WO2015080603A1

WO2015080603A1 - Glycoproteins

Info

Publication number: WO2015080603A1
Application number: PCT/NZ2014/050014
Authority: WO
Inventors: Antony John Fairbanks
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-11-28
Filing date: 2014-11-28
Publication date: 2015-06-04
Anticipated expiration: 2016-05-28

Abstract

The present invention relates generally to phosphorylated glycoproteins and glycopeptides, methods of chemoenzymatic synthesis of phosphorylated glycoproteins or glycopeptides, compositions comprising such phosphorylated glycoproteins or glycopeptides, therap eutic methods utilizing such phosphorylated glycoproteins or glycopeptides, and to the use of such phosphorylated glycoproteins or glycopeptides in the manufacture of medicaments.

Description

GLYCOPROTEINS

TECHNICAL FIELD

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.

BACKGROUND OF THE INVENTION

Lysosomal storage diseases (LSDs) 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 Enzyme replacement therapy (ERT) 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). The presence of terminal 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). 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.

Accordingly, there is a need in the art to develop new therapeutic compounds for ERT having reduced immunogenicity. The present invention goes at least some way towards addressing the above need in the art, and/or at least provides the public with a useful choice. In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

SUMMARY OF THE INVENTION

In one aspect 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.

In one embodiment the phosphorylated donor oligosaccharide is a synthetic

phosphorylated oligosaccharide.

In one embodiment the method further comprises isolating and/or purifying the phosphorylated glycoprotein or glycopeptide.

In one embodiment 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. Preferably 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). Preferably the ENGase is Endo A. In one embodiment the endoglycosidase is a modified or mutant Endo A, preferably E173H and N171Q. In one embodiment the endoglycosidase is Endo M or a modified or mutant Endo M. Preferably the mutant Endo M is N175Q.

In one embodiment the phosphorylated donor oligosaccharide is a phosphorylated donor oligosaccharide comprising monosaccharide residues linked by glycosidic linkages, and an anomeric leaving group. Preferably the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, and the leaving group is an oxazoline. In one embodiment 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. In one embodiment the phosphorylated donor oligosaccharide is a tetra- or hexasaccharide. In one embodiment the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, preferably a synthetic phosphorylated donor oligosaccharide oxazoline.

In one embodiment 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. Preferably 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. In one embodiment, the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues. Preferably, at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises a phosphorylated oligosaccharide comprising only glycosidic linkages. Preferably the glycosidic linkages are β 1-4 glycosidic linkages.

In one embodiment the acceptor protein or peptide and the phosphorylated

oligosaccharide are naturally linked to form a phosphorylated glycoprotein or glycopeptide. Preferably the natural linkages are β 1-4 glycosidic linkages.

In one embodiment the chemical bond between the at least one GlcNAc residue on the acceptor protein or peptide and the phosphorylated oligosaccharide is a β 1-4 glycosidic linkage as found in nature.

In one embodiment, 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. In one embodiment the acceptor protein or peptide is an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative of an enzyme. In one embodiment 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.

In one embodiment the enzyme, or modified enzyme is a lysosomal enzyme or a functional fragment, variant, analogue or derivative thereof. In one embodiment the fusion protein or peptide comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme.

Preferably the lysosomal enzyme is selected from the group consisting of an a-sialidase, cathepsin A, a-mannosidase, β-mannosidase, glycosylasparaginase, a-fucosidase, a-N- acetylglucosaminidase, β -galactosidase, β -hexosaminidase a -subunit, β - hexosaminidase β -subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase B, saposin B, formyl-glycin generating enzyme, β -galactosylceramidase, a -galactosidase A, iduonate sulfatase, a -iduronidase, heparan N-sulfatase, acetyl-CoA transferase, n-acetyl glucosaminidase, β -glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, ayaluronidase, a -glucosidase, a-sphingomyelinase, acid ceramidase, acid lipase, cathepsin , tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine,

phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NCI, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit. Preferably the lysosomal enzyme or modified lysosomal enzyme is an acid a-glucosidase or functional fragment, variant, analogue or derivative thereof. Preferably the acid a-glucosidase is alglucosidase-a-rch. In one embodiment, the lysosomal enzyme or modified lysosomal enzyme is a - galactosidase A or a modified form thereof including agalsidase alpha and/or agalsidase beta. In one embodiment, 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.

In another aspect 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.

In another aspect the invention 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. Preferably the natural linkage is a β 1-4 glycosidic linkage.

In one embodiment 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. In one embodiment 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. Preferably 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. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues. Preferably, at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.

In one embodiment, 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.

In another aspect 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. In another aspect 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. In one embodiment the composition is a pharmaceutical composition. In one

embodiment the pharmaceutical composition comprises a therapeutically effective amount of the phosphorylated glycoprotein or glycopeptide according to the invention.

In another aspect the invention relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use as a medicament.

In another aspect the invention relates to a pharmaceutical composition of the invention for use as a medicament.

In another aspect the invention relates to a phosphorylated glycoprotein or glycopeptide according to the invention for use in treating a disease or condition. In another aspect the invention relates to a pharmaceutical composition of the invention for use in treating a disease or condition.

In another aspect 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. In another aspect the invention relates to the use of a composition of the invention in the manufacture of a medicament for treating a disease or condition.

In one embodiment the disease or condition is a lysosomal storage disease (LSD) or disorder related thereto. In one embodiment the LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, β-mannosidosis,

aspartylglucosaminuria, fucosidosis, GM1 gangliosidosis, GM2-gangliosidosis (Tay- Sachs), GM2-gangliosidosis (Sandhoff), GM2-gangliosidosis, Gaucher disease, metachromatic leukodystrophy, multiple sulfatase deficiency, globoid cell leukodystrophy, Fabry disease, MPS II (Hunter), MPS 1 (Hurler, Scheie), MPS Ilia (Sanfilippo A), MPS IIIc (Sanfilippo C), MPS Illb (Sanfilippo B), MPS VII (Sly), MPS Hid (Sanfilippo D), MPS VI, MPS IVA (Morquio A), MPS IX, Pompe disease, Niemann Pick type A and B, Farber lipogranulomatosis, Wolman and cholesteryl ester storage disease,

pycnodystostosis, ceroide lipofuscinosis 2, ceroide lipofuscinosis 1, cystinosis, salla disease, mucolipidosis III (I-cell), mucolipidosis IV, Danon disease, Neimann Pick type C, ceroid lipofuscinosis, ceroid lipofuscinosis 6, ceroid lipofuscinosis, 8, Chediak-Higashi disease, Griscelli type 1, Griscelli type 2, Griscelli type 3, and Hermansky Pudliak 2 disease. In one embodiment the disease or condition is Fabry disease.

In another aspect the invention 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.

In another aspect the invention relates to a method for treating a lysosomal storage disease comprising administering a composition according to the invention to a subject in need thereof. In one embodiment the composition comprises a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide according to the invention.

In another aspect the invention 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.

Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

Certain statements that appear below are broader than what appears in the statements of the invention above. These statements are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which defines the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and with reference to the drawings in which:

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).

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)₂RNase with CI-MPR in HepG2 cells following 2 hours of incubation with 1 μΜ (M6P)₂RNase. 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.

Abbreviations: 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 μΜ 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). Abbreviations: DAPI, 4',6-diamidino-2-phenylindole ; CI-MPR, cation-independent mannose phosphate receptor; RNase, Ribonuclease.

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). Abbreviations: DAPI, 4',6-diamidino-2-phenylindole ; CI-MPR, cation- independent mannose phosphate receptor; RNase, Ribonuclease.

Figure 6 depicts the immunoprecipitation of (M6P)₂RNase from anti-CI-MPR coupled protein A. Abbreviations, IP CI-MPR, immunoprecipitation with anti-cation independent MPR antibody conjugated protein A, WB RNase, Western blot for RNase. Arrow indicates immunoprecipitated M6P- RNase with the anticipated molecular weight of 13.6kDa. Figure 7 depicts the glycosylation of de-glycosylated Fabrazyme with M6P- tetrasaccharide oxazoline donor.

DETAILED DESCRIPTION OF THE INVENTION Definitions Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in medicine, molecular biology and biochemistry can be found in: Dictionary of

Microbiology and Molecular Biology, Singleton et al, 2^nd edition, (1994); The

Encyclopedia of Molecular Biology, endrew et al. (Eds.), Blackwell Science Ltd.,

(1994); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (Ed.), VCH Publishers, Inc., (1995); The Dictionary of Cell & Molecular Biology, 4th Edition, Lackie. J (Ed.), Academic Press Inc (2007); and The Oxford

Dictionary of Biochemistry and Molecular Biology, 2^nd edition, Cammack et al. (Eds.), Oxford University Press Inc. (2006).

It is also believed that practice of the present invention can be performed using standard molecular biology and biochemistry protocols and procedures as known in the art, and as described, for example in Molecular Cloning: A Laboratory Manual, Maniatis et al. , Cold Spring Harbor Laboratory Press, (1982); Molecular Cloning: A Laboratory Manual (2 ed.), Sambrook et al , Cold Spring Harbor Laboratory Press, (1989); Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl (Eds.), Academic Press Inc., (1987); Protein Synthesis and Ribosome Structure: Translating the Genome, Nierhaus, K and Wilson D (eds.), Wiley-VCH Inc (2004); Synthetic Peptides: A User's Guide (Advances in Molecular Biology) 2ⁿ edition, Grant G. (Ed.), Oxford University Press (2002); Remington: The Science and Practice of Pharmacy 21 ^st edition, Beringer, P (Ed.), Lippincott Williams & Wilkins, (2005), pages 2393; and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.

The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

The term "phosphorylated donor oligosaccharide" as used herein 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. Preferably the suitable leaving group is an anomeric leaving group. For example, a phosphorylated donor

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. Preferably the anomeric leaving group is an oxazoline.

As used herein, a "suitable anomeric leaving group" refers to any anomeric leaving group that will serve as a donor in a transglycosylation reaction catalyzed by an

endohexosaminidase enzyme. Such leaving groups may include oxazoline, fluoride, para- nitrophenol, or even another carbohydrate. In one embodiment a phosphorylated donor oligosaccharide comprises at least one terminal monosaccharide residue that comprises at least one phosphate group. Preferably the at least one terminal monosaccharide residue is mannose. Preferably 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.

In one embodiment a phosphorylated donor oligosaccharide is an N-glycan

oligosaccharide selected from a group of N-linked oligosaccharides having a bi-, tri-, tetra-, penta-, hexa- or greater antennary structure. In one embodiment the N-glycan oligosaccharide has a bi-antennary structure. Preferably the phosphorylated donor oligosaccharide is a phosphorylated bi-antennary pentasaccharide. Preferably 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.

The term "donor oligosaccharide oxazoline" and variations thereof refers to a class of oligosaccharide compounds comprising an oxazoline functional group as known in the art. For example, 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.

The term "linked to" as used herein means chemically connected via at least one covalent bond. For example, following glycosylation with a bi-antennary oligosaccharide oxazoline as described herein, a GlcNAc residue on a protein or peptide of interest is linked to a GlcNAc residue on a synthetic phosphorylated donor oligosaccharide. The term "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. The term "modified" as used herein with reference to a protein or peptide (including therapeutic proteins, peptides and enzymes) 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.

The term "functional variant" as used herein with reference to a protein or peptide (including therapeutic proteins, peptides and enzymes) 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.

The term "functional fragment" as used herein with reference to a protein or peptide (including therapeutic proteins, peptides and enzymes) 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.

As used herein a "functional analogue" as used herein with reference to a protein or peptide (including therapeutic proteins, peptides and enzymes) 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.

The 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. Also included are 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.

The term "functional derivative" as used herein with reference to a protein or peptide (including therapeutic proteins, peptides and enzymes) 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 term "comprising" as used in this specification and claims means "consisting at least in part of. When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

Description

The present invention relates generally to the chemoenzymatic synthesis of

phosphorylated glycoproteins or glycopeptides comprising all natural linkages between the glycan and the protein. In some embodiments 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. Advantageously 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. In addition, 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. In some embodiments, 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 β 1-4 glycosidic linkages. In one embodiment, 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.

The use of glycoproteins and glycopeptides as systemic therapeutic agents is known, including the use of various glycoprotein conjugates comprising enzymes as in enzyme replacement therapy (ERT). However, enzymatic modification of a GlcNAc-peptide by an endohexosaminidase (ENGase) to add a phosphorylated oligosaccharide, creating a phosphorylated oligosaccharide-N-peptide having all naturally occurring linkages, has not previously been thought possible. Surprisingly, 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. In one embodiment the ENGase is a modified or mutant Endo A including E173H and N171Q.

Moreover, 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.

Accordingly, 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.

In particular, this invention allows for a method of synthesizing an ((M6P)n-bi-antennary octaoligosaccharide) - (enzyme) conjugate (where n = the number of mannose-6- phosphate residues having all natural chemical bonds), the conjugate being useful for the treatment of various lysosomal storage diseases. Preferably n = at least 6 M6P residues, preferably at least 5 M6P residues, preferably at least 4 M6P residues, preferably at least 3 M6P residues, preferably at least 2 M6P residues. In one embodiment, n = at least 2 M6P residues.

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.

In one embodiment the phosphorylated donor oligosaccharide is a synthetic

phosphorylated oligosaccharide. The term "synthetic" as used herein with reference to an oligosaccharide or other molecule takes its commonly held meaning in the art and refers to an oligosaccharide or other molecule that has been synthesized artificially, and that is not obtained from a natural source. In some embodiments the oligosaccharide or other molecule is also not obtainable from a natural source. In one embodiment 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.

In one embodiment 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. Preferably 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). Preferably the ENGase is Endo A. In one embodiment the endoglycosidase is a modified or mutant Endo A, preferably E173H and N171Q. In one embodiment the endoglycosidase is Endo M or a modified or mutant Endo M. Preferably 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.

According to the CAZy (http://www.cazy.org/fam/acc_GH.html) enzyme classification system all of the ENGases identified to date belong to either family 18 (GH18) or family 85 (GH85) of the superfamily of glycoside hydrolases. Several ENGases have shown useful synthetic glycosylation activity including Endo M from Mucor hiemalis, Endo A from Arthrobacter protophormiae, and Endo D from Streptococcus pneumoniae; all of which are members of the family GH85. Plasmids are available for both Endo A and Endo D which allow their expression, and also the production of mutant enzymes by site directed mutagenesis. Endo M, and its N175Q mutant, are commercially available enzymes marketed by Tokyo Chemical Industries Ltd (TCI).

Based on the known activity of Endo M to cleave negatively charged, sialic acid terminated, bi-antennary complex glycans, it was expected that 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. As seen in example 6, 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. Preferably the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide oxazoline.

In one embodiment 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. In one embodiment the phosphorylated donor oligosaccharide is a tetra- or hexa-saccharide. In one embodiment the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, preferably a synthetic phosphorylated donor oligosaccharide oxazoline.

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 identification of the number of potential glycosylation sites on a protein or peptide that can be comprised in a phosphorylated glycoprotein or glycopeptide according to, made by or useful in the invention is believed to be within the skill in the art. Preferably 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. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues. Preferably, at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises a phosphorylated oligosaccharide comprising only glycosidic linkages. Preferably the glycosidic linkages are β 1-4 glycosidic linkages.

In one embodiment the acceptor protein or peptide and the phosphorylated

In one embodiment the chemical bond between the at least one GlcNAc residue on the acceptor protein or peptide and the phosphorylated oligosaccharide is a β 1-4 glycosidic linkage as found in nature. In one embodiment, 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. In one embodiment the acceptor protein or peptide is an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative thereof. In one embodiment 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.

In one embodiment the enzyme, or modified enzyme is a lysosomal enzyme or modified lysosomal enzyme or functional fragment, variant, analogue or derivative thereof. In one embodiment the fusion protein or peptide comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme or modified lysosomal enzyme. Preferably the lysosomal enzyme is selected from the group consisting of an a- sialidase, cathepsin A, a-mannosidase, β-mannosidase, glycosylasparaginase, a- fucosidase, a-N-acetylglucosaminidase, β -galactosidase, β -hexosaminidase a -subunit, β -hexosaminidase β -subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase B, saposin B, formyl-glycin generating enzyme, β -galactosylceramidase, a -galactosidase A, iduonate sulfatase, a -iduronidase, heparan N-sulfatase, acetyl-CoA transferase, n-acetyl glucosaminidase, β -glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, ayaluronidase, a -glucosidase, a-sphingomyelinase, acid ceramidase, acid lipase, cathepsin , tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine,

phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NCI, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit. Preferably the lysosomal enzyme or modified lysosomal enzyme is an acid a-glucosidase or functional fragment, variant, analogue or derivative thereof. Preferably the acid a-glucosidase is alglucosidase-a-rch. In one embodiment, the lysosomal enzyme or modified lysosomal enzyme is a - galactosidase A or a modified form thereof including agalsidase alpha and/or agalsidase beta. In one embodiment, 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. Preferably, 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. In some embodiments the phosphorylated oligosaccharide is a synthetic phosphorylated oligosaccharide. In some embodiments the phosphorylated oligosaccharide is a synthetic phosphorylated oligosaccharide oxazoline. The resultant glycosylation yields a phosphorylated glycoprotein or glycopeptide according to the invention.

The production of the acceptor protein or peptide is believed to be within the skill in the art. For example, 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.

supra; Maniatis et al. supra). The glycosylated protein or peptide produced by standard protein expression can then be prepared for use in the present invention by

endohexosaminidase catalyzed hydrolysis, yielding an acceptor protein or peptide; i.e., a protein or peptide having at least one GlcNAc residue (Zou et al, J. Am. Chem. Soc. 2011, 133, 18975-18991 ; Schwarz et al. , Nat. Chem. Biol. 2010, 6, 264-266).

Alternatively, 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. Preferably the natural linkage is a β 1-4 glycosidic linkage.

In one embodiment 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.

In one embodiment 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. Preferably 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. In one embodiment the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues. Preferably, at least one mannose-6-phosphate (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. In some embodiments 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 comprising a phosphorylated glycoprotein or glycopeptide obtainable by a method of the invention and a suitable carrier, diluent or excipient.

In one embodiment the composition is a pharmaceutical composition. In one

embodiment the pharmaceutical composition comprises a therapeutically effective amount of the phosphorylated glycoprotein or glycopeptide according to the invention. The reader will appreciate that 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. For example, 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. For example, the phosphorylated glycoprotein or glycopeptide can be formulated for administration in 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. Such compositions and formulations can be used in accordance with the present invention. 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

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. 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. Such other components can be considered adjunct components as may be conventionally found in pharmaceutical compositions, at their art-established usage levels. Examples of 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.

In another aspect the invention 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. 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. In one embodiment the disease or condition is a lysosomal storage disease (LSD) or disorder related thereto. In one embodiment the LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, β-mannosidosis,

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. In one embodiment 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.

In the present invention, 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. Preferably 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.

Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

A person skilled in the art will be able to choose the appropriate mode of administration of a phosphorylated glycoprotein, glycopeptide or pharmaceutical composition according to the invention with reference to the literature and as described herein. By way of non- limiting example, a systemic application of MYOZYME® would be preferred for the treatment of Pompe disease following the recommendations of the manufacturer

(Genzyme, CA). In one embodiment, 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. In another embodiment, 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.

The formulation of pharmaceutical compositions comprising a phosphorylated glycoprotein or glycopeptide according to the invention, and their subsequent administration is believed to be within the skill of those in the art. For example, a formulation of a pharmaceutical composition according to the invention may comprise a phosphorylated glycoprotein or glycopeptide according to the invention at a

concentration of about 0.01 mg/mL to about 10 mg/mL, but is not limited thereto. 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.

In one embodiment, 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.

In one embodiment, 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.

Where a phosphorylated glycoprotein or glycopeptide according to the invention is formulated with an additional therapeutic agent, then 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. For example, 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 reader will also appreciate that 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. 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.

The invention will now be described by way of the following representative methods and examples which are provided to further illustrate the subject matter to which the invention relates. The use of any and all examples, or exemplary language (e.g., "such as" or "including") provided herein, is intended solely for the purposes of better describing the invention. The presence of examples and the use of exemplary language does not limit the scope of the invention as disclosed herein unless specifically otherwise indicated. No language used in the entirety of the disclosure of this application should be interpreted as indicating that any particular element or feature pertaining to the invention and as disclosed herein is essential to the practice of the invention, unless explicitly stated.

EXAMPLES

General Experimental Strategy

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. 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). Conversion of the JV-phthalimide to the JV-acetamide gave 8, and was followed by removal of the TIPS groups to produce diol 9. Phosphitylation of 9 with dibenzyl diisopropylphosphoramidate and immediate oxidation with meta- chloroperbenzoic acid (m-CBPA) gave the bis phosphate ester 10. Oxidative cleavage of the anomeric para-methoxyphenyl (PMP) protecting group then gave hemiacetal 11, which was globally de-protected by Birch reduction to yield free sugar 12 in very high yield. Finally, 12 was converted into the corresponding oxazoline 13 following the procedure of Noguchi (Noguchi, M. et. al., J Org. Chem. 2009, 74, 2210-2212) using 2- chloro-1,3 dimethylimidazolinium chloride (DMC) in water in very high yield.

Scheme 1. Synthesis of oxazoline (a) TIPSO, imidazole, tetrahydrofuran (THF), 0°C to rt, 24 h, 89%; (b) NaH, BnBr, THF, 60°C, 24 h, 78%; (c) 3, MeOTf, 3A mol. sieves, dichloromethane (DCM), tri-tert-butylpyrimidine (t-TBP), 0°C to rt, 18 h, 91%; (d) 5 Et₃SiH, PhBCl₂, 3A mol. sieves, DCM, -78°C, 50 min, 94%; (e) 3, MeOTf, 3A mol. sieves, DCM, t-TBP, 0°C to rt, 18 h, 79%; (f) (i) NH2CH2CH2NH2, MeOH, reflux, 16 h; (ii) Ac₂0, py, 24 h, 91% over 2 steps; (g) BF₃.OEt_2> DCM, 0°C, 1 h, 76%; (h) (BnO)₂PNz^'Pr₂, tetrazole, DCM, rt, 18 h, then add (m-CPBA), -78°C to rt, 2 h, 58% over two steps; (i) CAN, MeCN, H₂0, rt, 1 h, 78%; (j) Na, NH₃₍₁₎, -33°C, 1 h, 98%; (k) DMC,0 Et₃N, H₂0, rt, 95%.

General experimental protocols

All reactions involving moisture-sensitive reagents were performed under an atmosphere of argon or nitrogen via standard vacuum Schlenk-line techniques. All glassware for such reactions was flame-dried and cooled under an atmosphere of argon. Reactions conducted at -78°C were cooled by means of an acetone/dry ice bath; those conducted at -45°C were cooled by means of an acetonitrile/dry ice bath; those conducted at 0°C were cooled by means of an ice bath. Solvent was removed under reduced pressure using a rotary evaporator. Diethyl ether, toluene, dichloromethane and acetonitrile were dried by passing them through a column of activated basic alumina according to the Grubbs' procedure. 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. Thin Layer Chromatography (t.l.c.) was carried out on Merck ieselgel 6OF₂₅₄ pre-coated glass-backed plates. Visualisation of the plates was achieved using a UV lamp (λ_ΙΏ3Χ = 254 or 365 nm), and/or ammonium molybdate (5% in 2M H₂SO₄), and/or sulphuric acid (5% in EtOH) and/or iodine and/or vanillin and/or potassium permanganate. Flash column chromatography was carried out using Sorbsil C60 40/60 silica. Melting points were recorded on a ofler hot block and are uncorrected. Proton and carbon nuclear magnetic resonance (δπ, 5Q) spectra were recorded on Bruker DPX200 (200MHz), Bruker DPX250 (250MHz), Bruker DPX400 (400 MHz), Bruker AV400 (400MHz), Bruker AV500 (500MHz) and Bruker AV700 (700 MHz) spectrometers. All chemical shifts are quoted on the δ-scale in ppm using residual solvent as an internal standard. 1H and ¹³C spectra were assigned using COSY, DEPT, HSQC, HMBC, TOCSY and DPFGSE-TOCSY. Phosphorus nuclear magnetic resonance (δρ) 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^"). High resolution mass spectra were recorded by Mr Robin Procter, Dr Lingzhi Gong or Mr Colin Sparrow on ether a Walters 2790-Micromass LCT or a Bruker FT-ICR mass spectrometer using electrospray ionisation (ESI) or chemical ionisation (CI) techniques as stated. M/z values are reported in Daltons and are followed by their percentage abundance in parentheses. Optical rotations were measured on a Perkin-Elmer 241 polarimeter with a water-jacketed 1 cm cell with a path length of 1 dm, and are quoted in units of °.cm 2.g^" 1. Concentrations (c) are given in g / 100 cm ; solvent and temperature are recorded. Microanalyses were performed by the Inorganic Chemistry Laboratory Elemental Analysis service and obtained on an Elementar vario EL instrument. EXAMPLE 1

Ethyl 6-0-tri-/s0-propylsilyl-l-thio-a-D-mannopyranoside 2

Thioglycoside 1 (2.0 g, 8.90 mmol) and imidazole (1.52 g, 22.3 mmol) were dissolved in dimethylformamide (DMF) (20 mL). The mixture was cooled to 0°C and chloro-tri-wo- propylsilane (3.8 mL, 17.8 mmol) added. The reaction mixture was warmed to rt and stirred under an atmosphere of argon. After 24 h, t.l.c. (petroleum ethenethylacetate, 1 : 1) indicated formation of a major product (R_f 0.5) and consumption of starting material (R_f 0). The reaction mixture was diluted with ethyl acetate (50 mL), washed with ammonium chloride (2 x 25 mL of a saturated aqueous solution), dried (Na₂S0₄), 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

22 ether, 2: 1) to afford silyl ether 2 (3.0 g, 89%) as a white amorphous solid, [OC] D + 126 (c, 0.6 in CHC1₃); v_max ( Br disk) 3424 (br s, O-H); 5_H (500 MHz, CDC1₃) 1.07 (3H, s, CH(CH₃)₂), 1.08 (18H, s, CH(CH₃)₂), 1.29 (3H, t, J 7.4 Hz, SCH₂CH₃), 2.54-2.69 (2H, m, SCH₂CH₃), 2.68 (1H, d, J4.0 Hz, OH), 2.84 (1H, d, J 3.3 Hz, OH), 3.57 (1H, br s, OH), 3.82-3.88 (2H, m, H-3, H-4), 3.93-3.99 (2H, m, H-6, H-6'), 4.02-4.06 (2H, H-2, H- 5), 5.30 (1H, s, H-l); 5_C (125.8 MHz, CDCI₃) 1 1.9 (d, CH(CH₃)₂), 15.0 (q, CH₂CH₃),

18.0 (q, CH(CH₃)₂), 25.2 (t, CH₂CH₃), 66.1 (t, C-6), 70.1, 71.8, 72.3, 72.3 (4 x d, C-2, C- 3, C-4, C-5), 83.9 (d, C-l); m/z (ES⁺) 783 (M₂Na⁺, 100), 778 (MNH₄ ⁺, 47), 403 (MNa⁺, 18), 398 (MNH₄ ⁺, 27), 381 (MH⁺, 25%). HRMS (ES⁺) Calcd. For Ci₇H₃₆Na0₅SSi (MNa⁺) 403.1945. Found 403.1945. Found: C, 53.40; H, 9.69. C₁₇H₃₆0₅SSi requires C, 53.65; H, 9.53%.

Ethyl 2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-l-thio-a-D-mannopyranoside 3

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). Methanol (30 mL) was added and the solution stirred for 15 min. and then concentrated in vacuo. The residue was dissolved in ethyl acetate (50 mL), and washed with water (2 x 30 mL). The aqueous washings were re-extracted with DCM (2 x 30 mL). The organic extracts were combined, dried (Na₂S0₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ethenethyl acetate, 19: 1) to afford benzyl ether 3 (4.0 g, 78%) as a pale yellow oil, [a]_D ²² + 66 (c, 1.0 in CHC1₃); δ_Η (500 MHz, CDC1₃) 1.05 (3H, s, CH(CH₃)₂), 1.06 (18H, s, CH(CH₃)₂), 1.22 (3H, t, J 7.4 Hz, SCH₂CH₃), 2.51-2.64 (2H, m, SCH₂CH₃), 3.80 (1H, dd, Ji_;2 1.6 Hz, J_2>3 3.1 Hz, H-2), 3.86 (1H, dd, J_3>4 8.8 Hz, H-3), 3.92-3.99 (4H, m, H-4, H-5, H-6, H-6'), 4.58, 4.62 (2H, ABq, J 1 1.7 Hz, PhCH₂), 4.63, 4.92 (2H, ABq, J 10.9 Hz, PhCH₂), 4.67 (2H, br s,

PhCH₂), 5.34 (1H, d, H-1), 7.27-7.39 (15H, m, 15 x Ar-H); 5_C (125.8 MHz, CDC1₃) 12.1 (d, CH(CH₃)₂), 14.9 (q, CH₂CH₃), 18.1 (q, CH(CH₃)₂), 25.0 (t, CH₂CH₃), 63.2 (t, C-6), 72.1, 72.3, 75.3 (3 x t, 3 x PhCH₂), 73.8 (d, C-5), 75.2 (d, C-4), 76.8 (d, C-2), 80.6 (d, C- 3), 81.3 (d, C-l), 127.7, 127.8, 127.9, 128.0, 128.2, 128.4, 128.5, 128.5 (8 x d, 15 x Ar- C), 138.4, 138.5, 138.9 (3 x s, 3 x Ar-C); m/z (ES⁺) 1319 (M₂NH₄ ⁺, 32), 673 (MNa⁺, 12), 668 (MNH₄ ⁺, 100%); HRMS (ES⁺) Calcd. For C₃₈H₅₄Na0₅SSi₂ (MNa⁺) 673.3353. Found 673.3353. Found: C, 70.42; H, 8.62. C₃₈H₅₄0₅SSi requires C, 70.1 1 ; H, 8.36%.

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-D-manno pyranosyl- (1^3)-2-0-acetyl-4,6-0-benzylidene-p-D-mannopyranosyl-(1^4)-3,6-di-0-benzyl-2- deoxy-2-phthalimido-p-D-glucopyranoside 5

A solution of disaccharide 4 (1.00 g, 1.13 mmol), thioglycoside 3 (1.10 g, 1.69 mmol) and tri-tert-butylpyrimidine (1.26 g, 5.07 mmol) in DCM (40 mL) was added to a flame- dried round-bottom flask containing activated 3 A molecular sieves (3.0 g). The solution was cooled to 0°C under an atmosphere of argon, stirred for 20 min and methyl trifluoromethanesulfonate (0.574 mL, 5.07 mmol) added. After 16 h t.l.c. (petroleum ethenethyl acetate, 1 : 1) indicated formation of a major product (Rf 0.65) and complete consumption of the alcohol starting material 4 (Rf 0.25). Triethylamine (0.706 mL, 5.07 mmol) was added and the reaction mixture stirred for 10 min before being filtered through CELITE^® and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ethenethyl acetate, 2: 1) to afford trisaccharide 5 (1.52 g, 91 %) as a white foam, [a]_D ²⁵ + 1 1.0 (c, 0.5 in CHC1₃); v_max ( Br disk) 1751 , 1716 (s, C=0) cm^"1; 5_H (500 MHz, CDC1₃) 1.06 (3H, s, CH(CH₃)₂), 1.07 (18H, s, CH(CH₃)₂), 2.03 (3H, s, CH₃), 3.1 1 -3.15 (IH, m, H-5b), 3.56 (IH, at, J 10.4 Hz, H-6b), 3.64-3.70 (3H, m, H-2c, H-5a, H-5c), 3.71 (3H, s, OCH₃), 3.73 (IH, dd, J_2;3 2.6 Hz, J_3>4 9.9 Hz, H-3c), 3.77- 3.81 (3H, m, H-4b, H-6a, H-6'a), 3.87 (IH, dd, J_2>3 3.5 Hz, J_3>4 9.9 Hz, H-3b), 3.93 (IH, d, J 9.9 Hz, H-6c), 3.98 (IH, dd, J_5;6' 4.4 Hz, J_6>6' 1 1.1 Hz, H-6'c), 4.04 (I H, at, J 9.5 Hz, H-4c), 4.15-4.19 (2H, m, H-4a, H-6'b), 4.31 (IH, dd, J_2>3 10.6 Hz, J_3>4 8.5 Hz, H-3a), 4.37-4.46 (4H, m, H-2a, 3 x PhCH₂), 4.53, 4.74 (2H, ABq, J 12.0 Hz, PhCH₂), 4.55, 4.61 (2H, ABq, J 12.2 Hz, PhCH₂), 4.68, 4.91 (2H, ABq, J 1 1.2 Hz, PhCH₂), 4.76 (IH, s, H- lb), 4.84 (IH, d, J 12.2 Hz, PhCH₂), 5.24 (IH, s, H-l c), 5.40 (IH, d, J 3.5 Hz, H-2b), 5.46 (IH, s, PhCH(O)), 5.59 (IH, d, Ji_>2 8.4 Hz, H-l a), 6.68-6.70 (2H, m, 2 x Ar-H), 6.80-6.81 (2H, m, 2 x Ar-H), 6.90-6.96 (3H, m, 3 x Ar-H), 7.01 -7.02 (2H, m, 2 x Ar-H), 7.09-7.1 1 (2H, m, 2 x Ar-H), 7.14-7.19 (3H, m, 3 x Ar-H), 7.25-7.38 (18H, m, 18 x Aril), 7.43-7.45 (2H, m, 2 x Ar-H), 7.67-7.73 (4H, m, 4 x Ar-H); 5_C (125.8 MHz, CDC1₃) 12.2 (d, CH(CH₃)₂), 18.2 (q, CH(CH₃)₂), 21.0 (s, CH₃), 55.7 (q, OCH₃), 55.8 (d, C-2a), 63.2 (t, C-6c), 66.6 (d, C-5b), 68.3 (t, C-6a), 68.7 (t, C-6b), 71.1 (d, C-2b), 71.9, 72.2, 73.6, 74.7, 74.9 (5 x t, 5 x PhCH₂), 73.2 (d, C-3b), 74.1 (d, C-5c), 74.5 (d, C-4c), 74.9 (d, C-5a), 75.4 (d, C-2c), 77.2 (d, C-3a), 78.6 (d, C-4a), 79.1 (d, C-4b), 79.4 (d, C-3c), 97.8 (d, C-la), 98.7 (d, C-l c), 99.3 (d, C-lb), 101.9 (d, PhCH(O)), 1 14.5, 1 18.8, 126.2, 127.3,

127.4, 127.4, 127.5, 127.7, 127.8, 127.9, 128.0, 128.0, 128.0, 128.1, 128.2, 128.4, 128.4,

128.5, 128.5, 128.5, 128.6, 128.7, 129.4, 134.0, (24 x d, 38 x Ar-C), 137.4, 138.1, 138.6, 138.6, 138.9, 139.2, 151.0, 155.5 (8 x s, 10 x Ar-C), 169.6 (s, C=0); (ESI⁺) species observed (MNa⁺), (MNH₄ ⁺); (MNa⁺) peaks observed: 1498.61 (94), 1499.61 (100), 1500.61 (46), 1501.61 (16), 1502.62 (5), 1503.62 (2), peaks calculated for C₈₆H₉₇N0₁₉Si (MNa⁺): 1498.63 (100), 1499.63 (100), 1500.64 (57), 1501.64 (23), 1502.64 (7), 1503.64 (2%).

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-D-manno pyranosyl- (l→3)-2-0-acetyl-4-0-benzyl-p-D-mannopyranosyl-(l→4)-3,6-di-0-benzyl-2-deoxy- 2-phthalimido-p-D-glucopyranoside 6

A solution of benzylidene 5 (0.150 g, 0.102 mmol) in DCM (5 niL) was added to a flame- dried round-bottom flask containing activated 3A molecular sieves (0.30 g). The solution was stirred at rt under an atmosphere of argon for 30 min then cooled to -78°C. Triethyl- silane (49 μί, 0.31 mmol) and dichlorophenyl borane (45 μί, 0.35 mmol) were added and the reaction mixture stirred at 78°C. After 45 min t.l.c. (petroleum ethenethyl acetate, 3 :2) indicated formation of a single product (Rf 0.30) and consumption of starting material (Rf 0.50). Triethylamine (0.75 mL) and methanol (0.75 mL) were added, the reaction mixture diluted with DCM (10.0 mL), washed with sodium hydrogen carbonate (2 x 5 mL of a saturated solution), dried (MgS0₄), filtered and concentrated in vacuo. The resi- due was co-distilled with methanol five times at 50°C before being purified by flash column chromatography (3 :2 petroleum ethenethyl acetate) to afford alcohol 6 (0.143 g, 94%) as a white foam, [a]_D ²⁵ + 27 (c, 0.5 in CHC1₃); v_max ( Br disk) 3445 (s, O-H), 1749, 1716, 1636 (s, C=0)cm ^l; 5_H (500 MHz, CDC1₃) 1.07 (3H, s, CH(CH₃)₂), 1.08 (18H, s, CH(CH₃)₂), 2.07 (3H, s, CH₃), 3.07-3.09 (IH, m, H-5b), 3.42-3.44 (IH, m, H- 6b), 3.58-3.62 (3H, m, H-3b, H-4b, H-5c), 3.65-3.68 (3H, m, H-2c, H-5a, H-6'b), 3.71 (3H, s, OCH₃), 3.77 (IH, dd, J_5;6 2.9 Hz, J_6>6< 9.5 Hz, H-6a), 3.80-3.82 (2H, m, H-3c, H- 6'a), 3.92 (I H, dd, J_5;6 1 - 1 Hz, J_6>6' 1 1.3 Hz, H-6c), 4.03 (IH, dd, J_5;6' 3.2 Hz, H-6'c), 4.15 (IH, at, J 9.4 Hz, H-4a), 4.19 (IH, at, J 9.6 Hz, H-4c), 4.29 (IH, dd, J_2>3 10.6 Hz, J_3>4 9.7 Hz, H-3a), 4.37-4.42 (3H, m, H-2a, 2 x PhCH₂), 4.48-4.57 (5H, m, 5 x PhCH₂), 4.64 (IH, d, J 12.0 Hz, PhCH₂), 4.68 (IH, s, H- lb), 4.72-4.75 (2H, m, 2 x PhCH₂), 4.88 (2H, at, J 10.9 Hz, 2 x PhCH₂), 5.07 (IH, d, Ji_;2 1.7 Hz, H-l c), 5.35 (IH, d, J2.4 Hz, H-2b), 5.60 (IH, d, Ji_>2 8.5 Hz, H-l a), 6.69-6.71 (2H, m, 2 x Ar-H), 6.79-6.81 (2H, m, 2 x Ar-H), 6.92-6.98 (3H, m, 3 x Ar-H), 7.01 -7.03 (2H, m, 2 x Ar-H), 7.20-7.34 (25H, m, 25 x Ar- H), 7.67-7.79 (4H, m, 4 x Ar-H); 5_C (125.8 MHz, CDC1₃) 12.2 (d, CH(CH₃)₂), 18.2 (q, CH(CH₃)₂), 21.2 (s, CH₃), 55.7 (m, C-2a, OCH₃), 61.8 (t, C-6b), 62.6 (t, C-6c), 68.3 (t, C- 6a), 71.3 (d, C-2b), 72.4, 72.5, 73.7, 74.6, 74.7, 74.9 (6 x t, 6 x PhCH₂), 74.2 (d, C-5c), 74.3 (d, C-4c), 74.7 (d, C-4b), 74.9 (d, C-5a), 75.4 (d, C-5b), 76.5 (d, C-2c), 77.0 (d, C- 3a), 78.3 (d, C-4a), 78.3 (d, C-3b), 79.6 (d, C-3c), 97.7 (d, C-lb), 98.6 (d, C- l c), 100.7 (d, C-l a), 1 14.5, 1 18.8, 127.3, 127.3, 127.4, 127.5, 127.5, 127.5, 127.6, 127.7, 128.0, 128.0, 128.2, 128.2, 128.4, 128.4, 128.4, 128.6, 128.7, 133.7, (20 x d, 38 x Ar-C), 138.0,

138.1, 138.4, 138.7, 138.9, 139.2, 150.9, 155.5 (8 x s, 10 x Ar-C), 169.7 (s, C=0); (ESI⁺) species observed (MNa⁺), (MNH₄ ⁺); (MNa⁺) peaks observed: 1500.64 (99), 1501.65 (100), 1502.65 (55), 1503.65 (19), 1504.66 (4), peaks calculated for C₈₆H₉₉N0₁₉Si (MNa⁺): 1500.65 (100), 1501.65 (100), 1502.65 (57), 1503.66 (23), 1504.66 (8) 1505.66

(2%).

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-a-D-mannopyranosyl- (1^3)-[2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-a-D-mannopyranosyl-(1^6)]-2-i>- acetyl-4-0-benzyl-p-D-mannopyranosyl-(l— >4)-3,6-di-0-benzyl-2-deoxy-2- phthalimido-p-D-glucopyranoside 7

A solution of trisaccharide 6 (0.179 g, 0.121 mmol), thioglycoside 3 (0.1 18 g, 0.182 mmol) and tri-tert-butylpyrimidine (0.135 g, 0.545 mmol) in DCM (8 mL) was added to a flame-dried round-bottom flask containing activated 3A molecular sieves (0.3 g). The solution was cooled to 0°C under an atmosphere of argon, stirred for 20 min and methyl trifluoromethanesulfonate (62 μί, 0.55 mmol) added. After 16 h, t.l.c. (petroleum ethenethyl acetate, 2: 1) indicated formation of a major product (R_f 0.55) and complete consumption of the alcohol starting material 6 (R_f 0.20). Triethylamine (76 μί, 0.55 mmol) was added and the reaction mixture stirred for 10 min before being filtered through celite and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ethenethyl acetate, 2: 1) to afford tetrasaccharide 7 (0.198 g, 79%) as a white foam, [a]_D ²⁵ + 24 (c, 0.25 in CHC1₃); v_max ( Br disk) 1717, 1630 (s, C=0) ατ δ_Η ^ΟΟ MHz, CDC1₃) 1.00, 1.03 (6H, 2 x s, 2 x CH(CH₃)₂), 1.1 1, 1.12 (36H, 2 x s, 2 x CH(CH₃)₂), 2.06 (3H, s, CH₃), 3.1 1-3.22 (1H, m, H-5b), 3.59-3.76 (6H, m, H- 3b, H-5c, H-5d, H-6a, H-6'a, H-6b), 3.72 (3H, s, OCH₃), 3.78-3.89 (6H, m, H-2c, H-2d, H-4b, H-5a, H-6'b, H-6c), 3.94-4.00 (3H, m, H-3a, H-3d, H-6'c), 4.03-4.09 (2H, m, H- 3c, H-6d), 4.14-4.29 (4H, m, H-4a, H-4c, H-4d, H-6'd), 4.35-4.70 (14H, m, H-2a, 13 x PhCH₂), 4.73-4.78 (3H, m, H-lb, 2 x PhCH₂), 4.83-4.95 (4H, m, H-ld, 3 x PhCH₂), 5.08 (1H, s, H-lc), 5.39 (1H, s, H-2b), 5.58 (1H, d, Ji_>2 8.4 Hz, H-la), 6.68-6.76 (4H, m, 4 x Ar-H), 6.78-6.82 (4H, m, 4 x Ar-H), 6.93-6.98 (2H, m, 2 x Ar-H), 7.17-7.37 (39H, m, 39 x Ar-H), 7.62-7.67 (4H, m, 4 x Ar-H); 5_C (125.8 MHz, CDC1₃) 12.1, 12.2 (2 x d, 2 x CH(CH₃)₂), 18.0, 18.2 (2 x q, 2 x CH(CH₃)₂), 21.2 (s, CH₃), 55.7 (m, C-2a, OCH₃), 62.7 (t, C-6c), 62.9 (t, C-6d), 66.2 (t, C-6b), 68.3 (t, C-6a), 71.6 (d, C-2b), 71.6, 72.4, 72.5, 72.8, 74.4, 74.4, 74.5, 74.8, 74.9 (9 x t, 9 x PhCH₂), 73.4 (d, C-5c), 73.6 (d, C-4d), 73.9 (d, C-5b), 74.2 (d, C-4a), 74.4 (d, C-4c), 74.6 (d, C-5d), 74.9 (d, C-4b), 75.3 (d, C-5a), 75.5 (d, C-2d), 76.3 (d, C-2c), 77.2 (d, C-3a), 78.4 (d, C-3b), 79.2 (d, C-3d), 79.8 (d, C- 3c), 97.8 (d, C-la), 98.6 (d, C-ld), 99.6 (d, C-lb), 100.6 (d, C-lc), 1 14.4, 1 18.6, 126.6,

127.0, 127.1, 127.2, 127.4, 127.5, 127.6, 127.7, 127.7, 127.8, 127.9, 127.9, 127.9, 128.0,

128.1, 128.1, 128.2, 128.2, 128.3, 128.4, 128.5, 128.6, 128.6, 129.6, 129.6, 132.8, 133.4, 135.8, 135.8, 136.1 (32 x d, 53 x Ar-C), 133.7, 133.7, 134.0, 134.1, 138.0, 138.1, 138.6,

138.6, 138.8, 138.9, 139.2, 151.2, 155.5 (13 x s, 13 x Ar-C), 169.5, 169.6, 171.0 (3 x s, 3 x C=0); (ESI⁺) species observed (MNa⁺); (MNa⁺) peaks observed: 2088.91 (61), 2089.92 (100), 2090.92 (79), 2091.92 (38), 2092.92 (12), peaks calculated for Ci₂₂Hi4₇N0₂₄Si₂ (MNa ) 2088.97 (69), 2089.98 (100), 2090.98 (79), 2091.98 (44), 2092.98 (19), 2093.99

(6%).

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-a-D-mannopyranosyl - (1^3)-[2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-a-D-mannopyranosyl-(1^6)]-2-i>- acetyl-4-0-benzyl-p-D-mannopyranosyl-(l→4)-2-acetamido-3,6-di-0-benzyl-2- deoxy-p-D-glucopyranoside 8

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. After 20 h t.l.c. (petroleum ethenethyl acetate, 2: 1) indicated formation of a single product (R_f 0.40) and complete consumption of amine intermediate (R_f 0.25). The reaction mixture was concentrated in vacuo, the residue dissolved in DCM (10 mL), washed with water (10 mL), sodium hydrogen carbonate (2 x 10 mL of a saturated aqueous solution), dried (MgS0₄) and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ethenethyl

25

acetate, 3:2) to afford acetamide 8 (0.104 g, 91 %) as a pale yellow foam, [OC]D - 35 (c, 0.18 in CHC1₃); v_max ( Br disk) 3441 (s, N-H stretch), 1751, 1635 (s, C=0) cm^"1; 5_H (500 MHz, CDCI₃) 1.02 (6H, m, 2 x CH(CH₃)₂), 1.05, 1.06 (36H, 2 x s, CH(CH₃)₂), 1.89, 2.17 (6H, 2 x s, 2 x CH₃), 3.29-3.30 (IH, m, H-5b), 3.40-3.46 (2H, m, H-6b, H-6c), 3.56-3.79 (13H, m, H-2a, H-2c, H-2d, H-3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H- 6'c), 3.76 (3H, s, OCH₃), 3.86-3.90 (2H, m, H-3c, H-6d), 3.94 (IH, dd, J_5;6' 3.6 Hz, J_6>6' 1 1.0 Hz, H-6'd), 4.04 (IH, at, J 7.9 Hz, H-3a), 4.07-4.16 (3H, m, H-4a, H-4c, H-4d), 4.37 (IH, d, J 12.4 Hz, PhCH₂), 4.40 (IH, d, J 1 1.7 Hz, PhCH₂), 4.42 (IH, d, J 1 1.9 Hz, PhCH₂), 4.46-4.59 (10H, m, 10 x PhCH₂), 4.61 (IH, d, J 1 1.7 Hz, PhCH₂), 4.66 (IH, d, J 12.0 Hz, PhCH₂), 4.70 (IH, s, H-lb), 4.76 (IH, d, J 1 1.6 Hz, PhCH₂), 4.81 (IH, s, H-ld), 4.87 (IH, d, J 1 1.2 Hz, PhCH₂), 4.88 (IH, d, J 10.9 Hz, PhCH₂), 5.09 (IH, d, Ji_>2 1.3 Hz, H-lc), 5.27 (IH, d, J_2;NH 7.5 Hz, NH), 5.33 (IH, d, Ji_;2 6.9 Hz, H-la), 5.37 (IH, d, J 3.0 Hz, H-2b), 6.76-6.79 (2H, m, 2 x Ar-H), 6.88-6.90 (2H, m, 2 x Ar-H), 7.16-7.32 (45H, m, 45 x Ar-H); 5_C (125.8 MHz, CDC1₃) 12.2, 12.2 (2 x d, 2 x CH(CH₃)₂), 18.1, 18.2 (2 x q, 2 x CH(CH₃)₂), 21.0, 23.3 (2 x s, 2 x CH₃), 55.4 (d, C-2a), 55.8 (q, OCH₃), 62.5 (t, C-6c), 62.8 (t, C-6d), 65.9 (t, C-6b), 69.3 (t, C-6a), 71.4 (d, C-2b), 72.3, 72.4, 72.4, 72.6, 73.5, 74.3, 74.7, 74.8, 75.6 (9 x t, 9 x PhCH₂), 73.3 (d, C-5c), 73.9 (d, C-5a), 74.1 (d, C-5d), 74.3 (d, C-4d), 74.6 (d, C-5b), 75.1 (d, C-4a), 75.2 (d, C-4c), 75.4 (d, C-4b), 75.8 (d, C- 2d), 76.1 (d, C-2c), 77.3 (d, C-3a), 79.0 (d, C-3b), 79.6 (d, C-3d), 80.6 (d, C-3c), 97.4 (d, C-lb), 98.1 (d, C-ld), 98.8 (d, C-la), 100.4 (d, C-lc), 1 14.6, 1 18.5, 127.1, 127.4, 127.4, 127.5, 127.5, 127.6, 127.6, 127.7, 127.7, 127.8, 127.9, 127.9, 128.0, 128.2, 128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 128.5, 128.6 (24 x d, 49 x Ar-C), 138.0, 138.3, 138.6, 138.8, 138.9, 139.1, 139.1, 139.4, 151.6, 155.2 (10 x s, 1 1 x Ar-C), 169.8, 170.5 (2 x s, 2 x C=0); (ESI⁺) species observed (MNa⁺), (MNH₄ ⁺); (MNa⁺) peaks observed: 2000.87 (55), 2001.87 (100), 2002.87 (69), 2003.87 (33), 2004.86 (14), peaks calculated for C116H146NO23S12 (MNa ): 2000.98 (72), 2001.98 (100), 2002.99 (76), 2003.99 (41), 2004.99 (17), 2005.99 (5%).

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-a-D-mannopyranosyl-(l→3)-[2,3_?4-tri-0- benzyl-6--a-D-mannopyranosyl-(l→6)]-2-0-acetyl-4-0-benzyl-p-D-mannopyranosyl- (l→4)-2-acetamido-3,6-di-0-benzyl-2-deoxy-p-D-glucopyranoside 9

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 μί, 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). The reaction mixture was diluted with DCM (10 mL), washed with sodium hydrogen carbonate (10 mL of a saturated aqueous solution), dried (Na₂S0₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate, 1 : 1) to afford diol 9 (88 mg, 76%) as a white foam. [a]_D ²⁰ +32 (c, 1.0 in CHC1₃);

Vmax ( Br disk) 3428 (s, N-H stretch), 1746, 1739 (s, C=0) cm^"1; 5_H (500 MHz, CDC1₃) 1.58, 1.97 (6H, 2 x s, 2 x CH₃), 3.32-3.38 (1H, m, H-5b), 3.40-3.48 (2H, m, H-6b, H-6c), 3.56-3.79 (13H, m, H-2a, H-2c, H-2d, H-3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H-6'c), 3.76 (3H, s, OCH₃), 3.82-4.0 (4H, m, H-3c, H-6d ,Η-6'd, H-3a), 4.13-4.20 (3H, m, H-4a, H-4c, H-4d), 4.43-4.51 (3H, m, PhCH₂) 4.52-4.61 (10H, m, 10 x PhCH₂), 4.63-4.64 (2H, m, PhCH₂), 4.70 (IH, s, H-lb), 4.95 (IH, s, H-ld), 5.10 (IH, s, H-lc), 5.33 (IH, d, Ji,2 6.4 Hz, H-la), 5.37 (IH, d, J_2;3 3.0 Hz, H-2b), 6.77-6.79 (2H,m, 2 x Ar- H), 6.88-6.90 (2H, m, 2 x Ar-H), 7.19-7.32 (45H, m, 45 x Ar-H); 5_C (125 MHz, CDC1₃) 21.0, 23.3 (2 x s, 2 x CH₃), 55.64 (q, OCH₃), 56.62 (d, C-2a), 61.9 (t, C-6c), 62.3 (t, C- 6d), 66.4 (t, C-6b), 68.7 (t, C-6a), 71.2 (d, C-2b), 72.2, 72.3, 72.6, 72.7, 73.5, 74.2, 74.6, 74.7, 75.4 (9 x t, 9 x PhCH₂), 73.4 (d, C-5c), 74.1 (d, C-5a), 74.5 (d, C-5d), 75.0 (d, C- 4d), 75.2 (d, C-5b), 75.4 (d, C-4a), 75.5 (d, 4b), 75.6 (d, C-4c), 75.8 (d, C-2d), 76.0 (d, C- 2c), 77.3 (d, C-3a), 79.6 (d, C-3b), 79.7 (d, C-3d), 80.3 (d, C-3c), 97.5 (d, C-lb), 98.1 (d, C-ld), 98.7(d, C-la), 100.9 (d, C-lc), 1 14.4, 1 18.5, 126.9,127.1, 127.3, 127.4, 127.5, 127.6, 127.6, 127.7, 127.7, 127.8, 127.9, 127.9, 128.0, 128.2, 128.2, 128.3, 128.3,

128.4,128.4,128.5,128.5, 128.6 (24 x d, 49 x Ar-C) 137.7, 138.0, 138.1,138.3, 138.4, 138.5, 138.7, 139.0, 151.4, 155.1 (10 x s, 1 1 x Ar-C), 170.0, 170.4 (2 x s, 2 x C=0); HRMS (ES⁺) Calculated For C₉₈Hi₀₇NO₂₃ (M⁺) 1665.73. Found (MH⁺): 1666.73 (100).

/>-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-dibenzyloxyphosphoryl-a-D- mannopyranosyl-(l→3)- [2,3_?4-tri-0-benzyl-6-0-dibenzyloxyphosphoryl-a-D- mannopyranosyl-(l→6)]-2-0-acetyl-4-0-benzyl- -D-mannopyranosyl-(l→4)-2- acetamido-3,6-di-0-benzyl-2-deoxy-p-D-glucopyranoside 10

Dibenzyl Ν,Ν-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. (petrohethyl acetate, 1 : 1) indicated complete consumption of alcohol starting material (Rf 0.30) and formation of a major product (Rf 0.60). The reaction mixture was cooled to -78°C and m-chloroperbenzoic acid (0.035 g, 0.2 mmol) was added. The mixture was stirred for 2 h and then warmed to rt, after which time t.l.c. (petrohethyl acetate, 1 : 1) indicated complete consumption of the phosphine intermediate (Rf 0.60) and formation of a major product (Rf 0.25). The 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 (Na₂S0₄), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (petrohethyl acetate, 1 : 1) to afford phosphotriester 10 (0.25 g, 58% over two steps) as a white foam.[a]_D ²⁰ +41 (c, 1.0 in CHC1₃); δ_Η (400 MHz, CDC1₃) 1.61 , 1.89 (6H, 2 x s, 2 x CH₃), 3.29-3.30 (IH, m, H-5b), 3.42-3.48 (2H, m, H-6b, H-6c), 3.56-3.79 (13H, m, H-2a, H-2c, H-2d, H-3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H-6'c), 3.76 (3H, s, OCH₃), 3.86-3.94 (3H, m, H-3c, H-6d ,Η-6'd), 4.04-4.16 (4H, m, H-3a, H-4a, H-4c, H-4d), 4.37-4.42 (3H, m, PhCH₂), 4.46-4.59 (10H, m, 10 x PhCH₂), 4.61 -4.66 (2H, m, PhCH₂), 4.70 (IH, s, H- lb), 4.76 (IH, d, 10.6 Hz, PhCH₂), 4.81 (IH, s, H-l d), 4.87 (IH, d, 8 Hz, PhCH₂), 4.88 (IH, d, 10.2 Hz, PhCH₂), 5.10 (IH, s, H- l c), 5.21 (2H, m, PhCH₂), 5.33 (IH, d, Ji_;2 6.4 Hz, H- l a), 5.37 (IH, d, J_2;3 3.0 Hz, H-2b), 6.76-6.79 (2H,m, 2 x Ar-H), 6.88-6.90 (2H, m, 2 x Ar-H), 7.16-7.45 (65H, m, 65 x Ar-H); 5_C (100 MHz, CDC1₃) 21.0, 23.3 (2 x CH₃), 55.64 ( OCH₃), 56.62 (C-2a), 66.0 (C-6c), 66.8 (C- 6d), 67.1 (C-6b), 67.8 (C-6a), 71.2 (C-2b), 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 73.5, 74.0, 74.3, 74.6, 74.7, 75.0, 75.6 (13 x PhCH₂), 73.2 (C-5c), 74.1 (C-5a), 74.5 (C-5d), 75.1 (C- 4d), 75.2 (C-5b), 75.4 (C-4a), 75.5 (4b), 75.7 (C-4c), 75.8 (C-2d), 76.2 (C-2c), 77.3 (C- 3a), 78.1 (C-3b), 79.7 (C-3d), 80.7 (C-3c), 96.9 (C-lb), 97.9 (C-ld), 98.4(C-la), 100.2 (C-lc), 114.4, 118.4, 126.6, 126.9, 127.1, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.6, 127.6, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 128.0, 128.1 128.2, 128.2, 128.3, 128.3, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.7, 130.1, 133.1, 136.0 (41 x d, 69 x Ar-C) 135.8, 135.9, 136.0, 136.2, 137.7, 138.0, 138.1, 138.3, 138.4, 138.4, 138.5, 138.7, 139.0, 151.4, 155.1 (15 x Ar-C), 170.0, 170.5 (2 x s, 2 x C=0); δ_Ρ (162 MHz, CDC1₃) - 1.02, - 1.20; HRMS (ES⁺) Calculated For

Ci26Hi₃₃N0₂₉P2 (M⁺) 2185.84. Found (MH⁺): 2186.85. 2,3_?4-tri-0-benzyl-6-0-dibenzyloxyphosphoryl-a-D-mannopyranosyl-(l→3)-[2,3_?4- tri-0-benzyl-6-0-dibenzyloxyphosphoryl-a-D-mannopyranosyl-(l→6)]-2-0-acetyl-4- 0-benzyl- -D-mannopyranosyl-(l→4)-2-acetamido-3,6-di-0-benzyl-2-deoxy-D- glucopyranose 11.

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

consumption of starting material (R_f 0.25) and formation of a single product (R_f 0.15). 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 (Na₂S0₄), filtered, concentrated in vacuo, and the residue purified by flash column chromatography (ethyl acetate) to afford hemiacetal 11 (l lmg, 78%) as a pale yellow foam. 5_H (400 MHz, CDC1₃) 1.61, 1.89 (6H, 2 x s, 2 x CH₃), 3.29- 3.30 (IH, m, H-5b), 3.42-3.48 (2H, m, H-6b, H-6c), 3.56-3.79 (13H, m, H-2a, H-2c, H-2d, H-3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H-6'c), 3.76 (3H, s, OCH₃), 3.86-3.94 (3H, m, H-3c, H-6d ,Η-6'd), 4.04-4.16 (4H, m, H-3a, H-4a, H-4c, H-4d), 4.37- 4.42 (3H, m, PhCH₂) 4.46-4.59 (10H, m, 10 x PhCH₂), 4.61-4.66 (2H, m, PhCH₂), 4.70 (IH, s, H-lb), 4.76 (IH, d, 10.4 Hz, PhCH₂), 4.81 (IH, s, H-ld), 4.87 (IH, d, 11 Hz, PhCH₂), 4.88 (IH, d, 10.2 Hz, PhCH₂), 5.10 (IH, s, H-lc), 5.21 (2H, m, PhCH₂) 5.33 (IH, d, Ji_;2 6.4 Hz, H-la), 5.37 (IH, d, J_2;3 3.0 Hz, H-2b), 7.16-7.45 (65H, m, 65 x Ar-H); 5_C (100 MHz, CDC1₃) 21.0, 23.3 (2 x CH₃), 56.62 (C-2a), 66.0 (C-6c), 66.8 (C-6d), 67.1 (C- 6b), 67.8 (C-6a), 71.2 (C-2b), 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 73.5, 74.0, 74.3, 74.6, 74.7, 75.0, 75.6 (13 x PhCH₂), 73.2 (C-5c), 74.1 (C-5a), 74.5 (C-5d), 75.1 (C-4d), 75.2 (C-5b), 75.4 (C-4a), 75.5 (4b), 75.7 (C-4c), 75.8 (C-2d), 76.2 (C-2c), 77.3 (C-3a), 78.1 (C-3b), 79.7 (C-3d), 80.7 (C-3c), 96.9 (C-lb), 97.9 (C-ld), 98.4(C-la), 100.2 (C-lc),

126.6, 126.9, 127.1, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.6, 127.6, 127.7,

127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 128.0, 128.1 128.2, 128.2, 128.3, 128.3, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.7, 130.1, 133.1, 136.0 (39 x d, 65 x Ar-C) 135.8, 135.9, 136.0, 136.2, 137.7, 138.0, 138.1, 138.3, 138.4, 138.4, 138.5, 138.7, 139.0 (13 x s, 13 x Ar-C), 170.0, 170.5 (2 x s, 2 x C=0); 5_P (162 MHz, CDC1₃) - 1.04, - 1.10; HRMS (ES⁺) Calculated For Cn₉Hi₂₇N0₂₈P₂ (M⁺) 2079.80.

Found (MH⁺):2080.80.

6-0-Phosphate-a-D-mannopyranosyl-(l→3)-[6-0-phosphate-a-D-mannopyranosyl- (l→6)]- -D-mannopyranosyl-(l→4)-2-acetamido-2-deoxy-D-glucopyranose 12

Protected tetrasaccharide 11 (12 mg, 0.01 mmol) in THF (3 mL) was added to NH₃(/) (20 mL) at -33 °C. The minimum amount of sodium required to make the mixture turn deep blue was then added to the stirred mixture. After 30 min, MeOH (4 mL) was added, and reaction mixture was stirred for a further 1 h., warmed to rt, and the solvent was removed in vacuo. Gel filtration of the crude residue on a Sephadex G-10 column (eluting with 0.01% NH₃) afforded the deprotected tetrasaccharide 12 (4.9mg, 98%), as a white foam. 5_H (400 MHz, D₂0) 1.89 (3H, s, CH₃), 3.46-3.55 (1H, m, H-5b), 3.61-3.95 (16H, m, H-6b, H-6c, H-2a, H-2c, H-2d, H-3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H-6'c, H-3c, H-2b), 3.94-4.11 (2H, m, H-6d ,Η-6'd), 4.7-4.9 (4H, m, H-3a, H-4a, H-4c, H-4d), 5.0 (2H, m, H-lb, H-lc), 5.1 (1H, s, H-la); 5_C (100 MHz, D₂0) 21.9 (CH₃), 46.6 (C-2a), 60.0 (C-6c), 62.8 (C-6d), 63.0 (C-6b), 63.4 (C-6a), 65.1 (C-2b), 65.5 (C-5c), 65.7 (C-5a), 66.0 (C-5d), 66.3 (C-4d), 68.0 (C-5b), 68.9 (C-4a), 71.7 (4b), 71.9 (C-4c), 72.6 (C-2d), 73.8 (C-2c), 74.5 (C-3a), 79.8 (C-3b), 80.2 (C-3d), 80.5 (C-3c), 99.5 (C-lb), 99.7 (C-ld), 100.2 (C-la), 102.5 (C-lc), 174.5 (s, C=0); 5_P (162 MHz, D₂0) 4.70, 4.76; HRMS (ES⁺) Calculated For C₂₆H₄7N0₂₇P₂ (M⁺) 867.18. Found (MH⁺): 868.18.

2-Methyl [6-0-phosphate-a-D-mannopyranosyl-(l→3)-[6-0-phosphate-a-D- mannopyranosyl-(l→6)]- -D-mannopyranosyl-(l→4)-2-acetamido-l,2-deoxy-D- glucopyrano]-[2,l-^-oxazoline 13

Hemiacetal 12 (2 mg, 0.0023 mmol) and triethylamine (3 μί, 0.021 mmol) were dissolved in D₂0 (18.4 μί) 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% NH₃ afforded oxazoline 13 (1.8 mg, 95%) as a white foam. 5_H (400 MHz, D₂0) 1.89 (3H, s, CH₃), 2.8-2.95 (1H, m, H-5b), 3.26-3.95 (16H, m, H-6b, H-6c, H-2a, H-2c, H-2d, H- 3b, H-3d, H-4b, H-5a, H-5c, H-5d, H-6a, H-6'a, H-6'b, H-6'c, H-3c, H-2b), 3.96-4.1 1 (2H, m, , H-6d ,Η-6'd), 4.7-4.9 (6H, m, H-3a, H-4a, H-4c, H-4d, H-lb, H-lc), 5.95 (1H, d, H-la). δ_Ρ (162 MHz, CDC1₃) 4.38; HRMS (ES⁺) Calculated For C₂6H₄₅N0₂6P₂ (M+) 849.17. Found (MH⁺): 850.17. EXAMPLE 2

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).

Scheme 2. ENGase catalysed glycosylation of 14 (a) ENGase, phosphate buffer pH 6.5, H₂0, 37°C, 2 h.

Table 1. Activity of family GH85 ENGases for glycosylation of 14 by donor 13

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). General procedure for the enzymatic glycosylation of acceptor with oxazoline donor Endohexosaminidase-catalysed glycosylations were monitored by HPLC using a Dionex 1 HPLC instrument using Chromeleon software connected to a Dionex 1 variable wavelength detector at 254 nm wavelength. Analytical HPLC (Jupitor 5 μ C-18 column, 250 x 4.6 mm) was used to monitor the reactions, with 2 aliquots taken at appropriate time intervals. The column was eluted with 20% MeCN/H₂0. The yield was determined by integration of the product and acceptor peaks.

N⁴-6-0-Phosphate-a-D-mannopyranosyl-(l→3)-[6-0-phosphate-a-D- mannopyranosyl-(l→6)]-P-D-mannopyranosyl-(l→4)-2-acetamido-2-deoxy-P-D- glucopyranosyl-(l→4)-2-acetamido-2-deoxy-P-D-glucopyranosyl-l-methyl-iV -(9- fluorenylmethyloxycarbonyl)-L-asparagine 15

FmocHN

A solution of the tetrasaccharide oxazoline 13 (0.762 mg, 0.9μηιο1) and Fmoc- Asn(GlcNAc)-OH 14 (0.058 mg, Ο. ΐ μιηοΐ) was incubated with 2 mU of Endo A in 20 μΐ, of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C. After 2 h, RP-HPLC indicated the formation of a new product, which was then purified directly by RP-HPLC to give 15: (71 %, HPLC yield based on acceptor) as a white powder (0.88 mg); analytical HPLC: ¾ = 9.71 min; ESI-MS: calculated for C53H76N4O36P2: 1406.37. Found: 1407.38 (M+H)⁺; C₅3H7₆N₄Na0₃6P₂: 1429.36. Found: 1429.36 (M+Na). Interestingly 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.

The fact that complex N-glycans are terminated by negatively charged sialic acid residues suggests to the person of ordinary skill in the art that Endo M may be more tolerant of high mannose glycans terminated in negatively charged phosphates. However, a clearly unexpected result is that the expected higher tolerance of Endo M is seen where Endo M and N175Q are able to catalyze the synthesis of 7.9 (addition of the hexasaccharide to an acceptor), but not of 16 (addition of the tetrasaccharide to an acceptor). The N175Q gly- cosynthase mutant of Endo M (Umekawa et. al., J Biol. Chem. 2010, 285, 51 1-521) was also unable to effect glycosylation of 15, but as noted above, was able to catalyze the synthesis of 7.9 On the other hand, Endo D, which has considerably more specific structural requirements with respect to the N-glycans it will hydrolyse (Mizuochi et. al., J

Biochem. 1984, 95, 1209-1213) was able to catalyse the production of 16, albeit in a very low yield, yet another unexpected result.

It is apparent from the above that the ability of a given ENGase to catalyze the addition of high mannose glycans terminated in negatively charged phosphates to proteins or pep- tides to form phosphorylated glycoproteins or glycopeptides having all natural linkages is not predictable based solely on a knowledge of the hydrolytic activity of these enzymes.

EXAMPLE 3 In one non-limiting example, 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)₂RNase 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)₂RNase in considerably higher yield. Glycosylation of dRNase B.

A solution of the tetrasaccharide oxazoline 13 (0.229 mg, 0.27μηιο1) and dRNase B 16 (0.348 mg, 0.03μηιο1) was incubated with 2 mU of Endo-A in 20 of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C for 2h to give (M6P)₂RNase 17. ESI-MS: calculated for C₅3H76N₄036P₂-RNase B: 14734.46. Found: 14735.46 (M+H) ⁺.

Scheme 3 a) WT-Endo A, phosphate buffer pH 6.5, H₂0, 37°C, 2 h. EXAMPLE 4a

Evaluation of cation-independent mannose-6-phosphate receptor (CI-MPR) binding by (M6P)₂RNase.

Immunocytochemistry

A standard immunocytochemistry protocol was adapted to visualise the presence of cation-independent mannose-6-phosphate receptor (CI-MPR) and (M6P)₂RNase in cultured cells. Briefly, liver carcinoma cells (HepG2 cells) were cultured on chamber slides in the presence or absence of 1 μΜ (M6P)₂RNase for 1 -2 hours under standard cell culture conditions. HepG2 cells were also incubated in parallel with 1 μΜ 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.

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.

Immunoprecipitation of (M6P)₂RNase from immobilized CI-MPR HepG2 cells were treated with either (M6P)₂RNase or deglycosylated RNase as above and cell lysates prepared. Cell lysates were incubated with mouse anti-CI-MPR antibody-conjugated Protein A sepharose overnight at 4°C to capture (M6P)₂RNase bound to CI-MPR. The protein A sepharose was washed extensively and bound sample containing (M6P)₂RNase-CI-MPR was then eluted with reducing buffer containing sodium dodecyl sulphate and dithiothreitol. Eluate was subjected to Western blotting for detection of RNase A.

EXAMPLE 4b Evaluation of cation-independent mannose-6-phosphate receptor (CI-MPR) binding by (M6P)₂RNase.

Immunocytochemistry

Liver carcinoma cells (HepG2) were cultured on chamber slides in the presence or absence of 1 μΜ (M6P)₂RNase for 2 hours under standard cell culture conditions (37°C, 5% C0₂, minimal essential media, 1.5% BSA, 100 units/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin). HepG2's were also incubated in parallel with 1 μΜ 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). Cells were blocked with 3% non-fat dried milk/1% BSA in lx PBS, then two primary antibodies were applied to label the CI-MPR (mouse anti-cation independent mannose-6-phosphate receptor) and 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. Images were captured on a Leica TCS SP5 confocal microscope using a 63x objective (Figures 3-5). Appropriate controls were included in the experiment including: cells that had not been treated with RNase and stained as above; assay controls where primary and secondary antibodies are eliminated to control for non-specific fluorescence. Co-localization of CI-MPR and RNase signal was quantitated using Leica Application Suite Advanced Fluorescence Software (Leica) (Figure 3). Immunoprecipitation of (M6P)₂RNase from immobilized CI-MPR

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)₂RNase 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).

EXAMPLE 5

Synthesis of M6P hexasaccharide oxazoline donor

Reagents and Conditions a) 6.8, NIS, TfOH, 3A mol. sieves, DCM, -20°C, 30min, 65%; b) Et₃SiH, PhBCl₂, 3A mol. sieves, DCM, -78°C, 30 min, 78%; c) 6.8, NIS, TfOH, 3A mol. sieves, DCM, -20°C, 30min, 72%; d) (i) NH₂CH₂CH₂NH₂, MeOH, reflux, 18h; (ii) Ac₂0, py, 18h, 71%; e) BF₃.OEt₂ DCM, 0°C, lh, 68%, f) (BnO)₂PNz^'Pr₂, tetrazole, DCM, rt, 18h then m-CPBA, -78°C, 2h, 94%; g) CAN, MeCN, H₂0, rt, lh, 80%; h) Na, NH₃(1), -33°C, 83%; i) DMC, Et₃N, H₂0, rt, 30min, 95%.

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

ChemBioChem 2011, 12, 685 - 690. /)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-d-manno pyranosyl- (1— »2)- 3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l— »3)-2-0-acetyl-4,6-0- benzylidene-p-d mannopyranosyl-(l→4)-3,6-di-0-benzyl-2-deoxy-2-phthalimido-p- d-glucopyranoside 7.0

A solution of 3.6 (0.22 g, 0.25 mmol), and 6.8 (0.32 g, 0.29 mmol) in DCM (10 mL) was added to a flame-dried round bottom flask containing 3A molecular sieves (0.5 g). The solution was cooled to -20 °C under an atmosphere of nitrogen, stirred for 30 min and NIS (0.07 g, 0.30 mmol) was added. After 10 min, triflic acid (2.6 μί, 0.03 mmol) was added. After a further 30 min t.l.c. (petrohethyl acetate, 2: 1) indicated the formation of a major product (Rf 0.5) and the complete consumption of alcohol starting material 3.6 (Rf 0.3). 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 NaHC0₃ (10 mL), brine, dried over anhydrous Na₂S0₄, 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. [α]ο +2 (c, 1.0 in CHC1₃); v_max ( Br disk) 1753, 1712 (s, C=0) cm^"1; 5_H (400 MHz, CDC1₃) 1.02- 1.26 (21H, m, 3 x CH(CH₃)₂), 2.09 (3H, s, CH₃), 3.03-3.08 (IH, m, H-5b), 3.43-3.46 (IH, b, H-6b), 3.65-3.71 (5H, m, H-3b, H-5a, H-6c, H-6'c), 3.72 (3H, s, OCH₃), 3.72-3.89 (13H, m, H-4b, H-6a, H-6'a, H-3c, H-4c, H-5c, H-3d, H-4d, H-5d, H-6d, H-6'd), 3.97- 4.17 (5H, m, H-4a, H-6'b, H-2c, H-2d, H-3d), 4.28-4.34 (IH, m, H-3a), 4.40-4.68 (15H, m, H-2a, 14 x PhCH₂), 4.71 (IH, s, H-lc), 4.80-4.92 (4H, m, 4 x PhCH₂), 5.17 (IH, s, H- lb), 5.40 (IH, s, H-ld), 4.42-4.45( IH, m, H-2b), 5.50 (IH, s, PhCH(O)), 5.61 (IH, 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 (40H, m, 40 x Ar-H), 7.68-7.80 (4H, m, 4 x Ar-H); 5_C (100 MHz, CDC1₃) 12.0 (d, CH(CH₃)₂), 18.0 (q, CH(CH₃)₂), 21.1 (s, CH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 66.3 (d, C-5a), 66.5 (d, C-5b), 68.1 (t, C-6a), 68.4 (t, C-6b), 69.5 (t, C-6d), 71.0 (d, C-2b), 71.4, 71.5, 72.0, 72.3, 73.0, 73.7, 74.4 (8 x t, 8 x PhCH₂), 72.8 (d, C-5d), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.4 (d, C-4d), 74.5 (d, C-2c), 74.7 (d, C-4c), 75.1 (d, C-2d), 76.9 (d, C-3a), 78.5 (d, C-4a), 79.2 (d, C- 4b), 79.6 (d, C-2c), 80.2 (d, C-3d), 97.4 (d, C-la), 97.7 (d, C-ld), 99.2 (d, C-lc), 99.7 (d, C-lb), 101.3 (d, PhCH(O)), 114.3, 118.6, 123.4, 127.2, 125.63, 127.2, 127.3, 127.4, 127.4, 127.5, 127.5, 127.6, 127.7, 127.7, 127.8, 127.9, 128.0, 128.1, 128.1, 128.2, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.4, 129.6, 131.6, 133.8 (32 x d, 54 x Ar-C), 136.9, 137.1, 137.9, 138.0, 138.1, 138.4, 138.5, 138.6, 138.6, 138.8, 139.1 (11 x s, 12 x Ar-C), 151.0, 155.5, 169.6 (3 x s, 3 x C=0); HRMS (ES⁺) Calculated For CnsHnsNC^Si (MH⁺) 1908.84. Found (MH⁺) 1908.84.

/)-Methoxyphenyl 2,3_?4-tri-0-benzyl-6-0-tri-/50-propylsilyl-a-d-mannopyranosyl- (1— »2)- 3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l→3)-2-0-acetyl-4-0-benzyl-p-d mannopyranosyl-(l→4)-3,6-di-0-benzyl-2-deoxy-2-phthalimido-p-d- glucopyranoside 7.1

A solution of 7.0 (0.37 g, 0.20 mmol) in DCM (5 mL) was added to a flame-dried round bottom flask containing 3A molecular sieves (1.0 g). The solution was stirred for 30 min under an atmosphere of nitrogen, and then cooled to -78 °C. Triethylsilane (0.14 mL, 0.86 mmol) and dichlorophenyl borane (90 μί, 0.67 mmol) were added, and the reaction mixture was stirred at -78 °C. After 30 min, t.l.c. (petrol: ethyl acetate, 2: 1) showed the formation of a single product (Rf 0.3) and the consumption of starting material (Rf 0.5). Triethylamine (5 mL) and methanol (5 mL) were added, and the reaction mixture diluted with DCM (20 mL), washed with aqueous sodium hydrogen carbonate (2 x 10 mL of a saturated solution), dried (Na₂S0₄), filtered, and concentrated in vacuo. The residue was co-evaporated with methanol five times at 50 °C before it was purified by flash column chromatography (petrohethyl acetate, 2: 1) to afford compound 7.1 (0.29 g, 78%) as a white foam. [a]_D ^υ +3.5 (c, 1.0 in CHC1₃); v_max ( Br disk) 1747, 1715, 1634 (s, C=0) cm^" 5_H (400 MHz, CDC1₃) 1.02-1.26 (21H, m, 3 x CH(CH₃)₂), 2.09 (3H, s, CH₃), 3.03-3.08 (IH, m, H-5b), 3.43-3.46 (IH, b, H-6b), 3.65-3.71 (5H, m, H-3b, H-5a, H-6c, H-6'c), 3.72 (3H, s, OCH₃), 3.72-3.89 (13H, m, H-4b, H-6a, H-6'a, H-3c, H-4c, H-5c, H-3d, H- 4d, H-5d, H-6d, H-6'd), 3.97- 4.17 (5H, m, H-4a, H-6'b, H-2c, H-2d, H-3d), 4.28-4.34 (IH, m, H-3a), 4.40-4.68 (15H, m, H-2a, 14 x PhCH₂), 4.71 (IH, s, H-lc), 4.80-4.92 (4H, m, 4 x PhCH₂), 5.17 (IH, s, H-lb), 5.40 (IH, s, H-ld), 5.42-5.45( IH, m, H-2b), 5.50 (IH, s, PhCH(O)), 5.61 (IH, 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 (40H, m, 40 x Ar-H), 7.68-7.80 (4H, m, 4 x Ar-H); 5_C (100 MHz, CDC1₃) 12.0 (d, CH(CH₃)₂), 18.0 (q, CH(CH₃)₂), 21.1 (s, CH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 66.3 (d, C-5a), 66.5 (d, C-5b), 68.1 (t, C-6a), 68.4 (t, C-6b), 69.5 (t, C-6d), 71.0 (d, C-2b), 71.4, 71.5, 71.7, 72.0, 72.3, 73.0, 73.7, 74.1, 74.4 (9 x t, 9 x PhCH₂), 72.8 (d, C-5d), 73.2 (d, C- 3b), 74.3 (d, C-5c), 74.4 (d, C-4d), 74.5 (d, C-2c), 74.7 (d, C-4c), 75.1 (d, C-2d), 76.9 (d, C-3a), 78.5 (d, C-4a), 79.2 (d, C-4b), 79.6 (d, C-2c), 80.2 (d, C-3d), 97.6 (d, C-la), 98.2 (d, C-ld), 98.5 (d, C-lc), 101.4 (d, C-lb), 114.3, 118.6, 123.2, 123.3, 123.4, 127.2, 127.3, 127.4, 127.4, 127.5, 127.5, 127.6, 127.7, 127.7, 127.8, 127.9, 128.0, 128.1, 128.1, 128.2, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.4, 129.6, 131.6, 133.8 (32 x d, 54 x Ar-C), 136.9, 137.1, 137.9, 138.0, 138.1, 138.4, 138.5, 138.6, 138.6, 138.8, 139.1 (11 x s, 12 x Ar-C), 151.0, 155.5, 169.6 (3 x s, 3 x C=0); HRMS (ES⁺) Calculated For Cii₃Hi₂₇N0₂₄Si (MH⁺) 1910.85. Found (MH⁺) 1910.85.

/)-Methoxyphenyl 2,3,4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-d-manno pyranosyl- (1— »2)- 3,4,6- tri-0-benzyl-a-D-mannopyranosyl- (1— »6)-2-0-acetyl-4-0-benzyl- [2,3,4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-d-manno pyranosyl- (1— »2)- 3,4,6- tri-O- benzyl-a-D-mannopyranosyl-(l→3)]-p-d-mannopyranosyl-(l→4)-3,6-di-0-benzyl-2- deoxy-2-phthalimido-p-d-glucopyranoside 7.2

A solution of 7.1 (0.15 g, 0.08 mmol), and 6.8 (0.17 g, 0.16 mmol) in DCM (10 mL) was added to a flame-dried round bottom flask containing 3A molecular sieves (0.3 g). The solution was cooled to -20 °C under an atmosphere of nitrogen, stirred for 30 min and NIS (0.04 g, 0.19 mmol) was added. After 10 min, triflic acid (1.0 μί, 0.01 mmol) was added. After a further 30 min t.l.c. (petrol: ethyl acetate, 2: 1) indicated the formation of a major product (Rf 0.6) and the complete consumption of alcohol starting material 7.1 (Rf 0.3). 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 NaHC0₃ (10 mL), brine, dried over anhydrous Na₂S0₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (petrohethyl acetate, 2: 1) to afford compound 7.2 (0.24 g, 72%) as a white foam. [α]ο

+11 (c, 1.0 in CHC1₃); v_max ( Br disk) 1716, 1635 (s, C=0) cm^"1; 5_H (400 MHz, CDC1₃) 1.02-1.10 (42H, m, 6 x CH(CH₃)₂), 2.09 (3H, s, CH₃), 3.24-3.29 (1H, m, H-5b), 3.40- 3.43 (1H, m, H-6b), 3.49-4.19 (31H, m, H-4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H- 2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd'), 3.72 (3H, s, OCH₃), 4.23-4.29 (1H, m, H-3a), 4.30-4.57 (20H, m, H-2a, 19 x PhCH₂), 4.61- 4.69 (5H, m, 5 x PhCH₂) 4.71-4.82 (6H, m, H-lc, H-lc', 4 x PhCH₂), 4.86-4.92 (2H, m, 2 x

PhCH₂), 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, CDC1₃) 12.0 (d, CH(CH₃)₂), 18.0 (q, CH(CH₃)₂), 21.1 (s, CH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C- 6d'), 71.0 (d, C-2b), 71.4, 71.5, 71.8, 71.9, 72.0, 72.1, 72.2, 72.3, 72.3, 73.0, 73.7, 74.3, 74.5, 74.6, 74.7 (15 x t, 15 x PhCH₂), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C-4d), 74.5 (d, C-4d'), 74.5 (d, C-2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C- 4a), 79.6 (d, C-4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 80.2 (d, C-3d'), 97.4 (d, C-la), 98.2 (d, C-ld), 98.6 (d, C-ld'), 99.0 (d, C-l c), 99.6 (d, C-lc'), 101.6 (d, C-lb),

114.3, 118.6, 126.7, 126.8, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.3, 127.3, 127.3,

127.4, 127.4, 127.4, 127.5, 127,5, 127.6, 127.6, 127.7, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3,

128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.4, 129.6, 131.6, 133.8 (50 x d, 84 x Ar-C), 136.9, 137.1, 137.9, 138.0, 138.0, 138.1, 138.2, 138.2, 138.3, 138.4, 138.5, 138.6, 138.6, 138.7, 138.8, 139.0, 139.1 (17 x s, 18 x Ar-C), 151.0, 155.2, 169.6 (3 x s, 3 x C=0). />-Methoxyphenyl 2,3,4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-d-manno pyranosyl- (1— »2)- 3,4,6- tri-0-benzyl-a-D-mannopyranosyl- (1— »6)-2-0-acetyl-4-0-benzyl- [2,3,4-tri-0-benzyl-6-0-tri-/s0-propylsilyl-a-d-manno pyranosyl- (1— »2)- 3,4,6- tri-O- benzyl-a-D-mannopyranosyl-(l→3)]-p-d-mannopyranosyl-(l→4)-2-acetamido-3,6- di-O-benzyl-2-deoxy -β-d-glucopyranoside 7.3

Compound 7.2 (0.08 g, 0.03 mmol) was dissolved in a mixture of methanol (4 mL) and ethylene diamine (2 mL), and the solution was refluxed under an atmosphere of nitrogen. After 16 h, t.l.c. (petrohethyl acetate, 2: 1) indicated the formation of a single product (Rf 0.25) and the complete consumption of starting material (Rf 0.60). The reaction mixture was concentrated in vacuo and co-distilled five times with toluene (5 mL). The residue was dissolved in pyridine (2 mL), cooled to 0°C and acetic anhydride (0.7 mL) was added. The reaction mixture was then stirred at rt under an atmosphere of nitrogen. After 20 h, t.l.c. (petrohethyl acetate, 2: 1) indicated the formation of a single product (Rf 0.35) and the complete consumption of amine intermediate (Rf 0.25). The reaction mixture was concentrated in vacuo, the residue dissolved in DCM (10 mL), washed with water (10 mL), sodium hydrogen carbonate (2 x 10 mL of a saturated aqueous solution), dried (Na₂S0₄), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (petrol: ethyl acetate, 2: 1) to afford compound 7.3 (0.06 g, 71%) as a pale yellow foam. [a]_D ²⁰ +9 (c, 1.0 in CHC1₃); v_max ( Br disk) 1716, 1635 (s, C=0) cm^"1; 5_H (400 MHz, CDC1₃) 1.01-1.10 (42H, m, 6 x CH(CH₃)₂), 1.45 (3H, s, COCH₃), 1.85 (3H, s, CH₃), 3.21-3.27 (1H, m, H-5b), 3.32-3.43 (1H, m, H-6b), 3.49-4.19 (31H, m, H- 4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd'), 3.72 (3H, s, OCH₃), 4.23-4.29 (1H, m, H-3a), 4.30-4.57 (20H, m, H-2a, 19 x PhCH₂), 4.61- 4.69 (5H, m, 5 x PhCH₂) 4.71-4.82 (6H, m, H-lc, H- lc', 4 x PhCH₂), 4.86-4.92 (2H, m, 2 x PhCH₂), 5.14 (1H, s, H-lb), 5.20 (1H, s, H-ld'), 5.24 (1H, s, H-ld), 5.42-5.45 (2H, m, H-la, H-2b), 6.69-6.72 (2H, m, 2 x Ar-H), 6.79- 6.83 (2H, m, 2 x Ar-H), 7.10-7.37 (75H, m, 70 x Ar-H); 5_C (100 MHz, CDC1₃) 12.0 (d, CH(CH₃)₂), 18.0 (q, CH(CH₃)₂), 21.1 (s, CH₃), 23.4 (s, COCH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C-6d'), 71.0 (d, C-2b), 71.4, 71.5, 71.8, 71.9, 72.0, 72.1, 72.2, 72.3, 72.3, 73.0, 73.7, 74.3, 74.5, 74.6, 74.7 (15 x t, 15 x PhCH₂), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C-4d), 74.5 (d, C- 4d'), 74.5 (d, C-2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C-4a), 79.6 (d, C-4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 81.4 (d, C-3d'), 96.7 (d, C-la), 98.2 (d, C-ld), 98.4 (d, C-l d'), 98.6 (d, C- lc), 98.7 (d, C-lc'), 101.5 (d, C-lb), 114.3, 118.6, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.3, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127,5, 127.6, 127.6, 127.7, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8, 129.4, 129.6,

131.6, 133.8 (48 x d, 80 x Ar-C), 137.4, 138.0, 138.1, 138.3, 138.4, 138.5, 138.5, 138.6,

138.7, 138.8, 138.9, 139.0, 139.1, 139.2, 151.6, 155.1 (16 x s, 16 x Ar-C), 169.6, 170.5 (2 x s, 2 x C=0); HRMS (ES⁺) Calculated For C170H2Q3NO33S-2 (MH⁺) 2843.38. Found (MH⁺) 2843.38.

/>-Methoxyphenyl 2,3,4-tri-0-benzyl -oc-d-manno pyranosyl- (1— »2)- 3,4,6- tri-O- benzyl-a-D-mannopyranosyl- (l→6)-2-0-acetyl-4-0-benzyl-[2,3,4-tri-0-benzyl -a-d- nianno pyranosyl- (1— »2)- 3,4,6- tri-0-benzyl-a-D-mannopyranosyl- (1— »3)]-p-d- mannopyranosyl-(l— »4)-2-acetamido-3,6-di-0-benzyl-2-deoxy -β-d-glucopyranoside

7.4

Compound 7.3 (50 mg, 0.02 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 (22 μί, 0.18 mmol) was added dropwise. The reaction was stirred at 0 °C for 1 h, after which time t.l.c. (petrol: ethyl acetate, 1 : 1) indicated the complete consumption of starting material (Rf 0.5) and the formation of a single product (Rf 0.3). The reaction mixture was diluted with DCM (10 mL), washed with sodium hydrogen carbonate (10 mL of a saturated aqueous solution), dried (Na₂S0₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography

(petrohethyl acetate, 1 : 1) to afford compound 7.4 (30 mg, 68%) as a white foam. [α]ο +15 (c, 1.0 in CHCI₃); Vmax ( Br disk) 3428 (s, N-H stretch), 1746, 1739 (s, C=0) cm^"1; 5_H (400 MHz, CDCI₃) 1.60 (3H, s, COCH₃), 1.94 (3H, s, CH₃), 3.17-3.20 (1H, m, H-5b), 3.24-3.31 (1H, m, H-6b), 3.49-4.11 (31H, m, H-4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H- 3c', H-4c', H-5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd'), 3.72 (3H, s, OCH₃), 4.23-4.29 (1H, m, H-3a), 4.21-4.65 (25H, m, H-2a, 24 x PhCH₂), 4.67 (1H, s, Pile') 4.76- 4.89 (6H, m, 6 x PhCH₂), 4.77-4.89 (6H, m, 6 x PhCH₂), 4.95 (1H, s, H-lc), 5.01(1H, s, H-ld'), 5.04 (1H, s, H-ld), 5.16 (1H, s, H-lb), 5.31( 1H, d, Ji_>2 8.0 Hz, H-la), 5.38-5.40 (1H, m, H-2b), 6.69-6.72 (2H, m, 2 x Ar-H), 6.79-6.83 (2H, m, 2 x Ar-H), 7.10- 7.37 (75H, m, 70 x Ar-H); 5_C (100 MHz, CDC1₃) 21.0 (s, CH₃), 23.2 (s, COCH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C-6d'), 71.0 (d, C-2b), 71.4, 71.5, 71.8, 71.9, 72.0, 72.1, 72.2, 72.3, 72.3, 73.0, 73.7, 74.3, 74.5, 74.6, 74.7 (15 x t, 15 x PhCH₂), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C- 4d), 74.5 (d, C-4d'), 74.5 (d, C-2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C-4a), 79.6 (d, C-4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 81.4 (d, C-3d'), 97.1 (d, C-la), 98.7 (d, C-ld), 98.8 (d, C- ld'), 99.8 (d, C-lc), 100.0 (d, C-lc'), 101.5 (d, C-lb), 114.3, 118.6, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.3, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127,5, 127.6, 127.6, 127.7, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.8,

129.4, 129.6, 131.6, 133.8 (48 x d, 80 x Ar-C), 137.4, 138.0, 138.1, 138.3, 138.4, 138.5,

138.5, 138.6, 138.7, 138.8, 138.9, 139.0, 139.1, 139.2, 151.6, 155.1 (16 x s, 16 x Ar-C), 169.6, 170.5 (2 x s, 2 x C=0); HRMS (ES⁺) Calculated For Ci₅2Hi₆₃N0₃₃ (MH⁺) 2531.11. Found (MH⁺) 2531.11.

/>-Methoxyphenyl 2,3,4-tri-0-benzyl -oc-d-manno pyranosyl- (1— »2)- 3,4,6- tri-O- benzyl -6-0-dibenzyloxyphosphoryl-a-D-mannopyranosyl- (1— »6)-2-0-acetyl-4-0- benzyl-[2,3,4-tri-0-benzyl -6-0-dibenzyloxyphosphoryl -a-d-manno pyranosyl- (1— »2)- 3,4,6- tri-0-benzyl-a-D-mannopyranosyl-

il— »4)-2-acetamido-3,6-di-0-benzyl-2-deoxy -β-d-glucopyranoside 7.5

Diol 7.4 (13 mg, 0.005 mmol) and lH-tetrazole (0.08 mL of a 0.45 M solution in acetonitrile) were added to a flame-dried flask and dissolved in DCM (0. 50 mL). The solution was stirred at rt lh under nitrogen atmosphere. Dibenzyl N, N-diisopropyl phosphoramidate (0.014 mL, 0.042 mmol) was added, and the reaction stirred at rt for 18 h. After this time t.l.c. (MeOH:DCM, 1 :50) indicated complete consumption of the diol starting material (Rf 0.3) and the formation of a major product (Rf 0.60). The 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.

(MeOH:DCM, 1 :50) indicated complete consumption of the phosphine intermediate (Rf 0.60) and the formation of a major product (Rf 0.25). The 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

(MgS0₄), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH:DCM, 1 :50) to afford compound 7.5 (15 mg, 94% over two steps) as a white foam. [a]_D ²⁰ +12 (c, 1.0 in CHC1₃); v_max ( Br disk) 3430 (s, N-H stretch), 1744, 1740 (s, C=0) cm^"1; 5_H (400 MHz, CDC1₃) 1.60 (3H, s, COCH₃), 1.96 (3H, s, CH₃), 3.36-3.48 (2H, m, H-5b, H-6b), 3.60-4.15 (31H, m, H-4a, H-5a, H-6a, H-6'a, H- 3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H- 6'd'), 3.72 (3H, s, OCH₃), 4.23-4.29 (1H, m, H-3a), 4.21 -4.62 (16H, m, H-2a, 16 x PhCH₂), 4.01 -5.04 (20H, m, H- l c, H-l c', H- l d, H-l d', 16 x PhCH₂), 5.32 (1H, s, H-lb), 5.36( 1H, d, Ji_;2 8.0 Hz, H- l a), 5.58-5.59 (1H, m, H-2b), 6.69-6.72 (2H, m, 2 x Ar-H), 6.79-6.83 (2H, m, 2 x Ar-H), 7.10-7.37 (95H, m, 70 x Ar-H); 5_C (100 MHz, CDC1₃) 21.0 (s, CH₃), 23.2 (s, COCH₃), 55.6 (q, OCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C- 6d'), 71.0 (d, C-2b), 71.4, 71.5, 71.8, 71.9, 72.0, 72.1 , 72.2, 72.3, 72.3, 73.0, 73.7, 74.3, 74.5, 74.6, 74.7 (15 x t, 15 x PhCH₂), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C-4d), 74.5 (d, C-4d'), 74.5 (d, C-2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C- 4a), 79.6 (d, C-4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 81.4 (d, C-3d'), 97.1 (d, C-la), 98.7 (d, C-ld), 99.1 (d, C-ld'), 99.3 (d, C-l c), 99.8 (d, C-lc'), 101.2 (d, C-lb), 114.3, 118.6, 126.8, 126.9, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.2, 127.2, 127.3, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.5, 127.5, 127.6, 127.6, 127.6, 127.6, 127.6, 127.7, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9, 127.9, 127.9, 128.0, 128.0, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3, 128.3, 128.3, 128.4, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.6 (59 x d, 100 x Ar-C), 135.7, 135.8, 135.8, 135.9, 135.9, 135.9, 138.0, 138.1, 138.2, 138.2, 138.3, 138.3, 138.4, 138.5, 138.6, 138.7, 138.8, 139.0, 139.2, 151.7, 154.9 (21 x s, 21 x Ar-C), 169.6, 170.5 (2 x s, 2 x C=0); 5_P (162 MHz, CDC1₃) -1.25, -1.54; HRMS (ES⁺) Calculated For C180H189NO39P2 (M+2H)²⁺ 1527.13. Found [M+2H]²⁺ 1527.13. 2,3,4-tri-0-benzyl -oc-d-manno pyranosyl- (1— »2)- 3,4,6- tri-0-benzyl -6-0- dibenzyloxyphosphoryl-a-D-mannopyranosyl- (1— »6)-2-0-acetyl-4-0-benzyl- [2,3,4- tri-O-benzyl -6-0-dibenzyloxyphosphoryl -a-d-manno pyranosyl- (1— »2)- 3,4,6- tri- 0-benzyl-a-D-mannopyranosyl- (1— »3)]-p-d-mannopyranosyl-(l— »4)-2-acetamido- 3,6-di-0-benzyl-2-deoxy -β-d-glucopyranoside 7.6

PMP glycoside 7.5 (15 mg, 0.005 mmol) was suspended in a mixture of acetonitrile (0.8 mL) and water (0.21 mL). Ceric ammonium nitrate (14 mg, 0.025 mmol) was added, and the reaction mixture stirred at rt. After 1 h, t.l.c. (MeOH:DCM, 1 :50) indicated complete consumption of starting material (Rf 0.25) and the formation of a single product (Rf 0.15). 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 (Na₂S0₄), 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. v_max ( Br disk) 3430 (s, N-H stretch), 1740, 1735 (s, C=0) cm^"1; 5_H (400 MHz, CDC1₃) 1.65 (3H, s, COCH₃), 1.97 (3H, s, CH₃), 3.21 -4.15 (33H, m, H-5b, H-6b, H-4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H- 5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6' d, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd'), 3.72 (3H, s, OCH₃), 4.23-4.29 (1H, m, H-3a), 4.21 -4.62 (16H, m, H-2a, 16 x PhCH₂), 4.01 -5.04 (21H, m, H- l a, H- l c, H- l c', H-ld, H-ld', 16 x PhCH₂), 5.27 (1H, s, H-lb), 5.53 (1H, brs, H-2b), 7.10-7.37 (95H, m, 70 x Ar-H) 5c (100 MHz, CDC1₃) 21.0 (s, CH₃), 23.2 (s, COCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C-6d'), 71.0 (d, C-2b), 71.4, 71.5, 71.8, 71.9, 72.0, 72.1, 72.2, 72.3, 72.3, 73.0, 73.7, 74.3, 74.5, 74.6, 74.7 (15 x t, 15 x PhCH₂), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C-4d), 74.5 (d, C-4d'), 74.5 (d, C- 2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C-4a), 79.6 (d, C-4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 81.4 (d, C-3d'), 96.8 (d, C-la), 99.3 (d, C-ld), 99.4 (d, C-ld'), 99.5 (d, C-l c), 99.6 (d, C- lc'), 101.2 (d, C-lb), 126.8, 126.9, 126.9, 127.0, 127.1, 127.1, 127.2, 127.2, 127.2,

127.2, 127.3, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.5, 127.5, 127.6, 127.6, 127.6, 127.6, 127.6, 127.7, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9, 127.9, 127.9, 128.0, 128.0, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3,

128.3, 128.3, 128.4, 128.4, 128.4, 128.5, 128.5, 128.5, 128.6, 128.6 (57 x d, 95 x Ar-C), 135.7, 135.8, 135.8, 135.9, 135.9, 135.9, 138.0, 138.1, 138.1, 138.2, 138.2, 138.3, 138.3,

138.4, 138.5, 138.6, 138.7, 138.8, 139.2 (19 x s, 19 x Ar-C), 169.7, 171.3 (2 x s, 2 x C=0); δ_Ρ (162 MHz, CDC1₃) -1.37, -1.50; HRMS (ES⁺) Calculated For Ci₇₃Hi₈₄N0₃₈P₂ [M+2H]²⁺ 1474.11. Found (M+2H)²⁺ 1474.11.

6-0-Phosphate -a-d-manno pyranosyl- (1— »2) - a-D-mannopyranosyl- (1— »6)- [6-0- Phosphate -a-d-manno pyranosyl- (1— »2)-a-D-mannopyranosyl- (1— »3)]-p-d- mannopyranosyl-(l— »4) )-2-acetamido-2-deoxy-D-glucopyranose 7.7

A solution of protected hexasaccharide 7.6 (30 mg, 0.01 mmol) in THF (20 mL) was added to NH₃ (/) (20 mL) at -33 °C. The minimum amount of sodium required to make the mixture to turn deep blue was added to the stirred mixture. After 30 min MeOH (4 mL) was added, and reaction mixture was stirred for a futher 1 h, warmed to rt, and the solvent was removed in vacuo. Gel filtration of the crude residue on a Sephadex G-10 column (eluting with 0.01% NH₃) afforded deprotected hexasaccharide 7.7 (10.2 mg, 83%), as a white foam as a mixture of α:β anomers. δ_Η (400 MHz, CDC1₃) 1.98 (3H, s,

CH₃), 3.11-4.19 (35H, m, H-5b, H-6b, H-4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd', H-3a, H-2a), 4.92-5.09 (5H, m, H-la, H-lc, H-lc', H-ld, H-ld'), 5.13(1H, bs, H-la), 5.31 (1H, brs, H- lb); 5c (100 MHz, CDC1₃) 23.2 (s, COCH₃), 55.7 (d, C-2a), 62.5 (t, C-6c), 62.9 (t, C-6c'), 67.1 (d, C-5a), 68.1 (d, C-5b), 69.1 (t, C-6a), 69.2 (t, C-6b), 69.3 (t, C-6d), 69.4 ( t, C- 6d'), 71.0 (d, C-2b), 72.8 (d, C-5d), 73.1 (d, C-5d'), 73.2 (d, C-3b), 74.3 (d, C-5c), 74.3 (d, C-5c'), 74.4 (d, C-4d), 74.5 (d, C-4d'), 74.5 (d, C-2c), 74.6 (d, C-2c'), 74.7 (d, C-4c), 74.8 (d, C-4c'), 75.1 (d, C-2d), 76.3 (d, C-2d'), 76.9 (d, C-3a), 78.8 (d, C-4a), 79.6 (d, C- 4b), 79.7 (d, C-2c), 80.0 (d, C-2c'), 80.1 (d, C-3d), 81.4 (d, C-3d'), 96.7 (d, C-la), 99.3 (d, C-ld), 99.4 (d, C-ld'), 99.5 (d, C-lc), 99.6 (d, C-l c'), 101.2 (d, C-lb), 171.3 (s, C=0); 5_P (162 MHz, CDCI3) 3.91, 4.13; HRMS (ES⁺) Calculated For C₃₈H₆₈N0₃7P2 (MH⁺)

1192.29. Found (MH⁺) 1192.29.

2-Methyl [6-0-Phosphate -a-d-manno pyranosyl- (1— »2)- a-D-mannopyranosyl- (1— »6)- [6-0-Phosphate -a-d-manno pyranosyl- (1— »2)-a-D-mannopyranosyl- (1— »3)]-p-d-mannopyranosyl-(l— »4) )-2-acetamido-l,2-deoxy-D-glucopyrano]-[2,l- </]-oxazoline 7.8

Hemiacetal 7.7 (4 mg, 0.0034 mmol) and triethylamine (4.3 μί, 0.03 mmol) were dissolved in D₂0 (37 μί) 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% NH₃, afforded oxazoline 7.8 (1.8 mg, 95%) as a white foam. 5_H (400 MHz, CDC1₃) 1.98 (3H, s, CH₃), 3.11-4.19 (35H, m, H-5b, H-6b, H-4a, H-5a, H-6a, H-6'a, H-3b, H-4b, H-6'b, H-2c, H-3c, H-4c, H-5c, H-6c, H-6'c, H-2d, H-3d, H-4d, H-5d, H-6d, H-6'd, H-2c', H-3c', H4c', H5c', H-6c', H-6'c', H-2d', H-3d', H-4d', H-5d', H-6d', H-6'd', H-3a, H-2a), 4.92-5.09 (5H, m, H-la, H-lc, H-lc', H-ld, H-ld'), 5.31 (1H, brs, H-lb) 6.08 (1H, d, J_h2 7.3 Hz, H-la); 5_P (162 MHz, CDC1₃) 4.46; HRMS (ES⁺) Calculated For C₃₈H₆₇N0₃₇P2 [M- H₂0+H]⁺ 1192.29. Found (M-H₂0+H)⁺ 1192.29.

EXAMPLE 6

Glycosylation of the hexasaccharide oxazoline donor with Fmoc-Asn(GlcNAc)-OH with Endo A:

A solution of the hexasaccharide oxazoline 7.8 (0.34 mg, 0.30μηιο1) and Fmoc-

Asn(GlcNAc)-OH (0.06 mg, 0.10 μmol) was incubated with l μg of Endo-A in 20 μΐ, of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C. After 2 h, RP-HPLC indicated the formation of a new product, which was then purified directly by RP-HPLC to give 7.9 (12%, HPLC yield based on acceptor); ¾ = 10.6 min; ESI-MS: calculated for

C5oH₈₇N₄044P2 (M-Fmoc+H)⁺ : 1509.42. Found: 1509.42 (M-Fmoc+H)⁺

Glycosylation of the hexasaccharide oxazoline donor with Fmoc-Asn(GlcNAc)-OH using WT Endo M:

A solution of the hexasaccharide oxazoline 7.8 (0.66 mg, 0.50μηιο1) and Fmoc- Asn(GlcNAc)-OH (0.06 mg, 0.10 μιηοΐ) was incubated with ^g of WT Endo-M in 20 μΐ, of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C. After 2 h, RP-HPLC indicated the formation of a new product, which was then purified directly by RP-HPLC to give 7.9 (6%, HPLC yield based on acceptor); ¾ = 10.6 min; ESI-MS: calculated for C₅oH₈7N₄0₄₄P₂ (M-Fmoc+H)⁺ : 1509.42. Found: 1509.42(M-Fmoc+H)⁺

Glycosylation of the hexasaccharide oxazoline donor with Fmoc-Asn(GlcNAc)-OH using the N175Q Endo M mutant:

A solution of the hexasaccharide oxazoline 7.8 (0.66 mg, 0.50μηιο1) and Fmoc- Asn(GlcNAc)-OH (0.06 mg, 0.10 μιηοΐ) was incubated with ^g of Endo M N175Q in 20 μΐ, of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C. After 2 h, RP-HPLC indicated the formation of a new product, which was then purified directly by RP-HPLC to give 7.9 (8%, HPLC yield based on acceptor); t_R = 10.6 min; ESI-MS: calculated for C₅oH₈7N₄0₄₄P₂ (M-Fmoc+H) : 1509.42. Found: 1509.42 (M-Fmoc+H) . EXAMPLE 7

Glycosylation of de-glycosylated fabrazyme with the M6P-tetrasaccharide oxazoline donor Fabrazyme (5.0 mg) was deglycosylated with wild type Endo A (50 μg of Endo A) in 5 of sodium phosphate buffer (100 mM, pH 6.5) at 37 °C for 10 h (Figure 7). After Endo A mediated hydrolysis was complete the de-glycosylated Fabrazyme (dFab) was purified using RP-HPLC to give dFab.

A solution of the tetrasaccharide oxazoline 13 (0.24 mg, 0.28μηιο1) and dFab (0.21 mg, 0.005μηιο1) was incubated with ^g of Endo-A in 20 μΐ_^ of sodium phosphate buffer (100 mM, pH 6.5) at 23 °C for 2h to give double glycosylation of dFab with the addition of two tetrasaccharide units - M6P2Fab. HRMS: calculated for (2[M6P-tetra]-dFab+2H⁺): 48218.30; Found: 48218.30.

Although the invention has been described by way of example and with reference to particular embodiments, it is to be understood that modifications and/or improvements may be made without departing from the scope of the invention.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. The skilled person will appreciate that the invention as set forth and described herein is not limited solely to the aspects, embodiments, and examples as described, but also encompasses within the spirit and scope of the invention, those variations and modifications of the invention as would be obvious to the person of skill in the art (including the person of ordinary skill in the art) in view of the disclosures provided herein and the common general knowledge. INDUSTRIAL APPLICATION

The 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 according to the invention as disclosed herein all have industrial application for the production and therapeutic use of phosphorylated glycoproteins and glycopeptides.

REFERENCES

Zou et al, J. Am. Chem. Soc. 2011, 133, 18975-18991 ; Schwarz et al , Nat. Chem. Biol 2010, 6, 264-266

Schwarz et al, Nat. Chem. Biol 2010, 6, 264-266 Ueda et al , Bioorg. Med. Chem. Lett. 2010, 20 4631-4634 Huang et al , Org. Biomol. Chem. 2010, 8, 5224-5233 W02007/104789

Noguchi, M. et. al., J Org. Chem. 2009, 74, 2210-2212 Takegawa et. al, Appl Environ. Microbiol, 1989, 55, 3107-31 12 Yamamoto et. al., Biochem. Biophys. Res. Commun., 1994, 203, 244-252 Umekawa et. al., J. Biol Chem. 2010, 285, 51 1 -521) Mizuochi et. al., J Biochem. 1984, 95, 1209-1213 Yunpeng Liu, et. al., ChemBioChem 2011, 12, 685 - 690

Claims

WHAT WE CLAIM IS:

1. 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.

2. The method of claim 1, wherein the phosphorylated donor oligosaccharide is a synthetic phosphorylated oligosaccharide.

3. The method of claim 1 or 2, wherein the method further comprises isolating and/or purifying the phosphorylated glycoprotein or glycopeptide.

4. The method of any one of claims 1 to 3, wherein 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, wherein the ENGase, modified ENGase or functional fragment, variant, analogue or derivative thereof 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), preferably wherein the ENGase is Endo A, preferably wherein the Endo A is a modified or mutant Endo A, preferably the mutant Endo A is E173H or N171Q, or preferably where the

endoglycosidase is Endo M or a modified or mutant Endo M, preferably the mutant Endo M is N175Q.

5. The method of any one of claims 1 to 4, wherein the phosphorylated donor oligosaccharide is a phosphorylated donor oligosaccharide comprising monosaccharide residues linked by glycosidic linkages, and an anomeric leaving group, preferably wherein the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, and the leaving group is an oxazoline.

6. The method of any one of claims 1 to 5, wherein 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, preferably a tetra- or hexa-saccharide, and preferably wherein the phosphorylated donor oligosaccharide is a synthetic phosphorylated donor oligosaccharide, preferably a synthetic phosphorylated donor oligosaccharide oxazoline.

7. The method of any one of claims 1 to 6, wherein the phosphorylated

glycoprotein or glycopeptide comprises at least one phosphorylated oligosaccharide, preferably wherein 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.

8. The method of any one of claims 1 to 7, wherein 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.

9. The method of claim 8, wherein the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.

10. The method of claim 8 or 9, wherein at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.

1 1. The method of any one of claims 1 to 10, wherein the chemical bond between the at least one GlcNAc residue on the acceptor protein or peptide and the phosphorylated oligosaccharide is a β 1-4 glycosidic linkage as found in nature.

12. The method of any one of claims 1 to 11, wherein 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, preferably an enzyme, a modified enzyme, or a functional fragment, variant, analogue or derivative of an enzyme, preferably a fusion protein or peptide comprising an enzyme, a modified enzyme, or a functional portion, fragment, variant, analogue or derivative thereof.

13. The method of claim 12, wherein the enzyme, or modified enzyme is a lysosomal enzyme or a functional fragment, variant, analogue or derivative thereof.

14. The method of claim 12, wherein the fusion protein or peptide comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme.

15. The method of claim 13 or 14, wherein the lysosomal enzyme is selected from the group consisting of an a-sialidase, cathepsin A, a-mannosidase, β-mannosidase, glycosylasparaginase, a-fucosidase, a-N-acetylglucosaminidase, β -galactosidase, β - hexosaminidase a -subunit, β -hexosaminidase β -subunit, GM2 activator protein, glucocerebrosidase, saposin C, arylsulfatase B, saposin B, formyl-glycin generating enzyme, β -galactosylceramidase, a -galactosidase A, iduonate sulfatase, a -iduronidase, heparan N-sulfatase, acetyl-CoA transferase, N-acetyl glucosaminidase, β -glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6- sulfatase, ayaluronidase, a -glucosidase, a-sphingomyelinase, acid ceramidase, acid lipase, cathepsin , tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NCI, CLN3, CLN6, CLN8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit, preferably an acid a-glucosidase or functional fragment, variant, analogue or derivative thereof, preferably the acid a-glucosidase is alglucosidase-a-rch, preferably an a - galactosidase A or a modified form thereof, preferably agalsidase alpha or agalsidase beta.

16. The method of any one of claims 1 to 15, wherein 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.

17. A phosphorylated glycoprotein or glycopeptide made according to the method of any one of claims 1 to 16.

18. A phosphorylated glycoprotein or glycopeptide obtainable by a method of any one of claims 1 to 16.

19. 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, preferably wherein the natural linkage is a β 1-4 glycosidic linkage.

20. A phosphorylated glycoprotein or glycopeptide of claim 19, wherein the synthetic phosphorylated donor oligosaccharide is a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, or undeca-saccharide, preferably wherein the synthetic

phosphorylated donor oligosaccharide is a tetra- or hexa-saccharide.

21. A phosphorylated glycoprotein or glycopeptide of claim 19 or 20, wherein the phosphorylated glycoprotein or glycopeptide comprises at least one synthetic

phosphorylated oligosaccharide, preferably 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.

22. A phosphorylated glycoprotein or glycopeptide of any one of claims 19 to 21, wherein 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.

23. A phosphorylated glycoprotein or glycopeptide of claim 22, wherein the phosphorylated glycoprotein or glycopeptide comprises at least two M6P residues.

24. A phosphorylated glycoprotein or glycopeptide of claim 22 or claim 23, wherein preferably, at least one mannose-6-phosphate (M6P) residue is a terminal M6P residue.

25. A phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25, wherein 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, preferably wherein the acceptor protein or peptide is a lysosomal enzyme or functional fragment, variant, analogue or derivative thereof, or preferably wherein the acceptor protein or peptide comprise a fusion protein or peptide that comprises at least a functional portion, fragment, variant, analogue or derivative of a lysosomal enzyme.

26. A composition comprising a phosphorylated glycoprotein or glycopeptide made according to a method of any one of claims 1 to 16 and a suitable carrier, diluent or excipient.

27. A composition comprising a phosphorylated glycoprotein or glycopeptide obtainable by a method of any one of claims 1 to 16 and a suitable carrier, diluent or excipient.

28. A composition comprising the phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25, preferably wherein the composition is a pharmaceutical composition, preferably wherein the pharmaceutical composition comprises a therapeutically effective amount of the phosphorylated glycoproteion or glycopeptide.

29. A phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25 for use as a medicament.

30. A composition of any one of claims 26 to 28 for use as a medicament.

31. A phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25 for use in treating a disease or condition.

32. A composition of any one of claims 26 to 28 for use in treating a disease or condition.

33. Use of a phosphorylated glycoprotein or glycopeptide of any one of claims 17 to

25 in the manufacture of a medicament for treating a disease or condition.

34. Use of a composition of any one of claims 26 to 28 in the manufacture of a medicament for treating a disease or condition.

35. A use of claim 33 or 34, wherein the disease or condition is a lysosomal storage disease (LSD) or disorder related thereto.

36. A use of claim 35, wherein the LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, β-mannosidosis, aspartylglucosaminuria, fucosidosis, GMl gangliosidosis, GM2-gangliosidosis (Tay-Sachs), GM2-gangliosidosis (Sandhoff), GM2-gangliosidosis, Gaucher disease, metachromatic leukodystrophy, multiple sulfatase deficiency, globoid cell leukodystrophy, Fabry disease, MPS II

(Hunter), MPS 1 (Hurler, Scheie), MPS Ilia (Sanfilippo A), MPS IIIc (Sanfilippo C), MPS Illb (Sanfilippo B), MPS VII (Sly), MPS Hid (Sanfilippo D), MPS VI, MPS IVA (Morquio A), MPS IX, Pompe disease, Niemann Pick type A and B, Farber

lipogranulomatosis, Wolman and cholesteryl ester storage disease, pycnodystostosis, ceroide lipofuscinosis 2, ceroide lipofuscinosis 1 , cystinosis, salla disease, mucolipidosis III (I-cell), mucolipidosis IV, Danon disease, Neimann Pick type C, ceroid lipofuscinosis, ceroid lipofuscinosis 6, ceroid lipofuscinosis, 8, Chediak-Higashi disease, Griscelli type 1 , Griscelli type 2, Griscelli type 3, and Hermansky Pudliak 2 disease.

37. A use of any one of claims 33 to 36, wherein the disease or condition is Fabry disease.

38. A method for treating a lysosomal storage disease (LSD) comprising

administering a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25 to a subject in need thereof.

39. A method for treating a lysosomal storage disease comprising administering a composition of any one of claims 26 to 28 to a subject in need thereof.

40. A method of claim 39, wherein the composition comprises a therapeutically effective amount of a phosphorylated glycoprotein or glycopeptide.

41. A method of any one of claims 38 to 40, wherein the LSD is selected from the group consisting of sialidosis, galactosialidosis, a-mannosidosis, β-mannosidosis, aspartylglucosaminuria, fucosidosis, GM1 gangliosidosis, GM2-gangliosidosis (Tay- Sachs), GM2-gangliosidosis (Sandhoff), GM2-gangliosidosis, Gaucher disease, metachromatic leukodystrophy, multiple sulfatase deficiency, globoid cell leukodystrophy, Fabry disease, MPS II (Hunter), MPS 1 (Hurler, Scheie), MPS Ilia (Sanfilippo A), MPS IIIc (Sanfilippo C), MPS Illb (Sanfilippo B), MPS VII (Sly), MPS Hid (Sanfilippo D), MPS VI, MPS IVA (Morquio A), MPS IX, Pompe disease, Niemann Pick type A and B, Farber lipogranulomatosis, Wolman and cholesteryl ester storage disease,

pycnodystostosis, ceroide lipofuscinosis 2, ceroide lipofuscinosis 1, cystinosis, salla disease, mucolipidosis III (I-cell), mucolipidosis IV, Danon disease, Neimann Pick type C, ceroid lipofuscinosis, ceroid lipofuscinosis 6, ceroid lipofuscinosis, 8, Chediak-Higashi disease, Griscelli type 1, Griscelli type 2, Griscelli type 3, and Hermansky Pudliak 2 disease.

42. A method of any one of claims 38 to 41, wherein the LSD is Fabry disease.

43. A method for identifying a cell that expresses a M6P receptor, the method comprising a) contacting the cell with a phosphorylated glycoprotein or glycopeptide of any one of claims 17 to 25, 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.