HK1168024A - Oligosaccharide-protein conjugates - Google Patents

Description

Oligosaccharide-protein conjugates

This application claims priority from U.S. provisional application No.61/122,851, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to oligosaccharide-protein conjugates comprising specific oligosaccharides, and compositions comprising such conjugates. The invention also relates to methods of treating lysosomal storage diseases using the oligosaccharide-lysosomal enzyme conjugates.

Background

Lysosomal Storage Disorders (LSDs) are a class of rare metabolic disorders that include over forty genetic diseases, including defects in the activity of lysosomal hydrolases. A hallmark feature of LSD is the abnormal accumulation of lysosomal metabolites, which leads to the formation of large numbers of swollen lysosomes.

LSDs can be treated by administering an active form of the enzyme that is missing in the subject, a method known as Enzyme Replacement Therapy (ERT). The administered replacement enzyme with terminal mannose-6-phosphate (M6P) is taken up by the target cell and directed to the lysosome by cell-surface-associated cation-dependent M6P receptor (CI-MPR) -mediated endocytosis.

In general, poorly phosphorylated alternative enzymes are not efficiently internalized by M6P receptors on the cell surface and are therefore not directed to the lysosomes they function. Thus, a lower degree of mannose phosphorylation may have a significant adverse effect on the therapeutic effect of the replacement enzyme.

Various methods have been developed to increase the M6P content of alternative enzymes. For example, U.S. patent nos. 6,534,300; 6,670,165, respectively; and 6,861,242 describe enzymatic phosphorylation of terminal mannose residues. In another example, U.S. patent No.7,001,994 describes a method of coupling oligosaccharides and glycoproteins comprising M6P. It was found that conjugation of the lysosomal enzyme acid alpha-Glucosidase (GAA) and bis-M6P oligosaccharide prepared by this method reduced skeletal and myocardial glycogen more effectively than recombinant human GAA in a murine model of pompe disease, a muscle disease that is autosomal recessive, results from metabolic deletion of GAA, and is characterized by accumulation of lysosomal glycogen. Similarly, Zhu et al describe the coupling of synthetic bis-M6P oligosaccharide (formula A) and GAA. Zhu et al, biochem.J.389: 619-628(2005). Formula A native trilinear Man from N-linked glycans (formula B)₉Designing a core structure: removing one branch, shortening the other branch, anAnd phosphorylating the terminal mannose residue.

The resulting conjugate, which bound CI-MPR with enhanced affinity, was more efficiently internalized by L6 myoblasts and had approximately normal enzymatic activity. However, despite such success, it remains important to identify new oligosaccharides that can result in improved affinity for CI-MPR and/or more efficient cellular internalization when conjugated to lysosomal enzymes, while maintaining normal or near normal enzymatic activity. However, improving the uptake alone does not necessarily lead to better therapeutic results. Certain conjugation strategies and oligosaccharides result in conjugates with lower enzymatic activity. It is therefore desirable to identify oligosaccharides and conjugates that can improve the therapeutic outcome of subjects with LSD.

In addition, certain oligosaccharides such as those shown in formulas a and B are difficult and expensive to synthesize. In addition, the preparation of β -linked sugars in a stereoselective manner is a problem in carbohydrate chemistry. Alternative oligosaccharides and synthetic methods may be more practical on a commercial scale. There is also a need for optimized methods for the preparation of oligosaccharide-protein conjugates. In particular, for therapeutic purposes, the conjugate preparation should not be heterogenous in coverage, as this may lead to inconsistent biological function. Various aspects of the conjugate can affect the therapeutic effect, including the oligosaccharides and linkers used, conjugation methods, purification methods, and formulation.

Disclosure of Invention

Accordingly, certain embodiments of the present invention provide oligosaccharide-protein conjugates comprising (1) a protein and (2) an oligosaccharide of formula I:

wherein:

a ═ α 1, 2; α 1, 3; α 1, 4; or α 1, 6;

b ═ α 1, 2; α 1, 3; or α 1, 4;

c ═ α 1, 2; α 1, 3; α 1, 4; or α 1, 6; and

d ═ α, β, or a mixture of α and β.

Other embodiments of the invention provide oligosaccharide-protein conjugates comprising (1) a protein and (2) an oligosaccharide of formula II:

wherein:

e ═ α 1, 2; α 1, 3; α 1, 4; or α 1,6 and

f ═ α, β or a mixture of α and β,

provided that when e ═ α 1,6, f ═ α or a mixture of α and β.

Even other embodiments of the present invention provide oligosaccharide-protein conjugates comprising (1) a protein and (2) an oligosaccharide of formula III:

wherein:

g ═ α 1, 2; α 1, 3; or α 1, 4;

h ═ α 1, 2; α 1, 3; α 1, 4; or α 1, 6; and

i ═ α, β, or a mixture of α and β.

A further embodiment of the invention provides an oligosaccharide-protein conjugate comprising (1) a protein and (2) an oligosaccharide of formula IV:

wherein:

j is α 1, 2;

k is selected from α, β and mixtures of α and β;

x is 1,2 or 3; and

when x is 2 or 3, the linkage between each mannose is selected from α 1, 2; α 1, 3; α 1, 4; and α 1, 6.

A further embodiment provides an oligosaccharide-protein conjugate comprising (1) a protein and (2) an oligosaccharide of formula V:

wherein:

l is selected from α, β and mixtures of α and β.

Further embodiments provide oligosaccharide-protein conjugates comprising (1) a protein and (2) an oligosaccharide of formula VI:

wherein:

R^xand R^yEach independently selected from polyethylene glycol and C₁-C₁₀Alkyl radical, said C₁-C₁₀Alkyl is optionally substituted by oxo, nitro, halo, carboxy, cyano or lower alkyl and optionally interrupted by one or more substituents selected from N, O orOne or more heteroatoms of S;

z is selected from 0,1, 2, 3 or 4;

m is selected from α, β and mixtures of α and β; and

when y is 2, 3 or 4, the linkage between each mannose is selected from α 1, 2; α 1, 3; α 1, 4; and α 1, 6.

In a further embodiment, the present invention provides an oligosaccharide-protein conjugate comprising (1) a protein and (2) an oligosaccharide of formula a.

In certain embodiments, the conjugate comprises at least 2, 3, 4 or 5 moles of oligosaccharide of formula a per mole of protein.

In some embodiments, the oligosaccharide-protein conjugates of the invention comprise a linker between the oligosaccharide and protein components of the conjugate.

The present invention provides a pharmaceutical composition comprising: an oligosaccharide-protein conjugate of formula I, II, III, IV, V or VI; and fillers, disintegrants, buffers, stabilizers or excipients. The invention also provides methods of treating lysosomal storage diseases (such as those disclosed in table 1 below) using the oligosaccharide-protein conjugates of formula I, II, III, IV, V or VI, or pharmaceutical compositions comprising such oligosaccharide-protein conjugates. The lysosomal storage disorder can be selected from, for example, fabry disease, pompe disease, niemann-pick a disease, niemann-pick B disease, and mucopolysaccharidosis I. In a further embodiment, the present invention provides the use of an oligosaccharide-protein conjugate comprising (1) a protein and (2) an oligosaccharide of formula I, II, III, IV, V or VI in the manufacture of a medicament for treating a lysosomal storage disease in a subject in need thereof.

Further embodiments of the invention are discussed in this application. Other aspects, features and advantages of the present invention will become apparent from the following detailed description. Embodiments discussed in relation to one aspect of the invention apply to the other aspects of the invention and vice versa. The embodiments in the examples section are to be understood as embodiments of the invention applicable to all aspects of the invention.

It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this application.

Brief Description of Drawings

Figure 1 shows an exemplary reverse synthetic route set forth in the synthetic steps for oligosaccharide 82.

Figure 2 shows an exemplary synthesis of monosaccharide building blocks 2a that can be used in the synthesis of oligosaccharides as described herein.

Fig. 3 shows an exemplary synthesis of monosaccharide building blocks 1,2, 3 and 4 that can be used in the synthesis of oligosaccharides as described herein.

FIG. 4 shows a synthetic route to the ethylene linker.

Figure 5 shows a synthetic route for the assembly of a trisaccharide precursor to an oligosaccharide 82 using building blocks 2, 3 and 4.

Figure 6 shows a synthetic route to assemble protected heptasaccharides from the trisaccharide precursors described in figure 4.

Figure 7 shows a synthetic route to construct the protected heptasaccharide described in figure 5 to produce oligosaccharide 82.

Fig. 8A-E show synthetic routes to the use of dibutyltin to form a stereoselective intermediate and optionally thiol-reactive group formation to prepare β -linked hexoses of formula a.

Figure 9 shows the effect of oxidation level on the conjugation of NeoGAA β SAM 6. Fig. 9A shows the amounts of SAM2, SAM3, SAM4, linear SAM4, alpha SAM6, and beta SAM6 oligosaccharides conjugated with GAA at varying molar ratios. Fig. 9B shows the amounts of hexose (glycan) conjugated with varying amounts of periodate and oxidized rhGAA.

Figure 10 shows oxidation of sialic acid, fucose, galactose and mannose using varying amounts of periodate. Fig. 10A shows oxidation monitored by monosaccharide composition analysis (oxidation was inferred based on a decrease in the amount of monosaccharide quantified). FIG. 10B shows LTQ MS detection of positive-shadow AA-labeled SAM6 oligosaccharides. FIGS. 10C and 10D show MS/MS spectra corresponding to AA-labeled oxidized oligosaccharides. Figure 10E shows monosaccharide analysis of GAM conjugates titrated with various amounts of GAO.

Figure 11 shows HPLC analysis of oligosaccharides released from rhGAA and NeoGAA.

FIG. 12 shows peptide mapping LC/MS analysis of rhGAA treated with 2 and 22.5mM periodate. Highlighted in the box are elution positions for unoxidized, and mono-and di-oxidized tryptic peptides T13 (containing methionine 172 and 173).

Figure 13 shows Biacore binding analysis of NeoGAA and rhGAA to sCIMPR. Figure 13A induction graph shows association, dissociation, M6P elution, and regeneration phases for each sample injection. Figure 13B shows a representative 4-parameter fit of sensorgram data for NeoGAA samples and rhGAA control samples. Fig. 13C shows the M6P receptor affinity of NeoGAA samples prepared using 2mM vs.7.5mM periodate, spanning different conjugation levels for each preparation.

Figure 14 shows the specific activity of various NeoGAA conjugates.

Fig. 15A-E show elution of the NeoGAA conjugate from the M6P receptor column. Figure 15F shows tissue glycogen levels in GAAKO mice after administration of 4-week doses of SAM6 conjugate.

Figure 16 shows the results of the L6 myoblast uptake assay, confirming internalization of various NeoGAA conjugates.

Figure 17 shows glycogen clearance from heart, quadriceps and triceps of GAA knockout mice following treatment with SAM 2.

Figure 18 shows glycogen clearance from the heart and quadriceps of GAA knockout mice following treatment with SAM 4.

Figure 19 shows glycogen clearance from the heart and quadriceps of GAA knockout mice following treatment with SAM 6.

Figure 20 shows glycogen clearance from heart and quadriceps of GAA knockout mice following treatment with SAM6 and GAM 6.

Detailed Description

To aid in understanding the invention, certain terms are first defined. Additional definitions are provided throughout this application.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a process containing "a compound" includes mixtures of two or more compounds. The term "or" is used in its sense to generally include "and/or" unless the content clearly dictates otherwise.

I. Oligosaccharide-protein conjugates

In one embodiment, the present invention provides oligosaccharide-protein conjugates, which may further comprise a linker. Exemplary oligosaccharides, proteins, linkers, conjugation methods, and conjugates are disclosed.

A. Oligosaccharides

The oligosaccharide may be selected from a di-linear oligosaccharide derivative of formula B or formula VI, as shown above or a linear oligosaccharide derivative as shown in formulae IV and V. The two-wire oligosaccharide typically has two terminal M6P residues and in some embodiments may also comprise one or more penultimate M6P residues. Linear oligosaccharides have at least one M6P residue and may comprise a terminal M6P residue. Typically, the terminal M6P residues can be linked by α 1,2 linkage (see Distler et al, J.biol. chem.266: 21687-21692(1991), observing that α 1,2 linkage at the terminal M6P results in better binding to CI-MPR and cation-dependent MPR (CD-MPR) than α 1, 3 or α 1,6 linkages). In some embodiments, the terminal M6P residues are at their respective reductionsThe termini connect adjacent residues by α 1,2 ligation. In some embodiments, the two terminal M6P residues are greater than 5, 10, 15, 20, 25, 30, 35 orThe spacing, which is determined by, for example, X-ray crystallization, NMR and/or molecular modeling. For example, molecular modeling methods can be as described by Balaji et al, Glycobiology 4: 497, 515 (1994). In some embodiments, the oligosaccharide is selected such that the terminal M6P residue has relatively little steric hindrance. In some embodiments, the terminal M6P residue binds CI-MPR with greater affinity relative to an unhindered oligosaccharide than to an oligosaccharide that is hindered by the terminal M6P residue.

Typically, the oligosaccharides will bind CI-MPR. For example, the oligosaccharide may bind CI-MPR with a dissociation constant of less than, e.g., 500, 100, 50, 10, 5,1 or 0.1nM or less than, e.g., 100, 50, 10, 5,2 or 1 μ M. Both ligand-bound and unbound forms of the crystal structure of N-terminal domains 1-3 of CI-MPR are known. Olson et al, j.biol.chem.279: 34000-34009 (2004); olson et al, EMBO j.23: 2019-2028(2004). In addition, both ligand-bound and unbound forms of structurally related CD-MPRs are also known. Olson et al, j.biol.chem.274: 29889-29886 (1999); olson et al j.biol.chem.277: 10156-10161(2002). Thus, the skilled artisan is able to use the receptor structural information to select suitable oligosaccharides.

The oligosaccharide may be selected, for example, from any of the oligosaccharides of formulae I, II, III, IV, V or VI above, including oligosaccharides 1 to 127 described below. The oligosaccharides of formulae I-III are formally derived from formula B by: modifications to remove branching, remove and/or replace monosaccharide residues, and/or linkages between adjacent monosaccharide residues (e.g., α 1, 2; α 1, 3; α 1, 4; or α 1, 6). In certain embodiments, the oligosaccharide may have, for example, 1,2 or 3 additional mannose residues in one or both arms, as compared to any one of formulas I-VI via α 1, 2; α 1, 3; or α 1, 6.

Oligosaccharides may have, for example, 1,2 or 3M 6P residues. Oligosaccharides may have, for example, 1,2, 3, 4, 5,6, 7,8, 9 or 10 monosaccharide residues in total. In other embodiments, the oligosaccharide may have 1,2, 3, 4, 5,6, 7,8, 9, or 10 mannose residues, any of which may or may not be phosphorylated.

In some embodiments, the oligosaccharide is selected from oligosaccharides 1-96, which are substances of formula I:

in some embodiments, d is a mixture of α and β (i.e., the oligosaccharide is, for example, a mixture of oligosaccharides 1 and 2, 3 and 4, or 95 and 96).

In some embodiments, the oligosaccharide is selected from oligosaccharides 97-103, which are substances of formula II:

in some embodiments, f is a mixture of α and β (i.e., the oligosaccharide is, for example, a mixture of oligosaccharides 97 and 98, 99 and 100 or 101 and 102).

In some embodiments, the oligosaccharide is selected from oligosaccharide 104-127, which is a substance of formula III:

in some embodiments, i is a mixture of α and β (i.e., the oligosaccharide is, for example, a mixture of oligosaccharides 104 and 105, 106 and 107, or 126 and 127).

In some embodiments, the oligosaccharide is selected from the group consisting of oligosaccharide 128-133, which is a substance of formula IV:

in some embodiments, k is a mixture of α and β (i.e., the oligosaccharide is, for example, a mixture of oligosaccharides 128 and 129, 130 and 131, or 132 and 133).

In some embodiments, the oligosaccharide is selected from oligosaccharides 134 and 135, which are substances of formula V:

in some embodiments, the oligosaccharide is a mixture of oligosaccharides 134 and 135.

In some embodiments, the oligosaccharide is oligosaccharide 136, which is a substance of formula VI:

in some embodiments, the oligosaccharide may be isolated from a natural source. Oligosaccharides isolated from natural sources may be homologous or may be a heterologous mixture of related oligosaccharides.

In certain embodiments, the oligosaccharide is prepared by chemical and/or enzymatic synthesis. In some embodiments, the oligosaccharides may be prepared by chemical or enzymatic modification ("semi-synthesis") of oligosaccharides isolated from natural sources.

Oligosaccharides can be chemically and/or enzymatically synthesized as taught by: for example, FIGS. 1-7, Osborn et al, Oligosaccharides: their Synthesis and Biological rounds, Oxford university Press, 2000; wang et al (eds), Synthesis of Carbohydrates through Biotechnology, American Chemical Society, 2004; seeberger, Solid Support oligomeric synthesis and Combinatorial Carbohydrate Libraries, Wiley-Interscience, 2001; driguez et al, glycscience: synthesis of Oligosaccharides and Glycoconjugates, Springer, 1999; d ü ffels et al, chem. Eur. J.6: 1416-; hojo et al, CurrentProt. peptide Sci.1: 23-48 (2000); seeberger et a, Nature 446: 1046-1051 (2007); seeberger et al, Nature Rev. drug Discov.4: 751-763 (2005); srivastana et al, Carbohydrate Res.161: 195-; and Hagihara et al, chem.rec.6: 290-302(2006) and U.S. Pat. No.5,324,663; 6,156,547, respectively; 6,573,337, respectively; 6,723,843, respectively; 7,019,131, respectively; 7,160,517.

In some embodiments, the oligosaccharides may be synthesized by sequential introduction of monosaccharides. In certain embodiments, monosaccharides can be introduced at specific positions of an existing sugar (e.g., 2-O, 3-O, 4-O, or 6-O) by selective protection and deprotection. For example, the oligosaccharide 82 can be synthesized as described by the retrosynthetic analysis in FIG. 1 and the synthetic routes set forth in FIGS. 2-7. In some embodiments, building unit 2a may replace building unit 2 in the synthetic routes of fig. 3, 5, and 6. If building block 2a is used, removal of the benzylidene group of building block 2 at the heptasaccharide stage can be avoided.

Mannose residues may be enzymatically phosphorylated as taught, for example, in U.S. patent No.6,905,856. In certain embodiments, 1,2, or 3 of the M6P residues may be substituted with hydrolysis resistant enzyme M6P mimetics, such as, for example, malonyl ethers, malonates, and phosphonates, e.g., Berkowitz et al, org.lett.6: 4921 and 4924 (2004).

In certain embodiments, the linker may attach the saccharide by alpha or beta linkage. In some embodiments, the a β linkage may be formed by a method described in: crich et al, Tetrahedron, 54: 8321-8348 (1998); kim et al, j.am.chem.soc., 130: 8537-8547 (2008); srivasta et al, Tetrahedron Letters, 35: 3269-3272 (1979); hodosi et al, j.am.chem.soc., 119: 2335-2336 (1997); nicolaou et al, j.am.chem.soc., 119: 9057-9058(1997). In one embodiment, the a β linkage may be formed using dibutyltin oxide to form an intermediate that can react with a linker containing an unactivated leaving group.

One embodiment provides a method of preparing a compound having formula VII:

wherein:

R₁selected from hydrogen, hydroxy, optionally substituted lower alkyl, phosphate, sulfate, -OR₇Protecting groups and sugars;

R₂，R₃，R₄and R₅Each independently selected from hydrogen, sulfate, hydroxy, -OR₈Protecting groups and sugars;

R₆selected from hydrogen, hydroxy, carboxy, alkoxycarbonylA group, amino, amide, alkylamino, aminoalkyl, aminooxy, hydrazide, hydrazine, optionally substituted alkenyl and optionally substituted C₂-C₆An alkyl group;

R₇and R₈Each independently selected from acetyl and optionally substituted lower alkyl; and

n is an integer from 1 to 10;

the method comprises the following steps:

a) treating a compound having formula VIII:

wherein:

R₁to R₅As defined above; and

R₉and R₁₀Selected from hydrogen and hydroxy such that when R is₉And R₁₀When one of them is a hydroxyl group, the other is hydrogen;

and has the formula R₁₁R₁₂(Sn ═ O) to form a compound having formula IX:

wherein:

R₁to R₅As defined above; and

R₁₁and R₁₂Each independently selected from unsubstituted alkyl or R₁₁And R₁₂Taken together are unsubstituted alkylene groups;

and

b) optionally in the presence of a metalIn the case of halides, treating the compounds of the formula IX and of the formula R₆-(CH₂)_n-L to form a compound of formula VII,

wherein:

R₆and n is as defined above; and

l is halogen.

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" includes both "alkyl" and "substituted alkyl" as defined below. It will be appreciated by those skilled in the art that these groups do not intend to suggest any alternative or substitution impractical patterns, solids, synthesis infeasible and/or inherent instability for any group comprising one or more substitutions.

"alkyl" includes straight and branched chains, having the indicated number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 8 carbon atoms, for example 1 to 6 carbon atoms. E.g. C₁-C₆Alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-methylpentyl and the like. Alkylene is another subset of alkyl, referred to as the same residue of alkyl, but with two points of attachment. The alkylene group typically has 2 to 20 carbon atoms, for example 2 to 8 carbon atoms, for example 2 to 6 carbon atoms. E.g. C₀Alkylene represents a covalent bond and C₁Alkylene is methylene. When an alkyl residue having a specific number of carbon atoms is named, all numbers of carbon atoms having geometric isomers are intended to be encompassed, and thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and t-butyl; "propyl" includes n-propyl and isopropyl. "lower alkyl" refers to an alkyl group having 1 to 4 carbon atoms.

"alkenyl" means an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond removed from the parent alkyl group adjacent to the carbon atom by a hydrogen molecule. This group may be in either or both cis and trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, for example prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl; and the like. In some embodiments, alkenyl groups have from 2 to 20 carbon atoms, and in other embodiments, from 2 to 6 carbon atoms.

As used herein, the term "substituted" refers to any one or more designated hydrogen atoms or the provision that group S is represented by the same group, the designated atoms not exceeding the normal valency, alternative means being selected. When S on 2 hydrogen atoms of the substituent oxo (i.e., ═ O) is substituted. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds s or useful synthetic intermediates and the first stable compound or stable structure is intended to suggest that a compound is sufficiently robust to survive isolation from a reaction mixture as a practical at least agent. Unless otherwise indicated, the structure designated as the core is substituted. For example, it is understood that when a (cycloalkyl) alkyl substituent is marketed as one possibility, the alkyl moiety at the junction of the present substituted core structure.

Unless clearly defined otherwise, the term "substituted alkyl" refers to an alkyl group wherein one or more hydrogen atoms are substituted with a substituent independently selected from the group consisting of:

-R^a，-OR^b，-O(C₁-C₂alkyl) O- (e.g., methylenedioxy-), -SR^b，-NR^bR^cHalo, cyano, oxo, nitro, sulfate, phosphate, -COR^b，-CO₂R^b，-CONR^bR^c，-OCOR^b，-OCO₂R^a，-OCONR^bR^c，-NR^cCOR^b，-NR^cCO₂R^a，-NR^cCONR^bR^c，-SOR^a，-SO₂R^a，-SO₂NR^bR^c，-NR^cSO₂R^aEthylene glycol and polyethylene glycol (PEG).

If R is^aSelected from optionally substituted C₁-C₆Alkyl, optionally substituted aryl and optionally substituted heteroaryl;

R^bselected from hydrogen, -NH₂，-NHR^cOptionally substituted C₁-C₆An alkyl group, an optionally substituted aryl group, and an optionally substituted heteroaryl group; and

R^cselected from hydrogen and optionally substituted C₁-C₄An alkyl group; or

R^bAnd R^cAnd the nitrogen to which they are attached form an optionally substituted heterocycloalkyl; and

wherein each optionally substituted group may be unsubstituted or independently substituted with one or more substituents independently selected from C₁-C₄Alkyl, aryl, heteroaryl, aryl-C₁-C₄Alkyl-, heteroaryl-C₁-C₄Alkyl-, C₁-C₄Haloalkyl-, -OC₁-C₄Alkyl, -OC₁-C₄Alkylphenyl radical, -C₁-C₄alkyl-OH, -OC₁-C₄Haloalkyl, halo, -OH, -NH₂，-C₁-C₄alkyl-NH₂，-N(C₁-C₄Alkyl) (C₁-C₄Alkyl), -NH (C)₁-C₄Alkyl group), -N (C)₁-C₄Alkyl) (C₁-C₄Alkylphenyl), -NH (C)₁-C₄Alkylphenyl), cyano, nitro, oxo (substituents of heteroaryl), -CO₂H，-C(O)OC₁-C₄Alkyl, -CON (C)₁-C₄Alkyl) (C₁-C₄Alkyl), -CONH (C)₁-C₄Alkyl), -CONH₂，-NHC(O)(C₁-C₄Alkyl), -nhc (o) (phenyl), -N (C)₁-C₄Alkyl radical C (O) (C)₁-C₄Alkyl group), -N (C)₁-C₄Alkyl group C (O) (phenyl group), -C (O) C₁-C₄Alkyl, -C (O) C₁-C₄Phenyl, -C (O) C₁-C₄Haloalkyl, -OC (O) C₁-C₄Alkyl, -SO₂(C₁-C₄Alkyl), -SO₂(phenyl group), -SO₂(C₁-C₄Haloalkyl), -SO₂NH₂，-SO₂NH(C₁-C₄Alkyl), -SO₂NH (phenyl), -NHSO₂(C₁-C₄Alkyl), -NHSO₂(phenyl) and-NHSO₂(C₁-C₄Haloalkyl).

In some embodiments, R₁Selected from hydrogen, optionally substituted lower alkyl, phosphate, sulfate, -OR₇Protecting groups and sugars. In other embodiments, R₂，R₃，R₄And R₅Each independently selected from hydrogen, sulfate, hydroxy, -OR₈Protecting groups and sugars. In certain embodiments, R₇And R₈Each independently selected from acetyl and optionally substituted lower alkyl. In some embodiments may be, R₁，R₂，R₃，R₄And R₅Any or all of (a) may be selected from protecting groups which are selectively removable, including benzyl, silyl, and trityl ethers and esters (other than acetate). In some embodiments, R₁To R₅Two of which are linked to form a protecting group. In one embodiment, R₁Selected from-O-benzyl and-OCPh₃. In other embodiments, R₁To R₅At least one of which is-O-benzyl. In some embodiments, R₁，R₂And R₄Are both-O-benzyl. In yet another embodiment, R₁is-OCPh₃And R₄is-O-benzyl. In one embodiment, R₁₁And R₁₂The linking forms cyclohexane. In other embodiments, R₁₁And R₁₂Are all isopropyl groups. In yet another embodiment, R₁₁And R₁₂Are both hexyl groups.

In a further embodiment, the compound of formula VIII is selected from optionally protected mannose, rhamnose, idose and altrose. In one embodiment, the compound of formula VIII is optionally protected mannose. In some cases, mannose may be protected at one or more of the C-3, C-4 or C-6 positions. In some cases, a single protecting group may attach to both positions. For example, benzylidene groups can be used to protect the C-4 and C-6 positions.

In general, any protecting group on the saccharide that does not have a strong electro-electrophilicity or cross-reactivity with the compound can also be used. Suitable protecting groups include ethers such as optionally substituted benzyl dimethyl ether, trityl ether, allyl ether or silyl ether, esters such as optionally substituted acetate benzoate, chloroacetic acid, pivaloate or acetyl; and include benzylidene, isopropyl, butane diacetal, acetal, and the like. In addition, the protecting group may be selected from urethane and polyurethane. In some embodiments, the protecting groups may be selectively deleted, such as, for example, benzyl, silyl and trityl ethers and acetates, among others. The identity of the protecting group and the position of the protecting group can be determined according to different requirements of the final product. Additional protecting groups have been identified to those of ordinary skill in the art and may be used as specified in the embodiments.

In one embodiment, the compound of formula VIII is treated with a compound having the formula R₁₁R₁₂(Sn ═ O), wherein R₁₁And R₁₂Each independently selected from unsubstituted alkyl or R₁₁And R₁₂Linked, selected from unsubstituted alkylene. In some embodiments, R₁₁And R₁₂Is a butyl group. In one embodimentIn (1) has the formula R₁₁R₁₂(Sn ═ O) and a compound of formula VIII in a solvent such as toluene, benzene, dimethylformamide, isopropanol, methanol or xylene. In some embodiments, the reaction is carried out at elevated temperature, optionally refluxing, to form the compound of formula IX. In some embodiments, the reaction mixture is heated to at least 40, 50, 60, 70, or 80 ℃. In a further embodiment, of the formula R₁₁R₁₂(Sn ═ O) and the compound of formula VIII for at least 1,2, 5, 10, 15, or 20 hours.

In one embodiment, the compound of formula IX is copper formula R₆-(CH₂)_n-treatment of the compound L, optionally in the presence of a metal halide. In some embodiments, R₆Selected from the group consisting of hydrogen, hydroxy, carboxy, alkoxycarbonyl, amino, amide, alkylamino, aminoalkyl, aminooxy, hydrazide, hydrazine, optionally substituted alkenyl and optionally substituted C₂-C₆An alkyl group. In further embodiments, n is an integer from 1 to 10. In certain embodiments, n is 2, 3, 4, 5 or 6, R₆Is C₁-C₄An alkoxycarbonyl group. In one embodiment, n is 3 and R₆Is methoxycarbonyl. In some embodiments, L is an unactivated leaving group. Examples of activated leaving groups include triflate, sulfonate, mesylate and other types of groups. In some cases, the less reactive leaving group may be activated by a neighboring group, such as allyl. In certain embodiments, R₆No substituent activating the leaving group. In some embodiments, L is bromide, chloride, or iodide. In one embodiment, L is bromide. In other embodiments, formula R₆-(CH₂)_nThe compound of formula-L is methyl 4-bromobutyrate.

In one embodiment, the compound of formula VIII is optionally protected mannose, and formula R₆-(CH₂)_nThe compound of-L is methyl 4-bromobutyrate. In a further embodiment, the compound of formula VIII is selected from 3,4, 6-tri-O-benzyl-D-mannose and 3-O-allyl-6-O-trityl-D-mannose.

In some embodiments, the compound of formula IX is substituted with a compound of formula R₆-(CH₂)_n-treating the compound L in the presence of a metal halide. Certain embodiments of the metal halide include a metal fluoride. In some embodiments, the metal fluoride is selected from cesium fluoride, sodium fluoride, calcium fluoride, magnesium fluoride, lithium fluoride, and potassium fluoride. In one embodiment, the metal fluoride is cesium fluoride. In some embodiments, the compound of formula IX is substituted with a compound of formula R₆-(CH₂)_nThe treatment with the compound L also comprises the addition of a tetraalkylammonium halide. In some cases, the tetraalkylammonium halide is tetrabutylammonium iodide. In another embodiment, a metal halide may be used in the reaction. Examples of metal halides include alkali metal iodides such as sodium iodide.

In one embodiment, the compound of formula IX may be combined with formula R₆-(CH₂)_n-L compound in a polar aprotic solvent. Such solvents include dimethylformamide, dimethylacetamide, dimethylsulfoxide, nitromethane, hexamethylphosphoramide, N-methylpyrrolidone, acetone, acetonitrile, ethyl acetate, and methyl ethyl ketone, among others known to those of ordinary skill in the art.

In certain embodiments, the compound of formula IX may be combined with formula R at room temperature₆-(CH₂)_n-L compound. In other embodiments, the reactants are combined and heated to at least 50, 60, 70, or 80 ℃ to form the compound of formula VII. In further embodiments, the mixture is heated for at least 1,2, 5, 10, 15, or 20 hours.

In some embodiments, the methods described herein result in at least 50, 60, 70, 80, 90, 95, or 99% stereospecific products. In further embodiments, the yield of stereospecific products is at least 50, 60, 70, 75, 80, 85, 90, 95 or 99% of the maximum possible yield. In certain embodiments, the ratio of β to α linked products is at least 10: 1, 20: 1, 30: 1, 40: 1, 50: 1 or 100: 1.

In one embodiment, the compound of formula VII may be prepared on a large scale. In some embodiments, "large scale" refers to the administration of at least 50, 100, 500, or 1000 grams of starting materials, intermediates, or agents. In further embodiments, "large scale" includes the use of at least 10, 25, 50, 100, 250, or 500kg of starting materials, intermediates, or reagents.

An exemplary synthetic route to the compound of formula a using dibutyltin oxidation reagent is shown in figure 8.

B. Protein

The oligosaccharide-protein conjugates described herein can comprise any pure protein, partially purified protein or fragment thereof, including isolated proteins as well as recombinantly or synthetically prepared proteins. The terms "pure," "purified," and "isolated" refer to a molecule that is not substantially in its natural environment. For example, a pure protein is substantially free of cellular material and/or other proteins from the cell or tissue source from which the protein was obtained. The term refers, for example, to at least 70% to 80%, 80% to 90%, 90 to 95%, or at least 95%, 96%, 97%, 98%, 99% or 100% (w/w) pure preparation.

In other embodiments, the protein may be an enzyme having optimal activity at a pH of 1-7, e.g., 1-3, 2-5, 3-6, 4-5, 5-6, or 4-6, as measured by an activity assay. For example, the enzyme may have an optimal pH at a pH of 4-6.

In some embodiments, the protein may be an enzyme having an isoelectric point (pI) of 1 to 8, such as 1-3, 2-5, 3-8, 4-5, 5-6, 4-6, 5-8, 6-8, or 7-8. The PI of a protein can be measured using, for example, isoelectric focusing gel electrophoresis.

In certain embodiments, the protein itself has at least one oligosaccharide (i.e., it is a glycoprotein). In a particular embodiment, the protein is a therapeutic glycoprotein. For example, the therapeutic glycoprotein can be a lysosomal enzyme, including an ERT enzyme, such as one of the lysosomal hydrolases listed in table 1. In certain embodiments, the lysosomal enzyme is selected from, for example, alpha-Glucosidase (GAA), alpha-galactosidase a, acid sphingomyelinase, and alpha-L-iduronidase. In a particular embodiment, the lysosomal enzyme is GAA.

Table 1: examples of LSDs and corresponding lysosomal hydrolases

In some embodiments, the protein may be a glycoprotein having at least 1,2, 3, 4, 5 or more N-linked or O-linked oligosaccharides. In other embodiments, the protein may have 1,2, 3, 4, 5 or more consensus sites for N-linked or O-linked glycosylation, at least one glycosylation.

In certain embodiments, the protein may be a ligand for a receptor. For example, in some embodiments, the protein may be a glycoprotein that binds to a receptor that recognizes a sugar, such as mannose or mannose-6-phosphate, for example. In particular embodiments, the glycoprotein can bind, for example, a sialoglycoprotein receptor, CI-MPR, CD-MPR, or mannose receptor.

Suitable protein sequences are well known in the art. The skilled artisan can readily identify conserved regions by comparing significant functional subject related sequences to sequences comprising, for example, sequences from different species. Conserved amino acids are more likely to be important events, and conversely, this suggests that polypeptides that are not conserved amino acids are more likely to tolerate regions of variation. Following these guidelines, the skilled artisan may make no more effort than routine identification of more powerful variants. Furthermore, where the crystal structure is known, the skilled artisan can study the crystal structure and determine structures and/or functions that amino acids may be important, thereby reducing resistance to mutations. The skilled artisan will also be able to recognize that amino acid changes may be tolerated. In addition, the skilled artisan can evaluate the potential for mutations in known structural and functional relationships.

For example, the sequence and structure of α -galactosidase is well known. See, e.g., Garman et al, j.mol.biol., 337: 319-335 (2004); garman et al, mol, genet, metabol, 77: 3-11 (2002); matsuzawa et al, hum. genet.117: 317-328(2005). See also GenBank accession No. X05790. In further examples, the sequence of GAA is well known (see e.g.Martiniuk et al, Proc. Natl. Acad. Sci. USA 83: 9641-9644 (1986); Hoefsloot et al, biochem. J. 272: 493 497 (1990); Moreland et al, J. biol. chem. 280: 6780-6791 (2005); see also GenBank Accession No. NM-000152. furthermore, the crystal structure from E.coli homologous alpha-glycosidase is determined and can provide structural perspectives for other alpha-glycosidases. see Lovering et al, J.biol. chem. 280: 2105-2115 (2005); in the third example, the acid neurophatase sequence is well known (see e.g.Lansmann et al, Eur. J. Biochem. 270: 1076 (2003) and the phospholipase sequence is the key for acid neurophate.g.ash. chem. ash. 73: 2003-3172; see e.g.g.Bioshit. chem. Ser. No. 51. 3172; SEQ. ash. 3172; see also Toshij. Bioshij. Ser. No. 51. No. 31; 3172; see also SEQ. Bioshik. 93; see also SEQ. No. 32; SEQ ID.),752), the sequence of alpha-L-iduronidase is well known (see, e.g., Scott et al, Proc. Natl. Acad. Sci USA 88: 9695-. See, e.g., Scott et al, hum.mutat.6: 288-302 (1995); rempel et al, mol. Genet. Metab.85: 28-37 (2005); durand et al, Glycobiology 7: 277-284 (1997); beesley et al, hum. genet.109: 503-511 (2001); brooks et al, Glycobiology 11: 741-750 (2001); nieman et al, Biochemistry 42: 8054-8065(2003). In a further example, the sequence of iduronate-2-sulfatase is well known and is a disease-causing mutation. See, for example, Flomen et al, hum. 5-10 (1993); roberts et al, j.med.genet.26: 309-313 (1989); wilson et al, proc.natl.acad.sci.usa 87: 8531-8535 (1990); wilson et al, Genomics 17: 773. 775 (1993); Sukegwa-Hayasaka et al, J.Inherit.Metab.Dis 29: 755-761(2006) and incorporated herein. The structure of iduronate-2-sulfatase has been modeled. See, for example, Kim et al, hum. mutat.21: 193-201(2003). In another example, the sequence and structure of N-acetylgalactosamine-4-sulfatase (arylsulfatase B) is known, and this is a disease-causing mutation. See, for example, Litjens et al, hum.mut.1: 397-402 (1992); peters et al, j.biol.chem.265: 3374-3381 (1990); schuchman et al, Genomics 6: 149-158 (1990); bond et al, Structure 15: 277-289(1997).

C. Joint

In certain embodiments, the oligosaccharide-protein conjugates of the invention comprise a linker between the oligosaccharide and protein components of the conjugate. In other embodiments, the conjugate does not comprise a linker. In embodiments comprising a linker, any suitable linker known to those skilled in the art may be used so long as it does not interfere with the binding of the oligosaccharide and CI-MPR and/or hinder the activity (including, e.g., enzymatic activity) of the protein. For example, the joint may be U.S. patent No.4,671,958; 4,867,973, respectively; 5,691,154, respectively; 5,846,728, respectively; 6,472,506, respectively; 6,541,669, respectively; 7,141,676, respectively; 7,176,185 or 7,232,805 or one of the linkers disclosed in U.S. patent application publication No. 2006/0228348. In some embodiments, the linker is selected from the linkers disclosed in WO 2008/089403.

In some embodiments, the linker may have the formula:

wherein Z is selected from optionally substituted alkyl, alkenyl, alkynyl, aryl, glycol, polyethylene glycol (PEG), heteroaryl, and heterocyclyl, and is selected from hydrogen or an amino protecting group. As used herein, a chemical group on any aminooxy compound (e.g., such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy, and heterocyclyloxy) may be substituted or unsubstituted, and may be interrupted with one or more heteroatoms or chemical groups, unless otherwise specified. The inclusion of heteroatoms includes nitrogen, oxygen and sulfur. Substituents and intervening chemical groups may be selected from, for example, acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amido, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carbooxy, cyano, cycloalkyl, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, imino, nitro, oxo, phosphorylamino, sulfinyl, sulfamino, sulfonate, sulfonyl, thio, thioacylamino, thioureido, and ureido. The substituent groups themselves may be substituted or unsubstituted, and may be interrupted or capped with one or more heteroatoms, such as, for example, nitrogen, sulfur, and oxygen.

In one embodiment, the linker may be formed by reacting and forming:

in further embodiments, the linker may have the formula:

-Z-NH-P

wherein Z and P are as defined above.

In further embodiments, the linker may contain a disulfide linkage. Disulfide linkers can be used to attach oligosaccharides to the protein backbone, for example, through cysteine. In one embodiment, the linker may comprise or be formed from the reaction of:

generally, suitable lengths of the joints are such that: it avoids steric hindrance with the protein component of the oligosaccharide and conjugate, without interfering with the bound oligosaccharide and active (including for CI-multiplanar reconstruction and/or e.g. enzymatic activity) protein. For example, the linker may comprise 1-100, 1-60, 5-60, 5-40, 2-50, 2-20, 5-10, or 5-20 linear atoms, wherein the linker is attached by an additional means to esterify the protein with an oligosaccharide, amide, hydrazone, oxime, semicarbazone, ether, thioether, phosphorothioate, phosphonate, thioester, and/or disulfide. The remaining linear atoms in the linker are selected, for example, from carbon, oxygen, nitrogen and sulfur, any of which may be in a carbocyclic, heterocyclic, aryl or heteroaryl ring. The linear carbon atoms in the linker may alternatively be selected from halo, hydroxy, nitro, haloalkyl, alkyl, aryl base, aryl, arylalkyl, alkoxy, aryloxy substituents selected from amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbohydraxy, hydroxyalkyl, alkanesulfonyl, arsenesulfonyl, alkanesulfonamido, arsenosulfonamidoalkyl, and thioamido, alkylcarbonyl, acyloxy, cyano, and urea. The linear nitro atom may be optionally substituted at the linker with acyl, sulfonic acid, alkyl, aryl base, aryl alkyl, alkoxycarbonyl. A linear linker sulfur atom can be selectively oxidized.

In certain embodiments, the linker can be cleaved, as described, for example, in U.S. patent application publication No.2006/0228348 and U.S. patent No.4,867,973; 7,176,185, respectively; 7,232,805, respectively. In some embodiments, the linker can be cleaved under lysosomal conditions.

D. Process for preparing oligosaccharide-protein conjugates

Conjugates of the invention, e.g., conjugates comprising an oligosaccharide of formula I, II, III, IV, V or VI, can be prepared by any method known to one of ordinary skill in the art. In any of these methods, suitable linkers may be present in one or both of the oligosaccharide and protein. For example, the conjugate can be prepared as follows: for example, Zhu et al, biochem.J.389: 619-628 (2005); zhu et al, j.biol.chem.279: 50336 and 50341 (2004); U.S. patent nos. 5,153,312; 5,212,298, respectively; 5,280,113, respectively; 5,306,492, respectively; 5,521,290, respectively; 7,001,994, respectively; U.S. provisional patent application No.60/885,457 or 60/885,471.

In certain embodiments, the oligosaccharide may be conjugated to an amino acid of the protein, such as cysteine or lysine. For example, the saccharide may be conjugated via lysine by: the lysine residues were modified using a protein with succinimidyl 4-formylbenzoate. Alternatively, the saccharide can be modified by lysine conjugation using lysine and Traut' S reagents or linkers including disulfides such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP) or protected thiols such as N-succinimidyl-S-acetylthioacetate (SATA).

In further embodiments, the oligosaccharide may be conjugated to a glycan on the glycoprotein. In one embodiment, the oligosaccharide may be conjugated to sialic acid residues on the glycan. In other embodiments, the oligosaccharide may be conjugated to mannose, fucose, galactose and/or sialic acid residues on the glycan. To effect conjugation via galactose, the glycoprotein may be first treated with sialidase to remove sialic acid residues, and then treated with galactose oxidase prior to reaction with the oligosaccharide.

For example, oligosaccharide-protein conjugates, oligosaccharides can be prepared that have any functional group that may be present (including, for example, amines, thiols, carboxylic acids, hydroxyl groups) and/or that have the protein incorporated into a suitable reactive second functional group. For functional groups, it is well known in the art. For example, a method in which a glycoprotein has at least one carbonyl group introduced may result in oxidation of the glycoprotein, e.g., periodate (e.g., sodium periodate) or galactose oxidase. In another example, a carbonyl group may be introduced into an expression with an expanded genetic code system, as described in use, for example, Wang et al, proc.natl.acad.sci.usa100: 56-61(2003). See also, for example, U.S. patent application publication No.2006/0228348, which describes the introduction of reactive groups into glycoproteins.

In some embodiments, the glycoprotein is modified in the oligosaccharide conjugation-reactive group as a linker with a carbonyl group prior to oxidation with periodate. The carbonyl group of (a) is exemplified by the amino alkoxy group of the reactive group, with the exception of other hydrazines or hydrazides. In certain embodiments, the glycoprotein is oxidized to about 1,2, 3, 4, 5, 7.5, 10, or 22.5mm periodate. In certain embodiments, the gyco protein is oxidized under conditions sufficient to oxidize the glycoprotein glycan group of the sialic acid residue, reducing fucose and mannose oxidation. In exemplary embodiments, periodate levels of less than 2, 3, 4, or 5 millimeters are used. In one embodiment, the periodate salt is sodium periodate.

In certain embodiments, the conjugated form can be protein polymerized using various chromatographic methods. In one embodiment, Hydrophobic Interaction Chromatography (HIC) is employed. Examples of columns for HIC include butyl 650C and 650m, hexyl 650C, phenyl 6FF, full Capto octa-full Capto phenyl. In other embodiments, the total amount may be subjected to metal chelate chromatography, such as copper, nickel, cobalt or mercury. In one embodiment, a copper column may be used for the bind and-elute or flow-through mode. Exemplary elution buffers include glycine or imidazole. In some embodiments, aggregation is a10, 20, 30, 40, 50, 60, 70, 80, or 90% reduction. In further embodiments, the conjugate is comprised at less than 0.5, 1, 1.5, 2, 2.5 or 3% of the total.

E. Conjugates

The oligosaccharide and protein components of the conjugate can be, for example, any of the oligosaccharides and proteins described herein. In certain embodiments, the oligosaccharide-protein conjugate is an oligosaccharide-glycoprotein conjugate. In some embodiments, the oligosaccharide-protein conjugate is an oligosaccharide-lysosomal enzyme conjugate.

In some embodiments, the conjugate comprises an oligosaccharide selected from the group consisting of oligosaccharides of formulas I-VI. In certain embodiments, the conjugate comprises an average of 1,2, 3, 4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 1-2, 1-3, 1-4, 1-6, 2-4, 2-10, 2-12, 4-6, 3-8, 5-6, 5-10, 5-15, 5-20, 10-15, 10-20, 12-15, 12-18 or 15-20 molecules of oligosaccharide/glycoprotein. In some embodiments, the conjugate comprises at least 4, 5,6, 7,8, 9, or 10 molecules of oligosaccharide/molecule protein. In further embodiments, the conjugate comprises at least 2, 3, 4, 5,6, 7,8, 9 or 10 moles of M6P per mole of protein.

In certain embodiments, the conjugate exhibits sufficient activity (e.g., enzymatic activity) relative to the unconjugated protein. In other embodiments, the conjugate may exhibit at least, e.g., 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% activity relative to the unconjugated protein. Assays measure activity, including enzyme activity, as is well known in the art. See, e.g., Eisenthal et al, Enzyme Assays: a Practical Approach, Oxford University Press: new York, 2002. Assays for measuring lysosomal enzyme activity are described, for example, in Li et al, clin. 1785-1796 (2004); civallero et al, clin. chim. acta 372: 98-102(2006). An exemplary assay for measuring GAA activity is described in example 6. See also van Diggelen et al, j.inheit.meta.dis.28: 733-741(2005) (test describing acid sphingomyelinase activity); dowaning et al, Plant Biotechnol.4: 169-181(2006) (description)Iduronidase activity assay); voznyi et al, j.inheit.meta.dis.24: 675-80(2001) (describing iduronate-2-sulfatase activity assay); murray et al, mol. Genet. Metab.90: 307-312(2007) (describing the alpha-galactosidase A activity assay); brooks et al, j. 5-12(1991) (describing N-acetylgalactosamine-4-sulfatase activity assay).

In certain embodiments, the conjugate is more efficiently internalized by the target cell (e.g., by CI-MPR-mediated endocytosis) compared to the corresponding unconjugated protein. For example, the conjugate may internalize more efficiently than the unconjugated protein, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, or 200% (mol/mol), over a given period of time. In other embodiments, conjugates of at least 2, 3, 4, 5,6, 7,8, 9, 10, 20, 30, 40, 50, 100, or 1000fold (mol/mol) can be internalized by a Cell of the type of interest (e.g., such as, e.g., L6 myoblasts or Human Pompe fibrotic cells (NIGMS Human Genetic Cell repositivity, Cat. No. GM20005) within a given time frame relative to the unconjugated protein the reference time can be, e.g., 10, 30, 45 minutes or 1,2, 3, 5,6, 12, 24, 48, or 72 hours or more the in vitro uptake of L6 myoblasts can be determined as described in example 6 and Zhu et al, J.biol.Chem.279: 50336-.

In certain embodiments, the conjugate exhibits ascending CI binding multiplanar reconstitution relative to the unconjugated protein. For example, the conjugate may exhibit at least, e.g., a2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000 or 10000 fold increase to CI-MPR relative to the unconjugated protein, as determined, e.g., by comparison of the associated or dissociated conjugated and unconjugated protein constants. Binding to CI-MPR can be performed as described in example 5 and Zhu et al, J.biol.chem.279: 50336 the assay was performed as described in 50341 (2004).

In certain embodiments, the conjugate may exhibit no uptake of mannose receptors relative to the unconjugated protein or less than 5, 10, 15, 20, 30, with 40% or 50% increased uptake by mannose receptors relative to the unconjugated protein. Uptake of mannose receptors in rat alveolar macrophages can be measured in vitro, for example, by Zhu et al, biochem.j.389: 619-628 (2005).

In certain embodiments, the conjugate can exhibit, for example, at least a2, 3, 4, 5, 10, 20, 30, 40, 50, 100, or 1000-fold decrease in accumulated substrate levels using a metabolically deficient enzyme of a suitable animal model. For example, a decrease in glycogen levels in a Pompe mouse model may be measured as in Zhu et al, j.biol.chem.279: 50336 the assay was performed as described in 50341 (2004). Alternatively, the Pompe quali model is described, for example, in Kikuchi et al, J.Clin.invest.101: 827-33(1998), which can be used. In another example, reduction of stored glycosaminoglycans in the liver and spleen can be used in a cat model of mucopolysaccharidoses, such as Kakkis et al, mol. 199, 208 (2001). Furthermore, the reduction in the level of globotriosylcer amide can be used in Fabry mouse modelling, as for example, Ioannou et al, am.j.hum.genet.68: 14-25 (2001). In yet another example, the reduction in sphingomyelin levels can be measured in mouse models of the a and niemann-pick disease types, e.g., Horinouchi et al, nat. genet.10: 288-293 (1995).

Pharmaceutical compositions

In some embodiments, the present invention provides the use of an oligosaccharide-protein conjugate, comprising (1) protein a and (2) any oligosaccharide of formulae I-VI, in the preparation of a lysosomal storage disorder in a subject in need thereof.

The pharmaceutical compositions described herein comprise the oligosaccharide-protein conjugates described above and at least one additive, such as a filler, disintegrant, buffer, stabilizer, or excipient. In some embodiments, the pharmaceutical compositions of the invention comprise a conjugate comprising an oligosaccharide of any one of formulas I-VI and a lysosomal enzyme.

Standard pharmaceutical formulation techniques are well known to those skilled in the art (see, e.g., 2005 Physicians' Desk ReferenceThomson Healthcare: montvale, NJ, 2004; remington: the science and Practice of Pharmacy, 20th ed., Gennado et al, eds&Wilkins: philiadelphia, PA, 2000). Suitable pharmaceutical additives include, for example, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol stearic acid, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, and the like. May further comprise a pH buffering agent and a wetting or emulsifying agent componentPreservatives may or may not be included.

In some embodiments, the pharmaceutical composition comprises an α -galactosidase a conjugate, can comprise one or more excipients, such as, for example, mannitol, sodium phosphate mono-hydrate, and/or sodium phosphate dibasic heptahydrate. In some embodiments, the pharmaceutical composition, including the α -glucosidase of the conjugate, may include one or more of the following: mannitol, polysorbate 80, sodium phosphate dibasic heptahydrate, monobasic monohydrate, and sodium phosphate. In additional embodiments, the pharmaceutical composition, conjugate-glucosidase including a conjugate may include up to 2% glycine, up to 2% mannitol, up to 0.01% polysorbate 80 at 10mm histidine pH 6.5.

The pharmaceutical composition may include any of the conjugates, whether as the only effective compound or in combination with other compounds, ingredients or biological materials. For example, the pharmaceutical composition may further comprise one or more small molecules that are a LSD and/or that are useful in connection with LSD for the side effects of therapy. In some embodiments, the composition may include one or more compounds of miglustat and S, e.g., described in U.S. patent application publication nos. 2003/0050299, 2003/0153768, 2005/0222244; or 2005/0267094 in some embodiments, the pharmaceutical composition may further include one or more immunosuppressive agents.

The formulation of the pharmaceutical composition may vary depending on the following factors: the desired route of administration and other parameters (see, e.g., Rowe et al, Handbook of Pharmaceutical Excipients, 4th ed., APhA publications, 2003.). In some embodiments the composition may be a sterile, pyrogen-free, white or white-like lyophilized powder, cake or reconstituted by intravenous injection, sterile injectable, usp administration. The composition in other embodiments may be a sterile, pyrogen-free solution.

The pharmaceutical composition is not limited to any particular delivery system as described by the authorities and may include, but is not limited to, gastrointestinal (including subcutaneous, intravenous, intracranial, intramedullary, intraarticular, intramuscular, intrathecal or intraperitoneal injection), transdermal or oral (e.g. in capsules, suspensions or tablets). Government may occur in single dose or repeat administration to individuals and in the form of physiologically acceptable salts, and any kind and/or acceptable pharmaceutical carriers and/or additives as part of pharmaceutical compositions.

Pharmaceutically acceptable salts include, for example, acetic acid, benzene, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citric acid, dihydrochloride, edetate, edisylate, propionate laurylsulfate, ethanesulfonate, fumaric acid, glucoheptonate, gluconic acid, glutamate, glycolylglutathione, hexylresorcinol camphor, hydrabamine, hydrobromic acid, hydrochloride, hydroxynaphthalene, iodine, ibandronate, lactic acid, lactose, malic acid, maleic acid, mandelic acid, methanesulfonic acid, methylbromide, methylnitrate, methylsulfate, mucate, naphthalenesulfonate, nitrate, pamoate (pamoate), pantothenic acid, phosphate/diphosphate, polygalacturonate, salicylic acid, stearic acid, subacetate, succinic acid, methosulfate, tannic acid salts, tartaric acid and chlorotheophylline/trithiodianion; benzathine, chloroprocaine, choline, ethanolamine, ethylenediamine, glucamine and procaine (organic) cation sec; and aluminum, calcium, lithium, magnesium, potassium, sodium, zinc (metal) cations, e.g., Berge et al, j.pharm.sci.66: 1-19 (1977).

An amount of the conjugate that is therapeutically effective for administration. In general, a therapeutically effective amount will vary with the age, general condition, sex, and severity of the condition of the subject. The dosage can be determined by a physician and adjusted, if necessary, to accommodate observation of the therapeutic effect. Toxicity and therapeutic efficacy of such compounds may determine procedures in vitro and/or in vivo for standard pharmaceutical products. And as a ratio LD50/ED50, wherein the lethal dose LD50 is 50% of the population and the dose ED50 is therapeutically effective expressed in 50% of the population. The therapeutic index of the conjugates of the invention may occur at least, for example, 1, 1.5, 2, 3, 4, 5,6, 7,8, 9, 10 and 20.

Data obtained from in vitro and animal experiments, for example, can be used to formulate a range of dosage for human use. The compound is preferably administered at a level lying in the circulating concentration, which includes the ED50 range with low, low or no toxicity. The dosage may vary and be used in the form employed herein and by various routes of administration. Any therapeutically effective dose of the conjugate can be estimated initially, from in vitro experiments. One dose can be formulated in animal models to achieve an in vitro experimental study of the circulating blood concentration range, including that determined by its IC50 (i.e., half of the conjugate tested, achieving the maximum inhibitory concentration for symptoms). Can be measured in plasma, for example, by high performance liquid chromatography or by appropriate enzyme activity. The effect of any particular dose can be monitored for the appropriate biological activity at the endpoint.

Unless otherwise indicated, the conjugate may be administered at a dose of about 1 g/kg to 500 mg/kg, depending on the severity of the symptoms and the progression of the disease. For example, the conjugate can be administered by slow intravenous infusion set-up, e.g., 1,2, 3, 4, 5,6, 7,8, 9, by, e.g., weekly or more than 10 days or outpatient, bi-weekly, monthly or bi-monthly administration. The appropriate therapeutically effective dose of the compound is selected via clinical therapy and will range from 1 μ g/kg to 500 mg/kg, from 1 μ g/kg to 10 mg/kg, from 1 μ g/kg to 1 mg/kg from 10 μ g/kg to 100 g/kg, from 100 μ g to 1 mg/kg, from 500 μ g/kg to 5 mg/kg. In some embodiments, the appropriate therapeutic dose is selected from, for example, 0.1, 0.25, 0.5, 0.75, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70 and 100 mg/kg.

An alpha galactosidase conjugate, which can be administered by intravenous infusion, e.g., at a dose of 1.0 mg/kg, every two or four weeks, e.g., at an infusion rate of less than or equal to 10 body weights, 13, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 mg/hr). In another example, the conjugate includes the glucosidase, possibly in an intravenous tube, e.g., at a dose of 20 mg/kg or 40 mg/kg every two or four weeks for more than about, e.g., 1,2, 3, 4, 5,6, 7,8, 9, or 10 hours. In some embodiments, a discipline tolerance is established every 30 minutes, infusion rate, after an administration rate for glucosidase may begin, e.g., 1 mg/kg/hr, and then increased, e.g., 2 mg/kg/hr, until there is one, e.g., 7 mg/kg/hr maximum. The conjugate consists of N-acetylgalactosamine-4-sulfatase, which can be administered by intravenous infusion in a dosage of about mg/kg body weight per week, e.g., 1,2, 3, e.g., 1.0, 4, 5,6, 7,8, 9 or 10 hours. In addition, the Physicians' Desk Reference can be found in the case of specific dosages。

Methods of treating lysosomal storage disorders

In some embodiments, the present invention provides methods of treating lysosomal storage diseases, such as those disclosed in table 1. In some embodiments, the invention also provides methods of targeting protein lysosomes by conjugation of oligosaccharides comprising mannose-6-phosphate.

In certain embodiments, the methods comprise administering a subject (including a subject, e.g., a human mammal, e.g., cat, dog, mouse or bird, e.g., such as a quail) having a lysosomal storage disorder-the protein of the invention conjugated to a therapeutically effective amount of the compound. The oligosaccharide-protein conjugate may be a glycoprotein, such as a lysosomal enzyme (e.g., a lysosomal enzyme listed in table 1) conjugate, with an oligosaccharide comprising mannose-6-phosphate, such as a pair of any oligosaccharides of formulas I-VI. In one embodiment, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising at least one of the invention conjugates.

In certain embodiments, the method comprises administering a conjugate comprising: (1) proteins and (2) oligosaccharides are composed of mannose-6-phosphate, e.g., a pair of any oligosaccharides of formulas I-VI with one or more other therapies. One or more other therapies may be administered concurrently with (including concurrently with) administration, prior to or following administration of the conjugate.

In some embodiments, the methods comprise a subject of treatment (before, after or as described with a conjugate), with antipyretics, antihistamines, and/or immunosuppressive agents. In some embodiments, a subject matter can be viewed as having antipyretic, antihistamine, and/or immunosuppressive agents, with a pre-treatment oligosaccharide-for glycoprotein conjugate to reduce or avoid infusion-related reactions. For example, the subject may be one or more of pre-treated linaloon, thioprine, cyclophosphamide, cyclosporin a, diphenhydramine, methotrexate, mycophenolate mofetil, rapamycin oral steroids or more.

In some embodiments, the methods comprise treating the subject for or associated with one or more of the treatments with acetoneamine, nithioprine, cyclophosphamide, cyclosporine a, diphenhydramine, methotrexate, mycophenolate, rapamycin, oral steroids or more, e.g., for 0 and/or 12, 24, 36, 48, 60, 72, 96, 120 and 144 hours, e.g., item 1,2 (time of conjugate administration), 3, 4, 5,6, 7,8, 9, 10 or more. For example, in some embodiments, treatment with fabry's disease or the pompe's disease discipline can be considered treatment with methotrexate 0.1 (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5,6, 8, 10, 12, 15, 25, 30, 35, 40, 50, 60, 70, 80 mg/kg methotrexate or more) or thinking, e.g., t 0, 24 and 48 hours, e.g., 1,2, 3, 4, 5,6, 7, one conjugate for 8 weeks. In some embodiments, treatment of a conjugate that may induce immune tolerance with cyclosporin a and azathioprilin and a lysosomal storage disorder, e.g., such as mucopolysaccharidosis, are potentially in a discipline. For example, an object may be considered to be described in Kakkis et al, proc.natl.acad.sci.u.s.a.101: 829-834(2004).

In some embodiments, the methods include treatment of the subject (with a conjugate before, after, or during treatment) with small molecule therapy and/or gene therapy, including small molecule therapy and gene therapy directed lysosomal storage disorders for treatment. Small molecule therapies may include miglustat and/or one or more compound regimes in the visual field, such as described in U.S. patent application publication nos. 2003/0050299, 2003/0153768; 2005/0222244, respectively; and 2005/0267094. Gene therapy can be performed as described, for example, in U.S. patent nos. 5,952,516; 6,066,626, respectively; 6,071,890, respectively; and 6,287,857; and U.S. patent application publication No. 2003/0087868.

The terms "treatment", "method of treatment", and their cognate terms refer to both therapeutic and prophylactic treatment/prevention measures. Thus, where treatment is needed, it may include individuals already having a particular lysosomal storage disorder, as well as those at risk for the disease (i.e., who may ultimately acquire the symptoms of the disorder or some disease).

The results of the therapeutic approach, the results of prophylaxis or symptomatic or other desired biological modification, and may be improved in clinical symptoms or assessment of late-onset disease, increased activity of metabolically deficient enzymes, and/or decreased accumulation levels of metabolically deficient enzymes in the substrate.

In some embodiments, the method comprises administering a conjugate comprising: (1) a lysosomal enzyme and (2) a subject of any oligosaccharide to formulas I-VI, thereby increasing insufficient lysosomal enzyme activity, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 100% relative to endogenous activity. In some embodiments, the method comprises administering a conjugate comprising: (1) lysosomal enzymes and (2) a subject for any of the oligosaccharides of formulae I-VI, whereby the defective enzyme activity of the problem is increased is determined by, for example, at least a2, 3, 4, 5,6, 7,8, 9, 10, 20, 30, 40, 50, 100 or 1000fold increase in enzyme activity relative to endogenous active enzyme activity, which may be, for example, in clinical conditions, or by appropriate clinical trials or biological reductions.

In some embodiments, the methods comprise administering a family of GAA-conjugated compounds, including the compounds of the invention, thus treating Pompe disease (also known as acid glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, glyco known prometallosis II, and lysosomal α -glucosidase deficiency). in certain embodiments, recombinant human GAA hydrolase (rhGAA) in Chinese hamster ovary preparation (CHO cell) cells in other embodiments, possibly rhGAA conjugated, oligosaccharides selected from oligosaccharide 103, a mixture of formula 103; oligosaccharide 128, 129, 130, 131, 132, 133 or 136; and any combination thereof. In one embodiment, the oligosaccharide is selected from oligosaccharides 128 and 136. In another embodiment, the conjugate contains at least 5 moles of GAA per administered patient.

In certain embodiments, the patient is an adult with pompe disease. In other embodiments, the patient is a young child or child.

Increased GAA activity can be measured biologically (see, e.g., Zhu et al, J.biol. chem.279: 50336-50341(2004)) or by tissue observation to reduce lysosomal glycogen accumulation in, e.g., cardiomyocytes, skeletal muscle cells or dermal fibroblasts. GAA activity can also be measured, for example, in muscle biopsy samples, in lymphocytes of cultured skin fibroblasts, and in dried blood spots. For example, Umpathysivam and the like are described in the dry blood spot test. Clinically. And (4) chemical reaction. 47: 1378-1383 (2001), plum, etc. Clinically. And (4) chemical reaction. 50: 1785 1796 (2004). Treatment of pompe disease may also be assessed, for example, by serum creatinine kinase, as a measure of electrocardiogram, in the return of motor function (e.g., assessed by the alberta baby motion index), echocardiogram as a measure of changes in left ventricular mass index, and electrical heart activity. Government of gossypol conjugates may also result in relief of one or more symptoms of pompe diseases such as cardiac enlargement, cardiomyopathy, difficulty breathing during the day, difficulty breathing, retardation of growth, difficulty feeding, "fibrotic" gait abnormalities, headache, low muscle tone, swollen organs (e.g., swollen heart, tongue, liver), lordosis, balance, low back pain, headache in the morning, muscle weakness, respiratory insufficiency, winged shoulder blades, scoliosis, reduced loss of deep tendon reflexes, sleep apnea, susceptibility to respiratory infections and vomiting.

In other embodiments, the method comprises administering α -galactosidase a to a subject comprised of the conjugate, thereby treating fabry's disease. Fabry disease, or anderson, fabry disease, is a rare X-linkage, a consequence of accumulation of the lysosomal storage disorder with the defective marker α -galactosidase a, in cyclophosphamide (GL3) and in visceral and vascular endothelial tissue, abdominal and muscle cells of other neutral glycosphingolipids in lysosomes. Neutral glycosphingolipids accumulate in the narrowing and vasodilatation, the vascular consequences, and eventually ischemia and infarction.

Administration of a conjugate comprising an alpha galactosidase of the invention can result in one or more of fabry disease including, for example, acroparesthesia, angina, angiokeratoma, arrhythmia, gait instability, burning and/or stinging hands and feet with clinical symptoms alleviated, cataracts, chills, conduction abnormalities, corneal whorling, coronary artery disease, senile dementia, depression, diarrhea, cardiac dilatation, vertigo, cardiac enlargement, cardiomyopathy, diplopia, dysarthria, fatigue, elevated fever and blood sedimentation, hearing problems, heart disease, heart valve problems, aversion to heat, hemiparesis, hypohidrosis, impaired sweating, myocardial infarction, myocardial ischemia, joint pain, kidney disease, left ventricular hypertrophy, abnormal gating, lenticular opacity, lipiduria, muscle weakness, myocardial infarction, nausea, nystagmus, pain (e.g., severe pain all over the body, polydipsia, protein urban and rural junctures, postprandial pain, renal failure, retinal abnormalities, tinnitus, stomach pain, ST-T wave changes, stroke, uremia, valvular disease, vertigo, vomiting and weakness. The alpha conjugate may result in an increase in galactosidase, e.g., plasma, tear, leukocyte, or biopsy tissue culture skin fibroblast activity. Administration of a galactosidase a conjugate may also result in a reduced (e.g., at least 10%) or increased birefringent lipid globule deficiency histological finding. It may also result in a decrease in urinary sediment lipid globules, serum creatinine levels or creatinine clearance that improve renal function, and a decrease in protein urban and rural junctions. Administration of the galactosidase A conjugate may also result in a decrease in renal, cardiac, and cutaneous capillary endothelial cells in the GL3 inclusion bodies. Other experiments for measuring the efficacy of fabry disease treatments can be found, for example, MacDermott et al, j.med.genet.38: 750-760(2001).

In other embodiments, the methods comprise administering a compound of the invention comprising an acid sphingomyelinase as a host, thereby treating niemann-pick a or niemann-pick b disease or an acid sphingomyelinase deficiency. The government of acid sphingomyelinase conjugates may have resulted in alleviation of niemann-pick or niemann-pick b disease in one or more clinical conditions including, for example, abnormal cholesterol levels, dyslipidemia, dyskinesia, blood abnormalities, frequent lung infections in the eye, growth retardation, hepatosplenomegaly, platelets, lymphadenectasis, peripheral neuropathy, and few of the lung function, tachypnea problems, cherry red spots, skin pigmentation changes or xanthoma. In some embodiments, the conjugate can be administered intracranially.

In other embodiments, the methods comprise administering a compound of the invention conjugated to a subject comprising L iduronidase, thus treating mucopolysaccharidosis (including, e.g., project I in the form of Hurler and Hurler-Scheie). Government to L iduronidase conjugates may cause relief from one or more clinical symptoms of MPS including, for example, aortic regurgitation, aortic stenosis, carpal tunnel syndrome, chronic rhinitis, impaired conduction hearing, constipation, corneal opacity, delayed progression, diarrhea, abdominal distension, dorsolumbar humpback, dorsal gibbus malformations, hepatosplenomegaly, hydrocephalus, inguinal hernia, kyphosis, intellectual deficit, mitral regurgitation, mitral stenosis, nyctalopia, open angle glaucoma, poor hand function, progressive arthropathy, recurrent respiratory infections, respiratory insufficiency, retinal degeneration, scoliosis, neurohearing loss, severe back pain, rhinorrhea, sleep apnea, spinal cord compression, intersomatic fish atrophy, umbilical hernia, and upper respiratory compliance cations.

In further embodiments, the method comprises administering a subject matter of the conjugate, composition iduronate-2 invention-sulfatase, thus treating mucopolysaccharidosis two (hunter disease). Administration of iduronate-2-sulfatase conjugates may result in one or more of the clinical symptoms of MPS ii including, for example, heart valve disease, cardiopulmonary failure, carpal tunnel syndrome, chronic diarrhea, chronic papillary relieved, gross facial features, corneal haze, coronary stenosis, deafness, dysmorphism, dysplasia, ear infections, hearing impairment, hepatosplenomegaly, hydrocephalus, inguinal hernia, joint stiffness, scoliosis, mental deficits, cardiomyopathy, cardiac hypertrophy, pulmonary hypertension, retinal dysfunction, bone malformations, umbilical hernia formation, upper respiratory tract infections, and valve insufficiency.

In still other embodiments, the method comprises administering sulfatase (arylsulfatase B), a compound of the invention N-acetylgalactosamine-4, conjugated to a subject, thereby treating mucopolysaccharidosis six (maroteeaux-lamide syndrome). Administration of the sulfatase conjugate to N-acetylgalactosamine-4 may result in one or more of the clinical symptoms of blindness, cardiac abnormalities, cardiopulmonary disease, gross facial features, corneal haze, ear infections, growth retardation, hepatomegaly, hepatosplenomegaly, joint malformations, nerve compression symptoms, dyspnea, skeletal malformations, spinal cord compression, splenomegaly, joint stiffness, and upper airway obstruction in the sixth of MPS.

The foregoing and following are exemplary descriptions and illustrations only, and are not intended as limitations on the claimed invention.

Examples

Example 1 general procedure in oligosaccharide Synthesis

A. And (3) glycosylation:

the glycosylation reaction is performed by combining the donor and acceptor sugars using standard methods. Briefly, unless otherwise indicated, donor and acceptor compounds were dissolved in anhydrous DCM in the presence of thermally activated 4A molecular sieves in dry nitrogen. The solution was cooled and held at 0 ℃ for-30 minutes, then TMSOTf (1eq) was added slowly. The reaction was checked by TLC (silica gel) using hexane/EtOAc and quenched with TEA or Hunig's base (1.05 eq). The mixture was filtered and concentrated to a slurry, which was purified by flash column chromatography using a hexane/EtOAc gradient unless otherwise indicated,

B. acid catalyzed deacetylation:

in certain examples, the acetylated compound is deacetylated prior to glycosylation or other modification, such as phosphorylation. In the examples provided below, hydrogen chloride was generated by adding acetyl chloride to cold (0 ℃) dry methanol. The concentrated solution was added to a 1: 3 solution of the acetylated compound in DCM/methanol. The final concentration was 3% w/v relative to the methanol component of the solution. Performing deacetylation reaction: a) 18h for mono-substituted acetates and b) 48-64h for di-substituted acetates. Unless otherwise stated, the reaction was quenched with TEA or Hunig's base, followed by aqueous extraction from DCM or EtOAc.

C. Phosphorylation:

in some instances the saccharide is subjected to site-specific phosphorylation. To a solution of the sugar in dry acetonitrile was added 5-methyltetrazole (3.4eq) at room temperature and the mixture was stirred for 30 minutes under dry nitrogen. Dibenzyldiisopropylphosphoramidite (1.7eq/OH group) was added and stirred until the reaction was complete (. about.60 min.). The reaction was checked by TLC (silica gel) using hexane/EtOAc. The solution was cooled in ice/water for 15 minutes and 30% w/v hydrogen peroxide (2eq) was added. After-60 minutes the reaction was complete and an excess of saturated sodium thiosulfate was sandwiched. The mixture was concentrated to a gum and dissolved in EtOAc, washed with half saturated brine and dried over sodium sulfate. Unless otherwise indicated, the residue was purified by flash column chromatography using a hexane/EtOAc gradient.

Example 2: synthesis of disaccharide Aminooxyacetylaminopropyl 2-0- [ 6-O-phosphoryl-alpha-D-mannosyl ] -alpha-D-mannoside (17)

According to Pekari et al, j.org.chem.66: 7432(2001) to prepare allyl α -D-mannoside. To allyl- α -D-mannoside (8.68g, 39.4mmol) in dry methanol (100mL) was added 2, 3-butanedione (3.63mL, 86.1mmol), trimethyl orthoformate (16mL, 146mmol) and 10- (+) -camphorsulfonic acid (1.37g, 5.9 mmol). The mixture was heated at reflux under dry nitrogen for 9 h. The reaction mixture was quenched with TEA (1mL) and concentrated to a red slurry and purified by flash column chromatography on silica gel using 40-80% EtOAc in hexanes to give allyl 3-O, 4-O- [ dimethoxybut-2 ', 3' -diyl ] - α -D-mannoside 1 as a white solid (3.89g, 29.4%).

Compound 1(2.65g, 8mmol) was dissolved in dry pyridine (20mL), the solution was cooled in an ice/water bath, and t-butylbiphenyl silyl chloride (2.28mL, 8.7mmol) was added. After 18h, pyridine (20mL) and acetic anhydride (1.6mL, 16mmol) were added and the solution was heated to 50 ℃ for 16h, then concentrated to a slurry and stripped with toluene. The residue was dissolved in EtOAc (60mL) and washed with 1M HCl (2X50mL), saturated sodium bicarbonate (50mL) and dried over sodium sulfate. The mixture was filtered and concentrated in vacuo, and the solution was concentrated to give a slurry of allyl 2-O-acetyl-6-O-t-butyldiphenylsilyl-3-O, 4-O [ dimethoxybut-2 ', 3' -diyl ] - α -D-mannoside 2(5.0 g).

Palladium (II) chloride (0.425g, 2.4mmol) was added to a solution of 2(5.0g, 8mmol) in dry methanol (25mL) with stirring. After-3 h, the reaction was quenched with TEA (0.75mL, 4.8 mmol). Methanol was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel using 10-50% EtOAc in hexanes to give 2-O-acetyl-6-O-t-butyldiphenylsilyl-3-O, 4-O [ dimethoxybut-2 ', 3' -diyl ] - α -D-mannoside 3 as a white foam (2.72g, 59.2%).

Trichloroacetonitrile (1.75mL, 17.4mmol) and DBU (0.05mL, 0.35mmol) were added to a solution of 3(1.0g, 1.74mmol) in dry DCM (1 mL). After 75, the solution was purified directly by flash column chromatography on silica gel using EtOAc in hexanes (0 to 30%) to give 2-O-acetyl-6-O-t-butyldiphenylsilyl-3-O, 4-O [ dimethoxybut-2 ', 3' -diyl ] - α -D-mannose trichloroacetimidate 4 as a white foam (0.98g, 78.2%). According to the general glycosylation procedure of example 1, donor 4(0.98g, 1.36mmol) and acceptor 3-N-benzyloxycarbonylaminopropanol (0.284g, 1.36mmol) were converted to give N-benzyloxycarbonylaminopropyl 2-O-acetyl-6-O-t-butylbiphenylsilyl-3-O, 4-O [ dimethoxybut-2 ', 3' -diyl ] -a-D-mannoside 5 as a white foam (0.66g, 64%).

To a solution of 5(0.66g, 0.87mmol) in dry methanol (5mL) was added 25% w/v sodium methoxide in methanol (0.05mL, 0.22 mmol). After-1 h, the reaction was quenched with glacial acetic acid (0.025mL) and concentrated to a slurry. The product was dissolved in DCM (10mL), washed with half-saturated brine (5mL) and dried over sodium sulfate to give N-benzyloxycarbonylaminopropyl 6-O-t-butyldiphenylsilyl-3-O, 4-O [ dimethoxybut-2 ', 3' -diyl ] - α -D-mannoside 6(0.58g, 92%) as a white foam.

Allyl α -D-mannoside (3.15g, 14.3mmol) was dissolved in dry pyridine (20mL) and cooled in an ice/water bath. T-butylbiphenylsilyl chloride (4.03mL, 15.7mmol) was added and the solution was allowed to reach room temperature. After stirring for 18h benzoyl chloride (5.93mL, 51.5mmol) was added and after 24h the reaction was quenched with water (3mL) and stirred for 30 min. The solution was concentrated under vacuum and stripped with toluene (3 × 50 mL). The residue was dissolved in EtOAc (100mL) and washed with cold 1M HCl (50mL), half saturated brine (50mL), half saturated sodium bicarbonate (50mL), half saturated brine (50mL), dried over sodium sulfate, filtered and concentrated to a slurry. It was purified by flash column chromatography on silica gel using 0-50% EtOAc in hexanes to give allyl 2, 3, 4-tri-O-benzoyl-6-O-t-butyldiphenylsilyl- α -D-mannoside 7 as a white foam (8.62g, 78.2%).

Glacial acetic acid (11.1mL, 18.26mmol) and 1M tetrabutylammonium fluoride (18.26mL, 18.26mmol) were added to a solution of 7(12.77g, 16.6mmol) in dry THF (50 mL). Glacial acetic acid (0.15mL, 2.5mmol)) and 1M tetrabutylammonium fluoride (2.5mL, 2.5mmol) were added at 80 min, followed by addition of more glacial acetic acid (0.25mL, 4.15mmol) and 1M tetrabutylammonium fluoride (4.15mL, 4.15mmol) at 90 min. After 2h, the solution was concentrated to half volume and diluted with EtOAc (150 mL). The solution was washed with half saturated brine (2 × 150mL) and half saturated sodium bicarbonate (200mL), concentrated, and the residue was purified by flash column chromatography on silica gel using 10-50% EtOAc in hexanes to give allyl 2, 3, 4-tri-O-benzoyl- α -D-mannoside 8 as a white foam (6.75g, 76.4%). Compound 8(6.75g, y mmol) was converted according to the usual method for phosphorylation to give allyl 2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl- α -D-mannoside 9 as a white foam (9.52g, 95%).

Palladium (II) chloride (0.638g, 3.6mmol) was added to a solution of 9(9.52g, 12.1mmol) in dry methanol (50 mL). After-5 h more palladium (II) (0.145g) was added and the mixture was stored for 18 h. The solution was filtered, concentrated, and the residue was purified by flash column chromatography on silica gel with 20-70% EtOAc in hexanes to give 2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl- α -D-mannose 10 as a white foam (3.8g, 42.5%).

Trichloroacetonitrile (5.06mL, 5.1mmol) and DBU (0.15mL, 1mmol) were added to a solution of 10(3.8g) in dry DCM at 0 deg.C under dry nitrogen. After-90 minutes, the solution was purified directly by flash column chromatography on silica gel using 10-60% EtOAc in hexanes to give 2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl- α -D-mannoside trichloroacetimidate 11 as a white foam (3.3g, 72.1%).

According to the general glycosylation procedure of example 1, donor 6(2.62g, 2.97mmol) and acceptor 11(1.92g, 2.7mmol) were converted to give N-benzyloxycarbonylaminopropyl 2-O- [2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl- α -D-mannosyl ] -6-O-t-butylbiphenylsilyl-3-O, 4-O [ dimethoxybutane-2 ', 3' -diyl ] - α -D-mannoside 12 as a white foam (1.27g, 32.2%).

TFA/water 19: 1v/v (8.4mL) was added to a solution of 12(2.1g, 1.44mmol) in DCM (8mL) cooled in an ice/water bath. Consumption of starting material was shown by TLC after-2 h. Ethanol (25mL) was added to the solution, then concentrated and stripped with ethanol (3 × 25 mL). The residue was dissolved in dry methanol (10mL) and cooled in an ice/water bath. Acetyl chloride (0.4mL) was added to give a 3% w/v solution in HCl. The solution was allowed to reach room temperature. After-2 h, consumption of starting material was shown by TLC. The reaction was quenched with triethylamine (1mL), concentrated, and the residue was purified by flash column chromatography on silica gel with 30-100% EtOAc in hexanes to give 13 as a white foam (0.758g, 47.9%). To 13(0.758g, 0.69mmol) in dry methanol (10mL) was added 25% w/v sodium methoxide in methanol (0.15 mL). After-1 h, consumption of starting material was shown by TLC. The reaction was quenched with 1 MHCl. Thereby obtaining 14. Glacial acetic acid (25 μ L), wet 10% Pd/C (0.1g) were added to the solution and a hydrogen balloon was attached. After 6h reaction, the product was carbonized with 5% sulfuric acid/EtOH but not UV active. The mixture was filtered and concentrated to an oil, then dissolved in water (10mL) and lyophilized to give 3-aminopropyl 2-O- [ 6-O-phosphoryl- α -D-mannosyl ] - α -D-mannoside 15(0.300g, 91.3%, from 13).

A solution of 0.1M NaOH (.2mL) was added to a solution of 15(0.1g, 0.21mmol) in water (8.5mL) followed by N-t-butoxycarbonyl-aminooxyacetyl 2, 3, 5, 6-tetrafluorophenyl ether (0.14g, 0.42mmol) in THF (8.5 mL). After 18h, the solution was adjusted to pH 4 with 2M HCl and extracted with DCM (3 × 10 mL). Lyophilizing the aqueous phase to obtain N-t-butoxycarbonyl-aminooxyacetamidopropyl-2-O- [ 6-O-phosphoryl-alpha-D-mannosyl]- α -D-mannoside 16(0.12 g). Compound 16(0.12g) was dissolved in TFA/DCM 1: 1(10mL) and the solution was stirred for-30 min, then concentrated to an oil. It was dissolved in water (5mL) and the product was lyophilized to give a solid (0.45 g). The solid was applied to BiogelP2 was purified and eluted with water to give 17(0.063 g).

Example 3: synthesis of trisaccharide (35)

Such as Yamazaki et al, carb. res.201: 31(1990) 2-O-acetyl-3, 4, 6-tri-O-benzyl-alpha-D-mannose trichloroacetimidate 18.

E.g., Heng et al, j.carb.chem.20: 285(2001), preparation of 6-O-acetyl-3, 4, 6-tri-O-benzoyl-alpha-D-mannose trichloroacetimidate 19.

According to the usual glycosylation method, the donor 19(5.0g, 7.4mmol) and the acceptor N-9-fluorenylmethylcarbonylaminopropanol (2.41g, 8.1mmol) were converted to give N-9-fluorenylmethylcarbonylaminopropyl 6-O-acetyl-2, 3, 4-tri-O-benzoyl-alpha-D-mannoside 29 as a white foam (4.0g, 66.4%).

According to the usual procedure for acid catalyzed deacylation of the disubstituted acetate (b), compound 29(4.0g, 4.9mmol) was converted to give a white foam of N-9-fluorenylmethylcarbonylaminopropyl 2, 3, 4-tri-O-benzoyl- α -D-mannoside 30(3.3g, 87.3%). According to the usual glycosylation method, the donor 18(3.27g, 5.16mmol) and the acceptor 30(3.3g, 4.3mmol) were converted to give N-9-fluorenylmethylcarbonylaminopropyl 6-O- [ 2-O-acetyl-3, 4, 6-tri-O-benzylmannosyl ] -2, 3, 4-tri-O-benzoyl-alpha-D-mannoside 31 as a white foam (4.94g, 92.7%).

According to the usual procedure for acid catalyzed deacylation of the disubstituted acetate (b), compound 31(4.94g, 3.96mmol) was converted to give N-9-fluorenylmethylcarbonylaminopropyl 6-O- [3, 4, 6-tri-O-benzylmannosyl ] -2, 3, 4-tri-O-benzoyl-alpha-D-mannoside 32 as a white foam (1.73g, 36%). Compound 11 was prepared according to the procedure of example 2. According to the usual glycosylation method, donor 11(3.37g, 3.74mmol) and acceptor 32(1.73g, 1.44mmol) were converted to give 33 as a white foam (1.2g, 43.1%).

To a solution of 33(1.2g, 0.62mmol) in dry THF (10mL) was added dodecyl mercaptan (1.48mL, 6.2mmol) and diazabicycloundec-7-ene (DBU) (0.093mL, 0.62 mmol). Consumption of starting material was shown by TLC after 18 h. The solution was concentrated to a slurry and purified by flash column chromatography on silica gel using 0-20% methanol in DCM. To the product was added methanol/water 1: 1(20mL) and acetic acid (25. mu.L) with Pd/C (0.1g) and a hydrogen balloon attached. After 18h, the solution was filtered through Celite and concentrated to a white foam. The foam was dissolved in dry methanol (10mL), 25% w/v sodium methoxide in methanol (0.15mL), and after 6h the solution was concentrated in water (10mL) and washed with DCM (10 mL). The aqueous phase was lyophilized to give the disodium salt of aminopropyl 6-O- ([ alpha-D-mannosyl ] -2-O- [ 6-O-phosphoryl-alpha-D-mannosyl ]) -alpha-D-mannoside 34(0.27g, 63.5%, from 33).

To a solution of 34(0.17g, 0.3mmol) in water/DMSO 1: 1(10mL) was added N-t-butoxycarbonylamino-oxyacetyl ether (0.34g, 1.14mmol) in DMSO (2mL), followed by 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one (DHBT) (0.09g, 0.6mmol) in DMSO (1 mL). After 24h the solution was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking, and selected fractions were pooled and lyophilized to give a solid. It was dissolved in TFA/DCM (8mL) and stirred for 60 min, then concentrated to an oil. Water (5mL) was added and the product was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give 35(0.033g, 16.4% from 34).

Example 4: synthesis of tetrasaccharides

A. Aminoxyacetamido-1, 5-di-3-amidopropyl [2-O- [ 6-O-phosphoryl-alpha-D-mannose Base of]-alpha-D-mannosyl]Glutamate (28)

Compound 19 was prepared according to the procedure described in example 3. According to the usual glycosylation method, donor 19(15.49g, 24.3mmol) and acceptor 3-N-9-fluorenylmethoxycarbonylaminopropanol (8.7g, 29.19mmol) were converted to give N-9-fluorenylmethoxycarbonylaminopropyl 2-O-acetyl-3, 4, 6-tri-O-benzyl- α -D-mannoside 20 as a white foam (10.55g, 55%).

Acetyl chloride (4.8mL, 63mmol) was added dropwise over-30 min to a solution of 20(10.5g, y mmol) in dry DCM (50mL) and dry methanol (100mL) to give a 2.3% w/v solution of HCl in methanol. After 18H the reaction was quenched with Hunig's base (9.81mL, 63mmol) and an additional 1.5mL was added. The solution was concentrated to a slurry, stripped with chloroform (2 × 50mL), dissolved in DCM (100mL) and washed with half-saturated brine (100mL) and dried over sodium sulfate. The solution was concentrated and purified by flash column chromatography on silica gel using 0-100% EtOAc in hexanes to give N-9-fluorenylmethoxycarbonylaminopropyl 3, 4, 6-tri-O-benzyl- α -D-mannoside 21(6.11g, 61.2%) as a white foam. According to the usual glycosylation method, donor 21(6.11g, 13.4mmol) and acceptor 11(7.1g, 7.9mmol) were converted to give N-9-fluorenylmethoxycarbonylaminopropyl 2-O- [ 6-O-dibenzylphosphoryl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- α -D-mannoside 22 as a white foam (5.8g, 50.1%).

To a solution of 22(5.8g, 3.96mmol) in dry THF (75mL) was added dodecyl mercaptan (9.53mL, 40mmol) and DBU (0.6mL, 4 mmol). After-4 h the reaction was quenched with methanolic HCl (5.6mL, 8mmol) and concentrated to a slurry. The product was triturated with ether to give 3-aminopropyl 2-O- [2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- α -D-mannoside hydrochloride 23 as a gum (3.77g, 74.5%).

To a solution of 23(3.77g, 2.95mmol) in dry acetonitrile (50mL) were added N-benzyloxycarbonylglutamic acid (0.3661g, 1.3mmol), N-hydroxybenzotriazole (HOBt) (0.4g, 2.95mmol), DBU (4.6mL, 4mmol) and 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide (EDC) (0.75g, 8.85 mmol). After 16H Hunig's base (0.25mL) was added, followed by more EDC (0.75g, 8.85 mmol). After 21h the solution was concentrated to a slurry and purified by flash column chromatography on silica gel using 10% 2-propanol in DCM 0-50% relative to DCM to give N-benzyloxycarbonylamino 1, 5-bis- [ 3-amidopropyl 2-O- [2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- α -D-mannosyl ] glutamate 24 as a white foam (0.97g, 11.2%).

To a solution of 24(0.912g, 0.33mmol) in anhydrous DCM (20mL) and methanol (25mL) was added 25% w/v sodium methoxide in methanol (0.09mL, 0.41 mmol). After 6.5h the reaction was quenched with 1M HCl (0.41mL, 0.41mmol), concentrated to a slurry and purified by flash column chromatography on silica gel using 10% 2-propanol in DCM 0-100% relative to DCM to give N-benzyloxycarbonylamino 1, 5-bis-3-acylaminopropyl [2-O- [ 6-O-dibenzylphosphoryl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- α -D-mannosyl ] glutamate 25(0.306g, 44.4%).

To a stirred solution of 25(0.3g, 0.144mmol) in THF/water 2: 1v/v (75mL) was added wet 10% Pd/C (0.052g) with the attachment of a hydrogen balloon. Glacial acetic acid (25 μ L) was added after 16h and a fresh hydrogen balloon was attached. More 10% Pd/C (0.04g) and more hydrogen were added after 6 h. Fresh 5% Pc/C (0.05g) and more hydrogen was added after 18 h. After 24h, the product was carbonized with 5% sulfuric acid/EtOH but not UV-active. The mixture was filtered through celite, and the filter pad was washed, concentrated to-30% to remove THF, and then lyophilized to give 1, 5-di-3-amidopropyl [2-O- [ 6-O-phosphoryl- α -D-mannosyl ] glutamate 26(0.121g, 77.9%).

N-t-Butoxycarbonylaminoacetyl 2, 3, 5, 6-tetrafluorophenyl ether (0.19g, 0.57mmol) in DMSO (1mL) and 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine (DHBT) (0.052g, 0.3mmol) in DMSO (1mL) were added to a solution of 26(0.16g, 0.15mmol) in water/DMSO 1: 1(7.5 mL). After 18h, the solution was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give N-t-butoxycarbonylaminooxyethylamido-1, 5-di-3-amidopropyl [2-O- [ 6-O-phosphoryl- α -D-mannosyl ] glutamate 27(0.095g, 42.1%). To compound 27 was added TFA/DCM 1: 1(8mL) and the mixture was stirred until dissolved (. about.60 min) and then concentrated to an oil. Water (10mL) was added and the product was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give 28(0.048g, 54.2%).

B. Aminooxyacetoacetamidopropyl 6-O- ([ alpha-D-mannosyl)]-6-O- [ alpha-D-mannose Base of]-2-O- [ 6-O-phosphoryl-alpha-D-mannosyl]) -alpha-D-mannoside (47)

Compound 11 was prepared as described in example 2. According to the general glycosylation procedure of example 1, donor 11(5.0g, 7.4mmol) and acceptor 3-N-benzyloxycarbonylaminopropanol (1.93g, 9.25mmol) were converted to give a white foam. According to the usual method for acid catalyzed deacylation of mono-substituted acetates, the product was converted to yield a white foam of N-benzyloxycarbonylaminopropyl 2, 3, 4-tri-O-benzoyl- α -D-mannoside 36.

According to the general glycosylation procedure of example 1, donor 19(4.47g, 6.6mmol) and acceptor 36(3.6g, 5.3mmol) were converted to give N-benzyloxycarbonylaminopropyl 6-O- ([ 6-O-acetyl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl ]) -2, 3, 4-tri-O-benzoyl- α -D-mannoside 37 as a white foam (3.8g, 63.3%). According to the usual method for acid catalyzed deacylation of mono-substituted acetates, compound 37(3.8g, 3.17mmol) was converted to give N-benzyloxycarbonylaminopropyl 6-O- [2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -2, 3, 4-tri-O-benzoyl- α -D-mannoside 38 as a white foam (3.5g, 95.3%).

According to Yamazaki et al, carb.res.201: 31(1990) to prepare compound 18. According to the general glycosylation procedure of example 1, donor 18(2.41g, 3.75mmol) and acceptor 38(3.5g, 3mmol) were converted to give N-benzyloxycarbonylaminopropyl 6-O- ([2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -6-O- [ 2-O-acetyl-3, 4, 6-tri-O-benzyl- α -D-mannosyl ]) -2, 3, 4-tri-O-benzoyl- α -D-mannoside 39 as an oil (5.63 g). According to the usual procedure for the acid catalyzed deacylation of disubstituted acetates (see example 1), compound 39(5.83g) was converted to give a white foam of N-benzyloxycarbonyl-aminopropyl 6-O- ([2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2, 3, 4-tri-O-benzoyl- α -D-mannoside 40 (2.0g, 41.9%, from 38). According to the usual glycosylation method, the donor 19(1.07g, 1.63mmol) and the acceptor 41(2.0g, 1.3mmol) are converted, thus, a white foam of N-benzyloxycarbonylaminopropyl 6-O- ([2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2-O- [ 6-O-acetyl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -2, 3, 4-tri-O-benzoyl- α -D-mannoside 42(1.4g, 50.8%) was obtained. According to the usual procedure for acid catalyzed deacylation of mono-substituted acetates, compound 42(1.4g, 0.066mmol) was converted to give a white foam of N-benzyloxycarbonyl-aminopropyl 6-O- ([2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl-alpha-D-mannosyl ] -2-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl ] -2, 3, 4-tri-O-benzoyl-alpha-D-mannoside 43(1.0g, 80%). According to the usual method for phosphorylation, compound 43(1.0g, 0.048mmol) is converted, thus, a white foam (0.9g, 80.7%) of N-benzyloxycarbonylaminopropyl 6-O- ([2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2-O- [ 6-O-dibenzylphosphoryl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl ]) -2, 3, 4-tri-O-benzoyl- α -D-mannoside 44 was obtained.

To a solution of 44(0.9g, 0.039mmol) in dry methanol (20mL) was added 25% w/v sodium methoxide in methanol (0.09mL, 0.4 mmol). After-7 h the reaction was quenched with 1M HCl (0.5mL), 10% Pd/C (0.2g), and water (10 mL). The mixture was kept in hydrogen for 24h using a balloon. The mixture was filtered through celite and washed with EtOAc (20 mL). The solution was lyophilized to give N-benzyloxycarbonyl-aminopropyl 6-O- ([ α -D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2-O- [ 6-O-dibenzyl-phosphoryl- α -D-mannosyl ] - α -D-mannoside 45(0.23g, 74.7% from 44) as a white solid.

N-t-Butoxycarbonylaminoacetyl 2, 3, 5, 6-tetrafluorophenyl ether (0.375g, 1.14mmol) in DMSO (2mL) and DHBT (0.1g, 0.6mmol) in DMSO (1mL) were added to a solution of 45(0.23g, 0.3mmol) in water/DMSO 1: 1(10 mL). After 24h the solution was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking, and selected fractions were pooled and lyophilized. The product was re-acylated in water/DMSO 1: 1(10mL) using N-t-butoxycarbonylaminooxyacetyl 2, 3, 5, 6-tetrafluorophenyl ether (0.375g, 1.14mmol) in DMSO (2mL) and DHBT (0.1g, 0.6mmol) in DMSO (1 mL). After 24h the solution was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give N-t-butoxycarbonylaminooxyacetamidopropyl 6-O- ([ α -D-mannosyl ] -6-O- [ α -D-mannosyl ] -2-O- [ 6-O-phosphoryl- α -D-mannosyl ]) - α -D-mannoside 46(0.11g, 37.5%). Compound 46 dissolved in TFA/DCM (8mL) was added. The solution was stirred for 60 minutes and then concentrated to an oil. Water (5mL) was added and the product was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give 47(0.07g, 70.7%).

Example 5: synthesis of beta-linked hexoses

A. Tiglyl 3, 4, 6-tri-O-benzyl-beta-D-mannoside (51)

Such as Mayer et al, Eur J. org. chem.10: 2563(1999) 2-O-acetyl-3, 4, 6-tri-O-benzyl-alpha-D-mannose. 25% w/v sodium methoxide in methanol (0.5mL) was added to a solution of 2-O-acetyl-3, 4, 6-tri-O-benzyl- α -D-mannose (8.0g, 23.3mmol) in anhydrous methanol (50 mL). After-2 hours, the starting material disappeared as shown by Thin Layer Chromatography (TLC)And (4) consuming. The reaction was quenched with Amberlite IR120(H +) resin, filtered, and concentrated to a slurry to give 3, 4, 6-tri-O-benzyl- α -D-mannose 50(6.67g, 99%). To compound 50 was added toluene (150mL), followed by dibutyltin oxide (3.88g, 16.28mmol), and the mixture was heated at reflux for 3h using a Dean-Stark condenser. The resulting solution was cooled, concentrated to a slurry, and dissolved in dry DMF (50 mL). Cesium fluoride (2.28g, 12.1mmol), tetrabutylammonium iodide (5.47g, 14.8mmol) and methyl 4-bromocrotonate (2.46mL, 22.2mmol, technical grade) were added to the mixture and heated to-60 ℃ for 18 hours. The mixture was allowed to cool and the solids were filtered off. It was diluted with isopropyl ether/EtOAc3.7: 1(380mL) and washed with half-saturated sodium thiosulfate (240 mL). The aqueous phase was extracted with isopropyl ether/EtOAc 3.7: 1(2X190mL) and the organic layers were pooled and concentrated. The slurry was stripped with isopropanol (2 × 25mL) and purified by flash column chromatography using 0-50% EtOAc in hexanes to give a slurry of 51 (4.58g, 56.4%). 13C-NMR (100MHz)¹J_1C，1H(100MHz)，157Hz(β＜160Hz，α＞170Hz)

B. Methylbutyryl 3, 4, 6-tri-O-benzyl-beta-D-mannoside (52)

Compound 50 was prepared as described in example 2. To compound 50(9.4g, 21mmol) was added toluene (200mL), followed by dibutyltin oxide (5.49g, 22mmol), and the mixture was heated at reflux using a Dean-Stark condenser for 23 h. The resulting solution was cooled, concentrated to a slurry, and dissolved in dry DMF (100 mL). Methyl 4-bromobutyrate (4.23mL, 32mmol), tetrabutylammonium iodide (1.94g, 5.25mmol) and cesium fluoride (3.91g, 25.5mmol) were added and the mixture was heated to 60 ℃ for 2h followed by 18h at ambient temperature. The mixture was cooled and filtered through celite, washing with EtOAc (50 mL). It was concentrated to a gum, stripped with toluene (3 × 40mL), adsorbed onto silica and purified by flash column chromatography using 0-70% EtOAc in hexanes to give 52 as an oil (9.11g, 78.6%). 13C-NMR (100MHz)¹J_1C，1H(100MHz), 157.2Hz (. beta. < 160Hz,. alpha. > 170 Hz.) No observation was madeAlpha-linked products.

C. Methylbutyryl 2-O- [ 6-O-phosphoryl-alpha-D-mannosyl]-beta-D-mannoside (57)

Compound 52 was prepared as described in example 2, and was prepared according to Heng et al, j.carb.chem.20: 285(2001) preparation of 6-O-acetyl-3, 4, 6-tri-O-benzoyl-alpha-D-mannose trichloroacetimidate 19. According to the general glycosylation procedure of example 1, donor 19(4.29g, 6.36mmol) and acceptor 52(2.9g, 5.3mmol) were converted to yield a white foam of methylbutyryl 2-O- [ 6-O-acetyl-2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl ] -3, 4, 6-tri-O-benzyl-beta-D-mannoside 53(4.41g, 78.5%). According to the general procedure for acid catalyzed deacylation of mono-substituted acetates (described in example 1), compound 53(4.41g, 4.1mmol) was converted to yield a white foam of methylbutyryl 2-O- [2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- β -D-mannoside 54(2.25g, 53.7%). According to the general procedure for phosphorylation of example 1, compound 54(2.25g, 2.2mmol) was converted to give methylbutanoyl 2-O- [2, 3, 4-tri-O-benzoyl-6-O-dibenzylphosphoryl-alpha-D-mannosyl ] -3, 4, 6-tri-O-benzyl-beta-D-mannoside 55 as a white foam (2.2g, 77.7%).

To deprotect 55, 25% w/v sodium methoxide in methanol (0.2mL) was added to a solution of 55(2.2g, 1.7mmol) in anhydrous methanol (20 mL). Consumption of starting material was shown by TLC after-24 h. The reaction was quenched with Amberlite IR120(H +) and concentrated to a slurry. The solution was concentrated and purified by flash column chromatography on silica gel to give methylbutanoyl 2-O- [ 6-O-dibenzylphosphoryl- α -D-mannosyl ] -3, 4, 6-tri-O-benzyl- β -D-mannoside 56 as a white foam (1.10g, 67%).

To a stirred solution of 56(1.10g, 1.15mmol) in THF/water (20mL) was added glacial acetic acid (25. mu.L), wet 10% Pd/C (0.1g) and a hydrogen balloon attached. After 24h reaction, the product was carbonized with 5% sulfuric acid/EtOH but not visible under UV. The mixture was filtered through celite, and the filter pad was washed with water (20 mL). The solution was concentrated and dried in vacuo to give methylbutyryl 2-O- [ 6-O-phosphoryl- α -D-mannosyl ] - β -D-mannoside 57(0.6g, 98%).

D. Aminoxyacetamidohydrazinobutyl 2-O- [ 6-O-phosphoryl-alpha-D-mannosyl]-beta-D-mannose Glycoside (60)

Hydrazine hydrate (0.44mL, 5.75mmol) was added to 57(0.6g, 1.15mmol) in methanol (20mL) with stirring. After 30 minutes water (5mL) was added and the solution was stirred for 18 h. More hydrazine (0.44mL, 5.75mmol) was added and the mixture was stirred for 120 h. The solution was concentrated to a volume of-25%, stripped with water (2x10mL), and the product lyophilized to give a beige solid of hydrazinobutyl 2-O- [ 6-O-phosphoryl- α -D-mannosyl ] - β -D-mannoside 58 (0.6g, 99%).

To 58(0.2g, 0.38mmol) in DMSO/water 1: 1(10mL) was added a solution of N-t-butoxycarbonylaminooxyacetyl 2, 3, 5, 6-tetrafluorophenyl ether (0.49g, 1.52mmol) in DMSO (2mL) and DHBT (0.125g, 0.76mmol) in DMSO (2 mL). After 18h the solution was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give N-t-butoxycarbonylamino-oxyacetamido hydrazinobutyl 2-O- [ 6-O-phosphoryl- α -D-mannosyl ] - β -D-mannoside 59 as a beige solid (0.1g, 37.8%). Compound 59 was dissolved in TFA/DCM 1: 1(8 mL). The solution was stirred for-60 minutes and then concentrated to an oil. Water (10mL) was added and the product was purified on sephadex exclusion resin. Fractions were examined on silica gel plates by coking and selected fractions were pooled and lyophilized to give 60 as a beige solid (0.045g, 52.5%).

E. Large-Scale Synthesis of methylbutyryl 3-O-allyl-6-O-trityl-beta-D-mannoside (64)

50.0kg (128.09mol) of d-mannose pentaacetate in 100L of CH₂Cl₂The solution of (1) was treated with 26.8kg (243.2mol, 1.90eq.) of thiophenol and 27.3kg (192.3mol, 1.50eq.) of boron trifluoride diethyl etherate, and the resulting solution was stirred at 22 ℃ for 40h, after which the reaction was judged to be complete by HPLC analysis. 115L of 5N aqueous NaOH are then carefully added to the stirred reaction vessel, the phases are separated and the organic phase is washed more than once with 46L of 5N NaOH. Will CH₂Cl₂Removed by distillation under reduced pressure, and the residue was redissolved in 100kg of isopropanol at 60 ℃. After cooling to 9 ℃, the product F1 crystallized and could be isolated by filtration, then washed with isopropanol to yield 35.8kg (63%).

35.80kg of F1(88.28mol) were suspended in 143kg of MeOH at 22 ℃ and treated with 0.73kg of 30% methanol-sodium methoxide solution (4.05mol, 0.046eq.) before a clear solution was obtained. After the reaction was judged to be complete by TLC analysis, 0.49kg of acetic acid (8.14mol, 0.09eq.) was added and the solvent was removed under reduced pressure. The residue was suspended in toluene, concentrated again under reduced pressure and finally treated with acetone, and then the product phenyl- α -D-thiomannoside was crystallized. After filtration, washing and drying, a yield of 19.65kg (89%) was obtained.

Phenyl- α -D-thiomannoside (19.25kg, 70.69mol) in pyridine (43.8kg) was added to a solution of triphenylmethyl chloride (19.7kg, 70.66mol) in toluene (89kg) at 40 ℃ and stirred for 22 h. After the reaction was judged to be complete by HPLC analysis, the solvent was distilled under reduced pressure, and the residue was dissolved in toluene and re-concentrated. After dilution with more toluene, the solution was washed once with water. After filtration and drying, the product was precipitated by adding the toluene solution to a mixture of hexane (840L) and diisopropyl ether (250L) to give phenyl 6-O-trityl-1-thio- α -D-mannoside 61(32.30kg, 89%).

A mixture of 61(30.0kg, 58.29mol) and dibutyltin oxide (20.3kg, 81.5mol) in toluene (500kg) was heated at reflux for 2 hours until no further water separated from the condensed solvent vapors. The solution was cooled to 40 ℃ and DMF (34kg) was added. About half of the total solvent was distilled off under reduced pressure, then DMF (216kg) was added, and the solution was concentrated again to about half of its volume. More DMF (250kg) was added followed by caesium fluoride (8.9kg, 58.59mol), tetrabutylammonium iodide (23.6kg, 63.89mol) in DMF (65kg), and allyl bromide (21.1kg, 174.4 mol). The resulting mixture was stirred at 50 ℃ for 15 h. After the reaction was judged to be complete by HPLC analysis, the solid was removed from the reaction mixture by filtration and the filtrate was treated with a mixture of diisopropyl ether (136kg) and ethyl acetate (30kg), 10% w/w aqueous sodium thiosulfate (300 kg). After phase separation, the lower phase was re-extracted 4 times with a mixture of diisopropyl ether (136kg) and ethyl acetate (30kg), and the combined upper phases were washed three times with water (150 kg). The upper phase was concentrated under reduced pressure, and the residue was dissolved in ethanol (160kg) at 75 ℃. After cooling to 0 ℃, the product crystallizes and can be isolated by filtration. After washing and drying the filter cake, phenyl 3-O-allyl-6-O-trityl-1-thio- α -D-mannoside 62(15kg, 46%) was obtained.

A solution of 62(12.5kg, 22.53mol) in a mixture of THF (63kg) and pyridine (18kg, 227.5mol) was treated at 15 ℃ with a solution of toluenesulfonic acid monohydrate (16.7kg, 87.79mol) in water (10.7kg) and then with a solution of N-chlorosuccinimide (9.6kg, 71.89mol) in a mixture of water (17kg) and THF (83 kg). The resulting mixture was heated to 22 ℃ and stirred for 3 h. After the reaction was judged to be complete by HPLC analysis, a solution of sodium thiosulfate (4.6kg, 29.11mol) in water (15kg) was added to the reaction mixture. The phases were separated, the upper phase was concentrated under reduced pressure, and the residue was dissolved in toluene and concentrated again. The residue was redissolved in ethyl acetate, washed with water and then with 16% w/w aqueous sodium chloride. After evaporation of ethyl acetate, the crude product was purified by column chromatography on 52kg silica gel and eluted with a gradient of 3-10% v/v ethyl acetate in toluene to give 3-O-allyl-6-O-trityl- α -D-mannose 63(7.7kg, 73%).

A mixture of 63(7.7kg, 16.65mol) and dibutyltin oxide (4.56kg, 18.32mol) in methanol (61kg) was heated under reflux until a cloudy solution was obtained, then carried out for a further 1 h. The solution was cooled to 25 ℃, about half of the total solvent was distilled off under reduced pressure, then DMF (33kg) was added, and the solution was concentrated again to about half of its volume. More DMF (15kg) was added followed by caesium fluoride (2.53kg, 16.65mol), tetrabutylammonium iodide (6.15kg, 16.65mol) in DMF (17kg), and methyl 4-bromobutyrate (4.52kg, 24.97 mol). The resulting mixture was stirred at 80 ℃ for 5 h. The solids were removed from the reaction mixture by filtration and the filtrate was treated with a mixture of diisopropyl ether (41kg), ethyl acetate (14kg) and 10% w/w aqueous sodium thiosulfate solution (77 kg). After phase separation, the lower phase was re-extracted with a mixture of diisopropyl ether (41kg) and ethyl acetate (51kg) and the combined upper phases were washed with water (39 kg). The upper phase was concentrated under reduced pressure, and the residue was dissolved in methanol (35 kg).

The ligation reaction was repeated in order to improve the reaction yield. The dissolved residue was concentrated again under reduced pressure and then diluted with methanol (122 kg). Methanol (60L) was distilled again and the resulting solution was treated with dibutyltin oxide (2.28kg, 9.16mol) and the mixture was heated under reflux for 2 h. The solution was cooled to 29 ℃, about half of the total solvent was distilled off under reduced pressure, then DMF (37kg) was added, and the solution was concentrated again to about half of its volume. More DMF (15kg) was added followed by cesium fluoride (1.26kg, 8.29mol), tetrabutylammonium iodide (3.7kg, 10.02mol) in DMF (17kg), and methyl 4-bromobutyrate (3.06kg, 16.90mol) and the resulting mixture was stirred at 80 ℃ for 5 h. After the reaction was judged to be complete by HPLC analysis, the solid was removed from the reaction mixture by filtration and the filtrate was treated with a mixture of diisopropyl ether (25kg), ethyl acetate (32kg) and 10% w/w aqueous sodium thiosulfate solution (77 kg). After phase separation, the lower phase was re-extracted with a mixture of diisopropyl ether (25kg) and ethyl acetate (32kg) and the combined upper phases were washed with water (39 kg). The solution was concentrated under reduced pressure and the residue was redissolved in toluene (43kg) and finally concentrated to a final volume of about 30L. The crude methyl ester of 64 was then purified by column chromatography on 50kg silica gel and eluted with a gradient of 5% to 30% v/v ethyl acetate in toluene.

A solution of the purified methyl ester in methanol (50kg) was treated with a mixture of 30% w/w aqueous sodium hydroxide (3.29kg) and methanol (7.7kg) and the resulting solution was stirred for 14 h. After the reaction was judged to be complete by HPLC analysis, the reaction mixture was treated with a mixture of diisopropyl ether (41kg) and ethyl acetate (14kg), then with water (77 kg). The biphasic mixture was passed through a 1.2 μm filter column and the phases were separated. The lower phase was treated with a mixture of diisopropyl ether (41kg) and ethyl acetate (14kg) and the pH of the lower phase was lowered to 4.5-5 by the addition of 5% w/w aqueous citric acid (38L). The phases were separated and the lower phase was extracted with a mixture of diisopropyl ether (41kg) and ethyl acetate (14 kg). The combined upper phases were washed with water (39kg) and then concentrated under reduced pressure. The residue was combined with diisopropyl ether (39kg) and the solvent portion was concentrated to give a final volume of approximately 20L, then the product crystallized and could be isolated by filtration. After washing and drying of the filter cake, (3-O-allyl-6-O-trityl- β -D-mannosyl) -4-butyric acid 64(4.7kg, 51.5%) was obtained.

F. Large Scale Synthesis of Tetrasaccharide intermediate (69)

Compound 64 was prepared according to the procedure in part. The protecting group of 64 is modified between further glycosylation reactions. A solution of 64(4.25kg, 7.75mol) in THF (14kg) was carefully added to a stirred slurry of 60% sodium hydride dispersion (1.55kg, 38.75mol) in THF (45kg) and the resulting suspension was stirred until hydrogen generation ceased. A suspension of tetrabutylammonium iodide (0.29kg, 0.78mol) in THF (2kg) was added to the reaction vessel, followed by benzyl bromide (9.2kg, 53.79 mol). The mixture was stirred at 22 ℃ for 46h, then at 30 ℃ for 12h, and at 35 ℃ for 48 h. After the reaction was judged to be complete by HPLC analysis, the mixture was cooled to 0 deg.C and anhydrous methanol (0.7kg, 21.87mol), 30% w/w methanol-sodium methoxide (2.1kg, 11.66mol) were carefully added. Acetic acid (1.4kg), triethylamine (9.4kg, 92.89mol) were then added and the mixture was stirred for 18 h. To the resulting suspension was added water (31kg) and the two phases were separated. The upper phase was concentrated under reduced pressure, the residue was dissolved in toluene and concentrated here to a final volume of about 20L. The crude product was purified by column chromatography on 42kg silica gel eluting with a gradient of 5-15% ethyl acetate in hexane to give a solution of methylbutyryl 3-O-allyl-2, 4-di-O-benzyl-6-O-trityl- β -D-mannoside 65(4.4kg, 77%) in ethyl acetate.

To a solution of 65(4.4kg, 5.92mol) in methanol (19kg) was added a solution of toluene sulfonic acid monohydrate (0.9kg, 4.73mol) in methanol (6.3kg) at 37 ℃ and the resulting mixture was stirred for 1 h. After the reaction was judged to be complete by HPLC analysis, triethylamine (1.5kg, 14.82mol) was added and the solution was concentrated under reduced pressure. Toluene (28kg) was then added and the solution was washed with water (32 kg). The phases were separated and the upper phase was concentrated under reduced pressure. The crude product was purified by silica gel chromatography on silica gel (32kg) and eluted with a gradient of 9% then 17% then 50% v/v ethyl acetate in toluene to give a solution of methylbutyryl 3-O-allyl-2, 4-di-O-benzyl- β -D-mannoside 66(2.67kg, 90%) in toluene.

3.73kg (5.49mol, 1.10eq.) of 19 and 2.50kg (4.99mol) of 66 were dissolved in 34kg of dry toluene, of which 10L were evaporated under reduced pressure. The solution was then cooled to 0 ℃ and treated dropwise with 22g (0.099mol, 0.02eq.) of TMSOTf, so that the reaction temperature was kept < 5 ℃, stirring for 1h at 0 ℃ after the addition was complete. After the reaction was judged to be complete by HPLC analysis, 30g (0.296mol, 0.06eq.) Et was added₃And N neutralizing the mixture. Hexane (22L) was added, the resulting suspension was filtered and the filtrate was washed with 33L water and concentrated under reduced pressure. The residue was dissolved in 10L of toluene and concentrated again, and the process was repeated two more times. Column chromatography purification of the crude product was carried out on 50kg of silica gel and with a gradient of 9-13% v/v ethyl acetate in hexane: toluene 1: 1The ester was eluted, giving 4.23kg, 83% solution of compound 67 in toluene.

To 4.23kg (4.16mol)67 in 5.6L CH₂Cl₂And 40L of MeOH were added 0.72kg of 10% Pd/C followed by 0.12kg (0.63mmol, 0.15eq.) of a solution of toluenesulfonic acid monohydrate and the mixture was stirred at 22 ℃ for 24 h. After the reaction was judged to be complete by HPLC analysis, the palladium/carbon was removed by filtration and the filtrate was used in subsequent steps without further purification.

To the filtrate was added a solution of 2.97kg (15.6mol, 3.75eq.) of toluene sulfonic acid in 4L MeOH, and the resulting mixture was stirred at 22 ℃ for 16 h. After the reaction was judged to be complete by HPLC analysis, the mixture was cooled to 0 ℃ and neutralized by the addition of 1.64kg (16.2mol, 3.89eq.) of triethylamine. The solution was concentrated under reduced pressure and the residue was partitioned between 82L MTBE and 32L water. The organic phase is concentrated under reduced pressure, diluted with 10L of toluene and reconcentrated. This process was repeated two more times. Column chromatography purification of the crude product was performed on 38kg silica gel and eluted with a 23-26% v/v gradient of ethyl acetate in hexane: toluene 1: 1 to give 2.59kg of a solution of compound 68 (67% from 67) in toluene.

A solution of 2.90kg (3.10mol) of 68 and 5.24kg (8.23mol, 2.65eq.)18 in 30kg dry toluene was treated with 0.049kg (0.185mol, 0.06eq.) of TBDMSOTf at 0 ℃ and stirred for 4h at 0 ℃. After the reaction was judged to be complete by HPLC analysis, 0.104kg (1.03mol, 0.128eq.) of triethylamine was added followed by 33L of hexane. The resulting suspension was filtered and the filtrate was taken up in 28L of water and then 28L of 5% waterSexual Na₂CO₃And (6) washing. The toluene phase was concentrated under reduced pressure and the crude product was purified by column chromatography on 58kg silica gel and eluted with a gradient of 9-20% v/v ethyl acetate in hexane: toluene 1: 1 to give 4.40kg of a solution of tetrasaccharide intermediate 69 (75%) in toluene.

G. Synthesis of protected hexose (73)

Tetrasaccharide intermediate 69 was prepared according to the method described in example 7. To a solution of 9.7g (5.15mmol)69 in 60ml CH₂Cl₂To the solution of (a) was added 100ml of MeOH, then 27ml of a 5.7N solution of HCl in 1, 4-dioxane (0.154mol, 30eq.) was added dropwise so that the temperature of the mixture was kept below 30 ℃. The reaction mixture was then stirred at 22 ℃ for 40 h. After the reaction was judged to be complete by HPLC analysis, 32ml (22.9mmol, 44.7eq.) of triethylamine was carefully added so that the temperature of the mixture remained below 25 ℃. Water (250ml) and toluene (200ml) were added, the mixture was shaken and the phases separated. The lower phase was re-extracted with 50ml of toluene and the combined upper phases were washed with 50ml of water and concentrated under reduced pressure. The crude product was purified by column chromatography on 100g silica gel and eluted with a 30-50% v/v gradient of ethyl acetate in hexane to give methylbutyryl 3-O- ([3, 4, 6-tri-O-benzyl- α -D-mannosyl-l-3-O- ([3, 4, 6-tri-O-benzyl- α -D-mannosyl)]) - (6-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl-L-mannose]-6-O- [3, 4, 6-tri-O-benzyl-alpha-D-mannosyl]) -2, 4-di-O-benzyl- β -D-mannoside 70(6.35g, 69%).

A solution of 1.0g (0.56mmol) of 70 and 0.95g (1.40mmol, 2.5eq.)19 in 9ml of dry toluene was treated with 0.02g (0.075mmol, 0.14eq.) of TBDMSOTf at 0 ℃ and stirred for 1h at 0 ℃. After the reaction was judged to be complete by HPLC analysis, 22ml (0.158mmol, 0.28eq.) of triethylamine was added. The resulting mixture was washed twice with 10ml of water and concentrated under reduced pressure. The crude product was purified by column chromatography on 15g silica gel and eluted with a 15-25% v/v gradient of ethyl acetate in hexane: toluene 1: 1 to give 1.75g of the compound methylbutyryl 3-O- ([3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2-O- [ 6-O-acetyl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl ]) - (6-O- [2, 3, 4-tri-O-benzoyl- α -D-mannosyl ] -6-O- [3, 4, 6-tri-O-benzyl- α -D-mannosyl ] -2-O- [6-O- Acetyl-2, 3, 4-tri-O-benzoyl- α -D-mannosyl) -2, 4-di-O-benzoyl- β -D-mannoside 71, which contains residual toluene.

A solution of 10.0g (3.5mmol)71 in 40ml 1, 4-dioxane was treated with 60ml MeOH followed by 3.25g (14.0mmol, 4eq.) (+) -camphorsulfonic acid and the resulting solution was stirred at 22 ℃ for 100 h. After the reaction was judged to be complete by HPLC analysis, 3ml (21.5mmol, 6.2eq.) of triethylamine was added and the solvent was removed under reduced pressure. The residue was dissolved in 200ml MTBE and 200ml H₂O is shaken together. The phases were separated and the upper phase was concentrated under reduced pressure. The crude product was purified by chromatography on 114g silica gel and eluted with a gradient of 14-25% v/v ethyl acetate in toluene to give 8.24g methylbutyryl 3-O- ([3, 4, 6-tri-O-benzyl-alpha-D-mannosyl-mannose]-2-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl]) - (6-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl-L-mannose]-6-O- [3, 4, 6-tri-O-benzyl-alpha-D-mannosyl]-2-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl]) -2, 4-di-O-benzoyl- β -D-mannoside 72, which contains residual toluene.

To a solution of 0.965g (0.35mmol)72 in 4g dry acetonitrile 0.083g (0.70mmol, 2eq.)4, 5-dicyanoimidazole followed by 0.315g (0.91mmol, 2.6eq.) dibenzyldiisopropylphosphoramidite was added and the mixture stirred at 23 ℃ for 1 h. After the reaction was judged to be complete by TLC analysis, 0.5ml of water was added and the solution was stirred for 15 min. Water (9.5ml) and 10ml of MTBE were added and the resulting mixture was shaken, the phases separated and the lower phase shaken with 10ml of MTBE. The upper phases were combined and concentrated under reduced pressure to give a colorless oil. Dissolving the residue in CH₂Cl₂And cooled to-20 ℃ and treated with 0.247g (1.13mmol, 3.2eq.) of 70% 3-chloroperbenzoic acid. After the reaction was judged to be complete by TLC analysis, 10ml of 10% aqueous sodium thiosulfate solution was added and the mixture was heated to 23 ℃. The lower phase is separated and shaken with 10ml of water and concentrated under reduced pressure. The crude product was purified by column chromatography on 19g silica gel and eluted with a gradient of 25-50% v/v ethyl acetate in hexane to give 0.83g (72%) methylbutyryl 3-O- ([3, 4, 6-tri-O-benzyl- α -D-mannosyl-mannose]-2-O- [ 6-O-dibenzylphosphoryl-2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl]) - (6-O- [2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl-L-mannose]-6-O- [3, 4, 6-tri-O-benzyl-alpha-D-mannosyl]-2-O- [ 6-O-dibenzylphosphoryl-2, 3, 4-tri-O-benzoyl-alpha-D-mannosyl]) -2, 4-di-O-benzoyl- β -D-mannoside 73 as a colorless oil.

H. Aminoxyacetamidohydrazide 3-O- ([ alpha-D-mannosyl)]-2-O- [ 6-O-phosphoryl-alpha-D-mannose Sugar radical]) - (6-O- [ alpha-D-mannosyl)]-6-O- [ alpha-D-mannosyl]-2-O- [ 6-O-phosphoryl-alpha-D-mannose Sugar radical]) Synthesis of-beta-D-mannoside (77)

Compound 73 was prepared according to the procedure described in example 8. Glacial acetic acid (100. mu.L) was added to 73(64g, 19.6mmol) in methanol/THF 1: 1(600mL) and H-cube used at 50 ℃ CThe product was hydrogenated at 20% Pd (OH)₂On C, 50 bar H₂Pressure, flow rate 6mL/min, and circulation over the catalyst. After 20h, the reaction was checked by TLC for substantial completion and the solution was concentrated to give a 74 foam (39.88g, 93%).

Methanol (180mL) was added to 74(33.8g, 15.5mmol) while stirring until dissolved and the solution was cooled in an ice/water bath for 15 minutes. To the solution was added 64% hydrazine monohydrate (94ml, 1.24mol) while stirring. After 30 minutes water (120mL) was added and the solution was allowed to reach room temperature and stored for 18 h. The solution was concentrated to-100 mL and stripped with water (2x100mL), the final solution was adjusted to-180 mL with water. The solution was extracted with DCM (2 × 100mL) and then 60mL of 3 fractions were separated on a sephadex exclusion column. Fractions containing the purest material were pooled and lyophilized to give 75(15.5g, 80%).

DMSO (20mL) was added slowly to 75(2.5g, 2.0mmol) in water (30mL), followed by N-t-butoxycarbonylaminooxyacetyl 2, 3, 5, 6-tetrafluorophenyl ether (2.58g, 7.6mmol) in DMSO (6mL) and DHBT (0.65g, 4mmol) in DMSO (4 mL). After 18h the solution was purified on sephadex exclusion resin, the fractions were checked by coking on silica gel plates and the selected fractions were pooled and lyophilized to yield N-t-butoxycarbonylaminooxyacetamidoacylhydrazinobutyryl 3-O- ([ α -D-mannosyl ] -2-O- [ 6-O-phosphoryl- α -D-mannosyl ]) - (6-O- [ α -D-mannosyl ] -2-O- [ 6-O-phosphoryl- α -D-mannosyl ]) - β -D-mannoside 76(2.57g, 90%). DCM (30mL), then TFA (16mL) was added to compound 76(2.57g, 1.8 mmol). The mixture was stirred until dissolved (-60 min) and then concentrated to an oil. Water (20mL) was added, the product was purified on sephadex exclusion resin, fractions were checked on silica gel plates by coking, and selected fractions were pooled and lyophilized to give 77(1.6g, 67.1%).

Example 6: synthesis of hexoses with disulfide linkers

A. Preparation of hexoses in free acid form

Anhydrous MeOH was added to compound 73, followed by NaOMe and incubation for 4-18 h. The reaction was quenched with glacial acetic acid and the solution was concentrated to a slurry to give 78.

Compound 78 was dissolved in THF/methanol 1: 1 and hydrogenated Pd/C-H₂In (1). The solution was concentrated to a solid and dissolved in water, saponified with aqueous NaOH, pH adjusted to-4, and purified on sephadex g-10 to give the free acid 81.

B. Attachment of disulfide linkers

The crude fraction of compound 81 prepared by a different method (from biomera) was converted to Triethylamine (TEA) salt by mixing excess TEA followed by chromatography on Superdex Peptide (GE Healthcare) using 30% acetonitrile, 0.1% TEA bicarbonate as mobile phase. Pooled fractions were lyophilized and conjugated NEA: glycan containing NEA: EDAC: NHS: HOBt: TEA (1: 1.5: 1 mol: mol) in the following reaction and incubated overnight while gently shampooing. A portion (0.5mg) of the product was chromatographed on Superdex Peptide as before and lyophilized to give 82(0.28 mg).

Example 7: synthesis and oxidative optimization of alpha-glucosidase conjugates

A. Conjugation

The oligosaccharides were conjugated to recombinant human acid alpha-glucosidase (rhGAA) to form NeoGAA. Conjugates in which oligosaccharides are first attached by sialic acid residues on rhGAA are called "SAM", while those attached by galactose residues are called "GAM".

E.g., Zhu et al, Biochem J, 389(Pt 3): 619-628(2005) NeoGAA beta SAM6 was prepared essentially. The rhGAA samples used in this assay had-5.2 moles of sialic acid per mole of protein as determined by monosaccharide composition analysis. Briefly, 5mg/mL rhGAA (Genzyme Corp.) was buffer-exchanged into 100mM sodium acetate pH 5.6 and then reacted with sodium periodate (2, 7.5 or 22.5mM) on ice in the dark for 30 minutes. The reaction was quenched by addition of glycerol to 2% (vol/vol). Oxidized rhGAA was buffer-exchanged to remove small molecular weight byproducts from the oxidation reaction and conjugated with compound 77 (0-120-fold molar ratio vs. protein, as shown in fig. 9) at 37 ℃ for 6 hours. All conjugates were buffer-exchanged into 25mM sodium phosphate pH 6.25, containing 2% mannitol and 0.005% Tween-80.

Similar NeoGAA conjugates were prepared using SAM2 (compound 17, example 2), SAM3 (compound 35, example 3), SAM4 (compound 28, example 4A), linear SAM4 (compound 47, example 4B) and α SAM6 (oligosaccharide 103), using 7.5mM periodate and an altered molar ratio of oligosaccharide to rhGAA.

Alternative conjugation methods were also performed. Specifically, hexoses with aminooxy, hydrazide or thiol-reactive linkers attach rhGAA through Cys374, lysine, sialic acid or galactose residues.

Lysine conjugation was performed by modifying lysine residues in rhGAA using succinimidyl 4-formylbenzoate (SFB; Solulink Corp.) followed by conjugation to oligosaccharides. Briefly, rhGAA was first buffer-exchanged to 50mM sodium phosphate, pH7.2, which contained 150mM sodium chloride. The buffered rhGAA was then treated with freshly prepared (SFB) in a SFB to GAA 20: 1 molar ratio. The mixture was incubated at room temperature for 30min, after which it was buffer-exchanged to 100mM sodium acetate, pH 5.5, for conjugation of hydrazide hexoses at room temperature for 2 hours, or GAA modified with SFB was buffer-exchanged to 100mM sodium acetate, pH 5.6, for conjugation of aminoxyhexoses at 37 ℃ for 6 hours.

Cysteine-based conjugation was performed by reaction with thiol-reactive NEA-hexose 82 (example 6I). The NEA-modified hexose 82 was reconstituted in water and incubated with rhGAA (15: 1 molar ratio of neoglycan to rhGAA) in 50mM sodium phosphate and 50mM hydroxylamine pH7.2 at 25 ℃ for 2 hours. The pH was adjusted to 6.2 using 50mM sodium phosphate pH 4.1 and incubation continued overnight. The product was purified by centrifugal diafiltration against 25mM sodium phosphate pH 6.2. Less than 1 mol/mol of M6P was introduced.

Although direct conjugation through Cys374 was unsuccessful, spacer arms were testedHomobifunctional thiol-specific reagents 1, 4-bis- (3' - [ 2-pyridyldithiol)]Propionylamino) Butane (DPDPDPB) to be in the oligosaccharide before conjugationThe 374 position provides a more solvent accessible sulfhydryl group. A 60-fold molar excess of DPDPB and rhGAA was reacted under conditions of 10% DMSO or 10% propanol as co-solvent. This produces strong concentration, as detected by light scattering. The reaction in the presence of 20% acetonitrile also showed aggregation, but the absorbance of the ultrafiltrate of the reaction mixture at 344nm corresponded to the quantitative modification of cysteine. Lowering the acetonitrile concentration to 10% reduces the amount of aggregation, but results in a lower degree of modification.

An alternative thiol-based scheme is performed by introducing a thiol group at a lysine residue. The thiol protected is introduced on the lysine residue by: the enzyme was reacted with a 100-f fold molar excess of SATA-dPEG4-NHS (Quanta Biodesign) at 25 ℃ for 4 hours in sodium phosphate pH 6.2 and purified against the same buffer by dialysis overnight. The purified product and NEA-oligosaccharide 82 were then reacted under the conditions described above for cysteine-based conjugation to give lysine-thiol conjugates. This shows a 10-fold increase in Man-6P content (conjugated 5 glycans).

The stability of the hydrazide bearing lysine conjugates was evaluated at 37 ℃ for up to 14 days by measuring the intact protein molecular weight and M6P content. The conjugate was not stable because more than 50% of the neoglycan was lost within 14 days. Aminoxy conjugates were prepared via lysine using hexoses in molar excess relative to rhGAA 0, 16.6, 25, 33 and 40 as described above. The conjugate was saturable at 16.6-fold molar excess. Although only-31% (or 5 neoglycans conjugated) of total lysine was conjugated. High aggregation levels were also observed in several formulations. The pegylated form of SFB was tested with no reduction in aggregation.

Galactose conjugation (GAM) was performed by first pre-treating rhGAA6 hours (containing 2% mannitol and 0.005% Tween-80 in 25mM sodium phosphate, pH 6.25) with sialidase from closterium perfringens (20mU/mg) at 37 ℃. After desaturation, the protein was treated with galactose oxidase (GAO) at 1-10. mu.g/mg and catalase (Sigma) at 2U/mg in the same buffer overnight at 37 ℃ before Poros 50D was used(anion-exchange) chromatography to remove neuraminidase and catalase. The products treated with both enzymes were treated with the same volume of dH₂Diluted O and then applied to a Poros 50D column, pre-equilibrated with 10mM sodium phosphate buffer, pH 6.9. After the column was washed with 10mM sodium acetate buffer, pH 5.0, rhGAA was eluted with 150mM sodium acetate buffer, pH 5.0, and aminoxyhexoses were conjugated at 37 ℃ at various molar ratios for 6 hours.

The GAM conjugate was saturated with 6-7 glycan conjugated to GAA with a 16.6-f fold molar excess of hexose to GAA. The aggregation level was low. No sialic acid was detected after the desialytic acid, while a small amount of galactose was measured after galactose oxidase treatment. In some cases, 20-30% of the galactose residues are over-oxidized, thereby producing galacturonic acid, which is not conjugated to oligosaccharides. GAO was titrated, which showed that over 1 μ g/mg, GAO reduced glycan conjugation. There was a significant increase in the amount of galacturonic acid peroxidation product above 2. mu.g/mg GAO. After titration with GAO, the maximum amount of conjugation was achieved at 1-2 μ g GAO/mg rhGAA (fig. 10E-monosaccharide, including GalA content of Man-6P, Gal, GAM conjugates). Higher conjugation was observed at Man-6P content in the protein when 0.5 to 2ug gao/mg GAA was used. When lower or higher GAO is used, lower galactose or higher GalA is produced.

The amount of bis-M6P hexoglycan conjugated NeoGAA was quantified by M6P content analysis and MAL di-TOF. For M6P quantification, the samples were buffer exchanged using Amicon 4, 50,000MWCO centrifugal filter units (with 5 filtration cycles) to remove any potential excess glycans. 80 micrograms of each rhGAA or NeoGAA sample was hydrolyzed in 6.75M TFA for 1.5 hours at 100 ℃. The samples were cooled, dried in a Speed Vac, and reconstituted in 200 μ L of distilled water. The reconstituted sample was again dried in a Speed Vac and reconstituted with 200. mu.L of 50mM citrate pH 2.0. The sample was filtered through an S Mini H column (Sartorius) equilibrated in sodium citrate pH2.0 to remove impurities from the hydrolysate. For all samples and standards, ribose-5-phosphate was added as an internal standard. 50 μ L of the hydrolysate was injected onto a Dionex HPLC and analyzed by high pH anion exchange chromatography using pulsed amperometric detection (HPAEC-PAD). Quantification was performed using a standard curve constructed with hydrolysis standards for M6P. The degree of conjugation was then calculated based on the known molar ratio of 2 moles of M6P per mole of glycan.

MAL di-TOF MS analysis was performed in a linear fashion using a Voyager DE-PRO mass spectrometer. For all samples and standards 1: 5 was diluted into 0.1% formic acid in water followed by 1: 1 dilution into saturated sinapic acid in 50% acetonitrile/0.1% TFA. mu.L of this mixture was applied to the target. Samples, reference and BSA calibration controls were analyzed in triplicate. Two point calibrations were performed using (M + H) + and dimer ions of BSA. The degree of conjugation for each NeoGAA sample was evaluated based on the difference in molecular weight between the sample and the oxidized rhGAA control (no glycan introduced) taking into account the measured glycan molecular weight of 1323 g/mole.

Figure 9A shows the results of an assay using the above-described di-, tri-, tetra-and hexose conjugates.

Fig. 9B provides results for beta SAM6 conjugates prepared using different amounts of periodate. The level of saturated oligosaccharides needed to achieve conjugation reactions was proportional to the amount of periodate used in the oxidation step (fig. 9B, upper panel). Using 2mM periodate, rhGAA samples with-5.2 moles of sialic acid reached saturation at approximately 25-fold molar excess of hexose vs. protein (4.8-fold molar excess vs. sialic acid). Saturation was achieved at 33-fold molar excess of glycans using 7.5mM periodate. For rhGAA oxidized with 22.5mM periodate with 120-fold molar excess of glycans, saturation was approached but not achieved. The maximum conjugation levels achieved were also different for samples prepared using different levels of periodate. Approximately 8.5 and 10.5 moles of glycans per mole of protein were introduced using 7.5 and 22.5mM periodate, respectively. After oxidation with 2mM periodate, the achievable level of conjugation was approximately 5 moles of glycans per mole of protein, which is similar to the number of sialic acid residues in the starting material.

Glycan titration experiments were repeated with rhGAA with an initial sialic acid level of-7.2 moles/mole protein using 2mM periodate (fig. 9B, lower panel). At > 33-fold molar excess of glycan to protein (4.6-fold molar excess compared to sialic acid), conjugation levels were approximately 7 moles of glycan per mole of protein.

B. Aggregation reduction

Certain conjugation methods result in protein aggregation. Two approaches to reduce aggregation in neoGAA were developed: 1) hydrophobic Interaction Chromatography (HIC) using various HIC chromatography media and 2) metal chelation methods.

A 3g batch of NeoGAA was prepared and used to evaluate HIC and to remove aggregated copper columns. HIC columns were evaluated in a flow-through manner: butyl 650C and 650M, hexyl 650C, phenyl 6FF, Capto octyl and Capto phenyl. Hexyl and Capto phenyl gave comparable results with recoveries of 87.5% and 90.4% and aggregation reduced from 3.2% (initial level) to 1.4% and from 3.9% (initial) to 1.6%, respectively. See table 2.

Table 2: reduction of aggregation from conjugated GAA (3.2% agg) Using HIC column (8 ℃ C.)

The conditions for the copper chelating column (GE or Tosoh) run were also established in flow-through mode or bind-elute mode. A copper loaded 7ml metal chelating FF column (i.d., 7ml) was first evaluated in a bind-elute manner with 10mg/ml loaded conjugated GAA. When the column was eluted at RT with 175mM glycine, 100mM acetate, pH 5.5 as elution buffer, 87% of the NeoGAA was recovered, of which 1.2% was aggregated. At 8 ℃, glycine elution column above 175mM was required for satisfactory recovery. Good recovery of 92% was achieved in flow-through mode (table 3), with aggregation reduced from 3.2 to 1.2%. 150mM glycine, 100mM acetate, pH 5.5 was used as elution buffer.

TABLE 3 removal of aggregates from conjugated GAA (3.2% agg) using a copper 6FF column (RT, Ft mode)

Imidazole (7.5, 8 and 10mM) was also used as an elution buffer for metal chelating 6FF columns. About 8mM imidazole was required to elute the column. Since imidazole does not elute copper from the column, it is not necessary to condition the column or to allow EDTA in the upper portion of the column to reach a clear space. A column capacity of 15mg/ml NeoGAA was achieved.

The Toso AF-chelate 650M column was loaded with copper and also evaluated. In bind-and-elute mode, a column capacity of 15mg/ml was achieved using 8mM glycine to achieve 94.1% elution and 1.2% aggregation. In a flow-through manner, a capacity of 33.6mg/ml was achieved. 90.6% recovery and 1.2% aggregation were obtained using 50mM glycine in elution buffer.

C. Analysis of oligosaccharides

According to these experiments, the use of > 2mM periodate resulted in the introduction of NeoGAA glycans that exceeded the initial level of sialic acid in the protein, indicating non-sialic acid partial oxidation. To determine the level of oxidation at other carbohydrate sites by periodate, a series of periodate titration experiments were performed to monitor the levels of other monosaccharide residues.

To determine the sialic acid content, the samples were subjected to acidic hydrolysis using 0.5M formic acid at 80 ℃ for one hour. The liberated sialic acid was analyzed by high pH anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and carried out on a Dionex CarboPac PA1 column for 20 minutes using a 50-180mM sodium acetate gradient in 100mM sodium hydroxide. Results are expressed as moles of sialic acid (NANA or NGNA) per mole of rhGAA or NeoGAA and are determined from a standard curve of authentic commercially available sialic acid standards.

Levels of neutral monosaccharides, including fucose, galactose, GlcNAc and mannose, were determined by hydrolysis of 100. mu.g of rhGAA or NeoGAA in 1MTFA at 110 ℃ for 2 hours. After hydrolysis, the tubes were cooled on ice and centrifuged at 10,000rpm for 1 minute and evaporated to dryness by Speed Vac. The released monosaccharides were resuspended in 250 μ L of water and vortexed and filtered using a millipore ultra free-MC filter tube (10,000 MWCO). The released monosaccharides were analyzed by high pH anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and on a CarboPacPA1 column. Quantification was performed according to a standard curve of monosaccharides hydrolyzed in the same manner.

The results of the above test are shown in fig. 10A. The results indicate that sialic acid is the most easily oxidized monosaccharide. Low levels of fucose damage were detected at 2mM periodate and the levels were more detectable and reproducible at ≧ 5 mM. Slightly oxidized mannose was detectable only at > 7.5mM periodate.

To confirm the presence of oxidized sialic acid, fucose and mannose, fragment mass spectrometry was performed on the oligosaccharides released from rhGAA oxidized using 7.5mM periodate. The N-linked oligosaccharides in rhGAA and NeoGAA were released using an overnight digestion with PNGaseF at 50mM sodium phosphate pH 7.0 and 10mM B-mercaptoethanol. The released oligosaccharides were removed by means of a bio-dialysis (MWCO 500Da) in which water was exchanged several times. The dialyzed sample was dried in a high-speed concentrator and reconstituted using 110 μ L of 10mM ammonium formate pH 4.0 in 50% acetonitrile and 50% water. Samples were analyzed using a TSK Gel amide-80 column (100. mu.L injection to 2X100 mM, 5 μm particle size), using on-line MS detection (QStar tetrode flight time, LCT Premier flight time, and LTQ linear ion trap), using an acetonitrile-water gradient and 10mM ammonium formate pH 4.0.

For oligosaccharide structure analysis, oligosaccharides were dried and fluorescently labeled using Anthranilic Acid (AA). AA-labelled oligosaccharides were resolved by forward HPLC on a TSK gel amide 80 column using fluorescence detection and using an acetonitrile/water gradient. MS fragment analysis was performed on-line and using a cationic mode LTQ XL linear ion capture mass spectrometer. The spectral scan was from 400 to 2,000m/z, the normalized collision energy was set at 35 (error, unless specified herein) and the activation Q was set at 0.25.

Sialic acid:complete oxidation of sialic acid (C7, 8 and C8, 9 linkages) will result in a mass reduction of 62 daltons. Oxidation of fucose and mannose will initially result in a reduction of 2 daltons (C2, 3 or C3, 4 bond oxidation) followed by a reduction of 30 daltons upon oxidation of the remaining vicinal diols. Reductive amination of furfural with AA will result in a loss of oxygen, with a net addition of 121.1 daltons molecular weight per AA added. The theoretical and measured molecular weight changes of rhGAA oligosaccharides after sialic acid, fucose and mannose oxidations are shown in table 4.

Table 4 summary of target ions of AA-labeled SAM6 oligosaccharides for ms2 and ms3 analysis of "1 +" and "2 +", corresponding to singly and doubly charged cationic species, respectively. "2-" and "3-" correspond to doubly charged and triply charged anionic species, respectively.

Some ions were observed to correspond to oxidized and AA-derived oxidized sialic acid, fucose and mannose oligosaccharide species. Apart from derivatization of the reduced GlcNAc at all released oligosaccharides, derivatization of AA corresponds to the amount of reduced aldehyde species present, i.e., oxidized sialic acid is used in a 1: 1 ratio, and mannose and fucose are used in a 2: 1 ratio/oxidation.

The mass spectrum of fig. 10B shows the detection of 4 ions, which correspond to rhGAA oligosaccharides, with oxidation and AA-derivatization of sialic acid at C7. Two target ions (m/z 1057 and 1130) for sialic acid oxidation were selected for additional MS/MS fragment analysis. The fragmentation pattern for m/z 1057 is shown in FIG. 10C, with hypothetical identifications for each ion. Fragment ions m/z 716, 1032, 1235, 1397, 1584, and 1747 were selected for MS3 analysis. MS matched with hypothetical oligosaccharide structure³The spectra contain sialic acids oxidized and AA-labeled on the ends of the bilinear glycans. In particular, release of fragment m/z 351 confirmed AA attachment to sialic acid in the form of C7. In all samples analyzedOnly oxidized sialic acid in the form of C7 was observed; there was no evidence of oxidized sialic acid at C8.

Oxidation of fucose:table 5 lists the theoretical and measured masses of AA-derivatized, oxidized A1F and A2F

Table 5 theoretical and measured masses of AA-derivatized A1F and A2F, followed by oxidation of the 1 fucose residue and periodate. "2 +" corresponds to a doubly charged cationic species. Theoretical masses are based on conjugation of 4AA molecules/A1F oligosaccharide, and 5AA molecules/A2F

The fragmentation pattern of parent ion m/z 1250.4 is shown in fig. 10D and is consistent with that expected for AA-labeled monosialo singlet core fucosylated oligosaccharides containing oxidized sialic acid and oxidized fucose. The major fragment ions m/z 716, 1397, 1621 and 1783 were determined. These ion selections are for MS³Analyzed to confirm the identity of the hypothesis. MS at m/z 1621 and 1783³In the spectra, the ion fragmentation pattern was determined, with a loss of 195 daltons from the parent ions (1426 and 1588, from parent ions 1621 and 1783, respectively). The molecular weight change is consistent with cleavage between C1 and the oxygen atom of oxidized fucose, resulting in loss of the derivatized anthranilic acid attached to C4. In addition, further fragmentation and loss of the second derivatized anthranilic acid bound to C3 was observed as a loss of 386 daltons from the parent ion (1235 and 1397m/z from parent ions 1621 and 1783, respectively).

The MS fragment pattern of the oxidized rhGAA oligosaccharide A1F shows derivatization of C3, 4 in fucose with anthranilic acid, confirming the production of periodate oxidation. There is no evidence of oxidation of the C2-C3 bond. The conjugation of bis-M6P hexosan to oxidized rhGAA resulted in a net increase of 1305.3 daltons over the course of the condensation reaction.

To confirm the identification of the conjugated oligosaccharide structure, high quality precision MS analysis of native, released oligosaccharides was performed. Oligosaccharides from rhGAA and NeoGAA SAM6 were prepared using 2 and 7.5mM periodate, released using PNGase F, resolved by forward HPLC (TSK gel amide-80 column) using an acetonitrile-water gradient and 10mM ammonium formate pH 4.0. Mass spectrometric detection of glycans was performed on-line, anionically, using QStar or LCT time-of-flight mass spectrometers.

The table below provides the natural N-linked oligosaccharide peaks from rhGAA oxidized with 2 and 7.5mM periodate to identify SAM6, which is based on high precision MS/TOF analysis. "Ox" refers to the number of oxidation sites, the number of "Conj" conjugated bis-M6P hexoglycans. The theoretical mass is calculated from the monoisotopic molecular weight of the theoretical oligosaccharide structure and the theoretical and measured m/z corresponding to the charge state is shown below.

Mass accuracy > 20ppm determination was used for all oligosaccharide species. The MS results were consistent with the analysis of monosaccharide composition, which showed that oxidation of sialic acid was completed at ≧ 1mM periodate. Some oxidation of mannose and fucose also occurs. Conjugated ions corresponding to 1 and 2 moles of glycan/oxidized mannose and/or fucose were also measured, indicating that both aldehyde species of each were reduced to glycans, as used for AA assays.

Some of the conjugation of the high mannose structures (oligomannose 5 and 6) was detected in 2 and 7.5mM periodate-treated material. In the material conjugated with 2mM periodate, 0 or 1 conjugated glycans were observed in the a1 (monosialo) material, while ions corresponding to 0,1, 2, and 3 conjugated glycans were determined in the a1 material, which was in 7.5mM SAM 6. This result suggests that with 7.5mM periodate (but not 2mM), some oxidation and conjugation of core mannose and/or galactose residues occurred at the a1 structure.

For the A2 and A2F species (bi-sialylated, bi-linear, ± fucose), the mono-and bi-conjugated species were assayed at 2 and 7.5mM periodate samples. Evidence for tri-conjugation of oxidized mannose was observed only in 7.5mM periodate-treated samples, not in 2mM treatment, which is consistent with conjugation by fucose at increased periodate concentrations.

Figure 11A shows HPLC analysis of oligosaccharides released from rhGAA and NeoGAA. For the rhGAA control, most of the N-linked oligosaccharide material eluted at 11-13 minutes, which corresponds to the oligosaccharide without phosphorylation or conjugation. For NeoGAA oligosaccharides, the di-conjugated oligosaccharide species elutes at 19-20 minutes, the mono-conjugated oligosaccharide species elutes at 15-18 minutes, and the oxidized/unmodified oligosaccharides elute at 10-13 minutes. In the SAM6 sample prepared using 7.5mM periodate, about half of the oligosaccharides were found to elute in the region corresponding to the bis-conjugated species, whereas about 1/3 of the oligosaccharides from the 2mM periodate-treated sample were bis-conjugated. The elution characteristics of these samples were consistent with their levels of conjugation (-7 and 9 moles of glycan conjugate per mole of NeoGAA, SAM6 generated for 2 and 7.5mM periodate, respectively), as determined by MAL di-TOF and mannose-6-phosphate content analysis.

To improve better visualization and quantitative comparison of the structures in which oligosaccharides are present, the released oligosaccharides were analyzed by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Samples were run on a Dionex CarboPac PA100 column by HPAEC-PAD using a gradient of sodium acetate in 100mM sodium hydroxide. Oligosaccharide peak identification was confirmed by the following manner: the off-line fractions were collected, dialyzed vs. water, and analyzed by forward HPLC using on-line MS analysis.

Figure 11B shows a representative HPAEC-PAD curve with identified peaks (determined by MS). Figure 11C shows the oligosaccharide profile of HPAEC-PAD by 2 and 7.5mM periodate-treated rhGAA and NeoGAA SAM6 samples.

rhGAA protein backbone assay followed by periodate treatment

To observe potential modifications of the protein backbone of NeoGAA, peptide mapping LC/MS was performed. NeoGAA SAM6 produced using 0, 2, 7.5 and 22.5mM periodate was prepared using trypsin and analyzed by reverse phase HPLC with LCT time-of-flight mass spectrometer. Potential peptide modifications such as oxidation of cysteine, methionine, tryptophan, tyrosine and histidine residues and deamidation of asparagine were evaluated using BioPharmalynx software. The only significant modification detected was the oxidation of methionine at several different sites. The oxidation level of peptide T13 (containing methionine 172 and 173), followed by treatment with 0, 2 and 22.5mM periodate is shown in FIG. 12.

Significant levels of oxidation were found at methionine residues 122, 172 and 173. The oxidation of these methionine residues was confirmed by LC/MS/MS analysis. Low levels of oxidation were also observed to occur at methionine 363 in a periodate dependent manner, with periodate concentrations titrated to levels of 1 and 7.5mM in an attempt to minimize periodate oxidation at the most susceptible site (peptide T13) and monitored by LC/MS. Significant levels of methionine oxidation were observed at periodate concentrations greater than 1mM, indicating that methionine in rhGAA is readily oxidized to sialic acid residues.

During GAM conjugation, galactose oxidation by GAO resulted in-26% oxidation of Met 172/173. Methionine oxidation was eliminated when catalase was included in the oxidation reaction at 2 and 50 units/mg GAA.

Example 8: in vitro characterization of GAA conjugates

NeoGAA SAM2 was prepared as described in example 7 using compound 17 from example 2 and 7.5mM periodate. Galactose-conjugated NeoGAA GAM2 was prepared by treating rhGAA with galactose oxidase prior to conjugation with disaccharide 17. Similarly, trisaccharides NeoGAASAM3 (compound 35, example 3), tetrasaccharides NeoGAAs SAM4 (compound 28, example 4A) and linear SAM4 (compound 47, example 4B) were also prepared. In addition, hexose NeoGAA β SAM6 was prepared using compound 77 described in example 7, using 2 and 7.5mM periodate. Additional hexose conjugates α SAM6(α -linked, conjugated through sialic acid residues) and GAM6 (conjugated through galactose residues) were also prepared.

A. Specific activity:

the activity assay was performed by monitoring the rate of hydrolysis of the synthetic substrate p-nitrophenyl-D-alpha-glucopyranoside (p-NP), as catalyzed by rhGAA and NeoGAA. The released chromophore is a measure of absorption at 400 nm under basic conditions. One activity unit is defined as the amount of enzyme required to hydrolyze 1. mu. mol of p-nitrophenyl-D-alpha-glucopyranoside to p-nitrophenol at 37 ℃ per minute under defined assay conditions.

Specific activities of NeoGAAs SAM2, SAM3, SAM4, linear SAM4, alpha SAM6 and beta SAM6 are shown in fig. 13. In addition, the specific activity of the conjugate prepared by using SAM method vs. gam method was evaluated. There was a negative correlation between the amount of M6P/specific activity of GAA and NeoGAA conjugates, which were prepared on a small scale and had a 16.6-fold molar excess of oligosaccharide/GAA. The loss of GAA activity was also observed with increasing M6P content of SAM or GAM conjugates, respectively, conjugated during the NeoGAA concentration titration experiment (from 2.5 to 33-fold molar excess of oligosaccharides). For SAM, GAA activity 49-81% (compared to control), various NeoGAAs carry 6-8 oligosaccharide molecules/protein. SAM, and has 4-6 oligosaccharides/protein.

M6p receptor binding:

conjugation effects of function evaluation was performed to monitor NeoGAA binding of water soluble cation independent mannose-6-uptake by Biacore and M6P receptor columns affinity high performance liquid chromatography, phosphate receptor (siciprr) and in L6 myoblast cells. Ascimpr, purified from bovine serum, comprising the ectodomain of AX, but lacking the transmembrane portion.

For Biacore analysis, sCIMPR amine was coupled to CM-5 chips, 10 μ g/ml of NeoGAA was loaded onto the surface of the sample and mannose-6 concentration was increased to elute phosphate. Affinity is the concentration required for quantified M6P to replace the 50 NeoGAA% constraint (EC50 value). The method is used to monitor affinity conjugation and oxidation to the receptor.

Figure 14 shows the results of Biacore analysis. Injection into the ascimpr immobilized Biacore resulted in an increase of 10 μ g/ml NeoGAA in-250 chiplet reactions, while the same amount of rhGAA resulted in about 100 ru deflection. In addition, about 10 times as much M6P is required to elute a concentration ratio of rhGAA NeoGAA (about 0.1 vs. 1.0 mm EC50 values of rhGAA and NeoGAA, respectively). EC50 values between the linear relationship (R square > 0.95) and the conjugation level NeoGAA were prepared with 2 and 7.5mm periodate. Throughout the examination of conjugation ranges (1.6-2 mm periodate preparation and 1.04.7 moles of glycans per mole of NeoGAA-8.5 moles of glycans per mole of 7.5mm sodium periodate preparation), the effect of affinity on conjugation scale was similar to that of 2 and 7.5mm periodate preparation of NeoGAA.

M6P receptor binding high performance liquid chromatography was evaluated using a M6P receptor column supported on PorosEP resin, which was then incorporated into an analytical HPLC column package. rhGAA and NeoGAA elutions were prepared using 0.25, 0.85, 5 and 20mM M6P (fig. 15A and B). In one experiment, SAM2 and GAM2 were compared to β SAM6 and GAM6 (fig. 15C). SAM6 and GAM6 required that the material before 20mm M6P was mostly eluted (> 95% conjugate bound to the column). SAM2 and less tightly bound eluted most of the GAM2 at 5mm M6P (> 95% bound to the column).

In addition, NeoGAA conjugates SAM2, SAM3, SAM4, linear SAM4 and α SAM6 were evaluated for M6P receptor binding (fig. 15D). Most of SAM2, SAM3, were SAM4 conjugates eluted with 5mm of M6P, while SAM6 conjugates required 20mm of M6P.

NeoGAA conjugation was evaluated with different numbers of conjugates (FIG. 15E). The NeoGAA binding fraction ratio was consistently > 95% and > 2.0 moles per mole NeoGAA conjugate glycan these preparations were four at this lower level of conjugation (1.0-1.7 moles glycan) number fraction with binding fractions between 75-90%. The cross-section listed for NeoGAA M6P also shows the greatest number of species with higher affinity than rhGAA. Specifically, most of the bound rhGAA was eluted with 0.2 mm M6P, whereas 20mm M6P required elution of NeoGAA from the column. The overall effect of conjugation on the percent level of high affinity species was similar between the 2.0 and 7.5mm periodate treatments.

Fig. 15F shows the effect of the change amount M6P. The following amounts of M6P contained SAM6 conjugate: SAM6-1(4.9mol M6P/mol rhGAA), SAM6-2(7.4mol M6P/mol rhGAA), SAM6-3(10.5mol M6P/mol rhGAA), SAM6-4(11.2mol M6P/mol rhGAA) and SAM6-5(16.6mol M6P/mol rhGAA). Statistical differences are represented by: and, and represents p < 0.05 compared to 100mg/kg rhGAA, SAM6-1 and SAM6-3, respectively.

Internalization of L6 myoblasts

L6 myoblast uptake assays were performed, as described by Zhu et al, j.biol.chem.279: 50336 and 50341(2004) to confirm that rhGAA and NeoGAA target myoblasts via the cation-independent mannose-6-phosphate receptor (CIMPR) pathway. In the L6 myoblast uptake assay, rhGAA and NeoGAA were added +/-5mM M6P to the medium in the wells, containing L6 myoblasts, and incubated overnight for the scheduled. After incubation, cells were lysed and activity was determined using 4-MU glycoside substrate and protein concentration was determined by micro-BCA assay to generate an enzyme dose response curve.

The results of the L6 myoblast uptake assay are shown in FIG. 16. SAM2 and GAM2 conjugates showed significantly better uptake than unmodified rhGAA, but not as high as the bis-phosphorylated SAM6 or GAM6 conjugates (fig. 16, upper panel). The uptake of NeoGAAs SAM2, SAM3, SAM4, linear SAM4 and alpha SAM6 was also tested (fig. 16, lower panel). In a similar experiment, the lysine-thiol conjugate of example 7 produced an-8-fold increase in uptake.

Example 9: in vivo efficacy of novel GAA conjugates

The in vivo effects of certain NeoGAA conjugates were performed in GAA knockout mouse models, such as Raben et al j.biol.chem.273 (30): 19086-92 (1998). Groups of six mice were treated once a week for four weeks as follows:

group of	GAA	Dosage (mg/kg)
			1	Vehicle (repeat SAM2, SAM4 and SAM6 test)	--
2	Myozyme (repeat SAM2, SAM4 and SAM6 tests)	20
			3	Myozyme (repeat SAM2, SAM4 and SAM6 tests)	100
4	SAM2	4
			5	SAM2	20
6	SAM4	4
			7	SAM4	20
8	αSAM6	4
			9	αSAM6	20
10	Beta SAM6(7.5mM periodate)	4
			11	Beta SAM6(7.5mM periodate)	20
12	Beta SAM6(2mM periodate)	4
			13	Beta SAM6(2mM periodate)	20

Samples were taken from heart, quadriceps, and triceps and tissue glycogen content was measured SAM2, SAM 4. The results and SAM6 animals are shown in figures 17, 18 and 19, respectively. Experimental replicates using the animal population of 12SAM6 conjugate confirmed that SAM6 conjugate was more than five times more effective than unmodified rhGAA.

SAM and GAM hexose conjugates were compared using six groups of mice receiving vehicle, 20, 60 or 100 mg/rhGAA or 4, 12kg or 20 mg/kg once, four weeks SAM6 or GAM 6. Heart, quadriceps, triceps diaphragm and psoas muscle harvests and glycogen content were analyzed. Figure 20 shows the results of this study. Pharmacokinetic and pharmacodynamic studies: 30GAA knockout mice (male 15, female 15) at 3-6 months of age (obtained from Charles River Laboratories, Wilmington, Mass.). Animals were housed at 25 ℃ for a 12 hour light/dark cycle humidity hold. All animals were given free food (PicoLab)Rodent Diet 20) and water. Animals were randomly divided into 3 groups with 5 males and 5 females per group dose of 10 groups, and mice were totaled per dose group. 20 groups of rhGAA, free sub-alignment movement and SAM conjugate were received as single intravenous milligram/kg. Pharmacokinetic analysis blood samples were collected for 5,15, 30, 60, 120, 240 and 480 minutes post-ocular plexus dose in conscious mice. Serum rhGAA concentration, the GAA activity assay used was determined. The results are shown in FIG. 20B.

Example 10: synthesis of conjugates of acid sphingomyelinase

Recombinant human acid sphingomyelinase (rhASM) expressed in baculovirus expression systems or chinese hamster ovary cells has a C-terminal cysteine with a free thiol group. See Lansmann et al, eur.j. biochem.270: 1076 and 1088 (2003); qiu et al, j.biol.chem.278: 32744-32752(2003). rhASM can be coupled to any oligosaccharide 1-127 via this free thiol group, where the oligosaccharide contains a linker and a thiol-reactive group, according to the methods described in U.S. provisional patent application No.60/885,457 or example 6.

Example 11: synthesis of conjugates of alpha-l-iduronidase

Any of α -L-iduronidase-conjugated oligosaccharides 1-127, wherein the oligosaccharide comprises a linker, comprising a malondialdehyde reactive group, according to Lee et al, pharm. res.20: 818, 825 (2003). alpha-L-iduronidase and oligosaccharides were coupled in the presence of sodium cyanoborohydride as reducing agent at room temperature at pH 5.5 for 1 day. The small molecules are then removed from the reaction mixture by dialysis or ultrafiltration.

All references cited herein are incorporated by reference in their entirety. To the extent that the incorporated publications and patents or patent applications are inconsistent with the invention contained in the specification, this specification will supersede any conflicting material.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about," where about indicates, for example, ± 5%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant digits and by applying ordinary rounding techniques.

It will be apparent to those skilled in the art that many modifications and variations can be made to the present invention without departing from the spirit and scope thereof. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (18)

1. An oligosaccharide-protein conjugate comprising: (1) a protein and (2) an oligosaccharide of any one of formulae I-VI:

wherein:

a is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

b is selected from alpha 1, 2; α 1, 3; and α 1, 4;

c is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

d is selected from α, β and mixtures of α and β;

e is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

f is selected from α, β and mixtures of α and β;

g is selected from alpha 1, 2; α 1, 3; and α 1, 4;

h is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

i is selected from α, β and mixtures of α and β;

j is α 1, 2;

k is selected from α, β and mixtures of α and β;

x is 1,2 or 3;

l is selected from α, β and mixtures of α and β;

m is selected from α, β and mixtures of α and β;

R^xand R^yEach independently selected from polyethylene glycol and C₁-C₁₀Alkyl radical, said C₁-C₁₀Alkyl is optionally substituted by oxo, nitro, halo, carboxy, cyano or lower alkyl and optionally interrupted by one or more heteroatoms selected from N, O or S;

z is 0,1, 2, 3 or 4; and

when x is 2 or 3, or y is 2, 3 or 4, the linkage between each mannose of formula IV or formula VI is selected from α 1, 2; α 1, 3; α 1, 4; and α 1, 6;

provided that when e is α 1,6, f is selected from α and mixtures of α and β.

2. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide has two mannose-6-phosphate residues.

3. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide has three mannose-6-phosphate residues.

4. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide is oligosaccharide 82.

5. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide is oligosaccharide 128, oligosaccharide 129 or a mixture thereof.

6. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide is oligosaccharide 130, oligosaccharide 131 or a mixture thereof.

7. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide is oligosaccharide 132, oligosaccharide 133, or a mixture thereof.

8. The oligosaccharide-protein conjugate of claim 1, wherein the oligosaccharide is oligosaccharide 136.

9. The oligosaccharide-protein conjugate of claim 1, wherein the protein is a glycoprotein.

10. The oligosaccharide-protein conjugate of claim 9, wherein the glycoprotein is a lysosomal enzyme.

11. The oligosaccharide-protein conjugate of claim 10, wherein the lysosomal enzyme is acid alpha-glucosidase, alpha-galactosidase a, acid sphingomyelinase, alpha-L-iduronidase, iduronate-2-sulfatase, or N-acetylgalactosamine-4-sulfatase.

12. The oligosaccharide-protein conjugate of claim 10, wherein the lysosomal enzyme is acid alpha-glucosidase.

13. A pharmaceutical composition comprising the oligosaccharide-protein conjugate of claim 1 and an excipient.

14. Use of the conjugate of claim 1 in the manufacture of a medicament for treating a lysosomal storage disease in a subject in need thereof.

15. A method of treating a lysosomal storage disease comprising administering to a mammal an oligosaccharide-glycoprotein conjugate comprising: (1) a lysosomal enzyme and (2) an oligosaccharide of any one of formulae I-VI:

wherein:

a is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

b is selected from alpha 1, 2; α 1, 3; and α 1, 4;

c is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

d is selected from α, β and mixtures of α and β;

e is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

f is selected from α, β and mixtures of α and β;

g is selected from alpha 1, 2; α 1, 3; and α 1, 4;

h is selected from alpha 1, 2; α 1, 3; α 1, 4; and α 1, 6;

i is selected from α, β and mixtures of α and β;

j is α 1, 2;

k is selected from α, β and mixtures of α and β;

x is 1,2 or 3;

l is selected from α, β and mixtures of α and β;

m is selected from α, β and mixtures of α and β;

R^xand R^yEach independently selected from polyethylene glycol and C₁-C₁₀Alkyl radical, said C₁-C₁₀Alkyl is optionally substituted by oxo, nitro, halo, carboxy, cyano or lower alkyl, and optionally interrupted by one or more heteroatoms selected from N, O or S;

z is 0,1, 2, 3 or 4; and

when x is 2 or 3, or y is 2, 3 or 4, the linkage between each mannose of formula IV or formula VI is selected from α 1, 2; α 1, 3; α 1, 4; and α 1, 6;

provided that when e is α 1,6, f is selected from α and mixtures of α and β.

16. The method of claim 15, wherein the lysosomal storage disease is selected from the group consisting of fabry's disease, pompe's disease, niemann-pick a disease, niemann-pick B disease, mucopolysaccharidosis I, mucopolysaccharidosis II, and mucopolysaccharidosis VI.

17. The method of claim 16, wherein the lysosomal storage disorder is pompe disease.

18. The method of claim 15, further comprising administering methotrexate to the mammal before, after, or during treatment with the oligosaccharide-glycoprotein conjugate.