EP1861503A2

EP1861503A2 - Method for synthesizing oligosaccharides and glycosylation

Info

Publication number: EP1861503A2
Application number: EP06725216A
Authority: EP
Inventors: Klaus Buchholz; Jürgen SEIBEL; Hans-Joachim JÖRDENING; Raphael Beine
Original assignee: Technische Universitaet Braunschweig
Current assignee: Technische Universitaet Braunschweig
Priority date: 2005-03-22
Filing date: 2006-03-22
Publication date: 2007-12-05
Also published as: US20090076258A1; WO2006100253A3; WO2006100253A2

Abstract

The invention relates to an enzymatic method for synthesizing oligosaccharides, whereby one saccharide group of a sucrose analogue each is transferred onto an acceptor molecule, for example for glycosylating a hydroxyl compound, a saccharide, peptide, or a drug. According to the inventive method, an enzymatic synthesis of ß-D-fructofuranosyl-a-D-aldopyranoside is carried out in a first step, and in a second step one of the saccharide groups is enzymatically transferred onto the acceptor molecule.

Description

Process for the synthesis of oligosaccharides and glycosylation

The present invention relates to an enzymatic process for the synthesis of oligosaccharides. The method according to the invention is suitable for transferring in each case one saccharide group to an acceptor molecule, for example a hydroxyl compound, such as a saccharide, peptide, or a pharmaceutical.

In a first step, the process according to the invention preferably uses the enzymatic synthesis to prepare a β-D-fructofuranosyl-α-D-aldopyranoside from sucrose, which is used in a second step as a substrate for the enzymatic transfer of one of the saccharide residues to an acceptor molecule.

Moreover, the invention provides a combination of reactants and enzymes, with which the method according to the invention for the synthesis of oligosaccharides can be carried out.

State of the art

EP 0 130 054 discloses the use of a fructosyltransferase to transfer the fructosyl residue of sucrose. By way of example, galactose or a glucose derivative serves as the acceptor molecule for the fructosyl radical transferred from sucrose, so that fructose derivatized on the pyranoside radical is obtained. Subsequent halogenation produces a halogenated disaccharide which is used as a sweetener. The transfer of the Fructosylrestes of fructose by means of the fructosyltransferase should also be possible to other acceptor molecules, such as arabinose, lactose, maltose or glycerol.

Galacto, fructo, xylo and galacturono oligosaccharides are tested as alternatives to pregiotic oligosaccharides of human milk. Galacto-oligosaccharides are obtained by transglycosylation by β-galactosidase from lactose (Boehm et al., Nutrafoods 51-57 (2005)). Fructo-oligosaccharides are currently extracted mainly from chicory (Boehm et al., Acta Pediatrica 18-21 (Suppl. 449, 2005)

Furthermore, it is known to prepare oligosaccharides according to the natural biosynthesis pathway by using the transglycosylation of an acceptor molecule-containing saccharide with activated saccharide derivatives. These activated saccharide derivatives are activated by binding of a nucleoside diphosphate to the C 1. This synthetic method is disadvantageous in that the activated glycosides are very expensive and are not available in the large quantities required for industrial scale reactions.

General description of the invention

The process according to the invention uses the binding energy of β-D-fructofuranosyl-α-aldopyranosides, generally β-D-ketofuranosyl-α-D-aldopyranosides, as substrates for the enzymatically catalyzed transfer of one of the two glycosyl residues, namely the ketofuranosyl residue or the aldopyranosyl residue an acceptor molecule (see, for example, FIGS. 4 and 7). In the preferred embodiment, the .beta.-D-ketofuranosyl-.alpha.-D-aldopyranoside, from which a glycoside residue is transferred to an acceptor molecule, is prepared by enzymatically catalyzed transfer of the Generates glycoside residues on fructose, wherein the sucrose analog is generated and glucose is released (see, for example, Figure 3). In this way it is possible to obtain the binding energy contained in the sucrose analog to an extent sufficient for the transfer of a glycoside residue to an acceptor molecule. This transferred glycoside residue according to the invention is not originally contained in sucrose.

As an alternative to the synthesis of the analogues of sucrose, in which the natural fructosyl residue is replaced by another ketoanuranosyl residue, or the glucoside residue by another aldopyranoside residue, the synthesis may also be carried out in place of the sucrose with another sugar having sufficient binding energy of a ketofuranosyl residue an aldopyranoside to produce the analog, and then enzymatically transferring one of these residues to an acceptor. An example of such a sugar is the raffinose (α-D-galactopyranosyl- (l, 6) -α-D-glucopyranosyl- (1,2) -β-D-fructofuranoside) in which instead of the glucoside residue of the sucrose, a galactopyranosyl - (l, 6) -α-D-Glucopyranosylrest in 1,2-bond with the ß-D-Fructofuranosylrest is connected. In the description of the invention, all references to sucrose and sucrose analogs also apply to raffinose and its analogs.

Suitable acceptor molecules are (poly) hydroxyl compounds, for example saccharides, and thiol compounds, as well as peptides, proteins or pharmaceuticals. Both embodiments of the method according to the invention can be used by repeated application to an acceptor molecule to successively transfer several identical or different Ketofuranosylreste or Aldopyranosidreste so that oligosaccharides are produced with different molecular weight.

In a first embodiment, the method is for transfer of Ketofuranosylrestes on acceptor molecules having at least one hydroxyl group as an acceptor, but need not necessarily be pure glycoside compounds. As an example of substrate molecules which are glycosides, there may be mentioned β-D-ketofuranosyl-D-aldopyranosides and derivatives of β-D-fructofuranosyl compounds. In this first embodiment, according to the invention, the derivatized fructosyl residue is transferred to the acceptor molecule. Such a reaction is by ketofuranosyltransferases, so as catalyses fructosyltransferases, for example by the fructosyltransferase from Bacillus subtilis or by a glycosyltransferase.

In the present invention, it is preferable that the β-D-ketofuranosyl-D-aldopyranoside compound used as a substrate in which the ketofuranosyl group is an isomer of fructose in the form of a derivatized fructofuranosyl compound is enzymatically prepared from sucrose. This can be achieved by reacting sucrose with the desired fructose derivative to give a sucrose analogue in which the original fructosyl residue is replaced by a fructosyl derivative. For catalysis, a glycosyltransferase, e.g. a fructosyltransferase can be used.

For the purposes of the invention, the term derivatized fructofuranosyl compound or fructosyl derivative is taken to mean substituents which can replace the fructosyl radical of the sucrose, preferably in an enzymatically catalyzed reaction from sucrose. Examples of derivatized fructofuranosyl radicals are other ketofuranosyls, for example fructosyl derivatives and psicosyl, sorbosyl and tagatosyl radicals, and derivatives thereof.

In a second embodiment, the invention relates to the transfer of the D-aldopyranoside residue from β-D-ketofuranosyl-α-aldopyranosides onto an acceptor molecule. Suitable acceptor molecules are hydroxyl and thiol compounds, for example saccharides. The transfer of the aldopyranoside residue is catalyzed by a specific glycosyltransferase. Examples of specific glycosyltransferases are those which specifically confer a mannopyranosyl, galactopyranosyl, fucopyranosyl or rhamnopyranosyl radical linked in α 1-2 to a ketofuranosyl radical, for example fructofuranosyl radical, or a derivatized glycopyranosyl radical on acceptor molecules. Carbohydrates, peptides, proteins, alcohols, pharmaceuticals and natural products which carry a hydroxyl group and / or thiol group can be used as acceptor molecules.

In the second embodiment of the invention, it is preferred that the β-D-ketofuranosyl-D-aldopyranoside compound is produced enzymatically from sucrose, namely by reacting sucrose with the desired aldopyranose with catalysis by, for example, a fructosyltransferase (see, for example, Figure 3). That way the glucoside residue of the sucrose replaced by another aldopyranoside residue, which is transferred to the acceptor molecule in a subsequent enzymatic catalytic step (Figure 4).

The synthesis method according to the invention can be used in the second embodiment to selectively produce disaccharides and longer-chain oligosaccharides or polysaccharides, wherein an acceptor molecule, for example a mono- or disaccharide, is extended by the aldopyranoside residue of a β-D-ketofuranosyl-α-D-aldopyranoside , In this case, the aldopyranoside residue may be the same or different in successive reactions, so that an oligosaccharide chain is built up from identical and / or different aldopyranoside building blocks.

Accordingly, the synthesis method according to the invention can be used to selectively produce disaccharides and longer-chain oligosaccharides, wherein ß-D-Ketofuranosidreste or Aldopyranosidreste be transferred to an acceptor molecule. In successive reactions, identical or different derivatized ketofuranoside and / or aldopyranoside radicals can thus be transferred so that an oligosaccharide chain is built up from identical and / or different ketofuranosyl and / or aldofuranosyl units.

Oligosaccharides according to the invention, in particular fructo-oligosaccharides, are particularly suitable for use as dietary supplements with a prebiotic effect. Because unlike sucrose, maltose and other glycosides, the fructosyl oligosaccharides are not easily hydrolyzed in the acidic medium of the stomach or enzymatically, but at least get a significant portion of the small intestine into the colon. Here they can develop their prebiotic effect, since they are metabolized there by the probiotic bifidobacteria, as for example by the degradation to short-chain fatty acids. In this way, the oligosaccharides of the invention promote the survival and growth of Bifidobacterium, which supports and maintains important physiological functions of the digestive apparatus. Furthermore, it can be demonstrated that the absorption of minerals, for example calcium, iron (III) and zinc ions is promoted when incorporating inventive oligosaccharides in the human digestive system, which is a preventive effect for osteoporosis. Furthermore, in particular, fructose according to the invention Oligosaccharide on the fat metabolism and lead to a reduction in the plasma level of cholesterol, as well as to an increase in the HDL / LDL - cholesterol ratio, which leads to a reduction in the risk of arteriosclerotic vascular disease.

In summary, therefore, the prebiotic action of oligosaccharides according to the invention, in particular of fructosyl-oligosaccharides which preferably carry an aldopyranose as head group which is not glucose, leads to a health-promoting effect directly or via the promotion of the growth of probiotic bifidobacteria, in particular because the oligosaccharides according to the invention are essentially non-digestible are. Particularly preferred head groups are galactose, mannose, fucose, xylulose, in each case in the D or L configuration, L-glucose, L-rhamnose and, in particular, galactosylmelibiose.

Detailed description of the invention

In the first embodiment of the invention, there is provided a method and a set of substances for carrying out this process which comprises a ketofuranosyl radical, namely the fructosyl radical or a derivatized fructosyl radical, from a derivatized β-D-ketofuranosyl-α-D-aldopyranoside as sucrose. Analog, is transferred to an acceptor molecule. The acceptor molecule may be compounds containing hydroxyl groups or thiol groups, for example saccharides, including the sucrose analog itself, peptides, pharmaceuticals, synthetic or natural polymers. For the transferase reaction then the corresponding Ketofuranosylderivate the aldopyranoside, preferably the Glucopyranosids to use. If the specificity of the preferred fructosyltransferase should not be suitable for the transfer of a ketofuranosyl residue, ie a derivatized fructosyl residue, suitable specific ketofuranosyl transferases can be prepared and identified by mutagenesis and screening, as described below for the production and identification of Glycosyltransferases that are specific for aldopyranosides. In this case, the specific keto-furanosyl derivative is used as the substrate instead of the respective aldopyranoside derivative. Accordingly, the first embodiment also relates to oligosaccharides or polysaccharides which are obtainable by transfer of the ketofuranosyl radical. When sucrose analogs whose fructosyl radical is derivatized are used for the transferase reaction, an oligomer with derivatized fructosyl components is obtained. Compounds of the first embodiment according to the invention are therefore oligo- / poly- (keto-diuranosyl) compounds in which the ketofuranosyl groups are fructosyls and / or derivatized fructosyls.

If sucrose analogues whose aldopyranoside residue is not the glucose residue are reacted in the transferase reaction, the fructosyl residue can be transferred with suitable glycosyltransferases, so that an oligo- or polyfructoside with the respective aldopyranoside residue is obtained as a head group which is provided with fructosyl residues. This latter variant is suitable for producing oligo- or polyfructosyl-aldopyranoside compounds, which are also referred to as pyranosyloligofructoside or pyranosylpolyfructoside, with catalysis of a fructosyltransferase. In this method, enzyme activities outside the desired fructosyltransferase, unlike a polyfructosylation from sucrose, are not interfering, such as a dextran translase. The invention utilizes the fact that the more enzymatically active dextransucrase does not polymerize the derivatized or formally aldopyranoside residue exchanged for glucose, while the enzymatically less active fructosyltransferase transfers the fructosyl residue from the sucrose analog to an acceptor without substantial restriction. A suitable fructosyltransferase is known from Leuconostoc mesenteroides or Bacillus subtilis. Therefore, the compounds of the first embodiment according to the invention also include oligo- / polyfructosyl compounds which are obtainable by reacting a fructosyl-aldopyranoside in which the aldopyranoside is not glucose and is liberated.

Preferred pyranosyloligofructosides or pyranosylpolyfructosides contain in the range from 2 to 10 ⁶ , preferably 2 to 100, particularly preferably 5 to 20 or 10 fructosyl units. The fructosyl units are preferably glycosidically linked to one another in C2-C6 and / or C2-C1. For example, the C2-C1 linkage can be catalyzed by an inulosuccrase. The pyranoside residue is preferably terminally bonded in ß-1,2 to a fructose glycosidically. In a second, preferred embodiment of the present invention, a process for the synthesis of oligosaccharides or for the glycosylation of hydroxyl-containing and / or thiol-group-containing acceptor molecules is provided, with which an aldopyranoside residue is transferred from a β-D-keto-uranosyl-α-D-aldopyranoside compound. The preferred β-D-ketofuranosyl-α-D-aldopyranoside is the sucrose, preferably a sucrose derivative which contains, instead of the glucose residue as aldopyranoside, the residue of another aldopyranose or a glycoside derivative.

Accordingly, the second embodiment also relates to oligosaccharides or polysaccharides obtainable by transfer of the aldopyranoside residue. When sucrose analogues whose aldopyranoside residue is a derivatized glucoside residue or aldopyranoside residue other than glucose are used for the transferase reaction, an oligomer or polymer having the derivatized or other aldopyranoside constituents is obtained as glucose. Compounds according to the invention of the second embodiment are therefore oligo- / poly- (aldopyranosyl) compounds in which the aldopyranosyl radical is derivatized glucosyl or another aldopyranosyl as glucosyl.

For the purposes of the invention, derivatives of sucrose in which the fructosyl residue is formally replaced by a derivative of the fructosyl residue or another ketofuranoside residue and / or the glucoside residue by a derivative of the glucoside residue or another aldopyranoside residue are referred to as sucrose analogs.

The process according to the invention is based on the realization that the binding energy of the glycosidic bond of sucrose (-23 kJ / mol) is sufficient for transferring the ketofuranosyl radical or the aldopyranoside radical to an acceptor molecule in a kinetically controlled synthesis, even if it has been converted into the sucrose radical. Analogous only one of these two radicals is replaced by formal exchange of Ketofuranosylrests against a fructose isomer or a Fructosylderivat and / or the Glucopyranosidrest by another Aldopyranosidrest.

For this transferase reaction, therefore, the duration of the reaction and the concentrations of educts and products prevailing during the transferase reaction are to be adjusted such that the transferase reaction is essentially terminated after the highest concentration of product has been reached. These kinetically controlled transfer reactions for the first and second embodiment can be carried out and controlled in suitable reactor types, for example by using immobilized enzymes and adequate limited contact times, and / or controlling the concentrations of reactants and product (Boker et al., Biotech. Bioeng., 43: 856-864 (1994)). ,

The glycosyltransferase to be used for the method according to the invention which is specific for a ketofuranoside radical or an aldopyranoside radical or derivatives of these glycoside radicals, i. a desired ketofuranoside residue or an aldopyranoside residue from a β-D-ketofuranosyl-α-D-aldopyranoside compound, e.g. can transfer a sucrose analogue to an acceptor molecule can be identified in the screening method. Thus, known enzymes, for example fructosyltransferase or dextransucrase, can be identified for their specificity for a particular ketofuranoside residue or aldopyranoside residue in a β-D-ketofuranosyl-α-D-aldopyranoside by virtue of the fact that the enzyme in vitro contains the specific β-D-ketofuranosyl-α D-aldopyranoside is reacted. In the case of an enzyme with the specificity sought, the reaction results in the transfer of the ketofuranoside residue or the aldopyranoside residue to an acceptor molecule, for example for oligomerization with simultaneous release of the aldopyranoside residue or the ketofuranosyl residue of the sucrose analogue used as substrate. The activity of a specific enzyme can therefore be determined, for example, by colorimetric and / or photometric detection of the hydrolysis product. Upon release of fructose after transfer of an aldopyranoside residue from a β-D-fructofuranosyl-α-D-aldopyranoside, fructose may be used as a free hydrolysis product after isomerization by glucose isomerase to glucose, phosphorylation by hexokinase and oxidation by glucose-6-phosphate dehydrogenase with simultaneous Reduction of NADP to NADPH can be determined spectrophotometrically. This test system is applicable to various aldopyranoside derivatives, i. for example, sucrose analogs in which the glucoside residue is replaced, for example, by another aldopyranoside or a derivative of the glucoside residue.

In a further preferred embodiment of the invention, the .beta.-D-ketofuranosyl-.alpha.-D-aldopyranosides are produced enzymatically. In the second embodiment of the invention, preferably from sucrose, a desired β-D-ketofuranosyl-α-D-aldopyranoside is prepared as β-D-fructosyl-α-D-aldopyranoside in which the glucoside residue of the sucrose against the desired aldoside residue is exchanged. This can be catalyzed by a fructosyltransferase, for example fructosyltransferase from Bacillus subtilis (NCIMB 11871). Such .beta.-D-fructosyl-.alpha.-D-aldopyranosides may also be referred to as sucrose analogs because the saccharide linkage of the sucrose is retained but the original glucoside moiety has been replaced by a new pyranoside moiety.

For the first embodiment of the invention, it is preferable to enzymatically produce sucrose analogs in which the fructosyl moiety is formally substituted with a fructosyl derivative or another ketofuranosyl moiety. This reaction can be catalyzed by a glucosyltransferase.

Examples of sucrose analogs to be used according to the invention are those compounds in which the fructosyl radical of the sucrose is replaced by a radical selected from a ribulose, lyxose, xylulose, derivatized fructose, ribulose, psicose, sorbose, Tagatoserest or another to the C 1 of the glycoside residue glycosidically bound pentose, hexose, heptose, each in D- or L-configuration, or substituted derivatives of the aforementioned radicals.

Examples of sucrose analogues which can be used in the second embodiment of the invention are β-D-fructosyl compounds which have on the C2 of the fructosyl residue a ribose, arabinose, xylose, lyxose, allose, altrose, Galactose, mannose, gulose, idose, talose, fucose, 2-N-acetylglucosamine, 2-N-acetylgalactosamine, 2-deoxyglucose, 3-deoxyglucose, 4-deoxyglucose, 6-deoxyglucose , 2-deoxygalactose, 3-deoxygalactose, 3-ketoglucose, 4-ketoglucose, sialic acid, N-acetyl-neuraminic acid residue, each in D or L configuration, L-glucose, or another glycosidic bound pentose, hexose, heptose or substituted derivatives of the aforementioned radicals, eg have a derivatized glucose residue.

As acceptor molecules, alcoholic hydroxyl groups of carbohydrates, steroids, terpenes, polyketides, hydroxyamino acids, hydroxynitriles, other metabolic products of microorganisms, diglycerides, ceramides, enolic and / or phenolic hydroxyl groups of phenols, flavonoids and mercapto groups and amide groups, for example asparagine, threonine, serine, Cysteine, for example as part of peptides, purines, pyrimidines, benzimidazole or nicotinic acid amide. According to the invention it is provided in a preferred embodiment that the sucrose analogues which are used for the preparation of Pyranosyloligofructosiden or Pyranosylpolyfructosiden which are produced by enzyme-catalyzed reaction of the pyranose with sucrose, according to the other embodiments of the invention.

The invention will now be described in detail by way of example with reference to the figures in which:

FIG. 1 shows a schematic representation of the screening of suitable aldopyranoside transferases,

FIG. 2 shows a schematic representation of the screening of suitable ketofuranoside transferases,

Figure 3 is a schematic representation of the preferred synthesis of the sucrose analogs,

FIG. 4 is a schematic representation of the glycosylation of an acceptor molecule with a glycoside first converted to a sucrose analogue;

Figure 5 is a schematic representation of the transfer of a fructosyl residue from an aldosylfructoside to an alcohol,

FIG. 6 is a schematic representation of the transfer of a fructosyl residue from an aldosylfructoside to an amino acid derivative,

FIG. 7 is a schematic representation of the synthesis of a xylosyl oligofructoside from xyl-Fru with a Bacillus subtilis fructosyltransferase;

FIG. 8 is a schematic representation of the synthesis of a galacto-oligofructoside from the sucrose analog Gal-Fru with a Leuconostoc mesenteroides fructosyltransferase,

Figure 9 shows thin layer chromatograms over the course of the synthesis of polysaccharides (PS) according to the invention, under a) Gal-Fru- (Fru) _n , b) Gal-Fru- (Fru) "and c) Fuc-Fru- (Fru)" .

FIG. 10 shows the ESI-MS spectrum of a transfructosylation reaction according to the invention for xyl-Fru- (Fru) _n and its reaction scheme,

FIG. 11 shows a thin-layer chromatogram of the synthesis of mannosyl-oligofructoside from D-Man-Fru, FIG. 12 shows thin-layer chromatograms over the course of the synthesis of polysaccharides according to the invention,

FIG. 13 is a schematic representation of the glycosylation of an acceptor molecule immobilized on the polymeric support with subsequent detection of the synthesized oligosaccharide,

Figure 14 is a schematic representation of the transfer of a furanoside residue from a sucrose analog (galactosyl fructoside) to a saccharide to produce an oligosaccharide,

Figure 15 is a schematic representation of the oligomerization or polymerization of Pyranosidresten derived from a sucrose analogue, and

Figure 16 is a schematic representation of the oligomerization of furanoside residues derived from a sucrose analog.

Example 1: Production and Identification of a Specific Glucosyltransferase For the present invention, it is preferred to use a specific glycosyltransferase for the transfer of the ketofuranosyl residue or the aldopyranoside residue from a sucrose analog to an acceptor molecule. To transfer aldopyranoside residues from sucrose analogs, mutants of dextransucrase were first generated. For mutagenesis, the genes of the glycosyltransferase GtfR from Streptococcus oralis ATTC 10557, DsrS from Streptococcus mesenteroides (Fujiwara et al., 2000), GtfB and GtIC (Streptococcus mutans), were coupled with inducible promoters. The inducible promoter used was an IPTG-dependent promoter, alternatively a arabinose or dihydrotetracycline-regulatable promoter.

The glycosyltransferase genes were subjected to random mutagenesis, preferably by regiospecific PCR involving individual segments of the genes, for example, the domains involved in substrate binding. In this way, individual segments of the glycosyltransferase genes could be mutated, which subsequently formed a gene library of mutated gene sequences and were combined combinatorially with each other to form new glycosyltransferase genes. Alternatively, libraries of gene segments were used to replace the corresponding segment in the wild-type sequence. For screening the obtained substrate specificities of the mutated glycosyltransferase genes, these were expressed in E. coli (BL21). The screening was carried out by adding the respective sucrose analog and colorimetric or spectrophotometric determination of the liberated reducing sugar. Because in the presence of a specificity for the Fructosylderivatrest or Aldopyranosidrest a sucrose analogue is released as a byproduct of the untransferred saccharide residue. This liberated saccharide residue is reducing and can be determined spectrophotometrically by conventional methods.

By screening mutated glycosyltransferase genes, specific transferases could be found for the sucrose analogues described here in general and specifically, and in particular for the following sucrose analogues with high specific transferase activity, wherein only the saccharide radical formally replaced by sucrose is indicated: α-D -Mannoside, α-D-galactoside, α-D-xyloside (spectrophotometric measurement of released fructose or glucose).

For more accurate characterization of the resulting glycosyltransferase mutants, they were subjected to secondary screening in which transferases are incubated with an acceptor molecule and the specific substrate. Formed products are then analyzed by thin-layer chromatography, HPLC-MS and NMR.

For the identification of glycosyltransferases which synthesize lectin-binding oligosaccharides, a screening method can be used in which a lectin, for which a specific binding oligosaccharide is sought, is used to identify the oligosaccharide synthesized on a solid phase. For a simple detection method, the lectin is coupled to a reporter group, such as a fluorescent molecule. For solid-phase bound synthesis of the oligosaccharide synthesized from the glycosyltransferase from the sucrose analogs, an acceptor molecule is used which is coupled to a solid phase and has a free hydroxyl group. This may be, for example, the phenolic hydroxyl group, as shown schematically for the screening method in Figure 1.

For the identification of suitable glycosyltransferases, known enzymes or mutagenesis products thereof, such as dextransucrases, fructosyltransferases or mutants of glycosyltransferases, are screened. A screening method is schematically shown in FIG Figure 1, wherein the specificity of the transferase is identified by using a lectin with specificity for the desired oligosaccharide to identify the oligosaccharides synthesized from the sucrose analogs provided as co-substrates.

For easy analysis, the acceptor is solid-phase coupled so that the synthesized oligosaccharide is also linked to the solid phase and can be easily separated from the remaining components of the reaction. Upon reaction with the lectin, the specific binding can be detected by detection of a lectin-coupled group, such as a fluorescent molecule. In the case illustrated in FIG. 1, sucrose analogs are used in which the glucoside residue has been replaced by another constituent, for example by another aldopyranoside residue or a derivatized glucoside residue.

The screening method of FIG. 1 can also be carried out with sucrose analogs which, instead of the fructosyl residue, have a derivatized fructosyl residue or another ketofuranoside residue in order to identify a transferase which specifically transfers the respective ketofuranoside residue.

An example of screening for a fructosyltransferase with specificity for the transfer of a ketofuranoside residue from a sucrose analog is shown in FIG. The acceptor molecule is also immobilized by binding to a polymeric carrier and the specificity of the fructosyltransferase is analyzed by using a sucrose analog in which the fructosyl is replaced by another Ketofuranosidrest or a derivative of Fructosylrests, by subsequent hydrolysis of the am Acceptor molecule synthesized oligosaccharide reducing sugar can be determined.

This method of screening can also be used for the screening of glycosyltransferases specific for the particular residue of the sucrose analogue which is the original one, using sucrose analogues which have a different aldopyranose or a derivative of the glucoside residue instead of the glucoside residue Glucoside replaced. The example of glycosyltransferase R from ATCC 10557 (contained as SEQ ID NO: 5) (Fujiwara et al, Infect. Immun. 68: 2475-2483 (2000)), available at AB025228; BAA 95201.1 (EMBL) showed that single exchanges, insertions or deletions of amino acids could alter the substrate spectrum of the enzyme called wild-type glucosyltransferase. With the method for site-directed mutagenesis described above, variants of the wild-type sequence were produced which had different substrate specificities and are summarized in Table 1.

Table 1: New glucosyltransferases based on glucosyltransferase R from ATCC 10557

The numbering of the amino acids refers to the previously published wild-type sequence, the introduced mutations are underlined.

Example 2 Synthesis of a Disaccharide with Immobilized Glucosyltransferase For the immobilization of glycosyltransferases known carriers, for example Eupergit-C (Röhm & Haas) can be used. Another suitable method for immobilization is the inclusion in alginate, which is known to those skilled in the art as a method. Such immobilized enzymes can be used in flow-through reactors or batch reactors, as is well known in the art (Reischwitz et al., Enz. Microb. Technol. 19, 518-524 (1996)).

Example 3: Synthesis of sucrose analogs

For the enzymatic synthesis of β-D-fructofuranosyl-α- to be used according to the invention

D-Aldopyranosiden is the glucoside residue of sucrose by the desired Aldopyranoside replaced. This reaction was carried out by the Bacillus subtilis NCIB 11871 fructosyltransferase and it was shown that at sugar concentrations in the reaction solution of 10 to 400 g / L, preferably 100-300 g / L, sucrose analogs can be prepared. The products can be separated on ion exchangers.

The schematic sequence of the synthesis of sucrose analogues is shown in FIG. 3 using the example of the replacement of the glucosyl residue by a freely selectable aldopyranoside residue. In this case, sucrose is reacted with an aldopyranose as cosubstrate, catalyzed by fructosyltransferase, with liberation of glucose to aldopyranoside-1,2-.beta.-D-fructosylfuranoside.

Tables 1 and 2 below show the cosubstrates used for the conversion of sucrose and the sucrose analogs obtained with the fructosyltransferase reaction as disaccharides or trisaccharides:

Table 1: Sucrose-synthesized disaccharide-sucrose analogs

location Table 1:

Table 2: Sucrose-synthesized trisaccharide-sucrose analogs

location Table 2:

Example 4: Glycosylation of an acceptor by transfer of the pyranoside residue from a sucrose analog

FIG. 4 shows diagrammatically how, according to the invention, the aldopyranoside residue of a sucrose analogue is transferred to an acceptor which has a hydroxyl group here. With the aid of a known or mutagenesis-modified glycosyltransferase, the aldopyranoside residue is transferred to the hydroxyl group of the acceptor molecule, whereby fructose is liberated. As a result, an acceptor molecule glycosylated one to several times with the aldopyranoside residue is obtained as a product.

Example 5: Fructosylation by transfer of the fructoside residue from a sucrose analog to a hydroxyl group-containing acceptor

The preparation of a fructosyl derivative according to the invention is shown schematically in FIG. 5 by the example of the transfer of the fructose residue from the sucrose analog galactosylfructoside to alcohols. Under catalysis of β-glucosidase, the fructoside residue is transferred to the hydroxyl group of the cosubstrate alcohol to give a fructosylated alcohol. The galactoside residue is released as galactose. By continuing the reaction, an oligo- or polyfructosylation of the acceptor can be achieved, wherein in each case a further fructosyl group is transferred to the acceptor.

Example 6: Fructosylation by transfer of the fructoside residue from a sucrose analogue to an amino acid

FIG. 6 schematically shows the transfer of the fructosyl residue from a sucrose analogue, here a galactosylfructoside, to an amino acid. The amino acid is derivatized, namely serine, whose carboxyl and amine groups each have a protective group. This protected serine is shown to represent amino acids that are linked in a peptide and have reactive acceptor groups suitable for fructosylation. These may be thiol groups and amine groups in addition to the hydroxyl group shown. Upon catalysis of β-glucosidase, the fructosyl residue is transferred to the hydroxyl group of the serine to give a fructosylated serine derivative representative of fructosylated peptides.

EXAMPLE 7 Synthesis of Pyranosyl Oligofructosides The preparation of a pyranosyloligofructoside or pyranosyl polyfructoside according to the invention is shown schematically in FIG. 7 using the example of xylosyl di- or polyfructoside with 4 fructosyl units by reacting xylosylfructoside with fructosyltransferase from Bacillus subtilis. The fructosyl residue of the sucrose analogue xylosylfructoside is transferred by the fructosyltransferase to xylosylfructoside, so that, inter alia, the xylosyldifructoside shown, and upon continuation of the transferase reaction, a penta-glycoside having, for example, the structure shown of 4 fructosyl residues and 1 xylosyl. By continuing the reaction, longer fructosyl chains can be obtained which have at least 5 to 100 fructosyl units. The bonds of the fructosyl units are C2-C6, partly also C2-C1. The terminal pyranoside residue, in this case xylosyl, is bonded to the C2 of the next fructosyl unit in the β position according to the sucrose analogue in the α position on the Cl.

FIG. 8 shows schematically the transfer of the fructosyl residue from Gal-Fru with the aid of the fructosyltransferase from Leuconostoc mesenteroides. According to the synthesis of xylosyl oligo-fructoside, a galacto-oligo- or -polyfructoside is obtained.

For the synthesis of oligosaccharides according to the invention, fructosyl-aldopyranosides were used as the substrate, which were obtained by reacting sucrose with the aldopyranose replacing the glucose residue in each case. For catalysis, a fructosyltransferase was used.

In addition to D-Gal-Fru, D-Fuc-Fru was also used to generate D-GaI-FrU- (FrU) ₂₀ by means of L. mesenteroides (FTF-I) or B. subtilis (NCIMB 11871, FTF-2) fructosyltransferase _-100 and D-Gal-Fru- (Fru)> ₁₀₀ or D-Fuc-Fru- (Fru) 2 _O-1 oo ^un ^ D-Fuc-Fru- (Fru)> ₁₀₀ . Of the The course of the synthesis is shown in FIG. 9 on the basis of thin-layer chromatograms in which under a) the course of synthesis with FTF-I (137U / L) and under b) the synthesis with FTF-2 (2860 U / L) of the reaction of D-Gal -Fru is shown under c) the synthetic course with FTF-2 (2860 U / L) of the reaction of D-Fuc-Fru. It can be seen that over the time shown, the aforementioned polysaccharide (PS) is synthesized, oligosaccharides (OS) could not be separated and longer-chained PS at the point of application (inserted below in the chromatograms) remained.

Also synthesized with FTF-2 was XyI-FrU- (Fm) _1-S o from xyl-Fru, as shown schematically in FIG. The ESI-MS spectrum of XyI-FrU- (Fm) ₁ -So is shown in Figure 10, in which the signals 335.1, 497.1, 659.2, 821.2, 983.3, 1145, 3, 1307.4, 1469.4, 1631.4 ([M + Na] ⁺ ) show oligosaccharides of the type XyI- (Fm) _n with n 1 - 9. Higher molecular weights than those given in FIG. 10 were estimated in thin-layer chromatograms.

The particular advantage of the synthesis according to the invention is that addition of dextran glycation does not lead to the production of dextran. This is attributed to the fact that the sucrose analogs used in accordance with the invention whose glucoside residue has been derivatized or exchanged for another residue, for example against another hexose, do not constitute a substrate for dextran glycrase. Therefore, sucrose analogues can be used to prepare oligo- or polyfructosides without dextran or a polymer of the aldopyranoside radicals as impurities occurring as a result of side reactions.

Another example of the transfer of the fmctosyl residue from a sucrose analogue is the transfer of the Fmctosyl residue from D-Man-Fru with the FTF-2. For the preparation of a mannosyl-oligofructoside of the type (D-Man-Fru- (Fru) _1-8 ) according to the invention, D-Man-Fru was incubated with the FTF-2, this sucrose analogue functioning both as acceptor and as substrate for the transmission of the Fmctosylrests serves. In principle, one of the hydrolysis products, preferably the aldopyranoside, here D-mannose, may be added to suppress the side reaction which may occur as hydrolysis of the sucrose analogue. Under otherwise identical reaction conditions, the yield can be increased by this addition to the reaction mixture. The result of reacting D-Man-Fru for its fructosylation with the same sucrose analog as a substrate is shown in the thin-layer chromatogram in Figure 11, wherein the reference numeral 1 identifies the D-mannose and the reference 2 indicates the sucrose analog and at 3, the D-mannosyl oligofructosides (D-Man-Fru- (Fru) _1-8 ). The data for the individual traces of thin-layer chromatography indicate the reaction time in minutes. The section with reference numeral 4 indicates that even after a reaction time of three days, sucrose analogue can still be detected as a substrate.

These examples furthermore show that the binding energy of the sucrose analogs to be used according to the invention is sufficient to transfer in each case one of the two glycoside residues with liberation of the other glycoside residue.

EXAMPLE 8 Synthesis of Oligosaccharides According to the Invention from L-Aldopyranosyl Polvfructoside As an Example of Polyfructosides Which Maintained an Aldopyranose in L Configuration, L-Glucose, L-galactose, L-xylulose and L-Glucose-Fructoside (Comparative Example) catalysed by fructosyltransferase (FTF-2) by reacting the respective L-aldopyranose with sucrose.

The course of the synthesis is shown in FIG. 12 on the basis of thin-layer chromatograms, wherein a) shows the oligo fructosylation of L-Fuc-Fru (lanes 1, 5, 9, 13), L-gal-Fru-Fru (lanes 2, 6 , 10, 14), L-xyl-Fru (lanes 3, 7, 11, 15) and L-Glu-Fru (lanes 4, 8, 12, 16) after 0 min (lanes 1 to 4) 5 min (lanes 5 to 8), after 10 min (lanes 9 to 12) and after 20 min (lanes 13 to 16) branched, b) the same reactions after 30 min (lanes 1 to 4) after 60 min (lanes 5 to 8), after 120 min (lanes 9 to 12) and after 240 min (lanes 13 to 16) and under c) after 1220 min, in lane 1 L-Fuc-Fru, lane 2 L-Gal-Fru-Fru Lane 3 L-xyl-Fru and lane 4 L-Glu-Fru. These results were confirmed by ion exchange chromatography (Dionex).

Example 9: Transfer of the aldopyranoside residue from a sucrose analog to a peptide or natural product

FIG. 13 shows schematically the transfer of an aldopyranoside residue using the example of the glucosyl residue to a hydroxyl-containing compound which may be a peptide or another natural product. This acceptor molecule is for easier handling and control the transfer reaction coupled to a polymeric support. The aldopyranoside radical, here represented in the example as glucosyl radical, is to be replaced according to the invention by a derivative of the glucosyl radical or another aldopyranose.

The catalysis is made possible by a modified dextransucrase as glycosyltransferase, which simply or repeatedly transfers the aldopyranoside residue to the hydroxyl group of the peptide or natural product. The structure of the oligosaccharide on the peptide or natural product is controlled by washing after a limited reaction time of a first sucrose analogue containing a first aldopyranoside residue in order to stop the transfer reaction of the first aldopyranoside residue after a predetermined time. In this way, the desired number of transferred first Aldopyranosidreste can be predetermined based on the reaction time. In a second reaction step, a second aldopyranoside residue can then be transferred if the glycosyltransferase is provided with a second sucrose analogue cosubstrate having the second aldopyranoside residue. After washing again to remove the second cosubstrate, further identical or different aldopyranoside residues can be specifically built up by reaction with the particular sucrose analogue which has a certain aldopyranoside residue.

To cleave the provided with an oligosaccharide peptide or natural product from the polymeric carrier, the compound molecule was hydrolyzed and detected photometrically.

For analysis of the oligosaccharide chain synthesized on the peptide or natural product, NMR or MS can be used directly, or alternatively, after hydrolysis of the oligosaccharide from the peptide or natural product, the oligosaccharide can be analyzed. The hydrolysis of the oligosaccharide from the peptide or natural product can be carried out by aqueous sodium hydroxide, acid or glycosidases. The oligosaccharide can be analyzed spectrophotometrically, for example by reaction with glucose isomerase and hexokinase, glucose-6-phosphate dehydrogenase in the presence of NADP and ATP, so that NADPH ₂ can be measured spectrophotometrically. This analysis makes it possible to determine the substrate specificity of the transferase used, in particular in the case of a mixture of sucrose analogues as cosubstrate. Example 10: Synthesis of oligosaccharides by transfer of the aldopyranoside residue from β-D-fructosyl-α-D-galactoside

This example shows schematically in Figure 14 the synthesis of an oligo- or polyaldopyranosyl fructoside. Thus, .beta.-D-fructosyl-.alpha.-D-galactoside as a sucrose analog, prepared from sucrose and galactose according to Example 3, is reacted with a glycosyltransferase produced according to Example 1 by mutagenesis and screening from a glycosyltransferase gene. The analysis revealed that the galactoside residue was transferred to release fructose to obtain an oligogalacto-fructoside.

As schematically shown in Figure 15, catalysis by glycosyltransferase mutants to be used according to the invention, which are obtainable, for example, according to Example 1, respectively transfers the aldopyranoside residue, which is not a glucoside, of a sucrose analog to an oligosaccharide, releasing the fructosyl residue. For example, from the sucrose analogue, here Gal-Fru, with the dextransucrase from Bacillus subtilis, an oligoaldopyranosyl fructoside, here an oligogalactosyl fructoside, can be prepared.

Example 11: Synthesis of oligosaccharides by transfer of the ketofuranosyl residue from β-D-furanosyl-alpha-D-glucoside

The sucrose analogue β-D-ketofuranosyl-alpha-D-glucoside was obtained according to Example 3 by reacting sucrose with a ketofuranose. By catalysis of fructosyltransferase variants obtainable according to example 1, the ketofuranosyl radical can be transferred to an acceptor molecule, for example an oligosaccharide, with liberation of glucose. A scheme of this transfer reaction is shown generally in FIG. The product obtained is a glucosyl-oligo- or -polyfuranosid.

Example 12 Chromatographic Separation of Saccharides For the separation of starting materials and products both in the enzymatic synthesis of saccharide analogs and after the synthesis of oligosaccharides, remaining starting materials and product were obtained by chromatography on ion exchangers (commercially available Purolite, Lavatite, Amberlite XAD) separated. Elution was with distilled water (Berensmeier et al., Separation and Purification Technology, 38, 129-138 (2004)).

Claims

claims

1. Process for the synthesis of oligosaccharides or for glycosylation by enzyme-catalyzed transfer of the aldopyranoside residue or the furanosyl residue from a β-D-ketofuranosyl-α-D-aldopyranoside to an acceptor molecule, characterized in that the β-D-ketofuranosyl-α-D- Aldopyranoside is a sucrose analog wherein the ketofuranosyl moiety is other than the fructosyl moiety or the D-aldopyranoside moiety is other than the glucose moiety.

2. The method according to claim 1, characterized in that instead of a sucrose analogue, a raffinose analog is used.

3. The method according to any one of the preceding claims, characterized in that the sucrose analogue or raffinose analogue is synthesized by enzymatic reaction of the ketofuranose or aldopyranose with sucrose or raffinose.

4. The method according to any one of the preceding claims, characterized in that the pyranoside is selected from the group consisting of the ribose, arabinose, xylose, lyxose, allose, altrose, galactose, derivatized glucose, mannose , Gulose, idose, talose, fucose, rhamnose, 2-N-acetylglucosamine, 2-N-acetylgalactosamine, 2-deoxyglucose, 3-deoxyglucose, 4-deoxyglucose, 6-deoxyglucose, 2-deoxygalactose, 3-deoxygalactose, 3-ketoglucose, 4-ketoglucose, sialic acid, N-acetyl-neuraminic, maltose, isomaltose, melibiose, cellobiose, lactose, tagatose, and other glycosidic too binding pentoses, hexoses, heptoses, each independently in D or L configuration, L-glucose and substituted derivatives of the aforementioned radicals and mixtures thereof.

5. The method according to any one of the preceding claims, characterized in that the ß-D-Ketofuranosylrest is selected from the group comprising the ribulose, xylulose, derivatized fructose, ribulose, psicose, sorbose and Tagatoserest and others the C 1 of the glycoside residue glycosidically bound pentoses, hexoses, Heptoses and substituted derivatives of the aforementioned radicals and mixtures thereof.

6. The method according to any one of the preceding claims, characterized in that the enzyme-catalyzed transfer is effected by a glycosyltransferase specific for the aldopyranoside.

7. The method according to claim 6, characterized in that the glycosyltransferase is a Glucansucrase, ß-glucosidase or a mutant thereof.

8. The method according to any one of the preceding claims, characterized in that the enzyme-catalyzed transfer is effected by a glycosyltransferase specific for the ketofuranosyl radical.

9. The method according to claim 8, characterized in that the glycosyltransferase is a fructosyltransferase or a mutant thereof.

10. The method according to any one of the preceding claims, characterized in that the acceptor molecule has hydroxyl groups or thiol groups.

11. The method according to claim 10, characterized in that the acceptor molecule is selected from the group comprising carbohydrates, saccharides, steroids, terpenes, polyketides, hydroxyamino acids, hydroxynitriles, metabolites of microorganisms, diglycerides, ceramides, phenols, flavonoids, asparagine-containing peptides, purines , Pyrimidines, benzimidazoles, nicotinamide and compounds containing them.

12. The method according to any one of claims 10 or 11, characterized in that the acceptor molecule is the sucrose analog.

13. The method according to any one of claims 10 to 12, characterized in that the acceptor molecule is a ß-D-ketofuranosyl-α-D-aldopyranoside or a ß-D-ketofuranosyl-ß-L-aldopyranoside.

14. The method according to any one of the preceding claims, characterized in that the transferase is immobilized.

15. A process for the synthesis of an oligosaccharide, characterized by multiple stepwise application of a method according to any one of the preceding claims, in which stepwise different sucrose analogues or a mixture of these are used.

16. Pyranosyloligofructoside or pyranosylpolyfructoside obtainable by enzymatically catalyzed transfer of fructosyl residues from fructosylaldopyranosides, wherein the pyranosyl residue is selected from the group comprising the ribose, arabinose, xylose, lyxose, allose, altrose, galactose, derivatized glucose , Mannose, gulose, idose, talose, fucose, rhamnose, 2-N-acetylglucosamine, 2-N-acetylgalactosamine, 2-deoxyglucose, 3-deoxyglucose, 4-deoxyglucose , 6-deoxyglucose, 2-deoxygalactose, 3-deoxygalactose, 3-ketoglucose, 4-ketoglucose, sialic acid, N-acetyl-neuraminic acid, maltose, isomaltose, melibiose, cellobiose, lactose , Tagatoserest and other glycosidically bound tetroses, pentoses, hexoses, heptoses, each independently in D or L configuration and substituted derivatives of the aforementioned radicals and is obtainable by enzymatic reaction of aldopyranose with sucrose.

17. pyranosyloligofructoside or Pyranosylpolyfructosid according to claim 16, characterized in that the enzymatically catalyzed transfer and / or the enzymatic conversion of aldopyranose is catalysed with sucrose by fructosyltransferase.

18. Pyranosyloligofructosid or Pyranosylpolyfructosid according to claim 16 or 17, characterized by a number of Fructosylresten in the range of 2 to 10 ⁶ , 2 to 100, preferably 5 to 20.