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WO2000012747A1 - Method for enzymatic synthesis of glycosides, disaccharides, and oligosaccharides - Google Patents

Method for enzymatic synthesis of glycosides, disaccharides, and oligosaccharides Download PDF

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WO2000012747A1
WO2000012747A1 PCT/SE1999/001498 SE9901498W WO0012747A1 WO 2000012747 A1 WO2000012747 A1 WO 2000012747A1 SE 9901498 W SE9901498 W SE 9901498W WO 0012747 A1 WO0012747 A1 WO 0012747A1
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modified
enzyme
4glc
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Kurt Nilsson
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

Definitions

  • This invention relates to synthesis of Gal ⁇ l-3GlcNAc ⁇ l-3Gal ⁇ l-4Glc (below called LNT), derivatives and glycosides thereof, Gal ⁇ l-4GlcNAc ⁇ l-3Gal ⁇ l-4Glc (below called LNnt), derivatives and glycosides thereof and other biologically active saccharides.
  • LNT Gal ⁇ l-3GlcNAc ⁇ l-3Gal ⁇ l-4Glc
  • LNnt Gal ⁇ l-4GlcNAc ⁇ l-3Gal ⁇ l-4Glc
  • LNT and LNnt are present in human milk and also have a known antibacterial effect. Different methods to produce LNT and LNnt have been described, preferentially based on the isolation from human milk, or synthesis, preferentially using chemical methods.
  • This invention is based on enzymatic synthesis and enzymatic chemical methods for synthesis of LNT or LNnt, derivatives and glycosides thereof and other biologically active saccharides and derivatives, preferentially having antibacterial effect, or of other therapeutic or preventive interest.
  • LNT is synthesised in two enzymatic steps according to the scheme below:
  • ⁇ -hexosaminidase catalysed by ⁇ -hexosaminidase, or ⁇ -N-acetyl-D-glucosaminidase, or modified dito, for example ⁇ -hexosaminidsynthase or ⁇ -N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where R is -OH (i.e.
  • the donor is N-acetyl-D-glucosamine, that is equilibrium synthesis or reversed hydrolysis), or is an organic group, for example saccharide, or R is -OR, in which R is for example aliphatic, for example -Me, -All, etc, or aromatic, for example -Ph, -PhNO 2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglycon (so called transglycosylation).
  • R' is, in a corresponding manner, -OH (i.e.
  • lactose is used as acceptor), or is an organic group, for example -OR, in which R is for example aliphatic, for example, -Me, -All, -CH 2 Ph, -EtSiMe 3 , etc, or aromatic, for example, -Ph, -PhNO 2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon and
  • R" is -OH (i.e. donor is galactose; equilibrium synthesis or reversed hydrolysis), or is an organic, e.g. saccharide (i.e. lactose is used as donor) or R" is -O where R is e.g. aliphatic, e.g.
  • R' is -OH, or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All, -CH 2 Ph, -EtSiMe 3 , etc, or aromatic, e.g. -Ph, -PhNO 2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon.
  • acceptors can be used which are modified in one or several hydroxyl groups in addition to the anomeric position This gives for instance a possibility to produce analogs, employing, e.g conventional organic-chemical methods
  • the acceptor can be modified by chemical or enzymatic techniques For instance lactose can first be reacted with lipase and acyl donor to protect one or several hydroxyl groups Before this reaction, a derivative of lactose can be synthesised, which has a higher solubility in organic solvent Not limiting examples
  • HO-R is an appropriate alcohol according to this application, as for instance benzyl alcohol, allyl alcohol, trimetylsilyletanol or other substance, which after binding to glucose can be removed chemically,
  • reaction 2 can be catalysed by ⁇ -galactosidase, modified ⁇ -galactosidase or ⁇ - galactosynthase (for example modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006),
  • Step 4 and 5 Reaction of (Ac)nGal ⁇ l-4Glc ⁇ -OR (as acceptor), with any of the two enzymatic sequences wich are mentioned below, giving in the first step GlcNAc ⁇ l-3(Ac)nGal ⁇ l-4Glc ⁇ -OR, and in the next step,
  • Gal-R GlcNAc ⁇ l-3Gal ⁇ l-4Glc- R ' ⁇ Gal ⁇ l-3GlcNAc ⁇ -l-3Gal ⁇ l-4Glc-R + R"H catalysed by ⁇ -galactosidase from bovine testes or other appropriate enzyme, or where this enzyme first has been cloned and/or modified, (e.g ⁇ -galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where Gal-R" is lactose, or R" is glycosidically bound -OR, where R is aliphatic, e.g.
  • -Me -Me, -All, or aromatic, e.g. -Ph, -PhNO 2 -p, -PhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
  • ⁇ -hexosaminidase or ⁇ -N-acetyl-D-glucosaminidase from jack bean catalysed by ⁇ -hexosaminidase or ⁇ -N-acetyl-D-glucosaminidase from jack bean, or where this enzyme first has been cloned and/or modified, e.g. ⁇ -hexosaminidsynthase or ⁇ - N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean, where the hydrolysis ability completely, or more or less is missing) and where the donor is chitobiose, or where the donor is GlcNAc ⁇ -OPhNO 2 -p, and where R is -OH (i.e.
  • lactose is used as acceptor), or is an organic, e.g. -OR, where OR is e.g. aliphatic group, e.g. -OMe, -OA11 , -OCH 2 Ph, OEtSiMe 3 , etc, or an aromatic group, e.g. -OPh, OPhNO 2 -p, -OPhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon, and
  • Gal-R GlcNAc ⁇ l-3Gal ⁇ l-4Glc-R ' ⁇ Gal ⁇ 1-3 GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc-R + R' ⁇ catalysed by ⁇ -galactosidase from bovine testes or, other appropriate enzyme, or where this enzyme first has been cloned and/or modified, (e.g. ⁇ -galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where Gal-R" is lactose, or R" is glycosidically bound -OR, where R is an aliphatic group, e.g.
  • R" is an inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
  • ⁇ -galactosidase from e.g. bovine testes or, other appropriate enzyme, or modified dito, (e.g. ⁇ -galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e. the donor is galactose, i.e. equilibrium or reversed hydrolysis), or is an organic, e.g. saccharide (e.g. lactose is used as donor) or R" is -OR where -OR is an aliphatic group, e.g. -OMe, -OA11 , etc, or an aromatic group, e.g.
  • R' is in similar manner, -OH, or is an ⁇ - or a ⁇ -glycosidically bound organic group, e.g. -SEt, -SCr, -OBn, -OA11, or another for the purpose suitable group, and where R is an organic or inorganic group.
  • NR are e.g.
  • GlcNR-R' are, e.g., 2-amino-2-deoxy-glucosamine (GlcNH 2 ), GlcNH 2 ⁇ -SEt and GlcNHTroc ⁇ -SEt. Thereafter, chemical steps are used for the production of the end-substance (the conditions are chosen by the expert in the field and this does not limit the scope of the invention )
  • a chemically modified lactose-structure is used (3 ' -OH)(R” ) Gal ⁇ l-4Glc-R" (which has a free 3 ' -hydroxyl group) is used as acceptor, and (R' " ) symbolise the protection groups on the lactose-acceptor, e g benzyl groups, and R " is a glycosidically bound organic group, for instance a benzyl group
  • the conditions and the protection groups on the acceptor are chosen by the expert in the field, and this does not limit the scope of the invention.
  • step 4 above is, instead of a complete transformation to LNT-R", that the product after step 3 above can be used to make derivatives and analogs of LNT, employing conventional organic chemistry and/or enzymatic transformations
  • a N-Troc group be transformed to several different derivatives, where the amino group is modified, e g with an organic acyl group or with an alkyl group
  • other de ⁇ vatives of lactose-R ' can be used, which makes it possible to synthesise other derivatives or analogs of LNT
  • ⁇ -galactosidase catalysed by ⁇ -galactosidase, from e g bovine testes or, from another suitable enzyme source, or modified dito, (e g ⁇ -galactosynthase, genetically and/or chemically modified enzyme.
  • -R" is -OH (i e the donor is galactose, equilibrium or reversed hydrolysis), or is an organic group, e g saccharide (e g lactose is used as donor) or R" is -OR, where -OR is an aliphatic group, e g OMe, OA11, etc, or aromatic group, e g -OPh, -OPhNO 2 -p, -OPhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation) GlcNR-R ' is in this case GlcNR ⁇ 1-3 Gal ⁇ l-4Glc, where R is an organic or inorganic group
  • ⁇ -galactosidase catalysed by ⁇ -galactosidase, e g. from bovine testes or, another suitable enzyme source, or modified dito, (e g ⁇ -galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e.
  • the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, e g saccharide (e.g lactose is used as donor) or R" is -OR, where -OR is an aliphatic group, e.g OMe, OA11, etc, or aromatic group, e g -OPh, -OPhNO 2 -p, -OPhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation) R' is in this example a glycosidically bound nucleotide, -UDP, meaning that a nucleotide sugar, Gal ⁇ l-3GlcNR-UDP, is produced R in GlcNR-R', has been exemplified in the previous examples
  • reaction step 2 a glycosidase which hydrolyses unwanted regioisomers, e g 1-4, and/or 1-6 linkages, can be used
  • dimeric, oligomeric and polymeric products of LNT can be formed in several ways from the products synthesised above.
  • a crosslinker e g di-, or oligomeric PEG, peptides, proteins, e g KLH or serum albumin, polymers, e g polyacrylamide based system, or oligo- or polysaccharides
  • the product can be used in chemical/ biological/ food/ diagnostic/therapeutic applications and this does not limit the scope of the invention
  • LNnt is synthesised in two enzymatic steps, according to the scheme below
  • ⁇ -hexosaminidase catalysed by ⁇ -hexosaminidase, or ⁇ -N-acetyl-D-glucosaminidase, or modified dito, for example ⁇ -hexosaminidsynthase or ⁇ -N-acetyl-D-glucosaminidsynthase, (genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), where -R is -OH (i e the donor is N-acetyl-D-glucosamine, that is equilibrium synthesis or reversed hydrolysis), or is an organic, for example saccharide or R is -OR, in which R is for example aliphatic, for example -Me, -All, etc, or aromatic group, for example, -Ph, -PhNO 2 - ⁇ , -PhOMe-p, or inorganic (-F, etc) glycosidically bound
  • ⁇ -galactosidase catalysed by ⁇ -galactosidase, or modified dito, for example ⁇ -galactosynthase, (genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e. the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, e.g. saccharide (i.e. lactose is used as donor) or -OR where R is e.g. an aliphatic group, e.g. -Me, -All, etc, or aromatic group, e.g.
  • R' is -OH, or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All, -CH 2 Ph, - EtSiMe 3 , etc, or aromatic, e.g. -Ph, -PhNO 2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon.
  • R' is -OH, or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All, -CH 2 Ph, - EtSiMe 3 , etc, or aromatic, e.g. -Ph, -PhNO 2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon.
  • Reaction conditions and enzyme, purity of enzyme, isolation method are chosen by expert in the field, and this does not limit the scope of the invention.
  • acceptors can be used which are modified in one or several hydroxyl groups in addition to in the anomeric position.
  • the acceptor can be modified by chemical and/or enzymatic techniques.
  • lactose can first be reacted with lipase and acyl donor to protect one or several hydroxyl groups. Before this reaction, a derivative of lactose can be synthesised, which has a higher solubility in organic solvent.
  • HO-R is an appropriate alcohol, according to this application, as e.g. benzyl alcohol, allyl alcohol, trimetylsilyletanol or another substance wich after linking to glucose can be removed chemically,
  • reaction 2 can be catalysed by ⁇ -galactosidase, modified ⁇ -galactosidase or ⁇ - galactosynthase (for example modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006),
  • a lipase reaction where one or several of the OH-groups, for example the 6 OH group is modified with an acyl group (Ac above), e.g. not limiting example of Ac is an acetyl group.
  • Step 4 and 5 Reaction of (Ac)nGal ⁇ l-4Glc ⁇ -OR (as acceptor), with any of the two step enzymatic sequences wich are mentioned below, giving in the first step GlcNAc ⁇ 1 -3 (Ac)nGal ⁇ l-4Glc ⁇ -OR, and in the next step,
  • ⁇ -hexosaminidase catalysed by ⁇ -hexosaminidase, or ⁇ -N-acetyl-D-glucosaminidase from jack bean, or other enzymes which gives the desired binding, or where this enzyme first has been cloned and/or has been modified, e.g. ⁇ -hexosaminidsynthase or ⁇ -N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean or other appropriate enzyme, where the hydrolysis ability completely, or more or less is missing), and where the donor is N-acetyl-D-glucosamine (i.e.
  • R ' is -OH, (i.e. lactose is used as acceptor), or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All , -CH 2 Ph, -EtSiMe 3 , etc, or aromatic group, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon, and
  • ⁇ -galactosidase from Bullera singularis, Bacillus circulans, Clonezyme R 006 or catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified ⁇ - galactosidase from Bullera singularis, Bacillus circulans, Clonezyme R 006) or other suitable enzyme, or where this enzyme has been cloned and/or modified, (e.g.
  • Gal-R is lactose
  • Gal-R is a galactoside where R" is a glycosidically bound group as e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or glycosidically bound inorganic group (e.g. -F) glycosidically bound aglykon.
  • ⁇ -hexosaminidase catalysed by ⁇ -hexosaminidase, or ⁇ -N-acetyl-D-glucosaminidase from jack bean, or where this enzyme first has been cloned and/or modified, e.g.
  • ⁇ -hexosaminidsynthase or ⁇ -N-acetyl- D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean where the hydrolysis ability completely, or more or less is missing), and where the donor is chitobiose, or where the donor is GlcNAc ⁇ -OPhNO 2 -p, or R is glycosidically bound where -OR is aliphatic, e.g. -Me, -All, etc, or aromatic, e.g.
  • R' is -OH, (i.e. lactose is used as acceptor) or R' is an organic, e.g. -OR where -OR is e.g. an aliphatic e.g. -OMe, -OA11 , -OCH 2 Ph, -OetSiMe 3 etc, or an aromatic group, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or an inorganic (-F, etc) glycosidically bound aglycon, and
  • Gal-R lactose
  • Gal-R is a galactoside
  • R is a glycosidically bound group, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or a glycosidically bound inorganic group ( e.g. -F group)
  • ⁇ -galactosidase from e.g. Bullera singularis , Bacillus circulans, Clonezyme R 006 or catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified ⁇ - galactosidase from Bullera singularis, Bacillus circulans, Clonezyme R 006)or other suitable enzyme, or where this enzyme first has been cloned and/or modified, (e.g. ⁇ -galactosynthase, or other genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where R ' is -OH (i.e.
  • the donor is galactose; i.e. equilibrium synthesis or reversed hydrolysis
  • pr is an organic, e.g. saccharide (e.g. lactose is used as the donor), or R" is -OR, where OR is e.g. aliphatic group, e.g -OMe, -OAll, etc, or aromatic group, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or inorganic group (-F, etc) glycosidically bound aglycon (so called transglycosylation).
  • OR is e.g. aliphatic group, e.g -OMe, -OAll, etc, or aromatic group, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p; or inorganic group (-F, etc) glycosidically bound aglycon (so called transglycosylation).
  • R ' is in a corresponding manner, -OH or is an ⁇ - or a ⁇ -glycosidically bound organic group, e.g. -SEt, -SCr, -OBn, OAll, or for the purpose another suitable group and where R is an organic or inorganic group.
  • GlcNR ⁇ -R ' is e.g. 2-amino-2-deoxy-glucosamine (GlcNH 2 ), GlcNH 2 ⁇ -SEt and GlcNHTroc ⁇ -SEt.
  • a chemically modified lactose-structure is used (3 ' -OH)(R ' ")Gal ⁇ l-4Glc-R" (which have a free 3 ' -hydroxyl group) as acceptor, and (R ' " ) symbolise the protection groups on the lactose-acceptor, e.g. benzyl groups, and R" is a glycosidically bound organic group. for instance a benzyl group.
  • the conditions of the protection groups on the acceptor are chosen by the expert in the field and this does not limit the scope of the invention.
  • step 4 above is, instead of a complete transformation to LNnT-R", that the product after step 3 above can be used to make derivatives and analogs of LNnT, employing conventional organic chemistry and/or enzymatic transformations.
  • a N-Troc group be transformed to several different derivatives, where the amino group is modified, e.g. with an organic acyl group or with an alkyl group.
  • other derivatives of lactose-R" can be used, which makes it possible to synthesise other derivatives or analogs of LNnT.
  • the donor is galactose; equilibrium or reversed hydrolysis), or is an organic group, e.g. saccharide (e.g. lactose is used as donor), or R" is -OR, (where -OR is aliphatic, e.g. -OMe, -OAll, etc, or aromatic, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p), or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
  • -OR is aliphatic, e.g. -OMe, -OAll, etc, or aromatic, e.g. -OPh, -OPhNO 2 -p, -OPhOMe-p
  • GlcNR-R ' is in this case GlcNR ⁇ l-3Gal ⁇ l-4Glc, where R is a organic or inorganic group.
  • R is a organic or inorganic group.
  • ⁇ -galactosidase catalysed by ⁇ -galactosidase, e.g. from Bullera singularis , Bacillus circulans, Clonezyme R 006 or catalysed by modified ⁇ -galactosidase, or ⁇ -galactosynthase (e.g. modified ⁇ - galactosidase from Bullera singularis, Bacillus circulans, Clonezyme R 006) or from another suitable enzyme, or where this enzyme first has been cloned and/or modified, (e.g. ⁇ - galactosynthase, or other genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), and where R " is -OH, (i.e.
  • the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, (e.g. saccharide (i.e. lactose is used as donor) or R" is -OR, where -OR is aliphatic, e.g. -OMe, -OAll , etc, or aromatic, e.g., -OPh, -OPhNO 2 -p, -OPhOMe-p), or an inorganic (-F, etc) glycosidically bound aglykon, (so called transglycosylation).
  • -OR is aliphatic, e.g. -OMe, -OAll , etc, or aromatic, e.g., -OPh, -OPhNO 2 -p, -OPhOMe-p
  • R ' is in this example a glycosidically bound nucleotide, -UDP, meaning that a nucleotide sugar Gal ⁇ l-4GlcNR-UDP, is produced.
  • R in GlcNR-R' has been exemplified in the previous examples.
  • reaction step 2 a glycosidase which hydrolyses unwanted regioisomers, e.g.1-3- and or 1-6- linkages, can be used
  • dimeric, oligomeric and polymeric products of LNnT can be formed in several ways from the products above
  • di- or oligomeric PEG di- or oligomeric PEG, peptides, proteins, e g KLH or serum albumin, polymers, e.g. polyacrylamide-based system, oligo- or polysaccharides
  • the product can be used in chemical/ biological/food/ diagnostic/therapeutic applications and this does not limit the scope of the invention
  • glycosides and oligosaccharides So called transglycosylation reactions are often employed, where a glycoside, disaccharide, or higher oligosaccharide is used as glycosyl donor, in combination with a hydroxyl group containing organic substance (e g alcohol, amino acid, peptides, or saccharide) as acceptor
  • a glycoside, disaccharide, or higher oligosaccharide is used as glycosyl donor, in combination with a hydroxyl group containing organic substance (e g alcohol, amino acid, peptides, or saccharide) as acceptor
  • a modified glycosidase which completely or partially is lacking the ability for hydrolysis, but has the same, or more or less the same ability to catalyse transglycosylation compared to the natural glycosidase, can be used
  • As catalyst can be used a glycosidase which has a modified amino acid sequence compared with the enzyme present in Nature, on one or several sites, obtained via e g natural or in vitro genetic mutation of the natural DNA/RNA sequence of the enzyme, followed b ⁇ cloning/expression of the modified enzyme, or obtained via e g covalent modification of one or several of the amino acid side chains in the enzyme ' s amino acid sequence
  • So called retaining glycosidases often have two catalytic groups, which cooperate m hydrolysis and tranglycosylation, and gives a product with the same configuration as the donor
  • glycosidase in which a suitable catalytic group (for example, a catalytic carboxylic group) has been replaced with a catalytically inert group, e g glycine or alanine
  • a catalytically inert group e g glycine or alanine
  • so called glycosynthases have been obtained, which does not have the ability to catalyse hydrolysis, but which have ability to catalyse a transglycosylation/substitution of a so called leaving group (e g a (-F, group) on the donor saccharide, to one of the hydroxyl groups of the acceptor, so that a glycosidic linkage is formed between the donor and the acceptor
  • an ⁇ -donor glycoside is used with ⁇ -retaining enzyme to get ⁇ - glycosidic bond, and vice versa
  • this in the literature which descibes synthesis of Glc ⁇ l-Glc and Gal ⁇ 1 -Gal
  • An example of another genetic variant according to the invention is where the amino acid sequence of the enzyme is modified, so that one catalytic group get in a position where it cannot catalyse the hydrolysis reaction, or where the change of the amino acid sequence, leads to that the reaction of the acceptor is more or less favored relative to the reaction of water, via the modification of the amino acid sequence of the enzyme
  • glycosidases can be used according to the invention, where a catalytic group decisive for the hydrolysis reaction is chemically modified, or where the enzyme has been modified in such a manner that the reaction with the acceptor is increased relative to the hydrolysis reaction
  • the invention relates to synthesis of glycosidic linkages of different types with modified glycosidases/ glycosynthases of the type mentioned above
  • One aspect of the invention relates to the control of the regioselectivity of these genetically or chemically modified glycosidases/glycosynthases. This is obtained according to the invention, by the use of acceptor glycosides as acceptors of different aglykon structure and/or by the use of acceptor glycosides with either ⁇ - or ⁇ - configuration In this way can according to the invention, control of the regioselectivity of a given glycosynthase or modified glycosidase be obtained.
  • Non-limiting examples hereof are the synthesis of, more or less exclusively, Gal ⁇ l-3GalNAc ⁇ l-OPhNO 2 -p, with e g Gal ⁇ -F as donor and GalNAc ⁇ l-OPhNO 2 - ⁇ as acceptor, synthesis of, more or less exclusively, Gal ⁇ l-3GalNAc ⁇ l-OR, with e.g Gal ⁇ -F as donor and GalNAc ⁇ l-OR as acceptor (-OR is an organic group as e.g.
  • modified variant of bovine testes or bacteria enzyme such as galactosynthase or modified ⁇ -galactosidase as catalyst.
  • modified ⁇ -enzyme ⁇ -glycoside is used instead as the donor.
  • D-F D is a monosaccharide residue which is bound to a F-group and which consist of, e.g. a D-glycosyl, D-xylosyl, N- acetylneuraminyl-, D-mannosyl-, L-fucosyl-, N-acetyl-D-galactosaminyl-, or an N-acetyl-D- glucosaminyl-residue, and accepors, which e.g. are ⁇ - or ⁇ -glycoside,
  • A-OR is an glycosidically bound organic group e.g.
  • the product D-A-OR is formed from D-F + A-OR.
  • modified endo-glycosidases/endo-glycosynthases can be used with donors transferring di-, tri- or higher donor saccharide to the acceptor.
  • the enzym/enzymes is/are used in the invention in so called solid-phase synthesis.
  • -OR in A-OR is one to A- glycosidically bound spacer, which is covalently bound to a polymer.
  • the polymer is washed, and D-A is released through hydrolysis from the spacer.
  • control of the regioselectivity is obtained with the genetically or chemically modified enzymes, by the use of an enzyme which natural variant gives, more or less exclusively, the synthesis of the desired linkage, e.g. ⁇ l-3- or ⁇ l-6- linkage, or a ⁇ l-3-, ⁇ l-4- or a ⁇ l-6- linkage to the acceptor.
  • an enzyme which natural variant gives, more or less exclusively, the synthesis of the desired linkage, e.g. ⁇ l-3- or ⁇ l-6- linkage, or a ⁇ l-3-, ⁇ l-4- or a ⁇ l-6- linkage to the acceptor.
  • Non-limiting examples are modified ⁇ -galactosidase or ⁇ -galactosynthase, which natural enzyme variant gives predominant Gal ⁇ l-3GlcNAc (e.g. bovine testes), Gal ⁇ l-4GlcNAc (for example enzyme from Bacillus ciculans, Sporobolomyces singularis or Clonezyme R 006).
  • Gal ⁇ l-3GlcNAc e.g. bovine testes
  • Gal ⁇ l-4GlcNAc for example enzyme from Bacillus ciculans, Sporobolomyces singularis or Clonezyme R 006
  • LNnT Gal ⁇ l-4GlcNAc ⁇ l-3Gal ⁇ l-4Glc
  • the reaction Gal-R + GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006), where R of the donor, is of the type mentioned above (e.g. -F), where the acceptor have been obtained via e.g. the reaction: GlcNAc-R + Gal ⁇ l-4Glc ⁇ GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc
  • Gal ⁇ l-4GlcNH 2 ⁇ 1-3 Gal ⁇ l-4Glc may be mentioned the synthesis of Gal ⁇ l-4GlcNH 2 ⁇ 1-3 Gal ⁇ l-4Glc from e.g. the reaction Gal-R + GlcNH 2 ⁇ 1-3 Gal ⁇ l-4Glc, catalysed by modified ⁇ - galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006), where R of the donor, is of the type mentioned above ( e.g. -F).
  • modified ⁇ - galactosidase or ⁇ -galactosynthase e.g. modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006
  • R of the donor is of the type mentioned above (e.g. -F).
  • Gal ⁇ l-4GlcNH 2 may be mentioned the synthesis of Gal ⁇ l-4GlcNH 2 , from e.g. the reaction, Gal-R + GlcNH 2 , catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from Bullera singularis, Clonezyme R 006), where R of the donor, is of the type mentioned above (e.g. -F).
  • Gal ⁇ 1-3 GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc may be mentioned the synthesis of Gal ⁇ 1-3 GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc, from e.g. the reaction Gal-R + GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc, catalysed by modified ⁇ - galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from bovine testes), where R of the donor, is of the type mentioned above ( e.g. -F), via e.g. initially the reaction,
  • Gal ⁇ l-3GlcNH may be mentioned the synthesis of Gal ⁇ l-3GlcNH , via e.g. the reaction Gal-R + GlcNH 2 , catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from bovine testes), where R of the donor, is of the type mentioned above (e.g. -F).
  • modified ⁇ -galactosidase or ⁇ -galactosynthase e.g. modified enzyme from bovine testes
  • R of the donor is of the type mentioned above (e.g. -F).
  • donors and acceptors e.g. containing saccharides or glycosides thereof, where the monosaccharides consists of e.g. any or several of fucose, glucose, xylose, N-acetylneuraminic acid, mannose, and hexosamine.
  • modified endo-glycosidase/endo-glycosynthases can be used, with donors transferring di-, tri-, or higher saccharides to the acceptor, according to the invention.
  • the above mentioned type of enzyme is used for the synthesis of modified saccharides.
  • This is obtained by allowing, the enzyme to catalyse the synthesis between a donor saccharide, or glycoside, thereof and an acceptor, or acceptor glycoside, which is chemically modified with an organic or inorganic group, in at least one or several of the hydroxyl groups on C-2, C-3, C-4 or C-6 (hexopyranoses), and in the case when the acceptor is containing at least one amino-deoxy-saccharide or glycoside thereof, the acceptor can be modified in the amino group, and/or in any of the hydoxyl groups.
  • a modified D-GlcpNAc (2-amino-2-deoxy-N-acetyl-D- glucosaminopyranose) or ⁇ - or ⁇ -glycoside thereof, or a modified D-GalpNAc (2-amino-2- deoxy-N-acetyl-D-galactosaminopyranose), or ⁇ - or ⁇ -glycoside thereof, is used as acceptor.
  • modified acceptors/acceptor glycosides where e.g. the amino group or optionally any, or several of the amino groups, contains one amino group (e.g.
  • acceptors GlcNH 2> 2-amino-2-deoxy-D-glucosamine, and GalNH 2 , 2-amino-2-deoxy-D-galactosamine, and glycosides thereof, or saccharides with any or several of these groups), or where the amino group, modified with any or several other organic- or inorganic group than the acetyl group, e.g. non-limiting -NH-R, where R is a carbonyl- or carbonyloxy-containing aromatic or aliphatic group (C( O)R ' resp.
  • Modified ⁇ -D-galactosidase from e.g. Bullera singularis. or ⁇ -D-galactosynthase. catalyses the following:
  • Modified ⁇ -galactosidase from e.g. coffee beans, or ⁇ -galactosynthase catalyses the following:
  • the above type of enzyme is used for the synthesis of glycosides. This is obtained by allowing the enzyme to catalyse the synthesis between a donor saccharide, and an acceptor containing a nucleophile, such as a hydroxyl group, a thiol group or an amino group.
  • acceptors are e.g. aliphatic or aromatic alcohol, or hydroxyl group containing therapeutics.
  • Another non-imiting example is hydroxyl group containing amino acids e.g. L-serine and L- threonine, or derivatives thereof, protected with e.g. metoxycarbonyl-, allyloxycarbonyl-, benzyloxycarbonyl-, t-butyoxycarbonyl, or FMOC-group in the amino group, and/or with a free carboxyl group or with e.g. an esterified carboxyl group.
  • amino acids e.g. L-serine and L- threonine, or derivatives thereof, protected with e.g. metoxycarbonyl-, allyloxycarbonyl-, benzyloxycarbonyl-, t-butyoxycarbonyl, or FMOC-group in the amino group, and/or with a free carboxyl group or with e.g. an esterified carboxyl group.
  • amino acid glycosides are Tn-antigen and derivatives thereof, Sia ⁇ 2-6GalNAc ⁇ -Serine, GlcNAc ⁇ -Ser and derivatives thereof, and Gal ⁇ -Ser and derivatives thereof.
  • the type of enzymes mentioned above are used in the synthesis of saccharide linkages of proteins or of lipids.
  • the protein or the lipid can have one or several previously bound saccharides, or not.
  • the above type of enzyme can be used together with one or several glycosyltranferases for synthesis of glycosides, modified saccharides, disaccharides, or higher oligosaccharide or glycoside or thereof. This is obtained through the synthesis described above together with at least one glycosyltransferase reaction.
  • glycosyltransferases An extensive number of examples of glycosyltransferases, as well as cloning and production of these enzyme with genetic engineering techniques, are given in the literature.
  • Examples of the donor saccharides which are used with every type of glycosyltransferase are also given in the literature. Multi-enzyme systems which can be used simultaneously to generate the respective donor saccharide for the glycosyltransferase reaction, are also described in the literature.
  • Genetically manipulated cells can be used in connection with the above type of synthesis without previous enzyme isolation.
  • nucleotide sugar e.g. UDP-Gal or UDP-GlcNAc
  • UDP-Gal or UDP-GlcNAc can be generated by one type of genetically manipulated cell (from e.g.UTP generated by another genetically manipulated cell to which e.g. orotic acid is added)
  • the preformed nucleotide sugar can be used by a third type of genetically manipulated cell, in which a glycosyltransferase reaction takes place.
  • a glycosyltransferase reaction takes place.
  • Gal ⁇ l-4GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc by cells which express ⁇ (l-4)- galactosyltransferases from UDP-Gal as donor (produced in e.g. another cell in situ as above), and from GlcNAc ⁇ l-3Gal ⁇ l-4Glc, which has been produced according to any of the techniques above.
  • This sequence of reactions can be used e.g. when the regioselectivity is not high enough in a similar modified glycosidase-/glycosynthase reaction.
  • a disaccharide glycoside, a trisaccharide, or a trisaccharide glycoside, a tetrasaccharide or a higher oligosaccharide, or oligosaccharide derivatives can be obtained.
  • Gal ⁇ l-4GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc from, e.g. the reaction Gal-R + GlcNAc ⁇ 1-3 Gal ⁇ l-4Glc, catalysed by modified ⁇ -galactosidase or ⁇ -galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R 006), where R in the donor is of above mentioned type ( e.g. -F), where the acceptor could have been produced via e.g. the reaction
  • the literature describes this type of enzyme and reaction, and this do not limit the scope of the invention.
  • this reaction can e.g. this reaction be carried out by using a cell bound system, where UDP-GlcNAc is produced with one type of cell in situ and the synthesis of trisaccharide is catalysed by glycosyltransferase in another cell type.
  • the enzyme/enzymes mentioned in this invention are used in isolated form, in partially purified form, in situ, i.e. not separated from the cell, or in immobilised or entrapped form in batch, or in another type of reactor, and/or in a fermentation. In the latter case can e.g. the donor and initial acceptor be added to the fermentation medium.
  • xenotransplantation e.g. ⁇ -Gal-antibodies
  • allo-transplantation e.g. blood group A-, B- or O-antibodies
  • diagnostic reagents for determination of carbohydrate binding proteins e.g. ELISA or biosensor, as for example Biacore

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Abstract

The present invention describes a method for enzymatic synthesis of glucosides, disaccharides, oligosaccharides, modified disaccharides and oligosaccharides, preferably of the kind found in glycoproteins and especially of Lacto-N-tetraose (LNT) and of Lacto-N-neotetraose (LNnT).

Description

METHOD FOR ENZYMATIC SYNTHESIS OF GLYCOSIDES, DISACCHARIDES, AND OLIGOSACCHARIDES
This invention relates to synthesis of Galβl-3GlcNAcβl-3Galβl-4Glc (below called LNT), derivatives and glycosides thereof, Galβl-4GlcNAcβl-3Galβl-4Glc (below called LNnt), derivatives and glycosides thereof and other biologically active saccharides.
LNT and LNnt are present in human milk and also have a known antibacterial effect. Different methods to produce LNT and LNnt have been described, preferentially based on the isolation from human milk, or synthesis, preferentially using chemical methods.
This invention is based on enzymatic synthesis and enzymatic chemical methods for synthesis of LNT or LNnt, derivatives and glycosides thereof and other biologically active saccharides and derivatives, preferentially having antibacterial effect, or of other therapeutic or preventive interest.
In one aspect of the invention, LNT is synthesised in two enzymatic steps according to the scheme below:
1. GlcNAc-R + Galβl-4Glc-R' - GlcNAcβl-3Galβl-4Glc-R' + RH
catalysed by β-hexosaminidase, or β-N-acetyl-D-glucosaminidase, or modified dito, for example β-hexosaminidsynthase or β-N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where R is -OH (i.e. the donor is N-acetyl-D-glucosamine, that is equilibrium synthesis or reversed hydrolysis), or is an organic group, for example saccharide, or R is -OR, in which R is for example aliphatic, for example -Me, -All, etc, or aromatic, for example -Ph, -PhNO2-p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglycon (so called transglycosylation). R' is, in a corresponding manner, -OH (i.e. lactose is used as acceptor), or is an organic group, for example -OR, in which R is for example aliphatic, for example, -Me, -All, -CH2Ph, -EtSiMe3, etc, or aromatic, for example, -Ph, -PhNO2 -p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon and
2. Gal-R" + GlcNAcβl-3Galβl-4Glc-R' → Galβl-3GlcNAcβl-3Galβl-4Glc-R' + R"H
catalysed by β-galactosidase, or modified dito, for example β-galactosynthase (genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where R" is -OH (i.e. donor is galactose; equilibrium synthesis or reversed hydrolysis), or is an organic, e.g. saccharide (i.e. lactose is used as donor) or R" is -O where R is e.g. aliphatic, e.g. -Me, -All, etc, or aromatic, e.g., -Ph, -PhNO2-p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon (so called transglycosylation). R' is -OH, or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All, -CH2Ph, -EtSiMe3, etc, or aromatic, e.g. -Ph, -PhNO2-p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon.
Reaction conditions and enzyme are chosen by the expert in the field and this does not limit the scope of the invention. In the method according to above, acceptors can be used which are modified in one or several hydroxyl groups in addition to the anomeric position This gives for instance a possibility to produce analogs, employing, e.g conventional organic-chemical methods The acceptor can be modified by chemical or enzymatic techniques For instance lactose can first be reacted with lipase and acyl donor to protect one or several hydroxyl groups Before this reaction, a derivative of lactose can be synthesised, which has a higher solubility in organic solvent Not limiting examples
1 Cellobiose + HO-R → Glcβ-OR + Glc e g catalysed by Bullera stngidaris or enzyme therefrom, where HO-R is an appropriate alcohol according to this application, as for instance benzyl alcohol, allyl alcohol, trimetylsilyletanol or other substance, which after binding to glucose can be removed chemically,
2 Galβl-4Glc + Glcβ-OR -> Galβl-4Glcβ-OR + Glc
where reaction 2 can be catalysed by β-galactosidase, modified β-galactosidase or β- galactosynthase (for example modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R006),
3 Galβ 1 -4Glcβ-OR + ester → (Ac)nGalβ 1 -4Glcβ-OR + Glc
A lipase reaction where one or several of the OH-groups, for instance the 6 OH group is modified with an acyl group (Ac above) e g not limiting example of Ac is an acetyl group
Step 4 and 5 Reaction of (Ac)nGalβl-4Glcβ-OR (as acceptor), with any of the two enzymatic sequences wich are mentioned below, giving in the first step GlcNAcβl-3(Ac)nGalβl-4Glcβ-OR, and in the next step,
Galβl-3GlcNAcβl-3(Ac)nGalβl-4Glcβ-OR, which is deprotected conventionally with chemical methods, giving LNT
As a non-limitmg example of the method according to the invention can be mentioned
1 GlcNAc + Galβl-4Glc-R' → GlcNAcβ 1-3 Galβ l-4Glc-R' + RH
catalysed by β-hexosaminidase or β-N-acetyl-D-glucosammidase from jack bean, or other enzyme wich gives the desired linkage, or where this enzyme first has been cloned and/or has been modified, e g β-hexosaminidsynthase or β-N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean or other appropπate enzyme, where the hydrolysis ability completely, or more or less is missing), and where the donor is N-acetyl-D-glucosamine (i e equilibrium or reversed hydrolysis), and where R' is -OH (l e lactose is used as acceptor), or is an organic, e g -OR, where R is e g aliphatic, e g -Me, -All, -CH2Ph, -EtSiMe3, etc, or aromatic, e g -Ph, -PhNO2-p, -PhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, and 2. Gal-R" + GlcNAcβl-3Galβl-4Glc- R' → Galβl-3GlcNAcβ-l-3Galβl-4Glc-R + R"H catalysed by β-galactosidase from bovine testes or other appropriate enzyme, or where this enzyme first has been cloned and/or modified, (e.g β-galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where Gal-R" is lactose, or R" is glycosidically bound -OR, where R is aliphatic, e.g. -Me, -All, or aromatic, e.g. -Ph, -PhNO2-p, -PhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
As another non-limiting example can be mentioned:
1. GlcNAc-R + Galβl-4Glc-R' → GlcNAcβ 1-3 Galβ l-4Glc- R' + RH
catalysed by β-hexosaminidase or β-N-acetyl-D-glucosaminidase from jack bean, or where this enzyme first has been cloned and/or modified, e.g. β-hexosaminidsynthase or β- N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean, where the hydrolysis ability completely, or more or less is missing) and where the donor is chitobiose, or where the donor is GlcNAcβ-OPhNO2-p, and where R is -OH (i.e. lactose is used as acceptor), or is an organic, e.g. -OR, where OR is e.g. aliphatic group, e.g. -OMe, -OA11 , -OCH2Ph, OEtSiMe3, etc, or an aromatic group, e.g. -OPh, OPhNO2-p, -OPhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon, and
2. Gal-R" + GlcNAcβl-3Galβl-4Glc-R' → Galβ 1-3 GlcNAcβ 1-3 Galβ l-4Glc-R + R'Η catalysed by β-galactosidase from bovine testes or, other appropriate enzyme, or where this enzyme first has been cloned and/or modified, (e.g. β-galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where Gal-R" is lactose, or R" is glycosidically bound -OR, where R is an aliphatic group, e.g. -Me, -All, or aromatic group, e.g. -Ph, -PhNO2-p, -PhOMe-p; or R" is an inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
As another non-limiting example of the invention can the following be mentioned:
1. Gal-R" + GlcNR-R' → Galβ 1 -3GlcNR-R' + R"H
catalysed by β-galactosidase from e.g. bovine testes or, other appropriate enzyme, or modified dito, (e.g. β-galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e. the donor is galactose, i.e. equilibrium or reversed hydrolysis), or is an organic, e.g. saccharide (e.g. lactose is used as donor) or R" is -OR where -OR is an aliphatic group, e.g. -OMe, -OA11 , etc, or an aromatic group, e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation). R' is in similar manner, -OH, or is an α- or a β-glycosidically bound organic group, e.g. -SEt, -SCr, -OBn, -OA11, or another for the purpose suitable group, and where R is an organic or inorganic group. Non-limiting examples of NR are e.g. NH2, NHAc, or a so called NHTroc group, (NH-C(=O)-C-O- CH2CC13 group), or another amino protection group. Non-limiting examples of GlcNR-R' are, e.g., 2-amino-2-deoxy-glucosamine (GlcNH2), GlcNH2β-SEt and GlcNHTrocβ-SEt. Thereafter, chemical steps are used for the production of the end-substance (the conditions are chosen by the expert in the field and this does not limit the scope of the invention )
2. Peracetylation of Galβl-3GlcNRβ-R' with e g acetic acid anhydride in pyridine (the conditions are chosen by the expert in the field and this does not limit the scope of the invention), which gives (OAc)nGalβl-3GlcNRβ-R'
3. (OAc)nGalβl-3GlcNRβ-R' + (3 -OH)(R'")Galβl-4Glc-R" →
(OAc)n(Galβl-3GlcNAc) βl-3(R'") Galβl-4Glc-R" + R'
In this step, a chemically modified lactose-structure is used (3'-OH)(R" ) Galβl-4Glc-R" (which has a free 3 '-hydroxyl group) is used as acceptor, and (R'") symbolise the protection groups on the lactose-acceptor, e g benzyl groups, and R" is a glycosidically bound organic group, for instance a benzyl group The conditions and the protection groups on the acceptor, are chosen by the expert in the field, and this does not limit the scope of the invention.
4. Deblocking and, if a protection group is used on the amino group, acetylation of the amino group gives the end-substance Galβl-3GlcNAcβl-3Galβl-4Glc-R", where R"can be
-OH, or an aglykon, as e g mentioned above
The conditions are chosen by the expert in the field and this does not limit the scope of the invention
An alternative to step 4 above is, instead of a complete transformation to LNT-R", that the product after step 3 above can be used to make derivatives and analogs of LNT, employing conventional organic chemistry and/or enzymatic transformations For example can a N-Troc group be transformed to several different derivatives, where the amino group is modified, e g with an organic acyl group or with an alkyl group Moreover, other deπvatives of lactose-R' can be used, which makes it possible to synthesise other derivatives or analogs of LNT
As another non-limiting variant of the invention can be mentioned the following
1 Gal-R" + GlcNR-R' → Galβl-3GlcNR-R' + R"H
catalysed by β-galactosidase, from e g bovine testes or, from another suitable enzyme source, or modified dito, (e g β-galactosynthase, genetically and/or chemically modified enzyme. where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i e the donor is galactose, equilibrium or reversed hydrolysis), or is an organic group, e g saccharide (e g lactose is used as donor) or R" is -OR, where -OR is an aliphatic group, e g OMe, OA11, etc, or aromatic group, e g -OPh, -OPhNO2-p, -OPhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation) GlcNR-R' is in this case GlcNRβ 1-3 Galβ l-4Glc, where R is an organic or inorganic group Non-limiting example of NR is e g NH2, NHAc or a so called NHTroc group (NH-C(=O)-O-CH2CCl3 group), or another amino protection group As another non-limiting variant of the invention, the following can be mentioned
1 Gal-R" + GlcNR-R' → Galβ 1 -3GlcNR-R' + R"H
catalysed by β-galactosidase, e g. from bovine testes or, another suitable enzyme source, or modified dito, (e g β-galactosynthase, genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e. the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, e g saccharide (e.g lactose is used as donor) or R" is -OR, where -OR is an aliphatic group, e.g OMe, OA11, etc, or aromatic group, e g -OPh, -OPhNO2-p, -OPhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation) R' is in this example a glycosidically bound nucleotide, -UDP, meaning that a nucleotide sugar, Galβl-3GlcNR-UDP, is produced R in GlcNR-R', has been exemplified in the previous examples
2 Galβl-3GlcNR-UDP + lactose → Galβ 1-3 GlcNAcβ 1-3 Galβ l-4Glc + UDP catalysed by a glycosyltransferase, a N-acetyl-glucosaminyltransferase,
After the reactions above, except in the latest mentioned example, reaction step 2, a glycosidase which hydrolyses unwanted regioisomers, e g 1-4, and/or 1-6 linkages, can be used
Moreover, dimeric, oligomeric and polymeric products of LNT, alone or in combination with other saccharides, can be formed in several ways from the products synthesised above. For example, as bound via a crosslinker, e g di-, or oligomeric PEG, peptides, proteins, e g KLH or serum albumin, polymers, e g polyacrylamide based system, or oligo- or polysaccharides
The product can be used in chemical/ biological/ food/ diagnostic/therapeutic applications and this does not limit the scope of the invention
In one aspect of the invention, LNnt is synthesised in two enzymatic steps, according to the scheme below
1 GlcNAc-R + Galβl-4Glc-R - GlcNAcβ 1-3 Galβ l-4Glc-R' ^ RH
catalysed by β-hexosaminidase, or β-N-acetyl-D-glucosaminidase, or modified dito, for example β-hexosaminidsynthase or β-N-acetyl-D-glucosaminidsynthase, (genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), where -R is -OH (i e the donor is N-acetyl-D-glucosamine, that is equilibrium synthesis or reversed hydrolysis), or is an organic, for example saccharide or R is -OR, in which R is for example aliphatic, for example -Me, -All, etc, or aromatic group, for example, -Ph, -PhNO2-ρ, -PhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon (so called transglycosylation) R' is in a corresponding manner, -OH (i e lactose is used as acceptor) or is an organic, for example -OR, in which R is e g aliphatic, e.g -Me, -All, -CH2Ph, -EtSiMe3 etc, or aromatic, e.g -Ph, -PhNO2-p, -PhOMe-p, or inorganic (-F, etc) glycosidically bound aglykon, and 2. Gal-R" + GlcNAcβl-Galβl-4Glc-R' → Galβ l-4GlcNAcβ 1-3 Gal βl-4Glc-R' + R"H
catalysed by β-galactosidase, or modified dito, for example β-galactosynthase, (genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), where -R" is -OH (i.e. the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, e.g. saccharide (i.e. lactose is used as donor) or -OR where R is e.g. an aliphatic group, e.g. -Me, -All, etc, or aromatic group, e.g. -Ph, -PhNO2-p, -PhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon (so called tranglycosylation). R' is -OH, or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All, -CH2Ph, - EtSiMe3 , etc, or aromatic, e.g. -Ph, -PhNO2-p, -PhOMe-p; or an inorganic (-F, etc) glycosidically bound aglykon.
Reaction conditions and enzyme, purity of enzyme, isolation method, are chosen by expert in the field, and this does not limit the scope of the invention.
In the method according to above, acceptors can be used which are modified in one or several hydroxyl groups in addition to in the anomeric position. This gives for instance a possibility to produce analogs, employing, e.g. conventional organic-chemical methods. The acceptor can be modified by chemical and/or enzymatic techniques. For example lactose, can first be reacted with lipase and acyl donor to protect one or several hydroxyl groups. Before this reaction, a derivative of lactose can be synthesised, which has a higher solubility in organic solvent. Non-limiting example:
1. Cellobiose + HO-R → Glcβ-OR + Glc e.g. catalysed by Bullera singularis, or enzyme therefrom, where HO-R is an appropriate alcohol, according to this application, as e.g. benzyl alcohol, allyl alcohol, trimetylsilyletanol or another substance wich after linking to glucose can be removed chemically,
2. Galβl-4Glc + Glcβ-OR → Galβl-4Glcβ-OR + Glc
where reaction 2 can be catalysed by β-galactosidase, modified β-galactosidase or β- galactosynthase (for example modified enzyme from Bullera singularis, Bacillus circulans, Clonezyme R006),
3. Galβ 1 -4Glcβ-OR + ester → ( A Galβ 1 -4Glcβ-OR + Glc
A lipase reaction, where one or several of the OH-groups, for example the 6 OH group is modified with an acyl group (Ac above), e.g. not limiting example of Ac is an acetyl group.
Step 4 and 5: Reaction of (Ac)nGalβl-4Glcβ-OR (as acceptor), with any of the two step enzymatic sequences wich are mentioned below, giving in the first step GlcNAcβ 1 -3 (Ac)nGalβl-4Glcβ-OR, and in the next step,
Galβl-4GlcNAcβl-3(Ac)nGalβl-4Glcβ-OR, which is deprotected conventionally with chemical methods, giving LNnT.
As a non-limiting example of the method according to the invention can be mentioned: 1. GlcNAc + Galβl-4Glc-R' → GlcNAcβ 1-3 Galβ l-4Glc-R' + RH
catalysed by β-hexosaminidase, or β-N-acetyl-D-glucosaminidase from jack bean, or other enzymes which gives the desired binding, or where this enzyme first has been cloned and/or has been modified, e.g. β-hexosaminidsynthase or β-N-acetyl-D-glucosaminidsynthase (genetically and/or chemically modified enzyme from jack bean or other appropriate enzyme, where the hydrolysis ability completely, or more or less is missing), and where the donor is N-acetyl-D-glucosamine (i.e. equilibrium synthesis or reversed hydrolysis), and where R' is -OH, (i.e. lactose is used as acceptor), or is an organic, e.g. -OR, where R is e.g. aliphatic, e.g. -Me, -All , -CH2Ph, -EtSiMe3, etc, or aromatic group, e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon, and
2. Gal-R" + GlcNAcβ 1-3 Galβ l-4Glc- R' → Galβl-4GlcNAcβ-l-3Galβl-4Glc-R"H
catalysed by β-galactosidase from Bullera singularis, Bacillus circulans, Clonezyme R 006 or catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified β- galactosidase from Bullera singularis, Bacillus circulans, ClonezymeR006) or other suitable enzyme, or where this enzyme has been cloned and/or modified, (e.g. β-galactosynthase, or other genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where Gal-R" is lactose, or Gal-R" is a galactoside where R" is a glycosidically bound group as e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or glycosidically bound inorganic group (e.g. -F) glycosidically bound aglykon.
As another non-limiting example can be mentioned:
1. GlcNAc-R + Galβl-4Glc-R' → GlcNAcβ 1-3 Galβ l-4Glc- R' + RH
catalysed by β-hexosaminidase, or β-N-acetyl-D-glucosaminidase from jack bean, or where this enzyme first has been cloned and/or modified, e.g. β-hexosaminidsynthase or β-N-acetyl- D-glucosaminidsynthase, (genetically and/or chemically modified enzyme from jack bean where the hydrolysis ability completely, or more or less is missing), and where the donor is chitobiose, or where the donor is GlcNAcβ-OPhNO2-p, or R is glycosidically bound where -OR is aliphatic, e.g. -Me, -All, etc, or aromatic, e.g. -Ph, PhNO2-p, -PhOMe-p; or inorganic (-F, etc) glycosidically bound aglykon (so called tranglycosylation), and where R' is -OH, ( i.e. lactose is used as acceptor) or R' is an organic, e.g. -OR where -OR is e.g. an aliphatic e.g. -OMe, -OA11 , -OCH2Ph, -OetSiMe3 etc, or an aromatic group, e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or an inorganic (-F, etc) glycosidically bound aglycon, and
2. Gal-R" + GlcNAcβ 1-3 Galβ l-4Glc- R' → Galβl-4GlcNAcβ-l-3Galβl-4Glc-R"H
catalysed by β-galactosidase from Bullera singularis , Bacillus circulans, Clonezyme R 006 or catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified β- galactosidase from Bullera singularis, Bacillus circulans, ClonezymeR006) or other suitable enzyme, or where this enzyme has been cloned and/or modified, (e.g. β-galactosynthase, or other genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where Gal-R" is lactose, or Gal-R" is a galactoside, where R" is a glycosidically bound group, e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or a glycosidically bound inorganic group ( e.g. -F group)
As another non-limiting variant of the invention can be mentioned the following:
1. Gal-R" + GlcNR-R' → Galβl-4GlcNR-R' + R"H
catalysed by β-galactosidase from e.g. Bullera singularis , Bacillus circulans, Clonezyme R 006 or catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified β- galactosidase from Bullera singularis, Bacillus circulans, ClonezymeR006)or other suitable enzyme, or where this enzyme first has been cloned and/or modified, (e.g. β-galactosynthase, or other genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), and where R' is -OH (i.e. the donor is galactose; i.e. equilibrium synthesis or reversed hydrolysis) pr is an organic, e.g. saccharide (e.g. lactose is used as the donor), or R" is -OR, where OR is e.g. aliphatic group, e.g -OMe, -OAll, etc, or aromatic group, e.g. -OPh, -OPhNO2-p, -OPhOMe-p; or inorganic group (-F, etc) glycosidically bound aglycon (so called transglycosylation). R' is in a corresponding manner, -OH or is an α- or a β-glycosidically bound organic group, e.g. -SEt, -SCr, -OBn, OAll, or for the purpose another suitable group and where R is an organic or inorganic group. Non limiting examples of -NR is e.g. -NH2, -NHAc or a so called -NHTroc group (NH-C(=O)-O- CH2CCI3 group, or another amino protection group. Non-limiting example of GlcNRβ-R'is e.g. 2-amino-2-deoxy-glucosamine (GlcNH2), GlcNH2β-SEt and GlcNHTrocβ-SEt.
Chemical steps are thereafter used for the production of the end-substance (the conditions are chosen by the expert in the field and this does not limit the scope of the invention):
2. Peracetylation of Galβl-4GlcNR-R' with e.g. acetic acid anhydride in pyridine (the conditions are chosen by the expert in the field and this does not limit the scope of the invention.) which gives (OAc)nGalβl-4GlcNRβ-R\
3. (OAc)nGalβl-4GlcNRβ-R' + (3 '-OH)(R' ")Galβl-4Glc-R" →
(OAc)n(Galβl-4GlcNAc)βl-3(R' ") Galβl-4Glc-R" + R'
In this step a chemically modified lactose-structure is used (3 '-OH)(R'")Galβl-4Glc-R" (which have a free 3 '-hydroxyl group) as acceptor, and (R' ") symbolise the protection groups on the lactose-acceptor, e.g. benzyl groups, and R" is a glycosidically bound organic group. for instance a benzyl group. The conditions of the protection groups on the acceptor, are chosen by the expert in the field and this does not limit the scope of the invention.
4. Deblocking and, if a protection group is used on the amino group, acetylation of the amino group gives the end-substance Galβl-4GlcNAcβl-3Galβl-4Glc-R", where R "can be
-OH, or an aglycon such as e.g. mentioned above.
The conditions are chosen by the expert in the field and this does not limit the scope of the invention. An alternative to step 4 above is, instead of a complete transformation to LNnT-R", that the product after step 3 above can be used to make derivatives and analogs of LNnT, employing conventional organic chemistry and/or enzymatic transformations. For example can a N-Troc group be transformed to several different derivatives, where the amino group is modified, e.g. with an organic acyl group or with an alkyl group. Moreover, other derivatives of lactose-R" can be used, which makes it possible to synthesise other derivatives or analogs of LNnT.
As another non-limiting variant of the invention can be mentioned the following:
1. Gal-R" + GlcNR-R' - Galβ 1 -4GlcNR-R' + R"H
catalysed by β-galactosidase, from Bullera singularis, Bacillus circulans, ClonezymeR006 or catalysed by modified β-galactosidase or β-galactosynthase ( e.g. modified β-galactosidase from Bullera singularis, Bacillus circulans, ClonezymeR006) or another suitable enzyme source, or where this enzyme first has been cloned and/or modified, (e.g. β-galactosynthase, or other genetically and/or chemically modified enzyme, where the hydrolysis ability completely, or more or less is missing), where R'' is -OH (i.e. the donor is galactose; equilibrium or reversed hydrolysis), or is an organic group, e.g. saccharide (e.g. lactose is used as donor), or R" is -OR, (where -OR is aliphatic, e.g. -OMe, -OAll, etc, or aromatic, e.g. -OPh, -OPhNO2-p, -OPhOMe-p), or inorganic (-F, etc) glycosidically bound aglykon, (so called tranglycosylation).
GlcNR-R'is in this case GlcNRβl-3Galβl-4Glc, where R is a organic or inorganic group. Non limiting example of NR is e.g. -NH2, -NHAc or so called -NHTroc group (NH-C(=O)-O-CH2CCl3 group) or another amino protection group.
As another non-limiting variant of the invention the following can be mentioned:
1. Gal-R" + GlcNR-R' → Galβl-4GlcNR-R' + R"H
catalysed by β-galactosidase, e.g. from Bullera singularis , Bacillus circulans, Clonezyme R 006 or catalysed by modified β-galactosidase, or β-galactosynthase (e.g. modified β- galactosidase from Bullera singularis, Bacillus circulans, ClonezymeR006) or from another suitable enzyme, or where this enzyme first has been cloned and/or modified, (e.g. β- galactosynthase, or other genetically and/or chemically modified enzyme where the hydrolysis ability completely, or more or less is missing), and where R" is -OH, (i.e. the donor is galactose; equilibrium or reversed hydrolysis), or is an organic, (e.g. saccharide (i.e. lactose is used as donor) or R" is -OR, where -OR is aliphatic, e.g. -OMe, -OAll , etc, or aromatic, e.g., -OPh, -OPhNO2-p, -OPhOMe-p), or an inorganic (-F, etc) glycosidically bound aglykon, (so called transglycosylation). R' is in this example a glycosidically bound nucleotide, -UDP, meaning that a nucleotide sugar Galβl-4GlcNR-UDP, is produced. R in GlcNR-R' has been exemplified in the previous examples.
2. Galβl-4GlcNR-UDP + lactose → Galβ 1-3 GlcNAcβ 1-3 Galβ l-4Glc + UDP catalysed by a glycosyltransferase, a N-acetyl-glucosaminyltransferase. After the reactions above, except in the latest mentioned example, reaction step 2, a glycosidase which hydrolyses unwanted regioisomers, e.g.1-3- and or 1-6- linkages, can be used
Moreover dimeric, oligomeric and polymeric products of LNnT, alone or in combination with other saccharides, can be formed in several ways from the products above For example, as bound via a crosslinker, di- or oligomeric PEG, peptides, proteins, e g KLH or serum albumin, polymers, e.g. polyacrylamide-based system, oligo- or polysaccharides
The product can be used in chemical/ biological/food/ diagnostic/therapeutic applications and this does not limit the scope of the invention
Below, further examples are given on how the reactions above, and a number of other similar reactions, can be performed
Glycosidase-catalysed synthesis is used today for synthesis of glycosides and oligosaccharides So called transglycosylation reactions are often employed, where a glycoside, disaccharide, or higher oligosaccharide is used as glycosyl donor, in combination with a hydroxyl group containing organic substance (e g alcohol, amino acid, peptides, or saccharide) as acceptor
Hydrolysis of the donor and/or the product are often obtained as side reactions To minimize this, several techniques can be used, as e g high acceptor concentration, high concentration of other solvent than water, etc
In this invention a modified glycosidase, which completely or partially is lacking the ability for hydrolysis, but has the same, or more or less the same ability to catalyse transglycosylation compared to the natural glycosidase, can be used
As catalyst can be used a glycosidase which has a modified amino acid sequence compared with the enzyme present in Nature, on one or several sites, obtained via e g natural or in vitro genetic mutation of the natural DNA/RNA sequence of the enzyme, followed b\ cloning/expression of the modified enzyme, or obtained via e g covalent modification of one or several of the amino acid side chains in the enzyme's amino acid sequence
So called retaining glycosidases often have two catalytic groups, which cooperate m hydrolysis and tranglycosylation, and gives a product with the same configuration as the donor
An example of a genetic variant of this kind of enzyme is glycosidase in which a suitable catalytic group (for example, a catalytic carboxylic group) has been replaced with a catalytically inert group, e g glycine or alanine In this manner, so called glycosynthases have been obtained, which does not have the ability to catalyse hydrolysis, but which have ability to catalyse a transglycosylation/substitution of a so called leaving group (e g a (-F, group) on the donor saccharide, to one of the hydroxyl groups of the acceptor, so that a glycosidic linkage is formed between the donor and the acceptor In this later example, an α-donor glycoside is used with β-retaining enzyme to get β- glycosidic bond, and vice versa There is an example of this in the literature, which descibes synthesis of Glcβl-Glc and Galβ 1 -Gal di-, tri- and tetra saccharides, with a glucosidase in which one catalytic carboxylic group is genetically mutated to an alanine group and where Glcα-F or Galα-F is used as a donor
An example of another genetic variant according to the invention, is where the amino acid sequence of the enzyme is modified, so that one catalytic group get in a position where it cannot catalyse the hydrolysis reaction, or where the change of the amino acid sequence, leads to that the reaction of the acceptor is more or less favored relative to the reaction of water, via the modification of the amino acid sequence of the enzyme
Related chemically modified glycosidases can be used according to the invention, where a catalytic group decisive for the hydrolysis reaction is chemically modified, or where the enzyme has been modified in such a manner that the reaction with the acceptor is increased relative to the hydrolysis reaction
The invention relates to synthesis of glycosidic linkages of different types with modified glycosidases/ glycosynthases of the type mentioned above
One aspect of the invention relates to the control of the regioselectivity of these genetically or chemically modified glycosidases/glycosynthases. This is obtained according to the invention, by the use of acceptor glycosides as acceptors of different aglykon structure and/or by the use of acceptor glycosides with either α- or β- configuration In this way can according to the invention, control of the regioselectivity of a given glycosynthase or modified glycosidase be obtained.
In this way, it can be obtained as a non-limiting example, using one single enzyme, more or less exclusively, predominantially e g either αl-3 or αl-6 linkages to the acceptor glycoside (which can be e g α- or β-monosaccharide glycoside, an α- or β-linked di-, tri- or oligosaccharide, or glycoside thereof) with an α-enzyme, and βl-3- or βl-6- linkages to the acceptor glycoside (which can be e g α- or β-monosaccharide glycoside, an α- or β-linked di-, tri- or oligosaccharide, or glycoside thereof) with a β- enzyme
As non-limiting examples hereof, can be mentioned the synthesis of, more or less exclusively, Galαl-3Galαl-OPhNO2-p, with e g Galβ-F as donor and Galαl-OPhNO2 as acceptor, synthesis of, more or less exclusively, Gala 1-3 Gala 1 -OR with e g Galβ-F as donor and Galαl-OR as acceptor (-OR is a organic group, e g metoxy-, p-metyloxifenoxy-, etc) synthesis of, more or less exclusively, Gala l-6Galβl -OMe with e g Galβ-F as donor, and Galβ 1 -OMe as acceptor, etc, all with α-galactosidase, (e g cloned modified variant of coffee bean-, bacterial-, or yeast enzyme) as catalyst In some cases when using modified α-enzyme, α-glycoside is used instead as the donor
Other non-limiting examples hereof, are the synthesis of, more or less exclusively, Galβl-3GalNAcαl-OPhNO2-p, with e g Galα-F as donor and GalNAcαl-OPhNO2-ρ as acceptor, synthesis of, more or less exclusively, Galβl-3GalNAcαl-OR, with e.g Galα-F as donor and GalNAcαl-OR as acceptor (-OR is an organic group as e.g. O-L-serine, O-L- threonine, derivatives or peptides containing this group, bensyloxy-, allyloxy-, metoxy-, p-metyloxifenoxy-, etc), synthesis of a mixture of Galβl-3GalNAcβl-OMe and Galβl- 6GalNAcβl-OMe, with e.g. Galα-F as donor and GalNAcβl-OMe as acceptor, etc, all with a cloned or modified variant of β-galactosidase (e.g. cloned modified variant of bovine testes or bacteria enzyme), such as galactosynthase or modified β-galactosidase as catalyst. In some cases when using modified β-enzyme, β-glycoside is used instead as the donor.
Other non-limiting examples, are the use of other donors, D-F, where D is a monosaccharide residue which is bound to a F-group and which consist of, e.g. a D-glycosyl, D-xylosyl, N- acetylneuraminyl-, D-mannosyl-, L-fucosyl-, N-acetyl-D-galactosaminyl-, or an N-acetyl-D- glucosaminyl-residue, and accepors, which e.g. are α- or β-glycoside, A-OR (-OR is an glycosidically bound organic group e.g. O-L-Serine, O-L-Threonine, derivatives or peptides containing this group, bensyloxy-, allyloxy-, metoxy-,-p-metyloxifenoxy-, etc), where A is a mono-, di-, or oligosaccharide residue which is bound to an -OR group, and which consist of e.g. at least one D-glycosyl-, D-xylosyl-, N-acetylneuraminyl-, D-mannosyl-, L-fucosyl-, N- acetyl-D-galactosaminyl-, or a N-acetyl-D-glucosaminyl-residue. Thus, according to the invention, the product D-A-OR, is formed from D-F + A-OR.
Also, according to the invention, modified endo-glycosidases/endo-glycosynthases can be used with donors transferring di-, tri- or higher donor saccharide to the acceptor.
In another aspect of the invention, the enzym/enzymes is/are used in the invention in so called solid-phase synthesis. In this case -OR in A-OR, is one to A- glycosidically bound spacer, which is covalently bound to a polymer. When the synthesis is finished, the polymer is washed, and D-A is released through hydrolysis from the spacer. The literature is extensive concerning different types of spacers and polymers, and this does not limit the scope of the invention.
In another aspect of the invention, the control of the regioselectivity is obtained with the genetically or chemically modified enzymes, by the use of an enzyme which natural variant gives, more or less exclusively, the synthesis of the desired linkage, e.g. αl-3- or αl-6- linkage, or a βl-3-, βl-4- or a βl-6- linkage to the acceptor.
Non-limiting examples are modified β-galactosidase or β-galactosynthase, which natural enzyme variant gives predominant Galβl-3GlcNAc (e.g. bovine testes), Galβl-4GlcNAc (for example enzyme from Bacillus ciculans, Sporobolomyces singularis or ClonezymeR006).
As a further non-limiting example can be mentioned the synthesis, of e.g. Fucαl-3GlcNAc and of NeuAcα2-6GalNAc and NeuAcα2-3 lactose, using Fuc-R, and NeuAc-R, respectively, as donor, where R, is for example, above mentioned type of the donor glycoside.
As further example can be mentioned synthesis of LNnT, Galβl-4GlcNAcβl-3Galβl-4Glc, from as mentioned above, e.g. the reaction Gal-R + GlcNAcβ 1-3 Gal βl-4Glc, catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, ClonezymeR 006), where R of the donor, is of the type mentioned above (e.g. -F), where the acceptor have been obtained via e.g. the reaction: GlcNAc-R + Galβl-4Glc → GlcNAcβ 1-3 Galβ l-4Glc
catalysed by modified or non-modified β-glucosaminidase or β-glucosaminidsynthase.
As another example, may be mentioned the synthesis of Galβ l-4GlcNH2β 1-3 Galβ l-4Glc from e.g. the reaction Gal-R + GlcNH2β 1-3 Galβ l-4Glc, catalysed by modified β- galactosidase or β-galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, ClonezymeR 006), where R of the donor, is of the type mentioned above ( e.g. -F).
As another example, may be mentioned the synthesis of Galβl-4GlcNH2, from e.g. the reaction, Gal-R + GlcNH2, catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified enzyme from Bullera singularis, ClonezymeR 006), where R of the donor, is of the type mentioned above (e.g. -F).
As another example, may be mentioned the synthesis of Galβ 1-3 GlcNAcβ 1-3 Galβ l-4Glc, from e.g. the reaction Gal-R + GlcNAcβ 1-3 Galβ l-4Glc, catalysed by modified β- galactosidase or β-galactosynthase (e.g. modified enzyme from bovine testes), where R of the donor, is of the type mentioned above ( e.g. -F), via e.g. initially the reaction,
GlcNAc-R + Galβl-4Glc → GlcNAcβ 1-3 Galβ l-4Glc
catalysed by modified, or non-modified, β-glucosaminidase or β-glucosaminidsynthase.
As another example, may be mentioned the synthesis of Galβl-3GlcNH , via e.g. the reaction Gal-R + GlcNH2, catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified enzyme from bovine testes), where R of the donor, is of the type mentioned above (e.g. -F).
Other non-limiting examples of donors and acceptors, e.g. containing saccharides or glycosides thereof, where the monosaccharides consists of e.g. any or several of fucose, glucose, xylose, N-acetylneuraminic acid, mannose, and hexosamine.
Also, modified endo-glycosidase/endo-glycosynthases can be used, with donors transferring di-, tri-, or higher saccharides to the acceptor, according to the invention.
In another aspect of the invention, the above mentioned type of enzyme, is used for the synthesis of modified saccharides. This is obtained by allowing, the enzyme to catalyse the synthesis between a donor saccharide, or glycoside, thereof and an acceptor, or acceptor glycoside, which is chemically modified with an organic or inorganic group, in at least one or several of the hydroxyl groups on C-2, C-3, C-4 or C-6 (hexopyranoses), and in the case when the acceptor is containing at least one amino-deoxy-saccharide or glycoside thereof, the acceptor can be modified in the amino group, and/or in any of the hydoxyl groups. In the later case, can be mentioned that a modified D-GlcpNAc (2-amino-2-deoxy-N-acetyl-D- glucosaminopyranose) or α- or β-glycoside thereof, or a modified D-GalpNAc (2-amino-2- deoxy-N-acetyl-D-galactosaminopyranose), or α- or β-glycoside thereof, is used as acceptor. In this later case, can be mentioned modified acceptors/acceptor glycosides, where e.g. the amino group or optionally any, or several of the amino groups, contains one amino group (e.g. non-limiting examples of acceptors GlcNH2> 2-amino-2-deoxy-D-glucosamine, and GalNH2, 2-amino-2-deoxy-D-galactosamine, and glycosides thereof, or saccharides with any or several of these groups), or where the amino group, modified with any or several other organic- or inorganic group than the acetyl group, e.g. non-limiting -NH-R, where R is a carbonyl- or carbonyloxy-containing aromatic or aliphatic group (C(=O)R' resp. C(=O)-O-R', where R' is an organic, aromatic or aliphatic group), as non-limiting example, a so called NHTeoc- (N-trichloroetyloxy-) group, and as another non-limiting example, a N-phtalimido group. As non-limiting example can be mentioned, synthesis of, Galαl-3Galβl-4GlcNHTeocβ-SEt via, for example, the following two synthetic reactions :
1. Modified β-D-galactosidase from e.g. Bullera singularis. or β-D-galactosynthase. catalyses the following:
Gal-R + GlcNHTeocβ-SEt → Galβl-4GlcNHTeocβ-Set + RH
2. Modified α-galactosidase from e.g. coffee beans, or α-galactosynthase, catalyses the following:
Gal-R + Galβ l-4GlcNHTeocβ-SEt - Gala 1-3 Galβ l-4GlcNHTeocβ-SEt + RH
As further non-limiting examples can be mentioned the following synthesis:
GlcNAc-R + (6-R'-)Galβ-SEt → GlcNAcβl-3(6-R'-)Galβ-SEt catalysed by modified hexosaminidase, or hexosaminsynthase, and as another non-limiting example:
Gal-R + (2-R'-)Galα-OMe → Galα-l-3(2-R'-)Galα-OMe
catalysed by modified α-galactosidase or α-galactosynthase.
In another aspect of the invention, the above type of enzyme is used for the synthesis of glycosides. This is obtained by allowing the enzyme to catalyse the synthesis between a donor saccharide, and an acceptor containing a nucleophile, such as a hydroxyl group, a thiol group or an amino group. Examples of acceptors are e.g. aliphatic or aromatic alcohol, or hydroxyl group containing therapeutics.
Another non-imiting example is hydroxyl group containing amino acids e.g. L-serine and L- threonine, or derivatives thereof, protected with e.g. metoxycarbonyl-, allyloxycarbonyl-, benzyloxycarbonyl-, t-butyoxycarbonyl, or FMOC-group in the amino group, and/or with a free carboxyl group or with e.g. an esterified carboxyl group.
In this manner can e.g. allyl-, trimetylsilyl-, benzyl-, amino acid-, peptide glycosides, therapeutic glycosides be synthesised. Examples of amino acid glycosides are Tn-antigen and derivatives thereof, Siaα2-6GalNAcα-Serine, GlcNAcβ-Ser and derivatives thereof, and Galβ-Ser and derivatives thereof.
In another aspect of the invention, the type of enzymes mentioned above are used in the synthesis of saccharide linkages of proteins or of lipids. The protein or the lipid can have one or several previously bound saccharides, or not.
In another aspect of the invention, the above type of enzyme, can be used together with one or several glycosyltranferases for synthesis of glycosides, modified saccharides, disaccharides, or higher oligosaccharide or glycoside or thereof. This is obtained through the synthesis described above together with at least one glycosyltransferase reaction. An extensive number of examples of glycosyltransferases, as well as cloning and production of these enzyme with genetic engineering techniques, are given in the literature. Examples of the donor saccharides which are used with every type of glycosyltransferase are also given in the literature. Multi-enzyme systems which can be used simultaneously to generate the respective donor saccharide for the glycosyltransferase reaction, are also described in the literature.
Genetically manipulated cells can be used in connection with the above type of synthesis without previous enzyme isolation. In such a system, can e.g. three different genetically manipulated cells be used. For example, nucleotide sugar (e.g. UDP-Gal or UDP-GlcNAc), can be generated by one type of genetically manipulated cell (from e.g.UTP generated by another genetically manipulated cell to which e.g. orotic acid is added), and the preformed nucleotide sugar can be used by a third type of genetically manipulated cell, in which a glycosyltransferase reaction takes place. As non-limiting examples can be mentioned e.g. formation of Galβ l-4GlcNAcβ 1-3 Galβ l-4Glc by cells which express β(l-4)- galactosyltransferases from UDP-Gal as donor (produced in e.g. another cell in situ as above), and from GlcNAcβ l-3Galβl-4Glc, which has been produced according to any of the techniques above.
This sequence of reactions can be used e.g. when the regioselectivity is not high enough in a similar modified glycosidase-/glycosynthase reaction. In this way a disaccharide glycoside, a trisaccharide, or a trisaccharide glycoside, a tetrasaccharide or a higher oligosaccharide, or oligosaccharide derivatives, can be obtained.
As another non-limiting example can be mentioned the synthesis of Galβ l-4GlcNAcβ 1-3 Galβl-4Glc from, e.g. the reaction Gal-R + GlcNAcβ 1-3 Galβl-4Glc, catalysed by modified β-galactosidase or β-galactosynthase (e.g. modified enzyme from Bullera singularis, Bacillus circulans, ClonezymeR 006), where R in the donor is of above mentioned type ( e.g. -F), where the acceptor could have been produced via e.g. the reaction
UDP- GlcNAc + Galβl-4Glc → GlcNAcβ 1-3 Galβ l-4Glc + UDP catalysed by e.g. recombinant N-acetylglucosaminyltransferase, e.g. cloned from bacteria. The literature describes this type of enzyme and reaction, and this do not limit the scope of the invention. In a similar way, as described above, can e.g. this reaction be carried out by using a cell bound system, where UDP-GlcNAc is produced with one type of cell in situ and the synthesis of trisaccharide is catalysed by glycosyltransferase in another cell type. In another aspect of the invention, the enzyme/enzymes mentioned in this invention, are used in isolated form, in partially purified form, in situ, i.e. not separated from the cell, or in immobilised or entrapped form in batch, or in another type of reactor, and/or in a fermentation. In the latter case can e.g. the donor and initial acceptor be added to the fermentation medium.
These and other conditions, as for example concentrations, temperature, solvent, reaction time, methods for isolation, etc, are decided by the expert in the field and these factors do not limit the scope of the invention.
In another aspect of the invention, the application of the saccharides for e.g. injection, affinity chromatography of proteins, extra-corporal removal of proteins and antibodies, e.g. removal of antibodies from blood or plasma during xenotransplantation (e.g. α-Gal-antibodies), or allo-transplantation (e.g. blood group A-, B- or O-antibodies), diagnostic reagents for determination of carbohydrate binding proteins, enzymes, cells, virus, with e.g. ELISA or biosensor, as for example Biacore , application for inhibition of enzyme, protein, antibodies, pathogenic bacteria, virus, for vaccination, e.g. as protein conjugate, directed towards cancer cells or bacteria.

Claims

Claims:
1. Methods for enzymatic synthesis of glycosides, disaccharides, oligosaccharides, modified disaccharides and oligosaccharides, especially of the type present in glycoproteins and glycolipids.
PCT/SE1999/001498 1998-08-31 1999-08-31 Method for enzymatic synthesis of glycosides, disaccharides, and oligosaccharides Ceased WO2000012747A1 (en)

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