:3; and

- 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. Optionally, 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. To 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-C₄ alcohol solvent, preferably methanol, or a solvent mixture of C C₄ alcohols, or a ketone type solvent, preferably acetone, or a mixture of a ketone type solvent with a Ci-C₄ alcohol, preferably while heating to about 45-50 °C. After cooling, 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.

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. In this method 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. To a volume of the acidic aqueous solution is added about 2.5-3.5 fold volume of a CrC₄ alcohol solvent, preferably methanol, or a solvent mixture of C C₄ alcohols, preferably while heating to about 45-50 °C. Upon cooling the resultng acidic aqueous/alcoholic solution, the compound of formula 2, preferably LNnT-OBn, crystallizes out from it, preferably while stirring, within a period from 2 hours to overnight. 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. Optionally, 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 ). Doing so, 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, Na₂CO₃ or K₂CO₃. To a volume of the resulting basic aqueous solution is added about 3-5 volumes of a ketone type solvent, preferably acetone, preferably while heating to about 45-50 °C. Upon cooling the resulting basic aqueous/organic solution, a 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. 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.

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

EXAMPLES Abbreviations: Ac: acetyl, Bz: benzoyl, CIBz: 4-chlorobenzoyl, Bn: benzyl, Ph: phenyl, Piv: pivaloyl, NHCbz: benzyloxycarbonylamino, TCA: trichloroacetyl

1 . Donors of formulas 11 and 12

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 BF₃ OEt₂ 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-4,6-0-benzylidene-1-thio-a/ -D-glucopyranoside

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%).

TLC (petroleum ether/EtOAc 2:1 ) Rf=0.64 a-isomer, 0.15 β-isomer

Phenyl 2-azido-2-deoxy-3-0-acetyl-4,6-0-benzylidene-1-thio-a/B-D- glucopyranoside

Phenyl 2-azido-2-deoxy-4,6-O-benzylidene-1 -thio-a/3-D-glucopyranoside (5.50g, 14.3 mmol) was dissolved in pyridine/Ac₂O (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 MgSO₄ and the solvent was removed in vacuo. Flash chromatography (petroleum ether/EtOAc 3:1 ) yielded the title compound (5.90 g, 97%).

TLC (petroleum ether/EtOAc 3:1 ) Rf=0.38

[a]_D = + 22.3 (c = 1 , CHCI₃). ¹H-NMR (250 MHz, CDCI₃): δ= 2.15 (s, 3H, CH3OC); 4.15 (m, 3H, H-5, H-2, H-6a); 4.55 (m, 2H, H-6b, H-4); 5.15 (dd, 1 H, J3-4=3.4 Hz, J3-2=1 1 .1 Hz, H-3); 5.55 (s, 1 H, PhCH); 5.8 (d, 1 H, J1 -2=5,3 Hz, H-1 ); 7.2 (m, 10H, Ph).

MALDI-MS (positive Mode, Matrix DHB, THF): [M+Na]\ m/z = 450.2; found: m/z = 450.2. Combustion analysis:

Calc: C: 59.00; H: 4.95; N: 9.83 Found: C: 58.84; H: 5.02; N: 9.93

Phenyl 2-azido-2-deoxy-3,4-di-0-acetyl-6-0-benzyl-1-thio-a/B-D- glucopyranoside NaBH₃CN ( 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. ethereal HCI solution (~ 75 imL) waswas then added to the reaction mixture until the solution became a persistent pink. After 10 minutes at 0 °C, the reaction was quenched with a satd. aqueous NaHCO₃ solution (100 imL) and diluted with diethyl ether (250 imL). The aqueous phase was extracted with Et₂O (100 imL) and the combined organic layers were washed twice with water (100 imL), dried (MgSO₄), filtered, and concentrated. The residue was redissolved in 2 volumes of a mixture 1 /1 Ac₂O/pyridine and the reaction mixture stirred for 2h. The solvent was evaporated in vacuo; the residue was redissolved in EtOAc and washed 2 times with 1 N HCI and 3 times with H₂O. The organic solvent was evaporated in vacuo and the product was purified by flash chromatography (petroleum ether/EtOAc 4/1 to 7/3) to yield the title compoundcompound (17.1 mmol, 90%).

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

evaporated in vacuo. The intermediate product was dried 1 h in high vacuum, then the residue (7.4 mmol) was dissolved in dry DCM (30 imL), and imidazole (1 .8 equiv.) and TIPSCI (1 .2 equiv.). The reaction stirred 1 h at RT; the reaction waswas quenched with MeOH, and the solvent waswas evaporated in vacuo. Flash

chromatography (petroleum ether/EtOAc 8/1 ) yielded the sialylated product (6.81 mmol, 92%). The product was acetylated under standard conditions to yield 6.6 mmol (97%).

TLC (petroleum ether/EtOAc 5/1 ) Rf=0.61

2-Benzyloxycarbonylamino-2-deoxy-D-glucopyranose

To a mixture of D-glucosamineghydrochloride (50 g, 0.232 mol) and sodium

hydrogencarbonate (47.8 g, 0.569 mol) in 370 imL of water at 0-5 °C, benzyl chloroformate (55 imL, 0.358 mol, 95% (tech. grade)) was added drop wise under vigorously stirring. The reaction solution waswas allowed to reach RT overnight. The next morning the thick precipitate waswas filtered off and washed with cold water (2 x 250 ml_), EtOAc (300 ml_) and Et₂O (300 ml_). The solid was dried overnight to give 72 g of the CbZ-protected glucosamine derivative. Recrystallization from warm

MeOH/ water mixture (apprx. 1 :1 ) gave white crystals (54 g, 75%). M.p. 195 °C; changed structure; 205-207 °C; melted.

Phenyl 2-benzyloxycarbonylamino-2-deoxy-3,4,6-tri-0-acetyl-1-thio-a/B-D- glucopyranoside To a -5 °C solution of 2-benzyloxycarbonylamino-2-ceoxy-1 ,3,4,5-tetra-O-acetyl- glucopyranose in DCM (100 imL), tiophenol was added and the mixture was cooled with ice bath, then BF₃ OEt₂ was added drop wise and the reaction mixture stirred for 48h at this temp. The reaction mixture was neutralized with saturated bicarbonate solution and 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 thioglycoside (73%). The data was in coincidence with those reported in the literature. Phenyl 2-benzyloxycarbonylamino-2-deoxy-4,6-0-benzylidene-1-thio-a/B-D- glucopyranoside

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

dimethoxybenzaldehyde (6.4 mL, 42.52 mmol) was 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 wafer 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 to 1 /1 ) to give the title compound (23.24 mmol, 82%).

Phenyl 2-benzyloxycarbonyl-2-deoxy-3,4-di-0-acetyl-6-0-benzyl-1-thio-a/B-D- glucopyranoside

A mixture of previous compound (13.1 mmol), 3 A powdered molecular sieves (5 g) and Et₃SiH ( 21 mL, 132 mmol, 10 equiv.) in anhydrous CH₂CI₂ (150 mL) was cooled to 0 °C. Trifluoroacetic anhydride (TFAA, 10 mL, 130 mmol) was added, and the mixture was stirred for 1 h at 0 C. Et3N (3 mL) was added, and the mixture was diluted with CH₂CI₂ (100 mL), washed with water, saturated aq NaHCO₃ and water, dried (MgSO₄) and concentrated. The crude material was dissolved in Ac₂O/Pyridine mixture (2 volumes of a 1 /1 mixture) and the reaction mixture stirred for 2h at RT. The solvent was evaporated in vacuo; the residue was redissolved in EtOAc and washed 2 times with 1 N HCI and 3 times with H₂O. The organic solvent was evaporated in vacuo and the product was purified by flash chromatography

(petroleum ether/EtOAc 3/1 ) to yield the title compound (1 1 mmol, 85%).

Phenyl 2-deoxy-2-A/-trichloroacetamido 3-1 -D-thioglucosamine (75.6 g, 0.181 mol), 2,2-dimethoxypropane (70 mL) and p-TsOH x H₂O (2.6 g, 0.1 eq) were mixed in DMF (250 mL) and stirred for 2 hours at 40 °C under recUced pressure (250 mbar).

Quenched with Et3N and evaporated off reaction solution to get an oil. The crude product was re-dissolved in EtOAc (350 mL) by heating and made a hot extraction using water (350 mL). The layers were separated and the upper organic layer was placed on rotational evaporator to distill off ~ 100 mL of EtOAc. Warm heptane (250 mL) was added in big portions and then the solution was stirred against RT. The thick suspension formed was filtered and the crystals was washed once with MTBE (150 mL) and then dried to get isopropylidene protected compound (59 g, 0.134 mol, 74%).

¹³C-NMR (CDCI3, 300MHz) δ 19.8, 29.7, 57.6, 61 .9, 71 .8, 72.1 , 74.3, 87.3, 93.7, 99.7, 129.6-131 .0,

M.p. 190-192°C [a]_D = -12.8 (c 1 .0, DMSO)

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

whereupon acetic anhydride (100 mL) was added dropwise. The mixture was allowed to reach RT overnight and then evaporated off in vacuum at 50 °C. Dissolved the crude product in DCM (350 mL) and washed with aq. 1 M HCI (2 x 1 50 mL) and then sat. aq. sodium bicarbonate solution (2 x 150 mL, careful), dried over sodium sulfate, filtered and evaporated off all organic solvent. The product was re-crystallized from hot EtOAc (200 mL) to get 3-O-acetylated compound (47 g, 0.094 mol, 83%). ¹H-NMR (CDCI3, 300MHz) δ 1 .01 (s, 3H), 1 .43 (s, 3H), 2.04 (s, 3H), 3.62-3.82 (m, 3H), 3.95 (dd, 1 H, J = 5.1 10.5 Hz), 4.18 (ddd, 1 H, J = 9.6 9.9 10.5 Hz), 4.79 (d, 1 H, J = 10.5 Hz), 5.39 (dd, 1 H, J = 9.9 9.9 Hz), 7.26-7.30 (m, 3H), 7.44-7.47 (m, 2H), 7.80 (d, 1 H, J = 9.6 Hz). ¹³C-NMR (CDCI₃, 300MHz) δ 18.9, 20.8, 28.4, 54.3, 61 .7, 71 .4 (2C), 73.3, 87.9, 92.6, 99.7, 128.3, 128.9 (2C), 132.1 , 133.3 (2C), 162.0, 171 .8.

M.p. 216-219°C

[a]_D = -28.0 (c 1 .0, CHCI3)

To a solution of the isopropylidene compound (47 g, 0.094 mol) in DCM (300 mL) at 0-5 °C was added diluted aq. HCIO₄ (4 mL in total, 2 mL of water + 2 L of 70% aq. HCIO₄). The reaction was performed at this temperature for 3 hours when aq. sodium bicarbonate (250 mL) was added. The mixture was stirred vigorously for 30 min. and then transferred to separation funnel. The organic phase (lower) was evaporated off and then co-evaporated with toluene (150 mL) to get a white foam (37 g, 0.0807 mol, 86%) which was dried under reduced pressure before used in the acylations without further purifications.

¹H-NMR (CDCI₃, 300MHz) δ 2.01 (s, 3H), 3.03-3.18 (m, 1 H), 3.51 -3.57 (m, 1 H), 3.72- 3.91 (m, 3H), 3.96-4.06 (m, 2H), 4.92 (d, 1 H, J = 10.5 Hz), 5.36 (dd, 1 H, J = 10.2 10.2 Hz), 7.24-7.31 (3H), 7.40-7.47 (m, 3H).

¹³C-NMR (CDCI₃, 300MHz) δ 20.9, 54.4, 62.1 , 68.7, 75.9, 79.4, 86.4, 92.2, 128.2, 129.1 (2C), 132.2, 132.4 (2C), 162.1 , 172.0.

To 3-O-acetylated diol (4 g, 8.72 mmol) in pyridine (25 mL) cooled to 0°C was added pivaloyl chloride (1 .3 mL) over 30 minutes. MeOH (1 mL) was added next morning and the mixture was stirred for 1 hour before 10 mL of the reaction mixture was evaporated off. Then a mixture of pyridine and acetic anhydride was added (1 :1 , 20 mL) and the solution was stirred for 2 hours before concentrated to dryness. The crude product was re-dissolved in EtOAc and washed with with aq. 1 M HCI and then sat. aq. sodium bicarbonate solution. Reduced organic phase volume by distillation down to ~50 mL and then allowed to cool down to RT. Next morning filtered off crystals and washed with some n-hexane to get 3.3 g (65%) of the 6-O-Piv

compound as white crystals.

¹H-NMR (CDCI₃, 300MHz) δ 1 .23 (s, 9H), 2.00 (s, 3H), 2.01 (s, 3H), 3.76 (ddd, 1 H, J = 2.4 5.1 9.9 Hz), 3.95 (ddd, 1 H, J= 9.0 9.9 10.2), 4.16 (dd, 1 H, J = 5.1 12.3 Hz), 4.25 (dd, 1 H, J = 2.4 12.3 Hz), 4.87 (1 H, d, J = 10.2 Hz), 5.05 (dd, 1 H, J = 9.9 9.9 Hz), 5.31 (1 H, dd, J = 9.9 9.9 Hz), 6.80 (d, 1 H, J = 9.0 Hz), 7.27-7.40 8m, 3H), 7.50- 7.54 (m, 2H).

¹³C-NMR (CDCI₃, 300MHz) δ 20.4, 20.6, 27.1 , 38.8, 54.4, 62.3, 68.2, 73.2, 76.1 , 86.3, 92.2, 128.6, 129.0 (2C), 131 .4, 133.4 (2C), 1 61 .6, 169.0, 171 .1 , 178.1 .

To above 4,6-diol derivative (34.79 g, 76.18 mmol) in dry pyridine at 0-5°C was added p-chlorobenzoyl choride (10.3 mL, 80 mmol) during 30 min. The reaction solution was allowed to reach RT during 2 hours whereupon another portion of pCIBzCI (1 .5 L) was added dropwise. After totally 5 hours, MeOH (10 mL) was added and the solution was stirred for 20 minutes before applied vacuum to remove MeOH and some pyridine (20 mL in total). To this solution was added acetic anhydride (40 mL) and then the stirring was continued for 3 hours when all solvents were

evaporated off. The crude was dissolved in DCM (250 mL) and washed with 1 M aq. HCI. The phases were separated and the organic phase was washed once with brine and then DCM was distilled off (-75 mL) whereupon the product started to crystallize. Added n-hexane (50 mL) and then allowed the mixture to reach RT with stirring.

Another 2 hours stirring at ice-bath was followed by filtration and washing of the obtained solid with (n-hexane, 100 mL) to give the product as a white solid (25.5 g, 40 mmol). The mother liquid was evaporated to dryness and the obtained solid was dissolved in EtOAc (100 mL) and n-hexane (25 mL) was added. This procedure gave a 2^nd crop of product (10.2 g, 16 mmol, 74% in total).

¹H-NMR (CDCI₃, 300 MHz) δ 1 .95 (s, 3H), 2.01 (s, 3H), 3.90 (m, 1 H), 4.02 (ddd, 1 H, J= 9.3 10.2 10.5), 4.37 (dd, 1 H, J= 5.4 12.0 Hz), 4.56 (dd, 1 H, J = 2.4 12.3 Hz), 4.88 (d, 1Η, J= 10.5 Hz), 5.14 (t, 1H, J= 9.69.9 Hz), 5.37 (t, 1H, J=9.610.2 Hz), 6.91 (d, 1H, J=9.3 Hz), 7.15-7.51 (m, 7H), 7.95-8.00 (m, 2H).

¹³C-NMR (CDCI₃, 75.74 MHz) δ 20.4, 20.5, 54.4, 63.0, 68.4, 73.1, 76.0, 86.4, 92.2, 127.9, 128.7, 128.9, 128.9, 129.0, 131.1, 131.4, 133.5, 161.7, 165.2, 169.1, 171.1. M. p.219-221 °C

[a]_D=-11.0 (c 1.0, CHCI3)

To 3-O-acetylated diol (16 g, 35.03 mmol) in pyridine (80 mL) cooled to 0°C was added benzoyl chloride (3.85 mL, 33.33 mmol, 1.1 eq.) dropwise during 30 min. The reaction mixture was stirred for 3 hours when MeOH (5 mL) was added followed by stirring for 30 minutes at RT. Evaporated off 20 mL of the reaction mixture and then acetic anhydride (30 mL) was added together with pyridine (20 mL). After 1 hour all solvent was distilled off to get a foam which was dissolved in DCM (200 mL).

Washed with aq.1 M HCI (2 x 200 mL) and then sat. aq. sodium bicarbonate solution (2 x 150 mL) followed by evaporation to dryness. Re-crystallization from EtOAc (100 mL, hot) gave product (15g, 24.88 mmol, 71%) as a white crystalline compound.

¹H-NMR (CDCI₃, 300MHz) δ 1.93 (s, 3H), 2.01 (s, 3H), 3.92 (m, 1H), 4.05 (ddd, 1H, J = 8.49.310.2 Hz), 4.37 (dd, J= 5.712.3 Hz), 4.60 (dd, J= 2.412.3 Hz), 4.87 (d, 1H, J= 10.2 Hz), 5.16 (t, 1H, J=9.69.9 Hz), 5.38 (t, 1H, J=9.610.2 Hz), 6.97 (d, 1H, J = 8.4 Hz), 7.11 -7.64 (m, 8H), 8.04-8.07 (m, 2H).

¹³C-NMR (CDCI₃, 75.74 MHz) δ 20.3, 20.6, 54.2, 62.9, 68.6, 73.3, 76.0, 86.6, 128.5, 128.6, 128.9, 129.4, 129.7, 131.5, 133.4, 133.4, 161.7, 166.0, 169.1, 171.3.

M.p.203-206°C

[a]_D=-14.6 (c 1.0, CHCI3) 2. Glycosylations for the trisaccharides of formula 5

Case 1. Y=azido, R₃=group removable by hydrogenolysis or silyl, X₂ =SR₅

To a mixture of donor (1.3 equiv.) and acceptor of formula 10 (1 equiv.) in MeCN at - 40 °C the NBS (1.43 equiv.) is added followed by TDH (0.13 equiv.). The reaction mixture stirred at this temperature for 2 hours and the reaction is quenched with Et3N. The solvent is evaporated in vacuo and the residue is redissolved in EtOAc, washed with sat NaHCO₃, twice with Na₂S₂O₃ and water. The solvent is evaporated in vacuo.

Case 2. Y=NHCbz, X₂=SR₅ To a mixture of donor(1 .3 equiv.) and acceptor of formula 10 (1 equiv.) in DCM at - 5°C the NBS (1 .43 equiv.) is added followed by TfOH (0.13equiv.). The reaction mixture stirred at this temperature for 2 hours and the reaction is quenched with Et₃N. The solvent is evaporated in vacuo and the residue is redissolved in EtOAc, washed with sat NaHCO₃, twice with Na₂S₂O₃ and water. The solvent is evaporated in vacuo. Any of this two trisaccharidescan be converted into Y=NHAc by careful

hydrogenolysis under basic conditions followed by N-acetylation.

Case 3. Preparation of 3",4"-diol-2-deoxy-2-trichloroacetamido-lacto-A/-triose acceptors

R₆ = Ac, H

R₇ = Piv, CIBz, Bz To a solution of thioglycoside donor (wherein R₇ is Piv, 4-CIBz or Bz, R₆ is Ac, 10 mmol) and acceptor (6.7 mmol) in dry DCM (50 mL) was added NBS (12.5 mmol) and short thereafter triflic acid (0.5 mmol). The mixture was stirred for 2 hours at ambient and then cooled to 5 °C whereupon aq. NhiOH was added to quench the reaction. The mixture was allowed to reach ambient again and water was added (50 mL). The mixture was stirred vigorously for 1 hour and then the phases were allowed to separate. The organic phase was washed twice with brine and then the organic extract was evaporated to obtain a foam.

The crude product was re-dissolved in MeOH and DCM (1 :1 , 50 mL) and cooled to 0- 5 °C whereupon 5 mL cc. hfeSO₄ was added dropwise. The mixture was allowed to reach 25 °C and stirred for 18 hrs when E¾N was added to adjust the pH to ~8. The reaction mixture was concentrated and the diol acceptor was isolated by silica column purification to give the diol-lacto-A/-triose acceptors in typically 50-60% overall yield. R₇ = Piv, R₆ = Ac:

¹H-NMR (CDCI₃, 300MHz) δ 1.09 (s, 9H), 1.87 (s, 3H), 1.96 (s, 3H), 3.40-3.50 (m, 2H), 3.58-3.72 (m, 3H), 3.92 (dd, 1 H, J= 5.411.7 Hz), 3.99-4.11 (m, 3H), 4.17 (dd, 1H, J=2.412.3 Hz), 4.37-4.41 (m, 2H), 4.55 (d, 1H, J= 12.6 Hz), 4.57 (d, 1H, J = 7.8 Hz), 4.65 (d, 1 H, J= 7.8 Hz), 4.80 (d, 1 H, J =12.6 Hz), 4.87-4.93 (m, 2H, ), 5.24 (dd, 1 H, 9.010.5 Hz), 5.38 (dd, 1 H, J = 7.89.9 Hz), 5.45 (dd, 1 H, J = 7.89.9 Hz), 5.53-5.97 (m, 2H), 6.35 (d, 1 H, J= 7.8 Hz), 6.86-7.99 (m, 30H).

R₇= Piv, R₆ = H:

¹H-NMR (CDCI₃, 300MHz) δ 1.05 (s, 9H), 3.02-3.08 (m, 2H), 3.29-3.35 (m, 1H), 3.39 (dd, 1H, J=7.211.7 Hz), 3.65-3.85 (m, 3H), 3.93 (dd, 1H, J= 5.411.4 Hz), 3.99-

4.07 (m, 2H), 4.19-4.26 (m, 1H), 4.36-4.43 (m, 3H), 4.55 (d, 1H, J= 12.6 Hz), 4.58 (d, 1H, J=7.8 Hz), 4.66 (d, 1H, J=7.5 Hz), 4.80 (d, 1H, J=12.6 Hz), 4.82 (d, 1H, J =

7.8 Hz), 5.39 (dd, 1 H, J = 7.89.6 Hz), 5.47 (dd, 1 H, J= 7.89.9 Hz), 5.53-5.59 (m, 2H), 6.69 (d, 1 H, J= 6.0 Hz), 6.86-7.99 (m, 30H). R₇ = CIBz, R₆ = Ac:

¹H-NMR (CDCI₃, 300MHz) δ 1.87 (s, 3H), 1.94 (s, 3H), 3.31 (dd, 1H, J= 7.211.4 Hz), 3.46-3.55 (m, 1 H), 3.64-3.76 (m, 3H), 3.89 (dd, 1 H, J= 5.1 11.7 Hz), 3.98 (dd, 1 H, J = 9.39.3 Hz), 4.03 (dd, 1 H, J= 3.69.9 Hz), 4.35-4.40 (m, H), 4.55 (d, 1 H, J= 12.3 Hz), 4.56 (d, 1H, J=8.1 Hz), 4.64 (d, 1H, J= 7.8 Hz), 4.80 (d, 1 H, J= 12.3 Hz), 4.99 (d, 1H, J=7.5 Hz), 5.00 (dd, 1H, J= 9.39.3 Hz), 5.34 (dd, 1H, J=9.610.5 Hz), 5.38 (dd, 1 H, J = 7.810.2 Hz), 5.45 (dd, 1 H, J = 7.89.9 Hz), 5.54 (dd, 1 H, J = 9.39.3 Hz), 5.60 (d, 1 H, J= 3.3 Hz), 6.46 (d, 1 H, J= 7.8 Hz), 6.79-6.82 (m, 2H), 7.09-8.03 (m, 32H).

R₇ = CIBz, R₆ = H: ¹H-NMR (CDCI₃, 300MHz) δ 3.03-3.12 (m, 2H), 3.19-3.34 (m, 3H), 3.43-3.47 (m, 1 H), 3.68-3.72 (m, 2H), 3.88-4.05 (m, 4H), 4.35-4.42 (m, 2H), 4.55 (d, 1 H, J = 12.6 Hz), 4.57 (d, 1 H, J = 7.8 Hz), 4.65 (d, 1 H, J = 8.4 Hz), 4.71 -4.76 (m, 1 H), 4.80 (d, 1 H, J = 12.6 Hz), 4.96 (d, 1H, J= 8.1 Hz), 5.36-5.58 (m, 3H), 5.60 (d, 1H, J= 3.3 Hz), 6.69 (d, 1 H, J = 6.2 Hz), 6.86-7.99 (m, 34H). Case 4. Acidic trans-esterfication of protected N-acetamido lacto-N-triose derivative and silylation of the primary hydroxyl into a triol acceptor

Imidazole

THF

0 °C

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. H₂SO₄ (0.3 mL, 5.63 mmol) at room

temperature. The solid dissolved to give a yellow solution, which was stirred at room temperature for 18h. The solution was diluted with DCM (20 mL) and H₂O (10 mL) at room temperature then quenched with ammonia 25% in water (2.0 mL, 13.0 mmol) until pH was 10-1 1 . The phases were separated and the organic layer was

evaporated under reduced pressure to a syrup. MeOH (15 mL) was added and the obtained suspension was stirred at room temperature for 3h before filtration and washing with MeOH (10 mL), to afford 0.85 g of a white solid (94 %). R _f0.8

(toluene/acetone 3:7).

¹H-NMR (300 MHz, DMSO-afe) δ 2.15 - 2.45 (m, 4H), 2.47 - 2.57 (m, 1 H), 2.71 (dd, J = 1 1 .7, 5.1 Hz, 1 H), 2.86 (q, J = 9.9, 8.5 Hz, 3H), 3.10 (d, J = 10.5 Hz, 1 H), 3.16 -

3.31 (m, 2H), 3.37 (t, J = 9.5 Hz, 1 H), 3.48 (d, J = 7.2 Hz, 1 H), 3.52 - 3.63 (m, 4H), 3.73 - 3.93 (m, 3H), 4.08 (d, J = 3.2 Hz, 1 H), 4.14 - 4.38 (m, 3H), 4.79 (t, J = 9.3 Hz,

1 H), 6.16 - 6.95 (m, 23H), 6.95-7.23 (m, 7H).

¹³C-NMR (75 MHz, DMSO-afe) δ 21 .9, 48.7, 55.7, 60.8, 67.2, 70.0, 70.3, 70.4, 72.0, 72.2, 72.3, 72.8, 73.9, 75.4, 76.5, 80.6, 98.9, 100.6, 102.2, 127.3, 127.6, 128.1 , 128.3, 128.4, 128.7, 128.8, 128.9, 129.2, 129.3, 129.5, 129.7, 133.3, 133.5, 133.6, 133.7, 137.2, 140.8, 164.3, 164.7, 165.0, 165.3, 165.4, 168.6.

The above obtained material (4.2 g, 3.63 mmol) was stirred at 0-5 °C in THF (20 mL). To the mixture was added imidazole (3.5 g, 3.48 mmol) and then TBDMSCI (0.5 g,

3.32 mmol) in one portion, each at 0-5 °C. The mixtire was stirred at this

temperature for 30 min and then the solvent was evaporated under reduced pressure. To the residue was added DCM (100 mL) and washed once with H₂O (50 mL). The organic phase was concentrated under reduced pressure. Two successive purifications of the residue by flash chromatography (DCM/MeOH 95:5) afforded 2.0 g of a white foam (44 %). R ,0.5 (DCM/MeOH 9:1 ).

¹H-NMR (300 MHz, Methanol-^) δ 0.09-0.12 [m, 6H, Si(CH₃)₂], 0.74-1 .01 [m, 9H, C(CH₃)₃], 1 .47 (s, 3H, COCH₃), 3.13 - 3.40 (m, 4H), 3.46 - 3.97 (m, 7H), 3.99- 4.33(m, 3Η), 4.38 - 4.63 (m, 4H), 4.63 - 4.82 (m, 2H), 5.27 - 5.50 (m, 2H), 5.67 (dt, J = 15.8 Hz , J = 9.3 Hz, 1 H), 6.97-7.16 (m, 5H, H_arom), 7.16 - 7. 69 (m, 15H, H_arom), 7.78-8.12 (m, 10H, H_arom).

¹³C-NMR (75 MHz, MeOH-d₄) δ -5.1 , -5.0, 19.2, 22.7, 26.4, 26.5, 26.6, 57.2, 64.1 , 64.5, 70.0, 71 .6, 72.0, 72.4, 73.8, 74.0, 74.1 , 74.3, 74.6, 75.0, 77.5, 78.0, 81 .5, 100.6, 102.5, 104.1 , 128.8, 128.8, 129.3, 129.3, 129.6, 129.7, 129.8, 129.8, 130.5, 130.6, 130.7, 130.7, 130.9, 131 .0, 131 .2, 131 .4, 134.2, 134.5, 138.3, 166.3, 166.8, 167.3, 167.3, 173.9.

3. Glycosylations for the tetrasaccharides of formula 3 and 4 Case 1. X₁ =OC(NH)CCI₃, Y= NHCbz

To a -5 °C solution of a compound of formula6 (1 .2 equiv.) and 5 (1 equiv.) in DCM (5 volumes), TMSOTf (0.01 -0.02 equiv.) is added and the reaction mixture stirred for 1 h at this temperature. Triethylamine is added to neutralize and the organic solution is washed twice with water. The solvent is evaporated in vacuo to give a mixture of regioisomers.

Case 2. X₁ =SR₅, Y=NHCbz

To a -5°C solution of a compound of formula6 (1 .2 equiv.) and 5 (1 equiv.) in DCM (5 volumes), NBS (1 .1 equiv.) followed by TfOH (0.05 equiv.) is added and the reaction mixture stirred for 1 h at this temperature. A solution of 7% sodium thiosulfate in saturated bicarbonate is added to quench the reaction. Eventually this process could be done with aqueous ammonia. The organic solution is then washed twice with water. The solvent is evaporated in vacuo to give a mixture of regioisomers .

Case 3. X₁ =OC(NH)CCI₃ Y= N₃ or AcNH

To a 0°C solution of a compound of formula6 (1 .2 equiv.) and 5 (1 equiv.) in DCM (5 volumes), TMSOTf (0.01 -0.02 equiv.) is added and the cooling is removed. The reaction mixture stirred for 1 h. Triethylamine is added to neutralize and the organic solution is washed twice with water. The solvent is evaporated in vacuo to give a mixture of regioisomers.

Case 4. X₁ =SR₅, Y=N₃ or AcNH To a 0°C solution of a compound of formula6 (1 .2 equiv.) and 5 (1 equiv.) in DCM (5 volumes), NBS (1 .1 equiv.) followed by TfOH (0.05equiv.) is added and the cooling is removed. The reaction mixture stirred for 1 h. A solution of 7% sodium thiosulfate in saturated bicarbonate is added to quench the reaction. Eventually this process could be done with aqueous ammonia. The organic solution is then washed twice with water. The solvent is evaporated in vacuo to give a mixture of regioisomers. Case 5. Galactosylation of 3",4"-diol-2-deoxy-2-trichloroacetamido-lacto-A/-triose acceptor to the LNT/LNnT mixture

To a solution of 3",4"-diol-2-deoxy-2-trichloroacetamido-lacto-A/-triose acceptor (1 mmol) and acetylated a-trichloroacetimidate of galactose (1 .5 mmol) dissolved in dry DCM, was at RT added TMSOTf (0.1 mmol) in one portion. The mixture was stirred for 1 hour when Et₃N was used to quench the reaction and then followed by evaporation to dryness. The product mixture was purified by silica column

chromatography to get the separated protected "LNnT" compound and corresponding "LNT" tetrasaccharide both as foams. LNnT/LNT ratio is 3:1 as determined by HPLC. Protected LNT:

¹H-NMR (CDCI₃, 300MHz) δ 1 .92 (s, 3H), 1 .96 (s, 3H), 2.01 (s, 3H), 2.09 (s, 3H), 3.00-3.09 (m, 1 H), 3.29 (dd, 1 H, J = 7.5 1 1 .7 Hz), 3.47-3.52 (m, 2H), 3.61 -3.69 (m, 2H), 3.83-4.07 (m, 6H), 4.25-4.44 (m, 4H), 4.50-4.57 (m, 4H), 4.64 (d, 1 H, J = 7.5 Hz), 4.77-4.80(m, 1 H), 4.80 (d, 1 H, J = 12.3 Hz), 5.1 1 (d, 1 H, J = 8.1 Hz), 5.14 (dd, 1 H, J = 8.1 10.5 Hz), 5.30 (d, 1 H, J = 3.3 Hz), 5.38 (dd, 1 H, J = 7.8 9.9Hz), 5.44 (dd, 1 H, J = 7.5 9.9 Hz), 5.54 (dd, 1 H, J= 9.5 9.5 Hz), 5.63 (d, 1 H, J = 3.3 Hz), 6.78-6.83 (m, 3H), 7.09-8.03 (m, 32H).

Protected LNnT: ¹H-NMR (CDCI₃, 300MHz) δ 1 .93 (s, 6H), 2.06 (s, 3H), 2.1 1 (s, 3H), 3.15 (ddd, 1 H, J = 7.8 7.8 10.5 Hz), 3.26 (dd, 1 H, J= 7.5 1 1 .7 Hz), 3.40 (dd, 1 H, J = 7.8 9.6 Hz), 3.59- 3.69 (m, 3H), 3.82-3.90 (m, 2H), 3.95-4.1 1 (m, 5H), 4.17-4.22 (m, 2H), 4.35-4.43 (m, 3H), 4.54 (d, 1 H, J = 12.3 Hz), 4.56 (d, 1 H, J = 7.8 Hz), 4.63 (d, 1 H, J = 7.8 Hz), 4.79 (d, 1 H, J = 12.3 Hz), 4.86 (dd, 1 H, J= 3.3 10.5 Hz), 5.08 (d, 1 H, J = 8.1 Hz), 5.15 (dd, 1 H, J = 7.8 10.5 Hz), 5.29 (d, 1 H, J = 3.3 Hz), 5.37 (dd, 1 H, J = 7.8 9.9 Hz), 5.45 (dd, 1 H, J = 7.8 9.9 Hz), 5.53 (dd, 1 H, J = 9.9 9.9 Hz), 5.57 (d, 1 H, J= 3.3 Hz), 6.55 (d, 1 H, J = 6.9 Hz), 6.75-6.78 (m, 2H), 7.08-7.96 (m, 32H).

4.Deprotection to mixture of compounds of formula 1 and 2 Case 1. deprotection of R₃: acetal type group

The tetrasaccharide mixture obtained according to Example 3 having R₃: acetal type group is dissolved in DCM and cooled to 0-5 °C ushg. Diluted aq. HCIO₄ (1 part 70% aq. HCIO₄ 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. NaHCO₃ 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.

Case 2. deprotection of R₃: silyl group

A solution of the tetrasaccharide mixture obtained according to Example 3 having R₃: silyl group (2.5 mmol) in THF (containingl .2 equiv. of AcOH) is cooled to 0° and TBAF (2.7 imL, 1 .1 eq., 1 M solution) is added, and the reaction stirred at this temperature for 5h. The solution is diluted with DCM and washed with ammonium chloride saturated solution. The organic phase is died over sodium sulfate and the solvent is evaporated in vacuo. Flash chromatography yielded the 6-O deprotected compound (1 .87 mmol, 75%). Case 3. deprotection of O-acyl groups (R-i, R₄, optionally R2, R₃)

10 g of protected tetrasaccharide mixture (obtained in Example 3 or in Example 4 cases 1 or 2) is dissolved in MeOH (1 10 ml) and solution of NaOMe (1 M in MeOH) is added until pH 10. The solution is stirred at 40 °Cfor 5 h, then is neutralized by addition of Amberlite IR 120 H⁺ resin, the resin is filtered off, and the filtrate is evaporated to dryness. The residue is dissolved in warm DMF (10 ml) and added dropwise to 'Pr₂O (150 ml) and the suspension is stirred for additional 3 h. The precipitate is filtered off, washed with 'Pr₂O (2 x 20 ml) and dried to yield the O- acylated product in 90 % yield.

Case 4. conversion of Y: azido or NHCbz into Y: -NHAc

To a solution of ammonia (2 equiv.) in a mixture of MeOH/H₂O (3 volumes), the de- O-acylated material is added. To this mixture 10% Pd/C (10% of the substrate mass) is added and the reaction stirred under a H₂ atmosphere for cca. 1 h (continuous checking by TLC, an immediate quench of the reaction when de-O-benzylation is observed). The mixture is filtrated through a pad of celite and the solution is concentrated in vacuo. The residue is redissolved in a mixture MeOH/H₂O (3 volumes) with Et₃N as base (1 .5 equiv.) and to this solution Ac₂O (1 .2 equiv.) is added drop wise. The solvent mixture needs to be adjusted in order that the product precipitates out from the reaction mixture.

Case 5. de-O-acylation and conversion of Y: trifluoroacetylamino into Y: -NHAc

35 g of a protected tetrasaccharide mixture (obtained in Example 3 or in Example 4 cases 1 or 2) is dissolved in 1 10 ml of MeOH and 1 1 0 ml of aqueous KOH (7.5 g) solution and the mixture is stirred at rt. for 1 d. The mixture is then chilled with ice- bath, neutralized by HCI-gas and concentrated to dryness. For N-acetylation see Case 4 above.

Case 6.

LNnT-OBn

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 LNT/LNnT NHTCA-OBn tetrasaccharides in water was heated to 55°C and 2 eq of aq. NaOH (from 4M solution) was added. After 4 hours the reaction solution was cooled to15°C and MeOH was added. The precipitation of the product started immediately. Iso-propanol was added and after 30 min the filtration was started, he obtained cake was suspended in cold MeOH/ iso-propanol mixture (1 :1 ) and then filtered again. The solid was dried in at 50 °C/ 20 mbar for 16 hours to give the LNT/LNnT amine mixture in 87% yield as a white solid.

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. At this temperature acetone (2.5V) was addedusing dropping funnel and the product started to precipitate. After complete addition of the acetone the suspension was decreased to RT and stirred for 30 minutes. The compound was filtered and then suspended in MeOH (2V imL) and filtered again followed by drying at 50 °C / 20 mbar over night to get the final intermediate LNT/LNnT-OBn mixture in 86% yield as a solid.

5. Isolation of LNnT-OBn/LNT-OBn mixture by crystallization

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

essentially of a mixture of LNnT-OBn/LNT-OBn, is dried in vacuum and analyzed by NMR and HPLC to determine the ratio of LNnT-OBn/LNT-OBn.

6. Purification, enrichment, separation and isolation of LNnT-OBn from LNT-OBn, or LNT-OBn from LNnT-OBn, by crystallization

Case 1.

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. Then, 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.

Case 2.

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

acetone/water (3:1 ) to provide a solid LNT-OBn product.

Case 3.

To a solid mixture of LNT-OBn/LNnT-OBn (500 mg, 9:1 ) was added water (2 mL). The pH was checked and adjusted to be around 8-9 by addition of aq. NaOH. The thick mixture was stirred for 1 hour at ambient temperature before filtrated. The obtained solid was dried to give enriched LNT-OBn (HPLC analysis: from 90% to > 98%; relative purity by UV detection). Yield 350 mg. Case 4.

To a mixture of LNT-OBn/LNnT-OBn (360 mg, 5:1 ) was added 2 imL of water. The 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.

Case 5.

To a mixture of LNT-OBn/LNnT-OBn (1 .2 g, 1 :3) was added 2 imL of water. The pH can be adjusted to around 6 by the aid of aq. HCI. To the thick mixture was added MeOH (0.2 imL) and the suspension was heated to 45 °C and then allowed to reach RT. The suspension obtained was stirred for 2 hours at ambient temperature before filtrated. The obtained solid was washed with MeOH and dried to give LNT-OBn (HPLC analysis: from 25% to > 80%; relative purity by UV detection). Yield 250 mg. The pH of the mother liquor can be adjusted with aq. NaOH to between 7-8. The mother liquor was then heated to 45 °C whereupon acEtone/MeOH was added (2:1 , 5 imL) followed by seeding crystals of LNnT-OBn. The mixture was allowed to reach RT and stirred for 4 hours, filtrated and dried. Yield 700 mg (HPLC analysis: > 85%; relative purity by UV detection). Case 6.

To 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. To the resulting solution, 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.

Claims

ϋυ;\νθ 2013/091660 PCT/DK2012/050502 48 CLAIMS

1 . A mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2:

2 wherein R is a group removable by hydrogenolysis, preferably benzyl, 4- methylbenzyl or 4-chlorobenzyl, more preferably benzyl, and R3' is a group removable by hydrogenolysis or H, preferably H.

2. The mixture according to claim 1 that consists essentially of a compound of formula 1 wherein R is benzyl and R₃' is H (LNT-OBn), and a compound of formula 2 wherein R is benzyl and R3' is H (LNnT-OBn).

3. A method of obtaining a crystalline compound of formula 1 according to claim 1 and optionally a separate crystalline compound of formula 2 according to claim 1 , comprising the steps of: a) dissolving a mixture according to claim 1 in water, b) optionally adding 1 /5 to 1 /10 parts of a CrC₄ alcohol, or a solvent mixture of Ci-C₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a C C₄ 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

obtained in 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 volume, preferably at least a triple volume, of C C₄ alcohol solvent, or a solvent mixture of CrC₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a C C₄ alcohol, and, e) then crystallizing the compound of formula 2 from the mixture obtained in step d).

4. The method according to claim 3, wherein the compound of formula 1 is LNT- OBn, and the compound of formula 2 is LNnT-OBn.

5. The method according to claim 3 or 4, wherein the CrC₄ alcohol is methanol, and the ketone type solvent is acetone.

6. A method of obtaining a crystalline compound of formula 2 according to claim 1 and optionally a separate crystalline compound of formula 1 according to claim 1 , comprising the steps of: a) dissolving a mixture according to claim 1 in water, b) adding 2.5-3.5 fold volume of a CrC₄ alcohol, preferably methanol, or a solvent mixture of C C₄ 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 optionally d) removing the C C₄ 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).

7. The method according to claim 6, wherein the compound of formula 1 is LNT- OBn, and the compound of formula 2 is LNnT-OBn.

8. The method according to claim 6 or 7, wherein the CrC₄ alcohol is methanol, and the ketone type solvent is acetone.

9. A method for obtaining a crystalline mixture of, preferably a crystalline mixture consisting essentially of, compounds of formulas 1 and 2 according to claim 1 , comprising the steps of: ^ϋυ;\νθ 2013/091660 PCT/DK2012/050502

50 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 C₄ alcohol, or a solvent mixture of Ci-C₄ alcohols, or a ketone type solvent, or a mixture of a ketone type solvent with a CrC₄ 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).

10. The method according to claim 9, wherein the compound of formula 1 is LNT- OBn, and the compound of formula 2 is LNnT-OBn.

1 1 . The method according to claim 9 or 10, wherein the CrC₄ alcohol is methanol, and the ketone type solvent is acetone.

12. A method for the preparation of the mixture according to claim 1 , comprising the conversion by one or more deprotection steps of a mixture of, preferably a mixture consisting essentially of, a compound of formula 3 and a compound of formula 4 into the mixture according to claim 1

4 wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is acyl or H, R₃ is selected from a group removable by hydrogenolysis, acyl, silyl and an acetal type group, R₄ is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAc₂, benzamido, alkoxycarbonylamino,

haloalkoxycarbonylamino, benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3-diphenylmaleimido and 2,3-dimethylmaleimido.

A compound of formula 7

wherein R₆ is, independently, H or a residue of formula A

and wherein R is a group removable by hydrogenolysis, Ri is acyl, R₂ is acyl or H, R₃ is selected from a group removable by hydrogenolysis, acyl, silyl and an acetal type group, R₄ is acyl and Y is selected from alkanoylamido, haloalkanoylamido, -NAc₂, benzamido,

alkoxycarbonylamino, haloalkoxycarbonylamino,

benzyloxycarbonylamino, azido, phthalimido, tetrachlorophthalimido, 2,3- diphenylmaleimido and 2,3-dimethylmaleimido.

14. The compound according to claim 13 which is characterized by formula 3

or by formula 4

or by formula 5

5 wherein R, R-i , R₂, R3, R₄ and Y are as defined in claim 13.

15. A method for the preparation of a mixture of, preferably a mixture consisting essentially of, a compound of formula 3 according to claim 14 and a compound of formula 4 according to claim 14 comprising the step of: reacting a donor of formula 6

6 wherein R₄ is acyl, and Xi is selected from halogen, -OC(=NH)CCI₃, -OAc, -OBz and -SR5, and wherein R₅ is selected from alkyl, substituted phenyl and unsubstituted phenyl, with an acceptor of formula 5 according to claim 14.

16. A mixture consisting essentially of LNT and LNnT.

17. A process for the preparation of an LNnT/LNT mixture according to claim 16, comprising a step of subjecting a mixture of, preferably a mixture consisting essentially of, a compound of formula 1 and a compound of formula 2, according to claim 1 , to catalytic hydrogenolysis.

18. Use of the LNnT/LNT mixture according to claim 16 as a pharmaceutically active ingredient for the preparation of a pharmaceutical composition.

19. A pharmaceutical composition comprising the LNnT/LNT mixture according to claim 16 as a pharmaceutically ingredient and one or more pharmaceutically acceptable excipients.

20. Use of the LNnT/LNT mixture according to claim 16 as a nutritionally active ingredient for the preparation of a nutritional formulation.

21 . A nutritional formulation, particularly an infant formula, comprising the LNnT/LNT mixture according to claim 16 as a nutritionally active ingredient.