TRANSGLYCOLYTIC SYNTHESIS OF BRANCHED OLIGOSACCHARIDES

Abstract: The invention relates to a process exclusively relying on transglycolytic enzymes, for the synthesis of branched oligosaccharides. According to the described process, a donor saccharide is first incubated with an acceptor saccharide and with a suitable glycosidase. The resulting di- or oligosaccharide is then incubated with a second, different glycosidase and with a donor saccharide, to form a second glucosidic bond differing from the one formed in the first stage, thereby obtaining a branched oligosaccharide. The process is entirely and exclusively performed in water phase and allows to obtain, with high yield and specificity, branched oligosaccharides such as the trisaccharide Lewis-X.

Description: TRANSGLYCOLYTIC SYNTHESIS OF BRANCHED OLIGOSACCHARIDES FIELD OF THE INVENTION The present invention relates to the synthesis of branched oligosaccharides. A two-stage process for the preparation of branched oligosaccharides characterised by the exclusive use of transglycolitic enzymes. This invention implies the incubation of appropriate saccharide-acceptoFs and donors which are incubated with two giycosidases in a sequence, under particular reaction conditions. The whole process, which takes place exclusively in aqueous environment, can lead to branched oligosaccharides with high specificity STATE OF THE ART The branched oligosaccharides play a very important biological role, in particular in their conjugate forms (glycoproteins, glycolipids). All the cells have an oligosaccharidic layer composed of glycoproteins or glycolipids. The oligosaccharides on the cell surface can act as specific receptors for a variety of viruses, bacteria, parasites or as receptors for vegetal and bacterial toxins or act as antigenic determinants in auto and allo-immune responses. Changes in surface carbohydrates have been related to the oncologic differentiation and to the metastatic process. Many important glycoconjugates contain a fucose residue at their non-reducing end. Fucose-containing oligosaccharides have been detected in human milk, in glycoproteins, glycolipids specific of blood groups and in the human tumour cells of a neuroectodermic origin. The Lewis-X branched trisaccharide (Gal beta 1-4 [Fuc alpha 1-3]GlcNac) is an antigenic determinant present on the surface of a large variety of human tumours. Its sialylated form, the Lewis-X-sialyl, is expressed on the surface of activated granulocytes and is involved in the adhesion of leucocytes to the activated endothelium. The branched oligosaccharides play therefore a remarkable biological role in the study of the pathogenesis processes and in the setting up of drugs than can inhibit these processes. The studies mentioned above represent a breakthrough in the development of new strategies aimed at the effective synthesis of branched oligosaccharides. The basic approach consists in either the chemical or enzymatic synthesis. The chemical synthesis implies several obstacles due to the use of complex protection and deprotection strategies. If one considers that all carbohydrates contain one or more hydroxyl groups, the full protection of a simple monosaccharide requires from three to seven steps. Moreover, the chemical synthesis leads quite often to anomeric mixtures and the desired isomers must then be separated. For the enzymatic synthesis of oligosaccharides at least /two strategies are available : one requires the use of glycosyltransferases, the other requires the use of glycosidases. As far as the glycosidases are concerned, the large limitation due to the donor/acceptor couple, the fact that most of them are not available and the scarce possibility of handling the natural substrates (NDP-sugars) do not allow for the glycosyltransferases to be used on a large scale. In as far as the fucosyltransferases are concerned (Watkins, 1995 in Glycoproteins, Montreuil, Schachter and Vliegenthart eds., pp:313-390, Elsevier Science B.V.), these enzymes exhibit the disadvantage of having high preparation costs and the synthesis process based on them is equally costly. The latter enzymatic route is represented by transglycosylation reactions, catalysed by the use of glycosidases, which in this case, work the other way round, that is they promote the formation of glycosidic bonds instead of breaking them down . This behaviour is based on the control of the reaction balance which is conveniently shifted towards the reagents. In Biochem. Biophys. Res. Commun., 1996, 219, 6-13, the formation of the N- acetylactosamine disaccharide through transglycosylation catalysed by beta- galactosidases is described. WO89/09725 describes the use of glycosidase in the synthesis of oligosaccharides where part of the process is carried out by using a glycosyltransferase. Transglycosylation reactions are described also in W091/13164: all throughout this document, the authors must act under conditions of supersaturated reagents. The examples quoted do not go beyond the synthesis of disaccharides. WO93/03168 describes the formation of oligosaccharides by reactions of glycosidases with substrates which are chemically modified in one or more hydroxyls. In this case the use of protective groups is requested to overcome the aspecificity of glycosydases. In Carb. Res., 1992, 224, 291-299 the synthesis of alpha-L-fucosyl disaccharides by using alpha-fucosidases from different sources is described. The authors " work with purified alpha-fucosidase and use an organic solvent in the incubation mixture. In view of the above mentioned limitations, it is therefore necessary to find an effective process for the synthesis of branched oligosaccharides by transglycolytic route. In particular, it is necessary to get to a process enabling good regioselectivity without either resorting to protective groups or glycosyltransferases. DETAILED DESCRIPTION OF THE INVENTION The Applicant has surprisingly found that it is possible to obtain an effective synthesis of branched oligosaccharides by using only the transglycolytic route, without using either chemical protection/deprotection strategies or glycosyltransferases and also without resorting to steps of non-enzymatic chemical synthesis. The synthesis process is characterised by the following two steps: a) incubating with a first glycosidase at a temperature ranging from 15 °C to 45 °C for a time ranging from one hour to five days: a donor substance (DON-1 ) containing the first sugar to be transferred (S1 ) and an acceptor substance (ACC) containing the X-(S3)-Y structure where (S3) is the sugar desired as a branching point and where X and Y are the two different hydroxyls of this sugar from which the branching is to be developed; with formation of a (S1 )-X-(S3)-Y product characterised by a glycosidic selective bond between the hydroxyl in position 1 of the sugar (S1 ) and the hydroxyl X of sugar (S3); b)incubating with a second glycosidase which had been formerly purified: The (S1 )-X-(S3)-Y substance obtained in a. and second donor substance (DON2) containing the second sugar to be transferred (S2); with the formation of a (S1 )-X-(S3)-Y-(S2) branched product characterised by the selective glycosidic bond between the hydroxyl in position 1 of the sugar (S1 ) and the hydroxyl X of the sugar (S3) and between the hydroxyl in position 1 of the sugar (S2) and the hydroxyl Y of the sugar (S3), these bonds being of alpha or beta type. Schematically, the reaction is illustrated as follows: [DON-1] + [ACC] → [PRODUCT 1] (S1 ) X-(S3)-Y → (S1 ) X Y-(S3) [PRODUCT 1] + [DON-2] → [BRANCHED PRODUCT 2] (S1 ) (S1 ) X Y-(S3) (S2) (S2)-Y-(S3) According to the process described here, if the (S2) sugar is the same as (S1 ) sugar, the glycosidic linkages of (S1 ) and (S2) with (S3) are not at the same time of the alpha or beta type. The (S3) sugar contains, in addition to the hemiacetaiic hydroxyl on the carbon in position 1 , one or more free hydroxyls. The (S3) sugar can be any hexose or pentose and can be in a pyranosidic or furanosidic form. Examples of possible (S3) sugars are: alpha-L-fucose, alpha-D-galactose, alpha- D-glucose, alpha-D-xylose, alpha-D-mannose, beta-L-fucose, beta-D-galactose, beta-D-glucose, beta-D-xylose, beta-D-mannose, beta-D-galactosamine (2-deoxy, 2-amino beta-D-galactose ), beta-D-glucosamine (2-deoxy, 2-amino beta-D- glucose ), /V-acetyl-beta-D-galactosamine (2-deoxy, 2-acetamido beta-D- galactose), Λ/-acetyl-beta-D-giucosamine (2-deoxy, 2-acetamido beta-D-glucose). If one or more hydroxyls of the (S3) sugar different from X and Y are substituted, the substituents are of organic or inorganic type. Examples of organic substituents are the acyl group (preferably C2-C4 acyl), alkoxy , allyl, benzyl, p- metoxybenzyl. Examples of inorganic substituents are the sulphate, phosphate, hydroxylamino, azido groups. If present, the primary hydroxymethyl alcohol group located in position 5 of the pentose or in position 6 of the hexose (S3), can be free or substituted for example with halogens. Alternatively, it can be esterified for example with a phosphoric group. The hemiacetalic hydroxyl in position 1 can be free or substituted, for example in the form of N- or O-glucoside, or by esterification with an organic or inorganic acid. In this case, the hydroxyl can be present in an esterified form with alkanoic, carboxyalkanoic, alkenoic, optionally substituted benzoylic groups, phosphomonoester, phosphodiester, phosphodiester nucieotide (NDP), sulphuric groups. The (S3) sugar can be the (ACC) substance itself, or one of its derived structure. In the latter case, the (ACC) substance is a di-, tri-, or oligosaccharide containing the (S3) sugar, possibly in a non-reducing end position. Alternatively, the (ACC) substance is an O- or N- glycoside of the (S3) sugar. In this case, the aglycon will be, for example, represented by alkylic, aralkylic, aromatic, heterocyclic, vinylic, alkoxyalchilic, alkanoic or by a nucleoside. The donor substance (DON-1) is the (S1 ) sugar as such, or a di-, tri-, oligosaccharide or an 0- or /.-glucoside containing such sugar. Specific examples of possible (S1 ) sugars and of possible aglycons of glucoside are those quoted above in the definition of (S3). The donor substance (DON-2) is the (S2) sugar as such or is either a di-, tri-, oligosaccharide, or an O- or / .-glucoside containing this sugar. Specific examples of possible sugars (S2) and of possible aglycons of glucoside are those quoted above in the definition of (S3). In one case, the (S1 ) and (S2) sugars are the same. In this case, the bonds with (S3) will be sterically of a different kind. In other words, they will be one of an alpha and the other of a beta type. The glycosidases used in the present process can be obtained from several natural sources and are commercially available. Fucosidases, for example, can be obtained from pig liver, bovine testes or from bacterial species such as Corynebacterium, Fusarium, Aspergillus, and so on. Galactosidases can be obtained from bacterial species such as Bacillus and Escherichia. Transglycosilation reactions are carried out at a temperature ranging from 15 to 45 °C, for a time ranging from 1 hour to 5 days. Specifically, reactions of transfucosilation are preferably carried out at 30-40 °C, more preferably at 37 °C, for a period of time ranging from 3 to 5 days. Reactions using galactosidases are preferably carried out at room temperature (15-30 °C) for a period of time from 6 to 18 hours. Reactions using xylosidase are preferably performed at a temperature ranging from 30 to 40 °C, preferably at 37 °C, for a period of time from 1 to 12 hours. In one of the preferred implementations of the invention (hereinafter referred to as implementation A), sugar (S1 ), either in a free form or as being part of a di-, tri-, oligosaccharide or glucoside is represented by alpha-L-fucose or its derivatives, and the (S2) sugar in a free form or as being part of di-, tri-, oligosaccharides or glucosides is represented by beta-D-galactose or its derivatives. In a specific form of the implementation A, the donor substance, DON-1 , is the p- nitrophenyl glycoside of alpha-L-fucose; the substance (ACC) is the Λ/-acetyl-D- glucosamine (GlcNAc); the first glucosidase is alpha-L-fucosidase obtained from Aspergillus niger; the incubation is carried out at 37 °C for four days; the product of the former step (a) is the L-Fuc alpha 1-3 D-GlcNAc disaccharide; the second glycosidase is the beta-D-galactosidase obtained from Bacillus circulans; the donor (DON-2) is a disaccharide, the lactose, where the sugar to be transferred (S2) is the beta-D-galactose; the incubation is carried out at room temperature for 12 hours; the product obtained is a branched trisaccharide, D-Gal beta 1-4 [L-Fuc alpha 1-3] D-GlcNAc (Lewis-X). In another specific form of implementation A, the donor substance DON-1 is the p- nitrophenyl glycoside of alpha-L-fucose; the substance (ACC) is the /Vracetyl-D-glucosamine(GlcNAc), the first glucόsidase is alpha-L-fucosidase obtained from Corynebacterium sp.; the incubation is carried out at 37 °C for 4 days; the product of the former step (a) is the disaccharide L- Fuc alpha 1-4 D-GlcNAc; the second glycosidase is the beta-D-galactosidase obtained from bovine testes; the donor (DON-2) is a disaccharide, the lactose, where the sugar to be transferred (S2) is the beta-D-galactose; the incubation is carried out at room temperature for twelve hours; the product obtained is the branched trisaccharide D-Gal beta 1-3 [L-Fuc alpha 1-4] D-GlcNAc (Lewis-a). In a further specific form of implementation A, the donor substance DON-1 is the p-nitrophenyl glycoside of alpha-L-fucose; the substance (ACC) is glucose (Glc); the first glucosidase is alpha-L-fucosidase obtained from Aspergillus niger; the incubation is carried out at 37 °C for 4 days; the product of the first step (a) is the L-Fuc alpha 1-3 D-GIc disaccharide; the second glycosidase is the beta-D-galactosidase obtained from Bacillus circulans; the donor (DON-2) is a disaccharide, the lactose, where the sugar to be transferred (S2) is the beta-D-galactose; the incubation is carried out at room temperature for 12 hours; the product obtained is the D-Gal beta 1-4 [L-Fuc alpha 1-3] D-GIc (fucosyl- lactose) branched trisaccharide. The branched oligosaccharides produced according to the present invention can be separated from the other components of the incubation mixture by known techniques such as gel filtration chromatography. The branched oligosaccharides produced according to the present invention can either be used as such or can undergo further processes of derivatization or homologization according to the techniques available. In particular, we refer to the chemical or enzymatic addition of further saccharidic residues, with the obtainment of branched higher oligosaccharides and/or derivatization processes of hydroxyl functions such as esterification, etherification and so on. One of the favourite examples of processes of this kind is the sialylation process which, in the case of the Lewis-X trisaccharide, leads to the obtainment of the sialyl-Lewis-X antigenic determinant which has a remarkable biological importance. With respect to the existing processes, the synthesis process according to the present invention allows to obtain branched oligosaccharides containing fucose in high yields by exclusively transglycolytic way and in particular without resorting to glycosyltransferases: this objective is obtained without using protection/deprotection strategies. The claimed process takes place entirely in aqueous phase, i.e without the use of organic solvents or co-solvents which are normally required by the procedures described in literature (see Carb. Res. 1992, quoted above ).This aspect makes the synthetic procedure notably easier since it avoids further steps of extraction and recovery of the solvents. With reference to Carbohydr. Res, 1992, the transglycosylation reaction performed according to the present invention leads to the obtainment of comparable amounts of products, but with the use of very limited amounts of enzyme (0.6 against 70 mU). Moreover the process described in this document implies the pre-purification of fucosidases, which is not requested according to the present invention. According to one of the preferred implementation of the invention, another interesting aspect consists in the use of lactose as donor in phase b. The use of this product at a very low cost adds to the economy of the global reaction. The invention is hereafter described by the following examples: EXAMPLE 1 : Synthesis of the Lewis-x trisaccharide, D-Gal beta 1-4[L-Fuc alpha 1-3] D- GlcNAc. The process begins with the synthesis of the Fuc alpha 1-3 GlcNAc disaccharide. To this purpose, the acceptor GlcNAc (560 micromoles), and the donor Fuc- OPhN02-p (Fuc-PNP) (90 micromoles), were dissolved by heating (60 °C) in 2 ml of 50 mM, pH 5.0, sodium acetate buffer. 0.6 mU. (50 mg) of alpha-L-fucosidase from Aspergillus niger were added to the solution and the mixture obtained in this way was incubated at 37 °C for four days. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes, the supernatant was purified by reverse-phase chromatography on a C18 (2.5x6.0 cm) column, washed with 3 volumes of methanol, followed by washing with three volumes of water. With this first purification step, the polar compounds such as Fuc, GlcNAc and the Fuc alpha 1-3GlcNAc disaccharide were eluted by washing with water, while the less polar compounds such as Fuc-PNP and PNP were eluted with methanol. The volume of the aqueous phase was reduced to 3 ml by evaporation under vacuum. The residue underwent gel filtration chromatography on a Bio-Gel P2 column (2.0x100 cm) equilibrated in water. The fractions containing the disaccharide, Fuc alpha 1-3 GlcNAc, were collected and freeze-dried. The characterisation of the product was obtained by HPLC analysis, NMR (13C and 1 H) and ionspray mass spectrometry. The Fuc alpha 1-3 GlcNAc disaccharide, obtained from the previous reaction was used as acceptor during the next transglycosylation reaction and lactose was used as donor. To this purpose, Fuc alpha 1-3 GlcNAc (270 micromoles) and lactose (1460 micromoles) were dissolved by heating (60 °C) in 2 ml of 50 mM pH 5.0 sodium acetate buffer. 6U of beta-D-galactosidase from Bacillus circulans were added to the solution and the mixture obtained in this way was incubated at 25 °C for 12 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for ten minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes the supernatant underwent gel filtration chromatography on a Bio-Gel P2 (2.0x100 cm) column equilibrated in water. The fractions containing the D-Gal beta 1-4[L-Fuc alpha 1-3] D-GlcNAc trisaccharide, were then collected and freeze-dried. The characterisation of the product was obtained by HPLC, NMR (13C and 1 H) and ionspray mass spectrometry analyses. EXAMPLE 2 Synthesis of the Lewis-a trisaccharide, D-Gal Beta 1-3[L-Fuc alpha 1-4] D- GlcNAc. The process begins with the synthesis of the Fuc alpha 1-4GlcNAc disaccharide. To this purpose, the acceptor GlcNAc (560 moles), and the donor Fuc-OPh-NO2- p (Fuc-PNP) (90 moles), were dissolved by heating (60 °C) in 2 ml of 50 mM pH 5.0 sodium acetate buffer. 0.6 mU (50 mg) of alpha-L-fucosidase from Corynebacterium sp. were added to the solution and the mixture obtained in this way was incubated at 37 °C for four days. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. The purification and the characterisation of the product obtained were carried out as described in Example 1. The disaccharide, Fuc alpha 1-4 Glc, obtained from the previous reaction was used as acceptor in the next transglycosylation reaction by using lactose as donor. To this end the disaccharide, Fuc alpha 1-4 GlcNAc (270 micromoles) and the lactose (1460 micromoles) were dissolved by heating (60 °C) in 2 ml of sodium acetate buffer 50 mM pH 5.0. 6U of beta-D-galactosidase from bovine testes were then added to the solution and the mixture obtained in this way was incubated at 25 °C for 12 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. The purification and the characterisation of the D-Gal beta 1-3 [L-Fuc alpha 1-4] D-GlcNAc obtained in this way were then carried out as described in Example 1. EXAMPLE 3 Synthesis of the fucosyl-lactose trisaccharide, D-Gal beta 1 -4 [L-Fuc alpha 1- 3] D-GIc. The process begins with the synthesis of the disaccharide Fuc alpha 1-3 Glc. To this purpose, the acceptor Glc (560 micromoles), and the donor Fuc-OPh-NO2-p (Fuc-PNP) (90 micromoles), were dissolved by heating (60 °C) in 2 ml of sodium acetate buffer. 0.6 mU (50 mg) of alpha-L-fucosidases from Aspergillus niger were added to the solution and the mixture obtained in this way was incubated at 37 °C for four days. At the end of the incubation the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. The purification and characterisation of the product obtained were carried out as described in Example 1. The disaccharide, Fuc alpha 1-3 Glc, obtained from the previous reaction was used as acceptor in the next reaction of transglycosylation by using lactose as donor. To this end, Fuc alpha 1-3 Glc (270 micromoles) and lactose (1460 micromoles) were dissolved by heating (60 °C) in 2 ml of 50 mM, pH 5.0 sodium acetate buffer. 6 U of beta-D-galactosidase from Bacillus circulans were added to the solution and the mixture obtained in this way was incubated at 25 °C for 12 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. The purification and characterisation of the D-Gal beta 1-4 [L-Fuc alpha 1-3] D-GIc obtained in this way were carried out as described in Example 1. EXAMPLE 4 Synthesis of the D-Gal beta (1-4)-[D-Xyl beta (1-3)]-D-GlcNAc trisaccharide. The process begins with the synthesis of the Xy1 beta 1-3 GlcNAc disaccharide. To this end, GlcNAc (560 micromoles), as acceptor, and Xy1-OPhNO2-p (Xyl- PNP) (90 moles), as donor, were dissolved by heating (60 °C) in 2 ml of 50 mM pH 5.0 sodium acetate buffer. 1 U of beta-D-Xylosidase from Thermoanaerobacterium saccharolyticum was added to the solution and the mixture obtained in this way was incubated at 37 °C for 6 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. The purification and characterisation of the product obtained were carried out as described in Example 1. The disaccharide, Xy1 beta 1-3 GlcNAc, obtained from the previous reaction was used as acceptor in the next reaction of transglycosylation by using lactose as a donor. To this end, Xy1 beta 1-3 GlcNAc (270 moles) and lactose (1460) were dissolved by heating (60 °C) in 2 ml of 50 mM, pH 5.0 sodium acetate buffer. 6 U of beta-D-galactosidase from Bacillus circulans were added to the solution and the mixture obtained in this way was incubated at 25°C for 12 hours. At the end of the incubation, the mixture was heated to 100 °C for 10 minutes and then cooled in ice. The purification and characterisation of the D-Gal beta 1-4[D-xyl beta 1-3] D- GlcNAc obtained in this way were carried out as described in Example 1. EXAMPLE 5 Synthesis of the NeuAc 2-3 (Xylβ 1-6)Galβ O-methylsalicilate. The first step is the synthesis of 2-(1-carboxymethyl)phenyl β-D- galactopyranoside. Methyl salicilate (1.29 ml, 0.01 moles) was added dropwise to a solution of NaOH 1.25M (88 ml, 0.01 moles). 15 minutes later 2 ml of NaOH (1.25 M) were added to the solution. After 30 minutes, 1-α-bromo-tetraacetylgalactose (2.06 g, 0.005 moles, 0.5 eq.) and tetrabutylamonimun bromide (1.28 g, 0.004 moles, 0.4 eq.) dissolved in 20 ml of chloroform were carefully dropwise added during a period of time of 10 minutes. The mixture was then kept under reflux (70-80°C) for a period of 90 minutes. 1 gr of 1-α-bromo-tetraacetylgalactose (0.0025 moles, 0.25 eq.) dissolved in 5 ml of chloroform were added and the reaction mixture was further heated for 2 hours. This last step was repeated once again, and then the reaction mixture was cooled down to room temperature and left under stirring for 16 hours. The mixture was diluted with wateπchloroform (1 :1 , 100 ml). The aqueous phase was extracted with chloroform (3X 100 ml). All the organic phases were collected and put together and then washed with HCI 2N (100 ml) and water (100 ml). They were dried on NaSO4 and then under vacum. The substance obtained (5g) has an oily appearance with orange colour. By means of flash cromatography (hexane: ethylacetate, 6:4) the following compound was isolated (1.13 gr) from the mixture: 2-(1-carboxymethyl)phenyl β- D-2',3',4',6'-tetra-O-acetylgalactopyranoside. Its characterisation was carried out by NMR. This compound was dissolved in 100 ml of methanol. 60 ml of anhydrous methanol were added dropwise to the above solution during a period of 15 minutes. After two hours IRA-120 H+ resin were added and the mixture was kept under stirring at room temperature for 3 minutes until pH 5-6.The resin was filtered and washed with methanol, the solvent was evaporated under vacuum and the solid material obtained was dried in the oven at 40°C under vacuum. The product obtained (680 mg) was characterised by means of 1 H-NMR as being 2-(1-carboxymethyl)phenyl β-D-galactopyranoside (Galβ O-mSA). The acceptor Galβ O-methylsalicilate (63.6 micromoles), and the donor Xyl β O- N02-Ph (Xyl-PNP) (73.7 micromoles), were dissolved in 1 ml of 50 mM, pH 5.0, sodium acetate buffer. 0.038 U of beta-xylosidase from Aspergillus niger were added to the solution and the mixture so obtained was incubated at 37 °C for 24 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes, the supernatant was purified by reverse-phase chromatography on a C18 (1.0x6.0 cm) column, washed with 3 volumes of methanol, followed by washing with three volumes of water. With this first purification step, Xyl was eluted by washing with water, while the less polar compounds such as Xyl-PNP, PNP, Galβ O-mSA and Xylβ 1-6Galβ O- mSA were eluted with methanol. The methanolic phase was evaporated under vacuum. The residue was dissolved in 3 ml of water before carrying out the gel filtration chromatography on a Bio-Gel P2 column (2.0x100 cm) equilibrated in water. The fractions containing the disaccharide, Xylβ 1-6Galβ O-mSA, were collected and freeze-dried. The characterisation of the product was obtained by HPLC analysis, NMR (13C and 1 H) and ionspray mass spectrometry. The Xylβ 1-6Galβ O-mSA disaccharide, obtained from the previous reaction was used as acceptor during the next transglycosylation reaction and methylumbelliferyl-neuraminide (NeuAc-mU) was used as donor. To this purpose, Xylβ 1-6Ga1 β O-mSA (10 micromoles) and NeuAc-mU (10 micromoles) were dissolved in 0.5 ml of 100 mM pH 7.2 sodium phosphate saline buffer (PBS) containing 0.2% bovine serum albumine (BSA). 13U of trans-sialidase from Trypanosoma cruzi were added to the solution and the mixture so obtained was incubated at 37 °C for 24 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes the supernatant underwent gel filtration chromatography on a Bio-Gel P2 (2.0x100 cm) column equilibrated in water. The fractions containing the NeuAcα 2-3 (Xylβ1-6)Gal O-methylsalicilate were then collected and freeze-dried. The characterisation of the product was obtained by HPLC, NMR (13C and 1 H) and ionspray mass spectrometry analyses. EXAMPLE 6 Synthesis of the NeuAcα 2-3 (Xylβ 1-6)Galβ O-p-nitrophenyl. The process begins with the synthesis of Xylβ 1-6GalβO-p-NO2Ph. To this purpose, the acceptor GalβO-p-NO2Ph (Gal-PNP) (66 micromoles), and the donor XylβO-p-NO2-Ph (Xyl-PNP) (73.7 micromoles), were dissolved in 1 ml of 50 mM, pH 5.0, sodium acetate buffer. 0.038 U of beta-xylosidase from Aspergillus niger were added to the solution and the mixture so obtained was incubated at 37 °C for 24 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes, the supernatant was purified by reverse-phase chromatography on a C18 (1.0x6.0 cm) column, washed with 3 volumes of methanol, followed by washing with three volumes of water. With this first purification step, Xyl was eluted by washing with water, while the less polar compounds such as Xyl-PNP, PNP, and Xylβ1-6GalβO-pNO2Ph were eluted with methanol. The methanolic phase was evaporated under vacuum. The residue was dissolved in 3 ml of water before carrying out the gel filtration chromatography on a Bio-Gel P2 column (2.0x100 cm) equilibrated in water. The fractions containing the disaccharide, Xylβ 1-6 Galβ O-p-NO2Ph, were collected and freeze-dried. The characterisation of the product was obtained by HPLC analysis, NMR (13C and 1 H) and ionspray mass spectrometry. The Xylβ 1-6 Galβ O-p-NO2Ph disaccharide, obtained from the previous reaction, was used as acceptor during the next transglycosylation reaction and methylumbelliferyl-neuraminide (NeuAc-mU) was used as donor. To this purpose, Xylβ 1-6 Galβ 0-pNO2Ph (10 micromoles) and NeuAc-mU (10 micromoles) were dissolved in 0.5 ml of 100 mM pH 7.2 sodium phosphate saline buffer (PBS) containing 0.2% bovine serum albumine (BSA). 13U of trans-sialidase from Trypanosoma cruzi were added to the solution and the mixture obtained in this way was incubated at 37 °C for 24 hours. At the end of the incubation, the reaction mixture was heated at 100 °C for 10 minutes and then cooled in ice. After centrifugation at 11.000 rpm for 10 minutes the supernatant underwent gel filtration chromatography on a Bio-Gel P2 (2.0x100 cm) column equilibrated in water. The fractions containing the NeuAcα 2-3(Xylβ 1-6)Galβ O-p-NO2Ph were then collected and freeze-dried. The characterisation of the product was obtained by HPLC, NMR (13C and 1 H) and ionspray mass spectrometry analyses.

Claims: CLAIMS 1 1. A process, carried out exclusively in aqueous phase, for the highly 2 regioselective and stereospecific synthesis of branched oligosaccharides, the 3 process being characterised by the following steps: 4 incubating with a first glycosidase at a temperature ranging from 15 °C and 45 °C 5 for a time ranging from 1 hour to five days: 6 a donor substance (DON-1 ) containing the first sugar to be transferred (S1 ), and 7 an acceptor substance (ACC) containing the X-(S3)-Y structure where (S3) is the 8 sugar that is desired as a branching point and where X and Y are two different 9 hydroxyls of the above sugar from which the branching is to be developed; 10 with the formation of a product (S1 )-X-(S3)-Y characterised by the selective l i glycosidic linkage between the hydroxyl in position 1 of the sugar (S1 ) and the 12 hydroxyl X of the sugar (S3); 13 incubating with a second glycosidase previously purified: 14 the compound (S1 )-X-(S3)-Y obtained in a. and a second donor substance 15 (DON2) containing the second sugar to be transferred (S2); 16 with the formation of a product 17 18 (S1 ) 19 I 20 X 21 I 22 (S2) — Y— (S3) 23 24 25 characterised by the selective glycosidic linkage between the hydroxyl in position 26 1 of the sugar (S1 ) and the hydroxyl X of the sugar (S3) and between the hydroxyl 27 in position 1 of the sugar (S2) and the hydroxyl Y of the sugar (S3), these linkages 28 being of the alpha or beta type, 29 provided that if sugar (S2) is the same as sugar (S1 ), the two glycosidic linkages 30 of (S1 ) and (S2) with (S3) are not of the alpha or beta type at the same time; l 2. The process according to Claim 1 , where the sugar (S3) is hexose or pentose, in a pyranosidic or furanosidic form, and contains one or more non-substituted hydroxyls besides the hemiacetalic hydroxylic functions on the anomeric carbon atom in position 1 ; 1 3. The process according to Claims 1-2, where the acceptor substance (ACC) is a 2 di-, tri- or oligosaccharide containing the sugar (S3); l 4. The process according to Claims 1-2, where the acceptor substance (ACC) is . an O- or / .-glycoside of the sugar (S3); 1 5. The process according to Claim 1 , where the donor substance (DON1 ) is the 2 sugar (S1 ) to be transferred or a di-, tri-, or oligosaccharide containing this sugar 3 in a non-reducing end position; 1 6. The process according to Claim 1 , where the donor substance (DON1) is an O- 2 or Λ/-glycoside of the sugar (S1 ); 1 7. The process according to Claims 5-6 where the sugar (S1 ) to be transferred 2 either as such or contained in the di-, tri- or oligosaccharide or glycoside is the 3 alpha-L-fucose, alpha-D-galactose, alpha-D-glucose, alpha-D-xylose, alpha-D- 4 mannose, beta-L-fucose, beta-D-galactose, beta-D-glucose, beta-D-xylose, beta- 5 D-mannose, beta-D-galactosamine (2 deoxy, 2-amino b-D-galactose), beta-D- 6 glucosamine (2 deoxy, 2-amino beta-D-glucose), N-acetyl-beta-D-galactosamine 7 (2-deoxy, 2-acetamido beta-D-galactose), N-acetyl-beta-D-glucosamine (2-deoxy, 8 2-acetamido beta-D-glucose); 1 8. The process according to Claim 1 where the donor substance (DON2) is the 2 second sugar to be transferred (S2) or a di, tri- or oligosaccharide containing this 3 sugar in a non-reducing end position; 1 9. The process according to Claim 1 where the second donor substance (DON2) 2 is an O- or /.-glycoside of the desired sugar to be transferred (S2); 1 10. The process according to Claims 8-9 where the sugar (S2) to be transferred 2 either as such or as contained in the di-, tri- or oligosaccharide or glycoside is 3 alpha-L-fucose, alpha-D-galactose, alpha-D-glucose, alpha-D-xylose, alpha-D- 4 mannose, beta-L-fucose, beta-D-galactose, beta-D-glucose, beta-D-xylose, beta- 5 D-mannose, beta-D-galactosamine (2 deoxy, 2-amino beta-D-galactose), beta-D- 6 glucosamine (2 deoxy, 2-amino beta-D-glucose), N-acetyl-beta-D-galactosamine 7 (2-deoxy, 2-acetamido beta-D-galactose), N-acetyl-beta-D-glucosamine (2-deoxy, 2-acetamido beta-D-glucose); 11. The process according to Claims 1-10 where the first sugar to be transferred (S1 ) is either the alpha-L-fucose as such or as contained in a di-, tri- or oligosaccharide or as contained in its O- or in its Λ/-glycoside; and the second sugar to be transferred (S2) is either the beta-D-galactose as such or as contained in a di-, tri- or oligosaccharide where the beta-D-galactose is in a non-reducing position, or is contained in its O- or in its Λ/-glycoside; 12. The process according to Claim 11 , where (ACC) is the N-acetyl-D- glucosamine (2-deoxy, 2-acetamido D-glucose, GlcNAc); the first glycosidase is alpha-L-fucosidase extracted from Aspergillus niger; the incubation is carried out at 37 °C for four days; the disaccharide obtained is L-Fuc alpha 1->3 D-GlcNAc; the second glycosidase is the beta-D-galactosidase from Bacillus circulans; the incubation with beta-galactosidase is carried out at room temperature for 12 hours; the product obtained is the branched trisaccharide D-Gal beta 1->4 [L-Fuc alpha 1->3] D-GlcNAc (Lewis-x); 13. The process according to Claim 12, where the first donor substance (DON1 ) is the (p-nitro-phenyl) glycoside of the alpha-L-fucose; the second donor substance (DON2) is the disaccharide lactose, and the sugar (S2) to be transferred is the beta-D-galactose; 14. The process according to Claims 1-11 , where (ACC) is the N- acetylglucosamine (2-deoxy, 2 acetamido D-Glucose, GlcNAc); the first glycosidase is alpha-D-fucosidase extracted from Corinebacterium sp.; the incubation is carried out at 37 °C for 4 days: the disaccharide obtained is L-Fuc alpha 1-4 GlcNAc; the second glycosidase is the beta-D-galactosidase from bovine testes; the incubation with betagalactosidase is carried out at room temperature for 12 hours; the product obtained is the branched trisaccharide D-Gal beta 1-3 [L-Fuc alpha 1- 4]D-GlcNAc (Lewis a). 15. The process according to Claim 14 where: the first donor substance (DON1 ) is the (p-nitro-phenyl) glycoside of alpha-L-fucose; the second donor substance (DON2) containing the second sugar to be transferred is the disaccharide lactose, and where the sugar to be transferred (S2) is the beta-D-galactose. 16. The process according to Claims 1-11 , where (ACC) is the D-glucose (Glc); the first glycosidase is alpha-L-fucosidase obtained from Aspergillus niger; the incubation is carried out at 37 °C for 4 days; the disaccharide obtained is L-Fuc alpha 1->3 D-GIc; the second glycosidase is the beta-D-galactosidase extracted from Bacillus circulans; the incubation with beta-D-galactosidase is carried out at room temperature for 12 hours; the product obtained is the branched trisaccharide D- Gal beta 1->4 [L-Fuc alpha 1->3] D-GIc (fucosyl-lactose) 17. The process according to Claim 16 where: the first donor substance (DON1 ) is the (p-nitro-phenyl) glycoside of alpha-L-fucose; the second donor substance (DON2) is the disaccharide lactose, and where the sugar to be transferred (S2) is the beta-D-galactose. 18. The process according to Claims 1-10 where the first sugar to be transferred (S1 ) is the beta-D-xylose as such or as contained in a di-, tri- or oligosaccharide or as contained in its O- or in its Λ/-glycoside; and the second sugar to be transferred (S2) is the beta -D-galactose as such or as contained in a di-, tri- or oligosaccharide where the beta-D-galactose is in a non- reducing position or in its O- or in its /V-glycoside; 19. The process according to Claim 18, where (ACC) is the Λ/-acetylglucosamine (GlcNAc); the first glycosidase is beta-D-xylosidase extracted from Thermoanaerobacterium saccharolyticum; the incubation is carried out at 37 °C for six hours: the disaccharide obtained is D-Xyl beta 1-3 GlcNAc; the second glycosidase is the beta-D-galactosidase extracted from Bacillus circulans; the incubation with galactosidase is carried out at room temperature for 12 hours; the product obtained is the branched trisaccharide D-Gal beta 1-4 [D-Xyl beta 1-3]D-GlcNAc. 20. The process according to Claims 1-19, where the branched oligosaccharide obtained undergoes further chemical or enzymatic synthesis to obtain higher oligosaccharides or derivatives thereof; 21. The process according to Claim 20, where the further synthesis leads to the obtainment of sialylated saccharides; 22. The process according to Claim 21 , where the sialylated saccharides are sialyl-Lewis-X, sialyl-Lewis-a, sialyl-fucosyl-lactose, sialyl- D-Gal beta 1-4[D-Xyl beta 1-3] D-GINAc. 23. The branched trisaccharide D-gal beta 1-4[D-Xy1 beta 1-3] D-GlcNAc, or its sialylated derivative, as product of the process described in the previous claims.

Inventors: PAOLETTI SERGIO, VETERE AMEDEO, PAOLETTI, SERGIO, VETERE, AMEDEO
Applicants: COOPERATIVA CENTRO RICERCHE PO, PAOLETTI SERGIO, VETERE AMEDEO, PAOLETTI, SERGIO, SOCIETA' COOPERATIVA CENTRO RICERCHE POLY-TECH A RESPONSABILITA' LIMITATA, VETERE, AMEDEO
Application year: 1998
Main classification: C12P19
IPC codes: C07H3, C12P19