RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 60/628,829 filed November 16, 2004 and entitled "Synthesis and Use of Glycodendrimer O Reagents." This application is related to U.S. Patent Application 10/417,768 filed April 16, 2003, which in turn is a continuation-in-part of U.S. Patent Application 09/824,827, filed April 2, 2001, now granted as United States patent 6,627,744, and divisional United States Patent Application 10/629,976, which in turn is a continuation-in-part of U.S. Patent Application 09/347,029, filed July 2, 1999, now issued as United States patent 6,512,098 and s divisional United States Patent Application 10/062,970, which in turn claims the benefit of U.S. Provisional Applications 60/091,687, filed July 2, 1998 and 60/131,466, filed April 28, 1999, and 09/824,827 that is also of U.S. Patent Application 09/556,466, filed April 21, 2000, which in turn claims the benefit of U.S. Provisional Application 60/131,362, filed April 28, 1999, all of which are hereby incorporated by reference in their entirety for all purposes. 0

FIELD OF THE INVENTION

[0002] Disclosed herein are chemically modified mutant proteins having modified glycosylation patterns with respect to a precursor protein from which they are derived, hi particular, disclosed are chemically modified mutant proteins including a cysteine residue s substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate. Also disclosed are methods of producing the chemically modified mutant proteins and glycosylated methanethiosulfonate reagents. Further disclosed are methods of modifying the functional characteristics of a protein by reacting the protein with a 0 glycosylated methanethiosulfonate reagent. In addition, disclosed are methods of determining the structure-function relationships of chemically modified mutant proteins.

BACKGROUND OF THE INVENTION

[0003] Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish et al., Angew. Chem., Int. s Ed. Engl., 34:621-633 (1995)). However, the latter procedures are difficult to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.

[0004] Changing enzyme properties by chemical modification has been explored io previously, with the first report being in 1966 by the groups of Bender (Polgar, et al., J. Am. Chem. Soα, 88:3153-3154 (1966)) and Koshland (Neet et al., Proc. Natl. Acad. Sci. USA. 56:1606-1611 (1966)), who created a thiolsubtilisin by chemical transformation (CH ₂ OH — > CH ₂ SH) of the active site serine residue of subtilisin BPN' to cysteine. Interest in chemically produced artificial enzymes, including some with synthetic potential, was reviewed by Wu is (Wu et al., J. Am. Chem. Soα, 111:4514-4515 (1989); Bell et al., Biochemistry, 32:3754- 3762 (1993)) and Peterson (Peterson et al., Biochemistry, 34:6616-6620 (1995)), and more recently, Suckling (Suckling et al., Bioorg. Med. Chem. Lett., 3:531-534 (1993)).

[0005] Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural, wild-type, enzymes can never hope to accept all structures of synthetic

20 chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by

2s protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigued chemists for many years and continues to do so.

» [0006] U.S. Patent No. 5,208,158 to Bech et al. ("Bech") describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines. The cysteines are subsequently modified to confer upon the enzyme improved

stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with C _1-6 alkyl.

[0007] Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to develop one or s more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile, and surface binding properties for use in, for example, detergents or organic synthesis. In particular, enzymes, such as subtilisins, tailored for peptide synthesis would be desirable Enzymes useful for peptide synthesis have high esterase and low amidase activities. Generally, subtilisins do not

JO meet these requirements and the improvement of the esterase to amidase selectivities of subtilisins would be desirable. However, previous attempts to tailor enzymes for peptide synthesis by lowering amidase activity have generally resulted in dramatic decreases in both esterase and amidase activities. Previous strategies for lowering the amidase activity include the use of water-miscible organic solvents (Barbas et al., J. Am. Chem. Soc. 110:5162-5166

/j (1988); Wong et al., J. Am. Chem. Soc. 112:945-953 (1990); and Sears et al., Biotechnol. Prog., 12:423-433 (1996)) and site-directed mutagenesis (Abrahamsen et al., Biochemistry, 303:4151-4159 (1991); Bonneau et al., "Alteration of the Specificity of Subtilisin BPN' by Site-Directed Mutagenesis in its Sl and Sl' Binding -Sites," J. Am. Chem. Soc, 113:1026- 1030 (1991); and Graycar et al., Annal. RY. Acad. ScL. 67:71-79 (1992)). However, while

2o the ratios of esterase-to-amidase activities were improved by these approaches, the absolute esterase activities were lowered concomitantly. Abrahamsen, et al. Biochemistry, 30:4151- 4159 (1991). Chemical modification techniques (Neet et al., Proc Nat. Acad. Sci. USA. 54:1606 (1966); Polgar et al., J. Am. Chem. Soc, 88:3153-3154 (1966); Wu et al., J. Am. Chem. Soc, 111:4514-4515 (1980); and West et al., J. Am. Chem. Soc. 112:5313-5320

2s (1990), which permit the incorporation of unnatural amino acid moieties, have also been applied to improve the esterase to amidase selectivity of subtilisins. For example, chemical conversion of the catalytic triad serine (221) of subtilisin to cysteine (Neet et al., Proc Natl. Acad. Sci., 54:1606 (1966); Polgar et al., J. Am. Chem. Soc, 88:3153-3154 (1966); and Nakatsuka et al., J. Am. Chem. Soc. 109:3808-3810 (1987)) or to selenocysteine (Wu et al.,

30 J. Am. Chem. Soc, 111:4514=4515 (1989)), and methylation of the catalytic triad histidine (His57) of chymotrypsin (West et al., J. Am. Chem. Soc, 112:5313-5320 (1990)), effected substantial improvement in esterase-to-amidase selectivities. Unfortunately however, these

modifications were again accompanied by 50- to 1000-fold decreases in absolute esterase activity.

[0008] Surface glycoproteins act as markers in cell-cell communication events that determine microbial virulence (Sharon et al., Essays Biochem., 30:59-75 (1995)), s inflammation (Lasky, Annu. Rev. Biochem.. 64:113-139 (1995); Weis et al., Annu. Rev. Biochem., 65:441-473 (1996)), and host immune responses (Varki, GlvcobioL, 3:97-130 (1993); Dwek, Chem. Rev.. 96:683-720 (1996)). In addition, the correct glycosylation of proteins is critical to their expression and folding (Helenius, MoI. Biol. Cell. 5:253-265 (1994)) and increases their thermal and proteolytic stability (Opendakker et al., FASEB J.. O 7:1330-1337 (1993)). Glycoproteins occur naturally in a number of forms (glycoforms) (Rademacher et al., Annu. Rev. Biochem., 57:785-838 (1988)) that possess the same peptide backbone, but differ in both the nature and site of glycosylation. The differences exhibited (Rademacher et al., Annu. Rev. Biochem.. 57:785-838 (1988); Parekh et al., Biochem.. 28:7670 7679 ( 1989); Knight, Biotechnol.. 7:35-40 (1989)) by each component within these s microheterogeneous mixtures present regulatory difficulties (Liu, Trends Biotechnol., 10:114-120 (1992); Bill et al., Chem. Biol., 3:145-149 (1996)) and problems in determining exact function. To explore these key properties, there is a pressing need for methods that will not only allow the preparation of pure glycosylated proteins, but will also allow the preparation of non-natural variants for the determination of structure-function relationships, 0 such as structure- activity relationships (SARs). The few studies that have compared single glycoforms successfully have required abundant sources and extensive chromatographic separation (Rudd et al., Biochem., 33:17-22 (1994)).

[0009] Neoglycoproteins (Krantz et al, Biochem., 15:3963-3968 (1976)), formed via unnatural linkages between sugars and proteins, provide an invaluable alternative source s of carbohydrate-protein conjugates (For reviews see Stowell et al., Adv. Carbohvdr. Chem. Biochem., 37:225-281 (1980); Neoglvcoconiugates: Preparation and Applications. Lee et al., Eds., Academic Press, London (1994); Abelson et al., Methods Enzvmol., 242: (1994); Lee et al., Methods Enzvmol., 247: (1994); Bovin et al., Chem. Soc. Rev., 24:413-421 (1995)). In particular, chemical glycosylation allows control of the glycan structure and the nature of the 0 sugar-protein bond. However, despite these advantages, existing methods for their preparation (Stowell et al., Adv. Carbohvdr. Chem. Biochem., 37:225-281 (1980)) typically generate mixtures. In addition, these techniques may alter the overall charge of the protein

(Lemieux et al., J. Am. Chem. Soα, 97:4076-4083 ( 1975); Kobayashi et al., Methods Enzymol., 247:409-418 (1994)) or destroy the cyclic nature of glycans introduced (Gray, Arch. Biochem. Biophvs.. 163:426-428 (1974)). For example, the reductive amination of lactose with bovine serum albumin (BSA) caused indiscriminate modification of lysine s residues through the formation of acyclic amines introduced (Gray, Arch. Biochem. Biophvs.. 163:426-428 (1974)). Advances in the site-specific glycosylation of BSA have been made (Davis et al., Tetrahedron Lett.. 32:67936796 (1991); Wong et al., Biochem. J.. 300:843-850 (1994); Macindoe et al., J. Chem. Soc. Chem. Commun. 847-848 ( 1998)). However, these methods rely upon modification of an existing cysteine in BSA and, as such, allow no O flexibility in the choice of glycosylation site. Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate. To explore their properties, there is a need for homogenous sources of carbohydrate-protein conjugates. Existing methods typically generate product protein mixtures of poorly characterized composition, with little or no control over the site or level of glycosylation. s [0010] The present invention is directed to overcoming these deficiencies.

SUMMARY OF THE INVENTION

[0011] It is an object of the present invention to provide for novel glycosylated proteins. o [0012] It is a further object of the invention to provide for novel glycoslyated proteins that have improved functional characteristics.

[0013] It is a further object of the invention to provide for novel glycodendrimeric proteins.

[0014] It is a further object of the invention to provide for novel glycodendrimeric s proteins that have improved functional characteristics.

[0015] It is a further object of the invention to provide glycodendrimeric compositions.

[0016] It is a further object of the invention to exploit the glycodendrimeric compositions to target proteins toward specific receptors. o [0017] It is a further object of the invention to provide a method of producing glycosylated proteins which have well defined properties, for example, by having predetermined glycosylation patterns.

[0018] In one aspect of the present invention, a method is provided wherein the glycosylation pattern of a protein is modified in a predictable and repeatable manner. Generally, the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate. j- [0019] Thus, in one composition aspect of the invention, a chemically modified mutant ("CMM") protein is provided, wherein said mutant protein differs from a precursor protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate. Preferably, the glycosylated O thiosulfonate is an alkylthiosulfonate, most preferably a methanethiosulfonate.

[0020] In a method aspect of the present invention, a method of producing a chemically modified mutant protein is provided comprising the steps of: (a) providing a precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated s thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto. Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate. Also preferably, the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a o DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.

[0021] The present invention also relates to novel glycosylated thiosulfonates. In an embodiment, the glycosylated thiosulfonate is a methanethiosulfonate. In another embodiment, the glycosylated methanethiosulfonate comprises a chemical structure s including:

O

Il

H ₃ C-S-SR Il O where R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac) ₂ , -β-Glc(Ac) ₃ , -P-GIc(Ac) ₄ , -Et-OC-GIc(Ac) ₂ , -Et-OrGIc(Ac) ₃ , -Et-O-GIc(Ac) ₄ , -Et-β-Glc(Ac) ₂ , -Et-β-Glc(Ac) ₃ , -Et-β-Glc(Ac) ₄ , -Et-α-Man(Ac) ₃ , -Et-α-Man(Ac) ₄ , -Et-β-Gal(Ac) ₃ , o -Et-P-GaI(Ac) ₄ , -Et-Lac(Ac) _5> -Et-Lac(Ac) ₆ , or Et-Lac(Ac) ₇ .

[0022] Another aspect of the present invention is a method of modifying the functional characteristics of a protein including reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. Accordingly, the present invention s provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.

[0023] In another aspect, the present invention relates to methods of determining O the structure-function relationships of chemically modified mutant proteins. One method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified mutant s proteins and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins. Another method involves providing first and second chemically modified mutant proteins of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting said cysteine residue with 0 a glycosylated thiosulfonate, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.

[0024] The chemically modified mutant proteins of the present invention provide s an alternative to site-directed mutagenesis and chemical modification for introducing unnatural amino acids into proteins. Moreover, the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships ("SARs")) of non-natural variants of the 0 proteins.

[0025] An advantage of the present invention is that it is possible to introduce predetermined glycosylation patterns into proteins in a simple and repeatable manner. This

advantage provides an ability to modify critical protein characteristics such as partitioning, solubility, cell-signaling, catalytic activity, biological activity and pharmacological activity. Additionally, certain methods of the present invention provide for a mechanism of "masking" certain chemically or biologically important protein sites, for example, sites which are critical for immunological or allergenic responses or sites which are critical to proteolytic degradation of the modified protein.

[0026] Another advantage of the present invention is the ability to glycosylate a protein which is not generally glycosylated, or to modify the glycosylation pattern of a protein which is generally glycosylated. [0027] Another advantage of the present invention is improved synthetic methods for glycosylating a protein which is not generally glycosylated, or for modifying the glycosylation pattern of a protein which is generally glycosylated.

[0028] Another advantage of the present invention is novel reagents for glycosylating a protein which is not generally glycosylated, or for modifying the glycosylation pattern of a protein which is generally glycosylated.

[0029] Another advantage of the present invention is to produce enzymes that have altered catalytic activity. In one specific example, the inventors have shown that it is possible to modify the substrate specificity of a protease to increase its ability to degrade lectins, selectin or integrins or members of other adhesion receptor families. Degradation of these proteins may have many specific effects which include, but are not limited to, degradation of biofilms and especially pathogenic biofilms. As an example, pathogenic biofilms from the organism Pseudomonas may be degraded. In addition, production of enzymes with altered catalytic or substrate binding activity may have therapeutic uses related to cancers, as they relate, for example, to metastasis. Similarly, modifications of substrate specificity would be expected when utilizing the present invention with other enzymes.

[0030] These and other advantages of the present invention are described in more detail in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0031] Figure 1 shows dendrimer methanethiosulfonate ("MTS") reagents.

[0032] Figure 2 shows dendrimer methanethiosulfonate ("MTS") reagents and hybrid dendrimer methanethiosulfonate ("MTS") reagents.

[0033] Figure 3 shows a glycodendrimer protein binding to carbohydrate binding sites on a lectin, selectin, integrin or member of other adhesion receptor family. X, Y and Z represent optional carbohydrate linkers; the lengths of these linkers, if present, need not be equal to each other. s [0034] Figure 4 is a schematic illustration of a glycodendriprotein showing terminal carbohydrate moieties, optional linkers, disulfide linkages, dendrimer cores, and a model enzyme, SBL.

[0035] Figure 5 shows two different synthetic approaches for generating glycodendrimers, i.e., normal addition, and inverse addition. O [0036] Figure 6 shows several synthetic schemes for generating tethered and direct-linked carbohydrate MTS reagents.

[0037] Figure 7 shows additional synthetic schemes for generating direct-linked carbohydrate MTS reagents.

[0038] Figure 8 shows the X-ray crystal structure of the MTS reagent 5β. S [0039] Figure 9 shows the X-ray crystal structure of the MTS reagents 10a and lOβ.

[0040] Figure 10 illustrates the use of a glycodendriprotein to digest a lectin, selectin, integrin or member of other adhesion receptor family.

[0041] Figure 11 illustrates components of a glycodendriprotein. The Y shaped 0 dendrimer core illustrated is TREN-type, but also may represent Penta-E type, ArGal-type or other dendrimer core structures.

[0042] Figure 12 shows a normal addition synthetic scheme for producing two different first-generation glycodendrimer MTS reagents, and glycodendriproteins produced from these reagents. s [0043] Figure 13 shows a normal addition synthetic scheme for producing a glycodendriprotein.

[0044] Figure 14 shows a normal addition synthetic scheme for producing a second generation glycodendrimer reagent.

[0045] Figure 15 shows glyco MTS reagent 12β, and diglycosyl disulfides 18 and 0 19 resulting from the use of 12β or 5β in the in situ reduction approach described in Example 2. Figure 15 also illustrates an inverse addition synthesis scheme (Scheme 9) for a first generation glycodendrimer reagent.

[0046] Figure 16 shows an inverse addition synthesis scheme for a multi- generation glycodendrimer reagent.

[0047] Figure 17 shows Scheme 11, illustrating synthetic approaches for producing bis-MTS reagents; Scheme 12, for producing thioglycoses; and Scheme 13, s illustrating another synthetic method for generating a first generation glycodendrimer reagent.

[0048] Figure 18 shows another synthetic scheme for producing a glycodendrimer MTS reagent.

[0049] Figure 19 shows a synthetic scheme (Scheme 15) for producing a first O generation glycodendrimer MTS reagent, and a second generation hybrid glycodendrimer MTS reagent; and an improved synthesis scheme (Scheme 16) for producing sodium methanethiosulfonate ("NaMTS").

[0050] Figure 20 shows synthetic scheme 17 for producing ArGal-based glycodendrimer reagent 42; and synthetic scheme 18 for producing ArGal-based s glycodendrimer reagent 44, bearing two deprotected sugars.

[0051] Figure 21 illustrates synthetic scheme 19 and synthetic scheme 20 for producing glycodendrimer reagents.

[0052] Figure 22 illustrates synthetic scheme 21 for producing a glycodendriprotein. 0 [0053] Figure 23 illustrates a glycosylated variant of Bacillus lentus subtilisin mutant S 156C.

DETAILED DESCRIPTION

[0054] In one aspect, a method is provided wherein the glycosylation pattern of a s protein is modified in a predictable and repeatable manner. Generally, the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.

[0055] Thus, in one composition aspect of the present invention, a chemically modified mutant protein is provided, wherein said mutant protein differs from a precursor o protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by

reacting said cysteine residue with a glycosylated thiosulfonate. Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate.

[0056] In a method aspect of the present invention, a method of producing a chemically modified mutant protein is provided comprising the steps of: (a) providing a s precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto. Preferably, the glycosylated thiosulfonate is an o alkylthiosulfonate, most preferably, a methanethiosulfonate. Also preferably, the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein. The amino acid residues to be substituted with cysteine residues according to the present invention may be replaced using site-directed mutagenesis methods s or other methods well known in the art. See, for example, PCT Publication No. WO 95/10615, which is hereby incorporated by reference.

[0057] The present invention also relates to glycosylated thiosulfonate. Preferably, the glycosylated thiosulfonate comprises methanethiosulfonate. More preferably, the methanethiosulfonate comprises the chemical structure:

O

Il

H ₃ C-S-SR Il 0 ° where R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac) ₂ , -β-Glc(Ac) ₃ , -β-Glc(Ac) ₄ , -Et-O-GIc(Ac) ₂ , -Et-O-GIc(Ac) ₃ , -Et-O-GIc(Ac) ₄ , -Et-β-Glc(Ac) ₂ , -Et-β-Glc(Ac) ₃ , -Et-P-GIc(Ac) ₄ , -Et-α-Man(Ac) ₃ , -Et-α-Man(Ac) ₄ , -Et-β-Gal(Ac) ₃ , -Et-β-Gal(Ac) ₄ , -Et-LaC(Ac) ₅ , -Et-Lac(Ac) ₆₎ -Et-LaC(Ac) ₇ . s [0058] Another aspect of the present invention is a method of modifying the functional characteristics of a protein including providing a protein and reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein.

[0059] The functional characteristics of a protein which may be altered by the o present invention include, but are not limited to, enzymatic activity, the effect on a human or animal body, the ability to act as a vaccine, the tertiary structure (i.e., how the protein folds),

whether it is allergenic, its solubility, its signaling effects, its biological activity and its pharmacological activity (Paulson, "Glycoproteins: What are the sugar chains for?" Trends in Biochem. Sciences, 14:272-276 (1989), which is hereby incorporated by reference). The use of glycosylated thiosulfonates as thiol-specific modifying reagents in a method of the present s invention allows virtually unlimited alterations of protein residues. In addition, this method allows the production of pure glycoproteins with predetermined and unique structures and therefore, unique functional characteristics, with control over both the site and level of glycosylation.

[0060] In particular, the methods of modifying the functional characteristics of a O protein allow the preparation of single glycoforms through regio- and glycan-specific protein glycosylation at predetermined sites. Such advantages provide an array of options with respect to modification of protein properties which did not exist in the prior art. The ability to produce proteins having very specific and predictable glycosylation patterns enables production of proteins that have known and quantifiable effects in chemical, pharmaceutical, s immunological, or catalytic performance. For example, with knowledge of a specific problematic epitope, it is possible to construct a modified protein according to the present invention in which the epitope is masked by a carbohydrate moiety, thus reducing its allergenic or immunogenic response in a subject. As another example, where the solubility of a protein is problematic in terms of recovery or formulation in a pharmaceutical or industrial o application, it is possible, utilizing the present invention, to produce a protein that has altered solubility profiles thus producing a more desirable protein product. As another example, if a protein has a particular problem of being proteolytically unstable in the environment in which it is to be used, then it is possible to mask the proteolytic cleavage sites in the protein using the present invention to cover up such sites with a carbohydrate moiety. These examples are s merely a few of the many applications of the present invention to produce improved proteins.

[0061] Accordingly, the present invention provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein. As used herein, glycosylation pattern means o the composition of a carbohydrate moiety. The present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins. The first method includes providing first and second chemically modified mutant proteins of the

present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second s chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins. The second method involves providing a first and second chemically modified mutant protein of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting said cysteine residue with a glycosylated thiosulfonate evaluating a functional characteristic O of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.

[0062] The chemically modified mutant proteins of the present invention provide a valuable source of carbohydrate-protein conjugates. Moreover, the methods of the present S invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships ("SARs")) of non-natural variants of the proteins.

[0063] The protein of the invention may be any protein for which a modification o of the glycosylation pattern thereof may be desirable. For example, proteins which are naturally not glycosylated may be glycosylated via the invention. Similarly, proteins which exist in a naturally glycosylated form may be modified so that the glycosylation pattern confers improved or desirable properties to the protein. Specifically, proteins useful in the present invention are those in which glycosylation plays a role in functional characteristics s such as, for example, biological activity, chemical activity, pharmacological activity, or immunological activity.

[0064] Glycosylated proteins as referred to herein means moieties having carbohydrate components which are present on proteins, peptides, or amino acids. In the present invention, the glycosylation is provided, for example, as a result of reaction of the o glycosylated thiosulfonate with the thiol hydrogen of a cysteine residue thereby producing an amino acid residue which has bound thereto the carbohydrate component present on the glycosylated tliiosulfonate. Glycosylation also may be accomplished, according to the

present invention, by attachment of glycodendrimer reagents such as those described in the examples below. Such reagents comprise one or more dendrimer core portions, optionally a linker (or tether), and one or more carbohydrate moieties.

[0065] The invention provides for synthetic schemes for producing s glycodendrimer reagents. Said schemes include normal addition schemes in which a carbohydrate alkylthiosulfonate is reacted with a dendrimer core, said core comprising a free sulfhydryl group. Also included are inverse addition synthesis schemes in which a thioglycose is reacted with a dendrimer core alkylthiosulfonate. Further included are synthesis schemes for producing novel carbohydrate alkylthiosulfonates, including direct o linked and tethered carbohydrate alkylthiosulfonates. A synthesis scheme involves reacting a carbohydrate with an alkylthiosulfonate and a phase transfer catalyst under refluxing toluene conditions. In a specific scheme, the alkylthiosulfonate is a sodium salt of methanethiosulfonate, and the phase transfer catalyst is tetrabutylammonium iodide (Bu ₄ NI).

[0066] In one embodiment, the protein is an enzyme. The term "enzyme" s includes proteins that are capable of catalyzing chemical changes in other substances without being changed themselves. The enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include pullulanases, proteases, cellulases, amylases, isomerases, lipases, oxidases, and reductases. Preferably, the enzyme is a protease. The enzyme can be a wild-type or mutant protease. Wild-type proteases can be isolated 0 from, for example, Bacillus lentus or Bacillus amyloliquefaciens (also referred to as BPN'). Mutant proteases can be made according to the teachings of, for example, PCT Publication Nos. WO 95/10615 and WO 91/06637, which are hereby incorporated by reference. Functional characteristics of enzymes which are suitable for modification according to the present invention include, for example, enzymatic activity, solubility, partitioning, cell-cell s signaling, substrate specificity, substrate binding, stability to temperature and reagents, ability to mask an antigenic site, physiological functions, and pharmaceutical functions (Paulson, "Glycoproteins: What are the Sugar Chains For?" Trends in Biochem Sciences, 14:272-276 (1989), which is hereby incorporated by reference.)

[0067] In another embodiment the protein is modified so that a non-cysteine o residue is substituted with a cysteine residue, such as by recombinant means. For example, the amino acids replaced in the protein by cysteine are selected from the group consisting of

asparagine, leucine or serine. Orthogonal protection schemes that are well known in the art may be used when modification is to be carried out at more than one site within a protein.

[0068] The terms "thiol side chain group," "thiol containing group," and "thiol side chain" are terms which can be used interchangeably and include groups that are used to s replace the thiol hydrogen of a cysteine. In certain embodiments, the cysteine occurs in the native protein sequence, while in other embodiments, a cysteine replaces one or more amino acids in the protein. Commonly, the thiol side chain group includes a sulfur through which the thiol side chain groups defined above are attached to the thiol sulfur of the cysteine.

[0069] The glycosylated thiosulfonates of the invention are those which are O capable of reacting with a thiol hydrogen of a cysteine to produce a glycosylated amino acid residue. By glycosylated is meant that the thiosulfonate has bound thereto a sugar or carbohydrate moiety that can be transferred to a protein or dendrimer (which may be bound to a protein) pursuant to the present invention. Preferably, the glycosylated thiosulfonates are glycosylated alkylthiosulfonates, most preferably, glycosylated methanethiosulfonates. Such s glycosylated methanethiosulfonates have the general formula:

O Il

H ₃ C-S-SR Il O

In specific embodiments, the methanethiosulfonate R group comprises: -β-Glc, -Et-β-Gal, - Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac) ₂ , -β-Glc(Ac) ₃ , -β-Glc(Ac) ₄ , -Et-α- GIc(Ac) _2, -Et-OC-GIc(Ac) ₃ , -Et-OC-GIc(Ac) ₄ , -Et-β-Glc(Ac) ₂ , -Et-β-Glc(Ac) ₃ , -Et-β-Glc(Ac) ₄ , - 0 Et-α-Man(Ac) ₃ , -Et- α-Man( Ac) ₄ , -Et-β-Gal(Ac) ₃ , -Et-β-Gal(Ac) ₄ , -Et-Lac(Ac) _5, -Et- LaC(Ac) ₆ , -Et-Lac(Ac) ₇ .

[0070] In an embodiment, the carbohydrate moiety of the present invention is a dendrimer moiety. Multiple functionalization of chemically modified mutant proteins can be achieved by dendrimer approaches, whereby multiple-branched linking structures can be s employed to create poly-functionalized chemically modified mutant proteins.

[0071] Dendrimer moieties of the present invention may contain one or more branch points. As used in this specification, the term "first generation" refers to dendrimer moieties that contain a single branch point. The term "second generation" refers to a dendrimer moiety that contains multiple branch points. The number of branch points in a 0 second generation dendrimer will depend on the number of arms that can be attached to the central core of the dendrimer building block. In general, the number of branch points for a

dendrimer comprising central cores having the same number of arms is equal to

G

^ (N - I) ⁰ ^ where N equals the number of arms and G is the generation number. For

G=I example, the TREN-type dendrimers such as those illustrated in Fig. 1 have three arms attached to the central TREN core. Thus, N = 3, and the number of branch points for a first generation Tren-type structure = 1, for a second generation structure = 3, for a third generation structure = 7, etc. Similarly, for the Penta-E type dendrimers, such as those illustrated in Fig. 1, N = 4, and the number of branch points for a first generation Penta-E type structure =1, for a second generation structure = 4, for a third generation structure = 13, etc. [0072] Highly branched molecules or dendrimers first were synthesized by Vδgtle in 1978 (Buhleier et al., Synthesis, 155-158 (1978), which is hereby incorporated by reference). The attachment of identical building blocks that contain branching sites to a central core may be achieved with a high degree of homogeneity and control. Each branch contains a functional group which, after chemical alteration, may be connected to yet another branching building block. In this manner, layer after layer of branching rapidly generates highly-functionalized molecules.

[0073] For instance, multiple glycosylation, including multiple mannose- containing chemically modified mutant proteins, and varied sugar moieties can be created. The dendrimer reagent structures would include methanethiosulfonates with simple branching such as:

derived from pentaerythritol (i.e., "Penta-E"), to very complex branched dendrimer reagents (see Figure 1). Li particular, a first generation glycodendrimer reagent is synthesized as shown in Figure 12, Scheme 6. This approach can be extended to cover larger dendrimers. More specifically, by leaving one "arm" of the glycodendrimer free for conversion to a methanethiosulfonate, the remaining arms can be further branched to synthesize highly- functionalized glycodendrimer reagents as shown in Figure 14, Scheme 8.

[0074] In another aspect, the present invention relates to a method for producing a glycodendrimer, comprising the steps of providing at least a first dendrimer core building block, at least one alkylthiosulfonate, and at least one carbohydrate having a first sulfhydryl

group; reacting the first dendrimer core building block with the alkylthiosulfonate to produce a modified dendrimer core building block having an alkylthiosulfonate group; reacting the resultant modified dendrimer core building block with the carbohydrate to produce a glycodendrimer. s [0075] The carbohydrate is preferably a disaccharide, and may optionally be a monosaccharide or a polysaccharide. In certain embodiments, the alkylthiosulfonate is methanethiosulfonate. In other embodiments, the alkylthiosulfonate may be an ethanethiosulfonate, a propanethiosulfonate, or a C ₁ -C ₆ alkylthiosulfonate. In further embodiments, the methanethiosulfonate is a salt, such as alkali metal salt, e.g., lithium, o sodium, or potassium salt, or an alkali earth metal salt, e.g., magnesium or calcium salt.

[0076] hi some embodiments, the first dendrimer core building block is tris(2- aminoethyl)amine (TREN). In other embodiments, the first dendrimer core building block is pentaerythritol. hi yet other embodiments, the first dendrimer core building block is mesitylene or mesitylene tribromide. In some embodiments, the carbohydrate is directly s linked to the first sulfhydryl group, hi other embodiments, the carbohydrate is indirectly linked or, alternatively, tethered to the first sulfhydryl group.

[0077] hi some embodiments, the disaccharide is selected from the group consisting of Gal-Gal, Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and Glu-Man, and acetyl or benzoyl derivatives thereof, hi certain other embodiments, the polysaccharide is selected 0 from the group consisting of Gal-Lac, Glu-Lac, Man-Lac, and Lac-Lac, and acetyl or benzoyl derivatives thereof. "Gal" refers to the saccharide unit galactose. "GIu" refers to the saccharide unit glucose. "Man" refers to the saccharide unit manose. "Lac" refers to the disaccharide unit lactose, hi the above disaccharides, either the first or the second, or both, saccharide units may be linked to the dendrimer core. Further, the terms "Gal," "GIu," s "Man," and "Lac" are meant to encompass saccharide units that are mono-, di-, tri-, tetra-, or otherwise polysubstituted. Such substitution is preferably with acetyl or benzoyl groups.

[0078] hi some embodiments, the glycodendrimer produced by the above method is a first-generation glycodendrimer. hi further embodiments, the first-generation glycodendrimer comprises an alkylthiosulfonate group, hi some of these embodiments, the O alkylthiosulfonate group is methanethiosulfonate.

[0079] In additional embodiments, the above method for producing a glycodendrimer further comprises a step of reacting the modified dendrimer core building

block having an alkylthiosulfonate group with a second dendrimer core building block having a second sulfhydryl group to produce a multi-generation glycodendrimer. In some of these embodiments, the multi-generation glycodendrimer is a second-generation glycodendrimer.

[0080] In another aspect, the method for producing a glycodendrimer may s comprise the steps of providing a first dendrimer core building block and a carbohydrate having a first sulfhydryl group; and reacting said dendrimer core building block with said carbohydrate to produce a glycodendrimer; where the carbohydrate is a disaccharide.

[0081] The disaccharide may be any disaccharide, and is preferably selected from the group consisting of 4-O-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-2,3,6-tri -O- o benzoyl- 1-thio-β-D-galactopyranoside, Gal-Gal, Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and

Glu-Man, and acetyl or benzoyl derivatives thereof. In further embodiments, the dendrimer core building block is selected from the group consisting of TREN, TREN-Boc, TREN-Boc-

Ac(Cl) ₂ , CbZ ₂ -TREN, CbZ ₂ -TREN-B oc, and Cbz ₂ -TREN-CO-(CH ₂ ) ₄ -SH. In certain embodiments, the glycodendrimer is selected from the group consisting of TREN-Boc- s (SGaI) ₂ , TREN-(SGaI) ₂ , MTS-(CH ₂ ) ₄ -CO-TREN-(SGal) ₂ , (2,3,4,6-tetra-O-acetyl-

Galα(l,4)2,3,6-tri-0-benzoyl-GalβS) ₂ TREN-Boc, (Galα(l,4)GalβS) ₂ TREN-Boc,

(Galα(l,4)GalβS) ₂ TREN, and (GaIa(1, 4)GalβS) ₂ TREN-CO(CH ₂ ) ₄ MTS.

[0082] > Disclosed herein is a compound of the following formula

0 where

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ are each independently and optionally selected from the group consisting of hydrogen, alkyl, alkylacyl (alkyl-C(O)-), arylacyl (aryl-C(O)-) and arylalkylacyl (arylalkyl-C(O)-); and

R ₈ is selected from the group consisting of hydrogen, alkali metal, alkali earth metal, s aluminum, organic cation, and inorganic cation.

[0083] Disclosed herein is a compound selected from the group consisting of the following structures:

and esters and salts thereof.

[0084] Disclosed herein is a glycodendrimer reagent composition having the generic structure:

wherein a, b, c, and d are each independently selected from the group consisting of all integers from O to 10, inclusive, such as, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10,

X can be either SR or R, wherein R is a disaccharide, substituted disaccharide, polysaccharide, or substituted polysaccharide selected from the group consisting of Gal-Gal, Glu-Glu, Man-Man, Lac-Lac, Gal-Glu, Gal-Man, Gal-Lac, Glu-Man, and Glu-Lac.

[0085] X can be

and esters and salts thereof. Alternatively, X can be

and esters and salts thereof.

[0086] Additionally disclosed is a compound where a = 1, b =0, c = 1, and d = 1. Alternatively, disclosed is a compound where a = 3, b =0, c = 1, d = 1, and X is either

[0087] By way of example to illustrate some of the advantages of the methods and compounds disclosed, the following discussion focuses on certain proteases which are modified according to the methods of the present invention. Alkaline serine proteases (for example, subtilisins) are increasingly used in biocatalysis, particularly in chiral resolution, regioselective acylation of polyfunctional compounds, peptide coupling, and glycopeptide synthesis. As shown in Figure 5 of United States Patent Application Serial No. 09/347,029, subtilisins can be used to catalyze peptide bond formation. From an ester substrate, subtilisins can form an acyl enzyme intermediate which then reacts with a primary amine to form a peptide product. This reaction requires high esterase activity to promote acyl enzyme formation and low amidase activity to minimize hydrolysis of the peptide bond of the desired product. Generally, subtilisins do not meet these requirements. Nonetheless, improved

esterase to amidase selectivities are desired in the art. By using the methods described herein, it is now possible to produce subtilisins that have advantageous properties.

[0088] Site specific mutagenesis is described and used to modify certain residues and introduce additional cysteine residues within subtilisin. The mutated subtilisin then s serves to react with a glycosylated methanethiosulfonate or glycodendrimer reagent to produce a glycosylation point at the introduced cysteine. Bacillus lentus subtilisin is selected for illustrated purposes only, as it does not contain a natural cysteine and is not naturally glycosylated.

[0089] The substrate binding site of an enzyme consists of a series of subsites O across the surface of the enzyme. The portion of substrate that corresponds to the subsites are labeled P and the subsites are labeled S. By convention, the subsites are labeled S ₁ , S ₂ , S ₃ , Si', S ₂ ', etc. A discussion of subsites can be found in Berger et al., Phil Trans. Royl Soc. Lond. B. 257:249-264 (1970), Siezen et al., Protein Engineering, 4:719-737 (1991), and Fersht, Enzyme Structure and Mechanism, 2 ed., Freeman: New York, 29-30 (1985), which s were hereby incorporated by reference.

[0090] In the present illustration, any of the S ₁ , S ₁ ', or S ₂ subsites is selected as a suitable target for modification. In particular, the amino acid residues corresponding to N62, L217, S156, and!si66 in naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as Bacillus lentus subtilisin were 0 selected for modification to cysteine.

[0091] An amino acid residue of an enzyme is equivalent to a residue of a referenced enzyme (e.g., B. amyloliquefaciens subtilisin) if it is either homologous (corresponds in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (has the same or similar s functional capacity to combine, react, or interact chemically).

[0092] To establish homology to primary structure, the amino acid sequence of the subject enzyme (for example, a serine hydrolase, cysteine protease, aspartyl protease, metalloprotease, etc.) is directly compared to a reference enzyme (for example, B. amyloliquefaciens subtilisin in the case of a subtilisin type serine protease) primary sequence o and particularly to a set of residues known to be invariant in all enzymes of that family (e.g. subtilisins) for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the

elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of the reference enzyme (e.g., B. omyloliquefaciens subtilisin) are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of at least approximately 75% or at s least approximately 50% of conserved residues are also adequate to define equivalent residues. Conservation of a catalytic triad, (for example, Asp32/His64/Ser221) is preferably maintained for serine hydrolases.

[0093] The conserved residues may be used to define the corresponding amino acid residues in other related enzymes. For example, the two sequences (one a "reference" O and the other a "target") are aligned in order to produce the maximum homology of conserved residues. There may be a number of insertions and deletions in the "target" sequence as compared to the "reference" sequence. Thus, for example, a number of deletions are seen in the thermitase sequence as compared to B. amyloliquefaciens subtilisin (see, e.g. U.S. Patent 5,972,682). Thus, the corresponding amino acid of Tyr217 in B. s amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217 in Figure 5B-2 of the 5,972,682 patent.

[0094] The particular "corresponding" residues may be substituted by a different amino acid to produce a mutant carbonyl hydrolase because they the correspond in primary structures.

20 [0095] Corresponding residues may also be determined as homologous or corresponding at the level of tertiary structure for a particular enzyme. Tertiary structure may be determined by for example x-ray crystallography or other known techniques. Corresponding residues may be defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the reference sequence

?s (e.g., B. amyloliquefaciens subtilisin) and the sequence in question (target sequence) (N on N, CA on CA, C on C, and O on O) are within 0.2, 0.15, or 0.13 nm and preferably approximately 0.1 nm after alignment. Alignment may be achieved after the model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the enzyme in question to the reference sequence. The best model is the w crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

[0096] Corresponding residues may also be determined as functionally analogous to a specific residue of a reference sequence. In this embodiments, corresponding residues are defined as those amino acid residues, which may adopt a conformation such that they will

JT alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the reference sequence as described herein. Further, corresponding residues are those residues of the sequence (typically for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the main chain atoms of a given residue. Typically, the atomic coordinates of at io least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of the reference sequence residue(s). The three dimensional structures are to be aligned as described above. For an illustration of such an alignment procedure see U.S. Patent 5,972,682, which is incorporated by reference herein in its entirety.

[0097] The mutated subtilisins were produced through standard site directed

/s mutagenesis techniques and the obtained mutant subtilisin was reacted with certain glycosylated alkylthiosulfonates, particularly glycosylated methanethiosulfonates, as provided in the examples appended hereto.

[0098] Proteins or other compositions obtained using the methods described herein may be used in any application in which it is desired to use such proteins, especially w those in which having modified functional capabilities is advantageous. Proteins modified as provided herein may be used in the medical field for pharmaceutical compositions and in diagnostic preparations. Additionally, proteins, such as enzymes, that are modified as described herein may be used in applications which generally are known for such enzymes including industrial applications such as cleaning products, textile processing, feed

25 modification, food modification, brewing of grain beverages, starch processing, as antimicrobials, health care formulations and in personal care formulations. Moreover, the unique functionalities made possible by the methods described herein may result in new and unforeseen uses for proteins which have not heretofore been recognized.

W

EXAMPLES

Example 1: Synthesis and Characterization of Carbohydrate Methanethiosulfonate Reagents

[0099] Cationic carbohydrate-methanethiosulfonates have been prepared s previously, as described in United States Patent Application Serial No. 09/347,029 "Chemically Modified Proteins with a Carbohydrate Moiety," the entire disclosure of which is hereby incorporated by reference in its entirety. The following examples are those that have been prepared as novel compounds for use in glycodendriproteins.

[0100] The sugar-MTS reagents required for attachment to the tips of the O dendrimer were prepared. 2-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)ethyl methanethiosulfonate 3 was prepared as previously described (Fig. 6, Scheme 1). (Davis, B. G.; Maughan, M. A. T.; Green, M. P.; Ullman, A.; Jones, J. B. Tetrahedron Asymm. 2000, 11, 245).

[0101] The corresponding directly linked methanethiosulfonate 5β was unknown s and a synthesis needed to be developed. DMF has been used with limited success for the synthesis of other directly linked methanethiosulfonates. These reaction conditions were tried for the reaction of 1 with sodium methanethiosulfonate. (Fig. 6, Scheme T). A nicely crystalline product was obtained in high yield, but unfortunately, it was the acetyl migration, hydrolysis product 2. This is a known compound and was identified by comparison with the 0 literature. Chittenden, G. J. F. Carbohydr. Res. 1988, 183, 140.

[0102] The reaction of 1 with sodium methanethiosulfonate was attempted in refluxing toluene. Very little reaction occurs but all reagents appear to be stable under these conditions. A phase transfer catalyst (tetrabutylammonium iodide) was added to increase the solubility of the methanethiosulfonate salt. This gave 5β in 67% yield after chromatography s (Fig. 6, Scheme 2). Tetrabutylammonium salts of thiols have previously been used to synthesize α-thiogalactosides from the β-chloride. Blanc-Muesser, M.; Vigne, L.; Driguez, H. Tetrahedron Lett. 1990, 31 (27), 3869. There was initial uncertainty over the anomeric configuration of MTS reagent 5β. Cl-Hl NMR coupling suggested that the product was alpha anomeric stereochemistry ( ¹ Z _1CH = 165 Hz), (Bock, K.; Pedersen, C. /. Chem. Soc, O Perkin Trans. 1 1974, 293) but X-ray crystallography determined the absolute configuration to be the β-anomer. There are two molecules in the unit cell (Fig. 8). Details of the X-ray structure are given in the experimental section.

[0103] MTS reagent 3 could also be prepared from 2 using this tetrabutylammonium method, but the yields and rate were no improvement on that described in Scheme 1. This method was also used to prepare the directly linked glucofuranosyl MTS reagent 8 (Fig. 6, Scheme 3). The product was an inseparable anomeric mixture. No further s effort has yet been made to obtain pure 8α or 8β. Glucofuranose 6 is the first readily available crystalline peracylated glucofuranose. Furneaux, R.H.; Rendle, P.M.; Sims, LM. J. Chem. Soc, Perkin Trans. 1 2000, 2011. Glucose generally occurs in the pyranose form and glucofuranoses are very rarely, if ever seen naturally. Synthesis of 8 would allow a route to the addition of a readily available, non-natural sugar to a protein or glycodendriprotein. O Others have been investigating the preparation of furanosyl donors for this purpose. Ferrieres, V.; Bertho, J.-N.; Plusquellec, D. Carbohydr. Res. 1998, 311, 25.

[0104] This method has also allowed the preparation of novel directly-linked mannose MTS reagents 10a and lOβ (Fig. 7, Scheme 4), whose identity was again confirmed by X-ray crystallography (Fig. 9). In addition it allowed the more efficient preparation of β- s gluco MTS reagent 12β (Fig. 7, Scheme 5).

Experimental

2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosγl methanethiosulfonate 5β

[0105] Acetobromogalactose 1 (1.1 g, 2.68 mmol) and sodium o methanethiosulfonate (0.45 g, 3.35 mmol) in toluene (50 mL) were concentrated in vacuo to approximately 30 mL to remove any water as the azeotrope. The mixture was made up to 50 mL with more toluene and again concentrated to 30 mL. A catalytic amount of tetrabutylammonium iodide was added and the mixture heated at reflux for 75 minutes. After cooling, Celite (to stop the formation of a salt cake on top of the column) was added and the s whole mixture loaded directly on to a flash silica column. Elution with 40% ethyl acetate in petroleum ether and recrystallization from petroleum ether/ethyl acetate gave the title compound (787 mg, 67%) as colorless prisms; mp 118-119°C (petroleum ether/ethyl acetate);

[α] ² _D ³ = + 8.5 (c 1.0, CHCl ₃ ); IR (KBr) 1753 (C=O), 1325, 1138 (S-SO ₂ ) crn ¹ ; ¹ H NMR (400

MHz, CDCl ₃ ) δ 1.99 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.17 (s, 3H, Ac), 3.43 (s, o 3H, CH ₃ SO ₂ -), 4.05 (ddd, / 7.4, 4.2, 0.9 Hz, IH), 4.08 (dd, J 18.3, 7.5 Hz, IH), 4.20 (dd, J

10.8, 4.3 Hz, IH), 5.13 (dtd, J 10.7, 7.6, 3.4 Hz, IH), 5.26 (s, IH), 5.27 (dd, / 14.8, 10.3 Hz,

IH), 5.48 (dd, / 3.4, 0.8 Hz, IH); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 20.5, 20.6, 20.6, 20.6 (4 x

CH ₃ CO), 52.7 (CH ₃ SO ₂ -), 61.8 (C-6), 65.8, 67.0, 71.3, 75.3, 87.0 ( ¹ J _1CH 165 Hz, C-I), 169.7, 169.7, 170.0, 170.2 (4 x C=O); HRMS m/z (ES): found 460.0951; C ₁₅ H ₂₆ NO ₁₁ S ₂ requires 460.0947.

2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosylmethanethiosulf onate IQq, and 2,3,4,6-Tetra-O- acetyl-β-D-mannopyranosylmethanethiosulfonate 10β

[0106] The title compounds were prepared using essentially the same method as described above. Minor modifications to this method were made so that the silica plug mixture was purified using Flash silica column with eluting solvent 70:30 Petroleum Ether : Ethyl Acetate, moving to 60 :40 Petroleum Ether : Ethyl Acetate. This separated α/β mixture from the remaining bromide but two anomers could not be separated on the column. This was achieved through several recrystallizations from petroleum ether/ Ethyl Acetate. The two anomers are recovered off the column in a 50:50 mixture. The β anomer crystallizes first. The α anomer eventually crystallizes to give pure crystals. Yield = 63%.

2,3A6-Tetra-O-acetyl-β-D-glucopyranosyl methanethiosulfonate 12β

[0107] 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (Ig, 2.44mmol) and sodium methanethiosulfonate (0.4g, 3.05mmol) were placed under nitrogen. 30ml of anhydrous toluene was added followed by tetrabutylammonium bromide (69mg, 0.21mmol) and the mixture heated under reflux for 75 minutes. Part way through 6ml of DMF were added as it seemed that 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide was insoluble in toluene. Reaction continued to reflux despite the fact that solution not formed. Thin Layer Chromatography ("TLC") at end of reaction showed reaction to have gone to completion. Solution reduced on high pressure rotary evaporator to remove DMF. Product purified on flash silica gel column, reaction mixture added directly onto column (adding a small amount of Celite to reaction mixture sufficient to avoid salt cake forming on top of column.) Petroleum Ether: Ethyl Acetate 60:40 was used as the eluting solvent. Yield = 75%.

Example 2: TREN-based Glycodendrimer Synthesis Introduction [0108] Lectins, selectins, integrins and members of other adhesion receptor families are sugar binding proteins. The sugar binding sites are relatively shallow and hence binding is comparatively weak. Lectins, selectins, integrins and members of other adhesion receptor families, however often bind many saccharides of an oligosaccharide to give a strong, selective affinity between the lectin, selectin, integrin or members of another adhesion receptor family and a particular combination of saccharides. This is illustrated in Figure 3. Briefly, the aim is to attach many sugars to the surface of a dendrimeric structure that is in turn attached to a protein to mimic the natural system. The model protein we used is

subtilisin Bacillus lentus (SBL), a serine protease enzyme. If the glycodendrimer system synthesized has a strong affinity to the lectin, selectin, integrin or members of another adhesion receptor family being targeted, then the attached. SBL (being a protease) should start 'cutting' up the lectin, selectin, integrin or members of another adhesion receptor family. s This is shown schematically in Figure 10. The specific model glycodendrimer that is the initial synthetic target of this project is shown in Figure 11. SBL has no natural cysteines (and hence no thiols present). One can be introduced by way of site-directed mutagenesis. Methane thiosulfonate (MTS) reagents react specifically and quantitatively with thiols (Wynn, R.; Richards, F.M. Methods Enzymol 1995, 201, 351) giving an excellent method for O the attachment of the glycodendrimer to the protein.

Normal Addition

[0109] The following first-generation glycodendrimer reagents were prepared according to the methods described in Davis, B.G., "The controlled glycosylation of a protein s with a bivalent glycan: towards a new class of glycoconjugates, glycodendriproteins," Chem. Commun., 2001, 351-352, the entire disclosure of which is incorporated by reference. Fig. 12, Scheme 6.

[0110] Two different representative bivalent branched glycan MTS reagents, 7'a and 7'b, based on a trivalent tris(2-aminoethyl)amine (TREN) core were synthesized (Fig. 0 12, Scheme 6). 7'a bears at the end of its two glycan branches the same untethered peracetylglucose unit that had previously allowed dramatic enhancement of enzyme activity. Lloyd, R.C., Davis, B.G., and Jones, J.B., Bioorg. Med. Chem., 2000, 8, 1537. 7'b bears ethyl-tethered mannose moieties that had been used in the construction of previous glycoproteins mat had shown low levels of lectin binding. Davis, B. G., Hodgson, D., s Ullman, A., K. Khumtaveeporn, SaIa, R., Bott, R.R., and Jones, J.B. unpublished work. Lectin binding led to enhanced selectivity in the degradation of a mannose specific lectin by subtilisin bacillus lentus ("SBL") glycosylated with a single mannose residue. These two reagents therefore allow the introduction of multivalent, tethered or untethered, glycans with α or β anomeric stereochemistry from different parent carbohydrate systems. 0 [0111] After differentiation of one of the two amine termini of TREN 1 through selective protection as its mono-Boc derivative (Tecilla, P., Tonellato, U., Veronese, A., Felluga, F., and Scrimin, P., J. Org. Chem., 1997, 72, 7261), the two remaining free amine

teπnini were reacted with chloroacetic anhydride to give the corresponding bis-α- chloroamide. Treatment of this branched dichloride with the potassium salt of thioacetic acid gave the bis-thioester 2 in a good overall yield (58% over 3 steps from 1). One-pot selective deprotection and glycosylations of 2 were achieved by treatment with dilute aqueous NaOH solution to hydrolyze the labile thioacetates and then appropriate modification of the free thiol groups produced with the appropriate untethered β-gluco 3a or tethered α-manno 3b methanethiosulfonate reagents to yield the corresponding bivalent branched glycans 6'a or 6'b in 73% and 62% yield, respectively. It should be noted that the use of a basic TREN-core as a scaffold allowed the scavenging of 6'a,b from reaction mixtures using acidic ion exchange resin and therefore greatly simplified their purification. With the ability to introduce two distinct glycan endgroups a or b thus suitably demonstrated, 7 'a was deprotected through treatment with CF ₃ COOH and the free amine produced converted to the corresponding α-chloroamide. Displacement of the α-chloro group through treatment with NaSSO ₂ CH ₃ in DMF at 50°C proceeded smoothly and yielded the target bis-glycan MTS 7'a in good yield (52% over 3 steps from 6'a).

[0112] Modified syntheses of a first-generation and a second generation (and in a similar manner, multi-generation) glycodendrimers and their subsequent attachments to thiol- containing amino acid side-chains to form the corresponding glycodendriproteins are outlined in Scheme 7 (Fig. 13) and Scheme 8 (Fig. 14), respectively. [0113] The initial dendrimeric core building block was prepared from tris(2- aminoethyl)amine (TREN) 1 using literature methodology (Tecilla, P.; Tonellato, U.; Veronese, A. J. Org. Chem. 1997, 62, 7621) and that described in Scheme 7 (Fig. 13). Excess 1 is reacted with BoC ₂ O to selectively give the mono-Boc protected TREN. After chromatographic purification, the remaining amines were protected with chloroacetates and the chlorines displaced with thioacetates to give 2. The acetates were deprotected under mild basic conditions to give the dithiol 3. It had previously been noted that this product is slowly oxidized to the disulfide 4 on exposure to air making purification and subsequent use of 3 problematic. The dithiol was therefore deliberately oxidized directly to 4 by the addition of iodine. This gave a product that was much more amenable to storage and purification by chromatography.

[0114] The next step is the attachment of the sugars to the dendrimeric core. Because of the stability problems of the dithiol 3, many attempts were made to generate it in

situ. This type of coupling is one of the most important reactions in building of multi- generation glycodendriproteins (Scheme 8) and hence an elegant, high yielding reaction would be very useful. These attempts to generate the dithiol 3 in situ took two forms, either (a) reducing the disulfide 4 or (b) deprotecting the diacetate 2. s [0115] The problems associated with the former method are that only one equivalent of reducing reagent must be used to stop unwanted reduction of the product. In addition, the presence of the oxidized reductant may disrupt the coupling reaction. Ideally the latter method would utilize a base that was basic enough to deprotect the S-acetates but not basic enough to cleave the 0-acetates. o [0116] In both cases, care must be taken to avoid the oxidation of the dithiol 3 before it can couple with the MTS reagent. This includes 'degassing' the solvents to reduce the amount of oxygen present.

Reduction of disulfide 4 s [0117] Two methods were used to prepare dithiol 3 in situ by reduction. Disulfide

4 was treated with one equivalent of the 'organic' reductant, tributyl phosphine, and the resulting solution added dropwise to the MTS reagent in the presence of mild base. No coupling was observed by TLC and no coupled product isolated by chromatography. Some of the diglycosyl disulfide 18 or 19 (c.f. β-Gal-SS-β-Gal in: Kiefel, MJ.; Thomson, RJ.; o Radovanovic, M.; von Itzstein, M. J. Carbohydr. Chem. 1999, 18, 937) (depending on the MTS reagent used, 12β or 5β) was isolated (Fig. 15). This implies that thiols had been present in the reaction. Attempts with the 'inorganic' reductant, sodium metabisulfite, were also unsuccessful.

s Deprotection of diacetate 2

[0118] The first attempt using this approach involved reacting the diacetate 2 directly with MTS reagent 5β in the presence of excess triethylamine. Triethylamine is required for the coupling of the dithiol 3 with MTS reagent 5β anyway, and the hypothesis was this base could also deacetylate 2 to give the dithiol 3 in situ. Precedent for this is given o in: Greene. T. W., Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed. John Wiley & Sons Inc., 1991, New York. This was however unsuccessful. This reaction was

repeated but using the stronger base, diisopropylethylamine which had been shown previously (by TLC) to deacetylate 2. Again, no coupling product was observed.

[0119] The next step was to then try more conventional deacetylation reagents. Most deacetylations involve the use of catalytic amounts of the methoxide anion in methanol. j- Deprotection of ROAc with MeO ^" gives RO ^" and AcOMe. The solvent then protonates the deprotected alkoxide to give ROH and regenerates the catalyst MeO ^" . However in the deprotection of alkyl thioacetates, thiols are more acidic than alcohols and so the equilibrium lies in favor of RS ^~ rather than MeO ^" . Hence, for deprotection to go to completion, an excess of alkoxide per thioacetate is required. To avoid deprotection (for ease of purification) of the O resulting coupled product, any excess base ideally should be neutralized before coupling.

[0120] One source of methoxide is the use of anhydrous methanol saturated with anhydrous ammonia. This has the advantage that after deprotection of the diacetate 2, excess base can just be removed by concentration of the solution in vacuo. Several attempts were made to couple 12β with dithiol 3 generated in this way (i.e., according to Scheme 7), s however no coupling product was observed. Eventually all the MTS reagent would end up as the disulfide 18 (Fig. 15). The stability of MTS reagents (in this case, 12β) to ammonia was examined. To a CDC1 ₃ /CD ₃ OD solution of 12β was added a drop of aqueous ammonia. ¹ H NMR spectra of this sample before and after ammonia addition gave different spectra, suggestion the formation of an activated sugar-S-NH ₂ type species. However concentrating 0 the solution in vacuo gave back the MTS reagent 12β. Electrospray mass spectrometry was carried out on this solution which showed [M+Na] ⁺ for 12β, 18 and an unknown peak at m/z 436.

[0121] Many attempts to deprotect diacetate 2 with other basic conditions (for example, aqueous NaOH in methanol or sodium methoxide in methanol) followed by s reaction with an MTS reagent (either directly or after neutralization, or after neutralization and isolation of dithiol 3) failed to give high coupling yields. For example, 10% coupling was observed from deprotection with 1.1 equivalents of sodium methoxide in methanol and then direct reaction with a MTS reagent.

[0122] The difficulty observed with this coupling could in part be due to the facile o nature of the intramolecular disulfide formation of disulfide 4 (Fig. 13, Scheme 7). The above results lead to the suggestion that the coupling could be carried out in the reverse direction, i.e., with the MTS reagent on the dendrimer core and the free thiol on the sugar.

See Inverse Addition section below and Scheme 9, Fig. 15. The oxidative side reaction of the thiols to give a disulfide would now be an intermolecular process and known to not be competitive with the MTS coupling reaction. The inverse addition strategy proved to be efficient and actually requires fewer synthetic steps than the normal addition coupling s described in Schemes 6, 7 and 8, above.

Inverse Addition

[0123] This new inverse-addition approach for the synthesis of first- and multi- generation glycodendriproteins is outlined in Scheme 9 (Fig. 15) and Scheme 10 (Fig. 16). It o is based on the realization that improved coupling efficiency between the carbohydrate moiety and the dendrimer core is obtained by adding the methanethiosulfonate moiety to the ends of the dendrimer core, and reacting the dendrimer core with a sulfhydryl-bearing carbohydrate moiety.

[0124] The dichloroacetyl 20 was prepared as outlined in Scheme 9. Initially, we S attempted synthesis of 21 through the reaction of 20 with sodium methanethiosulfonate. (Scheme 9) This approach, however, did not give an appreciable yield of bis-MTS reagent 21. The similar reaction involving 31 (Scheme 11, Fig. 17) was also problematic. Substitution of a chloride α to a carbonyl does not appear to be as facile as for other alkyl halides. This has lead to the investigation of inserting a longer alkyl chain between the halide o and the amide (discussed later).

[0125] Thioglycoses 23, 24 and 27 (see Scheme 12, Fig. 17) all are available commercially or may be readily prepared. For example 24 was prepared as described in Scheme 12 (Fig. 17). Another direct method to a first generation glycodendrimer can be seen in Scheme 13 (Fig. 17). The deprotected sodium salt of 1-thiogalactose 24 readily reacts with s the bis-halide 20 to give the bis-galactoside 27 which can then be deprotected to give the amine 28. Purification of these comparatively high polarity compounds allowed the synthesis of 28 in good overall yields. The amine 28 was chloroacetylated to give 29 (Scheme 11, Fig. 17). However, as mentioned above, subsequent reaction with sodium methanethiosulfonate to give a glycodendrimer MTS reagent did not result in appreciable yields of the expected o product. It was thought that increasing the distance between the halide and amide should solve this problem. Precedent for this is supplied by preparation of MTS reagent 3 in excellent yield from bromide 2 (Scheme 1, Fig. 6).

[0126] The reaction of 3-bromopropionoyl chloride with amine 28 on a NMR scale gave the required product 30 (Scheme 11, Fig. 17). This acylating reagent {i.e., 3- bromopropionoyl chloride ) was also reacted with TREN 1 (Scheme 14, Fig. 8) with the aim of preparing the tris-MTS reagent 34 (n = 2). Initial attempts to acylate TREN 1 with 3- s bromopropionoyl chloride were done in the presence of base. This however led to elimination products being observed. Elimination of HBr is favorable due to the production of a conjugated α,β-unsaturated system. Repeating this acylation in DMF without any base present gives the required product 33 (n = 2). The identity of this product was confirmed by spectroscopic methods and by reacting with sodium methanethiosulfonate to give 34 (n = 2). O [0127] To avoid the problem of HBr elimination, the homologue acylating reagent

(4-bromobutyryl chloride) has also been investigated. Acylation of amine 28 gave 31 (Scheme 11, Fig. 17), however subsequent reaction with sodium methanethiosulfonate only, gave baseline material by TLC (10% saturated aqueous ammonia in methanol). TLC of the starting material (R _f = 0.3 in this solvent system) showed that it had since decomposed to s baseline material.

[0128] Preparation of 36 (Scheme 14, Fig. 18) involved acylation of TREN-Boc with excess reagent 4-bromobutyryl chloride. Addition of base (triethylamine) was required to get the reaction to go to completion. It is assumed that the HCl produced was giving the hydrochloride salt of the unreacted amines, halting reaction. The major product appeared to 0 be 33 (n = 3), suggesting that the acid production had cause deprotection of the Boc group. Subsequent reaction with sodium methanethiosulfonate however gave a product of the form XCO(CH ₂ ) ₃ SSO ₂ CH ₃ (that is, no TREN component observed), refuting this notion.

[0129] As stated above, a terminal amine functionality on the dendrimer core was converted to a terminal thiol by chloroacetylation, substitution with thioacetate and then s deacetylation. An alternative method for this terminal conversion is described by Blixt and Norberg. Blixt, O.; Norberg, T. J. Org. Chan. 1998, 63, 2705-2710. These authors report reacting 2-aminoethyl 2-acetamide-2-deoxy-β-D~glucopyranoside with γ-thiobutyrolactone in the presence of aqueous base and DTT (to stop disulfide formation) to give the corresponding ring-opened thiol in 71% yield. Amine 30 (Scheme 15, Fig. 19) was treated in the same way 0 to give the expected thiol 34 in moderate yield. This is a useful product for the preparation of second-generation glycodendrimers (by reaction with bis-MTS reagents of type 21). 34 was then used in the synthesis of the di-Gal-TREN-MTS 39 via a nitrosylation reaction and

reaction with methanesulfinate (Scheme 15, Fig. 19). 34 was also used to synthesize- the tetra-Gal-TREN/araGal hybrid-MTS 41 (Scheme 15, Fig. 19).

TREN-Boc disulfide 4 s [0130] TREN-SAc 2 (100 mg, 0.209 mmol) was dissolved in methanol (4.5 mL) and 2 M aqueous NaOH (0.5 mL). After 20 minutes deprotection was complete (assayed by TLC) and so the mixture was neutralized with acetic acid oxidized with iodine (60 mg, 0.236 mmol). After 1 hour, the mixture was concentrated in vacuo and purified using flash silica column chromatography and eluted with 10% methanol in ethyl acetate to give 4 in 90% O yield. ¹ H NMR (300 MHz, CD ₃ OD) δ 1.44 (s, 9H, (CH ₃ ) ₃ ), 2.68-2.76 (m, 6H, CH ₂ N(CHa) ₂ ), 3.22 (t, J 6.1 Hz, 2H, CH ₂ NHBoc), 3.29-3.35 (m, 4H, CH ₂ NHCOCH ₂ SS), 3.61 (s, 4H, CH ₂ SS).

B is { N- f 2-( 1 -thio- β-d- galactopyranosyPethano yli aminoethyl } - { N-tert- s butylcarbamoylaminoethyl } amine 27

[0131] 1-Thio-β-D-galactopyranose, sodium salt (417 mg, 1.91 mmol) was added to bis[N-(2-chloroethanoyl)aminoethyl]-[N-tert-butylcarbamoylam inoethyl]amine 20 (332 mg, 0.83 mmol) in DMF (15 mL). The suspension was stirred at room temperature. After three hours, the thiosugar had dissolved and a fine white precipitate had formed. The mixture 0 was concentrated in vacuo and the residue purified by flash silica column chromatography, eluting with chloroform/methanol/sat. aq. ammonia (60:30:8) to give the title compound as a colorless foam (528 mg, 88%); [α] ² _D ² = -26.6 (c 1.0, H ₂ O); ¹ H NMR (500 MHz, D ₂ O) δ 1.28

(s, 9H, Boc), 2.74 (br s, 6H, N(CH ₂ CHa) ₃ ), 3.09 (br s, 2H, NCH ₂ CH ₂ NHBoC), 3.26 (br s, 4H, NCH ₂ CH ₂ NHCO), 3.27 (d, J 15.3 Hz, 2H, COCHITS), 3.40 (d, J 15.3 Hz, 2H, COCHH ^{^} S), s 3.43 (dd, J 9.6, 9.4 Hz, 2H, H2 ^V ), 3.49 (dd, J 9.4, 3.2 Hz, 2H, HT), 3.53-3.59 (m, 6H, H5\ H6 ^V ), 3.82 (d, J 2.9 Hz, 2H, H4 ^V ), 4.33 (d, J 9.6 Hz, 2H, Hr); ¹³ C NMR (125 MHz, D ₂ O) δ 27.8 (NHBoc), 33.3 (SCH ₂ CO), 36.9 (N(CH ₂ CH ₂ ) ₃ ), 52.6 (NCH ₂ CH ₂ NHBoC), 53.0 (NCH ₂ CH ₂ NHCOCH ₂ ), 61.2 (C6 ^V ), 68.9 (C4 ^V ), 69.6 (CX), 73.9 (C3 ^* ), 79.2 (C5 ^V ), 81.4 (Boc), 85.7 (CO, 158.3 (NHCOO), 173.0 (NHCOCH ₂ S). o

2-(Bis{N-r2-(l-thio-β-d-galactopvranosvl)ethanovnaminoet hyl}amino)ethvlamine 30

[0132] Bis {N-[2-( 1 -thio-β-D-galactopyranosyl)ethanoyl]aminoethyl } - {N-tert- butylcarbamoylaminoethyl} amine 29 (1.35 g, 1.88 mmol) was stirred in trifluoroacetic acid (12 mL) and water (12 mL). After one hour, the solution was concentrated in vacuo and the residue loaded on to a Dowex 50W2-200 (H ⁺ ) column in water/methanol (1:1). The column s was washed with 80 mL volumes of methanol, water/methanol (1:1) and water and then the product removed by eluting with 15% aqueous ammonia to give the title compound as a colourless foam (1.06 g, 91%); [α] ² _β ² = -28.0 (c 1.0, H ₂ O); ¹ H NMR (500 MHz, D ₂ O) δ

(COCH ₂ S peaks not seen due to deuterium exchange) 2.48 (t, J 6.7 Hz, 2H, NCH ₂ CH ₂ NH ₂ ), 2.53 (t, J 6.7 Hz, 4H, NCH ₂ CH ₂ NHCOCH ₂ S), 2.61 (t, / 6.7 Hz, 2H, NCH ₂ CH ₂ NH ₂ ), 3.17 (t, o J 6.7 Hz, 4H, NCH ₂ CH ₂ NHCOCH ₂ S), 3.41 (dd, / 9.7, 9.4 Hz, H2 ^V ), 3.48 (dd, J 9.4, 3.3 Hz, H3 ^V ), 3.51-3.59 (m, 6H, H5\ H6 ^K ), 3.19 (d, / 3.2 Hz, 2H, H4 ^V ), 4.32 (d, / 9.7 Hz, 2H, UV); ¹³ C NMR (125 MHz, D ₂ O) δ 32.8 (COCH ₂ S), 37.4 (NCH ₂ CH ₂ NH), 37.8 (NCH ₂ CH ₂ NH ₂ ), 52.3 (NCH ₂ CH ₂ NH), 54.6 (NCH ₂ CH ₂ NH ₂ ), 61.2 (C6 ^V ), 68.9 (C4 ^S ), 69.6 (C2 ^V ), 73.9 (C3 ^V ), 79.2 (C5 ^V ), 85.6 (CX), 172.7 (NHCOCH ₂ S); HRMS m/z (ES): found 619.2320; s C ₂₂ H ₄₃ N ₄ O ₁₂ S ₂ [M+H] requires 619.2319.

N-r2-(Bis{N-r2-(l-thio-β-d-galactopyranosyl)ethanoyl1ami noethyl}amino ^' )ethvn-4- mercaptobutyramide 34

[0133] 2-(Bis{N-[2-(l-thio-β-D-galactopyranosyl)ethanoyl]aminoethy l}amino) 0 ethylamine 30 (241 mg, 0.39 mmol) was dissolved in a NaHCO ₃ aqueous solution (0.5 molL ^" \ 10 mL) and ethanol (3 mL). Dithiothreitol (300 mg, 1.95 mmol) and γ-thiobutyrolactone (337 μL, 3.90 mmol) were added and the mixture heated under nitrogen overnight at 50 ⁰ C. The resulting mixture was neutralized with HCl (2 molL ^"1 ) and concentrated in vacuo. The residue was purified by flash silica column chromatography, eluting with s chloroform/methanol/water/triethylamine (60:35:7:1), to give the product contaminated with triethylammonium chloride. This was loaded on to a Dowex 50W2-200 (H ⁺ ) column in water, washed with water and then the product removed by eluting with 10% aqueous ammonia to give title compound (174 mg, 62%) as a colourless foam; [α] ¹⁶ = -27.8 (c 0.6,

H ₂ O); ¹ H NMR (500 MHz, D ₂ O) 5 1.74 (tt, / 7.1, 7.3 Hz, 2H, CH ₂ CH ₂ SH), 2.24 (t, / 7.3 Hz, o 2H, CH ₂ (CH ₂ ) ₂ SH), 2.41 (t, J 7.1 Hz, 2H, CH ₂ SH), 2.56-2.62 (m, 6H, NCH ₂ ), 3.17 (t, J 6.5

Hz, 2H, NCH ₂ CH ₂ NHCO(CH ₂ ) ₃ SH), 3.20 (t, J 6.7 Hz, 2H, NCH ₂ CH ₂ NHCOCH ₂ S), 3.28 (d,

J 15.4 Hz, 2H, COCHff S), 3.41 (d, J 15.4 Hz, 2H, COCTttTS), 3.45 (dd, J 9.6, 9.4 Hz, 2H, H2 ^V ), 3.51 (dd, J 9.4, 3.1 Hz, H3 ^V ), 3.54-3.63 (m, 6H, H5\ H6 ^V ), 3.84 (d, J 3.1 Hz, 2H, H4 ^S ), 4.35 (d, / 9.6 Hz, 2H, HT); ¹³ C NMR (125 MHz, D ₂ O) δ 23.3 (CH ₂ SH), 29.6 (CH ₂ CH ₂ SH), 33.3 (NHCOCH ₂ S), 34.6 (CH ₂ CH ₂ CH ₂ SH), 37.0 (CH ₂ NHCO(CH ₂ ) ₃ SH), 37.4 s (SCH ₂ CONHCH ₂ ), 52.2 (NCH ₂ CH ₂ NHCOCH ₂ S), 52.3 (NCH ₂ CH ₂ NHCO(CH ₂ ) ₃ SH), 61.2 (C6 ^s ) _t 68.9 (C4 ^V ), 69.6 (C2 ^V ), 74.0 (C3 ^V ), 79.2 (C5 ^V ), 85.6 (CV), 172.6 (NHCOCH ₂ S), 176.2 (NHCO(CH ₂ ) ₃ SH); HRMS m/z (ES): found 721.2459; C ₂₆ H ₄₉ N ₄ O ₁₃ S ₃ [M+H] requires 721.2458.

O Second- generation Ralactodendrimer MTS reaRent tetra-Gal-TREN/AraGalhybrid-MTS 41:

[0134] Tris(methanethiosulfonatoinethyl)mesitylene 40 (26 mg, 0.05 mmol) and triethylamine (15 μL, 0.10 mmol) were dissolved in DMF (20 mL) in an ice/salt bath. A solution of N-^2-fBis{iV-[2-(l-thio-β-D-galactopyranosyl)ethanoyl]amino ethyl}amino)ethyl]- 4-mercaptobutyramide 34 (75 mg, 0.10 mmol) in water (20 mL) was added dropwise over 2 s hours. The resulting solution was allowed to warm to room temperature, left over night and then concentrated in vacuo. ESMS of the residue gives a spectrum consistent with the presence of the title compound.

Methanethiosulfonic acid S-{3-r2-N-r2-(Bis{N-r2-(l-thio-β-d- 0 galactopyranosvDethanoyllammoethyll amino)ethylcarbamovHpropyl| ester 39

[0135] N-/2-(Bis{iV-[2-(l-thio-β-D- galactopyranosyl)ethanoyl]aminoethyl}amino)ethyl] -4-mercaptobutyramide 34 (108 mg, 0.15 mmol) was dissolved in 2 M HCl (4 mL) and cooled to 0°C. Sodium nitrite (10 mg, 0.15 mmol) in water (1 mL) was added. After the addition, the now red solution was left at s 0°C for 15 mins and then at 4°C for a further 90 mins. Methanesulfinic acid, sodium salt (31 mg, 0.30 mmol) in a water (2 mL) was added and the solution left at room temperature for 4 hours by which stage most of the red color had gone. The solution was carefully neutralized with aqueous NaOH and concentrated in vacuo. ESMS of the residue gives a spectrum consistent with the presence of the title compound. 0

Example 3 - ArGal-based Glycodendrimer Synthesis Improved Synthesis for NaMTS

[0136] An alternative preparation of NaMTS (J.D. Macke, L. Field, /. Org. Chem. 1988, 53, 396-402) has been successfully tested, which is faster and avoids the tedious and lengthy separation of by-product from NaMTS as required in the Na ₂ SZMe ₃ SiCl method. NaMTS was synthesized in high yield by refluxing sodium sulfinate with sulphur in methanol s (Scheme 16, Fig. 19), described in further detail below). Although formation of small amounts of an unknown by-product was observed, it could be easily separated from NaMTS.

Inverse Addition Synthesis of Glycodendrimer Reagent

[0137] Two synthetic approaches for building block 42 were undertaken (Scheme o Yl, Fig. 20). Slow addition of KSAc under high dilution resulted in a 9% yield of 42. Better results were obtained under phase transfer conditions with catalytic amounts of Bu ₄ NI as phase transfer catalyst and toluene as solvent. In this case 42 was isolated in 17% yield.

[0138] A short synthesis of MTS reagents with two deprotected sugars on has been developed (Scheme 18, Fig. 20). The bromide 43 was reacted with NaMTS to yield the s methanethiosulfonate 40 in moderate yield, which was then treated with 2 equivalents of the sodium salt of 1-thio-β-D-galactose to afford the desired diGal-ArGal-MTS reagent 44 in 23% yield. As before, the main product was the trisubstituted compound.

Modification of Cysteine-Containing Protein with Glycodendrimer Reagent 0 [0139] A cysteine-containing mutant of subtilisin Bacillus lentus, S156C, was modified with the glycodendrimer reagent glycoMTS 44 to give the glycodendriprotein S156C-Mes(SS-β-Gal) ₂ ("di-gal protease")(Scheme 21, Fig. 22).

Synthesis of Sodium methanethiosulfonate (NaMTS) (J.D. Macke, L. Field, J. Org. Chem. s 1988, 53, 396-402)

[0140] A mixture of sodium methanesulfinate (5.43 g, 53 mmol) and sulphur (1.666 g, 52 mmol) in dry methanol (310 ml) was heated to reflux for 20 min, at which time almost all of the sulphur had dissolved. The hot solution was filtered and the filtrate concentrated to dryness. The off-white solid was stirred with a small amount of dry ethanol 0 at room temperature, filtered and concentrated. The trituration was repeated until ¹ H NMR of the white residue showed no more traces of sodium methanethiosulfonate. The filtrates were then combined and evaporated to dryness to yield the title compound (5.40 g, 77%) as fine

white needles; mp 271-272 ⁰ C (lit.[G.L. Kenyon, T. W. Bruice, Methods Enzymol. 1977, 47, 407-430.] 272-273.5 ⁰ C); ¹ H NMR (200 MHz, CDCl ₃ ) δ 3.18 (s, 3H, CH ₃ ); anal, calculated, for CH ₃ NaO ₂ S ₂ : C 8.95, H 2.25; found: C 8.86, H 2.55.

s Synthesis of l,3-Bis(acetylsulfanylmetfaylV5-broniomethyl-2,4,6-trimethyl -benzene (42) Synthesis A — DMF, slow addition, high dilution

[0141] A solution of potassium thioacetate (0.354 g, 3 mmol) in dry DMF (35 ml) was added dropwise over a period of 6 h to a solution of mesitylene tribromide (0.612 g, 1.5 mmol) in dry DMF (45 ml). After the end of the addition, stirring was continued over night at O room temperature. The reaction mixture was diluted with water (50 ml) and extracted with CH ₂ Cl ₂ (4x50 ml). The combined organic phases were washed with brine, dried over MgSO ₄ , and the solvents removed. The remaining beige solid was separated by flash chromatography (SiO ₂ , hexane : EtOAc, gradient elution, 10:1 to 5:1) to afford 3 products: 1- acetylsulfanylmethyl-3,5-bis(bromomethyl)-2,4,6-trimethyl-be nzene (0.032 g, 5%) as a white s solid, 42 (0.051 g, 9%) as a white solid, and tris-(acetylsulfanylmethyl)-mesitylene (0.215 g, 56%) as a white solid; analytical data for mono-SAc: MS m/z (EI+) 396 (7%), 394 (M ⁺ , 12%), 392 (6%), 315 (100%), 313 (97%), 239 (58%), 237 (59%), 191 (25%), 158 (43%); anal, calcd. for C ₁₄ H ₁₈ Br ₂ OS: C 42.66, H 4.60, Br 40.54, S 8.14; found: C 42.78, H 4.62, Br 40.59, S 8.14; analytical data for 42: MS m/z (EI+) 390 (M ⁺ , 14%), 388 (M ⁺ , 13%), 309 0 (100%), 233 (40%), 191 (20%), 157 (34%); analytical data for tris-SAc : MS m/z (EI+) 384 (M ⁺ , 31%), 309 (M ⁺ -SAc, 100%), 233 (58%), 157 (55%); anal, calcd. for C ₁₈ H ₂₄ O ₃ S ₃ : C 56.22, H 6.31, S 25.05; found: C 56.00, H 6.31, S 25.05.

Synthesis B — Bu4NI, toluene s [0142] A mixture of mesitylene tribromide (0.613 g, 1.5 mmol), Bu ₄ NI (0.055 g,

0.15 mmol) and potassium thioacetate (0.354 g, 3 mmol) in toluene (20 ml) was stirred for 4.5 h at room temperature. The reaction mixture was diluted with toluene (25 ml), washed with water (2x25 ml) and brine, dried over MgSO ₄ , and the solvent removed. The remaining residue was purified by flash chromatography (SiO ₂ , hexane : EtOAc, 10:1) to yield mono- 0 SAc (0.114 g, 21%) as a white solid, 42 (0.092 g, 17%) as a white solid, and tris-SAc; (0.184 g, 47%) as a white solid.

Svnthesis of Tris-fmetfaanthiosulfonatometfayD-mesitylene (40)

[0143] Sodium methanethiosulfonate (0.429 g, 3.15 mmol) and the bromide 43 (0.40Og, 1 mmol) were dissolved in dry DMF (20 ml) and stirred at 50°C under N ₂ over night. The reaction mixture was cooled to room temperature, diluted with water and extracted s with CH ₂ Cl ₂ (4x25 ml) and EtOAc (4x25 ml). The combined organic extracts were concentrated in vacuo and the crude product was chromatographed (SiO ₂ , EtOAc : hexane, 2 :1) to give 40 (0.277 g, 56%) as an off-white solid; ¹ H NMR (300 MHz, CDCl ₃ ) δ 2.44 (s, 3H, CH ₃ ), 3.32 (s, 3H, SO ₂ CH ₃ ), 4.43 (s, 2H, CH ₂ ); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 16.4 (CH ₃ ), 36.3 (CH ₂ ), 50.2 (SO ₂ CH ₃ ), 129.1 (aromat. C-2), 138.3 (aromat. C-I); ¹ H NMR (200 O MHz, acetone-d6) δ 2.55 (s, 3H, CH ₃ ), 3.55 (s, 3H, SO ₂ CH ₃ ), 4.64 (s, 2H, CH ₂ ); ¹³ C NMR (50 MHz, acetone-d6) δ 15.8 (CH ₃ ), 36.3 (CH ₂ ), 49.7 (SO ₂ CH ₃ ), 129.8 (aromat. C-2), 138.6 (aromat. C-I).

Synthesis of 1 ,3 -B is(thio- β-D- galactopyranos yldisulf anyrmethyl)-5 - s methanethiosulfonatomethyl-2,4,6-trimethyl-benzene (44 ^' )

[0144] A solution of the methanethiosulfonate 40 (0.238 g, 0.48 mmol) in DMF (20 ml) was cooled under N ₂ to 0°C and the sodium salt of 1-thio-β-D-galactose (0.209 g, 0.96 mmol) in water (10 ml) was added over a period of 2 h using a syringe pump. After warming to room temperature over night, the solvents were removed under reduced pressure, 0 and the resulting yellow oil was purified by flash chromatography (SiO ₂ , CHCl ₃ : MeOH : AcOH : H ₂ O, 60 : 30 : 3 :5) to afford three products: l,3-Bis(methanethiosulfonatomethyl)-5- thio-β-D-galactopyranosyldisulfanylmethyl-2,4,6-trimethyl-b enzene (0.048 g, 16%) as a colourless syrup, 44 (0.080 g, 23%) as a pale yellow solid, and tris-(thio-β-D- galactopyranosyldisulfanylmethyl)-mesitylene (0.210 g, 52%) as a white solid; analytical s data of mono-Gal: ¹ H NMR (250 MHz, CD ₃ OD) δ 2.52 (s, 3H, CH ₃ ), 2.56 (s, 6H, CH ₃ ), 3.48 (s, 6H, SO ₂ CH ₃ ), 3.59 (dd, IH, J 9.5 and 3.3 Hz, H-3'), 3.65 (t, IH, J 6.2 Hz, H-5 ¹ ), 3.79 (dd, IH, J. 11.3 and 5.2 Hz, H-6a') , 3.86 (dd, IH, J 11.2 and 6.7 Hz, H-6b'), 3.96-4.00 (m, 2H, H- 274'), 4.29 (d, IH, J 11.6 Hz, CHSS-GaI), 4.37 (d, IH, J 11.6 Hz, CHSS-GaI), 4.44 (d, IH, J 9.3 Hz, H-F), 4.59 (s, 4H, CH ₂ SSO ₂ ); ¹³ C NMR (63 MHz, CD ₃ OD) δ 17.4 (CH ₃ ), 17.8 o (CH ₃ ), 38.3 (CH ₂ SSO ₂ ), 43.2 (CH ₂ SS-GaI), 51.2 (SO ₂ CH ₃ ), 63.8 (CH ₂ OH), 70.8, 71.3, 77.0 (C-3 ¹ ), 82.0 (C-5 ¹ ), 93.8 (C-I'), 131.0, 134.9, 139.5, 140.5; analytical data of 44: ¹ H NMR (500 MHz, CD ₃ OD) δ 2.53 (s, 6H, CH ₃ ), 2.59 (s, 3H, CH ₃ ), 3.35 (s, 3H, SO ₂ CH ₃ ), 3.56 (dd,

2H, J 9.3 and 3.3 Hz, 2xH-3'), 3.62 (t, 2H, J 6.1 Hz, 2xH-5'), 3.77 (dd, 2H, J 11.3 and 5.6 Hz, 2xH-6a'), 3.82 (dd, 2H, J 11.4 and 6.5 Hz, 2xH-6b'), 3.93-3.97 (m, 4H, 2xH-2'/4'), 4.27 (d, 2H, J 11.4 Hz, 2xCHSS-Gal), 4.35 (d, 2H, 7 11.5 Hz, 2xCHSS-Gal), 4.40 (d, 2H, / 9.4 Hz, 2xH-l'), 4.58 (s, 2H, CH ₂ SSO ₂ ); ¹³ C NMR (125 MHz, CD ₃ OD) δ 16.7 (CH ₃ ), 17.3 (CH ₃ ), s 37.5 (CH ₂ SSO ₂ ), 42.4 (CH ₂ SS-GaI), 50.2 (SO ₂ CH ₃ ), 62.8 (CH ₂ OH), 69.8, 70.5, 76.2 (C-3 ¹ ), 81.1 (C-5 ¹ ), 92.9 (C-I ¹ ), 129.4, 133.4, 138.6, 139.7; HRMS m/z (TOF ES+) Found 747.0736 (M+Na ⁺ ), C ₂₅ H ₄₀ O ₁₂ S ₆ requires 747.0742; analytical data of tris-Gal: ¹ H NMR (300 MHz, D ₂ O) δ 2.65 (s, 3H, CH ₃ ), 3.51-3.69 (m, 5H), 3.85-3.86 (m, IH), - (m, 2H, CH ₂ OH), 4.24 (d, lH, / 9.4 Hz, 2xH-l'). O

Attempted synthesis of l,3-Bis( ^" methanethiosulfonatomethyl)-5-thioacetylmethyl-2,4,6- trimethyl-benzene ( ^" 47 or 48)

[0145] The bromide 43 (0.611 g, 1.5 mmol) was dissolved in dry DMF (30 ml) under argon. Sodium methanethiosulfonate (0.403 g, 3 mmol) in dry DMF (5 ml) and s potassium thioacetate (0.175 g, 1.5 mmol) in dry DMF (5 ml) were added simultaneously as fast as possible. After stirring for 35 h at room temperature, the solvent was removed under reduced pressure and the residue mixed with CH ₂ Cl ₂ (30 ml) and water (50 ml). The phases were separated and the aqueous phase was extracted with CH ₂ Cl ₂ (5x30 ml). The combined organic extracts were washed with brine, dried over MgSO ₄ , and evaporated. The residue was 0 chromatographed (SiO ₂ , hexane : EtOAc, gradient elution, 1:1 to 0:1) to afford the following products: the thioacetate 45 (0.102 g, 18%) as an off-white solid, the methanethiosulfonate 46 (0.322 g, 44%) as an off-white solid, and l,3-bis(thioacetylmethyl)-5- methanethiosulfonatomethyl-2,4,6-trimethyl-benzene (47; 0.096 g, 15%) as a colorless gum. A fourth isolated compound (white viscous foam, 0.088 g) is believed to be 48; analytical s data of 45: ¹ H NMR (300 MHz, CDCl ₃ ) δ 2.29 (s, 3H, CH ₃ ), 2.36 (s, 6H, COCH ₃ ), 2.39 (s, 6H, CH ₃ ), 3.36 (s, 3H, SO ₂ CH ₃ ), 4.20 (s, 4H, CH ₂ SAc), 4.48 (s, 2H, CH ₂ S); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 16.3 (CH ₃ ), 16.4 (CH ₃ ), 29.6 (CH ₂ SAc), 30.4 (CH ₃ CO), 36.7 (CH ₂ SSO ₂ ), 50.1 (SO ₂ CH ₃ ), 127.8 (aromat. C-4/6), 131.8 (aromat. C-l/3), 136.6 (aromat. C-2), 137.5 (aromat. C-5), 195.7 (C=O). Both spectra contain additional signals due to impurities. o

Svnthesis of 1,3,5-Trisdnethoxycarbonyl)benzene (SM. Dimick. S.C. Powell, S.A.

McMahon, D.N. Moothoo. J.H. Naismith. EJ. Toone. J. Am. Chem. Soc. 1999, 121, 10286-

10296)

[0146] In a flask equipped with a condenser and a drying tube 1,3,5- s benzenetricarboxylic acid 49 (22.12 g, 0.1 mol) was suspended in methanol (250 ml), concentrated sulphuric acid (25 ml) was added, and the mixture was refluxed over night.

After cooling to 0°C the white precipitate was filtered off, washed with cold water, dissolved in CHCl ₃ , and dried over MgSO ₄ . Removal of the solvent gave the desired product (22.05 g,

87%) as a white powder; mp 142-144°C (lit. 144-144.5°C); ¹ H NMR (300 MHz, CDCl ₃ ) δ o 3.99 (s, 3H, CH ₃ ), 8.85 (s, IH, aromat. H).

Synthesis of 1,3,5-Tris(hvdroxymethyl)benzene (5O)(Y. Yamagiwa, Y. Koreishi, S. Kiyozumi, M. Kobayashi, T. Kamikawa, M. Tsukino. H. Goi, M. Yamamaoto, M. Munakata, Bull. Chem. Soc. Jpn. 1996, 69, 3317-3323: J. Houk. G. Whitesides, J. Am. Chem. Soc. s 1987. 109, 6825-6836)

[0147] A solution of l,3,5-tris(methoxycarbonyl)benzene (10.09 g, 40 mmol) in dry THF (400 ml) was added over a period of 3h to a suspension of LiAlH ₄ (4.03 g, 0.1 mol) in dry THF (300 ml) under N ₂ . After stirring over night at room temperature, the reaction mixture was cooled to 0°C, hydrolysed with water (4 ml), 2M NaOH (4 ml) and water (12 0 ml), filtered, and the filter cake washed thoroughly with THF. The combined filtrates were concentrated and the crude product was recrystallized from hot (not boiling) ethanol to afford 50 (5.59 g, 83%) as white needles; mp 76-77 ⁰ C (lit. 77-78°C) ¹ H NMR (300 MHz, DMSO- d6) δ 4.49 (d, 3H, J 4.8 Hz, CH ₂ ), 5.30 (t, IH, J 5.4 Hz, OH), 7.14 (s, IH, aromat. H).

s Modification of SBL-S 156C with 44

[0148] In a polypropylene test-tube 16.1 mg of S156C was dissolved in 2.4 ml modification buffer (70 mM CHES, 5 mM MES, 2 mM CaCl ₂ , pH 9.5), mixed with 100 μl of a 0.25 M solution of 21 in water/CH ₃ CN (2/1), vortexed, and allowed to react using an end- over-end rotator at room temperature. After 30 min 10 μl of the reaction mixture were o withdrawn and tested for residual free thiol content by mixing with 10 μl Ellman's reagent (2.5*10 ^'2 M in pH 6.9 phosphate buffer). The absence of yellow color by visual inspection indicated complete reaction. An additional 100 μl of the 0.25 M solution of 21 was added and

the mixture permitted to react for a further 30 min. The reaction then was quenched by pouring the reaction mixture onto a pre-packed, pre-equilibrated G-25 Sephadex PDlO column and eluted with 3.5 ml of quench buffer (5 niM MES, 2 mM CaCl ₂ , pH 6.5). The eluant was dialyzed at 4°C against 10 mM MES, 2 mM CaCl ₂ , pH 5.8 (3x2 1, 3x60 min). The s resulting dialysate was aliquoted, flash frozen in liquid nitrogen, and stored at -18°C.

Example 4: Synthesis of disaccharide glvcodendrimers

[0149] Glycosylated macromolecules, also known as glycoconjugates, are present on the surfaces of all mammalian cells. Interactions of glycoconjugates with carbohydrate- io binding structures on other cells, viruses, bacteria or toxins are playing a crucial role in biological processes such as cell-cell recognition, inflammation, invasion by pathogens or the onset of cancer metastasis. Sugar-binding proteins, also known as lectins, show a highly specific affinity to complex carbohydrate structures. Sharon et al., Essays Biochem. 1995, 30, 59. Although the interaction between lectins and single carbohydrates is typically a weak is interaction (~ 10 ^"3 M), when more than one sugar is involved and several saccharides are properly arranged, specificity and affinity of the lectin-carbohydrate interaction rapidly increase, hi some cases, the increase in binding affinity is larger than expected due to increase in local concentration of the ligand. This increase in binding affinity is referred to as the "multivalent" or "glycoside cluster effect." Mammen et al., Angew. Chem. 1998, 110,

JO 2908-2953; Angew. Chem. Int. Ed. 1998, 37, 2754-2794.

[0150] One may present natural oligosaccharide structures to exploit lectin binding. Alternatively, one may present only the terminal structures of the multiantennary domains found in nature. Attaching saccharides to the outer sphere of dendrimers may be sufficient to achieve binding to lectins. A new class of structures is produced by ligation by a is dendrimeric supported carbohydrates to proteins: glycodendriproteins. The glycodendriproteins may be useful tools in the study of protein-carbohydrate interactions as well as in targeting lectins specifically with enzymes.

[0151] To synthesize glycodendriproteins, chemical synthesis of the glycodendrimer and site-directed mutagenesis of the enzyme were used, although other w methods may also be used. For ligation MTS reagents may be used. MTS reagents may be reacting in a specific and high yielding reaction with SH-functions introduced by site-directed

mutagenesis. Matsumoto et al., Chem. Commun. 2001, 903-904; Davis et al., Bioorg. Med. Chem. 2000, 8, 1527-1535.

Synthesis of Thiosugars [0152] One of the steps in the assembly of the protein modifying reagents is the attachment of the sugars via nucleophilic substitution. This step may be performed by using thio derivatives of sugars and halides of the branched core. Suitable sugar thiols include anomeric thiols of iV-acetylglucosamine, galactose and galabiose (galactose-α(l,4)-galactose) and a 4-thio derivative of mannose and were synthesized to achieve this end.

1 -Thio-galactopyranose

[0153] Acetobromo galactose 53 was converted to acetylated thiogalactose 55 via thiouronium salt 54 using thiourea in refluxing acetone, followed by treatment with sodium metabisulfite in a 62% yield over the two steps. Acetylated sugar 55 was deprotected under Zemplen conditions to yield the unprotected sodium salt 56 in very good yield (Scheme 22).

SCHEME 22 1-Thio-glucosamine

[0154] Using a synthetic method analogous to the synthesis described above a 1- thio derivative of glucosamine was synthesized. Specifically, N-acetylglucosamine 57 was acetylated and the anomeric chloride was formed in a one step procedure. The resulting chloride 58 was reacted with thiourea in acetone, followed by treatment with sodium metabisulfite to yield thiol 59 in 51% yield over these two steps. The yield, when compared to the galactose reaction, corresponds to the common observed yields in glucosamine reactions, and may be explained by the low solubility generally associated with glucosamine derivatives. Zemplen deacetylation of 59 led to the deprotected thio sugar 60 in almost quantitative yield (Scheme 23).

(51%, 2steps)

SCHEME 23

Thio- galabiose

[0155] The thiol of galabiose was synthesized from a mixture of acetyl 4-O- (2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-2,3,6,-O-tri- benzoyl-α-D-galactopyranoside 61 and the corresponding methyl glycoside methyl 4-O-(2,3,4,6-tetra-O-acetyl-α-D- galactopyranosyl)-2,3,6,-O-tri-benzoyl-α-D-galactopyranosid e 62 (Scheme 24).

[0156] This mixture was purified by column chromatography on silica and the acetyl glycoside 61 was isolated. Treatment with hydrobromic acid in acetic acid lead to the disaccharide bromide 63. Using thiourea, followed by sodium metabisulfite, acyl protected thiogalabioside 64 was prepared, which was deprotected to form 65.

61

61/62 62

SCHEME 24

[0157] A method of synthesizing the disaccharides includes starting from monosaccharides or simple sugars. D-Galactose 66 was converted to the corresponding α- methyl galactoside 67 using HCl in methanol. Benzoylation with benzoyl chloride in pyridine at O °C and subsequent warming to room temperature yielded the selectively 2,3,6 benzoate protected derivative 68, the glycosyl acceptor (Scheme 25).

66 67 68

SCHEME 25

[0158] To prepare the glycosyl donor, D-galactose 66 was acetylated, using acetic anhydride and pyridine (→ 69). The resulting peracetylated galactose 69 was treated with s thiophenol and BF ₃ -etherate to give thiophenyl galactoside 70. Removal of the acetate protective groups (— > 71) and benzylation using benzylbromide and sodium hydride gave the benzyl protected thio donor 72 (Scheme 26).

SCHEME 26 O [0159] Donor 72 was reacted with acceptor 68 to yield disaccharide 73 (Scheme

27). By using "alcohol free" dichloromethane yields of up to 66% can be obtained.

SCHEME 27

. [0160] Benzyl and benzoyl protected disaccharide 73 was debenzylated by catalytic hydrogenation with hydrogen (balloon) and palladium on carbon in methanol to s liberate four OH functions on the first sugar 74. An initial reaction time of three days was utilized, but could be reduced by increasing the reaction temperature to more than 20 ⁰ C (for example, 25 ⁰ C or 30 °C) and/or by switching the solvent from methanol to THF (a four hour reaction), yielding >95%.

[0161] Acetylation with acetic anhydride/pyridine gave acetyl/benzoyl protected O disaccharide 62 (85%); treatment with sulfuric acid in acetic anhydride and acetic acid gave a mixture of anomeric acetates 61 in 87% yield, followed by conversion to the α-bromo disaccharide 63 in 96% yield, using hydrobromic acid in acetic acid/acetic anhydride.

[0162] The resulting peracetylated bromo sugar was converted to the fully protected thiosugar 64 via the thiourea, sodium metabisulfite route (68%), discussed above. S

Synthesis of 4- Thiomannosides

[0163] Mannose 75 was converted to l,6-anhydro-2,3-0-isoρropylidene-mannose 77 via 1,6-anhydro mannose 76 following a known procedure (Fraser-Reid, J Org. Chern. 1989, 54, 6125-6127) (Scheme 28). Mesylation at 04 (→ 78), cleavage of the acetal protective group with acetic acid/water (-» 79) and base catalyzed (NaOMe/MeOH) intramolecular nucleophilic substitution yielded 1,6-3,4-anhydro-β-D-talose 80. A. Vasella, HeIv. Chirn. Acta 2001, 84, 2355-2367.

succesful

SCHEME 28

[0164] A thiobenzoate group was introduced at the 4-position to yield a 4-thio JO mannose derivative 81 by reaction with 80. In another test reaction 80 was treated with thioacetic acid/ pyridine (-» 82) (Scheme 29) to yield a thioacetate.

SCHEME 29

/$ ^■ Linker Syntliesis

[0165] A trivalent bifunctional core was chosen for the synthesis of the sugar displaying dendrons. This core was prepared following procedures described herein.

[0166] Tris(2-aminoethyl)amine (TREN) 83 was selectively mono-Boc-protected

(— > 84). Chloroacetates were introduced using chloroacetic anhydrides to give dichloro- zo compound 85. To improve the reactivity, TREN-Boc 84 was reacted with bromo acetyl bromide to synthesize dibromide 86. The product was characterized by electrospra ^' y mass spectrometry.

[0167] Alternative approach towards the synthesis of higher branched dendrons involved the strategy of synthesizing the branched core first and attaching the sugars later. zf TREN-Boc 84 was reacted with Cbz chloride to give Cbz ₂ TREN-Boc 87. Treatment with

TFA in dichloroniethane to remove the butyloxycarbonyl protecting group liberated one amino functionality (-> 88). Base catalyzed addition of γ-butyrolactone under ring-opening gave thiol 89 and the corresponding disulfide 90 in 43% and 27% yield respectively (Scheme 30).

H ₂ N. N,

:N ;N- -NHBoc

H ₂ N' N' 87

SCHEME 30

[0168] The disulfide 90 was reduced to 89 with tributyl phosphine (Scheme 31). Thiol 89 was coupled to the divalent TREN-chloride 85 using a large excess of DBU as base. This reaction led to the "tetravalent" dendron 91.

SCHEME 31

Synthesis of [Gali9,TREN-SI56C

[0169] The bivalent glycodendriprotein [Gal] ₂ TREN-SI56C 92 was synthesized according to a procedure analogous to that set forth below in Scheme 32.

[0170] Thiogalactose 56 was attached to TREN-Boc chloride 85 to yield 93. The B oc protected amine was deprotected by trifluoroacetic acid in dichloromethane to give 94. Coupling to the active ester of 4-carboxybutylmethanethiosulfonate 95 yielded the corresponding MTS reagent 96. MTS reagent 96 was successfully used to modify SBL- mutant S156C to the bivalent glycodendriprotein [Gal] ₂ TREN-SI56C 92.

SCHEME 32

Synthesis of FGaIa(1 ,4)Gal1?TREN-SI56C s [0171] An analogous synthesis of galabiose GaIa(1, 4)Gal was^ optimized as described above and galabiose was attached to the dendrimer core. The purification steps lead to the following synthesis of [GaIa(1, 4)Gal] ₂ TREN-SI56C (Schemes 33 and 34).

[0172] Reaction of the peracylated thiosugar 64 with (AcCl ₂ )TREN-Boc 85 and

Hϋnig's base yielded protected galabiose dendron 91 in 50% yield as a white foam. o Peracylated TRENB oc derivative 91 was deprotected in the standard way by treatment with sodium methoxide in anhydrous methanol. Neutralization with ion exchange resin (Dowex

50WX8-200 H ⁺ ) and lyophilization from water yielded pure deacylated compound 98 in very good yield. Cleavage of the Boc protecting group by trifluoroacetic acid in dichloromethane yielded free amine 99 which could be purified by loading on ion exchange resin (Dowex 50WX8-200 H ⁺ ), washing with water, then methanol, then water again and finally elution with aqueous ammonia (15%), concentration and lyophilization from water yielded pure deprotected GaIa(1, 4)Gal-TREN-NH ₂ 99.

SCHEME 33

[0173] Treatment of 99 with active ester reagent 95 (1.2 equivalents) in dry DMF in the presence of Hϋnig's base gave (after flash chromatography water/methanol/chloroform) MTS reagent 100 in 70% yield. After further purification over a

Cl 8 cartridge to remove remaining active ester or its hydrolyzed derivative (101), 100 was used in the ligation reaction with SBLS 156C.

SCHEME 34

[0174] Mass spectrometry showed the expected mass of 27770 Da for glycodendriprotein 102.

Mass Spectroscopical Studies

[0175] Peanut agglutinin (PNA) is a galactose binding lectin. Through native protein mass spectroscopic analysis, we observed binding between PNA and the bivalent galactose modified glycodendriprotein [GaI] ₂ TREN-S 156C 91. First the tetrameric PNA

complex was observed. Titration with the glycodendriprotein showed the formation of a complex between the two proteins and a micromolar K _D was extracted from this data.

2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (53) [0176] Acetobromogalactose was purchased from Fluka.

2,3,4, ό-tetra-O-acetyl-l-thio-β-D-galactopyranose (55)

[0177] Acetobromogalactose 53 (9.97 g, 24.3 mmol) and thiourea (1.95 g, 25.5 mmol) were dissolved in acetone and heated under reflux for 2 h. The reaction mixture was concentrated. Sodium metabisulfite (4.9 g, 25.5 mmol) in deionized water (50 mL) and dichloromethane (60 mL) were added. After stirring for 1 h at 45°C, the layers were separated and the aqueous layer was extracted with dichloromethane (2x50 mL). The combined extracts were dried over magnesium sulphate and concentrated in vacuo. Column chromatography yielded 55 as a colorless foam (5.46 g, 62%). [0178] NMR data is reported in, for example, Garegg, et al. Carbohydrate

Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9), 2335-2336;

Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

Sodium 1-thio-β-D-galactopyranoside (56) [0179] Acetylated sugar 55 (8.98g, 24.6mmol) was dissolved in 50 mL methanol

(anhydr.) and 1 M NaOMe in methanol (25 mL) was added. After stirring for 45 min at room temperature, TLC indicated completion of reaction, ethyl acetate (60 mL) was added and a white precipitate was formed. Filtering and subsequent washing of the precipitate with ethyl acetate and drying under high vacuo yielded sodium salt 56 (4.21 g, 78%). Treatment of the mother liquor with more EtOAc yielded another crop of the desired compound (0.84 g, 16%, total yield: 94%). NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosy l chloride (58)

[0180] N-acetylglucosamine (20 g, 90 mmol) and acetylchloride were stirred vigorously over night. Dichloromethane (50 mL) was added to the resulting amber syrup and

the solution was poured on ice water (100 mL). Layers were separated and the organic layer was poured on saturated sodium bicarbonate solution/ice (100 mL). When the neutralization was complete, the org. layer was dried over magnesium sulfate (10 g), concentrated in vacuo and crystallized from dry ether (140 mL). After 20 h at room temperature, solids were collected, washed with ether and dried under high vacuum (25.3 g, 77%).

[0181] NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

3, 4, β-tri-O-acetyl-l-acetamido-l-deoxy-l -thio-β-D-glucopyranose (59)

[0182] Acetochlorogalactose 58 (6.0 g, 16.2 mmol) and thiourea (2.0 g, 34 mmol) were dissolved in acetone and heated under reflux for 12 h. The reaction mixture was filtered, the collected solids were washed with dichloromethane and dried (5 g, 70%) followed by addition of sodium metabisulfite (2.8 g, 15 mmol) in deionized water (50 mL) and dichloromethane (100 mL). After stirring for 3 h at room temperature, TLC showed complete consumption of the starting material, the layers were separated and the aqueous layer was extracted with dichloromethane (2x50 mL). The combined extracts were washed with water (100 mL) and brine (100 mL), dried over magnesium sulfate and concentrated in vacuo (white solid 2.4 g). Recrystallization from EtOAc/PE yielded the desired thiol as white powder (1.95 g, 33% 2 steps).

[0183] NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

Sodium 2-acetamido-2-deoxy-l-thio-β-D-galactopyranoside (60)

[0184] Acetylated sugar 59 (770 mg, 2.12 mmol) was dissolved in 10 mL methanol (anhydr.) and 1 M NaOMe in methanol (2.15 mL) was added. After stirring for 60 min at room temperature, TLC indicated completion of reaction, ethyl acetate (40 mL) was added and a white precipitate was formed. Filtering and subsequent washing of the precipitate with ethyl acetate and drying under high vacuo yielded sodium salt 60 (530 mg, 96%).

[0185] NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

s Acetyl 4-O-(2, 3, 4, 6-tetra-0 -acetyl- a-D-galactopyranosyl)-2, 3, 6-tri-O-benzoyl- galactopyranoside (61)

[0186] Acyl protected galabiose 61 was obtained by purification of a mixture of the desired compound and the corresponding methyl glycoside by column chromatography

(petroleum ether/ethyl acetate 8:1 v/v). NMR data is reported in, for example, Garegg, et al. O Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9),

2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

Acetyl 4~O-(2,3,4,6-tetra-0-acetyl-a-D-galactopyranosyl)-2,3,6-tri- 0-benzoyl-a/β-D- galactopyranoside (61) s [0187] Methyl glycoside 62 (2.7 g) was dissolved in 25 mL acetic anhydride and cooled to 0 °C (ice bath). Acetic acid (15 mL) was added and subsequently 0.4 mL concentrated sulfuric acid were added dropwise. After 1 h the ice-bath was removed and the mixture was stirred at room temperature for another 4 h until TLC (petroleum ether/ethyl acetate 1:1) showed completion of the reaction. The reaction mixture was poured on 0 ice/saturated NaHCO ₃ and extracted with ethyl acetate (3x 150 mL), dried over magnesium sulfate and concentrated to yield 2.43 g of a white foam 61 (87%).

4-O-(2,3,4, 6-tetra-0 -acetyl- a-D-galactopyranosyl)-2, 3, 6-tri-O-benzoyl- OC-D- galactopyranosyl bromide (63) s [0188] A solution of acyl protected galabiose 61 in glacial acetic acid (3 mL) and acetic anhydride (3 mL) was cooled to 0 °C (ice bath) and 33% HBr/acetic acid (9 mL) was added dropwise over 30 min. The mixture was stirred at 0 ⁰ C for 60 min, than allowed to warm up to room temperature and was stirred for another 2 h at room temperature. The orange colored solution was poured on ice/sat. NaHCO ₃ and more NaHCO ₃ solution was 0 added until no more CO ₂ evolved. The aq. layer was extracted with dichloromethane (50 mL), the combined org. layers were washed with sat. NaHCθ ₃ solution (50 mL), dried over magnesium sulfate and concentrated to yield 63 as white foam (1.0 g, quant.).

4-O-(2, 3, 4, 6-tetra-0 -acetyl- a-D-galactopyranosyl)-2, 3, 6-tri-O-benzoyl-l-thio-β-D- galactopyranoside (64)

[0189] Bromide 63 (0.95 g, 1.1 mmol) and thiourea (120 mg, 1.58 mmol) were dissolved in acetone (20 mL) and heated under reflux for 12 h. The reaction mixture was concentrated and purified by flash chromatography (EtOAc/MeOH 95:5 v/v). The product containing fractions were concentrated and treated with sodium metabisulfite in water (20 mL) and dichloromethane (20 mL) at 40 °C under vigorous stirring (3 h). Layers were separated, the aq. layer was washed with brine (70 mL), dried over magnesium sulfate and concentrated. Column chromatography (PE/EtOAc 2: 1 — > 5:3 v /v) yielded 64 as a white foam (710 mg, 77%).

Methyl 2,3,6-tri-O-benzoyl-a-D-galactoside (68)

[0190] Galactose acceptor was synthesized following known procedures.

Thiophenyl 2,3,4,6-tetra-O-benzyl-β-D-galactoside (72)

[0191] Galactose donor was synthesized following known procedures.

1, 6-anhydro~4-deoxy-4-thio-4-S-acetyl-D-mannose (82) [0192] l,6:l,3-dianhydro-mannose (500 mg, 3.46 mmol), thioacetic acid (1.24 mL, 17.3 mmol), pyridine (1.67 mL, 20.75 mmol) and DMF (2 mL) were combined and stirred at room temperature for 5 h. The mixture was concentrated and purified by flash chromatography (FC) (CHCl ₃ ZMeOH 19:1), followed by 2 ^nd FC (CHCl ₃ /MeOH 39:1) and recrystallized from EtOAc (hot solution filtered through charcoal) to yield 115 mg (15%) colorless crystals. The mother liquor was concentrated and left at -18 ⁰ C for further crystallization.

TREN-Boc (84)

[0193] A solution of tris(2-aminoethyl)amine (TREN) (7 mL, 47 mmol) in dichloromethane (650 mL) was cooled to -78 °C and Boc anhydride in dichloromethane (160 mL) was added dropwise over 120 min. The mixture was allowed to warm up to room temperature and concentrated in vacuo. Flash chromatography (chloroform/MeOH/ aq.

ammonia 10:4:1 v/v/v) yielded mono Boc-protected TREN 84 as a pale yellow oil (2.25 g, 88%). NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.

S

[0194] A solution of TREN-Boc 84 (2.2 g, 8.9 mmol) in anhydrous dichloromethane (30 mL) and anhydrous pyridine (3.5 mL) was cooled to 0 °C (ice bath) and chloroacetic anhydride (3.7 g, 21.4 mmol) was added in portions. The mixture was allowed to O warm up to room temperature (90 min) and concentrated in vacuo. Flash chromatography (EtOAc/MeOH 19:1 ^' → 4:1 v/v) yielded bis(chloroacetamido)-TREN-Boc 85 as a pale yellow oil (1.47 g, 69%). NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75. S

[0195] TREN-Boc (1.14 g, 4.6 mmol) was dissolved in 10 mL water/dioxane 1:1. A solution of Cbz-chloride (1.43 mL, 10 mmol) in 5 mL dioxane was added drop wise alternating with drop wise addition of 3.5 M K ₂ CO ₃ to maintain a pH of 7. After addition was 0 complete the mixture was stirred for 1 h at pH = 7-8. Extraction with dichloromethane (2x50 mL) and washing of the organic layer with 1 M NaOH (30 mL) and water (30 mL), drying over MgSO ₄ , concentrating and FC (toluene/ethyl acetate 1:1) yielded 1 g (41 %) of a yellow oil.

[0196] ESI-MS: 515 (100) [M+H] ⁺ , 537 (30) [M+Naf. S

CbZ ₂ -TREN (88)

[0197] Cbz ₂ -TREN-Boc (100 mg, 0.19 mmol) was dissolved in 2 mL TFA/dichloromethane and stirred for 2 h at room temperature. Solvents were evaporated and the crude product was purified on Dowex 50WX2-100 H ⁺ to give 50 mg (65%) as a colorless 0 oil.

[0198] ESI-MS: 415 (100) [MH-H] ⁺ , 437 (5) [M+Na] ⁺ .

CbZ ₂ -TREN-CO-(CH ₂ USH (89)

[0199] CbZ ₂ -TREN (180 mg, 0.43 mmol) and 420 mg NaHCO ₃ were dissolved in 6 mL EtOH and 10 mL water. After degassing the solution (argon 30 min) DTT (330 mg, 2.16 mmol) was added and the reaction mixture was heated to 50 ⁰ C. After addition of s thiobutyrolactone (0.38 mL, 4.3 mmol) the reaction was stirred for 18 h, neutralized with 1 M HCl and concentrated. FC (EtOAc/MeOH 40:1-20:1) gave 95 mg (43%) of 89 and 60 mg (27%) of 90.

O [0200] A mixture of TREN-BOC-(ACCI) ₂ (85) (1.26 g, 3.15 mmol) and thiogalactose (1.58 g, 7.26 mmol) (56) in anhydrous DMF (40 mL) was stirred over night at room temperature and concentrated in vacuo.

[0201] Flash chromatography (chloroform/MeOH/aq. ammonia 4:4:1 v/v/v) yielded Boc-protected dithiogalactose compound (93) as a white foam (1.45 g, 64 %). S

TREN-(SGaI) ₂ (94)

[0202] A mixture of TREN-BoC-(SGaI) ₂ (93) (1.34 g, 1.86 mmol) and trifluoroacetic acid (12 mL) in dichloromethane (12 mL) was stirred for 3 h at room temperature (15 ⁰ C) and concentrated in vacuo. The crude product was dissolved in 0 water/methanol (50 mL 1:1), loaded on fresh activated Dowex 50X2-200 (H ⁺ ), washed with methanol (100 mL), methanol/water (100 mL 1:1), water (100 mL) and eluted with 10% aq. ammonia (200 mL). Evaporation of the solvents yielded amine (94) as a white foam (1.03 g, 89 %).

s MTS-(CH ₂ U-CO-TREN-(SGaI) ₂ (96)

[0203] Amine 94 (250 mg, 0.4 mmol) and active ester of 4-carboxybutyl methanethiosulfonate (125 mg, 0.4 mmol) were dissolved in dry DMF (20 mL) and stirred for 3 h at room temperature. Flash chromatography (chloroform/MeOH/water 6:3:5 v/v/v) yielded MTS dendrimer 96 as a colorless syrup (238 mg, 73%). 0

S56C-S-(CH ₂ ) ₄ -CO-TREN-(SGal) ₂ = S156Cdigal

[0204] S156C (10 mg) was dissolved in ligating buffer (1 mL; 70 mM CHES, 5 raM MES, 2 mM CaCl ₂ , pH 9.5) at room temperature and 100 μL of a 0.2 M solution of 53 in water was added, vortexed for 1 min and mixed on an end-over-end rotator for 40 min. The mixture was purified over a Pharmacia PD-10 column, dialyzed against MES-buffer (10 mM s MES, 2 mM CaCl ₂ , 3x (1 h, 1 L)) at 4 °C, than flash frozen and stored at -18 °C. A _{280 nm} = 0.146: C = 1.5 mg mL ^"1 LC/MS (positive mode): found: 27446 Da, calc: 27447 Da.

Sl 56C-S-(CH ₂ J ₄ -CO-TREN-(SGaI) ₂ = S156C-digal

[0205] S156C (85 mg) was dissolved in ligating buffer (4 mL; 70 mM CHES, 5 O mM MES, 2 mM CaCl ₂ , pH 9.5) at room temperature and 300 μL of a 0.2 M solution of (53) in water was added, vortexed for 1 min and mixed on an end-over-end rotator for 45 min. The mixture was purified over a Pharmacia PD-10 column, dialyzed against water, 3x (1 h, 1 L) at

4 ⁰ C, than flash frozen and stored at -18 ⁰ C.

[0206] LC/MS (positive mode): found: 27450 Da, calc: 27447 Da. S

Thio 4-0-(2,3,4,6-tetra-0-acetyl-a-D-galactopyranosyl)-2,3,6-tn-0 -benzoyl-a-O- galactopyranoside (64)

[0207] Bromide 53 (2.25 g, 2.63 mmol) was dissolved in 50 mL acetone and thiourea (0.22 g, 2.9 mmol) was added and the mixture was refluxed for 14 h until TLC 0 (petrol ether/ethyl acetate 1:1) showed complete consumption of the starting material (starting material R _f = 0.6; product baseline). Solvents were evaporated and the residue purified by flash chromatography over silica (ethyl acetate/methanol 19:1).

[0208] The obtained material was dissolved in dichloromethane (60 mL) and a solution of sodium metabisulfite (0.55 g) in water (60 mL) was added. The biphasic mixture s was stirred vigorously at 40 ⁰ C for 4 h. TLC: (ethyl acetate/petrol ether 1:1) R _f = 0.55. showed formation of a major product. Layers were separated, the aqueous layer was extracted with dichloromethane (2x50 mL). The organic layers were combined and washed with brine (100 mL), dried over magnesium sulfate and concentrated. Flash chromatography and recrystallization from ethyl acetate/ petrol gave thiodisaccharide 64 as colorless crystals (1.4 o g, 62%).

[0209] [α] _D = +131.0 (c = 1.0 in chloroform);

^{102101 1} H-NMR (200 MHz): δ = 2.56 (d, J = 10 Hz, 1 H; S-H), 3.61 (dd, J6,6' =

11 Hz, J5,6 = 6.1 Hz, 1 H; 6a-H), 3.81 (dd, J6,6' = HHz, J5,6' = 8 Hz, 1 H; 6'a-H), 4.14 (dd, J5,6' = 8 Hz, J5,6 = 6.1 Hz, 1 H; 5b-H), 4.41-4.62 (m, 3 H; 4b-H, 5b-H, 6b-H), 4.65-4.87 (m, 2 H; 2b-H, 6b'-H), 5.18-5.39 (m, 3 H; Ia-H, 2a-H, 4a-H), 5.43-5.56 (m, 2 H; 3a-H, 3b-H)" s 5.69 (dd, J),2 = 10 Hz, J = 10 Hz, 1 H; Ib-H), 7.3-7.65 (m, 18; Bz-H), 7.85-8.09 (m, 12 H; Bz-H).

(2,3, 4,6-tetra-O-acetyl-Gala(l, 4)2,3, 6-tri-0-benzoyl-GalβS) ₂ TREN-Boc (97)

[0211] Thiosaccharide 64 and chloride 85 were dried under high vacuum for 6 h. O 1.02 g of TREN-chloride 85 were dissolved in 5 mL dry and degassed DMF. 650 μL of this solution were added to 610 mg thiogalabiose 64 in 3 mL dry and degassed DMF under nitrogen. The mixture was stirred at room temperature until all solids were dissolved and 130 μL N-Ethyl-MiV-diisopropylamine were added. The reaction was stirred at 60 ⁰ C for 2 h under nitrogen. s [0212] Solvents were evaporated and flash chromatography (ethyl acetate/methanol 5%-30%) yielded 330 mg of colorless foam (50%) (97). [0213] TLC: R _f = 0.85 (ethyl acetate/methanol 9: 1)

^[0214] 1H-NMR (400 MHz): δ = 1.46 (s, 9 H; 3 Boc-Me), 1.85/1.91/2.01/2.09 (4 s, 12 H; 4 OAc), 2.39 (br, 6 H; N(CH ₂ CH ₂ N) ₃ ), 2.98 (br, 2 H; BoCHNCH ₂ ), 3.17 (br, 4 H; 2 o (CO)HNCH ₂ ), 3.39 (d, J = 15.6 Hz, 2 H; SCHH ¹ CO), 3.56 (d, J = 15.6 Hz, 2 H; SCHH ¹ CO), 3.66 (dd, J6,6' = 10.8 Hz, J5,6 = 6.4 Hz, 2 H; 6a-H), 3.86 (dd, J6,6' = 10.8 Hz, J5,6' = 7.0 Hz, 2 H; 6'a-H), 4.18 (dd, J5,6' = 6.4 Hz, J5,6 = 6.0 Hz, 2 H; 5b-H), 4.39-4.53 (m, 4 H; 4b-H, 6b- H), 4.62 (dd, J5,6 = 6.4 Hz, J5,6' = 7.0 Hz, 2 H; 5a-H), 4.69 (dd, J6,6' = 11.2 Hz, J5,6' = 6.4 Hz, 2 H; 6'b-H), 5.04 (d, J),2 = 9.6 Hz, 2 H; Ib-H), 5.22 (m, 2 H; 2a-H), 5.55 (d, J),2 = 3.2 s Hz, 2 H; Ia-H), 5.43 (dd, J2,3 = J3,4 = 10 Hz, 2 H; 3b-H), 5.49-5.54 (m, 4 H; 3a-H, 4a-H), 5.75 (dd, J),2 = 9.6 Hz, J2,3 = 10 Hz, 2 H; 2b-H), 7.3-7.65 (m, 18; Bz-H), 7.9-8.1 (m, 12 H; Bz-H).

[0215] MS (FAB+): calc. for C ₉₇ HmN ₄ O ₃₈ S ₂ ⁺ /: 2003.63 (90),2004.64 (100), 2005.64 (70), 2006.64 (40),2007.64 (15); found: 2003.22 (90), 2004.22 (100), 2005.21 o (75),2006.22 (40), 2007.22 (20)

(Galad,4)Galβ) ₂ TREN-Boc (98)

[0216] Protected dendrimer 97 (220 mg, 0.11 mmol) was dissolved in anhydrous methanol and 3 drops of a freshly prepared 1 M solution of NaOMe in methanol were added. The solution was stirred at room temperature and carefully monitored by TLC, which showed the formation of a single product after 18 h (chloroform/methanol/aqueous ammonia (15%) s 4:4: 1 ; R _f = 0.05). The mixture was neutralized with Dowex 50WX8- 100 H ⁺ , concentrated and lyophilized from H ₂ O to yield 98 as a colorless foam (105 mg, 95%).

[0217] [αfo = +30.0 (c = 0.5 in H ₂ O);

^[0218] 1H-NMR (400 MHz): δ = 1.32 (s, 9 H; 3 Boc-Me), 2.63 (br, 6 H;

N(CH ₂ CH ₂ N) ₃ )' 3.07 (br, 2 H; BocHNCH ₂ ), 3.23 (br, 4 H; 2 (CO)HNCH ₂ ), 3.30 (d, J = 15.0 o Hz, 2 H; SC HH'CO),3.45 (d, J = 15.0 Hz, 2 H; SCHH ¹ CO), 3.49 (dd, J),2 = 9.6 Hz, J2,3 = 9.2 Hz, 2 H; 2b-H), 3.55-3.85 (m, 18 H; 2a-H, 2b-H, 3a-H, 3b-H, 5b-H, 5a,5a'-H, 6a,6a'-H), 3.91 (bs, 2 H; 4a-H), 3.97 (bs, 2 H; 4b-H), 4.23 (dd, J = 6.4 Hz, J = 6.0 Hz, 2 H; 5a-H), 4.46 (d, J),2 = 9.6 Hz, 2 H; Ib-H), 4.84 (d, J),2 = 3.2 Hz, 2 H; Ia-H).

s (GaId l,4)Galβ) ₂ TREN ^' (50)

[0219] Deacylated dendrimer 98 was taken up in 1 mL of a mixture of trifluoroacetic acid and water (1:1) and stirred at room temperature for 45 min until TLC indicated complete consumption of the starting material. Solvents were evaporated and the residue was dissolved in water (3 mL) and loaded on Dowex 50WX8-100 (H ⁺ ), washed with

?o water, methanol and water (20 mL each) and eluted with 100 mL aqueous ammonia (15%). The filtrate was concentrated and lyophilized from water to yield 99 as 24 mg of a colorless foam (86%).

^[0220] 1H-NMR (400MHz): δ = 2.62 (t, J = 6.0 Hz, 4 H; N(CH ₂ CH ₂ N) ₂ COCH ₂ S),

2.73 (t, J = 5.6 Hz, 2 H; NCH ₂ CH ₂ NH ₂ ), 2.98 (t, J = 5.6 Hz, 2 H; NCH ₂ CH ₂ NH ₂ ), 3.23 (t, J = v 6.0 Hz, 4 H; N(CH ₂ CH ₂ N) ₂ COCH ₂ S), 3.32 (d, J = 15.6 Hz, 2 H; SCHH'CO), 3.46 (d, J = 15.6 Hz, 2 H; SCHH'CO), 3.51 (dd, J),2 = 9.6 Hz, 2 H; 2b-H), 3.55-3.86 (m, 18 H; 2a-H, 2b-H, 3a-H, 3b-H, 5b-H, 5a,5a'-H, 6a,6a'-H), 3.91 (bd, J = 1.6 Hz, 2 H; 4a-H), 3.97 (bd, J = 2.4 Hz, 2 H; 4b-H), 4.23 (dd, J = 6.4 Hz, J = 5.6 Hz, 2 H; 5a-H), 4.49 (d, J),2 = 9.6 Hz, 2 H; Ib-H), 4.86 (d, J),2 = 3.2 Hz, 2 H; Ia-H). w [0221] HRMS : calc. for C ₃₄ H ₆₃ N ₄ O ₂₂ S ₂ 943.3375 found 943.3356

(GaIa(1, 4)GalβS) ₂ TREN-CO(CH ₂ )MTS (100)

[0222] Amine 99 and 4 mg of active ester 95 were dissolved in 1 mL dry DMF and stirred under nitrogen at room temperature for 3.5 h. TLC (chloroform/methanol/water

6:4:1; R _f = 0.15) showed formation of a single product. Solvents were evaporated, the residue was chromatographed over a C18 cartridge with water/methanol 1-10%, concentrated and s lyophilized from water to yield 9 mg of a colorless foam (93%) (100).

[0223] NMR: sugar&TREN-part clearly assigned, MS (for C ₄₀ H ₇₂ N ₄ O _2S S ₄ 1036.34): 1137.9 [M+H] ⁺ , 1160.5 [M+Na] ⁺

Sl 56C-S-(CH ₂ )4-CO-TREN-(βGala( IA)GaI ₂ (102) o [0224] S 156C (5 mg) was dissolved in ligating buffer (0.5 mL; 7O mM CHES, 5 mM MES, 2 mM CaCl ₂ , pH 9.5) at room temperature and a solution of 2.2 mg (1.9 μmol) of 57 in water (100 μL) was added, vortexed for 20 sec and mixed on an end-over-end rotator for 40 min (Ellman's reagent negative). The mixture was purified over a Pharmacia PD-10 column (elution buffer: 5mM MES, 2mM CaCl ₂ ), dialyzed against water, 3 x (1 h, 2 L) at 4 s ⁰ C, then flash frozen and stored at -18 °C.

[0225] MS (positive mode): found: 27770 Da, calc: 27770 Da.

Methyl 4-O-(2, 3, 4, 6-tetra-0 -benzyl- a-D-galactopyranosyl)-2, 3, 6-tri-O-benzoyl- a-D- galactopyranoside o [0226] Fully protected disaccharide was synthesized following known procedures.

Methyl 4-0-(a-D-galactopyranosyl)-2,3,6-tri-0-benzoyl-a-D-galactopy ranoside

[0227] Protected disaccharide methyl 4-O-(2,3,4,6-tetra-O-benzyl-α-D- galactopyranosyl)-2,3,6-tri-O-benzoyl-α-D-galactopyranoside was dissolved in 500 mL s methanol and 500 mg Pd/C (10%) were added. The reaction flask was evacuated and flushed with hydrogen (3 times). The reaction mixture was stirred under hydrogen (balloon) at room temperature for 2 days. After filtering through Celite the reaction mixture was concentrated in vacuo; NMR and MS showed one remaining benzyl group. The crude product was taken up in methanol again and 500 mg Pd/C were added, the flask was evacuated and flushed with 0 hydrogen (3 times) and stirred under hydrogen over night. TLC (ethyl acetate/methanol 5:1) showed complete conversion. Work up as described above yielded 2.75 g (quant.) of debenzylated title compound.

[0228] Characterization: [a]ζ= +154 (c = 1, CDCl ₃ ); ¹ H-NMR (400 MHz, d4- methanol): δ = 3.27 (dd, J ₆₁₆ . = 10.7 Hz, / ₅ , ₆ = 5.2 Hz, 1 H, 6b-H), 3.33 (m, 1 H, 6'b-H), 3.48 (s, 3 H, OMe), 3.87 (dd, / _1>2 = 3.4 Hz, / ₂ , ₃ - 10.0 Hz, 1 H, 2b-H), 3.96 (dd, / ₃ ,4 = 2.7 Hz, / _2,3 = 10.0 Hz, 1 H, 3b-H), 4.02 (bs, 1 H, 4b-H), 4.16 (dd, / = 5.3 Hz, J = 5.6 Hz, 1 H, 5b-H), s 4.47 (dd, /5,6 = 5.0 Hz, / = 7.4 Hz, 1 H, 5a-H), 4.76 (dd, / ₅ , ₆ = 4.9 Hz, / ₆₎ 6' = 11.4 Hz, 1 H, 6a-H), 4.82-4.86 (m,l H, 6'a-H), 5.02 (d, 7 _1>2 = 3.4 Hz, 1 H, Ib-H), 5.23 (bs, 1 H, Ia-H), 5.76 (bs, 2 H, 2a-H, 3a-H), 7.35-7.67 (m, 9 H, Bz-H), 7.93 (d, J = 7.4 Hz, 2 H, Bz-ortho-H), 7.99 (d, J = 7.4 Hz, 2 H, Bzrortho-Η), 8.08 (d, J = 7.4 Hz, 2 H, Bz-ortho-H); ¹³ C-NMR (100 MHz, d4-methanol): δ = 54.8 (OMe), 61.1 (6b-C), 63.9 (6a-C), 69.3 (2b-C), 69.3 (5a-C), 69.4 (2a- 0 C), 70.0 (5b-C), 70.2 (2b-C), 70.9 (4b-C), 70.9 (3b-C), 71.0 (3a-C), 76.3 (4a-C), 97.9 (Ia-C), 101.6 (Ib-C), 128.6/128.6/128.7/129.5/129.6/129.8/129.9/130.1/133.4/133.6 (Bz-C), 166.3, 166.6, 166.7 (3 x CO); MS (ESI+): 669.3 [M+H] ⁺ , 691.3 [M+Naf.

Methyl ^O-il^A^-tetra-O-acetyl-a-D-galactopyranosyiyi^^-tri-O-benzo yl-a-D- s galactopyranoside (62)

[0229] "Tetraol" methyl 4-0-(α-D-galactopyranosyl)-2,3,6-tri-0-benzoyl-α-D- galactopyranoside was dissolved in 60 mL acetic anhydride and 30 mL pyridine and stirred at 35-20 °C (water bath) for 2 h. TLC (petroleum ether/ethyl acetate 1:1) indicated complete conversion. The mixture was concentrated and coevaporated with toluene (2x50 mL). 0 Purification by column chromatography (petroleum ether/ethyl acetate 2: 1) yielded acetylated compound 62 as a white foam (2.85 g; 85%).

[0230] ESI-MS: 854 [M+NHJ*, 859 [MH-Na] ⁺ .

Example 5 - Modification of Cysteine-Containing Protein with 2-(β-D- s galactopyranosyPethyl methanethiosulfonate

[0231] A cysteine-containing mutant of subtilisin Bacillus lentus, S156C, was modified with the glycoMTS reagent 2-(β-D-galactopyranosyl)ethyl methanethiosulfonate to give the glycoprotein S156C-SS-ethyl 2-(β-D-galactopyranose) following procedures outlined in Example 3. This resulted in a CMM enzyme ("gal-protease") illustrated in Fig. 18. 0

Preparation of 2-(β-d-Galactopyranosyl)ethyl methanethiosulfonate

[0232] A solution of NaOMe (0.104 m, 0.8 ml) was added to a solution of 2- (2,3,4,6-Tetra0-acetyl-β~D-galactopyranosyl)ethyl methanethiosulfonate 11 (778 nig, 1.71 mmol) in MeOH) (10 ml) under N ₂ . After 4 hours, the reaction solution was passed through a Dowex 50W(H ⁺ ) plug (3 x 1 cm, eluant MeOH), and the solvent removed to give 2- s bromoethyl β-D-galactopyranoside (450 mg, 92%) as a white solid which was used directly in the next step. NaSSO ₂ CH ₃ (180 mg, 1.34 mmol) was added to a solution of 2-bromoethyl β- D-galactopyranoside (290 mg, 1.01 mmol) in DMF (12 ml) under N ₂ and warmed to 5O ⁰ C. after 15 hours, the solution was cooled and the solvent removed. The residue was purified by flash chromatography (MeOH : ETOAc, 1:9 to give the title compound (229 mg., 71% as a o white foam). Characterization of the compound so produced is found in application serial number 09/347,029 "Chemically Modified Proteins with a Carbohydrate Moiety."

Example 5 - Catalytic Activity of Modified Enzymes

[0233] Lectin-mediated interactions between oral viridans group streptococci and s actinomyces may play an important role in microbial colonization of the tooth surface. Oral actinomycetes and streptococci freshly isolated from dental plaque are known to coaggregate via lactose-reversible cell-cell interactions. This finding suggests that the coaggregation is mediated by a network of lectin-carbohydrate interactions between complementary cell surface structures on the two cell types. Kolenbrander PE, Williams BL., "Lactose-reversible 0 coaggregation between oral actinomycetes and Streptococcus sanguis" Infect Immun. 1981 Jul;33(l):95-102.

[0234] The presence of two host-like motifs, either GaINAc betal~>3Gal (Gn) or Gal beta 1— >3 GaIN Ac (G), in the cell wall polysaccharides of five streptococcal strains, including S. sanguis, accounts for the lactose-sensitive coaggregations of these bacteria with s Actinomyces naeslundii. Cisar JO, Sandberg AL, Reddy GP, Abeygunawardana C, Bush CA, "Structural and antigenic types of cell wall polysaccharides from viridans group streptococci with receptors for oral actinomyces and streptococcal lectins," Infect Immun. 1997 Dec;65(12):5035-41. The S. sanguis receptor for the actinomyces lectin comprises repeating hexasaccharide units with Galactose, N-acetylgalactosamine (GaINAc) termini. The 0 agglutination of oral streptococci strains by the actinomyces lectin activity blocks attachment of actinomyces to epithelial cells, and this is thought to inhibit the killing of actinomyces by polymorphonuclear leukocytes. Mergenhagen SE, Sandberg AL, Chassy BM, Brennan MJ,

Yeung MK, Donkersloot JA,Cisar JO, "Molecular basis of bacterial adhesion in the oral cavity.'Ttev Infect Dis. 1987 Sep-0ct;9 Suppl 5:S467-74.

[0235] The ability of two different glycodendrimer proteins to inhibit the lectin activity of the bacteria A. naeslundii was tested to determine whether the attachment of one or two galactose moieties to the enzyme would modify the substrate specificity of the enzyme so that it can recognize and digest the lectin.

[0236] A coaggregation experiment was carried out according to methods similar to those described in Kolenbrander PE, Williams BL., "Lactose-reversible coaggregation between oral actinomycetes and Streptococcussanguis," Infect Immun. 1981 Jul;33(l):95- 102. A. naeslundii was pre-treated with subtilisin Bacillus lentus protease or S156C-SS-ethyl 2-(β-D-galactopyranose) ("gal-protease") (enzyme concentration 50 ug/ml) in the presence or absence of lactose (60-300 ug/ml), and the ability of the treated A. naeslundi to co-aggregate w/ S. sanguis was determined by microscopic evaluation. The amount of coaggregation (i.e., lectin activity), from highest to lowest is listed in Table 1, below.

Table 1 Co Aggregation of A. naeslundii and S. sanguis

A. naeslundii treated with protease untreated A. naeslundii (50 ug/ml) -

A. naeslundii treated with gal-protease (50 ug/ml)

A. naeslundii treated with protease (lactose in the enzyme reaction mix)

A. naeslundii treated with gal-protease (lactose in the enzyme reaction mix)

[0237] In a second experiment, the ability of the protease, the gal-protease and the di-gal protease to block attachment of A. naeslundii to human buccal epithelial cells was tested. The epithelial cells were treated with C. perfringens neuraminidase to remove terminal sialic acid residues (thus exposing galactose). A. naeslundii were incubated with one of the three proteases at a protease concentration of 10 ug/ml in the presence or absence of lactose. Attachment was assayed by labeling the bacteria with a fluorescein tag that is internalized by the bacteria (thereby not disturbing the bacteria's adhesive structures (i.e., the surface fimbrae). Following incubation of the buccal cells with the fluorescein-labeled bacteria, the number of bacteria adhering to the buccal cells was analyzed by running the reaction mix through a flow cytometer. The counts shown below in Table 2, are average

counts per buccal cell, and so roughly correspond to the number of bacteria attaching to each cell.

[0238] The results of this assay demonstrate that di-gal protease produces a greater reduction in the number of A. naeslundii adhering to the buccal epithelial cells, as compared to untreated bacteria and those treated with the control S156C protease. Interestingly, and in contrast to the coaggregation assay results, the presence of lactose appears to potentiate the binding of A. naeslundii to human buccal epithelial cells under the conditions of this assay.

[0239] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.