BACKGROUND OF THE INVENTION The biological relevance of oligosaccharide chains of glycoproteins and glycolipids is increasingly evident. Since both are found on cell surface membranes and circulating in biological fluids, these glycosidic residues act as recognition signals that mediate key events in normal cellular development and function. They are involved in embryogenesis, hormonal activities, neuronal development, inflammation, cellular proliferation, fertilization and the organization of different cell types into specific tissues. They are able to regulate the transportation of proteins between cells and should be regarde as signal substances in metabolism. They also participate in intracellular sorting and secretion of glycoproteins, as well as in the clearance of plasma glycoproteins from circulation, and numerus oligosaccharides are implicated in cell-cell recognition, cellular-immune response, cell oncogenic transformation and inflammation and other cell biology phenomena. See Sharon et al., Sci. Am. :268 82-89 (1993); Karlsson, Trends Pharm. Sci. , 12 (7): 265-272 (1991); Drickamer et al. , Curr. Opn. Strict Biol. , 2 (5): 653-654 (1992).

Oligosaccharides are also involved in the prevention and treatment of diseases.

Compound of this class include glycosamine and macrolide antibiotics, anthracycline and enediyne anticancer antibiotics. For instance, oligosaccharides on cell surfaces function as receptors for viruses, toxins, infectious bacteria, hormones, pathogens, enzymes, proteins, as well as more benign ligands. Cell surface carbohydrates, which had been modifie, were

implicated in tumorigenesis and metastasis. The oligosaccharide structures mediate migration of cells during embryo development, process of infection and other phenomena. <BR> <BR> <P>Rademacher et al. Annu. Rev. Biochem. , :57 785 (1988); Feizi et al. , TIBS, 24 (1985);<BR> <BR> Hakomori, TIBS, 45 (1984); Feizi TIBS, 84 (1991); Dennis and Laferte Cancer Res. , :45<BR> <BR> 6034 (1985); Markwell et al. Proc. Natl. Acad Sci. USA, :78 5406 (1981); Wiley and Skehel,<BR> <BR> J Annu. Rev. Biochem. , :56 365 (1987); Walz et al. , Science, 250 (1990); Kleinman et al.<BR> <BR> <P> Proc. Natl. Acad. Sci. USA, :76 3367 (1979).

There has been continuing efforts to develop products related to oligosaccharides, including drug delivery vehicles that recognize carbohydrate receptors, vaccines to block infection by viruses that recognize cell surface carbohydrates, diagnostic kits for identifying, detecting and analyzing carbohydrates associated with various diseases, and monoclonal antibodies, which recognize anormal carbohydrates, for use as drugs. The development of these and other carbohydrate-based biomedical products depends on the availability of technology to produce oligosaccharides and other glycoconjugates rapidly, efficiently, and in practical quantities for basic and development research.

More particularly, there is a need for methods that permit the rapid preparation of glycosidic libraries comprising mixtures of various oligosaccharides and other glycoconjugates, which could then be screened for a particular biological activity. For example, it has been shown that screening of mixtures of peptides is an efficient way of identifying active compound and elucidating structure-activity relationships. There are numerus ways to generate chemically diverse mixtures of peptides and determine active compound. See, for example, Zuckermann et al. , Proc. Natl. Acad. Sci. USA, 89: 4505 <BR> <BR> (1992); Lam et al. , Nature, :354 82 (1991); Houghten, Nature, :354 84 (1991); Petithory,<BR> <BR> <BR> Proc. Natl. Acad. Sci. USA, :88 11510 (1991); Geyse, Proc. Natl. Acad. Sci. USA, :81 3998<BR> <BR> (1984); Houghten, Proc. Natl. Acad. Sci. USA, :82 5131 (1985); Fodor, Science, :251 767 (1991).

Oligosaccharide synthesis is by far one of the most challenging fields in modem organic chemistry. Efficient construction of an oligosaccharide or a glycoconjugate involves attaching a glycosyl moiety to a specific position of a glycosyl acceptor not only in high yield but also with high stereocontrol. Based on this challenge, several glycosylation methods have been developed.

Classical donors in glycosylation are glycosyl halides. Nicolaou et al. , Preparative Carbohydrate Chemistry, Hanessian, S. ed., Marcel Dekker, Inc. (1997) reviewed oligosaccharide synthesis from glycosyl fluorides. One of the oldest methods of glycosylation is Koenigs-Knorr method, which inclues activation of the anomeric center by decomposition of the glycosidic halides, e. g., bromide or chloride, in the presence of salts of heavy metals such as silver or mercury. However, problems may arise in the conversion of many oligosaccharide derivatives such as glycosides or glycosyl esters into glycosyl halides resulting in low yielding steps quite far into synthetic sequence. In addition, due to harsh conditions needed for the generation of glycosyl halides, and their low thermal stability and high sensitivity to hydrolysis, other methods had to be developed, such as the use of trichloroacetamidate as a glycosyl donor under Lewis or protic acid conditions. The glycosylation proceeds with inversion of configuration, unless a participating group is present <BR> <BR> <BR> <BR> at C-2 in which case a ß-glycoside is obtained. Schmidt et al. , Tetrahedron Lett. , :25 821 (1984).

Several variations of glycal glycosidation method were pioneered by Lemieux et al., <BR> <BR> <BR> <BR> Can. J. Chem. , :43 2190 (1965); Thiem et al. , Synthesis, 696 (1978); Sinay et al. , J. Chem.<BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Soc. Chem. Commun., p. 572 (1981); Ogawa et al. , Tetrahedron Lett. , :28 2723 (1987) and<BR> <BR> <BR> <BR> <BR> <BR> <BR> Danishefsky et al. , J. Am. Chem. Soc. , :111 6661 (1989). In this method, an electrophile (E) is used to attack the electron rich glycal as a means to activate the anomeric center, which then reacts with the carbohydrate acceptor to afford the glycoside. Removal of the substituent at C-2 then leads to 2-deoxy glycoside.

The n-pentenyl glycoside method was introduced by Fraser-Reid et al., J Chem. Soc. <BR> <BR> <BR> <BR> <P>Chem. Commun., p. 823 (1988). This method depends on electrophilic addition to olefin followed by intramolecular displacement by the ring oxygen and eventual expulsion of pentenyl chain to form an oxonium species. Trapping with a glycoside acceptor leads to the desired glycoside.

Schmidt et al. , Tetrahedron Lett. , :33 6123 (1992) described a relatively new method which involves glycosyl phosphite as glycosyl donor. The method has found important applications in synthesis of sialyl derivatives.

Thioglycosides have attracted considerable attention as glycosyl donors. Thioalkyl or aryl groups offer efficient temporary protection of the anomeric center and, at the same time,

can be selectively activated under various conditions. Many glycosylation rections using thioglycosides have been reporte. Early attempts to use thioglycosides as glycosyl donors include activation by mercury (II) sulfate, Ferrier et al., J Glycoconjugate, :4 97-108 (1987); <BR> <BR> mercury (II) chloride, Tsai et al., Can. J Chem. , :62 1403-5 (1984); phenylmercury<BR> <BR> trifluoromethanesulfonate, Garegg et al., Carbohyd. Res. , :116 162-65 (1983); mercury (II)<BR> <BR> benzoate, van Cleve, Carbohyd. Res. , :116 162-65 (1983); mercury nitrate, Hanessian et al.,<BR> <BR> Carbohyd. Res. , :80 Cri7-22 (1980); silver triflate, Hanessian et al. , Carbohyd. Res. , :53 C13 (1977); copper (II) trifluoromethanesulfonate, Mukaiyama et al. , Chem. Lett. , 487-90 (1979); N-bromosuccinimide (NBS), Hanessian, supra and Nicolaou et al., J. Am. Chem. Soc., <BR> <BR> 105: 2430-34 (1982); lead (II) perchlorate, Woodward et al., J. Am. Chem. Soc. , :103 3215-17<BR> <BR> (1981); benzeneselenenyl triflate, Ito et al. , Tetrahed. Lett. , 1061-64 (1988), methyl triflate,<BR> <BR> Lonn, Carbohyd. Res. , :39 105-113 (1985); dimethyl (methylthio) sulfonium triflate (DMTST), Andersson et al. , Tetrahed. Lett. 3919-3922 (1986); benzenesulphonyl <BR> <BR> derivatives, Brown et al., Tetrahed. Lett., 29/38: 4873-4876 (1988); alkyl sulfonyl triflate,<BR> <BR> Dasgupta et al., Carbohyd. Research, :177 c13-c17 (1988). These various promoters did not yield results that led to their subsequent widespread use in oligosaccharide synthesis. Heavy metals are not reactive enough for general application. Using more reactive heterocyclic thioglycosides circumvented the problem. Hanessian et al. and Mukaiyama et al. supra.

A method of glycosidation using thioglycosides was developed by Kahne et al., in J <BR> <BR> Am. Chem. Soc., 111: 6881-2 (1989) in which the anomeric sulfide is first oxidized to the corresponding sulfoxide and then activated by the addition of triflic anhydride in the presence of glycosyl acceptor to yield glycosides. The triflic anhydride-activated glycosyl donors proved to be quite reactive in solution and could be used to glycosylate extremely unreactive substrats under mild conditions.

Kahne et al., U. S. 5,635,612 and U. S. 5,638,866, the contents of which are incorporated herein by reference, describe a method of forming multiple glycosidic linkages in solution in a single step using anomeric sugar sulfoxide as a glycosyl donor and for constructing sequential glycosidic linkages on solid phase, as well as solution phase. The activating agents include strong acids such as trifluoromethane sulfonic acid or triflic acid (TfOH), p-toluenesulfonic acid or methane sulfonic acid.

Recent work has shown that thioglycosides can be conveniently and reproducibly

activated in at least two other different techniques. The first is a"two step"activation which involves first forcing a glycosyl halide, and then further activating this with a halophilic ragent. Sato et al., Carbohyd Res. , :155 C6-10 (1986).

A two-stage activation procedure which employs combining glycosyl fluoride and sulfides for oligosaccharide synthesis was developed by Nicolaou et al., J. Am. Chem. Soc., <BR> <BR> <BR> <BR> 106: 4189 (1984). Glycosyl fluoride was described as a glycosyl donor by Mukaiyama et al.,<BR> <BR> <BR> <BR> <BR> <BR> <BR> Chem. Lett., p. 431 (1981). The method included activation and coupling of carbohydrate intermediates to glycosyl acceptors in the presence of silver perchlorate and tin dichloride.

The mechanism of activation is similar to Koenigs-Knorr process and so is the stereoselectivity of rection. In the presence of an equatorial participating group at C2, ß glycosides are formed. In absence of such moiety, a anomers are the predominant products. <BR> <BR> <BR> <BR> <P>Thioglycosides were described as glycosyl donors by Ferrier et al. , Carbohyd. Res. , :127 157<BR> <BR> <BR> <BR> <BR> <BR> <BR> (1984) using an ethylthio group at the anomeric position. Hanessian et al., Carbohyd Res.,<BR> <BR> <BR> <BR> <BR> <BR> <BR> 80: C17 (1980) demonstrated the use of 2-pyridylthioglycosides. The strategy of Nicolaou et<BR> <BR> <BR> <BR> <BR> <BR> <BR> al., J. Am. Chem. Soc. , :106 4189 (1984) employs a stable phenylthioglycoside as the key building block. The phenylthioglycoside is converted to the more reactive glycosyl fluoride by treatment with NBS and diethyl amino sulfur trifluoride (DAST). In the second activation, the glycosyl fluoride is coupled with the glycosyl acceptor which carries phenylthio at the anomeric position in the preparation of oligosaccharide chains. The two stage activation procedure is particularly suited for solid phase oligosaccharide synthesis.

The second technique involves a one step activation with a thiophilic reagent such as methyl triflate or dimethyl (methylthio) sulfonium trifluoromethane sulfonate (DMTST).

Complete stereospecificity is only achieved for the 1,2 trans bonds due to the use of neighboring group participation ftom 2-acyl substituent for controlling the steric outcome of the rection. DMTST is a highly thiophilic promoter in the synthesis of 1,2 trans glycosides using various thioglycosides with participating 2-substituents as glycosyl donors. Fugedi et al., J. Glycoconjugate, :4 97-108 (1987). It has also been used in the synthesis of 1,2 cis glycosides, Andersson et al., Tetrahed. Lett. , :27 3919-3922 (1986).

ß-selective glycosylation in the absence of neighboring group participation has been <BR> <BR> <BR> performed using insoluble silver catalysts. Garegg et al., Carbohyd. Res. :70 C13 (1979) or<BR> <BR> <BR> <BR> <BR> <BR> <BR> solvents with cation interacting ability such as acetonitrile, Hashimoto et al., Tetrahed. Lett.,

25: 1379 (1984).

The rection of sulfenate esters as glycosyl acceptors with benzylated methyl or phenyl l-thio-ß-glucopyranoside carried out in the presence of Lewis acids such as 0 CF3SO3SiMe3, BF3.Et2Oor at-35 C generally afforded α-ß mixtures. Ito et al., Tetrahed. Lett., 28:4701-4704 (1987). The stereoselectivity was highly dependent on the solvent.

Another method of thioglycoside activation developed by Sinay et al., Pure Appel.

Chem. 63: 519 (1991) involves electron transfer from sulfur to the activating agent tris (a- bromophenyl) ammoniumyl hexachloroantimonate (TBPA). The generated glycosyl radical cation suffers cleavage to a thiyl radical, leaving behind an oxonium species which then undergoes glycosidation.

Garegg et al., Carbohyd. Res., 116: 162-65 (1983) described the use of benzylated or acylated phenyl-l-thio-ß-D-glucopyranoside or l-thio-ß-D-galactopyranoside as glycosyl donors. Similarly, Van Cleve, Carbohyd. Res., 70: 161-164 (1979) described the use of phenyl-l-thio-ß-D-glucopyranoside as a glycosyl donor. Ferrer et al., J : Glycoconjugate, 4: 97-108 (1987) described the use of benzylated phenylthioglycoside as a glycosyl donor.

The preceding methods yield both the a-and ß-anomers.

Consequently, there is a need for improving the stereoselectivity of the glycosyl donors and stability and yield of the glycosylated product. The sulfoxide glycosylation method of the present invention has been shown to be successful in both solution and solid phases. It allows the glycosylation of sensitive and/or unreactive substrats at low temperatures and under essentially neutral conditions, with high degree of stereoselectivity in general.

SUMMARY OF THE INVENTION The present invention is generally directe to a process for the synthesis of ß- oligosaccharides, including (3-disaccharides and their conjugates, which process comprises reacting a glycosyl donor and a glycosyl acceptor. In particular, the invention is directe to a process for the synthesis of ß-oligosaccharides using alkylsulfenyl-or arylsulfenyl- 2-deoxy-2-N-trifluoroacetamido glycopyranose as a glycosyl donor in a sulfoxide-mediated glycosylation rection whether in solution or in the solid phase. Glycosyl donors, including

the preferred tert-butylsulfenyl-or phenylsulfenyl-2-deoxy-2-N-trifluoroacetamido glycopyranose, are described, along with methods for the preparation of same.

A further aspect of the invention is to provide a process for constructing glycosidic linkages using arylsulfenyl-or alkylsulfenyl-2-deoxy-2-N-trifluoroacetamidoglycopyranose as glycosyl donors. By utilizing selected conditions and starting materials, such as galactosamine hydrochloride and glucosamine hydrochloride, ß-anomers (i. e., ß-glycosidic linkages) can be produced on a solid phase using anomeric sugar sulfoxides as glycosyl donors. The process of the present invention may also be applied to the preparation of specific oligosaccharides or glycoconjugates or to the preparation of mixtures of various oligosaccharides or glycoconjugates for the creation of glycosidic chemical libraries that can subsequently be screened to detect compound having a desired biological activity.

In another aspect, the invention is directe to a process for the synthesis of ß-glycosides in high yield in the substantial absence of a-isomers.

In yet another aspect, the invention relates broadly to ß-selectivity in glycosylation using a glycosyl donor with a C-2 protecting group capable of neighboring group participation, such as an amide, an ester, an imide or a carbamate.

Another aspect of the invention relates to the design, construction and analysis of a combinatorial library comprising a plurality of the novel compound of the invention, including the salts and conjugates thereof, preferably one bound to a solid phase.

Other aspects of the present invention will be apparent to one of ordinary skill on consideration of the present disclosure.

BRIEF DESCRIPTION OF FIGURES Figure 1 illustrates a process for the synthesis of phenylsulfenyl-2-deoxy-2-N- trifluoroacetamido-3,4,6-tri-O-acetyl- (3-D-glucopyranose(6).

Figure 2 illustrates a process for the synthesis of phenylsulfenyl-2-deoxy-2-N- trifluoroacetamido-3,4, 6-tri-O-acetyl-ß-D-galactopyranose (12).

Figure 3 illustrates acceptors 13-15 immobilized on Rink Amide polystyrene resin.

Figure 4 illustrates characterized products 16-18 in the solid phase glycosylation rections.

Figure 5 illustrates'H NMR spectra of the rection products which indicated that only

the (3-disaccharide was formed (no trace of the corresponding a-isomer was observe).

Figure 6 illustrates the solid-phase derivatization of 16.

Figure 7 illustrates analytical characterization (HPLC and LC-MS) of intermediates and final product in the derivatization of 16.

Figure 8 illustrates the general structure and building blocks used in the combinatorial library based on 16.

Figure 9 illustrates the general structure and building blocks used in the combinatorial library based on 17.

Figure 10 illustrates the rection sequence used in the derivatization of 16 to generate a library.

Figure 11 illustrates the rection sequence used in the derivatization of 17 to generate alibrary.

Figure 12 illustrates the LC-MS spectrum of a representative member of the combinatorial library constructed around 16.

Figure 13 illustrates the analytical data obtained for the library constructed around 16.

Figure 14 illustrates the LC-MS spectrum of a representative member of the combinatorial library constructed around 17.

Figure 15 illustrates the analytical data obtained for the library constructed around 17.

DETAILED DESCRIPTION OF THE INVENTION The following glossary is provided as an aid to understand the use of certain terms used herein. The definitions provided are for explanatory, illustrative purposes only. They should not be used to narrowly construe or unduly limit the scope of the invention, which invention is limited only by the disclosure of the prior art.

Oligosaccharide: An oligomeric saccharide (or carbohydrate) containing more than one monosaccharide (or sugar) units linked through glycosidic bonds. An oligosaccharide may be called a disaccharide, a trisaccharide, tetrasaccharide etc. depending on the number of monosaccharides it will yield upon hydrolysis. As used herein, an oligosaccharide may include its conjugates (i. e., compound comprising the mono-, di-, tri-, etc. saccharide covalently bound to another non-sugar chemical moiety, including antitumor agents, macrolides, other natural products, amino acids, peptides, nucleosides, oligonucleotides and

the like).

Glycoside: Any sugar containing at least one pentose or hexose residue in which the anomeric carbon bears a non-hydrogen substituent. Typically, the non-hydrogen substituent is a heteroatom, such as nitrogen, oxygen, phosphorus, silicon, or sulfur.

Glycosidic link (also glycosidic bond or linkage): The link, bond, or linkage formed by the rection of a sugar, such as an aldose or ketose, in cyclic form with an alcohol ROH to form a mixed acetal or glycoside. When the alcohol is itself the hydroxyl group of a monosaccharide, the product is a disaccharide. This disaccharide may similarly react further to form a higher oligosaccharide and eventually a polysaccharide. Thus, the monosaccharide units of a polysaccharide are linked through glycoside bonds. The bond may be formed by rection of either the C-1 or C-2 hemiacetal hydroxyl with any of the hydroxyl groups of the other monosaccharide. The bond may be formed in such a way that the anomeric carbon has either configuration. Conventionally, in the standard ring orientation (the hemiacetal carbon atom lying at the extreme right-hand side of the structure) in a ß-glycosidic linkage, the link points above the plane of the ring and in an a-glycosidic linkage, the link points below the plane of the ring.

Activating agent: A chemical agent that on addition to a glycosyl sulfoxide reacts with the anomeric sulfoxide group, thus rendering the anomeric carbon susceptible to nucleophilic attack. In the case of bifunctional sugars or glycosidic residues, the activating agent is also able to deprotect a blocked nucleophilic group under the same conditions used to activate the anomeric sulfoxide group.

Glycosyl acceptor: Any compound that contains at least one nucleophilic group which, under the conditions of the process of the present invention, is able to form a covalent bond with the anomeric carbon of a glycosyl donor. As referred to herein, a glycosyl acceptor is any organic molecule, including a sugar, that contains unprotected hydroxyl, amino, or mercapto groups or such groups that are blocked by protecting groups that can be removed in situ, i. e., under the rection conditions of the present invention.

Glycosyl donor: A sugar or glycosidic residue that bears a sulfoxide group at the anomeric carbon, which group on activation renders the anomeric carbon susceptible to attack by the nucleophilic group of a glycosyl acceptor to form the glycosidic linkage. A preferred glycosyl donor is phenylSulfenyl-2-deoxy-2-N-trifluoroacetamidoglycopyranose. It is

important to point out that one of ordinary skill will appreciate that the donor glycoside may be a monosaccharide, a disaccharide, a trisaccharide, etc. Hence, the chemical libraries that can possibly be produced with the methods and compound of the present invention include all forms of oligosaccharides or their conjugates or glycoconjugates.

Glycosidic libraries: A mixture, collection, or a plurality of oligosaccharides of varying sequences which can be subjected to a screening procedure to identify compound or molecules that exhibit biological activity. Such chemical libraries may also include various conjugates or glycoconjugates.

Glycoconjugate: Any compound or molecule that comprises a non-sugar moiety that is covalently bound to a glycosidic residue. See, also, the definition of oligosaccharide, supra.

Protecting group: A blocking or protecting group that can be removed in situ, preferably, but not necessarily, under the same conditions used to activate an anomeric sulfoxide group. It refers to moities ordinarily used in oligosaccharide synthesis to prevent rection of the hydroxyl or amino groups in the rection being conducted. A preferred protecting group is the trifluoroacetyl moiety.

Blocking group: Similar to a protecting group, a blocking group is used to prevent the inappropriate rection of a functional group of interest. The terms protecting group or blocking group can be used interchangeably.

The term"substantially free"may refer to the glycosylated product formed under the conditions of the present invention, which in a preferred embodiment of the invention, exclues the presence of significant amounts of a-anomer.

In the present sulfoxide glycosylation method, the use of glycosyl sulfoxide donors based on galactosamine and glucosamine are investigated. More particularly, sulfoxide donors, which afford high ß-selectivity, are developed. Generally, ß-selectivity in glycosylation rections is achieved by using a glycosyl donor with a C-2 protecting group capable of neighboring group participation, including but not limited to amides, esters, imidos, or carbamates. In the case of glucosamine and galactosamine-based sulfoxides, the nature of this protecting group at C-2 is important not only because the sulfoxide should be reactive enough to glycosylate relatively unreactive nucleophiles, but also because this group should be easily removed to allow subsequent derivatization of the amino functionality.

In one embodiment of the process for the synthesis of ß-oligosaccharides, a glycosyl acceptor is allowed to react with a glycosyl donor, which is phenylsulfenyl-2-deoxy-2-N- trifluoroacetamido-3,4, 6-tri-O-acetyl-ß-D-glcoypranose, either in solution or in the solid phase.

Another embodiment of the invention is a process for the synthesis of a glycosyl donor, which is phenylsulfenyl-2-deoxy-2-N-trifluoroacetamido-3, 4, 6-tri-O-acetyl-p-D- glycopyranose. These donors are found to afford the ß-glycosides exclusively and in high yield. The trifluoroacetamido protecting groups are then removed under mild conditions and the resulting 2-amino groups are selectively derivatized as amides. Thus, this protecting group newly used in this chemistry adds flexibility to the sulfoxide glycosylation method.

In yet another embodiment of the invention, a process is provided for the synthesis of phenylsulfenyl-2-deoxy-2-N-trifluoroacetamido-3,4,6-tri-O-ac etyl-ß-D-glycopyranose comprising the steps of: a) reacting glycosamine hydrochloride with p-methoxybenzaldehyde in the presence of alkali to form 2-N-p-methoxybenzylidene glycosamine; b) acetylating 2-N-p-methoxybenzylidene glycosamine with acetic anhydride in the presence of pyridine and dimethylaminopyridine (DRAP) to form O-acetylated 2- N-p-methoxybenzylidene glycosamine; c) removing the p-methoxy benzaldehyde group with hydrochloric acid in acetone to form O-acetylated glycosamine hydrochloride; d) protecting the O-acetylated glycosamine hydrochloride with trifluoroacetic anhydride in the presence of pyridine and methylene chloride to form O-acetylated 2- N-trifluoroacetamido glycopyranose; e) subjecting the O-acetylated trifluoroacetamido glycopyranose to thiophenol, boron trifluoride etherate and methylene chloride to form phenyl-l-thio-O-acetylated 2-N-trifluoroacetamido glycopyranose; f) reacting phenyl-l-thio-O-acetylated 2-N-trifluoroacetamido glycopyranose with m-chloroperoxybenzoic acid to yield the corresponding phenylsulfenyl-2-deoxy- 2-N-trifluoroacetamido-3,4, 6-tri-O-acetyl-ß-D-glycopyranose ; and g) recovering the glycopyranose.

The preparation of the tert-butylsulfenyl counterpart can be accomplished in the same fashion by using thiotert-butanol reagent in place of thiophenol ragent. The rection solvent plays a role in the stereoselectivity of glycosylation in the absence of neighboring group participation. If a non-polar, aprotic solvent is used, the selectivity for a-glycosidic bond formation is increased while the use of a polar, aprotic solvent such as propionitrile increases selectivity for ß-glycosidic bond formation.

The protecting groups on the glycosyl donor also have an impact on the stereochemical course of the glycosylation rection. When the protecting group at the equatorial position of the C-2 center of the glycosyl donor is trifluoroacetamido, only ß- glycosidic bonds are formed in the glycosylation process, regardless of whether an aprotic, non-polar solvent or an aprotic, polar solvent is used for the rection. A large number of functionalities suitable for use as protecting groups of an amino group are disclosed in T. W.

Greene, Protecting Groups in Organic Synthesis, John Wiley & Sons. Suitable protecting groups include carbamates such as 9-fluorenylmethoxycarbonyl (Fmoc); and allyloxycarbonyl (Alloc); imides such as phthalimido (Phth) and tetrachlorophthalimido (PhthCl); or amides such as trifluoroacetamido (TFA). Of the many possible protecting groups for use on glycosyl donors, the present inventors found that TFA offered avantages which would not have been predicted. First, the corresponding glycosyl sulfoxides were shown to be very reactive and afford high glycosylation yields in both solution and solid phases. Second, analysis of the rection products indicated that the ß isomers were formed, with no obvious trace of the a isomers. Third, the trifluoroacetamido group was deprotected under mild conditions and the corresponding amino group was derivatized as an amide.

The TFA group is easily removed by treatment with LiOH in an anhydrous mixture of 50: 50 MeOH-THF. This procedure is compatible with solid phase procedures. Under the conditions of the present invention, all common ester groups are also removed. This is not a drawback since amines can be selectively transformed into amides in the presence of unprotected alcools.

Solution Methods for Obtaining Oligosaccharides The two general methods for obtaining oligosaccharides are: a) isolation from natural sources. This approach is limited to naturally occurring oligosaccharides that are produced in

large quantities; and b) enzymatic or chemical synthesis. Enzymatic synthesis is limited because enzymes are highly specific and can only accept certain substrates. In contrast, chemical synthesis is more flexible than enzymatic synthesis and has the potential to produce an enormous variety of oligosaccharides. The problem with chemical synthesis has been that it is extremely expensive in terms of time and labor.

Oligosaccharides are formed of monosaccharides connecte by glycosidic linkages.

In a typical solution-phase chemical synthesis of an oligosaccharide, a fully protected glycosyl donor is activated and allowed to react with a glycosyl acceptor (typically another monosaccharide having an unprotected hydroxyl group) in solution. The glycosylation rection itself can take anywhere from a few minutes to days, depending on the method used.

The coupled product is then purifie and chemically modifie to transform it into a glycosyl donor. Each purification is time consuming and can result in significant losses of material.

The new glycosyl donor, a disaccharide, is then coupled to another glycosyl acceptor. The product is then isolated and chemically modifie as before. It is not unusual for the synthesis of a trisaccharide to require ten or more steps from the component monosaccharides. Thus, the time and expense involved in the synthesis of oligosaccharides has been a serious obstacle to the development of carbohydrate drugs and other biomedical products.

Solid-Phase Synthesis of Oligosaccharides The solid phase synthesis of oligosaccharides eliminates the need for isolation and purification.

According to Frechet and Schuerch, J. Am. Chem. Soc, :93 492-496 (1971), solid phase synthesis of oligosaccharides requires: a) use of a saccharide derivative with a reactive leaving group at Cl; b) one hydroxyl group protected by a readily removable blocking group; c) the remaining hydroxyls protected by a stable blocking group; and d) a resin from which the formed oligosaccharide derivative can be separated without product degradation.

By previous attempts to synthesize oligosaccharides on insoluble resins, the coupling yields were low and the stereochemical control was inadequate, particularly for the construction of ß-glycosidic linkages. This is apparently because the rection kinetics on the solid phase are slower than they are in solution phase. See, Eby and Schuerch, Carbohydr. Res, :39 151-155 (1975). As a consequence, factors such as stereochemical control and yield

are affecte. Frechet and Schuerch found that two glycosylation rections, which both involve the displacement of an anomeric halide in the presence of a catalyst, gave predominantly the ß-anomer in solution but gave mixtures on the solid phase. It was concluded that it would be necessary to use neighboring group participation to form ß-glycosidic linkages on the solid phase.

However, it has been found that existing glycosylation methods could not be adapted to the solid phase because neighboring participating groups (NPGs) frequently deactivate glycosyl donor. Frequently, glycosyl donors would decompose in the resin mixture before glycosylation could take place. See, Eby and Schuerch, supra. In some instances, the resin has also been known to decompose due to the harshness of the conditions required for glycosylation. Furthermore, for many ester-type NPGs, there is a significant problem with acyl transfer from the glycosyl donors to the glycosyl acceptors on the resin. This side rection caps the resin and prevents further rection.

Frechet has reviewed the problems encountered in trying to implement a strategy for solid-phase oligosaccharide synthesis. See, Frechet, Polymer-supported Rections in Organic Synthesis, p. 407, P. Hodge and D. C. Sherrington, Eds., John Wiley & Sons, 1980.

Soluble resins were employed to overcome the unfavorable rection kinetics associated with solid-phase rections. Douglas et al., J. Am. Chem. Soc., 113: 5095 (1991) used a soluble polyethylene glycol resin with a succinic acid linker and achieved 85-95% coupling yields using the Koenigs-Knorr rection with excellent control of anomeric stereochemistry. Soluble resins may have avantages for some glycosylation rections because they offer a more"solution-like"environment. However, step-wise synthesis on soluble polymers requires that the intermediate be precipitated after each step and crystallized before another sugar residue can be couple.

Thus, there is need for a glycosylation method which provides for a rapid, efficient, and high yield solid phase synthesis of oligosaccharides.

Formation of Glycosidic Linkages in Solution A glycosyl donor having alkyl or aryl sulfoxides at the anomeric position and a glycosyl acceptor having one or more free hydroxyls and/or other nucleophilic groups (e. g., amines) and/or silyl ether protected hydroxyls are combine in a rection vessel.

The glycosyl donor is blocked by a suitable protecting group such as TFA at the C-2 position resulting in a 1,2-trans glycosidic bond.

A mixture of glycosyl donors and acceptors is dissolve under anhydrous conditions in a non-nucleophilic solvent, including, but not limited to toluene, ether, tetrahydrofuran (THF), methylene chloride, chloroform, propionitrile, ethyl acetate or mixtures thereof. It has been found that the choice of solvent influences the stereochemical outcome of glycosylation for rections in which neighboring group participation is not involved. In general, for a given donor/acceptor pair, the use of a non-polar solvent, such as toluene, results in the formation of a higher percentage of a-isomer, while the use of a more polar solvent, such as propionitrile, results in formation of a higher percentage of the ß-anomer.

Yet in other embodiments of the present invention, it may be desirable to include several different types of sugars in the rection mixture in order to generate a chemically diverse mixture of oligosaccharides or glycoconjugate products for the creation of libraries that may be screened for biological activity.

Formation of Glycosidic Linkages on the Solid Phase A glycosyl acceptor is attache to an insoluble support (hereafter terme the resin) through a linkage that can be readily cleaved at the end of the synthesis using conditions that do not damage glycosidic linkages. The resin may be any insoluble polymer that swells in organic solvents and has sites for attaching the glycosyl acceptor. Preferred resins inclue, but are not limited to, polystyrene resins, such as the Merrifield resins, and PEG-derivatized polystyrene resins, such as the TentaGelTm resins. The type of linkage depends on the type of functional sites available on the polymer phase and on the glycosyl acceptor.

The glycosyl acceptor may be any molecule having one or more reactive nucleophile including reactive hydroxyls, amines, and/or thiols, provided that it also has a suitable site for attachment to the resin. A reactive nucleophile is a free nucleophile or a nucleophile with a temporal protecting group that can be removed readily once the glycosyl acceptor is attache to the resin. The glycosyl acceptor may also have permanently protected nucleophiles, which are nucleophiles that cannot be deprotected under the conditions that are used to remove the temporal protecting groups. The glycosyl acceptor may be a sugar or some other nucleophile-bearing molecule, including, but not limited to, steroids, amino acids or peptides,

polar lipids, polycyclic aromatic compound, macrolides, natural products and the like.

Preferred acceptors (13-15) immobilized on Rink amide polystyrene are shown in Figure 3.

Protecting group schemes for sugars that permit selective protection and deprotection at any position are well known. See., e. g., Binkley, Modern Carbohydrate Chemistry, Marcel Dekker, Inc.: New York (1988).

After the potentially reactive nucleophile is attache to the resin, it is selectively deprotected, if necessary.

All of the cited patents, publications and literature are incorporated herein by reference. The following specific examples are provided to better assist the reader in the various aspects of practicing the present invention. As these specific examples are merely illustrative, nothing in the following descriptions should be construed as limiting the invention in any way.

EXAMPLES Synthesis of the Glycosyl Sulfoxide Donors The phenylsulfenyl 2-deoxy-2-N-trifluoroacetamido glycospyranoses are synthesized from commercially available glucosamine and galactosamine hydrochlorides, as shown on Figures 1 and 2. Both syntheses are performed in multigram scales. The corresponding sulfoxides can be easily synthesized from commercially available sugars and are stable for several weeks at room temperature.

Example 1. Synthesis of phenylsulfenyl-2-deoxy-2-N-trifluoroacetamido-3,4,6-tri-0- acetyl- (3-D-glucopyranose (6) 2-N-p-methoxvvbenzylidene-ßD-glucosamine(1).

The procedure is adapted from Bergman, M, and Zervas, L. , Chem. Ber. 975.1931, Glucosamine hydrochloride (50 g; 0.232 moles) is dissolve in 240 mL of 1M aqueous sodium hydroxide, forming a colorless solution. Anisaldehyde (28.5 mL; 0.235 moles) is added via syringe under intense stirring, forming a turbid solution. After several minutes of agitation, a white precipitate is formed. The system is kept in an ice bath for one hour to ensure complete precipitation. The solid is then collecte by filtration and washed consecutively with water (2 x 200 mL) and a 1: 1 mixture of methanol and ether (2 x 200 mol).

The precipitate is dried overnight under vacuum, affording 1 (50 g; 72% yield).'H NMR <BR> <BR> <BR> <BR> (300 MHz, DMSO-d6): 8 178. (s, 1H), 7.74 (d, 2H, J= 8.1 Hz), 7.04 (d, 2H, J= 7.8 Hz), 6.60 (d, 1H, J = 6.6 Hz), 4.99 (d, 1H, J = 4.5 Hz), 4.88 (d, 1H, J = 4.8 Hz), 4.75 (d, 1H, J = 7.2 Hz), 4.62 (t, 1H, J= 5.4 Hz), 3.85 (s, 3H), 3.69 (dd, 1H, J= 5.4,11.1 Hz), 3.58-3.42 (ion, 2H), <BR> <BR> <BR> 3.32-3.16 (ion, 2H), 2.85 (t, 1H, J = 8.7 Hz). 13C NMR (75.4 MHz, DMSO-d6): 8 24,161.

161.06,129.65,129.11,113.91,95.64,78.21,76.88,74.61,70.36 ,61.27,55.29. Anal. Calcd. for C14H19NO6 (297.31): C, 56.56%; H, 6.44%; N, 4.71%. Found: C, 55.97%; H, 6.38%; N, 4.56%. (dec).148-150°C 2-deoxy-2-N-p-methoxybenzylidene-1, 3,4,6-tetra-0-acetyl-ßD-glucopyranose (2).

1 (50 g; 0.168 moles) is treated with acetic anhydride (150 mL; 1.59 moles), pyridine (270 mL; 3.34 moles) and DMAP (0.5 g) in an ice-water bath. The solid slowly goes into solution, and the rection mixture is left at room temperature overnight. The solution is poured into 1.5 L ice, forming a white crystalline solid. The crystals are collecte by filtration, washed with water (2 x 100 mL) and ether (2 x 100 mL) and dried under vacuum to afford 2 (60 g; <BR> <BR> <BR> 77% yield). TLC R = 0.45 (50% ethyl acetate-hexane).'H NMR (300 MHz, CDCl3): 8 158.

(s, I H), 7.64 (d, 2H, J = 8.4 Hz), 6.91 (d, 2H, J = 8.7 Hz), 5.94 (d, 1H, J = 8.1 Hz), 5.42 (t, 1H, J = 9.3 Hz), 5.14 (t, 1 H, J 9.6 Hz), 4. 37 (dd, 1H, J = 4.5,12.3 Hz), 4.12 (dd, 1H, J = 2.1,12. 6 Hz), 3.97 (ddd, 1 H, J 2.4,4.8,9. 6 Hz), 3.84 (s, 3H), 3.44 (t, 1 H, J = 9. 6 Hz), 2.10 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.88 (s, 3H). I3C NMR (75.4 MHz, CDCl3) : # 69,170.

169.89,169.54,168.77,164.27,162.26,130.22,128.24,114.02,9 3.12,73.20,72.91,72.72, 67.98,61.78,55.39,20.79,20.67,20.49 (br). IR (neat, cmi'): 2948,2869,1752,1640,1606, 1508, 1372, 1223, 1039. FAB+ for calcdm/z466,foundm/z466;[MNa][MH] calcd m/z 488, found m/z 488. Anal. Calcd. for C,56.77%;H,5.85%;(465.46): N, 3.01 %. Found: C, 56.56%; H, 5.90%; N, 2.99%. mp 168-172°C (dec).

2-deoxy-2-amino-1,3,4,6-tetra-0-acetyl-ßD-glucopyranosyl hydrochloride (3).

2 (50 g; 0.108 moles) is dissolve in 250 mL of refluxing acetone and to this solution is added dropwise 25 mL of 5N HCl. After five minutes a white thick precipitate forms and the system is cooled to room temperature. The precipitate is filtered and washed with acetone

(100 mL) and ether (2 x 250 mL). The crude product 3 is dried under vacuum overnight, yielding 41.8 g (quantitative). 1H NMR (300 MHz, DMSO-d6) : 8 938. (s, br, 2.6H), 5.97 (d, 1H, J = 8.7 Hz), 5.42 (t, 1H, J = 9.9 Hz), 4.99 (t, 1H, J = 9.3 Hz), 4.25 (dd, 1H, J = 3.9,12 Hz), 4.11-4.03 (m, 2H), 3.62 (t, 1H, J= 9.3 Hz), 2.23 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H).'3C NMR (75.4 MHz, DMSO-d6) : # 98,169.78,169.32,168.67,90.08,71.59,169.

70.31,67.76,61.25,52.11,20.97,20.88,20.51,20.37. IR (neat, cmi'): 2805,2745,2683, 1757, 1595, 1519, 1366, 1247, 1208, 1084, 1060, 1040. FAB+ for C14H27NO9Cl: Cl]- calcd m/z 348, found m/z 348; HCl]calcdm/z370,foundm/z370.Anal.Calcd.for- C14H22NO9Cl (383.78): C, 43.81%; H, 5.78%; N, 3.65 %; Cl, 9.24%. Found: C, 43.80%; H, 5.80%; N, 3.57%; Cl, 9.15%. mp>200°C.

2-deoxy-2-N-trifluoroacetamido-1,3,4,6-tetra-O-acetyl-ß- D-glucopyranose(4).

3 (41.6 g; 0.108 moles) is suspende in pyridine (90 mL; 1.11 moles) and methylene chloride (90 mL). Trifluoroacetic anhydride (18.5 mL; 0.131 moles) is slowly added via syringe. The solid slowly goes into solution with slight rise in temperature. The rection mixture is concentrated in vacuo to dryness. The residue is dissolve in 100 mL methylene chloride and washed with 2N HCl (1 x 100 mL), aqueous NAHCO 3 (2 x 100 mL) and brine (1 x 50 mol), and dried over anhydrous Na SO. The clear solution is concentrated to dryness affording the off-yellow solid 4 5(48. g; quantitative). TLC Rf. = 0.47 (50% ethyl acetate-hexane). 1H <BR> <BR> <BR> NMR (300 MHz, CDC1) : 6 247. (d, 1H, J= 9.0 Hz), 5.75 (d, 1H, J= 9.0 Hz), 5.31 (t, 1H, J = 10.0 Hz), 5.13 (t, 1H, J = 9. 6 Hz), 4. 35 (q, 1H, J = 9.9 Hz), 4.27 (dd, 1H, J = 4.8,12.6 Hz), 4.15 (dd, 1H, J = 2.1,12.6 Hz), 3.90 (ddd, 1H, J= 2.1,4.8,9.9 Hz), 2.12 (s, 3H), 2.10 (s, <BR> <BR> <BR> <BR> 3H), 2.06 (s, 3H), 2.05 (s, 3H). 13C NMR (75.4 MHz, CDCl3) : 8 67,170.67,169.43,171.

169.34,157.50 (J = 38 Hz), 115.51 (J = 286 Hz), 91.78,72.95,72.00,67.76,61.54,53.15, 20.67-20.37 (br). IR (neat, em~l): 3326,3100,2952; 1748,1560,1374,1219,1079,1042.

FAB+ for C H NO F: [MNa] calcd mlz 466, found mlz 466. Anal. Calcd. for 16 20 10 3 C,43.35%;H,4.55%;N,3.16%.Found:C,43.16%;H,4.51%;N,C16H20NO10 F3(443.33); 3.15%.

phenyl-l-thio-2-deoxy-2-N-trifluoroacetamido-3,4, 6-tri-O-acetyl-ß-D-glucopyranose (5).

4 (48.25 g; 0.109 moles) is dissolve in 400 mL anhydrous methylene chloride and treated with thiophenol (17 mL; 0.166 moles) and boron trifluoride etherate (42 mL; 0.331 moles).

The rection mixture is left overnight at room temperature and then poured into a solution of 100 mL saturated aqueous NaHCO3, 100 mL aqueous Na2C03 and 50 mL brine. The organic layer is ftirther washed with a mixture of 50 mL saturated aqueous NAHCO 3 and 50 mL aqueous Na2CO3. The organic layer is dried over anhydrous Na2SO4and concentrated under vacuum. The resulting solid is washed with 300 mL boiling hexane and filtered. The filtrate is further washed with 300 mL ice-cold hexane and dried under vacuum to yield 5 (49.2 g; 92 <BR> <BR> <BR> % yield). TLC Rf=0. 54 (50% ethyl acetate-hexane). 1H NMR (300 MHz, CDCl3) : 6 51-7.

7.48 (ion, 2H), 7.33-7.26 (ion, 3H), 7.02 (d, 1H, J= 9.3 Hz), 5.28 (t, 1H, J= 9.9 Hz), 5.03 (t, 1H, J = 9.6 Hz), 4.78 (d, 1H, J= 10.2 Hz), 4.22-4.13 (ion, 2H), 4.08 (q, 1H, J= 10.2 Hz), 3.78 <BR> <BR> <BR> (m, 1H), 2.08 (s, 3H), 2.00 (s, 3H), 1.93 (s, 3H). I3C NMR (75.4 MHz, CDCl3) : 8 32,168.

167.55,166.15,154.03 (q, J = 38 Hz), 130.38,128.02,125.95,125.68,112.46 (q, J = 288 Hz), 82.87,72.82,70.23,65.13,59.12,50.12,17.64,17.34,17.29. IR (neat, cm-1) : 3302, 3103,2951,2879,1748,1706,1557,1371,1217,1178,1077,1037. FAB+ for calcdm/z516,foundm/z516.mp178-180°C(dec).C20H22NSO8F3:[MNa] phenysulfenyl-2-N-trifluoroacetamido-3,4,6-tri-O-acetyl-ß-D -glucopyranose(6).

4 (49 g; 0.099 moles) is dissolve in 500 mL CH2Cl2 and cooled to -78 °C. Sodium bicarbonate (0.5 g) and mCPBA (26.3 g; 68.7% pure; 0.104 moles) are added, and the temperature is slowly raised to-25 OC. As the rection progresses, the product precipitates out of solution. When the rection is judged complete by TLC, it is quenched with 1 mL dimethyl sulfide and allowed to reach room temperature. The rection mixture is diluted with 200 mL water, 200 mL aqueous 100mLCH2Cl2.Theorganiclayeriswashedand with a mixture of 100 mL aqueous Na2CO3 mLbrine,driedoveranhydrousNa2SO4100 and concentrated under vacuum. The solid is washed with 300 mL hot ether, filtered, further washed with 300 mL of ice-cold ether and dried under vacuum, yielding 5 (48 g; 95% yield).

TLC Rif = 0.15-0.26 (50% ethyl acetate-hexane).'H NMR (300 MHz, CDCl, diastereoisomers in 5: 1 ratio) : 8 378. (d, 0.16H, J= 7.8 Hz), 7.83 (d, 0.84H, J = 8.7 Hz), 7.68-7.41 (m, 5H), 5.65 (t, 0.16H, J= 9.9 Hz), 5.45 (t, 0.84H, J= 9.6 Hz), 5.02 (t, 0.16H, J= 9.9 Hz), 4.96-4.88 (m, 1.84H), 4.21-3.94 (m, 3H), 3.81 (ddd, 0.84H, J = 2.7,3.9,10.2 Hz), 3.68 (ddd, 0.16H, J = 2.4,6.0,9.6 Hz), 2.22-1.93 (m, 9H). 13C NMR (75.4 MHz, CDCl, <BR> <BR> <BR> <BR> <BR> diastereoisomers in 5: 1 ratio): 8 76,170.42,169.33,157.27170. (q, J = 38 Hz), 137.42, 137.00,131.970,131.88,129.08,125.60,125.40,115.23 (q, J= 288 Hz), 92.03,88.52,76.75, 76.28,72.30,71.53,68.23,67.58,61.81,61.33,51.51,50.17,20.60, 20.49,20.37. IR (neat, cm-1): 3232, 3070, 2955, 1750, 1372, 1222, 1182, 1110, 1037. FAB+ for C20H27NSO9F3: [MNa] calcd m/z 532, found m/z 532. Anal. Calcd. for C20H22NSO9F3 (509.45): C, 47.15%; H, 4.35%; N, 2.75%; S, 6.29%. Found: C, 47.02%; H, 4.34%; N, 2.70%; S, 6,21%. mp 134-140°C (dec).

Figure 1 illustrates the synthetic scheme to arrive at the compound (6).

Example 2. Synthesis of phenylsulfenyl-2-deoxy-2-N-trifluoroacetamido-3,4,6-tari-0- acetyl- (3-D-galactopyranose (12) 2-N-p-methoxybenzylidene-ßD-galactosamine (7).

The procedure is adapted from Bergman, M, and Zervas, L. , Chem. Ber. 975.1931, Galactosamine hydrochloride (5.0 g; 215.64 g/mole; 23.2 mmoles) is dissolve in 24 mL of 1 M aqueous sodium hydroxide (24 mmoles), forming a colorless solution. Anisaldehyde (2.85 mL; 3.2 g; 136 g/mole; p 1.119; 23.5 moles) is added via syringe under intense stirring, forming a turbid solution. After several minutes of agitation, a white precipitate is formed.

The system is kept in an ice bath for one hour to ensure complete precipitation. The solid is then collecte by filtration and washed with water (100 mL) and a 1: 1 mixture of methanol and ether (2 x 25 mL). The precipitate is dried overnight under vacuum, affording crude 7 <BR> <BR> <BR> <BR> (4.5 g; 297 g/mole).'H NMR (300 MHz, DMSO-d6): 8 188. (s, 1H), 7.73 (d, 2H, J= 8.7 Hz), 7.04 (d, 2H, J = 8.7 Hz), 6.52 (d, I H, J = 6.9 Hz), 4.72-4.67 (m, 2H), 4.61 (d, I H, J = 6.9 Hz), 4.53 (d, 1H, J= 4.5 Hz), 3.85 (s, 3H), 3.73 (t, 1H, J= 3.9 Hz), 3.67-3.49 (m, 4H), 3.15 <BR> <BR> <BR> (t, 1H, J = 7.8 Hz). 13C NMR (75.4 MHz, DMSO-d6): 8 161.21,160.97,129.54,129.20,

113.88, 96.15, 75.15, 74.51, 71.62, 67.18, 60.69 55.28. ES-MS for calcd[MH]+ m/zm/z198, found m/z 298; [M-H] calcd m/z 296, found m/z 296. Anal. Calcd. for C14N19NO6 (297.31): C, 56.56%; H, 6.44%; N, 4.71%. Found: C, 55.46%; H, 6.42%; N, 4.58%.

2-deoxy-2-N-p-methoxybenzylidene-1,3,4,6-tetra-0-acetyl-Ã ŸD-galactopyranose (8).

7 (4.5 g; 83% pure; 297 g/mole; 13 mmoles) is treated with acetic anhydride (15 mL; 102.09 g/mole; 1.082; 159 mmoles), pyridine (79.10 g/mole; p 0.978; 27 mL; 334 mmoles) and DMAP (0.05 g) in an ice-water bath. The solid slowly goes into solution and the rection mixture is left at room temperature for three hours. The solution is poured into 200 mL ice, forming a white crystalline solid. The crystals are collecte by filtration, washed with water (100 mL) and ether (100 mL) and dried under vacuum to afford 8 8(3. g; 465 g/mole; 63% yield). TLC Rf. = 0.71 (60% ethyl acetate-hexane).'H NMR (300 MHz, CDCl): 8 8.21 (s, 1H), 7.65 (d, 2H, J= 9.0 Hz), 6.92 (d, 2H, J= 8.4 Hz), 5.92 (d, 1H, J = 8.1 Hz), 5.45 (d, 1H, J= 3.3 Hz), 5.25 (dd, 1H, J= 3.3,10.5 Hz), 4.22-4.14 (ion, 3H), 3.84 (s, 3H), 3.61 (dd, 1H, J= <BR> <BR> <BR> 8.4,10.5 Hz), 2.17 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.88 (s, 3H). 13c NMR (75.4 MHz, CDCl3) : 8 170.45,170.12,169.69,168.75,164.47,162.17,130.16,128.36,11.9 7,93.45, 93.45, 71.71, 71.52, 68.78, 65.90, 61.29, 55.41, 20.71-20.54 (m). ES-MS for C22H27NO10: [MH]+ calcd m/z 466, found m/z 466; m/z524,foundm/z524.calcd 2-deoxy-2-amino-l, 3,4,6-tetra-0-acetyl-ßD-galactopyranosyl hydrochloride (9).

8 3(7. g; 465 g/mole; 15.7 mmoles) is dissolve in 37 mL of refluxing acetone and to this solution is added dropwise 3.65 mL of 5N HC1. After five minutes a white thick precipitate forms and the system is cooled to room temperature. The precipitate is filtered and washed with acetone (30 mL) and ether (60 mL). After more solid separates from this filtrate, it is united with the solid obtained from the first filtrate. The combine product is dried under vacuum overnight, yielding 5.8 g of crude 9 (96% yield).'H NMR (300 MHz, DMSO-d6): 8 8.81 (s, br, 2.46H), 5.95 (d, 1H, J= 8.7 Hz), 5. 36-5.33 (m, 2H), 4.35 (t, 1H, J= 6.3 Hz), 4.12- 4.073 (ion, 2H), 3.48 (br, 1H), 2.22 (s, 3H), 2.18 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H). 13C NMR (75.4 MHz, DMSO-d6): 8 169.98,169.89,169.33,168.65,90.258,71.04,68.80,65.76,61.18,

49.33, 20.85, 20.88, 20.70, 20.53, 20.36. ES-MS for C14H27NO9Cl: Cl]+calcdm/z- 348, found m/z 348; H]-calcd- m/z 382 and 384, found m/z 382 and 384.

2-deoxy-2-N-trifluoroacetamido-1, 3,4,6-tetra-0-acetyl-ßD-galactopyranose (10).

9 (5.6 g; 383. 5 g/mole; 14.6 môles) is suspende in pyridine (15 mL; 14.7 g; 79.1 g/mole; p 0.978; 185 mmoles) and methylene chloride (15 mL). Trifluoroacetic anhydride (2.5 mL; 3.7 g; 210.3 g/mole; p 1.487; 17.7 immoles) is slowly added via syringe. The solid slowly goes into solution with slight rise in temperature. The rection mixture is concentrated in vacuo to dryness. The residue is dissolve in methylene chloride (50 mL) and washed with 2N HCl (2 x 50 mL), aqueous NaHCO3 (50 mL) and brine (20 mL), and dried over anhydrous Na SO.

The clear solution is concentrated to dryness affording the off-yellow solid 10 (5.6 g; 443 g/mole; 12.6 mmoles; 87% yield). TLC R~=0. 36 (40% ethyl acetate-hexane).'H NMR (300 MHz, CDCl3) : 8 7.15 (d, I H, J = 9.6 Hz), 5.77 (d, I H, J = 8.7 Hz), 5.40 (d, I H, J = 2.7 Hz), 5.17 (dd, 1 H, J = 3.3,11.1 Hz), 4.48 (q, I H, J = 9.3 Hz), 4.19-4.14 (m, 2 H), 4.07 (q, 1H, J = <BR> <BR> <BR> <BR> 6.6 Hz), 2.19 (s, 3H), 2.13 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). 13C NMR (75.4 MHz, CDCl) : 8 170.81,170.64,170.14,169.61,157.66 (q, J= 38 Hz), 115.52 (J= 286 Hz), 92.22,71.97, 69.84, 67.92, 66.10, 61.33, 50.152 20.63-20.41 (m). ES-MS for [M+NH4]+ calcd m/z 461, found m/z 461; [M-H]- calcdm/zm/z 442, found m/z 442. Anal. Calcd. for C16H20NO10F3 (443.33): C, 43.35%; H, 4.55%; N, 3.16%. Found: C, 43.34%; H, 4.60%; N, 3.11%. phenyl-1-thio-2-deoxy-2-N-trifluoroacetamido-3,4,6-tri-O-ace tyl-ß-D-galactopyranose(11).

10 (5.55 g; 443 g/mole; 12.5 mmoles) is dissolve in anhydrous methylene chloride (20 ml) and treated with thiophenol (2.0 mL; 110.15 g/mole; p 1.073; 19.5 mmoles) and boron trifluoride etherate (4.8 mL; 141.93 g/mole; p 1.120; 37.9 mmoles). The rection mixture is left overnight at room temperature and then poured into a mixture of 25 mL methylene chloride, 15 mL saturated aqueous NaHCO, 15 mL aqueous Na CO and 15 mL brine. The organic layer is further washed with a mixture of 15 mL saturated aqueous NAHCO 3 and 15

mL aqueous Na2CO3. The organic layer is dried over anhydrous and concentrated under vacuum. The resulting solid is washed with 50 mL boiling hexane and filtered. The filtrate is further washed with 50 mL ice-cold hexane and dried under vacuum to yield 11 (5.75 g; 493 g/mole; 11.7 mmoles; 93 % yield). TLC Rf=0. 35 (40% ethyl acetate-hexane).

'H NMR (300 MHz, CDCl3) : 8 7.53-7.50 (m, 2H), 7.34-7.31 (m, 3H), 6.63 (d, 1H, J = 9.0 Hz), 5.39 (d, 1H, J= 3.0 Hz), 5.20 (dd, 1H, J= 3.3,10.8 Hz), 4.87 (d, 1H, J= 10.2 Hz), 4.30- 4.10 (m, 3H), 3.96 (t, 1H, J= 6.3 Hz), 2.13 (s, 3H), 2.040 (s, 3H), 1.97 (s, 3H). 13C NMR (75.4 MHz, CDCl3) : 8 170.63,170.50,170.10,157.24 (q, J= 37 Hz), 132.87,131.71,129.00, 128.48,115.48 (q, J = 286 Hz), 86.43,74.60,70.76,66.60,61.69,50.05,20.65-20.41 (br).

ES-MS for calcdm/z511,foundm/z511;[M-H]-calcdm/z492,[M+NH4]+ found m/z 492. Anal. Calcd. for C,48.68%;H,4.49%;N,2.84%;S,(493.45): 6.50%. Found: C, 48.43%; H, 4.37%; N, 2.75%; S, 6.40%. phenysulfenyl-2-deoxy-2-N-trifluoroacetamido-3,4,6-tri-O-ace tyl-ß-D-galactopyranose(12).

11 (5.85 g; 493 g/mole; 11.9 mmoles) is dissolve in 50 mL CHZCIz and cooled to -78°C.

Sodium bicarbonate (0.1 g) and mCPBA (3.13 g; 68.7% pure; 172.57 g/mole; 12.5 mmoles) are added, and the temperature is slowly raised to-25 °C. As the rection progresses, the product precipitates out of solution. When the rection is judged complete by TLC, it is quenched with 1 mL dimethyl sulfide and allowed to reach room temperature. The rection mixture is diluted with 50 mL aqueous NaHCO3 and 50 mL CHZCIz. The organic layer is washed with a mixture of 50 mL aqueous Na2CO3 and 10 mL brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The solid is washed with 75 mL hot ether, filtered, further washed with 100 mL of ice-cold ether and dried under vacuum, yielding 12 as a single diastereoisomer (4.75 g; 509 g/mole; 9.3 mmoles; 78% yield). TLC Rf. = 0.31 (60% ethyl acetate-hexane).'H NMR (300 MHz, CDCl3) : 8 7.72-7.53 (m, 6H), 5.40 (dd, 1H, J = 3.3, 10.8 Hz), 5.34 (d, 1H, J = 3.3 Hz), 4.84 (d, 1H, J= 10.5 Hz), 4.34 (q, 1H, J = 10.2 Hz), 4.09- <BR> <BR> <BR> 3.94 (m, 3H), 2.00-1.96 (m, 9H).'3C NMR (75.4 MHz, CDCl3) : 8 170.33,170.27,169.83, 157.46 (J = 38 Hz), 138.01,131.85,128.95,125.57,115.23 (J = 286 Hz), 92.33,74.95,

69.74, 66.21, 61.09, 46.80, 20.60-20.39 (br). ES-MS for C20H27NSO9F3: [M+H]+ calcd m/z 510, found m/z 510; [M+NH4]+ calcd m/z 527, found m/z 527; [M-H]- calcd m/z 508, found m/z 508. Anal. Calcd. for C20H22NSO9F3 (509.45): C, 47.15%; H, 4.35%; N, 2.75%; S, 6.29%. Found: C, 47.35%; H, 4.50%; N, 2.63%; S, 6.10%. mp 116-118°C.

Figure 2 illustrates the synthetic scheme to arrive at the compound (12).

Example 3. Synthesis of ß disaccharide or glycoconjugate on solid phase.

In a typical glycosylation procedure using the phenylsulfenyl 2-deoxy-2-N- trifluoroacetamido glycopyranosyl donors, the glycosyl acceptor immobilized on Rink Amide resin and containing a free hydroxyl group is dried under high vacuum and then kept under argon. To the resin is added a solution of the glycosyl donor (4 equivalents) and 2,6-di-t- butyl-4-methyl-pyridine (2 equivalents) in a solvent system compatible with the conditions of the sulfoxide glycosylation rection (generally a 9: 1 mixture of anhydrous methylene chloride and anhydrous ethyl acetate). The mixture is stirred at room temperature for 5 minutes and then cooled to-78 °C. Trifluoromethanesulfonic anhydride (4 equivalents) is slowly added and the system is kept at-70 °C for 1 hour. The rection mixture is then kept at -45°C to-40 °C for 3-16 hours, and quenched with a mixture of methanol and diisopropylethylamine. The rection mixture is allowed to warm to room temperature and the resin is washed with DMF (3x), tetrahydrofuran (2x), methanol (2x), and methylene chloride (2x). For analytical purposes, a sample of the resin is then cleaved with a 30% cocktail of trifluoroacetic acid and methylene chloride for half an hour. The supernatant is evaporated to dryness and the residue is dissolve and analyzed by HPLC and LC-MS.

Glycosyl donor (6) is coupled with glycosyl acceptors (13), (14) and (15) to obtain the corresponding ß-linked disaccharide (16), (17) and (18) respectively. The trifluoroacetamido group brings extra flexibility to the sulfoxide glycosylation. Once activated, the sulfoxides are reactive and glycosylate unreactive nucleophiles such as glycosyl acceptor (13). The high reactivity of glycosyl donors such as (6) and (12) can be appreciated by comparing them to other studied sulfoxides.

Glycosylation of glycosyl acceptors (13) at >90% conversion can be achieved with 4 equivalents of glycosyl donors (6) or (12). When the corresponding phthalimido-protected

sulfoxides are used, up to 8 equivalents of sulfoxides have to be used to achieve the same conversion level. Use of 4 equivalents of the related sulfoxides containing Alloc and Fmoc protecting groups afforded a maximum of 10% conversion to the corresponding disaccharides. Glycosyl donor (6) has been used to successfully glycosylate acceptors (13), (14) and (15), as shown in Figure 3. The glycosylated products (16), (17) and (18) (Figure 4) are obtained in >90% yield (as determined by cleaving the product from the resin with a TFA-CH Cl cocktail and analyzing it by HPLC). For each product, the stereochemistry of the glycosidic linkage is found to be (3 by'H NMR with no trace of a-anomer. (Figure 5) Glycosyl donor (12) is also shown to glycosylate acceptor (13) in >90% yield (as determined by HPLC).

For (3-disaccharide products (16) and (17), the trifluoroacetamido group is shown to be completely removed by treating the resin with LiOH in 1: 1 MeOH-THF solution. Under these conditions, the benzoate and acetate groups are also removed. Selective derivatization of the amino group with a carboxylic acid in the presence of unprotected alcools is possible using HATU-DIPEA coupling conditions. The (3-disaccharide product (16) is submitted to the rection sequence shown in Figure 6. The identity and purity of each rection product are determined by HPLC and LC-MS. (Figure 7) Solution Phase Glycosylation So far, investigation in the solution phase glycosylation chemistry of these glycosyl donors has been restricted to the glycosylation of isopropanol with glycosyl donor (6). Under standard glycosylation conditions, the ß-isopropoxy glycoside is isolated in 60% yield and characterized by mass spectrometry and'H NMR. No trace of the corresponding a-isomer is observe.

Solid Phase Libraries Following on the chemistry previously shown, combinatorial libraries are designed around 16 and 17. In the case of 16, the disaccharide core is derivatized with 8 different isocyanates and 12 different carboxylic acids (Figure 8). In this library, the anomeric group of the acceptor sugar is either a ß-thiophenyl group or a (x, p-hydroxy group (lactols). These three elements of diversity yields a 192-member combinatorial library. In the case of 17, the

disaccharide core is derivatized with 6 different isocyanates and 8 different carboxylic acids, yielding a 48-member combinatorial library (Figure 9).

Example 4. Construction of Libraries.

For each disaccharide library, the corresponding disaccharide immobilized on Rink Amide resin (16 or 17) is fully deprotected by treatment with LiOH in 1: 1 THF-MeOH. The deprotected resin is then suspende in a 4: 1 mixture of methylene chloride and tetrahydrofuran, and aliquots of this suspension are dispense into a Irori MicroKanT"" containing an RF microtag. The aliquots are calculated so that each MicroKan contains 15 mg resin (in the case of 16) or 20 mg resin (in the case of 17). Each MicroKanTm and its RF tag are scanned into the Irori synthesis software and assigne an identification number. The libraries are then synthesized according to the rection schemes shown in Figures 10 and 11.

The results of LC-MS analyses are consistent with the production of the desired library compound on the basis of their molecular weights. (See, Figures 14 and 15, below.) Once the synthetic steps are completed, the MicroKanTm containers, containing the derivatized resins, are placed in separate test tubes and treated with a 30% TFA-CHZC12 cocktail for 30 minutes. The supernatants are then transferred to a well of a microtiter plate and concentrated under vacuum using a Savant evaporator. The resulting residues are then reconstituted in 1 ml of DMSO and the solutions are aliquote for control by LC-MS analysis, antibacterial screens and compound storage.

For the library based on scaffold 16, the LC-MS trace for a representative product is shown in Figure 12. The analytical results obtained ftom the LC-MS analysis of this library are summarized in Figure 13.

For the library based on scaffold 17, the LC-MS trace for a representative product is shown in Figure 14. The analytical results obtained ftom the LC-MS analysis of this library are summarized in Figure 15.

The above examples have been depicted solely for the purpose of exemplification and are not intended to restrict the scope or embodiments of the invention. The invention is further illustrated with reference to the claims that follow hereto.