MODIFIED SUGAR SUBSTRATES AND METHODS OF USE

RELATED APPLICATIONS

This application claims the benefit of US Provisional application No. 61/027,782, filed on February 11, 2008. The entire contents of the aforementioned application are hereby incorporated herein by reference.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute

(NCI), Center for Cancer Research. This research has been funded in part with Federal funds from the NCI, NIH. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION The present invention relates to the field of glycobiology, and to novel carbohydrate substrates, e.g. sugar substrates, comprising bioactive agents that can be used to make glycoconjugates with therapeutic and diagnostic applications. The present invention provides methods of sythesis of novel carbohydrate substrates and their biological applications. Glycans can be classified as linear or branched sugars. Linear sugars are the glycosaminoglycans comprising polymers of sulfated disaccharide repeat units that are O- linked to a core protein, forming a proteoglycan aggregate (Raman et al, 2005). The branched glycans are found as N- and 0-linked sugars on glycoproteins or on glycolipids (Lowe et al., 2003). These carbohydrate moieties of the linear and branched glycans are synthesized by a superfamily of enzymes, the glycosyltransferases (GTs), which transfer a sugar moiety from a sugar donor to an acceptor molecule. Although GTs

catalyze chemically similar reactions in which a monosaccharide is transferred from an activated derivative, such as a UDP-sugar, to an acceptor, very few GTs bear similarity in primary structure.

Eukaryotic cells express several classes of oligosaccharides attached to proteins or lipids. Animal glycans can be N-linked via beta-GlcNAc to Asn (N-glycans), O-linked via -GaINAc to Ser/Thr (O-glycans), or can connect the carboxyl end of a protein to a phosphatidylinositol unit (GPI-anchors) via a common core glycan structure.

Thus, there is potential to develop carbohydrate substrates comprising bioactive agents that can be used to produce glycoconjugates carrying sugar moieties with bioactive agents. Such glycoconjugates have many therapeutic and diagnostic uses, e.g. in labeling or targeted delivery. Further, such glycoconjugates can be used in the assembly of bio-nanoparticles to develop targeted-drug delivery systems or contrast agents for medical uses.

Accordingly, carbohydrate substrates comprising bioactive agents have many applications in research and medicine, including in the development of pharmaceutical agents, and imaging and diagnostic tools that can be used to diagnose, prevent and treat disease.

SUMMARY OF THE INVENTION As described below, the present invention features methods and compositions for making and using functionalized sugars. The invention includes methods for forming a wide variety of products at a cell or in an in vitro environment, The products may provide a label, a binding site, a modulator of cell function such as a drug or toxin. These methods comprise the steps of making a glycoconjυgate using the novel functionalized sugars according to the invention and then contacting the functional group of the extracellularly expressed glycoconjugate with an agent which selectively reacts with the functional group to form a product.

In a first aspect, the invention features a composition comprising a sugar nucleotide and one or more functional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases.

In one embodiment, the glycosyitransferases are wild type glycosyltransferases. In another embodiment, the glycosyitransferases are altered glycosyltransferases.

In a further embodiment, the giycosyltransferases are selected from the group consisting of galactosy transferases, acetylgatactosy transferases and poiypeptidylgaiactosyltransferases. In a related embodiment, the galactosyltransferase is a beta galactosyltransferase or an alpha acetySgalactosaminyltransferase. In a further related embodiment, the glycosyltransferases are selected from the group consisting of: beta 1 ,4 galactosyltransferase, alpha 1,3 N-Acetylgalactosaminy transferase,

In another embodiment, the sugar nucleotide is selected from the group consisting of UDP-galactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues.

In a further embodiment, the sugar nucleotide comprises a chemical reactive group selected from the group consisting of an azido group, a keto group, an alkyne group or a thiol group. In a related embodiment, the azido group, the keto group, the alkyne group or the thiol group is substituted at the C2 position. In another further embodiment, the C2 position is used for the attachment of a functional group.

In another embodiment, the one or more functional groups is directly attached to the sugar nucleotide.

In a related embodiment, the functional group is selected from the group consisting of: chemical reactive groups, dyes, targeting agents, radiolabeis, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non- cleavable linkers, or a combination thereof.

In still another related embodiment, the one or more functional groups is transferred from the sugar donor nucleotide to an acceptor. In a further embodiment, the acceptor is selected from the group consisting of: a sugar acceptor, a polypeptide acceptor, and a lipid acceptor In a related embodiment, the sugar acceptor is selected from the group consisting of: a polypeptide, a glycopeptide, a glycan, and a lipid glycan. In still another related embodiment, the sugar acceptor is N-acetylglucosamine (GIcNAc). In another aspect, the invention features a method of making a sugar nucleotide comprising one or more functional groups, wherein the sugar nucleotide is a substrate of

one or more glycosyltransferases, the method comprising a step (a) of acylating g]ycosy!amine-1 -phosphate and a step (b) of coupling the product of step (a) with one or more nucleotides thereby making a sugar nucleotide comprising one or more functional groups. In one embodiment, the method further comprises the step of ion exchange.

In another aspect, the invention features a method of making a sugar nucleotide comprising one or more functional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases, the method comprising a step (a) of acylating glycosylamine-1 -phosphate and a step (b) of performing ion exchange; and a step (c) of coupling the product of step (a) or step (b) with one or more nucleotides; thereby making a sugar nucleotide comprising a functional group.

In one enbodiment of the above-mentioned methods, the functional group is selected from the group consisting of: chemical reactive groups, dyes, targeting agents, radiolabels, fluorescent labels, conjugated substances, probes, lipids, chelators, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non-cleavable linkers, or a combination thereof.

In aparticular embodiment, the glycosylamine-1 -phosphate intermediate is used in a method of labeling. In another aspect, the invention features a method of making a glycoprotein, oϋgoscaharide or glycolipid comprising incubating a reaction mixture comprising a sugar nucleotide and one or more functional groups with a glycotransferase and an acceptor.

In one embodiment, the acceptor is selected from the group consisting of: a sugar acceptor, a polypeptide acceptor, and a lipid acceptor. In a related embodiment, the sugar acceptor is selected from the group consisting of: a polypeptide, a glycopeptide, a glycan, and a lipid glycan.

In another particular embodiment, the glycosyltransferases are wild type glycosyltransferases. In still another embodiment, the glycosyltransferases are altered glycosyltransferases. In a related embodiment, the glycosyltransferases are selected from the group consisting of: galactosyltransferases, acetylgalactosyltransferases and polypeptidylgalactosyltransferases. In another further embodiment, the

galactosyltransferase is a beta galactosyltransferase or an alpha acetyigalactosaminy (transferase: In stilla nother further embodiment, the glycosyltransferases are selected from the group consisting of: beta 1,4 galactosyltransferase, alpha 1,3 N-Acetyigalactosaminyltransferase. In another embodiment, the sugar nucleotide is selected from the group consisting of UDP-galactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues.

In a particular embodiment, the sugar nucleotide comprises a chemical reactive group selected from the group consisting of: an azido group, a keto group, an alkyne group or a thiol group. In a further embodiment, the azido group, the keto group, the alkyne group or the thiol group is substituted at the C2 position. In a related embodiment, the C2 position is used for the attachment of functional group.

In one embodiment, the functional groυp is directly attached to the sugar ^" nucleotide.

In another embodiment, the functional group is selected from the group consisting of: chemical reactive groups, dyes, targeting agents, radiolabels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non-cleavable linkers, or a combination thereof. In another embodiment, the one or more functional groups is transferred from the sugar donor nucleotide to an acceptor,

In a related embodiment, the acceptor is selected from the group consisting of: a sugar acceptor, a polypeptide acceptor, and a lipid acceptor. In a further related embodiment, the sugar acceptor is selected from the group consisting of: a polypeptide, a glycopeptide, a glycan, and a lipid glycan. In still another embodiment, the sugar acceptor is N-acetylglucosamine (GIcNAc).

In another aspect, the invention features a method of coupling an agent to a carrier protein comprising incubating a reaction mixture comprising a sugar nucleotide and one or more ftinctional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases, with a sugar acceptor and a glycosytransferase.

In one embodiment, the sugar nucleotide is selected from the group consisting of UDP-gaiactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues. In a related embodiment, the sugar nucleotide comprises a chemical reactive group selected from; an azido group, a keto group, an alkyne group or a thiol group, In still another 5 related embodiment, the azido group, the keto group, the alkyne group or the thiol group is substituted at the C2 position. In a further embodiment, the C2 position is used for the attachment of functional group.

In one embodiment, the functional group is directly attached to the sugar nucleotide.

I O In another embodiment, the functional group is selected from the group consisting of: chemical reactive groups, dyes, targeting agents, radiolabels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, singSe chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non-cleavable 15 linkers, or a combination thereof.

In a related embodiment, the carrier protein is selected from the group consisting of: ovalbumin, single chain Abs and toxins.

In a further embodiment, the carrier protein is an IgG.

In another embodiment, the method farther comprises the steps of coupling a C2 20 UDP-gaiactose analogue to biotin for detection.

In one embodiment, the detection is by chemiluminescent assay.

In another embodiment, the contrast agent is a paramagnetic contrast agent.

In a further embodiment, the paramagnetic contrast agent is used in magnetic resonance imaging.

25 In another aspect, the invention features a method for the treatment of a subject suffering from a disease or disorder comprising administering to the subject an effective amount of a sugar nucleotide and one or more functional groups synthesized by a method comprising acylating glycosy!amlne-1 -phosphate; and coupling with one or more nucleotides; administering the sugar nucleotide and one or more functional groups to the 30 subject, thereby treating the subject.

In another aspect, the invention features a method for the diagnosis of a subject suffering from a disease or disorder comprising obtaining a sample from a subject; and contacting the sample with an effective amount of a sugar nucleotide and one or more functional groups synthesized by the methods described herein, and thereby diagnosing a subject as suffering from a disease or disorder,

In still another aspect, the invention features a method for imaging a target cell or tissue in a subject comprising administering to a subject an effective amount of a sugar and one or more functional groups synthesized by a method comprising acylating glycosylamine-1 -phosphate, and coupling with one or more nucleotides, administering the sugar nucleotide and one or more functional groups to the subject, thereby imaging a target eel! or tissue.

In one embodiment, the sugar nucleotide is selected from the group consisting of UDP-galactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues.

In another embodiment of any one of the above aspects, the sugar nucleotide comprises a chemical reactive group selected from: an azido group, a keto group, an aikyne group or a thiol group. In another related embodiment, the azido group, the keto group, the aikyne group or the thiol group is substituted at the C2 position. In still a further embodiment, the C2 position is used for the attachment of functional group.

In another particular embodiment, the functional group is directly attached to the sugar nucleotide.

In a further embodiment, the functional group is seSected from the group consisting of: chemical reactive groups, dyes, targeting agents, radio labels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non- cleavable linkers, or a combination thereof.

In another embodiment of any one of the above aspects, the one or more functional groups is transferred from the sugar donor nucleotide to an acceptor.

In a related embodiment, the acceptor is selected from the group consisting of: a sugar acceptor, a polypeptide acceptor, and a lipid acceptor. In a further embodiment, the sugar acceptor is selected from the group consisting of: wherein the sugar acceptor is

selected from the group consisting of: a polypeptide, a glycopeptide, a glycan, and a lipid glycan.

In another embodiment of any one of the above methods, the glycosyltransferases are wild type glycosyltransferases. In another embodiment of any one of the above methods, the glycosyltransferases are altered glycosyltransferases.

In still another embodiment of any one of the above aspects, the glycosyltransferases are selected from the group consisting of: galactosyltransferases, acetylgalactosyltransferases and polypeptidylgalactosyltransferases. In a related embodiment, the galactosyltransferase is a beta galactosyitransferase or an alpha acetylgalactosaminyltransferase. In another embodiment of any one of the above methods, the glycosyltransferases are selected from the group consisting of: beta 1 ,4 galactosyltransferase, alpha 1 ,3 N-Acetylgalactosaminyltransferase.

In still another embodiment of any one of the above aspects, the transfer occurs in the presence of magnesium. In another embodiment of any one of the above aspects, the acceptor is free or attached to a peptide of a glycopeptide.

In one embodiment, the imaging method is used in a diagnostic procedure.

In another embodiment, the imaging method is used in a prognostic procedure.

In one embodiment of any one of the above aspects, the method is used to determine the course of treatment.

In another aspect, the invention features kits comprising a sugar nucleotide and one of more functional groups, and a glycosy transferase, according to any one of the above-mentioned aspects.

In another embodiment of any one of the above methods, the glycosyltransferases are wild type glycosyltransferases. In another embodiment of any one of the above methods, the glycosyltransferases are altered glycosyltransferases.

In still another embodiment of any one of the above aspects, the glycosyltransferases are selected from the group consisting of galactosyltransferases, acetytgalactosyltransferases and polypeptidylgaSactosyltransferases, In a related embodiment, the galactosyltransferase is a beta galactosyltransferase or an alpha acetylgalactosaminyltraπsferase. In another embodiment of any one of the above

methods, the glycosyltransferases are selected from the group consisting of beta I ₁ 4 galactosyltransferase, and alpha 1 , 3 N-Acetylgaiactosaminyitraπsferase

In another embodiment, the sugar nucleotide is selected from the group consisting of UDP-galactose, UDP-GaINAc, UDP-Ga[NAc analogues or UDP-galactose analogues. In a related embodiment, the sugar nucleotide comprises a chemical reactive group selected from: an azido group, a keto group, an alkyne group or a thiol group

In a further related embodiment, the functional group is selected from the group consisting of- chemical reactive groups, dyes, targeting agents, radiolabels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non cleavable linkers, or a combination thereof

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic showing the efficient synthesis for useful sugar nucleotides application of functional ized carbohydrates substrates and/or inhibitors of glycosyltransferase affording new glycoconjugates for therapeutics and diagnostics, dyes, and biotinylated compounds

Figure 2 is a schematic showing efficient synthesis of UDP-α-GalNAz

Figure 3 is a schematic showing efficient synthesis of UDP-α-2-Keto-Gal

Figure 4 is a schematic showing the synthesis of mass-labeled, probes, fluoroprobes, biomolecules

Figure 5 is a schematic showing other alkyne substrates (top) and biotm probe and/or fluoroprobes substrate (bottom)

Figure 6 is a schematic showing synthesis of azido dyes and probes

Figure 7 shows ESI Mass spectra of glycans after transfer of 2-(But-3-ynoic acid amido)- GaI to the sugar acceptor chitotetrose GlcNAcβl, 4-GlcNAcβl, 4-GlcNAcβl , 4-GlcNAc.

Figure 8 shows ESl Mass spectra of glycans after transfer of 2-(But-3-ynoic acid amido)- GaI to the peptide acceptor PTTDSTTPA PTTK.

Figure 9 shows UDP-2-(Biotin arnido)-Gal Synthesis.

Figure 10 shows UDP-2-(Propynoic acid amido)-Gal Synthesis.

Figure 1 1 shows UDP-2-(pyruvic acid amido)-Gal Synthesis.

Figure 12 shows the synthesis of UDP~2-keto-GaI from galactal (Part 1 ).

Figure 13 shows the synthesis of UDP-2-keto-Gal (Part 2).

Figure 14 shows the final step of synthesis of the azido biotiπ product in the chemical synthesis of azido-biotin.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally features functional ized sugars and or sugar nucleotides and methods of making and using such sugars. The novel sugars described herein are used as substrates to make glycocoπjugates with therapeutic and diagnostic applications.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ai., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and

Technology (Walker ed., 198S); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),

I O

Springer Verlag (1991); and Haie & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

As used in the specification and claims, the singular form "a", "an" and "the" include plura! references unless the context clearly dictates otherwise. For example, the term "a ceil" includes a plurality of cells, including mixtures thereof. The term "a nucleic acid molecule" includes a plurality of nucleic acid molecules.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of, when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantia! method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

The term "acceptor" is meant to refer to a moiecuie or structure onto which a donor is actively linked through action of a catalytic domain of a galactosyltransferase, or altered thereof. Examples of acceptors include, but are not limited to, carbohydrates, glycoproteins, glycolipids.

The term "functional group" is meant to refer to any agent or biological agent, or any chemical or biological material or compound that is suitable for delivery that induces a desired effect in or on an organism, such as a biological or pharmacological effect, which may include, but is not limited to a prophylactic effect, alleviating a condition caused by a disease or a disorder, reducing or eliminating a disease or disorder. An agent or a bioactive agent refers to substances that are capable of exerting a biological effect in vitro and/ or in vivo. Examples include diagnostic agents, pharmaceuticals, drugs, synthetic organic molecules, proteins, peptides, vitamins, steroids, genetic material including nucleotides, nucleosides, polynucleotides, RNAs, siRNAs, shRNAs, anti-sense DNA or RNA.

1 !

The term "antibody" as used herein refers to both polyclonal and monoclonal antibody. The term can also refer to single chain antibodies, The term encompasses not only intact immunoglobulin molecules, but fragments and genetically engineered derivatives of immunoglobulin molecules as may be prepared by techniques known in the art, and which retains the binding specificity of the antigen binding site.

The term "donor" refers to a molecule that is actively linked to an acceptor molecule through the action of a catalytic domain of a galactosy transferase, or altered thereof. A donor, e.g. a donor nucleotide molecule can include a sugar, or a sugar derivative. Examples of donors include, but are not limited to, UDP-GaINAc, UDP- galactose or UDP-galNAc analogues, UDP-galactose analogues. Donors include sugar derivatives that include agents, biological agents, or active groups. Accordingly, oligosaccharides may be prepared according to the methods of the invention that include a sugar derivative having any desired characteristic.

The term "effective amount" is meant to refer to a sufficient amount that is capable of providing the desired local or systemic effect.

The term "homologous" is intended to include a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent amino acid residues or nucleotides, e.g., an amino acid residue which has a similar side chain, to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity.

The terms "oligosaccharide" and "polysaccharide" are used interchangeably herein. These terms refer to saccharide chains having two or more linked sugars. Oligosaccharides and polysaccharides may be homopolymers and heteropolymers having a random sugar sequence or a preselected sugar sequence. Additionally, oligosaccharides and polysaccharides may contain sugars that are normally found in nature, derivatives of sugars, and mixed polymers thereof. ''Saccharide" refers to any of a series of compounds of carbon, hydrogen, and oxygen in which the atoms of the latter two elements are in the ratio of 2: 1 , especially those containing the groupC6H 1 o05, including fructose, glucose, sucrose, lactose, maltose, galactose and arabinose.

The term "immunogenic" compound or composition as used herein refers to a compound or composition that is capable of stimulating production of a specific immunological response when administered to a suitable host, usually a mammal. The term "nucleic acid" is intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host ceϋ or can be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In one embodiment, the gene of polynucleotide segment is involved sugar transfer. A altered nucleic acid molecule or is intended to include a nucleic acid molecule or gene having a nucleotide sequence which includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide or polypeptide that can be encoded by said altered exhibits an activity that differs from the polypeptide or polypeptide encoded by the wild-type nucleic acid molecule or gene.

The terms "polypeptides" or "isolated polypeptide" and "proteins" are used interchangeably herein. Polypeptides and proteins can be expressed in vivo through use of prokaryotic or eukaryotic expression systems. Many such expressions systems are known in the art and are commercially available. (Clontech, Palo Alto, Calif; Stratagene, La JoMa, Calif.). Examples of such systems include, but are not limited to, the TV- expression system in prokaryotes and the bacculovirus expression system in eukaryotes. Polypeptides can also be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by in vitro transcription/translation systems. Such methods are described, for example, in U.S. Pat. Nos. 5,595,887; 5, 1 16,750; 5,168,049 and 5,053,133; Olson et al., Peptides, 9, 301 , 307 (1988). The solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc, 85 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; Bavaay and Merrifield, 'The

Peptides," eds. E. Gross and F. Meienhofer, Vo!. 2 (Academic Press, 1980) pp. 3-285;

and Clark-Lewis et al., Meth. Enzymol., 287, 233 ( 1997). These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography. The term an "isolated polypeptide" (e.g., an isolated or purified biosynthetic enzyme) is substantially free of cellular materia! or other contaminating polypeptides from the microorganism from which the polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized The polypeptides of the invention include polypeptides having amino acid exchanges, i.e., variant polypeptides, so long as the polypeptide variant is biologically active. The variant polypeptides include the exchange of at least one amino acid residue in the polypeptide for another amino acid residue, including exchanges that utilize the D rather than L form, as well as other well known amino acid analogs, e.g., N-alkyl amino acids, lactic acid, and the like. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, -tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruϋne, N-methyl-alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, and other similar amino acids and imino acids and tert- butylglycine.

Conservative amino acid exchanges are preferred and include, for example; aspartic-glutamic as acidic amino acids; lysine/argϊnine/histidine as basic amino acids; leucine/iso leucine, methionine/vaiine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid exchange also includes groupings based on side chains. Members in each group can be exchanged with another. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and iso leucine. These may be exchanged with one another. A group of amino acids having aliphatic-hydroxyl side chains is serine and threonine. A group of amino acids having amide-containing side chains is asparagine and glutamine. A group of amino acids having aromatic side chains is phenylalanine,

tyrosine, and tryptophan. A group of amino acids having basic side chains is lysine, arginine, and histidine. A group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid may be accomplished to produce a variant polypeptide of the invention.

The term "subject" as used herein refers to any animal, including mammals, preferably humans, to which the present invention may be applied.

The term "cancer" or "tumor" refers to an aggregate of abnormal cells and/or tissue which may be associated with diseased states that are characterized by uncontrolled cell proliferation. The disease states may involve a variety of cell types, including, for example, endothelial, epithelial and myocardial cells. Included among the disease states are neoplasms, cancer, leukemia and restenosis injuries.

Glycosyltrasferases

Beta 1, 4 Glycosyltrasferase

Specific glycosyltransferases synthesize oligosaccharides by the sequential transfer of the monosaccharide moiety of an activated sugar donor to an acceptor molecule. Members of the glycosyltransferase superfamily, which are often named after the sugar moiety that they transfer, are divided into subfamilies on the basis of linkage lhat is generated between the donor and acceptor. Transfer of the sugar residue occurs with either the retention (by retaining glycosyltransferases) or the inversion (by inverting glycosyltransferases) of the configuration at the anomeric Cl atom. beta-l,4-Galactosyltransferases (beta4Gal-T) are a Golgi resident, type Il membrane-bound family of enzymes (beta4Gal-Tl ~T7) that transfer galactose (Gal) in the presence of manganese ion (Mn 2+), from UDP-GaI to N-acetyiglucosamine (GIcNAc) ₁ either free or bound to an oligosaccharide of a glycoprotein or a glycolipid (Brew et al., 1968; Takase et al., J 984; Powell et al. 1976; HiIS, UCLA Forum Med. ScL, 21 : 63-86, 1979). This reaction allows galactose to be linked to an N-acety (glucosamine that may itself be linked to a variety of other molecules. Examples of these molecules include other sugars and proteins. The reaction can be used to make many types of

molecules of biological significance. For example, galactose- beta (1 ,4)-N- acetylglucosamine linkages are important for many recognition events that control how cells interact with each other in the body, and how cells interact with pathogens. In addition, numerous other linkages of this type are important for cellular recognition and binding events as well as cellular interactions with pathogens, such as viruses.

Sequences of beta galactosyltransferase I family members from human and other species are known, and family members exhibit a high level of sequence identity in their catalytic domains (Lo et al., 1998; Amado et al., 1998). DNA clones are available from commercial resources, for example, Open Biosources. The term "beta- 1 ,4 galactosyltransferase (beta 4GaI-Tl )" as used herein refers to enzymes substantially homologous to, and having substantially the same biological activity as, the enzyme encoded by the nucleotide sequence depicted in SEQ ID NO: 1 and the amino acid sequence depicted in SEQ ID NO: 2. This definition is intended to encompass natural allelic variations in the beta 4GaI-Tl sequence, and all references to beta 4GaI-Tl , and nucleotide and amino acid sequences thereof are intended to encompass such allelic variations, both naturally-occurring and man-made. The production of proteins such as the enzyme beta 4GaS-Tl from cloned genes by genetic engineering is well known.

( S EQ I D NO : 1 )

1 CTGCCCGCAT GCCCTGAGGA GTCCCCGCTG CTTGTGGGCC CCATGCTGAT 52 TGAGTTTAAC ATGCCTGTGG ACCTGGAGCT CGTGGCAAAG CAGAACCCAA 101 ATGTGAAGAT GGGCGGCCGC TATGCCCCCA GGGACTGCGT CTCTCCTCAC 151 AAGGTGGCCA TCATCATTCC ATTCCGCAAC CGGCAGGAGC ACCTCAAGTA 201 CTGGCTATAT TATTTGCACC CAGTCCTGCK GCGCCAGCAG CTGGACTATG 251 GCATCTATGT TATCAACCAG GCGGGAGACA CTATATTCAA TCGTGCTAAG 301 CTCCTCAATG TTGGCTTTCA AGAAGCCTTG AAGGACTATG ACTACACCTG 351 CTTTGTGTTT AGTGACGTGG ACCTCATTCC AATGAATGAC CATAATGCGT 401 ACAGGTGTTT TTCACAGCCA CGGCACATTT CCGTTGCAAT GGATAAGTTT 451[ GGATTCAGCC TACCTTATGT TCAGTTGTTT GGAGGTGTCT CTGCTCTAAG 501 TAAACAACAG TTTCTAACCA TCAATGGATT TCCTAATAAT TATTGGGGCT 5Sl GGGGAGGAGA AGATGATGAC ATTTTTAACA GATTAGTTTT TAGAGGCATG

601 TCTATATCTC GCCCAAATGC TGTGGTCGGG AGGACGCGTC ACATCCGCCA

651 CTCGAGAGAC AAGAAAAATG AACCCAATCC TCAGAGGTTT GACCGAATTG

701 CACACACAAA GGAGACAATG CTCTCTAATG GTTTGAACTC ACTCACCTAC

751 CAGGTGCTGG ATGTACAGAG ATACCCATTG TATACCCAAA TCACAGTGGA 801 CATCGGGACA CCGAGCTAG

(SEQ ID NO: 2)

127 LPACPEESPL LVGPMLIEFN MPVDLELVAK QNPNVKMGGR YAPRDCVSPH

177 KVAIIIPFRN RQEHLKYWLY YLHPVLQRQQ LDYGIYVINQ AGDTIFNRAK 227 LLNVGFQEAL KDYDYTCFVF SDVDLIPMND HNAYRCFSQP RHISVAMDKF

277 GFSLPYVQLF GGVSALSKQQ FLTINGFPNN YWGWGGEDDD IFNRLVFRGM

327 SISRPNAVVG RTRHIRHSRD KKNEPNPQRF DRIAHTKETM LSNGLNSLTY

377 QVLDVQRYPL YTQITVDIGT PS*

Glycosyltransferases show great structural similarity. They are all globular proteins with two types of fold, termed GT-A and GT-B, which each have an N-terminal and a C-termina! domain. The enzymes of the GT-A fold have two dissimilar domains. The ^" N-tenminal domain, which recognizes the sugar-nucleotide donor, comprises several b-strands that are each flanked by alpha-helices as in a Rossmann-iike fold, whereas the C-terminal domain, which contains the acceptor-binding site, consists largely of mixed b- sheets. By contrast, enzymes with the GT-B fold contain two similar Rossmann-like folds, with the N-terminaS domain providing the acceptor-binding site and the C-terminal domain providing the donor-binding site. In both types of enzyme, the two domains are connected by a linker region and the active site is located between the two domains. A metal-binding site is also located in the cleft in enzymes of both the GT-B and GT-A fold (Qasba et a!., 2005).

The methods of the invention are amenable to use with any beta 1,4 galactosyltransferase I. By any beta 1 ,4 galactosyltransferase I is meant from any species, for example, but not limited to, human, bovine, or mouse. Although they have the same donor sugar specificity, many of these are expected to transfer GaI to different oligosaccharides containing GIcNAc at their nonreducing end Although they have the same donor sugar specificity, many of these are expected to transfer Gal to different

BOS2 657374 ) ] 7

oligosaccharides containing GicNAc at their nonreducing end. Recent crystal lographic studies on beta4Gal-Tl have provided detailed information about the structure and function of the enzyme (Gastinel et al., 1999; Ramakrishnan et al., 2001 ; Ramakrishnan et al., 2001a; Ramakrishnan et a!., 2002; Ramakrishnan et al., 2002a; Ramakrishnan et a!,, 2003).

Structural studies on the beta -1,4-galactosy (transferase-] ( beta 4GaI-Tl) (Ramikrishnan et al, 2004a) and on other glycosyltraπsfεrases (Qasba et al. 2005) have shown that, upon binding the sugar-nucleotide donor substrate, flexible loops at the substrate binding site of these enzymes undergo a marked conformational change, from an open to a closed conformation (Qasba et al. 2005). This change creates an oligosaccharide acceptor-binding site in the enzyme that did not exist before. The loop then acts as a lid covering the bound donor substrate. After the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected, and the loop reverts to its native conformation to release the remaining nucleotide moiety. This conformational change in beta 4GaI-Tl also creates the binding site for beta -lactaSbumin, a protein produced in the mammary glands during lactation. The interaction of beta -lactalbumin with beta 4GaI-Tl changes the acceptor specificity of the enzyme from N -acetylglucosamine (GIcNAc) to glucose (GIc), which produces lactose that is secreted in milk. The conformational changes of these two loops are highly coordinated. Trp314 in the small loop plays a crucial role in the conformational state of the long loop, in the binding of the substrates, and in the catalytic mechanism of the enzyme (Ramakrishnan et al, 2001 ; Gunasekaran et al., 2003). In the unbound state (open conformation), the side chain of Trp is exposed to the solvent (Gastinel et al, 1999; Ramasamy et al. 2003), and the conformation of the long loop is such that the UDP-GaI and the metal binding sites are exposed. Once the substrate binds, the side chain of Trp314 moves into the catalytic pocket to lock the sugar, nucleotide in its binding site. Simultaneously, the long loop changes to its closed conformation, masking the sugar nucleotide binding site (Ramakrishnan et al, 2001 ; Ramakrishnan et al, 2003; Ramasamy et al., 2003), Furthermore, this conformational change in the long flexible loop repositions the amino acid residues at the N-terminal region, creating a metal ion binding site, and at the C-terminal region, creating an oligosaccharide-binding cavity that is also a protein-protein interaction site for R-

lactalbumin (LA) (Gasteinel et al., 1999; Ramakrishnan et al, 2001 ; Ramakrishnan et al, 2003). LA is a mammary gland-specific protein that modulates the acceptor specificity of the enzyme toward glucose (Brodbeck et al., 1967). LA binds at the extended sugar binding site, present only in the closed conformer of beta 4GaI-Tl , leaving the monosaccharide binding site of the enzyme available for the binding of GIc or GIcNAc. Since LA competes with the oligosaccharide for binding to the extended sugar binding site (Bell et a I, 1976; Powell et al., 1976), it is not possible to crystallize beta 4GaI-Tl in the presence of LA with a bound oligosaccharide acceptor. The wild-type enzyme also does not crystallize in the presence of UDP or UDPhexanolarπine, Mn2+, and oligosaccharides, thereby restricting our structural or biochemical studies on the interactions of oligosaccharides with beta 4GaI-Tl . It has previously been shown that the sugar moiety of the sugar nucleotide is essential for efficiently inducing a conformational change in beta 4GaI-Tl (Geren et a!., 1975).

The reaction catalyzed by these enzymes follows a kinetic mechanism in which the metal ion and sugar nucleotide bind to the enzyme first, followed by the acceptor.

After the glycosyl moiety of the sugar-nucleotide donor is transferred to the acceptor with the inversion or retention of the Cl configuration, the saccharide product is ejected. The release of the nucleotide and the metal ion follows, which returns the enzyme to its original state for a new round of catalysis. X-ray crystal structures of the catalytic domain of many giycosyltransferases, either free or bound to substrates, have been determined recently. These studies provide a structural basis for the ordered binding of the donor and acceptor and for the proposed catalytic mechanism of these enzymes (Unligil, U. M. and Rini, J. M. (2000); Berger, E.G. and Rohrer, J; Negishi, M. et al. (2003)). A three-residue motif, Asp-X-Asp (DXD) or Glu-X-Asp (EXD), or its equivalent generally participates in metal ion binding in enzymes of the GT-A fold. Enzymes of the GT-B fold such as the microbial giycosyltransferases MurG (Hu, Y. et al. (2003)) and GtfB (Mulichack et al. 2001), and BGT (Morera et al. 1999), do not have a DXD motif or its equivalent, even though some, BGT for example, require a metal ion for activity. In giycosyltransferases that require Mn2C ion as cofactor, the metal ion is bound in an octahedral coordination (Qasba et al. 2005). It interacts with one or both acidic residues

of the DXD or EXD motif and with two oxygen atoms from the a-phosphate and b- phosphate of UDP. To satisfy the octahedral geometry, the three remaining meta! ion links are made either to water molecules or to water in combination with other residues of the protein. In several glycosyltransferases only the first (Lobsanov, Y.D. et al. (2004)) or the second (Gastiπet et al. 1999; Ramakrishnan et a!. 2001 ; Ramakrishnan 2002; Unligil 2000) acidic residue of the motif coordinates directly with the metal ion. For example, in some enzymes, the first acidic residue of the motif either interacts directly with the sugar donor or the ribose moiety or interacts via the water molecules coordinated to the Mn2C ion. In blood group A and B and alpha 3GT transferases, by contrast, both aspartic acid residues of the DXD motif directly coordinate the metal ion.

The crystal structures of several glycosyltransferases of either the GT-A or GT-B fold show that at least one flexible loop region has a crucial role in the catalytic mechanism of the enzyme (Qasba et al. 2005). Although the exact location of this loop differs among the transferases, it is invariably located in the vicinity of the sugar nucleotide-binding site. Owing to the flexibility of this region, the loop structure cannot be traced in the apo form of the enzyme, which lacks bound substrate, ϊn the sugar- nucleotide-bound structures, the loop either is in a closed conformation covering the bound donor substrate or is found disordered in the vicinity of the sugar nucleotide- binding site. In a3GT, the C-terminai 1 1 -residue flexible loop changes its conformation when the sugar nucleotide donor is bound (Boix et al., 2001).

Of the six ligands that coordinate Mn2+, three are from bovine beta 4GaI-Tl : Asp254, Met344, and His347 (Ramakrishnan et a!, 2001 ; Ramakrishnan et al, 2003; Boeggeman et al., 2002). Residues Met344 and His347, separated by the hinge residue I!e345, are at the N-terminal region of the long flexible loop. The complete metal binding site is created oniy after His347 has moved during the conformational change to coordinate with the metal ion.

The beta- 1 , 4-galactosyltraπsferase enzyme can also a number of sugars, such GlcNac, N-acy!-substituted glucosamine and N-acetyl-D-mannosamine as substrates (Berliner, L. J. et al., MoI. CeIi. Biochem. , 62: 37-42 (1984)). The beta- 1 , 4- galactosyltransferase does not have an absolute requirement for the sugar donor UDP- GaI; it exhibits polymorphic donor specificity, in that it also transfers glucose (GIc), D-

deoxy-Glc, arabinose, GaINAc, and GicNAc from their UDP derivatives (Berliner, L. J. and Robinson, R. D., Biochemistry, 21 : 6340-6343 (1982); Andres, P. J. and Berliner L. J. , Biochim. Biophys. Acta, 544: 489-495 (1982); Do, K, Y. et al., J. Biol. Chem., 270: 18477- 18451 (1995); Palcic, M. M and Hindsgaul, O., Glycobiology, 1 : 205-209 (1991); Ramakrishnan, B. et al., J. Biol. Chem., 276: 37665-37671 (2001)). This reaction can be used to generate many types of molecules, as described herein, which have applications in research and medicine.

Application No, PCT/US2007/018656, incorporated by reference in its entirety herein describes BETA 1 ,4-GALACTOSYLTRANSFERASES with altered donor and acceptor specificities, compositions and methods of use.

Alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GaINAc-T) Specific glycosyltransferases synthesize oligosaccharides by the sequential transfer of the monosaccharide moiety of an activated sugar donor to an acceptor molecule. Members of the glycosyltraπsferase superfamily, which are often named after the sugar moiety that they transfer, are divided into subfamilies on the basis of linkage that is generated between the donor and acceptor. Transfer of the sugar residue occurs with either the retention (by retaining glycosyltransferases) or the inversion (by inverting glycosyltransferases) of the configuration at the anomeric C l atom. Giycosyltransferases show great structural similarity. They are all globular proteins with two types of fold, termed GT-A and GT-B, which each have an N-terminal and a C-terminal domain. The enzymes of the GT-A fold have two dissimilar domains. The N-terminal domain, which recognizes the sugar-nucleotjde donor, comprises several b-strands that are each flanked by a-helices as in a Rossmann-like fold, whereas the C-terminal domain, which contains the acceptor-binding site, consists largely of mixed b-sheets. By contrast, enzymes with the GT-B fold contain two similar Rossmann-like folds, with the N-termina) domain providing the acceptor-binding site and the C-terminal domain providing the donor- binding site. In both types of enzyme, the two domains are connected by a linker region and the active site is located between the two domains. A metal-binding site is also located in the cleft in enzymes of both the GT-B and GT-A fold (Qasba et al. 2005).

The alpha (l,3)-galactosyltransferase I (a3 GaI-T) enzyme mediates the formation of gal-alpha-gal moieties. A3 GaI-T uses UDP-ga!actose as a source of galactose, which it transfers to an acceptor oligosaccharide, usually GaI beta (l,4)GlcNAc (N-acetyl lactosamine). As used herein the term "alpha (l ,3)galactosyltraπsferase" and the abbreviation "alpha 1,3GT" refer to the enzyme, present in non-primate mammals, that catalyzes the formation of the Gal. alpha. (l,3)Gal determinant by attaching GaI in the .alpha. (1 , 3) position to the Gal.beta.(l ,4)GlcNAc acceptor, .alpha, 1 ,3GT has the Enzyme Commission designation EC 2.4.1.124.

The expression of alpha.1-3 galactosyltransferase is regulated both developmental Iy and in a tissue-specific manner. The cDNA for this enzyme has been isolated from many species, including pigs (Hoopes et al., poster presentation at the 1997 Xenotransplantation Conference, Nantes France; Katayama et al., J. Glycoconj., 15(6), 583-99 (1998); Sandrin et al., Xenotransplantation, 1, Sl -88 ( 1994), Strahan et al., Immunogenics, 41 , 101 -05 ( 1995)), mice (Joziasse et al., J. Bioi. Chem., 267, 5534-41 (1992)), and cows (Joziasse et al., J. Biol. Chem., 264, 14290-97 (1989). Some mammals do not express the GaJ alpha, (l,3)Gal product, an in these organisms the alpha 1,3GT locus is inactivated (Gaiiili et al., Proc. Natl. Acad. Sci. USA 15:7401, 1991). There are frameshift and nonsense substitutions within the locus, turning it into a non-functional, processed pseudogene (Laarsen et al., J, Biol. Chem. 265:7055, 1990; Joziasse et al,, J. Biol. Chem. 266:6991 , 1991 ). Larsen et al. (Proc. Natl. Acad. Sci. USA 86:8227, 19S9) isolated and characterized a cDNA encoding murine alpha.I,3GT. Joziasse et al. (J. Bioi. Chem. 267:5534, 1992) detected four distinct mRNA transcripts, which predict four different isoforms of the .alpha.1,3GT. The full-length mouse mRNA (including 5 ¹ untranslated mRNA) was reported to span at least 35-kB of genomic DNA, distributed over nine exons ranging from 36 base pairs to about.2600 base pairs in length.

Numbering in the 5' to 3' direction, the coding region is distributed over Exons 4 to 9. The four transcripts are formed by alternative splicing of the pre-mRNA. Joziasse et al. (J. Biol, Chem. 264: 14290, 1989) isolated and characterized a cDNA encoding bovine cDNA. The coding sequence was predicted to be a membrane-bound protein with a large glycosylated COOH-terminal domain, a transmembrane domain, and a short NH2 terminal domain.

The term Ga! alpha (l,3)Gal refers to an oligosaccharide determinant present on endothelial cells and other cells of most non-primate mammals, for which humans have a naturally occurring antibody. Except for Old World monkeys, apes and humans, most mammals carry glycoproteins on their eel! surfaces that contain galactose alpha 1,3- galactose (GaIIH et al., J. Biol. Chem. 263: 17755-17762, 1988). Humans, apes and Old World monkeys have a naturally occurring anti-alpha gal antibody that is produced in high quantity (Cooper et al., Lancet 342:682-683, 1993), It binds specifically to glycoproteins and glycolipids bearing galactose alpha- 1 ,3 galactose. In contrast, glycoproteins that contain galactose alpha 1,3-galactose are found in large amounts on cells of other mammals, such as pigs. This differential distribution of the "alpha-1 _; 3 GT epitope" and anti-Gal antibodies {i.e., antibodies binding to glycoproteins and glycolipids bearing galactose alρha-1 ,3 galactose) in mammals is the result of an evolutionary process which selected for species with inactivated (i.e. mutated) alpha- ^] ,3- galactosy transferase in ancestral Old World primates and humans. Thus, humans are "natural knockouts" of alphal,3GT. A direct outcome of this event is the rejection of xenografts, such as the rejection of pig organs transplanted into humans initially via HAR.

Application No, PCT/US07/ 18678, incorporated by reference in its entirety herein describes ALPHA 1 -3 N- GALACTOSYLTRANSFERASE with altered donor and acceptor specificities, compositions and methods of use.

Compositions

The invention relates generally to functionalized sugars , e.g. sugar nucleotides, and methods of making and using such sugars. The novel sugar nucleotides described herein are used as substrates to make glycoconjugates with therapeutic and diagnostic applications.

The invention features, generally, compositions comprising a sugar nucleotide and one or more functional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases. The sugar nucleotide can be any sugar nucleotide that is a substrate for a glycosy transferase.

Glycosyltransferases are enzymes that transfer a monosaccharide unit from an activated sugar phosphate to an acceptor. The result of glycosyl transfer can be a monosaccharide glycoside, an oligosaccharide, or a polysaccharide, although some glycosyltransferases catalyse transfer to inorganic phosphate or water. Glycosyl transfer can also occur to protein residues, usually to tyrosine, serine or threonine to give O- linked glycoproteins, or to asparagine to give N-linked glycoproteins. Mannosyl groups may be transfered to tryptophan to generate C- mannosyl tryptophan.

Glycosyltransferases are usually metal ion dependent with metals such as magnesium or manganese being found in the active site and acting as a Lewis acid by binding to the (di)ρhosphate leaving group.

In certain examples, the glycosyltransferases are wild type glycosyltransferases. The glycosyltransferases can also be altered glycosyltransferases, e.g. with mutaltion, deletion, substitution at one ormore residues.

Examptary glycosyitransferases for use in the invention include, but are not limited to, : galactosyltransferases, acetylgalactosyltransferases and polypeptidylgalactosyltransferases.

In certain examples, the galactosyltransferase is a beta galactosyltransferase or an alpha acetylgalactosaminyltransferase.

In other certain examples, the giycosy [transferases are selected from, but not limited to, beta 1 ,4 galactosyltransferase, alpha 1,3 N-Acetylgalactosaminyltransferase.

The compositions of the invention also include a sugar nucleotide. The sugar nucleotide can be, but is not limited to, UDP-galactose, UDP-GaINAc, UDP-Ga)NAc analogues or UDP-galactose analogues.

The sugar nucleotide comprises a chemically reactive group selected from, but not limited to, an azido group, a keto group, an alkyne group or a thiol group. The chemically reactive group is used an a handle for the attachment of a second group, e.g. a functional group. In this way, any chemically reactive group that is suitable for attachment to the sugar nucleotide is of use in the invention.

The chemically reactive group is substituted at the C2 position, which is used as the point of attachment for the functional group, as described herein.

Alternatively, the functional group may be directly attached to the sugar nucleotide, without the use of a chemically reactive group.

In the composition, any functional group that is of use to the method to be performed is possible, In the composition, any functional group that is envisioned by one skilled in the art is possible. In the composition, any functional group that can be attached, wither with a chemically reactive group or directly is possible.

In certain examples, the function group is selected from, but not limited to, chemical reactive groups, dyes, targeting agents, radiolabels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non-cleavab!e linkers, or a combination thereof.

Advantageoupsly, the one or more functional groups is transferred from the sugar donor nucleotide to an acceptor. The transfer creates giycocojugates comprising the functional group.

In certain examples, the acceptor is selected from the group consisting of: a sugar acceptor, a polypeptide acceptor, and a lipid acceptor. The sugar acceptor can be, for example, N-acetylglucosamine (GIcNAc).

Intermediates

In certain embodiments, intermediate compounds in the methods of synthesis of the sugars, e.g. the functionalized sugars, of the invention are useful.

An intermediate compound is, for example, produced in the method of making a sugar nucleotide. An intermediate compound can be produced by acylating glycosyiamine- 1 -phosphate. Thus, for example, an acylated giycosylamine-1 -phosphate, is an intermediate compound that has use in methods of the invention as described herein. For example, the acylated glycosylamine-I -phosphate is functionalized and used in methods of labeling.

In another example, in the synthesis of UDP-2-keto-Gal from galactal, the intermediates may have important uses. The intermediates of this synthesis maybe able to be used for metabolic cell surface engineering.

In certain preferred embodiments, the intermediate product is a UMP morpholidate pyrimidine as shown below.

form)

100% conversion 86% isolated BioGel P2

Methods of Making

The invention features methods of making a sugar nucleotide comprising one or more functional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases. Glycosylamine-1 -phosphate is a preferred starting material.

In particular examples, the method comprises acylating glycosylamine-1 - phosphate and coupling glycosylamine-1 -phosphate with one or more nucleotides, and thereby making a sugar nucleotide comprising one or more functional groups.

In certain examples, the method comprises a further step of ion exchange. A general scheme for synthesis is set forth below:

salt _

In certain aspects, the invention features methods of making a sugar comprising a functional group, wherein the sugar is a substrate of one or more glycosy transferases, the method comprising acylating g!ycosylamine-l -phosphate and coupling the glycosylamine -] -phosphate with one or more nucleotides, thereby making a sugar comprising a functional group. In certain embodiments of the invention the method comprises the step of performing ion exchange chromatography.

In certain preferred embodiments, the method is performed according to the schematic set forth below:

Glycosytamine-phosphate ^x . , Jϊ ^G ( Glycosylajnine-phosphate)Nr FG

AcySation (Ion-exchange ch rom atography); xs ■ ■ , , . j i, , j . . . , Nucleotide compline

X = acid, chloride, N-hydroxysucemirmde, reaction and/or other reactive group FQ = funtiDnal group comprising a chemical reactive group such as azide, alkyne, aldehyde, ketone, chelator, etc and/or biologically-active molecule, probe, dye, radioactive group, organic moiecuie, protein, giycan, πpid, nucleotide, and/or a combination of above

FG

The sugars and one or more functional groups, e.g. the sugar donors, as described herein, are transferred by glycosyl transferases, to a sugar acceptor. A sugar acceptor can 5 be selected from galactose beta 1 ,4 glcNac or galactose beta 1 ,4 glucose. Sugars that can be transferred include UDP-galactose, UDP- galactose analogues, UDP-GaINAc and UDP-GaINAc analogues. This reaction allows galactose to be linked to a sugar acceptor, for example galactose beta 1 ,4 glcNAc or galactose beta 1,4 glucose, that may itself be linked to a variety of other molecules, such as sugars and proteins, e. g., therapeutic0 agents, imaging agents, antibodies.

The methods as described herein provide the ability to conjugate multiple agents to compounds or compositions of the invention. An embodiment of the present invention provides a glycoconjugate in which one or more bioactive agents are bound to a modified saccharide residue, e. g., a modified sugar, for example a modified galactose, which is in S turn bound to a targeting compound, e. g., a compound capable of binding a receptor on a cell membrane. The 2' modified sugar, e.g. a modified galactose, can be used as a handle to deliver therapeutic agents to specific tissue sites. In this manner, many targeting glycoconjugates can be constructed. For example, a gene delivery system for genetic therapy can be produced by binding a nucleotide and a ligand or antibody to the modified0 sugar, A therapeutic compound for cancer can be produced by binding a chemotherapeutic agent and a ligand or antibody, e. g, , an antibody to a cancer antigen, to the modified sugar residue.

The glycoconjugates can be manufactured as designer glycoconjugates, according to therapeutic need. As such, the designer polypeptide itself can be used for the targeting and drug delivery. The glycoconjugates can be manufactured as nanoparticles. In certain examples, a biological substrate, such as a bioactive agent, for example a therapeutic agent, is used to engineer the nanoparticle. In other examples a second, third, fourth or more bioactive polypeptide is used in association with the nanoparticle to engineer multivalent nanoparticles. The bioactive agents do not have to be the same, for example a nanoparticle comprising three bioactive agents may comprise a chemotherapeutic, a tracking agent and a targeted delivery agent, such as an antibody, Nanoparticles of the invention have use in methods of treating diseases.

In other examples, the methods of the invention are used to engineer a glycoprotein from a magnetic resonance agent for use in diagnostic therapies. In these preferred examples, nanoparticles are engineered as described herein, where the nanoparticles are superparamagnetic nanoparticle. Polypeptide fragments of the invention having altered donor and acceptor specificity can be used to catalyze the linkage of numerous sugars from a donor to numerous acceptor sugars. Linkage of sugar derivatives can also achieved through use of the altered catalytic domains of the invention due to their expanded donor and acceptor specificity. The presence of modified sugar moieties on a glycoprotein makes it possible to link bioactive molecules via modified glycan chains, thereby assisting in the assembly of bionanoparticles that are useful for developing the targeted drug delivery system and contrast agents for example for use in imaging, e.g. magnetic resonance imaging. The reengineered recombinant glycosyltransferases as described herein also make it possible to remodel the oligosaccharide chains of glycoprotein drugs, and to synthesize oligosaccharides for vaccine development.

Targeted glycoconjugates

The sugars and one or more functional groups, e.g. the sugar donors, as described herein, are transferred by glycosyltransferases, to a sugar acceptor. A sugar acceptor can be selected from galactose beta 1 ,4 glcNac or galactose beta 1 ,4 glucose. Sugars that can

be transferred include U DP-ga lactose, UDP- galactose analogues, UDP-GaINAc and UDP-GaINAc analogues. This reaction allows galactose to be linked to a sugar acceptor, for example galactose beta 1,4 glcNAc or galactose beta 1,4 glucose, that may itself be linked to a variety of other molecules, such as sugars and proteins, e. g., therapeutic agents, imaging agents, antibodies.

In one embodiment of the invention, the donor sugar is modified so as to include a functional group at the C2 position of the sugar ring, preferably a ketone or an azido or a thiol functionality.

WO 2005/051429, incorporated by reference in its entirety herein, describes methods used to bind a bioactive agent to the modified sugar. The bioactive compounds may preferably include a functional group which may be useful, for example, in forming covalent bonds with the sugar residue, which are not generally critical for the activity of the bioactive agent. Examples of such functional groups include, for example, amino(~ NH : 2), hydroxy(--OH), carboxyl (-COOH), thiol(-SH), phosphate, phosphinate, ketone group, sulfate and sulfonate groups. If the bioactive compounds do not contain a useful group, one can be added to the bioactive compound by, for example, chemical synthetic means. Where necessary and/or desired, certain moieties on the components may be protected using blocking groups, as is known in the art, see, e. g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons)(l 991 ). Exemplary covalent bonds by which the bioactive compounds may be associated with the sugar residue include, for example, amide (---CONH-) ;thioamide (-CSNH-) ; ether (ROR', where R and R'may be the same or different and are other than hydrogen); ester (-COO-) ; thioester (-COS-) ;-- 0- ;-S- ;-Sn-, where π is greater than I ₁ preferably about 2 to about 8; carbamates ;— NH- ;-NR-, where R is aikyl, for example, alkyl of from about 1 to about 4 carbons; urethane; and substituted imidate; and combinations of two or more of these.

Covalent bonds between a bioactive agent and a modified sugar residue may be achieved through the use of molecules that may act, for example, as spacers to increase the conformational and topographical flexibility of the compound. Examples of such spacers include, for example, succinic acid, 1 ,6-hexanedioic acid, ] ,8-octanedioic acid,

and the like, as well as modified amino acids, such as, for example, 6-aminohexanoic acid, 4-aminobutanoic acid, and the like.

One of skill in the art can easily chose suitable compatible reactive groups for the bioactive agent and the modified sugar, so as to generate a covalent bond between the bioactive agent and the modified sugar. Also, while the glycoconjugates of the invention are generally described with the targeting agent as the acceptor molecule or structure onto which a donor molecule (e. g., UDP-galactose) is actively linked through the action of a catalytic domain of a galactosyltransferase the bioactive agent can also be an acceptor molecule. In certain embodiments, the instant method can be used to monitor glycosylation, for example the glycosylation of therapeutic glycoproteins and monoclonal antibodies. The potential of glycosyltransferase enzymes to produce glycoconjugates carrying sugar moieties with reactive groups may be a benefit to the glycotargeting of drugs to their site of action. Although a great number of pharmaceutical agents are discovered each year, the clinical application of these is many times hindered because of failure to reach the site of action. The methods described herein that include using reengineered glycosy transferases to transfer chemically reactive sugar residues for linking of other molecules via specific glycan chains may be used as an efficient drug delivery system.

Detection

The sugars comprising one or more functional groups, e.g. the sugar donors, as described herein have application in the detection of specific sugar residues on a glycan chain of a glycoconjugates and in the glycoconjugation and assembly of bio- nanoparticles for the targeted delivery of bioactive agents. Protein glycoslation is one of the most abundant posttranslationai modifications and plays a fundamental role in the control of biological systems and in disease.

Accordingly, glycosylation has been found to be a marker in disease. Additionally, carbohydrate modifications have been shown to be important for host- pathogen interactions, inflammation, development, and malignancy (Varki, 1993; Lasky, 1996;).

The methods described herein offer the advantages the modification occurs in a site directed manner, only where the carbohydrate is attached to the glycoprotein. Such specificity permits, for example, the use of site-directed immunotherapy without affecting the antigen binding affinity of the immunoglobulin. Such specificity permits, further, the potential use of this approach in developing a drug delivery system or biological probes.

Imaging

Included in the invention are methods for imaging a target cell or tissue in a subject. The methods as described herein comprise administering to a subject a polypeptide fragment synthesized by the method comprising incubating a reaction mixture comprising a sugar and one or more functional groups as described herein, wherein one or more imaging agents are linked to the sugar donor, a glycosyitransferase, and an sugar acceptor thereby imaging a target cell or tissue.

Coupling

The invention features methods coupling. For example, the invention features methods of coupling an agent to a carrier protein comprising incubating a reaction mixture comprising a sugar nucleotide and one or more functional groups, wherein the sugar nucleotide is a substrate of one or more glycosyltransferases, with a sugar acceptor and a giycosytransferase.

The sugar nucleotide, as described herein, can be any sugar nucleotide; however in certain preferred examples, the sugar nucleotide is selected from, but not limited to UDP-galactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues.

The sugar nucleotide in preferred embodiments comprises a chemically reactive group. The chemically reactive group is used for coupling to the carrier protein and can be selected from, but not limited to, an azido group, a keto group, an alkyne group or a thiol group. The azido group, the keto group, the alkyne group or the thiol group is substituted at the C2 position on the sugar.

The C2 position is preferred, in certain examples, for the attachment of functional group.

In other examples, the functional group, e.g. the carrier protein, is directly attached to the sugar nucleotide.

Methods of transfer of C2 modified sugar analogues, for example a C2 keto sugar from its UDP derivative to the GIcNAc residue on the N-g]ycan chain of ovalbumin or to an asialo-agalacto-ϊgG ] molecule have been described in the art, for example in WO 2005/051429, incorporated by reference in its entirety herein, A C2 modified galactose analogue, for example C2 keto galactose can be biotinyiated, thus allowing for biotinylation of carriers such as ovalbumin and IgG.

In addition to ovalbumin, single chain antibodies and toxins are also coupled using the methods described herein.

The method of coupling a target agent to a carrier protein via glycan chains, for example ovalbumin and IgGl , is advantageous over other cross-linking methods. In the instant method, the target agent is linked in a site-directed manner, only where the carbohydrate is attached to the glycoprotein, for example as in the IgG l molecule at the Fc domain, away from the antigen binding site. A problem encountered in previous approaches using monoclonal antibodies for immunotherapy is the lack of specificity of the reactions, resulting in heterologous labeling and a decrease in the antibody affinity for the antigen. The instant invention overcomes this problem.

Accordingly, the invention features methods of coupling an agent or agents to a carrier protein. The methods described herein comprise coupling an agent to a carrier protein comprising incubating a reaction mixture comprising a sugar nucleotide, wherein one or more targeting agents are linked to the sugar donor, a glycosyltransferase, and an sugar acceptor thereby imaging a target cell or tissue.

The carrier protein, in preferred examples, is ovalbumin, or an antibody (e.g. a single chain antibody) or toxin. The carrier protein, in other preferred examples, is an

IgG. In certain instances, it is advantageous to couple the C2 UDP-galactose analogue to biotin for detection. Subsequent detection of biotin can be carried out by chemiluminescent assay. The method as described herein is useful for imaging procedures, for example in magnetic resonance imaging.

Antibodies and Applications

As described herein, the targeting compound may be an antibody or a fragment thereof. The term "antibody" (Ab) or "monoclonal antibody" (M ab) is meant to include intact molecules as well as antibody portions (e. g., Fab and F (ab')2 portions and Fv fragments) which are capable of specifically binding to a cell surface marker. Such portions are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab portions) or pepsin (to produce F (ab')2 portions). Alternatively, antigen- binding portions can be produced through the application of recombinant DNA technology.

The immunoglobulin can be a "chimeric antibody" as that term is recognized in the art. Also, the immunoglobulin may be a bifunctional or a hybrid antibody, that is, an antibody which may have one arm having a specificity for one antigenic site, such as a tumor associated antigen, while the other arm recognizes a different target, for example, a hapten which is, or to which is bound, an agent lethal to the antigen-bearing tumor cell. Alternatively, the bifunctional antibody may be one in which each arm has specificity for a different epitope of a tumor associated antigen of the eel! to be therapeutically or biologically modified, ϊn any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state. Biological bifunctional antibodies are described, for example, in European Patent

Publication, EPA 0 105 360, which is incorporated herein by reference. Hybrid or bifunctional antibodies may be derived biologically, by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide bridge-forming reagents, and may be comprised of those antibodies and/or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed, for example, in PCT application W083/03679, published Oct. 27, 1983, and published European Application EPA 0 217 577, published Apr, 8, 1987, which are incorporated herein by reference. In one embodiment, the bifunctional antibodies are biologically prepared from a polydome or a quadroma, or are synthetically prepared with cross-linking agents such as bis- (maleimideo) -methyl ether("BMME"), or with other cross-linking agents familiar to those skilled in the art.

In addition, the immunogbbin may be a single chain antibody ("SCA"). These may consist of single chain Fv fragments ("scFv") in which the variable light ("V [L] ") and variable heavy ("V [H] ") domains are linked by a peptide bridge or by disulfide bonds. Also, the immunoglobulin may consist of single V [H] domains (dAbs) which possess antigen-binding activity. See, e. g. , G. Winter and C. Miistein, Nature, 349: 295 (1991); R. Glockshuber et a!., Biochemistry, 29: 1362 (1990); and, E. S. Ward et al. , Nature, 341 : 544 (1989).

The antibodies may, in certain embodiments, be chimeric monoclonal antibodies. As used herein, the term "chimeric antibody" refers to a monoclonal antibody comprising a variable region, i. e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques.

Chimeric antibodies comprising a murine variable region and a human constant region are preferred in certain applications of the invention, particularly human therapy, because such antibodies are readily prepared and may be less immunogenic than purely murine monoclonal antibodies. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of chimeric antibodies encompassed by the invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies. "Methods for producing chimeric antibodies involve conventional recombinant DNA and genetransfection techniques well known in the art. See, e. g. , Morrison, S. L. et al. , Proc.Nat'l Acad.ScL, 81 : 6851 (1984). Encompassed by the term "chimeric antibody" is the concept of "humanized antibody, "that is those antibodies in which the framework or "complementarity" determining regions ("CDR") have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. (See, e. g. , EPA 0 239 400 (published Sep. 30, 1987)) In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody." See, e. g. , L. Riechmann et al., Nature, 332: 323

( ^] 988); M. S. Neuberger et ai., Nature, 314: 268 (1985). Furthermore, the immunoglobulin (antibody), or fragment thereof, used in the present invention may be polyclonal or monoclonal in nature. Monoclonal antibodies are the preferred immunoglobulins, The preparation of such polyclonal or monoclonal antibodies is well known to those skilled in the art. See, e.g., G. Kohler and C. Milstein, Nature, 256: 495 (1975). The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the cell surface marker or an antigenic portion thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of protein is prepared and purified so as to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. However, the present invention should not be construed as limited in scope by any particular method of production of an antibody whether bifυnctional, chimeric, bifunctional- chimeric, humanized, or an antigen-recognizing fragment or derivative thereof. ϊn a preferred embodiment, the antibodies of the present invention are monoclonal antibodies (or portions thereof). Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al. Nature, 256: 495 (1975); Kohler et al, , Eur. J. Immunol., 6: 511 (1976); Kohler et al, Eur. J. Immunol., 6: 292 (1976); Hammerling et al., In : "Monoclonal Antibodies and T-CeII Hybridomas, "Elsevier, N. Y. , pp. 563- 681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a protein antigen or with a protein-expressing cell (suitable cells can be recognized by their capacity to bind antibody). The splenocytes of such immunized mice are extracted and fused with a suitable myeloma cell line. Any suitable myeϊoma cell line may be employed in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et a!., Gastroenterology, 80: 225-232 (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the antigen. In addition, hybridomas and/or monoclonal antibodies which are produced by such hybridomas and which are useful in

the practice of the present invention are publicly available from sources such as the American Type Culture Collection or commercial retailers. .

The antibodies of the present invention may be labeled, for example, for detection or diagnostic purposes, e. g. , imaging. Labels for the antibodies of the present invention include, but are not limited to, the following: examples of enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isornerase, yeast-alcohol dehydrogenase, alpha-glycero! phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, riboπuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase; examples of radioisotopic labels include 3H ₅ IIlIn, 1251, 1311, 32p, 35S, 14c, 51Cr, 57To _; 58Co, 59Fe, 75Se, 152Eu, 9OY, 67Cu ₁ 217Ci, 21 1 At ₁ 212Pb ₁ 47Sc, and 109Pd; examples of suitable non-radioactive isotopic labels includel57Gd, 55Mn,52Tr, and 56Fe ; examples of fluorescent labels include an 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label; examples of toxin labels include diphtheria toxin, ricin, and cholera toxin ; examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label; and examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and Fe,

Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al., Clin. Chim. Acta, 70: 1-31 (1976), and Schurs et al. , Clin. Chim. Acta.81 : 1-40 (1977), which are incorporated by reference In one embodiment, the glycoconjugates of the invention include monocSonal antibodies, such as those directed against tumor antigens, for use as cancer therapeutics. Generally, monoclonal antibodies have one N-linked bi- antennary oligosaccharide attached at the IgG-Fc region. The terminal sugars of the oligosaccharide moiety come in several glyco forms, for example, some are desiaiated, degalactosylated, with only terminal N-acety!glucosaminyl residues.

The monoclonal antibodies carrying only terminal N-acety!gucosamine on the bi- antennary oligosaccharide moieties, the Gogiycoform, can be generated by de-sialylation and de-gal actosyl at ion of the monoclonal antibodies. According to methods of the invention, a sugar moiety that has a chemically reactive group attached at the C2 position of the sugar, e.g. galactose, can then be transferred to Go glycoform of the monoclonal antibody. The chemically reactive group can include, for example, a ketone moiety that can serve as a neutral, yet versatile chemical handle to add other agents, such as bioactive agents, to the compound.

Methods of Treatment

The instant invention provides sugar nucleotides and methods that can be used to promote the chemical linkage of biologically important molecules that have previously been difficult to Sink, and thus provides a means to link agents for therapeutic application. Moreover, the instant invention provides a means to carry out the method in a physiological setting.

Accordingly, the invention features methods for the diagnosis or treatment of a subject suffering from a disease or disorder.

Disease states needing treatment are only limited by current available therapeutics. Methods of treatment comprise administering to the subject an effective amount of a sugar nucleotide and one or more functional groups synthesized by a method as described herein, and administering the sugar nucleotide and one or more functional groups to the subject, thereby treating the subject.

Methods of diagnosis comprise obtaining a sample from a subject; and contacting the sample with an effective amount of a sugar nucleotide and one or more functional groups synthesized by the methods as described herein, thereby diagnosing a subject as suffering from a disease or disorder .

As described herein any sugar nucleotide that is suitable for the methods can be used, with UDP-g a lactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues being preferable in certain methods.

Preferably, the sugar nucleotide comprises a chemical reactive group tha is used as a handle for the attachment of a functional group, e.g. an agent, e.g. a therapeutic or a diagnostic agent. In certain preferred examples, the chemically reactive group is an azido group, a keto group, an alkyne group or a thiol group. Preferably, the azido group, the keto group, the alkyne group or the thiol group is substituted at the C2 position, where the C2 position is used for the attachment of functional group.

Alternatively, the functional group is directly attached to the sugar nucleotide, One or more functional groups may be attached to the sugar nucleotide, The functional group, as described herein, may be any agent or bioactive agent that is useful to the method. For instance in a therapeutic method the functional group may be a drug to treat or prevent a disease or disorder.

As described herein, the methods of the invention are useful for engineering of nanoparticles, including multivalent nanoparticles, carrying any number of therapeutic agents. For example, the nanoparticles can be used to treat cancer, inflammatory disease, cardiovascular disease, obesity, ageing, bacterial infection, or any other disease amenable to therapy.

The glycoconjugates produced by the methods of the invention compositions of the invention can be used to treat and/or diagnose a variety of diseases and/or disorders. For example, the glycoconjugates compositions of the invention are used for specific, targeted delivery of bioactive agents, including toxic drugs, agents for imaging or diagnostics, (e. g. _> toxins, radionuclides), to therapeutically-relevant tissues or cells of the body, for exampJe, tumors. In another embodiment of the invention, the glycoconjugates compositions of the invention are used to deliver bioactive agents, including DNA vectors, to cells. As further examples, the glycoconjugates compositions of the invention are useful for the treatment of a number of diseases and/or disorders including, but not limited to: cancer, both solid tumors as well as blood-borne cancers, such as leukemia; hyperproliferative disorders that can be treated by the compounds of the invention include, but are not limited to, neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,

testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital

The glycoconjugates of the invention can be used to treat cardiovascular diseases and disorders including, but not limited to, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient isdiaemic attacks (TIA), peripheral vascular diseases, arteriosclerosis, angina, high blood pressure, high cholesterol, arrhythmia.

The glycoconjugates of the invention can be used to treat genetic diseases, such as enzyme deficiency diseases.

The glycoconjugates of the invention can be used to treat hyperproliferative disorders. Examples of such hyperproliferative disorders that can be treated by the gSycoconju gates of the invention are as described in Application WO 2005/051429, and are incorporated by reference in its entirety herein.

The glycoconjugates of the present invention are also useful for raising an immune response against infectious agents. Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated by the compounds of the invention. Examples of viruses that can cause disease or symptoms and that can be treated by the glycoconjugates of the invention are as described in Application WO 2005/051429, and are incorporated by reference in its entirety herein.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated by the glycoconjugates of the invention are as described in Application WO 2005/051429, and are incorporated by reference in its entirety herein.

Additionally, the glycoconjugates of the invention are useful for treating autoimmune diseases. An autoimmune disease is characterized by the attack by the immune system on the tissues of the victim. Autoimmune disease is characterized by the inability of the recocognitioπ of "self and the tissue of the afflicted subject is treated as a foreign target. The compounds of the present invention are therefore useful for treating autoimmune diseases by desensitizing the immune system to these self antigens by provided a TCR signal to T cells without a costimulatory signal or with an inhibitory signal. Examples of autoimmune diseases which may be treated using the glycoconjugates of the present invention are as described in Application WO 2005/051429, and are incorporated by reference in its entirety herein.

Simiiarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by giycoconjugates of the invention. Moreover, the glycoconjugates of the invention can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. The glycoconjugates of the invention which can inhibit an immune response are also useful for treating and/or preventing organ rejection or graft versus host disease, atherosclerosis; olitis; regional enteritis; adult respiratory distress syndrome; local manifestations of drug reactions, such as dermatitis, etc.; inflammation-associated or allergic reaction patterns of the skin; atopic dermatitis and infantile eczema; contact dermatitis; psoriasis; lichen planus; allergic enteropathies; allergic rhinitis; bronchial asthma; hypersensitivity or destructive responses to infectious agents; poststreptococcal diseases, e. g. cardiac manifestations of rheumatic fever, and the like.

Many bioactive metabolites possess unusual carbohydrates required for molecular recognition. (See for example, Liu, H.-w,; Thorson, J. S, Ann. Rev, Microbiol., 1994, 48, 223-256; Weymouth- Wilson, A. C. Nat. Prod. Rep, 1997, 14, 99-1 10; In Macrolide Antibiotics, Chemistry, Biology and Practice; Omura, S. Ed., Academic Press: New York; 1984; Johnson, D. A.; Liu, H.-w. Curr. Opin. Chem. Biol. 1998, 2, 642-649; and Trefzer, A.; Salas, J. A.; Bechthold, A. Nat. Prod. Rep. 1999, 16, 283-299.) In fact, roughly 70% of current lead compounds in modern drug discovery derive directly from natural products, many of which are glycosylated metabolites. (See Thorson, J. S. et al. Nature's Carbohydrate Chemists: The Enzymatic Glycosylation of Bioactive Bacterial Metabolites. Curr. Org. Chem. manuscript in press, (2000); and references therein and Weymouth-Wilson, A. C. The Role of Carbohydrates in Biologically Active Natural Products. Nat. Prod. Rep. 14, 99-1 10 (1997)). Examples of pharmaceutically important glycosylated metabolites include, for example, amphotericin, megalomicin/erythromycin, mithramycin, doxorubicin, vancomycin and calicheamicin. While it is known that the sugar moieties of these pharmaceutically important metabolites often define their corresponding biological activity, (see Weymouth-Wilson, A. C, The Role of Carbohydrates in Biologically Active Natural Products, Nat. Prod. Rep. 14, 99-1 10 (1997)), efficient methods to systematically alter these essential carbohydrate ligaπds are still lacking.

The present invention will broadly impact efforts to understand and exploit the biosynthesis of glycosylated bioactive natural products, many of which are pharmacologically useful. (See Thorson, J. S.; Shen, B.; Whitwam, R. E.; Liu, W.; Li, Y.; Ahlert, J. Bioorg. Chem., 1999, 27, 172-188; Whitwam, R. E.; Ahlert, J.; Holmaπ. T. R.; Ruppen, M ₁ ; Thorson, J. S. J. Am. Chem. Soc, 2000, 122, 1556-1557; Thorson, J. S.; Sievers, E. L.; Ahlert, J.; Shepard, E.; Whitwam, R. E.; Onwueme, K. C; Ruppen, M. Cur. Pharm. Des., 2000, manuscript in press; and J, S. Thorson, T. J. Hosted Jr., J, Jiang, J. B. Biggins, J. Ahlert, M. Ruppen, Curr. Org. Chem. 2000).

Vaccines

The invention also provides methods for eliciting an immune response in a mammal such as a human, including administering to a subject an immunological composition comprising a compound or composition as described herein. Therefore, one embodiment of the present invention is to use the glycoconjugates described herein in an immunological preparation.

The immunological composition according to the instant invention may be prepared by any method known in the art. For example, glycoconjugates of the present invention are prepared and are then injected into an appropriate animal. The compositions according to the present invention may be administered in a single dose or they may be administered in multiple doses, spaced over a suitable time scale to fully utilize the secondary immunization response. For exampfe, antibody titers may be maintained by administering boosters once a month. The vaccine may further comprise a pharmaceutically acceptable adjuvant, including, but not limited to Freund's complete adjuvant, Freund's incomplete adjuvant, lipopoiysaccharide, monophosphoryl lipid A, muramyl dipeptide, liposomes containing lipid A, alum.muramyl tripeptide- phosphatidylethanoloamine, keyhole and limpet hemocyanin.

Administration

The compositions of the present invention may be administered by any means that results in the contact of the bioactive agent with the agent's site or site (s) of action on or

in a subject, e, g., a patient. The compositions may be administered alone or in conjunction with one or more other therapies or treatments.

The targeted glycoconjugates produced according to the present invention, can be administered to a mammalian host by any route. Thus, as appropriate, administration can be orally, intravenously, recta] Iy, parenterallyjπtracistemally, intradermally,intravaginally, intraperitoneally, topically (as by powders, ointments, gels, creams, drops or transdermal patch), bucally, or as an orai or nasal spray. The term"parentera] ^! 'as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion. Parenteral administration in this respect includes administration by the following routes: intravenous, intramuscular, subcutaneous,intraocuJar,intrasynovial,transepiέhe!ia] including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol and rectal systemic. In addition, administration can be by periodic injections of a bolus of the therapeutic or can be made more continuous by intravenous or intraperitoneal administration from an external source. In certain embodiments, the therapeutics of the instant invention can be pharmaceutical- grade and incompliance with the standards of purity and quality control required for administration to humans. Veterinary applications are also within the intended meaning as used herein.

The formulations, both for veterinary and for human medical use, of the therapeutics according to the present invention typically include such therapeutics in association with a pharmaceutically acceptable carrier therefor and optionally other ingredient (s). The carrier (s) can be acceptable in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile

diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite ; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Useful solutions for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences. Formulations for parenteral administration also can include glycocholate for buccal administration, methoxysal icy late for rectal administration, or citric acid for vaginal administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Formulations of the present invention suitable for oral administration can be in the form of discrete units such as capsules, gelatin capsules, sachets, tablets, troches, or lozenges, each containing a predetermined amount of the drug; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil- in-water emulsion or a water-in-oil emulsion The therapeutic can also be administered in the form of a bo!us,electuary or paste. A tablet can be made by compressing or molding the drug optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the drug in a free-flowing form such as a powder or granules, optionally mixed by a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered drug and suitable carrier moistened with an inert liquid diluent.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients. Oral compositions prepared using a fluid carrier for use as a mouthwash include the compound in the fluid carrier and are applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules,

troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystaϊline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as algiπic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water,

Cremophor ELTM (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi, The carrier can be a solvent or dispersion medium containing, for example, water, ethano), polyol (for example, glycerol, propylene glycol, and liquid poiyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization, e. g. , filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from

those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient.

Formulations suitable for topical administration, including eye treatment, include liquid or semi-liquid preparations such as liniments, lotions, gets, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pasts; or solutions or suspensions such as drops. Formulations for topical administration to the skin surface can be prepared by dispersing the therapeutic with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap. In some embodiments, useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal.

For inhalation treatments, such as for asthma, inhalation of powder (self- propelling or spray formulations) dispensed with a spray can, a nebulizer, or an atomizer can be used. Such formulations can be in the form of a finely comminuted powder for pulmonary administration from a powder inhalation device or self-propelling powder- dispensing formulations. In the case of self- propelling solution and spray formulations, the effect can be achieved either by choice of a valve having the desired spray characteristics (i. e. , being capable of producing a spray having the desired particle size) or by incorporating the active ingredient as a suspended powder in controlled particle size. For administration by inhalation, the therapeutics also can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e. g. , a gas such as carbon dioxide, or a nebulizer. Nasal drops also can be used.

Systemic administration aiso can be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants generally are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and filsidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the therapeutics typically are formulated into ointments, salves, gels, or creams as generally known in the art.

The therapeutics can be prepared with carriers that will protect against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

The compounds of the invention may also suitably be administered by sustained- release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, e. g., films, or mirocapsules. Sustained-release matrices include polylactides (U. S. Pat. No. 3,773, 919, EP 58, 481), copolymers of L-glutamic acid and gamma- ethyl-L-glutamate (U. Sidman et a!. , Biopoiymers 22: 547-556 (1983) ), poly (2- hydroxyethyl methacrylate) (R. Langer et al. , J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12: 98-105

(1982)), ethylene vinyl acetate(R. Langer et al., Id. ) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomally entrapped compositions of the present invention (Epstein, et al, , Proc. Natl. Acad, Sci. USA 82: 3688- 3692 (1985); Hwang et al. , Proc. Natl. Acad. Sci. USA 77: 4030-4034(1980). The compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Generally, the therapeutics identified according to the invention can be formulated for administration to humans or other mammals, for example, in therapeutically effective amounts, e. g. , amounts which provide appropriate concentrations of the bioactive agent to target tissue/cells for a time sufficient to induce the desired effect Additionally, the therapeutics of the present invention can be administered alone or in combination with other molecules known to have a beneficial effect on the particular disease or indication of interest. By way of example only, useful

cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and antifungal agents and analgesics andanesthetics.

The effective concentration of the therapeutics identified according to the invention that is to be delivered in a therapeutic composition will vary depending upon a number of factors, including the final desired dosage of the drug to be administered and the route of administration. The preferred dosage to be administered aiso is likely to depend on such variables as the type and degree of the response to be achieved; the specific composition of another agent, if any, employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the composition; the duration of the treatment; bioactive agent (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts, In some embodiments, the therapeutics of this invention can be provided to an individual using typical dose units deduced from the earlier-described mammalian studies using non-human primates and rodents. As described above, a dosage unit refers to a unitary, i. e. a single dose which is capable of being administered to a patient, and which can be readily handled and packed, remaining as a physically and biologically stable unit dose comprising either the therapeutic as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers. Therapeutics of the invention also include "prodrug" derivatives. The term prodrug refers to a pharmacologically inactive (or partially inactive) derivative of a parent molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release or activate the active component. Prodrugs are variations or derivatives of the therapeutics of the invention which have groups cleavable under metabolic conditions. Prodrugs become the therapeutics of the invention which are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352- 401 , Academic Press, San Diego.Calif., 1992).

Therapeutic or Diagnostic Agents

A wide variety of agents may be included in the compounds of the present invention, such as any biologically active, therapeutic or diagnostic compound or composition. In general, the term bioactive agent includes, but is not limited to: polypeptides, including proteins and peptides (e. g. , insulin); releasing factors and releasing factor inhibitors, including Luteinizing Hormone Releasing Hormone (LHRH) and gonadotropin releasing hormone(GnRH) inhibitors; carbohydrates (e. g. , heparin); nucleic acids; vaccines; and pharmacologically active agents such as anti-infectives such as antibiotics and antiviral agents; anti-fungal agents; analgesics and analgesic combinations; anesthetics; anorexics ; anti-helminthics; anti-arthritic agents; respiratory drugs, including anti-asthmatic agents and drugs for preventing reactive airway disease; anticonvulsants; antidepressants ; anti-diabetic agentsjanti-diarrheals ; anticonvulsants; antihistamines; anti-inflammatory agents; toxins, anti-migraine preparations; anti- nauseants; anticancer agents, including anti-neoplastic drugs; anti-parkinsonism drugs; anti-pruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, alpha-blockers, cardioprotective agents; antiarrhythmics ; anti-hyperlipidemic agents; anti-hypertensives; diuretics; anti- diuretics; receptor agonists, antagonists, and/or mixed function agonist/antagonists; vasodilators including general coronary, peripheral and cerebral; centra! nervous system stimulants; vasoconstrictors; cough and cold preparations, including decongestants ; enzyme inhibitors; hormones such as estradiol, testosterone, progesterone and other steroids and derivatives and analogs, including corticosteroids ; hypnotics; hormonolytics ; immunosuppressive agents; muscle relaxants; parasympatholytics; central nervous system stimulants; diuretics; hypnotics leukotriene inhibitors; mitotic inhibitors; muscle relaxants; genetic material, including nucleic acid, RNA, DNA, recombinant RNA, recombinant DNA, antisense RNA, antisense DNA, hammerhead RNA, a ribozyme, a hammerheadribozyme, an antigene nucleic acid, a ribo-oligonucleotide, a deoxyribonucleotide, an antisense ribo- oligonucleotide, and/or an antisense deoxyribo- oligonucleotide; psychostimulants; sedatives; anabolic agents; vitamins ; herbal remedies;

anti- metabolic agents;anxiolytics ; attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD) drugs; neuroleptics ; and tranquilizers.

Application No. WO 2005/051429, incorporated by reference in its entirety herein, provides a list of exemplary agents that can be conjugated to the compositions of the instant invention,

Kits

Also included in the invention are kits. Preferably, kits comprise a packaging materia!, and a sugar comprising a functional group according to any one of the aspects of the invention as described herein.

The kits according to the invention can comprise a sugar nucleotide and one of more functional groups according to the invention as described herein. The kits also comprise a glycosy I transferase.

The glycosyltransferase can be any giycosyltransferase, as long as it is able to transfer a sugar from donor, e.g. donor nucleotide, to an acceptor.

The glycosyltransferase can be a wild type or a altered giycosyltransferase. The wild type glycosyltransferase is isolated and packaged in the kit,

In certain preferred examples, the gSycosyltransferases are selected from galactosyltransferases, acetylgalactosyltransferases and polypeptidylgalactosyltransferases. The galactosyltransferase can be a beta galactosy transferase or an alpha acetylgaϊactosaminyltransferase.

The glycosyitransferases can be selected from the group consisting of: beta 1 ,4 galactosyltransferase, alpha 1 ,3 N-Acetylgalactosaminyltransferase.

The kit preferably contains a sugar nucleotide that is selected from UDP- galactose, UDP-GaINAc, UDP-GaINAc analogues or UDP-galactose analogues.

The sugar nucleotide preferably comprises a chemically reactive group, selected from an azido group, a keto group, an alkyne group or a thiol group.

As described, the functional group can be any agent, e.g. a bioactive agent, that is useful in the methods as set forth herein. The functional group can be, in certain examples, chemical reactive groups, dyes, targeting agents, radio labels, fluorescent labels, conjugated substances, probes, lipids, chelators, contrast agents, magnetic

resonance imaging agents, mass labels, peptides, polymers, antibodies, single chain antibodies, bacterial toxins, growth factors, therapeutics, cleavable linkers, and non- cleavable linkers, or a combination thereof.

EXAMPLES

It should be appreciated that the invention shouid not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

Methods

The invention was carried out, in part, using the methods as described herein. Chemicals and reagents were used without further purification unless otherwise noted and were purchased from the commercia! supplier Sigma-Aldrich (St. Louis, MO). Analytic thin layer chromatography (TLC) was performed using Sorbent Technologies precoated TLC plates (silica gel XHL, 250 microns) containing a fluorescense indicator. The plates were visualized potassium permanganate stain (1.5 g of KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water) and/or by spraying with sugar spray reagent (5 mL of 4-methoxybenzaldehyde, 90 mL of ethanol, 5 mL of concentrated sulfuric acid, and 10 mL of glacial acectic acid) and subsequent heating. Biogel separation was performed on Biogel P2 (Bio-Rad) either with 0.25 M NH4HCO3 solution or water as the eluent. High pressure liquid chromatography (HPLC) was using Beckman Coulter, Inc. HPLC instrument with 128p semi-prepative pump and with 166p detector. ^" NMR spectra were obtained on Varian Inova 400 MHz spectrometer. High resolution mass spectra (MALDl-TOF MS) were obtained on an Applied Biosystem

QSTAR-XL hybrid triple-quad TOF mass spectrometer by Mr. Timothy J. Waybright of the Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., Frederick, MD 21702. The samples were typically infused at a flow rate of 5 uL/minute, data acquisition was done in either the positive or negative mode, acquisition time was typically 3-5 minutes. Prior to each HRMS analysis, the equipment was calibrated with either Renin substrate or taurochotic acid. Infrared spectroscopy data was obtained neat

with a Jasco FT-IR/615 spectrometer. The acceptor peptide PTTDSTTP APTTK was synthesized by GenScript Corporation (Piscatasway, NJ). The β 1 ,4-galactosy (transferase altered enzyme, Y289L GaI-Tl was expressed and purified as described previously. Human polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) was expressed and purified as described previously. All experiments with both Y289L GaI- Tl and ppGal ^" NAoT2 were kindly provided by and conducted with Dr. Elizabeth Boeggemaπ, SAIC-Frederick, Inc., Frederick, MD 21702. Analysis of chemical and enzymatic reactions were analyzed with liquid chromatography and mass spectometry (LC-MS) by Mr. Stephen D. Fox, SAIC-Frederick, Inc., Frederick, MD 21702. LC-MS had an Agilent Technologies (Wilmington, DE) 1 100 MSD ion trap mass spectrometer equipped with an electrospray ionization source, a KD Scientific (Holliston, MA) syringe pump, and a Dell Optiplex 170L workstation for control and data acquisition.

Example 1. Chemical Synthesis

Procedure A: Selective N-acylation, α-D-galactosamine-l -phosphate (1 equiv) was dissolved in water (0,2 M) and treated with a solution of the N-acyloxysuccinimide (1 equiv) in 5: 1 THF:water (0.2 M). The pH was adjusted to 7.0 using a 0.4 M solution of KOH in water. The resulting solution was stirred over night at room temperature, then was treated with some more of the N-aclyoxysuccinimide (0.8 equiv) in 5:1 THF: water solution (0.2 M) and subsequent pH adjustment to 7.0 and stirring at room temperature over night. The reaction was monitored with LC-MS to ascertain if the reaction was complete. Full conversion to product is essential for most of these compounds because otherwise separation of the desire acylated product is difficult to separate from the starting amine. If necessary, the reaction was treated with more of the N-acyloxysuccinimide (0.8 equiv) in 5: 1 THFiwater solution (0.2 M) and subsequent pH adjustment to 7.0 and stirring at room temperature over night. Keeping the reaction at pH = 7.0-8.0 proved to be helpful in improving the efficiency of these acylation reactions. The resulting solution had THF removed by evaporation and then the reaction was freezed-dried to afford a soMd. The resulting residue was taken up in water and purified on Biogel P2 with water as the eluent

and after freeze drying the factions containing product, this reaction yielded 80-90% of desired acylamido hexosyl phosphate. (Lazarevic, D., Thiem, J. Carbohydrate Research 2002, 2187; Lazarevic, D., Thiem, J, Carbohydrate Research 2006, 569.)

2-(But-3-yπoic acid amido)-2-deoxy-α-D-ga!actopyranosyl phosphate

Selective N-acylation. α-D-galactosamine-1 -phosphate was treated as mentioned in procedure A using but-3-ynoic acid iV-succinimidyl ester. The reaction was typicaSly complete after 72 hours. Upon purification with Biogel P2, the desired amide was isolated in 86% yield.

Procedure B: Triethylaniine salt formation.

The phosphates obtained by procedure A was dissolved in the minimum amount of water and passed through a column ( 1.5 x 8 cm) of Dowex 50W-X8 (triethyiammonium form) to give the phosphate in the form corresponding to triethylammonium salt in 88%-quantitative yield upon freeze drying the fractions.

^H r* ^{3 H} g ^Et3 2~(But-3-ynoic acid amido)-2-deoxy-α-D-galactopyraπosyl phosphate triethyJammonium salt

2-(But-3-ynoic acid amido)-2-deoxy-α-D-galactopyranosyl phosphate was transformed to the triethylammonium salt and achieved in 88% yield after freeze-drying the fractions.

Procedure C: Nucleotides by morphølidate coupling.

The triethylammonium phosphate salts (1 equiv) and uridine-5'- monophosphomorphoJtdate(4-moφlioline-N,N'-dicyciohexyt carboxamidiniurn salt (1.6 equiv) in anhydrous pyridine (0.02 M) was evaporated at ambient temperature under reduced pressure. After repeating this process for at ieast three times, the reaction was placed under dry argon and taken up in a 1 :1 anhydrous pyriodine: anhydrous DMF (0.06 M). This reaction was seaied under argon and stirred at room temperature for 5-7 days, After 3 days, the reaction was monitored by LC-MS but usually not observed to be complete. The solvents were removed under reduced pressure at ambient temperature. The product was then purified on Biogel P2 with first 0,25 M NH ₄ HCOi solution. Lyophilization of the desired fractions determined by HPLC (semi-preparative Varian Microsorb C 18, 100 rnM NH ₄ HCO ₃ , 5 mL/min).

Uridine-5'-diphospho-2-{But-3-ynoic acid amido)-2-deoxy-α-D-galactopyraπose dtammonium salt [UDP-2-(But-3-ynoic acid amido)-GaJ]

The titled compound was produced by procedure C. The reaction was completed in 7 days and in a greater than 40% yield upon Biogel P2 chromatography and HPLC purification and iyophilization. Procedure D offers more promise and will be tried in the future. Use of procedure D has decrease the reaction time as shown in the preparation of un ^' dine-5'-diphospho-2-(biotinyiamido)-2-deoxy-α-D-2-gaIactopy ranose which showed

reaction completion in 3-4 days as determined by LC-MS. Also, this reaction proved to be a much cleaner reaction than that of procedure C.

Procedure D: Nucleotides by morpholidate coupling The triethylammonium phosphate salts (1 equiv) and uridine-5'- monophosphomoφholidate(4~mθφholine-N,N'-dicyclohexyl carboxamidinium salt (1 .6 equiv) in anhydrous pyridine (0.02 M) was evaporated at ambient temperature under reduced pressure. After repeating this process for at least three times, the reaction was placed under dry argon and taken up in anhydrous pyridine (0.13 M). I H-Tetrazole (3 equiv) was added as a solid to the reaction flask; then, the reaction was sealed under argon and stirred at room temperature for 3-5 days. After 3 days, the reaction was monitored by LC-MS and conversion was nearly complete. The solvents were removed under reduced pressure at ambient temperature. The product was then purified on Biogel P2 with first 0.25 M NH ₄ HCO ₃ SOlUtIOn. Lyophilization of the desired fractions determined by HPLC (semi-preparative Varian Microsorb Cl 8, 100 mM NH ₄ HCO ₃ , 5 mL/min).

Example 2, Enzymatic Transfer Reactions

Y289L GaI-Tl and UDP-2-(But-3-ynoic acid amido)~Gal Reactions

For the reactions, a 25 μL incubation mixture containing 5 mM MnCb, 25 mM Tris-HCl, pH = 8 _; and using various concentration of the acceptor chitotetrose ; the sugar donor nucleotide UDP-2-(But-3-ynoic acid amido)-Gal; and the Y289L GaI-Tl enzyme as shown on Table 1 , below.

Example 3. LC-MS Analysis of Oligosaccharide

Reaction mixture solutions were diluted 20-fold in water and infυsed at a rate of l OuVmin into the electrospray ion source of the MS. The signal was acquired for approximated 3 minutes and the scans were summed to produce the averaged mass spectrum. Each reaction was monitored for oligosaccharide transferred product using LC- MS with an Agilent Technologies (Wilmington, DE) 1 100 MSD ion trap mass spectrometer equipped with an electrospray ionization source, a KD Scientific (Holliston, MA) syringe pump, and a Dell Optiplex 170L workstation for control and data acquisition. Operating conditions were as follows:

Ion polarity - negative

Ion profile - centroid

Scan window - 200 to 2000 m/z

Trap drive - 80.7 %

Capillary voltage - 4000

Drying gas temperature, flow - 300 ⁰ C, 8 l/min

The starting materials of sugar donor UDP-2-(But-3-ynoic acid amido)-Gai and acceptor chitotetrose were observed as expected in ail reactions even those in which the desired oligosaccharide transfer product was observed. Reaction M63 using 2 μg of Y289L GaI-T ^] was not any better than M67, M68, and M69 which used 4 μg, 8 μg, and 16 μg of Y289L GaI-TI respectively but increased enzyme did seem to give rise to more background in the analysis on the LC-MS so 2 μg of Y2S9L GaJ-Tl seemed to be the optimal amount of enzyme under these conditions. Further reaction optimization is ongoing to afford complete conversion. Figure 7 is the mass spectra of the transferred product.

ppGalNAc-T2 and UDP-2-(But-3-ynoic acid amido)-Gal Reactions

For the reactions, a 25 μL incubation mixture containing 5 mM MnCI ₂ , 25 mM Tris-HCl, pH = S, and using various concentration of the acceptor peptide PTTDSTTPAPTTK; the sugar donor nucleotide UDP-2-(But-3-ynoic acid amido)-Gal; and the enzyme ppGaϊNAc-T2 as shown on Table 2, below.

LC-MS Analysis of Oligosaccharide

Reaction mixture solutions were diluted 20-fold in water and infused at a rate of 30ui/miπ into the electrospray ion source of the MS. The signal was acquired for approximated 3 minutes and the scans were summed to produce the averaged mass spectrum. Each reaction was monitored for oligosaccharide transferred product using LC-

MS with an Agilent Technologies (Wilmington, DE) 1 100 MSD ion trap mass spectrometer equipped with an electrospray ionization source, a KD Scientific (Holliston, MA) syringe pump, and a Detl Optiplex 170L workstation for control and data acquisition. Operating conditions were as follows:

Ion polarity - negative Ion profi Ie - centroid Scan window - 200 to 2000 m/z Trap drive - 80.7 %

Capillary voltage - 4000

Drying gas temperature, flow - 300 ⁰ C, 8 1/min

The starting materials of sugar donor UDP-2-(But-3-ynoic acid amido)-Gal and acceptor peptide PTTD STTPAPTTK, were observed as expected in all reactions even those in which the desired glycopeptide transfer product was observed. Further reaction optimization is ongoing to afford complete conversion. Figure 8 is the mass spectra of the transferred product. Figure 8 shows the ESI Mass spectra of glycans after transfer of 2- (But-3-ynoic acid amido)-Gal to the peptide acceptor PTTDSTTP APTTK. Peak of 1579.6 m/z is the mass after addition of 2-(But-3-ynoic acid amido)-Ga! moiety.

Example 4. Other sugar substrates

Sugar donor nucleotide UDP-2-(But-3-ynoic acid amido)-Gal has be found to be a substrate for both altered Y289L GaI-Tl and ppGalNAc-T2 in which this new modified, unnatural sugar can be transferred to oligosaccharides and peptide acceptors, respectively. An azido-biotin probe is being made that should under go a 1,3 dipolar cycloaddition reaction via "click chemistry". These carbohydrate and glycopeptides products we be used to perform the subsequent reaction in which the chemical handle (e.g, the alkyne) can undergo a ciick reaction and specific deliver biotin to the oligosaccharide and glycoprotein. After this specific biotinylation, the reaction will be analyzed by MS as well as by blotting with streptavidin-HRP, followed by detection of chemiluminescence.

In addition, other substrates are prepared using procedure A-B and with either procedure C or D. The following substrates are underway. The dashed arrows reflect that these reaction are in progress or not performed to date. Otherwise the compounds are in hand and other studies are underway.

The synthesis of UDP-2-(biotin amido)-Gal sugar donor nucleotide is shown in Figure 9. This UDP-2-(biotin amido)-Gal sugar donor nucleotide has the probe directly attached to the sugar donor nucleotide so this eliminate the subsequent reaction if it transfers with the glycosyltransferases. In Figure 10, this UDP-2-(propynoic acid amido)-Gal has a smaller residue and may prove to transfer efficiently with our glycosyltransferases. This synthesis is underway

This sugar donor nucleotide, UDP-2-(pyruvic acid amido)-Gal as seen Figure 11 , is more attractive alternative to UDP-2-keto-Gal used previously (Figure 6-1 1 ). This UDP-2-keto-Gal was patented previously but has the drawback of a very long, poor yielding synthesis. ¹ This synthesis is three steps and should have an overall yield much higher than the eight-step synthesis of UDP-2-keto-Ga! which had less than 1 % overall yield (Figure 12 and 13). This ketone of the UDP-2-(pyruvic acid amido)-Gal should be amendable to the similar condensation reaction with aminooxy probes, aminooxy biotin, or other aminooxy biomoiecules (as seen previously). Iπvitrogen has now licensed the Y289L GaI-Tl as shown in molecular probes' kit (cat. # C33368). New synthesized, modified sugar donor nucleotides and intermediates would also be attractive substrate components for other such kits for glycosylatioπ detection.

Example 5. Intermediates

Figure 12 shows the synthesis of UDP-2-keto-Gal from galactat (Part 1), and

Figure 13 shows the synthesis of UDP-2-keto-Gal from galactal (Part 2). The intermediates of this synthesis may be able to be used for metabolic cell surface engineering (Hang, H. C, et al. 2001).

Example 6. Chemical synthesis of Azido-Biotiπ

2-Azido-ethylamine

To a solution of 2-chloro-l-ethylamine monohydrate (1 equiv) in water (0.9 M) was added sodium azide (3 equiv) and the reaction was sealed under argon and heated to 80C for 20 hours. The reaction was then cooled to OC. Then, diluted in half with diethyl ether. To cold solution was added KOFI (5.86 equiv, solid). The ethereal layer was

separated and the aqueous layer was extracted three more times with ether. The organic layer was dried with Na2SO4, filtered, and concentrated to give a volatile, cJear oi] in quantitative. The final step of synthesis of the azido biotin was previously described and is underway as shown in Figure 14. (Mayer, T. et al. 2007).

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any siπgJe element or combination (or subcombination) of iisted elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Ail patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

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