WO2015080603A1 - Glycoproteines - Google Patents

Glycoproteines Download PDF

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Publication number
WO2015080603A1
WO2015080603A1 PCT/NZ2014/050014 NZ2014050014W WO2015080603A1 WO 2015080603 A1 WO2015080603 A1 WO 2015080603A1 NZ 2014050014 W NZ2014050014 W NZ 2014050014W WO 2015080603 A1 WO2015080603 A1 WO 2015080603A1
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Prior art keywords
phosphorylated
glycopeptide
peptide
glycoprotein
residues
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PCT/NZ2014/050014
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English (en)
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Antony John Fairbanks
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Antony John Fairbanks
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Publication of WO2015080603A1 publication Critical patent/WO2015080603A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to phosphorylated glycoproteins and
  • glycopeptides the chemoenzymatic synthesis of phosphorylated glycoproteins or glycopeptides, compositions comprising phosphorylated glycoproteins or glycopeptides, methods utilizing such phosphorylated glycoproteins or glycopeptides, and uses of such.
  • Lysosomal storage diseases are a heterogeneous group of rare inherited disorders generally characterized by the accumulation of undigested or partially digested macromolecules. The accumulation of these macromolecules and partial digestion products results in various cellular dysfunctions and disease symptoms (clinical abnormality). Lysosomal storage diseases encompass enzyme deficiencies of the lysosomal hydrolases, as well as deficiencies or defects in the proteins required for normal post-translational modification of lysosomal enzymes, activator proteins, or proteins needed to direct intracellular trafficking to and from the lysosome. More than 50 different lysosomal storage diseases have been described in the literature.
  • These diseases are typically classified according to the substrate accumulated in the lysosome; for example, sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses (MPSs), lipoprotein storage disorders, lysosomal transport defects, and neuronal ceroid lipofuscinoses.
  • Enzyme Replacement Therapy is currently used, both safely and effectively for peripheral manifestations in patients having Fabry disease, Pompe disease, Gaucher disease types I and III, mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes), mucopolysaccharidosis II (Hunter syndrome), and mucopolysaccharidosis VI (Maroteaux-Lamy syndrome).
  • a current approach in ERT utilizes cellular receptors that recognize particular phosphorylated glycans to target uptake of therapeutic glycoproteins and glycopeptides.
  • One particular approach is the treatment of lysosomal storage diseases with glycoprotein conjugates comprising a modified lysosomal storage enzyme, such as an acid a- glucosidase linked by a non-naturally occurring chemical bond to a phosphorylated oligosaccharide comprising terminal mannose-6-phosphate residues (M6P).
  • M6P mannose-6-phosphate residues
  • M6P residues at the non- reducing termini of high mannose oligosaccharides is to facilitate the trafficking of proteins conjugated to such glycans to the lysosome via interaction with the mannose-6-phosphate receptor (M6PR).
  • M6PR mannose-6-phosphate receptor
  • a disadvantage of this current approach is the presence of an artificial or non-naturally occurring chemical bond between the MYOZYME® active and the phosphorylated oligosaccharide. Because enzyme replacement therapies are not curative, but rather provide long term therapeutic maintenance, there is an increased potential for patients undergoing ERT to develop immunogenic responses to therapeutic compounds.
  • the invention relates to a method of making a phosphorylated glycoprotein or glycopeptide, the method comprising contacting an acceptor protein or peptide comprising at least one GlcNAc residue with a phosphorylated donor oligosaccharide in the presence of an enzyme that catalyzes the addition of the phosphorylated donor oligosaccharide to the at least one GlcNAc residue on the acceptor protein or peptide to form the phosphorylated glycoprotein or glycopeptide.
  • the method further comprises isolating and/or purifying the phosphorylated glycoprotein or glycopeptide.
  • the enzyme that catalyzes the addition of the phosphorylated donor oligosaccharide to the at least one GlcNAc residue on the acceptor protein or peptide is an endoglycosidase enzyme (ENGase), a modified ENGase, or a functional fragment, variant, analogue or derivative thereof.
  • ENGase endoglycosidase enzyme
  • the ENGase is selected from the group consisting of an endoglycosidase A (Endo A), endoglycosidase Fl (EndoFl), endoglycosidase F2 (EndoF2), endoglycosidase F3 (Endo F3), endoglycosidase M (Endo M) and endoglycosidase S (Endo S).
  • the ENGase is Endo A.
  • the endoglycosidase is a modified or mutant Endo A, preferably E173H and N171Q.
  • the endoglycosidase is Endo M or a modified or mutant Endo M.
  • the mutant Endo M is N175Q.
  • the phosphorylated donor oligosaccharide is a phosphorylated donor oligosaccharide comprising monosaccharide residues linked by glycosidic linkages, and an anomeric leaving group.
  • the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, and the leaving group is an oxazoline.
  • the phosphorylated donor oligosaccharide is a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide, preferably a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide oxazoline.
  • the phosphorylated donor oligosaccharide is a tetra- or hexasaccharide.
  • the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, preferably a synthetic phosphorylated donor oligosaccharide oxazoline.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one phosphorylated oligosaccharide. In one embodiment the phosphorylated
  • glycoprotein or glycopeptide comprises at least two phosphorylated oligosaccharides, at least three phosphorylated oligosaccharides, at least four phosphorylated oligosaccharides, at least five phosphorylated oligosaccharides, at least six phosphorylated oligosaccharides, or at least seven or more phosphorylated oligosaccharides.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one phosphorylated oligosaccharide that comprises at least one phosphorylated mannose residue, preferably at least one mannose-6-phosphate (M6P) residue, preferably at least two M6P residues, at least three M6P residues, at least four M6P residues, at least five M6P residues, at least six M6P residues, at least seven M6P residues, at least eight M6P residues, at least nine M6P residues, or at least ten or more M6P residues.
  • the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.
  • At least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.
  • the phosphorylated glycoprotein or glycopeptide comprises a phosphorylated oligosaccharide comprising only glycosidic linkages.
  • the glycosidic linkages are ⁇ 1-4 glycosidic linkages.
  • oligosaccharide are naturally linked to form a phosphorylated glycoprotein or glycopeptide.
  • the natural linkages are ⁇ 1-4 glycosidic linkages.
  • the chemical bond between the at least one GlcNAc residue on the acceptor protein or peptide and the phosphorylated oligosaccharide is a ⁇ 1-4 glycosidic linkage as found in nature.
  • the acceptor protein or peptide is a therapeutic protein or peptide, a modified therapeutic protein or peptide, or a functional fragment, variant, analogue or derivative of a therapeutic protein or peptide.
  • the acceptor protein or peptide is an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative of an enzyme.
  • the acceptor protein or peptide is a fusion protein or peptide comprising an enzyme, a modified enzyme, or a functional portion, fragment, variant, analogue or derivative thereof.
  • the enzyme, or modified enzyme is a lysosomal enzyme or a functional fragment, variant, analogue or derivative thereof.
  • the fusion protein or peptide comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme.
  • the lysosomal enzyme is selected from the group consisting of an a-sialidase, cathepsin A, a-mannosidase, ⁇ -mannosidase, glycosylasparaginase, a-fucosidase, a-N- acetylglucosaminidase, ⁇ -galactosidase, ⁇ -hexosaminidase a -subunit, ⁇ - hexosaminidase ⁇ -subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase B, saposin B, formyl-glycin generating enzyme, ⁇ -galactosylceramidase, a -galactosidase A, iduonate sulfatase, a -iduronidase, heparan N-s
  • the lysosomal enzyme or modified lysosomal enzyme is an acid a-glucosidase or functional fragment, variant, analogue or derivative thereof.
  • the acid a-glucosidase is alglucosidase-a-rch.
  • the lysosomal enzyme or modified lysosomal enzyme is a - galactosidase A or a modified form thereof including agalsidase alpha and/or agalsidase beta.
  • the method optionally comprises an initial step of preparing an acceptor protein or peptide comprising at least one GlcNAc residue, the step comprising contacting a protein or peptide comprising at least one GlcNAc-GlcNAc bond with an enzyme that hydrolyzes at least a GlcNAc-GlcNAc bond between a first GlcNAc residue immediately adjacent the protein or peptide, and a second GlcNAc residue immediately adjacent the first GlcNAc residue, to form the acceptor protein or peptide comprising a single GlcNAc residue.
  • the invention relates to a phosphorylated glycoprotein or glycopeptide made according to a method of the invention. In another aspect the invention relates to a phosphorylated glycoprotein or glycopeptide obtainable by a method of the invention.
  • the invention in another aspect relates to a phosphorylated glycoprotein or glycopeptide comprising a synthetic phosphorylated oligosaccharide linked to an acceptor protein or peptide by a natural linkage between at least one GlcNAc residue on the protein or peptide and the phosphorylated oligosaccharide.
  • the natural linkage is a ⁇ 1-4 glycosidic linkage.
  • the synthetic phosphorylated donor oligosaccharide is a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide.
  • the synthetic phosphorylated donor oligosaccharide is a tetra- or hexa-saccharide.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one synthetic phosphorylated oligosaccharide.
  • the phosphorylated glycoprotein or glycopeptide comprises at least two synthetic phosphorylated oligosaccharides, at least three synthetic phosphorylated oligosaccharides, at least four synthetic phosphorylated oligosaccharides, at least five synthetic phosphorylated oligosaccharides, at least six synthetic phosphorylated oligosaccharides, or at least seven or more synthetic phosphorylated oligosaccharides.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one synthetic phosphorylated oligosaccharide that comprises at least one phosphorylated mannose residue, preferably at least one mannose-6-phosphate (M6P) residue, preferably at least two M6P residues, at least three M6P residues, at least four M6P residues, at least five M6P residues, at least six M6P residues, at least seven M6P residues, at least eight M6P residues, at least nine M6P residues, or at least ten or more M6P residues.
  • the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.
  • at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.
  • the acceptor protein or peptide is a therapeutic protein or peptide, a modified therapeutic protein or peptide, an enzyme, a modified enzyme, a fusion protein or a functional fragment, variant, analogue or derivative thereof as described above.
  • the invention relates to a composition comprising a phosphorylated glycoprotein or glycopeptide made according to a method of the invention and a suitable carrier, diluent or excipient.
  • the invention relates to a composition comprising a phosphorylated glycoprotein or glycopeptide obtainable by a method of the invention and a suitable carrier, diluent or excipient.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises a therapeutically effective amount of the phosphorylated glycoprotein or glycopeptide according to the invention.
  • the invention relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use as a medicament.
  • the invention in another aspect relates to a pharmaceutical composition of the invention for use as a medicament.
  • the invention relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use in treating a disease or condition.
  • the invention relates to a pharmaceutical composition of the invention for use in treating a disease or condition.
  • the invention relates to the use of a phosphorylated glycoprotein or glycopeptide according to the invention in the manufacture of a medicament for treating a disease or condition.
  • the invention relates to the use of a composition of the invention in the manufacture of a medicament for treating a disease or condition.
  • the disease or condition is a lysosomal storage disease (LSD) or disorder related thereto.
  • LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, ⁇ -mannosidosis,
  • the disease or condition is Fabry disease.
  • the invention in another aspect relates to a method for treating a lysosomal storage disease comprising administering a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide according to the invention to a subject in need thereof.
  • the invention in another aspect relates to a method for treating a lysosomal storage disease comprising administering a composition according to the invention to a subject in need thereof.
  • the composition comprises a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide according to the invention.
  • the invention in another aspect relates to a method for identifying a cell that expresses a M6P receptor, the method comprising a) contacting the cell with a phosphorylated glycoprotein or glycopeptide according to the invention, wherein the phosphorylated glycoprotein or glycopeptide further comprises a detectable label, and b) detecting the presence of the phosphorylated glycoprotein or glycopeptide bound to the cell.
  • Figure 1 depicts the chemoenzymatic synthesis of a phosphorylated glycoprotein or glycopeptide.
  • Hydrolysis of a heterogeneous mixture of protein glycoforms (A) is carried out using an ENGase (B) to yield a homogeneous glycoprotein bearing single GlcNAc residues at N-linked glycosylation sites (C).
  • the homogenous glycoprotein (C) is then combined with an N-glycan oxazoline with terminal mannose-6-phosphate (M6P) residues (D) in the presence an appropriate ENGase (E) which catalyzes the synthesis of a homogenous glycoprotein bearing mannose-6-phosphate terminated N-glycans at N- linked glycosylation sites (F).
  • M6P terminal mannose-6-phosphate
  • Figure 2 depicts the glycosylation of RNaseB over time: SDS-Page : Lane 1- Omin, Lane 2-15min, Lane 3-30min, Lane 4-lh, Lane 5-2h, Lane 6-4h, Lane 7-8h, Lane 8-24h, and Lane 9-24h.
  • Figure 3 depicts the co-localization of (M6P) 2 RNase with CI-MPR in HepG2 cells following 2 hours of incubation with 1 ⁇ (M6P) 2 RNase. Staining of HEPG2 cells post incubation for A, the nuclear stain DAPI, B, the CI-MPR, C, RNase , D, Merged image of A-C, arrows indicate examples of co-localization of CI-MPR with RNase.
  • DAPI 4',6-diamidino-2-phenylindole
  • CI-MPR cation-independent mannose phosphate receptor
  • RNase Ribonuclease
  • Figure 4 depicts the co-localization of deglycosylated RNase with CI-MPR in HepG 2 cells following 2 hours of incubation with 1 ⁇ deglycosylated RNase. Staining of HEPG2 cells post incubation for A, the nuclear stain DAPI, B, the CI-MPR, C, RNase, D, Merged image of A-C, No co-localization is noted (absence of arrows).
  • Figure 5 depicts CI-MPR in HepG 2 cells following 2 hours of incubation with buffer substituted for RNase (i.e., No RNase control). Staining of HEPG2 cells post incubation for A, the nuclear stain DAPI, B, the CI-MPR, C, Buffer), it is anticipated no signal should be present, D, Merged image of A-C, No co-localization is noted (absence of arrows).
  • FIG. 6 depicts the immunoprecipitation of (M6P) 2 RNase from anti-CI-MPR coupled protein A.
  • IP CI-MPR immunoprecipitation with anti-cation independent MPR antibody conjugated protein A
  • WB RNase Western blot for RNase.
  • Figure 7 depicts the glycosylation of de-glycosylated Fabrazyme with M6P- tetrasaccharide oxazoline donor.
  • phosphorylated donor oligosaccharide refers to a
  • phosphorylated N-linked oligosaccharide that may be any oligosaccharide that possesses a suitable leaving group at the reducing terminus that can serve as a donor in a transglycosylation reaction catalyzed by an ENGase.
  • the suitable leaving group is an anomeric leaving group.
  • oligosaccharide may be a glycosyl oxazoline, glycosyl fluoride, para-nitrophenyl glycoside or full length amino acid-linked N-glycan which can act a substrates for transglycosylation reactions.
  • the anomeric leaving group is an oxazoline.
  • a "suitable anomeric leaving group” refers to any anomeric leaving group that will serve as a donor in a transglycosylation reaction catalyzed by an
  • a phosphorylated donor oligosaccharide comprises at least one terminal monosaccharide residue that comprises at least one phosphate group.
  • the at least one terminal monosaccharide residue is mannose.
  • the at least one phosphate group is located at position 6 of the mannose. Accordingly, in a particular embodiment, a phosphorylated donor oligosaccharide comprises at least one terminal M6P residue.
  • the N-glycan oligosaccharide has a bi-antennary structure.
  • the phosphorylated donor oligosaccharide is a phosphorylated bi-antennary pentasaccharide.
  • the phosphorylated bi-antennary pentasaccharide comprises at least one phosphorylated mannose residue, preferably at least one phosphorylated terminal mannose residue, preferably at least one terminal M6P residue.
  • donor oligosaccharide oxazoline and variations thereof refers to a class of oligosaccharide compounds comprising an oxazoline functional group as known in the art.
  • these terms refer to any oligosaccharide with an oxazoline at the reducing terminus that can serve as a donor in a glycosylation reaction catalyzed by an ENGase.
  • linked to means chemically connected via at least one covalent bond.
  • a GlcNAc residue on a protein or peptide of interest is linked to a GlcNAc residue on a synthetic phosphorylated donor oligosaccharide.
  • natural linkage and “naturally linked” as used herein with reference to a type of chemical bond means that the type of chemical bond consists of a covalent linkage between molecules that is found in that type of chemical bond in nature.
  • modified refers to any modification made to the protein or peptide that results in a protein or peptide that differs in some aspect from a corresponding wild type protein or peptide, but that retains all, or substantially all of the biological activity of the wild type protein or peptide.
  • a protein or peptide refers to any variant of the protein or peptide that differs in some aspect from a corresponding wild type protein or peptide, but that retains all, or substantially all of the biological activity of the wild type protein or peptide.
  • telomere length refers to any fragment of the protein or peptide comprising a subsequence of contiguous amino acid residues that retains substantially all of the biological activity of the full length protein or peptide.
  • a "functional analogue” as used herein with reference to a protein or peptide refers to any analogue of the protein or peptide comprising an amino acid sequence where one or more amino acid residues is independently substituted, added or deleted as compared to the wild type amino acid sequence of the protein or peptide, and that retains substantially similar biological activity when compared to the amino acid sequence of the wild type protein or peptide comprised in the protein or peptide.
  • amino acid sequence of a functional analogue of a protein or peptide useful in the invention may differ from the amino acid sequence of the wild type protein or peptide comprised in the phosphorylated glycoprotein or glycopeptide, respectively by one or more conservative amino acid substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Non-conservative substitutions are also encompassed where functional equivalence is maintained, and will entail exchanging a member of one of these classes for a member of another class.
  • analogues that include residues other than naturally occurring L-amino acids, e.g.
  • D-amino acids or non-naturally occurring synthetic amino acids e.g. beta or gamma amino acids and cyclic analogues.
  • Substitutions, deletions, additions or insertions may be made by any method as known in the art. A skilled worker will also be aware of methods for making phenotypically silent amino acid substitutions.
  • a protein or peptide refers to any derivative of the protein or peptide in which one or more of the amino acid residues of the protein or peptide have been modified, for example, by glycosylation, amide formation, maleimide coupling, ester formation, alkylation, or acylation, but not limited thereto, and that retains substantially all of the biological activity of the full length protein or peptide.
  • the present invention relates generally to the chemoenzymatic synthesis of
  • the phosphorylated glycoproteins or glycopeptides comprising all natural linkages between the glycan and the protein.
  • the phosphorylated glycoproteins or glycopeptides comprise therapeutic proteins or peptides, including enzymes, modified forms of such proteins or peptides, and functional fragments, variants, analogues and derivatives thereof.
  • the phosphorylated glycoproteins or glycopeptides according to the invention may allow for prolonged, or even lifelong therapeutic use with reduced immunogenicity in a subject, as compared to phosphorylated glycoproteins or glycopeptides comprising non-natural or artificial chemical linkages.
  • phosphorylated glycoproteins or glycopeptides according to the invention may allow for increased the efficacy of the phosphorylated glycoprotein or glycopeptide for treating various diseases or conditions.
  • Some embodiments of the present invention provide a novel and inventive method of chemoenzymatic synthesis of a phosphorylated glycoprotein or glycopeptide comprising all natural linkages between the protein and the glycan.
  • the protein is linked to a phosphorylated N-glycan oligosaccharide comprising at least one terminal mannose-6-phosphate residue, where the phosphorylated N-glycan
  • oligosaccharide comprises monosaccharide residues naturally linked by glycosidic linkages, particularly ⁇ 1-4 glycosidic linkages.
  • the phosphorylated N-glycan oligosaccharide comprises at least two terminal mannose-6-phosphate residues.
  • the phosphorylated N-glycan can be any phosphorylated N-glycan having any structure including one selected from the group consisting of mono, bi, tri, tetra, penta, hexa- antennary and greater oligosaccharides, but not limited thereto.
  • glycoproteins and glycopeptides as systemic therapeutic agents is known, including the use of various glycoprotein conjugates comprising enzymes as in enzyme replacement therapy (ERT).
  • ERT enzyme replacement therapy
  • ENGase endohexosaminidase
  • a specific ENGase, Endo A has been shown to be particularly effective at catalyzing the transglycosylation reaction between the GlcNAc residue on a protein or peptide of interest, and a phosphorylated donor oligosaccharide oxazoline. This demonstration is particularly surprising given what is generally accepted in the art regarding ENGase substrate specificity.
  • the ENGase is a modified or mutant Endo A including E173H and N171Q.
  • a phosphorylated glycoprotein or glycopeptide comprising a phosphorylated bi, tri, tetra, penta, hexa-antennary or greater oligosaccharide conjugated to a therapeutic protein or peptide of interest by all natural linkages is expected to provide for increased therapeutic efficacy, particularly due to the reduced immunogenicity of the described phosphorylated glycoproteins and glycopeptides.
  • the present invention allows for a method of directly modifying a therapeutic protein or peptide of interest that contains at least one GlcNAc acceptor residue by chemoenzymatic addition of a phosphorylated oligosaccharide to the GlcNAc residue to create a phosphorylated glycoprotein or glycopeptide comprising all natural chemical bonds.
  • the present invention relates to a method of making a phosphorylated glycoprotein or glycopeptide, the method comprising contacting an acceptor protein or peptide comprising at least one GlcNAc residue with a phosphorylated donor oligosaccharide in the presence of an enzyme that catalyzes the addition of the phosphorylated donor oligosaccharide to the at least one GlcNAc residue on the acceptor protein or peptide to form the phosphorylated glycoprotein or glycopeptide.
  • the method further comprises isolating and/or purifying the phosphorylated glycoprotein or glycopeptide. Isolation and/or purification of a phosphorylated glycoprotein or glycopeptide is within the skill in the art.
  • the enzyme that catalyzes the addition of the phosphorylated donor oligosaccharide to the at least one GlcNAc residue on the acceptor protein or peptide is an endoglycosidase enzyme (a.k.a., endohexosaminidase, ENGase), a modified ENGase, or a functional fragment, variant, analogue or derivative thereof.
  • an endoglycosidase enzyme a.k.a., endohexosaminidase, ENGase
  • a modified ENGase a functional fragment, variant, analogue or derivative thereof.
  • the ENGase is selected from the group consisting of an endoglycosidase A (Endo A), endoglycosidase Fl (EndoFl), endoglycosidase F2 (EndoF2), endoglycosidase F3 (Endo F3),
  • the ENGase is Endo A.
  • the endoglycosidase is a modified or mutant Endo A, preferably E173H and N171Q.
  • the endoglycosidase is Endo M or a modified or mutant Endo M.
  • the mutant Endo M is N175Q.
  • Endohexosaminidases also known as endo-P-JV-acetylglucosaminidases (abbreviated as ENGases) are a class of enzyme that specifically cleave the chitobiose core [GlcNAc (l- 4)GlcNAc] of JV-linked glycans between the two JV-acetyl glucosamine residues.
  • Endo M and modified versions, variants and analogues thereof would be more efficient at catalyzing the synthesis of phosphorylated glycopeptides and glycoproteins bearing mannose-6- phosphate residues.
  • Endo M and a mutant thereof, N175Q were able to catalyze the transfer of a phosphorylated N-glycan hexasaccharide to a protein acceptor, although with relatively low yield.
  • a surprising aspect of the present invention is the determination that Endo A, which does not normally operate hydrolytically on N-glycans that bear negative charge, was also able to transfer the phosphorylated N-glycan oxazoline to the acceptor proteinln one embodiment the phosphorylated donor oligosaccharide is a phosphorylated donor oligosaccharide comprising monosaccharide residues linked by glycosidic linkages, and an anomeric leaving group.
  • the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide oxazoline.
  • the phosphorylated donor oligosaccharide is a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide, preferably a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide oxazoline.
  • the phosphorylated donor oligosaccharide is a tetra- or hexa-saccharide.
  • the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, preferably a synthetic phosphorylated donor oligosaccharide oxazoline.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one phosphorylated oligosaccharide. In one embodiment the phosphorylated
  • glycoprotein or glycopeptide comprises at least two phosphorylated oligosaccharides, at least three phosphorylated oligosaccharides, at least four phosphorylated oligosaccharides, at least five phosphorylated oligosaccharides, at least six phosphorylated oligosaccharides, or at least seven or more phosphorylated oligosaccharides.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one phosphorylated oligosaccharide that comprises at least one phosphorylated mannose residue, preferably at least one mannose-6-phosphate (M6P) residue, preferably at least two M6P residues, at least three M6P residues, at least four M6P residues, at least five M6P residues, at least six M6P residues, at least seven M6P residues, at least eight M6P residues, at least nine M6P residues, or at least ten or more M6P residues.
  • M6P mannose-6-phosphate
  • the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.
  • at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.
  • the phosphorylated glycoprotein or glycopeptide comprises a phosphorylated oligosaccharide comprising only glycosidic linkages.
  • the glycosidic linkages are ⁇ 1-4 glycosidic linkages.
  • oligosaccharide are naturally linked to form a phosphorylated glycoprotein or glycopeptide.
  • the natural linkages are ⁇ 1-4 glycosidic linkages.
  • the chemical bond between the at least one GlcNAc residue on the acceptor protein or peptide and the phosphorylated oligosaccharide is a ⁇ 1-4 glycosidic linkage as found in nature.
  • the acceptor protein or peptide is a therapeutic protein or peptide, a modified therapeutic protein or peptide, or a functional fragment, variant, analogue or derivative thereof.
  • the acceptor protein or peptide is an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative thereof.
  • the acceptor protein or peptide is a fusion protein or peptide comprising at least a functional portion, fragment, variant, analogue or derivative of an enzyme or modified enzyme.
  • the enzyme, or modified enzyme is a lysosomal enzyme or modified lysosomal enzyme or functional fragment, variant, analogue or derivative thereof.
  • the fusion protein or peptide comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme or modified lysosomal enzyme.
  • the lysosomal enzyme is selected from the group consisting of an a- sialidase, cathepsin A, a-mannosidase, ⁇ -mannosidase, glycosylasparaginase, a- fucosidase, a-N-acetylglucosaminidase, ⁇ -galactosidase, ⁇ -hexosaminidase a -subunit, ⁇ -hexosaminidase ⁇ -subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase B, saposin B, formyl-glycin generating enzyme, ⁇ -galactosylceramidase, a -galactosidase A, iduonate sulfatase, a -iduronidase, heparan N-sulfatas
  • the lysosomal enzyme or modified lysosomal enzyme is an acid a-glucosidase or functional fragment, variant, analogue or derivative thereof.
  • the acid a-glucosidase is alglucosidase-a-rch.
  • the lysosomal enzyme or modified lysosomal enzyme is a - galactosidase A or a modified form thereof including agalsidase alpha and/or agalsidase beta.
  • a method of the present invention optionally comprises an initial step of preparing an acceptor protein or peptide comprising at least one GlcNAc residue, the step comprising contacting a protein or peptide comprising at least one GlcNAc-GlcNAc bond with an enzyme that hydrolyzes at least a GlcNAc-GlcNAc bond between a first GlcNAc residue immediately adjacent the protein or peptide, and a second GlcNAc residue immediately adjacent the first GlcNAc residue, to form the acceptor protein or peptide comprising a single GlcNAc residue.
  • An acceptor protein or peptide useful in the invention may be prepared using any protein or peptide that comprises at least one amino acid residue comprising at least one N- acetylglucosamine (GlcNAc) residue as a side group.
  • the protein or peptide comprises one GlcNAc residue as a side group.
  • the acceptor protein or peptide is contacted with at least one phosphorylated donor oligosaccharide that is activated with an anomeric leaving group, and an endoglycosidase enzyme under reaction conditions that allow the enzyme to catalyze an N-glycosylation reaction between the at least one GlcNAc residue on the acceptor protein or peptide, and the at least one phosphorylated donor oligosaccharide.
  • the phosphorylated oligosaccharide is a synthetic phosphorylated oligosaccharide.
  • the phosphorylated oligosaccharide is a synthetic phosphorylated oligosaccharide oxazoline.
  • the acceptor protein or peptide can be produced as a glycosylated protein or peptide by standard methods of protein expression using an appropriate cell based system as known in the art, e.g., yeast or human cell culture (Sambrook et al.
  • glycosylated protein or peptide produced by standard protein expression can then be prepared for use in the present invention by
  • the acceptor protein or peptide bearing at least one GlcNAc residue for use in a method of the invention maybe produced synthetically as known in the art. Chemical synthetic production of protein and peptides bearing GlcNAc residues on particular amino acid residues can also be carried out by a person of skill in the art (Ueda et al. , Bioorg. Med. Chem. Lett. 2010, 20 4631-4634; Huang et al , Org. Biomol. Chem. 2010, 8, 5224-5233).
  • the present invention also relates to a phosphorylated glycoprotein or glycopeptide made according to a method of the invention.
  • the present invention also relates to a phosphorylated glycoprotein or glycopeptide obtainable by a method of the invention.
  • the present invention also relates to a phosphorylated glycoprotein or glycopeptide comprising a synthetic phosphorylated oligosaccharide linked to an acceptor protein or peptide by a natural linkage between at least one GlcNAc residue on the protein or peptide and the phosphorylated oligosaccharide.
  • the natural linkage is a ⁇ 1-4 glycosidic linkage.
  • the synthetic phosphorylated donor oligosaccharide is a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide. In one embodiment the synthetic phosphorylated donor oligosaccharide is a tetra- or hexa-saccharide.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one synthetic phosphorylated oligosaccharide. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises at least two synthetic phosphorylated
  • oligosaccharides at least three synthetic phosphorylated oligosaccharides, at least four synthetic phosphorylated oligosaccharides, at least five synthetic phosphorylated oligosaccharides, at least six synthetic phosphorylated oligosaccharides, or at least seven or more synthetic phosphorylated oligosaccharides.
  • the phosphorylated glycoprotein or glycopeptide comprises at least one synthetic phosphorylated oligosaccharide that comprises at least one phosphorylated mannose residue, preferably at least one mannose-6-phosphate (M6P) residue, preferably at least two M6P residues, at least three M6P residues, at least four M6P residues, at least five M6P residues, at least six M6P residues, at least seven M6P residues, at least eight M6P residues, at least nine M6P residues, or at least ten or more M6P residues.
  • M6P mannose-6-phosphate
  • the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.
  • M6P residue is a terminal M6P residue.
  • the acceptor protein or peptide may be any protein or peptide of interest including a therapeutic protein or peptide, a modified therapeutic protein or peptide, or a functional fragment, variant, analogue or derivative thereof as described herein.
  • the protein or peptide is an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative thereof as described herein.
  • the present invention also relates to a composition comprising a phosphorylated glycoprotein or glycopeptide made according a method of the invention and a suitable carrier, diluent or excipient.
  • the present invention also relates to a composition
  • a composition comprising a phosphorylated glycoprotein or glycopeptide obtainable by a method of the invention and a suitable carrier, diluent or excipient.
  • composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises a therapeutically effective amount of the phosphorylated glycoprotein or glycopeptide according to the invention.
  • a phosphorylated glycoprotein or glycopeptide according to the invention may be formulated, dosed and/or used for a variety of purposes that are suitable for the therapeutic purpose associated with, or know for, the protein or peptide portion of the phosphorylated glycoprotein or glycopeptide of the invention, and that it is within the skill in the art to determine such a suitable purpose and a corresponding suitable formulation, dosage and/or use for the phosphorylated glycoprotein or glycopeptide.
  • a phosphorylated glycoprotein or glycopeptide according to the invention can be formulated for therapeutic or prophylactic treatments in various ways as known and disclosed in the art.
  • the phosphorylated glycoprotein or glycopeptide can be formulated for therapeutic or prophylactic use as described in W02007/104789, the entire contents of which are specifically incorporated herein by reference.
  • the phosphorylated glycoprotein or glycopeptide can be formulated for administration in a pharmaceutical composition
  • a pharmaceutical composition comprising, but are not limited to, pharmaceutically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface agents, neutral or cationic lipids, lipid complexes, liposomes, carrier compounds and other pharmaceutically acceptable carriers and the like in addition to the agent.
  • a pharmaceutically acceptable carrier may be liquid or solid and is selected as known in the art, in view of a planned manner of administration.
  • a pharmaceutically acceptable carrier provides for the desired bulk, consistency, at least, of a pharmaceutical composition that is to be used or delivered in a particular context.
  • a pharmaceutically acceptable carrier typically includes binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl
  • methylcellulose and the like, fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, and sodium acetate); disintegrates (e.g., starch, sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate), but not limited thereto.
  • lubricants e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, and sodium acetate
  • disintegrates e.g., starch, sodium starch glyco
  • a phosphorylated glycoprotein or glycopeptide according to the invention can be formulated in pharmaceutical compositions that contain additional functional or therapeutic components or delivery reagent.
  • additional functional or therapeutic components or delivery reagent can be considered adjunct components as may be conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • Such components include compatible pharmaceutically-active materials, or other materials useful in physically formulating various dosage forms of a pharmaceutical composition may also be included, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • the invention relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use as a medicament.
  • the invention in another aspect relates to a pharmaceutical composition of the invention for use as a medicament.
  • the present invention also relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use in the treating a disease or condition.
  • the present invention also relates to a composition, preferably a pharmaceutical composition according to the invention, for use in treating a disease or condition.
  • a composition preferably a pharmaceutical composition according to the invention
  • the present invention also relates to the use of a phosphorylated glycoprotein or glycopeptide according to the invention in the manufacture of a medicament for treating a disease or condition.
  • the present invention also relates to the use of a composition of the invention in the manufacture of a medicament for treating a disease or condition.
  • the disease or condition is a lysosomal storage disease (LSD) or disorder related thereto.
  • the LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, ⁇ -mannosidosis,
  • the disease or condition is Fabry disease.
  • the present invention also relates to a method for treating a lysosomal storage disease comprising administering a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide according to the invention to a subject in need thereof.
  • the present invention also relates to a method for treating a lysosomal storage disease comprising administering a pharmaceutical composition according to the invention to a subject in need thereof.
  • the composition comprises a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide according to the invention.
  • a phosphorylated glycoprotein or glycopeptide of the present invention may be used in therapy according to conventional means as known and used in the art for treating lysosomal storage diseases and other related diseases and/or conditions.
  • phosphorylated glycoproteins or glycopeptides according to the invention, or pharmaceutical compositions thereof may be administered in a number of ways as known in the art to achieve a therapeutic effect.
  • Mode of administration may be as known in the art including topical, oral or parenteral.
  • Parenteral administration includes direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. Administration may be local or systemic.
  • administration is parenteral by subcutaneous injection or is by oral delivery.
  • Phosphorylated glycoproteins, glycopeptides, and pharmaceutical compositions according to the invention may be formulated for parenteral administration in any appropriate solution, including sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Phosphorylated glycoproteins, glycopeptides, and pharmaceutical compositions according to the invention may be formulated for oral administration in powders or granules, aqueous or non-aqueous suspensions or solutions, capsules, pills, lozenges or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Phosphorylated glycoproteins, glycopeptides and pharmaceutical compositions according to the invention may be formulated for topical or direct administration in transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders.
  • systemic application comprises the subcutaneous injection of a composition comprising a phosphorylated glycoprotein or glycopeptide according to the invention for the treatment and/or prevention of lysosomal storage diseases and/or related disorders.
  • systemic application comprises oral delivery of composition (in any form) comprising a phosphorylated glycoprotein or glycopeptide according the invention for the treatment and/or prevention of lysosomal storage diseases and/or related disorders.
  • a formulation of a pharmaceutical composition according to the invention may comprise a phosphorylated glycoprotein or glycopeptide according to the invention at a
  • a particular and effective dosage regime will be dependent on severity of the lysosomal storage disease and/or related condition to be treated and on the responsiveness of the treated subject to the course of treatment.
  • An effective treatment for a lysosomal storage disease and/or related condition may last the entire lifetime of the effected individual.
  • An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations and dosage regimes.
  • phosphorylated glycoprotein or glycopeptide according to the invention may be administered in conjunction with the administration of an additional therapeutic agent.
  • the additional therapeutic agent can be any appropriate therapeutic agent used to treat or prevent any symptom, side effect or other consequence of treatment, either as a result of the use of a phosphorylated glycoprotein or glycopeptide according to the invention, or for any other reason related to the desired treatment.
  • a suitable additional therapeutic agent may be an agent that provides an adjunct therapy, for example, an agent that alleviates a symptom associated with the use of a therapeutic phosphorylated glycoprotein or glycopeptide in an enzyme replacement therapy regime.
  • Suitable adjunct therapeutics include, but are not limited to, antihistamines,
  • corticosteroids and epinephrine as known in the art to alleviate symptoms associated with enzyme replacement therapies.
  • the use of such therapies is typically age-dependent, and may vary by physician choice, experience, or availability. All suitable combinations of the compounds according to the invention with one or more of the above-mentioned compounds and optionally one or more further pharmacologically active substances are contemplated herein.
  • the phosphorylated glycoprotein or glycopeptide or pharmaceutical composition according to the invention is administered separately, simultaneously or sequentially, with an adjunct therapeutic agent selected from the group consisting of acetaminophen, ibuprophen, chloropheniramine hydroxyzine, chloropheniramine (IV), hydrocortisone, epinephrine, albuterol and ranitidine.
  • an adjunct therapeutic agent selected from the group consisting of acetaminophen, ibuprophen, chloropheniramine hydroxyzine, chloropheniramine (IV), hydrocortisone, epinephrine, albuterol and ranitidine.
  • a phosphorylated glycoprotein or glycopeptide according to the invention is formulated with an additional therapeutic agent
  • the dosing of the phosphorylated glycoprotein or glycopeptide and the additional therapeutic agent can be separate, simultaneous or sequential, as is appropriate.
  • Repetition rates for dosing can be based on measured residence times and concentrations of a given agent in the cells, fluids or tissues of the subject that is being or that is to be treated. Maintenance therapy may be desirable in successfully treated patient in order to prevent recurrence. Determination of appropriate dosing of a phosphorylated glycoprotein or glycopeptide of the invention is believed to be within the skill of those in the art.
  • dosing may comprise administration of from about 0.01 mg/kg subject to about 50 mg/kg subject of the phosphorylated glycoprotein or glycopeptide from once to several times per day as required to achieve a tolerable maintenance dose.
  • the invention relates to the use of a phosphorylated glycoprotein or glycopeptide according to the invention in the manufacture of a medicament for the treatment of a disease or condition, particularly for the treatment of lysosomal storage diseases and/or conditions related thereto.
  • the present invention also relates to a method for identifying a cell that expresses a M6P receptor, the method comprising a) contacting the cell with a phosphorylated glycoprotein or glycopeptide according to the invention, wherein the phosphorylated glycoprotein or glycopeptide further comprises a detectable label, and b) detecting the presence of the phosphorylated glycoprotein or glycopeptide bound to the cell.
  • a phosphorylated glycoprotein or glycopeptide further comprises a detectable label
  • b) detecting the presence of the phosphorylated glycoprotein or glycopeptide bound to the cell According to the above method, presence of the phosphorylated glycoprotein or glycopeptide bound to the cell demonstrates for the tested cell type, the presence of cell surface receptors that recognize M6P and is useful for identifying potential therapeutic targets.
  • OH-6 of the known tetrol 1 was selectively protected by reaction with tri-wo-propylsilyl (TIPS) chloride to give triol 2, which was then benzylated to give thioglycoside 3, which was to serve as the donor for introduction of both the 3- and 6-branches of the core N- glycan oligosaccharide.
  • TIPS tri-wo-propylsilyl
  • thioglycoside 3 which was to serve as the donor for introduction of both the 3- and 6-branches of the core N- glycan oligosaccharide.
  • Glycosylation of the known acceptor disaccharide 4 with 3 produced trisaccharide 5.
  • Regioselective reductive cleavage of the 4,6-benzylidene gave the primary alcohol 6 and was followed by a second glycosylation with donor 3 to yield tetrasaccharide 7 (Scheme 1).
  • Tetrahydrofuran was distilled from sodium/benzophenone ketyl under an argon atmosphere prior to use.
  • N, N-Dimethylformamide, pyridine and methanol were purchased from Aldrich in sealed bottles. All other solvents were used as supplied (analytical or HPLC grade) without purification.
  • Petroleum ether refers to the fraction of light petroleum ether boiling in the range 40-60°C. Reagents were used as supplied without further purification unless otherwise stated: phosphorus trichloride, pyridine and N, N-di-wo-propylamine were purified by distillation prior to phosphine syntheses.
  • Phosphorus nuclear magnetic resonance ( ⁇ ) spectra were recorded on Bruker AV400 (400MHz) and Bruker DRX500 (500MHz) spectrometers. Low resolution mass spectra were recorded on a Micromass Platform 1 spectrometer or a Micromass LCT Premier spectrometer using atmospheric pressure electrospray ionisation in either positive or negative polarity (ES + and/or ES " ).
  • reaction mixture was diluted with ethyl acetate (50 mL), washed with ammonium chloride (2 x 25 mL of a saturated aqueous solution), dried (Na 2 S0 4 ), filtered, and concentrated in vacuo. Traces of DMF were removed by repetitive co-evaporation with toluene. The residue was purified by flash column chromatography (ethyl acetate :petroleum
  • Triol 2 (3.0 g, 7.88 mmol) was dissolved in THF (30 mL) and sodium hydride (1.89 g, 39.4 mmol of a 50% dispersion in mineral oil) was added in portions. The mixture was stirred under an atmosphere of argon for 10 min. and then benzyl bromide (5.66 mL, 47.3 mmol) was added in portions. The reaction mixture was heated to 60°C and stirred for a further 20 h, after which time t.l.c. (petroleum ethenethyl acetate, 9: 1) indicated the formation of a major product (R f 0.6) and the consumption of starting material (R f 0.0).
  • Phthalimide 7 (0.120 g, 0.0580 mmol) was dissolved in methanol (6 mL) and ethylene diamine (3 mL) and the solution heated to reflux under an atmosphere of argon. After 16 h, t.l.c. (petroleum ethenethyl acetate, 2: 1) indicated formation of a single product (R f 0.25) and complete consumption of starting material (R f 0.40). The reaction mixture was concentrated in vacuo and co-distilled five times with toluene. The residue was dissolved in pyridine (3 mL), cooled to 0°C and acetic anhydride (1 mL) added. The reaction mixture was stirred at rt under an atmosphere of argon.
  • Silyl ether 8 (140 mg, 0.07 mmol) was dissolved in anhydrous DCM (5 mL) under an atmosphere of nitrogen in a flame-dried flask. The solution was cooled to 0°C and boron trifluoride diethyl etherate (43 ⁇ , 0.35 mmol) was added dropwise. The reaction was stirred at 0°C for 1 h after which time t.l.c. (ethyl acetate) indicated complete consump- tion of starting material (R f 0.55) and formation of a single product (R f 0.2).
  • Dibenzyl ⁇ , ⁇ -diisopropyl phosphoramidate (0.053 niL, 0.16 mmol) and lH-tetrazole (0.48 mL of a 0.45 M solution in acetonitrile) were added to a flame-dried flask and dissolved in DCM (0.45 mL). The solution was stirred at rt 10 min under nitrogen atmosphere. Diol 9 (0.04 mg, 0.02 mmol) in DCM (0.45 mL) was added, and the reaction stirred for 18 h. After this time t.l.c.
  • reaction was quenched by addition of sodium bisulfite (5 mL of a 10% w/v aqueous solution), stirred for 10 min, and extracted with DCM (10 mL). The organic extracts were washed with sodium hydrogen carbonate (5 mL of a saturated aqueous solution) and brine (5 mL), dried (Na 2 S0 4 ), filtered and concentrated in vacuo.
  • Glycoside 10 (15 mg, 0.01 mmol) was suspended in a mixture of acetonitrile (0.8 mL) and water (0.21 mL). Ceric ammonium nitrate (27 mg, 0.05 mmol) was added, and the reaction mixture stirred at rt. After 1 h, t.l.c. (ethyl acetate) indicated complete
  • reaction mixture was diluted with DCM (10 mL) and washed with sodium hydrogen carbonate (2 x 5 mL of a saturated aqueous solution), sodium thiosulfate (2 x 5 mL of a 5% w/v aqueous solution), EDTA (2 x 5 mL of a 0.1 M aqueous solution) and water (5 mL).
  • Hemiacetal 12 (2 mg, 0.0023 mmol) and triethylamine (3 ⁇ , 0.021 mmol) were dissolved in D 2 0 (18.4 ⁇ ) and the resulting solution was cooled to 0 °C.
  • DMC (12 mg, 0.007 mmol) was added to the solution and the mixture was stirred for 15 min at the same temperature.
  • Gel filtration of the residue on a Sephadex G-10 column eluted with 0.01% NH 3 afforded oxazoline 13 (1.8 mg, 95%) as a white foam.
  • Oxazoline 13 was investigated as a substrate for a variety of the family GH85 ENGase enzymes, using the glycosyl amino acid 14 as acceptor (Scheme 2, Table 1).
  • WT Endo A (Takegawa et. a ⁇ ., Appl. Environ. Microbiol. , 1989, 55, 3107-31 12) was able to catalyse transfer of the oxazoline to the acceptor and the product 15 was produced in 73% yield. A full time course study of this particular reaction was undertaken, which revealed that although the product 15 was a hydrolytic substrate for WT Endo A, this occurred only slowly (Fig 2).
  • Endo M (Yamamoto et. al., Biochem. Biophys. Res. Commun. , 1994, 203, 244-252) was unable to catalyse the synthesis of 16, although both Endo M and the Endo M mutant, N175Q, were able to catalyze the synthesis of 7.9 (example 6). This result was unexpected as Endo M has a broader hydrolytic capability than Endo A; for example it is able to hydrolyse complex biantennary N-glycans as well as high mannose structures.
  • EXAMPLE 3 the synthesis method of the invention was carried out to produce a phosphorylated glycoprotein comprising the enzyme RNaseB.
  • RNaseB is commercially provided as a mixture of high mannose glycoforms and may be trimmed back to a single GlcNAc residue at the sole glycosylation site by treatment with Endo H, to give dRNase B 16.
  • Treatment of 16 with 3 equivalents of oxazoline 10 and WT Endo A then allowed the production of the phosphorylated glycoprotein (M6P) 2 RNase 17 (Scheme 3) as demonstrated by HRMS and SDS PAGE (Fig. 2).
  • Increasing the number of equivalents of oxazoline used from 3 to 10 allowed the production of (M6P) 2 RNase in considerably higher yield. Glycosylation of dRNase B.
  • liver carcinoma cells (HepG2 cells) were cultured on chamber slides in the presence or absence of 1 ⁇ (M6P) 2 RNase for 1 -2 hours under standard cell culture conditions. HepG2 cells were also incubated in parallel with 1 ⁇ deglycosylated RNase B to control for M6P-independent RNase binding to HepG2 cells. After incubation, cells were fixed (4% paraformaldehyde in lx phosphate buffered saline, PBS) and permeabilized (80% methanol in lx PBS). Cells were blocked with 3% non-fat dried milk/1% BSA in lx PBS, then two primary antibodies were applied to label CI- MPR (mouse anti-CI-MPR) and RNase (rabbit anti-RNase A).
  • Labeled cells were visualized using fluorophore-conjugated secondary antibodies targeted to the primary CI-MPR and RNase antibodies. Cells were stained with a nuclear stain and mounted with cover slips. Images were captured on a LECIA TCS SP5 confocal microscope using a 63x objective. Co-localization of CI-MPR and RNase signal was quantified using LECIA Application Suite Advanced Fluorescence Software (LECIA) (not shown). Appropriate controls were included in the experiment including: untreated (no RNase) and unstained cells. Assay controls were carried out in the absence of primary and secondary antibodies to control for background fluorescence.
  • LECIA LECIA Application Suite Advanced Fluorescence Software
  • the co-localization of the CI-MPR and RNase signals on HepG2 cells demonstrates that a phosphorylated glycoprotein or glycopeptide according to the invention can be specifically targeted to cells expressing cation independent mannose-6-phosphase receptors, leading to the subsequent internalization in the cell of the phosphorylated glycoprotein or glycopeptide.
  • HepG2 Liver carcinoma cells
  • 1 ⁇ (M6P) 2 RNase for 2 hours under standard cell culture conditions (37°C, 5% C0 2 , minimal essential media, 1.5% BSA, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 250 ng/ml amphotericin).
  • HepG2's were also incubated in parallel with 1 ⁇ deglycosylated RNase B to control for MPR-independent RNase binding. After incubation, cells were fixed (4% paraformaldehyde in lx phosphate buffered saline, PBS) and permeabilized (80% methanol in lx PBS).
  • CI-MPR mimouse anti-cation independent mannose-6-phosphate receptor
  • RNase rabbit anti- RNase A
  • Cells were then incubated with fluorophore-conjugated secondary antibodies which allow for visualization of CI-MPR and RNase (goat anti-mouse IgG-Alexa Fluor 488 and goat anti-rabbit IgG-Alexa Fluor 568 respectively) through their interaction with previously applied primary antibodies.
  • Cells were stained with a nuclear stain (4',6- diamidino-2-phenylindole, DAPI) and mounted with coverslips.
  • HepG2 cells were lysed in RIPA buffer, sonicated, centrifuged and the supernatant incubated overnight at 4°C with anti-CI-MPR conjugated protein A resin in order to capture MPR from the cell lysate.
  • the resin was then extensively washed and incubated with either (M6P) 2 RNase or deglycosylated RNase B overnight at 4°C.
  • the resin was extensively washed and eluted with reducing buffer. Eluted samples were subjected to SDS-PAGE followed by Western blotting for RNase using an anti-rabbit RNase antibody. A chromogenic development system was used to detect signal (Figure 6).
  • Disaccharide 6.8 was following the procedure described in: Yunpeng Liu, Yan Mei Chan, Jianhui Wu, Chen Chen, Alan Benesi, Jing Hu,Yanming Wang, and Gong Chen
  • Triethylamine (0.1 mL) was added and the reaction mixture stirred for 10 min before it was filtered through Celite ® .
  • the filtrate was diluted with DCM (10 mL) and washed with sat. aqueous NaHC0 3 (10 mL), brine, dried over anhydrous Na 2 S0 4 , filtered and concentrated in vacuo.
  • the residue was purified by flash column chromatography (petrol: ethyl acetate, 2: 1) to afford compound 7.0 (0.31 g, 65%) as a white foam.
  • PhCH 2 PhCH 2 ), 5.03(1H, s, H-lb), 5.10 (1H, s, H-ld'), 5.21 (1H, s, H-ld), 5.42-5.45( 1H, m, H- 2b), 5.50 (1H, d, Ji >2 8.2 Hz, H-la), 6.69-6.72 (2H, m, 2 x Ar-H), 6.79-6.83 (2H, m, 2 x Ar-H), 6.88-6.95 (3H, m, 3 x Ar-H), 7.02-7.04 (2H, m, 2 x Ar-H), 7.33-7.40 (70H, m, 70 x Ar-H), 7.68-7.80 (4H, m, 4 x Ar-H); 5 C (100 MHz, CDC1 3 ) 12.0 (d, CH(CH 3 ) 2 ), 18.0 (q, CH(CH 3 ) 2 ), 21.1 (s, CH 3 ), 55.6 (q, OCH 3
  • reaction mixture was diluted with DCM (10 mL), washed with sodium hydrogen carbonate (10 mL of a saturated aqueous solution), dried (Na 2 S0 4 ), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography
  • reaction mixture was cooled to -78 °C and m-chloroperbenzoic acid (0.01 g, 0.02 mmol) was added.
  • the mixture was stirred for 2 h and then warmed to rt, after which time t.l.c.
  • the reaction mixture was diluted with DCM (10 mL) and washed with sodium hydrogen carbonate (2 x 5 mL of a saturated aqueous solution), sodium thiosulfate (2 x 5 mL of a 5% w/v aqueous solution), EDTA (2 x 5 mL of a 0.1 M aqueous solution) and water (5 mL).
  • the organic extracts were dried (Na 2 S0 4 ), filtered, concentrated in vacuo, and the residue purified by flash column chromatography (MeOH:DCM, 1 :50) to afford hemiacetal 7.6 (11.6 mg, 80%) as a pale yellow foam.
  • Hemiacetal 7.7 (4 mg, 0.0034 mmol) and triethylamine (4.3 ⁇ , 0.03 mmol) were dissolved in D 2 0 (37 ⁇ ) and the resulting solution was cooled to 0 °C.
  • DMC (1.7 mg, 0.01 mmol) was added to the solution and the mixture was stirred for 30 min at 0 °C.
  • Gel filtration of the residue on a Sephadex G-10 column, eluting with 0.01% NH 3 afforded oxazoline 7.8 (1.8 mg, 95%) as a white foam.
  • the phosphorylated glycoproteins and glycopeptides have industrial application for the production and therapeutic use of phosphorylated glycoproteins and glycopeptides.

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Abstract

D'une manière générale, la présente invention concerne des glycoprotéines ou des glycopeptides phosphorylés, des procédés de synthèse chimio-enzymatique de glycoprotéines ou de glycopeptides phosphorylés, des compositions comportant de tels glycoprotéines ou glycopeptides phosphorylés, des procédés thérapeutiques mettant en œuvre de tels glycoprotéines ou glycopeptides phosphorylés, et l'utilisation de tels glycoprotéines ou glycopeptides phosphorylés dans la fabrication de médicaments.
PCT/NZ2014/050014 2013-11-28 2014-11-28 Glycoproteines WO2015080603A1 (fr)

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RU2741347C2 (ru) * 2015-11-13 2021-01-25 Тарлан МАММЕДОВ Получение in vivo n-дегликозилированных рекомбинантных белков посредством совместной экспрессии с endo h
US11041163B2 (en) 2015-11-13 2021-06-22 Tarlan Mammedov Production of in vivo N-deglycosylated recombinant proteins by co-expression with endo H
WO2017154938A1 (fr) * 2016-03-09 2017-09-14 株式会社糖鎖工学研究所 Procédé de production de sucre contenant un groupe sulfate et/ou un groupe phosphate
JPWO2017154938A1 (ja) * 2016-03-09 2019-01-10 株式会社糖鎖工学研究所 硫酸基および／またはリン酸基を有する糖の製造方法
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JP7401882B2 (ja) 2016-03-09 2023-12-20 株式会社糖鎖工学研究所 硫酸基を有する糖の製造方法
CN114574495A (zh) * 2020-12-01 2022-06-03 上海交通大学医学院附属仁济医院 核苷类衍生物改性的核酸适体r50
CN114574495B (zh) * 2020-12-01 2024-04-09 上海交通大学医学院附属仁济医院 核苷类衍生物改性的核酸适体r50
WO2022191313A1 (fr) * 2021-03-12 2022-09-15 第一三共株式会社 Glycane et procédé de production d'un médicament contenant un glycane
WO2022226425A1 (fr) * 2021-04-23 2022-10-27 WANG Lai Xi Remodelage glycane spécifique d'un site d'enzymes lysosomales et leurs applications
WO2023150388A1 (fr) * 2022-02-07 2023-08-10 M6P Therapeutics, Inc. Compositions comprenant de la glucocérébrocidase et leurs procédés d'utilisation
WO2024053574A1 (fr) * 2022-09-09 2024-03-14 第一三共株式会社 Nouvel oligosaccharide, intermédiaire de production pour un nouvel oligosaccharide, procédé de production d'un nouvel oligosaccharide et procédé de production d'un intermédiaire de production pour un nouvel oligosaccharide

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